Patent Application
20240327444
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The present invention relates to a method for purifying a nucleotide-based substance, a device for purifying a nucleotide-based substance, a hydrophobic reagent, and a hydrophobic substrate.
Nucleic acids such as DNA and RNA are important molecules in life science, and have been studied for many years in the medical field and the like. In recent years, pharmaceutical applications of mRNAs have attracted particular attention. In an mRNA drug, a therapeutic effect is obtained by synthesizing a protein in cells, unlike an antisense nucleic acid that inhibits protein synthesis, a small interfering RNA (siRNA) that causes RNA interference, and the like. Therefore, it is possible not only to inhibit abnormal proteins but also to replenish normal proteins, and the realization of mRNA drugs leads to expansion of therapeutic strategies using nucleic acid drugs. In addition, mRNAs do not need to be transported to the nucleus, and have no risk of genomic insertional mutation, and therefore have one aspect of being superior in safety. Furthermore, in recent years, as the development of nucleic acid delivery related technologies has progressed, it has become widely recognized that the low cell membrane permeability of an mRNA can be overcome eventually, and mRNA drugs can be realized.
High-performance liquid chromatography (HPLC) and denaturing polyacrylamide gel electrophoresis have been used for the isolation and purification of mRNAs. So far, an RNA purification method utilizing hydrophobicity has been known. For example, a method has been reported in which a dinitrobenzene protecting group, which is a hydrophobic tag, is introduced to an RNA end synthesized on a solid phase on the basis of the phosphoramidite method, and the RNA is purified by reverse-phase HPLC (see, for example, Pradere, U. et al., Chem. Eur. J. 2017, 23, 5210-5213, Non-Patent Literature 1, c of
On the other hand, the present inventors have developed primers that are used for amplification of nucleic acids and have a degradable protecting group (see, for example, WO 2021/020562 A, Patent Literature 1). This degradable protecting group is composed of a nitrobenzyl group or the like, and is introduced into a phosphate group at the 3′-position of the nucleotide at the intermediate position rather than an end position such as the 5′-end or the 3′-end among the nucleotides constituting the nucleic acid.
The method of Non-Patent Literature 1 is used only for purification of about 90 residues of chemically synthesized RNA. The dinitrobenzene protecting group in Non-Patent Literature 1 has two nitrobenzyl groups, and the carbon located therebetween is electrophilic and highly reactive. For this reason, the dinitrobenzene protecting group is easily attacked by ammonium or the like and is easily degraded, and therefore stability after introduction of the protecting group is poor, and the yield of an objective nucleotide-based substance is low. In addition, since the dinitrobenzene protecting group of this literature has two highly reactive nitrobenzyl groups in the molecule, it is explosive, and is difficult to handle and dangerous. Furthermore, in this literature, neither purification with a long RNA having 100 or more bases nor purification by a transcription method using a polymerase is described.
An object of the present invention is to provide a method for purifying a nucleotide-based substance and a device for purifying a nucleotide-based substance, with which a nucleotide-based substance with a protecting group introduced is stable, a yield of a nucleotide-based substance after deprotection is high, and safety is high. Another object of the present invention is to provide a hydrophobic reagent and a hydrophobic substrate to be used for such a method or a device for purifying a nucleotide-based substance.
The present inventors have intensively studied to solve the above problems. As a result, the present inventors have found that by employing mononitrobenzene as a protecting group, a nucleotide-based substance into which a protecting group has been introduced become stable, and the yield of a nucleotide-based substance attained through deprotection by light or by a reduction reaction is also improved, thereby having accomplished the present invention. The present invention is as follows.
[1] A method for purifying a nucleotide-based substance having at least one nucleotide and/or a derivative thereof as a constituent unit, the method comprising:
[2] The method for purifying a nucleotide-based substance according to the above [1], wherein the hydrophobic protecting group is represented by the following formula (P3),
[3] The method for purifying a nucleotide-based substance according to the above [1], wherein the protecting group introduction step introduces the hydrophobic protecting group into a nucleotide located at the 5′-end of the nucleotide-based substance.
[4] The method for purifying a nucleotide-based substance according to the above [3], wherein the protecting group introduction step introduces the hydrophobic protecting group into a 5′-phosphate group, a 2′-hydroxyl group, or a base of a sugar constituting the nucleotide.
[5] The method for purifying a nucleotide-based substance according to the above [1], wherein in the protecting group introduction step, a protecting group-introduced nucleotide is synthesized by introducing an amidite reagent represented by the following formula (CR1) into a nucleotide, and a mononucleotide is sequentially linked to a 3′-end side of the protecting group-introduced nucleotide by solid phase synthesis to synthesize the hydrophobic nucleotide-based substance,
[6] The method for purifying a nucleotide-based substance according to the above [1], wherein in the protecting group introduction step, the hydrophobic nucleotide-based substance is synthesized by employing, as a template, a nucleic acid having a sequence complementary to the nucleotide-based substance, employing, as substrates, a hydrophobic substrate represented by the following formula (ER1) and a nucleoside triphosphate, and transcribing the template by an RNA polymerase,
[7] The method for purifying a nucleotide-based substance according to the above [1], wherein in the protecting group introduction step, an adenylation reagent represented by the following formula (CA1) is reacted with a phosphate group at a 5′-end of an oligonucleotide containing the nucleotide-based substance to synthesize the hydrophobic nucleotide-based substance,
[8] The method for purifying a nucleotide-based substance according to the above [1], wherein in the protecting group introduction step, the hydrophobic nucleotide-based substance is synthesized by employing, as a template, a nucleic acid having a sequence complementary to the nucleotide-based substance, employing, as substrates, a hydrophobic substrate represented by the following formula (EA1) and a nucleoside triphosphate, and transcribing the template by an RNA polymerase,
[9] The method for purifying a nucleotide-based substance according to the above [1], wherein in the protecting group introduction step, a capping reagent selected from the group consisting of the following formulas (CC1) to (CC4) is reacted with a phosphate group at a 5′-end of an oligonucleotide containing the nucleotide-based substance to synthesize the hydrophobic nucleotide-based substance,
[10] The method for purifying a nucleotide-based substance according to the above [1], wherein in the protecting group introduction step, the hydrophobic nucleotide-based substance is synthesized by employing, as a template, a nucleic acid having a sequence complementary to the nucleotide-based substance, employing, as substrates, a hydrophobic substrate containing a structure of the following formula (EC0) and a nucleoside triphosphate, and transcribing the template by an RNA polymerase,
[11] The method for purifying a nucleotide-based substance according to the above [10], wherein the Nuc is a natural or non-natural nucleoside or a natural or non-natural nucleoside in which one or two natural or non-natural nucleotides are linked to the carbon at the 3′-position thereof.
[12] The method for purifying a nucleotide-based substance according to the above [10], wherein the hydrophobic substrate having the structure of the formula (EC0) is selected from the group consisting of the following formulas (EC1) to (EC4) and the following formulas (EC5) to (EC12),
[13] The method for purifying a nucleotide-based substance according to the above [10], wherein the hydrophobic substrate is a cap derivative represented by the formula (EC0), and
[14] The method for purifying a nucleotide-based substance according to the above [1], wherein in the isolation and purification step, the hydrophobic nucleotide-based substance is isolated and purified by high-performance liquid chromatography.
[15] The method for purifying a nucleotide-based substance according to the above [1], wherein in the deprotection step, the hydrophobic protecting group is deprotected by photoirradiation.
[16] A device for purifying a nucleotide-based substance having at least one nucleotide and/or a derivative thereof as a constituent unit, the device comprising:
[17] A hydrophobic reagent for synthesizing a hydrophobic nucleotide-based substance by chemical synthesis, wherein the hydrophobic reagent is selected from the group consisting of the following formulas (CR1), (CA1), and (CC1) to (CC4),
[18] A hydrophobic substrate for producing a hydrophobic nucleotide-based substance by an RNA polymerase, wherein the hydrophobic substrate is selected from the group consisting of the following formulas (ER1), (EA1), and (EC0),
[19] The hydrophobic substrate according to the above [18], wherein the Nuc is a natural or non-natural nucleoside or a natural or non-natural nucleoside in which one or two natural or non-natural nucleotides are linked to the carbon at the 3′-position thereof.
[20] An mRNA drug comprising a hydrophobic nucleotide-based substance in which a hydrophobic protecting group represented by the following formula (P1) or (P2) is introduced into a nucleotide-based substance having at least one nucleotide and/or a derivative thereof as a constituent unit,
[21] The mRNA drug according to [20], having a structure in which RNA is linked to carbon at a 3′-position of a nucleotide on a 3′-end side of a hydrophobic substrate containing a structure of the following formula (EC0),
[23] The mRNA drug according to the above [21], wherein the hydrophobic substrate containing the structure of the formula (EC0) is selected from the group consisting of the following formulas (EC1) to (EC12),
[24] A method for producing the mRnNA drug according to the above [20], the method comprising:
[25] The method for purifying the mRNA drug according to the above [21], wherein in the protecting group introduction step, the hydrophobic nucleotide-based substance is synthesized by employing, as a template, a nucleic acid having a sequence complementary to the nucleotide-based substance, employing, as substrates, a hydrophobic substrate having a structure of the formula (EC0) and a nucleoside triphosphate, and transcribing the template by an RNA polymerase.
[Opt 1] An mRNA drug of the above [21], wherein the hydrophobic substrate having the structure of the formula (EC0) is deprotected and Pro1 to Pro4 are hydrogen.
[Opt 2] The mRNA drug according to the above [Opt 1], wherein the hydrophobic substrate containing the structure of the formula (EC0) is selected from the group consisting of the formulas (EC1) to (EC12), the hydrophobic protecting group is deprotected, and Pro is hydrogen.
The present invention makes it possible to provide a method for purifying a nucleotide-based substance and a device for purifying a nucleotide-based substance, with which a nucleotide-based substance with a protecting group introduced is stable, a yield of a nucleotide-based substance after deprotection is high, and safety is high. In addition, the present invention makes it possible to provide a hydrophobic reagent and a hydrophobic substrate to be used for such a method or a device for purifying a nucleotide-based substance.
Hereinafter, the method for purifying a nucleotide-based substance of the present invention will be described. The method for purifying a nucleotide-based substance of the present invention is a method for purifying a nucleotide-based substance to be purified, and includes a protecting group introduction step, an isolation and purification step, and a deprotection step. Each will be described below. The “nucleotide-based substance” referred to in the present invention is any compound having at least one nucleotide and/or a derivative thereof as a constituent unit. Nucleotides are composed of a sugar, a base, and a phosphoric acid. Nucleotide-based substances include mononucleotides each composed of one nucleotide, oligonucleotides and polynucleotides in each of which a plurality of nucleotides are linked (the oligonucleotides and the polynucleotides are both referred to as “nucleic acids”), and derivatives thereof. Here, the term oligonucleotides mean nucleic acids (DNA, RNA) having 2 to 20 nucleotides, and the term polynucleotides means nucleic acids having 21 or more nucleotides. In addition, the nucleotide-based substances also include those having a branch structure in which the 5′-end of a nucleotide is branched into two or more, and examples of the branch structure include a bi-branched type structure and a tri-branched type structure.
In addition, examples of the derivative of at least one nucleotide include mononucleotide or nucleic acids (oligonucleotides, polynucleotides) subjected to modification or the like, for example, these nucleotides modified by methylation or the like, and those nucleotides whose 5′-end is adenylated or capped. The sugar constituting the nucleotide-based substance of the present invention may be ribose or deoxyribose. Examples of the base constituting the nucleotide-based substance of the present invention include adenine, guanine, cytosine, thymine, uracil, N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoadenine, 7-methylguanine, N-isobutyrylguanine, 5-fluorocytosine, 5-bromocytosine, 5-methylcytosine, 4-N-methylcytosine, 4-N,N-dimethylcytosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, and 5,6-dihydrouracil. Furthermore, examples of the phosphoric acid constituting the nucleotide-based substance of the present invention include monophosphoric acid, diphosphoric acid, and triphosphoric acid.
The protecting group introduction step is a step of introducing a hydrophobic protecting group (also referred to as “fat-soluble protecting group” or “purification tag”) represented by the following formula (P1) or (P2) into a nucleotide-based substance to produce a hydrophobic nucleotide-based substance,
Among them, R1 is a linear or branched alkyl group having 1 to 30 carbon atoms, and R4 is preferably hydrogen. In addition, from the viewpoint of stabilizing a hydrophobic protecting group, R1 is preferably a branched alkyl group, more preferably a secondary or tertiary alkyl group, and particularly preferably a tertiary alkyl group. On the other hand, the number of carbon atoms of R1 is preferably 5 or more, more preferably 10 or more from the viewpoint that the hydrophobicity of the hydrophobic protecting group is increased and the compound is easily separated from other compounds. In particular, from the viewpoint of increasing the degradation efficiency of the hydrophobic protecting group, R4 is preferably hydrogen when the hydrophobic protecting group is photodegradable, and R4 is preferably an alkyl group when the hydrophobic protecting group is degradable by a reductant.
In particular, as the hydrophobic protecting group, those having a tert-butyl group represented by the following formula (P3) are preferable,
In this case, in particular, R2 and R3 are preferably hydrogen.
When the nucleotide-based substance is a macromolecular compound such as an oligonucleotide or a polynucleotide, the hydrophobic protecting group can be introduced into any nucleotide, but it is particularly preferable to introduce the hydrophobic protecting group into a nucleotide located at the 5′-end of the nucleotide-based substance. The nucleotide located at the 5′-end is a nucleotide located at the most end on the 5′-side in the case of DNA, RNA, or the like. In addition, in the case of adenylation (AMP formation) or capping (CAP formation) described later, the nucleotide located at the 5′-end is the adenosine or the cap (7-methylguanylic acid) bonded to the nucleotide at the 5′-end of DNA or RNA.
In addition, the hydrophobic protecting group may be introduced into an arbitrary position of a nucleotide, and, in particular, is preferably introduced into a 5′-phosphate group, a 2′-hydroxyl group, or a base of a sugar.
When a hydrophobic protecting group is introduced in the 3′-phosphate group of a nucleotide at the intermediate position instead of a nucleotide located at an end as described in Patent Literature 1, the hydrophobic protecting group is present in a hydrophilic region composed of an oligonucleic acid, so that a hydrophobic effect is less likely to be exerted and purification is likely to be difficult. In addition, when a hydrophobic protecting group is introduced in the 3′-phosphate group of a nucleotide at the intermediate position instead of a nucleotide located at an end as described in Patent Literature 1, if the hydrophobic protecting group is degraded in the process until the nucleotide-based substance is purified under hydrophobic conditions, the nucleotide-based substance may be cleaved at an intermediate position thereof. Therefore, the hydrophobic protecting group is preferably introduced to a nucleotide located at the 5′-end, and specifically to a functional group that does not constitute a chain structure of a nucleic acid, such as a 5′-phosphate group, a 2′-hydroxy group, or a base. As a result of introducing a hydrophobic protecting group at an end position of such a nucleic acid, even a nucleic acid having a long hydrophilic moiety such as a long-chain RNA having 1000 or more bases can be easily purified by the hydrophobic protecting group.
When a hydrophobic protecting group is introduced into a phosphate group or a hydroxy group, the hydrophobic protecting group is preferably bonded directly to an oxygen atom constituting the phosphate group or the hydroxy group, or bonded via a linking group selected from —O—C— and —O—C(═O)—. When a hydrophobic protecting group is introduced into a base, the hydrophobic protecting group is preferably bonded to a nitrogen atom of an amino group constituting the base via a linking group selected from —O—C— and —O—C(═O)—. More specifically, from the viewpoint of the degree of the stability and the degree of ease of synthesis of the compound with a hydrophobic protecting group introduced therein, the position to which the hydrophobic protecting group is introduced is more preferably as follows.
The isolation and purification step is a step of isolating and purifying a hydrophobic nucleotide-based substance under a hydrophobic environment.
The deprotection step is a step of deprotecting a hydrophobic protecting group from a hydrophobic nucleotide-based substance to generate a nucleotide-based substance.
In Examples described later, examples of three types of purification methods, i.e., phosphorylation purification, adenylation purification, and capping purification, are described as the hydrophobic protecting group. Examples of the protecting group introduction step include a method of synthesizing a hydrophobic nucleotide-based substance through chemical synthesis using a hydrophobic reagent having a hydrophobic protecting group (chemical synthesis) and a method of synthesizing a hydrophobic nucleotide-based substance through an enzymatic reaction by a polymerase using a template and a substrate (enzyme synthesis). Hereinafter, chemical synthesis and enzyme synthesis will be described for each of the above three purification methods.
Phosphorylation purification is a method in which a hydrophobic protecting group (phosphorylation tag) is bonded to a phosphate group at the 5′-position of an endmost nucleotide on the 5′-side of a nucleotide-based substance, and the nucleotide-based substance is purified utilizing the hydrophobicity of the hydrophobic protecting group.
The phosphorylation purification tag is a hydrophobic protecting group that bonds to the phosphate group at the 5′-position of an endmost nucleotide on the 5′-side of a nucleotide-based substance. Examples of the phosphorylation purification include a method of introducing a hydrophobic protecting group through chemical synthesis and a method of introducing a hydrophobic protecting group through enzyme synthesis. In the following synthesis scheme (hereinafter, “phosphorylation purification scheme”), as one example of the hydrophobic protecting group, an example of using “bNB” in which R1 is a tert-butyl (t-Bu) group and R2 to R4 are hydrogen in the formula (P1) is disclosed. Note that bNB is merely one example of the hydrophobic protecting group of the present invention, and the present invention is not limited thereto, and can be appropriately replaced by another compound included in the formula (P1) or the formula (P2).
The upper part (a) of the phosphorylation purification scheme shows a scheme for synthesizing and purifying a hydrophobic nucleotide-based substance by a chemical method. In this method, a hydrophobic nucleotide-based substance having a hydrophobic protecting group of formula (P1) or formula (P2) is synthesized using an amidite reagent represented by the following formula (CR1) as a hydrophobic reagent.
The amidite reagent represented by the formula (CR1) can be obtained by reacting a phosphoramidite compound such as 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite with nitrobenzyl alcohol as a starting material.
In the method using the amidite reagent, a protecting group-introduced nucleotide is synthesized in the protecting group introduction step by introducing the amidite reagent into a nucleotide. Next, a mononucleotide is sequentially linked to the 3′-end side of a protecting group-introduced nucleotide by solid phase synthesis, and thereby a hydrophobic nucleotide-based substance is synthesized. The solid phase synthesis can be performed using a publicly known DNA synthesis apparatus. The hydrophobic nucleotide-based substance as a synthetic term can be isolated and purified from other nucleic acids using hydrophobicity (fat-solubility) by reverse-phase HPLC or the like, and further can be deprotected by photoirradiation or the like.
The lower part (b) of the phosphorylation purification scheme shows a scheme for synthesizing a hydrophobic nucleotide-based substance by an enzymatic method. When the nucleotide-based substance is an RNA, the enzyme to be used is preferably an RNA polymerase, and particularly preferably T7 RNA polymerase. The above scheme shows an example in which an RNA is synthesized using T7 RNA polymerase. In this case, in the protecting group introduction step, a nucleic acid (DNA) having a sequence complementary to a nucleotide-based substance on the downstream side of a T7 promoter sequence is used as a template. As substrates for T7 RNA polymerase, a hydrophobic substrate (guanosine monophosphate derivative) of the following formula (ER1) and a nucleoside triphosphate (NTP, i.e., ATP, GTP, CTP, or UTP) are used.
In the reaction of T7 RNA polymerase, transcription is initiated from GTP, and therefore when a guanosine monophosphate derivative and guanosine triphosphate (GTP) are used as substrates, two types of RNA, i.e., one having a guanosine monophosphate derivative at the 5′-end and one having guanosine triphosphate at the 5′-end, are generated in a mixed state. These two types of RNA can be separated according to hydrophobicity by using reverse-phase HPLC or the like, and only a hydrophobic nucleotide-based substance derived from the guanosine monophosphate derivative can be isolated and purified. The transcription of the template by the T7 RNA polymerase can be performed under appropriately set conditions, and for example, the reaction temperature may be set to 30 to 45° C., and the reaction time may be set to 1 to 5 hours. In general, T7, T3, and SP6 RNA polymerases initiate transcription from GTP. T7 RNA polymerase recognizes GTP, and an RNA chain extends to the 2′-hydroxyl group of the GTP. Therefore, when a hydrophobic substrate (guanosine monophosphate derivative) of the above formula (ER1) is used, not only T7 RNA polymerase but also T3 and SP6 RNA polymerases can be used. On the other hand, E. coli RNA polymerase, which is commercially available from New England Biolabs and so on, initiates transcription also from ATP. When this enzyme is used, an RNA chain can be extended from the 2′-hydroxyl group by using an adenosine monophosphate derivative having adenine instead of the guanine of ER1.
Adenylation purification is a method in which an adenylated RNA is generated by a transcription reaction using an adenylation purification tag and is isolated with high purity. An adenylation moiety is co-transcriptionally introduced into the 5′-end of an RNA transcript, and the resulting product can be separated from coexisting RNAs by reverse-phase HPLC or the like utilizing the hydrophobicity of a hydrophobic protecting group (adenylation purification tag) introduced into the adenyl group moiety. Thereafter, the protecting group is removed by photoirradiation or the like, and an objective adenylated RNA can thereby be obtained.
The adenylation purification tag is a hydrophobic tag for obtaining an adenylated nucleic acid. The adenylated nucleic acid has a structure in which an adenosine monophosphate derivative with a hydrophobic protecting group introduced therein is bonded to a nucleotide on the 3′-side with two phosphate groups interposed therebetween. Examples of the adenylation purification as well include a method of introducing a hydrophobic protecting group through chemical synthesis and a method of introducing a hydrophobic protecting group through enzyme synthesis. The following synthesis scheme (hereinafter, “adenylation purification scheme”) also shows an example using “bNB”. Also in this example, the bNB may be appropriately replaced by another compound of the formula (P1) or the formula (P2).
The adenylation reagent represented by the formula (CA1) can be synthesized by the following procedure. First, a nitrophenylimidazole compound is synthesized using iodonitrobenzene as a starting material, reacted with adenosine in which a hydroxy group or the like is protected with a protecting group such as tert-butyldimethylsilane, and then deprotected to synthesize an adenosine derivative to which a hydrophobic protecting group is bonded. This adenosine derivative is reacted with imidazole, dithiodypyridine, or the like, and thus an adenylation reagent represented by the formula (CA1) can be obtained.
The dinucleotide derivative represented by the formula (EA1) can be obtained by reacting guanosine 5′-phosphorimidazolide with the adenosine derivative of “(1) Chemical synthesis” described above.
When the dinucleotide derivative and guanosine triphosphate (GTP) are used as substrates, two types of RNA, i.e., an RNA having a protecting group-containing adenylated structure at the 5′-end and an RNA having a triphosphate structure, are generated in accordance with the mechanism of T7 RNA polymerase that initiates a transcription reaction from a guanosine. In the RNA having a protecting group-containing adenylated structure at the 5′-end, an adenosine derivative at the 5′-end and a nucleotide on the 3′-end side thereof are bonded with a diphosphate group interposed therebetween. These two types of RNA can be separated according to hydrophobicity by using reverse-phase HPLC or the like, and only the RNA having the protecting group-containing adenylated structure can be isolated and purified. The transcription of the template by the T7 RNA polymerase can be performed under appropriately set conditions, and for example, the reaction temperature may be set to 30 to 45° C., and the reaction time may be set to 1 to 5 hours.
Documents disclosing existing techniques related to adenylation purification include the following.
Yangming Wang and Scott K. Silverman “Efficient RNA 5′-adenylation by T4 DNA ligase to facilitate practical applications” RNA 2006, 12, 1142-1146. doi: 10.1261/rna.33106
Eukaryotic mRNA has a cap structure at the 5′-end. When a ribosome bonds to the cap structure, the initiation of translation of an mRNA is strongly promoted. Therefore, when an mRNA molecule is artificially produced, it is essential to provide the cap structure at the 5′-end of the molecule. Generally, a several thousands-base-long mRNA molecule is enzymatically synthesized from a template DNA by transcription with an RNA polymerase. By adding a cap analog compound that is a dinucleotide to the transcription reaction, a cap structure can be co-transcriptionally introduced into the 5′-end of the mRNA. However, since transcription is initiated also from the GTP of a coexisting transcribed substrate, the resulting RNA transcript is a mixture of a desired one having a cap analog at the 5′-end (5′-cap-RNA) and one having a triphosphate group (5′-ppp-RNA). It is known that the RNA having a triphosphate group at the 5′-end has an undesired immune response-inducing activity in vivo, and it is necessary to remove this.
5′-cap-RNA and 5′-ppp-RNA which are prepared using an existing cap compound are very similar in physical properties, and it is very difficult to separate them by a chromatography or the like. An RNA having a triphosphate group at an end (5′-ppp-RNA) can be degraded by an enzyme having a degrading activity against the RNA (RNA 5′-polyphosphatase (epicentre, RP8092H), RNA 5′-pyrophosphohydrolase (RppH) (New England Biolabs, M0356), and the like), and thus can be converted into 5′-monophosphorylated RNA using these enzymes. Furthermore, a monophosphorylated RNA can also be degraded by an enzyme such as XRN-1 (New England Biolabs, M0338) and removed (reference: Chem. Sci., 2021, 12, 4383-4388). Up to date, the isolation and purification of 5′-cap-RNA has taken time and effort as described above.
Then, in the present invention, a novel cap analog compound having a photodegradable or reductive degradable hydrophobic protecting group is used, and an objective capped RNA prepared in a co-transcriptional manner can be isolated from the above-mentioned contaminants by reverse-phase HPLC. The present method makes it possible to obtain an object having a higher purity more simply as compared especially with an enzymatic removal reaction.
The capping purification tag is a hydrophobic tag for obtaining a capped nucleic acid. The capped nucleic acid has a structure in which a cap structure with a hydrophobic protecting group introduced therein is bonded to a nucleotide on the 3′-side with three or four phosphate groups interposed therebetween. Examples of the capping purification as well include a method of introducing a hydrophobic protecting group through chemical synthesis and a method of introducing a hydrophobic protecting group through enzyme synthesis. The following synthesis scheme (hereinafter, “capping purification scheme”) also shows an example using “bNB”. Also in this example, the bNB may be appropriately replaced by another compound of the formula (P1) or the formula (P2). In the following capping purification scheme, only a scheme using a capping reagent of the formula (CC3) described later is shown. However, the present scheme is not limited thereto, and capping of a nucleic acid and purification of a hydrophobic nucleotide-based substance can be performed by a similar scheme even when a capping reagent of the formula (CC1), (CC2), or (CC4) is used.
The capping reagents represented by the formulas (CC1) to (CC4) can be synthesized by the following procedure. First, a nitrophenylimidazole compound is synthesized using iodonitrobenzene as a starting material, reacted with guanosine in which a hydroxy group or the like is protected with a protecting group such as tert-butyldimethylsilane, and then deprotected to synthesize a guanosine derivative to which a hydrophobic protecting group is bonded. Iodomethane, dimethyl sulfoxide (DMSO) or the like is reacted with the guanosine derivative to methylate the 7′ position of guanosine, and imidazole, dithiodypyridine or the like is reacted to bond imidazole to a phosphate group, whereby capping reagents represented by the formulas (CC1) to (CC4) can be obtained. Besides imidazole, various leaving groups can be used, and examples of such leaving groups include various imidazole derivatives and nitrogen-containing heteroaromatic compounds described in Japanese Patent Application No. 2020-032889 such as 1-methylimidazole and 4-methylimidazole.
(a) of the capping purification scheme shows a scheme in which an RNA having monophosphoric acid at the 5′-end is reacted with a diphosphate form capping reagent (n in the above formulas (CC1) to (CC4) is an integer of 2). In this case, the hydrophobic nucleotide-based substance to be generated has a structure in which the cap derivative at the 5′-end and the nucleoside at the 3′-end side are bonded with three phosphate groups interposed therebetween. On the other hand, (b) of the capping purification scheme shows a scheme in which an RNA having a triphosphate at the 5′-end is reacted with a monophosphate form capping reagent (n in the above formulas (CC1) to (CC4) is an integer of 1). In this case, the hydrophobic nucleotide-based substance to be generated has a structure in which the cap derivative at the 5′-end and the nucleoside at the 3′-end side are bonded with four phosphate groups interposed therebetween.
(c) of the capping purification scheme shows a scheme of synthesizing and purifying a hydrophobic nucleotide-based substance by an enzymatic method. This scheme shows an example of synthesizing an RNA using T7 RNA polymerase. In this case, in the protecting group introduction step, a nucleic acid (DNA) having a sequence complementary to a nucleotide-based substance on the downstream side of a T7 promoter sequence is used as a template. As substrates for T7 RNA polymerase, a hydrophobic substrate (cap derivative) containing a structure of the following formula (EC0) and a nucleoside triphosphate (NTP, i.e., ATP, GTP, CTP, or UTP) are used.
In US 20180273576 A1 [Compositions and methods for synthesizing 5′-capped RNAs], a transcription reaction is performed using a cap analog having a trinucleotide composition. It is possible to introduce a hydrophobic tag into this analog. Scheme (d) shows a method of introducing a cap purification tag through a transcription reaction using a trinucleotide as a substrate,
At least one of Pro1 to Pro4 is a hydrophobic protecting group represented by the above formula (P1) or (P2), and the rest are hydrogen. The number of hydrophobic protecting groups as Pro1 to Pro4 is preferably one or two.
The nucleoside constituting Nuc has a structure in which a base is bonded to the carbon at the 1′-position of a ribose, which is a sugar, or a derivative thereof. While a hydroxy group is bonded to the carbon at the 2′-position of the ribose, examples of a ribose derivative include a ribose derivative in which a hydroxy group is substituted with an alkyl group, and a ribose derivative in which oxygen bonded to the carbon at the 2′-position and carbon bonded to the carbon at the 4′-position are bonded to each other to form a heterocyclic ring. The base is the same as the Base described later, and is a natural nucleobase or a non-natural base. Examples of the natural nucleobase include adenine, cytosine, thymine, uracil, and guanine. Examples of the non-natural base may be N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoadenine, 7-methylguanine, N-isobutyrylguanine, 5-fluorocytosine, 5-bromocytosine, 5-methylcytosine, 4-N-methylcytosine, 4-N,N-dimethylcytosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, and 5,6-dihydrouracil.
To the carbon at the 3′-position of the nucleoside constituting Nuc may be linked one or two or more (preferably two) nucleotides. In this case, it is preferable that the carbon at the 3′-position of the nucleoside and the carbon at the 5′-position of the nucleotide are linked by a phosphodiester linkage. When there are two nucleotides, it is preferable that the nucleotides are 5′-3′-phosphodiester linked. The nucleotides are, similarly to the nucleoside constituting Nuc described above, composed of a sugar and a base, and the sugar is composed of a ribose or a derivative thereof, and the base is composed of a natural nucleobase or a non-natural base. Specific examples of Nuc include DNA, RNA, LNA, 2′OMe-RNA, 2′-methoxyethyl RNA, acyclic nucleosides, and linkers containing a heteroatom.
Examples of the hydrophobic substrate represented by the formula (EC0) include hydrophobic substrates selected from the group consisting of the following formulas (EC1) to (EC4) and the following formulas (EC5) to (EC12).
The cap derivatives represented by the formulas (EC1) to (EC4) can be obtained by reacting guanosine 5′-phosphorimidazolide with the formulas (CA1) to (CA4), which are capping reagents of the “(1) Chemical synthesis” described above, respectively.
As a cap derivative, compounds represented by the following formulas (EC5) to (EC12) can also be used.
Herein, examples of the natural nucleobase and the non-natural base include adenine, guanine, cytosine, thymine, uracil, N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoadenine, 7-methylguanine, N-isobutyrylguanine, 5-fluorocytosine, 5-bromocytosine, 5-methylcytosine, 4-N-methylcytosine, 4-N,N-dimethylcytosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, and 5,6-dihydrouracil.
When the cap derivative and guanosine triphosphate (GTP) are used as substrates, two types of RNA, i.e., an RNA having a protecting group-containing capped structure at the 5′-end and an RNA having a triphosphate group at the 5′-end, are generated. In the RNA having a protecting group-containing capped structure at the 5′-end, a cap structure at the 5′-end and a nucleotide on the 3′-end side thereof are bonded with a triphosphate group interposed therebetween. These two types of RNA can be separated according to hydrophobicity by using reverse-phase HPLC or the like, and only the RNA having the protecting group-containing capped structure can be isolated and purified. The transcription of the template by the T7 RNA polymerase can be performed under appropriately set conditions, and for example, the reaction temperature may be set to 30 to 45° C., and the reaction time may be set to 1 to 5 hours.
Among the cap derivatives described above, for the formulas (EC3) and (EC5) to (EC12), an mRNA in a state where a hydrophobic protecting group is bonded exhibits a higher translation activity than or equal to that of an mRNA in a state where no hydrophobic protecting group is bonded as shown in Examples described later. Therefore, when a cap derivative of the formula (EC3) is used, translation can be performed before the deprotection step by using a nucleotide-based substance in a state where a hydrophobic protecting group is bonded.
Next, the device for purifying a nucleotide-based substance of the present invention will be described. The device for purifying a nucleotide-based substance is a device for carrying out the above-described method for purifying a nucleotide-based substance, and comprises a protecting group introduction means, an isolation and purification means, and a deprotection means.
The protecting group introduction means is a means for carrying out the protecting group introduction step, and introduces a hydrophobic protecting group represented by the formula (P1) or (P2) into a nucleotide-based substance to generate a hydrophobic nucleotide-based substance. In the “chemical synthesis” of the above phosphorylation purification, examples of the protecting group introduction means include an amidite reagent represented by the formula (CR1), and various reagents, reaction devices, and so on to be used for phosphorylation purification. In the “enzyme synthesis” of the above phosphorylation purification, examples of the protecting group introduction means include a hydrophobic substrate represented by the formula (ER1), NTP, and various reagents, reaction devices, and so on to be used for the phosphorylation purification.
In the “chemical synthesis” of the adenylation purification, examples of the protecting group introduction means include adenylation reagents represented by the formulas (CA1) to (CA4), and various reagents, reaction devices, and so on to be used for adenylation purification. In the “enzyme synthesis” of the above adenylation purification, examples of the protecting group introduction means include a hydrophobic substrate represented by the formula (EA1), NTP, and various reagents, reaction devices, and so on to be used for the adenylation purification.
Further, in the “chemical synthesis” of the capping purification, examples of the protecting group introduction means include a capping reagent represented by the formula (CA1), and various reagents, reaction devices, and so on to be used for capping purification. In the “enzyme synthesis” of the above capping purification, examples of the protecting group introduction means include hydrophobic substrates having a structure of the formula (EC0), for example, hydrophobic substrates represented by the formulas (EC1) to (EC4) and the formulas (EC5) to (EC12), NTP, and various reagents, reaction devices, and so on to be used for capping purification.
The isolation and purification means is a means for performing the isolation and purification step described above, and isolates and purifies a hydrophobic nucleotide-based substance under a hydrophobic environment. The isolation and purification device is not particularly limited as long as it is under hydrophobic conditions, and examples thereof include liquid chromatography and membrane separation devices, and the liquid chromatography may be reverse-phase high-performance liquid chromatography.
The deprotection means is a means for performing the above deprotection step, and deprotects a hydrophobic protecting group from a hydrophobic nucleotide-based substance to generate a nucleotide-based substance. Examples of the deprotection means include a device that performs photoirradiation treatment, reduction treatment, or the like. Examples of the device that performs photoirradiation treatment include a light source device that emits light having a wavelength of 300 to 400 nm for 1 to 30 minutes. Examples of the device that performs reduction treatment include a reaction device in which a hydrophobic nucleotide-based substance is treated with a reducing agent such as sodium dithionite (Na2S2O4) at 25 to 80° C. for 1 to 30 minutes. When the reaction temperature is excessively high (50° C. or higher), the decomposition of sodium dithionite precedes, and the reduction reaction efficiency tends to decrease. Therefore, the reaction temperature is more preferably in the range of 25 to 50° C., and for example, 25° C. or 37° C. is particularly preferable. Similarly in the other treatments, deprotection is performed using a device for deprotecting a degradable protecting group (deprotection device).
6. mRNA Drug and Method for Producing Same
The hydrophobic nucleotide-based substance of the present invention is also useful as an mRNA drug. That is, the mRNA drug of the present invention is an mRNA drug comprising a hydrophobic nucleotide-based substance in which a hydrophobic protecting group represented by the following formula (P1) or (P2) is introduced into a nucleotide-based substance having at least one nucleotide and/or a derivative thereof as a constituent unit,
From the viewpoint of the degree of translation activity, the mRNA drug is particularly preferably composed of an mRNA having a cap structure (PureCap type mRNA of Examples described later). Some PureCap type mRNAs exhibit higher translation activity in cells than conventional mRNAs with methylpseudouridine introduced therein as described in
An mRNA drug having a cap structure has a structure in which an RNA is linked to the carbon at the 3′-position of the nucleotide on the 3′-end side of the hydrophobic substrate containing the structure of the formula (EC0),
An RNA has a sequence encoding a protein or peptide having a pharmaceutical effect, and is translated by a ribosome to generate such a useful protein or peptide.
Examples of the hydrophobic substrate represented by the formula (EC0) include hydrophobic substrates selected from the group consisting of the formulas (EC1) to (EC12),
A nucleotide-based substance resulting from deprotection of a hydrophobic protecting group of a hydrophobic nucleotide-based substance in a state where the hydrophobic protecting group described above is bonded thereto is also useful as a drug. That is, the mRNA drug of the present invention includes both a hydrophobic nucleotide-based substance and a nucleotide-based substance in a state where a hydrophobic protecting group is deprotected from the hydrophobic nucleotide-based substance. The latter nucleotide-based substance is specifically a nucleotide-based substance having at least one nucleotide and/or a derivative thereof as a constituent unit, and is a nucleotide-based substance in which a hydrophobic protecting group represented by the formula (P1), (P2), or (P3) is deprotected and hydrogen or a substituent is bonded to *, which is a bond with a nucleotide-based substance.
As such a nucleotide-based substance resulting from deprotection of a hydrophobic protecting group, the aforementioned mRNA drug having a cap structure is preferable from the viewpoint of the degree of high translation activity and the degree of low immune responsiveness. A specific example is a nucleotide-based substance in which an RNA is linked to the carbon at the 3′-position of the nucleotide on the 3′-end side of the hydrophobic substrate containing the structure of the formula (EC0), wherein all of Pro1 to Pro4 of the formula (EC0) are hydrogen as a result of deprotection of the hydrophobic protecting group.
Examples of the nucleotide-based substance represented by the formula (EC0) in a state where the formulas (EC1) to (EC12) are deprotected specifically include nucleotide-based substances of the formulas (EC1) to (EC12) each having a hydrophobic protecting group bonded, wherein Pro is hydrogen as a result of deprotection of the hydrophobic protecting group.
Furthermore, the present invention includes a method for producing the mRNA drug described above, and the production method comprises a protecting group introduction step of introducing a hydrophobic protecting group represented by the following formula (P1) or (P2) into a nucleotide-based substance to produce a hydrophobic nucleotide-based substance.
When the mRNA drug has a cap structure, the protecting group introduction step is a method of synthesizing a hydrophobic nucleotide-based substance by employing, as a template, a nucleic acid having a sequence complementary to the nucleotide-based substance, employing, as substrates, a hydrophobic substrate having a structure of the formula (EC0) and a nucleoside triphosphate, and transcribing the template by an RNA polymerase. Examples of the hydrophobic substrate having the structure of formula (EC0) include hydrophobic substrates selected from the group consisting of (EC1) to (EC12). Since the protecting group introduction step has already been described in detail, detailed description thereof is omitted here.
The mRNA drug of the present invention can be administered to a cell or a tissue by a method of including it in a carrier such as a solid lipid nanoparticle (SNP) or a lipid nanoparticle (LNP). The lipid nanoparticle is composed of a cationic lipid, a PEGylated lipid such as ALC-0159, DSPE-mPEG, or DMG-mPEG, a neutral phospholipid such as DSPC, DPPC, or DOPE, and cholesterol.
In mRNA synthesis by normal transcription, an uncapped RNA or a by-product generated by transcription may cause an immune response, leading to a decrease in the amount of protein synthesis. As shown in
As described above, a nucleotide-based substance resulting from deprotection of a hydrophobic protecting group is also useful as a drug, and thus the method for producing an mRNA drug of the present invention also includes a method of producing a deprotected nucleotide-based substance through the isolation and purification step and the deprotection step described above. In the deprotection step, the hydrophobic protecting group represented by the formula (P1), (P2) or (P3) is deprotected, and a nucleotide-based substance in which hydrogen or a substituent is bonded to *, which is a bond with a nucleotide-based substance is generated. In the case of an mRNA having a cap structure, a nucleotide-based substance in which all of Pro1 to Pro4 of the formula (EC0) are hydrogen is generated. As a nucleotide-based substance with the formulas (EC1) to (EC12) deprotected as a specific example, a nucleotide-based substance in which Pro is hydrogen is generated.
Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the object of the present invention. In the following Examples, the expression “%” is on a mass basis (mass percent) unless otherwise specified.
Four chemically phosphorylated amidite reagents (Compounds 1 to 4) having the following structures were synthesized.
WO2018/162610A1
A synthesis scheme of amidite reagent_1 is shown below.
To a solution of nitrobenzyl alcohol 1 (1.0 g, 4.8 mmol) in dry THF (19 mL) were added triethylamine (5.3 mL, 3.9 g, 38 mmol) and 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite (4.3 mL, 4.5 g, 11 mmol), and the mixture was stirred at room temperature for 4 hours. The reaction mixture was quenched with saturated aqueous sodium bicarbonate (100 mL). This solution was extracted three times with ethyl acetate (100 mL). The organic phase was dried over sodium sulfate, filtered and purified by silica gel column chromatography (hexane/ethyl acetate=3/1, containing 1% of triethylamine), affording the corresponding phosphoramidite 2 as a yellow oil (1.6 g, yield 83%).
1H NMR (400 MHz, CDCl3): δ7.77-7.70 (m, 2H), 7.61-7.53 (m, 1H), 7.41-7.36 (m, 1H), 5.45-5.40 (m, 1H), 3.90-3.49 (m, 4H), 2.76-2.62 (m, 1H), 2.36-2.17 (m, 1H), 1.28-1.16 (m, 2H), 1.18-1.13 (m, 6H), 1.00-0.98 (m, 9H), 0.87-0.86 (m, 4H) ppm.
13C NMR (400 MHz, CDCl3): δ149.78, 149.44, 135.63, 135.51, 131.78, 131.68, 131.07, 130.86, 128.09, 127.87, 123.86, 123.59, 117.75, 77.36, 77.21, 75.83, 75.71, 58.99, 58.76, 58.14, 57.91, 43.57, 43.42, 43.29, 37.23, 37.18, 37.12, 37.05, 25.89, 25.77, 24.83, 24.78, 24.73, 24.70, 24.67, 24.34, 24.27, 20.59, 20.53, 20.17, 20.10 ppm.
31P NMR (400 MHz, CDCl3): δ152.95, 148.78 ppm. ESI-TOF-MS calcd. for C20H32N3NaO4P, 432.2028 [M+Na]+; found, 432.1818.
A synthesis scheme of amidite reagent_2 is shown below.
1-Iodo-2-nitrobenzene (2.65 g, 10.66 mmol, 1.0 equiv.) was dissolved in THF (25 mL). Phenylmagnesium chloride 2 M in THF (5.86 mL, 11.73 mmol, 1.1 equiv.) was added dropwise at −48° C., and the mixture was stirred at −35° C. for 10 minutes. Raw material 3 (3.00 g, 10.12 mmol, 0.95 equiv.) dissolved in THF (15 mL) was added dropwise, and the reaction temperature was gradually raised to room temperature, then the mixture was stirred at room temperature for 15 minutes. A saturated aqueous NH4Cl solution (10 mL) was added, and liquid-liquid separation was performed using ethyl acetate (100 mL) and saturated saline (50 mL). The organic layer was collected and concentrated. The residue was purified by neutral flash column chromatography (hexane/ethyl acetate=15/1→10/1), affording 3.44 g of intermediate 4 (8.65 mmol, yield 81%).
1H-NMR (400 MHz, CDCl3) δ7.89 (dd, J=8.2 Hz, 1.6 Hz, 1H), 7.80 (dd, J=6.4 Hz, 1.6 Hz, 1H), 7.63 (td, J=6.4 Hz, 1.2 Hz, 1H), 7.41 (td, J=6.0 Hz, 1.6 Hz, 1H), 5.23 (q, J=4.0 Hz, 1H), 2.09-1.78 (m, 3H), 1.31-1.21 (m, 33H), 0.88 (t, J=7.2 Hz, 3H).
13C NMR (100 MHz, CDCl3) δ148.1, 140.3, 133.5, 128.1, 128.0, 124.4, 69.5, 38.3, 32.0, 29.8-29.4, 26.2, 22.8, 14.2.
ESI MS-HR calcd for C26H45NO3 (M+Na)+ 442.3297 found 442.3421.
To a solution of nitrobenzyl alcohol 3 (100 mg, 0.24 mmol) in dry THF (1.0 mL) were added diisopropylethylamine (2.5 mL, 250 mg, 1.3 mmol) and 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite (0.21 mL, 230 mg, 0.96 mmol), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated aqueous sodium bicarbonate (20 mL). This solution was extracted three times with ethyl acetate (20 mL). The organic phase was dried over sodium sulfate, filtered and purified by silica gel column chromatography (hexane/ethyl acetate=3/1, containing 1% of triethylamine), affording the corresponding phosphoramidite 5 as a yellow oil (66 mg, yield 45%).
1H NMR (400 MHz, CDCl3): δ7.89-7.77 (m, 2H), 7.66-7.58 (m, 1H), 7.42-7.36 (m, 1H), 5.44-5.33 (m, 1H), 3.94-3.80 (m, 1H), 3.67-3.42 (m, 3H), 2.72-2.61 (m, 1H), 2.45-2.30 (m, 1H), 1.86-1.70 (m, 2H), 1.55 (s, 2H), 1.36-1.08 (m, 40H), 0.90-0.86 (m, 7H) ppm.
31P NMR (400 MHz, CDCl3): δ150.68, 148.83 ppm. ESI-TOF-MS calcd. for C35H62KN3O4P, 658.4115 [M+K]+; found, 658.4246.
A synthesis scheme of amidite reagent_3 is shown below.
To a mixture of 3,4-dihydroxybenzaldehyde 1 (3.0 g, 22 mmol) and K2CO3 (12 g, 88 mmol) in dry DMF (50 mL) was added iodopentane (6.9 mL, 11 g, 53 mmol), and the resulting mixture was heated to 80° C. After stirring at 80° C. for 7 hours, the reaction mixture was cooled to room temperature. DMF was removed under reduced pressure and the residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over Na2SO4 and concentrated. The crude material was purified by silica gel column chromatography dissolved with 13→56% ethyl acetate/hexane, affording Intermediate 2 as a brown solid (6.2 g, quant.).
1H NMR (600 MHz, CDCl3): δ9.80 (s, 1H), 7.39-7.36 (m, 2H), 6.92 (d, J=8.4 Hz, 1H), 4.09-4.01 (m, 4H), 1.86-1.79 (m, 4H), 1.46-1.35 (m, 8H), 0.92-0.89 (m, 6H) ppm.
13C NMR (151 MHz, CDCl3): δ191.05, 154.73, 149.49, 129.93, 126.69, 111.78, 110.94, 69.16, 60.44, 28.83, 28.74, 28.24, 28.20, 22.51, 22.49, 14.25, 14.09 ppm.
ESI-TOF-MS calcd. for C17H26NaO3, 301.1774 [M+Na]+; found, 301.2054.
To a solution of Intermediate 2 (4.3 g, 16 mmol) in acetic acid (15 mL) was added HNO3 (15 mL, up to 70% purity) dropwise over 17 minutes. After stirring at room temperature for 4 hours, ice was added to quench the reaction mixture. The resulting precipitate was collected by filtration and washed with water. The resulting solid was suspended in ethanol and the resulting precipitate was collected by filtration and washed with ethanol, affording Intermediate 3 as a yellow powder (3.1 g, yield: 62%).
1H NMR (600 MHz, CDCl3): δ10.41 (s, 1H), 7.56 (s, 1H), 7.36 (s, 1H), 4.13-4.08 (m, 4H), 1.90-1.83 (m, 5H), 1.49-1.36 (m, 11H), 0.94-0.91 (m, 8H) ppm.
13C NMR (151 MHz, CDCl3): δ188.01, 153.27, 152.37, 151.85, 143.67, 125.34, 110.69, 108.20, 107.94, 70.26, 69.92, 69.78, 28.55, 28.52, 28.46, 28.11, 28.06, 22.42, 22.38, 14.05 ppm.
ESI-TOF-MS calcd. for C17H25NNaO5, 346.1630 [M+Na]+; found, 346.1862.
To a suspension of aldehyde 3 (2.5 g, 7.7 mmol) in dry methanol (40 mL) was slowly added NaBH4 (0.58 g, 15 mmol). After stirring at room temperature for 15 hours, the reaction mixture was concentrated. The residue was dissolved in dichloromethane and washed with water. The aqueous layer was extracted twice with dichloromethane. The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with hexane and then in 12→24% ethyl acetate/hexane, affording nitrobenzyl alcohol 4 as a brown solid (2.1 g, yield 84%).
1H NMR (600 MHz, CDCl3): δ7.65 (s, 1H), 7.10 (s, 1H), 4.89 (d, J=6.0 Hz, 2H), 4.10-4.01 (m, 4H), 1.87-1.81 (m, 4H), 1.47-1.36 (m, 8H), 0.93-0.90 (m, 6H) ppm.
13C NMR (151 MHz, CDCl3): δ154.23, 147.81, 139.52, 132.19, 112.21, 109.86, 69.65, 69.51, 62.97, 28.70, 28.67, 28.17, 28.14, 22.48, 22.45, 14.07 ppm.
ESI-TOF-MS calcd. for C17H27NNaO5, 348.1787 [M+Na]+; found, 348.1994.
To a solution of nitrobenzyl alcohol 4 (350 mg, 1.1 mmol) in dry THF (5.0 mL) were added triethylamine (1.2 mL, 870 mg, 8.6 mmol) and 2-cyanoethyl-N,N′-isopropylchlorophosphoramidite (0.94 mL, 1.0 g, 4.3 mmol). After stirring at room temperature for 6 hours, the reaction mixture was diluted with ethyl acetate and washed twice with a saturated aqueous NaHCO3 solution. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography by eluting with 1% triethylamine-containing hexanes, and subsequently 1% triethylamine-containing 2→6% ethyl acetate/hexane, affording the corresponding phosphoramidite 5 as a yellow oil (340 mg, yield 60%).
1H NMR (600 MHz, CD3CN): δ7.64 (s, 1H), 7.28 (s, 1H), 5.06-4.99 (m, 2H), 4.10-4.00 (m, 4H), 3.87-3.75 (m, 2H), 3.70-3.64 (m, 2H), 2.64-2.62 (m, 2H), 1.92-1.91 (m, 1H), 1.80-1.73 (m, 4H), 1.44-1.33 (m, 8H), 1.19-1.17 (m, 12H), 0.91-0.86 (m, 6H) ppm.
13C NMR (151 MHz, CD3CN) δ153.78, 147.33, 138.93, 130.88, 130.82, 118.52, 110.90, 109.64, 69.39, 69.23, 62.69, 62, 56, 58.69, 58.55, 43.23, 43.16, 28.53, 28.46, 27.97, 27.90, 24.16, 24.11, 23.96, 23.91, 22.20, 20.16, 20.11, 13.42 ppm.
31P NMR (243 MHz, CD3CN): δ149.03 ppm.
ESI-TOF-MS calcd. for C26H45N3O6P, 526.3041 [M+H]+; found, 526.3320; calcd. for C26H44N3NaO6P, 548.2865 [M+Na]+; found, 548.3153.
The synthesis scheme of amidite reagent_4 is shown below.
To a mixture of 3,4-dihydroxybenzaldehyde 1 (3.0 g, 22 mmol) and K2CO3 (12 g, 88 mmol) in dry DMF (50 mL) was added iodopentane (6.9 mL, 11 g, 53 mmol), and the resulting mixture was heated to 80° C. After stirring at 80° C. for 6 hours, the reaction mixture was cooled to room temperature. DMF was removed under reduced pressure and the residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over Na2SO4 and concentrated. The crude material was purified by silica gel column chromatography dissolved with 2→10% ethyl acetate/hexane, affording Intermediate 2 as a yellow solid (6.0 g, yield 88%).
1H NMR (400 MHz, CDCl3): δ9.89 (s, 1H), 7.61-7.12 (m, 2H), 6.98-6.88 (m, 1H), 4.25-3.83 (m, 4H), 2.06-1.18 (m, 14H), 1.02-0.84 (m, 6H) ppm.
ESI-TOF-MS calcd. for C17H26NaO3, 301.1774 [M+Na]+; found, 301.1832.
To a suspension of an aldehyde (Compound 2) (5 g, 18 mmol) in dry THF (60 mL) was slowly added tert-butylmagnesium chloride (2.4 g, 1.0 M, 20 mmol) at 0° C. After stirring at 0° C. for 30 minutes and at room temperature for 20 hours, 50 mL of an aqueous solution (sat. NH4Cl was added. The mixed solvent was extracted with ethyl acetate and washed twice with brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with 50% benzene/hexane and then with benzene, affording an alcohol (Compound 3) as a yellow liquid (3.2 g, yield 54%).
1H NMR (400 MHz, CDCl3): δ6.85-6.76 (m, 3H), 4.30 (s, 1H), 4.02-3.91 (m, 4H), 1.84-1.76 (m, 4H), 1.54-1.19 (m, 9H), 1.01-0.85 (m, 15H) ppm.
13C NMR (100 MHz, CDCl3): δ148.45, 148.33, 135.13, 120.20, 113.70, 112.91, 82.23, 77.47, 76.83, 69.39, 69.32, 35.75, 29.13, 28.33, 26.09, 22.59, 14.14 ppm.
ESI-TOF-MS calcd. for C21H36NaO3, 359.2664 [M+Na]+; found, 359.2709.
To a suspension of an alcohol (Compound 3) (1 g, 3 mmol) in dry CH2Cl2 (30 mL) was slowly added HNO3 (0.5 mL, up to 70% purity) at 0° C. After stirring at room temperature for 24 hours, 50 mL of water was added. The mixed solvent was extracted with CH2Cl2 and washed twice with brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with benzene, affording nitrobenzyl alcohol (Compound 4) as a yellow liquid (0.87 g, yield 77%).
1H NMR (400 MHz, CDCl3): δ7.41 (s, 1H), 7.18 (s, 1H), 5.59 (s, 1H), 5.29 (s, 1H), 4.12-4.00 (m, 4H), 1.87-1.78 (m, 4H), 1.49-1.34 (m, 8H), 0.94-0.84 (m, 15H) ppm.
13C NMR (100 MHz, CDCl3): δ152.23, 147.39, 141.43, 131.67, 112.35, 109.01, 77.48, 76.83, 74.08, 69.47, 69.31, 53.54, 37.05, 28.73, 28.19, 25.72, 22.49, 14.22, 14.06 ppm.
ESI-TOF-MS calcd. for C21H35NNaO5, 404.2515 [M+Na]+; found, 404.2754.
To a solution of nitrobenzyl alcohol (4) (300 mg, 0.79 mmol) in dry THF (10.0 mL) were added triethylamine (0.26 mL, 191 mg, 1.89 mmol) and 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite (0.23 mL, 223 mg, 1.9 mmol). After stirring at room temperature for 14 hours, the reaction mixture was diluted with ethyl acetate and washed twice with a saturated aqueous NaHCO3 solution. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography on in 3→10% ethyl acetate/hexane containing 1% of triethylamine, affording the corresponding phosphoramidite (Compound 5) as a yellow oil (238 mg, 52% yield).
ESI-TOF-MS calcd. for C30H52N3PNaO6, 604.3594 [M+Na]+; found, 604.3533.
A 19-base-long oligodeoxyribonucleotide 5′R-p-TAATACGACTCACTATAGG3′-(SEQ ID NO: 2) having a protected phosphate group at the 5′-end was synthesized by an automatic nucleic acid synthesizer NR-2A7MX (Nihon Techno Service Co., Ltd.) in accordance with a conventional method using amidite reagents_1, 2, and 3 and a commercially available phosphoramidite reagent (The ChemGenes Corporation). After completion of the synthesis, 1 mL of concentrated ammonia water was added to the solid phase carrier, and the solid phase carrier was heated at 55° C. for 16 hours to be deprotected. The supernatant was filtered through a Millex LH filter (0.45 μm, Merck), and then dried up under reduced pressure using a centrifugal evaporator. The oligonucleotide was re-dissolved in super deionized water, and the absorption at 260 nm was measured to determine the concentration. MALDI-TOF molecular weight measurement of the oligonucleotide was performed using 3-hydroxypicolinic acid as a matrix and using a positive mode of ultrafleXtreme (Bruker Corporation).
The 19-base-long oligodeoxyribonucleotide synthesized above was isolated and purified by reverse-phase HPLC. Conditions and so on of HPLC were described in “(2) Results” described later.
The deprotection reaction of a 5′-phosphate group by photoirradiation was performed as follows. An oligonucleotide (10 μM) was dissolved in a buffer containing 20 mM Tris-HCl (pH 8.5), 10 μL of this solution was added to a transparent 96-multiwell plate, and irradiated with 365 nm light at a quantity of light of 4 mW/cm2 for 10 minutes by a MAX-305 light source device (Asahi Spectra Co., Ltd.).
In (a) of the figure, a 19-base-long oligodeoxyribonucleotide 5′R-p-TAATACGACTCACTATAGG3′-(SEQ ID NO: 2) having a protected phosphate group at the 5′-end was synthesized using amidite reagent_1, and the object was isolated and purified by reverse-phase HPLC. The analysis results of the mixture before purification and the analysis results after isolation and purification are shown.
(b) of the figure shows the result of the MALDI-TOF molecular weight analysis of the oligodeoxynucleotide after isolation and purification. At the same time as a peak of the object was confirmed, a product resulting from the elimination of a protecting group by laser photoirradiation at the time of measurement was confirmed as the main peak.
(c) of the figure shows the results of the reverse-phase HPLC analysis of the oligodeoxynucleotide after the reverse-phase HPLC isolation and purification, and the oligonucleotide after irradiation with 365 nm light. Quantitative progress of the deprotection reaction was confirmed.
HPLC analysis conditions in (a) and (c) of the figure are as follows. System used: Chromaster (Hitachi High-Tech Corporation); column: YMC Hydrosphere C18 (250×4.6 mm I.D., YMC); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 0 to 60% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: room temperature; detection wavelength: 260 nm.
In (a) of the figure, a 19-base-long oligodeoxyribonucleotide 5′R-p-TAATACGACTCACTATAGG3′-(SEQ ID NO: 2) having a protected phosphate group at the 5′-end was synthesized using amidite reagent_2, and the object was isolated and purified by reverse-phase HPLC. The analysis results of the mixture before purification and the analysis results after isolation and purification are shown.
(b) of the figure shows the result of the MALDI-TOF molecular weight analysis of the oligodeoxynucleotide after isolation and purification. At the same time as a peak of the object was confirmed, a product resulting from the elimination of a protecting group by laser photoirradiation at the time of measurement was confirmed as the main peak.
(c) of the figure shows the results of the reverse-phase HPLC analysis of the oligodeoxynucleotide after the reverse-phase HPLC isolation and purification, and the oligonucleotide after irradiation with 365 nm light. Quantitative progress of the deprotection reaction was confirmed.
HPLC analysis conditions in (a) and (c) of the figure are as follows. System used: Chromaster (Hitachi High-Tech Corporation); column: YMC Hydrosphere C18 (250×4.6 mm I.D., YMC); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 0 to 100% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: room temperature; detection wavelength: 260 nm.
In (a) of the figure, a 19-base-long oligodeoxyribonucleotide 5′R-p-TAATACGACTCACTATAGG3′-(SEQ ID NO: 2) having a protected phosphate group at the 5′-end was synthesized using amidite reagent_3, and the object was isolated and purified by reverse-phase HPLC. The analysis results of the mixture before purification and the analysis results after isolation and purification are shown.
(b) of the figure shows the result of the MALDI-TOF molecular weight analysis of the oligodeoxynucleotide after isolation and purification. At the same time as a peak of the object was confirmed, a product resulting from the elimination of a protecting group by laser photoirradiation at the time of measurement was confirmed as the main peak.
HPLC analysis conditions in (a) of the figure are as follows. System used: Chromaster (Hitachi High-Tech Corporation); column: YMC Hydrosphere C18 (250×4.6 mm I.D., YMC); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 0 to 100% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: room temperature; detection wavelength: 260 nm.
(d) Comparison of results of amidite reagents_1 and 2 with known compounds (Comparative Examples)
(a) to (e) of the figure: a 19-base-long oligodeoxynucleotide 5′R-p-TAATACGACTCACTATAGG3′-(SEQ ID NO: 2) having a protected phosphate group at the 5′-end was synthesized using commercially available CPRI (a), commercially available CPRII (b), a compound known in literature (Chem. Eur. J. 23, 5210 (2017)), amidite reagent_1 (d), or amidite reagent_2 (e), and was deresinized and deprotected using concentrated aqueous ammonia in accordance with a conventional method (55° C., 16 hours). A mixture before purification containing a phosphate group-protected object was analyzed by reverse-phase HPLC to determine a target product content. HPLC analysis conditions are as follows. System used: Chromaster (Hitachi High-Tech Corporation); column: YMC Hydrosphere C18 (250×4.6 mm I.D., YMC); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 0 to 100% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: room temperature; detection wavelength: 260 nm. In the figure, a schematic diagram illustrating the chemical structure of the object, and its elution time and content % are described.
(f) of the figure is a table summarizing the elution time and the production yield (%) of the objective 5′-end protected phosphate group from the results shown in (a) to (e). * Chem. Eur. J. 23, 5210 (2017). From this result, it is seen that amidite reagent_1 and amidite reagent_2 of the present invention have a higher yield of the objective nucleic acid than the conventional compounds. In particular, when amidite reagent_1 and amidite reagent_2 are compared with each other, it is found that the yield of amidite reagent_1 in which R1 is a t-butyl group is higher than the yield of amidite reagent_2 in which R1 is a C19 linear alkyl group, and amidite reagent_1 is preferable to amidite reagent_2 in terms of yield.
107-base-long and 131-base-long oligoribonucleotides whose sequence are shown in the diagram were synthesized by an automatic nucleic acid synthesizer NR-2A7MX (Nihon Techno Service Co., Ltd.) in accordance with a conventional method using amidite reagent_1 and a commercially available phosphoramidite reagent (The ChemGenes Corporation). After completion of the synthesis, 1 mL of a 1:1 mixed solution of concentrated ammonia water and a 40% aqueous methylamine solution was added to the solid phase carrier, and the solid phase carrier was heated at 65° C. for 15 minutes to be deprotected. The supernatant was filtered through a Millex LH filter (0.45 μm, Merck), and then dried up under reduced pressure using a centrifugal evaporator. 1 mL of a 1 M TBAF solution in THF was added to the residue to dissolve, and the solution was heated at 35° C. overnight. 1 mL of a 1 M Tris-HCl (pH 7.5) buffer was added thereto and mixed, and the mixture was concentrated with a centrifugal evaporator and then desalted using an NAP-25 column (GE HealthCare). To 3 mL of the eluate were added 0.3 mL of a 3 M aqueous ammonium acetate solution (pH 5.2) and 3.3 mL of 2-propanol, and the mixture was cooled at −30° C. overnight, and then the precipitated RNA was centrifuged and collected. The RNA was re-dissolved in super deionized water, and the absorption at 260 nm was measured to determine the nucleic acid concentration.
The RNA synthesized above was isolated and purified by reverse-phase HPLC. Conditions and so on of HPLC were described in “(2) Results” described later.
The deprotection reaction of a 5′-phosphate group by photoirradiation was performed as follows. A solution (10 μL) of an RNA in 50 mM triethylammonium acetate (pH 7.0) was added to a transparent 96-multiwell plate, and irradiated with 365 nm light at a quantity of light of 4 mW/cm2 for 10 minutes using a MAX-305 light source device (Asahi Spectra Co., Ltd.).
(a) of the figure shows a chemically synthesized RNA sequence, and mG indicates that the hydroxy group at the 2′-position is methylated.
(b) of the figure shows the result of reverse-phase HPLC analysis of a mixture containing a 107-nt RNA after deprotection. The eluted solution was fractionated into three groups (Peak(s)_1, 2, and 3).
(c) of the figure shows the result of analysis of three groups of 5% modified polyacrylamide (containing 7 M urea) fractionated in (b). Peak(s)_2 contained the objective full-length 107-nt RNA. Peak(s)_1 contained by-products generated during synthesis that were not extended to the full length, and Peak(s)_3 contained unidentified by-products. The polyacrylamide gel after electrophoresis was stained with an SYBR Green II nucleic acid staining reagent to visualize an RNA.
(d) of the figure shows the result of reverse-phase HPLC analysis of the object (107-nt RNA) after purification using reverse-phase HPLC.
The HPLC analysis conditions used in (b) and (d) of the figure are as follows. System used: LaChrom Elite (Hitachi High-Tech Corporation); column: YMC Triat Bio C4 (250×4.6 mm I.D.); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 5 to 20% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: 50° C.; detection wavelength: 260 nm.
(a) of the figure shows a conceptual diagram of the deprotection reaction, and the protecting group of the 5-phosphate group is removed by irradiation with 365 nm light.
(b) and (c) of the figure show the results of reverse-phase HPLC analysis of a 107-nt RNA before photoirradiation (before deprotection); (b) shows the result before photoirradiation (before deprotection), and (c) shows the result after photoirradiation (after deprotection). From these results, quantitative deprotection by photoirradiation was confirmed.
The HPLC analysis conditions used in (b) and (c) of the figure are as follows. System used: LaChrom Elite (Hitachi High-Tech Corporation); column: YMC Hydrosphere C18 (250×4.6 mm I.D.); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 5 to 20% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: 50° C.; detection wavelength: 260 nm.
(a) of the figure shows a chemically synthesized RNA sequence, and mG indicates that the hydroxy group at the 2′-position is methylated.
(b) of the figure shows the result of reverse-phase HPLC analysis of a mixture containing a 131-nt RNA after deprotection. The eluted solution was fractionated into three groups (Peak(s)_1, 2, and 3) and purified.
(c) of the figure shows the result of analysis of three groups of modified polyacrylamides fractionated in (b). Peak(s)_2 contained the objective full-length 131-nt RNA. Peak(s)_1 contained by-products generated during synthesis that were not extended to the full length, and Peak(s)_3 contained unidentified by-products. The polyacrylamide gel after electrophoresis was stained with an SYBR Green II nucleic acid staining reagent to visualize an RNA.
(d) of the figure shows the result of reverse-phase HPLC analysis of the 5′-protected phosphate group (131-nt RNA) after purification using reverse-phase HPLC.
(e) of the figure shows the result of reverse-phase HPLC analysis of the object (131-nt RNA) purified using reverse-phase HPLC and then deprotected with 365-nm photoirradiation.
The HPLC analysis conditions used in (b) of the figure are as follows. System used: LaChrom Elite (Hitachi High-Tech Corporation); column: YMC Triat Bio C4 (250×4.6 mm I.D.); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 5 to 20% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: 50° C.; detection wavelength: 260 nm. The HPLC analysis conditions used are as follows.
System used: Chromaster (Hitachi High-Tech Corporation); column: YMC Hydrosphere C18 (250×4.6 mm I.D.); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 5 to 15% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: 50° C.; detection wavelength: 260 nm.
3. Transcriptional Synthesis and Isolation of 5′-End monophosphorylated RNA
In contrast, in the present invention, R-pG in which a protecting group is bonded to the 5′-end of guanosine is used. When the substrate NTP and the transcribed substrate R-pG coexist in a transcription reaction, the resulting transcript is a mixture of 5′-end triphosphate and protected (R)-monophosphate according to the mixing ratio of GTP/R-pG. Utilizing the hydrophobicity of the protecting group, it was possible to isolate the protected (R)-monophosphorylated RNA from the mixture by reverse-phase HPLC or the like. The protecting group of the phosphate group could be quantitatively removed by photoirradiation after isolation, and this showed that a 5′-end monophosphorylated RNA transcript (34, 100, 250, 650, or 1000-base-long) could be purified with high purity. The experiment and the results thereof are described below.
The analytical conditions of HPLC used in this experiment are shown below.
Column: Hydrosphere C18 (YMC) 4.6×250 mm or Triart C4 (YMC) 4.6×250 mm
Mobile phase: A) 50 mM TEAA buffer containing 5% acetonitrile (pH 7.0)
3′-TOM-ribo-guanosine CPG 1000 Å was used as a CPG and a transcribed substrate R-pG was synthesized in a nucleic acid synthesizer. After the synthesis, the CPG was transferred to a screw tube with a 1.5 mL O-ring (SARSTEDT), 1.0 mL of 40% aq. methylamine/28% aq. ammonia solution (1:1) was added thereto, and the mixture was incubated at 65° C. for 10 minutes. The mixture was allowed to cool to room temperature, then filtered using a Millex LH 0.45 μm filter. The filtrate was washed with sterile water (500 μL), and dried up with a centrifugal evaporator. The residue was dissolved in 115 μL of DMSO, and 60 μL of triethylamine was added thereto. 75 μL of triethylamine trihydrofluoride was added thereto, and the mixture was incubated at 65° C. for 2 hours. The reaction liquid was analyzed and purified by HPLC, then concentrated with a centrifugal evaporator, and subjected to absorbance measurement at 260 nm to determine the concentration (3.62 mM).
MS (ESI-MS, negativemode), [M−H]− calculated: 611.14 observed: 611.1318
HPLC conditions; column: C18, gradient: B0-80%, flow rate: 3 mL
An in vitro transcription reaction was performed using each template DNA. A transcription reaction liquid (template DNA 5 mg/L, 1×T7 RNA polymerase buffer (Takara) (Tris-HCl (pH 8.0) 40 mM, MgCl 28 mM, spermidine 2 mM), T7 RNA polymerase (Takara) 2.5 U/μL, DTT 5 mM, ATP 2 mM, UTP 2 mM, GTP 0.5 mM, GMP 2 mM or R-pG 2 mM, CTP 2 mM) was prepared and incubated at 37° C. for 2 hours. The reaction liquid was subjected to electrophoresis with a denaturing polyacrylamide gel to confirm the generation of an RNA. After the reaction, a protein was removed by phenol-chloroform extraction, and phenol was removed by chloroform extraction. Desalination and concentration were performed using Amicon Ultra (Merck Millipore, 0.5 mL, 10 K). The absorbance of the resulting RNA solution at 260 nm was measured to determine the concentration.
Using amidite reagent_1 and a CPG resin (Glen Research) carrying deoxyguanosine, a transcribed substrate R-pG was synthesized in accordance with a standard phosphoramidite method using an automatic nucleic acid synthesizer. After the synthesis, deprotection was performed, and then analysis and purification were performed by HPLC. The results are shown in
Using the purified RpG, RNAs having five lengths were synthesized through a transcription reaction. The RNAs synthesized by transcription have lengths of 34 nt, 100 nt, 250 nt, 650 nt, and 1078 nt, respectively. Each of the reaction liquid was analyzed by electrophoresis using a denaturing polyacrylamide gel to confirm an RNA having an objective chain length. The results are shown in
The RNA obtained by transcription was purified and then analyzed by HPLC. The analysis results of RNAs are shown in
The results summarizing the data are shown in the following table.
An RNA with a photoprotecting group introduced was purified by HPLC, and deprotection was performed by irradiating the RNA solution with 365 nm light for 10 minutes, followed by analysis by HPLC. The results are shown in
(a) and (b) of the figure illustrate synthesis schemes of bi- and tri-branched cap analog compounds. Compounds 1 and 4 were synthesized using an automatic nucleic acid synthesizer, and purified by reverse-phase HPLC using a hydrophobic protecting group of a novel phosphorylation reagent. The protecting group of the phosphate moiety was removed by photoirradiation, affording capped precursors 2 and 5.
(c) and (d) of the figure show the results of reverse-phase HPLC analysis of the mixtures containing Compounds 1 and 4 after deprotection and dialysis. Since the objects were contained in the peaks at retention times of 12.17 minutes (c) and 12.96 minutes (d), these were isolated.
(e) and (f) of the figure show the results of reverse-phase HPLC analysis of Compounds 2 and 5.
Guanosine derivatives 1 and 4 were synthesized using a nucleic acid synthesizer at a 1 micromole scale in accordance with a conventional phosphoramidite method. Commercially available branched amidite reagents (Symmetric Doubler Phosphoramidite, Glen Research, cat #10-1920: Trebler Phosphoramidite, Glen Research, cat #10-1922) were each bonded to a CPG solid phase carrier (ChemGenes, cat #N-3203-10) supporting a protected guanosine. Subsequently, rG nucleotide and a novel chemical phosphorylation reagent (Compound 1) were bonded, affording the intended bi-branched compound (1) and the objective tri-branched compound (4). 1 mL of an equal mixture of concentrated ammonia water and a 40% methylamine aqueous solution was added to the solid-phase carrier, and heated at 65° C. for 15 minutes. The supernatant was dried up under reduced pressure, the residue was dissolved in 100 μL of dimethyl sulfoxide, 125 μL of triethylamine trihydrofluoride was added thereto, and the mixture was heated at 65° C. for 2 hours. To the reaction liquid was added 1 mL of a 0.1 M triethylamine acetate buffer (pH 7.0), and dialysis was carried out overnight using a dialysis membrane (Biotech CE Tubing manufactured by Spectra/Pore; MWCO 500 to 1000 Da). The internal liquid was collected, and the object was isolated by reverse-phase HPLC. The analysis and purification conditions are as follows.
System used: LaChrom Elite (Hitachi High-Tech Corporation); column: YMC Triat Bio C4 (250×4.6 mm I.D.); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 5 to 80% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: 25° C.; detection wavelength: 260 nm.
The object isolated by reverse-phase HPLC was concentrated and then irradiated with 365 nm light to be deprotected. Dialysis was carried out overnight using a dialysis membrane (Biotech CE Tubing manufactured by Spectra/Pore; MWCO 500 to 1000 Da), and the objective Compounds 2 and 5 were obtained in yields of 72 nmol and 96 nmol, respectively. The purities of the objective Compounds 2 and 5 obtained were confirmed by reverse-phase HPLC. The analysis conditions are as follows.
System used: LaChrom Elite (Hitachi High-Tech Corporation); column: YMC Hydrosphere C18 (250×4.6 mm I.D.); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 0 to 20% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: 25° C.; detection wavelength: 260 nm.
The molecular weight of the obtained compound was measured using 3-hydroxypicolinic acid as a matrix and using a positive mode of an ultrafleXtreme MALDI-TOF/TOF mass spectrometer manufactured by Bruker Corporation. The analysis results are as follows.
Bi-branched guanosine derivative 2; [M+H]+ calcd: 1486.9, found: 1486.3 (−0.6). Tri-branched guanosine derivative 5; [M+H]+ calcd: 1932.2, found: 1931.3 (−0.9).
5. Development of adenylated RNA preparation method capable of isolation and purification: synthesis of AppG nucleotide analogue having photodegradable hydrophobic protecting group and introduction into RNA strand using RNA polymerase
A synthesis scheme of the AppG dinucleotide compound having a photodegradable hydrophobic protecting group (Compound 11) is shown below.
Adenosine (2.00 g, 7.48 mmol, 1.0 equiv.), tert-butyldimethylsilane (5.64 g, 37.4 mmol, 5.0 equiv.) and imidazole (6.11 g, 89.9 mmol, 12 equiv.) were dissolved in DMF (75.0 mL) and stirred at room temperature overnight. The reaction solution was dissolved in ethyl acetate, and washed with water and saturated saline, then the organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by neutral flash column chromatography (hexane/ethyl acetate=3/1), affording 3.30 g of Compound 2 (5.42 mmol, yield 72%).
[1H NMR] (400 MHZ, DMSO-d6, ppm) δ8.29 (s, 1H), 8.09 (s, 1H), 7.27 (s, 2H), 5.89 (d, 1H, J=6.4 Hz), 4.87 (dd, 1H, J=4.4, 6.4 Hz), 4.28 (d, 1H, J=4.4 Hz), 3.99˜3.94 (m, 2H), 3.70 (q, 1H, J=6.8 Hz), 0.87 (d, 18H, J=10.8 Hz), 0.67 (s, 9H), 0.08 (d, 6H, J=7.6 Hz), 0.04 (s, 6H), −0.15 (s, 3H), −0.40 (s, 3H)
[HRMS] (ESI) m/z Cal. for C28H55N5O4Si3Na+ [M+Na]+; 632.3454 found 632.3486.
THF (50 mL) added with 1-iodo-2-nitrobenzene (5.0 g, 20 mmol, 1.0 equiv.) was cooled to −40° C., a 2 M phenylmagnesium chloride THF solution (11 mL, 22 mmol, 1.1 equiv.) was slowly added dropwise thereto, and the mixture was stirred at −40° C. for 30 minutes. Pivalaldehyde (2.2 mL, 20 mmol, 1.0 equiv.) was slowly added dropwise and the resulting mixture was stirred at −40° C. for 3 hours. Saturated ammonium chloride water (100 mL) and water (100 mL) were added, and the mixture was returned to room temperature with stirring. Saturated saline (200 mL) was added, and the mixture was extracted with ethyl acetate (200 mL×3). The organic layer was dried over anhydrous sodium sulfate, and distilled off under reduced pressure. The residue was purified by neutral flash column chromatography (hexane/dichloromethane=2/1 →1/1→1/5), affording objective Compound 4 (2.9 g, 14 mmol, yield 69%).
[1H NMR] (400 MHz, CDCl3, ppm) δ7.78 (dd, 1H, J=8.0, 1.5 Hz), 7.71 (dd, 1H, J=8.2, 1.2 Hz), 7.54˜7.59 (m, 1H), 7.38 (ddd, 1H, J=8.4, 7.1, 1.0 Hz), 5.33 (d, 1H, J=3.1 Hz), 2.35 (d, 1H, J=3.6 Hz), 0.85 (s, 9H)
Compound 4 (0.300 g, 1.43 mmol, 1.0 equiv.) was azeotroped with benzene three times. 1,1′-Carbonyldiimidazole (0.626 g, 3.86 mmol, 2.7 equiv.) was added thereto, dissolved in dichloromethane (7.00 mL), and the mixture was stirred at room temperature for 6.5 hours. After washings with water and saturated saline, the organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Compound 2 (0.962 g, 1.58 mmol, 1.1 equiv.) and 18-crown-6 (0.455 g, 1.72 mmol, 1.2 equiv.) were added thereto, and the mixture was dissolved in dehydrated THF (7.0 mL). Furthermore, KH (0.0689 g, 1.72 mmol, 1.2 equiv.) was slowly added and the mixture was stirred at 50° C. overnight. After the solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate and the solution was washed with water and saturated saline. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by neutral flash column chromatography (hexane/ethyl acetate=3/1), affording 0.585 g of Compound 6 (0.692 mmol, yield 48%).
[1H NMR] (400 MHz, DMSO-d6, ppm) diastereomer mixture δ10.91 (s, 1H), 8.64˜8.57 (m, 2H), 7.96˜7.93 (m, 1H), 7.83˜7.81 (m, 1H), 7.63˜7.50 (m, 2H), 6.22 (s, 1H), 6.01˜6.00 (m, 1H), 4.88˜4.84 (m, 1H), 4.32˜4.30 (m, 1H), 4.01˜3.93 (m, 2H), 3.75˜3.72 (m, 1H), 0.88˜0.85 (m, 27H), 0.664˜0.661 (m, 9H), 0.11˜0.07 (m, 6H), 0.041˜0.039 (m, 6H), −0.14 (s, 3H), −0.415˜0.42 (m, 3H)
[13C NMR] (151 MHz, DMSO-d6, ppm) δ152.30, 152.07, 149.42, 143.14, 137.35, 133.62, 133.42, 133.08, 132.36, 131.81, 130.43, 130.10, 129.98, 129.80, 129.66, 128.97, 128.89, 128.52, 125.21, 125.15, 124.89, 123.74, 123.63, 87.60, 85.97, 80.23, 76.83, 74.93, 72.97, 72.68, 62.94, 36.64, 36.29, 18.55, 18.30, 17.98, −4.15, −4.31, −4.91, −5.01
[HRMS] (ESI) m/z Cal. for C40H68N6OSi3Na+ [M+Na]+; 867.4299 found 867.4305.
Compound 6 (0.585 g, 0.692 mmol, 1.0 equiv.) was dissolved in THF (7.00 mL), triethylamine and trihydrochloride (0.282 mL, 1.73 mmol, 2.5 equiv.) was added thereto, and the mixture was stirred at room temperature overnight. The reaction solution was dissolved in ethyl acetate, and washed with water and saturated saline, then the organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by neutral flash column chromatography (dichloromethane/MeOH=10/1), affording 0.226 g of Compound 7 (0.450 mmol, yield 65%).
[1H NMR] (400 MHz, DMSO-d6, ppm) diastereomer mixture 59.57˜9.53 (m, 1H), 8.35˜8.33 (m, 1H), 8.07˜8.05 (m, 1H), 7.81˜7.79 (m, 1H), 7.61˜7.51 (m, 2H), 7.35˜7.31 (m, 1H), 6.48˜6.46 (m, 1H), 5.83˜5.76 (m, 2H), 5.34 (s, 1H), 4.87˜4.72 (m, 2H), 4.33˜4.14 (m, 2H), 3.78˜3.62 (m, 2H), 0.90˜0.85 (m, 9H)
[13C NMR] (101 MHz, DMSO-d6, ppm) δ151.87, 150.89, 149.95, 149.49, 149.43, 149.18, 149.07, 143.15, 132.91, 132.65, 129.26, 128.76, 124.63, 122.46, 90.42, 87.04, 77.44, 74.25, 71.70, 62.57, 36.29, 25.77
[HRMS] (ESI) m/z Cal. for C22H26N6ONa+ [M+Na]+; 525.1704 found 525.1709.
The molar absorbance coefficient (ε260) of Compound 7 was calculated using a UV-vis spectrum. First, 0.990, 1.960, 2.913, and 3.846×10−4 [mol/L] of Compound 7 were prepared, and UV-vis spectra were measured. From ε260 at the respective concentrations, an average value was calculated, and ε260 of Compound 7 was determined to be 18717 L/mol·cm.
Compound 7 (0.100 g, 0.199 mmol, 1.0 equiv.) was dissolved in trimethylphosphate (2.00 mL), phosphoryl chloride (22.3 μL, 0.239 mmol, 1.2 equiv.) was added thereto, and then the mixture was stirred at room temperature for 6 hours. A TEAB buffer (2 M, 10.0 mL) was added thereto, the mixture was washed with chloroform, and then the solvent was distilled off under reduced pressure. The residue was purified using DEAE Sephadex A-25 (solvent: (A) water, (B) 1.5 M triethylammonium carbonate buffer 10% ACN, gradient: B conc. 0 to 200 min., 0 to 100%, flow rate: 1.0 mL/min., detection wavelength: 260 nm, size S), affording 8.82 mg of Compound 8 (11.2 μmol, yield 6%).
[1H NMR] (600 MHz, DMSO-d6, ppm) diastereomer mixture δ8.80 (s, 1H), 8.57 (s, 1H), 7.94˜7.93 (m, 1H), 7.80˜7.74 (m, 2H), 7.57˜7.54 (m, 1H), 6.23˜6.22 (m, 1H), 6.01˜5.99 (m, 1H), 4.68˜4.66 (m, 1H), 4.20˜4.19 (m, 1H), 4.06˜4.05 (m, 1H), 3.85˜3.81 (m, 2H), 2.47˜2.46 (m, 1H), 1.19 (s, 2H), 1.02˜0.89 (m, 9H)
[13C NMR] (151 MHz, DMSO-d6, ppm) δ152.52, 152.25, 151.75, 149.91, 149.41, 143.22, 133.50, 133.10, 129.79, 129.64, 124.88, 123.38, 87.45, 85.15, 76.72, 74.70, 71.73, 64.63, 36.42, 29.53, 25.98
[31P NMR] (243 MHz, DMSO-d6, ppm) δ0.79
[HRMS] (ESI) m/z Cal. for C22H26N6O11— [M−H]−; 581.1403 found 581.1381.
Guanosine 5′-phosphate disodium salt (0.500 g, 1.23 mmol, 1.0 equiv.) was dissolved in water and loaded onto a DOWEX 50W-X8 column in H+ type, and the column was washed with water. The eluate was added dropwise to an ethanol solution of triethylamine (0.685 mL, 4.92 mmol, 4.0 equiv.), and the solvent was distilled off under reduced pressure. Imidazole (0.837 g, 12.3 mmol, 10 equiv.) and 2,2′-dipyridyl disulfide (0.811 g, 3.68 mmol, 3.0 equiv.) were added thereto, and the mixture was dissolved in DMSO (10.0 mL). Further, triethylamine (0.372 mL, 3.68 mmol, 3.0 equiv.) and triphenylphosphine (0.965 g, 3.68 mmol, 3.0 equiv.) were added thereto, and the mixture was stirred at room temperature overnight. The reaction was traced by HPLC (column: CoresepSB; solvent: (A) 20 mM ammonium acetate 5% ACN, (B) 400 mM ammonium acetate 5% ACN; gradient: B conc. 2 to 22 min., 0 to 100%, flow rate: 1.0 mL/min., detection wavelength: 260 nm). A solution of sodium perchlorate (4.51 g, 36.8 mmol, 30 equiv.) in dehydrated acetone (200 mL) was added thereto, and the mixture was ice-cooled in an ice bath. The precipitate formed was collected by suction filtration, affording white powdery Compound 10 (0.530 g, 1.22 mmol, yield >99%). Various spectra agreed well with literature values[1].
Compound 8 (8.81 mg, 11.2 μmol, 1.0 equiv.) was dissolved in DMSO (1.00 mL), and guanosine 5′-phosphorimidazolide (24.4 mg, 56.0 μmol, 5.0 equiv.), 1-methylimidazole (4.42 μL, 56.0 μmol, 5.0 equiv.), and CaCl2) (12.4 mg, 112 μmol, 10 equiv.) were added, then the mixture was stirred at room temperature overnight. The reaction was traced by HPLC (column: Hydrosphere C18; solvent: (A) 20 mM triethylammonium acetate (pH 7.0), (B) ACN; gradient: B conc. 0 to 30 min., 0 to 80%, flow rate: 1.0 mL/min., detection wavelength: 260 nm). After confirming the completion of the reaction progress, purification was performed using HPLC (column: Hydrosphere C18; solvent: (A) 20 mM triethylammonium acetate (pH 7.0), (B) ACN; gradient: B conc. 0 to 30 min., 0 to 80%; flow rate: 10 mL/min.; detection wavelength: 260 nm), affording 2.15 mg of Compound 11 (1.90 μmol, yield 17%).
[1H NMR] (600 MHz, DMSO-d6, ppm) diastereomer mixture 58.34˜8.33 (m, 2H), 7.83˜7.81 (m, 1H), 7.69˜7.64 (m, 2H), 7.62˜7.57 (m, 1H), 7.43˜7.40 (m, 1H), 6.25˜6.24 (m, 1H), 5.96˜5.95 (m, 1H), 5.61˜5.57 (m, 1H), 4.50˜4.48 (m, 1H), 4.46˜4.43 (m, 1H), 4.33˜4.31 (m, 1H), 4.27˜4.26 (m, 1H), 4.23˜4.17 (m, 2H), 4.14˜4.11 (m, 2H), 4.08˜4.04 (m, 2H), 0.83˜0.82 (m, 9H)
[13C NMR] (151 MHz, DMSO-d6, ppm) δ157.35, 154.15, 152.14, 151.64, 149.82, 149.39, 142.97, 136.61, 133.52, 133.06, 129.80, 129.62, 124.85, 123.30, 88.12, 87.93, 83.84, 76.74, 74.81, 73.96, 70.57, 70.17, 64.62, 31.23, 27.59, 26.05, 25.97, 22.413
[31P NMR] (243 MHz, DMSO-d6, ppm) δ−10.66
[HRMS] (ESI) m/z Cal. for C32H38N11O18P2− [M−H]−; 926.1877 found 926.1916.
Compound 11 synthesized as described above was subjected to a transcription reaction by T7 RNA polymerase. A 1078-base-long protecting group-containing adenylated RNA was prepared by heating, at 37° C. for 2 hours, the following reaction liquid: 5 ng/μL dsDNA transcription template (PCR product of 1105-bp containing T7 promoter sequence 5′TAATACGACTCACTATAG3′-(SEQ ID NO: 1)), 2 mM ATP, 2 mM UTP, 2 mM CTP, 0.5 mM GTP, 2 mM Compound 11 (AppG dinucleotide compound having a photodegradable hydrophobic protecting group), 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 2.5 units/μL T7 RNA polymerase (Takara Bio Inc.). After the transcription reaction, the reaction liquid in any case was mixed with an equal mixture of TE saturated phenol and chloroform, followed by vigorous mixing, and then the resulting protein-like insolubles were removed. The aqueous layer was extracted with chloroform, then purified using an Amicon Ultra 10K ultrafiltration filter unit, and analyzed by reverse-phase HPLC.
(a) of the figure illustrates the outline of the experiment. The transcribed RNA is obtained as a mixture of one having a triphosphate structure at the 5′-end (ppp-RNA) and an objective protecting group-containing adenylated structure (R-App-RNA). Since the latter has hydrophobicity that is high and different from that of the former, it can be isolated by reverse-phase HPLC utilizing this property.
(b) of the figure shows the result of reverse-phase HPLC analysis of a 1078-base-long RNA transcription reaction.
Column: YMC-Triart Bio C4 (250 mm×4.6 mm I.D.); Solvent A: 50 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; Solvent B: acetonitrile; Linear Gradient: 0 to 20% Solvent B (0 to 20 min); Flow rate: 1 mL/min; Detection wavelength: 260 nm; Column temperature: 50° C.
A 250-base-long protecting group-containing adenylated RNA was prepared by heating, at 37° C. for 2 hours, the following reaction liquid: 5 ng/μL dsDNA transcription template (PCR product of 276-bp containing T7 promoter sequence 5′TAATACGACTCACTATAG3′-(SEQ ID NO: 1)), 2 mM ATP, 2 mM UTP, 2 mM CTP, 0.5 mM GTP, 2 mM Compound 11 (AppG dinucleotide compound having a photodegradable hydrophobic protecting group), 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 2.5 units/μL T7 RNA polymerase (Takara Bio Inc.). The reaction liquid after the transcription reaction was mixed with an equal mixture of TE saturated phenol and chloroform, followed by vigorous mixing, and then the resulting protein-like insolubles were removed. The aqueous layer was extracted with chloroform, then purified using an Amicon Ultra 10K ultrafiltration filter unit. This was analyzed by reverse-phase HPLC, and a photodegradable hydrophobic protecting group-containing adenylated RNA was fractionated and purified. The eluate containing the objective RNA was desalted using an Amicon Ultra 10K ultrafiltration filter unit. The protecting group-containing adenylated RNA obtained above was deprotected by photoirradiation. The RNA solution (16.5 ng RNA/μL, 100 μL) was added to a transparent 96-multiwell plate, and irradiated with 365 nm light at a quantity of light of 4 mW/cm2 for 15 minutes using a MAX-305 light source device (Asahi Spectra Co., Ltd.).
(a) of the figure illustrates the outline of the experiment.
(b) of the figure shows the results of reverse-phase HPLC analysis of a protecting group-containing adenylated RNA (after isolation).
(c) of the figure shows the result of reverse-phase HPLC analysis after irradiation of a protecting group-containing adenylated RNA (after isolation) with 365 nm light.
Column of (b) and (c): YMC-Triart Bio C4 (250 mm×4.6 mm I.D.); Solvent A: 50 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; Solvent B: acetonitrile; Linear Gradient: 0 to 20% Solvent B (0 to 20 min); Flow rate: 1 mL/min; Detection wavelength: 260 nm; Column temperature: 50° C.
Novel cap analog compounds having a photodegradable hydrophobic protecting group (Cap Analog_1 and Cap Analog_2) were synthesized.
The synthesis of (R/S)-tBu-(2-nitrobenzyl) alcohol (Compound 2) was carried out in accordance with literature. A mixture of 1-iodo-2-nitrobenzene (20 g, 80 mmol) in THF (150 mL) was cooled to −40° C. A solution of phenylmagnesium chloride in THF (2 M, 52 mL, 0.10 mol) was added and stirred at −40° C. for 1 hour. Trimethylacetaldehyde (12 mL, 9.6 g, 0.11 mol) was added to the reaction mixture and stirred at −40° C. for 36 m ci. The reaction mixture was gradually warmed to room temperature. After stirring for 4 hours, a saturated aqueous NH4Cl solution was added to stop the reaction. An aqueous NH4Cl solution was added to stop the reaction. The mixture was extracted three times with ethyl acetate. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with hexane and then with 17→25% ethyl acetate/hexane, affording Compound 2 as a dark brown solid (11 g, yield 65%). The spectral data of the compound were all consistent with those of the literature.
To a solution of (R/S)-1-(2-nitrophenyl)-2,2-dimethyl-1-propanol (Compound 2) (5.8 g, 28 mmol) in dichloromethane (60 mL) was added 1,1′-carbonyldiimidazole (6.8 g, 42 mmol). After stirring at room temperature for 5 hours, the reaction mixture was diluted with dichloromethane and washed with brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with 50→67% ethyl acetate/hexane, affording Compound 3 (8.5 g, quant.) as a brown oil.
1H NMR (400 MHz, CDCl3) δ8.11 (t, J=1.1 Hz, 1H), 7.87 (dd, J=8.2, 1.3 Hz, 1H), 7.59-7.48 (m, 2H), 7.46-7.35 (m, 2H), 7.05-6.98 (m, 1H), 6.62 (s, 1H), 0.96 (s, 9H) ppm
13C NMR (101 MHz, CDCl3) δ149.22, 147.85, 136.97, 132.83, 131.71, 131.01, 129.35, 128.58, 124.97, 117.09, 80.39, 36.51, 25.84. ESI-TOF-MS calcd. for C15H17N3NaO4, 326.1111 [M+Na]+; found, 326.1076.
Synthesis of 2′,3′,5′-O-trisTBDMS-guanosine (Compound 5) was performed in accordance with literature. To a suspension of guanosine (Compound 4: 10 g, 35 mmol) and imidazole (19 g, 0.28 mol) in N,N-dimethylformamide (70 mL) was added tert-butyldimethylsilyl chloride (21 g, 0.14 mol). After stirring at room temperature for 2 days, the reaction mixture was diluted with ethyl acetate and washed twice with an aqueous NH4Cl solution and twice with brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with dichloromethane and then with 1→3% methanol/dichloromethane, affording Compound 5 as a white solid (14 g, yield 62%). The spectral data of Compound 5 were all consistent with those of the literature.
To a solution of 2′,3′,5′-O-trisTBDMS-guanosine (Compound 5) (4.9 g, 7.9 mmol), Compound 3 (2.7 g, 8.7 mmol), and 18-crown-6 (2.8 g, 11 mmol) in THF (40 mL) was added 30% potassium hydride (1.4 g, 11 mmol) in mineral oil. After stirring at room temperature for 5 hours, the reaction mixture was diluted with ethyl acetate and washed with a 1:1 mixed liquid of brine and saturated aqueous NH4Cl solution. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with 25→50% ethyl acetate/hexane, affording Compound 6 as a brown foam (4.6 g, yield 68%).
1H NMR (600 MHz, CDCl3) δ11.17, 11.15 (2s, 1H), 8.22, 8.15 (2s, 1H), 8.00 (d, J=2.4 Hz, 1H), 7.90 (d, J=8.2 Hz, 1H), 7.62-7.50 (m, 2H), 7.49-7.42 (m, 1H), 6.53 (d, J=6.2 Hz, 1H), 5.83 (dd, J=5.9, 1.6 Hz, 1H), 4.37 (dd, J=5.8, 4.3, 1.6 Hz, 1H), 4.22 (dd, J=4.4, 2.7 Hz, 1H), 4.06-4.03 (m, 1H), 3.87 (dt, J=11.5, 3.0 Hz, 1H), 3.74 (dd, J=11.4, 2.5 Hz, 1H), 0.96-0.88 (m, 28H), 0.75 (d, J=12.9 Hz, 9H), 0.12-0.04 (m, 13H), −0.08 (d, J=6.9 Hz, 3H), −0.30 (d, J=14.6 Hz, 3H) ppm.
13C NMR (151 MHz, CDCl3) δ155.39, 152.80, 149.27, 149.23, 148.54, 146.38, 137.13, 132.65, 132.60, 132.11, 129.16, 128.77, 128.73, 124.90, 120.86, 87.24, 86.22, 79.15, 77.02, 72.57, 62.99, 60.52, 36.32, 26.15, 25.92, 25.77, 25.69, 21.17, 18.59, 18.16, 17.91, 14.27, −4.35, −4.50, −4.58, −5.11, −5.30, −5.38 ppm. ESI-TOF-MS calcd. for C40H68N6NaO9Si3, 883.4248 [M+Na]+; found, 883.4555.
To an ice-cooled solution of fully protected nucleoside 6 (2.0 g, 2.3 mmol) in THF (11 mL) was added 1 M tetrabutylammonium fluoride/THF (23 mL, 23 mmol) in an ice bath. After stirring in an ice bath at 0° C. for 3 hours, the reaction mixture was diluted with dichloromethane and washed three times with water. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with dichloromethane, and then dissolved with 1→17% methanol/dichloromethane. The product was suspended in dichloromethane and the solution was added dropwise to diethyl ether with stirring. The resulting precipitate was collected by filtration and washed with diethyl ether, affording Compound 7 (1.2 g, quant) as a white solid.
1H NMR (400 MHz, DMSO-d6) δ11.65 (s, 1H), 11.13 (s, 1H), 8.20 (s, 1H), 7.96 (dd, J=8.0, 1.3 Hz, 1H), 7.77 (td, J=7.6, 1.3 Hz, 1H), 7.65-7.52 (m, 2H), 6.23 (s, 1H), 5.75 (dd, J=5.7 Hz, 1H), 5.46 (dd, J=5.8, 3.1 Hz, 1H), 5.16 (d, J=4.6 Hz, 1H), 5.01 (t, J=5.4 Hz, 1H), 4.39 (q, J=5.5 Hz, 1H), 4.09 (q, J=4.3 Hz, 1H), 3.86 (q, J=4.0 Hz, 1H), 3.60 (dt, J=12.0, 4.8 Hz, 1H), 3.50 (dt, J=12.0, 4.6 Hz, 1H), 0.88 (s, 9H) ppm. 13C NMR (101 MHz, DMSO-d6) δ155.56, 154.36, 149.53, 149.31, 147.68, 138.15, 133.69, 132.07, 130.01, 129.33, 125.10, 120.42, 87.15, 85.83, 77.67, 74.46, 70.72, 61.67, 36.46, 25.83 ppm. ESI-TOF-MS calcd. for C22H26N6NaO9, 541.1654 [M+Na]+; found, 541.1887.
To a solution of guanosine derivative 7 (400 mg, 0.77 mmol) in trimethyl phosphate (7 mL) was added phosphoryl chloride (83 μL, 153 mg, 1.0 mmol) with ice cooling. The mixture was gradually warmed to room temperature and stirred at room temperature. After stirring at room temperature for 4 hours, a mixture of 0.5 M tetrabutylammonium dihydrogen phosphate/CH3CN (4.6 mL, 2.3 mmol) and tributylamine (1.6 mL, 1.3 g, 6.9 mmol) was added to the reaction mixture in an ice bath. The reaction mixture was warmed to room temperature and stirred at room temperature for 16 hours. Subsequently, a 0.2 M TEAB buffer (pH 7.9, 10 mL) was added to quench the reaction mixture. After stirring at room temperature for 1 to 2 hours, the clear solution was diluted with water and washed five times with dichloromethane. The aqueous layer was concentrated with a rotary evaporator at 50° C. The residue was purified by ion exchange chromatography on QAE-Sephadex in a linear 0 to 1.5 M gradient of TEAB buffer containing 0 to 10% CH3CN. Guanosine-5′-diphosphate (Compound 8) was obtained in the form of a triethylammonium salt as a white solid (121 mg, 18% yield).
1H NMR (600 MHz, D2O) δ8.15 (s, 1H), 7.84 (dd, J=8.1 Hz, 1H), 7.60 (dd, J=7.3, 3.6 Hz, 2H), 7.44 (dt, J=8.4, 4.3 Hz, 1H), 6.25 (s, 1H), 5.92 (dd, J=6.1, 2.1 Hz, 1H), 4.73-4.68 (m, 1H), 4.43 (dt, J=6.1, 3.1 Hz, 1H), 4.26-4.22 (m, 1H), 4.09 (q, J=4.0 Hz, 2H), 3.06 (q, J=7.4 Hz, 11H), 1.14 (t, J=7.3 Hz, 17H), 0.82 (s, 9H) ppm.
13C NMR (151 MHz, D2O) δ157.47, 154.72, 149.98, 148.55, 147.48, 139.55, 133.45, 133.41, 131.75, 129.33, 129.05, 124.69, 119.49, 87.22, 87.14, 84.11, 84.05, 79.23, 73.94, 73.85, 70.51, 65.17, 58.68, 46.71, 35.78, 24.80, 8.26 ppm.
31P NMR (243 MHz, D2O) δ−10.25 (1P), −10.69 (1P) ppm. ESI-TOF-MS calcd. for C22H27N6O15P2, 677.1015 [M−H]−; found, 677.1162.
To a solution of guanosine-5′-diphosphate (Compound 8) (72.0 mg, 82.0 μmol) in DMSO (1.82 mL) was added iodomethane (88.0 mg, 39.0 μL, 623 μmol). After stirring at room temperature for 24 hours, the reaction mixture was diluted with water and washed five times with diethyl ether. The aqueous phase was concentrated and the residue was dissolved in water. The crude product was purified by reverse-phase HPLC using a YMC-Triart C8 column (250×10.0 mm I.D., S-5 μm, 12 nm, flow rate: 3 mL/min, temperature: 50° C.) with a linear gradient of 10 to 80% CH3CN in a 0.1 M triethylammonium carbonate buffer (pH 7.9) over 25 minutes. The fractions containing the product were collected and acidified to pH 4.0 with the addition of a few drops of acetic acid. After removal of CH3CN under reduced pressure, the product was desalted with Wakosil (registered trademark) 25C18 (15 to 30 μm, spherical) and freeze-dried, affording N7-methyl-guanosine-5′-diphosphate (Compound 9) as a white solid (5.10 mg, 7.0% yield).
1H NMR (600 MHz, D2O) δ7.84 (d, J=8.2 Hz, 1H), 7.60 (s, 2H), 7.44 (d, J=7.2 Hz, 1H), 6.21 (s, 1H), 6.03 (d, J=4.9 Hz, 1H), 4.52 (d, J=15.6 Hz, 1H), 4.37 (s, 1H), 4.27 (s, 1H), 4.19 (d, J=11.7 Hz, 1H), 4.08 (s, 1H), 4.02 (s, 3H), 3.05 (q, J=7.1 Hz, 13H), 1.13 (t, J=7.3 Hz, 20H), 0.81 (s, 9H) ppm.
13C NMR (151 MHz, D2O) δ154.19, 148.63, 148.05, 133.34, 131.98, 129.23, 129.00, 124.64, 111.87, 89.95, 84.20, 78.79, 75.14, 69.16, 63.98, 46.69, 36.06, 35.73, 24.83, 8.25 ppm.
31P NMR (243 MHz, D2O) δ −9.24 (1P), −10.67 (1P) ppm. ESI-TOF-MS calcd. for C23H29N6O15P2, 691.1172 [M−H]−; found, 691.1395.
The synthesis of guanosine-5′-phosphorimidazolide (Compound 10) was carried out in accordance with literature. To a solution of guanosine 5′-monophosphate (300 mg, 0.53 mmol), imidazole (290 mg, 4.2 mmol), 2,2′-dithiodipyridine (350 mg, 1.6 mmol) in N,N-dimethylformamide (7.5 mL) was added triethylamine (150 μL, 110 mg, 1.1 mmol), and then triphenylphosphine (420 mg, 1.6 mmol) was added. After stirring at room temperature for 2 hours, the reaction mixture was added dropwise to a solution of sodium perchlorate (65 mg, 0.53 mmol) in acetone (60 mL) with stirring. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4,500 rpm for 10 minutes. The supernatant was discarded, and the precipitate was washed five times with acetone. The resulting solid was dried under reduced pressure in a desiccator over P2O5, affording guanosine 5′-phosphorimidiazolide monosodium salt (Compound 10) as a white solid (230 mg, quant.).
31P NMR (243 MHz, DMSO-d6) δ1.80 ppm.
N7-methyl-guanosine-5′-diphosphate (Compound 9) (1.30 mg, 1.45 μmol) and guanosine 5′-phosphorimidazolide (Compound 10) (2.10 mg, 4.79 μmol) were dissolved in a 0.59 M zinc chloride solution in DMSO (80 μL, ZnCl2: 47.2 μmol). The mixture was incubated at 37° C. for 2 days. The reaction mixture was quenched by the addition of 3 mM aq. EDTA (1.6 mL, 60 μmol) and diluted with a 0.2 M triethylammonium carbonate buffer (pH 7.9, 600 μL, pH adjusted to around 4.0). The mixture was purified by reverse-phase HPLC using a YMC-Triart C8 column (250×4.6 mm I.D., S-5 μm, 12 nm, flow rate: 1 mL/min, temperature: 50° C.) with a linear gradient of 5 to 80% CH3CN in a 0.1 M triethylammonium carbonate buffer (pH 7.9) over 25 minutes. The fractions containing the product were collected and acidified to pH 4.0 with the addition of a few drops of acetic acid. After removal of CH3CN under reduced pressure, the product was desalted with Wakosil® 25C18 (15 to 30 μm, spherical) and freeze-dried, affording N2-tBu-NB-m7GpppG (Compound 11) as a white solid (0.50 mg, 43% yield).
1H NMR (600 MHz, D2O) δ7.86-7.81 (m, 2H), 7.60 (dt, J=5.4 Hz, 2H), 7.43 (dt, J=8.5, 4.5 Hz, 1H), 6.23 (d, J=2.8 Hz, 1H), 5.90 (t, J=2.9 Hz, 1H), 5.64 (t, J=7.1 Hz, 1H), 4.57-4.51 (m, 1H), 4.44 (dt, J=13.6, 4.1 Hz, 1H), 4.31 (tt, J=9.6, 5.1 Hz, 2H), 4.25-4.20 (m, 2H), 4.18-4.04 (m, 5H), 3.95 (s, 3H), 3.06 (q, J=7.4 Hz, 16H), 1.13 (t, J=7.3 Hz, 24H), 0.82 (s, 9H) ppm.
13C NMR (151 MHz, D2O) δ158.52, 154.16, 153.87, 151.55, 148.53, 147.63, 137.35, 133.35, 131.77, 129.27, 129.01, 124.60, 115.94, 111.35, 89.88, 87.44, 86.60, 84.06, 83.81, 79.01, 75.14, 73.65, 70.46, 69.16, 65.47, 64.30, 46.67, 36.18, 35.76, 24.82, 8.24 ppm.
31P NMR (243 MHz, D2O) δ−11.02 (2P), −22.60 (1P) ppm. ESI-TOF-MS calcd. for C33H41N11O22P3, 1036.1646 [M−H]−; found, 1036.1643.
The synthesis of (R/S)-tBu-(2-nitrobenzyl) alcohol (2) was carried out in accordance with literature. A mixture of 1-iodo-2-nitrobenzene (20 g, 80 mmol) in THF (150 mL) was cooled to −40° C. A solution of phenylmagnesium chloride in THF (2 M, 52 mL, 0.10 mol) was added and stirred at −40° C. for 1 hour. Trimethylacetaldehyde (12 mL, 9.6 g, 0.11 mol) was added to the reaction mixture and stirred at −40° C. for 36 mi. The reaction mixture was gradually warmed to room temperature. After stirring for 4 hours, a saturated aqueous NH4Cl solution was added to stop the reaction. The mixture was extracted three times with ethyl acetate. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with hexane and then with 17→25% ethyl acetate/hexane, affording Compound 2 as a dark brown solid (11 g, yield 65%). The spectral data of the compound were all consistent with those of the literature.
To a solution of (R/S)-1-(2-nitrophenyl)-2,2-dimethyl-1-propanol (Compound 2) (3.00 g, 14.3 mmol) in acetic acid (34.4 g, 33.0 mL, 572 mmol) were added DMSO (22.3 g, 20.0 mL, 286 mmol) and acetic anhydride (29.2 g, 27.0 mL, 286 mmol). After stirring at room temperature for 5 days, a 10 M aqueous KOH solution (130 mL, 1.29 mol) was added dropwise to the reaction mixture in an ice bath. The mixture was stirred at room temperature for 2 hours and then extracted with ethyl acetate. The organic layer was washed with brine and dried over Na2SO4. The residue was dissolved in hexane and then subjected to silica gel column chromatography dissolved with 0 to 6.3% ethyl acetate/hexane, affording methylthioacetal 3 as a yellow oil (3.1 g, yield 79%).
1H NMR (600 MHz, CDCl3) δ7.71 (dd, J=8.2, 1.3 Hz, 1H), 7.64 (dd, J=7.9, 1.5 Hz, 1H), 7.57-7.51 (m, 1H), 7.40-7.33 (m, 1H), 5.28 (s, 1H), 4.62 (d, J=11.6 Hz, 1H), 4.27 (d, J=11.6 Hz, 1H), 2.11 (s, 3H), 0.83 (s, 9H) ppm.
13C NMR (151 MHz, CDCl3) δ151.20, 133.97, 131.94, 130.07, 128.35, 123.94, 79.15, 74.16, 36.41, 26.00, 14.58 ppm. ESI-TOF-MS calcd. for C13H19NNaO3S, 292.0978 [M+Na]+; found, 292.0981.
To a suspension of N2-isobutyryl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)guanosine (Compound 4) (2.96 g, 4.97 mmol), 2,2-dimethyl-(R/S)-1-(2-nitrophenyl)propylmethylthioacetal (Compound 3) (1.47 g, 5.47 mmol) and powdered molecular sieve 3 angstrom (2.00 g) were charged in tetrahydrofuran (50.0 mL), and N-iodosuccinimide (1.23 g, 5.47 mmol) was added, followed by cooling to −40° C. Trifluoromethanesulfonic acid (1.12 g, 662 μL, 7.46 mmol) was added to the cooled suspension, and the mixture was stirred at −40° C. for 19 hours. The reaction mixture was quenched by addition of triethylamine (24.0 mL), diluted with ethyl acetate, and washed twice with saturated aq. NaHCO3. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with 0 to 3.2% methanol/dichloromethane, affording Compound 5 as a yellow foam (3.12 g, yield 76%).
1H NMR (600 MHz, CDCl3) δ12.08 (s, 1H), 9.04 (s, 1H), 7.94 (s, 1H), 7.74 (dd, J=8.4 Hz, 1H), 7.61-7.52 (m, 2H), 7.40 (dd, J=8.3, 4.3 Hz, 1H), 5.92 (s, 1H), 5.19 (d, J=6.7 Hz, 1H), 4.61 (d, J=6.7 Hz, 1H), 4.52 (dd, J=9.4, 4.4 Hz, 1H), 4.28 (d, J=4.4 Hz, 1H), 4.16 (d, J=13.4 Hz, 1.5H), 4.02 (dd, J=9.5, 2.3 Hz, 1.5H), 3.93 (dd, J=13.4, 2.5 Hz, 2H), 2.73 (p, J=6.9 Hz, 1H), 1.23 (d, J=6.8 Hz, 3H), 1.18 (d, J=6.9 Hz, 3H), 1.12-0.91 (m, 78H), 0.91-0.82 (m, 7H), 0.65 (s, 9H) ppm.
13C NMR (151 MHz, CDCl3) δ179.11, 155.65, 150.82, 147.80, 147.47, 136.43, 133.14, 132.35, 130.10, 128.64, 124.11, 121.15, 91.47, 88.01, 80.98, 77.42, 77.21, 76.99, 76.25, 70.30, 59.48, 50.81, 36.54, 36.45, 25.78, 19.13, 18.92, 17.51, 17.44, 17.39, 17.35, 17.31, 17.27, 17.24, 17.17, 17.13, 17.02, 16.89, 13.47, 13.25, 13.10, 12.94, 12.70, 12.57 ppm. ESI-TOF-MS calcd. for C38H61N6O10Si2, 817.3982 [M+H]+; found, 817.3921.
A solution of compound 5 (850 mg, 1.04 mmol) in tetrahydrofuran (4.80 mL) was cooled in an ice bath, and 1 M TBAF/THF (10.0 mL, 10.4 mmol) was added. After stirring at 0° C. for 5 hours, the reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with 0 to 9.1% methanol/dichloromethane, affording Compound 6 as a colorless amorphous solid (450 mg, yield 75%).
1H NMR (600 MHz, CDCl3, mixture of stereoisomers) δ12.40 (s, 0.5H), 12.36 (s, 0.5H), 10.27 (m, 0.5H), 10.21 (m, 0.5H), 8.21 (s, 0.5H), 8.00 (s, 0.5H), 7.66 (d, J=8.8.21 (s, 0.5H), 8.00 (s, 0.5H), 7.66 (d, J=8.1 Hz, 0.5H), 7.61 (d, J=7.9 Hz, 0.5H), 7.53-7.45 (m, 2H), 7.35 (q, J=8.3 Hz, 1.5H), 7.25 (t, J=7.7 Hz, 0.5H), 5.98 (d, J=4.7 Hz, 0.5H), 5.80 (d, J=4.6 Hz, 0.5H), 5.10 (s, 0.5H), 4.99 (s, 0.5H), 4.84-4.71 (m, 2.5H), 4.67 (t, J=4.8 Hz, 1H), 4.58 (t, J=10.7 Hz, 1.5H), 4.13 (dd, J=10.4, 3.8 Hz, 1H), 3.91-3.84 (m, 1H), 3.76 (t, J=13.2 Hz, 1H), 3.39 (t, J=4.1 Hz, 1H), 2.90 (h, J=6.8 Hz, 1H), 1.24 (td, J=4.3, 2.0 Hz, 6H), 0.71 (s, 4.5H), 0.59 (s, 4.5H) ppm.
13C NMR (151 MHz, CDCl3, mixture of stereoisomers) δ180.28, 180.22, 155.67, 155.60, 150.52, 149.79, 148.38, 148.27, 148.16, 147.86, 139.26, 133.87, 133.51, 132.14, 131.81, 130.01, 129.92, 128.51, 128.32, 123.95, 123.77, 121.00, 120.94, 95.02, 93.66, 88.27, 86.19, 86.05, 80.47, 79.49, 79.41, 79.23, 77.38, 77.17, 76.96, 70.68, 70.50, 70.25, 62.00, 36.66, 36.21, 36.16, 26.57, 25.65, 25.60, 19.14, 19.06, 19.03 ppm. ESI-TOF-MS calcd. for C26H33N6O9, 573.2315 [M−H]−; found 573.2333.
A solution of compound 5 (850 mg, 1.04 mmol) in tetrahydrofuran (4.80 mL) was cooled in an ice bath, and 1 M TBAF/THF (10.0 mL, 10.4 mmol) was added. After stirring at 0° C. for 5 hours, the reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography dissolved with 0 to 9.1% methanol/dichloromethane, affording Compound 6 as a colorless amorphous solid (450 mg, yield 75%).
1H NMR (600 MHz, CDCl3, mixture of stereoisomers) δ12.40 (s, 0.5H), 12.36 (s, 0.5H), 10.27 (m, 0.5H), 10.21 (m, 0.5H), 8.21 (s, 0.5H), 8.00 (s, 0.5H), 7.66 (d, J=8.8.21 (s, 0.5H), 8.00 (s, 0.5H), 7.66 (d, J=8.1 Hz, 0.5H), 7.61 (d, J=7.9 Hz, 0.5H), 7.53-7.45 (m, 2H), 7.35 (q, J=8.3 Hz, 1.5H), 7.25 (t, J=7.7 Hz, 0.5H), 5.98 (d, J=4.7 Hz, 0.5H), 5.80 (d, J=4.6 Hz, 0.5H), 5.10 (s, 0.5H), 4.99 (s, 0.5H), 4.84-4.71 (m, 2.5H), 4.67 (t, J=4.8 Hz, 1H), 4.58 (t, J=10.7 Hz, 1.5H), 4.13 (dd, J=10.4, 3.8 Hz, 1H), 3.91-3.84 (m, 1H), 3.76 (t, J=13.2 Hz, 1H), 3.39 (t, J=4.1 Hz, 1H), 2.90 (h, J=6.8 Hz, 1H), 1.24 (td, J=4.3, 2.0 Hz, 6H), 0.71 (s, 4.5H), 0.59 (s, 4.5H) ppm.
13C NMR (151 MHz, CDCl3, mixture of stereoisomers) δ180.28, 180.22, 155.67, 155.60, 150.52, 149.79, 148.38, 148.27, 148.16, 147.86, 139.26, 133.87, 133.51, 132.14, 131.81, 130.01, 129.92, 128.51, 128.32, 123.95, 123.77, 121.00, 120.94, 95.02, 93.66, 88.27, 86.19, 86.05, 80.47, 79.49, 79.41, 79.23, 77.38, 77.17, 76.96, 70.68, 70.50, 70.25, 62.00, 36.66, 36.21, 36.16, 26.57, 25.65, 25.60, 19.14, 19.06, 19.03 ppm. ESI-TOF-MS calcd. for C26H33N6O9, 573.2315 [M−H]−; found 573.2333.
A 28% ammonium hydroxide solution (36.0 mL) was added to a solution of Compound 6 (1.08 g, 1.88 mmol) in acetonitrile (14.4 mL). The mixture was heated to 55° C. After stirring at 55° C. for 6 hours, the reaction mixture was concentrated and azeotroped with benzene. The residue was suspended in a 1:3 mixture of dichloromethane and diethyl ether. The resulting precipitate was collected by filtration and washed with a 1:3 mixture of dichloromethane and diethyl ether. The resulting solid was dried in a desiccator on P2O5 under vacuum, affording Compound 7 as a white foam (666 mg, 70% yield).
1H NMR (600 MHz, DMSO-d6, mixture of stereoisomers) δ10.61, 10.58 (2s, 1H), 7.88 (s, 0.5H), 7.79 (dd, J=8.0, 1.3 Hz, 0.5H), 7.70-7.62 (m, 1.5H), 7.53-7.46 (m, 1H), 7.41 (m, 1H), 7.31 (ddd, J=8.6, 6.6, 2.1 Hz, 0.5H), 6.37 (d, J=10.1 Hz, 2H), 5.81 (d, J=6.0 Hz, 0.5H), 5.59 (d, J=5.6 Hz, 0.5H), 5.21 (d, J=5.4 Hz, 0.5H), 5.15 (d, J=5.3 Hz, 0.5H), 5.10 (t, J=5.6 Hz, 0.5H), 5.03 (t, J=5.5 Hz, 0.5H), 4.95 (s, 0.5H), 4.82 (s, 0.5H), 4.71-4.67 (m, 1H), 4.67-4.61 (m, 1H), 4.47 (t, J=5.2 Hz, 0.5H), 4.38 (d, J=7.2 Hz, 0.5H), 4.23 (td, J=5.1, 3.8 Hz, 1H), 3.87 (q, J=3.6 Hz, 0.5H), 3.82 (q, J=3.7 Hz, 0.5H), 3.63-3.52 (m, 1H), 3.52-3.42 (m, 1H), 0.68 (s, 4.5H), 0.53 (s, 4.5H) ppm.
13C NMR (151 MHz, DMSO-d6, mixture of stereoisomers) 5157.25, 154.15, 154.11, 151.61, 151.48, 150.98, 150.00, 136.35, 135.70, 133.46, 133.00, 132.82, 132.39, 130.10, 129.99, 129.45, 128.93, 124.35, 124.11, 117.38, 117.18, 94.57, 92.51, 86.37, 86.04, 85.85, 85.39, 80.21, 78.95, 78.84, 78.72, 70.25, 70.06, 69.75, 62.05, 61.81, 40.46, 40.32, 40.19, 40.04, 39.91, 39.76, 39.62, 36.64, 36.04, 25.88, 25.83 ppm. ESI-TOF-MS calcd. for C22H28N6NaO8, 527.1861 [M+Na]+; found 527.1758.
To a solution of guanosine derivative 7 (200 mg, 0.396 mmol) in trimethyl phosphate (3.70 mL) was added phosphoryl chloride (48.0 μL, 79.0 mg, 0.515 mmol) with ice cooling. The mixture was gradually warmed to room temperature and stirred at room temperature. After stirring at room temperature for 2 hours, a mixture of 0.44 M tetrabutylammonium dihydrogen phosphate/CH3CN (1.80 mL, 0.792 mmol) and tributylamine (846 μL, 660 mg, 3.56 mmol) was added to the reaction mixture in an ice bath. The reaction mixture was warmed to room temperature and stirred at room temperature for 19 hours. Subsequently, a 0.2 M triethylammonium carbonate buffer (pH 7.9, 5.0 mL) was added to quench the reaction mixture. After stirring at room temperature for 1 hour, the mixture was diluted with water and washed five times with dichloromethane. The aqueous layer was concentrated with a rotary evaporator at 50° C. The residue was purified by ion exchange chromatography on QAE-Sephadex in a linear 0 to 1.5 M gradient of triethylammonium carbonate buffer containing 0 to 10% CH3CN. Guanosine-5′-diphosphate (Compound 8) was obtained as a triethylammonium salt in the form of a white solid (51.0 mg, 13% yield).
1H NMR (600 MHz, D2O, mixture of stereoisomers) δ7.87 (s, 0.5H), 7.58 (s, 0.5H), 7.52 (d, J=8.1 Hz, 0.5H), 7.47-7.38 (m, 1H), 7.38-7.29 (m, 1H), 7.28-7.17 (m, 1H), 7.01 (t, J=7.8 Hz, 0.5H), 5.73 (d, J=5.8 Hz, 0.5H), 5.48 (d, J=5.0 Hz, 0.5H), 4.96, 4.94 (2s, 1H), 4.82 (d, J=7.5 Hz, 0.5H), 4.76 (s, 0.5H), 4.71 (d, J=7.3 Hz, 0.5H), 4.62 (dd, J=18.0, 6.6 Hz, 1.5H), 4.52 (t, J=5.1 Hz, 0-.5H), 4.45 (t, J=5.0 Hz, 1H), 4.14 (d, J=4.1 Hz, 0.5H), 4.10-4.03 (m, 1.5H), 3.99 (d, J=4.8 Hz, 1H), 3.02 (q, J=7.3 Hz, 15H), 1.10 (t, J=7.3 Hz, 24H), 0.52 (s, 4.5H), 0.41 (s, 4.5H) ppm.
13C NMR (151 MHz, D2O, mixture of stereoisomers) δ158.95, 158.75, 153.89, 153.59, 151.59, 151.01, 149.74, 148.09, 138.02, 137.37, 133.49, 132.57, 132.47, 132.21, 129.86, 129.66, 128.65, 128.06, 123.80, 123.30, 116.33, 115.96, 95.44, 93.71, 86.20, 84.26, 84.20, 83.73, 83.67, 81.61, 80.72, 78.55, 76.97, 69.07, 68.94, 64.84, 64.81, 64.46, 46.63, 36.55, 35.65, 24.78, 24.68, 8.24 ppm.
31P NMR (243 MHz, D2O, mixture of stereoisomers) δ−8.54 (1P), −10.61 (1P) ppm. ESI-TOF-MS calcd. for C22H29N6O14P2, 663.1223 [M−H]−; found 663.1378.
To a solution of guanosine-5′-diphosphate (Compound 8) (4.3 mg, 4.4 μmol) in DMSO (100 μL) was added iodomethane (4.7 mg, 2.1 μL, 33 μmol). After stirring at room temperature for 39 hours, the reaction mixture was diluted with water and washed five times with diethyl ether. The aqueous phase was concentrated and the residue was dissolved in water. Crude purification was performed by reverse-phase HPLC using a YMC-Triart C8 column (250×4.6 mm I.D., S −5 μm, 12 nm, flow rate 1 mL/min, temperature 50° C.) with a linear gradient of 5 to 80% in concentration of CH3CN in a 0.1 M triethylammonium hydrogen carbonate buffer (pH 7.9) over 25 min. The fractions containing the product were collected and acidified to pH 4.0 with the addition of a few drops of acetic acid. After removal of CH3CN under reduced pressure, the product was desalted with Wakosil (registered trademark) 25C18 (15 to 30 μm, spherical) and freeze-dried, affording N7-methyl-guanosine-5′-diphosphate (Compound 9) as a white solid (0.60 mg, 18% yield, a mixture of two stereoisomers).
1H NMR (600 MHz, D2O, single stereoisomer isolated by HPLC) δ8.93 (s, 1H), 7.60 (d, J=8.2 Hz, 1H), 7.49 (dd, J=15.2, 8.1 Hz, 2H), 7.29 (t, J=7.6 Hz, 1H), 5.82 (s, 1H), 5.00 (d, J=4.1 Hz, 1H), 4.93 (d, J=8.0 Hz, 1H), 4.86 (d, J=7.2 Hz, 1H), 4.54-4.50 (m, 1H), 4.42 (t, J=5.2 Hz, 1H), 4.25 (s, 1H), 4.10 (d, J=11.2 Hz, 1H), 3.94 (d, J=3.7 Hz, 3H), 3.05 (t, J=7.3 Hz, 4H), 1.13 (t, J=7.1 Hz, 6H), 0.61 (s, 10H) ppm.
13C NMR (151 MHz, D2O, single stereoisomer isolated by HPLC) 5155.41, 154.99, 149.37, 148.61, 136.68, 133.90, 132.44, 130.24, 128.15, 123.51, 107.91, 95.16, 87.76, 85.95, 82.16, 79.32, 68.89, 64.78, 46.70, 36.52, 36.07, 24.76, 8.25 ppm.
31P NMR (243 MHz, D2O, single stereoisomer isolated by HPLC) δ−10.03 (1P), −10.71 (1P) ppm. ESI-TOF-MS calcd. for C23H31N6O14P2, 677.1379 [M−H]−; found 677.1374.
The synthesis of guanosine-5′-phosphorimidazolide (Compound 10) was carried out in accordance with literature. To a solution of guanosine 5′-monophosphate (300 mg, 0.53 mmol), imidazole (290 mg, 4.2 mmol), 2,2′-dithiodipyridine (350 mg, 1.6 mmol) in N,N-dimethylformamide (7.5 mL) was added triethylamine (150 μL, 110 mg, 1.1 mmol), and then triphenylphosphine (420 mg, 1.6 mmol) was added. After stirring at room temperature for 2 hours, the reaction mixture was added dropwise to a solution of sodium perchlorate (65 mg, 0.53 mmol) in acetone (60 mL) with stirring. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4,500 rpm for 10 minutes. The supernatant was discarded, and the precipitate was washed five times with acetone. The resulting solid was dried under reduced pressure in a desiccator over P2O5, affording guanosine 5′-phosphorimidiazolide monosodium salt (Compound 10) as a white solid (230 mg, quant.).
31P NMR (243 MHz, DMSO-d6) δ1.80 ppm.
N7-methyl-guanosine-5′-diphosphate (Compound 9) (1.1 mg, 1.3 μmol) and guanosine 5′-phosphorimidazolide (Compound 10) (1.9 mg, 4.3 μmol) were dissolved in 0.59 M zinc chloride solution in DMSO (80 μL, ZnCl2: 47 μmol). The mixture was incubated at 37° C. for 2 days. The reaction mixture was quenched by the addition of 38 mM aq. EDTA (1.6 mL, 60 μmol) and diluted with a 0.2 M TEAB buffer (pH 7.9, 600 μL, pH adjusted to around 4.0). The mixture was purified by reverse-phase HPLC using a YMC-Triart C8 column (250×4.6 mm I.D., S-5 μm, 12 nm, flow rate: 1 mL/min, temperature: 50° C.) with a linear gradient of 5 to 80% CH3CN in a 0.1 M triethylammonium carbonate buffer (pH 7.9) over 25 minutes. The fractions containing the product were collected and acidified to pH 4.0 with the addition of a few drops of acetic acid. After removal of CH3CN under reduced pressure, the product was desalted with Wakosil (registered trademark) 25C18 (15 to 30 μm, spherical) and freeze-dried, affording 2′-O-tBu-NB-m7GpppG (Compound 11) as a white solid (0.90 mg, 82% yield).
1H NMR (600 MHz, D2O, mixture of stereoisomers) δ7.85, 7.82 (2s, 1H), 7.66 (dd, J=8.1, 1.3 Hz, 0.5H), 7.57-7.51 (m, 1H), 7.49-7.41 (m, 1.5H), 7.38-7.32 (m, 0.5H), 7.24 (dd, J=8.4, 4.1 Hz, 0.5H), 5.81 (d, J=4.8 Hz, 0.5H), 5.66-5.59 (m, 1.5H), 4.95 (s, 0.5H), 4.87 (d, J=7.8 Hz, 1H), 4.80-4.70 (m, 1H), 4.55-4.45 (m, 2H), 4.41 (t, J=4.9 Hz, 0.5H), 4.35-4.25 (m, 2H), 4.25-4.04 (m, 8.5H), 3.95 (s, 1.5H), 3.90 (s, 1.5H), 3.06 (q, J=7.4 Hz, 13H), 1.14 (t, J=7.3 Hz, 20H), 0.56 (s, 5H), 0.53 (s, 6H) ppm.
13C NMR (151 MHz, D2O, mixture of stereoisomers) δ158.25, 155.45, 155.20, 154.66, 154.51, 153.89, 149.73, 149.40, 148.93, 148.54, 137.38, 136.90, 136.48, 133.82, 132.74, 132.41, 130.14, 130.00, 128.82, 128.08, 123.88, 123.40, 107.73, 107.39, 94.91, 93.58, 87.60, 87.04, 86.94, 85.62, 84.87, 83.72, 81.98, 80.56, 79.67, 79.24, 73.84, 73.74, 70.26, 68.68, 68.24, 65.37, 64.96, 64.56, 62.55, 46.68, 36.42, 36.18, 35.74, 24.84, 24.71, 8.25 ppm.
31P NMR (243 MHz, D2O, mixture of stereoisomers) δ−10.96 (2P), −22.54 (1P) ppm. ESI-TOF-MS calcd. for C33H43N11O21P3, 1022.1853 [M−H]−; found 1022.1851.
The cap analogs (Cap Analog_1 and Cap Analog_2) synthesized above were subjected to various analyses.
(a) of the figure illustrates the chemical structure of cap analog_1.
(b) of the figure shows the result of reverse-phase HPLC analysis after isolation and purification of Cap Analog_1. The analysis conditions are as follows.
Column: YMC-Triart C8 (250 mm×4.6 mm I.D.); Solvent A: 0.1 M triethylammonium carbonate buffer (pH 7.9); Solvent B: acetonitrile; Linear Gradient: 5 to 80% Solvent B (0 to 25 min); Flow rate: 1 mL/min; Detection wavelength: 280 nm; Column temperature: 50° C.
(c) of the figure shows the result of ESI-TOF MS molecular weight measurement.
(d) of the figure shows the result of 1H-NMR analysis, and (e) of the figure shows the result of 31P-NMR analysis.
(a) of the figure illustrates the chemical structure of the cap analog_2.
(b) of the figure shows the result of reverse-phase HPLC analysis after isolation and purification. The analysis conditions are as follows.
Column: YMC-Triart C8 (250 mm×4.6 mm I.D.); Solvent A: 0.1 M triethylammonium carbonate buffer (pH 7.9); Solvent B: acetonitrile; Linear Gradient: 5 to 80% Solvent B (0 to 25 min); Flow rate: 1 mL/min; Detection wavelength: 280 nm; Column temperature: 50° C.
(c) of the figure shows the result of ESI-TOF MS molecular weight measurement.
(d) of the figure shows the result of 1H-NMR analysis, and (e) of the figure shows the result of 31P-NMR analysis.
(4) Isolation and Purification of RNA with Novel Cap Analogue Introduced Therein
In (A) of the figure, a cap analog compound was added, a NanoLuc luciferase mRNA (650-base-long) was transcriptionally synthesized using T7 RNA polymerase, and the transcription reaction was analyzed by denaturing polyacrylamide gel (5%, containing 7.5 M urea as a denaturant) electrophoresis. The composition of the transcription reaction liquid and the reaction conditions are shown below.
15 ng/μL dsDNA transcription template (PCR product of 676-bp containing T7 promoter sequence 5′TAATACGACTCACTATAG3′-(SEQ ID NO: 1) and containing a NanoLuc luciferase gene coding region derived from a pNL1.1 TK vector (Promega)), 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP or [0.5 mM GTP+2 mM Cap Analog], 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 10 units/μL T7 RNA polymerase (Takara Bio Inc.).
The reaction liquid was warmed at 37° C. for 2 hours. After electrophoresis, the gel was stained with SYBR Green II stain and photographed with ChemiDoc MP imager (Bio-Rad), whereby the RNA was visualized.
(B) of the figure is a diagram showing the results of reverse-phase HPLC analysis and purification of a transcribed RNA.
(a) and (d) show the results of performing a transcription reaction by adding (a) Cap Analog_1 (CA1) or (d) Cap Analog_2 (CA2), subjecting an RNA to crude purification (deproteinization, alcohol precipitation, colloquial filtration), and analyzing the RNA by reverse-phase HPLC. A peak appearing around 15 minutes and a peak appearing thereafter were each fractionated and purified.
(b) of the figure shows the result of reanalysis performed by fractionating the eluted RNA around 17.9 minutes shown in (a).
(e) of the figure shows the result of reanalysis performed by fractionating the eluted RNA around 16.8 minutes shown in (d).
(c) of the figure shows the result of RNA analysis after irradiation with 365-nm light at 4 mW/cm2 for 10 minutes of the fractionated RNA eluted near 17.9 shown in (a). From the fact that the elution time of the RNA was completely shifted from around 17.9 minutes to around 15 minutes, it can be determined that the hydrophobic protecting group of the cap analog moiety was completely deprotected.
(f) of the figure shows the result of RNA analysis after irradiation with 365-nm light at 4 mW/cm2 for 10 minutes of the fractionated RNA eluted near 16.8 shown in (d). From the fact that the elution time of the RNA was completely shifted from around 16.8 minutes to around 15 minutes, it was determined that the hydrophobic protecting group of the cap analog moiety was completely deprotected.
The analytical conditions of HPLC are shown below.
Column: YMC-Triart Bio C4 (250 mm×4.6 mm I.D.); Solvent A, 50 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; Solvent B, acetonitrile; Linear Gradient 0 to 20% Solvent B (0 to 20 min); Flow rate: 1 mL/min; Detection wavelength: 260 nm; Column temperature: 50° C.
Cap Analog_1 has a form in which the hydrogen bonding of a base moiety is protected, and can completely inhibit translation activity in the protected state. Cap Analog_1 can be applied, through irradiation with light, to an mRNA that activates translation in vivo.
A synthesis scheme of a hydrophobic chemically capped compound (Compound 8) is shown below.
1-Eicosanol (3.00 g, 0.010 mmol, 1.0 equiv.) and Celite (4.0 g) were suspended in dehydrated dichloromethane (20 mL) at 0° C. Pyridinium chlorochromate (3.23 g, 0.015 mmol, 1.5 equiv.) was added thereto, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, a filtrate was collected by Celite filtration and concentrated. The residue was purified by column chromatography (neutral flash silica, dichloromethane), affording 2.80 g (0.095 mmol, yield 95%) of Compound 1 as a white solid.
1H-NMR (400 MHz, CDCl3) δ9.76 (t, J=1.6 Hz, 1H), 1.29-1.25 (m, 36H), 0.88 (t, J=7.2 Hz, 3H) ppm.
The other spectral data agreed well with literature values (Gray G. M., Macfarlane M. G., Biochem. J., 1961, 81, 480-488).
1-Iodo-2-nitrobenzene (2.65 g, 10.7 mmol, 1.0 equiv.) was dissolved in THF (25 mL). Phenylmagnesium chloride (2.0 M solution in THF, 5.86 mL, 11.7 mmol, 1.1 equiv.) was added dropwise at −48° C., and the mixture was stirred at −35° C. for 10 minutes. Compound 1 (3.00 g, 10.1 mmol, 0.95 equiv.) dissolved in THF (15 mL) was added dropwise, the reaction temperature was gradually raised to room temperature, and then the mixture was stirred at room temperature for 15 minutes. A saturated aqueous ammonium chloride solution (10 mL) was added thereto, and the resulting mixture was subjected to liquid-liquid separation twice with ethyl acetate (100 mL) and once with saturated saline (50 mL). The organic layer was collected and concentrated. The residue was purified by column chromatography (neutral flash silica, ethyl acetate/hexane=1/15→1/10), affording 3.44 g (8.65 mmol, yield 81%) of Compound 2 as a brown solid.
1H-NMR (400 MHz, CDCl3) δ7.89 (dd, J=8.2 Hz, 1.6 Hz, 1H), 7.80 (dd, J=6.4 Hz, 1.6 Hz, 1H), 7.63 (td, J=6.4 Hz, 1.2 Hz, 1H), 7.41 (td, J=6.0 Hz, 1.6 Hz, 1H), 5.23 (q, J=4.0 Hz, 1H), 2.09-1.78 (m, 3H), 1.31-1.21 (m, 33H), 0.88 (t, J=7.2 Hz, 3H) ppm.
13C-NMR (100 MHz, CDCl3) δ148.1, 140.3, 133.5, 128.1, 128.0, 124.4, 69.5, 38.3, 32.0, 29.8-29.4, 26.2, 22.8, 14.2 ppm.
ESI MS-HR calcd. for C26H45NNaO3+ (M+Na)+ 442.3297, found 442.3421.
Guanosine (2.00 g, 7.06 mmol, 1.0 equiv.) and tert-butyldimethylsilyl chloride (6.58 g, 43.6 mmol, 6.2 equiv.) were dissolved in dehydrated DMF (25 mL), and the solution was stirred at 0° C. for 10 minutes. Imidazole (3.75 g, 55.1 mmol, 7.8 equiv.) was added thereto, and the mixture was stirred at room temperature for 3 hours. Ethyl acetate (200 mL) was added thereto, the mixture was washed twice with water (50 mL), and the organic layer was collected and concentrated. The residue was purified by column chromatography (neutral flash silica, hexane/ethyl acetate=2/1→1/2), affording 3.29 g (5.26 mmol, yield 74%) of Compound 3 as a white solid.
1H-NMR (400 MHz, DMSO-d6) δ10.61 (s, 1H), 7.89 (s, 1H), 6.45 (s, 2H), 5.74 (d, J=6.8 Hz, 1H), 4.59 (dd, J=4.8 Hz, 2.4 Hz, 1H), 4.17 (dd, J=1.2 Hz, 3.2 Hz, 1H), 3.95 (t, J=3.6 Hz, 1H), 3.85 (dd, J=1.6 Hz, 2.0 Hz, 1H), 3.71 (dd, J=3.6 Hz, 8.0 Hz, 1H), 0.87 (s, 18H), 0.73 (s, 9H), 0.09-0.11 (m, 12H), −0.08 (s, 3H), −0.28 (s, 3H) ppm.
The other spectral data agreed well with literature values (Ogilvie K. K., Schifman A. L., Penney C. L., Can. J. Chem., 1979, 57(17), 2230-2238).
Nitrobenzyl alcohol derivative 2 (1.25 g, 2.98 mmol, 1.0 equiv.) and 1,1′-carbonyldiimidazole (966 mg, 5.96 mmol, 2.0 equiv.) were dissolved in dehydrated dichloromethane (12 mL), and the mixture was stirred at room temperature for 2 hours. The mixture was subjected once to liquid-liquid separation with saturated saline, and the organic layer was collected and concentrated. In the same flask, Compound 3 (2.23 g, 3.58 mmol, 1.2 equiv.) and 18-crown-6 (1.18 g, 4.47 mmol, 1.5 equiv.) were added and dissolved in dehydrated THF (15 mL). Potassium hydride (35% dispersion in mineral oil, 179 mg, 4.47 mmol, 1.5 equiv.) was gradually added thereto at 0° C., and the mixture was stirred at 40° C. for 2 hours. After the solvent was distilled off, ethyl acetate was added, and the mixture was washed with saturated saline solution: saturated ammonium chloride aqueous solution water (2:2:1). The organic layer was collected and concentrated, and then purified by column chromatography (neutral flash silica, ethyl acetate/hexane=1/5→1/2), affording 2.24 g (2.09 mmol, yield 70%) of Compound 4 as a white solid.
1H-NMR (400 MHz, CDCl3) δ11.14 (s, 1H), 8.13 (s, 1H), 8.00 (dt, J=8.4 Hz, 1.6 Hz, 1H), 7.65 (t, J=7.6 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.49 (t, J=8.8 Hz, 1H), 6.32 (m, 1H), 5.86 (dd, J=4.8 Hz, 1.2 Hz, 1H), 4.41 (q, J=4.4 Hz, 1H), 4.24 (t, J=3.2 Hz, 1H), 4.09 (d, J=1.6 Hz, 1H), 3.91 (dt, J=11.2 Hz, 2.8 Hz, 1H), 3.77 (dd, J=10.0 Hz, 1.6 Hz, 1H), 2.04-1.96 (m, 1H), 1.29-1.25 (m, 36H), 0.94 (s, 18H), 0.87 (t, J=6.8 Hz, 3H), 0.80 (d, J=7.2 Hz, 9H), 0.15-0.11 (m, 12H), −0.04 (d, J=4.0 Hz, 3H), −0.25 (d, J=10.8 Hz, 3H) ppm.
13C-NMR (100 MHz, CDCl3) δ154.4, 148.1, 137.0, 135.8, 133.5, 129.0, 127.2, 124.8, 87.5, 86.2, 75.1, 72.5, 62.9, 36.1, 32.0, 29.7-29.4, 26.1-25.7, 22.7, 18.6, 18.1, 14.1 ppm.
ESI MS-HR calcd. for C55H98N6NaO9Si3+ (M+Na)+ 1093.6601, found 1093.6626.
Compound 4 (2.20 g, 2.05 mmol, 1.0 equiv.) was dissolved in dehydrated THF (20 mL). Triethylamine hydrogen trifluoride (2.18 mL, 13.4 mmol, 6.5 equiv.) was added dropwise at 0° C., and the mixture was stirred at room temperature for 2 days. Ethyl acetate (50 mL) was added, and the mixture was washed once with saturated saline (25 mL), and the organic layer was collected and concentrated. The residue was purified by column chromatography (neutral flash silica, methanol/chloroform=1/15→1/8), affording 1.46 g (2.00 mmol, yield 98%) of Compound 5 as a white solid.
1H-NMR (400 MHz, DMSO-d6) δ11.56 (s, 1H), 11.10 (s, 1H), 8.18 (s, 1H), 8.00 (d, J=7.6 Hz, 1H), 7.79 (t, J=7.2 Hz, 1H), 7.68 (d, J=7.6 Hz, 1H), 7.57 (t, J=7.2 Hz, 1H), 6.08 (t, J=6.0 Hz, 1H), 5.72 (d, J=5.6 Hz, 1H), 5.42 (br, 1H), 5.14 (br, 1H), 4.96 (br, 1H), 4.40 (q, J=5.2 Hz, 1H), 4.09 (t, J=4.0 Hz, 1H), 3.85 (d, J=4.0 Hz, 1H), 3.54 (dd, J=22.8 Hz, 7.6 Hz, 2H), 1.22-1.13 (m, 36H), 0.84-0.77 (m, 3H) ppm.
13C-NMR (100 MHz, CDCl3) δ154.4, 148.1, 138.2, 136.0, 133.6, 128.9, 127.2, 124.2, 88.4, 85.5, 75.1, 75.0, 73.7, 70.4, 61.2, 35.7, 31.7, 29.4-29.2, 28.7, 25.4, 22.4, 13.1 ppm.
ESI MS-HR calcd. for C37H56N6NaO9+ (M+Na)+ 751.4001, found 751.4091.
Compound 5 (100 mg, 0.137 mmol, 1.0 equiv.) was dissolved in trimethyl phosphate (2.5 mL). Phosphoryl chloride (23.0 μL, 0.237 mmol, 1.8 equiv.) was added dropwise thereto at 0° C., and the mixture was stirred at −5° C. overnight. A mixed solution of a 0.35 m tributylammonium phosphate solution in DMF (2.34 mL, 0.824 mmol, 6.0 equiv.) and tributylamine (294 μL, 1.24 mmol, 9.0 equiv.) was added dropwise, and the mixture was stirred at −10° C. for 6 hours. After the reaction, a 0.2 M triethylammonium carbonate buffer (5 mL, pH 7.9) was added, and the mixture was stirred at room temperature for 30 minutes. The mixture was purified by reverse-phase HPLC (YMC Triart C8 (250×10 mm l.D., S-5 μm, 12 nm, TO12S05-2510WT), solvent: A) 0.1 M triethylammonium carbonate buffer (pH 7.97), B) acetonitrile, gradient: 40 to 100% B (0 to 35 min), column temperature: room temperature, flow rate: 3 mL/min, detection wavelength: 260 nm), affording 31.1 mg (35.0 μmol, yield 26%, triethylammonium salt) of Compound 6 as a white solid.
1H-NMR (600 MHz, DMSO-d6) δ8.11 (d, J=4.8 Hz, 1H), 8.01 (d, J=7.8 Hz, 1H), 7.81 (q, J=7.2 Hz, 1H), 7.57 (q, J=7.2 Hz, 1H), 6.13 (td, J=9.0 Hz, 4.2 Hz, 1H), 5.71 (d, J=6.0 Hz, 1H), 4.77 (dt, J=34.2 Hz, 6.0 Hz, 1H), 4.32 (dt, J=13.8 Hz, 3.0 Hz, 1H), 3.97 (t, J=3.6 Hz, 1H), 3.88 (m, 1H), 1.97-1.80 (m, 2H), 1.26-1.13 (m, 36H), 0.84 (t, J=6.6 Hz, 3H) ppm.
31P-NMR (241 MHz, DMSO-d6): δ−9.63 (d), −9.98 (d) ppm.
ESIMS-HR calcd. for C37H57N6O15P2− (M−H)− 887.3363, found 887.3563.
Diphosphate 6 (18.6 mg, 21.0 μmol, 1.0 equiv., triethylammonium salt) was dissolved in dehydrated DMSO (270 μL). Dimethyl sulfate (39.7 μL, 0.419 mmol, 20 equiv.) was added dropwise thereto, and the mixture was stirred at room temperature overnight. After the reaction, a 2 M triethylammonium carbonate buffer (2.0 mL, pH 7.97) was added, and the mixture was stirred at room temperature for 15 minutes. Water was added to the reaction solution, and the mixture was purified by reverse-phase HPLC (YMC Triart C8 (250×10 mm l.D., S-5 μm, 12 nm, T012S05-2510WT), solvent: A) 0.1 M triethylammonium carbonate buffer (pH 7.97), B) acetonitrile, gradient: 40 to 100% B (0 to 35 min), 100% B (35 to 40 min), column temperature: room temperature, flow rate: 3 mL/min, detection wavelength: 260 nm), affording 9.99 mg (11.0 μmol, yield 53%, triethylammonium salt) of a compound 21 as a white solid.
1H-NMR (600 MHz, DMSO-d6) δ9.67 (s, 1H), 7.82-7.78 (m, 2H), 7.76 (t, J=6.6 Hz, 1H), 6.01 (br, 1H), 5.87 (d, J=3.6 Hz, 1H), 4.24 (s, 1H), 4.27 (q, J=4.8 Hz, 1H), 4.08 (s, 1H), 4.06 (s, 3H), 4.02 (s, 1H), 1.83-1.82 (m, 2H), 1.22-1.13 (m, 36H), 0.85 (t, J=7.2 Hz, 3H).
31P-NMR (241 MHz, DMSO-d6) δ−9.90 (d), −10.61 (d) ppm.
ESI MS-HR calcd. for C38H58N6O15P2− (M−2H)− 901.3519, found 901.3562.
Compound 7 (11.0 mg, 11.1 μmol, 1.0 equiv., triethylammonium salt), imidazole (9.97 mg, 111 μmol, 10 equiv.) and 2,2′-dithiodipyridine (9.47 mg, 38.8 μmol, 3.5 equiv.) were dissolved in dehydrated DMSO (400 μL). Triethylamine (5.99 μL, 38.8 μmol, 3.5 equiv.) and triphenylphosphine (11.28 mg, 38.8 μmol, 3.5 equiv.) were added thereto, and the mixture was stirred at room temperature for 4 hours. After the reaction, sodium perchlorate (5.51 mg, 44.4 μmol, 4.0 equiv.) dissolved in dehydrated acetone (1.5 mL) was added, and the mixture was cooled at 0° C. for 15 minutes. The precipitate formed was collected by centrifugation and washed five times with dehydrated acetone (2.0 mL). The precipitate was dried up by freeze-drying, affording 9.90 mg (10.1 μmol, 85% yield, Na salt) of Compound 8 as a white solid.
1H-NMR (600 MHz, DMSO-d6) δ9.66 (s, 1H), 7.98 (d, J=5.6 Hz, 1H), 7.79 (d, J=4.4 Hz, 1H), 7.71 (s, 1H), 7.54 (t, J=4.4 Hz, 1H), 7.18 (s, 1H), 6.79 (s, 1H), 6.00 (br, 1H), 5.87 (d, J=2.4 Hz, 1H), 5.61 (m, 1H), 4.41 (s, 1H), 4.20 (q, J=2.0 Hz, 1H), 4.05 (s, 3H), 1.81 (q, J=4.4 Hz, 2H), 1.27-1.14 (m, 36H), 0.85 (t, J=4.8 Hz, 3H) ppm. 13C-NMR (101 MHz, CDCl3) δ154.8, 153.7, 148.0, 147.9, 138.6, 136.0, 133.7, 128.8, 127.2, 124.5, 74.6, 70.5, 61.5, 36.1, 31.8, 29.8-29.0, 25.7, 22.5, 13.9 ppm.
31P-NMR (241 MHz, DMSO-d6) δ−9.90 (d, J=16.6 Hz), −10.61 (d, J=19.8) ppm.
ESI MS-HR calcd. for C41H61N8O14P2− (M−2H)− 951.3788, found 951.4272.
(a) of the figure shows a conceptual diagram of the reaction.
(b) of the figure shows the result of reverse-phase HPLC analysis of the reaction of a 5′-monophosphorylated 19-base-long RNA with a hydrophobic chemical capping reagent.
(c) of the figure shows the result of denatured PAGE analysis.
A hydrophobic capping reagent (Compound 8) and a 19-base-long 5′-end phosphorylated RNA (5′-p-GAACGUGCGAAAGUCCACA-3′: SEQ ID NO: 15) were reacted as follows. 38 μL of an aqueous solution containing 2 nmol of the RNA and 500 nmol of calcium chloride was frozen and dried up by a freeze-drying method. 18.5 μL of dimethyl sulfoxide (DMSO), 26.5 μL of a hydrophobic capping reagent solution in DMSO (19 mM), and 5 μL of 1-methylimidazole were added thereto and mixed, and the mixture was heated at 55° C. for 5 hours. The final concentration of each component in the DMSO solution is as follows. 40 μM RNA, 10 mM hydrophobic capped compound, 10 mM calcium chloride, 1.25 M 1-methylimidazole. After completion of the reaction, the RNA was collected by alcohol precipitation, and analyzed by reverse-phase HPLC and denaturing polyacrylamide gel electrophoresis (PAGE). The HPLC analysis conditions shown in (b) of the figure are as follows. System used: Chromaster (Hitachi High-Tech Corporation); column: YMC Hydrosphere C18 (250×4.6 mm I.D., YMC); eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile, pH 7.0; eluent B: acetonitrile; gradient condition: 0 to 100% B (0 to 20 min); flow rate: 1.0 mL/min; column temperature: 50° C.; detection wavelength: 260 nm. In (c) of the figure, an RNA was migrated with 15% acrylamide (acrylamide:bis=19:1) containing 7.5 M urea as a denaturant, the gel was stained with a nucleic acid staining reagent SYBR Green II, and photographed with ChemiDoc XRS+imager (Bio-Rad).
7. Improved Synthesis of N7-Methyl-GDP and Synthesis of Chemical Capping Reagent with Hydrophobic Tag
The following shows an improved method of synthesizing 2′-O-modified-N7-methyl-GDP (Compound 4) and a synthetic scheme of chemical capping reagent (5).
To a solution of 2′-modified guanosine (1) (294 mg, 0.583 mmol) in pyridine (6.00 mL) was added dropwise a solution of p-toluenesulfonyl chloride (214 mg, 1.12 mmol) in pyridine (1.40 mL) in an ice bath. The mixture was stirred at room temperature for 3 to 4 days. Ice-cooled water was added to the reaction mixture to stop the reaction, and the mixture was extracted three times with dichloromethane. The organic layer was dried over Na2SO4 and distilled off. The residue was subjected to silica gel column chromatography and was eluted with 24 to 91% methanol/dichloromethane, affording Compound 2 (260 mg, 68% yield) as a yellow foam.
1H NMR (600 MHz, CDCl3, mixture of stereoisomers) δ11.92 (br, s), 7.76-7.63 (m, 4H), 7.57 (d, J=11.9 Hz, 1.5H), 7.46-7.35 (m, 2H), 7.27-7.23 (m, 2.5H), 6.03-5.76 (m, 1H), 5.68 (s, 1H), 5.26-5.02 (m, 2H), 4.90 (d, J=16.1 Hz, 2H), 4.76 (s, 2H), 4.47-4.30 (m, 1.5H), 4.21 (s, 2.5H), 2.37 (s, 3H), 0.77 (s, 4.5H), 0.74 (s, 4.5H) ppm.
13C NMR (151 MHz, CDCl3, mixture of stereoisomers) δ158.75, 154.18, 151.28, 151.01, 150.38, 145.30, 136.63, 133.76, 132.27, 131.92, 130.36, 130.10, 130.06, 129.95, 128.51, 128.02, 123.87, 117.14, 94.78, 81.80, 79.86, 78.56, 77.91, 70.70, 70.13, 69.94, 53.56, 36.68, 36.50, 29.78, 25.73, 25.67, 25.56, 21.72 ppm.
ESI-MS calcd. for C29H33N6O10S, 657.2 [M−H]−; found 657.4.
To a suspension of 5′-O-tosyl-2′-modified guanosine (2) (458 mg, 0.695 mmol) and powdered 3A molecular sieve (500 mg) in CH3CN (500 μL) was added 0.475 M tris-tetrabutylammonium pyrophosphate. After stirring in CH3CN (2.63 mL, 1.25 mmol) at 40° C. for 2 days, the reaction mixture was diluted with water and filtered through a membrane filter, whereby insolubles were removed. The filtrate was purified by ion exchange chromatography on QAE-Sephadex (φ=3 cm, h=10 cm, 70 cm3, 6 mL/min) in a linear 0 to 1.5 M gradient of triethylammonium carbonate buffer (pH 7.9) containing 0 to 10% acetonitrile. Guanosine-5′-diphosphate derivative (3) was obtained in the form of a triethylammonium salt (441 mg, 66% yield) as a yellow amorphous solid.
1H NMR (600 MHz, D2O, mixture of stereoisomers) δ7.87 (s, 0.5H), 7.58 (s, 0.5H), 7.52 (d, J=8.1 Hz, 0.5H), 7.47-7.38 (m, 1H), 7.38-7.29 (m, 1H), 7.28-7.17 (m, 1H), 7.01 (t, J=7.8 Hz, 0.5H), 5.73 (d, J=5.8 Hz, 0.5H), 5.48 (d, J=5.0 Hz, 0.5H), 4.96, 4.94 (2s, 1H), 4.82 (d, J=7.5 Hz, 0.5H), 4.76 (s, 0.5H), 4.71 (d, J=7.3 Hz, 0.5H), 4.62 (dd, J=18.0, 6.6 Hz, 1.5H), 4.52 (t, J=5.1 Hz, 0-.5H), 4.45 (t, J=5.0 Hz, 1H), 4.14 (d, J=4.1 Hz, 0.5H), 4.10-4.03 (m, 1.5H), 3.99 (d, J=4.8 Hz, 1H), 3.02 (q, J=7.3 Hz, 15H), 1.10 (t, J=7.3 Hz, 24H), 0.52 (s, 4.5H), 0.41 (s, 4.5H) ppm.
13C NMR (151 MHz, D2O, mixture of stereoisomers) δ158.95, 158.75, 153.89, 153.59, 151.59, 151.01, 149.74, 148.09, 138.02, 137.37, 133.49, 132.57, 132.47, 132.21, 129.86, 129.66, 128.65, 128.06, 123.80, 123.30, 116.33, 115.96, 95.44, 93.71, 86.20, 84.26, 84.20, 83.73, 83.67, 81.61, 80.72, 78.55, 76.97, 69.07, 68.94, 64.84, 64.81, 64.46, 46.63, 36.55, 35.65, 24.78, 24.68, 8.24 ppm.
31P NMR (243 MHz, D2O, mixture of stereoisomers) δ−9.09 (d, J=20.8 Hz, 1P), −10.29 (d, J=22.4 Hz, 1P) ppm.
ESI-TOF-MS calcd. for C22H29N6O14P2, 663.1223 [M−H]−; found 663.1378.
To a solution of guanosine-5′-diphosphate derivative (3) (441 mg, 0.509 mmol) in DMSO (4.24 mL) was added iodomethane (290 mg, 127 μL, 2.04 mmol). After stirring at room temperature for 10 hours, the reaction mixture was diluted with water and washed four times with dichloromethane. The aqueous layer was purified with a linear gradient of DEAE-Sephadex™ A-25 (φ=3 cm, h=10 cm, 70 cm3, 6 mL/min) in a 0 to 1.0 M triethylammonium acetate buffer (pH 6.0) containing 0 to 10% CH3CN. After removal of CH3CN under reduced pressure, the residue was desalted with Wakosil™ 25C18 (15 to 30 μm, spherical) and freeze-dried, affording N7-methyl-guanosine-5′-diphosphate derivative (4) (118 mg, 26% yield, a mixture of two stereoisomers) as a white solid.
1H NMR (600 MHz, D2O, single stereoisomer isolated by HPLC) δ8.93 (s, 1H), 7.60 (d, J=8.2 Hz, 1H), 7.49 (dd, J=15.2, 8.1 Hz, 2H), 7.29 (t, J=7.6 Hz, 1H), 5.82 (s, 1H), 5.00 (d, J=4.1 Hz, 1H), 4.93 (d, J=8.0 Hz, 1H), 4.86 (d, J=7.2 Hz, 1H), 4.54-4.50 (m, 1H), 4.42 (t, J=5.2 Hz, 1H), 4.25 (s, 1H), 4.10 (d, J=11.2 Hz, 1H), 3.94 (d, J=3.7 Hz, 3H), 3.05 (t, J=7.3 Hz, 4H), 1.13 (t, J=7.1 Hz, 6H), 0.61 (s, 10H) ppm.
13C NMR (151 MHz, D2O, single stereoisomer isolated by HPLC) δ155.41, 154.99, 149.37, 148.61, 136.68, 133.90, 132.44, 130.24, 128.15, 123.51, 107.91, 95.16, 87.76, 85.95, 82.16, 79.32, 68.89, 64.78, 46.70, 36.52, 36.07, 24.76, 8.25 ppm.
31P NMR (243 MHz, D2O, single stereoisomer isolated by HPLC) δ−10.03 (1P), −10.71 (1P) ppm.
ESI-TOF-MS calcd. for C23H31N6O14P2, 677.1379 [M−H]−; found 677.1374.
To a solution of N7-methyl-GDP derivative (4) (118 mg, 0.134 mmol), imidazole (72.8 mg, 1.07 mmol), and 2,2′-dithiodipyridine (88.6 mg, 0.402 mmol) in N,N-dimethylformamide (1.60 mL) was added triethylamine (37.5 μL, 27.1 mg, 0.268 mmol), and subsequently triphenylphosphine (105 mg, 0.402 mmol) was added. After stirring at room temperature for 5 hours, the reaction mixture was added dropwise to a solution of sodium perchlorate (39.4 mg, 0.322 mmol) in acetone (15 mL) with stirring. The resulting suspension was transferred to a centrifuge tube and centrifuged at 3,500 rpm for 10 minutes. The supernatant was removed and the precipitate was washed five times with acetone. The solid was dried under reduced pressure on P2O5 in a desiccator, affording guanosine 5′-diphosphate derivative imidazolide disodium salt (5) as a white solid.
1H NMR (600 MHz, D2O, mixture of stereoisomers) δ8.10 (s, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.68-7.01 (m, 6H), 6.88 (d, J=10.1 Hz, 1H), 5.98-5.63 (m, 1H), 5.03-4.71 (m, 3H), 4.52-4.08 (m, 3H), 3.95 (s, 1H), 3.93-3.80 (m, 3H), 0.70-0.19 (m, 9H) ppm.
13C NMR (151 MHz, D2O, mixture of stereoisomers) δ159.27, 149.76, 149.32, 148.44, 139.84, 134.64, 133.79, 132.74, 132.18, 130.00, 128.79, 128.33, 127.97, 123.79, 123.52, 120.43, 120.25, 108.52, 108.26, 95.13, 87.30, 85.53, 85.01, 81.91, 80.60, 79.17, 68.99, 68.59, 65.00, 36.45, 36.41, 35.88, 35.72, 30.25, 24.85, 24.75 ppm.
31P NMR (243 MHz, D2O, mixture of stereoisomers) δ−11.18 (d, J=19.7 Hz, 1P), −19.39 (d, J=19.7 Hz, 1P) ppm.
ESI-MS calcd. for C26H33N8O13P2, 727.2 [M−H]−; found 727.3.
The following shows a synthetic scheme of a novel cap compound (Compound 10).
Methanol (80.0 mL) was added to guanosine (Compound 1) (6.00 g, 21.2 mmol) to suspend the guanosine, and then N,N-dimethylformamide dimethyl acetal (18.9 mL, 159 mmol) was added thereto. After stirring at room temperature for 22 hours, the reaction solution was filtered and the solid formed was washed with ice-cooled methanol (80.0 mL). The solid collected by filtration was dried with a freeze-dryer, affording Compound 2 as a white solid. (6.81 g, 20.1 mmol, yield 95%)
1H NMR (400 MHz, DMSO-d6) δ11.35 (s, 1H), 8.53 (s, 1H), 8.04 (s, 1H), 5.79 (d, J=6.1 Hz, 1H), 5.43 (s, 1H), 5.19 (s, 1H), 5.04 (t, J=5.4 Hz, 1H), 4.48 (t, J=5.4 Hz, 1H), 4.11 (m, 1H), 3.90 (q, J=3.7 Hz, 1H), 3.58 (in, 2H) 3.15 (s, 3H), 3.03 (s, 3H) ppm
13C NMR (101 MHz, DMSO-d6) δ157.96, 157.63, 157.32, 150.02, 136.97, 119.79, 86.72, 85.45, 73.80, 70.51, 61.52, 40.68, 34.67 ppm
HRMS (ESI) m/z calcd. for C13H18N6NaO5+ (M+Na)+=361.1231, found 361.1221.
Dimethylformamide (90.0 mL) was added to Compound 2 (3.00 g, 8.87 mmol) and the compound is dissolved, and then imidazole (1.40 g, 21.3 mmol) and tert-butylmethylchlorosilane (1.60 g, 10.6 mmol) were added thereto. After stirring at room temperature for 20 hours, the reaction solution was diluted with chloroform and washed with water. The organic layer was dried over anhydrous sodium sulfate and then concentrated. The residue was purified by silica gel column chromatography (eluent: chloroform/methanol=10/1), affording Compound 3 as a white solid. (3.38 g, 7.47 mmol, yield 84%)
1H NMR (400 MHz, DMSO-d6) δ11.33 (s, 1H), 8.55 (s, 1H), 7.96 (s, 1H), 5.82 (d, J=5.2 Hz, 1H), 5.51 (d, J=3.6 Hz, 1H), 5.22 (d, J=3.4 Hz, 1H), 4.42 (d, J=3.6 Hz, 1H), 4.12 (s, 1H), 3.91 (d, J=3.9 Hz, 1H), 3.77 (m, 2H), 3.16 (s, 3H), 3.03 (s, 3H), 0.87 (s, 9H), 0.04 (s, 6H) ppm
13C NMR (101 MHz, DMSO-d6) δ157.91, 157.62, 157.31, 150.07, 136.28, 119.59, 86.45, 84.51, 73.94, 70.07, 63.09, 40.70, 34.71, 25.85, 18.08, −5.40, −5.43 ppm
HRMS (ESI) m/z calcd. for C19H32N6NaO5Si+ (M+Na)+=475.2096, found 475.2097.
Tetrahydrofuran (45 mL) was added to Compound 3 (2.00 g, 4.35 mmol) and Compound 12 (2.62 g, 9.57 mmol), and these compounds were dissolved. Thereafter, powdered molecular sieve 3 Å (2.10 g) and N-iodosuccinimide (2.15 g, 9.57 mmol) were added, and the mixture was cooled to −40° C. To this reaction solution was added trifluoromethanesulfonic acid (0.85 mL, 9.57 mmol), and the mixture was stirred at −40° C. for 2 hours. Thereafter, triethylamine (2.89 mL, 15.2 mmol) was added to stop the reaction. The resulting reaction solution was filtered with Celite to remove the powdery molecular sieve 3 Å. The filtrate was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and then concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate/methanol=100/3→20/1), affording Compound 4 as a brown solid. (1.72 g, 1.90 mmol, yield 44%)
1H NMR (400 MHz, DMSO-d6) δ11.15 (s, 1H), 8.46 (s, 1H), 7.99 (s, 1H), 7.63 (s, 1H), 7.56 (s, 1H), 7.19 (s, 1H), 6.79 (s, 1H), 6.01 (d, J=5.7 Hz, 1H), 4.99 (s, 1H), 4.91 (m, 6H), 4.71 (s, 2H), 4.49 (d, J=4.3 Hz, 1H), 4.11 (t, J=4.1 Hz, 1H), 3.81 (m, 16H), 3.08 (s, 3H), 2.98 (s, 3H), 0.82 (s, 9H), 0.01 (d, J=1.6 Hz, 6H) ppm
13C NMR (101 MHz, DMSO-d6) δ157.73, 157.16, 153.37, 153.24, 147.42, 147.20, 139.00, 138.13, 129.03, 128.92, 110.13, 109.93, 108.90, 108.78, 107.92, 107.78, 94.88, 94.41, 94.26, 85.40, 85.20, 83.87, 83.01, 79.06, 74.26, 66.50, 66.33, 66.22, 61.00, 59.75, 55.98, 55.93, 55.78, 40.58, 34.64, 25.81, 25.67, 20.76, 17.89, 14.09, −3.20, −5.61, −5.64 ppm
HRMS (ESI) m/z calcd. for C39H54N8NaO15Si+ (M+Na)+=925.3370, found 925.3337.
Tetrahydrofuran (10.0 mL) was added to Compound 4 (0.69 g, 0.76 mmol), and the compound was dissolved. Thereafter, triethylamine (0.32 mL, 2.29 mmol) and triethylamine hydrotrifluoride (0.37 mL, 2.29 mmol) were added, and the mixture was stirred at room temperature for 17 hours. The resulting reaction solution was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and then concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate/methanol=20/1→10/1), affording Compound 5 as a yellow solid. (0.50 g, 0.63 mmol, yield 83%)
1H NMR (400 MHz, DMSO-d6) δ11.10 (s, 1H), 8.44 (s, 1H), 8.08 (s, 1H), 7.63 (s, 1H), 7.56 (s, 1H), 7.21 (s, 1H), 6.78 (s, 1H), 5.99 (d, J=5.9 Hz, 1H), 5.22 (t, J=5.3 Hz, 1H), 4.98 (d, J=8.6 Hz, 1H), 4.89 (m, 6H), 4.67 (dd, J1=20.3 Hz, J2=15.3 Hz, 2H), 4.51 (m, 1H), 4.10 (q, J=3.5 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.77 (s, 3H), 3.63 (m, 2H), 3.08 (s, 3H), 2.98 (s, 3H) ppm
13C NMR (101 MHz, DMSO-d6) δ157.77, 157.25, 157.14, 153.42, 153.24, 149.61, 147.40, 147.17, 139.05, 138.06, 136.41, 129.05, 128.97, 119.63, 110.12, 108.80, 107.89, 107.79, 94.89, 94.41, 85.29, 83.84, 79.34, 79.19, 74.99, 66.42, 66.22, 61.06, 56.04, 55.99, 55.93, 55.79, 40.53, 34.55 ppm
HRMS (ESI) m/z calcd. for C33H40N8NaO15+ (M+Na)+=811.2505, found 811.2489.
To Compound 5 (0.50 g, 0.63 mmol) was added dichloromethane (6.00 mL), and the compound was dissolved. To this reaction solution was added a 7 M ammonia solution in methanol (6.30 mL, 44.4 mmol), and the mixture was stirred at 50° C. for 15 hours. The resulting reaction solution was diluted with chloroform and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: chloroform/methanol=20/1→10/1), affording Compound 6 as a pale yellow solid. (0.43 g, 0.59 mmol, yield 94%)
1H NMR (400 MHz, DMSO-d6) δ10.41 (s, 1H), 7.94 (s, 1H), 7.63 (s, 1H), 7.57 (s, 1H), 7.21 (s, 1H), 6.85 (s, 1H), 6.35 (s, 2H), 5.88 (d, J=7.2 Hz, 1H), 5.24 (t, J=5.5 Hz, 1H), 4.96 (m, 4H), 4.88 (d, J=7.0 Hz, 1H), 4.77 (d, J=6.7 Hz, 2H), 4.61 (dd, J1=35.9 Hz, J2=15.3 Hz, 2H), 4.45 (dd, J1=4.9 Hz, J2=2.0 Hz, 1H), 4.09 (dd, J1=5.9 Hz, J2=3.7 Hz, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.84 (s, 6H), 3.61 (m, 2H) ppm
13C NMR (101 MHz, DMSO-d6) δ156.36, 153.62, 153.38, 153.25, 151.03, 147.38, 147.17, 139.03, 138.16, 135.04, 129.04, 128.97, 116.53, 110.11, 108.99, 107.90, 107.80, 94.65, 94.44, 84.40, 84.13, 79.18, 79.03, 75.30, 66.24, 61.27, 56.06, 55.99, 55.92 ppm
HRMS (ESI) m/z calcd. for C30H35N7NaO15+ (M+Na)+=756.2083, found 756.2067.
Pyridine (2.00 mL) was added to Compound 6 (0.10 g, 0.14 mmol) and the compound was dissolved therein, and p-toluenesulfonyl chloride (0.04 g, 0.21 mmol) was added thereto under ice-cooling. After stirring at room temperature for 3 days, the mixture was diluted with chloroform and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: chloroform/methanol=100/3), affording Compound 7 as a yellow solid. (61.2 mg, 68.9 μmol, yield 49%)
1H NMR (400 MHz, DMSO-d6) δ10.48 (s, 1H), 7.73 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 7.65 (s, 1H), 7.58 (s, 1H), 7.35 (s, 1H), 7.33 (s, 1H), 7.19 (s, 1H), 6.86 (s, 1H), 6.36 (s, 2H), 5.84 (d, J=6.3 Hz, 1H), 4.92 (m, 4H), 4.86 (d, J=7.0 Hz, 1H), 4.79 (m, 2H), 4.64 (dd, J1=33.8 Hz, J2=15.1 Hz, 2H), 4.47 (m, 1H), 4.31 (m, 2H), 4.23 (m, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 2.35 (s, 3H) ppm
13C NMR (101 MHz, DMSO-d6) δ156.40, 153.61, 153.33, 153.26, 150.95, 147.42, 147.21, 145.11, 139.01, 138.28, 134.95, 131.91, 129.98, 128.89, 128.82, 127.49, 116.61, 110.05, 109.14, 107.87, 94.81, 84.92, 80.08, 79.18, 78.01, 74.86, 69.77, 66.43, 56.03, 55.91, 21.06 ppm
HRMS (ESI) m/z calcd. for C37H41N7NaO17S+ (M+Na)+=910.2172, found 910.2181.
To Compound 7 (50.0 mg, 56.3 μmol) was added acetonitrile (0.13 mL), and the compound was dissolved therein. Powdered molecular sieve 3 Å (50.0 mg) was added thereto, then a solution of bistetraammonium pyrophosphate in acetonitrile (0.475 M, 0.21 mL) was added, and the mixture was stirred at room temperature for 21 hours. After dilution with water, the precipitate was removed by centrifugation (3500 rpm, 15 min) and the supernatant was purified by ion exchange chromatography using QAE-Sephadex (eluent: 0 to 1.5 M triethylammonium carbonate buffer). Thereafter, the purified product was dried with a freeze dryer, affording Compound 8, which was a triethylammonium salt, as a yellow solid. (34.1 mg, 31.1 μmol, yield 55%)
1H NMR (600 MHz, D2O) δ8.01 (s, 1H), 7.44 (s, 1H), 7.42 (s, 1H), 7.01 (s, 1H), 6.53 (s, 1H), 5.82 (d, J=6.3 Hz, 1H), 5.04 (d, J=6.5 Hz, 1H), 4.92 (m, 6H), 4.66 (s, 1H), 4.53 (d, J=15.9 Hz, 1H), 4.43 (s, 1H), 4.33 (d, J=15.0 Hz, 1H), 4.18 (s, 2H), 3.85 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H), 3.75 (s, 3H), 3.46 (d, J=6.5 Hz, 2H), 3.16 (q, J=7.0 Hz, 10H), 1.24 (t, J=7.2 Hz, 18H) ppm
13C NMR (151 MHz, D2O) δ157.70, 153.27, 153.13, 151.10, 146.90, 146.50, 138.65, 137.00, 129.75, 129.20, 115.51, 109.94, 107.59, 107.29, 95.05, 94.94, 85.57, 82.89, 79.89, 76.06, 66.95, 66.38, 65.20, 56.14, 56.03, 55.87, 46.62, 8.22 ppm
31P NMR (243 MHz, D2O) δ−10.12 (d, J=21.2 Hz, 1P), −10.86 (d, J=21.2 Hz, 1P) ppm
HRMS (ESI) m/z calcd. for C30H36N7O21P2+ (M−H)−=892.1445, found 892.1431.
Dimethyl sulfoxide (0.76 mL) was added to Compound 8 (0.10 g, 91.2 μmol), and the compound was dissolved therein. Thereafter, iodomethane (46.0 μL, 0.73 mmol) was added, and the mixture was stirred at room temperature for 2 days. The reaction was stopped by adding a triethylammonium acetate buffer (pH 7.0) to the reaction solution, and the reaction solution was diluted with water. Thereafter, the mixture was washed with chloroform, and the aqueous layer was purified by ion exchange chromatography (eluent: 0 to 1.0 M triethylammonium acetate buffer) using QAE-Sephadex. Thereafter, the purified product was dried with a freeze dryer, affording Compound 9, which was a triethylammonium salt, as a pale yellow solid. (31.8 mg, 28.6 μmol, yield 31%)
1H NMR (600 MHz, D2O) δ7.28 (s, 1H), 7.20 (s, 1H), 6.80 (s, 1H), 6.68 (s, 1H), 6.01 (s, 1H), 4.95 (d, J=24.1 Hz, 4H), 4.74 (s, 1H), 4.65 (d, J=16.0 Hz, 1H), 4.56 (m, 4H), 4.47 (s, 1H), 4.26 (dd, J1=69.2 Hz, J2=8.0 Hz, 2H), 3.95 (s, 3H), 3.75 (s, 3H), 3.74 (s, 3H), 3.73 (s, 3H), 3.71 (s, 3H), 3.16 (q, J=7.3 Hz, 10H), 1.24 (t, J=7.3 Hz, 15H) ppm
13C NMR (151 MHz, D2O) δ155.49, 154.46, 153.08, 152.99, 149.40, 146.77, 146.65, 138.27, 137.51, 129.46, 129.02, 109.63, 107.88, 107.48, 107.30, 95.66, 94.63, 87.40, 83.35, 80.01, 75.17, 66.97, 66.58, 56.13, 56.06, 55.85, 55.78, 46.64, 35.91, 23.18, 8.24 ppm
31P NMR (243 MHz, D2O) δ−9.26 (d, J=19.7 Hz, 1P), −10.72 (d, J=26.3 Hz, 1P) ppm
HRMS (ESI) m/z calcd. for C31H38N7O21P2− (M−H)−=906.1601, found 906.1628.
To Compound 9 (30.0 mg, 27 μmol) and guanosine 5′-phosphorimidazolide (35.0 mg, 81.1 μmol) was added dimethyl sulfoxide (0.27 mL), and those compounds were dissolved. Thereafter, 2-nitroimidazole (9.20 mg, 81.1 μmol) was added to the reaction solution, and the mixture was stirred at 37° C. for 2 days. After dilution with water, the mixture was washed with chloroform and the aqueous layer was purified by reverse phase chromatography (Chromatorex C8 MB 100-40/75). (Eluent: 0 to 60% acetonitrile/100 to 40% 50 mM triethylammonium acetate buffer) Thereafter, the purified product was dried with a freeze dryer, affording Compound 10, which was a triethylammonium salt, as a pale yellow solid. (21.5 mg, 13.8 μmol, yield 51%)
1H NMR (600 MHz, D2O) δ7.88 (s, 1H), 7.25 (s, 1H), 7.17 (s, 1H), 6.66 (d, J=8.8 Hz, 1H), 5.92 (s, 1H), 5.62 (d, J=5.4 Hz, 1H), 5.11 (d, J=12.9 Hz, 1H), 4.98 (s, 1H), 4.90 (s, 2H), 4.64 (s, 2H), 4.52 (s, 2H), 4.49 (d, J=5.1 Hz, 1H), 4.47 (d, J=9.2 Hz, 1H), 4.41 (d, J=3.7 Hz, 4H), 4.23 (d, J=10.5 Hz, 4H), 3.97 (s, 3H), 3.72 (s, 3H), 3.70 (s, 3H), 3.69 (s, 3H), 3.68 (s, 3H), 3.16 (q, J=7.3 Hz, 15H), 1.23 (t, J=7.5 Hz, 23H) ppm
13C NMR (151 MHz, D2O) δ158.05, 155.10, 153.52, 153.03, 152.86, 150.97, 149.08, 146.63, 146.47, 138.08, 137.40, 136.85, 129.67, 128.87, 115.63, 109.50, 107.77, 107.59, 107.24, 96.03, 94.13, 87.53, 86.80, 83.49, 83.43, 82.50, 79.10, 74.55, 74.09, 70.11, 67.09, 66.75, 65.27, 64.01, 56.03, 55.94, 55.74, 46.65, 36.02, 23.25, 8.24 ppm
31P NMR (243 MHz, D2O) δ−10.87 (d, J=19.7 Hz, 1P), −11.09 (d, J=19.7 Hz, 1P), −22.41 (t, J=19.7 Hz, 1P) ppm
HRMS (ESI) m/z calcd. for C41H50N12O28P3− (M−H)−=1251.2081, found 1251.2123.
To Compound 11 (1.50 g, 7.04 mmol) were added acetic acid (16.1 mL, 281 mmol) and dimethyl sulfoxide (10.0 mL, 141 mmol), and the compound was dissolved. To the reaction solution was added acetic anhydride (13.3 mL, 141 mmol), and the mixture was stirred at room temperature for 3 days. Then, potassium hydroxide (10.0 M, 63.3 mL, 633 mmol) was added thereto at 0° C., and the mixture was stirred for 2 hours to stop the reaction. The reaction solution was dissolved in diethyl ether and washed with water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue obtained was suspended in a 1:3 mixture of ethyl acetate and hexane, and the suspension was filtered. The resulting solid was dried in a desiccator on P2O5 under vacuum, affording Compound 12 as a pale yellow solid. (1.20 g, 4.39 mmol, yield 62%)
1H NMR (400 MHz, CHLOROFORM-D) δ7.71 (s, 1H), 7.25 (s, 1H), 5.01 (s, 2H), 4.82 (s, 2H), 4.00 (s, 3H), 3.95 (s, 3H), 2.22 (s, 3H) ppm
13C NMR (101 MHz, CHLOROFORM-D) δ153.80, 147.76, 130.22, 109.71, 108.04, 77.48, 76.84, 75.68, 67.15, 56.57, 56.49, 14.54 ppm
HRMS (ESI) m/z calcd. for C11H15NNaO5S+ (M+Na)+=296.0563, found 296.0567.
The following shows a synthesis scheme of a nitrobenzyl derivative to be used for the synthesis of a 2′-O-methylated dinucleotide cap compound.
The following shows a synthesis scheme of the 2′-O-methylated dinucleotide cap compound.
The synthesis of Compound (2) was carried out in accordance with literature. In THF (150 mL), 1-iodo-2-nitrobenzene (1) (20 g, 80 mmol) was cooled to −40° C. To the mixture was added a solution of phenylmagnesium chloride in THF (2 M, 52 mL, 0.10 mol), and the resulting mixture was stirred at −40° C. for 1 hour. Trimethylacetaldehyde (12 mL, 9.6 g, 0.11 mol) was added to the reaction mixture, followed by stirring at −40° C. for 36 minutes. The reaction mixture was gradually warmed to room temperature. After stirring for 4 hours, the reaction was quenched by the addition of a saturated aqueous ammonium chloride solution. The mixture was extracted three times with ethyl acetate. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (17→25% ethyl acetate/hexane), affording Compound 2 (11 g, yield 65%) as a dark brown solid. All spectral data of the product agreed with those disclosed in literature (V. A. Litosh, et al., Nucleic Acids Res., 2011, 29(6), e39).
To a solution of Compound 2 (639 mg, 3.05 mmol) in THF (10.2 mL) were added N,N-diisopropylethylamine (986 mg, 1.30 mL, 7.63 mmol) and triphosgene (997 mg, 3.36 mmol) continuously. After stirring at room temperature for 23 hours, triphosgene (200 mg) and N,N-diisopropylethylamine (1.00 mL) were added to the mixture, and the resulting mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried over Na2SO4 and concentrated with a rotary evaporator, affording a chloroformate (3) as a dark brown oil (1.64 g, crude product). The crude product was purified by silica gel column chromatography (hexane: AcOEt=1.0, 6:1), affording Compound 3 (601 mg, 72.6% yield) as a yellow oil.
1H NMR (600 MHz, CDCl3) δ 7.90 (ddd, J=8.2, 3.3, 1.4 Hz, 1H), 7.70-7.54 (m, 2H), 7.46 (dddd, J=9.8, 7.1, 2.6, 1.5 Hz, 1H), 6.56 (d, J=1.5 Hz, 1H), 0.94 (d, J=2.5 Hz, 9H) ppm.
13C NMR (151 MHz, CDCl3) δ 149.97, 149.95, 149.04, 132.99, 131.13, 129.52, 128.80, 124.93, 84.30, 36.60, 25.56 ppm.
To a suspension of 2′-O-methylguanosine (4) (3.63 g, 12.2 mmol) in methanol (61.0 mL) was added N,N-dimethylformamide dimethyl acetal (7.27 g, 8.17 mL, 61.0 mmol). After stirring at room temperature for 21 hours, the reaction mixture was concentrated with a rotary evaporator. The oily residue was dissolved in methanol (10.0 mL) and a few drops of ether were added. The mixture was sonicated and then suspended by the addition of ether (150 mL). The resulting precipitate was collected by filtration and washed with ether. The solid was dried under reduced pressure, affording objective Compound 5 (4.08 g, 94.0) as a white powder. All spectral data agreed with those previous reported (T. Mukobata et al., Bioorg. Med. Chem. Lett., 2010, 20, 129-131).
To a cooled solution of Compound (5) (1.00 g, 2.84 mmol) in pyridine (22.7 mL) was added a solution of p-toluenesulfonyl chloride (812 mg, 4.26 mmol) in pyridine (5.70 mL) dropwise on an ice bath. The mixture was allowed to stand in a refrigerator at 4° C. for 17 hours, and then a solution of p-toluenesulfonyl chloride (271 mg, 1.42 mmol) in pyridine (1.00 mL) was added thereto. The mixture was placed in a refrigerator at 4° C. for 17 hours. The reaction mixture was quenched by pouring into water. The resulting mixture was extracted twice with dichloromethane. The organic layer was dried over Na2SO4 and evaporated. The residue was subjected to silica gel column chromatography eluted with 1.6 to 9.1% methanol/dichloromethane, affording 5′-O-tosylated compound 6 (682 mg, 47.4% yield) as a white foam.
1H NMR (400 MHz, CDCl3) δ 10.21-10.00 (m, 1H), 8.46 (s, 1H), 7.70 (s, 1H), 7.63 (d, J=8.4 Hz, 2H), 7.17 (d, J=8.4 Hz, 2H), 5.93 (d, J=4.1 Hz, 1H), 5.24 (s, 1H), 4.60 (t, J=5.4 Hz, 1H), 4.33-4.15 (m, 4H), 3.44 (s, 3H), 3.08 (s, 3H), 2.99 (s, 3H), 2.30 (s, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ 158.44, 158.38, 157.14, 150.29, 145.36, 136.69, 132.27, 129.94, 127.88, 120.31, 86.32, 83.03, 81.66, 69.41, 69.02, 58.82, 58.59, 53.62, 41.66, 41.48, 35.27, 31.00, 21.75, 21.65 ppm.
ESI-TOF-MS calcd for C21H27N6O7S, 507.1656 [M+H]+; found 507.1675, calcd for C21H26N6NaO7S, 529.1476 [M+Na]+; found 529.1495.
To a cooled solution of nitrobenzyl derivative (3) (107 mg, 0.394 mmol) in dichloromethane (985 μL) were added Compound 6 (100 mg, 0.197 mmol) and DMAP (48.1 mg, 0.394 mmol) in ice. After stirring at room temperature for 19.5 hours, the reaction mixture was diluted with dichloromethane and washed with a saturated aqueous NH4Cl solution. The organic layer was dried over Na2SO4 and concentrated. The residue was subjected to silica gel column chromatography eluted with 2.0 to 4.8% methanol/dichloromethane, affording target Compound 7 (126 mg, yield 93.3%) as a white solid.
1H NMR (600 MHz, CDCl3) δ 9.73-9.60 (m, 1H), 8.61 (s, 0.5H), 8.58 (s, 0.5H), 7.88 (ddd, J=8.8, 4.0, 1.2 Hz, 1H), 7.67-7.49 (m, 7H), 7.49-7.40 (m, 1H), 7.40-7.27 (m, 1H), 7.21-7.09 (m, 3H), 6.41 (s, 0.5H), 6.32 (s, 0.5H), 5.71 (td, J=11.0, 4.5 Hz, 1H), 5.67-5.61 (m, 1H), 4.68 (dd, J=5.7, 3.1 Hz, 0.5H), 4.58 (dd, J=5.7, 3.5 Hz, 0.5H), 4.33-4.13 (m, 4H), 3.37 (s, 2H), 3.21 (s, 1.5H), 3.08 (d, J=2.1 Hz, 3H), 3.03 (s, 1.5H), 2.92 (s, 1.5H), 2.33 (d, J=9.0 Hz, 3H), 0.95 (s, 4.5H), 0.93 (s, 4.5H) ppm.
13C NMR (151 MHz, CDCl3) δ 158.71, 158.66, 158.47, 158.15, 158.09, 157.24, 154.28, 154.02, 149.90, 149.31, 149.09, 145.91, 145.62, 145.51, 137.64, 132.93, 132.72, 132.59, 132.37, 132.13, 130.19, 129.93, 129.80, 129.76, 129.68, 129.26, 129.04, 129.00, 128.77, 128.01, 127.86, 127.80, 127.74, 124.81, 124.58, 121.22, 120.92, 88.19, 88.01, 87.36, 81.02, 80.93, 80.83, 80.78, 80.69, 80.60, 80.49, 80.16, 79.10, 78.94, 78.52, 78.20, 78.11, 75.84, 75.59, 75.20, 74.58, 74.07, 73.89, 67.70, 67.61, 67.55, 66.08, 65.30, 59.58, 59.40, 59.20, 58.94, 58.88, 53.58, 41.56, 41.43, 41.36, 41.05, 36.59, 36.34, 36.29, 36.25, 35.40, 35.34, 25.78, 25.74, 25.71, 25.60, 21.79, 21.68 ppm.
ESI-TOF-MS calcd for C33H40N7O11S, 742.2502 [M+H]+; found 742.2562, calcd for C33H39N7NaO11S, 764.2321 [M+Na]+; found 764.2313.
To a suspension of Compound 7 (591 mg, 0.797 mmol) in methanol (13.3 μL) and water (6.63 mL) was added trifluoroacetic acid (6.82 g, 4.68 mL, 59.8 mmol). After stirring at room temperature for 23 hours, the reaction mixture was concentrated to remove methanol. The residue was diluted with dichloromethane and washed with water. The organic layer was dried over Na2SO4 and then concentrated, affording objective Compound 8 (576 mg, quantitative) as a gray foam.
1H NMR (600 MHz, CDCl3) δ 7.91-7.86 (m, 1H), 7.75-7.66 (m, 3H), 7.66-7.60 (m, 2H), 7.29-7.21 (m, 3H), 6.43 (s, 0.5H), 6.30 (s, 0.5H), 5.75 (dd, J=6.3, 3.7 Hz, 1H), 5.29-5.21 (m, 1H), 4.73 (t, J=5.8 Hz, 0.5H), 4.63 (t, J=5.7 Hz, 0.5H), 4.40-4.35 (m, 1H), 4.31 (dd, J=7.9, 3.9 Hz, 1H), 4.29-4.20 (m, 1H), 3.31 (s, 1.5H), 2.97 (s, 1.5H), 2.36 (d, J=9.7 Hz, 3H), 0.95 (d, J=11.2 Hz, 9H) ppm.
13C NMR (151 MHz, CDCl3) δ 159.21, 154.10, 153.77, 151.66, 149.39, 149.25, 145.58, 136.77, 132.81, 132.70, 132.56, 132.41, 132.09, 130.17, 130.13, 130.10, 129.69, 129.27, 128.97, 128.89, 128.82, 128.07, 128.02, 127.99, 127.96, 126.45, 124.66, 120.91, 117.56, 86.95, 86.86, 80.88, 80.67, 80.54, 80.16, 80.10, 79.75, 79.69, 74.36, 74.21, 68.40, 59.66, 59.39, 59.21, 36.59, 36.32, 36.24, 35.44, 25.81, 25.73, 25.70, 25.61, 21.80, 21.71, 21.68 ppm.
ESI-TOF-MS calcd for C30H35N6O11S, 687.2079 [M+H]+; found 687.2091.
Compound 8 (53.3 mg, 71.9 μmol) and powdered 3A molecular sieve (55.0 mg) were suspended in acetonitrile (80.0 μL), and a 616 mM solution of tris-tetrabutylammonium pyrophosphate in acetonitrile (175 μL, 108 μmol) was added. After stirring at 40° C. for 18.5 hours, the reaction mixture was diluted with water. The resulting suspension was centrifuged at 4,500 rpm for 30 minutes. The supernatant was collected and the precipitate was resuspended in water. The resulting suspension was centrifuged at 4,500 rpm for 30 minutes. The supernatant was collected and purified by ion exchange chromatography (A: water, B: 1.5 M TEAB buffer (pH 7.9, containing 10% CH3CN), gradient: 0 to 100% B (270 min)) using QAE-Sephadex (φ=3.0 cm, h=10 cm, 70 cm3, 6 mL/min). The fractions containing the product were collected and concentrated, affording modified guanosine diphosphate 9 (56.9 mg, 57.1 mol, 79.7% yield) in the form of a triethylammonium salt. Quantification of the product by UV at 260 nm: 27.9 μmol (yield: 38.8%). The absorption coefficient (ε260)=15,780 M−1·cm−1 was used for the calculation.
1H NMR (400 MHz, CD3OD) δ 8.23-8.02 (m, 1H), 7.92 (d, J=8.2 Hz, 1H), 7.69 (q, J=6.3 Hz, 2H), 7.62-7.47 (m, 1H), 6.36 (s, 0.5H), 6.23 (s, 0.5H), 5.83 (dd, J=7.6, 2.9 Hz, 1H), 5.49 (dd, J=15.8, 5.0 Hz, 1H), 4.48-4.08 (m, 4H), 3.32 (s, 3H), 3.15 (q, J=7.3 Hz, 18H), 1.28 (q, J=11.2 Hz, 40H), 0.97 (d, J=8.8 Hz, 9H) ppm.
13C NMR (101 MHz, CD3OD) δ 158.15, 154.23, 153.93, 153.86, 149.52, 132.55, 132.16, 129.13, 129.05, 128.85, 126.03, 125.62, 124.27, 122.87, 85.84, 81.96, 80.80, 80.70, 80.23, 79.94, 75.54, 64.94, 58.41, 58.14, 58.03, 45.98, 42.24, 41.75, 36.10, 35.71, 30.37, 24.94, 24.77, 19.24, 10.25, 10.05, 7.75, 6.88 ppm.
31P NMR (162 MHz, CD3OD) δ −9.04 (1P), −9.44 (0.5P), −10.75 (0.5P) ppm.
ESI-TOF-MS calcd for C23H29N6O15P2, 691.1172 [M−H]−; found 691.1270.
To a solution of Compound 9 (180 μmol) in DMSO (1.50 mL) was added iodomethane (204 mg, 89.6 μL, 1.44 mmol). After stirring at room temperature for 13 hours, the reaction mixture was diluted with water and washed three times with dichloromethane. The crude product was purified by ion exchange chromatography (gradient, 0→0.8 M TEAA buffer (pH 6.0, 0→8% CH3CN (270 min)) using QAE-Sephadex TM A-25 (φ=4.5 cm, h=8.4 cm, 140 cm3, 12 mL/min). After removal of CH3CN under reduced pressure, the product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min), solvent A: water (20 min), solvent B: 90% CH3CN/water (20 min)). The fractions containing the product were concentrated, affording the objective Compound 10 (73.3 mg, 90.8 μmol, 50.4% yield) in the form of triethylammonium. The yield was calculated from the absorption at 260 nm. An absorption coefficient (ε260)=15,100 M1·cm−1 was used for the calculation.
1H NMR (600 MHz, D2O) δ 7.89-7.70 (m, 1H), 7.66-7.33 (m, 3H), 6.18 (s, 0.5H), 6.00 (d, J=11.2 Hz, 1.5H), 5.47 (s, 0.5H), 4.62 (d, J=5.6 Hz, 0.5), 4.67 (m, 0.5H), 4.55 (s, 0.5H), 4.50-4.38 (m, 1H), 4.18 (s, 0.5H), 4.06 (s, 1.5H), 3.99 (s, 3H), 3.30 (s, 1.5H), 3.07 (qd, J=7.4, 2.1 Hz, 6H), 2.99 (s, 1.5H), 1.14 (td, J=7.3, 2.2 Hz, 9H), 0.74 (d, J=12.1 Hz, 9H) ppm.
13CNMR (151 MHz, D2O) δ155.70, 155.63, 155.09, 154.94, 154.35, 153.94, 150.05, 149.87, 148.91, 148.65, 133.38, 133.05, 131.87, 131.18, 129.43, 129.22, 128.59, 124.70, 124.63, 108.16, 108.01, 87.55, 86.87, 83.55, 83.02, 82.51, 82.20, 81.55, 81.15, 75.27, 74.81, 69.65, 64.65, 64.44, 59.06, 58.99, 46.71, 36.20, 36.13, 35.58, 24.95, 24.92, 8.28 ppm. 31PNMR (243 MHz, D2O) δ−9.64 (1P), −10.72 (1P) ppm.
ESI-TOF-MS calcd for C24H31N6O15P2, 705.1328 [M−H]−; found 705.1404.
Compound 10 (7.60 mg, 9.40 μmol) and guanosine 5′-monophosphorimidazolide (12.3 mg, 28.2 μmol) were dissolved in a 0.59 M zinc chloride solution in DMSO (493 μL, ZnCl2: 291 μmol). After stirring at 37° C. for 2 days, 200 mM aqueous EDTA solution (pH 7.0, 1.60 mL) was added to quench the reaction mixture. After dilution with a 2.0 M TEAB buffer (pH 7.9, 100 μL), the mixture was purified by reverse-phase HPLC using a YMC-Triart-C8 column (250×4.6 mm I.D., S-5 μm, 12 nm, flow rate 1.0 mL/min, temperature: 50° C.). The fractions containing the product were concentrated, affording target Compound 11 (8.33 mg, 6.15 μmol, 65.4% yield) in the form of a triethylammonium salt. The product was freeze-dried and the resulting solid was re-dissolved in methanol (2.0 mL). A 190 mM NaClO4 (10 mL) solution in acetone was added to the mixture, and the resulting suspension was centrifuged (4,000 rpm, 20 min). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording a target cap compound 11 (3.46 mg, 3.10 μmol, 33.0% yield) in the form of a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. An absorption coefficient (ε260)=26,300 M1·cm−1 was used for the calculation. Triethylammonium salt
1H NMR (600 MHz, CD3OD, triethylammonium salt) δ 7.99 (d, J=5.4 Hz, 1H), 7.92 (q, J=8.0 Hz, 1H), 7.77-7.61 (m, 2H), 7.59-7.49 (m, 1H), 6.41-6.29 (m, 0.5H), 6.27-6.17 (m, 0.5H), 6.04-5.93 (m, 1H), 5.76 (s, 1H), 5.30 (s, 1H), 4.76-4.67 (m, 1H), 4.61 (s, 1H), 4.58-4.38 (m, 4H), 4.27 (s, 2H), 4.23-4.13 (m, 3H), 4.13-3.99 (m, 3H), 3.53-3.43 (m, 1.5H), 3.08 (d, J=7.9 Hz, 1.5H), 0.96 (d, J=8.4 Hz, 9H) ppm.
13C NMR (151 MHz, CD3OD, triethylammonium salt) δ 159.34, 157.66, 155.50, 155.17, 155.02, 153.03, 150.94, 150.78, 138.22, 133.97, 133.82, 133.27, 130.47, 130.35, 130.12, 125.63, 117.77, 108.69, 89.71, 89.29, 85.29, 84.35, 84.11, 84.02, 81.73, 81.48, 75.66, 75.20, 71.92, 66.63, 65.32, 60.04, 59.78, 47.43, 37.51, 37.09, 36.97, 26.28, 26.13, 23.46, 9.14 ppm.
31P NMR (243 MHz, CD3OD, triethylammonium salt) δ −10.53˜−11.77 (m, 2P), −22.19 (s, 1P) ppm.
1H NMR (600 MHz, D2O, sodium salt) δ 7.87-7.80 (m, 2H), 7.68-7.54 (m, 2H), 7.45 (t, J=7.7 Hz, 1H), 6.19 (s, 0.5H), 6.04 (s, 0.5H), 5.78 (d, J=5.4 Hz, 1H), 5.62 (t, J=6.4 Hz, 1H), 5.27-5.22 (m, 0.5H), 5.17 (t, J=4.3 Hz, 0.5H), 4.54-4.47 (m, 1H), 4.45-4.38 (m, 1H), 4.35 (t, J=5.4 Hz, 0.5H), 4.31 (q, J=5.4 Hz, 1H), 4.25-4.04 (m, 5.5H), 3.95 (d, J=5.4 Hz, 3H), 3.21 (s, 1.5H), 2.95 (s, 1.5H), 0.82 (d, J=13.4 Hz, 9H) ppm. 31P NMR (243 MHz, D2O, sodium salt) 5-10.97 (dd, J=36.2, 19.7 Hz), −22.56 (t, J=19.7 Hz) ppm.
The following shows a synthetic scheme of a 3′-O-methylated dinucleotide cap compound.
To a suspension of 2′-O-methylguanosine (1.50 g, 5.05 mmol) in methanol (25.3 mL) was added N,N-dimethylformamide dimethyl acetal (3.01 g, 3.39 mL, 25.3 mmol). After stirring at room temperature for 18 hours, the reaction mixture was concentrated with a rotary evaporator. The oily residue was dissolved in dichloromethane (10.0 mL) and suspended by the addition of ether. The resulting precipitate was collected by filtration and washed with ether. The solid was dried under reduced pressure, affording objective Compound 2 (1.72 g, yield 96.6%) as a white powder.
1H NMR (600 MHz, DMSO-d6) δ 11.35 (s, 1H), 8.54 (s, 1H), 8.04 (s, 1H), 5.78 (d, J=6.3 Hz, 1H), 5.52 (s, 1H), 5.15 (s, 1H), 4.68-4.63 (m, 1H), 3.99 (q, J=4.0 Hz, 1H), 3.84 (dd, J=5.0, 3.3 Hz, 1H), 3.66-3.60 (m, 1H), 3.53 (d, J=13.3 Hz, 1H), 3.41 (s, 3H), 3.16 (s, 3H), 3.03 (s, 3H) ppm.
13C NMR (151 MHz, DMSO-d6) δ 157.97, 157.45, 150.02, 136.87, 119.85, 86.88, 82.96, 79.86, 73.02, 64.93, 61.47, 57.61, 40.64, 34.65 ppm.
To a solution of N2-protected-3′-O-methylguanosine 2 (195 mg, 0.553 mmol) in pyridine (2.77 mL) was added DMTrCl (244 mg, 0.719 mmol). After stirring at room temperature for 12 hours, DMTrCl (93.9 mg, 0.277 mmol) was added. The reaction mixture was stirred at room temperature for 3.5 hours and then quenched by the addition of methanol (1.00 mL). The resulting mixture was diluted with dichloromethane and washed with a saturated aqueous sodium bicarbonate solution. The organic layer was dried over Na2SO4 and concentrated with a rotary evaporator. The crude product was subjected to silica gel column chromatography eluted with 1.6 to 4.8% methanol/dichloromethane containing 1.0% triethylamine, affording target Compound 3 (337 mg, 93.1% yield) as a white foam.
1H NMR (400 MHz, CDCl3, containing triethylamine) δ 8.37 (s, 1H), 7.66 (s, 1H), 7.36-7.29 (m, 2H), 7.25-7.04 (m, 7H), 6.75-6.67 (m, 4H), 5.91 (d, J=5.9 Hz, 1H), 4.73 (t, J=5.7 Hz, 1H), 4.17 (q, J=4.1 Hz, 1H), 3.93-3.87 (m, 1H), 3.66 (d, J=1.2 Hz, 6H), 3.37 (s, 3H), 3.32 (dd, J=10.6, 4.0 Hz, 1H), 3.22 (dd, J=10.6, 4.5 Hz, 1H), 2.87 (d, J=8.6 Hz, 6H), 2.43 (q, J=7.2 Hz, 15H), 0.92 (t, J=7.2 Hz, 22H) ppm.
13C NMR (101 MHz, CDCl3, containing triethylamine) δ 158.56, 158.16, 156.99, 150.69, 144.65, 136.21, 135.69, 130.04, 128.10, 127.95, 126.93, 120.26, 113.21, 87.65, 86.46, 81.54, 80.25, 73.84, 63.68, 58.29, 55.20, 46.15, 41.27, 35.09, 11.51 ppm.
To a cooled solution of protected guanosine 3 (531 mg, 0.811 mmol) and chloroformate (439 mg, 1.62 mmol) in dichloromethane (4.06 mL) was added DMAP (198 mg, 1.62 mmol) in an ice bath. After stirring at room temperature for 18 hours, the reaction mixture was diluted with dichloromethane and washed with a saturated aqueous ammonium chloride solution. The organic layer was dried over Na2SO4 and basified by the addition of triethylamine (4.00 mL, 4%). After distilling off the solvent, the residue was subjected to silica gel column chromatography eluted with 2.4 to 6.3% methanol/dichloromethane containing triethylamine, affording a target compound (745 mg, quantitative) as a white amorphous solid.
1H NMR (600 MHz, CDCl3) δ 10.20 (s, 1H), 8.56 (s, 0.4H), 8.47 (s, 0.6H), 7.87-7.80 (m, 1H), 7.70 (s, 0.4H), 7.66 (s, 0.6H), 7.60-7.53 (m, 0.8H), 7.52-7.49 (m, 1.2H), 7.42-7.34 (m, 3H), 7.28-7.24 (m, 4H), 7.23-7.19 (m, 2H), 7.17-7.12 (m, 1H), 6.79-6.73 (m, 4H), 6.45 (s, 0.6H), 6.30 (s, 0.4H), 6.03 (dd, J=6.8, 4.3 Hz, 1H), 6.02-5.94 (m, 1H), 4.23 (dt, J=5.6, 3.7 Hz, 1H), 4.13 (t, J=5.1 Hz, 0.6H), 4.00 (dd, J=6.1, 4.9 Hz, 0.4H), 3.72 (d, J=1.1 Hz, 6H), 3.45-3.40 (m, 1H), 3.33 (s, 1.8H), 3.27 (td, J=11.3, 4.1 Hz, 1H), 3.09 (s, 1.2H), 3.06-3.05 (m, 1.2H), 3.01-2.99 (m, 1.8H), 2.97 (s, 1.2H), 2.94 (s, 1.8H), 0.91 (d, J=5.8 Hz, 9H) ppm.
13C NMR (151 MHz, CDCl3) δ 158.75, 158.63, 158.51, 158.29, 158.26, 157.13, 157.08, 153.75, 153.56, 150.03, 149.80, 149.10, 148.91, 144.28, 136.42, 136.19, 135.48, 132.67, 132.45, 132.05, 129.96, 129.74, 129.08, 128.95, 128.71, 128.56, 128.07, 127.88, 126.90, 124.55, 124.35, 120.84, 120.76, 113.16, 86.47, 86.44, 86.15, 85.55, 81.74, 81.58, 80.77, 80.41, 78.23, 77.94, 76.31, 76.21, 62.84, 62.68, 58.83, 58.65, 55.17, 46.12, 41.33, 41.14, 36.51, 36.11, 35.12, 35.05, 25.72, 25.64, 25.44, 11.47 ppm.
ESI-TOF-MS calcd for C47H51N7NaO11, 912.3539 [M+Na]+; found 912.3644.
To a suspension of guanosine derivative 4 (56.9 mg, 63.9 μmol) in methanol (1.07 mL) and water (533 μL) was added trifluoroacetic acid (546 mg, 367 μL, 4.79 mmol). After stirring at room temperature for 19 hours, the reaction mixture was diluted with a mixture of methanol/water (2:1, v/v, 5.00 mL). The mixture was concentrated and purified by silica gel column chromatography with elution using 1.9 to 9.1% methanol/dichloromethane, affording the objective compound (24.3 mg, 71.5% yield) as a white solid.
1H NMR (600 MHz, DMSO-d6) δ 10.73 (d, J=9.5 Hz, 1H), 8.01-7.93 (m, 2H), 7.81-7.68 (m, 1H), 7.64-7.52 (m, 2H), 6.54 (s, 2H), 6.18 (s, 0.6H), 6.02 (s, 0.4H), 5.96 (d, J=5.5 Hz, 1H), 5.48 (q, J=5.1 Hz, 1H), 4.22 (t, J=4.7 Hz, 0.5H), 4.08-3.95 (m, 1.5H), 3.68-3.60 (m, 1H), 3.58-3.50 (m, 2H), 3.35 (s, 2H), 2.98 (s, 1H), 0.87 (d, J=3.7 Hz, 9H) ppm.
13C NMR (151 MHz, DMSO-d6) δ 156.61, 156.57, 153.91, 153.88, 153.10, 152.99, 150.98, 150.92, 149.08, 148.86, 135.66, 135.55, 133.05, 131.36, 130.78, 129.60, 129.47, 128.89, 128.51, 124.51, 124.48, 116.54, 116.47, 84.03, 82.96, 82.89, 79.93, 79.65, 77.90, 77.75, 77.27, 77.23, 60.91, 60.86, 58.05, 57.71, 36.02, 35.59, 25.44, 25.21 ppm.
To Compound 5 (290 mg, 0.545 mmol) was added a solution of bis(2-cyanoethyl) N,N-diisopropylphosphoramidite (222 mg, 0.818 mmol) in acetonitrile (5.45 mL). 1H-Tetrazole (57.3 mg, 0.818 mmol) was added to the mixture, followed by stirring at room temperature for 1.5 hours, and subsequently, to the reaction mixture was added 5 to 6 M tert-butyl hydroperoxide in a decane solution (218 μL, 1.09 to 1.31 mmol). The reaction mixture was further stirred at room temperature for 6 minutes and diluted with dichloromethane. The mixture was washed with a saturated aqueous sodium bicarbonate solution. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluted with 4.8 to 17% methanol/dichloromethane, affording the objective compound (317 mg, yield 80.9%) as a brown amorphous solid.
1H NMR (600 MHz, CDCl3) δ 12.25 (s, 0.5H), 7.88 (d, J=8.2 Hz, 0.5H), 7.81 (d, J=8.0 Hz, 0.5H), 7.74 (d, J=16.2 Hz, 1H), 7.66-7.60 (m, 1H), 7.59-7.50 (m, 1H), 7.46 (ddd, J=8.5, 5.9, 2.9 Hz, 0.5H), 7.40 (t, J=7.8 Hz, 0.6H), 6.60 (s, 1.5H), 6.35 (s, 0.5H), 6.22 (s, 0.5H), 5.95 (d, J=4.4 Hz, 1H), 5.73 (dd, J=10.3, 5.4 Hz, 1H), 4.53-4.32 (m, 2.5H), 4.25 (tq, J=11.5, 5.4 Hz, 5.5H), 3.44 (s, 1.5H), 3.03 (s, 1.5H), 2.75 (dq, J=12.6, 6.8 Hz, 4H), 0.94 (s, 9H) ppm.
13C NMR (151 MHz, CDCl3) δ 158.89, 154.01, 153.97, 153.81, 153.60, 151.23, 149.48, 149.26, 137.08, 132.74, 132.58, 131.98, 129.35, 129.10, 128.90, 124.62, 124.52, 117.55, 117.50, 117.12, 117.00, 86.69, 86.57, 80.98, 80.74, 78.01, 77.77, 76.37, 67.07, 62.85, 62.81, 59.18, 58.94, 36.52, 36.16, 25.83, 25.63, 19.69, 19.63 ppm.
31P NMR (243 MHz, CDCl3) δ −1.83 and 1.87 ppm.
HRESIMS calcd. for C29H35N8NaO12P, 741.2005 [M+Na]+; found 741.1980.
To a solution of nucleoside monophosphate 6 (317 mg, 0.441 mmol) in acetonitrile (4.41 mL) were added continuously N,O-bis(trimethylsilyl)acetamide (897 mg, 1.08 mL, 4.41 mmol) and 1,8-diazabicyclo[5.4. 0]undec-7-ene (268 mg, 263 μL, 1.76 mmol). After stirring at room temperature for 3 hours, the reaction mixture was quenched by the addition of a 1 M TEAA buffer (pH 6.0, 1.50 mL). The mixture was diluted with water (25.0 mL) and washed three times with dichloromethane (20.0 mL). The aqueous layer was purified using Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min)) (solvent A: 50 mM TEAA+0.5% CH3CN (pH 6.0), solvent B: CH3CN, 0 to 80% B gradient in 60 min). The fractions containing the objective product were combined, concentrated, and freeze-dried, affording the objective Compound 7 (347 mg, 89.9% yield; monotriethylammonium salt contained 3 equivalents of TEAA salt) as a brown powder.
1H NMR (600 MHz, CD3OD) δ 8.11-8.01 (m, 1H), 7.96-7.85 (m, 1H), 7.73 (t, J=7.7 Hz, 0.5H), 7.65 (dt, J=8.0, 5.8 Hz, 1H), 7.60-7.49 (m, 1.5H), 6.26 (s, 0.5H), 6.17 (s, 0.5H), 6.07 (dd, J=11.3, 5.6 Hz, 1H), 5.61 (q, J=5.1 Hz, 1H), 4.37 (t, J=4.4 Hz, 0.5H), 4.33-4.21 (m, 1.5H), 4.16 (q, J=7.3 Hz, 1H), 4.12-4.00 (m, 1H), 3.47 (s, 1.5H), 3.15 (q, J=7.4 Hz, 24H, triethylammonium), 3.11 (s, 1.5H), 1.93 (s, 9H, Acetate), 1.27 (t, J=7.4 Hz, 36H, triethylammonium), 0.92 (d, J=6.3 Hz, 9H) ppm.
13C NMR (151 MHz, CD3OD) δ 179.20, 159.53, 159.46, 155.91, 155.84, 154.88, 154.79, 152.96, 152.92, 150.76, 150.55, 138.06, 137.93, 133.90, 133.52, 133.15, 130.40, 130.34, 130.05, 130.03, 125.56, 125.51, 117.74, 117.53, 86.77, 86.50, 83.77, 83.72, 81.68, 81.53, 80.21, 79.86, 78.97, 78.80, 65.80, 65.77, 65.64, 59.17, 59.01, 47.10, 47.07, 37.24, 36.99, 26.19, 26.09, 23.81, 23.79, 9.05 ppm.
31P NMR (243 MHz, CD3OD) δ −1.53 and 1.48 ppm.
HRESIMS calcd. for C23H28N6O12P, 611.1508 [M−H]−; found 611.1504.
To a solution of 2′-modified guanosine monophosphate 7 (61.6 mg, 75.6 μmol) in DMSO (630 μL) was added iodomethane (85.9 mg, 37.7 μL, 605 μmol). After stirring at room temperature for 7 hours, the reaction mixture was diluted with water and washed three times with dichloromethane. The aqueous layer was purified using Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min)) (solvent A: 50 mM TEAA+0.5% CH3CN (pH 6.0), solvent B: CH3CN, 0 to 80% B gradient in 60 min). The fractions containing the objective product were combined, concentrated, and freeze-dried, affording the objective Compound 8 (18.3 mg, 33.3% yield) in the form of a triethylammonium salt.
1H NMR (600 MHz, D2O, triethylammonium salt) δ 7.86 (dd, J=8.2, 1.3 Hz, 0.4H), 7.79 (dd, J=8.2, 1.4 Hz, 0.6H), 7.73-7.67 (m, 0.4H), 7.64-7.59 (m, 1H), 7.57-7.44 (m, 1.6H), 6.19 (d, J=3.7 Hz, 0.4H), 6.09 (d, J=5.7 Hz, 0.6H), 6.04 (s, 0.5H), 5.96 (s, 0.5H), 5.62-5.55 (m, 1H), 4.44 (dt, J=8.8, 3.1 Hz, 1H), 4.35 (dd, J=5.0, 3.0 Hz, 1H), 4.27 (t, J=5.1 Hz, 0.5H), 4.16-4.10 (m, 0.5H), 4.08-4.02 (m, 3H), 3.98-3.92 (m, 1H), 3.49 (s, 2H), 3.17 (q, J=7.4 Hz, 18H triethylammonium), 3.14 (s, 1H), 1.89 (s, 3H, acetate), 1.25 (t, J=7.4 Hz, 27H, triethylammonium), 0.86 (d, J=7.0 Hz, 9H) ppm.
13C NMR (151 MHz, D2O, triethylammonium salt) δ 181.47, 160.00, 159.51, 154.23, 153.75, 149.74, 149.55, 149.03, 148.80, 133.35, 132.″8, 1′1.59, 131.05, 129.58, 129.39, 128.73, 128.50, 124.58, 124.16, 108.63, 108.02, 86.78, 85.62, 83.82, 83.76, 83.25, 81.89, 81.60, 78.86, 78.80, 78.09, 77.64, 63.23, 62.59, 58.52, 58.39, 46.68, 36.01, 35.95, 35.62, 24.92, 24.87, 23.32, 8.26 ppm.
31P NMR (243 MHz, D2O, triethylammonium salt) δ 4.17, 4.13 ppm.
HRESIMS calcd. for C24H30N6O12P, 625.1664 [M−H]−; found 625.1693.
To a solution of 2′-Nb-3′-OMe-N7m-guanosine monophosphate 8 (18.4 mg, 18.6 μmol) in DMSO (372 μL) were added guanosine 5′-phosphorimidazolide diphosphate (30.0 mg, 55.8 μmol) and zinc chloride (50.7 mg, 372 μmol). After stirring at 37° C. for 1.5 days, a 500 mM aqueous EDTA solution (pH 8.0, 968 μL, EDTA: 484 mmol) was added to quench the reaction mixture. The mixture was diluted with water (14.0 mL) and purified by reverse-phase HPLC (instrument: Shimadzu Prep, column: YMC-Actus Triart C8 (Preparative, 250×20.0 mm I.D., solvent A: 50 mM TEAA buffer (pH 6.0, containing 0.5% CH3CN), solvent B: CH3CN, 5 to 80% B gradient (25 min), flow rate: 10 ml/min, detection: 254 nm). The fractions containing the target product were collected, concentrated, and freeze-dried, affording a PureCap analog in the form of a triethylammonium salt. The product was re-dissolved in methanol (2.00 mL). 190 mM NaClO4 (12.0 mL) in acetone was added to the mixture and the resulting suspension was centrifuged (4,000 rpm, 20 min). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording Cap Analog 9 (14.9 mg, 13.3 μmol, 71.5% yield) in the form of a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=26,300 M1·cm−1 was used.
1H NMR (600 MHz, D2O) δ7.93 (s, 1H), 7.80 (d, J=8.2 Hz, 1H), 7.68-7.40 (m, 3H), 5.99 (s, 1H), 5.92 (d, J=5.7 Hz, 0.5H), 5.79 (s, 0.5H), 5.72 (d, J=5.9 Hz, 0.5H), 5.43 (s, 0.5H), 4.60 (s, 1H), 4.48-4.41 (m, 1H), 4.39-4.14 (m, 8H), 4.08 (s, 1H), 4.01 (s, 2H), 3.51-3.39 (m, 2H), 0.93-0.77 (m, 9H) ppm.
13C NMR (151 MHz, D2O) δ158.67, 154.06, 153.84, 151.39, 149.23, 148.87, 137.33, 133.00, 131.59, 130.94, 129.55, 129.27, 128.77, 124.21, 115.98, 108.52, 107.89, 86.67, 85.49, 83.68, 83.62, 82.51, 81.65, 78.20, 77.97, 73.79, 70.33, 65.53, 64.94, 58.55, 36.05, 35.67, 30.29, 24.80 ppm.
31P NMR (243 MHz, D2O) δ−10.96 (2P, dt, J=88.8, 23.0 Hz), −22.44 (1P, t, J=19.7 Hz) ppm.
The following shows a synthesis scheme of a nitrobenzyl derivative to be used for the synthesis of a long-chain-alkyl-modified nitrobenzyl dinucleotide cap compound.
The following shows a synthetic scheme of a long-chain-alkyl-modified nitrobenzyl dinucleotide cap compound.
A mixture of 1-iodo-2-nitrobenzene 1 (5.00 g, 20.1 mmol) in THF (41.9 mL) was cooled to −40° C. A solution of phenylmagnesium chloride in THF (2 M, 13.1 mL, 26.1 mmol) was added to the mixture, followed by stirring at −40° C. for 1 hour. Decanal (5.18 g, 6.24 mL, 28.1 mmol) was added to the reaction mixture, followed by stirring at −40° C. for 1 hour. The reaction mixture was quenched by the addition of a saturated aqueous ammonium chloride solution (20.0 mL). The mixture was extracted three times with dichloromethane (20.0 mL). The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluted with 17→50% ethyl acetate/hexane, affording the objective Compound 2 (6.08 g, yield 98.4%) as a brown oil.
1H NMR (60 MHz, CDCl3) δ 8.09-6.79 (m, 5H, overlapping with the signal of CHCl3), 5.59-4.86 (m, 1H), 1.34 (s, 20H), 0.99 (d, J=4.8 Hz, 3H) ppm.
13C NMR (15 MHz, CDCl3) δ 147.46, 140.76, 137.30, 133.11, 130.05, 129.25, 127.92, 127.57, 123.94, 119.79, 115.41, 68.96, 60.50, 38.59, 31.88, 29.63, 29.33, 26.10, 22.62, 13.97, 13.88 ppm.
Acetic acid (47.6 g, 45.3 mL, 792 mmol), DMSO (30.9 g, 28.1 mL, 396 mmol) and acetic anhydride (40.4 g, 37.4 mL, 396 mmol) were added to a nitrobenzyl alcohol derivative 2 (6.08 g, 19.8 mmol). After stirring at room temperature for 4 days, the reaction mixture was added dropwise to a 10 M aqueous KOH solution. (178 mL, 1.78 mol) In an ice bath. The mixture was stirred at room temperature for 4 hours and then extracted with ethyl acetate. The organic layer was washed with brine and dried over Na2SO4. The residue was dissolved in hexane and subjected to silica gel column chromatography eluted with 4.8 to 17% ethyl acetate/hexane, affording methyl thioacetal 3 (10.6 g, >100% yield) as a yellow oil.
1H NMR (600 MHz, CDCl3) δ 7.88 (dd, J=8.2, 1.3 Hz, 1H), 7.70 (dd, J=7.9, 1.5 Hz, 1H), 7.64-7.58 (m, 1H), 7.39 (ddd, J=8.2, 7.3, 1.5 Hz, 1H), 5.26 (dd, J=8.9, 3.4 Hz, 1H), 4.60 (d, J=11.6 Hz, 1H), 4.23 (d, J=11.6 Hz, 1H), 2.10 (s, 3H), 1.81-1.67 (m, 2H), 1.58-1.41 (m, 2H), 1.39-1.33 (m, 1H), 1.29-1.21 (m, 15H), 0.85 (t, J=7.1 Hz, 3H) ppm.
13C NMR (151 MHz, CDCl3) δ 148.81, 138.49, 133.35, 128.35, 128.10, 124.33, 73.92, 73.60, 37.92, 31.96, 29.70, 29.66, 29.64, 29.61, 29.38, 29.30, 26.18, 22.73, 14.23, 14.15 ppm.
(3) Synthesis of Compound 5 To a suspension of N2-isobutyryl-3′,5′-O-(1,1,3,3-tetrahydrofurandisiloxane-1,3-diyl) guanosine 4 (2.00 g, 3.36 mmol), a methylthioacetal derivative 3 (1.61 g, 4.37 mmol) and powdered molecular sieve 3 Å (1.50 g) in tetrahydrofuran (33.6 mL) was added N-iodosuccinimide (983 mg, 4.37 mmol), followed by cooling to −40° C. After 20 minutes at −40° C., trifluoromethanesulfonic acid (756 mg, 448 μL, 5.04 mmol) was added to the suspension, and the mixture was stirred at −40° C. for 16.5 hours. The reaction mixture was quenched by the addition of triethylamine (16.8 mL), and then diluted with ethyl acetate (100 mL). The mixture was washed with a saturated aqueous sodium bicarbonate solution (100 mL×2), sodium thiosulfate (100 mL×2), and brine (100 mL×1). The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography with elution using 0 to 4.8% methanol/dichloromethane, affording objective Compound 5 (2.53 g, 82.1% yield) as a brown foam.
1H NMR (600 MHz, CDCl3, mixture of the target compound and the starting material) δ 12.32-12.08 (m, 1.5H), 10.79 (s, 0.7H), 9.70 (s, 0.5H), 9.39 (s, 0.3H), 7.99 (s, 0.3H), 7.93-7.88 (m, 1H), 7.82 (s, 0.3H), 7.70 (dd, J=8.2, 1.4 Hz, 0.4H), 7.63 (dd, J=5.4, 2.0 Hz, 0.6H), 7.54 (dd, J=7.9, 1.5 Hz, 0.3H), 7.41 (dddd, J=10.6, 9.0, 6.9, 2.1 Hz, 0.6H), 7.23 (td, J=7.9, 1.5 Hz, 0.3H), 5.79 (s, 0.4H), 5.73 (s, 0.9H), 5.58 (s, 0.4H), 5.29 (dd, J=8.6, 3.3 Hz, 0.4H), 5.24 (s, 1.5H), 5.21 (dd, J=7.6, 4.6 Hz, 0.5H), 5.13 (d, J=6.5 Hz, 0.3H), 5.03 (d, J=6.6 Hz, 0.4H), 4.63 (d, J=6.6 Hz, 0.4H), 4.58 (d, J=6.5 Hz, 0.3H), 4.48 (dd, J=9.4, 4.2 Hz, 0.3H), 4.38 (dt, J=10.5, 5.4 Hz, 1.2H), 4.25 (d, J=5.2 Hz, 0.8H), 4.20-4.05 (m, 3.3H), 4.03-3.87 (m, 3.3H), 3.44 (s, 0.4H), 3.36 (d, J=5.9 Hz, 0.4H), 2.92 (p, J =6.8 Hz, 0.8H), 2.81 (p, J=6.9 Hz, 0.2H), 2.69 (p, J=6.8 Hz, 0.4H), 1.78-1.59 (m, 2H), 1.25-1.14 (m, 24H), 1.13-1.09 (m, 3H), 1.08-0.84 (m, 54H), 0.79 (t, J=7.1 Hz, 4H) ppm.
13C NMR (151 MHz, CDCl3, mixture of the target compound and the starting material) δ 180.13, 179.64, 179.13, 156.03, 155.80, 155.72, 148.50, 148.38, 148.02, 147.97, 147.58, 147.45, 138.31, 137.95, 136.56, 136.43, 136.37, 133.94, 133.27, 128.57, 128.51, 128.45, 128.07, 124.52, 124.00, 121.45, 121.35, 121.23, 93.62, 91.30, 89.08, 88.14, 87.38, 81.66, 81.25, 81.07, 79.04, 76.60, 75.30, 74.74, 72.28, 70.63, 70.03, 69.50, 69.37, 60.55, 59.57, 59.50, 53.55, 50.39, 37.93, 37.87, 36.45, 36.24, 36.05, 31.92, 29.68, 29.65, 29.62, 29.59, 29.55, 29.51, 29.49, 29.46, 29.43, 29.37, 29.33, 26.54, 25.81, 25.64, 22.69, 19.21, 19.08, 18.97, 18.95, 18.90, 17.49, 17.46, 17.40, 17.33, 17.26, 17.20, 17.17, 17.11, 17.08, 17.05, 17.00, 16.98, 16.96, 16.88, 16.84, 14.12, 13.44, 13.39, 13.08, 13.00, 12.93, 12.67, 12.54 ppm
A solution of a starting material 5 (2.53 g, 2.76 mmol) in tetrahydrofuran (13.8 mL) was cooled in an ice bath and 1 M TBAF/THF (27.6 mL, 27.6 mmol) was added thereto. After stirring at 0° C. for 4 hours, the reaction mixture was diluted with dichloromethane (120 mL) and washed with water (120 mL). The aqueous layer was extracted with dichloromethane (120 mL). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluted with 0 to 9.1% methanol/dichloromethane, affording target Compound 6 (1.00 g, 53.7% yield) as a white foam.
1H NMR (600 MHz, CDCl3) δ 12.15 (d, J=17.5 Hz, 1H), 10.71-10.47 (m, 1H), 7.81 (s, 0.5H), 7.69 (d, J=8.0 Hz, 0.5H), 7.62 (dd, J=8.2, 1.4 Hz, 0.5H), 7.49-7.42 (m, 2H), 7.28-7.19 (m, 1H), 7.16-7.10 (m, 0.5H), 5.71 (d, J=3.2 Hz, 0.5H), 5.30 (d, J=2.9 Hz, 0.5H), 5.10 (t, J=6.4 Hz, 0.5H), 5.02-4.92 (m, 1.5H), 4.82 (d, J=6.8 Hz, 0.5H), 4.76 (d, J=6.9 Hz, 0.5H), 4.72 (d, J=7.1 Hz, 0.5H), 4.60 (t, J=4.2 Hz, 0.5H), 4.50 (dd, J=4.9, 3.0 Hz, 0.5H), 4.44 (d, J=7.2 Hz, 0.5H), 4.23 (s, 1H), 3.92 (dt, J=6.5, 3.3 Hz, 0.5H), 3.87 (dt, J=7.0, 3.5 Hz, 0.5H), 3.78-3.59 (m, 2H), 3.27 (d, J=5.5 Hz, 0.5H), 3.24-3.17 (m, 6.5H), 3.05-2.93 (m, 1H), 1.56 (dq, J=16.3, 7.9 Hz, 8H), 1.50-1.39 (m, 1.5H), 1.30 (h, J=6.9 Hz, 7H), 1.17-1.01 (m, 23H), 1.01-0.92 (m, 3H), 0.92-0.88 (m, 1H), 0.85 (td, J=7.4, 1.3 Hz, 10H), 0.71 (td, J=7.1, 1.4 Hz, 3H) ppm.
13C NMR (151 MHz, CDCl3) δ 180.47, 180.28, 155.49, 155.47, 148.15, 147.87, 147.77, 147.73, 147.55, 147.51, 147.10, 138.97, 138.76, 138.44, 137.71, 133.25, 132.77, 128.26, 128.18, 127.97, 127.74, 123.89, 123.56, 121.42, 121.21, 94.72, 93.07, 88.61, 88.47, 84.22, 83.86, 78.84, 78.60, 74.87, 73.51, 70.30, 69.29, 69.11, 61.01, 60.87, 58.63, 37.49, 37.37, 35.53, 35.42, 31.59, 29.31, 29.29, 29.17, 29.08, 29.01, 26.22, 25.41, 25.26, 23.76, 22.37, 19.46, 18.97, 18.94, 18.83, 18.79, 13.84, 13.40 ppm.
A solution of a starting material 6 (970 mg, 1.44 mmol) in acetonitrile (9.40 mL) was added to a 28% ammonium hydroxide solution (18.8 mL). The mixture was heated to 55° C. After stirring at 55° C. for 19.5 hours, the reaction mixture was concentrated and azeotroped with benzene (x 1). The residue was dissolved in methanol (6.00 mL) and suspended by the addition of diethyl ether (200 mL). The resulting precipitate was collected by filtration and rinsed with ether. The resulting solid was dried under vacuum, affording objective Compound 7 (408 mg, yield 47.0%) as a white solid.
1H NMR (600 MHz, DMSO-d6) δ 10.23 (s, 0.5H), 7.93 (s, 0.5H), 7.88 (dd, J=8.2, 1.3 Hz, 0.5H), 7.84 (dt, J=8.1, 0.9 Hz, 0.5H), 7.78 (s, 0.5H), 7.70 (td, J=7.6, 1.4 Hz, 0.5H), 7.57 (dd, J=7.9, 1.5 Hz, 0.5H), 7.55-7.50 (m, 1.5H), 7.40 (ddd, J=8.1, 5.2, 3.6 Hz, 0.5H), 6.43 (s, 1.5H), 5.82 (d, J=6.3 Hz, 0.5H), 5.68 (d, J=5.9 Hz, 0.5H), 5.23 (d, J=5.3 Hz, 0.5H), 5.16 (d, J=5.0 Hz, 0.5H), 5.11 (t, J=5.5 Hz, 0.5H), 5.08 (t, J=5.4 Hz, 0.5H), 5.04 (dd, J=7.8, 4.4 Hz, 0.5H), 4.78 (dd, J=7.6, 4.9 Hz, 0.5H), 4.69-4.62 (m, 1.5H), 4.57 (d, J=7.0 Hz, 0.5H), 4.52-4.47 (m, 0.5H), 4.34 (d, J=7.3 Hz, 0.5H), 4.22 (dtd, J=16.7, 5.0, 3.4 Hz, 1H), 3.89 (q, J=3.7 Hz, 0.5H), 3.85 (q, J=3.7 Hz, 0.5H), 3.61 (tdd, J=12.5, 5.1, 3.6 Hz, 1H), 3.51 (dtd, J=12.0, 6.3, 3.9 Hz, 1H), 3.20-3.14 (m, 2H), 1.60-1.53 (m, 2H), 1.31 (p, J=7.4 Hz, 2H), 1.28-1.15 (m, 13H), 0.93 (t, J=7.4 Hz, 3H), 0.85 (td, J=7.1, 0.9 Hz, 3H) ppm.
13C NMR (151 MHz, DMSO-d6) δ 156.71, 153.69, 153.64, 151.13, 151.02, 148.51, 147.69, 137.19, 136.43, 135.59, 135.17, 133.42, 133.24, 128.74, 128.34, 128.10, 123.97, 123.91, 116.89, 116.67, 93.20, 91.53, 85.92, 85.60, 85.05, 84.77, 78.82, 77.97, 74.07, 73.01, 69.60, 69.09, 61.49, 61.27, 57.54, 37.09, 36.76, 31.31, 31.28, 29.01, 28.96, 28.93, 28.83, 28.72, 28.69, 28.63, 24.98, 24.92, 23.06, 22.10, 19.21, 13.96, 13.49 ppm.
To a solution of 2′-modified guanosine 7 (100 mg, 0.166 mmol) in DMF (1.33 mL) was added iodomethane (186 mg, 81.6 μL, 1.31 mmol). After stirring at room temperature for 12.5 hours, water (15.0 mL) was added to the reaction mixture, and the mixture was extracted four times with dichloromethane (15.0 mL). The organic layer was concentrated and the residue was dissolved in acetone (2.00 mL). The solution was added dropwise to ether (150 mL) and the resulting precipitate was collected by filtration. The solid was dried under reduced pressure, affording objective Compound 8 (107 mg, yield 75.4%) as a yellow solid.
1H NMR (600 MHz, CDCl3) δ 9.56 (d, J=7.3 Hz, 0.5H), 9.45 (d, J=7.6 Hz, 0.5H), 7.98 (d, J=7.1 Hz, 0.2H), 7.82 (q, J=8.7 Hz, 0.5H), 7.75 (t, J=7.8 Hz, 0.5H), 7.64 (dq, J=10.5, 5.5 Hz, 2H), 7.40 (dtd, J=14.8, 8.7, 5.4 Hz, 0.8H), 7.31-7.23 (m, 0.5H), 6.72 (s, 1H), 6.04 (q, J=3.6 Hz, 0.5H), 5.89 (q, J=3.4 Hz, 0.5H), 5.18-5.12 (m, 0.5H), 5.06 (q, J=6.6 Hz, 0.5H), 4.96 (t, J=7.0 Hz, 0.5H), 4.89 (t, J=7.1 Hz, 0.5H), 4.78 (t, J=7.1 Hz, 0.5H), 4.73 (t, J=7.0 Hz, 0.5H), 4.56 (qt, J=8.5, 4.8 Hz, 1H), 4.45 (dt, J=10.2, 5.0 Hz, 0.5H), 4.23-4.11 (m, 3H), 4.00-3.91 (m, 1H), 3.79 (dt, J=12.7, 5.9 Hz, 1H), 3.28 (dd, J=10.8, 6.2 Hz, 1.5H), 3.02-2.81 (m, 1H), 1.76-1.62 (m, 4H), 1.44 (hept, J=7.4 Hz, 2H), 1.27-1.13 (m, 14H), 1.00 (q, J =7.4 Hz, 2.5H), 0.82 (q, J=7.1 Hz, 2.5H) ppm.
13C NMR (151 MHz, CDCl3) δ 162.91, 156.24, 155.98, 154.16, 153.87, 148.90, 148.65, 148.53, 147.91, 137.62, 137.37, 136.28, 136.15, 133.73, 133.56, 128.68, 128.60, 128.45, 124.30, 124.02, 108.76, 108.55, 94.32, 94.12, 88.95, 86.85, 86.38, 80.38, 80.27, 75.20, 74.77, 69.30, 68.87, 60.50, 60.23, 59.26, 50.06, 49.91, 49.77, 49.63, 49.48, 37.63, 37.55, 37.04, 36.97, 36.74, 31.98, 29.73, 29.58, 29.54, 29.41, 25.70, 25.43, 24.29, 22.75, 19.88, 14.19, 13.80 ppm.
A suspension of 2′-C11Nb-N 7-methyl-guanosine (8) (53.2 mg, 62.3 μmol) in trimethyl phosphate (312 μL) was cooled to −10° C. 2,6-Lutidine (15.3 mg, 16.7 μL, 143 μmol) and phosphoryl chloride (21.9 mg, 13.4 μL, 143 μmol) were added continuously to the mixture. After stirring at −10° C. for 9 hours, a 0.2 M TEAB buffer (pH 7.9, 500 μL) was added at −10° C. to quench the reaction mixture. After warming to room temperature, the mixture was diluted with methanol (9.00 mL). The mixture was further diluted with 50% methanol/water (to 50 mL) and purified using a CHRMATREX C8 MB-100-40/75 column (column size: φ=3.0 cm, h=10 cm, 70 cm3 (7 mL/min) (solvent A: 50 mM TEAA buffer (pH 6.0, containing 0.5% CH3CN), solvent B: CH3CN, 0 to 90% B gradient over 60 minutes). The fractions containing the target product were combined, concentrated, and freeze-dried, affording the target Compound 9 (37.3 mg, yield 85.9%) as a white solid.
1H NMR (600 MHz, CD3OD) δ 7.88-7.79 (m, 1H), 7.74-7.62 (m, 2H), 7.49-7.41 (m, 1H), 6.12 (d, J=3.5 Hz, 0.5H), 5.96 (d, J=3.5 Hz, 0.5H), 5.22 (dd, J=7.6, 4.5 Hz, 0.5H), 5.12 (dd, J=7.6, 4.8 Hz, 0.5H), 5.01 (d, J=7.1 Hz, 0.5H), 4.77 (d, J=7.1 Hz, 0.5H), 4.70 (d, J=7.0 Hz, 0.5H), 4.61 (ddd, J=16.9, 4.8, 3.4 Hz, 1H), 4.47 (t, J=5.1 Hz, 0.5H), 4.41-4.34 (m, 0.5H), 4.24-4.16 (m, 1.5H), 4.13 (d, J=4.6 Hz, 2H), 4.06 (d, J=1.4 Hz, 1H), 4.04 (dd, J=4.7, 1.7 Hz, 0.5H), 4.02 (dt, J=4.7, 1.4 Hz, 0.5H), 3.24-3.19 (m, 3H), 3.16 (q, J=7.4 Hz, 16H, triethylammonium), 1.91 (s, 10H, acetate), 1.67-1.61 (m, 4H), 1.39 (h, J=7.4 Hz, 4H), 1.28 (t, J=7.4 Hz, 25H, triethylammonium), 1.22 (d, J=13.0 Hz, 14H), 1.00 (t, J=7.4 Hz, 5H), 0.86 (dd, J=7.6, 6.5 Hz, 3H) ppm.
13C NMR (151 MHz, CD3OD) δ 178.42 (acetate), 158.23, 158.05, 156.05, 155.91, 151.05, 150.87, 150.18, 149.47, 139.09, 138.56, 138.40, 134.49, 134.37, 129.79, 129.73, 129.67, 129.62, 129.37, 125.14, 125.06, 109.30, 109.10, 94.72, 94.62, 89.68, 89.63, 86.64, 86.40, 86.35, 81.47, 81.33, 80.84, 79.51, 79.29, 79.07, 75.88, 75.39, 69.89, 69.76, 64.26, 64.08, 59.49, 47.48, 38.63, 38.54, 36.70, 36.64, 33.04, 30.73, 30.70, 30.68, 30.64, 30.54, 30.49, 30.44, 26.64, 26.51, 24.76, 23.71, 22.96, 20.70, 14.45, 13.93, 9.10 ppm.
31P NMR (243 MHz, CD3OD) δ 1.65 ppm.
To a solution of 2′-Nb-N7-methyl-guanosine-5′-monophosphate (9) (37.3 mg, 53.5 μmol) in DMSO (1.07 mL) were added guanosine 5′-diphosphorimidazolide (115 mg, 214 μmol) and zinc chloride (146 mg, 1.07 mmol). The mixture was incubated at 37° C. for 2 days. The reaction mixture was quenched by the addition of a 500 mM aqueous EDTA solution (pH 8.0, 2.78 mL, EDTA: 1.39 mmol) and then diluted with water (10.0 mL). The crude product was purified using a CHRMATREX C8 MB-100-40/75 column (column size: φ=3.0 cm, h=10 cm, 70 cm3 (7 mL/min), solvent A: 50 mM TEAA buffer (pH 6.0, containing 0.5% CH3CN), solvent B: CH3CN, 0 to 90% B gradient over 60 min). The fractions containing the target product were combined, concentrated, and freeze-dried, affording the target compound in a triethylammonium form. The triethylammonium salt of the object was dissolved in methanol (1.00 mL). A solution of 0.19 M NaClO4 in acetone (12.0 mL) was added to the methanol solution, affording a white precipitate. The suspension was centrifuged at 4,500 rpm for 15 minutes. The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated three more times. The precipitate was dried under reduced pressure, affording target Cap Compound 10 as a sodium salt.
ESI-TOF-MS C40H56N11O21P3(M−2H)2− calcd. m/z 559.6438, found m/z 559.6.
The following shows a synthesis scheme of a nitrobenzyl derivative used for synthesis of a nitrobenzyl-modified dinucleotide cap compound that can be removed by reduction conditions.
The following shows a synthesis scheme of the nitrobenzyl-modified dinucleotide cap compound that can be removed by reduction conditions.
1-(4-Nitrophenyl)ethan-1-one (1) was azeotroped three times with toluene (25.0 mL) and the residue was dissolved in dichloromethane (100 mL). To the mixture was added Al(CH3)3 (15.6 mL, 21.8 mmol) at 0° C. After stirring at 0° C. for 4 hours, the reaction mixture was quenched by the addition of acetone (15.0 mL) and washed with a saturated aqueous ammonium chloride solution (100 mL). The mixture was diluted with dichloromethane and washed with water, a saturated aqueous NaHCO3 solution, and brine. The organic layer was dried over Na2SO4 nd concentrated. The crude product was purified by silica gel column chromatography eluted with 1-3% methanol/chloroform, affording the target Compound 2 (1.41 g, 7.78 mmol, 43% yield).
1H NMR (600 MHz, CDCl3) δ 8.19 (d, J=8.8 Hz, 2H), 7.67-7.64 (2H), 1.61 (s, 6H) ppm
To nitrobenzyl alcohol 2 (740 mg, 4.08 mmol) in dichloromethane (40.8 mL) was added 1,1′-carbonyldiimidazole (3.97 g, 24.5 mmol). After stirring overnight at room temperature, the reaction mixture was diluted with dichloromethane and washed with water and a saturated aqueous ammonium chloride solution. The organic layer was dried over Na2SO4 and concentrated, affording the target Compound 3 (1.08 g, 3.80 mmol, 95% yield).
1H NMR (400 MHz, CDCl3) δ 8.25 (dd, J=6.8, 2.0 Hz, 2H), 8.13 (d, J=2.0 Hz, 1H), 7.59 (dd, J=7.1, 2.0 Hz, 2H), 7.39 (t, J=1.4 Hz, 1H), 7.09 (q, J=0.8 Hz, 1H), 1.98 (s, 6H) ppm.
To a solution of N2 protected-2′-O-methylguanosine (4) (1.20 g, 3.50 mmol) in pyridine (34.6 mL) was added DMTrCl (1.40 g, 4.20 mmol). After stirring overnight at room temperature, the reaction mixture was diluted with dichloromethane and washed with water and brine. The organic layer was dried over Na2SO4 and concentrated with a rotary evaporator. The crude material was subjected to silica gel column chromatography eluted with 4.7% methanol/chloroform containing 1.0% triethylamine, affording the target Compound 5 (2.22 g, 3.40 mmol, 98% yield).
1H NMR (600 MHz, DMSO-d6) δ 11.39 (s, 1H), 8.50 (s, 1H), 7.94 (s, 1H), 7.19-7.37 (m, 9H), 6.82-6.86 (m, 4H), 5.96 (d, J=4.7 Hz, 1H), 5.29 (d, J=5.8 Hz, 1H), 4.33 (t, J=5.4 Hz, 1H), 4.26 (t, J=5.0 Hz, 1H), 4.01-4.03 (m, 1H), 3.73 (s, 6H), 3.38 (s, 3H), 3.24 (dd, J=10.4, 5.9 Hz, 1H), 3.15 (dd, J=7.4, 2.7 Hz, 1H), 3.10 (s, 3H), 3.03 (s, 3H) ppm
Protected nucleoside 5 (2.0 g, 3.1 mmol) was added to a solution of a carbonyl imidazole compound (1.1 g, 4.0 mmol) in dichloromethane (31 mL) and DBU (0.59 mL, 4.0 mmol). After stirring overnight at room temperature, the reaction mixture was diluted with dichloromethane and washed with water and a saturated aqueous ammonium chloride solution. The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by silica gel column chromatography eluted with 4.8% methanol/ethyl acetate, affording the target Compound 6 (1.6 g, 1.9 mmol, 60% yield).
1H NMR (600 MHz, DMSO-d6) δ 11.43 (s, 1H), 8.47 (s, 1H), 8.17-8.18 (m, 2H), 7.98 (s, 1H), 7.65-7.61 (2H), 7.27 (dd, J=8.3, 1.5 Hz, 2H), 7.19-7.23 (m, 3H), 7.13-7.15 (m, 4H), 6.77-6.80 (m, 4H), 5.98 (d, J=4.1 Hz, 1H), 5.44 (t, J=5.6 Hz, 1H), 4.74 (t, J=4.9 Hz, 1H), 4.17 (d, J=4.1 Hz, 1H), 3.72 (d, J=6.1 Hz, 6H), 3.28 (s, 3H), 3.13-3.21 (m, 2H), 2.96 (s, 3H), 2.89-2.82 (3H), 1.82-1.78 (3H), 1.75 (s, 3H) ppm.
To a mixture of protected guanosine 6 (2.1 g, 2.4 mmol) in THF (17.8 mL) and water (8.9 mL) was added trifluoroacetic acid (12.3 L, 160 mmol). After stirring at room temperature for 5 hours, the reaction mixture was diluted with chloroform and washed with water, and with saturated aqueous sodium bicarbonate solution. The organic layer was concentrated and purified by silica gel column chromatography eluted with 7.4% methanol/chloroform, affording the target Compound 7 (1.1 g, 2.2 mmol, 92% yield).
1H NMR (600 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.24 (dt, J=9.5, 2.3 Hz, 2H), 8.08 (s, 1H), 7.66 (dt, J=9.5, 2.3 Hz, 2H), 6.57 (s, 2H), 5.79 (d, J=7.1 Hz, 1H), 5.17 (q, J=2.3 Hz, 1H), 4.51 (dd, J=7.3, 4.9 Hz, 1H), 4.12 (td, J=4.0, 1.9 Hz, 1H), 3.56-3.61 (m, 2H), 3.18 (s, 3H), 1.82 (s, 3H), 1.75 (s, 3H) ppm
To a guanosine derivative (7) (290 mg, 570 μmol) was added a solution of bis(2-cyanoethyl) N,N-diisopropylphosphoramidite (230 mg, 850 μmol) in CH3CN (5.70 mL). 1H-Tetrazole (60.0 mg, 850 μmol) was added to the mixture, followed by stirring at room temperature for 3 hours, and then, to the reaction mixture was added 5 to 6 M of tert-butyl hydroperoxide in a decane solution (320 μL, 1.6 to 1.9 mmol). The reaction mixture was further stirred at room temperature for 1 hour and diluted with dichloromethane. The mixture was washed with a saturated aqueous sodium bicarbonate solution. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluted with 9.1% methanol/chloroform, affording the objective Compound 8 (280 mg, 410 μmol, 72% yield).
1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 8.23-8.25 (m, 2H), 7.94 (s, 1H), 7.67 (dd, J=7.0, 2.0 Hz, 2H), 6.53 (s, 2H), 5.83 (d, J=7.2 Hz, 1H), 5.25 (q, J=2.5 Hz, 1H), 4.54 (dd, J=7.2, 5.2 Hz, 1H), 4.34-4.37 (m, 1H), 4.28-4.31 (m, 2H), 4.14-4.21 (m, 4H), 3.21-3.16 (3H), 2.90 (ddd, J=16.4, 5.8, 0.9 Hz, 4H), 1.83 (s, 3H), 1.76 (s, 3H) ppm.
31P NMR (161 MHz, DMSO-d6) δ−1.60 (s, 1P) ppm
To a solution of a nucleoside monophosphate derivative (8) (250 mg, 0.360 mmol) in acetonitrile (3.60 mL), N,O-bis(trimethylsilyl)acetamide (0.890 mL, 3.60 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.220 mL, 1.45 mmol) were added continuously. After stirring at room temperature for 1 hour, the reaction mixture was quenched by the addition of a 2M TEAB buffer (pH 7.7, 0.730 mL). The mixture was diluted with water and washed three times with chloroform. The aqueous layer was purified using DEAE Sephadex A-25, (=4.5 cm, h=8.4 cm (140 cm3), flow rate: 12 mL/min, solvent A: water, solvent B: 1.5 MTEAB buffer (pH 7.7, containing 10% CH3CN). The fractions containing the objective product were collected and desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=1.3 cm, h=4.0 cm, 21 cm3), solvent A: water, solvent B: methanol). The fractions containing the product were combined and concentrated, affording the target Compound 9 (221 mg, 0.310 mmol, yield 86%).
1H NMR (400 MHz, D2O) δ8.25 (dd, J=7.0, 2.0 Hz, 2H), 8.12 (s, 1H), 7.68 (dd, J=7.1, 2.1 Hz, 2H), 5.84 (d, J=7.2 Hz, 1H), 5.40 (dd, J=5.0, 1.9 Hz, 1H), 4.66 (dd, J=7.3, 5.0 Hz, 1H), 4.46 (d, J=3.8 Hz, 1H), 4.04 (dd, J=4.7, 3.4 Hz, 2H), 3.25 (s, 3H), 3.19 (q, J=7.3 Hz, 8H), 1.90 (s, 3H), 1.81 (s, 3H), 1.27 (t, J=7.3 Hz, 12H) ppm.
31P NMR (161 MHz, D2O) δ1.66 ppm.
Iodomethane (73.0 μL, 1.17 mmol) was added to a solution of a modified guanosine monophosphoric acid triethylammonium salt 9 (100 mg, 0.150 mmol) in DMSO (1.22 mL). After stirring overnight at room temperature, the reaction mixture was diluted with water and washed three times with chloroform. The aqueous layer was purified using DEAE Sephadex A-25, Φ=3.0 cm, h=10 cm (70 cm3), flow rate: 6 mL/min, solvent A: water, solvent B: 1.0 M TEAA buffer (pH 7.0, containing 10% CH3CN). The fractions containing the objective product were collected and desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: Φ=1.3 cm, h=4.0 cm, 21 cm3), solvent A: water, solvent B: methanol). The fractions containing the product were combined and concentrated, affording the target Compound 10 (28.6 mg, 43.0 μmol, 33% yield).
1H NMR (600 MHz, D2O) δ8.27 (d, J=8.8 Hz, 2H), 7.69 (d, J=8.5 Hz, 2H), 6.07 (d, J=5.4 Hz, 1H), 5.32 (s, 1H), 4.55 (t, J=5.1 Hz, 2H), 4.12 (s, 1H), 4.10 (s, 3H), 3.99 (d, J=10.5 Hz, 1H), 3.34 (s, 3H), 3.19 (q, J=7.4 Hz, 4H), 1.89 (s, 3H), 1.80 (s, 3H), 1.27 (t, J=7.5 Hz, 6H) ppm.
31P NMR (243 MHz, D2O) δ2.49 (s, 1P) ppm
2-Nitroimidazole (10 mg, 90 μmol) was added to 2′-OMe-3′-Nb-N7m-guanosine monophosphate 10 (20 mg, 30 μmol) and GDP-imidazolide (48 mg, 90 μmol) in DMSO (300 μL). After stirring at 37° C. for 2 days, the reaction mixture was diluted with water and washed with chloroform. The aqueous layer was purified using DEAE Sephadex A-25, (=3.0 cm, h=10 cm (70 cm3), flow rate: 6 mL/min, solvent A: water, solvent B: 1.0 M TEAA buffer (pH 7.0, containing 10% CH3CN). The fractions containing the objective product were collected and desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=1.3 cm, h=4.0 cm, 21 cm3), solvent A: water, solvent B: methanol). The fractions containing the product were combined and concentrated, affording the target compound as a triethylammonium salt. An aqueous solution of the triethylammonium type as the objective compound was passed through a column filled with an H-type Dowex (registered trademark) 50 W-X2 resin, and was added dropwise to a mixture of triethylamine (10 equivalents) and ethanol (0.5 M triethylamine). After washing with water until the pH of the eluate was neutral, the mixture was concentrated and freeze-dried, affording the target Compound 11 in a sodium form (11.4 mg, 11.0 μmol, 37% yield). The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=28,293 M1·cm−1 was used.
1H NMR (600 MHz, D2O) δ8.25 (d, J=9.0 Hz, 2H), 7.94 (s, 1H), 7.67 (d, J=9.0 Hz, 2H), 5.86 (d, J=5.4 Hz, 1H), 5.73 (d, J=6.1 Hz, 1H), 5.24 (dd, J=4.9, 3.6 Hz, 1H), 4.62 (t, J=5.6 Hz, 1H), 4.52 (t, J=2.8 Hz, 1H), 4.43 (dd, J=5.2, 3.6 Hz, 1H), 4.38 (t, J=5.3 Hz, 1H), 4.31-4.35 (m, 1H), 4.28 (t, J=4.2 Hz, 1H), 4.23-4.25 (m, 1H), 4.21 (t, J=2.9 Hz, 1H), 4.17-4.19 (m, 1H), 4.07 (s, 3H), 3.29 (s, 3H), 1.88 (s, 3H), 1.79 (d, J=7.6 Hz, 3H) ppm.
31P NMR (161 MHz, D2O) δ−10.97 (t, J=23.3 Hz, 2P), −22.56 (t, J=21.2 Hz, 1P) ppm
The following shows the structure of the novel nitrobenzyl-modified nucleotide phosphorimidazolide compound.
To a solution (1.08 mL) of N7-methyl-guanosine-5′-diphosphate 6 (73.3 mg, 90.8 μmol), imidazole (49.4 mg, 726 μmol), and 2,2′-dithiodipyridine (59.9 mg, 272 μmol) in N,N-dimethylformamide was added triethylamine (25.4 (L, 18.4 mg, 182 (6 mol), and subsequently, triphenylphosphine (71.3 mg, 272 μmol) was added. After stirring at room temperature for 23 hours, a solution of sodium perchlorate (66.7 mg, 545 μmol) in 4% triethylamine/dry acetone (27.3 mL) was added dropwise while stirring the reaction mixture. The resulting suspension was centrifuged at 4,000 rpm for 20 minutes. The supernatant was discarded and the precipitate was rinsed four times with dry acetone. The solid was dried under reduced pressure, affording N7-methyl-guanosine 5′-diphosphorimidazolide disodium salt 1 (65.0 mg, 89.4% yield) as a white solid.
1H NMR (600 MHz, DMSO-d6) δ 9.82 (d, J=16.7 Hz, 1H), 7.98 (dd, J=8.4, 3.9 Hz, 1H), 7.81 (q, J=7.8 Hz, 1H), 7.69 (d, J=11.5 Hz, 1.5H), 7.62 (q, J=8.1 Hz, 1.5H), 7.17 (s, 1H), 6.78 (d, J=6.5 Hz, 1H), 6.25 (s, 0.5H), 6.10 (s, 0.5H), 5.95 (dd, J=14.7, 6.2 Hz, 1H), 5.36-5.23 (m, 1H), 4.76 (t, J=4.9 Hz, 0.2H), 4.66-4.55 (m, 0.8H), 4.41-4.31 (m, 1H), 4.02 (d, J=6.3 Hz, 3H), 3.92-3.75 (m, 2H), 3.45 (s, 1.5H), 3.00 (s, 1.5H), 0.92 (d, J=16.3 Hz, 9H) ppm.
13C NMR (151 MHz, DMSO-d6) δ 160.96, 153.28, 153.19, 149.91, 149.08, 148.90, 139.29, 134.79, 133.15, 131.57, 130.98, 129.62, 129.46, 129.12, 128.41, 127.94, 127.88, 124.61, 124.52, 120.14, 107.50, 85.74, 82.94, 82.08, 81.78, 79.82, 79.51, 75.71, 75.46, 68.54, 63.74, 58.67, 58.35, 55.85, 36.07, 35.67, 35.44, 32.14, 30.72, 29.62, 29.57, 25.53, 25.26 ppm.
31P NMR (243 MHz, DMSO-d6) δ−10.50 (d, 1P), −19.02 (d, 1P) ppm.
To a solution (233 μL) of N7-methyl-guanosine-5′-monophosphate 7 (12.3 mg, 19.6 μmol), imidazole (10.7 mg, 157 μmol), and 2,2′-dithiodipyridine (13.0 mg, 58.8 μmol) in N,N-dimethylformamide was added triethylamine (5.48 μL, 3.97 mg, 39.2 μmol), and subsequently, triphenylphosphine (15.4 mg, 58.8 μmol) was added. After stirring at room temperature for 23 hours, a solution of sodium perchlorate (14.4 mg, 118 μmol) in 4% triethylamine/dry acetone (5.90 mL) was added dropwise while stirring the reaction mixture. The resulting suspension was centrifuged at 4,000 rpm for 20 minutes. The supernatant was discarded and the precipitate was rinsed five times with dry acetone. The solid was dried under reduced pressure, affording N7-methyl-guanosine 5′-diphosphorimidazolide disodium salt 2 (11.6 mg, 85.0% yield) as a white solid.
1H NMR (600 MHz, DMSO-d6) δ 9.47-9.31 (m, 1H), 7.94 (d, J=8.2 Hz, 1H), 7.77 (dt, J=15.4, 7.8 Hz, 1H), 7.69 (d, J=9.9 Hz, 1H), 7.65-7.51 (m, 3H), 7.14-7.00 (m, 1H), 6.87-6.77 (m, 1H), 6.18 (s, 0.5H), 6.03 (s, 0.5H), 5.91 (dd, J=16.4, 6.0 Hz, 1H), 5.13-5.02 (m, 1H), 4.54 (d, J=11.6 Hz, 1H), 4.41 (t, J=5.3 Hz, 1H), 4.32-4.21 (m, 1H), 4.13-4.05 (m, 1H), 4.01-3.95 (m, 3H), 3.72 (s, 1H), 3.37 (s, 1.5H), 2.92 (s, 1.5H), 0.92-0.82 (2s, 6H), 0.75-0.62 (2s, 3H) ppm.
13C NMR (151 MHz, DMSO-d6) δ 161.78, 153.56, 149.58, 139.69, 133.64, 130.14, 129.04, 128.92, 125.03, 120.34, 108.52, 83.59, 81.93, 81.65, 80.36, 69.05, 59.28, 58.50, 56.37, 36.56, 36.16, 35.90, 32.65, 31.24, 30.14, 26.03, 25.92, 25.76 ppm.
31P NMR (243 MHz, DMSO-d6) δ −9.63 ppm.
Triethylamine (9.00 μL, 6.50 mg, 64.2 μmol) was added to a solution of N7-methyl-guanosine-5′-monophosphate 8 (19.2 mg, 32.1 μmol), imidazole (17.5 mg, 257 μmol), and 2,2′-dithiodipyridine (21.2 mg, 96.3 μmol) in N,N-dimethylformamide (382 μL), and subsequently, triphenylphosphine (25.3 mg, 96.3 μmol) was added. After stirring at room temperature for 16.5 hours, a solution of sodium perchlorate (23.6 mg, 193 μmol) in 4% triethylamine/dry acetone (9.65 mL) was added dropwise while stirring the reaction mixture. The resulting suspension was centrifuged at 4,500 rpm for 20 minutes. The supernatant was discarded and the precipitate was rinsed four times with dry acetone. The solid was dried under reduced pressure, affording N7-methyl-guanosine 5′-monophosphorimidazolide monosodium salt 3 (18.5 mg, 86.0% yield) as a pale yellow solid.
1H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 0.5H), 9.28 (s, 0.5H), 7.80 (dd, J=8.1, 1.4 Hz, 0.5H), 7.73-7.62 (m, 1H), 7.60 (dt, J =9.5, 1.1 Hz, 1.5H), 7.60-7.49 (m, 2.5H), 7.50 (ddd, J=8.5, 7.4, 1.5 Hz, 1H), 7.39 (ddd, J=8.4, 6.6, 2.1 Hz, 0.5H), 7.07 (dq, J=17.3, 1.4 Hz, 1H), 6.82 (dq, J=9.4, 1.3 Hz, 1H), 6.46 (s, 2.5H), 5.95 (d, J=5.4 Hz, 0.5H), 5.72 (d, J=4.3 Hz, 0.5H), 5.41 (d, J=5.9 Hz, 0.5H), 5.34 (d, J=4.8 Hz, 0.5H), 4.99 (s, 0.5H), 4.87-4.78 (m, 1H), 4.73-4.61 (m, 1.5H), 4.45 (d, J=7.1 Hz, 0.5H), 4.41 (t, J=4.5 Hz, 0.5H), 4.17 (q, J=5.0 Hz, 0.5H), 4.08-3.85 (m, 6.5H), 3.74-3.62 (m, 1H), 0.70 (s, 4H), 0.62 (s, 5H) ppm.
31P NMR (243 MHz, DMSO-d6) δ−9.53, −9.57 ppm.
HRESIMS calcd. for C26H32N8O10P, 647.1984 [M−H]−; found 647.2036.
The following shows a synthesis scheme of a nitrobenzyl-modified nucleotide phosphorimidazolide compound that can be removed by reduction conditions.
Triethylamine (41 μL, 70.29 mmol) was added to a solution of 3′-Nb-guanosine-5′-monophosphoric acid triethylammonium salt 9 (85 mg, 0.14 mmol), imidazole (79 mg, 1.16 mmol) and 2,2′-dithiodipyridine (96 mg, 0.47 mmol) in N,N-dimethylformamide (1.45 mL), and subsequently, triphenylphosphine (110 mg, 0.47 mmol) was added. After stirring at room temperature overnight, a solution of sodium perchlorate (110 mg, 0.87 mmol) in 4% triethylamine/dry acetone (44 mL) was added dropwise while stirring the reaction mixture. The suspension was centrifuged at 4,500 rpm for 20 minutes. The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated three more times. The solid was dried under reduced pressure, affording 3′-Nb-guanosine 5′-monophosphorphosphoimidazolide disodium salt 10 (70 mg, 0.11 mmol, yield of 79%).
1H NMR (600 MHz, DMSO-d6) δ 10.71 (s, 1H), 8.23 (d, J=9.2 Hz, 2H), 8.00 (s, 1H), 7.66 (d, J=2.0 Hz, 1H), 7.63-7.65 (m, 2H), 7.08 (s, 1H), 6.85 (s, 1H), 6.67 (s, 2H), 5.75-5.76 (m, 1H), 5.08 (dd, J=4.8, 1.0 Hz, 1H), 4.60 (dd, J=7.5, 4.8 Hz, 1H), 4.13 (t, J=4.1 Hz, 1H), 3.73-3.86 (m, 2H), 3.12 (s, 3H), 1.81 (s, 3H), 1.74 (s, 3H) ppm.
31P NMR (243 MHz, DMSO-d6) δ−9.90 ppm.
Triethylamine (58 μL, 0.41 mmol), zinc chloride (56 mg, 0.41 mmol), and a solution of tetrabutylammonium dihydrogen phosphate in CH3CN (0.1 M, 5.1 mL, 0.51 mmol) were added continuously to a solution of 3′-Nb-guanosine-monophosphrphosphoimidazolide 10 (34 mg, 51 μmol) in DMSO (0.51 mL). After stirring at room temperature for 2 days, the reaction mixture was quenched by the addition of 50 mM EDTA, and diluted with water. The mixture was washed with chloroform, and the aqueous layer was purified using DEAE Sephadex A-25, Φ=4.5 cm, h=8.4 cm (140 cm3), flow rate: 12 mL/min, solvent A: water, solvent B: 1.5 M TEAB buffer (pH 7.7, containing 10% CH3CN). The fractions containing the objective product were collected and desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=1.3 cm, h=4.0 cm, 21 cm3), solvent A: water, solvent B: methanol). The fractions containing the product were combined and concentrated, affording the target compound as triethylammonium salt 11 (20 mg, 23 μmol, 46% yield).
1H NMR (600 MHz, D2O) δ8.19 (s, 1H), 8.16 (d, J=8.8 Hz, 2H), 7.62 (d, J=8.8 Hz, 2H), 5.81 (d, J=7.5 Hz, 1H), 5.41 (dd, J=4.9, 1.5 Hz, 1H), 4.63 (dd, J=7.3, 4.9 Hz, 1H), 4.48 (s, 1H), 4.16 (d, J=3.7 Hz, 2H), 3.22 (s, 3H), 3.16 (q, J=7.4 Hz, 12H), 1.86 (s, 3H), 1.76 (s, 3H), 1.24 (t, J=7.5 Hz, 18H) ppm.
31P NMR (241 MHz, D2O) δ−10.19 (s, 1P), −10.92 (d, J=19.7 Hz, 1P) ppm.
Iodomethane (43 μL, 0.69 mmol) was added to a solution of modified guanosine diphosphoric acid triethylammonium salt 11 (60 mg, 69 μmol) in DMSO (0.58 mL). After stirring overnight at room temperature, the reaction mixture was diluted with water and washed three times with chloroform. The aqueous layer was purified using QAE Sephadex A-25, (=3.0 cm, h=10 cm (70 cm3), flow rate: 6 mL/min, solvent A: water, solvent B: 1.0 M TEAA buffer (pH 7.0, containing 10% CH3CN). The fractions containing the objective product were collected and desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=1.3 cm, h=4.0 cm, 21 cm3), solvent A: water, solvent B: methanol). The fractions containing the product were combined and concentrated, affording the target Compound 12 (30.5 mg, 39.0 μmol, 56% yield).
1H NMR (400 MHz, D2O) δ8.26 (d, J=9.0 Hz, 2H), 7.68 (d, J=9.0 Hz, 2H), 6.06 (d, J=5.4 Hz, 1H), 5.34 (dd, J=4.8, 3.3 Hz, 1H), 4.58 (d, J=3.8 Hz, 1H), 4.56 (d, J=5.2 Hz, 1H), 4.14-4.30 (m, 2H), 4.10 (s, 3H), 3.34 (s, 3H), 3.19 (q, J=7.3 Hz, 7H), 1.89 (s, 3H), 1.79 (s, 3H), 1.26 (t, J=7.3 Hz, 10H) ppm.
31P NMR (161 MHz, D2O) δ−9.96 (d, J=21.2 Hz, 1P), −10.84 (d, J=21.2 Hz, 1P) ppm.
Triethylamine (6.6 μL, 47 μmol) was added to 3′-Nb-N7-methyl-guanosine-5′-diphosphoric acid triethylammonium salt 12 (19 mg, 24 μmol), imidazole (13 mg, 0.19 mmol), and 2,2′-dithiodipyridine (16 mg, 71 μmol) in N, N-dimethylformamide (0.24 mL), and subsequently, triphenylphosphine (19 mg, 71 μmol) was added. After stirring at room temperature overnight, a solution of sodium perchlorate (17 mg, 71 μmol) in 4% triethylamine/dry acetone (7.1 mL) was added dropwise while stirring the reaction mixture. The suspension was centrifuged at 4,500 rpm for 20 minutes. The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated three more times. The solid was dried under reduced pressure, affording 3′-Nb-N7-methyl-guanosine 5′-diphosphorphosphoimidazolide disodium salt 4 (20 mg, 24 μmol, quantitative).
1H NMR (600 MHz, DMSO-d6) δ 9.78 (s, 1H), 8.24 (d, J=8.8 Hz, 2H), 7.71 (s, 1H), 7.68 (d, J=8.8 Hz, 2H), 7.17 (s, 1H), 6.85 (s, 2H), 6.80 (s, 1H), 5.97 (d, J=5.8 Hz, 1H), 5.17-5.19 (m, 1H), 4.60 (t, J=5.3 Hz, 1H), 4.34 (s, 1H), 4.02 (s, 3H), 3.77-3.92 (m, 2H), 3.28 (s, 3H), 1.83 (s, 3H), 1.76 (s, 3H) ppm.
31P NMR (243 MHz, DMSO-d6) δ−10.44 (d, J=21.2 Hz, 1P), −18.99 (d, J=21.2 Hz, 1P) ppm.
Triethylamine (10 μL, 72 μmol) was added to a solution of 3′-Nb-N7-methyl-guanosine-5′-monophosphoric acid triethylammonium salt 13 (22 mg, 36 μmol), imidazole (20 mg, 0.11 mmol), and 2,2′-dithiodipyridine (24 mg, 0.11 mmol) in N,N-dimethylformamide (0.37 mL), and subsequently, triphenylphosphine (29 mg, 0.11 mmol) was added. After stirring at room temperature overnight, a solution of sodium perchlorate (27 mg, 0.22 mmol) in 4% triethylamine/dry acetone (11 mL) was added dropwise while stirring the reaction mixture. The suspension was centrifuged at 4,500 rpm for 20 minutes. The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated three more times. The solid was dried under reduced pressure, affording 3′-Nb-N7-methyl-guanosine 5′-monophosphorphosphoimidazolide disodium salt 5 (21.4 mg, 32 μmol, 89% yield).
1H NMR (600 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.23 (d, J=8.8 Hz, 2H), 7.65 (d, J=8.5 Hz, 3H), 7.12-7.09 (1H), 6.86 (s, 1H), 6.71 (s, 2H), 5.97 (d, J=5.4 Hz, 1H), 5.02 (t, J=4.1 Hz, 1H), 4.50 (d, J=10.2 Hz, 1H), 4.29 (d, J=3.4 Hz, 1H), 4.03 (s, 3H), 3.99 (dd, J=7.1, 4.4 Hz, 1H), 3.76-3.79 (m, 1H), 3.26-3.24 (3H), 1.81 (s, 3H), 1.74 (s, 3H) ppm.
31P NMR (243 MHz, DMSO-d6) δ−9.64 (s, 1P) ppm.
The following shows the structure of a nitrobenzyl-modified trinucleotide-type cap compound.
The following shows the structure of a nitrobenzyl-modified tetranucleotide-type cap compound.
The following shows the structure of dinucleotide/trinucleotide required for the synthesis of the nitrobenzyl-modified trinucleotide/tetranucleotide-type cap compound.
The following shows the synthesis scheme of dinucleotides/trinucleotides (Compounds 14 to 26). R includes various chemical modifications.
Synthesis of dinucleotides and trinucleotides was performed using a DNA/RNA synthesizer NR-2A 7MX (Nihon Techno Service Co., Ltd., Ushiku, Ibaraki, Japan) with Primer Support 5G ribo G 300 (Cytiva, synthesis in 15.0 μmol scale). In the coupling step, 100 mM 2′-O-methyl-adenosine (n-benzoyl)-CE-phosphoramidite (ChemGenes), 100 mM 2′-O-methyl-N6-methyl-adenosine (n-benzoyl)-CE-phosphoramidite (synthesized in accordance with literature: Nucleic acid res., 2020, 48(4), 1607-1626) in CH3CN, 100 mM 2′-O-methyl-Guanosine (n-i-Bu)-CE-phosphoramidite (ChemGenes) in CH3CN, 10 mM 2′-F-adenosine (n-benzoyl)-CE-phosphoramidite (ChemGenes) in CH3CN, 100 mM LNA (MBN) adenosine (n-benzoyl)-CE-phosphoramidite (ChemGenes) in CH3CN, 100 mM 2′-formamide-adenosine (n-benzoyl)-CE-phosphoramidite in CH3CN, 100 mM 2′-OMe-2,6-diaminopurine-CE-phosphoramidite (Glen Research) in CH3CN, 50 to 70 mM 5′-DMTr-2′-TOM-ribo Adenosine (n-acetyl) OP (ChemGenes) in CH3CN, 150 mM bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite in CH3CN, and 0.3 M 5-(benzylthio)-1H-tetrazole (activator) in CH3CN were circulated through a column. A solution of 184 mM trichloroacetic acid/dichloromethane was used as a detritylation reagent, and 0.05 M 12 in pyridine/H2O (9:1, v/v), 10% Ac2O in THF/pyridine (8:1, v/v) as Cap A, and 10% 1-methylimidazole in THF as Cap B were used for oxidation. The synthesis was performed by applying a standard RNA synthesis protocol in a 10 μmol scale. After the last cycle of the synthesis, a solid support was treated with a 1:1 mixture of 28% ammonium hydroxide/40% aqueous methylamine solution at 65° C. for 1 to 2 hours (1.00 mL/15.0 μmol of solid support), whereby the nucleotide was cleaved from the solid support and the protecting group was removed. The resulting nucleotide was concentrated and dried up, and then re-dissolved in DMSO (1.00 mL/60.0 μmol). To this solution was added TEA-3HF (1.00 mL/60.0 μmol), and the mixture was incubated at 65° C. for 3 to 5 hours to remove a 2′-O-silyl protecting group. The reaction mixture was neutralized by adding a 250 mM aqueous Na2CO3 solution (10.0 mL/60.0 μmol). The crude product was purified by DEAE-Sephadex (φ=4.5 cm, h=8.4 cm, 140 cm3, 12.0 mL/min, solvent: 0 to 8 M TEAB buffer (pH 7.9, containing 0 to 8% CH3CN)). The fractions containing the product were collected and concentrated, affording di-/tri-nucleotides. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculations, the absorption coefficient (ε260)=25,000 or 18,100 M−1·cm−1 (for dinucleotides), and 35,100, 31,700 or 28,200 M−1·cm−1 (for trinucleotides) were used.
The following shows a synthesis scheme of a trinucleotide-type cap compound (Compounds 1 to 8). R1 and R2 include various chemical modifications.
The following shows a synthesis scheme of a tetranucleotide-type cap compound (Compounds 9 to 13). R1 and R2 include various chemical modifications.
2′-Nb-N7-methylguanosine diphosphorimidazolide disodium salt (2.00 equiv.) and 2-nitroimidazole (3.00 equiv.) were added to a solution of di-/trinucleotide triethylammonium salt (17.2 to 43.6 μmol) in DMSO (79.5 mM). After incubation at 37° C. for 1 to 2 days, 2′-Nb-N7-methylguanosine diphosphorimidazolide disodium salt (2.00 equiv.) and 2-nitroimidazole (3.00 equiv.) were added. The mixture was incubated at 37° C. for 2 to 3 days, and the reaction mixture was diluted with 18.5 to 38.6-fold amount of water. The crude product was purified by ion exchange chromatography on QAE-Sephadex (φ=3.0 cm, h=10 cm, 70 cm3, 6 mL/min). The fractions containing the product were collected and concentrated. The product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min)). The fractions containing the product were concentrated, affording the objective tri/tetranucleotide cap analog in the form of a triethylammonium salt. The product was re-dissolved in methanol (2.0 mL). A 190 mM NaClO4 (12 mL) solution in acetone was added to the mixture, and the resulting suspension was centrifuged (4,500 rpm, 20 min). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated three or four more times. The precipitate was dried under reduced pressure, affording the target tri-/tetranucleotide cap analog as a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculations, the absorption coefficient (ε260)=40,100 or 33,200 M−1·cm−1 (for trinucleotide cap analogs) and 50,200, 46,800 or 43,300 M−1·cm−1 (for tetranucleotide cap analogs) were used.
(5) Synthesis of Trinucleotide-Tetranucleotide-Type Cap compound (Method B)
2′-Nb-N7-methylguanosine diphosphorimidazolide disodium salt (3.00 equiv.) and zinc chloride (20.0 equiv.) were added to a solution of di-/trinucleotide triethylammonium salt (up to 106 μmol) in DMSO (50 mM). After stirring at 37° C. for 3 days, a 500 mM aqueous EDTA solution (pH 8.0, EDTA: 1.30 equiv. per molar amount of ZnCl2) was added to quench the reaction mixture. The mixture was diluted with 5.24 volumes of water, and purified by reverse-phase HPLC. The fractions containing the target compound were collected, concentrated, and freeze-dried, affording the target tri/tetranucleotide cap analog in the form of a triethylammonium salt. The product was re-dissolved in methanol (2.00 mL). A 190 mM NaClO4 (24.0 mL) solution in acetone was added to the mixture, and the resulting suspension was centrifuged (4,000 rpm, 20 min). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated three or four more times. The precipitate was dried under reduced pressure, affording the target tri-/tetranucleotide cap analog as a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculations, the absorption coefficient (ε260)=40,100 or 33,200 M1·cm−1 (for trinucleotide cap analogs) and 50,200, 46,800 or 43,300 M−1·cm−1 (for tetranucleotide cap analogs) were used.
(6) Synthesis of Trinucleotide Type Cap Compound Nb-m7GpppApG (Method A)
2′-Nitrobenzyl-modified N7-methylguanosine diphosphorimidazolide disodium salt (40.6 mg, 52.6 μmol) and 2-nitroimidazole (8.92 mg, 78.9 μmol) were added to a solution of dinucleotide triethylamnmonium salt 14 (26.2 mg, 26.3 μmol) in DMSO (331 μL). After incubation at 37° C. for 1 day, 2′-nitrobenzyl-modified N7-methylguanosine diphosphorimidazolide disodium salt (40.6 mg, 52.6 μmol) and 2-nitroimidazole (8.92 mg, 78.9 μmol) were added. The mixture was incubated at 37° C. for 3 days and the reaction mixture was diluted with water (10.0 mL). The crude product was purified by ion exchange chromatography on QAE-Sephadex (φ=3.0 cm, h=10 cm, 70 cm3, 6 mL/min). The fractions containing the product were collected and concentrated. The product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min). The fractions containing the product were concentrated, affording the target compound as a triethylammonium salt. The product was re-dissolved in methanol (2.0 mL). To the mixture was added 190 mM NaClO4 (12 mL) in acetone, and the resulting suspension was centrifuged (4,500 rpm, 20 minutes). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording the target Cap analog 1 (23.6 mg, 16.4 μmol, 62.3% yield) as a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=40,100 M−1·cm−1 was used.
1H NMR (600 MHz, D2O) δ 8.36 (s, 1H), 8.14-7.85 (m, 2H), 7.62-7.36 (m, 4H), 7.22 (s, 1H), 5.98-5.87 (m, 1.5H), 5.77 (s, 1H), 5.71 (s, 0.5H), 5.04-4.83 (m, 3H), 4.71 (s, 3H), 4.57 (s, 1H), 4.49 (d, J=14.2 Hz, 3H), 4.39-4.21 (m, 10H), 4.17 (s, 1H), 4.10 (t, J=7.0 Hz, 1H), 4.05-3.96 (m, 3H), 0.49 (s, 9H) ppm.
13C NMR (151 MHz, D2O) δ 158.30, 155.43, 155.24, 154.54, 154.35, 153.63, 151.85, 151.26, 149.67, 149.25, 149.06, 148.85, 148.72, 148.50, 139.66, 137.26, 133.59, 132.70, 132.51, 132.21, 129.97, 129.80, 128.71, 128.09, 123.75, 123.34, 118.19, 116.01, 107.57, 107.29, 94.61, 93.70, 87.76, 87.40, 87.10, 86.99, 85.08, 84.43, 83.41, 82.79, 81.24, 80.16, 79.56, 79.10, 74.53, 73.82, 70.19, 68.48, 68.13, 65.21, 64.98, 64.71, 64.37, 61.76, 48.96, 36.24, 36.20, 36.15, 35.68, 30.31, 25.10, 24.95, 24.84, 20.58, 13.32, 8.30 ppm.
31P NMR (243 MHz, D2O) δ0.07 (1P), −10.70 (2P), −21.98 (1P) ppm.
(7) Synthesis of Trinucleotide Type Cap Compound Nb-m7GpppA(2′OCH3)pG (Method A)
2′-O-methyl-modified dinucleotide triethylamnmonium salt 15 (17.4 mg, 17.2 μmol) in DMSO (216 μL). After incubating at 37° C. for 2 days, the reaction mixture was diluted with water (4.00 mL). The crude product was purified by ion exchange chromatography on DEAE-Sephadex (φ=4.5 cm, h=8.4 cm, 140 cm3, 12 mL/min). % CH3CN (270 mi). The fractions containing the product were collected and concentrated. After removal of CH3CN under reduced pressure, the product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min)). The fractions containing the product were concentrated, affording the objective compound as a triethylamnmonium salt. The product was re-dissolved in methanol (2.0 mL). To the mixture was added 190 mM NaClO4 (12 mL) in acetone, and the resulting suspension was centrifuged (4,500 rpm, 20 minutes). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording the target Cap analog 2 (8.02 mg, 5.51 μmol, 32.0% yield) as a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=40,100 M1·cm−1 was used.
1H NMR (600 MHz, D2O) δ8.51-8.28 (m, 1H), 8.17-7.88 (m, 2H), 7.63 (s, 0.5H), 7.54 (d, J=7.1 Hz, 1H), 7.44 (s, 1.5H), 7.37 (s, 0.5H), 7.28 (s, 0.5H), 5.98 (dd, J=17.3, 6.4 Hz, 1H), 5.88 (s, 1H), 5.79 (d, J=6.6 Hz, 1H), 5.70 (s, 1H), 5.00-4.87 (m, 3H), 4.72 (s, 1H), 4.61-4.55 (m, 0.5H), 4.49 (s, 2.5H), 4.42-4.14 (m, 10H), 4.11 (dt, J=15.6, 7.5 Hz, 1H), 4.08-3.94 (m, 3H), 3.39 (d, J=10.7 Hz, 3H), 0.72-0.46 (2s, 9H) ppm.
13C NMR (151 MHz, D2O) δ158.52, 155.75, 155.19, 154.97, 153.66, 152.74, 149.69, 149.27, 148.83, 148.62, 139.49, 133.63, 132.61, 132.25, 129.99, 129.86, 128.77, 128.08, 123.78, 123.35, 118.27, 116.26, 107.62, 107.30, 94.76, 93.54, 87.62, 87.37, 85.31, 85.03, 84.92, 84.67, 83.59, 83.53, 83.13, 82.16, 82.03, 81.66, 80.38, 79.49, 79.16, 73.53, 72.84, 70.32, 68.60, 68.24, 65.11, 64.84, 64.46, 61.77, 57.97, 36.31, 36.15, 36.12, 35.67, 35.54, 30.29, 24.87, 24.82, 24.74, 24.66, 20.56, 13.29 ppm.
31P NMR (243 MHz, D2O) δ−0.23 (1P), −10.81 (2P), −22.01 (1P) ppm.
(8) Synthesis of Trinucleotide Type Cap Compound Nb-m7Gpppm6A(2′OCH3)pG (Method A)
N7-methylguanosine diphosphorimidazolide disodium salt (67.4 mg, 87.2 μmol) and 2-nitroimidazole (14.8 mg, 131 μmol) were added to a solution of dinucleotide triethylammonium salt 16 (43.6 μmol) in DMSO (548 μL). After incubation at 37° C. for 1.5 days, N7-methylguanosine diphosphorimidazolide disodium salt (67.4 mg, 87.2 μmol) and 2-nitroimidazole (14.8 mg, 131 μmol) were added. The mixture was incubated at 37° C. for 3 days and the reaction mixture was diluted with water (14.0 mL). The crude product was purified by ion exchange chromatography on QAE-Sephadex (φ=3.0 cm, h=10 cm, 70 cm3, 6 mL/min). The fractions containing the product were collected and concentrated. The product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min)). The fractions containing the product were concentrated, affording the target compound as a triethylammonium salt. The product was re-dissolved in methanol (2.0 mL). To the mixture was added 190 mM NaClO4 (12 mL) in acetone, and the resulting suspension was centrifuged (4,500 rpm, 20 minutes). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording the target Cap analog 3 (37.9 mg, 25.8 μmol, 59.2% yield) as a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=40,100 M−1·cm−1 was used.
1H NMR (600 MHz, D2O) δ9.14 (s, 0.6H), 8.94 (s, 0.3H), 8.35 (d, J=17.1 Hz, 1H), 8.17-7.85 (m, 2H), 7.64-7.35 (m, 4H), 6.09-5.68 (m, 3H), 4.95 (s, 3H), 4.82 (s, 3H), 4.76 (s, 1H), 4.68 (s, 1H), 4.61-3.96 (m, 16H), 3.41 (d, J=8.6 Hz, 2H), 3.05-2.91 (m, 1H), 0.59-0.38 (m, 9H) ppm. 13CNMR (151 MHz, D2O) δ 158.30, 155.42, 155.22, 154.51, 154.39, 154.30, 153.64, 151.31, 149.89, 149.70, 149.26, 148.88, 148.80, 147.14, 139.20, 137.29, 136.32, 133.57, 132.75, 132.54, 132.25, 129.98, 128.72, 128.16, 123.75, 123.38, 118.63, 116.03, 107.89, 107.57, 107.28, 94.62, 93.76, 87.74, 87.49, 85.08, 84.44, 83.52, 83.09, 82.12, 81.26, 80.26, 79.62, 79.21, 73.71, 72.73, 70.28, 68.45, 68.13, 65.09, 64.66, 64.27, 61.76, 57.99, 57.49, 36.24, 36.20, 35.79, 35.70, 25.24, 25.14, 24.96, 24.86, 20.57, 16.85, 13.31 ppm.
31P NMR (243 MHz, D2O) δ−0.26 (1P), −10.84 (2P), −21.96 (1P) ppm.
ESI-TOF-MS calcd for C45H57N16O27P4, 459.0849 [M-3H]3−; found 459.0837.
(9) Synthesis of Trinucleotide Type Cap Compound Nb-m7GpppD(2′OCH3)pG (Method A)
N7-methylguanosine diphosphorimidazolide disodium salt (11.7 mg, 15.1 μmol) and 2-nitroimidazole (2.57 mg, 22.7 μmol) were added to a solution of dinucleotide triethylammonium salt 18 (7.74 mg, 7.55 μmol) in DMSO (95.0 μL). After incubation at 37° C. for 4 days, N7-methylguanosine diphosphorimidazolide disodium salt (11.7 mg, 15.1 nmol) and 2-nitroimidazole (2.57 mg, 22.7 μmol) were added. The mixture was incubated at 37° C. for 2.5 days and the reaction mixture was diluted with water (14.0 mL). The crude product was purified by ion exchange chromatography on QAE-Sephadex (φ=3.0 cm, h=10 cm, 70 cm3, 6 mL/min). The fractions containing the product were collected and concentrated. The product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min)). The fractions containing the target compound were collected and freeze-dried, affording the target compound as a triethylamnmonium salt. The product was re-dissolved in methanol (1.0 mL). To the mixture was added 190 mM NaClO4 (12 mL) in acetone, and the resulting suspension was centrifuged (4,000 rpm, 20 minutes). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording the target Cap analog 4 as a sodium salt.
(10) Synthesis of Trinucleotide Type Cap Compound Nb-m7GpppA (2′F)pG (Method B)
2′-Nb-Guanosine 5′-diphosphorphosphoimidazolide (46.4 mg, 60.0 μmol) and zinc chloride (54.5 mg, 400 μmol) were added to a solution of 5′-p-A(2′F)PG dinucleotide 19 (20.0 mg, 20.0 μmol, crude) in DMSO (400 μL). After stirring at 37° C. for 1.5 days, a 500 mM aqueous EDTA solution (pH 8.0, 1.04 mL, EDTA: 520 μmol) was added to quench the reaction mixture. The mixture was diluted with water (13.0 mL) and purified by reverse-phase HPLC (instrument: Shimadzu Prep, column: YMC-Actus Triart C8 (Preparative, 250×20.0 mm I.D., solvent A: 50 mM TEAA buffer (pH 6.0, 0.5% CH3CN), solvent B: CH3CN, 5 to 80% B linear gradient (25 min), flow rate: 10 ml/min, detection: 254 nm). The fractions containing the objective product were collected, concentrated, and freeze-dried, affording Pure Cap analog 5 as a triethylamnmonium salt (1.58 mg, 0.900 μmol, 4.50% yield, ε260=40,100 M−1·cm−1).
1H NMR (600 MHz, D2O) δ8.15 (s, 1H), 7.92 (s, 1H), 7.73 (s, 1H), 7.54-7.40 (m, 4H), 7.25 (t, J=7.6 Hz, 1H), 5.98 (d, J=16.3 Hz, 1H), 5.69-5.66 (m, 1H), 5.63 (d, J=5.8 Hz, 1H), 5.39 (s, 0.5H), 5.30 (s, 0.5H), 5.02 (s, 1H), 4.94 (d, J=7.6 Hz, 1H), 4.90 (d, J=7.6 Hz, 1H), 4.69 (t, J=5.8 Hz, 1H), 4.61 (s, 1H), 4.46 (d, J=12.7 Hz, 2H), 4.39 (s, 1H), 4.34-4.25 (in, 4.5H), 4.21 (d, J=10.5 Hz, 3.5H), 4.11 (d, J=10.5 Hz, 1H), 4.01 (s, 3H), 3.19 (qd, J=7.3, 1.2 Hz, 52H, triethylamnmonium), 1.89 (t, J=1.2 Hz, 12H, acetate), 1.26 (tt, J=7.3, 1.1 Hz, 80H, triethylamnmonium), 0.61 (s, 9H) ppm.
31P NMR (243 MHz, D2O) δ−0.63 (1P), −10.98 (2P), −22.33 (1P) ppm.
(11) Synthesis of Trinucleotide Type Cap Compound Nb-m7GpppA(NHCHO) pG (Method A)
N7-methylguanosine diphosphorimidazolide disodium salt (13.6 mg, 17.6 μmol) and 2-nitroimidazole (3.00 mg, 26.5 μmol) were added to a solution of dinucleotide triethylamnmonium salt 20 (8.82 μmol) in DMSO (111 μL). After 1.5 days of incubation at 37° C., N7-methylguanosine diphosphorimidazolide disodium salt (13.6 mg, 17.6 μmol) and 2-nitroimidazole (3.00 mg, 26.5 μmol) were added. The mixture was incubated at 37° C. for 2.5 days and the reaction mixture was diluted with water (10.0 mL). The crude product was purified by ion exchange chromatography on QAE-Sephadex (φ=3.0 cm, h=10 cm, 70 cm3, 6 mL/min). The fractions containing the product were collected and concentrated. The product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min)). The fractions containing the product were collected, and purified by reverse-phase HPLC using a YMC-Triart C8 column (250×4.6 mm I.D., S-5 μm, 12 nm, flow rate: 1 mL/min, temperature: 50° C.). The fractions containing the target compound were collected and freeze-dried, affording the target compound as a triethylammonium salt. The product was re-dissolved in methanol (1.0 mL). To the mixture was added 190 mM NaClO4 (12 mL) in acetone, and the resulting suspension was centrifuged (4,000 rpm, 20 minutes). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording the target Cap analog 6 (2.42 mg, 1.65 μmol, 18.7% yield) as a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=40,100 M1·cm−1 was used.
1H NMR (600 MHz, D2O) δ8.25 (s, 0.5H), 8.20 (s, 0.5H), 7.95 (s, 0.5H), 7.90 (s, 0.5H), 7.85 (s, 1H), 7.71 (d, J=5.6 Hz, 1H), 7.51 (s, 0.5H), 7.44 (s, 0.5H), 7.36 (s, 1H), 7.33-7.30 (m, 1H), 7.26 (s, 0.5H), 7.11 (s, 0.5H), 5.73 (s, 3.5H), 5.53 (s, 0.5H), 4.89 (s, 1.5H), 4.82 (s, 1.5H), 4.77 (s, 3H), 4.46 (s, 1H), 4.40 (s, 1H), 4.34 (s, 0.5H), 4.28 (s, 0.5H), 4.19 (s, 2.6H), 4.12 (s, 2.4H), 4.09-4.06 (m, 3H), 3.91 (d, J=5.1 Hz, 1.5H), 3.86 (d, J=5.1 Hz, 1.5H), 0.47 (s, 5H), 0.39 (s, 4H) ppm.
31P NMR (243 MHz, D2O) δ−0.30 (1P), −10.81 (2P), −21.96 (1P) ppm.
13C NMR (151 MHz, D2O) δ163.92, 160.09, 158.75, 155.35, 153.79, 152.81, 151.62, 149.63, 148.84, 148.17, 139.75, 137.70, 133.82, 132.53, 132.02, 129.91, 128.72, 127.74, 123.70, 123.42, 118.15, 116.39, 108.30, 107.99, 95.24, 93.61, 87.41, 86.98, 86.80, 85.06, 84.04, 83.36, 82.28, 80.68, 79.98, 79.23, 73.32, 70.25, 69.08, 68.71, 65.63, 65.42, 65.22, 54.24, 36.43, 35.93, 35.61, 30.29, 24.67, 24.61, 23.32 ppm.
(12) Synthesis of Trinucleotide Type Cap Compound Nb-m7GpppA(2′OCH3)psG (Method B)
2′-Nb-guanosine 5′-diphosphorimidazolide (34.1 mg, 44.1 μmol) and zinc chloride (40.1 mg, 294 μmol) were added to a solution of 5′-p-A(2′OMe)PSG dinucleotide 17 (14.7 μmol) in DMSO (294 μL). After stirring at 37° C. for 1.5 days, a 500 mM aqueous EDTA solution (pH 8.0, 764 μL, EDTA: 382 μmol) was added to quench the reaction mixture. The mixture was diluted with water (14.0 mL), and purified by reverse-phase HPLC (instrument: Shimadzu Prep., column: YMC-Actus Triart C8 (Preparative, 250×20.0 mm I.D.)). The fractions containing the objective product were collected, concentrated, and freeze-dried, affording Cap analog 7 (3.18 mg, 1.78 μmol, 12.1% yield) as a triethylammonium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=40,100 M−1·cm−1 was used.
1H NMR (600 MHz, D2O) δ 8.24 (s, 1H), 8.01-7.85 (m, 2H), 7.43 (d, J=7.8 Hz, 1H), 7.35 (s, 2H), 7.16 (s, 1H), 5.84 (d, J=12.2 Hz, 1H), 5.73-5.65 (m, 1H), 5.58 (s, 1H), 4.92 (d, J=9.4 Hz, 1H), 4.89 (s, 1H), 4.83 (d, J=7.1 Hz, 1H), 4.76 (d, J=7.6 Hz, 1H), 4.58 (s, 1H), 4.49-4.33 (m, 3H), 4.32-4.25 (m, 1H), 4.24-4.16 (m, 4H), 4.15-4.05 (m, 5H), 3.88 (d, J=8.8 Hz, 3H), 3.27 (d, J=15.4 Hz, 3H), 3.10-3.02 (m, 33H, triethylammonium), 1.80-1.74 (m, 7H, CH3COO−), 1.18-1.09 (m, 51H, triethylammonium), 0.54-0.47 (m, 9H) ppm.
31P NMR (243 MHz, D2O) δ 56.60 (1P, d, J=131.5 Hz), −10.81 (2P), −21.86 (1P) ppm.
(13) Synthesis of Trinucleotide Type Cap Compound Nb-m7GpppA(LNA)pG (Method B)
2′-Nb-Guanosine 5′-diphosphorphosphoimidazolide (60.7 mg, 78.6 μmol) and zinc chloride (71.4 mg, 524 μmol) were added to a solution of 5′-p-A(LNA)PG dinucleotide 21 (26.4 mg, 26.2 μmol) in DMSO (524 μL). After stirring at 37° C. for 2 days, a 500 mM aqueous EDTA solution (pH 8.0, 1.36 mL, EDTA: 681 μmol) was added to quench the reaction mixture. The mixture was diluted with water (13.0 mL), and purified by reverse-phase HPLC (instrument: Shimadzu Prep., column: YMC-Actus Triart C8 (Preparative, 250×20.0 mm I.D.)). The fractions containing the objective product were collected, concentrated, and freeze-dried, affording Pure Cap analog 8 as a triethylammonium salt (2.44 mg, 1.38 μmol, 5.27% yield, ε260=40,100 M−1·cm−1).
1H NMR (600 MHz, D2O) δ8.78 (s, 1H), 7.97-7.90 (m, 1H), 7.84 (d, J=4.6 Hz, 1H), 7.63 (s, 1H), 7.48 (dd, J=8.3, 4.0 Hz, 1H), 7.41 (s, 2H), 7.22 (s, 1H), 5.60-5.54 (m, 2H), 5.52 (d, J=3.9 Hz, 1H), 4.90 (d, J=4.4 Hz, 1H), 4.83 (d, J=4.0 Hz, 1.5H), 4.77 (dd, J=7.7, 3.8 Hz, 1.5H), 4.74 (d, J=4.6 Hz, 1.5H), 4.55 (s, 0.5H), 4.48 (d, J=3.6 Hz, 0.5H), 4.38 (s, 1H), 4.35 (s, 1H), 4.28 (d, J=4.0 Hz, 2.5H), 4.20 (t, J=4.9 Hz, 2H), 4.17-4.08 (m, 4H), 4.00-3.91 (m, 5H), 3.06 (dt, J=11.5, 7.4 Hz, 15H), 1.14 (td, J=7.4, 3.9 Hz, 23H), 0.53 (d, J=4.2 Hz, 9H) ppm. 31PNMR (243 MHz, D2O) δ−1.37 (1P), −11.02 (2P), −22.30 (1P) ppm.
(14) Synthesis of Tetranucleotide-Type Cap Compound Nb-m7GpppA(2′OCH3)pG(2′OCH3)pG (Method A)
2′-Nitrobenzyl-modified N7-methylguanosine diphosphorimidazolide disodium salt (31.8 mg, 41.2 μmol) and 2-nitroimidazole (6.99 mg, 61.8 μmol) were added to a solution of 2′-O-methyl-modi ied dinucleotide triethylamnmonium salt 22 (30.3 mg, 20.6 μmol) in DMSO (259 μL). After incubation at 37° C. for 1 day, 2′-nitrobenzyl-modified N7-methylguanosine diphosphorimidazolide disodium salt (31.8 mg, 41.2 μmol) and 2-nitroimidazole (6.99 mg, 61.8 μmol) were added. The mixture was incubated at 37° C. for 3 days and the reaction mixture was diluted with water (10.0 mL). The crude product was purified by ion exchange chromatography on QAE-Sephadex (φ=3.0 cm, h=10 cm, 70 cm3, 6 mL/min). The product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min). The fractions containing the product were concentrated, affording the target compound as a triethylammonium salt. The product was re-dissolved in methanol (2.0 mL). To the mixture was added 190 mM NaClO4 (12 mL) in acetone, and the resulting suspension was centrifuged (4,500 rpm, 20 minutes). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording the target Cap analog 9 (16.0 mg, 8.70 μmol, 42.2% yield) as a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=50,200 M1·cm−1 was used.
1H NMR (600 MHz, D2O) δ 8.42-8.34 (m, 1H), 8.06 (d, J=12.4 Hz, 1.5H), 7.97 (s, 1H), 7.84 (s, 1H), 7.67 (s, 0.5H), 7.57 (d, J=7.6 Hz, 1H), 7.51-7.36 (m, 2H), 7.29 (s, 1H), 6.88 (s, 1H), 5.94 (dd, J=11.6, 5.1 Hz, 2H), 5.88-5.80 (m, 1.5H), 5.73 (s, 1H), 5.68 (d, J=4.3 Hz, 0.5H), 4.89 (s, 4H), 4.58 (s, 2.5H), 4.52 (s, 0.5H), 4.46 (s, 3H), 4.38 (s, 3H), 4.34-4.24 (m, 8H), 4.21-4.08 (m, 11H), 4.05 (s, 1.5H), 4.00 (s, 1.5H), 3.83-3.47 (m, 6H), 0.64-0.46 (m, 9H) ppm.
13C NMR (151 MHz, D2O) δ159.70-158.2 (m), 153.71-150.8 (m), 149.70-148.00 (m), 132.07, 129.36, 128.17, 123.14, 117.26, 114.91, 107.66, 87.66, 84.99, 82.62, 80.56, 79.37, 75.68, 72.44, 68.13, 65.01, 61.77, 58.27, 57.91, 56.82, 48.94, 46.69, 36.13, 35.36, 30.30, 28.25, 24.76, 20.57, 16.84, 13.30, 8.32 ppm.
31P NMR (243 MHz, D2O) δ−0.26 (2P), −10.84 (2P), −22.09 (1P) ppm.
(15) Synthesis of Tetranucleotide-Type Cap Compound Nb-m7Gpppm6A(2′OCH3)pG(2′OCH3)pG (Method B)
Guanosine 5′-diphosphorimidazolide (246 mg, 0.318 mol) and zinc chloride (289 mg, 2.12 mmol) were added to a solution of 5′-p-m6A(2′OMe)PG(2′OMe)PG trinucleotide 23 (158 mg, 0.106 mmol) in DMSO (2.12 mL). After stirring at 37° C. for 3 days, a 500 mM aqueous EDTA solution (pH 8.0, 5.52 mL, EDTA: 2.76 mmol) was added to quench the reaction mixture. The mixture was diluted with water (40.0 mL), and purified by reverse-phase HPLC (instrument: Shimadzu Prep., column: YMC-Actus Triart C8 (Preparative, 250×20.0 mm I.D.)). The fractions containing the target product were collected, concentrated, and freeze-dried, affording Pure Cap analog 10 as a triethylammonium salt. The product was re-dissolved in methanol (2.00 mL). A 190 mM NaClO4 (24.0 mL) solution in acetone was added to the mixture, and the resulting suspension was centrifuged (4,000 rpm, 20 min). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording Cap analog 10 (70.7 mg, 38.2 μmol, 36.0% yield) as a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=50,200 M−1·cm−1 was used.
31P NMR (243 MHz, D2O) δ −0.38, −10.60, −21.60 ppm.
(16) Synthesis of Tetranucleotide-Type Cap Compound Nb-m7GpppG(2′OCH3)pG(2′OCH3)pG (Method B)
2′-Nb-Guanosine 5′-diphosphorphosphoimidazolide (35.0 mg, 45.5 μmol) and zinc chloride (41.0 mg, 300 μmol) were added to a solution of 5′-p-G(2′OMe)PG(2′OMe)PG trinucleotide 24 (21.0 mg, 15.0 μmol) in DMSO (300 μL). After stirring at 37° C. for 2 days, a 38 mM aqueous EDTA-NaOH solution (pH 8.0, 10.3 mL, EDTA: 391 μmol) was added to quench the reaction mixture. The mixture was purified by ion exchange chromatography using QAE-Sephadex (φ=3.0 cm, h=10 cm, 70 cm3, 6 mL/min). The fractions containing the product were collected and concentrated. The product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min). The fractions containing the product were concentrated, affording the target compound as a triethylamnmonium salt. The product was re-dissolved in methanol (2.0 mL). To the mixture was added 190 mM NaClO4 (12 mL) in acetone, and the resulting suspension was centrifuged (4,500 rpm, 20 minutes). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording the target Cap analog 11 (1.87 μmol, 12.5% yield) as a sodium salt. The yield was calculated using the absorbance of the product at 260 nm measured by NanoDrop. For the calculation, an absorption coefficient (ε260)=46,800 M−1·cm−1 was used.
1H NMR (600 MHz, D2O) δ 7.95-7.81 (m, 3H), 7.55 (d, J=6.8 Hz, 0.5H), 7.47 (t, J=7.0 Hz, 0.5H), 7.43-7.27 (m, 2.5H), 7.14 (t, J=7.4 Hz, 0.5H), 5.84-5.73 (m, 2.5H), 5.66 (dd, J=13.9, 6.5 Hz, 1H), 5.58 (d, J=5.8 Hz, 0.5H), 4.94 (s, 0.5H), 4.86 (s, 1.5H), 4.61 (s, 1H), 4.55 (s, 1H), 4.42-4.26 (m, 5.5H), 4.25-4.02 (m, 12H), 3.99 (t, J=1.6 Hz, 1H), 3.94-3.92 (m, 1.5H), 3.26 (dt, J=13.7, 1.7 Hz, 3H), 3.20 (dt, J=9.0, 1.7 Hz, 3H), 2.99 (q, J=7.4 Hz, triethylammonium), 1.12 (t, J=7.3 Hz, triethylammonium), 0.52-0.49 (m, 4.5H), 0.42-0.40 (m, 4.5H) ppm.
31P NMR (243 MHz, D2O, triethylammonium form) δ −0.22 (d, J=32.9 Hz), −11.00 (d, J=16.4 Hz), −22.43 (t, J=18.1 Hz) ppm.
(17) Synthesis of Tetranucleotide-Type Cap Compound Nb-m7GpppD(2′OCH3)pG(2′OCH3)pG (Method A)
N7-methylguanosine diphosphorimidazolide disodium salt (12.1 mg, 15.7 μmol) and 2-nitroimidazole (2.67 mg, 23.6 μmol) were added to a solution of trinucleotide triethylamnmonium salt (11.7 mg, 7.85 μmol) in DMSO (98.7 μL). After incubation at 37° C. for 4 days, N7-methylguanosine diphosphorimidazolide disodium salt (12.1 mg, 15.7 μmol) and 2-nitroimidazole (2.67 mg, 23.6 μmol) were added. The mixture was incubated at 37° C. for 2.5 days and the reaction mixture was diluted with water (14.0 mL). The crude product was purified by ion exchange chromatography on QAE-Sephadex (φ=3.0 cm, h=10 cm, 70 cm3, 6 mL/min). The fractions containing the product were collected and concentrated. The product was desalted with Wakosil (registered trademark) 25C18 (particle size: 15 to 30 μm (spherical), column size: φ=4.80 cm, h=10.2 cm, 184 cm3 (12 mL/min). The fractions containing the target compound were collected and freeze-dried, affording the target compound as a triethylammonium salt. The product was re-dissolved in methanol (1.0 mL). To the mixture was added 190 mM NaClO4 (12 mL) in acetone, and the resulting suspension was centrifuged (4,000 rpm, 20 minutes). The supernatant was discarded and the precipitate was resuspended in acetone. The suspension centrifugation process was repeated four more times. The precipitate was dried under reduced pressure, affording the target Cap analog 12 as a sodium salt.
(18) Synthesis of Tetranucleotide-Type Cap Compound Nb-m7GpppA(2′OCH3)psG(2′OCH3)psG (Method B)
2′-Nb-Guanosine 5′-diphosphorphosphoimidazolide (46.6 mg, 60.3 μmol) and zinc chloride (54.8 mg, 402 μmol) were added to a solution of 5′-p-A(2′OMe)PSG(2′OMe)PSG trinucleotide 26 (30.1 mg, 20.1 μmol) in DMSO (402 μL). After stirring at 37° C. for 2 days, a 500 mM aqueous EDTA solution (pH 8.0, 1.05 mL, EDTA: 523 μmol) was added to quench the reaction mixture. The mixture was diluted with water (13.0 mL), and purified by reverse-phase HPLC (instrument: Shimadzu Prep., column: YMC-Actus Triart C8 (Preparative, 250×20.0 mm I.D.)). The fractions containing the objective product were collected, concentrated, and freeze-dried, affording Cap analog 13 as a triethylamnmonium salt (3.31 mg, 1.46 μmol, 7.26% yield, ε260=50,200 M−1·cm−1).
1H NMR (600 MHz, D2O) δ 8.97 (d, J=7.5 Hz, 1H), 8.51-8.40 (m, 1H), 8.09 (d, J=2.7 Hz, 1H), 8.06-8.02 (m, 1H), 7.99-7.94 (m, 1H), 7.59 (q, J=7.0 Hz, 1H), 7.54-7.43 (m, 2H), 7.30 (t, J=6.9 Hz, 1H), 6.06-5.94 (m, 1H), 5.88-5.67 (m, 3H), 5.20-5.04 (m, 2H), 5.01 (t, J=5.7 Hz, 1H), 4.94 (d, J=7.1 Hz, 1H), 4.89-4.83 (m, 1.5H), 4.75-4.72 (m, 1H), 4.63-4.55 (m, 3H), 4.53-4.40 (m, 4.5H), 4.35-4.12 (m, 11H), 4.03 (dd, J=6.4, 3.4 Hz, 3H), 3.42-3.34 (m, 5H), 3.32 (d, J=5.5 Hz, 1H), 3.18 (qd, J=7.3, 2.1 Hz, 23H), 1.26 (td, J=7.3, 2.3 Hz, 36H), 0.66-0.52 (m, 9H) ppm.
31P NMR (243 MHz, D2O) δ57.92-55.14 (2P), −10.98 (2P), −22.24 (1P) ppm.
15. mRNA Synthesis by In Vitro Transcription Method Using Synthesized Novel Cap Analog and Evaluation of its Translation Activity
SEQ ID NO: 7 is a DNA sequence of a transcription synthesis template of Luc2 mRNA. SEQ ID NO: 8 is a sequence of Luc2 mRNA transcribed using the template DNA of SEQ ID NO: 7.
An RNA was transcriptionally synthesized with T7 RNA polymerase using a double-stranded DNA as a template. The double-stranded DNA of the template was obtained by amplifying pSP73-a-luc2 (described at the end of the experimental section) encoding the Luc2 gene by a PCR reaction using KOD-Plus-Neo (TOYOBO), Fw primer (cgcgttggccgattcatt: SEQ ID NO: 3), Rev primer (tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt ttttttttttttttttcagttagacgttgatcctgg: SEQ ID NO: 4) (1.0 ng/μL DNA vector, 1×KOD-Plus-Neo buffer, 0.2 mM dNTPs, 0.3 μM primers, 1.5 mM MgSO4, 0.02 units/μL polymerase).
A transcription reaction liquid (template DNA 15 ng/μL, 1×T7 RNA Polymerase buffer (Takara) (40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine), 10 U/μL T7 RNA polymerase (Takara), 5 mM DTT, 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 2 mM Nb_m7GpppApG or Nb-m7GpppA(2′OCH3)pG or Nb-m7GpppA(2′OCH3)pG(2′OCH3)pG or Nb-m7Gpppm6A(2′OCH3)pG(2′OCH3)pG) was prepared and incubated at 37° C. for 2 hours. Recombinant DNase (Takara) was added to the reaction liquid to 0.1 U/μL, and the mixture was incubated at 37° C. for 30 minutes to degrade the template DNA. After the reaction, phenol/chloroform extraction was carried out to perform alcohol precipitation. The product was purified by reverse-phase HPLC.
Column: YMC-Triart Bio C4 (250 mm×4.6 mm I.D.); Solvent A: 50 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; Solvent B: acetonitrile; Linear Gradient: 5 to 15% Solvent B (0 to 20 min); Flow rate: 1 mL/min; Detection wavelength: 260 nm; Column temperature: 50° C.
Deprotection by photoirradiation: The RNA solution was added to a transparent 96-multiwell plate, and irradiated with 365 nm light at a quantity of light of 4 mW/cm2 for 15 minutes using a MAX-305 light source device (Asahi Spectra Co., Ltd.). HPLC purification was performed. Column: YMC-Triart Bio C4 (250 mm×4.6 mm I.D.); Solvent A: 50 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; Solvent B: acetonitrile; Linear gradient: 5 to 15% Solvent B (0 to 20 min); Flow rate: 1 mL/min; Detection wavelength: 260 nm; Column temperature: 50° C.
The sequence information of pSP73-a-luc2 is as shown in SEQ ID NO: 5.
(2) Evaluation of Translation Activity of Luc2-Expressing mRNA
HeLa cells (RIKEN Cell Bank) were cultivated (37° C., 5% CO2) in Dulbecco's modified Eagle's medium (DMEM; WAKO) with addition of 10% fetal bovine serum (FBS; Invitrogen). HeLa cells were seeded in a 96-well plate the day before transfection (1.0×104 cells/well). The next day, the medium was removed and replaced with 90 μL/well Opti-MEM (registered trademark) (Thermo Fisher SCIENTIFIC). 0.15 μL Lipofectamine (registered trademark) Messenger MAX (registered trademark) and 25 ng mRNA were diluted in 10 μL Opti-MEM (registered trademark) and introduced into cells. After incubation at 37° C. for 5 hours, 20 μL/well 1× Cell Lysis Buffer (Promega) was added to dissolve the cells. Luminescence was measured using ONE-Glo (registered trademark) luciferase assay (registered trademark) (Promega).
Evaluation of intracellular translation activity (JAWS II cells): JAWS II cells (ATCC) were cultivated (37° C., 5% CO2) in Minimum Essential Medium α(MEM α; Thermo Fisher SCIENTIFIC) with addition of 10% fetal bovine serum (FBS; Invitrogen) and GM-CSF (5 ng/mL). JAWS II cells were seeded in a 96-well plate the day before transfection (3.0×104 cells/well). The next day, the medium was removed and replaced with 90 μL/well Opti-MEM (registered trademark) (Thermo Fisher SCIENTIFIC). 0.15 μL Lipofectamine (registered trademark) Messenger MAX (registered trademark) and 50 ng mRNA were diluted in 10 μL Opti-MEM (registered trademark) and introduced into cells. After incubation at 37° C. for 5 hours, 20 μL/well 1× Cell Lysis Buffer (Promega) was added to lyse the cells. Luminescence was measured using ONE-Glo (registered trademark) luciferase assay (registered trademark) (Promega).
The amino acid sequence information of Luc2 is as shown in SEQ ID NO: 6.
(3) Comparison of Translation Activity of NanoLuc Luciferase (Nluc) mRNA Synthesized Using Dinucleotide-Type Cap Analog
The template DNA sequence of NLuc mRNA is as shown in SEQ ID NO: 9. The sequence of NLuc mRNA (transcript) is as shown in SEQ ID NO: 10.
The capped mRNA used in
The elution time of the RNA transcript containing the hydrophobic protecting group-containing cap analog was 13.7 minutes to 14.0 minutes, whereas the elution time of the RNA transcript not containing the hydrophobic protecting group-containing cap analog was 15.5 minutes to 15.8 minutes, and both were clearly different. As a result, it was possible to separate and purify the mRNA containing the hydrophobic protecting group-containing cap analog. An RNA containing dephosphorylated ARCA (ARCA_AP) was prepared as follows. The reaction liquid (250 ng/μL ARCA-mRNA, 50 mM Bis-Tris-Propane HCl, 1 mM MgCl2, 0.1 mM ZnCl2, pH 6.0 @ 25° C., 0.25 U/UL Antarctic phosphatase (New England Biolabs), reaction liquid volume: 20 μL) was warmed at 37° C. for 30 minutes, and then extracted with an equal volume mixed solution of TE saturated phenol and chloroform, then extracted with chloroform, and then 1/10 amount of 3 M NaOAc (pH 5.2) buffer and an equal amount of 2-propanol were added. After cooling at −30° C. for 1 hour, the RNA was collected in the form of a pellet by centrifugation.
Deprotection by photoirradiation: After HPLC purification, an aqueous solution containing the RNA was added to a transparent 96-multiwell plate, and irradiated with 365 nm light at a quantity of light of 4 mW/cm2 for 10 minutes with a MAX-305 light source device (Asahi Spectra Co., Ltd.). Subsequently, the deprotected mRNA was isolated and purified by reverse-phase HPLC. Column: YMC-Triart Bio C4 (250 mm×4.6 mm I.D.); Solvent A: 50 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; Solvent B: acetonitrile; Linear gradient: 0 to 20% Solvent B (0 to 20 min); Flow rate: 1 mL/min; Detection wavelength: 260 nm; Column temperature: 50° C.
End analysis experiment: Nluc mRNA after purification was cleaved with DNAzyme (35-base-long DNA oligonucleotide) and converted into a mixture containing a 23-base-long 5′-end fragment, which was then analyzed by denaturing PAGE. The DNAzyme sequence used is as follows: 5′-TTCGAGGCCAGGCTAGCTACAACGAACGCGTCACC 3′-(SEQ ID NO: 11). The reaction liquid composition of the cleavage reaction is as follows. 104 ng/μL (0.5 μM) Nluc mRNA, 1 μM DNA, 5 mM Tris-HCl (pH 8.0), 5 mM magnesium chloride, reaction liquid volume 35 μL. The reaction liquid was warmed at 37° C. for 1 hour, then a 1/10 amount of a 3 M sodium acetate buffer (pH 5.2), an equal amount of 2-propanol, and 14 ng of glycogen (coprecipitation agent) were added, the mixture was cooled at −30° C. for 1 hour, and the RNA was collected in the form of a pellet by centrifugation. The resulting RNA was dissolved in 3.5 μL of water, and a 2.5 μL portion of the solution was analyzed by electrophoresis with 15% denaturing acrylamide gel containing 7.5 M urea as a denaturant. The gel after electrophoresis was stained with a nucleic acid staining reagent SYBR Gold, photographed by ChemiDoc MP gel imaging system (BioRad), and RNA bands were visualized. The molecular weight of the 5′-end RNA cleavage product was calculated using 1 μL of the RNA solution after alcohol precipitation as a substrate and using an MALDI-TOF mass spectrometer ultrafleXtreme (Bruker Corporation). Measurement was performed using 3-Hydroxy picolinic acid as a matrix and a Linear positive mode.
Translation experiment (HeLa cells): HeLa cells (RIKEN Cell Bank) were cultivated (37° C., 5% CO2) in a Dulbecco's modified Eagle's medium (DMEM; WAKO) with addition of 10% fetal bovine serum (FBS; Invitrogen). On the day before transfection, the culture medium was removed from the HeLa cells (5×103 cells/well) seeded in a 96-well plate and replaced with 40 μL/well Opti-MEM (registered trademark) (Thermo Fisher SCIENTIFIC), to which was added an mRNA (5 ng/well) mixed with Lipofectamine (registered trademark) MessengerMAX (registered trademark) (0.15 μL/well) and Opti-MEM (registered trademark) (10 μL/well). After incubation at 37° C. for 3 hours, DMEM culture medium containing 10% FBS was added at 100 μL/well, followed by further cultivation. After 12, 24, and 48 hours, a cell lysate was prepared using NanoGlo (registered trademark) Luciferase Assay System (Promega), and the amount of the NanoLuc luciferase protein contained in the lysate was evaluated by a luminescence measurement method.
Translation experiment (JAWS II cells): JAWS II cells (ATCC) were cultivated (37° C., 5% CO2) in an MEM α, nucleosides (Gibco) medium with addition of 10% fetal bovine serum (FBS; Invitrogen), 5 ng/mL. On the day before transfection, the culture medium was removed from the JAWS II cells (5×103 cells/well) seeded in a 96-well plate and replaced with 40 μL/well Opti-MEM (registered trademark) (Thermo Fisher SCIENTIFIC), to which was then added an mRNA (5 ng/well) mixed with Lipofectamine (registered trademark) MessengerMAX (registered trademark) (0.15 μL/well) and Opti-MEM (registered trademark) (10 μL). After incubation at 37° C. for 3 hours, an MEM α, nucleosides culture medium containing 10% FBS and 5 ng/mL GM-CSF was added at 100 μL/well, followed by further cultivation. After 24, 48, 72, and 120 hours, a cell lysate was prepared using NanoGlo (registered trademark) Luciferase Assay System (Promega), and the amount of the NanoLuc luciferase protein contained in the lysate was evaluated by a luminescence measurement method.
The amino acid sequence information of NLuc is as shown in SEQ ID NO: 12.
(3) Comparison of Translation Activity of NanoLuc Luciferase (Nluc) mRNAs Synthesized Using Trinucleotide-Type Cap Analog and Tetranucleotide-Type Cap Analog
The capped mRNA used in
The elution time of the RNA transcript containing the hydrophobic protecting group-containing cap analog was 13.7 minutes to 14.0 minutes, whereas the elution time of the RNA transcript not containing the hydrophobic protecting group-containing cap analog was 15.5 minutes to 15.8 minutes, and both were different. As a result, it was possible to separate and purify the mRNA containing the hydrophobic protecting group-containing cap analog.
Deprotection by photoirradiation: An aqueous solution of the RNA containing the hydrophobic protecting group-containing cap analog isolated by reverse-phase HPLC was added to a transparent 96-multiwell plate, and irradiated with 365 nm light at a quantity of light of 4 mW/cm2 for 10 minutes using a MAX-305 light source device (Asahi Spectra Co., Ltd.). Subsequently, the product was purified by reverse-phase HPLC under the following conditions. Column: YMC-Triart Bio C4 (250 mm×4.6 mm I.D.); Solvent A: 50 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; Solvent B: acetonitrile; Linear gradient: 0 to 20% Solvent B (0 to 20 min); Flow rate: 1 mL/min; Detection wavelength: 260 nm; Column temperature: 50° C.
End analysis: Nluc mRNA after purification was cleaved with DNAzyme (35-base-long DNA oligonucleotide) and converted into a mixture containing a 23-base-long 5′-end fragment, which was then analyzed by denaturing PAGE. The DNAzyme sequence used is as follows: 5′-TTCGAGGCCAGGCTAGCTACAACGAACGCGTCACC 3′-(SEQ ID NO: 11). The reaction liquid composition of the cleavage reaction is as follows. 104 ng/μL (0.5 μM) Nluc mRNA, 1 μM DNAzyme, 5 mM Tris-HCl (pH 8.0), 5 mM magnesium chloride, reaction liquid volume 40 μL. The reaction liquid was warmed at 37° C. for 1 hour, then a 1/10 amount of a 3 M sodium acetate buffer (pH 5.2), an equal amount of 2-propanol, and 14 ng of glycogen (coprecipitation agent) were added, the mixture was cooled at −30° C. for 1 hour, and the RNA was collected in the form of a pellet by centrifugation. The resulting RNA was dissolved in 4 μL of water, and a 3 μL portion of the solution was analyzed by electrophoresis with 15% denaturing acrylamide gel containing 7.5 M urea as a denaturant. The gel after electrophoresis was stained with a nucleic acid staining reagent SYBR Gold, photographed by ChemiDoc MP gel imaging system (BioRad), and RNA bands were visualized.
Translation experiment (HeLa cells): HeLa cells (RIKEN Cell Bank) were cultivated (37° C., 5% CO2) in a Dulbecco's modified Eagle's medium (DMEM; WAKO) with addition of 10% fetal bovine serum (FBS; Invitrogen). On the day before transfection, the culture medium was removed from the HeLa cells (5×103 cells/well) seeded in a 96-well plate and replaced with 40 μL/well Opti-MEM (registered trademark) (Thermo Fisher SCIENTIFIC), to which was added an mRNA (5 ng/well) mixed with Lipofectamine (registered trademark) MessengerMAX (registered trademark) (0.15 μL/well) and Opti-MEM (registered trademark) (10 μL/well). After incubation at 37° C. for 3 hours, DMEM culture medium containing 10% FBS was added at 100 μL/well, followed by further cultivation. After 12, 24, and 48 hours, a cell lysate was prepared using NanoGlo (registered trademark) Luciferase Assay System (Promega), and the amount of the NanoLuc luciferase protein contained in the lysate was evaluated by a luminescence measurement method.
Translation experiment (JAWS II cells): JAWS II cells (ATCC) were cultivated (37° C., 5% CO2) in an MEM α, nucleosides (Gibco) medium with addition of 10% fetal bovine serum (FBS; Invitrogen), 5 ng/mL. On the day before transfection, the culture medium was removed from the JAWS II cells (5×103 cells/well) seeded in a 96-well plate and replaced with 40 μL/well Opti-MEM (registered trademark) (Thermo Fisher SCIENTIFIC), to which was then added an mRNA (5 ng/well) mixed with Lipofectamine (registered trademark) MessengerMAX (registered trademark) (0.15 μL/well) and Opti-MEM (registered trademark) (10 μL). After incubation at 37° C. for 3 hours, an MEM α, nucleosides culture medium containing 10% FBS and 5 ng/mL GM-CSF was added at 100 μL/well, followed by further cultivation. After 24, 48, 72, and 120 hours, a cell lysate was prepared using NanoGlo (registered trademark) Luciferase Assay System (Promega), and the amount of the NanoLuc luciferase protein contained in the lysate was evaluated by a luminescence measurement method.
(4) Isolation and Purification of Longer Chain Capped mRNA by Reverse-Phase HPLC
The template DNA sequence (4375-bp) of Spike protein mRNA is as shown in SEQ ID NO: 13. The sequence (transcript) 4247 base of Spike protein mRNA is as shown in SEQ ID NO: 14.
The capped mRNA used in
The mRNA after purification was cleaved with DNAzyme (39-base-long DNA oligonucleotide) and converted into a mixture containing a 23-base-long 5′-end fragment, which was then analyzed by denaturing PAGE. The DNAzyme sequences used are as follows: 5′-TCTGTGGGGAGGCTAGCTACAACGACAGAAGAATACTAG 3′-(SEQ ID NO: 19). The reaction liquid composition of the cleavage reaction is as follows. 688 ng/μL mRNA (DiPure), 1 μM DNAzyme, 5 mM Tris-HCl (pH 8.0), 5 mM magnesium chloride, reaction liquid volume 9 μL. The reaction liquid was warmed at 37° C. for 1 hour, then 2 μL of a 3 M sodium acetate buffer (pH 5.2), 20 μL of 2-propanol, and 20 ng of glycogen (coprecipitation agent) were added, the mixture was cooled at −30° C. for 1 hour, and the RNA was collected in the form of a pellet by centrifugation. The resulting RNA was dissolved in 3 μL of water, and a 3 μL portion of the solution was analyzed by electrophoresis with 15% denaturing acrylamide gel containing 7.5 M urea as a denaturant. The gel after electrophoresis was stained with a nucleic acid staining reagent SYBR Gold, photographed by ChemiDoc MP gel imaging system (BioRad), and RNA bands were visualized.
(5) Advantages of PureCap Type mRNA in Comparison with Conventional mRNA
A 1Methylpseudouridine-modified mRNA and an unmodified mRNA were prepared as follows.
An mRNA was transcriptionally synthesized from a DNA template using a T7 RNA polymerase reaction under the following conditions. 15 ng/μL dsDNA transcription template (PCR product of 676-bp containing T7 promoter sequence 5′TAATACGACTCACTATAG3′-(SEQ ID NO: 1) and containing a NanoLuc luciferase gene coding region derived from a pNL1.1 TK vector (Promega)), 2 mM ATP, 2 mM 1 methylpseudo UTP or UTP, 2 mM CTP, 2 mM GTP, 2 mM PureCap-type cap analog or [0.5 mM GTP+2 mM ARCA cap analog (Jena Biosciences)], 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 5 mM DTT, 10 units/μL T7 RNA polymerase (Takara Bio Inc.). The reaction liquid was warmed at 37° C. for 2 hours, then DNase (Takara Bio Inc.) was added thereto such that a final concentration was 0.1 units/μL, and the mixture was warmed at 37° C. for 20 minutes. An equal volume mixed liquid of TE saturated phenol and chloroform was added to the reaction liquid in the same volume as the reaction liquid, and the mixture was vigorously stirred, then centrifuged, and the aqueous layer was taken. An equal amount of chloroform was added thereto, and the mixture was vigorously stirred again, then centrifuged, and the aqueous layer was taken. A 1/10 amount of a 3 M sodium acetate buffer (pH 5.2) and an equal amount of 2-propanol were added to the aqueous layer, and the mixture was cooled at minus 30° C. for 1 hour and then centrifuged, affording a crude purified product of an RNA. The crude purified product of the RNA was purified by reverse-phase HPLC under the following conditions. Column: YMC-Triart Bio C4 (250 mm×4.6 mm I.D.); Solvent A: 50 mM triethylammonium acetate (pH 7.0), 5% acetonitrile; Solvent B: acetonitrile; Linear gradient: 0 to 20% Solvent B (0 to 20 min); Flow rate: 1 mL/min; Detection wavelength: 260 nm; Column temperature: 50° C.
The elution time of the RNA transcript containing the hydrophobic protecting group-containing cap analog was 16.0 minutes to 16.8 minutes, whereas the elution time of the RNA transcript not containing the hydrophobic protecting group-containing cap analog was 13.4 minutes to 14.0 minutes, and both were different. As a result, it was possible to separate and purify the mRNA containing the hydrophobic protecting group-containing cap analog.
The translation activity of the mRNA prepared as described above was evaluated as follows. JAWS II cells (ATCC) were cultivated (37° C., 5% CO2) in an MEM α, nucleosides (Gibco) medium with addition of 10% fetal bovine serum (FBS; Invitrogen), 5 ng/mL. On the day before transfection, the culture medium was removed from the JAWS II cells (5×103 cells/well) seeded in a 96-well plate and replaced with 40 μL/well Opti-MEM (registered trademark) (Thermo Fisher SCIENTIFIC), to which was then added an mRNA (5 ng/well) mixed with Lipofectamine (registered trademark) MessengerMAX (registered trademark) (0.15 μL/well) and Opti-MEM (registered trademark) (10 μL). After incubation at 37° C. for 3 hours, an MEM α, nucleosides culture medium containing 10% FBS and 5 ng/mL GM-CSF was added at 100 μL/well, followed by further cultivation. After 24, 48, 72, and 120 hours, a cell lysate was prepared using NanoGlo (registered trademark) Luciferase Assay System (Promega), and the amount of the NanoLuc luciferase protein contained in the lysate was evaluated by a luminescence measurement method.
NF-κB reporter (Luc)-HEK 293 cells (BPS Bioscience Inc.) were cultivated (37° C., 5% CO2) in a Dulbecco's modified Eagle's medium (DMEM; WAKO) with addition of 10% fetal bovine serum (FBS; Invitrogen), 100 μg/ml of Hygromycin B. The day before transfection, NF-κB reporter (Luc)-HEK 293 cells were seeded in a 48-well plate (7.5×104 cells/well). The next day, the medium was removed and replaced with 225 μL/well Opti-MEM (registered trademark) (Thermo Fisher SCIENTIFIC). 0.375 μL Lipofectamine (registered trademark) Messenger MAX (registered trademark) and 12.5 ng mRNA (DiPure) were diluted in 25 μL Opti-MEM (registered trademark) and introduced into cells. After incubation at 37° C. for 5 hours, 50 μL/well 1× Cell Lysis Buffer (Promega) was added to lyse the cells. Luminescence was measured using ONE-Glo (registered trademark) luciferase assay (registered trademark) (Promega). The protein amount was quantified using Pierce (registered trademark) BCA Protein Assay Kit (Thermo Fisher SCIENTIFIC), and the luminescence value was corrected by the number of cells.
NF-κB reporter (Luc)-HEK 293 cells (BPS Bioscience Inc.) were cultivated (37° C., 5% CO2) in a Dulbecco's modified Eagle's medium (DMEM; WAKO) with addition of 10% fetal bovine serum (FBS; Invitrogen), 100 μg/ml of Hygromycin B. The day before transfection, NF-κB reporter (Luc)-HEK 293 cells were seeded in a 48-well plate (1.0×105 cells/well). The next day, the medium was removed and replaced with 240 μL/well Opti-MEM (registered trademark) (Thermo Fisher SCIENTIFIC). 0.15 μL Lipofectamine (registered trademark) Messenger MAX (registered trademark) and 100 ng mRNA were diluted in 10 μL Opti-MEM (registered trademark) and introduced into cells. After incubation at 37° C. for 24 hours, 50 μL/well 1× Cell Lysis Buffer (Promega) was added to lyse the cells. Luminescence was measured using ONE-Glo (registered trademark) luciferase assay (registered trademark) (Promega). The protein amount was quantified using Pierce (registered trademark) BCA Protein Assay Kit (Thermo Fisher SCIENTIFIC), and the luminescence value was corrected by the number of cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-111420 | Jul 2021 | JP | national |
This application is the United States national phase of International Application No. PCT/JP2022/026665 filed Jul. 5, 2022, and claims priority to Japanese Patent Application No. 2021-111420 filed Jul. 5, 2021, the disclosures of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/026665 | 7/5/2022 | WO |
