Patent Application
20090137789
The present invention relates to oligonucleotide probes, methods for their synthesis and intermediate compound beacons for said methods.
Numerous biotechnological applications use oligonucleotides because of their intrinsic capacity to selectively bond to complementary strands or to particular duplex using Watson and Crick or Hoogsteen type hydrogen bonds. These applications are largely facilitated because of the possibility of specifically recognizing oligonucleotides used as an “instrument” by the oligonucleotides present in the analytic sample. In order to do this, the oligonucleotide is “marked” with an easily recognizable group such as a radioactive isotope, for example, or a dye, or a fluorescent group. The oligonucleotide marked in this manner is referred to as an oligonucleotide probe.
Among main probe uses, is the localization of the probes themselves in the various cellular districts by means of fluorescent microscopy, and the detection of complementary sequences with “molecular beacons” for example (probes that quench their own fluorescence signal where are self-associated, but by hybridizing with the oligonucleotide of the target sequence they undergo conformational change returning to become fluorescent once more, revealing the presence of the target compound) when searching for alien DNA (viral or transgenic DNA) or modified DNA, or for amplification applications through PCR (polymerase chain reaction) etc.
For some time, fluorophors derived from fluorescein, have been used as fluorescent beacons for biological systems such as proteins, membranes and also nucleotides. In particular, it is usual practice to incorporate fluorescent beacons derived from fluorescein with oligonucleotides in order to obtain oligonucleotide probes.
Oligonucleotide probes derived from fluorescein present the following problems: relatively high “photobleaching”, (fluorescence loss); fluorescence dependent on pH, whose fluorescence is considerably reduced under a level of pH 7; a relatively wide emission spectrum that limits applications where different color emission is necessary (Handbook of fluorescent probes and research products, 9th Ed; Molecular Probes Inc.: Eugene, Oreg., 2002; Ch 1 Section 1.1 pg 47).
Certain applications where different color probes are required are: flow cytometry, DNA sequencing, fluorescent microscopy, etc.
An object of the present invention is to create oligonucleotide probes that are free of the problems described above. Further aims of the present invention are to supply synthesis methods for said oligonucleotide probes using compound beacons as well as realizing the compound beacons themselves.
According to the present invention oligonucleotide probes are created according to the general formula (I)
Onu-L-Thion (I)
as recited in the first-claim and, preferably, in any one of the claims dependent either directly or indirectly contingent on the first claim.
According to the present invention compounds are also provided according to the general formula III:
as recited in claim 16, and preferably, in any one of the subsequential claims dependent either directly or indirectly contingent to claim 16.
According to the present invention compounds are also provided according to the general formula V:
as recited in claim 30, and preferably, in any one of the subsequential claims dependent either directly or indirectly on claim 30.
According to the present invention compounds are also provided according to the general formula VI:
as recited in claim 38, and preferably, in any one of the subsequential claims dependent either directly or indirectly on claim 38.
According to the present invention compounds are also provided according to the general formula VII:
as recited in claim 46, and preferably, in any one of the claims dependent either directly or indirectly on claim 46.
According to the present invention methods are also provided, as described in the appended claims for the preparation of the probes according to the general formula I bonding oligonucleotides to compounds according to the general formulas III, V, VI and VII.
The invention is described below with reference to the appended drawings which relate to not limiting embodiments, wherein:
The definitions of the various chemical moieties will be introduced in the next few paragraphs, and are to be understood as being applied in the same manner throughout the whole text, including the claims, unless another definition is specifically set out.
The term “oligonucleotide” refers to a single strand DNA or RNA fragment composed of at least two nucleotides. In particular, an oligonucleotide comprises between two and two hundred nucleotides, preferably between 20 and 140 nucleotides and even more preferably between 20 and 60 nucleotides. This definition includes modified oligonucleotides, commonly used in biotechnological applications (such as phosphorothioates methylphosphonates, 2′-O-alkyl-RNA, 5′-alkylpyrimidine for example) whose synthesis occurs with the simple extension of standard protocols and whose transformation into fluorescent probes can be performed through simple extension of the methods described in the present text.
The term “thiophenic ring” refers to an aromatic ring with 5 members, wherein one of the members is a sulphur atom; optionally, the aromatic ring can have one or more substituents; optionally the sulphur can be present in the form of an oxide. Examples of thiophenic rings are the following:
The term “oligothiophene” refers to one or more thiophenic rings connected to each other in linear or branched mode, or fused together. Below are some examples of oligothiophenes:
The term “halogen” refers to a radical selected from the group consisting of: chlorine, fluorine, bromine, iodine.
The term “alkyl Cx-Cy” refers to at linear or branched monovalent alkyl group presenting a minimum of x carbon atoms and a maximum of y carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, ter-butyl and n-hexyl.
The term “alkenyl Cx-Cy” refers to a linear or branched monovalent alkenyl group presenting a minimum of x carbon atoms and a maximum of y carbon atoms and at least one unsaturated site. This term is exemplified by groups such as vinyl (—CH═CH2) andn-2-propenyl(allyl, —CH2CH═CH2).
The term “alkynyl Cx-Cy” refers to a linear or branched monovalent alkynyl group presenting a minimum of x carbon atoms and a maximum of y carbon atoms and at least one doubly unsaturated site. This term is exemplified by groups such as: ethynyl (—C≡CH) and propargyl (—CH2C≡CH).
The term “cycloalkyl Cx-Cy” refers to a saturated carbocyclic group presenting a minimum of four and a maximum of eight carbon atoms, and having a single ring (for example cyclohexyl) or condensed multiple rings.
The term “aliphatic Cx-Cy” refers to a monovalent group “cycloalkyl Cx-Cy”, “alkyl Cx-Cy”, “alkenyl Cx-Cy” or “alkynyl Cx-Cy”.
The term “alkylene Cx-Cy” refers to a bivalent group “cycloalkyl Cx-Cy”, “alkyl Cx-Cy”, “alkenyl Cx-Cy” or “alkynyl Cx-Cy”. As an option, the alkylene can contain one or more ether atoms, in particular N, O and S, each of which is bonded to two carbon atoms. This term is exemplified by groups such as —CH2—CH2—, —CH═CH—, —C≡C—, —CH2CH═CH—, —CH2—CH2—O—CH2—, —CH2—S—CH2— and —CH2—N—(CH3)—CH2—CH2—.
The term “saturated alkylene Cx-Cy” refers to a bivalent group “cycloalkyl Cx-Cy”, “alkyl Cx-Cy”. This term is exemplified by groups such as —CH2—CH2—, —(CH2)3—.
The term “arylic” or “aryl” refers to a phenyl group or a system of fused bicyclic or tricyclic rings, wherein at least one of the fused rings is a phenylic group. Examples of bicyclic and tricyclic systems are naphtalene and phenantrene. Optionally, the acrylic groups can be substituted by one to five substituents. In particular the substituents may be selected, each independently from one other, from the group consisting of: halogens, aliphatics C1-C12, carbonyls, alkanoyls, alkoxys, hydroxy (—OH), mercapto (—SH), alkylsulfonyls, alkylsulfanyls, cyano (—CN), nitro (—NO2), alkylcarboxys, amines (—NH2), alkylamines.
The term “alkanoyl” refers to an aliphatic group C3-C12 bonded to the remaining part of the molecule through a carbonyl group.
The term “alkoxy” “refers to an aliphatic group C1-C32 bonded to the remaining part of the molecule through an atom of oxygen.
The term “alkylsulfanyl”, refers to an aliphatic group or a substituted aliphatic group bonded to the remaining part of the molecule through a sulphur atom.
The term “alkylsulfonyl” refers to an aliphatic group C1-C12 bonded to the remaining part of the molecule through a group —SO2—.
The term “alkylcarboxy” refers to an alcoxy group bonded to the remaining part of the molecule through a carbonyl group.
The term “alkylammine” refers to one or two aliphatic groups C1-C12 bonded to the remaining part of the molecule through a nitrogen atom.
The term “substituted aliphatic Cx-Cy” refers to an aliphatic group Cx-Cy substituted by one or more substituents selected from a group composed of: halogens, carboxy groups, carbonyl groups, alkanoyl groups, alkoxy groups, hydroxyls, sulphydryl groups (—SH), alkoxy groups, alkylsuphonyl groups, alkylsulphanyl groups, cyano groups, nitro groups, alkylcarboxy, with the proviso that the number of substituents is always lower than the number of hydrogen atoms of the corresponding non-substituted aliphatic group.
According to a first aspect of the invention presented herein, an oligonucleotide probe having general formula (I) is provided:
Onu-L-Thion (I)
wherein Onu refers to an oligonucleotide residue; each Thio refers to a respective thiophenic ring, independently from the other Thio; Thion refers to a fluorescent oligothiophene containing n thiophenic rings; n is an integer lower than eight; L refers to at binder conceived to maintain Thion mobile in relation to Onu so that Thion is able to perform its fluorescent action correctly and Onu is able to hybridize freely with a complementary sequence; with the proviso that if n is equal to one, the sulphur of the thiophenic ring is bonded to two oxygens.
According to a further aspect of the invention presented herein, a beacon compound having a general formula III is provided:
wherein R14 is a protecting group of the phosphite ester and is selected so that R14 is removable through the action of a 30% aqueous ammonia solution; R15 and R16 being selected so that NR15R16 is removable through the action of the weak acids, in particular tetrazole; each Thio refers to a respective thiophenic ring, independently from the other Thio; Thion refers to a fluorescent oligothiophene containing n thiophenic rings; R17 being selected from the group consisting of: —R12— and —R9—C(O)—; R12 being selected from the group consisting of: an alkylene C1-C9, —R9—Si(R10)(R11)—; R10 and R11 representing, each independently from each other, a respective alkyl C1-C8, aryl, alkenyl C2-C8, alkynyl C2-C8; R9 representing an alkylene C1-C8.
According to certain embodiments, R15 and R16 are selected, each independently from each other, from the group consisting of: alkyl C1-C12, aryl, cycloalkyl C5-C10; R15 and R16 can be linked so that, together with N, they form a 5-6 member heterocyclic ring. Preferably, R15 and R16 represent, each independently from each other, a saturated alkyl C1-C3. Even more preferably, R15 and R36 represent, each independently from each other, a saturated alkyl C3. Particularly preferable are the embodiments wherein R15 and R16 represent, each, a respective isopropyl group.
Preferably, R14 is selected from the group consisting of: —(CH2)2CN and —CH3.
According to a further aspect of the invention presented herein a beacon compound is described presenting a general formula V:
each Thio refers to a respective thiophenic ring, independently from the other Thio; Thion refers to a fluorescent oligothiophene containing n thiophenic rings; R18 being selected from the group consisting of: —C(O)—O—, —R12—C(O)—O—, —C(O)—R9—C(O)—O—; R12 being selected from the group consisting of: an alkylene C1-C9, —R9—Si(R10)(R11)—; R10 and R11 representing, each independently from each other, a respective alkyl C1-C8, aryl, alkenyl C2-C8, alkynyl C2-C8; R9 representing an alkylene C1-C8.
Preferably the compound has the following formula:
According to a further aspect of the invention presented herein a beacon compound presenting a general formula VI is provided:
each Thio refers to a respective thiophenic ring, independently from the other Thio; Thion refers to a fluorescent oligothiophene containing n thiophenic rings; R18 being selected from the group consisting of: —C(O)—O—, —R12—C(O)—O—, —C(O)—R9—C(O)—O—; R12 being selected from the group consisting of: an alkylene C1-C9, —R9—Si(R10)(R11)—; R10 and R11 representing, each independently from each other, a respective alkyl C1-C8, aryl, alkenyl C2-C8, alkynyl C2-C8; R9 representing an alkylene C1-C8.
Preferably, the compound has the following formula:
According to a further aspect of the invention presented herein a beacon compound is described presenting a general formula VII:
each Thio refers to a respective thiophenic ring independently from the other Thio; Thion refers to a fluorescent oligothiophene containing n thiophenic rings; R17 being selected from the group consisting of: —R12— and —R9—C(O)—; R12 being selected from the group consisting of: an alkylene C1-C9, —R9—Si(R10)(R11)—; R10 and R11 representing, each independently from each other, an alkyl C1-C8, aryl, alkenyl C2-C8, alkynyl C2-C8; R9 representing an alkylene C1-C8. Preferably, R12 represents a saturated alkylene C2-C9 and R9 represents a saturated alkylene C1-C8. Even more preferably, R37 represents a saturated alkylene C2-C3.
The above-described compounds can be obtained by using the following general procedure. It should be understood that where typical or preferred experimental conditions are described (this refers to: temperature, reaction times, reactant moles, solvents etc.) other conditions could be used. Optimal reaction conditions can vary according to the particular reactants or solvents used.
According to a further aspect of the invention presented herein a method for the preparation of an oligonucleotide probe is also provided, having the general formula I as defined above, wherein L is selected from a group composed of —O—P(O)2—O—R12—, —O—P(O)2—O—R9—C(O)—; the method providing for a conjugation phase, wherein a compound having the general formula III as defined above is bonded to an oxygen in position 5′ of a nucleoside of an oligonucleotide residue Onu; during said conjugation phase NR13R16 being removed and P being oxidized; a removal phase, that occurs in basic conditions and during which R14 is removed. R12, R9, R15, R16 and R14 are defined as above.
Preferably, the conjugation phase occurs in a moderately acidic environment, in particular in the presence of tetrazole. Typically the conjugation phase is performed in the typical conditions of an automatic oligonucleotide synthesizer.
It is important to emphasize that, in this manner, using a normal oligonucleotide it is possible to incorporate the oligothiophene in position 5′ of the oligonucleotide residue Onu.
According to certain preferred embodiments, the compound having general formula III as defined above is obtained by means of nucleophilic sunstitution, wherein a first reactant, which has general formula Thion-R17—O−, is made to react with a second reactant having general formula IV:
LG1 being a leaving group. R17 is defined as above. Preferably, LG1 is a leaving group selected from the group consisting of: halogen and NR15R16.
Preferred embodiments of the above-described method can be schematically represented as follows:
According to a further aspect of the invention presented herein a method is provided for the synthesis of an oligonucleotide probe, having general formula I as defined above, wherein L is selected from the group consisting of R8—NH— (O)C—R9—C(O)—, —R8—NH— (O)C—R12—, —R8—NH— (O)C—; the method comprising a conjugation phase wherein a compound having the general formula V as defined above, is made to react with prearranged oligonucleotide residue having the formula Onu-R8—NH2, wherein Onu represents an oligonucleotide residue; R8 representing an alkylene C1-C8. Preferably, R8 is a hexyl group. R9 and R12 are defined as above.
Preferred embodiments of the above-described method can be schematically represented as follows:
wherein R18 is defined as above.
According to a further aspect of the invention presented herein a method is provided for the synthesis of an oligonucleotide probe, presenting the general formula I as defined above, wherein L is selected from the group consisting of R8—NH— (O)C—R9—C(O)—, —R8—NH—(O)C—R12—, —R8—NH— (O)C—; the method comprising a conjugation phase, wherein a compound having the general formula VI as defined above is made to react with a prearranged oligonucleotide residue having the formula Onu-R8—NH2, wherein Onu represents an oligonucleotide; R8 representing an alkylene C1-C8. Preferably, R8 is a hexyl group.
Preferred embodiments of the above-described method can be schematically represented as follows:
wherein R18 is defined as above.
According to a further aspect of the invention presented herein a method is provided for the preparation of an oligonucleotide probe, presenting the general formula I as defined above, wherein L is selected from the group consisting of;
the method providing for a conjugation phase, wherein a compound having the general formula VII as defined above is made to react with a prearranged oligonucleotide residue having the formula Onu-R8—SH, wherein Onu represents an oligonucleotide residue; R8 representing an alkylene C1-C8. Preferably, R8 is a hexyl group.
Preferably, the method comprises a nucleophilic substitution phase, wherein a first reactant, which is selected from the group consisting of Thio, —R12—O− Thion-C(O)—R9—O−, is made to react with
in order to obtain the compound presenting the general formula VII as defined above. Preferably the nucleophilic substitution occurs in the presence of PPh3, (CH3)3COH and CH3 CH2C(O)N2C(O)CH2CH3.
Preferred embodiments of the above-described method can be schematically represented as follows:
wherein R17 is defined as above. A possible alternative strategy for the synthesis of probes having the general formula I, wherein L is selected among —R8—NH—C(S)—NH—R12— and —R8—NH—C(S)—NH—R9—C(O)—, can be schematically represented as follows:
R9, R12, R17 and R8 are defined as above. Alo2 represents a halogen. Step 11 is a nucleophilic substitution and preferably occurs in acetone. Step 12 occurs, preferably in DMF/H2O.
Possible alternative strategies for the synthesis of probes presenting the general formula I, wherein L is selected from —R8—S—, —R8—S—R32— and —R8— S—R9—C(O)—, are schematically represented as follows:
Onu-R8—SH+X—R12-Thion→Onu-R8—S—R12-Thion
Onu-R8—SH+X—R9—C(O)-Thion→Onu-R8—S—R9—C(O)-Thion
Onu-R8—SH+X-Thion→Onu-R8—S-Thion
Onu-R8—X+HS—R12-Thion→Onu-R8—S—R12-Thion
Onu-R8—X+HS—R9—C(O)-Thion→Onu-R8—S—R9—C(O)-Thion
Onu-R8—X+HS-Thion→Onu-R8—S-Thion
X represents a bromine, an iodine or a chlorine. R8, R9 and R12 are defined as above.
A further strategy to incorporate an oligothiophene Thion in an Onu oligonucleotide residue consists in bonding the ologothiophene Thion to a single nucleoside and then modifying the nucleoside in order to obtain a beacon wherein the nucleoside with phosphoroamidite is bonded to the oligothiophene Thion (phosphoroamidite-nucleoside-Thion). As an alternative option, the nucleoside can be appropriately modified in order to present in position 5′, the hydroxy protected by a protecting group —PG, which is removable in an acid environment; in this case it is possible to incorporate the beacon in position 3′, in position 5, and in an intermediate position of the oligonucleotide. Wherever the beacon is to be incorporated in position 3′, the beacon will be attached to an automatic synthesizer column, then followed by the synthesis of the oligonucleotide. Wherever the beacon is to be incorporated in position 5′, incorporation will be carried out on completion of the synthesis (3′→5′) of the oligonucleotide. Wherever the beacon is to be incorporated in position 5′, incorporation will be carried out at an appropriate moment during the synthesis (3′→5′) of the oligonucleotide.
A possible synthesis strategy for the reagents Thion-R18—H is schematically represented as follows:
R12 represents an alkylene C2-C9. R9 and R18 are defined as above. Alo1 and Alo2 represent respective halogens, each independently from each other. Step 1 is an acilation and preferably occurs in the presence of AlCl3. Step 2 is a reduction and preferably occurs in the presence of AlCl3 and LiAlH4. Steps 4 and 3 are oxidations and involve treating the halogen derivates with KCN, in order to obtain a nucleophilic substitution with cyano, which through hydrolysis leads to carboxylic acids. Step 3 is schematically shown as follows:
According to a further approach, a possible strategy for synthesizing reagent Thion-R18—H is schematically shown as follows:
R9 and R18 are defined as above. Alo1 is defined as above, R20 represents an alkyl. Step 5 is an acilation and preferably, occurs in the presence of AlCl3. Step 6 is a hydrolysis and preferably, occurs in EtOH in presence of NaOH.
A possible strategy for synthesizing reagent −O—R17-Thion is schematically represented as follows:
R12 represents an alkylene C2-C9. R9, R17, Alo1 and Alo2 are defined as above. Step 7 is an acilation and preferably, occurs in the presence of AlCl3. Step 8 is a reduction and preferably, occurs in the presence of AlCl3 and LiAlH4. Steps 9 and 10 are nucleophilic substitutions and occur preferably, in a basic environment; even more preferably, steps 9 and 10 occur in the presence of N-Methylpyrrolidone (NMP).
A possible strategy for reactant synthesis Alo2—R12-Thion, wherein R12 represents —R9—Si(R10)(R11)—, is schematically represented as follows:
R9, R10, R11 and Alo2 are defined as above. Alo3 represents a halogen. Step 13 occurs preferably in presence of LDA (lithiumdiisopropylamine) o of n-butylthio.
A possible strategy for the synthesis of oligothiophene Thion is schematically represented as follows:
Thio and Thion are defined as above. Alo4 and Alo5 are, each independently from each other, halogens. Preferably, Alo4 and Alo5 represent, each, a respective bromine atom. Preferably, Step 14 is carried out in ethylic ether. Preferably, Step 15 is carried out in toluene. Even more preferably Step 15 carried out in presence of Pd(Ph3As)4 (F. Effenberger, F Wurthner, F steybe J. Org: Chem., 1995, 60, 2082-2091; G. Barbarella, M. Zambianchi, G. Sotgiu, A. Bongini Tetrahedron 1997, 53, 9401-9406).
A further possible strategy for oligothiophene Thion synthesis is also described in Jong, F. D.; Janssen, M. J. J. Org. Chem. 1971, 36, 1645-1648 and is shown in the example below:
A possible method for oxidizing the sulphur of a thiophenic ring involves using an oxidizing agent, in particular mCPBA. Even more preferably the reaction occurs in CH2Cl2.
With reference to what has been described above, according to preferred embodiments, independently from the other Thio, each Thio represents a respective thiophenic, ring presenting the general formula II:
wherein X is chosen from a free lone pair of S electrons and an oxygen radical; R1, R2, R3 are selected, each independently from one other, from the group consisting of: hydrogen, halogen, alkyl, C1-C12, aromatic, —CN, —NO2, -Thiom, —NR4R5, —SR4, —R5—SR4, —OR4, —R5—OR4, —C(O)R6, —NC(O)R6, —O—PG, —R7—O—PG; wherein R4 and R5 are selected, each independently from each other, from the group consisting of hydrogen, alkyl C1-C12, aromatic, Thiom; R6 is selected from the group consisting of hydrogen, halogen, alkyl C1-C12, aromatic, Thiom; m is an integer lower than n; —PG is a protecting group of hydroxy that is removable in an acidic environment; with the proviso that, if n is equal to 1, X represents an oxygen radical; each Thio group can be fused with 1 or 2 other Thio; R7 is a saturated alkylene C1-C8. Preferably, the aforesaid aromatic groups are acrylic groups.
Preferably, n is lower than 5. According to a preferred embodiment, R1, R2 and R3 are selected, each one in a manner separate from one other, from the group consisting of: alkyl C1-C8, —R5—S—R4, hydrogen, —SR4, —R7—O—PG; wherein R7, R4 and R5 represent each one in a manner separate from one other, a saturated alylene C1—Ce; —PG is defined as above and represents a protecting group of the hydroxy that is removable in an acidic environment.
Even more preferably, R1, R2, R3 are, each one in a manner separate from one other, selected from the group consisting of: hydrogen, —SR4, —R7—O—PG. Particularly preferable are the embodiments wherein R1, R2, R3 represent, each, a respective hydrogen.
Preferably, X represents a lone pair of electrons of S. Preferably, —PG is selected from the group consisting of: dimethoxytrityl, monomethoxytrityl, methoxy ethoxy methyl (MEM), methoxy methyl (MOM).
Particularly preferable are the embodiments, wherein Thion represents:
With reference to what has been described above, according to preferred embodiments, L is selected from the group consisting of: alkylene C2-C8, —R8—C(O)—, —R8—O—C(O)—, —R8—C(O)—R9—, —R′—O—C(O)—R9—, —R8—O—R9—C(O)—, —R8—O—R12—, —R8—S—, —R8—S—R9—C(O)—, —R8—S—R12, —R8—NH—R9—C(O)—, —R8—NH—R12—, —R8—Si (R10)(R11)—, —O—P(O)2—O—R12—, —O—P(O)2—O—R9—C(O)—, —R8—NH—(O)C—R9—C(O)—, —R8—NH—(O)C—R12—, —R8—NH— (O)C—, —R8—NH—C(S)—NH—R12—, —R8—NH—C(S)—NH—R9—C(O)—,
Preferably, L is selected from the group consisting of: saturated alkylene C2-C8, —R8—C(O)—, —R8—O—C(O)—, R8—C(O)—R9—, —R8—O—C(O)—R9—, —R8—O—R9—C(O)—, —R8—O—R12—, —R8—S—R9—C(O)—, —R8—S—R12—, —R8—NH—R9—C(O)—, —R8—NH—R12—, —R8—Si(R10)(R11)—, —O—P(O)2—O—R12—, —O—P(O)2—O—R9—C(O)—, —R8—NH—(O)C—R9—C(O)—, —R8—NH— (O)C—R12—, —R8—NH—(O)C—, —R8—NH—NH—C(S)—NH—R12—, —R8—NH—C(S)—NH—R9—C(O)—,
Even more preferably, L is selected from the group consisting of: —R8—S—R9—C(O)—, —R8—S—R12—, —O—P(O)2—O—R12—, —O—P(O)2—O—R9—C(O)—, —R8—NH—(O)C—R9—C(O)—, —R8—NH—(O)C—R12—, —R8—NH— (O)C—, —R8—NH—C(S)—NH—R12—, —R8—NH—C(S)—NH—R9—C(O)—,
Even more preferably, L is selected from the group consisting of: —O—P(O)2—O—R12—, —O—P(O)2—O—R9—C(O)—, —R8—NH—(O)C—R9—C(O)—, —R8—NH—(O)C—R12—, —R8—NH— (O)C—;
Particularly preferable are the embodiments wherein L is selected from the group consisting of: —R8—NH— (O)C—, —O—P(O)2—O_R12—.
With reference to what has been described above, according to preferred embodiments R10 and R11 represent, each independently from each other, a respective alkyl C1-C6. Preferably, R10 and R11 represent, each independently from each other, a respective saturated alkyl.
According to preferred embodiments, R8 and R9 represent, each independently from each other, a respective alkylene C1-C6. Preferably, R8 and R9 represent, each independently from each other, a respective saturated alkylene. Even more preferably, R8 and R9 represent, each independently from each other, a respective linear alkylene. Particularly preferable are the embodiments wherein R8 represents a hexyl.
Preferably, R12 is selected from the group consisting of: alkylene C2-C9, —R9—Si (R10)(R11)—. More preferably, R12 represents an alkylene C2-C7. Even more preferably, R12 represents an alkylene C2-C3. According to preferred embodiments, R12 represents a saturated and preferably linear alkylene.
From the descriptions provided above it is obvious that the compounds presenting the general formulas III, V, VI and VII can be used not only as beacons for oligonucleotide probes but also as beacons for any other type of application including pharmacology.
Oligonucleotide probes having the general formula I, have the following advantages compared to known oligonucleotide probes:
Further characteristics of the present invention will be made clear from the following description of certain examples provided simply as illustrations and not to be considered in any way limiting.
The examples from 1 to 7 are schematically illustrated in the scheme 1 shown below wherein R represents a hydrogen:
Bromobutyrylchloride (2.35 mmol, 0.27 mL) is added to a solution of aluminium chloride (2.82 mmol, 375 mg) in 20 mL of methylene chloride at 0° C. The mixture is stirred for an hour and added a drop at a time to a solution of dithien[3,2-b; 2′,3′-d]thiophene (2.35 mmol, 460 mg) dissolved in 25 mL of methylene chloride at 0° C. The reaction mixture is left to be mixed at room temperature overnight and is later quenched with a solution of hydrochloric acid 0.1M. The product is extracted using ether and methylene chloride and the organic phases are anhydrified with anhydrous sodium sulphate. The solvent is eliminated using a rotavapor and the residue purified through crystallization from pentane. 649 mg of a light green powder is obtained (Yield 80%): mp 104° C.; MS m/e 346 (M+); FTIR (neat) vCO 1649 cm−1; 1H NMR (CDCl3, TMS/ppm) δ 7.972 (s, 1H), 7.535 (d, 3J=5.2 Hz, 1H), 7.332 (d, 3J=5.2 Hz, 1H), 3.565 (t, 3J=6.4 Hz, 2H), 3.174 (t, 3J=6.8 Hz, 2H), 2.349 (m, 2H); 13C NMR (CDCl3, TMS/ppm) δ 192.154, 145.437, 143.927, 141.453, 137.47, 131.056, 129.501, 126.253, 121.183, 36.978, 33.730, 27.409
A solution of 4-bromo-1-dithien[3,2-b;2′,3′-d]thien-2-yl-butane-1-one (1) (0.0030 mol, 1 g) in 20 mL of methylene chloride is added a drop at a time to a solution of aluminium chloride (0.015 mol, 2.04 g) and borane tributylamine (0.031 mol, 2.66 g) in 30 mL of methylene chloride at 0° C. The reaction mixture is left to be stirred for 4 hours at room temperature and is then quenched with a solution of hydrochloric acid 0.1M. the product is extracted with diethyl ether and methylene chloride, the organic phase anhydrified with sodium sulphate and the solvent eliminated with a rotavapor. The residue is purified through flash-chromatography on aluminia using the mixture of pentane: methylene chloride 9:1, as an eluent, to obtain 1.20 g of yellow oil product (Yield 71%): MS m/e 332 (M+); 1H NMR (CDCl3, TMS/ppm) δ 7.308 (d, 3J=4.8 Hz, 1H), 7.264 (d, 3J=4.8 Hz, 1H), 6.991 (t, 4J=1.2 Hz, 1H), 3.442 (t, 3, J=6.4 Hz, 2H), 2.946 (t, 3J=6.4 Hz, 2H), 1.931 (m, 4H); 13C NMR (CDCl3, TMS/ppm) δ 145.814, 140.714, 140.236, 131.091, 128.860, 125.119, 120.664, 117.933, 33.287, 31.722, 30.176, 29.918
A solution of m-chloro perbenzoic acid (5.438 mmol, 1.22 g), previously anhydrified with magnesium sulphate is added a drop at a time to a solution of 2-(4-bromo-butyl)-dithien[3,2-b;2′,3′-d]thiophene (2) (1.807 mmol, 0.6 g) in 25 mL of methylene chloride. The mixture is stirred overnight at room temperature, after which it is first washed with distilled water. KOHaq at 10% and lastly NaHCO3aq at 10% and extracted using methylene chloride. The organic phase is anhydrified on sodium sulphate, the solvent eliminated using the rotavapor and the residue purified using flash-chromatography on aluminia and using as an eluent the mixture ether: methylene chloride:ethyl acetate 6:1:3. 436 mg of a yellow solid are obtained (Yield 66%): mp 110° C.; MS m/e 364 (M+); FTIR (neat) vSO2 1307, 1139 cm−1; 1H NMR (CDCl3, TMS/ppm) δ7.306 (d, 3J=5.2 Hz, 1H), 7.197 (d, 3J=5.2 Hz, 1H), 6.928 (t, 4J=1.2 Hz, 1H), 3.434 (t, 3J=6.4 Hz, 2H), 2.871 (t, 3J=8.4 Hz, 2H), 1.902 (m, 4H); 13C NMR (CDCl3, TMS/ppm) δ 150.566, 142.606, 142.097, 136.489, 133.362, 128.923, 120.173, 117.153, 32.895, 31.551, 29.707, 29.707
A solution of 2-(4-bromo-butyl)-dithien[3,2-b;2′,3′-d]thiophene-4,4-dioxide (3) (0.137 mmol, 50 mg) and sodium thiocyanate (2.747 mmol, 222.5 mg) is placed in a 5 mL wheaton V-Vial in 5 mL of distilled acetone. The mixture is stirred strongly at 200° C. for 4 hours. After cooling at room temperature, the synthesis crude is filtered on a silica plug to remove excess sodium salt, and is then purified through crystallization from toluene/pentane to produce 40 mg (Yield 85%) of a yellow ochre solid: MS m/e 341 (M+); FTIR (neat) vSO2 1306, 1139 cm−1; vNCS 2149 cm−1; λmax (CH2Cl2)=364λem (CH2Cl2)=456 nm; ε (CH2Cl2)=8523 mol−1*cm−1; 1H NMR (CDCl3, TMS/ppm) δ 7.315 (d, 3J=5.2 Hz, 1H), 7.196 (d, 3J=5.2 Hz, 1H), 6.932 (t, 4J=1.2 Hz, 1H), 2.980 (t, 3J=6.8 Hz, 2H), 2.894 (t, 3J=7.6 Hz, 2H), 1.889 (m, 4H); 13C NMR (CDCl3, TMS/ppm) δ 149.898, 142.643, 142.127, 136.352, 133.506, 129.074, 120.165, 117.258, 111.961, 33.456, 29.866, 29.525, 29.039
154 mg (0.424 mmol) of 2-(4-bromo-butyl)-dithien[3,2-b;2′,3′-d]thiophene-4,4-dioxide (3) is dissolved in 2 mL of N-Methylpyrrolidone (NMP) with the addition of 0.5 mL of distilled water. The mixture is brought to reflux and left to react for 12 hours. It is washed several times with water then with methylene chloride. The organic phase is anhydrified and the solvent eliminated by the rotavapor. The residue, a brown oil, is distilled to eliminate residue NMP and then purified using flash-chromatography on silica, eluting with ether:ethyl acetate: methylene chloride: 5:3:2. This produces 93 mg (Yield 74%) of a yellow crystalline solid: mp 115° C.; MS m/e 300 (M+); FTIR (neat) vSO2 1304, 1135 cm−1, vOH 3368 cm−1, vCO 1063 cm−1; λmax(CH2Cl2)=363 nm; λem(CH2Cl2)=457 nm 1H NMR (CDCl3, TMS/ppm) δ 7.293 (d, 3J=5.2 Hz, 1H), 7.188 (d, 3=5.2 Hz, 1H), 6.918 (t, 4J=1.2 Hz, 1H), 3.683 (t, 3J=6.4 Hz, 2H), 2.870 (t, 3J=8.4 Hz, 2H), 1.682 (m, 4H); 13C NMR (CDCl3, TMS/ppm) δ 151.340, 142.545, 142.006, 136.610, 133.173, 128.817, 120.143, 116.993, 62.233, 31.680, 30.352, 27.628
In solution of CH2Cl2 4-bromo-1-dithien[3,2-b;2′,3′-d]thien-2-yl-butane-1-one(1) is made to react with mCPBA. A pale orange powder is obtained: mp 187° C.; MS m/e 378 (M+); FTIR (neat) vSO2 1303, 1141 cm−1, vCO 1646 cm−1; λmax (CH2Cl2)=374 nm; λem (CH2Cl2)=454 nm; ε (CH2Cl2)=14000 mol−1*cm−1; 1H NMR (CDCl3, TMS/ppm) δ 7.769 (s, 1H), 7.543 (d, 3J=4.8 Hz, 1H), 7.298 (d, 3J=4.8 Hz, 1H), 3.117 (m, 4H), 2.316 (m, 2H); 13C NMR (CDCl3, TMS/ppm) δ 191.067, 147.461, 144.957, 143.159, 141.914, 135.084, 132.420, 123.610, 120.703, 36.726, 32.886, 26.610
4-bromo-1-(4,4-dioxy-dithien[3,2-b;2′,3′-d]thien-2-yl)-butane-1-one (5) is made to react with NaSCN in acetone. A red solid is obtained: mp 155° C.; MS m/e 355 (M+); FTIR (neat) vSO21302, 1138 cm−1, vCO 1661 cm−1, vNCS 2152 cm−1; λmax (CH2Cl2)=480 nm; λem (CH2Cl2)=586 nm; ε (CH2Cl2)=44480 mol−1*cm−1; 1H NMR (CDCl3, TMS/ppm) δ 7.761 (s, 1H), 7.537 (d, 3J=5.2 Hz, 1H), 7.291 (d, 3J=5.2 Hz, 1H), 2.498 (s, 4H), 0.535 (s, 12H); 13C NMR (CDCl3, TMS/ppm) δ 142.908, 142.840, 141.922, 137.588, 136.784, 134.318, 134.196, 133.225, 125.932, 125.864, 125.165, 115.611, 113.927, 18.634, −2.592
The examples from 8 to 10 are schematically illustrated in the scheme 2 shown below wherein R represents at hydrogen:
N-bromosuccinimide (0.493 mmol, 87.7 mg) is added in small doses to a solution of 2-(4-bromo-butyl)-dithien[3,2-b;2′,3′-d]thiophene-4,4-dioxide (0.448 mmol, 163 mg) dissolved in 20 mL of a solution 1:1 of methylene chloride: acetic acid at −20° C. and wrapped in tin foil to protect it from the light. The mixture is stirred at room temperature overnight and then quenched with water. The aqueous phase is extracted using methylene chloride, the organic phases are washed with KOHaq at 10% k and NaHCO3aq at 10%. Anhydrification is carried out with sodium sulphate, then the solvent is eliminated and the residue is crystallized from toluene/pentane to obtain 188 mg of yellow powder product (95% of Yield): mp 165° C.; MS m/e 442 (M+); 1H NMR (CDCl3, TMS/ppm) δ 7.259 (s, 1H), 6.930 (t, 4J=1.2 Hz, 1H), 3.434 (t, 3J=6.4 Hz, 2H), 2.868 (t, 3J=8.0 Hz, 2H), 1.903 (m, 4H); 13C NMR (CDCl3, TMS/ppm) δ 151.137, 141.431, 141.044, 136.331, 132.780, 122.558, 117.223, 115.773, 32.828, 31.545, 29.777, 29.686
2-(4-bromo-butyl)-6-bromo-dithien[3,2-b;2′,3′-d]thiophene-4,4-dioxide (0.424 mmol, 188 mg) is dissolved in 5 mL of toluene followed by the addition of the catalyst generated in situ Pd(Ph3As)4 0.011 mmol. The mixture is brought to reflux and the 2-tributylstannyl-5-octylsulfanyl-thiophene (0.488 mmol; 252 mg) is added a drop at a time, using a syringe. The mixture is left at reflux for another 5 hours, then the solvent is eliminated using the rotavapor and the residue purified using a chromatographic column on silica, with the mixture pentane:ethyl acetate: methylene chloride 6:3:1 as eluent, the product obtained recrystallizes from isopropylic alcohol to produce 194 mg (Yield 78%) of a micro-crystalline orange solid: mp 98° C.; MS m/e 590 (M+); FTIR (neat) vSO21302, 1134 cm−1; λmax (CH2Cl2)=421 nm; λem (CH2Cl2)=550 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.172 (s, 1H), 7.048 (d, 3J=3.6 Hz, 1H), 7.001 (d, 3J=3.6 Hz, 1H), 6.934 (t, 4J=0.8 Hz, 1H), 3.438 (t, 3J=6.4 Hz, 2H), 2.859 (m, 4H), 1.907 (m, 4H), 1.641 (m, 2H), 1.399 (m, 2H), 1.267 (bs, 8H), 0.875 (t, 6.8 Hz, 3H); 13C NMR (CDCl3, TMS/ppm) δ 150.801, 142.461, 141.771, 141.194, 137.521, 137.225, 133.772, 133.469, 133.378, 125.091, 117.251, 115.574, 38.768, 32.864, 31.749, 31.574, 29.783, 29.723, 29.373, 29.131, 29.055, 28.410, 22.612, 14.082
2-(4-bromo-butyl)-6-(5-octylsulfanyl-thien-2-yl)-dithien[3,2-b;2′,3′-d]thiophene-4,4-dioxide (9) is made to react with NaSCN in a solution of acetone. An orange solid is obtained: mp 102° C.; MS m/e 622 (M+); FTIR (neat) vso21302, 1134 cm−1, vNCS 2154 cm−1; λmax (CH2Cl2)=421 nm; λem (CH2Cl2)=550 nm; ε(CH2Cl2)=28417 mol−1*cm−1; 1H NMR (CDCl3, TMS/ppm) δ 7.176 (s, 1H), 7.054 (d, 3J=4.0 Hz, 1H), 7.004 (d, 3J=3.6 Hz, 1H), 6.943 (t, 4J=1.2 Hz, 1H), 2.988 (t, 3J=6.8 Hz, 2H), 2.905 (t, 3J=7.6 Hz, 2H), 2.842 (t, 3J=7.2 Hz, 2H), 1.913 (m, 4H), 1.642 (m, 2H), 1.400 (m, 2H), 1.268 (bs, 8H), 0.875 (t, 6.8 Hz, 3H); 13C NMR (CDCl3, TMS/ppm) δ 150.080, 142.529, 141.823, 141.337, 137.459, 137.308, 133.627, 133.551, 133.460, 125.135, 117.387, 115.573, 111.923, 38.767, 33.486, 31.740, 29.964, 29.562, 29.365, 29.122, 29.084, 29.054, 28.409, 22.611, 14.081
The examples from 11 to 13 are schematically illustrated in the scheme 3 shown below wherein R represents a hydrogen:
Starting with 4-bromo-1-(4,4-dioxy)-dithien[3,2-b;2′,3′-d]thien-2-yl-butane-1-one (5) and following a similar procedure to that described in example 8 a yellow solid is obtained: mp 210° C.; MS m/e 456 (M+); λmax (CH2Cl2)=385 nm; FTIR (neat) vSO21311, 1156 cm−1, vCO 1651 cm−1; 1H NMR (CDCl3, TMS/ppm) δ 7.777 (s, 1H), 7.292 (s, 1H), 3.526 (t, 3J=6.4 Hz, 2H), 3.106 (t, 3J=6.8 Hz, 2H), 2.311 (m, 2H); 13C NMR (CDCl3, TMS/ppm) δ 151.137, 141.431, 141.044, 136.331, 132.780, 122.558, 117.223, 115.773, 32.828, 31.545, 29.777, 29.686
Staring with 4-bromo-1-(4,4-dioxy-6-bromo-dithien[3,2-b;2′,3′-d]thien-2-yl)-butane-1-one (11) and following a similar procedure to that described in example 9 a red solid is obtained: mp 155° C.; MS m/e 604 (M+); FTIR (neat) vSO21306, 1139 cm−1, vCO 1654 cm−1; λmax (CH2Cl2)=440 nm; λem (CH2Cl2)=600 nm; ε (CH2Cl2)=14187 mol−1*cm−1, 1H NMR (CDCl3, TMS/ppm) δ 7.284 (d, 3J=3.6 Hz, 2H), 7.246 (d, 3J=3.6 Hz, 2H), 7.245 (s, 2H), 7.140 (bs, 4H), 2.966 (s, 4H), 0.486 (s, 12H); 13C NMR (CDCl31 TMS/ppm) δ 142.894, 142.227, 142.079, 138.053, 136.315, 135.943, 133.947, 133.196, 125.933, 125.758, 124.924, 115.574, 30.327, −3.505
Starting with 4-bromo-1-(4,4-dioxy-6-(5′-octysulfanyl-thien-2′-yl)-dithien[3,2-b;2′,3′-d]thien-2-yl)-butane-1-one (12) and following a similar procedure to that described in example 10, a red solid is obtained: MS m/e 581 (M+); FTIR (neat) vSO21305, 1140 cm−1, vCO 1656 cm−1, vNCS 2152 cm−1; λmax (CH2Cl2)=480 nm; λem (CH2Cl2)=586 nm; ε (CH2Cl2)=44480 mol−1*cm−1; 1H NMR (CDCl3, TMS/ppm) δ 7.761 (s, 1H), 7.537 (d, 3J=5.2 Hz, 1H), 7.291 (d, 3J=5.2 Hz, 1H), 7.118 (bs, 4H), 2.498 (s, 4H), 0.535 (s, 12H); 13C NMR (CDCl3, TMS/ppm) δ 190.308, 146.771, 145.603, 145.079, 142.377, 142.157, 139.038, 136.564, 133.195, 131.707, 126.092, 123.618, 115.718, 111.681, 38.661, 35.816, 33.069, 31.763, 29.373, 29.145, 29.062, 28.439, 23.924, 22.634, 14.097
Triphenylphosphine (0.374 mmol, 0.098 g) is placed in a flask under nitrogen in 5 mL of anhydrous THF. The flask is cooled to −78° C. and diethylazodicarboxylate (0.374 mmol, 0.06 mL) is added. It is left to be stirred for 5 min then 2-[2,2′;5′,2″]terthien-5-yl-ethanol (0.34 mmol, 0.100 g) is added leaving it to stir for another 5 min. Neopentyl alcohol (0.175 mmol, 0.015 g) and maleimide (0.374 mmol, 0.037 g) are added. This is left at −78° C. for a further 5 minutes then at room temperature for the whole night. The reaction crude is purified using a chromatographic column on silica gel eluting with petroleum ether: ethyl acetate 7:3. A yellow-orange solid is obtained.
1H NMR (CDCl3, TMS/ppm) δ 7.21 (dd, 3J=5.2 Hz, 4J=1.4 Hz, 1H), 7.16 (dd, 3J=3.8 Hz, 4J=1.2 Hz, 1H), 7.02 (m, 4H), 6.74 (d, 3J=3.6 Hz, 1H), 6.70 (s, 2H), 3.82 (t, 2H), 3.11 (t, 2H); 13C NMR (CDCl3, TMS/ppm) δ 170.42, 139.15, 137.126, 136.10, 135.90, 135.93, 134.15, 127.86, 126.52, 124.42, 124.25, 123.94, 123.63, 123.52, 38.94, 28.70.
A bromine derivative (0.5 moles) is added to a solution of Pd(PPh3)4 prepared in situ (0.015 mmoles) in toluene (5 mL) in an inert atmosphere. The mixture is heated to 80° C., followed by the introduction of stannum derivative (0.5 mmoles) dissolved in toluene (3 mL). After 2 hours the reaction mixture is left to cool at room temperature, the solvent is removed and the compound purified using flash-chromatography (silica gel, petroleum ether-ethyl acetate 1:1) in order to obtain a white microcrystalline solid. Yield: 127 mg (83%), pf 159-160° C.; EI-MS m/z 307 (M+); λmax (CH2Cl2) 347 nm; εmax 24000; λem (CH2Cl2) 418 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.91 (d, 3J=4.0 Hz, 1H), 7.36 (dd, 3J=5.0 Hz, 4J=0.8 Hz, 1H), 7.33 (dd, 3J=3.6 Hz, 4J=0.8 Hz, 1H), 7.21 (d, 3J=4.0 Hz, 1H), 7.07 (dd, 3J=3.6 Hz, 3J=5.0 Hz, 1H), 2.89 (s, 4H); 13C NMR (CDCl3, TMS/ppm) δ 169.14, 157.09, 147.71, 137.52, 135.41, 128.32, 127.13, 126.19, 124.26, 124.05, 25.58; Anal. calculated for C13H9NO4S2 (307, 34): C, 50.80; H, 2.95. Found: C, 50.92; H, 3.02
ester 2,5-dioxy-pyrrolidin-1ylic of 2,2′;5′,2′-terthiophene-5-carboxylic acid
Following a procedure similar to that described in example 15 an amorphous lemon yellow solid is obtained. Yield: 185 mg (95%), pf 223-224° C.; EI-MS m/z 389 (M+); m, (CH2Cl2) 395 nm; εmax 36900; λem (CH2Cl2) 482 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.92 (d, 3J=4.0 Hz, 1H), 7.28 (dd, 3J=5.2 Hz, 4J=1.2 Hz, 1H), 7.26 (d, 3J=4.0 Hz, 1H), 7.23 (dd, 3J=4.0 Hz, 4J=1.2 Hz, 1H), 7.20 (d, 3J=4.0 Hz, 1H), 7.13 (d, 3J=4.0 Hz, 1H), 7.05 (dd, 3J=4.0 Hz, 3J=5.2 Hz, 1H), 2.90 (s, 4H); 13C NMR (CDCl3, TMS/ppm) δ 169.14, 157.08, 147.41, 139.22, 137.61, 136.31, 133.89, 128.07, 126.93, 125.43, 124.63, 124.54, 124.05, 123.95, 25.62; Anal. calculated for C17H11NO4S3 (389.47): C, 52.43; H, 2.85; found: C, 52.54; H, 2.98.
Following a procedure similar to that described in example 15 an amorphous orange coloured solid is obtained. Yield: 188 mg (80%), pf 263-264° C.; EI-MS m/z 471 (M+); λmax (CH2Cl2) 421 nm; εmax 45100; λem (CH2Cl2) 536 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.93 (d, 3J=4.0 Hz, 1H), 7.26 (d, 3J=4.0 Hz, 1H), 7.24 (dd, 3J=4.0 Hz, 4J=1.2 Hz, 1H), 7.21 (d, 3J=4.0 Hz, 1H), 7.20 (dd, 3J=5.2 Hz, 4J=1.2 Hz, 1H), 7.12 (m, 3H), 7.04 (dd, 3J=4.0, 3J=5.2, 1H), 2.90 (s, 4H); 13C NMR (CDCl3, TMS/ppm) δ 169.10, 157.08, 147.32, 138.93, 137.60, 137.45, 137.39, 136.76, 134.97, 133.97, 127.97, 127.02, 125.17, 124.91, 124.53, 124.49, 124.10, 124.07, 25.64; Anal. calculated for C27H25NO7S4 (4713.59): C, 53.48; H, 2.78. Found: C, 53.54; H, 2.83.
Following a procedure similar to that described in example 15 a light yellow oil is obtained. Yield; 180-mg (82%), EI-MS m/z 439 (M+); λmax (CH2Cl2) 359 nm; δmax 26700; λem (CH2Cl2) 432 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.89 (d, 3J=4.0 Hz, 1H), 7.15 (d, 3J=4.0 Hz, 1H), 7.12 (d, 3J=4.0 Hz, 1H), 6.80 (d, 3J=4.0 Hz, 1H), 4.75 (s, 2H), 3.81 (t, J=6.0 Hz, 2H), 3.67 (m, 2H), 3.53 (m, 2H), 3.37 (s, 3H), 3.09 (t, J=6.0 Hz, 2H), 2.88 (s, 4H); 13C NMR (CDCl3, TMS/ppm) δ 169.14, 157.06, 148.09, 144.12, 137.48, 133.67, 126.57, 125.91, 123.59, 123.41, 95.43, 71.61, 67.70, 66.85, 58.89, 30.70, 25.52; Anal. calculated for C19H21NO7S2 (439.50): C, 51.92; H, 4.82. Found: C, 51.95; H, 4.96.
Following a procedure similar to that described in example 15 a yellow-orange poly-crystalline solid is obtained. Yield: 209 mg (80%), pf 119-120° C.; EI-MS m/z 521 (M+); λmax (CH2Cl2) 404 nm; εmax 33700; λem (CH2Cl2) 498 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.88 (d, 3J=4.0 Hz, 1H), 7.21 (d, 3J=4.0 Hz, 1H), 7.16 (d, 3J=4.0 Hz, 1H), 7.02 (d, 3J=4.0 Hz, 2H), 6.77 (d, 3J=3.6 Hz, 1H), 4.74 (s, 2H), 3.80 (t, J=6.2 Hz, 2H), 3.66 (m, 2H), 3.52 (m, 2H), 3.36 (s, 3H), 3.07 (t, J=6.2 Hz, 2H), 2.87 (s, 4H); 13C NMR (CDCl3, TMS/ppm) δ 169.14, 157.02, 147.47, 142.08, 139.54, 137.55, 134.54, 133.32, 126.88, 126.24, 124.17, 123.97, 123.87, 123.70, 95.46, 71.66, 67.91, 66.85, 58.95, 30.70, 25.58; Anal. calculated for C23H23NO7S3 (521.63): C, 52.96; H, 4.44. Found: C, 52.98; H, 4.49.
Following a similar procedure to that described in example 15 an amorphous orange coloured solid is obtained. Yield: 260 mg (86%), pf 141-142° C.; EI-MS m/z 603 (M+); λmax (CH2Cl2) 427 nm; εmax 61900; λem (CH2Cl2) 552 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.91 (d, 3J=4.0 Hz, 1H), 7.24 (d, 3J=4.0 Hz, 1H), 7.19 (d, 3J=4.0 Hz, 1H), 7.09 (m, 2H), 7.01 (m, 2H), 6.77 (d, 3J=4.0, 1H), 4.75 (s, 2H), 3.81 (t, J=6.6 Hz, 2H), 3.68 (m, 2H), 3.54 (m, 2H), 3.38 (s, 3H), 3.08 (t, J=6.6 Hz, 2H), 2.89 (s, 4H); 13C NMR (CDCl3, TMS/ppm) δ 169.10, 157.06, 147.34, 141.52, 139.03, 137.74, 137.59, 135.03, 134.45, 133.79, 127.00, 126.18, 125.13, 124.37, 124.04, 123.98, 123.87, 123.72, 95.55, 71.74, 68.05, 66.92, 59.00, 30.78, 25.63; Anal. calculated for C27H25NO7S4 (603.05): C, 53.71; H, 4.17. Found: C, 53.82; H, 4.23.
Following a similar procedure to that described in example 15 a lemon yellow microcrystalline solid is obtained. Yield: 143 mg (81%), pf 181-182° C.; EI-MS m/z 353 (M+); λmax (CH2Cl2) 370 nm; εmax 27300; λem (CH2Cl2) 479 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.90 (d, 3J=4.0 Hz, 1H), 7.18 (d, 3J=4.0 Hz, 1H), 7.15 (d, 3J=4.0 Hz, 1H), 6.99 (d, 3J=4.0 Hz, 1H), 2.90 (s, 4H), 2.55 (B, 3H); 13C NMR (CDCl3, TMS/ppm) δ 169.08, 157.09, 147.21, 140.57, 137.53, 136.60, 130.84, 126.37, 124.08, 124.02, 25.63, 21.44; Anal. calculated for C14H11NO4S3 (353.44): C, 47.58; H, 3.14; found: C, 47.76; H, 3.26.
Following a similar procedure to that described in example 15 a yellow-orange amorphous solid is obtained. Yield: 55 mg (26%), pf 239-240° C.; EI-MS m/z 419 (M+); λmax (CH2Cl2) 396 nm; 480 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.95 (d, 3J=4.0 Hz, 1H), 7.55 (s, 1H), 7.44 (d, 3J=5.2 Hz, 1H), 7.31 (d, 3J=5.2 Hz, 1H), 7.26 (d, 3J=4.0 Hz, 1H), 2.91 (9, 4H); Anal. calculated for C17H9NO4S4 (418.94): C, 48.67; H, 2.16. Found: C, 48.76; H, 2.22.
Following a similar procedure to that described in example 15 an orange amorphous solid is obtained. Yield: 140 mg (62%), pf 260-261° C.; EI-MS m/z 307 (M+); λmax (CH2Cl2) 405 nm; εmax 32400; λem (CH2Cl2) 491 nm; 1H NMR (CDCl3, TMS/ppm) δ 7.95 (d, 3J=3.6 Hz, 1H), 7.44 (d, 3J=4.8 Hz, 1H), 7.42 (s, 1H), 7.28 (d, 3J=3.6 Hz, 1H), 7.26 (d, 3J=4.8 Hz, 1H), 2.92 (s, 4H); 13C NMR (CDCl3, TMS/ppm) δ 168.95, 156.77, 144.63, 144.05, 143.23, 139.58, 137.47, 135.84, 135.40, 130.75, 126.33, 125.55, 120.56, 118.14, 25.64; Anal. calculated for C17H9NO6S4 (450.93): C, 45.22; H, 2.01. Found: C, 45.25; H, 2.09.
4-dimethylamminopyridine (2%) and methoxyethoxylmethoxyl chloride are added to a mixture of 2-thiophen-2-yl-ethanol (2.56 g 0.02 mol) and N,N-diisopropylethylammine (3.87 g 0.03 mol) dissolved in 30 mL of methylene chloride, a drop at a time at room temperature. The reaction mixture is left to react overnight, then washed several times with a saturated solution of NaHCO3 and extracted using methylene chloride. The organic phase is anhydrified with sodium sulphate and the solvent removed using the rotavapor. The residue is purified using flash-chromatography on silica eluting with petroleum ether:ethyl acetate 7:3. 4.32 g of yellow oil is obtained (Yield 98%):
MS m/e 141 (M+); 1H NMR (CDCl3, TMS/ppm) δ 7.125 (dd, 3J=6.4 Hz, 1H), 6.918 (dd, 3J=8.6 Hz, 1H), 6.846 (m, 1H), 4.732 (B, 1H), 3.796 (t, 2H), 3.654 (m, 2H) 3.529 (m, 2H), 3.373 (8, 3H), 3.106 (t, 2H);
The compound
that for simplicity is referred to in this case as A/MZ292, emits in yellow and presents an absorbency spectrum in H2O/DMSO as shown in
Under nitrogen atmosphere, 5 ml of CH2Cl2 and 0.129 ml of DIPEA are added to the residue and mixed for 5 minutes; using a syringe, B is added a drop at a time; it is left to react, controlling the reaction progress through TLC (eluent mixture: CH2Cl2/EtOAc/Et3N; 45/45/10).
After 30 minutes the reaction is considered completed and WORK UP is started with a saturated solution of NaHCO3 and then with water. Anhydrification is carried out with anhydrous Na2SO4, followed by solution filtering and concentration.
The purification of the reaction crude occurs through preparative TLC on a plate (eluent mixture: CH2Cl2/EtOAc/Et3N; 45/45/10).
The phosphoroammidite obtained was used for synthesis, on an automatic synthesizer for oligonucleotides, with two conjugates
whose absorption profile in H2O is shown in
A/MZ292-T4 has an emission profile shown in
La
wherein Oligo1 represents 5′ACCACCCTTCGAACCACAC 3′. A/MZ292-Oligol presents the absorption profile shown in
The compound
which for simplicity, is referred to in this case as A/MZ03, emits in yellow and presents an absorbency spectrum as shown in
Process: Oligo2 is dissolved in water, A/MZ-03 and DMF are added. Since the fluorescent compound does not dissolve, a further 60.8 μl of DMF is added. The sample is placed in an oven at 46° C. for 16 h. It is controlled to ensure that conjugation has occurred by means of injection in HPLC in inverse phase.
The excess A/MZ-03 is extracted using a mixture of CH2Cl2/MeOH (85/15) until the organic phase remains without any color. The conjugated Oligo2-A/MZ03 extracted in water emits fluorescence in yellow when irradiated at 360 nm.
| Number | Date | Country | Kind |
|---|---|---|---|
| BO2004A000697 | Nov 2004 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB05/03369 | 11/10/2005 | WO | 00 | 8/11/2008 |
