Patent Application
20250215494
The present application relates to dyes containing a tricyclic polymethine core and their uses as fluorescent labels in nucleic acid sequencing applications.
Non-radioactive detection of nucleic acids bearing fluorescent labels is an important technology in molecular biology. Many procedures employed in recombinant DNA technology previously relied on the use of nucleotides or polynucleotides radioactively labeled with, for example 32P. Radioactive compounds permit sensitive detection of nucleic acids and other molecules of interest. However, there are serious limitations in the use of radioactive isotopes such as their expense, limited shelf life, insufficient sensitivity, and, more importantly, safety considerations. Eliminating the need for radioactive labels reduces both the safety risks and the environmental impact and costs associated with, for example, reagent disposal. Methods amenable to non-radioactive fluorescent detection include by way of non-limiting examples, automated DNA sequencing, hybridization methods, real-time detection of polymerase-chain-reaction products, and immunoassays.
For many applications, it is desirable to employ multiple spectrally distinguishable fluorescent labels to achieve independent detection of a plurality of spatially-overlapping analytes. In such multiplex methods, the number of reaction vessels may be reduced, simplifying experimental protocols and facilitating the production of application-specific reagent kits. In multi-color automated DNA sequencing systems for example, multiplex fluorescent detection allows for the analysis of multiple nucleotide bases in a single electrophoresis lane, thereby increasing throughput over single-color methods, and reducing uncertainties associated with inter-lane electrophoretic mobility variations.
However, multiplex fluorescent detection can be problematic and there are a number of important factors that constrain selection of appropriate fluorescent labels. First, it may be difficult to find dye compounds with substantially resolved absorption and emission spectra in a given application. In addition, when several fluorescent dyes are used together, generating fluorescence signals in distinguishable spectral regions by simultaneous excitation may be complicated because absorption bands of the dyes are usually widely separated, so it is difficult to achieve comparable fluorescence excitation efficiencies even for two dyes. Many excitation methods use high power light sources like lasers and therefore the dye must have sufficient photo-stability to withstand such excitation. A final consideration of particular importance to molecular biology methods is the extent to which the fluorescent dyes must be compatible with reagent chemistries such as, for example, DNA synthesis solvents and reagents, buffers, polymerase enzymes, and ligase enzymes.
Fluorescent dye molecules with improved fluorescence properties such as suitable fluorescence intensity, shape, and wavelength maximum of fluorescence band can improve the speed and accuracy of nucleic acid sequencing. Strong fluorescence signals are especially important when measurements are made in water-based biological buffers and at higher temperatures as the fluorescence intensities of most organic dyes are significantly lower under such conditions. Moreover, the nature of the base to which a dye is attached also affects the fluorescence maximum, fluorescence intensity, and others spectral dye properties. The sequence-specific interactions between the nucleobases and the fluorescent dyes can be tailored by specific design of the fluorescent dyes. Optimization of the structure of the fluorescent dyes can improve the efficiency of nucleotide incorporation, reduce the level of sequencing errors, and decrease the usage of reagents in, and therefore the costs of, nucleic acid sequencing.
Improvements in the detection of multiple fluorescent labels can be achieved using fluorescent dyes with different Stokes shifts. The Stokes shift is the difference between the absorption maximum wavelength and the emission maximum wavelength for the same electron transition. Most fluorescent dyes in the visible region have a Stokes shift less than 40 nm, meaning the most efficient excitation wavelength and maximum of emission wavelength are relatively close together. Compounds with longer Stokes shifts have better signal to noise ratio as the emission and excitation wavelength are further apart. Long Stokes shift dyes also allow two different labels to be separately detected using the same emission channel but with different excitation wavelengths. For example, a detection measurement can be recorded between 550-570 nm and can detect signals arising from a first label with a short Stokes shift which is excited at 523 nm, and a second label which has a long Stokes shift and is excited at say 450 nm. Similarly, in one-excitation two-channel sequencing by synthesis (1Ex-2Ch SBS), the best sequencing performance could be obtained when all three sets of dyes (one short Stokes shift, one medium Stokes shift and one long Stokes shift) have little or no emission spectrum overlap (crosstalk).
Increasing cluster density in sequencing applications requires dyes with high quantum efficiencies that are chemical robust and resistant to photobleaching. However, designing new dyes with appropriate adsorption, good chemical stability, and tailored Stokes shifts as nucleic acid labels for sequencing applications remains challenging.
One aspect of the present disclosure relates to a compound of formula (I):
or a salt or a mesomeric form thereof,
In some embodiments, X2 is CR5bR6b and the compound of formula (I) is represented by formula (I-A):
In some embodiments, the compound of formula (I) or (I-A) is also represented by Formula (Ia):
or a salt or a mesomeric form thereof,
In some further embodiments, the compound of formula (Ia) is also represented by formula (Ia-1) or (Ia-2):
or a salt or a mesomeric form thereof,
In some other embodiments, the compound of formula (I) or (I-A) is also represented by formula (Ib):
or a salt or a mesomeric form thereof,
In some further embodiments, the compound of formula (Ib) is also represented by formula (Ib-1) or (Ib-2):
a salt or a mesomeric form thereof,
In some other embodiments, the compound of formula (I) or (I-A) is also represented by formula (Ic) or (Id):
In some other embodiments, the compound of formula (I) is also represented by formula (I-B) or (I-E):
or a salt or a mesomeric form thereof.
In some other embodiments, the compound of formula (I) is also represented by formula (I-C) or (I-D):
or a salt or a mesomeric form thereof.
In some aspect, a compound of the present disclosure is labeled or conjugated with a substrate moiety such as, for example, a nucleoside, nucleotide, polynucleotide, polypeptide, carbohydrate, ligand, particle, cell, semi-solid surface (e.g., gel), or solid surface. The labeling or conjugation may be carried out via a carboxyl group, which can be reacted using methods known in the art with an amino or hydroxy group on a moiety (such as a nucleotide) or a linker bound thereto, to form an amide or ester.
Another aspect of the present disclosure relates to dye compounds comprising linker groups to enable, for example, covalent attachment to a substrate moiety (such as a nucleotide). Linking may be carried out at any position of the dye. In some embodiments, linking may be carried out via a carboxyl group of formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic) or (Id).
A further aspect of the present disclosure provides a labeled nucleoside or nucleotide compound defined by the formula:
N-L-Dye
Another aspect of the present disclosure relates to an oligonucleotide or polynucleotide comprising a nucleotide labeled with a compound of formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic) or (Id) incorporated therein.
A further aspect of the present disclosure relate to a kit comprising the tricyclic polymethine dye described herein, or the dye labeled nucleotides or nucleosides that may be used in various immunological assays, oligonucleotide or nucleic acid labeling, or for DNA sequencing by synthesis (SBS), particularly suited to cycles of SBS on an automated instrument platform. In some aspect, are kits containing one or more nucleotides where at least one nucleotide is a labeled nucleotide described herein.
A further aspect of the disclosure relates to a method of determining the sequences of a plurality of target polynucleotides, comprising:
Embodiments of the present disclosure relate to dyes containing tricyclic polymethine cores (e.g., cyanine dyes with a locked polymethine bridge in the form of a fused tricyclic structure) with enhanced fluorescent intensity, high quantum efficiency under green light excitation, and improved chemical and thermal stability. In some embodiments, Stokes shifts of the dyes described herein range from about 5 nm to 50 nm, or from 8 nm to about 20 nm, or from about 10 nm to about 15 nm. The polymethine dyes described herein may be used in Illumina's sequencing platforms, for example, MiSeq® with two-channel detection and one light excitation (e.g., green light at about 520 nm to about 525 nm, or about 523 nm).
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
It is noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless expressly and unequivocally limited to one referent. It will be apparent to those skilled in the art that various modifications and variations can be made to various embodiments described herein without departing from the spirit or scope of the present teachings. Thus, it is intended that the various embodiments described herein cover other modifications and variations within the scope of the appended claims and their equivalents.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have”, “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.
As used herein, common organic abbreviations are defined as follows:
As used herein, the term “array” refers to a population of different probe molecules that are attached to one or more substrates such that the different probe molecules can be differentiated from each other according to relative location. An array can include different probe molecules that are each located at a different addressable location on a substrate. Alternatively, or additionally, an array can include separate substrates each bearing a different probe molecule, wherein the different probe molecules can be identified according to the locations of the substrates on a surface to which the substrates are attached or according to the locations of the substrates in a liquid. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those including beads in wells as described, for example, in U.S. Pat. No. 6,355,431 B1, US 2002/0102578 and PCT Publication No. WO 00/63437. Exemplary formats that can be used in the invention to distinguish beads in a liquid array, for example, using a microfluidic device, such as a fluorescent activated cell sorter (FACS), are described, for example, in U.S. Pat. No. 6,524,793. Further examples of arrays that can be used in the invention include, without limitation, those described in U.S. Pat. Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413; 6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93/17126; WO 95/11995; WO 95/35505; EP 742 287; and EP 799 897.
As used herein, the term “covalently attached” or “covalently bonded” refers to the forming of a chemical bonding that is characterized by the sharing of pairs of electrons between atoms. For example, a covalently attached polymer coating refers to a polymer coating that forms chemical bonds with a functionalized surface of a substrate, as compared to attachment to the surface via other means, for example, adhesion or electrostatic interaction. It will be appreciated that polymers that are attached covalently to a surface can also be bonded via other means in addition to covalent attachment.
The term “halogen” or “halo,” as used herein, means any one of the radio-stable atoms of column 7 of the Periodic Table of the Elements, e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.
As used herein, “Ca to Cb” in which “a” and “b” are integers refer to the number of carbon atoms in an alkyl, alkenyl or alkynyl group, or the number of ring atoms of a cycloalkyl or aryl group. That is, the alkyl, the alkenyl, the alkynyl, the ring of the cycloalkyl, and ring of the aryl can contain from “a” to “b”, inclusive, carbon atoms. For example, a “C1 to C4 alkyl” group refers to all alkyl groups having from 1 to 4 carbons, that is, CH3—, CH3CH2—, CH3CH2CH2—, (CH3)2CH—, CH3CH2CH2CH2—, CH3CH2CH(CH3)— and (CH3)3C—; a C3 to C4 cycloalkyl group refers to all cycloalkyl groups having from 3 to 4 carbon atoms, that is, cyclopropyl and cyclobutyl. Similarly, a “4 to 6 membered heterocyclyl” group refers to all heterocyclyl groups with 4 to 6 total ring atoms, for example, azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine, and the like. If no “a” and “b” are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl, or aryl group, the broadest range described in these definitions is to be assumed. As used herein, the term “C1-C6” includes C1, C2, C3, C4, C5 and C6, and a range defined by any of the two numbers. For example, C1-C6 alkyl includes C1, C2, C3, C4, C5 and C6 alkyl, C2-C6 alkyl, C1-C3 alkyl, etc. Similarly, C2-C6 alkenyl includes C2, C3, C4, C5 and C6 alkenyl, C2-C5 alkenyl, C3-C4 alkenyl, etc.; and C2-C6 alkynyl includes C2, C3, C4, C5 and C6 alkynyl, C2-C5 alkynyl, C3-C4 alkynyl, etc. C3-C8 cycloalkyl each includes hydrocarbon ring containing 3, 4, 5, 6, 7 and 8 carbon atoms, or a range defined by any of the two numbers, such as C3-C7 cycloalkyl or C5-C6 cycloalkyl.
As used herein, “alkyl” refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated). The alkyl group may also be a medium size alkyl having 1 to 9 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 6 carbon atoms. By way of example only, “C1-6 alkyl” or “C1-C6 alkyl” indicates that there are one to six carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.
As used herein, “alkoxy” refers to the formula —OR wherein R is an alkyl as is defined above, such as “C1-9 alkoxy” or “C1-C9 alkoxy”, including but not limited to methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy, and the like.
As used herein, “alkenyl” refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term “alkenyl” where no numerical range is designated. The alkenyl group may also be a medium size alkenyl having 2 to 9 carbon atoms. The alkenyl group could also be a lower alkenyl having 2 to 6 carbon atoms. By way of example only, “C2-C6 alkenyl” or “C2-6 alkenyl” indicates that there are two to six carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and the like.
As used herein, “alkynyl” refers to a straight or branched hydrocarbon chain containing one or more triple bonds. The alkynyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term “alkynyl” where no numerical range is designated. The alkynyl group may also be a medium size alkynyl having 2 to 9 carbon atoms. The alkynyl group could also be a lower alkynyl having 2 to 6 carbon atoms. By way of example only, “C2-6 alkynyl” or “C2-C6 alkenyl” indicates that there are two to six carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynyl groups include, but are in no way limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.
The term “aromatic” refers to a ring or ring system having a conjugated pi electron system and includes both carbocyclic aromatic (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of atoms) groups provided that the entire ring system is aromatic.
As used herein, “aryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) containing only carbon in the ring backbone. When the aryl is a ring system, every ring in the system is aromatic. The aryl group may have 6 to 18 carbon atoms, although the present definition also covers the occurrence of the term “aryl” where no numerical range is designated. In some embodiments, the aryl group has 6 to 10 carbon atoms. The aryl group may be designated as “C6-C10 aryl,” “C6 or C10 aryl,” or similar designations. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.
An “aralkyl” or “arylalkyl” is an aryl group connected, as a substituent, via an alkylene group, such as “C7-14 aralkyl” and the like, including but not limited to benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a C1-6 alkylene group).
As used herein, “heteroaryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the ring backbone. When the heteroaryl is a ring system, every ring in the system is aromatic. The heteroaryl group may have 5-18 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heteroaryl” where no numerical range is designated. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl group may be designated as “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similar designations. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.
A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, as a substituent, via an alkylene group. Examples include but are not limited to 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a C1-6 alkylene group).
As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When the carbocyclyl is a ring system, two or more rings may be joined together in a fused, bridged or spiro-connected fashion. Carbocyclyls may have any degree of saturation provided that at least one ring in a ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20 carbon atoms, although the present definition also covers the occurrence of the term “carbocyclyl” where no numerical range is designated. The carbocyclyl group may also be a medium size carbocyclyl having 3 to 10 carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3 to 6 carbon atoms. The carbocyclyl group may be designated as “C3-6 carbocyclyl”, “C3-C6 carbocyclyl” or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.
As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in a fused, bridged or spiro-connected fashion. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. The heteroatom(s) may be present in either a non-aromatic or aromatic ring in the ring system. The heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heterocyclyl” where no numerical range is designated. The heterocyclyl group may also be a medium size heterocyclyl having 3 to 10 ring members. The heterocyclyl group could also be a heterocyclyl having 3 to 6 ring members. The heterocyclyl group may be designated as “3-6 membered heterocyclyl” or similar designations. In preferred six membered monocyclic heterocyclyls, the heteroatom(s) are selected from one up to three of O, N or S, and in preferred five membered monocyclic heterocyclyls, the heteroatom(s) are selected from one or two heteroatoms selected from O, N, or S. Examples of heterocyclyl rings include, but are not limited to, aziridinyl, azetidinyl, azepanyl, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.
As used herein, (cycloalkyl)alkyl refers to a cycloalkyl group connected via an alkylene group, such as (C3-C7 cycloalkyl)C1-C6 alkyl. Non-limiting examples include cyclohexyl-(CH2)1-6—, cyclopentyl-(CH2)1-6—, or cyclopropyl-(CH2)1-6—.
As used herein, (aryl)alkyl refers to an aryl group connected via an alkylene group, such as (C6-C10 aryl)C1-C6 alkyl. Non-limiting example includes phenyl-(CH2)1-6.
As used herein, (heteroaryl)alkyl refers to a heteroaryl group connected via an alkylene group, such as (5 to 10 membered heteroaryl)C1-C6 alkyl. Non-limiting examples include pyridyl-(CH2)1-6—, pyrimidinyl-(CH2)1-6—, or pyrrolyl-(CH2)1-6—.
As used herein, (heterocyclyl)alkyl refers to a heterocyclyl group connected via an alkylene group, such as (3 to 10 membered heterocyclyl)C1-C6 alkyl. Non-limiting examples include morpholinyl-(CH2)1-6—, piperidinyl-(CH2)1-6—, piperazinyl-(CH2)1-6—, pyrrolidinyl-(CH2)1-6—, or azetidinyl-(CH2)1-6—.
As used herein, “alkoxyalkyl” or “(alkoxy)alkyl” refers to an alkoxy group connected via an alkylene group, such as C2-C8 alkoxyalkyl, or (C1-C6 alkoxy)C1-C6 alkyl, for example, —(CH2)1-3—OCH3.
An “O-carboxy” group refers to a “—OC(═O)R” group in which R is selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
A “C-carboxy” group refers to a “—C(═O)OR” group in which R is selected from the group consisting of hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. A non-limiting example includes carboxyl (i.e., —C(═O)OH).
As used herein, “carboxylate” refers to a deprotonated carboxyl group “—C(═O)O”.
A “sulfonyl” group refers to an “—SO2R” group in which R is selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
A “sulfino” group refers to a “—S(═O)OH” group.
A “sulfo” group refers to a “—S(═O)2OH” or “—SO3H” group.
A “sulfonate” group refers to a “—SO3−” group.
A “sulfate” group refers to “—SO4−” group.
A “S-sulfonamido” group refers to a “—SO2NRARB” group in which RA and RB are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
An “N-sulfonamido” group refers to a “—N(RA)SO2RB” group in which RA and Rb are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
A “C-amido” group refers to a “—C(═O)NRARB” group in which RA and RB are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
An “N-amido” group refers to a “—N(RA)C(═O)RB” group in which RA and RB are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
An “amino” group refers to a “—NRARB” group in which RA and RB are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. A non-limiting example includes free amino (i.e., —NH2).
An “aminoalkyl” group refers to an amino group connected via an alkylene group.
As used herein, “—OAc” or “—O-acyl” refers to acetyloxy with the structure —O—C(═O)CH3.
When a group is described as “optionally substituted” it may be either unsubstituted or substituted. Likewise, when a group is described as being “substituted”, the substituent may be selected from one or more of the indicated substituents. As used herein, a substituted group is derived from the unsubstituted parent group in which there has been an exchange of one or more hydrogen atoms for another atom or group. Unless otherwise indicated, when a group is deemed to be “substituted,” it is meant that the group is substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5-10 membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5-10 membered heteroaryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), halo, —CN, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g., —CF3), halo(C1-C6)alkoxy (e.g., —OCF3), C1-C6 alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, —SO3H, sulfonate, sulfate, sulfino, —OSO2C1-C4alkyl, and oxo (═O). Wherever a group is described as “optionally substituted” that group can be substituted with the above substituents. In some embodiments, when an alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl or heterocyclyl group is substituted, each is independently substituted with one or more substituents selected from the group consisting of halo, —CN, —SO3−, —OSO3−, —SO3H, —SRA, —ORA, —NRBRC oxo, —CONRBRC, —SO2NRBRC, —COOH, and —COORB, where RA, RB and RC are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.
As understood by one of ordinary skill in the art, a compound described herein may exist in ionized form, e.g., —CO2−, —SO3− or —O—SO3−. If a compound contains a positively or negatively charged substituent group, for example, —SO3, it may also contain a negatively or positively charged counterion such that the compound as a whole is neutral. For example, a compound may contain both —CO2 and a quaternary ammonium cation, or both —SO3 and a quaternary ammonium cation. In other aspects, the compound may exist in a salt form, where the counterion is provided by a conjugate acid or base. It is understood that when the compound described herein is substituted with —SO3H, such substituent may exist in its anionic form —SO3 in aqueous solution.
It is to be understood that certain radical naming conventions can include either a mono-radical or a di-radical, depending on the context. For example, where a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a di-radical. For example, a substituent identified as alkyl that requires two points of attachment includes di-radicals such as —CH2—, —CH2CH2—, —CH2CH(CH3)CH2—, and the like. Other radical naming conventions clearly indicate that the radical is a di-radical such as “alkylene” or “alkenylene.”
When two “adjacent” R groups are said to form a ring “together with the atom to which they are attached,” it is meant that the collective unit of the atoms, intervening bonds, and the two R groups are the recited ring. For example, when the following substructure is present:
Wherever a substituent is depicted as a di-radical (i.e., has two points of attachment to the rest of the molecule), it is to be understood that the substituent can be attached in any directional configuration unless otherwise indicated. Thus, for example, a substituent depicted as -AE- or
includes the substituent being oriented such that the A is attached at the leftmost attachment point of the molecule as well as the case in which A is attached at the rightmost attachment point of the molecule. In addition, if a group or substituent is depicted as
and L is defined an optionally present linker moiety; when L is not present (or absent), such group or substituent is equivalent to
As used herein, a “nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. They are monomeric units of a nucleic acid sequence. In RNA, the sugar is a ribose, and in DNA a deoxyribose, i.e. a sugar lacking a hydroxy group that is present in ribose. The nitrogen containing heterocyclic base can be purine, deazapurine, or pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof, such as 7-deaza adenine or 7-deaza guanine. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine.
As used herein, a “nucleoside” is structurally similar to a nucleotide, but is missing the phosphate moieties. An example of a nucleoside analogue would be one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule. The term “nucleoside” is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside comprising a ribose moiety and a deoxyribonucleoside comprising a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced with a carbon and/or a carbon has been replaced with a sulfur or an oxygen atom. A “nucleoside” is a monomer that can have a substituted base and/or sugar moiety. Additionally, a nucleoside can be incorporated into larger DNA and/or RNA polymers and oligomers.
The term “purine base” is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term “pyrimidine base” is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. A non-limiting list of optionally substituted purine-bases includes purine, adenine, guanine, deazapurine, 7-deaza adenine, 7-deaza guanine. hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (e.g., 5-methylcytosine).
As used herein, when an oligonucleotide or polynucleotide is described as “comprising” a nucleoside or nucleotide described herein, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. Similarly, when a nucleoside or nucleotide is described as part of an oligonucleotide or polynucleotide, such as “incorporated into” an oligonucleotide or polynucleotide, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. In some such embodiments, the covalent bond is formed between a 3′ hydroxy group of the oligonucleotide or polynucleotide with the 5′ phosphate group of a nucleotide described herein as a phosphodiester bond between the 3′ carbon atom of the oligonucleotide or polynucleotide and the 5′ carbon atom of the nucleotide.
As used herein, the term “cleavable linker” is not meant to imply that the whole linker is required to be removed. The cleavage site can be located at a position on the linker that ensures that part of the linker remains attached to the detectable label and/or nucleoside or nucleotide moiety after cleavage.
As used herein, “derivative” or “analog” means a synthetic nucleotide or nucleoside derivative having modified base moieties and/or modified sugar moieties. Such derivatives and analogs are discussed in, e.g., Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprise modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate and phosphoramidate linkages. “Derivative”, “analog” and “modified” as used herein, may be used interchangeably, and are encompassed by the terms “nucleotide” and “nucleoside” defined herein.
As used herein, the term “phosphate” is used in its ordinary sense as understood by those skilled in the art, and includes its protonated forms (for example,
As used herein, the terms “monophosphate,” “diphosphate,” and “triphosphate” are used in their ordinary sense as understood by those skilled in the art, and include protonated forms.
As used herein, the term “phasing” refers to a phenomenon in SBS that is caused by incomplete removal of the 3′ terminators and fluorophores, and/or failure to complete the incorporation of a portion of DNA strands within clusters by polymerases at a given sequencing cycle. Prephasing is caused by the incorporation of nucleotides without effective 3′ terminators, wherein the incorporation event goes 1 cycle ahead due to a termination failure. Phasing and prephasing cause the measured signal intensities for a specific cycle to consist of the signal from the current cycle as well as noise from the preceding and following cycles. As the number of cycles increases, the fraction of sequences per cluster affected by phasing and prephasing increases, hampering the identification of the correct base. Prephasing can be caused by the presence of a trace amount of unprotected or unblocked 3′-OH nucleotides during sequencing by synthesis (SBS). The unprotected 3′-OH nucleotides could be generated during the manufacturing processes or possibly during the storage and reagent handling processes. Accordingly, the discovery of nucleotide analogues which decrease the incidence of prephasing is surprising and provides a great advantage in SBS applications over existing nucleotide analogues. For example, the nucleotide analogues provided can result in faster SBS cycle time, lower phasing and prephasing values, and longer sequencing read lengths.
Some aspects of the disclosure relate to tricyclic polymethine dyes of formula (I), or salts or mesomeric forms thereof:
or a salt or a mesomeric form thereof,
In some embodiments of the compound of formula (I), X2 is CR5bR6b, and the compound is also represented by formula (I-A):
In some other embodiments, both X1 and X2 are O, and the compound is also represented by formula (I-B):
In some other embodiments, both X1 and X2 are S, and the compound is also represented by formula (I-C):
In some other embodiments, X1 is O and X2 is S, and the compound is also represented by formula (I-D):
In still other embodiments, X2 is O, S or N(CH3). In some other embodiments, X1 is NR11 and X2 is NR12 and the compound is also represented by formula (I-E):
In some embodiments of the compounds of formula (I), (I-A), (I-B), (I-C), (I-D) or (I-E), each of R1, R2, R3, R4, R7, R8, R9 and R10 is H. In other embodiments, at least one of R1, R2, R3, R4, R7, R8, R9 and R10 is carboxyl; C1-C6 haloalkyl; halo; sulfo; sulfonate; —SO2NH2; —C(═O)NRBRC; —ORE; unsubstituted 5 or 6 membered heteroaryl containing 1 to 3 heteroatoms selected from O, N and S; unsubstituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; substituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; unsubstituted C1-C6 alkoxy; substituted C1-C6 alkoxy; substituted C2-C6 alkenyl; or substituted C1-C6 alkyl; wherein the substituted C1-C6 alkoxy, substituted C2-C6 alkenyl, or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, —C(═O)NRBRC or —SO2NH2. In some embodiments, R2 is sulfo, sulfonate or —SO2NH2 or R8 is sulfo, sulfonate or —SO2NH2. In other embodiments, R2 and R8 are each independently sulfo, sulfonate or —SO2NH2. In some other embodiments, at least one of R2 and R8 is independently —CF3, —F, or C1-C6 alkoxy (e.g., methoxy or ethoxy). In some other embodiments, at least one of R2 and R8 is independently C1, C2, C3, C4 or C5 alkoxy substituted with sulfo, sulfonate, or carboxyl (e.g., —O(CH2)3SO3H, —O(CH2)4SO3− or —O(CH2)5COOH). In some other embodiments, at least one of R2 and R8 is independently —C(═O)NRBRC, wherein each RB and RC is independently H, unsubstituted C1-C4 alkyl (e.g., methyl or ethyl), or C1-C4 alkyl substituted with sulfo, sulfonate, or carboxyl. In further embodiments, at least one of R2 and R8 is independently —C(═O)NMe2, —C(═O)NEt2, C(O)NH(CH2)2SO3H, or —C(O)NH(CH2)2SO3−). In some other embodiments, at least one of R2 and R8 is independently optionally substituted phenyl, optionally substituted phenoxy, optionally substituted pyridyl, or optionally substituted furanyl. In some other embodiments, at least one of R2 and R8 is independently —CH═CH—C(═O)NRBRC, wherein each RB and RC is independently H, unsubstituted C1-C4 alkyl (e.g., methyl or ethyl), or C1-C4 alkyl substituted with sulfo, sulfonate, or carboxyl. In further embodiment, at least one of R2 and R8 is independently —CH═CH—C(═O)NMe2. In other embodiments, at least one of R2 and R8 is independently C1-C5 alkyl substituted with sulfo, sulfonate, or carboxyl (e.g., —CH2COOH). In some other embodiments, at least one of R2 and R8 is independently unsubstituted or substituted 5 or 6 membered heterocyclyl containing one or two heteroatoms selected from N and O. In further embodiment, at least one of R2 and R8 is independently piperidyl, N-methyl piperidyl, morpholinyl, N-methyl morpholinyl, each optionally substituted with oxo (═O). In some such embodiments, each of R1, R3, R4, R7, R9 and R10 is H. In other embodiments, R1 or R7 is C1-C3 alkoxy (e.g., methoxy or ethoxy), or —C(═O)NRBRC, wherein each RB and RC is independently H, unsubstituted C1-C4 alkyl (e.g., methyl or ethyl), or C1-C4 alkyl substituted with sulfo, sulfonate, or carboxyl. In other embodiments, R3 or R9 is C1, C2, C3, C4 or C5 alkoxy substituted with sulfo, sulfonate, or carboxyl (e.g., —O(CH2)3SO3H, —O(CH2)4SO3− or —O(CH2)5COOH).
In some embodiments of the compounds of formula (I), (I-A), (I-B), (I-C), (I-D) or (I-E), each of R1, R4, R7, R8, R9 and R10 is independently H; carboxyl; unsubstituted C1-C6 haloalkyl; halo; sulfo; sulfonate; —SO2NH2; —C(═O)NRBRC; —ORE; unsubstituted 5 or 6 membered heteroaryl containing 1 to 3 heteroatoms selected from O, N and S; unsubstituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; substituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; unsubstituted C1-C6 alkoxy; substituted C1-C6 alkoxy, substituted C2-C6 alkenyl, or substituted C1-C6 alkyl; wherein the substituted C1-C6 alkoxy, substituted C2-C6 alkenyl, or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, —C(═O)NRBRC or —SO2NH2; and R2 and R3 together with the atoms to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl containing 1 or 2 heteroatoms selected from the group consisting of N, O, and S, for example,
In some other embodiments, R8 is —CF3, —F, or C1-C6 alkoxy (e.g., methoxy or ethoxy). In some other embodiments, R8 is C1, C2, C3, C4 or C5 alkoxy substituted with sulfo, sulfonate, or carboxyl (e.g., —O(CH2)3SO3H, —O(CH2)4SO3− or —O(CH2)5COOH). In some other embodiments, R8 is —C(═O)NRBRC, wherein each RB and RC is independently H, unsubstituted C1-C4 alkyl (e.g., methyl or ethyl), or C1-C4 alkyl substituted with sulfo, sulfonate, or carboxyl. In further embodiments, R8 is —C(═O)NMe2, —C(═O)NEt2, C(O)NH(CH2)2SO3H, or —C(O)NH(CH2)2SO3−). In some other embodiments, R8 is optionally substituted phenyl, optionally substituted phenoxy, optionally substituted pyridyl, or optionally substituted furanyl. In some other embodiments, R8 is —CH═CH—C(═O)NRBRC, wherein each RB and RC is independently H, unsubstituted C1-C4 alkyl (e.g., methyl or ethyl), or C1-C4 alkyl substituted with sulfo, sulfonate, or carboxyl. In further embodiments, R8 is —CH═CH—C(═O)NMe2. In other embodiments, R8 is C1-C5 alkyl substituted with sulfo, sulfonate, or carboxyl (e.g., —CH2COOH). In some other embodiments, R8 is unsubstituted or substituted 5 or 6 membered heterocyclyl containing one or two heteroatoms selected from N and O. In further embodiment, R8 is piperidyl, N-methyl piperidyl, morpholinyl, N-methyl morpholinyl, each optionally substituted with oxo (═O). In some such embodiments, each of R1, R4, R7, R9 and R10 is H. In other embodiments, R1 or R7 is C1-C3 alkoxy (e.g., methoxy or ethoxy), or —C(═O)NRBRC, wherein each RB and RC is independently H, unsubstituted C1-C4 alkyl (e.g., methyl or ethyl), or C1-C4 alkyl substituted with sulfo, sulfonate, or carboxyl. In other embodiments, R9 is C1, C2, C3, C4 or C5 alkoxy substituted with sulfo, sulfonate, or carboxyl (e.g., —O(CH2)3SO3H, —O(CH2)4SO3− or —O(CH2)5COOH).
In other embodiments of the compounds of formula (I), (I-A), (I-B), (I-C), (I-D) or (I-E), each of R1, R2, R3, R4, R7 and R10 is independently H; carboxyl; unsubstituted C1-C6 haloalkyl; halo; sulfo; sulfonate; —SO2NH2, —C(═O)NRBRC; —ORE; unsubstituted 5 or 6 membered heteroaryl containing 1 to 3 heteroatoms selected from O, N and S; unsubstituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; substituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy, substituted C2-C6 alkenyl, or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy, substituted C2-C6 alkenyl or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, —C(═O)NRBRC or —SO2NH2; and R1 and R9 together with the atoms to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl containing 1 or 2 heteroatoms selected from the group consisting of N, O, and S, for example,
In some other embodiments, R2 is —CF3, —F, or C1-C6 alkoxy (e.g., methoxy or ethoxy). In some other embodiments, R2 is C1, C2, C3, C4 or C5 alkoxy substituted with sulfo, sulfonate, or carboxyl (e.g., —O(CH2)3SO3H, —O(CH2)4SO3− or —O(CH2)5COOH). In some other embodiments, R2 is —C(═O)NRBRC, wherein each RB and RC is independently H, unsubstituted C1-C4 alkyl (e.g., methyl or ethyl), or C1-C4 alkyl substituted with sulfo, sulfonate, or carboxyl. In further embodiments, R2 is —C(═O)NMe2, —C(═O)NEt2, C(O)NH(CH2)2SO3H, or —C(O)NH(CH2)2SO3−). In some other embodiments, R2 is optionally substituted phenyl, optionally substituted phenoxy, optionally substituted pyridyl, or optionally substituted furanyl. In some other embodiments, R2 is —CH═CH—C(═O)NRBRC, wherein each RB and RC is independently H, unsubstituted C1-C4 alkyl (e.g., methyl or ethyl), or C1-C4 alkyl substituted with sulfo, sulfonate, or carboxyl. In further embodiments, R8 is —CH═CH—C(═O)NMe2. In other embodiments, R2 is C1-C5 alkyl substituted with sulfo, sulfonate, or carboxyl (e.g., —CH2COOH). In some other embodiments, R2 is unsubstituted or substituted 5 or 6 membered heterocyclyl containing one or two heteroatoms selected from N and O. In further embodiment, R2 is piperidyl, N-methyl piperidyl, morpholinyl, N-methyl morpholinyl, each optionally substituted with oxo (═O). In some such embodiments, each of R1, R3, R4, R7, and R10 is H. In other embodiments, R1 or R7 is C1-C3 alkoxy (e.g., methoxy or ethoxy), or —C(═O)NRBRC, wherein each RB and RC is independently H, unsubstituted C1-C4 alkyl (e.g., methyl or ethyl), or C1-C4 alkyl substituted with sulfo, sulfonate, or carboxyl. In other embodiments, R3 is C1, C2, C3, C4 or C5 alkoxy substituted with sulfo, sulfonate, or carboxyl (e.g., —O(CH2)3SO3H, —O(CH2)4SO3− or —O(CH2)5COOH).
In some embodiments of the compounds of formula (I) or (I-A), R1 and R2 together with the atoms to which they are attached form an optionally substituted C6-C10 aryl to provide compounds of formula (Ia):
or a salt or a mesomeric form thereof,
In some embodiments of the compounds of formula (Ia), each of R7, R8, R9 and R10 is H. In other embodiments, at least one of R7, R8, R9 and R10 is carboxyl, halo, sulfo, sulfonate, —SO2NH2, unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, or —SO2NH2. In some such embodiments, R8 is sulfo, sulfonate, —SO2NH2 or —CH2COOH. In some embodiments, each of R7 and R8 is H. In some other embodiments, R7 and R8 together with the atoms to which they are attached form an optionally substituted C6-C10 aryl to provide compounds having the structure of formula (Ia-1):
or a salt or a mesomeric form thereof, wherein each Ry is independently unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, (C1-C6 alkoxy)C1-C6 alkyl, —O—(C1-C6 alkoxy)C1-C6 alkyl, halo, cyano, unsubstituted or substituted amino, carboxyl, hydroxy, nitro, sulfonyl, sulfino, sulfo, sulfonate, S-sulfonamido, N-sulfonamido, —C(═O)ORA, —C(═O)NRBRC or —SO3RD; and n is 0, 1, 2, or 3. In other embodiments, R7 and R8 together with the atoms to which they are attached form an optionally substituted 5 or 6 membered heteroaryl containing one, two or three heteroatoms selected from N, O and S (e.g., pyridyl, pyrimidyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl or triazolyl).
In some embodiments of the compounds of formula (Ia), each of R9 and R10 is H. In some other embodiments, R9 and R10 together with the atoms to which they are attached form an optionally substituted C6-C10 aryl to provide compounds having the structure of formula (Ia-2):
or a salt or mesomeric form thereof, wherein each Ry is independently unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, (C1-C6 alkoxy)C1-C6 alkyl, —O—(C1-C6 alkoxy)C1-C6 alkyl, halo, cyano, unsubstituted or substituted amino, carboxyl, hydroxy, nitro, sulfonyl, sulfino, sulfo, sulfonate, S-sulfonamido, N-sulfonamido, —C(═O)ORA, —C(═O)NRBRC or —SO3RD; and n is 0, 1, 2, or 3. In some embodiments, each of R7 and R8 is H. In other embodiments, R9 and R10 together with the atoms to which they are attached form an optionally substituted 5 or 6 membered heteroaryl containing one, two or three heteroatoms selected from N, O and S (e.g., pyridyl, pyrimidyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl or triazolyl).
In some embodiments of the compounds of formulas (Ia-1) or (Ia-2), n is 1 or 2 and each Ry is independently carboxyl, halo, sulfo, sulfonate, —SO2NH2, or C1-C6 alkyl substituted with carboxyl, carboxylate, sulfo, sulfonate, or —SO2NH2.
In some embodiments of the compounds of formulas (Ia), (Ia-1) or (Ia-2), each of R3 and R4 is H. In some embodiments, m is 1 or 2, and each Rx is independently carboxyl, sulfo, sulfonate, —SO2NH2, unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, or —SO2NH2.
In some embodiments of the compounds of formula (I), R3 and R4 together with the atoms to which they are attached form an optionally substituted C6-C10 aryl to provide compounds of formula (Ib):
or a salt or mesomeric form thereof, wherein each Rx is independently unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, (C1-C6 alkoxy)C1-C6 alkyl, —O—(C1-C6 alkoxy)C1-C6 alkyl, halo, cyano, unsubstituted or substituted amino, carboxyl, hydroxy, nitro, sulfonyl, sulfino, sulfo, sulfonate, S-sulfonamido, N-sulfonamido, —C(═O)ORA, —C(═O)NRBRC or —SO3RD; and m is 0, 1, 2, or 3. In other embodiments, R3 and R4 together with the atoms to which they are attached form an optionally substituted 5 or 6 membered heteroaryl containing one, two or three heteroatoms selected from N, O and S (e.g., pyridyl, pyrimidyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl or triazolyl). In some embodiments, each of R7, R8, R9 and R10 is H. In other embodiments, at least one of R7, R8, R9 and R10 is carboxyl, halo, sulfo, sulfonate, —SO2NH2, unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy, or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, or —SO2NH2. In some such embodiments, R8 is sulfo, sulfonate, —SO2NH2, or —CH2COOH.
In some embodiments of the compounds of formula (Ib), each of R7 and R8 is H. In other embodiments, R7 and R8 together with the atoms to which they are attached form an optionally substituted C6-C10 aryl to provide compounds having the structure of formula (Ib-1):
or a salt or a mesomeric form thereof, wherein each Ry is independently unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, (C1-C6 alkoxy)C1-C6 alkyl, —O—(C1-C6 alkoxy)C1-C6 alkyl, halo, cyano, unsubstituted or substituted amino, carboxyl, hydroxy, nitro, sulfonyl, sulfino, sulfo, sulfonate, S-sulfonamido, N-sulfonamido, —C(═O)ORA, —C(═O)NRBRC or —SO3RD; and n is 0, 1, 2, or 3. In some embodiments, each of R9 and R10 is H. In other embodiments, R7 and R8 together with the atoms to which they are attached form an optionally substituted 5 or 6 membered heteroaryl containing one, two or three heteroatoms selected from N, O and S (e.g., pyridyl, pyrimidyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl or triazolyl).
In some embodiments of the compounds of formula (Ib), each of R9 and R10 is H. In other embodiments, R9 and R10 together with the atoms to which they are attached form an optionally substituted C6-C10 aryl to provide compounds having the structure of formula (Ib-2):
or a salt or mesomeric form thereof, wherein each Ry is independently unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, (C1-C6 alkoxy)C1-C6 alkyl, —O—(C1-C6 alkoxy)C1-C6 alkyl, halo, cyano, unsubstituted or substituted amino, carboxyl, hydroxy, nitro, sulfonyl, sulfino, sulfo, sulfonate, S-sulfonamido, N-sulfonamido, —C(═O)ORA, —C(═O)NRBRC or —SO3RD; and n is 0, 1, 2, or 3. In some embodiments, each of R7 and R8 is H. In other embodiments, R9 and R10 together with the atoms to which they are attached form an optionally substituted 5 or 6 membered heteroaryl containing one, two or three heteroatoms selected from N, O and S (e.g., pyridyl, pyrimidyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl or triazolyl).
In some embodiments of the compounds of formulas (Ib-1) or (Ib-2), n is 1 or 2 and each Ry is independently carboxyl, halo, sulfo, sulfonate, —SO2NH2, unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy, or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, or —SO2NH2.
In some embodiments of the compounds of formulas (Ib), (Ib-1) or (Ib-2), each of R1 and R1 is H. In some embodiments, m is 1 or 2, and each Rx is independently carboxyl, halo, sulfo, sulfonate, —SO2NH2, unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy, or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, or —SO2NH2.
In some embodiments of the compounds of formula (I), the compounds have the structure of formulas (Ic) or (Id):
In some embodiments of the compounds of formula (Ic) or (Id), each of R1, R2, R3, and R4 is H. In other embodiments, at least one of R1, R2, R3, and R4 is carboxyl, halo, sulfo, sulfonate, or —SO2NH2, unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy, or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, or —SO2NH2.
In some embodiments of the compounds of formula (Ic) or (Id), n is 1 or 2 and each Ry is independently carboxyl, halo, sulfo, sulfonate, —SO2NH2, unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy, or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy, or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, or —SO2NH2. In some embodiments, each of R7, R8, R9, and R10 is H.
In some embodiments of the compounds of formulas (I), (I-A), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic), or (Id), X1 is CR5aR6a, and each of R5a and R6a is unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl). In some embodiments, each of R5a and R6a is methyl. In some other embodiments, one of R5a and R6a is H or unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl), and the other one of R5a and R6a is C1—C alkyl (e.g., a straight chain C3, C4 or C5 alkyl) substituted with carboxyl, sulfo, sulfonate, —C(═O)ORA, —C(═O)NRBRC, or —SO3RD. In some such embodiments, one of R5a and R6a is H or methyl. In other embodiments, R5a and R6a together with the carbon atom to which they are attached form an unsubstituted or substituted five or six membered heterocyclyl comprising one or two heteroatoms selected from O, N and S, for example,
In some embodiments of the compounds of formulas (I), (I-A), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic), or (Id), X2 is CR5bR6b, and each of R5b and R6b is unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl). In some embodiments, each of R5b and R6b is methyl. In some other embodiments, one of R5b and R6b is H or unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl), and the other one of R5b and R6b is C1-C6 alkyl (e.g., a straight-chain C3, C4 or C5 alkyl) substituted with carboxyl, sulfo, sulfonate, —C(═O)ORA, —C(═O)NRBRC, or —SO3RD. In some such embodiments, one of R5b and R6b is H or methyl. In other embodiments, R5b and R6b together with the carbon atom to which they are attached form an unsubstituted or substituted five or six membered heterocyclyl comprising one or two heteroatoms selected from O, N and S, for example,
In some embodiments of the compounds of formulas (I), (I-A), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic), or (Id), X1 is O or S. In some other embodiments, X1 is NR11. In some such embodiments, R11 is unsubstituted C1-C4 alkyl, or C1-C6 alkyl substituted with carboxyl, carboxylate, sulfo, sulfonate, —C(═O)ORA, —C(═O)NRBRC or —SO3RD.
In some other embodiments of the compounds of formula (I), X1 is NR11 and X2 is S, O or NR12. In further embodiments, X1 is NR11, X2 is NR12 and the compounds have the structure of formula (I-E):
or a salt or a mesomeric form thereof. In some embodiments, each of R1, R2, R3, R4, R7, R8, R9 and R10 is H. In other embodiments, at least one of R1, R2, R3, R4, R7, R8, R9 and R10 is carboxyl; halo; C1-C6 haloalkyl; sulfo; sulfonate; —SO2NH2; —C(═O)NRBRC; —ORE; unsubstituted or substituted 5 or 6 membered heteroaryl containing 1 to 3 heteroatoms selected from O, N and S; unsubstituted or substituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; unsubstituted or substituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; unsubstituted C1-C6 alkoxy; substituted C1-C6 alkoxy; substituted C2-C6 alkenyl or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy, substituted C2-C6 alkenyl, or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, —C(═O)NRBRC or —SO2NH2. In further embodiments, at least one of R1, R2, R3, R4, R7, R8, R9 and R10 is carboxyl, carboxylate, halo, sulfo, sulfonate, —SO2NH2, unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy or substituted C1-C6 alkyl is substituted with carboxyl, carboxylate, sulfo, sulfonate or —SO2NH2. In some embodiments, each of R11 and R12 is independently unsubstituted C1-C4 alkyl, or C1-C6 alkyl substituted with carboxyl, carboxylate, sulfo, sulfonate, —C(═O)ORA, —C(═O)NRBRC or —SO3RD.
In some embodiments of the compounds of formula (I), X1 is S and X2 is S, and the compounds have the structure of formula (I-C):
or a salt or a mesomeric form thereof. In some embodiments, each of R1, R2, R3, R4, R7, R8, R9 and R10 is H. In other embodiments, at least one of R1, R2, R3, R4, R7, R8, R9 and R10 is carboxyl; halo; C1-C6 haloalkyl; sulfo; sulfonate; —SO2NH2; —C(═O)NRBRC; —ORE; unsubstituted or substituted 5 or 6 membered heteroaryl containing 1 to 3 heteroatoms selected from O, N and S; unsubstituted or substituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; unsubstituted or substituted 5 or 6 membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S; unsubstituted C1-C6 alkoxy; substituted C1-C6 alkoxy; substituted C2-C6 alkenyl or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy, substituted C2-C6 alkenyl, or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate, —C(═O)NRBRC or —SO2NH2. In further embodiments, at least one of R1, R2, R3, R4, R7, R8, R9 and R10 is carboxyl, carboxylate, halo, sulfo, sulfonate, —SO2NH2, unsubstituted C1-C6 alkoxy, substituted C1-C6 alkoxy or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkoxy or substituted C1-C6 alkyl is independently substituted with carboxyl, carboxylate, sulfo, sulfonate or —SO2NH2.
Additional embodiments of the compound of formula (I) include, but are not limited to, those described below, and salts, esters, and mesomeric forms thereof. Non-limiting examples of esters include corresponding C1-C6 alkyl carboxylic esters (such as methyl esters, ethyl esters, isopropyl esters, and t-butyl esters formed from the carboxylic group of the compounds).
and salts and mesomeric forms thereof. In some further embodiments, the tricyclic polymethine dyes of formula (I) do not include the following compounds:
and their salts, amides (e.g., the reaction product between the carboxyl group of the dye and an compound containing an amino group), and esters (e.g., C1-C6 alkyl ester or activated esters such as NHS ester formed from the carboxyl group of the compound) thereof.
In any embodiments of the tricyclic polymethine compounds described herein, the compound may be further modified to introduce a photo-protecting moiety covalently bonded thereto, for example, a photo-protecting cyclooctatetraene (COT) moiety as described in U.S. Appl. No. 2021/0155983 A1, which is incorporated by reference in its entirety. For example, the tricyclic polymethine compounds of formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic), or (Id) may be covalently attached to a COT moiety via an amide bond, wherein the photo-protecting COT moiety comprises the structure pf formula (II):
is optionally replaced with O, S, or N, provided that when said carbon atom is replaced with O or S, then Rx and Ry are both absent; when said carbon atom is replaced with N, then Ry is absent; and
In some further embodiments, the photo-protecting COT moiety may comprise the following structures:
The COT moiety may be covalently attached to the tricyclic polymethine dye described herein by reaction between a functional group of the tricyclic polymethine dye described herein (e.g., a carboxyl group) and an amino group of a COT derivative to form an amide bond (where the carbonyl group of the amide bond is not shown). Alternatively, the tricyclic polymethine dye described herein may have an amino group (instead of a carboxyl group) that forms the amide bond with a carboxyl group of the COT derivative. Non-limiting exemplary tricyclic polymethine dye COT derivatives include:
Additional photoprotective moieties for use as triplet state quencher (TSQ) or triplet-triplet annihilator (TTA) including 1,4-substituted COT moiety, vinyl-fluorene moieties, or stilbene moieties are described in U.S. Publication No. 2024/0140939 A1, which is also incorporated by reference in its entirety.
According to an aspect of the disclosure, dye compounds described herein are suitable for attachment to substrate moieties, particularly comprising linker groups to enable attachment to substrate moieties. Substrate moieties can be virtually any molecule or substance to which the dyes of the disclosure can be conjugated, and, by way of non-limiting example, may include nucleosides, nucleotides, polynucleotides, carbohydrates, ligands, particles, solid surfaces, organic and inorganic polymers, chromosomes, nuclei, living cells, and combinations or assemblages thereof. The dyes can be conjugated by an optional linker by a variety of means including hydrophobic attraction, ionic attraction, and covalent attachment. In some aspect, the dyes are conjugated to the substrate by covalent attachment. More particularly, the covalent attachment is by means of a linker group. In some instances, such labeled nucleotides are also referred to as “modified nucleotides.”
Some aspects of the present disclosure relate to a nucleotide labeled with a dye of formulas (I), (I-A), (I-B), (I-C), (I-D), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic), (Id), or (Ie), or a salt or mesomeric form thereof as described herein, or a derivative thereof containing a photo-protecting moiety (e.g., COT) as described herein. The labeled nucleotide or oligonucleotide may be attached to the dye compound disclosed herein via a carboxyl (—CO2H) or an alkyl-carboxyl group to form an amide or alkyl-amide bond. In some further embodiments, the carboxyl group may be in the form of an activated form of a carboxyl group, for example, an amide or ester, which may be used for attachment to an amino or hydroxy group of the nucleotide or oligonucleotide. The term “activated ester” as used herein, refers to a carboxyl group derivative which is capable of reacting in mild conditions, for example, with a compound containing an amino group. Non-limiting examples of activated esters include, but are not limited to, p-nitrophenyl, pentafluorophenyl and succinimido esters.
For example, the dye compound of formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic), or (Id), may be attached to the nucleotide via a carboxyl group of the compound and the attachment forms an amide moiety between the carboxyl functional group of the dye compound and the amino functional group of a nucleotide or a nucleotide linker. In some further embodiments, R6b of the compound of formula (I), (I-A), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic), (Id) comprises a carboxyl functional group. In some further embodiments, R2 of the compound of formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (Ib), (Ib-1), (Ib-2), (Ic) or (Id) comprises a carboxyl functional group. In yet still other embodiments, R8 of the compound of formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (Ia), (Ia-2), (Ib), (Ib-2), or (Id) comprises a carboxyl functional group. In some embodiments, R11 or R12 of the compound of formula (I-E) comprises a carboxyl functional group.
In some embodiments, the dye compound may be covalently attached to the nucleotide via the nucleotide base. In some embodiments, the labeled nucleotide may have the dye attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base, optionally through a linker moiety. For example, the nucleobase may be 7-deaza adenine, and the dye is attached to the 7-deaza adenine at the C7 position, optionally through a linker. The nucleobase may be 7-deaza guanine, and the dye is attached to the 7-deaza guanine at the C7 position, optionally through a linker. The nucleobase may be cytosine, and the dye is attached to the cytosine at the C5 position, optionally through a linker. As another example, the nucleobase may be thymine or uracil and the dye is attached to the thymine or uracil at the C5 position, optionally through a linker.
The labeled nucleotide may also have a blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide. The blocking group may be attached at any position on the ribose or deoxyribose sugar. In particular embodiments, the blocking group is at the 3′ OH position of the ribose or deoxyribose sugar of the nucleotide. Various 3′ OH blocking group are disclosed in WO2004/018497 and WO2014/139596, which are hereby incorporated by references. For example, the blocking group may be azidomethyl (—CH2N3) or substituted azidomethyl (e.g., —CH(CHF2)N3 or CH(CH2F)N3), or allyl connecting to the 3′ oxygen atom of the ribose or deoxyribose moiety. In some embodiments, the 3′ blocking group is azidomethyl, forming 3′-OCH2N3 with the 3′ carbon of the ribose or deoxyribose.
Additional 3′ blocking groups are disclosed in U.S. Publication No. 2020/0216891 A1, which is incorporated by reference in its entirety. Non-limiting examples of the 3′ blocking group include:
each covalently attached to the 3′ carbon of the ribose or deoxyribose.
In some embodiments, the 3′ blocking group may be removed or deprotected by a chemical reagent to generate a free hydroxy group, for example, in the presence of a water-soluble phosphine reagent. Non-limiting examples include tris(hydroxymethyl)phosphine (THMP), tris(hydroxyethyl)phosphine (THEP) or tris(hydroxylpropyl)phosphine (THP or THPP). 3′-acetal blocking groups described herein may be removed or cleaved under various chemical conditions. For 3′ acetal blocking groups such as
non-limiting cleaving conditions include a Pd(II) complex, such as Pd(OAc)2 or allylPd(II) chloride dimer, in the presence of a phosphine ligand, for example tris(hydroxymethyl)phosphine (THMP), or tris(hydroxylpropyl)phosphine (THP or THPP). For those blocking groups containing an alkynyl group (e.g., an ethynyl), they may also be removed by a Pd(II) complex (e.g., Pd(OAc)2 or allyl Pd(II) chloride dimer) in the presence of a phosphine ligand (e.g., THP or THMP).
In some other embodiments, the 3′ blocking group described herein such as allyl or AOM may be cleaved by a palladium catalyst. In some such embodiments, the Pd catalyst is water soluble. In some such embodiments, is a Pd(0) complex (e.g., Tris(3,3′,3″-phosphinidynetris(benzenesulfonato)palladium(0) nonasodium salt nonahydrate). In some instances, the Pd(0) complex may be generated in situ from reduction of a Pd(II) complex by reagents such as alkenes, alcohols, amines, phosphines, or metal hydrides. Suitable palladium sources include Na2PdCl4, Li2PdCl4, Pd(CH3CN)2Cl2, (PdCl(C3H5))2, [Pd(C3H5)(THP)]Cl, [Pd(C3H5)(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd(Acac)2, PdCl2(COD), Pd(TFA)2, Na2PdBr4, K2PdBr4, PdCl2, PdBr2, and Pd(NO3)2. In one such embodiment, the Pd(0) complex is generated in situ from Na2PdCl4 or K2PdCl4. In another embodiment, the palladium source is allyl palladium(II) chloride dimer[(PdCl(C3H5))2]. In some embodiments, the Pd(0) complex is generated in an aqueous solution by mixing a Pd(II) complex with a phosphine. Suitable phosphines include water soluble phosphines, such as THP, THMP, PTA, TCEP, bis(p-sulfonatophenyl)phenylphosphine dihydrate potassium salt, or triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt.
In some embodiments, the palladium catalyst is prepared by mixing [(Allyl)PdCl]2 with THP in situ. The molar ratio of [(Allyl)PdCl]2 and the THP may be about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10. In one embodiment, the molar ratio of [(Allyl)PdCl]2 to THP is 1:10. In some other embodiment, the palladium catalyst is prepared by mixing a water soluble Pd reagent such as Na2PdCl4 or K2PdCl4 with THP in situ. The molar ratio of Na2PdCl4 or K2PdCl4 and TIP may be about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10. In one embodiment, the molar ratio of Na2PdCl4 or K2PdCl4 to THP is about 1:3. In another embodiment, the molar ratio of Na2PdCl4 or K2PdCl4 to THP is about 1:3.5. In yet another embodiment, the molar ratio of Na2PdCl4 orK2PdCl4 to THP is about 1:2.5. In some further embodiments, one or more reducing agents may be added, such as ascorbic acid or a salt thereof (e.g., sodium ascorbate). In some embodiments, the cleavage mixture may contain additional buffer reagents, such as a primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In some further embodiments, the buffer reagent comprises ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylethanolamine (DMEA), 2-diethylethanolamine (DEEA), N,N,N′,N′-tetramethylethylenediamine (TEMED), N,N,N′,N′-tetraethylethylenediamine (TEEDA), or 2-piperidine ethanol (also known as (2-hydroxyethyl)piperidine, having the structure
or combinations thereof. In one embodiment, the buffer reagent comprises or is DEEA. In another embodiment, the buffer reagent comprises or is (2-hydroxyethyl)piperidine. In another embodiment, the buffer reagent contains one or more inorganic salts such as a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In one embodiment, the inorganic salt is a sodium salt.
The dye compounds as disclosed herein may include a reactive linker group at one of the substituent positions for covalent attachment of the compound to a substrate or another molecule. Reactive linking groups are moieties capable of forming a bond (e.g., a covalent or non-covalent bond), in particular a covalent bond. In a particular embodiment the linker may be a cleavable linker. Use of the term “cleavable linker” is not meant to imply that the whole linker is required to be removed. The cleavage site can be located at a position on the linker that ensures that part of the linker remains attached to the dye and/or substrate moiety after cleavage. Cleavable linkers may be, by way of non-limiting example, electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavable under reductive conditions (for example disulfide or azide containing linkers), oxidative conditions, cleavable via use of safety-catch linkers and cleavable by elimination mechanisms. The use of a cleavable linker to attach the dye compound to a substrate moiety ensures that the label can, if required, be removed after detection, avoiding any interfering signal in downstream steps.
Useful linker groups may be found in PCT Publication No. WO 2004/018493 (herein incorporated by reference), examples of which include linkers that may be cleaved using water-soluble phosphines or water-soluble transition metal catalysts formed from a transition metal and at least partially water-soluble ligands. In aqueous solution the latter form at least partially water-soluble transition metal complexes. Such cleavable linkers can be used to connect bases of nucleotides to labels such as the dyes set forth herein.
Particular linkers include those disclosed in PCT Publication No. WO 2004/018493 (herein incorporated by reference) such as those that include moieties of the formulae:
(wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C1-C10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C1-C10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). In some aspect, the linkers connect the bases of nucleotides to labels such as, for example, the dye compounds described herein.
Additional examples of linkers include those disclosed in U.S. Publication No. 2016/0040225 (herein incorporated by reference), such as those include moieties of the formulae:
(wherein * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). The linker moieties illustrated herein may comprise the whole or partial linker structure between the nucleotides/nucleosides and the labels. The linker moieties illustrated herein may comprise the whole or partial linker structure between the nucleotides/nucleosides and the labels.
Additional examples of linkers are disclosed in U.S. Publication No. 2020/0216891 A1, which is incorporated by reference in its entirety:
wherein B is a nucleobase; n is 1, 2, 3, 4, 5; s is an integer of 1, 2, 3, 4 5, 6, 7, 8, 9 or 10; Z is —N3 (azido), —O—C1-C6 alkyl, —O—C2-C6 alkenyl, or —O—C2-C6 alkynyl; and Fl comprises a dye moiety, which may contain additional linker structure. One of ordinary skill in the art understands that the dye compound described herein is covalently bounded to the linker by reacting a functional group of the dye compound (e.g., carboxyl) with a functional group of the linker (e.g., amino). In one embodiment, the cleavable linker comprises
where Z is —O-allyl. In some embodiments, s is 1. In other embodiments, the nucleotide may contain multiple cleavable linkers repeating units (e.g., s is 2, 3, 4 5, 6, 7, 8, 9 or 10).
In particular embodiments, the length of the linker between a fluorescent dye (fluorophore) and a guanine base can be altered, for example, by introducing a polyethylene glycol spacer group, thereby increasing the fluorescence intensity compared to the same fluorophore attached to the guanine base through other linkages known in the art. Exemplary linkers and their properties are set forth in PCT Publication No. WO 2007/020457 (herein incorporated by reference). The design of linkers, and especially their increased length, can allow improvements in the brightness of fluorophores attached to the guanine bases of guanosine nucleotides when incorporated into polynucleotides such as DNA. Thus, when the dye is for use in any method of analysis which requires detection of a fluorescent dye label attached to a guanine-containing nucleotide, it is advantageous if the linker comprises a spacer group of formula —((CH2)2O)n— (PEG linker), wherein n is an integer between 2 and 50, as described in WO 2007/020457. A PEG linker may be introduced to a dye compound described herein by reacting the carboxyl group of the dye with a PEG containing reagent with a terminal amino group to form amide bonding (e.g., H2N—(CH2)2O)2-10—(CH2)2C(O)OH).
Nucleosides and nucleotides may be labeled at sites on the sugar or nucleobase. As known in the art, a “nucleotide” consists of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA, the sugar is ribose and in DNA is a deoxyribose, i.e., a sugar lacking a hydroxy group that is present in ribose. The nitrogenous base is a derivative of purine or pyrimidine. The purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T) or in the context of RNA, uracil (U). The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. A nucleotide is also a phosphate ester of a nucleoside, with esterification occurring on the hydroxy group attached to the C-3 or C-5 of the sugar. Nucleotides are usually mono, di- or triphosphates.
A “nucleoside” is structurally similar to a nucleotide but is missing the phosphate moieties. An example of a nucleoside analog would be one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule.
Although the base is usually referred to as a purine or pyrimidine, the skilled person will appreciate that derivatives and analogues are available which do not alter the capability of the nucleotide or nucleoside to undergo Watson-Crick base pairing. “Derivative” or “analogue” means a compound or molecule whose core structure is the same as, or closely resembles that of a parent compound but which has a chemical or physical modification, such as, for example, a different or additional side group, which allows the derivative nucleotide or nucleoside to be linked to another molecule. For example, the base may be a deazapurine. In particular embodiments, the derivatives should be capable of undergoing Watson-Crick pairing. “Derivative” and “analogue” also include, for example, a synthetic nucleotide or nucleoside derivative having modified base moieties and/or modified sugar moieties. Such derivatives and analogues are discussed in, for example, Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogues can also comprise modified phosphodiester linkages including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate, phosphoramidate linkages and the like.
A dye may be attached to any position on the nucleotide base, for example, through a linker. In particular embodiments, Watson-Crick base pairing can still be carried out for the resulting analog. Particular nucleobase labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base. As described above a linker group may be used to covalently attach a dye to the nucleoside or nucleotide.
In particular embodiments the labeled nucleotide or oligonucleotide may be enzymatically incorporable and enzymatically extendable. Accordingly, a linker moiety may be of sufficient length to connect the nucleotide to the compound such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by a nucleic acid replication enzyme. Thus, the linker can also comprise a spacer unit. The spacer distances, for example, the nucleotide base from a cleavage site or label.
Nucleosides or nucleotides labeled with the dyes described herein may have the formula:
where Dye is a tricyclic polymethine dye compound (label) moiety described herein (after covalent bonding between a functional group of the dye and a functional group of the linker “L”); B is a nucleobase, such as, for example uracil, thymine, cytosine, adenine, 7-deaza adenine, guanine, 7-deaza guanine, and the like; L is an optional linker which may or may not be present; R′ can be H, or —OR′ is monophosphate, diphosphate, triphosphate, thiophosphate, a phosphate ester analog, —O— attached to a reactive phosphorous containing group, or —O— protected by a blocking group; R″ is H or OH; and R″′ is H, a 3′ blocking group described herein, or —OR″′ forms a phosphoramidite. Where —OR″′ is phosphoramidite, R′ is an acid-cleavable hydroxyl protecting group which allows subsequent monomer coupling under automated synthesis conditions. In some further embodiments, B comprises
or optionally substituted derivatives and analogs thereof. In some further embodiments, the labeled nucleobase comprises the structure
In a particular embodiment, the blocking group is separate and independent of the dye compound, i.e., not attached to it. Alternatively, the dye may comprise all or part of the 3′-OH blocking group. Thus R″′ can be a 3′ OH blocking group which may or may not comprise the dye compound.
In yet another alternative embodiment, there is no blocking group on the 3′ carbon of the pentose sugar and the dye (or dye and linker construct) attached to the base, for example, can be of a size or structure sufficient to act as a block to the incorporation of a further nucleotide. Thus, the block can be due to steric hindrance or can be due to a combination of size, charge and structure, whether or not the dye is attached to the 3′ position of the sugar.
In still yet another alternative embodiment, the blocking group is present on the 2′ or 4′ carbon of the pentose sugar and can be of a size or structure sufficient to act as a block to the incorporation of a further nucleotide.
The use of a blocking group allows polymerization to be controlled, such as by stopping extension when a labeled nucleotide is incorporated. If the blocking effect is reversible, for example, by way of non-limiting example by changing chemical conditions or by removal of a chemical block, extension can be stopped at certain points and then allowed to continue.
In a particular embodiment, the linker (between dye and nucleotide) and blocking group are both present and are separate moieties. In particular embodiments, the linker and blocking group are both cleavable under the same or substantially similar conditions. Thus, deprotection and deblocking processes may be more efficient because only a single treatment will be required to remove both the dye compound and the blocking group. However, in some embodiments a linker and blocking group need not be cleavable under similar conditions, instead being individually cleavable under distinct conditions.
The disclosure also encompasses polynucleotides incorporating dye compounds. Such polynucleotides may be DNA or RNA comprised respectively of deoxyribonucleotides or ribonucleotides joined in phosphodiester linkage. Polynucleotides may comprise naturally occurring nucleotides, non-naturally occurring (or modified) nucleotides other than the labeled nucleotides described herein or any combination thereof, in combination with at least one modified nucleotide (e.g., labeled with a dye compound) as set forth herein. Polynucleotides according to the disclosure may also include non-natural backbone linkages and/or non-nucleotide chemical modifications. Chimeric structures comprised of mixtures of ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide are also contemplated.
Non-limiting exemplary labeled nucleotides as described herein include:
wherein L represents a linker and R represents a ribose or deoxyribose moiety as described above, or a ribose or deoxyribose moiety with the 5′ position substituted with mono-, di- or tri-phosphates.
In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below:
refers to the connection point of the Dye with the cleavable linker as a result of a reaction between an amino group of the linker moiety and the carboxyl group of the Dye. In any embodiments of the labeled nucleotide described herein, the nucleotide is a nucleotide triphosphate.
Additional aspects of the present disclosure relate to an oligonucleotide or polynucleotide comprising or incorporating a labeled nucleotide described herein. In some embodiments, the oligonucleotide or polynucleotide is hybridized to at least a portion of a target polynucleotide. In some embodiments, the target polynucleotide is immobilized on a solid support. In some further embodiments, the solid support comprises an array or a plurality of different immobilized target polynucleotides. In further embodiments, the solid support comprises a patterned flow cell. In further embodiments, the patterned flow cell comprises a plurality of nanowells. In further embodiments, the solid support comprises at least 5,000,000 spatially distinguishable sites/cm2 that comprise multiple copies of target polynucleotides.
Additional aspects of the present disclosure relate to a protein tag or an antibody comprising one or more tricyclic polymethine dyes described herein. In particular, the protein tag or an antibody may comprise multiple copies of the same dye for increased fluorescent intensity. The protein tag or antibody may be used as an affinity reagent that binds superficially to a particular type of unlabeled 3′ blocked nucleotide.
Also provided herein are kits including a first type of nucleotide labeled with a tricyclic polymethine compound of the present disclosure of formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (Ia), (Ia-1), (Ia-2), (Ib), (Ib-1), (Ib-2), (Ic) or (Id) (i.e., a first label). In some embodiments, the kit also comprises a second type of labeled nucleotide, which is labeled with a second compound that is different than the tricyclic polymethine dye in the first type of labeled nucleotide (i.e., a second label). In some embodiments, the first and second type labeled nucleotides are excitable using a single excitation source, which may be a first light source having a first excitation wavelength. For example, the excitation bands for the first and the second labels may be at least partially overlapping such that excitation in the overlap region of the spectrum causes both labels to emit fluorescence. In some other embodiments, the second type of labeled nucleotides is excitable using a second excitation source, which may be a second light source having a second excitation wavelength that is different from the first excitation wavelength. In some further embodiments, the kit may include a third type nucleotide, wherein the third nucleotide is labeled with a third compound that is different from the first and the second labels (i.e., a third label). Alternatively, the third type of nucleotide is a mixture of the third type of nucleotide labeled with the first label and the third type of nucleotide labeled with the second label. In some such embodiments, the third type labeled nucleotide is excitable using the first light source having the first excitation wavelength. That is, each of the first type, second type and the third type of nucleotide is excitable using the same light source with a single wavelength. In some other embodiments, the third type labeled nucleotide is excitable using both the first light source having the first excitation wavelength, or the second light source having the second excitation wavelength. In still other embodiments, the third type of nucleotide is excitable using a third light source with a third excitation wavelength. In some further embodiments, the kit may further comprise a fourth type of nucleotide. In some such embodiments, the fourth nucleotide is unlabeled (dark). In other embodiments, the fourth nucleotide is labeled with a different compound than the first, second and the third nucleotide, and each label has a distinct absorbance maximum that is distinguishable from the other labels. In still other embodiments, the fourth nucleotide is unlabeled. In some embodiments, the first light source has an excitation wavelength from about 400 nm to about 490 nm, from about 420 nm to about 470 nm, or from 450 nm to about 460 nm (e.g., 450 nm or 488 nm). In some other embodiments, the first excitation light source has a wavelength from about 500 nm to about 550 nm, from about 510 to about 540 nm, or from about 520 to about 530 nm (e.g., 523 nm or 525 nm). In other embodiments, when two excitation light sources are used, the first excitation light source has an excitation wavelength from about 400 nm to about 490 nm, from about 420 nm to about 470 nm, or from 450 nm to about 460 nm (e.g., 450 nm or 488 nm), and the second excitation light source a wavelength from about 500 nm to about 550 nm, from about 510 to about 540 nm, or from about 520 to about 530 nm (e.g., 523 nm or 525 nm). In some embodiments, the emissions of the first type of labeled nucleotide, the second type of labeled nucleotide and the third type of labeled nucleotide are detectable in two detection channels with different wavelengths (e.g., at blue region with a wavelength ranging from about 472 to about 520 nm, and at a green region with a wavelength ranging from about 540 nm to about 640 nm). In some other embodiments, the emissions of the first type of labeled nucleotide, the second type of labeled nucleotide and the third type of labeled nucleotide are detectable in two detection channels with different wavelengths (e.g., at a green region with a wavelength ranging from about 540 nm to about 585 nm, and a second green region with a wavelength ranging from about 585 nm to about 640 nm). In other embodiments, each of the first type, the second type and the third type of nucleotide has an emission spectrum that can be collected in a single emission collection filter or channel.
In some embodiments, the kit may contain four types of labeled nucleotides (A, C, G and T or U), where the first of the four nucleotides is labeled with a compound as disclosed herein. In such a kit, each of the four nucleotides can be labeled with a compound that is the same or different from the label on the other three nucleotides. Alternatively, a first of the four nucleotides is a labeled nucleotide describe herein, a second of the four nucleotides carries a second label, a third nucleotide carries a third label, and a fourth nucleotide is unlabeled (dark). As another example, a first of the four nucleotides is a labeled nucleotide described herein, a second of the four nucleotides carries a second label, a third nucleotide carries a mixture of two labels, and a fourth nucleotide is unlabeled (dark). Thus, one or more of the label compounds can have a distinct absorbance maximum and/or emission maximum such that the compound(s) is(are) distinguishable from other compounds. For example, each compound can have a distinct absorbance maximum and/or emission maximum such that each of the compounds is spectrally distinguishable from the other three compounds (or two compounds if the fourth nucleotide is unlabeled). It will be understood that parts of the absorbance spectrum and/or emission spectrum other than the maxima can differ and these differences can be exploited to distinguish the compounds. The kit may be such that two or more of the compounds have a distinct absorbance maximum.
The compounds, nucleotides, or kits that are set forth herein may be used to detect, measure, or identify a biological system (including, for example, processes or components thereof). Exemplary techniques that can employ the compounds, nucleotides or kits include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cellular assay (e.g., cell binding or cell function analysis), or protein assay (e.g., protein binding assay or protein activity assay). The use may be on an automated instrument for carrying out a particular technique, such as an automated sequencing instrument. The sequencing instrument may contain two light sources operating at different wavelengths.
In a particular embodiment, the labeled nucleotide(s) described herein may be supplied in combination with unlabeled or native nucleotides, or any combination thereof. Combinations of nucleotides may be provided as separate individual components (e.g., one nucleotide type per vessel or tube) or as nucleotide mixtures (e.g., two or more nucleotides mixed in the same vessel or tube).
Where kits comprise a plurality, particularly two, or three, or more particularly four, nucleotides, the different nucleotides may be labeled with different dye compounds, or one may be dark, with no dye compounds. Where the different nucleotides are labeled with different dye compounds, it is a feature of the kits that the dye compounds are spectrally distinguishable fluorescent dyes. As used herein, the term “spectrally distinguishable fluorescent dyes” refers to fluorescent dyes that emit fluorescent energy at wavelengths that can be distinguished by fluorescent detection equipment (for example, a commercial capillary-based DNA sequencing platform) when two or more such dyes are present in one sample. When two nucleotides labeled with fluorescent dye compounds are supplied in kit form, it is a feature of some embodiments that the spectrally distinguishable fluorescent dyes can be excited at the same wavelength, such as, for example by the same light source. When four nucleotides labeled with fluorescent dye compounds are supplied in kit form, it is a feature of some embodiments that two of the spectrally distinguishable fluorescent dyes can both be excited at one wavelength and the other two spectrally distinguishable dyes can both be excited at another wavelength. Particular excitation wavelengths for the dyes are 450 nm to 460 nm, 480-490 nm (e.g., 488 nm), or 500 nm to 540 nm (e.g., 523 nm).
In some embodiments, the kits may contain a labeled nucleotide having a short Stokes shift between about 20 nm to about 50 nm, a labeled nucleotide having a long Stokes shift between about 100 nm to about 130 nm, and a labeled nucleotide having a medium Stokes shift between about 60 nm to about 90 nm (where “Stokes shift” is the distance between the peak absorption and peak emission wavelengths). In further embodiments, each of the first type, second type and third type of nucleotides are excitable by a single light source (e.g., a green light having a wavelength of between about 500 nm to about 540 nm, or about 520 nm to about 530 nm, or about 523 nm). In some embodiments, the emission spectra of the first type, second type and third type of nucleotides are collectable in two different detection channels or regions (e.g., at one region with a wavelength ranging from about 472 to about 530 nm, and at another region with a wavelength ranging from about 540 nm to about 640 nm).
Although kits are exemplified herein in regard to configurations having different nucleotides that are labeled with different dye compounds, it will be understood that kits can include 2, 3, 4 or more different nucleotides that have the same dye compound.
In addition to the labeled nucleotides, the kit may comprise together at least one additional component. The further component(s) may be one or more of the components identified in a method set forth herein or in the Examples section below. Some non-limiting examples of components that can be combined into a kit of the present disclosure are set forth below. In some embodiments, the kit further comprises a DNA polymerase (such as a mutant 9° N DNA polymerase) and one or more buffer compositions. Non-limiting examples of DNA polymerase may be used in the present disclosure include those disclosed in WO 2005/024010, U.S. Publication Nos. 2020/0131484 A1, 2020/0181587 A1, and 2024/0141427, each of which is incorporated by reference herein in its entirety. Exemplary polymerases include but not limited to Pol 812, Pol 1901, Pol 1558, Pol 963, or Pol A. The amino acid sequences of Pol 812, Pol 1901, Pol 1558 or Pol 963 DNA polymerases are described, for example, in U.S. Patent Publication Nos. 2020/0131484 A1 and 2020/0181587 A1. The amino acid sequence of Pol A is disclosed as SEQ ID NO:5 of U.S. Publication No. 2024/0141427. One buffer composition may comprise antioxidants such as ascorbic acid or sodium ascorbate, which can be used to protect the dye compounds from photo damage during detection. Additional buffer composition may comprise a reagent can may be used to cleave the 3′ blocking group and/or the cleavable linker. For example, a water-soluble phosphines or water-soluble transition metal catalysts formed from a transition metal and at least partially water-soluble ligands, such as a palladium complex. Various components of the kit may be provided in a concentrated form to be diluted prior to use. In such embodiments a suitable dilution buffer may also be included. Again, one or more of the components identified in a method set forth herein can be included in a kit of the present disclosure. In any embodiments of the nucleotide or labeled nucleotide described herein, the nucleotide contains a 3′ blocking group.
Nucleotides comprising a dye compound according to the present disclosure may be used in any method of analysis such as method that include detection of a fluorescent label attached to such nucleotide, whether on its own or incorporated into or associated with a larger molecular structure or conjugate. In this context the term “incorporated into a polynucleotide” can mean that the 5′ phosphate is joined in phosphodiester linkage to the 3′ hydroxyl group of a second nucleotide, which may itself form part of a longer polynucleotide chain. The 3′ end of a nucleotide set forth herein may or may not be joined in phosphodiester linkage to the 5′ phosphate of a further nucleotide. Thus, in one non-limiting embodiment, the disclosure provides a method of detecting a labeled nucleotide incorporated into a polynucleotide which comprises: (a) incorporating at least one labeled nucleotide of the disclosure into a polynucleotide and (b) determining the identity of the nucleotide(s) incorporated into the polynucleotide by detecting the fluorescent signal from the dye compound attached to said nucleotide(s). Particular embodiments of the method of sequencing utilizes a one-excitation, two-channel detection system (also known as 1Ex-2Ch). Detailed disclosures are provided in WO 2018/165099 and U.S. Publication No. 2022/0403450 A1, each of which is incorporated by reference in its entirety. In particular, the 1Ex-2Ch may contain a green excitation light source having a wavelength between about 500 to about 540 nm, or about 520 nm to about 525 nm, and two separate collection channels at two separate optic regions (e.g., at a green region with a wavelength ranging from about 540 nm to about 585 nm, and at a green region with a wavelength ranging from about 585 nm to about 640 nm). In other embodiments, the 1Ex-2Ch may contain a blue excitation light source having a wavelength between about 400-500 nm or 450-460 nm, and two separate collection channels at two separate optic regions (e.g., at a blue region with a wavelength ranging from about 472 nm to about 520 nm, and at a green region with a wavelength ranging from about 540 nm to about 640 nm). In further embodiments, the method may also be used in two-excitation, two-channel detection system (also known as 2Ex-2Ch).
This method can include: a synthetic step (a) in which one or more labeled nucleotides according to the disclosure are incorporated into a polynucleotide and a detection step (b) in which one or more labeled nucleotide(s) incorporated into the polynucleotide are detected by detecting or quantitatively measuring their fluorescence.
Some embodiments of the present application are directed to a method for determining the sequences of a plurality of different target polynucleotides, comprising:
In some further embodiments, the method is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises a single light source operating with a green laser at about 500 nm to about 540 nm, or about 520 to about 525 nm. The incorporation of the first type of the nucleotide is determined by detection a first optical detection channel/region, and the incorporation of the second type of nucleotide is determined by detection in the second optic detection channel/region. The incorporation of the third type of nucleotide is determined by detection in both the first and the second optic detection channels/regions. The incorporation of the fourth type of nucleotide is determined by no detection in either the first or the second detection channel/region.
In other embodiments, the automatic sequencing instrument may comprise two light sources operating at different wavelengths (e.g., at 450-460 nm and 520-540 nm). The incorporation of the first type of the nucleotide conjugates is determined by a signal state in the first imaging event and a dark state in the second imaging event. The incorporation of the second type of the nucleotide conjugates is determined by a dark state in the first imaging event and a signal state in the second imaging event. The incorporation of the third type of the nucleotide conjugates is determined by a signal state in both the first imaging event and the second imaging event. The incorporation of the fourth type of the nucleotide conjugates is determined by a dark state in both the first imaging event and the second imaging event.
In some embodiments, at least one nucleotide is incorporated into a polynucleotide (such as a single stranded primer polynucleotide described herein) in the synthetic step by the action of a polymerase enzyme. However, other methods of joining nucleotides to polynucleotides, such as, for example, chemical oligonucleotide synthesis or ligation of labeled oligonucleotides to unlabeled oligonucleotides, can be used. Therefore, the term “incorporating,” when used in reference to a nucleotide and polynucleotide, can encompass polynucleotide synthesis by chemical methods as well as enzymatic methods.
In a specific embodiment, a synthetic step is carried out and may optionally comprise incubating a template or target polynucleotide strand with a reaction mixture comprising fluorescently labeled nucleotides of the disclosure. A polymerase can also be provided under conditions which permit formation of a phosphodiester linkage between a free 3′ hydroxyl group on a polynucleotide strand annealed to the template or target polynucleotide strand and a 5′ phosphate group on the labeled nucleotide. Thus, a synthetic step can include formation of a polynucleotide strand as directed by complementary base pairing of nucleotides to a template/target strand.
In all embodiments of the methods, the detection step may be carried out while the polynucleotide strand into which the labeled nucleotides are incorporated is annealed to a template/target strand, or after a denaturation step in which the two strands are separated. Further steps, for example chemical or enzymatic reaction steps or purification steps, may be included between the synthetic step and the detection step. In particular, the polynucleotide strand incorporating the labeled nucleotide(s) may be isolated or purified and then processed further or used in a subsequent analysis. By way of example, polynucleotide strand incorporating the labeled nucleotide(s) as described herein in a synthetic step may be subsequently used as labeled probes or primers. In other embodiments, the product of the synthetic step set forth herein may be subject to further reaction steps and, if desired, the product of these subsequent steps purified or isolated.
Suitable conditions for the synthetic step will be well known to those familiar with standard molecular biology techniques. In one embodiment, a synthetic step may be analogous to a standard primer extension reaction using nucleotide precursors, including the labeled nucleotides as described herein, to form an extended polynucleotide strand (primer polynucleotide strand) complementary to the template/target strand in the presence of a suitable polymerase enzyme. In other embodiments, the synthetic step may itself form part of an amplification reaction producing a labeled double stranded amplification product comprised of annealed complementary strands derived from copying of the primer and template polynucleotide strands. Other exemplary synthetic steps include nick translation, strand displacement polymerization, random primed DNA labeling, etc. A particularly useful polymerase enzyme for a synthetic step is one that is capable of catalyzing the incorporation of the labeled nucleotides as set forth herein. A variety of naturally occurring or mutant/modified polymerases can be used. By way of example, a thermostable polymerase can be used for a synthetic reaction that is carried out using thermocycling conditions, whereas a thermostable polymerase may not be desired for isothermal primer extension reactions. Suitable thermostable polymerases which are capable of incorporating the labeled nucleotides according to the disclosure include those described in WO 2005/024010 or WO06120433, each of which is incorporated herein by reference. In synthetic reactions which are carried out at lower temperatures such as 37° C., polymerase enzymes need not necessarily be thermostable polymerases, therefore the choice of polymerase will depend on a number of factors such as reaction temperature, pH, strand-displacing activity and the like.
In specific non-limiting embodiments, the disclosure encompasses methods of nucleic acid sequencing, re-sequencing, whole genome sequencing, single nucleotide polymorphism scoring, any other application involving the detection of the modified nucleotide or nucleoside labeled with dyes set forth herein when incorporated into a polynucleotide.
A particular embodiment of the disclosure provides use of labeled nucleotides comprising dye moiety according to the disclosure in a polynucleotide sequencing-by-synthesis reaction. Sequencing-by-synthesis generally involves sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5′ to 3′ direction using a polymerase or ligase in order to form an extended polynucleotide chain complementary to the template/target nucleic acid to be sequenced. The identity of the base present in one or more of the added nucleotide(s) can be determined in a detection or “imaging” step. The identity of the added base may be determined after each nucleotide incorporation step. The sequence of the template may then be inferred using conventional Watson-Crick base-pairing rules. The use of the nucleotides labeled with dyes set forth herein for determination of the identity of a single base may be useful, for example, in the scoring of single nucleotide polymorphisms, and such single base extension reactions are within the scope of this disclosure.
In an embodiment of the present disclosure, the sequence of a template/target polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent strand complementary to the template polynucleotide to be sequenced through the detection of fluorescent label(s) attached to the incorporated nucleotide(s). Sequencing of the template polynucleotide can be primed with a suitable primer (or prepared as a hairpin construct which will contain the primer as part of the hairpin), and the nascent chain is extended in a stepwise manner by addition of nucleotides to the 3′ end of the primer in a polymerase-catalyzed reaction.
In particular embodiments, each of the different nucleotide triphosphates (A, T, G and C) may be labeled with a unique fluorophore and also comprises a blocking group at the 3′ position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides may be unlabeled (dark). The polymerase enzyme incorporates a nucleotide into the nascent chain complementary to the template/target polynucleotide, and the blocking group prevents further incorporation of nucleotides. Any unincorporated nucleotides can be washed away and the fluorescent signal from each incorporated nucleotide can be “read” optically by suitable means, such as a charge-coupled device using light source excitation and suitable emission filters. The 3′ blocking group and fluorescent dye compounds can then be removed (deprotected) (simultaneously or sequentially) to expose the nascent chain for further nucleotide incorporation. Typically, the identity of the incorporated nucleotide will be determined after each incorporation step, but this is not strictly essential. Similarly, U.S. Pat. No. 5,302,509 (which is incorporated herein by reference) discloses a method to sequence polynucleotides immobilized on a solid support.
The method, as exemplified above, utilizes the incorporation of fluorescently labeled, 3′-blocked nucleotides A, G, C, and T into a growing strand complementary to the immobilized polynucleotide, in the presence of DNA polymerase. The polymerase incorporates a base complementary to the target polynucleotide but is prevented from further addition by the 3′-blocking group. The label of the incorporated nucleotide can then be determined, and the blocking group removed by chemical cleavage to allow further polymerization to occur. The nucleic acid template to be sequenced in a sequencing-by-synthesis reaction may be any polynucleotide that it is desired to sequence. The nucleic acid template for a sequencing reaction will typically comprise a double stranded region having a free 3′ hydroxyl group that serves as a primer or initiation point for the addition of further nucleotides in the sequencing reaction. The region of the template to be sequenced will overhang this free 3′ hydroxyl group on the complementary strand. The overhanging region of the template to be sequenced may be single stranded but can be double-stranded, provided that a “nick is present” on the strand complementary to the template strand to be sequenced to provide a free 3′ OH group for initiation of the sequencing reaction. In such embodiments, sequencing may proceed by strand displacement. In certain embodiments, a primer bearing the free 3′ hydroxyl group may be added as a separate component (e.g., a short oligonucleotide) that hybridizes to a single-stranded region of the template to be sequenced. Alternatively, the primer and the template strand to be sequenced may each form part of a partially self-complementary nucleic acid strand capable of forming an intra-molecular duplex, such as for example a hairpin loop structure. Hairpin polynucleotides and methods by which they may be attached to solid supports are disclosed in PCT Publication Nos. WO0157248 and WO2005/047301, each of which is incorporated herein by reference. Nucleotides can be added successively to a growing primer, resulting in synthesis of a polynucleotide chain in the 5′ to 3′ direction. The nature of the base which has been added may be determined, particularly but not necessarily after each nucleotide addition, thus providing sequence information for the nucleic acid template. Thus, a nucleotide is incorporated into a nucleic acid strand (or polynucleotide) by joining of the nucleotide to the free 3′ hydroxyl group of the nucleic acid strand via formation of a phosphodiester linkage with the 5′ phosphate group of the nucleotide.
The nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid molecule comprised of deoxynucleotides and ribonucleotides. The nucleic acid template may comprise naturally occurring and/or non-naturally occurring nucleotides and natural or non-natural backbone linkages, provided that these do not prevent copying of the template in the sequencing reaction.
In certain embodiments, the nucleic acid template to be sequenced may be attached to a solid support via any suitable linkage method known in the art, for example via covalent attachment. In certain embodiments template polynucleotides may be attached directly to a solid support (e.g., a silica-based support). However, in other embodiments of the disclosure the surface of the solid support may be modified in some way so as to allow either direct covalent attachment of template polynucleotides, or to immobilize the template polynucleotides through a hydrogel or polyelectrolyte multilayer, which may itself be non-covalently attached to the solid support.
Arrays in which polynucleotides have been directly attached to a support (for example, silica-based supports such as those disclosed in WO00/06770 (incorporated herein by reference), wherein polynucleotides are immobilized on a glass support by reaction between a pendant epoxide group on the glass with an internal amino group on the polynucleotide. In addition, polynucleotides can be attached to a solid support by reaction of a sulfur-based nucleophile with the solid support, for example, as described in WO2005/047301 (incorporated herein by reference). A still further example of solid-supported template polynucleotides is where the template polynucleotides are attached to hydrogel supported upon silica-based or other solid supports, for example, as described in WO00/31148, WO01/01143, WO02/12566, WO03/014392, U.S. Pat. No. 6,465,178 and WO00/53812, each of which is incorporated herein by reference.
A particular surface to which template polynucleotides may be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and in WO2005/065814, which is incorporated herein by reference. Specific hydrogels that may be used include those described in WO2005/065814 and U.S. Pub. No. 2014/0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide)).
DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Pat. No. 6,172,218 (which is incorporated herein by reference). Attachment to beads or microparticles can be useful for sequencing applications. Bead libraries can be prepared where each bead contains different DNA sequences. Exemplary libraries and methods for their creation are described in Nature, 437, 376-380 (2005); Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. Sequencing of arrays of such beads using nucleotides set forth herein is within the scope of the disclosure.
Template(s) that are to be sequenced may form part of an “array” on a solid support, in which case the array may take any convenient form. Thus, the method of the disclosure is applicable to all types of high-density arrays, including single-molecule arrays, clustered arrays, and bead arrays. Nucleotides labeled with dye compounds of the present disclosure may be used for sequencing templates on essentially any type of array, including but not limited to those formed by immobilization of nucleic acid molecules on a solid support.
However, nucleotides labeled with dye compounds of the disclosure are particularly advantageous in the context of sequencing of clustered arrays. In clustered arrays, distinct regions on the array (often referred to as sites, or features) comprise multiple polynucleotide template molecules. Generally, the multiple polynucleotide molecules are not individually resolvable by optical means and are instead detected as an ensemble. Depending on how the array is formed, each site on the array may comprise multiple copies of one individual polynucleotide molecule (e.g., the site is homogenous for a particular single- or double-stranded nucleic acid species) or even multiple copies of a small number of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species). Clustered arrays of nucleic acid molecules may be produced using techniques generally known in the art. By way of example, WO 98/44151 and WO00/18957, each of which is incorporated herein, describe methods of amplification of nucleic acids wherein both the template and amplification products remain immobilized on a solid support in order to form arrays comprised of clusters or “colonies” of immobilized nucleic acid molecules. The nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for sequencing using nucleotides labeled with dye compounds of the disclosure.
Nucleotides labeled with dye compounds of the present disclosure are also useful in sequencing of templates on single molecule arrays. The term “single molecule array” or “SMA” as used herein refers to a population of polynucleotide molecules, distributed (or arrayed) over a solid support, wherein the spacing of any individual polynucleotide from all others of the population is such that it is possible to individually resolve the individual polynucleotide molecules. The target nucleic acid molecules immobilized onto the surface of the solid support can thus be capable of being resolved by optical means in some embodiments. This means that one or more distinct signals, each representing one polynucleotide, will occur within the resolvable area of the particular imaging device used.
Single molecule detection may be achieved wherein the spacing between adjacent polynucleotide molecules on an array is at least 100 nm, more particularly at least 250 nm, still more particularly at least 300 nm, even more particularly at least 350 nm. Thus, each molecule is individually resolvable and detectable as a single molecule fluorescent point, and fluorescence from said single molecule fluorescent point also exhibits single step photobleaching.
The terms “individually resolved” and “individual resolution” are used herein to specify that, when visualized, it is possible to distinguish one molecule on the array from its neighboring molecules. Separation between individual molecules on the array will be determined, in part, by the particular technique used to resolve the individual molecules. The general features of single molecule arrays will be understood by reference to published applications WO00/06770 and WO01/57248, each of which is incorporated herein by reference. Although one use of the labeled nucleotides of the disclosure is in sequencing-by-synthesis reactions, the utility of such nucleotides is not limited to such methods. In fact, the labeled nucleotides described herein may be used advantageously in any sequencing methodology which requires detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide.
In particular, nucleotides labeled with dye compounds of the disclosure may be used in automated fluorescent sequencing protocols, particularly fluorescent dye-terminator cycle sequencing based on the chain termination sequencing method of Sanger and co-workers. Such methods generally use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides in a primer extension sequencing reaction. So-called Sanger sequencing methods, and related protocols (Sanger-type), utilize randomized chain termination with labeled dideoxynucleotides.
Thus, the present disclosure also encompasses nucleotides labeled with dye compounds which are dideoxynucleotides lacking hydroxyl groups at both of the 3′ and 2′ positions, such modified dideoxynucleotides being suitable for use in Sanger type sequencing methods and the like.
Nucleotides labeled with dye compounds of the present disclosure incorporating 3′ blocking groups, it will be recognized, may also be of utility in Sanger methods and related protocols since the same effect achieved by using dideoxy nucleotides may be achieved by using nucleotides having 3′ OH blocking groups: both prevent incorporation of subsequent nucleotides. Where nucleotides according to the present disclosure, and having a 3′ blocking group are to be used in Sanger-type sequencing methods it will be appreciated that the dye compounds or detectable labels attached to the nucleotides need not be connected via cleavable linkers, since in each instance where a labeled nucleotide of the disclosure is incorporated; no nucleotides need to be subsequently incorporated and thus the label need not be removed from the nucleotide.
Alternatively, the sequencing methods described herein may also be carried out using unlabeled nucleotides and affinity reagents containing a tricyclic polymethine dye described herein. For example, one, two, three or each of the four different types of nucleotides (e.g., dATP, dCTP, dGTP and dTTP or dUTP) in the incorporation mixture of step (a) may be unlabeled. Each of the four types of nucleotides (e.g., dNTPs) has a 3′ blocking group to ensure that only a single base can be added by a polymerase to the 3′ end of the primer polynucleotide. After incorporation of an unlabeled nucleotide in step (b), the remaining unincorporated nucleotides are washed away. An affinity/labeling reagent is then introduced that specifically recognizes and binds to the incorporated dNTP to provide a labeled extension product comprising the incorporated dNTP. Uses of unlabeled nucleotides and affinity/labeling reagents in sequencing by synthesis have been disclosed in WO 2018/129214 and WO 2020/097607, as well as U.S. Publication No. 2023/0383342 A1 and U.S. Ser. No. 18/820,008, each of which is incorporated by reference in its entirety. A modified sequencing method of the present disclosure using unlabeled nucleotides may include the following steps:
In some embodiments of the modified sequencing method described herein, the method further comprises removing the affinity reagents from the incorporated nucleotides. In still further embodiments, the 3′ blocking group and the affinity reagent are removed in the same reaction. In some embodiments, the method further comprises a step (f) washing the solid support with an aqueous wash solution. In further embodiments, steps (b′) through (f) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles to determine the target polynucleotide sequences. In some embodiments, the set of affinity reagents may comprise a first affinity reagent that binds specifically to the first type of nucleotide, a second affinity reagent that binds specifically to the second type of nucleotide, and a third affinity reagent that binds specifically to the third type of nucleotide. In some further embodiments, each of the first, second and the third affinity reagents comprises a detectable labeled that is spectrally distinguishable. In some embodiments, the affinity reagents may include protein tags, antibodies (including but not limited to binding fragments of antibodies, single chain antibodies, bispecific antibodies, and the like), aptamers, knottins, affimers, or any other known agent that binds an incorporated nucleotide with a suitable specificity and affinity. In one embodiment, at least one affinity reagent is an antibody or a protein tag. In another embodiment, at least one of the first type, the second type, and the third type of affinity reagents is an antibody or a protein tag comprising one or more detectable labels (e.g., multiple copies of the same detectable label), wherein the detectable label is or comprises a tricyclic polymethine dye moiety described herein.
Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.
All experiments that were sensitive to moisture or air were performed under a positive pressure of Ar gas in flame-dried glassware equipped with a rubber septum. Solvent and liquid reagents were transferred using Ar-flushed syringes or cannulae. Unless otherwise noted, commercial reagents were purchased from Aldrich, Alfa Aesar, fluorochem and other commercial suppliers and were used as received. All reactions were monitored using TLC Merck 60 F254 pre-coated silica gel plates. Flash column chromatography was conducted on biotage column. Nuclear magnetic resonance spectra (1H, 13C NMR) were recorded on Bruker AC spectrometer. Chemical shift for 1H NMR spectra is reported as δ in parts per million (ppm) down field from tetramethylsilane (δ 0.0 ppm) using the residual solvent signal as an internal standard: chloroform-d (δ 7.26 ppm, singlet), dimethyl sulfoxide-d6 (δ 2.49 ppm, quintet), methanol-d4 (δ 3.3 ppm, quintet), water-d2 (δ 4.67 ppm, singlet). Multiplicities are given as: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), dt (doublet of triplet), m (multiplet). Coupling constants J are reported in hertz. The number n of protons for a given resonance absorbance is indicated by nH. Carbon nuclear magnetic resonance spectra (13C NMR) are reported as δ in part per million relatives to solvent signal: chloroform-d (δ 77.7 ppm, triplet), dimethyl sulfoxide-d6 (δ 39.5 ppm, septet), methanol-d4 (δ 49.0 ppm, septet). Electrospray ionisation mass spectrometry (ESI-MS) analyses were carried out on an ACQUITY H-Class PLUS Bio System with SQD 2 mass detector in an ESI mode. UV/vis absorption spectra were obtained using an Agilent Technology, Cary Series UV-vis Spectra. Fluorescence spectra were recorded on an Agilent Eclipse Fluorescence Spectra fluorimeter with a 10 mm cuvette.
Imidazole, compound 1, was dissolved in DMF (the volume calculated to yield 0.5M solution). After cooling to 0° C., sodium hydride (1.2 eq.) was added slowly. The reaction mixture was stirred for 30 min, and 2-(2-bromoethyl)-1,3-dioxolane (1.1 eq.) was added. The reaction was stirred at ambient temperature under nitrogen for 16 h. After completion, the reaction mixture was diluted with water and extracted three times with DCM. The organic phase was dried over magnesium sulfate and evaporated, yielding compound 2. The product was used without further purification.
Compound 2 was dissolved in acetonitrile and 1,3-propanesultone (1.1 eq) was added. After refluxing for 24-48 h, the crude mixture was diluted with water and purified using RP chromatography (C18 column) to afford compound 3a.
Alternatively, Compound 2 was dissolved in acetonitrile and 6-bromohexanoic acid (1.1 eq) was added. After refluxing for 24-48 h, the crude mixture was diluted with water and purified using RP chromatography (C18 column) to give compound 3b.
Compound 4 was mixed with N,N′-diphenylformamidine (1.1 eq., CAS 622-15-1) and 1-ethyl-3-methylimidazolium chloride. The mixture was stirred at 105° C. until conversion of compound 4 into compound 5 is complete (2-8 h). Afterwards, either compound 3a or compound 3b was added to the flask, followed by NaOAc (4 eq.), and the mixture was stirred at 135° C. for 4-16 h. The target product compound 6 was purified using RP chromatography (C18 column).
Dye 6 was dissolved in DCM (1 mg/1 mL) and a 1:1 (by volume) mixture of sulfuric acid and water was added. The mixture was stirred vigorously for 45 min, DCM was removed under reduced pressure and the residue was dissolved in water. The excess of sulfuric acid was quenched with a calculated amount of sodium hydroxide until pH ˜10. The dye, compound 7, was purified using RP. The purity of the dyes was confirmed by HPLC and LCMS.
Reagent and conditions: (a) CH3CO2H, 130° C., 12-14 h; (b) 2-(2-bromoethyl)-1,3-dioxolane, KI, DMF, 72 h, 50° C.; (c) (i) N,N′-diphenylformamidine, (CH3CO)2O, 80° C., 2-3 h, (ii) NaOAc, CH3CO2H, 80° C., 5-6 h; (d) CHCl3, 50% H2SO4/H2O, 1 h, RT.
Synthesis of Compound 10: 2-Naphthylhydrazine hydrochloride (1.0 g, 5.13 mmol), compound 9 (1.4 g, 6.73 mmol) and 10 mL of acetic acid were taken in a 50 mL round bottom flask and the resulting solution was stirred at 130° C. for 16-18 h. Then the reaction was allowed to cool to room temperature and acetic acid was removed to concentrate by rotary evaporator. Next, the solution was neutralized by saturated NaHCO3 solution (5 mL), and the aqueous solution was poured into EtOAc to get a precipitate. The precipitate was filtered and washed with Et2O (100 mL) and hexane (150 mL). The solid precipitate was dried in air to afford the desired product 10 as a brown solid (1.43 g, 84%). 1H NMR (500 MHz, CD3OD, 293 K, δ): 8.12-8.08 (m, 1H), 7.99-7.96 (m, 1H), 7.91-7.88 (dd, J=8.0, 4.0 Hz, 1H), 7.79-7.72 (m, 1H), 7.69-7.58 (m, 1H); 7.48-7.45 (m, 1H), 2.93-2.77 (m, 2H), 2.57-2.52 (m, 2H), 2.39 (s, 3H), 2.19-2.12 (m, 2H), 2.00-1.92 (m, 2H), 1.29 (s, 3H).
Synthesis of Compound 11: To a solution of compound 10 (1.0 g, 3.02 mmol) in 5 mL of sulfolane, tBuOK (340 mg, 3.02 mmol), KI (50 mg, 0.3 mmol) and 2-(2-Bromoethyl)-1,3-dioxolane (1.8 mL, 15.3 mmol) were taken in a 50 mL round-bottom flask equipped with magnetic stir bar. The reaction was heated to 50° C. for 72 h under argon atmosphere. Then, the reaction was stopped and allowed to cool to room temperature. Finally, it was subjected to flash chromatography on silica gel (eluent=CH2Cl2/MeOH (10:1)) to afford the desired product 11 as a brown solid (365 mg, 28%). 1H NMR (300 MHz, CD3OD, 293 K, δ): 8.31-8.29 (m, 1H), 8.24-8.22 (d, J=8.0 Hz, 1H), 8.16-8.14 (d, J=8.0 Hz, 1H), 7.98-7.96 (d, J=4 Hz, 1H), 7.82-7.78 (m, 1H), 7.73-7.67 (m, 1H), 5.06-5.03 (t, J=4.0 Hz, 1H), 4.79-4.75 (t, J=8.0 Hz, 2H), 4.57 (s, 2H), 3.90-3.80 (m, 4H), 3.30 (s, 3H), 2.82-2.74 (m, 2H), 2.64-2.42 (m, 6H), 1.78 (s, 3H). LCMS (ESI+)=432.28.
Synthesis of Compound 15: Compound 15 was prepared from compound 14 following the same procedure as described above for synthesis of compound 11. It was purified by flash chromatography on silica gel (eluent=CH2Cl2/MeOH (10:1)) to afford the desired product 15 as a brown solid (72%). 1H NMR (400 MHz, CD3OD, 293 K, δ): 7.90-7.88 (m, 1H), 7.77-7.74 (m, 1H), 7.67-7.65 (m, 1H), 5.03-5.01 (t, J=4.0 Hz, 1H), 4.72-4.69 (t, J=8.0 Hz, 2H), 3.88-3.86 (m, 2H), 3.82-3.79 (m, 2H), 3.30-3.28 (m, 1H), 3.24-3.19 (dd, J=8.0, 1H), 2.40-2.38 (m, 2H), 2.20 (s, 3H), 1.59 (s, 3H), 1.33-1.30 (t, J=4.0 Hz, 2H), 1.11-0.91 (m, 2H). LCMS (ESI+)=332.26.
Synthesis of Compound 16: Compound 15 (200 mg, 0.6 mmol) and N,N′-diphenylformamidine (120 mg, 0.6 mmol) were taken in a 25 mL round-bottom flask equipped with a magnetic stir bar, next 3 mL of Ac2O was added to it. The reaction mixture was heated to 80° C. for 3 h. Next, compound 11 (312 mg, 0.72 mmol), NaOAc (246 mg, 3.01 mmol) and 3 mL of CH3CO2H were added to the reaction mixture and the whole mixture was heated further 5-6 h at 80° C. under argon atmosphere. Then, the reaction was allowed to cool to room temperature. Finally, it was subjected to flash chromatography on silica gel (eluent=CH2Cl2/MeOH (10:3)) to afford the desired product, which contained two isomers as the product has 2 chiral centers. The product mixture was a pink solid (52 mg, 11%) which was used in the next step without purifying the single isomer. LCMS (ESI+)=773.70.
Synthesis of Compound I-1: Compound 16 (20 mg, 0.02 mmol) was dissolved in 15 mL of CHCl3 and next 10 mL of 50% aq. H2SO4 was added slowly. Next, the resulting solution was stirred for 30 min. Next, the mixture was extracted using CHCl3/MeOH (20/1). The organic part was concentrated. After that, the residue was subjected to preparative RP C18 column to afford four isomers; the desired product I-1 was isolated with retention time 16.06 min (pink solid). HPLC (ELSD) tR=16.06 min, purity 100%; LCMS (ESI+)=667.54.
Synthesis of Compound 17:
Compound 17 was prepared according to standard procedure of Fisher indole synthesis as above, as a dark brown solid (72%). 1H NMR (400 MHz, CDCl3, 293 K, δ): 7.97-7.93 (m, 2H), 7.85-7.83 (m, 1H), 7.79-7.96 (m, 1H), 7.56-7.51 (m, 1H), 7.46-7.42 (m, 1H), 2.35 (s, 3H), 2.15-2.11 (m, 2H), 2.03-1.94 (m, 2H), 1.58-1.54 (m, 2H), 1.51 (s, 3H), 1.42-1.34 (m, 2H), 1.15-1.10 (m, 2H). LCMS (ESI+)=310.48.
Synthesis of Compound 18:
Compound 18 was prepared according to same procedure as described above; the desired product was a pink solid (32%). 1H NMR (400 MHz, CD3CN, 293 K, δ): 8.25-8.15 (m, 2H), 7.98-7.87 (m, 1H), 7.79-7.70 (m, 2H), 7.57-7.47 (m, 1H), 4.62-4.60 (t, J=4.0 Hz, 1H), 3.83-3.80 (m, 2H), 3.75-3.72 (m, 2H), 3.67-3.62 (m, 4H), 3.19-3.15 (m, 2H), 2.18-2.14 (m, 2H), 1.90-1.85 (m, 2H), 1.83-1.77 (m, 5H), 1.72 (s, 3H), 1.64-1.60 (m, 2H). LCMS (ESI+)=410.59.
Synthesis of Compound 19:
Compound 19 was prepared like the above described procedure by mixing compound 18 and compound 11, as a pink solid (yield=23%) and directly proceeded to next step without isolating the single isomer.
Synthesis of Compound I-2:
Compound I-2 was prepared according to the same procedure described in the synthesis of Compound I-1. LCMS (ESI+)=745.76.
Synthesis of Compound 20:
Compound 20 was prepared according to similar procedure as described above, as a purple solid (29%). LCMS (ESI−)=390.17.
Synthesis of Compound 21:
Compound 21 was prepared according to the same procedure described above by reacting Compound 18 with Compound 20; as a pink solid (18%).
Synthesis of Compound I-3:
Compound I-3 was prepared according to the same procedure described in the synthesis of Compound I-1. LCMS (ESI+)=703.65.
Synthesis of Compound 22:
Compound 22 was prepared according to same procedure as described above as a pink solid (38%). 1H NMR (400 MHz, CD3CN3, 293 K, δ): 7.74-7.72 (d, J=8.0 Hz, 1H), 7.67-7.66 (d, J=4.0 Hz, 1H), 7.54-7.51 (dd, J=8.0, 4.0 Hz, 1H), 5.00-4.98 (t, J=4.0 Hz, 1H), 4.56-4.53 (t, J=4.0 Hz, 2H), 3.84-3.74 (m, 6H), 2.76 (s, 3H), 2.34-2.30 (m, 2H), 1.52 (s, 6H). LCMS (ESI+)=318.19.
Synthesis of Compound I-4:
Compound I-4 was prepared according to the same procedure described in the synthesis of Compound I-1 from Compound 15. LCMS (ESI+)=611.38.
Synthesis of Compound 23:
Compound 23 was prepared according to the same procedure described above as a purple solid (55%). LCMS (ESI+)=360.45.
Additional dyes, shown in Table 1, can be prepared according to the methods described herein.
Synthesis of Compound 25: 2-methyl-benzothiazole, was dissolved in sulfolane (the volume calculated to yield a 0.5M solution) in a sealed tube fitted with a magnetic stir bar. To the reaction tube was added 2-(2-bromethyl)-1-3-dioxolane (1.5 eq). The reaction was stirred and heated to 50° C. for 16 h to give Compound 25. After completion, the reaction mixture was loaded on to a Si column and purified by DCM/MeOH (0%-20% MeOH) Yield: 75%. 1H NMR (400 MHz, CDCl3) δ 8.29 (ddd, J=8.2, 1.2, 0.6 Hz, 1H), 8.06 (dt, J=8.6, 0.9 Hz, 1H), 7.83 (ddd, J=8.5, 7.3, 1.2 Hz, 1H), 7.72 (ddd, J=8.3, 7.3, 1.0 Hz, 1H), 5.11-4.88 (m, 3H), 4.01-3.91 (m, 2H), 3.91-3.80 (m, 2H), 3.45 (s, 3H), 2.49-2.33 (m, 2H).
Synthesis of Compound 27: Compound 25 [3-(2-(1,3-dioxolan-2-yl)ethyl)-2-methylbenzo[d]thiazol-3-ium](above) was mixed with Compound 26 (3,3-dimethyl indole hemicyanine 5-acetic acid), NaOAc, AcOH and Ac2O in a reaction tube. The reaction tube was sealed and then heated to 90° C. for 3 h to give Compound 27. The target compound was purified using RP chromatography (0.1M TEABaq/ACN). Yield: 20%
Synthesis of Compound I-36: To the reaction flask containing the unlocked dye, Compound 27, was added DCM (100 eq), sulfuric acid (10 eq), and water (10 eq). The reaction mixture was stirred at room temperature. The brightly colored solution became clear, leaving portions of the desired product stuck to the walls of the flask. Enough NaOH (4M) was added to neutralize the reaction before drying down the reaction in vacuo. The material was redissolved in 50% ACN/H2O and filtered before purification by RP Prep HPLC. The absorbance of Dye I-36 in 50% EtOH/H2O is about 568 nm.
Compound I-37 was prepared in a similar manner according to the methods described herein. First, Compound 15 was mixed with N,N′-diphenylformadine and acetic anhydride at 80° C. for 2-3 h to give Compound 28. Compound 28 was mixed with Compound 29 (3-(3,3-diethoxypropyl)-2-methylbenzo[d]thiazol-3-ium]), NaOAc, AcOH and Ac2O in a reaction tube. The reaction tube was sealed and then heated to 100° C. for 3 h to give Compound 30. To the reaction flask containing the unlocked dye, Compound 30, was added DCM, sulfuric acid and water. The reaction mixture was stirred at room temperature for 1 h and purified in a similar manner to Compound I-36 to yield Compound I-37.
To a sealed reaction vial were added 2-methylbenzothiazol-5-ol, potassium carbonate (1.3 eq), ethyl-6-bromohexanoate and DMF (the volume calculated to yield a 0.5M solution). The mixture was heated at 50° C. for 18 h. The cooled reaction mixture was separated into ethyl acetate and washed with water in a separatory funnel. The organic layer was dried over magnesium sulfate and filtered. The material was dried down in vacuo to yield a clear oil. The material was purified by normal phase Si chromatography Petroleum Ether/EA (5%-95%) to yield Compound 32 as a clear colorless oil. Yield: 89%. 1H NMR (400 MHz, DMSO) δ 7.86 (dd, J=8.8, 0.4 Hz, 1H), 7.43 (d, J=2.4 Hz, 1H), 7.01 (dd, J=8.8, 2.5 Hz, 1H), 4.07 (q, J=6.9 Hz, 2H), 4.02 (t, J=6.5 Hz, 2H), 2.77 (s, 3H), 2.32 (t, J=7.3 Hz, 2H), 1.74 (dt, J=8.0, 6.6 Hz, 2H), 1.58 (dt, 2H), 1.46 (dt, 2H), 1.18 (t, J=7.1 Hz, 3H). Compound 32 was used to prepare Compound I-38 according to the methods described herein.
General Procedure of Fully Functionalized Nucleotides (ffNs) Coupled with Rigid Polymethine Dyes:
The corresponding dye (10 mol) was dissolved in anhydrous DMA (1 mL). DIPEA (5 eq.) and TNTU (1.1 eq.) were added consecutively. After confirming the formation of an activated ester, pppN (10 mol) in 200 L of 0.1M TEAB was added to the mixture. After stirring for 16 h at room temperature, the sample was diluted with 15 mL of 0.1 M TEAB and purified via ion-exchange and RP-C18 chromatography. The purified samples were diluted to 100 μM concentration and stored at −80° C.
ffT-db-AOL-I-2, ffT-db-AOL-I-3 and ffT-db-AOL-I-4 were also prepared according to same procedure as described above. Each ffT contains a 3′-AOM blocking group. ffT-db-AOL-I-1 has demonstrated much higher fluorescence intensity as compared to SulfoCy3.5 and Cy3.5, two known green polymethine dyes.
The fluorescent spectra of other exemplary ffNs prepared from dyes disclosed herein are summarized in Table 3 below. All dyes showed increased brightness in solution compared to their unlocked analogues.
The ffNs labeled with the tricyclic polymethine dyes described herein were tested on an Illumina MiSeq™ instrument, which was set up to take the first image with a green excitation light (˜523 nm) and the second image with a green excitation light (˜523 nm). Images were taken simultaneously through two separate optic collection channels, which are in blue (540-585 nm) and green (585-640 nm). It is also referred to as 1ex-2ch green/green sequencing.
Sequencing run on 1Ex-2Ch green/green MiSeq™: an incorporation mix containing the following four ffNs with 3′-AOM blocking group, each in 2 μM was used: ffA-db-AOL-Cy3B (a known green dye); ffC-db-(AOL)2-NR7180A, dark G, and ffT-db-AOL-I-1 in 50 mM glycine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4 and a DNA polymerase. The scatterplot in
The structures of ffC-db-AOL-I-10 and ffA-db-AOL-Cy3B are:
The structures of ffC-db-(AOL)3-NR7180A and ffT-db-AOL-I-1 are:
This application claims the benefit of priority to U.S. Provisional Application Nos. 63/616,289 and 63/681,387, filed Dec. 29, 2023 and Aug. 9, 2024 respectively, each of which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63681387 | Aug 2024 | US | |
| 63616289 | Dec 2023 | US |
