阅读说明：本技术 环外胺取代的香豆素化合物及其作为荧光标记物的用途 (Exocyclic amine substituted coumarin compound and application thereof as fluorescent marker ) 是由 N.罗马诺夫 C.阿纳斯塔西 P.麦考利 于 2020-02-28 设计创作，主要内容包括：本申请涉及环外胺-取代的香豆素衍生物以及它们作为荧光标记物的用途。这些化合物可用作核酸测序应用中核苷酸的荧光标记物。(The present application relates to exocyclic amine-substituted coumarin derivatives and their use as fluorescent labels. These compounds are useful as fluorescent labels for nucleotides in nucleic acid sequencing applications.)

1. A compound of formula (I), a salt or meso form thereof:

wherein:

x is O, S, Se or NRⁿWherein R isⁿIs H, C_1-6Alkyl, or C_6-10An aryl group;

ring a is a 3-to 10-membered heterocyclyl;

R、R¹、R²and R⁴Each independently of the others being H, halogen, -CN, -CO₂H. Amino, -OH, C-amido, N-amido, -NO₂、-SO₃H、-SO₂NR^aR^bOptionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;

each R is³Independently halogen, -CN, -CO₂H、-(CH₂)_p-CO₂R^c、-(CH₂)_q-C(O)NR^dR^eAmino, -OH, C-amido, N-amido, -NO₂、-SO₃H、-SO₂NR^aR^bOptionally substituted C_1-6Alkyl, optionally substituted C_2-6Alkenyl, optionally substituted C_2-6Alkynyl, optionally substitutedCarbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R³Oxo (═ O);

wherein p and q are each 1, 2, 3 or 4;

each R is⁵Independently halogen, -CN, -CO₂R^fAmino, -OH, C-amido, N-amido, -NO₂、-SO₃H、-SO₂NR^aR^bOptionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;

each R is^aAnd R^bIndependently is H or optionally substituted C_1-6An alkyl group;

each R is^c、R^d、R^eAnd R^fIndependently is H, optionally substituted C_1-6An alkyl group, an optionally substituted carbocyclyl group, an optionally substituted heterocyclyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group;

m is 0, 1, 2, 3 or 4; and is

n is 0, 1, 2, 3, 4 or 5.

2. The compound of claim 1, wherein X is O or S.

3. The compound of claim 1, wherein X is NRⁿ。

4. A compound according to any one of claims 1 to 3, wherein R is H, halogen, -CO₂H. Amino, -OH, C-amido, N-amido, -NO₂、-SO₃H、-SO₂NH₂Optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

5. The compound of claim 4, wherein R is H, halogen, or C_1-6An alkyl group.

6. The compound of claim 5, wherein R is H.

7. The compound of any one of claims 1 to 6, wherein R¹Is H, halogen, or C_1-6An alkyl group.

8. The compound of claim 7, wherein R¹Is H.

9. The compound of any one of claims 1 to 8, wherein R²Is H, -SO₃H. Optionally substituted alkyl, or optionally substituted with-CO₂H or-SO₃C of H_1-4An alkyl group.

10. The compound of claim 9, wherein R²Is H or-SO₃H。

11. The compound of any one of claims 1 to 10, wherein R⁴Is H, -SO₃H. Optionally substituted alkyl, or optionally substituted with-CO₂H or-SO₃C of H_1-4An alkyl group.

12. The compound of claim 11, wherein R⁴Is H or-SO₃H。

13. The compound of any one of claims 1 to 12, wherein ring a is a 3 to 7 membered monocyclic heterocycle comprising one nitrogen atom.

14. The compound of any one of claims 1 to 13, wherein ring a is

15. The compound of any one of claims 1 to 14, wherein n is 1, 2 or 3, and wherein each R is³Independently is-CO₂H、-SO₃H. Optionally substituted by-CO₂H or-SO₃C of H_1-4Alkyl, - (CH)₂)_p-CO₂R^cOr optionally substituted C_1-6An alkyl group.

16. The compound of claim 15, wherein n is 1.

17. The compound of claim 16, wherein R³is-CO₂H or- (CH)₂)_p-CO₂R^c。

18. The compound of any one of claims 1 to 17, wherein each R⁵Is halogen, -CN, -CO₂H、-SO₃H、-SO₂NR^aR^bOr optionally substituted C_1-6An alkyl group.

19. The compound of claim 18, wherein R⁵is-CO₂H、-SO₃H、-SO₂NH₂Or substituted by-CO₂H、-SO₃H or-SO₂NH₂C of (A)_1-6An alkyl group.

20. The compound of claim 1, wherein X is O, S or NH; each R, R¹、R²And R⁴Is H; ring A isn is 0 or 1; r³is-CO₂H or- (CH)₂)_p-CO₂R^c(ii) a m is 0 or 1; and R is⁵Is halogen, -CO₂H、-SO₃H、-SO₂NH₂Or substituted by-SO₃H or-SO₂NH₂C of (A)_1-6An alkyl group.

21. The compound of claim 1, and salts and meso forms thereof, selected from the group consisting of:

22. the compound according to claims 1 to 21, wherein said compound is attached to a nucleotide or oligonucleotide.

23. A nucleotide or oligonucleotide labelled with a compound according to any one of claims 1 to 21.

24. A labelled nucleotide or oligonucleotide according to claim 23 wherein the compound is formed by the reaction of R of formula (I)³Attaching the nucleotide or oligonucleotide.

25. The labeled nucleotide or oligonucleotide according to claim 24, wherein R of formula (I)³is-CO₂H or- (CH)₂)_p-CO₂H and the attachment uses-CO₂The H group forms an amide.

26. A labelled nucleotide or oligonucleotide according to claim 23 wherein the compound is formed by the reaction of R of formula (I)⁵Attaching the nucleotide or oligonucleotide.

27. A labelled nucleotide or oligonucleotide according to claim 26 wherein R of formula (I)⁵is-CO₂H and the attachment uses-CO₂H radical formTo form amide.

28. A labelled nucleotide or oligonucleotide according to any one of claims 23 to 27, wherein the compound is attached to the C5 position of the pyrimidine base or the C7 position of the 7-deaza purine base through a linker moiety.

29. A labelled nucleotide or oligonucleotide according to any one of claims 23 to 28, further comprising a 3' OH blocking group covalently linked to the ribose or deoxyribose sugar of the nucleotide.

30. A kit comprising a first labeled nucleotide and a second labeled nucleotide according to any one of claims 23 to 29.

31. The kit of claim 30, wherein the second labeled nucleotide is labeled with a different compound than the first labeled nucleotide.

32. The kit of claim 31, wherein the first and second labeled nucleotides are excitable using a single laser wavelength.

33. The kit of claim 31 or 32, further comprising a third nucleotide and a fourth nucleotide, wherein each of the second, third, and fourth nucleotides is labeled with a different compound, wherein each label has a different maximum absorbance that is distinguishable from the other labels.

34. The kit of claim 30, wherein the kit comprises four nucleotides, wherein a first of the four nucleotides is a labeled nucleotide according to any one of claims 23 to 29, a second of the four nucleotides carries a second label, a third nucleotide carries a third label, and a fourth nucleotide is unlabeled (black).

35. The kit according to claim 30, wherein the kit comprises four nucleotides, wherein a first of the four nucleotides is a labelled nucleotide according to any one of claims 23 to 29, a second of the four nucleotides carries a second label, a third nucleotide carries a mixture of two labels, and a fourth nucleotide is unlabelled (black).

36. Use of a compound according to any one of claims 1 to 21, a labelled nucleotide or oligonucleotide according to claims 23 to 29, or a kit according to any one of claims 30 to 35 in sequencing, expression analysis, hybridisation analysis, genetic analysis, RNA analysis, or protein binding assays.

37. The use of claim 36, performed on an automated sequencer, wherein the automated sequencer comprises two lasers operating at different wavelengths.

38. A method of sequencing comprising incorporating a labelled nucleotide according to any one of claims 23 to 29 in a sequencing test.

39. The method of claim 38, further comprising detecting the labeled nucleotide.

40. The method of claim 38 or 39, wherein the sequencing test is performed on an automated sequencer, and wherein the automated sequencer comprises two light sources operating at different wavelengths.

41. A method of synthesis of a compound of formula (I) as described herein.

Technical Field

The present disclosure relates to exocyclic amine substituted coumarin derivatives and their use as fluorescent markers. In particular, the compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.

Background

Several publications and patent documents are cited in this application in order to more fully describe the state of the art to which this disclosure pertains. The disclosures of each of these publications and documents are incorporated herein by reference.

Non-radioactive detection of nucleic acids carrying fluorescent labels is an important technique in molecular biology. Many of the procedures employed in recombinant DNA technology have previously relied upon using, for example³²P use of a radioactively labeled nucleotide or polynucleotide. Radioactive compounds allow sensitive detection of nucleic acids and other molecules of interest. However, there are serious limitations in the use of radioisotopes such as their expense, limited shelf life, inadequate sensitivity and, more importantly, safety considerations. Eliminating the need for radioactive labels reduces both safety risks and environmental impact and costs associated with, for example, reagent disposal. Suitable methods for non-radioactive fluorescence detection include, by way of non-limiting example, 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 enable independent detection of multiple spatially overlapping analytes. In such a multiplex method, the number of reaction vessels can be reduced, which simplifies the experimental protocol and facilitates the production of kits for specific applications. In a multicolor automated DNA sequencing system, for example, multiplex fluorescence detection allows analysis of multiple nucleotide bases in a single electrophoretic lane, thereby increasing throughput (throughput) and reducing uncertainty associated with electrophoretic mobility changes between lanes compared to a monochromatic method.

However, multiplex fluorescence detection can be problematic and there are a number of important factors that limit the selection of suitable fluorescent labels. First, it can be difficult to find dye compounds with absorption and emission spectra that are well resolved in a given application. Furthermore, when several fluorescent dyes are used together, the generation of fluorescent signals in distinguishable spectral regions by simultaneous excitation can be complicated by the fact that the absorption bands of these dyes are usually evacuated, so that comparable fluorescence excitation efficiencies are difficult to achieve even for two dyes. Many excitation methods use high power light sources like lasers and therefore the dye must have sufficient photostability to survive such excitation. A final consideration of particular importance to molecular biology methods is the extent to which the fluorescent dyes must be compatible with chemical reagents such as, for example, DNA synthesis solvents and reagents, buffers, polymerases, and ligases.

As sequencing technology advances, a need has developed for additional fluorescent dye compounds, nucleic acid conjugates thereof, and multiple fluorescent dye sets that satisfy all of the above limitations and are particularly suited for high-throughput molecular methods such as solid-phase sequencing and the like.

Fluorescent dye molecules with improved fluorescent properties, such as suitable fluorescence intensity, shape of fluorescence, and wavelength maxima of fluorescence, can improve the speed and accuracy of nucleic acid sequencing. A strong fluorescence signal is particularly important when the measurement is performed in a water-based biological buffer and at higher temperatures, since the fluorescence intensity of most dyes is significantly lower under such conditions. In addition, the nature of the base to which the dye is attached also affects the fluorescence maximum, fluorescence intensity and other spectral properties of the dye (spectral properties). The sequence specific interaction between the nucleobase and the fluorescent dye can be tuned by the specific design of the fluorescent dye. Optimization of the structure of the fluorescent dye can improve the efficiency of nucleotide incorporation, reduce the level of sequencing errors, and reduce the use of reagents in nucleic acid sequencing, and thus reduce the cost of nucleic acid sequencing.

Some optical and technical developments have resulted in greatly improved image quality, but are ultimately limited by poor optical resolution. Typically, the optical resolution of an optical microscope is limited to objects spaced at about half the wavelength of the light used. In practice, however, only objects placed at great distances (at least 200nm to 350nm) can be resolved by optical microscopy. One way to increase the image resolution and increase the number of resolvable objects per unit surface area is to use shorter wavelengths of excitation light. For example, under the same optics, if the wavelength of light is shortened by Δ λ -100 nm, the resolution will be better (about Δ 50 nm/(about 15%)), less distorted images will be recorded, and the density of objects on the recognizable area will increase by about 35%.

Certain nucleic acid sequencing methods employ lasers to excite and detect dye-labeled nucleotides. These instruments use longer wavelength light, such as a red laser, and a suitable dye that can be excited at 660 nm. To detect more densely packed nucleic acid sequencing clusters while maintaining usable resolution, a shorter wavelength blue light source (450nm-460nm) can be used. In this case, the optical resolution will not be limited by the emission wavelength of the longer wavelength red fluorescent dye, but by the emission of the dye that can be excited by the next longest wavelength light source, e.g., by the "green laser" at 532 nm. Therefore, there is a need for blue dye labels for fluorescence detection in sequencing applications.

Although blue dye chemistry and related laser technology have been improved, for example, to produce dyes for DVD and Blu-ray disc (Blu-ray disc), these compounds are not suitable biomarkers and cannot be used as biomarkers.

Unfortunately, commercially available blue dyes with strong fluorescence suitable for nucleotide labeling are still very rare. Described herein are novel fluorescent compounds suitable for nucleotide labeling that exhibit strong fluorescence under blue light excitation.

Disclosure of Invention

The present invention relates to exocyclic amine-substituted coumarin derivatives. The compounds can be used as fluorescent labels, particularly for nucleotide labeling in nucleic acid sequencing applications. In some aspects, the dye absorbs light at short wavelength light, optimally absorbing light at wavelengths between 450nm and 460nm, and is particularly advantageous where a blue wavelength excitation source having a wavelength between 450nm and 460nm is used. Blue wavelength excitation allows detection and resolution of higher density features per unit area due to the shorter wavelength of fluorescence emission. When such dyes are used in conjugates of nucleotides, improvements in the length, strength, accuracy and quality of sequencing reads obtained during nucleic acid sequencing methods can be seen.

Some embodiments of the present invention relate to compounds of formula (I), or a salt thereof:

wherein X is O, S, Se or NRⁿWherein R isⁿIs H, C_1-6Alkyl or C_6-10An aryl group;

ring a is a 3-to 10-membered heterocyclyl;

R、R¹、R²and R⁴Each independently of the others being H, halogen, -CN, -CO₂H. Amino, -OH, C-amido, N-amido, -NO₂、-SO₃H、-SO₂NR^aR^bOptionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;

each R is³Independently halogen, -CN, -CO₂H、-(CH₂)_p-CO₂R^c、-(CH₂)_q-C(O)NR^dR^eAmino, -OH, C-amido, N-amido, -NO₂、-SO₃H、-SO₂NR^aR^bOptionally substituted C_1-6Alkyl, optionally substituted C_2-6Alkenyl, optionally substituted C_2-6Alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R³Oxo (═ O); wherein p and q are each 1, 2, 3 or 4;

each R is⁵Independently halogen, -CN, -CO₂R^fAmino, -OH, C-amido, N-amido, -NO₂、-SO₃H、-SO₂NR^aR^bOptionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;

each R is^aAnd R^bIndependently is H or optionally substituted C_1-6An alkyl group;

each R is^c、R^d、R^eAnd R^fIndependently is H, optionally substituted C_1-6An alkyl group, an optionally substituted carbocyclyl group, an optionally substituted heterocyclyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group;

m is 0, 1, 2, 3 or 4; and n is 0, 1, 2, 3, 4 or 5.

In some aspects, at least one of m or n is not 0. In some other aspects, when R, R¹、R²And R⁴When each is H, at least one of m or n is not 0. For example, when R, R¹、R²And R⁴Each is H, and when m is 0, n is 1, 2, 3, 4 or 5. When R, R¹、R²And R⁴Is H, when n is 0, then m is 1, 2, 3 or 4. In some aspects, when m is 1; r⁵is-CO₂H；R、R¹、R²、R⁴Each is H; ring A isWhen the current is over; then X is O, Se or NRⁿ。

In some other embodiments, the compounds of the present disclosure are conjugated to 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. Conjugation may be via a carboxyl group (-CO)₂H) The carboxyl group may be reacted with an amino group or a hydroxyl group on a moiety (such as a nucleotide) or a linker bound to the moiety to form an amide or ester using methods known in the art.

Some other aspects of the present disclosure relate to dye compounds comprising a linker group to enable, for example, covalent attachment to a substrate moiety. Attachment can be made at any position of the dye, including on any R group. In some embodiments, R of formula (I) may be replaced by³Or R⁵The connection is made.

Other aspects of the disclosure provide nucleoside or nucleotide compounds defined by the formula:

N-L-dyes

Wherein N is a nucleotide;

l is an optional linker moiety; and is

The dye is a fluorescent compound according to the present invention.

Some other embodiments described herein relate to moieties, in particular nucleotides or oligonucleotides, labelled with compounds of formula (I).

Some other disclosures provide methods of sequencing using the dye compounds of the present invention.

According to further aspects, the present disclosure also provides kits comprising dye compounds (free or in conjugate form) that can be used for various immunoassays, oligonucleotide or nucleic acid labeling, or for synthesis-by-synthesis DNA sequencing. In yet another aspect, the present disclosure provides kits comprising a "set" of dyes that are particularly suited for cycles of sequencing-by-synthesis on an automated instrument platform. In some aspects are kits comprising one or more nucleotides, wherein at least one nucleotide is a labeled nucleotide described herein.

A further aspect of the present disclosure is the chemical preparation of compounds of the present disclosure including exocyclic amine-substituted coumarin dyes and moieties such as nucleotides labeled with such dyes.

A further aspect is a sequencing method comprising incorporating a labeled nucleotide described herein into a polynucleotide in a sequencing assay, and detecting the incorporated labeled nucleotide.

Drawings

FIG. 1 is a scatter plot showing the availability of fully functionalized A nucleotides labeled with dye I-4 described herein in a two-channel sequencing analysis.

FIG. 2 is a scatter plot showing the availability of fully functionalized A nucleotides labeled with dye I-5 described herein in a two-channel sequencing assay.

FIG. 3 is a scatter plot showing the availability of fully functionalized A nucleotides labeled with dye I-6 described herein in a two-channel sequencing analysis.

Detailed Description

The present disclosure provides exocyclic amine-substituted coumarin compounds that are particularly useful in fluorescence detection and sequencing-by-synthesis methods. Embodiments described herein relate to dyes of the structure of formula (I) and derivatives thereof, salts and meso forms thereof.

In some aspects, X is O. In some aspects, X is S. In some aspects, X is Se. In some aspects, X is NRⁿWherein R isⁿIs H, C_1-6Alkyl, or C_6-10Aryl, and in one aspect, RⁿIs H. In some other embodiments, when m is 1; r⁵is-CO₂H；R、R¹、R²、R⁴Each is H; ring A isThen X is O, Se or NRⁿ. In some other embodiments, when n is 0; ring A isR、R¹、R²、R⁴Each is H; when X is O; then m is 1, 2, 3 or 4. In some aspects, when n is 0, then m is 1, 2, 3, or 4 and at least one R⁵is-CO₂H. In some other aspects, when n is 1 and R³is-CO₂When H, then m is 0 or R⁵Is not-CO₂H。

In some aspects, R is H, halo, -CO₂H. Amino, -OH, C-amido, N-amido, -NO₂、-SO₃H、-SO₂NH₂Optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In one aspect, R is H. In another aspect, R is halogen. In some aspects, R is optionally substituted C_1-6An alkyl group. In some aspects, R is-CO₂H. In some aspects, R is-SO₃H. In some aspects, R is-SO₂NR^aR^bWherein R is^aAnd R^bIndependently is H or optionally substituted C_1-6An alkyl group. In one aspect, R is-SO₂NH₂. In some aspects, R is not-CN.

In some aspects, R¹Is H. In some aspects, R¹Is halogen. In some aspects, R¹is-CN. In some aspects, R¹Is C_1-6An alkyl group. In some aspects, R¹is-SO₂NR^aR^bWherein R is^aAnd R^bIndependently is H or optionally substituted C_1-6An alkyl group. In one aspect, R¹is-SO₂NH₂In some aspects, R¹Is not-CN.

In some aspects, R²Is H. In some aspects, R²Is halogen. In some aspects, R²is-SO₃H. In some aspects, R²Is optionally substituted alkyl, e.g. C_1-6An alkyl group. In some other embodiments, R²Is optionally substituted by-CO₂H or-SO₃C of H_1-4An alkyl group.

In some aspects, R⁴Is H. In some aspects, R⁴is-SO₃H. In some aspects, R⁴Is optionally substituted alkyl, e.g. C_1-6An alkyl group. In some other embodiments, R⁴Optionally substituted by-CO₂H or-SO₃C of H_1-4An alkyl group.

In some aspects, ring a is a 3 to 7 membered monocyclic heterocycle. In some other embodiments, the 3 to 7 membered monocyclic heterocycle contains one or more heteroatoms selected from nitrogen, oxygen, and sulfur. In some other embodiments, the 3-to 7-membered monocyclic heterocycle comprises one nitrogen atom. In some aspects, ring A isIn one such embodiment, ring a isIn some aspects, ring A isIn one such embodiment, ring a isIn some aspects, ring A isIn one such embodiment, ring a isIn some aspects of ring a described herein, n is 0. In some aspects of ring a described herein, n is 1. In some aspects of ring a described herein, n is 2 or 3. In some casesIn aspect, each R³Independently is-CO₂H、-SO₃H. Optionally substituted by-CO₂H or-SO₃C of H_1-4Alkyl, - (CH)₂)_p-CO₂R^cOr optionally substituted C_1-6An alkyl group. In some aspects, R3 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl. In other aspects, R³Is substituted C_1-4An alkyl group. In some aspects, R³To be substituted by-CO₂H or-SO₃C of H_1-4Alkyl or C_2-6An alkyl group. In some other embodiments, n is 1 and R³is-CO₂H or- (CH)₂)_p-CO₂R^c. In some other embodiments, R^cIs H or C_1-4An alkyl group.

Of formula (I)Part of the phenyl rings being optionally represented as R in one, two, three or four positions⁵Is substituted with the substituent(s). When m is 0, the phenyl ring is unsubstituted. When m is greater than 1, each R⁵May be the same or different. In some aspects, m is 0. In other aspects, m is 1. In other aspects, m is 2. In some aspects, m is 1, 2, or 3, and each R⁵Independently halogen, -CN, -CO₂R^fAmino, -OH, -SO₃H、-SO₂NR^aR^bOr optionally substituted C_1-6Alkyl radical, wherein R^fIs H or C_1-4An alkyl group. In some other embodiments, R⁵is-CO₂H、-SO₃H、-SO₂NH₂Or substituted by-CO₂H、-SO₃H or-SO₂NH₂C of (A)_1-6An alkyl group. In some other embodiments, R⁵Is- (CH)₂)_xCOOH, wherein x is 2, 3, 4, 5 or 6. In some embodiments, when R, R¹、R²、R⁴Each is H; n is 0; when m is 1; thenSubstitution at the following positions: in one embodiment, R⁵is-CO₂H。

Specific examples of compounds of formula (I) include those wherein X is O, S or NH; each R, R¹、R²And R⁴Is H; ring A isn is 0 or 1; r³is-CO₂H or- (CH)₂)_p-CO₂R^c(ii) a p is 1, 2, 3 or 4; r^cIs H or C_1-6An alkyl group; m is 0 or 1; and R is⁵Is halogen, -CO₂R^f、-SO₃H、-SO₂NR^aR^bOr substituted by-SO₃H or-SO₂NR^aR^bC of (A)_1-6An alkyl group. In some embodiments, R^aAnd R^bAt least one or two of them being H or C_1-6An alkyl group. In some other embodiments, R^fIs H or C_1-4An alkyl group. In some other embodiments, when m is 0, then n is 1; or when n is 0, m is 1. In one embodiment, m and n are both 1. In some other embodiments, when m is 1,substitution at the following positions: in one embodiment, R⁵is-CO₂H. In another embodiment, R⁵Is halogen, such as chlorine, or-SO₃H. In another embodiment, R⁵is-SO₂NR^aR^bWherein R is^aAnd R^bAt least one or two of them being H or C_1-6An alkyl group.

Specific examples of compounds of formula (I) include those wherein X is O, S or NH; each R, R¹、R²And R⁴Is H; ring A isn is 0 or 1; r³is-CO₂H or- (CH)₂)_p-CO₂R^c(ii) a p is 1, 2, 3 or 4; r^cIs H or C_1-6An alkyl group; m is 0 or 1; and R is⁵Is halogen, -CO₂R^f、-SO₃H、-SO₂NR^aR^bOr substituted by-SO₃H or-SO₂NR^aR^bC of (A)_1-6An alkyl group. In some embodiments, R^aAnd R^bAt least one or two of them being H or C_1-6An alkyl group. In some other embodiments, R^fIs H or C_1-4An alkyl group. In some other embodiments, when m is 0, then n is 1; or when n is 0, m is 1. In one embodiment, m and n are both 1. In some other embodiments, when m is 1,substitution at the following positions: in one embodiment, R⁵is-CO₂H. In another embodiment, R⁵Is halogen, e.g. chlorine, or-SO₃H. In another embodiment, R⁵is-SO₂NR^aR^bWherein R is^aAnd R^bAt least one or two of them being H or C_1-6An alkyl group.

Specific examples of compounds of formula (I) include those wherein X is O, S or NH; each R, R¹、R²And R⁴Is H; ring A isn is 0 or 1; r³is-CO₂H or- (CH)₂)_p-CO₂R^c(ii) a p is 1, 2, 3 or 4; r^cIs H or C_1-6An alkyl group; m is 0 or 1; and R is⁵Is halogen, -CO₂R^f、-SO₃H、-SO₂NR^aR^bOr substituted by-SO₃H or-SO₂NR^aR^bC of (A)_1-6An alkyl group. In some embodiments, R^aAnd R^bAt least one or two of them being H or C_1-6An alkyl group. In some other embodiments, R^fIs H or C_1-4An alkyl group. In some other embodiments, when m is 0, then n is 1; or when n is 0, m is 1. In one embodiment, m and n are both 1. In some other embodiments, when m is 1,substitution at the following positions: in one embodiment, R⁵is-CO₂H. In another embodiment, R⁵Is halogen, e.g. chlorine, or-SO₃H. In another embodiment, R⁵is-SO₂NR^aR^bWherein R is^aAnd R^bAt least one or two of them being H or C_1-6An alkyl group.

Specific examples of exocyclic amine-substituted coumarin dyes include:

and salts and meso forms thereof.

Particularly useful compounds are nucleotides or oligonucleotides labeled with the dyes described herein. The labeled nucleotides or oligonucleotides may be attached to the dye compounds disclosed herein through a carboxyl or alkyl-carboxyl group to form an amide or alkyl-amide. For example, the dye compounds disclosed herein are prepared by reacting R of formula (I)³Or R⁵Attached to a nucleotide or oligonucleotide. In some embodiments, R of formula (I)³is-CO₂H or- (CH)₂)_p-CO₂H and the attachment uses-CO₂The H group forms an amide. In some embodiments, R of formula (I)⁵is-CO₂H and the attachment uses-CO₂The H group forms an amide. The labeled nucleotide or oligonucleotide may have the label attached to the C5 position of the pyrimidine base or the C7 position of the 7-deaza purine base through a linker moiety.

The labeled nucleotide or oligonucleotide 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 of the ribose or deoxyribose. In a particular embodiment, the blocking group is at the 3' OH position of the ribose or deoxyribose sugar of the nucleotide.

Provided herein are kits comprising two or more nucleotides, wherein at least one nucleotide is a nucleotide labeled with a compound of the disclosure. The kit may comprise two or more labeled nucleotides. These nucleotides may be labeled with two or more fluorescent labels. Two or more of the labels may be excited using a single excitation source, which may be a laser. For example, the excitation bands of two or more labels may be at least partially overlapping, such that excitation in the overlapping region of the spectrum causes both labels to emit fluorescence. In particular embodiments, the emission from two or more labels will occur in different regions of the spectrum, such that the presence of at least one of the labels can be determined by optically distinguishing the emissions.

The kit may comprise four labeled nucleotides, wherein a first nucleotide of the four nucleotides is labeled with a compound as disclosed herein. In such a kit, each of the four nucleotides may be labeled with a compound that is the same as or different from the labels on the other three nucleotides. Thus, one or more of the compounds may have a different absorption maximum and/or emission maximum, such that the compound is distinguishable from the other compounds. For example, each compound may have a different absorption maximum and/or emission maximum, such that each of the compounds is distinguishable from the other three compounds. It will be appreciated that the portions of the absorption spectrum and/or emission spectrum other than the maxima may be different and these differences may be exploited to distinguish the compounds. The kit may be such that two or more of the compounds have different absorption maxima. The compounds of the invention generally absorb light in the region below 500 nm.

The compounds, nucleotides, or kits described herein can be used to detect, measure, or identify a biological system (including, e.g., a process or component thereof). Exemplary techniques that can employ these compounds, nucleotides, or kits include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cellular assays (e.g., cell binding or cell function analysis), or protein assays (e.g., protein binding assays or protein activity assays). The use can be on an automated instrument (such as an automated sequencing instrument) for performing a particular technique. The sequencing instrument may include two lasers operating at different wavelengths.

Disclosed herein are methods of synthesizing compounds of the present disclosure. Dyes according to the present disclosure can be synthesized from a variety of different suitable starting materials. For example, compounds of formula (I) may be prepared by reacting a compound of formula (II) with an optionally substituted cyclic amine of formula (III):

wherein each variable, X, R, R¹、R²、R³、R⁴、R⁵Ring A, m and n are as defined herein. The reaction may be carried out in an organic solvent at ambient or elevated temperature.

Definition of

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

It should be 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 the 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 commonly understood by one of ordinary skill in the art. The use of the term "including" as well as other forms such as "includes," "including," and "including" is non-limiting. The use of the term "having" as well as other forms such as "having", "having" and "having" is non-limiting. As used in this specification, the terms "comprises" and "comprising," whether in transitional phrases or in the main body of the claims, should be interpreted to have an open-ended meaning. That is, the above terms should be interpreted synonymously with the wording "having at least" or "including at least". For example, when used in the context of a process flow, the term "comprising" means that the process flow includes at least the recited steps, but may include other steps. The term "comprising" when used in the context of a compound, composition or device means that the compound, composition or device includes at least the recited features or components, but may also include other features or components.

As used herein, the term "covalently attached" or "covalently bound" refers to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently attached polymeric coating refers to a polymeric coating that forms a chemical bond with a functionalized surface of a substrate as compared to attachment to the surface via other means, such as adhesion or electrostatic interaction. It will be appreciated that the polymers covalently attached to the surface may also be bound via means other than covalent attachment.

As used herein, the term "halogen" or "halo" means any of the radio-stable atoms (halogens) of column 7 of the periodic table of elements, such as fluorine, chlorine, bromine or iodine, with fluorine and chlorine being preferred.

As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., does not contain double or triple bonds). An alkyl group can have from 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as "1 to 20" refers to each integer within the given range; e.g., "1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, and the like, up to and including 20 carbon atoms, although the present definition also covers the term "alkyl" appearing where no numerical range is specified). The alkyl group may also be a medium size alkyl group having 1 to 9 carbon atoms. The alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group may be designated as "C_1-4Alkyl "or similar names. By way of example only, "C_1-6Alkyl "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, isopropyl, n-butyl, isobutyl, sec-butylAnd a tertiary butyl group. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like.

As used herein, "alkoxy" refers to the formula-OR, wherein R is alkyl as defined above, such as "C_1-9Alkoxy "includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and the like.

As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. An alkenyl group may have 2 to 20 carbon atoms, but the present definition also covers the term "alkenyl" when no numerical range is specified. The alkenyl group may also be a medium size alkenyl group having 2 to 9 carbon atoms. The alkenyl group may also be a lower alkenyl group having 2 to 6 carbon atoms. An alkenyl group may be designated as "C_2-6Alkenyl "or similar names. By way of example only, "C_2-6Alkenyl "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: vinyl, 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, but-1, 3-dienyl, but-1, 2-dienyl and but-1, 2-dien-4-yl. Typical alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. Alkynyl groups may have 2 to 20 carbon atoms, but the present definition also covers the term "alkynyl" as it appears without a numerical range being specified. The alkynyl group can also be a medium-sized alkynyl having 2 to 9 carbon atoms. The alkynyl group may also be a lower alkynyl having 2 to 6 carbon atoms. Alkynyl groups may be designated "C_2-6Alkynyl "or similar names. By way of example only, "C_2-6Alkynyl "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, hexynyl, and the like.

As used herein, "heteroalkyl" refers to a straight or branched hydrocarbon chain containing one or more heteroatoms (i.e., elements other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur) in the chain backbone. A heteroalkyl group may have from 1 to 20 carbon atoms, but the present definition also covers the term "heteroalkyl" as it appears without a numerical range being specified. The heteroalkyl group may also be a medium size heteroalkyl having from 1 to 9 carbon atoms. The heteroalkyl group may also be a lower heteroalkyl group having from 1 to 6 carbon atoms. The heteroalkyl group may be designated as "C_1-6Heteroalkyl "or similar names. The heteroalkyl group may contain one or more heteroatoms. By way of example only, "C_4-6Heteroalkyl "indicates that there are four to six carbon atoms in the heteroalkyl chain and one or more heteroatoms additionally present in the backbone of the chain.

The term "aromatic" refers to rings or ring systems having conjugated pi-electron systems and includes both carbocyclic aromatic groups (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term includes monocyclic groups or fused-ring polycyclic (i.e., rings that share adjacent pairs of atoms) groups, provided that the entire ring system is aromatic.

As used herein, "aryl" refers to an aromatic ring or aromatic ring system (i.e., two or more fused rings sharing two adjacent carbon atoms) that contains only carbon in the ring backbone. When the aryl group is a ring system, each ring in the system is aromatic. An aryl group may have from 6 to 18 carbon atoms, but the present definition also covers the term "aryl" when no numerical range is specified. In some embodiments, the aryl group has 6 to 10 carbon atoms. The aryl group may be designated as "C_6-10Aryl group "," C₆Aryl or C₁₀Aryl "or similar names. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl (azulenyl), and anthracenyl.

"aralkyl" or "arylalkyl" is an aryl group attached as a substituent via an alkylene group, such as "C_7-14Aralkyl "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., C)_1-6An alkylene group).

As used herein, "heteroaryl" refers to an aromatic ring or aromatic ring system (i.e., two or more fused rings that share two adjacent atoms) that contains one or more heteroatoms (i.e., elements other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur) in the ring backbone. When the heteroaryl group is a ring system, each ring in the system is aromatic. Heteroaryl groups may have from 5 to 18 ring members (i.e., the number of atoms making up the ring backbone, including carbon and heteroatoms), but this definition also covers the term "heteroaryl" as it occurs where no numerical range is specified. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. Heteroaryl groups may be designated as "5-to 7-membered heteroaryl", "5-to 10-membered heteroaryl", or similar names. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

"heteroarylalkyl" or "heteroarylalkyl" is a heteroaryl group attached as a substituent via an alkylene group. Examples include, but are not limited to, 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl, pyrrolidinylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, it is possible to use,the alkylene group being a lower alkylene group (i.e. C)_1-6An alkylene group).

As used herein, "carbocyclyl" means a non-aromatic ring or non-aromatic ring system that contains only carbon atoms in the backbone of the ring system. When a carbocyclyl group is a ring system, two or more rings may be joined together in a fused, bridged or spiro-linked manner. The carbocyclyl group may have any degree of saturation provided that at least one ring in the ring system is not aromatic. Thus, carbocyclyl includes cycloalkyl, cycloalkenyl, and cycloalkynyl. Carbocyclyl groups may have 3 to 20 carbon atoms, but the present definition also covers the term "carbocyclyl" when no numerical range is specified. The carbocyclyl group may also be a medium size carbocyclyl having 3 to 10 carbon atoms. The carbocyclyl group may also be a carbocyclyl having 3 to 6 carbon atoms. The carbocyclyl group may be designated as "C_3-6Carbocyclyl "or similar names. Examples of carbocyclic rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2, 3-dihydro-indene, bicyclo [2.2.2]Octyl, adamantyl and spiro [4.4 ]]Nonyl radical.

As used herein, "cycloalkyl" means a fully saturated carbocyclic ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, "heterocyclyl" means a non-aromatic ring or non-aromatic ring system containing at least one heteroatom in the ring backbone. The heterocyclic groups may be joined together in a fused, bridged or spiro-linked manner. The heterocyclyl group may have any degree of saturation provided that at least one ring in the ring system is not aromatic. Heteroatoms may be present in non-aromatic or aromatic rings in the ring system. A heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon and heteroatoms), but this definition also covers the term "heterocyclyl" as it occurs where no numerical range is specified. The heterocyclyl group may also be a medium-sized heterocyclyl having 3 to 10 ring members. The heterocyclyl radical may also be a hetero ring having 3 to 6 ring membersA cyclic group. A heterocyclyl group may be designated as a "3-to 6-membered heterocyclyl" or similar name. In preferred six membered monocyclic heterocyclic groups, the heteroatom is selected from one up to three of O, N or S, and in preferred five membered monocyclic heterocyclic groups, the heteroatom is selected from one or two heteroatoms selected from O, N or S. Examples of heterocyclyl rings include, but are not limited to, azaAn azolyl group (azepinyl), an acridinyl group, a carbazolyl group, a cinnolinyl group, a dioxolanyl group, an imidazolinyl group, an imidazolidinyl group, a morpholinyl group, an oxiranyl group, an oxepanyl group (oxepanyl group), a thiepanyl group (thiepanyl group), a piperidyl group, a piperazinyl group, a dioxopiperazinyl group, a pyrrolidinyl group, a pyrrolidonyl group (pyrrolidonyl group), a pyrrolindionyl group (pyrrolidonyl group), a 4-piperidonyl group, a pyrazolinyl group, a pyrazolidinyl group, a1, 3-dioxinyl group, a1, 3-dioxanyl group, a1, 4-dioxinyl group, a1, 4-dioxanyl group, a1, 3-oxathianyl group, a1, 4-oxathiadienyl group, a 2H-1, 2-oxazinyl group, a trioxanyl group, a hexahydro-1, 3, 5-triazinyl group, a1, 3-dioxolyl group, a morpholinyl group, a pyrrolidyl group, a thiazin, 1, 3-dioxolane, 1, 3-dithiolane, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidonyl, thiazolinyl, thiazolidinyl, 1, 3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydro-1, 4-thiazinyl, thiomorpholinyl (thiomorpholinyl), dihydrobenzofuranyl, benzimidazolinyl, and tetrahydroquinoline.

As defined herein, an "O-carboxy" group refers to an "-OC (═ O) R" group, wherein R is selected from hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Carbocyclyl, C_6-10Aryl, 5-to 10-membered heteroaryl, and 3-to 10-membered heterocyclyl.

As defined herein, a "C-carboxy" group refers to a "-C (═ O) OR" group, wherein R is selected from hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Carbocyclyl, C_6-10Aryl, 5-to 10-membered heteroaryl, and 3-to 10-membered heterocyclyl. Non-limiting examples include carboxyl (i.e., -C (═ O) OH).

As defined herein, a "sulfonyl" group refers to "-SO₂R' group, wherein R is selected from hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Carbocyclyl, C_6-10Aryl, 5-to 10-membered heteroaryl, and 3-to 10-membered heterocyclyl.

A "sulfinyl" group refers to an "-S (═ O) OH" group.

As defined herein, an "S-sulfonylamino" group refers to "-SO₂NR_AR_B"group, wherein R_AAnd R_BEach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Carbocyclyl, C_6-10Aryl, 5-to 10-membered heteroaryl, and 3-to 10-membered heterocyclyl.

As defined herein, an "N-sulfonamido" group refers to "-N (R)_A)SO₂R_B"group, wherein R_AAnd R_BEach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Carbocyclyl, C_6-10Aryl, 5-to 10-membered heteroaryl, and 3-to 10-membered heterocyclyl.

As defined herein, a "C-amido" group refers to "-C (═ O) NR_AR_B"group, wherein R_AAnd R_BEach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Carbocyclyl, C_6-10Aryl, 5-to 10-membered heteroaryl, and 3-to 10-membered heterocyclyl.

As defined herein, an "N-amido" group refers to a "-N (R)_A)C(＝O)R_B"group, wherein R_AAnd R_BEach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Carbocyclyl, C_6-10Aryl, 5-to 10-membered heteroaryl andand a 3-to 10-membered heterocyclic group.

As defined herein, an "amino" group refers to a "-NR" group_AR_B"group, wherein R_AAnd R_BEach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Carbocyclyl, C_6-10Aryl, 5-to 10-membered heteroaryl, and 3-to 10-membered heterocyclyl. Non-limiting examples include free amino (i.e., -NH)₂)。

As defined herein, an "aminoalkyl" group refers to an amino group attached via an alkylene group.

As defined herein, an "alkoxyalkyl" group refers to an alkoxy group attached via an alkylene group, such as "C_2-8Alkoxyalkyl groups "and the like.

As used herein, a substituted group is derived from an unsubstituted parent group (parent group) in which one or more hydrogen atoms have been exchanged for another atom or group. Unless otherwise indicated, when a group is considered "substituted," it means that the group is substituted with one or more substituents independently selected from the group consisting of: c₁-C₆Alkyl radical, C₁-C₆Alkenyl radical, C₁-C₆Alkynyl, C₁-C₆Heteroalkyl group, C₃-C₇Carbocyclyl (optionally substituted by halogen, C)₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl, and C₁-C₆Haloalkoxy substituted), C₃-C₇-carbocyclyl-C₁-C₆Alkyl (optionally substituted by halogen, C)₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl, and C₁-C₆Haloalkoxy substituted), 3-to 10-membered heterocyclyl (optionally substituted with halogen, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl, and C₁-C₆Haloalkoxy substituted), 3-to 10-membered heterocyclyl-C₁-C₆-alkyl (or)Optionally substituted by halogen, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl, and C₁-C₆Haloalkoxy substituted), aryl (optionally substituted by halogen, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl, and C₁-C₆Haloalkoxy substituted), aryl (C)₁-C₆) Alkyl (optionally substituted by halogen, C)₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl, and C₁-C₆Haloalkoxy substituted), 5-to 10-membered heteroaryl (optionally substituted with halogen, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl, and C₁-C₆Haloalkoxy substituted), 5-to 10-membered heteroaryl (C)₁-C₆) Alkyl (optionally substituted by halogen, C)₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl, and C₁-C₆Haloalkoxy substituted), halogen, -CN, hydroxy, C₁-C₆Alkoxy radical, C₁-C₆Alkoxy (C)₁-C₆) Alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halogen (C)₁-C₆) Alkyl (e.g., -CF)₃) Halogen (C)₁-C₆) Alkoxy (e.g., -OCF)₃)、C₁-C₆Alkylthio, arylthio, amino (C)₁-C₆) Alkyl, nitro, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, S-sulphonamido, N-sulphonamido, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, -SO₃H. Sulfino, -OSO₂C_1-4Alkyl, and oxo (═ O). In any case where a group is described as "optionally substituted", the group may be substituted with the substituents described above.

In some casesIn embodiments, a substituted alkyl group, alkenyl group, or alkynyl group selected from the group consisting of halogen, -CN, SO₃ ^-、-SO₃H、-SR^A、-OR^A、-NR^BR^COxo, -CONR^BR^C、-SO₂NR^BR^C-COOH and-COOR^BIs substituted with one or more substituents of the group consisting of R^A、R^BAnd R^CEach independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

The compounds described herein may be represented as several meso forms. In the case of drawing a single structure, the intention is any of the relevant meso forms. The coumarin compounds described herein are represented by a single structure but may equally be represented as any of the relevant meso forms. For formula (I), exemplary meso structures are shown below:

in each case where a single meso form of a compound described herein is shown, alternative meso forms are likewise contemplated.

As understood by one of ordinary skill in the art, the compounds described herein can be in ionized form, e.g., -CO₂ ^-or-SO₃ ^-Are present. If the compound contains a positively or negatively charged substituent group, e.g. SO₃ ^-When used, the compound may also contain a negatively or positively charged counterion, such that the compound is neutral as a whole. In other aspects, the compounds may be present in the form of a salt, wherein the counter ion is provided by a conjugate acid or conjugate base.

It should be understood that certain radical naming conventions (naming conventions) may include single or double radicals, depending on the context. For example, two attachments to a substituent are requiredAt the point of the remainder of the molecule, it is understood that the substituent is a diradical. For example, substituents identified as alkyl requiring two attachment points include diradicals, such as-CH₂-、-CH₂CH₂-、-CH₂CH(CH₃)CH₂-and the like. Other group naming conventions clearly indicate that the group is a diradical, such as "alkylene" or "alkenylene".

When two "adjacent" R groups are said to form a ring "together with the atoms to which they are attached" it is meant that the collective units of these atoms, intervening bonds, and the two R groups are the recited ring. For example, when the following substructure is present:

and R is¹And R²Is defined as selected from the group consisting of hydrogen and alkyl, or R¹And R²Together with the atom to which they are attached form an aryl or carbocyclyl group, means R¹And R²May be selected from hydrogen or alkyl, or alternatively, the substructure has the following structure:

wherein A is an aryl ring or a carbocyclic group containing the double bond.

Labelled nucleotides

According to an aspect of the present disclosure, there is provided a dye compound suitable for attachment to a substrate portion, the dye compound in particular comprising a linker group capable of attachment to a substrate portion. The base portion can be virtually any molecule or substance to which the dyes of the present disclosure can be conjugated, and by way of non-limiting example, the base portion can include nucleosides, nucleotides, polynucleotides, carbohydrates, ligands, particles, solid surfaces, organic and inorganic polymers, chromosomes, nuclei, living cells, and combinations or assemblies thereof (assembly). The dye may be conjugated through an optional linker, through a variety of means including hydrophobic attraction, ionic attraction, and covalent attachment. In some aspects, the dye is conjugated to the substrate by covalent attachment. More particularly, the covalent attachment is via a linker group. In some cases, such labeled nucleotides are also referred to as "modified nucleotides".

The present disclosure also provides conjugates of nucleosides and nucleotides (modified nucleotides) labeled with one or more of the dyes set forth herein. Labeled nucleosides and nucleotides can be used to label polynucleotides such as, by way of non-limiting example, formed via enzymatic synthesis in PCR amplification, isothermal amplification, solid phase amplification, polynucleotide sequencing (e.g., solid phase sequencing), nick translation reactions, and the like.

Attachment to biomolecules may be via R, R of a compound of formula (I)¹、R²、R³、R⁴、R⁵Or the X position. In some aspects, the linkage is via R of formula (I)³Or R⁵A group. In some embodiments, the substituent group is carboxy or substituted alkyl, e.g., with-CO₂H or an activated form of a carboxyl group, such as an amide or ester substituted alkyl group, which may be used to attach to an amino group or a hydroxyl group of a biomolecule. The term "activated ester" as used herein refers to a derivative of a carboxyl group that is capable of reacting under mild conditions, for example, with a compound comprising an amino group. Non-limiting examples of activated esters include, but are not limited to, p-nitrophenyl, pentafluorophenyl, and succinimidyl esters.

In some embodiments, the dye compound may be covalently attached to the oligonucleotide or nucleotide via a nucleotide base. For example, a labeled nucleotide or oligonucleotide may have a label attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety. The labeled nucleotide or oligonucleotide may also have a 3' -OH blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide.

A particularly advantageous application of the novel fluorescent dyes as described herein is for labeling biomolecules, such as nucleotides or oligonucleotides. Some embodiments of the present application relate to nucleotides or oligonucleotides labeled with novel fluorescent compounds as described herein.

Linking group

Dye compounds as disclosed herein may include a reactive linker group on one of the substitution positions for covalent attachment of the compound to a substrate or another molecule. A reactive linker group is a moiety capable of forming a bond (e.g., a covalent bond or a non-covalent bond), particularly a covalent bond. In particular embodiments, the linker may be a cleavable linker. The use of the term "cleavable linker" is not meant to imply that the entire linker needs to be removed. The cleavage site may be located on the linker to ensure that a portion of the linker remains attached to the dye and/or substrate moiety after cleavage. By way of non-limiting example, a cleavable linker can be an electrophilically cleavable linker, a nucleophilically cleavable linker, a photocleavable linker, a linker that is cleavable under reducing conditions (e.g., a linker comprising a disulfide or azide), a linker that is cleavable under oxidizing conditions, cleavable via the use of a safe capture linker, and cleavable by a removal mechanism. The use of a cleavable linker to attach the dye compound to the substrate portion ensures that the label can be removed after detection if necessary, avoiding any signal interference in downstream steps.

Useful linker groups can be found in PCT publication No. WO2004/018493 (incorporated herein by reference), examples of which include linkers that can be cleaved using a water-soluble phosphine or a water-soluble transition metal catalyst formed from a transition metal and an at least partially water-soluble ligand. In aqueous solution, the latter form transition metal complexes which are at least partially soluble in water. Such cleavable linkers can be used to link the base of a nucleotide to a label, such as a dye set forth herein.

Specific linkers include those disclosed in PCT publication No. WO2004/018493 (incorporated herein by reference), such as those comprising a moiety of the formula:

(wherein X is selected from the group consisting of O, S, NH and NQ, wherein Q is a C1-10 substituted or unsubstituted alkyl group, Y is selected from the group consisting of O, S, NH and N (allyl), and T is hydrogen or C₁-C₁₀Substituted or unsubstituted alkyl group, and indicates the position at which the moiety is attached to the remainder of the nucleotide or nucleoside). In some aspects, a linker connects the base of the nucleotide to a label, such as, for example, a dye compound described herein.

Additional examples of linkers include those disclosed in U.S. publication nos. 2016/0040225 and 2019/0017111 (incorporated herein by reference), such as those linkers that comprise a moiety of the formula:

the linker moieties set forth herein may include all or part of the linker structure between the nucleotide/nucleoside and the tag.

In particular embodiments, the length of the linker between the fluorescent dye (fluorophore) and the guanine base can be varied, for example by introducing a polyethylene glycol spacer group, to increase fluorescence intensity compared to the same fluorophore attached to the guanine base by other linkages known in the art. Exemplary linkers and their properties are set forth in PCT publication No. WO2007020457 (incorporated herein by reference). The design of the linker and in particular its increased length may allow for an increase in the brightness of the fluorophore attached to the guanine base of a guanosine nucleotide when incorporated into a polynucleotide such as DNA. Thus, when the dye is used in any place where it is desired to detect a fluorescent dye label attached to a nucleotide comprising guanineWhen used in any assay, if the linker comprises the formula- ((CH)₂)₂O) n-where n is an integer between 2 and 50, as described in WO 2007/020457, are advantageous.

Nucleosides and nucleotides can 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 the sugar is deoxyribose, i.e. a sugar lacking the hydroxyl groups present in ribose. The nitrogenous base is a derivative of a purine or pyrimidine. Purines are adenine (a) and guanine (G), and pyrimidines are cytosine (C) and thymine (T) or uracil (U) in the context of RNA. The C-1 atom of the deoxyribose is bonded to the N-1 of the pyrimidine or the N-9 of the purine. Nucleotides are also phosphate esters of nucleosides, in which esterification occurs on a hydroxyl group attached to C-3 or C-5 of a sugar. Nucleotides are typically monophosphates, bisphosphates or triphosphates.

A "nucleoside" is similar in structure to a nucleotide but lacks a phosphate moiety. An example of a nucleoside analogue would be one in which the label is attached to the base and no phosphate group is attached to the sugar molecule

Although the base is commonly referred to as a purine or pyrimidine, the skilled artisan will appreciate that derivatives and analogs that do not alter the ability of a nucleotide or nucleoside to undergo Watson-Crick base pairing (Watson-Crick base pairing) are useful. By "derivative" or "analog" is meant a compound or molecule whose core structure is the same as or very similar to that of the parent compound, but which has chemical or physical modifications (such as, for example, different or additional side groups which allow the derivatized 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 Watson-Crick pairing. "derivatives" and "analogs" also include, for example, synthetic nucleotide derivatives or nucleoside derivatives having a modified base moiety and/or a modified sugar moiety. Such derivatives and analogues are described, for example, in Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al, Chemical Reviews 90: 543 and 584, 1990. Nucleotide analogs may also contain modified phosphodiester linkages including phosphorothioate linkages, phosphorodithioate linkages, alkylphosphonate linkages, phosphoranilate linkages, phosphoramidate linkages, and the like.

The dye may be attached to any position on the nucleotide base, for example, by a linker. In particular embodiments, Watson-Crick base pairing can still be performed on the resulting analogs. Specific nucleobase marker sites include the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base. As described above, a linker group may be used to covalently attach the dye to the nucleoside or nucleotide.

In particular embodiments, the labeled nucleoside or nucleotide may be enzyme incorporable and enzyme extendable. Thus, the linker moiety may be of sufficient length to link the nucleotide to the compound such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by the nucleic acid replicase. Thus, the linker may also comprise a spacer unit. For example, a spacer (spacer) keeps the nucleotide base away from the cleavage site or label.

The nucleoside or nucleotide labeled using the dyes described herein may have the formula:

wherein the dye is a dye compound; b is a nucleobase such as, for example, uracil, thymine, cytosine, adenine, guanine, and the like; l is an optional linker group which may or may not be present; r may be H, monophosphate, diphosphate, triphosphate, phosphorothioate, phosphate analog, O-which is attached to a reactive phosphorus-containing group, or O-which is protected by a blocking group; r ' can be H, OH, phosphoramidite, or a 3 ' -OH blocking group, and R ' is H or OH. Where R "is a phosphoramidite, R' is an acid cleavable hydroxyl protecting group that allows for subsequent monomer coupling under automated synthesis conditions.

In a particular embodiment, the blocking group is separate and independent of the dye compound, i.e. not attached to the dye compound. Alternatively, the dye may comprise all or a portion of the 3' -OH blocking group. Thus, R 'may be a 3' -OH blocking group that may or may not include a dye compound.

In yet another alternative embodiment, no blocking group is present on the 3' carbon of the pentose sugar and, for example, the dye (or dye and linker construct) attached to the base may have a size or structure sufficient to act as a blocker of the incorporation of additional nucleotides. Thus, blocking may be due to steric hindrance or may be due to a combination of size, charge and structure, whether or not the dye is attached to the 3' position of the saccharide.

In yet another alternative embodiment, the blocking group is present on the 2 'carbon or the 4' carbon of the pentose and may have a size or structure sufficient to act as a blocker of the incorporation of additional nucleotides.

The use of blocking groups allows for control of polymerization, such as by terminating extension, when modified nucleotides are incorporated. If the blocking effect is reversible, for example, by way of non-limiting example, by changing the chemical conditions or by removing the chemical blocker, the extension may be stopped at some point and then allowed to continue.

In yet another particular embodiment, the 3' -OH blocking group will comprise the moieties disclosed in WO2004/018497 and WO2014/139596, both of which are incorporated herein by reference. For example, the blocking group may be azidomethyl (-CH)₂N₃) Or substituted azidomethyl (e.g., -CH (CHF)₂)N₃Or CH (CH)₂F)N₃) Or allyl.

In particular embodiments, both the linker (between the dye and the nucleotide) and the blocking group are present and are separate moieties. In particular embodiments, both the linker and the blocking group are cleavable under substantially similar conditions. Thus, the deprotection and deblocking process may be more efficient, as only a single treatment would be required to remove both the dye compound and the blocking group. However, in some embodiments, the linker and blocking group need not be cleavable under similar conditions, but are separately cleavable under different conditions.

The present disclosure also encompasses polynucleotides incorporating dye compounds. Such polynucleotides may be DNA or RNA comprising deoxyribonucleotides or ribonucleotides, respectively, linked by phosphodiester linkages. A polynucleotide may comprise a combination of a naturally occurring nucleotide, a non-naturally occurring (or modified) nucleotide other than a labeled nucleotide described herein, or any combination thereof, and at least one modified nucleotide set forth herein (e.g., labeled with a dye compound). Polynucleotides according to the present disclosure may also include non-natural backbone linkages and/or non-nucleotide chemical modifications. Chimeric structures comprising a mixture of ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide are also contemplated.

By way of non-limiting example, labeled nucleotides described herein comprise:

wherein L represents a linker and R represents a sugar residue as described above.

In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below:

reagent kit

The present disclosure also provides kits comprising modified nucleosides and/or nucleotides labeled with dyes. Such kits will typically comprise at least one modified nucleotide or nucleoside labeled with a dye set forth herein and at least one additional component. The additional component may be one or more of the components identified in the methods set forth above or in the examples section below. Some non-limiting examples of components that may be combined into the kits of the present disclosure are set forth below.

In particular embodiments, the kit may comprise at least one modified nucleotide or nucleoside and a modified or unmodified nucleotide or nucleoside labeled with a dye set forth herein. For example, a modified nucleotide labeled with a dye according to the present disclosure may be supplied in combination with: unlabeled or natural nucleotides and/or fluorescently labeled nucleotides or any combination thereof. Thus, the kit may comprise modified nucleotides labeled with dyes according to the present disclosure and modified nucleotides labeled with other, e.g., prior art dye compounds. Combinations of nucleotides can be provided as separate individual components (e.g., one nucleotide type per container or tube) or as a mixture of nucleotides (e.g., two or more nucleotides mixed in the same container or tube).

In case the kit comprises more than one modified nucleotide, in particular two, or three, or more in particular four modified nucleotides, which are labeled with dye compounds, different nucleotides may be labeled with different dye compounds, or one nucleotide may be black (without dye compounds). In the case where different nucleotides are labeled with different dye compounds, the kit is characterized in that the dye compounds are spectrally distinguishable fluorescent dyes. As used herein, the term "spectrally distinguishable fluorochromes" refers to fluorochromes that, when two or more such dyes are present in a sample, emit fluorescent energy at wavelengths that can be distinguished by a fluorescence detection device (e.g., a commercial capillary-based DNA sequencing platform). When two modified nucleotides labeled with a fluorescent dye compound are provided in a kit form, some embodiments feature that spectrally distinguishable fluorescent dyes can be excited at the same wavelength (such as, for example, by the same laser). When four modified nucleotides labeled with fluorescent dye compounds are provided in kit form, some embodiments feature 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. Specific excitation wavelengths are 488nm and 532 nm.

In one embodiment, the kit comprises a modified nucleotide labeled with a compound of the present disclosure and a second modified nucleotide labeled with a second dye, wherein the dyes have a difference in absorption maxima of at least 10nm, particularly 20nm to 50 nm. More particularly, the two dye compounds have a Stokes shift between 15nm and 40nm, where "Stokes shift" is the distance between the peak absorption wavelength and the peak emission wavelength.

In a further embodiment, the kit may further comprise two further modified nucleotides labeled with fluorescent dyes, wherein the dyes are excited by the same laser at 532 nm. The dye may have a difference in absorption maxima of at least 10nm, in particular 20nm to 50 nm. More particularly, the two dye compounds may have a Stokes shift between 20nm and 40 nm. Particular dyes that are spectrally distinguishable from the dyes of the present disclosure and meet the above criteria are polymethine analogs as described in U.S. patent No. 5,268,486 (e.g., Cy3) or WO 0226891(Alexa 532; Molecular Probes a20106) or asymmetric polymethines as disclosed in U.S. patent No. 6,924,372, each of which is incorporated herein by reference. Alternative dyes include rhodamine analogs, such as tetramethyl rhodamine and its analogs.

In alternative embodiments, the kits of the present disclosure may comprise nucleotides in which the same base is labeled with two different compounds. The first nucleotide may be labeled with a compound of the disclosure. The second nucleotide may be labelled with a spectrally different compound (e.g. a 'green' dye that absorbs at less than 600 nm). The third nucleotide may be labeled with a mixture of a compound of the present disclosure and a spectrally distinct compound, and the fourth nucleotide may be 'black' and contain no label. Thus, in brief, nucleotides 1-4 can be labeled 'blue', 'green', 'blue/green', and black. To further simplify the instrument, four nucleotides may be labeled with two dyes excited by a single laser, and thus the labels of nucleotides 1-4 may be 'blue 1', 'blue 2', 'blue 1/blue 2' and black.

The nucleotide may comprise two dyes of the present disclosure. The kit may comprise two or more nucleotides labeled with a dye of the present disclosure. The kit may comprise further nucleotides, wherein the nucleotides are labeled with a dye that absorbs in the region of 520nm to 560 nm. The kit may also comprise unlabeled nucleotides.

Although the kits are exemplified herein with respect to configurations having different nucleotides labeled with different dye compounds, it is understood that the kits can contain 2, 3, 4, or more different nucleotides having the same dye compound.

In particular embodiments, the kit can comprise a polymerase capable of catalyzing the incorporation of the modified nucleotide into the polynucleotide. Other components to be included in such kits may include buffers and the like. Modified nucleotides labeled with dyes according to the present disclosure and any other nucleotide components including mixtures of different nucleotides may be provided in a kit 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 the methods set forth herein may be included in a kit of the present disclosure.

Sequencing method

Modified nucleotides (or nucleosides) comprising dye compounds according to the present disclosure can be used in any analytical method, such as a method that includes detecting a fluorescent label attached to a nucleotide or nucleoside, whether by itself alone or incorporated into or associated with a larger molecular structure or conjugate. In this context, the term "incorporated into a polynucleotide" may mean that the 5 'phosphate is phosphodiester-bonded to the 3' hydroxyl group of a second (modified or unmodified) nucleotide which may itself form part of a longer polynucleotide chain. The 3 'terminus of a modified nucleotide set forth herein may or may not be phosphodiester-linked to the 5' phosphate of another (modified or unmodified) nucleotide. Thus, in one non-limiting embodiment, the present disclosure provides a method of detecting a modified nucleotide incorporated into a polynucleotide, the method comprising: (a) incorporating at least one modified nucleotide of the present disclosure into a polynucleotide, and (b) detecting the modified nucleotide incorporated into the polynucleotide by detecting a fluorescent signal from a dye compound attached to the modified nucleotide.

The method may include: a synthesis step (a) in which one or more modified nucleotides according to the present disclosure are incorporated into a polynucleotide; and a detection step (b) in which the one or more modified nucleotides incorporated into the polynucleotide are detected by detecting or quantitatively measuring their fluorescence.

Some embodiments of the present application relate to a sequencing method comprising: (a) incorporating at least one labeled nucleotide as described herein into a polynucleotide; and (b) detecting the labeled nucleotide incorporated into the polynucleotide by detecting a fluorescent signal from the new fluorescent dye attached to the modified nucleotide.

In one embodiment, at least one modified nucleotide is incorporated into the polynucleotide by the action of a polymerase during the synthesis step. However, other methods of linking modified nucleotides to polynucleotides may be used, such as, for example, chemical oligonucleotide synthesis or linking of labeled oligonucleotides to unlabeled oligonucleotides. Thus, when used in reference to nucleotides and polynucleotides, the term "incorporation" can encompass polynucleotide synthesis by chemical as well as enzymatic methods.

In particular embodiments, the synthesis step is performed and may optionally include incubating the template polynucleotide strand with a reaction mixture comprising the fluorescently labeled modified nucleotides of the present disclosure. The polymerase may also be provided under conditions that allow for the formation of a phosphodiester bond between a free 3 'hydroxyl group on the polynucleotide strand that anneals to the template polynucleotide strand and a 5' phosphate group on the modified nucleotide. Thus, the step of synthesizing may comprise polynucleotide strand formation as directed by complementary base pairing of nucleotides to the template strand.

In all embodiments of the method, the detection step may be performed at the same time as the polynucleotide strand into which the labelled nucleotide is incorporated is annealed to the template strand, or after a denaturation step in which the two strands are separated. Additional steps may be included between the synthesis step and the detection step, such as chemical or enzymatic reaction steps or purification steps. In particular, target strands incorporating labeled nucleotides can be isolated or purified and then further processed or used in subsequent assays. For example, a modified nucleotide-tagged target polynucleotide as described herein in a synthetic step may subsequently be used as a labeled probe or primer. In other embodiments, the products of the synthetic steps set forth herein may be subjected to additional reaction steps, and the products of these subsequent steps purified or isolated, if desired.

Suitable conditions for the synthetic steps will be well known to those familiar with standard molecular biology techniques. In one embodiment, the synthesis step may be similar to a standard primer extension reaction that uses nucleotide precursors including modified nucleotides as described herein to form an extended target strand complementary to the template strand in the presence of a suitable polymerase. In other embodiments, the synthesis step itself may form part of an amplification reaction that produces a labeled double-stranded amplification product comprising annealed complementary strands obtained by copying the target and template polynucleotide strands. Other exemplary synthetic steps include nick translation, strand displacement polymerization, randomly primed DNA labeling, and the like. Particularly useful polymerases for the synthetic step are polymerases capable of catalyzing the incorporation of modified nucleotides as set forth herein. A variety of naturally occurring or modified polymerases can be used. For example, thermostable polymerases may be used for synthesis reactions performed using thermocycling conditions, however thermostable polymerases may not be desirable for isothermal primer extension reactions. Suitable thermostable polymerases capable of incorporating modified nucleotides according to the present disclosure include those described in WO2005/024010 or WO06120433 (each of which is incorporated herein by reference). In synthesis reactions carried out at lower temperatures, such as 37 ℃, the polymerase does not necessarily have to be a thermostable polymerase, and thus the choice of polymerase will depend on many factors, such as reaction temperature, pH, strand displacement activity, etc.

In certain non-limiting embodiments, the present disclosure encompasses methods of nucleic acid sequencing, re-sequencing, whole genome sequencing, single nucleotide polymorphism scoring, any other application involving the detection of modified nucleotides or nucleosides labeled with a dye set forth herein when incorporated into a polynucleotide. Any of a variety of other applications that benefit from the use of polynucleotides labeled with modified nucleotides comprising fluorescent dyes may use modified nucleotides or nucleosides having the dyes set forth herein.

In a particular embodiment, the present disclosure provides the use of a modified nucleotide comprising a dye compound according to the present disclosure in a polynucleotide sequencing-by-synthesis reaction. Sequencing-by-synthesis typically involves the sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide strand in the 5 'to 3' direction using a polymerase or ligase so as to form an extended polynucleotide strand complementary to the template nucleic acid to be sequenced. The identity of the base present as one or more added nucleotides may be determined in the detection step or "imaging" step. The identity of the added base can be determined after each nucleotide incorporation step. The sequence of the template can then be deduced using conventional Watson-Crick base-pairing rules. The use of modified nucleotides labeled with the dyes set forth herein for determining 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 the present disclosure.

In embodiments of the present disclosure, the sequence of the template polynucleotide is determined by detecting the incorporation of one or more nucleotides into the nascent strand complementary to the template polynucleotide to be sequenced via detection of a fluorescent label attached to the incorporated nucleotide. Sequencing of the template polynucleotide may be primed with an appropriate primer (or prepared as a hairpin construct that will contain the primer as part of the hairpin), and the nascent strand is extended in a stepwise manner by the 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) can be labeled with a unique fluorophore and also contain a blocking group at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides may be unlabeled (black). The polymerase incorporates the nucleotide into the nascent strand complementary to the template polynucleotide, and the blocking group prevents further incorporation of the nucleotide. Any unincorporated nucleotides can be washed away and the fluorescent signal from each incorporated nucleotide can be optically "read" by suitable means, such as a charge coupled device using laser excitation and suitable emission filters. The 3' blocking group and the fluorescent dye compound can then be removed (deprotected) (either simultaneously or sequentially) to expose the nascent strand for additional nucleotide incorporation. Typically, the identity of the incorporated nucleotide will be determined after each incorporation step, but this is not strictly necessary. Similarly, U.S. Pat. No. 5,302,509, which is incorporated herein by reference, discloses a method of sequencing polynucleotides immobilized on a solid support.

As exemplified above, the method utilizes incorporation of fluorescently labeled, 3' blocked nucleotide A, G, C and T into a growing strand complementary to an immobilized polynucleotide in the presence of a DNA polymerase. The polymerase incorporates bases complementary to the target polynucleotide, but prevents further addition by a 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 for which sequencing is desired. The nucleic acid template used for the sequencing reaction will typically comprise a double stranded region with a free 3' hydroxyl group which 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 have the free 3' hydroxyl group pendant to the complementary strand. The overhanging region of the template to be sequenced may be single stranded, but may 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 priming the sequencing reaction. In such embodiments, sequencing may be performed by strand displacement. In certain embodiments, a primer bearing a free 3' hydroxyl group can 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 intramolecular duplex (duplex), such as, for example, a hairpin loop structure. Hairpin polynucleotides and methods by which they can be attached to a solid support are disclosed in PCT publications No. WO0157248 and No. WO2005/047301, each of which is incorporated herein by reference. Nucleotides may be added sequentially to the growing primer, resulting in the synthesis of a polynucleotide strand in the 5 'to 3' direction. The nature of the bases that have been added may be determined specifically, 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 the nucleotide to a free 3 'hydroxyl group of the nucleic acid strand via a phosphodiester bond formation 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 (hybrid molecule) comprising deoxynucleotides and ribonucleotides. Nucleic acid templates may comprise naturally occurring nucleotides and/or non-naturally occurring nucleotides as well as natural backbone linkages or non-natural backbone linkages, provided that these do not prevent replication of the template in a sequencing reaction.

In certain embodiments, the nucleic acid template to be sequenced may be attached to the solid support via any suitable attachment method known in the art (e.g., via covalent attachment). In certain embodiments, the template polynucleotide may be directly attached to a solid support (e.g., a silica-based support). However, in other embodiments of the present disclosure, the surface of the solid support may be modified in a manner so as to allow direct covalent attachment of the template polynucleotide, or so as to immobilize the template polynucleotide through a hydrogel or polyelectrolyte multilayer that may itself be non-covalently attached to the solid support.

Arrays in which polynucleotides have been attached directly to a silica-based support are for example those disclosed in WO00006770 (incorporated herein by reference), in which polynucleotides are immobilised on a glass support by reaction between pendant epoxy groups on the glass and internal amino groups on the polynucleotides. Furthermore, the polynucleotide may be attached to the solid support by reaction of a sulfur-based nucleophile with the solid support, for example, as described in W02005/047301 (incorporated herein by reference). Yet another further example of a solid-supported template polynucleotide is a hydrogel in which the template polynucleotide is attached to a silica-based or other solid support, for example, as described in W000/31148, W001/01143, W002/12566, W003/014392, U.S. patent nos. 6,465,178 and WO00/53812, each of which is incorporated herein by reference.

The specific surface on which the template polynucleotide can 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 can be used include those described in WO2005/065814 and U.S. publication No. 2014/0079923. In one embodiment, the hydrogel is PAZAM (poly (N- (5-azidoacetamidopentyl) acrylamide-co-acrylamide)).

DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. patent No. 6,172,218 (which is incorporated herein by reference). Attachment to beads or microparticles may be useful for sequencing applications. A library of beads can be prepared, wherein each bead comprises a different DNA sequence. Exemplary libraries and methods for producing them are described in Nature, 437, 376-380 (2005); science, 309, 5741, 1728-. Sequencing of such arrays of beads using the nucleotides set forth herein is within the scope of the present disclosure.

The template 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 methods of the present disclosure are applicable to all types of high density arrays, including single molecule arrays, clustered arrays, and bead arrays. Modified nucleotides labeled with the dye compounds of the present disclosure can be used to sequence templates on essentially any type of array, including but not limited to arrays formed by immobilizing nucleic acid molecules on a solid support.

However, modified nucleotides labeled with the dye compounds of the present disclosure are particularly beneficial in the context of sequencing clustered arrays. In a clustered array, different regions (often referred to as sites or features) on the array contain multiple polynucleotide template molecules. Typically, multiple polynucleotide molecules cannot be resolved by optical means individually but are instead detected as a population. Depending on how the array is formed, each site on the array may comprise multiple copies of a single polynucleotide molecule (e.g., the site is homogeneous for a particular single-stranded nucleic acid species or double-stranded nucleic acid species) or even a small number of multiple copies of a different polynucleotide molecule (e.g., multiple copies of two different nucleic acid species). Clustered arrays of nucleic acid molecules can be generated using techniques generally known in the art. For example, WO98/44151 and WO00/18957 (each of which is incorporated herein) describe a method of nucleic acid amplification in which both the template and the amplification products are held immobilised on a solid support so as to form an array comprising clusters or "clones" of immobilised nucleic acid molecules. Nucleic acid molecules present on clustered arrays prepared according to these methods are suitable templates for sequencing modified nucleotides labeled with the dye compounds of the present disclosure.

Modified nucleotides labeled with the dye compounds of the present disclosure are also useful in sequencing templates on single molecule arrays. The term "single molecule array" or "SMA" as used herein refers to a population of polynucleotide molecules distributed (or arranged) on a solid support, wherein the spacing of any individual polynucleotide from any individual polynucleotide in the population is such that it is possible for an individual to distinguish the individual polynucleotide molecules. Thus, in some embodiments, target nucleic acid molecules immobilized to the surface of a solid support may be capable of being resolved by optical means. This means that one or more different signals, each representing a polynucleotide, will appear within a distinguishable region of the particular imaging device used.

Single molecule detection can be achieved where the spacing between adjacent polynucleotide molecules on the array is at least 100nm, more particularly at least 250nm, still more particularly at least 300nm, even more particularly at least 350 nm. Thus, each molecule is individually resolvable and detectable as a single-molecule fluorescent dot, and fluorescence from the single-molecule fluorescent dot also exhibits single-step photobleaching.

The terms "individually resolved" and "individually resolved" are used herein to illustrate that, when visualized, it is possible to distinguish one molecule on an array from its neighboring molecules. The separation between individual molecules on the array will be determined in part by the particular technique used to resolve the individual molecules. The general characteristics 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 modified nucleotides of the present disclosure is in sequencing-by-synthesis reactions, the utility of the modified nucleotides is not limited to such methods. In fact, the nucleotides can be advantageously used in any sequencing method that requires the detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide.

In particular, modified nucleotides labeled with the dye compounds of the present disclosure can be used in automated fluorescent sequencing protocols, particularly fluorescent dye terminator cycle sequencing based on Sanger and co-workers' chain termination sequencing methods. Such methods typically use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides into primer extension sequencing reactions. The so-called Sanger sequencing method and related protocols (Sanger type) utilize randomized chain termination of labeled dideoxynucleotides.

Thus, the present disclosure also encompasses modified nucleotides labeled with dye compounds, which are dideoxynucleotides lacking a hydroxyl group at both the 3 'and 2' positions, such modified dideoxynucleotides being suitable for use in methods such as the Sanger-type sequencing method.

It will be appreciated that modified nucleotides incorporating a 3 'blocking group labeled with the dye compounds of the present disclosure may also have utility in the Sanger method and related protocols, as the same effect achieved by using modified dideoxynucleotides may be achieved by using modified nucleotides having a 3' -OH blocking group: both of which prevent subsequent incorporation of nucleotides. In the case where nucleotides according to the present disclosure and having a 3' blocking group are to be used in a Sanger-type sequencing method, it will be appreciated that the dye compound or detectable label attached to the nucleotide need not be linked via a cleavable linker, as in each case the labeled nucleotides of the present disclosure are incorporated; the nucleotide need not be subsequently incorporated and thus the label need not be removed from the nucleotide.

Examples

Further embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the claims in any way.

Example 1: compound I-1:7- (3-carboxyazetidin-1-yl) -3- (5-chloro-benzoxazol-2-yl) incense Bean extract

3- (5-chloro-benzoxazol-2-yl) -7-fluoro-coumarin (0.32g, 1mmol) and 3-carboxy azetidine (0.2g, 2mmol) were added to anhydrous dimethyl sulfoxide (DMSO, 5mL) in a round bottom flask. The mixture was stirred at room temperature for a few minutes, then DIPEA (0.52g, 4mmol) was added. After stirring at 120 ℃ for 7 hours and standing at room temperature for 1 hour, the mixture was diluted with water (15mL) and stirred overnight. The resulting precipitate was collected by suction filtration. Yield 0.25g (63%). The purity, structure and composition of the product was confirmed by HPLC, NMR and LCMS. Ms (duis): MW calculated 396.05. Measured value m/z: (+)397(M +1)⁺；(-)395(M-1)^-.

Example 2. Compound I-2: 7- (3-carboxyazetidin-1-yl) -3- (benzoxazol-2-yl) coumarin

3- (benzoxazol-2-yl) -7-fluoro-coumarin (0.56h, 2mmol) and 3-carboxy azetidine (0.3g, 3mmol) were added to anhydrous dimethyl sulfoxide (DMSO, 5mL) in a round bottom flask. The mixture was stirred at room temperature for a few minutes, then DIPEA (0.52g, 4mmol) was added. After stirring at 125 ℃ for 9 hours and standing at room temperature for 1 hour, the reaction mixture was diluted with water (10mL) and stirred overnight. The resulting precipitate was collected by suction filtration. Yield 0.41g (56%). The purity, structure and composition of the product was confirmed by HPLC, NMR and LCMS. Ms (duis): MW calculated 362.09. Measured value m/z: (+)363(M +1)⁺.

Example 3. Compound I-3: 7- (3-carboxyazetidin-1-yl) -3- (benzimidazol-2-yl) coumarin

3- (benzimidazol-2-yl) -7-fluoro-coumarin (FC-2, 0.56g, 2mmol, 1eq.) and 3-carboxy azetidine (AC-C4, 0.3g, 3mmol, 1.5eq) were added to anhydrous dimethyl sulfoxide (DMSO, 5mL) in a round bottom flask. The mixture was stirred at room temperature for a few minutes, then DIPEA (0.52g, 4mmol) was added. The mixture was stirred at 120 ℃ for 9 hours. An additional portion of 3-carboxyazetidine (0.3g, 3mmol) and DIPEA (0.26g, 2mmol) was added. After stirring at 120 ℃ for another 3 hours and standing at room temperature for 1 hour, the reaction mixture was diluted with water (10mL) and stirred overnight. The resulting precipitate was collected by suction filtration. Yield 0.26g (36%). The purity, structure and composition of the product was confirmed by HPLC, NMR and LCMS. Ms (duis): MW calculated 361.11. Measured value m/z: (+)362(M +1)⁺；(-)360(M-1)^-.

Example 4. Compound I-4: 7- (3-carboxyazetidin-1-yl) -3- (benzothiazol-2-yl) coumarin

3- (benzothiazol-2-yl) -7-fluoro-coumarin (0.30g, 1mmol) and 3-carboxyazetidine (0.2g, 2mmol) were added to anhydrous dimethyl sulfoxide (DMSO, 5mL) in a round-bottom flask. The mixture was stirred at room temperature for a few minutes, then DIPEA (0.52g, 4mmol) was added. After stirring at 120 ℃ for 8 hours and standing at room temperature for 1 hour, the reaction mixture was diluted with water (10mL) and stirred overnight. The resulting precipitate was collected by suction filtration. Yield 0.28g (75%). The purity, structure and composition of the product was confirmed by HPLC, NMR and LCMS. Ms (duis): MW calculated 378.07. Measured value m/z: (+)379(M +1)⁺；(-)377(M-1)^-.

Example 5. Compound I-5: 7- (3-carboxypyrrolidin-1-yl) -3- (benzothiazol-2-yl) coumarin

3- (benzothiazol-2-yl) -7-fluoro-coumarin (0.30g, 1mmol) and 3-carboxypyrrolidine (0.23g, 2mmol) were added to anhydrous dimethyl sulfoxide (DMSO, 5mL) in a round bottom flask. The mixture was stirred at room temperature for a few minutes, then DIPEA (0.52g, 4mmol) was added. After stirring at 120 ℃ for 6 hours and standing at room temperature for 1 hour, the reaction mixture was diluted with water (20mL) and stirred overnight. The resulting precipitate was collected by suction filtration. Yield 0.31g (80%). The purity, structure and composition of the product was confirmed by HPLC, NMR and LCMS. Ms (duis): MW calculated 392.08. Measured value m/z: (+)393(M +1)⁺；(-)391(M-1)^-.

Example 6. Compound I-6: 7- (4-carboxypiperidin-1-yl) -3- (benzothiazol-2-yl) coumarin

3- (benzothiazol-2-yl) -7-fluoro-coumarin (0.30g, 1mmol) and hexahydroisonicotinic acid (0.26g, 2mmol) were added to anhydrous dimethyl sulfoxide (DMSO, 5mL) in a round-bottom flask. The mixture was stirred at room temperature for a few minutes, then DIPEA (0.52g, 4mmol) was added. After stirring at 120 ℃ for 6 hours and standing at room temperature for 1 hour, the reaction mixture was diluted with water (20mL) and stirred overnight. The resulting precipitate was collected by suction filtration. Yield 0.34g (83%). The purity, structure and composition of the product was confirmed by HPLC, NMR and LCMS. Ms (duis): MW calculated 406.10. Measured value m/z: (+)407(M +1)⁺；(-)405(M-1)^-。

Example 7. Compound I-7: 7- (3-Carboxyazetidin-1-yl) -3- (6-sulfo-benzothiazol-2-yl) Coumarin compound

7- (3-Carboxyazetidin-1-yl) -3- (benzothiazol-2-yl) coumarin (0.38g, 1mmol) was added to 20% oleum (0.5mL) at about-5 ℃. The mixture was stirred for several hours under cooling, then for 3 hours at room temperature. After stirring at 80 ℃ for 1 hour and standing at room temperature for 1 hour, the reaction mixture was diluted with anhydrous ether (10mL) and stirred overnight. The resulting precipitate was collected by suction filtration. The product was purified by HPLC. Yield 0.1g (22%). The purity, structure and composition of the product was confirmed by HPLC, NMR and LCMS. Ms (duis): MW calculated 458.02. Measured value m/z: (+)459(M +1)⁺.

Example 8, Compound I-8: 7- (3-carboxyazetidin-1-yl) -3- (6-sulfonamido-benzoxazole-2-one- Alkyl) coumarin

3- (6-sulfonamido-benzoxazol-2-yl) -7-fluoro-coumarin (0.36g, 1mmol) and 3-carboxy azetidine (0.3g, 3mmol) were added to anhydrous dimethyl sulfoxide (DMSO, 5mL) in a round bottom flask. The mixture was stirred at room temperature for a few minutes, then DIPEA (0.52g, 4mmol) was added. After stirring at 125 ℃ for 9 hours and standing at room temperature for 1 hour, the reaction mixture was diluted with water (10mL) and stirred overnight. The resulting precipitate was collected by suction filtration. Yield 0.26g (60%). The purity, structure and composition of the product was confirmed by HPLC, NMR and LCMS. Ms (duis): MW calculated 441.06. Measured value m/z: (+)442(M +1)⁺。

Example 9 comparison of fluorescence intensities

The fluorescence intensity of an exemplary dye solution (maximum excitation wavelength 450nm) was compared to a standard dye for the same spectral range. The results obtained are shown in table 1 and demonstrate the significant advantages of the exemplary dyes for fluorescence-based analytical applications.

Table 1. spectral properties of the novel fluorescent dyes disclosed in the examples.

Example 10 general procedure for the Synthesis of fully functionalized nucleotide conjugates

The coumarin fluorescent dyes disclosed herein are combined with suitable amino-substituted adenine (A) and cytosine (C) nucleotide derivatives A-LN3-NH₂Or C-LN3-NH₂Coupling:

after activation of the carboxyl group of the dye with a suitable reagent according to the following adenine exemplary protocol

The general products for adenine coupling are shown below:

ffA-LN 3-dye refers to a fully functionalized adenine (A) nucleotide having a LN3 linker and labeled with the coumarin dye disclosed herein. The R group in each structure refers to the coumarin dye moiety after coupling.

The dye (10. mu. mol) was dried by placing it in a 5mL round bottom flask, redissolving it in anhydrous dimethylformamide (DMF, 1mL), and then evaporating the solvent in vacuo. This process was repeated twice. The dried dye was dissolved in anhydrous N, N-dimethylacetamide (DMA, 0.2mL) at room temperature. N, N, N ', N' -tetramethyl-O- (N-succinimidyl) uronium tetrafluoroborate (TSTU, 1.5 equiv., 15. mu. mol, 4.5mg) was added to the dye solution, and DIPEA (3 equiv., 30. mu. mol, 3.8mg, 5.2. mu.L) was added to the solution via a micropipette. Under nitrogenThe reaction flask was sealed. The progress of the reaction was monitored by TLC (eluent acetonitrile-water 1: 9) and HPLC. At the same time, a solution of an appropriate amino-substituted nucleotide derivative (A-LN 3-NH)₂20mM, 1.5 equiv, 15. mu. mol, 0.75mL) was concentrated in vacuo and redissolved in water (20. mu.L). The solution of activated dye in DMA was transferred to a flask containing N-LN3-NH₂The solution of (1). More DIPEA (3eq, 30. mu. mol, 3.8mg, 5.2. mu.L) and triethylamine (1. mu.L) were added. The progress of the coupling was monitored hourly by TLC, HPLC and LCMS. When the reaction was complete, triethylammonium bicarbonate buffer (TEAB, 0.05M-3 mL) was added to the reaction mixture via pipette. The primary purification of the fully functionalized nucleotides is by passing the quenched reaction mixture through a DEAE-Sephadex column to remove most of the dye that has not yet reacted. For example, willPoured into an empty 25g Biotage packed column (cartridge) and the solvent system TEAB/MeCN. The solution from the Sephadex column was concentrated under vacuum. The remaining material was redissolved in a minimum volume of water and acetonitrile and then filtered through a20 μm Nylon filter. The filtered solution was purified via preparative-HPLC. The composition of the prepared compound was determined by LCMS.

The general product of coupling with cytosine is shown below, followed by a similar procedure as described above.

ffC-LN 3-dye refers to a fully functionalized cytosine (C) nucleotide having a LN3 linker and labeled with the coumarin dyes disclosed herein. The R group in each structure refers to the coumarin dye moiety after coupling.

EXAMPLE 11 preparation of amide derivatives of the Compound of formula (I)

Some further embodiments described herein relate to amide derivatives of compounds of formula (I) and processes for their preparation, which processes comprise converting a compound of formula (Ia) to a compound of formula (Ia') by carboxylic acid activation:

and reacting the compound of formula (Ia') with a primary or secondary amine of formula (Am) to give an amide derivative of formula (Ib):

wherein variable X, R, R¹、R²、R³、R⁴And n is as defined herein; r' is the residue moiety of a carboxyl activator (such as N-hydroxysuccinimide, nitrophenol, pentafluorophenol, HOBt, BOP, PyBOP, DCC, etc.); r_AAnd R_BEach of (A) is independently hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Carbocyclyl, C_6-10Aryl, 5-to 10-membered heteroaryl, 3-to 10-membered heterocyclyl, aralkyl, heteroaralkyl, or (heterocyclyl) alkyl.

General procedure for the preparation of Compounds of formula (Ib)

The appropriate dye of formula (Ia) (0.001mol) was dissolved in an appropriate anhydrous organic solvent (DMF, 1.5 mL). To this solution is added a carboxyl activating agent such as TSTU, BOP or PyBOP. The reaction mixture was stirred at room temperature for about 20min, then the appropriate amine derivative was added. The reaction mixture was stirred overnight, filtered and the excess activator quenched with 0.1M aqueous TEAB. The solvent was evaporated in vacuo and the residue was redissolved in TEAB solution and purified by HPLC.

Example 12 two-channel sequencing applications

The efficiency of a nucleotides labeled with the novel dyes described herein in sequencing applications was demonstrated in a two-pass detection method. With respect to the two-channel method described herein, nucleic acids can be sequenced using the methods and systems described in U.S. patent application No. 2013/0079232, the disclosure of which is incorporated herein by reference in its entirety.

In a dual channel assay, a nucleic acid can be sequenced by providing a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first and second channels, and a fourth nucleotide type lacking a label that is not detected or minimally detected in both channels. Scatter plots were generated by RTA2.0.93 analysis of the experiments. The scatter plots shown in fig. 1-3 are for cycle 5 of each of the 26 runs.

FIG. 1 shows a scatter plot of a mixture of fully functionalized nucleotides (ffN) comprising: A-I-4 (0.5. mu.M), A-NR550S0 (1.5. mu.M), C-NR440 (2. mu.M), black G (2. mu.M) and T-AF550POPOS0 (2. mu.M) in a blending buffer with Pol 812. Blue exposure (channel 1) for 500ms, green exposure (channel 2) for 1000 ms; scanning in a scan mix).

FIG. 2 shows a scatter plot of a mixture of fully functionalized nucleotides (ffN) comprising: A-I-5 (1. mu.M), A-NR550S0(1. mu.M), C-NR440 (2. mu.M), black G (2. mu.M) and T-AF550POPOS0 (2. mu.M) in a blending buffer with Pol 812. Blue exposure (channel 1) for 500ms, green exposure (channel 2) for 1000 ms; scanning in a scan mixture.

FIG. 3 shows a scatter plot of a mixture of fully functionalized nucleotides (ffN) comprising: A-I-6 (1. mu.M), A-NR550S0(1. mu.M), C-NR440 (2. mu.M), black G (2. mu.M) and T-AF550POPOS0 (2. mu.M) in a blending buffer with Pol 812. Blue exposure (channel 1) for 500ms, green exposure (channel 2) for 1000 ms; scanning in a scan mixture.

In each of FIGS. 1-3, the "G" nucleotide is unlabeled and is shown as the lower left black spot (cloud) ("Black G"). The signals from the mixture of "a" nucleotides labeled with the novel dyes described herein and the green dye (NR550S0) are shown as the upper right black spots in figures 1-3, respectively. The signal from the "T" nucleotide labelled with the dye AF550POPOS0 is indicated by the upper left black spot, whereas the signal from the "C" nucleotide labelled with the dye NR440 is indicated by the lower right black spot. The X-axis shows the signal strength of one (blue) channel and the Y-axis shows the signal strength of the other (green) channel. The chemical structures of NR440, AF550POPOS0, and NR550S0 are disclosed in PCT publication nos. WO2018060482a1, WO2017051201a1, and WO2014135221a1, respectively, all of which are incorporated herein by reference.

FIGS. 1-3 each show that fully functional A-nucleotide conjugates labeled with the novel dyes described herein provide adequate signal intensity and excellent black spot separation.