阅读说明：本技术 杂环叠氮化物单元及其在聚合物涂层中的用途 (Heterocyclic azide units and their use in polymeric coatings ) 是由 韦恩·N·乔治 吴晓琳 安德鲁·A·布朗 唐纳德·威灵斯 于 2019-12-09 设计创作，主要内容包括：本文描述了含杂环叠氮化物的单体单元,包含这样的含杂环叠氮化物的单体单元的共聚物,基底结合的共聚物和寡核苷酸结合的共聚物,用于制备这样的共聚物并使它们与基底和/或寡核苷酸反应的方法,以及使用这样的共聚物用于将寡核苷酸固定至基底的方法,例如用于DNA测序或其他诊断应用的用途。(Heterocyclic azide-containing monomer units, copolymers comprising such heterocyclic azide-containing monomer units, substrate-bound copolymers and oligonucleotide-bound copolymers, methods for preparing such copolymers and reacting them with a substrate and/or an oligonucleotide, and uses of such copolymers for immobilizing oligonucleotides to a substrate, e.g., for DNA sequencing or other diagnostic applications, are described herein.)

1. A compound of formula (I)

Wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

l is a linker comprising a straight chain having 2 to 20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on carbon and any nitrogen atoms in the chain;

e is a straight chain having 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group; and is

Z is a nitrogen-containing heterocycle.

2. The compound of claim 1, wherein E is optionally substituted C_1-4An alkylene group.

3. The compound of claim 1 or 2, wherein the compound has formula (Ia)

Wherein p is 1,2, 3 or 4.

4. A compound according to any one of claims 1 to 3, wherein the nitrogen-containing heterocycle comprises a 6-membered ring.

5. The compound of claim 4, wherein A and E are joined at positions 2 and 5 of the 6-membered ring.

6. A compound according to any one of claims 1 to 3, wherein the nitrogen-containing heterocycle comprises a 5-membered ring.

7. The compound of any one of claims 1 to 6, wherein the nitrogen-containing heterocycle is aromatic.

8. The compound of any one of claims 1 to 6, wherein the nitrogen-containing heterocycle is saturated.

9. The compound of any one of claims 1,2, 3, 4, or 5, wherein the compound has formula (Ib)

Wherein X is CH or N.

10. The compound of any one of claims 1,2, 3, or 6, wherein the compound is of formula (Ic)

11. The compound of any one of claims 1 to 10, wherein the compound is of formula (Id)

12. The compound of any one of claims 1 to 10, wherein the compound is of formula (Ie)

13. The compound of claim 1, wherein the compound is of formula (If)

14. The compound of claim 1, wherein the compound has formula (Ig)

15. The compound of claim 1, wherein the compound has formula (Ih)

16. The compound of claim 1, wherein the compound is of formula (Ij)

17. The compound of any one of claims 1,2, 3, 4, 5, 7, 9, or 11, wherein the compound is of formula (Ik):

wherein X is CH or N.

18. The compound of claim 17, wherein the compound of formula (Ik) is a compound of formula (Im):

wherein n is an integer from 2 to 20.

19. A copolymer comprising repeating monomer units of formula (II):

wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

l is a linker comprising a straight chain having 2 to 20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on carbon and any nitrogen atoms in the chain;

e is a straight chain having 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group; and is

Z is a nitrogen-containing heterocycle; and

at least one other repeating monomer unit.

20. The copolymer of claim 19, wherein the repeating monomer unit of formula (II) is a repeating monomer unit of formula (IIa)

Wherein p is 1,2, 3 or 4.

21. The copolymer of claim 20, wherein the repeating monomer unit of formula (IIa) is a compound of formula (IIb), formula (IIc), formula (IIe), or formula (IIf):

wherein n in formula (IIc) is an integer from 2 to 20.

22. The copolymer of any one of claims 19 to 21, wherein the at least one other repeating monomer unit is a compound of formula (III):

wherein

R⁴Is H or C_1-4An alkyl group;

R⁵is H or C_1-4An alkyl group; and is

R⁶Is H or C_1-4An alkyl group.

23. The copolymer of claim 22, wherein the copolymer comprises a repeating monomer unit of formula (IIb), formula (IIc), formula (IId), or formula (IIe), a repeating monomer unit of formula (III), and at least one other repeating monomer unit.

24. A substrate-copolymer product formed from the reaction of the copolymer of any one of claims 19 to 23 with a substrate.

25. The substrate-copolymer product of claim 24, wherein the substrate copolymer product is formed by reaction of an azido group of the copolymer with an alkenyl or alkynyl containing group on a surface of the substrate.

26. A structure comprising a copolymer covalently bonded to a substrate, wherein the substrate-bonded copolymer comprises repeating monomer units covalently bonded to the substrate, wherein the repeating, covalently bonded monomer units are compounds of formula (IV):

wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

l is a linker comprising a straight chain consisting of 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

e is a straight chain having 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group;

z is a nitrogen-containing heterocycle;

is a single or double bond; and is

The triazole or triazoline is covalently bound to the substrate through one or both triazole/triazoline ring carbon atoms either directly or indirectly through a linker.

27. The structure of claim 26, wherein the repeating, covalently bound monomer unit is a compound of formula (IVa):

wherein p is 1,2, 3 or 4.

28. The structure of claim 26, wherein the repeating, covalently bound monomer unit is a compound of formula (V):

wherein G is a linking group between the triazole/triazoline ring and the substrate.

29. The structure of claim 28, wherein the repeating, covalently bound monomer unit is a compound of formula (Va):

wherein p is 1,2, 3 or 4.

30. The structure of claim 27, wherein the repeating, covalently bound monomer unit is a compound of formula (IVb), formula (IVc), formula (IVd), or formula (IVe):

wherein

X is CH or N; and is

N in formula (IVc) is an integer from 2 to 20.

31. The structure of any one of claims 26 to 30, wherein the repeating, covalently bound monomer unit is a compound of formula (Vb), formula (Vc), formula (Vd), or formula (Ve):

wherein G is a linking group between the triazole/triazoline ring and the substrate.

32. A copolymer-oligonucleotide product formed from a reaction between a copolymer according to any one of claims 19 to 23 and one or more oligonucleotides.

33. The copolymer-oligonucleotide product of claim 32, wherein the copolymer oligonucleotide product is formed from the reaction of an azido group of the copolymer and an alkene or alkyne functionalized oligonucleotide.

34. An oligonucleotide-conjugated copolymer comprising an oligonucleotide-conjugated repeating monomer unit of formula (IV):

wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

l is a linker comprising a straight chain consisting of 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

e is a straight chain having 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group;

z is a nitrogen-containing heterocycle;

is a single or double bond; and is

The copolymer is covalently bound to the oligonucleotide through one or both triazole/triazoline ring carbon atoms, either directly or indirectly through a linker.

35. The oligonucleotide-conjugated copolymer according to claim 34, wherein the oligonucleotide-conjugated repeating monomer unit has formula (IVa):

wherein p is 1,2, 3 or 4.

36. The oligonucleotide-conjugated copolymer according to claim 34, wherein the oligonucleotide-conjugated repeating monomer unit has formula (VI):

37. the oligonucleotide-conjugated copolymer of claim 36, wherein the oligonucleotide-conjugated repeating monomer unit has formula (VIa):

wherein p is 1,2, 3 or 4.

38. The oligonucleotide-conjugated copolymer of claim 36, wherein the oligonucleotide-conjugated repeating monomer unit has formula (IVb), formula (IVc), formula (IVd), or formula (IVe):

wherein X is CH or N; p is 1,2, 3 or 4; and n in formula (IVc) is an integer from 2 to 20.

39. The oligonucleotide-conjugated copolymer of any one of claims 34-38, wherein the oligonucleotide-conjugated repeating monomer unit has formula (VIb), formula (VIc), formula (VIa), or formula (VIb):

wherein X is CH or N; and n in formula (IVc) is an integer from 2 to 20.

40. A copolymer-substrate-oligonucleotide product formed from the reaction of a copolymer according to any one of claims 19 to 23 with a substrate and one or more oligonucleotides.

41. A method of immobilizing a copolymer of any one of claims 19 to 23 to a substrate, the method comprising reacting an azido group of the copolymer with more than one alkenyl-or alkynyl-containing functional group on a surface of the substrate to form a covalent bond between the copolymer and the surface.

42. A method of grafting more than one oligonucleotide to a copolymer of any one of claims 19 to 23, the method comprising reacting an azido group of the copolymer with the more than one oligonucleotide.

43. The method of claim 42, wherein the more than one oligonucleotide comprises an alkenyl group or an alkynyl group and the reaction occurs between the azido group of the copolymer and the alkenyl group or alkynyl group of the oligonucleotide.

44. A process for preparing a compound of formula (I):

the process comprises reacting a compound of formula (X) with an acrylate of formula (XI):

wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

l is a linker comprising a straight chain consisting of 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

e is a straight chain having 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group;

z is a nitrogen-containing heterocycle; and is

Hal is Cl or F

To form the compound of formula (I).

45. A process for preparing a compound of formula (Ik):

the method comprises reacting a compound of formula (Xa):

with an acrylate of formula (XI):

wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

l is a linker comprising a straight chain consisting of 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

p is 1,2, 3 or 4;

x is CH or N; and is

Hal is Cl or F

To form a compound of formula (Ik).

46. A process as in claim 45, further comprising reacting a compound of formula (XII):

with a compound of formula (XIII):

wherein

Pg is H or an amino protecting group; and is

Y is-OH or-Cl;

to form said compound of formula (Xa).

47. A process for preparing a compound of formula (Ik), the process comprising reacting a compound of formula (XIV):

wherein Y is-OH or-Cl;

with a compound of formula (XV):

to form said compound of formula (Ik).

Wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

l is a linker comprising a straight chain consisting of 6 to 8 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

p is 1,2, 3 or 4; and is

X is CH or N.

48. A process according to claim 47, further comprising reacting a compound of formula (XII) with a cyclic anhydride to form the compound of formula (XIV).

49. A compound of formula (Ik):

wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

R³is H or C_1-4An alkyl group;

x is CH or N;

p is 1,2, 3 or 4; and is

L is a linker having a straight chain consisting of 2 to 20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on carbon and nitrogen atoms in the chain.

50. A compound according to claim 49, wherein the compound of formula (Ik) is a compound of formula (Im):

wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

R³is H or C_1-4An alkyl group;

x is CH or N;

p is 1,2, 3 or 4; and is

n is an integer from 2 to 20.

51. A copolymer comprising repeating monomer units of formula (IIb), formula (IIc), formula (IId), or formula (IIe):

wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

R³is H or C_1-4An alkyl group;

x is CH or N;

p is 1,2, 3 or 4;

l in formula (IIb) is a linker having a straight chain consisting of 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and nitrogen atoms in the chain; and is

N in formula (IIc) is an integer from 2 to 20;

and at least one other repeating monomer unit.

52. The copolymer of claim 51, comprising recurring monomeric units of formula (IIb), formula (IIc), formula (IId), or formula (IIe) and recurring monomeric units of formula (III):

wherein

R⁴Is H or C_1-4An alkyl group;

R⁵is H or C_1-4An alkyl group; and is

R⁶Is H or C_1-4An alkyl group.

53. The copolymer of claim 52, wherein the copolymer comprises a repeating monomer unit of formula (IIb), formula (IIc), formula (IId), or formula (IIe), a repeating monomer unit of formula (III), and at least one other repeating monomer unit.

54. A substrate comprising a copolymer comprising a repeating monomer unit of formula (IIb), formula (IIc), formula (IId), or formula (IIe) and at least one other repeating monomer unit, wherein the copolymer is covalently bonded to the substrate.

55. The substrate of claim 54, wherein covalent attachment is by reaction of an azido group of a monomeric unit of formula (IIb), formula (IIc), formula (IId), or formula (IIe) with an alkenyl or alkynyl containing group on the surface of the substrate.

56. The substrate of claim 54, wherein the substrate-bound copolymer further comprises repeating monomer units covalently bound to the substrate, wherein the repeating, covalently bound monomer units are represented by formula (IVb), formula (IVc), formula (IVd), or formula (IVe):

wherein R is¹、R²、R³L, p and n are as defined herein,is a single or double bond, and the triazole or triazoline is covalently bound via one or two triazole/triazoline ring carbon atoms, either directly or indirectly via a linking groupIs bonded to the substrate.

57. The substrate of claim 56, wherein the copolymer comprises repeating, substrate-bound monomer units represented by formula (Vb), formula (Vc), formula (Vd), or formula (Ve):

wherein G is a linking group between the triazole/triazoline ring and the substrate.

58. The copolymer of claim 51, wherein the copolymer is covalently bound to an oligonucleotide.

59. The copolymer of claim 58, wherein the oligonucleotide is covalently bound to the copolymer by reaction of the azido groups of the repeating monomeric units of formula (IIb), formula (IIc), formula (IId), or formula (IIe) with an alkene-or alkyne-functionalized oligonucleotide.

60. The copolymer of claim 59, wherein the oligonucleotide-conjugated copolymer comprises repeating monomer units of formula (IVb), formula (IVc), formula (IVd), or formula (IVe), wherein R¹、R²、R³L, p and n are as defined herein,is a single or double bond, and the copolymer is covalently bonded to the oligonucleotide through one or both triazole/triazoline ring carbon atoms, either directly or indirectly through a linker.

61. The copolymer of claim 60, comprising oligonucleotide-bound repeat monomer units of formula (VIb), formula (VIc), formula (VId), or formula (VIe):

wherein the variables are as defined herein.

62. The copolymer of claim 51, wherein the copolymer is covalently bound to a substrate and covalently bound to an oligonucleotide.

63. The copolymer of claim 62, wherein the copolymer comprises repeating monomer units of one or more of formula (Vb), formula (VIc), formula (VId), and formula (VIe).

64. A method of immobilizing a copolymer comprising recurring monomeric units of formula (IIb), (IIc), (IId), or (IIe) to a substrate, the method comprising reacting an azido group of a copolymer comprising recurring monomeric units of formula (IIb), (IIc), (IId), or (IIe) with more than one alkenyl-or alkynyl-containing functional group on a surface of the substrate to form a covalent bond between the copolymer and the surface.

65. A method of grafting more than one oligonucleotide to a copolymer comprising repeating monomer units of formula (IIb), formula (IIc), formula (IId), or formula (IIe), the method comprising reacting the azido groups of the copolymer with the oligonucleotide.

66. The method of claim 65, wherein the oligonucleotide comprises an alkenyl group or an alkynyl group, and the reaction occurs between the azido group of the copolymer and the oligonucleotide.

67. A process for preparing a compound of formula (Ik):

the method comprises reacting a compound of formula (Xa):

with an acrylate of formula (XI):

wherein R is¹、R²、R³L, p and X are as defined herein and Hal is C1 or F to form a compound of formula (Ik).

68. A process as in claim 67, further comprising reacting a compound of formula (XII):

wherein p and R³And X is as defined herein;

with a compound of formula (XIII):

wherein

Pg is H or an amino protecting group; and is

Y is-OH or-Cl;

to form said compound of formula (Xa).

69. A process for preparing a compound of formula (Ik), the process comprising reacting a compound of formula (XIV):

wherein Y is-OH or-Cl;

with a compound of formula (XV):

to form said compound of formula (Ik).

70. A process according to claim 69, further comprising reacting a compound of formula (XII) with a cyclic anhydride to form the compound of formula (XIV).

Background

Polymer coated substrates are used in many technical applications. For example, an implantable medical device (implantable medical device) may be coated with a biologically inert polymer (biomedical polymer). In another example, the polymer coated substrate is used for the preparation and/or analysis of biomolecules. Molecular analysis such as certain nucleic acid sequencing methods rely on the attachment of nucleic acid strands to a polymer-coated surface of a substrate. The sequence of the attached nucleic acid strand can then be determined by a number of different methods known in the art.

In certain sequencing methods, such as sequencing-by-synthesis (SBS), the surface of a substrate (such as a flow cell) is coated with a polymer, and then primers (single-stranded DNA or ssDNA) are grafted to the polymer. In the array method, the beads are coated with a polymer that is also functionalized to capture target oligonucleotides.

The polymer surface (and its preparation) is generally compatible with a wide range of sequencing and detection methods, including different chemical conditions, temperatures, optical detection methods, capture moiety density and other parameters, and is generally stable under a variety of storage and transport conditions. Certain polymeric materials used in these molecular biological methods use pendant azido groups that react in a copper-mediated cycloaddition reaction with olefin or alkyne groups on the surface of the substrate to be grafted and/or of the oligonucleotide. However, the residual copper may have cytotoxic effects in biologically relevant environments. With respect to DNA sequencing applications, in some cases, copper can damage DNA, thereby reducing sequencing yield (sequencing yield) and data quality. Furthermore, copper-catalyzed reactions are typically copper-intensive and therefore expensive, and may not be able to run efficiently or quickly enough to ensure adequate polymer attachment and positioning on the substrate surface. Thus, there is a need for surface polymer coatings with improved properties, such as increased reaction efficiency and resulting in reduced residual copper.

SUMMARY

The examples provided herein relate to polymers comprising heterocyclic azide units, optionally with oligonucleotides grafted thereto, substrates with the polymers attached thereto, and uses of the polymers and substrates in applications such as molecular biology methods, such as DNA sequencing and other diagnostic applications. Methods of making the heterocyclic azide polymers and substrates and methods of using the compositions are also disclosed.

In some examples herein, monomers and copolymers are provided that use a heterocycloazide group that reacts efficiently in a cycloaddition reaction with reduced copper loading and, thus, less residual copper.

In one aspect, provided herein is a compound of formula (I)

Wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; l is a linker comprising a straight chain containing 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; e is a straight chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group; and Z is a nitrogen-containing heterocycle.

In some examples, E may be optionally substituted C_1-4An alkylene group.

In some examples, the compound of formula (I) may be a compound of formula (Ia)

Wherein p is 1,2, 3 or 4.

In some examples, the nitrogen-containing heterocycle may include a 6-membered ring. Illustratively, A and E (e.g., alkyleneazido groups) are bonded at the 2-and 5-positions of a 6-membered ring. In other examples, the nitrogen-containing heterocycle includes a 5-membered ring.

Additionally or alternatively, the nitrogen-containing heterocycle is optionally aromatic, or optionally saturated.

Additionally or alternatively, the compound is optionally of formula (Ib)

Wherein X is CH or N.

Alternatively, the compound is optionally of formula (Ic)

Alternatively, the compound is optionally of formula (Id)

Alternatively, the compound is optionally of formula (Ie)

In another example, the compound has formula (If)

In another example, the compound has the formula (Ig)

In another example, the compound has the formula (Ih)

In another example, the compound has formula (Ij)

In another example, the compound has formula (Ik)

Wherein X is CH or N.

Illustratively, the compound of formula (Ik) is a compound of formula (Im):

wherein n is an integer from 2 to 20.

In another aspect, provided herein is a copolymer comprising repeating monomer units of formula (II)

Wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; l is a linker comprising a straight chain containing 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; e is a straight chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group; and Z is a nitrogen-containing heterocycle; and at least one other repeating monomer unit.

In some examples, the repeating monomer unit of formula (II) is a repeating monomer unit of formula (IIa):

wherein p is 1,2, 3 or 4.

In some examples, the repeating monomer unit of formula (IIa) is a repeating monomer unit of formula (IIb), formula (IIc), formula (IId), or formula (IIe):

wherein n in formula (IIc) is an integer from 2 to 20. In some examples, at least one other repeating monomer unit is a compound of formula (III):

wherein R is⁴Is H or C_1-4An alkyl group; r⁵Is H or C_1-4An alkyl group; and R is⁶Is H or C_1-4An alkyl group.

Illustratively, the copolymer comprises a repeating monomer unit of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe), a repeating monomer unit of formula (III), and at least one other repeating monomer unit.

In another aspect, provided herein is a substrate-copolymer product formed from the reaction of a copolymer as set forth above with a substrate. In some examples, the substrate copolymer product is formed by the reaction of an azido group of the copolymer with an alkenyl or alkynyl containing group on the surface of the substrate.

In another aspect, provided herein is a structure comprising a copolymer covalently bonded to a substrate, wherein the substrate-bonded copolymer comprises repeating monomer units covalently bonded to the substrate, wherein the repeating, covalently bonded monomer units are compounds of formula (IV):

wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; l is a linking group comprising 2 to 20 groups selected from the group consisting of carbon, oxygen and nitrogenA linear chain of atoms of the group and optional substituents on carbon and any nitrogen atom in the chain; e is a straight chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group; z is a nitrogen-containing heterocycle;is a single or double bond; and the triazole or triazoline is covalently bound to the substrate through one or both triazole/triazoline ring carbon atoms, either directly or indirectly through a linker.

In some examples, the recurring, covalently bound monomer unit is a compound of formula (IVa):

wherein p is 1,2, 3 or 4. .

In some examples, the recurring, covalently bound monomer unit is a compound of formula (V):

wherein G is a linking group between the triazole/triazoline ring and the substrate. In some examples, the repeating, covalently bound monomer unit is a compound of formula (Va):

wherein p is 1,2, 3 or 4.

In some examples, the recurring, covalently bound monomer unit is a compound of formula (IVb), formula (IVc), formula (IVd), or formula (IVe):

wherein X is CH or N; and n in formula (IVc) is an integer from 2 to 20.

Alternatively or additionally, the repeating, covalently bound monomer unit is optionally a compound of formula (Vb), formula (Vc), formula (Vd) or formula (Ve):

wherein G is a linking group between the triazole/triazoline ring and the substrate.

In another aspect, provided herein is a copolymer-oligonucleotide product formed from a reaction between a copolymer as set forth above and one or more oligonucleotides.

In some examples, the copolymer oligonucleotide product is formed from the reaction of the azido group of the copolymer and an alkene or alkyne functionalized oligonucleotide.

In another aspect, provided herein is an oligonucleotide-conjugated copolymer comprising a repeating monomer unit of formula (IV):

wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; l is a linker comprising a straight chain containing 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; e is a straight chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group; z is a nitrogen-containing heterocycle;is a single or double bond; and the copolymer is covalently bound to the oligonucleotide through one or both triazole/triazoline ring carbon atoms, either directly or indirectly through a linker.

In some examples, the oligonucleotide-bound repeating monomer unit has formula (IVa):

wherein p is 1,2, 3 or 4.

In some examples, the oligonucleotide-bound repeat monomer units have formula (VI):

in some examples, the oligonucleotide-binding repeat monomer units have formula (VIa):

wherein p is 1,2, 3 or 4.

In some examples, the oligonucleotide-binding repeat monomer unit has formula (IVb), formula (IVc), formula (IVd), or formula (IVe):

wherein X is CH or N; and n in formula (IVc) is an integer from 2 to 20.

Additionally or alternatively, the oligonucleotide-binding repeat monomer units optionally have formula (VIb), formula (VIc), formula (VId), or formula (VIe):

wherein X is CH or N; and n in formula (IVc) is an integer from 2 to 20.

In another aspect, provided herein is a copolymer-substrate-oligonucleotide product formed by reaction of a copolymer according to any set forth above with a substrate and one or more oligonucleotides.

In another aspect, provided herein is a method of immobilizing a copolymer according to any statement set forth above to a substrate, the method comprising reacting an azido group of the copolymer with more than one alkenyl-or alkynyl-containing functional group on a surface of the substrate to form a covalent bond between the copolymer and the surface of the substrate.

In another aspect, provided herein is a method of grafting more than one oligonucleotide to a copolymer according to any statement as set forth above, the method comprising reacting the azido group of the copolymer with more than one oligonucleotide. In some examples, more than one oligonucleotide comprises an alkenyl group or an alkynyl group, and the reaction occurs between the azido group of the copolymer and the alkenyl group or alkynyl group of the oligonucleotide.

In another aspect, provided herein is a process for preparing a compound of formula (I):

the process comprises reacting a compound of formula (X) with an acrylate of formula (XI):

wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; l is a linker comprising a straight chain containing 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; e is a compound containing 1 to 4 members selected from the group consisting of carbon, oxygen and nitrogenStraight chain of atoms and optional substituents on carbon and any nitrogen atoms in the chain; a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group; z is a nitrogen-containing heterocycle; and Hal is Cl or F to form the compound of formula (I).

In another aspect, provided herein is a process for preparing a compound of formula (Ik):

the method comprises reacting a compound of formula (Xa):

with an acrylate of formula (XI):

wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; l is a linker comprising a straight chain containing 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; p is 1,2, 3 or 4; x is CH or N; and Hal is Cl or F to form a compound of formula (Ik).

In some examples, the method further comprises reacting a compound of formula (XII):

with a compound of formula (XIII):

wherein Pg is H or an amino protecting group; and Y is-OH or-Cl; to form a compound of formula (Xa).

In another aspect, provided herein is a method of preparing a compound of formula (Ik), comprising reacting a compound of formula (XIV):

wherein Y is-OH or-Cl; with a compound of formula (XV):

to form a compound of formula (Ik).

Wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; l is a linker comprising a straight chain containing 6 to 8 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; p is 1,2, 3 or 4; and X is CH or N.

In some examples, the method further comprises reacting the compound of formula (XII) with a cyclic anhydride to form the compound of formula (XIV).

Some examples provided herein relate to compounds of formula (Ik):

wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; r³Is H or C_1-4An alkyl group; x isCH or N; p is 1,2, 3 or 4; and L is a linker having a straight chain consisting of 2 to 20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on carbon and nitrogen atoms in the chain.

In some aspects, the compound of formula (Ik) is a compound of formula (Im):

wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; r³Is H or C_1-4An alkyl group; x is CH or N; p is 1,2, 3 or 4; and n is an integer from 2 to 20.

Some examples provided herein relate to a copolymer comprising repeating monomer units of formula (IIb), formula (IIc), formula (IId), or formula (IIe):

wherein R is¹Is H or C_1-4An alkyl group; r²Is H or C_1-4An alkyl group; r³Is H or C_1-4An alkyl group; x is CH or N; p is 1,2, 3 or 4; and L in formula (IIb) is a linker having a straight chain consisting of 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and nitrogen atoms in the chain; and n in formula (IIc) is an integer from 2 to 20; and at least one other repeating monomer unit.

The copolymer may be linear, branched or crosslinked. The repeating units may be present in the copolymer in any arrangement, such as block, alternating, or random. The copolymer may also comprise one or more additional repeating monomer units.

In some aspects, the copolymer comprises repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and repeating monomer units of formula (III):

wherein R is⁴Is H or C_1-4An alkyl group; r⁵Is H or C_1-4An alkyl group; and R is⁶Is H or C_1-4An alkyl group.

In some aspects, the copolymer comprises a repeating monomer unit of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe), a repeating monomer unit of formula (III), and at least one other repeating monomer unit.

Some examples provided herein relate to a substrate having a copolymer as described herein covalently bonded to the substrate. In some examples, the covalent attachment is by reaction of the azido groups of the monomeric units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) with alkenyl or alkynyl containing groups on the surface of the substrate. Such examples are substrate-bound copolymers comprising repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId) or formula (IIe), since not all of the azido groups in the copolymer have to be reacted. In some examples, the substrate-bound copolymer further comprises repeating monomer units covalently bound to the substrate. The repeating, covalently bound monomer units are represented by formula (IVb), formula (IVc), formula (IVd), or formula (IVe):

wherein R is¹、R²、R³L, p and n are as defined herein,is a single or double bond, and the triazole or triazoline is bonded to the ring carbon atom (directly) through one (e.g., where the substrate-bound alkenyl or alkynyl group is acyclic) or two (e.g., where the substrate-bound alkenyl or alkynyl group is part of a ring) triazole/triazoline ring carbon atomsEither indirectly or through a linker) to a substrate.

Thus, some exemplary substrate-bound monomeric units are represented by formula (Vb), formula (Vc), formula (Vd), or formula (Ve):

wherein G is a linking group between the triazole/triazoline ring and the substrate.

The copolymers described herein may also be covalently bound to oligonucleotides. In some examples, the oligonucleotide is covalently bound to the copolymer by reaction of the azido groups of the repeating monomeric units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) with an alkene-or alkyne-functionalized oligonucleotide. Because not all of the azido groups in the copolymer need to be reacted, some examples provided herein relate to oligonucleotide-conjugated copolymers comprising repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe). In some examples, the oligonucleotide-bound copolymer further comprises a repeating monomer unit covalently bound to the oligonucleotide. In some examples, the oligonucleotide-conjugated copolymer comprises repeating monomer units of formula (IV), formula (IVa), formula (IVb), formula (IVc), formula (IVd), or formula (IVe), wherein R is¹、R²、R³L, p and n are as defined herein,is a single or double bond, and the copolymer is covalently bound to the oligonucleotide through one or both triazole/triazoline ring carbon atoms (either directly or indirectly through a linker). Examples of such structures are oligonucleotide-bound repeating monomer units of formula (VIb), formula (VIc), formula (VId), or formula (VIe):

wherein the variables are as defined herein.

Some examples provided herein also relate to copolymers that are covalently bound to both the substrate and the oligonucleotide. Thus, in some examples are copolymers comprising repeating monomer units of one or more of formula (V), formula (Va), formula (Vb), formula (VIc), formula (VId), and formula (VIe).

Some examples provided herein relate to methods of preparing compounds of formula (Ik), methods of preparing copolymers comprising recurring monomeric units of formula (IIb), and methods of preparing copolymers comprising recurring monomeric units of formula (IIb) and recurring monomeric units of formula (III).

Some examples provided herein relate to a method of immobilizing a copolymer as described herein to a substrate, the method comprising reacting an azido group of the copolymer comprising a repeating monomer unit of formula (IIb) with more than one alkenyl-or alkynyl-containing functional group on a surface of the substrate to form a covalent bond between the copolymer and the surface.

Some examples provided herein relate to methods of grafting more than one oligonucleotide to a copolymer as described herein, the methods comprising reacting an azido group of the copolymer with the oligonucleotide. In some examples, the oligonucleotide comprises an alkenyl group or an alkynyl group. In some examples, the oligonucleotide comprises an alkynyl group. In some examples, the oligonucleotide is reacted with the copolymer prior to immobilizing the copolymer on the surface of the substrate. In some examples, the oligonucleotide is reacted with the copolymer after the copolymer is immobilized on the surface of the substrate.

It will be understood that any respective features/examples of each aspect of the disclosure as described herein may be implemented together in any suitable combination, and any features/examples from any one or more of these aspects may be implemented together with any features of the other aspects as described herein in any suitable combination.

Detailed Description

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 "including", "includes" and "included" is not limiting. The use of the term "having" as well as other forms such as "having", "having" and "having" is not limiting. As used in this specification, the terms "comprises" and "comprising," whether in transitional phrases or in the bodies of the claims, should be interpreted to have an open-ended meaning. That is, the above terms should be interpreted synonymously with the phrase "having at least" or "including at least". For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. 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 additional features or components.

The terms "substantially", "about" and "about" are used throughout this specification to describe and account for small fluctuations such as due to variations in processing. For example, they may refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

As used herein, the term "array" refers to a population of different probe molecules that are attached to one or more substrates such that the different probe molecules can be distinguished from each other according to relative position. The array may comprise different probe molecules each located at a different addressable location on the substrate. Alternatively or additionally, the array may comprise separate substrates each carrying different probe molecules, wherein the different probe molecules may be identified according to the position of the substrate on the surface to which the substrate is attached or according to the position of the substrate in the liquid.

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, "C" wherein "a" and "b" are integers_aTo C_b"or" C_a-b"refers to the number of carbon atoms in a given group. That is, the group may contain from "a" to "b" (inclusive) carbon atoms. Thus, for example, "C₁To C₄Alkyl "or" C_1-4Alkyl "or" C_1-4Alkyl "groups refer to all alkyl groups having from 1 to 4 carbons, i.e., CH₃-、CH₃CH₂-、CH₃CH₂CH₂-、(CH₃)₂CH-、CH₃CH₂CH₂CH₂-、CH₃CH₂CH(CH₃) -and (CH)₃)₃C-。

As used herein, the term "halogen" or "halogen (halo)" means fluorine, chlorine, bromine or iodine, with fluorine and chlorine being examples.

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 may 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; for example, "1 to 20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the term "alkyl" appearing without a specified numerical range). 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 4 carbon atoms. The alkyl group may be designated as "C_1-4Alkyl "or similar names. By way of example only, "C_1-4Alkyl "or" C_1-4Alkyl "indicates that there are one to four 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-butyl, and tert-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, 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, although 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 4 carbon atoms. An alkenyl group may be designated as "C_2-4Alkenyl "or similar names. By way of example only, "C_2-4Alkenyl "indicates that there are two to four 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, and hexenyl, and the like.

The group comprising alkenyl groups includes optionally substituted alkenyl, cycloalkenyl and heterocycloalkenyl groups.

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, although 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 4 carbon atoms. Alkynyl radicalA clique may be designated as "C_2-4Alkynyl "or similar names. By way of example only, "C_2-4Alkynyl "or" C_2-4Alkynyl "indicates that there are two to four carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of: ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl and 2-butynyl. Typical alkynyl groups include, but are in no way limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl groups, and the like.

Groups that include alkynyl groups include optionally substituted alkynyl, cycloalkynyl, and heterocyclic alkynyl groups.

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, although the present definition also covers the term "aryl" when no numerical range is specified. In some examples, the aryl group has 6 to 10 carbon atoms. The aryl group may be designated as "C_6-10Aryl group "," C₆Or C₁₀Aryl "or similar names. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

As used herein, "heterocycle" refers to a cyclic compound that includes a carbon atom as well as another atom (heteroatom), such as nitrogen, oxygen, or sulfur. Heterocycles can be aromatic (heteroaryl) or aliphatic. The aliphatic heterocyclic ring may be fully saturated or may contain one or more or two or more double bonds, for example the heterocyclic ring may be heterocycloalkyl. The heterocyclic ring may include a single heterocyclic ring or a plurality of condensed heterocyclic rings.

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 includes 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), although this definition also covers the term "heteroaryl" as it occurs without a range of numerical values being specified. In some examples, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. Heteroaryl groups may be designated as "5-7 membered heteroaryl", "5-10 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.

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

As used herein, "cycloalkenyl" or "cycloalkene" means a carbocyclic ring or ring system having at least one double bond, wherein none of the rings in the ring system are aromatic. Examples are cyclohexenyl or cyclohexene. Another example is norbornene or norbornenyl.

As used herein, "heterocycloalkenyl" or "heterocycloalkene" means a carbocyclic ring or a carbocyclic ring system having at least one double bond, having at least one heteroatom in the ring backbone, wherein no ring in the ring system is aromatic. In some examples, the heterocycloalkenyl or heterocycloalkenyl ring or ring system is 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered.

As used herein, "cycloalkynyl" or "cycloalkyne" means a carbocyclic ring or ring system having at least one triple bond, wherein no ring in the ring system is aromatic. An example is cyclooctyne. Another example is bicyclononylyne.

As used herein, "heterocycloalkynyl" or "heterocycloalkyne" means a carbocyclic ring or ring system having at least one triple bond with at least one heteroatom in the ring backbone, wherein no ring in the ring system is aromatic. In some examples, the heterocycloalkynyl or heterocycloalkyne ring or ring system is 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered.

As used herein, "heterocycloalkyl" means a non-aromatic ring or non-aromatic ring system that contains at least one heteroatom in the ring backbone. The heterocycloalkyl groups may be joined together in a fused, bridged or spiro-linked manner. The heterocycloalkyl group can have any degree of saturation, provided that at least one of the heterocyclic rings in the ring system is not aromatic. A heterocycloalkyl group can have from 3 to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon and heteroatoms), although this definition also covers the term "heterocycloalkyl" as it appears without a numerical range being specified. The heterocycloalkyl group can also be a medium size heterocycloalkyl having 3 to 10 ring members. The heterocycloalkyl group can also be a heterocycloalkyl group having from 3 to 6 ring members. A heterocycloalkyl group can be designated as "3-6 membered heterocycloalkyl" or a similar name. In some six-membered monocyclic heterocycloalkyl, the heteroatom is selected from one up to three of O, N or S, and in some five-membered monocyclic heterocycloalkyl, the heteroatom is selected from one or two heteroatoms selected from O, N or S. Examples of heterocycloalkyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1, 3-dioxinyl, 1, 3-dioxanyl, 1, 4-dioxinyl, 1, 4-dioxanyl, 1,3-oxathianyl (1, 3-oxathinyl), 1, 4-oxathianyl, 2H-1, 2-oxazinyl, trioxanyl, 2H-oxazinyl, and oxathianyl, Hexahydro-1, 3, 5-triazinyl, 1,3-dioxolyl (1,3-dioxolyl), 1,3-dioxolanyl (1,3-dioxolyl), 1,3-dithiolyl (1,3-dithiolyl), 1,3-dithiolyl (1, 3-dithiolan), isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl (1,3-oxathiolanyl), indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydro-1, 4-thiazinyl, thiomorpholinyl (thiomorpholinyl), dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

As used herein, the term "azido" refers to-N₃A group.

As used herein, a substituted group is derived from an unsubstituted parent group (parent group) in which there has been an exchange of one or more hydrogen atoms for another atom or group. Unless otherwise indicated, when a group is 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), 5-10 membered heterocyclyl (optionally substituted with halogen, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl and C₁-C₆Haloalkoxy substituted), 5-10 membered heterocyclyl-C₁-C₆Alkyl (optionally substituted by halogen, C)₁-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), 5-10 membered heteroaryl (optionally substituted with halogen, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl and C₁-C₆Haloalkoxy substituted), 5-10 membered heteroaryl (C)₁-C₆) Alkyl (optionally substituted by halogen, C)₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Haloalkyl and C₁-C₆Haloalkoxy substituted), halogen, cyano, hydroxy, C₁-C₆Alkoxy radical, C₁-C₆Alkoxy (C)₁-C₆) Alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halo (C)₁-C₆) Alkyl (e.g., -CF3), halo (C)₁-C₆) Alkoxy (e.g., -OCF)₃)、C₁-C₆Alkylthio (alkylthio), arylthio (arylthio), amino (C)₁-C₆) Alkyl, nitro, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl and oxo (═ O). In any case where a group is described as "optionally substituted," the group may be substituted with the above substituents.

It is understood that certain radical naming conventions (naming conventions) may include single or double radicals, depending on the context. For example, where a substituent requires two attachment points to the remainder of the molecule, it is understood that this is the caseThe substituents are diradicals. 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".

In any case where a substituent is depicted as a diradical (i.e., having two attachment points to the remainder of the molecule), it is to be understood that the substituent can be attached in any directional configuration unless otherwise indicated. Thus, for example, depicted as-AE-orIncludes the case where the substituents are oriented such that a is attached at the leftmost attachment point of the molecule, and where a is attached at the rightmost attachment point of the molecule.

Where the compounds disclosed herein have at least one stereocenter, the compounds may exist as individual enantiomers and diastereomers or mixtures of such isomers (including racemates). The separation of the individual isomers or the selective synthesis of the individual isomers is accomplished by the application of a variety of methods well known to practitioners in the art. Where a compound disclosed herein is understood to exist in tautomeric forms, all tautomeric forms are included within the scope of the structure as depicted. Unless otherwise indicated, all such isomers and mixtures thereof are included within the scope of the compounds disclosed herein. Furthermore, the compounds disclosed herein may exist in one or more crystalline or amorphous forms. Unless otherwise indicated, all such forms are included within the scope of the compounds disclosed herein, including any polymorphic form. In addition, some of the compounds disclosed herein may form solvates with water (i.e., hydrates) or common organic solvents. Unless otherwise indicated, such solvates are included within the scope of the compounds disclosed herein.

As used herein, "nucleotide" includes nitrogen-containing heterocyclic bases, sugars, and one or more phosphate groups. They may be monomeric units (whether precursors or linked monomers) of the nucleic acid sequence. In RNA, the sugar is ribose, and in DNA the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present at the 2' position in ribose. The nitrogen-containing heterocyclic base may be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G) and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of the deoxyribose is bonded to the N-1 of the pyrimidine or the N-9 of the purine.

As used herein, the term "polynucleotide" or "oligonucleotide" refers to a nucleic acid that generally includes DNA (e.g., genomic DNA or cDNA), RNA (e.g., mRNA), synthetic oligonucleotides, and synthetic nucleic acid analogs, such as protected, locked, or bridged nucleic acids. Polynucleotides may include natural or non-natural bases or combinations thereof and natural or non-natural backbone linkages, such as phosphorothioate (phosphorothionate), PNA or 2' -O-methyl-RNA, or combinations thereof.

As used herein, the term "primer" is defined as a nucleic acid having a single strand with a free 3' OH group. The primer may also have a modification at the 5' end to allow a coupling reaction or to couple the primer to another moiety. The primer length can be any number of bases long and can include a variety of non-natural nucleotides. As used herein, "BCN primer" or "BCN modified primer" refers to a primer comprising a covalently attached bicyclo [6.1.0] non-4-yne at the 5' end. In some examples, the primer is appended with a terminal alkynyl group at the 5' terminus.

In some examples, the primers used on the substrate surface are P5 primer and P7 primer for sequencing as used in commercial flow cells sold by Illumina, inc. Primer sequences are described in U.S. patent publication No. 2011/0059865a1, which is incorporated herein by reference in its entirety. The P5 and P7 primer sequences may include the following:

paired end set (paired end set):

p5: paired terminal 5 '→ 3'

P7: paired terminal 5 '→ 3'

Single read set (single read set):

p5: single-reading: 5 '→ 3'

P7: read only 5 '→ 3'

Wherein G is 8-oxoguanine.

In some examples, the attached oligonucleotide (such as a primer or P5 primer or P7 primer) comprises a linker or spacer at the 5' end. Such a linker or spacer may be included in order to allow chemical or enzymatic cleavage or to confer some other desired property, e.g. being able to covalently attach to a polymer or solid support, or to act as a spacer to position the cleavage site at an optimal distance from the solid support. In certain instances, 10 spacer nucleotides may be positioned between the attachment points of the P5 primer or the P7 primer to the polymer or solid support. In some examples, poly-t (polyt) spacers are used, although other nucleotides and combinations thereof may also be used. In one example, the spacer is a 6T to 10T spacer. In some examples, the linker comprises a cleavable nucleotide comprising a chemically cleavable functional group, such as vicinal diol or allyl T.

As used herein, the term "silane" refers to an organic or inorganic compound containing one or more silicon atoms. A non-limiting example of an inorganic silane compound is SiH₄Or halogenated SiH₄Wherein hydrogen is replaced by one or more halogen atoms. Non-limiting examples of organosilane compounds are X-R^C-Si(OR^D)₃Wherein X is a non-hydrolysable organic group such as amino, vinyl, epoxy, methacrylate, sulphur, alkyl, alkenyl or alkynyl; r^CIs a spacer, e.g. - (CH)₂)_n-, where n is 0 to 1000; each R^DIndependently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted 5-10 membered heteroaryl, and optionally substituted 5-10 membered heterocyclyl, as defined herein. In some examples, the silane may be crosslinked such that X-R^C-Si(OR^D)₃OR of^DThe oxygen atoms of the radicals being attached to adjacent organosilane compounds X-R^C-Si(OR^D)₃Silicon atom(s) of (a). Further, the silane compound may be represented by X-R^C-Si(OR^D)₃Moieties are covalently bound to oxygen atoms on the surface to attach to the substrate surface. Thus, in some examples, the described silanes include the following structure:

as used herein, the term "silane" may include mixtures of different silane compounds. In some examples, X is a norbornenyl group. In some examples, X is a bicyclic nonynyl group. In some examples, X is an alkene or alkyne-containing group. In some examples, X is an alkene or alkyne. In some examples, R^CThe linking group being C_2-6An alkylene group.

As used herein, the term "substrate" refers to a material that serves as a support for the copolymers described herein. In the array method, the beads are coated with a polymer that is also functionalized to capture target oligonucleotides. Polymeric materials for sequencing have been described in U.S. patent publication nos. 2014/0079923 and 2016/0122816, both of which are incorporated by reference in their entirety. In examples described herein, the substrate material may include glass, silicon dioxide, plastic, quartz, metal oxide, organosilicate (e.g., polyhedral organic silsesquioxane (POSS)), polyacrylate, tantalum oxide, Complementary Metal Oxide Semiconductor (CMOS), or a combination thereof. An example of a POSS may be the POSS described in Kehagias et al, Microelectronic Engineering 86(2009), pp 776-778, which is incorporated by reference in its entirety. In some examples, substrates used in the present application include silica-based substrates, such as glass, fused silica, and other silica-containing materials. In some examples, the silicon dioxide based substrate may be silicon, silicon dioxide, silicon nitride, silicon hydride (silicon hydride). In some examples, substrates used in the present application include plastic materials or components such as polyethylene, polystyrene, poly (vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly (methyl methacrylate). Exemplary plastic materials include poly (methyl methacrylate) substrates, polystyrene substrates, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or a plastic material or a combination thereof. In particular examples, the substrate has at least one surface comprising a glass or silica-based polymer. In some examples, the substrate comprises glass. In some examples, the substrate may be, or may contain or include, a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface comprising a metal oxide. In one example, the surface comprises tantalum oxide or tin oxide. Acrylamide, ketene or acrylate may also be used as a base material or component. Other substrate materials may include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramics, polyimides, quartz, resins, polymers, and copolymers. In some examples, the substrate and/or substrate surface may be or include quartz. In some other examples, the substrate and/or substrate surface may be or include a semiconductor, such as GaAs or ITO. The foregoing list is intended to illustrate the present application but not to limit the present application. The substrate may comprise a single material or more than one different material. The substrate may be a composite or a laminate. In some examples, the substrate includes an organosilicate material.

The substrate may be flat, round, spherical, rod-like, or any other suitable shape. The substrate may be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

The substrate may be unpatterned, textured or patterned on one or more surfaces of the substrate. In some examples, the substrate is patterned. Such patterns may include pillars, pads, wells, ridges, channels, or other three-dimensional concave or convex structures. The pattern across the surface of the substrate may be regular or irregular. The pattern may be formed, for example, by nanoimprint lithography or by using, for example, metal pads that form features on a non-metallic surface.

In some examples, the surface of the substrate includes both copolymer-coated regions and inert regions. In some examples, the surface of the substrate may include both functionalized silane coated regions and inert regions. For examples using a patterned substrate, the copolymer or silane may be selectively attached to the pattern features (e.g., may be attached to a metal pad or the gel may be attached to the interior of the well), or alternatively the copolymer or silane may be uniformly attached across both the pattern features and the interstitial regions, and then optionally removed from the interstitial regions.

In some examples, a substrate described herein forms at least a portion of a flow cell or is located in a flow cell. In some such examples, the flow cell further comprises an oligonucleotide attached to the surface of the substrate via a copolymer coating. In such an example, the surface of the flow cell body to which the oligonucleotides are attached is considered to be the substrate. In other examples, a separate substrate (e.g., bead) having a copolymer-coated surface is inserted into the body of the flow cell. In a preferred example, the flow cell is a flow chamber divided into more than one lane (lane) or more than one segment, wherein one or more of the more than one lane or more than one segment comprises a surface coated with a copolymer coating described herein. Exemplary flow cells and substrates for use in making flow cells that can be used in the methods or compositions set forth herein include, but are not limited to, those commercially available from Illumina, Inc.

As used herein, the term "structure" refers to a compound, such as a copolymer, that is bound to a substrate. The copolymer can be covalently bound to the substrate, such as via an azido group, for example.

As used herein, the term "polymer" refers to a molecule that includes a plurality of repeating subunits (repeat subunits) or repeating units (recurring units). Non-limiting examples of polymer structures include linear polymers, branched polymers, or hyperbranched polymers. Non-limiting examples of linear polymers include block copolymers or random copolymers. Non-limiting examples of branched polymers include star polymers, star polymers or star block polymers comprising both hydrophobic and hydrophilic segments, H-polymers comprising both hydrophobic and hydrophilic segments, dumbbell polymers, comb polymers, brush polymers, dendrimers, ladders, and dendrimers. The polymer may be crosslinked or lightly crosslinked. The polymers as described herein may be linear, branched, hyperbranched or dendritic. The polymers described herein may also be in the form of polymer nanoparticles. Other examples of polymer architectures include, but are not limited to, ring block polymers (ring block polymers) and coil-type polymers (coil-cycle-coil polymers). Polymers having more than one type of repeat unit may be arranged as block copolymers, random copolymers, or alternating copolymers, or mixtures thereof. The final copolymer structure may be in different configurations including, for example, a random copolymer configuration, a block copolymer configuration, a comb polymer configuration, or a star polymer configuration. Different classes of polymer backbones include, but are not limited to, polyacrylamides, polyacrylates, polyurethanes, silicones, polyacrylaldehydes, polyphosphazenes, polyisocyanates, polyols, polysaccharides, and combinations thereof. In some examples, the polymer includes a polyacrylamide backbone. In some other examples, the polymer includes a polyacrylate backbone. In still other examples, the polymer includes a polyurethane backbone. In still other examples, the polymer includes a polyphosphazene backbone. In still other examples, the polymer comprises a dendrimer backbone.

As used herein, the prefix "light" or "photo-" is meant to refer to light or electromagnetic radiation. The term may encompass all or part of the electromagnetic spectrum including, but not limited to, one or more of the following ranges: the part of the spectrum usually referred to as radio part, the microwave part, the infrared part, the visible part, the ultraviolet part, the X-ray part or the gamma-ray part. This portion of the spectrum may be that portion blocked by metallic regions of the surface (such as those metals set forth herein). Alternatively or additionally, the portion of the spectrum may be a portion of a gap region through the surface (such as a region made of glass, plastic, silicon dioxide, or other materials set forth herein). In a particular example, radiation that is capable of passing through metal may be used. Alternatively or additionally, radiation masked by glass, plastic, silicon dioxide, or other materials set forth herein may be used.

As used herein, the term "YES process" refers to a chemical vapor deposition tool provided by the yield engineering system ("YES") developed by Illumina, inc. It includes three different vapor deposition systems. The automated YES-VertaCoat silane gas phase system is designed for volume production (volume production) flexible wafer handling modules that can accommodate 200mm or 300mm wafers. The manual loading YES-1224P silane gas phase system is designed for diversified mass production with its configurable large volume chamber. Yes-LabKote is a low cost desktop version (tabletop version) ideal for feasibility studies and R & D.

In an aspect, there is provided a compound according to formula (I):

wherein

R¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

l is a linker comprising a straight chain containing 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

e is a straight chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group; and is

Z is a nitrogen-containing heterocycle.

The compounds according to formula (I) can be used as polymerizable monomers. For example, the presence of an acryloyl group may provide a polymerizable moiety, optionally allowing more than one such compound (monomer) to polymerize with each other, and optionally with one or more other types of compounds (monomers). Additionally or alternatively, the azido groups present in the compounds of formula (I) optionally may undergo cycloaddition reactions with one or more suitable compounds, such as one or more suitable alkene or alkyne-containing compounds. Illustratively, it has been found that the presence of a nitrogen-containing heterocyclic group proximate to an azido group can result in an increase in the rate of copper-mediated cycloaddition reactions, for example, by a factor of 20-38, as compared to compounds that do not comprise a nitrogen-containing heterocyclic group proximate to an azido group. This means that less copper catalyst is involved in catalyzing the cycloaddition reaction. Without wishing to be bound by any theory, it is believed that the nitrogen within the nitrogen-containing heterocycle may coordinate (e.g., chelate) with the copper catalyst such that the copper center of the copper catalyst may be bound in a position sufficiently close to the azido group to more readily catalyze the cycloaddition reaction between the azido group and the alkene or alkyne-containing compound, thereby increasing the reaction rate and reducing the consumption of the copper catalyst. In some examples, the nitrogen within the nitrogen-containing heterocycle is located at a distance of about 5nm or less from the azido group, e.g., a distance of about 1 to 5nm, or a distance of about 2 to 2nm, or a distance of about 5 to 1nm, in order to facilitate coordination to the copper center of the copper catalyst. The monomeric compounds disclosed herein may optionally form polymers or copolymers, which may optionally be used to coat a substrate, thereby functionalizing the substrate. For example, the polymer or copolymer coated substrate may be reacted with an alkene or alkyne functionalized biomolecule.

E may be, for example, optionally substituted C_1-4Alkylene, each carbon optionally substituted by one or more groups selected from, for example, -C_1-4Alkyl, -OH, -OC_1-4Alkyl or a substituent of ═ O. E may be, for example, unsubstituted C_1-4Alkylene radicals, e.g. CH₂、(CH₂)₂、(CH₂)₃Or (CH)₂)₄。

Optionally, E may comprise an ether, an ester or an amide. For example, E may comprise-CH₂CH₂OCH₂-、-COCNHCH₂-or-CH₂COOCH₂-。

R¹May be, for example, H or a straight or branched chain alkyl group having between 1 and 4 carbon atoms. For example, R¹It may be H or methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl. Illustratively, R¹May be H or methyl.

R²Can be, for example, H or a linear or branched alkyl radical having between 1 and 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl. For example, R²May be H or methyl.

R³Can be, for example, H or a linear or branched alkyl group having between 1 and 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl. For example, R³May be H or methyl.

In some examples, L can be a linker comprising-C_2-20Alkylene-or a linear chain of 3 to 20 atoms of linear heteroalkylene, each optionally substituted by one or more groups selected from-C_1-4Alkyl, -OH, -OC_1-4Alkyl or a substituent of the group consisting of ═ O. L may be a linker having a structure of-C_2-20Straight chain alkylene-, optionally substituted by one or more-C_1-4Alkyl, -OH, -OC_1-4Alkyl or ═ O substituents. L may be unsubstituted-C_2-6Alkylene- (also drawn as- (CH)₂)_2-6-, for example L may be unsubstituted-C_3-4Alkylene-, e.g. - (CH)₂)₃-or- (CH)₂)₄-. L may be a linker comprising a linear chain of linear heteroalkylene groups of 3 to 20 atoms, optionally substituted with one or more groups selected from-C_1-4Alkyl, -OH, -OC_1-4Alkyl or a substituent of the group consisting of ═ O. Illustratively, L may comprise one or more ethylene glycol units. L may be-CH₂CH₂(OCH₂CH₂)_x-OCH₂CH₂-, where x is 0 to 10. In one non-limiting example, x is 1,2, 3, 4, 5, or 6. L may comprise one or more amide groups. For example, L may be-C_2-6alkyl-NHC (O) -C_2-6Alkyl-, or L may be- (CH)₂)₂-NHC(O)-(CH₂)₂-or- (CH)₂)₃-NHC(O)-(CH₂)₂-. L may comprise one or more natural or unnatural amino acids, e.g., L may comprise one or more natural amino acids, e.g., L may comprise one or more amino acids selected from the group consisting of: glycine, alanineAcid, valine, isoleucine, leucine, lysine, serine, threonine, cysteine, asparagine, or glutamine. In some examples, L may comprise 1,2 or 3 amino acid units.

The N-substituted amide a can be bound to L and Z in two possible configurations, e.g., the carbonyl carbon of a can be bound to L, and the amide nitrogen of a can be bound to Z. Alternatively, the carbonyl carbon of a may be bound to Z, and the amide nitrogen of a may be bound to L. Examples of these two configurations are set forth below in formulas (Ic) and (Id).

Z may include a nitrogen-containing heterocyclic ring having from 5 to 10 ring members, such as a 5 to 10 membered heterocyclic ring, wherein the ring members are atoms that form the backbone of the heterocyclic ring. Z may comprise a single ring structure or a fused structure comprising two or more ring systems. In the case of a monocyclic structure, Z may comprise 5 or 6 ring members, for example Z may be a 5 or 6 membered heterocyclic ring. In the case of fused structures, Z may comprise 9 or 10 ring members. The nitrogen-containing heterocycle may contain more than one heteroatom, such as one or more additional nitrogen heteroatoms, or one or more oxygen heteroatoms, or one or more sulfur heteroatoms, or any suitable combination of such heteroatoms. The nitrogen-containing heterocycle may be aromatic, such as pyridyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, indolyl, quinolinyl, quinazolinyl. The nitrogen-containing heterocycle may be aliphatic, such as cycloalkyl. The aliphatic nitrogen-containing heterocycle may be saturated or may contain one or more double bonds, but is not aromatic. In one example, the aliphatic nitrogen-containing heterocycle may be pyrrolidinyl.

Where Z is a 6 membered heterocyclic ring, the compound of formula (I) may optionally be configured such that a is bound at the 2 position of the 6 membered ring (N constituting the 1 position) and the alkylazido group is bound at the 5 position of the 6 membered ring. Such a configuration may be considered to provide a1, 4 substitution pattern relative to a 6-membered heterocyclic ring.

In some options, the compound of formula (I) may have (Ia):

wherein R is¹、R²L, A and Z are as defined above for formula (I), and p is 1,2, 3 or 4.

In some options, the compound of formula (Ia) may have (Ib)

Wherein R is¹、R²L, A and p are as defined above for formula (I) and formula (Ia), and X is CH or N.

The compound of formula (Ia) may optionally be a compound of formula (Ic)

Wherein R is¹、R²L, A and p are as defined above for formula (I) and formula (Ia).

As mentioned above, the compound of formula (Ia) may optionally be a compound of formula (Id) or formula (Ie)

Wherein R is¹、R²L, A and p are as defined above for formula (I) and formula (Ia). That is, a may have any suitable configuration.

In some examples, the compounds of formula (Ib) and formula (Id) may be compounds of formula (Ik)

Wherein R is¹、R²、R³X and p are as defined above for formula (I), formula (Ia) and formula (Ib).

The compound of formula (Ik) optionally may be a compound of formula (Im)

Wherein R is¹、R²、R³X and p are as defined above for formula (I), formula (Ia) and formula (Ib), and n is an integer from 2 to 20. For example, n may be an integer between 1 and 10, such as between 1 and 6, for example 2, 3, 4, 5 or 6. For example, n may be 3 or 4.

In a specific and non-limiting example, the compounds of formula (I), formula (Ia), formula (Ib) and formula (Ie) may be compounds having formula (If)

In another specific and non-limiting example, the compounds of formula (I), formula (Ia), formula (Ic) and formula (Ie) may be compounds having formula (Ig)

In still other non-limiting examples, the compounds of formula (I), formula (Ia), formula (Ib), and formula (Id) may be compounds having formula (Ih) or formula (Ij):

in some aspects, a copolymer is provided that is formed by reacting a compound as defined by formula (I), formula (Ia), formula (Ib), formula (Ic), formula (Id), formula (Ie), formula (If), formula (Ig), formula (Ih), formula (Ij), formula (Ik), or formula (Im) with at least one other repeating monomer unit.

In some aspects, a copolymer is provided comprising repeating monomer units of formula (II)

Wherein R is¹Is H or C_1-4An alkyl group;

R²is H or C_1-4An alkyl group;

l is a linker comprising a straight chain containing 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

e is a straight chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain;

a is of the formulaN-substituted amides of (a), wherein R³Is H or C_1-4An alkyl group; and is

Z is a nitrogen-containing heterocycle; and

at least one other repeating monomer unit.

It will be understood that R¹、R²L, A, Z and E can be defined as set forth above with respect to formula (I).

It will be appreciated that the repeating monomeric units of formula (II) may be derived from compounds of formula (I). The carbon-carbon double bond in the acryloyl group of the compound of formula (I) may be polymerized, thereby producing a copolymer comprising repeating monomer units of formula (I). It will be appreciated that the repeating monomeric unit of formula (II) may be derived from any of the compounds of formula (I), formula (Ia), formula (Ib), formula (Ic), formula (Id), formula (Ie), formula (If), formula (Ig), formula (Ih), formula (Ij), formula (Ik) or formula (Im).

Optionally, the at least one other repeating monomer unit may be selected from the group consisting of: polyacrylamide, polyacrylate, polyurethane, polysiloxane, silicone, polyacrolein, polyphosphazene, polyisocyanate, polyol, and polysaccharide, and any combination thereof.

The copolymer may, for example, comprise repeating monomer units of formula (IIa):

wherein R is¹、R²L, A, Z and p are as defined above for formula (I) and formula (Ia).

The copolymer may, for example, comprise repeating monomer units of formula (IIb), formula (IIc), formula (IId), or formula (IIe):

wherein R is¹、R²And X is as defined above for formula (I), formula (Ia) and formula (Ib), and n in formula (IIc) is an integer from 2 to 20.

At least one other repeating monomer unit optionally may be a compound of formula (III)

Wherein

R⁴Is H or C_1-4An alkyl group;

R⁵is H or C_1-4An alkyl group; and is

R⁶Is H or C_1-4An alkyl group.

R⁴May be, for example, H or a straight or branched chain alkyl group having between 1 and 4 carbon atoms. For example, R⁴It may be H or methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl. Illustratively, R⁴May be H or methyl.

R⁵Can be, for example, H or a linear or branched alkyl radical having between 1 and 4 carbon atoms, e.g. methyl, ethylPropyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl. For example, R⁵May be H or methyl.

R⁶Can be, for example, H or a linear or branched alkyl group having between 1 and 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl. For example, R⁶May be H or methyl.

The copolymer can comprise a repeating monomer unit of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe), a repeating monomer unit of formula (III), and at least one other repeating monomer unit.

In another aspect, there is provided a substrate-copolymer product formed from the reaction of a copolymer as set forth above with reference to formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) with a substrate, e.g., a substrate as described herein. The substrate copolymer product may be formed by reaction of an azido group of the copolymer with an alkenyl or alkynyl containing group on the surface of the substrate.

Reacting a substrate with a copolymer as set forth above can result in a functionalized substrate in which the pendant azido groups are configured to react with alkenyl or alkynyl groups found in a target molecule (e.g., a biomolecule or functionalized biomolecule). The functionalized substrates can be used for applications such as sequencing or molecular analysis.

In another aspect, a structure is provided that includes a copolymer covalently bonded to a substrate, wherein the substrate-bonded copolymer comprises repeating monomer units covalently bonded to the substrate, wherein the repeating, covalently bonded monomer units are compounds of formula (IV):

wherein R is¹、R²L, A, Z, E are as defined above in relation to formula (I),is a single or double bond, and the triazole or triazoline is covalently bound to the substrate through one or both triazole/triazoline ring carbon atoms, either directly or indirectly through a linker. It will be appreciated that the repeating covalently bound monomeric units of formula (IV) may be derived from a compound of formula (I). It will be appreciated that the repeating monomeric unit of formula (IV) may be derived from any of the compounds of formula (I), formula (Ia), formula (Ib), formula (Ic), formula (Id), formula (Ie), formula (If), formula (Ig), formula (Ih), formula (Ij), formula (Ik) or formula (Im).

The recurring, covalently bound monomer unit can be a compound of formula (IVa),

wherein R is¹、R²L, A, Z and p are as defined above for formula (I) and formula (Ia),is a single or double bond, and the triazole or triazoline is covalently bound to the substrate through one or both triazole/triazoline ring carbon atoms, either directly or indirectly through a linker.

The repeating, covalently bound monomer unit may be a compound of formula (IVb), formula (IVc), formula (IVd) or formula (IVe):

wherein R is¹、R²、R³X and p are as defined above for formula (I), formula (Ia) and formula (Ib),is a single or double bond, and the triazole or triazoline is covalently bound to the substrate through one or both triazole/triazoline ring carbon atoms, either directly or indirectly through a linker; and n in formula (IVc) is an integer from 2 to 20. For example, n may be an integer between 1 and 10, such as between 1 and 6, for example 2, 3, 4, 5 or 6, for example n may be 3 or 4.

The repeating, covalently bound monomer unit may be a compound of formula (V)

Wherein R is¹、R²L, A, Z and E are as defined above for formula (I), and wherein G is a linking group between the triazole/triazoline ring and the substrate.

G may be, for example, a silane linker. For example, G may be-X' -R^C-Si(O-)₃Wherein X' is the product of the reaction of the group X of the silane linker precursor with the azido group of the copolymer. X may be alkenyl, alkynyl, norbornenyl or bicyclononyl. X' may be a single bond, a double bond,wherein the dotted 5-membered ring is a triazoline unit, orWherein the dashed 5-membered ring is a triazoline unit. In some examples, G is-cycloalkyl-CH₂CH₂-Si(O-)₃Wherein the cycloalkyl group is fused to the triazoline unit. In some examples, G is-cycloalkenyl-CH₂CH₂-Si(O-)₃Wherein the cycloalkenyl is fused to the triazole unit and shares a double bond with the triazole unit.

The repeating, covalently bound monomer unit may be a compound of formula (Va)

Wherein R is¹、R²L, A, Z and p are as defined above for formula (I) and formula (Ia), and G is as defined above for formula (V).

The repeating, covalently bound monomer unit may be a compound of formula (Vb), formula (Vc), formula (Vd) or formula (Ve):

wherein R is¹、R²、R³X and p are as defined above for formula (I), formula (Ia) and formula (Ib); g is as defined above for formula (V); and n in formula (Vc) is an integer from 2 to 20. For example, n may be an integer between 1 and 10, such as between 1 and 6, for example 2, 3, 4, 5 or 6, for example n may be 3 or 4.

In an aspect, a copolymer-oligonucleotide product is provided that is formed from a reaction between a copolymer as defined by formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and one or more oligonucleotides. The copolymer oligonucleotide product can be formed from the reaction of the azido group of the copolymer and an alkene or alkyne functionalized oligonucleotide.

Illustratively, the oligonucleotide may be or include DNA, RNA, genomic DNA, a template DNA fragment, or a primer such as an amplification primer. The amplification primers may be P5 or P7 sequences for sequencing-by-synthesis systems.

In another aspect, an oligonucleotide-conjugated copolymer is provided, the oligonucleotide-conjugated copolymer comprising a repeating monomer unit of formula (IV):

wherein R is¹、R²、R³L, A, Z and E are as defined above for formula (I);

is a single or double bond; and is

The copolymer is covalently bound to the oligonucleotide through one or both triazole/triazoline ring carbon atoms, either directly or indirectly through a linker.

It will be appreciated that the repeating monomeric units of formula (IV) may be derived from a compound of formula (I). It will be appreciated that the repeating monomeric unit of formula (IV) may be derived from any of the compounds of formula (I), formula (Ia), formula (Ib), formula (Ic), formula (Id), formula (Ie), formula (If), formula (Ig), formula (Ih), formula (Ij), formula (Ik) or formula (Im).

For example, the oligonucleotide-binding repeat monomer unit can have formula (IVa):

wherein R is¹、R²、R³L, A, Z and p are as defined above for formula (I) and formula (Ia);

is a single or double bond; and is

The copolymer is covalently bound to the oligonucleotide through one or both triazole/triazoline ring carbon atoms, either directly or indirectly through a linker.

For example, the oligonucleotide-binding repeat monomer unit can have formula (IVb), formula (IVc), formula (IVd), or formula (IVe):

wherein R is¹、R²P and X are as defined above for formula (I), formula (Ia) and formula (Ib), and n in formula (IVc) is an integer from 2 to 20. For example, n may be an integer between 1 and 10, such as between 1 and 6, for example 2, 3, 4, 5 or 6, for example n may be 3 or 4.

In some examples, the oligonucleotide-bound repeat monomer units can have formula (VI):

wherein R is¹、R²L, A, Z and E are as defined above for formula (I);is a single or double bond; and G is as defined above for formula (V).

The oligonucleotide-binding repeat monomer units may have the formula (VIa):

wherein R is¹、R²L, A, Z and p are as defined above for formula (I) and formula (Ia), and G is as defined above for formula (V).

In some examples, the oligonucleotide-binding repeat monomer units may optionally have formula (VIb) or formula (VIc), formula (VId), or formula (VIe):

wherein R is¹、R²、R³X and p are as defined above for formula (I), formula (Ia) and formula (Ib); g is as defined above for formula (V); and n in formula (VIc) is an integer from 2 to 20. For example, n may be an integer between 1 and 10, such as between 1 and 6, for example 2, 3, 4, 5 or 6, for example n may be 3 or 4.

In another aspect, a copolymer-substrate-oligonucleotide product is provided, which is formed from the reaction of a copolymer as set forth above with reference to formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) with a substrate and one or more oligonucleotides. The one or more oligonucleotides may comprise DNA, RNA, genomic DNA, a template DNA fragment, or a primer such as an amplification primer. The amplification primers may be P5 or P7 sequences for sequencing-by-synthesis systems.

In another aspect, there is provided a method of immobilizing a copolymer as set forth above with reference to formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) to a substrate, the method comprising reacting the azido groups of the copolymer with more than one alkenyl-or alkynyl-containing functional group on the surface of the substrate to form covalent bonds between the copolymer and the surface of the substrate. The alkene-or alkyne-containing functional group can be, for example, alkenyl, alkynyl, cycloalkynyl, or cycloalkenyl. In some specific non-limiting examples, the alkene or alkyne-containing group is a norbornenyl group. In some specific non-limiting examples, the alkene or alkyne-containing group is a silane. In some specific non-limiting examples, the alkene or alkyne-containing group is a silane comprising a norbornene group. The method may further comprise applying the copolymer to a surface of a substrate prior to fixing. For example, the copolymer may be applied to the surface by applying a copolymer precursor solution to the surface of the substrate and curing the precursor solution to form the copolymer. The copolymer precursor solution or copolymer can be applied to the substrate using any known surface application technique known to those skilled in the art, such as flow-through, spin coating, spray coating, dip coating, or ink jet coating. After the copolymer is applied to the substrate, excess copolymer may be removed by a polishing step (e.g., from interstitial regions of the patterned substrate, leaving the copolymer at patterned regions, such as wells).

In some examples, the method of immobilizing a copolymer further comprises functionalizing the substrate with an alkenyl or alkynyl containing group prior to immobilizing the copolymer on the substrate. In some examples, the surface of the substrate is pretreated with an alkene-or alkyne-containing silane. For example, the functionalized silane can be deposited onto the surface by a Chemical Vapor Deposition (CVD) process. In some examples, the functionalized silane can be applied to the first surface by a CVD process using a Yield Engineering System (YES) oven.

In an aspect, there is provided a method of grafting more than one oligonucleotide to a copolymer as defined above by formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId) or formula (IIe), the method comprising reacting the azido groups of the copolymer with more than one oligonucleotide. More than one oligonucleotide comprises an alkenyl group or an alkynyl group, and the reaction occurs between the azido group of the copolymer and the alkenyl group or alkynyl group of the oligonucleotide. The alkene-or alkyne-containing group can be an alkenyl, alkynyl, cycloalkenyl, or cycloalkynyl group, or substituted variants thereof. For example, alkene-or alkyne-containing groups include cycloalkynyl groups, such as bicyclo [6.1.0] non-4-yne (BCN). The alkene or alkyne-containing group can include an alkynyl group.

The oligonucleotide may be grafted to the copolymer prior to fixing the copolymer to the substrate. For example, the oligonucleotides may be grafted to the copolymer after the copolymer is immobilized to the substrate. The alkene or alkyne-containing groups on the substrate and the oligonucleotide may be the same or different. The method may further comprise a washing step to remove unreacted (ungrafted) oligonucleotides. The method may further comprise a drying step.

In another aspect, there is provided a process for preparing a compound of formula (I):

the process comprises reacting a compound of formula (X) with an acrylate of formula (XI):

to form a compound of formula (I); wherein R is¹、R²L, A, Z and E are as set forth above for formula (I); and Hal is Cl or F.

Also provided is a process for preparing a compound of formula (Ik):

the method comprises reacting a compound of formula (Xa):

with an acrylate of formula (XI):

wherein R is¹、R²、R³L, X and p are as set forth above for formula (I), formula (Ia) and formula (Ib); and Hal is Cl or F to form a compound of formula (Ij).

The method may further comprise reacting a compound of formula (XII):

with a compound of formula (XIII):

thereby forming a compound of formula (Xa); wherein R is³X and p are as set forth above for formula (I) and formula (Ia); pg is H or an amino protecting group; and Y is-OH or-Cl.

Also provided is a method of preparing a compound of formula (Ik), the method comprising reacting a compound of formula (XIV):

with a compound of formula (XV):

to form a compound of formula (Ik)

Wherein R is¹、R²、R³L, X and p are as set forth above for formula (I), formula (Ia) and formula (Ik); y is-OH or-Cl.

The method of preparing compound of formula (XIV) may further comprise reacting a compound of formula (XII) with a cyclic anhydride, such as succinic anhydride or glutaric anhydride, to form a compound of formula (XIV).

Also provided is a method of forming a compound according to formula (Ie)

Wherein the method comprises reacting a compound of formula (Xb):

with an acrylate of formula (XI):

wherein R is¹、R²、R³L, Z and p are as defined above for formula (I) and formula (Ia); and Hal is Cl or F;

to form the compound of formula (Ie).

The compound according to formula (Xb) may be formed by a method comprising reacting a compound of formula (XVI)

With a compound of the formula (XVII)

To form a compound of (Xb)

Wherein R is¹、R²、R³L, Z and p are as defined above for formula (I) and formula (Ia); pg is H or an amino protecting group.

Oligonucleotide-conjugated copolymers as described herein can be used in a variety of amplification techniques. Exemplary techniques that may be used include, but are not limited to, Polymerase Chain Reaction (PCR), Rolling Circle Amplification (RCA), Multiple Displacement Amplification (MDA), or Random Primer Amplification (RPA), or combinations thereof. In some examples, one or more primers for amplification are attached to a copolymer coating on a substrate. Bridge amplification can be performed using a format of two attached primers, because the double-stranded amplicon forms a bridge-like structure between the two attached primers flanking the template sequence that has been copied. Amplification in which one amplification primer is attached to the copolymer coating and the second primer is in solution (e.g., emulsion PCR), or amplification of both primers in solution, can also be performed.

The copolymer-coated substrates described herein can be used in methods for determining the nucleotide sequence of a target nucleic acid. For example, the method comprises:

hybridizing a target nucleic acid to one of more than one primer covalently bound to a substrate VIa a copolymer as described herein (or to an oligonucleotide of repeating monomer units of formula (VI), formula (VIa), formula (VIb), formula (VIc), formula (VId), or formula (VIe));

amplifying the hybridized target nucleic acids using more than one primer to form clusters of substrate-bound amplicons;

treating the cluster of substrate-bound amplicons with labeled nucleotides and a polymerase such that a detectable signal is generated when the nucleotides are incorporated by the polymerase; and detecting the signal, thereby determining the nucleotide sequence of the target nucleic acid.

For one or more of the polynucleotides attached to the polymer coating on the surface of the substrate present in the flow cell, the determination of the one or more nucleotides incorporated during each flow step is achieved by detecting the signal generated at or near the template. In some examples, the detectable signal comprises an optical signal. In other examples, the detectable signal comprises a non-optical signal. In such instances, the non-optical signal includes a change in pH or current at or near the one or more nucleic acids being sequenced.

Suitable sequencing methods include, but are not limited to, sequencing-by-synthesis, pyrosequencing, ligation sequencing, and other methods known in the art.

In a sequencing-by-synthesis method, one or more nucleotides are provided to a template polynucleotide associated with a polynucleotide polymerase. The polynucleotide polymerase incorporates one or more nucleotides into a newly synthesized nucleic acid strand that is complementary to the polynucleotide template. Synthesis begins with an oligonucleotide primer that is complementary to a portion of the template polynucleotide or to a portion of a universal nucleic acid or a non-variable nucleic acid that is covalently bound at one end of the template polynucleotide. As nucleotides are incorporated against the template polynucleotide, detectable signals are generated that allow determination of which nucleotide has been incorporated during each step of the sequencing method. In this manner, a nucleic acid sequence complementary to at least a portion of the template polynucleotide can be generated, thereby allowing the nucleotide sequence of at least a portion of the template polynucleotide to be determined.

Other useful techniques that can use the copolymers and substrates of the invention include real-time monitoring of DNA polymerase activity, SBS techniques that detect proton release upon nucleotide incorporation (e.g., Ion Torrent, Thermo Fisher), and gene expression analysis. For example, nucleotide incorporation can be detected by Fluorescence Resonance Energy Transfer (FRET) interaction between a fluorophore-bearing polymerase and a gamma-phosphate labeled nucleotide, or with Zero Mode Waveguiding (ZMW). Gene expression can be detected or quantified using RNA sequencing techniques, such as those known as digital RNA sequencing. RNA sequencing techniques can be performed using sequencing methods known in the art, such as those set forth above. Gene expression can also be detected or quantified using hybridization techniques by direct hybridization of the array or multiplex assays where the products of the assay are detected on the array.

In some examples of compounds of formula (I) -formula (Ik), repeating monomer units of formula (II) -formula (IIe), repeating monomer units of formula (IV) -formula (IVd), repeating monomer units of formula (V) -formula (Vd), and repeating monomer units of formula (VI) -formula (VId), R¹Is H. In some examples, R¹Is H or methyl. In some examples, R¹Is C_1-4An alkyl group. In some examples, R¹Is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some examples, R²Is H. In some examples, R²Is H or methyl. In some examples, R²Is C_1-4An alkyl group. In some examples, R²Is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some examples, R³Is H. In some examples, R³Is H or methyl. In some examples, R³Is C_1-4An alkyl group. In some examples, R³Is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some examples, X is CH. In some examples, X is N.

In some examples, p is 1. In some examples, p is 1 or 2.

In some examples, L is a linker having a structure of-C_2-20Alkylene-or a linear chain of 3 to 20 atoms of linear heteroalkylene, each optionally substituted by one or more groups selected from-C_1-4Alkyl, -OH, -OC_1-4Alkyl or a substituent of the group consisting of ═ O. In some examples, L is a linker having a structure of-C_2-20Straight chain alkylene-, optionally substituted by one or more-C_1-4Alkyl, -OH, -OC_1-4Alkyl or ═ OSubstituent groups. In some examples, L is unsubstituted-C_2-6Alkylene- (also drawn as- (CH)₂)_2-6-). In some examples, L is unsubstituted-C_3-4Alkylene-. In some examples, L is- (CH)₂)₃-. In some examples, L is- (CH)₂)₄-。

In some examples, L is a linker having a linear chain of linear heteroalkylene groups of 3 to 20 atoms, optionally substituted with one or more groups selected from-C_1-4Alkyl, -OH, -OC_1-4Alkyl or a substituent of the group consisting of ═ O. In some examples, L comprises one or more ethylene glycol units. In some examples, L is-CH₂CH₂(OCH₂CH₂)_x-OCH₂CH₂-, where x is 0 to 10. In some examples, x is 1,2, 3, 4, 5, or 6. In some examples, L comprises one or more amide groups. In some examples, L is-C_2-6alkyl-NHC (O) -C_2-6An alkyl group-. In some examples, L is- (CH)₂)₂-NHC(O)-(CH₂)₂-or- (CH)₂)₃-NHC(O)-(CH₂)₂-. In some examples, L comprises one or more natural or unnatural amino acids. In some examples, L comprises one or more natural amino acids. In some examples, L comprises one or more amino acids selected from the group consisting of: glycine, alanine, valine, isoleucine, leucine, lysine, serine, threonine, cysteine, asparagine, or glutamine. In some examples, L comprises 1,2, or 3 amino acid units.

In some examples, the compound of formula (Ik) is a compound of formula (Im). In some examples, the recurring monomeric unit of formula (IIb) is a recurring monomeric unit of formula (IIc). In some examples, the recurring monomeric unit of formula (IVb) is a recurring monomeric unit of formula (IVc). In some examples, the recurring monomeric unit of formula (Vb) is a recurring monomeric unit of formula (Vc). In some examples, the recurring monomeric unit of formula (VIb) is a recurring monomeric unit of formula (VIc). At each oneIn some examples of the independent formula, R¹Is H. In some examples, R¹Is H or methyl. In some examples, R¹Is C_1-4An alkyl group. In some examples, R¹Is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some examples, R²Is H. In some examples, R²Is H or methyl. In some examples, R²Is C_1-4An alkyl group. In some examples, R²Is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some examples, R³Is H. In some examples, R³Is H or methyl. In some examples, R³Is C_1-4An alkyl group. In some examples, R³Is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some examples, X is CH. In some examples, X is N.

In some examples, n is 1-10, 1-6, or 2, 3, 4, 5, or 6. In some examples, n is 3 or 4.

In some examples, p is 1. In some examples, p is 1 or 2.

In some examples, of formula (IV), formula (IVa), formula (IVb), formula (IVc), formula (IVd) or formula (IVe)Is a single bond. In some examples, it is a double bond. In some examples, the triazole of formula (IV), formula (IVa), formula (IVb), formula (IVc), formula (IVd) or formula (IVe) is covalently bound to the surface through one or two triazole/triazoline ring carbon atoms (either directly or indirectly through a linker).

In some examples of formula (V), formula (Va), formula (Vb), formula (Vc), formula (Vd) or formula (Ve) or formula (VI), formula (VIa), formula (VIb), formula (VIc), formula (VId) or formula (VIe), G is a linking group between the triazole/triazoline ring and the substrate. In some examples, G is a silane linker. In some examples, G is-X' -R^C-Si(O-)₃Wherein X' is the product of the reaction of the group X of the silane linker precursor with the azido group of the copolymer. In some examples, X is alkenyl, alkynyl, norbornenyl, or bicyclononyl. Thus, in some examples, X' is a single bond, a double bond,wherein the dotted 5-membered ring is a triazoline unit, orWherein the dashed 5-membered ring is a triazoline unit. In some examples, G is-cycloalkyl-CH₂CH₂-Si(O-)₃Wherein the cycloalkyl group is fused to the triazoline unit. In some examples, G is-cycloalkenyl-CH₂CH₂-Si(O-)₃Wherein the cycloalkenyl is fused to the triazole unit and shares a double bond with the triazole unit.

In some examples are copolymers comprising repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and at least one other repeating monomer unit. At least one other repeating monomer unit is selected from the group consisting of: polyacrylamides, polyacrylates, polyurethanes, polysiloxanes, silicones, polyacroleins, polyphosphazenes, polyisocyanates, polyols and polysaccharides and combinations thereof. In some examples, the copolymer comprises repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and repeating monomer units of formula (III). In some aspects, the copolymer comprises a repeating monomer unit of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe), a repeating monomer unit of formula (III), and at least one other repeating monomer unit, as described above.

In some examples of the repeating monomer unit of formula (III), R⁴Is H. In some examples, R⁴Is C_1-4An alkyl group. In some aspects, R⁴Is H or methyl. In some examples, R⁴Is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some examples, R⁵Is H. In some examples, R⁵Is C_1-4An alkyl group. In some aspects, R⁵Is H or methyl. In some examples, R⁵Is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some examples, R⁶Is H. In some examples, R⁶Is C_1-4An alkyl group. In some aspects, R⁶Is H or methyl. In some examples, R⁶Is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

Some examples provided herein relate to a substrate having a copolymer as described herein covalently bonded to the substrate, wherein the substrate-bonded copolymer comprises repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe). Thus, in some examples, the substrate-bound copolymer comprises a repeating monomer unit of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and a repeating monomer unit covalently bound (directly or indirectly via a linker) to the substrate. In some examples, the substrate-bound copolymer comprises repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and repeating monomer units of formula (IVb), formula (IVc), formula (IVd), or formula (IVe):

wherein R is¹、R²、R³X, L, p and n are as defined herein,is a single or double bond, and the triazole or triazoline is co-joined (directly or indirectly through a linker) to one (e.g., where the substrate-bound alkenyl or alkynyl group is acyclic) or two (e.g., where the substrate-bound alkenyl or alkynyl group is part of a ring) triazole/triazoline ring carbon atomsIs covalently bonded to the substrate.

In some examples, the substrate-bound copolymer comprises a repeating monomer unit of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and a repeating monomer unit of formula (Vb), formula (Vc), formula (Vd), or formula (Ve):

wherein R is¹、R²、R³X, n, p and G are as defined herein.

Described herein is a copolymer comprising repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe), wherein the copolymer is covalently bound to an oligonucleotide. Thus, in some examples, the oligonucleotide-conjugated copolymer comprises a repeating monomer unit of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and a repeating monomer unit covalently bound to the oligonucleotide. In some examples, the oligonucleotide-conjugated copolymer comprises a repeating monomer unit of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and a repeating monomer unit of formula (IV), formula (IVa), formula (IVb), formula (IVc), formula (IVd), or formula (IVe), wherein R is¹、R²、R³L, p and n are as defined herein,is a single or double bond, and the copolymer is covalently bound to the oligonucleotide through one or both triazole/triazoline ring carbon atoms (either directly or indirectly through a linker). In some examples, the oligonucleotide-conjugated copolymer comprises repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) and repeating monomer units of formula (VIb), formula (VIc), formula (VId), or formula (VIe):

wherein R is¹、R²、R³X, n, p and G are each as defined herein.

In some examples, the attached oligonucleotide is DNA, RNA, genomic DNA, a template DNA fragment, or a primer such as an amplification primer. In some examples, the oligonucleotide is a primer. In some examples, the primers are P5 and P7 sequences for a sequencing-by-synthesis system.

In some examples, the copolymers described herein are covalently bound to both the substrate and the oligonucleotide. Thus, in some examples, the copolymer comprises repeating monomer units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe), repeating monomer units of formula (V), formula (Va), formula (Vb), formula (Vc), formula (Vd), or formula (Ve), and repeating monomer units of formula (VI), formula (VIa), formula (VIb), formula (VIc), formula (VId), or formula (VIe).

Some examples provided herein relate to methods of preparing compounds of formula (Ik), methods of preparing copolymers comprising recurring monomeric units of formula (IIb), and methods of preparing copolymers comprising recurring monomeric units of formula (IIb) and recurring monomeric units of formula (III).

Described herein is a process for preparing a compound of formula (Ik):

the method comprises reacting a compound of formula (Xa):

with an acrylate of formula (XI):

wherein R is¹、R²、R³L, p and X are as defined herein and Hal is C1 or F.

In some examples, the method comprises reacting a compound of formula (XII):

wherein p and R³And X is as defined herein;

with a compound of formula (XIII):

wherein

Pg is H or an amino protecting group; and is

Y is-OH or-Cl;

to form a compound of formula (Xa).

In other aspects is a process for preparing a compound of formula (I), the process comprising reacting a compound of formula (XIV):

wherein Y is-OH or-Cl;

with a compound of formula (XV):

to form a compound of formula (Ik).

Such methods may further comprise reacting a compound of formula (XII) with a cyclic anhydride, such as succinic anhydride or glutaric anhydride, to form a compound of formula (XIV).

Some examples provided herein relate to a method of immobilizing a copolymer (or oligonucleotide-conjugated copolymer) as described herein on a substrate, the method comprising reacting an azido group of the copolymer comprising recurring monomeric units of formula (II), formula (IIa), formula (IIb), formula (IIc), formula (IId), or formula (IIe) with an alkenyl-or alkynyl-containing group on the substrate to form a covalent bond between the copolymer and the substrate. In some examples, the alkene or alkyne-containing group is an alkenyl, alkynyl or cycloalkynyl, cycloalkenyl group. In some examples, the alkene or alkyne-containing group is a norbornenyl group. In some examples, the alkene or alkyne-containing group is a silane. In some examples, the alkene or alkyne-containing group is a silane comprising a norbornene group. In some examples, the method further comprises applying the copolymer to a surface of a substrate prior to fixing. In other examples, the copolymer is applied to the surface by applying a copolymer precursor solution to the surface of the substrate and curing the precursor solution to form the copolymer. The copolymer precursor solution or copolymer can be applied to the substrate using any known surface application technique known to those skilled in the art, such as flow-through, spin coating, spray coating, dip coating, or ink jet coating. In some examples, after the copolymer is applied to the substrate, excess copolymer is removed by a polishing step (e.g., from interstitial regions of the patterned substrate, leaving the copolymer at patterned regions, such as wells).

In some examples, the method of immobilizing a copolymer further comprises functionalizing the substrate with an alkenyl or alkynyl containing group prior to immobilizing the copolymer on the substrate. In some examples, the surface of the substrate is pretreated with an alkene-or alkyne-containing silane. In some examples, the functionalized silane is deposited onto the surface by a Chemical Vapor Deposition (CVD) process. In some such examples, the functionalized silane can be applied to the first surface by a CVD process using a Yield Engineering System (YES) oven.

A method of grafting an oligonucleotide to a copolymer as described herein includes reacting an azido group of the copolymer with an alkenyl-or alkynyl-containing group on the oligonucleotide to form an oligonucleotide-bound copolymer. In some examples, the alkene or alkyne-containing group is an alkenyl, alkynyl, cycloalkenyl, or cycloalkynyl group or substituted variants thereof. In some examples, the alkene-or alkyne-containing group includes a cycloalkynyl group, such as bicyclo [6.1.0] non-4-yne (BCN). In some other examples, the alkene or alkyne-containing group comprises an alkynyl group. In some examples, the oligonucleotide is grafted to the copolymer prior to immobilizing the copolymer to the substrate. In some examples, the oligonucleotides are grafted to the copolymer after the copolymer is immobilized to the substrate. The alkene or alkyne-containing groups on the substrate and the oligonucleotide may be the same or different. In some examples, the method further comprises a washing step to remove unreacted (ungrafted) oligonucleotides. In some examples, the method further comprises a drying step.

Sequencing applications

Oligonucleotide arrays as described herein can be used in a variety of amplification techniques. Exemplary techniques that may be used include, but are not limited to, Polymerase Chain Reaction (PCR), Rolling Circle Amplification (RCA), Multiple Displacement Amplification (MDA), or Random Primer Amplification (RPA), or combinations thereof. In a particular example, one or more primers for amplification are attached to a copolymer coating on a substrate. Bridge amplification can be performed using a format of two attached primers, because the double-stranded amplicon forms a bridge-like structure between the two attached primers flanking the template sequence that has been copied. Amplification in which one amplification primer is attached to the copolymer coating and the second primer is in solution (e.g., emulsion PCR), or amplification of both primers in solution, can also be performed.

In some examples, the copolymer-coated substrates described herein can be used in methods for determining the nucleotide sequence of a target nucleic acid. In such an example, the method comprises:

hybridizing a target nucleic acid to one of more than one primer covalently bound to a substrate VIa a copolymer as described herein (or to an oligonucleotide of repeating monomer units of formula (VI), formula (VIa), formula (VIb), formula (VIc), formula (VId), or formula (VIe));

amplifying the hybridized target nucleic acids using more than one primer to form clusters of substrate-bound amplicons;

treating the cluster of substrate-bound amplicons with labeled nucleotides and a polymerase such that a detectable signal is generated when the nucleotides are incorporated by the polymerase; and detecting the signal, thereby determining the nucleotide sequence of the target nucleic acid.

For one or more of the polynucleotides attached to the polymer coating on the surface of the substrate present in the flow cell, the determination of the one or more nucleotides incorporated during each flow step is achieved by detecting the signal generated at or near the template. In some examples, the detectable signal comprises an optical signal. In other examples, the detectable signal comprises a non-optical signal. In such instances, the non-optical signal includes a change in pH or current at or near the one or more nucleic acids being sequenced.

Suitable sequencing methods include, but are not limited to, sequencing-by-synthesis, pyrosequencing, ligation sequencing, and other methods known in the art.

In sequencing by synthesis, one or more nucleotides are provided to a template polynucleotide associated with a polynucleotide polymerase. The polynucleotide polymerase incorporates one or more nucleotides into a newly synthesized nucleic acid strand that is complementary to the polynucleotide template. Synthesis begins with an oligonucleotide primer that is complementary to a portion of the template polynucleotide or to a portion of a universal nucleic acid or a non-variable nucleic acid that is covalently bound at one end of the template polynucleotide. As nucleotides are incorporated against the template polynucleotide, detectable signals are generated that allow determination of which nucleotide has been incorporated during each step of the sequencing method. In this manner, a nucleic acid sequence complementary to at least a portion of the template polynucleotide can be generated, thereby allowing the nucleotide sequence of at least a portion of the template polynucleotide to be determined.

Other useful techniques that can use the copolymers and substrates of the invention include real-time monitoring of DNA polymerase activity, SBS techniques that detect proton release upon nucleotide incorporation (e.g., Ion Torrent, Thermo Fisher), and gene expression analysis. For example, nucleotide incorporation can be detected by Fluorescence Resonance Energy Transfer (FRET) interaction between a fluorophore-bearing polymerase and a gamma-phosphate labeled nucleotide, or with Zero Mode Waveguiding (ZMW). Gene expression can be detected or quantified using RNA sequencing techniques, such as those known as digital RNA sequencing. RNA sequencing techniques can be performed using sequencing methods known in the art, such as those set forth above. Gene expression can also be detected or quantified using hybridization techniques by direct hybridization of the array or multiplex assays where the products of the assay are detected on the array.

It will be understood that any respective features/examples of each aspect of the disclosure as described herein may be implemented together in any suitable combination, and any features/examples from any one or more of these aspects may be implemented together with any features of the other aspects as described herein in any suitable combination.

Further embodiments

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 1.4-acrylamido-N- (6- (azidomethyl) pyridin-3-yl) butanamide.

Step 1. tert-butyl (4- ((6- (azidomethyl) pyridin-3-yl) amino) -4-oxobutyl) carbamate. To a solution of 6- (azidomethyl) pyridin-3-amine and 4- ((tert-butoxycarbonyl) amino) butyric acid is added a suitable amide coupling reagent (such as EDC/DMAP or EDC/HOBt) in a polar solvent such as dichloromethane or DMF. The resulting mixture was stirred at rt. The product was isolated and purified.

Step 2.4-amino-N- (6- (azidomethyl) pyridin-3-yl) butanamide. To a solution of the product from step 1 in dichloromethane was added TFA. The resulting mixture was stirred at rt (rt) to remove the Boc group. The product was isolated and purified.

Step 3. solution of the product of step 2 in dichloromethane with acryloyl chloride and Et₃N, and the resulting mixture is stirred at rt. The product was isolated and purified.

Example 2.5-acrylamido-N- (6- (azidomethyl) pyridin-3-yl) pentanamide.

The title compound was prepared as described in example 1 using 5- ((tert-butoxycarbonyl) amino) pentanoic acid instead of 4- ((tert-butoxycarbonyl) amino) butanoic acid.

_6-20Example 3.5-acrylamido-N- (6- (azidomethyl) pyridin-3-yl) -C-alkylamide (z ═ 1, 2. 3, 4, 5, 6, 7 or 8).

The title compound was prepared as described in example 1 using the appropriate acid instead of 4- ((tert-butoxycarbonyl) amino) butanoic acid.

¹Example 4N- (6- (azidomethyl) pyridin-3-yl) -N4- (2-methacrylamidoethyl) succinyl An imine.

Step 1.4- ((6- (azidomethyl) pyridin-3-yl) amino) -4-oxobutanoic acid. A solution of 6- (azidomethyl) pyridin-3-amine and succinic anhydride in dichloromethane was stirred at rt. The product was isolated and purified.

Step 2. to a solution of the product of step 1 in dichloromethane or DMF is added N- (2-aminoethyl) methacrylamide and a suitable amide coupling reagent (such as EDC/DMAP or EDC/HOBt). The resulting mixture was stirred at rt. The product was isolated and purified.

¹Example 5N- (6- (azidomethyl) pyridin-3-yl) -N4- (3-methacrylamidopropan)Yl) succinyl An imine.

The title compound was prepared as described in example 4 substituting N- (3-aminopropyl) methacrylamide for N- (2-aminoethyl) methacrylamide.

Example 6.

Step 1. use of a solution of 5-hydroxymethyl-L-proline in dichloromethane (PhO)₂PON₃Treatment followed by treatment with aqueous sodium hydroxide in methanol to form the azido compound, which is isolated and purified.

Step 2. A solution of the product of step 1 and tert-butoxycarbonyl (tBOC) protected butane-1, 4-diamine was treated with thionyl chloride in dichloromethane and the mixture was stirred at 0 ℃ for 1 hour. The mixture was then treated with trimethylamine in dichloromethane and cooled for 4 hours. The product was isolated and purified.

Step 3. the product of step 2 is treated with a suitable deprotecting agent such as trifluoroacetic acid in dichloromethane. The product is then isolated and purified.

Step 4. the solution of the product of step 3 and acryloyl chloride was treated with aqueous sodium hydroxide at 0 ℃ for 2 hours, followed by stirring at room temperature for 10 hours. The solution was then treated with aqueous hydrochloric acid and the pH was adjusted to 7. The product was isolated and purified as N- [3- [ (1-oxo-2-propen-1-yl) amino ] butyl ] -5- (azidomethyl) -2-pyrrolidine.

Example 7

Step 1. use of a solution of 6-hydroxymethyl-nicotinic acid methyl ester in dichloromethane (PhO)₂PON₃Treatment to form the azido compound, which is isolated and purified.

Step 2. the solution of the product of step 1 was treated with aqueous sodium hydroxide and ethanol and stirred at room temperature for 12 hours. The pH of the mixture was adjusted to 7. The product was isolated and purified.

Step 3. A solution of the product of step 2 and tert-butoxycarbonyl (tBOC) protected butane-1, 4-diamine was treated with thionyl chloride in dichloromethane and stirred at 0 ℃ for 1 hour. The reaction mixture was then treated with trimethylamine in dichloromethane and cooled for 4 hours. The product was isolated and purified.

Step 4. treat the product of step 3 with a suitable deprotecting agent such as trifluoroacetic acid in dichloromethane.

Step 5. the solution of the product of step 4 and acryloyl chloride was treated with aqueous sodium hydroxide and stirred at 0 ℃ for 2 hours, the temperature of the reaction mixture was raised to room temperature, and the mixture was stirred at room temperature for 10 hours. The solution was then treated with aqueous hydrochloric acid and the pH was adjusted to 7. The product was isolated and purified as N- [3- [ (1-oxo-2-propen-1-yl) amino ] butyl ] -6- (azidomethyl) -3-pyridinecarboxamide.

Example 8

Step 1. Add appropriate amide coupling reagents, such as TSTU and N, N-diisopropylethylamine in DMF to 4-aminobenzol and t-Boc-N-amido-PEG 2-CH₂CO₂A solution of H. The resulting mixture was stirred at room temperature and the BOC protected amide was isolated and purified.

Step 2. the product from step 1 was treated with diphenyl phosphorazidate (diphenylphosphate azide) and DBU in DMF and the resulting mixture was stirred at room temperature.

Step 3. the product of step 2 is deprotected by treatment with a suitable deprotecting agent such as trifluoroacetic acid in dichloromethane.

Step 4. the solution of the product of step 3 was treated with acryloyl chloride in pyridine and the resulting mixture was stirred at 4 ℃. Separating and purifying the product into

Example 9 copolymer formation.

The solutions of example 1 (2% -50%) and acrylamide were polymerized in the presence of AIBN (or other water soluble form, such as Vazo 56) or KPS/TMED, or under Controlled Radical Polymerization (CRP) conditions (e.g., RAFT, ATRP, NMP).

Example 10. fixing the copolymer to the substrate.

A solution of the copolymer as described herein is applied to the norbornene silane derived substrate surface by spin coating. The substrate was incubated at 60 ℃ for 1h to adhere the copolymer to the substrate.

Example 11 grafting of copolymer with oligonucleotide.

The copolymer as described in example 9 or example 10 was treated with the alkyne-functionalized oligonucleotide, the copper (I) catalyst and the copper ligand in an aqueous buffer.

The mixture is applied to a substrate surface (e.g., a flow cell channel), and the substrate is incubated and then washed with a buffer. Grafting is accomplished at lower copper loadings than grafting with polymers that do not contain heterocyclic azide units (e.g., alkyl azides, phenyl azides, or benzyl azides). Lower copper loadings are advantageous for ease of manufacture (less toxic reagents), reduced DNA damage to grafted oligonucleotides, reduced copper present during downstream amplification and in sequencing protocols to reduce damage to template nucleic acids, improved grafting reaction kinetics, and milder grafting conditions, since rapid, additive-free reactions are beneficial during complex manufacturing processes.

While various illustrative embodiments have been described above, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the invention. It is intended that the appended claims cover all such changes and modifications that fall within the true spirit and scope of this present invention.