The invention provides novel sulforhodamine dye phosphoramidite compounds useful as automated oligonucleotide synthesis-compatible red dyes.

Fluorescent dyes are among the most commonly used tags for modifying oligonucleotides because they offer sensitive detection in a wide variety of applications ranging from PCR to sequencing. Rhodamine dyes are widely used in oligonucleotide labeling because they offer longer wavelength emission maxima than other dyes such as fluoresceins and provide opportunities for multicolor labeling and staining. Additionally, rhodamines exhibit higher photostability than fluoresceins and coumarins.

Preparation of fluorescent dye-labeled polynucleotides is typically done by post-synthetic conjugation, for example, by reacting an activated dye intermediate with an amino derivative of a polynucleotide. This approach suffers from certain drawbacks, including low conjugation yields and the need for additional purification of the conjugated product. Incorporation of fluorescent dyes into synthetic polynucleotides via automated phosphoramidite synthesis offers a more convenient approach. However, few phosphoramidite derivatives of fluorescent dyes which allow the fluorescent dye to be added to the polynucleotide as part of the automated solid phase synthesis are commercially available.

One example of a popular oligonucleotide labeling dye is Sulforhodamine 101 sulfonyl chloride (Texas Red® dye). However, Texas Red® dye can only be introduced into an oligonucleotide via post-synthetic coupling as no phosphoramidite derivatives of Texas Red® dye are known. Because Texas Red® dye is unstable, it gives much lower coupling yields than carboxyrhodamine dyes (such as 5-TAMRA, SE) which increases the costs associated with preparation of Texas Red®-labeled oligonucleotides.

To be a viable alternative to Texas Red® dye for automated oligonucleotide synthesis, a fluorophore should have spectral characteristics close to those of Texas Red® dye. Such fluorophore should be stable to standard oligonucleotide synthesis conditions (e.g., iodine treatment, capping, and acid deprotection conditions) and amenable to incorporation at any position in the oligonucleotide. Additionally, for PCR applications, such a fluorophore's brightness should be insensitive to changes in the biologically relevant pH range, and the dye needs to be quenched well to produce high End-Point

Fluorescence (EPF), which is measured as a difference between the fluorescence of a quenched probe and a cleaved probe or an unquenched probe (e.g., a hybridized probe).

Previously known alternatives of Texas Red® dye that are compatible with oligo synthesis have certain drawbacks. For instance, CAL Fluor Red® 610 dye is a phosphoramidite that fluoresces in the orange-red region of the visible spectrum and can be used for the 5' labeling of fluorogenic probes, such as probes used in 5' nuclease assays, molecular beacons, and similar detection assays. However, this dye does not contain a protected hydroxyl group and thus can only be added to the 5' terminus of an oligonucleotide, limiting its applications. Another family of dyes, AquaPhluor®, includes a red dye that is highly fluorescent with an absorption maximum at 593 nm and an emission maximum at 613 nm. However, due to its structural limitations, this dye can only be incorporated at the 5' end or the 3' end of an oligonucleotide.

Thus, a need still exists for red fluorescent dyes that are compatible with the conditions of automated phosphoramidite oligonucleotide synthesis, can be incorporated into any position of a polynucleotide, have emission and absorption maxima compatible with the existing PCR instrumentation, and provide a high endpoint fluorescence signal in PCR applications.

SUMMARY

In one aspect, provided herein is a compound having a structure represented by

Formula I:

or a stereoisomer, tautomer, or salt thereof, wherein:

R2 is halogen or S02NH2,

R3 is H or halogen;

R4, R7, R8, and R11 when taken alone are independently H, halogen, or optionally substituted C^-Cg alkyl,

R5, R6, R9, and R10 when taken alone are independently H or optionally substituted C j-C6 alkyl;

R4 and R5 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R4 and R5 are attached;

R6 and R7 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R6 and R7 are attached;

R8 and R9 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R8 and R9 are attached;

R10 and R11 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R10 and R11 are attached;

R5 and R6, when taken together with the nitrogen atom to which R5 and R6 are attached, form a 5-membered or a 6-membered unsaturated ring;

R9 and R10, when taken together with the nitrogen atom to which R9 and R10 are attached, form a 5-membered or a 6-membered unsaturated ring;

L1 is an optionally substituted C2-Cl0 alkylene or optionally substituted C2-C2o heteroalkylene;

X is an activated ester, N3, propynyl, maleimido, or -0-P(0CH2CH2CN)NR12R13 or X is:

i— z

, wherein

L3 and L4 are independently an optionally substituted C2-C1Q alkylene or optionally substituted C2-C30 heteroalkylene;

Q is a hydroxyl protecting group;

Z is CH, N, NHC(0)N, or 0C(0)N; and

R12 and R13 are independently optionally substituted Cj-Cg alkyl.

In some embodiments, R2 is Cl and R3 is H. In some embodiments, X is

-OP(OCH2CH2CN)N(i-Pr)2. In some embodiments, L1 is a PEG2-10 linker. In certain embodiments, L1 is -CH2CH2OCH2CH2-.

In some embodiments, R4 and R5 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R4 and R5 are attached; R10 and R11 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R10 and R11 are attached; R6 and R7 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R6 and R7 are attached; and R8 and R9 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R8 and R9 are attached. In some embodiments, R4 and R5 taken together are a propylene chain connecting the atoms to which R4 and R5 are attached; R10 and R11 taken together are a propylene chain connecting the atoms to which R10 and R11 are attached; R6 and R7 taken together are a propylene chain connecting the atoms to which R6 and R7 are attached; and R8 and R9 taken together are a propylene chain connecting the atoms to which R8 and R9 are attached. In some embodiments, R5, R6, R9, and R10 are each methyl.

In some embodiments, Q is an acid-labile hydroxyl protecting group. In some embodiments, Q is a trityl or dimethoxytrityl group.

In some embodiments, C2-C6 alkylene or -(OCH2CH2)m-, wherein m is an integer ranging from 2 to 6. In some embodiments, L3 and L4 are -0ί20¾-. In some embodiments, X is:

In some embodiments, the compound has a structure represented by Formula IA:

or a stereoisomer, tautomer, or salt thereof, wherein R1 is L * X and R3 is H or halogen. In some embodiments, X is -OP(OCH2CH2CN)N(i-Pr)2.

In some embodiments, the compound is:

In a second aspect, provided herein is a compound represented by Formula II:

or a stereoisomer, tautomer, or salt thereof, wherein:

R1 is Cj-Cg alkyl;

R2 is H, halogen, or SO2NH2,

R3 is H or halogen;

R14 and R16 when taken alone are independently H, halogen, or optionally substituted C j-Cg alkyl;

R15 and R17 when taken alone are independently H or optionally substituted CrC6 alkyl;

R14 and R15 when taken together form an optionally substituted C2-C3 alkylene chain connecting the atoms to which R14 and R15 attached;

R16 and R17 when taken together form an optionally substituted C2-C3 alkylene chain connecting the atoms to which R16 and R17 are attached;

Q is a hydroxyl protecting group; and

L1 and L2 are independently an optionally substituted C2-Cl0 alkylene or optionally substituted C2-C20 heteroalkylene.

In some embodiments, R3 is H. In some embodiments, R2 is H. In some embodiments, R2 is Cl.

In some embodiments, the compound is represented by Formula (IIA):

or a stereoisomer, tautomer, or salt thereof, wherein:

R1 is Cj-Cg alkyl;

R2 is H, halogen, or SC^NF^; and

R3 is H or halogen.

In some embodiments, the compound is:

In a third aspect, provided herein is a compound having a structure of Formula (III):

or a stereoisomer, tautomer, or salt thereof, wherein:

R2 is H, halogen or S02NH2;

R3 is halogen or H;

R4, R7, R8, and R11 when taken alone are independently H, halogen, or optionally substituted C^-Cg alkyl,

R5, R6, R9 and R10 when taken alone are independently H or optionally substituted C j- , alkyl;

R4 and R5 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R4 and R5 are attached;

R6 and R7 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R6 and R7 are attached;

R8 and R9 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R8 and R9 are attached;

R10 and R11 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R10 and R11 are attached;

R5 and R6, when taken together with the nitrogen atom to which R5 and R6 are attached, form a 5-membered or a 6-membered unsaturated ring;

R9 and R10, when taken together with the nitrogen atom to which R9 and R10 are attached, form a 5-membered or a 6-membered unsaturated ring;

n is an integer from 1 to 9;

Y is:

wherein:

Q is a hydroxyl protecting group; and

R12 and R13 are independently optionally substituted C j- , alkyl.

In some embodiments, R2 is Cl and R3 is H.

In some embodiments, R4 and R5 taken together are an optionally substituted

C3 alkylene chain connecting the atoms to which R4 and R5 are attached; R10 and R11 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R10 and R11 are attached; R6 and R7 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R6 and R7 are attached; and R8 and R9 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R8 and R9 are attached. In some embodiments, R4 and R5 taken together are a propylene chain connecting the atoms to which R4 and R5 are attached; R10 and R11 taken together are a propylene chain connecting the atoms to which R10 and R11 are attached; R6 and R7 taken together are a propylene chain connecting the atoms to which R6 and R7 are attached; and R8 and R9 taken together are a propylene chain connecting the atoms to which R8 and R9 are attached.

In some embodiments, Q is an acid-labile hydroxyl protecting group. In some embodiments, Q is a trityl or dimethoxytrityl group. In yet other embodiments, R12 and R13 are each isopropyl.

In some embodiments, the compound is:

In a fourth aspect, provided herein is a compound represented by Formula VI:

or a stereoisomer, tautomer, or salt thereof, wherein:

R1 is I C;

R2, R3, R4, and R5 are independently H, halogen, Cj-Cg alkyl, or S02NH2;

R6 and R9 are independently H or optionally substituted C j-Cg alkyl;

R7, R8, R10, R11, R14, and R15 are independently H or optionally substituted C j-C6 alkyl;

L1 is an optionally substituted C2-C | () alkylene or optionally substituted C2-C2() heteroalkylene;

X is an activated ester, N3, propynyl, maleimido, or

-0-P(0CH2CH2CN)NR12R13, or X is:

R 12

-N

R 13

LJ-0

i— z

wherein

L3 and L4 are independently an optionally substituted C2-CJO alkylene or optionally substituted C2-C30 heteroalkylene;

Q is a hydroxyl protecting group;

Z is CH, N, NHC(0)N, or 0C(0)N; and

R12 and R13 are independently optionally substituted Cj-Cg alkyl.

In some embodiments of Formula VI, R7, R8, R10, R11, R14, and R15 are methyl. In certain embodiments, X is -OP(OCH2CH2CN)N(i-Pr)2. In some embodiments, L1 is a PEG,.,,, linker, and in other embodiments, L1 is -CH2CH2OCH2CH2-. In certain embodiments, R2, R3, R4, and R5 are H.

In some embodiments, R6 is a C j- , alkyl optionally substituted with OH or Cl- C6 acyloxy, for example, R6 is an ethyl substituted with OH or OAc. In some embodiments, R9 is a C^-Cg alkyl optionally substituted with OH or C1-C6 acyloxy, for example, R9 is an ethyl substituted with OH or OAc.

In some embodiments of Formula VI, the compound is:

or a stereoisomer, tautomer, or salt thereof, wherein:

R1, R2, R3, R4, and R5 are as described for above for Formula VI; and

Q1 and Q2 are independently H or C1-C6 acyl.

In some embodiments, R2, R3, R4, and R5 are H. In some embodiments, Q1 and

Q2 are acetyl.

In some embodiments of Formula VI, the compound is:

wherein R1 is as defined above for Formula VI.

In some embodiments of Formula VI, the compound is:

In a fifth aspect, provided herein is a labeled polynucleotide comprising a compound of formula IV:

or a stereoisomer, tautomer, or salt thereof, wherein:

R2 is halogen or S02NH2;

R3 is H or halogen;

R4, R7, R8, and R11 when taken alone are independently H, halogen, or optionally substituted C^-Cg alkyl,

R5, R6, R9, and R10 when taken alone are independently H or optionally sub stituted C 3 -Cg alkyl ;

R4 and R5 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R4 and R5are attached;

R6 and R7 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R6 and R7 are attached;

R8 and R9 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R8 and R9 are attached;

R10 and R11 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R10 and R11 are attached;

R5 and R6, when taken together with the nitrogen atom to which R5 and R6 are attached, form a 5-membered or a 6-membered unsaturated ring;

R9 and R10, when taken together with the nitrogen atom to which R9 and R10 are attached, form a 5-membered or a 6-membered unsaturated ring;

n is an integer from 1 to 9;
CLAIMS

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1 A compound represented by Formula I:

or a stereoisomer, tautomer, or salt thereof, wherein:

R2 is halogen or S02NH2,

R3 is H or halogen;

R4, R7, R8, and R11 when taken alone are independently H, halogen, or optionally substituted Cj-Cg alkyl,

R5, R6, R9, and R10 when taken alone are independently H or optionally substituted C j-C8 alkyl;

R4 and R5 when taken together are an optionally substituted C2-C2 alkylene chain connecting the atoms to which R4 and R5 are attached;

R6 and R7 when taken together are an optionally substituted C2-C2 alkylene chain connecting the atoms to which R6 and R7 are attached;

R8 and R9 when taken together are an optionally substituted C2-C2 alkylene chain connecting the atoms to which R8 and R9 are attached;

R10 and R11 when taken together are an optionally substituted C2-C2 alkylene chain connecting the atoms to which R10 and R11 are attached;

R5 and R6, when taken together with the nitrogen atom to which R5 and R6 are attached, form a 5-membered or a 6-membered unsaturated ring;

R9 and R10, when taken together with the nitrogen atom to which R9 and R10 are attached, form a 5-membered or a 6-membered unsaturated ring;

L1 is an optionally substituted C2-Cl0 alkylene or optionally substituted C2-C20 heteroalkylene;

X is an activated ester, N3, propynyl, maleimido, or -0-P(0CH2CH2CN)NR12R13, or X is:

, wherein

L3 and L4 are independently an optionally substituted C2-C1Q alkylene or optionally substituted C2-C30 heteroalkylene;

Q is a hydroxyl protecting group;

Z is CH, N, NHC(0)N, or 0C(0)N; and

R12 and R13 are independently optionally substituted Cj-Cg alkyl.

2. The compound of Claim 1, wherein R2 is Cl and R3 is H.

3. The compound of Claim 1, wherein X is -OP(OCH2CH2CN)N(i-Pr)2.

4. The compound of Claim 1, wherein L1 is a PEG2.10 linker.

5. The compound of Claim 1, wherein L1 is -CH2CH2OCH2CH2-.

6. The compound of Claim 1, wherein R4 and R5 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R4 and R5 are attached; R10 and R11 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R10 and R11 are attached; R6 and R7 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R6 and R7 are attached; and R8 and R9 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R8 and R9 are attached.

7. The compound of Claim 1, wherein R4 and R5 taken together are a propylene chain connecting the atoms to which R4 and R5 are attached; R10 and R11 taken together are a propylene chain connecting the atoms to which R10 and R11 are attached; R6 and R7 taken together are a propylene chain connecting the atoms to which R6 and R7 are attached; and R8 and R9 taken together are a propylene chain connecting the atoms to which R8 and R9 are attached.

8. The compound of Claim 1, wherein R5, R6, R9 and R10 are each methyl.

9. The compound of Claim 1, wherein Q is an acid-labile hydroxyl protecting group.

10. The compound of Claim 1, wherein Q is a trityl or dimethoxytrityl group.

11. The compound of Claim 1, wherein L3 and L4 are independently C2-C6 alkylene or -(OCH2CH2)m- wherein m is an integer ranging from 2 to 6.

12. The compound of Claim 1, wherein L3 and L4 are independently CH2CH2.

13. The compound of Claim 1, wherein X is:

14. The compound of Claim 1, wherein the compound is represented by Formula I A:

or a stereoisomer, tautomer, or salt thereof, wherein R1 is L3X and R3 is H or halogen.

15. The compound of Claim 1 or Claim 14, wherein X is -OP(OCH2CH2CN)N(i-Pr)2.

16. The compound of Claim 1, wherein the compound is:

17. A compound represented by Formula II:

or a stereoisomer, tautomer, or salt thereof, wherein:

R1 is C^-Cg alkyl;

R2 is H, halogen, or S02NH2,

R3 is H or halogen;

R14 and R16 when taken alone are independently H, halogen, or optionally substituted C j- , alkyl;

R15 and R17 when taken alone are independently H or optionally substituted CrC6 alkyl;

R14 and R15 when taken together form an optionally substituted C2-C3 alkylene chain connecting the atoms to which R14 and R15 attached;

R16 and R17 when taken together form an optionally substituted C2-C3 alkylene chain connecting the atoms to which R16 and R17 are attached;

Q is a hydroxyl protecting group; and

L1 and L2 are independently an optionally substituted C2-Cl0 alkylene or optionally substituted C2-C2o heteroalkylene.

18. The compound of Claim 17, wherein R3 is H.

19. The compound of Claim 17, wherein R2 is H.

20. The compound of Claim 17, wherein R2 is Cl.

21. The compound of Claim 17, wherein the compound is represented by

Formula (II A):

22. The compound of Claim 17, wherein the compound is:

23. A compound represented by Formula (III):

or a stereoisomer, tautomer, or salt thereof, wherein:

R2 is H, halogen or SO2NH2;

R3 is halogen or H;

R4, R7, R8, and R11 when taken alone are independently H, halogen, or optionally substituted C^-Cg alkyl,

R5, R6, R9, and R10 when taken alone are independently H or optionally substituted C j- , alkyl;

R4 and R5 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R4 and R5 are attached;

R6 and R7 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R6 and R7 are attached;

R8 and R9 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R8 and R9 are attached;

R10 and R11 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R10 and R11 are attached;

R5 and R6, when taken together with the nitrogen atom to which R5 and R6 are attached, form a 5-membered or a 6-membered unsaturated ring;

R9 and R10, when taken together with the nitrogen atom to which R9 and R10 are attached, form a 5-membered or a 6-membered unsaturated ring;

n is an integer from 1 to 9;

Y is:

wherein:

Q is a hydroxyl protecting group; and

R12 and R13 are independently optionally substituted C j- , alkyl.

24. The compound of Claim 23, wherein R2 is Cl and R3 is H.

25. The compound of Claim 23, wherein R4 and R5 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R4 and R5 are attached; R10 and R11 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R10 and R11 are attached; R6 and R7 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R6 and R7 are attached; and R8 and R9 taken together are an optionally substituted C3 alkylene chain connecting the atoms to which R8 and R9 are attached.

26. The compound of Claim 23, wherein R4 and R5 taken together are a propylene chain connecting the atoms to which R4 and R5 are attached; R10 and R11 taken together are a propylene chain connecting the atoms to which R10 and R11 are attached; R6 and R7 taken together are a propylene chain connecting the atoms to which R6 and R7 are attached; and R8 and R9 taken together are a propylene chain connecting the atoms to which R8 and R9 are attached.

27. The compound of Claim 23, wherein Q is an acid-labile hydroxyl protecting group.

28. The compound of Claim 23, wherein Q is a trityl or dimethoxytrityl group.

29. The compound of Claim 23, wherein R12 and R13 are each isopropyl.

30. The compound of Claim 23, wherein the compound is:

31. A labeled polynucleotide comprising a compound of formula IV :

or a stereoisomer, tautomer, or salt thereof, wherein:

R2 is halogen or S02NH2;

R3 is H or halogen;

R4, R7, R8, and R11 when taken alone are independently H, halogen, or optionally substituted C^-Cg alkyl,

R5, R6, R9 and R10 when taken alone are independently H or optionally substituted C j- , alkyl;

R4 and R5 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R6 and R7 are attached;

R6 and R7 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R6 and R7 are attached;

R8 and R9 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R8 and R9 are attached;

R10 and R11 when taken together are an optionally substituted C2-C3 alkylene chain connecting the atoms to which R10 and R11 are attached;

R5 and R6, when taken together with the nitrogen atom to which R5 and R6 are attached, form a 5-membered or a 6-membered unsaturated ring;

R9 and R10, when taken together with the nitrogen atom to which R9 and R10 are attached, form a 5-membered or a 6-membered unsaturated ring;

n is an integer from 1 to 9;

Y is:

wherein:

the wavy line is the point of attachment to Formula III;

R12 and R13 are independently optionally substituted C j- , alkyl;

W is H or IAO-Z2;

L3, L4, and L5 are independently an optionally substituted C -C Q alkylene or optionally substituted C2-C30 heteroalkylene;

Z is CH, N, NHC(0)N, or 0C(0)N;

Z1 is a nucleotide or oligonucleotide; and

Z2 is a nucleotide, oligonucleotide, or H.

32. The labeled polynucleotide of Claim 31, wherein R2 is Cl and R3 is H.

33. The labeled polynucleotide of Claim 31, wherein R4 and R5 are an optionally substituted C3 alkylene chain connecting the atoms to which R4 and R5 are attached; R10 and R1 1 are an optionally substituted C3 alkylene chain connecting the atoms to which R10 and R1 1 are attached; R6 and R7 are an optionally substituted C3 alkylene chain connecting the atoms to which R6 and R7 are attached; and R8 and R9 are an optionally substituted C3 alkylene chain connecting the atoms to which R8 and R9 are attached.

34. The labeled polynucleotide of Claim 31, wherein the labeled polynucleotide is a 5'-nuclease PCR probe.

35. The labeled polynucleotide of Claim 31, wherein the labeled polynucleotide further comprises a fluorescence quencher.

36. The labeled polynucleotide of Claim 31, wherein the labeled polynucleotide is attached to a solid support.

37. The labeled polynucleotide of Claim 31, wherein the solid support is a controlled pore glass bead, polystyrene bead, magnetic bead, or microwell plate.

38. A compound represented by Formula VI:

or a stereoisomer, tautomer, or salt thereof, wherein:

R1 is I C;

R2, R3, R4, and R5 are independently H, halogen, C3-C6 alkyl, or S02NH2;

R6 and R9 are independently H or optionally substituted C j-Cg alkyl;

R7, R8, R10, R11, R14, and R15 are independently H or optionally substituted C3-C6 alkyl;

L1 is an optionally substituted C2-Cl0 alkylene or optionally substituted C2-C20 heteroalkylene;

X is an activated ester, N3, propynyl, maleimido, or -0-P(0CH2CH2CN)NR12R13, or X is:

wherein

L3 and L4 are independently an optionally substituted C2-Cl0 alkylene or optionally substituted C2-C3Q heteroalkylene;

Q is a hydroxyl protecting group;

Z is CH, N, NHC(0)N, or 0C(0)N; and

R12 and R13 are independently optionally substituted C j -Cg alkyl.

39. The compound of Claim 38, wherein R7, R8, R10, R11, R14, and R15 are methyl.

40. The compound of Claim 38, wherein X is -OP(OCH2CH2CN)N(i-Pr)2.

41. The compound of Claim 38, wherein L1 is a PEG2-10 linker.

42. The compound of Claim 38, wherein L1 is -CH2CH2OCH2CH2-.

43. The compound of Claim 38, wherein R2, R3, R4, and R5 are H.

44. The compound of Claim 38, wherein R6 is a Cj-Cg alkyl optionally substituted with OH or C1-C6 acyloxy.

45. The compound of Claim 38, wherein R9 is a Cj-Cg alkyl optionally substituted with OH or C1-C6 acyloxy.

46. The compound of Claim 38, wherein R6 is an ethyl substituted with OH or

OAc.

47. The compound of Claim 38, wherein R9 is an ethyl substituted with OH or

OAc.

48. The compound of Claim 38, wherein the compound is:

or a stereoisomer, tautomer, or salt thereof, wherein:

R1, R2, R3, R4, and R5 are as described for Compound of Claim 43; and

Q1 and Q2 are independently H or C1-C6 acyl.

49. The compound of Claim 48, wherein R2, R3, R4, and R5 are H.

50. The compound of Claim 48, wherein Q1 and Q2 are acetyl.

51. The compound of Claim 38, wherein the compound is:

wherein R1 is as defined for compound for Claim 43.

52. The compound of Claim 38, wherein the compound is:

53. A method for preparing a labeled conjugate of a ligand comprising contacting a ligand with a compound of any one of Claim 1 to Claim 30 or Claims 38 to 52 in a suitable solvent under conditions sufficient to covalently attach the compound to the ligand thereby forming the labeled conjugate.

54. The method of Claim 53, wherein the ligand is a polynucleotide, a protein, a peptide, a polysaccharide, a polymer with an ethylenic backbone, or a solid support.

55. The method of Claim 53, wherein the ligand is a polynucleotide.

56. The method of Claim 53, wherein the conditions sufficient to covalently attach the compound to the ligand are automated phosphoramidite oligonucleotide synthesis conditions.