Search

US-12624391-B2 - Cyanine dyes

US12624391B2US 12624391 B2US12624391 B2US 12624391B2US-12624391-B2

Abstract

The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.

Inventors

  • Stephen Yue
  • Gene Shen
  • Wei-Chuan (David) SUN

Assignees

  • PACIFIC BIOSCIENCES OF CALIFORNIA, INC.

Dates

Publication Date
20260512
Application Date
20230314

Claims (13)

  1. 1 . A method of synthesizing a first polynucleotide having a first nucleic acid sequence, the method comprising: under conditions of a template dependent polymerization reaction, contacting a nucleic acid polymerase complexed with a second polynucleotide having a second nucleic acid sequence complementary to the first nucleic acid sequence with a cyanine compound of Formula X: wherein R f , R g , R h , and R i are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heteroalkyl; R c , and R d are independently selected from alkyl and heteroalkyl, substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, and phosphoric acid; a and e are independently selected from selected from the integers 0, 1, 2, 3 and 4, with the proviso that at least one member selected from a and e is 2 or greater, such that when a is 2 or greater, each R a is independently selected, and when e is 2 or greater, each R e is independently selected; wherein when a is 2 or greater, ring A is selected from phenyl substituted at least at the 4- and 6-positions with (R a ) a, and substituted or unsubstituted napthyl; and when e is 2, or more ring E is selected from phenyl substituted at least at the 4- and 6-positions with (R e ) e , and substituted or unsubstituted naphthyl; Q is a member selected from wherein n is selected from the integers from 1 to 3; each R a and R e , is independently selected from C(O)R 9 , OR 12 , NR 12 R 13 , CR 12 C(O)R 13 , NR 12 C(O) 2 R 13 , SO 3 H, and C(O) NR 12 R 13 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, a bond to M, and a ring structure formed by joining a member selected from two R a moieties together with the atoms they are attached, two Re moieties together with the atoms they are attached, and a combination thereof, to form said ring structure, which is a member selected from a substituted or unsubstituted aryl and a substituted or unsubstituted heteroaryl, wherein R 9 is a member selected from OR 10 , and a bond to M wherein R 10 is a member selected from H and substituted or unsubstituted alkyl; R 11 is a nucleic acid moiety, which is a substrate for the polymerase; and R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl, and a bond to M, wherein M comprises a linker bound to a nucleic acid carrier, which is a substrate for the polymerase, and at least one of R a , R c , R d , R e , R f , R g , R h , R i , R 9 , R 12 and R 13 is a bond to M, thereby synthesizing the first polynucleotide.
  2. 2 . The method according to claim 1 , wherein M comprises a nucleic acid polyphosphate.
  3. 3 . The method according to claim 2 , wherein at least one of R a , R b , R c , R d , R e , R f , R g , R h , R i , R 9 , R 12 and R 13 is attached to a 5′-oxygen of the nucleic acid.
  4. 4 . The method according to claim 1 , wherein R 11 comprises: wherein u is selected from the integers from 1 to 8; and Y is a nucleobase.
  5. 5 . The method according to claim 1 , wherein the synthesizing is performed by template directed DNA synthesis.
  6. 6 . The method according to claim 5 , wherein the synthesizing is a component of a single molecule DNA sequencing analysis.
  7. 7 . The method according to claim 1 , wherein the nucleic acid polymerase complexed with the second polynucleotide is immobilized within an optical confinement.
  8. 8 . The method according to claim 7 , wherein the optical confinement is a zero-mode waveguide.
  9. 9 . The method of synthesizing a first polynucleotide having a first nucleic acid sequence according to claim 1 , wherein the compound of Formula X is a member selected from:
  10. 10 . The method of synthesizing a first polynucleotide having a first nucleic acid sequence according to claim 1 , wherein the compound of Formula X is selected from: wherein n is an integer selected from 1, 2, and 3; a, e, and j are independently selected from the integers 1, 2, 3, and 4, such that when a is 2 or greater, each R a is independently selected, when e is 2 or greater, each R e is independently selected, and when j is 2 or greater, each R i is independently selected; b and e′ are independently selected from the integers 0, 1, and 2, such that when b is 2, each R b is independently selected, and when e′ is 2, each R e′ is independently selected; R a , R b , R e , R e′ , and R i , are independently selected from C(O)R 9 , OR 12 , NR 12 R 13 , CR 12 C(O)R 13 , NR 12 C(O) 2 R 13 , SO 3 H, and C(O) NR 12 R 13 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and a bond to M; R f , R g , R h , and R i are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heteroalkyl, and a bond to M; R e , and R d are independently selected from a bond to M, and alkyl substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, and phosphoric acid, and heteroalkyl substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, and phosphoric acid; wherein R 9 is a member selected from OR 10 , and a bond to M; R 10 is a member selected from H and substituted or unsubstituted alkyl; and R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and a bond to M, wherein M comprises a linker bound to a nucleic acid carrier, which is a substrate for the polymerase, and at least one of R a , R b , R c , R d , R e , R e′ , R f , R g , R h , R i , R i , R 9 , R 12 and R 13 is a bond to M, thereby synthesizing the first polynucleotide.
  11. 11 . The method of synthesizing a first polynucleotide having a first nucleic acid sequence according to claim 1 , wherein the compound of Formula X is: wherein n is selected from the integers 1 and 2; R a is selected from H, a moiety according to Formula II: and a moiety joined with R b , together with the atoms to which R a and R b are attached, forming substituted phenyl ring C: wherein each y is an independently selected integer from 0 to 10; R u and R v are independently selected from —SO 3 H, a moiety according to Formula II, and wherein R 8 is selected from H and substituted or unsubstituted alkyl; x is an integer from 1 to 10 R b is selected from H, and a moiety joined with R a forming, together with the atoms to which R a and R b are attached, substituted phenyl ring C; z is an integer from 0 to 10; R 8 is selected from H and substituted or unsubstituted alkyl; R c is selected from H, q is an integer from 0 to 10; wherein R a , R b and R c are not all H; R d is selected from H and OCH 3 ; R e and R e′ are independently selected from wherein t is an integer from 1 to 10; R a′ is selected from H, C(O)-M, a moiety according to Formula II, and a moiety joined with R b′ forming, together with the atoms to which R a′ and R b′ are attached, substituted phenyl ring C′; R u′ and R v′ are independently selected from H, a moiety according to Formula II, —SO 3 H, and wherein each x is an integer independently selected from 0 to 10; R 10 is selected from H and substituted or unsubstituted alkyl; R w′ is selected from H and a moiety according to Formula II; R b′ is selected from H, C(O)-M, a moiety according to Formula II, a moiety joined with R a′ , together with the atoms to which R a′ and R b′ are attached, forming substituted phenyl ring C′, and wherein p is an integer from 0 to 10; R c′ is selected from H, C(O)-M and —SO 3 H, wherein R a′ , R b′ and R e′ are not all H; R d′ is selected from H and OCH 3 ; and R f , R g , R f′ and R g′ are independently selected from CH 3 , wherein each t is an independently selected integer from 1 to 10; M is a nucleic acid, and at least one of R a′ , R b′ and R c′ , R e , R e′ , R f , R g , R f′ and R g′ comprises M.
  12. 12 . A method of sequencing a first polynucleotide having a first nucleic sequence, the method of sequencing comprising: under conditions of a template dependent polymerization reaction, contacting a nucleic acid polymerase complexed with a second polynucleotide having a second nucleic acid sequence complementary to the first nucleic acid sequence with a cyanine compound of Formula X: wherein R f , R g , R h , and R i are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heteroalkyl; R c , and R d are independently selected from alkyl and heteroalkyl, substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, and phosphoric acid; a and e are independently selected from selected from the integers 0, 1, 2, 3 and 4, with the proviso that at least one member selected from a and e is 2 or greater, such that when a is 2 or greater, each R a is independently selected, and when e is 2 or greater, each R e is independently selected; wherein when a is 2 or greater, ring A is selected from phenyl substituted at least at the 4- and 6-positions with (R a ) a , and substituted or unsubstituted napthyl; and when e is 2, or more ring E is selected from phenyl substituted at least at the 4- and 6-positions with (R e ) e , and substituted or unsubstituted naphthyl; Q is a member selected from wherein n is selected from the integers from 1 to 3; R a and R e , are independently selected from C(O)R 9 , OR 12 , NR 12 R 13 , CR 12 C(O)R 13 , NR 12 C(O) 2 R 13 , SO 3 H, and C(O) NR 12 R 13 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and a ring structure formed by joining a member selected from two R a moieties together with the atoms they are attached, two R e moieties together with the atoms they are attached, and a combination thereof, to form said ring structure, which is a member selected from a substituted or unsubstituted aryl and a substituted or unsubstituted heteroaryl wherein R 9 is a member selected from OR 10 , and a bond to M wherein R 10 is a member selected from H and substituted or unsubstituted alkyl; R 11 is a nucleic acid moiety, which is a substrate for the polymerase; and R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl, and a bond to M, wherein M comprises a linker bound to a nucleic acid carrier, which is a substrate for the polymerase, and at least one of R a , R c , R d , R e , R f , R g , R h , R i , R 9 , R 12 and R 13 is a bond to M, such that under the conditions of the template dependent polymerization reaction the cyanine compound is released from the nucleic acid moiety which is a substrate for the polymerase, and is detected, thereby sequencing the first polynucleotide.
  13. 13 . The method of synthesizing a first polynucleotide having a first nucleic acid sequence according to claim 1 , wherein the compound of Formula X is selected from: wherein R f , R g , R h , and R i are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heteroalkyl; R c , and R d are independently selected from alkyl and heteroalkyl, substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, and phosphoric acid; a and e are independently selected from selected from the integers 0, 1, 2, 3 and 4, with the proviso that at least one member selected from a and e is 2 or greater, such that when a is 2 or greater, each R a is independently selected, and when e is 2 or greater, each R e is independently selected; wherein when a is 2 or greater, ring A is selected from phenyl substituted at least at the 4- and 6-positions with (R a ) a, and substituted or unsubstituted napthyl; and when e is 2, or more ring E is selected from phenyl substituted at least at the 4- and 6-positions with (R e ) e , and substituted or unsubstituted naphthyl; Q is a member selected from wherein n is selected from the integers from 1 to 3; R a and R e , are independently selected from C(O)R 9 , OR 12 , NR 12 R 13 , CR 12 C(O)R 13 , NR 12 C(O) 2 R 13 , SO 3 H, and C(O) NR 12 R 13 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and a ring structure formed by joining a member selected from two R a moieties together with the atoms they are attached, two R e moieties together with the atoms they are attached, and a combination thereof, to form said ring structure, which is a member selected from a substituted or unsubstituted aryl and a substituted or unsubstituted heteroaryl wherein R 9 is a member selected from OR 10 , and a bond to M wherein R 10 is a member selected from H and substituted or unsubstituted alkyl; R 11 is a nucleic acid moiety, which is a substrate for the polymerase; and R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl, and a bond to M, wherein M comprises a linker bound to a nucleic acid carrier, which is a substrate for the polymerase, and at least one of R a , R b , R c , R d , R e , R f , R g , R h , R i , R 9 , R 12 and R 13 is a bond to M, thereby synthesizing the first polynucleotide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 16/545,900 filed Aug. 20, 2019, which is a continuation of U.S. application Ser. No. 15/262,546 filed Sep. 12, 2016, which is a continuation of U.S. application Ser. No. 14/489,143 filed Sep. 17, 2014, which is a continuation of U.S. application Ser. No. 13/218,428 filed Aug. 25, 2011, which claims priority to Provisional Application Nos. 61/377,004, filed on Aug. 25, 2010, 61/377,022, filed on Aug. 25, 2010, 61/377,031, filed on Aug. 25, 2010, 61/377,038, filed on Aug. 25, 2010, and 61/377,048, filed on Aug. 25, 2010 the disclosures of which are incorporated herein by reference in their entirety for all purposes. FIELD OF INVENTION The present invention relates generally to the synthesis of fluorescent compounds that are analogues of cyanine dyes. The compounds of the invention are fluorophores that are derivatized to allow their facile attachment to another moiety. The invention also relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. An exemplary method involves detection of single molecules of fluorescent labels released from a nucleic acid during synthesis of an oligonucleotide. BACKGROUND There is a continuous and expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances as analytes in research and diagnostic mixtures. Of particular value are methods for measuring small quantities of nucleic acids, peptides, saccharides, pharmaceuticals, metabolites, microorganisms and other materials of diagnostic value. Examples of such materials include narcotics and poisons, drugs administered for therapeutic purposes, hormones, pathogenic microorganisms and viruses, peptides, e.g., antibodies and enzymes, and nucleic acids, particularly those implicated in disease states. The presence of a particular analyte can often be determined by binding methods that exploit the high degree of specificity, which characterizes many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. In these methods, the existence of a complex of diagnostic value is typically indicated by the presence or absence of an observable “label” which is attached to one or more of the interacting materials. The specific labeling method chosen often dictates the usefulness and versatility of a particular system for detecting an analyte of interest. Preferred labels are inexpensive, safe, and capable of being attached efficiently to a wide variety of chemical, biochemical, and biological materials without significantly altering the important binding characteristics of those materials. The label should give a highly characteristic signal, and should be rarely, and preferably never, found in nature. The label should be stable and detectable in aqueous systems over periods of time ranging up to months. Detection of the label is preferably rapid, sensitive, and reproducible without the need for expensive, specialized facilities or the need for special precautions to protect personnel. Quantification of the label is preferably relatively independent of variables such as temperature and the composition of the mixture to be assayed. A wide variety of labels have been developed, each with particular advantages and disadvantages. For example, radioactive labels are quite versatile, and can be detected at very low concentrations. However, such labels are expensive, hazardous, and their use requires sophisticated equipment and trained personnel. Thus, there is wide interest in non-radioactive labels, particularly in labels that are observable by spectrophotometric, spin resonance, and luminescence techniques, and reactive materials, such as enzymes that produce such molecules. Labels that are detectable using fluorescence spectroscopy are of particular interest because of the large number of such labels that are known in the art. Moreover, as discussed below, the literature is replete with syntheses of fluorescent labels that are derivatized to allow their attachment to other molecules, and many such fluorescent labels are commercially available. Fluorescent nucleic acid probes are important tools for genetic analysis, in both genomic research and development, and in clinical medicine. As information from the Human Genome Project accumulates, the level of genetic interrogation mediated by fluorescent probes will expand enormously. One particularly useful class of fluorescent probes includes self-quenching probes, also known as fluorescence energy transfer probes, or FET probes. The design of different probes using this motif may vary in detail. In an exemplary FET probe, both a fluorophore and a quencher are tethered to a nucleic acid. The probe is configured such that the fluorophore is proximate to the quencher a