Source: http://www.google.com/patents/US6864059?ie=ISO-8859-1&dq=inassignee:integral+inassignee:peripherals
Timestamp: 2015-04-25 01:01:20
Document Index: 360150654

Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'arts 2', 'art 4', 'ART 1', 'ART 2', 'ART 4']

Patent US6864059 - Coupling a nucleic acid labeling compound (a biotin compound attached to a ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsNucleic acid labeling compounds including the following structure are disclosed: wherein A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+; X is O; Y is OH; Z is OH; L is selected from the group consisting of �CH═CH�C(O)�NH�CH2�CH2�NH�C(O)�...http://www.google.com/patents/US6864059?utm_source=gb-gplus-sharePatent US6864059 - Coupling a nucleic acid labeling compound (a biotin compound attached to a nucleic acid base derivative containing triphosphate) with a nucleic acidAdvanced Patent SearchPublication numberUS6864059 B2Publication typeGrantApplication numberUS 10/314,012Publication dateMar 8, 2005Filing dateDec 5, 2002Priority dateJan 23, 1996Fee statusPaidAlso published asUS20030180757Publication number10314012, 314012, US 6864059 B2, US 6864059B2, US-B2-6864059, US6864059 B2, US6864059B2InventorsGlenn McGall, Anthony D. BaroneOriginal AssigneeAffymetrix, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (66), Non-Patent Citations (93), Referenced by (8), Classifications (29), Legal Events (6) External Links: USPTO, USPTO Assignment, EspacenetCoupling a nucleic acid labeling compound (a biotin compound attached to a nucleic acid base derivative containing triphosphate) with a nucleic acid
US 6864059 B2Abstract
Nucleic acid labeling compounds including the following structure are disclosed: wherein A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+; X is O; Y is OH; Z is OH; L is selected from the group consisting of �CH═CH�C(O)�NH�CH2�CH2�NH�C(O)� and �CH2�CH2�C(O)�NH�CH2�CH2�NH�C(O); M is �(CH2)45�NH�, n is 1 and Q is biotin having the structure: The labeling compounds are suitable for enzymatic attachment to a nucleic acid, either terminally or internally, to provide a mechanism of nucleic acid detection.
1. A nucleic acid labeling compound of the following structure: wherein A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+; X is O; Y is OH; Z is OH; L is selected from the group consisting of �CH═CH�C(O)�NH�CH2�CH2�NH�C(O)� and �CH2�CH2�C(O)�NH�CH2�CH2�NH�C(O); M is �(CH2)45�NH�, n is 1 and Q is biotin having the structure: 2. A nucleic acid labeling compound according to claim 1 wherein L is �CH═CH�C(O)�NH�CH2�CH2�NH�C(O)�.
3. A nucleic acid derivative produced by coupling a nucleic acid labeling compound according to claim 1 with a nucleic acid.
4. A hybridization product, wherein the hybridization product comprises the nucleic acid derivative according to claim 3 bound to a complementary probe.
5. The hybridization product according to claim 4, wherein the probe is attached to a glass chip.
6. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 1 to a nucleic acid.
7. A method of detecting a nucleic acid comprising incubating a nucleic acid derivative according to claim 3 with a probe.
8. A method according to claim 7, wherein the probe is attached to a glass chip.
9. A nucleic acid labeling compound of the following structure: wherein A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+; X is O; Y is OH; Z is OH; L is selected from the group consisting of �CH═CH�C(O)�NH�CH2�CH2�NH�C(O)� and �CH2�CH2�C(O)�NH�CH2�CH2�NH�C(O); M is �(CH2)45�NH�, n is 1 and Q is biotin having the structure: 10. A nucleic acid labeling compound according to claim 9 wherein L is �CH═CH�C(O)�NH�CH2�CH2�NH�C(O)�.
11. A nucleic acid derivative produced by coupling a nucleic acid labeling compound according to claim 9 with a nucleic acid.
12. A hybridization product, wherein the hybridization product comprises the nucleic acid derivative according to claim 11 bound to a complementary probe.
13. The hybridization product according to claim 12, wherein the probe is attached to a glass chip.
14. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 9 to a nucleic acid.
15. A method of detecting a nucleic acid comprising incubating a nucleic acid derivative according to claim 11 with a probe.
16. A method according to claim 15, wherein the probe is attached to a glass chip.
17. A nucleic acid labeling compound having the structure wherein A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+, Y is OH; Z is OH; L is �C(O)NH(CH2)2NH�, M is selected from the group consisting of �C(O)(CH2)5NH� and �C(O)((CH2)2O)4(CH2)2NH�, n is 1 and Q is biotin, having the structure: 18. A nucleic acid labeling compound according to claim 17 wherein M is �C(O)(CH2)5NH�.
19. A nucleic acid derivative produced by coupling a nucleic acid labeling compound according to claim 17 with a nucleic acid.
20. A hybridization product, wherein the hybridization product comprises the nucleic acid derivative according to claim 19 bound to a complementary probe.
21. The hybridization product according to claim 20, wherein the probe is attached to a glass chip.
22. A hybridization product, wherein the hybridization product comprises the nucleic acid derivative according to claim 18 bound to a complementary probe.
23. The hybridization product according to claim 22, wherein the probe is attached to a glass chip.
24. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 17 to a nucleic acid.
25. A method of detecting a nucleic acid comprising incubating a nucleic acid derivative according to claim 19 with a probe.
26. A method according to claim 25, wherein the probe is attached to a glass chip.
This application is a continuation-in-part of U.S. application Ser. No. 10/097,113, filed Mar. 12, 2002; and a continuation-in-part of U.S. application Ser. No. 09/952,387, filed Sep. 11, 2001; Ser. No. 09/780,574, filed Feb. 9, 2000, now U.S. Pat. No. 6,596,856 issued Jul. 22, 2003; U.S. application Ser. No. 09/126,645, filed Jul. 31, 1998, now abandoned; and a continuation-in-part of U.S. Ser. No.: 08/882,649, Filed: Jun. 25, 1997, now U.S. Pat. No. 6,344,316 issued Feb. 5, 2002 which is a continuation of PCT/US97/01603, filed on Jan. 22, 1997 designating the Unites States of America, which claims priority from U.S. Provisional Application No. 60/010,471 filed on Jan. 23, 1996 and U.S. Provisional Application No. 60/035,170, filed on Jan. 9, 1997, all of which are herein incorporated by reference for all purposes.
The labeling of a nucleic acid is typically performed by covalently attaching a detectable group (label) to either an internal or terminal position. Scientists have reported a number of detectable nucleotide analogues that have been enzymatically incorporated into an oligo- or polynucleotide. Langer et al., for example, disclosed analogues of dUTP and UTP that contain a covalently bound biotin moiety. Proc. Natl. Acad. Sci. USA 1981, 78, 6633-6637. The analogues, shown below, possess an allylamine linker arm that is attached to the C-5 position of the pyrimidine ring. The dUTP and UTP analogues, wherein R is H or OH, were incorporated into a polynucleotide. Petrie et al. disclosed a dATP analogue, 3-[5-[(N-biotinyl-6-aminocaproyl)-amino]pentyl]-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine-5′-triphosphate. Bioconjugate Chem. 1991, 2, 441-446. The analogue, shown below, is modified at the 3-position with a linker arm that is attached to a biotin moiety. Petrie et al. reported that the compound wherein R is biotin is incorporated into DNA by nick translation. Prober et al. disclosed a set of four dideoxynucleotides, each containing a succinylfluorescein dye. Science 1987, 238, 336-341. The dideoxynucleotides, one of which is shown below, were enzymatically incorporated into an oligonucleotide through a template directed extension of a primer. The compounds provided for a DNA sequencing method based on gel migration. Herrlein et al. disclosed modified nucleoside trisphosphates of the four DNA bases. Helv. Chim. Acta 1994, 77, 586-596. The compounds, one of which is shown below, contain a 3′-amino group containing radioactive or fluorescent moieties. Herrlein et al. further described the use of the nucleoside analogues as DNA chain terminators. Cech et al. disclosed 3′-amino-functionalized nucleoside triphosphates. Collect. Czech. Chem. Commun. 1996, 61, S297-S300. The compounds, one of which is shown below, contain a fluorescein attached to the 3′-position through an amino linker. Cech et al. proposed that the described functionalized nucleosides would be useful as terminators for DNA sequencing. SUMMARY OF THE INVENTION
A�O�CH2�T�Hc�L�(M)m�Q
X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is is amido alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3. In another embodiment, A is H or H4O9P3�; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is �C(O)NH(CH2)nNH�, wherein n is an integer ranging from about 2 to about 10; Q is biotin or a carboxyfluorescein; and, M is �CO(CH2)5NH�, wherein m is 1 or 0.
In one embodiment, the nucleic acid labeling compounds have the following structures: wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is functionalized alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H4O9P3�; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is �(CH2)nC(O)�, wherein n is an integer ranging from about 1 to about 10; Q is biotin or fluorescein; and, M is �NH(CH2CH2O)kNH�, wherein, k is an integer from 1 to about 5, wherein m is 1 or 0. preferably k is 1 or 2;
In another embodiment, Y is H or OH; Z is H or OH; L is �CH2�C(O)�; Q is a carboxyfluorescein or biotin; and M is �NH(CH2CH2O)kNH�, wherein, k is 2 and m is 1.
In another embodiment, Y is OH; Z is OH; L is �CH2�C(O)�; Q is biotin; and M is �NH(CH2CH2O)kNH�, wherein, k is 2 and m is 1.
In another embodiment,; L is �CH═CHCH2NH�; Q is a carboxyfluorescein; and M is �NH(CH2CH2O)kNH�, wherein, k is 2 and m is 1.
X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is functionalized alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3. In another embodiment, A is H or H4O9P3�; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is �(CH2)nC(O)�, wherein n is an integer ranging from about 1 to about 10; Q is biotin or a fluorescein; and, a first M is �NH(CH2)nNH�, wherein n is an integer from about 2 to about 10, and a second M is �CO(CH2)5NH�, wherein m is 1 or 2.
In another embodiment, Y is H or OH; Z is H or OH; L is �(CH2)2C(O)�, Q is biotin or a carboxyfluorescein; and a first M is �NH(CH2)2NH�, and a second M is �CO(CH2)5NH�, wherein m is 2.
In another embodiment, Y is OH; Z is OH; L is �(CH2)2C(O)�, Q is a carboxyfluorescein; and, a first M is �NH(CH2)2NH�, and a second M is �CO(CH2)5NH�, wherein m is 2.
In another embodiment, Y is OH; Z is OH; L is �(CH2)2C(O)�, Q is or biotin; and, a first M is �NH(CH2)2NH�, and a second M is �CO(CH2)5NH�, wherein m is 2.
In another embodiment, wherein A is a functional group the permits the attachment of the nucleic acid labeling compound to a nucleic acid; preferably, A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+; X is O; Y is OH; Z is OH; L is selected from the group consisting of �CH═CH�C(O)�NH�CH2�CH2�NH�C(O)� and �CH2�CH2�C(O)�NH�CH2�CH2�NH�C(O); M is �(CH2)4�NH� and Q is biotin having the structure: In one embodiment, the nucleic acid labeling compounds have the following structures: wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;
X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is amido alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3. In another embodiment, A is H or H4O9P3�; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is �C(O)NH(CH2)nNH�, wherein n is an integer ranging from about 2 to about 10; Q is biotin or a fluorescein; wherein m is 0, 1, or 2.
In another embodiment, Y is H or OH; Z is H or OH; L is �C(O)NH(CH2)4NH�; and Q is biotin or a carboxyfluorescein.
In another embodiment, Y is OH; Z is H; L is �C(O)NH(CH2)4NH�; Q is biotin.
In another embodiment, Y is OH; Z is H; L is �C(O)NH(CH2)4NH�; and Q is a carboxyfluorescein.
In another embodiment, wherein A is a functional group the permits the attachment of the nucleic acid labeling compound to a nucleic acid; preferably, A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+, Y is OH, Z is H or OH, L is �C(O)NH(CH2)2NH�, M is �C(O)(CH2)5NH�, n is 1 and Q is biotin, having the structure: In another embodiment, wherein A is a functional group the permits the attachment of the nucleic acid labeling compound to a nucleic acid; preferably, A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+, Y is OH, Z is H or OH, L is �C(O)NH(CH2)2NH�, M is �C(O)((CH2)2O)4(CH2)2NH�, n is 1 and Q is biotin, having the structure: In another aspect of the present invention, a method for preparing a labeled nucleic acid sample is provided having the steps of: providing a nucleic acid sample, the nucleic acid sample having DNA; reacting the nucleic acid sample in the presence of an enzymatic quantity of the enzyme terminal transferase with the preceding nucleic acid labeling compound. Preferably, according to the instant invention that nucleic acid sample is cDNA.
In one embodiment, the nucleic acid labeling compounds used in the coupling have the following structures: wherein A is H or H4O9P3�; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is �C(O)NH(CH2)nNH�, wherein n is an integer ranging from about 2 to about 10; Q is biotin or a carboxyfluorescein; and, M is �CO(CH2)5NH�, wherein m is 1 or 0.
FIG. 3 shows a synthetic route to fluorescein and biotin labeled i-(2,3-dideoxy-D-glycero-pentafuranosyl)imidazole-4-carboxamide nucleotides.
FIG. 11 shows graphical comparisons of observed hybridization fluorescence intensities using Biotin-M-ddITP (wherein M=aminocaproyl) and Biotin-N-6-ddATP.
FIG. 16 shows a schematic for the preparation of Ni-labeled 5-(β-D-ribofuranosyl)-2,4[1H,3H]-pyrimidinedione 5′-triphosphate.
FIG. 18 a shows various labeling reagents. FIG. 18 b shows still other labeling reagents. FIG. 18 c shows non-ribose or non-2′-deoxyribose-containing labels. FIG. 18 d shows sugar-modified nucleotide analogue labels 18 d. FIG. 19 shows HIV array data for analog 42a (T7 labeling of RNA target).
The connecting groups (M)m may serve to covalently attach the linker group (L) to the detectable moiety (Q). Each M group can be the same or different and can independently be any suitable structure that will not interfere with the function of the labeling compound. Nonlimiting examples of M groups include the following: amino alkyl, �CO(CH2)5NH�, �CO�, �CO(O)�, �CO(NH)�, �CO(CH2)5NHCO(CH2)5NH�, �NH(CH2CH2O)kNH�, and �CO(CH2)5�; wherein, k is an integer from 1 to about 5, preferably k is 1 or 2; m is an integer ranging from 0 to about 5, preferably 0 to about 3.
In one embodiment, the nucleic acid labeling compounds of the present invention are of the following structure: wherein L is a linker moiety; Q is a detectable moiety; X is O, S, NR1 or CHR2; Y is H, N3, F, OR9, SR9 or NHR9; Z is H, N3, F or OR10; H, is a heterocyclic group; A is H or a functional group that permits the attachment of the nucleic acid label to a nucleic acid; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3. The substituents R1, R2, R9 and R10 are independent of one another and are H, alkyl or aryl.
In a preferred embodiment, the heterocyclic group (H,) is a C3 substituted 4-aminopyrazolo[3,4-d]pyrimidine and the linking group is an alkynyl alkyl: wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; n is an integer ranging from 1 to about 10; R5 is O or NH; A is hydrogen or H4O9P3�; Q is biotin or carboxyfluorescein; M is �CO(CH2)5NH�, wherein m is 1 or 0. More preferably, Y and Z are OH; n is 1; R5 is NH; A is H4O9P3�; and, Q is biotin or 5- or 6-carboxyfluorescein, wherein m is 1.
In a preferred embodiment, the heterocyclic group (H,) is an N4 substituted 4-amino-pyrazolo[3,4-d]pyrimidine and the linking group is an amino alkyl: wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; n is an integer ranging from about 2 to about 10; A is hydrogen or H4O9P3�; Q is biotin or carboxyfluorescein; M is �CO(CH2)5NH� or �CO(CH2)5NHCO(CH2)5NH�, wherein m is 1 or 0. More preferably, Y and Z are hydrogen; n is 4; A is H4O9P3�; Q is biotin or 5- or 6-carboxyfluorescein, wherein m is 0.
In a preferred embodiment, the nucleic acid labeling compounds have the formulas: wherein Q is biotin or a carboxyfluorescein.
In another embodiment, the nucleic acid labeling compounds have the formulas: wherein R11 is hydrogen, hydroxyl, a phosphate linkage, or a phosphate group; R12 is hydrogen or hydroxyl; R13 is hydrogen, hydroxyl, a phosphate linkage, or a phosphate group; and R14 is a coupled labeled moiety.
In another embodiment, wherein A is a functional group the permits the attachment of the nucleic acid labeling compound to a nucleic acid; preferably, A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+, Y is OH, Z is H or OH, L is �C(O)NH(CH2)2NH�, M is �C(O)(CH2)5NH�, n is 1 and Q is biotin, having the structure: In another embodiment, wherein A is a functional group the permits the attachment of the nucleic acid labeling compound to a nucleic acid; preferably, A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+, Y is OH, Z is H or OH, L is �C(O)NH(CH2)2NH�, M is �C(O)((CH2)2O)4(CH2)2NH�, n is 1 and Q is biotin, having the structure: Synthesis of Nucleic Acid Labeling Compounds
FIG. 3 shows a synthetic route to nucleic acid labeling compounds 8a and 8b, in which the heterocyclic group (Hc) is an imidazole and the linker moiety (L) is an amido alkyl. The silyl protected imidazole (2) was added to pentofuranose (1) to provide a mixture of carboethoxyimidazole dideoxyriboside isomers (3a-3d). The isomers were separated to afford purified 3c. The carboethoxy group of 3c was converted into an amino carboxamide (4) upon treatment with a diamine. The terminal amine of 4 was protected to give the trifluoroacetylated product 5. The silyl protecting group of 5 was removed, providing the primary alcohol 6. Compound 6 was converted into a 5′-triphosphate to afford 7. The trifluoroacetyl protecting group of 7 was removed, and the deprotected amine was reacted with biotin-NH(CH2)5CO�NHS or 5-carboxyfluorescein-NHS giving, respectively, nucleic acid labeling compounds 8a and 8b.
Nucleoside 6 was converted to a 5′-triphosphate, deprotected, reacted with biotin-NH(CH2)5CO�NHS or 5-carboxyfluorescein-NHS and purified according to procedures reported elsewhere (see, Prober, J. M., et al., 1988, PCT 0 252 683 A2) to give the labeled nucleotides 8a,b in >95% purity by HPLC, 31P-NMR.
The synthesis of 3-iodo-4-aminopyrazolo[3,4-d]pyrimidine ribofuranside (9) was carried out as described by H. B. Cottam, et al. 1993, J. Med. Chem. 36:3424. Using the appropriate deoxyfuranoside precursors, both the 2′-deoxy and 2′,3′-dideoxy nucleosides are prepared using analogous procedures. See, e.g., U. Neidballa & H. Vorbruggen 1974, J. Org. Chem. 39:3654; K. L. Duehom & E. B. Pederson 1992, Synthesis 1992: 1). Alternatively, these are prepared by deoxygenation of ribofuranoside 9 according to established procedures. See, M. J. Robins et al. 1983 J. Am. Chem. Soc. 103:4059; and, C. K. Chu, et al. 1989 J. Org. Chem. 54:2217.
A protected propargylamine linker was added to the 4-aminopyrazolo[3,4-d]pyrimidine nucleoside (9) via organopalladium-mediated substitution to the 3-position of 4-aminopyrazolo[3,4-d]pyrimidine riboside using the procedure described by Hobbs (J. Org. Chem. 54: 3420; Science 238: 336.). Copper iodide (38 mg; 0.2 mmole), triethylamine (560 uL; 4.0 mmole), N-trifluoroacetyl-3-aminopropyne (700 uL; 6.0 mmole) and 3-iodo-4-aminopyrazolo[3,4-d]pyrimidine ∃-D-ribofuranoside (9) (H. B. Cottam, et al., 1993, J. Med. Chem. 36: 3424.) (786 mg; 2.0 mmole) were combined in 5 ml of dry DMF under argon. To the stirring mixture was added tetrakis(triphenylphosphine) palladium(0) (232 mg; 0.2 mmole). The solution became homogeneous within 10 minutes, and was left stirring for an additional 4 hours in the dark, at which time the reaction was diluted with 20 mL of MeOH-DCM (1:1), 3.3 g of Dowex AG-1 anion exchange resin (bicarbonate form) was added, and stirring was continued for another 15 minutes. The resin was removed by filtration and washed with MeOH-DCM (1:1), and the combined filtrates were evaporated to dryness. The residue was dissolved in 4 mL of hot MeOH, then 15 mL DCM was added and the mixture kept warm to maintain a homogeneous solution while it was loaded onto a 5 cm�25 cm column of silica gel that had been packed in 1:9 MeOH-DCM. The product (Rf�0.4, 6:3:1:1 DCM-EtOAc-MeOH-HOAc) was eluted with a 10-15-20% MeOH-DCM step gradient. The resulting pale yellow solid was washed 3� with 2.5 ml of ice-cold acetonitrile, then 2� with ether and dried in vacuo to obtain 630 mg (75%) of 4-amino-3-(N-trifluoroacetyl-3-aminopropynyl)pyrazolo[3,4-d]pyrimidine β-D-ribofuranoside (10). Identity of the product was confirmed by 1H-nmr, mass spectrometry and elemental analysis.
The nucleoside was converted to the triphosphate using the Eckstein phosphorylation procedure (Ludwig, J. L.; Eckstein, F. J. Org. Chem. 1989, 54, 631-635) followed by HPLC purification on a ResourceQ anion exchange column (buffer A is 20 mM Tri pH 8, 20% CH3CN and buffer B is 20 mM Tris pH 8, 1 M NaCl, 20% CH3CN). 31P-NMR, UV and MS data were consistent with the structure of the triphosphate. The trifluoroacetyl-protecting group was removed by treatment with excess NH4OH at 55� C. for 1 hr. followed by evaporation to dryness. The mass spectral data were consistent with the aminobutyl nucleotide 17. Without further purification, the nucleotide was treated with either Biotin-NHS esters or 5-Carboxyfluorescein-NHS as described elsewhere (Prober, J. M., et al., 1988, PCT 0 252 683 A2) to form the labeled nucleotides 18a-18d, respectively, which were purified by HPLC as described (Prober, J. M., et al., 1988, PCT 0 252 683 A2) except that, in the case of 18a, the buffer was 20 mM sodium phosphate pH 6. The 31P-NMR and UV data were consistent with the structure of the labeled analogs.
Nucleoside 30 is converted to a 5′-triphosphate, deprotected, reacted with oxysuccinimidyl-(N-biotinoyl-6-amino)hexanoate or oxysuccinimidyl-(N-(fluorescein-5-carboxy)-6-amino)hexanoate and purified according to procedures reported elsewhere (Prober, J. M., et al. 1988, PCT 0 252 683 A2), giving, respectively, the biotin- and fluorescein-labeled nucleotides 31a and 31b.
Labeling Effeciency
% Labeling Effecieny
�C(O)(CH2)5NH- Biotin
�C(O)(CH2)5NH- trifluoroacetyl
control OH
R = 5-carboxyfluoroscein
R = -biotin 5-carboxyfluoroscein
analogs: H
R = -biotin CO(CH2)5NH-biotin �CO(CH2)5NHCO(CH2)5NH- biotin 5-carboxyfluoroscein
48 41 57 58
R = -biotin 5-carboxyfluoroscein 6-carboxyfluoroscein
25 53 37
84 97 86 OH
R = �CO(CH2)5NH-biotin 5-carboxyfluoroscein 6-carboxyfluoroscein
100 94 73
max12 97 99
R = -biotin �CO(CH2)5NH-biotin �CO(CH2)5NHCO(CH2)5NH- biotin 5-carboxyfluoroscein
85 98 93
R = �CO(CH2)5NH-biotin �CO(CH2)5NH-fluoroscein
R = �CO(CH2)5NH-biotin 5-carboxyfluoroscein
Hybridization Studies of Labeled Imidazole Carboxamide (�ITP�) and 4-aminopyrazolo[3,4-d]pyrimidine (�APPTP) Nucleotides.
The performance of the labeled imidazolecarboxamide and 4-aminopyrazolo[3,4-d]pyrimidine nucleotides was evaluated in a p53 assay using standard GeneChip� product protocols (Affymetrix, Inc., Santa Clara, Calif.), which are described, for example, in detail in the GeneChip� p53 assay package insert. The sample DNA used in these experiments was the plasmid �p53mut248.� The labeled nucleotide analog was substituted for the usual labeling reagent (Fluorescein-N-6-ddATP or Biotin-M-N-6-ddATP (wherein M=aminocaproyl), from NEN, part #'s NEL-503 and NEL-508, respectively). Labeling reactions were carried out using both the standard amount of TdT enzyme specified in the assay protocol (25 U) and with 100 U of enzyme. After labeling, Fluorescein-labeled targets were hybridized to the arrays and scanned directly. In experiments using the biotin-labeled targets, the GeneChip� chips were stained in a post-hybridization step with a phycoerythrin-streptavidin conjugate (PE-SA), prior to scanning, according to described procedures (Science 280:1077-1082 (1998)).
FIG. 9 shows comparisons of the observed hybridization fluorescence intensities for the 1300 bases called in the �Unit-2� part of the chip. In the lower plot, intensities for the Fluorescein-ddITP (8b) labeled targets are plotted against those for the standard Fluorescein-N6-ddATP labeled targets (control), both at 25 U of TdT. The observed slope of �0.75 indicates that the labeling efficiency of 8b was about 75% of that of Fluorescein-N6-ddATP under these conditions. In the upper plot, the same comparison is made, except that 100 U of TdT was used in the 8b labeling reaction. The slope of �1.1 indicates equivalent or slightly better labeling than the standard Fluorescein-N6-ddATP/25 U control reaction.
FIG. 10 shows comparisons of the observed hybridization fluorescence intensities for the 1300 bases called in the �Unit-2� part of the chip. Intensities for the Biotin-(M)2-ddAPPTP (18c, M=aminocaproyl linker; referred to as Biotin-N4-ddAPPTP in FIG. 10) labeled targets (after PE-SA staining) are plotted against those for the standard Biotin-M-N6-ddATP labeled targets (control), both at 25 U of TdT. The observed slope of −0.3 indicates that the labeling efficiency with Biotin-(M)2-ddAPPTP (18c) was about 30% of that of Biotin-M-N6-ddATP under these conditions.
FIG. 11 shows comparisons of the observed hybridization fluorescence intensities for the 1300 bases called in the �Unit-2� part of the chip. In the lower plot, intensities for the Biotin-M-ddITP (8a, M=aminocaproyl; referred to as Bio-ddITP in FIG. 11) labeled targets are plotted against those for the standard Biotin-M-N6-ddATP labeled control targets, both at 25 U of TdT. The observed slope of �0.4 indicates that the labeling efficiency with 8a was about 40% of that of Biotin-M-N6-ddATP under these conditions. In the upper plot, the same comparison is made, except that 100 U of TdT was used in the 8a labeling reaction. The slope of �1.1 indicates equivalent or slightly better labeling than the standard Biotin-M-N6-ddATP/25 U control reaction.
FIG. 12 shows a comparison of the overall re-sequencing (base-calling) accuracy, for both strands, obtained using Fluorescein-ddITP labeled targets at both 25 U and 100 U of TdT, as well as the standard Fluorescein-N6-ddATP/25 U TdT labeled �control� targets. FIG. 13 shows a similar comparison for the targets labeled with biotin-M-ddITP (8a; referred to as Biotin-ddITP in FIG. 13) and biotin-M-N6-ddATP �control,� followed by PE-SA staining. FIG. 14 shows a comparison of re-sequencing accuracy using Biotin-(M)2-ddAPPTP/100 U TdT and Biotin-M-N6-ddATP/25 U TdT. These data indicate that labeled imidazolecarboxamide and 4-aminopyrazolo[3,4-d]pyrimidine dideoxynucleotide analogs can be used for DNA target labeling in hybridization-based assays and give equivalent performance to the standard labeled-N-6-ddATP reagent.
The performance of the biotin-labeled imidazolecarboxamide and 4-aminopyrazolo[3,4-d]pyrimidine nucleotides (�biotin-M-ITP� (8a) and �biotin-(M)2-APPTP� (18c)) was evaluated using a single-nucleotide polymorphism genotyping GeneChip� chip array. Published protocols (D. G. Wang, et al., 1998, Science 280: 1077-82.) were used in these experiments, except for the following variations: 1) labeling reactions were carried out using both the standard amount of TdT enzyme specified in the published protocol (15U), or three-fold (45 U) enzyme; 2) substitution of the labeled nucleotide analog for the standard labeling reagent (Biotin-N6-ddATP, from NEN: P/N NEL-508); 3) the labeled nucleotide analog was used at either twice the standard concentration specified in the published protocol (25 uM), or at six-fold (75 uM). After labeling, biotin-labeled targets were hybridized to the arrays, stained with a phycoerythrin-streptavidin conjugate (PE-SA), and the array was scanned and analyzed according to the published procedure.
(M)2- ddAppTP
It was determined that TdT was generally tolerant of base substitutions, and that ribonucleotides were about as efficiently incorporated as 2′-deoxy, and 2′, 3′-dideoxynucleotides. In contrast, T7 was relatively intolerant of heterocyclic base substitutions with the exception of the 5-(1,3-pyrimidine-2,4-dione), i.e. the pseudo-uridine analog. Two new reagents, a C4-labeled 1-(2′,3′-didexoy-∃-D-ribofuranosyl) imidazole-4 carboxamide 5′-triphophate and an N1-labeled pseudo-uridine 5′-triphophate, were found to be excellent substrates for TdT and T7, respectively. These new analogs prove array assay performance equivalent to that obtained using conventional labeling reagents.
To 0.5 μmoles (50 μL of a 10 mM solution) of the amino-derivatized nucleotide triphosphate, 3′amino-3′deoxythymidinetriphosphate (1) or 2′-amino-2′-deoxyuridine triphosphate (2), in a 0.5 ml ependorf tube was added 25 μL of 11 M aqueous solution of sodium borate, pH 7, 87 μL of methanol, and 88 μL (10 μmol, 20 wquiv) of a 100 mM solution of 5-carboxyfluorescein-X�NHS ester in methanol. The mixture was vortexed briefly and allowed to stand at room temperature in the dark for 15 hours. The sample was then purified by ion-exchange HPLC to afford the fluoresceinated derivatives Formula 3 or Formula 4, below, in about 78-84% yield. Experiments suggest that these molecules are not substrates for terminal transferase (TdT). It is believed, however, that these molecules would be sutstrates for a polymerase, such as klenow fragment.
The analogs as-triazine-3,5[2H,4H]-dione (�6-aza-pyrimidine�) nucleotides (see, FIG. 23 a) are synthesized by methods similar to those used by Petrie, et al., Bioconj. Chem. 2: 441 (1991).
Other suitable labels include non-ribose or non-2′-deoxyribose-containing structures some of which are illustrated in FIG. 23 c and sugar-modified nucleotide analogues such as are illustrated in FIG. 23 d. Using the guidance provided herein, the methods for the synthesis of reagents and methods (enzymatic or otherwise) of label incorporation useful in practicing the invention will be apparent to those skilled in the art. See, for example, Chemistry of Nucleosides and Nucleotides 3, Townsend, L. B. ed., Plenum Press, New York, at chpt. 4, Gordon, S. The Synthesis and Chemistry of Imidazole and Benzamidizole Nucleosides and Nucleotides (1994); Gen Chem. Chemistry of Nucleosides and Nucleotides 3, Townsend, L. B. ed., Plenum Press, New York (1994); can be made by methods simliar to those set forth in Chemistry of Nucleosides and Nucleotides 3, Townsend, L. B. ed., Plenum Press, New York, at chpt. 4, Gordon, S. �The Synthesis and Chemistry of Imidazole and Benzamidizole Nucleosides and Nucleotides (1994); Lopez-Canovas, L. Et al., Arch. Med. Res 25: 189-192 (1994); Li, X., et al., Cytometry 20: 172-180 (1995); Boultwood, J. Et al., J. Pathol. 148: 61 ff. (1986); Traincard, et al., Ann. Immunol. 1340: 399-405 (1983).
To 5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 39 (100 mg, 0.41 mmol, 1 eq.) in acetonitrile (5 ml) was added 1 M TEAB, pH 9 (5 ml) followed by methyl acrylate (5.5 ml, 61 mmol, 150 eq). The reaction was stirred at room temperature overnight. The solvents were evaporated, and the residue was coevaporated with water (3�, 5 ml) yielding 135 mg of acrylate 40. The acrylate 40 was then treated with neat ethylenediamine (2 ml, excess) and two drops of TEA and heated to 100� C. After 1 hour the excess EDA was evaporated, yielding 146 mg of the free amine (quantitative). The crude residue was then co-evaporated with pyridine (3�, 5 ml, insoluble), resuspended in a mixture of pyridine and DMF and was cooled to 0� C. To this mixture was added TFA-imidazole (73.8 mg, 1.1 eq.). The reaction was then allowed to warm to room temperature and stirred overnight. An additional 1 eq. of TFA-imidazole was added at this time and the reaction was stirred an additional 15 minutes. The solvent was then evaporated, and the residue was co-evaporated with water(2�, 5 ml) and dissolved in 5 ml of water. The white precipitate that formed was removed by filtration. The mother liquor, which contained the TFA-protected nucleoside 3, was separated into two aliquots and purified by reverse phase HPLC. The fractions were then pooled and evaporated to yield 20% (35 mg) of pure 41, which was verified by ′H NMR. Using standard procedures (eg., Prober, et al., EP 0252683), compound 41 was converted to the triphosphate, which was then conjugated to biotin and fluorescein to afford 42a and 42b.
The IVT incorporation efficiency (the number of labeled analogs incorporated per transcript) of the N1-fluorescein-X-5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5′-triphosphate 42a was measured by HPLC (diode array UV detection at 260 nm and 495 nm) in an IVT amplification of a 1.24 kb transcript. See U.S. patent application Ser. No. 09/126,645 for additional details on test methods used. Table 1 summarizes the data obtained using different ratios of UTP/5 At a ratio of 1:5, the incorporation and relative yield (measured relative to the yield obtained with UTP only) of transcript are optimal. This transcript was compared in a hybridization assay to transcript labeled using fluorescein. The preliminary results indicated that the N1-fluorescein-X-5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5′-triphosphate (42a) performed equivalently in a hybridization assay in terms of number of correct calls and in hybridization intensity (Charts 2 and 3). The hybridization assay used for this purpose was the Affymetrix HIV-PRT GeneChip assay (see Kozal, et al. Nature Medicine 1996, 2: 753-9.).
Similarly, the efficiency of DNA 3′-end labeling of a polythymidylate oligonucleotide (T16) using terminal deoxynucleotidyl transferase and N1-fluorescein and biotin-labeled 5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5′-triphosphate, was determined by HPLC. In this analysis, the percent conversion of oligo-T16 to the 3′-end labeled T16-Fl, is determined by AX-HPLC (see U.S. patent application Ser. No. 09/126,645 for detailed procedures). The data is summarized in Chart 4. The incorporation of the biotin and fluorescein triphosphates was very efficient as determined by HPLC. CHART 1
Incorporation efficiency of N1-fluorescein-labeled 5-(β-D-ribofuranosyl)-
2,4(1H,3H)-pyrimidinedione 5′-triphosphate 42a, determined by HPLC
CHART 2 Call accuracy of N1-fluorescein-labeled 5-(β-D-ribofuranosyl)-2,4(1H, 3H)-pyrimidinedione 5′-triphosphate 42a. Data was obtained from Affymetrix HIV-PRT GeneChip hybridization assay (see Kozal, et al. Nature Medicine 1996, 2: 753-9.).
Data was obtained from hybridization of labeled transcript to the Affymetrix HIV-PRT GeneChip array (see Kozal, et al. Nature Medicine 1996, 2: 753-9.).
CHART 4 TdT labeling efficiency of Fluorescein and Biotin labeled 5-(βD-ribofuranosyl)- 2,4(1H,3H)-pyrimidinedione 5′-triphosphate 42a and 42b, determined by HPLC. Reaction conditions: TdT (40 units), 20 uM U*TP and 3.2 uM T16 oligo in 50 ul of water. Heated at 37� C. for 1 hour and 70� C. for 10 min., followed by 1 ul of 100 mM EDTA. HPLC analysis was performed on a Dionex DNAPac� PA-100 column.
99 X=OH, Y=NH2 Z=H
98 X=NH2, Y=H, Z=CH3 97 X=OH, Y=NHCO(CH2)5NHCOFL, Z=H
96 X=NHCO(CH2)5NHCOFL, Y=H, Z=CH3 To 0.5 umoles (50 uL of a 10 mM solution) of the amino nucleotide triphosphate (1 or 2) in a 0.5 mI ependorf tube was added 25 ul of a 1 M aqueous solution of sodium borate, pH 8, 87 uL of methanol, and 88 uL (10 mmol, 20 equiv) of a 100 mM solution of 5-carboxyfluorescein-X�NHS ester in methanol. The mixture was vortexed briefly and allowed to stand at room temperature in the dark for 15 hours. The sample was then purified by ion-exchange HPLC to afford the fluoresceinated derivatives 3 or 4 in about 78-84% yield. Relative efficiencies of incorporation of these compounds by TdT are shown in Table 5.
NHCO(CH2)5NH�(CO�FL)
O(CH2)6NHCO�(CH2)6-NHCO�
N1-(di-O-acetylfluorescein-5-carboxamido)ethyl-3-deoxy-4-O-acetyl-6-O-dimethoxytrityl allonamide 43 (U.S. patent application Ser. No. 08/574,461) was detritylated with 80% acetic acid, and the crude product was purified on a small silica gel column to obtain N1-(di-O-acetylfluorescein-5-carboxamido)ethyl-3-deoxy-4-O-acetyl allonamide 44. The allonamide was phosphorylated using POCl3 followed by reaction with pyrophosphate (Bogachev, Russ. J. Bioorg. Chem. 1996, 22: 559-604). The crude product was treated with NH4OH to remove the acetyl protecting groups, then purified using a preparative Source QTM AX-HPLC column. Pure fractions (analysed by analytical ion-exchange HPLC) were pooled and evaporated to near-dryness. The triphosphate salt 45a was precipitated with MeOH-acetone and dried under high vacuum to obtain a product which was 98% pure by ion-exchange HPLC and 31p NMR.
Morpholino-uracil tosylate salt 1 (30 mg) was co-evaporated with pyridine (3�3 ml) and dissolved in 2 ml of pyridine and cooled to 0� C. Trifluoroacetic anhydride (30 uL) was added and stirred for 1 hour. The reaction was followed by HPLC until complete. The pyridine was removed and the residue was dissolved in 1 ml of water and filtered. The product was purified by HPLC on a Waters C-18 bondapak cartridge (Buffer: A=50 mM TEM pH 7.0; B=acetonitrile) using a gradient of 0-25% B in 30 minutes (retention time=22 min.). The product was desalted on a Sep-Pak cartridge and freeze-dried to give 15I mg of 2. Phosphorylation of 2 using the POCl3 method gave 3. The removal of the trifluoroacetyl group with conc. NH4OH at 50� C. for 30 min to 4 followed by conjugation to 5-carboxyfluoroscein-aminocaproic acid N-hydroxysucciimide (Fl-X-NHS) under standard conditions gave thE amide 5. The mass spectral and NMR data for compounds 1-5 were consistent with the proposed structures. Example 17
Synthesis of Biotin-ΨisoCTP, Propenamide-linked (Scheme 30)
Peracetylated Pseudoisocytidine (2)
Pseudoisocytidine (1) (2.5 g, 9 mmoles) was dissolved in 40 ml dry pyridine. Acetic anhydride (8.5 ml, 90 mmoles) was added and the mixture was stirred under argon for at least 4 hours at room temperature. The reaction can be monitored by HPLC (C18 column, buffer A: 0.1M TEAA, pH 7.5; buffer B: acetonitrile; gradient: 5-95% B over 20 minutes). The pyridine was removed under vacuum and the residual oil was dissolved in 500 ml of ethyl acetate. More ethyl acetate may be added to get a clear solution since the product has limited solubility in ethyl acetate. The organic phase was washed three times with brine and dried over anhydrous Na2SO4, filtered and the solvent removed. The white solid was recrystallized from ethyl acetate/hexane yielding 3.2 g (85%) of 2.
Compound 3 (0.85 g, 1.7 mmoles) was dissolved in chloroform (5 ml) and aqueous concentrated hydrochloric acid (conc., 10 ml) was added. The rosy red solution turned a lemon yellow instantly. The reaction was stirred at room temperature for an additional 48 hours or until the reaction was complete as determined by RP-HPLC (C18 column, buffer A: 0.1M TEAA, pH 7.5; B, acetonitrile; gradient: 0% B for 9 minutes, 0 to 90% B over 10 minutes). The solvent and water were removed by rotary-evaporation. The product was purified by precipitation from methanol/acetonitrile and dried under vacuum to afford 500 mg (94%) of 4.
Compound 4 (500 mg, 1.6 mmoles) and a buffered solution of ethylenediamine in water (8 ml of 2.0 M ethylenediamine in MES buffer, pH 5.5, containing 16 mmoles of EDA) were mixed and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2 g, 16 mmoles) was added to the reaction with vigorous stirring. After 1 hour the reaction was analyzed by LC/MS and determined to be complete. The compound was purified by preparative HPLC: PRP-1, 30�250 mm column; flow rate 25 ml/min; buffers: A, 0.1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 9 minutes, 0 to 90% B over 10 minutes. Salts were removed with a retention time of about 4 min. and the compound eluted from 6 to 7.5 minutes. The collected fractions were pooled and the solvent removed under vacuum. The residue which contained triethylammonium acetate was co-evaporated with water several times and finally the product was precipitated from methanol/acetonitrile to afford 290 mg (51%) of 5.
Compound 5 (280 mg, 0.79 mmoles) was dissolved in dry DMF (5 ml) followed by the addition of triethylamine (160 mg, 220 μl, 1.58 mmoles). The pH of the solution was adjusted to 7.5 with the addition of more triethylamine, if necessary. Biotin-X-NHS ester (358 mg, 0.79 mmoles,) was then added to the mixture with stirring. After 1.5 hours the solvent volume was reduced under vacuum to about 1 ml. Caution: do not vacuum to dryness because this compound tends to aggregate and it will be difficult to redissolve. The compound was purified by preparative HPLC: PRP-1, 30�250 mm column; flow rate 25 ml/min; buffers: A, 0.1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 8 minutes, then 0 to 95% B over 20 minutes. Fractions were collected across the peak from 16-17 min and the solution of pooled fractions was quantitated for the presence of product spectrophotometrically (λ289, assuming ε=8000). The solvent was removed under vacuum and the residue was co-evaporated with water (30 ml) three times and methanol (50 ml) two times. The product was precipitated from methanol/acetonitrile yielding 379 mg (69%) of 6.
Synthesis of Biotin-ΨUTP, Propenamide-linked (Scheme 31)
Compound 1 (2.5 g, 10.2 mmoles) and dimethylaminopyridine (1.25 g, 10.2 mmoles) were dissolved in 125 ml dry DMF under argon. Methyl propiolate (0.943 g, 1.0 ml, 11.2 mmoles) was added and the solution was stirred at room temperature for 24 hours. The reaction turned from a colorless to amber colored solution. The reaction was followed by HPLC until no more product was produced. The solvent was removed by roto-evaporation and the residue was dissolved in 10 ml methanol-acetonitrile (1:1 volume). It was purified by preparative PRP-1, 30�250 mm column using water as buffer A and acetonitrile as buffer B with a flow rate 25 ml/min. Eluting from 5 to 95% B in 15 minutes. Collect the fraction from 9 to 10 minute. Remove solvent to afford 1.1 g (33%) as a white solid.
Compound 2 (1.1 g, 3.35 mmoles) was dissolved in 80 ml 1.0 N HCl and heated to 60� C. for 88 hours when LC-MS indicated the starting material is completed converted. The reaction mixture was evaporated to an oily residual by rotary-evaporation and redissolve in minimum amount of methanol. Add the methanol solution slowly to acetonitrile (at least 200 ml) to precipitate the free acid. Collect the solid and dried under vacuum to afford 1.0 g (94%) of white solid.
Compound 3 (1.0 g, 3.18 mmoles) and a buffered solution of ethylenediamine in water (16 ml of 2.0 M ethylenediamine in 0.1 M MES buffer, pH 5.5, containing 32 mmoles of EDA) were mixed and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (4 g, 32 mmoles) was added to the reaction with vigorous stirring. After 1 hour the reaction was analyzed by LC/MS and determined to be complete. Note: desalt a sample for LC-MS. The compound was purified by preparative HPLC: PRP-1, 30�250 mm column; flow rate 25 ml/min; buffers: A, 0.1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 9 minutes, 0 to 90% B over 10 minutes. Salts were removed with a retention time of about 4 min. and the compound eluted from 6 to 7.5 minutes. The fractions were pooled and the solvent removed under vacuum. The residue which contained triethylammonium acetate was co-evaporated with water several times and finally the product was precipitated from methanol/acetonitrile to afford 700 mg (62%) of 4.
Compound 4 (102 mg, 0.286 mmoles) was co-evaporated with dry DMF twice (5 ml each) and then dissolved in dry DMF (1.5 ml) followed by the addition of triethylamine (29 mg, 40 μl, 0.286 mmoles). The pH of the solution was adjusted to 7.5 with the addition of more triethylamine, if necessary. Biotin-X-NHS ester (0.286 mmoles, 130 mg) was then added to the mixture with stirring. After 1.0 hour, the reaction was monitored by HPLC for completion. The solvent volume was reduced under vacuum to about 1 ml. Caution: do not vacuum to dryness because this compound tends to aggregate and it will be difficult to redissolve. The residual was redissolved in 5 ml water and 1 ml methanol.
Compound 5 (130 mg, 0.187 mmoles) was dried over P2O5 under vacuum for 24 hours and then dissolved in trimethyl phosphate (dried over molecular sieves, 20 ml) with gentle heating to about 60� C. Once the material dissolved the solution was cooled to ambient temperature and a trap-pack (ABI Trap-pak, P#GEN 084034) was added and allowed to gently stir overnight. The solution turned into a little cloudy when chilled on ice. The trap-pack was removed and to the solution at 0� C. under argon was added POCl3 (115 mg, 70 μl, 0.748 mmoles). The reaction was monitored by AX-HPLC for the conversion to the monophosphate, and after 4 hours, an additional one equivalent of POCl3 were added and the reaction was allowed to stir for 2 more hours (90% conversion). While monitoring the reaction, a solution of dry tetra(tri-N-butylammonium)pyrophosphate (0.187�5�3.3=3.1 mmoles) in 6 ml dry DMF was prepared. Then the reaction was added drop wise to the pyrophosphate solution with vigorous stirring. After 5 minutes, triethylammonium bicarbonate (1.0 M, pH 7.5, 23 ml) was added to quench the reaction. The mixture was stirred on ice for 30 minutes and placed in a fridge overnight. The mixture was then analyzed by HPLC (70% triphosphate) and purified using standard TriLink procedures on DEAE.
To prepare tetra(tri-N-butylammonium)pyrophosphate, TBA-PPi (Aldrich, P-8533, 1.5 TBA per PPi, 1.4 g, 3.1 mmoles) was dissolved in 5 ml dry DMF. Add TBA 287 mg, 364 μl, 1.55 mmoles). Co-evaporate with 5 ml dry DMF at least three times. Redissolve in 5 ml anhydrous DMF. Add TBA (1.46 ml, 3.1 mmoles). Handle the materials in a glove box filled with Ar.
At this point, the nucleoside was phosphorylated to 3 using standard conditions for the preparation of dexyribonucleotide triphosphates2. Example 33
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS3352849Oct 24, 1965Nov 14, 1967Merck & Co Inc6-aza-2'-deoxyuridinesUS3817837Nov 6, 1972Jun 18, 1974Syva CorpEnzyme amplification assayUS3850752Oct 29, 1971Nov 26, 1974Akzona IncProcess for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specificallyUS3891623May 3, 1972Jun 24, 1975Schering AgProcess for preparing cytidinesUS3939350Apr 29, 1974Feb 17, 1976Board Of Trustees Of The Leland Stanford Junior UniversityFluorescent immunoassay employing total reflection for activationUS3996345Jun 30, 1975Dec 7, 1976Syva CompanyFluorescence quenching with immunological pairs in immunoassaysUS4275149Nov 24, 1978Jun 23, 1981Syva CompanyImmunoassayUS4277437Dec 10, 1979Jul 7, 1981Syva CompanyChemiluminescent source, quencher moleculeUS4366241Aug 7, 1980Dec 28, 1982Syva CompanyConcentrating zone method in heterogeneous immunoassaysUS4594339Jun 18, 1984Jun 10, 1986Sloan-Kettering Institute For Cancer ResearchAnti-herpes virus compositions containing 5-substituted 1-2'(deoxy-2-'-substituted-β-d-arabinofuranosyl)pyrimedene nucleosidesUS4981783Apr 16, 1986Jan 1, 1991Montefiore Medical CenterMethod for detecting pathological conditionsUS4997928Sep 15, 1988Mar 5, 1991E. I. Du Pont De Nemours And CompanyFluorescent reagents for the preparation of 5'-tagged oligonucleotidesUS5002867Oct 24, 1988Mar 26, 1991Macevicz Stephen CNucleic acid sequence determination by multiple mixed oligonucleotide probesUS5143854Mar 7, 1990Sep 1, 1992Affymax Technologies N.V.Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereofUS5151507Jun 12, 1991Sep 29, 1992E. I. Du Pont De Nemours And CompanyDNA sequencesUS5202231Jun 18, 1991Apr 13, 1993Drmanac Radoje TDetecting probes which are exactly complementary to part of nucleic acid fragment being analyzed, then computerized comparison for sequence determinationUS5242796Oct 22, 1991Sep 7, 1993E. I. Du Pont De Nemours And CompanyMethod, system and reagents for DNA sequencingUS5262536Dec 5, 1990Nov 16, 1993E. I. Du Pont De Nemours And CompanyReagents for the preparation of 5'-tagged oligonucleotidesUS5324633Nov 22, 1991Jun 28, 1994Affymax Technologies N.V.Exposing immobilized polymers to a fluorescently-labelled receptor in solutions of varying concentrationUS5332666Oct 22, 1991Jul 26, 1994E. I. Du Pont De Nemours And CompanyMethod, system and reagents for DNA sequencingUS5422241Jul 3, 1991Jun 6, 1995Ambion, Inc.A ribonuclease protective assays comprising two reagents, one reagent containing a enzyme-resistant carrier promoting the quantitative precipitation of RNA, second reagent containing a chaotropic agent deactivating nucleaseUS5424186Dec 6, 1991Jun 13, 1995Affymax Technologies N.V.Very large scale immobilized polymer synthesisUS5445934Sep 30, 1992Aug 29, 1995Affymax Technologies N.V.Selective photoremovable groups on surfaceUS5543292Jun 14, 1993Aug 6, 1996Hitachi, Ltd.Process for the measurement of nucleic acidsUS5571639May 24, 1994Nov 5, 1996Affymax Technologies N.V.Computer-aided engineering system for design of sequence arrays and lithographic masksUS5608063Mar 28, 1995Mar 4, 1997E. I. Du Pont De Nemours And CompanyMethod, system and reagents for DNA sequencingUS5744305Jun 6, 1995Apr 28, 1998Affymetrix, Inc.Arrays of materials attached to a substrateUS6174998Sep 15, 1997Jan 16, 2001Roche Diagnostics GmbhC-nucleoside derivatives and their use in the detection of nucleic acidsUS6211158Jun 26, 1992Apr 3, 2001Roche Diagnostics GmbhDesazapurine-nucleotide derivatives, processes for the preparation thereof, pharmaceutical compositions containing them and the use thereof for nucleic acid sequencing and as antiviral agentsDE19509038A1Mar 14, 1995Sep 19, 1996Boehringer Mannheim GmbhC-Nukleosid-Derivate und deren Verwendung in der Detektion von Nukleins�urenEP0132621A2Jun 28, 1984Feb 13, 1985Fuji Photo Film Co., Ltd.Autoradiographic gene-screening methodEP0159719A2Apr 26, 1985Oct 30, 1985Enzo Biochem, Inc.Hybridization method for the detection of genetic materialsEP0252683A2Jul 1, 1987Jan 13, 1988E.I. Du Pont De Nemours And CompanyProcess and reagents for DNA sequence analysisEP0266787A2Nov 6, 1987May 11, 1988Max-Planck-Gesellschaft zur F�rderung der Wissenschaften e.V.Process for the detection of restriction fragment length polymorphisms in eukaryotic genomesEP0320308A2Dec 12, 1988Jun 14, 1989Abbott LaboratoriesMethod for detecting a target nucleic acid sequenceEP0322311A2Dec 21, 1988Jun 28, 1989Applied Biosystems, Inc.Method and kit for detecting a nucleic acid sequenceEP0336731A2Apr 5, 1989Oct 11, 1989City Of HopeMethod of amplifying and detecting nucleic acid sequencesEP0535242A1Mar 18, 1992Apr 7, 1993Institut Molekulyarnoi Biologii Imeni V.A. Engelgardta Akademii Nauk SssrMethod and device for determining nucleotide sequence of dnaEP0717113A2Oct 20, 1995Jun 19, 1996Affymax Technologies N.V.Computer-aided visualization and analysis system for nucleic acid sequence evaluationEP0721016A2Oct 20, 1995Jul 10, 1996Affymax Technologies N.V.Nucleic acid library arrays, methods for synthesizing them and methods for sequencing and sample screening using themJPS61109797A Title not availableWO1989010977A1May 2, 1989Nov 16, 1989Isis InnovationAnalysing polynucleotide sequencesWO1990000626A1Jul 3, 1989Jan 25, 1990Baylor College MedicineSolid phase assembly and reconstruction of biopolymersWO1990003370A1Sep 26, 1989Apr 5, 1990Microprobe CorpDERIVATIVES OF PYRAZOLO[3,4-d]PYRIMIDINEWO1990004652A1Oct 23, 1989May 3, 1990Dnax Research Inst Of MoleculaDna sequencing by multiple mixed oligonucleotide probesWO1990015070A1Jun 7, 1990Dec 13, 1990Affymax Tech NvVery large scale immobilized peptide synthesisWO1992002258A1Jul 1, 1991Feb 20, 1992Isis Pharmaceuticals IncNuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expressionWO1992010092A1Nov 20, 1991Jun 7, 1992Affymax Tech NvVery large scale immobilized polymer synthesisWO1992010588A1Dec 6, 1991Jun 25, 1992Affymax Tech NvSequencing by hybridization of a target nucleic acid to a matrix of defined oligonucleotidesWO1993016094A2Feb 12, 1993Aug 19, 1993Chromagen IncApplications of fluorescent n-nucleosides and fluorescent structural analogs of n-nucleosidesWO1993017126A1Feb 19, 1993Sep 2, 1993New York Health Res InstNovel oligonucleotide arrays and their use for sorting, isolating, sequencing, and manipulating nucleic acidsWO1995000530A1Jun 24, 1994Jan 5, 1995Affymax Tech NvHybridization and sequencing of nucleic acidsWO1995004594A1Aug 5, 1994Feb 16, 1995Inst Molekulyarnoi Biolog Im VMethod of dispensing micro-doses of aqueous solutions of substances onto a carrier and a device for carrying out said methodWO1995004833A1Aug 5, 1994Feb 16, 1995Ershov Gennady MMethod of immobilizing water-soluble bio-organic compounds on a capillary-porous carrierWO1995004834A1Aug 5, 1994Feb 16, 1995Ershov Gennady MMethod of manufacturing a matrix for the detection of mismatchesWO1995011995A1Oct 26, 1994May 4, 1995Affymax Tech NvArrays of nucleic acid probes on biological chipsWO1995020681A1Jan 27, 1995Aug 3, 1995Incyte Pharma IncComparative gene transcript analysisWO1995030774A1Apr 24, 1995Nov 16, 1995Beckman Instruments IncOligonucleotide repeat arraysWO1995035505A1Jun 16, 1995Dec 28, 1995Univ Leland Stanford JuniorMethod and apparatus for fabricating microarrays of biological samplesWO1996028460A1Mar 12, 1996Sep 19, 1996Boehringer Mannheim GmbhC-nucleoside derivatives and their use in nucleic acid detectionWO1997010365A1Sep 13, 1996Mar 20, 1997Affymax Tech NvExpression monitoring by hybridization to high density oligonucleotide arraysWO1997027317A1Jan 22, 1997Jul 31, 1997Affymetrix IncNucleic acid analysis techniquesWO1997028176A1Feb 3, 1997Aug 7, 1997Amersham Int PlcNucleoside analoguesWO1997029212A1Feb 7, 1997Aug 14, 1997Affymetrix IncChip-based speciation and phenotypic characterization of microorganismsWO1998011104A1Sep 11, 1997Mar 19, 1998Boehringer Mannheim GmbhHeterocyclic compounds and their use for isolating nucleic acidsWO2000006771A2Jul 20, 1999Feb 10, 2000Affymetrix IncNucleic acid labeling compoundsNon-Patent CitationsReference1Akita, Y., et al., "Cross-Coupling Reaction of Chloropyrazines with Acetylenes", Chemical & Pharmaceutical Bulletin, 34 (4), (Apr. 1986), pp. 1447-1458.2Aoyagi, M., et al., "Nucleosides and Nucleotides. 115. Synthesis of 3-Alkyl-3-Deazainosines via Palladium-Catalyzed Intramolecular Cyclization: A New Conformational Lock with the Alkyl Group at the 3-Position of the 3-Deazainosine in Anti-Conformation", Tetrahedron Letters, 34 (1), (1993), pp. 103-106.3Avila, J.L., et al., "Biological Action of Pyrazolopyrimidine Derivatives Against Trypanosoma Cruzi. Studies In Vitro and In Vivo", Comp. Biochem. Physiol.., 86C (1), (1987), pp. 49-54.4Barringer, et al., "Blunt-end and single-strand litigations by Escherichia coli ligase: influence on an in vitro amplification scheme", Gene, 89, (1990), pp. 117-122.5Basnak, I., et al., "Some 6-Aza-5-Substituted-2'-Deoxyuridines Show Potent and Selective Inhibition of Herpes Simplex Virus Type 1 Thymidine Kinase", Nucleosides & Nucleotides, 17 (1-3), (1998), pp. 187-206.6Beabealashvilli, R.S., et al., "Nucleoside 5'-triphosphates modified at sugar residues as substrates for calf thymus terminal deoxynucleotidyl transferase and for AMV reverse transcriptase", Biochimica et Biophysica Acta, 868, (1986), pp. 136-144.7Bergeron, et al., "Reagents for the stepwise functionalization of spermine", J. Org. Chem., 53, (1998), pp. 3108-3111.8Bergstrom, D.E., et al., "Design and Synthesis of Heterocyclic Carboxamides as Natural Nucleic Acid Base Mimics", Nucleosides & Nucleotides, 15 (1-3), (1996), pp. 59-68.9Bobek, M., et al., "Nucleic Acids Components and their Analogues. XCVII. Synthesis of 5-Hydroxymethyl-6-Aza-2'-Deoxyuridine and 5-Hydroxymethyl-6-Aza-2'-Deoxycytidine", Collection Czechoslov. Chem. Commun., 32, (1967), pp. 3581-3586.10Brody, R.S., et al., "The Purification of Orotidine-5'-phosphate Decarboxylase from Yeast by Affinity Chromatography", The Journal of Biological Chemistry 254 (10), (1979), pp. 4238-4244.11Broude, N.E., et al., "Enhanced DNA sequencing by hyridization", PNAS, 91 (1994), 3072-3076.12Canard, B., et al., "Catalytic editing properties of DNA polymerases", PNAS, 92, (Nov. 1995), pp. 10859-10863.13Cech, D., et al., "New Approaches Toward the Synthesis of Non-Radioactively Labeled Nucleoside Triphosphates as Terminators for DNA Sequencing", Collect. Czech. Chem. Commun., 61, Special Issue, (1996), pp. S297-S300.14Chee, M., et al., "Accessing Genetic Information with High-Density DNA Arrays", Science, 274, (Oct. 25, 1996), pp. 610-614.15Chernetskii, V.P., et al., "Anomalous nucleoside and related compounds, XIV. Derivatives of 6-azacytidine.", Chemical Abstracts, 74 (21), Abstract No. 112367j, (1971).16Chidgeavadez, Z.G., et al., "2', 3'-Dideox-3' aminonucleoside 5'-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases", Nucleic Acids Research, 12 (3), (1984), pp. 1671-1686.17Chu, et al., "General Synthesis of 2', 3'-dideoxynucleosides and 2', 3'-didehydro-2', 3'-dideoxynucleosides", J. Org. Chem., 54, (1989), pp. 2217-2225.18Cottam, Howard, B., et al., "New Adenosine Kinase Inhibitors with Oral Antiinflammatory Activity: Synthesis and Biological Evaluation", Journal of Medicinal Chemistry, 36 (22), Oct. 29, 1993), pp. 3424-3430.19Curriden, M., "A New Evidence Tool-First Use of Mitochondrial DNA Test in a U.S. criminal trial", ABA Journal, (Nov. 1996), 1 p.20Danscher, et al., "Autometallographic silver amplification of colloidal gold", J. Histotech., 16, (1993), pp. 201-207.21Depelley, J., et al., "New Non-Aromatic Triazinic Nucleosides Synthesis and Antiretroviral Evaluation of Beta-Ribosylamine Nucleoside Analogs", Nucleosides & Nucleotides 15 (5), (1996), pp. 995-1008.22Dueholm, et al., "2-3-dideoxy-furanoses in convergent synthesis of 2', 3'-dideoxy nucleosides", Synthesis, (1992), pp. 1-22.23Edo, K., et al., "Studies on Pyrimidine Derivatives. IX. Coupling Reaction of Mono-substituted Acetylenes with Iodopyrimidines", Chemical & Pharmaceutical Bulletin, 26(12), (Dec. 1978), pp. 3843-3850.24Eggers, M., et al., "A Microchip for Quantitative Detection of Molecules Utilizing Luminescent and Radioisotope Reporter Groups", Bio Techniques, 17 (3), (1994), 516-525.25Feldman, W., et al., "Gray Code Masks for Sequencing by Hybridization", Genomics, 23 (1994), 233-235.26Fodor, et al., "Light-directed, spatially addressable parallel chemical synthesis" Science, 251, (1991), 767-773.27Freskos, J.M., "Synthesis of 2'-Deoxypyrimidine Nucleosides Via Copper (1) Iodine Catalysis", Nucleosides & Nucleotides, 8 (4), (1989), pp. 549-555.28Galushko, S.V., et al., "Relationship between retention parameters in reversed-phase high performance liquid chromatography and antitumor activity of some pyrimidine bases and nucleosides", Journal of Chromatography, 547 (1991), pp. 161-166.29Galushko, S.V., et al., "Reversed-phase HPLC of N4-and O'derivatives of 6-azacytidine", Chemical Abstracts, 111 (26), Abstract No. 243960u, (1990).30Grzybowski, et al., "Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups", Nucleic Acids Research, 21 (8), 1(1993), pp. 1705-1712.31Guatelli, et al., "Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication", PNAS, 87m, (1990), pp. 1874-1878.32Hamilton, H., et al., "C4-Substituted 1-beta-D-Ribofuranosyllpyrazolo [3, 4-d]pyrimidens as Adenosine Agonist Analogues", J. Med. Chem., 26 (11), (1993, pp. 1601-1606).33Herrlein, et al., "57.3'-amino-modified nucleotides useful as potent chain terminators for current DNA sequencing method", Helv. Chim. Acta. 77, (1994), pp. 588-596.34Hobbs, Jr., F.W., et al., "Palladium-catalyzed synthesis of alkynylamino nucleosides. A universal linker for nucleic acids", J. Org. Chem., 54, (1980), pp. 3420-3422.35Hoheisel, J.D., "Application of Hybridization Techniques to Genome Mapping and . . . ", Trend in Genetics, 10 (3), (1994), 79-83.36Holy, A., et al., "Oligonucleotidic Compounds, XVII. Synthesis of Oligonucleotides Containing 6-Azauriding and 6-Azacytidine", Collectino Czechoslov. Chem. Commun., 32 (1967), pp. 2980-2997.37Izuta, S., et al., "Chain Termination with Sugar-Modified Nucleotide Analogs in the DNA Synthesis by DNA Polymerase Y", Nucleosides & Nucleotides, 15 (1-3), (1996), pp. 683-692.38Johnson, W.T., et al., "The Synthesis and stability of oligodeoxyribonucleotides containing the deoxyadenosine mimic 1-(2'-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide", Nucleic Acids Research, 25 (3), (1997), pp. 559-567.39Kallioniemi, A., et al., "Comparative Genomic Hybridization for Molecular Cytogenetic", Science, 258, (1992), 818-821.40Khrapko, K.R., et al., "An Oligonucleotide Hybridization Approach to DNA Sequencing", Hybridization Approach to DNA Sequencing, FEBS Letters, 6, (1989), 118-122.41Kohler, P. et al., "264. Nucleoside und Nucleotide. Teil 15. Synthese von Desoxyribonucleosid-monophosphaten und-triphosphaten mit 2 (1 H)-Pyrimidinon, 2 (1 H)-Pyridinon und 4-Amino-2 (1 H)-pyridinon als Basen", Helvetica Chimica Acta, 63; (1980), pp. 2488-2494.42Kutateladze, T., et al., "3'-Deoxy-3'-aminonucleoside 5'-triphosphates-Terminators of RNA synthesis, catalyzed by DNA-dependent RNA polymerase from Escherichia coli", FEBS Letters, 153 (2), Mar. 1983), pp. 420-426.43Kwoh, et al., "Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format", PNAS, 86, (1989), LPP.1173-1177.44Landegren, et al., "A ligase-mediated gene detection technique", Science, 241, (1988), pp. 1077-1080.45Langer, et al., "Enzymatic synthesis of biotin-labeled polynucleotides: Novel nucleic acid affinity probes", PNAS, 78, (1981), pp. 6633-6637.46Lazurkevich, Z.V., et al., "Growth activity of 6-substituted azauracils", Chemical Abstracts, 102 (25), Abstract No. 216761w, (1985).47Le Bec, C., et al., "Derivatives of Imidazole-4-Carboxamide as Substrates for Various DNA Polymerases", Nucleosides & Nucleotides, 167 (7-9), (1997), pp. 1301-1302.48Lennon, G.G., et al., "Hybridization analyses of arrayed cDna libraries", Trends in genetics, 7 (10), (1991), 314-317.49Lipshutz, R.J., et al., "Using Oligonucleotide Prode Arrays to access Genetic Diversity", Biotechniques, 19 (3), (1995), 442-447.50Lockhart, D.J., et al., "Expression monitoring by hybridization to high-density ", Nature Biotechnology, 14 (13), (1996), 1675-1680.51Ludwig, et al., "Rapid and efficient synthesis of nucleoside 5'-O-(1-thiotriphosphates), 5'-triphosphates and 2',3'-cyclophosphorothioates using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one", J. Org. Chem., 54, (1989), pp. 631-635.52Mikkelsen, et al., "Genetics of the malignant progression of astrocytoma", J. Cell. Biochem., 46, (1991), pp. 3-8.53Mirkin, et al., "A DNA-based method for rationally assembling nanoparticles into macroscopic materials", Nature, 382, (1996), pp. 607-609.54Misura, K., et al., "Biotinyl and phosphotyrosinyl phosphoramidite derivatives useful in the incorporation of multiple reporter groups on synthetic oligonucleotides", Nucleic Acids Research, 18 (15), pp. 4345-4354.55Mitchell, et al., "Synthesis and antiviral properties of 5-(2-substituted vinyl)-6-aza-2'-deoxyuridines", J. Chem. Med., 29, (1986), pp. 809-816.56Nelson, P.S., et al., "Oligonucleotide labeling methods 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1, 3-propanediol backbone", Nucleic Acids Research, 20 (23), (1992), pp. 6253-6259.57 *Nelson, Paul S.; Muthini, Sylvester; Kent, Mark A.; Smith, Thomas H., Nucleosides & Nucleotides, 16(10 & 11), 1951-1959 (English) 1997.*58Niedballa, et al., "A general synthesis of N-glycosides. I. Synthesis of pyrimidine nucleosides", J. Org. Chem., 39, (1974), pp. 3654-3660.59Nishida, M., et al., "Facile Perfluoralkylation of Uraciles and Uridines at the C-5 Position", Journal of Fluorine Chemistry, 63, (1993, pp. 43-52.60Ohsawa, A., et al., "Alkynylation of Halopyridazines and Their N-Oxides", Chemical & Pharmaceutical Bulletin, 28 (12), (Dec. 1980), pp. 3488-3493.61Petrie, et al., "A novel biotinylated adenylate analogue derived from pyrazolo [3,4-d]pyrimidine for labeling DNA probes", Bioconjugage Chem., 2, (1991), pp. 441-446.62Pevzner, P.A., et al., "Improved Chips for Sequencing by Hybridization", Journal of Biomolecular Structure, (1991), 339-410.63Pirrung, et al., "A convenient procedure for the deprotection of silylated nucleosides and nucleotides using triethylamine trihydrofluoride", Biorg. Med. Chem. Lett., 4, (1994), pp. 1345-1346.64Pochet, et al., "Enzymatic synthesis of 1-(2-deoxy-beta-D-ribofuranosyl) imidazole-4-carboxamide, a simplified DNA building block", Bioorg. Med. Chem. Lett., 5, (1995, pp. 1679-1684.65Pochet, S., et al., "Ambiguous Base Pairing of 1-(2-Deoxy-beta-D-Ribofuranosyl) Imidazole-4-Carboxamide During PCR", Nucleosides & Nucleotides, 16 (7-9), (1997), pp. 1749-1752.66Pochet, S., et al., "Synthesis and enzymatic polmerisation of 5-amino-1-(2'-depxy-beta-D-ribofuranosyl) imidazole-4- carboxamide-5'triphosphate", Nucleic Acids Research, 18.(23), (Dec. 11, 1990), pp. 7127-7131.67Prober, James, M., et al., "A System for Rapid DNA Sequencing with Fluorescent Chain-Terminating Dideoxynucleotides", Science, 238, (Oct. 16, 1987), pp. 336-341.68Prystas, M., et al., "Nucleic Acids Components and their Analogues. CXXI. Glycosylation of 6-Azathymine by the Silylation Process", Collection Czechoslov. Chem. Commun., 34, (1969), pp. 1104-1107.69Rambrook, et al., "Bacteriophage T4 RNA ligase (Bacteriophase T4-infected E. coli)", In: Molecular Cloning: A Laboratory Manual, 2<nd >Edition, Cold Spring Harbor Laboratory Press, (1989), pp. 5.66-5.69.70Rideout, J.L., et al., "Pyrazolo[3,4-d]pyrimidine Ribonucleosides as Anticoccidials. 2. Synthesis and Activity of Some Nucleosides of 4-(Alkylamino)-1H-Pyrazolo[3,4-d]pyrimidines", J. Med. Chem., 25 (9), pp. 1040-1044.71Robins, et al., "Nucleic acid related compounds. 42. A general procedure for the efficient deoxygeneration of secondary alcohols. Reglospecific and steroselective conversion of ribonucleosides to 2'-deoxynucleosides", J. Amer. Chem. Soc., 105, (1983), pp. 4059-4065.72Robins, et al., "Potential purine antagonists. I. Synthesis of some 4,6-substituted pyrazolo [3,4-d]pyrimidines", J. Amer. Chem. Soc., 78, (1995), pp. 784-790.73Rosemeyer, H., et al., "Stereoelectronic Effects of Modified Purines on the Sugar Conformation of Nucleosides and Fluorescence properties", Nucleosides & Nucleotides, 16 (5&6), (1997), pp. 831-828.74Sala, M., et al., "Ambiguous base pairing of the purine analogue 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide during PCR", Nucleic Acids Research, 24 (17), (1996), pp. 3302-3306.75Schena, M., et al., "Parallel human gemone analysis: Microarray-based expression", PNAS, 93, (1996), 10614-10619.76Seela, F., et al., "131.8-Aza-7-deaza-2'-deoxyguanosine: Phosphoramidite Synthesis and Properties of Octanucleotides", Helvetica Chimica ACta, 71, (1988), pp. 1191-1198.77Seela, F., et al., "193. 8-Aza-7 deazaadenine N8- and N9- (Beta-D-2'-Deoxyribofuranosides): Building Blocks for Automated DNA Synthesis and Properties of Oligodeoxyribonucleotides", Helvetica Chimica Acta, 71, (1988), pp. 1813-1823.78Seela, F., et al., "Alternating d(G-C)3 and d(C-G)3 hexanucleotides containing 7-deaza-2'deoxyguanosine or 8-aza-7-deaza-2'-deoxyguanosine in place of dG" Nucleic Acids Research, 17 (3), (1989), pp. 901-910.79Seela, F., et al., "Synthesis of 7-alkynlated 8-aza-7-deaza-2'-deoxyadenosines via the Pd-catalysed cross-coupling reaction", J. Chem. Soc., Perkin Trans., 1, (1998), pp. 3233-3239.80Seela, F., et al., "Synthesis of oligonucleotides containing pyrazolo[3,4-d]pyrimidines: The influence of 7-substituted 8-aza-7-deazaadenines of the duplex structure and stability", J. Chem. Soc., Perkin Trans., 1, (1999), pp. 479-488.81Southern, E.M., et al., "Arrays of complementary oligonucleotides for analyzing the . . . ", Nucleic Acids Research, 22 (8), (1994), 1368-1373.82Southern, et al., "Analyzing and comparing nucleic acid sequences by hybridization", Genomics 1, (1992), 1008-1017.83Stille, J.K., et al., "Stereospecific Palladium-Catalyzed Coupling Reactions of Vinyl iodides with Acetylenic Tin Reagents", Journal of the American Chemical Society, 109 (7), (Apr. 1, 1987), pp. 2138-2152.84Stimpson, D.I., et al., "Real-Time Detection of DNA Hybridization and Melting on oligonucleotide arrays by using optical wave guides", PNAS, 92, (1995), 6379-6383.85Tanji, K., et al., "Studies on Pyrimidine Derivatives. XXVII. Synthesis of 2- and 4- Pyrimidinyl Ketones by Means of the Hydration of Alkynylprimidines", Chemical & Pharmaceutical Bulletin, 30 (5), (May 1982), pp. 1865-1867.86Theisen, P., et al., "Fluorescent Dye Phosphoramidite Labeling of Oligonucleotides", Nucleic Acids Symposium Series, 27, (1992), pp. 99-102.87Uhlenbeck, et al., "T4 RNA ligase", In: The Enzymes, XV, Nucleic Acids, Part B, Boyer, P.D., (Ed.), Academic Press, (1982), pp. 31-58.88Wages, J.M., et al., "High-Performance Liquid Chromatography Analysis of PCR Products", In: PCR Strategies, Chapter 11, Academic Press, Inc., (1995), pp. 140-153.89Wang, et al., "Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome", Science, 280, (1998), pp. 1077-1082.90Wojczewski, C., et al., "Synthesis and Application of 3'-Amino-Dye-Terminators for DNA Sequencing", Nucleosides & Nucleotides, 16 (5-6), (1997), pp. 751-754.91Wu, et al., "The Ligation amplification reaction (LAR)-Amplification of specific DNA sequences using sequential rounds of template-dependent ligation", Genomics, 4, (1989), pp. 560-569.92Yu, et al., "Cyanine dye dUTP analogs for enzymatic labeling of DNA probes", Nucleic Acids Research, 22, (1994), pp. 3226-3232.93Zhu, Z., et al., "Directly labeled DNA probes using Fluorescent nucleotides with different length linkers", Nucleic Acids Research, 22 (16), (1994), pp. 3418-3422.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS7179905 *Jun 2, 2003Feb 20, 2007Affymetrix, Inc.Nucleic acid labeling compoundsUS7282327 *Aug 15, 2003Oct 16, 2007Affymetrix, Inc.Pyrimidine base containing compound suitable for enzymatic attachment to a nucleic acid, to provide a mechanism of nucleic acid detectionUS7291463Dec 23, 2003Nov 6, 2007Affymetrix, Inc.Incubating/condensating pseudoisocytidine molecule with (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to provide a pseudouridine molecule; oncology diagnosisUS7423143 *May 10, 2005Sep 9, 2008Affymetrix. Inc.Coupling via incubating with transferase; solid phase synthesisUS7468243 *Mar 12, 2002Dec 23, 2008Affymetrix, Inc.2-aminopyrimidin-4-one nucleic acid labeling compoundsUS8026057Jun 27, 2008Sep 27, 2011Affymetrix, Inc.Nucleic acid labeling compoundsUS8076072Sep 28, 2010Dec 13, 2011Affymetrix, Inc.Nucleic acid labeling compoundsUS8497064Nov 1, 2011Jul 30, 2013Affymetrix, Inc.Nucleic acid labelling compounds* Cited by examinerClassifications U.S. Classification435/6.14, 536/25.1, 536/23.1, 435/6.12International ClassificationC07H19/04, C12Q1/68, C07H19/12, C07B61/00, C07H7/06, C07H19/06, C07H19/052, C07H21/00, C07H7/00Cooperative ClassificationC07H19/052, C40B40/00, C07H19/04, C12Q2600/156, C07H19/06, C07H21/00, C07H19/12, C07H7/00, C07H7/06European ClassificationC07H21/00, C07H19/12, C07H19/06, C07H19/052, C07H7/06, C07H19/04, C07H7/00Legal EventsDateCodeEventDescriptionSep 10, 2012FPAYFee paymentYear of fee payment: 8Jun 27, 2012ASAssignmentEffective date: 20120625Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT, MAFree format text: SECURITY AGREEMENT;ASSIGNOR:AFFYMETRIX, INC.;REEL/FRAME:028465/0541Sep 15, 2008REMIMaintenance fee reminder mailedSep 8, 2008FPAYFee paymentYear of fee payment: 4Jul 19, 2005CCCertificate of correctionSep 5, 2003ASAssignmentOwner name: AFFMETRIX, INC., CALIFORNIAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MCGALL, GLENN H.;BARONE, ANTHONY D.;REEL/FRAME:013950/0743;SIGNING DATES FROM 20030212 TO 20030219Owner name: AFFMETRIX, INC. 3380 CENTRAL EXPRESSWAYSANTA CLARAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MCGALL, GLENN H. /AR;REEL/FRAME:013950/0743;SIGNING DATES FROM 20030212 TO 20030219RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services