Design and Synthesis of a Photocleavable Fluorescent Nucleotide
`3¢-O-Allyl-dGTP-PC-Bodipy-FL-510 as a Reversible Terminator for
`DNA Sequencing by Synthesis
`
`Qinglin Meng,†,‡,§ Dae Hyun Kim,†,| Xiaopeng Bai,†,‡,§ Lanrong Bi,†,‡
`Nicholas J. Turro,‡,§ and Jingyue Ju*,†,‡
`Columbia Genome Center, Columbia UniVersity College of Physicians and Surgeons,
`New York, New York 10032, and Departments of Chemical Engineering, Chemistry, and
`Biomedical Engineering, Columbia UniVersity, New York, New York 10027
`
`ju@genomecenter.columbia.edu
`ReceiVed February 14, 2006
`
`DNA sequencing by synthesis (SBS) using reversible fluorescent nucleotide terminators is potentially an
`efficient approach to address the limitations of current DNA sequencing techniques. Here, we report the
`design and synthesis of a 3¢-O-allyl photocleavable fluorescent nucleotide analogue, 3 ¢-O-allyl-dGTP-
`PC-Bodipy-FL-510, as a reversible terminator for SBS. The nucleotide is efficiently incorporated by
`DNA polymerase into a growing DNA strand to terminate the polymerase reaction. After that, the
`fluorophore is photocleaved quantitatively by irradiation at 355 nm, and the allyl group is rapidly and
`efficiently removed by using a Pd-catalyzed reaction under DNA-compatible conditions to regenerate a
`free 3¢-OH group to reinitiate the polymerase reaction. Two cycles of such steps were successfully
`demonstrated to sequence a homopolymeric region of a DNA template, facilitating the development of
`SBS as a viable approach for high-throughput DNA sequencing.
`
`Introduction
`
`DNA sequencing is a fundamental tool for biological research
`and medical diagnostics, driving disease gene discovery and
`gene function studies. DNA sequencing by synthesis (SBS)
`using reversible fluorescent nucleotide terminators1 is a poten-
`tially efficient approach to address the limitations of current
`DNA sequencing techniques, such as throughput and data
`accuracy. We have previously reported the design and synthesis
`of a 3¢-O-allyl photocleavable (PC) fluorescent nucleotide
`analogue, 3¢-O-allyl-dUTP-PC-Bodipy-FL-510, as a reversible
`terminator for SBS.2 The nucleotide can be efficiently incor-
`porated by DNA polymerase into a growing DNA strand to
`
`* To whom correspondence should be addressed. Phone: 212-851-5172.
`Fax: 212-851-5215.
`† Columbia Genome Center, Columbia University College of Physicians and
`Surgeons.
`‡ Department of Chemical Engineering, Columbia University.
`§ Department of Chemistry, Columbia University.
`| Department of Biomedical Engineering, Columbia University.
`(1) Ju, J.; Li, Z.; Edwards, J.; Itagaki, Y. U.S. Patent 6,664,079, 2003.
`
`3248
`
`J. Org. Chem. 2006, 71, 3248-3252
`
`terminate the polymerase reaction. After that, the fluorophore
`can be photocleaved quantitatively by irradiation at 355 nm,
`and the allyl group is rapidly and efficiently removed by using
`a Pd-catalyzed reaction in water to regenerate a free 3¢ -OH group
`to reinitiate the polymerase reaction.
`To the best of our knowledge, using 3¢-modified dGTP as a
`reversible terminator for SBS has not been reported, partly due
`to the difficulty of modifying the 3¢-OH of guanosine by a
`suitable capping group without protecting the guanine base.
`Here, we describe the design and synthesis of a 3¢-O-allyl
`photocleavable fluorescent 7-deaza-dGTP, 3¢ -O-allyl-dGTP-PC-
`Bodipy-FL-510 (10), with a fluorophore attached to the 7
`position of the modified guanine base and its successful
`application as a reversible terminator to determine two repeated
`nucleotide sequences in SBS. Previously, the Pd-catalyzed
`deallylation to regenerate a free 3¢-OH of the DNA extension
`product was carried out in pure water,2 which can destabilize
`
`(2) Ruparel, H.; Bi, L.; Li, Z.; Bai, X.; Kim, D. H.; Turro, N. J.; Ju, J.
`Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5932-5937.
`
`10.1021/jo060300k CCC: $33.50 © 2006 American Chemical Society
`Published on Web 03/24/2006
`
`
`
`Illumina Ex. 1084
`IPR Petition - USP 10,435,742
`
`

`

`Design and Synthesis of a PhotocleaVable Fluorescent Nucleotide
`
`Synthesis of
`SCHEME 1.
`3¢ -O-Allyl-dGTP-PC-Bodipy-FL-510 (10)
`
`SCHEME 2. DNA Polymerase Extension Reaction Using
`3¢ -O-Allyl-dGTP-PC-Bodipy-FL-510 (10) as a Reversible
`Terminator
`
`the primer-template duplex. We report here the identification
`of a new condition for rapid quantitative deallylation in a buffer
`solution at pH 8.8, which is commonly used for polymerase
`reaction. The successful synthesis of compound 10 as a
`reversible terminator to sequence through a homopolymer
`sequence and the identification of the new deallylation condition
`will facilitate the development of SBS as a viable approach for
`de novo DNA sequencing.
`
`Results and Discussion
`2-Amino-6-methoxy-9-((cid:226)-D-2¢ -deoxyribofuranosyl)-7-deaza-
`purine 1 was chosen as the starting material for the synthesis
`of 3¢ -O-allyl-7-(3-aminoprop-1-ynyl)-7-deaza-dGTP 9 (Scheme
`1). Compound 1 was first protected by isobutyryl chloride to
`yield 2 quantitatively.3 Compound 2 was iodinated at
`the
`7-position by N-iodosuccinimide (NIS) to produce a single
`product 3 in 90% yield, as the 2-acylamino group in the
`nucleobase promotes the formation of 7-substituted product.4
`Compound 3 was deprotected by methanolic sodium methoxide
`to afford 4 in 94% yield. 5¢ -OH of 4 was protected by tert-
`butyldimethylsilyl chloride to produce 5 in 88% yield.5 3¢ -OH
`of 5 was subsequently allylated in CH2Cl2 and 40% aqueous
`
`(3) Seela, F.; Driller, H. Nucl. Nucl. 1989, 8, 1-21.
`(4) Ramzaeva, N.; Seela, F. Hel. Chim. Acta 1995, 78, 1083-1090.
`(5) Ryu, E. K.; Ross, R. J.; Matsushita, T.; MacCoss, M.; Hong, C. I.;
`West, C. R. J. Med. Chem. 1982, 25, 1322-1329.
`
`NaOH solution using tetrabutylammonium bromide as phase-
`transfer catalyst to give a 92% yield of 6 without N2-allylated
`product. Sonogashira cross-coupling reaction of 6 with the
`terminal alkyne catalyzed by Pd(0)/Cu(I) formed 7 in 94%
`yield.6 Next, a one-pot procedure of demethylation7 and
`desilylation of 7 gave a moderate 34% yield of 8. Finally 8
`was transformed into the corresponding triphosphate 3¢ -O-allyl-
`dGTP 9 following established procedures.8 Coupling 9 with PC-
`Bodipy-FL-510 NHS ester9 yielded the target compound, 3¢ -
`O-allyl-dGTP-PC-Bodipy-FL-510 (10).
`For 3¢ -O-allyl-dGTP-PC-Bodipy-FL-510 (10) to act as a
`reversible terminator for SBS, it is important to establish that
`10 can be used to determine a repeated DNA sequence in a
`polymerase reaction. To this end, we performed a polymerase
`DNA extension reaction using 10 as a substrate in solution as
`shown in Scheme 2. This allows the isolation of the DNA
`product at each step for detailed molecular structure character-
`ization by using MALDI-TOF mass spectrometry (MS) as
`shown in Figure 1.
`
`(6) Hobbs, F. W. J. Org. Chem. 1989, 54, 3420-3422.
`(7) Ramasamy, K.; Imarura, N.; Robins, R. K.; Revankar, G. R. J.
`Heterocycl. Chem. 1988, 25, 1893-1898.
`(8) Lee, S. E.; Sidorov, A.; Gourlain, T.; Mignet, N.; Thorpe, S. J.;
`Brazier, J. A.; Dickman, M. J.; Hornby, D. P.; Grasby, J. A.; Williams, D.
`M. Nucleic Acids Res. 2001, 29, 1565-1573.
`(9) Seo, T. S.; Bai, X.; Kim, D. H.; Meng, Q.; Shi, S.; Ruparel, H.; Li,
`Z.; Turro, N. J.; Ju, J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5926-
`5931.
`
`J. Org. Chem, Vol. 71, No. 8, 2006 3249
`
`

`

`Meng et al.
`
`peak corresponding to the photocleavage product 12 appears
`as the sole dominant peak at m/z 6552. Figure 1C shows a single
`peak at m/z 6512, which corresponds to a deallylated photo-
`cleavage product 13. The absence of a peak at m/z 6552 proves
`that the deallylation reaction was completed with high efficiency.
`The next extension reaction was carried out by using this
`deallylated photocleavage product 13 as a primer along with
`3¢ -O-allyl-dGTP-PC-Bodipy-FL-510 (10) to yield an extension
`product 14 (Figure 1D). DNA products (15 and 16) from
`photocleavage (Figure 1E) and deallylation (Figure 1F), respec-
`tively, were obtained in a similar manner as described previ-
`ously, thereby completing two entire polymerase extension
`cycles to sequence a homopolymeric region of a template using
`10 as a reversible terminator.
`In summary, we have developed a successful strategy for the
`synthesis of a 3¢ -O-allyl-modified 7-deaza-dGTP bearing a
`photocleavable fluorophore at
`the 7 position. This novel
`nucleotide analogue is shown to be an excellent substrate for
`9(cid:176) N DNA polymerase A485L/Y409V and can be incorporated
`with high efficiency in a polymerase extension reaction. We
`have also demonstrated that complete photocleavage to remove
`the fluorophore is achieved in 10 s on these DNA products.
`Furthermore, we have shown that deallylation can be swiftly
`achieved to near completion under mild reaction conditions in
`an aqueous environment by using a palladium catalyst. Finally,
`we have established that the deallylated DNA product can be
`used as a primer to continue the polymerase reaction and that
`extension, photocleavage, and deallylation can be performed
`with high efficiency. These results provide further proof of the
`feasibility of using the allyl group as a reversible capping moiety
`for the 3¢ -OH of the photocleavable nucleotide analogues for
`SBS, validating the approach we had previously proposed.1
`Thus, the successful engineering of the building blocks of DNA
`with synthetic chemistry will facilitate the development of SBS
`for high-throughput DNA sequencing and genotyping.
`
`Experimental Section
`2-(2-Methylpropanoyl)amino-6-methoxy-9-[3¢ ,5¢ -bis-O-(2-
`methylpropanoyl)-(cid:226)-D-2¢ -deoxyribofuranosyl]-7-deazapurine (2).
`To a stirred suspension of 1 (1.00 g; 3.57 mmol) in anhydrous
`pyridine (35 mL) was added slowly isobutyryl chloride (3.40 mL;
`32.2 mmol) at 0 (cid:176) C. The reaction mixture was stirred at 0 (cid:176) C for
`1 h. Methanol (2 mL) was then added, and the reaction mixture
`was stirred for another 10 min. Then most solvent was removed
`under vacuum. Ethyl acetate (200 mL) and saturated aqueous
`NaHCO3 (50 mL) were added to the residue. The organic layer
`was separated, washed with saturated aqueous NaHCO3 and NaCl,
`respectively, and dried over anhydrous Na2SO4. After evaporation
`of the solvent, the residue was purified by flash column chroma-
`tography over silica gel using ethyl acetate-hexane (1:3(cid:24)2) as the
`eluent to afford 2 as white foam (1.75 g; 99% yield): 1H NMR
`(400 MHz, CD3OD) (cid:228) 7.28 (d, J ) 3.7 Hz, 1H), 6.66 (dd, J ) 5.9,
`8.6 Hz, 1H), 6.51 (d, J ) 3.7 Hz, 1H), 5.41 (m, 1H), 4.33-4.36
`(m, 2H), 4.22 (m, 1H), 4.08 (s, 3H), 2.83-2.96 (m, 2H), 2.54-
`2.70 (m, 2H), 2.48-2.54 (ddd, J ) 2.0, 5.9, 14.2 Hz, 1H), 1.15-
`1.23 (m, 18H); 13C NMR (100 MHz, CD3OD) (cid:228) 178.2, 177.7, 177.4,
`164.2, 153.4, 152.5, 123.4, 103.5, 100.7, 85.2, 83.0, 75.9, 65.0,
`54.4, 37.9, 36.6, 35.0, 34.9, 19.9, 19.3-19.4 (four peaks); HRMS
`(FAB+) calcd for C24H35O7N4 (M + H+) 491.2506, found
`491.2503.
`2-(2-Methylpropanoyl)amino-6-methoxy-7-iodo-9-[3¢ ,5¢ -bis-
`O-(2-methylpropanoyl)-(cid:226)-D-2¢ -deoxyribofuranosyl]-7-deaza-
`purine (3). To a vigorously stirred solution of 2 (1.75 g; 3.57 mmol)
`in anhydrous DMF (27 mL) was added 95% N-iodosuccimide (NIS)
`
`FIGURE 1. Continuous polymerase extension scheme (left) and
`MALDI-TOF MS spectra of the resulting DNA products (right).
`
`A synthetic 100-mer DNA corresponding to a portion of exon
`7 of the human p53 gene was used as a template to perform the
`extension. The sequence in the template immediately adjacent
`to the annealing site of the primer had a repeating sequence of
`3¢ -CC-5¢ . First, a polymerase extension reaction using 10 as a
`terminator along with a primer and the above template was
`performed. After the reaction, a small portion of the DNA
`extension product was characterized by MALDI-TOF MS. The
`rest of the product was irradiated at 355 nm for 10 s to cleave
`the fluorophore from the DNA and then analyzed by MALDI-
`TOF MS. After photocleavage, the DNA product was added to
`a deallylation cocktail [1X Thermopol reaction buffer/Na2PdCl4/
`P(PhSO3Na)3] to remove the 3¢ -allyl group in 30 s to yield
`quantitatively deallylated DNA product. The deallylated DNA
`product with a free 3¢ -OH group regenerated was then used as
`a primer to incorporate 10 in a subsequent second extension
`reaction.
`Figure 1 (right panel) shows a sequential mass spectrum at
`each step of DNA SBS using 10 as a reversible terminator. As
`can be seen from Figure 1A, the MALDI-TOF MS spectrum
`consists of a distinct peak at m/z 7048 corresponding to the
`single-base DNA extension product 11 with 100% incorporation
`efficiency, confirming that the reversible terminator 10 can be
`incorporated base-specifically by DNA polymerase into a
`growing DNA strand. The small peak at m/z 6552 corresponding
`to the photocleavage product is due to the partial cleavage
`caused by the nitrogen laser pulse (337 nm) used for ionization
`in MALDI-TOF MS. Figure 1B shows the photocleavage result
`after 10 s irradiation of the DNA extension product at 355 nm.
`The peak at m/z 7048 has completely disappeared, whereas the
`
`3250 J. Org. Chem., Vol. 71, No. 8, 2006
`
`

`

`Design and Synthesis of a PhotocleaVable Fluorescent Nucleotide
`
`(866 mg; 3.66 mmol). The reaction mixture was stirred at room
`temperature for 22 h, and then most solvent was removed under
`vacuum. Diethyl ether (200 mL) and saturated aqueous NaHCO3
`(50 mL) were added. The organic layer was separated, washed with
`saturated aqueous NaCl, and dried over anhydrous Na2SO4. After
`evaporation of the solvent, the residue was purified by flash column
`chromatography over silica gel using ethyl acetate-hexane (1:3)
`as the eluent to afford 3 as white solid (1.98 g; 90% yield): 1H
`NMR (400 MHz, CD3OD) (cid:228) 7.43 (s, 1H), 6.63 (dd, J ) 6.0, 8.2
`Hz, 1H), 5.41 (m, 1H), 4.33-4.36 (m, 2H), 4.23 (m, 1H), 4.09 (s,
`3H), 2.78-2.94 (m, 2H), 2.57-2.70 (m, 2H), 2.50-2.57 (ddd, J
`) 2.3, 6.0, 14.2 Hz, 1H), 1.17-1.24 (m, 18H); 13C NMR (100
`MHz, CD3OD) (cid:228) 178.3, 177.8, 177.5, 164.3, 153.3, 152.8, 128.6,
`105.2, 85.3, 83.3, 75.8, 65.0, 54.4, 51.8, 38.2, 36.8, 35.2, 35.1, 19.9,
`19.3-19.5 (four peaks); HRMS (FAB+) calcd for C24H34O7N4I
`(M + H+) 617.1472, found 617.1464.
`2-Amino-6-methoxy-7-iodo-9-((cid:226)-D-2¢ -deoxyribofuranosyl)-7-
`deazapurine (4). Compound 3 (1.98 g; 3.21 mmol) was dissolved
`in 0.5 M methanolic CH3ONa (50 mL) and stirred at 65 (cid:176) C for 12
`h. Saturated aqueous NaHCO3 (20 mL) was added, and the mixture
`was stirred for 10 min. Then most of methanol was evaporated,
`and the residue was extracted by ethyl acetate (150 mL). The
`organic layer was washed with saturated aqueous NaHCO3 and
`NaCl, respectively, and dried over anhydrous Na2SO4. After
`evaporation of the solvent, the residue was purified by flash column
`chromatography over silica gel using CH3OH-CH2Cl2 (1:30-15)
`as the eluent to afford 4 as a white solid (1.23 g; 94% yield): 1H
`NMR (400 MHz, CD3OD) (cid:228) 7.17 (s, 1H), 6.36 (dd, J ) 6.0, 8.4
`Hz, 1H), 4.47 (m, 1H), 3.99 (s, 3H), 3.96 (m, 1H), 3.77 (dd, J )
`3.4, 12.0 Hz, 1H), 3.70 (dd, J ) 3.7, 12.0 Hz, 1H), 2.55-2.64
`(ddd, J ) 6.0, 8.4, 13.4 Hz, 1H), 2.20-2.26 (ddd, J ) 2.4, 5.9,
`13.4 Hz, 1H); 13C NMR (100 MHz, CD3OD) (cid:228) 164.7, 160.6, 154.3,
`126.5, 101.6, 88.7, 86.0, 73.0, 63.7, 53.7, 51.3, 41.1; HRMS
`(FAB+) calcd for C12H16O4N4I (M + H+) 407.0216, found
`407.0213.
`2-Amino-6-methoxy-7-iodo-9-[(cid:226)-D-5¢ -O-(tert-butyldimethyl-
`silyl)-2¢ -deoxyribofuranosyl]-7-deazapurine (5). To a stirred
`solution of 4 (1.23 g; 3.02 mmol) and imidazole (494 mg; 7.24
`mmol) in anhydrous DMF (15 mL) was added tert-butyldimethyl-
`silyl chloride (TBDMSCl) (545 mg; 3.51 mmol). The reaction
`mixture was stirred at room temperature for 20 h. Then most solvent
`was removed under vacuum, and the residue was purified by flash
`column chromatography over silica gel using ethyl acetate-hexane
`(1:2(cid:24)0.5) as the eluent to afford 5 as a white foam (1.38 g; 88%
`yield): 1H NMR (400 MHz, CD3OD) (cid:228) 7.23 (s, 1H), 6.49 (dd, J
`) 6.1, 7.7 Hz, 1H), 4.46 (m, 1H), 3.99 (s, 3H), 3.94 (m, 1H), 3.79-
`3.87 (m, 2H), 2.36-2.44 (ddd, J ) 5.8, 7.7, 13.3 Hz, 1H), 2.24-
`2.31 (ddd, J ) 3.1, 6.0, 13.3 Hz, 1H), 0.96 (s, 9H), 0.14 (s, 3H),
`0.13 (s, 3H); 13C NMR (100 MHz, CD3OD) (cid:228) 164.6, 160.7, 154.7,
`125.1, 101.0, 88.2, 84.2, 72.7, 64.7, 53.7, 51.7, 41.9, 26.7, 19.4,
`-5.0, -5.1; HRMS (FAB+) calcd for C18H30O4N4SiI (M + H+)
`521.1081, found 521.1068.
`2-Amino-6-methoxy-7-iodo-9-[(cid:226)-D-3¢ -O-allyl-5¢ -O-(tert-butyl-
`dimethylsilyl)-2¢ -deoxyribofuranosyl]-7-deazapurine (6). To a
`stirred solution of 5 (1.38 g; 2.66 mmol) in CH2Cl2 (80 mL) were
`added tetrabutylammonium bromide (TBAB) (437 mg; 1.33 mmol),
`allyl bromide (1.85 mL, 21.4 mmol), and 40% aqueous NaOH
`solution (40 mL). The reaction mixture was stirred at room
`temperature for 1 h. Ethyl acetate (200 mL) was added, and the
`organic layer was separated. The aqueous layer was extracted with
`ethyl acetate (2 (cid:2) 50 mL). The combined organic layer was washed
`with saturated aqueous NaHCO3 and NaCl, respectively, and dried
`over anhydrous Na2SO4. After evaporation of the solvent, the
`residue was purified by flash column chromatography over silica
`gel using ethyl acetate-hexane (1:3) as the eluent to afford 6 as a
`white solid (1.37 g; 92% yield): 1H NMR (400 MHz, CD3OD) (cid:228)
`7.20 (s, 1H), 6.43 (dd, J ) 6.2, 7.9 Hz, 1H), 5.89-5.99 (m, 1H),
`5.29-5.35 (dm, J ) 17.3 Hz, 1H), 5.16-5.21 (dm, J ) 10.5 Hz,
`1H), 4.24 (m, 1H), 4.01-4.11 (m, 3H), 3.99 (s, 3H), 3.76-3.84
`
`(m, 2H), 2.32-2.44 (m, 2H), 0.95 (s, 9H), 0.14 (s, 3H), 0.13 (s,
`3H); 13C NMR (100 MHz, CDCl3) (cid:228) 163.3, 158.6, 153.6, 134.1,
`123.7, 116.9, 100.6, 84.4, 83.0, 79.1, 70.0, 63.6, 53.3, 51.1, 38.1,
`26.1, 18.5, -5.1, -5.3; HRMS (FAB+) calcd for C21H34O4N4SiI
`(M + H+) 561.1394, found 561.1390.
`2-Amino-6-methoxy-7-{3-[(trifluoroacetyl)amino]prop-1-
`ynyl]-9-[(cid:226)-D-3¢ -O-allyl-5¢ -O-(tert-butyldimethylsilyl)-2¢ -deoxyribo-
`furanosyl}-7-deazapurine (7). To a stirred solution of 6 (1.37 g;
`2.45 mmol) in anhydrous DMF (11 mL) were added tetrakis-
`(triphenylphosphine)palladium(0) (286 mg; 0.245 mmol) and CuI
`(101 mg; 0.532 mmol). The solution was stirred at room temperature
`for 10 min. Then N-propargyltrifluoroacetamide (1.12 g; 7.43 mmol)
`and triethylamine (0.68 mL; 4.90 mmol) were added. The reaction
`was stirred at room temperature for 13 h with exclusion of air and
`light. Most DMF was removed under vacuum, and the residue was
`dissolved in ethyl acetate (100 mL). The solution was washed with
`saturated aqueous NaHCO3 and NaCl, respectively, and dried over
`anhydrous Na2SO4. After evaporation of the solvent, the residue
`was purified by flash column chromatography over silica gel using
`ethyl acetate-hexane (1:3-1.5) and CH3OH-CH2Cl2 (1:30),
`respectively, as the eluent to afford 7 as yellow solid (1.34 g; 94%
`yield): 1H NMR (400 MHz, CD3OD) (cid:228) 7.34 (s, 1H), 6.42 (dd, J
`) 6.2, 7.7 Hz, 1H), 5.88-5.99 (m, 1H), 5.28-5.35 (dm, J ) 17.3
`Hz, 1H), 5.16-5.21 (dm, J ) 10.5 Hz, 1H), 4.29 (s, 2H), 4.24 (m,
`1H), 4.00-4.09 (m, 3H), 3.98 (s, 3H), 3.76-3.84 (m, 2H), 2.32-
`2.45 (m, 2H), 0.94 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H); 13C NMR
`(100 MHz, CD3OD) (cid:228) 165.0, 161.2, 158.1 (q, J ) 36 Hz), 154.2,
`135.6, 125.0, 117.2 (q, J ) 284 Hz), 117.0, 99.2, 97.3, 86.0, 84.6,
`84.5, 80.3, 78.0, 71.0, 64.8, 53.8, 39.0, 30.9, 26.5, 19.3, -5.1, -5.2;
`HRMS (FAB+) calcd for C26H37O5N5F3Si (M + H+) 584.2516,
`found 584.2491.
`3¢ -O-Allyl-7-{3-[(trifluoroacetyl)amino]prop-1-ynyl}-7-deaza-
`2¢ -deoxyguanosine (8). To a stirred solution of 7 (1.34 g; 2.30
`mmol) in anhydrous CH3CN (86 mL) were added NaI (363 mg;
`2.42 mmol) and chlorotrimethylsilane (TMSCl) (0.306 mL; 2.42
`mmol). The reaction was stirred at room temperature for 1 h and
`then at 50 (cid:176) C for 12 h. The solvent was evaporated, and the residue
`was dissolved in anhydrous THF (76 mL). Tetrabutylammonium
`fluoride (TBAF) (1 M) in THF solution (4.80 mL; 4.80 mmol) was
`added, and the reaction was stirred at room temperature for 1 h.
`The solvent was evaporated, and the residue was dissolved in ethyl
`acetate (150 mL). The solution was washed with saturated aqueous
`NaCl and dried over anhydrous Na2SO4. After evaporation of the
`solvent, the residue was purified by flash column chromatography
`over silica gel using CH3OH-ethyl acetate (1:30) as the eluent to
`afford 8 as yellow solid (356 mg; 34% yield): 1H NMR (400 MHz,
`CD3OD) (cid:228) 7.21 (s, 1H), 6.30 (dd, J ) 6.0, 8.4 Hz, 1H), 5.88-5.99
`(m, 1H), 5.28-5.35 (dm, J ) 17.3 Hz, 1H), 5.15-5.20 (dm, J )
`10.5 Hz, 1H), 4.29 (s, 2H), 4.23 (m, 1H), 4.00-4.10 (m, 3H), 3.65-
`3.75 (m, 2H), 2.41-2.49 (ddd, J ) 5.8, 8.4, 13.6 Hz, 1H), 2.34-
`2.40 (ddd, J ) 2.3, 6.0, 13.6 Hz, 1H); 13C NMR (100 MHz,
`CD3OD) (cid:228) 160.9, 158.0 (q, J ) 36 Hz), 154.1, 151.8, 135.6, 124.4,
`117.2 (q, J ) 284 Hz), 117.0, 101.4, 99.7, 86.4, 85.5, 84.8, 80.7,
`78.0, 71.0, 63.7, 38.5, 31.2; HRMS (FAB+) calcd for C19H21O5N5F3
`(M + H+) 456.1495, found 456.1493.
`3¢ -O-Allyl-7-(3-aminoprop-1-ynyl)-7-deaza-2¢ -deoxyguanosine-
`5¢ -triphosphate (9). The procedure is the same as that of preparing
`3¢ -O-allyl-5-(3-aminoprop-1-ynyl)-2¢ -deoxyuridine-5¢ -triphos-
`phate in ref 2 to yield 9 as a colorless syrup: 1H NMR (300 MHz,
`D2O) (cid:228) 7.56 (s, 1H), 6.37 (t, J ) 7.3 Hz, 1H), 5.89-6.02 (m, 1H),
`5.31-5.39 (dm, J ) 17.3 Hz, 1H), 5.21-5.28 (dm, J ) 10.5 Hz,
`1H), 4.49 (s, 2H), 4.32 (m, 1H), 4.06-4.18 (m, 3H), 3.92-3.99
`(m, 2H), 2.44-2.60 (m, 2H);31P NMR (121.4 MHz, D2O) (cid:228) -6.1
`(d, J ) 20.8 Hz, 1P), -10.8 (d, J ) 18.9 Hz, 1P), -21.9 (t, J )
`19.8 Hz, 1P).
`3¢ -O-Allyl-dGTP-PC-Bodipy-FL-510 (10). PC-Bodipy-FL-510
`NHS ester (prepared by the same procedure as in ref 9) (7.2 mg,
`12 (cid:237)mol) in 300 (cid:237)L of acetonitrile was added to a solution of 9 (2
`mg, 3.4 (cid:237)mol) in 300 (cid:237)L of Na2CO3-NaHCO3 aqueous buffer (0.1
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`J. Org. Chem, Vol. 71, No. 8, 2006 3251
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`

`

`M, pH 8.5). The reaction mixture was stirred at room temperature
`for 3 h. A preparative silica gel TLC plate was used to separate
`the unreacted PC-Bodipy-FL-510 NHS ester from the fraction
`containing 10 with CHCl3-CH3OH (85:15) as the eluent. The
`product was concentrated further under vacuum and purified with
`reversed-phase HPLC on a 150 (cid:2) 4.6-mm C18 column to obtain
`the pure product 10 (retention time of 34 min). Mobile phase: A,
`8.6 mM triethylamine/100 mM hexafluoroisopropyl alcohol in water
`(pH 8.1); B, methanol. Elution was performed with 100% A
`isocratic over 10 min, followed by a linear gradient of 0-50% B
`for 20 min and then 50% B isocratic over another 20 min. 3¢ -O-
`Allyl-dGTP-PC-Bodipy-FL-510 10 was characterized by the fol-
`lowing primer extension reaction and characterization by MALDI-
`TOF MS.
`Primer Extension Using 3¢ -O-Allyl-dGTP-PC-Bodipy-FL-510
`(10) and Photocleavage of the Extension Product 11. The
`polymerase extension reaction mixture consisted of 60 pmol of
`(5¢ -GTTGATGTACACATTGTCAA-3¢ ), 80 pmol of
`primer
`template (5¢ -TACCCGGAGGCCAAGTACGGCGGG-
`100-mer
`T A C G T C C T T G A C A A T G T G T A C A T C A A C A T -
`C A C C T A C C A C C A T G T C A G T C T C G G T T G -
`GATCCTCTATTGTGTCCGGG-3¢ ), 120 pmol of 3¢ -O-allyl-
`dGTP-PC-Bodipy-FL-510, 1X Thermopol reaction buffer (20 mM
`Tris-HCl/10 mM (NH4)2SO4/10 mM KCl/2 mM MgSO4/0.1%
`Triton X-100, pH 8.8, New England Biolabs), and 6 units of 9(cid:176) N
`Polymerase (exo-)A485L/Y409V in a total volume of 20 (cid:237)L. The
`reaction consisted of 20 cycles at 94 (cid:176) C for 20 s, 46 (cid:176) C for 40 s,
`and 60 (cid:176) C for 90 s. After the reaction, a small portion of the DNA
`extension product was desalted by using ZipTip and analyzed by
`MALDI-TOF MS, which shows a dominant peak at m/z 7048
`corresponding to the DNA product 11. The rest of the product
`mixture was freeze-dried, resuspended in 200 (cid:237)L of deionized water,
`and irradiated at 355 nm for 10 s to cleave the fluorophore from
`the DNA to yield product 12 and then analyzed by MALDI-TOF
`MS (m/z 6552).
`Deallyation of Photocleaved DNA Extension Product 12. DNA
`product 12 (20 pmol) was added to a mixture of degassed 1X
`Thermopol reaction buffer (20 mM Tris-HCl/10 mM (NH4)2SO4/
`
`Meng et al.
`
`10 mM KCl/2 mM MgSO4/0.1% Triton X-100, pH 8.8, 1 (cid:237)L),
`Na2PdCl4 in degassed H2O (7 (cid:237)L, 23 nmol), and P(PhSO3Na)3 in
`degassed H2O (10 (cid:237)L, 176 nmol) to perform the deallylation. The
`reaction mixture was then placed in a heating block and incubated
`at 70 (cid:176) C for 30 s to yield quantitatively deallylated DNA product
`13 and analyzed by MALDI-TOF MS (m/z 6512).
`Primer Extension Reaction Performed with the Deallylated
`DNA Product. The deallylated DNA product 13 was used as a
`primer in a single-base extension reaction. The 20 (cid:237)L: reaction
`mixture consisted of 60 pmol of the deallylated product 13, 80 pmol
`of the 100-mer template (5¢ -TACCCGGAGGCCAAGTACGGC-
`GGGTACGTCCTTGACAATGTGTACATCAACATCACCTACCA-
`CCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGGG-3¢ ),
`120 pmol of 3¢ -O-allyl-dGTP-PC-Bodipy-FL-510 (10), and 6 units
`of 9(cid:176) N Polymerase (exo-)A485L/Y409V in a total volume of 20
`(cid:237)L. The reaction consisted of 20 cycles at 94 (cid:176) C for 20 s, 46 (cid:176) C
`for 40 s, and 60 (cid:176) C for 90 s. The DNA extension product 14 was
`desalted by using the ZipTip protocol, and a small portion was
`analyzed by using MALDI-TOF MS (m/z 7429). The remaining
`product was then irradiated with near-UV light (355 nm) for 10 s
`to cleave the fluorophore from the extended DNA product. The
`resulting photocleavage product 15 (m/z 6933) was analyzed by
`using MALDI-TOF MS. Finally, deallylation of the photocleavage
`product 15 was performed using a Pd-catalyzed deallylation reaction
`resulting in a deallylated DNA product 16, which was then analyzed
`by MALDI-TOF MS (m/z 6893).
`
`Acknowledgment. We thank New England Biolabs for
`providing the 9(cid:176) N DNA polymerase. This work was supported
`by NIH Grant Nos. P50 HG002806 and R01 HG003582 and
`the Packard Fellowship for Science and Engineering.
`
`Supporting Information Available: Spectral data for 2-9
`(1H, 13C, and 31P NMR) and general experimental procedures.
`This material
`is available free of charge via the Internet at
`http://pubs.acs.org.
`
`JO060300K
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`3252 J. Org. Chem., Vol. 71, No. 8, 2006
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