NUCLEOSIDES & NUCLEOTIDES, 18(2), 197-201 (1999)
`
`SYNTHESIS OF FLUORESCENT, PHOTOLABILE 3'-O(cid:173)
`PROTECTED NUCLEOSIDE TRIPHOSPHATES FOR THE
`BASE ADDITION SEQUENCING SCHEME
`
`Mike B. Welch and Kevin Burgess*
`
`Department of Chemistry, Texas A & M University, PO Box 300012,
`College Station, TX 77842-3012
`
`ABSTRACT: The dansylated nucleoside triphosphates la and lb were
`prepared as a prelude to investigating sequencing of DNA via a scheme that
`does not involve electrophoresis.
`
`Throughput of sequence data is critical in The Human Genome
`
`Project and similar ventures. Gel electrophoresis is a "bottle-neck" in high
`
`throughput sequencing of DNA hence it is desirable to develop methods that
`
`avoid this technique. Several groups have been interested in an approach
`
`that we call, "The Base Addition Sequencing Scheme", BASS.I-6 This
`
`involves modified nucleotides I that are: (i) incorporated by DNA
`
`replicating enzymes; (ii) spectroscopically distinct (at P*) so that the parent
`
`base (A, T, G, and C) can be differentiated; and, (iii) labile at the 3' -position
`
`such that the 3'-hydroxyl terminus of a polynucleotide can be regenerated.
`
`This scheme may be slower and sequence less bases per experiment than
`
`existing methods but, unlike methods involving gels, there is potential for
`
`multiple experiments to be run in parallel. Such combinatorial sequencing
`
`schemes may lead to significantly improved throughput. A major challenge
`
`in development of BASS, however, is that the 3'-blocking group must be
`
`197
`
`Copyright O I 999 by Marcel Dekker. Inc.
`
`www .dekker.com
`
`Columbia Ex. 2006
`Illumina, Inc. v. The Trustees
`of Columbia University
`in the City of New York
`IPR2020-01177
`
`
`
`SYNTHESIS OF FLUORESCENT, PHOTOLABILE 3'-O(cid:173)
`PROTECTED NUCLEOSIDE TRIPHOSPHATES FOR THE
`BASE ADDITION SEQUENCING SCHEME
`
`Mike B. Welch and Kevin Burgess*
`
`Department of Chemistry, Texas A & M University, PO Box 300012,
`College Station, TX 77842-3012
`
`ABSTRACT: The dansylated nucleoside triphosphates la and lb were
`prepared as a prelude to investigating sequencing of DNA via a scheme that
`does not involve electrophoresis.
`
`Throughput of sequence data is critical in The Human Genome
`
`Project and similar ventures. Gel electrophoresis is a "bottle-neck" in high
`
`throughput sequencing of DNA hence it is desirable to develop methods that
`
`avoid this technique. Several groups have been interested in an approach
`
`that we call, "The Base Addition Sequencing Scheme", BASS.I-6 This
`
`involves modified nucleotides I that are: (i) incorporated by DNA
`
`replicating enzymes; (ii) spectroscopically distinct (at P*) so that the parent
`
`base (A, T, G, and C) can be differentiated; and, (iii) labile at the 3' -position
`
`such that the 3'-hydroxyl terminus of a polynucleotide can be regenerated.
`
`This scheme may be slower and sequence less bases per experiment than
`
`existing methods but, unlike methods involving gels, there is potential for
`
`multiple experiments to be run in parallel. Such combinatorial sequencing
`
`schemes may lead to significantly improved throughput. A major challenge
`
`in development of BASS, however, is that the 3'-blocking group must be
`
`197
`
`Copyright O I 999 by Marcel Dekker. Inc.
`
`www .dekker.com
`
`Columbia Ex. 2006
`Illumina, Inc. v. The Trustees
`of Columbia University
`in the City of New York
`IPR2020-01177
`
`
`
`NUCLEOSIDES & NUCLEOTIDES, 18(2), 197-201 (1999)
`
`SYNTHESIS OF FLUORESCENT, PHOTOLABILE 3'-O(cid:173)
`PROTECTED NUCLEOSIDE TRIPHOSPHATES FOR THE
`BASE ADDITION SEQUENCING SCHEME
`
`Mike B. Welch and Kevin Burgess*
`
`Department of Chemistry, Texas A & M University, PO Box 300012,
`College Station, TX 77842-3012
`
`ABSTRACT: The dansylated nucleoside triphosphates la and lb were
`prepared as a prelude to investigating sequencing of DNA via a scheme that
`does not involve electrophoresis.
`
`Throughput of sequence data is critical in The Human Genome
`
`Project and similar ventures. Gel electrophoresis is a "bottle-neck" in high
`
`throughput sequencing of DNA hence it is desirable to develop methods that
`
`avoid this technique. Several groups have been interested in an approach
`
`that we call, "The Base Addition Sequencing Scheme", BASS.I-6 This
`
`involves modified nucleotides I that are: (i) incorporated by DNA
`
`replicating enzymes; (ii) spectroscopically distinct (at P*) so that the parent
`
`base (A, T, G, and C) can be differentiated; and, (iii) labile at the 3' -position
`
`such that the 3'-hydroxyl terminus of a polynucleotide can be regenerated.
`
`This scheme may be slower and sequence less bases per experiment than
`
`existing methods but, unlike methods involving gels, there is potential for
`
`multiple experiments to be run in parallel. Such combinatorial sequencing
`
`schemes may lead to significantly improved throughput. A major challenge
`
`in development of BASS, however, is that the 3'-blocking group must be
`
`197
`
`Copyright O I 999 by Marcel Dekker. Inc.
`
`www .dekker.com
`
`
`
`198
`
`WELCH AND BURGESS
`
`B = adenine, A; thymine, T
`guanine, G; cytosine, C
`
`dansyl=
`
`1a B=A
`1b8=T
`
`Diagram 1
`
`removed in an aqueous medium using reagents/conditions that do not
`
`denature or modify double strand DNA. To address this issue, the focus of
`
`our efforts has been on photolabile protection. Earlier work from these labs
`
`proved that 3'-0-(2"-nitrobenzyl)adenosine triphosphate II could be
`
`incorporated by a DNA polymerase.I In an extension of these studies, we
`now describe syntheses of two systems 1a and lb containing a 3 '-0-(2-
`nitrobenzyl) group modified to carry a fluorescent label (Diagram l ).
`The synthetic approach to target molecules 1a and lb has parallels
`with chemistry developed for solid phase photolabile linkers.7,8 Details of
`
`the synthetic route used are given in Scheme 1. The most difficult step in
`this procedure was coupling of a benzylic halide to the 3'-hydroxyl group of
`
`a nucleoside derivative. Development of conditions for this step required
`
`considerable experimentation.
`
`Details of the synthesis shown in Scheme 1 are as follows. The
`allyloxycarbonyl (ALLOC) protected bromide 2 was used to alkylate the
`
`vanillin derivative 3 to give the aldehyde 4. Nitration then borohydride
`
`
`
`FLUORESCENT 3'-0-PROTECTED NUCLEOSIDE TRIPHOSPHATES
`
`199
`
`~ ( +
`
`Meo~ ""t-1::NHALLOC
`OH
`5
`
`3
`
`2
`
`ii, iii
`
`iv, V
`
`OH N02
`
`.,,,,;,
`
`MeO
`
`°11:NHALLOC
`
`5
`
`5
`
`'8,P1>S;~~NHALLOC
`
`vi - viii
`
`N0 2
`
`6
`
`~NHdansyl
`5
`
`B = T and A82
`
`Scheme 1. Reagents and conditions: i, I½CO 1, KI, MeCN, reflux, 12 h (>95 %); ii,
`HNO,, Acp, 0 °C; iii, NaBH4, EtOH, 0 °C, 2 h then 25 'C, 2 h (53 % for ii+ iii); iv,
`CBr4 , PPh 3, EtOAc, 25 °C, 1 h; v, 5'-O-tert-butyldiphenilsilylnucleoside, NaI, Bu4NOH,
`CH2CI2, 1 M NaOH, 25 °C, 16 h (iv+ v, 49 % for B = A ', 88 % for B = T); vi, cat.
`Pd(PPh3)t THF, HNEt2 , 25 °C, 2.5 h; vii, dansyl-Cl, NEt3, cat. 4-DMAP, PhMe/fHF
`(1: 1 ), 25 C, 1 h; viii, Bu 4NF, THF, 25 ·c, 11 min for T, and 10 min at O ·c then 1 h at 25
`·c for A 8 '; (vi - viii, 69 % for B = AB', 66 % for B = T).
`
`
`
`200
`
`WELCH AND BURGESS
`
`reduction of this gave the benzylic alcohol 5, which emerged as a key
`intermediate in this work. Coupling of benzyl alcohol 5 to the 3 '-hydroxyl
`
`group of nucleoside derivatives was difficult. After considerable
`
`experimentation, it was found that conversion to the benzylic bromide, then
`alkylation under phase transfer conditions worked whereas many obvious
`alternative approaches did not. Removal of the ALLOC group from the
`coupled products 6, dansylation, then removal of the 5' -silyl protecting
`group gave the desired nucleosides 7.
`Tri phosphorylation of nucleosides 7, and of many other unnatural
`
`nucleosides prepared in these labs, is experimentally difficult and tends to
`
`give poor yields. Ultimately, we settled on the protocol developed by
`
`Eckstein and co-workers,9 although none of the methods attempted were
`
`entirely satisfactory. Fortunately, only small amounts of the product are
`
`required for feasibility tests in bioassays. Debenzoylation of the adenosine
`derivative 7a was performed after the triphosphorylation sequence (NH,.OH,
`60 •c, 3 h). Both final products, la and lb, were purified via
`chromatography, first on DEA cellulose, then by RP HPLC.
`Preliminary testsl of compounds la and lb as substrates for
`polymerases (Klenow, rTth DNA polymerase, and Vent (exo-) DNA
`
`polymerase) did not show evidence for incorporation. Further experiments
`are in progress, including modeling studies to attempt to dock la and 1 b in
`the active sites of polymerases. The latter experiments should highlight bad
`
`interactions that must be avoided to advance this work further.
`
`Acknowledgments. We thank Richard Gibbs of Baylor College
`
`of Medicine for valuable advice. Support for this work was provided by The
`
`Texas Technology Program, The Robert A. Welch Foundation, and The
`
`National Institutes of Health (HG/GM0l 745-01). KB thanks the NIH
`
`Research Career Development Award, and The Alfred P. Sloan Foundation
`
`for a fellowship.
`
`
`
`FLUORESCENT 3' -0-PROTECTED NUCLEOSIDE TRIPHOSPHATES
`
`201
`
`REFERENCES
`
`1. Metzker, M. L.; Raghavachari, R.; Richards, S.; Jacutin, S. E.;
`Civitello, A.; Burgess, K.; Gibbs, R. A. Nucl. Acids Res. 1994, 22,
`
`4259.
`
`2. Tsien, R. Y.; Ross, P.; Fahnestock, M.; Johnston, A. J. WO Patent
`
`91/06678, 1991.
`
`3. Cheeseman, P. C. US Patent 5,302,509, 1994.
`
`4. Wilhelm, A. Germany PatentDE4141178 Al, 1993.
`
`5. Rosenthal, A.; Close, K.; Brenner, S. WO Patent 93/21340, 1993.
`6. Canard, B.; Sarfati, R. S. Gene 1994, 148, 1.
`7. Yoo, D. J.; Greenberg, M. M. J. Org. Chem. 1995, 60, 3358.
`8. Holmes, C. P. J. Org. Chem. 1997, 62, 2370.
`9. Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54,631.
`
`received 7/16/98
`accepted 11/23/98
`
`
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