A diversity oriented synthesis of 3′-O-modified nucleoside triphosphates for DNA 'sequencing by synthesis'

Article abstract of DOI:10.1016/j.bmcl.2006.05.035

The nucleoside triphosphates 1, containing a photochemically cleavable group, and 2, having one that may be cleaved via palladium catalysis, were prepared as a prelude to investigating sequencing of DNA via sequencing by synthesis.

Full text of DOI:10.1016/j.bmcl.2006.05.035

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3902–3905
A diversity oriented synthesis of 30-O-modified nucleoside
triphosphates for DNA ‘sequencing by synthesis’
Genliang Lu and Kevin Burgess*
Department of Chemistry, Texas A&M University, PO Box 300012, College Station, TX 77842-3012, USA
Received 29 March 2006; revised 10 May 2006; accepted 12 May 2006
Available online 6 June 2006
Abstract—The nucleoside triphosphates 1, containing a photochemically cleavable group, and 2, having one that may be cleaved via
palladium catalysis, were prepared as a prelude to investigating sequencing of DNA via sequencing by synthesis.
Ó 2006 Elsevier Ltd. All rights reserved.
With notable exceptions,1 throughput in practical DNA
sequencing technologies tends to be limited by the
requirement of electrophoresis.2 In response, several
groups,3–11 including ours,12,13 have been motivated to
investigate ‘sequencing by synthesis’ (SBS) methods that
circumvent electrophoresis. A typical embodiment of
this idea would feature derivatives of the natural nucle-
oside triphosphates (dATP, dTTP, dCTP, and dGTP)
modified such that the 30-hydroxyl functionalities carry
a cleavable group attached to a fluorescent label, that
is compound types I.
plementary base analog; (ii) detection of the fluorescent
label (thereby revealing the complementary base on the
template); (iii) rupture of the cleavable group giving a
natural 30-hydroxy terminus; and, (iv) repetition of the
process (Fig. 1).
Four nucleobase analogs I would be required, each with
resolvable fluorescent labels that reveal the nucleobase
with which they are associated. Sequencing would then
be achieved via the following, iterative, process: (i) poly-
merase-mediated incorporation of the appropriate, com-
Keywords: DNA; Sequencing; Nucleoside; Triphosphate; Photolabile.
*
Corresponding author. Tel.: +1 979 845 4345; fax: +1 979 845
1881; e-mail: burgess@tamu.edu
Figure 1. The concept of sequencing by synthesis.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.035

G. Lu, K. Burgess / Bioorg. Med. Chem. Lett. 16 (2006) 3902–3905
3903
The main obstacle to reducing Figure 1 to practice is
that polymerase enzymes are extremely intolerant of
modifications at the 30-position: they tend not to incor-
porate these bases efficiently. Until the problem of incor-
poration is solved, other critical questions like the
fidelity of the incorporation cannot be addressed. Con-
sequently, it is clear that successful implementation of
sequencing by synthesis as shown in Figure 1 will re-
quire libraries of compounds I to be screened against li-
braries of polymerase mutants that are engineered to
tolerate changes at the nucleotide 30 site.
group, so if these surrogates are not incorporated by
the polymerases then ones containing fluorescent dyes
are unlikely to be incorporated too.
Compounds 1 and 2 have another important attribute,
but this relates to the overall strategy rather than to
the compound structures. The triazole functionality en-
ables single advanced precursors to be prepared, then
derivatized15 with a range of fluorescent dyes that have
pendant terminal alkynes. In other words, the synthesis
is diversity oriented.
The problem of producing even small numbers of nucle-
oside triphosphates I is non-trivial for several reasons.
Formation of nucleoside triphosphates tends to be mod-
erate-to-low-yielding reactions requiring HPLC purifi-
cation.14 Purification is necessary because tests for
incorporation by polymerases can give deceptive results
(both false positives and negatives) if small amounts of
impurities are present in the triphosphate sample.
The synthesis of compound 1 is described in Scheme 1.
The valeraldehyde-derived alcohol 3 was alkylated with
(i)
TsO
N3
OH
NO2
O
5
4
K2CO3, CH3CN, reflux, 40 h
(ii) PBr3, Et2O, 25 ˚C, 1 h
MeO
OH
The considerations outlined above led us to consider
methods for syntheses of the compounds 1 and 2
(Chart 1). These model compounds have the following
attributes. First, both compounds have groups that
could be cleaved efficiently under conditions that
should not disrupt ds (double strand) DNA. Second,
they both have oligoethylene glycol linkers that remove
the steric bulk of the dye (or dye surrogate in this case)
from the nucleoside component. Third, instead of
including a fluorescent dye, molecules 1 and 2 each
have a phenyl group. All the fluorescent dyes that are
usable in this methodology are larger than a phenyl
3
O
N
O
O
N
Ph
TBSO
O
Br
OH
6
NO2
Bu4NOH, NaOH, cat. NaI
CHCl3/H2O, 25 ˚C, 12 h
MeO
O
N3
O
5
5
79 %
O
O
N
N
O
O
NH
Ph
TPO
(i) TBAF, THF, 25 ˚C, 2 h
TBSO
N
O
(ii) NH4OH, EtOH, 25 ˚C, 1 h
O
O
(iii)PhC CH, Cu, CuSO4
THF/H2O, 22 h
O
photocleavable
O
group
NO2
NO2
dye
surrogate
Ph
N
MeO
MeO
O
N
N
O
N3
O
5
O
linker
5
7
48 %
1
O
O
NH
NH
HO
O
N
O
TPO
N
O
O
O
(i)
O
P
O
dye
Cl
O
surrogate
O
O
Pd-cleavable
group
1
NO2
O
DMF, pyridine, 25 ˚C, 15 min
(ii) (Bu3NH)2H2P2O7
Ph
O
60 %
Ph
N
Bu3N, 25 ˚C, 20 min
N
N
MeO
N
(iii) I2, py/H2O, 25 ˚C, 20 min
O
3
O
N
N
linker
5
2
8
17 %
Chart 1. Target nucleoside triphosphates 1 and 2.
Scheme 1. A synthesis of nucleoside triphosphate 1.

3904
G. Lu, K. Burgess / Bioorg. Med. Chem. Lett. 16 (2006) 3902–3905
an azido ethylene glycol linker 416 giving an oligoether
that was then converted into the benzylic bromide 5.
N-Protection of the thymidine base is essential for
derivatization of the 30-oxygen, otherwise the base is
alkylated preferentially.17,18 Consequently, the known
N-benzyl thymidine derivative 6 was coupled with the
benzylic bromide 5 under phase transfer conditions to
give the 30-ether 7. Desilylation of the 50-O-TBS and
hydrolysis of the 3-N-benzoyl group afforded a nucleo-
side ready for copper-mediated cycloaddition. This pro-
ceeded efficiently to give a nucleoside that was then
triphosphorylated using the Ludwig–Eckstein method,
that is phosphorylation with 2-chloro-4H-1,3,2-benzodi-
oxaphosphorin-4-one followed by pyrophosphate addi-
tion and oxidation.19
A synthesis of nucleotide triphosphate 2 was completed as
shown in Scheme 2. A triethylene glycol chlorocarbonate
was constructed as shown, then coupled to a 50-protected
thymidine derivative. In this case protection of the thymi-
dine part is not necessary because the chlorocarbonate
adds to the 30-hydroxyl selectively. Desilylation and cop-
per-mediated cycloaddition chemistry with phenylethyne
(our dye surrogate) gave the nucleoside 13 that was then
triphosphorylated as described above.
PPh3, CBr4
O
O
N3
OH
N3
Br
2
2
25 ˚C, 2 h
9
10 78 %
TBSO
It is important that 30-protection of the nucleoside tri-
phosphates 1 and 2 can be cleaved under relatively mild
conditions that would not perturb dsDNA. Photolysis
of compound 8 was performed using a relatively simple
strip light designed to emit at 360 nm (Southern New
England Ultraviolet Company). Acetonitrile was used
as solvent and ethanolamine as an additive. HPLC anal-
(i)
HO
OH
NaH, THF, 25 ˚C, 23 h
N3
O
3
(ii) TBAF, THF, 25 ˚C, 5 h
11 42 %
O
NH
TBSO
N
O
(i) phosgene, THF, 25 ˚C, 1 h
O
(ii) NaH, THF, 25 ˚C, 24 h
O
O
O
O
NH
N3
TBSO
O
3
N
O
O
12 47 %
OH
O
NH
HO
N
O
(i) TBAF, THF, 25˚C, 5 h
O
(ii) PhC CH, Cu, CuSO4
THF/H2O, 14 h
O
O
Ph
O
N
N
N
O
3
13 59 %
O
O
O
(i)
NH
P
Cl
O
TPO
N
O
DMF, pyridine, 25 ˚C, 15 min
O
(ii) (Bu3NH)2H2P2O7
Bu3N, 25 ˚C, 20 min
(iii) I2, py/H2O, 25 ˚C, 20 min
O
O
Ph
O
N
N
N
O
3
Figure 2. HPLC data from the deprotection of nucleoside 13: (a)
deoxythymidine; (b) nucleoside (13); (c) deprotection of 13 under
Pd(OAc)2 and PPh3; (d) deprotection of 13 under Pd[P(C6H4SO3Na)3]4.
The unlabeled peaks were unidentified.
2 69 %
Scheme 2. A synthesis of nucleoside triphosphate 2.

G. Lu, K. Burgess / Bioorg. Med. Chem. Lett. 16 (2006) 3902–3905
3905
ysis showed that the 30-O-photolabile group of deoxy-
thymidine 8 was completely deprotected in 15 min giv-
ing dT. Ethanolamine was necessary, however, since
without it the deprotection was only partial under other-
wise identical conditions.20 Deprotection of 13 was
examined under two sets of conditions involving palladi-
um catalysis (1, HCOOH/BuNH2 (1:1), Pd(OAc)2, PPh3,
H2O/CH3CN (1:1); and, 2, Pd[P(C6H4SO3Na)3]4, H2O).
Both these methods resulted in complete removal of the
ALLOC-based protecting group of 13 in fewer than
10 min and neutral pH giving dT also. Figure 2 shows
illustrative HPLC data from the palladium catalysis
deprotection.
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Much work needs to be done to reach a viable sequenc-
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tants is at least as demanding as the synthetic
challenges surrounding the triphosphates to be tested.
However, the work summarized here represents a signif-
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photolabile system 8 and the Pd-cleavable system 13
can potentially be combined with a series of fluorescent
dyes containing alkyne handles, then converted to tri-
phosphates. This approach will facilitate the arduous
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30-protected nucleoside triphosphates for testing in SBS.
Acknowledgments
Support for this project was provided by The NIH:
HG003573 and GM72041, and The Robert Welch
Foundation.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.05.035.
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17. Welch, M.; Martinez, C.; Zhang, A.; Jin, S.; Gibbs, R.;
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