Syntheses of nucleosides designed for combinatorial DNA sequencing

Article abstract of DOI:10.1002/(SICI)1521-3765(19990301)5:3<951::AID-CHEM951>3.0.CO;2-G

Nucleoside triphosphates I with 3′-O-blocking groups that are both photolabile and fluorescent were required to investigate the viability of a strategy for sequencing DNA in a combinatorial fashion (see Figure 1). Four compounds were prepared to realize this goal. Two of them, 14a and 14t, had dansyl-functionalized, 3′-O-(2″-nitrobenzyl) ether groups, while the other two, 18a and 18t, had similar pendant carbonate groups. Tests for incorporation of these analogues were performed by using five different DNA replicating enzymes, but the analogues were not incorporated. These results were surprising in view of the fact that previous studies had shown that 3′-O-(2″-nitrobenzyl)adenosine triphosphate II was incorporated by Bst DNA polymerase I. However, molecular simulations with the coordinates of a T7 polymerase crystal structure as a model demonstrates that analogues 14a, 14t, 18a and 18t are too large to fit into the enzyme active site, whereas accommodation of the unsubstituted 2-nitrobenzyl compound II is much less demanding. We conclude that both the nucleoside triphosphates and the DNA polymerase enzyme must be modified if the proposed DNA sequencing scheme is to be viable.

Full text of DOI:10.1002/(SICI)1521-3765(19990301)5:3<951::AID-CHEM951>3.0.CO;2-G

FULL PAPER
Syntheses of Nucleosides Designed for Combinatorial DNA Sequencing
Mike B. Welch,[a] Carlos I. Martinez,[b] Alex J. Zhang,[a] Song Jin,[a]
Richard Gibbs,[b] and Kevin Burgess*[a]
Abstract: Nucleoside triphosphates
I
by using five different DNA replicating
enzymes, but the analogues were not
incorporated. These results were sur-
prising in view of the fact that previous
studies had shown that 3'-O-(2''-nitro-
benzyl)adenosine triphosphate II was
incorporated by Bst DNA polymerase
I. However, molecular simulations with
the coordinates of a T7 polymerase
crystal structure as a model demon-
strates that analogues 14a, 14t, 18a
and 18t are too large to fit into the
enzyme active site, whereas accommo-
dation of the unsubstituted 2-nitroben-
zyl compound II is much less demand-
ing. We conclude that both the nucleo-
side triphosphates and the DNA
polymerase enzyme must be modified
if the proposed DNA sequencing
scheme is to be viable.
with 3'-O-blocking groups that are both
photolabile and fluorescent were re-
quired to investigate the viability of a
strategy for sequencing DNA in a com-
binatorial fashion (see Figure 1). Four
compounds were prepared to realize this
goal. Two of them, 14a and 14t, had
dansyl-functionalized,
3'-O-(2''-nitro-
benzyl) ether groups, while the other
two, 18a and 18t, had similar pendant
carbonate groups. Tests for incorpora-
tion of these analogues were performed
Keywords: DNA sequencing ´ en-
zyme catalysis ´ nucleotides
Other advances in sequencing focus on simultaneous
processing of data. The simplest and most widely applied
form of such multiplexing is separations of combinations of
Sanger sequencing reactions in single gel lanes.[17] Schemes
involving combination of two sets of Sanger sequencing
reactions have also been proposed,[17] but are not used
frequently. Multichannel capillary electrophoresis has also
been explored, and shows considerable promise.[8, 18±20] Other
forms of multiplex sequencing involve oligonucleotide probes
to visualize fragments after they have been transferred to a
nylon membrane.[21±23] However, efficiency enhancements
from any one of these methods is unlikely to raise the
throughput of sequence data by more than one or two orders
of magnitude.
There are few fundamentally new approaches to sequenc-
ing. Novel methodologies include those involving scanning
tunneling microscopy,[24] single molecule detection,[25, 26] meth-
ods based on detection of the pyrophosphate liberated in each
addition step,[27, 28] mass spectrometry,[29] and hybridization
(SBH-techniques).[30±32] These protocols may offer significant
increases in efficiency over the established procedures. They
generally do not require gel electrophoresis, therefore some
can potentially process larger numbers of samples without
concomitant increases in equipment, reagents, or time. How-
ever, at this stage these procedures are largely unproven, and
some have obvious disadvantages. Scanning tunneling micro-
scopy and other single molecule detection methods, for
instance, have not evolved to the level required for reliable
Introduction
Complete DNA sequence analysis of the human genome is a
costly and time-consuming project. Accelerated methods for
sequencing large DNA strands are therefore highly desirable.
Most of the current efforts to improve sequencing are
technological improvements of the Sanger[1, 2] or Maxam ±
Gilbert[3] schemes.[4] These include adaptation of robotic
systems for processing fluorescent dideoxy-terminated nu-
cleotides on commercially available DNA sequencing ma-
chines[5, 6] coupled with ultra thin,[7] or capillary gel,[8±11]
electrophoresis to improve efficiency. Conventional gel elec-
trophoresis is not required for some of these approaches,
nevertheless they are unlikely to reduce the cost and time
factors to acceptable levels. Other modifications of conven-
tional sequencing schemes focus on the primer, but still
require gel electrophoresis.[6, 12±14] For instance, contiguous
hexamer strings may be used in primer walking methods
wherein the appropriate primers are drawn from an oligonu-
cleotide library,[15, 16] but the feasibility of this methodology
remains to be proven for large-scale projects.
[a] Prof. Dr. K. Burgess, M. B. Welch, A. J. Zhang, S. Jin
Texas A & M University, Chemistry Department
P. O. Box 300012, College Station, Texas 77842 (USA)
[b] C. I. Martinez, R. Gibbs
Department of Molecular and Human Genetics
Baylor College of Medicine, One Baylor Plaza
Houston, TX77030 (USA)
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sequencing, and the techniques based on pyrophosphate
liberation and hybridization cannot be used to characterize
repeated sequences. The main applications of SBH-techni-
ques appear to be for detecting mutations.[33±35] Numerous
other schemes have been proposed but work on these has
apparently ceased.[36, 37]
Our group and others have been exploring another way to
facilitate parallel analyses of multiple samples in an array
without gel electrophoresis.[38±43] We call this the Base
Addition Sequencing Scheme or BASS. Central to this
approach is a set of four nucleoside triphosphates I that have
3'-O-blocking groups that are both labile and fluorescent. The
nucleotide triphosphate analogue I by a DNA replicating
enzyme(s); 2) spectroscopic identification of the base ana-
logue incorporated; and, 3) removal of the blocking group P*
to regenerate a 3'-hydroxy terminus on the (now elongated)
polynucleotide chain.
If realized, base addition sequencing would have several
significant advantages over the methodologies currently used
for sequencing DNA. First, results from each DNA sample
will be distinguished by direct analysis of the array, so addition
of more samples would not proportionately increase the
amount of effort or materials required. This compares
favorably with procedures wherein each DNA template must
be handled separately, and for which every additional sample
requires a new gel lane. Consequently, the proposed scheme
potentially has a much greater capacity than conventional
sequencing methods. Second, if the experiment could be
arranged in such a way that millions of primed DNA
fragments were analyzed simultaneously, then it would not
be necessary to characterize each primer. Instead the primers
could be generated by a combinatorial method, and the
arrangement of sequences would be deduced from overlaps in
the data. It is possible that the extent of multiplexing would be
such that the method would be viable even if a relatively short
read of DNA sequence was obtained from each individual
experiment (for example 10 ± 50 bases). Moreover, the ease of
primer synthesis would represent a highly significant cost/time
saving advantage. Finally, the method could have incidental
benefits like circumvention of artifacts due to gel compres-
sions (frequently associated with G,C-rich strings in the
sequence).
fluorescence of the protecting group should enable the parent
base on the ribose skeleton (A, T, G, or C) to be identified. A
cycle in the proposed sequencing scheme would consist of the
following steps (Figure 1): 1) incorporation of the appropriate
In preliminary work we found that 3'-O-(2''-nitrobenzyl)-
adenosine triphosphate II could be incorporated by a DNA
polymerase, and that photodeprotection of the 3'-hydroxy was
possible facilitating DNA replication.[38] This encouraging
result indicated that 3'-modifications could be tolerated by
DNA replicating enzymes. Herein we describe syntheses of
nucleosides protected with photolabile 3'-O-blocking groups
that are also fluorescent, and report preliminary tests for
incorporation by DNA polymerase enzymes.
Results and Discussion
Syntheses of nucleoside triphosphates with 3'-ether linkages:
Initial attempts to prepare 3'-O-protected nucleosides fo-
cused on the use of nitrobenzoic acid derivatives as illus-
trated in Scheme 1. The readily available starting material
1
[44] was coupled with (N-allyloxycarbonyl)pentan-5-ol amine,
via the acid chloride, to give the ester 2. A phase transfer
catalyst was used to form the critical ether linkage; devel-
opment of conditions for this step required considerable
experimentation. Removal of the 5'-silyl protecting group,
triphosphorylation,[45] and removal of the allyloxycarbonyl
group then gave the nucleotide amine 6. A potentially
attractive feature of this route was that addition of fluorescent
labels at the very end of the synthesis would allow one ad-
vanced intermediate to be transformed into several com-
pounds. Unfortunately, labeling of the triphosphate with
BODIPY-SE 503/512 was unsuccessful, due to the small
Figure 1. The base addition sequencing scheme (BASS).
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Nucleosides Designed for Combinatorial Sequencing
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Coupling of derivatives III (R ˆ H) to nucleosides was
problematic, but eventually, suitable conditions were devel-
oped. Scheme 2 outlines syntheses of derivatives of adenosine
Scheme 1. Synthesis of nucleoside amine 4.
amounts of material and difficulty in handling triphosphates.
A disadvantage of the design 6 is that 2-nitrobenzyl groups
substituted with carboxy functionalities cleave less readily
under photolytic conditions than comparatively electron-rich
systems.[46] This factor, combined with the experimental
difficulties associated with the triphosphorylation and label-
ing steps, led us to investigate alternative routes featuring
more photosensitive molecules.
The generic structure III represents the photolabile con-
nection sought in the next phase of this work. Photodecom-
position of the methyl substituted compounds III, R ˆ Me,
gives nitroso ketones, whereas the corresponding compounds
without this methyl substituent (R ˆ H) give nitroso alde-
hydes.[47] Nitroso ketones are less reactive by-products hence
initial efforts focused on the methyl-substituted compounds.
However, the secondary benzylic alcohol III where R ˆ Me
did not undergo coupling with the 3'-hydroxy of the nucleo-
side under a variety of conditions.
Scheme 2. Preparation of 14a and 14t.
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K. Burgess et al.
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and thymidine. The synthesis began with alkylation of vanillin
to install a linker for the fluorescent reporter. Nitration
followed by reduction provided the primary benzylic alcohol
9. The critical coupling step involved conversion of the
benzylic alcohol 9 into the corresponding benzyl bromide,
then phase transfer catalyzed reaction with a 5'-protected
nucleoside under biphasic conditions. Numerous other ap-
proaches were attempted, but only phase transfer based
methods gave positive results. In preparation for the con-
version to the nucleotide, the amine was deprotected then
dansylated; 5'-desilation then afforded nucleosides 13a and
13t.
Triphosphorylation of nucleosides 13 and 17, and of many
other unnatural nucleosides prepared in our laboratories, has
proved to be experimentally difficult and tends to give poor
yields. The protocol developed by Eckstein et al. was the best
out of several approaches attempted,[45] although none of
those were entirely satisfactory. Fortunately, only small
amounts of the product are required for feasibility tests in
bioassays. Debenzoylation of the adenosine derivative 13a
and 17a was performed after the triphosphorylation sequence
(NH4OH, 608C, 3 h). Both pairs of final products, 14a/14t and
18a/18t, were purified by chromatography, first on diethyl-
aminoethyl (DEAE) cellulose, then by RP HPLC.
Syntheses of nucleoside triphosphates with 3'-carbonate link-
ages: Difficulties encountered in the syntheses of the ether
linked derivatives, as outlined above, led us to explore
preparations of structurally similar, but hopefully more
accessible compounds. Syntheses of structural variants would
also help probe the tolerance of DNA replicating enzymes to
unnatural nucleosides. Consequently, two carbonate-linked
compounds were constructed as described in Scheme 3. Alter-
native routes to the same compounds were attempted, for
instance, by forming a chlorocarbonate functionality from the
nucleoside 3'-hydroxy, but none worked as well as that shown.
Tests for incorporation of 3'-blocked nucleoside triphos-
phates: Analogues 14a, 14t, 18a, and 18t were tested as
substrates for a series of commercially available DNA
replicating enzymes. The protocol used for these experiments
was based on a procedure we have reported previously.[38]
Briefly, 5'-fluorescein-labeled universal primer was annealed
Scheme 3. Preparation of carbonate-linked compounds.
to
a
synthetic oligo template, 5'-TACGGAGGTG-
14a, 14t, 18a, or 18t. After incubation the reactions were
stopped and loaded on a 20% acrylamide gel, subjected to gel
electrophoresis, the gel was scanned, and the results were
visualized with a fragment analysis software.
Table 1 shows the results for the incorporation assays. The
enzymes tested were unable to recognize the nucleotide
GACTGGCCGTCGTTTTACA (italic sequence indicates
the replication region). The reactions were carried out in
the presence of a mixture containing the corresponding
enzyme, some dNTPs, and no other added nucleotides
(control), a ddNTP (positive control), or a sample of analogue
Table 1. Tests of analogues 14a, 14t, 18a, 18t as substrates for DNA polymerases.
Analogue
Polymerase
Klenow
rTth DNA Pol.
Vent (exo-) DNA Pol.
Ampli Taq DNA Pol.
Ampli Taq FS
14a
14t
inhibition
(100 mm)
no incorporation
inhibition
(10 mm)
nonselective inhibition
(6.5 mm)
inhibition
(100 mm)
no incorporation
nonselective inhibition
(100 mm)
no incorporation
nonselective inhibition
(100 mm)
no incorporation
18a
18t
no incorporation
no incorporation
no incorporation
no incorporation
no incorporation
no incorporation
no incorporation
inhibition (1.8mm)
no incorporation
no incorporation
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Nucleosides Designed for Combinatorial Sequencing
951±960
analogues as substrates for the termination of the DNA
amplification under the conditions studied. No incorporation
was observed in most of the experiments, but in some there
was evidence that the analogue being tested inhibited the
polymerase under study, that is no incorporation was ob-
served and only a band corresponding to the unreacted primer
could be seen. This was the case for Ampli Taq DNA
polymerase for which a 1.18mm final concentration of the
thymidine carbonate 18t caused complete inhibition. Klenow
Fragment, rTth, and Vent (exo ) DNA polymerases ex-
hibited the same behavior but at 100mm of the adenosine ether
14a. Nonspecific inhibition (termination of the amplification
reaction at different positions along the template with no
specificity) was observed for Ampli Taq DNA polymerase, FS
and Ampli Taq DNA polymerase when a 100mm of 14a was
used (Figure 2).
Figure 2. Illustrative data from incorporation assay. Attempted incorpo-
ration of 14a by Ampli Taq DNA polymerase, FS. Arrow marks bands
corresponding to no incorporation. Template amplification sequence is
shown on the right of the gel. Fluorescein-labeled universal primer was
annealed to
a complementary oligo template (5'-TACGGAGGTG-
GACTGGCCGTCGTTTTACA). Lane 1 contained no dNTPs or ddNTPs.
Lanes 2 ± 8 contained 0.1mm dCTP, in addition lanes 3 and 4 contained
0.5mm ddATP and 0.1mm dATP, 0.1mm dTTP, respectively. Lanes 5 to 8
contained 0.1mm, 2mm, 10mm and 100mm D*ATP, respectively.
Molecular simulations of 3'-blocked nucleoside triphosphates
in the active sites of DNA replicating enzymes: Molecular
simulations were performed to rationalize the lack of
incorporation of the analogues prepared in the course of the
work described above. It was perplexing that compound 3'-O-
(2''-nitrobenzyl)adenosine triphosphate II was previously
incorporated by a DNA polymerase, whereas similar ana-
logues prepared in the current study were not.
Coordinates for a crystal structure of T7 DNA polymerase
encapsulating a primed template and ddGTP were down-
loaded from the Protein Data Bank and used as a model for
this study. This particular set of coordinates was used since
they include all the components (enzyme, primed template,
and nucleoside triphosphate) and because the data set was
recorded at high resolution (2.2 Š). An expansion of the
active site of this enzyme complex is shown in Figure 3 top.
Removal of the ddGTP entity gave a vacant active site, and
several conformers of 3'-O-(2''-nitrobenzyl)adenosine tri-
phosphate II were fitted in this void by visually docking to
form reasonable contacts and avoid unfavorable interactions.
A few orientations seemed reasonable, and one illustrative
representation is shown in Figure 3 middle. Conversely,
Figure 3. a) Active site of T7 polymerase highlighting two magnesium
atoms coordinated to dideoxyguanosine triphosphate with critical side-
chains of the protein (grey) and terminus of an encapsulated primer (blue)
highlighted; b) as above but with 3'-O-(2''-nitrobenzyl)adenosine triphos-
phate encapsulated; c) as in a) but with the nucleoside triphosphate 18a
encapsulated.
attempts to fit the analogues with fluorescent groups and a
methoxy-substituent attached to the aromatic ring were less
successful. Figure 3 bottom shows a representation of 18a in
the active site; several interactions were involved that would
not be permissible in reality. These docking experiments are
too crude to allow detailed conclusions to be formulated, but
it does seem clear that 3'-O-(2''-nitrobenzyl)adenosine tri-
phosphate II is much more easily accommodated in this
particular enzyme than any of the analogues prepared in this
study.
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K. Burgess et al.
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J ˆ 10.5, 1.5 Hz, 1H), 4.78 (brs, 1H), 4.55 ± 4.50 (m, 3H), 4.30 (t, J ˆ
6.3 Hz, 2H), 4.00 ± 3.80 (m, 3H), 3.16 (q, J ˆ 6.6 Hz, 2H), 2.35 (ddd, J ˆ
13.2, 5.7, 2.4 Hz, 1H), 2.22 ± 2.12 (m, 1H), 1.75 (p, J ˆ 7.2 Hz, 2H), 1.63 (s,
3H), 1.59 ± 1.38 (m, 4H), 1.06 (s, 9H); 13C NMR (CDCl3, 75 MHz): d ˆ
164.3, 163.2, 156.3, 150.7, 148.8, 136.9, 135.5, 135.2, 134.1, 133.8, 132.8, 132.2,
130.5, 130.2, 130.1, 128.4, 128.0, 127.9, 125.9, 117.6, 110.3, 87.0, 85.4, 72.0,
65.5, 64.0, 41.6, 41.1, 40.8, 29.6, 28.2, 27.0, 23.1, 19.3, 12.8; HRMS (positive-
ion FAB, nitrobenzyl alcohol (NBA)): calcd for C43H52N4O11SiNa 851.3299,
found 851.3327.
Conclusions
3'-O-Blocked nucleoside triphosphates, wherein the 3'-protect-
ing group is both photolabile and fluorescent, are syntheti-
cally accessible. However, they tend to be too big to fit into
the active site of DNA polymerases as evidenced by the data
from the activity screens and the molecular-simulation experi-
ments. It seems clear that modifications to the polymerase
enzyme as well as to the nucleoside triphosphate are required
if BASS is to be developed into a viable sequencing scheme.
There are indications that modern methods for combinatorial
mutagenesis of proteins, gene shuffling,[48] have the potential
to overcome this obstacle, but those methods must be refined
further before this goal can be realized.
3'-O-[(4''-(N-Allyloxycarbonyl)-5'''-aminopent-1-yl-oxycarbonyl)-2''-nitro-
phenylmethyl]thymidine (4): Compound 3 (700 mg, 0.84 mmol), THF
(8 mL), and terabutylammonium fluoride (TBAF) (1.26 mL, 1.26 mmol)
were stirred at 258C for 15 min. The reaction was concentrated and purified
directly by flash chromatography with a gradient of 100% EtOAc, then 1 to
5% MeOH in EtOAc as the eluant to give 4 as a foam (589 mg, 88% yield).
Rf ˆ 0.11 (75% EtOAc/hexanes); IR (neat): nÄ ˆ 3479, 2947, 1720,
1
1642 cm
;
1H NMR (CDCl3, 300 MHz): d ˆ 8.54 (d, J ˆ 1.8 Hz, 1H),
8.06 (dd, J ˆ 8.1, 1.5 Hz, 1H), 7.60 (s, 1H), 7.24 (d, J ˆ 8.1 Hz, 1H), 6.17 (t,
J ˆ 6.6 Hz, 1H), 5.89 ± 5.76 (m, 1H), 5.44 (s, 2H), 5.21 (d, J ˆ 17.4 Hz, 1H),
5.12 (d, J ˆ 10.5 Hz, 1H), 4.97 (br, 1H), 4.47 ± 4.41 (m, 3H), 4.28 (t, J ˆ
6.3 Hz, 2H), 4.00 (t, J ˆ 6.6 Hz, 2H), 3.89 (br, 1H), 3.81 ± 3.69 (m, 2H), 3.12
(q, J ˆ 6.6 Hz, 2H), 3.95 (br, 1H), 2.25 ± 2.19 (m, 2H), 1.99 (s, 3H), 1.74 (p,
J ˆ 7.5 Hz, 2H), 1.58 ± 1.35 (m, 4H); 13C NMR (CDCl3, 75 MHz): d ˆ 164.3,
163.2, 156.4, 150.7, 148.7, 136.8, 135.5, 133.9, 132.8, 130.5, 128.4, 125.8, 117.5,
110.1, 87.0, 86.5, 71.1, 65.6, 65.4, 62.1, 41.5, 40.7, 40.3, 29.5, 28.2, 23.0, 13.1;
HRMS (positive-ion FAB, NBA): calcd for C27H34N4O11Na 613.2122, found
613.2122.
Experimental Section
General procedures: High-field NMR spectra were recorded on a Unity‡
1
300 spectrometer (1H at 300 MHz, 13C at 75 MHz), H chemical shifts are
reported in d relative to CHCl3 (7.23 ppm) as internal standard, and 13C
chemical shifts are reported in ppm relative to CDCl3 (77.0 ppm) unless
1
otherwise specified. Multiplicities in H NMR are reported as (br) broad,
3'-O-[(4''-(N-Allyloxycarbonyl)-5'''-aminopent-1-yl-oxycarbonyl)-2''-nitro-
phenylmethyl]thymidine-5'-O-triphosphate (5): Compound 4 was azeo-
troped with PhH (3 Â 5 mL) and placed in a high vacuum dessicator
overnight. Compound 4 (369 mg, 0.46 mmol), 1,8-bis(dimethylamino)naph-
thalene (199 mg, 0.93 mmol), and trimethyl phosphate (4.6 mL) were
cooled to 08C under a nitrogen atmosphere, and phosphorus oxychloride
(142 mg, 0.93 mmol) was added in one portion. This mixture was stirred at
08C for 140 min. A premixed solution of tributylammonium pyrophosphate
(894 mg, 1.6 mmol) and tributylamine (296 mg, 1.60 mmol) in DMF
(2.0 mL) was then added. The reaction was stirred for 5 min and then
quenched at 08C with triethylammonium bicarbonate (1m, pH ˆ 5.7). The
reaction mixture was allowed to warm to 258C, stirred for an additional 1 h,
diluted with deionized H2O (10 mL), lyophilized, taken up in EtOH (3 Â
15 mL), and concentrated at 258C on a high vacuum rotary evaporator.
Purification by ion exchange chromatography with DEAE-sephadex A-25
resin, eluting with a 0 to 1.0m triethylammonium bicarbonate (500 mL
each) gradient gave the product 5. This was detected by monitoring the
absorbance at 302 nm. Appropriate fractions were then combined,
concentrated, redissolved in deionized water (5 mL), and concentrated
again to yield compound 5 (34 mg, 8.9% yield). 31P NMR (D2O, 121 MHz):
d ˆ 3.82 (d, J ˆ 24.3 Hz), 5.40 (d, J ˆ 21.4 Hz), 19.0 (t, J ˆ 20.8 Hz);
MS (MALDI-TOF): calcd for C27H35N4O20P3 828, found 828.
(s) singlet, (d) doublet, (t) triplet, (q) quartet, (p) quintet and (m) mul-
tiplet. Thin-layer chromatography was performed on silica gel 60F254
plates from Whatman. Flash chromatography was performed on SP silica
gel 60 (230 ± 600-mesh ASTM). BODIPY 503/512 was purchased from
Molecular Probes. Other chemicals were purchased from commercial
suppliers and used as received.
(N-Allyloxycarbonyl)-5-aminopent-1-yl 4-bromomethyl-3-nitrobenzoate
(2): 4-Bromomethyl-3-nitrobenzoic acid (2.0 g, 7.7 mmol), CH2Cl2
(38 mL), and DMF (0.1 mL) were cooled to 08C under a nitrogen
atmosphere. Oxalyl chloride (1.95 g, 15.4 mmol, 1.3 mL) was added,
resulting in a vigorous evolution of gas. The reaction mixture was stirred
for 3 h, concentrated, and taken up in CH2Cl2 (38 mL). A solution of
triethylamine (1.56 g, 15.4 mmol, 2.15 mL), catalytic DMAP, and (N-
allyloxycarbonyl)-5-aminopentanol (1.44 g, 7.7 mmol) in CH2Cl2 (10 mL)
was added to the above acid chloride, and the mixture was stirred for 3 h.
The reaction mixture was then diluted with CH2Cl2 (200 mL), washed with
HCl (0.5m 2 Â 250 mL), and the aqueous layer was back extracted with
CH2Cl2 (50 mL). Purification of the crude product by flash chromatography
with a gradient of 25% to 30% EtOAc/hexanes as the eluant gave 2 as a
thick liquid with an orange tinge (2.9 g, 87% yield). Rf ˆ 0.66 (50% EtOAc/
1
hexanes); H NMR (CDCl3, 300 MHz): d ˆ 8.57 (d, J ˆ 1.5 Hz, 1H), 8.21
(dd, J ˆ 8.1, 1.5 Hz, 1H), 7.74 (d, J ˆ 8.1 Hz, 1H), 5.90 ± 5.77 (m, 1H), 5.20
(d, J ˆ 17.1 Hz, 1H), 5.12 (d, J ˆ 10.2 Hz, 1H), 4.94 (s, 2H), 4.88 (brs, 1H),
4.47 (d, J ˆ 5.1 Hz, 2H), 4.31 (t, J ˆ 6.6 Hz, 2H), 3.14 (q, J ˆ 6.6 Hz, 2H),
1.76 (p, J ˆ 7.5 Hz, 2H), 1.58 ± 1.36 (m, 4H); 13C NMR (CDCl3, 75 MHz)
d ˆ 163.9, 156.2, 147.8, 136.5, 134.0, 132.7, 131.7, 126.0, 117.4, 65.7, 65.3, 42.2,
40.6, 29.5, 28.1, 27.9, 23.0.
3'-O-[4''-(5'''-Aminopent-1-yl-oxycarbonyl)-2''-nitrophenylmethyl]thymi-
dine-5'-O-triphosphate (6): Compound 5 (81 mg, 0.10 mmol), degassed
HPLC grade H2O (1.0 mL, 0.1m), [Pd(PPh3)4] (11 mg, 0.01 mmol), and
degassed morpholine (85 mg, 0.98 mmol) were stirred for 19 h at 258C. The
reaction was then filtered through a 45 mm HPLC filter. Purification was
performed by C18 RP-HPLC (analytical) with a gradient of 0% B (time t ˆ
3'-O-[(4''-(N-Allyloxycarbonyl)-5'''-aminopent-1-yl-oxycarbonyl)-2''-nitro-
phenylmethyl]-5'-O-(tert-butyldiphenylsilyl)thymidine (3): A solution of 2
(676 mg, 1.57 mmol) in CH2Cl2 (4.0 mL, 0.39m) was added dropwise over
0 min); 50%
B
(t ˆ 20 min); 70%
B
(t ˆ 30 min) where
A is 0.1m
triethylammonium acetate, and B is 70% MeCN/A. This procedure was
repeated 15 times for small portions of the reaction mixture on an
analytical instrument. Fractions containing the product 6 were assayed by
UV absorption at 302 nm, combined, and lyophilized to yield a white
hygroscopic powder (5.1 mg, 7% yield). MS (MALDI-TOF): calcd for
C23H32N4O18P3 745, found 745.
10 min to
a stirred mixture of 5'-O-(tert-butyldiphenylsilyl)thymidine
(771 mg, 1.60 mmol), aqueous Bu4NOH (70%, 0.5 mL), NaI (cat), CH2Cl2
(5 mL), H2O (5 mL), NaOH (1m, 5 mL), and stirred for 17 h at 258C. The
reaction mixture was diluted with CH2Cl2 (100 mL) and extracted with HCl
(0.5m, 2 Â 75 mL). The combined aqueous layers were then back extracted
with CH2Cl2 (50 mL). Purification of the crude product was by flash
chromatography with a gradient of 4:1:5 EtOAc/hexanes/CH2Cl2 increased
to 100% EtOAc as the eluting solvent. Appropriate fractions were
combined and concentrated to yield 3 as a yellow foam (717 mg, 54%
yield). Rf ˆ 0.28 (50% EtOAc/hexanes); IR (neat): nÄ ˆ 3448, 2934, 2859,
1715, 1668 cm 1; 1H NMR (CDCl3, 300 MHz): d ˆ 8.59 (d, J ˆ 1.8 Hz, 1H),
8.10 (dd, J ˆ 8.1, 1.5 Hz, 1H), 7.64 ± 7.61 (m, 4H), 7.54 (d, J ˆ 1.2 Hz, 1H),
7.45 ± 7.37 (m, 6H), 7.25 (d, J ˆ 8.1 Hz, 1H), 6.34 (dd, J ˆ 7.8, 5.4 Hz, 1H),
5.93 ± 5.80 (m, 1H), 5.49 (s, 2H), 5.24 (dd, J ˆ 17.1, 1.5 Hz, 1H), 5.16 (dd,
4-[(N-Allyloxycarbonyl)-5-aminopent-1-yl-oxy]-3-methoxybenzaldehyde (8):
Compound
7 (3.35 g, 13 mmol), MeCN (40 mL), vanillin (1.98 g,
0.01 mmol), KI (cat), and K2CO3 (5.6 g, 0.04 mol) were refluxed for 12 h.
The crude reaction mixture was filtered through a plug of silica gel on
Celite and diluted with EtOAc (100 mL). The organic layer was extracted
with water (2 Â 50 mL), HCl (0.5m, 2 Â 50 mL), and brine (25 mL), dried
over Na2SO4, filtered, and concentrated to yield 8 as a thick orange liquid
(4.2 g, 100% yield). Rf ˆ 0.38 (50% EtOAc/hexanes); IR (neat): nÄ ˆ 3454,
2945, 2866, 1700, 1266 cm 1; 1H NMR (CDCl3, 300 MHz): d ˆ 9.75 (s, 1H),
956
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Chem. Eur. J. 1999, 5, No. 3

Nucleosides Designed for Combinatorial Sequencing
951±960
7.35 (dd, J ˆ 8.1, 1.8 Hz, 1H), 7.32 (s, 1H), 6.87 (d, J ˆ 1.8 Hz, 1H), 5.89 ±
5.76 (m, 1H), 5.20 (dd, J ˆ 17.1, 1.2 Hz, 1H), 5.11 (dd, J ˆ 10.2, 0.9 Hz,
1H), 4.94 (brs, 1H), 4,46 (d, J ˆ 5.4 Hz, 2H), 4.01 (t, J ˆ 6.6 Hz, 2H), 3.83
(s, 3H), 3.13 (q, J ˆ 6.3 Hz, 2H), 1.82 (p, J ˆ 6.9, 2H), 1.57 ± 1.41 (m, 4H);
13C NMR (CDCl3, 75 MHz): d ˆ 190.8, 156.2, 153.9, 149.6, 132.8, 129.7,
126.6, 117.3, 111.2, 109.0, 68.6, 65.2, 55.8, 40.6, 29.5, 28.3, 23.0; HRMS
(positive-ion FAB, NBA): calcd for C17H24N1O5 322.1654, found 322.1646.
6.9 Hz, 2H), 1.64 (d, J ˆ 0.9 Hz, 3H), 1.56 ± 1.42 (m, 4H), 1.05 (s, 9H);
13C NMR (CDCl3, 75 MHz): d ˆ 163.4, 156.3, 153.5, 150.8, 147.3, 141.9,
135.5, 135.3, 133.9, 133.0, 132.9, 132.4, 130.2, 130.1, 128.0, 128.0, 126.7, 117.5,
114.0, 110.9, 109.5, 86.9, 85.5, 72.0, 69.2, 65.4, 64.0, 56.2, 41.4, 41.0, 29.6, 28.5,
27.0, 23.1, 19.3, 12.8; HRMS (positive-ion FAB, NBA): calcd for
C43H54N4O11SiNa 853.3456, found 853.3479.
3'-O-[4''-((N-Allyloxycarbonyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-
nitrophenylmethyl]-2'-deoxy-5'-O-(tert-butyldiphenylsilyl)-N-6-benzoyl-
adenosine (11a): Compound 11a was prepared by a similar procedure to
that used for 11t but with 10 (409 mg, 0.95 mmol) in CHCl3 (3.2 mL), 6-N-
benzoyl-5'-O-tert-butyldiphenylsilyl-2'-deoxy-N-6-benzoyladenosine
(564 mg, 0.95 mmol), Bu4NOH (0.5 mL), NaI (cat), NaOH (1m, 3.0 mL),
and CHCl3 (3.2 mL) for a reaction time of 23 h. Chromatography with 60 to
70% EtOAc/hexanes as the eluant gave 11a (384 mg, 43% yield) as a
yellow foam. Rf ˆ 0.34 (70% EtOAc/hexanes); IR (CDCl3): nÄ ˆ 3451, 2943,
2863, 1713, 1578, 1520 cm 1; 1H NMR (CDCl3, 300 MHz): d ˆ 8.49 (s, 1H),
8.02 (s, 1H), 7.62 ± 7.54 (m, 5H), 7.44 ± 7.20 (m, 11H), 7.13 ± 7.08 (m, 2H),
6.34 (t, J ˆ 6.6 Hz, 1H), 5.96 (s, 2H), 5.93 ± 5.80 (m, 1H), 5.23 (dq, J ˆ 17.1,
1.5 Hz, 1H), 5.14 (dq, J ˆ 10.2, 1.5 Hz, 1H), 4.77 (brs, 1H), 4.66 ± 4.59
(brm, 1H), 4.50 (d, J ˆ 5.1 Hz, 2H), 4.03 ± 3.93 (m, 3H), 3.86 ± 3.77 (m,
2H), 3.75 (s, 3H), 3.15 (t, J ˆ 6.3 Hz, 2H), 2.65 ± 2.56 (m, 1H), 2.48 ± 2.40
(m, 1H), 2.35 (brs, 1H), 1.79 (p, J ˆ 7.5 Hz, 2H), 1.57 ± 1.38 (m, 4H), 1.00 (s,
9H); 13C NMR (CDCl3, 75 MHz): d ˆ 181.7, 172, 156.3, 154.1, 152.3, 146.8,
142.1, 140.2, 135.5, 135.4, 133.0, 132.7, 130.9, 130.0, 130.0, 128.7, 127.9, 127.2,
117.5, 110.5, 109.6, 87.0, 84.4, 71.8, 69.2, 65.4, 63.8, 56.4, 49.7, 40.8, 40.3, 29.6,
28.5, 26.9, 23.1, 19.2; HRMS (positive-iom FAB, NBA): calcd for
C50H57N7O10SiNa 966.3834, found 966.3892.
1-[(N-Allyloxycarbonyl)-5'-aminopent-1-yl-oxy]-4-(hydroxymethyl)-2-meth-
oxy-5-nitrobenzene (9): Compound 8 (4.23 g, 13.2 mmol) in Ac2O (17 mL)
was slowly added to a stirred mixture of HNO3 (66 mL) and Ac2O (17 mL)
at 08C. After 2 h the reaction was allowed to warm to 258C and stirred for
an additional 2 h. The resulting solution was diluted with EtOAc (200 mL),
washed with brine (4 Â 100 mL), NaHCO3 (6 Â 100 mL), brine (100 mL),
dried over Na2SO4, filtered through a plug of silica gel on Celite, and
concentrated yielding a thick yellow oil. This crude material was dissolved
in absolute EtOH (66 mL) and cooled to 08C. Solid NaBH4 (1.0 g,
26 mmol) was added in about 50 mg portions; the reduction was monitored
by TLC until starting material was completely consumed (approximately
4 h). The reaction was then quenched with saturated aqueous NH4Cl,
allowed to warm to room temperature, and diluted with EtOAc (200 mL).
The crude reaction mixture was extracted with water (2 Â 100 mL), brine
(100 mL), dried over Na2SO4, filtered through a plug of silica gel on Celite,
and concentrated. The crude product was purified by flash chromatography
with a gradient of 50 to 60% EtOAc/hexanes as the eluting solvent yielding
9 (0.73 g, 53% yield over two steps) as a yellow oil which crystallized upon
standing. Rf ˆ 0.36 (3  30% EtOAc/hexanes); IR (neat): nÄ ˆ 3439, 2943,
2862, 1711, 1576, 1323, 1064 cm 1; 1H NMR (CDCl3, 300 MHz): d ˆ 7.65 (s,
1H), 7.15 (s, 1H), 5.93 ± 5.80 (m, 1H), 5.24 (dd, J ˆ 17.4, 1.5 Hz, 1H), 5.14
(dd, J ˆ 10.2, 1.2 Hz, 1H), 4.91 (s, 2H), 4.85 (br, 1H), 4.49 (d, J ˆ 5.4 Hz,
2H), 4.02 (t, J ˆ 6.6 Hz, 2H), 3.93 (s, 3H), 3.16 (q, J ˆ 6.3 Hz, 2H), 3.10 ±
2.85 (br, 1H), 1.84 (p, J ˆ 6.9 Hz, 2H), 1.60 ± 1.42 (m, 4H); 13C NMR
(CDCl3, 75 MHz): d ˆ 156.3, 154.1, 147.2, 139.4, 132.8, 132.0, 117.6, 110.8,
109.1, 69.1, 65.4, 62.6, 56.3, 40.7, 29.6, 28.4, 23.0; HRMS (positive-ion FAB,
NBA): calcd for C17H25N2O7 369.1661, found 369.1666.
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methyl]-5'-O-(tert-butyldiphenylsilyl)thymidine (12t): Compound 11t
(681 mg, 0.82 mmol) was dissolved in freshly distilled THF (4.0 mL) at
258C under N2 atmosphere. Degassed distilled diethylamine (1.2 g,
16.4 mmol) and [Pd(PPh3)4] (95 mg, 0.82 mmol) were added and the
solution was stirred at 258C for 75 min. The reaction was concentrated,
dissolved in CHCl3 (5 mL), and concentrated again. This cycle was
repeated two more times to remove residual diethylamine. The reaction
mixture was charged with THF (4 mL), TEA (triethylamine; 331 mg,
3.3 mmol), dimethylaminopyridine (DMAP) (cat), and stirred at 258C. A
slurry of dansyl chloride (221 mg, 0.82 mmol) in THF/DMF (2:1 mL) was
added to the above reaction mixture, and this was then stirred for 6 h at
258C. The crude product was purified by flash chromatography with
2.5:2.5:1 to 3:2:1 EtOAc/hexanes/CH2Cl2 as eluant and gave 12t as a off-
white foam (582 mg, 73% yield). Rf ˆ 0.57 (70% EtOAc/hexanes);
1H NMR (CDCl3, 300 MHz): d ˆ 8.46 (d, J ˆ 8.7 Hz, 1H), 8.24 (d, J ˆ
8.7 Hz, 1H), 8.18 (dd, J ˆ 7.2, 1.2 Hz, 1H), 7.94 (s, 2H), 7.62 ± 7.30 (m,
12H), 7.10 (d, J ˆ 7.8 Hz, 1H), 6.61 (s, 1H), 6.36 (dd, J ˆ 8.1, 5.7 Hz, 1H),
5.46 (s, 2H), 5.21 (t, J ˆ 6 Hz, 1H), 4.56 ± 4.50 (brm, 1H), 4.00 ± 3.75 (m,
5H), 3.74 (s, 3H), 3.54 (d, J ˆ 3.6 Hz, 1H) 2.86 (brm, 2H), 2.82 (s, 3H), 2.81
(s, 3H), 2.35 (ddd, J ˆ 13.2, 5.7, 2.1 Hz, 1H), 2.16 ± 2.07 (m, 1H), 1.61 (s,
3H), 1.55 (p, J ˆ 7.5 Hz, 2H), 1.37 (p, J ˆ 6.9 Hz, 2H), 1.28 ± 1.20 (m, 2H),
1.03 (s, 9H); 13C NMR (CDCl3, 75 MHz): d ˆ 163.3, 153.4, 151.9, 150.7,
147.1, 141.7, 135.4, 135.2, 134.0, 132.9, 132.4, 132.0, 131.9, 130.2, 130.0, 129.9,
129.8, 129.6, 129.3, 128.5, 128.3, 128.2, 127.8, 127.8, 126.8, 123.0, 118.7, 115.1,
110.2, 110.0, 109.3, 87.0, 85.4, 71.7, 68.9, 64.0, 56.1, 45.2, 42.9, 41.3, 40.9, 28.9,
28.0, 26.9, 22.7, 19.2, 12.7; HRMS (positive-ion FAB, NBA): calcd for
C51H61N5O11SiSNa 1002.3755, found 1002.3791.
1-[(N-Allyloxycarbonyl)-5'-aminopent-1-yl-oxy]-4-bromomethyl-2-meth-
oxy-5-nitrobenzene (10): Compound 9 (114 mg, 0.31 mmol), in an oven-
dried flask, was azeotroped with PhH (2 mL), charged with distilled EtOAc
(1.5 mL), triphenylphosphine (122 mg, 0.46 mmol), and stirred until
homogeneous at 258C. Carbon tetrabromide (154 mg, 0.46 mmol) was
added and the resulting orange-red solution was stirred for 1 h. The
reaction was exothermic and a gummy material formed. The crude reaction
mixture was filtered through a plug of silica gel on Celite and washed with
EtOAc (50 mL). Purification of the crude product by flash chromatography
with a gradient of 30 to 40% EtOAc/hexanes as the eluting solvent gave
compound 10 (117 mg, 88% yield), as an off-white solid. M.p. 107 ± 1088C;
Rf ˆ 0.36 (40% EtOAc/hexanes); IR (CDCl3): nÄ ˆ 3452, 2942, 1718, 1526,
1
1282 cm
;
1H NMR (CDCl3, 300 MHz): d ˆ 7.60 (s, 1H), 6.88 (s, 1H),
5.93 ± 5.81 (m, 1H), 5.25 (ddd, J ˆ 17.4, 3.0, 1.2 Hz, 1H), 5.16 (ddd, J ˆ 10.2,
2.7, 1.5 Hz, 1H), 4.82 (s, 2H), 4.83 ± 4.76 (brs, 1H), 4.50 (d, 5.7 Hz, 2H), 4.03
(t, J ˆ 6.3 Hz, 2H), 3.93 (s, 3H), 3.17 (q, J ˆ 6.3 Hz, 2H), 1.85 (dq, J ˆ
6.6 Hz, 2H), 1.61 ± 1.41 (m, 4H); 13C NMR (CDCl3, 75 MHz): d ˆ 156.2,
153.4, 148.3, 140.0, 132.9, 127.2, 117.5, 113.7, 109.4, 69.1, 65.4, 56.4, 40.7, 30.2,
29.6, 28.3, 23.0; HRMS (positive-ion FAB, NBA): calcd for C17H24N2O6Br
431.0817, found 431.0830.
3'-O-[4''-((N-Allyloxycarbonyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-
nitrophenylmethyl]-5'-O-(tert-butyldiphenylsilyl)thymidine (11t): A solu-
tion of 10 (800 mg, 1.85 mmol) in CHCl3 (5.0 mL, 0.37m) was added over
10 min to a vigorously stirred solution of 5'-O-(tert-butyldiphenylsilyl)-
thymidine (891 mg, 1.85 mmol), Bu4NOH (0.5 mL), NaI (cat), NaOH (1m,
5.0 mL), and CHCl3 (5.0 mL). After 16 h the reaction was extracted with
H2O (20 mL), and brine (20 mL). Material in the combined aqueous
fractions was back extracted into CHCl3 (2 Â 10 mL), dried (Na2SO4),
filtered, and concentrated. The crude product was purified by flash
chromatography with 50 to 60% EtOAc/hexanes as eluant giving 11t as an
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methyl]-2'-deoxy-5'-O-(tert-butyldiphenylsilyl)-N-6-benzoyladenosine (12a):
Compound 12a was prepared by a similar procedure to that used for 12t
but with compound 11a (174 mg, 0.18 mmol), diethylamine (0.27 g,
3.6 mmol, 381 mL), [Pd(PPh3)4] (21 mg, 0.82 mmol), THF (1.0 mL) for a
reaction time of 75 min. After the reaction mixture was concentrated to
dryness, it was then dissolved in THF (1.0 mL), triethylamine (75 mg,
0.74 mmol), (cat) DMAP, dansyl chloride (50 mg, 0.18 mmol) in 2:1 THF/
DMF (1.0 mL) for a reaction time of 6 h. Flash chromatography with 3:2:1
to 3.5:1.5:1 EtOAc/hexanes/CH2Cl2 as eluant gave 12a (47.8 mg, 24%
yield) as a yellow foam. Rf ˆ 0.56 (70% EtOAc/hexanes); 1H NMR
(CDCl3, 300 MHz): d ˆ 8.50 (s, 1H), 8.50 ± 8.46 (m, 1H), 8.19 (dd, J ˆ
1.2 Hz, 1H), 8.01 (d, J ˆ 7.2 Hz, 1H), 7.62 ± 7.08 (m, 21H), 6.31 (dd, J ˆ 8.7,
6.0 Hz, 1H), 5.98 (s, 2H), 5.62 ± 5.58 (m, 1H), 4.72 (t, J ˆ 6.3 Hz, 1H),
4.20 ± 4.15 (m, 1H), 3.85 (t, J ˆ 3.3 Hz, 2H), 3.79 ± 3.71 (m, 2H), 3.75 (s,
3H), 2.92 ± 2.86 (m, 2H), 2.83 (s, 3H), 2.80 ± 2.70 (m, 1H), 2.54 ± 2.46 (m,
off-white foam (1.56 g, 49% yield). Rf ˆ 0.30 (60% EtOAc/hexanes); IR
1
(neat): nÄ ˆ 3425, 3271, 2937, 1713, 1646, 1520 cm
;
1H NMR (CDCl3,
300 MHz): d ˆ 7.64 ± 7.61 (m, 4H), 7.51 (s, 2H), 7.45 ± 7.33 (m, 6H), 6.64 (s,
1H), 6.34 (dd, J ˆ 8.1, 6.0 Hz, 1H), 5.94 ± 5.81 (m, 1H), 5.46 (d, J ˆ 2.7 Hz,
2H), 5.24 (dd, J ˆ 17.1, 1.5 Hz, 1H), 5.15 (dd, J ˆ 10.5, 1.2 Hz, 1H), 4.84 ±
4.76 (brm, 1H), 4.56 ± 4.48 (brm, 3H), 4.01 ± 3.80 (m, 6H), 3.77 (s, 3H), 3.16
(q, J ˆ 6.3 Hz, 2H), 2.39 ± 2.32 (m, 1H), 2.19 ± 2.10 (m, 1H), 1.82 (p, J ˆ
Chem. Eur. J. 1999, 5, No. 3
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0947-6539/99/0503-0957 $ 17.50+.50/0
957

K. Burgess et al.
FULL PAPER
1H), 1.60 ± 1.50 (m, 2H), 1.40 ± 1.32 (m, 2H), 1.29 ± 1.20 (m, 2H), 0.99 (s,
9H); 13C NMR (CDCl3, 75 MHz): d ˆ 172.2, 159.9, 153.9, 153.8, 152.4,
152.1, 146.6, 141.8, 139.9, 135.5, 135.4, 134.6, 132.5, 132.3, 131.1, 130.4, 130.0,
129.8, 129.6, 129.5, 128.9, 128.7, 128.3, 127.9, 127.9, 127.0, 123.1, 118.5, 115.1,
110.1, 109.2, 85.0, 84.2, 74.0, 68.8, 63.4, 56.4, 49.8, 45.3, 42.9, 37.9, 29.0, 28.4,
27.9, 26.8, 22.7, 19.1.
before ion exchange purification. The product was isolated as an off-white
solid (21 mg, 13% yield). 31P NMR (D2O, 121 MHz): d ˆ 7.02 (brs),
8.09 (brs), 19.60 (brs); MS (MALDI-TOF): calcd for C35H43N8O18P3S
988 , found 988.
3'-O-[4''-((N-Allyloxycarbonyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-
nitrophenylmethoxycarbonyl]-5'-O-(tert-butyldiphenylsilyl)thymidine (15t):
Compound 9 (144 mg, 0.39 mmol), THF (2 mL), and a solution of phosgene
(2.1 mL, 1.9m) in toluene (3.91 mmol) were stirred at 258C for 1.5 h. The
mixture was concentrated using a rotary evaporator in a hood (CAUTION:
phosgene is a toxic gas), and azeotroped with benzene (2 Â 2 mL). The
residual chlorocarbonate formed was dissolved in THF (4 mL) and cooled
to 08C. In a separate flame-dried flask, 5'-O-(tert-butyldiphenylsilyl)thy-
midine (169 mg, 0.35 mmol), was dissolved in THF (2 mL), and NaH
(17 mg, 0.70 mmol, 60% in oil) was added (without removal of oil), and this
slurry was stirred at 258C for 1 h. The anion was transferred slowly to the
chlorocarbonate solution via a cannula, over a 4 min period, then washed in
with THF (2 Â 1 mL). The reaction mixture was stirred at 08C for 30 min
then at 258C for an additional 1.5 h. The product was purified by flash
chromatography with 4:1 CH2Cl2/EtOAc eluant which gave 15t (336 mg,
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methyl]thymidine (13t): Compound 12t (570 mg, 0.58 mmol), THF
(2.9 mL), and TBAF in THF (1m, 870 mL) were stirred for 45 min at
258C. The reaction mixture was concentrated, diluted with CH2Cl2
(50 mL), then washed with HCl (0.5m, 20 mL), H2O (20 mL), and brine
(20 mL). The organic layer was dried (Na2SO4) filtered, and concentrated.
The crude product was purified by flash chromatography with EtOAc
eluant giving 13t (397 mg, 92% yield) as a yellow foam. Rf ˆ 0.32 (100%
1
EtOAc); IR (CDCl3): nÄ ˆ 3502, 2937, 1703, 1527, 1247 cm
;
1H NMR
([D6]acetone, 300 MHz): d ˆ 8.50 (dt, J ˆ 5.1, 1.2 Hz, 1H), 8.38 (dt, J ˆ 8.7,
0.9 Hz, 1H), 8.20 (dd, J ˆ 7.2, 1.2 Hz, 1H), 7.99 (s, 1H), 7.95 (d, J ˆ 1.5 Hz,
1H), 7.72 ± 7.49 (m, 3H), 7.19 (dd, J ˆ 7.8, 0.9 Hz, 1H), 6.76 (t, J ˆ 6.0 Hz,
1H), 6.72 (s, 1H), 6.32 (t, J ˆ 6.9 Hz, 1H), 5.41 (s, 2H), 4.52 ± 4.46 (brs,
1H), 4.38 ± 4.32 (brs, 1H), 3.97 ± 3.93 (brm, 1H), 3.83 (t, J ˆ 6.6 Hz, 2H),
3.82 ± 3.76 (m, 2H), 3.74 (s, 3H), 2.86 ± 2.94 (m, 2H), 2.81 (s, 6H), 2.29 ± 2.23
(m, 2H), 1.85 (d, J ˆ 0.9 Hz, 3H), 1.55 (p, J ˆ 6.9 Hz, 2H), 1.47 ± 1.25 (m,
4H); 13C NMR ([D6]acetone, 75 MHz): d ˆ 163.8, 154.3, 152.6, 151.7, 148.0,
142.3, 137.1, 136.1, 132.6, 130.5, 129.6, 128.5, 127.2, 124.0, 120.0, 115.8, 110.6,
109.7, 109.7, 88.6, 86.6, 71.8, 69.4, 62.5, 56.4, 45.5, 43.4, 41.6, 41.2, 28.9, 23.4,
13.2; HRHRMS (positive-ion FAB, NBA): calcd for C35H43N5O11SNa
764.2577, found 764.2605.
97% yield) as a white foam. Rf ˆ 0.35 (3  50% EtOAc/hexanes); IR
1
(neat): nÄ ˆ 3574, 3499, 3297, 2966, 2858, 1961, 1717, 1456 cm
;
1H NMR
(CDCl3, 300 MHz): d ˆ 9.61 (d, J ˆ 5.7 Hz, 1H), 7.67 (s, 1H), 7.64 ± 7.59 (m,
4H), 7.44 (d, J ˆ 1.2 Hz, 1H), 7.40 ± 7.32 (m, 6H), 7.03 (s, 1H), 6.41 (dd, J ˆ
9.3, 5.1 Hz, 1H), 5.93 ± 5.80 (m, 1H), 5.54 (s, 2H), 5.36 (d, J ˆ 5.7 Hz, 1H),
5.23 (d, J ˆ 17.1 Hz, 1H), 5.13 (d, J ˆ 10.5 Hz, 1H), 4.96 (brs, 1H), 4.55
(brs, 1H), 4.50 (d, J ˆ 5.4 Hz, 1H), 4.16 (s, 1H), 4.03 (t, J ˆ 6.0 Hz, 2H),
4.00 ± 3.90 (m, 2H), 3.95 (s, 3H), 3.17 (q, J ˆ 6.3 Hz, 2H), 2.52 (dd, J ˆ 14.1,
5.1 Hz, 1H), 2.31 ± 2.21 (m, 1H), 1.84 (p, J ˆ 7.2 Hz, 2H), 1.58 ± 1.45 (m,
4H), 1.51 (s, 3H), 1.06 (s, 9H); 13C NMR (CDCl3, 75 MHz): d ˆ 163.8,
156.2, 154.0, 153.9, 150.5, 147.7, 139.3, 135.4, 135.0, 134.7, 132.9, 132.7, 131.8,
130.1, 130.0, 128.0, 127.9, 125.9, 117.4, 111.5, 109.7, 109.1, 84.7, 84.1, 78.7, 69.1,
66.6, 65.3, 64.2, 56.5, 40.6, 37.8, 29.5, 28.3, 26.9, 23.0, 19.3, 11.8; HRMS
(positive-ion FAB, NBA): calcd for C44H54N4O13SiNa 897.3354, found
897.3369.
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methyl]-2'-deoxy-N-6-benzoyladenosine (13a): Compound 13a was pre-
pared by a similar procedure to that used for 13t but with compound 12a
(40 mg, 0.04 mmol), THF (0.5 mL), and TBAF (1m) in THF (55 mL) for a
reaction time of 45 min. Chromatography with 100% EtOAc to 10%
MeOH/EtOAc as eluant gave 13a (32 mg, 100% yield) as a yellow foam.
Rf ˆ 0.41 (100% EtOAc); IR (CDCl3): nÄ ˆ 3513, 2942, 1701, 1579,
1
1252 cm
;
1H NMR ([D6]acetone, 300 MHz): d ˆ 8.58 (s, 1H), 8.51 (s,
3'-O-[4''-((N-Allyloxycarbonyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-
nitrophenylmethoxycarbonyl]-2'-deoxy-5'-O-(tert-butyldiphenylsilyl)-N-6-
1H), 8.49 (dt, J ˆ 8.4, 1.2 Hz, 1H), 8.36 (dt, J ˆ 8.7, 0.9 Hz, 1H), 8.18 (dd,
J ˆ 7.5, 1.5 Hz, 1H), 8.00 (s, 1H), 7.64 ± 7.47 (m, 4H), 7.39 ± 7.30 (m, 2H),
7.25 ± 7.18 (m, 3H), 6.70 (t, J ˆ 6.0 Hz, 1H), 6.44 (dd, J ˆ 7.8, 6.0 Hz, 1H),
5.93 (s, 2H), 4.61 ± 4.56 (m, 1H), 4.50 ± 4.45 (m, 1H), 4.04 ± 4.00 (m, 2H),
3.82 ± 3.74 (m, 2H), 3.76 (s, 3H), 2.94 ± 2.87 (m, 3H), 2.80 (s, 6H), 2.78 ± 2.65
(m, 1H), 2.38 ± 2.31 (m, 1H), 1.53 (p, J ˆ 6.9 Hz, 2H), 1.40 (p, J ˆ 7.5 Hz,
2H), 1.32 ± 1.25 (m, 2H); 13C NMR ([D6]acetone, 75 MHz): d ˆ 172.5,
154.9, 154.8, 154.6, 153.2, 152.7, 152.3, 147.9, 141.0, 137.2, 136.8, 131.7, 130.0,
130.5, 129.7, 129.5, 129.3, 128.7, 128.6, 128.3, 124.1, 120.3, 115.9, 111.1, 109.9,
89.6, 86.4, 79.1, 72.6, 69.5, 63.2, 56.7, 53.7, 49.9, 45.5, 43.5, 41.2, 26.2, 23.4,
20.4, 13.8; HRMS (positive-ion FAB, NBA): calcd for C42H46N8O10SNa
877.2955, found 877.2955.
benzoyladenosine (15a): Compound 15a was prepared by
a similar
procedure to that used for 15t but with compound 9 (150 mg, 0.41 mmol),
THF (2 mL), phosgene (2.2 mL, 4.1 mmol) for a reaction time of 1.5 h. 6-N-
Benzoyl-5'-O-tert-butyldiphenylsilyl-2'-deoxyadenosine (75.5 mg, 0.13 mmol),
NaH (15 mg, 0.25 mmol, 60% in oil), and THF (2.0 mL) were mixed in
another flask and stirred for 30 min. The nucleoside anion was added slowly
with a cannula to the chlorocarbonate solution for a reaction time of 17 h.
Chromatography with a 3:1 to 1:2 CH2Cl2/EtOAc eluant gave 15a (57 mg,
1
41% yield) as a yellow foam. Rf ˆ 0.20 (20% EtOAc/hexanes); H NMR
(CDCl3, 300 MHz): d ˆ 9.12 (brs, 1H), 8.69 (s, 1H), 8.18 (s, 1H), 7.98 (d,
J ˆ 7.2 Hz, 2H), 7.69 (s, 1H), 7.63 ± 7.29 (m, 13H), 7.03 (s, 1H), 6.52 (dd, J ˆ
8.7, 5.4 Hz, 1H), 5.94 ± 5.81 (m, 1H), 5.57 (s, 2H), 5.50 (d, J ˆ 6.0 Hz, 1H),
5.24 (dd, J ˆ 17.4, 1.5 Hz, 1H), 5.15 (dd, J ˆ 10.5, 1.2 Hz, 1H), 4.83 (br,
1H), 4.51 (d, J ˆ 5.4 Hz, 2H), 4.30 (br, 1H), 4.05 (t, J ˆ 6.3 Hz, 2H), 3.99 ±
3.87 (m, 2H), 3.96 (s, 3H), 3.18 (q, J ˆ 6.3 Hz, 2H), 2.94 ± 2.84 (m, 1H), 2.73
(dd, J ˆ 13.2, 5.4 Hz, 1H), 1.86 (p, J ˆ 7.2, 2H), 1.59-1.42 (m, 4H), 1.03 (s,
9H); 13C NMR (CDCl3, 75 MHz): d ˆ 165.5, 155.9, 153.6, 153.5, 152.1,
151.1, 149.3, 147.5, 140.6, 139.1, 135.2, 135.1, 135.0, 133.1, 132.5, 132.2, 132.1,
131.9, 129.6, 128.2, 127.5, 127.5, 127.4, 125.3, 123.0, 117.0, 109.8, 108.8, 84.7,
83.9, 78.3, 68.7, 64.9, 63.4, 56.1, 40.3, 37.6, 29.2, 28.0, 26.5, 22.7, 18.8; HRMS
(positive-ion FAB, NBA): calcd for C51H57N7O12SiNa 1010.3732, found
1010.3711.
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methyl]thymidine 5'-O-triphosphate (14t): Compound 13t was azeotroped
with anhydrous pyridine (3 Â 1.0 mL) and stored for 20 h in a vacuum
dessicator over P2O5. A solution of 2-chloro-4H-1,3,2-benzodioxaphos-
phorin-4-one (1.0m 4.3 mg, 21.2 mmol) in dioxane (21 mL) was added to 13t
(14.3 mg, 19.3 mmol) in pyridine (38 mL) and dioxane (116 mL), and the
reaction was then stirred at 258C under a nitrogen atmosphere for 10 min.
A solution of tributylammonium pyrophosphate (0.5m, 13.2 mg, 29 mmol)
and tributylamine (14.8 mg, 79 mmol, 19 mL) in DMF (158 mL, 0.5m) was
prepared in a glove box. This was added to the 13t solution and stirred for
15 min. A solution of 1% I2 (7.8 mg, 31 mmol) in pyridine (764 mL) and H2O
(16 mL) was added and the resulting solution was stirred for 15 min, then
quenched with saturated aqueous NaHSO3 (added until the red color
dissipated, approximately four drops). The reaction was concentrated to
dryness, taken up in H2O (2 mL) and concentrated to dryness again. The
residue was purified by ion exchange chromatography with DEAE-
sephadex A-25 and a gradient of H2O (300 mL) to triethylammonium
acetate pH ˆ 7.5 (1.0m, 300 mL) giving 14t (2.1 mg, 13% yield). 31P NMR
(D2O, 121 MHz): d ˆ 3.70 (brs), 8.1 (brs), 19.00 (brs); MS (MALDI-
TOF): calcd for C35H44N5O20P3S 978, found 978.
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methoxycarbonyl]-5'-O-(tert-butyldiphenylsilyl)thymidine (16t): Com-
pound 15t (821 mg, 0.94 mmol), THF (4.7 mL, 0.26m), HNEt2 (686 mg,
9.4 mmol, 970 mL), and [Pd(PPh3)4] (108 mg, 0.09 mmol, 10 mol%) was
stirred at 258C for 2.5 h and concentrated giving the deprotected amine. A
sample of the crude amine prepared as above (16 mg, 0.02 mmol) was
dissolved in 1:1 toluene/THF (100 mL); NEt3 (16 mL, 0.12 mmol) and
DMAP (cat.) was added, and the solution was stirred at 258C for 15 min. A
solution of dansyl chloride (5.5 mg, 0.02 mmol) in 1:1 toluene/THF
(0.5 mL) was added, and the solution was stirred at 258C for 1 h, then
concentrated to dryness. The residue was purified by flash chromatography
with 1:1:2 to 6:4:5 EtOAc/hexanes/CH2Cl2 eluant which gave 16t (19 mg,
92% yield) as a yellow foam. Rf ˆ 0.36 (60% EtOAc/hexanes); IR (neat):
3'-O-[4''-((N-Dansyl)-5'''-aminopent-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methyl]-2'-deoxy-N-6-benzoyladenosine 5'-O-triphosphate (14a): Com-
pound 13a was prepared by a similar procedure to that used for 14t,
except that the solution was heated to 608C with NH4OH (5.0 mL) for 5.5 h
958
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0947-6539/99/0503-0958 $ 17.50+.50/0
Chem. Eur. J. 1999, 5, No. 3

Nucleosides Designed for Combinatorial Sequencing
951±960
1
nÄ ˆ 3387, 2939, 1751, 1682, 1519, 1274 cm
;
1H NMR (CDCl3, 300 MHz):
(dd, J ˆ 9.6, 5.4 Hz, 1H), 5.92 (brd, J ˆ 9.3 Hz, 1H), 5.56 (s, 2H), 5.50 (d,
J ˆ 5.4 Hz, 1H), 4.82 (t, J ˆ 5.7 Hz, 1H), 4.37 (s, 1H), 3.96 (s, 3H), 4.00 ±
3.87 (m, 2H), 3.88 (t, J ˆ 6.6 Hz, 2H), 3.25 ± 3.50 (m, 1H), 2.90 (q, J ˆ
6.3 Hz, 2H), 2.85 (s, 6H), 2.54 (dd, J ˆ 13.8, 5.1 Hz, 1H), 1.90-1.70 (brs,
1H), 1.66 (p, J ˆ 6.9 Hz, 2H), 1.49 ± 1.30 (m, 4H); 13C NMR (CDCl3,
75 MHz): d 153.9, 153.8, 152.1, 150.3, 148.0, 142.5, 139.9, 134.6, 133.3, 133.0,
130.4, 129.8, 129.6, 128.9, 128.3, 127.9, 125.4, 123.2, 118.6, 115.1, 111.0, 109.2,
87.5, 87.1, 80.1, 69.0, 67.0, 63.2, 56.5, 45.4, 42.9, 37.7, 29.1, 28.0, 22.8; HRMS
(positive-ion FAB, NBA); calcd for C43H46N8O12SNa 921.2853, found
921.2858.
d ˆ 9.13 ± 9.04 (brm, 1H), 8.48 (d, J ˆ 8.7 Hz, 1H), 8.25 (d, J ˆ 8.7 Hz,
1H), 8.21 (dd, J ˆ 7.5, 1.2 Hz, 1H), 7.65 ± 7.60 (m, 5H), 7.52 ± 7.34 (m, 9H),
7.13 (d, J ˆ 7.5 Hz, 1H), 7.02 (s,1H), 6.42 (dd, J ˆ 6.9, 5.1 Hz, 1H), 5.52 (s,
2H), 5.38 (d, J ˆ 6.0 Hz, 1H), 4.99 ± 4.92 (brm, 1H), 4.17 (d, J ˆ 1.2 Hz,
1H), 3.97 (qd, J ˆ 11.4, 1.8 Hz, 2H), 3.95 (s, 3H), 3.85 (t, J ˆ 6.3 Hz, 2H),
2.90 (q, J ˆ 6.6 Hz, 2H), 2.84 (s, 6H), 2.53 (dd, J ˆ 13.8, 5.1 Hz, 1H), 2.32-
2.22 (m, 1H), 1.64 (p, J ˆ 6.9 Hz, 2H), 1.52 (s, 3H), 1.43 (p, J ˆ 6.9 Hz, 2H),
1.36 ± 1.29 (m, 2H), 1.07 (s, 9H); 13C NMR (CDCl3, 75 MHz): d ˆ 163.6,
154.0, 152.0, 150.4, 147.7, 139.3, 135.4, 135.1, 134.8, 134.7, 132.7, 131.8, 130.3,
130.2, 130.1, 129.8, 129.6, 128.3, 128.1, 128.0, 125.9, 123.1, 118.6, 115.1, 111.6,
109.8, 109.1, 84.7, 84.2, 78.8, 68.9, 66.7, 64.3, 56.6, 45.3, 42.9, 37.9, 29.1, 28.0,
26.9, 22.7, 19.3, 11.9; HRMS (positive-ion FAB, NBA): calcd for
C52H61N5O13SiSNa 1046.3650, found 1046.3662.
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methoxycarbonyl]thymidine 5'-O-triphosphate (18t): Compound 17t
(35 mg, 0.04 mmol) was azeotroped with benzene (3 Â 2 mL) and stored
overnight in a desiccator over P2O5. 2-Chloro-4H-1,3,2-benzodioxaphos-
phorin-4-one (10 mg, 0.05 mmol), DMF (160 mL) then pyridine (64 mL)
were added and the solution was stirred at 258C for 15 min. A solution of
tributylammonium pyrophosphate (0.5m, 30 mg, 0.07 mmol) in DMF
(132 mL) and tributylamine (44 mL, 0.19 mmol) were added simultaneously,
and the mixture was stirred at 258C for 11 min. A solution of 1% iodine
(18 mg, 0.07 mmol) in pyridine/water (98/2) (1760 mL/36 mL) was added,
and the mixture was stirred at 258C for 11 min. The reaction was quenched
by dropwise addition of 5% aqueous NaHSO3, until the red color
disappeared. The reaction mixture was stirred for 1 h at 258C after the
quench, then concentrated under a high vacuum. The residue was purified
by semi-prep C18 RP-HPLC with a gradient of 0 to 100% B (over 70 min)
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methoxycarbonyl]-2'-deoxy-5'-O-(tert-butyldiphenylsilyl)-N-6-benzoyl-
adenosine (16a): Compound 16a was prepared by a similar procedure to
that used for 16t but with compound 15a (260 mg, 0.26 mmol), HNEt2
(192 mg, 2.6 mmol, 272 mL), Pd(PPh3)4 (30 mg, 0.03 mmol) in THF
(1.3 mL) for a reaction time of 3 h. A portion of the amine prepared
above (134 mg, 0.15 mmol) was combined with NEt3 (90 mg, 0.89 mmol,
124 mL), DMAP (cat.), and dansyl chloride (40 mg, 0.15 mmol) in THF
(1.3 mL) and stirred at 258C for 17 h. Chromatography with 60 to 90%
EtOAc/hexanes eluant, gave 16a (38 mg, 23% yield) as an off-white foam.
Rf ˆ 0.34 (70% EtOAc/hexanes); IR (neat): nÄ ˆ 3162, 2934, 2863, 1754,
1612, 1527 cm 1; 1H NMR (CDCl3, 300 MHz): d ˆ 9.12 (s, 1H), 8.71 (s, 1H),
8.49 (d, J ˆ 8.7 Hz, 1H), 8.25 (d, J ˆ 9.0 Hz, 1H), 8.21 (dd, J ˆ 7.2, 1.2 Hz,
1H), 8.19 (s, 1H), 7.99 (d, J ˆ 7.2 Hz, 2H), 7.67 ± 7.30 (m, 16H), 7.12 (d, J ˆ
6.9 Hz, 1H), 5.52 (dd, J ˆ 9.0, 5.4 Hz, 1H), 5.58 (s, 2H), 5.50 (d, J ˆ 5.7 Hz,
1H), 4.92 (t, J ˆ 6.0 Hz, 1H), 4.34 ± 4.28 (m, 1H), 3.97 (s, 3H), 4.00 ± 3.85
(m, 4H), 2.90 (q, J ˆ 6.6 Hz, 2H), 2.84 (s, 6H), 2.76 (d, J ˆ 4.5 Hz, 1H),
2.71 (d, J ˆ 5.7 Hz, 1H), 1.95 (brs, 1H), 1.65 (p, J ˆ 7.2 Hz, 2H), 1.95 (brs,
1H), 1.65 (p, J ˆ 7.2 Hz, 2H), 1.48 ± 1.28 (m, 4H), 1.04 (s, 9H); 13C NMR
(CDCl3, 75 MHz): d ˆ 164.6, 153.9, 152.7, 152.0, 151.5, 149.6, 147.8, 140.8,
139.5, 135.5, 135.4, 134.7, 133.1, 132.8, 132.5, 132.3, 132.1, 132.0, 132.0, 131.9,
130.4, 130.0, 129.8, 129.9, 128.8, 128.5, 128.4, 128.3, 127.9, 125.8, 123.2, 118.6,
115.1, 110.2, 109.1, 85.2, 84.2, 78.8, 69.0, 66.8, 63.8, 56.6, 45.3, 42.9, 38.3, 29.1,
28.0, 26.9, 22.8, 19.2; HRMS (positive-ion FAB, NBA): calcd for
C59H64N8O12SiSNa 1159.4030, found 1159.4058.
1
at 10 mLmin pump rate. The product (Rt ˆ 47 min) 18t (10.3 mg, 23%
yield) was isolated as a pale orange fluffy powder. MS (positive-ion FAB,
NBA): calcd for C36H44N5O22P3S 1023, found 1023; 31P NMR (D2O,
121 MHz) d ˆ 1.46 (d, J ˆ 20.8 Hz), 6.83 (d, J ˆ 18.9 Hz), 17.62 (t,
J ˆ 20.2 Hz).
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methoxycarbonyl]-2'-deoxyadenosine 5'-O-triphosphate (18a): Compound
18a was prepared by a similar procedure to that used for 18t but with
compound 17a (22 mg, 25 mmol), 2-Chloro-4H-1,3,2-benzodioxaphosphor-
in-4-one (5.5 mg, 27 mmol), DMF (100 mL), and pyridine (40 mL) for a
reaction time of 11 min. A solution of tributylammonium pyrophosphate
(0.5m, 17 mg, 37 mmol,) in DMF (80 mL) and tributylamine (30 mL,
126 mmol) were added simultaneously and the resulting solution was
stirred for 14 min. A solution of 1% iodine (11 mg, 39 mmol) in 98:2
pyridine/water was added, and the solution was stirred for 15 min. The
reaction was quenched by dropwise addition of 10% aqueous NaHSO3,
until the red color disappeared. The solution was stirred at 258C for 10 min
after the quench, then concentrated under a high vacuum. The residue was
dissolved in 29% NH4OH (10 mL) and heated to 608C for 2 h then
concentrated to dryness. Purification of the residue was by semi-prep C18
RP-HPLC with 0 to 100% B over 70 min at 10 mLmin 1. The product 18a
was collected as an off-white solid (4 mg, 18% yield). MS (MALDI-TOF):
calcd for C36H43N8O20P3S 1032, found 1032.
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methoxycarbonyl]thymidine (17t): Compound 16t (55.9 mg, 0.06 mmol),
THF (275 mL), and TBAF (61 mL) were stirred for 1 h at 08C then allowed
to warm to room temperature. The reaction was concentrated to dryness,
dissolved in CH2Cl2 (30 mL), then extracted with aqueous HCl (0.5m, 2 Â
30 mL), and brine (30 mL), dried over Na2SO4, filtered, and concentrated.
The residue was purified by flash chromatography with 30:4:1 to 45:5:1
EtOAc/hexanes/CH2Cl2 eluant which gave 17t as a yellow foam (32 mg,
75% yield). Rf ˆ 0.53 (90% EtOAc/hexanes), 0.28 (80% EtOAc/hexanes);
1
IR (neat): nÄ ˆ 2925, 2860, 1760, 1695, 1515, 1285 cm
;
1H NMR (CDCl3,
Incorporation assays: Klenow fragment of DNA polymerase
I was
300 MHz): d ˆ 9.04 (br, 1H), 8.49 (d, J ˆ 8.4 Hz, 1H), 8.23 (d, J ˆ 9 Hz,
1H), 8.20 (d, J ˆ 7.2 Hz, 1H), 7.60 (s, 1H), 7.49 (m, 3H), 7.12 (d, J ˆ 7.8 Hz,
1H), 7.00 (s, 1H), 6.23 (t, J ˆ 7.2 Hz, 1H), 5.53 (s, 2H), 5.31 (brm, 1H), 4.96
(t, J ˆ 6.0 Hz, 1H), 4.17 (brm, 1H), 3.49 (s, 3H), 3.90 (br, 2H), 3.87 (m,
2H), 2.89 (q, J ˆ 6.3 Hz, 2H), 2.80 (br, 1H), 2.83 (s, 6H), 2.44 (m, 2H), 1.86
(s, 3H), 1.64 (p, J ˆ 7.2 Hz, 2H), 1.41 (m, 2H), 1.32 (m, 2H); 13C NMR
(CDCl3, 75 MHz): d ˆ 163.7, 154.1, 152.0, 150.4, 147.8, 139.5, 136.5, 134.6,
130.4, 129.8, 129.6, 128.3, 125.7, 123.2, 118.6, 115.0, 111.4, 110.2, 109.2, 86.2,
84.9, 78.9, 69.0, 66.8, 62.6, 56.6, 45.3, 42.9, 37.1, 29.1, 28.0, 22.8, 12.5; HRMS
(positive-ion FAB, NBA): calcd for C36H43N5O13SNa 808.2476, found
808.2510.
purchased from Boehringer Mannheim. Bst DNA polymerase, Moloney
Murine Leukemia Virus (M-MuLV) reverse transcriptase, and VentR
(exo ) DNA polymerase were purchased from New England Bio-Labs.
rTth DNA polymerase was purchased from Pharmacia. Ampli Taq DNA
polymerase and Ampli Taq DNA polymerase, FS were purchased from
Perkin Elmer. Fluorescein-labeled universal primer (5'-TGTAAAAC-
GACGGCCAGT) and oligonucleotide template (5'-TACGGAGGTG-
GACTGGCCGTCGTTTTACA) were purchased from Life Technologies
(italic sequence represents that part of the oligonucleotide that remains for
amplification).
The single-stranded template (1 pmol) was mixed with 5'-fluorescein
tagged universal primer (1 pmol) in the corresponding buffer solution. This
mixture was warmed to 808C for 7 min and then allowed to reach room
temperature slowly. Extension of the primer-template complex was
performed in the absence of the natural nucleotide when either a ddNTP
or a 3'-modified nucleotide was tested. Each reaction mixture contained
5 mL of the annealed DNA duplex and 5 mL of the corresponding mixtures
containing the enzyme and the nucleotides in the specific buffer.
Conditions for all the enzymatic reactions can be found in reference 38
except for those of Ampli Taq DNA polymerase, FS. In this case, the buffer
3'-O-[4''-((N-Dansyl)-5'''-aminopent-1-yl-oxy)-5''-methoxy-2''-nitrophenyl-
methoxycarbonyl]-2'-deoxy-N-6-benzoyladenosine (17a): Compound 17a
was prepared by a similar procedure to that used for 17t but with
compound 16a (37 mg, 0.03 mmol), THF (330 mL), TBAF (33 mL) for a
reaction time of 18 min. Chromatography with 90% EtOAc/hexanes then
EtOAc then 5 to 10% MeOH/EtOAc as eluant gave 17a (22 mg, 75%
yield) as an off-white foam. Rf ˆ 0.13 (70% EtOAc/hexanes), 0.52 (100%
EtOAc); IR (neat): nÄ ˆ 3322, 2989, 2961, 2887, 1766 cm 1; 1H NMR (CDCl3,
300 MHz): d ˆ 9.09 (br, 1H), 8.74 (s, 1H), 8.50 (d, J ˆ 8.4 Hz, 1H), 8.22 (t,
J ˆ 9.0 Hz, 2H), 8.06 (s, 1H), 7.99 (d, J ˆ 7.2 Hz, 2H), 7.62 (s, 1H), 7.62 ±
7.56 (m, 1H), 7.52 ± 7.46 (m, 4H), 7.14 (d, J ˆ 7.5 Hz, 1H), 7.00 (s, 1H), 6.34
was 80mm Tris, 2mm MgCl2, pH 9.0; dNTPs were used at
a final
concentration of 0.1mm, and ddNTPs at a final concentration of 0.5mm.
Chem. Eur. J. 1999, 5, No. 3
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0947-6539/99/0503-0959 $ 17.50+.50/0
959

K. Burgess et al.
FULL PAPER
The incubation temperature was 658C in the presence of 1 unit of the
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Received: August 21, 1998 [F1317]
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