Preparation of base-deuterated 2′-deoxyadenosine nucleosides and their site-specific incorporation into DNA

Article abstract of DOI:10.1081/NCN-100108321

We report a four-step synthesis of 2′-deoxy-2-deuteroadenosine from 2′-deoxyadenosine in 38% overall yield. The more accessible 2′-deoxy-8-deuteroadenosine was also prepared and incorporated into DNA by automated solid phase synthesis (80% deuterium) using N⁶-benzoyl-2′-deoxy-8-deuteroadenosine-3′-O- (2-cyanoethyl-N,N-diisopropylphos-phoramidite) in combination with acetyl-protected deoxycytidine and phenoxyacetyl-protected purine phosphoramidites.

Full text of DOI:10.1081/NCN-100108321

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PREPARATION OF BASE-DEUTERATED 2′-
DEOXYADENOSINE NUCLEOSIDES AND THEIR SITE-
SPECIFIC INCORPORATION INTO DNA
Panadda Chirakul a , Justin R. Litzer a & Snorri Th. Sigurdsson b
a Department of Chemistry , University of Washington , Seattle, WA, 98195-1700, U.S.A.
b Department of Chemistry , University of Washington , Seattle, WA, 98195-1700, U.S.A.
Published online: 17 Aug 2006.
To cite this article: Panadda Chirakul , Justin R. Litzer & Snorri Th. Sigurdsson (2001) PREPARATION OF BASE-DEUTERATED 2′-
DEOXYADENOSINE NUCLEOSIDES AND THEIR SITE-SPECIFIC INCORPORATION INTO DNA, Nucleosides, Nucleotides and Nucleic
Acids, 20:12, 1903-1913
To link to this article: http://dx.doi.org/10.1081/NCN-100108321
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(12), 1903–1913 (2001)
PREPARATION OF BASE-DEUTERATED
20-DEOXYADENOSINE NUCLEOSIDES AND
THEIR SITE-SPECIFIC INCORPORATION
INTO DNA
Panadda Chirakul, Justin R. Litzer, and Snorri Th. Sigurdsson*
Department of Chemistry, University of Washington,
Seattle, WA 98195-1700, USA
ABSTRACT
We report a four-step synthesis of 20-deoxy-2-deuteroadenosine from
20-deoxyadenosine in 38% overall yield. The more accessible 20-deoxy-
8-deuteroadenosine was also prepared and incorporated into DNA
by automated solid phase synthesis (80% deuterium) using N6-benzoyl-
20-deoxy-8-deuteroadenosine-30-O-(2-cyanoethyl-N,N-diisopropylphos-
phoramidite) in combination with acetyl-protected deoxycytidine and
phenoxyacetyl-protected purine phosphoramidites.
INTRODUCTION
It is widely accepted that the mechanistic understanding of biomolecular
function is based on structure. However, it has become clear that many
reported structures represent ground states that may bear little resemblance
to the structures of the transition states. Therefore, structural information
about ground states needs to be augmented with knowledge about the
movements of functionally important residues. Solid state deuterium NMR is
a valuable tool to study motions of specific sites in nucleic acids1–3
.
*Corresponding author.
1903
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1904
CHIRAKUL, LITZER, AND SIGURDSSON
For example, solid state deuterium NMR was used to detect large amplitude
motions in the sugar-phosphate backbone at the GC-AT duplex junction of
the EcoR1 restriction site 50-d(CGAATTCG)2, precisely where this enzyme
cuts3. This cleavage reaction can be suppressed by base-methylation at
position 5 of 20-deoxycytidine (dC) and interestingly, the methylation reduces
the amplitudes of motion of the sugar-phosphate backbone3. It is possible
that the origin of the selectivity of EcoR1 for the non-methylated sequence
resides in the change of conformational flexibility at the cleavage site.
We are interested in studying the role of dynamics in the interactions of
proteins with DNA. The study of DNA dynamics by solid state deuterium
NMR requires site-specific incorporation of deuterium-labeled nucleotides.
The 50- and 20-positions of thymidine (T), dC and 20-deoxyadenosine (dA)
have been used for sugar-labeling4–6 and the 5- and 6-positions have been
used for base-labeling of dC and T1. The methyl groups of T and 5-methyl
dC have also been deuterated3. To extend the studies of nucleic acid
dynamics by solid state deuterium NMR, we intend to study the base-
motions of dA in DNA. The required incorporation of deuterium into either
the 2- or 8-position of the adenine base is outlined here.
We focused initially on the 2-position of dA because the proton in the 8-
position can exchange with protic solvents1. There has been only one report
of the synthesis of 20-deoxy-2-deuteroadenosine (1), by oxidation of 20-
deoxyadenosine, followed by ring-opening and subsequent ring-closure using
a deuterated derivative of formic acid7. However, six synthetic steps were
required, giving 1 in only 16% overall yield. Another ring-opening=ring-
closure strategy has been reported for the synthesis of 2-deuteroadenosine in
four steps via 1-N6-ethenoadenosine, yielding the desired product in 55%
overall yield8. We describe here the application of this approach to the
preparation of 1 and the corresponding phosphoramidite for its site-specific
incorporation into DNA.
Given the synthetic effort required for the preparation of 1 we have also
investigated the incorporation of 20-deoxy-8-deuteroadenosine (2) into DNA.
Compound 2 can be readily prepared by a proton-exchange reaction in the
presence of triethylamine1. While this represents an easy route to base-
deuterated 20-deoxyadenosine, the deuteron in the 8-position can proton-
exchange with solvent during deprotection and purification of the DNA. We
show that this strategy, when coupled with the use of phenoxyacetyl-pro-
tected purine nucleosides and acetyl-protected dC, allows incorporation of 2
into DNA with 80% incorporation of deuterium.
RESULTS AND DISCUSSION
Our approach for the synthesis of 1 was to use a ring-opening=ring-
closing strategy, similar to that reported for 2-deuteroadenosine8.

BASE-DEUTERATED 20-DEOXYADENOSINE NUCLEOSIDES
1905
Scheme 1. Synthesis of 20-deoxy-2-deuteroadenosine.
The synthetic route (Scheme 1) starts with 1,N6-ethenodeoxyadenosine (3),
prepared by the reaction of 20-deoxyadenosine with chloroacetaldehyde using
a slight modification of a previously published protocol9. Reaction of 3 with
NaOH at 23ꢀC resulted in ring opening to yield 4. We found the optimal
concentration of NaOH to be 0.8 M, with a reaction time of 3 h. Reaction of
4 with 1,1,1-triethoxy-1-deuteromethane10 in N,N-dimethylacetamide (DMA)
resulted in ring closure with incorporation of deuterium (94%) to give 5. The
last step entailed the removal of the etheno-modification using ammonium
persulfate8. It is critical to maintain the pH close to neutral during this
reaction to avoid depurination. We therefore used a high concentration
(1 M) of phosphate buffer. The overall yield for the four-step synthesis of 1
from 20-deoxyadenosine was 38%, a considerable improvement over the six-
step synthesis (16% overall yield) reported by Fujii and co-workers7. Com-
pound 1 was subsequently converted to a protected phosphoramidite as
described in Scheme 1 for 8-deutero-20-deoxyadenosine.
We also explored the possibility of using 8-deutero-20-deoxyadenosine
(2) for solid state deuterium NMR studies. Compound 2, which has been
incorporated into DNA for the chemical shift assignment of triplet repeats,1
can be conveniently prepared in one step that does not require purification.
This approach required using ND4OD, instead of NH4OH, for the depro-
tection of the oligomer. In the solid state NMR experiments, however, the
deuterium atom functions as the reporter. Therefore, the ND4OD depro-
tection protocol cannot be used due to incorporation of deuterium into other
nucleotides. We investigated how much deuterium would be lost by proton-
exchange during the synthesis and incorporation of 2 into DNA using non-
deuterated reagents.
Compound 2 was prepared by incubation of 20-deoxyadenosine (6) in
D2O in the presence of triethylamine (Scheme 2)1. Compound 2 was

1906
CHIRAKUL, LITZER, AND SIGURDSSON
Scheme 2. Synthesis of 20-deoxy-8-deuteroadenosine phosphoramidite.
converted to 20-deoxy-8-deutero-N6-benzoyladenosine (7) using a transient
protection protocol where the hydroxyl groups were protected as tri-
methylsilyl (TMS) ethers during acylation of the exocyclic amino group11.
The 50-alcohol was subsequently protected as a dimethoxytrityl (DMT)
ether and the 30-alcohol phosphitylated to yield phosphoramidite 8. The
level of deuterium incorporation into the phosphoramidite was 90%, as
1
determined by H NMR.
During a standard DNA synthesis protocol, the oligomer is deprotected
in aqueous ammonia at 55ꢀC, during which time the deuterium atom in 2
would exchange with the solvent. Alternatively, DNA can be synthesized
with commercially available phosphoramidites that can be deprotected under
much milder conditions (in a methanolic solution of potassium carbonate at
25ꢀC for 2 h)12. These phosphoramidites contain phenoxyacetyl(PAC)-pro-
tecting groups for the purine nucleosides and an acetyl protecting group for
dC. However, the PAC phosphoramidites are not as easy to prepare and are
somewhat unstable. Of the normal protecting groups used for automated
DNA synthesis, the N6-benzoyl group of dA is the most easily removed.
Therefore, we investigated the possibility of using phosphoramidite 8 in
combination with PAC phosphoramidites to reduce the amount of time
required for deprotection of the DNA.
The rate of deprotection of the benzoyl group was compared to the loss
of deuterium label during the same time period. 20-Deoxy-N6-benzoylade-
nosine was heated at 55ꢀC in aqueous ammonia and shown by RP-HPLC
analysis to be > 97% deprotected after 3 h (data not shown). To determine if
the rate of deprotection was the same in DNA, the oligomer 50-d(TTABzTT)
was prepared and incubated for varying lengths of time under the same

BASE-DEUTERATED 20-DEOXYADENOSINE NUCLEOSIDES
1907
conditions. The ammonia-treated oligomer samples were enzymatically
digested with snake venom phosphodiesterase and alkaline phosphatase. The
resulting mixture of nucleosides was subsequently analyzed by RP-HPLC
(data not shown). Interestingly, the benzoyl group was removed faster in the
oligomer, where deprotection was complete after 1 h (data not shown).
The rate of exchange for the deuteron in position 8 was determined by
incubation of 2 in aqueous ammonia at 55ꢀC for different periods of time
1
(Fig. 1). The level of deuterium in the samples was determined by H-NMR
analysis. There was still 78% of deuterium left in the 8-position of dA after
incubation for 3 h. To verify that the same results would be obtained in
DNA, the oligomer 50-d(TTABzTT) was synthesized using phosphoramidite
1
8 and deprotected as described above. H-NMR analysis showed that there
was indeed 80% deuterium present in the oligomer, which makes this a
practical approach for site-specific deuteration of deoxyadenosine nucleo-
tides in DNA. The same procedure was also used to make sequences con-
taining A, T, C, and G (data not shown).
In conclusion, we have described an improved synthesis of 1 for
incorporation into DNA. This nucleoside will be valuable for solid state
NMR studies that require prolonged incubation under conditions where
there might be appreciable proton exchange in the 8-position. However, most
experiments can be performed with deuterated DNA prepared from 2, which
is substantially easier to prepare. We have shown that site-specifically
deuterated DNA can be synthesized by using a combination of an N6-ben-
zoyl-protected phosphoramidite of 2, and phosphoramidites that can be
deprotected under mild conditions, with 80% incorporation of deuterium.
The results of solid state deuterium NMR studies of samples prepared by the
protocols described here will be reported in due course.
Figure 1. Deuterium exchange from 2 in aqueous ammonia at 55ꢀC.

1908
CHIRAKUL, LITZER, AND SIGURDSSON
EXPERIMENTAL SECTION
Materialsand Methods
20-Deoxyadenosine was purchased from Biosearch Research Chemicals.
Chloroacetaldehyde (50% wt. solution in water), N, N-dimethylacetamide
(DMA), and ammonium persulfate were purchased from Aldrich. Aqueous
ammonia (29.4%) was purchased from Fisher. CH2Cl2 and pyridine were
distilled from calcium hydride. All reactions were run under anhydrous
conditions and an inert argon atmosphere unless otherwise noted. DNA
oligomers were synthesized on an ABI 394 synthesizer, using phosphor-
amidites purchased from Glen Research. Flash column chromatography was
carried out with EM type 60 (230–400 mesh) silica gel and neutral alumina,
1
Brockmann activity grade 1 (50–200 mm). H NMR and 13C NMR were
recorded on a Bruker DRX 500 MHz spectrometer. Chemical shifts are
1
reported in ppm relative to DMSO-d6 (2.49 ppm for H and 39.5 ppm for
1
13C) or CDCl3 (7.24 ppm for H and 77.0 ppm for 13C), unless otherwise
noted. High resolution (HRMS), fast atom bombardment (FAB) mass
spectra were obtained on a JEOL HX-110 mass spectrometer for new com-
pounds (4 and 5). UV spectra were recorded on a Perkin Elmer, Lambda
BIO 20 spectrophotometer.
Preparative RP-HPLC separations were performed using a Dynamax
C18 column (Rainin, 25 cm, 4.6 mm ID, 5 mm, 300 A) and the solvent
gradient was run at 10 mL=min as follows: Solvent A 100 mM Et3NHOAc
(pH 7.0); solvent B, acetonitrile; isocratic 98% A for 13 min, 12 min linear
gradient to 85% A, 10 min linear gradient to 75% A, isocratic for 10 min,
5 min linear gradient to initial conditions. Analytical RP-HPLC analysis was
performed using an Econosphere C18 column (Alltech, 25 cm, 2.4 mm ID,
5 mm, 300 A) and the solvent gradient was run at 1 mL=min as follows:
Solvent A 50 mM Et3NHOAc (pH 7.0); solvent B, 30% solvent A in acet-
onitrile; isocratic 88.6% A for 7 min, 10 min linear gradient to 57.1% A,
5 min linear gradient to 14.3% A, 5 min linear gradient to 0.0% A, isocratic
for 5 min, then 3 min linear gradient to initial conditions.
Deprotection of N6-benzoyl-20-deoxyadenosine
a) Nucleoside. Twelve different solutions containing N6-Benzoyl-20-
deoxyadenosine (1.0 mg each, 0.0028 mmol) in aqueous ammonia (0.25 mL)
were heated at 55ꢀC. Every half-hour (over a 5.5 h period) a sample was
removed from the heat block and the solvent evaporated in vacuo, followed
by HPLC analysis. b) Oligomer. The oligomer 50-d(TTABzTT) was prepared
by automated DNA 50-DMT-on synthesis on a 1-mmol scale. The oligomer
was not cleaved from the resin. Seven different mixtures containing resin-
bound DNA (3.0 mg, 5% of a 1 mmol synthesis) in aqueous ammonia

BASE-DEUTERATED 20-DEOXYADENOSINE NUCLEOSIDES
1909
(0.25 mL) were incubated at 55ꢀC. Time points (10 min, 30 min, 1 h, 2 h,
3 h, 4 h and 5 h) were collected by removing a sample from the heat block,
filtering off the resin and evaporating the solvent in vacuo. Each sample was
purified by preparative RP-HPLC, the DMT group removed (see below),
and the oligomer enzymatically digested (see below) and analyzed by ana-
lytical RP-HPLC.
Deuterium Exchange of 20-deoxy-8-deuteroadenosine with Aqueous Ammonia
Twelve solutions containing 20-deoxy-8-deuteroadenosine (7.5 mg each,
0.0297 mmol) in aqueous ammonia (0.63 mL) were heated at 55ꢀC. Every
0.5 h (over a 5.5 h period) a sample was removed from the heat block, cooled
1
down to 0ꢀC and the solvent evaporated in vacuo, followed by H-NMR
analysis. For the 0 h time point, the aqueous ammonia was added and
immediately evaporated.
Determination of Deuterium Incorporation into DNA
The oligomer 50-d(TTATT) containing a 50-DMT group was synthe-
sized on a 2-mmol scale using phosphoramidite 8 and deprotected in aqueous
ammonia (2.0 mL) at 55ꢀC for 3 h. The sample was then filtered and the
solvent removed in vacuo. After purification by RP-HPLC, the DMT group
was removed by treatment with aqueous acetic acid (0.5 mL, 80%) for
15 min at 23ꢀC, followed by removal of the solvent in vacuo. The oligomer
was dissolved in water (300 mL), extracted with CHCl3 (3 X 300 mL) and the
solvent removed in vacuo from the aqueous phase. The DNA was dissolved
1
in D2O and the H NMR spectrum recorded using HOD as an internal
standard at 4.8 ppm. Integration of the H8 proton of dA (d 8.36), relative to
the H2 proton (d 8.06), revealed 80% incorporation of deuterium.
Enzymatic Digestion
The oligonucleotide (1.3 nmol) in a TRIS buffer (50 mM, 50 mL, pH 8.5
@ 20ꢀC) containing MgCl2 (1 mM) was digested with snake venom phos-
phodiesterase I (0.4 U) and calf intestinal alkaline phosphatase (6.4 U) for
1 h at 37ꢀC, followed by RP-HPLC analysis.
20-Deoxy-1,N6-ethenoadenosine (3). 20-Deoxyadenosine (2.51 g, 10.0
mmol) was stirred at 30ꢀC for 12 h in a 2 M aqueous solution of chlor-
oacetaldehyde (5.4 mL, 10.7 mmol). The reaction mixture was maintained at
pH 4.5 by the addition of aqueous NaOH (4 M). After completion of the
reaction, the pH was adjusted to 7.2 and the solvent was removed in vacuo.

1910
CHIRAKUL, LITZER, AND SIGURDSSON
The residue was purified by flash column chromatography on silica gel with
1
MeOH=CH2Cl2 (3:17) to give 3 in 92% yield as a white solid. H NMR
(DMSO-d6): d 9.30 (1H, s, H2), 8.53 (1H, s, H8), 8.08 (1H, d, J ¼ 1.5 Hz,
H11), 7.55 (1H, d, J ¼ 1.5 Hz, H10), 6.48 (1H, t, J ¼ 7.0 Hz, H10), 5.37 (1H, d,
J ¼ 4.0 Hz, 30-OH), 4.97 (1H, t, J ¼ 5.5 Hz, 50-OH), 4.44 (1H, m, H30), 3.90
(1H, m, H40), 3.62 (1H, m, H50or H500), 3.54 (1H, m, H50or H500), 2.72 (1H, m,
H20or H200), 2.38 (1H, m, H20or H200).
3-b-D-20-Deoxyribofuranosyl-4-amino-5-(imidazol-2-yl)imidazole (4).
Compound 3 (0.500 g, 1.82 mmol) in aqueous NaOH (0.8 N, 12.5 mL,
9.99 mmol), was stirred at 23ꢀC for 3 h. The reaction mixture was cooled to
0ꢀC and neutralized by dropwise addition of concentrated hydrochloric acid.
The solvent was removed in vacuo and the solid residue was purified by flash
column chromatography on neutral alumina with MeOH=CH2Cl2 (3:7) to
give 4 in 76% yield as a pale yellow solid. 1H NMR (DMSO-d6): d 7.46 (1H,
s, H2), 6.98 (2H, s, H8 and H9), 5.97 (1H, t, J ¼ 7.5 Hz, H10), 5.76 (2H, bs,
NH2), 5.31 (1H, d, J ¼ 4.0 Hz, OH), 5.12 (1H, t, J ¼ 4.0 Hz, OH), 4.35 (1H,
m, H30), 3.52 (1H, m, H40), 3.82 (2H, m, H50 and H500), 2.48 (1H, m, H20 or
H200), 2.17 (1H, m, H20 or H200); 13C NMR (DMSO-d6): d 141.0, 139.5, 130.0,
114.5, 89.0, 85.5, 81.5, 62.0, 64.0; FAB-HRMS: m=z 266.1255 (MþH)þ
(calcd. 266.1255 for C11H15N5O3).
2-Deutero-20-deoxy-1,N6-ethenoadenosine (5). 1,1,1-Triethoxy-1-deu-
teromethane (1.961 mL, 11.7 mmol) was added dropwise to a stirred solution
of imidazole 4 (0.7348 g, 2.769 mmol) in N,N-dimethylacetamide (DMA)
(12 mL) at 23ꢀC. The reaction mixture was stirred at 100ꢀC for 12 h. The
solvent was removed in vacuo and the crude solid was purified by flash
column chromatography on neutral alumina with MeOH=CH2Cl2 (1:19 to
1
3:7) to give 5 in 77% yield as a white solid (94% deuterium). H NMR
(DMSO-d6): d 9.30 (0.06H, s, H2), 8.53 (1H, s, H8), 8.08 (1H, d, J ¼ 1.5 Hz,
H11), 7.55 (1H, d, J ¼ 1.5 Hz, H10), 6.48 (1H, t, J ¼ 7.0 Hz, H10), 5.37 (1H, d,
J ¼ 4.0 Hz, 30-OH), 4.97 (1H, t, J ¼ 5.5 Hz, 50-OH), 4.44 (1H, m, H30), 3.90
(1H, m, H40), 3.62 (1H, m, H50or H500), 3.54 (1H, m, H50or H500), 2.72 (1H, m,
H20or H200), 2.38 (1H, m, H20or H200); 13C NMR (DMSO-d6): d 141.4, 140.5,
140.4, 138.9, 133.6, 123.4, 113.0, 88.8, 84.7, 71.5, 62.4; FAB-HRMS: m=z
277.1159 (MþH)þ (calcd. 277.1159 for C12H13N5O3).
2-Deutero-20-deoxyadenosine (1). Compound 5 (0.1326 g, 0.4799 mmol)
and ammonium persulfate (0.2190 g, 0.9597 mmol) were dissolved in an
aqueous phosphate buffer (10 mL, 1 M, pH 7.2 @ 23ꢀC) and stirred at
80ꢀC for 3 h. The solvent was removed in vacuo and the solid residue was
purified by flash column chromatography on silica with MeOH=CH2Cl2
1
(5:95) to give 1 as a white solid in 71% yield (94% deuterium). H NMR
(DMSO-d6): d 8.33 (1H, s, H8), 8.15 (0.06H, s, H2), 7.31 (2H, s, NH2),

BASE-DEUTERATED 20-DEOXYADENOSINE NUCLEOSIDES
1911
6.48 (1H, m, H10), 5.37 (1H, d, J ¼ 4.0 Hz, 30-OH), 5.25 (1H, t, J ¼ 6 Hz,
50-OH), 4.41 (1H, m, H30), 3.88 (1H, m, H40), 3.65 (1H, m, H50 or H500),
3.55 (1H, m, H50 or H500), 2.71 (1H, m, H20 or H200), 2.35 (1H, m, H20 or H200).
20-Deoxy-8-deuteroadenosine
(2). 20-Deoxyadenosine
(5.0043 g,
18.589 mmol), deuterium oxide (250 mL), and triethylamine (2.58 mL,
18.6 mmol) were stirred at 65ꢀC for 65 h. The solvent was removed in vacuo
to give 2 as a white solid in quantitative yield ( > 98% deuterium). 1H NMR
(DMSO-d6): d 8.08 (1H, s, H2), 7.30 (2H, s, NH2), 6.33 (1H, m, H10), 5.27
(1H, d, J ¼ 4.0 Hz, 30-OH), 5.24 (1H, d, J ¼ 6 Hz, 50-OH), 4.42 (1H, m, H30),
3.87 (1H, m, H40), 3.58 (2H, m H50 and H500), 2.69 (1H, m, H20 or H200), 2.35
(1H, m, H20 or H200).
N6-Benzoyl-8-deutero-20-deoxyadenosine (7). Compound 2 (5.0029 g,
19.84 mmol) was co-evaporated with pyridine (3620 mL) and suspended in
50 mL of pyridine. The mixture was cooled to 0ꢀC and chlorotrimethylsilane
(12.5 mL, 98.5 mmol) was slowly added. The reaction mixture was stirred for
30 min at 0ꢀC, followed by addition of benzoyl chloride (11.0 mL,
94.5 mmol). The ice bath was removed and the reaction was stirred at 23ꢀC
for 2 h. The mixture was cooled again in an ice-bath and water (20 mL) was
added. After 20 min, aqueous ammonia (40 mL) was added, the ice bath
removed and the reaction mixture stirred for 30 min. The solvent was re-
moved in vacuo and the residue was dissolved in water (100 mL) and washed
with ethyl acetate (40 mL). The aqueous solution was refrigerated (4ꢀC) for
2 h and the white solid collected and dried to give 7 in 72% yield (97%
1
deuterium). H NMR (CDCl3): d 8.82 (1H, s, H2), 8.15 (0.03H, s, H8), 6.86
(5H, m, Bz), 6.51 (1H, m, H10), 4.74 (1H, s, H30), 4.17 (1H, s, H40), 3.45 (2H,
m, H50 and H500), 2.93 (1H, m, H20 or H200), 2.62 (1H, m, H20 or H200).
50-O-(Dimethoxytrityl)-N6-benzoyl-8-deutero-20-deoxyadenosine. Com-
pound 7 (0.4397 g, 1.235 mmol) was co-evaporated with pyridine (365 mL),
suspended in pyridine (8 mL), and cooled to 0ꢀC. 4,4-Dimethoxytrityl
chloride (0.5023 g, 1.482 mmol) was added and the solution was allowed to
warm up to 23ꢀC. After 12 h, the solution was cooled to 0ꢀC and methanol
(2 mL) was added. The solvent was removed in vacuo and the crude product
was purified by flash column chromatography on silica gel with
MeOH=CH2Cl2 (0:10 to 3:7) giving the desired compound as a white solid in
1
83% yield (90% deuterium). H NMR (CDCl3): d 8.75 (1H, s, H2), 8.18
(0.1H, s, H8), 8.04–6.82 (18H, ArH), 6.51 (1H, m, 1H0), 4.73 (1H, m, H30),
4.18 (1H, m, H40), 3.81 (3H, s, OCH3), 3.44 (2H, m, H50 or H500), 2.93 (1H, m,
H20 or H200), 2.56 (1H, m, H20 or H200).
50-O-(Dimethoxytrityl)-N6-benzoyl-8-deutero-20-deoxyadenosine-30 -O-
(2-cyanoethyl-N,N-diisopropylphosphoramidite) (8). The trityl derivative of

1912
CHIRAKUL, LITZER, AND SIGURDSSON
compound 7 (0.3640 g, 0.5527 mmol) was dried by co-evaporation with
pyridine (365 mL) and stored in vacuo overnight. The residue was dissolved
in CH2Cl2 (5 mL) was treated dropwise with N, N-diisopropyl ethylamine
(0.21 mL, 1.2 mmol) and 2-cyanoethyl diisopropylchloro-phosphoramidite
(0.14 mL, 0.61 mmol) while stirring at 23ꢀC. After 20 min, the reaction
mixture was diluted with CH2Cl2 (20 mL) and washed sequentially with sa-
turated aqueous sodium bicarbonate (25 mL) and brine (20 mL). The aqu-
eous solutions were extracted with CH2Cl2 (25 mL). The organic phases were
combined, dried over sodium sulfate, and concentrated in vacuo. The solid
residue was purified by flash column chromatography on silica gel with
hexane=EtOAc =Et3N (20:80:0.5) to give 8 as a white solid in 73% yield (91%
deuterium). 1H NMR (CDCl3): d 8.92 (1H, s, H2, diasteromer 1), 8.77 (1H, s,
H2, diasteromer 2), 8.23 (0.09H, s, H8, diasteromer 1), 8.21 (0.09H, s, H8,
diastereomer 2), 8.04–6.82 (18H, ArH), 6.53 (1H, m, H10), 4.82 (1H, m, H30),
4.37 (1H, m, H40), 3.80 (6H, s, OCH3), 3.63 (4H, m, C2H4CN), 3.42 (2H, m,
CH), 3.44 (2H, m, H50), 2.93, (1H, m, H20 or H200), 2.56 (1H, m, H20 or H200),
1.28 (12H, m, CH3).
ACKNOWLEDGMENTS
We thank the National Institutes of Health (GM58914) for financial
support, Dr. M. Sadilek for HRMS-FAB analyses, Dr. G. P. Drobny for
helpful discussions, T. H. Madden, A. G. Singer, and members of the
Sigurdsson research group for critical reading of the manuscript.
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Received April 11, 2001
Accepted August 22, 2001