Incorporation of 8-histaminyldeoxyadenosine 18-(2-(4-imidazolyl)ethylamino)-2'-deoxyriboadenosine] into oligodeoxyribonucleotides by solid phase phosphoramidite coupling.

Article abstract of DOI:10.1081/NCN-120015723

The 3'phosphoramidite of 8-histaminyl deoxyadenosine has been prepared and successfully incorporated into a short oligodeoxyribonucleotide. The synthetic methodology leading to this preparation is given and the implications for developing new DNAzymes as

Full text of DOI:10.1081/NCN-120015723

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INCORPORATION OF 8-HISTAMINYL-DEOXYADENOSINE
[8-(2-(4-IMIDAZOLYL)ETHYLAMINO)-2′-
DEOXYRIBOADENOSINE] INTO
OLIGODEOXYRIBONUCLEOTIDES BY SOLID PHASE
PHOSPHORAMIDITE COUPLING
Leonard Lermer a , John Hobbs b & David M. Perrin a
a Department of Chemistry , University of British Columbia , 2036 Main Mall, Vancouver,
B.C., V6T-1Z1, Canada
b NAPS (Nucleic Acid and Peptide Sequencing) Unit , University of British Columbia , 6174
University Blvd., Vancouver, B.C., V6T-1Z1, Canada
Published online: 17 Aug 2006.
To cite this article: Leonard Lermer , John Hobbs & David M. Perrin (2002) INCORPORATION OF 8-HISTAMINYL-
DEOXYADENOSINE [8-(2-(4-IMIDAZOLYL)ETHYLAMINO)-2′-DEOXYRIBOADENOSINE] INTO OLIGODEOXYRIBONUCLEOTIDES BY
SOLID PHASE PHOSPHORAMIDITE COUPLING, Nucleosides, Nucleotides and Nucleic Acids, 21:10, 651-664, DOI: 10.1081/
NCN-120015723
To link to this article: http://dx.doi.org/10.1081/NCN-120015723
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MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016
©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.
NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, No. 10, pp. 651–664, 2002
INCORPORATION OF 8-HISTAMINYL-
DEOXYADENOSINE
[8-(2-(4-IMIDAZOLYL)ETHYLAMINO)-20-
DEOXYRIBOADENOSINE] INTO
OLIGODEOXYRIBONUCLEOTIDES
BY SOLID PHASE PHOSPHORAMIDITE
COUPLING
Leonard Lermer,1 John Hobbs,2 and David M. Perrin1,*
1Department of Chemistry, 2036 Main Mall, and
2NAPS (Nucleic Acid and Peptide Sequencing) Unit,
6174 University Blvd., University of British Columbia,
Vancouver, B. C., V6T-1Z1 Canada
ABSTRACT
The 30phosphoramidite of 8-histaminyl deoxyadenosine has been pre-
pared and successfully incorporated into a short oligodeoxyribonucleo-
tide. The synthetic methodology leading to this preparation is given and
the implications for developing new DNAzymes as well as probing
unusual nucleic acid structures are discussed.
*Corresponding author. E-mail: dperrin@chem.ubc.ca
651
DOI: 10.1081=NCN-120015723
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

652
LERMER, HOBBS, AND PERRIN
INTRODUCTION
The imidazole functionality is of special interest to structural biologists,
enzymologists, and medicinal chemists alike as this functionality plays a
unique and important role in terms of catalysis, recognition, and drug
delivery in part because of its pKa that lies in the range of 6–7. The imidazole
is found almost ubiquitously at the active sites of enzymes, especially those
that operate on phosphate-derived substrates where it chelates metals and=or
plays the role of a general acid and=or a general base.[1] In other enzymes,
it strongly chelates redox active metals so as to position them in proximity
to substrate and=or alter their redox potentials.[2,3] In the case of Zn-finger
proteins, imidazole side-chains of histidine chelate divalent zinc to properly
structure the DNA binding domain so as to permit sequence specific high
affinity recognition of DNA sequences.[4] When DNA is condensed with
cationic co-block polymers prepared from imidazole-modified monomers,
cellular penetration and uptake of DNA is enhanced.[5–7] The imidazole has
been appended directly to oligonucleotides in the study of pH dependent
uptake and cellular fate[8] and to develop oligonucleotides that can recognize
mRNA complements and effect site-specific cleavage via an RNaseA-like
activity.[9–15] Finally imidazoles have been appended to monomer nucleotide
triphosphate that can be enzymatically polymerized to give modified DNA,
which in turn can be PCR amplified.[16–20] Such methodology would indicate
the possibility of introducing this very important functionality in a combi-
natorial manner for the discovery of modified oligonucleotides that may use
imidazoles for activities ranging from recognition to catalysis to cellular
penetration. In particular, one of the first of such methodological reports
(and the first to report on the simultaneous incorporation of two modified
nucleotides) involved an examination of the enzymatic uptake of 8-histami-
nyl-dATP i.e., 8-(2-(4-Imidazolyl)ethylamino)-20-deoxyriboadenosine tri-
phosphate.[21] As the incorporation of imidazoles into biopolymers is of
great interest and relates to many diverse subjects, we wish to report the
synthesis of the phosphoramidite corresponding to 8-histaminyl-dATP:
(6N-benzoyl-8-(2-(4-Imidazolyl)ethylamino)adenyl))-30-O-(2-cyanoethyl-N,N-
diisopropylphosphoramidyl)-50-O-(4,40-dimethoxytrityl)-20-deoxy-b-D-ribofu-
ranose (Fig. 1) and its incorporation into oligonucleotides by standard solid
phase oligonucleotide chemistries.
Although a great number of modified pyrimidine and purine derivatives
have been investigated with linkages to both the base and sugar moieties,[22–28]
very few reports exist of an 8-amino-linked purine that presents an imidazole
for use in solid phase synthesis.[29] Besides the immediate interest in a solid
phase synthesis of a recently reported metal-independent DNAzyme that
mimics RNaseA,[30] this report expands the general methodologies for
positioning imidazoles at different positions on the purine and sugar moi-
eties. Of further interest are the recent reports of novel triplex and parallel

DEVELOPING OLIGODEOXYRIBONUCLEOTIDES
653
Figure 1.
duplex structures that are induced by 8-amino-substituted purines via
Hoogsteen base pairing.[31,32]
RESULTS
After investigating several alternate approaches to protection, the
phosphoramidite shown in Fig. 1 was ultimately synthesized in the order
shown in Sch. 1. Compounds 6 & 7 are addressed in the discussion pertaining
to the formulation of the synthetic route. This scheme differs from our initial
approach which had been to i) condense 8-bromo-deoxyadenosine[33] with
histamine, ii) perbenzoylate, iii) selectively saponify the 30,50 benzoyl
esters,[34] iv) dimethoxytritylate and v) install the phosphoramidite. Initially,
this seemed to be the most straightforward approach, particularly since
displacement of the unprotected bromide with 5 eq. histamine normally
proceeded over a period of 3–7 days in refluxing ethanol, and such conditions
we suspected might otherwise have cleaved the N6-benzoyl group.[35] In the
initial synthetic route, the DMT and benzoyl groups were installed after
histamine displacement. This initial strategy was guided in part by i) a desire
to monitor reaction completion by a characteristic l-max shift from 263 nm
to 280 nm, and ii) to take advantage of the very large Rf difference that was
observed between the unprotected Br-dA and the corresponding histaminyl
adduct, which we thought would aid in purification. Following displacement
by histamine, treatment with benzoyl chloride gave a monobenzoylated
1
histaminyl adduct. H-NMR revealed a broad singlet at 9.05 ppm char-
acteristic of an amide proton thus indicating benzoylation had transpired at
the intended N6 position and not at the N8 position, as has been observed by
others in the context of C8-NH guanine derivatives.[36]
The N6-benzoyl-8-histaminyl-dA rapidly reacted with dimethoxytrityl
chloride in pyridine to yield a chromatographically pure compound that was

654
LERMER, HOBBS, AND PERRIN
initially taken to be 4 as ESI indicated the presence of one dimethoxytrityl,
one benzoyl, and one histaminyl group, each on the parent deoxy-
riboadenosine. As DMT can be used to protect imidazoles,[37,38] we enter-
tained the possibility that the DMT had reacted exclusively on the imidazole
to give an unwanted structural isomer 7. Our suspicions in this regard were
further aroused when the product was treated with 2-cyanoethyldiisopro-
pylchlorophosphoramidite as two phosphorus adducts were detected by ESI
and 4 singlets detected by 31P NMR (data not shown).
Initially, two hypotheses were developed to explain this observation: 1)
The dimethoxytrityl chloride reacted with the imidazole leaving two free
hydroxyls to react with the oxophilic phosphonamidyl chloride or 2) the
dimethoxytrityl was indeed properly installed on the 50OH but both the 30OH
as well as the imidazole were phosphatidylated. Previous reports by Bashkin
et al. used DMT-protected nucleosides that were subsequently appended
with peptides containing NBoc-, FMOC- and DMT-protected histidines,
presumably to avoid reaction of the imidazole with the phosphatidylating
reagent used to prepare peptide-modified phosphoramidite monomers.[39]
These early studies guided our thinking in part. More recent work by Joyce
et al. however described the preparation of a urocanamido-allyl-dU phos-
phoramidite that did not require imidazole protection.[40] A doubly DMT-
protected compound, similar to those investigated by Bashkin and coworkers
also would have been amenable to phosphoramidite synthesis and sub-
sequent oligonucleotide coupling, and moreover would have obviated con-
cerns regarding the placement of the first DMT. We attempted to prepare
such a derivative by retreating the product in question with excess DMT-
chloride, however this only resulted in decomposition and further attempts to
protect both the 50OH and the imidazole were abandoned. Given the large
number of aromatic protons on the target, we were unable to readily confirm
the position of the DMT group by 1H-NMR. For example, in d6-DMSO, the
50-methylene protons were found to overlap with the methylene protons of
the histamine such that coupling to the nonexchangeable 50OH proton (and
subsequent decoupling by D2O addition) was not easily detected. Never-
theless the two D2O-exchangeable protons were detected in the alcohol
region (t, 5.74 ppm J ¼ 3.8 Hz, 1H; d 5.34 ppm J ¼ 4.1 Hz).
The questions regarding the chemoselectivity of dimethoxytritylation
caused us to reformulate the final synthetic strategy shown in Sch. 1. In this
case, 3 was reacted with histamine to give 4. Remarkably, this reaction went
to completion within minutes rather than days as had been observed with the
free bromide. Continued refluxing in ethanol with excess histamine did in fact
result in a debenzoylation (data not shown). Given that histamine displace-
ment on the unprotected bromide proceeded over a period of days, it was
unexpected that the displacement on 3 would be i) so dramatically acceler-
ated by the introduction of protecting groups, and ii) so selective for attack at
the 8 position without any observable debenzoylation (little if any 50-DMT-8-

DEVELOPING OLIGODEOXYRIBONUCLEOTIDES
655
bromo-deoxyadenosine was detected prior to complete displacement by
histamine at the 8 position). With 4 in hand, detailed HMQC=HMBC NMR
analysis finally indicated that reaction of 6 with DMT-Cl gave exclusive
protection of the imidazole resulting in an unwanted structural isomer 7.
Briefly stated, the final synthetic approach (Sch. 1) involved bromina-
tion, benzoylation, limited saponification, dimethoxytritylation, bromide
displacement, and finally phosphatidylation. Product 4 was found to have a
very different Rf from the previously obtained DMT-protected material 7
that was initially thought to be 4. Chemical correlation distinguished 4 from
7 and phosphatidylation proceeded cleanly to give one product with two
closely spaced singlets in the 31P-NMR spectrum indicative of one diaster-
eomeric trivalent phosphorus atom. It was fortuitous that the bromide dis-
placement occurred rapidly and without debenzoylation on the fully
protected bromide. At the outset of this undertaking, there appeared to be
several ways to assemble the desired target, and no way of a-priori deciding
Scheme 1. a) Excess benzoylchloride=pyridine, b) aqueous NH4OH, c) 1.1 eq. DMT-Cl=
pyridine, d) 5 eq. histamine=ethanol, e) 1.1–1.3 eq. cyanoethyldiisopropylchlorophosphor-
amidite= 3 eq. ethyldiisopropylamine, f) aq. K2CO3:CH2Cl2 extraction.

656
LERMER, HOBBS, AND PERRIN
on the most efficacious strategy. We hope other investigators seeking to
prepare imidazole-modified oligonucleotides will find the preceding discus-
sion germane to their efforts. As such, we have characterized both the ulti-
mate target of this work as well as the unanticipated structural isomer.
Once in hand, the phosphoramidite was successfully introduced into a
small oligonucleotide of sequence d(TTTTAAAimAimT) on a 40 nmol scale
using standard CPG oligonucleotide synthesis procedures on an ABI auto-
mated synthesizer with 10 min coupling times: tetrazole activation, coupling,
iodine-collidine-water oxidation, acetic anhydride capping, and DCA detri-
tylation. The dimethoxytrityl cation in the efflux of each coupling step was
quantified by reactivation with addition of toluenesulfonic acid and absor-
bance noted at 450 nm, Yields were found to be greater than 90% per cou-
pling step based on UV-vis absorbance analysis (498 nm) of the detritylation
fractions and no appreciable difference was observed when the modified
phosphoramidite was introduced. The oligonucleotide was cleaved from the
CPG by overnight treatment with concentrated ammonium hydroxide at
55ꢀC. Following lyophilization, the crude oligonucleotide was subjected to
MALDI analysis (negative ion mode) shown in Fig. 2.
Although a small amount of an internally deleted product was detected
by MALDI (2506.510), it was not observed by 20% D-PAGE (vide infra).
The signal at 1859.269 represents a fragmentation product of either the three
bases at the 30 end of the full-length product or of the last two bases at the 30
end of an n-1 internally deleted product. The 20% D-PAGE analysis (29:1
monomer:bis, 1X TBE pH 8.3) on the crude (Fig. 3) indicated no impurity by
UV shadowing. The Rf values in the gel are given in centimeters measured
from the top of the well, with lanes 1 and 3 containing only marker dyes as
shown and lane 2 containing only product.
CONCLUSIONS AND DISCUSSION
Phosphoramidite 5 will allow for the solid phase synthetic scale-up of
imidazole-modified oligonucleotides with altered recognition and uptake
properties as well as catalytic activities. More specifically 5 will allow the
synthesis of a candidate DNAzyme that had been initially polymerized in
picomol amounts by DNA polymerase in the presence of 8-histaminyl-dATP
in lieu of dATP.[41] This polymerization in conjunction with a combinatorial
selection has resulted in the report of a putative RNaseA activity.[42] The
phosphoramidite reported herein will likely contribute to defining interesting
structure-function relationships on which this hydrolytic activity appears
to depend. While this activity is possibly the first such RNaseA mimic,
its discovery also indicated some of the methodological limitations that are
encountered when using modified nucleotides to expand the catalytic
repertoire of nucleic acids. One of these constraints is polymerase uptake.
The polymerization yields of Im-dATP using Sequenase 2.0TM diminished

DEVELOPING OLIGODEOXYRIBONUCLEOTIDES
657
Figure 2.
Figure 3.

658
LERMER, HOBBS, AND PERRIN
Figure 4.
precipitously after 3–5 isosequential incorporations. Polymerizations using
Klenow enzymes exoþ= ꢁ did not exhibit incorporation beyond two con-
secutive bases (unpublished observations). This represents a serious limita-
tion in terms of the number of potential sequences that may be interrogated
during the combinatorial selection of DNAzymes. One hypothesis for
explaining this observation is that the N8 delivers 3 hydrogen bonds between
the template thymidine and the Hoogsteen face of the Im-dA as shown
schematically in Fig. 4.
Such Hoogsteen bonding, which would be favored by the C8-NH
proton, has been observed in the context of triple helices.[43] More recently,
strands rich in 8-amino purines were found to recognize their complements in
a preferred parallel orientation.[44] If this Hoogsteen structure were to occur
either within the active site of the polymerase during polymerization, or
when polymerase transiently dissociates from the template, non-extendable
dead-end complexes will result, thus explaining the limited incorporation of
the corresponding triphosphate. Biophysical studies including gel-retardation
and hyperchromic melting studies using synthetically prepared oligonucleo-
tides generated from the aforementioned Im-dA phosphoramidite will also
provide evidence for or against this hypothesis. In addition, primers as well as
templates containing varying proportions of this modification vs. unmodified
adenosine can now be prepared to address fine-structure kinetic aspects of
polymerase uptake. In addition, the ability of this modified nucleotide to
confer novel properties to oligonucleotides in the context of double and triple
helices, and to cell up-take studies will be investigated.
MATERIALS AND METHODS
Materials
Deoxyadenosine, bromine, dimethoxytrityl chloride, benzoyl chloride,
histamine, diisopropylethylamine, pyridine, and acetonitrile were purchased

DEVELOPING OLIGODEOXYRIBONUCLEOTIDES
659
from Sigma-Aldrich. 2-cyanoethyldiisopropylchlorophosphoramidite was
obtained from Lancaster. Flash chromatography was carried out using Silica
gel 60, 230–400 mesh, supplied by E. Merck Co. Silica gel 60 F254 on alu-
minum sheets (E. Merck, type 5554) was used for TLC analysis. Nuclear
Magnetic Resonance spectra (1H, 13C and 31P) were obtained on a Bruker
AV-300 or Bruker AV-400 instrument. Solvent, signal multiplicity, coupling
constants, and integration ratios are indicated in parentheses. An external
standard of 85% H3PO4 was used for 31P NMR. (ESI) MS were obtained on
Bruker Esquire-LC instrument. Maldi-MS were obtained on Bruker Biflex
instrument in negative ion mode. All reactions were carried out under
positive argon pressure and dark conditions. Solvents used in modified
phosphoramidite synthesis were distilled to dryness. Unmodified phosphor-
amidites were purchased in conjunction with the NAPS (Nucleic Acid Pep-
tide Sequencing) Unit of UBC. Compounds 1, 2 and 3 were prepared
following a literature procedure.[45,46] The syntheses of compounds 4, 5, and 7
are described.
6N-Benzoyl-50-O-(4,40-dimethoxytrityl)-8-(2-(4-Imidazolyl)ethylamino)-
20deoxyadenosine (4): A solution of Compound 3 0.9081 g (1.234 mmol)
and 0.6886 g (6.168, 5 eq.) of histamine in 40 mL of absolute ethanol was
degassed prior to warming to 80ꢀC. The reaction was monitored by TLC
developed with 10% MeOH in CHCl3 (Rf ¼ 0.12). After 2 h the reaction
mixture was concentrated and purification by silica gel flash chromato-
graphy, eluted with 10% MeOH in CHCl3 to afford 0.6805 g (72%) of
1
compound 4 as a solid white foam. H NMR (CD3OD, 400 ppm): 8.28 (s,
1H, H2), 8.02 (d, J ¼ 7.5 Hz, 2H, o-Bz-H), 7.54 (t, J ¼ 7.5 Hz, 1H, p-Bz-H),
7.51 (s, 1H, Im-H2), 7.45 (t, J ¼ 7.5 Hz, 2H, m-Bz-H), 7.29 (d, J ¼ 7.2 Hz, 2H,
Ph-DMT), 7.16 (d, J ¼ 8.68 Hz, 4H, PhOMe(DMT)), 7.16-7.06 (m, 3H, Ph-
DMT), 6.75 (s, 1H, Im-H5), 6.71 (d, 8.6 Hz, 2H, PhOMe(DMT)), 6.68 (t,
J ¼ 8.68 Hz, 2H, PhOMe(DMT)), 6.21 (dd, J ¼ 6.4, 6.7 Hz, 1H, H10), 4.75
(ddd, J ¼ 4.27, 4.27, 6.1 Hz, 1H, H30), 4.09 (ddd, 3.8, 4.27, 5.2 Hz, 1H, H40),
3.65 (s, 3H, -OMe), 3.64 (s, 3H, -OMe), 3.57 (m, 2H, N-CH2-), 3.47 (ddd,
J ¼ 6.1, 6.4, 13.2 Hz, 1H, H20), 3.33 (dd, J ¼ 3.8, 10.3 Hz, 1H, H50), 3.24 (dd,
J ¼ 5.2, 10.3 Hz, 1H, H50), 2.89 (t, J ¼ 7.2 Hz, 2H, -CH2-Im), 2.30 (ddd,
J ¼ 4.7, 6.7, 13.2 Hz, 1H, H20) 13C NMR (CD3OD, 400 ppm): 167.96 (car-
bonyl), 160.98 (C-p-Ph-OMe(DMT)), 159.94 (C-p-Ph-OMe(DMT)), 155.87
(C8), 154.45 (C4), 149.04 (C2), 146.04 (C-Ph-DMT), 144.59 (C6), 137.03 (C-
DMT), 136.92 (C-DMT), 136.03 (Im-C2), 135.31 (Im-C4), 133.50 (C-p-Bz),
131.24 (C-Ph-OMe(DMT)), 131.10 (C-Ph-OMe(DMT)), 129.61 (C-m-Bz),
129.30 (C-Ph-DMT), 129.13 (C-o-Bz), 128.67 (C-Ph-DMT), 127.81 (C-Ph-
DMT), 124.67 (C5), 118.30 (Im-C5), 113 (C-Ph-OMe(DMT)), 87.50
(Ph3C(DMT)), 87.44 (C40), 85.30 (C10), 79.42, 72.56 (C30), 64.64 (C50), 55.67
(-OMe), 43.63 (N-CH2-), 38.25 (C20), 27.54 (-CH2-Im). Mass: 789(MþNaþ),
767 (Mþ).

660
LERMER, HOBBS, AND PERRIN
(6N-benzoyl-8-(2-(4-Imidazolyl)ethylamino)adenyl))-30-O-(2-cyanoethyl-
N,N-diisopropylphosphoramidyl)-50-O-(4,40-dimethoxytrityl)-20-deoxy-b-D-
ribofuranose (5): Compound 4 0.2508 g (0.327 mmol) was dried overnight
by co-evaporation from a 5 mL solution of 10% pyridine in CH2Cl2. After
dissolving the sample in 18 mL of CH2Cl2, adding 100 mL of N,N-diisopro-
pylethyl amine, and cooling to ꢁ78ꢀC, a 72.9 mL (0.327 mmol) sample of 2-
Cyanoethyl-N,N-diisopropylchloro-phosphoramidite was added. The clear
colorless solution was allowed to warm to room temperature after 5 min.
After 12 h the reaction was quenched by the addition of 0.1 mL of MeOH,
diluted in CHCl3 and washed with 10% aqueous sodium bicarbonate. The
organic layer was dried with sodium sulfate filtered and concentrated under
reduced pressure to afford a white foam. The foam was dissolved in a
minimal amount of EtOAc and precipitated by the addition of 15–20
volumes of hexane. The precipitate was centrifuged and the supernatant was
removed. The further addition of approximately 5 volumes of hexane to the
supernatant afforded a second crop of precipitate. The precipitates were
combined and further purified by silica gel chromatography eluted with 6%
MeOH=2% Et3N in CHCl3 afforded compound 5 0.2843 g (89.9%) as a
white foam. The reaction was monitored by TLC developed with 10%
MeOH, ꢂ1.5% TEA in CHCl3 (Rf ¼ 0.36). 1H NMR (CDCl3, 300 ppm): 8.67
(broad s, NH-amide), 8.42 (s, 1H, H-2), 8.03 (d, J ¼ 7.3 Hz, 2H, o-Bz-H), 7.59
(t, J ¼ 7.3 Hz, 1H, p-Bz-H), 7.51 (t, J ¼ 7.3 Hz, 2H, m-Bz-H), 7.38–7.20 (9H),
6.82 (m, 4H, PhOMe(DMT)), 6.57 (m, 1H), 6.52 ( s, 1H, Im-H5), 6.30 (m,
1H, H10), 4.79 (m, 1H, H30), 4.20 (d, J ¼ 3.1 Hz, 0.45H, H40), 4.15 (d,
J ¼ 3.1 Hz, 0.55H, H40), 3.77 (s, 6H, -OMe), 3.86-3.48 (m, 7H), 3.37 (td, J ¼ 3,
10.8 Hz, 1H), 3.1–2.78 (m, 3H), 2.60 (t, J ¼ 6.2 Hz, 1.1 H,
-CH2-CN), 2.6-2.40 (m, 2H), 2.37 (t, J ¼ 6.2 Hz, 0.9H, -CH2-CN), 1.16
(d, J ¼ 6.94 Hz, 6H), 1.15 (d, J ¼ 6.94 Hz, 3H), 1.01 (d, J ¼ 6.94 Hz, 3H) 31P
NMR (CDCl3): 149.70, 149.21 Mass (ESI): 967 (Mþ).
6N-Benzoyl-8-(2-(4-(1-N-(4,40-dimethoxytrityl)-Imidazolyl)ethyla-
mino)-20deoxyadenosine (7): Compound 6 was refluxed with DMT-Cl in
pyridine. The reaction was monitored by TLC developed with 10% MeOH in
1
CHCl3 (Rf ¼ 0.27). H NMR (400 MHz, CDCl3) ppm: 9.05 (broad s, NH-
amide) 8.44 (s, 1H, H2), 7.94 (d, J ¼ 7.5 Hz, 2H, o-Bz-H), 7.49 (t, J ¼ 7.2 Hz,
1H, p-Bz-H), 7.41 (t, J ¼ 7.5 Hz, 2H, m-Bz-H) 7.29 (s, 1H, Im-H2), 7.24 (t,
J ¼ 3.1 Hz, 3H, Ph-DMT), 7.00 (dd, J ¼ 5.9, 7.0 Hz, 2H, Ph-DMT), 6.94 (d,
J ¼ 8.9 Hz, 4 H, PhOMe(DMT)), 6.77 (d, J ¼ 8.9 Hz, 4 H, PhOMe(DMT)),
6.59 (t, J ¼ 7.5 Hz, 1H, H10), 6.59 (s, 1H, Im-H5), 4.67 (d, J ¼ 6.2 Hz, 1H,
H30), 3.98 (s, 1H, H40), 3.79 (m, 2H, H50), 3.74 (s, 6H, -OMe), 3.64 (m,
2H,-N-CH2-), 2.89 (td, J ¼ 5.5, 15.3 Hz, 1H, -CH2-Im), 2.83 (td, J ¼ 5.5,
15.3 Hz, 1H, -CH2-Im), 2.61 (ddd, J ¼ 13.7, 9.3, 6.4 Hz, 1H, H20), 2.10 (dd,
J ¼ 13.1, 5.7 Hz, 1H, H20) 13C NMR (75 MHz, CD3OD) ppm: 165.0 (car-
bonyl), 159.2 (C-PhOMe(DMT)), 153.0 (C-4), 152.8 (C-8), 148.3 (C-2), 143.5

DEVELOPING OLIGODEOXYRIBONUCLEOTIDES
661
(C-6), 142.6 (C-DMT), 138.7 (Im-C4), 137.9 (Im-C2), 134.5 (C-Bz), 134.4 (C-
Ph-OMe(DMT)), 132.1 (C-p-Bz), 130.9 (C-PhOMe(DMT)), 129.4 (C-
Ph(DMT)), 129.4 (C-Ph(DMT)), 128.6 (C-m-Bz), 128.0 (C-m-Ph(DMT)),
127.8 (C-o-Bz), 122.7 (C-5), 119.0 (Im-C5), 113.3 (C-Ph-OMe(DMT)), 87.6
(C40), 83.8 (C10), 74.8 (Ph3C(DMT)), 71.9 (C30), 61.0 (C50), 55.3 (-OMe), 43.5
(-N-CH2-), 39.1 (C20), 26.9 (-CH2-Im).
ACKNOWLEDGMENT
This work was supported by UBC Start-up funds.
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Received April 15, 2002
Accepted July 5, 2002