Synthesis and hybridization properties of RNA containing 8-chloroadenosine.

Article abstract of DOI:10.1081/NCN-120015071

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3'- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by approximately 5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.

Full text of DOI:10.1081/NCN-120015071

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SYNTHESIS AND HYBRIDIZATION PROPERTIES OF RNA
CONTAINING 8-CHLOROADENOSINE
Lisa S. Chen a & Terry L. Sheppard a
a Department of Chemistry , The Robert H. Lurie Comprehensive Cancer Center ,
Northwestern University , 2145 Sheridan Road, Evanston, IL, 60208-3113, U.S.A.
Published online: 07 Feb 2007.
To cite this article: Lisa S. Chen & Terry L. Sheppard (2002) SYNTHESIS AND HYBRIDIZATION PROPERTIES OF RNA CONTAINING
8-CHLOROADENOSINE, Nucleosides, Nucleotides and Nucleic Acids, 21:8-9, 599-617, DOI: 10.1081/NCN-120015071
To link to this article: http://dx.doi.org/10.1081/NCN-120015071
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©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, Nos. 8 & 9, pp. 599–617, 2002
SYNTHESIS AND HYBRIDIZATION PROPERTIES
OF RNA CONTAINING 8-CHLOROADENOSINE
Lisa S. Chen and Terry L. Sheppard*
Department of Chemistry and The Robert H. Lurie Comprehensive
Cancer Center, Northwestern University, 2145 Sheridan Road,
Evanston, IL 60208-3113
ABSTRACT
8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemother-
apeutic agent for the treatment of multiple myeloma and certain leuke-
mias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and
incorporation of the analog into cellular RNA in malignant cells. To
investigate the effects of 8-Cl-Ado modifications on RNA structure and
function, an 8-Cl-Ado phosphoramidite and controlled-pore glass
support were synthesized and used to introduce 8-Cl-Ado at internal and
30- terminal positions, respectively. RNA oligonucleotides containing
8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with
complementary RNA strands. Circular dichroism spectroscopy of the
resulting RNA duplexes revealed that the modified nucleobase does not
perturb the overall A-form helix geometry. The thermal stabilities of
8-Cl-Ado modified duplexes were determined by UV thermal denatura-
tion analysis and were compared with analogous natural duplexes con-
taining standard and mismatched base pairs. The 8-Cl-Ado modification
destabilizes RNA duplexes by ꢀ 5 kcal=mole, approximately as much as
a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may
result from perturbation of Watson-Crick base pairing induced by con-
formational preferences of 8-halogenated nucleosides.
*Corresponding author. Fax: (847) 491-7713; E-mail: sheppard@chem.northwestern.edu
599
DOI: 10.1081=NCN-120015071
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

600
CHEN AND SHEPPARD
Key Words: Nucleoside; RNA synthesis; RNA duplex stability; Phos-
phoramidite; 8-Chloroadenosine
INTRODUCTION
Nucleoside analogs represent a growing class of medicinal agents that
have been used extensively in anti-viral and cancer chemotherapy. By
resembling the natural building blocks of nucleic acids, these analogs are
taken up by cells, activated by phosphorylation, and incorporated into DNA
or RNA by polymerases. Structurally altered nucleosides frequently inhibit
enzymes or inactivate nucleic acid replication, repair, and processing path-
ways. Nucleoside analogs have been used for decades in combating viral
infections. For example, the azidothymidine derivative, AZT (1, Fig. 1),
which acts by inhibiting HIV reverse transcriptase (HIV-RT), served as an
early treatment for HIV infections and currently is used in therapeutic
cocktails.[1] Halogenated nucleosides, such as fludarabine (2, Fig. 1) and
gemcitabine (3, Fig. 1), also have been employed for the treatment of various
forms of cancer. These analogs are taken up by cells and converted to the
corresponding 50-triphosphate derivatives, the apparent cytotoxic agents.[2,3]
The halogenated nucleoside, 8-chloroadenosine (8-Cl-Ado, Fig. 1, 4),
recently has shown potential for the treatment of multiple myeloma (MM)
and other leukemias. Early experiments demonstrated that 8-chloro-
adenosine 30,50-cyclic monophosphate (8-Cl-cAMP) induced apoptosis in
several cancer cell lines.[4–9] However, the anti-proliferative effects of 8-Cl-
cAMP were dependent on its enzymatic conversion into 8-Cl-Ado.[4,5,8,10–12]
More recently, 8-Cl-Ado has been shown to induce apoptosis in drug resis-
tant MM and leukemia cell lines.[11] Investigations of cellular nucleotide
metabolism have demonstrated that MM cells treated with 8-Cl-Ado rapidly
accumulate the 50-triphosphate derivative, 8-Cl-ATP, with a concomitant
depletion of natural ATP levels.[12] Furthermore, RNA levels in treated
myeloma cells decrease to 50% of untreated control after 12 h, while DNA
levels remain largely unaltered.[12]
The emerging picture of 8-Cl-Ado action includes cellular uptake of 8-
Cl-Ado, enzymatic conversion to 8-Cl-ATP, and subsequent depletion of
Figure 1. Nucleoside analogs used in chemotherapeutic approaches.

RNA CONTAINING 8-CHLOROADENOSINE
601
cellular RNA and ATP levels. However, the molecular mechanism for
apoptosis induction remains unclear. To gain a better understanding of the
RNA-targeted mechanism of 8-Cl-Ado action, a chemical method for the
incorporation of 8-Cl-Ado nucleotides within RNA sequences was devel-
oped. Phosphoramidite and controlled-pore glass support derivatives of 8-Cl-
Ado were synthesized. These building blocks were used to synthesize RNA
oligonucleotides containing 8-Cl-A bases, and the base pairing properties of
these modified RNA sequences were assessed by circular dichroism (CD) and
UV thermal denaturation spectroscopies. The addition of a chlorine atom at
the 8-position of adenine does not perturb global RNA helical structure, but
destabilizes RNA duplexes approximately as much as a U:U mismatch in
double stranded RNA.
RESULTS AND DISCUSSION
Synthesis of 8-Cl-Ado Phosphoramidite and Solid-Support
The introduction of 8-Cl-Ado into RNA was based on standard auto-
mated RNA synthesis with 20-O-silyl nucleoside phosphoramidites.[13]
Thus, 8-Cl-Ado phosphoramidite (10, Sch. 1) and solid-support derivative
Scheme 1. Synthesis of 8-chloroadenosine phosphoramidite 10. Reagents and conditions: (a)
HCl, MCPBA, DMA, 25ꢁC, 2.5 h, 39%. (b) (CH3)3Si-Cl, pyridine then phenoxyacetylchloride,
1,2,4-triazole, pyridine, CH3CN, 55ꢁC, 7 h, 70%. (c) DMTCl, DMAP, pyridine, 25ꢁC,
12 h, 58%. (d) TBDMSCl, AgNO3, Et3N, pyridine, THF, 25ꢁC, 2.5 h, 16% (8) and 38% (9). (e)
8 and [(i-Pr)2N]2POClCH2CH2CN, N-methylimidazole, 2,4,6-collidine, THF, 25ꢁC, 4 h, 50%.

602
CHEN AND SHEPPARD
Scheme 2. Synthesis of an 8-chloroadenosine-derivatized controlled-pore glass support.
Reagents and conditions: (a) Succinic anhydride, DMAP, 12 h, 25ꢁC, 71% (b) DCC, 4-ni-
trophenol, pyridine, dioxane, DMF, 12 h, 25ꢁC. Final loading: 22 mmol=g.
(12, Sch. 2) were synthesized. Phosphoramidite 10 utilized standard 4,40-
dimethoxytrityl (DMT) and tert-butyldimethylsilyl (TBDMS) protecting
groups for the sugar hydroxyls, and a phenoxyacetyl (PAC) group for blocking
of the exocyclic amine. Isomeric 30-O-silyl nucleoside 9 was used for construc-
tion of a controlled-pore glass (CPG) support modified with 8-Cl-Ado (12).
The 8-Cl-Ado phosphoramidite building block 10 was prepared as
described in Sch. 1. 8-Chloroadenosine (4) was synthesized by oxidative
chlorination of adenosine (5) using a modification of a literature proce-
dure.[14] 8-Cl-Ado (4) was subjected to transient trimethylsilyl protection,[15]
and reacted with phenoxyacetyl chloride in the presence of 1,2,4-triazole to
produce the phenoxyacetyl-protected nucleoside 6.[16] The N-phenoxyacetyl
nucleoside 6 was readily converted to the DMT derivative 7 in 58% yield.[17]
The tritylated intermediate 7 then was silylated with TBDMS-Cl in the
presence of silver nitrate to form the 20-silyl (8) and 30-silyl (9) isomers. The
30-silyl isomer 9 (38% yield) was formed preferentially over the 20-silyl isomer
8 (16% yield[18,19]). To complete the synthesis, compound 8 was converted to
the desired phosphoramidite 10 by standard methods.[13]
An 8-Cl-Ado-derivatized solid support (12) was synthesized as outlined
in Sch. 2. The 30-silyl nucleoside byproduct (9, Sch. 1) was reacted with
succinic anhydride to produce the carboxylic acid derivative 11. The resulting
succinic acid 11 was activated as a nitrophenol ester and coupled to 1000 A
alkyl amine-derivatized CPG using previously described methods.[20]
Unreacted CPG amine sites then were capped using acetic anhydride. The
final loading of the 8-Cl-Ado nucleoside on support 12 was 22 mmol=g as
determined by dimethoxytrityl cation release.
RNA Oligonucleotide Synthesis, Deprotection, and Characterization
A series of RNA oligonucleotides containing single or multiple 8-Cl-
Ado substitutions were synthesized and are listed with their complementary

RNA CONTAINING 8-CHLOROADENOSINE
603
Figure 2. 8-Chloroadenosine-modified and standard RNA oligonucleotide sequences.
strands in Fig. 2. Sequences containing internal 8-Cl-Ado residues were
prepared by standard solid-phase synthesis using phosphoramidite 10, which
was inserted into RNA oligonucleotides with 99% coupling yields. Three
oligonucleotides were synthesized with one internal 8-Cl-A base (13, 21, 27),
and one sequence identical to 27 was prepared with two internal 8-Cl-Ado
modifications (28). A 30-terminally 8-Cl-Ado modified sequence (24) was
synthesized, which contained the same sequence as 13 upstream of the
modified base, to compare the effects of terminal 8-Cl-Ado substitution in an
identical sequence context. For comparison, unmodified RNA sequences
were prepared with an adenosine residue substituting for the 8-Cl-Ado
nucleotide. After synthesis, modified RNA oligonucleotides were deprotected
using standard methods.[13] Oligonucleotides were deblocked and cleaved
from the resin by treatment with 3:1 aqueous ammonia in ethanol and
subsequently were desilylated with tetrabutylammonium fluoride (TBAF) in
THF.[21] RNA oligonucleotides were purified by polyacrylamide gel elec-
trophoresis (PAGE) and characterized using MALDI-TOF mass spectro-
metry. Experimentally determined masses were in excellent agreement with
calculated masses for each RNA sequence.
Spectroscopic Analysis of 8-Cl-Ado-Modified
RNA Duplex Structure and Stability
Circular dichroism (CD) spectroscopy and UV thermal denaturation
analysis were used to assess the effect of 8-Cl-Ado substitution on the

604
CHEN AND SHEPPARD
stability of RNA-RNA duplexes. 8-Cl-Ado modified strands (13, 21, 24, 27
and 28) were annealed to the corresponding complementary strands (Fig. 2).
The properties of the modified duplexes were compared with natural RNA-
RNA duplexes constructed from unmodified RNA oligonucleotides (Fig. 2).
Spectroscopic analyses were performed on 2 mM solutions of each RNA
oligonucleotide in 10 mM phosphate buffer, 150 mM NaCl, 0.1 mM EDTA,
at a final pH of 7.0.
Circular dichroism (CD) spectroscopy indicated that RNA duplexes
containing internal 8-Cl-A substitutions adopt A-form RNA helices, char-
acteristic of natural RNA duplexes. Representative CD spectra for modified
and natural RNA duplexes are shown in Fig. 3A. As illustrated in Fig. 3A,
the complex between unmodified 17-mer RNA strands 29^30 (squares)
produced a CD spectrum with a positive absorption peak centered on 262 nm
and a strong negative peak below 250 nm, which is diagnostic of A-form
RNA duplexes.[22] Hybridization of 27, which contained a single substitution
of an adenosine residue with 8-Cl-Ado, with 30 (Fig. 3A, circles), produced
an RNA duplex with a CD spectrum similar to that of the natural 29^30
duplex. The introduction of two 8-Cl-Ado nucleotides into the RNA duplex
(28^30 Figure 3A, triangles) resulted in a CD spectrum similar to the native
RNA duplex (29^30). The CD spectrum of a shorter RNA duplex (24^26),
where the 8-Cl-Ado occupied a 30-terminal position, resembled the spectrum
of 25^26, which adopts A-form RNA structure (not shown).[23] Thus, CD
spectroscopy suggests that 8-Cl-A bases at internal or terminal positions do
not significantly alter the global RNA A-form helical structure.
Although RNA duplexes containing single or multiple 8-Cl-Ado sub-
stitutions adopt a normal RNA helical conformation, these nucleobase
modifications destabilize duplex RNA. To assess the relative thermal stabi-
lities of 8-Cl-Ado substitutions, samples used for CD spectroscopy were
subjected to UV thermal denaturation analysis. As shown in Fig. 3B, the UV
absorbances of modified duplexes 27^30 and 28^30 were monitored at
260 nm as the temperature was increased at a rate of 0.5ꢁC=min. The results
were compared to the unmodified control sequences, 29^30. All samples
showed a similar hyperchromicity change ( ꢀ 25%) over the range of 30 to
90ꢁC in 10 mM phosphate buffer, 150 mM NaCl, 0.1 mM EDTA, pH 7.0.
The melting temperature (Tm) for each RNA duplex was calculated from the
first derivative of the melting profile, and the results are given in Table 1. The
unmodified RNA duplex 29^30 (Fig. 3B, squares) displayed a Tm of 75.3ꢁC.
Substitution of a single adenine base with 8-Cl-A (27^30, Fig. 3B, circles)
produced an RNA duplex that melts at 73.1ꢁC under identical conditions.
The destabilization of ꢀ 2–3ꢁC for a single 8-Cl-Ado substitution was
additive: the insertion of a second 8-Cl-A destabilized duplex 28^30 (Fig. 3B,
triangles) by 5ꢁC relative to the natural duplex, 29^30.
To assess the effect of neighboring sequences on the duplex stability
of 8-Cl-Ado-containing RNAs, two alternate sequences (13 and 21), each

RNA CONTAINING 8-CHLOROADENOSINE
605
Figure 3. Spectroscopic characterization of modified RNA duplexes containing single (27^30
circles) and double (28^30, triangles) 8-Cl-Ado modifications and comparison with an un-
modified RNA-RNA hybrid (29^30, squares). A. Circular dichroism spectroscopy. B. UV
thermal denaturation curves at 0.5ꢁC=min. Conditions: 2 mM of each RNA strand in 10 mM
phosphate buffer, 150 mM NaCl, 0.1 mM EDTA, pH 7.0.
containing a single 8-Cl-Ado positioned centrally in a 17-mer duplex, were
studied using UV thermal denaturation methods. As summarized in Table 1,
the natural RNA sequence containing an A:U base pair (14 ^17) displayed a
Tm of 83.6ꢁC, while the analogous 8-Cl-Ado analog duplex (13^17) showed a
Tm of 77.9ꢁC, corresponding to a 5.7ꢁC decrease in thermal stability. A
related sequence exhibited a similar trend, with 8-Cl-Ado modified duplex
21^23 showing a Tm value of 4.4ꢁC lower than that of the natural duplex
(22^23). The effect of a 30-terminal 8-Cl-Ado substitution on duplex stability
also was evaluated. Comparison of a duplex with a 30-terminal 8-Cl-A:U pair
(24^26) with one containing a 30-terminal A:U pair (25^26) indicated that

606
CHEN AND SHEPPARD
Table 1. Melting Temperature Comparisons of Control and 8-Chloroadenosine Containing
Duplexesa
Duplex
Base Pair
Tm (ꢁC)a
Duplex
Base Pair
Tm (ꢁC)a
Internal modification
13^17
13^18
ACl:U
77.9 ꢂ 0.7
73.8 ꢂ 0.3
71.5 ꢂ 0.3
74.4 ꢂ 0.8
77.5 ꢂ 0.4
75.2 ꢂ 0.6
74.9 ꢂ 0.2
73.1 ꢂ 0.2
70.3 ꢂ 0.5
14^17
14^18
14^19
14^20
16^17
16^18
16^20
22^23
29^30
A:U
A:A
A:C
A:G
G:U
G:A
G:G
A:U
A:U
83.6 ꢂ 0.9
75.2 ꢂ 1.0
77.1 ꢂ 0.2
76.2 ꢂ 0.2
82.5 ꢂ 0.1
77.1 ꢂ 0.4
77.9 ꢂ 0.1
79.3 ꢂ 0.1
75.3 ꢂ 0.3
ACl:A
13^19
13^20
15^17
15^19
21^23
27^30
ACl:C
ACl:G
U:U
U:C
ACl:U
A:U=ACl:U
ACl:U= ACl:U
28^30
Terminal modification
24^26
ACl:U
58.8 ꢂ 0.4
25^26
A:U
62.9 ꢂ 0.2
aMelting temperatures (Tm’s) were determined from the average of three independent
experiments. Ranges ( ꢂ ) are given as the standard deviation of the three runs. Conditions:
2 mM of each RNA strand in 10 mM sodium phosphate, 0.1 mM EDTA, 150 mM NaCl,
pH 7.0.
the chlorinated analog destabilizes the duplex by 4.1ꢁC in the context of a 9-
mer double helical construct.
The propensity of 8-Cl-A bases to pair with other nucleobases (A, G, C)
was assessed and compared with the destabilization induced by mismatch
pairs within natural RNA duplexes (Table 1). Of the four natural nucleo-
bases, 8-Cl-A formed the most stable pair with uracil (Tm ¼ 77.9ꢁC, 13^17).
8-Cl-A paired equally well with guanine (Tm ¼ 74.4ꢁC, 13^20) and adenine
(Tm ¼ 73.8ꢁC, 13^18), and least well with cytosine (Tm ¼ 71.5ꢁC, 13^19). In
contrast, in the same sequence context (14), the natural adenine base formed
the most stable pair with uracil (17), followed by cytosine (19), guanine (20),
then adenine (18). Finally, a series of duplexes were constructed that con-
tained the same sequence as 13 or 14, but introduced natural base mis-
matches at the central base pair. Interestingly, the 8-Cl-A:U base pair (13^17,
Tm ¼ 77.9ꢁC) was more destabilizing than a G:U wobble base pair (16^17,
Tm ¼ 82.5ꢁC). The 8-Cl-A:U (13^17) pair destabilized duplexes to the same
extent as a U:U mismatch (15^17, Tm ¼ 77.5ꢁC) or a G:purine mismatch
(G:A, 16^18, Tm ¼ 77.1ꢁC or G:G, 16^20, Tm ¼ 77.9ꢁC). However, the
8-Cl-A:U base pair (13^17) was thermally more stable than the mismatched
U:C base pair (15^19), Tm ¼ 75.2ꢁC).
Taken together, the CD spectroscopy and UV thermal denaturation
studies revealed that the substitution of adenosine with 8-Cl-Ado in
RNA leads to a destabilization of RNA duplexes by 2–6ꢁC in 17 mer RNA
oligonucleotides, which corresponds to an ꢀ 5 kcal=mole (DDGꢁ
)
298

RNA CONTAINING 8-CHLOROADENOSINE
607
destabilization per internal 8-Cl-Ado substitution. The magnitude of helix
stability perturbation depends on the flanking sequence context (compare
13^17 with 27^30) and the location of the 8-Cl-Ado relative to the duplex
termini, consistent with extensive thermodynamic data on mismatches in
duplex RNA.[24,25]
Characterization of the stabilities of RNA duplexes containing
8-Cl-Ado provides insight into the effects of potential nucleoside therapeutic
agents on nucleic acid structure. Few 8-modified ribonucleotides have been
introduced into RNA duplexes and characterized by biophysical meth-
ods.[26,27] However, DNA oligonucleotides containing C-8 purine deoxy
nucleotide modifications, including 8-methoxy-,[28] 8-bromo-,[29] and
8-oxo-20-deoxyadenosine,[30] and the guanosine derivatives, 8-methyl-,[31] 8-
bromo-,[32] and 8-oxo-20-deoxyguanosine[33] have been synthesized. Biophy-
sical characterization of these modified DNAs revealed that C-8 purine
substitution generally resulted in DNA duplex destabilization in Watson-
Crick pairing contexts.[28–33] However, 8-modified purines generally stabi-
lized DNA complex formation in cases where syn glycosidic torsion angles
were preferred, such as G-quadruplexes[32] or A:G mismatches.[28] In this
context, it is interesting that in our system, converting an A:U base pair (14 ^
17, Tm ¼ 83.6ꢁC) to an A:G base pair (14^20, Tm ¼ 76.2ꢁC) lowers the Tm by
7.4ꢁC in a 17 mer RNA context. In contrast, converting an 8-Cl-A:U pair to
an 8-Cl-A:purine pair is less disruptive: the melting temperature of a 8-Cl-
A:G ‘‘mismatch’’ (13^20, Tm ¼ 74.4ꢁC) is only 3.5ꢁC lower than the 8-Cl-
A:U pair (13^17, Tm ¼ 77.9ꢁC) in the same sequence context.
The structural basis for duplex destabilization by 8-modified bases
correlates with the preference of C-8-substituted purines to adopt a syn
glycosidic torsion angle rather than the anti conformation found in A-form
and B-form duplexes. X-ray structural analysis[34] and CD studies[35] of
8-bromoadenosine and its 50-monophosphate derivative demonstrated that
substitution of the C-8 hydrogen of adenosine with a sterically-demanding
bromine atom forces the nucleobase to adopt exclusively the syn conforma-
tion. The syn preference of 8-bromoguanosine (8-Br-G) was demonstrated
1
for the dinucleotide, Cp-8-Br-G, using H-NMR,[27] and for 8-substituted
adenosine derivatives of sequence ApUpG, using UV and CD spectro-
scopy.[26] Thus, we currently believe that RNA helix destabilization caused
by 8-Cl-Ado substitution is the result of an altered syn glycosidic bond tor-
sion angle induced by the chlorine substitution at C-8. The outcome of a syn
glycosidic torsion angle preference would be the presentation of a different
hydrogen bonding face to potential pairing partners, and potentially,
perturbation of the sugar pucker of 8-Cl-Ado nucleosides. This model is
consistent with the relatively lower destabilizing effect of a 8-Cl-Ado:purine
mismatch compared with a A:A or A:G mismatch. Although a chlorine
atom (van der Waals radius 1.8 A) is somewhat smaller than a bromine
atom (1.95 A), both are more sterically demanding than a hydrogen atom

608
CHEN AND SHEPPARD
(1.2 A).[36] While theoretical calculations suggest that the syn conformer of
8-chloroadenosine is favored significantly over the anti conformer,[37] struc-
tural verification of these predicted effects has not been reported. As a result,
NMR structural analyses of 8-Cl-Ado and RNA containing this chlorinated
analog are underway in our laboratory.
EXPERIMENTAL SECTION
General Methods
All reactions were performed in oven dried (140ꢁC) glassware using
anhydrous Aldrich Sure-SealTM solvents and N2 as an inert atmosphere.
Unless otherwise noted, reagents were obtained from Aldrich and used
without further purification. Amine-derivatized 1000 A long chain alkyl
amine controlled pore glass (LCAA-CPG) was obtained from Chem Genes
Corporation. EM Science Kieselgel 60 F254 plates (0.25 mm) were used for
thin-layer chromatography. Compounds on TLC were visualized using a
short-wave UV lamp and stained with p-anisaldehyde=sulfuric acid=acetic
acid. Silica gel (0.040–0.063 mm, EM Science) was used for flash column
1
chromatography. H- and 13C-NMR spectra were recorded at 500 and
125 MHz respectively, on a Varian 500 instrument, unless otherwise noted,
and are referenced in parts per million (ppm) in the following format: che-
mical shift (multiplicity, integration, coupling constant in Hertz, proton
1
1
assignment). Proton assignments for H spectra were determined using H
-1H NMR COSY data. 31P-NMR data were recorded at 162 MHz on a
Varian 400 spectrometer and were referenced to an external standard of 85%
aqueous phosphoric acid. High resolution data were obtained using analy-
tically pure materials.
8-Chloroadenosine (4). m-Chloroperoxybenzoic acid (MCPBA, 25 g,
77%) was suspended in 120 mL of CH2Cl2 and stirred for 4 h at 25ꢁC. The
water layer was removed, and the CH2Cl2 layer was dried over MgSO4 and
filtered. To the filtrate solution was added 110 mL of anhydrous dimethyla-
cetamide (DMA), and the resulting solution was concentrated in vacuo to
120 mL (final concentration 0.9 M MCPBA=DMA). A hydrochloric acid
solution was prepared by bubbling HCl gas through 100 mL of anhydrous
DMA at 0ꢁC for 10 min. The solution then was diluted with 100 mL of
anhydrous DMA to give a 1.5 M HCl=DMA solution. Adenosine (5 g,
18.7 mmol) was taken up in 25 mL of anhydrous DMA, and 21 mL
(31.5 mmol) of 1.5 M HCl=DMA solution was added. The reaction was
stirred at 0ꢁC for 5 min, then 35 mL (32 mmol) of a 0.9 M MCPBA=DMA
solution was added dropwise over 30 min while the reaction was kept between
0 ꢃ 10ꢁC. The reaction was stirred under nitrogen, gradually warming to

RNA CONTAINING 8-CHLOROADENOSINE
609
25ꢁC over 2.5 h. The reaction was quenched by dropwise addition of a
solution of 3.5 g of NaHCO3 in 7.5 mL of H2O. The reaction was con-
centrated in vacuo at 45ꢁC to 25 mL The resulting syrup was co-evaporated
twice with 60 mL 2:1 xylenes=EtOH, then 60 mL MeOH and 167 mL of H2O
was added to the syrup. The resulting solution then was filtered, extracted
with Et2O (36100 mL), and the aqueous layer was concentrated in vacuo to
an orange solid. The product was purified by column chromatography (10%
MeOH=CHCl3) to give 3.8 g of crude product, which was recrystallized from
absolute EtOH to give 2.2 g of 8-chloroadenosine (4), (17.6 mmol), 39%
yield). mp 189–191ꢁC (lit.[14] 188–190ꢁC),1H NMR (DMSO-d6) d: 8.15 (s,
1H, H2), 7.59 (s, 2H, NH2), 5.84 (d, 1H, J1& ,2 ¼ 6 Hz, H10), 5.53 (d, 1H,
0
0
0
0
0
0
0
0
0
0
J2 OH,2 ¼ 6 Hz 2 OH), 5.48 (q, 1H,J5 OH,5 a ¼ 8 Hz, J5 OH,5b ¼ 8.4 Hz, 5 OH),
0
0
0
0
0
0
0
5.29 (d, 1H, J3 OH,3 ¼ 4.4 Hz, 3 OH), 5.04 (q, 1H, J1 ,2 ¼ 6 Hz, J2 ,3
¼
11.6 Hz, H20), 4.18 (s, 1H, H30), 3.97 (d, 1H, J ¼ 1.6 Hz, H40), 3.67 (m, 1H,
H50a), 3.52 (m, 1H, H50b). 13C NMR (DMSO-d6) d: 56.7, 71.5, 71.9, 87.4,
89.9, 118.6, 137.7, 150.3, 153.3, 155.9. HRMS-ESI (m=z): [M þ H]þ calcd,
302.0656; found, 302.0656.
N-6-Phenoxyacetyl-8-chloroadenosine (6). To a stirred solution of 4
(0.600 g, 1.99 mmol) in 10 mL anhydrous pyridine, 1.89 mL (14.9 mmol) of
chlorotrimethylsilane was added by syringe. The resulting white suspension
was stirred for 30 min at 25ꢁC. In a separate flask, 0.206 g (2.98 mmol) of
1,2,4-triazole and 0.55 mL (3.98 mmol) phenoxyacetyl chloride were dis-
solved in 5 mL each of anhydrous pyridine and anhydrous CH3CN. The
phenoxyacetyl chloride solution was added dropwise over 2 h to the 8-
chloroadenosine solution while the temperature was gradually raised to 55ꢁC
over 1 h. The reaction was stirred for another 5 h at 55ꢁC, then cooled to
25ꢁC. The reaction was quenched by addition of 2 mL of water and 1.5 mL of
28% NH4OH (aq). The solution then was evaporated to a syrup. Water
(15 mL) and CH2Cl2 (15 mL) were added to the syrup, the layers were
separated, and the aqueous layer was back-extracted with CH2Cl2
(4615 mL). The combined CH2Cl2 layers were washed with water
(2620 mL, dried over Na2SO4, filtered and concentrated to an orange foam.
The product was purified by column chromatography (3–7% MeOH=CHCl3)
1
to give 0.611 g (71% yield) of pale orange foam (6). H NMR (DMSO-d6)
d: 11.09 (s, 2H, NH2), 8.71 (s, 1H, H2), 7.30 (t, 2H, J ¼ 7.75 Hz, O-Ph), 6.96
0
0
0
0
0
(m, 3H, O-Ph), 5.93 (d, 1H, J1 ,2 ¼ 6 Hz, H1 ), 5.53 (d, 1H, J2 ,2 OH ¼ 5.5 Hz,
20OH), 5.29 (d, 1H, J3 ,3 OH ¼ 4.5 Hz, 30OH), 5.15 (q, 1H, J1 ,2 ¼ 6 Hz, J2 ,3
¼
0
0
0
0
0
0
11.5 Hz, H20), 5.00 (s, 2H, -CH2-OPh), 4.97 (t, 1H, J5 a,5 OH ¼ 5 Hz,
0
0
J5 b,5 OH ¼ 6.5 Hz, 50OH), 4.27 (q, 1H, J3 ,3 OH ¼ 4.5 Hz, J3 ,4 ¼ 8 Hz, H3 ),
0
0
0
0
0
0
0
0
0
0
0
0
0
3.97(q, 1H, J3 ,4 ¼ 8 Hz, J4 ,5 ¼ 4.75 Hz, H4 ), 3.68 (m, 1H, H5 a), 3.53
(m, 1H, H50b). 13C NMR (DMSO-d6) d: 167.4, 157.8, 152.2, 148.3, 141.5,
129.5, 122.3, 121.1, 114.6, 94.0, 86.3, 67.1, 25.2. HRMS-FAB (m=z): [M þ
Na]þ calcd, 458.0843; found, 458.0842.

610
CHEN AND SHEPPARD
50-O-Dimethoxytrityl-N-6-phenoxyacetyl-8-chloroadenosine (7). Nucleo-
side 6 (0.241 g, 0.552 mmol) was co-evaporated with anhydrous THF
(265 mL), then dried in vacuo for 12 h. The dried starting material was
dissolved in 1.4 mL anhydrous pyridine and 4,4-dimethylaminopyridine
(DMAP, 0.0120 g, 0.16 mmol) was added. The reaction was stirred for 5 min,
then 4,40-dimethoxytritylchloride (DMTCl, 0.387 g, 1.14 mmol) was added in
three equal portions over 3 h, and the reaction was stirred for 16 h. The
reaction was quenched with 2 mL saturated aqueous NaHCO3, 5 mL H2O,
and 5 mL CH2Cl2. The layers were separated and the aqueous layer was
back-extracted with CH2Cl2 (465 mL). The combined CH2Cl2 layers were
washed successively with 10 mL each of 10% aqueous NaHCO3, H2O, and
brine. The organic layer then was dried over Na2SO4, filtered, and con-
centrated in vacuo to a brown oil. The residue was coevaporated with 10 mL
toluene and purified by column chromatography (5% MeOH=CHCl3) to give
0.238 g (58% yield) of golden foam.[7] 1H NMR (500 MHz, DMSO-d6)
d: 11.08 (s, 1H, NH), 8.57 (s, 1H, H2), 7.29 (m, 4H, DMT, O-Ph), 7.15 (m,
7H, DMT), 6.960 (m, 3H, O-Ph), 6.78 (m, 4H, DMT), 5.97 (d, 1H,
J1 ,2 ¼ 4.5 Hz, H1 ), 5.59 (d, 1H, J2 ,2 OH ¼ 4.5 Hz, 20OH), 5.30 (d, 1H,
0
0
0
0
J3 ,3 OH ¼ 5.5 Hz,30OH), 5.25 (q, 1H, J2 ,2 OH ¼ 4.5 Hz, J2 ,3 ¼ 10 Hz, H2 ),
0
0
0
0
0
0
0
0
0
0
0
0
5.01 (s, 2H, -CH2-OPh), 4.54 (q, 1H, J3 ,3 OH ¼ 5.5 Hz, J2 ,3 ¼ 10 Hz, H3 ),
0
0
0
0
0
4.11 (q, 1H, J3 ,4 ¼ 9.5 Hz, J4 ,5 ¼ 4.75 Hz, H4 ), 3.67 (s, 6H, -OCH3), 3.25
(dd, 1H, J4 ,5 a ¼ 3.5 Hz, J5 a,5 b ¼ 10.5 Hz, H50a), 3.16 (dd, 1H, J4 ,5 b ¼ 5.5 Hz,
0
0
0
0
0
0
þ
J5 a,5 b ¼ 10.5 Hz, H50b). HRMS-FAB (m=z): [M þ Na] calcd, 760.2149;
0
0
found, 760.2147.
20-O-tert-Butyldimethylsilyl-50-O-dimethoxytrityl-N-6-phenoxyacetyl-8-
chloroadenosine (8). Compound 7 (0.595 g, 0.806 mmol) was coevaporated
with anhydrous THF (362 mL) and dried in vacuo for 12 h. The starting
material then was dissolved in 8 mL anhydrous, THF, and subsequently,
anhydrous pyridine (0.240 mL, 2.98 mmol) and Et3N (0.110 mL, 0.806 mmol)
were added by syringe. Silver nitrate (0.164 g, 0.967 mmol) was added, and
the reaction was stirred at 25ꢁC for 15 min in the absence of light. tert-
Butyldimethylsilylchloride (TBDMSCl, 0.158 g, 1.04 mmol) then was added,
and the reaction was stirred for 2.5 h. Then, Et3N (0.17 mL, 1.5 equiv),
AgNO3 (0.137 g, 1 equiv), and TBDMS-Cl (0.103 g, 0.85 equiv) were added
sequentially, and the reaction was stirred for another 20 min. The reaction
was diluted with 15 mL CH2Cl2 and filtered through Celite. The Celite was
rinsed with 50 mL CH2Cl2 and the combined CH2Cl2 layers were washed
with a 5% aqueous NaHCO3 solution (2625 mL). The aqueous layer was
back-extracted with CH2Cl2 (2625 mL), and the combined organics were
dried over Na2SO4, filtered, and concentrated in vacuo to a yellow oil. The
product was purified by column chromatography (3% EtOAc=CHCl3) to
give (8) (0.110 g, 16% yield) and (9) (0.259 g, 38% yield) as pale yellow
1
foams. Compound (8): H NMR (DMSO-d6) d: 11.10 (s, 1H, NH), 8.54

RNA CONTAINING 8-CHLOROADENOSINE
611
(s, 1H, H2), 7.30 (m, 4H, DMT, O-Ph), 7.20 (m, 7H, DMT), 6.96 (m, 3H,
0
0
0
O-Ph), 6.79 (m, 4H, DMT), 5.96 (d, 1H, J1 ,2 ¼ 6.5 Hz, H1 ), 5.29 (t, 2H,
0
0
0
0
0
0
J1 ,2 ¼ 6.5 Hz, J2 ,3 ¼ 6 Hz, H2 , 3 OH), 5.00 (s, 2H, -CH2-OPh), 4.45 (q, 1H,
0
0
0
0
0
0
0
0
0
J2 ,3 ¼ 6 Hz, J3 ,4 ¼ 10.5 Hz, H3 ), 4.13 (q, 1H, J3 ,4 ¼ 10.5 Hz, J4 ,5 ¼ 5.5 Hz,
H40), 3.69 (s, 6H, -OCH3), 3.29 (dd, 1H, J4 ,5 a ¼ 5 Hz, J5 a,5 b ¼ 10.5 Hz,
0
0
0
0
H50a), 3.18 (dd, 1H, J4 ,5 b ¼ 6 Hz, J5 a,5 b ¼ 10.5 Hz, H50b), 0.725 (s, 9H,
t-Bu), ꢃ 0.05 (s, 3H, Si-CH3), ꢃ 0.18 (s, 3H, Si-CH3). 13C NMR (DMSO-d6)
d: 158.7, 158.5, 152.7, 152.6, 149.1, 149.0, 145.6, 142.0, 140.9, 136.2, 136.2,
130.4, 130.3, 130.2, 129.6, 128.4, 128.3, 128.1, 127.3, 127.1, 122.6, 121.8,
115.2, 113.7, 113.5, 90.2, 86.0, 84.6, 80.6, 79.87, 73.3, 70.6, 67.8, 63.5, 26.2,
18.5, ꢃ 4.1, ꢃ 4.7. HRMS-FAB (m=z): [M þ H]þ calcd, 852.3195; found,
852.3195.
0
0
0
0
30-O-tert-Butyldimethylsilyl-50-O-dimethoxytrityl-N-6-phenoxyacetyl-8-
1
chloroadenosine (9). Isolated from synthesis of 8. H NMR (400 MHz,
DMSO-d6) d: 11.11 (s, 1H, NH), 8.65 (s, 1H, H2), 7.29 (t, 2H, J ¼ 6.4 Hz, O-
Ph), 7.18 (m, 4H, DMT), 7.06 (m, 4H, DMT), 6.95 (m, 3H, O-Ph), 6.75 (m,
0
0
0
0
0
3H, DMT), 5.93 (d, 1H, J1 ,2 ¼ 3 Hz, H1 ), 5.53 (d, 1H, J2 ,2 OH ¼ 4.4 Hz,
H20OH), 5.34 (d, 1H, J1 ,2 ¼ 3 Hz H3 ), 4.99 (s, 2H, -CH2-OPh), 4.90 (t, 1H,
0
0
0
0
0
0
0
0
J3 ,4 ¼ 4 Hz, H3’), 4.07 (d, 1H, J3 ,4 ¼ 4 Hz, H4 ), 3.69 (s, 3H, -OCHa3), 3.68
(s, 3H, O-CHb3), 2.93 (dd, 1H, J4 ,5 a ¼ 2.6 Hz, J5 a,5 b ¼ 10.25 Hz, H50a), 0.83
(s, 9H, t-Bu), 0.11 (s, 3H, Si-CHa3), 0.07 (s, 3H, Si-CHb3). HRMS-FAB
(m=z): [M þ H]þ calcd, 852.3195; found, 852.3195.
0
0
0
0
20-O-tert-Butyldimethylsilyl-30-O-(2-cyanoethyl-N,N-diisopropylphosphor-
amidite)-50-O-dimethoxytrityl-N-6-phenoxyacetyl-8-chloroadenosine (10).
Nucleoside 8 (0.108 g, 0.127 mmol) was coevaporated with anhydrous THF
(262 mL), dried in vacuo, and dissolved in 0.39 mL anhydrous THF. 2,4,6-
Collidine (0.110 mL, 0.954mmol) and N-methylimidazole (0.005 mL,
0.064 mmol) were added by syringe, followed by dropwise addition
of N,N-diisopropylaminocyanoethylphosphoramidic chloride (0.071 mL,
0.318 mmol) . The reaction was stirred at 25ꢁC for 4 h, then diluted with 5 mL
EtOAc, and washed sequentially with 5 mL of cold 5% aqueous NaHCO3
solution, and 5 mL of H2O. The organic layer was dried over Na2SO4, filtered,
and concentrated in vacuo to a yellow oil. The products were purified by
column chromatography (5% hexanes=10% EtOAc=CH2Cl2) to give 0.067 g
(50% yield) of isomers 10a and 10b. HRMS-FAB (m=z): [M þ H]þ calcd,
1052.4274; found, 1052.4278. Compound 10a. 1H NMR (CD3CN) d: 9.23 (s,
1H, NH), 8.50 (s, 1H, H2), 7.28 (m, 11H, DMT, O-Ph), 7.03 (m, 3H, O-Ph),
0
0
0
0
6.79 (m, 4H, DMT), 6.00 (d, 1H, J1 ,2 ¼ 5 Hz, H1 ), 5.55 (t, 1H,J ¼ 5 Hz, H2 ),
0
0
0
4.96 (s, 2H, -CH2-OPh), 4.73 (m, 1H, J3 ,4 ¼ 4.4 Hz, J ¼ 10 Hz, H3 ), 4.43 (q,
0
0
0
1H, J3 ,4 ¼ 4.4 Hz, H4 ), 3.76 (s, 6H, -OCH3), 3.68 (m, 4H, O-CH2CH2-CN,
0
0
0
0
N-CH(CH3)2), 3.51 (dd, 1H, J4
¼ 4 Hz, J5 a,5 b ¼ 10 Hz, H50a), 3.30 (dd,
,5 a
0
0
0
0
1H, J4
¼ 4.8 Hz, J5 a,5 b ¼ 10 Hz, H50b), 2.47 (td, 2H, J ¼ 2 Hz, 6 Hz,
,5 b

612
CHEN AND SHEPPARD
O-CH2CH2-CN), 1.22 (d, 6H, J ¼ 6.8 Hz, i-Pra), 1.20 (d, 6H, J ¼ 6.4 Hz, i-
Prb), 0.80 s 9H, t-Bu), 0.01 (s, 3H, Si-CHa3), ꢃ 0.16 (s, 3H, Si-CHb3). 13C
NMR (CD3CN) d: 167.11, 158.87, 158.84, 157.87, 152.40, 152.19, 148.03,
145.42, 142.50, 136.30, 136.16, 130.32, 130.24, 129.96, 128.25, 127.96, 127.0,
122.06, 118.50, 115.00, 113.20, 113.18, 89.97, 86.22, 83.93, 72.89, 72.81, 72.39,
68.19, 43.50, 43.40, 43.06, 25.53, 25.27, 24.49, 24.43, 24.19, 24.14, 20.25, 20.20,
1
17.80, ꢃ 4.97, ꢃ 5.63. 31P NMR (CD3CN) d: 151.19. Compound 10b. H
NMR (400 MHz, CD3CN) d: 9.21 (s, 1H, NH), 8.45 (s, 1H, H2), 7.28 (m,
11H, DMT, O-Ph), 7.02 (m, 3H, O-Ph), 06.79 (m, 4H, DMT), 6.00 (d, 1H,
0
0
0
J1 ,2 ¼ 6 Hz, H1 ), 5.44 (t, 1H,J ¼ 5 Hz, H2 ), 4.94 (s, 2H, -CH2-OPh), 4.64 (td,
1H, J ¼ 4 Hz, H30), 4.34 (s, 1H, J ¼ 3.2 Hz, H40), 3.73 (s, 6H, -OCH3), 3.66 (m,
4H, O-CH2CH2-CN, N-CH(CH3)2), 3.49 (m, 1H, H50a), 3.38 (m, 1H, H50b),
2.68 (m, 2H, O-CH2CH2-CN), 1.18 (d, 6H, J ¼ 7 Hz i-Pra), 1.10 (d, 6H,
J ¼ 7 Hz, i-Prb), 0.75 (s, 9H, t-Bu), ꢃ 0.04 (s, 3H, Si-CHa3), ꢃ 0.25 (s, 3H, Si-
CHb3). 31P NMR (CD3CN) d: 149.34.
20-O-Succinyl-30-O-tert-butyldimethylsilyl-50-O-dimethoxytrityl-N-6-phe-
noxyacetyl-8-chloroadenosine (11). To a stirred solution of 9 (0.177 g,
0.207 mmol) dissolved in 0.90 mL anhydrous pyridine was added DMAP
(0.0165 g, 0.135 mmol) and succinic anhydride (0.0332 g, 0.332 mmol) . The
solution was stirred under N2 for 12 h, then diluted with 5 mL EtOAc, and
washed with 5 mL H2O. The aqueous layer was back-extracted with EtOAc
(2 6 5 mL), then the combined organics were washed sequentially with 10 mL
satd. aqueous KH2PO4 (pH ¼ 4.13), 10 mL brine, and dried over Na2SO4.
The organic layer was concentrated in vacuo to an orange oil, and the pro-
duct was purified by column chromatography (5% MeOH=CHCl3) to give 11
(0.140 g, 71% yield). 1H NMR (400 MHz, acetonitrile-d3) d: 9.25 (s, 1H, NH),
8.76 (s, 1H, H2), 8.759 (s,1H, OH), 7.35 (m, 3H, O-Ph), 7.11 (m, 11H, DMT,
0
0
0
0
O-Ph), 6.75 (m, 4H, DMT, O-Ph), 6.51 (dd, 1H, J1 ,2 ¼ 1.6 Hz, J2 ,3 ¼ 5 Hz,
H20), 6.12 (d, 1H, J1 ,2 ¼ 1.6 Hz, H1 ), 5.40 (dd, 1H, J2
¼ 5 Hz,
0
,30
0
0
0
0
0
0
0
J3 ,4 ¼ 7.6 Hz, H3 ), 4.94 (s, 2H, -CH2-OPh), 4.12 (m, 1H, H4 ), 3.75 (s, 3H,
0
0
0
0
-OCHa3), 3.74 (s, 3H, O-CHb3), 3.48 (dd, 1H, J4
¼ 2.4 Hz, J5 a,5 b ¼
,5 a
11 Hz, H50a), 3.02 (dd, 1H, J4
¼ 3.4 Hz, J5 a,5 b ¼ 11 Hz, H50b), 0.84 (s,
0
0
0
0
,5 a
9H, t-Bu), 0.15 (s, 3H, Si-CH3), 0.06 (s, 3H, Si-CH3). HRMS-FAB (m=z):
[M þ Na]þ calcd, 974.3175; found, 974.3174.
Controlled Pore Glass Linked 20-O-Succinyl-30-O-tert-Butyldimethylsilyl
-50-O-dimethoxytrityl-N-6-phenoxyacetyl-8-chloroadenosine (12). Succiny-
lated nucleoside 11 (0.128 g, 0.134 mmol) was dissolved in 0.67 mL anhydrous
1,4-dioxane. To this solution was added anhydrous pyridine (0.028 mL,
0.348 mmol), p-nitrophenol (0.021 g, 0.148 mmol), and dicyclohex-
ylcarbodiimide (0.083 g, 0.402 mmol) . The solution was stirred for 30 min
under N2, and the resulting precipitate was filtered. Then, 1000 A LCAA-cpg
(0.500 g, 54 mmol amino groups) was suspended in 3 mL anhydrous DMF and

RNA CONTAINING 8-CHLOROADENOSINE
613
0.20 mL Et3N, and the filtrate solution was added. The mixture was shaken
for 12 h, and the CPG was filtered and washed with 5 mL each of DMF,
MeOH and Et2O, and dried in vacuo. The underivatized amino groups were
capped by placing the CPG in a solution containing 4.5 mL anhydrous pyr-
idine, acetic anhydride (0.5 mL, 4.5 mmol), and DMAP (0.055 g, 0.45 mmol).
The solution was shaken for 12 h, then the CPG was rinsed with 5 mL each of
DMF, MeOH, and Et2O. The final loading of 22 mmol=g was determined by
UV quantitation of dimethoxytrityl cation release.
RNA Synthesis, Deprotection, and Purification
All unmodified RNA oligonucleotides were purchased from Dharmacon
Research Inc. (Boulder, CO) and were deprotected at 37ꢁC for 30 min with
400 mL of 100 mM acetic acid, adjusted to pH 3.8 with tetramethylethylene-
diamine (TEMED). Modified oligonucleotides were synthesized by automated
methods on a Pharmacia Gene Assembler on a 1.0 mmol scale using RNA
phosphoramidites obtained from Glen Research (Sterling, VA) with labile
base protecting groups for the natural nucleosides (Pac-A-CE, Ac-C-CE, iPr-
Pac-G-CE, U-CE). Phosphoramidites 10a and 10b were coupled using 0.1 M
anhydrous CH3CN solutions (coupling time, 10 min). After synthesis, the
oligonucleotide was deprotected by treatment with 1 mL 1:3 EtOH=NH4OH
for 20 h at 37ꢁC. The EtOH=NH4OH solution was removed using a Speedvac
concentrator and the resulting powder was desilylated with 600 mL of 1.0 M
TBAF in THF for 24 h at 25ꢁC. The product was dried using a Speedvac
concentrator, redissolved in 500 mL double-distilled water (ddH2O), and
desalted using a G-25 Sephadex column (NAP, Amersham). The coupling
yield for 10 was 99%, as determined by dimethoxytrityl cation release.
Synthesis of 30–terminally modified sequences using solid support 12 pro-
ceeded with a 77% coupling yield for the first 30–nucleotide, and with a yield of
91–96% for subsequent bases. RNA oligonucleotides were purified by 20%
denaturing polyacrylamide gel electrophoresis (PAGE) as described.[38]
Purified oligonucleotide solutions were quantitated by UV absorption at
260 nm using extinction coefficients based on nearest-neighbors method.[39]
The extinction coefficient of 8-chloroadenosine was assumed to be equal to
adenosine.
Matrix-Assisted Laser Desorption Ionization Time-of-Flight
(MALDI-TOF) Mass Spectrometry
8-Cl-Ado-modified and unmodified RNA oligonucleotides were
characterized using a PerSeptive Biosystems, Inc. (Foster City, CA), Voyager-
DETM PRO BiospectrometryTM Workstation MALDI-TOF mass spectro-
meter. A N2 laser was used (337 nm wavelength, 3 ns pulse), and spectra were

614
CHEN AND SHEPPARD
acquired in the negative ion mode averaging 128 shots. A matrix consisting of
2,4,6-trihydroxyacetophenone=ammonium citrate in CH3CN=H2O was used,
and poly(dT)10 and poly(dT)18 sequences obtained from Integrated DNA
Technologies were used for internal calibration. All masses found were in
agreement with calculated masses. 50-UGU GCC CGACl CUU GCC GU-30
(13): m=z [M]ꢃ calcd,ꢃ5387.6; found, 5386.8. 50-UGU GCC AGACl CUU GCC
GU-30 (21): m=z [M] calcd, 5411.6; found, 5411.0. 50-UGU GCC CGACl-30
(24): m=z [M]ꢃ calcd,ꢃ2865.2; found, 2865.6.50-AGU GCA CGU UAC AClGC
CG-30 (27): m=z [M] calcd, 5458.7; found, 5458.0. 50-AGU GCACl CGU
UAC AClGC CG-30 (28): m=z [M]ꢃ calcd, 5493.2; found, 5493.4.
Circular Dichroism Spectroscopy
CD spectra were recorded using a JASCO J-715 CD spectropolarimeter.
Oligonucleotide duplex samples containing 2 mM of each strand were pre-
pared in 10 mM sodium phosphate, 0.1 mM EDTA, 150 mM NaCl, pH ¼ 7.0.
Samples were annealed by heating to 95ꢁC for 3 min, followed by gradual
cooling to 25ꢁC over 2 h. Spectra were recorded from 200 to 350 nm at 25ꢁC
in a 2 mm cuvette. Each spectrum represents a baseline corrected average of
four accumulations per scan. All data were corrected by subtracting the
buffer reference signal.
UV Thermal Denaturation Analysis
Thermal denaturation data were acquired on a Cary 500 spectro-
photometer equipped with a multi-cuvette thermoelectric controller in 10 mm
quartz cuvettes. Solutions of 2 mM duplex oligonucleotides in pH 7.0 buffer
consisting of 10 mM sodium phosphate, 0.1 mM EDTA, 150 mM NaCl were
annealed by heating to 95ꢁC for 5 min and cooling to 25ꢁC over 2 h. The
samples were degassed under vacuum for 3 min prior to melting analysis. The
absorbance was measured at 260 nm at 0.1ꢁC intervals as the samples were
heated from 30ꢁC to 90ꢁC at 0.5ꢁC=min. Data sets for the downward tran-
sition from 90ꢁC to 30ꢁC were also collected at 0.5ꢁC=min; all melting pro-
files were fully reversible to within 1.0ꢁC. Melting temperatures (Tm’s) were
determined from plots of dA260=dT vs. T as described[40], and are derived
from replicate melting experiments using three independent samples.
ACKNOWLEDGMENTS
We thank our collaborators Dr. Steven Rosen, Dr. Nancy Krett, and
Dr. Varsha Gandhi for stimulating discussions. We acknowledge the Keck
Biophysics Facility and the Analytical Services Laboratory of Northwestern
University, and Dr. Steven Mullen at the University of Illinois, Urbana-
Champaign Mass Spectrometry Laboratory. This work was supported by

RNA CONTAINING 8-CHLOROADENOSINE
615
research grants from the National Institutes of Health (RO1 CA85915–02)
and the Leukemia and Lymphoma Society (6505–00).
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of Fludarabine Triphosphate in Chronic Lymphocytic-Leukemia Cells During
Fludarabine Therapy. Leuk. Lymphoma 1993, 10, 49–56.
3. Gandhi, V.; Legha, J.; Chen, F.; Hertel, L. W.; Plunkett, W. Excision of 20,20-
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Received April 15, 2002
Accepted June 25, 2002