Incorporation of 2'-deoxy-2'-mercaptocytidine into oligonucleotides via phosphoramidite chemistry

Full text of DOI:10.1021/jo970096o

J . Org. Chem. 1997, 62, 3415-3420
3415
Substitution of the 2′-hydroxyl group in RNA by a
mercapto (SH) group has received relatively little atten-
tion despite the potential use of 2′-deoxy-2′-mercapto-
nucleosides as probes for the role of the 2′-hydroxyl in
RNA structure and function. Although the first 2′-deoxy-
2′-mercaptonucleosides were synthesized more than two
decades ago,9 only recently has the synthesis of an RNA
dinucleotide containing 2′-deoxy-2′-mercaptouridine been
reported.10 The synthesis employed phosphate triester
methodology in which 5′-O-(9-phenylxanth-9-yl)-2′-deoxy-
2′-(9-(p-anisyl)xanthen-9-ylthio)uridine-3′-O-(2-chlorophen-
yl)phosphate was coupled to 2′,3′-di-O-acetyluridine.
This approach used relatively strong acidic conditions (0.1
M HCl) to remove the 2′-[9-(p-anisyl)xanthen-9-yl] sulfur
protecting group and may therefore be problematic in the
synthesis of longer oligonucleotides due to the tendency
of adenosine and guanosine to depurinate under acidic
conditions.11
In cor p or a tion of
2′-Deoxy-2′-m er ca p tocytid in e in to
Oligon u cleotid es via P h osp h or a m id ite
Ch em istr y
Michelle L. Hamm and J oseph A. Piccirilli*
Howard Hughes Medical Institute,
Department of Biochemistry and Molecular Biology, and
Department of Chemistry, The University of Chicago,
5841 South Maryland Avenue, Room N104,
Chicago, Illinois 60637
Received J anuary 17, 1997
Modified oligonucleotides that contain a sulfur atom
in place of an oxygen atom have proven enormously
valuable in studies of nucleic acid structure and func-
tion,1-3 protein-nucleic acid interactions,4-6 and anti-
sense gene therapy.7 Virtually every oxygen atom on the
bases,1,4 the sugar,2,5,7 and the phosphoryl group3,6 has
been replaced by sulfur. Some of these nucleotide
analogues have provided insight into the most intricate
details of biological function.8
Although phosphate triester methodology is useful for
large scale solution synthesis of oligonucleotides, solid
phase phosphoramidite methodology is generally the
method of choice for most biochemical investigations
because it enjoys near quantitative coupling yields and
fewer side products.12 Thus, the development of chem-
istry for the incorporation of 2′-mercaptonucleosides into
RNA and DNA via the phosphoramidite approach would
facilitate further investigation of nucleic acid structure
and function. Herein, we report the synthesis of ap-
propriately protected phosphoramidites that are suitable
for the incorporation of 2′-deoxy-2′-mercaptocytidine and
2′-deoxy-2′-mercaptouridine into oligonucleotides. Meth-
ods for the postsynthetic protection and deprotection of
the mercapto group via disulfide exchange chemistry are
also described.
* To whom correspondence should be addressed. Tel.: (773) 702-
9312. Fax: (773) 702-0271. E-mail: jpicciri@midway.uchicago.edu.
(1) For examples of sulfur modifications in the heterocyclic base
see: (a) Sontheimer, E. J . Mol. Biol. Rep. 1994, 20, 35. (b) Rao, T. S.;
Durland, R. H.; Seth, D. M.; Myrick, M. A.; Bodepudi, V.; Revankar,
G. R. Biochemistry 1995, 34, 765. (c) Xu, Y.-Z.; Zheng, Q.; Swann, P.
F. Tetrahedron Lett. 1992, 33, 5837. (d) Kuimelis, R. G.; Nambiar, K.
P. Nucleic Acids Res. 1994, 22, 1429. (e) Woisard, A.; Favre, A.; Clivio,
P.; Fourrey, J .-L. J . Am. Chem. Soc. 1992, 114, 10072. (f) Favre, A.;
Fourrey, J .-L. Acc. Chem. Res. 1995, 28, 375. (g) Kutyavin, I. V.;
Rhinehart, R. L.; Lukhtanov, E. A.; Gorn, V. V.; Meyer, R. B.; Gamper,
H. B. Biochemistry 1996, 35, 11170.
(2) For examples of sulfur modifications in the sugar see: (a)
Piccirilli, J . A.; Vyle, J . S.; Caruthers, M. H.; Cech, T. R. Nature 1993,
361, 85. (b) Zhou, D.-M.; Usman, N.; Wincott, F. E.; Matulic-Adamic,
J .; Orita, M.; Zhang, L.-H.; Komiyama, M.; Kumar, P. K. R.; Taira, K.
J . Am. Chem. Soc. 1996, 118, 5862. (c) Kuimelis, R. G.; McLaughlin,
L. W. Biochemistry 1996, 35, 5308. (d) Kuimelis, R. G.; McLaughlin,
L. W. J . Am. Chem. Soc. 1995, 117, 11019. (e) Kuimelis, R. G.;
McLaughlin, L. W. Nucleic Acids Res. 1995, 23, 4753. (f) Weinstein,
L. B.; Earnshaw, D. J .; Cosstick, R.; Cech, T. R. J . Am. Chem. Soc.
1996, 118, 10341. (g) Bellon, L.; Morvan, F.; Barascut J .-L.; Imbach,
J .-L. Biochem. Biophys. Res. Commun. 1992, 184, 797. (h) Hancox, E.
L.; Connolly, B. A.; Walker, R. T. Nucleic Acids Res. 1993, 21, 3485.
(3) For examples of phosphorothioate modifications see: (a)
Herschlag, D.; Piccirilli, J . A.; Cech, T. R. Biochemistry 1991, 30, 4844.
(b) Dahm, S.-A. C.; Uhlenbeck, O. C. Biochemistry 1991, 30, 9464. (c)
Slim, G.; Gait, M. J . Nucleic Acids Res. 1991, 19, 1183. (d) van Tol,
H.; Buzayan, J . M.; Feldstein, P. A.; Eckstein, F.; Bruening, G. Nucleic
Acids Res. 1990, 18, 1971. (e) McSwiggen, J . A.; Cech, T. R. Science
1989, 244, 679. (f) Rajagopal, J .; Doudna, J . A.; Szostak, J . W. Science
1989, 244, 692. (g) Ruffner, D. E.; Uhlenbeck, O. C. Nucleic Acids Res.
1990, 18, 6025. (h) Christian, E. L.; Yarus, M. Biochemistry 1993, 32,
4475. (i) Hardt, W.-D.; Erdmann, V. A.; Hartman, R. K. RNA 1996, 2,
1189. (j) Eckstein, F. Annu. Rev. Biochem. 1985, 54, 367.
(4) For examples of sulfur modifications in the heterocyclic base
see: (a) Mishima, Y.; Steitz, J . A. EMBO 1995, 14, 2679. (b) McGregor,
A.; Vaman Rao, M.; Duckworth, G.; Stockley, P. G.; Connolly, B. A.
Nucleic Acids Res. 1996, 24, 3173.
(5) For examples of sulfur modifications in the sugar see: (a) Coves,
J .; Le Hir de Fallois, L.; Le Pape, L.; Decout, J .-L.; Fontecave, M.
Biochemistry 1996, 35, 8595. (b) Vyle, J . S.; Connolly, B. A.; Kemp,
D.; Cosstick, R. Biochemistry 1992, 31, 3012. (c) Bellon, L.; Barascut,
J .-L.; Maury G.; Divita, G.; Goody, R.; Imbach, J .- L. Nucleic Acids
Res. 1993, 21, 1587. (d) Szczelkun, M. D.; Connolly, B. A. Biochemistry
1995, 34, 10724.
(6) For examples of phosphorothioate modifications see: (a) J affe,
E. K.; Cohn, M. J . Biol. Chem. 1979, 254, 10839. (b) Milligan, J . F.;
Uhlenbeck, O. C. Biochemistry 1989, 28, 2849. (c) Grasby, J . A.;
Connolly, B. A. Biochemistry 1992, 31, 7855.
(7) (a) Secrist, J . A.; Riggs, R. M.; Tiwari, K. N.; Montgomery, J . A.
J . Med. Chem. 1992, 35, 533. (b) van Draanen, N. A.; Freeman, G. A.;
Short, S. A.; Harvey, R.; J ansen, R.; Szczech, G.; Koszalka, G. W. J .
Med. Chem. 1996, 39, 538. (c) Rahim, S. G.; Trivedi, N.; Bogunovic-
Batchelor, M. V.; Hardy, G. W.; Mills, G.; Selway, J . W. T.; Snowden,
W.; Littler, E.; Coe, P. L.; Basnak, I.; Whale, R. F.; Walker, R. T. J .
Med. Chem. 1996, 39, 789.
Standard preparation of nucleoside synthons used in
solid phase synthesis of oligonucleotides includes protec-
tion of the 2′- and 5′-hydroxyls as tert-butyldimethylsilyl
and dimethoxytrityl ethers, respectively, protection of the
heterocyclic amines as amides, and phosphitylation of the
3′-oxygen to the â-cyanoethyl N,N-diisopropylphosphor-
amidite.13 Protection of a 2′-mercapto group during solid
phase synthesis is necessary to prevent side reactions due
to the redox and nucleophilic properties of sulfur. Fur-
thermore, because of the potential intramolecular SN
reactions at either the 1′-carbon14 or 3′-phosphorus15
under basic conditions, it is important that the sulfur
protecting group remain stable during removal of the
base labile groups. The most commonly used 2′-hydroxyl
protecting group, tert-butyldimethylsilyl, is not suitable
for sulfur protection.16 However, protection of the mer-
(8) See for example, references 1a, 2a, 3a-i, 4a, and 6a-c.
(9) (a) Ryan, K. J .; Acton, E. M.; Goodman, L. J . Org. Chem. 1971,
36, 2646. (b) Imazawa, M.; Ueda, T.; Ukita, T. Chem. Pharm. Bull.
1975, 23, 604. (c) Patel, A. D.; Schrier, W. H.; Nagyvary, J . J . Org.
Chem. 1980, 45, 4830. (d) Divakar, K. J .; Mottoh, A.; Reese, C. B.;
Sanghvi, Y. S. J . Chem. Soc., Perkin Trans. 1 1990, 969. (e) Marriott,
J . H.; Moltahedeh, M.; Reese C. B. Carbohydr. Res. 1991, 216, 257.
(10) Reese, C. B.; Simons, C.; Zhang, P.-Z. J . Chem. Soc., Chem.
Commun. 1994, 1809.
(11) Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980, 65, 499.
(12) Caruthers, M. H. In Synthesis and Applications of DNA and
RNA; Narang, S. A., Ed.; Academic Press Inc.: Orlando, 1978; p 52.
(13) (a) Usman, N.; Ogilvie, K. K.; J iang, M. Y.; Cedergren, R. J . J .
Am. Chem. Soc. 1987, 109, 7845. (b) Hakimelahi, G. H.; Proba, Z. A.;
Ogilvie, K. K. Can. J . Chem. 1982, 60, 1106.
(14) J ohnson, R.; Reese, C. B.; Pei-Zhuo, Z. Tetrahedron 1995, 51,
5093.
(15) Dantzman, C. L.; Kiessling, L. L. J . Am. Chem. Soc. 1996, 118,
11715.
S0022-3263(97)00096-0 CCC: $14.00 © 1997 American Chemical Society

3416 J . Org. Chem., Vol. 62, No. 10, 1997
Notes
Sch em e 1a
a
Conditions: (i) Ac2O/pyridine; (ii) NEt3/PCl3/1,2,4-triazole/
CH3CN; (iii) CH2Cl2/NH3; (iv) MeOH/NH3; (v) Bz2O/DMF/pyridine;
(vi) DMTrCl/DMAP/pyridine; (vii) NCCH2CH2OP(Cl)Ni-Pr2/1-Me-
imidazole/i-Pr2NEt/CH2Cl2.
capto group as a disulfide17 or triphenylmethyl (trityl)
ether18 has proven suitable in the past; both are stable
under basic conditions but can be removed under mild
conditions. For example, 2′-deoxy-2′-(tritylthio)uridine
(2a ) is stable under strongly basic or mildly acidic
conditions, but the trityl protecting group can be readily
removed by treatment with silver nitrate.9d
F igu r e 1. Reverse phase HPLC data for the dinucleotides
dC2′SXpG. HPLC-purified trityl-protected dimer 7a (A) was
treated with silver nitrate plus DTT or 2,2′-dipyridine disulfide
to yield the 2′-mercapto derivative 10a (B) or the 2′-(2-
pyridyldithio) derivative 9a (C), respectively. An HPLC, puri-
fied sample of 9a was treated with DTT and gave the
chromatogram shown in D.
Phosphoramidites for the incorporation of 2′-deoxy-2′-
mercaptocytidine and 2′-deoxy-2′-mercaptouridine into
oligonucleotides were prepared from the corresponding
2′-deoxy- 2′-(tritylthio)nucleosides (Scheme 1). 2′-Deoxy-
2′-(tritylthio)cytidine (6a ) was synthesized directly from
2′-deoxy-2′-(tritylthio)uridine (2a ), analogous to previous
work with 2′-deoxy-2′-(9-phenylxanthen-9-ylthio)uridine.9d
The uridine derivative 2a was first reacted with acetic
anhydride to protect the 3′- and 5′-hydroxyl groups and
then with the highly labile intermediate tris(1,2,4-triazol-
1-yl)phosphine19 to afford the 4-triazolyl pyrimidine
derivative 3. Nucleoside 3 was converted to the corre-
sponding cytidine derivative by treatment with ammonia-
saturated CH2Cl2. Subsequent treatment with half-
saturated methanolic ammonia removed the acetyl
protecting groups. This four-step sequence gave the
cytosine nucleoside 6a in 66% overall yield. The 4-amino
and 5′-hydroxy groups were protected as the benzoyl
amide20 and the dimethoxytrityl ether,13b respectively, to
afford 6c in 65% yield. Phosphitylation of the 3′-hydroxyl
of 6c with â-cyanoethyl N,N-diisopropylchlorophosphor-
amidite13a produced phosphoramidite 5 which was char-
acterized by HRMS and 31P NMR. Analogous 5′ protec-
tion and 3′ activation reactions with starting material
2a gave the 2′-deoxy-2′-(tritylthio)uridine phosphoramid-
ite 4 in 92% yield over the two steps.
To demonstrate the suitability of these phosphoramid-
ites as building blocks for the incorporation of 2′-mer-
captopyrimidines into oligonucleotides, we synthesized
the dinucleotide 2′-deoxy- 2′-(tritylthio)cytidylyl (3′f5′)
2′-deoxyguanosine (5′dC2′STrpG3′, 7a ) and characterized it
by reverse phase HPLC (Figure 1A) and mass spectrom-
etry (Figure 2A).21 The trityl-protected dinucleotide 7a
was deprotected by treatment with aqueous silver nitrate
followed by addition of dithiothreitol (DTT) which pre-
cipitates Ag+ as the salt of DTT (Scheme 2).18b The
resulting species had shorter retention time by HPLC
relative to 7a (Figure 1A and B), and its mass spectrum
(Figure 2B) gave a molecular ion mass consistent with
the desired 2′-mercapto dinucleotide 5′dC2′SHpG3′ (10a ).
The Ag+/dimer complex 8a can also be readily reacted to
a mixed disulfide by disulfide exchange chemistry. Thus,
treatment of the 2′-tritylthio derivative 7a with silver
nitrate and 2,2′-dipyridine disulfide resulted in quantita-
tive conversion to a new product that had interme-
diate mobility on HPLC relative to 7a and the 2′-
mercapto derivative 10a (Figure 1C), and a mass spec-
trum consistent with the 2′-(2-pyridyldithio) derivative
5′dC2′S-SpyrpG3′ (9a ; Figure 2C). Treatment of 9a with
DTT22 quantitatively reduced the disulfide to thiopyri-
done and 10a (Figure 1D). This methodology can also
(16) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 2nd ed.; J ohn Wiley and Sons Inc.: New York, 1992; p 297.
(17) See for example: (a) Goodwin, J . T.; Glick, G. D. Tetrahedron
Lett. 1994, 35, 1647. (b) Sun S.; Tang, X.-Q.; Merchant, A.; Anjaneyulu,
P. S. R.; Piccirilli, J . A. J . Org. Chem. 1996, 61, 5708.
(18) (a) Manoharan, M.; Tivel, K. L.; Ross, B.; Cook, P. D. Gene 1994,
149, 147. (b) Connolly, B. A. Nucleic Acids Res. 1985, 12, 4485.
(19) McGee, D. P. C.; Vaughn-Settle, A.; Vargeese, C.; Zhai, Y. J .
Org. Chem. 1996, 61, 781.
(21) We also synthesized the dimer 2′-deoxy-2′-(tritylthio)uridylyl
(3′f5′) thymidine via solid phase methods using phosphoramidite 4.
Mass spectroscopy gave the expected molecular ion (M- ) 805 m/z).
(22) Cleland, W. W. Biochemistry 1963, 3, 480.
(20) Sasaki, T.; Mizuno, Y. Chem. Pharm. Bull. 1967, 15, 894.

Notes
J . Org. Chem., Vol. 62, No. 10, 1997 3417
F igu r e 2. Mass spectral data of the dinucleotides dC2′SXpG.
Trityl-protected dimer 7a (A), 2′-mercapto 10a (B), and 2′-
disulfide 9a (C) all display appropriate M+ m/z peaks. In B,
the peaks at 551 and 643 arise from the matrix used for
analysis.
F igu r e 3. Analysis of the stability of 18mer oligonucleotides
via electrophoresis. Input (Inp) oligonucleotides 11 (lane 4),
10c (lane 7), 9c (lane 9), and 7c (lane 11) were incubated for
7 days at 25 °C, pH 7.5 (lanes 5, 8, 10, and 12, respectively).
S1 nuclease digestions of 11 and 10c are shown in lanes 3
and 6, respectively. Products from S1 nuclease-catalyzed
cleavage to the 3′ side of C2′X in 11 (X ) OH) and 10c (X )
SH) show identical mobilities as expected and are labeled at
the left of the gel. The identity of the fragmentation product
from 10c (lane 8) was established as p-dGTCGTCGCCp*Co
by comparison to an independently synthesized standard (lane
2). Both the 10c-derived fragment and the independently
synthesized oligonucleotide have identical electrophoretic
mobilities and S1 nuclease digestion patterns (data not shown).
In lane 6 the band that comigrates with p-dGTCGTCGCCp*Co
(lane 2) may be due to decomposition of 10c (lane 8) and not
S1 nuclease activity.
Sch em e 2
ample, when 3′-radiolabeled oligonucleotide 7c was treated
with silver nitrate followed by DTT, a significant portion
of radioactivity remained in the precipitate and could not
be recovered by washing with 0.1 M triethylammonium
acetate (TEAAC). We found it more convenient to
convert the 2′-tritylthio oligonucleotide directly to the 2′-
(2-pyridyldithio) derivative by treatment with silver
nitrate and 2,2′-dipyridine disulfide followed by HPLC
purification, thereby circumventing the precipitation
step. The oligonucleotide could then be stored as the 2′-
disulfide and deprotected with DTT22 or tris(carboxyeth-
yl)phosphine (TCEP)23 to generate the 2′-mercapto de-
rivative just prior to use. Figure 3 shows the electro-
phoretic mobilities and stabilities of 7c (X ) STr), 9c (X
) S-Spyr), and 10c (X ) SH; 5′dGGGAACGTC2′X
-
GTCGTCGCCp*Co3′)24 relative to the corresponding oli-
gonucleotide containing ribocytidine at the internal
position, 5′dGGGAACGTC2′OHGTCGTCGCCp*Co3′ (11).
The relative mobilities of 7c, 9c, and 10c parallel the
relative HPLC retention times for the corresponding
dinucleotide derivatives (7a , 9a , and 10a ). Thus, the
be applied to longer oligonucleotides as described in the
Experimental Section.
We next examined the feasibility of 2′-mercaptocytidyl
oligonucleotides for biochemical investigations by incor-
poration of phosphoramidite 5 into an internal position
of an 18mer oligonucleotide (7b). Although the Ag+/DTT
deprotection scheme described above could be used to
obtain 2′-mercaptocytidyl oligonucleotides, precipitation
of the Ag+/DTT complex always resulted in significant
loss of oligonucleotide from the supernatant. For ex-
(23) Burns, J . A.; Butler, J . C.; Moran, J .; Whitesides, G. M. J . Org.
Chem. 1991, 56, 2648.
(24) p* indicates the presence of a radioactive isotope (32P) in the
phosphate moiety; Co ) 3′-deoxyadenosine (cordycepin).

3418 J . Org. Chem., Vol. 62, No. 10, 1997
Notes
(Strem) were prepared fresh daily and stored in the dark.
Solutions of DTT and 2,2′-dipyridine disulfide were stored at
-20 °C and discarded after two months of use. Merck silica gel,
8983 grade, 230-400 mesh, 60 Å (Aldrich) was used for column
chromatography. Silica gel on glass with fluorescent indicator
(Sigma) was used for TLC. Reverse phase HPLC was performed
using a Beckman Ultrasphere ODS C18 column (10 × 250 mm).
HPLC solvent gradients were performed with 0.1 M TEAAC (pH
6.5; solvent A) and CH3CN (solvent B). Two gradient programs
were used: for dinucleotides 7a -10a , 0-12% B from 0-24 min
and 12-50% B from 24-42 min, and for oligonucleotides 7b-
10b, 100% A from 0-5 min, 0-12% B from 5-20 min, and 12-
30% B from 20-35 min. 1H, 31P, and 13C spectra were obtained
trityl derivative 7c has reduced electrophoretic mobility
(Figure 3, lane 11) compared to the 2′-mercapto deriva-
tive 10c (lane 7) which has similar mobility to its
2′-hydroxyl counterpart 11 (lane 4). The disulfide deriva-
tive 9c has intermediate mobility (lane 9).
Early work reported that 2′-mercaptocytidine is quite
labile (t1/2 ) 29.5 min at pH 7.0, 37 °C) due to intramo-
lecular SN attack by sulfur on the adjacent 1′-carbon of
the ribose, displacing the heterocyclic base.9c However,
a more recent investigation reported that 2′-mercapto-
cytidine does not undergo glycosidic cleavage very readily,
even at high pH.14 The stability of 2′-mercaptocytidine
as part of an internucleotide linkage has not yet been
investigated. However, 2′-mercaptouridylyl (3′f5′) uri-
dine and 2′-mercaptouridine 3′-(p-nitrophenyl)phosphate
undergo fragmentation at high pH to give uracil and
uridine 5′-phosphate10 or p-nitrophenyl phosphate,15
respectively. The latter products presumably result from
further decomposition of the initial abasic product that
results from glycosidic bond cleavage via the intramo-
lecular SN reaction. Incubation of the 2′-mercapto de-
on
a
GE 500 MHz NMR spectrometer. 31P spectra were
referenced to aqueous phosphoric acid, and 1H and 13C spectra
were referenced to TMS, CH2Cl2, or DMSO. Mass spectral data
were obtained from the Mass Spectral Analysis Lab at the
University of California, Riverside, on a VG ZAB high resolution
mass spectrometer. Elemental analyses were obtained from
Atlantic Microlabs Inc. Polyacrylamide gel electrophoresis
(PAGE) was performed using 20% polyacrylamide (Fisher;
acrylamide:bis-acrylamide, 29:1) with 7 M urea. Gel loading
buffer contained 8 M urea (Fisher), 50 mM EDTA (pH 8.0,
Fisher), 0.02% bromophenol blue (EM Science), and 0.02% xylene
cyanol FF (Kodak).
o
rivative 10c at pH 7.5 and 25 C for seven days (in the
1-[3′,5′-Di-O-a cet yl-2′-d eoxy-2′-(t r it ylt h io)-â-D-r ib ofu r -
a n osyl]-4-(1,2,4-tr ia zol-1-yl)p yr im id in -2-(1H)-on e (3). The
uridine nucleoside 2a 9d (2.7 g, 5.4 mmol) was dried by azeotropic
distillation with pyridine (3 × 5 mL) and redissolved in
anhydrous pyridine (10 mL). Acetic anhydride (2.0 mL, 21.5
mmol) was added, and the solution was stirred for 4 h under
Ar. The reaction mixture was concentrated and purified by
column chromatography (1-3% MeOH in CHCl3) to afford a
white foam, presumably 3′,5′-di-O-acetyl-2′-deoxy-2′-(tritylthio)-
uridine, 2b (3.06 g, 97%), which was used directly in the next
reaction. 2b (2.7 g, 4.6 mmol) was dissolved in anhydrous CH3-
CN (10 mL) under Ar. In a separate flask, triazole (2.6 g, 37.6
mmol), NEt3 (5 mL), and PCl3 (1.0 mL, 11.5 mmol) were added
to anhydrous CH3CN (10 mL) and stirred under Ar. Within 5
min a white precipitate formed. This suspension was filtered
directly into the solution containing 2b, and the remaining solid
was washed with CH3CN (5 mL). After the reaction was stirred
under Ar for 12 h, NEt3 (18 mL), water (3 mL), and saturated
aqueous NaHCO3 (100 mL) were added, and the mixture was
extracted with CHCl3 (2 × 100 mL). The organic phases were
combined, dried with MgSO4, and evaporated to yield 3 (2.49 g,
85%) as a white foam. The compound was analyzed spectro-
scopically and used in the next reaction without further purifica-
tion. 1H NMR (CDCl3) δ: 9.36 (s, 1H), 8.21 (s, 1H), 7.76 (d, J )
7.4, 1H), 7.37 (m, 6H), 7.17 (m, 9H), 7.04 (d, J ) 7.4, 1H), 6.49
(d, J ) 10.1, 1H), 4.15 (dd, J ) 11.9, 1H), 4.08 (s, 1H), 3.96 (dd,
J ) 11.9, 1H), 3.60 (d, J ) 5.0, 1H), 3.02 (dd, J ) 5.0, 10.1, 1H),
2.13 (s, 3H), 1.92 (s, 3H). 13C NMR (CDCl3) δ: 167.0, 169.7,
159.2, 154.5, 154.3, 146.1, 143.7, 143.4, 129.0, 128.2, 127.2, 95.8,
87.7, 82.4, 73.8, 67.8, 63.7, 51.0, 20.8, 20.6. HRMS (MNa+) calcd
for C34H31N5O7S1Na1 660.1893, found 660.1905.
2′-Deoxy-2′-(tr itylth io)cytid in e (6a ). The triazole deriva-
tive 3 (200 mg, 0.3 mmol) was dissolved in ammonia saturated
CH2Cl2 (5 mL) and stirred for 36 h. The solution was evapo-
rated, and the residue was redissolved in half-saturated metha-
nolic ammonia (5 mL). After 48 h the solution was evaporated
to dryness, and the residue was recrystallized from methanol
to afford compound 6a (126 mg, 80%) as white crystals. 1H NMR
(DMSO-d6) δ: 7.32 (d, 1H), 7.27 (m, 7H), 7.17 (m, 9H), 6.07 (d,
J ) 10.1, 1H), 5.61 (d, 1H), 4.98 (d, J ) 4.3, 1H), 4.72 (t, J )
4.3, 1H), 3.67 (s, 1H), 3.18 (m, 2H), 2.79 (dd, J ) 3.7, 10.1, 1H),
2.71 (m, J ) 3.7, 4.3, 1H). 13C NMR (DMSO-d6) δ: 165.7, 155.7,
144.6, 141.2, 129.3, 128.2, 126.9, 95.4, 86.6, 96.1, 71.7, 67.1, 62.1,
53.0. Anal. Calcd for C28H27N3O4S1: C, 67.0; H, 5.4; N, 8.4; S,
6.3. Found: C, 66.9; H, 5.5; N, 8.4; S, 6.3. HRMS (MNa+) calcd
524.1620, found 524.1639.
presence of TCEP to prevent oxidation) resulted in
similar fragmentation to give the product 5′p-dGT-
CCGTCGCCp*Co3′ (Figure 3, lane 8); the calculated half-
life was 3000 h.25 In contrast, the blocked mercapto
derivatives 7c and 9c and the 2′-hydroxyl derivative 11
showed no tendency to fragment (Figure 3, lanes 12, 10,
and 5, respectively) under similar conditions and times.26
These observations are consistent with a mechanism of
decomposition that involves nucleophilic attack by the
2′-sulfur.
In conclusion, we have demonstrated the incorporation
of 2′-deoxy-2′-mercaptocytidine into DNA oligonucleotides
via the phosphoramidite approach. Protection of the
sulfur as a trityl ether allows solid phase synthesis using
standard reagents and protocols and requires no special
precautions. Following oligonucleotide synthesis and
deprotection, the tritylthio moiety is readily cleaved with
silver nitrate followed by DTT, or is converted to the 2′-
(2-pyridyldithio) derivative via treatment with silver
nitrate and 2,2′-dipyridine disulfide. Synthesis of RNA
oligonucleotides containing the modified nucleoside is
also feasible (data not shown). Under mildly reducing
conditions, oligonucleotides containing 2′-mercaptocyti-
dine are stable at physiological pH on the time scale of
most biochemical experiments and are therefore suitable
as probes to explore the biological structure and function
of nucleic acids.
Exp er im en ta l Section
Gen er a l. All reactions were preformed at rt unless otherwise
noted. All reagents and anhydrous solvents were from Aldrich
and all drying reagents and other solvents were from Fisher
unless otherwise noted. All nucleotide modifying enzymes were
from U.S. Biochemicals. [R-32P]-3′-Deoxyadenosine triphosphate
([R-32P]cordycepin triphosphate or [R-32P]CoTP) was obtained
from New England Nuclear. Solutions of AgNO3 and TCEP
(25) The half-life (t1/2) was determined as follows: Aliquots (1 µL)
were removed from the reaction at 0, 3, 10, 26, 63, 109, and 168 h,
quenched with gel loading buffer (3 µL), and stored at -20 °C before
PAGE. The degree of decomposition was visualized and quantitated
with a Molecular Dynamics Phosphorimager. The data were plotted
as log [St/So] vs time, where [St/So] is the fraction of 10c remaining at
time t. The observed rate constant for decomposition (kobs) was
determined from the slope (m) of a linear fit to the data as m ) -k/
4-N-Ben zoyl-5′-O-(d im et h oxyt r it yl)-2′-d eoxy-2′-(t r it yl-
t h io)cyt id in e (6c). The cytidine derivative 6a (675 mg, 1.4
mmol) was coevaporated with pyridine (3 × 10 mL) and
redissolved in a mixture of anhydrous DMF (4.5 mL) and
anhydrous pyridine (10.5 mL). Benzoic anhydride (379 mg, 1.7
mmol) was added under Ar, and the solution stirred at 100 °C
for 4 h. After evaporation of the solvent, the residue was
2.303. The half life was calculated as t1/2 ) 0.693/kobs
.
(26) TCEP was absent from incubations containing 9c.

Notes
J . Org. Chem., Vol. 62, No. 10, 1997 3419
redissolved in CHCl3 (40 mL) and extracted first with saturated
aqueous NaHCO3 (2 × 20 mL) and then with water (20 mL).
The organic layer was dried with MgSO4 and concentrated to
afford a white foam, presumably 4-N-benzoyl-2′-deoxy-2′-(tri-
tylthio)cytidine (6b) (630 mg, 77%), which was converted without
further purification to the dimethoxytrityl derivative as follows.
6b (353 mg, 0.6 mmol) and DMAP (36 mg, 0.3 mmol) were
coevaporated with pyridine (3 × 5 mL) and redissolved in
anhydrous pyridine (10 mL). Dimethoxytrityl chloride (396 mg,
1.2 mmol) was added, and the reaction stirred for 30 h under
Ar. The solution was concentrated and purified by column
chromatography (0-2% MeOH in CHCl3) to yield 6c (452 mg,
85% yield) as a yellow foam. 1H NMR (CDCl3) δ: 8.85 (s, 1H),
8.21 (m, 1H), 7.99 (m, 2H), 7.64 (t, J ) 7.3, 1H), 7.55 (t, J ) 7.3,
2H), 7.40 (m, 6H), 7.20 (m, 20H), 6.67 (dd, 4H), 6.47 (d, 1H),
3.99 (s, 1H), 3.78 (s, 6H), 3.41 (dd, J ) 4.4, 1H), 3.24 (m, 2H),
2.51 (d, J ) 4.4, 1H). 13C NMR (CDCl3) δ: 158.7, 158.7, 143.9,
143.1, 135.4, 135.1, 133.3, 130.0, 129.9, 129.2, 128.6, 128.2, 127.9,
127.1, 113.2, 87.0, 86.3, 85.4, 70.7, 67.2, 63.9, 56.8, 56.8, 55.3,
55.3. HRMS (MNa+) calcd for C56H49N3O7S1Na1 930.3189, found
930.3176.
4-N-Ben zoyl-5′-O-(d im et h oxyt r it yl)-2′-d eoxy-2′-(t r it yl-
th io)cytid in -3′-yl â-Cya n oeth yl N,N-Diisop r op ylp h osp h or -
a m id ite (5). Fully protected nucleoside 6c (300 mg, 0.3 mmol)
was coevaporated with pyridine (3 × 8 mL) and redissolved in
CH2Cl2 (10 mL) that had been freshly distilled over CaH2.
Diisopropylethylamine (115 µL, 0.7 mmol), â-cyanoethyl N,N-
diisopropylchlorophosphoramidite (150 µL, 0.7 mmol) and re-
distilled 1-methylimidazole (13 µL, 0.2 mmol) were then added,
and the reaction was stirred for 2 h under Ar. The solution was
concentrated and purified by column chromatography (0.2%
NEt3 and 2% acetone in CH2Cl2; both the acetone and CH2Cl2
were filtered through basic alumina before mixing) to afford 5
(335 mg, 92%) as a white foam. 31P NMR (CDCl3) δ: 153.1,
150.0. HRMS (MNa+) calcd for C65H66N5O8S1P1Na1 1130.4267,
found 1130.4333.
5′-O-(Dim eth oxytr ityl)-2′-d eoxy-2′-(tr itylth io)u r id in e (1).
Nucleoside 2a 9d (1.47 g, 2.9 mmol) and DMAP (178 mg, 1.6
mmol) were coevaporated with pyridine (3 × 15 mL) and
redissolved in anhydrous pyridine (15 mL). Dimethoxytrityl
chloride (2.03 g, 6.0 mmol) was added, and the reaction was
stirred for 4 h under Ar. The reaction mixture was concentrated
and purified by column chromatography (CHCl3) to afford 1 (2.25
g, 96%) as a yellow foam. 1H NMR (CDCl3) δ: 7.92 (d, J ) 8.1,
1H), 7.48 (d, 6H), 7.27 (m, 9H), 7.14 (m, 6H), 7.05 (dd, J ) 3.9,
8.9, 4H), 6.68 (d, J ) 8.9, 2H), 6.63 (d, J ) 8.9, 2H), 6.25 (d, J
) 9.7, 1H), 6.32 (d, J ) 8.1, 1H), 3.97 (s, 1H), 3.78 (s, 3H), 3.77
(s, 3H), 3.43 (dd, J ) 4.7, 9.7, 1H), 3.18 (d, J ) 10.9, 1H), 3.10
(d, J ) 10.9, 1H), 2.52 (d, J ) 4.7, 1H). 13C NMR (CDCl3) δ:
163.0, 158.7, 158.6, 150.5, 143.8, 143.7, 140.9, 135.2, 134.8, 130.0,
129.8, 129.0, 128.6, 128.0, 127.8, 127.2, 127.1, 113.1, 102.9, 87.1,
85.8, 85.2, 70.6, 67.3, 63.9, 55.7, 55.3. HRMS (MNa+) calcd for
THF solution (0.5 mL), gently shaken in the dark for 24 h,
neutralized with 0.1 M TEAAC (pH 6.9), and purified by elution
through a NAP-10 gel-filtration column (Pharmacia Biotech).
Deprotected oligonucleotides were purified by reverse phase
HPLC and stored at -20 °C in unbuffered water. Overall yields
of oligonucleotides containing the 2′-modification were compa-
rable to those for oligonucleotides lacking the modification.
Gen er a l P r oced u r e for Dep r otection to 2′-Deoxy-2′-
m er captocytidyl Oligon u cleotides (10). (a) Fr om 2′-Deoxy-
2′-(tr itylth io)cytid yl Oligon u cleotid es. Dinucleotide 7a or
oligonucleotide 7b (5 nmol) was treated with a solution of AgNO3
(150 nmol) in aqueous 0.1 M TEAAC (pH 6.5; 48 µL) for 30 min.
Aqueous DTT (0.1 M; 2 µL) was added, and the mixture was
incubated for 5 min before centrifugation for 5 min. The
supernatant was removed from the Ag+/DTT precipitate and
analyzed by HPLC. Conversion appeared quantitative by UV/
HPLC. Dinucleotide 10a had a retention time of 22 min (Figure
1B).
(b) F r om 2′-Deoxy-2′-(2-p yr id yld ith io)cytid yl Oligon u -
cleotid es. Dinucleotide 9a or oligonucleotide 9b (5 nmol) was
treated for 15 min with DTT (200 nmol) in aqueous 50 mM Tris-
HCl (pH 8.0; 50 µL). The solution was centrifuged for 5 min
and analyzed by HPLC. Conversion appeared quantitative by
UV/HPLC. Thiopyridone and dinucleotide 10a had retention
times of 13 min and 22 min, respectively (Figure 1D).
Gen er a l P r oced u r e for P r ep a r a tion of 2′-Deoxy-2′-(2-
p yr id yld ith io)cytid yl Oligon u cleotid es (9). (a ) F r om 2′-
Deoxy-2′-(tr itylth io)cytid yl Oligon u cleotid es. Dinucleotide
7a or oligonucleotide 7b (5 nmol) was treated for 30 min with
AgNO3 (150 nmol) in unbuffered water (43 µL). Treatment of
this mixture with methanolic 2,2′-dipyridine disulfide (0.1 M; 2
µL) and aqueous 0.5 M Tris-HCl (pH 8.0; 5 µL) resulted in the
formation of a yellow precipitate. After incubation for 1 h, the
suspension was centrifuged for 5 min, and the supernatant was
analyzed by HPLC. Conversion appeared quantitative by UV/
HPLC. Dinucleotide 9a had a retention time of 27 min (Figure
1C).
(b) F r om 2′-Deoxy-2′-m er ca p tocytid yl Oligon u cleotid es.
Dinucleotide 10a (<1 nmol), methanolic 2,2′-dipyridine disulfide
(0.1 M; 2 µL), aqueous 0.5 M Tris-HCl (pH 8.0; 5 µL), and
unbuffered water were combined to a final volume of 50 µL. After
incubation for 1 h, the solution was centrifuged for 5 min and
analyzed by HPLC. Conversion appeared quantitative by UV/
HPLC.
Ra d iola belin g of Oligon u cleotid es. Oligonucleotides were
3′-end labeled with [R-32P]CoTP and terminal deoxynucleotidyl
transferase (TDT). Reactions included [R-32P]CoTP (30 µCi),
TDT (17 units), oligonucleotide (20 pmol), and cobalt(II) chloride
(20 nmol) in a 10 µL volume. After 1 h incubation at 37 °C, the
reactions were quenched with gel loading buffer (8 µL), and the
components were separated by PAGE. Products were visualized
by autoradiography, excised from the gel, and eluted at 4 °C
with water. The eluent was applied to a Sep-Pac C18 cartridge
(Waters) which was then washed with water (2 mL), followed
by 50% aqueous CH3CN (2 mL). Fractions containing oligo-
nucleotide were evaporated to dryness and brought up in water
to a concentration of 20 nM as determined by specific radioactiv-
ity.
To obtain p-dGTCGTCGCCp*Co (Figure 3, lane 2) as a
marker for the fragmentation product from 10c, dGTCGTCGCC
was 3′ end labeled as described above (lane 1) and then
5′-phosphorylated with T4 Polynucleotide Kinase (PNK) and
adenosine triphosphate (ATP) by addition of the following
ingredients directly to the TDT reaction: 0.5 M Tris-HCl (pH
7.5, 1.5 µL), 0.1 M MgCl2 (1.5 µL), 50 mM ATP (1.7 µL), and 30
units/µL PNK (0.3 µL). The reaction mixture was incubated at
37 °C for 30 min before quenching with gel loading buffer. The
product was purified by PAGE/Sep-Pac C18 as described above.
C
49H44N2O7S1Na1 827.2767, found 827.2746.
5′-O-(Dim e t h oxyt r it yl)-2′-d e oxy-2′-(t r it ylt h io)u r id in -
3′-yl â-Cya n oeth yl N,N-Diisop r op ylp h osp h or a m id ite (4).
Protected nucleoside 1 (100 mg, 0.1 mmol) was coevaporated
with pyridine (3 × 3 mL) and redissolved in CH2Cl2 (5 mL) that
had been freshly distilled over CaH2. Diisopropylethylamine (53
µL, 0.3 mmol), â-cyanoethyl N,N-diisopropylchlorophosphor-
amidite (100 µL, 0.5 mmol), and redistilled 1-methylimidazole
(6 µL, 0.1 mmol) were then added, and the reaction was stirred
for 6 h under Ar. The solution was concentrated and purified
by column chromatography (1% NEt3 in CH2Cl2 that had been
filtered through basic alumina) to afford 4 (125 mg, 96%) as a
yellow foam. 31P NMR (CDCl3) δ: 152.6, 149.6. HRMS (MNa+)
calcd for C58H61N4O8P1S1Na1 1027.3845, found 1027.3852.
In cor p or a tion of 2′-Deoxy-2′-(tr itylth io)cytid in e in to
Oligon u cleotid es by Solid P h a se Syn th esis (7). Oligonucle-
otides were synthesized on
a 1 µmol scale with standard
Digestion of Oligon u cleotid es w ith S1 Nu clea se. Ra-
diolabeled oligonucleotide 9c or 11 (20 fmol) was incubated for
5 min with S1 nuclease (25 units) in aqueous buffer (10 µL)
containing 50 mM sodium acetate (pH 5.2), 1 mM ZnCl2, 0.25
M NaCl, and 1 mM TCEP. The reaction was stopped by the
addition of gel loading buffer (10 µL) followed by cooling on dry
ice.
phosphoramidites (Perseptive Biosystems) using a Millipore
Expidite Nucleic Acid Synthesis System and standard DNA and
RNA protocols. Phosphoramidite 5 was coupled according to the
standard RNA synthesis protocol and gave yields comparable
to those of the standard phosphoramidites. Synthesized oligo-
nucleotides were deprotected at 55 °C for 14-18
h with
concentrated aqueous NH3 and evaporated to dryness. Oligo-
nucleotides that contained ribonucleotides were desilylated by
treatment with 1.0 M tetrabutylammonium fluoride (TBAF)/
Sta bility of Oligon u cleotid es. Radiolabeled oligonucle-
otides 7c, 9c, 10c, and 11 (20 fmol) were incubated for 7 days

3420 J . Org. Chem., Vol. 62, No. 10, 1997
Notes
at 25 °C in aqueous 50 mM Tris-HCl (pH 7.5, 10 µL). The 2′-
mercapto derivative 10c was obtained by reduction of the 2′-(2-
pyridyldithio) derivative 9c with TCEP. For consistency, all
reaction mixtures except 9c initially contained 1mM TCEP.
Mildly reducing conditions were maintained throughout the 7
day incubation by adding fresh TCEP (0.1 µL of a solution
containing 0.1 M TCEP and 50 mM Tris, pH 7.5) after 2, 4, and
6 days. After 7 days, the reaction mixtures were diluted with
gel loading buffer (10 µL) and analyzed by PAGE (Figure 3).
reading of the manuscript. M.L.H. is supported by a
G.A.A.N.N. fellowship, and J .A.P. is an Assistant In-
vestigator of the Howard Hughes Medical Institute.
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H, 13C,
and 32P NMR spectra (10 pages). This material is contained
in libraries on microfiche, immediately follows this article in
the microfilm version of the journal, and can be ordered from
the ACS; see any current masthead page for ordering
information.
Ack n ow led gm en t. We thank Larissa Munishkina
for assistance with oligonucleotide synthesis. We also
thank Drs. Erik Sontheimer and Sengen Sun for critical
J O970096O