Independent generation and characterization of a C2′-oxidized abasic site in chemically synthesized oligonucleotides

Article abstract of DOI:10.1021/jo049033d

Abasic lesions, which are formed endogenously and as a consequence of exogenous agents, are lethal and mutagenic. Hydrogen atom abstraction from C2′ in DNA under aerobic conditions produces an oxidized abasic lesion (C2-AP), along with other forms of DNA

Full text of DOI:10.1021/jo049033d

In d ep en d en t Gen er a tion a n d Ch a r a cter iza tion of a C2′-Oxid ized
Aba sic Site in Ch em ica lly Syn th esized Oligon u cleotid es
J aeseung Kim, Yvonne N. Weledji, and Marc M. Greenberg*
Department of Chemistry, J ohns Hopkins University, 3400 N. Charles St., Baltimore, Maryland 21218
mgreenberg@jhu.edu
Received J une 8, 2004
Abasic lesions, which are formed endogenously and as a consequence of exogenous agents, are lethal
and mutagenic. Hydrogen atom abstraction from C2′ in DNA under aerobic conditions produces
an oxidized abasic lesion (C2-AP), along with other forms of DNA damage. The effects of C2-AP on
DNA structure and function are not well understood. A method for the solid-phase synthesis of
oligonucleotides containing C2-AP lesions is reported. The lesion is released via periodate oxidation
of a triol containing a vicinal diol. The triol is introduced via a phosphoramidite that is compatible
with standard oligonucleotide synthesis and deprotection conditions. UV-melting studies indicate
that the C2-AP lesion has a comparable effect on the thermal stability of duplex DNA as other
abasic lesions. The C2-AP lesion is rapidly cleaved by piperidine at 90 °C. However, cleavage by
NaOH (0.1 M, 37 °C) shows that C2-AP is considerably less labile (t1/2 ) 3.3 ( 0.2 h) than other
abasic lesions.
SCHEME 1
DNA damage is involved in aging and a variety of
diseases, including cancer.1-4 Chemistry plays an impor-
tant role in identifying and characterizing endogenously
and/or exogenously produced lesions, which induce sig-
nificant biological effects. Abasic sites, which as their
name suggests are missing their nucleobases, are
an important family of lesions. It is estimated that
∼10,000 AP sites are produced per cell each day.5,6
Oxidized AP sites (e.g., C4-AP, L) are also produced in
varying amounts depending upon the damaging agent.7-10
of oxidized abasic lesions on DNA replication and repair
are distinct from those of AP lesions and each other.11-13
The oxidized abasic site resulting from formal hydrogen
atom abstraction from the C2′-nucleotide position (C2-
AP) is observed during γ-irradiation of DNA and pho-
tolysis of DNA containing 5-halopyrimidines (Scheme
1).10,14-18 Very little is known about the effects of this
lesion on DNA structure and function. We describe herein
the synthesis and characterization of oligonucleotides
containing C2-AP, which will facilitate evaluation of the
lesion’s biochemical and biological effects.
Recent in vitro and in vivo studies reveal that the effects
Abasic lesions have often been referred to as nonin-
structive lesions because they cannot form Watson-Crick
* To whom correspondence should be addressed. Phone: 410-516-
8095. Fax: 410-516-7044.
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Published on Web 08/12/2004
6100
J . Org. Chem. 2004, 69, 6100-6104

Independent Generation of C2′-Oxidized Abasic Sites
SCHEME 2
hydrogen bonds with nucleotides. However, studies on
2-deoxyribonolactone (L) have shown that the oxidized
abasic site and AP interact differently with individual
polymerases involved in replication in E. coli.11 Replica-
tion of L does not follow the “A-rule”, an empirical rule
developed in response to studies on AP lesions in E. coli.19
Furthermore, the lactone lesion (L) exhibits distinct
behavior in its interactions with some base excision
repair enzymes, which is manifested in the formation of
DNA-protein cross-links.20-22 These experiments suggest
that the term noninstructive should not be used when
describing the biochemistry of abasic lesions. Moreover,
it suggests that detailed studies on other abasic lesions,
such as C4-AP and C2-AP, could provide important
information concerning the biological effects of these
lesions.23
SCHEME 3a
Studies on DNA lesions are greatly facilitated by
synthesis of oligonucleotides containing them at defined
sites.24-26 Synthesis of oligonucleotides containing abasic
sites is more complex because the lesions are alkali-labile.
Successful strategies for the synthesis of L- or C4-AP-
containing oligonucleotides unmask the lesions in a final
photochemical step.27-30 The latter has also been incor-
porated chemoenzymatically in oligonucleotides.31 AP
lesions have been incorporated into oligonucleotides
chemoenzymatically by exploiting the deglycosylation of
2′-deoxyuridine by uracil DNA glycosylase.32 More re-
cently, oligonucleotides containing AP lesions were pre-
pared via mild periodate oxidation in the final step.33
a
(a) (i) aq AcOH (30%), reflux, (ii) NaBH4, MeOH, 0 f 25 °C;
(b) TBSOTf, 2,6-lutidine, CH2Cl2, 0 °C; (c) H2 (50 psi), Pd(OH)2/C,
EtOAc, 25 °C; (d) DMTCl, pyridine, 25 °C; (e) 2-cyanoethyl
diisopropylchlorophosphoramidite (5 equiv), diisopropylethylamine
(8 equiv), CH2Cl2, reflux.
sites.33 The aldehyde is unmasked by mild sodium
periodate oxidation, a common method for introducing
aldehydes in chemically synthesized RNA and DNA.34-37
The diol and hydroxyl group derived from the C4′-position
of the nucleotide are in turn protected as their silyl
ethers, which is a common protecting group for the 2′-
hydroxyl during the chemical synthesis of RNA.38 This
is desirable because the hydroxyl groups remain pro-
tected during the alkaline ammonia treatment, which
prevents cleavage and/or phosphoryl migration due to
intramolecular attack by the hydroxyl group(s) on the
phosphate diesters.
Implementation of this synthetic approach was effected
by the synthesis of phosphoramidite 1 (Scheme 3). This
was accomplished from triol (3), which was obtained in
high yield from 3,5-dibenzyl-1,2-isopropylidene ribofura-
nose (2) using a previously reported procedure for the
C3-epimer of 2.39 The ribofuranose isopropylidene (2) was
previously prepared from the 1,2-isopropylidene of xylose
(7).40 In this project 2 was prepared from commercially
available 7 via the silyl ether (Scheme 4).41 Stereochem-
ical inversion of the C3-hydroxyl was accomplished
Resu lts a n d Discu ssion
The C2-AP has been shown to undergo a retroaldol
reaction under mild alkaline conditions.16 Consequently,
chemical synthesis of oligonucleotides containing this
molecule using standard 5′-dimethoxytrityl, â-cyanoethyl
phosphoramidites required disguising the lesion until
after the protecting groups are removed and the biopoly-
mer is cleaved from its solid-phase synthesis support
(Scheme 2). We chose to disguise C2-AP by introducing
the latent aldehyde as a vicinal diol. This strategy is
analogous to that employed by J ohnson in his recent
chemical synthesis of oligonucleotides containing AP
(19) Strauss, B. S. BioEssays 1991, 13, 79-84.
(20) Hashimoto, M.; Greenberg, M. M.; Kow, Y. W.; Hwang, J .-T.;
Cunningham, R. P. J . Am. Chem. Soc. 2001, 123, 3161-3162.
(21) Kroeger, K. M.; Hashimoto, M.; Kow, Y. W.; Greenberg, M. M.
Biochemistry 2003, 42, 2449-2455.
(22) DeMott, M. S.; Beyret, E.; Wong, D.; Bales, B. C.; Hwang, J .-
T.; Greenberg, M. M.; Demple, B. J . Biol. Chem. 2002, 277, 7637-
7640.
(23) Greenberg, M. M.; Weledji, Y. N.; Kroeger, K. M.; Kim, J .;
Goodman, M. F. Biochemistry 2004, 43, 2656-2663.
(24) Wang, D.; Kreutzer, D. A.; Essigmann, J . M. Mutat. Res. 1998,
400, 99-115.
(25) Kamiya, H. Nucleic Acids Res. 2003, 31, 517-531.
(26) Butenandt, J .; Burgdorf, L. T.; Carell, T. Synthesis 1999, 1085-
1105.
(27) Zheng, Y.; Sheppard, T. L. Chem. Res. Toxicol. 2004, 17, 197-
207.
(28) Kotera, M.; Bourdat, A.-G.; Defrancq, E.; Lhomme, J . J . Am.
Chem. Soc. 1998, 120, 11810-11811.
(29) Kim, J .; Gil, J . M.; Greenberg, M. M. Angew. Chem., Int. Ed.
2003, 42, 5882-5885.
(34) Bellon, L.; Workman, C.; Scherrer, J .; Usman, N.; Wincott, F.
J . Am. Chem. Soc. 1996, 118, 3771-2.
(35) Tilquin, J . M.; Dechamps, M.; Sonveaux, E. Bioconjugate Chem.
2001, 12, 451-457.
(36) Forget, D.; Renaudet, O.; Boturyn, D.; Defrancq, E.; Dumy, P.
Tetrahedron Lett. 2001, 42, 9171-9174.
(37) Forget, D.; Boturyn, D.; Defrancq, E.; Lhomme, J .; Dumy, P.
Chem. Eur. J . 2001, 7, 3976-3984.
(38) Wincott, F.; DiRenzo, A.; Shaffer, C.; Grimm, S.; Tracz, D.;
Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe, S.; Usman, N.
Nucleic Acids Res. 1995, 23, 2677-84.
(30) Goodman, B. K.; Greenberg, M. M. J . Org. Chem. 1996, 61, 2-3.
(31) Chen, J .; Stubbe, J . Biochemistry 2004, 43, 5278-5286.
(32) Shibutani, S.; Takeshita, M.; Grollman, A. P. J . Biol. Chem.
1997, 272, 13916-13922.
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Tetrahedron 1997, 53, 6509-6522.
J . Org. Chem, Vol. 69, No. 18, 2004 6101

Kim et al.
SCHEME 4a
a
(a) TBDPSCl, imidazole, DMF, 25 °C; (b) DMSO, Ac2O, 25 °C;
(c) NaBH4, EtOH/H2O, 25 °C; (d) TBAF, THF, 25 °C; (e) NaH,
BnBr, THF, reflux.
via oxidation to the ketone and selective reduction from
the less hindered â-face. Transformation of 3 into the
desired phosphoramidite (1) was accomplished by a series
of hydroxyl group deprotection and functionalization
steps. The diastereomers of 1 were chromatographically
separable. However, there was no advantage to this (vide
infra), and the phosphoramidite (1) was routinely em-
ployed as a mixture of diastereomers in solid-phase
synthesis.
F IGURE 1. Cleavage of C2-AP (15b) as a function of time
upon treatment with NaOH (0.1 M, 37 °C).
tion). MS analysis of these oligonucleotides showed no
evidence for the triol starting materials, indicating that
the reactions had proceeded to completion.
Oligonucleotides containing up to 30 nucleotides (12-
15) were prepared using 1 and commercially available
The C2-AP lesion had previously been shown to be
alkaline-labile.16 The lesion contains â-hydroxy phosphate
groups as are present in RNA and is also capable of
undergoing a retroaldol reaction. As is the case with
many alkali-labile lesions, 5′-32P-15b is completely cleaved
by 1 M piperidine (90 °C, 20 min). However, C2-AP is
significantly more stable to NaOH than other abasic
lesions. AP, L, or C4-AP are completely cleaved upon
treatment with NaOH (0.1 M, 37 °C, 20 min).7,29,42 C2-
AP is cleaved with first-order kinetics (Figure 1), but the
half-life is considerable (3.3 ( 0.2 h, 5.9 ( 0.2 × 10-5
s-1).
The effect of C2-AP on the thermal stability of duplex
DNA was analyzed by UV-melting. Despite the relative
robust behavior of this lesion toward alkali treatment,
care was taken to prevent adventitious cleavage during
hybridization. Duplex 16 was formed by hybridizing 12b
with its complement at 37 °C for 30 min, followed by
cooling at 25 °C for 2 h.29 The Tm was determined to be
38.6 °C. The Tm for 16 was within 1.5 °C of those
measured for C4-AP and the tetrahydrofuran analogue
of AP in otherwise identical duplexes.29
“fast deprotecting” phosphoramidites (N-acetyl-2′-deoxy-
cytidine, N-phenoxyacetyl-2′-deoxyadenosine, N-isopro-
pylphenoxyacetyl-2′-deoxyguanosine). Standard cou-
pling cycles provided by the manufacturer were employed
except when coupling 1, which required additional ex-
perimentation. Extending the coupling time of 1 to 5 min
resulted in a 50% yield according to trityl cation mea-
surement. Surprisingly, carrying out a double-coupling
of 1 did not improve the yield. Furthermore, solid-phase
synthesis coupling yields were not any higher using
individual diastereomers of 1 epimeric at the phosphorus
center. Despite the lower than typical coupling yield for
phosphoramidite 1, oligonucleotides 12a-15a were readily
purified by denaturing polyacrylamide gel electrophoresis
following a two-step deprotection. Desilylation was car-
ried out using Et3N‚3HF for 3 h following cleavage from
the solid-phase support and deprotection of the phosphate
and exocyclic amine groups using concentrated am-
monium hydroxide.38 The four triol-containing oligonucle-
otides exhibited the expected m/z ratio for the fully
protonated and single sodium adduct molecules (see
Supporting Information). Periodate oxidation was carried
out on 2-6 nmol of oligonucleotides using buffered (0.1
M NaOAc, pH 6.0) sodium periodate (5 M). The reactions
were quenched by desalting using C-18 Sep pak car-
tridges after 30 min. No further purification was neces-
sary, as evidenced by ESI-MS (see Supporting Informa-
Con clu sion
We have developed a method for the chemical synthesis
of oligonucleotides containing C2-AP, an oxidized abasic
lesion. The method is compatible with standard solid-
phase synthesis reagents and as such is not restricted
in terms of the sequences that can be prepared. Prelimi-
nary studies suggest that the C2-AP lesion reduces the
thermal stability of duplex DNA by an amount compa-
rable to reduction by other abasic lesions. DNA contain-
ing C2-AP is considerably more resistant to strand
(42) Roupioz, Y.; Lhomme, J .; Kotera, M. J . Am. Chem. Soc. 2002,
124, 9129-9135.
6102 J . Org. Chem., Vol. 69, No. 18, 2004

Independent Generation of C2′-Oxidized Abasic Sites
rated NaCl. The organic layer was dried over Na2SO4, filtered,
and concentrated on a rotary evaporator. The crude material
was purified by column chromatography (oven-dried silica gel,
20% Et2O in hexanes) to afford a diastereomeric mixture of
phosphoramidite 1 (485 mg, 77.8%). A small amount of sample
was rechromatographed to separate the phosphoramidite
diastereomers for analytical characterization. Lower Rf 0.18,
major isomer, 1:9 EtOAc/hexanes: IR (film) 2930, 2856, 1608,
scission than other abasic lesions, suggesting that it will
be necessary for repair enzymes to excise it.
Exp er im en ta l Section
(2S,3R,4R)-3,5-O-Diben zyloxy-1,2,4-O-tr i-ter t-bu tyld i-
m eth ylsilyloxyp en ta n e (4). TBSOTf (1.95 g, 7.36 mmol) was
added to (2S,3S,4R)-3,5-O-dibenzyloxy-1,2,4-pentanetriol39 (3,
546 mg, 1.64 mmol) and 2,6-lutidine (1.06 g, 9.87 mmol) in
CH2Cl2 (17 mL) at 0 °C. After 1 h at 0 °C, the solution was
poured into cold saturated NH4Cl solution, extracted with
EtOAc, and then washed with brine. The organic layer was
dried with Na2SO4. The solution was filtered and concentrated
on a rotary evaporator. The crude material was purified by
column chromatography (2-5% EtOAc in hexanes) to give 3
(1.08 g, 97.6%): Rf 0.62 (1:9 EtOAc/hexanes); IR (neat) 3032,
2929, 2857, 1472, 1361, 1254, 1095, 836 cm-1; 1H NMR (CDCl3)
δ 7.36-7.33 (m, 10H), 4.80 (dd, J ) 11.2, 19.2 Hz, 2H), 4.54
(d, J ) 1.2 Hz, 2H), 4.22-4.18 (m, 1H), 3.96-3.93 (m, 1H),
3.80-3.69 (m, 4H), 3.60 (dd, J ) 6.4, 10.0 Hz, 1H), 0.96 (s,
9H), 0.95 (s, 9H), 0.94 (s, 9H), 0.14 (s, 3H), 0.12 (s, 6H), 0.11
(s, 3H), 0.08 (s, 6H); 13C NMR (CDCl3) δ 139.1, 138.7, 128.2,
128.1, 127.8, 127.6, 127.4, 127.2, 82.8, 74.5, 74.1, 73.3, 72.7,
72.3, 64.8, 26.0, 25.9, 18.3, 18.2, 18.1, -4.2, -4.3, -4.8, -5.3,
-5.4; HRMS (MALDI-TOF) M + Na+ calcd for C37H66O5NaSi3
697.4110, found 697.4094.
1
1510, 1464, 1363, 1252, 1077, 834 cm-1; H NMR (CDCl3) δ
7.46-7.20 (m, 9H), 6.82 (dd, J ) 1.2, 9.2 Hz, 4H), 4.23-4.19
(m, 1H), 4.02-3.98 (m, 1H), 3.79 (s, 6H), 3.77-3.69 (m, 2H),
3.68-3.49 (m, 5H), 3.28 (dd, J ) 4.4, 10.0 Hz, 1H), 3.16 (dd, J
) 7.2, 10.4 Hz, 1H), 2.88-2.24 (m, 2H), 1.15 (d, J ) 7.2 Hz,
12H), 0.90 (s, 9H), 0.88 (s, 9H), 0.86 (s, 9H), 0.16 (s, 3H), 0.09
(s, 3H), 0.02 (s, 6H), 0.00 (s, 3H), -0.06 (s, 3H); 13C NMR
(CDCl3) δ 158.3, 145.1, 136.4, 130.3, 130.2, 128.4, 127.6, 126.6,
117.6, 112.8, 86.3, 79.8 (d, J ) 12.9 Hz), 73.9 (d, J ) 4.5 Hz),
73.6, 66.2, 65.1, 57.9 (d, J ) 21.2 Hz), 55.1, 43.1 (d, J ) 12.9
Hz), 26.1, 24.8, 24.7, 24.5, 19.9, 18.4, 18.2, 18.1, -4.0, -4.5,
-4.7, -5.1, -5.3; 31P NMR (CDCl3) δ 152.3; HRMS (MALDI-
TOF) M + Na+ calcd for C53H89N2O8NaSi3P 1019.5557, found
1019.5608. Higher Rf 0.23, minor isomer, 1:9 EtOAc/hexanes:
1H NMR (CDCl3) δ 7.47-7.17 (m, 9H), 6.77 (d, J ) 8.8 Hz,
4H), 4.42 (dd, J ) 1.2, 7.2 Hz, 1H), 4.00 (dd, J ) 1.6, 10.8 Hz,
1H), 3.78 (s, 6H), 3.74-3.60 (m, 3H), 3.47-3.24 (m, 5H), 2.98
(dd, J ) 2.0, 10.0 Hz, 1H), 2.54 (t, J ) 6.4 Hz, 2H), 1.08 (d, J
) 6.0 Hz, 6H), 0.97 (s, 9H), 0.93 (d, J ) 6.8 Hz, 6H), 0.87 (s,
9H), 0.86 (s, 9H), 0.20 (s, 3H), 0.17 (s, 3H), 0.01 (s, 9H), -0.13
(s, 3H); 13C NMR (CDCl3) δ 158.2, 145.0, 136.6, 136.3, 130.3,
130.2, 128.4, 127.5, 126.4, 117.4, 112.8, 85.9, 79.3 (d, J ) 9.1
Hz), 75.3, 72.7, 66.4, 65.4, 57.6 (d, J ) 22.8 Hz), 55.1, 42.8 (d,
J ) 12.9 Hz), 26.1, 24.9, 24.8, 24.4, 20.3, 20.2, 18.5, 18.2, -3.9,
-4.1, -4.3, -5.1, -5.3; 31P NMR (CDCl3) δ 151.8.
Oligon u cleotid e Syn th esis, Dep r otection , a n d P u r ifi-
ca tion . Standard cycles (ABI 394) were used for incorporating
native nucleotides, which were introduced using commercially
available fast deprotecting phosphoramidites. The coupling
time allowed for phosphoramidite 1 was increased to 5 min.
Deprotection of the exocyclic amine protecting groups and
â-cyanoethyl groups and cleavage from the solid-phase syn-
thesis support was carried out using concentrated aqueous
NH4OH for 2 h at 55 °C. The TBDMS groups were removed
under conditions reported by Wincott for deprotecting the 2′-
hydroxyl group in chemically synthesized RNA (250 µL of a
solution containing 1.5 mL N-methylpyrrolidinone, 750 µL of
Et3N, and 1.0 mL of Et3N‚3HF; 3 h at 65 °C). Fully deprotected
oligonucleotides containing the triol were purified by 20%
denaturing PAGE.38
Oxid a tive Clea va ge of Oligon u cleotid es u sin g Na IO4.
The oligonucleotides (12-15a , 2-6 nmol, 35-100 µM) were
treated with 50-60 µL of 5 mM NaIO4 in 0.1 M sodium acetate
buffer (pH 6.0) for 30 min at room temperature and then
desalted on a Sep-Pak C18 cartridge (Waters).
Tr ea tm en t of Sin gle Str a n d Oligon u cleotid e Con ta in -
in g C2-AP (15b) w ith Na OH a n d P ip er id in e. A solution
(25 µL) containing 0.1 µM 15b was added to 25 µL of NaOH
(0.2 M), and the reaction incubated at 37 °C for 360 min.
Aliquots (5 µL) were removed at 30, 60, 120, 180, 240, and
360 min. The aliquots were neutralized immediately with 5
µL of HCl (0.1 M), and diluted in 95% formamide loading buffer
containing 10 mM EDTA (20 µL). The kinetic analysis was
carried out using three reactions. The rate constant reported
is the average of three such experiments. The result of one
experiment (three replicates) is shown in Figure 1).
(2S,3R,4R)-3,5-Dih yd r oxy-1,2,4-O-tr i-ter t-bu tyld im eth -
ylsilyloxyp en ta n e (5). 2S,3R,4R-3,5-O-Dibenzyl-1,2,4-O-tri-
tert-butyldimethylsilyloxypentane 4 (741 mg, 1.10 mmol) and
20% Pd(OH)2/C (240 mg) were stirred in EtOAc (25 mL) under
H2 (50 psi) overnight. After the reaction mixture was filtered
through a Celite pad, which was then washed with EtOAc,
the filtrate was concentrated on a rotary evaporator. The crude
material was purified by column chromatography (1-2%
MeOH in CH2Cl2) to give (2S,3R,4R)-3,5-dihydroxy-1,2,4-O-
tri-tert-butyldimethylsilyloxypentane 5 (507 mg, 92.7%): Rf
0.50 (1:50 MeOH/ CH2Cl2); IR (neat) 3452, 2930, 2858, 1463,
1362, 1256, 1083, 836 cm-1; 1H NMR (CDCl3) δ 3.92-3.81 (m,
3H), 3.76-3.62 (m, 4H), 2.98 (brs, 2H), 0.89 (s, 27H), 0.11 (s,
3H), 0.10 (s, 6H), 0.09 (s, 3H), 0.06 (s, 6H); 13C NMR (CDCl3)
δ 76.1, 73.1, 72.1, 64.7, 63.4, 25.9, 25.8, 18.3, 18.1, 18.0, -4.4,
-4.5, -4.8, -4.9, -5.4, -5.5; HRMS (FAB) M + H+ calcd for
C
23H55O5Si3 495.3357, found 495.3361.
(2S,3R,4R)-5-O-DMT-1,2,4-O-tr i-ter t-bu tyld im eth ylsil-
yloxyp en ta n -3-ol (6). 4,4′-Dimethoxytrityl chloride (360 mg,
1.06 mmol) was added to a solution of 5 (438 mg, 0.88 mmol)
in pyridine (4.4 mL) at room temperature. After 6 h, the
reaction mixture was concentrated on a rotary evaporator. The
crude material was dissolved in EtOAc (100 mL) and then
washed sequentially with saturated NaHCO3, H2O, and satu-
rated NaCl. The organic layer was dried over Na2SO4, filtered,
and concentrated on a rotary evaporator. The crude material
was purified by column chromatography (oven-dried silica gel,
5% EtOAc in hexanes) to afford 6 (678 mg, 96.6%): Rf 0.33
(1:9 EtOAc/hexanes); IR (film) 3508, 2929, 2856, 1608, 1509,
1
1471, 1252, 1177, 1081, 833 cm-1; H NMR (CDCl3) δ 7.51-
7.22 (m, 9H), 6.84 (d, J ) 8.8 Hz, 4H), 4.14 (dd, J ) 4.4, 8.8
Hz, 1H), 3.80 (s, 6H), 3.76-3.74 (m, 1H), 3.66 (dd, J ) 5.2,
10.4 Hz, 1H), 3.56-3.52 (m, 1H), 3.29 (d, J ) 4.0 Hz, 2H), 3.14
(d, J ) 4.0 Hz, 1H), 0.97 (s, 9H), 0.92 (s, 9H), 0.84 (s, 9H),
0.16 (s, 3H), 0.12 (s, 3H), 0.07 (s, 6H), 0.03 (s, 3H), -0.14 (s,
3H); 13C NMR (CDCl3) δ 158.4, 144.7, 136.0, 135.9, 130.2,
130.1, 128.3, 127.7, 126.6, 113.0, 86.3, 75.7, 73.9, 71.8, 65.4,
65.2, 55.1, 25.9, 25.8, 18.3, 18.1, 18.0, -4.1, -4.4, -4.8, -5.3,
-5.4; HRMS (MALDI-TOF) M + Na+ calcd for C44H72O7NaSi3
819.4478, found 819.4463.
For treatment with piperidine, 25 µL of 15b (0.1 µM) was
added to 25 µL of piperidine (2 M), and the reaction incubated
at 90 °C for 20 min, at which time the samples were placed in
a dry ice/ethanol cold bath. The samples were then placed in
a Savant speed vacuum at medium heat for 10 min to remove
the piperidine before resuspending in 95% formamide loading
buffer containing 10 mM EDTA (20 µL).
P h osp h or a m id ite 1. 2-Cyanoethyl diisopropylchlorophos-
phoramidite (753 mg, 3.18 mmol) was added to a solution of 6
(500 mg, 0.63 mmol) and DIPEA (649 mg, 5.02 mmol) in CH2-
Cl2 (10 mL). After refluxing for 5 h, the solution was cooled to
room temperature, diluted with CH2Cl2 (90 mL), and then
washed sequentially with saturated NaHCO3, H2O, and satu-
All samples were separated on a 20% denaturing PAGE.
J . Org. Chem, Vol. 69, No. 18, 2004 6103

Kim et al.
MS, UV-melting experiment (16), and NaOH cleavage of 15b;
NMR spectra of 1-6 and 8-11; ESI-MS of 12-15a ,b. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Ack n ow led gm en t. We are grateful to the National
Institute of General Medical Sciences (NIH GM-063028)
for financial support.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for the synthesis of 3, preparation of samples for ESI-
J O049033D
6104 J . Org. Chem., Vol. 69, No. 18, 2004