Efficient synthesis of 2′,3′-dideoxy-2′-amino-3′- thiouridine

Article abstract of DOI:10.1021/ol049366x

Metal ion rescue experiments provide a powerful approach to establish the presence and role of divalent metal ions in the biological function of RNA. The utility of this approach depends on the availability of suitable nucleoside analogues. To expand the

Full text of DOI:10.1021/ol049366x

ORGANIC
LETTERS
2004
Vol. 6, No. 13
2169-2172
Efficient Synthesis of
2′,3′-Dideoxy-2′-amino-3′-thiouridine
Qing Dai† and Joseph A. Piccirilli*
Howard Hughes Medical Institute, Department of Biochemistry & Molecular Biology,
and Department of Chemistry, The UniVersity of Chicago,
5841 South Maryland AVenue, MC 1028, Chicago, Illinois 60637
jpicciri@midway.uchicago.edu
Received April 7, 2004
ABSTRACT
Metal ion rescue experiments provide a powerful approach to establish the presence and role of divalent metal ions in the biological function
of RNA. The utility of this approach depends on the availability of suitable nucleoside analogues. To expand the range of this experimental
strategy, we describe the first synthesis of 2′,3′-dideoxy-2′-amino-3′-thiouridine (12) in 19.5% overall yield starting from 2,2′-anhydrouridine
(1).
RNA molecules adopt complex tertiary architectures that
perform biological functions.1 The negatively charged phos-
phodiester backbone of RNA endows this biopolymer with
unique structural and functional properties and may impose
a strict requirement for divalent metal ions.2 To understand
how RNA molecules perform their biological functions, the
locations and roles of metal ions must be defined. Metal ion
rescue experiments provide a powerful means to identify and
distinguish specific metal ions, assign their ligands, and test
hypotheses regarding the structural and functional roles the
metal ions play.3 Metal ions have varying abilities to
coordinate oxygen, nitrogen, and sulfur, and these coordina-
tion preferences reflect a metal ion’s “hardness” or “soft-
ness”.4 Consequently, atomically perturbed RNAs containing
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Harris, M. E. EMBO J. 2002, 21, 2253-2262. (c) Kuo, L. Y.; Piccirilli, J.
A. Biochim. Biophys. Acta 2001, 1522, 158-166. (d) Shan, S.; Kravchuk,
A. V.; Piccirilli, J. A.; Herschlag, D. Biochemistry 2001, 40, 5161-5171.
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Christian, E. L.; Kaye, N. M.; Harris, M. E. RNA 2000, 6, 511-519. (g)
Gordon, P. M.; Sontheimer, E. J.; Piccirilli, J. A. Biochemistry 2000, 39,
12939-12952. (h) Gordon, P. M.; Sontheimer, E. J.; Piccirilli, J. A. RNA
2000, 6, 199-205. (i) Yoshida, A.; Shan, S.; Herschlag, D.; Piccirilli, J. A.
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884. (l) Warnecke, J. M.; Sontheimer, E. J.; Piccirilli, J. A.; Hartmann, R.
K. Nucleic Acids Res. 2000, 28, 720-727. (m) Shan, S.; Herschlag, D.
Biochemistry 1999, 38, 10958-10975. (n) Yoshida, A.; Sun, S.; Piccirilli,
J. A. Nat. Struct. Biol. 1999, 6, 318-321. (o) Sontheimer, E. J.; Gordon,
P. M.; Piccirilli, J. A. Genes DeV. 1999, 13, 1729-1741. (p) Shan, S.;
Yoshida, A.; Sun, S.; Piccirilli, J. A.; Herschlag, D. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 12299-12304. (q) Brautigam, C. A.; Sun, S.; Piccirilli, J.
A.; Steitz, Thomas A. Biochemistry 1999, 38, 696-704. (r) Weinstein, L.
B., Jones, B. C., Cosstick, R.; Cech, T. R. Nature 1997, 388, 805-808.
(s). Curley, J. F.; Joyce, C. M.; Piccirilli, J. A. J. Am. Chem. Soc. 1997, 51,
12691-12692. (t) Sontheimer, E. J.; Sun, S.; Piccirilli, J. A. Nature 1997,
388, 801-805. (u) Warnecke, J. M.; Furste, J. P., Hardt, W. D.; Erdmann,
V. A.; Hartmann, R. K. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8924-
8928. (v) Piccirilli, J. A., Vyle, J. S.; Caruthers, M. H.; Cech, T. R. Nature
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† Present address: The Ben May Institute for Cancer Research, The
University of Chicago, 5841 S. Maryland Ave. MC 6027, Chicago, IL,
60637.
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10.1021/ol049366x CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/27/2004

sulfur or nitrogen atom substitutions of single oxygen atoms
often exhibit rescue behavior by “soft” metal ions,3 thereby
implicating the substituted oxygen atom as a metal ligand.
However, the small number of nucleoside analogues currently
available5 limits the scope and applicability of these metal
ion rescue experiments.
We previously conducted metal ion rescue experiments
together with quantitative analysis to study the exon-ligation
step of group II intron self-splicing.3g,h,o Using 2′-amino-
uridine and 3′-thiouridine, we obtained evidence that both
the 2′- and 3′-oxygen atoms at the cleavage site (U877 at the
3′-terminus of the ai5γ group II intron) coordinate divalent
metal ions in the transition state3g (Figure 1). However,
(or 3′)-amino7 (or thio8) single-modified uridines and 2′,3′-
doubly modified nucleosides such as 2′,3′-dideoxy-2′,3′-
diamino-,9 2′,3′-dideoxy-2′,3′-dithio-,10 and 2′-deoxy-2′-thio-
3′-deoxy-3′-aminouridines11 have been reported, the 2′,3′-
dideoxy-2′-amino-3′-thio nucleosides remain unknown. Here,
we describe a convenient synthesis of the uridine analogue.
Our synthesis of 2′,3′-dideoxy-2′-amino-3′-thiouridine 12
begins with the commercially available 2,2′-anhydrouridine
1 (Scheme 1). We used the method of McGee et al. to prepare
2′-deoxy-2′-amino-5′-O-(4,4′-dimethoxy-trityl)uridine (2).12
Successive mesylation/displacement reactions have enabled
installation of a 3′-R-sulfur atom into uridine previously.13
To employ this strategy in our synthesis, the amino group
of 2 must bear a protecting group that fulfills several
criteria: (1) it must weaken the nucleophilicity of the 2′
amine so as to allow selective mesylation of the 3′-R
hydroxyl group, (2) it must remain unaffected under strongly
basic conditions (1-6 N NaOH in ethanol) used for
displacement of the 3′-R mesylate, and (3) it must undergo
facile removal under appropriate conditions. Based on these
considerations, we chose tert-butoxycarbonyl (Boc), well-
known for its use in peptide chemistry but used infrequently
in nucleic acid chemistry. To obtain the Boc-protected
nucleoside, 2′-deoxy-2′-amino-5′-O-(4,4′-dimethoxytrityl)-
uridine (2) was treated at 40 °C overnight with 2-(tert-
butoxycarbonyloxyimino)-2-phenylacetonitrile (Boc-ON)14 in
dioxane to afford 3 selectively in 94% yield. Other reagents,
such as di-tert-butyl dicarbonate,15 or other solvents, such
as methanol (MeOH) or acetonitrile (CH3CN), gave 3 in
significantly lower yields.
Figure 1. Metal ion ligands in the transition state for the exon
ligation step of group II intron self-splicing. R1 and R2 are exon 1
and exon 2, respectively. The 3′-hydroxyl group of exon 1 (OR1)
attacks the 5′-splice site, giving rise to spliced exons and releasing
the excised intron. Both the 2′- and 3′-oxygen atoms of the cleavage
site uridine interact with metal ions,3g,h,o but it remains unknown
whether two metal ions (as in A) or a single metal ion (as in B)
mediate(s) these interactions. Dots symbolize metal ion coordina-
tion.
To test whether the Boc group meets the aforementioned
requirements, we conducted the following control experi-
ments: (i) 3 was treated with methanesulfonyl chloride in
pyridine to generate compound 4a selectively in 90% yield.16
We could detect no mesylation of the 2′-NHBoc group under
these conditions. (ii) Overnight treatment of compound 3 with
6 N NaOH in ethanol (1:2 v/v) at room temperature gave
no reaction, suggesting that the Boc group would remain
whether two distinct metal ions mediate these interactions
(Figure 1A) or a single metal ion mediates both interactions
(Figure 1B) remains unknown. Metal ion rescue experiments
using an oligonucleotide substrate containing doubly modi-
fied 2′,3′-dideoxy-2′-amino-3′-thiouridine residue at the
cleavage site could help to answer this fundamental mecha-
nistic question.6 Preparation of such a substrate requires
synthesis of the parent nucleoside 12 and its incorporation
into an oligonucleotide. Although numerous syntheses of 2′
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2170
Org. Lett., Vol. 6, No. 13, 2004

5′-dimethoxytrityl (DMTr) group instantly, but the Boc group
survived overnight exposure. In contrast, 30% trifluoroacetic
acid (TFA) in dichloromethane (CH2Cl2) removed both the
DMTr and Boc groups within 1 h to give 2′-deoxy-2′-
aminouridine12 quantitatively. These control experiments
supported our reasoning that Boc protection of the amino
group would enable us to access our target.
Scheme 1a
We attempted to generate the 3′-â-OH analogue from
methanesulfonyl derivative 4a by treatment with 1 or 6 N
ethanolic NaOH (1:1 v/v) at room temperature.17 No reaction
occurred after overnight treatment; overnight reflux with 6
N ethanolic NaOH (1:1 v/v) removed both the Ms and Boc
groups, regenerating 2. As an alternative, we activated 3 as
the more reactive trifluoromethanesulfonylate ester5f,18 by
overnight exposure to triflic anhydride in pyridine and
CH2Cl2 (1:1), initially at -78 °C followed by gradual
warming to room temperature. Thin-layer chromatography
(TLC) showed that 3 was completely consumed, apparently
giving rise to one major product. However, standard workup
and column chromatography resulted in a low yield (40%)
of 4b, suggesting that decomposition may have occurred
during the purification process. Incubation with ethanolic
NaOH (1.0 N, 1.1 equiv) converted 4b to the 3′-anhydro-
uridine derivative 5 in 80% yield. Further exposure to
ethanolic NaOH (1.0 N, 2.0 equiv) converted 5 to the xylose
derivative 6 in 90% yield.19
To minimize the possible decomposition of 4b during
isolation, we explored the transformation of 3 to 6 in a “one-
pot” reaction. After 3 was completely consumed during
trifluoromethanesulfonylation (as monitored by TLC), we
quenched the reaction by adding MeOH and removed solvent
at low temperature (<30 °C). Without further purification,
we treated the residue with ethanolic NaOH, monitoring by
TLC the gradual formation of 5 and then 6. The yields of 6
varied with the number of NaOH equivalents added, presum-
ably due to the formation of a byproduct by E2 elimination.5f,20
When 2.5 equiv of NaOH was used, the “one-pot” reaction
gave 6 in 65% overall yield.21
To install the 3′ sulfur on the R face of the nucleoside,
we converted 6 to the corresponding 3′-â-mesylate ester 7
in 91% yield. To avoid spectral overlap, NOE experiments
were conducted on compound 7 instead of 6. Irradiation of
H-3′ enhanced H-4′ strongly (8.6%) and H-2′ weakly (1.8%),
confirming that the 3′-OMs resides on the â-face of the ribose
ring. Mesylate 7 reacted with 2-quinolinethiol in the presence
of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) at 80 °C to
afford 8a in 70% yield22 or with sodium thiobenzoate in N,N-
a Reaction conditions: (a) (1) DMTr-Cl, DMAP, pyridine, 16
h, (2) Cl3CCN, Et3N, reflux, 24 h, (3) 6 N NaOH, EtOH, reflux,
16 h; (b) Boc-ON, Et3N, dioxane, 40 °C, 16 h; (c) for 4a, MeSO2Cl,
pyridine; for 4b, (CF3SO2)2O, pyridine, CH2Cl2, -78 °C to rt; (d)
6 N NaOH (1.1 equiv), EtOH, rt, 16 h; (e) 6 N NaOH (2.0 equiv),
EtOH, rt, 16 h; (f) (CF3SO2)2O, pyridine, CH2Cl2, -78 °C to rt;
(d) 6 N NaOH (1.1 equiv), EtOH, rt, 16 h; 6 N NaOH (1.4 equiv),
EtOH, rt, 16 h; (g) MeSO2Cl, pyridine, rt, 16 h; (h) for 8a,
2-quinolinethiol and DBU, DMF, 80 °C, 16 h; for 8b, PhC(O)SNa,
DMF, 100 °C, 8 h; (i) (1) 40% aqueous MeNH2, rt, 16 h, (2)
aldrithiol, DMF, 60 °C, 16 h; (j) 3% trichloroacetic acid in
acetonitrile, rt, 0.5 h; (k) 30% trifluoroacetic acid in CH2Cl2, rt, 1
h; (l) dithiothreitol, THF, rt, 1 h; (m) atmospheric O2, rt, 3 days.
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(21) 1 equiv was added dropwise to the reaction. After 24 h, TLC showed
that 4b reacted entirely to 5, and another 1.4 equiv of 1 N NaOH was added
dropwise.
intact under the strongly basic conditions required in the
subsequent steps. (iii) Treatment of 3 with weak acid (3%
trichloroacetic acid in dichloromethane, TCA) removed the
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Org. Lett., Vol. 6, No. 13, 2004
2171

dimethylformamide (DMF) at 100 °C to afford 8b in 92%
yield.23 Attempts to generate the 3′-thionucleoside directly
from 2′-amino-3′-anhydrouridine 5 by treatment with tert-
butyl thiol24 or sodium thiobenzoate gave no reaction.
We attempted to acquire the target nucleoside 12 by
removing the protecting groups from 8a. TCA in acetonitrile
(3%) selectively removed the 5′-DMTr to afford 10a, which
upon exposure to TFA in CH2Cl2 (30%) removed the Boc
group and generated 11a. However, attempts at various
temperatures to remove the quinoline moiety by reduction
with NaBH3CN in acetic acid and subsequent hydrolysis22
failed to give 2′,3′-dideoxy-2′-amino-3′-thiouridine 12.
The 3′-thiobenzoyl derivative 8b allowed access to the
target nucleoside. We converted 8b to the 3′-pyridyl disulfide
9b before removing the Boc group, thereby circumventing
benzoyl migration to the amino group.25 Compound 8b was
treated with 40% aqueous methylamine to generate the 3′-
mercaptan analogue.26 Solvent was removed, and the residue
was treated with aldrithiol at 60 °C for 16 h in DMF27 to
give the disulfide 9b in 80% yield. Incubation with 30% TFA
in CH2Cl2 removed DMTr immediately followed by Boc
within 1 h to give the corresponding trifluoroacetate salt.
Addition of triethylamine (Et3N) to neutralize the nucleoside
caused partial regeneration of the 5′-DMTr ether. To
eliminate this problem, we removed DMTr selectively by
treatment with 3% TCA in CH3CN for 0.5 h and isolated
10b in 92% yield. Subsequent incubation of 10b with 30%
TFA in CH2Cl2 for 1 h removed the Boc group.28 Following
evaporation of the volatile components, we treated the residue
with Et3N in CH2Cl2 to afford the neutral nucleoside 11b in
80% yield. Subsequent reduction of the disulfide bond with
dithiothreitol (DTT) gave the target nucleoside, 2′,3′-dideoxy-
2′-amino-3′-thiouridine 12 in 95% yield.29 In dimethyl
sulfoxide (DMSO), the nucleoside 12 undergoes gradual
aerial oxidation to its dimeric disulfide 13.30 In the presence
of DTT, 13 undergoes quantitative reduction back to 12.
In conclusion, we have developed an efficient method for
the first synthesis of 2′,3′-dideoxy-2′-amino-3′-thiouridine
12 in 19.5% overall yield starting from 2,2′-anhydrouridine.
Our synthetic strategy included four stages: (i) conversion
of the starting material to 5′-DMTr-2′-deoxy-2′-aminouridine
using the method of McGee et al.;12 (ii) protection of the 2′
amine as the tert-butoxycarbamate; (iii) installation of the
3′ sulfur by stereochemical inversion of the 3′-hydroxyl
group, mesylation and displacement with thiobenzoate and
(iv) deprotection by consecutive removal of the protecting
groups to generate the target nucleoside. This work expands
the arsenal of nucleoside analogues with which to investigate
the role of metal ions in RNA catalysis. Current efforts are
directed toward incorporating 12 into the 3′-splice site of
the group II intron.
Acknowledgment. Q.D. was a Research Associate and
J.A.P. is an Associate Investigator of the Howard Hughes
Medical Institute. We thank C. Cortez and L. Elias for
technical support, R. Fong for assistance with the figure, and
Dr. J. Ye, Dr. N. Li, Dr. S. R. Das, and J. Hougland for
helpful discussions and critical comments on the manuscript.
Supporting Information Available: Full experimental
1
and analytical data and the H and 13C NMR spectra for
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1545.
compounds 2, 3, 4b, 5-7, 8b-11b, 12, and 13. This material
is available free of charge via the Internet at http://pubs.acs.org.
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OL049366X
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(30) Immediately after column purification, compound 12 was dissolved
in DMSO-d6. 1H NMR indicated that the sample already contained
detectable amounts of the disulfide 13. After the solution was exposed to
the air for 48 h, only 10% of 12 remained. After 72 h, 12 had oxidized
quantitatively to 13.
2172
Org. Lett., Vol. 6, No. 13, 2004