Synthesis and properties of 2'-deoxy-2'-α-C-branched nucleosides and nucleotides

Article abstract of DOI:10.1021/jo9614607

Four functionalized 2'-deoxy-2'-α-C-branched nucleosides, namely, 2'-deoxy-2)-α-C-(carboxymethyl)uridine, 2'-deoxy-2'-α-C-acetamidouridine, 2'-deoxy-2'-α-C-(hydroxyethyl)uridine, and 2'-deoxy-2'α-C-(2,3-dihydroxypropyl)uridine, have been prepared. Conversion of these nucleosides to their appropriately protected phosphoramidites, followed by tetrazole-induced reaction with 2',3'-di-O-acetyluridine, oxidation, and subsequent deprotection furnished the corresponding dinucleoside monophosphates. During the oxidation of the amide-derived phosphite, partial dehydration occurred to give a mixture of the amide- and nitrile-containing dimers. Interestingly, the ratio of amide to nitrile could be largely controlled by choice of oxidant. The hydroxyethyl- and dihydroxypropyl-modified dimers were particularly resistant to snake venom phosphodiesterase-catalyzed hydrolysis (relative half-lives of 129 and 120, respectively, in comparison to UpU). It is anticipated that these nucleoside analogues will ultimately be used in the construction of ribozymes containing enhanced functionality and nuclease resistance.

Full text of DOI:10.1021/jo9614607

J . Org. Chem. 1996, 61, 9213-9222
9213
Syn th esis a n d P r op er ties of 2′-Deoxy-2′-r-C-br a n ch ed Nu cleosid es
a n d Nu cleotid es
Anthony J . Lawrence, J ohn B. J . Pavey, Richard Cosstick,* and Ian A. O’Neil*
Robert Robinson Laboratories, Department of Chemistry, University of Liverpool, P.O. Box 147,
Liverpool L69 3BX, U.K.
Received J uly 30, 1996X
Four functionalized 2′-deoxy-2′-R-C-branched nucleosides, namely, 2′-deoxy-2′-R-C-(carboxymethyl)-
uridine, 2′-deoxy-2′-R-C-acetamidouridine, 2′-deoxy-2′-R-C-(hydroxyethyl)uridine, and 2′-deoxy-2′-
R-C-(2,3-dihydroxypropyl)uridine, have been prepared. Conversion of these nucleosides to their
appropriately protected phosphoramidites, followed by tetrazole-induced reaction with 2′,3′-di-O-
acetyluridine, oxidation, and subsequent deprotection furnished the corresponding dinucleoside
monophosphates. During the oxidation of the amide-derived phosphite, partial dehydration occurred
to give a mixture of the amide- and nitrile-containing dimers. Interestingly, the ratio of amide to
nitrile could be largely controlled by choice of oxidant. The hydroxyethyl- and dihydroxypropyl-
modified dimers were particularly resistant to snake venom phosphodiesterase-catalyzed hydrolysis
(relative half-lives of 129 and 120, respectively, in comparison to UpU). It is anticipated that these
nucleoside analogues will ultimately be used in the construction of ribozymes containing enhanced
functionality and nuclease resistance.
In tr od u ction
boxylic acid (1), a primary amide (2), a primary alcohol
(3), or a 1,2-diol (4). While the carboxyl, amide, and
alcohol groups have a direct parallel in amino acids, the
diol function was chosen as a means to introduce func-
tionality that would have the potential to cleave a
phosphodiester bond in the presence of metal ions such
as lanthanides. Recently, alcohols such as glycerol and
gluconate have been shown to accelerate the hydrolysis
of DNA by lanthanide cations.5 Since metal ions are an
absolute requirement for efficient ribozyme catalysis, it
may prove possible to create metalloribozymes capable
of catalyzing new reactions (e.g., redox reactions) by
engineering transition metal binding sites into RNA.6
There is increasing circumstantial evidence for the
existence of early life forms that were characterized by
RNA-catalyzed replication, a situation that has been
termed the RNA world.1 Robertson and Miller have
shown that under prebiotic conditions uracil reacts with
formaldehyde and simple nucleophiles to give 5-substi-
tuted uracils with the side chains of most of the 20
genetically-coded amino acids.2 The authors proposed
that ribozymes in the RNA world would have contained
the functional groups that are currently found in proteins
and consequently may have possessed greater catalytic
activity than those currently in existence. It is certainly
true that the variety of reactions that naturally occurring
ribozymes are known to catalyze is extremely narrow in
comparison to protein enzymes and is restricted to
transformations that involve the RNA phosphodiester
backbone.3 It is therefore interesting to speculate as to
whether the catalytic activity and substrate diversity of
ribozymes might be enhanced by the inclusion of func-
tionality that is known to be important for protein
enzymes.
In a recent paper we described the synthesis of the 2′-
R-C-branched nucleosides 1-3.7 We now report the full
details of syntheses of 1-3, the preparation of the
dihydroxypropyl nucleoside 4, the incorporation of these
compounds into dinucleoside monophosphates, and stud-
ies on the hydrolysis of the dimers. To our knowledge,
this is the first systematic approach to the synthesis of
nucleic acids containing catalytically useful functional
groups, although 2′-deoxy-2′-amino nucleosides,8 2′-thio-
uridine,9 and 2′-R-C-(hydroxymethyl)thymidine10 have all
been incorporated into oligoribo- or oligodeoxyribonucle-
As the first step toward addressing this issue, func-
tionalized 2′-R-C-branched nucleosides have been pre-
pared for incorporation into oligoribonucleotides. The 2′-
position was chosen as the site of modification for the
following reasons. (i) It enables the regular 3′-5′ phos-
phodiester linkages and the Watson-Crick hydrogen
bonding scheme to be maintained. (ii) The same modi-
fication can be introduced into both purine and pyrimi-
dine nucleosides. (iii) It has been shown that the
introduction of 2′-R-alkyl substituents into oligodeoxyri-
bonucleotides increases their resistance to degradation
by nucleases.4 Modified nucleosides were designed to
contain one of the following functional groups: a car-
(5) Rammo, J .; Hettich, R.; Roigk, A.; Schneider, H.-J . J . Chem. Soc.,
Chem. Commun. 1996, 105.
(6) Pyle, A. M. Science 1993, 261, 709.
(7) Lawrence, A. J .; Pavey, J . B. J .; O’Neil, I. A.; Cosstick, R.
Tetrahedron Lett. 1995, 36, 6341.
(8) Aurup, H.; Tuschl, T.; Benseler, F.; Ludwig, J .; Eckstein, F.
Nucleic Acids Res. 1994, 22, 701.
(9) Reese, C. B.; Simons, C.; Zhang, P. Z. J . Chem. Soc., Chem.
Commun. 1994, 1809.
X Abstract published in Advance ACS Abstracts, December 1, 1996.
(1) J oyce, G. J .; Orgel, L. E. In The RNA World; Gesteland, R.,
Atkins, J ., Eds.; Cold Spring Harbor Press: Cold Spring Harbor, NY,
1993; pp 1-25.
(2) Robertson, M. P.; Miller, S. L. Science 1995, 268, 702.
(3) Wilson, C.; Szostak, J . W. Curr. Opin. Struct. Biol. 1992, 2, 749.
(4) De Mesmaeker, A.; Ha¨ner, R.; Martin, P.; Moser, H. E. Acc.
Chem. Res. 1995, 28, 366.
(10) Schmit, C.; Be´vierre, M.-O.; De Mesmaeker, A.; Altman, K.-H.
Bioorg. Med. Chem. Lett. 1994, 4, 1969.
S0022-3263(96)01460-0 CCC: $12.00 © 1996 American Chemical Society

9214 J . Org. Chem., Vol. 61, No. 26, 1996
Lawrence et al.
Sch em e 1a
Sch em e 2a
a
Key: (i) NaClO2, KH2PO4, 2-methyl-2-butene, 2-methyl-2-
propanol; (ii) 8, H2CdCHCH2OH, DCC, DMAP, THF; 9, Ph2CN2,
acetone; 10, (a) N-hydroxysuccinimide, DCC, DMAP, 1,4-dioxane,
(b) NH3 (aq).
otides, chiefly with the aim of stabilizing oligomers to
nuclease digestion.
a
Key (i) NaBH4, MeOH; (ii) Fpmp vinyl ether, camphorsulfonic
Resu lts a n d Discu ssion
acid, CH2Cl2; (iii) OsO4, (DHQD)2PYR, K3Fe(CN)6, K2CO3, t-BuOH/
water; (iv) Ac2O, pyridine.
The synthesis of all four 2′-R-C-branched nucleosides
started from the previously described 2′-R-C-allyl nucleo-
side 5.11 Oxidative cleavage of the allylic side chain using
OsO4 in the presence of N-methylmorpholine oxide,
followed by treatment with NaIO4, gave the 3′,5′-tetra-
isopropyldisiloxanediyl (TIPS)-protected aldehyde 6,11
which served as the starting material for the synthesis
of 1-3.
treatment of the carboxylic acid 7 with diphenyldiazo-
methane in acetone gave the benzhydryl ester 9 (75%).
Conversion of the acid 7 to the primary amide 10 was
achieved in a two-step procedure via the intermediate
N-hydroxysuccinimide ester.14 Treatment of acid 7 with
N-hydroxysuccinimide and DCC in dioxane, followed by
addition of concentrated aqueous ammonia, furnished 10
(79%).
For the preparation of a suitably protected derivative
of the hydroxyethyl nucleoside (3) the TIPS-protected
aldehyde (6) was reduced using NaBH4 in MeOH to give
the alcohol (11, Scheme 2) in quantitative yield. The 1-(2-
fluorophenyl)-4-methoxypiperidinyl (Fpmp) group was
initially chosen to protect the primary hydroxyl group.15
This achiral acetal group has been extensively used in
RNA synthesis, and unlike the rival tert-butyldimethyl-
silyl group, it is compatible with the removal of the cyclic
TIPS ether. Thus, treatment of the hydroxyethyl nucleo-
side (11) with excess 1-(2-fluorophenyl)-4-methoxy-1,2,3,6-
tetrahydropyridine in the presence of camphorsulfonic
acid gave the Fpmp-protected nucleoside (12) in 73%
yield.
The TIPS-protected diol (13) was accessible through
an osmium-catalyzed dihydroxylation of the allyl nucleo-
side (5, Scheme 2). To assist the characterization and
isolation of the TIPS-protected diol (13), and its subse-
quent derivatives, it was considered desirable to obtain
this compound as a single diastereoisomer. In an initial
experiment to assess whether the chirality of the sugar
would impose a significant diastereomeric bias on the
reaction, a standard dihydroxylation procedure using
OsO4 in the presence of N-methylmorpholine oxide was
performed and 13 isolated in 81% yield with a slight
predominance of one diastereoisomer (ratio 57:43). The
diastereomeric composition of 13 could not be determined
P r ep a r a tion of 2′-r-C-Br a n ch ed Nu cleosid es 1-4.
For the preparation of the carboxymethyl nucleoside (1)
the TIPS-protected aldehyde (6) was oxidized with Na-
ClO2 in the presence of KH2PO4 and 2-methyl-2-butene
in aqueous 2-methyl-2-propanol, to give the required
carboxylic acid 7 (95%) (Scheme 1). For incorporation of
this analogue into oligomers a protective group for the
carboxylic acid function was required. There is no
precedent for the protection of a comparable carboxylic
acid function in oligonucleotide chemistry, but since it is
possible that this functionality could assist the hydrolysis
of the adjacent phosphodiester bond at extremes of pH,
it was considered prudent to use a blocking group that
could be removed under neutral conditions. With these
considerations in mind we first examined the allyl group,
which can be removed under mild conditions using
palladium(0).12 Treatment of the carboxylic acid 7 with
allyl alcohol and DCC gave the TIPS-protected allyl ester
(8) in 80% yield. However, subsequent attempts (see
Scheme 3) to remove the TIPS group using NEt3‚3HF
gave an inseparable mixture of the desilylated allyl ester
(15) and the γ-butyrolactone (19). It seemed likely that
lactonization could be reduced by increasing the steric
bulk of the protecting group. Diphenylmethyl (benzhy-
dryl) esters are readily prepared from diphenyldiazo-
methane, and the carboxyl group can be unmasked by
either catalytic hydrogenolysis or acid hydrolysis.13 Thus,
(11) De Mesmaeker, A.; Lebreton, J .; Hoffman, P.; Freir, S. M.
Synlett 1993, 677.
(12) (a) Wakabyashi, S.; Kato, H.; Noyori, R. J . Am. Chem. Soc. 1990,
112, 1691. (b) Kuyl-Yeheskiely, E.; Tromp, C. M.; Lefeber, A. W. M.;
van der Marel, G. A.; van Boom, J . H. Tetrahedron 1988, 44, 6515.
(13) Stelakatos, G. C.; Paganou, A.; Zervas, L. J . Chem. Soc. C 1966,
1373.
(14) Galaverna, G.; Corrandini, R.; Dossena, A.; Marchelli, R. Int.
J . Peptide Protein Res. 1993, 42, 53.
(15) (a) Capaldi, D. C.; Reese, C. B. Nucleic Acid Res. 1994, 22, 2209.
(b) Rao, M. V.; Reese, C. B.; Schehlman, V.; Yu, P. K. J . Chem. Soc.,
Perkin Trans. 1 1993, 43.

Synthesis of 2′-Deoxy-2′-R-C-branched Nucleosides
J . Org. Chem., Vol. 61, No. 26, 1996 9215
Sch em e 3a
suitably protected equivalents 16-18. In all cases
removal of the TIPS group was accomplished using
NEt3‚3HF, and the products were generally isolated in
good yield (75-80%). In specific instances this desily-
lation reaction requires further comment. While the
formation of 5-membered ring lactones is extremely
favorable the carboxymethyl nucleoside 1 was relatively
stable and was readily isolated as the triethylammonium
salt following silica gel chromatography. However, under
acidic conditions conversion to the γ-butyrolactone (19)
occurred effortlessly and the carboxymethyl nucleoside
was additionally characterized as the lactone.7 Gratify-
ingly, the lactonization that accompanied desilylation of
the allyl ester (8) was not observed with the benzhydryl
ester 9, and 16 was isolated in 87% yield. Compounds 2
and 16-18 were subsequently converted to their 5′-O-
dimethoxytrityl derivatives (20a -d ), in preparation for
oligonucleotide synthesis. While there is increasing use
of DMAP/NEt3 as a catalyst for tritylation reactions,19
we found that reactions performed in pyridine/CH2Cl2
were the most rapid and gave the highest yields. The
inclusion of DMAP/NEt3 was particularly deleterious in
the preparation of ester 20c and resulted in partial
lactonization.
P r ep a r a tion of Din u cleosid e Mon op h osp h a tes
Con ta in in g 1-4. The 5′-O-DMT-protected hydroxyethyl
(20a ), dihydroxypropyl (20b), carboxymethyl (20c), and
acetamido (20d ) monomers were prepared for solid-
phase-style coupling by converting them to their 3′-O-
phosphoramidites 21a -d , respectively, using 2-cyano-
ethyl N,N,N′,N′-tetraisopropylphosphorodiamidite under
standard conditions (Scheme 4).20 The phosphoramidites
were then coupled with 2′,3′-di-O-acetyluridine in aceto-
nitrile in the presence of 1H-tetrazole under conditions
analogous to those used in solid-phase synthesis.21 The
reactions were followed by 31P NMR, which showed the
formation of the intermediate diastereomeric dinucleoside
phosphites (22a , 144.93/144.53; 22b, 141.26/141.51; 22c,
141.27/141.67; and 22d , 141.70/141.55 ppm). Without
isolation, the phosphites were subsequently oxidized to
the corresponding phosphates (23) by excess addition of
a 4% solution (w/v) of I2 in THF/2,6-lutidine/H2O (8:1:1).
The coupling reaction employing the hydroxyethyl de-
rivative (21a ) proceeded as expected to give the corre-
sponding fully protected dinucleoside monophosphate
(23a ) in 58% yield. Complete deprotection to furnish 24a
was achieved by sequential treatment with methanolic
ammonia to remove the acetyl and cyanoethyl groups,
acetate buffer (pH 3‚25) to cleave the 1-(2-fluorophenyl)-
4-methoxypiperidinyl (Fpmp) group,15a and finally AcOH:
H2O (8:2) to completely remove the DMT group. An
analogous coupling reaction performed on the dihydrox-
ypropyl monomer (21b) gave the fully protected dimer
23b (87% yield), which was subsequently deprotected
using methanolic ammonia followed by aqueous AcOH
to yield 24b. Likewise, the carboxymethyl monomer 21c
was converted to the fully protected dimer 23c in 33%
yield. Following the methanolic ammonia and aqueous
AcOH deprotection steps, the benzhydryl blocking group
was removed by hydrogenolysis using palladium on
charcoal in THF/MeOH. This final deprotection step
a
Key: (i) NEt3‚3HF, THF; (ii) DMTCl, pyridine, CH2Cl2.
from analysis of the proton NMR spectrum as the
resonances for the two diastereoisomers were almost
totally superimposed. However, following removal of the
TIPS group the composition could be accurately deter-
mined by reversed-phase HPLC. In an attempt to obtain
a more heavily biased diastereomeric ratio, Sharpless
asymmetric dihydroxylation (ADH) reactions16 were per-
formed using the commercially available ligands (DHQD)2-
PYR and (DQD)2PYR.17 Literature precedent suggested
that these ligands would give the greatest chiral induc-
tion in the case of a terminal alkene. The reaction using
the former ligand gave an 86% yield of the required diol
as an 86:14 mixture of diastereoisomers, whereas the
latter gave the diol in 72% yield as a 71:29 mixture with
the same diastereoisomer predominating in each case.
The fact that each of the matched ligands gave the same
predominating diastereoisomer and neither reaction was
as selective as examples reported in the literature
suggests that the chiral sugar moiety interferes with
ligand binding in the reaction complex. The diol mixture
from the ADH reaction using (DHQD)2PYR, which was
obtained as an oil, was subsequently converted to the
crystalline 2,4-dinitrobenzoate mono ester, recrystallized,
and treated with methanolic ammonia to give the diol
as a single diastereoisomer (de >99%). In preparation
for oligonucleotide synthesis, the purified diol was sub-
sequently protected as the bisacetyl derivative (14).18 To
our knowledge, this is the first reported instance of the
ADH reaction being applied to a nucleoside derivative.
Scheme 3 shows the removal of the cyclic TIPS protect-
ing group to give the nucleosides 1-4, as well as their
(16) Sharpless, K. B.; Hartung, J .; J eong, K. S.; Kwong, H. L.;
Morikawa, K.; Wang, Z. M.; Xu, D.; Zhang, X. L. J . Org. Chem. 1992,
57, 2768.
(17) Crispino, G. A.; J eong, K. S.; Kolb, H. C.; Wang, Z. M.; Xu, D.;
Sharpless, K. B. J . Org. Chem. 1993, 58, 3785.
(19) Chauhary, S. K.; Hernandez, O. Tetrahedron Lett. 1979, 20,
95.
(20) (a) Nielsen, J .; Taagaard, Marugg, J . E.; van Boom, J . H.; Dahl,
O. Nucleic Acids Res. 1986, 14, 7391. (b) Kierzek, R.; Caruthers, M.
H.; Longfellow, C. E.; Swinton, D.; Turner, D. H.; Frier, S. M.
Biochemistry 1986, 25, 7840.
(18) The chronological order in which the work described in this
paper was performed was such that studies on the base-catalyzed
hydrolysis of the closely related hydroxyethyl-containing dimer (24a )
had already established that conditions necessary for the removal of
the acetyl groups would not be deleterious to the phosphodiester
linkage.
(21) Sinha, N. D.; Biernat, J .; McManus, J .; Ko¨ster, H. Nucleic Acids
Res. 1984, 12, 4539.

9216 J . Org. Chem., Vol. 61, No. 26, 1996
Lawrence et al.
Sch em e 4a
Sch em e 55
a
Key: (i) (CF3CO)2O, pyridine, 1,4-dioxane; (ii) NEt3‚3HF, THF.
Sch em e 6. P r op osed Mech a n ism for th e
Deh yd r a tion of Am id e 23d
Additionally, hydrolysis of 24d using SVPD and analysis
of the resultant digestion mixture by HPLC showed the
presence of the two expected products, UMP and 2′-
deoxy-2′-C-acetamidouridine (2). The 13C NMR spectrum
of the major product, derived from deprotection of 23e,
showed no amide carbonyl resonance, but it did show a
new resonance with the expected chemical shift for a
nitrile carbon (119.71 ppm). Both the FAB mass spec-
1
trum and H NMR spectrum were also consistent with
a
Key: (i) 2-cyanoethyl bis(N,N-diisopropylamino)phosphor-
24e being the nitrile-containing product. Digestion of
24e with SVPD and HPLC analysis of the hydrolysate
showed the formation of two products, UMP and a new,
unidentified compound that was presumed to be the
nitrile (26). To confirm the identity of this product the
chemical synthesis of 26 (Scheme 5) was performed. The
(TIPS)-protected amide (10) was treated with trifluoro-
acetic anhydride and pyridine22 to give the protected
nitrile-containing nucleoside (25) in 78% yield. Subse-
quent removal of the TIPS protecting group using NEt3‚
3HF gave 2′-deoxy-2′-C-(cyanomethyl)uridine (26), which
had an HPLC retention time identical to that of the
unidentified product from the SVPD digestion of 24e.
To explain this dehydration reaction, we propose the
mechanism shown in Scheme 6. Involvement of the
activated phosphonium species is indirectly supported by
the following observations. (i) Treatment of the TIPS-
protected amide (10) under exactly the same conditions
as those involved in the coupling and oxidation procedure
gave no dehydration product. (ii) The 31P NMR spectrum
of the intermediate dinucleoside phosphite (22d ) shows
the presence of a single diastereomeric product, suggest-
ing that dehydration occurred during the subsequent
oxidation step. Additionally, a procedure for the dehy-
amidite, diisopropylammonium tetrazolide, CH2Cl2; (ii) 2′,3′-di-
O-acetyluridine, tetrazole, CH3CN; (iii) I2, lutidine, water, THF;
(iv) 23b,d ,e, (a) NH3, MeOH, (b) AcOH/water (4:1); 23a , (a) NH3,
MeOH, (b) pH 3.5 buffer, (c) AcOH/water (4:1), 23c, (a) NH3,
MeOH, (b) AcOH/water (4:1), (c) H2, Pd/C, THF/MeOH (3:1).
could be monitored by reversed-phase HPLC with reten-
tion times of 17.03 and 28.10 min, respectively, for 24c
and its benzhydryl derivative. The spectroscopic data
obtained on the three dimers 24a -c were absolutely
consistent with their proposed structures. Additional
characterization was obtained from digestion with SVPD
and HPLC analysis of the hydrolysate. In each case only
the two expected hydrolysis products were observed,
namely uridine 5′-monophosphate (UMP) and either 3,
4, or 1, respectively, for the dimers 23a -c.
The coupling reaction and subsequent oxidation per-
formed with the amide 21d yielded two fully protected
dinucleoside monophosphates that were isolated by col-
umn chromatography. The minor product (∼15% isolated
yield) had both a FAB mass spectrum and a 1H NMR
spectrum that were consistent with the anticipated amide
dimer 23d . However, spectroscopic analysis of the major
product (∼33% isolated yield) was consistent with the
amide undergoing dehydration to yield the nitrile 23e.
In particular, both the FAB and electrospray mass
spectra gave pseudomolecular ions that were concordant
with the loss of water from 23d . In order to unequivo-
cally establish the identity of the two products, both 23d
and 23e were deprotected by treatment with methanolic
ammonia followed by 80% aqueous acetic acid. Spectro-
scopic data on the minor product, derived from 23d , were
absolutely consistent with it being the amide-containing
dimer 24d . In particular, the 13C spectra showed a
characteristic amide carbonyl resonance at 176.80 ppm.
23
dration of amides using triphenylphosphine and CCl4
has been reported previously and involves the formation
of a phosphonium salt that acts as the dehydrating agent;
it seems probable that a similar species is formed on
treatment of the phosphite (22d ) with iodine. This
phosphonium species is then attacked either by the amide
functionality, which results in formation of the nitrile
(22) Campagna, F.; Carotti, A.; Casini, G. Tetrahedron Lett. 1977,
21, 1813.
(23) Yamato, E.; Sugasawa, S. Tetrahedron Lett. 1970, 4383.

Synthesis of 2′-Deoxy-2′-R-C-branched Nucleosides
J . Org. Chem., Vol. 61, No. 26, 1996 9217
Ta ble 1. Oxid a tion of In ter m ed ia te P h osp h ite 22d u n d er
Differ en t Con d ition s
tance is introduced on the basis of specific functional
groups rather than relying on steric bulk, which results
in enhanced hydrolytic stability at the expense of hy-
bridization affinity.29
entry
oxidation condns
amide:nitrilea
1
2
3
4% I2 in THF/lutidine/H2O (8:1:1)
4% I2 in THF/lutidine (9:1)
t-BuOOH/H2O (7:3), 10% tetrabutyl-
ammonium Oxone
1.5:1
1:8.7
21.9:1
The dimers were also tested as substrates for nuclease
P1 and ribonuclease A. Once again, the enzymatic
digestions were performed at pH 6.5 to eliminate back-
ground hydrolysis.30 None of the modified dimers were
substrates for either nuclease P1 (half-life for UpU ) 12
min) or ribonuclease A (half-life for UpU ) 29 min). The
inability of the hydroxyethyl-containing dimer to act as
a substrate for ribonuclease A is of interest in light of
previous studies. Schmit et al. have attributed the very
small (3-fold) increase in stability toward 3′-exonucleases
present in fetal calf serum, which is observed for oligo-
nucleotides containing 2′-R-CH2OH substituents to the
“RNA-like structure” of this modification, and hence
possible attack by RNA degrading enzymes.10 However,
the susceptibility of these substrates to hydrolysis by
specific ribonucleases was not examined. The consider-
able nuclease stability observed for hydroxyethyl and
dihydroxypropyl modifications suggests that these ana-
logs may have potential application to the antisense field;
however, previous studies have shown that simple 2′-
alkyl-substituted oligonucleotides exhibit reduced binding
affinity for complementary RNA and DNA due to their
predominant 2′-endo sugar conformation.10 The large
J 1′-2′ coupling constants (range 8.8-9.4 Hz) for nucleo-
sides 1-4 are also very indicative of a 2′-endo conforma-
tion,10 and on this basis, oligonucleotides containing these
modified residues are also likely to have a lower affinity
for a complementary strand than the corresponding
unmodified oligomer.
The hydroxyethyl-containing dimer, which can be
considered as a homologue of UpU, was also shown to
be very stable to base-catalyzed hydrolysis and under-
went no observable decomposition (less than 1%) when
treated with 2 M NaOH at 80 °C for 1 h. In comparison,
UpU had a half-life of less than 30 min in 0.1 M NaOH
at 20 °C. The base-catalyzed hydrolysis of RNA proceeds
through the formation of a strained five-membered cyclic
phosphate that results from nucleophilic attack of the
vicinal hydroxyl group.31 The resistance of 24a to base-
catalyzed hydrolysis is similar to that observed for
deoxyribonucleotide oligomers and is consistent with the
fact that the hydroxyl function on the proximal 2′-ethyl
side chain is unable to assist hydrolysis.32 The ineffectual
nature of the hydroxyethyl side chain is probably at-
tributable to two major factors. Firstly, base-catalyzed
hydrolysis through the classical mechanism would in-
volve the formation of an entropically less favorable
7-membered cyclic phosphate. Secondly, a diminution in
the nucleophilicity of the hydroxyl group on the ethyl side
chain of 24a is expected relative to that of the 2′-hydroxyl
group of UpU on the basis of their pKa’s. Christensen et
al.33 determined the pKa of the 2′-hydroxyl group of
uridine to be 12.59, whereas that of the hydroxyl group
on the ethyl side chain is likely to be closer to that of
ethanol (15.9). As expected, none of the other modified
4
in CH3CN, 10% tetrabutyl-
ammonium periodate
in CH3CN
20.3:1
4.7:1
5
a
Ratio of amide (24d ):nitrile (24e) was determined after
deprotection and analysis by reversed-phase HPLC.
(23e), or by water to give the expected amide (23d ). In
order to ascertain whether the oxidation could be ma-
nipulated to yield either the amide or nitrile preferen-
tially, several reagents were investigated for this reaction
(Table 1). Using I2 in THF/2,6-lutidine/H2O (Table 1,
entry 1), the amide:nitrile ratio was approximately 1.5:
1. As expected, in the absence of H2O (entry 2) the ratio
was heavily biased in favor of the nitrile (amide:nitrile
1:8.7). Interestingly, using either t-BuOOH24 or tetrabu-
tylammonium Oxone25 (Table 1, entries 3 and 4, respec-
tively) gave amide:nitrile ratios of greater than 20:1.
Much poorer selectivity was achieved with tetrabutylam-
monium periodate26 (Table 1, entry 5). While these
reactions have not been extensively optimized, the results
indicate that sufficient bias can be introduced into the
oxidation step to enable the preparation of oligomers
containing either a single 2′-deoxy-2′-C-(cyanomethyl)-
uridine or a 2′-deoxy-2′-C-acetamidouridine residue from
the same acetamido phosphoramidite 21d .
Hyd r olysis Stu d ies on th e Mod ified Dim er s. To
ascertain whether the 2′-substituent conferred nuclease
resistance to the dimers, their half-lives, relative to UpU,
for hydrolysis with SVPD were measured. Hydrolysis
was performed at pH 6.5 (see Experimental Section for
conditions) in order to completely eliminate background
base-catalyzed hydrolysis of UpU.27 Under these condi-
tions, UpU was hydrolyzed at a rapid but measurable
rate (half-life ) 5 min). The relative half-lives were found
to be 1, 129, 120, 29, 84, and 18 for UpU and 24a -e,
respectively. Hydrolysis by SVPD is known to proceed
through a covalent phosphoryl intermediate involving an
active site threonine residue.28 On the basis of this
mechanism, it is not unexpected that the introduction of
substituents that are bulky, in comparison to the hy-
droxyl group of the wild-type substrate, result in a
reduced rate of hydrolysis. However, the observations
that (i) the cyanomethyl group is more than 7-fold less
stable than the comparably-sized hydroxyethyl substitu-
ent and (ii) the hydroxyethyl substituent confers margin-
ally greater nuclease resistance than the larger dihy-
droxypropyl group suggest that factors other than steric
bulk also require consideration. In particular, it is noted
that the two dimers containing the alcoholic substituents
show the greatest resistance to SVPD-catalyzed hydroly-
sis. These observations may be useful in the design of
2′-modified antisense oligomers in which nuclease resis-
(24) Hayakawa, Y.; Uchiyama, M.; Noyori, R. Tetrahedron Lett.
1986, 27, 4191.
(25) Cosstick, R.; Vyle, J . S. Nucleic Acids Res. 1990, 18, 829.
(26) Fourrey, J .-L.; Varenne, J . Tetrahedron Lett. 1985, 26, 1217.
(27) While the pH optimum for snake venom phosphodiesterases is
normally reported as 9.0, it is dependent on the nature of the substrate
(Bernardi, A.; Bernardi, G. The Enzymes; Boyer, P. D., Ed; Academic
Press: New York, 1971; Vol. IV, p 329). In general, the enzyme is active
over the pH range 6-9, and some substrates are more rapidly
hydrolyzed at pH 6.
(29) Iribarren, A. M.; Sproat, B. S.; Neuner, P.; Sulston, I.; Lamond,
A. I. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 7747.
(30) The pH optimum for this enzyme is dependent on the substrate,
but it is generally used within the pH range 4.0-7.2 (Shishido, K;
Ando, T. Nucleases; Lin, S. M., Roberts, R. J ., Eds.; Cold Spring
Harbor: New York, 1983; p 155).
(31) Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70.
(32) Dugas, H. Bioorganic Chemistry, 2nd Ed.; Springer-Verlag:
New York, 1989; Chapter 3.
(28) Culp, J . S.; Butler, L. G. Arch. Biochem. Biophys. 1986, 246,
245.
(33) Christensen, J . J .; Rytting, J . H. J . Phys. Chem. 1967, 71, 2700.

9218 J . Org. Chem., Vol. 61, No. 26, 1996
Lawrence et al.
clease P1 (Penicillium citrium), ribonuclease A (bovine pan-
creas), uridine 5′-monophosphate, and uridine 2′/3′-mono-
phosphate were purchased from Sigma. Snake venom phos-
phodiesterase (Crotalus durissuss) was purchased from Boe-
hringer Mannheim. 1-(2-Fluorophenyl)-4,4-dimethoxypiperi-
dine was kindly donated by Cruachem, Glasgow, U.K.
dimers underwent phosphodiester bond cleavage when
incubated in 2 M NaOH at 25 °C over a period of 5 days.
However, under these conditions both the amide (24d )
and nitrile (24e) dimers were quantitatively hydrolyzed
to the carboxymethyl dimer (24c) within 4 days.
The hydroxyethyl-containing dimer was also resistant
to acid-catalyzed hydrolysis. Treatment of 24a with 1.5
M HCl at 50 °C for 42 h resulted in about 30% hydrolysis
to give uridine and uracil as the major products. Under
identical conditions UpU was completely hydrolyzed
within 18 h to give uridine, a mixture of uridine 2′- and
3′-phosphates, and less than 5% uracil (the uracil peak
in the HPLC trace was relatively broad and was not
reliably integrated).
The hydrolysis of salicyl phosphates has been exten-
sively investigated by Kirby et al.34 Their results have
shown that between pH 3 and 6 the presence of the
adjacent carboxyl functionality greatly increases the rate
of hydrolysis of the neighboring phosphate by acting as
an intramolecular general acid catalyst. We were inter-
ested to see if the carboxyl functionalized dimer (24c)
could catalyze the cleavage of the adjacent phosphodi-
ester bond in a similar fashion, and thus, its hydrolysis
was compared to UpU at both pH 3 and pH 4. After 4
days at 25 °C, HPLC analysis revealed that at both pH
values neither UpU or 24c had undergone any measur-
able cleavage of the phosphodiester bond. Even under
more forcing conditions (1.5 M HCl, 50 °C, 24 h) no
cleavage of the phosphodiester bond (determined by
release of uridine) was observed.
Our present studies show that dinucleoside monophos-
phates containing 2′-C- (hydroxyethyl), (carboxymethyl),
-acetamido, and (dihydroxypropyl) groups are more re-
sistant to both enzyme- and acid/base-catalyzed hydroysis
than the natural substrates. It is anticipated that these
nucleoside analogues will ultimately be used in the
construction of ribozymes containing enhanced function-
ality and nuclease resistance. In order to explore the
possibility of using in vitro selection techniques35 to
investigate the potential of the 2′-C-functionalized nu-
cleosides as components in RNA catalysts and aptamers,
work is beginning on the synthesis of the 5′-triphosphates
of these analogs to ascertain whether they are substrates
for RNA polymerases.
2′-Deoxy-2′-r-C-(2-oxoeth yl)-3′,5′-O-(1,1,3,3-tetr a isop r o-
p yld isiloxy)u r id in e (6). 2′-C-(2-Propenyl)-3′,5′-O-(1,1,3,3-
tetraisopropyldisiloxy)uridine (5) (4.31 g, 8.45 mmol) was
dissolved in aqueous acetone (45 mL acetone:12 mL water)
along with N-methylmorpholine N-oxide (1.09 g, 9.39 mmol),
and to this was added a 1% (w/v) aqueous solution of OsO4
(10.78 mL, 0.42 mmol). The mixture was stirred vigorously
for 1.5 h and then quenched by the addition of saturated
aqueous sodium thiosulfate solution (15 mL) and water (15
mL) and diluted with ethyl acetate (100 mL). The mixture
was concentrated in vacuo to approximately one-third of the
volume and rediluted ethyl acetate (100 mL) before being
washed with saturated aqueous NaHCO3 solution (2 × 75 mL)
and brine (75 mL). The organic layer was dried (MgSO4) and
solvent removed in vacuo, affording the crude diol as a white
foam (4.58 g, 99.6% crude yield). The crude product (4.58 g,
8.42 mmol) was dissolved in aqueous dioxane (90 mL dioxane:
30 mL water), and to this solution was added NaIO4 (3.58 g,
16.83 mmol). The mixture was stirred vigorously for 10 min,
diluted with ethyl acetate (200 mL), washed successively with
saturated aqueous NaHCO3 solution (2 × 150 mL) and brine
(150 mL), dried (MgSO4), and reduced in vacuo to a thick
colorless oil. The residue was purified by column chromatog-
raphy (CH2Cl2 containing an increasing gradient of MeOH
from 0-3%) yielding the pure product as a white foam (3.72
g, 81% yield, two steps): 1H NMR (400 MHz, CDCl3) δ 1.00-
1.09 (28H, m), 2.66 (1H, dd, J ) 9.3, 21.2 Hz), 2.93-2.97 (2H,
m), 3.93 (1H, ddd, J ) 3.1, 4.0, 7.2 Hz), 4.03 (1H, dd, J ) 3.1,
12.9 Hz), 4.09 (1H, dd, J ) 4.0, 12.9 Hz), 4.56 (1H, t, J ) 7.2
Hz), 5.74-5.79 (2H, m), 7.67 (1H, d, J ) 8.1 Hz), 9.81 (1H, s),
10.18 (1H, br s); 13C NMR (50.4 MHz, CDCl3) δ 12.56-13.26,
16.85-17.31, 40.27, 43.43, 61.03, 69.28, 83.83, 88.31, 102.24,
139.07, 150.52, 163.70, 199.61; FAB HRMS m/ z found (M +
H)+ 513.2448, C23H41N2O7Si2 requires (M + H)+ 513.2452.
Anal. Calcd for C23H40N2O7Si2: C, 53.93; H, 7.87; N, 5.47.
Found: C, 53.49; H, 7.84; N, 5.38.
2′-Deoxy-2′-r-C-(ca r b oxym et h yl)-3′,5′-O-(1,1,3,3-t et r a -
isop r op yld isiloxy)u r id in e (7). To a solution of 2′-C-(2-
oxoethyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxyl)uridine (6) (10.32
g, 20.15 mmol) and 2-methyl-2-butene (35 mL, 330 mmol) in
2-methyl-2-propanol (300 mL) was added an aqueous solution
of NaClO2 and KH2PO4 (27.45 g, 300 mmol, and 30.18 g, 220
mmol, respectively, in 100 mL of water), and the mixture was
stirred vigorously for 1 h. The organic solvents were removed
in vacuo and the resultant residue diluted with ethyl acetate
(250 mL), washed with saturated aqueous NaHCO3 (2 × 200
mL), water (200 mL), and brine (200 mL), dried (MgSO4), and
evaporated to yield the carboxylic acid as a pure white foam
(10.00 g, 94% yield): 1H NMR (200 MHz, CDCl3) δ 1.01-1.08
(28H, m), 2.49 (1H, dd, J ) 12.1, 19.8 Hz), 2.78-2.84 (2H, m),
3.91 (1H, m), 4.05 (2H, m), 4.53 (1H, t, J ) 7.2 Hz), 5.77 (1H,
d, J ) 8.8 Hz), 5.90 (1H, d, J ) 2.5 Hz), 7.66 (1H, d, J ) 8.8
Hz), 10.69 (1H, br s); 13C NMR (50.4 MHz, CDCl3) δ 12.39-
13.17, 16.65-17.24, 30.64, 45.30, 61.08, 69.21, 83.55, 88.30,
102.18, 139.48, 150.78, 164.04, 175.15; FAB HRMS m/ z found
(M + H)+ 529.2393, C23H41N2O8Si2 requires (M + H)+ 529.2401.
Exp er im en ta l Section
Gen er a l P r oced u r es. Most of the general analytical
procedures have been decribed in an earlier publication.36
NMR spectra were recorded at the field strength indicated,
and peaks displaying multiplicity due to the presence of two
diastereomers are denoted with an asterisk. HPLC was
conducted using a Varian Star 9010 liquid chromatograph
equipped with a Varian Star 9050 variable-wavelength UV
detector recording at 260 nm for analytical puposes and 290
nm for preparative work. Unless stated otherwise, analyses
were performed on a Nucleosil C18 reversed-phase column,
using a gradient of 0 f 10% MeCN in 50 mM triethylammo-
nium acetate (pH 6.5) over 20 min, with a flow rate of 1 mL/
min. Data were recorded using a Varian 4400 recording
integrator.
2′-Deoxy-2′-r-C-[[(a llyloxy)ca r b on yl]m et h yl]-3′,5′-O-
(1,1,3,3-tetr a isop r op yld isiloxy)u r id in e (8). The acid 7
(0.55 g, 0.97 mmol), dicyclohexylcarbodiimide (0.24 g, 1.16
mmol), and 4-(dimethylamino)pyridine (15 mg, 0.12 mmol)
were dissolved in dry THF (20 mL), and to this was added
allyl alcohol (0.08 mL, 1.14 mmol). The solution was stirred
for 16 h, poured onto ether (100 mL), filtered, washed succes-
sively with saturated aqueous NaHCO3 (2 × 50 mL), water
(50 mL), brine (50 mL), dried (MgSO4), and evaporated in
vacuo. The resultant oil was purified by column chromatog-
raphy (75% petroleum ether/25% ethyl acetate), yielding the
allyl ester as a white foam (0.47 g, 80% yield): 1H NMR (200
MHz, CDCl3) δ 1.01-1.08 (28H, m), 2.56 (1H, dd, J ) 9.4, 20.0
Hz), 2.61-2.90 (2H, m), 3.92 (1H, m), 4.03 (2H, d, J ) 3.9 Hz),
4.57 (3H, m), 5.19-5.37 (2H, m), 5.74 (1H, d, J ) 8.0 Hz),
Hydroquinine 2,5-diphenyl-4,6-pyrimidinediyl diether
[(DHQ)2PYR], hydroquinidine 2,5-diphenyl-4,6-pyrimidinediyl
diether [(DHQD)2PYR], and 2-cyanoethyl bis(N,N′-diisopro-
pylamino)phosphoramidite were obtained from Aldrich. Nu-
(34) Bromilow, R. H.; Kirby, A. J . J . Chem. Soc., Perkin Trans. 2
1972, 149.
(35) (a) Lorsch, J . R.; Szostak, J . W. Acc. Chem. Res. 1996, 29, 103.
(b) Tuerk, C.; Gold, L. Science 1990, 249, 505.
(36) Li, X.; Scott; G. K.; Baxter; A. D.; Taylor, R. J .; Vyle, J . S.;
Cosstick, R. J . Chem. Soc., Perkin Trans. 1 1994, 2123.

Synthesis of 2′-Deoxy-2′-R-C-branched Nucleosides
J . Org. Chem., Vol. 61, No. 26, 1996 9219
5.81-6.01 (2H, m), 7.58 (1H, d, J ) 8.3 Hz); 13C NMR (50.4
MHz, CDCl3) δ 12.51-13.21, 16.69-17.34, 31.84, 44.83, 61.26,
65.46, 69.66, 83.81, 87.91, 102.13, 118.52, 131.82, 139.48,
150.34, 164.03, 171.00; FAB HRMS m/ z found (M + H)+
569.2728, C26H45N2O8Si2 requires (M + H)+ 569.2714.
2′-Deoxy-2′-r-C-[2-[[1-(2-flu or op h en yl)-4-m et h oxyp i-
p er id in -4-yl]oxy]et h yl]]-3′,5′-O-(1,1,3,3-t et r a isop r op yl-
d isiloxy)u r id in e (12). Alcohol 11 (1.5 g, 2.92 mmol) and 1-(2-
fluorophenyl)-4-methoxy-1,2,5,6-tetrahydropyridine (Fpmp vi-
nyl ether) [1.0 g, 4.8 mmol, prepared from 1-(2-fluorophenyl)-
4,4-dimethoxypiperidine as previously described37] were
dissolved in 50 mL of CH2Cl2. A solution of camphor-10-
sulfonic acid (450 mg, 1.46 mmol) in CH2Cl2 (15 mL) was added
dropwise over 15 min and the resulting solution stirred under
nitrogen for 16 h. TLC indicated that some starting material
was still present; therefore, a further portion of the Fpmp vinyl
ether (0.5 g, 2.4 mmol) and camphor-10-sulfonic acid (225 mg,
1.2 mmol) were added to the reaction mixture. After a further
4 h, the mixture was washed with saturated bicarbonate
solution (2 × 30 mL) and brine (2 × 20 mL). The organic layer
was separated, dried (Na2SO4), and filtered and the solvent
removed under reduced pressure. The required product was
isolated from the resulting residue by column chromatography,
eluting with CH2Cl2/MeOH (0-2%) to afford 12 as a pale
yellow foam (1.52 g, 73%): 1H NMR (400 MHz, CDCl3) δ 1.08-
1.01 (28H, m), 1.71-1.69 (1H, m), 1.93 (4H, br s), 2.31-2.26
(1H, m), 2.36-2.32 (1H, m), 3.06 (4H, br s), 3.21 (3H, s), 3.67
(2H, m), 3.94 (1H, m), 4.00 (1H, d, J )13.1 Hz), 4.13 (1H, d, J
) 13.1 Hz), 4.45 (1H, t, J ) 7.1 Hz), 5.69 (1H, d, J ) 8.1 Hz),
5.83 (1H, s), 7.02-6.95 (4H, m), 7.80 (1H, d, J ) 8.1 Hz), 10.14
(1H, s); 13C NMR (75.5 MHz, CDCl3) δ 11.20, 11.72, 12.08,
15.70, 15.62, 15.86, 15.96, 25.13, 32.97, 45.34, 47.38, 47.76,
57.49, 60.40, 68.46, 83.06, 89.07, 97.23, 101.65, 115.85 (d, J )
21.0 Hz), 119.33, 122.17, 124.25, 139.60, 140.35, 150.28, 154.85
(d, J ) 245.5 Hz), 163.93; FAB HRMS m/ z found M+ 721.3599,
C35H56N3O8FSi2 requires M+ 721.3590.
2′-Deoxy-2′-r-C-(2,3-d ih yd r oxyp r op yl)-3′,5′-O-(1,1,3,3-
tetr a isop r op yld isiloxy)u r id in e (13) (Mixtu r e of Dia ste-
r eoisom er s). (DHQD)2PYR (28 mg, 0.032 mmol), K2CO3
(1.321 g, 9.6 mmol) and K2Fe(CN)6 (3.150 g, 9.6 mmol), were
dissolved in 2-methyl-2-propanol (20 mL) and distilled water
(20 mL) and the mixture cooled to 0 °C. Osmium tetraoxide
(8.1 mg, 0.032 mmol, as a 1% solution in water) and 2′-C-allyl
nucleoside 5 (1.628 g, 3.2 mmol) were added to the mixture
with vigorous stirring. After 1.5 h, ethyl acetate (3 × 50 mL)
was added to the reaction mixture and the volume of solvent
reduced under reduced pressure to give a biphasic mixture.
Saturated sodium thiosulfate (2 × 15 mL) was added and the
mixture shaken vigorously. The organic layer was separated
and washed with saturated sodium bicarbonate (2 × 20 mL)
and brine (2 × 20 mL), dried (MgSO4), and filtered and the
solvent removed under reduced pressure to give a pale yellow
oil. Column chromatography over silica gel eluting with CH2-
Cl2 and methanol (0-4%) afforded the required diol as a white
foam 1.50 g (86% yield). Approximately 1 mg of this material
was treated with tetrabutylammonium fluoride (1 mL). HPLC
of the resulting solution indicated that the reaction gave a 82:
18 mixture of diastereoisomers (retention times of 9.56 and
10.01 min for the major and minor diastereoisomers, respec-
tively) 1H NMR (200 MHz, CDCl3) δ 1.22-0.77 (28H, m), 1.57-
1.32 (1 H, m), 2.44-2.31 (1 H, m), 2.75-2.60 (1 H, m), 3.75-
3.45 (2H, m), 4.14-3.85 (2H, m), 4.32-4.21 (2H, m), 4.39 (1
H, m), 5.71 (1 H, d, J ) 8.1 Hz), 5.78 (1 H, s), 7.96 (1 H, d, J
) 8.1 Hz), 9.83 (1 H, s); 13C NMR (50.4 MHz, CDCl3) δ 12.42,
12.88, 13.05, 13.37, 16.84, 16.92, 16.98, 17.06, 17.19, 17.30,
17.38, 17.49, 28.22, 44.77, 59.76, 67.17, 67.39, 68.63, 83.05,
88.93, 102.08, 139.58, 151.57, 164.03; CI HRMS m/ z found
(M + H)+ 545.2713, C24H45N2O8Si2 requires (M + H)+ 545.2714.
2′-Deoxy-2′-r-C-(2,3-d ia cet oxyp r op yl)-3′,5′-O-(1,1,3,3-
tetr a isop r op yld isiloxy)u r id in e (14). Diol 13 (1.00 g, 1.8
mmol) was dried by coevaporation with distilled pyridine (3
× 30 mL) and dissolved in pyridine (40 mL). The solution was
cooled to -5 °C and treated with 3,5-dinitrobenzoyl chloride
(0.610 g, 2.6 mol) under an atmosphere of nitrogen. After 2 h
the reaction was quenched by addition of methanol (10 mL)
and the solvent removed under reduced pressure. The residue
was coevaporated with toluene (3 × 15 mL) and dissolved in
CH2Cl2 (40 mL). The resulting solution was washed with
saturated sodium bicarbonate (2 × 20 mL) and brine (2 × 20
2′-D e o x y -2′-r-C-[[(d ip h e n y lm e t h y l)o x y ]c a r b o n y l]-
m eth yl]-3′,5′-O-(1,1,3,3-tetr aisopr opyldisiloxy)u r idin e (9).
The acid 7 (0.40 g, 0.76 mmol) was dissolved in CH2Cl2 (20
mL), washed with 1 N HCl (15 mL), and dried (MgSO4), and
the solvent was removed in vacuo. Diphenyldiazomethane
(0.20 g, 1.03 mmol) was placed into the same flask, and acetone
was added dropwise (∼0.3 mL) until all the solid had dissolved.
The mixture was left until bubbling had ceased (4 h), diluted
with ethyl acetate (50 mL), washed with saturated aqueous
NaHCO3 (2 × 30 mL) and brine (30 mL), dried (MgSO4), and
evaporated in vacuo to give a purple oil. The oil was purified
by column chromatography (CH2Cl2 containing an increasing
gradient of MeOH from 0-1%) yielding the required ester as
a white foam (0.53 g, 75% yield): 1H NMR (400 MHz, CDCl3)
0.92-1.06 (28H, m), 2.61 (1H, dd, J ) 6.2, 16.9 Hz), 2.76 (1H,
m), 2.95 (1H, dd, J ) 7.7, 16.8 Hz), 3.91 (1H, m), 3.97 (1H, dd,
J ) 5.4, 12.6 Hz), 4.02 (1H, dd, J ) 3.4, 12.2 Hz), 4.58 (1H, t,
J ) 6.3 Hz), 5.67 (1H, dd, J ) 2.2, 8.1 Hz), 5.82 (1H, d, J )
4.9 Hz), 6.86 (1H, s), 7.32 (10H, m), 7.43 (1H, d, J ) 8.1 Hz),
8.27 (1H, bs); 13C NMR (50.4 MHz, CDCl3) δ 12.25-13.09,
16.59-17.29, 31.64, 44.41, 61.72, 70.32, 77.13, 84.20, 87.84,
102.28, 126.87, 127.62, 128.27, 139.08, 139.97, 150.21, 163.34,
170.51; FAB HRMS m/ z found (M
+
Na)+ 717.3020,
C36H50N2O8Si2Na requires (M + Na)+ 717.3003.
2′-Deoxy-2′-r-C-a ceta m id o-3′,5′-O-(1,1,3,3-tetr a isop r o-
p yld isiloxy)u r id in e (10). The acid 7 (1.02 g, 1.93 mmol),
dicyclohexylcarbodiimide (0.51 g, 2.47 mmol), 4-(dimethylami-
no)pyridine (70 mg, 0.57 mmol), and N-hydroxysuccinimide
(0.29 g, 2.52 mmol) were dissolved in dry dioxane (26 mL) and
the mixture stirred for 16 h. The solution was filtered and
the residue washed with dry dioxane (10 mL). To the
combined filtrates was added excess concentrated aqueous NH3
(2.5 mL, 33% solution) and the solution stirred. After 10 min,
the reaction mixture was diluted with CH2Cl2 (150 mL),
washed with saturated aqueous NaHCO3 (2 × 100 mL) and
brine (100 mL), dried (MgSO4), and evaporated in vacuo. The
residue was purified by column chromatography (CH2Cl2
containing an increasing gradient of MeOH from 0-5%),
yielding the required product as a white foam (0.80 g, 78%
yield): 1H NMR (400 MHz, CDCl3) δ 1.02-1.09 (28H, m), 2.48
(1H, dd, J ) 10.4, 17.0 Hz), 2.66-2.75 (2H, m), 3.91 (1H, dd,
J ) 7.3, 11.6 Hz), 3.99 (1H, ddd, J ) 3.3, 5.0, 7.3 Hz), 4.08
(1H, dd, J ) 3.3, 11.6 Hz), 4.52 (1H, dd, J ) 5.0, 7.1 Hz), 5.75
(1H, d, J ) 8.1 Hz), 5.97 (1H, d, J ) 6.0 Hz), 6.30 (1H, br s),
7.08 (1H, br s), 7.48 (1H, d, J ) 8.1 Hz), 10.66 (1H, br s); 13C
NMR (50.4 MHz, CDCl3) δ 13.01-13.33, 16.75-17.40, 31.55,
44.73, 62.71, 72.04, 85.17, 87.84, 103.07, 139.03, 151.51,
163.45, 174.09; FAB HRMS m/ z found (M + H)+ 528.2562,
C23H42N3O7Si2 requires (M + H)+ 528.2561.
2′-Deoxy-2′-r-C-(2-h yd r oxyeth yl)-3′,5′-O-(1,1,3,3-tetr a -
isop r op yld isiloxy)u r id in e (11). Aldehyde 6 (3.0 g, 5.9
mmol) was dissolved in cold methanol (10 mL) and added,
dropwise over 5 min with stirring, to a suspension of sodium
borohydride (0.664 g, 17.5 mmol) in methanol (20 mL) at 0
°C. After 20 min the reaction mixture was neutralized with
citric acid and the solvent evaporated in vacuo. The residue
was taken up in CH2Cl2 (100 mL) and washed with water (2
× 30 mL), saturated sodium bicarbonate solution (2 × 30 mL)
and brine (2 × 20 mL). The organic layer was separated, dried
(MgSO4), and filtered and the solvent removed in vacuo to
afford the required product as a white foam (3.06 g, 100%
yield): 1H NMR (400 MHz, CDCl3) δ 1.26-1.00 (28H), 1.68-
1.59 (1H, m), 2.17-2.08 (1H, m), 2.50-2.46 (1H, m), 3.96-
3.92 (3H, m), 4.40 (1H, dd, J ) 13.4, 2.5 Hz), 4.24 (1H, d, J )
13.4 Hz), 4.43 (1H, dd, J ) 7.4, 8.9 Hz), 5.73 (1H, d, J ) 8.1
Hz), 5.88 (1H, s), 7.98 (1H, d, J ) 8.1 Hz), 10.38 (1H, s); 13C
NMR (50.4 MHz; CDCl3) δ 12.48, 12.85, 13.04, 13.34, 16.82,
16.87, 16.96, 17.03, 17.17, 17.28, 17.36, 17.46, 27.79, 46.26,
59.90, 60.25, 67.47, 82.99, 88.98, 101.81, 139.57, 151.22,
164.11; FAB HRMS m/ z found (M + H)+ 515.2612, C23H43N2O7-
Si2 requires 515.2609. Anal. Calcd for C23H42O7Si2N2‚H2O:
C, 51.85; H, 8.32; N, 5.26. Found: C, 51.58; H, 7.95; N, 5.06.
(37) Reese, C. B.; Thompson, E. A. J . Chem. Soc., Perkin Trans. 1
1988, 2881.

9220 J . Org. Chem., Vol. 61, No. 26, 1996
Lawrence et al.
mL). The organic layer was separated, dried (MgSO4), and
filtered and the solvent removed to give an orange oil. The
crude product was recrystallized (pentane/ethyl acetate) to
afford the 3,5-dinitrobenzoate monoester (713 mg, 64%, based
on starting material being only 82% of one diastereoisomer).
[Approximately 1 mg of this material was treated with
methanolic ammonia (1 mL). After 1 h the solvent was
evaporated and the residue treated with tetrabutlyammonium
fluoride (1 mL); HPLC analysis of the resulting solution
showed the product to be a single diastereoisomer.] The
dinitrobenzoate (685 mg, 0.93 mmol) was dissolved in satu-
rated methanolic ammonia (20 mL). After 16 h the solvent
was evaporated and the residue coevaporated with dry pyri-
dine (3 × 20 mL). The resulting black residue was dissolved
in pyridine (20 mL) and treated with acetic anhydride (1.73
mL, 18.6 mmol). After 20 h the solvent was evaporated, and
traces of pyridine were removed by coevaporation with toluene
(3 × 30 mL). The residue was taken up in CH2Cl2 (30 mL),
washed with saturated sodium bicarbonate (2 × 20 mL) and
brine (2 × 20 mL), dried (MgSO4), and filtered and the solvent
removed under reduced pressure. Column chromatography
over silica gel eluting with CH2Cl2 and methanol (0-2%)
afforded the required product 14 as a white foam (573 mg,
98% yield, 52% from diol 13): 1H NMR (400 MHz, CDCl3) δ
1.10-1.03 (28H, m), 1.77-1.70 (1H, m), 2.03 (3H, s), 2.07 (3H,
s), 2.23-2.17 (1H, m), 2.35-2.32 (1H, m), 3.93-3.87 (1H, m),
3.99 (1H, dd, J ) 2.88, 13.2 Hz), 4.13 (1H, dd, J ) 2.5, 13.2
Hz), 4.18 (1H, dd, J ) 5.1, 12.0 Hz), 4.29 (1H, dd, J ) 3.7 Hz,
12.0), 4.46 (1H, t, J ) 7.7 Hz), 5.34-5.29 (1H, m), 5.69 (1H,
dd, J ) 2.1, 8.2 Hz), 5.77 (1H, d, J ) 2.1 Hz), 7.74 (1H, d, J )
8.1 Hz), 9.08 (1H, s); 13C NMR (50.4 MHz, CDCl3) δ 12.30,
12.68, 12.74, 13.08, 16.56, 16.61, 16.67, 16.72, 16.91, 16.99,
17.02, 17.15, 20.34, 20.60, 26.80, 44.11, 60.34, 64.47, 68.54,
69.29, 82.96, 88.70, 101.64, 139.05, 149.94, 163.34, 169.97,
pound was formally characterized by conversion to u r id in e
2′-r-C-3′-O-γ-bu tyr ola cton e (19). 2′-Deoxy-2′-R-C-carboxy-
methyluridine (292 mg, 1.09 mmol) was dissolved in acetic
acid/methanol (4:1; 29.2 mL) and heated at 60 °C for 3 h. The
solution was evaporated in vacuo, and the residual acetic acid
was coevaporated with water (2 × 30 mL) and dried over P2O5
for 48 h. The product was isolated as a pure white crystalline
solid (273 mg, 96% yield): mp 221-222 °C. 1H NMR (400
MHz, d6-DMSO) 2.64 (1H, dd, J ) 2.3, 18.2 Hz), 2.92 (1H, dd,
J ) 9.2, 18.2 Hz), 3.17 (1H, m), 3.65 (3H, d, J ) 3.9 Hz), 4.22
(1H, m), 5.04 (1H, dd, J ) 2.4, 7.3 Hz), 5.22 (1H, t, J ) 5.1
Hz), 5.68 (1H, d, J ) 8.0 Hz), 5.86 (1H, d, J ) 6.4 Hz), 7.83
(1H, d, J ) 8.0 Hz), 11.39 (1H, bs); 13C NMR (100.6 MHz, d6-
DMSO) 31.75, 44.76, 61.02, 74.87, 84.08, 84.73, 90.24, 101.96,
140.51, 150.63, 163.16, 175.67; HRMS m/ z found (M + H)+
269.0754, C11H13N2O6 requires (M + H)+ 269.0774; IR νmax
/
cm-1 (KBr) 1780 (CdO lactone). Anal. Calcd for C11H12N2O6:
C, 49.26; H, 4.51; N, 10.44. Found: C, 49.07; H, 4.55; N, 10.39.
2′-Deoxy-2′-r-C-a ceta m id ou r id in e (2): 78% yield; 1H
NMR (400 MHz, d6-DMSO) δ 2.10 (1H, dd, J ) 6.1, 15.7 Hz),
2.43 (1H, dd, J ) 7.7, 15.7 Hz), 2.58 (1H, m), 3.55 (2H, d, J )
3.8 Hz), 3.85 (1H, dd, J ) 3.1, 3.8 Hz), 4.15 (1H, d, J ) 5.3
Hz), 5.11 (1H, br s), 5.35 (1H, br s), 5.67 (1H, d, J ) 8.2 Hz),
5.91 (1H, d, J ) 9.4 Hz), 6.82 (1H, s), 7.34 (1H, s), 7.84 (1H, d,
J ) 8.2 Hz); 13C NMR (100.6 MHz, CD3OD) δ 33.68, 46.87,
63.79, 74.56, 89.28, 89.68, 103.46, 142.96, 152.95, 166.36,
176.80; FAB HRMS m/ z found (M + H)+ 286.1031, C11H16N3O6
requires (M + H)+ 286.1039; HPLC retention time 9.19 min.
2′-Deoxy-2′-r-C-(2-h yd r oxyeth yl)u r id in e (3): 88% yield;
1H NMR (400 MHz, D3COD) δ 1.64-1.51 (1H, m), 2.01-1.84
(1H, m), 2.48-2.33 (1H, m), 3.60 (2H, t, J ) 6.3 Hz), 3.73 (2H,
d, J ) 3.6 Hz), 3.99-3.97 (1H, m), 4.29 (1H, d, J ) 5.4 Hz),
5.72 (1H, d, J ) 8.1 Hz), 6.04 (1H, d J ) 9.2 Hz), 7.97 (1H, d,
J ) 8.1 Hz); 13C NMR (50.4 MHz, D3COD) δ 28.03, 47.89,
60.86, 63.56, 73.77, 88.89, 89.84, 103.18, 142.59, 152.69,
165.96; FAB HRMS m/ z found (M + 2H)+ 274.1156, C11H18N2O6
requires (M + 2H)+ 274.1165; HPLC retention time 10.64 min.
2′-Deoxy-2′-r-C-(2,3-d ih yd r oxyp r op yl)u r id in e (4): 80%
170.40; CI HRMS m/ z found (M + H)+ 629.2920, C28H49N2O10
-
Si2 requires (M + H)+ 629.2926.
2′-Deoxy-2′-r-C-(cya n om eth yl)-3′,5′-O-(1,1,3,3-tetr a iso-
p r op yld isiloxy)u r id in e (25). To a stirred solution of 2′-C-
acetamido-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxy)uridine (10)
(130 mg, 0.247 mmol) in dry dioxane (2 mL) at 0 °C was added
dry pyridine (0.04 mL, 0.494 mmol) followed by dropwise
addition of trifluoroacetic anhydride (0.04 mL, 0.288 mmol)
over 10 min. The reaction mixture was left to warm to room
temperature over 30 min and then diluted with CH2Cl2 (30
mL), washed with water (2 × 20 mL) and brine (20 mL), dried
(MgSO4), and concentrated in vacuo. Following column chro-
matography (CH2Cl2 containing an increasing gradient of
MeOH from 0-3%), the pure product was isolated as a white
foam (96 mg, 76% yield): 1H NMR (400 MHz, CDCl3) δ 1.05-
1.09 (28H, m), 2.63 (1H, m), 2.71 (1H, dd, J ) 5.4, 16.9 Hz),
2.81 (1H, dd, J ) 7.8, 16.9 Hz), 4.02-4.12 (3H, m), 4.58 (1H,
t, J ) 7.8 Hz), 5.75 (1H, d, J ) 8.2 Hz), 5.80 (1H, d, J ) 2.7
Hz), 7.67 (1H, d, J ) 8.2 Hz), 9.73 (1H, br s); 13C NMR (50.4
MHz, CDCl3) δ 12.52-13.25, 14.65, 16.70-17.32, 45.33, 60.50,
68.54, 83.28, 88.11, 102.28, 117.84, 138.81, 150.35, 163.55;
found FAB HRMS m/ z found (M + H)+ 510.2454, C23H40N3O6-
Si2 requires (M + H)+ 510.2456.
Gen er a l P r oced u r e for th e Rem ova l of th e 1,1,3,3-
Tetr a isop r op yld isiloxy P r otectin g Gr ou p . The TIPS-
protected nucleoside (0.45 mmol) was dissolved in dry THF
(30 mL), and to this was added NEt3‚3HF (0.08 mL, 5 mmol).
The solution was left for 8-16 h and monitored by TLC. The
solution was concentrated in vacuo, diluted with methanol (20
mL), and evaporated onto silica gel; the product was subse-
quently isolated by column chromatography (CH2Cl2 contain-
ing an increasing gradient of MeOH from 0-10%) as a colorless
oil or white foam.
1
yield; H NMR (200 MHz, CD3OD) 1.42-1.74 (2H, m), 2.31-
2.45 (1H, m), 3.33 (2H, s), 3.44-3.56 (1H, m), 3.62 (1H, s), 3.64
(1H, s), 3.87-3.90 (1H, m), 4.23 (1H, dd, J ) 1.5 Hz, J ′ ) 4.8
Hz), 5.62 (1H, d, J ) 8.1 Hz), 5.92 (1H, d, J ) 8.9 Hz), 7.87
(1H, d, J ) 8.1 Hz); 13C NMR (50.4 MHz, CD3OD) 28.89, 47.24,
63.53, 67.51, 71.61, 74.58, 88.30, 89.90, 103.18, 142.64, 152.67,
165.96; FAB HRMS m/ z found (M
+
Na)+ 325.1018,
C12H18N2O7Na requires 325.1012; HPLC retention time 10.01
min. Pure samples of 4 were most readily obtained by
deprotection of 20b.
2′-Deoxy-2′-r-C-(O-d ip h en ylm et h yl ca r b oxym et h yl)-
1
u r id in e (16): 87% yield; H NMR (400 MHz, CDCl3) δ 3.09
(1H, dd, J ) 5.9, 15.9 Hz), 3.26 (1H, m), 3.37 (1H, dd, J ) 6.9,
15.9 Hz), 4.22 (2H, m), 4.47 (1H, m), 4.82 (1H, dd, J ) 1.4, 5.5
Hz), 6.10 (1H, d, J ) 8.1 Hz), 6.53 (1H, d, J ) 8.5 Hz), 7.24
(1H, s), 7.72 (10H, m), 8.37 (1H, d, J ) 8.1 Hz); 13C NMR (50.4
MHz, CD3OD) δ 30.78, 46.03, 63.34, 73.99, 78.71, 88.92, 89.54,
103.13, 127.98, 128.87, 129.49, 141.59, 142.43, 152.59, 165.96,
172.59; FAB HRMS m/ z found (M + H)+ 453.1630, C24H25N2O7
requires (M + H)+ 453.1662.
2′-Deoxy-2′-r-C-[2-[[1-(2-flu or op h en yl)-4-m et h oxyp i-
p er id in -4-yl]oxy]eth yl]u r id in e (17): 76% yield; 1H NMR
(400 MHz, CDCl3) δ 1.60-1.59 (1H, m), 2.05-1.82 (5H, m),
2.47-2.38 (1H, m), 3.10-2.93 (4H, m), 3.11 (3H, s), 3.45-3.43
(1H, m), 3.72-3.61 (2H, m), 3.80 (1H, d, J ) 11.5 Hz, 3.91
(1H, d, J ) 11.5 Hz), 4.46 (1H, d, J ) 5.1 Hz), 5.76 (1H, d, J
) 8.1 Hz), 5.93 (1H, d, J ) 9.0 Hz), 7.05-6.91 (4H, m), 7.73
(1H, d, J ) 8.1 Hz); 13C NMR (75.5 MHz, CDCl3) δ 24.46, 32.81,
33.42, 47.98, 49.27, 58.88, 63.30, 73.43, 86.34, 90.35, 99.01,
102.92, 116.90 (d, J ) 21.1 Hz), 119.50, 122.70 (d, J ) 8.0 Hz),
124.46 (d, J ) 3.0 Hz), 139.99 (d, J ) 9.1 Hz), 141.31, 150.78,
155.79 (d, J ) 245.5 Hz), 163.43; FAB HRMS m/ z found M+
479.2036, C23H30N3O7F requires M+ 479.2068.
2′-Deoxy-2′-r-C-(ca r b oxym et h yl)u r id in e t r iet h yla m -
1
m on iu m sa lt (1): 85% yield; H NMR (400 MHz, CD3OD) δ
1.30 (9H, t, J ) 7.2 Hz), 2.31 (1H, dd, J ) 5.8, 15.5 Hz), 2.62
(1H, dd, J ) 7.9, 15.5 Hz), 2.70 (1H, m), 3.17 (6H, q, J ) 7.2
Hz), 3.75 (2H, d, J ) 3.5 Hz), 4.00 (1H, m), 4.35 (1H, m), 5.73
(1H, dd, J ) 8.1, 2.2 Hz), 6.04 (1H, d, J ) 8.8 Hz) 7.98 (1H, d,
J ) 8.1 Hz); 13C NMR (50.4 MHz, CD3OD) δ 9.34, 33.10, 47.34,
47.70, 63.85, 74.62, 89.00, 89.87, 103.27, 143.07, 152.81,
166.24, 178.59; FAB+ m/ z 287 (M + H+, 3.5), 269 [M + H -
H2O+ (lactone)]; HPLC retention time 9.41 min. This com-
2′-Deoxy-2′-r-C-(2,3-d ia cetoxyp r op yl)u r id in e (18): 81%
1
yield; H NMR (400 MHz, CD3OD) δ 1.84-1.75 (1H, m), 1.97
(3H, s), 2.00 (3H, s), 2.12-2.03 (1H, m), 2.42-2.40 (1H, m),
3.73 (1H, s), 3.74 (1H, s), 4.02-3.97 (3H, m), 4.28-4.24 (2H,
m), 5.72 (1H, dd, J ) 8.1, 0.5 Hz), 6.06 (1H, d, J ) 9.1 Hz),
7.96 (1H, d, J ) 8.1 Hz); 13C NMR (50.4 MHz, CDCl3) δ 20.42,

Synthesis of 2′-Deoxy-2′-R-C-branched Nucleosides
J . Org. Chem., Vol. 61, No. 26, 1996 9221
20.78, 26.57, 45.47, 63.31, 65.76, 70.75, 74.50, 88.76, 89.62,
103.45, 142.18, 152.48, 165.77, 171.92, 172.27; CI HRMS m/ z
found (M + H)+ 387.1404, C16H23N2O9 requires (M + H)+
387.1403.
2′-Deoxy-2′-r-C-(cya n om eth yl)u r id in e (26): 81% yield;
1H NMR (200 MHz, CD3OD) δ 2.53-2.81 (3H, m), 3.71 (2H, d,
J ) 3.0 Hz), 3.98 (1H, m), 4.29 (1H, dd, J ) 1.7 Hz, J ) 3.3
Hz), 5.70 (1H, d, J ) 8.1 Hz), 6.02 (1H, d, J ) 8.5 Hz), 7.92
(1H, d, J ) 8.1 Hz); 13C NMR (50.4 MHz, CD3OD) δ 13.69,
46.68, 63.12, 73.50, 88.87, 89.03, 103.45, 119.57, 142.08,
152.64, 165.99; FAB HRMS m/ z found (M + H)+ 268.0933,
C11H14N3O5 requires (M + H)+ 268.0934; HPLC retention time
13.47 min.
13C NMR (100.6 MHz, CD3OD) δ 31.83, 47.40, 55.89, 64.47,
74.06, 86.59, 87.49, 88.82, 103.57, 112.67, 113.95, 127.71,
128.65, 128.82, 130.76, 135.96, 136.14, 141.02, 144.98, 152.36,
159.26, 164.45, 175.74; FAB HRMS m/ z found (M + H)+
588.2330, C32H34N3O8 requires (M + H)+ 588.2346.
Gen er a l Meth od for th e P r ep a r a tion of Nu cleosid e 3′-
[(2-Cya n oeth yl)-N,N-d iisop r op ylp h osp h or a m id ites). The
5′-O-tritylated nucleoside (0.613 mmol) and diisopropylam-
monium tetrazolide (53 mg, 0.307 mmol) were dissolved in dry
CH2Cl2 and the solution purged with nitrogen. To this was
added 2-cyanoethyl bis(N,N-diisopropylamino)phosphoramid-
ite (0.22 mL, 0.674 mmol) and the reaction left for 16 h. The
mixture was diluted with CH2Cl2 (20 mL) and washed with
saturated aqueous NaHCO3 (3 × 6 mL) and brine (6 mL), the
aqueous layers were combined and back-extracted with CH2-
Cl2 (2 × 2 mL), and the organics were dried (Na2SO4) and
evaporated. The crude residue was purified by column chro-
matography, the eluent system depending on the polarity of
the compound, to yield the product, a mixture of diastereo-
isomers, as a white foam.
2′-Deoxy-2′-r-C-[2-[[1-(2-flu or op h en yl)-4-m et h oxyp i-
p er id in -4-yl]oxy]eth yl]-5′-O-(d im eth oxytr ityl)u r id in e 3′-
(2-cya n oeth yl N,N-d iisop r op ylp h osp h or a m id ite) (21a ):
eluent, hexane/CH2Cl2/NEt3 (10:9:1); yield 70%; 1H NMR (200
MHz, CDCl3) δ 1.23-0.89 (12H, m), 2.20-1.89 (7H, m), 2.34-
2.21 (1H, m), 2.71-2.82 (1H, m), 3.15-2.89 (4H, m), 3.18*,
3.20* (3H, s), 3.75-3.27 (9H, m), 3.77 (6H, s), 4.73-4.52 (1H,
m), 5.35*, 5.40* (1H, d, J ) 8.8 Hz, J ′ ) 9.0 Hz), 6.10*, 6.15*
(1H, d, J ) 8.8 Hz, J ′ ) 9.0 Hz), 7.40-6.79 (17H, m), 7.80*,
7.86* (1H, d, J ) 8.1 Hz, J ′ ) 8.1 Hz); 31P NMR (25 MHz,
CDCl3) δ 148.64, 151.47; m/ z (FAB+) 981 (M+, 100).
Gen er a l Meth od for th e P r ep a r a tion of Nu cleosid e 5′-
O-d im Eth oxytr ityl Eth er s. The nucleoside (0.30 mmol) was
dried by coevaporation with dry pyridine (2 × 3 mL) and
dissolved in the same solvent (1.3 mL). Dimethoxytrityl
chloride (170 mg, 0.50 mmol) was dissolved in a mixture of
dry pyridine and dichloromethane (0.2 and 0.75 mL, respec-
tively) and added to the nucleoside solution dropwise over 30
min, and the mixture was stirred for a further 1 h. Methanol
(0.1 mL) was added, and after a further 15 min saturated
aqueous NaHCO3 (0.5 mL) was also added; the mixture was
then concentrated in vacuo to a thick oil. The residue was
coevaporated with toluene (2 × 20 mL), diluted with CH2Cl2
(5 mL), and washed with saturated aqueous NaHCO3 (2 × 2.5
mL) and brine (2.5 mL), the aqueous layers were combined
and back-extracted with CH2Cl2 (5 mL), and the organics were
dried (MgSO4) and evaporated. The crude product was puri-
fied by column chromatography (CH2Cl2 containing an in-
creasing gradient of MeOH from 0 to 3%) to yield the required
DMT derivative as a white/cream foam.
2′-Deoxy-2′-r-C-[2-[[1-(2-flu or op h en yl)-4-m et h oxyp i-
p er id in -4-yl]oxy]et h yl]-5′-O-(d im et h oxyt r it yl)u r id in e
(20a ): 75% yield. 1H NMR (400 MHz, CDCl3) δ 1.79-1.89
(1H, m), 1.91-2.18 (5H, m), 2.33-2.45 (1H, m), 3.12-3.02 (4H,
m), 3.24 (3H, s), 3.43-3.53 (2H, m), 3.58-3.46 (1H, m), 3.76-
3.74 (8H, m), 4.19 (1H, s), 4.58 (1H, d, J ) 4.9 Hz), 5.43 (1H,
d, J ) 8.1 Hz), 6.13 (1H, d, J ) 8.8 Hz), 7.02-6.79 (8H, m),
7.19-7.36 (9H, m), 7.75 (1H, d, J ) 8.1 Hz); 13C NMR (75.5
MHz, CDCl3) δ 22.65, 24.29, 31.58, 33.03, 33.32, 47.84, 47.93,
50.38, 52.82, 55.20, 58.99, 64.26, 73.71, 85.39, 85.93, 88.27,
98.99, 102.79, 98.99, 102.79, 113.28, 116.03 (d, J ) 21.1 Hz),
119.38 (d, J ) 2.0 Hz), 122.48 (d, J ) 8.0 Hz), 124.45 (d, J )
3.0 Hz), 127.13, 127.98, 128.05, 130.05, 135.25, 135.40, 139.95
(d, J ) 9.1 Hz), 140.30, 144.44, 150.94, 154.48, 157.81 (d, J )
245.5 Hz), 163.47; FAB HRMS m/ z found M+ 781.3383,
C44H48N3O9F requires M+ 781.3375.
2′-Deoxy-2′-r-C-(2,3-d ia cetoxyp r op yl)-5′-O-(d im eth ox-
ytr ityl)u r id in e (20b): 94% yield; 1H NMR (400 MHz, CDCl3)
δ 1.89-1.70 (1H, m), 2.20-1.97 (7H, m), 2.49-2.34 (1H, m),
3.41 (2H, s), 3.75 (6H, s), 4.32-4.03 (4H, m), 4.48 (1H, d, J )
6.0), 5.07-4.88 (1H, br s), 5.36 (1H, d, J ) 8.0 Hz), 6.07 (1H,
d, J ) 8.5 Hz), 7.39-6.76 (13H, m), 7.68 (1H, d, J ) 8.0 Hz);
13C NMR (50.4 MHz, CDCl3) δ 20.39, 20.79, 22.36, 24.92, 45.83,
46.32, 54.98, 63.82, 64.06, 70.56, 73.04, 85.76, 86.89, 87.53,
102.61, 113.09, 126.92, 127.75, 127.89, 129.86, 134.90, 135.06,
139.88, 143.98, 150.67, 158.51, 163.06, 170.48, 170.83; CI
HRMS m/ z found M+ 688.2614, C37H40N2O11 requires 688.2632.
2′-De oxy-2′-r-C-[[[(d ip h e n ylm e t h yl)oxy]ca r b on yl]-
m eth yl]-5′-O-(d im eth oxytr ityl)u r id in e (20c): 86% yield:
1H NMR (400 MHz, CDCl3) 2.62-2.77 (2H, m), 2.94 (1H, dd,
J ) 11.5, 17.6 Hz), 3.40 (2H, m), 3.79 (3H, s), 4.10 (1H, m),
4.54 (1H, dd, J ) 2.2, 5.2 Hz), 5.39 (1H, dd, J ) 2.2, 8.2 Hz),
6.05 (1H, d, J ) 8.1 Hz), 6.82 (4H, d, J ) 8.7 Hz), 6.88 (1H, s),
7.24-7.35 (19H, m), 7.64 (1H, d, J ) 8.2 Hz), 8.20 (1H, bs);
13C NMR (75.5 MHz, CDCl3) 29.88, 45.96, 55.12, 63.42, 72.98,
77.95, 85.65, 86.96, 87.64, 102.80, 113.31, 126.91, 127.18,
128.01, 128.17, 128.64, 130.11, 134.78, 139.44, 139.63, 144.10,
150.31, 158.51, 162.23, 171.12; FAB HRMS m/z found M+
754.2897, C45H42N2O9 requires M+ 754.2890.
2′-Deoxy-2′-r-C-(2,3-d ia cetoxyp r op yl)-5′-O-(d im eth ox-
yt r it yl)u r id in e 3′-(2-cya n oet h yl N,N-d iisop r op ylp h os-
p h or a m id ite) (21b): eluent, hexane/CH2Cl2/NEt3 (10:9:1);
1
yield 73%; H NMR (200 MHz, CDCl3) δ 1.30-1.03 (12H, m),
1.83-1.65 (1H, m), 1.95*, 1.97* (3H, s), 1.99*, 2.01* (3H, s),
2.78-2.27 (4H, m), 3.71-3.35 (6H, m), 3.76 (6H, s), 4.36-3.90
(4H, m), 4.63-4.38 (1H, m), 5.30*, 5.49* (1H, d, J ) 8.1 Hz, J ′
) 8.1 Hz), 6.06*, 6.10* (1H, d, J ) 6.0 Hz, J ′′ ) 6.1 Hz), 7.38-
6.76 (13H, m), 7.68*, 7.73* (1H, d, J ) 8.1 Hz, J ′ ) 8.1); 31P
NMR (101 MHz, CDCl3) δ 147.00, 149.11; CI HRMS m/ z found
(M + H)+ 889.3756, C46H58N4O12P requires 889.3789.
2′-De oxy-2′-r-C-[[[(d ip h e n ylm e t h yl)oxy]ca r b on yl]-
m et h yl]-5′-O-(d im et h oxyt r it yl)u r id in e 3′-(2-cya n oet h yl
N,N-d iisop r op ylp h osp h or a m id ite) (21c): eluent, hexane/
EtOAc/NEt3 (10:9:1); 94% yield; 1H NMR (200 MHz, CDCl3) δ
1.06-1.16 (12H, m), 2.20-2.35 (2H, m), 2.58 (1H, m), 2.87-
3.00 (2H, m), 3.40-3.70 (6H, m), 3.78 (6H, s), 4.11-4.34 (1H,
m), 4.63*, 4.76* (1H, dd, J ) 5.0, 9.6, 4.0, 11.6 Hz), 5.35*, 5.39*
(1H, d, J ) 8.4, 6.6 Hz), 6.07*, 6.11* (1H, d, J ) 8.8, 5.9 Hz),
6.80-6.85 (4H, m), 6.88*, 6.94* (1H, s), 7.24-7.42 (19H, m,),
7.59*, 7.64* (1H, d, J ) 8.2, 8.2 Hz); 31P NMR (101 MHz,
CDCl3) δ 150.01, 151.27; FAB+ m/ z 977 (M + Na+, 3.3), 955
(M + H+, 9.6).
2′-Deoxy-2′-r-C-a cet a m id o-5′-O-(d im et h oxyt r it yl)u r i-
d in e 3′-(2-cya n oeth yl N,N-d iisop r op ylp h osp h or a m id ite)
(21d ): eluent, EtOAc/CH2Cl2/NEt3 (9:9:2); 29% yield; 1H NMR
(200 MHz, CDCl3) δ 1.12-1.26 (12H, m), 1.95-2.35 (2H, m),
2.40-3.09 (3H, m), 3.37-3.65 (6H, m), 3.80 (6H, s), 4.26*, 4.39*
(1H, m), 4.44*, 4.54* (1H, dd, J ) 8.2, 14.0, 7.6, 15.7 Hz), 5.45*,
5.46* (1H, d, J ) 7.9, 6.0 Hz), 5.68*, 5.96* (1H, br s), 6.15*,
6.17* (1H, d, J ) 9.5, 9.9 Hz), 6.72*, 6.84-6.86 (5H, br s, m),
7.27-7.43 (9H, m), 7.62*, 7.70* (1H, d, J ) 8.1, 8.2 Hz); 31P
NMR (81 MHz, D2O) δ 152.27, 152.77; m/ z FAB+ m/z 826 (M
+ Na + O+, 6.0), 810 (M + Na+, 1.6).
Gen er a l Meth od for th e P r ep a r a tion of th e 2′-Mod ified
Din u cleosid e Mon op h osp h a tes. 2′,3′-Di-O-acetyluridine38
(22 mg, 0.067 mmol) and freshly sublimed tetrazole (23 mg,
0.329 mmol) were dissolved in dry CH3CN and purged with
argon, and the mixture was stirred to effect solution. To this
an argon-purged solution of the nucleoside phosphoramidite
(0.100 mmol) in dry CH3CN (2 mL) was added dropwise over
10 min. After being stirred for a further 50 min, the reaction
mixture was analyzed by 31P NMR. A 4% (w/v) solution of I2
2′-Deoxy-2′-r-C-a cet a m id o-5′-O-(d im et h oxyt r it yl)u r i-
d in e (20d ): 56% yield; 1H NMR (400 MHz, CDCl3) δ 2.56 (1H,
m), 2.69 (1H, m), 3.37 (2H, br s), 3.74 (3H, s), 4.15 (1H, br s),
4.46 (1H, br s), 5.40 (1H, d, J ) 8.1 Hz), 6.07 (1H, d, J ) 7.6
Hz), 6.58 (1H, br s), 6.81 (4H, d, J ) 8.8 Hz), 6.93 (1H, br s),
7.17-7.39 (9H, m), 7.65 (1H, d, J ) 8.1 Hz), 8.34 (1H, br s);
(38) Kenner, G. W.; Todd, A. R.; Webb, R. F.; Weymouth, F. J . J .
Chem. Soc. 1954, 2288.

9222 J . Org. Chem., Vol. 61, No. 26, 1996
Lawrence et al.
in 2,6-lutidine/water/THF (1:1:8) was then added dropwise to
the mixture until a deep red color persisted. The solution was
diluted with CH2Cl2 (30 mL) and washed with 10% Na2S2O4
solution (2 × 20 mL) and brine (20 mL), the aqueous layers
were combined and back-extracted with CH2Cl2 (15 mL), and
the organics were dried (MgSO4) and evaporated in vacuo. The
crude product was purified by column chromatography (CH2-
Cl2 containing an increasing gradient of MeOH from 0-3%)
to yield the required dinucleoside monophosphate, which was
immediately deprotected.
Dep r otection of th e Din u cleosid e Mon op h osp h a tes:
(i) Gen er a l P r oced u r e for th e Rem ova l of Acetyl, 2-Cy-
a n oeth yl, a n d DMT P r otectin g Gr ou p s. The protected
dinucleoside monophosphate (∼0.02 mmol) was dissolved in
saturated methanolic NH3 (10 mL) and left overnight. Solvent
was removed in vacuo and the residue coevaporated twice with
methanol (2 × 10 mL). The dimer was subsequently taken
up into 80% aqueous acetic acid and left for a further 2 h before
solvent was again removed in vacuo and the residue coevapo-
rated twice with water (2 × 10 mL). The product was dissolved
in water (10 mL), and extracted with EtOAc (3 × 10 mL) and
the aqueous layer evaporated in vacuo to yield the crude
product as a colorless oil.
(ii) Rem ova l of th e F p m p P r otectin g Gr ou p . Following
treatment with methanolic ammonia, the dimer was taken up
into acetate buffer (1 mL, pH 3.25, 100 mM, supplied by
Cruachem, Glasgow, U.K.) and stirred at room temperature
for 5 h. After this period, glacial acetic acid (1.5 mL) was
added and the suspension stirred for a further 1 h. The solvent
was evaporated and the residue coevaporated and extracted
as described above.
(iii) Rem ova l of th e Ben zh yd r yl P r otectin g Gr ou p .
Following treatment with methanolic ammonia and 80%
aqueous acetic acid, the residue was taken up into MeOH/THF
(3:1; 1 mL), and Pd/C catalyst was suspended in the solution
by vigorous stirring. The reaction vessel was evacuated and
purged with H2 three times and the mixture left for 1.5 h. The
solution was diluted with MeOH/THF (3:1, 3 mL), filtered
through Celite, and evaporated to dryness in vacuo.
Purification of the deprotected dinucleoside monophosphates
was carried out by HPLC on a Nucleosil C18 reversed-phase
column, using a gradient of 0 f 10% MeCN in 50 mM
triethylammonium bicarbonate (pH 8.0) over 20 min, with a
flow rate of 1 mL/min.
64.67, 67.83, 72.56, 76.57, 80.53, 85.93, 88.58, 91.32, 92.03,
105.57, 105.82, 144.66, 144.81, 154.74, 168.99, 179.74; 31P
NMR (101 MHz, D2O) δ 1.27; FAB- m/ z (glycerol matrix) 591
(M-, 17.4); HPLC retention time 17.03 min. HPLC analysis
of SVPD hydrolysate gave only two products: 4.89 min (UMP),
9.41 min (2′-deoxy-2′-R-C-(carboxymethyl)uridine).
2′-Deoxy-2′-r-C-a ceta m id ou r id ylyl(3′-5′)u r id in e (24d ).
The product was greater than 94% pure as determined by
HPLC: 1H NMR (400 MHz, D2O) δ 2.48 (1H, dd, J ) 8.7, 15.8
Hz), 2.71 (1H, dd, J ) 5.5, 15.8 Hz), 2.88 (1H, m), 3.80 (2H, d,
J ) 3.9 Hz), 4.10 (1H, ddd, J ) 3.9, 5.9, 11.7 Hz), 4.14-4.25
(3H, m), 4.31 (1H, t, J ) 5.2 Hz), 4.34-4.38 (2H, m), 5.89-
5.93 (3H, m), 6.08 (1H, d, J ) 9.3 Hz), 7.83 (1H, d, J ) 8.2
Hz), 7.87 (1H, d, J ) 8.1 Hz); 13C NMR (100.6 MHz, D2O) δ
33.14, 46.48, 64.42, 67.75, 72.32, 76.39, 80.48, 85.87, 88.38,
90.77, 92.09, 105.32, 105.77, 144.53, 144.58, 154.57, 169.00,
178.42; 31P NMR (101 MHz, D2O) δ -0.71; FAB- m/ z (glycerol
matrix) 590 (M-, 100); HPLC retention time 17.41 min. HPLC
analysis of SVPD hydrolysate gave only two products: 4.94
min (UMP), 9.19 min (2′-deoxy-2′-R-C-(acetamidomethyl)-
uridine).
2′-Deoxy-2′-r-C-(cya n om et h yl)u r id ylyl(3′-5′)u r id in e
(24e). The product was greater than 96% pure as determined
by HPLC: 1H NMR (400 MHz, D2O) δ 2.78-2.94 (3H, m), 3.80
(2H, d, J ) 3.2 Hz), 4.09-4.38 (7H, m), 5.89-5.93 (3H, m),
6.14 (1H, d, J ) 6.1 Hz), 7.86 (2H, d, J ) 8.1 Hz); 13C NMR
(100.6 MHz, D2O) δ 13.83, 44.40, 62.10, 65.77, 70.24, 74.36,
77.30, 83.52, 86.32, 88.15, 90.18, 103.26, 103.98, 119.71,
142.12, 142.64, 152.56, 152.69, 166.80, 167.01; 31P NMR (101
MHz, D2O) δ -0.91; FAB- m/ z (glycerol matrix) 572 (M-, 100);
HPLC retention time 19.17 min. HPLC analysis of SVPD
hydrolysate gave only two products: 4.67 min (UMP), 13.47
min (2′-deoxy-2′-R-C-(cyanomethyl)uridine).
E n zym a t ic Digest ion of Din u cleosid e Mon op h os-
p h a tes. (i) SVP D. Digestion mixtures contained dinucleo-
side monophosphate (8 OD260 units/mL), potassium phosphate
buffer (40 mM, pH 6.5), and SVPD (C. durissuss) (0.12 mg/
mL) in a total of 250 µL. Reaction mixtures were incubated
at 25 °C and analyzed at appropriate time intervals by HPLC.
Digestion products were identified by coelution with authentic
samples.
(ii) Nu clea se P 1 a n d r ibon u clea se A. Digestion mixtures
contained dinucleoside monophosphate (3.5 OD260 units/mL),
potassium phosphate buffer (50 mM, pH 6.5), and enzyme (67
µg/mL) in a total of 250 µL. Reaction mixtures were incubated
at 25 °C and analyzed at appropriate time intervals by HPLC.
Digestion products were identified by coelution with authentic
samples.
Acid /ba se Hyd r olysis Rea ction s. Reaction mixtures
contained dinucleoside monophosphate (3.5 OD260 units/mL)
in the following solutions: 1.5 M HCl; 2.0 M NaOH; 40 mM
citrate buffer (pH 3.0); 56 mM citrate buffer (pH 4.0), in a total
of 250 µL. Reaction mixtures were incubated at temperatures
described in the Results and Discussion and analyzed at
appropriate time intervals by HPLC.
2′-De oxy-2′-r-C-(2-h yd r oxye t h yl)u r id ylyl(3′-5′)u r i-
d in e (24a ). The product was greater than 96% pure as
determined by HPLC: 1H NMR (400 MHz, D2O) δ 1.70-1.61
(1H, m), 2.00-1.93 (1H, m), 3.35-3.29 (1H, m), 3.65-3.59 (2H,
m), 3.80 (2H, d, J ) 4.1 Hz), 4.38-4.14 (7H, m), 5.96-5.92
(3H, m), 6.06 (1H, d, J ) 9.4 Hz), 7.89 (1H, d, J ) 8.8 Hz),
7.91 (1H, d, J ) 8.3 Hz); 31P NMR (60 MHz, D2O) δ 0.71; m/ z
(FAB-, glycerol matrix) 577 (M-, 5.5), 91 (100); HPLC reten-
tion time 17.75 min. HPLC analysis of SVPD hydrolysate gave
only two products: 4.52 min (UMP), 10.64 min [2′-deoxy-2′-
R-C-(2-hydroxyethyl)uridine].
2′-Deoxy-2′-r-C-(2,3-d ih yd r oxyp r op yl)u r id ylyl(3′-5′)-
u r id in e (24b). The product was greater than 98% pure as
determined by HPLC: 1H NMR (400 MHz, D2O) δ 1.42-1.40
(1H, m), 1.54-1.50 (1H, m), 2.42-2.30 (1H, m), 3.50 (2H, d, J
) 4.0 Hz), 3.83-4.07 (7H, m), 5.61 (1H, d, J ) 8.1 Hz), 5.63
(1H, d, J ) 8.1 Hz), 5.64 (1H, d, J ) 4.4 Hz), 5.78 (1H, d, J )
Ack n ow led gm en t . We thank the BBSRC and
EPSRC for provision of studentships to A.J .L. and
J .B.J .P., respectively, and are also grateful to Mr. Allan
Mills (Liverpool) and the EPSRC service at Swansea for
obtaining FAB mass spectra and Dr. Paul Leonard for
assistance with NMR spectroscopy. We also wish to
thank Drs. M. V. Rao and J . S. Vyle (Cruachem Ltd)
for the provision of the Fpmp protecting group and
helpful discussions on its use.
9.2 Hz), 7.60 (1H, d, J ) 8.1 Hz), 7.61 (1H, d, J ) 8.1 Hz); 31
P
NMR (101 MHz, D2O) δ -0.59. FAB- m/ z (glycerol matrix)
607 (M-, 100); HPLC retention time 17.86 min. HPLC
analysis of SVPD hydrolysate gave only two products: 4.05
min (UMP), 9.72 min [2′-deoxy-2′-R-C-(2,3-dihydroxypropyl)-
uridine].
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
2′-De oxy-2′-r-C-(ca r b oxym e t h yl)u r id ylyl(3′-5′)u r i-
d in e (24c). The product was greater than 97% pure as
determined by HPLC: 1H NMR (400 MHz, D2O) δ 2.42 (1H,
dd, J ) 7.8, 16.7 Hz), 2.69 (1H, dd, J ) 6.0, 16.7 Hz), 2.81
(1H, m), 3.76 (2H, d, J ) 3.9 Hz), 4.05 (1H, m), 4.11-4.23 (3H,
m), 4.26 (1H, t, J ) 5.2 Hz), 4.30-4.33 (2H, m), 5.85-5.89 (3H,
m), 6.01 (1H, d, J ) 9.3 Hz), 7.80 (1H, d, J ) 8.2 Hz), 7.83
(1H, d, J ) 8.2 Hz); 13C NMR 100.6 MHz, D2O) δ 33.86, 46.90,
spectra for compounds 4, 6-14, 17, 18, 20a -d , 21a -d , 25,
and 26 and 13C NMR spectra for compounds 16 and 19 (27
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
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J O9614607