Synthesis and nucleic-acid-binding properties of sulfamide- and 3′-N-sulfamate-modified DNA

Article abstract of DOI:10.1039/b110603c

A novel synthetic route for the preparation of sulfamide- and 3′-N-sulfamate-modified dinucleosides has been developed. The synthesis utilises 4-nitrophenyl chlorosulfate to prepare 4-nitrophenyl 3′- or 5′-sulfamates (e.g.,18 and 27), which couple smoothl

Full text of DOI:10.1039/b110603c

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Synthesis and nucleic-acid-binding properties of sulfamide- and
3Ј-N-sulfamate-modified DNA†
Kevin J. Fettes,a,b Nigel Howard,b David T. Hickman,a,b Steven Adah,c Mark R. Player,c
Paul F. Torrenced and Jason Micklefield*a
1
a Department of Chemistry, University of Manchester Institute of Science and Technology,
Faraday Building, PO Box 88, Manchester, UK M60 1QD
b Department of Chemistry, Birkbeck College, University of London, 29 Gordon Square,
London, UK WC1H 0PP
c Laboratory of Medicinal Chemistry, NIDDK, NIH Bethesda, MD, 20892-0805, USA
d Department of Chemistry, Northern Arizona University, Flagstaff, AZ, 86011-5698, USA
Received (in Cambridge, UK) 19th November 2001, Accepted 13th December 2001
First published as an Advance Article on the web 29th January 2002
A novel synthetic route for the preparation of sulfamide- and 3Ј-N-sulfamate-modified dinucleosides has been
developed. The synthesis utilises 4-nitrophenyl chlorosulfate to prepare 4-nitrophenyl 3Ј- or 5Ј-sulfamates (e.g., 18
and 27), which couple smoothly with the alcohol or amine functionalities of other nucleosides. The conformational
properties of the sulfamide- and 3Ј-N-sulfamate-modified dinucleosides d(TnsnT) and d(TnsoT) were compared with
the native dinucleotide d(TpT) using NMR and CD spectroscopy. Whilst both modifications result in a shift in the
conformational equilibrium of the 5Ј-terminal ribose rings from C2Ј-endo to a preferred C3Ј-endo conformation,
only the 3Ј-N-sulfamate-modified dimer exhibits an increased propensity to adopt a base-stacked helical
conformation. Incorporation of the sulfamide- and 3Ј-N-sulfamate modifications into the DNA sequence
d(GCGT10GCG) allowed the duplex melting temperature to be determined using UV thermal denaturation
experiments. This reveals that the sulfamide modification significantly destabilises duplexes with both
complementary DNA and RNA. However, the 3Ј-N-sulfamate modification has little effect on duplex stability
and even stabilises DNA duplexes at low salt concentration. These results indicate that the 3Ј-N-sulfamate group
is one of the most promising neutral replacements of the phosphodiester group in nucleic acids, that have been
developed to date, for therapeutic and other important applications.
stable to nuclease digestion and increase the lipophilicity of
Introduction
backbone, possibly aiding cellular uptake.1–3 In addition it was
Nucleic acids have been extensively modified by replacing
anticipated that introducing neutral linkages would improve
affinity for complementary DNA and RNA, by reducing
electrostatic repulsion between stands. One of the earliest
the nucleobase, sugar, phosphodiester linkage or the whole
backbone with alternative structures.1–5 Evaluating the effects
of the various structural modifications on the conformation
and recognition properties of nucleic acids can provide an
examples of this class are the methylphosphonate-modified
oligonucleotides 1 (Scheme 1).14,15 Replacement of one of
alternative perspective from which the structure and function
of native DNA and RNA can be better understood. More-
over, if the modifications investigated could potentially
have arisen under prebiotic conditions then it is possible to
speculate about the origins of the genetic material.6,7 Modified
oligonucleotides have also been developed as antisense or
antigene therapeutic agents.1–3 Similarly, modification strategies
can be used to stabilise aptamers8,9 or ribozymes10,11 for
use in vivo via exogenous delivery. Furthermore, modified
nucleic acids also have significant potential as diagnostic
agents or bio-analytical tools which depend upon hybridis-
ation.12 For example, novel microarrays have been developed
which utilise peptide nucleic acid probes.13 Here improved
stability, affinity and sequence specificity can also be
Scheme 1
advantageous.
In antisense and antigene research there has been consider-
able interest in the replacement of the phosphodiester group
of oligonucleotides with alternative neutral linkages that are
the non-bridging oxygen atoms with a methyl group in 1 results
in a chiral phosphorus atom. Although the R-configured
methylphosphonates form duplexes which are as stable as iso-
sequential native duplexes, oligonucleotides consisting of a
mixture of diastereoisomers have relatively poor affinity for
complementary nucleic acids.14 Despite this, efficient synthetic
methodology for the enantioselective synthesis of mixed-
† Electronic supplementary information (ESI) available: typical melting
curves obtained from thermal denaturation of modified oligonucleo-
tides with complementary DNA and RNA. See http://www.rsc.org/
suppdata/p1/b1/b110603c/
DOI: 10.1039/b110603c
J. Chem. Soc., Perkin Trans. 1, 2002, 485–495
This journal is © The Royal Society of Chemistry 2002
485

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sequence oligomers with all R stereocentres has yet to be
developed.15 Several neutral achiral phosphodiester replace-
ments have also been investigated, including methylene-
(methylimino) (MMI) 2,16 amide 3,2 and 3Ј-thioformacetal 4,17
which exhibit good affinity for nucleic acid targets and hold
more promise for the development of improved antisense
or antigene agents. Other neutral backbone modifications
have also been used in the development of oligonucleotides
with potentially different modes of therapeutic action. For
example, dsDNA possessing an Eco RV restriction site was
modified with a single neutral dimethylenesulfone linkage
6.18 The resulting duplex adopts a bent conformation and
functions as a transition-state analogue inhibiting cleavage
of the native dsDNA substrate. Extension of this strategy
could lead to modified oligonucleotide inhibitors of other
enzymes that process native DNA substrates. Furthermore
a thrombin binding DNA aptamer, generated by in vitro
selection, was modified with formacetal linkages 5.9 The
modified chimeric oligonucleotide aptamer, which adopts a
chair-like structure, retains high affinity for the protein target
but has considerably improved stability and anticoagulant
activity in vivo. Finally, methylphosphonate modifications 1
have also been used to probe protein–nucleic acid inter-
actions using a template-directed interference footprinting
approach.19
Results and discussion
Synthesis of sulfamide- and 3Ј-N-sulfamate-modified
dinucleosides
Previously we have shown that the 2-hydroxyphenyl sulfamate 9
(Scheme 2), derived from catechol sulfate, can be coupled with
5Ј-amino nucleosides to give sulfamide-linked dinucleosides.22
However, these coupling reactions are slow, requiring heating
under reflux in 1,4-dioxane for extended periods and give only
moderate yields of sulfamide products. Moreover, treatment
of the 2-hydroxyphenyl sulfamate 9 with nucleoside 5Ј-alcohols
in the presence of base fails to give any sulfamate products.
Aryl sulfamates 10 are thought to undergo elimination in the
presence of base to form N-sulfonylamines 12 via the anion 11
by an E1cB mechanism or directly through an E2-type pro-
cess.24 The highly electrophilic N-sulfonylamine 12 is then
trapped by nucleophiles, for example amines or alcohols,
resulting in sulfamides 13 or sulfamates 14, respectively. Given
that catechol is a relatively poor leaving group (pKa1 ≈ 9.1)25 it
seems likely that, in the case of the 2-hydroxyphenyl sulfamate
9, the equilibrium between the sulfamate 10 or the conjugate
base 11 and N-sulfonylamine 12 lies on the side of the starting
material. As a consequence of this only more nucleophilic
amines can compete with catecholate, in the reverse reaction,
and trap the low concentration of N-sulfonylamine to give
sulfamide products 13. With this in mind we reasoned that
more reactive aryl sulfamates 10, sulfamoyl chlorides (RNH-
SO2Cl) or sulfamoyl azides (RNHSO2N3), would be required as
substrates for coupling with alcohols to give sulfamates 14.
Nucleosides with 5Ј-sulfamoyl azide groups have been
employed previously in the synthesis of 5Ј-N-sulfamate
dinucleosides.26 Nevertheless we chose to avoid this chemistry
given that it necessitates the use the explosive reagent sulfuryl
chloride azide and yields were low. Also, earlier attempts to
synthesise the sulfamoyl chloride 17 (Scheme 3) from 3Ј-amino
Recently we have been involved in the synthesis and evalu-
ation of backbone-modified nucleic acids.20–22 In particular we
have sought to develop modified oligonucleotides with neutral
sulfamide 7 and 3Ј-N-sulfamate 8 groups replacing the
negatively charged phosphodiester linkage (Scheme 2).21,22 In
Scheme 2
Scheme 3
addition to possessing many of the advantageous properties
common to other neutral modifications 1–6, these linkages
should be easier to prepare and more closely resemble the
phosphodiester group both sterically and conformationally. In
the case of native nucleic acids anomeric or gauche effects are,
at least in part, responsible for the conformational preferences
of the phosphodiester P–Oester bonds.23 Similar stereoelectronic
effects are likely to control the conformational properties of
the sulfamate and sulfamide linkages in 7 and 8. In addition the
tetrahedral nitrogen atoms, in these linkages, can potentially
invert configuration, resulting in different orientations of the
N–H bonds.23 In this paper we describe in detail the novel
synthesis of 3Ј-N-sulfamate- and sulfamide-modified oligo-
nucleotides and examine the effects of these modification on
the backbone conformation and affinity for complementary
DNA and RNA.
nucleoside 15 using sulfuryl dichloride had failed. We therefore
chose to investigate the reaction of the 3Ј-amine 15 with 4-
nitrophenyl chlorosulfate 16, anticipating the production of
either the sulfamoyl chloride 17 or the 4-nitrophenyl sulfamate
18. Accordingly, 4-nitrophenyl chlorosulfate 16 was prepared
using a modification of an earlier method which involves treat-
ing 4-nitrophenol with sulfuryl dichloride and pyridine in
diethyl ether at low temperature.27 The resulting product 16, is
a crystalline solid which is relatively stable and can be safely
stored for several months under an inert atmosphere. Although
4-nitrophenyl chlorosulfate 16 has been known since 1948,28 it
has found little practical use in synthesis. Despite this the
mechanism of nucleophilic substitution reactions of 16 and
other aryl chlorosulfates has been investigated.29 These studies
486
J. Chem. Soc., Perkin Trans. 1, 2002, 485–495

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show that nucleophilic attack at sulfur can result in either S–Cl
or S–OAr bond scission, with S–OAr bond cleavage the major
reaction pathway. Yet we found that the reaction of 3Ј-amine 15
with a slight excess of 16 and triethylamine at room temper-
ature, or at Ϫ78 ЊC, in a variety of different solvents consist-
ently gave the symmetrical sulfamide dimer 19 as the major
product (ca. 58% yield). A minor amount of the 4-nitrophenyl
sulfamate 18 (19% yield) was also produced, but none of the
sulfamoyl chloride 17 was ever isolated. It seems likely, if
S–OAr bond cleavage is the major reaction pathway, that the
sulfamoyl chloride 17 generated in situ reacts with the 3Ј-amine
15 to give the dimer 19. Presumably the presence of triethyl-
amine, which is necessary to avoid deprotection of the acid-
labile dimethoxytrityl group, results in elimination of chloride
from the more reactive sulfamoyl chloride 17 to give the
N-sulfonylamine, which is trapped by the 3Ј-amine even at
Ϫ78 ЊC. Thus the less reactive 4-nitrophenyl sulfamate 18,
which was isolated, is probably derived from the minor reaction
pathway involving S–Cl bond cleavage. Attempts to prepare 17
were therefore abandoned. Instead we sought to increase the
yield of 4-nitrophenyl sulfamate 18 in order to investigate
coupling reactions with the 5Ј-hydroxy group of other nucleo-
sides. We argued that by adding excess of 4-nitrophenol and
triethylamine to the reaction of 15 with 16 it may be possible to
intercept any N-sulfonylamine with 4-nitrophenoxide, thus pre-
venting formation of the unwanted symmetrical dimer 19. This
proved to be the case and 4-nitrophenyl sulfamate 18 could be
prepared in 68% yield, with no dimer 19, upon addition of 2
equivalents of chlorosulfate 16 to the 3Ј-amine 15 along with a
large excess (10 equivalents) of nitrophenol and triethylamine.
The 4-nitrophenyl sulfamate 18 was then treated with a two-
fold excess of 5Ј-alcohol 23 (Scheme 4) and 5 equivalents
Scheme 5
well as 3Ј-N-sulfamate or sulfamide modifications on DNA
duplex stability we also prepared the 3Ј-azido-2Ј-fluorouridine
derivative 20.30 Reduction of the azide to the amine 21 was
achieved in near quantitative yield with H2S in aq. pyridine.31
Generally we have found this method to be superior, to hydro-
genolysis or other methods commonly used in the reduction of
5Ј- and 3Ј-azido nucleosides. Sulfomylation of the 3Ј-amino-2Ј-
fluorouridine derivative 21, with the 4-nitrophenyl chlorosulfate
16 as described above, was more problematic, resulting in only
59% of sulfamate 22. For reasons that remain unclear all
attempts to couple sulfamate 22 with 5Ј-alcohol 23 failed to give
any 2Ј-fluoro-3Ј-N-sulfamate dinucleoside 25. Despite this we
were able to prepare the 2Ј-fluorosulfamide dinucleoside 29
(Scheme 5). This was achieved by sulfonylation of 5Ј-amino
nucleoside 26 with 4-nitrophenyl chlorosulfate 16 (78% yield) to
give the 5Ј-sulfamate 27 followed by coupling with 3Ј-amino-2Ј-
fluorouridine 21 in the presence of triethylamine (80% yield).
Notably, the aminolysis of 4-nitrophenyl sulfamate 27 was
complete within 3 h at room temperature in contrast to the
rather more vigorous conditions required for aminolysis of
the corresponding 2-hydroxyphenyl sulfamate.22 Similarly, the
sulfamide dinucleoside 28 was prepared in 83% yield from
4-nitrophenyl sulfamate 27 and 3Ј-amine 15. This was a con-
siderable improvement on the 56% yield obtained previously for
the coupling reaction between 3Ј-amine 15 and the analogous
2-hydroxyphenyl sulfamate.22 Overall, these findings clearly
indicate that 4-nitrophenyl sulfamate nucleosides (e.g., 18 and
27) are significantly superior building blocks, compared with
the 2-hydroxyphenyl sulfamates, for the synthesis of sulfamide-
as well as sulfamate-modified dinucleosides.
The synthesis was completed by desilylation of 3Ј-N-
sulfamate and 2Ј-fluorosulfamide dinucleosides 24 and 29, with
TBAF, to give 3Ј-alcohols 32 and 33 (Scheme 6), which were
phosphitylated using 2-cyanoethyl N,N,N Ј,NЈ-tetraisopropyl-
phosphorodiamidite and 4,5-dicyanoimidazole resulting in the
required phosphoramidites 35 and 36. These, along with the
sulfamide phosphoramidite 37, which was previously derived
from alcohol 34,22 were used in the subsequent solid-phase syn-
thesis of modified oligonucleotides. In addition a small portion
of the 3Ј-N-sulfamate dinucleoside 32 was detritylated, using
dichloroacetic acid, to give the dinucleoside d(TnsoT) 30 which
was required for conformational studies.
Scheme 4
of triethylamine, resulting in a near quantitative yield of the
sulfamate dinucleoside 24 after stirring at room temperature for
12 h. Clearly, nitrophenoxide (pKa = 7.1)25 is a superior leaving
group compared with catecholate and this has a dramatic effect
on the rate of nucleophilic substitution. This is consistent
with elimination of the aryloxide being the rate-determining
step in such reactions.24 Furthermore, the inclusion of dry 4Å
molecular sieves is also critical to the success of this coupling
reaction, particularly when performed on a small scale. Without
sieves yields were considerably diminished, presumably due to
competing hydrolysis and formation of a sulfamic acid side
product which was not isolated.
Conformational properties of sulfamide- and 3Ј-sulfamate-
modified dinucleosides
The conformational properties of the sulfamide dimer d-
(TnsnT) 31 were previously compared with those of the native
dinucleotide d(TpT) using NMR spectroscopy.22 Here the
In order to investigate the combined effects of 2Ј-fluoro as
J. Chem. Soc., Perkin Trans. 1, 2002, 485–495
487

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Table 1 Conformation of the sugar rings in 3Ј-N-sulfamate dinucleo-
side d(TnsoT) 30, between 70 and 10 ЊC, compared with the sulfamide
dinucleoside d(TnsnT) 31 and the native dinucleotide d(TpT)22
Σ1Јa/Hz (%N)b
Temp./ЊC
70
50
30
10
d(T nsoT)c 30
d(TnsoT )
d(T nsnT) 3122
d(TnsnT )
d(T pT)
12.0a (63)b
12.9 (47)
12.3 (58)
13.4 (39)
13.7 (34)
13.6 (36)
11.8 (66)
13.1 (44)
12.1 (61)
13.3 (40)
13.7 (34)
13.6 (36)
11.6 (69)
13.1 (44)
11.8 (66)
13.2 (42)
13.6 (36)
13.6 (36)
11.1 (78)
12.7 (51)
11.2 (76)
13.1 (44)
13.4 (39)
13.6 (36)
d(TpT )
a The sum of the H1Ј vicinal coupling constants Σ1Ј = J1Ј2Ј ϩ J1Ј2Љ b The
.
% of N-conformer in the N–S conformational equilibrium was deter-
mined from the Altona sum rule32 % S = 100(Σ1Ј Ϫ 9.8)/5.9. c Each row
refers to the sugar ring italicised with the 5Ј- and 3Ј-terminal sugars
indicated to the left and right, respectively.
d(TnsnT) and d(TpT) were recorded across a range of different
temperatures (Fig. 1). The spectra for all three dimers were
similar with a strong positive band at 280 nm, a strong negative
Scheme 6
conformational effects of replacing the phosphodiester group
of d(TpT) with a 3Ј-N-sulfamate group (30) are similarly
examined. Accordingly the 1H NMR spectrum of d(TnsoT) 30,
in D2O, was fully assigned with the aid of COSY and NOESY
experiments. From this the vicinal coupling constants J1Ј2Ј
and J1Ј2Љ were then measured at various temperatures. The sum
of these coupling constants (Σ1Ј) were used to determine the
approximate extent of the equilibrium, between the northern
(N) or C3Ј-endo and the southern (S) or C2Ј-endo conform-
ation of the sugar rings using the method of Altona.32 This
revealed (Table 1) that the 5Ј-terminal ribose ring of d(T nsoT)
exists predominantly in a northern sugar conformation (69% N
at 30 ЊC), which was also found to be the case with d(T nsnT)
(66% N at 30 ЊC). In contrast the 5Ј-terminal ribose unit of the
native dinucleoside phosphate d(T pT) preferentially adopts a
southern conformation (36% N at 30 ЊC). The observed shift
towards a preferred N-conformation is normally attributed to a
reduction in the gauche effect between the 3Ј-substituent and
the deoxyribose O4Ј atom, given that the 3Ј-amino group
in d(T nsoT) and d(T nsnT) is more electropositive than the
3Ј-oxygen atom of d(T pT),33–35 although other factors, such as
aqueous solvation, may also contribute to this conformational
effect.23 The 3Ј-terminal ring of d(TnsoT ) was 44% N at 30 ЊC,
which is similar to that observed previously for d(TnsnT ) (42%
at 30 ЊC) and slightly higher than for d(TpT ) (36% at 30 ЊC).
Furthermore we observed that the % of N-conformer for the
5Ј-terminal ring of d(T nsoT) increases upon lowering the tem-
perature from 63% N at 70 ЊC to 78% N at 10 ЊC. A similar
increase in % N had previously been observed for d(T nsnT).
This was attributed to a possible increased propensity of
the sulfamide modified dinucleoside to adopt a base-stacked
conformation at lower temperatures, given that intramolecular
base stacking is known to favour the N-ribose conformation in
dinucleoside phosphates.36,37
Fig. 1 The effects of changing temperature on the CD spectra of
d(TnsoT) 30, d(TnsnT) 31 compared with that of the native d(TpT).
band at 255 nm, and a weak positive band at 225 nm. This
suggests that all of the dinucleosides adopt similar overall
conformations. In addition for each sample the major bands at
280 and 255 nm showed a significant decrease in intensity as the
temperature was increased. This shift is attributed to a change
from a predominately base-stacked conformation at lower
temperature to a random conformation, with little or no base
stacking, at higher temperatures.39 Significantly the change in
the signal intensity of the major bands for d(TnsoT) was twice
the magnitude of that observed for the native d(TpT) across a
similar temperature range. On the other hand the change in
intensity of the bands for d(TnsnT) was very similar to d(TpT).
Thus d(TnsoT) appears to have a greater propensity to adopt a
base-stacked helical conformation at lower temperature than do
either d(TnsnT) or d(TpT). In summary both 3Ј-N-sulfamate
To further investigate the hypothesis that base stacking
occurs in the modified dinucleosides, circular dichrosim (CD)
spectroscopy was employed. It is well established that the inten-
sity of the CD signal of di- and oligonucleotides is highly
dependent on the proportion of molecules that exist in an
helical base-stacked conformation, as opposed to a random
conformation.38,39 Accordingly the CD spectra of d(TnsoT),
488
J. Chem. Soc., Perkin Trans. 1, 2002, 485–495

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Duplex melting temperatures of 3Ј-N-sulfamate and
sulfamide-modified oligonucleotides with complementary DNA and
Table 2 Electrospray mass spectrometry data for native 38 and
modified oligonucleotides 39–45
Table 3
RNA
Electrospray
MS/Da
T m a (∆T m/mod.)b/ЊC
Oligo.
(Calc.d) (Found)
DNAc
RNAd
Oligo. 0.02 M [Naϩ]
0.1 M [Naϩ]
1.0 M [Naϩ]
0.1 M [Naϩ]
38 d(GCGT10GCG)
4875.2 4875.3
4874.3 4874.2
4872.5 4872.5
39 d(GCGT4TnsoTT4GCG)a
40 d(GCGTnsoTTTTnsoTTTTnsoTGCG)
38
39
40
41
42
43
44
45
43.2a
55.0
66.4
49.0
41 d(GCGTnsoTTnsoTTnsoTTnsoTTnsoTGCG) 4870.7 4870.6
43.6a (ϩ0.4)b
44.0 (ϩ0.3)
44.8 (ϩ0.3)
40.0 (Ϫ3.2)
33.7 (Ϫ3.2)
N.D.
55.1 (ϩ0.1)
55.5 (ϩ0.2)
53.3 (Ϫ0.3)
51.7 (Ϫ3.3)
45.0 (Ϫ3.3)
49.2 (Ϫ5.8)
35.8 (Ϫ6.4)
66.0 (Ϫ0.4)
47.8 (Ϫ1.2)
48.0 (Ϫ0.3)
46.0 (Ϫ0.6)
45.9 (Ϫ3.1)
41.0 (Ϫ2.7)
N.D.
42 d(GCGT4TnsnTT4GCG)b
4873.3 4872.8
4869.5 4869.7
4877.3 4876.7
4881.4 4881.1
N.D.e
43 d(GCGTnsnTTTTnsnTTTTnsnTGCG)
N.D.
44 d(GCGT4UFnsnTT4GCG)c
60.9 (Ϫ5.5)
54.6 (Ϫ3.9)
N.D.
45 d(GCGUFnsnTTTUFnsnTTTUFnsnTGCG)
a A single 3Ј-N-sulfamate nucleoside linkage is indicated by the abbrevi-
N.D.
N.D.
N.D.
ation nso. b Sulfamide linkage (nsn). c 2Ј-Fluorouridine (UF).
a Duplex melting temperature (T m). b Figures in parentheses represent
the change in T m per modification (∆T m/mod.) compared with the
native duplexes. c Data recorded with complementary DNA d(CG-
CA10CGC) at low, intermediate and high salt concentration. d Data
recorded with complementary RNA r(CGCA10CGC) at intermediate
salt concentration. e N.D.: T m not determined.
and sulfamide modification of the dinucleoside phosphate d-
(TpT) result in a shift towards a preferred C3Ј-endo deoxyribose
conformation. However, only the 3Ј-N-sulfamate modification
appears to increase the extent with which the bases can adopt a
stacked conformation. It is now well established that modifi-
cations to oligodeoxynucleotides that increase the proportion
of C3Ј-endo deoxyribose rings and increase the propensity of
the single strand to base stack are better preorganised into
an helical conformation required for the formation of
stable hybrid duplexes with complementary RNA as well as
DNA.1 It can therefore be expected that sulfamide- and,
particularly, 3Ј-N-sulfamate-modified oligodeoxynucleotides
will be well disposed to form duplexes with complementary
nucleic acids.
concentration. Each UV melting curve exhibited typical hyper-
chromicity, between 10 and 25%, with a single transition con-
sistent with the melting of a duplex to single strands. Typical
melting curves obtained from thermal denaturation with
complementary DNA and RNA are shown in Fig. 2 and 3,
respectively, in the electronic supplementary information that
accompanies this paper.†The maximum of the first derivative
of these curves was then used to determine the duplex melting
temperatures (T ms) (Table 3). This revealed that modified
oligonucleotides with 1 and 3 central sulfamide linkages (42
and 43) form less stable duplexes with complementary DNA,
exhibiting a drop in T m per modification (∆T m/mod.), com-
pared with the native duplex, of ca. Ϫ3.2 ЊC at 0.02 and 0.1 M
salt concentration [Naϩ]. Increasing the salt concentration
to 1.0 M Naϩ results in a more marked drop in T m of the
sulfamide-modified duplexes, compared with the native duplex,
of up to Ϫ5.5 ЊC ∆T m/mod. Furthermore, 2Ј-fluorouridine-
sulfamide modifications in oligonucleotide 44 and 45 produced
an even larger drop in T m of between Ϫ5.8 and Ϫ6.4 ЊC per
modification with DNA at 0.1 M Naϩ. In contrast, oligo-
nucleotides with 1, 3 or 5 sulfamate modifications (39, 40
and 41) formed duplexes with complementary DNA that were
moderately more stable than the native duplex at low salt con-
centration (0.02 M Naϩ) with an observed increase in T m of
ϩ0.3 ЊC per modification. At 0.1 M Naϩ the 3Ј-N-sulfamate-
modified oligonucleotides 39–41 formed duplexes of com-
parable stability to the native duplex, whilst at higher salt
concentration (1.0 M Naϩ) the T m dropped by Ϫ0.4 ЊC for one
3Ј-sulfamate modification.
The observed changes in ∆T m per modification with change
in salt concentration for 3Ј-N-sulfamate-modified DNA
duplexes can be explained by the reduction in net negative
charge of the oligonucleotide backbone upon introduction
of neutral linkages. The resulting reduction in electrostatic
repulsion between complementary strands will, when all other
things are equal, stabilise the duplex that is formed. Clearly
this effect will be more significant at low salt concentration
when fewer sodium cations are present to mask the negatively
charged phosphodiester groups. In the case of the sulfamide
modifications (42 and 43) any favourable effects due to reduc-
tion in backbone negative charge are probably outweighed by
an unfavourable change in conformation, which results in a
suboptimal structure for formation of a Watson–Crick base-
paired duplex. Nevertheless, the destabilisation of the duplex
formed between sulfamide-modified oligonucleotides 42 or 43
and DNA is still greatest at higher salt concentration when
electrostatic repulsion is less significant.
Modified-oligonucleotide synthesis and nucleic-acid-binding
studies
To investigate the effects of 3Ј-N-sulfamate and sulfamide
modifications on the stability of nucleic acid duplexes we chose
to incorporate the modified linkages into the DNA sequence
d(GCGT10GCG) using the block-synthesis approach. This
sequence has been used extensively by others and gives fairly
representative results for comparison of the effects of modifi-
cations on duplex melting temperatures.40 Accordingly, chim-
eric oligodeoxynucleotide 16mers, with between one and five
central 3Ј-N-sulfamate (nso) or sulfamide (nsn) linkages 39–43
(Table 2), were synthesised. The synthesis was carried out using
an automated DNA synthesiser with standard nucleoside
phosphoramidites and the modified dimer phosphoramidites
35 and 37. The 2Ј-fluorouridine-modified sulfamide dimer 36
was also used to synthesise oligonucleotides 44 and 45 con-
taining 1 and 3 central 2Ј-fluorouridine-sulfamide modifi-
cations (UFnsnT). Standard phosphoramidite protocols for
DNA synthesis were used throughout, with the added pre-
caution of increased coupling times of 15 minutes for the
modified dimers. Coupling efficiencies for monomer and modi-
fied dimers was in excess of 98% as determined by measuring
the release of the dimethoxytrityl cation spectrophoto-
metrically. Following deprotection and cleavage from the solid
support, with aq. ethanolic ammonia, the oligonucleotide
ammonium salts were purified by RP-HPLC and converted to
the sodium forms using an ion-exchange resin. All the modified
oligonucleotides were subjected to electrospray mass spec-
trometry and the observed masses of the parent ions were all in
close agreement with the calculated values (Table 2).
Following this the UV absorption at 260 nm vs. temperature
curves for equimolar mixtures (3 µM strand concentrations) of
the native d(GCGT10GCG) 38 and modified oligonucleotides
(39–45) with complementary DNA and RNA (CGCA10CGC)
were recorded. All mixtures were prepared in phosphate buffer
(pH 7.0) adjusted to either 0.02, 0.1 or 1.0 M total sodium ion
J. Chem. Soc., Perkin Trans. 1, 2002, 485–495
489

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The sulfamide modifications in oligonugleotides 42 and 43
has a similar effect on duplex stability with complementary
RNA at 0.1 M salt concentration, resulting in a drop in T m of
ca. Ϫ3.0 ЊC per modification. In the case of the 3Ј-N-sulfamate-
modified oligonucleotides the heteroduplexes formed with
RNA were slightly less stable than the isosequential modified
DNA duplexes. At intermediate salt concentration with 3 and 5
sulfamate modifications a moderate drop in T m per modifi-
cation of Ϫ0.3 and Ϫ0.6 ЊC, respectively, was observed with a
larger drop in T m of Ϫ1.2 ЊC recorded for one modification. In
light of the earlier NMR observations with 3Ј-N-sulfamate
dinucleoside d(TnsoT), it seems likely that the more 3Ј-N-
sulfamate linkages incorporated into the oligodeoxynucleotide
the higher the proportion of C3Ј-endo deoxyribose rings that
are present. As a consequence of this, oligonucleotides which
are more highly substituted with 3Ј-N-sulfamate groups are
more likely to adopt an overall A-type conformation which is
preferred for binding to complementary RNA. Furthermore we
anticipate that oligodeoxynucleotides that are fully modified
with, or possess a longer continuous stretch of, 3Ј-N-sulfamate
modifications will have greater conformational homogeneity
and increased affinity for RNA.
Taken overall these results clearly show that 3Ј-N-sulfamate-
modified oligonucleotides 39–41 hybridise with both comple-
mentary RNA and DNA with similar binding affinity as native
DNA. On the other hand the sulfamide congeners 42 and 43
have significantly lower affinity for both complementary nucleic
acids. Given that both modifications are neutral and result in a
similar shift in the sugar pucker towards a preferred C3Ј-endo
conformation it seems that other conformational factors
are responsible for the significant differences in affinity. Indeed
the additional incorporation of an highly electronegative 2Ј-
fluorine substituent in sulfamide-modified oligonucleotides 44
and 45 would be expected to increase further the bias of the
modified sugar rings towards a locked C3Ј-endo conformation.
Yet this combination of modifications proved to be most detri-
mental to the formation of duplexes with complementary
DNA. Furthermore CD spectra recorded for both sulfamide
and 3Ј-N-sulfamate dinucleosides 30 and 31 indicate that the
3Ј-sulfamate-modified oligonucleotides are probably better
preorganised into a base-stacked helical conformation, in the
single stranded state. This would reduce the loss of entropy on
binding to complementary RNA and DNA and account for the
greater stability of 3Ј-N-sulfamate-modified duplexes. Also the
nitrogen atoms in the 3Ј-N-sulfamate and -sulfamide linkages
are likely to be tetrahedral in geometry and thus are able to
adopt different configurations that will differ in the orientation
of the N–H bonds.23 Given that there are two such nitrogen
atoms in the sulfamide linkage and given that the energy barrier
to inversion of configuration is likely to be low,23 it is tempting
to postulate that the sulfamide linkage will be intrinsically
more flexible than the 3Ј-sulfamate linkage. If that were the
case it would also point towards there being a larger loss of
entropy on formation of a duplex with sulfamide modified
oligonucleotides.
pair or form duplexes under any conditions.35,41 A number of
explanations have been put forward to account for this. First,
modelling studies suggest that the 5Ј-NH group is less well
solvated than the 3Ј-NH group.41 Secondly, from X-ray crystal-
lographic data of a 3Ј-N-phosphoramidate-modified duplex, it
is postulated that a steric clash between the 5Ј-amino hydrogen
atom and the H2Ј atom of an adjacent deoxyribose ring would
destabilise a typical A-type duplex.35 Similarly a steric clash of
5Ј-NH with the pyrimidine H6 or deoxyribose O4Ј atoms would
destabilise a B-type duplex.
Conclusions
Following on from earlier work22 we have developed an
improved synthesis of sulfamide dinucleosides and a new syn-
thetic route for the preparation of 3Ј-N-sulfamate-modified
dinucleosides. The synthesis uses 4-nitrophenyl chlorosulfate, a
simple yet hitherto little utilised reagent, to prepare nucleosides
with 4-nitrophenyl 3Ј- and 5Ј-sulfamate groups (18, 22 and 27)
which couple smoothly with alcohol or amine functionalities
of other nucleosides. Whilst yields are typically high, coupling
times with alcohols were in excess of 12 hours. As a result of
this it is likely that more reactive aryl sulfamates, for example
2,4-dinitrophenyl sulfamates corresponding to 18, are required
as monomers for future iterative solid-phase synthesis of the
most promising 3Ј-N-sulfamate-modified oligonucleotides.
Using NMR and CD we have also established the effects of
both sulfamide and 3Ј-N-sulfamate modifications on dinucleo-
tide conformation. Notably both modifications result in a shift
from a preferred C2Ј-endo to C3Ј-endo sugar conformation, but
only the 3Ј-N-sulfamate modification results in an increase in
the proportion of base-stacked dimers.
Modified sulfamide and 3Ј-N-sulfamate dinucleoside phos-
phoramidites were incorporated into a DNA 16mer using the
block-synthesis approach. This enabled the effects of both
modifications on duplex stability to be determined using
UV thermal denaturation experiments. Whilst the sulfamide
modification results in a significant destabilisation of duplexes,
the 3Ј-N-sulfamate group has little effect compared with the
native duplexes and even stabilises DNA duplexes at low
salt concentration. Moreover the affinity of 3Ј-N-sulfamate-
modified oligonucleotides for complementary DNA and RNA
compares favourably with other neutral linkages that have been
used to replace the phosphodiester group in oligonucleotides.
In fact the methylene(methylimino)- (MMI)16 2, amide-2 3 and
3Ј-thioformacetal-17 4 modified oligonucleotides (Scheme 1),
which are amongst the best neutral modifications of this type
that have been developed, show similar or only moderately
higher affinity for complementary nucleic acids.40 Moreover the
chemistry described in this paper indicates that 3Ј-N-sulfamate-
modified oligonucleotides can be prepared more easily and
economically than some of the other modified oligonucleotides
with neutral linkages. Thus 3Ј-N-sulfamate-modified oligo-
nucleotides are worthy of further development for application
as antisense or antigene therapeutic agents. In addition the
3Ј-N-sulfamate group is sterically, electronically and probably
conformationally more similar to the phosphodiester group
than are all the other neutral linkages that have been developed
so far. This is reflected in minimal disruption of nucleic acid
duplexes as evidenced from the T m results presented here.
As a consequence the 3Ј-N-sulfamate modification may find
use as a general phosphodiester replacement for other appli-
cations. For example, it might be possible to probe important
contacts between the nucleic acid backbones and specific
amino acid residues in nucleic acid–protein complexes by
selectively replacing phosphodiester groups with the 3Ј-N-
sulfamate linkages.19 Alternatively, aptamers selected in vitro
for binding to therapeutically relevant protein targets could be
stabilised against nuclease digestion by replacing phospho-
diester groups, which were not involved in electrostatic contacts
It is also interesting to note that 3Ј-N-sulfamate-modified
oligonucleotides display higher affinity for complementary
DNA than do oligonucleotides incorporating isomeric 5Ј-N-
sulfamate linkages,26 with a 5Ј-amino rather than a 3Ј-amino
substituent. According to an earlier report a DNA duplex
with 5Ј-N-sulfamate modifications exhibited in a drop in T m of
Ϫ1.5 ЊC per modification.26 It is likely that the 5Ј-amino
substituent common to both sulfamide and 5Ј-N-sulfamate
modifications is responsible for the destabilisation of duplexes
with complementary nucleic acids. A similar but more dramatic
effect has been observed with isosteric and isoelectronic
phosphoramidate DNA.35,41 3Ј-N-Phosphoramidate oligo-
nucleotides form very stable duplexes with complementary
DNA and RNA, but the isomeric 5Ј-N-phosphoramidates with
the linkage in the opposite orientation are unable to base
490
J. Chem. Soc., Perkin Trans. 1, 2002, 485–495

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with the protein residues, with 3Ј-N-sulfamate groups.8,9 Simi-
larly it may be possible to stabilise hammerhead ribozymes
using 3Ј-N-sulfamate groups, whilst retaining catalytic activity,
for use in vivo.10,11
containing the oligonucleotide products 38–45 were evaporated
to dryness and then re-dissolved in Ultrapure water and
desalted on a Sep-Pak C18 cartridge. The desalted oligonucleo-
tide was then exchanged for the Naϩ-form using Dowex®
50WX8 wet ion-exchange resin stirring for 45 min at 4 ЊC before
being filtered and evaporated to dryness. Two ten-fold dilutions
were then carried out for each sample, and the absorbance (A)
was determined using a UV spectrophotometer. Electrospray
ionisation mass spectrometry of oligonucleotides was per-
formed on a Q-Tof (Micromass, Altrincham, Manchester,
UK). The electrospray capillary potential was 2.5–3.2 kV and
the cone was held at 47 V. All samples were introduced via
constant infusion at 0.5 µL minϪ1 using a syringe driver
(Harvard Apparatus). Samples were dissolved in a water–
acetonitrile solution (typically 1 : 1 v/v) with 0.1% ammonium
acetate. For all infusion experiments a scan rate of 100 m/z-
units per second was used and the data were collected in ‘multi-
channel analyser’ mode. Data were typically accumulated for
2 minutes depending on the signal intensity.
Experimental
General procedures
1H NMR spectra were recorded at 270, 400 or 500 MHz. 13C
NMR spectra were recorded at 67.9, 100.6 or 125.7 MHz.
Chemical shifts are reported in parts per million relative to
Me4Si or residual solvent signal. J-values are given in Hz. 19F
NMR spectra were recorded at 188.2 MHz unreferenced.
31P NMR spectra were recorded at 161.9 or 202.4 MHz with
H3PO4 as external standard. FAB mass spectra were obtained
on a ZAB-SE VG Analytical Fisons Instrument. IR spectra
were acquired on a Perkin-Elmer 783 or 1710 FT instrument
for samples as KBr disks or CHCl3 solutions. Melting
points were determined using a Cambridge Instrumental
microscope with a Reichert-Jung heating mantle and are
uncorrected. Flash silica column chromatography was carried
out over Merck Kieselgel 60 (230–400 mesh), and Merck
Kieselgel 60 F254 0.25 mm plates were used for analytical
TLC. Reactions involving anhydrous conditions were carried
out in flame-dried glassware under a positive pressure of
argon. All solvents were distilled before use. Reagents were
purified and solvents dried using standard procedures.42
Petroleum ether refers to the fraction with distillation range
40–60 ЊC.
UV thermal denaturation experiments
Phosphate buffer solutions were prepared in doubly distilled
water and consisted of 10 mM NaH2PO4 (pH 7.0), 0.1 mM
EDTA and a total sodium ion concentration of either
0.02, 0.1 or 1.0 M, following addition of NaCl. Buffers for
experiments involving RNA were prepared in water pretreated
with diethyl pyrocarbonate (DEPC) and heated at 180 ЊC
overnight. For all UV thermal denaturation experiments
9 nmol of each strand was mixed and the solution was
evaporated. Samples were then reconstituted in the appropriate
buffer (3 mL) and transferred to a 1 cm quartz cuvette con-
taining a stirrer bar. A thin layer of silicone oil was placed
over the surface of the sample to eliminate evaporation.
Absorbance vs temperature profiles were then recorded on an
HP845A diode array spectrophotometer with an HP89089A
temperature-control unit connected to a Peltier heating block
controlled via a temperature probe placed directly into sample.
Samples were heated at 1.0 ЊC minϪ1 and absorbance values at
260 nm were collected every 0.1 ЊC. The first derivative of
the absorption vs. temperature curve was calculated and used
to determine duplex melting temperatures (T ms). All data
acquisition and manipulation was carried out using HP UV
Chemstation software.
NMR and CD studies of modified and native dinucleotides
1D and 2D NMR spectra for the 3Ј-N-sulfamate-modified
dinucleoside d(TnsoT) 30 were recorded at 500 MHz and
assigned in an analogous fashion to that described in our earlier
paper.22 CD spectra were measured with either a JASCO
600 or 720 spectropolarimeter complemented with ordinary
UV adsorption measurements employing an AVIV 17DS
spectrophotometer. Sample temperatures were controlled by a
Hewlett-Packard 89090A temperature unit via a temperature
probe. Samples were dissolved in buffer adjusted to pH 7.0 con-
taining 3.75 mM NaH2PO4, 1 mM EDTA and 0.1 M NaCl.
4-Nitrophenyl chlorosulfate 1627,28
Oligonucleotide synthesis and electrospray mass spectrometry
A solution of 4-nitrophenol (1.39 g, 10 mmol) and pyridine
(0.81 mL, 10 mmol) in Et2O (10 mL) was added dropwise to
a solution of SO2Cl2 (0.80 mL, 10 mmol) in Et2O (10 mL) at
Ϫ78 ЊC under argon. The mixture was allowed to warm to room
temperature, stirred for 4 h, and then filtered through Celite.
The solvent was evaporated under reduced pressure and the
resulting orange crystalline solid was recrystallised at Ϫ78 ЊC
from a mixture of CH2Cl2 and petroleum ether to give 16 (1.82
g) as a low-melting, pale yellow solid. The mother liquor was
evaporated and the residue was purified by flash chromato-
graphy (CH2Cl2–petroleum ether 1 : 1) to give further product
16 (147 mg) in 83% overall yield: νmax(KBr)/cmϪ1 3080, 3120
(Ar–CH), 1535 (NO2), 1490 (Ar), 1430 (SO2), 1350 (NO2), 1210,
1180, 1150 (SO2); δH (270 MHz; CDCl3) 7.61 (2H, d, J 9.2,
H2/6), 8.39 (2H, d, J 9.2, H3/5); δC (67.9 MHz; CDCl3) 122.9
(C2/6), 126.0 (C3/5), 147.5 (C4), 153.5 (C1); m/z (FABϩ) 238
([M(35Cl) ϩ H]ϩ, 100%), 240 ([M(37Cl) ϩ H]ϩ, 35); HRMS m/z
(FABϩ) 237.9590 ([M(35Cl) ϩ H]ϩ, [C6H5ClNO5S35]ϩ requires
m/z, 237.9577).
Solid-phase supports and standard nucleoside phosphoramid-
ites were purchased from Glen Research (Virginia, USA).
0.1 M Phosphoramidite solutions in anhydrous acetonitrile
were prepared and oligonucleotides were synthesised auto-
matically on an ABI (Foster City, CA, USA) 392 DNA/RNA
synthesiser using standard procedures. 1 µM Syntheses were
performed for native and 0.2 µM for oligonucleotides incor-
porating modified dimers. Coupling wait times were extended
to 60 s for native monomer phosphoramidites and 15 min
for modified dimer phosphoramidites 35–37. 4,5-Dicyano-
imidazole (0.25 M in acetonitrile) was used as the activator.
Coupling efficiencies for oligonucleotide synthesis were
measured using an on-line dimethoxytrityl cation detector.
Final deprotection was carried out using a mixture of conc. aq.
ammonia and absolute ethanol (3 : 1 v/v) for 4 hours at 55 ЊC.
Solutions were evaporated to dryness before being recon-
stituted in Ultrapure water and filtered using a Millex®-GS
0.22 µm filter unit. Purification was achieved by HPLC using a
Hamilton polystyrene reversed-phase PRP-1 HPLC column
(250 × 21.5 mm) and the following elution protocol: Solvent A
was 10 mM aq. tetrabutylammonium phosphate (TBAP),
pH 7.5; solvent B was 10 mM TBAP, pH 7.5, in MeCN–water
(8 : 2, v/v); elution with a convex gradient of 5–95% of solvent
B in A in for 40 min at a flow rate of 5 mL/minϪ1. Fractions
4-Nitrophenyl 5Ј-O-(4,4Ј-dimethoxytrityl)-3Ј-deoxythymidine-
3Ј-sulfamate 18
A solution of 3Ј-amine 15 (54 mg, 0.10 mmol), 4-nitrophenol
(139 mg, 1.00 mmol) and Et3N (170 µL, 1.22 mmol) in CH2Cl2
J. Chem. Soc., Perkin Trans. 1, 2002, 485–495
491

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(0.4 mL) was added dropwise to a solution of chlorosulfate 16
(48 mg, 0.20 mmol) in CH2Cl2 (0.1 mL) at Ϫ78 ЊC under argon.
After being stirred for 30 min the reaction mixture was allowed
to warm to room temperature, diluted with CH2Cl2 (5 mL), and
washed with 1 M aq. NaH2PO4 (3 × 5 mL). The aqueous layers
were then re-extracted with CH2Cl2 (2 × 5 mL) and the com-
bined organic extracts were dried over MgSO4 and evaporated
under reduced. The resulting residue was purified by flash
chromatography, eluting with 10% EtOAc in CH2Cl2 followed
by 1 2% MeOH in CH2Cl2 to give sulfamate 18 (51 mg, 68%)
as a white foam, νmax(KBr)/cmϪ1 2940–3400 br (NH), 1693 br
4-Nitrophenyl 5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-fluoro-2Ј,3Ј-
dideoxyuridine-3Ј-sulfamate 22
A solution of 4-nitrophenyl chlorosulfate 16 (261 mg, 1.10
mmol) in dry CH2Cl2 (0.5 mL) was added rapidly to a mixture
of 2Ј-fluoro-3Ј-amine 21 (275 mg, 0.50 mmol), 4-nitrophenol
(696 mg, 5 mmol), Et3N (0.83 mL, 6 mmol) and activated 4Å
molecular sieves (≈ 0.5 g) in dry CH2Cl2 (2.5 mL) under argon.
After stirring of the mixture at room temperature for 1 h an
additional equivalent of 4-nitrophenyl chlorosulfate (119 mg,
0.50 mmol) and Et3N (70 µL, 0.5 mmol) in dry CH2Cl2 (230 µL)
was added to the reaction mixture. After a further 2.5 h of
stirring at room temperature, CH2Cl2 (20 mL) was added and
the mixture was washed with 1 M aq. NaH2PO4 (3 × 30 mL).
The aqueous layers were then re-extracted with CH2Cl2 (2 ×
50 mL) and the combined organic extracts were dried over
MgSO4 and evaporated under reduced. Purification by flash
chromatography, eluting with 10% EtOAc in CH2Cl2 followed
by a gradient of 10 50% acetone in CH2Cl2, gave 2Ј-fluoro-3Ј-
sulfamate 22 as a pale yellow foam (154 mg, 59% based on
unrecovered starting material 21), νmax(KBr)/cmϪ1 3310–2860
(C᎐O), 1530, 1349 (NO ), 1255, 1180 (SO ); δ (500 MHz;
᎐
2
2
H
CDCl3) 1.49 (3H, d, J 1.1, CH3), 2.43 (1H, ddd, J 13.5, 5.4 and
1.4, Ha2Ј), 2.50 (1H, ddd, J 13.5, 9.4 and 7.3, Hb2Ј), 3.40 (1H,
dd, J 10.8 and 2.5, Hb5Ј), 3.56 (1H, dd, J 10.8 and 2.8, Ha5Ј),
3.78 (6H, s, 2 × OCH3), 4.34 (1H, ddd, J 2.8, 2.5 and 2.0 Hz,
H4Ј), 4.38 (1H, ddd, J 7.3, 2.0 and 1.4, H3Ј), 6.55 (1H, dd, J 9.4
and 5.4, H1Ј), 6.82–6.87 (4H, m, ArH), 7.23–7.33 (9H, m,
ArH), 7.37–7.41 (2H, m, ArH), 7.59 (1H, d, J 1.1, H6), 8.20
(2H, d, J 9.1, ArH), 8.37 and 8.88 (each 1H, br, NH); δC (125.7
MHz; CDCl3) 11.8 (CH3), 37.9 (C2Ј), 55.3 (2 × OCH3),
56.1 (C3Ј), 64.1 (C5Ј), 84.2, 85.0, 87.5 [C1Ј, C4Ј, OC(Ar)3],
113.2 (C5), 113.4, 122.6, 125.5, 127.3, 127.8, 128.0, 128.1,
129.1, 130.0, 130.1, 134.5, 135.0, 135.1 (12 × ArC and C6),
144.1, 146.0 (2 × ArC), 151.5 (C2), 154.1, 158.9 (2 × ArC),
162.5 (C4).
br (NH), 1690 (C᎐O), 1530, 1350 (NO ), 1255, 1180 (SO );
᎐
2
2
δH (500 MHz; CDCl3) 3.61 (1H, dd, J 11.4 and 2.8, Hb5Ј),
3.76 (1H, dd, J 11.4 and 1.8, Ha5Ј), 3.78 (3H, s, OCH3), 3.79
(3H, s, OCH3), 4.12 (1H, ddd, J 10.0, 2.8 and 1.8, H4Ј), 4.73
3
3
(1H, ddd, JHF 25.2, JHH 9.8 and 4.3, H3Ј), 5.26 (1H, dd,
3
2JHF 52.0, JHH 4.3, H2Ј), 5.29 (1H, d, J 8.2, H5), 5.99 (1H, d,
3JHF 18.6, H1Ј), 6.82 (4H, m, ArH), 7.23–7.40 (11H, m,
ArH), 7.89 (1H, d, J 8.2, H6), 8.21 (2H, d, J 9.1, ArH),
Symmetrical dimer 19
2
In the absence of excess of Et3N and 4-nitrophenol the reaction
of 3Ј-amine 15 and chlorosulfate 16, according to the procedure
described above, gave the symmetrical dimer 19 as the major
product (58% yield), νmax(KBr)/cmϪ1 2860 br (NH), 1700 br
8.74 (1H, br s, NH); δC (125.7 MHz; CDCl3) 53.9 (d, JCF
16.9, C3Ј), 55.2 (OCH3), 60.2 (C5Ј), 80.7, 87.5 [C3Ј, C(Ar)3],
2
1
89.3 (d, JCF 35.4, C1Ј), 93.3 (d, JCF 183.3, C2Ј), 102.8
(C5), 113.2, 113.3, 122.2, 125.6, 127.3, 128.1, 128.2, 130.2,
130.3, 134.8, 134.9, 140.2, 144.0, 146.1, 149.8, 154.0, 158.8,
158.9 (16 × ArC, C5, C6), 162.7 (C4); δF (188.2 MHz;
CDCl3) Ϫ194.6 (ddd, J 52.0, 25.2 and 18.6); m/z (FABϩ)
881 ([M ϩ Cs]ϩ, 100%), 749 ([M ϩ H]ϩ, 75); HRMS m/z
(FABϩ) 881.0852 ([M ϩ Cs]ϩ, C36H33FN4O11SؒCsϩ requires
m/z, 881.0905).
(C᎐O), 1610, 1510 (Ar), 1255 (SO ); δ (400 MHz; CDCl3)
᎐
2
H
1.59 (6H, s, 2 × CH3), 2.45 (4H, m, 2 × H2Ј/H2Љ), 3.20 (2H, m,
2 × Ha5Ј), 3.40 (2H, m, 2 × Hb5Ј), 3.73 (12H, s, 4 × OCH3), 3.80
(2H, m, 2 × H3Ј), 4.08 (2H, m, 2 × H4Ј), 6.25 (2H, br s,
NHSO2NH), 6.40 (2H, m, 2 × H1Ј), 6.79–7.37 (26H, m,
DMTr), 7.56 (2H, 2 × H6), 9.38 (2H, 2 × imide NH); δC (100.6
MHz; CDCl3) 12.2 (2 × CH3), 30.1 (2 × C2Ј), 55.0 (2 × C3Ј),
55.6 (4 × OCH3), 64 (2 × C5Ј), 85.0 (br, 2 × C1Ј, 2 × C4Ј), 87.4
(2 × OCAr3), 113.6, 113.7, 127.5, 128.1, 128.2, 128.4, 129.5,
130.4, 135.6, 144.6 (Ar, 2 × C5, 2 × C6), 151.8 (2 × C2), 159.1
(2 × Ar), 164.0 (C4); m/z (FABϩ) 1172 ([M ϩ Na]ϩ, 35%), 1149
([M ϩ H]ϩ, 35) and 303 (Ar3Cϩ, 100); HRMS m/z (FABϩ)
1171.4019 ([M ϩ Na]ϩ, C62H64N6O14SؒNaϩ requires m/z,
1171.4099).
5Ј-O-(4,4Ј-Dimethoxytrityl)-3Ј-deoxythymidine-3Ј-sulfamoyl-
[3Ј(N) 5Ј(O)]-3Ј-O-(tert-butyldimethylsilyl)thymidine 24
A mixture of sulfamate 18 (32 mg, 0.043 mmol), 5Ј-alcohol 23
(31 mg, 0.087 mmol) and activated 4Å molecular sieves
(≈ 50 mg) in dry CH2Cl2 (110 µL) was stirred at room tem-
perature for 1 h in a sealed Wheaton vial under argon. Et3N
(31 µL, 0.22 mmol) was then added and the reaction mixture
immediately developed a deep yellow colour. After 12 h the
reaction mixture was filtered, and purified by flash chrom-
atography, eluting with 10% EtOAc in CH2Cl2 followed by a
gradient of 1 3% MeOH in CH2Cl2, to give the sulfamate
dimer 24 (40 mg, 97%) as a white foam, νmax(KBr)/cmϪ1 3470–
3Ј-Amino-5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-fluoro-2Ј,3Ј-dideoxy-
uridine 21
A solution of 2Ј-fluoro-3Ј-azide 2030 (900 mg, 1.57 mmol) in
60% aq. pyridine (20 mL) was saturated with H2S gas and left
for 18 h at room temperature. CH3OH (15 mL) was then added
and the reaction mixture was filtered through Celite, evaporated
under reduced pressure, and purified by flash chromatography,
eluting with a gradient of 0 3% CH3OH in CH2Cl2, to give 2Ј-
fluoro-3Ј-amine 21 (851 mg, 99%) as a yellow solid, mp 107–
109 ЊC; δH (270 MHz; CDCl3) 3.46 (1H, dd, J 11.0 and 2.0 Hz,
Hb5Ј), 3.60–3.69 (2H, m, H3Ј and H4Ј), 3.72 (6H, s, 2 × OCH3),
3.82 (1H, d, J 11.0, H3Ј), 4.78 (1H, dd, 2JHF 52.4, 3JHH 3.9, H2Ј),
5.26 (1H, d, J 8.4, H5), 5.94 (1H, d, J 16.4, H1Ј), 6.78 (4H, d,
J 9.0, ArH), 7.17–7.34 (9H, m, ArH), 8.01 (1H, d, J 8.4, H6);
δC (67.9 MHz; CDCl3) 51.8 (d, 2JCF 18.3, C3Ј), 55.2 (2 × OCH3),
60.5 (C5Ј), 83.3 (C4Ј), 87.0 (CAr3), 88.5 (d, 2JCF 36.7, C1Ј), 95.9
3191 br (NH), 1698 (C᎐O str), 1610, 1510 (Ar), 1275 (Si–C),
᎐
1252, 1178 (SO2); δH (500 MHz; CDCl3) 0.07 and 0.08 (each 3H,
s, SiCH3), 0.88 [9H, s, C(CH3)3], 1.42 (3H, d, J 1.2, CH3), 1.76
(3H, d, J 1.2, CH3), 2.21 (1H, ddd, J 13.5, 7.3 and 3.9, Ha2Ј or
Hb2Ј), 2.41 (1H, ddd, J 13.5, 9.1 and 7.5, Hb2Ј or Ha2Ј), 2.54
(1H, ddd, J 13.5, 5.4 and 1.6, Ha2Ј or Hb2Ј), 2.65 (1H, ddd,
J 13.5, 6.7 and 6.7, Hb2Ј or Ha2Ј), 3.36 (2H, m, Ha5Ј/Hb5Ј), 3.78
(6H, s, 2 × OCH3), 4.06 (1H, ddd, J 5.3, 3.9 and 3.9, H4Ј), 4.26
(2H, m, H4Ј and Hb5Ј), 4.32 (1H, br s, H3Ј), 4.35 (1H, dd, J 10.7
and 4.0, Ha5Ј), 4.53 (1H, ddd, J 7.7, 3.9 and 3.3, H3Ј), 5.72 (1H,
dd, J 6.7 and 6.7, H1Ј), 6.48 (1H, dd, J 9.1 and 5.4, HЈ), 6.82
(4H, m, ArH), 6.96 (1H, d, J 1.2, H6), 7.21–7.31 (9H, m,
ArH), 7.51 (1H, d, J 1.2, H6), 7.61 (1H, br s, SO2NH), 9.64
and 9.83 (each 1H, br s, imide NH); δC (125.7 MHz; CDCl3)
Ϫ4.9, Ϫ4.8 (2 × SiCH3), 11.7, 12.2 (2 × CH3), 17.9
[SiC(CH3)3], 25.7 [SiC(CH3)3], 38.1, 39.1 (2 × C2Ј), 55.2 (2 ×
OCH3), 55.7 (HNC3Ј), 64.0, 69.0 (2 × C5Ј), 72.3 (OC3Ј),
84.2, 84.3, 85.0, 87.2, 90.9 [2 × C4Ј, 2 × C1Ј, OC(Ar)3], 110.8,
1
(d, JCF 183.3, C2Ј), 102.5 (C5), 113.3, 123.7, 127.1, 127.9,
128.1, 130.1, 135.2, 135.3, 135.9, 139.8, 144.3, 149.6, 150.1,
158.7 (12 × ArC, C2, C6), 163.3 (C4); m/z (FABϩ) 680 ([M ϩ
Cs]ϩ, 16%), 570 ([M ϩ Na]ϩ, 27), 548 ([M ϩ H]ϩ, 100), 303
(100); HRMS m/z (FABϩ) 548.2187 ([M ϩ H]ϩ, [C30H31FN3-
O6]ϩ requires m/z, 548.2197).
492
J. Chem. Soc., Perkin Trans. 1, 2002, 485–495

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112.5 (2 × C5), 113.3, 127.2, 128.0, 128.1, 130.0, 135.0, 135.1,
135.2, 138.6, 144.2, 150.4, 151.5, 158.8 (9 × ArC, 2 × C2, 2 ×
C6), 163.5, 163.9 (2 × C4); m/z (FABϩ) 984 ([M ϩ Na]ϩ,
52%), 961 (Mϩ, 17) and 303 (CAr3ϩ, 100); HRMS m/z
(FABϩ) 984.3470 ([M ϩ Na]ϩ, C47H59N5O13SSiؒNaϩ requires
m/z, 984.3497).
5Ј-O-(4,4Ј-Dimethoxytrityl)-3Ј-deoxythymidin-3Ј-ylsulfamido-
[3Ј(N) 5Ј(N)]-3Ј-O-(tert-butyldimethylsilyl)-5Ј-deoxythymidine
2822
A mixture of sulfamate 27 (50 mg, 0.09 mmol), 3Ј-amine 15 (98
mg, 0.18 mmol) and activated 4Å molecular sieves (≈ 75 mg)
in dry CH2Cl2 (225 µL) was sealed in a Wheaton vial under
argon. After being stirred for 12 h the reaction mixture was
filtered and subjected directly to flash chromatography, eluting
with EtOAc–CH2Cl2 (1 : 1), to give sulfamide dimer 28 (72 mg,
83%) as a white foam which was identical to a sample that had
been prepared by an alternative route.22
4-Nitrophenyl 3Ј-O-(tert-butyldimethylsilyl)-5Ј-deoxythymidine-
5Ј-sulfamate 27
A solution of 5Ј-amine 26 (71 mg, 0.20 mmol), 4-nitrophenol
(278 mg, 2.00 mmol) and Et3N (334 µL, 2.40 mmol) in dry
CH2Cl2 (0.8 mL) was added dropwise over a period of 5 min
to a solution of chlorosulfate 16 (95 mg, 0.40 mmol) in dry
CH2Cl2 (0.2 mL) at Ϫ78 ЊC under argon. After 1 h the reaction
mixture was allowed to warm to room temperature, diluted
with CH2Cl2 (5 mL), and washed with 1 M aq. NaH2PO4 (2 ×
25 mL). The aqueous layers were then re-extracted with CH2Cl2
(3 × 15 mL) and the combined organic extracts were dried
over MgSO4 and evaporated under reduced pressure. Purifi-
cation by flash chromatography, eluting with EtOAc–
petroleum ether (1 : 4 1 : 1) gave sulfamate 27 (76 mg, 68%)
5Ј-O-(4,4Ј-Dimethoxytrityl)-3Ј-deoxythymidin-3Ј-ylsulfamoyl-
[3Ј(N) 5Ј(O)]-thymidine 32
A solution of 1 M TBAF in THF (1.68 mL, 1.68 mmol) was
added to a solution of sulfamate dimer 24 (538 mg, 0.56 mmol)
in dry THF (1 mL) and the resulting mixture was stirred for 5 h.
EtOAc (60 mL) was then added and the mixture was washed
with 1 M aq. KH2PO4 (3 × 40 mL). The aqueous layers were
then re-extracted with EtOAc (2 × 50 mL) and the combined
organic extracts were dried over MgSO4 and evaporated under
reduced pressure. Purification by flash chromatography, eluting
with 1 5% MeOH in CH2Cl2, gave dimer-3Ј-alcohol 32 (444
mg, 94%) as a white foam, νmax(KBr)/cmϪ1 3450 (OH), 3196
as a white foam, νmax(CHCl3)/cmϪ1 3675 br (NH), 1696 (C᎐
᎐
O), 1521, 1350 (NO2) 1180 (SO2); δH (270 MHz; CDCl3) 0.04
and 0.06 (each 3H, s, SiCH3), 0.86 [9H, s, C(CH3)3], 1.85 (3H,
d, J 0.8, CH3), 2.17 (1H, ddd, J 13.7, 7.0 and 3.9, Ha2Ј), 2.62
(1H, ddd, J 13.7, 6.9 and 6.9, Hb2Ј), 3.50 (2H, m, H25Ј), 3.99
(1H, dd, J 7.6 and 3.8, H4Ј), 4.47 (1H, ddd, J 7.4, 3.9 and
3.8, H3Ј), 5.78 (1H, dd, J 7.0 and 6.9, H1Ј), 7.01 (1H, d,
J 0.8, H6), 7.43 (2H, d, J 9.4, ArH), 8.22 (2H, d, J 9.4, ArH); δC
(67.9 MHz; CDCl3) Ϫ4.9, Ϫ4.7 [2 × Si(CH3)2], 12.2 (CH3), 17.8
[SiC(CH3)3], 25.6 [SiC(CH3)3], 39.1 (C2Ј), 45.1 (C5Ј), 72.2
(C3Ј), 85.1, 90.1 (C1Ј and C4Ј), 111.5 (C5), 122.4, 125.4 (ArC),
138.6 (C6), 145.8 (ArC), 150.4 (C2), 154.7 (ArC), 163.6
(C4); m/z (FABϩ) 579 ([M ϩ Na]ϩ, 11%), 557 ([M ϩ H]ϩ,
34), 431 ([M Ϫ C5H5N2O2]ϩ, 28), 418 ([M Ϫ C6H4NO3]ϩ, 7);
HRMS m/z (FABϩ) 557.1766 ([M ϩ H]ϩ, [C22H32N4O9SSi]ϩ
requires m/z, 557.1738).
(NH), 1700 (C᎐O), 1610, 1510 (Ar), 1254 and 1177 (SO );
᎐
2
δH (500 MHz; acetone-d6) 1.48 (3H, d, J 1.2, CH3), 1.78 (3H, d,
J 1.2, CH3), 2.25 (2H, dd, J 6.9 and 5.1, H22Ј), 2.55 (1H, ddd,
J 14.0, 6.7 and 5.2, Ha2Ј), 2.61 (1H, ddd, J 14.0, 8.1 and 6.7,
Hb2Ј), 3.43 (1H, dd, J 10.7 and 2.9, Hb5Ј), 3.45 (1H, dd, J 10.7
and 3.7, Ha5Ј), 4.07 (1H, ddd, J 4.8, 3.7 and 3.5, H4Ј), 4.19 (1H,
m, H4Ј), 4.27 (1H, dd, J 10.9 and 4.8, Hb5Ј), 4.35 (1H, dd,
J 10.9 and 3.5, Ha5Ј), 4.40–4.46 (2H, m, 2 × H3Ј), 6.25–6.30
(2H, m, 2 × H1Ј), 6.89 (4H, m, ArH), 7.21–7.39 (7H, m, ArH),
7.45 (1H, d, J 1.3, H6), 7.49 (2H, m, ArH), 7.59 (1H, d, J 1.2,
H6); δC (125.7 MHz; acetone-d6) 12.2, 12.5 (2 × CH3), 38.8, 40.2
(2 × C2Ј), 55.0 (HNC3Ј), 55.5 (2 × OCH3), 64.0, 70.4 (2 × C5Ј),
71.8 (OC3Ј), 84.2, 84.7, 85.0, 85.8 (2 × C4Ј, 2 × C1Ј), 87.5
[OC(Ar)3], 111.1, 111.2 (2 × C5), 114.0, 127.7, 128.7, 129.0,
131.0 (5 × ArC), 136.3, 136.5, 136.5, 136.6 (2 × ArC, 2 × C6),
145.8 (ArC), 151.2 (2 × C2), 159.7 (ArC), 164.1 (2 × C4); m/z
(FABϩ) 870 ([M ϩ Na]ϩ, 25%), 847 ([M ϩ H]ϩ, 15); HRMS
m/z (FABϩ) 870.2650 ([M ϩ Na]ϩ, C41H45N5O13SؒNaϩ requires
m/z, 870.2632).
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-fluoro-2Ј,3Ј-dideoxyuridin-3Ј-
ylsulfamido-[3Ј(N) 5Ј(N)]-3Ј-O-(tert-butyldimethylsilyl)-5Ј-
deoxythymidine 29
Et3N (5 µL, 0.036 mmol) was added to a solution of 2Ј-fluoro-
3Ј-amine 21 (20 mg, 0.036 mmol) and 5Ј-sulfamate 27 (29 mg,
0.052 mmol) in dry THF (200 µL) sealed in a Wheaton vial
under argon. After 3 h the reaction mixture was purified by
flash chromatography, eluting with EtOAc–CH2Cl2 (1 : 1), to
give sulfamide dimer 29 (28 mg, 80%) as a white solid, mp 135–
137 ЊC; δH (400 MHz; CD3OD) 0.16 (6H, s, 2 × SiCH3), 0.95
[9H, s, C(CH3)3], 1.90 (3H, d, J 1.0, CH3), 2.18–2.30 (2H, m,
T-H22Ј), 3.15 (1H, dd, J 14.1 and 5.0, T-Hb5Ј), 3.21 (1H, dd,
J 14.1 and 4.5, T-Ha5Ј), 3.59 (2H, m, U-H25Ј), 3.80 (3H, s,
OCH3), 3.81 (3H, s, OCH3), 3.94 (1H, m, T-H4Ј), 4.14 (1H,
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-fluoro-2Ј,3Ј-dideoxyuridin-3Ј-
ylsulfamido-[3Ј(N) 5Ј(N)]-5Ј-deoxythymidine 33
A solution of sulfamide dinucleoside 29 (319 mg, 0.331 mmol)
in dry THF (3 mL) was treated with 1 M TBAF in THF (1 mL,
1 mmol). After being stirred for 1.5 h the reaction mixture was
worked up and purified as described above (24 32) to give the
dimer-3Ј-alcohol 33 (272 mg, 97%) as a white solid, mp 205–208
ЊC; δH (270 MHz; CDCl3) 1.86 (3H, s, CH3), 2.24 (2H, m, H22Ј),
3.10 (1H, dd, J 14.1 and 5.1, Hb5Ј), 3.45 (1H, dd, J 14.1 and
3.9, Ha5Ј), 3.56 (2H, br s, H25Ј), 3.75 (6H, s, 2 × OCH3), 3.87
(1H, dd, J 8.8 and 4.5, T-H4Ј), 4.06 (1H, m, U-H4Ј), 4.25–4.44
3
3
m, U-H4Ј), 4.43 (1H, ddd, JHF 27.1, JHH 10.6 and 4.0,
U-H3Ј), 4.50 (1H, m, T-H3Ј), 5.17 (1H, d, J 8.0, U-H5), 5.30
2
3
3
(1H, dd, JHF 52.0, JHH 4.0, U-H2Ј), 5.98 (1H, d, JHF 17.6,
U-H1Ј), 6.22 (1H, dd, J 6.5 and 7.5, T-H1Ј), 6.89 (4H, d,
J 9.0, ArH), 7.21–7.49 (9H, m, ArH), 7.54 (1H, d, J 1.0, T-H6),
8.13 (1H, d, J 8.0, U-H6); δC (100.6 MHz; CD3OD) Ϫ4.1
(2 × SiCH3), 12.9 (T-CH3), 19.2 [SiC(CH3)3], 26.7 [SiC(CH3)3],
2
(2H, m, 2 × H3Ј), 5.14 (1H, d, J 7.8, H5), 5.21 (1H, dd, JHF
3
3
52.0, JHH 3.9, U-H2Ј), 5.94 (1H, d, JHF 17.6, U-H1Ј), 6.12
(1H, t, J 6.6, T-H1Ј), 6.80–6.83 (4H, d, J 9.0, ArH), 7.16–7.41
(10H, m, ArH and T-H6), 8.04 (1H, d, J 7.8, U-H6); δC (67.9
MHz; CDCl3) 11.2 (CH3), 38.7 (T-C2Ј), 43.5 (T-C5Ј), 52.1
2
41.1 (T-C2Ј), 45.5 (T-C5Ј), 54.4 (d, JCF 18.3, U-C3Ј), 56.2
(2 × OCH3), 61.9 (U-C5Ј), 74.3, 82.2, 87.1, 87.3 (T-C3Ј, U-C4Ј,
T-C4Ј and T-C1Ј), 88.8 [C(Ar)3], 91.5 (d, 2JCF 34.6, U-C1Ј), 94.7
1
2
(d, JCF 183.4, U-C2Ј), 102.9, 112.3 (T-C5 and U-C5) 114.7,
(d, JCF 18.3, U-C3Ј), 54.4 (2 × OCH3), 59.6 (U-C5Ј), 70.5
116.9, 127.5, 128.5, 129.4, 129.9, 131.9, 132.0, 136.9, 137.1,
138.7, 142.8, 146.3 (11 × ArC, U-C6 and T-C6), 152.1, 152.8
(2 × C2), 160.6, 160.7 (2 × ArC), 166.4, 166.8 (2 × C4); m/z
(FABϩ) 987 ([M ϩ Na]ϩ, 71%), 964 ([M]ϩ, 37); HRMS
m/z (FABϩ) 964.3474 (Mϩ, C46H57FN6O12SSiϩ requires M,
964.3509).
(T-C3Ј), 80.1, 84.0, 85.2, 86.6 (T-C1Ј, 2 × C4Ј, OCAr3), 88.9
2
1
(d, JCF 36.7, U-C1Ј), 92.5 (d, JCF 187.0, U-C2Ј), 101.4, 110.5
(2 × C5), 112.7, 126.5, 127.3, 127.7, 129.7, 129.8, 134.6, 134.8
(6 × ArC, 2 × C6), 136.4, 140.1, 143.8 (3 × ArC), 149.9 and
150.4 (2 × C2), 158.2, 158.3 (2 × ArC), 163.9, 164.4 (2 × C4);
m/z (FABϩ) 873 ([M ϩ Na]ϩ, 15%), 850 (Mϩ, 40); HRMS m/z
J. Chem. Soc., Perkin Trans. 1, 2002, 485–495
493

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(FABϩ) 873.2549 ([M ϩ Na]ϩ, C40H43FN6O12SؒNaϩ requires
m/z, 873.2541).
159.0, 159.1, 162.7 (ArC); δp (202.4 MHz; CDCl3; 287 K) 149.2,
150.8 (P diastereoisomers); m/z (FABϩ) 1089 ([M ϩ K]ϩ, 90%),
303 (CAr3ϩ, 100); HRMS m/z (FABϩ) 1089.3377 ([M ϩ K]ϩ,
C49H60N8O13FPSؒKϩ requires m/z, 1089.3359).
5Ј-O-(4,4Ј-Dimethoxytrityl)-3Ј-deoxythymidin-3Ј-ylsulfamoyl-
[3Ј(N) 5Ј(O)]-3Ј-O-[(2-cyanoethoxy)(diisopropylamino)phos-
phino]thymidine 35
3Ј-Deoxythymidin-3Ј-ylsulfamoyl-[3Ј(N) 5Ј(O)]-thymidine
d(TnsoT) 30
A solution of 0.375 M 2-cyanoethyl N,N,N Ј,NЈ-tetraisopropyl-
phosphoramidite in CH2Cl2 (1.76 mL, 0.66 mmol) was added
to a mixture of 3Ј-alcohol 32 (335 mg, 0.40 mmol) and 4,5-
dicyanoimidazole (47 mg, 0.40 mmol) under argon. After
stirring of the mixture for 12 h CH2Cl2 (20 mL) was added and
the mixture was washed with saturated aq. NaHCO3 (3 ×
20 mL) followed by saturated aq. NaCl (20 mL). The aqueous
layers were then re-extracted with CH2Cl2 (2 × 60 mL) and
the combined organic extracts were dried over MgSO4 and
evaporated under reduced pressure. Purification by flash
chromatography, eluting with 1% Et3N in CH2Cl2 then 1% Et3N
in EtOAc, gave a mixture of phosphoramidite diastereoisomers
35 (307 mg, 74%), as a white powder; νmax(KBr)/cmϪ1 3190
3% CHCl2CO2H in dry CH2Cl2 (0.5 mL) was added to the
3Ј-alcohol 32 (35 mg, 0.041 mmol) under argon. The orange
reaction mixture was purified immediately by flash chromato-
graphy, eluting with a gradient of 0 10% MeOH in CH2Cl2,
to give d(TnsoT) 30 (17.5 mg, 78%) as a white foam, νmax(KBr)/
cmϪ1 3420 (OH), 3211 (NH), 1691 (C᎐O), 1275, 1180 (SO );
᎐
2
δH (500 MHz; D2O; 303 K) 1.84 (3H, s, CH3 5Ј-TnsoT -3Ј), 1.85
(3H, s, CH3 5Ј-T nsoT-3Ј), 2.40 (2H, m, H22Ј TnsoT ), 2.45–2.50
(2H, m, H22Ј T nsoT), 3.78 (1H, dd, J 12.8 and 3.7, Hb5Ј
T nsoT), 3.91 (1H, dd, J 12.8 and 2.7, Ha5Ј T nsoT), 3.99–4.06
(2H, m, H3Ј and H4Ј T nsoT), 4.19 (1H, ddd, J 4.9, 3.8 and 2.6,
H4Ј TnsoT ), 4.39 (1H, dd, J 11.1 and 3.8, Hb5Ј TnsoT ), 4.46
(1H, dd, J 11.1 and 2.6, Ha5Ј TnsoT ), 4.58 (1H, ddd, J 5.7, 5.7
and 4.9, H3Ј TnsoT ), 6.07 (1H, dd, J 6.5 and 5.1, H1Ј T nsoT),
6.28 (1H, dd, J 6.5 and 6.5, H1Ј TnsoT ), 7.51 (1H, s, H6
TnsoT ), 7.68 (1H, s, H6 T nsoT); δC (125.7 MHz; D2O) 11.5,
11.6 (2 × CH3), 37.1, 38.3 (2 × C2Ј), 51.8 (NHC3Ј), 59.6, 68.7
(2 × C5Ј), 69.5 (OC3Ј), 82.9, 83.8 (2 × C4Ј), 85.0 (2 × C1Ј
coincident), 111.0, 111.3 (2 × C5), 137.0, 137.1 (2 × C6),
151.1, 151.4 (2 × C2), 165.8, 166.1 (2 × C4); m/z (FABϩ) 568
([M ϩ Na]ϩ, 35%); HRMS m/z (FABϩ) 568.1350 ([M ϩ Na]ϩ,
C20H27N5O11SؒNaϩ requires m/z, 568.1325).
(NH), 2255 (CN), 1690 (C᎐O), 1610, 1512 (Ar), 1255, 1180
᎐
(SO2); δH (500 MHz; CDCl3) 1.18 [12H, m, 2 × NCH(CH3)2],
1.46 (3H, br s, 5-CH3), 1.80 (3H, d, 5-CH3), 2.36–2.73 (6H,
m, CH2CN, 2 × H22Ј), 3.39 (2H, m, H25Ј), 3.56–3.64 [2H, m,
2 × NCH(CH3)2], 3.69–3.75 (1H, m, OCHaHbCH2CN), 3.80
(6H, s, 2 × OCH3), 3.84–3.91 (1H, m, OCHaHbCH2CN), 4.23–
4.29 (2H, m, 2 × H4Ј), 4.31–4.46 (3H, m, HNC3ЈH, H25Ј),
4.61–4.69 (1H, m, OC3ЈH), 5.84 and 5.89 (each 0.5H, dd,
H1Ј diastereoisomers), 6.42–6.47 (1H, m, H1Ј), 6.92 (4H, d,
J 8.8, ArH), 7.02 and 7.05 (each 0.5H, s, H6 diastereoisomers),
7.22–7.42 (9H, m, ArH), 7.51 (1H, s, H6); δC (100.6 MHz;
CDCl3) 11.6, 11.9 (2 × 5-CH3), 20.5 (CH2CN), 24.7, 25.2
[2 × NCH(CH3)2], 38.3, 38.5 (2 × C2Ј), 43.2, 43.4, 43.5 [2 ×
NCH(CH3)2 diastereoisomers], 55.4 (2 × OCH3 and NC3Ј),
58.1, 58.3 (OCH2CH2CN diastereoisomers), 64.1, 69.4 (2 ×
OC5Ј), 73.5, 73.6 (OC3Ј diastereoisomers), 84.0, 84.4, 87.3,
89.5 [2 × C1Ј, 2 × C4Ј, C(Ar)3], 111.1, 111.3, 112.5 (2 × C5
diastereoisomers), 113.5 (ArC), 118.2 (CN), 127.3, 128.2, 130.2
(4 × ArC), 135.1, 135.3, 135.4 (2 × C6, ArC), 144.4 (ArC),
150.7, 151.5 (2 × C2), 158.9 (ArC), 163.6, 163.9, 164.1 (C4
diastereoisomers); δp (161.9 MHz; CDCl3) 149.2, 149.9 (P
diastereoisomers); m/z (FABϩ) 1086 ([M ϩ K]ϩ, 55%), 303
(CAr3ϩ, 100); HRMS m/z (FABϩ) 1086.3446 ([M ϩ K]ϩ,
C50H62N7O14PSؒKϩ requires m/z, 1086.3450).
Acknowledgements
We thank the EPSRC for studentships to K. J. F. and D. T. H.,
the EPSRC National Chiroptical Spectroscopy Service
(Kings College, London), the Michael Baber centre for
Mass Spectrometry, and Dr Polkit Sangvanich (UMIST) for
assistance with electrospray mass spectrometry.
References
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5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-fluoro-2Ј,3Ј-dideoxyuridin-3Ј-
ylsulfamido-[3Ј(N) 5Ј(N)]-3Ј-O-[(2-cyanoethoxy)(diisopropyl-
amino)phosphino]-5Ј-deoxythymidine 36
A solution of 0.375 M 2-cyanoethyl N,N,N Ј,NЈ-tetraisopropyl-
phosphoramidite in CH2Cl2 (0.895 mL, 0.34 mmol) was added
to a mixture of 3Ј-alcohol 33 (168 mg, 0.197 mmol) and 4,5-
dicyanoimidazole (23 mg, 0.197 mmol). After stirring of the
solution for 12 h CH2Cl2 (10 mL) was added and the mixture
was worked up and purified as described above (32 35) to give
a mixture of phosphoramidite diastereoisomers 36 (102 mg,
49%) as a white powder, νmax(KBr)/cmϪ1 3240 (NH), 2260 (CN),
9 G.-X. He, J. P. Williams, M. J. Postich, S. Swaminathan, R. G. Shea,
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1690 (C᎐O), 1610, 1512 (Ar), 1255, 1180 (SO ); δ (500 MHz;
᎐
2
H
CDCl3; 323 K) 1.12–1.20 [12H, m, 2 × NCH(CH3)2], 1.82 (3H,
br s, CH3], 2.37–2.70 (4H, m, CH2CN, H22Ј), 3.40–3.65 [6H, m,
2 × H25Ј and 2 × NCH(CH3)2], 3.72 (7H, m, 2 × OCH3 and
OCHaHbCH2CN), 3.88 (1H, m, H4Ј), 4.00 (2H, m, NHC3ЈH,
OCHaHbCH2CN), 4.38 (1H, br d, H3Ј), 4.70 (1H, br d, H3Ј),
5.21 (1H, m, H5), 5.26 (1H, d, 2JHF 52, H2Ј), 5.93 (1H, m, H1Ј),
6.15 (1H, m, H1Ј), 6.82 (4H, d, ArH), 7.05–7.45 (9H, m,
ArH), 7.87 (1H, m, H6); δC (100.6 MHz; CDCl3; 298 K) partial
spectrum 12.5 (5-CH3), 20.4 (CH2CN), 24.6 [NCH(CH3)2],
43.2, 43.3 [2 × NCH(CH3)2, 45.8 (T-C5Ј), 52.4 (U-C3Ј), 55.2
(2 × OCH3), 57.8, 59.8 (OCH2CH2CN and U-C5Ј), 80.9, 83.9,
87.2, 88.9 (T-C3Ј, U-C4Ј, T-C4Ј, T-C1Ј, CAr3), 102.1 (C5),
113.7, 127.5, 128.4, 128.6, 130.5, 130.7, 134.5, 144.2, 150.4,
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