A conformationally locked tricyclic nucleoside. Synthesis, crystal structure and incorporation into oligonucleotides

Article abstract of DOI:10.1039/b104438a

A tricyclic nucleoside is synthesised from a bicyclic nucleoside precursor by applying a stereoselective dihydroxylation, a regioselective tosylation and an intramolecular ether formation. This tricyclic nucleoside is constructed as a conformationally loc

Full text of DOI:10.1039/b104438a

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A conformationally locked tricyclic nucleoside. Synthesis,
crystal structure and incorporation into oligonucleotides
Jacob Ravn,a Niels Thorupb and Poul Nielsen*a
1
a Department of Chemistry, University of Southern Denmark, Odense University, Odense,
DK-5230, Denmark
b Department of Chemistry, Technical University of Denmark, DTU 207, Lyngby, DK-2800,
Denmark
Received (in Cambridge, UK) 21st May 2001, Accepted 22nd June 2001
First published as an Advance Article on the web 23rd July 2001
A tricyclic nucleoside is synthesised from a bicyclic nucleoside precursor by applying a stereoselective
dihydroxylation, a regioselective tosylation and an intramolecular ether formation. This tricyclic nucleoside
is constructed as a conformationally locked thymidine analogue and has been analysed by X-ray crystallography.
Thus, the furanose ring of this nucleoside adopts a perfect S-type conformation and the torsion angle γ, describing
the C4Ј–C5Ј bond is restricted in the ϩac range. The tricyclic nucleoside is incorporated into two nonameric
oligonucleotide sequences displaying strongly decreased affinity towards complementary DNA and RNA
when compared to the corresponding unmodified oligodeoxynucleotide sequences.
Introduction
The demand for oligonucleotides with efficient and selective
recognition of complementary nucleic acid sequences for
diagnostic or therapeutic purposes has recently motivated
significant research activity.1,2 Conformationally restricted
nucleosides have proven to be a very successful approach for
the construction of oligonucleotides preorganised for efficient
nucleic acid recognition.2,3 As a prime example, LNA (locked
nucleic acid) has recently been introduced4 as a nucleic acid
analogue displaying unprecedented recognition of comple-
mentary DNA and RNA due to its nucleoside monomers
being conformationally locked in an N-type (C3Ј-endo) con-
formation5 by a bicyclic carbohydrate moiety.4 Other bi- and
tricyclic nucleoside analogues have been introduced in this
context3 including the pioneer example 1 by Leumann and
co-workers (Fig. 1).6 This nucleoside structure has been found
to be restricted in an S-type (C1Ј-exo) conformation. However,
the C4Ј–C5Ј bond is also restricted and the torsion angle
γ describing this bond5 is found to prefer the ϩac/ϩap range,
which is known to be untypical in natural duplexes. Sub-
Fig. 1 Bicyclic and tricyclic nucleosides.
sequently, oligonucleotides containing 1 have been shown to
prefer the Hoogsteen base-pairing mode over the usual
Watson–Crick base-pairing.7 The binding affinities towards
complementary nucleic acid sequences were slightly improved
compared to unmodified oligodeoxynucleotides but were very
dependent on the nucleotide sequences.6,7 Improved binding
affinities have been introduced with a 5Ј–6Ј cyclopropanated
tricyclic analogue of 1 in which γ is restricted to the less
unfavourable ϩac range.8
Another bicyclic nucleoside with interesting properties is 2
which in a fully modified sequence demonstrated increased
affinity for complementary RNA but not DNA.9 This nucleo-
side has been shown by molecular modelling of the monomer,10
as well as in NMR11 and X-ray studies12 of duplexes containing
this modification, to adopt an E-type (O4Ј-endo) conform-
ation.5 The bicyclic nucleoside 3 has also been synthesised13
and incorporated into oligonucleotides. However, very large
decreases in duplex stability were observed.14 This nucleoside
might be suggested to be strongly restricted in a perfect S-type
conformation but with γ in an unfavourable ϩap conform-
ation due to a chair conformation of the six-membered ring.
Furthermore, the 6Ј-carbon atom might sterically prevent the
nucleobase from taking a proper anti-position5 relative to the
furanose ring.
Recently, we have introduced the syntheses of bicyclic ribo-
nucleoside analogues by an RCM (ring-closing metathesis)
method.15 Thus, an olefinic moiety connecting the C3Ј and the
C5Ј atoms provided the opportunity for the construction of
analogues of 1 as well as e.g. tricyclic nucleosides.15 Following
up these results, we hereby introduce a nucleoside combining
the ring-systems of the three bicyclic nucleosides 1, 2, and 3 in a
tricyclic structure 4. Thus, interesting properties concerning the
recognition of complementary nucleic acids might be obtained.
Thus, each of the three bicyclic ring systems in 1, 2, and 3 retain
some degree of flexibility but are hereby strongly conform-
ationally restricted, apparently towards more favourable con-
formations. Hence, simple modelling suggests that this tricyclic
nucleoside 4 will be conformationally locked due to the very
rigid [2.2.1]bicyclic system connected to the furanose ring.
Furthermore, this furanose should adopt a perfect S-type con-
formation and γ should prefer a conformation that is less
unfavourable than the ranges found for 1 and 3.
DOI: 10.1039/b104438a
J. Chem. Soc., Perkin Trans. 1, 2001, 1855–1861
This journal is © The Royal Society of Chemistry 2001
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In order to perform a ring-closing reaction between the 2Ј-
OH group and C6Ј, the 6Ј-OH group was converted to an
appropriate sulfonate as a potential leaving group. Taking
advantage of the sterically demanding toluene-p-sulfonate
group, the selective esterification between secondary alcohols
has been observed before avoiding tosylation of the 2Ј-OH on
a similar arabino-nucleoside substrate.17 Thus, treatment of 8
with 7 equivalents of toluene-p-sulfonyl chloride in pyridine at
room temperature resulted in a completely regioselective tosyl-
ation of 6Ј-OH to give the monotosylate 9 in 77% yield together
with a small amount of unreacted starting material 8. We sug-
gest that the first sulfonic ester sterically prevents the formation
of a second ester on the neighbouring 7Ј-OH group. Cyclisation
of 9 by treatment with NaH in DMF and heating did not give
the expected tricyclic compound. Instead, an analysis of the
crude product indicated cyclisation between O2 of the thymine
nucleobase and C6Ј to give a 2,6Ј-anhydro compound. In order
to avoid this unwanted reaction, we decided to replace the
imide proton of the nucleobase with a benzyloxymethyl (BOM)
group, thereby preventing a nucleophilic attack from the
adjacent O2 carbonyl group. Surprisingly, this protection of
N3 using benzyloxymethyl chloride and 1,8-diazabicyclo[5.4.0]-
undec-7-ene (DBU) in CH3CN did not afford the N-BOM-
protected analogue of 9, but instead a single product, which
was identified as the cyclised compound 10, was obtained in
76% yield. Thus, after BOM-protection DBU must have been
able to promote the immediate cyclisation between 2Ј-OH and
C6Ј. Finally, treatment of 10 with hydrogen in the presence of
20% Pd(OH)2–C in methanol afforded the fully deprotected
tricyclic nucleoside 4 in 65% yield. The structure of this
novel nucleoside was confirmed by NMR, mass spectrometry
and X-ray crystallographic data (vide infra).
Results
Synthesis of the tricyclic nucleoside 4
The tricyclic nucleoside 4 and the corresponding phosphor-
amidite building block 14 were synthesised from the benzyl-
protected bicyclic nucleoside 5 as outlined in Scheme 1. The
Alternative approaches
In addition to the successful synthetic strategy shown (Scheme
1), two other strategies for the construction of 4 have been
investigated (Scheme 2). Thus, conversion of the olefinic moiety
Scheme 1 Reagents and conditions: i, MsCl, pyridine; ii, aq. NaOH,
EtOH; iii, OsO4, NMO, aq. THF, t-BuOH; iv, TsCl, pyridine; v, BOM-
Cl, DBU, CH3CN; vi, BzCl, pyridine; vii, H2, Pd(OH)2–C, MeOH; viii,
DMT-OTf, DMAP, pyridine; ix, NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2,
CH2Cl2.
synthesis of 5 in 13 steps and 36% overall yield from diacetone-
α--glucose, taking advantage of an RCM strategy, has recently
been described by us.15 Inversion of configuration at C2Ј was
achieved using a 2,2Ј-anhydro approach.9,16 Thus, conversion of
the 2Ј-OH of 5 to the corresponding methanesulfonic ester to
give 6 and subsequent treatment with aq. base in ethanol
afforded the arabino-configured nucleoside 7 in 82% yield over
the two steps. The alkene moiety of the bicyclic system was
treated with a catalytic amount of osmium tetraoxide in the
presence of N-methylmorpholine N-oxide (NMO) as a co-
oxidant to give the dihydroxylated nucleoside 8 in 83% yield.
The reaction proceeded with complete stereoselectivity giving
only a single product. The stereochemistry of this product 8
Scheme 2 Reagents and conditions: i, H2O2, K2CO3, PhCN, MeOH;
ii, NaH, DMF; iii, SOCl2, Et3N, CH2Cl2.
3
was indicated by a large coupling constant JH5ЈH6Ј = 7.5 Hz
indicating the two protons to be in a trans position on the
cyclopentane ring. This result was expected, since osmium
tetraoxide should approach the double bond from the less
hindered, convex side of the bicyclic system. Unfortunately, the
reaction is relatively slow giving the product in 84% yield after
stirring at 50 ЊC for at least 5 days. Raising the temperature
above 50 ЊC did not speed up the reaction but led to a varying
amount of by-products resulting from dihydroxylation of the
thymine double bond.
of 7 to an epoxide was expected to proceed stereoselectively
from the convex side of the bicyclic nucleoside affording a rea-
sonable substrate for intramolecular ether formation. However,
this potential was recognised for both the target structure 4 as
well as for an alternative, but still interesting, tricyclic nucleo-
side containing a four-membered ring connecting 2Ј-O and C7Ј.
After a standard epoxidation using MCPBA failed to give any
product, the epoxide 15 was obtained as the only product in
68% yield from alkene 7 using the protocol of Payne et al. with
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Synthesis of oligonucleotides
The tricyclic nucleoside 4 was incorporated into oligonucleotide
sequences by using the standard automated solid-phase phos-
phoramidite methodology.19 As a prerequisite, the 7Ј-hydroxy
group should be appropriately protected with e.g. a base-labile
protecting group, complying with the standard methods for
oligonucleotide synthesis. Thus, the hydroxy group of 10
(Scheme 1) was esterified with benzoyl chloride in pyridine to
give 11 in 83% yield, and subsequent debenzylation under
standard hydrogenation conditions afforded the diol nucleoside
12 in a reasonable 57% yield, but surprisingly, also 20% yield
of the fully deprotected nucleoside 4. For the conversion of
nucleoside 12 into an appropriate phosphoramidite, the 5Ј-
hydroxy group was protected as its 4,4Ј-dimethoxytrityl (DMT)
ether. The product 13 was successfully obtained in 64% yield
using 4,4Ј-dimethoxytrityl triflate (DMT-OTf ) in pyridine in
the presence of dimethylaminopyridine (DMAP). This reagent
has been developed by Leumann and co-workers20 and used
for the tritylation of secondary and tertiary alcohols of other
bi- and tricyclic nucleosides.20,21 Despite the predicted steric
hindrance of the secondary 5Ј-hydroxy group of 12, we
expected the tritylation to be selective for this position. The
Fig. 2 Molecular structure (ORTEP-plot) of the tricyclic nucleoside 4.
Table 1 Important torsion angles
Torsion
Definitiona
Measured angleb/Њ
ν0
ν1
ν2
ν3
ν4
δ
C4Ј–O4Ј–C1Ј–C2Ј
O4Ј–C1Ј–C2Ј–C3Ј
C1Ј–C2Ј–C3Ј–C4Ј
C2Ј–C3Ј–C4Ј–O4Ј
C3Ј–C4Ј–O4Ј–C1Ј
O3Ј–C3Ј–C4Ј–C5Ј
C3Ј–C4Ј–C5Ј–O5Ј
O4Ј–C1Ј–N–C2
O3Ј–C3Ј–C7Ј–O7Ј
Ref. 5
Ϫ12.3
35.9
Ϫ42.8
36.8
Ϫ16.4
152.5
137.4
Ϫ151.2
Ϫ61.6
182.2
42.8
1
structure of the only product 13 was verified by its H NMR
1
spectrum. Thus, in the H NMR spectrum of 12 (recorded in
DMSO-d6) the 3Ј-OH signal occurs as a singlet at 6.72 ppm
whereas the 5Ј-OH signal occurs as a doublet at 5.88 ppm. The
fact that only a singlet at 6.79 ppm occurs in the corresponding
spectrum of the DMT-protected analogue 13 strongly indicates
the DMT-group to be situated at O5Ј. This compound was
phosphorylated to give the phosphoramidite building block 14
as an impure material in quantitative yield. Thus, traces of
hydrolysed phosphitylation reagent as well as a phosphonate
were detected in 31P NMR spectra. Nevertheless, this com-
pound could be conveniently used in an automated solid-phase
synthesis of oligodeoxynucleotides using the phosphoramidite
approach.19 Thus, two nonameric mixed sequences incorp-
orating 4 (via phosphoramidite 14) once and three times,
respectively, were synthesised (Table 2). The use of standard
1H-tetrazole activation for the coupling reaction of 14 afforded
only low coupling yields, whereas pyridinium hydrochloride
improved the stepwise coupling yield of 14 to >85%. The modi-
fied oligonucleotides were synthesised by using the DMT-ON
mode and purified by using disposable reversed-phase chrom-
atography cartridges (Cruachem). The purities of the syn-
thesised oligonucleotides were found to be >95% by capillary
gel electrophoresis. The compositions of the sequences were
verified by MALDI-MS spectra.
γ
χ
P
Φmax
ν2/cos P
a Torsion angles defined according to ref. 5. b Torsion angles measured
from crystallographic data.
H2O2 and benzonitrile.18 Nevertheless, subsequent treatment of
15 with NaH in DMF and heating did not give any traceable
cyclisation, but rather a slow decomposition of the starting
material. As an alternative, the same reaction conditions were
applied to the cyclic sulfite 16 synthesised in 43% yield from
the dihydroxylated nucleoside 8 by reaction with thionyl
chloride, but again no cyclisation was observed. As no cyclis-
ation involving the nucleobase was indicated with either 15 or
16, the same BOM-protecting strategy as that successfully
applied before with 9 was not attempted.
Crystal structure of 4
The molecular structure of the tricyclic nucleoside 4 was
determined by a single-crystal X-ray diffraction study of the
dihydrate. The molecules in the crystal are connected through a
network of hydrogen bonds, especially O–H ؒ ؒ ؒ O interactions
involving two hydrate water molecules. A plot of the structure,
as shown in Fig. 2, demonstrates the expected configuration of
the nucleoside analogue and its constrained tricyclic nature.
About the only flexibility of the molecule lies in the torsion
around the anomeric N–C1Ј bond. The remainder of the mol-
ecule is the expected rigid arrangement of interlocked rings
leading to some strain as reflected in several of the bond angles
around C1Ј to C7Ј that deviate considerably from the ideal
tetrahedral values. The most notable deviation is found for
C6Ј–C7Ј–C3Ј, which is 93.4Њ. The pyrimidine ring is planar as
expected, having the attached methyl carbon 0.67 Å out of
that plane. The most relevant torsional angles measured are
displayed in Table 1. Thus, the torsions describing the furanose
ring ν0–ν4 were measured, and from the general definition,5 the
pseudorotational angle P, as well as the puckering amplitude
Φmax, were calculated. Also, the torsions describing the back-
bone angles δ and γ, as well as the torsions describing the
anomeric bond χ and the vicinal diol moiety of the C3Ј–C7Ј
bond, are shown in Table 1.
Melting properties of oligonucleotides
The modified sequences 18 and 19 were mixed with their
complementary DNA and RNA sequences 20 and 21 and the
thermal stabilities of the duplexes were determined (Table 2).
When compared to the corresponding unmodified duplexes
obtained by mixing the unmodified sequence 17 with the same
complements, large decreases in thermal stability of 9 and 10 ЊC
were observed for the sequence 18 with one tricyclic nucleoside
incorporated. For the sequence 19 with three tricyclic nucleo-
sides 4 incorporated, no stable duplexes with either comple-
mentary DNA or RNA could be observed.
Discussion
The target tricyclic nucleoside 4 has been obtained in 6 steps
and 26% overall yield from 5 through an efficient synthetic
strategy including a selective tosylation and subsequent ring-
closing reaction. Including the procedures towards 5,15 4 has
been obtained in 19 steps and 9% overall yield from the cheap
starting material diacetone--glucose taking advantage from
a very efficient RCM-protocol.15 The nucleoside has been
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Table 2 Hybridisation data
T m/ЊCa
DNA complement
20 5Ј-GCATATCAG-3Ј
RNA complement
21 5Ј-GCAUAUCAG-3Ј
Oligonucleotides
17
18
19
5Ј-CTGATATGC-3Ј
5Ј-CTGAXATGC-3Ј
5Ј-CXGAXAXGC-3Ј
29
20
<8
27
17
<8
a Melting temperatures (T m) obtained from the maxima of the first derivatives of the melting curve [A260 (absorbance at 260 nm) vs. temperature]
recorded in a buffer containing 10 mM Na2HPO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0 using 1.5 µM concentrations of the two complementary
sequences assuming identical absorption coefficients for all the thymine nucleotides. X corresponds to 4 incorporated via 14.
efficiently incorporated into oligonucleotides through the
phosphoramidite 14. Thus, the problems with a phosphor-
amidite functionality positioned on a tertiary 3Ј-hydroxy
functionality as described earlier22 were not observed in this
case. Neither was this expected, as the present tricyclic system
should not be able to adopt the planar geometry of a potential
3Ј-cation and, therefore, no cleavage of the C3Ј–O bond
occurred during oxidation.22 On the other hand, rearrangement
of the oligomers during deprotection in the form of a migration
of the sequence in the 3Ј-end of the tricycle from 3Ј-OH to
the free 7Ј-OH cannot be completely excluded from the
MALDI-MS and electrophoretic data. However, we consider
this migration to be very unlikely due to the relatively large
distance (3.0 Å) between the two vicinal hydroxy functionalities.
Thus, the constrained tricyclic system dictates an inflexible
torsion angle O3Ј–C3Ј–C7Ј–O7Ј of 62Њ (Table 1). Furthermore,
no mixtures of different (including shorter) sequences have
been observed, and the sequences 18 and 19 were obtained in
yields as expected from the detected stepwise coupling yields.
The crystallographic data of 4 prove the nucleoside to be
restricted in a typical S conformation with a pseudorotational
angle P = 182Њ and a puckering amplitude Φmax = 43Њ (Table 1).
Hence, this nucleoside can be considered as a perfect mimic
of nucleosides in S-type conformations. The relatively large
puckering amplitude further verifies the conformational rigidity
of the tricyclic system. The C4Ј–C5Ј bond is very strongly
restricted or locked in γ = 137Њ, i.e. in the ϩac range. However,
this is not typically found in nucleic acid duplexes. Nevertheless,
the deviation from the typical ϩsc range (around 60Њ) is smaller
than that observed for another tricyclic nucleoside recently
described by us (178Њ)10 or the bicyclic nucleosides 1 (140–
160Њ),6,7 but is larger compared to the more successful tricyclic
nucleosides constructed by Steffens and Leumann (110–130Њ).8
The hybridisation data (Table 2), however, demonstrate the tri-
cyclic nucleoside structure 4 to be very unfavourable for duplex
formation in contrary to those of the bicyclic nucleoside 16,7
and its 5Ј–6Ј cyclopropanated tricyclic analogue.8 At least,
higher affinity for complementary DNA than for RNA might
have been expected as the S-type conformation dictates a
B-type duplex as typically found in DNA–DNA duplexes. One
reason for the strong general destabilisation of duplexes might
be found in the highly locked structure of 4 in combination with
an imperfect angle γ. Hence, no small adjustments during
hybridisation are allowed. On the other hand, both X-ray data
and simple modelling also suggest the anomeric bond described
by the torsion angle χ to be less flexible than expected and,
perhaps, unfavourably restricted. Apparently, the nucleobase is
prevented from adopting a typical anti position relative to the
furanose ring due to steric hindrance from the 2Ј-O atom and
the 5Ј-O atom both irreversibly locked in their positions by
the tricyclic ring system. Thus, the torsion angle found,
Ϫ151Њ (Table 1), deviates slightly from the values of around
Ϫ130Њ typically found for nucleosides in C2Ј-endo (S-type)
conformations.5 With the free rotation around the anomeric
bond prohibited, the correct position of the nucleobase for
favourable base-pairing and duplex formation cannot be found.
Finally, steric destabilisation or crucial disturbance of the
duplex hydration pattern by the additional rings and the
secondary alcohol moiety (7Ј-OH) cannot be excluded.
The present results suggest that even though strong con-
formational restriction in locked nucleoside structures can be
extremely useful as with LNA,4 the same strategy can also be
extremely unfavourable for duplex formation, if an imperfect
conformation is favoured. In the present case, the effect of
adding a few additional atoms to the natural deoxynucleoside
structure, thereby restricting its conformational freedom, is
at the same level of destabilisation as formerly demonstrated
by increasing the same conformational freedom through the
introduction of acyclic nucleoside moieties.23 Nevertheless, the
possibility of obtaining high-affinity nucleic acid recognition by
using nucleosides that are locked in S-type conformations is
still an obvious opportunity. However, the C4Ј–C5Ј bond
should be either flexible or, perhaps even better, restricted in a
more favourable ϩsc conformational range. Likewise, a proper
anti-positioning of the nucleobase should be allowed.
Conclusions
A tricyclic nucleoside has been efficiently synthesised through
a bicyclic precursor which has been conveniently obtained via
an RCM strategy. From X-ray crystallography, the tricyclic
nucleoside has been demonstrated to adopt a perfect S-type
conformation but with both the C4Ј–C5Ј bond and the
anomeric bond restricted in partly unfavourable conformations.
Subsequently, the tricyclic nucleoside was found to strongly
decrease the affinity of oligodeoxynucleotides for comple-
mentary DNA and RNA sequences. Nevertheless, the results
give new insight into the process of duplex formation and
describe the limits for the design of future conformationally
restricted nucleosides. Alternatively, the present tricyclic nucleo-
side structure might be a significant tool in the study of con-
formational preferences of nucleoside/nucleotide converting
enzymes or receptors. Further work on the design of conform-
ationally restricted bi- and tricyclic nucleosides for the general
study of the scopes and limitations of high-affinity nucleic acid
recognition is in progress in our laboratory.
Experimental
All commercial reagents were used as supplied except for
toluene-p-sulfonyl chloride, which was recrystallised from
chloroform, and thionyl chloride, which was distilled before
use. All reactions were performed under an atmosphere of
nitrogen. Column chromatography was carried out on glass
columns using Silica gel 60 (0.040–0.063 mm). NMR spectra
were obtained on a Bruker AC250, a Varian Gemini 2000 or a
Varian Unity 500 spectrometer. 1H NMR spectra were recorded
at 250, 300 or 500 MHz and 13C NMR spectra were recorded at
62.5 or 75 MHz. Values for δ are in ppm relative to tetramethyl-
silane as internal standard. A 31P NMR spectrum was recorded
1
at 121.5 MHz with 85% H3PO4 as external standard. H–1H-
COSY spectra were recorded for compounds 4, 8, 9, 10, 11, 12,
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15 and 16 and a 1H–13C-COSY spectrum was recorded for
compound 10. Assignments of NMR signals follow standard
carbohydrate and nucleoside style. However, the compound
names are given according to von Baeyer nomenclature. FAB
mass spectra were recorded in positive ion mode on a Kratos
MS50TC spectrometer, and EI mass spectra were recorded
on a SSQ710 Finnigan MAT spectrometer. Microanalyses were
performed at the Microanalytical Laboratory, Department of
Chemistry, University of Copenhagen.
pressure. The residue was extracted with EtOAc (4 × 50 cm3)
and the combined organic extracts were dried (MgSO4). The
solvent was removed by distillation under reduced pressure and
the residue purified by column chromatography [3–8% (v/v)
MeOH in CH2Cl2] to give the product 8 (761 mg, 83%) as a
white solid material (Found: C, 61.51; H, 5.62; N, 5.58.
C26H28N2O8ؒ¹H2O requires C, 61.77; H, 5.78; N, 5.54%);
¯
²
δH (DMSO-d6) 1.61 (3H, s, CH3), 3.99 (1H, dd, J 7.5 and 6.6,
5Ј-H), 4.07–4.20 (2H, m, 6Ј-H and 7Ј-H), 4.27–4.33 (2H, m, 2Ј-
H, 4Ј-H), 4.53 (1H, d, J 11.6, CH2Ph), 4.55 (1H, d, J 11.5,
CH2Ph), 4.66 (1H, d, J 11.6, CH2Ph), 4.82 (1H, d, J 11.5,
CH2Ph), 4.91 (1H, d, J 4.3, 6Ј-OH), 5.05 (1H, d, J 6.8, 7Ј-OH),
5.95–6.00 (2H, m, 1Ј-H and 2Ј-OH), 7.26–7.44 (10H, m, Ph),
7.58 (1H, s, 6-H), 11.34 (1H, s, NH); δC (DMSO-d6) 12.4, 66.3,
69.1, 71.1, 72.0, 75.8, 81.0, 82.2, 86.2, 92.1, 106.9, 127.8, 128.0,
128.2, 128.4, 128.5, 128.6, 137.8, 138.5, 138.7, 150.1, 163.8;
FAB-MS m/z 497 [M ϩ Hϩ].
(1R,3R,4R,5R,8R)-5,8-Bis(benzyloxy)-4-methylsulfonyloxy-3-
(thymin-1-yl)-2-oxabicyclo[3.3.0]oct-6-ene 6
Methanesulfonyl chloride (0.8 cm3, 10.3 mmol) was added
dropwise to a stirred solution of 515 (1.21 g, 2.62 mmol) in
anhydrous pyridine (15 cm3) at 0 ЊC. The reaction mixture was
stirred for 3 h at room temperature, quenched with ice-cold
water (30 cm3), and extracted with CH2Cl2 (3 × 40 cm3). The
combined extracts were washed with saturated aq. NaHCO3
(3 × 20 cm3) and then dried (MgSO4). The solvent was removed
by distillation under reduced pressure and the residue was
co-evaporated with toluene (2 × 50 cm3) and then purified by
column chromatography [0–2% (v/v) MeOH in CH2Cl2] to give
the product 6 (1.36 g, 96%) as a white solid material (Found: C,
60.54; H, 5.58; N, 5.28. C27H28N2O8S requires C, 59.99; H, 5.22;
N, 5.18%); δH (CDCl3) 1.66 (3H, d, J 1.2, CH3), 3.22 (3H, s,
SO2CH3), 4.38 (1H, d, J 11.4, CH2Ph), 4.58 (1H, d, J 11.5,
CH2Ph), 4.63 (1H, d, J 11.5, CH2Ph), 4.72 (1H, m, 5Ј-H), 4.76
(1H, d, J 11.4, CH2Ph), 4.81 (1H, d, J 4.8, 4Ј-H), 5.18 (1H, d,
J 3.5, 2Ј-H), 6.03 (1H, d, J 5.9, 7Ј-H), 6.17 (1H, d, J 5.9, 6Ј-H),
6.17 (1H, d, J 3.5, 1Ј-H), 7.26–7.36 (10H, m, Ph), 7.74 (1H, d,
J 1.2, 6-H), 9.26 (1H, br s, NH); δC (CDCl3) 12.2, 39.2, 67.6,
72.4, 81.6, 82.9, 84.4, 91.3, 92.8, 110.9, 127.6, 127.8, 127.9,
128.2, 128.4, 128.6, 131.2, 135.7, 137.1, 137.3, 138.9, 150.8,
163.6; FAB-MS m/z 541 [M ϩ Hϩ].
(1R,3R,4S,5R,6R,7R,8R)-5,8-Bis(benzyloxy)-4,6-dihydroxy-3-
(thymin-1-yl)-7-(4-tolylsulfonyloxy)-2-oxabicyclo[3.3.0]octane 9
To a solution of 8 (750 mg, 1.51 mmol) in anhydrous pyridine
(50 cm3) was added toluene-p-sulfonyl chloride (2.0 g, 10.5
mmol) and the mixture was stirred at room temperature for 48
h. The reaction was quenched with H2O (50 cm3) and extracted
with CH2Cl2 (3 × 100 cm3). The combined extracts were washed
with saturated aq. NaHCO3 (2 × 150 cm3) and then dried
(MgSO4). The solvent was removed by distillation under
reduced pressure and the residue was co-evaporated with tolu-
ene (2 × 50 cm3) and then purified by column chromatography
[1–3% (v/v) MeOH in CH2Cl2] to give unreacted starting
material 8 (53 mg, 7%) and the product 9 (759 mg, 77%) as
white solid materials (Found: C, 60.99; H, 5.24; N, 4.29.
C33H34N2O10S requires C, 60.91; H, 5.27; N, 4.31%); δH (CDCl3)
1.48 (3H, s, CH3), 2.43 (3H, s, PhCH3), 4.24–4.56 (7H, m, 4Ј-H,
5Ј-H, 7Ј-H, CH2Ph), 4.88 (1H, m, 2Ј-H), 5.29 (1H, dd, J 8.2 and
3.6, 6Ј-H), 6.15 (1H, d, J 2.8, 1Ј-H), 7.35–7.20 (12H, m, Bn, Ts),
7.68 (1H, s, 6-H), 7.80 (2H, d, J 8.1, Ts), 11.32 (1H, br s, NH);
δC (CDCl3) 11.9, 21.6, 67.5, 68.5, 71.5, 72.4, 79.0, 82.4, 85.6,
88.8, 93.3, 107.6, 127.8, 127.8, 127.9, 128.0, 128.1, 128.3, 128.6,
128.6, 129.8, 133.6, 137.2, 137.3, 145.1, 150.5, 166.6; FAB-MS
m/z 651 [M ϩ Hϩ].
(1R,3R,4S,5R,8R)-5,8-Bis(benzyloxy)-4-hydroxy-3-(thymin-1-
yl)-2-oxabicyclo[3.3.0]oct-6-ene 7
To a solution of 6 (1.05 g, 1.94 mmol) in EtOH (25 cm3) and
H2O (25 cm3) was added 2.0 M aq. NaOH (4 cm3, 8 mmol) and
the reaction mixture was stirred under reflux for 16 h. After
cooling to room temperature, the mixture was neutralised with
1 M aq. HCl and the solvent was partly removed by distillation
under reduced pressure. The residue was saturated with NaCl
and then extracted with CH2Cl2 (3 × 40 cm3). The combined
extracts were washed with saturated aq. NaHCO3 (2 × 50 cm3)
and then dried (MgSO4). The solvent was removed by distill-
ation under reduced pressure and the residue purified by
column chromatography [0–2% (v/v) MeOH in CH2Cl2] to give
the product 7 (761 mg, 85%) as a white solid material (Found:
C, 67.32; H, 5.77; N, 6.11. C26H26N2O6 requires C, 67.52; H,
5.67; N, 6.06%); δH (CDCl3) 1.52 (3H, s, CH3), 4.00 (1H, d,
J 7.7, OH), 4.43 (1H, d, J 11.4, CH2Ph), 4.54 (1H, d, J 5.6, 5Ј-
H), 4.60 (1H, d, J 11.4, CH2Ph), 4.61 (1H, m, 2Ј-H), 4.62 (1H,
d, J 11.4, CH2Ph), 4.68 (1H, d, J 5.6, 4Ј-H), 4.79 (1H, d, J 11.4,
CH2Ph), 6.21 (2H, br s, 6Ј-H and 7Ј-H), 6.35 (1H, d, J 3.9,
1Ј-H), 7.25–7.34 (10H, m, Ph), 7.57 (1H, s, 6-H), 9.58 (1H, br s,
NH); δC (CDCl3) 12.1, 67.2, 72.4, 74.8, 80.4, 82.5, 89.2, 99.6,
108.7, 127.4, 127.7, 127.8, 128.1, 128.4, 128.6, 131.9, 136.8,
137.2, 137.8, 138.5, 150.6, 163.5.
(1S,3S,4R,6R,7R,8R,9R)-7,9-Bis(benzyloxy)-8-hydroxy-4-(3-N-
benzyloxymethylthymin-1-yl)-2,5-dioxatricyclo[4.2.1.03,7]nonane
10
To a solution of 9 (592 mg, 0.91 mmol) in anhydrous CH3CN
(10 cm3) were added benzyl chloromethyl ether (0.2 cm3, 1.5
mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.3 cm3,
2.0 mmol) and the mixture was stirred at room temperature for
16 h. After adding additional benzyl chloromethyl ether (0.1
cm3, 0.73 mmol) and DBU (0.15 cm3, 1.0 mmol) the mixture
was stirred for a further 6 h. The mixture was concentrated by
distillation under reduced pressure and the residue was purified
by column chromatography [0–2% (v/v) MeOH in CH2Cl2] to
give the product 10 (415 mg, 76%) as a white solid material
1
–
(Found: C, 67.52; H, 5.79; N, 4.68. C34H34N2O8ؒ3H2O requires
C, 67.54; H, 5.78; N, 4.63%); δH (CDCl3) 1.34 (3H, s, CH3), 2.92
(1H, br s, OH), 4.07 (1H, m, 6Ј-H), 4.38 (1H, dd, J 6.6 and 1.1,
5Ј-H), 4.43 (1H, d, J 1.7, 2Ј-H), 4.48 (1H, br s, 7Ј-H), 4.55 (1H,
d, J 10.7, CH2Ph), 4.66 (2H, s, CH2Ph), 4.70 (1H, d, J 10.7,
CH2Ph), 4.72 (1H, d, J 11.7, CH2Ph), 4.72 (1H, d, J 6.6, 4Ј-H),
4.78 (1H, d, J 11.7, CH2Ph), 5.44 (2H, AB, J 9.9, NCH2O), 6.30
(1H, d, J 1.7, 1Ј-H), 7.21–7.42 (15H, m, Ph), 8.36 (1H, s, 6-H);
δC (CDCl3) 12.3 (CH3), 68.1 (CH2Ph), 70.4 (NCH2O), 71.9,
72.1, 72.3 (4Ј-C, 2 × CH2Ph), 76.7 (5Ј-C), 76.7 (2Ј-C), 79.2
(7Ј-C), 79.3 (6Ј-C), 86.5 (1Ј-C), 94.7 (3Ј-C), 108.3 (5-C), 127.3,
127.6, 127.7, 128.2, 128.2, 128.3, 128.4, 128.6, 128.7 (3 × Ph),
136.9, 137.5, 137.8 (3 × Ph), 138.3 (6-C), 151.1 (2-C), 163.8
(4-C); FAB-MS m/z 599 [M ϩ Hϩ].
(1R,3R,4S,5R,6R,7R,8R)-5,8-Bis(benzyloxy)-4,6,7-trihydroxy-
3-(thymin-1-yl)-2-oxabicyclo[3.3.0]octane 8
To a solution of 7 (840 mg, 1.82 mmol) in THF (20 cm3) and
H2O (20 cm3) were added N-methylmorpholine N-oxide (640
mg, 5.45 mmol) and a 2.5% w/w solution of OsO4 in tert-butyl
alcohol (1.0 cm3, 0.077 mmol), and the reaction mixture was
stirred at 50 ЊC for 6 days. After cooling to room temperature
the reaction was quenched with 5% aq. Na2S2O5 (10 cm3) and
the solvent was partly removed by distillation under reduced
J. Chem. Soc., Perkin Trans. 1, 2001, 1855–1861
1859

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(1S,3S,4R,6R,7R,8R,9R)-7,8,9-Trihydroxy-4-(thymin-1-yl)-2,5-
dioxatricyclo[4.2.1.03,7]nonane 4
(0.5 cm3) were added dimethylaminopyridine (DMAP) (18 mg,
0.15 mmol) and 4,4Ј-dimethoxytrityl triflate (DMT-OTf ) (135
mg, 0.30 mmol), and the mixture was stirred for 3 h at 60 ЊC.
Additional DMT-OTf (45 mg, 0.10 mmol) was added and the
mixture was stirred for a further 16 h. The reaction was
quenched by the addition of saturated aq. NaHCO3 (3 cm3) and
extracted with CH2Cl2 (3 × 8 cm3). The combined extracts were
dried (MgSO4) and the solvent was removed by distillation
under reduced pressure and the residue purified by column
chromatography [0–4% (v/v) MeOH and 0.25% pyridine (v/v) in
CH2Cl2] to give the product 13 (45 mg, 64%) as a white solid
material; δH (DMSO-d6) 1.34 (3H, s), 3.00 (1H, br s), 3.70 (3H,
s), 3.71 (3H, s), 4.10–4.23 (3H, m), 5.04 (1H, d, J 2.5), 6.22 (1H,
br s), 6.79 (1H, s, 3Ј-OH), 6.80–6.88 (2H, m), 7.16–7.38 (8H,
m), 7.38–7.52 (4H, m), 7.64–7.72 (2H, m), 8.46 (1H, s), 11.35
(1H, s); δC (DMSO-d6) 12.0, 55.1, 74.2, 74.6, 75.0, 78.4, 81.8,
84.3, 87.1, 88.3, 107.8, 113.5, 127.0, 127.4, 128.1, 128.2, 128.5,
128.8, 128.9, 129.0, 129.5, 129.5, 134.0, 135.1, 135.5, 138.4,
144.6, 150.5, 158.4, 163.7, 164.4; FAB-MS m/z 705 [M ϩ Hϩ].
A solution of 10 (31 mg, 0.022 mmol) in methanol (1.0 cm3) was
added 20% Pd(OH)2–C (20 mg) and the mixture was degassed
with argon and flushed with H2 for 5 min. After stirring under
an atmosphere of H2 for 16 h the mixture was filtered through a
pad of Celite and the solvent was removed by distillation under
reduced pressure to give the product 4 (10 mg, 65%) as a white
solid; single crystals were grown by slow evaporation of a solu-
tion of 4 in methanol; δH (CD3OD) 1.84 (3H, d, J 1.1, CH3),
3.87 (1H, dd, J 2.9 and 1.3, 6Ј-H), 4.12 (1H, d, J 1.8, 2Ј-H), 4.19
(1H, d, J 2.9, 7Ј-H), 4.28 (1H, d, J 7.0, 4Ј-H), 4.51 (1H, dd, J 7.0
and 1.3, 5Ј-H), 6.13 (1H, d, J 1.8, 1Ј-H), 8.82 (1H, d, J 1.1, 6-H);
δC (CD3OD) 12.5, 73.4, 74.8, 80.0, 80.7, 84.0, 87.5, 91.0, 109.9,
142.1, 152.4, 166.7; FAB-MS m/z 299 [M ϩ Hϩ].
(1S,3S,4R,6R,7R,8R,9R)-8-Benzoyloxy-7,9-bis(benzyloxy)-4-
(3-benzyloxymethylthymin-1-yl)-2,5-dioxatricyclo[4.2.1.03,7]-
nonane 11
To a solution of 10 (308 mg, 0.515 mmol) in anhydrous pyridine
(10 cm3) was added benzoyl chloride (0.2 cm3, 1.7 mmol), and
the mixture was stirred for 1 h at room temperature. The reac-
tion was quenched with H2O (10 cm3) and extracted with
CH2Cl2 (3 × 25 cm3). The combined extracts were washed with
saturated aq. NaHCO3 (2 × 50 cm3) and then dried (MgSO4).
The solvent was removed by distillation under reduced pressure
and the residue was co-evaporated with toluene (2 × 30 cm3)
and then purified by column chromatography [0–1% (v/v)
MeOH in CH2Cl2] to give the product 11 (303 mg, 83%) as a
white solid material (Found: C, 69.47; H, 5.28; N, 4.16.
C41H38N2O9ؒ¼H2O requires C, 69.63; H, 5.49; N, 3.96%); δH
(CDCl3) 1.38 (3H, s, CH3), 4.25 (1H, dd, J 6.6 and 1.1, 5Ј-H),
4.42 (1H, dd, J 3.0 and 1.1, 6Ј-H), 4.61 (1H, d, J 10.4, CH2Ph),
4.63 (1H, d, J 1.8, 2Ј-H), 4.69 (2H, s, CH2Ph), 4.72 (1H, d,
J 11.5, CH2Ph), 4.74 (1H, d, J 10.4, CH2Ph), 4.80 (1H, d, J 11.5,
CH2Ph), 4.81 (1H, d, J 6.6, 4Ј-H), 5.48 (2H, AB, J 9.8,
NCH2O), 5.70 (1H, d, J 3.0, 7Ј-H), 6.39 (1H, d, J 1.8, 1Ј-H),
7.22–7.40 (15H, m, Ph), 7.57 (2H, m, Bz), 7.71 (1H, m, Bz), 7.95
(2H, m, Bz), 8.30 (1H, d, J 1.1, 6-H); δC (CDCl3) 12.4, 68.6,
70.4, 71.9, 72.1, 72.5, 74.8, 75.8, 78.9, 79.7, 86.4, 93.7, 108.7,
127.6, 127.7, 127.7, 128.3, 128.3, 128.5, 128.5, 128.6, 128.6,
128.7, 128.7, 129.7, 134.0, 136.4, 136.5, 137.8, 138.0, 151.3,
163.6, 164.9; FAB-MS m/z 703 [M ϩ Hϩ].
(1S,3S,4R,6R,7R,8R,9R)-8-Benzoyloxy-7-[2-cyanoethoxy-
(diisopropylamino)phosphanyloxy]-9-(4,4Ј-dimethoxytrityloxy)-
4-(thymin-1-yl)-2,5-dioxatricyclo[4.2.1.03,7]nonane 14
2-Cyanoethyl N,N-diisopropylphosphoramidochloridite (0.02
cm3, 0.09 mmol) was added dropwise to a solution of 13 (45 mg,
0.064 mmol) in anhydrous CH2Cl2 (0.5 cm3) and diisopropyl-
ethylamine (0.05 cm3). After stirring at room temperature for
3 h, EtOAc (4 cm3) was added to the mixture, which was
washed with saturated aq. NaHCO3 (2 × 3 cm3) and brine
(2 × 3 cm3) and then dried (MgSO4). The solvent was removed
by distillation under reduced pressure and the residue purified
by column chromatography [0.5% (v/v) triethylamine in
CH2Cl2] to give a yellow oil. This oil was subsequently dissolved
in a minimum of toluene and added dropwise to petroleum
ether (20 cm3, Ϫ40 ЊC). The yellow precipitate was filtered off
and re-dissolved in CH3CN and the solvent was removed by
distillation under reduced pressure to give the product 14
(64 mg) as an impure yellow oil which was used without
further purification for automated oligonucleotide synthesis;
δP (CDCl3) 146.4.
(1R,2R,4R,5R,6R,8R,9S)-1,5-Bis(benzyloxy)-9-hydroxy-8-
(thymin-1-yl)-3,7-dioxatricyclo[4.3.0.02,4]nonane 15
To a solution of 7 (33 mg, 0.070 mmol) in MeOH (3 cm3) were
added benzonitrile (85 mg, 0.83 mmol), a 35% aq. solution of
H2O2 (80 mg, 0.82 mmol) and K2CO3 (10 mg, 0.0725 mmol)
and the mixture was stirred at room temperature for 3 h. The
mixture was diluted with Et2O (6 cm3) and H2O (3 cm3) and
extracted with CH2Cl2 (3 × 10 cm3). The combined extracts
were washed with brine (10 cm3) and then dried (MgSO4). The
solvent was removed by distillation under reduced pressure and
the residue purified by column chromatography [1–2% (v/v)
MeOH in CH2Cl2] to give the product 15 (23 mg, 68%) as a
white solid material. δH (CDCl3) 1.85 (3H, s, CH3), 3.87 and
3.68 (2H, 2 × br s, 6Ј-H and 7Ј-H), 4.15 (1H, d, J 6.0, 5Ј-H),
4.28 (1H, d, J 6.0, 4Ј-H), 4.60 (1H, d, J 11.9, CH2Ph), 4.76 (1H,
d, J 11.2, CH2Ph), 4.79 (1H, d, J 11.9, CH2Ph), 4.86 (1H, d,
J 11.2, CH2Ph), 4.95 (1H, d, J 3.8, OH), 5.18 (1H, dd, J 5.6 and
3.8, 2Ј-H), 6.27 (1H, d, J 5.6, 1Ј-H), 7.25–7.40 (10H, m, Ph),
8.05 (1H, s, 6-H), 10.71 (1H, br s, NH); δC (CDCl3) 12.4, 53.5,
57.4, 68.1, 72.4, 75.7, 77.9, 79.7, 86.1, 91.7, 108.2, 127.6, 128.0,
128.1, 128.5, 128.5, 128.6, 137.4, 137.6, 141.5, 151.0, 165.8;
FAB-MS m/z 479 [M ϩ Hϩ].
(1S,3S,4R,6R,7R,8R,9R)-8-Benzoyloxy-7,9-dihydroxy-4-
(thymin-1-yl)-2,5-dioxatricyclo[4.2.1.03,7]nonane 12
To a solution of 11 (271 mg, 0.386 mmol) in methanol (5 cm3)
was added 20% Pd(OH)2–C (100 mg), and the mixture was
degassed with argon and flushed with H2 for 5 min. After stir-
ring under an atmosphere of H2 for 16 h, additional catalyst (30
mg) was added and the mixture was stirred for a further 48 h.
The mixture was filtered through a pad of Celite and the solvent
was removed by distillation under reduced pressure and the
residue was purified by column chromatography [1–10% (v/v)
MeOH in CH2Cl2] to give 4 (23 mg, 20%) and the product 12
(89 mg, 57%) as a white solid material; δH (DMSO-d6) 1.76 (3H,
d, J 1.0, CH3), 4.15 (1H, m, 6Ј-H), 4.23 (1H, d, J 1.8, 2Ј-H), 4.34
(1H, dd, J 6.8 and 3.9, 5Ј-H), 4.40 (1H, d, J 6.8, 4Ј-H), 5.32 (1H,
d, J 2.8, 7Ј-H), 5.88 (1H, d, J 3.9, 5Ј-OH), 6.12 (1H, d, J 1.8,
1Ј-H), 6.72 (1H, s, 3Ј-OH), 7.57 (2H, m, Bz), 7.71 (1H, m, Bz),
8.02 (2H, m, Bz), 8.76 (1H, d, J 1.0, 6-H), 11.33 (1H, s, NH);
δC (DMSO-d6) 13.0, 71.7, 75.0, 76.4, 78.8, 82.5, 85.5, 88.6,
107.4, 129.1, 129.4, 130.1, 134.4, 140.0, 151.0, 164.3, 165.4;
FAB-MS m/z 403 [M ϩ Hϩ].
(4-RS/SS-1R,2R,6R,7R,8R,10R,11S)-1,7-Bis(benzyloxy)-11-
hydroxy-4-oxo-10-(thymin-1-yl)-3,5,9-trioxa-4-thiatricyclo-
[6.3.0.02,6]undecane 16
(1S,3S,4R,6R,7R,8R,9R)-8-Benzoyloxy-9-(4,4Ј-dimethoxytrit-
yloxy)-7-hydroxy-4-(thymin-1-yl)-2,5-dioxatricyclo[4.2.1.03,7]-
nonane 13
A stirred solution of 8 (34 mg, 0.069 mmol) in CH2Cl2 (2 cm3)
To a solution of 12 (40 mg, 0.10 mmol) in anhydrous pyridine
and triethylamine (0.1 cm3) was cooled to 0 ЊC, and a 0.41 M
1860
J. Chem. Soc., Perkin Trans. 1, 2001, 1855–1861

View Article Online
solution of thionyl chloride in CH2Cl2 (0.2 cm3, 0.081 mmol)
was added dropwise. The reaction mixture was stirred at 0 ЊC
for 30 min and then quenched with H2O (4 cm3). The phases
were separated and the aqueous phase was extracted with
CH2Cl2 (3 × 10 cm3). The combined extracts were washed with
saturated aq. NaCl (2 × 15 cm3) and then dried (MgSO4). The
solvent was removed by distillation under reduced pressure and
the residue purified by column chromatography [1–2% (v/v)
MeOH in CH2Cl2] to give the product 16 (16 mg, 43%) as a
mixture of isomers (1 : 1). The product is a white solid material;
δH (CDCl3) 1.17 (s, CH3), 1.21 (s, CH3), 4.00 (dd, J 8.6 and 4.2,
5Ј-H), 4.27 (m, 2Ј-H), 4.42 (m, 2Ј-H), 4.55 (d, J 8.6, 4Ј-H), 4.63
(s, CH2Ph), 4.66 (s, CH2Ph), 4.67 (dd, J 8.7 and 3.6, 5Ј-H), 4.80
(d, J 8.7, 4Ј-H), 5.09 (d, J 5.8, 7Ј-H), 5.28 (dd, J 5.8 and 3.6,
6Ј-H), 5.37 (t, J 4.2, 6Ј-H), 5.48 (br s, 7Ј-H), 5.68 (br s, 2Ј-OH),
5.83 (br s, 2Ј-OH), 6.10 (m, 1Ј-H), 7.16–7.45 (m, Ph), 7.79 (s,
6-H), 7.90 (s, 6-H), 11.48 (br s, NH); FAB-MS m/z 543
[M ϩ Hϩ].
maximum of the first derivatives of the absorbance vs. temper-
ature curve.
Acknowledgements
The Danish Natural Science Research Council is thanked
for financial support. Ms Britta M. Dahl, Department of
Chemistry, University of Copenhagen is thanked for the
synthesis of oligonucleotides. Dr M. Petersen and Dr J. P.
Jacobsen are thanked for assistance with molecular modelling.
References
1 E. Uhlmann, Curr. Opin. Drug Discovery Dev., 2000, 3, 203.
2 (a) P. Herdewijn, Liebigs Ann., 1996, 1337; (b) P. Herdewijn,
Biochim. Biophys. Acta, 1999, 1489, 167; (c) E. T. Kool, Chem. Rev.,
1997, 97, 1473.
3 The use of bicyclic nucleosides in oligonucleotides has recently been
reviewed by M. Meldgaard and J. Wengel, J. Chem. Soc., Perkin
Trans. 1, 2000, 3539.
4 (a) S. K. Singh, P. Nielsen, A. A. Koshkin and J. Wengel, Chem.
Commun., 1998, 455; (b) A. A. Koshkin, S. K. Singh, P. Nielsen,
V. K. Rajwanshi, R. Kumar, M. Meldgaard, C. E. Olsen and
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Y. Hari, J. Andoh, K. Morio, T. Doi and T. Imanishi, Tetrahedron
Lett., 1998, 39, 5401; (d ) J. Wengel, Acc. Chem. Res., 1999, 32, 301;
(e) V. K. Rajwanshi, A. E. HÅkansson, M. D. Sørensen, S. Pitsch,
S. K. Singh, R. Kumar, P. Nielsen and J. Wengel, Angew. Chem., Int.
Ed., 2000, 39, 1656.
5 For definitions, nomenclature and conformational behaviour of
nucleosides and nucleotides see W. Saenger, Principles of Nucleic
Acid Structure, Springer, New York, 1984.
6 (a) M. Tarköy, M. Bolli, B. Schweizer and C. Leumann, Helv. Chim.
Acta, 1993, 76, 481; (b) M. Tarköy and C. Leumann, Angew. Chem.,
Int. Ed. Engl., 1993, 32, 1432.
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1996, 24, 4660; (b) M. Bolli, J. C. Litten, R. Schütz and C. J.
Leumann, Chem. Biol., 1996, 3, 197.
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121, 3249.
Crystallographic data of 4†
C12H14N2O7ؒ2H2O, M = 334.28, monoclinic, a = 24.555(3),
b = 8.5576(11), c = 6.7140(8) Å, β = 98.416(10), V = 1395.6(3)
Å3, space group C2 (no. 5), Z = 4, Dx = 1.591 g cmϪ3
,
F(000) = 704, graphite monochromated Mo-Kα radiation,
λ = 0.71073 Å, µ = 0.138 mmϪ1, T = 120 K. Crystal size
0.45 × 0.43 × 0.19 mm, colourless plates. The intensities of
8619 reflections were measured on a Siemens/Bruker SMART
1K CCD diffractometer to θmax = 29.79Њ and were merged to
3464 unique reflections (Rint = 0.0145). Data collection, inte-
gration of frame data and conversion to intensities were
performed using the programs SMART,24 SAINT24 and
SADABS.25 Structure solution, refinement and analysis of the
structure, and production of crystallographic illustrations were
carried out using the programs SHELXTL26 and PLATON.27
The refinement using 280 parameters converged at R1 = 0.0262
[for Fo > 4σ(Fo)].
9 (a) P. Nielsen, H. M. Pfundheller and J. Wengel, Chem. Commun.,
1997, 825; (b) P. Nielsen, H. M. Pfundheller, C. E. Olsen and
J. Wengel, J. Chem. Soc., Perkin Trans. 1, 1997, 3423.
10 P. Nielsen, M. Petersen and J. P. Jacobsen, J. Chem. Soc., Perkin
Trans. 1, 2000, 3706.
11 L. B. Jørgensen, P. Nielsen, J. Wengel and J. P. Jacobsen, J. Biomol.
Struct. Dyn., 2000, 18, 45.
12 G. Minasov, M. Teplova, P. Nielsen, J. Wengel and M. Egli,
Biochemistry, 2000, 39, 3525.
13 V. K. Rajwanshi, R. Kumar, M. Kofod-Hansen and J. Wengel,
J. Chem. Soc., Perkin Trans. 1, 1999, 1407.
14 R. Kumar and J. Wengel, personal communication.
15 J. Ravn and P. Nielsen, J. Chem. Soc., Perkin Trans. 1, 2001, 985.
16 J. J. Fox and N. C. Miller, J. Org. Chem., 1963, 28, 936.
17 M. Raunkjaer, C. E. Olsen and J. Wengel, J. Chem. Soc., Perkin
Trans. 1, 1999, 2543.
18 (a) G. B. Payne and P. Williams, J. Org. Chem., 1961, 26, 651; (b)
G. B. Payne, P. H. Deming and P. Williams, J. Org. Chem., 1961, 26,
659.
Synthesis of oligodeoxynucleotides
Synthesis of oligonucleotides 17–20 was performed on a 0.2
µmol scale using commercially available 2-cyanoethyl phos-
phoramidites and compound 14. The synthesis followed the
regular protocol for the DNA-synthesiser. However, for com-
pound 14 a prolonged coupling time of 10 min was used
and the coupling efficiency was ∼30% using 1H-tetrazole and
∼85% using pyridinium hydrochloride. Coupling yields for
unmodified 2-cyanoethyl phosphoramidites were >99%. The
5Ј-O-DMT-ON oligonucleotides were removed from the solid
support by treatment with conc. ammonia at 50 ЊC for 16 h,
which also removes the protecting groups. Subsequent purific-
ation using disposable reversed-phase cartridges, including 5Ј-O
detritylation, afforded the pure oligonucleotides 18 and 19.
MALDI-MS [M ϩ Hϩ] gave the following results: 18, 2772.7
(calc. 2768.8) and 19 2881.6 (calc. 2880.9).
19 M. H. Caruthers, Acc. Chem. Res., 1991, 24, 278.
20 M. Tarköy, M. Bolli and C. Leumann, Helv. Chim. Acta, 1994, 77,
716.
Melting experiments
21 R. Steffens and C. J. Leumann, Helv. Chim. Acta, 1997, 80, 2426.
22 C. Scheuer-Larsen, B. M. Dahl, J. Wengel and O. Dahl, Tetrahedron
Lett., 1998, 39, 8361.
23 P. Nielsen, L. Dreiøe and J. Wengel, Bioorg. Med. Chem., 1995, 3, 19.
24 SMART and SAINT. Area Detector Control and Integration
Software. Version 5.054, Bruker Analytical X-Ray Instruments Inc.,
Madison, Wisconsin, USA, 1998.
25 G. M. Sheldrick, SADABS. Version 2.01. Program for Empirical
Correction of Area Detector Data, University of Göttingen,
Germany, 2000.
26 G. M. Sheldrick, SHELXTL. Structure Determination Programs.
Version 5.10, Bruker Analytical X-Ray Instruments Inc., Madison,
Wisconsin, USA, 1997.
Melting experiments were carried out in a medium salt buffer
containing 10 mM Na2HPO4, 100 mM NaCl, 0.1 mM EDTA,
pH 7.0 using 1.5 µM concentrations of the two complementary
sequences. The absorption coefficient was calculated to be 77.7
OD µmolϪ1 assuming the absorption coefficient to be identical
for all the thymine nucleotides. The increase in absorbance at
260 nm as a function of time was recorded while the temper-
ature was raised linearly from 8 to 60 ЊC at a rate of 0.5 ЊC
minϪ1. The melting temperature was determined as the local
† CCDC reference number 164511. See http://www.rsc.org/suppdata/
p1/b1/b104438a/ for crystallographic files in .cif or other electronic
format.
27 A. L. Spek, Acta Crystallogr., Sect. A: Fundam. Crystallogr., 1990,
46, C34.
J. Chem. Soc., Perkin Trans. 1, 2001, 1855–1861
1861