Synthesis and duplex stability of oligodeoxyribonucleotides containing a 2′→5′-amide linkage

Article abstract of DOI:10.1039/a808505f

2′-Deoxy-2′-α-C-carboxymethyl-3′-O-tert- butyldimethylsilyl-5′-O-dimethoxytrityluridine (11a) and the corresponding 3′-deoxy derivative 2′,3′-dideoxy-2′-α-C-carboxymethyl-5′-O- dimethoxytrityluridine (11b) have been condensed with 5′-amino-5′-deoxythymidi

Full text of DOI:10.1039/a808505f

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Synthesis and duplex stability of oligodeoxyribonucleotides
containing a 2Ј→5Ј-amide linkage
Mai-Yee Chan,a Robin A. Fairhurst,b Stephen P. Collingwood,b Julie Fisher,c
John R. P. Arnold,d Richard Cosstick*a and Ian A. O’Neila
a Robert Robinson Laboratories, Department of Chemistry, University of Liverpool,
Crown Street, Liverpool, UK L69 7ZD. E-mail: rcosstic@liverpool.ac.uk
b Novartis Pharmaceuticals UK Limited, Horsham Research Centre, Wimblehurst Road,
Horsham, West Sussex, UK SK10 2NX
c School of Chemistry and d School of Biology, University of Leeds, Leeds UK LS2 9JT
Received (in Cambridge) 2nd November 1998, Accepted 30th November 1998
2Ј-Deoxy-2Ј-α-C-carboxymethyl-3Ј-O-tert-butyldimethylsilyl-5Ј-O-dimethoxytrityluridine (11a) and the
corresponding 3Ј-deoxy derivative 2Ј,3Ј-dideoxy-2Ј-α-C-carboxymethyl-5Ј-O-dimethoxytrityluridine (11b)
have been condensed with 5Ј-amino-5Ј-deoxythymidine to prepare dinucleotide analogues (5a and 5b) which
contain a 2Ј→5Ј-amide linkage. These dimer units have been incorporated into deoxynucleotide dodecamers
using solid-phase phosphoramidite chemistry. Thermal melting studies show that a single 2Ј→5Ј-amide linkage
in a deoxyoligonucleotide has a considerable destabilising effect on duplexes formed with both the DNA and
RNA complementary sequences. Interestingly, the amide linkage has a significantly greater destabilising influence
in the DNA duplexes than in the RNA hybrids.
Introduction
R1O
O
U
T
O
O
DNA or RNA bearing exclusively 2Ј→5Ј phosphodiester bonds
(isoDNA/RNA) has been shown to associate with itself and
both DNA and RNA in a manner that is consistent with duplex
formation through standard Watson–Crick base pairs.1–6
Intriguingly, isoDNA forms a heteroduplex with RNA that is
almost as stable as the comparable natural DNA–RNA duplex,
yet it binds only relatively weakly with complementary DNA
and isoDNA.7,8 Although the isoDNA–RNA duplexes are not
substrates for E. coli RNase H, the selective binding of isoDNA
to RNA makes it a strong candidate for therapeutic antisense
applications. Indeed, chimeric 2Ј→5Ј linked oligonucleotides
containing seven contiguous and centrally placed 3Ј→5Ј phos-
phorothioate residues, as a putative RNase H recognition site,
show the ability to inhibit the expression of steroid 5 alpha
reductase in cell culture.9
To date relatively little work on oligonucleotides bearing
dephospho 2Ј→5Ј linkages has appeared in the scientific liter-
ature. Pudlo et al. have investigated the duplex stability of
oligonucleotides containing either 2Ј→5Ј formacetal (1) or
thioformacetal (2) linkages and shown that the former linkage
has RNA binding properties that are almost comparable to the
3Ј→5Ј-phosphodiester control.10 Dinucleotide analogues have
also been prepared with a 2Ј-O-carboxylic ester linkage11 and
previously we have prepared a 2Ј-C-acetamido linked analogue
of a dinucleoside monophosphate (3a) by opening uridine 2Ј-C,
3Ј-O-γ-butyrolactone with 5Ј-amino-5Ј-deoxythymidine.12 It is
worth noting that the analogous 3Ј→5Ј-amide linkage (4) has
been shown to be one of the most successful phosphodiester
replacements in terms of a potential therapeutic antisense
agent.13 The amide linkages offer some degree of rigidity which
leads to preorganisation of the backbone in a conformation
that is favourably disposed for duplex formation and as a result
2Ј-deoxyoligonucleotides containing this linkage exhibit RNA
binding properties that are very similar to the natural DNA
oligomers.
O
X
O
R2
HN
T
O
T
O
O
OH
1, X=O
2, X=S
3a, R1=H, R2=OH
3b, R1=R2=H
5a, R1=DMT, R2=OTBDMS
5b, R1=DMT, R2=H
O
T
O
O
T=thymin-1-yl
U=uracil-1-yl
HN
T
O
O
4
incorporation of these dimer units into a dodecadeoxynucleo-
tide and Tm studies on duplexes formed with complementary
ribo- and deoxyribo-oligonucleotides.
Results and discussion
An initial consideration at the inception of this study was un-
certainty as to whether dimer 3a would undergo lactonisation
(Scheme 1) during oligonucleotide synthesis–deprotection,
resulting in chain cleavage of the oligomer at the amide
linkage. Our concern originated from an earlier observation14
that the amide 6 undergoes slow lactonisation in solution and
a report by Rosenthal and Baker15 that a related dimethyl
amide was susceptible to thermal lactonisation at 60 ЊC. In a
more recent study we have shown that in the case of dimer 3a,
We now describe the synthesis of two suitably protected
2Ј→5Ј-acetamido linked analogues (5a, 5b) of UpT, the
J. Chem. Soc., Perkin Trans. 1, 1999, 315–320
315

View Article Online
HO
us to use established protocols for the deprotection of oligo-
nucleotides containing 5a. Treatment of alcohol 8 with excess
TBDMS triflate (3 equiv.) in dry pyridine gave the fully pro-
tected nucleoside 9a in a low yield of 44% following isolation by
flash chromatography.
U
HO
O
U
O
O
i
NH2R
HO
O
O
NHR
In the case of the 3Ј-deoxy dimer 5b deoxygenation was
achieved by a standard Barton-type procedure. Thus, alcohol
8 was converted to the thiocarbonate 10 in 67% yield by reac-
tion with O-phenyl chlorothioformate and was subsequently
reduced by treatment with excess tributyltin hydride and AIBN
in toluene, to yield the 3Ј-deoxy derivative 9b in 88% yield. The
C3Ј atom of 9b was observed at 35.36 ppm in the 13C NMR
spectrum and was shifted significantly upfield from its position
(83.98 ppm) in the thiocarbonate 10. Removal of the benz-
hydryl group from 9a and 9b was achieved by hydrogenolysis
using 10% palladium on charcoal in MeOH–THF, to give the
carboxylic acids 11a and 11b in yields of 77 and 67%, respect-
ively. 5Ј-Amino-5Ј-deoxythymidine, which was required for
the lower half of the dimers, was prepared in two steps from
thymidine by an established procedure.16,17
Previous studies on 3Ј→5Ј-amide linked dinucleosides had
revealed that amide formation could be achieved very efficiently
from TBDMS-protected nucleoside carboxylic acids, using
TBTU (O-benzotriazol-1-yl-N,N,NЈ,NЈ-tetramethyluronium
tetrafluoroborate).18 The condensation of 11a with 5Ј-amino-
5Ј-deoxythymidine was initially investigated using TBTU as
previously described.19 Using these conditions a poor isolated
yield (27%) of the dimer 5a was obtained and the paucity of the
product was attributed to partial loss of the dimethoxytrityl
(DMT) group. Subsequently much higher yields were obtained
(90% for 5a and 86% for 5b) using dicyclohexylcarbodiimide
and N-hydroxysuccinimide to effect the condensation. Dimers
5a and 5b were made ready for solid-phase synthesis by conver-
sion to their 3Ј-O-phosphonamidites 12a and 12b respectively,
using 2-cyanoethoxy bis(N,N-diisopropylamino)phosphine
and diisopropylammonium tetrazolide under standard con-
ditions.20,21 The 31P NMR spectra of both 12a and 12b showed a
characteristic pair of singlets at around 149 ppm resulting from
the diastereomeric phosphorus centre.
6, R=H
3a, R=5'-deoxythymidin-5'-yl
Scheme 1 Reagents and conditions: i, acetic acid–water (8:2) at 50 ЊC,
half-life 115 and 930 min for 6 and 3a, respectively.
the lactonisation process is slow, even when heated in aqueous
acetic acid.12 However, it was deemed desirable to also prepare
the 2Ј→5Ј-acetamido linked dinucleoside without a 3Ј-hydroxy
group (3b) in order to establish whether the absence of this
group would have any subtle effect on duplex stability.
Synthesis of acetamide-linked dinucleosides 5a and 5b
The synthesis of the suitably protected dimers (5a and 5b)
started from the previously reported 2Ј-deoxy-2Ј-α-C-(diphenyl-
methoxycarbonylmethyl)uridine (7, Scheme 2). Tritylation of
HO
DMTO
U
U
O
O
i
O
HO
O
HO
OCHPh2
OCHPh2
8
7
iii
ii
DMTO
U
O
DMTO
U
O
iv
O
O
R1
O
S
OCHPh2
OR2
OPh
9a, R1=OTBDMS, R2=CHPh2
9b, R1=H, R2=CHPh2
10
v
Synthesis of and studies on oligonucleotide duplexes containing
2Ј→5Ј amide linkages
11a, R1=OTBDMS, R2=H
11b, R1=R2=H
The duplexes that were selected to assess the effect of a single
amide linkage on duplex stability are shown in Table 1 and are
based on a previously studied non-self-complementary DNA
duplex (duplex G in Table 1). Fawthrop et al.22 have completely
vi
DMTO
DMTO
U
O
U
O
1
assigned the H NMR spectrum of this duplex and these data
would be of considerable assistance in providing structural
information on the duplexes containing the amide linkage. As
the amide linkage was constructed from a uridine nucleoside, a
corresponding uracil base was incorporated into the top strand
of the control duplexes (entries A and D) to enable the direct
comparison of results. Oligonucleotides were assembled using
procedures based on standard protocols although modific-
ations to the coupling times and deprotection conditions were
introduced (see Experimental section). Oligonucleotides con-
taining amide linkages were characterised by MALDI-TOF
mass spectrometry and gave the expected molecular ions
[3529.46 for d(CCT AAA TU × T GCC) and 3545.46 for
d(CCT AAA TUOH × T GCC)].
As anticipated, melting curves for duplexes A–F showed a
single transition and Tm values were determined from the aver-
age of three experiments. Comparison of the data for the two
control duplexes showed that the DNA–DNA duplex (entry
D, Table 1) had a higher melting temperature than the DNA/
RNA hybrid duplex (entry A, Table 1). This is in agreement
with data previously published for duplexes containing mixed
pyrimidine–purine sequences.23–25 Entries A–C show Tm values
for duplexes containing the complementary RNA strand.
O
R
O
R
vii
T
HN
HN
T
O
O
OH
O
O(CH2)2CN
P
5a, R=OTBDMS
5b, R=H
N(iPr)2
12a, R=OTBDMS
12b, R=H
Scheme 2 Reagents and conditions: i, 4Ј,4Ј-dimethoxytrityl chloride,
pyridine, CH2Cl2; ii, TBDMS triflate, pyridine; iii, PhOC(S)Cl, DMAP,
NEt3, CH2Cl2; iv, Bu3SnH (3 equiv.), AIBN, toluene, 65 ЊC; v, H2, Pd–C,
THF–MeOH (3:1); vi, N-hydroxysuccinimide, DCC, DMAP, 5Ј-
amino-5Ј-deoxythymidine, 1,4-dioxane; vii, 2-cyanoethoxy bis(N,N-
diisopropylamino)phosphine,
CH2Cl2.
diisopropylammonium
tetrazolide,
7 under standard conditions gave the dimethoxytrityl derivative
8 in good yield. For the preparation of dimer 5a the tert-
butyldimethylsilyl (TBDMS) group was chosen to protect the
3Ј-position of the uridine moiety. The use of this blocking
group which is routinely used in RNA synthesis would enable
316
J. Chem. Soc., Perkin Trans. 1, 1999, 315–320

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Table 1 Tm values for duplexes prepared in this study
G with preliminary spectra acquired for the analogous amide-
containing duplex E indicates that the two structures are almost
identical in the regions flanking the modification. However, at
the immediate site of the modification the spectra show that
there is some degree of structural heterogeneity consistent with
the amide-linkage producing increased conformational flexi-
bility. A more detailed structural analysis of duplex E will be
reported at a later date.
Entry
Duplexes with RNA
Tm/ЊCa
∆Tm/ЊC
A (control) 5Ј-d(CCT AAA TUT GCC)
3Ј-r(GGA UUU AAA CGG)
38.9
—
B
C
5Ј-d(CCT AAA TUT × GCC)
3Ј-r(GGA UUU AA A CGG)
31.2
Ϫ7.7
5Ј-d(CCT AAA TUOH × T GCC) 30.9
3Ј-r(GGA UUU AA A CGG)
Ϫ8.0
Experimental
General experimental procedures and spectroscopic instru-
mentation are as previously reported.12 Peaks in NMR spectra
displaying obvious diastereomeric splitting are denoted with an
asterisk.
Duplexes with DNA
D (control) 5Ј-d(CCT AAA TUT GCC)
3Ј-d(GCA TTT AAA CGG)
45.4
32.7
—
Ϫ12.7
Ϫ13.4
—
E
F
5Ј-d(CCT AAA TU × T GCC)
3Ј-d(GGA TTT AA A CGG)
2Ј-Deoxy-2Ј-á-C-(diphenylmethoxycarbonylmethyl)-5Ј-O-
dimethoxytrityluridine (8)
5Ј-d(CCT AAA TUOH × T GCC) 32.0
3Ј-d(GGA TTT AA A CGG)
2Ј-Deoxy-2Ј-α-C-(diphenylmethoxycarbonylmethyl)uridine
(3.18 g, 7.03 mmol) was dried by coevaporation with dry pyri-
dine and dissolved in the same solvent (32 cm3). Dimethoxy-
trityl chloride (4.04 g, 11.93 mmol) was dissolved in a mixture
of dry pyridine and dry dichloromethane (4.5 cm3 :16.1 cm3
respectively), added to the nucleoside solution dropwise over
30 min and the mixture stirred for a further 1 hour. Methanol
(0.5 cm3) was added and after a further 15 min saturated aq.
NaHCO3 (1.0 cm3) was also added and the mixture was then
concentrated in vacuo to a thick oil. The residue was coevapo-
rated twice with toluene (2 × 100 cm3), diluted with CH2Cl2
(100 cm3) and washed with saturated aq. NaHCO3 (2 × 50 cm3)
and brine (50 cm3). The aqueous layers were combined and
back extracted with CH2Cl2 (100 cm3) and the organic layers
dried (MgSO4) and evaporated. The crude product was purified
by column chromatography (CH2Cl2 containing an increasing
gradient of MeOH from 0–2%) to yield the required DMT
derivative as a white foam (4.35 g, 82%). δH (400 MHz, CDCl3)
2.62–2.77 (2H, m, H2Ј, H6Ј), 2.94 (1H, dd, J6Љ-2Ј 11.5, J6Љ-6Ј 17.6,
H6Љ), 3.40 (2H, m, H5Ј, H5Љ), 3.79 (6H, s, OCH3), 4.10 (1H, m,
H4Ј), 4.54 (1H, dd, J 2.2, 5.2, H3Ј), 5.39 (1H, dd, J 2.2, J5-6 8.2,
H5), 6.05 (1H, d, J1Ј-2Ј 8.1, H1Ј), 6.82 (4H, d, J 8.7, ArH
o-OCH3), 6.88 (1H, s, OCHPh2), 7.24–7.35 (19H, m, ArH), 7.64
(1H, d, J6-5 8.2), 8.20 (1H, br s, NH); δC (75.5 MHz, CDCl3)
29.88 (C6Ј), 45.96 (C2Ј), 55.12 (OCH3), 63.42 (C5Ј), 72.98
(C3Ј), 77.95 (CHPh2), 85.65 (OCAr3), 86.96 (C4Ј), 87.64 (C1Ј),
102.80 (C5), 113.31 (ArC), 126.91 (ArC), 127.18 (ArC), 128.01
(ArC), 128.17 (ArC), 128.64 (ArC), 130.11 (ArC), 134.78
(ArC), 139.44 (C6), 139.63 (ArC), 144.10 (ArC), 150.31 (C2),
158.51 (ArC), 162.23 (C4), 171.12 (C7Ј); m/z (FABϩ) 777
(M ϩ Naϩ, 0.4%), 755 (M ϩ Hϩ, 1.9%), 754 (Mϩ, 2.2%), 303
(DMTϩ, 91.8%), 167 (Ph2CHϩ, 100%) (Found: C, 71.17; H,
5.61; N, 3.68. C45H42N2O9 requires C, 71.60; H, 5.61; N, 3.76%).
G
5Ј-d(CCT AAA TTT GCC)
3Ј-d(GGA TTT AAA CGG)
—
O
U
O
O
U
O
O
O
HO
HN
HN
T
O
T
O
O
UOH×T
O
U×T
a For details of experimental conditions see Experimental section.
Comparison of the data for these three duplexes reveals the
following: i) considerable destabilisation of the duplex structure
results from the incorporation of a single 2Ј→5Ј amide linkage;
ii) the presence (entry C, Tm 30.9 ЊC) or absence (entry B,
Tm 31.2 ЊC) of a 3Ј-hydroxy group adjacent to the amide linkage
does not have a significant influence on duplex stability.
For the duplexes formed with the DNA complement (entries
D–F) the trends in the Tm data are similar to those observed
with the RNA hybrids. Interestingly, comparison of entries B
and E and also C and F reveal that the amide linkage
has a significantly greater destabilising influence in the DNA
duplexes than in the RNA hybrids. Comparison of entries E
and F suggests that the presence of the 3Ј-hydroxy group
adjacent to the amide exerts a small destabilising effect
(Ϫ0.7 ЊC) and could result from the hydroxy group inducing
unfavourable steric interactions and/or adversely affecting
the sugar conformation. Indeed molecular modelling studies
performed by Pudlo et al.10 suggest that in comparison to the
3Ј→5Ј linkage, there is reduced steric space for a 2Ј→5Ј connec-
tion and the introduction of a 3Ј-OH group may result in
unfavourable steric interactions. The results discussed in this
study are necessarily derived from a restricted set of oligo-
nucleotides and it is therefore difficult to make general assump-
tions about the effect of the amide modifications. However,
preliminary studies incorporating the dimer 3a into other
DNA sequences suggest that the extent of the destabilisation
is dependent on the sequence and, as expected, the reduction
in the Tm per modification is reduced in longer duplexes.26 In
the case of double substitution the destabilisation was approxi-
mately additive.
2Ј-Deoxy-2Ј-á-C-(diphenylmethoxycarbonylmethyl)-3Ј-O-tert-
butyldimethylsilyl-5Ј-O-dimethoxytrityluridine (9a)
2Ј-Deoxy-2Ј-α-C-(diphenylmethoxycarbonylmethyl)-5Ј-O-
dimethoxytrityluridine (100 mg, 0.13 mmol) was coevaporated
with dry pyridine (2 × 5 cm3) and then dissolved in the same
solvent (1 cm3). To a stirred solution of the nucleoside, tert-
butyldimethylsilyl triflate (90 µl, 0.40 mmol) was added under a
nitrogen atmosphere. The mixture was left stirring for 5 hours,
then the solvent was removed in vacuo to leave an oily residue.
The residue was diluted with CH2Cl2 (10 cm3) and washed
with saturated aq. NaHCO3 (2 × 5 cm3), H2O (5 cm3), dried
(MgSO4) and evaporated in vacuo to yield the crude product
which was purified by column chromatography (CH2Cl2 con-
taining an increasing gradient of MeOH from 0–1%) to yield a
white foam (52 mg, 44%). δH (400 MHz, CDCl3) Ϫ0.16 (3H, s,
SiCH3), Ϫ0.10 (3H, s, SiCH3), 0.80 (9H, s, t-Bu), 2.53 (1H, dd,
J6Ј-2Ј 4.3, J6Ј-6Љ 17.8, H6Ј), 2.73 (1H, m, H2Ј), 2.88 (1H, dd, J6Љ-2Ј
In order to improve the design of future antisense constructs
it is important to recognise specific features that cause a modi-
fication to either stabilise or destabilise the duplex structure.
Comparison of 1H NMR data previously obtained22 for duplex
J. Chem. Soc., Perkin Trans. 1, 1999, 315–320
317

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9.0, J6Љ-6Ј 17.7, H6Љ), 3.36 (2H, m, H5Ј, H5Љ), 3.78 (6H, s, OCH3),
4.04 (1H, m, H4Ј), 4.52 (1H, d, J 5.1, H3Ј), 5.41 (1H, d, J5-6 8.0,
H5), 6.06 (1H, d, J1Ј-2Ј 9.3, H1Ј), 6.81 (4H, d, J 8.7, ArH o-CH3),
6.88 (1H, s, OCHPh2), 7.18–7.42 (19H, m, ArH), 7.59 (1H, d,
J6-5 8.0, H6), 8.52 (1H, br s, NH); δC (100.6 MHz, CDCl3) Ϫ5.35
(SiCH3), Ϫ4.66 (SiCH3), 17.83 (Si-C(CH3)2), 25.64 (C(CH3)3),
29.66 (C6Ј), 45.84 (C2Ј), 55.22 (OCH3), 63.63 (C5Ј), 74.11
(C3Ј), 77.30 (CHPh2), 87.08 (OCAr3), 87.14 (C4Ј), 87.57 (C1Ј),
102.72 (C5), 113.30 (ArC), 127.07 (ArC), 127.17 (ArC), 127.92
(ArC), 128.11 (ArC), 128.50 (ArC), 130.04 (ArC), 135.29
(ArC), 139.89 (C6), 140.08 (ArC), 144.08 (ArC), 150.53 (C2),
158.67 (ArC), 162.70 (C4), 170.82 (C7Ј) (Found: FAB HRMS
m/z (M Ϫ H)Ϫ, 867.3702. C51H55N2O9Si requires (M Ϫ H)Ϫ,
867.3677).
NH); δC (75.5 MHz, CDCl3) 30.94 (C6Ј), 35.36 (C3Ј), 40.12
(C2Ј), 54.40 (OCH3), 63.49 (C5Ј), 78.48 (CHPh2), 83.80
(OCAr3), 88.70 (C4Ј), 89.84 (C1Ј), 101.81 (C5), 112.62 (ArC),
126.38 (ArC), 126.52 (ArC), 127.17 (ArC), 127.40 (ArC),
127.53 (ArC), 128.38 (ArC), 128.52 (ArC), 129.46 (ArC),
130.02 (ArC), 135.85 (ArC), 139.20 (C6), 139.95 (ArC), 150.03
(C2), 158.10 (ArC), 162.30 (C4), 170.03 (C7Ј); m/z (FABϩ) 739
(M ϩ Hϩ, 2.58%), 738 (Mϩ, 2.39%), 303 (DMTϩ, 96.15%), 167
(Ph2CHϩ, 100%) (Found: HRMS (FABϩ) m/z (M ϩ Hϩ),
739.3038. C45H43N2O8 requires (M ϩ Hϩ), 739.3019).
General procedure for the removal of the diphenylmethyl
protecting group
The ester (0.27 mmol) was dissolved in MeOH–THF (9:3 cm3)
contained in a flask (50 cm3) with a side arm. The mixture was
degassed with N2 before addition of (10%) Pd–C (≈10 mg).
The flask was evacuated using the water pump, and then
charged with H2; this operation was repeated twice and after the
third time the flask was left stirring under a H2 atmosphere.
After 4 hours, the flask was evacuated and flushed with N2. The
charcoal was filtered through Celite and the solvent removed
in vacuo to give a clear oil which was purified by column
chromatography (CH2Cl2 containing an increasing gradient
of MeOH from 0–5%).
2Ј-Deoxy-2Ј-á-C-(diphenylmethoxycarbonylmethyl)-3Ј-O-phen-
oxythiocarbonyl-5Ј-O-dimethoxytrityluridine (10)
To a stirred, ice-cold, nitrogen-flushed solution of 2Ј-deoxy-2Ј-
α-C-(diphenylmethoxycarbonylmethyl)-5Ј-O-dimethoxytrityl-
uridine (8) (133 mg, 0.18 mmol) in dry CH2Cl2 (1.86 cm3)
was added triethylamine (27 µl, 0.19 mmol) and 4-(N,N-
dimethylamino)pyridine (24 mg, 0.19 mmol). O-Phenyl chloro-
thioformate (37 µl, 0.26 mmol) was subsequently added
dropwise under nitrogen and the mixture was then left to react
at room temperature for 16 hours. The solvent was evaporated
to leave an oily residue which was dissolved in ethyl acetate
(5 cm3). The organic solution was washed with saturated aq.
NaHCO3 (3 cm3) and brine (3 cm3), dried (MgSO4) and evapor-
ated in vacuo to yield the crude product as a yellow oil. The
required compound was obtained following purification by
column chromatography (CH2Cl2 containing an increasing
gradient of MeOH from 0–0.5%) yielding the pure product as a
pale yellow foam (105 mg, 67%). δH (400 MHz, CDCl3) 2.75
(1H, dd, J6Ј-2Ј 5.78, J6Ј-6Љ 17.6, H6Ј), 2.97 (1H, dd, J6Љ-2Ј 7.87, J6Љ-6Ј
17.6, H6Љ), 3.24 (1H, m, H2Ј), 3.40 (2H, m, H5Ј, H5Љ), 3.77 (6H,
s, OCH3), 4.41 (1H, m, H4Ј), 5.37 (1H, d, J5-6 8.03, H5), 6.06
(1H, d, J1Ј-2Ј 5.46, H1Ј), 6.20 (1H, d, J 9.15, H3Ј), 6.80 (4H, d,
J 8.77, ArH, o-CH3), 6.88 (1H, s, OCHPh2), 6.93 (2H, m, o-
ArH), 7.19–7.41 (22H, m, ArH), 7.65 (1H, d, J6-5 8.19, H6), 8.28
(1H, br s, NH); δC (75.5 MHz, CDCl3) 28.61 (C6Ј), 43.63 (C2Ј),
54.48 (OCH3), 63.48 (C5Ј), 78.00 (CHPh2), 83.47 (OCAr3),
83.70 (C4Ј), 83.98 (C3Ј), 86.74 (C1Ј), 102.33 (C5), 112.76 (ArC),
121.03 (ArC), 126.19 (ArC), 126.41 (p-ArC), 126.52 (ArC),
126.68 (ArC), 127.36 (ArC), 127.46 (ArC), 127.97 (ArC),
128.99 (ArC), 129.46 (m-Ar), 134.32 (ArC), 139.06 (C6), 143.44
(ArC), 152.61 (C2), 158.20 (ArC), 162.26 (C4), 169.52 (C7Ј),
2Ј-Deoxy-2Ј-á-C-carboxymethyl-3Ј-O-tert-butyldimethylsilyl-
5Ј-O-dimethoxytrityluridine (11a). White foam (77%); δH (400
MHz, CD3OD) Ϫ0.12 (3H, s, SiCH3), Ϫ0.05 (3H, s, SiCH3),
0.77 (9H, s, t-Bu), 2.26 (1H, dd, J6Љ-2Ј 4.69, J6Љ-6Ј 17.0, H6Љ), 2.52
(1H, dd, J6Ј-2Ј 8.81, J6Ј-6Љ 17.0, H6Ј), 2.77 (1H, m, H2Ј), 3.28 (2H,
m, H5Ј, H5Љ), 3.65 (6H, s, OCH3), 3.91 (1H, m, H4Ј), 4.45 (1H,
d, J 4.11, H3Ј), 5.30 (1H, d, J5-6 8.22, H5), 5.88 (1H, d, J1Ј-2Ј
9.39, H1Ј), 6.75 (4H, d, J 8.22, ArH, o-OCH3), 7.11–7.34 (9H,
m, ArH), 7.65 (1H, d, J6-5 8.22, H6); δC (75.5 MHz, CDCl3)
Ϫ5.25 (SiCH3), 17.85 (SiC(CH3)2), 25.62 (C(CH3)3), 29.58
(C6Ј), 46.05 (C2Ј), 55.20 (OCH3), 63.27 (C5Ј), 75.24 (C3Ј),
86.56 (OCAr3), 87.17 (C4Ј), 89.04 (C1Ј), 103.36 (C5), 113.34
(ArC), 127.19 (ArC), 128.03 (ArC), 128.16 (ArC), 130.04
(ArC), 135.26 (ArC), 139.69 (C6), 144.02 (ArC), 152.50 (C2),
158.79 (ArC), 163.15 (C4), 177.50 (C7Ј); m/z (FABϪ) 701
(M Ϫ HϪ, 5.2%), 569 (M Ϫ H Ϫ HTBDMSOϪ, 8.92%), 111
(uracilϪ) (Found: HRMS (FABϪ) m/z (M Ϫ HϪ), 701.2911.
C38H45N2O9Si requires (M Ϫ HϪ), 701.2894).
2Ј,3Ј-Dideoxy-2Ј-á-C-carboxymethyl-5Ј-O-dimethoxytrityl-
uridine (11b). White foam (67%); δH (400 MHz, CDCl3) 1.83
(1H, m, H3Ј), 2.30 (1H, m, H3Ј), 2.59–2.80 (3H, m, H2Ј, H6Ј,
H6Љ), 3.10 (1H, dd, J5Ј-4Ј 3.58, J5Ј-5Љ 10.45, H5Ј), 3.29 (1H, dd,
J5Љ-4Ј 2.75, J5Љ-5Ј 10.45, H5Љ), 4.29 (1H, m, H4Ј), 5.35 (1H, d,
J5-6 8.25, H5), 5.80 (1H, d, J1Ј-2Ј 4.95, H1Ј), 6.80 (4H, d, J 8.8,
ArH, o-OCH3), 7.25–7.36 (9H, m, ArH), 7.74 (1H, d, J6–5 8.25,
H6), 8.35 (1H, br s, NH); δC (75.5 MHz, CDCl3) 31.74 (C6Ј),
36.16 (C3Ј), 40.92 (C2Ј), 55.20 (OCH3), 64.29 (C5Ј), 84.60
(OCAr3), 87.50 (C4Ј), 88.64 (C1Ј), 101.51 (C5), 113.36 (ArC),
126.91 (ArC), 127.71 (ArC), 128.10 (ArC), 128.28 (ArC),
130.20 (ArC), 135.96 (ArC), 136.14 (ArC), 141.02 (C6), 144.28
(ArC), 152.36 (C2), 158.84 (ArC), 162.23 (C4), 172.12 (C7Ј);
m/z (FABϩ) 595 (M ϩ Naϩ, 0.75%), 573 (M ϩ Hϩ, 2.31%), 572
(Mϩ, 2.91%), 303 (DMTϩ, 100%) (Found: HRMS (FABϩ)
m/z (M ϩ Hϩ), 573.2246. C32H33N2O8 requires (M ϩ Hϩ),
573.2237).
193.39 (C᎐S); m/z (FABϩ) 891 (M ϩ Hϩ, 0.73%), 890 (Mϩ,
᎐
1.86%), 303 (DMTϩ, 100%), 167 (Ph2CHϩ, 93.61%), 154
(OC(S)OPhϩ, 4.21%) (Found: C, 69.70; H, 5.05; N, 2.94.
C52H46N2O10S requires C, 70.10; H, 5.20; N, 3.14%).
2Ј,3Ј-Dideoxy-2Ј-á-C-(diphenylmethoxycarbonylmethyl)-5Ј-O-
dimethoxytrityluridine (9b)
2Ј-Deoxy-2Ј-α-C-(diphenylmethoxycarbonylmethyl)-3Ј-O-
phenoxythiocarbonyl-5Ј-O-dimethoxytrityluridine (100 mg,
0.11 mmol) was dissolved in dry toluene (3.5 cm3). Tributyltin
hydride (0.15 cm3, 0.56 mmol) and AIBN (15 mg, 0.09 mmol)
were added to the solution and the flask was degassed with N2.
The mixture was heated for 8 hours at 65 ЊC. The solvent was
removed in vacuo and the crude product purified by column
chromatography (100% petroleum ether (bp 60–80 ЊC) then
60% petroleum ether–40% ethyl acetate) to give the required
product as a white foam (73 mg, 88%). δH (200 MHz, CDCl3)
1.77 (1H, m, H3Ј), 2.30 (1H, m, H3Љ), 2.56–2.89 (3H, m, H2Ј,
H6Ј, H6Љ), 3.21 (1H, dd, J5Ј-4Ј 3.85, J5Ј-5Љ 10.72, H5Ј), 3.36 (1H,
dd, J5Љ-4Ј 2.75, J5Љ-5Ј 10.45, H5Љ), 3.78 (6H, s, OCH3), 4.29 (1H, m,
H4Ј), 5.35 (1H, d, J5-6 8.24, H5), 5.80 (1H, d, J1Ј-2Ј 4.95, H1Ј),
6.80 (4H, d, J 8.8, ArH, o-OCH3), 6.87 (1H, s, OCHPh2), 7.18–
7.45 (19H, m, ArH), 7.73 (1H, d, J6-5 8.25, H6), 8.46 (1H, br s,
General procedure for the synthesis of the 2Ј→5Ј-linked amide
dimers
The carboxylic acid (0.65 mmol), 1,3-dicyclohexylcarbodiimide
(0.85 mmol), DMAP (0.2 mmol), and N-hydroxysuccinimide
(0.85 mmol) were dissolved in dry dioxane (11 cm3) and stirred
overnight under N2. The dicyclohexylurea was removed by
filtration and washed with dioxane. To the filtrate was added
318
J. Chem. Soc., Perkin Trans. 1, 1999, 315–320

View Article Online
5Ј-amino-5Ј-deoxythymidine (0.98 mmol) dissolved in pyridine
(2 cm3) and the mixture stirred. After 2.5 hours the mixture was
diluted with CH2Cl2 (50 cm3), and the solution washed with
aq. NaHCO3 (75 cm3), brine (50 cm3) and dried (MgSO4). The
aqueous layers were back extracted with CH2Cl2 (2 × 50 cm3)
and dried (MgSO4). The organic solvent was removed in vacuo
and coevaporated with toluene. The crude amide dimer was
purified by column chromatography (CH2Cl2 containing an
increasing gradient of MeOH 0–3%).
was dissolved in CH2Cl2 (20 cm3) and washed with saturated aq.
NaHCO3 (10 cm3) and the organic layer dried (MgSO4). The
product was precipitated by addition of the CH2Cl2 solution to
pentane (60 cm3) and isolated by filtration.
5Ј-O-(Dimethoxytrityl)-3Ј-O-tert-butyldimethylsilyl-2Ј-deoxy-
2Ј-á-C-{N-[5Ј-deoxy-3Ј-O-(2-cyanoethoxy-N,N-diisopropyl-
amino)phosphinothymidin-5Ј-yl]carbamoylmethyl}uridine (12a).
White amorphous solid (91%); δH (400 MHz, d6-acetone) 0.05
(3H, s, SiCH3), 0.08 (3H, s, SiCH3), 0.83 (9H, s, t-Bu), 1.12
(12H, m, 4 × CH3), 1.22 (2H, m, CH2CN), 1.77 (3H, s, CH3),
2.05 (4H, m, 2 × Me2CHN, NCCH2CH2O), 2.32 (2H, m, H2ЈT,
H2ЉT), 2.58 (2H, m, H6ЈU, H6ЉU), 2.90 (1H, m, H2ЈU), 3.32
(2H, m, H5ЈT, H5ЉT), 3.45 (1H, m, H2ЈU), 3.65 (2H, m, H5ЈU,
H5ЉU), 3.76 (6H, s, OCH3), 4.01 (1H, m, H4ЈU), 4.49 (1H, m,
H3ЈT), 4.62 (1H, m, H3ЈU), 5.35 (1H, d, J5-6 8.04, H5U), 5.86*,
5.92* (1H, t, J 6.91, 7.92, H1ЈT), 6.16 (1H, d, J1Ј-2Ј 6.75, H1ЈU),
6.87 (4H, d, J 8.66, ArH, o-OCH3), 7.21–7.48 (9H, m, ArH),
7.16*, 7.18* (1H, s, H6T), 7.60*, 7.62* (1H, d, J 8.2, H6U);
δP (162 MHz, d6-acetone) 149.60, 149.67; m/z (ESϩ) 1149
(M ϩ Naϩ, 12.1%), 1126 (M ϩ Hϩ, 100%), 303 (DMTϩ, 11%)
(Found: C, 59.57; H, 6.82; N, 8.48. C57H76N7O13PSiؒH2O
requires C, 59.82; H, 6.88; N, 8.57%).
5Ј-O-(Dimethoxytrityl)-3Ј-(O-tert-butyldimethylsilyl)-2Ј-
deoxy-2Ј-á-C-[N-(5Ј-deoxythymidin-5Ј-yl)carbamoylmethyl]-
uridine (5a). White foam (90%); δH (400 MHz, CD3OD) Ϫ0.11
(3H, s, SiCH3), 0.05 (3H, s, SiCH3), 0.77 (9H, s, t-Bu), 1.84 (3H,
s, CH3), 2.24 (2H, m, H2Ј, H2ЉT), 2.38 (1H, dd, J6Ј-2Ј 6.4, J6Ј-6Љ
15.4, H6ЈU), 2.42 (1H, dd, J6Љ-2Ј 6.9, J6Љ-6Ј 15.4, H6ЉU), 2.96 (1H,
m, H2ЈU), 3.39 (2H, m, H5Ј, H5ЉT), 3.76 (6H, s, OCH3), 3.84
(3H, m, H4ЈT, H5ЈU, H5ЉU), 4.01 (1H, m, H4Ј), 4.22 (1H, m,
H3ЈT), 4.52 (1H, m, H3ЈU), 5.38 (1H, d, J5-6 8.17, H5U), 6.02
(1H, d, J1Ј-2Ј 8.75, H1ЈU), 6.16 (1H, t, J1Ј-2Ј 6.74, H1ЈT), 6.80
(4H, d, J 8.9, ArH, o-OCH3), 7.20–7.50 (9H, m, ArH) 7.52
(1H, d, J 1.2, H6T), 7.76 (1H, d, J6-5 8.16, H6U); δC (75.5 MHz,
CDCl3) Ϫ5.00 (SiCH3), Ϫ5.37 (SiCH3), 12.21 (CH3T), 17.94
(SiC(CH3)3), 25.76 (C(CH3)3), 31.47 (C6ЈU), 38.80 (C2ЉT),
40.50 (C5ЉT), 46.03 (C2ЈU), 55.38 (OCH3), 63.81 (C5ЈU), 70.62
(C3Ј), 75.20 (C3Ј), 85.10 (C4Ј), 86.08 (C4Ј), 87.18 (OCAr3),
87.32 (C1Ј), 88.60 (C1Ј), 102.82 (C5U), 111.18 (C5T), 113.62
(ArC), 128.30 (ArC), 128.43 (ArC), 130.34 (ArC), 135.56
(ArC), 138.90 (ArC), 140.87 (C6), 144.49 (C6), 151.23 (C2),
151.40 (C2), 159.02 (ArC), 164.17 (C4), 164.31 (C4), 172.02
(C7ЈU); m/z (FABϩ) 948 (M ϩ Naϩ, 3.79%), 926 (M ϩ Hϩ,
7.15%) (Found: HRMS (FABϩ) m/z (M ϩ Hϩ), 926.4031.
C48H60N5O12Si requires (M ϩ Hϩ), 926.4008).
5Ј-O-(Dimethoxytrityl)-2Ј,3Ј-dideoxy-2Ј-á-C-{N-[5Ј-deoxy-
3Ј-O-(2-cyanoethoxy-N,N-diisopropylamino)phosphinothy-
midin-5Ј-yl]carbamoylmethyl}uridine (12b). White amorphous
solid (82%); δH (300 MHz, CDCl3) 1.18 (12H, m, 4 × CH3),
1.22 (2H, m, CH2CN), 1.82 (3H, s, CH3T), 2.03 (5H, m,
2 × Me2CHN, NCCH2CH2O, H3ЈU), 2.35 (3H, m, H2ЈT, H2ЉT,
H3ЉU), 2.54 (2H, m, H6ЈU, H6ЉU), 2.86 (1H, m, H2ЈU), 3.36
(2H, m, H5ЈT, H5ЉT), 3.54 (2H, m, H5ЈU, H5ЉU), 3.81 (6H, s,
OCH3), 3.87 (1H, m, H4ЈT), 4.08 (1H, m, H4ЈU), 4.38 (1H, m,
H3ЈT), 5.39 (1H, d, J5-6 8.10, H5U), 5.70*, 5.78* (1H, d, J 4.8,
J 3, H1ЈU), 6.20*, 6.26* (1H, t, J 7.2, H1ЈT), 6.82 (4H, d, J 9.0,
ArH, o-OCH3), 7.09*, 7.14* (1H, s, H6T), 7.19–7.42 (9H, m,
ArH), 7.80*, 7.83* (1H, d, J 8.2, H6U); δP (162 MHz, d6-
acetone) 149.94, 150.21; m/z (FABϩ) 996 (M ϩ Hϩ, 13%), 895
(M Ϫ iPr2Nϩ, 29%) (Found: C, 60.41; H, 6.38; N, 9.65.
C51H62N7O12PؒH2O requires C, 60.40; H, 6.36; N, 9.67%).
5Ј-O-(Dimethoxytrityl)-2Ј,3Ј-dideoxy-2Ј-á-C-[N-(5Ј-deoxy-
thymidin-5Ј-yl)carbamoylmethyl]uridine (5b). White foam,
(86%); δH (400 MHz, CD3OD) 1.87 (3H, s, CH3), 1.91 (1H, m,
H3ЈU), 2.24 (2H, m, H2Ј, H2ЉT), 2.31 (1H, m, H3ЉU), 2.42 (1H,
dd, J6Ј-2Ј 8.14, J6Ј-6Љ 14.61, H6ЈU), 2.52 (1H, dd, J6Љ-2Ј 8.00, J6Љ-6Ј
14.61, H6ЉU), 2.83 (1H, m, H2ЈU), 3.29 (2H, m, H5Ј, H5ЉT),
3.46 (2H, m, H5Ј, H5ЉU), 3.76 (6H, s, OCH3), 3.90 (1H, m,
H4ЈT), 4.25 (1H, m, H4ЈU), 4.32 (1H, m, H3ЈT), 5.29 (1H, d,
J5-6 8.10, H5U), 5.81 (1H, d, J1Ј-2Ј 5.31, H1ЈU), 6.14 (1H, t, J1Ј-2Ј
6.91, H1ЈT), 6.84 (4H, d, J 8.9, ArH, o-OCH3), 7.21–7.43 (9H,
m, ArH), 7.51 (1H, d, J 1.2, H6T), 7.88 (1H, d, J6-5 8.10, H6U);
δC (101.6 MHz, CDCl3) 12.27 (CH3), 31.82 (C6ЈU), 37.56
(C3ЈU), 39.44 (C2ЈT), 40.90 (C5ЈT), 41.97 (C2ЈU), 55.16
(OCH3), 64.74 (C5ЈU), 71.44 (C3ЈT), 83.51 (C4Ј), 84.91 (C4Ј),
86.55 (OCAr3), 88.75 (C1Ј), 89.77 (C1Ј), 102.35 (C5U), 111.13
(C5T), 111.45 (ArC), 113.18 (ArC), 126.94 (ArC), 127.90
(ArC), 128.09 (ArC), 130.04 (ArC), 135.37 (ArC), 137.10 (C6),
140.35 (C6), 144.41 (ArC), 150.89 (C2), 151.27 (C2), 158.52
(ArC), 163.92 (C4), 164.20 (C4), 171.55 (C7ЈU); m/z (FABϩ)
796 (M ϩ Hϩ, 1.45%), 303 (DMTϩ, 95.68%) (Found: HRMS
(FABϩ) m/z (M ϩ Hϩ), 796.3176. C42H46N5O11 requires
(M ϩ Hϩ), 796.3194).
Synthesis and purification of oligonucleotides
Solid-phase synthesis of the oligodeoxynucleotides and oligo-
ribonucleotides were carried out on a 1 µmol scale using pro-
cedures based on those previously reported22 although modifi-
cations to the coupling times and deprotection conditions were
introduced. Coupling times for the dimer phosphonamidites
12a,b were extended to 10 min. For the removal of the 2Ј-O-
TBDMS groups from the RNA oligonucleotides the dried
oligomers were dissolved in DMSO (150 µl) and Et3Nؒ3HF
(750 µl) and heated at 50 ЊC for 4 h. The RNA was precipitated
by the addition of a few drops of 1 M NH4Cl and after removal
of the supernatant, the precipitate was dissolved in water
(1 cm3) in preparation for ion-exchange chromatography.
For the removal of the 3Ј-O-TBDMS group from the oligo-
nucleotide derived from amidite 12a, the dried oligomer was
dissolved in DMSO (150 µl) and Et3Nؒ3HF (150 µl) and heated
at 50 ЊC for 4 h. Triethylammonium salts were removed by
reversed-phase HPLC prior to purification.
General procedure for the preparation of dinucleoside-3Ј-O-
phosphonamidites
2-Cyanoethoxy bis(N,N-diisopropylamino)phosphine (1.59
mmol) was added to a solution of the dinucleoside (0.53 mmol)
and diisopropylammonium tetrazolide (2.65 mmol) in CH2Cl2
(16 cm3) at 0 ЊC. The reaction mixture was stirred overnight at
room temperature. The mixture was diluted with CH2Cl2 (20
cm3) and washed with saturated aq. NaHCO3 (2 × 10 cm3), the
aqueous layers were combined and back extracted with CH2Cl2
(2 × 10 cm3) and the organic layers dried (MgSO4) and evapor-
ated. The crude nucleoside phosphoramidite was purified by
column chromatography (ethyl acetate containing 0.1% tri-
ethylamine) to yield the product as a white foam. The product
All oligonucleotides were purified by ion-exchange chrom-
atography using a Dionex Nucleopac PA-100 column main-
tained at 55 ЊC, eluting with a gradient of 0.05–0.6 M aqueous
NH4Cl (pH 5.3) over 20 min with a flow rate of 1 cm3 minϪ1
.
Oligonucleotides were desalted by filtration through Centricon
filters (1000 Da cut-off). The final concentration of oligonucleo-
tide solutions was determined by UV absorption and from
calculated extinction coefficients.27
The modified oligonucleotides were characterised by
MALDI-TOF mass spectrometry using a Hewlett Packard
J. Chem. Soc., Perkin Trans. 1, 1999, 315–320
319

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1793.
G2025A LD TOF mass spectrometer and diammonium hydro-
gen citrate–2,6-dihydroxyacetophenone as an ionisation matrix;
m/z 3529.46 for d(CCT AAA TU × T GCC) (C117H151N41O69-
P10) and 3545.46 for d(CCT AAA TUOH × T GCC) (C117H151
N41O68P10).
-
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Melting studies
UV melting curves were determined using a Hewlett Packard
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HP89090A Peltier unit, controlled via a PC. The sample temp-
erature was monitored by a probe immersed in the sample.
The sample was contained in a quartz cell of path length 1 cm
and volume of 1300 µl. Average heating and cooling rates were
around 0.2 ЊC minϪ1. The melts were performed at a duplex
concentration of 3.0 µM in 20 mM phosphate buffer (pH 6.8)
and 100 mM KCl. All values are averages from at least 3
experiments. The absolute error in the Tm values is 0.5 ЊC.
17 W. S. Mungall, G. L. Greene, G. A. Heavner and R. L. Letsinger,
J. Org. Chem., 1975, 40, 1659.
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Acknowledgements
We thank the EPSRC (M.-Y. C.) for provision of a studentship,
Novartis Pharmaceuticals UK Limited for financial support
(M.-Y. C.) and the Wellcome Trust. JRPA is a Wellcome Senior
Fellow. We are also grateful to Mr Allan Mills (Liverpool)
and the EPSRC service at Swansea for obtaining mass spectra
and Dr Paul Leonard (Liverpool) for assistance with NMR
spectroscopy. We also thank Dr David Earnshaw (MRC,
Cambridge) for obtaining MALDI-TOF mass spectra.
23 K. B. Hall and L. W. McLaughlin, Biochemistry, 1991, 30, 10606.
24 L. Ratmeyer, R. Vinyak, Y. Y. Zhong, G. Zon and W. D. Wilson,
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I. A. O’Neil, unpublished observations.
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