Novel uridin-2′-yl carbamates: Synthesis, incorporation into oligodeoxyribonucleotides, and remarkable fluorescence properties of 2′-pyren-1-ylmethylcarbamate

Article abstract of DOI:10.1039/b111434b

A convenient method for the preparation of uridin-2′-yl carbamate derivatives is described. The stable 2′-O-(imidazol-1-ylcarbonyl)-3′,5′-O- (tetraisopropyldisiloxane-1,3-diyl)uridine, prepared from uridine, was treated with primary or secondary aliphatic

Full text of DOI:10.1039/b111434b

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Novel uridin-2Ј-yl carbamates: synthesis, incorporation into
oligodeoxyribonucleotides, and remarkable fluorescence properties
of 2Ј-pyren-1-ylmethylcarbamate†
1
Vladimir A. Korshun,‡ Dmitry A. Stetsenko and Michael J. Gait*
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge,
UK CB2 2QH. E-mail: mgait@mrc-lmb.cam.ac.uk; Fax: (44) 1223 402070;
Tel: (44) 1223 248011
Received (in Cambridge, UK) 14th December 2001, Accepted 6th March 2002
First published as an Advance Article on the web 25th March 2002
A convenient method for the preparation of uridin-2Ј-yl carbamate derivatives is described. The stable 2Ј-O-
(imidazol-1-ylcarbonyl)-3Ј,5Ј-O-(tetraisopropyldisiloxane-1,3-diyl)uridine, prepared from uridine, was treated
with primary or secondary aliphatic amines to give 2Ј-carbamates in high yield. After 3Ј,5Ј-O-deprotection with
triethylamine trihydrofluoride, 5Ј-O-dimethoxytritylation, and 3Ј-O-phosphitylation with bis(N,N-diisopropyl-
amino)-2-cyanoethoxyphosphine, modified phosphoramidites were obtained and used in machine-assisted synthesis of
modifiedoligodeoxynucleotidescontaininguridin-2Ј-ylcarbamateresiduesbearingvariousN-substituents.Theinfluence
of uridin-2Ј-yl carbamate moieties on the stability of modified oligonucleotide duplexes with complementary DNA
and RNA was studied by thermal denaturation experiments. Pyrene-modified oligonucleotides showed a considerable
increase in fluorescence intensity upon hybridisation to complementary RNA and interesting binding properties
when hybridised to a mismatched DNA.
stable under hot (55 ЊC) ammoniacal deprotection conditions in
Introduction§
Oligonucleotides conjugated with various ligands are the
subject of extensive studies and applications in bioorganic
chemistry, molecular biology and biotechnology. The particular
site of ligand attachment depends on the nature of the bio-
target. Studies we present here were motivated originally by a
contrast to aniline-derived nucleoside 3Ј-carbamates.6
Oligonucleotides containing nucleoside 2Ј-carbamates have
been reported previously.7 Cytidin-2Ј-yl carbamate was
obtained by CDI activation followed by amine treatment.7b
Some uridin-2Ј-yl carbamate modifications were found to be
detrimental to the stability of DNA–RNA duplexes.7c,e How-
need to develop a convenient method for the introduction of
ever, systematic studies and full synthetic details have not been
various ligands into ribozymes. For example, attachment of
reported yet. Moreover, the merit of 2Ј-carbamate functionalis-
ation in many nucleic acid applications remained unexplored.
Here we describe the preparation of several uridin-2Ј-yl
ligands such as polyamines to the hairpin ribozyme might
enhance its RNA cleavage potential.1 Since nucleobases in
ribozyme core regions are often involved in various non-
carbamates and their introduction into oligodeoxyribonucleo-
covalent interactions important for maintaining structure and
tides and report on their hybridisation with complementary
function,2 the sugar part of nucleosides, particularly the 2Ј-
DNA and RNA. It was found that 2Ј-pyren-1-ylmethyl-
hydroxy group, seemed to be an attractive position for ligand
carbamate-modified oligonucleotides showed remarkable DNA
attachment, especially because 2Ј-ligand functionalisation has
mismatch affinity and enhanced fluorescent properties when
already proved valuable in applications such as fluorescence
bound to a complementary RNA.
monitoring of the tertiary folding of RNAs.3
There are numerous examples of 2Ј-O-alkyl derivatives of
nucleosides and their introduction into oligonucleotides.4
A
Results and discussion
general and frequent shortcoming is a poor yield in the key step,
2Ј-O-alkylation. An alternative opportunity for 2Ј-functionalis-
ation is via a carbamate function, easily generated from
primary or secondary alcohols by successive treatment with
1,1Ј-carbonyldiimidazole (CDI) and an aliphatic amine. This
chemistry has been used successfully for 5Ј-modification of
oligonucleotides.5 Aliphatic 5Ј-carbamates were reported to be
Synthesis of modified phosphoramidites
A general approach to the preparation of uridin-2Ј-yl carb-
amates and their corresponding phosphoramidites is outlined
in Scheme 1. The starting 3Ј,5Ј-O-(tetraisopropyldisiloxane-1,3-
diyl)uridine 1 was obtained from uridine as reported previ-
ously8 and purified by column chromatography on silica gel in
EtOAc–CHCl3 (1 : 9, then 1 : 2 v/v). Subsequent reaction of 1
with CDI in dry DCM gave 2Ј-O-(imidazol-1-ylcarbonyl)-3Ј,5Ј-
O-(tetraisopropyldisiloxane-1,3-diyl)uridine 2 in nearly quanti-
tative yield. DCM proved to be the best solvent for imidazolide
formation, whereas use of the more polar MeCN led to a
much longer reaction time. Imidazolide 2 is stable during
aqueous extraction, but is prone to hydrolysis under acidic or
alkaline conditions. Some decomposition was observed also
during silica gel chromatography. Note that the MALDI-TOF
mass spectrum of compound 2 was obtained using the neutral
† Electronic supplementary information (ESI) available: Additional
experimental data for compounds 3, 5 and 6. See http://www.rsc.org/
suppdata/p1/b1/b111434b/
‡ Present address: Shemyakin-Ovchinnikov Institute of Bioorganic
Chemistry, Miklukho-Maklaya 16/10, Moscow 117997, Russia.
§ Abbreviations:CDI1,1Ј-carbonyldiimidazole, DIEAN,N-diisopropyl-
ethylamine, Dmt 4,4Ј-dimethoxytrityl, Piv pivaloyl = 2,2-dimethyl-
propanoyl, Tfa trifluoroacetyl, 2,4,6-THAP 2,4,6-trihydroxy-
acetophenone, 2,5-DHBA 2,5-dihydroxybenzoic acid, MALDI-TOF
matrix-assisted laser desorption ionization time-of-flight.
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J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b111434b

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Scheme 1
matrix 2,4,6-trihydroxyacetophenone (2,4,6-THAP) rather
amine) reacted rapidly in DCM in less than an hour, N-methyl-
than the acidic 2,5-dihydroxybenzoic acid (2,5-DHBA), which
is the standard matrix used for other 3Ј,5Ј-O-(tetraisopropyl-
disiloxane-1,3-diyl)uridine derivatives.
In many cases imidazolide 2 need not be isolated, but instead
reacted in situ with an excess of the appropriate amine in dry
DCM. In the case of amine hydrochlorides, 1.1 equivalents of
DIEA was added to the reaction mixture. Reaction times dif-
fered vastly from one amine to another, and were found also to
be solvent dependent. Whilst propargylamine (prop-2-ynyl-
propargylamine, a secondary amine, required overnight reac-
tion. 2-Aminomethyl-15-crown-5 reacted very slowly in
DCM. To accelerate the reaction, the solvent was changed to
MeCN and temperature increased to 55 ЊC. 3Ј,5Ј-O-Protected
uridin-2Ј-yl carbamates 3 were isolated by column chromato-
graphy.
The reaction with polyamines, N-(3-aminopropyl)propane-
1,3-diamine and spermine, gave exclusively primary amine-
substituted products. This observation is in good agreement
J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104
1093

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1
with the recently reported high selectivity of the reaction of
imidazolecarbonyl derivatives of alcohols with primary vs.
secondary amines.9
The remaining amino functions in compounds 3i, 3k, 3n were
protected by the trifluoroacetyl group, which is used commonly
for protection of aliphatic amino group in various phosphor-
amidite reagents, e.g. nucleoside reagents carrying polyamines.10
The hydroxy group in compound 3g was acylated with pivaloyl
chloride to give ester 3h.
column chromatography. H NMR showed that the signal set
obtained from 9 is similar to that obtained from 5b, thus con-
firming that 5b and 9 are isomers. MALDI-TOF mass spectral
analysis also showed identical molecular masses.
For an independent route of synthesis, 5Ј-O-dimethoxy-
trityluridine 712 was treated with CDI in dry DCM to give
cyclic 2Ј,3Ј-carbonate 8 in quantitative yield (Scheme 2). The
Initially we used TBAF in THF to remove the Markiewicz’
3Ј,5Ј-O-silyl group from 3. After 15–20 min, TLC showed
smooth silyl deprotection, but also the appearance of a by-
product with similar mobility. After several hours of TBAF
treatment, an equilibrium was reached where a 1 : 1 ratio of
desired product to by-product was obtained. The by-product
was assumed to be the regioisomeric 3Ј-carbamate, generated
by carbamoyl migration in the vicinal diol system. The possi-
bility of carbamoyl migration has been discussed in a similar
case,7a but the 3Ј-carbamate itself was not detected. Interest-
ingly, these authors7a used a buffered TBAF–AcOH deprotec-
tion mixture. In other cases7b,d TBAF solution in THF was
used. However in our hands, TBAF in THF always gave a mix-
ture of products. Therefore we tried the alternative desilylating
reagent, triethylamine trihydrofluoride. This gave only the
desired 2Ј-carbamate product after overnight deprotection.
We were unable to obtain crystalline samples of uridin-2Ј-yl
carbamates 4, except in the case of aromatic derivatives 4c and
4d. Therefore crude compounds 4 (viscous oils) were used in the
subsequent 5Ј-dimethoxytritylation to give derivatives 5 in high
yield. The bulky 2Ј-carbamate moiety prevented subsequent
dimethoxytritylation of the neighbouring 3Ј-hydroxy group,
and 5Ј,3Ј-bis-dimethoxytritylated product was isolated only in
the case of 15-crown-5 carbamate. No 2Ј–3Ј migration was
detected when pyridine was used as the solvent for dimethoxy-
tritylation or in most cases during the column chromatography
in the presence of MeOH and triethylamine. However, isolated
compounds 5 showed a slow 2Ј–3Ј migration under basic condi-
tions or in protic solvents. Noteworthy, the pyrene derivative 5d
and the dipeptide derivative 5e showed a considerable rate of
isomerisation during column chromatography in the presence
of triethylamine. Therefore pyridine was used as a neutraliz-
ation additive during their purifications on silica gel.
Scheme 2
formation of 2Ј,3Ј-carbonates from CDI and 5Ј-O-tritylated
nucleosides has been described before.13 Carbonate 8 was
reacted with N-methylpropargylamine to give a mixture of
two regioisomeric carbamates 5b and 9. These were separated
by column chromatography and proved to be identical to the
samples obtained by the first route.
Oligonucleotide synthesis and thermal denaturation studies
5Ј-O-Dimethoxytrityluridin-2Ј-yl carbamates 5 were then
2Ј-Modified nucleoside phosphoramidites 6 were used in solid
phase oligodeoxyribonucleotide synthesis where the coupling
time was increased to 10 min. The sequence chosen was a non-
self-complementary 15-mer of mixed sequence d(CTCCCAG-
GCTCAAAT) (ON2). A deoxyuridine or a 2Ј-modified uridine
was introduced in place of the internal thymidine (ON4–
ON14), the thymidine close to the 5Ј-end (ON15–ON23), or at
both sites (ON24–ON31). Also prepared were the comple-
mentary unmodified oligodeoxyribonucleotide d(ATTT-
GAGCCTGGGAG) (ON1) and oligoribonucleotide r(AU-
UUGAGCCUGGGAG) (ON3) as hybridisation targets. These
syntheses allowed model DNA–DNA (ON2–ON1, T m 57.0 ЊC)
and DNA–RNA (ON2–ON3, T m 59.2 ЊC) duplexes to be
studied, into which 2Ј-modifications were incorporated into the
same DNA strand. The series of 2Ј-modified oligonucleotides
(Table 1) was characterised by MALDI-TOF mass spec-
trometry. In all cases, a 2Ј-carbamate function was found to be
completely stable to the conditions of oligonucleotide synthesis
as well as to final deprotection with concentrated aqueous
ammonia (55 ЊC, 8–16 h). The pivalate ester obtained by use of
phosphoramidite 3h proved to be unexpectedly stable. Thus
only after 4 days of hot ammoniacal treatment did the
MALDI-TOF spectrum show complete ester hydrolysis in the
doubly modified oligomer ON28.
phosphitylated
with
bis(N,N-diisopropylamino)-2-cyano-
ethoxyphosphine in DCM in the presence of diisopropyl-
ammonium tetrazolide to give phosphoramidites 6 which were
isolated by column chromatography. None of the starting
carbamate compounds 5 gave rise to isomerisation product
during the phosphitylation reaction.
2Ј–3Ј Migration of the carbamoyl group
Carbamates 4 and 5, containing an adjacent 3Ј-OH group, are
prone to isomerisation under alkaline conditions.11 5Ј-O-Dmt
derivatives 5 upon ammonia treatment in aqueous MeOH gave
1 : 1 mixtures of two tritylated compounds with rather similar
TLC mobilities. One spot was always the starting compound
and the other was clearly different from 5Ј-O-dimethoxy-
trityluridine 7. To confirm that the compound generated under
migration conditions is the 3Ј-carbamate we set out to isolate it,
confirm its structure by NMR, and prepare it by an independ-
ent method. Complete separation of the by-product by column
chromatography appeared to be a difficult task. However, in
the case of a carbamate derived from the secondary amine
N-methylpropargylamine, the difference in chromatographic
mobilities of the 5Ј-O-Dmt derivatives was sufficient for com-
plete resolution on a preparative silica gel column. Therefore, a
sample of compound 3b was desilylated with TBAF and the
resulting mixture was treated with DmtCl in pyridine to give
5Ј-O-Dmt-compounds 5b and 9, which were separated by
The negative effect of a 2Ј-carbamate function on the
thermal stability of a DNA–RNA duplex has been reported
recently.7c Our data (Table 1) confirmed this tendency both in
the DNA–DNA and DNA–RNA series. Incorporation of
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J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104

Table 1 Properties of modified oligonucleotides and their duplexes with complementary DNA and RNA
Duplexes with
ON1; d(ATTTGAGCCTGGGAG)
∆T m/ЊC
ON3; r(AUUUGAGCCUGGGAG)
∆Tm/ЊC
MALDI MS [M ϩ H]ϩ
(found/calculated)
#
Sequence, 5Ј
3Ј
U = U-2Ј-R; R =
T m/ ЊC
(per modification)
T m/ ЊC
(per modification)
ON4
H
OMe
4482.2/4483.9
4515.9/4513.9
4581.2/4581.0
4594.5/4595.0
4759.9/4759.0
4756.1/4757.2
4772.3/4775.2
4628.2/4631.1
4744.3/4746.2
46.57.7/4657.1
4927.9/4728.3
56.4
55.0
50.0
49.0
49.9
54.7
49.8
50.1
49.8
51.5
51.4
59.1
59.6
54.5
53.2
54.7
57.8
53.9
53.7
53.9
53.4
55.1
ON5
Ϫ1.4
Ϫ6.4
Ϫ7.4
Ϫ6.5
Ϫ1.7
Ϫ6.6
Ϫ6.3
Ϫ6.6
Ϫ4.9
Ϫ5.0
ϩ0.4
Ϫ4.7
Ϫ6.0
Ϫ4.5
Ϫ1.4
Ϫ5.3
Ϫ5.5
Ϫ5.3
Ϫ5.8
Ϫ4.0
᎐
ON6
OCONHCH2C᎐CH
OCON(Me)CH2C᎐CH
OCONHCH2(4-iodophenyl)
OCONHCH2(pyren-1-yl)
OCONHCH2([15-crown-5]-1-yl)
OCONHCH2CH2OCH2CH2OH
OCONH(CH2)3[O(CH2)2]3O(CH2)3NH2
OCONH(CH2)3NH(CH2)3NH2
OCONH(CH2)3NH(CH2)4NH(CH2)3NH2
᎐
᎐
ON7
᎐
ON8
ON9
CTCCCAGGCUCAAAT
ON10
ON11
ON12
ON13
ON14
ON15
ON16
ON17
ON18
ON19
ON20
ON21
ON22
ON23
H
OMe
4482.0/4483.9
4516.1/4513.9
4759.6/4759.0
4756.3/4757.2
4772.3/4775.2
4627.3/4631.1
4744.2/4746.2
4657.3/4657.1
4729.7/4728.3
56.2
55.1
53.4
57.5
53.5
52.8
52.6
53.1
54.1
58.8
59.4
54.4
55.5
54.8
56.2
55.5
55.9
59.0
Ϫ1.1
Ϫ2.8
ϩ1.3
Ϫ2.8
Ϫ3.4
Ϫ3.6
Ϫ3.1
Ϫ2.3
ϩ0.6
Ϫ4.4
Ϫ3.3
Ϫ4.0
Ϫ2.6
Ϫ3.3
Ϫ2.9
ϩ0.2
OCONHCH2(4-iodophenyl)
OCONHCH2(pyren-1-yl)
OCONHCH2([15-crown-5]-1-yl)
OCONHCH2CH2OCH2CH2OH
OCONH(CH2)3[O(CH2)2]3O(CH2)3NH2
OCONH(CH2)3NH(CH2)3NH2
OCONH(CH2)3NH(CH2)4NH(CH2)3NH2
CUCCCAGGCTCAAAT
CUCCCAGGCUCAAAT
ON24
ON25
ON26
ON27
ON28
ON29
ON30
ON31
H
OMe
4468.4/4469.9
4531.5/4529.9
5015.6/5016.5
5050.2/5050.0
4759.3/4764.2
4995.7/4994.5
4818.2/4816.3
4958.2/4958.6
56.1
52.5
52.6
45.2
43.9
45.0
46.3
47.3
58.1
60.7
50.1
48.6
49.8
49.5
50.6
50.1
Ϫ1.8
Ϫ1.8
Ϫ5.5
Ϫ6.6
Ϫ5.6
Ϫ4.9
Ϫ4.4
ϩ1.3
Ϫ4.0
Ϫ4.8
Ϫ4.2
Ϫ4.3
Ϫ3.8
Ϫ4.0
OCONHCH2(pyren-1-yl)
OCONHCH2([15-crown-5]-1-yl)
OCONH(CH2CH2OCH2CH2OH
OCONH(CH2)3[O(CH2)2]3O(CH2)3NH2
OCONH(CH2)3NH(CH2)3NH2
OCONH(CH2)3NH(CH2)4NH(CH2)3NH2

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Table 2 Thermal stabilities of mismatched duplexes with DNA
DNA mismatch
ON35; d(ATTTGCGCCTGGGAG)
∆T m/*ЊCa
ON36; d(ATTTGAGCCTGGGCG)
#
T m/ЊC
T m/ЊC
∆T m*/ЊC
ON4
47.0
Ϫ9.4
49.8
46.5
38.4
37.3
37.9
44.7
37.8
38.5
38.2
39.5
39.8
50.1
50.6
50.4
55.9
50.1
49.5
50.0
49.7
50.2
49.6
46.6
50.3
39.2
37.8
39.6
40.8
41.2
Ϫ6.6
Ϫ8.5
Ϫ11.6
Ϫ11.7
Ϫ12.0
Ϫ10.0
Ϫ12.0
Ϫ11.6
Ϫ11.6
Ϫ12.0
Ϫ11.6
Ϫ6.1
Ϫ4.5
Ϫ3.0
Ϫ1.6
Ϫ2.4
Ϫ3.3
Ϫ2.6
Ϫ3.4
Ϫ3.9
Ϫ6.5
Ϫ5.9
Ϫ2.3
Ϫ6.0
Ϫ6.1
Ϫ5.4
Ϫ4.5
Ϫ6.1
ON5
45.2
42.9
42.8
46.4
52.9
43.8
42.9
43.1
44.7
45.9
46.4
44.2
40.9
44.8
41.4
41.0
40.9
40.9
40.9
46.1
42.4
51.6
37.3
36.7
37.4
38.6
40.2
Ϫ9.8
Ϫ7.1
Ϫ6.2
Ϫ3.5
Ϫ1.8
Ϫ6.0
Ϫ7.2
Ϫ6.7
Ϫ6.8
Ϫ5.5
Ϫ9.8
Ϫ10.9
Ϫ12.5
Ϫ12.7
Ϫ12.1
Ϫ11.8
Ϫ11.7
Ϫ12.2
Ϫ13.2
Ϫ10.0
Ϫ10.1
Ϫ1.0
Ϫ7.9
Ϫ7.2
Ϫ7.6
Ϫ7.7
Ϫ7.1
ON6
ON7
ON8
ON9
ON10
ON11
ON12
ON13
ON14
ON15
ON16
ON17
ON18
ON19
ON20
ON21
ON22
ON23
ON24
ON25
ON26
ON27
ON28
ON29
ON30
ON31
a ∆T m* = ∆T m of mismatch contribution.
2Ј-O-methyluridine (oligomers ON5, ON16 and ON25) in
place of deoxyuridine was also somewhat destabilising for
DNA–DNA duplexes, but slightly stabilising for DNA–
RNA complexes. Most carbamate modifications result in a
considerable decrease in T m values of the duplexes but there
was no correlation between the physical size of the carbamate
N-substituent and the T m of the corresponding duplexes. The
highest destabilising effect was observed for the tertiary
carbamate of N-methylpropargylamine (oligomer ON7)
whereas the least destabilising was found for pyren-1-ylmethyl
carbamate, especially in the case of the DNA–DNA duplex.
As expected, a single modification close to the 5Ј-terminal
position of the 15-mer (ON17–ON23) caused less destabilis-
ation than one in the middle (ON8–ON14). In general, internal
carbamate modifications are less destabilising for DNA–RNA
than for DNA–DNA duplexes. By contrast, near-terminal
carbamate modification is less destabilising for DNA–DNA
duplexes.
Both 1-pyrenylmethyl and 4-iodobenzyl substituents are
residues of a similar nature (arylmethyl) and have comparable
molecular masses. It is interesting to compare data for oligo-
nucleotides bearing these modifications. Not surprisingly,
ON9–ON1 is 4.8 ЊC more stable than ON8–ON1 (internal pos-
ition, DNA–DNA), ON18–ON1 is 4.1 ЊC more stable than
ON17–ON1 (near-terminal position, DNA–DNA), ON9–ON3
is 3.1 ЊC more stable than ON8–ON3 (internal position, DNA–
RNA) and ON18–ON3 is 1.1 ЊC more stable than ON17–ON3
(near-terminal position, DNA–RNA). Thus, despite the chem-
ical similarity of pyrenyl and iodophenyl moieties, pyrene-
modified duplexes are always more stable than those of iodo-
phenyl (as well as most other substituents). The difference lies
apparently in the planar polyaromatic structure of pyrene
which is evidently more suitable for binding to nucleic acid
duplexes. The nature of the interaction leading to partial
compensation of carbamate destabilisation remains unclear.
Pyrene itself is often thought of as a DNA intercalator and its
interactions with nucleic acids have been studied spectroscopic-
ally.14 However, at least three modes of free pyrene binding to
DNA have been postulated,14a with groove binding being
particularly favoured.14c From consideration of the B-DNA
duplex structure, stacking between two adjacent base pairs
(intercalation) and, more obviously, minor groove binding are
both in principle feasible for a pyren-1-ylmethyl-2Ј-carbamate
substituent. That pyrene is more stabilising than iodophenyl in
the case of DNA–DNA rather than DNA–RNA duplexes,
might suggest the better fit of pyrene into the DNA–DNA
minor groove. In comparison with uridin-2Ј-ylpyren-1-yl-
methylcarbamate modification with 2Ј-deoxyuridine, very little
difference is seen for internally located pyrene substitution on
DNA–DNA and DNA–RNA duplex stability (1.4 and 1.7 ЊC
decrease in T m, respectively). Near-terminal pyrene location,
by contrast, gives ϩ1.4 ЊC stabilisation for DNA–DNA and
Ϫ3.3 ЊC destabilisation for DNA–RNA duplexes. These data
show that the effect of near-terminal pyrene for DNA–DNA
and DNA–RNA duplexes is very different. Thus pyrene is
better able to exert its DNA–DNA binding effect near the end
of the duplex, presumably because the greater flexibility in this
region allows better minor groove placement of the pyrene.
A number of studies of pyrene attached to the 2Ј-position of
individual nucleosides within duplexes have been published
recently.3,15 Pyrene as well as other polyaromatic compounds
often show greater stabilising effect in DNA–DNA duplexes
than in DNA–RNA duplexes.15b,d The pyrene 2Ј-carbamate
modification is not an exception, giving a clear stabilising effect
of pyrene superimposed on the destabilising influence of the
carbamate linker.
A similar situation occurs in the case of polyamines, where a
T m increase due to phosphate charge neutralisation might be
expected. In fact, the duplex-disturbing influence of carbamate
is predominant, so that only in the case of near-terminal
location of spermine in the RNA–DNA (ON23–ON3) duplex
was there no decrease in T m observed.
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Doubly modified duplexes are more than additively (“cooper-
atively”) destabilised. Again, in this case pyrene showed the
lowest destabilising influence, especially for the DNA–
DNA duplex, where it was of the same level as 2Ј-O-methyl
substitution.
To evaluate the selectivity of binding of 2Ј-carbamate-
modified oligonucleotides to a complementary DNA we carried
out melting experiments using two mismatched oligodeoxy-
ribonucleotides ON35 and ON36 as templates (Table 2) con-
taining deoxycytidine in place of the normal deoxyadenosine.
The overall effect of a dC : dU mismatch in the unmodified
series (ON4, ON15 and ON24) was found to be in the range of
6–10 ЊC, those in the middle of a sequence being considerably
more destabilising. The incorporation of a single uridin-2Ј-yl
carbamate directly opposite a deoxycytidine in otherwise
complementary DNA led to a further decrease in stability,
again more prominent when the mismatch is internally situated.
However, most interesting behaviour was demonstrated by the
pyren-1-ylmethyl carbamate, which appeared to selectively
stabilise dC : U mismatch when placed directly opposite
(U stands for uridinyl carbamate). In the case of internal pyrene
(ON9–ON35), the pure mismatch effect is about 2 ЊC, whereas
for near-terminal pyrene (ON18–ON36) the overall destabilis-
ation is even less pronounced as compared to the unmodified
duplex (Table 2). Our data further show this unusual stabilis-
ation disappearing when the mismatch is situated elsewhere
(ON9–ON36 and ON18–ON35 duplexes show a T m decrease
of 10–13 ЊC). The dC mismatch was also well-tolerated in
doubly pyrenylated duplexes ON26–ON35 and ON26–ON36.
These results may be ascribed to 2Ј-pyrene actually replacing
uridine in stacking with neighboring nucleobases. This proposal
is confirmed by an efficient fluorescence quenching in the case
of pyrene mismatches which is characteristic of pyrene being
able to interact with DNA heterocycles (data not shown).
Thus, uridin-2Ј-yl carbamate modification in a DNA strand
is usually destabilising for both DNA–DNA and DNA–RNA
duplexes and therefore might be less suitable for antisense
applications where higher stability of a duplex is preferential.
However, the 2Ј-carbamate modification affords a convenient
way to place various ligands in the minor groove of a DNA–
DNA duplex (e.g. crown ether for metal ion binding, an
aliphatic amino group for labelling with activated derivatives
of fluorescent dyes). Other possible applications include the
specific minor groove delivery of reactive functions (e.g. for
directed cross-linking or conjugation with peptides), as well as
modifications of flexible parts of structured RNA molecules,
such as ribozymes, for studies of catalysis1 or folding.3 In this
respect, we have incorporated some 2Ј-carbamate modified
uridines into synthetic oligoribonucleotides (data not shown)
and studies on such modified ribozymes will be reported later.
Fig. 1 (a) UV spectra of pyrene-modified oligonucleotides ON9 (1),
ON18 (2) and ON26 (3) in water, normalized by pyrene absorbance.
(b) Comparison of the UV spectrum of pyrene-modified nucleoside 4d
(1) and the difference between UV spectra of oligonucleotides ON26
and ON18 (2) in water.
Pyrene absorbance and fluorescence in single-stranded
oligonucleotides and duplexes
The introduction of a pyrene chromophore into oligonucleo-
tides can be easily confirmed by the characteristic UV spectrum
of modified oligonucleotides, showing pyrene absorbance
bands in the region of 320–360 nm (Fig. 1a). The oligo-
nucleotide-bound pyrene is spectrally distinct from the single
nucleoside-bound. Curve 1 shown in Fig. 1b depicts the UV
difference spectrum of oligonucleotides ON26 and ON18, i.e.
formally corresponds to the spectrum of a single oligo-
nucleotide-bound pyrene. It can be seen clearly that the pyrene
maxima either in pyrenyl oligonucleotides (Fig. 1a), or in this
“oligonucleotide-bound pyrene” are shifted by 5–8 nm in com-
parison to pyrene in the pyrenyl nucleoside 4d (Fig. 1b). Such a
bathochromic shift correlates well with previously published
data.14c
Fig. 2 Thermal denaturation curves of the duplexes ON26 × ON1
(1,3) and ON26 × ON3 (2,4) in hybridisation buffer detected at 260
(1,2) and 349 nm (3,4).
an increase of nucleoside absorbance at 260 nm, but also with a
decrease in pyrene absorbance at 349 nm (Fig. 2). For the DNA–
DNA duplex the inversion point of the curve at 52.3 ЊC is con-
sistent with that of the nucleoside absorbance curve (52.6 ЊC).
This result can be explained by the average distance between
two chromophores in the flexible single stranded oligomer
ON26 being less than in the more rigid, extended duplex
ON26–ON1. The increase of pyrene–pyrene distance in the
duplex leads to an increase in their total “photon capture
The melting of the duplexes carrying two pyrene residues
(ON26–ON1 and ON26–ON3) can be observed not only with
J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104
1097

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section” and, as a consequence, an increase in the absorbance.
In this simplified explanation, it is also assumed that pyrene
molecules both in a single strand and in a duplex are capable of
random rotation, which may not necessarily be the case here.
For the DNA–RNA duplex T m values calculated from nucleo-
side (50.1 ЊC) and pyrene absorbance (51.1 ЊC) are also in
close agreement. Noteworthy, the “pyrene” melting curves for
DNA–DNA and DNA–RNA duplexes have different shape
thus confirming a different mode of interaction of a pyrene
residue with a nucleic acid framework.
The total fluorescence intensity upon duplex binding of
singly pyrene-labelled oligomers with complementary DNA
does not change very much (Fig. 4). By contrast, the binding to
The fluorescence spectra of single-stranded pyrene oligo-
nucleotides (ON9, ON18; Fig. 3a) show that fluorescence inten-
sity (which correlates with quantum yield) is dependent on the
modification site: Internally bound pyrene fluoresces approxi-
mately three times more intensively than near-terminal pyrene
(note that the nucleobase context is the same in both cases).
The spectrum of the doubly-modified oligomer ON26 has a
distinct broad band of excimer fluorescence in the region
around 480 nm. This provides evidence for a conformation with
a close coplanar arrangement of two pyrene residues (e.g. a
metastable hairpin with two base pairs in the stem). When
bound to a complementary DNA oligomer, the pyrene residues
in ON26 are unable to be brought closely together and the
excimer fluorescence completely disappears (Fig. 3b).
Fig. 4 (a) Fluorescence spectra of single stranded pyrene-modified
oligonucleotide ON9 (1) and its duplex with complementary DNA
ON9 × ON1 (2) and RNA ON9 × ON3 (3). (b) Fluorescence spectra of
single stranded pyrene-modified oligonucleotide ON18 (1) and its
duplex with complementary DNA ON18 × ON1 (2) and RNA ON18 ×
ON3 (3). (c) Fluorescence spectra of single stranded pyrene-modified
oligonucleotide ON26 (1) and its duplex with complementary DNA
ON26 × ON1 (2) and RNA ON26 × ON3 (3). Conditions: hybridis-
ation buffer (see Experimental section), λex 339 nm, concentrations
2 × 10Ϫ7 M.
Fig. 3 (a) Fluorescence spectra of single stranded pyrene-modified
oligonucleotides ON9 (1, ON18 (2) and ON26 (3). (b) Fluorescence
spectra of single stranded double-pyrene-modified oligonucleotides
ON26 (1) and is duplexed with complementary oligodeoxynucleotide
ON26 × ON1 (2). Conditions: hybridisation buffer (see Experimental
section), λex 339 nm, concentrations 2 × 10Ϫ7 M.
a complementary RNA gives a 5 (internal pyrene) to 30 fold
(near-terminal pyrene) increase in fluorescence for singly
pyrene-labelled (Fig. 4a–b) and 30-fold for doubly pyrene-
labelled oligonucleotide (Fig. 4c). This provides evidence that
the excited pyrene fluorophore (linked through 2Ј-carbamate) is
effectively quenched by nucleobases in single-stranded DNA
oligomers and in DNA–DNA duplexes, but not in DNA–RNA
duplexes. Similar examples of fluorescence increases upon
hybridisation with complementary RNA are demonstrated for
probes containing 2Ј-O-attached pyrene.15b,e
This effect could be applied to the detection of comple-
mentary sequences by use of an excimer-forming double
pyrene-labelled probe. This might be sequence-dependent and
would require extensive experiments on probe structure opti-
misation (to design probes with maximal excimer fluores-
cence) and thorough controls (study of the influence of non-
complementary and partly complementary sequences on
excimer fluorescence).
To conclude, a convenient method of oligonucleotide 2Ј-
modification is described. Most interesting properties were
demonstrated by the 2Ј-O-(pyren-1-ylmethylcarbamoyl)-
uridine-containing DNA oligomers. The pyrene modification
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J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104

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was able to stabilise selectively a duplex with a mismatched
deoxycytidine directly opposite the carbamate-modified nucleo-
side. Moreover, the fluorophore showed a considerable fluor-
escence increase upon hybridisation with complementary RNA.
The remarkable features of the 2Ј-O-(pyren-1-ylmethylcarb-
amoyl)uridine may therefore find application in the detection of
RNA and possibly detection of point mutations in DNA.
showed the conversion of the starting nucleoside into an imid-
azolide with lower mobility on TLC is complete after 0.5–2 h.
The solution was washed with water (2 × 50 cm3), dried
(Na2SO4), evaporated, coevaporated with dry DCM, and the
residue was dried in vacuo to give the imidazole derivative
(2.872 g, 98.9%) as a white crystalline powder, pure according
to TLC and NMR. Rf 0.38 (EtOAc), mp 193–195 ЊC (EtOAc–
CHCl3). MALDI-TOF (2,4,6-THAP): [M ϩ Na]ϩ calc. 603.77,
found 604.58, [M ϩ K]ϩ calc. 619.88, found 620.55. 1H-NMR:
11.44 (s, 1H, H-3), 8.39 (s, 1H, imidazole), 7.69 (s, 1H, imid-
azole), 7.64 (d, 1H, J5,6 = 8.0 Hz, H-6), 7.09 (s, 1H, imidazole),
5.85 (s, 1H, H-1Ј), 5.79 (d, 1H, J2Ј,3Ј = 5.4 Hz, H-2Ј), 5.61 (d, 1H,
J5,6 = 8.0 Hz, H-5), 4.74 (m, 1H, H-3Ј), 4.13–3.92 (m, 3H, H-4Ј,
H-5Ј), 1.10–0.80 (m, 28H, Pri).
Experimental
S-Ethyl trifluorothioacetate and 4-iodobenzylamine were
from Lancaster Synthesis; N,N-diisopropylethylamine (DIEA),
propargylamine, N-methylpropargylamine, 1-pyrenylmethyl-
amine hydrochloride, 1-aminomethyl-15-crown-5, 2-(2-amino-
ethoxy)ethanol, 4,7,10-trioxatridecane-1,13-diamine, spermine,
N-(3-aminopropyl)propane-1,3-diamine, Et3N, triethylamine
trihydrofluoride, and pivaloyl chloride were from Aldrich; 1,1-
carbonyldiimidazole (CDI) was from Sigma, leucylphenyl-
alaninamide hydrochloride was from Bachem, 4,4Ј-dimethoxy-
tritylchloride (DmtCl) was purchased from Avocado; and
3Ј,5Ј-O-(Tetraisopropyldisiloxane-1,3-diyl)uridin-2Ј-yl
carbamates (3)
A general procedure. To a solution of 2, prepared as above from
5 mmol of 1, the corresponding amine was added and the com-
pletion of reaction (from 1 h to several days) was monitored by
TLC (EtOAc). The reaction mixture was diluted with DCM
(50 cm3), washed with water (100 cm3), 5% citric acid (100 cm3),
and water (100 cm3), then dried (Na2SO4), evaporated, and the
residue was chromatographed on a silica gel column in an
appropriate solvent system. Fractions containing product were
combined, evaporated, and the residue was dried in vacuo to
afford compounds listed below as white foamy solids.
bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine
from
Fluka. 3Ј,5Ј-O-(Tetraisopropyldisiloxane-1,3-diyl)uridine,8b 5Ј-
O-(4,4Ј-dimethoxytrityl)uridine,12 and diisopropylammonium
tetrazolide16 were prepared as described.
DCM (Fisher) was always used freshly distilled over CaH2.
Anhydrous pyridine was from Aldrich; THF (BDH) was freshly
distilled over powdered LiAlH4 and stored over 4 Å molecular
sieves under nitrogen. Other solvents, toluene (BDH), chloro-
form, ethyl acetate, acetone, acetonitrile, hexane, absolute
ethanol (Fisher) and methanol (Fluka) were used as received.
300 MHz 1H and 121 MHz 31P NMR spectra were recorded
on a Bruker DRX 300 spectrometer. Chemical shifts (δ) for 1H
and 31P are referenced to internal solvent resonances and
reported relative to SiMe4 and 85% aq. H3PO4, respectively. 1H
NMR coupling constants are reported in Hz and refer to
apparent multiplicities. MALDI-TOF mass spectra were
obtained on a Voyager-DE BioSpectrometry Workstation
(PerSeptive Biosystems) in a positive ion mode. High-resolution
ESI mass spectra were provided by the Department of
Chemistry, University of Cambridge. TLC and column
chromatographies were carried out with Macherey-Nagel silica
gel (ALUGRAM SIL G/UV254 and Kieselgel 60 0.040–0.063
mm). Thermal denaturation experiments with oligonucleotide
duplexes were performed on a Perkin Elmer Lambda 40 UV/
VIS Spectrometer with PTP 6 (Peltier Temperature Program-
mer). Fluorescence spectra were obtained using a Perkin Elmer
LS 50B Luminescence Spectrometer.
Oligonucleotide Synthesis was carried out on a ABI 380B
DNA/RNA synthesizer either on 0.2 or 1 µmol scale using
2Ј-deoxynucleoside phosphoramidites from Cruachem
(Scotland) and 2Ј-TOM-ribonucleoside phosphoramidites from
Glen Research (via Cambio), and standard synthetic pro-
cedures.17 Ion exchange HPLC purification of oligoribo-
nucleotide ON3 was carried out using a NucleoPac PA-100
(Dionex) (9 × 250 mm) using gradients of sodium perchlorate
(1 mM–400 mM) in a buffer of 20 mM Tris HCl (pH 6.8), 25%
formamide, and at 280 nm wavelength. The mass of each oligo-
nucleotide was checked by MALDI-TOF mass spectrometry on
a Perseptive Biosystems Voyager DE mass spectrometer in posi-
tive ion mode using a mixture (1 : 1 v/v) of 2,6-dihydroxy-
acetophenone (40 mg cmϪ3 in MeOH) and aqueous diam-
monium hydrogen citrate (80 mg cmϪ3) as a matrix premixed
just before loading the samples onto a plate. Duplex stability
studies were done in a buffer containing 100 mM NaCl, 10 mM
Na–phosphate, 0.1 mM EDTA, pH 7.0.
2Ј-O-(Propargylaminocarbonyl)-3Ј,5Ј-O-(tetraisopropyl-
disiloxane-1,3-diyl)uridine (3a). Chromatography: stepwise
gradient of 33 40% (v/v) EtOAc in CHCl3. Yield 2.780 g
(97.9%). Rf 0.49 (CHCl3–EtOAc 1 : 1 v/v). ESI MS: [M ϩ Na]ϩ
1
calc. 590.23, found 590.23. H-NMR: 11.42 (s, 1H, H-3), 7.84
(t, 1H, J = 5.4 Hz, OCONH ), 7.69 (d, 1H, J5,6 = 8.0 Hz, H-6),
5.64 (s, 1H, H-1Ј), 5.59 (d, 1H, J5,6 = 8.0 Hz, H-5), 5.34 (d, 1H,
J2Ј,3Ј = 4.9 Hz, H-2Ј), 4.50 (m, 1H, H-3Ј), 4.34–3.88 (m, 2H,
᎐
H-5Ј), 3.85–3.69 (m, 3H, CH N, H-4Ј), 3.06 (s, 1H, ᎐CH ), 1.10–
᎐
2
0.80 (m, 28H, Pri).
2Ј-O-(N-Methyl-N-propargylaminocarbonyl)-3Ј,5Ј-O-(tetra-
isopropyldisiloxane-1,3-diyl)uridine (3b). Chromatography:
EtOAc–CHCl3, 1 : 2 v/v. Yield 2.827 g (97.2%). Rf 0.56 (CHCl3–
EtOAc, 1 : 1 v/v). ESI MS: [M ϩ Na]ϩ calc. 604.25, found
604.24. 1H-NMR: 11.41 (s, 1H, H-3), 7.66 (d, 1H, J5,6 = 8.1 Hz,
H-6), 5.67 (s, 1H, H-1Ј), 5.59 (d, 1H, J5,6 = 8.1 Hz, H-5), 5.35
(m, 1H, H-2Ј), 4.53 (dd, 1H, J2Ј,3Ј = 6.0 Hz, J3Ј,4Ј = 7.9 Hz, H-3Ј),
᎐
4.17–3.83 (m, 5H, H-4Ј, H-5Ј, CH N), 3.21 (s, 1H, ᎐CH ), 2.93
᎐
2
(s, 1.8H), 2.84 (s, 1.2H) (NCH3, rotamers), 1.07–0.82 (m, 28H,
Pri).
2Ј-O-(4-Iodobenzylaminocarbonyl)-3Ј,5Ј-O-(tetraisopropyl-
disiloxane-1,3-diyl)uridine (3c). Chromatography: stepwise
gradient of 25 33% (v/v) EtOAc in CHCl3. Yield 3.491 g
(93.6%). Rf 0.50 (CHCl3–EtOAc, 1 : 1 v/v). MALDI-TOF (2,5-
DHBA): [M ϩ Na]ϩ calc. 768.74, found 769.30, [M ϩ K]ϩ calc.
1
784.85, found 785.37. H-NMR: 11.41 (s, 1H, H-3), 7.96 (br t,
1H, OCONH ), 7.68 (d, 1H, J5,6 = 8.0 Hz, H-6), 7.62 (d, 2H,
J = 7.6 Hz, ArH ), 7.05 (d, 2H, J = 7.6 Hz, ArH ), 5.65 (s, 1H,
H-1Ј), 5.58 (d, 1H, J5,6 = 8.0 Hz, H-5), 5.35 (d, 1H, J2Ј,3Ј = 5.4
Hz, H-2Ј), 4.48 (m, 1H, H-3Ј), 4.20–3.80 (m, 5H, CH2Ar, H-4Ј,
H-5Ј), 1.08–0.88 (m, 28H, Pri).
2Ј-O-(Pyren-1-ylmethylaminocarbonyl)-3Ј,5Ј-O-(tetra-
isopropyldisiloxane-1,3-diyl)uridine (3d). Chromatography:
stepwise gradient of 25 30 33% (v/v) EtOAc in CHCl3.
Yield 3.492 g (93.9%). Rf 0.54 (CHCl3–EtOAc, 1 : 1). MALDI-
2Ј-O-(Imidazol-1-ylcarbonyl)-3Ј,5Ј-O-(tetraisopropyldisiloxane-
1,3-diyl)uridine (2)
To a solution of 1 (2.434 g, 5.0 mmol) in dry DCM (50 cm3),
1
TOF (2,5-DHBA): [M ϩ Na]ϩ calc. 766.98, found 768.31. H-
NMR: 11.43 (s, 1H, H-3), 8.42 (d, 1H, J9Љ,10Ј = 9.3 Hz,
CDI (852 mg, 5.25 mmol) was added in one portion. TLC
pyrene H-10), 8.31–7.97 (m, 9H, ArH, OCONH ), 7.69 (d, 1H,
J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104
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J5,6 = 8.1 Hz, H-6), 5.68 (s, 1H, H-1Ј), 5.59 (d, 1H, J5,6 = 8.1 Hz,
v/v). ESI MS: [M ϩ Na]ϩ calc. 724.33, found 724.32. 1H-NMR:
11.41 (s, 1H, H-3), 7.69 (d, 1H, J5,6 = 8.0 Hz, H-6), 7.37 (br t,
1H, J = 4.6 Hz, OCONH ), 5.64 (s, 1H, H-1Ј), 5.59 (d, 1H, J5,6
H-5), 5.40 (d, 1H, J2Ј,3Ј = 5.8 Hz, H-2Ј), 4.91 (m, 2H, CH2Ar),
2
4.66 (m, 1H, H-3Ј), 4.04 (m, 1H, J5Јa,5Јb = 12.6 Hz, J4Ј,5Јa
=
=
3.2 Hz, H-5Јa), 3.93–3.77 (m, 2H, H-4Ј, H-5Јb), 1.04–0.67 (m,
8.0 Hz, H-5), 5.31 (d, 1H, J2Ј,3Ј = 4.9 Hz, H-2Ј), 4.50 (m, 1H,
H-3Ј), 4.17–3.78 (m, 5H, H-4Ј, H-5Ј, CH2OPiv), 3.59 (m, 2H,
CH2OCH2), 3.40 (m, 2H¶, CH2N), 3.10 (m, 2H, CH2OCH2),
1.12 (s, 9H, But), 1.07–0.83 (m, 28H, Pri).
28H, Pri).
2Ј-O-(1,4,7,10,13-Pentaoxacyclopentadecan-2-ylmethyl)-
aminocarbonyl-3Ј,5Ј-O-(tetraisopropyldisiloxane-1,3-diyl)uridine
(3e). The solution of 2 (1.162 g, 2.0 mmol) and 2-aminomethyl-
15-crown-5 (514 mg, 2.0 mmol) in MeCN (25 cm3) was kept at
55 ЊC for 96 h, then cooled, evaporated to dryness, and the
residue was dissolved in EtOAc (100 cm3), washed with water
(100 cm3), 5% citric acid (100 cm3), and water (100 cm3), dried
(Na2SO4) and evaporated. The residue was chromatographed
on silica gel (a stepwise gradient of 2 to 10% (v/v) MeOH in
CHCl3). Yield 1.165 g (76.4%). Rf 0.32 (CHCl3–MeOH, 17 : 3).
ESI MS: [M ϩ H]ϩ calc. 762.36, found 762.37. 1H-NMR:
11.41 (s, 1H, H-3), 7.68 (d, 1H, J5,6 = 8.0 Hz, H-6), 7.40 (t, 1H,
2Ј-O-[13-(Trifluoroacetylamino)-4,7,10-trioxatridecan-1-yl-
aminocarbonyl]-3Ј,5Ј-O-(tetraisopropyldisiloxane-1,3-diyl)-
uridine (3j). Compound 2 was prepared from 1 (1.703 g,
3.5 mmol) and CDI (592 mg, 3.65 mmol) in dry DCM (30 cm3).
The solution was added dropwise to the magnetically stirred,
ice-cooled solution of 4,7,10-trioxatridecane-1,13-diamine
(3.9 cm3, 17.5 mmol) in DCM (100 cm3). After 2 h, cooling was
removed, and the mixture was kept overnight at 20 ЊC, then
washed with water (150 cm3), 5% NaHCO3 (150 cm3), and water
(150 cm3), dried (Na2SO4), and evaporated to dryness. The
residue was dissolved in dry DCM (10 cm3), and S-ethyl tri-
fluorothioacetate (3.0 cm3, 24 mmol) was added in one portion.
The mixture was left overnight, then evaporated to dryness
(STENCH!), and the residue was chromatographed on silica gel
(CHCl3–EtOAc, stepwise gradient 1 : 1 1 : 2 1 : 3 1 : 4,
v/v) to give the product as a colourless oil (1.888 g, 65.1%). Rf
0.42 (EtOAc). ESI MS: [M ϩ Na]ϩ calc. 851.35, found 851.35.
1H-NMR: 11.41 (s, 1H, H-3), 9.36 (br s, 1H, NHTFA), 7.69 (d,
1H, J5,6 = 8.0 Hz, H-6), 7.36 (t, 1H, J = 5.7 Hz, OCONH ), 5.64
(d, 1H, J1Ј,2Ј = 1.8 Hz, H-1Ј), 5.59 (d, 1H, J5,6 = 8.0 Hz, H-5),
J = 5.7 Hz, OCONH ), 5.64 (s, 1H, H-1Ј), 5.59 (d, 1H, J5,6
=
8.0 Hz, H-5), 5.33 (d, 1H, J2Ј,3Ј = 5.7 Hz, H-2Ј), 4.48 (dd, 1H,
2
J2Ј,3Ј = 5.7 Hz, J3Ј,4Ј = 8.2 Hz, H-3Ј), 3.99 (m, 2H, J5Јa,5Јb = 12.9
Hz, J4Ј,5Јa = 2.7 Hz, J4Ј,5Јb = 4.1 Hz, H-5Ј), 3.84 (m, 1H, H-4Ј),
3.64–3.36 (m, 19H¶, CH(CH2OCH2)4CH2), 3.00 (m, 2H,
CH2N), 1.06–0.82 (m, 28H, Pri).
Nꢀ-[3Ј,5Ј-O-(Tetraisopropyldisiloxane-1,3-diyl)uridin-2Ј-O-
ylcarbonyl]-L-leucyl-L-phenylalaninamide (3f ). The solution of
2 (1.162 g, 2.0 mmol), H-Leu-Phe-NH2 hydrochloride (942 mg,
3.0 mmol) and DIEA (0.61 cm3, 3.5 mmol) in MeCN (25 cm3)
was kept at 55 ЊC for 72 h, then cooled, evaporated to dryness,
and the residue was dissolved in EtOAc (100 cm3), washed with
water (100 cm3), 5% citric acid (100 cm3), and water (100 cm3),
dried (Na2SO4) and evaporated. The residue was chromato-
graphed on silica gel (a stepwise gradient of 33 50% EtOAc in
CHCl3, then 33 50% acetone in CHCl3–EtOAc (1 : 1 v/v)).
Yield 610 mg (38.6%). Rf 0.45 (EtOAc). ESI MS: [M ϩ H]ϩ
5.31 (m, 1H, H-2Ј), 4.49 (dd, 1H, J2Ј,3Ј = 5.0 Hz, J3Ј,4Ј = 7.9 Hz,
2
H-3Ј), 3.99 (m, 2H, J5Јa,5Јb = 13.0 Hz, J4Ј,5Јa = 2.7 Hz, J4Ј,5Јb
=
4.4 Hz, H-5Ј), 3.82 (m, 1H, H-4Ј), 3.55–3.30 (m, 12H¶,
(CH2OCH2)3), 3.22 (apparent q, 2H, J = 6.5 Hz, CH2NHTFA),
3.00 (m, 2H, OCONHCH2), 1.69 (apparent quintet, 2H,
J = 6.8 Hz, CH2), 1.60 (apparent quintet, 2H, J = 6.6 Hz, CH2),
1.06–0.81 (m, 28H, Pri).
1
calc. 790.39, found 790.43. H-NMR: 11.43 (s, 1H, H-3), 7.87
2Ј-O-[4-Trifluoroacetyl-7-(trifluoroacetylamino)-4-azaheptan-
1-ylaminocarbonyl]-3Ј,5Ј-O-(tetraisopropyldisiloxane-1,3-diyl)-
uridine (3m). A solution of 2, prepared from 1 (2.434 g,
5.0 mmol) and CDI (852 mg, 5.25 mmol) in dry DCM (50 cm3),
was added dropwise to the magnetically stirred, ice-cooled
solution of N-(3-aminopropyl)propane-1,3-diamine (5.6 cm3,
40 mmol) in DCM (100 cm3). After 2 h cooling was removed,
and the mixture was kept overnight at 20 ЊC, then washed
with water (200 cm3), 5% NaHCO3 (200 cm3), and water
(200 cm3), dried (Na2SO4), and evaporated to dryness. The resi-
due was coevaporated with THF (30 cm3), dissolved in dry
THF (10 cm3), and S-ethyl trifluorothioacetate (5.0 cm3,
39 mmol) was added in one portion. After 20 h TLC (EtOAc)
showed formation of one predominant product. The mixture
was evaporated to dryness (STENCH!) and the residue was
chromatographed on silica gel (CHCl3–EtOAc, stepwise gradi-
ent 1 : 1 1 : 2 1 : 3 v/v) to give the product as a white foam
(2.558 g, 61.2%). A sample of the product was triturated in
DCM–hexane to give colourless crystals, mp 74–77 ЊC. Rf 0.69
(EtOAc). ESI MS: [M ϩ Na]ϩ calc. 858.30, found 858.30.
1H-NMR: 11.42 (s, 1H, H-3), 9.51, 9.43 (2 br t, 1H, NHTFA,
rotamers), 7.69 (d, 1H, J5,6 = 8.1 Hz, H-6), 7.51, 7.45 (2m, 1H,
OCONH, rotamers), 5.65 (d, 1H, J1Ј,2Ј = 1.8 Hz, H-1Ј), 5.59 (d,
1H, J5,6 = 8.1 Hz, H-5), 5.32 (m, 1H, H-2Ј), 4.50 (m, 1H, H-3Ј),
(d, 1H, J = 8.2 Hz, NHCHCH2Ph), 7.70 (d, 1H, J5,6 = 8.0 Hz,
H-6), 7.44 (d, 1H, J = 8.4 Hz, OCONH ), 7.39 (br s, 1H, NH2),
7.19 (m, 5H, Ph), 7.06 (br s, 1H, NH2), 5.64 (s, 1H, H-1Ј), 5.59
(d, 1H, J5,6 = 8.0 Hz, H-5), 5.35 (d, 1H, J2Ј,3Ј = 5.1 Hz, H-2Ј),
4.47 (m, 2H, H-3Ј, CHCH2Ph), 4.15–3.83 (m, 4H, H-4Ј, H-5Ј,
2
CHBui), 3.03–2.73 (m, 2H, J = 13.4 Hz, JAX = 4.7 Hz, JBX
=
9.1 Hz, CH2Ph), 1.49 (m, 1H, CH2CHMe2), 1.29 (m, 2H,
CH2Pri), 1.10–0.72 (m, 34H, SiCH(CH3)2, CH2CH(CH3)2).
2Ј-O-[2-(2-Hydroxyethoxy)ethylaminocarbonyl]-3Ј,5Ј-O-
(tetraisopropyldisiloxane-1,3-diyl)uridine
(3g).
Chromato-
graphy: CHCl3–EtOAc–acetone, stepwise gradient 2 : 1 : 1 1 :
1 : 1 1 : 1 : 2 (v/v/v). Yield 2.269 g (73.5%). Rf 0.28 (EtOAc).
ESI MS: [M ϩ Na]ϩ calc. 640.27, found 640.26. 1H-NMR:
11.41 (s, 1H, H-3), 7.68 (d, 1H, J5,6 = 7.9 Hz, H-6), 7.40 (br s,
1H, OCONH ), 5.64 (s, 1H, H-1Ј), 5.59 (d, 1H, J5,6 = 7.9 Hz,
H-5), 5.31 (d, 1H, J2Ј,3Ј = 5.4 Hz, H-2Ј), 4.55 (m, 2H, H-3Ј,
OH ), 4.12–3.79 (m, 3H, H-4Ј, H-5Ј), 3.53–3.27 (m, 6H¶,
CH2OCH2CH2), 3.12 (m, 2H, CH2N), 1.10–0.82 (m, 28H, Pri).
2Ј-O-[2-(2-Pivaloyloxyethoxy)ethylaminocarbonyl]-3Ј,5Ј-O-
(tetraisopropyldisiloxane-1,3-diyl)uridine (3h). To the stirred
solution of 3g (2.162 g, 3.5 mmol) in dry DCM (20 cm3), pyrid-
ine (1.0 cm3, 12.5 mmol) and pivaloyl chloride (0.615 cm3,
5 mmol) were added, and the reaction was kept at 20 ЊC until
the starting compound disappeared (ca. 20 h). The mixture was
diluted with DCM (100 cm3), washed with water (100 cm3), 5%
NaHCO3 (100 cm3), and water (100 cm3), dried (Na2SO4),
evaporated to dryness, and the residue was chromatographed
on silica gel (stepwise gradient of 10 to 50% (v/v) EtOAc in
CHCl3). Yield 1.977 g (80.5%). Rf 0.34 (CHCl3–EtOAc, 1 : 1
2
3.97 (m, 2H, J5Јa,5Јb = 12.5 Hz, J4Ј,5Јa = 4.2 Hz, J4Ј,5Јb = 2.4 Hz,
H-5Ј), 3.83 (m, 1H, H-4Ј), 3.33 (m, 4H¶ CH2NCH2), 3.19 (m,
2H, CH2NHTFA), 2.98 (m, 2H, OCONHCH2), 1.74 (m, 4H,
CH2), 1.10–0.85 (m, 28H, Pri).
2Ј-O-[4,9-Bis(trifluoroacetyl)-12-(trifluoroacetylamino)-4,9-
diazadodecan-1-ylaminocarbonyl]-3Ј,5Ј-O-(tetraisopropyl-
disiloxane-1,3-diyl)uridine (3p). A solution of 2 (5 mmol,
prepared in situ as above) in dry DCM (50 cm3) was added
dropwise to a stirred, ice-cooled solution of spermine (2.63 g,
¶ Calculated value; the signal of water is also present in the region.
1100
J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104

View Article Online
13 mmol) in DCM (20 cm3). After 2 h the cooling was removed
and the mixture was stirred overnight, diluted with CHCl3
(150 cm3), washed with 20% NaCl (2 × 200 cm3), dried
(Na2SO4), and evaporated to dryness. The residue was trifluoro-
acetylated as above, and the desired compound was isolated
using chromatography on silica gel (stepwise gradient of
50 30 20 0% CHCl3 in EtOAc, v/v). Yield 2.457 g (49.0%),
Rf 0.67 (EtOAc). MALDI-TOF (2,5-DHBA): [M ϩ H]ϩ calc.
1004.07, found 1004.02, [M ϩ Na]ϩ calc. 1025.35, found
mixture was diluted with CHCl3 (100 cm3), washed with water
(100 cm3), 5% NaHCO3 (100 cm3), and water (100 cm3), then
dried (Na2SO4), evaporated, coevaporated with toluene (3 × 25
cm3) and the residue was chromatographed on silica gel column
in an appropriate solvent system. Fractions containing product
were combined, evaporated, and the residue was dried in vacuo
to afford compounds listed below as amorphous solids.
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-(propargylaminocarbonyl)-
1
1025.64, [M ϩ K]ϩ calc. 1041.33, found 1041.36. H-NMR:
uridine (5a). Chromatography: stepwise gradient of 1
3
5%
11.42 (s, 1H, H-3), 9.51, 9.44 (2 br t, 1H, NHTFA, rotamers),
7.69 (d, 1H, J5,6 = 8.0 Hz, H-6), 7.51, 7.45 (2t, 1H, J = 5.2 Hz,
MeOH in CHCl3 ϩ 0.5% Et3N (v/v/v). Yield 1.156 g (92.1%), Rf
0.60 (EtOAc). ESI MS: [M ϩ Na]ϩ calc. 650.21, found 650.20.
1H-NMR: 11.38 (br s, 1H, H-3), 7.85 (t, 1H, J = 5.7 Hz,
OCONH ), 7.66 (m, 1H, H-6), 7.40–7.19 (m, 9H, ArH ), 6.89 (d,
4H, J = 8.7 Hz, ArH ), 5.79 (d, 1H, J = 5.7 Hz, 3Ј-OH ), 5.75 (s,
1H, H-1Ј), 5.39 (d, 1H, J5,6 = 8.1 Hz, H-5), 5.11 (m, 1H, H-2Ј),
OCONH, rotamers), 5.65 (s, 1H, H-1Ј), 5.59 (d, 1H, J5,6
=
8.0 Hz, H-5), 5.32 (m, 1H, H-2Ј), 4.49 (m, 1H, H-3Ј), 3.98 (m,
2H, 2J5Јa,5Јb = 12.6 Hz, J4Ј,5Јa = 4.2 Hz, H-5Ј), 3.83 (m, 1H, H-4Ј),
3.40–3.28 (m, 8H¶, CH2N(TFA)CH2), 3.20 (m, 2H, CH2-
NHTFA), 2.98 (m, 2H, OCONHCH2), 1.86–1.60 (m, 4H,
CH2CH2NH), 1.51 (m, 4H, CH2CH2CH2CH2), 1.06–0.80 (m,
28H, Pri).
4.35 (m, 1H, H-3Ј), 4.06 (m, 1H, H-4Ј), 3.79 (m, 2H, CH2N),
᎐
3.73 (s, 6H, CH ), 3.40–3.15 (m, 3H¶, H-5Ј, ᎐CH ).
᎐
3
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-[N-methyl-N-propargyl-
aminocarbonyl]uridine (5b). Chromatography: stepwise gradient
of 1 2% MeOH in CHCl3–EtOAc (1 : 1) ϩ 0.5% Et3N (v/v/v/
v). Yield 1.097 g (85.5%), Rf 0.73 (EtOAc). ESI MS: [M ϩ Na]ϩ
Uridin-2Ј-yl carbamates (4)
A general procedure.
To a solution of 3Ј,5Ј-O-(tetraisopropyldisiloxane-1,3-diyl)-
uridin-2Ј-yl carbamate (2 mmol) in THF (5 cm3) in a screw-top
Teflon flask (Nalgene) was added triethylamine trihydrofluoride
(0.814 cm3, 5 mmol) and the mixture was left for 6 h or over-
night at room temperature (the completion of deprotection was
checked by TLC (15% MeOH in CHCl3, v/v)), then diluted
with hexane (25 cm3). The upper layer was discarded, and the
residue (oil or solid) was washed with 1 : 1 (v/v) toluene–hexane
mixture (3 × 25 cm3) by decantation, triturated in absolute
ethanol (5 cm3), and the crystalline product was filtered
off, washed with EtOH (5 cm3), Et2O (10 cm3), and dried
in vacuo.
1
calc. 664.23, found 664.22. H-NMR: 11.39 (s, 1H, H-3), 7.70
(d, 1H, J5,6 = 8.0 Hz, H-6), 7.41–7.19 (m, 9H, ArH ), 6.89 (d,
4H, J = 8.6 Hz, ArH ), 5.89 (d, 1H, J1Ј,2Ј = 3.3 Hz, H-1Ј), 5.54 (m,
1H, 3Ј-OH ), 5.39 (d, 1H, J5,6 = 8.0 Hz, H-5), 5.15 (m, 1H, H-2Ј),
4.32 (m, 1H, H-3Ј), 4.12–3.93 (m, 3H, H-4Ј, CH2N), 3.73 (s, 6H,
᎐
OCH3), 3.35–3.16 (m, 3H¶, H-5Ј, ᎐CH ), 2.92 (s, 1.8H), 2.85
᎐
(s, 1.2H), (NCH3, rotamers).
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-(4-iodobenzylamino-
carbonyl)uridine (5c). Chromatography: stepwise gradient of
0.5
1
1.5 2% MeOH in CHCl3–EtOAc (1 : 1) ϩ 0.5%
Et3N (v/v/v/v). Yield 1.378 g (85.5%), Rf 0.75 (EtOAc). ESI MS:
[M ϩ Na]ϩ calc. 828.14, found 828.14. 1H-NMR: 11.42 (s, 1H,
2Ј-O-(4-Iodobenzylaminocarbonyl)uridine (4c). Yield 993 mg
(98.6%). Rf 0.34 (CHCl3–MeOH, 17 : 3), mp 192–197 ЊC
(EtOH). MALDI-TOF (2,5-DHBA): [M ϩ Na]ϩ calc. 526.24,
found 527.55. 1H-NMR: 11.36 (s, 1H, H-3), 7.90 (m, 2H,
H-6, OCONH ), 7.65 (d, 2H, J = 7.4 Hz, ArH ), 7.02 (d, 2H,
J = 7.4 Hz, ArH ), 5.99 (m, 1H, H-1Ј), 5.66 (d, 1H, J5,6 = 8.0 Hz,
H-5), 5.52 (d, 1H, J = 4.4 Hz, 3Ј-OH ), 5.18 (m, 1H, 5Ј-OH ),
5.05 (m, 1H, H-2Ј), 4.25–4.05 (m, 3H, H-3Ј, CH2N), 3.88 (m,
1H, H-4Ј), 3.59 (m, 2H, H-5Ј).
H-3), 7.95 (t, 1H, J = 6.0 Hz, OCONH ), 7.70 (d, 1H, J5,6
=
8.1 Hz, H-6), 7.66 (d, 1H, J = 8.2 Hz, ArH ), 7.40–7.18 (m, 9H,
ArH ), 7.06 (d, 2H, J = 8.2 Hz, ArH ), 6.88 (d, 4H, J = 8.8 Hz,
ArH ), 5.92 (d, 1H, J1Ј,2Ј = 4.8 Hz, H-1Ј), 5.61 (d, 1H, J3Ј,OH
=
5.6 Hz, 3Ј-OH ), 5.38 (d, 1H, J5,6 = 8.1 Hz, H-5), 5.17 (apparent
t, 1H, J1Ј,2Ј = J2Ј,3Ј = 4.8 Hz, H-2Ј), 4.33 (m, 1H, H-3Ј), 4.14 (d,
2H, J = 6.0 Hz, NCH2), 3.98 (m, 1H, H-4Ј), 3.73 (s, 6H, CH3),
3.35–3.16 (m, 2H¶, H-5Ј).
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-(pyren-1-ylmethylamino-
carbonyl)uridine (5d). Chromatography: stepwise gradient of
2Ј-O-(Pyren-1-ylmethylaminocarbonyl)uridine (4d). Yield 467
mg (93.5%). Rf 0.37 (CHCl3–MeOH, 17 : 3), mp 200–208 ЊC
(EtOH). MALDI-TOF (2,5-DHBA): [M ϩ H]ϩ calc. 502.50,
found 502.34, [M ϩ Na]ϩ calc. 524.48, found 525.60, [M ϩ K]ϩ
calc. 540.59, found 541.68. 1H-NMR: 11.38 (s, 1H, H-3),
0.5
1
1.5% MeOH in CHCl3 ϩ 0.5% pyridine (v/v/v). Yield
1.482 g (92.2%), Rf 0.75 (EtOAc). ESI MS: [M ϩ Na]ϩ calc.
826.27, found 826.28. 1H-NMR: 11.44 (s, 1H, H-3), 8.41 (d, 1H,
J9Љ,10Љ = 9.3 Hz, H-10Љ), 8.32–8.00 (m, 9H, ArH, OCONH ), 7.73
(d, 1H, J5,6 = 8.0 Hz, H-6), 7.42–7.10 (m, 9H, ArH ), 6.88
(d, 4H, J = 8.6 Hz, ArH ), 5.97 (d, 1H, J1Ј,2Ј = 4.6 Hz, H-1Ј), 5.65
(d, 1H, J3Ј,OH = 5.6 Hz, 3Ј-OH ), 5.39 (d, 1H, J5,6 = 8.0 Hz, H-5),
5.26 (m, 1H, H-2Ј), 4.95 (d, 2H, J = 5.3 Hz, NCH2), 4.38
(m, 1H, H-3Ј), 3.99 (m, 1H, H-4Ј), 3.72 (s, 6H, CH3), 3.35–3.15
(m, 2H¶, H-5Ј).
8.42–7.90 (m, 11H, H-6, OCONH, AH ), 6.03 (d, 1H, J1Ј,2Ј
=
5.7 Hz, H-1Ј), 5.68 (d, 1H, J5,6 = 7.8 Hz, H-5), 5.57 (m, 1H,
3Ј-OH ), 5.21 (m, 1H, 5Ј-OH ), 5.14 (m, 1H, H-2Ј), 4.91 (m,
2H, CH2Ar), 4.24 (m, 1H, H-3Ј), 3.91 (m, 1H, H-4Ј), 3.60
(m, 2H, H-5Ј).
5Ј-O-(4,4Ј-Dimethoxytrityl)uridin-2Ј-yl carbamates (5)
A general procedure.
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-(1,4,7,10,13-pentaoxa-
The crude oily uridin-2Ј-yl carbamates from the general pro-
cedure above (washed with 1 : 1 (v/v) toluene–hexane mixture)
were coevaporated with toluene (3 × 20 cm3), pyridine (3 ×
20 cm3), dissolved in dry pyridine (15 cm3), cooled in an ice
bath, and DmtCl (845 mg, 2.5 mmol) was added in one portion.
The reaction was monitored by TLC and further portions of
DmtCl (170 mg, 0.5 mmol) were added every 4 h until the start-
ing nucleoside disappears (the amount of DmtCl is dependent
on the residual HF removal rate and varied usually from 3 to
4 mmol). After completion of the reaction, the excess of DmtCl
was quenched with MeOH (1 cm3), and after 10 min the
cyclopentadecan-2-ylmethylaminocarbonyl)uridine (5e). Chro-
matography: stepwise gradient of 3 4.5
6
9% MeOH in
CHCl3–EtOAc (2 : 1) ϩ 0.5% Et3N (v/v/v/v). Yield 1.248 g
(85.5%), Rf 0.29 (CHCl3–MeOH, 17 : 3). ESI MS: [M ϩ Na]ϩ
1
calc. 844.33, found 844.32. H-NMR: 11.39 (s, 1H, H-3), 7.69
(d, 1H, J5,6 = 8.0 Hz, H-6), 7.41–7.12 (m, 10H, ArH, OCONH ),
6.89 (d, 4H, J = 8.8 Hz, ArH ), 5.90 (d, 1H, J1Ј,2Ј = 4.7 Hz, H-1Ј),
5.53 (m, 1H, 3Ј-OH ), 5.38 (d, 1H, J5,6 = 8.0 Hz, H-5), 5.15 (m,
1H, H-2Ј), 4.32 (m, 1H, H-3Ј), 3.97 (m, 1H, H-4Ј), 3.73 (s, 6H,
CH3), 3.69–3.16 (m, 21H¶, H-5Ј, CH(CH2OCH2)4CH2), 3.02
(m, 2H, CH2N).
J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104
1101

View Article Online
Nꢀ-[5Ј-O-(4,4Ј-Dimethoxytrityl)uridin-2Ј-O-ylcarbonyl]-L-
leucyl-L-phenylalaninamide (5f ). Chromatography: stepwise
1H, H-2Ј), 4.34 (m, 1H, H-3Ј), 3.96 (m, 1H, H-4Ј), 3.73 (s, 6H,
CH3), 3.44–3.14 (m, 12H¶, H-5Ј, CH2NTFA), 3.01 (m, 2H,
OCONHCH2), 1.73 (m, 4H, CH2CH2NH), 1.52 (m, 4H,
CH2CH2CH2CH2).
gradient of 1
2
3% MeOH in CHCl3–EtOAc (2 : 1) ϩ 0.5%
pyridine (v/v/v/v). Yield 706 mg (83.1%), Rf 0.45 (EtOAc).
MALDI-TOF (2,4,6-THAP): [M ϩ Na]ϩ calc. 872.35, found
1
872.62, [M ϩ K]ϩ calc. 888.32, found 888.62. H-NMR: 11.45
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-O-
(4,4Ј-dimethoxytrityl)uridin-2Ј-yl carbamates (6)
(s, 1H, H-3), 7.90 (d, 1H, J = 8.4 Hz, NHCHCH2Ph), 7.72 (d,
1H, J5,6 = 8.1 Hz, H-6), 7.48–7.10 (m, 12H, ArH, NH2,
A general procedure.
OCONH ), 6.89 (d, 4H, J = 8.8 Hz, ArH ), 5.90 (d, 1H, J1Ј,2Ј
=
5Ј-O-(4,4Ј-Dimethoxytrityl)uridin-2Ј-yl carbamates 5 (1.0
mmol) were coevaporated with dry DCM (2 × 20 cm3), dis-
solved in dry DCM, diisopropylammonium tetrazolide (171
mg, 1.0 mmol) and bis(N,N-diisopropylamino)-2-cyanoethoxy-
phosphine (0.38 cm3, 1.2 mmol) were added, and the mixture
was stirred under nitrogen for 2 h. After conversion of the start-
ing compound 5 is complete (monitoring by TLC, 25% acetone
in CHCl3 ϩ 1% Et3N v/v/v, pair of diastereomers), the mixture
was diluted with CHCl3, washed with 5% NaHCO3 (100 cm3),
20% NaCl (100 cm3), dried over Na2SO4, evaporated to dryness,
and the residue was chromatographed on a silica gel column in
an appropriate solvent system. Fractions containing product
were combined, evaporated, and the residue was dried in vacuo
to afford compounds listed below as white amorphous solids.
4.4 Hz, H-1Ј), 5.52 (d, 1H, J3Ј,OH = 5.9 Hz, 3Ј-OH ), 5.43 (d, 1H,
J5,6 = 8.1 Hz, H-5), 5.13 (m, 1H, H-2Ј), 4.47 (m, 2H, H-3Ј,
CHCH2Ph), 4.37 (m, 1H, H-3Ј), 4.17 (m, 1H, CHBui), 3.98
(m, 1H, H-4Ј), 3.73 (s, 6H, OCH3), 3.52–3.10 (m, 2H, H-5Ј),
3.05–2.70 (m, 2H, CH2Ph), 1.57–1.22 (m, 3H, CH2CHMe2),
0.86–0.69 (m, 6H, CH(CH3)2).
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-[2-(2-pivaloyloxyethoxy)-
ethylaminocarbonyl]uridine (5h). Chromatography: stepwise
gradient of 0.5
1
1.5 2% MeOH in CHCl3–EtOAc (2 : 1)
ϩ 0.5% Et3N (v/v/v/v). Yield 1.377 g (90.4%), Rf 0.60 (EtOAc).
ESI MS: [M ϩ Na]ϩ calc. 784.31, found 784.30. 1H-NMR:
11.40 (s, 1H, H-3), 7.69 (d, 1H, J5,6 = 8.1 Hz, H-6), 7.41–7.19
(m, 10H, ArH, OCONH ), 6.89 (d, 4H, J = 8.8 Hz, ArH ), 5.90
(d, 1H, J1Ј,2Ј = 5.0 Hz, H-1Ј), 5.53 (d, 1H, J3Ј,OH = 5.7 Hz,
3Ј-OH ), 5.38 (d, 1H, J5,6 = 8.1 Hz, H-5), 5.12 (apparent t, 1H,
J1Ј,2Ј = J2Ј,3Ј = 5.0 Hz, H-2Ј), 4.32 (m, 1H, H-3Ј), 4.11 (m, 2H,
CH2OPiv), 3.97 (m, 1H, H-4Ј), 3.73 (s, 6H, OCH3), 3.57, 3.43
(2m, 4H, CH2OCH2), 3.27–3.02 (m, 4H, H-5Ј, CH2N), 1.11
(s, 9H, But).
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-(propargylaminocarbonyl)uridine
(6a). Chromatography: 33 50 100% EtOAc in CHCl3 ϩ 1%
Et3N (v/v/v). Yield 1.029 g (82.8%). “Faster moving” diaster-
eomer: Rf 0.56. MALDI-TOF (2,6-DHAP-citrate): [M ϩ H]ϩ
1
calc. 828.87, found 828.67. H-NMR: 11.45 (s, 1H, H-3), 7.95
(m, 1H, OCONH ), 7.72 (d, 1H, J5,6 = 8.1 Hz, H-6), 7.41–7.18
(m, 9H, ArH ), 6.88 (d, 4H, J = 8.5 Hz, ArH ), 5.89 (d, 1H,
J1Ј,2Ј = 5.0 Hz, H-1Ј), 5.43 (d, 1H, J5,6 = 8.1 Hz, H-5), 5.33
(apparent t, 1H, J1Ј,2Ј = J2Ј,3Ј = 5.0 Hz, H-2Ј), 4.56 (m, 1H, H-3Ј),
4.15 (m, 1H, H-4Ј), 3.85–3.68 (m, 8H, OCH3, CH2N), 3.63–3.44
(m, 4H, POCH2, CHCH3), 3.34–3.22 (m, 2H¶, H-5Ј), 3.09 (t,
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-[13-(trifluoroacetylamino)-
4,7,10-trioxatridecan-1-ylaminocarbonyl]uridine (5j). Chrom-
atography: stepwise gradient of 1
2
3
3.5% MeOH in
CHCl3–EtOAc (2 : 1) ϩ 0.5% Et3N (v/v/v/v). Yield 1.427 g
(80.3%), Rf 0.22 (EtOAc). ESI MS: [M ϩ Na]ϩ calc. 911.33,
1
found 911.33. H-NMR: 11.39 (s, 1H, H-3), 9.36 (br s, 1H,
4
᎐
1H, J = 2.3 Hz, ᎐CH ), 2.58 (t, 2H, J = 6.1 Hz, CH CN), 1.19–
᎐
2
NHTFA), 7.70 (d, 1H, J5,6 = 8.1 Hz, H-6), 7.41–7.19 (m, 10H,
ArH, OCONH ), 6.89 (d, 4H, J = 8.8 Hz, ArH ), 5.89 (d, 1H,
J1Ј,2Ј = 5.0 Hz, H-1Ј), 5.54 (d, 1H, J3Ј,OH = 5.6 Hz, 3Ј-OH ), 5.38
0.95 (m, 12H, CHCH3). 31P NMR (CD3CN): 150.784. “Slower
moving” diastereomer: Rf 0.41. MALDI-TOF (2,6-DHAP-
citrate): [M ϩ H]ϩ calc. 828.87, found 828.76. 1H-NMR: 11.44
(s, 1H, H-3), 7.90 (t, 1H, J = 5.7 Hz, OCONH ), 7.73 (d, 1H,
J5,6 = 8.1 Hz, H-6), 7.41–7.18 (m, 9H, ArH ), 6.87 (m, 4H,
ArH ), 5.91 (d, 1H, J1Ј,2Ј = 5.1 Hz, H-1Ј), 5.42 (d, 1H, J5,6 = 8.1
Hz, H-5), 5.36 (apparent t, 1H, J1Ј,2Ј = J2Ј,3Ј = 5.1 Hz, H-2Ј), 4.55
(m, 1H, H-3Ј), 4.10 (m, 1H, H-4Ј), 3.82–3.68 (m, 10 H, OCH3,
(d, 1H, J5,6 = 8.1 Hz, H-5), 5.13 (apparent t, 1H, J1Ј,2Ј = J2Ј,3Ј
=
5.0 Hz, H-2Ј), 4.32 (m, 1H, H-3Ј), 3.96 (m, 1H, H-4Ј), 3.73 (s,
6H, CH3), 3.53–3.36 (m, 12H, (CH2OCH2)3), 3.28–3.16 (m, 4H,
H-5Ј, CH2NHTFA), 3.02 (m, 2H, OCONHCH2), 1.75–1.56 (m,
4H, CH2CH2CH2).
CH2N, POCH2), 3.46 (m, 2H, CHCH3), 3.35–3.21 (m, 2H¶,
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-[4-trifluoroacetyl-7-
(trifluoroacetylamino)-4-azaheptan-1-ylaminocarbonyl]uridine
(5m). Compound 5m was isolated by chromatography using a
᎐
H-5Ј), 3.12 (m, 1H, ᎐CH ), 2.74 (t, 2H, J = 5.8 Hz, CH CN),
᎐
2
1.19–0.89 (m, 12H, CHCH3). 31P NMR (CD3CN): 150.729.
stepwise gradient of 1
2
4% MeOH in CHCl3–EtOAc (2 : 1)
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-(N-methyl-N-propargylamino-
carbonyl)uridine (6b). Chromatography: 30 50 70% EtOAc
in CHCl3 ϩ 1% Et3N (v/v/v). Yield 1.004 g (79.5%), Rf 0.58,
0.69. MALDI-TOF (2,6-DHAP-citrate): [M ϩ H]ϩ calc.
ϩ 0.5% Et3N (v/v/v/v); yield 1.556 g (86.8%), white amorphous
solid, Rf 0.49 (EtOAc). ESI MS: [M ϩ Na]ϩ calc. 918.28, found
1
918.27. H-NMR: 11.37 (br s, 1 H, H-3) (exchangeable with
D2O), 9.51, 9.46 (2 br s, 1 H, NHTFA, rotamers), 7.69 (m, 1 H,
H-6), 7.53–7.19 (m, 10 H, ArH (Dmt), OCONH ), 6.89 (m, 4 H,
ArH (Dmt)), 5.92 (m, 1 H, H-1Ј), 5.56 (m, 1 H, 3Ј-OH)
(exchangeable with D2O), 5.37 (m, 1 H, H-5), 5.15 (m, 1 H,
H-2Ј), 4.34 (m, 1 H, H-3Ј), 3.97 (m, 1 H, H-4Ј), 3.73 (s, 6 H,
CH3), 3.40–3.14 (m, 8 H¶, H-5Ј, CH2NTFA), 3.00 (m, 2 H,
OCONHCH2), 1.73 (m, 4 H, CH2CH2CH2).
1
842.89, found 842.89. H-NMR: 11.42 (s, 1H, H-3), 7.70 (m,
1H, H-6), 7.42–7.16 (m, 9H, ArH ), 6.87 (m, 4H, ArH ), 5.90
(m, 1H, H-1Ј), 5.47–5.18 (m, 2H, H-5, H-2Ј), 4.55 (m, 1H,
H-3Ј), 4.20–3.92 (m, 3H, H-4Ј, CH2N), 3.73 (s, 6H, OCH3),
᎐
3.62–3.18 (m, 7H¶, POCH , CHCH , H-5Ј, ᎐CH ), 2.94–2.78
᎐
2
3
(m, 3H, NCH3), 2.74, 2.56 (2m, 2H, CH2CN), 1.20–0.88 (m,
12H, CHCH3). 31P NMR (CD3CN): 150.892 (26%), 150.764
(30%), 150.391 (44%), diastereomers and rotamers.
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-[4,9-bis(trifluoroacetyl)-12-
(trifluoroacetylamino)-4,9-diazadodecan-1-ylaminocarbonyl]-
uridine (5p). Chromatography: stepwise gradient of 1
2
5%
MeOH in CHCl3–EtOAc (2 : 1) ϩ 0.5% Et3N (v/v/v/v). Yield
1.773 g (83.4%), Rf 0.50 (EtOAc). MALDI-TOF (2,4,6-THAP):
[M ϩ Na]ϩ calc. 1085.33, found 1085.28, [M ϩ K]ϩ calc.
1101.31, found 1100.91. 1H-NMR: 11.40 (s, 1H, H-3), 9.51, 9.44
(2m, 1H, NHTFA rotamers), 7.69 (m, 1H, H-6), 7.51–7.18 (m,
10H, ArH, OCONH ), 6.88 (d, 4H, J = 8.8 Hz, ArH ), 5.92 (m,
1H, H-1Ј), 5.56 (m, 1H, 3Ј-OH ), 5.37 (m, 1H, H-5), 5.15 (m,
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-(4-iodobenzylaminocarbonyl)-
uridine (6c). Chromatography: stepwise gradient of 20 25%
acetone in CHCl3 ϩ 1% Et3N (v/v/v). Yield 674 mg (67%), Rf
0.47, 0.62. MALDI-TOF (2,6-DHAP-citrate): [M ϩ H]ϩ calc.
1006.84, found 1008.17. 1H-NMR: 11.46 (s, 1H, H-3), 8.04 (m,
1H, OCONH ), 7.73–7.61 (m, 3H, ArH, H-6), 7.42–7.18 (m,
1102
J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104

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9H, ArH ), 7.07 (m, 2H, ArH ), 6.87 (m, 4H, ArH ), 5.93 (m,
1H, H-1Ј), 5.46–5.28 (m, 2H, H-5, H-2Ј), 4.53 (m, 1H, H-3Ј),
4.12 (m, 3H, H-4Ј, NCH2), 3.78–3.19 (m, 12H¶, POCH2, CHN,
H-5Ј, OCH3), 2.68, 2.58 (2m, 2H, CH2CN), 1.23–0.89 (m, 12H,
CHCH3). 31P NMR (CD3CN): 149.274.
(v/v/v). Yield 1.396 g (93.8%), Rf 0.14, 0.25. MALDI-TOF
(2,6-DHAP-citrate): [M ϩ H]ϩ calc. 1097.02, found 1097.45.
1H-NMR: 11.40 (br s, 1H, H-3), 9.52, 9.44 (2 br s, 1H, NHTFA,
rotamers), 7.70 (m, 1H, H-6), 7.53 (m, 1H, OCONH ), 7.41–
7.15 (m, 9H, ArH ), 6.87 (m, 4H, ArH ), 5.92 (m, 1H, H-1Ј),
5.45–5.22 (m, 2H, H-5, H-2Ј), 4.54 (m, 1H, H-3Ј), 4.12 (m, 1H,
H-4Ј), 3.80–3.09 (m, 18H¶, OCH3, POCH2, CHN, H-5Ј,
CH2NTFA), 2.99 (m, 2H, OCONHCH2), 2.74, 2.57 (2m, 2H,
CH2CN), 1.73 (m, 4H, CH2CH2CH2), 1.20–0.87 (m, 12H,
CHCH3). 31P NMR (CD3CN): 150.741 (54%), 150.658 (22%),
150.581 (24%), diastereomers and rotamers.
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-(pyren-1-ylmethylamino-
carbonyl)uridine (6d). Chromatography:
5
10 20 25%
acetone in CHCl3 ϩ 1% Et3N (v/v/v). Yield 978 mg (97.4%), Rf
0.55, 0.68. MALDI-TOF (2,6-DHAP-citrate): [M ϩ H]ϩ calc.
1
1005.08, found 1006.67. H-NMR: 11.47 (s, 1H, H-3), 8.41 (d,
1H, J9Љ,10Љ = 9.3 Hz, H-10Љ), 8.36–7.98 (m, 9H, pyrene H-2Љ–H-
9Љ, OCONH ), 7.73 (d, 1H, J5,6 = 8.0 Hz, H-6), 7.43–7.17 (m,
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-[4,9-bis(trifluoroacetyl)-12-
(trifluoroacetylamino)-4,9-diazadodecan-1-ylaminocarbonyl]-
uridine (6p). Chromatography: stepwise gradient of 20 50%
acetone in CHCl3 ϩ 1% Et3N (v/v/v). Yield 1.242 g (98.3%), Rf
0.18, 0.32. MALDI-TOF (2,6-DHAP-citrate): [M ϩ H]ϩ calc.
9H, ArH ), 6.87 (d, 4H, J = 8.5 Hz, ArH ), 5.95 (d, 1H, J1Ј,2Ј
=
5.1 Hz, H-1Ј), 5.42 (m, 2H, H-5, H-2Ј), 4.91 (d, 2H, J = 5.3 Hz,
NCH2), 4.57 (m, 1H, H-3Ј), 4.17 (m, 1H, H-4Ј), 3.71 (s, 6H,
OCH3), 3.42–3.35 (m, 6H, POCH2, CHN, H-5Ј), 2.88, 2.58 (2t,
2H, J = 5.8 Hz, CH2CN, diastereomers), 1.17, 1.02–0.89 (2m,
12H, CHCH3). 31P NMR (CD3CN): 149.295.
1
1264.15, found 1264.67. H-NMR: 11.36 (br s, 1H, H-3), 9.52,
9.45 (2 br s, 1H, NHTFA, rotamers), 7.69 (m, 1H, H-6), 7.53
(m, 1H, OCONH ), 7.41–7.16 (m, 9H, ArH ), 6.87 (m, 4H,
ArH ), 5.92 (m, 1H, H-1Ј), 5.44–5.21 (m, 2H, H-5, H-2Ј), 4.53
(m, 1H, H-3Ј), 4.12 (m, 1H, H-4Ј), 3.80–3.12 (m, 22H¶, OCH3,
POCH2, CHN, H-5Ј, CH2NTFA), 2.99 (m, 2H, OCONHCH2),
2.74, 2.57 (2m, 2H, CH2CN), 1.74 (m, 4H, CH2CH2NH), 1.51
(m, 4H, CH2CH2CH2CH2), 1.19–0.86 (m, 12H, CHCH3). 31P
NMR (CD3CN): 150.758 (56%), 150.651 (20%), 150.584 (24%),
diastereomers and rotamers.
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-(1,4,7,10,13-pentaoxacyclo-
pentadecan-2-ylmethylaminocarbonyl)uridine (6e). Chromato-
graphy: stepwise gradient of 20 50 66% acetone in CHCl3 ϩ
1% Et3N (v/v/v). Yield 955 mg (93.4%), Rf 0.11, 0.24. MALDI-
TOF (2,6-DHAP-citrate): [M ϩ H]ϩ calc. 1023.09, found
1024.61, [M ϩ Na]ϩ calc. 1045.07, found 1046.01, [M ϩ K]ϩ
1
calc. 1061.18, found 1062.02. H-NMR: 11.44 (br s, 1H, H-3),
7.70 (m, 1H, H-6), 7.49–7.15 (m, 10H, ArH, OCONH ), 6.87
(m, 4H, ArH ), 5.91 (m, 1H, H-1Ј), 5.46–5.25 (m, 2H, H-5,
H-2Ј), 4.53 (m, 1H, H-3Ј), 4.11 (m, 1H, H-4Ј), 3.80–3.19 (m,
31H¶, POCH2, CHN, H-5Ј, CH(CH2OCH2)4CH2, OCH3), 3.00
(m, 2H, CH2N), 2.75, 2.59 (2m, 2H, CH2CN), 1.20–0.90
(m, 12H, CHCH3). 31P NMR (CD3CN): 149.280, 149.275
(diastereomers).
5Ј-O-(4,4Ј-Dimethoxytrityl)uridine-2Ј,3Ј-cyclic carbonate (8)
To a solution of 5Ј-O-(4,4Ј-dimethoxytrityl)uridine (2.733 g,
5.0 mmol) in DCM (50 cm3), CDI (892 mg, 5.5 mmol) was
added in one portion. After 2 h TLC (EtOAc) showed com-
pletion of the reaction. The mixture was diluted with CHCl3
(100 cm3), washed with water (150 cm3), 5% citric acid (150
cm3), and water (150 cm3), dried (Na2SO4), evaporated, and the
residue was chromatographed on a short silica gel column
(stepwise gradient of EtOAc 1 : 1 3 : 2 2 : 1 in CHCl3 ϩ
0.5% pyridine, v/v/v). Yield 2.857 g (99.8%), white solid. ESI
MS: [M ϩ Na]ϩ calc. 595.55, found 595.17. 1H-NMR: 11.45 (s,
1H, H-3), 7.76 (d, 1H, J5,6 = 8.0 Hz, H-6), 7.39–7.16 (m, 9H,
ArH ), 6.86 (m, 4H, ArH ), 5.99 (d, 1H, J1Ј,2Ј = 1.6 Hz, H-1Ј),
5.64 (d, 1H, J5,6 = 8.0 Hz, H-5), 5.59 (dd, 1H, J1Ј,2Ј = 1.6 Hz,
J2Ј,3Ј = 7.6 Hz, H-2Ј), 5.20 (dd, 1H, J2Ј,3Ј = 7.6 Hz, J3Ј,4Ј = 4.2 Hz,
H-3Ј), 4.41 (m, 1H, H-4Ј), 3.72 (s, 6H, OCH3), 3.37 (m, 1H¶,
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-[2-(2-pivaloyloxyethoxy)ethyl-
aminocarbonyl]uridine (6h). Chromatography: acetone–CHCl3
(1 : 1) ϩ 1% Et3N (v/v/v). Yield 947 mg (98.4%), Rf 0.43, 0.57.
MALDI-TOF (2,6-DHAP-citrate): [M ϩ H]ϩ calc. 963.04,
1
found 964.82. H-NMR: 11.44 (s, 1H, H-3), 7.70 (d, 1H, J5,6
=
8.0 Hz, H-6), 7.47–7.19 (m, 10H, ArH, OCONH ), 6.88 (m, 4H,
ArH ), 5.90 (m, 1H, H-1Ј), 5.43 (d, 1H, J5,6 = 8.0 Hz, H-5), 5.30
(m, 1H, H-2Ј), 4.55 (m, 1H, H-3Ј), 4.10 (m, 3H, H-4Ј,
CH2OCO), 3.72 (s, 6H, OCH3), 3.62–3.01 (m, 12H¶, POCH2,
CHN, H-5Ј, NCH2CH2OCH2), 2.74, 2.57 (2m, 2H, CH2CN),
1.23–0.90 (m, 21H, CHCH3, But). 31P NMR (CD3CN): 149.488,
149.416, 149.351 (diastereomers and rotamers).
2J5Јa,5Јb = 10.0 Hz, J4Ј,5Јa = 7.4 Hz, H-5Јa), 3.13 (m, 1H, 2J5Јa,5Јb
10.0 Hz, J4Ј,5Јb = 4.6 Hz, H-5Јb).
=
5Ј-O-(4,4Ј-Dimethoxytrityl)-3Ј-O-(N-methyl-N-propargylamino-
carbonyl)uridine (9)
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-[13-(trifluoroacetylamino)-
4,7,10-trioxatridecan-1-ylaminocarbonyl]uridine (6j). Chroma-
tography: stepwise gradient of 20 33 50 66% acetone in
CHCl3 ϩ 1% Et3N (v/v/v). Yield 1.069 g (98.2%), Rf 0.20, 0.34.
MALDI-TOF (2,6-DHAP-citrate): [M ϩ H]ϩ calc. 1090.11,
To a solution of 8 (1.145 g, 2.0 mmol) in dry DCM (30 cm3)
N-methylpropargylamine (0.422 cm3, 5.0 mmol) was added.
The reaction was monitored by TLC (CHCl3–acetone, 4 : 1 v/v).
After one week the starting carbonate was consumed. The
mixture was diluted with CHCl3 (50 cm3), washed with water
(100 cm3), 5% citric acid (100 cm3), and water (100 cm3), dried
(Na2SO4), evaporated, and the residue was chromatographed
on silica gel using a stepwise gradient of 0.5 1.0 1.5 3.0%
MeOH in CHCl3–EtOAc (1 : 1) ϩ 0.5% Et3N (v/v/v/v).
Compound 5b (131 mg, 10.2%) was eluted first. Compound 9
(1.058 g, 82.4%) was obtained as a white amorphous solid, Rf
0.64 (EtOAc). ESI MS: [M ϩ Na]ϩ calc. 664.23, found 664.22.
1H-NMR: 11.37 (br s, 1H, H-3), 7.69 (d, 1H, J5,6 = 8.0 Hz, H-6),
7.40–7.27 (m, 5H, ArH ), 7.22 (d, 4H, J = 8.6 Hz, ArH ), 6.89 (d,
4H, J = 8.6 Hz, ArH ), 5.89–5.71 (m, 2H, 2Ј-OH, H-1Ј), 5.42 (d,
1H, J5,6 = 8.0 Hz, H-5), 5.07 (m, 1H, H-3Ј), 4.40–3.95 (m, 4H,
1
found 1092.09. H-NMR: 11.44 (s, 1H, H-3), 9.37 (br s, 1H,
NHTFA), 7.71 (d, 1H, J5,6 = 8.0 Hz, H-6), 7.47–7.19 (m, 10H,
ArH, OCONH ), 6.87 (m, 4H, ArH ), 5.91 (m, 1H, H-1Ј), 5.42
(d, 1H, J5,6 = 8.0 Hz, H-5), 5.31 (m, 1H, H-2Ј), 4.55 (m, 1H,
H-3Ј), 4.12 (m, 1H, H-4Ј), 3.80–3.16 (m, 24H¶, (CH2OCH2)3,
POCH2, CHN, H-5Ј, OCH3), 3.01 (m, 2H, OCONHCH2), 2.74,
2.57 (2m, 2H, CH2CN), 1.74–1.55 (m, 4H, CH2CH2CH2), 1.18–
0.92 (m, 12H, CHCH3). 31P NMR (CD3CN): 149.346, 149.275
(diastereomers).
3Ј-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-[4-trifluoroacetyl-7-(trifluoro-
acetylamino)-4-azaheptan-1-ylaminocarbonyl]uridine
Chromatography: 20 33 66% acetone in CHCl3 ϩ 1% Et3N
(6m).
H-2Ј, H-4Ј, CH2N), 3.70 (s, 6H, OCH3), 3.38–3.15 (m, 3H¶,
᎐
H-5Ј, ᎐CH ), 2.90, 2.86 (2s, 3H, NCH , rotamers).
᎐
3
J. Chem. Soc., Perkin Trans. 1, 2002, 1092–1104
1103

View Article Online
P. D. Cook and M. Manoharan, US Pat., 2000, 6,166,188;
(e) M. Prhavc, E. A. Lesnik, V. Mohan and M. Manoharan,
Tetrahedron Lett., 2001, 42, 8777.
Acknowledgements
This work was supported in part by a grant (to V. A. K.) from
Ribozyme Pharmaceuticals Inc. We thank Donna Williams
and Richard Grenfell for oligonucleotide assemblies and
purification of oligoribonucleotide ON3.
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