2′-O-Carbamate-containing oligonucleotides: Synthesis and properties

Article abstract of DOI:10.1016/S0040-4039(01)01908-6

In order to evaluate the effect of a new 2′-carbohydrate modification on the hybridization properties of oligonucleotides, uridine 2′-O-carbamates were synthesized and incorporated into DNA strands. The key intermediate in the synthesis, a mixed succinimide carbonate 2, was treated with various amines to give 2′-O-carbamates 3. Thermal melting studies of modified oligonucleotides revealed that the presence of the 2′-O-carbamate modification significantly destabilized DNA/RNA duplexes. A molecular-modeling study indicated that unfavorable steric interactions between the hydrogen of the NH group from the carbamate substituent and the anomeric hydrogen of the sugar residue on the same strand of the duplex may be the contributing factor causing destabilization.

Full text of DOI:10.1016/S0040-4039(01)01908-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 8777–8780
2%-O-Carbamate-containing oligonucleotides:
synthesis and properties
Marija Prhavc, Elena A. Lesnik, V. Mohan and Muthiah Manoharan*
Department of Medicinal Chemistry, Isis Pharmaceuticals, 2292 Faraday Ave., Carlsbad, CA 92008, USA
Received 23 August 2001; revised 9 October 2001; accepted 10 October 2001
Abstract—In order to evaluate the effect of a new 2%-carbohydrate modification on the hybridization properties of oligonucleo-
tides, uridine 2%-O-carbamates were synthesized and incorporated into DNA strands. The key intermediate in the synthesis, a
mixed succinimide carbonate 2, was treated with various amines to give 2%-O-carbamates 3. Thermal melting studies of modified
oligonucleotides revealed that the presence of the 2%-O-carbamate modification significantly destabilized DNA/RNA duplexes. A
molecular-modeling study indicated that unfavorable steric interactions between the hydrogen of the NH group from the
carbamate substituent and the anomeric hydrogen of the sugar residue on the same strand of the duplex may be the contributing
factor causing destabilization. © 2001 Elsevier Science Ltd. All rights reserved.
Introducing different functional groups at the 2%-position
of antisense oligonucleotides gives rise to a class of
compounds collectively known as ‘RNA mimetics’.1,2
These modifications usually result in a C3%-endo sugar
conformation leading to favorable binding affinity for the
target RNA, improved nuclease resistance, altered phar-
macokinetics, and increased chemical stability during
oligonucleotide synthesis.1–3 In addition, these modified
oligonucleotides have improved oral absorption proper-
ties relative to phosphorothioate DNA as there is little
possibility of depurination in the acidic pH of the gut.
Some RNA mimics with these desirable properties are
2%-deoxy-2%-fluoro (2%-F),4 2%-O-(2-methoxyethyl) (2%-O-
MOE),5,6 2%-O-(3-aminopropyl) (2%-O-AP)7,8 and 2%-O-[2-
(N,N-dimethylaminooxy)ethyl] (2%-O-DMAOE)9 modi-
fied oligonucleotides (Fig. 1).
stituent; however, no binding affinity data is available for
this class of compounds.12
The 2%-O-carbamate modification (Fig. 2) is a simple,
chemically feasible modification at the 2%-position of the
sugar, which can be achieved via a 2%-O-carbonate
intermediate. Various 2%-O-carbamates can be synthe-
sized from a single precursor by using different amines.
In addition, if a substituent with a free amino terminal
group is used, further 2%-conjugation can be carried out.
Although carbamate-containing backbone linkages13 and
solid supports are known,14 sugar modifications are not.15
R
(2'-F)
F
B
O
O
However, not all 2%-modifications stabilize hybrid duplex
formation. Numerous destabilizing 2%-modifications have
also been reported in the literature.10 In such cases,
enhanced nuclease resistance and lipophilicity, for exam-
ple, may make these modifications useful. Herdewijn and
his group have shown that the incorporation of 2%-acet-
amido-2%-deoxynucleosides (Fig. 1) in RNA and DNA
strands destabilized RNA–RNA, DNA–DNA and mixed
duplexes.11 Polushin introduced various 2%-methoxyox-
alamido functionalities (Fig. 1) on the 2%-amino sub-
O
O
(2'-O-MOE)
O
R
O
(2'-O-AP)
NH3
N
O
O
(2'-O-DMAOE)
O
O
(2'-Acetamido)
N
H
O
(2'-Methoxyoxalamido)
N
H
O
Keywords: carbonates; carbamates; nucleosides; antisense; oligonucle-
otides.
* Corresponding author. E-mail: mmanoharan@isisph.com
Figure 1.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01908-6

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M. Prha6c et al. / Tetrahedron Letters 42 (2001) 8777–8780
(Scheme 2). Compound 6 was used in oligonucleotide
R
B
synthesis in an automated DNA synthesizer at 0.5 M
concentration in MeCN under standard conditions.
Oligonucleotides were cleaved from solid support and
deprotected after an overnight treatment in 30%
aqueous ammonia at 55°C. They were then purified by
RP HPLC, followed by size-exclusion chromatography,
and were analyzed by HPLC, CE and ESMS. The
properties of the different oligomers synthesized, all of
which had phosphodiester backbones, are summarized
in Table 1.
O
O
Me
NMe2
NH2
O
O
O
NHR
Figure 2.
The synthesis of uridinyl-2%-carbamate is summarized in
Scheme 1. 5%-O-Dimethoxytrityl-3%-O-tert-butyldime-
thylsilyluridine (1) was transformed into the 2%-O-car-
bonate 2 with di-(N-succinimidyl)carbonate (DSC) and
Et3N according to the procedure by Ghosh et al.16,17
The carbonate 2 was further treated with an amine to
give the 2%-O-carbamate 3 in good yield. If the carbon-
ate 2 is treated with TBAF, the cyclic carbonate 4 is
formed. A short treatment of 3 with TBAF in THF at
room temperature afforded the 3%-O-deprotected
nucleoside 5, which was further phosphitylated into the
Helix to coil transition melting temperatures were mea-
sured for duplexes formed with a complementary RNA
oligonucleotide (Table 1). In all cases, a substantial
decrease in Tm was observed in comparison to
unmodified oligonucleotides indicating that carbamate
modifications lead to a destabilization of the duplex.
Possible reasons for the loss of affinity are lack of
rotational freedom of the carbamate substituent due to
C-sp2 hybridization, van der Waals repulsion (Fig. 3)
and steric bulk due to the carbamate group.
phosphoramidite
6
using conventional procedures
O
O
O
N
NH
NH
NH
N
O
N
O
O
DMTO
DMTO
i
ii
DMTO
O
O
O
53-67%
90%
TBDMSO
O
O
TBDMSO
OH
TBDMSO
O
O
O
NHR
1
O
N
3
2
O
TBAF/THF
0 oC, 1 h
3
a
R
Me
O
N
N
NH
b
c
O
N
O
DMTO
O
O
O
O
O
4
Scheme 1. (i) Disuccinimidyl carbonate/Et3N/MeCN/rt/5 h; (ii) RNH2/Et3N/MeCN/rt/1 h.
O
O
NH
NH
N
O
N
O
O
i
DMTO
DMTO
TBAF, THF
rt, 0.5 h
O
O
3a,b
75-81%
iPr
OH
O
O
N
O
O
iPr
P
86%
NHR
O
NHR
5a,b
NC
6a,b
Scheme 2. (i) 2-Cyanoethyl N,N-bisdiisopropylaminophosphorodiamidite/1H-tetrazole/iPr2NH/CH2Cl2/rt/16 h.

M. Prha6c et al. / Tetrahedron Letters 42 (2001) 8777–8780
Table 1. Properties of 2%-O-carbamate-containing oligonucleotides
8779
Oligo
Sequence
(5%“3%)
Modif. Ua
6
Retention time ESMS calcd
(min)b
ESMS found
Tm (°C)
Tm (°C)
DTm/mod. (°C)
(unmodif.)c,d
I
CTC GTA
Ua
Ua
Ua
Ua
Ub
18.68
17.47
17.74
18.67
18.66
5437.3
5069.9
5629.3
5466.0
5494.3
5435.7
58.3
64.0
60.6
60.7
50.2
64.0
−5.76
−2.34
−3.17
CCU
CGG TCC
CC AGG
6 TTC
II
U
6
5070.4
51.3
U
6
GU
6
6
CCG
C
CAU
III
IV
V
CTC GTA
CUU UUC
CGG TCC
GCG UUU
UUU UUU
5629.4
48.0
5465.9
NCe
U
6
GC G
CTC GTA
CCU TTC
CGG TCC
5495.3
58.3
−5.68
6
a Ua: 2%-O-(N-methylcarbamoyl)uridine, Ub: 2%-O-[N-(2-N,N-dimethylaminoethyl)carbamoyl]uridine.
b Column: C18 Waters Delta Pak (15 mm; 3.8×300 mm); buffer A: 50 mM triethylammonium acetate pH 7.0; buffer B: MeCN; a linear gradient
from 5 to 60% B in 60 min.
c Melting temperature of the parent unmodified oligonucleotide (U
6 =T).
d Ref. 10.
e Non-cooperative transition was observed.
Figure 3. Energy minimized molecular-modeling diagram of 2%-O-(N-methylcarbamte) containing uridine (right) base paired with
adenosine in the A form of an RNA/DNA duplex.
lations and structural studies will be required to dissect
the roles of the various energetic contributions (hydra-
tion, dipole moment of the 2%-substituent, etc.) that give
rise to the observed duplex instability.
Molecular-modeling calculations were carried out to
understand the molecular origins of the instability of an
RNA:DNA duplex containing an RNA r(GCGAGCG)
strand and DNA d(CGCU6 CGC) strand with and with-
out the 2%-O-(N-methylcarbamate) (Ua) modification.
The 7-mer RNA–DNA duplex was constructed in the
A-form geometry and the energy was minimized
(AMBER force filed, MSI software package) for com-
parison purposes with an RNA:DNA duplex contain-
ing the 2%-modified nucleotide. The modified nucleotide
was incorporated at the center of the DNA strand and
energy minimization was performed. Fig. 3 shows the
base pair between the uridine with the carbamate mod-
ification and the adenine on the RNA strand. The
model suggests that there is a steric clash between the
anomeric hydrogen and the hydrogen of the NH group
It is interesting to note that while a single Ua modifica-
tion in oligomer I leads to a destabilization of 5.8°C,
multiple substitutions (as in the oligomers II and III)
lead to less destabilization per modification. While we
do not have any structural data to explain these differ-
ences, one possible explanation is that the introduction
of ‘kinks’ at the junction point of the modification
generates maximum steric perturbation with a single
modification, and the helix accommodates such pertur-
bations better with multiple modifications leading to
less drastic changes. It is worth pointing out that most
of the ‘destabilizing’ 2%-modifications (such as 2%-sulfur-
,
from the carbamate substituent (2.3 A). Further calcu-

8780
M. Prha6c et al. / Tetrahedron Letters 42 (2001) 8777–8780
or 2%-a-alkyl-linked modifications) studied so far exhibit
Biol. 1999, 6, 535–539.
a similar trend.10
6. Martin, P. Helv. Chim. Acta 1995, 78, 486–504.
7. Griffey, R. H.; Monia, B. P.; Cummins, L. L.; Freier, S.;
Greig, M. J.; Guinosso, C. J.; Lesnik, E.; Manalili, S. M.;
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Symons, A. M.; Cook, P. D.; Manoharan, M.; Egli, M.
Proc. Natl. Acad. Sci. USA 1999, 96, 14240–14245.
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15. The present work has appeared in: (a) Prhavc, M.;
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1999, 64, 6468–6472.
In summary, a simple chemical approach for the syn-
thesis of 2%-O-carbamate-containing nucleosides and
oligonucleotides is presented. The modified oligonucleo-
tides destabilize the duplexes formed with the target
RNA as compared to unmodified controls. Neverthe-
less, the modification is worth evaluating for the follow-
ing reasons. The therapeutic properties of nucleosides
with 2%-O-carbamates are not known yet. In addition,
the 2%-O-carbamate chemistry may be used (with an
intermediate such as compound 3c) for further conjuga-
tion of ligands to oligonucleotides, either for enhance-
ment of antisense properties or for use as diagnostic
agents. Finally, placement of a 2%-O-carbamate moiety
at the 5%- or 3%-terminus may not cause extensive desta-
bilization due to the ‘fraying’ of the duplex, but might
enhance nuclease resistance.
References
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Bristol, J. A., Ed. Second-generation antisense oligonu-
cleotides: 2%-modifications; Academic Press: San Diego,
1998; Vol. 33, pp. 313–325.
2. Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117–
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3. Crooke, S. T. Handb. Exp. Pharmacol. 1998, 131, 1–50.
4. Kawasaki, A. M.; Casper, M. D.; Prakash, T. P.; Man-
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5. Teplova, M.; Minasov, G.; Tereshko, V.; Inamati, G. B.;
Cook, P. D.; Manoharan, M.; Egli, M. Nature Struct.