Synthesis of locked pyranosyl nucleic acid (LpNA)

Article abstract of DOI:10.1016/j.bmcl.2011.10.005

A new locked pyranosyl nucleoside was synthesized by phenylsulfinyl- assisted chemistry. The novel building block was inserted into oligonucleotides and provides new insight on conformational restricted pyranosyl nucleosides on duplex formation

Full text of DOI:10.1016/j.bmcl.2011.10.005

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7376–7378
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Synthesis of locked pyranosyl nucleic acid (LpNA)
⇑
Niels Bomholt, Per T. Jørgensen, Erik B. Pedersen
Nucleic Acid Center , Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
A new locked pyranosyl nucleoside was synthesized by phenylsulfinyl-assisted chemistry. The novel
building block was inserted into oligonucleotides and provides new insight on conformational restricted
pyranosyl nucleosides on duplex formation
Received 12 September 2011
Revised 3 October 2011
Accepted 4 October 2011
Available online 8 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Carbohydrates
Locked pyranosyl nucleic acid
Nucleic acids
Nucleosides
Ozonolysis
Conformational restriction of nucleotides is currently being
used to control sugar conformation and thereby influence the func-
tion of the modified nucleic acids. The inspiration for synthesizing
a locked pyranose nucleic acid (LpNA, Fig. 1a) came from the work
by the groups of Imanishi1 and Wengel,2 who simultaneously re-
ported the synthesis of locked nucleic acid (LNA, Fig. 1b) in
1998,3,4 which has been shown to hybridize strongly with RNA
and DNA.5,6 Since then, numerous analogues comprising different
linkages and substitution patterns have been synthesized and eval-
uated,7 however, only very few of these have concerned covalently
constrained nucleotides comprising a six-membered sugar-moiety.
The rationale seemed to be that the locking of the pyranose nucleo-
sides is not needed because the pyranose ring is less flexible than
the furanose ring, and as a result thereof its conformation is to
some degree restricted to a chair conformation. However, a com-
pletely different spatial arrangement of its substituents is achieved
if the pyranose ring is locked into the corresponding boat confor-
mation and such nucleosides seem only to have been reported by
the group of Chattopadhyaya with the hydroxymethyl group trans-
posed from the 50 to the 40-position.8,9
(a)
HO
HO
HO
MeO
U
T
O
O
HO
O
O
1
2
LpNA monomers
(b)
HO
(c)
OH
B
O
O
LNA monomer
Figure 1. (a) LpNA monomers 1 and 2. (b) LNA monomer. (c) Overlay of LNA
(indicated with grey carbon atoms) and 30-deoxy-LpNA monomer (indicated with
green carbon atoms).
view of the reaction sequence for the successful synthesis of a 30-
methoxy LpNA (2, Fig. 1a).
We did molecular modeling before starting the synthesis of an
LpNA and we found a surprisingly high degree of similarity of
the spatial arrangement of the substituents in an LpNA with those
in an LNA monomer (Fig. 1c).
Huge amount of experimental knowledge is known about the
chemistry of hexoses and therefore we thought it easy to develop
a convergent synthesis of an LpNA monomer by coupling an appro-
priate carbohydrate with a nucleobase. However, during this pro-
ject we ran into numerous synthetic dead ends which are
reported in the Supplementary data and here we shall give an over-
During the preparation of this paper describing the synthesis of
what we thought was the first example of a pyranose sugar locked
into a boat-type conformation by an O20 to C50 methylene linkage,
a patent from ISIS Pharmaceuticals was disclosed in early 2011
describing the synthesis of the 30-deoxy locked uridine derivative
⇑
Corresponding author. Tel.: +45 65502583; fax: +45 66158760.
E-mail address: ebp@ifk.sdu.dk (E.B. Pedersen).
The Nucleic Acid Center is funded by The Danish National Research Foundation
for studies on nucleic chemical biology.
(1, Fig. 1a).10 Their synthesis of
1 involved a
2,20-anhydro
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.005

N. Bomholt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7376–7378
7377
O
O
HO
AcO
O
O
O
O
O
AcO
TBDMSO
OAc
a-d
e
MeO
OAc
O
O
HO
MeO
4
5
3
Scheme 1. Reagents and conditions: (a) KOH, Bu4NBr, MeI, acetone, 0 °C to rt to reflux, 27 h, 74%; (b) 60% aq AcOH, 60 °C, 2 h, 84%; (c) TBDMS-Cl, imidazole, DMF, rt, 4 h, 69%;
(d) (i) DMSO, (COCl)2, NEt3, DCM, ꢀ78 °C, 2 h, (ii) CH2CHMgBr, THF, rt, 1 h, 73%; (e) (i) IR-120 H+ resin, 1,4-dioxane/H2O (1:1, v/v), 80 °C, 3 days, (ii) Ac2O, pyridine, rt, 3.5 h,
83%. DCM = dichloromethane, TBDMS = t-butyldimethylsilyl.
AcO
AcO
AcO
AcO
Ph
O
O
O
O
OAc
SPh
SPh
O
a
b
MeO
MeO
MeO
OAc
OAc
OH
6
7
5
Scheme 2. Reagents and conditions: (a) PhSH, BF3OEt2, DCM, rt, 3.5 h, 81%; (b) (i) CH3ONa, MeOH, rt, 1 h, IR-120 H+ resin, 1 h, (ii) PhCH(OMe)2, pTsOHꢁH2O, DMF, 70 °C, 3 h,
76%. DCM = dichloromethane, Ts = toluene-4-sulfonyl.
O
SPh
Ph
O
Ph
O
Ph
O
O
O
O
SPh
a
SPh
O
O
O
MeO
MeO
MeO
+
OH
OH
OH
OH
OH
9
8 (65 % after 3 min)
(50 % after 1 h)
7
Scheme 3. Reagents and conditions: (a) (i) O3, DCM, ꢀ78 °C, 3–60 min, then quenched with Me2S, (ii) NaBH4, abs EtOH (8) or MeOH (9), 0 °C to rt, 2 h.
DCM = dichloromethane.
nucleoside derivative in the key step, whereas we were looking for
a convergent strategy for the nucleosides that normally allows all
natural nucleobases to be introduced.2
The synthesis of compound 5 was performed according to liter-
ature procedures in five steps (Scheme 1)11,12 with transformation
of the furanose sugar 4 into the pyranose sugar 5 in the last step as
the key step in achieving a suitable starting material for the pyra-
nose nucleoside 2.
It was possible to couple 5 with silylated thymine, but we failed
to transform the synthesized nucleoside into an LpNA monomer
(Scheme S7). By making the anomeric substituent orthogonal to
other substituents concerning deprotection, it opens up other
routes to an LpNA monomer and we therefore turned to thioglyco-
sides as key intermediates. Fernández and Castillón have shown
that C1-thiophenyl hexopyranose derivatives can be used for the
synthesis of nucleosides using N-bromosuccinimide (NBS) as acti-
vator.13 Consequently, we synthesized the 3-methoxy-1-thiophe-
nylglycoside 7 (Scheme 2) in a similar procedure as reported by
Blériot et al. who used 3-O-benzyl derivative in the synthesis of a
locked glucoside.14
thiophenyl by ozonolysis has not been reported in the literature
(Scheme 3).
Chanteloup and Beau have previously shown that phenylsulfi-
nyl ribofuranosides are highly suitable for nucleoside coupling
reactions.15 Scheme 4 summarizes the eight-step route to the
DMT-protected phosphoramidite 16 starting from compound 9.
The diacetylated sulfinyl derivative 10 was used in a stereose-
lective Vorbrüggen coupling reaction using in situ silylation of thy-
mine by N,O-bistrimethylsilyl acetamide (BSA) before addition of
the acetylated compound 10 and trimethylsilyl trifluoro-methane-
sulfonate (TMSOTf) as a Lewis acid catalyst. The b-nucleoside 11
was isolated in 81% yield after 4 h at room temperature. To our
knowledge, this is the first example of a nucleoside coupling reac-
tion using a phenylsulfinyl-hexopyranoside derivative. The cycliza-
tion reaction was successfully performed on the tosyl derivative 13
using NaH in DMF affording the corresponding locked b-nucleoside
14 in 74% yield. The DMT protected derivative 15 was also con-
verted into the corresponding 5-methylcytosine nucleoside as pre-
viously
performed
using
DMT-protected
nucleosides
(Scheme S13).16
However, when we proceeded according to the procedure of
Blériot et al. with ozonolysis of the vinyl group in compound 7,
we succeeded to synthesize 8 in a low scale synthesis, only
(Scheme 3). It was indeed possible to synthesize the corresponding
locked glycoside from 8 and to use it in a low yield coupling reac-
tion with silylated thymine to give an anomeric mixture of LpNA
monomers (Scheme S11). Alternatively, when we made the nucle-
oside from 8 before locking it into an LpNA monomer, then we also
ran into experimental difficulties (Scheme S12). Therefore we
decided to take advantage of the observation that phenylsulfinyl
glycoside 9 is formed in a large scale synthesis in the ozonolysis
of compound 7. In a large scale synthesis an increased reaction
time was required in order to saturate the solution with ozone.
We presume that the formed ozonide oxidizes the C1-thiophenyl
via an intra- or intermolecular reaction, since direct oxidation of
It was also attempted to synthesize the corresponding 30-deoxy
nucleosides by starting out from the 30-deoxy derivative of 3, but in
the step analogous to formation of the branched glucoside 5 from
4, a mixture of the corresponding furanose derivative and an 1,6-
anhydro pyranose derivative was obtained (Scheme S2).
Although structural resemblance of LpNA to LNA was expected
as mentioned above, incorporation of the thymine monomer once
or twice into a 21mer DNA or RNA resulted for the corresponding
DNA/DNA or DNA/RNA duplex in a 6–7 °C lower melting tempera-
ture per modification (Table 1). Further lowering of melting tem-
perature in mismatch studies (Tables S3 and S4) confirmed the
base-pairing capability of LpNA.
Molecular modeling of the modified B-type duplexes shows that
the axial position of the C30-OMe group cause a distortion of the
backbone. Moreover, the position of the neighbouring nucleobase

7378
N. Bomholt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7376–7378
O
SPh
O
SPh
Ph
O
Ph
O
Ph
O
O
O
O
T
T
O
O
O
a
b
MeO
MeO
MeO
OH
OAc
OAc
OH
OAc
OAc
9
10
11
Ph
O
Ph
O
Ph
O
O
O
O
c
T
T
O
d
O
e
O
11
MeO
MeO
MeO
OH
OH
OH
O
OTs
12
13
14
NC
HO
DMTO
DMTO
O
O
P
T
O
O
T
O
T
HO
MeO
HO
g
h
f
N
MeO
MeO
14
O
O
O
2
15
16
Scheme 4. Reagents and conditions: (a) Ac2O, pyridine, rt, 23 h; 98%; (b) thymine, BSA, TMSOTf, DCE, rt, 4 h, 81%; (c) satd methanolic NH3, rt, 7 h, 89%; (d) TsCl, pyridine,
70 °C, 29 h, 70%; (e) NaH, DMF, rt, 20 h, 74%; (f) H2, Pd/C 10%, EtOAc, rt, 2 h, 100%; (g) DMTCl, pyridine, rt, 14 h, 87%; (h) DIPEA, 2-cyanoethyl N,N-diisopropyl-
chlorophosphordiamidite, DCE, rt, 4 h, 85%. BSA = N,O-bistrimethylsilyl acetamide, TMSOTf = trimethylsilyl trifluoro-methanesulfonate, DCE = 1,2-dicloroethane, Ts = tolu-
ene-4-sulfonyl, DMT = dimethoxytrityl, DIPEA = N,N-diisopropylethylamine, T = thymin-1-yl.
Table 1
Tm (°C) data for matched DNA/DNA and DNA/RNA melting, taken from UV melting curves (k = 260 nm)a
DNAb 30-ACGTGACATACAGACATGGTA ON4
RNAb 30-ACGUGACAUACAGACAUGGUA ON5
Entry
Sequence
Tm (°C)
D
Tm
Ref.
ꢀ6.0
ꢀ14.5
Tm (°C)
D
Tm
Ref.
ꢀ6.5
ꢀ14.5
ON1
ON2
ON3
50-TGCACTGTATGTCTGTACCAT
50-TGCACTGTATPGTCTGTACCAT
50-TGCACTGTATPGTPCTGTACCAT
62.5
56.5
48.0
63.0
56.5
48.5
a
1
l
M of each ON in 10 mM NaH2PO4/Na2HPO4, 100 mM NaCl, 0.1 mM EDTA at pH 7.0.
b
Target for monomer Tp (ON2) is underlined.
is affected resulting in less intrastrand nucleobase overlap at the 4-
end and thereby loss of stabilizing stacking (Fig. S4). Unfortu-
References and notes
p–p
nately, it is difficult to make conclusions about the effect of the C30-
OMe group as melting was not reported for the wild type oligo in
the patent about the corresponding 2-deoxy oligonucleotides.10
In conclusion, we have described the synthesis and incorpora-
tion of a novel six-membered bicyclic nucleic acid (LpNA) into oli-
gonucleotides using standard automated DNA synthesis. The 30-
methoxy-LpNA was synthesized in 2% overall yield over 16 steps
1. Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T.
Tetrahedron Lett. 1997, 38, 8735.
2. Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard,
M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607.
3. Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. Commun. 1998, 455.
4. Obika, S.; Morio, K.; Nanbu, D.; Imanishi, T. Chem. Commun. 1997, 1643.
5. Vester, B.; Wengel, J. Biochemistry 2004, 43, 13233.
6. Singh, S. K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63, 10035.
7. Zhou, C. Z.; Chattopadhyaya, J. Curr. Opin. Drug Discovery Dev. 2009, 12, 876.
8. Zhou, C. Z.; Plashkevych, O.; Liu, Y.; Badgujar, N.; Chattopadhyaya, J.
Heterocycles 2009, 78, 1715.
starting from commercially available diacetone-D-glucose.
9. Li, Q.; Yuan, F.; Zhou, C.; Plashkevych, O.; Chattopadhaya, J. J. Org. Chem. 2010,
75, 6122.
10. Migawa, M. T.; Punit, P. P.; Swayze, E. E.; Ross, B. S.; Song, Q.; Han, M. (ISIS
Pharmaceuticals), WO2011/017521A2, 2011.
11. Das, S. K.; Mallet, J. M.; Esnault, J.; Driguez, P. A.; Duchaussoy, P.; Sizun, P.;
Herault, J. P.; Herbert, J. M.; Petitou, M.; Sinaÿ, P. Chem. Eur. J. 2001, 7, 4821.
12. Yan, S. H.; Klemm, D. Tetrahedron 2002, 58, 10065.
Acknowledgments
We gratefully acknowledge the financial support from The Dan-
ish National Research Foundation.
13. Fernández, R.; Castillón, S. Tetrahedron 1999, 55, 8497.
14. Blériot, Y.; Vadivel, S. K.; Herrera, A. J.; Greig, I. R.; Kirby, A. J.; Sinaÿ, P.
Tetrahedron 2004, 60, 6813.
Supplementary data
15. Chanteloup, L.; Beau, J. M. Tetrahedron Lett. 1992, 33, 5347.
16. Ross, B. S.; Han, M. M.; Ravikumar, V. T. Nucleosides Nucleotides Nucleic Acids
2006, 25, 765.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.005.