Synthesis of triazole-linked analogues of RNA (TLRNA)

Article abstract of DOI:10.1246/cl.2012.403

A triazole-linked analogue of RNA (TLRNA) has been developed. Monomeric ribonucleoside analogues bearing natural nucleobases were prepared from D-xylose to afford the elongating units in gram quantities. The puckering of the monomeric analogues was similar to that of natural ribonucleosides and preferred the North-type conformation. An efficient elongation reaction via the copper-catalyzed Huisgen cycloaddition on a solid support was used for the synthesis of trinucleotides.

Full text of DOI:10.1246/cl.2012.403

doi:10.1246/cl.2012.403
Published on the web March 24, 2012
403
Synthesis of Triazole-linked Analogues of RNA (TLRNA)
Tomoko Fujino, Kenta Endo, Naomi Yamazaki, and Hiroyuki Isobe*
Department of Chemistry, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578
(Received January 12, 2012; CL-120030; E-mail: isobe@m.tohoku.ac.jp)
A triazole-linked analogue of RNA (TLRNA) has been
developed. Monomeric ribonucleoside analogues bearing natural
nucleobases were prepared from D-xylose to afford the
elongating units in gram quantities. The puckering of the
monomeric analogues was similar to that of natural ribonu-
cleosides and preferred the North-type conformation. An
efficient elongation reaction via the copper-catalyzed Huisgen
cycloaddition on a solid support was used for the synthesis of
trinucleotides.
reaction via solid-phase synthesis was demonstrated for the
preparation of trinucleotides.
The synthesis of ribonucleoside analogues turned out to be
more robust than that of deoxyribonucleoside analogues. Unlike
the deoxyribonucleoside analogues, which required both rela-
tively expensive deoxythymidine for the starting material and
nonselective transglycosylation for the nucleobase replace-
ment,4,6,7 the ribonucleoside analogues were readily accessible
from inexpensive D-xylose8 and required one common glyco-
sydic donor for the introduction of various nucleobases. Oxetane
1 was obtained through a 4-step synthetic procedure from D-
xylose9 and was converted into glycosidic donor 2 through a
synthetic route similar to that for deoxyribonucleoside analogues
(Scheme 1).4,10 The subsequent glycosylation of the nucleobases
proceeded in a stereoselective manner as the result of the
anchimeric assistance of the acetoxy group at the pseudo-2¤-
position. The pyrimidine analogues 3U and 3C were obtained
in good yield (92% and 90%) using a TMSOTf-promoted
glycosylation reaction of silylated nucleobases.6,11 Although the
silylation of the guanine derivative required an alternate
reagent,12,13 the glycosylation proceeded after silylation with
N,O-bis(trimethylsilyl)acetamide (BSA) to afford the corre-
sponding analogue 3G in 71% yield. The glycosylation reaction
of N6-octanoyladenine proceeded in a similar manner but gave
the adenosine analogue in poorer yield (42% with TMSCl/
TMSOTf). We therefore switched to the SnCl4-promoted
Albeit subtle at a glance, the presence of the hydroxy group
at the 2¤-position in a furanose ring is essential for the
discrimination of the structures and functions of RNA from
those of DNA (Figure 1).1 For the design of artificial variants
with modified internucleotide linkages, it is therefore important
to decide whether to incorporate the 2¤-hydroxy group. Pres-
ently, ribonucleic analogues have been much less explored than
deoxyribonucleic analogues.2 In nature, both types of mono-
meric nucleosides are readily available, as the removal of the 2¤-
hydroxy groups of ribonucleosides is readily accessible through
enzymatic dehydroxylation.3 In contrast, the reverse transfor-
mation, i.e., the 2¤-hydroxylation of deoxyribonucleosides, is not
synthetically accessible, which forced us to return to the starting
point of the synthesis of ribonucleic analogues after the
development of a triazole-linked analogue of DNA (TLDNA).4
We herein report the synthesis of triazole-linked analogues of
RNA (TLRNA) that possess 2¤-hydroxy groups in the nucleosidic
structures in addition to the six-bond periodicity in the
oligonucleotide form.4,5 The monomeric ribonucleoside ana-
logues, each of which can bear one of the four natural
nucleobases, were prepared from a natural monosaccharide,
D-xylose, through a common glycosylation reaction of the
corresponding nucleobases. The presence of a pseudo-2¤-
hydroxy group was advantageous for the ¢-selective introduc-
tion of nucleobases and also for maintaining the North-type
puckering of the monomeric analogues. An efficient elongation
•
Si(i-Pr)3, t-BuLi, BF3 OEt2,
1)
THF, –78 °C, 90%
2) Tf2O, pyridine, CH2Cl2, 0 °C, 95%
O
O
D-xylose
3) NaN3, DMF, rt, 57%
4) aq. CF3CO2H, rt
5) Ac2O, DMAP, Et3N, CH2Cl2, rt,
98% (2 steps)
O
O
1
Si(i-Pr)3
Si(i-Pr)3
Base-H
glycosylationa
O
O
3U: 92% (Base = U)
3C: 90% (Base = C)
3G: 71% (Base = GPG
3A: 77% (Base = A)
OAc
OAc
Base
OAc
)
N3
N3
2
NH2
O
OCONPh2
N
NH2
N
N
N
N
N
NH
Base =
N
O
N
O
N
N
NHAc
N
U
C
GPG
A
Scheme 1. Synthesis of ribonucleoside analogues. aFor
Base = U and C: 1) TMSCl, NH(SiMe3)2, reflux, 2) TMSOTf,
ClCH2CH2Cl, reflux. For Base = GPG: 1) BSA, ClCH2CH2Cl,
reflux, 2) TMSOTf, ClCH2CH2Cl, reflux. For Base = A: SnCl4,
CH3CN, rt.
Figure 1. Structures of DNA, RNA, TLDNA, and TLRNA.
Chem. Lett. 2012, 41, 403-405
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

404
Table 1. Vicinal coupling constants and puckering population
in DMSO
O
O
N
NH
Analogue
3JH1¤,H2¤/Hz
3JH3¤,H4¤/Hz
%North
O
3U
3C
3G
3A
4.9
4.4
2.6
4.7
6.1
6.3
7.0
6.0
55
59
73
60
O
H
N
O
O
4
•
1) 3U, CuBr SMe2,
t-BuOH/dioxane/H2O
(1:2:3 v/v), 80 °C
2) n-Bu4NF, THF, rt,
96% (2 steps)
Si(i-Pr)3
glycosylation of adenine14 and obtained 3A in 77% yield. All
glycosylation reactions afforded the desired ¢-anomers exclu-
sively.
O
Base
(U, C, G, A)
N N
OH
O
Analysis of the ribonucleoside analogues by 1H NMR
showed that the puckering preference was similar to that of
the natural congeners. One of the important structural dif-
ferences between ribo- and deoxyribonucleosides is found in
their puckering conformations: ribonucleosides prefer a North-
type (3¤-endo) conformation, whereas deoxyribonucleosides
prefer a South-type (2¤-endo) conformation.1,15 In our previous
experience with TLDNA, deoxyribonucleoside analogues, de-
spite bearing nonnatural acetylene/azide linking moieties, also
prefer the South-type conformation in solution.6 When we
O
N
N
NH
•
1) 3, CuBr SMe2,
O
O
t-BuOH/dioxane/H2O
(1:2:3 v/v), 80 °C
O
N
NH
N
N
N
OAc
O
2) aq. NH4OH, 80 °C
N N
N
OH
O
O
N
NH
O
O
O
N
NH
O
H
N
O
O
HO
O
UtUtdT: 99%a
CtUtdT: 92%a
GtUtdT: 95%a
AtUtdT: 95%a
UtdT
1
recorded the H NMR spectra of the ribonucleoside analogues 3
in DMSO at 25 °C, all of them showed smaller vicinal coupling
constants (3J) for H1¤ and H2¤ than for H3¤ and H4¤ (Table 1).
This observation indicated the preference for the North-type
conformation, and the Altona-Sundaralingam analysis of the 3J-
values indeed showed a larger population of furanose rings with
the North-type conformation, with the values of 55% for 3U,
59% for 3C, 73% for 3G, and 60% for 3A.15,16 The results may
indicate the dominant contribution of the 2¤-hydroxy group to
the puckering preference of the furanose ring.1,17
Scheme 2. Solid-phase synthesis of trinucleotide analogues.
aYield after the two-step synthetic operation from UtdT.
instance, RNA interference, that require the enzymatic recog-
nition of the oligonucleotides. The design and synthesis of such
oligonucleotides will be investigated in the near future.
Finally, we examined the elongation reaction with the
ribonucleoside analogues using a deoxythymidine analogue on
polystyrene resins as a 3¤-terminus substrate 4.7 The elonga-
tion reaction, i.e., the copper-catalyzed Huisgen cycloaddition
between acetylene and azide, proceeded with 3U and 4, and the
dinucleotide UtdT was obtained in 96% yield (Scheme 2).18,19
The elongation reaction between ribonucleoside analogues was
then conducted with UtdT and each of the four ribonucleoside
analogues, and the corresponding trinucleotides were obtained in
excellent yields: The fully deprotected trinucleotides after the
hydrolytic cleavage from the resins were obtained in 99% yield
for UtUtdT, 92% yield for CtUtdT, 95% yield for GtUtdT, and
95% yield for AtUtdT. The elongation efficiency was compa-
rable to that of deoxyribonucleic analogues,4,7 a result that
shows that neither the nucleobases nor the 2¤-acetoxy group
severely hampers the click coupling reactions for the extension
of triazole-linked oligonucleotides.
This study was partly supported by KAKENHI (Nos.
20108015 and 23550041), the Takeda Science Foundation and
the Astellas Foundation. N.Y. thanks JSPS for a predoctoral
fellowship.
References and Notes
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In conclusion, we have developed a new ribonucleic
analogue, TLRNA. Starting from an abundantly available
monosaccharide, D-xylose, the synthesis of ribonucleoside
analogues is sufficiently concise to be conducted on a gram
scale. The puckering preference, which is similar to that of
natural ribonucleosides, may help TLRNA mimic the functions
of RNA. Moreover, as TLDNA has been demonstrated to be
a competent enzymatic substrates,20-22 the biologically non-
degradable linkage of TLRNA may provide an intriguing
opportunity to explore the sophisticated function of RNA, for
7
8
T. Fujino, N. Yamazaki, A. Hasome, K. Endo, H. Isobe,
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10 Supporting Information is available electronically on the
Chem. Lett. 2012, 41, 403-405
© 2012 The Chemical Society of Japan www.csj.jp/journals/chem-lett/

405
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19 We used “t” for the descriptors of nonnatural triazole
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Chem. Lett. 2012, 41, 403-405
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/