Solid-phase synthesis of a 5′-terminal TMG-capped trinucleotide block of U1 snRNA

Article abstract of DOI:10.1016/S0040-4039(01)01941-4

A 5′-terminal 2,2,7-trimethylguanosine-capped trinucleotide block (m₃^2,2,7G^5′pppAmpUmpA) was successfully synthesized on a highly cross-linked polystyrene resin by a new method for introduction of the first nucleoside onto the resin and a newly developed pyrophosphorylating agent having a benzotriazolyloxy substitutent as the leaving group.

Full text of DOI:10.1016/S0040-4039(01)01941-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 8853–8856
Solid-phase synthesis of a 5%-terminal TMG-capped trinucleotide
block of U1 snRNA
Michinori Kadokura,a Takeshi Wada,a Kohji Seio,a Tomohisa Moriguchi,a Jochen Huber,b
Reinhard Lu¨hrmannb and Mitsuo Sekinea,*
aDepartment of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan
bInstitut fu¨r Molekularbiologie und Tumorforschung, Philipps-Universita¨t Marburg, Emil-Mannkopff-Straße 2,
D-3550 Marburg, Germany
Received 31 May 2001; revised 10 October 2001; accepted 12 October 2001
Abstract—A 5%-terminal 2,2,7-trimethylguanosine-capped trinucleotide block (m32,2,7G5%pppAmpUmpA) was successfully synthe-
sized on a highly cross-linked polystyrene resin by a new method for introduction of the first nucleoside onto the resin and a newly
developed pyrophosphorylating agent having a benzotriazolyloxy substitutent as the leaving group. © 2001 Published by Elsevier
Science Ltd.
In the current oligonucleotide synthesis, RNA fragments
have been successfully obtained by extensive application
of the phosphoramidite approach to the solid-phase
synthesis.1 Now, a variety of base-modified RNA frag-
ments have also been synthesized by the solid-phase
strategy.2 However, this straightforward approach has
never been applied to the synthesis of extremely base-
labile 7-methylguanosine (MMG)-capped mRNAs and
2,2,7-trimethylguanosine (TMG)-capped U snRNAs.3
This is a major problem to be solved in the organic
synthesis of nucleic acids. Here, we report the first
successfulsolid-phasesynthesisofm32,2,7G5%pppAmUmA4
1, i.e. the 5%-terminal site of U1 snRNA (Fig. 1)5 by
exploring a new method for pyrophosphorylation.
group but becomes acid-labile once the R% group is
eliminated.7 The nature of the inherent dramatic change
in this linker allows us to carry out successively: (1)
elongation of the RNA chain; (2) 5%-terminal phosphoryl-
ation; (3) deprotection of all base-labile protecting
groups; (4) 5%-terminal pyrophosphorylation; (5) addition
of a cap structure to the terminal site of the pyrophos-
phate group; (6) release of the synthetic RNA oligomer
derivative by the action of acids, and (7) dephosphoryla-
tion of the 3%-terminal phosphate group. Our strategy for
the synthesis of 1 on a highly cross-linked polystyrene
resin is outlined, as shown in Schemes 1 and 2.
Am
C
G
C
U
U
To realize the solid-phase synthesis of 1, we considered
that a TMG-capping reaction should be carried out at
a later stage than the deprotection of all base-labile
protecting groups, since the cap structure is extremely
fragile under basic conditions.6 As a possible choice to
allow the release of capped RNAs from the solid support
without damage of the TMG-cap structure, we used the
following phosphoramidate linker between the 3%-termi-
nal phosphate of the RNA chain and the amino group
on solid supports. Previously, Letsinger reported that the
PꢀN bond of ROP(O)(OR%)(NHR¦) is sufficiently stable
under conditions prescribed for removal of the DMTr
U
C
CH3
N+
O
N
A
C
C
C
U
U
G
G
A
G
HN
N
CH3
N
O
U
U
C
G
C
U
CH3
G
G
A
G
C
G
G
G
A
C
O
U1 snRNA
HO
OH
G
A
C
m3G (TMG)
C
C
G
C
G
U
C
C
U
G
U
G
C
A
G
G
C
G
U
A C C A
G C
A
C
U
A
A
G G
U
U U U C C
U
C
U
CG A UUUCCC
G C
A
C
U
G
A
C
U
U
C
C
C
C
G
G G
G
A
U
A
AAAG G G
U
C
U
U
A
U
A
G
G
G
G
G
G
G
A
C
G G
G
C Ψ Ψ AC C U
AU U U G U G G
G
C A U A
U A
U
G
U
m3G pppAmUmA
1
Keywords: TMG-cap; solid-phase synthesis of RNA; BDCP; U1
snRNA.
* Corresponding author. Tel.: +81-45-924-5706; fax: +81-45-924-5772;
e-mail: msekine@bio.titech.ac.jp
Figure 1. The secondary structure of U1 snRNA with a
2,2,7-trimethylguanosine cap.
0040-4039/01/$ - see front matter © 2001 Published by Elsevier Science Ltd.
PII: S0040-4039(01)01941-4

8854
M. Kadokura et al. / Tetrahedron Letters 42 (2001) 8853–8856
Bz
N
HN
N
Br
Cl
N
N
ABz
ABz
Br
O
P
ABz
O
DMTrO
DMTrO
H
DMTrO
Cl
O
O
DMTrO
Cl
Br
O
O
c, d, e
a
O
OTBDMS
BDCP
O
P
OTBDMS
O
O
P
OTBDMS
CN
OTBDMS
CN
O
P
O P O
CN
(iPr2N)2
b
HN
O
O
CN
5
3
4
2
Scheme 1. Synthesis of an adenosine-loaded resin by use of BDCP. (a) 1 equiv. of 1H-tetrazole, H2O–pyridine (1:20, v/v), rt, 30
min; (b) 3 equiv. of BDCP (30 mmol), pyridine, rt, 5 min; (c) compound 4 (30 mmol), highly cross-linked aminopropyl-polystyrene
resin (1 mmol, 35 mmol/g of NH2 group), rt, 5 min; (d) 17 mM of I2, 50 mM pyridine, H2O–THF (9:91, v/v), 15 s×3; (e)
Ac2O–pyridine (1:9, v/v), rt, 6 h.
Abz
U
Abz
Abz
Abz
U
OTBDMS
O
OCH3
O
OCH3
O
OCH3
OCH3
O
OTBDMS
chain
O
P
O-
elongation
O
P
O
RO
d
O P
O
O
NH
O P
O
P
RO
O
O
O
5
O
P
O-
O
O
P
O-
NH
a
O-
OCE
OCE
OCE
10: R = H
12: R =
6: R = DMTr
7: R = H
O
e
11
O
b
O P
S
O
O
O
DMTrO
O-
c
S
P
O
9: R = DMTrO
O
OCE
U
A
A
A
U
A
m3G
OCH3 OCH3
OTBDMS
O
O P NH
O-
O
OCH3
O
P
OCH3
O
OTBDMS
O
HO
HO
O
P
O
f
h
O
P
O
P
O-
O
O P
O
RO P O
O-
O
O
O
O P
O
O
O
O
O
P O
O P
O-
O
O
P OH
O-
O-
O-
O-
O-
O-
O-
16
13: R = H
i
g
14
m3G
15: R =
HO
HO
O
m32,2,7G5'pppAmpUmpAp
O P
O-
17
j
CH3
O
O
N
OCH2CH2CN
NH2
N
N
O
N
CH3
N
+
N
O
8
O
P
+
NH
S
DMTrO
DMTrO
O
P O
O-
HN
H3C N
CH3
N(iPr2)2
N
N CH3
CH3
O
N
N
O
O
O
N
O
B
N
N
O
N
O P O P O P O
O- O- O-
O
OH HO
O
NH
O
O
O
P
O-
O
O
OCH3
O
N
O
N
N
NH2
N
S
Ph
O P O
O-
O
N
N
O
14
NO2
O
OCH3
N
F3C
O P
O-
O
O
1: m32,2,7G5'pppAmpUmpA
11
HO OH
Scheme 2. Solid-phase synthesis of m32,2,7G5%pppAmpUmpA. (a) (1) Detritylation: 2.5% dichloroacetic acid in CH2Cl2, 15 s×3, (2)
washing: CH2Cl2, CH3CN, (3) condensation: 0.05 M amidite, 0.5 M 1H-tetrazole, CH3CN, 10 min, (4) washing: pyridine, (5)
capping: 10% Ac2O, 0.1 M DMAP, pyridine, 2 min, (6) washing: pyridine, CH2Cl2, (7) oxidation: 17 mM I2, 50 mM pyridine,
H2O–THF (9:91, v/v); (b) detritylation: 2.5% dichloroacetic acid in CH2Cl2; (c) compound 8, 1H-tetrazole, CH3CN, 5 min; (d)
(1) 10% DBU/pyridine–BSA (1:1, v/v), rt, 10 min, (2) Et3N–MeOH (1:9, v/v); (e) 0.1 M of 11, pyridine, 2 h; (f) conc. NH3–EtOH
(3:1, v/v), 55°C, 4 h; (g) 0.1 M of 14, pyridine, 24 h; (h) 80% AcOH, rt, 24 h; (i) HCl (pH 2.0), rt, 12 h; (j) calf intestinal alkaline
phosphatase, Tris–HCl buffer (pH 7.0), 10 mM MgCl2, 37°C, 1 h.

M. Kadokura et al. / Tetrahedron Letters 42 (2001) 8853–8856
8855
First, we encountered much difficulty in synthesizing an
adenosine-linked polymer 5 by the reaction of an
adenosine 3%-phosphoramidite derivative 2 with a highly
cross-linked aminomethylpolystyrene support8 (35
mmol/g) in the presence of 1H-tetrazole. The loading
amount of the adenosine unit was only 7 mmol/g, unlike
that of the deoxy counterpart.7 This low efficiency
might be due to the steric hindrance arising from the
2%-O-TBDMS group and neutralization of the amino
group with the activator.
Therefore, we developed a new method for the synthe-
sis of 5. Reaction of the H-phosphonate derivative 3
derived
from
2
with
tris(2,4,6-tribromophen-
oxy)dichlorophosphorane (BDCP)9,10 gave a highly
reactive chlorophosphite intermediate 4, which, in turn,
was allowed to react with the same resin to give 5 with
a loading amount of 31.5 mmol/g (Scheme 1).
Figure 2. Anion-exchange HPLC profile of the mixture
obtained by acid treatment of 17.
ond phosphorylation and capping steps should be
improved in the future.
A 5%-phosphosphorylated trimer block 9 was synthe-
sized with the average coupling yield of 99% by the
standard phosphoramidite approach11 using the 2%-O-
methyluridine and N-benzoyl-2%-O-methyladenosine
phosphoramidite units and the phosphalink agent 812
via the intermediates 6–7, as shown in Scheme 2. All
the cyanoethyl and sulfonylethyl groups were promptly
and simultaneously deprotected by using DBU–
bis(trimethylsilyl)acetamide (BSA)13 to give the 5%-ter-
minal free product 10. Pyrophosphorylation of 10 with
a new reagent 1114 in pyridine gave the product 12 in
80% yield. Removal of the remaining N-benzoyl groups
from 12 was successively performed by treatment with
ammonia–EtOH15 to afford the product 13. For
triphosphate bond formation, coupling reaction of 13
with the boranylated 2,2,7-trimethylguanosine 5%-phos-
phorimidazolide derivative 14,16 which was synthesized
to improve the solubility via a one-pot reaction from
N,N-dimethylguanosine 5%-phosphate, gave the capped
product 15.
Acknowledgements
This work was supported by a Grant from ‘Research
for the Future’ Program of the Japan Society for the
Promotion of Science (JSPS-RFTF97I00301) and a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology,
Japan.
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