Benzimidazolium triflate as an efficient promoter for nucleotide synthesis via the phosphoramidite method

Full text of DOI:10.1021/jo961569e

7996
J . Org. Chem. 1996, 61, 7996-7997
Sch em e 1
Ben zim id a zoliu m Tr ifla te a s a n Efficien t
P r om oter for Nu cleotid e Syn th esis via th e
P h osp h or a m id ite Meth od
Yoshihiro Hayakawa,*,† Masanori Kataoka,† and
Ryoji Noyori*,‡
Laboratory of Bioorganic Chemistry, Graduate School of
Human Informatics, and Department of Chemistry and
Molecular Chirality Research Unit, Nagoya University,
Chikusa, Nagoya 464-01, J apan
Received August 13, 1996
slight excess of the phosphoramidite and the promoter
(1.2 equiv each) to the nucleoside (1 equiv) was employed
The condensation of a nucleoside phosphoramidite
and a nucleoside is an essential step in oligonucleotide
synthesis.1 This reaction usually requires an azole pro-
moter such as 1H-tetrazole,2 5-(p-nitrophenyl)-1H-tetra-
zole (NPT),3 and 5-(ethylthio)-1H-tetrazole.4 None of
these reagents, however, is sufficiently effective for nu-
cleoside phosphoramidites such as arylated deoxyribo-
nucleoside N,N-diisopropylphosphoramidites or phospho-
romorpholidites and sterically crowded ribonucleoside
N,N-diisopropylphosphoramidites. The lack of reactivity
of these amidites prevents the preparation of some
oligonucleotides with modified backbones that are at-
tractive as antisense molecules.5 Thus, the invention of
new reagents with a potent promotion ability is highly
desirable. We disclose here that benzimidazolium triflate
(1)6 perfectly meets such a requirement (Scheme 1).
The reagent 1, mp 188-190 °C, was quantitatively ob-
tained by mixing benzimidazole and trifluoromethane-
sulfonic acid in equimolar amounts in dichloromethane
at room temperature. This reagent can be stored at am-
bient temperature under atmosphere without decomposi-
tion. This salt has good solubility in acetonitrile, ca. 0.4
mol/L, which is comparable to that of 1H-tetrazole (ca.
0.5 mol/L) and higher than that of NPT (ca. 0.1 mol/L).3
The triflate 1 is an extremely reactive promoter,
generally accomplishing the condensation of a nucleoside
phosphoramidite and a nucleoside within 1 min at 25 °C,
even with normally weakly reactive amidites such as
arylated deoxyribonucleoside 3′-phosphoromorpholidites
or sterically crowded ribonucleoside N,N-diisopropylphos-
phoramidites. Table 1 lists several examples of the
preparation of phosphates via the condensation of the
amidites, 2-6 and 10-12, and the nucleosides, 13-15,
followed by tert-butyl hydroperoxide (TBHP) oxidation.7
The high reactivity of 1 allowed rapid condensation of
phosphoramidites with an electron-withdrawing sub-
stituent such as o-chlorophenyl.8,9 The sterically crowded
ribonucleoside phosphoramidites could also be used. A
† Laboratory of Bioorganic Chemistry, Graduate School of Human
Informatics.
to produce a reasonable rate of reaction.10 The benzimi-
dazolium reagent activated the phosphoramidite in a
chemoselective manner without noticeable deterioration
to effect the reaction of the substrates having an (allyl-
oxy)carbonyl,11 tert-butyldimethylsilyl, or even mono-
‡ Department of Chemistry and Molecular Chirality Research Unit.
(1) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123-6194
and references cited therein.
(2) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22,
1859-1862.
(3) (a) Froehler, B. C.; Matteucci, M. D. Tetrahedron Lett. 1983,
24, 3171-3174. (b) Pon, R. T. Tetrahedron Lett. 1987, 28, 3643- 3646.
(4) Vinayak, R.; Colonna, F.; Tsou, D.; Mullah, B.; Andrus, A.;
Sproat, B. Nucleic Acids Symp. Ser. 1994, 31, 165-166.
(5) (a) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543-584. (b)
Milligane, J . F., Matteucci, M. D.; Martin, J . C. J . Med. Chem. 1993,
36, 1923-1937. (c) Varma, R. S. Synlett 1993, 621-637. (d) Stec, W.
J .; Wilk, A. Angew. Chem., Int. Ed. Engl. 1994, 33, 709-722.
(6) Pyridinium tetrafluoroborates and N-methylimidazolium trif-
luoroacetate are used as the activator for the weakly reactive amidites
including the phosphorothioamidite, but these reagents cause consider-
able cleavage of the dimethoxytrityl protector. See: Brill, W. K.-D.;
Nielsen, J .; Caruthers, M. H. J . Am. Chem. Soc. 1991, 113, 3972-
3980.
(7) Hayakawa, Y.; Uchiyama, M.; Noyori, R. Tetrahedron Lett. 1986,
27, 4191-4194.
(8) Use of benzimidazolium mesylate, mp 214-215 °C, less soluble
in acetonitrile (<0.1 mol/L), usually completes the reaction of o-
chlorophenyl phosphoramidites within 45 min. Benzimidazolium to-
sylate, mp 222-223 °C, is scarcely soluble in acetonitrile.
(9) (a) Dahl, B. H.; Nielsen, J .; Dahl, O. Nucleic Acids Res. 1987,
15, 1729-1743. (b) Schwarz, M. W.; Pfleiderer, W. Tetrahedron Lett.
1984, 25, 5513-5516.
(10) In usual cases, the reaction requires 2-4 equiv of a promoter
for gaining an acceptable reaction rate. See: Stec, W. J .; Zon, G.
Tetrahedron Lett. 1984, 25, 5279-5282.
S0022-3263(96)01569-1 CCC: $12.00 © 1996 American Chemical Society

Communications
Ta ble 1. Syn th esis of Din u cleosid e P h osp h a tesa
product yield,b,c
amidite nucleoside product reagent 1 NPT 1H-tetrazole
J . Org. Chem., Vol. 61, No. 23, 1996 7997
%
2
3
4
5
6
10
11
12
13
13
13
13
13
14
15
15
16
17
18
19
20
21
22
23
97
98
97
98
99
99
99
98
71 (45)
72 (45)
68 (43)
72 (47)
98 (85)
99 (88)
88 (74)
87 (71)
29 (13)
32 (16)
26 (10)
30 (12)
69 (44)
75 (49)
49 (32)
51 (29)
a
Condensation was carried out using amidite, nucleoside, and
promoter in a 1.2:1:1.2 molar ratio in a promoter-saturated (1, ca.
0.4 M; NPT, ca. 0.1 M; 1H-tetrazole, ca. 0.5 M) acetonitrile solution
at 25 °C. The resulting phosphite was directly oxidized by tert-
butyl hydroperoxide (in acetonitrile-toluene, 25 °C, 5 min) to the
b
phosphate. Isolated yield obtained via the phosphitylation with
1, NPT, and 1H-tetrazole for 1, 30, and 60 min, respectively, unless
otherwise stated. c Yields in parentheses were obtained via 1-min
condensation.
methoxy- or dimethoxytrityl protector. No depurination
occurred in the reaction of the deoxyadenosine and
deoxyguanosine derivatives.
The following is a typical procedure for the preparation
of dinucleoside phosphates. The salt 1 (0.644 g, 2.40
mmol), the phosphoramidite 5 (1.89 g, 2.40 mmol), and
the nucleoside 13 (0.713 g, 2.00 mmol) were dissolved in
dry acetonitrile (6.0 mL) and the resulting solution was
stirred at room temperature for 1 min. To this was added
a 1.0 M toluene solution of TBHP (4.00 mL, 4.00 mmol),
and stirring was continued for 5 min. The reaction
mixture was diluted with ethyl acetate and washed with
brine. The organic layer was concentrated to give an oil,
which was chromatographed on silica gel (40 g) with a
1:5 mixture of hexane and ethyl acetate to afford 19 (2.10
g, 98% yield) as an amorphous solid.
When 5 and 13 were combined with a conventional
tetrazole reagent, NPT or 1H-tetrazole, with similar
stoichiometry and maximum promoter concentration, the
reaction proceeded much slower. In the reaction using
NPT, for example, the complete consumption of the
amidite 5 needed ca. 60 min. The reaction with 1H-
tetrazole did not finish even after 180 min. In these
cases, the highest yields of 19 after TBHP oxidation were
72% after 30 min (NPT) and 30% after 60 min (1H-
tetrazole), despite the remaining unreacted amidite; the
longer reaction caused decomposition of the product,
which decreased the yield.
1
zole showing an H singlet at δ 8.28 ppm due to H-2. The
reagent 1 satisfies the crucial requirements for an
efficient promoter. It has sufficient acidity13 to activate
the phosphoramidite, whereas benzimidazole, the con-
jugate base, possesses a high nucleophilicity displacing
the dialkylamine from the phosphorus atom, giving the
benzimidazolidite intermediate. Neutral benzimidazole
is too weak an acid13 to be an activator of the amidite.
Imidazolium triflate is less acidic13 than 1, resulting in
slower condensation. For example, the complete forma-
tion of 25 (a 31P singlet at δ 127.1 ppm) from 10 needed
5 min. The reaction using NPT or NPT together with
benzimidazole was much slower, while a mixture of triflic
acid and NPT caused various side reactions that gave
none of the desired product. No reaction took place by
the use of a 1H-tetrazole-benzimidazole mixture.
The benzimidazolium reagent 1 overcomes several
drawbacks of the currently employed tetrazole promoters
including explosiveness and harmfulness to health.14 We
highly recommend the use of 1 based on its favorable
properties.
Notably, the new promoter 1 can be used for the solid-
phase synthesis of oligodeoxyribonucleotides. For ex-
ample, d(ACGTACGTAT) was prepared in 92% overall
yield (99.1% average coupling yield) according to the
reported procedures11b using 7-10 as building blocks.
Ack n ow led gm en t. This work was partially sup-
ported by a Grant-in-Aid for Scientific Research (No.
08454200) (Y.H.) and a Grant-in-Aid for J SPS Fellows
(No. 80003104) (M.K.) from the Ministry of Education,
Science, Sports and Culture, J apan, and the Takeda
Science Foundation (Y.H.).
NMR studies revealed that the condensation of Scheme
1 proceeds via phosphorobenzimidazolidite intermediates.
The reaction of equimolar amounts of the phosphoramid-
ite 10 (diastereomers; 31P singlets: δ 147.3 and 147.6
ppm) and 1 in the absence of any nucleoside in CD3CN
Su p p or tin g In for m a tion Ava ila ble: Characterization
data for all new compounds (33 pages).
J O961569E
1
formed 24 within 1 min, with an H singlet due to theH-2
of the imidazole ring at δ 8.31 ppm and two 31P singlets
at δ 128.9 and 130.2 ppm due to the two diastereomers
(dinucleoside phosphorotetrazolidites: δ ∼127 ppm12).
Quantitative formation of diisopropylammonium triflate
(11) (a) Hayakawa, Y.; Kato, H.; Uchiyama, M.; Noyori, R. J . Org.
Chem. 1986, 51, 2400-2402. (b) Hayakawa, Y.; Wakabayashi, S.; Kato,
H.; Noyori, R. J . Am. Chem. Soc. 1990, 112, 1691-1696.
(12) (a) Barone, A. D.; Tang, J . Y.; Caruthers, M. H. Nucleic Acids
Res. 1984, 12, 4051-4061. (b) Berner, S.; Mu¨hlegger, K.; Seliger, H.
Nucleic Acids Res. 1989, 17, 853-864.
(13) The pKa values in aqueous solvents are as follows: NPT, 3.7
(1:1 ethanol-H2O); benzimidazolium triflate, 4.5 (1:1 ethanol-H2O);
1H-tetrazole, 4.8 (H2O); imidazolium triflate, 6.9 (H2O); benzimidazole,
13 (H2O).
1
was proved by the H signals at δ 3.54 (heptet) and at δ
1.36 ppm (doublet) due to (CH3)2CH and (CH3)2CH,
respectively. Addition of 1 equiv of the nucleoside 14 to
this mixture instantaneously consumed 24 to give the
dinucleoside phosphite 26, displaying two 31P singlets at
δ 139.9 and 140.5 ppm (diastereomers) and benzimida-
(14) Sigma-Aldrich Library of Chemical Safety 2; Sigma-Aldrich:
Milwaukee, 1990; p 3313D (Sigma-Aldrich Material Safety Data
Sheets; Product No. 33644-0).