Use of 2-cyano-1-t-butylethyl or 2-cyano-1-(1,1-diethyl-3-butenyl)ethyl as a phosphorus protecting group in oligonucleotide synthesis via in situ phosphoramidite methods

Article abstract of DOI:10.1246/cl.2000.1134

2-Cyano-1-t-butylethyl or 2-cyano-1-(1,1-diethyl-3-butenyl)ethyl 3′-O-phosphorimidazolidite of 5′-O-protected nucleoside was prepared in situ by the use of 2-cyano-1-t-butylethyl or 2-cyano-1-(1,1-diethyl-3-butenyl)ethyl phosphorobisimidazolidite as a new

Full text of DOI:10.1246/cl.2000.1134

1134
Chemistry Letters 2000
Use of 2-Cyano-1-t-butylethyl or 2-Cyano-1-(1,1-diethyl-3-butenyl)ethyl
as a Phosphorus Protecting Group in Oligonucleotide Synthesis
via in situ Phosphoramidite Methods
Akinori Kitamura, Yoji Horie, and Tadao Yoshida*
Bioscience Research Department, Tsukuba Research Laboratory, Toagosei Co., Ltd., 2 Ohkubo, Tsukuba, Ibaraki 300-2611
(Received June 16, 2000; CL-000592)
2-Cyano-1-t-butylethyl or 2-cyano-1-(1,1-diethyl-3-
butenyl)ethyl 3'-O-phosphorimidazolidite of 5'-O-protected
nucleoside was prepared in situ by the use of 2-cyano-1-t-
butylethyl or 2-cyano-1-(1,1-diethyl-3-butenyl)ethyl phospho-
robisimidazolidite as a new phosphitylating reagent. The phos-
phorimidazolidites were found to be key intermediates for
preparing 5'-O-protected nucleoside 3'-O-monoalkylphospho-
ramidites in situ useful in the solid-phase oligonucleotide syn-
thesis, and for synthesizing conveniently 3'-OH free dinucleo-
side phosphates and phosphorothioates in solution.
The phosphoramidite method is most widely used for the
synthesis of oligonucleotide in solution and on a solid support.1
In this approach, the 2-cyanoethyl group has been proved to be
an useful protecting group for the internucleotide linkage.2
We wish to present here three developmental works for
broadening the scope in the 2-cyanoethyl phosphoramidite
chemistry: (i) a new phosphitylating reagent, 2-cyano-1-t-butyl-
ethyl or 2-cyano-1-(1,1-diethyl-3-butenyl)ethyl phosphorobis-
imidazolidite 1, is used in in situ generation of 5’-O-protected
nucleoside 3'-O-phosphorimidazolidite 3; (ii) the phosphorimi-
dazolidite 3 serves as a key intermediate for the preparation of a
new type of the nucleoside 3'-O-phoshoromonoalkylamidite 4;
(iii) the imidazolidite 3 is used for the selective introduction of
the 3'-5' internucleotide linkage by the reaction with a 3',5'-
O,O-unprotected nucleoside 5.
A general method of preparation of 1 and 3 is represented
in Scheme 1. The reaction of the phosphorodichloridite 6 (R1 =
t-butyl or 1,1-diethyl-3-butenyl)3 with 1-(trimethylsilyl)imida-
zole in a 1 : 2.2 ratio in toluene, followed by evaporation under
reduced pressure to dryness, gave 14 quantitatively as an oil.5
The treatment of 1 with 1.03 equivalents of 5'-O-(4,4'-
dimethoxytrityl(DMTr))-nucleoside 2 for 1 h at room tempera-
ture produced the corresponding imidazolidite 3 in ca. 97%
yield (based on 1).6 This indicates that the reaction of 1 with 2
proceeds selectively before the produced 3 reacts with 2 to give
the 3'-3' dinucleoside phosphite. This would be caused by the
steric hindrance of the phosphorus protecting group.7
1.1-1.3 equivalents of 5 in chloroform/pyridine (1/2, v/v) (room
temperature, several hours) afforded 7 in good yields (based on
3). The phosphite triester 7 was readily oxidized with
iodine/water to give the phosphate derivatives. Thus, the treat-
ment of crude 7 prepared as mentioned above with 1.2 equiva-
lents of iodine in water/THF (1/10, v/v) for 0.5 h at room tem-
perature produced the phosphate 8 essentially quantitatively.11
Furthermore, the reaction of crude 7 with elemental sulfur gave
the phosphorothioate derivatives 9 (Table 1).12
The 2-cyano-1-t-butylethyl or 2-cyano-1-(1,1-diethyl-3-
butenyl)ethyl group of the internucleotide phosphate and phos-
phorothioate was removed as readily as the 2-cyanoethyl group
in concetrated aqueous ammonia/pyridine (1/1, v/v) (room tem-
perature, < 1 h).
The compound 3 was converted to the phosphoromono-
alkylamidite 4 quantitatively by adding 1 equivalent of the cor-
responding primary amine (Scheme 1). The monoalkylamidite
4 obtained was useful for the solid-phase oligonucleotide syn-
thesis using an automated synthesizer8 without any purification.
For example, the thymidine icosamer (d-T20) was synthesized
in an average coupling yield of 99.1% by the use of in situ pre-
pared 4 (R1 = t-C4H9, R2 = i-C3H7)9 as a monomer unit.
In addition, 3 was selectively coupled with the 5'-hydroxyl
group of a 3',5'-O,O-unprotected nucleoside 5 to give the corre-
sponding triester 710 (Table 1). Thus, the reaction of 3 with
Copyright © 2000 The Chemical Society of Japan

Chemistry Letters 2000
1135
5
6
31P NMR (161.7 MHz, CDCl3, (MeO)3P): 1 (R1 = t-C4H9) δ
109.4; 1 (R1 = C(C2H5)2CH2CHCH2) δ 109.4.
31P NMR of the reaction mixture displayed essentially four
peaks (127–135 ppm, ca. 97%) for the desired imidazolidites
3, and two signals (140-142 ppm, ca. 3%) for the 3'-3' dinu-
cleoside phosphites formed from 1 and excess 2. 31P NMR
(161.7 MHz, CDCl3, (MeO)3P): 3 (B1 = T, R1 = t-C4H9) δ
127.3, 127.5, 128.4, 130.6;
3 = =
(B1 T, R1
C(C2H5)2CH2CHCH2) δ 126.9, 129.5, 130.2, 133.5; 3 (B1 =
CBz, R1 = C(C2H5)2CH2CHCH2) δ 127.5, 131.1, 131.8,
134.8; 3 (B1 = ABz, R1 = C(C2H5)2CH2CHCH2) δ 129.5,
130.3, 131.1, 134.3; 3 (B1 = GiBu, R1 = C(C2H5)2CH2CHCH2)
δ 128.3, 128.6, 131.8, 132.6.
R. L. Letsinger, E. P. Groody, N. Lander, and T. Tanaka,
Tetrahedron, 40, 137 (1984); J. E. Marugg, C. E. Dreet, G.
A. van der Marel, and J. H. van Boom, Recl. Trav. Chim.
Pays-Bas, 103, 97 (1984).
The chain elongation was achieved on a 0.2 µmol scale fol-
lowing the standard protocol by using a PerSeptive
Biosystems ExpediteTM 8909 automated synthesizer. The
reagents except for 4 and solvents used were purchased from
PerSeptive Biosystems, Inc.
7
8
9
The reagent 4 (B1 = T, R1 = t-C4H9, R2 = i-C3H7) used in
solid-phase synthesis was prepared by the reaction of 3 (B1 =
T, R1 = t-C4H9) with i-propylamine in a 1 : 1 ratio in chloro-
form followed by dilution to a 0.1 mol dm–3 solution with
acetonitrile. The reagent 4 was stable for at least one month
when stored at –20 °C under an inert atmosphere. 31P NMR
(161.7 MHz, CDCl3, (MeO)3P): δ 141.8, 142.4, 144.5, 145.8.
10 The related reaction, in which the nucleoside 3'-O-phospho-
rochloridites were treated with the 3',5'-O,O-unprotected
nucleosides in the presence of a base, followed by oxidation
with iodine/water, afforded the 3'-OH free dinucleoside
phosphates in ca. 65% yields.7
11 After the usual work-up, the crude product (B1 = B2 = T, R1
= t-C4H9, 4.5 mmol based on 1) was chromatographed on a
column of silica gel (150 g) with chloroform/methanol
(100/1 to 100/7, v/v) to give 8 (3.49 g, 81%): 31P NMR
(161.7 MHz, CDCl3, (MeO)3P): δ –4.2, –3.4; Rf silica (chlo-
roform/methanol = 10/1, v/v): 0.33.
In conclusion, the in situ preparation of the phosphitylating
reagents and the high selectivities in the phosphitylating reac-
tions would make this methodology afford a new route to a
facile oligonucleotide synthesis.
References and Notes
1
For example: S. L. Beaucage and R. P. Iyer, Tetrahedron, 49,
6123 (1993) and references cited therein.
12 In a typical case, elemental sulfur (0.26 g, 8 mmol) was
added to the reaction mixture of 7 (B1 = T, B2 = GiBu, R1 = t-
C4H9, 4 mmol based on 1) prepared as mentioned above.
The resulting mixture was stirred for 2 h at room tempera-
ture, poured into water (100 mL) and then extracted with
chloroform (100 mL × 2). The combined organic layer was
washed with 5% NaHCO3 (40 mL × 2) and brine (50 mL).
The organic phase was dried over anhydrous Na2SO4, filtered
and concentrated under reduced pressure. The residue was
chromatographed on a column of silica gel (100 g) with chlo-
roform/methanol (100/1 to 100/7, v/v) to give 9 (3.85 g,
90%): 31P NMR (161.7 MHz, CDCl3, (MeO)3P): 9 (B1 = T,
B2 = GiBu, R1 = t-C4H9): δ 65.6, 66.1, 66.2; Rf silica (chloro-
form/methanol = 7/1, v/v): 0.42. 31P NMR and TLC analysis
data for the other dinucleoside phosphorothioates 9 were as
follows: 9 (B1 = ABz, B2 = GiBu, R1 = t-C4H9), (CDCl3): δ
65.4, 65.9, 66.0, 66.2; Rf silica (chloroform/methanol = 7/1,
v/v): 0.38, 9 (B1 = B2 = ABz, R1 = C(C2H5)2CH2CHCH2),
(CDCl3/pyridine = 1/2, v/v): δ 65.2, 65.5, 65.8, 65.9; Rf silica
(chloroform/methanol = 7/1, v/v): 0.48.
2
For example: N. D. Sinha, J. Biernat, and H. Köster,
Tetrahedron Lett., 24, 5843 (1983); S. L. Beaucage and R. P.
Iyer, Tetrahedron, 48, 2223 (1992) and references cited therein.
2-Cyano-1-t-butylethanol was first treated with phosphorus
trichloride, but desired 6 (R1 = t-C4H9) was not obtained in
high purity. Compound 6 was satisfactorily synthesized by
the method of Hata et al., where phosphorus trichloride was
allowed to react with the corresponding alkoxytrimethylsi-
lanes. See: H. Nagai, T. Fujiwara, M. Fujii, M. Sekine, and
T. Hata, Nucleic Acids Res., 17, 8581 (1989). 6 (R1 = t-
C4H9): yield 89%; bp 79–80 °C/0.1 mmHg (1 mmHg =
133.322 Pa); 31P NMR (161.7 MHz, CDCl3, (MeO)3P) δ
177.9. 6 (R1 = C(C2H5)2CH2CHCH2): yield 78%; bp 119-
120 °C/0.1 mmHg; 31P NMR (161.7 MHz, CDCl3, (MeO)3P)
δ 181.9.
3
4
Alkyl phosphorobisimidazolidite was previously used by
Hata et al. for the preparation of alkyl nucleoside 3'-O-phos-
phonates. See: T. Wada, R. Kato, and T. Hata, J. Org.
Chem., 56, 1243 (1991).