- Email: [email protected]
A versatile approach to the synthesis of dinucleotide analogs containing neutral 5′-thioformacetal internucleoside linkages
8)
T~trahedro,.
Pergamon
I.e/un. Vol. 36, No. 37, pp. 6643-6646. 1995 Elsevier Science Ltd Printed in Gteat Britain 0040403919S $9.50+0.00
~039(9S)01393-8
A Versatile Approach to the Synthesis of Dinucleotide Analogs Containing Neutral S'·Thioforrnacetal Internucleoside Linkages Yves Ducharme· and Kimberly A. Harrison t Merck. Frosst Centre/or Therapeutic Reuarch. P.O. 80% }OO5. Poi1l1e Claire-Dorval. Quebec. Canada H9R 4P8
Abstract: Activation of nucleoside donors 5 by sulfuryl chloride followed by the addition of S'·thionucleoside acceptors 3 yields S' ·thiofonnacetal dinucleotide analogs 6 with in situ trapping of liberated methanesulfenyl chloride with cyclohexene. Purine as wen as pyrimidine derivatives can be part of a coupling reaction as nucleoside donors or acceptors. The dimethoxylrilyl proleCting group is compatible with the presenl coupling methodology allowing differential 3',5'-end protection wilh concomilallt orthogonal base proleCtion.
In view of their potential use as antisense agents,l numerous efforts have recently been devoted to the synthesis of oligonucleotide analogs with modified backbones. 2 In the course of our study on the molecular recognition properties of oligonucleotide analogs, we required access to a variety of dinucleotide derivatives 1 containing a neutral S'·thioformacetal intemucleoside linkage, acid labile protecting groups PI and P2' and where the nucleic bases Bland B2 would be indiscriminately thymine, cytosine, guanine or adenine. PI0L-o __ ~
~I 0'1
S'---o-J ~2 OP2
The only previous synthesis of a S'-thioformacetal dinucleotide analog was reported by Matteucci et al. 3 who have prepared dimer 4 by activating nucleoside donor 2 wilh molecular bromine followed by the addition of nucleoside acceptor 3t (eq I),
HS
+
pCM.
DMT"'1
__
Lo 1
_0_
~
0...,
3t
4
~ Bz
(I)
S
OTBDMS
\=} OTBDMS
6643
6644
While this method is very useful for the coupling of nucleoside derivatives bearing pyrimidine bases, it is inefficient for the preparation ofdinucleotide analogs containing purine bases. We now repon a versatile method for the synthesis of 5' -thioformacetal dinucleotide analogs containing purine as well as pyrimidine nucleic bases. The methylthiomethyl ether function found in nucleoside donor 2 is known to be transformed into a a• chloromethyl ether upon treatment with sulfuryl chloride. 4 A previous reporr on the use of this reaction in the context of nucleic acid chemistry suggested to us its potential utility for the synthesis of 5'-thioformacetal dinucleotide analogs. In a preliminary experiment. the coupling of nucleoside donor Sa, a purine derivative, with nucleoside acceptor 3t by sulfuryl chloride activation was attempted (Scheme 1) and afforded the desired dinucleotide analog 6at in a low 19% yield. A careful analysis of the reaction mixture revealed the presence of asynunetric methyl disulfide 7 (25%). The formation of this unwanted disulfide results from the liberation of highly electtophilic methanesulfenyl chloride upon reaction of methylthiomethyl ether Sa with sulfuryl chloride. Reaction of mercaptan 3t with methanesulfenyl chloride forms disulfide 7.
TBDMS~
_0_
C~CI
~ Bz
~~
+S02 +
[BDMSO~Bz] ~ O......,CI
0_ SCH 3 3t
5a
8
TBDMS~Bz 0
1
6at1s"!o~ OTBDMS
3SS'L.-o_l
H C
~ OTBOMS
725%
Scheme 1 The yield of desired dinucleotide analog 6at is greatly improved by trapping the liberated methanesulfenyl chloride with cyclohexene" prior to the addition of nucleoside acceptor 3t. In a typical reaction, sulfuryl chloride (1.3 equiv) is added dropwise to a O°C mixture of nucleoside donor Sa (1 equiv), N,N-diisopropylethylamine (1.4 equiv) and 3 A molecular sieves in dichloromethane. After 2 minutes, cyclohexene (2 equiv) is added and the reaction mixture is allowed to reach room temperature. Ten minutes later, a solution of nucleoside acceptor 3t (1.3 equiv) and N,N-diisopropylethylamine (1.4 equiv) in dichloromethane is added and the reaction mixture is stirred under nitrogen for 3 hours. After an aqueous work-up and a purification by flash chromatography, the desired dinucleotide analog 6at is obtained in 68% yield. The versatility of the method is demonstrated by the results presented in Table 1. Purine as well as pyrimidine derivatives can be part of a coupling reaction as nucleoside donors 5 or acceptors 3. Noteworthy, S'-thioformacetal dinucleotide analogs containing two purines such as 6aa and 6ag are easily obtained.
6645
Table 1. Preparation of S'·Thiofonnacetal Dinucleotide Analogs 6
_n_ ~
TBDMSa..,
~'
o Sa
B, _ABz
51
B, - T
0'1
S
3a 3c 3g 31
8Z. A
OTBDMS
82. CBz 82. a lBu 82- T
5
Nucleoside Donor
Nucleoside Acceptor
Dinucleotide Analog
B,
B2
5a 5a 5a
3a 3c
5aa 6ac 6ag 5at 51a 61c 61g
ABz ABz ABz ABz
ABz CBz alBu
3g
31
58
3a
51 51 51 51
3c 3g
31
\=)2
Bz
T
ABz CBz a lBu
T
T
T T
611
T
Yield (0/0) 64 53 49
tll
51
53
:n
00
The dimethoxytrityl protecting group is compatible with the present coupling methodology as shown by the coupling of donor 9 6 with acceptor 31 to afford 3',s'-differentiaIly protected dinucleotide analog 10 in 58% yield (eq 2). DMT1.-0-l
~Q~~ s~
(2)
5B%
9
10
OTBDMS 7
The required nucleoside donors and acceptors were prepared in the following ways. A Pummerer reaction on Mi-benzoyl-S'-O-t-butyldimethylsilyl-2'-deoxyadenosine (11)1 gave access to donor Sa (Scheme 2). Nucleoside donor St was prepared by the alkylation of S'·O.t-butyldimethylsilylthymidine (12)8 with chloromethyl methyl sulfide.
6646
TBDMS~Bz
TBDMS~
PC 20, AcOH •
NaH' Nal CH;tSCH 2CI
DMSO 70%
OH
5t
8)0/0
OH 12
11
Scheme 2 The four nucleoside acceptors 3a, 3c, 3g and 3t were prepared by the method of Kawai et al.' (Table 2). Mitsunobu coupling of suitably base protected deoxynucleosides 13 t 0 with thiolacetic acid afforded the 5'-8• acetyl nucleoside derivatives 14. Silylation of the secondary alcohols with t-butyldimethylsilyl chloride gave fully protected nucleosides 15. Methanolysis of the thioesters fmally provided the desired nucleoside acceptors
3.
Table 2. Preparation of Nucleoside Acceptors 3 H\:=}
PPh3' DIAD
ACS\=}
TBDMSCI
ACS\=}
AcSH
-
NaOH
3
MeOH
OH
OH 13
OTBDMS 15
14
Entry
B
a
ABz CBz
c g t
GiBu T
Yields (%)
14 76
41 94 42
15
91 92
78 98
3
52
56 64
82
References and Notes
t 1. 2.
3. 4. 5. 6. 7. 8. 9. 10.
Undergraduate Coop Student, University of Waterloo. (a) Uhlmann, E.; Peyman, A. Chem.Rev. 1990,90, 543. (b) Milligan, J. F.; Matteucci, M.D.; Martin, J.C. J. Med. Chern. 1993, 36, 1923. (a) Vanna, R. S. Synlett 1993, 621. (b) Sanghvi, Yogesh S.; Cook, P. Dan; Carbohydrates: Synthetic Methods and Applications in Antisense Therapeutics. In Carbohydrate Modifications in Antisense Research; Sanghvi, Yogesh S.; Cook, P. Dan Eds.; ACS Symposium Series No. 580; American Chemical Society, 1994; pp. 1-22. Matteucci, M. D.; Lin, K.-Y.; Butcher, S.: Moulds, C. J. Am. Chem. Soc. 1991,1l3, 7767. (a) Benneche. T.; Undheim. K. Acta Chem. Scand. B 1983.37. 93 (b) Benneche, T.; Strande, P.: Undheim, K. Synthesis 1983, 762. zavgorodny, S.; Polianski, M.; Besidsky. E.; Kriukov, V.; Sanin. A.; Pokrovskaya, M.; Gurskaya, G.; Ulnnberg, H.; Azhayev, A. Tetrahedron Lett. 1991,32,7593. Matteucci, M. Tetrahedron Lett. 1990,31,2385. Pojer, P.M.; Angyal, S.J. Tetrahedron Lett. 1976,17,3067. Ogilvie, K.K. Can. J. Chem. 1973,51,3799. Kawai, S.H.: Wang. D.: Just, G. Can. J. Chem. 1992,70. 1573. Ti, G.S.; Gaffney, B.L.; Jones, R.A. J. Am. Chem. Soc. 1982,104,1316.
(Received in USA 5 July 1995; accepted 20 July 1995)
T~trahedro,.
Pergamon
I.e/un. Vol. 36, No. 37, pp. 6643-6646. 1995 Elsevier Science Ltd Printed in Gteat Britain 0040403919S $9.50+0.00
~039(9S)01393-8
A Versatile Approach to the Synthesis of Dinucleotide Analogs Containing Neutral S'·Thioforrnacetal Internucleoside Linkages Yves Ducharme· and Kimberly A. Harrison t Merck. Frosst Centre/or Therapeutic Reuarch. P.O. 80% }OO5. Poi1l1e Claire-Dorval. Quebec. Canada H9R 4P8
Abstract: Activation of nucleoside donors 5 by sulfuryl chloride followed by the addition of S'·thionucleoside acceptors 3 yields S' ·thiofonnacetal dinucleotide analogs 6 with in situ trapping of liberated methanesulfenyl chloride with cyclohexene. Purine as wen as pyrimidine derivatives can be part of a coupling reaction as nucleoside donors or acceptors. The dimethoxylrilyl proleCting group is compatible with the presenl coupling methodology allowing differential 3',5'-end protection wilh concomilallt orthogonal base proleCtion.
In view of their potential use as antisense agents,l numerous efforts have recently been devoted to the synthesis of oligonucleotide analogs with modified backbones. 2 In the course of our study on the molecular recognition properties of oligonucleotide analogs, we required access to a variety of dinucleotide derivatives 1 containing a neutral S'·thioformacetal intemucleoside linkage, acid labile protecting groups PI and P2' and where the nucleic bases Bland B2 would be indiscriminately thymine, cytosine, guanine or adenine. PI0L-o __ ~
~I 0'1
S'---o-J ~2 OP2
The only previous synthesis of a S'-thioformacetal dinucleotide analog was reported by Matteucci et al. 3 who have prepared dimer 4 by activating nucleoside donor 2 wilh molecular bromine followed by the addition of nucleoside acceptor 3t (eq I),
HS
+
pCM.
DMT"'1
__
Lo 1
_0_
~
0...,
3t
4
~ Bz
(I)
S
OTBDMS
\=} OTBDMS
6643
6644
While this method is very useful for the coupling of nucleoside derivatives bearing pyrimidine bases, it is inefficient for the preparation ofdinucleotide analogs containing purine bases. We now repon a versatile method for the synthesis of 5' -thioformacetal dinucleotide analogs containing purine as well as pyrimidine nucleic bases. The methylthiomethyl ether function found in nucleoside donor 2 is known to be transformed into a a• chloromethyl ether upon treatment with sulfuryl chloride. 4 A previous reporr on the use of this reaction in the context of nucleic acid chemistry suggested to us its potential utility for the synthesis of 5'-thioformacetal dinucleotide analogs. In a preliminary experiment. the coupling of nucleoside donor Sa, a purine derivative, with nucleoside acceptor 3t by sulfuryl chloride activation was attempted (Scheme 1) and afforded the desired dinucleotide analog 6at in a low 19% yield. A careful analysis of the reaction mixture revealed the presence of asynunetric methyl disulfide 7 (25%). The formation of this unwanted disulfide results from the liberation of highly electtophilic methanesulfenyl chloride upon reaction of methylthiomethyl ether Sa with sulfuryl chloride. Reaction of mercaptan 3t with methanesulfenyl chloride forms disulfide 7.
TBDMS~
_0_
C~CI
~ Bz
~~
+S02 +
[BDMSO~Bz] ~ O......,CI
0_ SCH 3 3t
5a
8
TBDMS~Bz 0
1
6at1s"!o~ OTBDMS
3SS'L.-o_l
H C
~ OTBOMS
725%
Scheme 1 The yield of desired dinucleotide analog 6at is greatly improved by trapping the liberated methanesulfenyl chloride with cyclohexene" prior to the addition of nucleoside acceptor 3t. In a typical reaction, sulfuryl chloride (1.3 equiv) is added dropwise to a O°C mixture of nucleoside donor Sa (1 equiv), N,N-diisopropylethylamine (1.4 equiv) and 3 A molecular sieves in dichloromethane. After 2 minutes, cyclohexene (2 equiv) is added and the reaction mixture is allowed to reach room temperature. Ten minutes later, a solution of nucleoside acceptor 3t (1.3 equiv) and N,N-diisopropylethylamine (1.4 equiv) in dichloromethane is added and the reaction mixture is stirred under nitrogen for 3 hours. After an aqueous work-up and a purification by flash chromatography, the desired dinucleotide analog 6at is obtained in 68% yield. The versatility of the method is demonstrated by the results presented in Table 1. Purine as well as pyrimidine derivatives can be part of a coupling reaction as nucleoside donors 5 or acceptors 3. Noteworthy, S'-thioformacetal dinucleotide analogs containing two purines such as 6aa and 6ag are easily obtained.
6645
Table 1. Preparation of S'·Thiofonnacetal Dinucleotide Analogs 6
_n_ ~
TBDMSa..,
~'
o Sa
B, _ABz
51
B, - T
0'1
S
3a 3c 3g 31
8Z. A
OTBDMS
82. CBz 82. a lBu 82- T
5
Nucleoside Donor
Nucleoside Acceptor
Dinucleotide Analog
B,
B2
5a 5a 5a
3a 3c
5aa 6ac 6ag 5at 51a 61c 61g
ABz ABz ABz ABz
ABz CBz alBu
3g
31
58
3a
51 51 51 51
3c 3g
31
\=)2
Bz
T
ABz CBz a lBu
T
T
T T
611
T
Yield (0/0) 64 53 49
tll
51
53
:n
00
The dimethoxytrityl protecting group is compatible with the present coupling methodology as shown by the coupling of donor 9 6 with acceptor 31 to afford 3',s'-differentiaIly protected dinucleotide analog 10 in 58% yield (eq 2). DMT1.-0-l
~Q~~ s~
(2)
5B%
9
10
OTBDMS 7
The required nucleoside donors and acceptors were prepared in the following ways. A Pummerer reaction on Mi-benzoyl-S'-O-t-butyldimethylsilyl-2'-deoxyadenosine (11)1 gave access to donor Sa (Scheme 2). Nucleoside donor St was prepared by the alkylation of S'·O.t-butyldimethylsilylthymidine (12)8 with chloromethyl methyl sulfide.
6646
TBDMS~Bz
TBDMS~
PC 20, AcOH •
NaH' Nal CH;tSCH 2CI
DMSO 70%
OH
5t
8)0/0
OH 12
11
Scheme 2 The four nucleoside acceptors 3a, 3c, 3g and 3t were prepared by the method of Kawai et al.' (Table 2). Mitsunobu coupling of suitably base protected deoxynucleosides 13 t 0 with thiolacetic acid afforded the 5'-8• acetyl nucleoside derivatives 14. Silylation of the secondary alcohols with t-butyldimethylsilyl chloride gave fully protected nucleosides 15. Methanolysis of the thioesters fmally provided the desired nucleoside acceptors
3.
Table 2. Preparation of Nucleoside Acceptors 3 H\:=}
PPh3' DIAD
ACS\=}
TBDMSCI
ACS\=}
AcSH
-
NaOH
3
MeOH
OH
OH 13
OTBDMS 15
14
Entry
B
a
ABz CBz
c g t
GiBu T
Yields (%)
14 76
41 94 42
15
91 92
78 98
3
52
56 64
82
References and Notes
t 1. 2.
3. 4. 5. 6. 7. 8. 9. 10.
Undergraduate Coop Student, University of Waterloo. (a) Uhlmann, E.; Peyman, A. Chem.Rev. 1990,90, 543. (b) Milligan, J. F.; Matteucci, M.D.; Martin, J.C. J. Med. Chern. 1993, 36, 1923. (a) Vanna, R. S. Synlett 1993, 621. (b) Sanghvi, Yogesh S.; Cook, P. Dan; Carbohydrates: Synthetic Methods and Applications in Antisense Therapeutics. In Carbohydrate Modifications in Antisense Research; Sanghvi, Yogesh S.; Cook, P. Dan Eds.; ACS Symposium Series No. 580; American Chemical Society, 1994; pp. 1-22. Matteucci, M. D.; Lin, K.-Y.; Butcher, S.: Moulds, C. J. Am. Chem. Soc. 1991,1l3, 7767. (a) Benneche. T.; Undheim. K. Acta Chem. Scand. B 1983.37. 93 (b) Benneche, T.; Strande, P.: Undheim, K. Synthesis 1983, 762. zavgorodny, S.; Polianski, M.; Besidsky. E.; Kriukov, V.; Sanin. A.; Pokrovskaya, M.; Gurskaya, G.; Ulnnberg, H.; Azhayev, A. Tetrahedron Lett. 1991,32,7593. Matteucci, M. Tetrahedron Lett. 1990,31,2385. Pojer, P.M.; Angyal, S.J. Tetrahedron Lett. 1976,17,3067. Ogilvie, K.K. Can. J. Chem. 1973,51,3799. Kawai, S.H.: Wang. D.: Just, G. Can. J. Chem. 1992,70. 1573. Ti, G.S.; Gaffney, B.L.; Jones, R.A. J. Am. Chem. Soc. 1982,104,1316.
(Received in USA 5 July 1995; accepted 20 July 1995)