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Rapid synthesis of oligonucleotides by the phosphotriester method
BIOCtlIMIE, 1985, 67, 791-795
Rapid synthesis of oligonucleotides by the phosphotriester method. V.A. EFIMOV and O.G. CHAKHMAKHCHEVA.
USSR Academy of Sciences, Shemyakin Institute of Bioorganic Chemistry, U1. Vavilova, 32 117988 Moscow, B-334 USSR. (Refu le 19-3-1985, accept~ le 24-4-1985).
R~sum~ - - Nous avons ddveloppd une mdthodologie efficace de phosphotriester, basde sur l'utilisation d'agents de condensation en prdsence de divers catalyseurs O-nucldophiliques. Mots-cl~s : oligonucl~otides / phosphotriester / synth~se.
Summary -- An efficient phosphotriester methodology based on the use of condensing agents in the presence of several O-nucleophilic catalysts has been developed. Key-words : oligonueleotides / phosphotriester / synthesis.
Recently, we have developed a rapid variant of the phosphotriester approach based on the use of arylsulfonyl chlorides in the presence of N-methylimidazole as coupling reagents for internucleotide bond formation [1]. These reagents enable to perform internucleotide condensations not only in pyridine, which is a traditional solvent for the oligonucleotide synthesis, but also in other organic solvents, such as acetonitrile, methylene chloride, dioxane, chloroform, ethylene chloride, etc. The efficiency of this rapid method in homogeneous solution and on solid phase has been demonstrated by the successful synthesis of more than 100 deoxyribooligonucleotides of 12-62 nucleotides in length [2]. Now we describe an improved rapid phosphotriester method based on the use of some O-nucleophilic eatalysts. It had been shown that pyridine N-oxide and its derivatives are effective nucleophilic catalysts in acylations, sulfonylations and phosphorylations [3, 4]. We found that the use of condensing
agents (MSCI, TPSCI and MSNT)* in conjunction with 4-N,N-disubstituted derivatives of pyridine N-oxide (III) and quinoline N-oxide (IV) (Fig. l) leads to further increase of the rate of phosphotriester bond formation (Fig. 2). Compounds (III) and (IV) were obtained as described [5]. In the presence of these compounds, condensations in solution achieve completion in 0.5-3 min under the action of such condensing agents as MSCI, MSNT, or TPSCI. On polymer supports (pore glass or paper disks), the coupling reactions are complete in 2-5 min. Thus, the overall time for addition of each nucleotide to a growing chain on solid phase is about 10min (Table I). Furthermore, these catalysts increase three fold the rate of pbosphotriester bond formation in the hydroxybenzotriazole approach [6] (Fig. 2). At the same time, the use of these catalysts minimizes the amount of by-products caused by the modification of heterocyclic bases, in particular guanine (Table II).
* Abbreviations : MSCI : mesitylene sulfonyl chloride
TPSCI : MSNT :
triisopropylsulfonyl chloride mesitylene sulfonyl nitrotriazol.
10
V.A. Efimov and O.G. Chakhmakhcheva
792
e[(,.o)2~].~c_(cl~)
A
X
N
0 0
N
÷ 0
CH3
\CH3
0
\C2H5
(~)
(I'rl) 'I}'SCl +(lII)
100
.~+~cl
n
/
/
80
/
(Ill}
{II) /EH3 /C2H5
(I)
+ a~-e(~.) 3 o l v ~ t - C~2C~1-2
° FIll~ ' ,.I
N
(b)
(ll~....,~,
cB~ + ( I )
+ (I)~,~
v%'1
/ / ~ + J
P
(I) /
/
/
o
I (C)
.--.---
+ ( I ~ ) ~ .
J/ ,/
/ /j,
/
ff / .
"
I
I
(d)
(el
J
d
1
. J
4
2
5
6
T
8
(IV) IZZeL
IOO
0 o
B
o.c~
RO-P-OH---[---- RO-P-O-{i
~R"I 9
Pr-SCI
o
o~}-x
-
X
R'OH
0
J / ,p
.- R0_[~-0a"
ORy_
y
I
/
= ~r-~OO-kr-%
iI
f
j,,
,,,,.. " " I Y s
/
'
I
CI-
R = - C6HtEl
40
l
I~,l:g. protected nucleoside Y = El-, ArS020- or R'OR0(O)PO-
FIG. 1. - - Nucleophilic catalysts for rapid phosphotriester
method.
I
I I !
~rw~(mtn) F[o. 2. -- Rates of coupling reaction using different condensing reagents attd nucleophilic catalysts. A) 0.075 mmole of OH-component, 0.1 mmole of P-component and 0.2 mmole of coupling agent in the presence of 0.4 mmole of nucleophilic catalyst were enabled to react in 1 ml of dry solvent. B) 0.05 M solution of OH-component, 0.025 M solution of P-component, 0.075 M solution of TPSCI
and 0.275 M solution of nucleophilic catalist were used. Aliquots were spotted at specific time intervals on silica gel tic and fractionated using !:9 (v/v) methanol-chloroform mixture. The components were eluted and quantitated by UV-spectroscopy. CBT=d[(MeO)2Tr]anC-(CIPh, BT), BTbenzotriazole.
Rapid synthesis of oligonueleotides by the phosphotriester method
The above p r o c e d u r e has been applied to the synthesis o f several oligodeoxyribonucleotides in solution and on p o l y m e r supports. The H P L C analysis and the M a x a m - G i l b e r t sequencing o f the synthetic 17- and 20-mer, obtained by solidphase synthesis using one-solvent procedure [1], are s h o w n in Figure 3.
TABLE I
Reaction cyclefor chemical synthesis of oligonucleotides on polymer supports. Step
1 2 3 4
Reagents and solvents ta~ 3 % dichloroacetic acid in C2H4Ci2 1,2-dichloroethane Coupling mixture in 1,2-dichloroethane t~) (stop flow) 1,2-dichloroethane
793
Time(min) t° I min
T h e application o f pyridine N-oxide derivatives ( I I I ) a n d quinoline N-oxide derivatives (IV) as nucleophilic catalysts for phosphotriester oligonucleotide synthesis has substantially reduced the time needed to p e r f o r m each internucleotide condensation. As a result, the rate o f synthesis b y this i m p r o v e d rapid phosphotriester m e t h o d is n o w practically the same as that with using the phosphite approach. With the p r o p o s e d nucleophilic catalysts, the yields o f oligonucleoticles are not inferior to those obtained with N-methylimidazole. It should also be noted that, apparently, there is n o necessity to i n t r o d u c e additional blocking groups into nucleotide heterocycles in view o f the low degree o f modification of heterocyclic bases in the presence o f substances ( I I I ) and (IV).
2 min 2-5 min depending on condensing agent 2 min
(a) Other solvents, dichloromethane, pyridine, acetonitrile, etc., can be used as effective solvents at different steps of the synthesis. (b) The nucleotide component (5-10-fold molar excess over polymerbound Y-OH component) is dried by coevaporation with C2H4C12,then the solution of arylsulfonyl chloride (or MSNT) (8-10 equiv.) and nucleophilic catalyst (16-20 equiv.) in dry solvent is added, and the reaction solution is injected into the reaction vessel. (c) The synthesis is performed in a column connected to a continuous flow system with the flow rate of 1-2 ml/min, or in a syringe.
TABLE II
Comparison of the time period for the completion of condensation reactions and rates of modification of N2-isobutyryl guanine residue using different condensing and phosphorylating agentst°~. condensing and phosphorylating agents (solvent) TPSC1 (Py) TPSCI + (I) (Py) TPSC! + (I) (CH~Cl2) TPSCl + (1)+NEh (CH2CI2) TPSCI + (II) (CH,Ch) TPSCI + (III) (CH2C12) MSCI + ( I I I ) (CH2CI2) MSNT (Py) MSNT + (I) (Py) MSNT + (I) (CH2C12) MSNT + (III) (CH2Ci2) d[(MeO)2Tr] anC--(CIPh, BT)+(I) (dioxane) d [(MeO)2Tr] anC --(CIPh, B'I) + (I) + NEt3 (dioxane) d [(MeO)2Tr] anC--(CIPh, BT) + (III) (dioxane)
Reaction time (min)
Percent of modification Percent of modification of d[(Ac) ibG(Ac)] during of d [(Ac) ibG (Ac)] the reaction time per raintb~
1200 I0 15 12 10 2 1 40 10 12 1.0 9
0.05 0.05 2.0 19.5 32.5 0.34 0.62 0.46 1.35 0.90 0.45 0.30
6 2.5
0.80 0.05
54 40 30 100 I00 0.50 0.60 18.4 13.5 10.8 0.45 2.7 4.8 0.125
(a) dl(Ac)ibG(Ac)] (0.05 M) was allowed to react with d[(MeO)2Trl anC--(CIPh) (0.1 M) activated by condensing agent (0.2 M), or with dl(MeO)2Tr]anC--(CIPh, BT) (0.1 M), in the presence of nucleophilic catalyst (0.6 M). (b) The yields of modification reactions were determined according to disappearance of ld(Ac) ibG(Ac)] which was measured with the help of tic and UV-spectroscopy (see also legend to Fig. 2).
P.A. Efimov and O.G. Chakhmakhcheva
794
20-met
17-met
c~
FIG. 3a. - - The HPLC analysis on Zorbax C-8 column of the purified by gel electrophoresis synthetic 20-met and 17-mer.
L
, 10
2O
3o 4°TIHE (rain)
20
40
30
Pv
C
G Pu
C T A T T
G
G G G G 8
A
A A
T
IIA G
°
E
O~
G
T
T
~8
T
G
.C 8 T T T AT
.C ..............:TA ~O-mer FIG. 3b. - -
17-mer Sequence analysis fo 5"-~2P-labeledoligonucleotides.
Rapid synthesis of oligonucleotides by the phosphotriester method REFERENCES 1. Efimov, V.A., Reverdatto, S.V. & Chakhmakhcheva, O.G. (1982) Nucleic Acids Res., 10, 6675. 2. Efimov, V.A., Buryakova, A.A., Reverdatto, S.V., Chakhmakhcheva, O.G. & Ovchinnikov, Yu.A. (1983) Nucleic Acids Res., 11, 8369. 3. Savelova, V.A., Belousova, I.A., Litvinenko, L.M. &
795
Yakovetz, A.A. (1984) Dokl. USSR Acad. of Sciences, 274, 1393. 4. Mizuno, Y. & Endo, T. (1978) J. Org. Chem., 43, N 4, 684. 5. Ochiai, E. (1952) J. Org. Chem., 18, 534. 6. Marugg, J.E., Traup, M., Jhurani, P., Hoyng, C.F., van der Marel, G.A. & van Boom, J.H. (1984) Tetrahedron, 40, 73.
Rapid synthesis of oligonucleotides by the phosphotriester method. V.A. EFIMOV and O.G. CHAKHMAKHCHEVA.
USSR Academy of Sciences, Shemyakin Institute of Bioorganic Chemistry, U1. Vavilova, 32 117988 Moscow, B-334 USSR. (Refu le 19-3-1985, accept~ le 24-4-1985).
R~sum~ - - Nous avons ddveloppd une mdthodologie efficace de phosphotriester, basde sur l'utilisation d'agents de condensation en prdsence de divers catalyseurs O-nucldophiliques. Mots-cl~s : oligonucl~otides / phosphotriester / synth~se.
Summary -- An efficient phosphotriester methodology based on the use of condensing agents in the presence of several O-nucleophilic catalysts has been developed. Key-words : oligonueleotides / phosphotriester / synthesis.
Recently, we have developed a rapid variant of the phosphotriester approach based on the use of arylsulfonyl chlorides in the presence of N-methylimidazole as coupling reagents for internucleotide bond formation [1]. These reagents enable to perform internucleotide condensations not only in pyridine, which is a traditional solvent for the oligonucleotide synthesis, but also in other organic solvents, such as acetonitrile, methylene chloride, dioxane, chloroform, ethylene chloride, etc. The efficiency of this rapid method in homogeneous solution and on solid phase has been demonstrated by the successful synthesis of more than 100 deoxyribooligonucleotides of 12-62 nucleotides in length [2]. Now we describe an improved rapid phosphotriester method based on the use of some O-nucleophilic eatalysts. It had been shown that pyridine N-oxide and its derivatives are effective nucleophilic catalysts in acylations, sulfonylations and phosphorylations [3, 4]. We found that the use of condensing
agents (MSCI, TPSCI and MSNT)* in conjunction with 4-N,N-disubstituted derivatives of pyridine N-oxide (III) and quinoline N-oxide (IV) (Fig. l) leads to further increase of the rate of phosphotriester bond formation (Fig. 2). Compounds (III) and (IV) were obtained as described [5]. In the presence of these compounds, condensations in solution achieve completion in 0.5-3 min under the action of such condensing agents as MSCI, MSNT, or TPSCI. On polymer supports (pore glass or paper disks), the coupling reactions are complete in 2-5 min. Thus, the overall time for addition of each nucleotide to a growing chain on solid phase is about 10min (Table I). Furthermore, these catalysts increase three fold the rate of pbosphotriester bond formation in the hydroxybenzotriazole approach [6] (Fig. 2). At the same time, the use of these catalysts minimizes the amount of by-products caused by the modification of heterocyclic bases, in particular guanine (Table II).
* Abbreviations : MSCI : mesitylene sulfonyl chloride
TPSCI : MSNT :
triisopropylsulfonyl chloride mesitylene sulfonyl nitrotriazol.
10
V.A. Efimov and O.G. Chakhmakhcheva
792
e[(,.o)2~].~c_(cl~)
A
X
N
0 0
N
÷ 0
CH3
\CH3
0
\C2H5
(~)
(I'rl) 'I}'SCl +(lII)
100
.~+~cl
n
/
/
80
/
(Ill}
{II) /EH3 /C2H5
(I)
+ a~-e(~.) 3 o l v ~ t - C~2C~1-2
° FIll~ ' ,.I
N
(b)
(ll~....,~,
cB~ + ( I )
+ (I)~,~
v%'1
/ / ~ + J
P
(I) /
/
/
o
I (C)
.--.---
+ ( I ~ ) ~ .
J/ ,/
/ /j,
/
ff / .
"
I
I
(d)
(el
J
d
1
. J
4
2
5
6
T
8
(IV) IZZeL
IOO
0 o
B
o.c~
RO-P-OH---[---- RO-P-O-{i
~R"I 9
Pr-SCI
o
o~}-x
-
X
R'OH
0
J / ,p
.- R0_[~-0a"
ORy_
y
I
/
= ~r-~OO-kr-%
iI
f
j,,
,,,,.. " " I Y s
/
'
I
CI-
R = - C6HtEl
40
l
I~,l:g. protected nucleoside Y = El-, ArS020- or R'OR0(O)PO-
FIG. 1. - - Nucleophilic catalysts for rapid phosphotriester
method.
I
I I !
~rw~(mtn) F[o. 2. -- Rates of coupling reaction using different condensing reagents attd nucleophilic catalysts. A) 0.075 mmole of OH-component, 0.1 mmole of P-component and 0.2 mmole of coupling agent in the presence of 0.4 mmole of nucleophilic catalyst were enabled to react in 1 ml of dry solvent. B) 0.05 M solution of OH-component, 0.025 M solution of P-component, 0.075 M solution of TPSCI
and 0.275 M solution of nucleophilic catalist were used. Aliquots were spotted at specific time intervals on silica gel tic and fractionated using !:9 (v/v) methanol-chloroform mixture. The components were eluted and quantitated by UV-spectroscopy. CBT=d[(MeO)2Tr]anC-(CIPh, BT), BTbenzotriazole.
Rapid synthesis of oligonueleotides by the phosphotriester method
The above p r o c e d u r e has been applied to the synthesis o f several oligodeoxyribonucleotides in solution and on p o l y m e r supports. The H P L C analysis and the M a x a m - G i l b e r t sequencing o f the synthetic 17- and 20-mer, obtained by solidphase synthesis using one-solvent procedure [1], are s h o w n in Figure 3.
TABLE I
Reaction cyclefor chemical synthesis of oligonucleotides on polymer supports. Step
1 2 3 4
Reagents and solvents ta~ 3 % dichloroacetic acid in C2H4Ci2 1,2-dichloroethane Coupling mixture in 1,2-dichloroethane t~) (stop flow) 1,2-dichloroethane
793
Time(min) t° I min
T h e application o f pyridine N-oxide derivatives ( I I I ) a n d quinoline N-oxide derivatives (IV) as nucleophilic catalysts for phosphotriester oligonucleotide synthesis has substantially reduced the time needed to p e r f o r m each internucleotide condensation. As a result, the rate o f synthesis b y this i m p r o v e d rapid phosphotriester m e t h o d is n o w practically the same as that with using the phosphite approach. With the p r o p o s e d nucleophilic catalysts, the yields o f oligonucleoticles are not inferior to those obtained with N-methylimidazole. It should also be noted that, apparently, there is n o necessity to i n t r o d u c e additional blocking groups into nucleotide heterocycles in view o f the low degree o f modification of heterocyclic bases in the presence o f substances ( I I I ) and (IV).
2 min 2-5 min depending on condensing agent 2 min
(a) Other solvents, dichloromethane, pyridine, acetonitrile, etc., can be used as effective solvents at different steps of the synthesis. (b) The nucleotide component (5-10-fold molar excess over polymerbound Y-OH component) is dried by coevaporation with C2H4C12,then the solution of arylsulfonyl chloride (or MSNT) (8-10 equiv.) and nucleophilic catalyst (16-20 equiv.) in dry solvent is added, and the reaction solution is injected into the reaction vessel. (c) The synthesis is performed in a column connected to a continuous flow system with the flow rate of 1-2 ml/min, or in a syringe.
TABLE II
Comparison of the time period for the completion of condensation reactions and rates of modification of N2-isobutyryl guanine residue using different condensing and phosphorylating agentst°~. condensing and phosphorylating agents (solvent) TPSC1 (Py) TPSCI + (I) (Py) TPSC! + (I) (CH~Cl2) TPSCl + (1)+NEh (CH2CI2) TPSCI + (II) (CH,Ch) TPSCI + (III) (CH2C12) MSCI + ( I I I ) (CH2CI2) MSNT (Py) MSNT + (I) (Py) MSNT + (I) (CH2C12) MSNT + (III) (CH2Ci2) d[(MeO)2Tr] anC--(CIPh, BT)+(I) (dioxane) d [(MeO)2Tr] anC --(CIPh, B'I) + (I) + NEt3 (dioxane) d [(MeO)2Tr] anC--(CIPh, BT) + (III) (dioxane)
Reaction time (min)
Percent of modification Percent of modification of d[(Ac) ibG(Ac)] during of d [(Ac) ibG (Ac)] the reaction time per raintb~
1200 I0 15 12 10 2 1 40 10 12 1.0 9
0.05 0.05 2.0 19.5 32.5 0.34 0.62 0.46 1.35 0.90 0.45 0.30
6 2.5
0.80 0.05
54 40 30 100 I00 0.50 0.60 18.4 13.5 10.8 0.45 2.7 4.8 0.125
(a) dl(Ac)ibG(Ac)] (0.05 M) was allowed to react with d[(MeO)2Trl anC--(CIPh) (0.1 M) activated by condensing agent (0.2 M), or with dl(MeO)2Tr]anC--(CIPh, BT) (0.1 M), in the presence of nucleophilic catalyst (0.6 M). (b) The yields of modification reactions were determined according to disappearance of ld(Ac) ibG(Ac)] which was measured with the help of tic and UV-spectroscopy (see also legend to Fig. 2).
P.A. Efimov and O.G. Chakhmakhcheva
794
20-met
17-met
c~
FIG. 3a. - - The HPLC analysis on Zorbax C-8 column of the purified by gel electrophoresis synthetic 20-met and 17-mer.
L
, 10
2O
3o 4°TIHE (rain)
20
40
30
Pv
C
G Pu
C T A T T
G
G G G G 8
A
A A
T
IIA G
°
E
O~
G
T
T
~8
T
G
.C 8 T T T AT
.C ..............:TA ~O-mer FIG. 3b. - -
17-mer Sequence analysis fo 5"-~2P-labeledoligonucleotides.
Rapid synthesis of oligonucleotides by the phosphotriester method REFERENCES 1. Efimov, V.A., Reverdatto, S.V. & Chakhmakhcheva, O.G. (1982) Nucleic Acids Res., 10, 6675. 2. Efimov, V.A., Buryakova, A.A., Reverdatto, S.V., Chakhmakhcheva, O.G. & Ovchinnikov, Yu.A. (1983) Nucleic Acids Res., 11, 8369. 3. Savelova, V.A., Belousova, I.A., Litvinenko, L.M. &
795
Yakovetz, A.A. (1984) Dokl. USSR Acad. of Sciences, 274, 1393. 4. Mizuno, Y. & Endo, T. (1978) J. Org. Chem., 43, N 4, 684. 5. Ochiai, E. (1952) J. Org. Chem., 18, 534. 6. Marugg, J.E., Traup, M., Jhurani, P., Hoyng, C.F., van der Marel, G.A. & van Boom, J.H. (1984) Tetrahedron, 40, 73.