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To cite this article: M. G. Ivanovskaya , M. B. Gottikh & Z. A. Shabarova (1987): Modification of Oligo
(Poly) Nucleotide Phosphomonoester Groups in Aqueous Solutions, Nucleosides and Nucleotides, 6:5,
913-934
To link to this article: http://dx.doi.org/10.1080/15257778708073437
I v a n o v s k a y a , M.B.
G o t t i k h and Z.A.
Shabarova
O l i g o n u c l e o t i d e d e r i v a t i v e s , c o n t a i n i n g v a r i o u s non-nucleot i d e g r o u p s c o v a l e n t l y bound t o t e r m i n a l p h o s p h a t e g r o u p s , a r e
f i n d i n g e v e r wider a p p l i c a t i o n as chemical p r o b e s [ I ] , markers
[2]
a f f i n i t y r e a g e n t s [ 3 ] , a g e n t s f o r c h e m i c a l l i g a t i o n 141
s o l u t i o n s [7-91.
The u s e o f a n a n h y d r o u s medium l i m i t e d t h e
a p p l i c a b i l i t y o f t h e method b e c a u s e u n p r o t e c t e d o l i g o - a n d
p o l y n u c l e o t i d e s , D N A s , RNAs do n o t d i s s o l v e i n o r g a n i c s o l v e n t s .
Furthermore, such s i d e p r o c e s s e s a s a l k y l a t i o n of h e t e r o c y c l i c
913
Copyright 0 1987 by Marcel Dekker, Inc.
914
(PA)
( P U ) were
~ ~ from SRCTI (SD AS USSR). EDC, MES, MeIm 1121 ,
Lichrosorb-NH2 (10 wm); aniline, imidazole, morpholine, benzylamine, n-butylamine, ethylenediamine and 4-nitrophenol were
obtained from "Werck"; 2-hydroxypyridine - from "Aldrich";
glycine methyl ester, serine methyl ester, tyrosine methyl ester, cysteine methyl ester - from "Koch-Light" : N-hydroxybenzotriazole, 3-hydroxypropionitrile, Dans-chloride [121 - from
"Fluka", Nucleosil Cl8 (5 vm)
915
Yield of
80
20
20
3.0
3.0
4.5
5.0
butylamine
3.0
3 .O
3.0
6.5
4.5
5.0
4.5
4-n itrophenol
N-hydroxybenzo
2-hydroxypyridine
3.0
1.5
triazole
4.0
4.5
4.5
2- (N-Dans)-aminoethanol
90
90
2
5
95
10
95
24
10
5
40
24
10
90
3.0
4.5
n-propanol
3-hydroxypropinonitrile
80
20
3.0
20
80
85
2
2
20
20
6 .O
methanol
ethanol
4.5
90
4.5
~ _ _ _ _
85
20
3.0
5.0
85
morpholine
benzylamine
90
20
3.0
3.0
6 .O
4.5
95
tive,
p A deriva-
20
Process
time,
hours
20
TC
3.0
Nuc leophile
concentration,
4.5
PH
aniline
imidazole
Nucleophilic agent
TABLE 1 .
917
in 50
p1
containing 2 M
( P a 3o
(PA)1 1
0.1
0.1
4.0
4.5
4.5
4.5
N-hydroxybensotriazole
ethylenediamine
ethylenediamine
4.5
6.5
4.5
0.1
0.1
4-nitrophenol
ethy lenediamine
0.2
6.0
4.5
4.5
4.5
imidazole
ethylenediamine
ethanol
ethyleneg 1yco1
2- (N-Dans)aminoethanol
N-hydroxybenzotriazole
0.03
0.1
0.1
0.2
0.2
0.1
0.2
4.5
d (TGGCCAACGTp)
0.2
0.2
0.2
0.2
aniline
imidaz o le
morpholine
1,4-diaminobutane
2-hydroxyethyl ester
of bromoacetic acid
d ( TTGAATGp)
pH
4.5
6.0
5.0
4.5
Nucleophilic
agent
Oligo- or
polynucleotide
Nucleotide
concentration (per
monomeric
union1 ,mM
3.0
3.0
3 -0
24
2
90
70
95
95
100
4
3.0
3.0
30
95
90
95
98
95
80
95
90
95
90
24
4
4
2
6
6
4
6
8
~~~~
Conversion
of phosphomonoester
groups, %
1.5
3.0
3.0
3.0
6-0
6 .O
3.0
3.0
3.0
3.0
3.0
Nucleophi- Process
le contime,
centration, hours
M
TABLE 2.
x-
v1
z
0
.c:
C
P
919
Preparation of N-hydroxybenzotriazole esters of (PU)~,d(TGGCCAAGCTp). 0 . 0 1 - 0 . 0 2 pmol of the oligonucleotide was dissolved in 40 &l of DMFA - 0.4 M MES-buffer, containing 2 M
MgC12 (pH 4 . 5 - 5 . 5 ) 3 : l (v/v). The reagents were added as described above or hydroxybenzotriazole ester of mononucleotides.
The reaction mixture was incubated at 4C for 3 h. The products
were isolated by gel-filtration on Bioqel P-2 with 90% yields.
c2H5N=c=N>
HN
H+
/ \
/ \
H,C
CH,
c,n,Nn-$NHJ i
c2
HN,=<,!
JH
H,C
N
/\
HjC
CH,
TCH,
For this reason EDC-induced reactions are usually pH-dependent [ 1 5 ] . We have found pH-dependence of phosphomonoester group
modification to be individual for each class of nucleophilic
agents (amines, alcohols, phenols). In the case of EDC-induced
modification of pA by amines we established the forming of phosphoamidates to take place in a wide pH-range (from 2 to 1 0 ,
Table 3 ) . The maximal efficiency and rate of modification for
amines of different basicity were observed at pH from 3 to 5
(Table 3 ) . Under these conditions EDC predominantly exists in
the most reactive form I. The modification efficiency sharply
decreases if pH is brought down to 1 and below, because a phos-
920
I V A N O V S K A Y A , GOTTIKH, A N D SHABAROVA
0 0 0
0 0 0
0 0 0 0
0 0 0 0
- 7 . 7 .
v r - 7
0 0 0
0 0 0
o o o m
U
7
7 - c
0 0 0 0
0 0 0 0
omco mm
omtn
- m o o a i m o & w e
1 1 1 0 1 1 0 1 1 1
m o m - m m - m o o
corn
m a
r-wv
Ln
0 0 0 I
r r - 0
0 0 0
0 0 0
7 - 7
O\O
0 s
.rl
.!Ju
Ln
0 0 0
0 0 0
7 - 7
ooow
0 0 0
-t--o
Lnm
o o m c o
0 0 1 I
r - 0 0
mm
0 0
m m
o o w m
rd
0
-4
a
0
E r .
o o m
0 0 I
- 7 0
w
0
r-
a , I
a,
tn
o o w m
001 I
r r m m
r-m
o m w m
o m 1 I
W N
r r m
a,
w m
mo
0 0 I
o o m
0
0
1 I
- - 0 0
o m
~
m
o m
01
- - 0
- 0
m m o
m e w
ale
I1
4
.rl
e x
rda
m
0
m
I
- ~co r n mmmmr r 1 ~
L n o o o o o o o o m
0 0 -
n
I
o o o m o m m m m o
921
+ /CH3
NuH - a
nucleophilic agent
This assumption is confirmed by inhibition of the EDC-induced modification at pH>10 (Table 3 ) when the tertiary amine
group becomes unprotonated. Besides, carbodiimides whose structure lacks the protonodonor group, for example, l-cyclohexyl- 3 - (2-morpholinoethyl) carbodiimide metho.!! -toluenesulfonate,
do not induce phosphomonoester group modification at pH>6 [ 1 6 ] .
The proposed scheme is in accordance with I.T. Ibrahim and
A. Williams data about reactivity of the acyclic form of EDC in
a basic medium [14].
An investigation of the pA reaction with hydroxycompounds in
the presence of EDC has shown the optimal pH range to be dependent on the hydroxycompound nature.
In the case of simple alcohols, such as methanol, ethanol,
n-propanol and others, the reaction proceeds at pH from 2 to 6
(Table 4) and phosphodiesters are formed. The increase of pHvalue to 7 and higher results in pyrophosphate as the only reac-
922
TABLE 4
EFFICIENCY OF EDC-INDUCED MODIFICATION O F PA BY HYDROXYCOMPOUNDS AT VARIOUS pH VALUES (CONCENTRATION OF p A - 0 . 0 3 M ,
HYDROXYCOMPOUND
3 M , EDC - 0 . 5 M , TEMPERATURE - 20C)
Hydroxycompound
pH
n-propanol"
1 .O
2.0
3.2
4.5
5.5
6 .O
7.0
8.0
4-nitrophenol
Degree of pA m o d i f i c a t i o n ,
6 h
16 h
0.5 h
2 h
0
30-35
30-35
25-30
15-20
10-15
0
100
95
90-95
0
0
0
100
100
100
100
100
0
0
2.5
4.0
6.0
7.0
8.0
45-50
60-65
50-55
5-10
0
15-20
0
90-95
90-95
90-95
35-40
0
3.0
5.0
7.:
8.0
9.0
75-80
65-70
20-25
10-15
3-5
100
100
90-95
70-75
60-65
100
100
78-83
30-35
15-20
3.5
5.0
6.5
7.0
8.0
8.4
pKa = 7 . 1 5
0
80-85
95-100
N-hydroxybenzotriazole
pKa = 4 . 0 0
2-hydroxypyridine
= 11.65
pKa
--
24 h
0
100
100
100
100
100
3-5
0
0
100
100
100
100
100
5-10
0
3-5
15-20
90-95
60-65
45-50
10-15
5-1 0
20-25
100
90-95
60-65
15-20
--
100
100
95-97
35-40
0
100
100
85-90
47-52
40-45
A similar dependence i s o b s e r v e d f o r m e t h a n o l , e t h a n o l a n d
o t h e r simple alcohols.
reacts f i r s t of a l l w i t h t h e P A - c a r b o d i i m i d e a d d u c t .
P h e n o l s , u n l i k e a l c o h o l s , are n o t n u c l e o p h i l i c i n t h e noni o n i z e d form.
g e s t n u c l e o p h i l i c a g e n t s a n d so t h e r e a c t i o n w i t h p h e n o l s beco-
m e s p o s s i b l e a t p H >, pK of p h e n o l . W e s u c c e e d e d i n s y n t h e s i z i n g
4-nitrophenyl
e s t e r of PA.
l y a t pH 6 . 5 - 7 . 0
The r e a c t i o n p r o c e e d s m o s t e f f i c i e n t -
However, p r e p a r a t i o n of p A d e r i v a t i v e s w i t h p h e n o l ( p K = l o )
a
a n d N-Ac-Tyr m e t h y l ester ( p K a = 1 0 . 5 ) h a s proved t o be impos-
923
s i b l e a p p a r e n t l y a s a r e s u l t of EDC i n a c t i v a t i o n a t pH
>
Moreover, t h e n u c l e o p h i l i c s u b s t i t u t i o n i n PA-EDC-adduct
10.
by
p h e n o l a t e a n i o n i s impeded by e l e c t r o s t a t i c r e p u l s i o n o f t h e
same c h a r g e d p a r t i c l e s . T h i s e f f e c t a c c o u n t s f o r a d e c r e a s e d
r a t e of t h e r e a c t i o n o f pA w i t h 4 - n i t r o p h e n o l
i n comparison
w i t h h i g h y i e l d s i n a wide pH-range
(from 2 t o 8 , T a b l e 4 ) .
on t h e c o n c e n t r a t i o n o f r e a g e n t s h a s r e v e a l e d t h a t a s u f f i c i e n t l y
h i g h c o n c e n t r a t i o n o f a n u c l e o p h i l i c a g e n t (1-3 M) a n d EDC
( 0 . 5 M) i s r e q u i r e d ( T a b l e 5 ) .
A r i s e i n t h e t e m p e r a t u r e from 4'C
t o 50C l e a d s t o a n i n -
c r e a s e i n t h e m o d i f i c a t i o n r a t e ; y e t t h e y i e l d s of t h e d e r i v a t i v e s d e c r e a s e owing t o a c c e l e r a t i o n of EDC c o m p e t i t i v e h y d r o l y s i s .
W e have found t h e m o d i f i c a t i o n of o t h e r deoxyribo- and r i b o -
n u c l e o t i d e s t o o c c u r t h e same a s PA.
However, i t i s n e c e s s a r y
t o t a k e i n t o a c c o u n t t h e p o s s i b i l i t y of t h e h e t e r o c y c l i c b a s e s
(U, T , G ) m o d i f i c a t i o n by EDC. W e h a v e f o u n d no h e t e r o c y c l i c
b a s e s m o d i f i c a t i o n a t pH<6 ( 4 8 h , 2 O o C ) . A t pH > 6 t h i s m o d i f i c a t i o n may b e d e t e c t e d a f t e r 2 h i n c u b a t i o n a t 2OoC. The a b o v e
m o d i f i c a t i o n o f U , T a n d G i s r e v e r s i b l e , and w e recommend t h e
f o l l o w i n g p r o c e d u r e f o r i t s e l i m i n a t i o n : t r e a t m e n t by 0 . 2 M
Na2C03 s o l u t i o n (pH 1 0 . 5 ) f o r 6-10 h a t 37OC ( o r 2 4 h a t 2 0 C ) .
Consequently, we have determined t h e o p t i m a l c o n d i t i o n s f o r
t h e m o d i f i c a t i o n of t h e phosphomonoester g r o u p s i n m o n o n u c l e o t i d e s . I t i s recommended t o c o u p l e m o n o n u c l e o t i d e s t o a m i n e s ,
s i m p l e a l c o h o l s , N-hydroxybenzotriazole and 2-hydroxypyridine
a t pH of t h e r e a c t i o n m i x t u r e from 4 . 5 t o 5 . 5 . Lower pH v a l u e s
a r e n o t a d v i s a b l e f o r t h e r e a c t i o n of d e o x y r i b o n u c l e o t i d e s i n
v i e w of t h e i r p o s s i b l e a p u r i n i z a t i o n . The o p t i m a l c o n c e n t r a t i o n s
a r e a s f o l l o w s : EDC
0.5 M; n u c l e o p h i l i c a g e n t
3 M ( f o r ami-
n e s it i s p o s s i b l e t o d e c r e a s e c o n c e n t r a t i o n t o 1-0.5 M); a n d
t h e o p t i m a l t e m p e r a t u r e i s 4-20C
(Table 1 ) . For t h e r e a c t i o n
w i t h 4 - n i t r o p h e n o l t h e o p t i m a l pH v a l u e i s 6.5-7.0,
while
o t h e r c o n d i t i o n s a r e s i m i l a r t o t h o s e i n d i c a t e d above ( T a b l e 1 ) .
R e a c t i o n s of m o n o n u c l e o t i d e s w i t h b i f u n c t i o n a l a g e n t s
I t was i n t e r e s t i n g t o s t u d y EDC-induced
phosphomonoester
g r o u p s m o d i f i c a t i o n by b i f u n c t i o n a l a g e n t s c o n t a i n i n g b o t h
s i m i l a r and d i f f e r e n t f u n c t i o n a l groups.
I n c a s e of r e a g e n t s
3
0.3
ethylenediamine
benzylamine
butylamine
3
0.3
aniline
3
1
0.3
0.3
3
3
3
1
1
1
1
0.3
0.3
ethanol
M i n e or
alcohol
Amine o r
alcohol
concentration,M
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.3
0.3
0.3
0.1
EDC concentration, M
20
20
20
20
20
20
20
20
20
4
20
50
20
50
20
50
20
20
TOC
30-35
10-15
5
75-80
25-30
100
80-85
95
60-65
55-60
45-50
85-90
30-35
100
90
100
95
100
42-47
90-1 0 0
70-75
45-50
47-52
40-45
36-4 1
20-25
2 h
30-35
50-55
70-75
35-40
45-50
30-35
35-40
15-20
l h
80-85
60-65
50-55
45-50
100
90
100
100
95
90-95
100
70-75
55-60
50-52
50-55
36-42
30-35
4 h
95-100
100
55-60
100
90
100
95
100
100
70-75
60-65
55-60
51-56
40-45
35-40
0
6 h
Degree of pA m o d i f i c a t i o n ,
~~~~
100
95
80-85
100
85-90
100
90
100
95
100
100
70-75
70-75
55-60
52-57
43-48
36-42
0
~~
24 h
TABLE 5
925
95
100
70
P-N
P-N
P-N
2
2
6
1.5
5 .O
2.0
5.0
8.0
3.0
3.0
1.5
3.0
85
30
P-N
P-N
P-N
6
2
24
6.0
95
90
P-N
P-N
2.0
4.5
95
85
95
P-0
5.0
97
Yield of PA
derivative,
4.5
P-N
P-N
3.0
Process
time,
hours
4.5
pH
6.0
3.0
Reagent
concentration,
M
ethylenediamine
1,4-diaminobutane
ethyleneglycol
Bifunctional reagent
TABLE 6
rj
H
rj
OLIGO(P0LY)NUCLEOTIDE
PHOSPHOMONOESTER GROUPS
I
I
46000
I
I
42000
927
78 000
f 4 000
I
70000
928
46000
42000
I
I
78000
foooo
74000
y, C M '
) , starting pA (-2-(N-Dans)-a~ninoethanol ( -)
--
ester
and
. We
assume t h i s i n c r e a s e of unde-
s i r a b l e m o d i f i c a t i o n may b e c a u s e d b y t h e p o l y e l e c t r o l y t e e f f e c t
of a p o l v a n i o n o l i q o n u c l e o t i d e . T h e c a t i o n r e a g e n t
EDC
owing
t o e l e c t r o s t a t i c i n t e r a c t i o n s seems t o b e c o n c e n t r a t e d n e a r t h e
n o l y a n i o n s u g a r - p h o s p h a t e b o n e . The r e s u l t i s a n i n c r e a s e i n EDC
l o c a l c o n c e n t r a t i o n n e a r h e t e r o c y c l i c b a s e s and m o d i f i c a t i o n acc e l e r a t i o n . Remarkably, no s u c h a c c e l e r a t i o n of h e t e r o c y c l i c bases m o d i f i c a t i o n i s o b s e r v e d d u r i n g o l i q o n u c l e o t i d e ahosphoamid a t e p r e u a r a t i o n . P r o b a b l y , w r o t o n a t e d amine ( i t s c o n c e n t r a t i o n
e x c e e d s 6-7
t i m e s t h e c a r b o d i i m i d e o n e ) forms a p o l y e l e c t r o l y t e
l a y e r n e a r t h e o l i g o n u c l e o t i d e a n d p r o t e c t s it a g a i n s t m o d i f i c a tion.
T o lower t h e d e g r e e of u n d e s i r a b l e m o d i f i c a t i o n i n t h e s y n t h e -
s i s of o l i g o n u c l e o t i d e n h o s p h o d i e s t e r s w e have u s e d t h e p r o p e r t y
929
I
I
46000
42 000
78 000
y.000
000
2+
9 30
74
4,O
95
0
I
46 000
42000
74 000
78 000
000
9,CM-
type hydrox~~compounds
including formerly virtually inaccessible
.w
4-nitrophenyl esters , N-hydrgenzot r iazole and 2 -hydroxypyridine
derivatives of unprotected oligonucleotides. Besides, an oligonucleotide affinity reaqent, containing the bromoacetic acid residue, and fluorescent N-Dans-aminoethyl ester of the decanucleotide have been synthesized (Table 2).
Isolation of oligo(po1y)nucleotide derivatives
Phosphoamidates and Dhosphodiesters of oligonucleotides were
separated from the excess of reagents by gel-filtration on biogel
P-2 followed by chromatoqraphy on LichrosorS-!W2 or Nucleosil C 1 8 .
All synthesized derivatives are homoneneous in chromatography on
ion-exchange and reversed-phase carriers. Chromatography of the
reaction mixtures after synthesis of aminoethvlamide aqd N-hydroxybenzotriazole ester of ( P U ) ~is shown in Firlure 5. The formation of phosphoamide or phosphodiester linkaqe at the terminal pho-s-
931
a
Downloaded by [University of Hong Kong Libraries] at 05:03 25 March 2013
932
p h a t e of o l i g o n u c l e o t i d e s was c o n f i r m e d by t h e l a c k o f h y d r o l y s i s
o f t h e s e s u b s t a n c e s by b a c t e r i a l a l k a l i n e p h o s p h a t a s e .
CONCLUSION
I n t h e p r e s e n t communication a q e n e r a l e f f i c i e n t method f o r
m o d i f i c a t i o n of t h e phosphomonoester g r o u p s of u n p r o t e c t e d o l i g o n u c l e o t i d e s o f p r a c t i c a l l y any l e n g t h and c o m p o s i t i o n i s p r o p o s e d
The f o l l o w i n g m o d i f i c a t i o n c h a r a c t e r i s t i c s h a v e b e e n f o u n d :
1 ) M o d i f i c a t i o n p r o c e e d s a t a h i g h e r r a t e i n a wide pli i n t e r -
v a l 2-9.
T h i s i s due t o t h e n a t u r e of t h e carbodiim.i.de u s e d ,
c o n t a i n i n g a p r o t o n donor group.
2 ) The o p t i m a l p H r a n g e d e p e n d s on t h e n a t u r e of a n u c l e o p h i l i c aqent.
3 ) The s t r o n g e s t n u c l e o p h i l i c a g e n t p r e s e n t i n t h e r e a c t i o n
m i x t u r e i s a l w a y s i n v o l v e d i n t h e c o n d e n s a t i o n . A l l o u r d a t a about
r e a c t i v i t y of d i f f e r e n t n u c l e o p h i l i c a g e n t s a l l o w u s t o a r r a n g e
t h e investiqated nucleophiles according t o t h e i r a b i l i t y t o attack
t h e EDC-phosphate a d d u c t i n t h e f o l l o w i n g o r d e r : a m i n e s , N-hydro-
x y b e n z o t r i a z o l e , 2-hydroxypyridine
phate
s i t i v i t y " t o t h e s t r e n g t h of a n u c l e o n h i l e e n a b l e s u s t o s u q q e s t
a r e a c t i o n p r o c e e d i n g a c c o r d i n q t o t h e SN2 mechanism v i a a t r a n s i t i o n s t a t e of t h e t r i q o n a l hipyramid s t r u c t u r e [ 1 9 ] .
The d e v e l o p e d method e x h i b i t s h i g h s e l e c t i v i t y of m o d i f i c a t i o n .
Owinq t o t h e f a c t t h a t i n t e r n u c l e o t i d e p h o s n h a t e s a r e n o t a f f e c t e d , n o t o n l v 3 ' - and 5 ' - t e r m i n a l p h o s p h a t e s of o l i q o d e o x y r i b o n u c l e o t i d e s h u t a l s o 5 ' - t e r m i n a l Dhosphates of o l i g o r i b o n u c l e o t i d e s
may b e e a s i l y m o d i f i e d .
The s i m p l i c i t y and t h e o n e - s t e p c h a r a c t e r o f t h e method a l l o w
u s t o recommend it f o r d e r i v a t i z a t i o n o f s y n t h e t i c and n a t u r a l
D N A s and R N A s a l o n g w i t h t h e method b a s e d on i m i d a z o l i d e i n t e r m e -
d a i t e s [ 2 0 1 . Moreover, t h e u s e of t h e w a t e r - s o l u b l e c a r b o d i i m i d e
a s a c o n d e n s i n q a g e n t i n o l i g o - o r p o l y n u c l e o t i d e c h e m i s t r y seems
t o b e more p e r s p e c t i v e , b e c a u s e i t p e r m i t s t o prenare n o t o n l y
phos?horamidate b u t n h o s p h o d i e s t e r d e r i v a t i v e s i n c l u d i n g d e r i v a t i v e s w i t h polymer s u p p o r t s [ 2 1 , 2 2 1
REFERENCES
1 . L.V.
2.
Mochalova, I . N .
J.
Mol. R i o l .
A.
Chollet, E.H.
(1985).
S h a t s k y , A.A.
159, 6 3 7 - 6 5 0
Boqdanov, V . D .
Vasiliev,
(1982).
Kawashima, N u c l . A c i d s R e s .
2, 1529-1541
933
3. V . V .
V l a s s o v , S.A.
Gaidamakov, V.V.
FEBS L e t t . l&,
415-418
4 . Z.A.
Shabarova, M.G.
Lett.
154, 288-292
Gorn, S.A.
I v a n o v s k a y a , M.G.
I s a g u l i a n t s , FEBS
(1983).
5. G . L a n c e l o t , U. A s s e l i n e , N . T .
m i s t r y 2,2521-2529 (1985).
Thuong, C. Helene, B i o c h e -
Some R e c e n t Development i n t h e C h e m i s t r y o f
6 . H.G,Khorana,
P h o s p h a t e E s t e r of B i o l o g i c a l I n t e r e s t , Ed.
Shabarova,
Z.A.
1964, p p . 146-162.
M i r , MOSCOW,
Grachev,
(1985).
1977,
Ja-
46,
669-676.
pp.
8. M.
W i l c h e k , Academic P r e s s , N e w York,
Colowick, N . O .
5,
pp. 645-669.
MeIm,
1-methylimidazole:
-sulfonyl;
13. N.V.
nov,
Bulychev, A.V.
Yu.E.
Dans, 5-dimethylaminonaphthalene-l-
DMFA, N,N-dimethylformamide.
Lebedev, L . Z .
N e s t e r i k h i n , S.Yu.
Benimetskaya, A.L.
Novozhilov, S.G.
Kozio-
Raytian,
15. W . J .
Rkzyzosiak, J. B i e r n a t , J. C i e s i o k a , P. G o r n i c k i , M.Wi-
ewiorowski, Nucl.
16. G.A.
Dobronravova, S.A.
B i o o r g a n . Khimiya
17. V . V .
Acids R e s .
8,
3,
1663-1674 (1979).
Zykov, A . A .
Mustaev, G.A.
Nevinski,
1349-1357 (1982).
Shumyantseva, N . I .
Acids R e s .
2,
Sokolova, Z.A.
S h a b a r o v a , Nucl.
903-916 (1976).
O r g e l , Nucl.
Acids R e s .
It,
9 34
I V A N O V S K A Y A , GOTTIKH, A N D S H A B A R O V A
21.
P.T.
22.
N.W.Y.
Gilham, Biochemistry
Ho, R . E .
67 (1981).
2,
2809-2813
(1968).
2, 6 4 -