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Nucleosides and Nucleotides


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Modification of Oligo (Poly) Nucleotide


Phosphomonoester Groups in Aqueous
Solutions
a

M. G. Ivanovskaya , M. B. Gottikh & Z. A. Shabarova

Department of Chemistry, and A.N. Belozersky Laboratory


of molecular Biology and Bioorganic Chemistry, Moscow State
University, Moscow, 119899, USSR
Version of record first published: 13 Dec 2006.

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

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NUCLEOSIDES & NUCLEOTIDES, 6(5), 913-934 (1987)

MODIFICATION O F OLIGO(P0LY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS


I N AQUEOUS SOLUTIONS
M.G.

I v a n o v s k a y a , M.B.

G o t t i k h and Z.A.

Shabarova

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Department o f C h e m i s t r y , and A . N . B e l o z e r s k y L a b o r a t o r y o f Mol e c u l a r B i o l o g y and B i o o r g a n i c C h e m i s t r y , Moscow S t a t e U n i v e r s i t y , Moscow 119899, U S S R


ABSTRACT. S e l e c t i v e m o d i f i c a t i o n o f o l i g o ( p o 1 y ) n u c l e o t i d e phosphoc,onoester g r o u p s i n an aqueous medium by N- (3-dimethylaminopropy1)-N'-ethylcarbodiimide i n t h e p r e s e n c e of v a r i o u s n u c l e o p h i l i c a g e n t s h a s been i n v e s t i g a t e d . Optimal c o n d i t i o n s o f t h e
m o d i f i c a t i o n by amino- and hydroxycompounds h a v e been f o u n d .
Based on t h e s e s t u d i e s a g e n e r a l e f f i c i e n t method f o r p r e p a r a t i o n of o l i g o ( p o 1 y ) n u c l e o t i d e phosphoamidates and p h o s p h o d i e s ters i n a n aqueous s o l u t i o n h a s been d e v e l o p e d . The method a l lows t o p r e p a r e b o t h oligodeoxyribonucleotide d e r i v a t i v e s a t
3 ' - and 5 ' - t e r m i n a l p h o s p h a t e g r o u p s and o l i g o r i b o n u c l e o t i d e
d e r i v a t i v e s a t 5 ' - t e r m i n a l p h o s p h a t e g r o u p s w i t h 80-100%
yields.
INTRODUCTION

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

and f o r t e r m i n a t i o n o f t r a n s c r i p t i o n [51. For e x t e n s i v e a p p l i c a t i o n o f t h e above 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 i n d i f f e r e n t


m o l e c u l a r b i o l o g y i n v e s t i g a t i o n s it i s n e c e s s a r y t o e l a b o r a t e
a n e f f i c i e n t and r a t h e r s i m p l e method f o r i n t r o d u c t i o n o f v a r i o u s n o n - n u c l e o t i d e r e s i d u e s i n t o o l i g o n u c l e o t i d e s . The c r u c i a l
s t e p i n preparing oligonucleotide derivatives a t the terminal
p h o s p h a t e c o n s i s t s i n s e l e c t i v e a c t i v a t i o n o f phosphomonoester
g r o u p s of o l i g o ( p o 1 y ) n u c l e o t i d e s .
The c a r b o d i i m i d e method, e v o l v e d by H. Khorana 161, a p p e a r s
t o b e t h e most s u i t a b l e p r o c e d u r e f o r m o d i f i c a t i o n of phosphomonoester g r o u p s . K h o r a n a ' s methodology w a s b a s e d on u t i l i z a t i o n
of dicyclohexylcarbodiimide i n a n h y d r o u s o r s l i g h t l y a q u e o u s

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

IVANOVSKAYA, GOTTIKH, A N D SHABAROVA

bases [lo], cleavage and izomerization of internucleotide bonds


in RNA [ I l l take place in an anhydrous medium. To solve these
problems it appeared tempting to evolve a carbodiimide activation strategy for modification of phosphomonoesters in an
aqueous medium.
In the present communication a thorough investigation of
oligonucleotide phosphomonoester groups modification,induced
by N- (3-dimethylaminopropyl)-N'-ethylcarbodiirni.de hydrochlori-

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de (EDC), in the presence of different nucleophilic agents is


described. This investigation has made it possible to suggest
a general and effective method for synthesizing a wide range
of oligonucleotide derivatives in an aqueous medium.
MATERIALS AND XETHODS
Reagents and enzymes. Nucleoside-5'-phosphates were obtained
from "Sigma"; d (TGGCCAAGCTp), d (TTGAATGp) were kindly provided
by T.S. Oretskaya (Moscow State University) ; (pU)

(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)

from "Chemapol" ; methanol,

ethanol, n-propanol, 2-aminoethanol, ethyleneglycol - from


"Soyuzkhimreaktiv", USSR. 2-(N-Dans)-aminoethanol was synthesized as described earlier [13]. Bacterial alkaline phosphatase
and snake venom phosphodiesterase ( P D E ) were obtained from
"Worthington Biochemicals Corporation".
Chromatography and electrophoresis. Electrophoresis was performed on FN-1 paper at 900-1000 v in 0.05 M triethylammonium
bicarbonate buffer, pH 7 . 5 , for 1.5-2 h using "Labor" apparatus.
Chromatography was carried out on Lichrosorb-NH2 columns
(1x50 rim) in a linear gradient of sodium phosphate, pH 7 . 5 ,
in 7 M urea, and on Nucleosil C18 columns (2x62 mm) in a linear
gradient of methanol in 0.1 M ammonium acetate, pH 6.0, using
HPL chromatograph "Milichrom", USSR. Gel-filtration was accomplished on Biogel P-2 (200-400 mesh, "Bio Rad"). Paper chromatography was carried out on FN1 paper in the systemS:ethanol-I M
ammonium acetate, p H 7 . 5 (7:3 v/v) (System A) and ethanol-I M
ammonium acetate, pH 7 . 5 (3:2 v/v) (System B ) .

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OLIGO(P0LY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS

915

Buffers. To maintain stable pH values during modification


procedures the following buffers were used: pH 1- 0.1 N aqueous
HC1; pH 2 - 0.01 M aqueous HC1; pH 3-6 - 0.4 Iy MES-buffers,
pH 7-9 - 0.4 M MeIm-buffer.
General procedure for preparation of mononucleotide phosphoamidates.
An aqueous solution of amine (100 wl of 3 M solution) pretitrated to pH 4.5-5.5 by 6 N aqueous HC1 was added
to a water-soluble salt of the mononucleotide (1-5 wmol). Then
9.6 mg (50 pmol) of EDC was added and the reaction mixture was
stored at 2OoC for the time given in Table 1. The phosphoamidates
were isolated by paper electrophoresis or by paper chrsmatography in system A. The yields of pA phosphoamidates are given in
Table 1.
General procedure for preparation of mononucleotide derivatives with simple alcohols. An alcohol solution ( 1 0 0 p l of 3-6 M
solution) in 0.4 M MES buffer (pH 4.5-5.5) was added to watersoluble salt of the mononucleotide (1-5 wmol). Then 9.6 mg
(50 pmol) of EDC was added and the reaction mixture was stored
at 2OoC for the time given in Table 1. The mononucleotide phosphodiesters were isolated by paper electrophoresis. The yields
of PA phosphodiesters are given in Table 1.
Preparation of 2-(N-Dans)-aminoethy1 esters of mononucleotides. 44 mg (0.15 mmol) of 2-(N-Dans)-aminoethanol, dissolved
in 30 p l of DMFA [12], was adjusted to pH 3.0 by adding 20 wl
of 6 N aqueous HC1. This solution was added to a water-soluble
salt of a mononucleotide (1-5 pmol) in 40 p1 of 0.4 M MES buffer,
pH 5.5. The resultant solution (pH 4.5-5.0) was supplemented
with 9.6 mg (50 pmol) of EDC and incubated at 10C for 24 hours.
The 2-(N-Dans)-aminoethyl esters of mononucleotides were isolated by paper chromatography in system B. For 2-(N-Dans)-aminoethyl ester of pA R was 0.79, and the yield - 40%.
f
Preparation of 4-nitrophenyl esters of mononucleotides.
The solution of 4-nitrophenol (50 p1 of 6 M solution in DMFA-water (1:1, V/V) , pretitrated to pH 6.5-7.0 by 6 N aqueous HC1,
was added dropwise to a water-soluble salt of a mononucleotide
(1-5 pmol) dissolved in 50 p 1 of DMFA-water (l:l, v/v). The resulting mixture was supplemented with 9.6 mg (50 wmol) of EDC
and allowed to stay at 10C for 20-24 h. 4-Nitrophenyl esters
of pA and pC wereisolated by paper chromatography in System B.
The yields were 95-100%. When the reaction of p U , dpT and pG with
4-nitrophenol was over, the excess of 4-nitrophenol was extracted by chloroform and the water fraction was evaporated in vacuum, dissolved in 0.2 M of Na2C03 (pH 10.5) and incubated for

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

2-hydroxyethyl ester of bromoacetic


acid

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

glycine methyl ester

3.0

Nuc leophile
concentration,

4.5

PH

aniline
imidazole

Nucleophilic agent

PREPARATION OF p A PHOSPHOAMIDATES AND PHOSPHODIESTERS UNDER OPTIMAL REACTION CONDITIONS


(CONCENTRATION OF p A - 0.02 M , EDC - 0.5 M )

TABLE 1 .

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917

OLIGO(POLY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS

24 h at 2OoC (or for 6-10 h at 37OC). 4-Nitrophenyl esters


of pU, pT and pG were isolated by paper chromatography in system B. The yields were 75-80%.
Preparation of N-hydroxybenzotriazole esters of mononucleotides. 40 mg (0.3 mmol) of N-hydroxybezotriazole was dissolved

of DMFA-water mixture (4:1, v/v). Then 6 ell of 2 N


NaOH was added under stirring to adjust pH of the solution to
4.0-4.5. This mixture was added to a solution of a water-soluble
salt of the nucleotide (1-5 hmol) dissolved in 40 h l DMFA-water

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in 50

p1

( 3 : 1 , v/v). The reaction mixture was shaked, supplemented with

EDC (9.6 mg, 50 p m o l ) and allowed to stay at 5OC for 3.5-4.0


hours. N-hydroxybenzotriazole esters of mononucleotides were
isolated by paper chromatography in System B. The yields were
90-95%.
General procedure for preparation of oligo(po1y)nucleotide
phosphoamidates. A water solution of amine (50 p l of a 3 M
solution) pretitrated by 6 N aqueous HC1 to pH 4.5-5.5 was
added to a water-soluble salt of the oligo(po1y)nucleotide

pmol). The reaction mixture was supplemented with


4.8 mg (25 bmol) of EDC and allowed to stay at 4C for the time
indicated in Table 2 . The excess of the reagents was separated
by gel-filtration on Biogel P-2, and the oligonucleotide phos(O.OO?-O.Ol

phoamidates were isolated by HPLC on nucleosil C18 columns.


Conversion of phosphomonoester groups of oligonucleotides is presented in Table 2.
General procedures for preparation of oligonucleotide derivatives with simple alcohols. A water-soluble salt of oligo(p01y)nucleotide (0.001-0.1 bmol) was supplemented with 50 1.11of a
3-6 M alcohol solution in 0.4 M MES-buffer,

containing 2 M

MgC12, pH 4.5-5.5, and 4.8 mg (25 bmol) of EDC. The reaction


mixture was incubated at 4'C for the time indicated in Table 2.
After gel-filtration on Biogel P-2, the oligonucleotide phosphodiesters were isolated by HPLC on Nucleosil C18. Conversion of
phosphomonoester groups of oligonucleotides is presented in
Table 2.
Preparation of 2-(N-Dans)-aminoethyl ester of d(TGGCCAAGCTp).
d(TGGCCAAGCTp) (0.03 bmol) was dissolved in 40 1.11 of 0.4 M MESbuffer, containing 2 M MgC12, p H 4.0-4.5. The reagents were
added as described above for 2-(N-Dans)-aminoethyl ester of mononucleotides. The reaction mixture was incubated at 4C for
24 h. The reaction product was isolated by gel-filtration on
Biogel P-2 followed by HPLC on Nucleosil C18. The yield was 30%.

( 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

PREPARATION OF OLIGO(P0LY)NUCLEOTIDE PHOSPHOAMIDATES AND PHOSPHODIESTERS UNDER OPTIMAL


REACTION CONDITIONS (CONCENTRATION OF EDC - 0.5 M , TEMPERATURE -4OC)

TABLE 2.

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x-

v1

z
0
.c:

C
P

919

OLIGO(P0LY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS

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.

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RESULTS AND DISCUSSION


Reaction of mononucleotides with amino- and hydroxycompounds
Mononucleotide pA was selected as an object for a detailed
study of the EDC-induced modification of phosphomonoester groups. The heterocyclic base of pA is not modified by carbodiimides
and therefore it becomes possible to investigate only the conversions of the phosphomonoester group.
The characteristic feature of EDC is its ability to exist in
different tautometric forms depending on pH of the reaction medium [ I 4 1 :

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

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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

* . . m9 .mo.wo.r 0-. om.tbn.wm.rm-.

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

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OLIGO(POLY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS

phomonoester group becames wholly undissociate (pK1=l.O, pK2 =


6.5) and thus unable to form an EDC-adduct. The distinguishing
feature of EDC is its ability to induce phosphomonoester group
modification at p H higher than 6 (Table 3 ) . We suggest this ability may be explained by the presence of a strongly basic tertiary aminogroup (pK = 11.1 [I41 ) in its structure. This aminoa
group is always protonated under modification conditions. Apparently at pH > 6 EDC can be activated in consequence of the intramolecular proton transfer from the tertiary nitrogen atom to
the imide one. In this case the phosphomonoester modification
appears to proceed according to the following scheme:

+ /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-

IVANOVSKAYA, GOTTIKH, AND SHABAROVA

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)

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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.

t i o n product. This sharp a l t e r a t i o n i n t h e reaction course i s


c a u s e d b y t h e a p p e a r a n c e i n t h e r e a c t i o n medium o f a d i a n i o n phosphate which is a s t r o n g e r n u c l e o p h i l e t h a n a l c o h o l a n d

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.

I n c o n t r a s t , t h e phenolate i o n is one of t h e stron-

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 -

with no pyrophosphate being formed (Table 4 ) .

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

OLIGO(P0LY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS

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 amines and a l c o h o l s (Table 4 ) .


EDC-induced phosphomonoester g r o u p m o d i f i c a t i o n i n t h e pres e n c e of N-hydroxybenzotriazole and 2-hydroxypyridine p r o c e e d s

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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 ) .

The v e r y h i g h a c t i v i t y o f t h e a b o v e compounds may b e due t o


t h e i r specific electron structure.
An i n v e s t i g a t i o n o f t h e d e p e n d e n c e o f m o d i f i c a t i o n e f f i c i e n c y

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

EFFICIENCY OF EDC-INDUCED MOCIFICATION OF pA BY AMINES AND ALCOHOLS AT VARIOUS REAGENT


CONCENTRATIONS AND TEMPERATURE (PA CONCENTRATION - 0 . 0 2 M, pH - 4 . 5 )

TABLE 5

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OLIGO(P0LY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS

925

with similar functional groups (diamines,glycols) only one


functional group reacts, while the other remains intact (Table
6 ) . The presence of an unsubstituted amino- or hydroxygroup in
such derivatives allows to use them for further derivatization,
for instance, acylation or alkylation. One should point to an
unusual reaction course under pA coupling to ethylenglycol.
In this case not only 2-hydroxy ethyl ester of pA (yield 80-85%)
but also by-product - nucleoside A was formed (yield 15-20%).
An analogous process of the dephosphorylation takes place also
under preparation of oligonucleotide 2-hydroxyethylesters.
At the same time isolated 2-hydroxyethyl esters of mono(o1igo)nucleotides are stable in neutral and slightly acidic solutions.
The dephosphorylation seems to occur at the stage of interaction
of ethylenglycol with EDC-PA-adduct.
As for bifunctional reagents containing different functional
groups, the strongest nucleophilic group interacts first of all
with a preactivated phosphomonoester group. For example, in an
EDC-induced coupling of pA to tyrosine methyl ester or cysteine
methyl ester, only phosphoamidates but not phosphodiester or
phosphothiol are formed. No variations in modification conditions - pH values of the reaction mixtures or the reagent concentrations - altered the reaction course.
Isolation of mononucleotide derivatives, their structure
Phosphoamide and phosphodiester derivatives of pA were isolated by electrophoresis on paper at pH 7.5 or by paper chromatography. The anilide, imidazolide, morpholide, benzylamide,
butylamide of PA; and methyl, ethyl, propyl esters of pA were
found identical to compounds previously synthesized in our laboratory 1171. The presence of an unsubstituted amino group in
aminoethylamide and aminobutylamide of pA was proved by a colour
reaction with ninhydrin.
The structure of FA derivatives with amino acid methyl esters
was proved by acidic hydrolysis followed by amino acid assay on
an amino acid analyzer. The nucleotide amount was determined
spectrophotometrically. The nucleotide - amino acid molar ratio
was close to an equimolar one.
The structure of 2-cyanoethvl ester of pA was confirmed by alkaline hydrolysis (conc. ammonium hydroxyd, 37"C, 24h) and by
snake venom PDE hydrolysis. The structure of 4-nitrophenyl,
2-(N-Dans)-aminoethyl esters of pA and also of pA derivatives
with 2-hydroxvpyridine and N-hydroxybenzotriazole was determined
by snake venom PDE hydrolysis followed by separation of the hyd-

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

serine methyl ester

tyrosine methyl ester

cysteine methyl ester

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

Type of synthesized bond

3.0

Process
time,
hours

4.5

pH

6.0

3.0

Reagent
concentration,
M

ethylenediamine
1,4-diaminobutane
ethyleneglycol

Bifunctional reagent

EDC-INDUCED MODIFICATION OF PA IN THE PRESENCE OF BIFUNCTIONAL REAGENTS (CONCENTRATION OF


p A - 0.02 M , EDC - 0.5 M, TEMPERATURE - 20C)

TABLE 6

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rj
H

rj

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OLIGO(P0LY)NUCLEOTIDE

PHOSPHOMONOESTER GROUPS

I
I
46000

I
I
42000

927

78 000

f 4 000

I
70000

Figure 1. UV-spectra of 4-nitrophenyl ester of pA


() , starting PA (--and
+nitrophenol (- - - -)

rolyzate hy Daner electrophoresis. The pA - alcohol molar ratios


determined spectrophotometrically were 1:0.97, 1:0.98, 1:0.37,
1 : 1 . 0 5 consequently. UV spectra of synthesized pA phosphodiesters are shown in Figures 1-4.
Reactions of oligonucleotides with amino- and hydroxycompounds

The chemical nature of phosphomonoester groups in mono- and


oliqonucleotides is identical, and therefore the results obtained
for mononucleotides have been used as a basis for selective modification of oligo(po1y)nucleotide.s. Reaction conditions determined
for mononucleotides have proved to be wholly applicable to EDCinduced couplinq of oligonucleotides to amines. A number of deoxyribo- and ribooliqonucleotide phosphoamidates have been synthesized under above conditions (Table 2).
In contrast to the reaction with amines, the reaction with alcohols has proved to be more complex. Noticeable modification of

IVANOVSKAYA, GOTTIKH, A N D SHABAROVA

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928

46000

42000

I
I
78000

foooo

74000

y, C M '

Figure 2 . UV-spectra of 2-(N-Dans)-arninoethyl


Of pA (

) , starting pA (-2-(N-Dans)-a~ninoethanol ( -)

--

ester

and

h e t e r o c y c l i c b a s e s (U,T and G ) i n o l i g o n u c l e o t i d e s by EDC t a k e s


p l a c e u n d e r c o n d i t i o n s when t h e s e b a s e s a r e not m o d i f i e d i n monon u c l e o t i d e s (pH 5 . 5 , 4OC, 16 h )

. 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

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OLIGO(POLY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS

I
I
46000

42 000

78 000

y.000

000

Figure 3. UV-spectra of pA derivative with 2-hydroxypyridine ( ) , starting PA (---)


and 2-hydroxypyridine ( - - - )

2+

of oligonucleotides to form tiqht ionic p i r s with Mg


ions [18].
If the reaction is carried out at ?H 4 5.5, but in the presence
of 2 M MqC12 and at 4C, heterocyclic bases modification does not
exceed 5-10% after 24 h. To synthesize 4-nitrophenyl esters of
oligonucleotides the reaction has to be carried out at H > 6
(see the mononucleotide reactions). In this case partial heterocyclic bases modification takes place. For oligodeoxvribonucleotides it is possible to eliminate this modification at conditions
found for mononucleotides. Since oligoribonucleotides incubated
at p H 10.5 are hydrolvzed, a different approach is needed for
preparation of 4-nitrophenyl esters of oliqoribonucleotides containing U and G residues.
The developed method is the only one for preparation of o l i g o nucleotide phosphodiester in an aqueous medium. We have synthesized a wide number of oligonucleotide derivatives with different

IVANOVSKAYA, GOTTIKH, AND SHABAROVA

9 30

74

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4,O

95

0
I

46 000

42000

74 000

78 000

000

9,CM-

Figure 4. UV-spectra of pA derivative with N-hydroxybenzotriazole (


) , starting pA (--)
and N-hydroxybenzotriazole ( - - - )

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

OLIGO(POLY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS

a
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Figure 5. Chromatography of the reaction mixtures after


synthesis of N-hydroxybenzotriazole ester of
( P U ) ~on Nucleosil C 1 8 (a) and aminoethylamide of (pU)5 on Lichrosorb-NH2 (b)
(---)
chromatography of the starting ( P U ) ~ ;
(-)
chromatography OP the reaction mixtures,
peak 1
N-hydroxybenzotriazole ester of (pU)*,
peak 2
aminoethylamide of (pUI5.

932

IVANOVSKAYA, G O T T I K H , AND SHABAROVA

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 -

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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-

> 4-nitrophenol > dianionphos> a l c o h o l > w a t e r > monoanionphosphate. T h i s r e a c t i o n " s e n -

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 .

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