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Modification of Oligo (Poly) NucleotidePhosphomonoester Groups in AqueousSolutionsM. G. Ivanovskaya a , M. B. Gottikh a & Z. A. Shabarova aa Department of Chemistry, and A.N. Belozersky Laboratoryof molecular Biology and Bioorganic Chemistry, Moscow StateUniversity, Moscow, 119899, USSRVersion 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

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

MODIFICATION OF OLIGO(P0LY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS I N AQUEOUS SOLUTIONS

M.G. Ivanovskaya, M.B. G o t t i k h and Z.A. Shabarova

Department o f Chemistry, and A . N . Belozersky Labora to ry o f Mo- l e c u l a r Biology and B ioorgan ic Chemistry, Moscow State Univer- 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 phos- phoc,onoester groups i n an aqueous medium by N- (3-dimethylamino- propy1)-N'-ethylcarbodiimide i n t h e p r e s e n c e of v a r i o u s nuc leo - 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 have been found. 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 repa ra - 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 phosphodies- ters i n an aqueous s o l u t i o n h a s been developed. 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 phosphate g roups 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 phosphate groups w i t h 80-100% y i e l d s .

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-nucleo- t i d e groups c o v a l e n t l y bound t o t e r m i n a l phosphate 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 chemica l 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 chemica 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 mo lecu la 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 an e f f i c i e n t and r a t h e r s imple method f o r i n t r o d u c t i o n o f v a r i - ous non-nuc leo t ide 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 p r e p a r i n g 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 a t t h e t e r m i n a l phospha te 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 groups of o l i g o ( p o 1 y ) n u c l e o t i d e s .

The ca rbod i imide method, evo lved by H. Khorana 161, a p p e a r s t o be t h e most s u i t a b l e p rocedure f o r m o d i f i c a t i o n of phospho- monoester g roups . Khorana 's methodology wasbased on u t i l i z a t i o n of dicyclohexylcarbodiimide i n anhydrous o r s l i g h t l y aqueous

s o l u t i o n s [ 7 - 9 1 . The u s e o f an anhydrous 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 because u n p r o t e c t e d o l i g o - and 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 o f h e t e r o c y c l i c

913

Copyright 0 1987 by Marcel Dekker, Inc.

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914 IVANOVSKAYA, GOTTIKH, AND 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 activa- tion 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- 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 amine, n-butylamine, ethylenediamine and 4-nitrophenol were obtained from "Werck" ; 2-hydroxypyridine - from "Aldrich" ; glycine methyl ester, serine methyl ester, tyrosine methyl es- ter, cysteine methyl ester - from "Koch-Light" : N-hydroxyben- zotriazole, 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 synthesi- zed as described earlier [13]. Bacterial alkaline phosphatase and snake venom phosphodiesterase (PDE) were obtained from "Worthington Biochemicals Corporation".

(10 wm); aniline, imidazole, morpholine, benzyl-

Chromatography and electrophoresis. Electrophoresis was per- formed 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 accomp- lished on Biogel P-2 (200-400 mesh, "Bio Rad"). Paper chromato- graphy 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 phospho- amidates. An aqueous solution of amine (100 wl of 3 M solu- tion) 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 chrsmatogra- phy in system A. The yields of pA phosphoamidates are given in Table 1.

General procedure for preparation of mononucleotide deriva- tives with simple alcohols. An alcohol solution (100 p l of 3-6 M solution) in 0.4 M MES buffer (pH 4.5-5.5) was added to 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 mononucleotide phos- phodiesters were isolated by paper electrophoresis. The yields of PA phosphodiesters are given in Table 1.

Preparation of 2-(N-Dans)-aminoethy1 esters of mononucleoti- - des. 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 10°C for 24 hours. The 2-(N-Dans)-aminoethyl esters of mononucleotides were isola- ted by paper chromatography in system B. For 2-(N-Dans)-amino- ethyl 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-wa- ter (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 re- sulting mixture was supplemented with 9.6 mg (50 wmol) of EDC and allowed to stay at 10°C 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 extrac- ted by chloroform and the water fraction was evaporated in va- cuum, dissolved in 0.2 M of Na2C03 (pH 10.5) and incubated for

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TABLE

1.

PREP

ARAT

ION OF

pA

PHO

SPHOAMIDATES AND PHOSPHODIESTERS UNDER OPTIMAL

REAC

TION CONDITI

ONS

(CONCENTRATION OF

pA

- 0

.02

M

, EDC

- 0.

5 M)

Nucl

eoph

ilic

agent

Nuc leophile

Proc

ess

Yield of

PH

concentration,

T°C

time

, p

A deriva-

M

hours

tive

, %

anil

ine

imid

azol

e gl

ycin

e me

thyl

est

er

morp

holi

ne

benz

ylam

ine

buty

lami

ne

4.5

3.0

6 .O

3.0

4.5

3.0

5.0

3

.0

4.5

3.0

5.0

3.

0

20

1

20

1

20

1

20

2

20

4

20

6

95

90

85

90

85

80

~

__

__

meth

anol

et

hano

l n-

prop

anol

3-

hydr

oxyp

ropi

noni

trile

2- (N-

Dans

) -am

inoe

than

ol

4-n it rop

heno

l N-

hydr

oxyb

enzo

tr

iazole

2-hy

drox

ypyr

idin

e 2-

hydr

oxye

thyl

est

er of

bromoacetic

acid

4.5

4.5

4.5

4.5

4.5

6.5

4.5

5.0

4.5

6 .O

3.0

3.0

4.0

1.5

3.0

3 .O

3.0

3.0

20

20

20

20

10

10

5 5

10

2 2 2

4

24

24

4 2 6

80

85

80

90

40

95

95

90

90

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OLIGO(POLY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS 91 7

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 sys- tem B. The yields were 75-80%.

Preparation of N-hydroxybenzotriazole esters of mononucleo- tides. 40 mg (0.3 mmol) of N-hydroxybezotriazole was dissolved in 50 p 1 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 ( 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 pl 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 (O.OO?-O.Ol pmol). The reaction mixture was supplemented with 4.8 mg (25 bmol) of EDC and allowed to stay at 4°C 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-

phoamidates were isolated by HPLC on nucleosil C18 columns. Conversion of phosphomonoester groups of oligonucleotides is pre- sented in Table 2.

General procedures for preparation of oligonucleotide deriva- tives with simple alcohols. A water-soluble salt of oligo(p0- 1y)nucleotide (0.001-0.1 bmol) was supplemented with 50 1.11 of 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 phospho- diesters 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 MES- buffer, containing 2 M MgC12, p H 4.0-4.5. The reagents were added as described above for 2-(N-Dans)-aminoethyl ester of mo- nonucleotides. The reaction mixture was incubated at 4°C 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%.

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TABLE

2.

PREP

ARAT

ION

OF OL

IGO(

P0LY)NUCLEOTIDE PHOSPHOAMIDATES AND

PHOSPHODIEST

ERS UN

DER

OPTIMAL

REAC

TION

CONDITIONS

(CONCENTRATION OF

EDC

- 0.5

M, TEMPE

RATU

RE -4

OC)

Olig

o- o

r

Nucleophilic

poly

nucl

eoti

de

agent

d ( TTG

AATG

p)

aniline

imida z

o le

morpholine

1,4-diaminobutane

2-hydroxyethyl ester

of b

romoacetic acid

Nucleotide

concentra-

pH

tion (per

monomeric

union1 ,m

M

4.5

0.2

6.0

0.2

5.0

0.2

4.5

0.2

~ ~

~~~~

Nucleoph

i- Pr

oces

s Co

nver

sion

le con-

time,

of p

hospho

- centrati

on, hour

s mono

este

r M

grou

ps,

%

3.0

2 95

3.0

4 90

3.0

6

95

3.0

8

90

4.5

0.2

3.0

6

80

d (T

GGCC

AACG

Tp)

imidazole

6.0

0.2

3.0

4 90

ethylenediamine

4.5

0.1

3.0

2 95

ethanol

4.5

0.1

6 -0

6 98

ethylene g 1 yc o 1

4.5

0.1

6 .O

6

95

2- (N-Dans) amino-

ethanol

4.0

0.1

1.5

24

30

N-hydroxybenzotriazole

4.5

0.1

3.0

4 95

N-hydroxybensotriazole

4.5

0.2

3.0

4 95

ethylenediamine

4.5

0.2

3.0

2 100

(PA)

11

4-nitrophenol

6.5

0.1

3.0

24

70

e thy lenediamine

4.5

0.1

3 -0

2 95

(Pa 3o

et

hylenediamine

4.5

0.03

3.0

2 90

H

C

P z

0

.c:

v1 x x- 4

P

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OLIGO(P0LY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS 919

Preparation of N-hydroxybenzotriazole esters of (PU)~,- d(TGGCCAAGCTp). 0.01-0.02 pmol of the oligonucleotide was dis- solved 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 descri- bed above €or hydroxybenzotriazole ester of mononucleotides. The reaction mixture was incubated at 4°C for 3 h. The products were isolated by gel-filtration on Bioqel P-2 with 90% yields.

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 gro- ups. The heterocyclic base of pA is not modified by carbodiimides and therefore it becomes possible to investigate only the con- versions of the phosphomonoester group.

The characteristic feature of EDC is its ability to exist in different tautometric forms depending on pH of the reaction me- dium [ I 4 1 :

c2H5N=c=N> HN + H+ c,n,Nn-$NHJ ‘i

/ \ HjC CH,

/ \

H,C CH, c 2 H,N=,<! HJ N T - / \

H,C CH,

For this reason EDC-induced reactions are usually pH-depen- dent [ 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 phos- phoamidates 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-

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920

O\O

c 0 s .rl .!Ju rd 0 -4 w 4 a 0 5

E r .

% w 0 a , I a, h 7 tn a, n

IVANOVSKAYA, GOTTIKH, AND SHABAROVA

I 7 P

I U 7

I U

C

I

0 0 0 0 0 0 - 7 . 7 .

0 0 0 0 0 0 7 - c

0 0 0 0 0 0 7 - 7

m o o m

m

0 0 I - 7 0

r- o o m

m 0 0 I r r m

m 0 1 I - 0

o m

m

m m o * . . m e w

m W

a l e c 4 I1 4 .rl a e x rda

0 0 0 0 0 0 0 0 v r - 7

Ln o o o m

m

0 0 0 I r r - 0

Ln ooow 0 0 0 I - t - -o

W

0 0 o o w m 0 0 1 I r r m m

r - m

m o o m w m o m 1 I - o m ~

W N

I I I I

m o o 0 9 m w r - . . . .

0 0 0 0 0 0 0 0

0 0 0 0 0 0 I 7 - 7

Lnm o o m c o

m m 0 0 1 I r - 0 0

m m o o w m 0 0 1 I - - 0 0

w m

m 0 0 - 0 0 1 I - - 0

e

m

o t n m m . . . . m b w r -

o m c o m m o m t n - m o o a i m o & w e

1 1 1 0 1 1 0 1 1 1 0

co rn m a r - w v m o m - m m - m o o

I I I I I I I I I I I

I I I I I I I I I I

o o o m o m m m m o - ~ c o r n m m m m r r 1 ~

1 1 1 1 1 1 1 1 1 1 0 L n o o o o o o o o m

I I I I I I I I I I I

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OLIGO(POLY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS 921

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 abi- lity may be explained by the presence of a strongly basic ter- tiary aminogroup (pK = 11.1 [I41 ) in its structure. This amino- group is always protonated under modification conditions. Appa- rently at pH > 6 EDC can be activated in consequence of the int- ramolecular proton transfer from the tertiary nitrogen atom to the imide one. In this case the phosphomonoester modification

a

appears to proceed according to the following scheme:

+ /CH3

NuH - a nucleophilic agent

This assumption is confirmed by inhibition of the EDC-indu- ced modification at pH>10 (Table 3 ) when the tertiary amine group becomes unprotonated. Besides, carbodiimides whose struc- ture 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 depen- dent 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 pH- value to 7 and higher results in pyrophosphate as the only reac-

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9 22 I V A N O V S K A Y A , GOTTIKH, AND SHABAROVA

TABLE 4

EFFICIENCY OF EDC-INDUCED MODIFICATION O F PA BY HYDROXY- COMPOUNDS AT VARIOUS pH VALUES (CONCENTRATION OF p A - 0 . 0 3 M , HYDROXYCOMPOUND - 3 M, EDC - 0 . 5 M, TEMPERATURE - 20°C)

Degree of pA m o d i f i c a t i o n , % 0 . 5 h 2 h 6 h 16 h 24 h Hydroxycompound p H

n - p r o p a n o l " 1 .O 2.0 3.2 4 . 5 5 . 5 6 .O 7.0 8 .0

0 30-35 30-35 25-30 15-20 10-15

0 0

0 0 80-85 1 0 0 95-100 100

100 100 9 5 1 0 0

90-95 100 0 0 0 0

0 1 0 0 100 100 1 0 0 100 3-5

0

0 100 100 100 100 100 5-10

0

- - - 4 - n i t r o p h e n o l 3 . 5 3-5 5-1 0 5 .0 - - - 15-20 20-25

- - - 90-95 100 6 . 5 pKa =7.15 7 . 0 - - - 60-65 90-95

- - - 45-50 60-65 8 . 0 - - - 10-15 15-20 8 . 4 ~

N-hydroxybenzo- 2 .5 45-50 60-65 90-95 - 100 t r i a z o l e 4 . 0 - 90-95 1 0 0 - - pKa = 4.00 6 . 0 50-55 - 90-95 - 95-97

7 .0 5-10 15-20 35-40 - 35-40 8 . 0 0 0 0 0 -

2 - h y d r o x y p y r i d i n e 3 .0 75-80 100 100 - 100 p K a = 11.65 5.0 65-70 100 1 0 0 - 100

7.: 20-25 90-95 78-83 - 85-90 8 . 0 10-15 70-75 30-35 - 47-52 9 . 0 3-5 60-65 15-20 - 40-45

* A similar d e p e n d e n c e 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 s i m p l e a l c o h o l s .

t i o n p r o d u c t . T h i s s h a r p a l t e r a t i o n i n t h e r e a c t i o n c o u r s e 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 -

p h o s p h a t e w h i c h i s 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 non-

i o n i z e d form. I n c o n t r a s t , t h e p h e n o l a t e i o n i s o n e o f t h e s t r o n -

gest 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 - n i t r o p h e n y l ester of PA. 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 -

l y a t pH 6 .5-7 .0 w i t h n o p y r o p h o s p h a t e b e i n g f o r m e d ( T a b l e 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 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-

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OLIGO(P0LY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS 923

s i b l e a p p a r e n t l y as a r e s u l t of EDC i n a c t i v a t i o n a t pH > 10.

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 by

p h e n o l a t e a n i o n i s impeded by e lec t ros ta t ic r e p u l s i o n o f t h e

same cha rged 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 of pA w i t h 4 -n i t ropheno l i n comparison

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

EDC-induced phosphomonoester g roup m o d i f i c a t i o n i n t h e pre- sence of N-hydroxybenzotr iazole and 2-hydroxypyridine p roceeds

w i t h h igh y i e l d s i n a wide pH-range (from 2 t o 8 , Tab le 4 ) .

The ve ry h i g h a c t i v i t y of t h e above compounds may b e due t o t h e i r s p e c i f i c e l e c t r o n s t r u c t u r e .

An i n v e s t i g a t i o n of t h e dependence 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 of 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 of a n u c l e o p h i l i c a g e n t (1-3 M) and EDC

( 0 . 5 M) i s r e q u i r e d (Tab le 5 ) .

A r ise i n the t e m p e r a t u r e from 4'C t o 50°C 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 -

ves 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, it is n e c e s s a r y

t o t a k e i n t o accoun 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 have found 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 (48 h , 2 O o C ) . A t pH > 6 t h i s mod i f i -

c a t i o n may be 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 above

m o d i f i c a t i o n o f U , T and 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 rocedure 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 24 h a t 20°C).

t h e m o d i f i c a t i o n of t h e phosphomonoester groups i n mononucleot i -

d e s . It i s recommended t o coup le mononucleot ides t o amines ,

s imp le a l c o h o l s , N-hydroxybenzotr iazole 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

view 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); and

t h e o p t i m a l t e m p e r a t u r e i s 4-20°C (Tab le 1 ) . For t h e r e a c t i o n

wi th 4 -n i t ropheno l t h e o p t i m a l p H v a l u e i s 6.5-7.0, wh i l e 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 ) .

Consequent ly , w e have de te rmined t h e o p t i m a l c o n d i t i o n s f o r

Reac t ions of mononucleot ides 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 groups 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

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TABL

E 5

EFFI

CIEN

CY OF

ED

C-IN

DUCE

D MO

CIFI

CATI

ON OF

pA

BY

AM

INES

A

ND

AL

COHO

LS AT V

ARIO

US RE

AGEN

T CO

NCEN

TRAT

IONS

AN

D TE

MPER

ATUR

E (P

A CO

NCEN

TRAT

ION

- 0

.02

M,

pH

- 4

.5)

Am

ine

or

EDC

con-

M

ine

or

alc

oh

ol

ce

ntr

a-

alc

oh

ol

con

cen

t-

tio

n, M

rati

on

,M

eth

an

ol

3 0

.5

3 0

.5

3 0

.5

1 0

.5

1 0

.5

1 0

.3

1 0

.3

0.3

0

.3

0.3

0.

1

TOC

4 20

50

20

50

20

50

20

2

0

~ ~~

~~

Deg

ree

of

pA m

od

ific

ati

on

, %

lh

2

h

4h

6

h

24

h

30-3

5 50

-55

70-7

5 35

-40

45-5

0 30

-35

35-4

0 15

-20

0

42-4

7 90

-1 0

0

70-7

5 45

-50

47-5

2 40

-45

36-4

1

20-2

5 0

90-9

5 1

00

70

-75

55-6

0 50

-52

50-5

5 36

-42

30-3

5 0

10

0

10

0

70-7

5 60

-65

55-6

0 51

-56

40-4

5 35

-40

0

10

0

10

0

70-7

5 70

-75

55-6

0 52

-57

43-4

8 36

-42

0 ~

~~

an

ilin

e

3 0

.5

20

10

0

10

0

10

0

10

0

10

0

0.3

0

.5

20

95

9

5

95

95

9

5

eth

yle

ned

iam

ine

3 0

.5

20

100

10

0

10

0

10

0

10

0

0.3

0

.5

20

80

-85

90

90

90

90

ben

zyla

min

e 3

0.5

20

75

-80

85-9

0 1

00

1

00

10

0 0

.3

0.5

20

25

-30

30-3

5 45

-50

55-6

0 85

-90

bu

tyla

min

e 3

0.5

2

0

30-3

5 6

0-6

5

80

-85

9

5-1

00

10

0 1

0.5

20

10

-15

55-6

0 60

-65

95

0

.3

0.5

2

0

5 45

-50

50-5

5 - -

80-8

5 h

z

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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 conditi- ons - pH values of the reaction mixtures or the reagent concent- rations - altered the reaction course.

Isolation of mononucleotide derivatives, their structure Phosphoamide and phosphodiester derivatives of pA were iso-

lated by electrophoresis on paper at pH 7.5 or by paper chroma- tography. The anilide, imidazolide, morpholide, benzylamide, butylamide of PA; and methyl, ethyl, propyl esters of pA were found identical to compounds previously synthesized in our labo- ratory 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.

kaline 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-

The structure of 2-cyanoethvl ester of pA was confirmed by al-

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TABLE 6

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

pA

- 0.02

M, EDC -

0.5

M, TEMPERATURE -

20°C)

Reagent

Process

Type of

synthe-

Yield of

PA

time,

sized bond

derivative,

% Bifunctional reagent

concentration,

pH

M hours

ethylenediamine

3.0

4

.5

1,4-diaminobutane

3.0

4.5

serine methyl ester

3.0

2.0

ethyleneglycol

6.0

5.0

4.5

6.0

tyrosine methyl ester

3.0

1.5

1.5

5 .O

cysteine methyl ester

3.0

2.0

5.0

8.0

1 6 6 2 2 6

2 24 2 2 6

P-N

P-N

P-0

P-N

P-N

P-N

P -N

P-N

P-N

P-N

P-N

97

95

85

95

90

95

85

30

95

100

70

0

0

rj

rj

H

x

2

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OLIGO(P0LY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS 927

I I I I I I I I I I 46000 42000 78 000 f 4 000 70 000

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 .05 consequently. UV spectra of synthesized pA phosphodies- ters 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 modi- fication of oligo(po1y)nucleotide.s’. Reaction conditions determined for mononucleotides have proved to be wholly applicable to EDC- induced couplinq of oligonucleotides to amines. A number of deo- xyribo- and ribooliqonucleotide phosphoamidates have been synthe- sized under above conditions (Table 2).

In contrast to the reaction with amines, the reaction with al- cohols has proved to be more complex. Noticeable modification of

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928 IVANOVSKAYA, GOTTIKH, AND SHABAROVA

I I 1 I I I I I I 1 J 46000 42000 78000 74000 foooo - y, C M '

Figure 2. UV-spectra of 2-(N-Dans)-arninoethyl ester Of pA ( - ) , starting pA (-- 4 and 2-(N-Dans)-a~ninoethanol ( - -- -) .

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 under 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 modif ied i n mono-

n 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 be caused by 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 po lvan ion 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

no lyan ion sugar-phosphate bone. The r e s u l t i s an 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 ac- c e l e r a t i o n . Remarkably, no such 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 ba-

ses m o d i f i c a t i o n i s obse rved d u r i n g o l i q o n u c l e o t i d e ahosphoami- d a t e p r e u a r a t i o n . P robab ly , wro tona ted amine ( i t s c o n c e n t r a t i o n

exceeds 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 and p r o t e c t s it a g a i n s t mod i f i ca - t i o n .

T o lower t h e deg ree 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 syn the -

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 used t h e p r o p e r t y

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OLIGO(POLY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS 929

I I I I I I I I I 1 46000 42 000 78 000 y.000 p 000

L

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

2+ of oligonucleotides to form tiqht ionic pirs 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 4’C, 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 hetero- cyclic bases modification takes place. For oligodeoxvribonucleoti- des 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 con- taining U and G residues.

The developed method is the only one for preparation of ol igo-

nucleotide phosphodiester in an aqueous medium. We have synthesi- zed a wide number of oligonucleotide derivatives with different

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9 30

7 4

4,O

9 5

0

IVANOVSKAYA, GOTTIKH, AND SHABAROVA

I I I I I I 1 I I I

46 000 42 000 78 000 74 000 p 000 - 9, CM-’

Figure 4. UV-spectra of pA derivative with N-hydroxy- benzotriazole ( - ) , starting pA (--- ) and N-hydroxybenzotriazole ( - - - - ) .

type hydrox~~compounds including formerly virtually inaccessible 4 -nitrophenyl esters , N-hydrgenzot r ia zole and 2 -hydroxypyr idine derivatives of unprotected oligonucleotides. Besides, an oligo- nucleotide affinity reaqent, containing the bromoacetic acid resi- due, and fluorescent N-Dans-aminoethyl ester of the decanucleoti- de have been synthesized (Table 2).

.w

- 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-hydro- xybenzotriazole ester of ( P U ) ~ is shown in Firlure 5. The formati- on of phosphoamide or phosphodiester linkaqe at the terminal pho-s-

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OLIGO(POLY)NUCLEOTIDE PHOSPHOMONOESTER GROUPS 931

a 1 I I

Figure 5. Chromatography of the reaction mixtures after synthesis of N-hydroxybenzotriazole ester of ( P U ) ~ on Nucleosil C18 (a) and aminoethylami- de of (pU) 5 on Lichrosorb-NH2 (b) . (---) chromatography of the starting (PU)~; (-) chromatography OP the reaction mixtures, peak 1 - N-hydroxybenzotriazole ester of (pU)*, peak 2 - aminoethylamide of (pUI5.

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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 confirmed by t h e l a c k of h y d r o l y s i s

of 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 phospha ta se .

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 groups 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 compos i t ion i s p r o p o s e d

The fo l lowing 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 have been found:

1 ) Modi f i ca t ion p roceeds 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 . Th 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 ange depends on t h e n a t u r e of a nuc leoph i -

l i c a q e n t .

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

mix tu re i s always invo lved 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 i n v e s t i q a t e d n u c l e o p h i l e s a c c o r d i n g t o t h e i r a b i l i t y t o a t t ack

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 : amines, N-hydro-

x y b e n z o t r i a z o l e , 2-hydroxypyridine > 4-n i t ropheno l > d ian ionphos -

p h a t e > a l c o h o l > wate r > monoanionphosphate. T h i s r e a c t i o n "sen-

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 roceed ing acco rd inq 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 developed 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 phosnha te 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 phospha te s of ol iqodeoxyribonuc-

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 be e a s i l y modif ied.

The s i m p l i c i t y and t h e one - s t ep 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 of 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 long w i t h t h e method based on i m i d a z o l i d e in t e rme-

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 condensinq 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 be more p e r s p e c t i v e , because it 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 -

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