Eur. J. Biochem. 24 (1971) 4-11

Monofunctional Substrates of Polynucleotide Phosphorylase The Monoaddition of 2’(3’)-O-Isovaleryl-nucleoside Diphosphate

to an Initiator Oligonucleotide

Gabriel KAUFMANN, Matityahu FRIDKIN, Aliza ZUTRA, and Uriel Z. LITTAUER

Departments of Biochemistry and Biophysics, The Weizmann Institute of Science, Rehovot

(Received August 12/0ctober 2,1971)

Polynucleotide phosphorylase from Escherichia coli cells catalyzes the transfer of a single nucleotidyl residue from each of the 2’(3’)-O-isovalerylesters of ADP, GDP, CDP and UDP to the initiator oligonucleotide, A-A-A. The products of these reactions are identified as the 2‘(3’)-O-isovaleryl esters of the corresponding tetranucleotides A-A-A-A, A-A-A-G, A-A-A-C and A-A-A-U. The 2‘(3‘)-O-isovaleryl residue can be removed from the product by treatment with aqueous methanolic ammonia under conditions that do not significantly damage the oligo- nucleotide chains. The monoaddition of 2‘(3’)-0-acyl esters of nucleoside diphosphates to an oligonucleotide acceptor is proposed as a method for the stepwise synthesis of oligoribonucleotides of defined sequence.

Polynucleotide phosphorylase catalyzes the rever- sible polymerization of ribonucleoside &phosphates. Two functional sites can be defined in the nucleoside- diphosphate substrate. The first site resides in the pyrophosphate moiety of the NDP which is attacked during the polymerization reaction by the terminal 3’-hydroxyl group of the growing end of the poly- nucleotide chain. As a result, inorganic phosphate is released and the chains is lengthened by one unit. The second site is the free 3’-hydroxyl group of the incoming NDP which becomes, in turn, the new accepting terminus [ 11.

We have previously suggested that certain modi- fications of the sugar moiety of NDP may convert these nucleotides to “monofunctional” substrates of polynucleotide phosphorylase [ 11. Such NDP derivatives, blocked in their 3’-hydroxyl-function, should not sustain de novo polymerization but should be able to transfer one nucleotidyl residue to an oligo- nucleotide initiator. Thus, the monofunctional sub- strates should behave as chain terminators which could serve for the stepwise synthesis of polyribo- nucleotides of defined sequence.

Unusual Abbreviations. iVa-ADP, iVa-GDP, iVa-UDP and iVa-CDP, the 2’(3’)O-isovaleryl esters of ADP, GDP, UDP and CDP.

Enzymes. Polynucleotide phosphorylase or nucleoside- diphosphate : polynucleotide nucleotidyl transferase (EC 2.7.7.8) ; spleen phosphodiesterase or orthophosphoric-diester phosphohydrolase (EC 3.1.4.1); alkaline phosphatase or orthophosphoric-monoester phosphohydrolase (EC 3.1.3.1).

DerL)(.(tiou. A,,, unit, the quantity of matcrial contained in 1 ml of a solution which has an absorbance of 1 a t 260 nm, when measured in a 1-cm pathlength cell.

Recent studies have shown that several NDP analogs modified in their sugar moiety can indeed serve as substrates for the reactions catalyzed by polynucleotide phosphorylase. Thus, 2’-O-methyl- ADP and 2’-O-methyl-CDP are polymerized by poly- nucleotide phosphorylase [2,3]. It was also found that dADP participates in the NDP-Pi exchange and in the polymerization reaction of polynucleotide phosphorylase. The polymerization reaction of dADP is limited, however, to the addition of a few deoxyadenylyl residues onto an oligoribonucleotide initiator [4,5]. I n addition, ik was shown that amino- acyl- and N-blocked aminoacyl-tRNAs are phos- phorolyzed by polynucleotide phosphorylase yield- ing the corresponding 2’(3’)-O-aminoacyl and N- blocked aminoacyl derivatives of ADP [i]. The latter finding indicated that 2’(3‘)-0-acyl derivatives of NDP may serve as substrates in the polymerization reaction. Since these derivatives exist a t neutral pH as a mixture of the 2‘ and 3’ isomers [6,7], one cannot expect effective blocking of the 3‘-OH site by direct substitution. Therefore, 2’(3’)-O-acyl-NDP’s should not serve as monofunctional substrates unless the acyl groups is bulky enough to interfere with the 3‘-OH function even when esterified to the vicinal 2’-OH groups.

In the present report we describe the synthesis of various 2’(3’)-O-acyl-NDP7s and their ability to serve as monofunctional substrates for polynucleotide phosphorylase. The polymerization of some of these derivatives was found to be limited to the transfer of a single mononucleotide residue to an oligonucleotide

Vo1.34, No. 1, 1971 G. KAUFMANN, M. FRIDKIN, A. ZUTRA, and U. Z. LITTAUER 5

acceptor. Furthermore, the acyl-blocking group could be easily removed from the oligonucleotide product. These findings show that these derivatives can be used for the stepwise synthesis of oligonucleotides of defined sequence.

phase was then diluted with 0.5 ml of 50°/, acetic acid, applied to paper (Schleicher & Schull, No. 589, green ribbon) and chromatographed for 8 h in sol- vent B. The iVa-ADP band (RF = 0.48) was excised and eluted with water. The solution was passed through a Dowex-EiOXS-H+ column (1 x 5 cm), lyo- phylized to dryness, dissolved in water and adjusted MATERIALS AND METHODS

Solvent Systems for Chromatography Solvent A: ethanol-1 M ammonium acetate

pH 5.5 (50:50, v/v); solvent B : n-butanol-acetic acid-water (5:2:3, by vol.); solvent C: n-butanol- formic acid-water ( 7 : 1 :2, by vol.). High-voltage paper-electrophoresis was performed in “Savant” tanks using “Varsol”, as coolant. pH 1.9 “buffer” contained 7.5O/, (v/v) acetic acid and 2.5O/, (v/v) formic acid. pH 3.5 buffer contained 5 O / , acetic acid which was brought to pH 3.5 with ammonia.

Materials GDP, UDP, CDP and ADP were purchased from

Schwarz BioResearch Inc. while N,N’-carbonyldi- imidazole was obtained from K & K laboratories Inc. Polynucleotide phosphorylase was prepared from Escherichia coli cells according to Kimhi and Lit- tauer [8]. The definition of an enzyme unit as well as the enzyme assay were previously described by these authors [8]. Bovine-spleen phosphodiesterase and E. coli alkaline phosphatase were both obtained from Worthington Biochemical Corp. (Ap),A, (Ap),A and (Ap),A were prepared by mild alkaline hydrolysis of poly(A) [9] followed by removal of the 3’-terminal phosphate by E. coli alkaline phosphatase. The re- sulting (Ap),A mixture was fractionated by paper chromatography using solvent A. The various oligo- nucleotides were further purified by paper electro- phoresis a t pH 1.9. pApA was prepared from poly(A) by sheep-kidney-nuclease digestion [lo].

Synthesis of Esters The synthesis of 2’(3’)-O-isovaleryl NDPs was

adapted from the procedure of Kraevskii et al. [11] for the synthesis of aminoacyl-NMP. The synthesis of 2‘(3’)-O-isovaleryl-ADP was carried out as follows : 102 mg (1 mmol) isovaleric acid in 0.2 ml dimethyl- formamide was reacted with 162 mg ( I mmol) N,N’- carbonyldiimidazole and left for 10 min a t 25 “C after which 0.4 ml of 0.25 M aqueous Li,ADP was added and the incubation was continued for 3-5 h.

The acylation of ADP was followed by thin- layer chromatography on cellulose-coated plates (Riedel de Haen AG-DC plates CEF). Samples of 0.05-0.1 y1 were applied to 1 x5 cm plates which were developed for 5 min in solvent B. iVa-ADP has an RF = 0.5 in this system whereas the unacylated ADP remains close to the origin. At the end of the incubation the reaction mixture was extracted 3 times with 2-ml portions of dry diethyl-ether. The aqueous

- - to pH 6.0 with 1 N NaOH. The yield of iVa-ADP based on A,,, units was 60°/,. The product did not react with periodate and contained one mol hydro- xamate-forming acyl-ester per mol nucleotide [ 121.

The 2’(3’)-0-isovaleryl esters of GDP, UDP and CDP were prepared and characterized by the same procedure as was just described. The yields of the monoacylated derivative of these nucleotides were : 30°/, for iVa-GDP, 30°/, for iVa-UDP and 40°/, for iVa-CDP. Considerable amounts of the diacylated derivatives of GDP, UDP and CDP were obtained in these syntheses but were removed by chromatog- raphy.

The synthesis of 2’(3’)-0-acetyl, propionyl, butyryl, isobutyryl and pivaloyl-NDP was adapted from the procedure of Jencks [I31 for the synthesis of Z’(3‘)-0- acyl-5-adenylates. The synthesis of 2’(3‘)-0-isobutyryl ADP was carried out as follows. To 1 ml of 0.1 M Li,ADP in 2 M imidazole, 50 y1 isobutyric anhydride were addedin 10-20-yl portions over 20-min intervals and the reaction mixture was stirred a t room temper- ature. Acylationwasfollowed by thin-layer chromatog- raphy as described above. Three ultraviolet absorb- ing spots were detected: ADP a t the origin, Z’(3‘)-0- isobutyryl-ADP with an RB = 0.5 and 2’,3’-O-diiso- butyryl-ADP with an RF = 0.8. The reaction was stopped by the addition of 0.1 ml of glacial acetic- acid when the yield of the monoacylated derivative reached a maximum. Further steps were carried out as described for the synthesis of 2’(3‘)-O-isovaleryl- ADP. The yield of 2’(3’)-0-isobutyryl-ADP was 400/,.

The 2’(3’)-O-isovaleryl nucleosides were prepared from the corresponding iVa-NDP’s by treatment with E. coli alkaline phosphatase. The reaction mixture contained 5-10 mM iVa-NDP, 25 mM Tris-HC1 pH 7.5 and 3 unitslml alkaline phosphatase. The mixture was incubated for 1-3 h a t room temperature after which it was acidified with acetic acid.

Oligonucleotide Hydrolysis Complete alkaline hydrolysis of oligonucleotides

was performed as follows: 0.5-1.0 A,,, units of the oligonucleotide were dissolved in 3-5 y1 of 0.3 N NaOH and incubated for 16 h at 37 “C in 0.1 ml polypropylene test-tubes. The digest was neutralized with acetic acid and separated by paper electro- phoresis a t pH 1.9, or by paper chromatography using ethanol and 1 M ammonium acetate, pH 3.8, in a ratio of 5 : 2 . The products were eluted from the paper with 0.4 ml of 0.01 M HCl and their absorbance a t

6 Monofunctional Substrates of Polynucleotide Phosphorylase Eur. J. Biochem.

260, 280 and 290 nm was measured against suitable paper blanks.

RESULTS Monofunctional-Substrate-Assay System

Monofunctional substrates of polynucleotide phos- phorylase should meet the following requirements in order to be used for the stepwise synthesis of oligoribonucleotides of defined sequence (a) they should be completely blocked in the 3'-OH function, (b) unimpaired in the pyrophosphate function, (c) stable under polymerization conditions and (d) their blocking group should be removable by a mild treat- ment that does not damage the oligonucleotide chain.

One class of derivatives that seemed to fulfill these requirements were the 2'(3')-O-monoacyl- NDP's. These derivatives may be easily and rapidly prepared from a wide range of available carboxylic acids, they are relatively stable at neutral pH but can be hydrolyzed by mild alkali. Although these deriva- tives exist a t neutral pH as a mixture of the 2' and 3' isomers [6,7], we surmised that a bulky acyl group might affect the 3'-OH function even when esterified to the vicinal 2'-hydroxyl group.

Various 2'(3')-O-monoacyl NDP's such as the acetyl, propionyl, butyryl, valeryl, isobutyryl, isovale- ryl and pivaloyl esters were prepared as described in Methods. and tested as monofunctional substrates of polynucleotide phosphorylase. The assay was carried out as follows. A reaction mixture containing the NDP derivative, an oligonucleotide initiator, Tris-HC1 buffer, MnCl, and purified E . coli poly nucleotide phosphorylase was incubated a t 37 "C. Samples from the mixture were removed and analyzed by paper electrophoresis. A derivative whose poly- merization was limited to the addition of a single nucleotide residue to the initiator chain and gave good yields was considered for further studies. Out of the several acyl NDP's mentioned above, the iso- valeryl and isobutyryl derivatives were found to be good monofunctional substrates. We have tested, as initiators, the oligonucleotides ApA, pApA, (Ap),A, (AP)~A and (Ap),A. Among these pApA was found to be the smallest oligonucleotide that supported de- tectable monoadditions with isovaleryl-NDP deriva- tives while ApA was not active.

In the following sections we describe the synthesis of four tetranucleotides- the 2'(3')-O-isovaleryl esters of (Ap),A, (Ap),G, (Ap),C and (Ap),U-by the re- spective monoaddition of the 2'(3')-O-isovaleryl esters of ADP, GDP, CDP and UDP to (Ap),A.

Synthesis of A-A-A-A-iVa from 8-8-8 and iVa-ADP

The reaction mixture (0.1 ml) contained 20 mM iVa-ADP, 2mM (Ap),A, 100mM Tris-HC1 pH 8.5,

2 mM MnCl, and 4 units of purified E . coli polynucleo- tide phorphorylase (105 units/mg protein). Following incubation a t 37 "C, the formation of higher oligo- nucleotides was followed by paper electrophoresis a t pH 1.9. Fig. 1 A demonstrates that as a result of this reaction the oligonucleotide (Ap),A was converted mainly to a new oligonucleotide which migrated like (Ap),A. This product was eluted from the paper and further chromatographed on paper using solvent A. In this solvent it travelled faster than (Ap),A, sug- gesting a more hydrophobic nature(Fig. 2 A, spotNo. I ) The new oligonucleotide was shown to be (Ap),A- iVa by the following analyses (a) Mild alkaline treat- ment converted it to (Ap),A (Fig.2A, spot no. 2 and 3). (b) Complete alkaline hydrolysis of the new product [or of the material derived from (Ap),A-iVa by mild alkaline treatment] yields Ado and Ap in a ratio of 1.0:3.0. (c) Complete digestion with spleen phosphodiesterase converted the oligonucleotide to iVa-Ado and Ap (Fig.3A, spot no. 3). It was not easy, however, to completely recover iVa-Ado by this di- gestion procedure. The ratios of iVa-Ado to Ap ob- tained in all the spleen-phosphodiesterases digests ranged between 0.6-0.8, to 3.0.

I n addition to iVa-Ado, the reaction mixture contained small amounts of ApA and (Ap),A-iVa (Fig. 1 A). ApA and (Ap),A-iVa were found in equi- molar ratio and probably arose by transnucleotida- tion of (Ap),A to ApA and (Ap),A [I41 followed by addition of an iVa-AMP residue to the tetranucleotide. The amount of ApA and (Ap),A-iVa was minimized by replacement of Mg2+ by Mn2+ and by employing a high NDP : oligonucleotide ratio. Under these conditions the convertion of (Ap),)A to (Ap),A-iVa reached 70°/,.

Synthesis of 2'(3')-O-isovaleryl Esters of (AP)3G, (AP13C a d (AP)3U

The 2'(3')-O-isovaleryl esters of GDP, UDP and CDP were also tested as monofunctional substrates of polynucleotide phosphorylase. The conditions of these monoaddition reactions differed somewhat from those described for the monoaddition of iVa-ADP to (Ap),A. Thus, the pH was raised to 8.8 in the iVa- GDP system while the amounts of enzyme were doubled in the iVa-UDP and iVa-CDP systems.

The electrophoretic migration of the products resulting from reaction of iVa-GDP with (Ap),A is depicted in Fig.1B. In this reaction (AP)~A was converted mainly to a new, slower-migrating oligo- nucleotide which was later shown to be (Ap),G-iVa. This material was found to be contaminated by trace amounts of (Ap),G-iVa which could be removed by paper chromatography in solvent A. The results of the analyses of (Ap),G-iVa are described in Fig. 2 B and 3B. Small amounts of the slower moving (Ap),GpG- iVa were also detected among the reaction products.

Vo1.24. No. 1. 1971 G. KAUFMANN, M. FRIDKIN, A. ZUTRA, and U. Z. LITTAUER 7

A 0

'0 2 5 10 20 30 '60 120 (ApInA m o r k crs

Time (min)

*.

Orig in

0 5 30 120 (Ap),A markers

T ime (min)

Fig.i. Monoaddition of ( A ) iVa-ADP, ( B ) iVa-GDP, ( C ) iVa-CDP and (0) iVa-UDP to (Ap) ,A . (A) The reaction mixture (0.1 ml) contained 20 mM iVa-ADP, 2 mM (AP)~A, 100 mM Tris-HCI p H 8.5,2 mM MnCl, and 4 units of purified E. wli polynucleotide-phosphorylase. Incubation was car- ried at 37 "C. Samples of 2 pl were withdrawn and applied to Whatman No. 1 Damr a t the indicated times. SeDaration

3 0 APA

0 0 2 5 10 2 0 35 6 0 l A p ) , A

m o r l t c r * Time (min)

f

0 2 5 10 2 0 5 0 6 0 ( A p i , A m o r k c f s

Time ( m i n )

mixture and the electrophoretic separation were similar to those described in (A) except that iVa-GDP replaced iVa-ADP and the pH was raised to 8.8. (C) The conditions were similar to those described in (A) except that iVa-CDP replaced iVa-ADP and the amount of the enzvme was dou- bled. (D) The reaction mixture was similar to tha t described in (A) except that iVa-UDP replaced iVa-ADP and the amount of the enzyme was doubled. Electrophoretic separa- tion was carried out in the pH-3.5 system for 60 min a t

was performed i i 2he pH-1.9 electrophoresis syt&em for 80 min at 30 V/cm using (AP)~A markers. (B) The reaction

30 V/cm using (Ap),A markers

8 Monofunctional Substrates of Polynucleotide Phosphorylase Eur. J. Biochem.

A ’:I

0 0 I I I I I I 1 2 3 4 5 6

I l l 1 2 3

S p o t n u m b e r

1 2 3 4 5 6 1 2 3 4 5 6

S p o t n u m b e r

Fig.2. Mild alkaline hydrolysis of the 2’(3’) -0-isovaleryl

and (Ap),C and ( D ) ( A P ) ~ U and (Ap),U. (A) The 2 (3 )- 0-isovaleryl esters of (Ap),A and (Ap),A were isolated as follows. The bulk of the reaction mixture described in the legend to Fig.lA was separated by paper electro- phoresis (pH 1.9, 3-MM Whatman paper) followed by paper chromatography (Whatman No. 52) using solvent A. 1.0 A,,, unit of each of the purified products was dissolved in 10 pl of 1 N NH,OH and incubated at 37 “C. 2 p1 aliquots were withdrawn at the indicated intervals, applied to Whatman No. 52 paper and chromatographed for 24 h with solvent A using (AP)~A markers. Chromatograms Nos. 1, 2 and 3 re- present mild alkaline treatment of (Ap),A-iVA at 0, 1 and 4 h, respectively, while Nos. 4, 5 and 6 represent mild alka- line treatment of (Ap),A-iVa at 0, 1 and 4 h, respectively. (B) 2’(3’)-O-Isovaleryl (Ap),G was synthesized as described in Fig. 1 B and isolated by electrophoresis on Whatman 3-MM paper in the pH 1.9 system, followed by chromatog- raphy on Whatman paper No. 52 using solvent A. 1.0 A,,, unit of the purified material was treated with 1 N NH,OH

esters 01 ( A ) (AP),A and (AP),A, ( B ) (AP3P9 ( C ) (Ap)sC

This material was formed by a second nucleotide transfer probably after some of the isovaleryl esters had been hydrolyzed. The rate of iVa-GDP mono-

and chromatographed as described in (A). Chromatograms Nos. 1, 2 and 3 represent 0, 1 and 4 h of incubation, respec- tively. (C) The 2’(3’)-isovaleryl esters of (Ap),C and (Ap),C were synthesized as described in Fig.lC and isolated by paper electrophoresis at pH 1.9 followed by paper chromatog- raphy with solvent A. The purified oligonucleotides were treated with 1 N NH,OH and chromatographed as described in (A). Chromatograms 1, 2, and 3 represent mild alkaline treatment of (Ap),C-iVa at 0, 1 and 4 h, respectively, while Nos, 4, 5, and 6 represent mild alkaline treatment of (Ap),C- iVa at 0 , l and 4 h, respectively. Broken circles indicate traces of nonhydrolyzed material. (D) The 2’(3’)-O-isovaleryl esters of (Ap),U and (Ap),U were synthesized as described in Fig. 1D and isolated by paper electrophoresis a t pH 3.5 followed by paper chromatography with solvent A. The purified oligonucleotides were treated with 1 N NH,OH and chro- matographed as described in (A). Chromatograms Nos. 1, 2 and 3 represent mild alkaline treatment of (Ap),U-iVa at 0 , l and 4 h, respectively, while Nos. 4 ,5 and 6 represent mild alkaline treatment of (Ap),U-iVa a t 0 , l and 4 h, respectively. The broken circle indicates traces of nonhydrolyzed material

addition to (AP)~A was several-fold faster than that of iVa-ADP and the molar yield of (Ap),G-iVa was 85O/,.

Vo1.24, No.l,1871 0. KAUFUNN, M. FRIDKIN, A. ZUTRA, and U. Z. LITTAUER 9

Front I-

I 2 3 4 1 2

S p o t n u m b e r

Fig.3. Spleen-p~8p~diesterase digest of (A) (Ap),A-iVa and (Ap),A-iVa, ( B ) (Ap),G-iVa, ( C ) (Ap),C-iVa and (Ap),C-iVa and (0) (Ap)3U-.iVa. (Ap),A-iVa was pre- pared and isolated as described in Fig.1A and 2A. 0.6 A,,, unit were dissolved in 3 p1 of a solution containing 0.2 mg/ ml of spleen phosphodiesterase in 0.1 M ammonium acetate, pH 7.5 and O.O5O/, Tween 80. After incubation for 4 h at room temperature, the digest was chromatographed on Whatman No. 52 paper with solvent C using undigested (Ap),A-iVa, Ap, Ado and iVa-Ado markers. The products were eluted from the paper and their amounts determined by their absorbance at 260,280 and 290 nm. (1) (Ap),A:iVa; (2) (Ap),A-iVa; (3) (Ap),A-iVa digest; (4) (Ap),-AiVa hgest. (B) (Ap),G-iVa was prepared and isolated as described in

The reaction of (Ap),A with iVa-CDP is shown in Fig. 1 C. Three products were detected on the paper and were shown by subsequent analyses to be ApA, (Ap,)C-iVa and (Ap),C-iVa (Fig. 2 C and 3 C). The rate of iVa-CDP monoaddition to (Ap),A under these conditions was much slower than the rates observed with iVa-GDP and iVa-ADP though the iVa-CDP was reacted with a double amount of enzyme. Trans- nucleotidation was more extensive in this system yielding significant quantities of ApA and (AP)~A thus lowering the yield of (Ap),C-iVa to 30-400/,.

The reaction of iVa-UDP with (AP)~A was carried out in a manner similar to the synthesis described above. In this case the products were analyzed by paper electrophoresis at pH 3.5. The results of this reaction are shown in Fig. 1 D. During the incubation period (Ap),A disappears while a new, faster-migrat- ing product appears. In addition, small amounts of ApA (spot near the origin) were detected. The faster- migrating material was chromatographed on paper using solvent A. Two oligonucleotides were separated and subsequently shown to be the 2'(3')-O-isovaleryl

20 .

15 .

t

Spot number

Fig. 1 B and 2B. 0.6 Azao unit was digested with spleen phosphodiesterase and chromatographed as described in (A) except that (Ap),G-iVa, Ap, Guo and iVa-Guo served as markers. (1) (Ap),G-iVa; (2) (Ap),G-iVa digest. (C) (Ap)&- iVa and (Ap),C-iVa were prepared and isolated as described in Fig. 1 C and 2C. 1.0 A,,, unit was digested and then sepa- rated as described in (A) except that (Ap),C-iVa, (Ap),C-iVa, Ap, Cyd and iVa-Cyd served as markers. (1) (Ap),C-iVa; (2) (Ap),C-iVa; (3) (Ap),C-iVa digest; (4) (Ap),C-iVa digest. (D) The preparation and isolation of (Ap),U-iVa were as described in Fig.1D and 2D. 0.6 A,, unit were digested and chromatographed as described in (A) except that (Ap),U-iVa, Ap, Urd, and iVa-Urd served as markers. (1)

(Ap),U-iVa; (2) (Ap),U-iVa digest

esters of (Ap),U and (Ap),U (Fig.1D and 3D). On longer incubation periods a small amount of a still- faster-migrating compound was detected (marked with a broken line). This spot probably contained a mixture of (Ap),U, and (Ap),U, derivatives which were formed by a second-nucleotide transfer, probably after some of the isovaleryl esters had been hydrolyzed. The small amounts of these side products prevented the analyses of their structure. The molar yield of (Ap),U-iVa was 45 Ole.

Removal of the Isovaleryl-Blocking Group from the Oligonucleotide Product

The 2'(3')-O-isovaleryl ester prevents further poly- merization from taking place on the 3'-terminus of the oligonucleotide product. In order to use the prod- uct as an initiator in a subsequent monoaddition re- action the isovaleryl blocking groups must first be removed. Fig.2 shows that treatment of the blocked oligonucleotides with 1 N NH,OH for 1-4 h at 37 "C completely removes the isovaleryl group. This

10 Monofunctional Substrates of Polynucleotide Phosphorylase Em. J. Biochem.

I I I I 5 10

Incubation time (hours)

Fig.4. Removal of the iswaleryl-blocking group from A-A-A- G-i Va. A-A-A-G-iVa was prepared from [14C](Ap)zA and iVa-GDP as described in Results. Amounts containing 500 counts/min (specific activity 5000 counts x min-' x A , unit-') of [l*C]A-A-A-G-iVa were dissolved in 0.2 ml of 50°/0 aqueous methanol saturated with ammonia and incubated at room temperature. At the indicated time intervals the solvent was removed by rapid evaporation in vmw at room temperature. The residue waa then dissolved in 10 pl acetic acid and applied to chromatography paper (Whatman No. 52). The chromatograms were developed with solvent A for 8 h. A-A-A-G-iVa, A-A-A-G, A-A-G, Ap, (Ap), and (Ap), served as markers. The chromatogram of each sample was cut in two: one piece waa from the origin up to and including A-A-A-G, while the second comprised the rest of the chro- matogram and contained A-A-A-G-iVa and all its possible phosphodiester-breakage products aa well aa those of A-A- A-G-. The graph represents the fraction of radioactivity in the first part of the chromatogram as a function of the time

of incubation

treatment is accompanied, however, by some breakage of phosphodiester bonds. In order to estab- lish conditions for the selective removal of the 2'(3')- 0-isovaleryl ester we have followed the conversion of W-labeled A-A-A-G-iVa to A-A-A-G by incubation with various ammonical solvents. The best results were obtained with 50°/, aqueous methanol saturated with ammonia. The experiment was conducted as follows. Samples of l*C-labeled A-A-A-G-iVa (labeled in the oligonucleotide moiety) were dissolved in 0.2 ml of the ammonical solvent and kept a t room tempera- ture in stoppered test tubes. At the end of the incu- bation the solvent was removed in vacm a t room temperature and the residue was dissolved in lop1 0.1 N acetic acid and chromatographed on Whatman paper No. 52 with solvent A for 8 h. The chromato- grams of each sample were cut into two areas; one from the origin up to and including the A-A-A-G spot, the second, which comprised the rest of the chro- matogram, contained A-A-A-G-iVa and all the pos- sible breakdown products of A-A-A-G. The fraction of radioactivity in the first area represented the yield of A-A-A-G. Fig.4 shows a semilogarithmic plot of A-A-A-G liberation as a function of the time of in- cubation in 50°/, aqueous methanol saturated with

ammonia. The curve is biphasic; the first phase re- presents the rapid ammonolysis of the isovaleryl ester with a t:/, of 20 min while the second shows the slow phosphodiester hydrolysis which reduces the yield of A-A-A-G by 1-2O/, per hour of incubation. A 9501, yield of A-A-A-G was obtained after 2-3 h of incubation. Thus, the ammonical treatment en- abled recovery of the deblocked oligonucleotide in a good yield.

DISCUSSION It was demonstrated in this work that the poly-

merization of nucleoside diphosphates by polynucleo- tide phosphorylase can be restricted to the addition of a single nucleotide residue to preformed oligonucleo- tides. This was achieved by suitable rnodihation of the sugar moiety of the nucleoside &phosphate. Thus, 2'(3')-O-isovaleryl derivatives of ADP, GDP, UDP and CDP reacted with (Ap)& to yield the cor- responding 2'(3')-O-isovaleryl esters of (Ap),A, (Ap),G, (Ap),U and (Ap),C according to the following equation

-+ polynucleotide phosphorylase

A-A-A + ppN-iVa A-A-A-N-iVa + Pi

The interference of the 2'(3')-O-isovaleryl substi- tuent with further polymerization can be explained by (a) reduced afhi ty of the product oligonucleotide to the enzyme or (b) by direct inhibition of the 3'-OH function, either by its substitution or by a steric effect ofthe 2'-OH vicinal substituent. The first explanation is less likely since the product seems to be a strong inhibitor of the monoaddition reaction (unpublished results). The blocked reactivity of the 3'-OH function is, therefore, the more likely mechanism. However, this assumption has to be verSed by further studies.

It is interesting to note that the reactivity of modi- fied NDP's varies with the type of modification in- troduced in the 2'-OH. Thus, 2'-methoxy ADP is polymerized de nwo by polynucleotide phosphorylase to yield poly 2-'methoxy adenylic acid [2], while polymerization of 2'-deoxy ADP is limited to the ad- dition of only a few mononucleotide residues to the oligonucleotide initiator [4,5]. On the other hand, the polymerization of 2'(3')-O-isovaleryl-NDP's was seen to be strictly limited to the transfer of a single residue to the initiator oligonucleotide. Successive additions of mononucleotide residues were only de- tected upon hydrolysis of the 2'(3')-O-isovaleryl esters of both NDP and oligonucleotide. Such an hy- drolysis occurred after very long incubation periods or when the pH of the reaction was raised.

At neutral pH the isovaleryl esters of NDP are likely to be a rapidly equilibrating mixture of 2' and 3' isomers [6,7]. The manner in which the iso- valeryl residue in the 2'-isomer inhibits the 3'-OH function is not clear. This inhibition could be due to

Vo1.24, No.l,1071 G. K A ~ , M. FBIDKIN, A. ZUTRA, and U. 2. LZITAUER I1

a masking effect of the bulky acyl group or to the aquisition of an unfavored conformation by the base of the substrate analogue, as was suggested for 8BrGDP [15].

The monoaddition reactions described in this paper were accompanied by transnucleotidation of the initia- tor oligonucleotide. The rate and the extent of this side reaction were reduced in the presence of iVa- NDP’s, were inversely related to the iVa-NDP : oligo- nucleotide ratio and depended on the type of iVa- NDP employed. Thus, transnucleotidation was ex- tensive in the presence of iVa-CDP (which is a poor substrate for monoaddition) but was less pronounced in the presence of iVa-GDP. These observations may be interpreted as reflecting competition between the nucleoside diphosphate and the 3‘-terminal nucleotide residue of the oligonucleotide for a common site on the enzyme.

The monoaddition of the four 2’(3’)-O-isovaleryl- NDP’s to (Ap)& indicates that these substrates may be used for the stepwise synthesis of ribooligonucleo- tides of defined sequence. The deacylated oligonucleo- tide can now serve as an initiator in a second mono- addition reaction. Indeed this method has recently been applied by us for the synthesis of the hepta- nucleotide U-U-U-G-A-A-G [ 161.

We wish to thank Dr Y. Kimhi and Mr Y. Tichauer for supplying us with a purified polynucleotide-phosphorylase preparation.

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G. Kaufmann, A. Zutra, and U. Z .Littauer Biochemistry Department, Weizmann Institute of Science P.O. Box 26, Rehovot, Israel

M. Fridkin Department of Biophysics, Weizmann Institute of Science P.O. Box 26, Rehovot, Israel