Volume 2 number 6 June 1975

Nucleic Acids Research

Cyclization of uridine monophosphate by diethyl pyrocarbonate

F.Solymosy*, L. Ehrenberg**, I.Fedorcsdk***

*Biological Research Center, Hungarian Academy of Sciences, 6701 Szeged, P.Q. Box 521, Hungary. Received 1 May 1975

ABSTRACT Reaction between diethyl pyrocarbonate and uridine 2'-phosphate or uridine 3'-phosphate leads to the formation in high yields of uridine 21:3'-cyclic phosphate. This reaction product was identified in experiments involving (a) ultraviolet spectrophotometry, (b) paper chromatography, (c) high voltage paper electrophoresis at both pH 3.5 and 7.4, (d) acid hydrolysis, and (e) digestion with pancreatic ribonuclease. INTRODUCTION In experiments aimed at the determination of the average chain length of yeast RNA treated with diethyl pyrocarbonate ((EtOCO)2CQ it was found that in the treated samples part of the terminal phosphates became inaccessible to alkaline phosphatase. Therefore determinations of free, non-esterified phosphate liberated by alkaline phosphatase revealed a lower phosphorus value for (EtOCO)20-treated RNA than for the untreated control. Carbethoxylation of terminal phosphate groups or formation of cyclic phosphate esters from the 3'-terminal phosphates upon treatment of the RNA with (EtOCO)20 would explain this observation. In the present paper experimental evidence wil I be presented for the formation in high yields of uridine 2':3'-cyclic phosphate (2':3' UMP) from either uridine 2'-phosphate (2'-UMP) or uridine 3'phosphate (3'-UMP) upon treatment with (EtOCO)20. MATERIALS AND METHODS Materials. 2'-UMP (di-lithium salt), 3'-UMP (di-sodium salt), 5'- UMP(di-sodium salt), pancreatic ribonucl ease and E. col i al kal ine phosphatase were purchased from Sigma Chemical Co., Saint Louis, Mo., U.S.A. Diethyl pyrocarbonate ("Baycovin") was a generous gift of Farbenfabriken Bayer, Leverkusen, West Germany. All reagents were of analytical grade. Treatment of uridine monqphosphate with -EtOCO)20. 2'-UMP, 3'-UMP or 5'-UMP was dissolved in distilled water to a final concentration of 10 mM. To 7 mlaliquots of the nucleotide solutions 490 /ul (EtOCO)20 was added in 70 iul portions, first at 0 time and every 30 min thereafter, for 3 hours. These amounts of the chemical are assumed to ensure a fairly uniform saturation level of (EtOCO)20 during the time of 985

Nucleic Acids Research treatment,. calculating with a mean life tm = 17 min for a saturated, 40mM (EtOCO)20 solution at 370C2. The samples were shaken on a horizontal shaker at 370C for 4 hours. The other 7 ml-aliquots of the samples were treated in the same way,, except that instead of 70 /ul portions of (EtOCO)20, 56 /ul portions of ethanol were added, i. e. the amount of ethanol formed by the decomposition of 70 /,ul (EtOCO)20. Determination of terminal osphorus in 0.1 ml aliquots taken at given time intervals from the samples was done according to Seaman3. Paper Chromatograehy. 30 1 al iquots of 10 mM or 60 aliquots of 5 mM nucleotide solutions were spotted on Whatman No. 1 paper and subjected to descending chromatography using the solvent system (isopropyl alcohol: ammonia: water, 70: 10: 20, v/v/v) described by Tener and Khorana4. Ultraviolet absorbing spots and proper blanks were cut from the paper, eluted with 5 ml 0.1 N HCI ovemight and then the ultraviolet spectra of the eluaotes were recorded. Htah _XLtagopaeer elecrhol !st was carried out in a Shandon Model L24 apparatus using Whatman No. 3 MM paper, 20/ul aliquots of 10 mM nudeotide solutions and either 0. 05 M amlmonium formate buffer, pH 3. 5 ,or 0. 05 M Na-phosphate buffer, pH 7.45. The conditions of the run were the following: 53.6 V/cm, 30 mA, 3.5 hours (for the formate buffer) or 26.8 V/cm, 40 mA, 4 hours (for the phosphate buffer). Quantitative evaluation of the electrophoretograms was done as described for the chromatograms, but the volume of the eluent was 3 ml. Acid hydrolysis. 30 /ul of the 10 mM nucleotide solution was diluted 1:1 with 0.2 N HCI and kept at room temperature for 4 hours. The acid-treated samples were quantitatively transferred onto Whatman No. 3MM paper for chromatographic analysis. Treatment with pancreatic ribonuclease. To 30 ul of a 10 mM nucleotide solution 15 /A of 0.4 M Tris- Ha buffer, pH 7.5, and 15 1u of a pancreatic ribonuclease solution (10 mg ribonuclease/ml water) were added. The mixture was incubated at 37°C ovemight and then quantitatively transferred onto Whatman No. 3MM paper for chromatographic analysis. RESLLTS As shown in Fig. i treatment with (EtOCO)20 of 2'-UMP, 3'-UMP or 5'-UMP led to a time-depoendent disappearance of terminal phosphate accessible to E. coli alkaline phosphatase. The reaction resulting in the formation of non-detectable phosphorus was the fastest with 2'- UMP and slowest with 5'-UMP. The major product carrying the "inaccessible" phosphate was identified as 2': 3' UMP in the (EtOCO)20treated 2'-UMP and 3'-UMP solutions on the basis of the following evidence: a) The ultraviolet absorption spectra of standard 2'-UMP, standard 2': 3' UMP ,

and the

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(EtOCO)20-treated 2'-UMPsolutions at identical concentrations could be

Nucleic Acids Research

10

0

60

240 120 TIME (MIN)

Fig. 1. Amounts of inorganic phosphate liberated by E. coli alkaline phosphatase from o ) and 2'- UMP (A -A) 0), 3'- UMP ( o 5'- UMP ( treated with (EtOCO)20 for the times indicated on the abscissa. For details see tMterials and Methods.

0.7 ¢0.4 m 0. ql~0.1

300

250 270 WAVELENGTH (nm)

230

), Ultraviolet absorption spectra of 55 ,uM aqeous solutions of 2'-UMP ( 2': 3' UMP (-----) and 2'-UM(AP treated with (EtOCO)20 for 4 hours ( -*- --) as described in Materidis and Methods. interpreted as indicating that the (EtOCO)20-treated 2'-UMP solution contained a mixture of 2'-UMP and 2':3' UMP (Fig. 2). The same was true of the (EtOCO)20treated 3'-UMP.

Fig. 2.

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Nucleic Acids Research b) Both 2'- UMP and 3'-UMP, upon treatment with (EtOCO)20, gave rise to a product which had the same mobility as the standard 2': 3' UMP in both the chromatographic and electrophoretic (both at pH 3.5 and 7.4) systems (Figs 3, 4 and 5). c) Acid or enzymatic hydrolysis of the (EtOCO)20-treated 2'- UMP and 3'- UMP resulted in the disappearance of the component which had the same mobility on the chromatogram as the standard 2': 3' UMP with a concomitant intensification of the spot representing the starting material (2'- UMP or 3'- UMP). The same was true of the chromatographic behaviour of acid-, or ribonuclease-treated standard 2': 3' UMP.

Front

1~~

'I II

III

rV V

VI

VII

Vill

Start

Fig. 3. Chromatograms of 2':3' UMP (1), (EtOCO) 0-treated 2'-UMP (11), (EtOCO) 0-treated 3'-UMP (111), 5'-UMFi(lV), (EtOCO) 0-treated 5'-UMP (V), 2'- 3MP (VI), 3'- UMP (VI ), and 3': Y UMP (VIII). Re nucleotides from the spots numbered 1- to 4 were eluted and quantitatively analyzed (see Table 1). For more details see Materials and Methods.

d) A quantitative evaluation of the chromatograms and electrophoretograms indicated the presence of about 77 % and about 57 % 2': 3' UMP in the (EtOCO)20treated 2'-UMP and 3'-UMP samples, respectively (compare Table 1 and Fig. 1). 988

Nucleic Acids Research 03

+

sogO

+1

Qo

sO

34

19

13:'.

IS 170

12 '%7

16

11 "

10 I

I

III

IV

v VI

Start VII

ViII

I

q. #

0 20 i

11 III IV v VI vil Vil

Fig. 4. (Left) Electrophoretic mobilities at pH 3.5 of 3': 5' UMP (1), 2'- UMP (I1), (EtOCO)20-treated 2'- UMP (11l), 3'-UMP (IV), 2': 3' UMP (V), (EtOCO) 0-treated 3'-UMP (VI), (EtOCO) 0-treated 5'-UMP (VIl) and 5'- UMP ?VIl1). The nucleotides from the spo?s numbered 5 to 9 were eluted and quantitatively analyzed (see Table 1). For more details see Materials and Methods. Fig. 5.(Right) Electrophoretic mobilities at pH 7.4 of 3': 5' UMP (1), 2'-UMP (11), (EtOCO)20-treated 2'-UMP (111), 3'-UMP (IV), 2': 3' UMP (V), (EtOCO) 0-treated 3'-UMP (VI), (EtOCO)20-treated 5'-UMP (VIl) and 5'-L 4P (VIII). The nucleotides from the spots numbered 10 to 21 were eluted and quantitatively analyzed (see Table 1). For more details see Materials and Methods. Among the reaction products of 5'-UMP treated with (EtOCO)20 no cyclic phosphate esters could be detected (cf. Figs 2, 3 and 4), although about 23 % of phosphorus became inaccessible to E. col i al kal ine phosphatase.

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Nucleic Acids Research Table 1. Percentage distribution of products formed from 2'-UMP, 3'-UMP, and 5'- UMP upon reaction with (EtOCO)20

System Chromatography

2'- UMP Expt. Expt. ot No.x No.1 No.2 1 23 24.7 2 77 75.3 3 4

Electrophoresis pH 3.5

5 6 7 8 9

22.2 77.8

Electrophoresis pH 7.4

10 11 12 13 14 15 16 17 18

11.6 77.2 2.3 3.7 5.2

19

20 21 X

Per Cent Reaction Products from 3' UMP 5' UMP Expt. Expt. Expt. Expt. Expt. Expt. No.3 No.1 No.2 Nv.3 No.1 No.2 32 68 43 42 45 57 58 55 42.2 1.6 56.8

15 72 i U 5

25.9 24 60.3 64 1. 6f 9.5 12

2.7L

88.3 11.7

86 14

See Figs 3, 4 and 5

DISCUSSI ON

Spectrophotometric, chromatographic, electrophoretic and chemical evidence has indicated that (EtOCO)20 reacts with 2'-UMP or 3'-UMP to give rise to 2': 3' UMP. Although this reaction escaped the attention of Leonard's group7 despite their detailed and extended studies of the reactions of nucleic acid components (including adenosine 2'(3') phosphate) with (EtOCO)20, its occurrence is not surprising at all, because other acylating agents, such as acetic anhydride and trifluoroacetic anhydride are known to react in the same way, and the reaction pattern of (EtOCO)2O corresponds to that of alkanoic anhydrides in general2. In fact, the carbethoxylating agent, ethyl chloroformate, has been shown also to give rise to 2': 3' cyclization10 Of the nucleophilic groups of 2'(3' )-UMP the phosphate group is expected to have a relatively high reactivity towards (EtOCO)20, and the cyclization is supposed 990

Nucleic Acids Research to be the consequence of an intramolecular phosphorylation of the vicinal hydroxyl by the mixed ethoxycarbonic phosphoric anhydride. This reaction mechanism explains why no cyclic product (3': 5' UMP) is found upon treatment of 5'-UMP with (EtOCO)20. Besides the 2':3' UMP, at least three additional components are found in the reaction of (EtOCO)20 with 2'- or 3'-UMP, as shown in the Wlectropherogram (Fig. 5). The chemical nature of these products has not yet been studied. Products of 2'-, 3'- or 5'- UMP carbethoxylated on riboe hydroxyl groups are expected to be formed7, and their occurrence was in fact indicated by a slightly raised C/N ratio in the reaction product. (A determination of C, N and P of the samples revealed that treatment with (EtOCO)20 of 2'-UMP caused an increase of the C/2N ratio from the expected value of 9.0 to that of 9.7. This increase corresponds to the introduction of the three-carbon EtOCO group in 23 % of the final product. ) Stable reaction products containing carbethoxy phosphate groups or carbethoxylated groups of uracil (especially N-3 is reactive11) are not supposed to be recovered. Due to their lability these products are expected to be rapidly hydrolyzed or, to a minor extent, to give rise to bimolecular 9 The formation of intramolecular reaction products such as diuridine pyrophosphate. products, such as cyclic nucleotides, in comparison with intermolecular products, such as the mentioned ;yrophosphate, is favoured by working at a lower initial concentration of the nucleotide . The three other common nucleoside 3'-phosphates (3'-CMP, 3'-AMP and 3'-GMP), if treated with (EtOCO)20, also gave rise to products (in yields similar to that obtained with 3'-UMP) carrying the phosphate group in a state inaccessible to al kal ine phosphatase. This suggests that cyclization of 2' or 3' nucleoside phosphates is a generally occurring reaction with (EtOCO)20. This reaction might be useful for the analysis of the position of terminal phosphates in a polynucleotide chain.

ACKNOWLEDGEMENT Thanks are due to Dr. J. Tomasz for helpful discussions and to Mrs. Marie-Louise Hanngren as well as Miss Inga-Lise Kinell for skillful technical assistance. The work was done within the frame of collaboration with the Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary. It was supported financially by the Swedish Natural Science Research Council.

**Department of Radiobiology, University of Stockholm, Wallenberg Laboratory, Lilla Frescati, 104 05 Stockholm, Sweden.

***Department of Genetics,

EOtvOs University, Budapest, Hungary.

REFERENCES 1 Fedorcs6k, I., Ehrenberg, L. and Solymosy, F. (1975) Bioche'm. Biophys. Res. Commun. (Submitted for publication). 2 Ehrenberg, L., Fedorcsdk, 1. and Solymosy, F. (1975) Progress in Nucleic Acid Research and Molecular Biology. Vol. 16 (In press). 991

Nucleic Acids Research 3 Seaman, Edna (1968) in Methods in Enzymology. Vol. 12 B. pp. 218 - 220, Academic Press, New York. 4 Tener, G.M. and Khorana, M.G. (1955) J. Am. Chem. Soc. 77, 5349- 5351. 5 IMarkham, R. and Smith, J. D. (1952) Biochem. J. 52, 552 - 557. 6 Heppel, L.A., Whitfeld, P.R. and tMrkham, R. (1955) Biochem. J. 60, 8- 15. 7 Henderson, R. E. L., Kirkegaard, L. H. and Leonard, N.J. (1973) Biofim. Biophys. Acta 294, 356- 364. 8 Rammler, D. H., Lapidot, Y. and Khorana, H, G. (1963) J. Am. Chem. Soc. 85, 1989- 1997. 9 Brown, D.M.., Magrath, D.l. and Todd, A.R. (1952) J. Chem. Soc., pp. 2708 - 2714. 10 Letters, R. -and Michelson, A. M. (1962) J. Chem. Soc., pp. 71 - 76. 11 Vincze, A., Henderson, R. E. L., McDonald, J. J. and Leonard, N. J. (1973) J. Am. Chem. Soc. 95, 2677 - 2682.

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