A M I N O A C I D E S T E R S OF 9-(2',3'-DIHYDROXYPROPYL-I')-ADENINE

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I N H I B I T O R S OF P R O T E I N S Y N T H E S I S ON R I B O S O M E S

B. P. GOTTIKH, A. A. KRAYEVSKY, M. K. KUKHANOVA, A. A. JATSYNA, A. M. KRITZYN, AND C. L. FLORENTIEV

Institute of Molecular Biology, the U.S.S.R. Academy of Sciences, Moscow, U.S.S.R.

(Received 3 August, 1973; in revised form 12 September, 1973) ABSTRACT. Peptide acceptor properties of phenylalanine and glycine esters of 9'-(2',3'-dihydroxypropyl-l')-adenine and 1-(2',3'-dihydroxypropyl-l')-4-thiouracyl were investigated. All these esters appeared to be powerful inhibitors of polyphenylalanine synthesis in E. coli MRE-600 ribosomes charged with poly U. Like puromycin, esters of adenine derivatives accepted the AcPhe residue from Ac-[ ~4C] Phe-tRNA in a ribosomal system charged with polyU. However, peptidyl esters of 9-(2',3'-dihydroxypropyl-l')-adenine remained bound with ribosomes. The structure of the peptide esters synthesized was ascertained after dissociation of ribosomes into subparticles by direct comparison with the synthetic specimens. ABBREVIATIONS. AcPhe = acetyl-L-phenylalanine; HP-Ade = 9-(2',Y-dihydroxypropyl-l')-adenine; Phe-HP-Ade and Gly-HP-Ade = L-phenylalanine and glycine esters of HP-Ade; Phe-HPTUra = L-phenylalartine ester l-(2',3'-dihydroxypropyl-l')-4-thiouracyl; AcPhePhe-HP-Ade and AcPheGly-HP-Ade = acetyl-L-diphenylalanineand acetyl-L-phenylalanylglycineesters of HP-Ade respectively; AcPhe-puromycin = acetyl-L-phenylalanyl-puromycin. I. INTRODUCTION Amino acid esters of nucleosides [i] and nucleotides [2] react on ribosomes as acceptors of the peptidyl residue, i.e., as puromycin. The peptide acceptor activity of those esters depends on the nature of bases [3] and of amino acid residues [4, 2]. The structure of' sugar residue is also important in the peptide acceptor activity of the model acceptors since amino acid esters of 2'dehydroxyadertosine are inactive [3] whereas the same esters of 2'-o-methyladenosine show the acceptor capacity similar to that of puromycin [5]. The sugar part of phenylalanine esters of the adenosine molecule can be replaced by a 2,3-dihydroxycyclopentanicresidue with partial preserving of the acceptor activity [6]. At the same time esters of 9-(l'-fl-o-arabinosyl)-adenine are totally inactive [7]. It is shown here that amino acid esters of 9-(2',Y-dihydroxypropyl-l')-adenine possess high peptide acceptor capacity, but their mechanism of action differs from that of well-known peptide acceptors. 173 Molecular Biology Reports 1 (1973) 173-178. All Rights Reserved Copyright 9 1973 by D. Reidel Publishing Company, Dordrecht-Holland

II. MATERIALS A N D M E T H O D S Esters Phe-HP-Ade and Gly-HP-Ade were synthesized by condensation of HP-Ade with the appropriate N-tert-butyloxycarbonyl aminoacyl N-imidazoles, followed by removal of the N H 2protecting group according to Gottikh et al. [8]. Similarly, Phe-HP-TUra was obtained by condensation of N-salicyliden phenylalanyl N-imidozole with 1-(2',3'-dihydroxypropyl-l')-4thiouracyl. The isolation of all compounds synthesized was achieved by means of electrophoresis on Whatmann 3 MM paper in 6~o acetic acid at 23 V cm -1 and their structure was established by usual methods [9]. E. coli MRE-600 ribosomes [10] and E. coli B tRNA containing 1 8 ~ t R N A Phe were used. [x4C] Phe-tRNA was prepared by a known method [11]. Radioactive [14C] phenylalanine (220 mCi mmo1-1) was supplied by UVVVR, Czechoslovakia. AC-[14C] Phe-tRNA was synthesized from [14C] Phe-tRNA and acetyloxysuccinimide according to Lapidot et al. [12]. Radioactivity of the samples was measured in the SL-40 Liquid Scintillation Spectrometer (Intertechnique, France). Inhibition of polyphenylalanine synthesis by Phe-HP-Ade, Cly-HP-Ade and Phe-HP-TUra was investigated in a cell-free system according to Maden et al. [13]. Testing of the accepter activity of these esters was performed in the system containing ribosomes charged with Ac-[~4C] PhetRNA, either in the presence of polyU [4] or in a template-free system in the presence of alcohol [14]. After incubating the polyU.Ac-[14C] Phe-tRNA.ribosome complex with acceptors dissociation of the ribosomes was achieved by dilution of the incubation mixture with 1 ml of 1 x 10- 2 M tris. Hcl, buffer pH 7, containing 5 x 10- 2 M KCI, followed by incubation during 3 hr at 2 ~ Substances isolated by ethyl acetate extraction were compared chromatographically and electrophoretically with synthetic 3'-o-acetyl-dipeptidyl esters of 2',3'-dihydroxypropyl adenine. TABLE I Inhibition of polyU-directed polyphenylalanine synthesis by amino acid esters of 9-(2',3'-dihydroxypropyl-l')-adenine and 1-(2',3'-dihydroxypropyl-l')-4-thiouracyl Inhibitor, M x 10 3

Polyphenylalanine synthesized, pmoles a

Inhibition of polyphenylalanine synthesis,

puromycin 0.5 chloramphenicol 1.7 Phe-HP-Ade 0.6 Gly-HP-Ade 1.0 Phe-HP-TUra 0.6 HO-HP-Ade 0.35

26.5 7.9 15.0 1.54 5.47 3.8 23.8

0 70.0 43.4 94.2 79.4 85.8 10.2

a These values are given without nonspecifical adsorption, that consists about 7-8 % from control. - Incubation mixture: each complete 0.15 ml reaction mixture contained ribosomes 3.0 A260units; protein fraction 1.0 A2a0units; polyU 0.8 A2a0 units; Ac-[laC]Phe-tRNA 25-50 pmoles; tris.HCl pH 7.6,5 lamoles; MgCI~ 2 Bmoles;ATP 1.5 pmoles; GTP 1 Bmole.After incubation at 37 ~for 40 min, reaction was stopped by adding 0.1 m110% trichloroacetic acid, heating at 100~ for 20 min and then filtering through nitrocellulose filters. Radioactivity was measured in toluene scintillation liquid. 174

III. RESULTS A N D D I S C U S S I O N The structure of the substances synthesized is shown below. OH

Phe-HP-Ade Gly-HP-Ade Phe-HP-TUra

I

NH2CHRCOOCH2CHCH2B

R =CH2C6H5, B = adenine R = H, B = adenine R =CHzC6Hs, B = 4-thiouracyl.

The inhibitory effect of Phe-HP-Ade, GIy-HP-Ade and Phe-HP-TUra was tested in a polyUdirected polyphenylalanine synthesizing system and the results obtained are summarized in Table I. As may be seen from this table all compounds tested effectively inhibit polyphenylalanine synthesis. The degree of inhibitory activity displayed by the different compounds was approximately the same, and this inhibition is stronger than with puromycin or chloramphenicol. The adenine derivative lacking amino acid (HP-Ade) was practically inactive. The peptide acceptor activity of Phe-HP-Ade and Gly-HP-Ade was tested in a cell-free system with ribosomes, p o l y U and Ac-[14C] Phe-tRNA acting as a peptide donor. No radioactivity was extracted by ethyl acetate after incubation of the ribosomal complex with both esters. On the other hand it was shown that Phe-HP-Ade and GIy-HP-Ade compete with puromycin in this system. A typical curve for Phe-HP-Ade is shown in Fig. 1. There are two possible explanations for these facts. First, Phe-HP-Ade and GIy-HP-Ade may bind with the same ribosomal site as puromycin, i.e. with the acceptor site. However they cannot accept a peptidyl residue as reported for aminophenethyl phosphonium adenosine [15]. Second, %

60

4O

25 20

0

0.4

I

I

I

I

I

0.6

0.8

1.0

1.2

1.4

C o n c e n t r a t i o n of Phe-HP-Ade, M xlO 3

Fig. 1. Inhibition of transfer of the AcPhe residue from AcPhe-tRNA to puromycin by the phenylalanine ester of 9-(2',Y-dihydroxypropyl-l')-adenine. One test tube contained in 0.1 ml 3.0 A2e0 units of ribosomes; 0.8 Az6o units of polyU; Ac-[14C]Phe-tRNA25 pmoles; tris-HCl 5 ~tmoles, pH 7.6; MgCI~ 1 lamoles; KC1 5 lamoles.After 10 min of incubation at 30~ puromycin up to the 5 • 10-4 M and increasing concentrations of Phe-HP-Ade were added and incubation was continued for additional 15 min. The reaction was stopped by addition of 1 ml of tris.HCl buffer, pH 7.0 and 3 ml of ethyl acetate. After extraction, the radioactivity ofa 2 ml aliquot of the organic phase was counted in 10 ml toluene scintillation liquid mixed with 5 ml of methoxy ethanol. 175

one might suppose that AcPhePhe-HP-Ade and AcPheGly-HP-Ade are formed, but they do not separate from the ribosomes. In order to confirm directly the mechanism of action of Phe-HP-Ade and Gly-HP-Ade, ribosomes were incubated with esters, and then dissociated into subunits. The substances released were then extracted with ethyl acetate and analoged by electrophoresis and paper chromatography using appropriate controls (Fig. 2). It seems likely that Phe-HP-Ade and Gly-HP-Ade were able to accept an AcPhe residue on ribosomes, but the products formed could not leave the ribosomes.

0

0

0

0

0

0

0

0 0

0

0 1

2

3

4

5

6

7

8

0

1

+

2

3

8 +

Fig. 2. Paper electrophoretic (A, in 6 % acetic acid, pH 2.5, at 800 V for 1 hr) and chromatographic (B, in nbutanol-acetic acid-water 78:5:17 v/v) analysis of reaction products formed by transfer of the AcPhe-residue from AcPhe-tRNA to acceptor substrates Phe-HP-Ade, GIy-HP-Ade and puromycin. - 1. Phe-HP-Ade; 2. Ac-[14C] PhePhe-HP-Ade; 3. Synthetic AcPhePhe-HP-Ade; 4. GIy-HP-Ade; 5. Ac-p4C]PheGIy-HP-Ade; 6. Puromycin; 7. Ac-[14C]Phe-puromycin; 8. Ac-p4C]Phe.

All esters examined (Phe-HP-Ade, GIy-HP-Ade and Phe-HP-TUra) inhibited the transfer of the AcPhe residue from tRNA to ethanol. This effect was tested in a cell-free system with ribosomes in the presence of ethanol but without template [14, 16]. A typical experiment is depicted in Table II. The results reported here provide evidence that amino acid esters of nucleosides with dihydroxypropyl group replacing the ribose moiety are strong inhibitors of peptide synthesis in E. coli ribosomes. Their mechanism of action differs from that of well-known inhibitors like puromycin or amino acid esters of adenosine. Amino acid esters of dihydroxypropyl adenine accept a peptidyl residue. However, the reaction products are released from the ribosomes. The data presented in Table I and II show that both phenylalanine and glycine esters of 9(2',Y-dihydroxypropyl-l')-adenine are active as peptide acceptors. At the same time phenylalanine ester of adenosine is totally inactive [4, 2]. Similarly, Phe-HP-TUra is active, while the phenylatanine ester of uridine is inactive [3]. We suggest that the activity of the peptide acceptors depends on the general conformation of the acceptor molecule. Amino acid esters of dihydroxypropyl adenine and dihydroxypropyl 4-thiouracyl have additional conformational possibilities in 176

TABLE I1 Inhibition of peptidyl transfer reaction to ethanol by phenylalanine and glycine esters of dihydroxypropyl-adenine and dihydro-xypropyl-4thiouracyl in ribosomes without a template Inhibitor, M x 10 a

Radioactivity in ethyl acetate, pmoles

Inhibition, %

Complete system + chloramphenicol 3.2 + Phe-HP-Ade 1.0 + GIy-HP-Ade 1.6 + Phe-HP-TUra 1.2 + HO-HP-Ade 0.7 Complete system without ethanol

3.45 2.43 1.60 1.87 2.46 3.44

0 30 53.5 66 29 0

0.21

Conditions of experiment: Each complete 0.10 ml reaction mixture contained ribosomes 3.0 A2n0units; Ac-[~aC]Phe-tRNA17.5 pmoles; tris.HCl pH 8.0, 5.7 ~tmoles; MgClg 2 lamoles; KCI 40 Bmoles. The reaction was initiated by addition of 0.05 ml ethyl alcohol. After incubation at 20 ~ for 1 hr, 1 ml of 0.1 M tris.HCl buffer pH 7.0 and 3 ml ethyl acetate were added. After extraction all operations were similar to those described in the legend to Fig. 1. comparison with those of nucleoside esters. Therefore it is possible that the ribosomes per se give Phe-HP-Ade, GIy-HP-Ade and Phe-HP-TUra the conformation necessary for their functions. The high peptide acceptor activity of all esters investigated shows that their binding with ribosomes is not determined solely by the structure of the base or that of the amino acid residue. More probably their activity depends on the general conformation of the acceptor molecule during the interaction with a ribosome or its ability to acquire this conformation. We suggest that substances like amino acid esters of 9-(2',3'-dihydroxypropyl-l')-adenine may serve as a new tool for ribosome investigations. REFERENCES 1. Waller, J. P., Erd6s, T., Lemoine, F., Guttemann, S., and Sandrin, E., Biochim. Biophys. Acta 119, 566 (1966). 2. Gottikh, B. P., Nikolayeva, L. V., Krayevsky, A. A., and Kisselev, L. L., FEBS Letters 7, 112 (1970). 3. t~erna, J., Rychlik, I., ~emli~ka, J., and Chladek, S., Biochim. Biophys. Acta 204, 203 (1970). 4. Rychlik, I., t~erna, J., Chladek, S., Pulkrabek, P., and ~emli~ka, J., Europ. J. Biochem. 16, 136 (1970). 5. Pozdnyakov, V. A., Mitin, Yu. V., Kukhanova, M. K., Nikolayeva, L. V., Krayevsky, A. A., and Gottikh, B. P., FEBS Letters 24, 177 (1972). 6. Vince, R., Daluge, S., and Palm, M., Biochem. Biophys. Res. Commun. 46, 866 (1972). 7. Fisher, L. V., Lee, W. W., and Goodman, L., J. Med. Chem. 13, 775 (1970). 8. Gottikh, B. P., Krayevsky, A. A., Purygin, P. P., Tsilevich, T. L., Belova, Z. S., and Rudzite, L. N., Izvestia A N S.S.S.R., Ser. Chimich., 1967, p. 2571. 177

9. Krayevsky, A. A., Purygin, P. P., Rudzite, L. N., Belova, Z. S., and Gottikh, B. P., Izvestia A N S.S.S.R., Ser. Chimich., 1968, p. 378. 10. Gavrilova, L. P. and Smolyaninov, V. V., Molekularnaya Biologia 5, 883 (1971). 11. Lewin, J. C. and Nirenberg, M., J. Mol. BioL 34, 467 (1968). 12. Lapidot, Y., de Groot, N., and Fry-Shafrir, I., Biochim. Biophys. Acta 145, 292 (1967). 13. Maden, B. E. H., Traut, R. R., and Monro, R. E., J. Mol. Biol. 35, 333 (1968). 14. Weissbach, H., Reifield, B., and Brot, N., Arch. Biochem. Biophys. 127, 705 (1968). 15. ~emli~ka, J. and Chladek, S., Collect. Czechosl. Chem. Communs. 34, 1007 (1969). 16. Scolnick, E., Milman, G., Rosman, M., and Caskey, T., Nature 225, 152 (1970).

178