Int. J. Peptide Protein Rex 14, 1979,34-40 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

SYNTHETIC ENKEPHALINS Addicting Properties and Conformational Studies in Solution

BRUNO FILIPPI’, PIER0 CIUSTI’, LORENZO CIMA’, GIANFRANCO BORIN’, FERNANDA RICCHELLI’ and FERNANDO MARCHIORI’

Riopolymer Research Center, CNR, Institute o f Organic Chemistry, Padua, ’Institute o f Pharmacology, Institute o f Animal Riolbgy, University of Padua, Italy

Received 6 September, accepted for publication 14 December 1978

The addicting properties of [Leus]-enkephalin in mice are conserved in the ~ - S e analogue r~ and lost both in the L-Ser’ analogue and in all the ~ - C h ade~ rivatives of the above peptides. Fluorescence measurements in water show the presence of hydrogen-bonded tyrosyl OH groups in [Leu’l-enkephalin and in its LSer’ analogue. The Phe4/Cha replacements d o not influence these equilibria, but they affect the near U.V. dichroism o f the hydrogen bonded tyrosyl residues. I n the peptide absorption region in water solution, only [Leu’l-enkephalin and its cyclohexylalanyl derivative show a positive dichroism towards high frequencies, which is maintained in 8 M urea. N o clear relation is found between conformation(s) in solution and biological activity. A d 0-turn, with residues in positions 2 and 3 at the corners, is suggested for the conformation o f enkephalin bound to the receptors involved in the bioassay here used. Key words: addicting properties; enkephalins; circular dichroism; conformation;

fluorescence.

Enkephalins, two naturally occurring pentapeptides with morphine-like action (Hughes et ul., 1975), and enkephalin analogues have been widely studied to determine the influence of the constituent residues on the biological activity (Coy et ul., 1976; Terenius et ul., 1976; Bajusz et al., 1976; Agarwal et al., 1977), their conforrnational properties in solution by n.m.r. measurements (Jonesetal., 1976,1977; Garbay34

0367-8377/79/050034-07

Jaureguiberry et ul., 1976; Bleich et al., 1976; Khaled et al., 1977) and conformational preferences by theoretical calculations (De Cohen et al., 1977; Isogai et al., 1977; Momany, 1977; Humblet & De Cohen, 1977). Discrepancies resulted both among the n.m.r. data and among the results obtained by theoretical calculations. Furthermore, the @ and )I torsion angles at both Gly residues in crystalline [LeuS]enke-

$02.00/0 0 1979 Munksgaard, Copenhagen

SYNTHETIC ENKEPHALINS TABLE 1 Amino acid analyses were performed on a Garlo Erba analyser on samples hydrolyzed in 6 N hydrochloric acid for 22 h at 110" (upper numbers) and with aminopeptidase M (EC 3.4.11.2) at 3 7' for 48 h in 0.1 M Tris buffer, p H Z 75 (lower numbers)

Peptide

Amino acid ratios ~~

Enkephalin Cha'

Ser'

Ser', Cha' Ser

Ser3, Cha4

Tyr, 0.99; 1.01; Tyr, 0.99; 1.02; Tyr, 1.03; 1.01 Tyr, 1 .OO; 1.01; Tyr, 1.01; 1.02; Tyr, 0.99; 1.01;

Gly, 2.01; 1.97; Gly, 2.03; 1.99; Gly, 1.00; 1.oo Gly, 1.03; 1.00; Gly, 1.00; 1.01 ; Gly, 1.03; 1.01;

phalin define a conformation incompatible with L-amino acids (Smith & Griffin, 1978). This can explain the biological activity of (Walker et al., 1977; Wei, 1978), the D-&' D&r2 (Woodroff el al., 1978) and D-Met' (Szbkely et al., 1978) analogues, but not the lack of activity of the D-&i3 one (Coy er a!., 1976). Therefore, in addition to the difficulty of defining the conformation of enkephalin in solution, there is the difficulty of correlating it to the conformation needed at the receptors. We report here the synthesis of [Leu']-enkephalin and of its LSer' and ~ S e r 'analogues, together with their m a 4 (cyclohexylalanine) derivatives; further we present their addicting properties in mice and conformational studies in solution by fluorescence and CD techniques. MATERIALS AND METHODS

[Leu'] enkephalin was prepared by coupling in solution ZGlyGly-NHNH2 and H-F%e-Leu-OBut using the azide procedure. The resulting tetrapeptide was deprotected at the amino group by hydrogenolysis, then one fraction of the sample was acylated by Z-Tyr-NHNH2by means of the

~

~

Phe, 0.98; 1.03; cha, 1.01; 0.98; Ser, 0.95; 0.94 Ser, 0.98; 0.94; Ser, 0.96; 0.97; Ser, 0.96; 0.96;

Leu, 1.02;

1 .oo; Leu, 1.00; 0.99; Phe, 1.00; 1.03 Cha, 0.99; 0.96; Phe, 1.00; 1 .oo;

Cha, 1.01; 1.01;

Leu, 1.00; 0.99;

Leu, 1.oo; 0.99; Leu, 1.00; 0.99; Leu, 1.01; 1.02;

azide procedure, and the remaining were hydrogenated under 7 atm over rhodium to convert phenylalanine into cyclohexylalanine and successively acylated by Z-Tyr(Z)-OSU. After removal of the protecting groups, the products were purified by gel-filtration on Sephadex G-10 (eluted with 10%acetic acid) and on Sephadex LH-20(eluted with twicedistilled water). The same procedure was used for the synthesis of all the other derivatives. The products were chromatographically homogeneous in several t1.c. systems and the amino acid analysis showed the expected amino acid composition after acid end enzymic hydrolysis (Table 1). Circular dichroism (CD) measurements were performed with a Cary 61 dichrograph, calibrated with epiandrosterone. An MPF 4 PerkinElmer spectrophotofluorimeter was used to obtain fluorescence spectra, which were recorded in the ratio mode; solutions having optical densities of 0.05 h t the excitation wavelength were used. Fluorescence yields, relative to tyrosinamide, were determined from the height of the emission maximum (302 nm). Preliminary spectroscopic investigations showed that the reproducibility of the results

35

B. FILIPPI ET AL.

was dependent both upon concentration and upon the time required by the solutions to reach equilibrium. In aqueous solution, at concentraM, light scattering was tions higher than observed in the absorption spectra towards the visible region, indicating aggregation of the peptides. At the concentrations used in Tris M) and for fluoresbuffer for CD (1.42 X cence (3.55 X lo-' M) measurements, the lack of scattering is taken as evidence for the monomeric state of the peptides in solution, in agreement with the results of Khaled et d. (1977). The diluted solutions undergo a time-dependent conformational transition monitored by changes of the tyrosyl CD in the near U.V. region; the largest effect was found for the L-Ser'enkephalin, for which an inversion of the CD sign was also observed. Generally, no more than 24 h at room temperature were needed to reach the equilibrium, the longer time being required for solutions prepared with lyophilized samples stored in the solid state for a long period of time. When these precautions were observed, independent measurements gave highly reproducible results, which therefore concern equilibrium conditions of the monomeric peptides. Physical dependence was obtained in mice pretreated with a single S.C. injection of sustained release preparation obtained by suspending [Leu'] enkephalin or its derivatives in 0.75 ml sorbitan monooleate and 4.25 ml light liquid paraffin and emulsifying this oily phase with 5 ml saline. Injected dose: 150 mg/kg morphine or 500 mg/kg of [Leu'] enkephalin or equimolar amounts of its derivatives in a 0.2 ml volume. The dependence rate was quantified by counting the number of jumps elicited up to 60 min by standard dose of naloxone (50 mg/kg i.p.) in the hot plate test.

RESULTS AND DISCUSSION The data reported in Fig. 1 show that the addicting properties of [Leu'] enkephalin are maintained in the case of the Gly'/~-Ser replacement and lost as a consequence both of the Gly2/LSer and of all the Phe4/Cha replacements. These effects may II priori depend indirectly upon changes induced in the backbone conformation 36

or directly on the modified side chains (i.e. the side chain introduced in position 2 and the thick cyclohexyl ring in position 4 may prevent the binding to the receptor by steric hindrance). From the spectra at 25", reported in Fig. 2, the fluorescence yields related to tyrosinamide are 0.9, 1 and 0.85 for [Leu'lenkephalin and its [Ser3]- and [Ser'] -analogues, respectively, and are independent of the Phe4/Cha replacements in the parent peptides. On lowering the temperature to 14O, the relative yields become 0.87, 1 and 0.83 (the differences of the relative values at the two temperatures are small but real, as they have been confirmed in independent experiments). Since diffusion or collisional processes (and also their temperature dependence at the two values investigated) are exactly the same for tyrosinamide and [Ser3]enkephalin, the former product results in a very good model for the fluorescence behaviour of the tyrosyl part of the upper peptides interacting only with solvent molecules. Therefore the decreased fluorescence at 25" of enkephalin, and to a greater extent of its Ser'-analogue strongly support the presence of intramolecular hydrogen bonded tyrosyl OH groups. The increased relative quenching of tyrosyl fluorescence of enkephalin and of its Ser2analogue on lowering the temperature is consistent with an increased population of the hydrogen-bonded species, as the result of the temperature effect on this equilibrium. On the basis of theoretical conformational analysis for uncharged [Met'] enkephalin, Isogai et al. (1977) found that one conformation, involving a 11' bend at Gly3-Phe4and a hydrogen bond between the tyrosyl OH and the carbonyl group of Gly3 or Phe4, was more stable by 5 kcal/mol than a large group of different conformations. The great calculated energy of stabilization seems overestimated in the light of the partial quenching of tyrosine fluorescence in [Leu5]enkephalin, nevertheless there is some agreement between the proposed picture and the fluorescence results. In fact, the increased quenching of the Ser' analogue in comparison with the native peptide and the lack of quenching for the Ser3 one are in agreement with the requirement of a 11' &turn at Gly3-Phe4,according to the rules of Venkatachalam (1968). In a 11' &turn, the first corner position must be occu-

SYNTHETIC ENKEPHALINS

150

I 100

7

i 50

24

48

72

144

96

Hours

FIGURE 1

Naloxone responsiveness of mice treated S.C. with sustained release preparations of [ Leus] enkephalin morphine and (Ser’ , Leus ] enkephalin Q. None of the other synthetic derivatives reported in this work cause addiction.

1

100-

75 -

50

-

25-

I

1

I

280

300

320

340

Wavelength,

nrn

FIGURE 2 Fluorescence spectra of: (1) tyrosinamide, [Ser’, Leu’] - and [Ser’, Cha‘, Leu’] enkephalin; (2) [Leu’]and [Cha‘, Leus]enkephalin; (3) [Ser*, Leu5]- and [Sera, Cha‘, Leu’lenkephalin. lO-’M Tris buffer, pH 7; 2 5 ” ; h exc = 274.8 nm; excitation and emission slits 7 and 8 nm, respectively.

pied by a D-amino acid: Gly3 can play this role, and LSer’ may reinforce this conformation, which is prevented by L-Ser3. Further conformational information was obtained by studying the CD properties in solution of the products. In the near U.V. region, from the spectra in water solution reported in Fig. 3, it is evident that the phenylalanyl bands, located in the 250-270 nm region, are weaker for the Ser’ -analogue than those of [Leu’ ] -and [Ser’] enkephalin. This result may indicate an increased conformational heterogeneity of the C-terminal part of the molecule. The positive CD values in the 270-300 nm interval, originated by the tyrosyl residues, parallel, at least qualitatively, the decrements of hydrogen bonded tyrosyl groups. The values are not affected by the Phe4/Cha replacement for the Ser3 analogue and are affected to a low extent for the other two peptides. This indicates that in water solution the Phe4/Cha replacement has no or little influence on the conformational equilibria at the N-terminus of the molecules, particularly if we take into account the possibility of interactions between transitions of phenylalanyl and hydrogen bonded tyrosyl residues, which should be located in proximity. In a further searching of conformational relationships, the spectra in 8 M urea were obtained (Fig. 3). In comparison with the spectra in water, the intensity of the phenylalanyl bands is decreased for the Ser3 analog, essentially conserved for [Leu’] enkephalin, and increased for the Ser‘analogue: in these conditions the conformation at the C-terminus in the latter two products seems to be similar. The Phe4/Cha replacement has again no effect on the tyrosyl dichroism of [~er’]enkephalin, a little one for the Ser’analogue and a greater one for enkephalin. Some of the features observed in the near U.V. region are common to the spectra in the peptide absorption region (Fig. 4). In fact, on passing from water to 8 M urea solution, all the spectra show a red shift, even if the extent of shift is different for each case. The CD spectra in the fat U.V. region are not useful for a definition of the conformational preferences, both because of the presence of aromatic residues whose dichroism overlaps the peptide one and because of the lack of sure reference values for the possible conformational states (extended or 37

B. FILIPPI ET AL.

01

-

2 1

FIGURE 3

CD spectra in water (left side; 0

pH 7; c = 1.42 X lo-' M; 5 cm path length) and in 8 M urea (right side, subscript A ; C = 7.1 X lo-' M; lo-' M Tris buffer, pH 7; 1 cm path length) of Phe4enkephalins (original spectra) and of their Cha4 derivatives (solid lines); (1) [Leu']-enkephalins; (2) [Serz]enkephalins; (3) [Ser3]enkephalins.

CD spectra in water (solid line; pH 7; c = 1.42 X lo-' M) and in 8 M urea (dashed'line; 7.1 X lo-' M; lo-' M Tris buffer, pH 7) of [Leu']enkephalin fl), [Ser3, Leu5]enkephalin (2), [Sera,Leu']enkephalin (3). [Cha', Leu']cnkephalii (4), [Ser', Cha', Leu'] enkephalin (51, [Sera, Cha', LeuS]enkephalii (6).

38

SYNTHETIC ENKEPHALINS

&turn). However the comparison of the spectra is of interest. AU the spectra show only positive dichroism in the region investigated, when the phenylalanyl residue is present (Fig. 4, nos. 1 , 2 and 3). That this feature is dependent upon the aromatic residues is particularly indicated by the presence of shoulders or maxima at 227 nm (tyrosine) and 220 nm (phenylalanine). This is quite evident in spectra numbers 1 and 3, and is substantiated by the finding that the reduction of phenylalanine to cyclohexylalanine in all cases shifts the dichroism towards negative values, leaving a positive maximum at 227 nm, as expected for a tyrosyl residue. On passing from water to 8 M urea solution, the essential features of the spectra are maintained, even if the changes may be in the opposite directions if the phenylalanyl residue is present (Fig. 4, nos. 1, 2 and 3). When it is replaced by cyclohexylalanine, the dependence of the spectra on the solvent change is more differentiated. Taking as reference the behaviour of [L-Ser3, Cha4]enkephalin, for which only a solvent-induced red shift results, its spectrum in 8 M urea is approached by the LSer’ analogue (Fig. 4, no. 5 ) but not by the [Cha4]enkephalin (Fig. 4, no. 4). In the last two examples a conformational change seems to occur on passing from water to 8 M urea, as indicated by the CD changes in the region near 215 nm. However, the [Leu’] enkephalin shows positive CD values towards 200 nm which is in contrast to the behaviour of both the seryl analogues and indicates peculiar conformational properties. All the CD and fluorescence results show that there is no evident relationship with the biological activity data. The tendency to have intramolecular hydrogen bonded tyrosyl OH groups could explain the inability of the LSer’ analogue to cause physical dependence, but this is excluded by the finding that the natural enkephalin, still having hydrogen bonded tyrosyl groups, is biologically active. Further, the CD properties of the products indicate a greater similarity between the two seryl analogues than between the two biological active products. Therefore there is no direct way to correlate the conformational properties of the monomeric enkephalins in solution with their biological responses. General principles of binding and conformational compatibility may give useful indications.

The inability of giving physical dependence produced by the Phe4/Cha replacements, in otherwise active compounds, can be explained as the result of having transformed the planar benzene ring into the thicker and non-planar cyclohexyl ring. This replacement, at the level of Phe’ in the S-peptide, produced a decreased binding to S-protein and a distortion in the complex, as evidenced by the decreased activity (Borin et al., 1977). Since the S-peptide/ S-protein system has the formal character of an hormone/receptor complex, the same effects, depending upon the Phe4/Cha substitution in enkephalin, can explain the lack of biological activity. The different effects produced by the replacement of the enkephalin glycyl residues by L-serine seem to involve directly the backbone conformation. There are several examples in the literature proving that the substitution of Gly2 by amino acids increases the activity in different biological tests, while the substitution by Lamino acids reduces the activity to insignificant values. The lack of physical dependence here reported for the LSer2 analogue is in line with the general behaviour of Gly’ as a D-aminO acid. Different replacements of Gly3 by both Dand L-amino acids were always found to strongly reduce the biological activity of the resulting derivatives. The full activity of the LSer3 analogue reported here seems therefore limited to the biological test used. This result, together with the inactivity shown by the L-Ser’ analogue, allows a definition of the conformation of enkephalin bound to the receptors responsible for the physical dependence phenomenon, independently of the conformation(s) of monomeric enkephalin in solution. At the receptors, the qb and $ torsion angles of Gly’ and Cly3 determine a conformation which is allowed only to D- and L-amino acids, respectively. Therefore in the light of the stereochemical criteria (Venkatachalam, 1968), the above permitted configuration indicates a 11’ &turn, centered at the two glycyl residues. A &turn in this position was the first proposed ordered conformation for enkephalin at the receptors (Bradbury et al., 1976). This proposal originated from the striking similarity to the spatial structure of oripavine, a potent derivative of morphine. 39

B. FILIPPI ET AL,

To explain the finding that the replacement of Gly3by L-amino acids, in different bioassays, produces inactive derivatives one must assume that either the introduced side chain can not fit at the specific receptors or that a different conformation is required at the different receptors. Of course, glycine has larger conformational possibilities than any other amino acid, and the minimum side chain steric hindrance. ACKNOWLEDGMENTS Our thanks are due to Mr. U. Anselmi for the amino acid analyses and t o Mrs. F. De Zuane for typewriting the manuscript.

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Humblet, G. & De Cohen, J.L. (1977) in Peptides: Proceedings of the 5th American Peptide Symposium (Goodman, M. & Meienhofer, J., eds.), pp. 88-91, John Wiley & Sons,New York Isogai, Y., Nemethy, G. & Scheraga, H.A. (1977) Proc. Natl. Acad. Sci. US 14,414-418 Jones, C.R., Gibbons, W.A. & Garsky, V. (1976) Nature 262,779-782 Jones, C.R., Alper, J.B., Kuo, M.C. & Gibbons, W.A. (1977) in Peptides: Proceedings of the 5th American Peptide Symposium (Goodman, M. & Meienhofer, J., eds.), pp. 329-332, John Wdey & Sons, New York Khaled, M.H., Long, M.M., Thompson, W.D., Bradley, R.J.,Brown, G.B. & Urry, D.W. (1977) Biochem Biophys. Res. Commun. 16,224-231 Momany, F.A. (1977) Biochem. Biophys. Res. Commun. 75,1098-1 103 Smith, G.D. & Griffii, J.F. (1978) Science 199, 1214-1216

Agarwal, S.N., Hruby, V.J., Katz, R., Klee, W. & Niremberg, M. (1977) Bwchem. Biophys. Res. Commun. 16,129-135 Bajusz, S., Ronh, A.Z., Szkkely, J.I., Dunai-Kovks, Zs., Berzktei, I. & Grif, L. (1976) Acta Biochim. Biophys. Acad. Sci. Hung. 11,305-309 Bleich, H.E., Cutnell, J.D., Day, A.R., Freer, R.J., Glasel, J.A. & McKelvy, J.F. (1976) F’roc. Natl. Acad. Sci. US 13,2589-2593 Borin, G., Filippi, B., Moroder, L., Santoni, G. & Marchiori, F. (1977) Int. J. Peptide Rotein Res. 10,27-38

Bradbury, A.F., Smith, D.G. & Snell, C.R. (1976) Nature 260,165-166 Coy, D.H., Kastin, AJ.. Schally, A.V., Morin, O., Caron, N.G., Labrie, F., Walker, J.M., Fertel, R., Berntson, G.G. & Sandman, C.A. (1976)Biochem. Biophys. Res. Commun. 13,632-638 De Cohen, J.L., Humblet, C. & Kock, M.H.J.(1977) FEBS Lett. 13,38-42 Garbay-Jaureguiberry , C., Roques, B.P., Oberlii, R., Anteunis, M. & Lala, A.K. (1976) Biochem. Bwphys. Res. Commun. 71,558-565 Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A. & Moms, H.R. (1975) Nature 258,577-579

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Szkkely, J.I., Ron;, A.Z., Dunai-Ko~ks,Z., Miglkcz, E., Berzktei, I., Bajujz, S. & Grif, L. (1978) in 7th International Congress of Pharmacology, Paris. Abstr. No. 276 Terenius, L., Wahlstron, A., Lindeberg, G., Karlsson, S. & Ragnarsson,V. (1976) Biochem. Biophys. Res. Commun. 71,175-179 Venkatachalam, C.M. (1968) Bwpolymers 6, 14251436

Walker, J.M., Berntson, G.G., Sandman, C.A., Coy, D.H., Schally, A.V. & Kastin, A.J. (1977) Science 196,86-87

Wei, E. (1978) in 7th International Congress of Pharmacology, Pans. Abstr. No. 273 Woodroff, G.N., McCarthy, P.S., Turnbull, M.J. & Shaw, J.S. (1978) in 7th International Congress o f Pharmacology, Pans. Abstr. No. 441 Address: Dr. Fernando Marchiori Istituto di Chimica Organics Via Marzolo, 1 35100 Padova Italy