5889 was added to a solution of 3D, Ser4 1-12 (0.186 g, 0.096 mmol), in a mixture of 90% acetic acid (8 ml), 1 N hydrochloric acid (0.4 ml), and saturated sodium chloride solution (0.8 ml) at -10". After stirring for 15 min at -lo", precooled 20% sodium chloride solution (80 ml) was added, and the resulting precipitate was collected and washed with ice-cold water. The still-wet material was dissolved in D M F (20 ml) at -10" and dried over sodium sulfate. The drying agent was filtered off, and a solution of methionylaspartylserylserylthreonylserylalanylalanine15 (3E, 13-20; 0.17 g, 0.192 mmol, as monoacetate, trihydrate) in D M F ( 5 ml) and triethylamine (0.06 ml) was added. The reaction mixture was stirred for 7 days at 5 " and for 1 day at room temperature, filtered, and concentrated to 10 ml under reduced pressure and water was added (150 ml). The resultant precipitate was centrifuged, washed with water and ether, and dried, yielding 0.14 g (58%). The crude material (3F, Ser4 1-20) (0.132 g) was dissolved in anhydrous TFA (1.3 ml), and the solution was kept for 150 min at room temperature. An excess of ice-cold ether was added and after 30 min at - 10" the peptide was collected by centrifugation, washed with ether, and dried. The residue, dissolved in 0.2 M sodium phosphate buffer (pH 6.4), was purified by passing through an Amberlite C G 50 column, desalted by gel filtration on a Sephadex G-25 column, and lyophilized as described previously. The product (3G, ~ +C 1 " (c 0.117, water), Ser4 1-20) (0.02 g, 19%) had [ a ] * *-70.6 single ninhydrin- and Pauly-positive component on paper electrophoresis at pH 1.9, 3.5, 6.4, and 9.5; amino acid ratios in acid hydrolysate: Lys?,ooOrnl.10G1u2. 8jThr2.ooAla3, soPheo. s7Hisa. oiMeto.98A ~ p 0 . 9 8 S e r ~amino . ~ ~ ; acid ratios in AP-M digest: Lysl,070rnl,ol-
+
Glul,o?Thrl.ssAlas,o2Phel,02HisO.96Meto.g j A ~ ~ ~ . 9 5 ( SGln)4, e r 80. The [Ser4,0rnlO]-S-peptide gave, after recombination in 1 : 1 molar ratio with S-protein, a 40% active partially synthetic ribonuclease. Lysylglutamylthreonylalanylserylalanyllysylphenylalanylglutamylornithylglutaminylhistidylmethionylaspart~lserylserylthreonyls erylalanylalanine (3G, Serb 1-20). The condensation of 3D, Serb 1-12 (0.315 g, 0.164 mmol), with 3E, 13-2Olb (0.29 g, 0.328 mmol, as monoacetate, trihydrate), by the azide procedure was carried out as described above for 3G, Ser4 1-20, and gave the partially protected [Ser6,Orn'O]-S-peptide (3F, Ser5 1-20; 0.357 g; 82 %). Treatment of 0.15 g of crude product with anhydrous TFA (1.5 ml), purification on Amberlite CG 50 and on Sephadex G-25, followed by lyophilization (3G, Serb 1-20; 0.04 g, 33%); gave the pure [Ser5,0rn1@]-S-peptide [aI2*D-71.4 + 1" (c 0.115, water); single ninhydrin- and Paulypositive component on paper electrophoresis at pH 1.9, 3.5, 6.4, and 9.5; amino acid ratios in acid hydrolysate: Lys2,0r,G~u2,9s,Thrl,8i, A l a ~ . ~ 2 , 0 r n , , o l , P h e l , o ~ , H i s ~ , ~ o , M e t ~ , ~ ~amino , A s pacid ~ , ~ ratios 6,Ser~,~7; . stlThrl.pjAlaa.020rnl, ajPhel,@?Hiso. 86in AP-M digest : L y s ~ojGlul, Metl.02Aspo, !,e(Ser Gln)j,oo. The [Ser5,0rn"JI-S-peptide gave, after recombination in 1 :1 molar ratio with S-protein, a 50% active partially synthetic ribonuclease.
+
Acknowledgment. The authors wish to thank Dr. E. Celon for carrying out the microanalyses, Dr. L. Carpino for reading the manuscript, and Mr. U. Anselmi and Mr. D. Stivanello for the skillful technical assistance.
Synthesis of Peptide Analogs of the N-Terminal Eicosapeptide Sequence of Ribonuclease A. IX. Synthesis of [ SerB,Ornlo] - and [P r 0 ' , 0 r n ~-eicosapeptides'.' ~] Fernando Marchiori, Raniero Rocchi, Luigi Moroder, Angelo Fontana, and Ernest0 Scoffone Contribution f r o m Istituto di Chimica Organica dell' Unicersita', Sezione VIII del Centro Nazionale di Chimica delle Macromolecole del C N R , Padua, Italy. Receiued June 3, 1968
Abstract: Syntheses are described of two analogs of the S-peptide i n which the arginyl residue in position 10 is replaced by ornithine a n d the alanyl residue in position 6 by either serine or proline. T h e stereochemical homogeneity of these peptides, Le., lysylglutamylthreonylalanylalanylseryllysylphenylalanylglutamylornithylglutaminylhistidylmethionylaspartylserylserylthreonylserylalanylalanine a n d lysylglutamylthreonylalanylalanylprolyllysylphenylalanylglutamylornithylglutaminylhistidylmethionylaspartylserylserylthreonylserylalanylalanine, was assessed by digestion with aminopeptidase M followed b y quantitative a m i n o acid analysis. The enzymic properties of the two synthetic eicosapeptides were checked, with RNA, after recombination with S-protein. T h e [Ser6,0rn10]S-peptide a n d the [ P r ~ ~ , O r n ~ ~ ] - S - p e pftoi rdm e respectively a 40 a n d 15 active partially synthetic ribonuclease a t a molar ratio of 1 : 1 with S-protein.
In
a previous communication we described the syntheses of [Ser4,0rn10]-and [Ser5,0rn10]-eicosapeptide analogs of the N-terminal sequence of bovine pancreatic ribonuclease A 4 and we reported that these (1) The peptides and peptide derivatives mentioned have the L configuration, For a simpler description the customary L designation for individual amino acid residues is omitted. The following abbreviations (IUPAC-IUB Commission on Biochemical Nomenclature, J . B i d . Chem., 241, 2491 (1966)) are used: Z = benzyloxycarbonyl, Boc = t-butyloxycarbonyl, OMe = methyl ester, OEt = ethyl ester, OBut = t-butyl ester, ONp = p-nitrophenyl ester, DMF = dimethylformamide, TFA = trifluoroacetic acid. (2) Some of the results recorded in this paper have been presented at the IXth European Peptide Symposium, Orsay, France, April 15, 1968: E. Scoffone, F. Marchiori, L. Moroder, R . Rocchi, and A. Scatturin, Proceedings of the Symposium, in press. (3) Part VIII: R. Rocchi, L. Moroder, F. Marchiori, E. Ferrarese, and E. Scoffone, J . A m . Chem. Soc., 90,5885 (1968).
synthetic modified S-peptides show high ribonuclease activity after recombination with S-protein in a 1 : l molar ratio when tested with RNA as substrate. The present study deals with the synthesis of two new eicosapeptide analogs where the arginyl residue in position 10 has been replaced by ornithine and the alanyl residue in position 6 either by serine or proline. (4) (a) F. M Richards, Proc. Nutl. Acad. Sci. U.S., 44, 162 (1958); RNase A, the principal chromatographic component of beef pancreatic ribonuclease; RNase, S, subtilisin-modified RNase A; S-protein, the protein component obtained from RNase S; S-peptide, the eicosapeptide obtained from RNase S ; RNase S', the reconstituted enzyme obtained by mixing equimolar amounts of S-peptide and S-protein. (b) According to M. S. Dosher and C. H. W. Hirs, Federation Proc., 25, 527 (1966), natural S-peptide is a mixture of at least [1-20]-S-peptide and [1-2l]-S-peptide.
Scoffone, et al. 1 Synthesis of [Ser6,0rn10]and [Pro6,0rn10]-eicosapeptides
5890 Chart I. Amino Acid Sequences of S-Peptide and Its Synthetic Analogs
S-Peptide 1
2
4
3
5
7
6
8
10 11 12 13
9
14 15 16 17 18 19 20
Lys-Glu-Thr-AIa-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Met-Asp-Ser-Ser-Thr-Ser-Ala-Ala [Ornlo]-S-Peptide 9 10 11 12 13 14 15 16 17 18 19 20 Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Orn-Gln-His-Met-Asp-Ser-Ser-Thr-Ser-Ala-Ala 1
2
3
4
5
7
6
8
[Ser6,0rn10]-S-Peptide 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Lys-GIu-Thr-Ala-Ala-Ser-Lys-Phe-Glu-Orn-Gln-His-Met-Asp-Ser-Ser-Thr-Ser-Ala-Ala 1
2
3
[Pro6,0rn1a]-S-Pep tide 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Lys-Glu-Thr-Ala-Ala-Pro-Lys-Phe-Glu-Orn-Gln-His-Met-Asp-Ser-Ser-Thr-Ser-Ala-Ala 1
Chart 11. Synthesis of the 1-6 Sequences
2
1
5
4
3
28 2c
Z i Ala
-
HA Ala
Z W O N p BOC
B o c d Lys Boc
-0Me
XLOMe
OBuf
!
I
2D
6
H-OMe
Z-I-ONp
2A
G1
XFOMe 7 Thr
7NHNH2
Z--/ Ala
Ala
OBuf
I
2E
Bwd Lys Boc I1
2F
B o c i Lqs Boc I
2G
H A Ala
?alA
X
OMe
X
OMe
OBul I I
Glu
A l a ?
Thr
Ala
OBul t
B o c dT Lys Glu
h Ala X
=
r Ala
t X NHNH?
Ser or Pro
The X-ray structure of RNase-S5 shows that the section of the S-peptide molecule which contains the sequence 2-12 is in an a-helical conformation and that the alanyl residue in position 4 appears to be involved in the hydrophobic interaction responsible for the correct S-peptide-S-protein association. The results of our substitution at positions 4 and 5 showed that direct hydrophobic interactions between the alanyl-4 or -5 side-chain residue and groups in the protein appears to be of little significance. In order to see how the alanyl residue in position 6 affects the ability of the S-peptide to assume its correct conformation when it is bound to S-protein, we synthesized the [Ser6,Orn1O]-S-peptide(Chart I). The findings of Beintema,Gthat an aspartyl residue replaces alanine-6 in the rat ribonuclease sequence, makes probable that the substitution of the apolar residue in position 6 by an amino acid with a polar side chain does not significantly affect the catalytic function. In order to explore the influence of the dimensions of the helical portion on the S-peptide’s ability to bind and to activate the S-protein, we carried out the synthesis of the [Pro6,0rn10]-S-peptide(Chart I). The steric restrictions of proline prevent such a residue from taking part in an a-helical conformation. The expected consequence of the replacement of alanine-6 with pro-
line is then a shortening or the disappearance of the helical section of the S-peptide, which could affect its capacity to regenerate ribonuclease activity with the S protein. The activity data show that both synthetic analogs are able to catalyze the depolymerization of RNA, after recombination with S-protein in a 1 : l molar ratio. The [SerG,Orn1O]-and the [Pro6,0rn10]-RNase S’ show about 65 and 25 %, respectively, of the enzymic activity of [OrnlO]-RNase S’ (40 and 15 in comparison with RNase S’). Since the alanyl residues in positions 4, 5, or 6 can be replaced by the seryl residue without significantly affecting the potential catalytic properties of the resulting S-peptide analogs, it is possible to conclude that the hydrophobic character of these residues is not an important feature for the correct peptide-protein association. Moreover, the potential catalytic activity of the synthetic analog containing, in position 6, an imino acid (proline) in place of an amino acid (alanine) shows that such a modified eicosapeptide still posseses those basic structural features which are responsible for the activation of the S-protein in spite of the considerable difference in conformation which undoubtedly is found in the polypeptide backbone.
( 5 ) H. w. Wyckoff, K. D. Hardman, N. M. Allewell, T. Inagami, L. N. Johnson, and F. M. Richards, J, Bid, Chem., 242,3984 (1967). (6) J. J. Beintema and M. G. Gruber, Biochim. Biophys. Acta, 147, 612 (1967).
(7) (a) E. Scoffone, F. Marchiori, R. Rocchi, G. Vidali, A. M. Tamburro, A. Scatturin, and A . Marzotto, Tetrahedron Letfers, 943 (1966); (b) E. Scoffone, R. Rocchi, F. Marchior, A. Marzotto, A. Scatturin, A . M. Tamburro, and G. Vidali, J . Chem. Soc., C, 606 (1967).
Journal of the American Chemical Society
90.21
’
October 9, 1968
5891 Chart 111. Synthesis ot the 1-2U Sequences
4
1 2 3 Boc OBut
5
6
Boc OBul 3B B o c i L y s G
l
m
a
q
BOC ?But
3C B o c i Lys Glu Thr Ala Ala X Boc OBuf
3D BocA Lys Glu Thr Ala Ala X Boc OBuf 3E Boc
N
7 8 Boc
9
Boc
OBuf Boc
13 14 15 16 17 18 19 20
10 11 12 OBu'Boc
3 H--/ Lys Phe Glu BOC
OB.' BOC
Lys Phe Glu Orn Gln His F O M e Boc
OBuf Boc
Lys Phe Glu Orn Gln His L N H N H t Boc
OBu'Boc
Lys Glu Thr Ala AI
N3
H
Met Asp Ser Ser Thr Ser Ala Ala
OH
BOC OBul
BOC
3F Boc-
Lys Glu Thr Ala Ala X
Lys Phe Glu Orn Gln His
Met Asp Ser Ser Thr Ser Ala Ala -OH
H-
Lys Glu Thr Ala Ala X
Lys Phe Glu Orn Gln His
Met Asp Ser Ser Thr Ser Ala Ala -OH
3G
OBu' BOC
X = Ser or Pro
These findings agree with Hofmann's8 observation that the heptapeptide W-formyllysylphenylalanylglutamylarginylglutaminylhistidylmethionine still possesses the ability to activate the S-protein, with R N A as substrate (although at high peptide :protein molar ratios). We demonstratedg that the S-peptide is essentially randomly coiled in the absence of the partner S-protein while it undergoes a coil-to-helix transition in the presence of the protein. If one assumes that the existence of a helical portion in the S-peptide, stabilized by a cooperative effect of S-protein, is essential to facilitate the hydrophobic interactions which allow the association process, it is possible to deduce, from the above-mentioned findings, that only the helical section including residue 7-12 is essential for the correct peptide-protein binding, while residue 2-6 could be in a different conformation without a remarkable effect on the affinity of synthetic peptides for S-protein. A more detailed examination of the enzymic properties of [Ser6,0rn10]and [Pro6,0rn10]-S-peptide including the evaluation of their conformational stability will be reported in a forthcoming paper. Peptide Syntheses The synthetic route to [Ser6,0rn10]- and [Pro6,Orn'O]-S-peptide, illustrated in Charts I1 and 111, is similar to that which was used for the preparation of other analogs. 3 , 7 , lo The sequence 1-6 was prepared by condensing the azide corresponding to the Na,Nf-di-tbutyloxycarbonyllysyl-7-t-butylglutamylthreoninehy(8) F. M. Finn and I
