Short communication
Int. J. Peptide Protein Res. 7 , 1975, 503-504 Published by Munksgaard, Copenhagen. Denmark No part may be reproduced by any process without written permission from the author(s)
P R E P A R A T I O N O F F U R T H E R 2-PHENYLISOPROPYLOXYCARBONYL A M I N O A C I D S F O R A P P L I C A T I O N I N S O L I D PHASE P E P T I D E SYNTHESIS BENGTE. B. SANDBERG and ULF RAGNARSSON Institute of Biochemistry, University of Uppsala, Uppsala, Sweden
Received 16 May 1975 Proper selection of protecting groups is necessary for successful application of solid phase peptide synthesis (SPPS)(1). This was recently discussed in detail by Erickson & Merrifield (2). The problem is of equal importance in peptide synthesis using conventional techniques. The SPPS scheme as commonly employed calls for an acidic reagent to expose the a-amino group, which has so far, in most cases, been protected by a t-butyloxycarbonyl group. Today a few extremely acid labile a-amino protecting groups are available,
the most important one being 24p-biphenyl)isopropyloxycarbonyl (Bpoc) (3), which make (temporary) a-amino deblocking possible with much less premature loss of other protecting groups of permanent type. As an alternative to Bpoc-amino acids, we recently prepared a few NE-2-phenylisopropyloxycarbonyl amino acid derivatives, for simplicity called Ppoc-amino acids, and tested them in the preparation of bradykinin (4). These derivatives have the advantages of Bpoc-amino acids but
TABLE1 Preparation and characterization of some new Na-Ppoc-amino acids NQ-Ppof amino acids
talb" m.p. "Ce (c = 1, MeOH)
Rtd A
B
0.62
0.34
+3.9
0.61
0.23
+9.8
0.62
0.38
+4.4
0.63
0.50
f19.4
0.63
0.41
+5.0
0.64
0.35
-20.4
0.68
0.44
Formula
Mol. wt.
Yieldb
C,6H,aNz04
332.61
42
112-114
Arflos)
C,.H,.N,O,S
490.59
60
122.5-123.5
Asn(Mbh)
C,,H.IN,O,
520.59
90
amorph.
Cys(Bz1) DCHA
C.,H,.N;O,S
554.80
41
134-135
Glu(y-0BzI) CHA
C,,Ha.N.O,
498.62
37
134-136
Gln(Mbh)
C.,H,,N,OI
534.61
61
amorph.
Leu
C,,H,.NO,
293.37
34
108-110
LYs(2)DCHA
C*.H,,NsO.
623.84
64
81-89
+12.7
0.10
0.44
Thr(BzI) CHA
C,,HalN,Ol
470.62
42
131-133
+29.0
0.71
0.44
Met
C,,H,,NO,S
311.41
62
75.5-17
-18.2
0.68
0.31
Ala DCHA
-3.9
Elementary analysis CalcdIFound C H N S 69.41 9.32 6.48 69.31 9.39 6.21 56.32 6.16 11.42 6.54 56.10 6.18 11.24 6.58 66.91 6.20 5.38 66.54 6.14 4.99 69.28 8.36 5.05 5.78 69.09 8.43 5.04 5.72 67.45 7.68 5.62 61.42 1.64 5.61 67.40 6.41 5.24 66.69 6.43 5.10 65.51 7.90 4.77 65.50 7.89 4.10 69.31 8.56 6.14 69.39 8.55 6.75 68.91 8.14 5.95 68.70 8.11 5.94 57.86 6.80 4.50 10.30 57.92 6.85 4.53 10.09
* All amino acids were obtained from Ajinomoto Co. Inc.. Tokyo, Japan, or Tanabe Sciyaku Co.. Osaka, Japan. Exccpt for glycine, they were of Lsonfiguration. In the case of trifunctional amino acids, the sources of the required starting material were as follows: Argqos) (9). Asn(Mbh) (lo), Cys(Bzl) (11). Glu ( y - O M )(12), Gln(Mbh) (10). Lys(Z) (13). and Thr(BzI) (14). b Reaction time 42-50 h, except for Gln(Mbh) 29 h and Leu 19 h. c Melting points were taken in open capillaries and arc uncomaed. d Thin-layer chromatography was performed on precoated silica plates (Merck DC Fertigplatten Kinelgel 60 F,..). Solvent system A: BuOH/AcOH/Pyridine/H,O (30 : 6 : 20: 24). Solvent system B: CHCI,/MeOH (9 : 4). Spots were revealed by ninhydrin after brief heating.
503
BENGT E. B. SANDBERG A N D ULF RAGNARSSON
are more easily prepared, since the corresponding alcohol is a common one. The results obtained in this synthesis were quite promising. We therefore decided to test the usefulness of Ppocamino acids for peptides exceeding bradykinin size, especially in conjunction with some other side-chain protecting groups not used in the bradykinin synthesis mentioned. Such an attempt has now been made on apamin (5-7), which is a neurotoxic component of bee venom. To our present knowledge there has been no report on attempts to synthesize this peptide. Apamin is the smallest neurotoxic polypeptide known, and it is the only one whose interaction with the central nervous system is well established (8). The peptide consists of eighteen amino acids and has two disulphide bridges. Our attempt at its synthesis involved straight-forward SPPS like that for bradykinin (4). After complete reduction and reoxidation followed by gel filtration (G-25) and ion-exchange chromatography (CM-cellulose and IRC-SO), the product was tested by intraperitoneal injection in mice. The very typical symptoms caused by the synthetic peptide preparation were identical with those of fresh natural apamin. The toxicity was about 60% of that of natural apamin. Methods for further purification of the synthetic product are now under development. Preparation of Ppoc-amino acids. The new Ppoc-
amino acids used in the synthesis above were prepared by reaction of the corresponding amino acids or derivatives with 2-phenylisopropyl phenyl carbonate using tetramethylguanidine as the base as described earlier (4). Data on the new compounds are collected in Table 1. In a few cases the work-up had to be modified. Thus, in the case of Ppoc-Arg(Tos), precipitation of the product from the ether-ethyl acetate solution occurred on drying over MgS04. To increase the solubility, CHCI, should be added in this case. Ppoc-Asn(Mbh) was crystallized from an oil by adding petroleum ether (b.p. 40-60°C). To start precipitation of the DCHA salt of Ppoc-Lys(Z) a small amount of petroleum ether had to be added to the ether solution.
504
PCKNOWLEDGEMENTS
Financial support was provided by The Swedish Natural Science Research Council, Knut and Alice Wallenberg's Foundation and Stiftelsen Bengt Lundqvists Minne.
REFERENCES
1. MERRIFIELD, R. B. (1963) J. Amer. Chem. SOC.85, 2 149-21 54. B. W. & MERRIFIELD, R. B. (1973) 2. ERICKSON, J. Amer. Chem. SOC.95, 3750-3756. 3. SIEBER, P. & ISELIN, B. (1968) Helv. Chim. Acta 51, 622-63;!. 4. SANDBERG, B. E. B. & RAGNARSSON, U. (1974) Int. J . Pept. Prot. Res. 6, 11 1-1 19. 5 . HAUX,P., SAWERTHAL, H. & HABERMANN, E. (1967) Hoppe-Seyler's Z . Physiol. Chem. 348, 737-738. R., BRADBURY, A. F., CALLEWAERT, 6. SHIPOLINI, G. L. & VERNON, C. A. (1967) J.C.S. Chem. Comm. 679-680. 7. CALLEWAERT, G. L., SHIPOLINI, R. & VERNON, C. A. (1968) FEBSLetters 1, 111-113. 8. HABERMANI'I, E. (1972) Science 177,314-322. J. & LI, C. H. (1962) J. Org. 9. RAMACHANDRAN, Chem. 27,40064009. 10. KONIG,W. & GEIGER, R. (1970) Chem. Eer. 103, 204-2051. 11. FRANKEL, M., GERTNER, D., JACOBSON, H. & ZILKHA, A. (1960) J. Chem. SOC.139C1393. T.,HARADA. K. & Fox,S.W. (1966) 12. HAYAKAWA, Bull. Chem. SOC.Japan 39,391-395. 13. KUWATA, S. & WATANABE, H. (1965) Bull. Chem. SOC.Japan 38,676-671. T., LEVIN, G., WOOLLEY,D. W. & 14. MIZCGUCHI, STEWART, J. M. (1968) J. Org. Chem. 33,903-904.
Address : Bengt E. B. Satidberg
Institute of Biochemistry, BMC Box 576 S-751 23 Uppsala
Sweden
Int. J. Peptide Protein Res. 7 , 1975, 503-504 Published by Munksgaard, Copenhagen. Denmark No part may be reproduced by any process without written permission from the author(s)
P R E P A R A T I O N O F F U R T H E R 2-PHENYLISOPROPYLOXYCARBONYL A M I N O A C I D S F O R A P P L I C A T I O N I N S O L I D PHASE P E P T I D E SYNTHESIS BENGTE. B. SANDBERG and ULF RAGNARSSON Institute of Biochemistry, University of Uppsala, Uppsala, Sweden
Received 16 May 1975 Proper selection of protecting groups is necessary for successful application of solid phase peptide synthesis (SPPS)(1). This was recently discussed in detail by Erickson & Merrifield (2). The problem is of equal importance in peptide synthesis using conventional techniques. The SPPS scheme as commonly employed calls for an acidic reagent to expose the a-amino group, which has so far, in most cases, been protected by a t-butyloxycarbonyl group. Today a few extremely acid labile a-amino protecting groups are available,
the most important one being 24p-biphenyl)isopropyloxycarbonyl (Bpoc) (3), which make (temporary) a-amino deblocking possible with much less premature loss of other protecting groups of permanent type. As an alternative to Bpoc-amino acids, we recently prepared a few NE-2-phenylisopropyloxycarbonyl amino acid derivatives, for simplicity called Ppoc-amino acids, and tested them in the preparation of bradykinin (4). These derivatives have the advantages of Bpoc-amino acids but
TABLE1 Preparation and characterization of some new Na-Ppoc-amino acids NQ-Ppof amino acids
talb" m.p. "Ce (c = 1, MeOH)
Rtd A
B
0.62
0.34
+3.9
0.61
0.23
+9.8
0.62
0.38
+4.4
0.63
0.50
f19.4
0.63
0.41
+5.0
0.64
0.35
-20.4
0.68
0.44
Formula
Mol. wt.
Yieldb
C,6H,aNz04
332.61
42
112-114
Arflos)
C,.H,.N,O,S
490.59
60
122.5-123.5
Asn(Mbh)
C,,H.IN,O,
520.59
90
amorph.
Cys(Bz1) DCHA
C.,H,.N;O,S
554.80
41
134-135
Glu(y-0BzI) CHA
C,,Ha.N.O,
498.62
37
134-136
Gln(Mbh)
C.,H,,N,OI
534.61
61
amorph.
Leu
C,,H,.NO,
293.37
34
108-110
LYs(2)DCHA
C*.H,,NsO.
623.84
64
81-89
+12.7
0.10
0.44
Thr(BzI) CHA
C,,HalN,Ol
470.62
42
131-133
+29.0
0.71
0.44
Met
C,,H,,NO,S
311.41
62
75.5-17
-18.2
0.68
0.31
Ala DCHA
-3.9
Elementary analysis CalcdIFound C H N S 69.41 9.32 6.48 69.31 9.39 6.21 56.32 6.16 11.42 6.54 56.10 6.18 11.24 6.58 66.91 6.20 5.38 66.54 6.14 4.99 69.28 8.36 5.05 5.78 69.09 8.43 5.04 5.72 67.45 7.68 5.62 61.42 1.64 5.61 67.40 6.41 5.24 66.69 6.43 5.10 65.51 7.90 4.77 65.50 7.89 4.10 69.31 8.56 6.14 69.39 8.55 6.75 68.91 8.14 5.95 68.70 8.11 5.94 57.86 6.80 4.50 10.30 57.92 6.85 4.53 10.09
* All amino acids were obtained from Ajinomoto Co. Inc.. Tokyo, Japan, or Tanabe Sciyaku Co.. Osaka, Japan. Exccpt for glycine, they were of Lsonfiguration. In the case of trifunctional amino acids, the sources of the required starting material were as follows: Argqos) (9). Asn(Mbh) (lo), Cys(Bzl) (11). Glu ( y - O M )(12), Gln(Mbh) (10). Lys(Z) (13). and Thr(BzI) (14). b Reaction time 42-50 h, except for Gln(Mbh) 29 h and Leu 19 h. c Melting points were taken in open capillaries and arc uncomaed. d Thin-layer chromatography was performed on precoated silica plates (Merck DC Fertigplatten Kinelgel 60 F,..). Solvent system A: BuOH/AcOH/Pyridine/H,O (30 : 6 : 20: 24). Solvent system B: CHCI,/MeOH (9 : 4). Spots were revealed by ninhydrin after brief heating.
503
BENGT E. B. SANDBERG A N D ULF RAGNARSSON
are more easily prepared, since the corresponding alcohol is a common one. The results obtained in this synthesis were quite promising. We therefore decided to test the usefulness of Ppocamino acids for peptides exceeding bradykinin size, especially in conjunction with some other side-chain protecting groups not used in the bradykinin synthesis mentioned. Such an attempt has now been made on apamin (5-7), which is a neurotoxic component of bee venom. To our present knowledge there has been no report on attempts to synthesize this peptide. Apamin is the smallest neurotoxic polypeptide known, and it is the only one whose interaction with the central nervous system is well established (8). The peptide consists of eighteen amino acids and has two disulphide bridges. Our attempt at its synthesis involved straight-forward SPPS like that for bradykinin (4). After complete reduction and reoxidation followed by gel filtration (G-25) and ion-exchange chromatography (CM-cellulose and IRC-SO), the product was tested by intraperitoneal injection in mice. The very typical symptoms caused by the synthetic peptide preparation were identical with those of fresh natural apamin. The toxicity was about 60% of that of natural apamin. Methods for further purification of the synthetic product are now under development. Preparation of Ppoc-amino acids. The new Ppoc-
amino acids used in the synthesis above were prepared by reaction of the corresponding amino acids or derivatives with 2-phenylisopropyl phenyl carbonate using tetramethylguanidine as the base as described earlier (4). Data on the new compounds are collected in Table 1. In a few cases the work-up had to be modified. Thus, in the case of Ppoc-Arg(Tos), precipitation of the product from the ether-ethyl acetate solution occurred on drying over MgS04. To increase the solubility, CHCI, should be added in this case. Ppoc-Asn(Mbh) was crystallized from an oil by adding petroleum ether (b.p. 40-60°C). To start precipitation of the DCHA salt of Ppoc-Lys(Z) a small amount of petroleum ether had to be added to the ether solution.
504
PCKNOWLEDGEMENTS
Financial support was provided by The Swedish Natural Science Research Council, Knut and Alice Wallenberg's Foundation and Stiftelsen Bengt Lundqvists Minne.
REFERENCES
1. MERRIFIELD, R. B. (1963) J. Amer. Chem. SOC.85, 2 149-21 54. B. W. & MERRIFIELD, R. B. (1973) 2. ERICKSON, J. Amer. Chem. SOC.95, 3750-3756. 3. SIEBER, P. & ISELIN, B. (1968) Helv. Chim. Acta 51, 622-63;!. 4. SANDBERG, B. E. B. & RAGNARSSON, U. (1974) Int. J . Pept. Prot. Res. 6, 11 1-1 19. 5 . HAUX,P., SAWERTHAL, H. & HABERMANN, E. (1967) Hoppe-Seyler's Z . Physiol. Chem. 348, 737-738. R., BRADBURY, A. F., CALLEWAERT, 6. SHIPOLINI, G. L. & VERNON, C. A. (1967) J.C.S. Chem. Comm. 679-680. 7. CALLEWAERT, G. L., SHIPOLINI, R. & VERNON, C. A. (1968) FEBSLetters 1, 111-113. 8. HABERMANI'I, E. (1972) Science 177,314-322. J. & LI, C. H. (1962) J. Org. 9. RAMACHANDRAN, Chem. 27,40064009. 10. KONIG,W. & GEIGER, R. (1970) Chem. Eer. 103, 204-2051. 11. FRANKEL, M., GERTNER, D., JACOBSON, H. & ZILKHA, A. (1960) J. Chem. SOC.139C1393. T.,HARADA. K. & Fox,S.W. (1966) 12. HAYAKAWA, Bull. Chem. SOC.Japan 39,391-395. 13. KUWATA, S. & WATANABE, H. (1965) Bull. Chem. SOC.Japan 38,676-671. T., LEVIN, G., WOOLLEY,D. W. & 14. MIZCGUCHI, STEWART, J. M. (1968) J. Org. Chem. 33,903-904.
Address : Bengt E. B. Satidberg
Institute of Biochemistry, BMC Box 576 S-751 23 Uppsala
Sweden
