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Activation of Diketopiperazine Formation by Alkylammonium Carboxylate Salts and Aprotic Dipolar Protophobic Solvents
Peptides, Vol. 19, No. 2, pp. 389 –391, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(97)00325-2
BRIEF COMMUNICATION
Activation of Diketopiperazine Formation by Alkylammonium Carboxylate Salts and Aprotic Dipolar Protophobic Solvents SANTE CAPASSO*‡1 AND LELIO MAZZARELLA*§ *Centro di Studio di Biocristallografia, C.N.R., Napoli, Italy, ‡Facolta` di Scienze Ambientali, Seconda Universita` di Napoli, Caserta, Italy, §Dipartimento di Chimica, Universita` di Napoli ‘‘Federico II,’’ Napoli, Italy Received 10 July 1997; Accepted 25 September 1997 CAPASSO, S. AND L. MAZZARELLA. Activation of diketopiperazine formation by alkylammonium carboxylate salts and aprotic dipolar protophobic solvents. PEPTIDES 19(2) 389 –391, 1998.—Diketopiperazine formation from the N-terminal residues of a peptide chain is accelerated by aprotic dipolar protophobic solvents and catalyzed in organic solvents by alkylammonium carboxylate salts. The t1/2 for the first-order reaction of H-Ala-Pro-NH2zTFA falls from 20 d in methanol to 3.6 min in acetonitrile containing 0.02 mol dm23 triethylammonium acetate; for H-Ala-Ala-NH2 z TFA in the same reaction media t1/2 falls from an unmeasurably long time to 1.3 d. © 1998 Elsevier Science Inc. Diketopiperazine
Cyclization
Solvent effect
Catalysis
DIKETOPIPERAZINES (2,5-dioxopiperazines; DKPs) have long raised the interest of peptide chemists, for their frequent occurrence during the storage and manipulation of peptides as well as for their biological interest. In water and organic solvents, the N-terminal deprotonated amino group of a polypeptide chain can attack the carbonyl carbon atom of the second residue, causing the breakdown of the chain and formation of a DKP (Reference 9; Scheme 1). It has been shown, for example, that the main pathway of the spontaneous chemical degradation in solution of the undecapeptide substance P (7), a neuropeptide implicated in several physiological processes, and of the recombinant DNA-derivative growth hormone consists in the release of N-terminal dipeptides via the corresponding DKPs (1). On the other hand, many DKPs, encompassing a wide range of biological activities, have been found in a variety of tissues and body fluids (6,10,12). Moreover, DKPs are
conformationally constrained cyclic peptides, typically stable to proteolysis, with side-chains oriented in a spatially defined manner. These properties make DKPs and their derivatives of particular interest in drug design and in the study of structure º biological activity relationship (5). An efficient route for the synthesis of DKPs on a solid support (13) and the kinetics and mechanism of DKP formation from the N-terminal residues of a peptide chain in aqueous solution have recently been described (Capasso, S; Vergara, A; Mazzarella, L. Mechanism of 2,5-dioxopiperazine formation, submitted for publication, 1997). By contrast, there is no exhaustive description of the influence of organic solvents and the catalytic effects of cosolutes, though DKP formation has frequently been documented as a side reaction during peptide synthesis (2). The present communication documents that alkylammonium carboxylate salts are very efficient catalysts of DKP
1
Requests for reprints should be addressed to Sante Capasso, Centro di Studio di Biocristallografia, C.N.R., via Mezzocannone 4, 80134 Napoli, Italy. E-mail: [email protected]
389
390
CAPASSO AND MAZZARELLA
C H¤N HN R0
O
O
R
H
N
R9
R
C N
R H
C O
C
R9 N
1 NH¤©R9 R
O
R9 5 H or an alkyl residue Scheme 1.
formation and that aprotic dipolar protophobic solvents (according to the classification of Kolthoff; Reference 8) increase the reaction rate of several orders of magnitude with respect to the rate in polar solvents. We studied DKP formation from the peptides H-AlaPro-NH2 z TFA and H-Ala-Ala-NH2 z TFA in a variety of solvents covering a wide range of polarity, hydrogen-bonding, and acid-base ability, and containing as cosolutes, acids, bases, and salts. In the first peptide, the N-alkylated cyclic residue in second position should ensure a measurable rate of DKP formation also under conditions unfavorable to this reaction. The trifluoroacetate salts of the peptides have been chosen because this is usually the form obtained by HPLC purification of N-unprotected peptides. Furthermore, the stability of this form of peptides is of particular interest because it is widely accepted that the protonation of the N-terminal amino group prevents the formation of DKP. METHOD The peptide H-Ala-Pro-NH2 z TFA was synthesized by standard liquid-phase method (2). The DKP cyclo(-AlaPro-) was synthesized and characterized, as reported formerly (Capasso, S; Vergara, A; Mazzarella, L. Mechanism of 2,5-dioxopiperazine formation, submitted for publication, 1997). Cyclo(-Ala-Ala-) and Ala-Ala-NH2 z HCl were purchased from Bachem, the latter peptide being converted into the TFA form by a Dower 2x1 resin. All the peptides gave the expected NMR and FAB-mass spectra. Solutions of the peptides, 0.5 mmol dm23, were filtered through a 0.45-mm membered filter and then stored in a thermostatted bath at 20.0°C. At preselected times, an aliquot was removed and analyzed by HPLC on a C8 reversedphase column. The rate constants were calculated by leastsquares analysis, assuming the rate to be first order in the concentration of the reacting peptide. For all the samples, the fitting of the experimental data was satisfactory. RESULTS The values of the observed pseudo-first-order rate constant (kobs) for the cyclization of H-Ala-Pro-NH2zTFA as a func-
FIG. 1. Values of the observed pseudo-first-order rate constant (kobs) for diketopiperazine formation from H-Ala-Pro-NH2 z TFA as function of the relative dielectric constant (er) of the solvents, T 5 20°C.
tion of the relative dielectric constant (er) of several solvents are reported in Figure 1. The results clearly indicate that there is no direct correlation between kobs and er, and that in acetonitrile, acetone, and nitromethane, solvents characterized by an aprotic dipolar protophobic character, kobs is relatively high. As regards the catalysis of DKP formation, Figure 2 shows the effect of triethylammonium acetate, acetic acid, and triethylamine on kobs for the reaction from H-Ala-ProNH2 z TFA in acetonitrile. Interestingly, triethylammonium acetate proves to be an efficient catalyst. At the concentration 0.02 mol dm23, this salt increases kobs by a factor of thousand: the half-life value from 1.7 d in pure acetonitrile falls to 3.6 min in the presence of the salt. The constant value of kobs (Fig. 2) at higher concentrations of the salt
FIG. 2. Dependence of the observed pseudo-first-order-rate constant (kobs) for diketopipeazine formation from H-Ala-Pro-NH2z TFA in acetonitrile on the concentration of triethylamine acetate (■), triethylamine (F) and acetic acid (Œ), T 5 20°C.
ACTIVATION OF DIKETOPIPERAZINE FORMATION
probably reflects a change of the rate-limiting step of the reaction. It is worth noting that the excellent catalytic performance of triethylammonium acetate is related to the acid-base properties of both the anion and the cation. In fact, the replacement of acetate with chloride or of triethylammonium with sodium ion markedly reduces the efficacy of the catalysis (data not shown). On the other hand, the replacement of acetate with propionate causes no significant variation of the catalytic properties of the alkylammonium salt. The acid and base catalysis of DKP formation have already been reported in the literature (3,11); the data in Figure 2 show that these have a lower efficiency than the catalysis by triethylammonium acetate. Moreover, the kobs for the acetic acid, and to a lesser extent for triethylamine, shows an initial rapid increase with the concentration of catalyst, followed by a slow decrease at higher concentrations. The reaction of DKP formation from H-Ala-AlaNH2 z TFA is unmeasurably slow, except in acetonitrile, acetone, and nitromethane. In these solvents, however, as
391
for the Pro-containing peptide, triethylammonium acetate is an efficient catalyst: at the salt concentration of 0.02 mol dm23 in acetonitrile, t1/2 is 1.3 d. DISCUSSION The data obtained on H-Ala-Pro-NH2 z TFA and H-Ala-AlaNH2 z TFA show that the DKP formation from peptides having unprotected the N-terminal amino group is markedly promoted by aprotic dipolar protophobic and alkylammonium carboxylate salts. Under these conditions, the formation of DKP can occur at a significant rate, also for peptides whose sequence does not promote this reaction. These results must be taken into account for the manipulation and storage of peptides. Moreover, they show a convenient route for the synthesis of DKPs. ACKNOWLEDGEMENT This work was supported by grants from the Italian Ministero dell’ Universita` e della Ricerca Scientifica e Tecnologica and Consiglio Nazionale delle Ricerche, Progetto Strategico di Biologia Strutturale.
REFERENCES 1. Battersby, J. E.; Hancock, W. S.; Canova-Davis, E.; Oeswein, J.; O’Connor, B. Diketopiperazine formation and N-terminal degradation in recombinant human growth hormone. Int. J. Peptide Protein Res. 41:207–221; 1993. 2. Bodanszky, M. Principles of Peptide Synthesis. Berlin: Springer-Verlag; 1984. 3. Capasso, S.; Sica, F.; Mazzarella, L.; Balboni, G.; Guerini, R.; Salvadori, S. Acid catalysis in the formation of dioxopiperazines from peptides containing tetrahydroisoquinoline-3-carboxylic acid at position 2. Int. J. Peptide Protein Res. 45:567–73; 1995. 4. Crescenzi, O.; Fraternali, F.; Picone, D.; Tancredi, T.; Balboni, G.; Guerrini, R.; Lazarus, L. H.; Salvadori, S.; Temussi, P. A. Design and solution structure of a partially rigid opioid antagonist lacking of the basic center. Eur. J. Biochem. 247: 66 –73; 1997. 5. Ie´naga, K.; Nakamura, K.; Kurohashi, M.; Nakanishi, T.; Ichii, T. Hydroxyproline-containing diketopiperazines inducing drought resistance in rice. Phytochemistry. 29:35–9; 1990. 6. Kertescher, U.; Bienert, M.; Krause, E.; Sepetov, N.; Mehlis, B. Spontaneous chemical degradation of substance P in the solid phase and in solution. Int. J. Peptide Protein Res. 41: 207–11; 1993.
7. Kolthoff, I. M. Acid-Base Equilibria in Dipolar Aprotic Solvents. Anal. Chem. 46:1992–2003; 1974. 8. Marsden, B. J.; Nguyen, T. M.-D.; Schiller, P. W. Spontaneous degradation via diketopiperazine formation of peptides containing a tetrahydroisoquinoline-3-carboxylic acid residue in the 2-position of the peptide sequence. Int. J. Peptide Protein Res. 41:313–16; 1993. 9. Moyroud, J.; Gelin, J.; Cheˆne, A.; Mortier, J. Synthese d’analogues structuraux de thaxtomines, Phytotoxines responsables de la gala de la pomme de terre. Tetrahedron. 52:8525– 34; 1996. 10. Møss, J.; Bundgaard, H. Kinetics and mechanism of the facile cyclization of histidyl-prolineamide to cyclo (his-pro) in aqueous solution and the competitive influence of human plasma. J. Pharm. Pharmacol. 42:7–12; 1990. 11. Shiba, T.; Uratani, H.; Kubota, I.; Sumi, Y. Some aspects of the relationship between the structure of a bitter diketopiperazine and its receptor. Biopolymers. 20:1985– 87; 1981. 12. Szardenings, A. K.; Burkoth, T. S.; Lu, H. H.; Tien, D. W.; Campbell, D. A. A simple procedure for the solid phase synthesis of diketopiperazine and diketomorpholine derivatives. Tetrahedron. 53:6573–93; 1997.
PII S0196-9781(97)00325-2
BRIEF COMMUNICATION
Activation of Diketopiperazine Formation by Alkylammonium Carboxylate Salts and Aprotic Dipolar Protophobic Solvents SANTE CAPASSO*‡1 AND LELIO MAZZARELLA*§ *Centro di Studio di Biocristallografia, C.N.R., Napoli, Italy, ‡Facolta` di Scienze Ambientali, Seconda Universita` di Napoli, Caserta, Italy, §Dipartimento di Chimica, Universita` di Napoli ‘‘Federico II,’’ Napoli, Italy Received 10 July 1997; Accepted 25 September 1997 CAPASSO, S. AND L. MAZZARELLA. Activation of diketopiperazine formation by alkylammonium carboxylate salts and aprotic dipolar protophobic solvents. PEPTIDES 19(2) 389 –391, 1998.—Diketopiperazine formation from the N-terminal residues of a peptide chain is accelerated by aprotic dipolar protophobic solvents and catalyzed in organic solvents by alkylammonium carboxylate salts. The t1/2 for the first-order reaction of H-Ala-Pro-NH2zTFA falls from 20 d in methanol to 3.6 min in acetonitrile containing 0.02 mol dm23 triethylammonium acetate; for H-Ala-Ala-NH2 z TFA in the same reaction media t1/2 falls from an unmeasurably long time to 1.3 d. © 1998 Elsevier Science Inc. Diketopiperazine
Cyclization
Solvent effect
Catalysis
DIKETOPIPERAZINES (2,5-dioxopiperazines; DKPs) have long raised the interest of peptide chemists, for their frequent occurrence during the storage and manipulation of peptides as well as for their biological interest. In water and organic solvents, the N-terminal deprotonated amino group of a polypeptide chain can attack the carbonyl carbon atom of the second residue, causing the breakdown of the chain and formation of a DKP (Reference 9; Scheme 1). It has been shown, for example, that the main pathway of the spontaneous chemical degradation in solution of the undecapeptide substance P (7), a neuropeptide implicated in several physiological processes, and of the recombinant DNA-derivative growth hormone consists in the release of N-terminal dipeptides via the corresponding DKPs (1). On the other hand, many DKPs, encompassing a wide range of biological activities, have been found in a variety of tissues and body fluids (6,10,12). Moreover, DKPs are
conformationally constrained cyclic peptides, typically stable to proteolysis, with side-chains oriented in a spatially defined manner. These properties make DKPs and their derivatives of particular interest in drug design and in the study of structure º biological activity relationship (5). An efficient route for the synthesis of DKPs on a solid support (13) and the kinetics and mechanism of DKP formation from the N-terminal residues of a peptide chain in aqueous solution have recently been described (Capasso, S; Vergara, A; Mazzarella, L. Mechanism of 2,5-dioxopiperazine formation, submitted for publication, 1997). By contrast, there is no exhaustive description of the influence of organic solvents and the catalytic effects of cosolutes, though DKP formation has frequently been documented as a side reaction during peptide synthesis (2). The present communication documents that alkylammonium carboxylate salts are very efficient catalysts of DKP
1
Requests for reprints should be addressed to Sante Capasso, Centro di Studio di Biocristallografia, C.N.R., via Mezzocannone 4, 80134 Napoli, Italy. E-mail: [email protected]
389
390
CAPASSO AND MAZZARELLA
C H¤N HN R0
O
O
R
H
N
R9
R
C N
R H
C O
C
R9 N
1 NH¤©R9 R
O
R9 5 H or an alkyl residue Scheme 1.
formation and that aprotic dipolar protophobic solvents (according to the classification of Kolthoff; Reference 8) increase the reaction rate of several orders of magnitude with respect to the rate in polar solvents. We studied DKP formation from the peptides H-AlaPro-NH2 z TFA and H-Ala-Ala-NH2 z TFA in a variety of solvents covering a wide range of polarity, hydrogen-bonding, and acid-base ability, and containing as cosolutes, acids, bases, and salts. In the first peptide, the N-alkylated cyclic residue in second position should ensure a measurable rate of DKP formation also under conditions unfavorable to this reaction. The trifluoroacetate salts of the peptides have been chosen because this is usually the form obtained by HPLC purification of N-unprotected peptides. Furthermore, the stability of this form of peptides is of particular interest because it is widely accepted that the protonation of the N-terminal amino group prevents the formation of DKP. METHOD The peptide H-Ala-Pro-NH2 z TFA was synthesized by standard liquid-phase method (2). The DKP cyclo(-AlaPro-) was synthesized and characterized, as reported formerly (Capasso, S; Vergara, A; Mazzarella, L. Mechanism of 2,5-dioxopiperazine formation, submitted for publication, 1997). Cyclo(-Ala-Ala-) and Ala-Ala-NH2 z HCl were purchased from Bachem, the latter peptide being converted into the TFA form by a Dower 2x1 resin. All the peptides gave the expected NMR and FAB-mass spectra. Solutions of the peptides, 0.5 mmol dm23, were filtered through a 0.45-mm membered filter and then stored in a thermostatted bath at 20.0°C. At preselected times, an aliquot was removed and analyzed by HPLC on a C8 reversedphase column. The rate constants were calculated by leastsquares analysis, assuming the rate to be first order in the concentration of the reacting peptide. For all the samples, the fitting of the experimental data was satisfactory. RESULTS The values of the observed pseudo-first-order rate constant (kobs) for the cyclization of H-Ala-Pro-NH2zTFA as a func-
FIG. 1. Values of the observed pseudo-first-order rate constant (kobs) for diketopiperazine formation from H-Ala-Pro-NH2 z TFA as function of the relative dielectric constant (er) of the solvents, T 5 20°C.
tion of the relative dielectric constant (er) of several solvents are reported in Figure 1. The results clearly indicate that there is no direct correlation between kobs and er, and that in acetonitrile, acetone, and nitromethane, solvents characterized by an aprotic dipolar protophobic character, kobs is relatively high. As regards the catalysis of DKP formation, Figure 2 shows the effect of triethylammonium acetate, acetic acid, and triethylamine on kobs for the reaction from H-Ala-ProNH2 z TFA in acetonitrile. Interestingly, triethylammonium acetate proves to be an efficient catalyst. At the concentration 0.02 mol dm23, this salt increases kobs by a factor of thousand: the half-life value from 1.7 d in pure acetonitrile falls to 3.6 min in the presence of the salt. The constant value of kobs (Fig. 2) at higher concentrations of the salt
FIG. 2. Dependence of the observed pseudo-first-order-rate constant (kobs) for diketopipeazine formation from H-Ala-Pro-NH2z TFA in acetonitrile on the concentration of triethylamine acetate (■), triethylamine (F) and acetic acid (Œ), T 5 20°C.
ACTIVATION OF DIKETOPIPERAZINE FORMATION
probably reflects a change of the rate-limiting step of the reaction. It is worth noting that the excellent catalytic performance of triethylammonium acetate is related to the acid-base properties of both the anion and the cation. In fact, the replacement of acetate with chloride or of triethylammonium with sodium ion markedly reduces the efficacy of the catalysis (data not shown). On the other hand, the replacement of acetate with propionate causes no significant variation of the catalytic properties of the alkylammonium salt. The acid and base catalysis of DKP formation have already been reported in the literature (3,11); the data in Figure 2 show that these have a lower efficiency than the catalysis by triethylammonium acetate. Moreover, the kobs for the acetic acid, and to a lesser extent for triethylamine, shows an initial rapid increase with the concentration of catalyst, followed by a slow decrease at higher concentrations. The reaction of DKP formation from H-Ala-AlaNH2 z TFA is unmeasurably slow, except in acetonitrile, acetone, and nitromethane. In these solvents, however, as
391
for the Pro-containing peptide, triethylammonium acetate is an efficient catalyst: at the salt concentration of 0.02 mol dm23 in acetonitrile, t1/2 is 1.3 d. DISCUSSION The data obtained on H-Ala-Pro-NH2 z TFA and H-Ala-AlaNH2 z TFA show that the DKP formation from peptides having unprotected the N-terminal amino group is markedly promoted by aprotic dipolar protophobic and alkylammonium carboxylate salts. Under these conditions, the formation of DKP can occur at a significant rate, also for peptides whose sequence does not promote this reaction. These results must be taken into account for the manipulation and storage of peptides. Moreover, they show a convenient route for the synthesis of DKPs. ACKNOWLEDGEMENT This work was supported by grants from the Italian Ministero dell’ Universita` e della Ricerca Scientifica e Tecnologica and Consiglio Nazionale delle Ricerche, Progetto Strategico di Biologia Strutturale.
REFERENCES 1. Battersby, J. E.; Hancock, W. S.; Canova-Davis, E.; Oeswein, J.; O’Connor, B. Diketopiperazine formation and N-terminal degradation in recombinant human growth hormone. Int. J. Peptide Protein Res. 41:207–221; 1993. 2. Bodanszky, M. Principles of Peptide Synthesis. Berlin: Springer-Verlag; 1984. 3. Capasso, S.; Sica, F.; Mazzarella, L.; Balboni, G.; Guerini, R.; Salvadori, S. Acid catalysis in the formation of dioxopiperazines from peptides containing tetrahydroisoquinoline-3-carboxylic acid at position 2. Int. J. Peptide Protein Res. 45:567–73; 1995. 4. Crescenzi, O.; Fraternali, F.; Picone, D.; Tancredi, T.; Balboni, G.; Guerrini, R.; Lazarus, L. H.; Salvadori, S.; Temussi, P. A. Design and solution structure of a partially rigid opioid antagonist lacking of the basic center. Eur. J. Biochem. 247: 66 –73; 1997. 5. Ie´naga, K.; Nakamura, K.; Kurohashi, M.; Nakanishi, T.; Ichii, T. Hydroxyproline-containing diketopiperazines inducing drought resistance in rice. Phytochemistry. 29:35–9; 1990. 6. Kertescher, U.; Bienert, M.; Krause, E.; Sepetov, N.; Mehlis, B. Spontaneous chemical degradation of substance P in the solid phase and in solution. Int. J. Peptide Protein Res. 41: 207–11; 1993.
7. Kolthoff, I. M. Acid-Base Equilibria in Dipolar Aprotic Solvents. Anal. Chem. 46:1992–2003; 1974. 8. Marsden, B. J.; Nguyen, T. M.-D.; Schiller, P. W. Spontaneous degradation via diketopiperazine formation of peptides containing a tetrahydroisoquinoline-3-carboxylic acid residue in the 2-position of the peptide sequence. Int. J. Peptide Protein Res. 41:313–16; 1993. 9. Moyroud, J.; Gelin, J.; Cheˆne, A.; Mortier, J. Synthese d’analogues structuraux de thaxtomines, Phytotoxines responsables de la gala de la pomme de terre. Tetrahedron. 52:8525– 34; 1996. 10. Møss, J.; Bundgaard, H. Kinetics and mechanism of the facile cyclization of histidyl-prolineamide to cyclo (his-pro) in aqueous solution and the competitive influence of human plasma. J. Pharm. Pharmacol. 42:7–12; 1990. 11. Shiba, T.; Uratani, H.; Kubota, I.; Sumi, Y. Some aspects of the relationship between the structure of a bitter diketopiperazine and its receptor. Biopolymers. 20:1985– 87; 1981. 12. Szardenings, A. K.; Burkoth, T. S.; Lu, H. H.; Tien, D. W.; Campbell, D. A. A simple procedure for the solid phase synthesis of diketopiperazine and diketomorpholine derivatives. Tetrahedron. 53:6573–93; 1997.