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Kinetics of Diketopiperazine Formation Using Model Peptides
Kinetics of Diketopiperazine Formation Using Model Peptides CHIMANLALL GOOLCHARRAN
AND
RONALD T. BORCHARDT*
Contribution from The Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047 Received August 15, 1997. Final revised manuscript received December 15, 1997. Accepted for publication December 16, 1997. Abstract 0 The intramolecular aminolysis of Phe-Pro-p-nitroaniline (Phe-Pro-pNA) to Phe-Pro-diketopiperazine (Phe-Pro-DKP) was studied as a function of pH, temperature, buffer concentration, and buffer species using an HPLC assay that permits simultaneous analysis of the disappearance of the starting material and the appearance of degradation products. The degradation followed pseudo-first-order kinetics and showed significant dependence on pH. Phosphate (pH 5−8) and glycine (pH 9−10) buffers exhibit general base catalysis. The pH−rate profile suggested that the rate of Phe-Pro-DKP formation depends on the degree of ionization of the N-terminal amino group, with the unprotonated reactant being more reactive than the protonated form. The pKa value of 6.1, determined kinetically, and three microscopic rate constants were adequate to describe the shape of the pH−rate profile. In the pH range studied, Phe-Pro-DKP was the only product generated upon degradation of Phe-Pro-pNA. At pH values between 3 and 8, Phe-Pro-DKP was stable, while at pH less than 3 and greater than 8 it undergoes hydrolysis to the dipeptide, Phe-Pro-OH. Sequence inversion, a reaction normally associated with DKP formation, was not observed. The influence of primary sequence on the formation of DKP was also investigated using X-Pro-pNA analogues, where X ) Gly, Ala, Val, Phe, β-cyclohexylalanine, and Arg. Changing the amino acid preceding the proline residue had a significant effect on the rate of DKP formation at pH 7.0.
Introduction Peptides and proteins that possess the N-terminal sequence in which proline is the penultimate residue undergo nonenzymatic aminolysis, yielding a diketopiperazine (DKP), which arises from the first two amino acids, and a truncated sequence.1-3 The mechanism of DKP formation involves nucleophilic attack of the N-terminal nitrogen on the amide carbonyl between the second and third amino acids. This intramolecular aminolysis reaction occurs readily in aqueous solution;4-6 it is known to play an important role in the biosynthetic pathway of biologically active cyclic dipeptides such as cyclo(His-Pro), which are found throughout the central nervous system, peripheral tissues, and body fluids.7 These cyclic dipeptides play important roles in neurophysiological functioning.8 Diketopiperazines also have implications in the diagnosis of proteins in fossils and thermal processing of food.9 It is well-documented in the literature that dipeptide esters and amides readily cyclize to the DKP and that the rate of cyclization competes favorably with hydrolysis.4,9,10 Sequence inversion and racemization have also been associated with diketopiperazine formation and greatly complicate the kinetics of such reactions.10 The cyclization of dipeptide esters containing a C-terminal proline residue * Tel: (785) 864-4820. Fax: (785) 864-5736. E-mail: Borchardt@ smissman.hbc.ukans.edu.
© 1998, American Chemical Society and American Pharmaceutical Association
has been shown to occur at a faster rate than in dipeptides that contain other C-terminal amino acids.9 In fact, the composition and configuration of the peptide plays a major role in determining the rate of the intramolecular aminolysis reaction.11 During peptide synthesis, this intramolecular aminolysis reaction occurs readily and creates problems in both solution and solid-phase synthesis techniques.11-16 The cyclization was shown to be catalyzed by acids17 and bases.3 In addition to the pH of the solution, the nature of the buffer species may also influence the rate of DKP formation.18 Although the formation of diketopiperazine has been shown to be a prevalent side reaction in peptide synthesis, it has not been studied in any detail as a degradation pathway of pharmaceutically important peptides and proteins in solution. Recently, several workers observed DKP formation upon storage of their protein pharmaceuticals.1,6,18,19 This observation led to a renewed interest in the intramolecular aminolysis reaction as a significant degradation pathway for peptides and proteins that contain a proline as the penultimate residue. In this study, the kinetics of DKP formation from the dipeptide analogue Phe-Pro-p-nitroaniline (Phe-Pro-pNA) was investigated in aqueous solution at 37 °C as a function of pH, temperature, buffer species, and concentration. The p-nitroaniline amides were selected for these studies because they undergo DKP formation more rapidly than amino acid amides (i.e., tripeptides), thus making the kinetic studies more convenient. In addition, the influence of primary sequence on the kinetics was addressed. For the latter study, we synthesized analogues in which the amino acid preceding the proline residue was varied.
Materials and Methods MaterialssAll t-Boc amino acid derivatives and proline-pnitroaniline (Pro-pNA) were purchased from Bachem, Bioscience Inc., Philadelphia, PA. HPLC grade acetonitrile and trifluoroacetic acid were obtained from Fisher Scientific (Fairlawn, NJ). 1-[3(Dimethylamino)propyl]-3-ethylcarbodiimide (EDC), 1-hydroxylbenzotriazide (HOBT), sodium phosphate monobasic, sodium phosphate dibasic, and sodium chloride were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of analytical grade and used as received. The water used in all studies was from a Millipore MILLI-Q water system. The dipeptide analogues and their degradation products (Scheme 1) were manually synthesized using a solution phase peptide synthesis technique11 utilizing EDC as the activating reagent. The peptide syntheses were monitored by TLC using Analtech, Inc., silica gel plates, which were developed using a mixture of hexane (60%), ethyl acetate (35%), and glacial acetic acid (5%). Plates were visualized with iodine vapor. Purification was performed using HPLC on a preparative Rainin Dynamax C18 column (10 µm, 22.5 × 250 mm). Kinetic MeasurementssThe following buffers at concentrations of 200, 100, and 50 mM were used for the kinetic experiments: pH 1.0, HCl; pH 2.0-3.0, glycine; pH 4.0-5.0, acetate; pH 5.5-8.0, phosphate; and pH 9.5-10.0, glycine. A constant ionic strength of 1.0 M was maintained for each buffer by adding the
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Journal of Pharmaceutical Sciences / 283 Vol. 87, No. 3, March 1998
Figure 1sTime course for the disappearance of Phe-Pro-pNA (0) and the appearance of Phe-Pro-DKP (O) and p-nitroaniline (]) at pH 5.0, 0.1 M acetate buffer (I ) 1.0) at 37 °C.
Scheme 1 appropriate amount of sodium chloride. The buffers were prepared at the experimental temperature and the pH value for a given solution remained unchanged throughout the investigation. An Orion (420A) pH meter equipped with an Orion automatic temperature compensation (ATC) electrode was used to measure the pH ((0.05) of the buffer solutions. All kinetic experiments were carried out in aqueous buffer solutions at 37 ( 1 °C unless otherwise indicated. The purified peptide analogues were dissolved in the appropriate buffer solution to yield an initial concentration of about 1 mM. Aliquots of 200 µL of the resulting solution were transferred to 250 µL HPLC vials, which were then sealed and stored in a 37 °C incubator. At various times, a vial was removed, cooled to room temperature, and analyzed by HPLC. All reactions were carried out for three or more half-lives. HPLC AnalysissThe HPLC system consisted of Rainin Dynamax SD-200 pumps, a Dynamax automatic sample injector (Al1A), and a Dynamax absorbance detector (UV-1), which were controlled by PC Acquisition HPLC-PDA (version 1.8) software. Analysis of the initial peptide and its degradation products was performed on a Rainin Dynamax 300A C18 column (5 µm, 4.6 × 250 mm) at room temperature. The solvent systems consisted of Milli-Q water:acetonitrile:trifluoroacetic acid in the ratios of 95: 5:0.1 (solvent A) and 10:90:0.1 (solvent B). Elution was accomplished utilizing an isocratic method of 15% solvent B for 10 min followed by a linear gradient to 80% solvent B in 15 min. The flow rate was maintained at 1.0 mL/min and the detection wavelength was 214 nm. The degradation products were identified by comparing their retention times with those of authentic samples.
Results and Discussion Kinetics of Diketopiperazine FormationsThe two possible pathways for the degradation of Phe-Pro-pNA are intramolecular aminolysis, yielding the corresponding PhePro-DKP, and direct hydrolysis, which produces the dipeptide Phe-Pro-OH (Scheme 1). Phe-Pro-DKP could then undergo further hydrolysis at either of the two carbonyl groups, producing two different dipeptides as shown in Scheme 1. Other reactions that have been observed to occur during DKP formation are sequence inversion and 284 / Journal of Pharmaceutical Sciences Vol. 87, No. 3, March 1998
Figure 2sKinetic profile for the degradation of Phe-Pro-pNA (0) at pH 9.0, I ) 1.0 and 37 °C. On the ordinate: O, Phe-Pro-DKP; ], p-nitroaniline; and 4, Phe-Pro-OH. The points represent experimental data and the lines were calculated by non-least-squares curve-fitting to eqs 1−3 and the degradation pathway shown in Scheme 2.
racemization.7,10 In these studies, formation of Pro-PheOH was not observed, indicating that sequence inversion did not occur. The possibility of racemization of the dipeptide was not investigated in this study. The kinetics of the Phe-Pro-pNA degradation were studied in aqueous buffer solutions at 37 °C over the pH range 1-10. The disappearance of the Phe-Pro-pNA and appearance of the degradation products were monitored by a stability-indicating HPLC method (see Materials and Methods). Pseudo-first-order kinetics was observed in all cases, and the rate constants, kobs, for the formation of DKP were obtained by linear regression of semilogarithmic firstorder plots. Figure 1 shows a typical time course of the disappearance of Phe-Pro-pNA and the appearance of PhePro-DKP and p-nitroaniline. In the pH range 3-8, PhePro-DKP was the only degradation product observed; formation of Phe-Pro-OH was not seen in this pH range. However, at pH values below 3 and above 8 detectable levels of Phe-Pro-OH were observed. The Phe-Pro-OH was formed after an initial lag phase that corresponded to about two half-lives of Phe-Pro-pNA (Figure 2). This lag time suggests that Phe-Pro-OH was possibly formed from PhePro-DKP rather than from Phe-Pro-pNA. This hypothesis was tested by modeling the degradation curves (i.e., Figure
Scheme 2
2) to the rate equations that describe the reaction pathway shown in Scheme 2.
A ) A0 exp(-kamt)
(1)
B ) {(A0kam)/(kdkp - kam)}{exp(-kamt) - exp(-kdkpt)} (2) C ) A0 +{A0/(kam - kdkp)}{kdkp exp(-kamt) - kam exp(-kdkpt)} (3) The disappearance of Phe-Pro-pNA and the appearance of Phe-Pro-DKP and Phe-Pro-OH were fitted with a correlation coefficient of 0.9954 (Figure 2). In separate experiments, Phe-Pro-DKP was found to be stable between pH 3 and 8, but it hydrolyzed to Phe-Pro-OH at pH values below 3 and above 8 (data not shown). These results indicate that the Phe-Pro-OH that is formed during the degradation of Phe-Pro-pNA must be due to the hydrolysis of Phe-Pro-DKP and not the hydrolysis of Phe-Pro-pNA. This is consistent with previous investigations that demonstrated that ester and amide functional groups of dipeptides undergo intramolecular aminolysis at a much faster rate than hydrolysis.4,20 Buffer CatalysissThe possible catalytic effect of the buffer was determined by measuring the rate of Phe-ProDKP formation from Phe-Pro-pNA at constant pH, ionic strength, and temperature while varying the buffer concentration. Plots of kobs values versus total buffer concentrations, [B]t, were fit to eq 4
kobs ) ko + kcat[B]t
(4)
where ko is the observed rate constant at zero buffer concentration and kcat is the catalytic rate constant. At pH below 5.0, kobs does not increase with increasing buffer concentration, suggesting no buffer catalysis (data not shown). However, at higher pH values (i.e., above 5.0), there is a significant increase in kobs as the phosphate and glycine buffer concentrations increase, indicating the presence of buffer catalysis (Figure 3a,b). The apparent rate constants of the buffer-catalyzed reaction, kcat, were used to determine the presence of general acid or general base catalysis. For the glycine buffer, the rate of Phe-Pro-DKP formation increases linearly with increasing fraction of uncharged buffer species, suggesting that the uncharged form of the buffer is the effective catalyst. On the other hand, the phosphate buffer exhibits a nonlinear relationship between the apparent rate constant of the buffer catalyzed reaction, kcat, and the fraction of HPO42- (Figure 4). There are two possible explanations for this nonlinearity: (i) the fractional species of Phe-Pro-pNA is changing and the buffer is acting on different ionic forms or (ii) there is a change in the rate-determining step of the reaction.21 pH DependencesThe rate constants at zero buffer concentration, ko, were obtained from the intercepts of plots of kobs versus total buffer concentration (i.e., Figure 3). The pH dependence of the rate of Phe-Pro-DKP formation at 37 °C and I ) 1.0 is shown in Figure 5. A possible explanation for the unusual pH profile is shown in Scheme 3, where AH2+ represents the protonated reactant and AH the neutral form. The overall pH-rate profile can be adequately described by eq 5, where ko is the pH-dependent first-order constant, Ka is the apparent dissociation con-
Figure 3sA plot of pseudo-first-order rate constants versus total buffer concentrations for the degradation of Phe-Pro-pNA in aqueous solution at 37 °C.
stant for the N-terminal amino group, k1 and k2 are microscopic first-order rate constants for the water or neutral catalysis of the protonated and unprotonated species, respectively, and k3 is the second-order rate constant for specific base catalysis.
ko ) k1[[H]/([H] + Ka)] + (k2 + k3[OH])[ka/(ka + [H])] (5) The kinetic rate constants and dissociation constant used to generate the theoretical pH-rate profile, the solid line in Figure 5, are the following: k1 ) 3.5((0.4) × 10-4 h-1, k2 ) 2.7((0.5) × 10-2 h-1, k3 ) 1.2((0.08) × 103 M-1 h-1, and Ka ) 7.9((0.6) × 10-6 (pKa ) 6.1). The shape of the pH-rate profile suggests that the degradation kinetics and mechanism of Phe-Pro-pNA are affected by the ionization of the N-terminal amino group. It is also apparent that the unprotonated reactant is much more reactive than the protonated form. At or pH below 3, where the reactant exists predominantly in the protonated form, AH2+, the rate of degradation is independent of pH. From pH 4-6, the rate increases with pH, but it is not first-order in hydroxide ion (approximate slope ) 0.73). This may due to the complexity of the mechanism involving different ionic species and buffer catalysis. Between pH 6 and 8 there is a plateau. In this region, the free N-terminal amino group is available for cyclization, but the hydroxide ion concentration is not high enough, so degradation occurs by the neutral-catalyzed pathway. In the pH region 9-10, the slope of the pH-rate profile is unit positive, indicating specific hydroxide ion catalysis. It should be pointed out Journal of Pharmaceutical Sciences / 285 Vol. 87, No. 3, March 1998
Figure 4sA plot of the catalytic rate constants versus the fraction of buffer in the base form for Phe-Pro-DKP formation in phosphate buffer at various pH values.
Figure 6sEyring plot for the formation of Phe-Pro-DKP at pH 2.0, 7.0, and 10.0 at 0.05 M buffer and I ) 1.0. Table 1sSummary of Rate Constant for Diketopiperazine Formation of X-Pro-pNA at pH 7.0 (37 °C, I ) 1.0)
Figure 5sThe pH−rate profile for the formation of Phe-Pro-DKP in aqueous solution at 37 °C. The solid line represents the theoretical profile based on the nonlinear regression fit to eq 5.
Scheme 3
that the reaction described by the neutral catalysis of the protonated species is kinetically equivalent to the acid catalysis of the unprotonated species and, similarly, the reaction for the neutral catalysis for the unprotonated species is kinetically equivalent to the base-catalyzed reaction of the protonated species. From the pH-rate profile and the fact that the unprotonated form of the reactant is more reactive than the protonated form, the water-catalyzed reaction was chosen for both reactions described above, although no strict basis exists for the preference of one over the other. Effect of TemperaturesThe rate of Phe-Pro-DKP formation from Phe-Pro-pNA was studied at pH 7.0 (0.05 M phosphate buffer, I ) 1.0) from 37 to 70 °C. Similar experiments were carried out at pH 2.0 and 10.0 (0.05 M glycine buffer, I ) 1.0). At all temperatures and pH values, the disappearance of the dipeptide analogue followed firstorder kinetics. For pH 7.0, at all temperatures, Phe-ProDKP was the only product formed; no hydrolytic product 286 / Journal of Pharmaceutical Sciences Vol. 87, No. 3, March 1998
X for analogs
(Rate (h-1) ± SD) × 102
Gly Ala Val Phe β-cyclohexylalanine Arg
0.57 ± 0.15 2.57 ± 0.13 1.16 ± 0.20 5.82 ± 0.24 3.50 ± 0.21 3.73 ± 0.10
was observed. However, at pH 2.0 and 10.0, Phe-Pro-OH was also produced, due to the further degradation of PhePro-DKP. At all pH values, the rate of DKP formation increased with increasing temperature, obeying the Arrhenius equation. From the Arrhenius plot, apparent activation energies of 102.8 ( 5.4, 73.1 ( 4.8, and 57.3 ( 5.9 kJ mol-1 were calculated for pH 2.0, 7.0, and 10.0, respectively. The other activation parameters calculated from the Eyring plot (Figure 6) were as follows: ∆Hq ) 100.2 ( 7.2 kJ mol-1 and ∆Sq ) 9.3 ( 5.1 J K-1 mol-1 for pH 2.0, ∆Hq ) 70.4 ( 5.4 kJ mol-1 and ∆Sq ) -46.3 ( 10.2 J K-1 mol-1 for pH 7.0, and ∆Hq ) 56.4 ( 6.1 kJ mol-1 and ∆Sq ) -77.3 ( 9.8 J K-1 mol-1 for pH 10.0. The positive value of entropy of activation at pH 2.0 suggests an increase in entropy at the transition state. This increase is due to a larger contribution of the positive entropy from the expulsion of water molecules of the highly solvated charged reactant than from the negative entropy value upon cyclization. Effect of Primary SequencesThe effect of the Nterminal residue on the rate of DKP formation at pH 7.0 was examined by studying the kinetics of degradation of X-Pro-pNA analogues, where X ) Gly, Ala, Val, Arg, Phe, and β-cyclohexylalanine. Varying the amino acid located on the N-terminal side of the proline residue has a significant effect on the rate of degradation (Table 1). The differences observed in the rate of DKP formation can be explained by (i) differences in the pKa values of the terminal amino group of the analogues, (ii) differences in the steric bulk, (iii) the ability of the X-Pro peptide bond to undergo cis-trans isomerization, and/or (iv) the conformational stability of the resulting DKPs. The shape of the pH-rate profile around pH 7.0 does not change as changes are made in the N-terminal amino acid (data not shown). This suggests that the pKa of the terminal amino group does not change dramatically and, therefore, does not influence the rate of DKP formation for the analogues studied.
The effect of alkyl and aryl substituents in intramolecular reactions has been well documented, with the results suggesting that an increase in the bulkiness of the substituent increases the rate of cyclization.22-25 This does not seem to be true for the series studied herein, as the Ala analogue cyclizes twice as fast as the bulkier Val analogue. Thus, simple steric bulk cannot be used to explain the data. Unlike other peptide bonds, those between Pro and its preceding amino acid (X-Pro) can exist as a mixture of cis and trans isomers in solution.26 Usually the trans isomer is favored over the cis in the absence of ordered structure, but structural constraints can stabilize one isomer over the other.27 In addition, the cis-trans equilibrium depends on the nature of the flanking amino acids and on the charge distribution around the X-Pro-peptide bond.26,28 In DKPs, the two side chains are located on the opposite sides of the general plane of the ring, which greatly reduces the strain applied to the six-member ring. On the basis of spacefilling models of dipeptide esters containing a proline residue, the cis isomer has been shown from a geometrical consideration to adapt a conformation in which the attacking N-terminal amino nitrogen atom is in proximity to the amide carbonyl, favoring ring closure.4 Thus, one would expect the cis isomer to undergo aminolysis more rapidly than its trans counterpart. There are no correlations between the bulkiness of the side chain of the amino acid and the occurrence of a cis peptide bond.29 Investigations are currently underway in our laboratory to determine the ability of these dipeptide analogues to undergo cis-trans isomerization. The conformation of the Phe-Pro-DKP in aqueous solution has been determined,30,31 and it was shown that the DKP ring adopted a boat-shaped conformation with the Phe aromatic ring stacked over it. Ciarkowsi et al.32 argued that the presence of the ring-ring interaction in a cyclic dipeptide composed of an aromatic amino acid residue adopted a more energetically favored conformation, in which the aromatic ring is stacked over the DKP ring, and that the intramolecular dipole-induced dipole should stabilize this form. There is also the possibility of an aromatic-aromatic interaction between the Phe side chain and the pNA leaving group, which could stabilize the tetrahedral intermediate and lead to an enhanced rate of cyclization of Phe-Pro-pNA. To check this hypothesis, the aromatic Phe residue was replaced with β-cyclohexylalanine. As shown in Table 1, the rate decreased by a factor of approximately 1.6, suggesting that an aromaticaromatic interaction may be one of many factors responsible for the intramolecular reaction. Further studies are needed to elucidate the contribution of these different factors.
Conclusions These results indicate that DKP formation is sensitive to the pH of the solution, the buffer species present at high pH, the temperature, and the N-terminal amino acid residue. The rapid rate at which the intramolecular reaction occurs at neutral and basic pH suggests that DKP formation may play an integral role in the degradation of peptides and proteins that contain a proline residue at position 2 under these conditions. The results of these studies will hopefully give scientists involved in the formulation of therapeutically important peptides and proteins insight into the multitude of endogenous (e.g., primary sequence) and exogenous (e.g., pH, buffer species) factors that can influence the rate of diketopiperazine formation in aqueous solution.
References and Notes 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. Pept. Protein Res. 1994, 44, 215-222. 2. Kertscher, U.; Bienert, M.; Krause, E.; Sepetov, N. F.; Mehlis, B. Spontaneous chemical degradation of substance P in the solid phase and in solution. Int. J. Pept. Protein Res. 1993, 41, 207-221. 3. Beyermann, M.; Bienert, M.; Niedrich, H.; Carpino, L. A.; Sadat-Aalace, D. Rapid continuous peptide synthesis via FMOC amino acid chloride coupling and 4-(aminomethyl)piperidine deblocking. J. Org. Chem. 1990, 55, 721-728. 4. Purdie, J. E.; Benoiton, N. L. Piperazinedione formation from esters of dipeptides containing glycine, alanine, and sarcosine: The kinetics in aqueous solution. J. Chem. Soc. Perkin Trans. 2 1973, 13, 1845-1852. 5. Skwierczynski, R. D.; Connors, K. A. Demethylation kinetics of aspartame and L-phenylalanine methyl ester in aqueous solution. Pharm. Res. 1993, 10, 1174-1180. 6. Gu, L.; Strickley, R. G. Diketopiperazine formation, hydrolysis, and epimerization of the new dipeptide angiotensinconverting enzyme inhibitor RS-10085. Pharm. Res. 1987, 4, 392-397. 7. Moss, 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. 1990, 42, 7-12. 8. Miyashita, K.; Murakami, M.; Yamada, M.; Iriuchijima, T.; Mori, M. Histidyl-proline diketopiperazine. J. Biol. Chem. 1993, 268, 20863-20865. 9. Steinberg, S. M.; Bada, J. L. Peptide decomposition in the neutral pH region via the formation of diketopiperazines. J. Org. Chem. 1983, 48, 2295-2298. 10. Steinberg, S. M.; Bada, J. L. Diketopiperazine formation during investigations of amino acid racemization in dipeptides. Science 1981, 213, 544-545. 11. Bodanszky, M.; Martinez, J. In The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhoffer, J., Eds.; Academic Press Inc.: New York, 1983; Vol. 5, pp 120-123, 193-196. 12. Gerig, J. T.; McLeod, R. S. Attempted synthesis of 2-methylalanyl-L-prolyl-L-tryptophan. An unexpected result. J. Org. Chem. 1976, 41, 1653-1655. 13. Gisin, B. F.; Merrifield, R. B. Carboxyl-catalyzed intramolecular aminolysis. A side reaction in solid-phase peptide synthesis. J. Am. Chem. Soc. 1972, 94, 3102-3106. 14. Mazurov, A. A.; Andronati, S. A.; Korotenko, T. I.; Gorbatyuk, V. Y.; Shapiro, Y. E. Formation of pyroglutamylglutamine (or asparagine) diketopiperazine in ‘non- classical’ conditions: A side reaction in peptide synthesis. Int. J. Pept. Protein Res. 1993, 42, 14-19. 15. Meienhoffer, J. Syntheses of actinomycin and analogues. III. A total synthesis of actinomycin D (C1) via peptide cyclization between proline and sarcosine. J. Am. Chem. Soc. 1970, 92, 3771-3777. 16. Fields, G. B.; Noble, R. L. Solid-phase peptide synthesis utilizing 9- fluorenylmethoxycarbonyl amino acid. Int. J. Pept. Protein Res. 1990, 35, 16-214. 17. Sepetov, N. F.; Krymsky, M. A.; Ovchinnikov, M. V.; Bespalova, Z. D.; Isakova, O. L.; Soueek, M.; Lebl, M. Rearrangement, racemization and decomposition of peptides in aqueous solution. Pept. Res. 1991, 4, 308-313. 18. Straub, J. A.; Akiyama, A.; Parmar, P.; Musso, G. F. Chemical pathways of the bradykinin analogue, RMP-7. Pharm. Res. 1995, 12, 305-308. 19. Oyler, A. R.; Naldi, R. E.; Lloyd, J. R.; Graden, D. A.; Shaw, C. J. Characterization of the solution degradation products of Histrelin, a gonadotropin releasing hormone (LH/RH) agonist. J. Pharm. Sci. 1991, 80, 271-275. 20. Radzicka, A.; Wolfenden, R. Rates of uncatalyzed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. J. Am. Chem. Soc. 1996, 118, 61056109. 21. Jenks, W. P. In Catalysis in Chemistry and Enzymology; McGraw-Hill: New York, 1969; pp 163-195. 22. Bruice, T. C.; Bradbury, W. C. The gem effect. The influence of 3-mono- and 3,3-disubstitution on the rates of solvolysis of mono-p-bromophenyl glutarate. J. Am. Chem. Soc. 1965, 87, 4846-4850. 23. Higuchi, T.; Eberson, L.; McRae, J. D. Acid anhydride-free acid equilibria in water in some substituted succinic scid systems and their interaction with aniline. J. Am. Chem. Soc. 1967, 89, 3001-3004. 24. Borchardt, R. T.; Cohen, L. A. Stereopopulation Control. II. Rate enhancement of intramolecular nucleophilic displacement. J. Am. Chem. Soc. 1972, 94, 9166-9174.
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25. Borchardt, R. T.; Cohen, L. A. Stereopopulation Control. III. Facilitation of intramolecular conjugate addition of the carboxyl group. J. Am. Chem. Soc. 1972, 94, 9175-9182. 26. Levitt, M. Effect of proline residues on protein folding. J. Mol. Biol. 1981, 145, 251-263. 27. Stein, R. L. In Advances in Protein Chemistry; Afinsen, C. B., Edsall, J. T., Richards, F. M., Eisenberg, D. S., Eds.; Academic Press, Inc.: New York, 1993; Vol. 44. 28. Ramachandran, G. N.; Mitra, A. K. An explanation for the rare occurrence of cis peptide units in proteins and polypeptides. J. Mol. Biol. 1976, 107, 85-92. 29. Stewart, D. E.; Sarkar, A.; Wampler, J. E. Occurrence and role of cis peptide bonds in protein structures. J. Mol. Biol. 1990, 214, 253-260. 30. Ciarkowski, J.; Gdaniec, M.; Kolodziejczyk, A.; Liberek, B.; Borremans, F. A. M.; Anteunis, M. J. O. Conformation of cyclo-(D-phenylalanyl-trans-4-fluoro-D-prolyl). Int. J. Pept. Protein Res. 1990, 36, 285-291.
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31. Budesinsky, M.; Symersky, J.; Jency, J.; VanHecke, J.; Hosten, N.; Anteunis, M.; Borremans, F. A. M. Cyclo (Lpropyl-L-N-methylphenylalanyl). Conformation in solution and in the crystal. Int. J. Pept. Protein Res. 1992, 39, 123130. 32. Liwo, A.; Ciakowski, J. Origin of the ring-ring interaction in cyclic dipeptides incorporating an aromatic amino acid. Tetrahedron Lett. 1985, 26, 1873-1876.
Acknowledgments This work was supported by the United States Public Health Service (GM-088359). We would also like to thank Drs. Richard Schowen and Suman Patel for their advice.
JS970325M
AND
RONALD T. BORCHARDT*
Contribution from The Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047 Received August 15, 1997. Final revised manuscript received December 15, 1997. Accepted for publication December 16, 1997. Abstract 0 The intramolecular aminolysis of Phe-Pro-p-nitroaniline (Phe-Pro-pNA) to Phe-Pro-diketopiperazine (Phe-Pro-DKP) was studied as a function of pH, temperature, buffer concentration, and buffer species using an HPLC assay that permits simultaneous analysis of the disappearance of the starting material and the appearance of degradation products. The degradation followed pseudo-first-order kinetics and showed significant dependence on pH. Phosphate (pH 5−8) and glycine (pH 9−10) buffers exhibit general base catalysis. The pH−rate profile suggested that the rate of Phe-Pro-DKP formation depends on the degree of ionization of the N-terminal amino group, with the unprotonated reactant being more reactive than the protonated form. The pKa value of 6.1, determined kinetically, and three microscopic rate constants were adequate to describe the shape of the pH−rate profile. In the pH range studied, Phe-Pro-DKP was the only product generated upon degradation of Phe-Pro-pNA. At pH values between 3 and 8, Phe-Pro-DKP was stable, while at pH less than 3 and greater than 8 it undergoes hydrolysis to the dipeptide, Phe-Pro-OH. Sequence inversion, a reaction normally associated with DKP formation, was not observed. The influence of primary sequence on the formation of DKP was also investigated using X-Pro-pNA analogues, where X ) Gly, Ala, Val, Phe, β-cyclohexylalanine, and Arg. Changing the amino acid preceding the proline residue had a significant effect on the rate of DKP formation at pH 7.0.
Introduction Peptides and proteins that possess the N-terminal sequence in which proline is the penultimate residue undergo nonenzymatic aminolysis, yielding a diketopiperazine (DKP), which arises from the first two amino acids, and a truncated sequence.1-3 The mechanism of DKP formation involves nucleophilic attack of the N-terminal nitrogen on the amide carbonyl between the second and third amino acids. This intramolecular aminolysis reaction occurs readily in aqueous solution;4-6 it is known to play an important role in the biosynthetic pathway of biologically active cyclic dipeptides such as cyclo(His-Pro), which are found throughout the central nervous system, peripheral tissues, and body fluids.7 These cyclic dipeptides play important roles in neurophysiological functioning.8 Diketopiperazines also have implications in the diagnosis of proteins in fossils and thermal processing of food.9 It is well-documented in the literature that dipeptide esters and amides readily cyclize to the DKP and that the rate of cyclization competes favorably with hydrolysis.4,9,10 Sequence inversion and racemization have also been associated with diketopiperazine formation and greatly complicate the kinetics of such reactions.10 The cyclization of dipeptide esters containing a C-terminal proline residue * Tel: (785) 864-4820. Fax: (785) 864-5736. E-mail: Borchardt@ smissman.hbc.ukans.edu.
© 1998, American Chemical Society and American Pharmaceutical Association
has been shown to occur at a faster rate than in dipeptides that contain other C-terminal amino acids.9 In fact, the composition and configuration of the peptide plays a major role in determining the rate of the intramolecular aminolysis reaction.11 During peptide synthesis, this intramolecular aminolysis reaction occurs readily and creates problems in both solution and solid-phase synthesis techniques.11-16 The cyclization was shown to be catalyzed by acids17 and bases.3 In addition to the pH of the solution, the nature of the buffer species may also influence the rate of DKP formation.18 Although the formation of diketopiperazine has been shown to be a prevalent side reaction in peptide synthesis, it has not been studied in any detail as a degradation pathway of pharmaceutically important peptides and proteins in solution. Recently, several workers observed DKP formation upon storage of their protein pharmaceuticals.1,6,18,19 This observation led to a renewed interest in the intramolecular aminolysis reaction as a significant degradation pathway for peptides and proteins that contain a proline as the penultimate residue. In this study, the kinetics of DKP formation from the dipeptide analogue Phe-Pro-p-nitroaniline (Phe-Pro-pNA) was investigated in aqueous solution at 37 °C as a function of pH, temperature, buffer species, and concentration. The p-nitroaniline amides were selected for these studies because they undergo DKP formation more rapidly than amino acid amides (i.e., tripeptides), thus making the kinetic studies more convenient. In addition, the influence of primary sequence on the kinetics was addressed. For the latter study, we synthesized analogues in which the amino acid preceding the proline residue was varied.
Materials and Methods MaterialssAll t-Boc amino acid derivatives and proline-pnitroaniline (Pro-pNA) were purchased from Bachem, Bioscience Inc., Philadelphia, PA. HPLC grade acetonitrile and trifluoroacetic acid were obtained from Fisher Scientific (Fairlawn, NJ). 1-[3(Dimethylamino)propyl]-3-ethylcarbodiimide (EDC), 1-hydroxylbenzotriazide (HOBT), sodium phosphate monobasic, sodium phosphate dibasic, and sodium chloride were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of analytical grade and used as received. The water used in all studies was from a Millipore MILLI-Q water system. The dipeptide analogues and their degradation products (Scheme 1) were manually synthesized using a solution phase peptide synthesis technique11 utilizing EDC as the activating reagent. The peptide syntheses were monitored by TLC using Analtech, Inc., silica gel plates, which were developed using a mixture of hexane (60%), ethyl acetate (35%), and glacial acetic acid (5%). Plates were visualized with iodine vapor. Purification was performed using HPLC on a preparative Rainin Dynamax C18 column (10 µm, 22.5 × 250 mm). Kinetic MeasurementssThe following buffers at concentrations of 200, 100, and 50 mM were used for the kinetic experiments: pH 1.0, HCl; pH 2.0-3.0, glycine; pH 4.0-5.0, acetate; pH 5.5-8.0, phosphate; and pH 9.5-10.0, glycine. A constant ionic strength of 1.0 M was maintained for each buffer by adding the
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Journal of Pharmaceutical Sciences / 283 Vol. 87, No. 3, March 1998
Figure 1sTime course for the disappearance of Phe-Pro-pNA (0) and the appearance of Phe-Pro-DKP (O) and p-nitroaniline (]) at pH 5.0, 0.1 M acetate buffer (I ) 1.0) at 37 °C.
Scheme 1 appropriate amount of sodium chloride. The buffers were prepared at the experimental temperature and the pH value for a given solution remained unchanged throughout the investigation. An Orion (420A) pH meter equipped with an Orion automatic temperature compensation (ATC) electrode was used to measure the pH ((0.05) of the buffer solutions. All kinetic experiments were carried out in aqueous buffer solutions at 37 ( 1 °C unless otherwise indicated. The purified peptide analogues were dissolved in the appropriate buffer solution to yield an initial concentration of about 1 mM. Aliquots of 200 µL of the resulting solution were transferred to 250 µL HPLC vials, which were then sealed and stored in a 37 °C incubator. At various times, a vial was removed, cooled to room temperature, and analyzed by HPLC. All reactions were carried out for three or more half-lives. HPLC AnalysissThe HPLC system consisted of Rainin Dynamax SD-200 pumps, a Dynamax automatic sample injector (Al1A), and a Dynamax absorbance detector (UV-1), which were controlled by PC Acquisition HPLC-PDA (version 1.8) software. Analysis of the initial peptide and its degradation products was performed on a Rainin Dynamax 300A C18 column (5 µm, 4.6 × 250 mm) at room temperature. The solvent systems consisted of Milli-Q water:acetonitrile:trifluoroacetic acid in the ratios of 95: 5:0.1 (solvent A) and 10:90:0.1 (solvent B). Elution was accomplished utilizing an isocratic method of 15% solvent B for 10 min followed by a linear gradient to 80% solvent B in 15 min. The flow rate was maintained at 1.0 mL/min and the detection wavelength was 214 nm. The degradation products were identified by comparing their retention times with those of authentic samples.
Results and Discussion Kinetics of Diketopiperazine FormationsThe two possible pathways for the degradation of Phe-Pro-pNA are intramolecular aminolysis, yielding the corresponding PhePro-DKP, and direct hydrolysis, which produces the dipeptide Phe-Pro-OH (Scheme 1). Phe-Pro-DKP could then undergo further hydrolysis at either of the two carbonyl groups, producing two different dipeptides as shown in Scheme 1. Other reactions that have been observed to occur during DKP formation are sequence inversion and 284 / Journal of Pharmaceutical Sciences Vol. 87, No. 3, March 1998
Figure 2sKinetic profile for the degradation of Phe-Pro-pNA (0) at pH 9.0, I ) 1.0 and 37 °C. On the ordinate: O, Phe-Pro-DKP; ], p-nitroaniline; and 4, Phe-Pro-OH. The points represent experimental data and the lines were calculated by non-least-squares curve-fitting to eqs 1−3 and the degradation pathway shown in Scheme 2.
racemization.7,10 In these studies, formation of Pro-PheOH was not observed, indicating that sequence inversion did not occur. The possibility of racemization of the dipeptide was not investigated in this study. The kinetics of the Phe-Pro-pNA degradation were studied in aqueous buffer solutions at 37 °C over the pH range 1-10. The disappearance of the Phe-Pro-pNA and appearance of the degradation products were monitored by a stability-indicating HPLC method (see Materials and Methods). Pseudo-first-order kinetics was observed in all cases, and the rate constants, kobs, for the formation of DKP were obtained by linear regression of semilogarithmic firstorder plots. Figure 1 shows a typical time course of the disappearance of Phe-Pro-pNA and the appearance of PhePro-DKP and p-nitroaniline. In the pH range 3-8, PhePro-DKP was the only degradation product observed; formation of Phe-Pro-OH was not seen in this pH range. However, at pH values below 3 and above 8 detectable levels of Phe-Pro-OH were observed. The Phe-Pro-OH was formed after an initial lag phase that corresponded to about two half-lives of Phe-Pro-pNA (Figure 2). This lag time suggests that Phe-Pro-OH was possibly formed from PhePro-DKP rather than from Phe-Pro-pNA. This hypothesis was tested by modeling the degradation curves (i.e., Figure
Scheme 2
2) to the rate equations that describe the reaction pathway shown in Scheme 2.
A ) A0 exp(-kamt)
(1)
B ) {(A0kam)/(kdkp - kam)}{exp(-kamt) - exp(-kdkpt)} (2) C ) A0 +{A0/(kam - kdkp)}{kdkp exp(-kamt) - kam exp(-kdkpt)} (3) The disappearance of Phe-Pro-pNA and the appearance of Phe-Pro-DKP and Phe-Pro-OH were fitted with a correlation coefficient of 0.9954 (Figure 2). In separate experiments, Phe-Pro-DKP was found to be stable between pH 3 and 8, but it hydrolyzed to Phe-Pro-OH at pH values below 3 and above 8 (data not shown). These results indicate that the Phe-Pro-OH that is formed during the degradation of Phe-Pro-pNA must be due to the hydrolysis of Phe-Pro-DKP and not the hydrolysis of Phe-Pro-pNA. This is consistent with previous investigations that demonstrated that ester and amide functional groups of dipeptides undergo intramolecular aminolysis at a much faster rate than hydrolysis.4,20 Buffer CatalysissThe possible catalytic effect of the buffer was determined by measuring the rate of Phe-ProDKP formation from Phe-Pro-pNA at constant pH, ionic strength, and temperature while varying the buffer concentration. Plots of kobs values versus total buffer concentrations, [B]t, were fit to eq 4
kobs ) ko + kcat[B]t
(4)
where ko is the observed rate constant at zero buffer concentration and kcat is the catalytic rate constant. At pH below 5.0, kobs does not increase with increasing buffer concentration, suggesting no buffer catalysis (data not shown). However, at higher pH values (i.e., above 5.0), there is a significant increase in kobs as the phosphate and glycine buffer concentrations increase, indicating the presence of buffer catalysis (Figure 3a,b). The apparent rate constants of the buffer-catalyzed reaction, kcat, were used to determine the presence of general acid or general base catalysis. For the glycine buffer, the rate of Phe-Pro-DKP formation increases linearly with increasing fraction of uncharged buffer species, suggesting that the uncharged form of the buffer is the effective catalyst. On the other hand, the phosphate buffer exhibits a nonlinear relationship between the apparent rate constant of the buffer catalyzed reaction, kcat, and the fraction of HPO42- (Figure 4). There are two possible explanations for this nonlinearity: (i) the fractional species of Phe-Pro-pNA is changing and the buffer is acting on different ionic forms or (ii) there is a change in the rate-determining step of the reaction.21 pH DependencesThe rate constants at zero buffer concentration, ko, were obtained from the intercepts of plots of kobs versus total buffer concentration (i.e., Figure 3). The pH dependence of the rate of Phe-Pro-DKP formation at 37 °C and I ) 1.0 is shown in Figure 5. A possible explanation for the unusual pH profile is shown in Scheme 3, where AH2+ represents the protonated reactant and AH the neutral form. The overall pH-rate profile can be adequately described by eq 5, where ko is the pH-dependent first-order constant, Ka is the apparent dissociation con-
Figure 3sA plot of pseudo-first-order rate constants versus total buffer concentrations for the degradation of Phe-Pro-pNA in aqueous solution at 37 °C.
stant for the N-terminal amino group, k1 and k2 are microscopic first-order rate constants for the water or neutral catalysis of the protonated and unprotonated species, respectively, and k3 is the second-order rate constant for specific base catalysis.
ko ) k1[[H]/([H] + Ka)] + (k2 + k3[OH])[ka/(ka + [H])] (5) The kinetic rate constants and dissociation constant used to generate the theoretical pH-rate profile, the solid line in Figure 5, are the following: k1 ) 3.5((0.4) × 10-4 h-1, k2 ) 2.7((0.5) × 10-2 h-1, k3 ) 1.2((0.08) × 103 M-1 h-1, and Ka ) 7.9((0.6) × 10-6 (pKa ) 6.1). The shape of the pH-rate profile suggests that the degradation kinetics and mechanism of Phe-Pro-pNA are affected by the ionization of the N-terminal amino group. It is also apparent that the unprotonated reactant is much more reactive than the protonated form. At or pH below 3, where the reactant exists predominantly in the protonated form, AH2+, the rate of degradation is independent of pH. From pH 4-6, the rate increases with pH, but it is not first-order in hydroxide ion (approximate slope ) 0.73). This may due to the complexity of the mechanism involving different ionic species and buffer catalysis. Between pH 6 and 8 there is a plateau. In this region, the free N-terminal amino group is available for cyclization, but the hydroxide ion concentration is not high enough, so degradation occurs by the neutral-catalyzed pathway. In the pH region 9-10, the slope of the pH-rate profile is unit positive, indicating specific hydroxide ion catalysis. It should be pointed out Journal of Pharmaceutical Sciences / 285 Vol. 87, No. 3, March 1998
Figure 4sA plot of the catalytic rate constants versus the fraction of buffer in the base form for Phe-Pro-DKP formation in phosphate buffer at various pH values.
Figure 6sEyring plot for the formation of Phe-Pro-DKP at pH 2.0, 7.0, and 10.0 at 0.05 M buffer and I ) 1.0. Table 1sSummary of Rate Constant for Diketopiperazine Formation of X-Pro-pNA at pH 7.0 (37 °C, I ) 1.0)
Figure 5sThe pH−rate profile for the formation of Phe-Pro-DKP in aqueous solution at 37 °C. The solid line represents the theoretical profile based on the nonlinear regression fit to eq 5.
Scheme 3
that the reaction described by the neutral catalysis of the protonated species is kinetically equivalent to the acid catalysis of the unprotonated species and, similarly, the reaction for the neutral catalysis for the unprotonated species is kinetically equivalent to the base-catalyzed reaction of the protonated species. From the pH-rate profile and the fact that the unprotonated form of the reactant is more reactive than the protonated form, the water-catalyzed reaction was chosen for both reactions described above, although no strict basis exists for the preference of one over the other. Effect of TemperaturesThe rate of Phe-Pro-DKP formation from Phe-Pro-pNA was studied at pH 7.0 (0.05 M phosphate buffer, I ) 1.0) from 37 to 70 °C. Similar experiments were carried out at pH 2.0 and 10.0 (0.05 M glycine buffer, I ) 1.0). At all temperatures and pH values, the disappearance of the dipeptide analogue followed firstorder kinetics. For pH 7.0, at all temperatures, Phe-ProDKP was the only product formed; no hydrolytic product 286 / Journal of Pharmaceutical Sciences Vol. 87, No. 3, March 1998
X for analogs
(Rate (h-1) ± SD) × 102
Gly Ala Val Phe β-cyclohexylalanine Arg
0.57 ± 0.15 2.57 ± 0.13 1.16 ± 0.20 5.82 ± 0.24 3.50 ± 0.21 3.73 ± 0.10
was observed. However, at pH 2.0 and 10.0, Phe-Pro-OH was also produced, due to the further degradation of PhePro-DKP. At all pH values, the rate of DKP formation increased with increasing temperature, obeying the Arrhenius equation. From the Arrhenius plot, apparent activation energies of 102.8 ( 5.4, 73.1 ( 4.8, and 57.3 ( 5.9 kJ mol-1 were calculated for pH 2.0, 7.0, and 10.0, respectively. The other activation parameters calculated from the Eyring plot (Figure 6) were as follows: ∆Hq ) 100.2 ( 7.2 kJ mol-1 and ∆Sq ) 9.3 ( 5.1 J K-1 mol-1 for pH 2.0, ∆Hq ) 70.4 ( 5.4 kJ mol-1 and ∆Sq ) -46.3 ( 10.2 J K-1 mol-1 for pH 7.0, and ∆Hq ) 56.4 ( 6.1 kJ mol-1 and ∆Sq ) -77.3 ( 9.8 J K-1 mol-1 for pH 10.0. The positive value of entropy of activation at pH 2.0 suggests an increase in entropy at the transition state. This increase is due to a larger contribution of the positive entropy from the expulsion of water molecules of the highly solvated charged reactant than from the negative entropy value upon cyclization. Effect of Primary SequencesThe effect of the Nterminal residue on the rate of DKP formation at pH 7.0 was examined by studying the kinetics of degradation of X-Pro-pNA analogues, where X ) Gly, Ala, Val, Arg, Phe, and β-cyclohexylalanine. Varying the amino acid located on the N-terminal side of the proline residue has a significant effect on the rate of degradation (Table 1). The differences observed in the rate of DKP formation can be explained by (i) differences in the pKa values of the terminal amino group of the analogues, (ii) differences in the steric bulk, (iii) the ability of the X-Pro peptide bond to undergo cis-trans isomerization, and/or (iv) the conformational stability of the resulting DKPs. The shape of the pH-rate profile around pH 7.0 does not change as changes are made in the N-terminal amino acid (data not shown). This suggests that the pKa of the terminal amino group does not change dramatically and, therefore, does not influence the rate of DKP formation for the analogues studied.
The effect of alkyl and aryl substituents in intramolecular reactions has been well documented, with the results suggesting that an increase in the bulkiness of the substituent increases the rate of cyclization.22-25 This does not seem to be true for the series studied herein, as the Ala analogue cyclizes twice as fast as the bulkier Val analogue. Thus, simple steric bulk cannot be used to explain the data. Unlike other peptide bonds, those between Pro and its preceding amino acid (X-Pro) can exist as a mixture of cis and trans isomers in solution.26 Usually the trans isomer is favored over the cis in the absence of ordered structure, but structural constraints can stabilize one isomer over the other.27 In addition, the cis-trans equilibrium depends on the nature of the flanking amino acids and on the charge distribution around the X-Pro-peptide bond.26,28 In DKPs, the two side chains are located on the opposite sides of the general plane of the ring, which greatly reduces the strain applied to the six-member ring. On the basis of spacefilling models of dipeptide esters containing a proline residue, the cis isomer has been shown from a geometrical consideration to adapt a conformation in which the attacking N-terminal amino nitrogen atom is in proximity to the amide carbonyl, favoring ring closure.4 Thus, one would expect the cis isomer to undergo aminolysis more rapidly than its trans counterpart. There are no correlations between the bulkiness of the side chain of the amino acid and the occurrence of a cis peptide bond.29 Investigations are currently underway in our laboratory to determine the ability of these dipeptide analogues to undergo cis-trans isomerization. The conformation of the Phe-Pro-DKP in aqueous solution has been determined,30,31 and it was shown that the DKP ring adopted a boat-shaped conformation with the Phe aromatic ring stacked over it. Ciarkowsi et al.32 argued that the presence of the ring-ring interaction in a cyclic dipeptide composed of an aromatic amino acid residue adopted a more energetically favored conformation, in which the aromatic ring is stacked over the DKP ring, and that the intramolecular dipole-induced dipole should stabilize this form. There is also the possibility of an aromatic-aromatic interaction between the Phe side chain and the pNA leaving group, which could stabilize the tetrahedral intermediate and lead to an enhanced rate of cyclization of Phe-Pro-pNA. To check this hypothesis, the aromatic Phe residue was replaced with β-cyclohexylalanine. As shown in Table 1, the rate decreased by a factor of approximately 1.6, suggesting that an aromaticaromatic interaction may be one of many factors responsible for the intramolecular reaction. Further studies are needed to elucidate the contribution of these different factors.
Conclusions These results indicate that DKP formation is sensitive to the pH of the solution, the buffer species present at high pH, the temperature, and the N-terminal amino acid residue. The rapid rate at which the intramolecular reaction occurs at neutral and basic pH suggests that DKP formation may play an integral role in the degradation of peptides and proteins that contain a proline residue at position 2 under these conditions. The results of these studies will hopefully give scientists involved in the formulation of therapeutically important peptides and proteins insight into the multitude of endogenous (e.g., primary sequence) and exogenous (e.g., pH, buffer species) factors that can influence the rate of diketopiperazine formation in aqueous solution.
References and Notes 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. Pept. Protein Res. 1994, 44, 215-222. 2. Kertscher, U.; Bienert, M.; Krause, E.; Sepetov, N. F.; Mehlis, B. Spontaneous chemical degradation of substance P in the solid phase and in solution. Int. J. Pept. Protein Res. 1993, 41, 207-221. 3. Beyermann, M.; Bienert, M.; Niedrich, H.; Carpino, L. A.; Sadat-Aalace, D. Rapid continuous peptide synthesis via FMOC amino acid chloride coupling and 4-(aminomethyl)piperidine deblocking. J. Org. Chem. 1990, 55, 721-728. 4. Purdie, J. E.; Benoiton, N. L. Piperazinedione formation from esters of dipeptides containing glycine, alanine, and sarcosine: The kinetics in aqueous solution. J. Chem. Soc. Perkin Trans. 2 1973, 13, 1845-1852. 5. Skwierczynski, R. D.; Connors, K. A. Demethylation kinetics of aspartame and L-phenylalanine methyl ester in aqueous solution. Pharm. Res. 1993, 10, 1174-1180. 6. Gu, L.; Strickley, R. G. Diketopiperazine formation, hydrolysis, and epimerization of the new dipeptide angiotensinconverting enzyme inhibitor RS-10085. Pharm. Res. 1987, 4, 392-397. 7. Moss, 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. 1990, 42, 7-12. 8. Miyashita, K.; Murakami, M.; Yamada, M.; Iriuchijima, T.; Mori, M. Histidyl-proline diketopiperazine. J. Biol. Chem. 1993, 268, 20863-20865. 9. Steinberg, S. M.; Bada, J. L. Peptide decomposition in the neutral pH region via the formation of diketopiperazines. J. Org. Chem. 1983, 48, 2295-2298. 10. Steinberg, S. M.; Bada, J. L. Diketopiperazine formation during investigations of amino acid racemization in dipeptides. Science 1981, 213, 544-545. 11. Bodanszky, M.; Martinez, J. In The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhoffer, J., Eds.; Academic Press Inc.: New York, 1983; Vol. 5, pp 120-123, 193-196. 12. Gerig, J. T.; McLeod, R. S. Attempted synthesis of 2-methylalanyl-L-prolyl-L-tryptophan. An unexpected result. J. Org. Chem. 1976, 41, 1653-1655. 13. Gisin, B. F.; Merrifield, R. B. Carboxyl-catalyzed intramolecular aminolysis. A side reaction in solid-phase peptide synthesis. J. Am. Chem. Soc. 1972, 94, 3102-3106. 14. Mazurov, A. A.; Andronati, S. A.; Korotenko, T. I.; Gorbatyuk, V. Y.; Shapiro, Y. E. Formation of pyroglutamylglutamine (or asparagine) diketopiperazine in ‘non- classical’ conditions: A side reaction in peptide synthesis. Int. J. Pept. Protein Res. 1993, 42, 14-19. 15. Meienhoffer, J. Syntheses of actinomycin and analogues. III. A total synthesis of actinomycin D (C1) via peptide cyclization between proline and sarcosine. J. Am. Chem. Soc. 1970, 92, 3771-3777. 16. Fields, G. B.; Noble, R. L. Solid-phase peptide synthesis utilizing 9- fluorenylmethoxycarbonyl amino acid. Int. J. Pept. Protein Res. 1990, 35, 16-214. 17. Sepetov, N. F.; Krymsky, M. A.; Ovchinnikov, M. V.; Bespalova, Z. D.; Isakova, O. L.; Soueek, M.; Lebl, M. Rearrangement, racemization and decomposition of peptides in aqueous solution. Pept. Res. 1991, 4, 308-313. 18. Straub, J. A.; Akiyama, A.; Parmar, P.; Musso, G. F. Chemical pathways of the bradykinin analogue, RMP-7. Pharm. Res. 1995, 12, 305-308. 19. Oyler, A. R.; Naldi, R. E.; Lloyd, J. R.; Graden, D. A.; Shaw, C. J. Characterization of the solution degradation products of Histrelin, a gonadotropin releasing hormone (LH/RH) agonist. J. Pharm. Sci. 1991, 80, 271-275. 20. Radzicka, A.; Wolfenden, R. Rates of uncatalyzed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. J. Am. Chem. Soc. 1996, 118, 61056109. 21. Jenks, W. P. In Catalysis in Chemistry and Enzymology; McGraw-Hill: New York, 1969; pp 163-195. 22. Bruice, T. C.; Bradbury, W. C. The gem effect. The influence of 3-mono- and 3,3-disubstitution on the rates of solvolysis of mono-p-bromophenyl glutarate. J. Am. Chem. Soc. 1965, 87, 4846-4850. 23. Higuchi, T.; Eberson, L.; McRae, J. D. Acid anhydride-free acid equilibria in water in some substituted succinic scid systems and their interaction with aniline. J. Am. Chem. Soc. 1967, 89, 3001-3004. 24. Borchardt, R. T.; Cohen, L. A. Stereopopulation Control. II. Rate enhancement of intramolecular nucleophilic displacement. J. Am. Chem. Soc. 1972, 94, 9166-9174.
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25. Borchardt, R. T.; Cohen, L. A. Stereopopulation Control. III. Facilitation of intramolecular conjugate addition of the carboxyl group. J. Am. Chem. Soc. 1972, 94, 9175-9182. 26. Levitt, M. Effect of proline residues on protein folding. J. Mol. Biol. 1981, 145, 251-263. 27. Stein, R. L. In Advances in Protein Chemistry; Afinsen, C. B., Edsall, J. T., Richards, F. M., Eisenberg, D. S., Eds.; Academic Press, Inc.: New York, 1993; Vol. 44. 28. Ramachandran, G. N.; Mitra, A. K. An explanation for the rare occurrence of cis peptide units in proteins and polypeptides. J. Mol. Biol. 1976, 107, 85-92. 29. Stewart, D. E.; Sarkar, A.; Wampler, J. E. Occurrence and role of cis peptide bonds in protein structures. J. Mol. Biol. 1990, 214, 253-260. 30. Ciarkowski, J.; Gdaniec, M.; Kolodziejczyk, A.; Liberek, B.; Borremans, F. A. M.; Anteunis, M. J. O. Conformation of cyclo-(D-phenylalanyl-trans-4-fluoro-D-prolyl). Int. J. Pept. Protein Res. 1990, 36, 285-291.
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Acknowledgments This work was supported by the United States Public Health Service (GM-088359). We would also like to thank Drs. Richard Schowen and Suman Patel for their advice.
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