1990
J. Am. Chem. Soc. 1998, 120, 1990-1995
Mechanism of 2,5-Dioxopiperazine Formation
Sante Capasso,*,†,‡ Alessandro Vergara,§ and Lelio Mazzarella†,§
Contribution from the Centro di Studio di Biocristallografia, C.N.R., Via Mezzocannone 4,
80134 Napoli, Italy, Facolta` di Scienze Ambientali, Seconda UniVersita` di Napoli, Via Arena 22,
81100 Caserta, Italy, and Dipartimento di Chimica, UniVersita` di Napoli “Federico II”,
Via Mezzocannone 4, 80134 Napoli, Italy
ReceiVed June 23, 1997
Abstract: The cyclization of H-Ala-Pro-NH2 to the 2,5-dioxopiperazine (DKP) has been studied as a model
for the spontaneous cleavage of the peptide bond with concomitant formation of 2,5-dioxopiperazine that can
occur at the N-terminus of a polypeptide chain. The reaction involves pre-equilibrium attack of the N-terminal
amino group on the carbonyl carbon of the second residue giving a zwitterionic cyclic intermediate, T(, which
is in acid-base equilibrium with various forms characterized by different grades of protonation, T0, T+ and
T-. The Brønsted plot for the base-catalysis and the pH-rate profile give pKa ∼ 7 and ∼ 13 for the equilibria
T- + H+ h T( and T- + H+ h T0, respectively. The reaction is subject to general base and general acid
catalysis, acting on different steps. Departure of the amino group from T0 and T- by two parallel routes gives
the product. The bifunctional acid catalyst HCO3- strongly increases the reaction rate and at high concentrations
causes a change of the rate-limiting step. At high pH, the overall reaction rate is limited by the trans f cis
isomerization of the Ala-Pro peptide bond.
Introduction
the design of conformationally restricted cyclic peptides,10 to
be employed in the study of structure-biological activity
relationship.11 Moreover, it has been suggested12 that the
posttranslational N-acetylation of proteins in higher organism
is an evolutionary protection against spontaneous degradation
Via formation of DKPs.
The nucleophilic addition of amino groups to carbonyl carbon
atoms has been the subject of extensive investigation, since it
is involved in many reactions such as the formation of peptides,
imines and so forth. It has been observed that the attack of the
nitrogen atom on the carbonyl carbon usually occurs along a
preferred direction,13 producing a zwitterionic unstable inter-
mediate which, by acid-base reactions, gives intermediates with
various grades of protonation.14-16 As concerns the formation
of DKPs from the N-terminal residues of a polypeptide chain,
the current literature mostly deals with the influence of acids
and bases on the reaction occurring during peptide synthesis in
organic solvents.17 Recently, an efficient novel route for the
synthesis of DKPs on a solid support has been described.18 The
2,5-Dioxopiperazine (diketopiperazine, DKP) formation from
the N-terminal residues of a peptide chain commonly occurs as
a disturbing reaction in the synthesis1 and long-term storage of
peptides.2,3 In addition, many DKPs, encompassing a wide
range of biological activities, have been found in a variety of
tissues and body fluids.4 The nitrogen atom of the N-terminal
deprotonated amino group can attack the carbonyl carbon atom
of the second residue causing the breakdown of the chain and
formation of DKPs.5 This reaction is known to occur easily in
dipeptide esters6 because of the presence of good leaving groups
(alcohol molecules) and in dipeptides with alternate chirality1
because of the higher stability of the resulting DKPs. For D/L
DKPs the amino acid side chains will lie on opposite sides of
the plane of the cycle. As in the dioxopiperazine rings both
peptide bonds must be in cis conformation, the cyclization
reaction is promoted by N-alkylated cyclic residues in second
position because of their high propensity to form a cis peptide
bond with the preceding residue.7,8 The recent interest in the
stability of N-alkylated residues3,9 is mainly due to their use in
(7) (a) Stewart, D. E.; Sarkar, A.; Wampler, J. E. J. Mol. Biol. 1990,
214, 253-260. (b) Ramachandean, G. N.; Mitra, A. K. J. Mol. Biol. 1976,
107, 85-92.
† Centro di Studio di Biocristallografia, C.N.R.
‡ Seconda Universita` di Napoli.
(8) Grathwohl, C.; Wu¨thrich, K. Biopolymers 1976, 15, 2043-2057.
(9) Capasso, S.; Sica, F.; Mazzarella, L.; Balboni, G.; Guerrini, R.;
Sanvadori, S. Int. J. Peptide Prot. Res. 1995, 45, 567-573.
(10) Toniolo, C. Int. J. Peptide Prot. Res. 1990, 35, 287-300.
(11) Lomize, A. L.; Pogozheva, I. D.; Mosberg, H. I. Biopolymers 1996,
38, 221-234.
§ Universita` di Napoli “Federico II”.
(1) Bodanszky, M. Principles of Peptide Synthesis; Springer-Verlag:
Berlin, 1984; pp 158-201.
(2) (a) Battersby, J. E.; Hancock, W. S.; Canova-Davis, E.; Oeswein, J.;
O’Connor, B. Int. J. Peptide Prot. Res. 1994, 44, 215-222. (b) Kertescher,
U.; Bienert, M.; Krause, E.; Sepetov, N.; Mehlis, B. Int. J. Peptide Prot.
Res. 1993, 41, 207-211. (c) Steinberg, S.; Bada, J. L. Science 1981, 213,
544-545.
(3) Marsden, B. J.; Nguyen, T. M.-D.; Schiller, P. W. Int. J. Peptide
Prot. Res. 1993, 41, 313-316.
(4) Mortier, J.; Chene, A.; Gelin, J.; Moyroud, J. Tetrahedron 1996, 52,
8525-8534.
(5) Møss, J.; Bundgaard, H. J. Pharm. Pharmacol. 1990, 42, 7-12.
(6) (a) Field, G. B.; Noble, R. L. Int. J. Peptide Prot. Res. 1990, 35,
161-214. (b) Beyermann, M.; Bienert, M.; Niedrich, H.; Carpino, L. A.;
Sadat-Aalee, D. J. Org Chem. 1990, 55, 721-728.
(12) Radzicka, A.; Wolfenden, R. J. Am. Chem. Soc. 1996, 118, 6105-
6109.
(13) (a) Dunitz, J. D. X-ray analysis and the structure of organic
molecules; Cornell University Press: Ithaca and London, 1979; pp 301-
384. (b) Kirby, A. J. AdV. Phys. Org Chem. 1994, 29, 87-183.
(14) (a) Jencks, W. P. Acc. Chem. Res. 1976, 9, 425-432. (b) Camilleri,
P.; Ellul, R.; Kirby, A. J.; Mujahid, T. G. J. Chem. Soc., Perkin Trans. 2
1979, 1617-1620.
(15) Kirby, A. J.; Mujahid, T. G.; Camilleri, P. J. Chem. Soc., Perkin
Trans. 2 1979, 1610-1616.
(16) Fox, J. P.; Jencks, W. P. J. Am. Chem. Soc. 1974, 96, 1436-1449.
S0002-7863(97)02051-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/20/1998