Alkaline and acidic hydrolysis of the β-lactam ring

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1 ELECTRONIC JOURNAL OF THEORETICAL CHEMISTRY, VOL. 2, (1997) Alkaline and acidic hydrolysis of the β-lactam ring JUAN FRAU 1, MIGUEL COLL 1, JOSEFA DONOSO 1, FRANCISCO MUÑOZ 1,BARTOLOMÉ VILANOVA 1 AND FRANCISCO GARCÍA-BLANCO 2 1 Departament de Química, Universitat de les Illes Balears, Palma de Mallorca, Spain 2 Departamento de Química-Física, Facultad de Farmacia, Instituto Pluridisciplinar UCM, Universidad Complutense, Madrid, Spain SUMMARY A complete study of the alkaline and acidic hydrolysis of the β-lactam ring of azetidin-2-one was carried out using ab initio molecular orbital calculations at the RHF/6 31+G* and RHF/6 31G** levels, respectively. Alkaline hydrolysis has been studied through a B AC2 mechanism characterized by a nucleophilic attack on the β-lactam carbonyl group, formation of the tetrahedral intermediate and cleavage of the C N bond until the formation of the final product of the reaction, this being the limiting step of the reaction. On the other hand, the acidic hydrolysis has been studied by means of a A-1 type unimolecular mechanism, characterized by a nitrogen-protonation followed by an opening of the ring and further addition of water to the carbonyl group. The system involving the azetidin-2-one ring, the H 3O + ion and a water molecule has been considered. Three transition states have been identified; the barriers corresponding to the addition of H + to the nitrogen and the addition of water to the carbonyl group are practically negligible (first and third reaction steps, respectively). Received 9 October 1996; Accepted 1 November 1996 Electron. J. Theor. Chem., Vol.2, (1997) No. of Figures: 5 No. of Tables: 2 No. of References: 40 KEY WORDS β-lactam; alkaline hydrolysis; acidic hydrolysis; ab initio INTRODUCTION Since their discovery in the 1920s, β-lactam antibiotics have played a prominent role in the fight against bacterial diseases. Carboxyl and transpeptidase enzymes catalyze and control, in its final stages, the biosynthesis of bacterial cell-wall peptidoglycan. β-lactam antibiotics act as inhibitors of the transpeptidase enzyme, because they mimic the active conformation of the terminal D-alanyl-D-alanine of the peptidoglycan [1]. Many authors have suggested that the mechanism of enzymatic hydrolysis is similar to that of alkaline hydrolysis [2]. The knowledge of the stability of the former molecules in acidic medium is of great interest. As an example, acidic hydrolysis takes place in the human body when the antibiotic (amoxycillin, penicillin V, cloxacillin, ampicillin) is orally ingested [3]. For these reasons is very importantto havea good knowledgeof the mechanisms of the action ofβ-lactam antibiotics, which in turn has facilitated the determination of new structures with a similar chemical reactivity in addition to resistance to bacterial defense mechanisms. Correspondence to: J. Frau; dqujfm0@ps.uib.es Contract grant sponsor: Spanish Government; Contract grant number: DGICYT, Project PB ; Contract grant number: DGICYT, Project PB CCC /97/ $ by John Wiley & Sons, Ltd.

2 ALKALINE AND ACIDIC HYDROLYSIS OF THE β-lactam RING 57 During the last ten years a great amount of theoretical studies on β-lactam antibiotics have been performed. Ab initio calculations were recently used to determine structural parameters for various β-lactam compounds [4 8]. On the other hand, the chemical reactivity (basically alkaline hydrolysis) of β-lactam antibiotics has been studied by using semi-empirical methods preferentially [9 13], with the exception of the early investigations of Petrongolo and coworkers [14,15] and more recent studies with a high-quality basis set [16]. A large number of kinetic studies regarding penicillin and cephalosporin degradation in acidic medium are found in the literature [17 19]; however, at present, there are still important doubts concerning the reaction pathway [20 22]. Theoretical studies on neutral and acidic hydrolysis of amidic and similar systems [23 29] have been carried out. Nevertheless, the reactivity of these molecules is essentially different to those of β- lactam antibiotics since oxygen-protonationis thermodynamically favored against nitrogen-protonation [20 25], circumstances which, on the other hand, do not take place in β-lactam compounds [19 22]. There are no theoretical studies on the acidic hydrolysis reaction of the structures selected as models of β-lactam compounds. This paper reports a comprehensive theoretical study of the alkaline and acidic hydrolysis of the azetidin-2-one ring. Alkaline hydrolysis has been studied through a B AC2 mechanism characterized by a nucleophilic attack on the carbonyl group followed by cleavage of the C N bond. Acidic hydrolysis consists of a A-1 type mechanism [20,21]. The first reaction step is that of nitrogen-protonationfollowed by the opening of the ring and the addition of water to the carbonyl group. The whole system has been stabilized by means of an additional water molecule in order to introduce a certain solvent effect. All calculations were based on 6 31+G and 6 31G basis sets. METHODOLOGY Azetidin-2-one was the model compound used to study the β-lactam ring. Previously elucidated semiempirical structures [10,30] were used as the starting points for the ab initio calculations in both reactions, which were done on the 6 31+G and 6 31G basis sets. In the alkaline hydrolysis, a polarized and a diffuse basis for heavy atoms were included in order to ensure reliable results for small charge-localized anions [31]. In those cases, where the geometry derived from the semi-empirical calculations was inappropriate (particularly as regards saddle points), reaction coordinates were used with full optimization of every parameter until the desired steady-state point was reached. Calculations were performed on an ALPHA DEC AXP computer running the program GAMESS [32] as modified by Schmidt and coworkers [33]. All structures were characterized by vibrational analysis. RESULTS Alkaline hydrolysis As noted earlier, the basic hydrolysis of β-lactam compounds takes place via a B AC2 mechanism that involves a nucleophilic attack on the amide carbon of the β-lactam ring. In this mechanism (Scheme 1a) the nucleophilic attack gives rise to a tetrahedral intermediate that is cleft at the C 2 N 3 bond to two potential products in which the hydrogen is bound to oxygen (d) or nitrogen (h). Figure 1 depicts the reaction profile derived for the gas-phase reaction. Table 1 shows the main geometric parameters and energy for the stationary points of the reaction and Figure 2 shows some of these structures.

3 58 J. FRAU ET AL. Scheme 1. (a) Alkaline hydrolysis of the azetidin-2-one ring. B AC2 mechanism. (b) Acidic hydrolysis of azetidin- 2-one ring. A-1 mechanism Figure 1. RHF/6 31+G* energy profile along the B AC2 reaction pathway for the reaction between the hydroxyl ion and the β-lactam ring. Energies relative to the final product (h) in kcal mol 1

4 ALKALINE AND ACIDIC HYDROLYSIS OF THE β-lactam RING 59 Figure 2. Structures corresponding to the reactants, intermediates, transition states and final product of the reaction during alkaline hydrolysis A nucleophilic attack of an hydroxyl ion on a carbonyl group in the gas phase is known to take place with no potential barrier between the reactant (a) and the tetrahedral intermediate formed (b) provided the attack pathway is roughly normal to the plane of the β-lactam ring. However, the situation is different if variations in the attack pathway are allowed for (see the dotted line in Figure 1). The low stability of the hydroxyl ion in the gas phase results in the reaction pathway deviating from verticality in order to stabilize the charge by interacting with the different protons of the β-lactam ring, even though it always remains at the same distance from the carbonyl group. The intermediate structures formed in the process (a1 and a2) are difficult to characterize as they rapidly evolve to form a water molecule by elimination and transfer the negative charge to the β-lactam ring. Structures of this type were previously observed for other systems [31,34]. The tetrahedral intermediate (b) can undergo ring opening to reach the transition state c, with a C 2 N 3 distance of Å. An important feature of transition state c is that the proton of the hydroxyl

5 60 J. FRAU ET AL. Table 1. RHF/6 31+G* energy and selected geometric parameters for the stationary points. All energy quantities are relative to the final structure (h) in kcal mol 1 ; E SCF is a.u. ZPEs are in hartrees and bond lengths in Å a b c d e f O 1 C C 2 N N 3 C C 4 C C 2 C C 2 O O 1C 2N O 1C 2C C 2N 3C N 3C 4C O 11C 2N O 1C 2 N 3C C 2N 3 C 4C H 12O 11 C 2O Energy (SCF) a b ZPE c d a RHF/6 31+G* energy. b These energies are the sums of the total electronic energies of the β-lactam ring and the hydroxyl ion. c ZPE for the β-lactam ring. d ZPE for the hydroxyl ion. ion (H 12 ) is Å from N 3, which allows it to be readily transferred. The vibrational analysis of this structure revealed a single negative constant involving the atoms C 2,N 3 and H 12. This confirms the hypothesis that point c may be the transition state for the opening of the β-lactam ring and also for the transfer of a proton to the β-lactam nitrogen. As a result, this structure can evolve via the two above-mentioned pathways to the end-point h (see the broken line in Figure 1). On the other hand, structure c can also evolve via a single pathway involving the cleavage of the C 2 N 3 bond to point d,wheretheβ-lactam ring is fully open and H 12 has not yet been transferred. Such a proton is Å from the β-lactam ring and in an inappropriate spatial orientation for direct transfer. This problem was previously detected when the present reaction was studied using semi-empirical methods [9]. For proton H 12 to be properly orientated, the acid group (or only the hydroxyl group) must rotate to a structure where the proton can be more readily transferred. Semi-empirical methods have shown this alternative pathway to involve a lower energy. The last step would be the proton transfer (via a transition state, TS) to the final product of the reaction, the most stable in the whole reaction pathway. Following the same methodology employed for the semi-empirical calculations, the OH group was rotated until the TS, called structure e (with a single, clear-cut negative force constant), was reached. However, after this structure the proton was immediately transferred to the nitrogen atom and the reaction end-product (h) was directly formed and no more structures were obtained. This behavior was previously observed in studying the alkaline hydrolysis of penicillin G using AM1 methods [35]. As can clearly be seen from Figure 1, the rate-determining step of the process in the gas phase is the cleavage of the C N bond (the step from b to c), which contradicts the experimental results obtained in solution, where the formation of the tetrahedral intermediate is the limiting step. The potential barrier

6 ALKALINE AND ACIDIC HYDROLYSIS OF THE β-lactam RING 61 obtained was kcal mol 1 ( hartrees), which, for the above-mentioned reasons, departs from the experimental value. Acidic hydrolysis Scheme 1b shows the different reaction intermediates involved in the acidic hydrolysis of β-lactam antibiotics via a A-1 unimolecular mechanism. Figure 3 gives the profile corresponding to the reaction obtained for the studied hydrolysis in the gas phase and Figure 4 shows the most relevant structures involved in the reaction pathway. Table 2 lists the values of the most significant geometric parameters and total energies corresponding to the different structures. Initially the β-lactam ring of the azetidin-2-one (a) is practically flat and the C 2 N 3 distance is Å. The N 3 H 10 bond is located on the same plane in relation to the ring (the H 10 N 3 C 2 C 5 dihedral angle is 180 ).ThenegativechargeofN 3 is only slightly higher than that of O 1 ( against , respectively). In the study of several amidic systems it can be observed that the oxygen-protonation is thermodynamically favored [19]. The pk a values for the oxygen-protonated and nitrogen-protonated amides varies between 0 and 3 [36,37] and 7and 8[38,39], respectively. However, β-lactam compounds are less basic than the normal amides with regard to oxygen-protonation, the reaction being initiated by nitrogen-protonation (pk a should be lower than 5). This behavior takes place in both bicyclic and monocyclic β-lactams [22]. Due to experimental evidence, although the negative charges corresponding to N 3 and O 1 are practically identical, the studies have been focused on nitrogen-protonation. In the structure (b) the system is stabilized due to the existence of hydrogen bonding between azetidin-2-one, the water molecule and the H 3 O + ion (the N 3 H 11,O 15 H 14 and O 1 H 16 distances are 1.710, and Å, respectively). In this structure of minimum energy a slight increase in the bond length of C 2 N 3 can be observed (from to Å), together with a decrease in the O 1 C 2 N 3 angle (from to ). This tendency occurs throughout the reaction, except when reaching the final product. The ring is then no longer flat, and the value corresponding to the H 10 N 3 C 2 C 5 dihedral varies considerably (from 180 to ), which implies the loss of the trigonal hybridization regarding N 3 (however, a complete tetrahedral structure is not achieved). The approach of H 11 + to N 3 allows a transition state (c) characterized by a N 3 H 11 distance of Å to be determined, and its main characteristic is the increase in the tetrahedral character of the nitrogen (H 10 N 3 C 2 C 5 dihedral presents a value of ) although the ring remains practically flat (C 2 N 3 C 4 C 5 dihedral angle is 2.5 ). The hydrogen bonds which are responsible for the stabilization of the system remain; however, the O 1 H 10 distance has considerably increased (the water molecule has moved away from the ring) from to Å. The energy barrier regarding this process is only 0.82 kcal mol 1 (practically negligible), probably due to the solvent absence. This behavior is likewise similar to that described in the studies on alkaline hydrolysis in the absence of solvent [10,11]. The tetrahedral intermediate (d) is characterized by an initial relaxation of the C 2 N 3 bond and by the flatness of the ring, with a dihedral angle among the C 2 N 3 C 4 C 5 atoms of 0.9. The structure is stabilized by the presence of two hydrogen bonds between the H 11 O 12 (1.583 Å) and O 15 H 14 (1.718 Å) atoms. This intermediate evolves with the opening of the ring and a loss of its planar character, and a TS is detected at a C 2 N 3 distance of Å(e). The energy barrier corresponding to the former process is kcal mol 1. The value for this barrier is probably similar to that obtained when studying this reaction in the aqueous phase, since no addition is included in this reaction step (breaking of the solvation barriers would be implied in the former reaction).

7 62 J. FRAU ET AL. Figure 3. Reaction profile of the acidic hydrolysis of the azetidin-2-one ring. Energies relative to the final product (h) in kcal mol 1 Figure 4. Structures corresponding to the reactants, intermediates, transition states and final product of the reaction during acidic hydrolysis

8 ALKALINE AND ACIDIC HYDROLYSIS OF THE β-lactam RING 63 Table 2. RHF/6 31G** energy and selected geometric parameters for the stationary points. All energy quantities are relative to the final structure (h) in kcal mol 1 ;E SCF is a.u. ZPE energies are in hartrees and bond lengths in Å a b c d e f g h O 1 C C 2 N N 3 C C 4 C C 2 C N 3 H C 2 O O 12 H H 14 O O 1 H H 11 O O 1C 2N O 1C 2C C 2N 3C N 3C 4C O 15C 2N H 10N 3 C 2C H 11N 3 C 2C O 1C 2 N 3C C 2N 3 C 4C O 12H 11 N 3C O 15C 2 C 5C Energy a b ZPE c d e a RHF/6 31G** energy. b These energies are the sums of the total electronic energies of azetidin-2-one, H 2OandH 3O +. c ZPE for the β-lactam ring. d ZPE for the water molecule. e ZPE for the H 3O + ion. The reaction evolves until structure f, where the ring is completely open (the C 2 N 3 distance is Å), is formed. The positive charge is located on C 2, being therefore the best candidate to be involved in nucleophilic attack by a water molecule (the C 2 O 15 distance is Å). A new hydrogen bond appears between H 7 (linked to C 5 )ando 15, with a length of Å. From the intermediate f the reaction evolves with the addition of water to the carbonyl group. The approach of the water molecule is via a transition state (g) characterized by a C 2 N 3 distance of Å, ac 2 O 15 distance of Å and a greater torsion regarding the main chain (the C 2 N 3 C 4 C 5 dihedral angle is 27.1 ). The final product obtained (h) presents a protonated amine group which is stabilized by the presence of a water molecule (O 12 H 10 distance is Å). The molecule is subjected to the presence of an intramolecular hydrogen bond between the H 17 O 15 atoms (1.953 Å). The energy barrier regarding this process is 1.57 kcal mol 1, far smaller than the former, which reflects how easily the latter process takes place, which, on the other hand, is not the limiting step of the reaction. The ease of adding water to the carbonyl group (in the gas phase) has also been described in the acidic hydrolysis reaction with nitrogen-protonation of formamide [25]. Nevertheless, several authors have proposed the addition of water to the carbonyl group as a limiting step of the reaction [19], whereas other authors propose nitrogen-protonation instead [18]. The presence of the solvent would force the water molecule to break the solvation barrier, and therefore the height of the barriers corresponding to the first and third reaction steps would be considerably increased.

9 64 J. FRAU ET AL. CONCLUSIONS The reaction pathway for the base-catalyzed hydrolysis of azetidin-2-one has been investigated using ab initio calculations at the RHF/6 31+G level of theory. This study shows that the nucleophilic attack is barrierless; however some structures can be detected in this step due to the low stability of the hydroxyl ion in the gas phase. The opening of the tetrahedral intermediate is the limiting step of this reaction, which can take place simultaneously with the transfer of a proton to the β-lactam nitrogen until the final product is formed. On the other hand, the acidic hydrolysis of the azetidin-2-one has been investigated using ab initio calculations at the RHF/6 31G level. In this study, practically negligible barriers have been obtained for both nitrogen-protonation and water addition. The only relevant barrier is that related to the opening of the ring, being therefore the limiting step of the reaction. It is clear that the solvent effects will be important in these kind of reactions, specially in the approach of the hydroxyl ion at the carbonyl group in the alkaline reaction. Further work is in progress to assess this aspect of the reaction [16,40]. Acknowledgments The authors thank Dr M. Schmidt for kindly supplying the current version of the program GAMESS. We also thank the Centre de Càlcul de la Universitat de les Illes Balears for access to their computer facilities. The support of the Spanish Government is gratefully acknowledged (DGICYT, Project PB and PB ). REFERENCES 1. D. J. Tipper and J. L. Strominger, Proc. Natl. Acad. Sci. USA, 54, 75 (1965). 2. J. Fisher, Antimicrobial Drug Resistance: β-lactam Resistant to Hydrolysis by the β-lactamases, Academic Press, New York, 1984, Ch. 2, p J. Floŕez, J. A. Armijo and A. Mediavilla, Farmacologia Humana, Vol. 2, Edunsa, 1988, pp K. M. Marstokk, H. Mollendal, S. Samdal and E. Uggerud, Acta Chemica Scand., 43, 351 (1989). 5. E. Sedano, J. M. Ugalde, F. P. Cossio and C. Palomo, J. Mol. Struct. (Theochem), 166, 481 (1988). 6. S. Vishveshwara and V. S. R. Rao, J. Mol. Struct. (Theochem), 92, 19 (1993). 7. M. Alcolea Palafox, J. L. Nuñez and M. Gil, J. Phys. Chem., 99, 1124 (1995). 8. B. Fernández, L. Carballeira and M. A. Rios, Biopolymers, 32, 97 (1992). 9. Y. G. Smeyers, A. Hernández-Laguna and R. González-Jonte, J. Mol. Struct. (Theochem), 287, 261 (1993). 10. J. Frau, J. Donoso, F. Muñoz and F. García Blanco, J. Comput. Chem., 13, 681 (1992). 11. J. Frau, J. Donoso, B Vilanova, F. Muñoz and F. García Blanco, Theor. Chim. Acta, 86, 229 (1993). 12. J. Frau, J. Donoso, F. Muñoz and F. García Blanco, J. Comput. Chem., 14, 1545 (1993). 13. J. Frau, J. Donoso, F. Muñoz and F. García Blanco, Helv. Chim. Acta, 77, 1557 (1994). 14. C. Petrongolo, G. Ranghino and R. Scordamaglia, Chem. Phys., 45, 279 (1980). 15. C. Petrongolo, E. Pescatori, G. Ranghino and R. Scordamaglia, Chem. Phys., 45, 291 (1980). 16. J. Frau, J. Donoso, F. Muñoz and F. García Blanco, Helv. Chim. Acta, 79, 353 (1996). 17. M. A. Schwartz, J. Pharm. Sci., 54, 472 (1965). 18. J. L. Londridge and D. Timms, J. Chem. Soc. B, 852 (1971). 19. M. L. Sinott, Adv. Phys. Org. Chem., 24, 113 (1988). 20. M. I. Page, Acc. Chem. Res., 17, 144 (1984). 21. M. I. Page, Adv. Phys. Org. Chem., 23, 165 (1987). 22. S. Wolfe, C. K. Kim and K. Yang, Can. J. Chem., 72, 1033 (1994). 23. I. Lee, C. K. Kim and B. Lee, J. Phys. Org. Chem., 3, 397 (1990). 24. O. N. Ventura, E. L. Coitiño, A. Lledós and J. Bertran, J. Comp. Chem., 13, 1037 (1992). 25. J. P. Krug, P. L. A. Popelier and R. F. W. Bader, J. Phys. Chem., 96, 7604 (1992). 26. S. Antonczak, M. F. Ruiz-Lopez and J. L. Rivail, J. Am. Chem. Soc., 116, 3912 (1994).

10 ALKALINE AND ACIDIC HYDROLYSIS OF THE β-lactam RING T. Katagi, J. Comp. Chem., 11, 1094 (1990). 28. I. Lee, C. K. Kim and B. S. Lee, Bull. Korea Chem. Soc., 11, 194 (1990). 29. I. Lee, C. K. Kim and B. C. Lee, J. Phys. Org. Chem., 2, 281 (1989). 30. M. Coll, J. Donoso, B. Vilanova, J. Frau, A. Llinas and F. Munõz, Electronic Conference on Heterocyclic Chemistry (ECHET96), 1996, poster J. D. Madura and W. L. Jorgensen, J. Am. Chem. Soc., 108, 2517 (1986). 32. GAMESS US. M. Despuis, D. Spangler and J. J. Wendoloski, National Resources for Computations in Chemistry, Software Catalog, Vol. 1 Program QG01, Lawrence Berkeley Laboratory, USDOE, California, USA, M. W. Schmidt, K. K. Baldridge, J. A. Boatz, J. H. Jensen, S. Koseki, M. S. Gordon, K. A. Nguyen, T. L. Windus and T. S. Elbert, QCPE Bulletin, 10, 52 (1990). 34. J. Pranata, J. Phys. Chem., 98, 1180 (1994). 35. J. Frau, J. Donoso, F. Muñoz and F. García Blanco, J. Mol. Struct. (Theochem), 390, 255 (1997). 36. M. Liler, J. Chem. Soc. R., 385 (1969). 37. K. Yates and J. B. Stevens, Com. J. Chem., 43, 529 (1965). 38. A. Williams, J. Am. Chem. Soc., 97, 6278 (1975). 39. A. R. Fersht, J. Am. Chem. Soc., 93, 3504 (1971). 40. J. Frau, J. Donoso, F. Muñoz and F. García Blanco, J. Mol. Struct. (Theochem), in press.