High-performance liquid chromatographic determination of a new beta-lactamase inhibitor and its metabolite in combination therapy with piperacillin in biological materials

[2S-(2 alpha,3 beta,5 alpha)]-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-yl- methyl)-4-thia-1-azabicyclo [3.2.0]-heptane-2-carboxylic acid 4,4-dioxide (YTR-830H) is a new beta-lactamase inhibitor and the combination therapy of this compound with piperacillin is now under study. For the determination of the beta-lactamase inhibitor and piperacillin in biological materials, plasma and visceral tissue homogenates were deproteinized, whereas diluted urine and filtered faeces homogenates were treated with a Sep-Pak C18 cartridge. In order to assay the inactive metabolite of beta-lactamase inhibitor, each sample was treated with a Sep-Pak C18 cartridge. Aliquots of each preparation were chromatographed using ion-pair and reversed-phase chromatographic techniques on a high-performance liquid chromatograph equipped with a UV detector, set at 220 nm. The detection limits of beta-lactamase inhibitor and piperacillin were 0.2 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 0.2-0.5 microgram/g in visceral tissue and faeces. Those of the metabolite were 1.0 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 1.0 microgram/g in visceral tissue and faeces. A precise and sensitive assay for the determination of the beta-lactamase inhibitor, its metabolite and piperacillin is described, and their stabilities in several media are reported.     

A theoretical study on the substrate deacylation mechanism of class C beta-lactamase

The whole reaction of the deacylation of class C beta-lactamase was investigated by performing quantum chemical calculations under physiological conditions. In this study, the X-ray crystallographic structure of the inhibitor moxalactam-bound class C beta-lactamase (Patera et al. J. Am. Chem. Soc. 2000, 122, 10504-10512.) was utilized and moxalactam was changed into the substrate cefaclor. A model for quantum chemical calculations was constructed using an energy-minimized structure of the substrate-bound enzyme obtained by molecular mechanics calculation, in which the enzyme was soaked in thousands of TIP3P water molecules. It was found that the deacylation reaction consisted of two elementary processes. The first process was formation of a tetrahedral intermediate, which was initiated by the activation of catalytic water by Tyr150, and the second process was detachment of the hydroxylated substrate from the enzyme, which associated with proton transfer from the side chain of Lys67 to Ser64O(gamma). The first process is a rate-determining process, and the activation energy was estimated to be 30.47 kcal/mol from density functional theory calculations considering electron correlation (B3LYP/6-31G**). The side chain of Tyr150 was initially in a deprotonated state and was stably present in the active site of the acyl-enzyme complex, being held by Lys67 and Lys315 cooperatively.     

Positive effect of natural and negatively charged cyclodextrins on the stabilization of penicillins towards beta-lactamase degradation due to inclusion and external guest-host association. An NMR and MS study

The complexation of penicillin (1a-c) and cephalosporin (2a,b) antibiotics with cyclodextrins (CDs), both natural [beta-CD (3b) and gamma-CD (3c)] and carboxylated [heptakis(6-oxycarbonylethylthio-6-deoxy)-beta-CD sodium salt (4b) and octakis(6-oxycarbonylethylthio-6-deoxy)-gamma-CD (4c) sodium salt], has been studied at neutral pH. Penicillins [ampicillin (1a), amoxicillin (1b) and dicloxacillin (1c) form inclusion complexes with the above CDs, as was shown by extensive NMR spectroscopic studies, whereas cephalosporins (cephalexin, cefadroxil) do not. Inclusion of the penicillins into either 3b or 4b was not accompanied by significant chemical shift changes in the 1H NMR spectra. On the contrary, with the wider 3c and its derivative 4c inclusion was evidenced by both chemical shift displacements of the cavity protons and intermolecular interactions, indicating the formation of primarily 1:1 guest-host inclusion complexes. The binding constants for 1a/3c, 1a/4c and 1c/3c were calculated as 19 +/- 4, 17 +/- 0.9 and 622 +/- 200 M(-1), respectively. With 4c, a 1:2 stoichiometry was also found. In addition, simultaneous formation of aggregates by external association takes place in solution, as shown by the ESI-mass spectrometric data. Studies on the hydrolysis of ampicillin under pseudo-first order conditions using an excess of 3c, 4c and of linear maltoheptaose at pH 7 showed that the drug hydrolysed at a similar rate in all cases. In the presence, however, of beta-lactamase enzyme and the carboxylated host 4c, ampicillin degraded twice as slowly (0.008 h(-1)) as in the presence of beta-lactamase alone (0.017 h(-1)). This was explained by the effective protection provided by both inclusion and external association of the host. The interaction, therefore, of penicillins with carboxylated CDs may present a means to lessen the chemical instability of these drugs in the presence of beta-lactamase enzymes.     

Development of a method for the detection of beta-lactamases in milk samples

With the rapid growth of the dairy industry and the establishment of strict antimicrobial residue limits in the People's Republic of China's (PRC) milk supply, a beta-lactamase product known as "antimicrobial destroyer" was introduced into dairy production without regulatory review. We developed a method for detecting this product in milk samples based on a modified cylinder plate method. The presence of beta-lactamase is defined as a difference between the inhibitory zones of the test samples (supplemented with 25 microg/mL sulbactam plus 0.5 microg/mL penicillin G) and control samples (supplemented only with 0.5 microg/mL penicillin G) > or = 3 mm. Using this method, 77 individually packaged milk samples were randomly collected from 5 retail stores in 3 cities over a 4-month period (May to August 2006). Of the 77 samples, 49 were found to be beta-lactamase-positive. In 2 undiluted milk samples showing extremely high beta-lactamase activity, 25 microg/mL sulbactam could not inhibit penicillin G activity. Because there is a lack of safety data on beta-lactamases in milk products, these data indicated a potentially serious safety concern for the dairy industry in the PRC.     

Inhibition of class A beta-lactamases by carbapenems: crystallographic observation of two conformations of meropenem in SHV-1

Carbapenem antibiotics are often the "last resort" in the treatment of infections caused by bacteria resistant to penicillins and cephalosporins. To understand why meropenem is resistant to hydrolysis by the SHV-1 class A beta-lactamase, the atomic structure of meropenem inactivated SHV-1 was solved to 1.05 A resolution. Two conformations of the Ser70 acylated intermediate are observed in the SHV-1-meropenem complex; the meropenem carbonyl oxygen atom of the acyl-enzyme is in the oxyanion hole in one conformation, while in the other conformation it is not. Although the structures of the SHV-1 apoenzyme and the SHV-1-meropenem complex are very similar (0.29 A rmsd for Calpha atoms), the orientation of the conserved Ser130 is different. Notably, the Ser130-OH group of the SHV-1-meropenem complex is directed toward Lys234Nz, while the Ser130-OH of the apo enzyme is oriented toward the Lys73 amino group. This altered position may affect proton transfer via Ser130 and the rate of hydrolysis. A most intriguing finding is the crystallographic detection of protonation of the Glu166 known to be involved in the deacylation mechanism. The critical deacylation water molecule has an additional hydrogen-bonding interaction with the OH group of meropenem's 6alpha-1 R-hydroxyethyl substituent. This interaction may weaken the nucleophilicity and/or change the direction of the lone pair of electrons of the water molecule and result in poor turnover of meropenem by the SHV-1 beta-lactamase. Using timed mass spectrometry, we further show that meropenem is covalently attached to SHV-1 beta-lactamase for at least 60 min. These observations explain key properties of meropenem's ability to resist hydrolysis by SHV-1 and lead to important insights regarding future carbapenem and beta-lactamase inhibitor design.     

Chromophoric spin-labeled beta-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C beta-lactamases

The chromophoric spin-label substrate 6-N-[3-(2,2,5,5-tetramethyl-1-oxypyrrolin-3-yl)-propen-2-oyl]penicillanic acid (SLPPEN) was synthesized by acylation of 6-aminopenicillanic acid with the acid chloride of 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)-2-propenoic acid and characterized by physical methods. By application of angle-selected electron nuclear double resonance (ENDOR), we have determined the molecular structure of SLPPEN in solution. SLPPEN exhibited UV absorption properties that allowed accurate monitoring of the kinetics of its enzyme-catalyzed hydrolysis. The maximum value of the (substrate-product) difference extinction coefficient was 2824 M(-1) cm(-1) at 275 nm compared to 670 M(-1) cm(-1) at 232 nm for SLPEN [J. Am. Chem. Soc. 117 (1995) 6739]. For SLPPEN, the steady-state kinetic parameters kcat and kcat/KM, determined under initial velocity conditions, were 637 +/- 36 s(-1) and 13.8 +/- 1.4 x 10(6) M(-1) s(-1), respectively, for hydrolysis catalyzed by TEM-1 beta-lactamase of E. coli, and 0.5 +/- 0.04 s(-1) and 3.9 +/- 0.4 x 10(4) M(-1) s(-1) for hydrolysis catalyzed by the beta-lactamase of Enterobacter cloacae P99. We have also observed "burst kinetics" for the hydrolysis of SLPPEN with P99 beta-lactamase, indicative of formation of an acylenzyme reaction intermediate. In DMSO:H2O (30:70, v:v) cryosolvent mixtures buffered to pH* 7.0, the half-life of the acylenzyme intermediate formed with the P99 enzyme at -5 degrees C was > or = 3 min, suitable for optical characterization. The observation of burst kinetics in the hydrolysis of SLPPEN catalyzed by P99 beta-lactamase suggests that this chromophoric spin-labeled substrate is differentially sensitive to active site interactions underlying the cephalosporinase and penicillinase reactivity of this class C enzyme.     

Screening of bacterial genes responsible for resistance to beta-lactam antibiotics using microarrays with enzymatic detection

The method of hybridization analysis on microarrays with enzymatic detection based on horseradish peroxidase is applied to screen infectious agents of nosocomial and community-acquired infections for beta-lactamase genes causing resistance to beta-lactam antibiotics. The advantages of using this method for the rapid identification of genes are demonstrated. Similarities and differences in the distribution of beta-lactamase genes identified in the infectious agents of nosocomial and community-acquired infections are revealed. The most common type of extended-spectrum beta-lactamases (ESBLs) is CTX-M. The high prevalence of extended-spectrum beta-lactamases, particularly of the TEM-1 beta-lactamase, is demonstrated. Individually or in combination with genes of TEM-1 and SVH-1 beta-lactamases, the genes of subgroup CTX-M-1 beta-lactamases were the most frequently identified in community-acquired infectious agents. There were no cases of the simultaneous detection of multiple ESBLs in community-acquired infectious agents. Much more varied combinations of beta-lactamases were identified in nosocomial infectious agents: a combination of extended-spectrum beta-lactamases and broad-spectrum beta-lactamases was identified in 62% of strains and the simultaneous presence of two different types of ESBLs was identified in 18% of strains.     

Crystal structure of BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, in complex with Enterobacter cloacae 908R beta-lactamase: evidence for a stereoselective mechanism from docking studies

BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, is an active-site-directed inactivator of bacterial beta-lactamases. The crystal structure of Enterobacter cloacae 908R class C beta-lactamase in complex with BRL 42715, docking, and energy minimization studies explain stereoselectivity of the binding of C6-(heterocyclic methylene)penems against class C beta-lactamase.     

Communication between the active site and the allosteric site in class A beta-lactamases

Bacterial production of beta-lactamases, which hydrolyze beta-lactam type antibiotics, is a common antibiotic resistance mechanism. Antibiotic resistance is a high priority intervention area and one strategy to overcome resistance is to administer antibiotics with beta-lactamase inhibitors in the treatment of infectious diseases. Unfortunately, beta-lactamases are evolving at a rapid pace with new inhibitor resistant mutants emerging every day, driving the design and development of novel beta-lactamase inhibitors. Here, we examined the inhibitor recognition mechanism of two common beta-lactamases using molecular dynamics simulations. Binding of beta-lactamase inhibitor protein (BLIP) caused changes in the flexibility of regions away from the binding site. One of these regions was the H10 helix, which was previously identified to form a lid over an allosteric inhibitor binding site. Closer examination of the H10 helix using sequence and structure comparisons with other beta-lactamases revealed the presence of a highly conserved Trp229 residue, which forms a stacking interaction with two conserved proline residues. Molecular dynamics simulations on the Trp229Ala mutants of TEM-1 and SHV-1 resulted in decreased stability in the apo form, possibly due to loss of the stacking interaction as a result of the mutation. The mutant TEM-1 beta-lactamase had higher H10 fluctuations in the presence of BLIP, higher affinity to BLIP and higher cross-correlations with BLIP. Our results suggest that the H10 helix and specifically W229 are important modulators of the allosteric communication between the active site and the allosteric site.     

Synthesis and evaluation of ketophosph(on)ates as beta-lactamase inhibitors

A series of amidoketophosph(on)ates of general structure PhCH2OCONHCH(R)COCHR'(CH2)n(O)P(O2-)(O)R' (R = H, CH3; R' = H, CH3; n = 0, 1; R' = H, CH3, Et, Ph) have been prepared as a potential source of beta-lactamase inhibitors. The phosphonates (n = 0) were obtained by means of the Arbuzov reaction while most of the phosphates were achieved from reaction of phosph(or/on)ic acids with the appropriate diazoketone PhCH2OCONHCH(R)COCR'N2. The electrophilicity of the carbonyl group in the resulting phosph(on)ates was assessed by the degree of hydration in aqueous solution, determined from NMR spectra. These compounds inhibited typical class C and class D beta-lactamases, particularly the latter group, but showed no activity against class A enzymes. To enhance the carbonyl electrophilicity, an alpha-difluorinated analogue (R = H, CHR' = CF2, n = 0, R' = Et) was also prepared, but no enhanced inhibitory activity was observed. All evidence suggested that these compounds inhibited in the carbonyl form rather than by formation of tetrahedral adducts at the beta-lactamase active site. They show promise as leads to specific class D beta-lactamase inhibitors.     

Inhibition of Beta-Lactamase by 1,4-Naphthalenedione from the Plant <Emphasis Type="Italic">Holoptelea integrifolia</Emphasis>

The most important mechanism of the beta-lactam antibiotic resistance is the destruction of the antibiotics by the enzyme beta-lactamase. Use of beta-lactamase inhibitors in combination with antibiotics is one of the successful antibacterial strategies. The inhibitory effect of a phytochemical, 1,4-naphthalenedione, isolated from the plant Holoptelea integrifolia on beta-lactamase is reported here. This compound was found to have a synergistic effect with the antibiotic amoxicillin against a resistant strain of Staphylococcus aureus. The enzyme was purified from the organism and incubated with the compound. An assay showed that the compound can inhibit the enzymatic activity of beta-lactamase. Modeling and molecular docking studies indicated that the compound can fit into the active site of beta-lactamase. Hence, the compound can serve as a potential lead compound for the development of effective beta-lactamase inhibitor that can be used against beta-lactam-resistant microbial strains.     

Mixed quantum mechanical/molecular mechanical (QM/MM) study of the deacylation reaction in a penicillin binding protein (PBP) versus in a class C beta-lactamase

The origin of the substantial difference in deacylation rates for acyl-enzyme intermediates in penicillin-binding proteins (PBPs) and beta-lactamases has remained an unsolved puzzle whose solution is of great importance to understanding bacterial antibiotic resistance. In this work, accurate, large-scale mixed ab initio quantum mechanical/molecular mechanical (QM/MM) calculations have been used to study the hydrolysis of acyl-enzyme intermediates formed between cephalothin and the dd-peptidase of Streptomyces sp. R61, a PBP, and the Enterobacter cloacae P99 cephalosporinase, a class C beta-lactamase. Qualitative and, in the case of P99, quantitative agreement was achieved with experimental kinetics. The faster rate of deacylation in the beta-lactamase is attributed to a more favorable electrostatic environment around Tyr150 in P99 (as compared to that for Tyr159 in R61) which facilitates this residue's function as the general base. This is found to be in large part accomplished by the ability of P99 to covalently bind the ligand without concurrent elimination of hydrogen bonds to Tyr150, which proves not to be the case with Tyr159 in R61. This work provides an essential foundation for further work in this area, such as selecting mutations capable of converting the PBP into a beta-lactamase.     

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by cyclic acyl phosph(on)ates: rescue by return.

As previously described (Pratt, R. F.; Hammar, N. J. J. Am. Chem. Soc. 1998, 120, 3004.), 1-hydroxy-4,5-benzo-2,6-dioxaphosphorinone(3)-1-oxide (salicyloyl cyclic phosphate) inactivates the class C beta-lactamase of Enterobacter cloacae P99 in a covalent fashion. The inactivated enzyme slowly reverts to the active form. This paper shows that reactivation involves a recyclization reaction that regenerates salicyloyl cyclic phosphate rather than hydrolysis of the covalent intermediate. The inactivation, therefore, is a slowly reversible covalent modification of the active site. The thermodynamic dissociation constant of the inhibitor from the inactivated enzyme is 0.16 microM. Treatment of the inactivated enzyme with alkali does not produce salicylic acid but does, after subsequent acid hydrolysis, yield one molar equivalent of lysinoalanine. This result proves that salicyloyl cyclic phosphate inactivates the enzyme by (slowly reversible) phosphorylation of the active site serine residue. This result contrasts sharply with the behavior of acyclic acyl phosphates which transiently inactivate the P99 beta-lactamase by acylation (Li, N.; Pratt, R. F. J. Am. Chem. Soc. 1998, 120, 4264.). This chemoselectivity difference is explored by means of molecular modeling. Rather counterintuitively, in view of the relative susceptibility of phosphates and phosphonates to nucleophilic attack at phosphorus, 1-hydroxy-4,5-benzo-2-oxaphosphorinanone(3)-1-oxide, the phosphonate analogue of salicyloyl cyclic phosphate, did not appear to inactivate the P99 beta-lactamase in a time-dependent fashion. It was found, however, to act as a fast reversible inhibitor (K(i) = 10 microM). A closer examination of the kinetics of inhibition revealed that both on and off rates (9.8 x 10(3) s(-1) x M(-1) and 0.098 s(-1), respectively) were much slower than expected for noncovalent binding. This result strongly indicates that the inhibition reaction of the phosphonate also involves phosphylation of the active site. Hence, unlike the situation with bacterial DD-peptidases covalently inactivated by beta-lactams, the P99 beta-lactamase inactivated by the above cyclic acyl phosph(on)ates can be rescued by return. Elimination of the recyclization reaction would lead to more effective inhibitors.     

Structural aspects for evolution of beta-lactamases from penicillin-binding proteins

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and beta-lactamases, resistance enzymes to beta-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for beta-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7beta-[N-Acetyl-L-alanyl-gamma-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporin bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC beta-lactamase from Escherichia coli was solved at 1.71 A resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the -lactamase active site. Furthermore, insertion of a peptide in the beta-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the beta-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.     

Ab initio QM/MM study of class A beta-lactamase acylation: dual participation of Glu166 and Lys73 in a concerted base promotion of Ser70

Beta-lactamase acquisition is the most prevalent basis for Gram-negative bacteria resistance to the beta-lactam antibiotics. The mechanism used by the most common class A Gram-negative beta-lactamases is serine acylation followed by hydrolytic deacylation, destroying the beta-lactam. The ab initio quantum mechanical/molecular mechanical (QM/MM) calculations, augmented by extensive molecular dynamics simulations reported herein, describe the serine acylation mechanism for the class A TEM-1 beta-lactamase with penicillanic acid as substrate. Potential energy surfaces (based on approximately 350 MP2/6-31+G calculations) reveal the proton movements that govern Ser70 tetrahedral formation and then collapse to the acyl-enzyme. A remarkable duality of mechanism for tetrahedral formation is implicated. Following substrate binding, the pathway initiates by a low energy barrier (5 kcal mol(-1)) and an energetically favorable transfer of a proton from Lys73 to Glu166, through the catalytic water molecule and Ser70. This gives unprotonated Lys73 and protonated Glu166. Tetrahedral formation ensues in a concerted general base process, with Lys73 promoting Ser70 addition to the beta-lactam carbonyl. Moreover, the three-dimensional potential energy surface also shows that the previously proposed pathway, involving Glu166 as the general base promoting Ser70 through a conserved water molecule, exists in competition with the Lys73 process. The existence of two routes to the tetrahedral species is fully consistent with experimental data for mutant variants of the TEM beta-lactamase.     

Binding of D- and L-captopril inhibitors to metallo-beta-lactamase studied by polarizable molecular mechanics and quantum mechanics

The bacterial Zn2+ metallo-beta-lactamase from B. fragilis is a zinc-enzyme with two potential metal ion binding sites. It cleaves the lactam ring of antibiotics, thus contributing to the acquired resistance of bacteria against antibiotics. The present study bears on the binuclear form of the enzyme. We compare several possible binding modes of captopril, a mercaptocarboxamide inhibitor of several zinc-metalloenzymes. Two diastereoisomers of captopril were considered, with either a D- or an L-proline residue. We have used the polarizable molecular mechanics procedure SIBFA (Sum of Interactions Between Fragments ab initio computed). Two beta-lactamase models were considered, encompassing 104 and 188 residues, respectively. The energy balances included the inter and intramolecular interaction energies as well as the contribution from solvation computed using a continuum reaction field procedure. The thiolate ion of the inhibitor is binding to both metal ions, expelling the bridging solvent molecule from the uncomplexed enzyme. Different competing binding modes of captopril were considered, either where the inhibitor binds in a monodentate mode to the zinc cations only with its thiolate ion, or in bidentate modes involving additional zinc binding by its carboxylate or ketone carbonyl groups. The additional coordination by the inhibitor's carboxylate or carbonyl group always occurs at the zinc ion, which is bound by a histidine, a cysteine, and an aspartate side chain. For both diastereomers, the energy balances favor monodentate binding of captopril via S-. The preference over bidentate binding is small. The interaction energies were recomputed in model sites restricted to captopril, the Zn2+ cations, and their coordinating end side chains from beta-lactamase (98 atoms). The interaction energies and their ranking among competing arrangements were consistent with those computed by ab initio HF and DFT procedures.     

Novel fluorogenic substrates for imaging beta-lactamase gene expression

A new class of small nonfluorescent fluorogenic substrates becomes brightly fluorescent after beta-lactamase hydrolysis with up to 153-fold enhancement in the fluorescence intensity. Less than 500 fM of beta-lactamase in cell lysates can be readily detected, and beta-lactamase expression in living cells can be imaged with a red fluorescence derivative. These new fluorogenic substrates should find uses in clinical diagnostics and facilitate the applications of beta-lactamase as a biosensor.     

Structures of protonated arginine dimer and bradykinin investigated by density functional theory: further support for stable gas-phase salt bridges

The gas-phase structures and energetics of both protonated arginine dimer and protonated bradykinin were investigated using a combination of molecular mechanics with conformational searching to identify candidate low-energy structures, and density functional theory for subsequent minimization and energy calculations. For protonated arginine dimer, a good correlation (R = 0.88) was obtained between the molecular mechanics and EDF1 6-31+G* energies, indicating that mechanics with MMFF is suitable for finding low-energy conformers. For this ion, the salt-bridge or ion-zwitterion form was found to be 5.7 and 7.2 kcal/mol more stable than the simple protonated or ion-molecule form at the EDF1 6-31++G** and B3LYP 6-311++G** levels. For bradykinin, the correlation between the molecular mechanics and DFT energies was poor (R = 0.28), indicating that many low-energy structures are likely passed over in the mechanics conformational searching. This result suggests that structures of this larger peptide ion obtained using mechanics calculations alone are not necessarily reliable. The lowest energy structure of the salt-bridge form of bradykinin is 10.6 kcal/mol lower in energy (EDF1) than the lowest energy simple protonated form at the 6-311G* level. Similarly, the average energy of all salt-bridge structures investigated is 13.6 kcal/mol lower than the average of all the protonated forms investigated. To the extent that a sufficient number of structures are investigated, these results provide some additional support for the salt-bridge form of bradykinin in the gas phase.