Structural Features of Vancomycin

Transcription

1 REVIEWS OF INFECTIOUS DISEASES VOL. 3, SUPPLEMENT NOVEMBER-DECEMBER by The University of Chicago. All rights reserved /81/ $02.00 Structural Features of Vancomycin Ralph R. Pfeiffer From the Lilly Research Laboratories, Indianapolis, Indiana Recent analytical methods have advanced knowledge of the structure of vancomycin from a description of only several molecular fragments to a complete understanding of the intact molecule. The molecular weight is 1,448. The molecule consists of a sevenmembered peptide chain that is formed by parts of three phenylglycine systems, two chlorinated tyrosine units, aspartic acid, and N-methylleucine. Two ether bonds and a carbon-carbon bond join the various substituents on the peptide chain into three large rings. A disaccharide, composed of glucose and vancosamine, is also present but is not part of the cyclic structure. Details of the vancomycin structure have been related to hydrogen bonding between the antibiotic and bacterial cell-wall precursors that have a D-alanyl-D-alanine carboxyl terminus; such bonding would provide a molecular basis for the cell-wall mode of action for vancomycin. With one carboxyl, two amino, and three phenolic groups, vancomycin undergoes a variety of ionic interactions in solutions of different ph and composition. The renewal of clinical interest in vancomycin and the determination of its complete structure are relatively recent occurrences. Nevertheless, the two events are entirely coincidental. Vancomycin was developed in 1956 and was widely used as an antistaphylococcal agent until displaced by semisynthetic f3-lactamase-resistant penicillins and cephalospirins. While the use of vancomycin declined, little effort was made to explore its clinical value, which present studies are bringing to light. In contrast, the complexity of the molecule was appreciated by early workers who identified many of its fragments by classical degradation procedures. However, the problems of how these fragments are linked and what their spatial relationships are in the intact molecule defied resolution until recently, when the required analytical procedures became available and the complete structure could be described. Molecular Structure Early chemical studies indicated that vancomycin was unlike any previously described antibiotic. On the basis of its carbohydrate and peptide content, vancomycin was classified as a glycopeptide antibiotic. It was the first member of this new class. Later, other glycopeptide antibiotics, such as ac- Please address requests for reprints to Dr. Ralph R. Pfeiffer, Lilly Research Laboratories, 307 East McCarty Street, Indianapolis, Indiana tinoidin [1] and ristocetin [2], were discovered, but vancomycin remains the only glycopeptide antibiotic in wide clinical use. Glycopeptide antibiotics differ widely in the number and kinds of sugars and other substituents that they contain and in the complexity of their cyclic structures but have in common the presence of both a carbohydrate and a peptide portion. Members of this class have molecular weights well above those of the penicillins, cephalosporins, tetracyclines, aminoglycosides, and macrolides. In the first structure studies, relatively mild degradation procedures served to identify glucose, aspartic acid, N-methylleucine, and a-chlorophenol as fragments of the vancomycin molecule [3-5]. However, more aggressive chemical degradations yielded complex mixtures which could not be analyzed by any method that revealed further structural details of the molecule. Mild degradations also yielded several crystalline products of unknown structure that represented the major portions of the vancomycin molecule. Attempts to degrade these products again resulted in complex mixtures that were of little value in elucidating the structure of vancomycin. With the exception of the discovery and characterization of a second carbohydrate fragment, the novel amino sugar vancosamine [6], further degradation studies identified no new fragments, and the bulk of the vancomycin structure remained unknown. Renewal of interest in the structure of vancomycin was stimulated a decade later by several important advances in analytical methodology. Better S205

2 5206 Pfeiffer chromatographic methods made possible the direct isolation of pure degradation fragments. Refined mass spectrometric technology simplified the identification of fragments. Continued improvements in nuclear magnetic resonance spectrometry permitted the application of this method to ever larger fragments and eventually to the intact vancomycin molecule. Investigations using these new techniques revealed that the vancomycin molecule contains five benzene rings [7] that are in the form of two chlorinated f3-hydroxytyrosine and three phenylglycine units [8]. It was postulated that the molecule is polycyclic [7]. In 1976, a study by Williams and Kalman [9] finally provided a description of virtually every detail of the vancomycin structure. In the absence of suitable crystals of vancomycin itself, X-ray diffraction studies were made on a crystalline degradation product, CDP-l [5], and were reported in 1978 [10]. CDP-l contains a carboxyl group in place of the amide on the aspartic Vancosamine..... Disaccharide acid residue in vancomycin. Thus, all the essential features of the vancomycin molecule are revealed in the structure of CDP-l. These X-ray studies confirmed the nuclear magnetic resonance work and showed that the molecular conformation is the same in the solid state as in solution. The empirical formula of vancomycin is C66H7SClzN9024, and the molecular weight is 1,448. The structure is shown in figure 1. Despite its complexity, the molecule lends itself to a reasonably concise summary; vancomycin is a tricyclic glycopeptide in which two chlorinated f3-hydroxytyrosine units, three substituted phenylglycine systems, N-methylleucine, and aspartic acid amide are interconnected in a seven-membered peptide chain. Two ether bonds and a carbon-carbon bond join the substituents to form three large rings. In addition, one of the phenylglycine units bears a disaccharide composed of glucose and the unique amino sugar vancosamine. The disaccharide is not part of the tricyclic structure. N-methylleucine Aspartic acid amide Figure 1. The structure of vancomycin, according to X-ray analysis of the crystalline degradation product, CDP-l [10], consists of a disaccharide (vancosamine and glucose), two fj-hydroxychlorotyrosine units, three substituted phenylglycine systems, N-methylleucine, and aspartic acid amide. The peptide backbone is shown in heavier type. The atoms involved in hydrogen bonding with acetyl-n-alanyl-n-alanine (see figure 2) are indicated by asterisks.

3 Structural Features of Vancomycin S207 Figure 2. Space-filling model of vancomycin-acetyl-n-alanyl-n-alanine complex as proposed by Sheldrick et al. [10]. The vancomycin molecule is oriented on the page as in figure 1. The dipeptide suspended above the molecule obscures the disaccharide. The heavy lines identify the atoms involved in hydrogen bonding, although only a small crescent of the hydrogen on the far left shows in this view. The cleft, which can accommodate the dipeptide, is particularly evident at the left portion of the vancomycin molecule. (Space-filling models were available through the courtesy of Dr. Noel D. Jones.) Mode of Action and Binding Studies Multiple modes of antimicrobial action have been ascribed to vancomycin. These include a direct effect on the cytoplasmic membrane [11], inhibition of ribonucleic acid synthesis [12], and inhibition ofcell-wall mucopeptide synthesis [13]. The action of vancomycin on the cell wall, which occurs at a stage preceding that of penicillins and cephalosporins, prevents polymerization of the phosphodisaccharide-pentapeptide-lipid complex. Of the three mechanisms of antimicrobial action, only interference with cell-wall synthesis has been related to the structural details of the vancomycin molecule. Before the structure of vancomycin was known, Chatterjee and Perkins [14], during the course of paper chromatography studies, found evidence for the formation of complexes between vancomycin and several pentapeptides isolated from bacterial cultures. Perkins [15] and Nieto and Perkins [16,17] then measured the effect of various peptides on the ultraviolet absorption spectrum of vancomycin and calculated stability constants for the complexes. This work correlated peptide structure and strength of binding to vancomycin and provided a basis for much speculation on the vancomycin peptide complexes. High-resolution nuclear magnetic resonance spectroscopy was later utilized in several studies [9, 18, 19] to pinpoint

4 S208 Pfeiffer the features of the participating molecules that would account for the specificity of the interaction. By mixing vancomycin with various peptides and observing shifts in resonance peaks assigned in previous structural studies to specific protons of the respective component molecules, it was found that the strongest binding occurs when the peptide has a D-alanyl-D-alanine carboxyl terminus. In contrast, peptides with an L-alanyl-Dalanine terminus failed to bind measurably to vancomycin [19]. Structural variations in the peptide farther from the terminal carboxyl appear to be less critical and probably have only a directing effect on the peptide as it approaches the binding site. The model in figure 2 of a vancomycin-acetyl D-alanyl-D-alanine complex, proposed by Sheldrick et al. [10], is one interpretation that takes into account the accumulated information. The interaction requires the formation of three hydrogen bonds, each of which arises from the exact positioning of an NH of one molecule with respect to an 0 of the other molecular species to form an NH-O bond. Moreover, the area of the vancomycin molecule where such multiple hydrogen bonding is known to occur is in the shape of a cleft that can spatially accommodate the D-ala-Dala "tail" and thus strengthen the entrapment and provide additional selectivity to the interaction. The ultimate proof of this proposal, or of similar ones, may require the determination of structure by X-ray diffraction on crystals of a vancomycinpeptide complex. Chelation of Metal Ions A different kind of binding occurs between vancomycin and certain metal ions, such as copper, iron, and cadmium. Although no evidence is available to imply that chelation of metal ions is a mode of action of vancomycin, the ability of vancomycin to chelate copper ion is exploited during the isolation and purification of the commercial product. Addition of a copper salt to an impure solution of vancomycin causes the formation of a violet vancomycin-copper complex that is precipitated by ethanol. The copper is subsequently removed as the highly insoluble copper sulfide by precipitation from aqueous solution of the metal complex. Antimicrobial Activity of Derivatives Unlike the penicillins, cephalosporins, tetracyclines, and macrolides, which have been extensively modified in efforts to alter their antimicrobial properties, vancomycin has been subjected to relatively few chemical modifications. Removal of the disaccharide from the vancomycin molecule results in only a fractional loss of antibacterial activity [5]. Some activity is also retained when vancomycin is acetylated or formylated [15]. These active derivatives were all found to retain their ability to bind peptides. In contrast, substitution of a carboxyl group for the amide group in the aspartic acid substituent, i.e., CDP-I, results in total loss of antibacterial activity [5]. The loss of activity is paradoxical inasmuch as CDP-I retains the molecular features required for binding of peptides, as in the Sheldrick model [10] of the CDP-I peptide complex (figure 2). One must speculate, therefore, that the absence of the amide group in CDP-l or, more likely, the presence of a second carboxyl group, interferes with a critical aspect of the binding process. With knowledge of the chem- 6.. en 5 c G) 4 'i > "Sc:r UI + % Net Molecular Charge ph Figure 3. Changes in net molecular charge during titration of vancomycin.

5 StructuralFeatures of Vancomycin S209 ical structure of vancomycin now available, information on new derivatives may be forthcoming. Pharmaceutical Chemistry The manner in which vancomycin in aqueous solution reacts to changes in ph is described in figure 3. The titration curve shows that the net molecular charge varies continuously from + 2 to - 4 as each of the ionizable groups (one carboxyl, two amino, and three phenolic), gains or loses a proton. Vancomycin is very soluble at the ph of reconstitution (ph 4). As ph increases, the solubility of vancomycin decreases rapidly and passes through a minimum ("'15 mg/ml) near neutrality, where the molecular charge is zero. Vancomycin is soluble but unstable in alkaline solution. While these properties contraindicate the mixing of concentrated solutions of vancomycin intended for parenteral use with any substance that alters the ph toward neutrality or into the alkaline range, they indicate that vancomycin remains in solution at concentrations encountered in body fluids, regardless of ph. The variety of charged species of vancomycin that exists at different ph values also provides myriad possibilities for the formation of insoluble salts; hence, admixture of vancomycin with substances not known to be compatible should be avoided. Vancomycin is preferably reconstituted with water or 0.85% NaCI as described in the package literature. References 1. Brazhnikova, M. G., Lomakina, N. N., Yurina, M. S., Lavrova, M. F. Antibiotic actinoidin: its isolation and chemical properties. Antibiot. Chemother. II : , Philip, J. E., Schenck, J. R., Hargie, M. P. Ristocetins A and B, two new antibiotics: isolation and properties. Antibiotics Annual, Medical Encyclopedia, Inc., New York, 1957, p McCormick, M. H., Stark, W. M., Pittenger, G. E., Pittenger, R. C.; McGuire, J. M. Vancomycin, a new antibiotic. I. Chemical and biologic properties. Antibiotics Annual, Medical Encyclopedia, Inc., New York, 1956, p Higgins, H. M., Harrison, W. H., Wild, G. M., Bungay, H. R., McCormick, M. H. Vancomycin, a new antibiotic. VI. Purification and properties of vancomycin. Antibiotics Annual, Medical Encyclopedia, Inc., New York, 1958, p Marshall, F. J. Structure studies on vancomycin. J. Med. Chern. 8:18-22, Smith, R. M., Johnson, A. W., Guthrie, R. D. Vancosamine: a novel branched chain amino sugar from the antibiotic vancomycin. J. Chern. Soc. [Perkin I] 6: , Smith, G. A., Smith, K. A., Williams, D. H. Structural studies on the antibiotic vancomycin: evidence for the presence of modified phenylglycine and beta-hydroxytyrosine units. J. Chern. Soc. [Perkin I] 21: , Smith, K. A., Williams, D. H., Smith, G. A. Structural studies on the antibiotic vancomycin: the nature of the aromatic rings. J. Chern. Soc. [Perkin I] 20: , Williams, D. H., Kalman, J. R. Structural and mode of action studies on the antibiotic vancomycin. Evidence from 270-MHz proton magnetic resonance. J. Am. Chern. Soc. 99: , Sheldrick, G. M., Jones, P. G., Kennard, 0., Williams, D. H., Smith, G. A. Structure of vancomycin and its complex with acetyl-n-alanyl-n-alanine. Nature 271: , Jordan, D. C. Effect of vancomycin on the synthesis of the cell wall and cytoplasmic membrane of Staphylococcus aureus. Can. J. Microbiol. 11: , Jordan, D. C.; Innis, W. E. Selective inhibition of ribonucleic acid synthesis in Staphylococcus aureus by vancomycin [letter]. Nature 184: , Jordan, D. C. Effect of vancomycin on the synthesis of cell wall mucopeptide of Staphylococcus aureus. Biochern. Biophys. Res. Commun. 6: , Chatterjee, A. N., Perkins, H. R. Compounds formed between nucleotides related to the biosynthesis of bacterial cell wall and vancomycin. Biochem. Biophys. Res. Commun. 24: , Perkins, H. R. Specificity of combination between mucopeptide precursors and vancomycin or ristocetin. Biochem. J. 1l1: , Nieto, M., Perkins, H. R. Physiochemical properties of vancomycin and iodovancomycin and their complexes with diacetyl-l-lysyl-d-alanyl-d-alanine. Biochem. J. 123: , Nieto, M., Perkins, H. R. Modifications of the acyl-nalanyl-n-alanine terminus affecting complex formation with vancomycin. Biochem. J. 123: , Brown, J. P., Terenius, L., Feeney, J., Burgen, A. S. V. A nuclear magnetic resonance study of the interaction between vancomycin and acetyl-d-alanyl-d-alanine in aqeous solution. Mol. Pharmacol. 11: , Brown, J. P., Terenius, L., Feeney, J., Burgen, A. S. V. A structure-activity study by nuclear magnetic resonance of peptide interactions with vancomycin. Mol. Pharmacol. 11: , 1975.