Structure of Ristocetin A

Abstract

Acid hydrolysis of ristocetin A yields a number of amixo acids of unusual structure. One set of diastereoisomeric pairs has a molecular weight of 362 and an empirical formula of C1711sN207. Mass spectral and nuclear magnetic resonance (NMR) evidence suggest a diphenyl ether with hydroxyl and a-amino acid [-CH-(NH2)COOH] groups on one ring, and methyl, hydroxyl and a-amino acid groups on the other ring. Coupling in the NMR and nuclear Overhauser effect experiments favor certain substitution patterns, which are shown. Another set of diastereoisomers seems to be a lower homolog of the previous set, without the aromatic methyl group.Ristocetins (1) A and B have antibacterial properties (2), a molecular weight of several thousand, and yield 10 and 7 identified monosaccharide units, respectively, on mild acid hydrolysis (3). The aglycone is a polypeptide yielding amino acids of novel, but unknown, structure on strong acid hydrolysis; it has been recognized, however, that phenolic aamino acids are present. The mode of antibacterial action of the ristocetins has been studied extensively (4). The present communication describes information about the structure of the aglycone of ristocetin A.By hydrolysis of 1 mM ristocetin with 6 N HCl in a degassed sealed tube, followed by analysis with an amino acid analyzer (5), we have found five distinct amino acids (I, II, III, IV, V) giving positive ninhydrin tests; the elution of ninhydrin-active material runs parallel to increases in the ultraviolet absorption at 260 nm, so that separation of the five can be followed conveniently by spectrophotometry (Uviscan and recorder). The sample of hydrolyzate was placed on a 150-cm column containing Aminex Q-150S resin at 30°. Upon elution with pH 3.25 buffer at about 60 ml/hr, I came off at 5 hr 40 min, II at 6 hr 30 min and III at 7 hr 50 min.After a change to pH 4.25 buffer, IV came off in 3 hr 40 min and V. at 4 hr 30 min. Hydrolysis of 400 mg of ristocetin yielded a total, after separation and desalting, of about 75 mg of amino acids I-V.We found that acid II is converted to a mixture of I and II, II predominating, and IV is converted to a 50-50 mixture of IV and V by heating with HCl; each pair appears to consist of two diastereoisomers, with almost identical NMR spectra for each pair. Acid III is obtained in very small amounts.Evidence for the presence of Ar(H2N)CHCOOH groups comes from the ninhydrin reaction, ester formation, the NMR spectra, the formation of t-BOC and thiol-t-BOC derivatives with di-t-butyl dicarbonate and its sulfur analog (6), as well as mass spectra. Phenolic groups are shown by a bathochromic shift of the wavelength of maximal absorption in base, by ease of oxidation with Tollens reagent, by empirical formulas requiring aromatic rings, and by NMR. The empirical formula of IV (and V) is C17H18N207, based on peaks at m/e for the bis-t-BOC bis-ethyl ester, 1, of 618

Comparative Study on Collagen-Binding Enzyme-Linked Immunosorbent Assay and Ristocetin Cofactor Activity Assays for Detection of Functional Activity of von Willebrand Factor

“…Ristocetin A is an antibiotic from the actinomycete Nocardia lurida with a molecular mass of 2063 D, 17,18 and it exists as a dimer in aqueous solution. 19 Its structure 18,20 is shown in Figure 2.…”

The Interaction of Botrocetin with Normal or Variant von Willebrand Factor (Types IIA and IIB) and Its Inhibition by Monoclonal Antibodies that Block Receptor Binding

“…It has been postulated that positively charged ristocetin binds to sites on the platelet surface and reduces the platelet's negative charge by which vWF can bind to GP lb (2). However, it is not fully unterstood whether its modulation takes place by interacting with only the platelet receptor or with vWF, even though the biochemical structure of ristocetin has been partially elucidated (3,4). As an alternative, the snake venom platelet coagglutinin, botrocetin, was partially purified and characterized by Read et al (5) and by Brinkhous et al (6).…”

Amino acid constituents of ristocetin A

“…At present we believe that the peptide portion of ristocetin A is made up of ristomycinic acid (12), actinoidinic acid (17), and dechlorovancomycinic acid (23a), although the presence of a low molecular weight amino acid, particularly an aromatic one, could possibly have escaped detection. It should be pointed out that only single stereoisomers of the amino acids are thought to occur in the native antibiotic; epimerization of phenylglycines occurs during both basic and acidic hydrolyses.…”

“…Ristomycinic Acid (12). The studies by Fehlner et al12 of the diphenyl ether type bis(amino acid) had led them to conclude that the compound contained two phenylglycines linked via an oxygen bridge between the aromatic rings, that each ring bore one hydroxyl group, and that one ring also was methylated.…”