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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

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Primary hydrogen isotope effects on the rates of bimolecular elimination reactions of some cyclohexyl tosylates and bromides are small (kH/kD = 2-3) for very E2C-like reactions, pass through a maximum of 6 for more E2H-like reactions, and are small again (2-3) for very E2H-like reactions. Movement from the E2C to the E2H side of the spectrum has been achieved by increasing the acidity of the substrate, by increasing the hydrogen basicity of the base, and by changing the leaving group from tosylate to bromide. Differences between kH/kD values, as measured by intramolecular competition vs. intermolecular rate comparisons, may be due to a secondary hyperconjugative isotope effect.The primary hydrogen isotope effects, kH/kD, on the rates of /3-eliminations promoted by weak hydrogen bases, B, but strong carbon nucleophiles, such as chloride ion (E2C-like reactions), are low at 25-75°, i.e., 2.3-3.2.2-4 They are considerably less than kH/kD of 5-7, commonly observed for /3-elimination induced by strong hydrogen bases, such as alkoxides (E2H-like reactions).5 This observation is true for the limited number of eliminations studied, and, although the topic has been reviewed,6•7 a representative study of kH/k^through the E2C-E2H spectrum has not been made.The changes in kn/kP can in themselves be explained by any one of several sets of variable transition states, but opinions differ as to which set to use. Certainly there must be variable transition states for E2 reactions,8 because only in this way can we explain the large changes in kH/kD which are observed under different conditions. In this paper we have chosen to discuss the isotope effects in terms of the E2C-E2H spectrum of transition states. However, recent work in our laboratories on the effect of conjugative substituents9 has caused us to reexamine our ideas10 about the structure of the E2C extreme transition state. Although we still favor the "loose" structure I, with a well-developed double bond, and very loose C