Structure of Alamethicin in solution. One- and two-dimensional 1H nuclear magnetic resonance studies at 500 MHz.

We report here the 500 MHz 1H nuclear magnetic resonance spectra of Alamethicin, an icosapeptide antibiotic isolated from Trichoderma viride, in methanol, water and methanol/water mixtures. At this frequency, resonances from all the protons are well-resolved in methanol and may be assigned unambiguously. Spectral assignments were made using two-dimensional spin-echo correlated spectroscopy and by spin-decoupling experiments. The amide coupling constants (JNH-alpha CH) facilitated conformational predictions, which were confirmed in part by two-dimensional nuclear Overhauser experiments. On the basis of these data, we propose a secondary structure for Alamethicin that is alpha-helical toward the N terminus and extended beta-sheet at the C-terminal end. This structure is consistent with earlier circular dichroism measurements (McMullen et al., 1971), infrared attenuated total reflection spectroscopy studies (Fringeli & Fringeli, 1979) and proton exchange data (Davis & Gisin, 1981). The proposed structure is a tightly bound dimer, wherein the beta-sheet is stabilized by intermolecular hydrogen-bonds between opposing molecules. An interesting feature of this structure is that it exhibits both a hydrophobic and a hydrophilic surface. This highly amphiphilic nature of the dimer structure may account for the extensive further aggregation of Alamethicin in water. The 1H n.m.r. spectrum of Alamethicin in water is broad, suggesting extensive association. However, spectral assignments and amide coupling constant measurements in water, which were accomplished by titration of methanolic solution of Alamethicin by water, revealed no gross changes in the basic secondary structure of the molecule.

Synthesis, base-catalyzed hydrolytic reactivity, and anticancer evaluation of O-aryl phosphorodiamidates as a novel class of pro(phosphorodiamidic acid mustards).

Bis(2-chloroethyl)phosphoramidic dichloride [MP(O)Cl2, M = N(CH2CH2Cl)2] has been used as the starting material for the synthesis of O-aryl phosphorodiamidates having the general structure MP(O)(NHR)OAr: 9, R = H, Ar = 4-NO2C6H4; 10, R = H, Ar = C6F5; 11, R = C6H5, Ar = C6F5; 12, R = 4-MeC6H4, Ar = C6F5; and 13, R = 4-EtOC6H4, Ar = C6F5. The phosphorodiamidic chloride precursor to 13 (14) was also isolated. Kinetics for the base-catalyzed hydrolysis of compounds 9--13 were investigated by UV and NMR methods and are considered in connection with service of these compounds as pro(phosphorodiamidic acid mustards) [MP(O)(NHR)OAr leads to MP(O)(NHR)OH] via an E1cB mechanism involving the intermediacy of a mustard-bearing metaphosphorodiimide [MP(O)=NR]. Anticancer screening tests against L1210 lymphoid leukemia in mice indicated that 9--14 are inactive; similar negative results were obtained with the KB cell culture, except in the case of 14 which was marginally active.

Synthesis and study of a spin-labeled cyclophosphamide analogue, 3-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide.

3-(1-Oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (7) was isolated in 36% yield following H2O2-Na2WO4 oxidation of 3-(2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (6), which was synthesized in three steps (25% yield) starting with 4-amino-2,2,6,6-tetramethylpiperidine. Binding of 7 to mouse liver microsomes was investigated by optical and electron spin resonance spectroscopy. Compared with the mouse liver microsomal metabolism of 1, separate incubations of 6 and an ca. 1:1 mixture of 1 and 6 gave approximately 90 and 60% less acrolein, respectively. A spin-labeled metabolite of 7, viz., N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)phosphoramide mustard (9), was synthesized and its intramolecular O-alkylation at pH 7.4, 37 degrees C, was studied by 31P NMR spectroscopy. Compounds 7 and 9 were inactive in screening tests against L1210 lymphoid leukemia in mice.

Mass-spectral studies of isomeric D-ribofuranosylribitol disaccharides from the capsular polysaccharides of Haemophilus influenzae type b and Escherichia coli K 100.

Two isomeric-D-ribofuranosylribitols, derived from capsular polysaccharides of Haemophilus influenzae type b and Escherichia coli K 100, were methylated or acetylated, and the products analyzed by gas-liquid chromatography-mass spectrometry. The marked difference in the mass spectra of the methyl ethers of these disaccharides allowed clear distinction between 1- and 2-O-D-ribofuranosylribitol was characteristic for this disaccharide; its isomer, the (1 leads to 2)-linked species, has a base peak at m/e 57. The difference in the base peaks is attributable to fragmentation of the methylated ribitol, as both spectra display common ions characteristic of the methylated D-ribofuranosyl group. For the acetylated disaccharides, the mass spectra displayed common ions characteristic of the acetylated D-ribofuranosyl group. However, no ions similar to those found for the methylated ribitol allowed ready differentiation between the two acetates. Instead, their spectra displayed similar ions, differing somewhat in relative abundance; the M-1 ion, m/e 577, was obtained for both. Comparison of the relative abundance of m/e 139, 259, and 303 in the spectra of the two acetates did allow distinction between them.

Structural studies of the Haemophilus influenzae type e capsular polysaccharide.

The structure of the Haemophilus influenzae type e capsular polysaccharide has been determined by a combination of chemical and spectroscopic methods. The structure of the repeating unit of the polymer was found to be leads to 3)-beta-D-GlcNAc-(1 leads to 4)-beta-D-ManANAc-(1 leads to ; both sugars were present in the pyranoid form.

Structural and immunological studies of the Escherichia coli K7 (K56) capsular polysaccharide.

The structure of the Escherichia coli K7 capsular polysaccharide has been investigated by a combination of chemical and spectroscopic methods. The Structure of the repeating unit of the polymer was found to be goes to 3)-beta-D-ManNAcA-(1 leads to 4)-beta-D-Glc-(1 goes to ; the O-6 atom of the D-glucosyl residue in the repeating unit is acetylated. The K7 polysaccharide is cross-reactive with the Streptococcus pneumoniae type 3 polysaccharide, the structure of which had previously been determined; our n.m.r. studies of the S. pneumoniae type 3 polysaccharide are in accord with this structure. The E. coli K7 and K56 capsular antigens have been shown by serology and 13C-n.m.r. spectroscopy to be identical.

Determination of the structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with the capsule from type b Haemophilus influenzae.

The structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with that from type b Haemophilus influenzae, was determined by using a combination of chemical and spectroscopic techniques. The structure of the K100 repeating unit was found to be----3)-beta-D-Ribf-(1----2)-D-ribitol-5-(PO4----. The K100 polysaccharide is thus identical in composition to, but different in linkage from, the H. influenzae type b capsular polysaccharide, which has beta-D-Ribf-(1----1)-D-ribitol linkages.

Structural and immunological studies of the Haemophilus influenzae type d capsular polysaccharide.

Employing a combination of chemical and spectroscopic techniques, the structure of the Haemophilus influenzae type d capsular polysaccharide was found to be leads to 4)-beta-D-GlcNAc-(1 leads to 3)-beta-D-ManNAcA-(1 leads to. L-Alanine, L-serine, and L-threonine, in the molar ratios of approximately 1.0:1.0:0.3, were linked to C-6 of the D-mannosyluronic residue as amides; the (serine + alanine + threonine) to ManNAcA ratio was approximately 0.95:1.0. Removal of the amino acids by mild hydrolysis with sodium hydroxide resulted in a material that was cross-reactive with the native, type d polymer. The base-treated, type d polysaccharide was not observed to cross-react with either the H. influenzae type e or Escherichia coli K7 capsular polysaccharide, both of which are structurally similar to type d.

Serological, chemical, and structural analyses of the Escherichia coli cross-reactive capsular polysaccharides K13, K20, and K23.

The Escherichia coli K13, K20, and K23 capsular polysaccharide antigens are serologically related. All of these polysaccharides contain ribose and 2-keto-3-deoxyoctonate in equimolar quantities. The K13 and K20 polysaccharides are partially O-acetylated. A comparison of these polysaccharides after O-deacetylation, by nuclear magnetic resonance and permethylation analysis, showed that these polysaccharides contained the disaccharide repeat unit leads to)-beta-ribofuranosyl-(1 leads to 7)-beta-2-keto-3-deoxyoctonate. They differed in the presence and location of an acetyl moiety. The K13 polysaccharide was O-acetylated at C-4 of the 2-keto-3-deoxyoctonate. The K20 antigen was O-acetylated at C-5 of the ribose moiety. The K23 polymer was nonacetylated. The cross-reactivity of these antigens was demonstrated by tandem-crossed immunoelectrophoresis. Antibodies to K23 could be completely absorbed from OK K23 serum by K13, K20, and K23 antigenic extracts. The K13 and K20 antibodies could be completely absorbed from their respective antisera only by homologous antigenic extracts. Monoclonal antibodies were prepared against a protein conjugate of the K13 polysaccharide. Analyses of the reactions of these antibodies with the three polysaccharides suggest that the K13 polysaccharide has at least three antigenic sites, one of which is common to the K13, K20, and K23 polysaccharides.