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Cytokine-induced neutrophil chemoattractant (CINC/Gro) is a chemotactic factor for neutrophils in the rat and a member of the CXC chemokine family. The refined three-dimensional structure of CINC/Gro was derived with the aid of heteronuclear magnetic resonance spectroscopy and a hybrid method of distance geometry and simulated annealing. The backbone atomic r.m.s. differences for 19 structures about the mean coordinates for residues 7 to 70 are 0.69+/-0.15 A for the dimer and 0.56+/-0.13 A for the monomer. The N terminal region containing an ELR sequence, an essential motif for chemotactic activity, is disordered in solution, as in other CXC chemokines. The dimer structure consists of a six-stranded anti-parallel beta sheet with two C-terminal alpha helices on the beta sheet. The overall dimer structure of CINC/Gro is similar to that of human MGSA, though the backbone r.m.s.d.s between CINC/Gro and the two MGSA structures were high (1.81 and 2.34 A for the monomer) in spite of the high sequence homology (67%). The major difference resides in the relative position of the C-terminal alpha helix with respect to the beta sheet. This results in a difference of interhelical distances in the dimer: wider (15 A) in CINC/Gro, as in IL-8, than in MGSA (11.7 and 10 A).

CS-670, (±)-2-[4-(2-oxocyclohexylidenemethyl) phenyl] propionic acid, is a novel derivative of 2-arylpropionic acid non-steroidal anti-inflammatory drugs (profen NSAIDs). The major urinary metabolite of this drug from dogs was isolated and its chemical structure was determined by MS and NMR spectroscopy. The metabolite was identified as a taurine conjugate of the trans-OH form (trans-OH-taurine) which was first generated by stereoselective reduction of the double bond and the carbonyl function of the CS-670 molecule. The taurine conjugate was excreted in urine as the main metabolite, regardless of the optical configuration of CS-670 administered [(2R)-enantiomer : 47.2% of the dose, (2S)-enantiomer : 70.9% of the dose]. The trans-OH-taurine was hydrolyzed by refluxing it in 6N HCl without racemization. The released trans-OH was derivatized to diastereoamides with (+)-(R)-1-(1-naphthyl) ethylamine to examine the stereochemical properties of the 2-arylpropionic acid side chain. It was found that the configuration of the 2-carbon of the trans-OH-taurine was almost entirely (S). As the CoA thioesters are obligate intermediates for amino acid conjugation, the results suggest that the (2S)-enantiomer of the trans-OH metabolite serves as a substrate for canine acyl CoA ligase (EC 6.2.1.3) as well as the (2R)-enantiomer, but only the CoA thioester with a (2S)-configuration is a substrate for taurine N-acyl transferase. It is interesting to note that these results are not consistent with the chiral inversion mechanism by which the (2R)-enantiomers of profen NSAIDs are stereospecifically converted to CoA thioester intermediates.

The glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen was treated with pepsin and glycopeptidase A to release asparagine-linked oligosaccharides. The reducing ends of the oligosaccharides were aminated with the fluorescent reagent 2-aminopyridine. The oligosaccharide derivatives were purified by gel permeation chromatography and reversed-phase HPLC. Their structures were determined by sequential exoglycosidase digestion and 500 MHz 1H-NMR spectroscopy. Four oligosaccharide structures, A, B, C, and D, were identified as the xylose-containing complex-type. They were present at a molar ratio of 8:1:6:1. By amino acid sequence analyses of the tryptic peptides, Asn-170 and Asn-333 of Cry j I were found to carry asparagine-linked oligosaccharides. [formula: see text]

The structures of matlystatins, novel type IV collagenase inhibitors isolated from Actinomadura atramentaria, have been determined by a systematic application of homo- and heteronuclear 2D NMR and FAB-MS/MS techniques. Their structures were characterized by the presence of piperazic acid and hydroxamic acid moieties, structural motifs often seen in protease inhibitors.

Synthesis of 5-epi-trehazolin (trehalostatin) (2) was accomplished via the crucial intermediate, epoxide (6 alpha), from D-glucose. The stereochemistry of epoxide (6 alpha) and its isomer (6 beta) which were obtained from Sharpless epoxidation, was determined by comparison between the NMR relaxation times of relevant protons.

Thiomarinol, an antimicrobial antibiotic, was isolated from the culture broth of a marine bacterium, Alteromonas rava sp. nov. SANK 73390. Its structure was deduced as a hybrid composed of a pseudomonic acid analogue and holothin by NMR spectral analysis and chemical degradation. Antimicrobial activity against Gram-positive and Gram-negative bacteria of thiomarinol was stronger than both of pseudomonic acids and pyrrothine antibiotics.

A new inhibitor of mammalian adenosine triphosphatases, designated aquastatin A, has been isolated from a fungus identified as Fusarium aquaeductuum. The structure of this compound has been determined by MS and NMR analyses. It inhibits Na+/K(+)-ATPase with an IC50 value of 7.1 microM, and H+/K(+)-ATPase with an apparent IC50 value of 6.2 microM.

Trehalamine, (3aR, 4R, 5S, 6S, 6aS)-2-amino-4-(hydroxymethyl)-3a, 5, 6, 6a-tetrahydro-4H-cyclopent[d]oxazole-4, 5, 6-triol (1) and D-glucose were obtained by acid hydrolysis of trehazolin (3), a trehalase inhibitor produced by actinomycetes. More vigorous hydrolytic treatment of trehazolin afforded an aminocyclitol, (1R, 2S, 3R, 4S, 5R)-5-ammo-l-(hydroxymethyl)cyclopentane-1, 2, 3, 4-tetraol (2). Trehalamine, the aglycon of trehazolin, was also found in the culture broths of two trehazolin producing strains, Micromonospora sp. SANK 62390 and Amycolatopsis sp. SANK 60791. These trehazolin related compounds trehalamine (1) and 2 were poor inhibitors of trehalase (1; IC50 1.8 × 10-4 M, 2; > 5.0 × 10-4 M). On the other hand they inhibited more potently rat intestinal sucrase (1; IC50 6.8 × 10-5M) and sweet almond β-glucosidase (2; IC50 5.6× 10-6M) than trehazolin.

The structure of a novel herbicide, hydantocidin isolated from the fermentation broth of Streptomyces hygroscopics SANK 63584, was determined by the combined analysis of MS and 1H NMR spectra. Hydantocidin is a novel spiro compound containing a subofuranoid ring, at the anomeric position of which a hydantoin ring is used such that the C(1)–N(1) linkage is β. The relative configuration and the conformation in solution was determined by quantitative analysis of the NOE spectra and T1 values. In CD3OD and [2H6]DMSO (dimethyl sulphoxide) solutions, the ribofuranose moiety of hydantocidin was found to be fixed in a C2-endo conformation, probably due to the rigidity of the spiro structure and hydrogen bonding between 3-OH and the carbonyl group at C-4′.

The chemical structure of quercetin 3-glucosyl(1 ⇒ 2)gentiobioside ,isolated from the violet blue flowers of primura polyantha was determined to be quercetin 3-O(β-D-glucopyranosyl(1→2)-O-β-D-glucopyranosyl(1→6))-β―D-glucopyranoside unambiguously based on the spectroscopic data

With proton nuclear magnetic resonance spectroscopy at 22{degree}C and pD 4.5, individual exchange rates in the range from 2 {times} 10{sup {minus}5} to 1 {times} 10{sup {minus}1} min{sup {minus}1} were observed for 23 amide protons in recombinant desulfatohirudin. The remaining 38 backbone amide protons exchange more rapidly than 1 {times} 10{sup {minus}1} min{sup {minus}1}. All 23 slowly exchanging protons are located in the polypeptide segment from residue 4 to residue 42, which forms a well-defined globular domain. Three different breathing modes of this molecular region are manifested in the exchange data, which appear to be correlated with the location of the three disulfide bonds. Chemical shift changes larger than 0.15 ppm between pH 2.5 and pH 5.0 arising from through-space interactions with carboxyl groups were observed for seven backbone amide protons. Two of these shifts can be explained by hydrogen bonds in the core of the protein, Gly 25 NH-Glu 43 O{sup {epsilon}} and Ser 32 NH-Asp 33 O{sup {delta}}, and two others by intraresidual NH-O{sup {epsilon}} interactions in Glu 61 and Glu 62. The remaining three pH shifts for Glu 35, Cys 39, and Ile 59 imply the existence of transient interactions between the molecular core and the flexiblemore » C-terminal segment 49-65, which have so far not been characterized by nuclear Overhauser effects or other conformational constraints.« less

Two active metabolites of ML-236B, 4β, 6α-dihydroxy ML-236B (M3, 2), and 3β-hydroxy ML-236B (M4, 3), were isolated from the urine of ML-236B-treated dogs. The structures of M3 and M4 were elucidated on the basis of spectral data and confirmed by X-ray analysis.

Proton nuclear magnetic resonance (1H-NMR) relaxation of saframycin A in C6D6 has been studied. Quantitative analysis of spin-lattice relaxation times and nuclear Overhauser effect (NOE) factors allowed the determination of the α-axial orientation of the nitrile group at C21, (previously unknown). The conformation of saframycin A in C6D6, which was deduced from the relaxation data, was compared with that of saframycin C in the crystal state.

The three-dimensional structure of recombinant desulfatohirudin in aqueous solution was determined by {sup 1}H nuclear magnetic resonance at 600 Mhz and distance geometry calculations with the program DISMAN. The input for the structure calculations was prepared on the basis of complete sequence-specific resonance assignments at pH 4.5 and 22{degree}C and consisted of 425 distance constraints from nuclear Overhauser enhancements and 159 supplementary constraints from spin-spin coupling constants and from the identification of intramolecular hydrogen bonds. Residues 3-30 and 37-48 form a molecular core with two antiparallel {beta}-sheets and several well-defined turns. The three disulfide bonds 6-14, 16-28, and 22-39 were identified by NMR. In contrast to this well-defined molecular core, with an average root mean square distance for the polypeptide backbone of 0.8 {angstrom} for a group of nine DISMAN solutions, no preferred conformation was found for the C-terminal segment 49-65, and a loop consisting of residues 31-36 is not uniquely constrained by the NMR data either. These structural properties of recombinant desulfatohirudin coincide closely with the previously described solution conformation of natural hirudin, but the presence of localized differences is indicated by chemical shift differences for residues Asp 5, Ser 9, Leu 15, Asp 53, Gly 54, and Aspmore » 55.« less

The isolation and structural elucidation of plaunolide, a new furanoid diterpene, from Croton sublyratus are described.