Lachrymatory (Z)-butanethial S-oxide along with several 1-butenyl thiosulfinates was detected by DART mass spectrometry upon cutting Allium siculum , a popular ornamental Allium species used in some cultures as a spice. (Z)-Butanethial S-oxide isolated from the plant was shown to be identical to a synthetic sample. Its likely precursor, (R(S),R(C),E)-S-(1-butenyl)cysteine S-oxide (homoisoalliin), was isolated from homogenates of A. siculum, and a closely related species Allium tripedale , and fully characterized. Through use of LC-MS, a series of related gamma-glutamyl derivatives were tentatively identified in A. siculum and A. tripedale homogenates, including gamma-glutamyl-(E)-S-(1-butenyl)cysteine and its S-oxide, gamma-glutamyl-S-butylcysteine and its S-oxide, and gamma-glutamyl-S-methylcysteine and its S-oxide. Because compounds containing the 1-butenyl group have not been previously identified in genus Allium species, this work extends the range of known Allium sulfur compounds. The general applicability of DART mass spectrometry in identifying naturally occurring, thermally fragile thial S-oxides and thiosulfinates is illustrated with onion, Allium cepa , as well as a plant from a different genus, Petiveria alliacea .

The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.

We have characterized the primary structures of the predominant N-linked oligosaccharides on cellobiohydrolase I from the filamentous fungus Trichoderma reesei RUTC30. Different enzymatic and chromatographic techniques were used to analyze six oligosaccharides. The combined data showed that the fungal carbohydrates have a core structure that is identical to the mammalian N-linked core. In the bulk of the N-glycans, the u-1,3 arm is extended with two mannoses and a glucose, suggesting incomplete processing of the oligosaccharides in the endoplasmic reticulum. The u-1,6 arm shows a remarkable heterogeneity: in addition to a-1,2-Man and a-1,6-Man, the presence of a terminal mannose a-1,6-phosphodiester was observed. This latter substituent has not been characterized before on mannosidase-processed N-glycan and its function and synthesis pathway are entirely unknown. The predominant Nglycans on cellobiohydrolase I can be represented as follows : GlcMan,GIcNAc,, GlcMan,GlcNAc,, Man,GlcNAc,, ManPGlcMan,GlcNAc,, GlcMan,GlcNAc, and Man,GlcNAc,.Keywords: cellobiohydrolase I ; fungus ; N-glycans ; NMR ; Trichoderma reesei.Trichoderma reesei is a filamentous fungus capable of secreting high amounts of cellulose-degrading enzymes (up to 40 g/l by manipulated strains). This remarkable secretory capacity led to the idea to use this organism as a candidate host for the production of heterologous proteins [I]. However, it is not clear whether T. reesei can be used for production of pharmaceutically important glycoproteins since post-translational modifications by the fungus, such as glycosylation, could be a major problem for application. Oligosaccharides lacking sialic acid as terminal sugar residues are easily recognized by lectins circulating in blood and present on cells of the reticulo-endothelial system. This recognition leads to fast clearance, which prevents the therapeutic proteins reaching their target organs [2, 31. Furthermore, it has been demonstrated that foreign glycosyl structures often elicit an antigenic response [4].Only few data concerning glycosylation in filamentous fungi are available. They indicate high-mannose-type glycans of limited sizes compared with these synthesized by yeast [S-121. The identification by Salovuori et al. 1121 of Man,GlcNAc, and Man,GlcNAc, as the major N-glycans on 7: reesei cellobiohydrolase I (CBH I) suggested a possible analogy between fungal and mammalian oligosaccharide structures. For identical primary structures, in vitro and eventually in vivo extension of oligosaccharides on proteins secreted from this fungus to the complex type should be possible.Correspondence to R. Contreras Enzyme. a-mannosidase (EC 3.2.1.24).During the eukaryotic secretion process, proteins receive in the endoplasmic reticulum a common precursor oligosaccharide, namely Glc,Man,GlcNAc,. This precursor is transferred by an oligosaccharyl transferase to asparagine residues in the consensus sequence Asn-Xaa-(Ser or Thr), with Xaa representing any amino acid except proline. A battery of glycosidases and gl...

ClinicalTrials.gov identifier NCT00378690.

The cyclohexane nucleosides with a 1,4-relationship between nucleoside base and hydroxymethyl moiety were synthesized using a conjugated addition reaction of the nucleobases to ethyl 1,3-cyclohexadiene-1-carboxylate and hydroboration of the cyclohexenyl precursor. The lack of antiviral activity of the compounds was correlated with the conformation of these nucleosides as deduced from NMR and X-ray analysis.

Two novel, structurally unusual cysteine derivatives were isolated from the bulbs of Allium stipitatum (Allium subg. Melanocrommyum) and shown to be S-(2-pyridyl)cysteine N-oxide and S-(2-pyridyl)glutathione N-oxide. The former compound is the first example of a naturally occurring alliinase substrate that contains an N-oxide functionality instead of the S-oxide group. In addition, S-methylcysteine S-oxide (methiin) and S-(methylthiomethyl)cysteine 4-oxide (marasmin) were found in the bulbs. Presented data suggest that the previously reported identification of S-(2-pyridyl)cysteine S-oxide was most likely erroneous. The alliinase-mediated formation of pyridyl-containing compounds following disruption of A. stipitatum bulbs was studied by a combination of HPLC-MS, HPLC-PDA, DART-MS, and NMR techniques. It was found that no pyridyl-containing thiosulfinates are present in homogenized bulbs in detectable quantities. Instead, various pyridine N-oxide derivatives are formed, including N-hydroxypyridine-2(1H)-thione (pyrithione), 2-(methyldithio)pyridine N-oxide, 2-[(methylthio)methyldithio]pyridine N-oxide, di(2-pyridyl) disulfide N-oxide, and di(2-pyridyl) disulfide N,N'-dioxide. This represents the first report of pyrithione formation as a natural product.

Two pulse sequences applicable to the determination of relative signs of coupling constants, gHSQC-RELAY(P) and gHSQC-RELAY(D), were developed and tested. These sequences are suitable for determination of relative signs of long-range coupling constants (<2 Hz) between two heteronuclei of low abundance (such as (29)Si and (13)C), and are applicable even to cases in which one of the heteronuclei ((29)Si) does not exhibit coupling with some of the detected protons ((1)H). The two sequences differ in the manner in which they suppress undesirable homonuclear coherence transfers. Each of the sequences can be combined with an isotope filter for better suppression of the centerlines arising from more abundant NMR-inactive isotopes. The sequences were tested on ethoxytrimethylsilane and (E)-(buta-1,3-dienyloxy)trimethylsilane, and we conclude that (2)J((29)Si-O-(13)C) is positive while (3)J((29)Si-O-C-(13)C) is negative in both compounds.

Mammalian serine racemase (SR) is a pyridoxal-5'-phosphate (PLP) dependent enzyme responsible for the biosynthesis of the neurotransmitter D-serine, which activates N-methyl-D-aspartate (NMDA) receptors in the CNS. Aberrant regulation of NMDA receptor signaling has been implicated in a variety of neuropathologies, and inhibitors of SR would therefore be a worthwhile tool for further investigation or treatment of such conditions. Here, we identify a series of small aliphatic hydroxamic acids (HAs) that act as potent SR inhibitors. However, specificity studies showed that some of these HAs can act as nonspecific inhibitors of PLP-dependent enzymes. We employed NMR, MS, and UV/vis spectroscopic techniques to reveal that the nonspecific effect is likely due to irreversible interaction of the HA moiety with PLP to form aldoxime species. We also characterize L-aspartic acid beta-hydroxamate as a competitive and selective SR inhibitor that could be used as a scaffold for further inhibitor development.