Chemical Assignment of Structural Isomers of Sulfur-Containing Metabolites in Garlic by Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry.

BACKGROUND: The chemical assignment of metabolites is crucial to understanding the relation between food composition and biological activity. OBJECTIVE: This study was designed to detect and chemically assign sulfur-containing metabolites by using LC-Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) in Allium plants. METHODS: Ultrahigh resolution (>250,000 full width at half-maximum) and mass accuracy (<1 mDa) by FTICR-MS allowed us to distinguish ions containing sulfur isotopes ((32)S and (34)S). RESULTS: Putative 69 S-containing monoisotopic ions (S-ions) were extracted from the metabolome data of onion (Allium cepa), green onion (Allium fistulosum), and garlic (Allium sativum) on the basis of theoretical mass differences between (32)S-ions and their (34)S-substituted counterparts and on the natural abundance of (34)S. Eight S-ions were chemically assigned by using the reference data according to the guidelines of the Metabolomics Standards Initiative. Three ions detected in garlic were assigned as derived from the isomers γ-glutamyl-S-1-propenylcysteine and γ-glutamyl-S-2-propenylcysteine and as S-2-propenylmercaptoglutathione on the basis of differences in key product ions identified in reference tandem MS spectra. CONCLUSION: The ability to discriminate between such geometric isomers will be extremely useful for the chemical assignment of unknown metabolites in MS-based metabolomics.

Proton transfer in a reaction catalyzed by onion lachrymatory factor synthase.

We produced a single deuterated lachrymatory factor (propanthial S-oxide, m/z = 91) in a model reaction system comprising purified alliinase, lachrymatory factor synthase (LFS), and (E)-(+)-S-(1-propenyl)-L-cysteine sulfoxide ((E)-PRENCSO) in D(2)O. Onion LFS reacted with the degraded products of (E)-PRENCSO by alliinase, but not with those of (Z)-PRENCSO. These findings indicate that onion LFS is an (E)-1-propenylsulfenic acid isomerase.

Novel bisthiolane polysulfides from lachrymatory factor synthase-suppressed onion and their in vitro cyclooxygenase-1 inhibitory activity.

Two novel bisthiolane polysulfides (compounds 1 and 2), trivially named thiolanotrisulfide and thiolanotetrasulfide, were isolated from a reaction model of tearless onion (in which lachrymatory factor synthase is suppressed), and the presence of another novel bisthiolane polysulfide (3), trivially named thiolanopentasulfide, was confirmed. On the basis of spectroscopic and mass spectrometric analyses, it was found that these bisthiolane polysulfides were bis(5-hydroxy-3,4-dimethylthiolan-2-yl)-tri/tetra/pentasulfide with the general formulas of C12H22O2S5 (tri-), C12H22O2S6 (tetra-) and C12H22O2S7 (penta-), and they were confirmed to exist in authentic tearless onion juice. Thiolanotrisulfide (1) and thiolanotetrasulfide (2) inhibited cyclooxygenase-1 activity with IC50 values of 720 ± 78 and 464 ± 48 µM respectively, compared with 3282 ± 188 µM for aspirin.

Structures and reactions of compounds involved in pink discolouration of onion.

In "pinking" of onion, E-(+)-S-(1-propenyl)-L-cysteine sulfoxide is first cleaved by alliinase to yield colour developers (CDs), which react with amino acids, such as valine, to form pigment precursors (PPs). The PPs react with naturally occurring carbonyls (NOCs) to form pigments. By inducing a PP from previously isolated cepathiolanes and L-valine, it was confirmed that cepathiolanes constitute at least a part of the CDs. From the PP and formaldehyde as a NOC, two colourless and two pink compounds were derived. The structure of one of the colourless compounds was established as 2-(2-(1-(1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-yl)methyl-3,4-dimethyl-1H-pyrrol-1-yl)-3-methylbutanoic acid. The structures of the other colourless compound and the pink pigments were predicted based on their molecular formula and the MS(n) spectral data. A trimeric pigment structure was predicted for one of the pink pigments, which was believed to be the first to be reported in the literature. With these, a new reaction scheme for "pinking" of onion is proposed.

Structure and bioactivity of thiosulfinates resulting from suppression of lachrymatory factor synthase in onion.

In normal onion (Allium cepa), trans-S-1-propenyl-L-cysteine sulfoxide is transformed via 1-propenesulfenic acid into propanethial S-oxide, a lachrymatory factor, through successive reactions catalyzed by alliinase and lachrymatory factor synthase (LFS). A recent report showed that suppression of the LFS activity caused a dramatic increase in thiosulfinates previously reported as "zwiebelane isomers". After purification by recycle high-performance liquid chromatography and subsequent analyses, we established the planar structure of the putative "zwiebelane isomers" as S-3,4-dimethyl-5-hydroxythiolane-2-yl 1-propenethiosulfinate, in which two of the three molecules of 1-propenesulfenic acid involved in the formation gave the thiolane backbone, and the third molecule gave the thiosulfinate structure. Of at least three stereoisomers observed, one in the (2'R,3'R,4'R,5'R)-configuration was collected as an isolated fraction, and the other isomers were collected as a combined fraction because spontaneous tautomerization prevented further purification. Both fractions showed inhibitory activities against cyclooxygenase-1 and α-glucosidase in vitro.

Silencing onion lachrymatory factor synthase causes a significant change in the sulfur secondary metabolite profile.

Through a single genetic transformation in onion (Allium cepa), a crop recalcitrant to genetic transformation, we suppressed the lachrymatory factor synthase gene using RNA interference silencing in six plants. This reduced lachrymatory synthase activity by up to 1,544-fold, so that when wounded the onions produced significantly reduced levels of tear-inducing lachrymatory factor. We then confirmed, through a novel colorimetric assay, that this silencing had shifted the trans-S-1-propenyl-l-cysteine sulfoxide breakdown pathway so that more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of this raised thiosulfinate level was a marked increase in the downstream production of a nonenzymatically produced zwiebelane isomer and other volatile sulfur compounds, di-1-propenyl disulfide and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophene, which had previously been reported in trace amounts or had not been detected in onion. The consequences of this dramatic simultaneous down- and up-regulation of secondary sulfur products on the health and flavor attributes of the onion are discussed.