Jürgen Conrad

Formation Pathways for Lysine-Arginine Cross-links Derived from Hexoses and Pentoses by Maillard Processes UNRAVELING THE STRUCTURE OF A PENTOSIDINE PRECURSOR

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates and their formation pathways are largely unknown. Synthesis and unequivocal structural characterization are reported for the lysine-arginine cross-linksN 6-{2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-5-[(2S,3R)-2,3,4- trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene}-l-lysinate (DOGDIC 12),N 6-{2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-5-[(2S)-2,3-dihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene}-l-lysinate (DOPDIC 13), and 6-((6S)-2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl] amino}-6-hydroxy-5,6,7,7a-tetrahydro-4H-imidazo[4,5-b] pyridin-4-yl)-l-norleucinate (pentosinane 10). For these compounds, as well as for glucosepane 9 and pentosidine 11, the formation pathways could be established by starting from native carbohydrates, Amadori products, and 3-deoxyosones, respectively. Pentosinane 10 was unequivocally proven to be an important precursor of pentosidine 11, which is a well established fluorescent indicator for advanced glycation processesin vivo. The Amadori products are shown to be the pivots in the formation of the various cross-links 9–13. The bicyclic structures 9–11 are directly derived from aminoketoses, whereas 12 and 13 stem from reaction with the 3-deoxyosones. All products 9–13 were identified and quantified from incubations of bovine serum albumin with the respective 3-deoxyosone or carbohydrate. From these results it seems fully justified to expect both glucosepane 9 and DOGDIC 12 to constitute important in vivo cross-links.

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.

Elution order of quercetin glycosides from apple pomace extracts on a new HPLC stationary phase with hydrophilic endcapping

A stationary phase with hydrophilic endcapping was used for the separation of seven quercetin (Q) glycosides by high-performance liquid chromatography. The elution order was established as Q 3-rutinoside, Q 3-galactoside, Q 3-glucoside, Q 3-xyloside, Q 3-arabinopyranoside, Q 3-arabinofuranoside, and Q 3-rhamnoside. Since a recent study had revealed that a commercially available reference compound (Q 3-arap) was erroneously labeled as avicularin (Q 3-araf), standards of the two isomers of Q 3-ara were characterized by 1 D and 2D NMR spectroscopy, and by optical rotation. The predominant Q glycosides detected in apple pomace were Q 3-gal, Q 3-araf, Q 3-rha and Q 3-xyl, whereas considerably lower amounts of Q 3-glc and Q 3-arap were found. Total content of Q glycosides was approximately 900 mg/kg on a dry matter basis, indicating that apple pomace is a good source of natural antioxidants.

Two triterpene esters from Terminalia macroptera bark

Abstract Investigation of the bark from Terminalia macroptera led to the isolation of two novel esterified triterpenes, identified as 23-galloylarjunolic acid and its β- d -glucopyranosyl ester. In addition terminolic acid, arjunic acid, arjungenin, arjunglucoside 1 , sericic acid and sericoside were isolated and tested in a number of bioassays (antibacterial, antifungal and anthelminthic activity as well as haemolytic property).

Laccase-Catalyzed Domino Reactions between Hydroquinones and Cyclic 1,3-Dicarbonyls for the Regioselective Synthesis of Substituted p-Benzoquinones

Highly substituted p-benzoquinones were obtained in yields ranging from 39% to 98% by laccase-catalyzed domino reactions between hydroquinones and cyclic 1,3-dicarbonyls using aerial oxygen as the oxidant. In almost all reactions bis-adducts with two adjacent 1,3-dicarbonyl substituents on the quinone moiety were formed selectively. The transformations can be regarded as domino oxidation/1,4-addition/oxidation/1,4-addition/oxidation processes. With unsubstituted hydroquinone as the substrate 2,3-disubstituted p-benzoquinones were isolated. Bis-adducts were also formed exclusively upon reaction with monosubstituted hydroquinones. In almost all cases the 2,3,5-trisubstituted p-benzoquinones were obtained. When 2,3-disubstituted hydroquinones were employed as starting materials the 2,3,5,6-tetrasubstituted p-benzoquinones were isolated. The unambiguous structure elucidation of all products has been achieved by NMR spectroscopic methods including spin pattern analysis of the long-range coupled C═O carbons and (13)C satellites analysis in (1)H NMR spectra.

Formation pathways for lysine–arginine cross-links derived from hexoses and pentoses by Maillard processes

Abstract α-Dicarbonyl compounds are crucial intermediates in the cross-linking of proteins by reducing sugars in the course of the Maillard reaction. The novel dideoxyosones N6-(2,3-dihydroxy-5,6-dioxohexyl)lysine (9a,b) and N6-(2-hydroxy-4,5-dioxopentyl)lysine (12) were unequivocally identified via their triazine derivatives and these α-diketo compounds established as precursors of the major in vivo cross-links glucosepane 11 and pentosidine 16, respectively. This new dideoxyosone class is formed in high yield in the model incubations and the underlying reaction sequence will improve our understanding of protein cross-linking and of Maillard chemistry in general.

Oxidative Dimerization of (E)- and (Z)-2-Propenylsesamol with O2 in the Presence and Absence of Laccases and Other Catalysts: Selective Formation of Carpanones and Benzopyrans under Different Reaction Conditions

The oxidative dimerization of 2-propenylsesamol to carpanone with O(2) as the oxidant, which probably proceeds as a domino phenol oxidation/anti-β,β-radical coupling/intramolecular hetero Diels-Alder reaction, can be efficiently catalyzed by laccases. Experiments with laccases and other catalysts like a Co(salen) type catalyst and PdCl(2) clearly demonstrate that the diastereoselectivity of the carpanone formation does not depend on the catalyst but on the double-bond geometry of the substrate. With (E)-2-propenylsesamol as the substrate, carpanone and a so far unknown carpanone diastereoisomer are formed in a 9:1 ratio. When (Z)-2-propenylsesamol is used as starting material, carpanone is accompanied by two carpanone diastereoisomers unknown so far in a 5:1:4 ratio. All three carpanone diastereoisomers have been separated by HPLC, and their structures have been elucidated unambiguously by NMR spectroscopy, DFT calculations, and spin work analysis. When the oxidation of 2-propenylsesamol with O(2) is performed in the absence of any catalyst two diastereoisomeric benzopyrans are formed, probably as the result of a domino oxidation/intermolecular hetero Diels-Alder reaction. Under these conditions, carpanone is formed in trace amounts only.

Identification and quantification of flavonoids and ellagic acid derivatives in therapeutically important Drosera species by LC–DAD, LC–NMR, NMR, and LC–MS

AbstractDroserae herba is a drug commonly used for treatment of convulsive or whooping cough since the seventeenth century. Because of the contribution of flavonoids and ellagic acid derivatives to the therapeutic activity of Droserae herba, an LC–DAD method has been developed for quantification of these analytes in four Drosera species used in medicine (Drosera anglica, D. intermedia, D. madagascariensis, and D. rotundifolia). During elaboration of the method 13 compounds, including three substances not previously described for Drosera species, were detected and unambiguously identified by means of extensive LC–MS and LC–NMR experiments and by off-line heteronuclear 2D NMR after targeted isolation. The most prominent component of D. rotundifolia and D. anglica, 2″-O-galloylhyperoside, with myricetin-3-O-β-glucopyranoside and kaempferol-3-O-(2″-O-galloyl)-β-galactopyranoside, were identified for the very first time in this genus. The LC–DAD method for quantification was thoroughly validated, and enables, for the first time, separation and precise analysis of these analytes in Droserae herba. Simple sample preparation and use of a narrow-bore column guarantee low cost and simplicity of the suggested system, which is excellently suited to quality control of the drug or herbal medicinal products containing this drug. Figure2″-O-galloylhyperoside - a major compound in Drosera anglica and Drosera rotundifolia

Sesquiterpene lactones from glandular trichomes of Viguiera radula (Heliantheae; Asteraceae).

In addition to known compounds, the floral parts of Viguiera radula afforded two new sesquiterpene lactones. All compounds were detected in glandular trichomes, which were micromechanically collected from the anther appendages and analyzed by HPLC. Structure identification was performed by NMR and MS techniques.

Investigations into the Phenolic Constituents of Dog's Mercury (Mercurialis perennis L.) by LC-MS/MS and GC-MS analyses

INTRODUCTION Dogs mercury (Mercurialis perennis L.) is a traditional European medicinal plant considered as a rich source of bioactive natural products. Yet phytochemical data of the plant are scant. OBJECTIVE This study aimed to identify the hydrophilic phenolic constituents from M. perennis by aqueous and hydroalcoholic extraction. METHODOLOGY Extracts of herbal parts were investigated in-depth by HPLC(DAD)-MS/MS and GC/MS analyses. In addition, a novel compound was isolated and fully characterised by 1- and 2D-NMR experiments. RESULTS Several conjugates of caffeic, p-coumaric and ferulic acids together with glucaric or 2-hydroxyglutaric acids (depsides) were detected in the aqueous extracts from aerial plant parts by use of LC-MS/MS techniques as well UV-spectral data. By implementation of preparative chromatography on polyamide pretreated with formic acid followed by vacuum liquid chromatography on reversed-phase C(18) -silica, one of the predominant depsides was isolated as a pure compound. The NMR spectra ((1) H and (13) C NMR) together with 2D-hetereonuclear multiple bond correlation NMR experiments (gHMBC and gHSQC) and chiral GC investigation, allowed identification of this compound as (-)-(E)-caffeoyl-2-(R)-oxoglutarate. This structure was additionally supported by GC/MS data after silylation and methylation reactions. The hydroalcoholic extract from aerial parts was separated by solvent partition between ethyl acetate and n-butanol. The latter fraction (n-butanol) yielded a mixture of mono- and oligo-glycosides of kaempferol and quercetin, all of them being assigned by LC-MS/MS. CONCLUSIONS The present investigation constitutes the first comprehensive report on the hydrophilic constituents of the rarely studied plant Mercurialis and thus completes the phytochemical knowledge on M. perennis.