Reinhard Meusinger

Triazole bridge: disulfide-bond replacement by ruthenium-catalyzed formation of 1,5-disubstituted 1,2,3-triazoles.

About one fourth of the peptidic macromolecular structures deposited in the protein data base (PDB) contain at least one disulfide bridge. In nature, disulfide bonds are formed in a milieu where oxidizing conditions prevail, for example, on the cell surface or in the extracellular matrix. Many proteins benefit from disulfide contributions to their conformational stability. In particular, the defined tertiary folding of oligopeptides smaller than 30 residues essentially relies on macrocyclization through the cystine motif because of the restricted number of noncovalent intramolecular interactions available. Moreover, formation of the disulfide pattern results in structural rigidity of the peptidic framework, as for example, in the family of cystine knot miniproteins, leading to conformationally constrained scaffolds with extraordinary thermal stability and resistance against proteolytic degradation. Hence, the discovery and development of disulfidebridged peptides suitable for diagnostic and therapeutic applications remains a field of intense research. The in-vitro generation of disulfide bonds in peptides is usually achieved post-synthetically and mediated by DMSO, air oxygen, or other oxidizing agents. Although this reaction step can be achieved under relatively mild conditions in solution, it remains one of the most demanding obstacles towards high-yield peptide synthesis, especially for disulfiderich species in which the controlled regiospecific formation of several disulfide bonds is not trivial to control. In addition, to suppress unwanted intermolecular reactions of the thiol groups of individual peptides, oxidative folding usually has to be conducted in highly diluted solutions. In spite of the use of gluthathione-based redox buffers, polymer-supported oxidation systems, macrocyclization on the solid support and/or orthogonal protecting groups, control over the topology of the disulfide bridges formed is still a challenge. 5] In view of these difficulties and to improve the redox stability of bridged peptides, several routes towards synthetic disulfide surrogates have been developed. Straightforward approaches usually employ thioether, olefin, or alkane-based isosters. However, cystathione bridges require multiple synthetic steps and careful choice of orthogonal protection, and dicarba bridges give cis/trans isomers during ring-closing metathesis (RCM). Only an additional purification step or the subsequent palladium-catalyzed hydrogenation of the unsaturated species to the corresponding alkane leads to a construct with defined configuration. In 2004, Meldal et al. described the utility of copper(I)catalyzed azide–alkyne cycloaddition (CuAAC) for a triazole-based disulfide replacement. Owing to the compelling characteristics of this prototypic “click” reaction, it has been extensively applied in peptide chemistry exploiting the almost perfect orthogonality to side-chain reactivities. The introduction of 1,4-disubstituted 1,2,3-triazoles into peptides has also been used to mimic and rigidify conformations of the amide backbone. Moreover, a variety of examples of CuAAC-based macrocyclizations of peptides in solution and on solid supports has been reported. Using the same azideand alkyne-functionalized buidling blocks, 1,5-disubstituted 1,2,3-triazoles can be generated in the ruthenium(II)-catalyzed variant (RuAAC) of the CuAAC. This reaction expands the range of peptidomimetic structures selectively accessible from the same precursor and having different biological activities governed by the architecture of the incorporated triazole. To our knowledge, 1,5-disubstitiuted 1,2,3-triazoles have not been taken into consideration as disulfide mimics to date. Herein, we report the facile introduction of 1,4and 1,5disubstituted 1,2,3-triazoles into a monocyclic variant of the sunflower trypsin inhibitor-I (SFTI-1[1,14], 1; Figure 1) and show that the macrocyclic peptidomimeticum 2 with the “1,5” substitution pattern retains nearly full biological activity in contrast to the “1,4” variants 3 and 4. The choice of 1 as the model peptide for the investigation of triazole-based disulfide replacements had several reasons. SFTI-1 is a small, though very potent, inhibitor of trypsin. Therefore, the influence of different modes of macrocyclization on the bioactivity of the corresponding synthetic variant can be routinely examined by serine protease inhibition assays. [*] M. Empting, Dr. O. Avrutina, Dr. R. Meusinger, S. Fabritz, M. Reinwarth, Prof. Dr. H. Kolmar Clemens-Sch pf-Institut f r Organische Chemie und Biochemie Technische Universit t Darmstadt Petersenstrasse 22, 64287 Darmstadt (Germany) Fax: (+49)6151-16-5399 E-mail: kolmar@biochemie-tud.de Homepage: http://www.chemie.tu-darmstadt.de/kolmar

Storage and release of hydrogen cyanide in a chelicerate (Oribatula tibialis)

Significance Hydrogen cyanide (HCN) is highly volatile and among the most toxic substances known, being lethal to humans at a dosage of 1–2 mg/kg body weight. HCN blocks the respiratory chain and prevents aerobic organisms from using oxygen. In nature, HCN is produced by numerous plants that store it mainly as glycosides. Among animals, cyanogenesis is a defensive strategy that has seemed restricted to a few mandibulate arthropods (certain insects, millipedes, and centipedes), which evolved ways to store HCN in the form of stable and less volatile molecules. We found an instance of cyanogenesis in the phylogenetically distant group Chelicerata (“spider-like” arthropods), involving an aromatic ester for stable HCN storage and two degradation pathways that release HCN. Cyanogenesis denotes a chemical defensive strategy where hydrogen cyanide (HCN, hydrocyanic or prussic acid) is produced, stored, and released toward an attacking enemy. The high toxicity and volatility of HCN requires both chemical stabilization for storage and prevention of accidental self-poisoning. The few known cyanogenic animals are exclusively mandibulate arthropods (certain myriapods and insects) that store HCN as cyanogenic glycosides, lipids, or cyanohydrins. Here, we show that cyanogenesis has also evolved in the speciose Chelicerata. The oribatid mite Oribatula tibialis uses the cyanogenic aromatic ester mandelonitrile hexanoate (MNH) for HCN storage, which degrades via two different pathways, both of which release HCN. MNH is emitted from exocrine opisthonotal oil glands, which are potent organs for chemical defense in most oribatid mites.

Characterization and analysis of structural isomers of dimethyl methoxypyrazines in cork stoppers and ladybugs (Harmonia axyridis and Coccinella septempunctata)

The three constitutional isomers of dimethyl-substituted methoxypyrazines: 3,5-dimethyl-2-methoxypyrazine 1; 2,5-dimethyl-3-methoxypyrazine 2; and 2,3-dimethyl-5-methoxypyrazine 3 are potent flavor compounds with similar mass spectrometric, gas chromatographic, and nuclear magnetic resonance spectroscopic behavior. Therefore, unambiguous analytical determination is critical, particularly in complex matrices. The unequivocal identification of 1–3 could be achieved by homo- and heteronuclear NMR correlation experiments. The observed mass fragmentation for 1–3 is proposed and discussed, benefitting from synthesized partially deuterated 1 and 2. On common polar and apolar stationary phases used in gas chromatography (GC) 1 and 2 show similar behavior whereas 3 can be separated. In our focus on off-flavor analysis with respect to wine aroma, 1 has been described as a “moldy” off-flavor compound in cork and 2 as a constituent in Harmonia axyridis contributing to the so-called “ladybug taint,” whereas 3 has not yet been described as a constituent of wine aroma. A successful separation of 1 and 2 could be achieved on octakis-(2,3-di-O-pentyl-6-O-methyl)-γ-cyclodextrin as stationary phase in GC. Applying heart-cut multidimensional GC analysis with tandem mass spectrometric detection we could confirm the presence of 1 as a “moldy” off-flavor compound in cork. However, in the case of Harmonia axyridis, a previous identification of 2 has to be reconsidered. In our experiments we identified the constitutional isomer 1, which was also found in Coccinella septempunctata, another species discussed with respect to the “ladybug taint.” The analysis of such structurally related compounds is a demonstrative example for the importance of a chromatographic separation, as mass spectrometric data by itself could not guarantee the unequivocal identification.

Nuclear Overhauser effect challenge.

We would like to invite you to participate in the Analytical Challenge, a series of puzzles to entertain and challenge our readers. This special ABC feature has established itself as a truly unique quiz series, with a new scientific puzzle published every other month. Readers can access the complete collection of published problems with their solutions on the Analytical and Bioanalytical Chemistry homepage at http://www.springer.com/abc. Test your knowledge and tease your wits in diverse areas of analytical and bioanalytical chemistry by viewing this collection. In the present challenge the nuclear Overhauser effect is the topic. And please note that there is a prize to be won (a Springer book of your choice up to a value of € 75). Please read on...

NMR hide-and-seek challenge

We would like to invite you to participate in the Analytical Challenge, a series of puzzles to entertain and challenge our readers. This special feature of “Analytical and Bioanalytical Chemistry” has established itself as a truly unique quiz series, with a new scientific puzzle published every other month. Readers can access the complete collection of published problems with their solutions on the ABC homepage at http://www.springer.com/abc. Test your knowledge and tease your wits in diverse areas of analytical and bioanalytical chemistry by viewing this collection. In the present challenge, NMR is the topic. And please note that there is a prize to be won (a Springer book of your choice up to a value of €100). Please read on...

Ariadne’s thread NMR challenge

We would like to invite you to participate in the Analytical Challenge, a series of puzzles to entertain and challenge our readers. This special feature of “Analytical and Bioanalytical Chemistry” has established itself as a truly unique quiz series, with a new scientific puzzle published every other month. Readers can access the complete collection of published problems with their solutions on the ABC homepage at http://www.springer.com/abc. Test your knowledge and tease your wits in diverse areas of analytical and bioanalytical chemistry by viewing this collection. In the present challenge, nuclear magnetic resonance (NMR) is the topic. And please note that there is a prize to be won (a Springer book of your choice up to a value of €100). Please read on...

Angels’ share challenge

We would like to invite you to participate in the Analytical Challenge, a series of puzzles to entertain and challenge our readers. This special feature of “Analytical and Bioanalytical Chemistry” (ABC) has established itself as a truly unique quiz series, with a new scientific puzzle published every other month. Readers can access the complete collection of published problems with their solutions on the ABC homepage at http://www.springer.com/abc. Test your knowledge and tease your wits in diverse areas of analytical and bioanalytical chemistry by viewing this collection. In this challenge, flavorants are the topic. And please note that there is a prize to be won (a Springer book of your choice up to a value of €100). Please read on ...

The Olympic Games challenge

We would like to invite you to participate in the Analytical Challenge, a series of puzzles to entertain and challenge our readers. This special ABC feature has established itself as a truly unique quiz series, with a new scientific puzzle published every other month. Readers can gain access to the complete collection of published problems, with their solutions, on the Analytical and Bioanalytical Chemistry homepage at http://www.springer.com/abc. Test your knowledge and tease your wits in diverse areas of analytical and bioanalytical chemistry by viewing this collection. In the present challenge, the topic is spectroscopy and the Olympic Games. And please note that there is a prize to be won (a Springer book of your choice up to a value of € 75). Please read on...

Mechanistic study on –C–O– and –C–C– hydrogenolysis over Cu catalysts: identification of reaction pathways and key intermediates

Important petro-based polyol compounds with a longer carbon chain, such as oligohydroxy hexanes (e.g. 1,2- and 1,6-hexanediol or 1,2,6-hexanetriol), require at least three to four synthesis steps. Replacing this complex chemistry by a one-pot reaction via –C–O– bond cleavage from sugars would be a significant breakthrough for the use of renewable feedstocks. Cu is known for its dehydroxylation (deoxygenation) properties, yielding the desired products from sugars. In this joint research between academic and industrial chemistry, we have identified so far unknown intermediate products and present the first mechanism that explains the selective cleavage of OH-groups over copper. Strong interactions between polyols, unsaturated species and the copper surface are observed. Stable five-membered rings are formed with Cu via two vicinal OH-groups of the polyol reactant that makes these OH-groups inert to –C–O– bond cleavage. Adjacent free OH-groups in close proximity to the catalyst are dehydroxylated (deoxygenated). We further show that degradation of polyols not only occurs via commonly cited retro-aldol reactions. The formation of acid intermediates with subsequent decarboxylation is validated as a new pathway for –C–C– bond cleavage to short-chain polyols and CO2. The proposed mechanisms for –C–O– and –C–C– bond cleavage elucidate why hydrogenolysis reactions require high hydrogen pressure (up to 200 bar) to suppress the degradation of sugars and obtain high yields of deoxy C6 products. With this knowledge, the improvement of a standard commercial Cu-RANEY® catalyst under optimized reaction conditions was shown. In contrast to alumina-supported Cu, the Cu–Al alloy in a RANEY®-type catalyst shows selective –C–O– bond cleavage properties while maintaining the C6 carbon chain. These new insights into the transformation of sugars to value added commodities show the potential for new approaches in future biorefinery concepts.

Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone.

Dimethylmenaquinone (DMMK), a prevalent menaquinone (MK) derivative of uncertain function, is characteristic for members of the class Coriobacteriia. Such bacteria are frequently present in intestinal microbiomes and comprise several pathogenic species. The coriobacterial model organism Adlercreutzia equolifaciens was used to investigate the enzymology of DMMK biosynthesis. A HemN-like class C radical S-adenosylmethionine methyltransferase (MenK2) from A. equolifaciens was produced in Wolinella succinogenes or Escherichia coli cells and found to methylate MK specifically at position C-7. In combination with a previously described MK methyltransferase (MqnK/MenK) dedicated to MK methylation at C-8, 7,8-DMMK6 was produced in W. succinogenes. The position of the two methyl groups was confirmed by two-dimensional NMR and midpoint redox potentials of 7-MMK6, 8-MMK6 and 7,8-DMMK6 were determined by cyclic voltammetry. A phylogenetic tree of MenK, MenK2 and HemN proteins revealed a Coriobacteriia-specific MenK2 clade. Using chimeric A. equolifaciens MenK/MenK2 proteins produced in E. coli it was shown that the combined linker and HemN domains determined the site-specificity of methylation. The results suggest that the use of MenK2 as a biomarker allows predicting the ability of DMMK synthesis in microbial species.