Stereochemistry and Conformation of Skyllamycin,a Non‐Ribosomally Synthesized Peptide from Streptomyces sp. Acta 2897

Skyllamycin is a non‐ribosomally synthesized cyclic depsipeptide from Streptomyces sp. Acta 2897 that inhibits PDGF‐signaling. The peptide scaffold contains an N‐terminal cinnamoyl moiety, a β‐methylation of aspartic acid, three β‐hydroxylated amino acids and one rarely occurring α‐hydroxy glycine. With the exception of α‐hydroxy glycine, the stereochemistry of the amino acids was assigned by comparison to synthetic reference amino acids applying chiral GC‐MS and Marfey‐HPLC analysis. The stereochemistry of α‐hydroxy glycine, which is unstable under basic and acidic conditions, was determined by conformational analysis, employing a combination of data from NOESY‐NMR spectroscopy, simulated annealing and free MD simulations. The simulation procedures were applied for both R‐ and S‐configured α‐hydroxy glycine of the skyllamycin structure and compared to the NOESY data. Both methods, simulated annealing and free MD simulations independently support S‐configured α‐hydroxy glycine thus enabling the assignment of all stereocenters in the structure of skyllamycin and devising the role of two‐component flavin dependent monooxygenase (Sky39) as S‐selective.     

A Hydrolase-Catalyzed Cyclization Forms the Fused Bicyclic β-Lactone in Vibralactone

Vibralactone is isolated from the basidiomycete fungus Boreostereum vibrans as one of the strongest lipase inhibitors. Its unusual β-lactone-fused bicycle is derived from an aryl ring moiety by an oxidative ring-expansion prior to an intramolecular cyclization. Herein, we report the discovery of the cyclase VibC which belongs to the α/β-hydrolase superfamily and is involved in the vibralactone biosynthesis. Biochemical and crystal studies suggest that VibC may catalyze an aldol or an electrocyclic reaction initiated by the Ser-His-Asp catalytic triad. For the aldol and pericyclic chemistry in living cells, VibC is a unique hydrolase performing the carbocycle formation of an oxepinone to a fused bicyclic β-lactone. This presents a naturally occurring, new enzymatic reaction in both aldol and hydrolase (bio)chemistry that will guide future exploitation of these enzymes in synthetic biology for chemical-diversity expansion of natural products.     

Purete enantiomerique et configuration absolue de l'acide β-hydroxy β-trifluoromethyl β-phenyl propionique obtenu par synthese asymetrique

The enantiomeric purity of β-hydroxy β-trifluoromethyl β-phenyl propionic acid, prepared by asymmetric synthesis, was determined by NMR with a chiral europium complex. Its absolute configuration was established by correlation with α-hydroxy α-trifluoromethyl α-phenyl acetic acid. At the same time, the absolute configurations of 2-hydroxy 2-trifluoromethyl 2-phenyl ethylamine, 3-hydroxy 3-trifluoromethyl 3-phenyl 1-amino propane, and 2-trifluoromethyl 2-phenyl 1,2 ethanediol were determined.     

Low-temperature formation of cubic β-PbF2: precursor-based synthesis and first-principles phase stability study

A precursor-based approach to the cubic β-phase of PbF(2) was developed and allowed the preparation of this high-temperature phase well below the temperature for transition from the orthorhombic α- to the cubic β-phase. The formation of β-PbF(2) from the molecular precursors Pb[Se(C(6)H(2)(CF(3))(3))](2) and Pb(C(6)H(2)(CF(3))(3))(2) is facilitated by the presence of several short PbF contacts in these molecules. The cubic form of PbF(2) was obtained as macroscopic crystals as well as nanoparticulate powder. Its formation at relatively low temperature suggested a theoretical re-investigation of the phase stabilities of the two polymorphs. The theoretical results from the Kohn-Sham density functional theory indicate that the energy content for the β-phase is slightly lower than the one for the α-phase, by 0.5-1.7 kJ mol(-1) depending on the density functional used (zero-point vibrational energy correction included).     

Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-α-hydroxyglycine ester catalyzed by serine β-lactamases and dd-peptidases

The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate () is a quite effective substrate of serine β-lactamases and low molecular mass dd-peptidases. The class C P99 and ampC β-lactamases catalyze the hydrolysis of both enantiomers of , although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and Actinomadura R39 dd-peptidases catalyze hydrolysis of only one enantiomer of at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since β-lactamases usually prefer l(S) enantiomers and dd-peptidases d(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is d(R). Thus, it is the β-lactamases that have switched preference rather than the dd-peptidases. Molecular modeling of the P99 β-lactamase active site suggests that the α-hydroxyl of may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the β-lactamase active site, although their effects are not additive; this may also be true for inhibitors.     

Taxol biosynthesis: tyrocidine synthetase A catalyzes the production of phenylisoserinyl CoA and other amino phenylpropanoyl thioesters

In Taxus plants the biosynthesis of the pharmaceutical paclitaxel includes the transfer of β-amino phenylpropanoyls from coenzyme A to the diterpenoid baccatin III by an acyl CoA-dependent acyltransferase. Several enzymes on the pathway are known, yet a few remain unidentified, including the putative ligase that biosynthesizes key β-amino phenylpropanoyl CoAs. The multienzyme, nonribosomal peptide synthetase that produces tyrocidines contains a tridomain starter module tyrocidine synthetase A that normally activates (S)-α-Phe to an adenylate anhydride in the adenylation domain. The Phe moiety is then thioesterified by the pendent pantetheine of the adjacent thiolation domain. Herein, the adenylation domain was found to function as a CoA ligase, making α-, β-phenylalanyl, and phenylisoserinyl CoA. The latter two are substrates of a phenylpropanoyltransferase on the biosynthetic pathway of the antimitotic paclitaxel.     

用桑蚕丝素蛋白固定脲酶的研究及脲酶电极的制备

提出了一种新的酶固定化方法, 即通过甲醇处理, 使蚕丝素蛋白膜的构象由random coil向β-sheet发生根本性的变化, 从而将酶固定在β-sheet所特有的分子间氢键中。利用此方法所制成的脲酶电极, 在合适的操作条件下, 各项响应指标均令人满意, 并且脲酶的耐温性能被大大提高, 电极的有效使用寿命长达三个月以上。此种酶固定化方法原则上能够应用于其他不破坏蚕丝素蛋白分子结构的可溶性酶。     

Asymmetric epoxidation of styrene derivatives by styrene monooxygenase from Pseudomonas sp. LQ26: effects of α- and β-substituents

Recombinant Escherichia coli expressing a styrene monooxygenase, StyAB2, from Pseudomonas sp. LQ26 was applied to synthesize a range of chiral epoxides from conjugated styrene derivatives with excellent (>99%) enantioselectivity in most cases. The substrate preference was studied with a special focus on the steric effect of α- and β-substituents.     

N^2-（1-Methoxycarbonylethyl）guanosine, a new nucleoside coupled with an amino acid derivative from Amanita exitialis

A new purine nucleoside coupled with an amino acid derivative,N~2-(1-methoxycarbonylethyl)guanosine 1,along withβ-carboline and russulaceramide was isolated from the fruiting bodies of Amanita exitialis,a newly described poisonous mushroom. Its structure was elucidated by spectroscopic methods.This is the first report of naturally occurring nucleosides in which anα-amino acid derivative is bonded through itsα-amino nitrogen to a nucleobase aglycone by a C-N bond.The new compound was found to be toxic in ...     

Characterisation of Specific Activities and Hydrolytic Properties of Cell-Wall-Degrading Enzymes Produced by Trichoderma reesei Rut C30 on Different Carbon Sources

Conversion of lignocellulosic substrates is limited by several factors, in terms of both the enzymes and the substrates. Better understanding of the hydrolysis mechanisms and the factors determining their performance is crucial for commercial lignocelluloses-based processes. Enzymes produced on various carbon sources (Solka Floc 200, lactose and steam-pre-treated corn stover) by Trichoderma reesei Rut C30 were characterised by their enzyme profile and hydrolytic performance. The results showed that there was a clear correlation between the secreted amount of xylanase and mannanase enzymes and that their production was induced by the presence of xylan in the carbon source. Co-secretion of α-arabinosidase and α-galactosidase was also observed. Secretion of β-glucosidase was found to be clearly dependent on the composition of the carbon source, and in the case of lactose, 2-fold higher specific activity was observed compared to Solka Floc and steam-pre-treated corn stover. Hydrolysis experiments showed a clear connection between glucan and xylan conversion and highlighted the importance of β-glucosidase and xylanase activities. When hydrolysis was performed using additional purified β-glucosidase and xylanase, the addition of β-glucosidase was found to significantly improve both the xylan and glucan conversion.     

Active-site structure of a β-hydroxylase in antibiotic biosynthesis

X-ray absorption and resonance Raman spectroscopies show that CmlA, the β-hydroxylase of the chloramphenicol biosynthetic pathway, contains a (μ-oxo)-(μ-1,3-carboxylato)diiron(III) cluster with 6-coordinate iron centers and 3 - 4 His ligands. This active site is found within a unique β-lactamase fold and is distinct from those of soluble methane monooxygenase and related enzymes that utilize a highly conserved diiron cluster with a 2-His-4-carboxylate ligand set within a 4-helix bundle motif. These structural differences may have an impact on the nature of the activated oxygen species of the reaction cycle.     

Optically Active Amino Acid Synthesis by Artificial Transaminase Enzymes

The conversion of α-ketoacids to α-aminoacids is performed in some enzyme models by transamination, using pyridoxamine derivatives. When the pyridoxamine is attached to β-cyclodextrin the simple enzyme mimic shows substrate selectivity and also the induction of optical activity in the products. When base catalytic groups are attached to pyridoxamine the proton transfers are catalyzed by the basic groups; if they are mounted chirally on a rigid framework they also induce optical activity in the products. When these lines are combined, artificial enzymes are produced that bind a substrate selectively and then perform a chiral proton transfer to produce optically active amino acids with e.e.'s ranging from 74% to 96%.Two types of combination have been examined. In one of them the optical induction is only fairly good, but in the other the selectivity is excellent. The key seems to be the use of a particular bifunctional basic catalytic group, an ethylenediamine. This group is the best yet examined at catalyzing proton transfers in transamination reactions. Its use in a chiral artificial enzyme system leads to the observed high enantioselectivity.     

The Structure of a Transient Complex of a Nonribosomal Peptide Synthetase and a Cytochrome P450 Monooxygenase

Studying the interplay between nonribosomal peptide synthetases (NRPS), a major source of secondary metabolites, and crucial external modifying enzymes is a challenging task since the interactions involved are often transient in nature. By applying a range of synthetic inhibitor‐type compounds, a stabilized complex appropriate for structural analysis was generated for such a tailoring enzyme and an NRPS domain. The complex studied comprises an NRPS peptidyl carrier protein (PCP) domain bound to the Cytochrome P450 enzyme that is crucial for the provision of β‐hydroxylated amino acid precursors in the biosynthesis of the cyclic depsipeptide skyllamycin. The structure reveals that complex formation is governed by hydrophobic interactions, the presence of which can be controlled through minor alterations in PCP structure that enable selectivity amongst multiple highly similar PCP domains.     

Nucleosides and nucleotides. Part 7. Four dithymidine monophosphates with different anomeric configurations, their synthesis and behaviour towards phosphodiesterases

The four isomeric dithymidine monophosphates βT_d-βT_d ( 1 ), αT_d-βT_d (2), βT_d-αT_d ( 3 ) and αT_d-αT_d ( 4 ), differing only by the anomeric configurations of the nucleoside units, were prepared from the suitably protected nucleosides and nucleotides. The four dinucleoside monophosphates were tested as substrates of snake venom phosphodiesterase and spleen phosphodiesterase. Both enzymes accept all four compounds as substrates, however, the rate of the hydrolysis is considerably smaller if the enzymic attack takes place at the α-nucleoside moiety of the dinucleoside monophosphate.     

Esterase-mediated synthesis of optically active GABA analogues containing a stereogenic all-carbon quaternary carbon atom

Esterase from Horse Liver (HLAP) was able to hydrolyze a series of linear and cyclic β,β-dialkyl-γ-nitroesters, in spite of the well-known reluctance of hydrolytic enzymes to recognize and transform hindered substrates, such as those possessing a stereogenic quaternary carbon atom next to the reaction site. The resulting optically active γ-nitroesters gave access to optically active β,β-disubstituted γ-aminoacids as well as α,α-disubstituted succinic acids, both being biologically relevant compounds.     

Chemoselective mono halogenation of β-keto-sulfones using potassium halide and hydrogen peroxide; synthesis of halomethyl sulfones and dihalomethyl sulfones

The synthesis of α-halo β-keto-sulfones using potassium halide and hydrogen peroxide as a chemoselective mono halogenation reagent and the synthesis of α,α-symmetrical and asymmetrical dihalo β-keto-sulfones and α-halo, α-alkyl and β-keto-sulfones is described. Base induced cleavage of α-halo β-keto-sulfones, α,α-dihalo β-keto-sulfones, and α-halo, α-alkyl β-keto-sulfones afforded the corresponding halomethyl sulfones, dihalomethyl sulfones and haloalkyl sulfones.     

Cobalt-Catalyzed α-Arylation of Substituted α-Bromo α-Fluoro β-Lactams with Diaryl Zinc Reagents: Generalization to Functionalized Bromo Derivatives

A cobalt-catalyzed cross-coupling of α-bromo α-fluoro β-lactams with diarylzinc or diallylzinc reagents is herein disclosed. The protocol proved to be general, chemoselective and operationally simple allowing the C4 functionalization of β-lactams. The substrate scope was expanded to α-bromo lactams and amides, α-bromo lactones and esters as well as N - and O-containing heterocycles.     

An Artificial Hemoprotein with Inducible Peroxidase- and Monooxygenase-Like Activities

A novel inducible artificial metalloenzyme obtained by covalent attachment of a manganese(III)-tetraphenylporphyrin (MnTPP) to the artificial bidomain repeat protein, (A3A3′)Y26C, is reported. The protein is part of the αRep family. The biohybrid was fully characterized by MALDI-ToF mass spectrometry, circular dichroism and UV/Vis spectroscopies. The peroxidase and monooxygenase activities were evaluated on the original and modified scaffolds including those that have a) an additional imidazole, b) a specific αRep bA3-2 that is known to induce the opening of the (A3A3′) interdomain region and c) a derivative of the αRep bA3-2 inducer extended with a His₆-Tag (His₆-bA3-2). Catalytic profiles are highly dependent on the presence of co-catalysts with the best activity obtained with His₆-bA3-2. The entire mechanism was rationalized by an integrative molecular modeling study that includes protein–ligand docking and large-scale molecular dynamics. This constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways.     

A convenient,rapid, and general synthesis of α-oxo thiocyanates using clay supported ammonium thiocyanate

A very rapid, convenient, and general method for the synthesis of α-oxo thiocyanates has been described by using clay supported ammonium thiocyanate. The procedure avoids the use of additional catalyst, solvent, aqueous work-up and the yields are high. Moreover, the method is applicable for a variety of aryl, heteroaryl, alkyl α-halo carbonyls, β-keto tosylates, α-halo β-dicarbonyl, α-tosyl, β-dicarbonyl, alkyl halide, and alkyl tosylates.     

Enzymatic methods for the preparation of enantiopure malic and aspartic acid derivatives in organic solvents

The kinetic resolution of malic and aspartic acid diesters as well as of its N-butanoyl dimethyl ester by highly regioselective lipases and acylase I enzymes were studied. Candida antarctica lipase A on Celite catalyzed the enantioselective acylation of the hydroxyl and amino groups. Acylase I from Aspergillus melleus and Candida antarctica lipase B catalyzed the enantioselective alcoholyses of the ester groups at the α- and β-positions, respectively.