The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Modulating the folding stability and ligand binding affinity of Pin1 WW domain by proline ring puckering

The pyrrolidine side chain makes proline play a unique role in protein structure and function. The C^γ ring pucker preference and the cis– trans peptidyl bond ratio can be mediated via stereoelectronic effects. Here we used a compact triple‐stranded antiparallel β‐sheet protein, the human Pin1 WW domain, to study the consequences of implanting a preorganized C^γ ring pucker on protein structure and function. The conserved Pro37 is a key residue involved in one hydrophobic core, plays an important role in the WW domain, and adopts a C^γ‐endo ring pucker in the native structure. Pro37 was replaced with C^γ‐exo biased pucker derivatives: (2S,4R)‐4‐hydroxyproline (4R‐Hyp), (2S,4R)‐4‐fluoroproline (4R‐Flp), (2S,4R)‐4‐methoxyproline (4R‐Mop), and C^γ‐endo biased pucker derivatives: (2S,4S)‐4‐hydroxyproline (4S‐hyp), (2S,4S)‐4‐fluoroproline (4S‐flp), (2S,4S)‐4‐methoxyproline (4S‐mop) to examine how a preorganized pucker affects the folding stability and ligand‐binding affinity. Circular dichroism measurements indicate that among the variants, only the one with 4S‐flp substitution (P37flp) is more stable than the wild type, suggesting that the stabilization effects originated from preorganization of the backbone conformation and the hydrophobicity of C? F group. Analysis of ligand‐binding affinity using isothermal titration calorimetry revealed that only P37flp has a stronger ligand affinity than the wild type, showing that 4S‐flp can stabilize the WW domain and increase its ligand affinity. Together we have used 4‐substituted proline derivatives and the WW domain to demonstrate that proline ring puckering can be a key factor in determining the folding stability of a protein but the choice of the derivative groups is also critical. Proteins 2014; 82:67–76. © 2013 Wiley Periodicals, Inc.     

Production of elemental sulfur by green and purple sulfur bacteria

The utilization of sulfide by phototrophic sulfur bacteria temporarily results in the accumulation of elemental sulfur. In the green sulfur bacteria (Chlorobiaceae), the sulfur is deposited outside the cells, whereas in the purple sulfur bacteria (Chromatiaceae) sulfur is found intracellularly. Consequently, in the latter case, sulfur is unattainable for other individuals. Attempts were made to analyze the impact of the formation of extracellular elemental sulfur compared to the deposition of intracellular sulfur.According to the theory of the continuous cultivation of microorganisms, the steady-state concentration of the limiting substrate is unaffected by the reservoir concentration (S _R).It was observed in sulfide-limited continuous cultures ofChlorobium limicola f.thiosulfatophilum that higherS _R values not only resulted in higher steady-state population densities, but also in increased steady-state concentrations of elemental sulfur. Similar phenomena were observed in sulfide-limited cultures ofChromatium vinosum.It was concluded that the elemental sulfur produced byChlorobium, althouth being deposited extracellularly, is not easily available for other individuals, and apparently remains (in part) attached to the cells. The ecological significance of the data is discussed.Non-standard abbreviations RP reducing power - BChl bacteriochlorophyll - N_cell cell material - specific growth rate - {ie52-1} maximal specific growth rate - D dilution rate - K _s saturation constant - s concentration of limiting substrate - S _R same ass but in reservoir bottle - Y yield factor - iS^o intracellular elemental sulfur - eS^o extracellular elemental sulfur - PHB poly-beta-hydroxybutyric acid     

Use of isotopically chiral [4′-13C]penciclovir and 13C NMR to determine the specificity and absolute configuration of penciclovir phosphate esters formed in HSV-1- and HSV-2-infected cells and by HSV-1-encoded thymidine kinase

Penciclovir is a potent antiherpesvirus agent which is highly selective due to its phosphorylation only in virus infected cells. Phosphorylation of one of the hydroxymethyl groups of penciclovir (PCV) creates a chiral centre leading to the possible formation of (R)- and (S)-enantiomers. The absolute configuration and stereospecificity of the PCV-phosphates produced in cells infected with herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), as well as by HSV-1-encoded thymidine kinase, were determined using isotopically chiral [4′-¹³C]PCV precursors and ¹³C NMR spectroscopy of the isolated metabolites. The absolute configuration of penciclovir-triphosphate (PCV-TP) produced in HSV-1-infected cells was shown to be S with an enantiomeric purity of greater than 95%. However, in contrast to HSV-1-infected cells in which none of the (R) enantiomer was detected, about 10% of (R)-PCV-TP was produced in HSV-2-infected cells. Phosphorylation of PCV by HSV-1-encoded thymidine kinase was found to give 75% (S)- and 25% (R)-PCV-monophosphate. The proportion of the (S)-isomer appears to be amplified in the subsequent phosphorylations leading to the triphosphate. © 1993 Wiley-Liss, Inc.     

Absolute stereochemistry of methanopterin and 5,6,7,8-tetrahydromethanopterin

The configuration at the C-9 of methanopterin (MPT) has been determined by comparing the circular dichroism (CD) spectra of MPT and its hydrolytic fragment, 1-[4-[[1-(2-amino-7-methyl-4-hydroxy-6-pteridinyl)-ethyl]amino]phenyl]-1-deoxy-D -ribitol (HP-1), with the CD spectra of a series of model compounds of known stereochemistry. These compounds included (S)-6-[1-(4-carboxymethylanilino)ethyl]pterin, (S-6(1-hydroxyethyl)-7-methylpterin, (S-6-(1-hydroxyethyl)pterin, (R)-6-(1-phenoxyethyl)pterin, D (+)-neopterin, and L -biopterin. From this comparison it was concluded that MPT has the R configuration at C-9 and is thus configurationally related to D (+)-neopterin, which has the S configuration at C-1. From previous work establishing the relative stereochemistry at C-6, C-7, and C-9 of N⁵-N¹⁰-methenyl-5,6,7,8-tetrahydromethanopterin (N⁵-N¹⁰-methenyl-H₄MPT) as R, S, and R, respectively, it is clear that the remaining asymmetric carbons at C-6 and C-7 of H₄MPT have the S and S configuration, respectively. Comparison of these latter two positions to the equivalent carbons in 5,6,7,8-tetrahydrofolate (H₄folate) show that the steps involved in the biological reduction of MPT to H₄MPT occur with the same stereochemical outcome as those involved in the biological reduction of folate to H₄folate. © 1996 Wiley-Liss, Inc.     

Compartment Analysis of Disease Progression in Time. I. Unrestricted Disease Increase

Disease progression in time through presporulating (latent), sporulating (infectious) and postporulating (removal) stages has been modelled as a three-compartment system in the case where no upper bound for total disease increase is considered. Deterministic compartment analysis has been performed based on Jeger's linked differential equations. Some important, well-established epidemilogical principles and concepts have been ‘rediscovered’, clarified or redefined. A new variable, defined as total inoculum reservoir of the host, is suggested as an additional epidemiological concept. Three rate parameters with phytopathological meaning were included in the system: R the constant intrinsic infection rate comparable to the Vanderplankian R_,c, h pathogen's ‘hatching rate’, related to the mean latent period p and g the intrinsic removal rate related to the infectious period i. Another parameter λ₁ is empirical and comparable to the logarithmic, infection rate, sensu Vanderplank if time becomes very large. In the compartment system iR= 0 is a threshold condition for total disease to increase over initial disease level and iR= 1 is a threshold condition for an ‘explosive’ total disease increase that may induce a large epidemic rather than a threshold for total disease to start or to exist. When iR < 1, the total disease level approaches α asymptotically, a finite value, while when iR > 1, the total disease, increase is explosive; only where λ₁=√dh does this increase follows the exponential law at larger t, otherwise the exponential law with the same rate parameter λ₁ underestimates or overstimates the amount of total disease at any time t. Some further implications are discussed and compared with those derived from the Vanderplankian system of epidemiological analysis.     

Two New Ergosterol Derivatives from the Basidiomycete Cortinarius glaucopus

Two new sterols 1 and 2 and five known ones 3 – 7 were isolated for the first time from the fruiting bodies of Cortinarius glaucopus. Their structures were established by 1‐ and 2D‐NMR spectra and HR‐FABS‐MS. The relative configuration of 1 was firmly determined by comparison of the observed ¹H–¹H couplings and NOESY correlations, with those predicted for the computed geometries of the conformers. Calculations were performed by means of DFT with the B3LYP functional at 6‐31 + G(d,p) level of theory, in CHCl₃ as the solvent. The structures of the new ergosterol derivatives, called glaucoposterol A ( 1 ) and B ( 2 ), were thus established as (3S,5R,7R,10R,13R,17R,20S,22R,23R,24R)‐5,6‐epoxy‐3,7,23‐trihydroxystrophast‐8‐en‐14‐one and (22E,3S,5S,9S,10R,13R,17R,20R,24R)‐3,5‐dihydroxyergosta‐6,8(14),22‐trien‐15‐one, respectively. Moreover, the configuration of known strophasterol C ( 3 ) was determined as (3S,5R,6S,7R,10R,13R,17R,20S,22S,24R). Glaucoposterol A ( 1 ) and strophasterol C ( 3 ) represent the second finding in nature of steroids with the rare strophastane skeleton.     

Synthesis and evaluation of in vivo anti‐hypothermic effect of all stereoisomers of the thyrotropin‐releasing hormone mimetic: Rovatirelin Hydrate

We discovered the orally active thyrotropin‐releasing hormone (TRH) mimetic: (4S,5S)‐5‐methyl‐N‐{(2S)‐1‐[(2R)‐2‐methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide 1 (rovatirelin). The central nervous system (CNS) effect of rovatirelin after intravenous (iv) administration is 100‐fold higher than that of TRH. As 1 has four asymmetric carbons in its molecule, there are 16 stereoisomers. We synthesized and evaluated the anti‐hypothermic effect of all stereoisomers of 1 , which has the (4S),(5S),(2S),(2R) configuration from the N‐terminus to the C‐terminus, in order to clarify the structure?activity relationship (SAR) of stereoisomers. The (4R),(5R),(2R),(2S)‐isomer 16 did not show any anti‐hypothermic effect. Only the (4S),(5S),(2S),(2S)‐isomer 10 , which has the (2S)‐2‐methylpyrrolidine moiety at the C‐terminus showed the anti‐hypothermic effect similar to 1 . Stereoisomers, which have the (5R) configuration of the oxazolidinone at the N‐terminus and the (2R) configuration at the middle‐part, showed a much lower anti‐hypothermic effect than that of 1 . On the other hand, stereoisomers, which have the (4R) configuration of the oxazolidinone at the N‐terminus or the (2S) configuration of the C‐terminus, have little influence on the anti‐hypothermic effect.     

Circularly polarized luminescence of Sm (III) and Eu (III) complexes with chiral ligand (R/S)‐BINAPO

Luminescent lanthanide (III) ions have been exploited for circularly polarized luminescence (CPL) for decades. However, very few of these studies have involved chiral samarium (III) complexes. Complexes are prepared by mixing axial chiral ligands (R/S))‐2,2’‐bis(diphenylphosphoryl)‐1,1′‐binaphthyl (BINAPO) with europium and samarium Tris (trifluoromethane sulfonate) (Eu (OTf)₃ and Sm (OTf)₃). Luminescence‐based titration shows that the complex formed is Ln((R/S)‐BINAPO)₂(OTf)₃, where Ln = Eu or Sm. The CPL spectra are reported for Eu((R/S)‐BINAPO)₂(OTf)₃ and Sm((R/S)‐BINAPO)₂(OTf)₃. The sign of the dissymmetry factors, g_em, was dependent upon the chirality of the BINAPO ligand, and the magnitudes were relatively large. Of all of the complexes in this study, Sm((S)‐BINAPO)₂(OTf)₃ has the largest g_em = 0.272, which is one of the largest recorded for a chiral Sm³⁺ complex. A theoretical three‐dimensional structural model of the complex that is consistent with the experimental observations is developed and refined. This report also shows that (R/S)‐BINAPO are the only reported ligands where g_em (Sm³⁺) > g_em (Eu³⁺).     

A New Biosensor for Chiral Recognition Using Goat and Rabbit Serum Albumin Self‐Assembled Quartz Crystal Microbalance

Quartz crystal microbalance (QCM) biosensor was used for the chiral recognition of five pairs of enantiomers by using goat serum albumin (GSA) and rabbit serum albumin (RbSA) as chiral selectors. Serum albumin (SA) was immobilized on the QCM through the self‐assembled monolayer technique, and the surface concentration of GSA and RbSA were 8.8 × 10^?12 mol cm^?2 and 1.2 × 10^?11 mol cm^?2, respectively. The QCM biosensors showed excellent sensitivity and selectivity. Meanwhile, the chiral recognition of SA sensors was quite species dependent. There were differences between GSA and RbSA sensors in the ability and the preference of chiral recognition. To R,S‐1,2,3,4‐tetrahydro‐1‐naphthylamine (R,S‐1‐TNA), R,S‐1‐(4‐methoxyphenyl)ethylamine (R,S‐4‐MPEA), and R,S‐1‐(3‐methoxyphenyl)ethylamine (R,S‐3‐MPEA), the preference of the stereoselective SA‐drug binding of the two kinds of SA sensors were consistent. However, to R,S‐2‐octanol (R, S‐2‐OT) and R,S‐methyl lactate (R,S‐MEL), the two kinds of SA sensors had opposite chiral recognition preference. Moreover, the interactions of SA and the five pairs of enantiomers have been further investigated through ultraviolet (UV) and fluorescent (FL) spectra. The UV/FL results were in accordance with the consequence of QCM. Chirality 24:804–809, 2012. © 2012 Wiley Periodicals, Inc.     

Preparative Enantioseparation of (RS)‐Baclofen: Determination of Molecular Dissymmetry

The present work reports preparative enantioseparation of (RS)‐baclofen using thin‐layer chromatography (TLC) and high‐performance liquid chromatography (HPLC). Diastereomers were synthesized using a new monochloro‐s‐triazine‐based chiral derivatizing reagent (CDR), namely, N‐(4‐chloro‐6‐piperidinyl‐[1,3,5]‐triazine‐2‐yl)‐L‐phenylalanine, under microwave irradiation. Acetonitrile‐0.1% aq. triflouroacetic acid in gradient elution mode and CH₃OH‐CH₂Cl₂ (4:5; v/v) were successful as mobile phase in HPLC and TLC, respectively. The two diastereomers were isolated by preparative TLC. Molecular dissymmetry was established by developing the lowest energy optimized structures of the diastereomers based on Density Functional Theory and with the help of ¹H NMR showing anisotropic effect associated with aromatic ring of s‐triazine (in the CDR). The configuration of diastereomers was established as [L‐Phe‐(R)‐Bac] and [L‐Phe‐(S)‐Bac], where the first notation refers to the configuration of chiral auxiliary (of the CDR) and the second to that of the analyte Bac. Limits of detection were found to be 0.056 and 0.061 ng mL^?1, respectively, for the two diastereomers. Determination of absolute configuration of the two diastereomers lent support to the elution order and separation mechanism.Chirality 27:299–305, 2015. © 2015 Wiley Periodicals, Inc.     

Asymmetric ammonolysis of (R/S)-mandelic acid by immobilized lipases via direct amidation of mandelic acid in biphasic media

Abstract

We have investigated the direct enantioselective amidation of mandelic acid with ammonia, catalyzed by a variety of commercial lipases including those from Candida rugosa, Mucor miehei, Pseudomonas sp., Rhizomucor miehei, and Thermomyces lanuginosus covalently immobilized onto Florisil^® support via glutaraldehyde and polysuccinimide spacer arms. All the immobilized lipase preparations tested preferentially amidated the R isomer of mandelic acid. The highest amide yields were obtained for immobilized Pseudomonas sp. lipase preparations under the optimized reaction conditions. After 24 h of amidation, the reaction had proceeded with an excellent yield (50%) and enantiopurity (> 99%). The immobilized Pseudomonas sp. lipase preparations catalyzed the amidation reaction with the same yield and enantioselectivity. The enzyme immobilized via a glutaraldehyde spacer arm showed better reusability than that immobilized via a polysuccinimide spacer arm.

In view of these results, it is revealed that the direct amidation of mandelic acid catalyzed by the immobilized Pseudomonas sp. lipases is a facile and effective methodology for obtaining (S)-mandelic acid and (R)-mandelamide.     

Circularly polarized luminescence of Eu(III) complexes with chiral 1,1′-bi-2-naphtol-derived bisphosphate ligands

The interest for lanthanide circularly polarized luminescence (CPL) has been quickly growing for 10 years. However, very few of these studies have involved correlation between the dissymmetry factor (g_lum) and the chemical modifications in a series of chiral ligands. Four polymeric compounds of Eu(III) were prepared by using a series of binaphtyl derivatives for which the size of the π system as well as the number of stereogenic elements (i.e., the binaphtyl moiety) are modulated. The resulting {[Eu(hfac)₃((S)/(R)-L^x)]}_n (x = 1 and 3) and {[Eu(hfac)₃((S,S,S)/(R,R,R)-L^x)]}_n (x = 2 and 4) have been characterized by powder X-ray diffraction by comparison with the X-ray structures on single crystal of the Dy(III) analogs. In solution, the structure of the complexes is deeply modified and becomes monomeric. The nature of the ligand induces change in the shape of the CPL spectra in CH₂Cl₂ solution. Furthermore, a large |g_lum| = 0.12 of the magnetic-dipole transition for the [Eu(hfac)₃((S,S,S)/(R,R,R)-L²)] complex involving the ligand with three stereogenic elements and an extended ?? system has been measured. This report also shows CPL measurements in solid state for the series of {[Eu(hfac)₃((S)/(R)-L^x)]}_n (x = 1 and 3) and {[Eu(hfac)₃((S,S,S)/(R,R,R)-L^x)]}_n (x = 2 and 4) polymers.     

Substituted α-mercaptoketones,new types of specific neprilysin inhibitors

New neprilysin inhibitors containing an α-mercaptoketone HSC(R¹R²)CO group, as zinc ligand were designed. Two parameters were explored for potency optimization: the size of the inhibitor which could interact with the S₁, S₁′ or S₂′ domain of the enzyme and the nature of the substituents R¹, R² of the mercaptoketone group. Introduction of a cyclohexyl chain in R¹, R² position and a (3-thiophen)benzyl group in position R³ (compound 12n) yielded to the most potent inhibitor of this series with a Ki value of 2 ± 0.3 nM. This result suggests that this new inhibitor interacts within the S₁, S₁′ domain of NEP allowing a pentacoordination of the catalytic Zn²⁺ ion by the mercaptoketone moiety.     

Production of Cephalosporin Antibiotics by Strains Belonging to the Genera Arachnomyces,Anixiopsis and Spiroidium

The absolute configuration of a phytoalexin “Safynol” (3E, 11E)-3, 11-tridecadiene-5, 7, 9-triyne-l, 2-diol (I), separated from Phytophthora drechsleri-infected safflower (Carthamus tinctorius L.) was confirmed. (R) and (S)-safynol were synthesized from 2, 3-O-isopropyli- dene-(S)- and (R)-glyceraldehyde respectively, and the (R) configuration was assigned to the natural product. The inhibitory effects of (R)- and (S)-safynol on mycelial growth of five fungi were almost the same and their ED₅₀ values ranged from 6 to 70 ppm.     

Absolute configurations and CD spectra of axially chiral biphenyls prepared in a facile manner by crystallization‐induced configuration transformation

Axially chiral biphenyls such as (M,S)‐ 3k have been conveniently obtained by crystallization of their diastereomeric mixtures, which were synthesized from racemic 4,4′‐dimethoxy‐5,6,5′,6′‐bis(methylenedioxy)‐2‐carboxylester‐2′‐carboxyl‐biphenyls 4 and chiral amino alcohols (R)‐alaninol, (S)‐alaninol, (S)‐valinol, and (S)‐phenylalaninol. A crystallization‐induced configuration transformation of the biphenyls was thus achieved. It was found that amide formation of an (S)‐valinol or (S)‐phenylalaninol at the 2′‐position of the biphenyl usually induced the deposition of crystals with the (M)‐configuration from ethanol in yields higher than 50%. The absolute configurations (ACs) of two crystalline biphenyls have been determined by X‐ray crystallographic analysis. The ACs of nine biphenyls have been assigned based on their CD spectra. Further, stability investigation of these axially chiral biphenyls revealed that the ACs could revert upon redissolution. The energy barrier to epimerization between (P,R)‐ 3b and (M,R)‐ 3b was measured as ΔG^# = 21.45 kcal/mol and the half‐life in ethanol at 301 K was 17.1 h. Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Stereochemistry of octopine and of its isomers and their enzymatic properties

The four isomers of octopine were prepared from pyruvic acid and l- or d-arginine and from α-keto δ-guanidinovaleric acid and l- or d-alanine by reduction with sodium cyanoborohydride. The absolute configuration of d-octopine, the natural occurring isomer being S(l) at the arginine center, and R(d) at the alanine center, was confirmed enzymatically. d-Octopine is the only isomer oxidized by NAD⁺ in the presence of octopine dehydrogenase from Pecten maximus L. The isomer with configuration S(l) at the alanine center is found to be a competitive inhibitor. Isomers with R(d) configuration at the arginine center show no detectable effect on the enzymatic reaction.