Necessary and sufficient conditions for the asymmetric synthesis of chiral amines using ω‐aminotransferases

The half reactions of ω‐aminotransferase (ω‐AT) from Vibrio fluvialis JS17 (ω‐ATVf) were carried out using purified pyridoxal 5′‐phosphate‐enzyme (PLP‐Enz) and pyridoxamine 5′‐phosphate‐enzyme (PMP‐Enz) complexes to investigate the relative activities of substrates. In the reaction generating PMP‐Enz from PLP‐Enz using L ‐alanine as an amine donor, L ‐alanine showed about 70% of the initial reaction rate of (S)‐α‐methylbenzylamine ((S)‐α‐MBA). However, in the subsequent half reaction recycling PLP‐Enz from PMP‐Enz using acetophenone as an amine acceptor, acetophenone showed nearly negligible reactivity compared to pyruvate. These results indicate that the main bottleneck in the asymmetric synthesis of (S)‐α‐MBA lies not in the amination of PLP by alanine, but in the amination of acetophenone by PMP‐Enz, where conformational restraints of the enzyme structure is likely to be the main reason for limiting the amine group transfer from PMP‐Enz to acetophenone. Based upon those half reaction experiments using the two amino acceptors of different activity, it appears that the relative activities of the two amine donors and the two acceptors involved in the ω‐AT reactions can roughly determine the asymmetric synthesis yield of the target chiral amine compound. Predicted conversion yields of several target chiral amines were calculated and compared with the experimental conversion yields. Approximately, a positive linear correlation (Pearson's correlation coefficient = 0.92) was observed between the calculated values and the experimental conversion yields. To overcome the low (S)‐α‐MBA productivity of ω‐ATVf caused by the possible disadvantageous structural constraints for acetophenone, new ω‐ATs showing higher affinity to benzene ring of acetophenone than ω‐ATVf were computationally screened using comparative modeling and protein‐ligand docking. ω‐ATs from Streptomyces avermitilis MA‐4680 (SAV2612) and Agrobacterium tumefaciens str. C58 (Atu4761) were selected, and the two screened ω‐ATs showed higher asymmetric synthesis reaction rate of (S)‐α‐MBA and lower (S)‐α‐MBA degradation reaction rate than ω‐ATVf. To verify the higher conversion yield of the variants of ω‐ATs, the reaction with 50 mM acetophenone and 50 mM alanine was performed with coupling of lactate dehydrogenase and two‐phase reaction system. SAV2612 and Atu4761 showed 70% and 59% enhanced yield in the synthesis of (S)‐α‐MBA compared to that of ω‐ATVf, respectively. Biotechnol. Bioeng. 2011;108: 253–263. © 2010 Wiley Periodicals, Inc.     

Resolution of (±)‐β‐methylphenylethylamine by a novel chiral stationary phase for Pirkle‐type column chromatography

In this study, a new Pirkle‐type chiral column stationary phase for resolution of β‐methylphenylethyl amine was described by using activated Sepharose 4B as a matrix, L ‐tyrosine as a spacer arm, and an aromatic amine derivative of L ‐glutamic acid as a ligand. The binding capacities of the stationary phase were determined at different pH values (pH = 6, 7, and 8) using buffer solutions as mobile phase, and enantiomeric excess (ee) was determined by HPLC equipped with chiral column. The ee was found to be 47%. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Conformational transitions driven by pyridoxal‐5′‐phosphate uptake in the psychrophilic serine hydroxymethyltransferase from Psychromonas ingrahamii

Serine hydroxymethyltransferase (SHMT) is a pyridoxal‐5′‐phosphate (PLP)‐dependent enzyme belonging to the fold type I superfamily, which catalyzes in vivo the reversible conversion of l ‐serine and tetrahydropteroylglutamate (H₄PteGlu) to glycine and 5,10‐methylenetetrahydropteroylglutamate (5,10‐CH₂‐H₄PteGlu). The SHMT from the psychrophilic bacterium Psychromonas ingrahamii (piSHMT) had been recently purified and characterized. This enzyme was shown to display catalytic and stability properties typical of psychrophilic enzymes, namely high catalytic activity at low temperature and thermolability. To gain deeper insights into the structure–function relationship of piSHMT, the three‐dimensional structure of its apo form was determined by X‐ray crystallography. Homology modeling techniques were applied to build a model of the piSHMT holo form. Comparison of the two forms unraveled the conformation modifications that take place when the apo enzyme binds its cofactor. Our results show that the apo form is in an “open” conformation and possesses four (or five, in chain A) disordered loops whose electron density is not visible by X‐ray crystallography. These loops contain residues that interact with the PLP cofactor and three of them are localized in the major domain that, along with the small domain, constitutes the single subunit of the SHMT homodimer. Cofactor binding triggers a rearrangement of the small domain that moves toward the large domain and screens the PLP binding site at the solvent side. Comparison to the mesophilic apo SHMT from Salmonella typhimurium suggests that the backbone conformational changes are wider in psychrophilic SHMT. Proteins 2014; 82:2831–2841. © 2014 Wiley Periodicals, Inc.     

Role of active‐site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C‐S lyase

In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C‐S lyase from Corynebacterium diphtheriae is a pyridoxal 5′‐phosphate‐dependent enzyme in the transsulfuration pathway that catalyzes the α,β‐elimination of sulfur‐containing amino acids, such as l ‐cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L ‐methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active‐site residues, Y55F and Y114F, were characterized by UV‐visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady‐state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130‐fold decrease in at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP‐binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of l ‐cystathionine, and causes a change in substrate specificity towards l ‐serine and O‐acetyl‐l ‐serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C‐S lyase, which seems to be regulated by active‐site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors. Proteins 2015; 83:78–90. © 2014 Wiley Periodicals, Inc.     

The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

Recent studies have demonstrated that the O‐antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N‐formylated sugars (3‐formamido‐3,6‐dideoxy‐d ‐glucose or 4‐formamido‐4,6‐dideoxy‐d ‐glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6‐dehydratase, a pyridoxal 5'‐phosphate or PLP‐dependent aminotransferase, and an N‐formyltransferase. To date, there have been no published reports of N‐formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N‐formyltransferase. Given that M. tuberculosis produces l ‐rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6‐dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N‐formylated sugar in M. tuberculosis, namely a PLP‐dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.     

Molecular characterization of novel pyridoxal‐5′‐phosphate‐dependent enzymes from the human microbiome

Pyridoxal‐5′‐phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP‐dependent enzymes account for ～1.5% of most prokaryotic genomes and are estimated to be involved in ～4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP‐dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram‐positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP‐dependent enzyme fold and structure‐guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.     

Molecular dynamics simulations of apo,holo, and inactivator bound GABA‐at reveal the role of active site residues in PLP dependent enzymes

The pyridoxal 5‐phosphate (PLP) cofactor is a significant organic molecule in medicinal chemistry. It is often found covalently bound to lysine residues in proteins to form PLP dependent enzymes. An example of this family of PLP dependent enzymes is γ‐aminobutyric acid aminotransferase (GABA‐AT) which is responsible for the degradation of the neurotransmitter GABA. Its inhibition or inactivation can be used to prevent the reduction of GABA concentration in brain which is the source of several neurological disorders. As a test case for PLP dependent enzymes, we have performed molecular dynamics simulations of GABA‐AT to reveal the roles of the protein residues and its cofactor. Three different states have been considered: the apoenzyme, the holoenzyme, and the inactive state obtained after the suicide inhibition by vigabatrin. Different protonation states have also been considered for PLP and two key active site residues: Asp298 and His190. Together, 24 independent molecular dynamics trajectories have been simulated for a cumulative total of 2.88 µs. Our results indicate that, unlike in aqueous solution, the PLP pyridine moiety is protonated in GABA‐AT. This is a consequence of a pK_a shift triggered by a strong charge–charge interaction with an ionic “diad” formed by Asp298 and His190 that would help the activation of the first half‐reaction of the catalytic mechanism in GABA‐AT: the conversion of PLP to free pyridoxamine phosphate (PMP). In addition, our MD simulations exhibit additional strong hydrogen bond networks between the protein and PLP: the phosphate group is held in place by the donation of at least three hydrogen bonds while the carbonyl oxygen of the pyridine ring interacts with Gln301; Phe181 forms a π–π stacking interaction with the pyridine ring and works as a gate keeper with the assistance of Val300. All these interactions are hypothesized to help maintain free PMP in place inside the protein active site to facilitate the second half‐reaction in GABA‐AT: the regeneration of PLP‐bound GABA‐AT (i.e., the holoenzyme). Proteins 2016; 84:875–891. © 2016 Wiley Periodicals, Inc.     

Molecular tweezers for enantiodiscrimination in NMR: Di‐(R,R)‐1‐[10‐(1‐hydroxy‐2,2,2‐trifluoroethyl)‐9‐anthryl]‐2,2,2‐trifluoroethyl benzenedicarboxylates

A series of new chiral molecular tweezers, di‐(R,R)‐1‐[10‐(1‐hydroxy‐2,2,2‐trifluoroethyl)‐9‐anthryl]‐2,2,2‐trifluoroethyl phthalate (2), isophthalate (3) and terephthalate (4), were synthesized and their structure studied by NMR and molecular mechanics. Their effectiveness as chiral solvating agents for the determination of the enantiomeric purity of chiral compounds using NMR was demonstrated. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Vibrational circular dichroism of 3‐(trifluoroacetyl)‐camphor and its interaction with chiral amines

Vibrational circular dichroism (VCD) spectroscopy and density functional theory (DFT) calculations are used to investigate the keto–enol equilibrium of 3‐(trifluoroacetyl)‐camphor (TFC) and to study the interaction of TFC with chiral amines in deuterated Chloroform. It is shown that the VCD spectra of the enol‐ and keto forms of TFC can clearly be distinguished and that the enol form is favored. By deprotonation of the TFC enol with chiral amines, no indication of a mutual diasteriomeric influence on the VCD spectra induced by transfer of stereochemical information between the chiral ionic species is found, neither experimentally nor theoretically. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

Structural determinants in bacterial 2‐keto‐3‐deoxy‐D‐gluconate dehydrogenase KduD for dual‐coenzyme specificity

Short‐chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2‐keto‐3‐deoxy‐d ‐gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG‐producing SDR enzymes (NADPH‐dependent A1‐R and NADH‐dependent A1‐R′) involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes—KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)—prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5‐diketo‐3‐deoxy‐d ‐gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X‐ray crystallography. Both PcaKduD and SpyDhuD consist of a three‐layered structure, α/β/α, with a coenzyme‐binding site in the Rossmann fold; similar to enzymes A1‐R and A1‐R′, both arrange the two short and long loops close to the coenzyme‐binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1‐R′ but not A1‐R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2′ coenzyme region were determined to be structurally crucial for dual‐coenzyme specificity in PcaKduD by structural comparison of the NADH‐ and NADPH‐specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual‐coenzyme specificity. Proteins 2016; 84:934–947. © 2016 Wiley Periodicals, Inc.     

Purification and characterization of intracellular and extracellular α‐glucosidases from Geobacillus toebii strain E134

Two different α‐glucosidase‐producing thermophilic E134 strains were isolated from a hot spring in Kozakli, Turkey. Based on the phenotypic, phylogenetic and chemotaxonomic evidence, the strain was proposed to be a species of G. toebii. Its thermostable exo‐α‐1,4‐glucosidases also were characterized and compared, which were purified from the intracellular and extracellular fractions with estimated molecular weights of 65 and 45 kDa. The intracellular and extracellular α‐glucosidases showed optimal activity at 65 °C, pH 7·0, and at 70 °C, pH 6·8, with 3·65 and 0·83 K_m values for the pNPG substrate, respectively. Both enzymes remained active over temperature and pH ranges of 35–70 °C and 4·5–11·0. They retained 82 and 84% of their activities when incubated at 60 °C for 5 h. Their relative activities were 45–75% and 45–60% at pH 4·5 and 11·0 values for 15 h at 35 °C. They could hydrolyse the α‐1,3 and α‐1,4 bonds on substrates in addition to a high transglycosylation activity, although the intracellular enzyme had more affinity to the substrates both in hydrolysis and transglycosylation reactions. Furthermore, although sodium dodecyl sulfate behaved as an activator for both of them at 60 °C, urea and ethanol only increased the activity of the extracellular α‐glucosidase. By this study, G. toebii E134 strain was introduced, which might have a potential in biotechnological processes when the conformational stability of its enzymes to heat, pH and denaturants were considered. Copyright © 2011 John Wiley & Sons, Ltd.     

Immobilization of Escherichia coli containing ω-transaminase activity in LentiKats®

Whole Escherichia coli cells overexpressing ω‐transaminase (ω‐TA) and immobilized cells entrapped in LentiKats® were used as biocatalysts in the asymmetric synthesis of the aromatic chiral amines 1‐phenylethylamine (PEA) and 3‐amino‐1‐phenylbutane (APB). Whole cells were permeabilized with different concentrations of cetrimonium bromide (CTAB) and ethanol; the best results were obtained with CTAB 0.1% which resulted in an increase in reaction rate by 40% compared to the whole cells. The synthesis of PEA was carried out using isopropyl amine (IPA) and L ‐alanine (Ala) as amino donors. Using whole cell biocatalysis, the reaction with IPA was one order of magnitude faster than with Ala. No reaction was detected when permeabilized E. coli cells containing ω‐TA were employed using Ala as the amino donor. Additionally, the synthesis of APB from 4‐phenyl‐2‐butanone and IPA was studied. Whole and permeabilized cells containing ω‐TA and their immobilized LentiKats® counterparts showed similar initial reactions rates and yields in the reaction systems, indicating 100% of immobilization efficiency (observed activity/activity immobilized) and absence of diffusional limitations (due to the immobilization). Immobilization of whole and permeabilized cells containing ω‐TA in LentiKats® allowed improved stability as the biocatalyst was shown to be efficiently reused for five reaction cycles, retaining around 80% of original activity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Automated docking studies provide insights into molecular determinants of ligand recognition by N‐acetyl‐1‐d‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d‐glucopyranoside deacetylase (MshB)

The metal‐dependent deacetylase N‐acetyl‐1‐d ‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d ‐glucopyranoside deacetylase (MshB) catalyzes the deacetylation of N‐acetyl‐1‐d ‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d ‐glucopyranoside (GlcNAc‐Ins), the committed step in mycothiol (MSH) biosynthesis. MSH is the thiol redox buffer used by mycobacteria to protect against oxidative damage and is involved in the detoxification of xenobiotics. As such, MshB is a target for the discovery of new drugs to treat tuberculosis (TB). While MshB substrate specificity and inhibitor activity have been probed extensively using enzyme kinetics, information regarding the molecular basis for the observed differences in substrate specificity and inhibitor activity is lacking. Herein we begin to examine the molecular determinants of MshB substrate specificity using automated docking studies with a set of known MshB substrates. Results from these studies offer insights into molecular recognition by MshB via identification of side chains and dynamic loops that may play roles in ligand binding. Additionally, results from these studies suggest that a hydrophobic cavity adjacent to the active site may be one important determinant of MshB substrate specificity. Importantly, this hydrophobic cavity may be advantageous for the design of MshB inhibitors with high affinity and specificity as potential TB drugs. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 406–417, 2014.     

Structural and functional analyses of human tryptophan 2,3‐dioxygenase

Tryptophan 2,3‐dioxygenase (TDO), one of the two key enzymes in the kynurenine pathway, catalyzes the indole ring cleavage at the C2‐C3 bond of l ‐tryptophan. This is a rate‐limiting step in the regulation of tryptophan concentration in vivo, and is thus important in drug discovery for cancer and immune diseases. Here, we report the crystal structure of human TDO (hTDO) without the heme cofactor to 2.90 Å resolution. The overall fold and the tertiary assembly of hTDO into a tetramer, as well as the active site architecture, are well conserved and similar to the structures of known orthologues. Kinetic and mutational studies confirmed that eight residues play critical roles in l ‐tryptophan oxidation. Proteins 2014; 82:3210–3216. © 2014 Wiley Periodicals, Inc.     

Structure of the external aldimine form of PglE,an aminotransferase required for N,N'‐diacetylbacillosamine biosynthesis

N,N'‐diacetylbacillosamine is a novel sugar that plays a key role in bacterial glycosylation. Three enzymes are required for its biosynthesis in Campylobacter jejuni starting from UDP‐GlcNAc. The focus of this investigation, PglE, catalyzes the second step in the pathway. It is a PLP‐dependent aminotransferase that converts UDP‐2‐acetamido‐4‐keto‐2,4,6‐trideoxy‐d ‐glucose to UDP‐2‐acetamido‐4‐amino‐2,4,6‐trideoxy‐d ‐glucose. For this investigation, the structure of PglE in complex with an external aldimine was determined to a nominal resolution of 2.0 Å. A comparison of its structure with those of other sugar aminotransferases reveals a remarkable difference in the manner by which PglE accommodates its nucleotide‐linked sugar substrate.     

CD‐sensitive Zn‐porphyrin tweezer host‐guest complexes,part 1: MC/OPLS‐2005 computational approach for predicting preferred interporphyrin helicity

This article describes a computational study on dimeric zinc porphyrin tweezer complexes with primary/secondary amines and secondary alcohols that validates the use of Optimized Potential for Liquid Simulations (OPLS‐2005) as the lead computational choice for assisting the tweezer methodology in the absolute configurational assignment of organic compounds. A supramolecular, microscale approach known as the tweezer method has been widely applied in the past decade for determining the absolute configuration of chiral substrates that are difficult to study by other readily available methods. The method relies on a host/guest complexation mechanism between a porphyrin tweezer moiety and a substrate, after its conversion into a bidentate conjugate. The formation of 1:1 complexes is a stereodifferentiating process: upon complexation, the originally achiral tweezer adopts a preferential interporphyrin helicity, dictated by the absolute configuration of the chiral substrate. By correctly predicting the sign of the interporphyrin helicity in the complex, OPLS‐2005 provides a correlation between the observed circular dichroism (CD) signal and the absolute configuration of the substrate. It also offers a great degree of insight into the structural factors responsible for chiral recognition and the amplitude of exciton couplets. Moreover, the preferential binding sites between the Zn‐tweezer and secondary amine conjugates were revealed by using the new computational approach. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Structural studies on KijD1, a sugar C‐3′‐methyltransferase

Kijanimicin is an antitumor antibiotic isolated from Actinomadura kijaniata. It is composed of three distinct moieties: a pentacyclic core, a monosaccharide referred to as d ‐kijanose, and a tetrasaccharide chain composed of l ‐digitoxose units. d ‐Kijanose is a highly unusual nitro‐containing tetradeoxysugar, which requires at least ten enzymes for its production. Here we describe a structural analysis of one of these enzymes, namely KijD1, which functions as a C‐3′‐methyltransferase using S‐adenosylmethionine as its cofactor. For this investigation, two ternary complexes of KijD1, determined in the presence of S‐adenosylhomocysteine (SAH) and dTDP or SAH and dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐3‐methyl‐d ‐glucose, were solved to 1.7 or 1.6 Å resolution, respectively. Unexpectedly, these structures, as well as additional biochemical analyses, demonstrated that the quaternary structure of KijD1 is a dimer. Indeed, this is in sharp contrast to that previously observed for the sugar C‐3′‐methyltransferase isolated from Micromonospora chalcea. By the judicious use of site‐directed mutagenesis, it was possible to convert the dimeric form of KijD1 into a monomeric version. The quaternary structure of KijD1 could not have been deduced based solely on bioinformatics approaches, and thus this investigation highlights the continuing need for experimental validation.     

Crystal structure of the apo form of a β‐transaminase from Mesorhizobium sp. strain LUK

Pyridoxal 5′‐phosphate (PLP)‐dependent β‐transaminases (βTAs) reversibly catalyze transamination reactions by recognizing amino groups linked to the β‐carbon atoms of their substrates. Although several βTA structures have been determined as holo forms containing PLP, little is known about the effect of PLP on the conversion of the apo structure to the holo structure. We determined the crystal structure of the apo form of a βTA from Mesorhizobium sp. strain LUK at 2.2 Å resolution to elucidate how PLP affects the βTA structure. The structure revealed three major disordered regions near the active site. Structural comparison with the holo form also showed that the disordered regions in the apo form are ordered and partially adopt secondary structures in the holo form. These findings suggest that PLP incorporation into the active site contributes to the structural stability of the active site architecture, thereby forming the complete active site. Our results provide novel structural insights into the role of PLP in terms of active site formation.     

Effect of one D‐Leu residue on right‐handed helical ‐L‐Leu‐Aib‐ peptides in the crystal state

Four diastereomeric‐Leu‐Leu‐Aib‐Leu‐Leu‐Aib‐peptides, Boc‐D ‐Leu‐L ‐Leu‐Aib‐L ‐Leu‐L ‐Leu‐Aib‐OMe (1), Boc‐L ‐Leu‐D ‐Leu‐Aib‐L ‐Leu‐L ‐Leu‐Aib‐OMe (2), Boc‐L ‐Leu‐L ‐Leu‐Aib‐D ‐Leu‐L ‐Leu‐Aib‐OMe (3), and Boc‐L ‐Leu‐L ‐Leu‐Aib‐L ‐Leu‐D ‐Leu‐Aib‐OMe (4), were synthesized. The crystals of the four hexapeptides were characterized by X‐ray crystallographic analysis. Two diastereomeric hexapeptides 1 and 2 having D ‐Leu(1) or D ‐Leu(2) were folded into right‐handed (P) 3 ₁₀ ‐helical structures, while peptide 3 having D ‐Leu(4) was folded into a turn structure nucleated by type III′ and I$' \bf{\beta}$ ‐turns, and peptide 4 having D ‐Leu(5) was folded into a left‐handed (M) 3 ₁₀ ‐helical structure. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.