R-mandelic Acid Research Articles

Amino Acid-Based Molecular and Membranous Chiral Tools for Enantiomeric Recognition.

Given the need, both academic and industrial, for new approaches and technologies for chiral discrimination of enantiomers, the present work demonstrates the development through rational design and integration of two new chiral platforms (molecular and membranous) for enantioselective recognition through visual as well as microscopic observation. The molecular platform (TPT) is based on the tryptophan derivative developed through the condensation of two tryptophan units with terepthaloyl chloride. While TPT based on l-tryptophan recognizes R-mandelic acid over the S-isomer, the host with reverse chirality (TPDT) recognizes S-mandelic acid over R-isomer. The role of chemical functionality in this sensitive recognition process was established experimentally by developing an analogue of TPT and by judiciously using different chiral analytes. Importantly, a detailed theoretical study at the molecular level revealed the U-shaped conformation of TPT, creating a cavity for accommodating a chiral guest with selective functional interaction resulting in the discrimination of enantiomers. Finally, a chiral polymeric mat of poly(methyl methacrylate) (PMMA)/polyacrylonitrile (PAN) (2:3) impregnated with TPT was developed via electrospinning. The resulting fibrous mat was successfully utilized for chiral recognition through microscopic and architectural observation. Hence, the present work reports simple chiral tools for enantiomeric recognition.

Highly sensitive enantioselective spectrofluorimetric determination of R-/S-mandelic acid using l-tryptophan-modified amino-functional silica-coated N-doped carbon dots as novel high-throughput chiral nanoprobes

Amino-functional silica-coated N-doped carbon dots (NH2–SiO2-CDs) were covalently modified by l-tryptophan (chiral selector) by producing an amide bond between carboxyl groups of L-try and amino groups of NH2–SiO2-CDs to develop a novel high throughput chiral nanoprobes (L-try–CONH–SiO2-CDs) for highly sensitive and enantioselective quantification of S-/R-mandelic acid (S-/R-Man). The method showed a great difference between S- and R-isomers (enantioselectivity coefficient = 4.17) due to the ultra-stability of the Meisenheimer complex that was formed between S-isomer and nanoprobe (KS-Man/KR-man = 2122.7, where K is the binding-constant). At optimal experimental conditions, two linear ranges of 0.5–25.0 (LOD of 0.05 μM) and 0.5–22.0 μM (LOD of 0.27 μM) for S- and R-Man, respectively, along with an enhanced sensitivity toward S-isomer (about 5.7-fold higher than R-isomer) were attained. High selectivity for the determination of mandelic acid was achieved compared to metal ions, amino acids, and sugars that commonly coexist with it. Intra-day as well as inter-day assays, respectively, showed RSD values of about 3.2 and 3.9%. The mechanistic studies were performed for proving the enantioselective behavior of the developed nanoprobe. The method was then used for S-/R-mandelic acid determination in bio-samples. The figures of merit for the method were found to be better than those already reported for enantioselective detection of R-/S-Man.

Measurement of Minute Liquid Volumes of Chiral Molecules Using In-Fiber Polarimetry.

We report an optofluidic method that enables to efficiently measure the enantiomeric excess of chiral molecules at low concentrations. The approach is to monitor the optical activity induced by a Kagome-lattice hollow-core photonic crystal fiber filled with a sub-μL volume of chiral compounds. The technique also allows monitoring the enzymatic racemization of R-mandelic acid.

Efficient biosynthesis of (R)-mandelic acid from styrene oxide by an adaptive evolutionary Gluconobacter oxydans STA

Background(R)-mandelic acid (R-MA) is a highly valuable hydroxyl acid in the pharmaceutical industry. However, biosynthesis of optically pure R-MA remains significant challenges, including the lack of suitable catalysts and high toxicity to host strains. Adaptive laboratory evolution (ALE) was a promising and powerful strategy to obtain specially evolved strains.ResultsHerein, we report a new cell factory of the Gluconobacter oxydans to biocatalytic styrene oxide into R-MA by utilizing the G. oxydans endogenous efficiently incomplete oxidization and the epoxide hydrolase (SpEH) heterologous expressed in G. oxydans. With a new screened strong endogenous promoter P12780, the production of R-MA was improved to 10.26 g/L compared to 7.36 g/L of using Plac. As R-MA showed great inhibition for the reaction and toxicity to cell growth, adaptive laboratory evolution (ALE) strategy was introduced to improve the cellular R-MA tolerance. The adapted strain that can tolerate 6 g/L R-MA was isolated (named G. oxydans STA), while the wild-type strain cannot grow under this stress. The conversion rate was increased from 0.366 g/L/h of wild type to 0.703 g/L/h by the recombinant STA, and the final R-MA titer reached 14.06 g/L. Whole-genome sequencing revealed multiple gene-mutations in STA, in combination with transcriptome analysis under R-MA stress condition, we identified five critical genes that were associated with R-MA tolerance, among which AcrA overexpression could further improve R-MA titer to 15.70 g/L, the highest titer reported from bulk styrene oxide substrate.ConclusionsThe microbial engineering with systematic combination of static regulation, ALE, and transcriptome analysis strategy provides valuable solutions for high-efficient chemical biosynthesis, and our evolved G. oxydans would be better to serve as a chassis cell for hydroxyl acid production.

Photodetachment of Deprotonated R-Mandelic Acid: The Role of Proton Delocalization on the Radical Stability.

The photodetachment and stability of R-Mandelate, the deprotonated form of the R-Mandelic acid, was investigated by observing the neutral species issued from either simple photodetachment or dissociative photodetachment in a cold anions set-up. R-Mandalate has the possibility to form an intramolecular ionic hydrogen-bond between adjacent hydroxyl and carboxylate groups. The potential energy surface along the proton transfer (PT) coordinate between both groups (O- …H+ …- OCO) features a single local minima, with the proton localized on the O- group (OH…- OCO). However, the structure with the proton localized on the - OCO group (O- …HOCO) is also observed because it falls within the extremity of the vibrational wavefunction of the OH…- OCO isomer along the PT coordinate. The stability of the corresponding radicals, produced upon photodetachment, is strongly dependent on the position of the proton in the anion: the radicals produced from the OH…- OCO isomer decarboxylate without barrier, while the radicals produced from the O- …HOCO isomer are stable.

Optimization of the medium composition and product extraction for R-mandelic acid using recombinant Escherichia coli expressing Alcaligenes sp. nitrilase

The aim of this study is to investigate the effects of the components of the medium on nitrilase expression in recombinant Escherichia coli ( E. coli), which is applied in the hydrolysis of racemic mandelonitrile (MN) to R-mandelic acid (MA). In addition, the separation of R-MA from the reaction mixture is studied. The glycerol medium is screened, and compositions are optimized in single-factor experiments. The nitrilase activity is 1.96-fold higher than before using the optimal medium containing peptone 15 g/L, yeast extract 12 g/L, NaCl 10 g/L, glycerol 15 g/L, (NH4)2SO4 5 g/L, KH2PO4 10 mM, K2HPO4·3H2O 10 mM, and MgSO4·7H2O 15 mM. During the acid-base extraction of R-MA, the optimal alkaline pH, the alkaline extractant ratio, the optimal acidic pH, the acidic extractant ratio and the amount of activated carbon are 10.0, 1:0.5 (once), 2.50, 1:2 (twice) and 1%, respectively. This study provides a basis for the industrial production of R-MA.

Magnetic Molecularly Imprinted Polymers Based on Dehydroabietylamine as Chiral Monomers for the Enantioseparation of RS-Mandelic Acid.

Stereoselective adsorption of the enantiomers shows potential in the resolution of a racemate. In this work, we synthesized novel magnetic surface molecularly imprinted polymers (MIPs) on the surface of the γ-methacryloxypropyltrimethoxysilane (MPS)-modified Fe3O4@SiO2 particles to utilize chiral dehydroabietylamine (DHA) as a functional monomer and R-mandelic acid as a template molecule (DHA-MIPs). We performed the resolution of mandelic acid racemate (RS-MA) via adsorption on the as-prepared MIPs. The results revealed that the MIPs have good affinity and high adsorption capacity for R-MA and show better enantioselective adsorption ability for R-MA than that for S-MA. One-stage adsorption of RS-MA on the MIPs can achieve up to 53.7% enantiomeric excess (ee) for R-MA. These help us to improve the chiral separation ability of the traditional MIPs using a chiral rather than an achiral monomer in MIP preparation. The MIPs can be employed as an economic and efficient adsorbent for chiral separation of MA racemate.

Constitutive secretory expression and characterization of nitrilase from Alcaligenes faecalis in Pichia pastoris for production of R-mandelic acid.

Nitrilases can directly hydrolyze nitrile compounds into carboxylic acids and ammonium. To solve the current problems of bioconversions using nitrilases, including the difficult separation of products from the resting cells used as the catalyst and high costs of chemical inducers, a nitrilase from Alcaligenes faecalis was heterologously expressed in Pichia pastoris X33. The stable nitrilase-expressing strain No.39-6-4 was obtained after three rounds of screening based on a combined detection method including dot-blot, SDS-PAGE, and western blot analyses, which confirmed the presence of recombinant nitrilase with a molecular mass of about 50 kDa. The temperature and pH optima of the nitrilase were 45°C and pH 7.5, respectively. Cu2+ , Zn2+ , and Tween 80 strongly inhibited the enzyme activity, but the optical purity of the product R-mandelic acid (R-MA) was stable, with practically 100% enantiomeric excess (ee). The nitrilase-producing P. pastoris strain developed in this study provides a basis for further research on the enzyme.

Adsorption of mandelic acid enantiomers on chiral stationary phase with grafted antibiotic eremomycin: The effect of the eluent pH

The adsorption of mandelic acid enantiomers on the Nautilus-E chiral stationary phase with eremomycin as a grafted antibiotic, as affected by value of the water-ethanol eluent under conditions of an overloaded column, was studied. Adsorption equilibrium under experimental conditions, when mandelic acid dissociates both completely and then very weakly, was studied. The adsorption isotherms of S- and R-mandelic acid were plotted by the Glueckauf method and inverse method using bi-Langmuir model, as well as the tri-Langmuir model assuming independent adsorption on three types of adsorption centers, with two of them being high-energy enantioselective (ion-exchange and molecular adsorption) and the third – the low-energy nonselective types of sites. This model takes into account the ionization of mandelic acid in the eluent and a change in the value in the mass transfer zone caused by its ionization. The saturated capacity and binding energy of all the types of adsorption sites depend on of the eluent. Nonselective interactions contribute to the adsorption of mandelic acid mainly. To determine the adsorption isotherms of mandelic acid enantiomers on Nautilus-E CSP in the absence of its dissociation and distortion of the elution profiles, the Glueckauf method and the bi-Langmuir model were identified as an acceptable choice.

Upscale production of (R)-mandelic acid with a stereospecific nitrilase in an aqueous system.

(R)-Mandelic acid (R-MA) is a key precursor for the synthesis of semi-synthetic penicillin, cephalosporin, anti-obesity drugs, antitumor agents, and chiral resolving agents for the resolution of racemic alcohols and amines. In this study, an enzymatic method for the large-scale production of R-MA by a stereospecific nitrilase in an aqueous system was developed. The nitrilase activity ofthe Escherichia coli BL21(DE3)/pET-Nit whole cells reached 138.6U/g in a 20,000-L fermentor. Using recombinant E. coli cells as catalyst, 500mM R,S-mandelonitrile (R,S-MN) was resolved into 426mM (64.85g/L) R-MA within 8h, and the enantiomeric excess (ee) value of R-MA reached 99%. During the purification process, pure R-MA with a recovery rate of 78.8% was obtained after concentration and crystallization. This study paved the foundation for the upscale production of R-MA using E. coli whole cells as biocatalyst.

Discovery of a diverse set of esterases from hot spring microbial mat and sea sediment metagenomes

Esterases are an important group of biocatalysts for synthetic organic chemistry. Functional metagenomics allows discovery of novel biocatalysts by providing access to the gene pool of the microbial community of a habitat. Two metagenomic libraries representing the gene pool of sea sediment and hot spring microbial mat were constructed. Functional screening of these libraries resulted in the isolation of total 8 clones with tributyrin hydrolytic activity. Sequence analysis revealed 10 putative lipolytic proteins with 42–99% homology to the protein sequences in the databases, nine of which represented six known esterase families. Four of the encoded proteins represented Family V and amongst others, one each represented the Family VIII, pectin acetylesterase, enterobactin esterase, G-D-S-L family and OsmC domain containing esterase. One unusual lipolytic protein possessed poly-(3-hydroxybutyrate) depolymerase domain fused to lipase/esterase domain. Two phylogenetically related esterases (MLC3 and SLC5) belonging to family V were expressed and purified to homogeneity. The enzymes exhibited environment-adapted temperature optimum and thermostability. MLC3 was able to stereoselectively hydrolyze R-methyl mandelate to produce R-mandelic acid, an important chiral building block, which suggests MLC3 has potential commercial application.

Adsorption of mandelic acid enantiomers on a chiral stationary phase with a grafted antibiotic eremomycin

Adsorption of mandelic acid enantiomers on the Nautilus-E chiral stationary phase with eremomycin as a grafted antibiotic, as influenced upon by composition of the water–ethanol mobile phase, was studied. The surface of the adsorbent was evinced to be heterogeneous and to have two types of adsorption sites: the enantioselective and nonselective ones. Nonselective interactions contribute to adsorption of mandelic acid mainly. With the ethanol fraction in the eluent increased from 40 vol% to 60 vol%, contribution of nonselective interactions to adsorption was increase. The adsorption isotherms of S- and R-mandelic acid were plotted by the Glueckauf method and described by the bi-Langmuir and Henry–Langmuir isotherm models. The Henry–Langmuir model was found out to adequately describe the experimental equilibrium findings in the concentration range under study. The temperature effect on the adsorption equilibrium was discussed. The thermodynamic characteristics of enantioselective adsorption sites were characterized by their adsorption constants, saturation capacities as influenced upon by temperature. A significant increase in the true enantioselectivity at the high-energy adsorption sites with an increase in temperature was revealed. Retention and recognition of mandelic acid enantiomers on the enantioselective adsorption sites were governed by the entropy term.

Enantioselective separation of RS-mandelic acid using β-cyclodextrin modified Fe3O4@SiO2/Au microspheres.

Chiral magnetic microspheres show great potential in direct enantioseparation. Herein novel chiral magnetic microspheres were prepared by self-assembling β-cyclodextrin on magnetic Fe3O4@SiO2/Au composite microspheres. The prepared materials were characterized by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), vibrating sample magnetometry (VSM) and Fourier transform infrared spectroscopy (FTIR). The results showed that the functional microspheres have a three-ply structure and high saturation magnetization. β-Cyclodextrin-immobilized magnetic Fe3O4@SiO2/Au microspheres (Fe3O4@SiO2/Au/β-CD) were applied in the direct chiral separation of RS-mandelic acid. The analysis results of HPLC revealed that the Fe3O4@SiO2/Au/β-CD magnetic microspheres exhibited stronger enantioselective adsorption for R-mandelic acid than S-mandelic acid. The chiral selector functionalized magnetic microspheres are therefore expected to be an efficient and economical chiral separation method for use in further research.

Separation of chiral compounds using magnetic molecularly imprinted polymer nanoparticles as stationary phase by microchip capillary electrochromatography.

In this work, a simple and rapid approach was developed for separation and detection of chiral compounds based on a magnetic molecularly imprinted polymer modified poly(dimethylsiloxane) (PDMS) microchip coupled with electrochemical detection. Molecularly imprinted polymers were prepared employing Fe3 O4 nanoparticles (NPs) as the supporting substrate and norepinephrine as the functional monomer in the presence of template molecule in a weak alkaline solution. After extracting the embedded template molecules, Fe3 O4 @polynorepinephrine NPs (MIP-Fe3 O4 @PNE NPs) showed specific molecular recognition selectivity and high affinity towards the template molecule, which were then used as stationary phase of microchip capillary electrochromatography for chiral compounds separation. Mandelic acid and histidine enantiomers were used as model compounds to test the chiral stationary phase. By using R-mandelic acid as the template molecule, mandelic acid enantiomer was effectively separated and detected on the MIP-Fe3 O4 @PNE NPs modified PDMS microchip. Moreover, the successful separation of histidine enantiomers on the MIP-Fe3 O4 @PNE NPs modified microchip using L-histidine as template molecule was also achieved.

Production of R-Mandelic Acid Using Nitrilase from Recombinant E. coli Cells Immobilized with Tris(Hydroxymethyl)Phosphine.

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH8.0 and 55°C. The half-lives of THP-immobilized cells measured at 35, 40, and 50°C were 1800, 965, and 163h, respectively. The concentration of R-mandelic acid (R-MA) reached 358mM after merely 1-h conversion by the immobilized cells with 500mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307gL-1day-1 and the space-time productivity of 143.2mmolL-1h-1g-1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100mM R,S-MN as substrate at 40°C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.

Enantioselective electrochemical sensor for R-mandelic acid based on a glassy carbon electrode modified with multi-layers of biotin-loaded overoxidized polypyrrole and nanosheets of reduced graphene oxide

The authors describe an electrochemical method for quantitation of R-mandelic acid (R-MA). A glassy carbon electrode (GCE) was modified by electrochemical deposition of several layers of D-(+)-biotin-loaded overoxidized polypyrrole (OPPy-biot) on nanosheets of reduced graphene oxide. The deposited film was characterized by scanning electron microscopy, differential pulse voltammetry, cyclic voltammetry and electrochemical impedance spectroscopy. The modified GCE is shown to enable stereoselective recognition of R-MA even in the presence of high concentrations of S-mandelic acid (S-MA), best at a voltage of around 1.5 V (vs. Ag/AgCl). The analytical performance of the electrode was studied by differential pulse voltammetry which revealed a linear range that extends over the 5 to 80 mM R-MA concentration range, a 1.5 mM detection limit, and an enanotiospecific characteristic toward R-MA. Under optimum conditions, the method displays high reproducibility and stability. It was applied to the determination of R-MA in a synthetic mixture and no interference was observed by S-MA.

Total Synthesis of the Bis-silyl Ether of (+)-15-epi-Aetheramide A.

Synthesis of the macrolactone depsipeptide aetheramide A was attempted by three different approaches. The first approach to form the macrolactone involving macrolactonization to form the C1-C21 bond and the second approach using a ring-closing metathesis (RCM) strategy to form the C10-C11 olefinic bond failed. The third approach starting from R-mandelic acid, involving the RCM reaction to install the C18-C19 ring junction, was successful in assembling the macrolactone.

R-mandelic acid production with immobilized recombinant Escherichia coli cells in a recirculating packed bed reactor

A recirculating packed bed reactor (RPBR) was used for efficient production of R-mandelic acid (R-MA) by kinetic resolution of racemic R,S-mandelonitrile (R,S-MN) using the recombinant E. coli cells crosslinked with diatomite (DA)/glutaraldehyde (GA)/polyethyleneimine (PEI). The performance and productivity of RPBR were evaluated by several parameters, including cell load, substrate feeding rate, height diameter (H/D) ratio, reactor structures, and operation stability. The kinetic resolution process showed higher initial reaction rate (1.52 mM/min) and yield (100%) by recycling 100 mL of substrate solution (70 mM) through RPBR packed with 6.0 g immobilized cells at a substrate-feeding rate of 19 mL/min while the H/D ratio was 2.8. The immobilized cells were successfully applied into kinetic resolution of R,S-MN in the RPBR for 50 batches with an average productivity of 4.12 g/L/h for R-MA with >99% of enantiomeric excess.

Salt or cocrystal of salt? Probing the nature of multicomponent crystal forms with infrared spectroscopy

The recognition of the nature of a multicomponent crystal form (solvate, salt, cocrystal or cocrystal of salt) is of great importance for pharmaceutical industry because it is directly related to the performance of a pharmaceutical ingredient, since there is interdependence between the structure, its energy and its physical properties. In this context, here we have identified the nature of multicomponent crystal forms of the anti-HIV drug lamivudine with mandelic acid through infrared spectroscopy. These investigated crystal forms were the known S-mandelic acid cocrystal of lamivudine R-mandelate trihydrate (1), a cocrystal of salt, and lamivudine R-mandelate (2), a salt. This approach also supports the identification and distinction of both ionized and unionized forms of mandelic acid in the infrared spectrum of 1. In this way, infrared spectroscopy can be useful to distinguish a cocrystal of salt from either salt or cocrystal forms. In the course of this study, for the first time we have also characterized and determined the crystal structure of R-mandelic acid cocrystal of sodium R-mandelate (3).

Racemization of undesired enantiomers: Immobilization of mandelate racemase and application in a fixed bed reactor.

Production of optically pure products can be based on simple unselective synthesis of racemic mixtures combined with a subsequent separation of the enantiomers; however, this approach suffers from a 50% yield limitation which can be overcome by racemization of the undesired enantiomer and recycling. Application of biocatalyst for the racemization steps offers an attractive option for high-yield manufacturing of commercially valuable compounds. Our work focuses on exploiting the potential of racemization with immobilized mandelate racemase. Immobilization of crude mandelate racemase via covalent attachment was optimized for two supports: Eupergit(®) CM and CNBr-activated Sepharose 4 Fast Flow. To allow coupling of enzymatic reaction with enantioselective chromatography, a mobile phase composition compatible with both processes was used in enzymatic reactor. Kinetic parameters obtained analyzing experiments carried out in a batch reactor could be successfully used to predict fixed-bed reactor performance. The applicability of the immobilized enzyme and the determined kinetic parameters were validated in transient experiments recording responses to pulse injections of R-mandelic acid. The approach investigated can be used for futher design and optimization of high yield combined resolution processes. The characterized fixed-bed enzymatic reactor can be integrated e.g. with chromatographic single- or multicolumn steps in various configurations.