Determination of L- and D-enantiomers of methotrexate using amperometric biosensors

In order to determine the enantiopurity of methotrexate (Mtx), seven biosensors were proposed for the assay of l-Mtx and three biosensors for the assay of d-Mtx. The biosensors were designed using physical and chemical immobilization of glutamate oxidase and/or l-amino acid oxidase (l-AAOD) and/or horseradish peroxidase (HRP) for the assay of l-methotherexate, and d-amino acid oxidase (d-AAOD) and HRP for the assay of d-Mtx. Electrode characteristics were obtained and compared for the different carbon paste based biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol l⁻¹ to pmol l⁻¹, magnitude order with limits of detection in the fmol l⁻¹ to nmol l⁻¹ concentration range. All biosensors were successful for the determination of the enantiopurity of Mtx as raw material, and in its pharmaceutical formulations (tablets and injections).     

Chirality conversion and enantioselective extraction of amino acids by imidazolium-based binol-aldehyde

A novel imidazolium-based binol receptor 4 has been synthesized and used as a chirality conversion reagent for general amino acids with higher d-form selectivity compared to other guanidinium-based receptors. Favorable solubility in chloroform enabled 4 as an effective chiral extractant for the resolution of racemic amino acids.     

A d,l-proline catalyzed diastereoselective trimolecular condensation: an approach to the one-pot synthesis of perhydrofuro[3,2-b]pyran-5-ones

d,l-Proline was found to catalyze efficiently the one-pot trimolecular condensation of indoles, a sugar hydroxyaldehyde, and Meldrum’s acid followed by intramolecular cyclization with evolution of carbon dioxide and elimination of acetone to afford 7-(1H-3-indolyl)-2,3-dimethoxyperhydrofuro[3,2-b]pyran-5-ones. The reaction proceeded cleanly at ambient temperature to afford the products in good yields with high diastereoselectivity.     

Selection of synthetic receptors capable of sensing the difference in binding of d-Ala-d-Ala or d-Ala-d-Lac ligands by screening of a combinatorial CTV-based library

Screening of a combinatorial CTV-based artificial, synthetic receptor library 1 {1-13, 1-13, 1-13} for binding of a variety d-Ala-d-Ala and d-Ala-d-Lac containing ligands (6-11) was carried out in phosphate buffer (0.1 N, pH=7.0). After screening and Edman sequencing, synthetic receptors were found containing amino acid sequences, which are either characteristic for binding dye labeled d-Ala-d-Ala or d-Ala-d-Lac containing ligands. For example, receptors capable of binding d-Ala-d-Ala containing ligands 6, 7, 9 and 11 contained—almost in all cases—at least one basic amino acid residue—predominantly Lys—in their arms. This was really a striking difference with the arms of the receptors capable of binding d-Ala-d-Lac containing ligands 8 and 10, which usually contained a significant number of polar amino acids (Gln and Ser), especially in ligand 8, but hardly any basic amino acids. Use of different (fluorescent) dye labels showed that the label has a profound, albeit not decisive, influence on the binding by the receptor. A hit from the screening of the CTV-library with FITC-peptidoglycan (6) was selected for resynthesis and validation.     

A molecular half-subtractor based on a fluorescence and absorption dual-modal sensor for copper ions

A l-phenyl alanine derivative (L) could be used as a fluorescence and absorption dual-modal sensor, which was highly sensitive and selective to copper ion at physiological pH. We have drawn attention to the use of its copper complex to acquire a half-subtractor with parallel operating INHIBIT and XOR logic gates, by monitoring, fluorescence and absorbance as output signals, respectively.     

Highly selective recognition of monosaccharide based on two-component system in aqueous solution

A highly selective switch for d-fructose was formed by water-soluble conjugated polymer PP-S-BINOL and tetraboronic acid functionalized benzyl viologen ToBV. The two-component system showed a high selectivity and sensitivity only for d-fructose in familiar d-monosaccharides. The high selectivity of the sensing system for d-fructose may be depended on stable pyranose ester form of d-fructose with ToBV. A desirable linear response of the sensing system to low concentrations of d-fructose (<10.0 mM) was observed with 0.9936 linear dependent coefficient at pH 7.4.     

Novel practical synthesis of d-cycloserine

The synthesis of d-cycloserine has been successfully accomplished from the readily available d-serine through three simple and efficient routes. In each synthetic strategy, cyclization reactions are involved as the key step, and one-pot processes are employed. The simple treatment and mild reaction conditions are attractive features in this methodology.     

Improved synthesis of 6-amino-6-deoxy-d-galactono-1,6-lactam and d-mannono-1,6-lactam from corresponding unprotected d-hexono-1,4-lactones

Regioselective bromination of unprotected d-galactono-1,4-lactone and d-mannono-1,4-lactone with PPh₃/CBr₄ led to 6-bromo-6-deoxy derivatives. These intermediates were treated with LiN₃ and hydrogenated to give 6-amino-6-deoxy-d-galactono-1,6-lactam (8) and 6-amino-6-deoxy-d-mannono-1,6-lactam (13) in 74 and 67% overall yield, respectively.     

A convenient synthesis of l-ribose from d-fructose

An efficient method for the stereoselective synthesis of l-ribose was accomplished starting from commercially inexpensive d-fructose. The intermediates in the process can serve as versatile precursors for the preparation of l-nucleoside analogues.     

A Simple Route for Synthesis of 4-Phospho-D-Erythronate

4-Phospho-d-erythronate is an intermediate in the synthesis of pyridoxal 5′-phosphate in some bacteria and an inhibitor of ribose 5-phosphate isomerase. Previous synthetic schemes for the preparation of 4-phospho-d-erythronate required expensive precursors and typically gave low yields. We report a straightforward synthesis of 4-phospho-d-erythronate from the inexpensive precursor d-erythronolactone in five steps with a preparatively useful yield of 22%.     

l-Proline-catalyzed diastereoselective synthesis of cis-isoquinolonic acids and evaluation of their neuroprotective effects

l-Proline efficiently catalyzes the one-pot, three-component reaction of homophthalic anhydride, amines, and aldehydes in CH₃CN at room temperature, to afford the corresponding cis-isoquinolonic acids in high yields and with excellent diastereoselectivities. The synthesized compounds are screened for neuroprotective activities.     

Concise and practical synthesis of (2S,3R,4R,5R) and (2S,3R,4R,5S)-1,6-dideoxy-1,6-iminosugars

The syntheses of (2S,3R,4R,5R) and (2S,3R,4R,5S)-1,6-dideoxy-1,6 iminosugars 1a and 1b, respectively, from d-glucose are described. The key transformations in this reaction sequence include regio-selective epoxide ring opening with N-benzylamine followed by intramolecular reductive amination of amino-aldehyde.     

One-pot inversion of d-mannono-1,4-lactone for the practical synthesis of l-ribose

l-Ribose was synthesized in a concise manner from d-mannono-1,4-lactone using one-pot inversion conditions. Treatment of d-mannono-1,4-lactone with piperidine, followed by mesylation-induced S_N2-type O-alkylation, afforded the desired one-pot inversion in an optimum yield, and the following straightforward transformations provided l-ribose in good yields.     

Combinatorial solid-phase synthesis and screening of a diverse tripodal triazacyclophane (TAC)-based synthetic receptor library showing a remarkable selectivity towards a d-Ala-d-Ala containing ligand

A large and diverse library of a TAC-based tripodal synthetic receptor library (6) has been prepared by split-mix synthesis on the solid phase. Each receptor of the 46,656-member TAC-based library (6) is attached to a solid support bead and contains three different dipeptide arms. On-bead screening for binding of a d-Ala-d-Ala containing ligand (7) by the TAC-based library (6) was performed in phosphate buffer (0.2 N, pH=7.0). Remarkable selectivity for particular library members was observed. The best binding members from the screening were manually selected using fluorescence microscopy and subjected to structural analysis by sequencing. The thus determined binding sequences showed a high consensus.     

Solid-phase synthesis of peptide ribonucleic acids (PRNA)

The range of available peptide ribonucleic acid (PRNA) monomers was fully expanded for the use in solid-phase synthesis of PRNA oligomers, which were designed to reversibly control the recognition and complexation behavior of the complementary DNA/RNA by external factors. A couple of PRNA 12-mers with desired purine-pyrimidine mixed sequences were prepared indeed in high yields by the solid-phase synthesis.     

Microbial screening in hydroxylation of l-proline

Microbial screening of 250 wild type strains resulted in identification of five strains with the activity of prolyl hydroxylase. All five strains hydroxylated regioselectively and enantioselectively l-proline into 4(R)-trans-hydroxy-l-proline 1. The best conversions were obtained with a wild type of Aeromonas caviae. 3-Hydroxylase activity was not detected.     

Studies on the Conformation of Polyhydroxyalkylpyrazolo (3,4-b) Quinoxalines

¹H NMR spectral studies on polyhydroxyalkylpyrazolo-(3,4-b)-quinoxaline derivatives of sugars having five to seven carbon atoms have shown that the polyhydroxyalkyl chain exists in a conformation in which a hydrogen bond is formed between the l'-hydroxyl group and N-2 of the pyrazolo(3,4-b)-quinoxaline base moiety. The polyhydroxyalkyl chain exists in the planar zigzag conformation, where no 1,3 syn axial interaction is present. Substitution in the base moiety did not change the extent of the population of the planar zigzag conformation.     

Macrocyclic antibiotics as chiral selectors in the design of enantioselective, potentiometric membrane electrodes for the determination of l- and d-enantiomers of methotrexate

Three enantioselective, potentiometric membrane electrodes (EPMEs) based on macrocyclic glycopeptide antibiotics—vancomycin and teicoplanin (modified or not with acetonitrile)—were proposed for the determination of l- and d-enantiomers of methotrexate (Mtx). The linear concentration ranges for the proposed enantioselective membrane electrodes were between 10⁻⁶ and 10⁻³ mol l⁻¹ for l- and d- methotrexate. The slopes of the electrodes were 58.00 mV/pl-Mtx for vancomycin-based electrode; 57.60 mV/pd-Mtx for teicoplanin-based electrode and 55.40 mV/pd-Mtx for teicoplanin modified with acetonitrile-based electrode. The detection limits of the proposed electrodes were of 10⁻⁸ mol l⁻¹ magnitude order. The surfaces of the electrodes are stable and easily renewable by polishing on alumina paper. All proposed electrodes proved to be successful for the determination of the enantiopurity of Mtx as raw material and of its pharmaceutical formulations (tablets and injections).     

Effects of molecular weight and small amounts of d-lactide units on hydrolytic degradation of poly(l-lactic acid)s

Films of poly(l-lactic acid) (PLLA) with different number-average molecular weights (M_n) and d-lactide unit contents (X_d) were made amorphous and the effects of molecular weight and small amounts of d-lactide units on the hydrolytic degradation behavior in phosphate-buffered solution at 37 °C of PLLA were investigated. The degraded films were investigated using gravimetry, gel permeation chromatography, polarimetry, differential scanning calorimetry, X-ray diffractometry, and tensile testing. To exclude the effects of crystallinity on the hydrolytic degradation, the films were made amorphous by melt-quenching. The incorporation of small amounts of d-lactide units drastically enhanced the hydrolytic degradation of PLLA. In the period of 0-32 weeks, the hydrolytic degradation rate constant (k) of PLLA films increased with increasing X_d, while the k values did not depend on M_n. This means that the effects of X_d on the hydrolytic degradation rate of the films are higher than those of M_n. In contrast, in the period of 32-60 weeks neither X_d nor M_n was a crucial parameter to determine k values, probably because in addition to these parameters the differences in the amount of catalytic oligomers accumulated in films and crystallinity affect the hydrolytic degradation behavior of the films. The initially amorphous PLLA films remained amorphous even after the hydrolytic degradation for 60 weeks.     

d-Poly(lactide) and LHRH decapeptide stereointeractions investigated by vibrational spectroscopy

The interactions between poly(d-lactic acid) (PDLA) and l-configured leuprolide (LHRH) to form heterocomplexes were investigated by a combination of infrared (IR), Raman and near infrared spectroscopy (NIR). It was found that an α crystal with 10₃ helical conformation of PDLA is formed in the PDLA/LHRH heterocomplexes with various LHRH loadings, whereas the secondary structures of LHRH in these heterocompelexes are greatly affected by the blend ratio of LHRH and PDLA. Based on the analysis of IR, Raman and NIR spectra of the various heterocomplexes, it is suggested that stereoselective Van der Waals interactions, consisting of interwined α helices of PDLA and LHRH, is responsible for the driving force of PDLA and LHRH stereocomplexation. To clarify the solid structure and the potential interaction in these heterocomplexes with various LHRH loadings, temperature-dependent IR spectral measurements were also employed. On the basis of the results presented, a model for the PDLA and LHRH stereointeraction process was proposed.