D-pipecolic Acid Research Articles

Joint analysis of transcriptome and metabolome revealed the difference in carbohydrate metabolism between super hybrid rice and conventional rice under salt stress

With the increasingly severe problem of soil salinization worldwide, exploring the molecular mechanism of salt tolerance of super hybrid rice has become an important scientific issue. In this study, the super hybrid rice Chaoyou1000 was used as the focus of attention, and the conventional rice Huanghuazhan was used as the control to explore the molecular mechanism of salt tolerance in super hybrid rice. The joint analysis of transcriptome and metabolome found 4661 DEGs, of which 2130 were up-regulated and 2531 were down-regulated; 70 named differential metabolites were found in the positive ion mode, and 32 were found in the negative ion mode. KEGG enrichment analysis showed that the carbohydrate metabolism-related pathways enriched by DEGs and differential metabolites were “starch and sucrose metabolism”, “galactose metabolism”, “glyoxylate and dicarboxylate metabolism”, and “citrate cycle (TCA cycle)”. Specifically, we found that some critical genes involved in carbohydrate metabolism, such as LOC4330753, LOC4325448, and LOC4341069, were up-regulated; some key metabolites, such as cis-aconitate, sucrose, and raffinose, were up-regulated. The difference in the expressions of these critical genes and metabolites between the two varieties is one of the most important reasons for the high resistance of Chaoyou1000. In addition, this study found that a large number of DEGs had a strong correlation with D-pipecolic acid, niveusin C, Gly-Val Ala-Ile, 2-(3-hydroxyphenyl)ethanol 1′-glucoside, 2,6-dimethyl-7-octene-1,6-diol 8-O-glucoside, (+)-syringaresinol, 2-indanone, 2-keto-6-acetamidocaproate, 3-dehydroquinate, IRETOL, and estrone glucuronide, suggesting that these genes and corresponding metabolites may exist with functional associations and regulatory relationships. The results of this study will provide a reference for the breeding of salt-tolerant rice.

Effects of different harvest frequencies on microbial community and metabolomic properties of annual ryegrass silage.

In this study, we analyzed the fermentation quality, microbial community, and metabolome characteristics of ryegrass silage from different harvests (first harvest-AK, second harvest-BK, and third harvest-CK) and analyzed the correlation between fermentative bacteria and metabolites. The bacterial community and metabolomic characteristics were analyzed by single-molecule real-time (SMRT) sequencing and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS), respectively. After 60 days of ensiling, the pH of BK was significantly lower than those of AK and CK, and its lactic acid content was significantly higher than those of AK and CK. Lactiplantibacillus and Enterococcus genera dominate the microbiota of silage obtained from ryegrass harvested at three different harvests. In addition, the BK group had the highest abundance of Lactiplantibacillus plantarum (58.66%), and the CK group had the highest abundance of Enterococcus faecalis (42.88%). The most annotated metabolites among the differential metabolites of different harvests were peptides, and eight amino acids were dominant in the composition of the identified peptides. In the ryegrass silage, arginine, alanine, aspartate, and glutamate biosynthesis had the highest enrichment ratio in the metabolic pathway of KEGG pathway enrichment analysis. Valyl-isoleucine and glutamylvaline were positively correlated with Lactiplantibacillus plantarum. D-Pipecolic acid and L-glutamic acid were positively correlated with Levilactobacillus brevis. L-phenylalanyl-L-proline, 3,4,5-trihydroxy-6-(2-methoxybenzoyloxy) oxane-2-carboxylic acid, and shikimic acid were negatively correlated with Levilactobacillus brevis. In conclusion, this study explains the effects of different harvest frequencies on the fermentation quality, microbial community, and metabolites of ryegrass, and improves our understanding of the ensiling mechanisms associated with different ryegrass harvesting frequencies.

Enzymatic Synthesis of D-pipecolic Acid by Engineering the Substrate Specificity of Trypanosoma cruzi Proline Racemase and Its Molecular Docking Study

Pipecolic acid is an unnatural amino acid mostly used for pharmaceutical purposes. Pipecolic acid has two types of enantiomers with different roles in the synthesis of drugs. The development of efficient catalytic methods for the production of enantiopure pipecolic acid is currently a crucial topic of research. Few chemo- or biosynthetic methods have been proposed for the synthesis of pure enantiomers; however, enzymatic conversion of the chirality of pipecolic acid has not been demonstrated because no pipecolic acid racemase has been reported yet. In this work, we attempted to engineer pipecolic acid racemase activity into Trypanosoma cruzi proline racemase (TcPRAC) for the enzymatic synthesis of D-pipecolic acid from L-pipecolic acid. For the binding of pipecolic acid (C6 ring) into the active site of TcPRAC, which was optimized for the original substrate proline (C5 ring), four bulky aromatic residues (Phe102, Phe120, Phe220, and Phe 290) of TcPRAC were mutated to smaller hydrophobic residues. Among the mutants, six single-point mutants (F102A, F102I, F102L, F102V, F290L, and F290V) exhibited significant racemase activity against L-pipecolic acid. The most efficient variant, F102V, showed 74% racemization. Molecular docking simulations revealed that lowering the binding energy of L-pipecolic acid to the active site was important for achieving high racemization activity of TcPRAC mutant proteins.

Asymmetric Synthesis of d-Proline, d-Pipecolic Acid, (2R,3S,4R)-3,4-Dihydroxyproline, and 1,4-Dideoxy-1,4-imino-d-talitol from a Common Precursor

Methodology involving stereoselective aza-Michael addition and ring-closing metathesis as key steps has been developed for the preparation of (2R)-pipecolic acid, (2R)-proline, (2R,3S,4R)-3,4-dihydroxyproline, and the known glycosidase inhibiting azasugar 1,4-dideoxy-1,4-imino- d -talitol from a common starting material namely (R)-cyclohexylideneglyceraldehyde in good overall yields.

Multimode Sensors Based on Nanostructured Materials for Simultaneous Screening of Biological Fluids for Specific Breast Cancer and Hepatitis B Biomarkers

Maltodextrin with dextrose equivalence between 4 and 7 was proposed for the design of a multimode sensor. The multimode term was introduced for the sensors that can be used in more than one electrochemical method. The selection of the materials was based on its nanostructure nature (for stochastic mode), and its catalytic activity (for amperometric mode). The qualitative analysis was performed using the stochastic mode, while the quantitative analysis of the antigens was performed using both stochastic and amperometric modes. The biomarkers specific for hepatitis B and breast cancer were determined directly from the biological fluids (e.g., whole blood). The response characteristics of the new multimode sensors for CA15–3, and hepatitis B biomarkers (HBV antigen and D-pipecolic acid) (linear concentration ranges, limits of determination) as well as their recovery tests performed using whole blood samples proved that they can be used for screening tests of whole blood with high reliability.

New Multimode Sensors Based on Nanostrucured Materials for Simultaneous Screening of Biological Fluids for Specific Breast Cancer and Hepatitis B Biomarkers

New materials (e.g., dextrins) were proposed for the design of multimode sensors for screening of biological fluids for specific biomarkers for breast cancer antigen and hepatitis B. The selection of the materials was based on its nanostructure (for stochastic mode), and its catalytic activity (for amperometric mode). The qualitative analysis was performed using the stochastic mode, and it was followed by the quantitative analysis of the antigens using stochastic mode and amperometric mode. The biomarkers were determined directly from the biological fluids (e.g., whole blood). The response characteristics of the new multimode sensors for CA15-3, and hepatitis B biomarkers (HBV antigen and D-pipecolic acid) as well as their recovery in the biological fluid of the patients are shown.

Normalization of Proliferation and Tight Junction Formation in Bladder Epithelial Cells from Patients with Interstitial Cystitis/Painful Bladder Syndrome by d‐Proline and d‐Pipecolic Acid Derivatives of Antiproliferative Factor

Interstitial cystitis/painful bladder syndrome is a chronic bladder disorder with epithelial thinning or ulceration, pain, urinary frequency and urgency, for which there is no reliably effective therapy. We previously reported that interstitial cystitis/painful bladder syndrome bladder epithelial cells make a glycopeptide antiproliferative factor or 'APF' (Neu5Acα2-3Galβ1-3GalNAcα-O-TVPAAVVVA) that induces abnormalities in normal cells similar to those in interstitial cystitis/painful bladder syndrome cells in vitro, including decreased proliferation, decreased tight junction formation, and increased paracellular permeability. We screened inactive APF derivatives for their ability to block antiproliferative activity of asialylated-APF ('as-APF') in normal bladder cells and determined the ability of as-APF-blocking derivatives to normalize tight junction protein expression, paracellular permeability, and/or proliferation of interstitial cystitis/painful bladder syndrome cells. Only two of these derivatives [Galβ1-3GalNAcα-O-TV-(d-pipecolic acid)-AAVVVA and Galβ1-3GalNAcα-O-TV-(d-proline)-AAVVVA] blocked as-APF antiproliferative activity in normal cells (p < 0.001 for both). Both of these antagonists also 1) significantly increased mRNA expression of ZO-1, occludin, and claudins 1, 4, 8, and 12 in interstitial cystitis/painful bladder syndrome cells by qRT-PCR; 2) normalized interstitial cystitis/painful bladder syndrome epithelial cell tight junction protein expression and tight junction formation by confocal immunofluorescence microscopy; and 3) decreased paracellular permeability of (14) C-mannitol and (3) H-inulin between confluent interstitial cystitis/painful bladder syndrome epithelial cells on Transwell plates, suggesting that these potent APF antagonists may be useful for the development as interstitial cystitis/painful bladder syndrome therapies.

N-Ethoxycarbonylation combined with ( S)-1-phenylethylamidation for enantioseparation of amino acids by achiral gas chromatography and gas chromatography–mass spectrometry

N-Ethoxycarbonylation was combined with ( S)-1-phenylethylamidation for enantioseparation of amino acids by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) on achiral capillary columns. The method provided complete enantioseparations of 12 amino acids as diastereomeric N-ethoxycarbonyl/( S)-1-phenylethylamides with exceptional resolutions for proline ( R s ≥ 9.9) and pipecolic acid ( R s ≥ 10.2). GC–MS analysis in selected ion monitoring mode employing standard addition method, facilitated quantitation of d-pipecolic acid in kidney bean (0.95 μg/10 mg) and adzuki bean (0.14 μg/10 mg). The peak area ratios indicated that they had the identical chiral composition at 2.5% for d-pipecolic acid and 97.5% for l-pipecolic acid.

Sequential Injection Analysis utilizing Amperometric Biosensors as Detectors for the Simultaneous Determination of L- and D-Pipecolic Acid

Enantioanalysis of biomedical substances is very important for clinical analysis because the L- and D-enantiomers of the same chiral compound may be markers for different illnesses. A sequential injection analysis (SIA) system using amperometric biosensors as detectors is proposed for the simultaneous determination of L- and D-pipecolic acid (PA). The amperometric biosensors were based on L-amino acid oxidase (L-AAOD) or/and horseradish peroxidase (HRP) for the assay of L-PA and D-amino acid oxidase (D-AAOD) and/or HRP for the assay of D-PA. The linear concentration ranges are of pmol/L to nmol/L magnitude order, with very low limits of detection. The SIA/biosensors system can be used reliably on-line for the simultaneous assay of L- and D-PA from serum samples, with a frequency of 34 samples per hour.

New Amperometric Biosensors Based on Diamond Paste for the Assay of L‐ and D‐Pipecolic Acids in Serum Samples

Monocrystalline natural diamond, L‐amino acid oxidase (L‐AAOD), D‐amino acid oxidase (D‐AAOD), and paraffin oil were used for the design of the modified diamond paste. The technique used for the direct voltammetric assay was differential pulse voltammetry (DPV) with applied potential pulse amplitude of 25 mV vs. Ag/AgCl. Using the new amperometric biosensors L‐pipecolic acid (L‐PA) and D‐pipecolic acid (D‐PA) were determined reliably from serum samples at 700 and 200 mV vs. Ag/AgCl, respectively, with low limits of detection.

Enantioselective, potentiometric membrane electrode based on vancomycin: Its application for the determination of d-pipecolic acid

The enantioselective, potentiometric membrane electrode based on vancomycin-was proposed for the assay of d-pipecolic acid. The linear concentration range for the proposed enantioselective membrane electrode is 10 −9 to 10 −6 mol l −1 with the slope of electrode function 60.2 mV/ p( d-pipecolic acid). The enantioselectivity was determined over l-pipecolic acid. The proposed electrode could be reliably employed for the assay of d-pipecolic acid in serum samples. The surface of the electrode is stable and easily renewable by polishing on alumina paper.

Semisynthesis and Degradation of the Tubulin Inhibitors Epothilone and Tubulysin

AbstractFor Abstract see ChemInform Abstract in Full Text.

Biosensors for the enantioselective analysis of pipecolic acid

Mono- and bienzyme-based amperometric biosensors were used for the direct enantioanalysis of l- and d-pipecolic acid in serum samples. The biosensors were designed using physical immobilization of l-amino acid oxidase and/or horseradish peroxidase for the assay of l-pipecolic acid, and d-amino acid oxidase and horseradish peroxidase for the assay of d-pipecolic acid. Electrode characteristics were obtained and compared for the different carbon paste biosensors at different working potentials. The linear concentration ranges for the proposed biosensors were in the fmol/l to μmol/l magnitude order with limits of detection of fmol/l to pmol/l magnitude order. The results obtained for enantioanalysis of l- and d-pipecolic acid in serum samples are correlating very good with those obtained using a standard method of analysis.

Diamond Paste Based Amperometric Biosensors for the Determination of l- and d-Pipecolic Acid

Diamond paste based amperometric biosensors were designed for the direct assay of l- and d-pipecolic acid in serum samples. l-Amino acid oxidase and d-amino acid oxidase were used for the design of the amperometric biosensor used for the assay of l- and d-pipecolic acid, respectively. Monocrystallin diamond powder and paraffin oil were mixed with the enzyme solution to form the modified diamond paste. The linear concentration ranges for the proposed biosensors were in the fmol/L to pmol/L magnitude order. The biosensors proved high reliability for the enantioanalysis of pipecolic acid in serum samples.

Semisynthesis and degradation of the tubulin inhibitors epothilone and tubulysin

Abstract The structure-activity relationships of epothilones indicate that major modifications are only tolerated in the western ring segment. In particular, C2 methyl of the thiazole ring appears to be most flexible. Its broad modification started from epothilone F, which was obtained from natural epothilone B by hydroxylation via the N-oxide. Some of the prepared derivatives exhibit improved esterase stability in addition to high cytotoxic activity. For these and other favorable properties, amine (BMS-310705) was recently introduced in clinical trials. In an alternative approach, modified side chains were introduced by replacement of the C12,C15 ring segment via ring-opening olefin metathesis (ROM) of epothilone C in the presence of ethylene to 12,13-seco-epothilone C, introduction of a synthetic building block followed by ring-closing olefin metathesis (RCM), and epoxidation to the 16-alkyne analog of epothilone A. The structure of the tetrapeptide tubulysin D was confirmed by total hydrolysis to N-methyl d-pipecolic acid, l-isoleucine, tubuvaline (Tuv), tubuphenylalanine (Tup), formaldehyde, and 3-methylbutyric acid. Mild acidic hydrolysis to cyclo-tubulysin andoxidative degradation to l-valine allowed the assignment of the stereocenters of Tuv, hydrazinolysis, and comparison with synthetic reference samples to that of Tup. The absolute configuration of tubulysin D is: (R)-Mep, (2,3S)-Ile, (1'R ,3'R)-Tuv, and (2S,4R)-Tup.

Determination of l-Pipecolic Acid in Plasma Using Chiral Liquid Chromatography-Electrospray Tandem Mass Spectrometry

L-pipecolic acid (L-PA), an important biochemical marker for the diagnosis of peroxisomal disorders, is usually determined as the racemate. We developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of L-PA in plasma. We used a narrow bore chiral macrocyclic glycopeptide teicoplanin column for the enantioseparation of D-pipecolic acid (D-PA) and L-PA and interfaced the column directly to the electrospray source of a tandem mass spectrometer. We used phenylalanine-d5 as internal standard added to 50 microL of plasma followed by deproteinization, evaporation, and injection. The analysis was performed in the selected-reaction monitoring mode using two transitions: m/z 130-->m/z 84 for PA, and m/z 171-->m/z 125 for phenylalanine-d5. L-PA eluted at 7 min, and D-PA eluted at 11.7 min, whereas phenylalanine-d5 eluted at 6 min. The turnaround time for the assay was 20 min. Linear calibration curves were obtained in the range of 0.5-80 micromol/L. At a plasma concentration of 1.0 micromol/L, the signal-to-noise ratio was 50:1. The intra- and interassay variations were 3.1-7.9% and 5.7-13%, respectively, at concentrations of 1-50 micromol/L. Mean recoveries of L-PA added to plasma were 95% (5 micromol/L) and 102% (50 micromol/L). The method clearly distinguished between healthy individuals and peroxisomal disease patients. The novel LC-MS/MS method is simple, rapid, and stereoselective, and uses only 50 microL of plasma, no derivatizing reagents, and a commercially available internal standard. Sample preparation is not complex and is faster than for all other methods.

Design of P1‘ and P3‘ Residues of Trivalent Thrombin Inhibitors and Their Crystal Structures

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1‘ and P3‘ residues of the linker with thrombin S1‘ and S3‘ subsites, respectively, were identified using the “Methyl Scan” method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494−13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(d-pipecolic acid)-Xaa-Gly-Yaa-Gly-βAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-(β-cyclohexylalanine)-(d-Glu)-OH, in which nonpolar P1‘ residue Xaa or P3‘ residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3‘ residue with d-phenylglycine or d-Phe improved the Ki value to (9.5 ± 0.6) × 10-14 or 1.3 ± 0.5 × 10-13 M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (Ki = (2.4 ± 0.5) × 10-11 M). Similarly, substitution of the P1‘ residue with l-norleucine or l-β-(2-thienyl)alanine lowered the Ki values to (8.2 ± 0.6) × 10-14 or (5.1 ± 0.4) × 10-14 M, respectively. The linker Gly-Gly-Gly-βAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvment of the Ki values to (3.8 ± 0.6) × 10-14 or (1.7 ± 0.4) × 10-14 M, respectively. These Ki values are equivalent to that of natural hirudin (2.2 × 10-14 M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with l-norleucine or l-β-(2-thienyl)alanine at the P1‘ residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human α-thrombin. The crystal structures of these complexes were solved and refined to 2.1 Å resolution. The Lys60F side chain of thrombin moved significantly and formed a large nonpolar S1‘ subsite to accommodate the bulky P1‘ residue.

Synthesis of chiral nonracemic 4- trans-substituted pipecolic acid derivatives

The syntheses and resolutions of enantiomerically enriched 4-phenyl, 4- tert-butyl, and 4-isopropyl pipecolic acids are described. Optically active diastereomers were prepared by diastereomeric salt formation with the chiral base, l-tyrosine hydrazide, to provide Cbz or Boc protected 4- cis- d-pipecolic acid derivatives in >98% ee. Subsequent esterification followed by sodium methoxide catalyzed epimerization provided the isomeric 4- trans- l-pipecolic esters. In addition, an efficient synthesis of 4-phenyl- cis-pipecolic acid is described.

Asymmetric synthesis and pharmacology of methylphenidate and its para-substituted derivatives.

We report the first asymmetric synthesis of the individual enantiomers of methylphenidate (1). From d-pipecolic acid, the (2R,2'R) and (2S,2'R) enantiomers of 1 were obtained in > 99% optical purity while the (2S,2'S) and (2R,2'S) enantiomers of 1 were derived from l-pipecolic acid in 96% optical purity. The versatility of this methodology is demonstrated with the synthesis of the (2R,2'R) and (2S,2'S) enantiomers of p-bromo and p-methoxy derivatives in similar yields and enantiomeric purities. Comparative neurochemical assessments of these synthesized enantiomers at purported dopamine, norepinephrine, and serotonin uptake sites along with locomotor activity studies in rats are also reported.

Nonpolar Interactions of Thrombin and Its Inhibitors at the Fibrinogen Recognition Exosite: Thermodynamic Analysis

Nonpolar interactions play a major role in the association of the fibrinogen recognition exosite of thrombin with the C-terminal fragment (55-65), Asp-Phe-Glu-IIe-Pro-Glu-Glu-Tyr-Leu-Gln, of hirudin, which is a naturally occurring thrombin inhibitor. The thermodynamic details (free energy, enthalpy, entropy, and heat capacity) of the molecular recognition are studied by using five analogs of a synthetic bivalent thrombin inhibitor (P552), tert-butylbenzensulfonyl-Arg-(D-pipecolic acid)-(12-amino-dodecanoic acid)-(gamma-aminobutyric acid)-hirudin55-65. The residue of PheH56, IleH59, ProH60, TyrH63, or LeuH64 in hirudin 55-65 segment is substituted by Gly in each analog in order to elucidate the contributions of these nonpolar side chains. The results show that the interactions of these nonpolar side chains with thrombin are enthalpy-driven, except for the contribution of the PheH56 side chain which is entropy-driven. Interestingly, molecular modeling predicts a large conformational change due to the Gly substitution of PheH56. In analyzing the correlation among the thermodynamic and structural properties of the nonpolar interaction, a good correlation is observed between the binding free energy and the hydrophobicity of the molecular surface; i.e., tighter binding is observed as more nonpolar atoms are buried and more polar atoms are exposed upon molecular association.