Engineered C–N Lyase: Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners

Aspartic acid derivatives with branched N-alkyl or N-arylalkyl substituents are valuable precursors to artificial dipeptide sweeteners such as neotame and advantame. The development of a biocatalyst to synthesize these compounds in a single asymmetric step is an as yet unmet challenge. Reported here is an enantioselective biocatalytic synthesis of various difficult N-substituted aspartic acids, including N-(3,3-dimethylbutyl)-l -aspartic acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-l -aspartic acid, precursors to neotame and advantame, respectively, using an engineered variant of ethylenediamine-N,N′-disuccinic acid (EDDS) lyase from Chelativorans sp. BNC1. This engineered C–N lyase (mutant D290M/Y320M) displayed a remarkable 1140-fold increase in activity for the selective hydroamination of fumarate compared to that of the wild-type enzyme. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step-economic synthesis of an important class of food additives.     

Conformation and structure of bis­(glycyl‐l‐aspartic acid) oxalate 0.4‐hydrate

The title bis­(glycyl‐l ‐aspartic acid) oxalate complex {systematic name: bis­[2‐(2‐ammonio­acetamido)butane­dioic acid] oxalate 0.4‐hydrate}, 2C₆H₁₁N₂O₅⁺·C₂O₄²⁻·4H₂O, crystallizes in a triclinic space group with the planar peptide unit in a trans conformation. The asymmetric unit consists of two glycyl‐l ‐aspartic acid mol­ecules with positively charged amino groups and neutral carboxyl groups, and an oxalate dianion. The twist around the C—Cα bond indicates that both the peptide mol­ecules adopt extended conformations, while the twist around the N—Cα bond shows that one has a folded and the other a semi‐extended state. The present complex can be described as an inclusion compound with the dipeptide mol­ecule as the host and the oxalate anion as the guest. The usual head‐to‐tail sequence of aggregation is not observed in this complex, as is also the case with the glycyl‐l ‐aspartic acid dihydrate mol­ecule. The study of aggregation and inter­action patterns in binary systems is the first step towards understanding more complex phenomena. This further leads to results that are of general interest in bimolecular aggregation.     

Total Synthesis of Aurantoside G,an N‐β‐Glycosylated 3‐Oligoenoyltetramic Acid from Theonella swinhoei

The first synthesis of a natural N‐glycosylated 3‐acyltetramic acid is reported. Aurantoside G ( 1 g ), a deep‐red metabolite of the marine sponge Theonella swinhoei, is highly delicate in the pure state. It features a chlorinated dodecapentaenoyl side chain at an l ‐asparagine‐derived tetramic acid, the ring nitrogen atom of which is linked to a β‐configured d ‐xylose. The side chain was built through consecutive Wittig and HWE reactions and used to N‐acylate the amino group of an asparaginate that had already been N‐xylosylated through a Fukuyama–Mitsunobu reaction. This N‐acylation step fixes the β‐configuration of the xylose, which is essential for the antifungal activity, but only if the sugar carries bulky, electron‐rich protecting groups such as PMB. In the final step, the heterocycle was closed quantitatively through a basic Lacey–Dieckmann condensation of an entirely unprotected precursor.     

Determination of kynurenic acid levels in rat brain microdialysis samples and changes in kynurenic acid levels induced by N‐acetylaspartic acid

The levels of kynurenic acid, an endogenous antagonist of α₇ nicotinic acetylcholine and N‐methyl‐D ‐aspartate receptors, were measured in microdialysis samples obtained from the prefrontal cortices of rats using column‐switching high‐performance liquid chromatography with fluorescence detection. When the perfusate was constantly infused at a rate of 1.0 μ/min, the in vitro recovery of kynurenic acid through the dialysis membrane was approximately 20.4%, and the precision was within 1.31%. Endogenous kynurenic acid in the microdialysis sample was clearly detected using column‐switching high‐performance liquid chromatography. As an application study, N‐acetyl‐L ‐aspartic acid, an endogenous metabolite and precursor of N‐acetyl‐L ‐aspartyl‐L ‐glutamic acid, which is an agonist of metabotropic glutamate receptors, was infused for 120 min through the microdialysis probe. The kynurenic acid level significantly increased during the infusion of N‐acetyl‐L ‐aspartic acid, suggesting that kynurenic acid might have some association with N‐acetyl‐L ‐aspartic acid in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

One‐step synthesis of poly(N‐vinylpyrrolidone)‐b‐poly(l‐lactide) block copolymers using a dual initiator for RAFT polymerization and ROP

Amphiphilic, biocompatible poly(N‐vinylpyrrolidone)‐b‐poly(l ‐lactide) (PVP‐b‐PLLA) block polymers were synthesized at 60 °C using a hydroxyl‐functionalized N,N‐diphenyldithiocarbamate reversible addition–fragmentation chain transfer (RAFT) agent, 2‐hydroxyethyl 2‐(N,N‐diphenylcarbamothioylthio)propanoate (HDPCP), as a dual initiator for RAFT polymerization and ring‐opening polymerization (ROP) in a one‐step procedure. 4‐Dimethylamino pyridine was used as the ROP catalyst for l ‐lactide. The two polymerization reactions proceeded in a controlled manner, but their polymerization rates were affected by the other polymerization process. This one‐step procedure is believed to be the most convenient method for synthesizing PVP‐b‐PLLA block copolymers. HDPCP can also be used for the one‐step synthesis of poly(N‐vinylcarbazole)‐b‐PLLA block copolymers. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 1607–1613     

Simultaneous determination of NG‐monomethyl‐l‐arginine,NG,NG‐dimethyl‐l‐arginine,NG,NG′‐dimethyl‐l‐arginine,and l‐arginine using monolithic silica disk‐packed spin columns and a monolithic silica column

We have developed and validated a high‐performance liquid chromatography method that uses monolithic silica disk‐packed spin columns and a monolithic silica column for the simultaneous determination of N^G‐monomethyl‐l ‐arginine, N^G,N^G‐dimethyl‐l ‐arginine, and N^G,N^G′‐dimethyl‐l ‐arginine in human plasma. For solid‐phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole, and then the derivatives are separated on a monolithic silica column. l ‐Arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2–1.0 μM for methylated arginines and 40–200 μM for l ‐arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r²). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3–4.8% (intraday) and 3.0–6.8% (interday). When this method was applied to samples from six healthy donors, the detected concentrations of N^G‐monomethyl‐l ‐arginine, N^G,N^G‐dimethyl‐l ‐arginine, N^G,N^G′‐dimethyl‐l ‐arginine and l ‐arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 μM (n = 6), respectively.     

Enantioselective Recognition of Aspartic Acids by Chiral Ligand Exchange Potentiometry

Enantioselective resolution is realized by combining potentiometry with ligand exchange (CE) in a new method called chiral ligand exchange potentiometry (CLEP). A chiral selector, N‐carbobenzoxy‐L ‐aspartic acid (N‐CBZ‐L‐Asp), preferentially recognizes D ‐aspartic acid (D‐Asp) and undergoes ligand exchange with the enantiomeric labile coordination complexes of [Cu(II)(D‐Asp)₂] or [Cu(II)(L‐Asp)₂] to form a diastereoisomeric complex [(D‐Asp)Cu(II)(N‐CBZ‐L‐Asp)] (a) or [(L‐Asp)Cu(II)(N‐CBZ‐L‐Asp)] (b). Considerable stereoselectivity occurs in the formation of these diastereoisomeric complexes, and their net charges were ?2 (a) and 0 (b), respectively, resulting in different Nernst factor (electrode slope), thus enabling chiral D‐Asp to be distinguished by potentiometry without any pre‐ or postseparation processes.     

Resolution and quantification of arginine,monomethylarginine, asymmetric dimethylarginine,and symmetric dimethylarginine in plasma using HPLC with internal calibration

N^G,N^G‐dimethyl‐l ‐arginine (asymmetric dimethylarginine, ADMA),N^G‐monomethyl‐l ‐arginine (l ‐NMMA) and N^G,N^G’‐dimethyl‐l ‐arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l ‐NMMA inhibit nitric oxide synthase by competing with l ‐arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high‐performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l ‐NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed‐mode cation‐exchange columns, using synthetic monoethyl‐l ‐arginine as an internal standard. Metabolites were derivatized with ortho‐phthaldialdeyhde and 3‐mercaptopropionic acid, separated by reverse‐phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter‐day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l ‐NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd.     

Cyclic lipoundecapeptide amphisin from Pseudomonas sp. strain DSS73

The crystal structure of the lipoundecapeptide amphisin, presented here as the tetrahydrate, C₆₆H₁₁₄N₁₂O₂₀·4H₂O, originating from non‐ribosomal biosynthesis by Pseudomonas sp. strain DSS73, has been solved to a resolution of 0.65 Å. The primary structure of amphisin is β‐hydroxy­decanoyl‐d ‐Leu‐d ‐Asp‐d ‐allo‐Thr‐d ‐Leu‐d ‐Leu‐d ‐Ser‐l ‐Leu‐d ‐Gln‐l ‐Leu‐l ‐Ile‐l ‐Asp (Leu is leucine, Asp is aspartic acid, Thr is threonine, Ser is serine, Gln is glut­amine and Ile is isoleucine). The peptide is a lactone, linking Thr4 O_γ to the C‐terminal. The stereochemistry of the β‐hydroxy acid is R. The peptide is a close analogue of the cyclic lipopeptides tensin and pholipeptin produced by Pseudomonas fluorescens. The structure of amphisin is mainly helical (3₁₀‐helix), with the cyclic peptide wrapping around a hydrogen‐bonded water mol­ecule. This lipopeptide is amphiphilic and has biosurfactant and antifungal properties.     

A Combined Effect of Thermal and Enzymatic Racemization for d‐Aspartic Acid to l‐Aspartic Acid by High‐Performance Liquid Chromatography Assay

The racemization of d ‐aspartic acid to l ‐aspartic acid has been successfully performed with a coupled enzyme system at 90 °C and a pH of about 4.0 by the assay of high‐performance liquid chromatography. This coupled enzymatic racemization is a successive two‐step reaction first induced by d ‐amino acid oxidase and a subsequent coupled reaction by an aminotransferase clonezyme with the help of coenzyme pyridoxal 5′‐phosphate and cosubstrate l ‐glutamate. Due to the very high temperature, part of the l ‐aspartic acid is produced by the thermal effect. In fact the thermal racemization for aspartic acid can proceed from either d ‐ or l ‐aspartic acid via an intermediate fumaric acid and leads to the formation of d ,l ‐malic acid. The formation of α‐oxalacetic acid formed irreversibly from d ‐aspartic acid with d ‐amino acid oxidase can induce a side reaction to l ‐alanine. The thermal effect may also be responsible for the production of d ‐, and l ‐alanine.     

An Improved Synthesis of 2‐Acetamido‐2‐deoxy‐ D‐gluconohydroximolactone (PUGNAc), A Strong Inhibitor of β‐N‐Acetylglucosaminidases

O‐(2‐Acetamido‐3,4,6‐tri‐O‐acetyl‐D ‐glucopyranosylidene)amino N‐phenylcarbamate ( 1 ), an established inhibitor of β‐N‐acetylglucosaminidases, has been prepared by an improved six‐step synthesis from N‐acetyl‐D ‐glucosamine.     

Simultaneous detection of stable isotope‐labeled and unlabeled l‐tryptophan and of its main metabolites,l‐kynurenine,serotonin and quinolinic acid,by gas chromatography/negative ion chemical ionization mass spectrometry

A method for the detection of unlabeled and ¹⁵N₂‐labeled l ‐tryptophan (l ‐Trp), l ‐kynurenine (l ‐Kyn), serotonin (5‐HT) and quinolinic acid (QA) in human and rat plasma by GC/MS is described. Labeled and unlabeled versions of these four products were analyzed as their acyl substitution derivatives using pentafluoropropionic anhydride and 2,2,3,3,3‐pentafluoro‐1‐propanol. Products were then separated by GC and analyzed by selected ion monitoring using negative ion chemical ionization mass spectrometry. l ‐[¹³C₁₁, ¹⁵N₂]‐Trp, methyl‐serotonin and 3,5‐pyridinedicarboxylic acid were used as internal standards for this method. The coefficients of variation for inter‐assay repeatability were found to be approximately 5.2% for l ‐Trp and ¹⁵N₂‐Trp, 17.1% for l ‐Kyn, 16.9% for 5‐HT and 5.8% for QA (n = 2). We used this method to determine isotope enrichments in plasma l ‐Trp over the course of a continuous, intravenous infusion of l ‐[¹⁵N₂]Trp in pregnant rat in the fasting state. Plasma ¹⁵N₂‐Trp enrichment reached a plateau at 120 min. The free Trp appearance rate (Ra) into plasma was 49.5 ± 3.35 µmol/kg/h. The GC/MS method was applied to determine the enrichment of ¹⁵N‐labeled l ‐Trp, l ‐Kyn, 5‐HT and QA concurrently with the concentration of non‐labeled l ‐Trp, l ‐Kyn, 5‐HT and QA in plasma. This method may help improve our understanding on l ‐Trp metabolism in vivo in animals and humans and potentially reveal the relative contribution of the four pathways of l ‐Trp metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Biosynthesis of the N–N‐Bond‐Containing Compound l‐Alanosine

The formation of a N?N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N?N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate‐containing amino acid l ‐alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l ‐alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl‐carrier‐protein‐based mechanism for activation of the precursor l ‐diaminopropionate, and we also show that nitric oxide can participate in non‐enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N‐terminal cupin domain and a C‐terminal AraC‐like DNA‐binding domain, is required for alanosine biosynthesis.     

Synthesis and Sequential Deprotection of Triblock Copolypept(o)ides Using Orthogonal Protective Group Chemistry

The synthesis of triblock copolymers based on polysarcosine, poly‐N‐ε‐t‐butyloxycarbonyl‐l ‐lysine, and poly‐N‐ε‐t‐trifluoroacetyl‐l ‐lysine by ring‐opening polymerization of the corresponding α‐amino acid N‐carboxyanhydrides (NCAs) is described. For the synthesis of N‐ε‐t‐butyloxycarbonyl‐l ‐lysine (lysine(Boc)) NCAs, an acid‐free method using trimethylsilylchloride/triethylamine as hydrochloric acid (HCl) scavengers is presented. This approach enables the synthesis of lysine(Boc) NCA of high purity (melting point 138.3 °C) in high yields. For triblock copolypept(o)ides, the degree of polymerization (X_n) of the polysarcosine block is varied between 200 and 600; poly‐N‐ε‐t‐butyloxycarbonyl‐l ‐lysine and poly‐N‐ε‐t‐trifluoroacetyl‐l ‐lysine blocks are designed to have a X_n in the range of 10–50. The polypeptide‐polypeptoid hybrids (polypept(o)ides) can be synthesized with precise control of molecular weight, high end group integrity, and dispersities indices between 1.1 and 1.2. But more important, the use of tert‐butyloxycarbonyl‐ and trifluoroacetyl‐protecting groups allows the selective, orthogonal deprotection of both blocks, which enables further postpolymerization modification reactions in a block‐selective manner. Therefore, the presented synthetic approach provides a versatile pathway to triblock copolypept(o)ides, in which functionalities can be separated in specific blocks.

     

Modified Jiu Wei Qiang Huo decoction improves dysfunctional metabolomics in influenza A pneumonia‐infected mice

In order to study the effective mechanism of a traditional Chinese medicine (TCM), modified Jiu Wei Qiang Huo decoction (MJWQH), against H1N1‐induced pneumonia in mice, we chose a holistic approach. A reverse‐phase liquid chromatography with quadruple time‐of‐flight mass spectrometry (LC‐Q‐TOF‐MS) was developed to determine metabolomic biomarkers in mouse serum for the MJWQH effects. Thirteen biomarkers of H1N1‐induced pneumonia in mice serum were identified, which comprised l ‐valine, lauroylcarnitine, palmitoyl‐l ‐carnitine, l ‐ornithine, uric acid, taurine, O‐succinyl‐l ‐homoserine, l ‐leucine, l ‐phenylalanine, PGF2α, 20‐ethyl‐PGE2, arachidonic acid, and glycerophospho‐N‐arachidonoyl ethanolamine. Among them, metabolites of amino acids, fatty acids and arachidonic acid had the most relevant changes in mice with H1N1‐induced pneumonia. MJWQH effectively improved weight loss, lung index, biomarkers and inflammatory mediators such as prostaglandin E2 and phospholipase A2 in the infected mice. Importantly, MJWQH reversed the elevated biomarkers to the control levels from the infection, which provided a systematic view and a theoretical basis for its prevention or treatment. The results suggest that the protective effect of MJWQH against H1N1‐induced pneumonia is possibly through regulation of pathways for amino acid, fatty acid and arachidonic acid metabolism. They also suggest that the LC‐MS‐based metabolomic strategy is a powerful tool for elucidation of the mechanisms of TCM. Copyright © 2013 John Wiley & Sons, Ltd.     

Expedient Synthesis of (+)‐Lycopalhine A

Two amino acids play a key role in the first total synthesis of lycopalhine A. l ‐glutamic acid serves as a convenient chiral starting material for the 13‐step synthesis, and l ‐proline promotes an unusual 5‐endo‐trig Mannich cyclization that generates the central pyrrolidine ring of the Lycopodium alkaloid. The bicyclo[3.3.0]octanol moiety of the molecule is formed through an intramolecular aldol addition that may occur spontaneously in nature.     

N‐(l‐2‐Aminobutyryl)‐l‐alanine and 2‐(l‐alanylamino)‐l‐butyric acid 0.33‐hydrate

The crystal structure of N‐(l ‐2‐amino­butyryl)‐l ‐alanine, C₇H₁₄N₂O₃, is closely related to the structure of l ‐alanyl‐l ‐alanine, both being tetragonal, while the retro‐analogue 2‐(l ‐alanyl­amino)‐l ‐butyric acid 0.33‐hydrate, C₇H₁₄N₂O₃·­0.33H₂O, forms a new type of molecular columnar structure with three peptide mol­ecules in the asymmetric unit.     

Microstructural characterization of terpolymers of leucine, β‐benzyl aspartate,and valine

Three different characterization methods—¹³C NMR spectroscopy, a terminal terpolymerization model, and a probability analysis based on the Poisson distribution—were used to determine the microstructure of random terpolymers. The methods were used to determine the amino acid sequence distribution of random terpolymers prepared from the polymerization of N‐carboxyanhydrides that contained L ‐leucine, β‐benzyl‐L ‐aspartate, and L ‐valine. Poly(L ‐leucine‐L ‐aspartic acid‐L ‐valine) [poly(LDV)] was designed as a target specific substrate for the α₄β₁ integrin that recognizes the tripeptide sequence leucine‐aspartic acid‐valine (LDV). The presence of the tripeptide sequence LDV within the polymer was determined to be eight LDV triad sequences on average in terpolymers of approximately 100 kDa. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 4328–4337, 2006     

Determination of GABA, glutamate and carbamathione in brain microdialysis samples by capillary electrophoresis with fluorescence detection

Disulfiram has been used as a deterrent in the treatment of alcohol abuse for almost 60 years. Our laboratory has shown that a disulfiram metabolite, S‐(N,N‐diethylcarbamoyl) glutathione (carbamathione), is formed from disulfiram and appears in the brain after the administration of disulfiram. Carbamathione does not inhibit aldehyde dehydrogenase but has been shown to be a partial non‐competitive inhibitor of the N‐methyl‐D ‐aspartic acid glutamate (Glu) receptor. In light of disulfiram's apparent clinical effectiveness in cocaine dependence, and carbamathione's effect on the N‐methyl‐D ‐aspartic acid receptor, the effect of carbamathione on brain Glu and γ‐aminobutyric acid (GABA) needs to be further examined. A CE‐LIF method based on derivatization with napthalene‐2,3‐dicarboxyaldehyde to simultaneously detect both neurotransmitter amino acids and carbamathione in brain microdialysis samples is described. The separation of Glu, GABA and carbamathione was carried out using a 50 mmol/L boric acid buffer (pH 9.6) on a 75 cm×50 μm id fused‐silica capillary (60 cm effective) at +27.5 kV voltage with a run time of 11 min. The detection limits for Glu, GABA and carbamathione were 6, 10 and 15 nmol/L, respectively. This method was used to monitor carbamathione and the amino acid neurotransmitters in brain microdialysis samples from the nucleus accumbens after the administration of an intravenous dose of the drug (200 mg/kg) and revealed a carbamathione‐induced change in GABA and Glu levels. This method demonstrates a simple, rapid and accurate measurement of two amino acid neurotransmitters and carbamathione for in vivo monitoring in the brain using microdialysis sampling.     

Total Synthesis,Absolute Configuration,and Biological Activity of Xyloallenoide A

The novel natural product xyloallenoide A, isolated from the marine mangrove endophytic fungus from the South China Sea, and its diastereoisomer xyloallenoide A1, which contain N‐methyl‐substituted amino acids, were synthesized. The absolute configurations of the amino acid units of xyloallenoide A were finally confirmed to be L ‐Lys, Me‐D ‐Val, and Me‐L ‐Ala. This report represents a practical and attractive alternative for the synthesis of N‐methyl‐substituted cyclotripeptides. In the preliminary bioassay, synthetic xyloallenoide A showed marginal activities against KB (IC₅₀=9.6 μM ) and KBv200 cells (IC₅₀=10.3 μM ), and xyloallenoide A1 was inactive against KB and KBv200 cells.