l‐Phenyl­alanyl‐l‐alanine dihydrate

A new type of molecular arrangement for dipeptides is observed in the crystal structure of l ‐phenyl­alanyl‐l ‐alanine dihydrate, C₁₂H₁₆N₂O₃·2H₂O. Two l ‐Phe and two l ‐Ala side chains aggregate into large hydro­phobic columns within a three‐dimensional hydrogen‐bond network.     

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.     

The monohydrates of the four polar dipeptides l‐seryl‐l‐asparagine,l‐seryl‐l‐tyrosine,l‐tryptophanyl‐l‐serine and l‐tyrosyl‐l‐tryptophan

The crystal structures of the four dipeptides l ‐seryl‐l ‐asparagine monohydrate, C₇H₁₃N₃O₅·H₂O, l ‐seryl‐l ‐tyrosine monohydrate, C₁₂H₁₆N₂O₅·H₂O, l ‐tryptophanyl‐l ‐serine monohydrate, C₁₄H₁₇N₃O₄·H₂O, and l ‐tyrosyl‐l ‐tryptophan monohydrate, C₂₀H₂₁N₃O₄·H₂O, are dominated by extensive hydrogen‐bonding networks that include cocrystallized solvent water molecules. Side‐chain conformations are discussed on the basis of previous observations in dipeptides. These four dipeptide structures greatly expand our knowledge on dipeptides incorporating polar residues such as serine, asparagine, threonine, tyrosine and tryptophan.     

Rose Bengal‐Sensitized Photooxidation of the Dipeptides l‐Tryptophyl‐l‐Phenylalanine,l‐Tryptophyl‐l‐Tyrosine and l‐Tryptophyl‐l‐Tryptophan: Kinetics,Mechanism and Photoproducts¶

The Rose Bengal‐sensitized photooxidations of the dipeptides l ‐tryptophyl‐l ‐phenylalanine (Trp‐Phe), l ‐tryptophyl‐l ‐tyrosine (Trp‐Tyr) and l ‐tryptophyl‐l ‐tryptophan (Trp‐Trp) have been studied in pH 7 water solution using static photolysis and time‐resolved methods. Kinetic results indicate that the tryptophan (Trp) moiety interacts with singlet molecular oxygen (O₂(¹Δ_g)) both through chemical reaction and through physical quenching, and that the photooxidations can be compared with those of equimolecular mixtures of the corresponding free amino acids, with minimum, if any, influence of the peptide bond on the chemical reaction. This is not a common behavior in other di‐ and polypeptides of photooxidizable amino acids. The ratio between chemical (k_r) and overall (k_t) rate constants for the interaction O₂(¹Δ_g)‐dipeptide indicates that Trp‐Phe and Trp‐Trp are good candidates to suffer photodynamic action, with k_rlk_t values of 0.72 and 0.60, respectively (0.65 for free Trp). In the case of Trp‐Tyr, a lower k_rlk_t value (0.18) has been found, likely as a result of the high component of physical deactivation of O₂(¹Δ_g) by the tyrosine moiety. The analysis of the photooxidation products shows that the main target for O₂(¹Δ_g) attack is the Trp group and suggests a much lower accumulation of kynurenine‐type products, as compared with free Trp. This is possibly because of the occurrence of another accepted alternative pathway of oxidation that gives rise to 3a‐oxidized hydrogenated pyrrolo[2,3‐b]indoles.     

l‐Isoleucyl‐l‐serine 0.33‐hydrate,l‐phenyl­alanyl‐l‐serine and l‐methionyl‐l‐serine 0.34‐hydrate

The structures of the title dipeptides, C₉H₁₈N₂O₄·0.33H₂O, C₁₂H₁₆N₂O₄ and C₈H₁₆N₂O₄S·0.34H₂O, complete a series of investigations focused on l ‐Xaa‐l ‐serine peptides, where Xaa is a hydro­phobic residue. All three structures are divided into hydro­philic and hydro­phobic layers. The hydro­philic layers are thin for l ‐phenyl­alanyl‐l ‐serine, rendered possible by an unusual peptide conformation, and thick for l ‐isoleucyl‐l ‐serine and l ‐methionyl‐l ‐serine, which include cocrystallized water mol­ecules on the twofold axes.     

N‐Acetyl‐l‐tyrosine methyl ester monohydrate at 293 and 123 K

The crystal structure of a protected l ‐tyrosine, namely N‐acetyl‐l ‐tyrosine methyl ester monohydrate, C₁₂H₁₅NO₄·H₂O, was determined at both 293 (2) and 123 (2) K. The structure exhibits a network of O—H...O and N—H...O hydrogen bonds, in which the water molecule plays a crucial role as an acceptor of one and a donor of two hydrogen bonds. Molecules of water and of the protected l ‐tyrosine form hydrogen‐bonded layers perpendicular to [001]. C—H...π interactions are observed in the hydrophobic regions of the structure. The structure is similar to that of N‐acetyl‐l ‐tyrosine ethyl ester monohydrate [Soriano‐García (1993). Acta Cryst. C 49 , 96–97].     

Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method for the simultaneous determination of l‐valine,l‐leucine,l‐isoleucine,l‐phenylalanine,and l‐tyrosine in human serum

l ‐Valine, l ‐leucine, l ‐isoleucine, l ‐phenylalanine, and l ‐tyrosine are important proposed biomarkers for the early detection and diagnosis of type 2 diabetes. A simple and selective hydrophilic interaction chromatography with tandem mass spectrometry method was developed for the simultaneous determination of these amino acids in human serum, using stable isotope‐labeled amino acids as internal standards. Chromatographic separation was carried out on a Syncronis HILIC column (150 mm × 2.1 mm, 5 μm) with the column temperature of 35°C and a mobile phase consisted of acetonitrile/120 mM ammonium acetate (89:11, v/v), and the run time was 11.0 min. The mass spectrometric analysis was performed using a QTRAP 5500 mass spectrometer coupled with an electrospray ionization source in positive ion mode. As these five amino acids are endogenous compounds in serum, we used the corresponding stable isotope‐labeled amino acids to evaluate the matrix effect and recovery in serum. The matrix effect was 98.7–107.3%, and the recovery was 92.7–102.3%. Calibration curves spiked unlabeled amino acids in water were linear over the range of 0.200–100 μg/mL. The accuracy, inter‐, and intraday precision were below 10.2%. Analytes were stable during the study. This assay method has been validated and applied to the early diagnosis research of type 2 diabetes.     

Expression,Purification and Characterization of Human α‐l‐Fucosidase

α‐l ‐Fucosidases (EC 3.2.1.51) are exo‐glycosidases. On the basis of the multi‐alignment of amino acid sequence, α‐l ‐fucosidases were classified into two families of glycoside hydrolases, GH‐29 and GH‐95. They are responsible for the removal of l ‐fucosyl residues from the non‐reducing end of glycoconjugates. Deficiency of α‐l ‐fucosidase results in Fucosidosis due to the accumulation of fucose‐containing glycolipids, glycoproteins and oligosaccharides in various tissues. Recent studies discovered that the fucosylation levels are increased on the membrane surfaces of many carcinomas, indicating the biological function of α‐l ‐fucosidases may relate to this abnormal cell physiology. Although the gene of human α‐l ‐fucosidase (h‐fuc) was cloned, the recombinant enzyme has rarely been overexpressed as a soluble and active from. We report herein that, with carefully control on the growing condition, an active human α‐l ‐fucosidases (h‐Fuc) was successfully expressed in Escherichia coli for the first time. After a series steps of ion‐exchange and gel‐filtration chromatographic purification, the recombinant h‐Fuc with 95% homogeneity was obtained. The molecular weight of the enzyme was analyzed by SDS‐PAGE (～50 kDa) and confirmed by ESI mass (50895 Da). The recombinant h‐Fuc was stable up to 55 °C with incubation at pH 6.8 for 2 h; the optimum temperature for h‐Fuc is approximately 55 °C. The enzyme was stable at pH 2.5–7.0 for 2 h; the enzyme activity decreased greatly for pH greater than 8.0 or less than 2.0. The K_m and k_cat values of the recombinant h‐Fuc (at pH 6.8) were determined to be 0.28 mM and 17.1 s^?1, respectively. The study of pH‐dependent activity showed that the recombinant enzyme exhibited optimum activity at two regions near at pH 4.5 and pH 6.5. These features of the recombinant h‐Fuc are comparable to the native enzyme purified directly from human liver. Studies on the transfucosylation and common intermediate of the enzymatic reaction by NMR support that h‐Fuc functions as a retaining enzyme catalyzing the hydrolysis of substrate via a two‐step, double displacement mechanism.     

Biosynthesis of l‐4‐Chlorokynurenine,an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

l ‐4‐Chlorokynurenine (l ‐4‐Cl‐Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l ‐tryptophan into l ‐4‐Cl‐Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ‐490. We used genetic, biochemical, structural, and analytical techniques to establish l ‐4‐Cl‐Kyn biosynthesis, which is initiated by the flavin‐dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3‐dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.     

Introduction of d‐Amino Acids in Minimalistic Peptide Substrates by an S‐Adenosyl‐l‐Methionine Radical Epimerase

Post‐translational modifying enzymes from the S‐adenosyl‐l ‐methionine (AdoMet) radical superfamily garner attention due to their ability to accomplish challenging biochemical reactions. Among them, a family of AdoMet radical epimerases catalyze irreversible l ‐ to d ‐amino acid transformations of diverse residues, including 18 sites in the complex sponge‐derived polytheonamide toxins. Herein, the in vitro activity of the model epimerase OspD is reported and its catalytic mechanism and substrate flexibility is investigated. The wild‐type enzyme was capable of leader‐independent epimerization of not only the stand‐alone core peptide, but also truncated and cyclic core variants. Introduction of d ‐amino acids can drastically alter the stability, structure, and activity of peptides; thus, epimerases offer opportunities in peptide bioengineering.     

Biosynthesis of l‐4‐Chlorokynurenine,an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

l ‐4‐Chlorokynurenine (l ‐4‐Cl‐Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l ‐tryptophan into l ‐4‐Cl‐Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ‐490. We used genetic, biochemical, structural, and analytical techniques to establish l ‐4‐Cl‐Kyn biosynthesis, which is initiated by the flavin‐dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3‐dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.     

Mechanistic Insights into the Radical S‐adenosyl‐l‐methionine Enzyme NosL From a Substrate Analogue and the Shunt Products

The radical S‐adenosyl‐l ‐methionine (SAM) enzyme NosL catalyzes the transformation of l ‐tryptophan into 3‐methyl‐2‐indolic acid (MIA), which is a key intermediate in the biosynthesis of a clinically interesting antibiotic nosiheptide. NosL catalysis was investigated by using the substrate analogue 2‐methyl‐3‐(indol‐3‐yl)propanoic acid (MIPA), which can be converted into MIA by NosL. Biochemical assays with different MIPA isotopomers in D₂O and H₂O unambiguously indicated that the 5′‐deoxyadenosyl (dAdo)‐radical‐mediated hydrogen abstraction is from the amino group of l ‐tryptophan and not a protein residue. Surprisingly, the dAdo‐radical‐mediated hydrogen abstraction occurs at two different sites of MIPA, thereby partitioning the substrate into different reaction pathways. Together with identification of an α,β‐unsaturated ketone shunt product, our study provides valuable mechanistic insight into NosL catalysis and highlights the remarkable catalytic flexibility of radical SAM enzymes.     

Melt polycondensation approach for reduction degradable helical polyester based on l‐cystine

Melt polycondensation approach is developed for new classes of reduction responsive disulfide containing functional polyesters based on l ‐cystine amino acid resources under solvent free process. l ‐Cystine was converted into multi‐functional ester‐urethane monomer and subjected to thermoselective transesterification at 120 °C with commercial diols in the presence of Ti(OBu)₄ to produce polyesters with urethane side chains. The polymers were produced in moderate to high molecular weights and the polymers were found to be thermally stable up to 250 °C. The β‐sheet hydrogen bonding interaction among the side chain urethane unit facilitated the self‐assembly of the polyester into amyloid‐like fibrils. The deprotection of urethane unit into amine functionality modified the polymers into water soluble cationic polyester spherical nanoparticles. The reduction degradation of disulfide bond was studied using DTT as a reducing agent and the high molecular weight polymers chains were found be chopped into low molecular weight oligomers. The cytotoxicity of cationic disulfide nanoparticle was studied in MCF‐7 cells and they were found to be biocompatible and non‐toxic to cells upto 50 μg/mL. The custom designed reduction degradable and highly biocompatible disulfide polyesters from l ‐cystine are useful for futuristic biomedical applications. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 2864–2875     

l‐Leucinium perchlorate

Crystals of l ‐leucinium perchlorate, C₆H₁₄NO₂⁺·ClO₄⁻, are built up from protonated l ‐leucinium cations and perchlorate anions. l ‐Leucinium cations related by a twofold screw axis are inter­connected by N—H⋯O hydrogen bonds into zigzag chains parallel to [010]. The O atoms of the perchlorate anions act as acceptors of hydrogen bonds that link the l ‐leucinium chains into separated but inter­acting two‐dimensional layers parallel to (001). Since the title compound crystallizes in a non‐centrosymmetric space group, it can be useful as a material for non‐linear optics. The efficiency of second harmonic generation is about twice that of K₂[HPO₄].     

Methyl 4‐O‐β‐l‐galactopyranosyl‐β‐d‐glucopyranoside (methyl β‐l‐lactoside)

Methyl β‐l ‐lactoside, C₁₃H₂₄O₁₁, (II), is described by glycosidic torsion angles ϕ (O5_Gal—C1_Gal—O4_Glc—C4_Glc) and ψ (C1_Gal—O1_Gal—C4_Glc—C5_Glc) of 93.89 (13) and −127.43 (13)°, respectively, where the ring atom numbering conforms to the convention in which C1 is the anomeric C atom and C6 is the exocyclic hydroxy­methyl (CH₂OH) C atom in both residues (Gal is galactose and Glc is glucose). Substitution of l ‐Gal for d ‐Gal in the biologically relevant disaccharide, methyl β‐lactoside [Stenutz, Shang & Serianni (1999). Acta Cryst. C 55 , 1719–1721], (I), significantly alters the glycosidic linkage inter­face. In the crystal structure of (I), one inter‐residue (intra­molecular) hydrogen bond is observed between atoms H3O_Glc and O5_Gal. In contrast, in the crystal structure of (II), inter‐residue hydrogen bonds are observed between atoms H6O_Glc and O5_Gal, H6O_Glc and O6_Gal, and H3O_Glc and O2_Gal, with H6O_Glc serving as a donor with two intra­molecular acceptors.     

Two modifications of l‐alanyl‐l‐tyrosyl‐l‐alanine with different solvent mol­ecules in the crystal lattice

The low‐temperature crystal and mol­ecular structure analyses of two modifications of l ‐alanyl‐l ‐tyrosyl‐l ‐alanine with water, C₁₅H₂₁N₃O₅·2.63H₂O [(I), at 9 K], and ethanol, C₁₅H₂₁N₃O₅·C₂H₅O [(II), at 20 K], solvent mol­ecules in the crystal lattice show that the overall conformations of both modifications of the title tripeptide are practically the same. Moreover, despite the presence of different solvent mol­ecules in the crystal lattice, the specific inter­molecular inter­actions characteristic for individual tripeptide mol­ecules of (I) and (II) are conserved. The crystal packing of the two modifications of Ala‐Tyr‐Ala differ from each other only in the solvent region. The tight arrangements of tripeptide mol­ecules seem to be responsible for similar displacement parameters for all non‐H atoms, despite the different distances from the mol­ecular centre of mass. Comparison of the displacement parameters between the room‐ and low‐temperature structures shows that an average U_eq value decrease of about 80% takes place at 9 K [for (I)] and 20 K [for (II)] with respect to room temperature.     

A pipecolic acid (Pip)‐containing dipeptide,Boc‐d‐Ala‐l‐Pip‐NHiPr

The title dipeptide, 1‐(tert‐butoxy­carbonyl‐d ‐alanyl)‐N‐iso­propyl‐l ‐pipecol­amide or Boc‐d ‐Ala‐l ‐Pip‐NHⁱPr (H‐Pip‐OH is pipecolic acid or piperidine‐2‐carboxylic acid), C₁₇H₃₁N₃­O₄, with a d –l heterochiral sequence, adopts a type II′β‐­turn conformation, with all‐trans amide functions, where the C‐terminal amide NH group interacts with the Boc carbonyl O atom to form a classical i+3 i intramolecular hydrogen bond. The C^α substituent takes an axial position [H^α (Pip) equatorial] and the trans pipecolamide function is nearly planar.     

l‐Cysteinium semioxalate

The title salt, C₃H₈NO₂⁺·C₂HO₄⁻, formed between l ‐cysteine and oxalic acid, was studied as part of a comparison of the structures and properties of pure amino acids and their cocrystals. The structure of the title salt is very different from that formed by oxalic acid and equivalent amounts of d ‐ and l ‐cysteine molecules. The asymmetric unit contains an l ‐cysteinium cation and a semioxalate anion. The oxalate anion is only singly deprotonated, in contrast with the double deprotonation in the crystal structure of bis(dl ‐cysteinium) oxalate. The oxalate anion is not planar. The conformation of the l ‐cysteinium cation differs from that of the neutral cysteine zwitterion in the monoclinic and orthorhombic polymorphs of l ‐cysteine, but is similar to that of the cysteinium cation in bis(dl ‐cysteinium) oxalate. The structure of the title salt can be described as a three‐dimensional framework formed by ions linked by strong O—H...O and N—H...O and weak S—H...O hydrogen bonds, with channels running along the crystallographic a axis containing the bulky –CH₂SH side chains of the cysteinium cations. The cations are only linked through hydrogen bonds via semioxalate anions. There are no direct cation–cation interactions via N—H...O hydrogen bonds between the ammonium and carboxylate groups, or via weaker S—H...S or S—H...O hydrogen bonds.     

Microchip CE‐LIF method for the hydrolysis of L‐glutamine by using L‐asparaginase enzyme reactor based on gold nanoparticle

l ‐Asparaginase (l ‐Asnase) can suppress the growth of malignant cells by rapid depletion of two essential amino acids, l ‐glutamine (l ‐Gln) and l ‐asparagine (l ‐Asn). To study the cytotoxic effect and the secondary complications of l ‐Asnase in the treatment of acute lymphoblastic leukemia, the development of a novel enzyme reactor of l ‐Asnase for the hydrolysis of l ‐Gln, employing the enzyme‐gold nanoparticle conjugates in capillary, was reported in this work. First, a microchip CE (MCE)‐LIF was established for the separation of l ‐amino acids (l ‐Gln and l ‐glutamic acid) and studying the hydrolysis of l ‐Gln by using l ‐Asnase enzyme reactor. Then, using l ‐Gln as target analyte, the enzyme kinetics of l ‐Asnase in free solution, enzyme‐gold nanoparticle conjugates (E‐GNP), and the enzyme‐gold nanoparticle conjugates immobilized in capillary (E‐GNP‐C) were investigated in detail with the proposed MCE‐LIF method. Moreover, for optimizing the enzymatic reaction efficiency, three important parameters, including the length of capillary, the enzyme concentration reacted with gold nanoparticle and the amount of l ‐Asnase immobilized on the gold nanoparticle, have been studied. Owing to the high specific activity, the E‐GNP‐C enzyme reactor exhibited the best performance for the hydrolysis of l ‐Gln.     

catena‐Poly[copper(II)‐μ‐l‐tyrosyl‐l‐leucinato]

In the title compound, [Cu(C₁₅H₂₀N₂O₄)]_n, the copper(II) coordination is square planar. The anionic l ‐tyrosyl‐l ‐leucinate ligand binds in an N,N′,O‐tridentate mode to one Cu^II cation on one side and in an O‐monodentate mode to a second Cu^II cation on the other side, thus defining –Cu—O—C—O—Cu′– chains which run along the a axis. These chains are held together by a strong hydrogen bond involving the hydroxy H atom.