Ω-amino Acids Research Articles

Enhancement through Mutagenesis of the Binding of the Isolated Kringle 2 Domain of Human Plasminogen to ω-Amino Acid Ligands and to an Internal Sequence of a Streptococcal Surface Protein

In the background of the recombinant K2 module of human plasminogen (K2(Pg)), a triple mutant, K2(Pg)[C4G/E56D/L72Y], was generated and expressed in Pichia pastoris cells in yields exceeding 100 mg/liter. The binding affinities of a series of lysine analogs, viz. 4-aminobutyric acid, 5-aminopentanoic acid, epsilon-aminocaproic acid, 7-aminoheptanoic acid, and t-4-aminomethylcyclohexane-1-carboxylic acid, to this mutant were measured and showed up to a 15-fold tighter interaction, as compared with wild-type K2(Pg) (K2(Pg)[C4G]). The variant, K2(Pg)[C4G/E56D], afforded up to a 4-fold increase in the binding affinity to these same ligands, whereas the K2(Pg)[C4G/L72Y] mutant decreased the same affinities up to 5-fold, as compared with K2(Pg)[C4G]. The thermal stability of K2(Pg)[C4G/E56D/L72Y] was increased by approximately 13 degrees C, as compared with K2(Pg)[C4G]. The functional consequence of up-regulating the lysine binding property of K2(Pg) was explored, as reflected by its ability to interact with an internal sequence of a plasminogen-binding protein (PAM) on the surface of group A streptococci. A 30-mer peptide of PAM, containing its K2(Pg)-specific binding region, was synthesized, and its binding to each mutant of K2(Pg) was assessed. Only a slight enhancement in peptide binding was observed for K2(Pg)[C4G/E56D], compared with K2(Pg)[C4G] (K(d) = 460 nM). A 5-fold decrease in binding affinity was observed for K2(Pg)[C4G/L72Y] (K(d) = 2200 nM). However, a 12-fold enhancement in binding to this peptide was observed for K2(Pg)[C4G/E56D/L72Y] (K(d) = 37 nM). Results of these PAM peptide binding studies parallel results of omega-amino acid binding to these K2(Pg) mutants, indicating that the high affinity PAM binding by plasminogen, mediated exclusively through K2(Pg), occurs through its lysine-binding site. This conclusion is supported by the 100-fold decrease in PAM peptide binding to K2(Pg)[C4G/E56D/L72Y] in the presence of 50 mM 6-aminohexanoic acid. Finally, a thermodynamic analysis of PAM peptide binding to each of these mutants reveals that the positions Asp(56) and Tyr(72) in the K2(Pg)[C4G/E56D/L72Y] mutant are synergistically coupled in terms of their contribution to the enhancement of PAM peptide binding.

Apparent molar volume of some ω-amino acids in aqueous electrolyte systems

Apparent molar volumes (Vϕ) of some ω-amino acids have been determined in aqueous guanidine hydrochloride (6 m GuHCl) and sodium sulphate (2 m Na2SO4) solutions at 288.15 and 298.15 K using a vibrating tube digital densimeter. The transfer volumes of amino acids from water to aqueous electrolyte systems have been reported. The transfer properties are interpreted in terms of strong interactions, based on a cosphere overlap model of guanidine hydrochloride/sodium sulphate molecules with the charged centers of the zwitterions (amino acid molecules), as compared to ion - nonpolar group interactions.Key words: amino acids, denaturation, guanidine hydrochloride, sodium sulphate, apparent molar volume, zwitterions, limiting apparent molar volume.

Incorporation of glycine residues in even–even polyamides. Part II: Nylons 6,10 and 12,10

Sequential copolymers of glycine and nylons 6,10 and 12,10 have been prepared by incorporating single glycine units at both ends of the diamine. These polymers have been obtained by interfacial polymerization and subsequently characterized by infrared and n.m.r. spectroscopies together with thermal analysis. The structure and morphology of lamellar crystals have also been investigated by X-ray diffraction and transmission electron microscopy, both imaging and diffraction. Additional data have been obtained from uniaxially oriented fibres. Results described in this paper complete previous studies on the incorporation of glycine units in nylons derived either from ω-amino acids or from diamines and diacids.

Apparent molar volume of some ω-amino acids in aqueous electrolyte systems

Apparent molar volume of some ω-amino acids in aqueous electrolyte systems

Synthesis of ω-Nitro Acids and ω-Amino Acids by Ring Cleavage of α-Nitrocycloalkanones

The reaction of various α-nitrocycloalkanones 1 with aqueous 0.05 M NaOH, at 80 °C, in the presence of cetyltrimethylammonium chloride (CTACl) as a cationic surfactant, produces ω-nitro acids 2 in good yields. Reduction of the latter with HCOONH4/Pd-C, in methanol, at 80 °C affords ω-amino acids 3. The synthesis of methyl 9-oxodecanoate (8) is also reported.

ω-Amino acids and lactams with chiral biaryl axis

The optically active amino acids ( R)- 7 and ( S)- 8 were prepared as the first representatives of ω-amino acids possessing a biaryl axis as the sole element of chirality.

Incorporation of glycine residues in even–even nylons disrupts their characteristic all- trans conformation

A series of regular polymers derived from the inclusion of glycine in even–even nylons (2,4, 2,6, 4,4 and 4,6) has been prepared by the active ester method. The structure and morphology of lamellar crystals have also been investigated by using transmission electron microscopy, selected-area electron diffraction and X-ray diffraction. The results indicate that at room temperature the structure of these nylons is different from the typical forms ( α and β) of even–even polyamides. Observations described in this paper are complementary to previous work about the incorporation of glycine units in nylons derived from ω-amino acids and indicate that glycine residues always tend to adopt their characteristic polyglycine II conformation.

Crystal structure of tryptophanase

The X-ray structure of tryptophanase (Tnase) reveals the interactions responsible for binding of the pyridoxal 5′-phosphate (PLP) and atomic details of the K + binding site essential for catalysis. The structure of holo Tnase from Proteus vulgaris (space group P2 12 12 1 with a = 115.0 Å, b = 118.2 Å, c = 153.7 Å) has been determined at 2.1 Å resolution by molecular replacement using tyrosine phenol-lyase (TPL) coordinates. The final model of Tnase, refined to an R-factor of 18.7%, ( R free = 22.8%) suggests that the PLP-enzyme form observed in the structure is a ketoenamine. PLP is bound in a cleft formed by both the small and large domains of one subunit and the large domain of the adjacent subunit in the so-called “catalytic” dimer. The K + cations are located on the interface of the subunits in the dimer. The structure of the catalytic dimer and mode of PLP binding in Tnase resemble those found in aspartate aminotransferase, TPL, ω-amino acid pyruvate aminotransferase, dialkylglycine decarboxylase (DGD), cystathionine β-lyase and ornithine decarboxylase. No structural similarity has been detected between Tnase and the β 2 dimer of tryptophan synthase which catalyses the same β-replacement reaction. The single monovalent cation binding site of Tnase is similar to that of TPL, but differs from either of those in DGD.

ω-Amino Acids in Peptide Design. Crystal Structures and Solution Conformations of Peptide Helices Containing a β-Alanyl-γ-Aminobutyryl Segment

Insertion of achiral ω-amino acids into peptide sequences results in replacement of scissile peptide bonds by proteolytically stable C−C bonds. This provides a convenient means of creating peptidomimetics. The present study establishes the preservation of helical structures in octa- and undecapeptides with centrally located β- and γ-amino acids in the sequence. X-ray diffraction analyses of single crystals and NMR studies have been used to investigate the extent of perturbations of a regular 310- or α-helix by the introduction of (−CH2−)n groups into the backbone by the use of the β-Ala-γ-Abu segment (β-Ala = β-alanine, γ-Abu = γ-aminobutyric acid), which is formally homomorphous with a (Gly)3 segment. In crystals, the octapeptide Boc-Leu-Aib-Val-β-Ala-γ-Abu-Leu-Aib-Val-OMe (1) and the undecapeptide Boc-Leu-Aib-Val-β-Ala-γ-Abu-Leu-Aib-Val-Ala-Leu-Aib-OMe (2) retain their helical motifs with minor bulges. Five new types of 4 → 1, 5 → 1, and 6 → 1 hydrogen bond rings are formed with up to three extra CH2 moieties. Cell parameters for peptide 1 are space group P212121 with a = 11.506 (1) Å, b = 16.600 (1) Å, c = 27.362(1) Å, and R = 6.1% for 2696 data measured >4σ(F); for the undecapeptide 2, the space group is P21 with a = 8.605 (3) Å, b = 22.806 (4) Å, c = 19.014 (3) Å, β = 101.47(2)°, and R = 7.5% for 3797 data measured >4σ(F). Helical conformations in solution are also maintained for peptide 2 as is evident from NMR studies in CDCl3, which suggest that the centrally positioned, flexible β-Ala-γ-Abu segment can be comfortably accommodated into helical structures adopting gauche conformations about specific C−C bonds of the poly(methylene) units. Twenty structures for backbone conformations generated from MD simulations using NMR-derived contraints, superpose with a low RMSD value (0.78 ± 0.05 Å), further indicating that in these peptides the conformational flexibility of the β-Ala-γ-Abu segment is limited and confined to largely helical conformations.

Apparent Molar Volume of some ω-Amino Acids in Aqueous Guanidine Hydrochloride Solutions at 15 and 25°

Apparent Molar Volume of some ω-Amino Acids in Aqueous Guanidine Hydrochloride Solutions at 15 and 25°

Asymmetric Synthesis of α-Alkyl-ω-amino Acids and α-Alkyllactams

A versatile access to optically active α-branched ω-amino acids 8 and 9 and corresponding lactams 11 was developed allowing the synthesis of either enantiomer of these products. This asymmetric synthesis is based on amino alcohols 3 as chiral auxiliaries, which were converted to 2-(ω-benzenesulfonylaminoalkyl)oxazolines 4 by reaction with ω-amino-imido esters 2 or derivatives of lactams 1. The 2-(ω-benzenesulfonylaminoalkyl)oxazolines 4 were deprotonated by LDA and α-alkylated by alkyl halides 5. Final hydrolytic cleavage of the oxazoline ring afforded the amino acids 8 and 9 or lactams 11. The method does not follow Meyers model of α-alkylation of 2-alkyloxazolines and requires BEt3 as additive in order to achieve complete stereoselectivity.

Maillard Reaction of Free and Nucleic Acid-Bound 2-Deoxy-d-ribose and d-Ribose with ω-Amino Acids

The Maillard reaction of free and nucleic acid-bound 2-deoxy-d-ribose and d-ribose with ω-amino acids (4-aminobutyric acid, 6-aminocaproic acid) was investigated under both stringent and mild conditions. Without (with) amines 2-deoxy-d-ribose (d-ribose) displays the strongest browning activity, and DNA is much more reactive than RNA. From stringent reaction between 2-deoxy-d-ribose (or DNA) and methyl 4-aminobutyrate, methyl 4-[2-[(oxopyrrolidinyl)methyl]-1-pyrrolyl]butyrate (12) was identified by GC/MS and NMR as a new 2-deoxy-d-ribose specific key compound trapped by pyrrolidone formation. Levulinic acid-related N-substituted lactames 13−15 were identified as predominant products from DNA with amino acids, whereas RNA paralleled the reaction with d-ribose. α-Angelica lactone (2), a significant degradation product of DNA, and thiols leads under mild conditions to new addition products (e.g., 17 with glutathione). Probable reaction pathways considering activating effects of the polyphosphate backbone of nucleic acids are discussed. Keywords: Maillard reaction of nucleic acids/2-deoxy-d-ribose; levulinic acid; α-angelica lactone; trapping of 2-(hydroxymethyl)pyrrole; pyrroles from ω-amino acids and riboses/nucleic acids

A New Facile and Rapid Synthesis of Aliphatic Polyamides by Microwave Assisted Polycondensation of ω-Amino Acids and Nylon Salts

A new facile method for the rapid synthesis of aliphatic polyamides was developed by using a domestic microwave oven by the polycondensation of both ω-amino acids and nylon salts in the presence of a small amount of a polar organic medium that acts as a primary microwave absorber. Suitable organic media were tetramethylene sulfone, amide-type solvents such as N-cyclohexyl-2-pyrrolidone and 1,3-dimethyl-2-imidazolidone, and phenolic solvents like m-cresol and o-chlorophenol. The polycondensation proceeded rapidly, compared with the conventional melt polycondensation, and was almost complete within 5 min, producing a series of polyamides with inherent viscosities around 0.5 dl g−1.

Crystal Structures of Apolipoprotein(a) Kringle IV37 Free and Complexed with 6-Aminohexanoic Acid and with p-Aminomethylbenzoic Acid: Existence of Novel and Expected Binding Modes

Kringles are protein modules found within a wide variety of fibrinolytic and coagulation-related proteins that show binding affinity for lysine, lysine analogs and for fibrin. We report here the crystal structures of apolipoprotein(a) kringle IV37 (apo(a) K4 37) in its free state and in separate complexes with two ω-amino acids, 6-aminohexanoic acid (6AHA) and p-aminomethylbenzoic acid (PAMBA). The structures of the unliganded form and of both complexes have been determined and refined by restrained least-squares methods to about 2.0 Å. The overall kringle architecture is essentially identical with that determined in other kringles but it shows some small significant structural changes in the lysine binding site. There is virtually no difference in conformation between the unliganded and complexed forms, suggesting that apo(a) K4 37does not undergo any conformational rearrangement upon binding. The 6AHA molecule binds to apo(a) K4 37in a completely different way from that observed with the kringle 4 of plasminogen (PGK4). Its amino group makes an ion pair interaction with the two aspartate residues (Asp55/Asp57) of the anionic center and its carboxylate group faces out into the solvent making water-mediated contacts with the protein. The mode of binding of PAMBA resembles more that described for 6AHA when bound to PGK4. The PAMBA molecule is bound by ion pair interactions with the two aspartate residues (Asp55/Asp57) and with Arg71 from the cationic center and by van der Waals contacts. The relative importance of the cationic center from kringles for binding zwitterionic ligands is discussed.

Novel method of synthesizing poly(succinimide) and its copolymeric derivatives by acid-catalysed polycondensation of l-aspartic acid

High molecular weight poly(succinimide) was quantitatively synthesized by polycondensation of l-aspartic acid using a catalytic amount of o-phosphoric acid in the mixed solvent of mesitylene and sulfolane. In addition, under similar conditions, l-aspartic acid and ω-amino acid produced an organic solvent-soluble poly(succinimide-co-ω-amino acid), which melted below the decomposition temperature.

Synthesis of enantiomerically pure ω-amino acids by asymmetric α-alkylation of chiral ω-aminoalkyloxazolines

Enantiomerically pure α-alkyl-ω-aminocarboxylic acids 5, 6 and the corresponding α-alkyllactams 7 are synthesized starting from lactams 1 by ring transformation with a chiral aminoalcohol 2, asymmetric α-alkylation of the resulting 2-(ω-aminoalkyl)-oxazolines 3 and final hydrolysis.

Thermodynamics of the interaction of cyclodextrins with aromatic and α, ω-amino acids in aqueous solutions: a calorimetric study at 25°C

The interaction in water of α- and β-cyclodextrins with l-phenylalanine, l-tyrosine, l-tryptophan, and l-histidine has been studied calorimetrically at 25°C in pure water and in a phosphate buffer (pH 11.3). The interaction in water of α-cyclodextrin with some α, ω-amino acids was also studied. When a complex forms, calorimetry allows the calculation of both the enthalpy and the association constant, from which the free energy and the entropy of the process can be obtained. Aromatic amino acids form 1:1 inclusion complexes, characterized by low values of the association constants. The association occurs through the insertion of the guest's aromatic ring into the host's cavity, and is stronger at pH 11.3 than in pure water. For α, ω-amino acids the association constants increase with increasing lengths of the alkyl chains between the functional groups. For this class of substances the association is supposed to occur through an interaction mechanism different from inclusion, which involves mainly the exterior of cyclodextrin. The analysis of the signs and values of the thermodynamic parameters obtained permits hypotheses to be made about the forces involved in the association process.

Intramolecular hydrogen bonding of ?-hydroxypentanoic acid

Six symmetry-unique local minima of δ-hydroxypentanoic acid, which are stabilized by an intramolecular O HOCO hydrogen bond, were located by ab initio self-consistent field (restricted Hartree–Fock) calculations. The geometry data and hydrogen bond parameters of these conformers are presented together with the potential barriers for the various reaction paths. These data are compared to the previous studies on the smaller homologues and the results of investigations on ω-amino acids of the same size. © 1995 John Wiley & Sons, Inc.

Synthesis of bridged thiazolium salts as models for thiamin

Bridged thiazolium salts 16b and c have been synthesized in a short procedure starting with ω-amino acids. Condensation with carbon disulfide and α-chloro ketone 19 provided thiazole-2(3H)-thiones 14b and c and the bridge was then formed by standard macrolactonization procedures. Oxidation of the thiones with hydrogen peroxide then gave the thiazolium salts. The same cyclization procedures failed to yield the shorter bridged compound 15a but gave the cyclic dimer and trimer. Starting with protected lysine derivatives 31b and c, thiazole-2(3H)-thiones 36b and c were obtained. In both cases the cyclization reaction yielded two separable atropisomers depending on which side of the ring the bridge was formed. The catalytic reactions of thiazolium salts 16b and c were compared with unbridged analogues and it was found that, whereas the longer bridged one behaved normally, the shorter bridged salt was unable to catalyse the benzoin condensation. A novel 2-benzoyl-2,3-dihydrothiazole 44 was isolated from this reaction mixture.

Structural and Mechanistic Analysis of Two Refined Crystal Structures of the Pyridoxal Phosphate-dependent Enzyme Dialkylglycine Decarboxylase

Two refined structures, differing in alkali metal ion content, of the bifunctional, pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase (DGD) are presented in detail. The enzyme is an α tetramer, built up as a dimer of dimers, with a subunit molecular mass of 46.5 kDa. The fold of DGD is similar to those of aspartate aminotransferase, ω-amino acid aminotransferease and tyrosine phenol-lyase. The structure has two binding sites for alkali metal ions. GDG with potassium in site 1 (near the active site) and sodium in site 2 (at the surface of the molecule) has been refined against 2.6Å resolution data ( R-factor=17.6%), and DGD with sodium at both sites has been refined against 2.1 Å resolution data ( R-factor=17.8%). The proximity of site 1 to the active site accounts for the dependence of enzyme activity on potassium ions, and the observed active site structural changes caused by ion exchange at this site explain the inhibition of activity by sodium. DGD catalyzes both the decarboxylation of dialkylglycine species and the transamination of l-amino acids in its normal catalytic cycle. The active site structure of DGD is moderately homologous to that of aspartate aminotransferase, which catalyzes only transamination; both the differences and similarities provide mechanistic guidelines for the DGD-catalyzed reactions. Models of the l-isovaline and l-alanine external aldimine intermediates suggest mechanisms by which the decarboxylation and transamination reactions could be accomplished within the single active site. Decarboxylatio is proposed to be at least partially catalyzed by stereo-electronic activation of the C α-carboxylate bond achieved by orienting this bond perpendicular to the plane of the pyridinium ring in the dialkylglycine external aldimine intermediate. Transamination is proposed to be catalyzed by a similar effect on the C α-H bond of the l-amino acid external aldimine intermediate, combined with general base catalysis provided by Lys272, in analogy to the mechanism of as partate aminotransferase.