Helix–coil stability constants for the naturally occurring amino acids in water. XXI. Glutamine parameters from random poly(hydroxypropylglutamine-co-L-glutamine) and poly(hydroxybutylglutamine-co-L-glutamine)

Water-soluble, random copolymers containing L -glutamine and either N⁵-(3-hydroxypropyl)-L -glutamine or N⁵-(4-hydroxybutyl)-L -glutamine were synthesized, fractionated, and characterized. The thermally induced helix–coil transitions of these copolymers were studied in water. A short-range interaction theory was used to deduce the Zimm-Bragg parameters σ and s for the helix–coil transition in poly(L -glutamine) in water from an analysis of the melting curves of the copolymers in the manner described in earlier papers. The computed values of s indicate that L -glutamine is helix-indifferent at low temperature and a helix-destabilizing residue at high temperature in water. At all temperatures in the range of 0–70°C, the glutamine residue promotes helix–coil boundaries since the computed value of σ is large.     

Effect of hydrophobic side chains on the pressure-induced changes in the NMR spectra of water-soluble biopolymers

Measurements have been made of the high-resolution nmr spectra of the polyamino acids poly[N⁵-(2-hydroxyethyl)-L -glutamine] and poly[N⁵-(4-hydroxybutyl)-L -glutamine] in mixed deuterium oxide and water solvent at varying pressures from 1.03 to 3163.7 kg/cm². The results are compared with previously reported results for the polymer poly[N⁵-(3-hydroxypropyl)-L -glutamine] under similar conditions. The significance of the behaviour of the polymers is considered in terms of the effect of the presence of hydrophobic residues in their side chains.     

Helix-coil stability constants for the naturally occurring amino acids in water. XII. Asparagine parameters from random poly(hydroxybutylglutamine-co-L-asparagine)

The synthesis and characterization of water-soluble random copolymers containing L -asparagine with N⁵-(4-hydroxybutyl)-L -glutamine, and the thermally induced helix-coil transitions of these copolymers in water, are described. The incorporation of L -asparagine was found to decrease the helix content of the polymers in water at all temperatures. The Zimm-Bragg parameters σ and s for the helix-coil transition in poly(L -asparagine) in water were deduced from an analysis of the copolymer melting curves in the manner described in earlier papers. The computed values of s indicate that asparagine destabilizes helical sequences at all temperatures in the range 0–60°C.     

Helix-coil stability constants for the naturally occuring amino acids in water. XIV. Methionine parameters from random poly(hydroxypropylglutamine,L-Methionine)

Water-soluble, random copolymers containing L -methionine and N⁵-(3-hydroxypropyl)-L -glutamine have been prepared, fractionated, and characterized. The thermally induced helix-coil transitions of these copolymers in water have been investigated, and it has been found that incorporation of L-methionine increases the helix content of the polymers at all temperatures in the range of 0–60°C. The Zimm-Bragg parameters σ and s for the helix-coil transition in poly(L -methionine) in water were deduced from an analysis of the melting curves of the copolymers using the methods described in earlier papers.     

Rigidity of α-helical polypeptides. A new method of obtaining the persistence length from viscosimetric data

The hydrodynamic properties of α-helical poly(L -glutamic acid), (Glu)_n in aqueous solutions and in mixtures of water with organic solvents have been interpreted in terms of the persistence length of the macromolecule. A modification of the method of Vitovskaya and Tsvetkov has been proposed in order to allow a more accurate determination of this parameter. The addition of an organic solvent increases strongly the rigidity of the helical conformation of (Glu)_n. A comparison is made with some data of the literature of poly[N⁵-(3-hydroxy propyl)L -glutamine], [Gln(CH₂)₃OH]_n, and poly(γ-benzyl-L -glutamate), [Glu(OBzl)]_n.     

Solution properties of synthetic polypeptides. VI. Helix-coil transition of poly-N5-(3-hydroxypropyl)-L-glutamine

A new procedure for evaluating u and σ characterizing σ-helix-forming polypeptides in solution was derived from Nagai's theory for the helix–coil transition of such polymers. Here u is the activity for helix formation from random coil, and σ is the helix initiation parameter. The necessary data are the helical content f_N at fixed solvent and temperature as a function of N, where N is the degree of polymerization of the polypeptide sample. Such data were obtained from ORD measurements on a number of fractionated samples of poly-N⁵-(3-hydroxypropyl)-L -glutamine (PHPG) in mixtures of water and methanol covering the complete range of composition and at various termperatures (5–40°C). When analyzed in terms of the proposed procedure, they yielded values of σ which were in the range (3.2 ± 0.6) × 10^?4, substantially independent of solvent composition and temperature. These values were much larger than those obtained recently for σ of poly(β-benzyl-L -aspartate) in m-cresol and in a mixture of chloroform and DCA. The data for [η] and s₀ (limiting sedimentation coefficient) as functions of molecular weight indicated that the molecular shape of PHPG in pure methanol is essentially rodlike, whereas that in pure water is not entirely randomly coiled but rather may be regarded as an interrupted helix. These indications were consistent with the results from ORD measurements. When plotted against the corresponding values of f_N, the values of [η] and [s₀] for PHPG in mixtures of water and methanol of various compositions and temperatures formed smooth composite curves, and we attributed these phenomena to the fact that σ of PHPG was nearly constant under these solvent conditions. Here [s₀] stands for a reduced limiting sedimentation coefficient which is equal to the inverse friction factor of the solute molecule.     

Isolation of N-acetylglutamine, pyroglutamic acid and the butyl ester of pyroglutamic acid from human brain

N-acetyl-l -glutamine, pyroglutamic acid, and the butyl ester of pyroglutamic acid were isolated in pure form from an aqueous extract of human brain. These compounds were isolated by combination of paper and ion exchange chromatography. The isolated substance identified as N-acetyl-l -glutamine did not react with the ninhydrin reagent but yielded glutamic acid and ammonia upon acid hydrolysis. An acetyl hydrazide was identified by paper chromatography from hydrazinolysates of the isolated substance. The glutamic acid liberated by hydrolysis had the l -configuration. The results of elementary analysis of the isolated compound were in full accord with the analysis calculated for synthetic N-acetyl-l -glutamine. A large amount of pyroglutamic acid and a substance identical with the butyl ester of pyroglutamic acid were isolated in pure form. The results of our studies suggest that pyroglutamic and the butyl ester derivative were artifacts formed during the isolation and purification procedures.     

The random copolymerization of γ-benzyl-L-glutamate and L-valine N-carboxyanhydrides: Reactivity ratio and heteogeneity studies

The random copolymerization of the N-carboxyhydrides of γ-benzyl-L -glutamate and L -valine using triethylamine as the initiator in low dielectric media reults in high-molecular-weight copolymers at low convenrson. This behavior makes it possible to apply the monomer reactivity ration theory, which was dervied for addition polymerizations, and from the use of the copolymer composition equation, the respective monomer reactivity ratios, the average and incremental copolymer compositions, and the monomer feed ratio at any conversion can be determined. A comparison of the reactivity ratios for the copolymerization of γ-benzyl-L -glutamate NCA and L -valine NCA in benzene/methylene chloride (r_G = 2.1, r_V = 0.6) with those obtained using dioxane (r_G = 2.7, r_V = 0.3) indicates that the interchain compositional heterogeneity is greater for copolymers prepared in the dioxane. For Example, at 100% conversion of the monomeric NCAs, Poly[Glu(OBzl)⁵⁰Val⁵⁰] prepared in dioxance has an interchain composition ranging from 74 to 0 mol % γ-benzyl-L -glutamate, whereas in benzene/methylene chloride the interchain composition of γ-benzyl-L -glutamae ranges from 65 to 0 mol %. Once the reactivity ratios are obtained for any pair of α-amino and N-carboxyanhydrides, the use of the aforementioned parameters relating to interchain composition can give insight into the compositional heterogeneity between chains as a function of conversion and provide a basis for the preparation of random α-amino acid copolymers that are homogeneous.     

Poly(L-histidyl-L-alanyl-α-L-glutamic acid). I. Synthesis

Poly(L -histidyl-L -alanyl-α-L -glutamic acid) has been prepared in order to test the acid–base catalytic ability of a carboxyl-imidazole hydrogen-bonded system. Two different blocked histidyl-alanyl-glutamic acid monomers were used in the polymerization step. The imidazole ring was blocked with either a dinitrophenyl or a t-butyloxycarbonyl group. The γ-carboxyl of glutamic acid was protected as the benzyl ester. Both the coupling reactions and the polymerization step were via the N-hydroxysuccinimide active ester method. Thiolysis removed the dinitrophenyl group, while hydrogen bromide removed the t-butyloxycarbonyl and the benzyl groups. The water-soluble unblocked polymers obtained were fractionated on Sephadex G-50 or Bio-Gel P30. Fractions within a range of average molecular weights of 2,000 to 25,000 were isolated. Enzymatic oxidation of the acid hydrolyzate of the polymers revealed that no detectable racemization had occurred.     

Kinetic studies of the helix–coil transition in aqueous solutions of poly(L-lysine)

The rate of conformational change of aqueous poly(α-L -lysine) solutions was measured using the electric field pulse relaxation method with conductivity detection. The relaxation time as a function of pH exhibits two maxima. One is assigned to a proton transfer reaction and the other to the helix–coil conformational transition. The helix nucleation parameter and the maximum relaxation time yield the rate constant of helix growth process (k_F) according to Schwarz's kinetic theory as k_F = 2 × 10⁷ sec^?1, which is comparable to that of the poly(glutamic acid) solution. The thermodynamic parameters of the helix growth process are compared with those of poly(glutamic acid).     

On the Manner of Cyclization of <Emphasis Type="Italic">N</Emphasis>-Acylated Aspartic and Glutamic Acid Derivatives

Abstract  

When synthesizing arylpiperazine library modified with N-acylated amino acid derivatives (e.g., cyclized aspartic acid, cyclized glutamic acid, proline) we wished to rapidly determine the way of cyclization of N-acylated glutamic acid derivatives. During concomitant cleavage and cyclization two alternative routes were possible—either formation of six-member imide (glutarimide) or five-member lactam. Application of MS/MS and ¹H NMR method allowed us to establish that cyclization of N-acylated glutamic acid derivatives preceded to lactams—N-acylated pyroglutamic acid derivatives.     

The infuence of interchain compositional heterogeneity on the conformation in random copolymers of γ-benzyl-L-glutamate and L-valine

Variation in the solvent used for the copolymerization of γ-benzyl-L -glutamate and L -valine N-carboxyanhydrides provides copolymers which have variable interchain compositions, and this variation in interchain compositional heterogeneity is reflected in the solid-state conformations of the respective copolymers. Poly[Glu(OBzl)²⁹Val⁷¹] prepared in dioxane exhibits a β-structure, whereas a copolymer of the same average composition prepared in benzene/methylene chloride shows predominantly an β-helix conformation with a small amount of β-structure. The use of the monomer reactivity ratio permits the calculation of the average and incremental copolymer compositions at any conversion; thus, correlations between conformation and interchain compositional heterogeneity can be made. In general, copolymers prepared in dioxane show a greater distribution of chain composition and therefore permit a wider variety of conformation than copolymers prepared in benzene/methylene chloride under identical conditions.     

15N-NMR spectroscopy. 34. Stereospecificity of the polymerization of D,L-leucine N-carboxyanhydride and γ-OMe-D,L-glutamic acid N-carboxyanhydride

D ,L -Leucine-N-carboxyanhydride (D ,L -Leu-NCA) and γ-methyl-D ,L -glutamic acid N-carboxyanhydride (γ-OMe-D ,L -Glu-NCA) were synthesized with ca. 2.5% ¹⁵N enrichment. Their polymerizations were conducted under a variety of conditions using benzylamine, triethylamine potassium tert-butanolate, and pyridine as initiators. The 40.55-MHz ¹⁵N-nmr spectra of the resulting stereocopolypeptides measured in trifluoroacetic acid display at least four signals, representing the isotatic, syndiotactic, and two heterotactic triads. From the signal intensities it was concluded that these NCAs behave nearly identically. With benzylamine initiation the formation of isotactic blocks is slightly favored, and they are still more predominant when strong bases are used as initiators. Initiation by pyridine favors the formation of syndiotactic sequences. However, in all cases the average lengths of the stereoblocks never exceeded 4 monomer units. The low stereospecificity of most polymerizations of D ,L -NCAs is confirmed by the high degree of solubility of the resulting poly(D ,L -amino acids) in aprotic solvents. Penultimate effects are weak or absent, so that most polymerizations follow Bernoullian type statistics. Deviations from these statistics were found for polymerizations in pyridine.     

Correlation between the sequence-length distribution and structure of statistical copolymers of L-valine and γ-benzyl-L-glutamate

Statistical copolymers were prepared from N-carboxyanhydrides of L -valine and γ-benzyl-L -glutamate in dioxan with triethylamine as an initiator. The copolymerization conversion was determined by ir spectroscopy, the copolymer composition by amino acid analysis, and the molecular weights by light scattering. The monomer reactivity ratios were found to be r_Val = 0.14 and r_Glu(OBzl) = 6.4. High-molecular-weight copolymers are formed even at low conversions. The content of β-structure in the copolymers was estimated from the ir spectra in copolymerization mixtures. The sequence-length distribution of L -valine and γ-benzyl-L -glutamate copolymers was calculated and its dependence on copolymerization conversion is discussed. Relations between the sequence-length distribution and the content of β-structure were studied. It was found that the content of β-structure in samples with the same composition is different for low- and high-conversion copolymers. The formation of β-structure in copolymers in the copolymerization mixture requires a certain minimal sequence length, which has been found to be about 6 valine units.     

Solid-state conformation of copolymers of β-benzyl-L-aspartate with L-alanine,L-leucine,L-valine, γ-benzyl-L-glutamate,or ϵ-carbobenzoxy-L-lysine

The solid-state conformation of copolymers of β-benzyl-L -aspartate [L -Asp(OBzl)] with L -leucine (L -Leu), L -alanine (L -Ala), L -valine (L -Val), γ-benzyl-L -glutamate [L -Glu(OBzl)], or ?-carbobenzoxy-L -lysine (Cbz-L -Lys) has been studied by ir spectroscopy and circular dichroism (CD). The ir spectra in the region of the amide I and II bands and in the region of 700–250 cm^?1 have been determined. The results from the ir studies are in good agreement with data obtained by CD experiments. Incorporation of the amino acid residues mentioned above into poly[L -Asp(OBzl)] induces a change from the left-handed into the right-handed α-helix. This conformational change for the poly[L -Asp(OBzl)] copolymers was observed in the following composition ranges: L -Leu, 0–15 mol %; L -Ala, 0–32 mol %; L -Val, 0–8 mol %; L -Glu(OBzl), 3–10 mol %; and Cbz-L -Lys, 0–9 mol %.     

Cytokinins induce direc somatic embryogenesis of <Emphasis Type="Italic">Dendrobium</Emphasis> chiengmai pink and subsequent plant regeneration

Summary Four auxins (2,4-dichlorophenoxyacetic acid [2,4-D], indole-3-acetic acid [IAA], indole-3-butyric acid [IBA], and naphthaleneacetic acid [NAA]), and five cytokinins (N ⁶-[2-isopentenyl]-adenine [2iP], N ⁶-benzyladenine [BA], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-[4-hydroxy-3-methylbut-2-enylamino]purine [zeatin]) were examined for their effects on direct embryo induction from leaf explants of Dendrobium cv. Chiengmai Pink cultured on 1/2 Murashige and Skoog (MS) medium. Whether in light or darkness, explants easily became necrotic and no embryos were obtained on growth regulator-free or auxin-containing media after 60 d of culture. By contrast, five cytokinins tested induced direct embryo formation from leaf explants, and explants cultured in light had a higher embryogenic response compared with those cultured in darkness. The best condition for direct embryo induction was at 18.16 μM TDZ cultured in light for 60 d, where 33% of explants formed a mean number of 33.6 embryos per explant. During subculture on growth regulator-free 1/2 MS medium, embryos gradually developed into plantlets. Secondary embryogenesis was occasionally found on sheath leaves of embryos. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment.     

Identification of Volicitin-related Compounds from the Regurgitant of Lepidopteran Caterpillars

Volicitin-related compounds were found in the oral secretion of the three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, and one sphingid species, Agrius convolvuli. Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine], N-(17-hydroxy-linoleoyl)-glutamine, N-linolenoylglutamine and N-linoleoylglutamine were identified in the secretion from the noctuid larvae. In secretions from the sphingid larvae, N-linolenoylglutamine and N-linoleoylglutamine were the main components. Furthermore, there were significant differences in the amounts of the N-acylamino acid conjugates in the secretions from the three noctuid species. These results suggest that the proportion of volicitin-related compounds in the regurgitant was species-specific.     

Spectroscopic and equilibrium dialysis studies of poly(α-L-glutamic acid)–Cu(II) complexes in the pH range 4–7

The formation of complex between the Cu²⁺ ion and poly(α-L -glutamic acid) [poly(Glu)] in 150 mM NaCl solutions was studied by uv–visible absorption and equilibrium dialysis methods at the mixing ratios of Glu residues to Cu²⁺, R, of 32, 16, and 8 and in the pH range 4–7. The results showed that more than 90% of Cu²⁺ ions bind to the poly(Glu) at pH > 4.9, but the bound Cu(II) begins to dissociate with a decrease in pH. The absorption spectra of bound Cu(II) varied with pH and R in a complicated manner. Three different component spectra were disclosed from the analysis of the pH dependence of the bound spectra. We concluded that poly(Glu)–Cu(II) complexes fall into three classes in the pH range 4–7, with the proportions of these complexes varying with both pH and R. The three complexes predominate either in the helix or extended-coil region, in the helix–coil transition region, or in the helix-aggregate region. The stability constant and binding mode of each Cu(II)–Glu complex were estimated from the dialysis data. With these results, the possible structure of each complex is discussed.     

Experimental characterization of poly(hydroxyalkyl-L-glutamine) conformations in aqueous calcium chloride and sodium perchlorate solutions

Poly(hydroxyalkyl-L -glutamine) (alkyl = ethyl, propyl, butyl) solutions have been studied by CD as functions of temperature and activity of calcium chloride and sodium perchlorate. Helical content is altered by changes in salt activity and temperature. The helicity of poly(hydroxybutyl-L -glutamine) and poly(hydroxypropyl-L -glutamine) falls to zero in a monotonic fashion with increasing calcium chloride activity. A nonzero helicity reappears at activities in excess of 5–50 mol kg^?1. Poly(hydroxypropyl-L -glutamine) is much more sensitive to calcium chloride than is poly(hydroxybutyl-L -glutamine), and both polypeptides are more sensitive to calcium chloride than are typical proteins. Markedly different behavior is observed with sodium perchlorate. This salt acts as a helix stabilizer at low activities but becomes a destabilizer at activities higher than 0.3–1.0 mol kg^?1. In this respect the effect of sodium perchlorate on nonionic poly(hydroxyalkyl-L -glutamines) resembles that seen with cationic poly(L -lysine) and poly(L -arginine). Helix stabilization at low sodium perchlorate activity is moderate for poly(hydroxybutyl-L -glutamine) and large for poly(hydroxypropyl-L -glutamine) and poly(hydroxyethyl-L -glutamine).     

Syntheses and conformational studies of poly(Nε-methyl-L-lysine), poly(Nδ-methyl-L-ornithine), poly(Nδ-ethyl-L-ornithine), and their carbobenzoxy derivatives

The helix–coil transitions of poly(N^ε-methyl, N^ε-carbobenzoxy-L -lysine), poly(N^δ-methyl, N^δ-carbobenzoxy-L -ornithine), and poly(N^δ-ethyl, N^δ-carbobenzoxy-L -ornithine) in chloroform–dichloroacetic acid and their corresponding decarbobenzoxylated polypeptides in alkaline solutions were followed by optical rotation measurements. The introduction of a methyl or an ethyl group to the side chains of the carbobenzoxy derivatives of poly(L -lysine) and poly(L -ornithine) appeared to weaken the helical conformation at 25°C. The thermodynamic quantities of the three water-soluble polypeptides were calculated from the data on potentiometric titrations at several temperatures. For uncharged coil-to-helix transition, ΔH = ?370 cal/mol and ΔS = ?1.1 eu/mol for poly(N^ε-methyl-L -lysine), and ΔH = ?540 cal/mol and ΔS = ?1.6 eu/mol for poly(N^δ-ethyl-L -ornithine) (all on molar residue basis). The absolute values of ΔH and ΔS dropped in the region of pH-induced transition and eventually both quantities became positive. The initiation factor σ was about 2 × 10^?3, which was essentially independent of temperature. For poly(N^δ-methyl-L -ornithine) the coil-to-helix transition was not complete even when the polymer was uncharged at high pH.