Synthesis of sequential polypeptides containing L-β-3,4-dihydroxyphenyl-α-alanine (DOPA) and L-glutamic acid

Sequential polypeptides with the repeating units L -glutamyl-L -DOPA, L -DOPA-L -glutamyl-L -DOPA, L -glutamyl-L -glutamyl-L -DOPA, L -DOPA-L -DOPA-L -glutamyl-L -DOPA, and L -glutamyl-L -glutamyl-L -glutamyl-L -DOPA have been synthesized by solution polymerization of the p-nitrophenyl esters of the corresponding di-, tri-, and tetrapeptides. The O, O′-dimethyl and γ-methyl groups were used to protect side chains of L -DOPA and L -glutamic acid. The monomers for the polytripeptides and polytetrapeptides were prepared by stepwise elongation, using the dicyclohexylcarbodiimide coupling method. Moderately high molecular weight sequential polypeptides were obtained. The protected groups of the side chain were removed simultaneously by use of boron tribromide in chloroform. Trimethylphosphate-soluble sequential polypeptides containing L -DOPA were obtained.     

Helix–coil transition in copolypeptides. II. Poly(γ-benzyl L-glutamate-co-ε-carbobenzoxy-L-lysine)

The thermal helix–coil transition of poly(γ-benzyl L -glutamate-co-ε-carbobenzoxy-L -lysine) copolypeptides was studied in solvent mixtures of different compositions. The cooperativity parameter v changes linearly with polymer (and solvent) composition, whereas the heat of the transition shows a very pronounced minimum as a function of polymer composition. This minimum cannot be due only or mainly to the solvent changes and must be attributed to the effect on the transition of the side chains of the polypeptides.     

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.     

Conformational studies of sequential polypeptides containing lysine and tyrosine

The conformation of three sequential copolypeptides, poly(L -tyrosyl-L -lysine), poly(L -tyrosyl-L -lysyl-L -lysine), and poly[L -tyrosyl-(L -lysyl)₂-L -lysine] have been studied by a variety of techniques, including CD, ir spectroscopy, analytical ultracentrifugation, and x-ray diffraction. Depending upon the pH and sovent composition, poly(L -tyrosyl-L lysyl-L -lysine) and poly [L -tyrosyl-(L lysyl)₂-L -lysine] can adopt either the α-helical or random-coil conformation, while poly(L -tyrosyl-L -lysine) forms either inter- or intramolecular β-structures.     

Structure of branched poly-α-amino acids in dimethylformamide. I. Light scattering

Light scattering of multichain poly-α-amino acids was studied in dimethylformamide (DMF). The polymers consisted of a backbone of poly-L -lysine of degree of polymerization n with side chains of benzyl L -glutamate and benzyl L -aspartate of degree of polymerization, m, on each ε-amino group. The backbone length n is known and m is obtained by amino acid analysis. The results on a series of such materials confirm this structure and show that the molecules are dissolved in highly compacted conformations. It was found that DMF is a poor solvent for these polymers. In the case of the higher molecular weight polymers, the solutions initially were not molecularly disperse. The aggregates were resistant to dilution in the experimental range. Mild heat treatment, however, disaggregated the solutions irreversibly, and the light-scattering data indicated that a structural rearrangement of the molecules had occurred.     

Photoresponsive polymers: Azobenzene-containing poly(L-lysine)

Poly(L -lysine) was reacted with various azo-reagents, including p-phenylazobenzoic acid, p-phenylazobenzoyl chloride, and p-phenylazobenzoic N-hydroxy-succinimide ester, to give polypeptides containing 5–44 mol % azobenzene units in the side chains. The conformation of the azo-modified polypeptides was investigated in connection with their photochromic behavior caused by the trans ? cis photoisomerization of the azo groups present in the side chains. In methanol/water solvent mixture, the 20% azo-poly(L -lysine) adopts the α-helix conformation. The helix stability was found to be higher when the azo side chains are in cis than when they are in trans configuration. So irradiation at 340 nm (trans-to-cis isomerization), and alternately at 450 nm (cis-to-trans isomerization), produced reversible variations of the α-helix content. In hexafluoro-2-propanol/water/sodium dodecyl sulfate mixture, the 43% azo-poly(L -lysine) adopts a β-structure, as indicated by CD spectra. Irradiation at 340 nm caused the disruption of the β-structure and promoted the α-helix conformation. The effect was reversed upon irradiation at 450 nm. The photoinduced β ? helix change was explained on the basis of the different geometry and hydrophobic character of the trans and the cis azobenzene units.     

Spin-label studies of the pH-induced random coil to α-helix conformational transition in poly(L-lysine)

Poly(L -lysine) of various molecular weights between 2700 and 475,000 was spin-labeled. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined by calculation of the rotational correlation time as the poly(L -lysine) underwent the pH-induced random coil to α-helix conformational transition. In general, the rotational correlation time of the nitroxide increased as the pH was increased, indicating a more restricted environment for the spin label when poly(L -lysine) is deprotonated. For the high-molecular-weight poly(L -lysine) this corresponds to the formation of the α-helix and indicates that the side chain–side chain interaction and decreased segmental motion of the backbone (slightly) restricts the motion of the spin label. For the 2700-molecular-weight poly(L -lysine), previously shown not to assume a helical conformation at high pH, the increase in the rotational correlation time of the spin label indicates that the side chain–side chain interaction takes place after deprotonation but without helix formation. This may indicate that helix formation per se is not needed to produce the observed effect even with the high-molecular-weight polymers. The rotational correlation time of the spin label at a particular pH did not depend on the molecular weight of the poly(L -lysine) over the 200-fold range of molecular weights. This indicates that the rotational correlation time reflects the rotational mobility of the spin label in a localized environment and not the rotational diffusion of the entire macromolecule.     

Viscosity and potentiometric measurements of poly(L-histidyl-L-alanyl-α-L-glutamic acid) and poly(L-lysyl-L-alanyl-α-L-glutamic acid)

Poly(His-Ala-Glu) and poly(Lys-Ala-Glu) were examined by viscosity and potentiometric titration. These measurements were interpreted in terms of the hydrodynamic size of the above sequential polypeptides. Effects of polymer, size and concentration, and solution-salt concentration were demonstrated. Although the sequential polypeptides generally behave like polyampholytes, they do demonstrate some differences. These differences my be attributed to the ability of ionized side chains three residues apart to repel themselves, in the order His < Glu < Lys.     

Estimation of the circular dichroism exhibited by statistical coils of poly(L-alanine) and unionized poly(L-lysine) in water

The circular dichroism of Ac-(Ala)_x-OMe and H-Lys-(Lys)_x-OH with x = 1, 2, 3, and 4 has been measured in aqueous solutions. The oligomers with x = 4 show similar circular dichroism spectra in water when the lysyl amino groups are protonated, and they respond in similar fashion to heating and to sodium perchlorate. Both oligomers at 15°C exhibit a positive circular dichroism band at 217–218 nm, which is eliminated by the isothermal addition of 4 M sodium perchlorate or by heating. The positive circular dichroism of the lysine oligomer is also eliminated when the pH is elevated to deprotonate the amino groups. Positive circular dichroism is still observed for Ac-(Ala)₄-OMe at elevated pH. Circular dichroism spectra have been estimated for poly(L -alanine) and poly(L -lysine) as statistical coils under the above conditions, based on the trends established with the oligomers. Poly(L -lysine) and poly(L -alanine) are predicted to exhibit similar circular dichroism behavior in aqueous solution so long as the lysyl amino groups are protonated. The circular dichroism of the statistical coil of poly(L -lysine), but not poly(L -alanine), is predicted to change when the pH is elevated sufficiently to deprotonate the lysyl amino groups. These results suggest that the unionized lysyl side chains participate in interactions that are not available to poly(L -alanine). Hydrophobic interactions may occur between the unionized lysyl side chains. Protonation of the lysyl amino groups is proposed to disrupt these interactions, causing poly(L -alanine) and protonated poly(L -lysine) to have similar circular dichroism properties.     

Stepwise synthesis of oligopeptides with N-carboxy α-amino acid anhydrides. II. Oligopeptides with some polar side chains

The reaction of NCA's with some amino acids having a nucleophilic functional group on the side chain was studied in a heterogeneous reaction medium (acetonitrile-water). Glutamic acid and aspartic acid, having a free carboxyl group on the side chain, were successfully used to synthesize oligopeptides without interactions of the γ- and β-carboxyl group with NCA's. Two products were obtained by the reaction of NCA with L -lysine, which contains a free amino group on the side chain. ε-Protected lysine was used to prepare α-peptides as a nucleophile in the reaction. No racemization was observed in the synthesis of peptides by the NCA method in the heterogeneous solvent system. Oligopeptides with some polar side chains were synthesized by the NCA method.     

Raman spectroscopic study of poly (β-benzyl-L-aspartate) and sequential polypeptides

Poly-β-benzyl-L -aspartate (poly[Asp(OBzl)]) forms either a lefthanded α-helix, β-sheet, ω-helix, or random coil under appropriate conditions. In this paper the Raman spectra of the above poly[Asp(OBzl)] conformations are compared. The Raman active amide I line shifts from 1663 cm^?1 to 1679 cm^?1 upon thermal conversion of poly[Asp(OBzl)] from the α-helical to β-sheet conformation while an intense line appearing at 890 cm^?1 in the spectrum of the α-helix decreases in intensity. The 890 cm^?1 line also displays weak intensity when the polymer is dissolved in chloroform–dichloroacetic acid solution and therefore is converted to the random coil. This line probably arises from a skeletal vibration and is expected to be conformationally sensitive. Similar behavior in the intensity of skeletal vibrations is discussed for other polypeptides undergoing conformational transitions. The Raman spectra of two cross-β-sheet copolypeptides, poly(Ala-Gly) and poly(Ser-Gly), are examined. These sequential polypeptides are model compounds for the crystalline regions of Bombyx mori silk fibroin which forms an extensive β-sheet structure. The amide I, III, and skeletal vibrations appeared in the Raman spectra of these polypeptides at the frequencies and intensities associated with β-sheet homopolypeptides. Since the sequential copolypeptides are intermediate in complexity between the homopolypeptides and the proteins, these results indicate that Raman structure–frequency correlations obtained from homopolypeptide studies can now be applied to protein spectra with greater confidence. The perturbation scheme developed by Krimm and Abe for explaining the frequency splitting of the amide I vibrations in β-sheet polyglycine is applied to poly(L -valine), poly-(Ala-Gly), poly(Ser-Gly), and poly[Asp(OBzl)]. The value of the “unperturbed” frequency, V₀, for poly[Asp(OBzl)] was significantly greater than the corresponding values for the other polypeptides. A structural origin for this difference may be displacement of adjacent hydrogen-bonded chains relative to the standard β-sheet conformation.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-β- -galactopyranoside and methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-β- -galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β- -galactopyranoside (4) gave a fully acetylated (1→6)-β- -galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α- -galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β- -galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β- -galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Packing constraints of hydrophobic side chains in (α/β)8 barrels

An analysis of possible tight packing of hydrophobic groups simultaneously at the both surfaces of β-hyperboloid-8 was conducted. This analysis shows that the disposition of amino acid side chains at the real β-structure's surface is unique. If we sign the mean distance between adjacent β-strands as “a,” and the mean distance along β-strand between C_α atoms, whose side chains are directed to one side of the β-sheet, as “b,” the ratio b/a = √2 very precisely. This ratio ensures the most efficient packing of side hydrophobic groups at the outer surface of β-hyperboloid-8, forming, at the same time, the second by efficiency packing at its inner surface. © 1995 Wiley-Liss, Inc.     

Synthesis and β-conformation of sequential polypeptides containing arginine,histidine, and leucine

Alternating poly(Arg-Leu) and copolypeptides with Arg-Leu and His-Leu sequences were prepared by condensation of the corresponding p-nitrophenyl dipeptide esters in the presence of 1-hydroxybenzotriazole. Arginine was used without any protection and histidine side chains were protected using π-benzyloxymethyl group recently introduced in peptide chemistry. The ability of these polypeptides with alternating hydrophilic and hydrophobic residues to form water-soluble β-sheets was investigated by CD.     

Effect of temperature and pH on the β–helix transition of poly(L-lysine) in sodium dodecyl sulfate solution

The conformational phase diagram of poly(L -lysine) (4.6 × 10^?4 M, residue) in sodium dodecyl sulfate (1.6 × 10^?2 M) solution was constructed from circular dichroism results at various temperatures and pH's. Poly(L -lysine)–sodium dodecyl sulfate complexes undergo a β–helix transition upon raising the pH of the solution. The transition pH tends to shift downward at elevated temperatures. No helix–β transition can be detected for poly(L -lysine) in sodium dodecyl sulfate solution (pH > 11) even after 1-hr heating at 70°C. This is in marked contrast with uncharged poly(L -lysine) solution without sodium dodecyl sulfate, which is converted into the β-form upon mild heating of the solution above 50°C.     

Synthesis of methyl glycosides of β-(1→6)-linked -galactobiose, galactotriose, and galactotetraose having a 3-deoxy-3-fluoro-β- -galactopyranoside end-residue

Methyl 2,4-di-O-acetyl-3-deoxy-3-fluoro-β- -galactopyranoside was synthesized by sequential tritylation, acetylation, and detritylation of methyl 3-deoxy-3-fluoro-β- -galactopyranoside, and used as the initial nucleophile in the synthesis of methyl β-glycosides of (1→6)-β- -galacto-biose, -triose (20), and -tetraose (22) having a 3-deoxy-3-fluoro-β- -galactopyranoside end-residue. The extension of the oligosaccharide chais, to form the internal units in 20 and 22, was achieved by use of 2,3,4-tri-O-acetyl-6-O-bromoacetyl-α- -galactopyranosyl bromide as a glycosyl donor, and mercuric cyanide or silver triflate as the promotor. While fewer by-products were formed in the reactions involving mercuric cyanide, the reactions catalyzed by silver triflate were stereospecific and yielded only the desired β (trans) products.     

L-3-(3,4-Dihydroxyphenyl)alanine (<Emphasis Type="SmallCaps">L</Emphasis>-DOPA), an allelochemical exuded from velvetbean (<Emphasis Type="Italic">Mucuna pruriens</Emphasis>) roots

We investigated whether or not lettuce growth was inhibited by diffused L-3-(3,4-dihydroxyphenyl)alanine (L-DOPA), an allelochemical exuded from the roots of velvetbean (Mucuna pruriens (L.) DC. var. utilis) cultivars using a modified plant-box bioassay. For all the cultivars and one accession examined L-DOPA diffused from the roots and caused radicle and hypocotyl growth inhibition. A high correlation co-efficient (r = 0.838 to 0.982) was observed between L-DOPA concentration and lettuce seed sowing distance. L-DOPA diffused equally in all directions from roots at 0 mm position (close to root surface) in the plant-box, while the inhibition (%) of lettuce radicle growth gradually decreased with distance from the roots. For all cultivars the concentration of L-DOPA was significantly different at 0 mm position: being highest in cv. preta (167 g/ml) and lowest in cv. jaspeada and cv. ana (13 g/ml). The correlation between lettuce radicle growth inhibition and concentration of diffused L-DOPA was high (r = 0.856 to 0.966) in all cultivars and accession examined. However, the concentration of diffused L-DOPA did not correlate with the fresh weight concentration of L-DOPA measured in roots. The lettuce radicle growth inhibition from mucuna diffused L-DOPA was very similar that induced by synthetic L-DOPA, suggesting that diffused L-DOPA was the allelochemical responsible for growth inhibition.     

Synthesis, and carbon-13 n.m.r. study, of O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose and O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl (1→3)- -rhamnopyranose, constituents of bacterial, cell-wall polysaccharides

O-α- -Rhamnopyranosyl-(1→3)- -rhamnopyranose (19) and O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose were obtained by reaction of benzyl 2,4- (7) and 3,4-di-O-benzyl-α- -rhamnopyranoside (8) with 2,3,4-tri-O-acetyl-α- -rhamnopyranosyl bromide, followed by deprotection. The per-O-acetyl α-bromide (18) of 19 yielded, by reaction with 8 and 7, the protected derivatives of the title trisaccharides (25 and 23, respectively), from which 25 and 23 were obtained by Zemplén deacetylation and catalytic hydrogenolysis, With benzyl 2,3,4-tri-O-benzyl-β- -galactopyranoside, compound 18 gave an ≈3:2 mixture of benzyl 2,3,4-tri-O-benzyl-6-O-[2,4-di-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-α- -rhamnopyranosyl]-β- -galactopyranoside and 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-β- -rhamnopyranose 1,2-(1,2,3,4-tetra-O-benzyl-β- -galactopyranose-6-yl (orthoacetate). The downfield shift at the α-carbon atom induced by α- -rhamnopyranosylation at HO-2 or -3 of a free α- -rhamnopyranose is 7.4-8.2 p.p.m., ≈1 p.p.m. higher than when the (reducing-end) rhamnose residue is benzyl-protected (6.6-6.9 p.p.m.). α- -Rhamnopyranosylation of HO-6 of gb- -galactopyranose deshields the C-6 atom by 5.7 p.p.m. The 1 2-orthoester ring structure [O₂,C(me)OR] gives characteristic resonances at 24.5 ±0.2 p.p.m. for the methyl, and at 124.0 ±0.5 p.p.m. for the quaternary, carbon atom.     

Poly(ε-L-lysine): Synthesis and conformation

The synthesis of poly(ε-L -lysine) is described. This is a poly(ε-amino acid) in which the ε-amino group of lysine is condensed with the α-carboxyl group to produce a chain backbone that is a variant of the usual one seen in proteins and the side chain is the α-amino group. Conformational studies of poly(ε-L -lysine) and its t-butyloxycarbonyl derivative suggest the likelihood of a chain order that is formally similar to the antiparallel pleated-sheet conformation of proteins.     

Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: Chondroitin 4-sulfate and dermatan sulfate

Circular dichroism spectroscopy has been used to study the interactions of both dermatan sulfate and chondroitin 4-sulfate with the cationic polypeptides; poly(L -arginine), poly(L -lysine), and poly(L -ornithine). The results indicate that the mucopolysaccharides have a conformation directing effect on both poly(L -arginine) and poly-(L -lysine) such that these polypeptides adopt the α-helical conformation. The extent of interaction in each polypeptide-polysaccharide system can be judged by the degree of induced helicity and the “melting temperature” at which the interaction is disrupted On comparison of these results with those previously obtained for chondroitin 6-sulfate-polypeptide mixtures, the extent of interaction can be seen to depend on the length of the amino acid side chain and the positions of the anionic groups on the mucopolysaccharide chain. Such considerations place the three mucopolysaccharides in order of increasing interaction: chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate. These results are correlated with observations that dermatan sulfate is bound more tightly to collagen in connective tissues than are the other two polysaccharides.