Syntheses of poly[beta-(l)-menthyl D- and L-aspartates] and their secondary structures

β-(l)-Menthyl D - and L -aspartates were prepared by a fusion reaction of N-phthalyl D - and L -aspartic anhydrides with l-menthol, followed by hydrazinolysis. The monomers were then polymerized to poly[β-(l)-menthyl D - and L -aspartates] by the N-carboxyanhydride method. These polymers were soluble in many organic solvents, such as diethyl ether, tetrahydrofuran, chloroform, n-bexane, and dioxane. From the results obtained by a study of the optical rotatory dispersions and circular dichroisms, poly [β-(l)-menthyl D -aspartate] was found to be a β form structure in solution. On the other hand, poly[β-(l)-menthyl L -aspartate] was a random-coil structure. These results suggest that the asymmetry of the l-menthyl chromophore in the side chain interacts with the polypeptide main chain and causes an extraordinary optical rotation.     

Syntheses and conformational studies of poly- ,N-alkyl L-asparagines

Several β,N-alkyl L -asparagines were prepared from the phthalyl and benzyloxycarbonyl derivatives. High-molecular-weight poly-β,N-benzyl L -asparagine and poly-β,N-(1)-phenethyl L -asparagine were prepared from the corresponding N-carboxyanhy-drides. From the results obtained by a study of the infrared absorption spectra and the optical rotatory dispersion, poly-β-N-benzyl L -asparagine was found to be a random coil structure in dichloroacetic acid and the optical rotatory dispersion curves gradually changed into the left-handed α-helix structure when chloroform was added to the solution. The coil-to-helix transition was observed in the vicinity of 20% dichloroacetic acid in a dichloroacetic acid-chloroform mixture. Poly-β,N-(d), (l), and (d + l, 1:1)

1 (d + l, 1:1): mixed polymer containing the same weighed poly-β,N-(d) and (l)-(1)-phenethyl L -asparagines.

-(1)-phenethyl L -asparagines showed a nearly constant specific rotation in the dichloroacetic acidchloroform solvent system. Poly-β,N-(dl)-(1)-phenethyl L -asparagine caused a gradual folding of the helix at dichloroacetic acid content of less than 20%.     

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.     

Poly( -p-nitrobenzyl-L-glutamate): synthetic aspects, sedimentation, and viscosity studies

A series of poly(γ-p-nitrobenzyl-L -glutamates), PNBG, has been synthesized by the polymerization of N-carboxyanhydride (NCA) derivatives of γ-p-nitrobenzyl-L -glutamate, NBG, using triethylamine as an initiator. We studied the influence of (a) the solvents dioxane, nitrobenzene, dimethylformamide (DMF), and DMF–1,2-dichloroethane mixture and (b) the anhydride–initiator ratio (A/I) for the polymerization in nitrobenzene (A/I varying from 50 to 750) on the properties of the polymers obtained. In order to improve its synthesis, NBG, was prepared by three different methods. Ten samples of PNBG, ranging in M_w from 10,000 to 50,000, were examined viscometrically in DMF and dichloroacetic acid (DCA) and by ultracentrifugation in DMF. The data for [η] and S_o (limiting sedimentation coefficient) as functions of M_w for PNBG in DMF were utilized, applying theories of Kuhn and Kuhn,¹³ Schachman,¹⁴ and Perrin, ¹⁵ for the estimation of the length per monomeric residue h. Viscosity data gave a h value of about 2.3 Å, Whereas sedimentation yielded 1.5 Å. Treating viscosity and sedimentation data for poly(γ-benzyl-L -glutamate), PBLG, in the same way leads to somewhat higher hvalues (2.4 Å and 1.7 Å, respectively). Although a nitroaromatic effect was shown to be absent for PNBG in DMF, it can be concluded that in this medium PNBG has a somewhat more compact structure than PBLG.     

Synthesis and conformational study of poly(0,0′-dicarbobenzoxy-L-β-3,4-dihydroxyphenyl-α-alanine)

High-molecular-weight poly(0,0′-dicarbobenzoxy-L -β-3,4-dihydroxyphenyl-α-alanine) was prepared by the N-carboxyanhydride method. From the results obtained by a study of the optical rotation, nuclear magnetic resonance, and solution infrared absorption, the conformation of poly(0,0′-dicarbobenzoxy-L -β-3,4-dihydroxyphenyl-α-alanine) depended greatly on the solvent taking a right-handed helix with [θ]₂₂₅ = ?13,600 ～ ?18,900 in alkyl halides, a left-handed helix with [θ]₂₂₈ = 22,100 ～ 24,800 in cyclic ethers or trimethylphosphate, and a random coil structure in dichloroacetic acid, trifluoroacetic acid, or hexafluoroacetone sesquihydrate. The polypeptide underwent a right-handed helix-coil transition in chloroform/dichloroacetic acid (or trifluoroacetic acid) mixed solvents and a left-handed helix-coil transition in dioxane/dichloroacetic acid (or trifluoroacetic acid) mixed solvents. The results were compared with those of poly(0-carbobenzoxy-L -tyrosine).     

Synthesis and conformational study of poly(N -benzyl-L-lysine) and its benzyloxycarbonyl derivative

The solvent-and pH-induced conformational changes are examined in order to investigate the influence of benzyl group. Polymer was prepared via N^?-benzyloxycarbonyl, N^?-benzyl-N^α-carboxy-L -lysine anhydride. The resulting poly (N^?-benzyloxycarbonyl, N^?-benzyl-L -lysine) was obtained in high yield and had a high molecular weight. The protected polymer was removed into poly (N^?-benzyl-L -lysine) by treating it with hydrogen bromide. From the results of the ORD and CD, the protected polymer has a righthanded α-helix, showing [m′]₂₃₃ = –10,300, [θ]₂₂₀ = –27,600 and [θ]₂₀₇ = –25,100 in dioxane. The breakdown of the helical conformation is found to occur at 8% dichloroacetic acid in chloroform-dichloroacetic acid mixture. In the pH range 3.35–6.90, poly (N^?-benzyl-L -lysine) is in a random coil structure. In the pH range 7.50–13.0, the polypeptide has a right-handed α-helix structure; [m′]₂₃₃ = –12,000, [0]₂₂₀ = –27,200, and [0]₂₀₇ = –27,000. In comparison with poly-L -lysine, the coil-to-helix transition is observed at lower pH range in 50% n-propanol. Above pH 8 by heating, the α ? β transition of poly (N^?-benzyl-L -lysine) is not observed in an aqueous media.     

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.     

Relative stability of the α-helix of deuterated poly(γ-benzyl-L-glutamate)

The coil-to-helix transition temperatures of hydrogen bearing and deuterated poly(γ-benzyl-L -glutamate) in 1,3-dichlorotetrafluoroacetone/H₂O and/D₂O mixtures, respectively, have been determined. Together with previously obtained data for the conformational transition of this polypeptide in normal and deuterated dichloroacetic acid, these results have been used in an analysis of the effect of deuterium substitution on the intrinsic stability of the α-helical form of poly(γ-benzyl-L -glutamate). The findings, consistent for both solvent systems, showed that the deuterated polypeptide is some 5% more stable than the normal protonated poly(γ-benzyl-L -glutamate), while the polypeptide-active solvent interaction enthalpy is also slightly increased by deuterium substitution in the respective molecules. A consideration of available data for poly(β-benzyl-L -aspartate) reveals an anomaly with respect to the present analysis.     

13C and 1H NMR studies of helix-coil transition of poly(β-benzyl-L-aspartate) and poly(γ-benzyl-L-glutamate): Behavior in nonprotonating solvent mixtures,and origin of solvent-induced chemical shift

From the results of ¹³C-nmr measurement of poly(β-benzyl-L -aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(β-benzyl-L -aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the α-helix. The chemical shift differences in the benzyl group carbons of poly(γ-benzyl-L -glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (C^β, C^γ) originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(β-benzyl-L -aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.     

Conformational studies of poly(L-tyrosine). A helix–coil transition in dimethyl sulfoxide–dichloroacetic acid mixtures

Poly(L -tyrosine) is a random coil in dimethyl sulfoxide. Upon addition of dichloroacetic acid, poly(L -tyrosine) undergoes a conformational transition centered at about 10% dichloroacetic acid. The transition is nearly complete at 20% dichloroacetic acid. Further addition of dichloroacetic acid leads to precipitation of poly(L -tyrosine). We have characterized this transition by optical rotation, viscosity, circular dichroism, and infrared. The optical rotation at 350 nm and the intrinsic viscosity increase sharply to values that are consistent with a transition to the α-helix conformation. The circular dichroism of poly(L -tyrosine) in dimethyl sulfoxide and in dimethyl sulfoxide/dichloroacetic acid (80:20 v/v) agrees with previous reports for random-coil and α-helix conformations, respectively. The infrared spectrum of poly(L -tyrosine) in dimethyl sulfoxide/dichloroacetic acid (80:20 v/v) shows no evidence of β-structure. We conclude that the transition on going from dimethyl sulfoxide to 20% dichloroacetic acid in dimethyl sulfoxide is a coil → α-helix transition. The amide-I band of poly(L -tyrosine) in dimethyl sulfoxide/dichloroacetic acid (80:20) is found to be at 1662 cm^?1. It has been suggested that this high frequency may be indicative of a left-handed α-helix. However, this high amide-I frequency is consistent with conformational energy calculations of Scheraga and co-workers. The mechanism of the dichloroacetic acid-induced transition to an α-helix is discussed. Dichloroacetic acid and dimethyl sulfoxide interact strongly and the transition presumably involves a marked decrease in the ability of dimethyl sulfoxide to solvate the peptide backbone and aromatic side chains upon complex formation with dichloroacetic acid.     

Study by circular dichroism and optical rotatory dispersion of polypeptides with aromatic side-chain chromophores

Poly(ortho-, meta-, and para-γ-nitrobenzyl-L -glutamates) were studied by circular dichroism (CD) and optical rotatory dispersion (ORD) in two helicogenic solvents, hexafluoroisopropanol (HFIP) and dichloroethane (EDC), and two non-helicogenic solvents, dichloracetic acid (DCA) and trifluoroacetic acid (TFA). The corresponding glutamates were also studied in DCA and TFA. The symmetric nitrobenzylic chromophore is optically active when the polymers are in solution in DCA and TFA. The corresponding glutamates are also optically active under the same conditions. Thus, it was not possible to explain the origin of the optical activity of the side-chain chromophore when the polymer is in solution in a helicogenic solvent. Nevertheless, from a side-chain dichroic band, a helix–coil transition curve was determined and the stability of each poly(γ-nitrobenzyl-L -glutamate) given; this stability depends on the position of the nitro substituent on the aromatic ring.     

Side-chain conformation in poly(γ-phenethyl-L-glutamate)

X-ray diffraction and energy-minimization results are reported for poly(γ-phenethyl-L -glutamate). Orthorhombic unit-cell parameters of drawn fibers are a = 15.4 Å, b = 26.6 Å, c = 54.4 Å. Atomic coordinates are derived for an α-helix peptide conformation that corresponds to a calculated side-chain internal energy minimum. The side-chain conformation correlates well with the electron density projection; the side chains wrap around the α-helical main chain with the phenethyl ester group directed toward the N-terminus. The para-axis of the benzene ring is inclined at an angle nearly nearly normal to the helix axis. The x-ray structure factors calculated for this model, when compared to the 10 observed structure factors, yield a crystallographic reliability index of R = 0.23.     

Alkylated poly(amino acids). I. Conformational properties of poly(Nε-trimethyl-L-lysine) and poly(Nδ-trimethyl-L-ornithine)

Poly(N^ε-trimethyl-L -lysine), [Lys(Me₃)]_n, and poly(N^δ-trimethyl-L -ornithine), [Orn(Me₃)]_n, in sodium dodecylsulfate do not assume the β-structure or α-helix, respectively, of their parent polymers. In 0.5M Ca(ClO₄)₂ both [Lys(Me₃)]_n and [Orn(Me₃)]_n are aggregated and display CD spectra indicative of a regular, perhaps helical, structure. For [Lys]_n and [Lys(Me₃)]_n, the T₁ of the α-hydrogens are 0.379 and 0.230 sec, respectively, indicating greater rigidity for [Lys(Me₃)]_n. The CD spectrum of [Lys(Me₃)]_n at pH 8 is more heat resistant than that of [Lys]_n. It is suggested that apolar interactions are more important in the methylated polymers than in the parent polymers.     

Stability of helical polyamino acids in solution at high temperatures

Optical rotatory dispersion studies have been carried out at temperatures up to 150 °C. on poly(γ-benzyl L -glutamate) in α-chloronaphthalene and N-methylcaprolactam, and on poly-ε-carbobenzoxy-L -lysine, poly-δ-carbobenzoxy-L -ornithine, and poly(L -glutamic acid) in N-methylacetamide. The Moffitt-Yang ?b₀ values were large in all cases, but significant decreases in ?b₀ were observed at the upper temperature limits of the study suggesting that a transition region was being entered. Polymer degradation generally precluded examination of the systems through the suggested transition region.     

Optical and other properties of a hydrocarbon-soluble polypeptide, poly-gamma-(N-dodecyl)-L-glutamate

The polypeptide poly-γ-(n-dodecyl)-L -glutamate (PDLG) is soluble in hydrocarbon solvents such as hexane, cyclohexane, and dodecane. The CD spectra of PLDG in these solvents are reported here. These spectra are typical α-helix spectra and show none of the wavelength shifts and magnitude changes displayed by partially helical proteins in membrane preparations. This observation rules out the possibility that the membrane protein CD spectra result from solvent effects. The PDLG helix is stable in dodecane up to at least 70 °C. However, it is easily disrupted by trifluoroacetic acid, with the helix–coil transition centered at 3% TFA in hexane. Viscosity measurements of PDLG in dry dichloroacetic acid exhibit polyelectrolyte effects which can be suppressed by addition of several percent water.     

Étude thermodynamique de la transition hélice–pelote statistique en solvants mixtes de différents esters de l'acide poly(L-glutamique)

Helix–coil transition of poly(γ-methyl-L -glutamate), poly(γ-ethyl-L -glutamate), and poly(γ-benzyl-L -glutamate) has been studied in mixed solvents by calorimetry, polarimetry, and viscometry. The experimental data have allowed the evaluation of solvation enthalpy Δh_b, equilibrium constant K for hydrogen bond formation between the active solvent component and CO and NH groups, and the cooperativity parameter σ. The conformational transition of polypeptides in solution in a mixed solvent containing enough active solvent to maintain the coiled conformation has been produced by dilution with the helix-supporting solvent for the measurements of enthalpy of transition Δh_s. The average value for Δh_s is 3550 ± 300 J/mol and is practically independent of the nature of the side chain for the dichloroacetic acid-ethylene dichloride solvent pair at 25°C. A noticeable concentration effect exists in the case of poly(γ-benzyl-L -glutamate). The helical conformation is less stable for poly(γ-ethyl-L -glutamate), and this is explained by a steric effect hindering the access of dichloroacetic acid to side chains. Constant K has been calculated using polarimetric data and also from values of Δh_s obtained at different temperatures using the Bixon and Lifson theory on the one hand and that of Sayama and coworkers on the other hand. Values of σ for poly(γ-ethyl-L -glutamate) have been calculated according to both theories mentioned, and the results show that the two sets of values are quite similar. The constant σ depends on the nature of the active solvent, on temperature, and on the binary-solvent composition. These conclusions are confirmed by viscometric results. Values of Δh_b calculated from constant K are 5230 J/mol when Bixon and Lifson theory is used and 5569 J/mol when the theory at Sayama and coworkers is used. In both cases the value for Δh_b is much lower than that of an intramolecular hydrogen bond. Experimental results suggest that the solvation mechanism would proceed in a manner so that mechanisms described in both theories are involved.     

The viscosity behavior and conformations of low-molecular-weight poly-γ-benzyl-L-glutamate in various solvents

Reduced viscosity and infrared spectra of low-molecular-weight poly-γ-benzyl-L -glutamate (which was prepared by polymerization of the N-carboxyanhydride with n-hexylamine initiation at [A]/[I] 3, 4, and 8) have been measured in various organic solvents. Infrared spectra indicate that the polypeptide molecules consist of a series of residues of two forms, the solvated σ-form and the hydrogen-bonded β-form, and relative abundance of the two forms depends on solvent species and polypeptide concentration. An approximate method is developed for estimating the content of β-structure from a single spectrum of dissolved polypeptide. The reduced viscosity of some solutions is scarcely dependent in polypeptide concentration, in which a single conformation is predominantly kept over the concentration range. In the other solutions the reduced viscosity displays a strong concentration dependence or some anomalous behavior. The observed viscosity behavior has been attributed to the changes in size and shape of aggregates, which are determined by the number of hydrogen bonds in the aggregate. This unusual behavior is exhibited by solutions of the polypeptides which have a moderate content of β-structure at a finite concentration. Both the content of β-structure and the extent of association increase in the following solvents, ranked in order of effectiveness: dimethylformamide, trifluoroethanol < trimethyl phosphate < chloroform < dioxane < ethylene dichloride < ethylene dibromide. Infrared spectra suggest that the conformation of the polypeptide in dichloroacetic acid differs from either the σ- or the β-conformation.     

Intra- and intermolecular charge-transfer interactions between terminal electron donor and acceptor attached to poly(γ-benzyl-L-glutamate) in solution

Poly(γ-benzyl-L -glutamate) having a terminal dimethylaminoanilide group as an electron donor (D) and a terminal 3,5-dinitrobenzoyl group as an electron acceptor (A) (A-[Glu(OBzl)]_n-D) was synthesized by the N-carboxyanhydride method. Polymer samples were fractionated by gel chromatography and their number-average degrees of polymerization n were determined by the absorbances of the terminal chromophores. These polymers in chloroform and dimethylformamide solutions showed a charge-transfer (CT) absorption band around 455 nm, and the fraction of the polymer forming the CT complex was evaluated as a function of the chain length. CT absorption for short chains (n = 5 ～ 20) was attributed to intramolecular CT complex in which the A-[Glu(OBzl)]_n-D chain takes cyclic conformations. An optimum chain length for the intramolecular CT was found to be n ? 10, where the [Glu(OBzl)]_n chain may most easily bend back to form cyclic conformations. Stronger CT absorption observed for longer chains than n = 20 was shown to be intermolecular, and an intermolecular head-to-tail aggregation was found to be a cause of the strong CT interaction. All helical A-[Glu(OBzl)]_n-D chains were found to form the head-to-tail dimers in chloroform solution.     

Occurrence of beta-chain conformation in poly-gamma-methyl glutamate membranes

Molecular chain conformations of poly-γ-methyl-L -glutamate, poly-γ-methyl-D -glutamate, and poly-γ-methyl-D ,L -glutamate in membranes prepared by using mainly trifluoroacetic acid and formic acid as solvents were investigated by infrared, X-ray diffraction, and optical rotatory dispersion measurements. It was pointed that these polymers exist in the α-helix form in membranes cast from trifluoroacetic acid solutions, but in the β-chain form in membrances swollen in formic acid. The β-chain structure was also observed in crystals precipitated from dilute solutions including formic acid. The formation of the β-chain structure was discussed.     

Electrophoretic identification of poly-γ-glutamate chain-lengths of 5,10-methylenetetrahydrofolate using thymidylate synthetase complexes

A method has been developed to characterize the poly-γ-glutamates of 5,10-methyl-enetetrahydrofolate. Incorporation of 5,10-methylenetetrahydrofolates into a ternary complex with L. casei thymidylate synthetase and 5-fluoro-2-deoxy[³H]uridylate stabilizes the reduced folate against oxidation, loss of the one carbon moiety, and poly-γ-glutamate degradation. The covalent ternary complexes, containing 5,10-methylenetetrahydrofolate polyglutamates, were resolved electrophoretically. Electrophoretic mobility was shown to be a linear function of polyglutamate chain-length. The method can potentially be applied to analysis of chemically prepared folate polyglutamates, the monitoring of enzyme-mediated interconversions of polyglutamates and characterization of tissue extract polyglutamates.