Synthesis and characterization of stereoregular poly(D-methionyl-L-methionine) and poly(L-methionyl-D-methionyl-L-methionine)

By use of a polycondensation procedure free of racemization, stereoregular polymethionines have been synthesized from C-activated D -methionyl-L -methionine and L -methionyl-D -methionyl-L -methionine. The poly(D -methionyl-L -methionine) and poly(L -methionyl-D -methionyl-L -methionine) so prepared are soluble in chloroform and can be purified through dissolution in this solvent and precipitation by ligroin. Poly(D -Met-L -Met)which is obtained in a 25% yield, is about 5000 in average molecular weight. It has no discernible optical activity when examined between 400 and 600 nm in a trifluoroacetic acid solution. Poly(L -Met-D -Met-L -Met) (40% yield, M. W. = 10,000) is an optically active polymer. [α]₄₃₆²⁴ ≈ + 170° for a chloroformic solution (c = 0.2 CHCl₃).     

Conformation of oligopeptides with L-leucyl-L-leucyl-L-alanyl and L-methionyl-L-methionyl-L-leucyl repeating units

The conformation of oligopeptides with hydrophobic side chains, Nps-(L -Leu-L -Leu-L -Ala)_n-OEt and Nps-(L -Met-L -Met-L -Leu)_n-OEt(n = 1–6), in the solid state, obtained either by evaporation of the solvent or by precipitation with diethyl ether from a 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solution, has been studied with ir spectroscopy and x-ray powder-diffraction measurements. The conformation of these peptides in the HFIP solution has been studied by CD spectroscopy. Due to a strong preference of the amino acids to form an α helix, the peptides begin forming α helices at the dodecapeptide in the HFIP solution, and in the solid state by evaporation. In the solid state, with precipitation, the α-helical conformation is first observed at the octadecapeptide and the lower peptides assume a β structure. The conformational change, from the α helix to the β structure of the peptides with 12 to 15 amino acid residues, during the precipitation process, is due to a strong tendency of the amino acids to form the β-structure in rather short peptide lengths.     

Conformation of poly(L-homoarginine)

Poly(L -arginine) assumes the α-helix in the presence of the tetrahedral-type anions or some polyanions by forming the “ringed-structure bridge” between guanidinium groups and anions which is stabilized by a pair of hydrogen bonds and electrostatic interaction [Ichimura, S., Mita, K. & Zama, M. (1978) Biopolymers 17 , 2769–2782; Mita, K., Ichimura, S. & Zama, M. (1978) Biopolymers 17 , 2783–2798]. This paper describes the parallel CD studies on the conformational effects on poly (L -homoarginine) of various mono-, di-, polyvalent anions and some polyanions, as well as alcohol and sodium dodecylsulfate. The random coil to α-helix transition of poly(L -homoarginine) occurred only in NaClO₄ solution or in the presence of high content of ethanol or methanol. The divalent and polyvalent anions of the tetrahedral type (SO, HPO, and P₂O), which are strong α-helix-forming agents for poly(L -arginine), failed to induce the α-helical conformation of poly(L -homoarginine). By complexing with poly(L -glutamic acid) or with polyacrylate, which is also a strong α-helix-forming agent for poly(L -arginine), poly(L -homoarginine) only partially formed the α-helical conformation. Monovalent anions (OH^?, Cl^?, F^?, and H₂PO) did not change poly(L -homoarginine) to the α-helix, and in the range of pH 2–11, the polypeptide remained in an unordered conformation. In sodium dodecylsulfate, poly(L -homoarginine) exhibited the remarkably enlarged CD spectrum of an extended conformation, while poly(L -arginine) forms the α-helix by interacting with the agent. Thus poly(L -homoarginine), compared with poly(L -arginine), has a much lower ability to form the α-helical conformation by interacting with anions. The stronger hydrophobicity of homoarginine residue in comparison with the arginine residue would provide unfavorable conditions to maintain the α-helical conformation.     

Solid-state modifications of poly(γ-ethyl-L-glutamate)

Several modification of the arrangements of α-helical molecules were found in the solid films of poly (γ-ethyl-L -glutamate), depending on the casting solvent and the temperature. The helical conformation is somewhat looser than the normal 18-residue, 5-turn α-helix. Using x-ray diffraction, the types of molecular arrangements were classified into tetragonal, pseudohexagonal, and hexagonal ones. Tetragonal packing was observed in the filmm (form T) prepared by casting the solution in trifluorethanol or dichlorethane. The sample obtained from chloroform solution is a well-ordered, pseudohexagonal modification (form I). Forms I and T change into a poorly crystalline form III by annealing at temperatures above 130° C. It is particularly noteworthy that the less-ordered form III exhibits a thermoreversible transition around 110°C into a well-ordered form H with the hexagonal molecular packing.     

Structure and properties of two plastic peptide triblock,ABA, copolymers of poly(γ-benzyl-l-glutamate), A,and poly(butadiene/acrylonitrile), B

Two prototype triblock (ABA) copolymers of poly[(γ-benzyl-l-glutamate) _x (butadiene/acrylonitrile)_y (γ-benzyl-l-glutamate)_x] have been synthesized and characterized. They were prepared by reacting a primary amine capped butadiene/acrylonitrile (ATBN) polymer with the $\text{N-}\text{carboxy}$ anhydride of γ-benzyl-l-glutamate. The copolymers were ～38 000 (copolymer 1) and 74 000 (copolymer II) molecular weight. X-ray diffraction and Fourier Transform infrared spectroscopy of films cast from dioxane (preferential for PBLG) and chloroform (non-preferential) show the benzyl glutamate segments to be predominantly α-helical and disordered α-helical, respectively. Electron microscopy of osmium tetroxide strained films cast from dioxane revealed lamellar domain formation indicative of phase separation. The midblock butadiene layers were ～150 Å thick while the alternating benzyl glutamate layers were 300 and 500 Å thick for copolymers I and II, respectively. Films cast from chloroform exhibit a nearly homogeneous morphology, indicative of considerable phase mixing. Dynamic mechanical spectroscopy of the copolymers also revealed a dependence on morphology. The side chain transition of the benzyl glutamate appeared as a single peak when the copolymers were cast from dioxane and a double peak when the copolymers were cast from chloroform.     

Conformational studies on concentrated solutions of poly(gamma-benzyl L-glutamate)

We have studied the effect of polypeptide concentration on the helix–coil transition of poly(γ-benzyl L -glutamate) (PBLG) in both dichloroacetic acid (DCA) and DCA–chloroform (CHF) mixtures. In agreement with other reports, we find the van't Hoff transition enthalpy to be strongly dependent on PBLG concentration. Also, an apparent effect of polypeptide concentration was noted on the transition temperature; however, corrections for finite PBLG concentration on the mole fraction of DCA seem to remove this effect. In order to explain our data, as well as some calorimetric data in the literature, we consider the transition free energy and enthalpy as a sum of three partial terms. These represent the thermodynamic parameters associated with: (1) conformational changes of the polypeptide, e.g. formation or disruption of intramolecular hydrogen bonds; (2) binding by the strong acid to the nonhelical segments of the polypeptide; (3) an overall (weak) interaction of the polypeptide with the nonbound solvent giving rise to dilution parameters that are dependent on the polypeptide conformation. The latter effect is generally ignored, since it is assumed that solvent interactions, other than specific binding, are similar for both the helical and the nonhelical conformation. Striking effects of water (small amounts) and solution aging on the formation of PBLG helices was observed. Water, as expected, acts as a helicogenic solvent when combined with DCA. The processes occurring during solution aging are not known, although the net effect is to stabilize the helical conformation. Finally, we present some rather unique thermally induced transitions of concentrated PBLG (about 200 mg/ml) in DCA. At low temperatures the soluble randomly coiled conformation is present. Heating produces first an isotropic gel, followed at higher temperatures by an isotropic solution consisting of about 70% α-helicity.     

Conformation of poly(L-arginine). II. Complexes with polyanions

Conformaitons of poly(L -arginine)/polyanion complexes were studies by CD measurements. The polyanions were the homoplolypeptides poly(L -glutamic acid) and poly(L -aspartic acid); the synthetic polyelectrolytes and polyethylenesulfonate; and the polynucleotides were native DNA, denatured DNA, and poly(U). It was found that poly(L -arginine) forms the α-helical conformation by interacting with the acidic homopolypeptides and the synthetic anionic polyelectrolytes. In each complex, poly(L -glutamic acid) is in the α-helical conformation, whereas poly(L -aspartic acid) is mostly in the random structure. The poly(L -glutamic acid) complex changed into the β-sheet structure at the transition temperature about 65°C in 0.01M cacodylate buffer (pH 7). Even in the presence of 5M urea, this complex remained in the α-helical conformation at room temperature. The existence of the stable complex of α-helical poly(L -arginine) and α-helical poly(L -glutamic acid) was successfully supported by the model building study of the complex. The α-helix of poly(L -arginine) induced by binding with polyacrylate was the most stable of the poly(L -arginine)-polyanion complexes examined as evidenced by thermal and urea effects. The lower helical content of the polyethylenesulfonate-complexed poly(L -aginine) was explained in terms of the higher charge density of the polyanion. On the other hand, native DNA, denatured DNA, and poly(U) were not effective in stabilizing the helical structure of poly(L -arginine). This may be due to the rigidity of polyanions and to the steric hindrance of bases. Furthermore, the distinitive structual behavior of poly(L -arginine) and poly(L -lysine) regarding polyanion interaction has been noticed throughout the study.     

Spectroscopic studies of random chain and -helical polypeptides in hexafluoroisopropanol

The infrared, ultraviolet, circular dichroism, and optical rotatory dispersion spectra of five synthetic polypeptides dissolved in hexafluoroisopropanol are reported. This solvent is useful because it dissolves most proteins and non-ionic polypeptides and also is transparent in spectral regions critical for polypeptide conformational diagnoses. Poly-γ-morpholinylethyl-L -glutamamide has random chain type spectra in this solvent, whereas the spectra of poly-γ-methyl-L -glutamate, poly-L -methionine, poly-ε, N -Carbo-benzoxy-L -lysine, and poly-L -homoserine indicate that these four polypeptides are α-helical. Small but significant variability between the different α-helical polypeptides is seen in their circular dichroism spectra and optical rotatory dispersions. An argument is presented that these differences may be due to slight geometry differences between different α-helices.     

The complete chirospectroscopic signature of the peptide 3(10)-helix in aqueous solution

We synthesized by solution methods a water-soluble, terminally blocked heptapeptide based on five markedly helicogenic, C(alpha)-tetrasubstituted alpha-amino acids C(alpha)-methyl-L-norvalines and two strongly hydrophilic 2-amino-3-[1-(1,4,7-triazacyclononane)]-L-propanoic acid residues at positions 2 and 5. A Fourier transform infrared absorption and NMR analysis in deuterated chloroform and aqueous solutions of the heptapeptide and two side-chain protected synthetic precursors confirmed our working hypothesis that all oligomers are folded in the 3(10)-helical conformation. Based on these findings, we exploited this heptapeptide as a chiral reference compound for detailed electronic CD, vibrational CD, and Raman optical activity characterizations of the 3(10)-helix in aqueous solution.     

Synthesis and characterization of poly(LysAla3)

The synthesis and characterization of poly(LysAla₃) are described. The polytetrapeptide is a model for short sequences found in proelastin, and is presumably involved in desmosine or isodesmosine cross-link formation in the native protein. Poly(LysAla₃) is found to possess a mixture of conformations in aqueous solution dependent on molecular weight and pH. Low-molecular-weight (ca. 3000) material appears to be a mixture of random and extended helix at neutral pH. However, as the molecular weight is increased an increasing amount of α-helix is observed rising to >50% for mol wt = 21,000. The α-helical chain segments are thermally stable, melting to a mixture of extended and random forms at T_m = 25°C. High pH (10.5) promotes further α-helix formation but at pH >11.0 the polypeptide becomes insoluble. The inference is that short chain segments of the peptide in elastin are unlikely to be α-helical in the equilibrium state but may fluctuate through such a conformation.     

Microcalorimetric studies of solvent-induced conformational change of sodium and cesium salts of poly(L-glutamic acid) in aqueous media

Organic solvent-induced coil → helix conformational change of poly(sodium) L -glutamate (NaPLG) and poly(cesium L -glutamate) (CsPLG) in solution in aqueous mixed solvents have been studied at 25°C. Heats of dilution of NaPLG in the water–dioxane pair have been measured as a function of polymer concentration and solvent composition. The results indicate that the overall chain conformation in the disordered form is not too different from that in the α-helical form. Heat capacity measurements by flow microcalorimetry have also been done. The apparent monomolar heat capacity at constant pressure of the polymer, C_{p, ?}, decreases with dilution similarly to other strong polyelectrolytes in aqueous media. In the water–dioxane pair, C_{p, ?} increases with the dioxane content due to partial desolvation of ionic species resulting from increasing ionic association. In the case of the water-2-chloroethanol (CE) pair, the transition takes place at low CE content and results show a fast decrease in C_{p, ?} when the α-helical conformation predominates. It is believed carboxylate groups and CE molecules associate themselves into a complex formation responsible for the transition. The size of the cation plays a significant role in the thermodynamic properties of these polyelectrolytes in solution since sodium ions are more strongly bound to the chain than cesium ions.     

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.     

Solvent effects on the orientation of naphthalene rings in the side chain of poly-gamma-1-naphthylmethyl-L-glutamate

The orientation of naphthalene rings in the side chain of poly-γ-1-naphthylmethyl-L -glutamate (PNLG) in mixed solvents of dichoroethane (DCE) and hexafluoroisopropanol (HFIP) has been studied together with its conformation by infrared, circular dichroism, and fluorescence spectra. The CD pattern of PNLG varies with the solvent composition while it maintains the α-helical conformation. The fluorescence spectra of PNLG in solution show excimer emission of the naphthalene chromophores. The ratio of intensity of the excimer emission to that of the normal fluorescence decreases as the HFIP component in the solvent increases. It is suggested that the naphthalene rings in the side chain of α-helical PNLG are more rigidly orientated in the solvents of higher HFIP ratio.     

Dielectric studies of aqueous solutions of poly(L-glutamic acid)

The dielectric features of poly(L -glutamic acid) are studied by the Fourier synthesized pseudorandom noise method in a time domain combined with a four-electrode cell. Polymer concentration dependence, the effect of the solvent viscosity, salt effects, and pH dependence are studied concomitantly with measurements of CD. A helix-to-coil transition occurs near pH 5.6 for a salt-free solution; at higher pH values, the polymer has an ionized random-coil conformation, and at lower pH, it has a deionized α-helical conformation. When it is in the ionized random-coil conformation, with the usual features of an electrolytic polymer, the solution shows a relaxation spectrum with a large dielectric increment at low frequencies. In the deionized α-helical state, no distinct relaxation curves are obtained, which does not deny the existence of a permanent peptide dipole. The pH dependence of the dielectric increment does not mainly correspond to the conformational change from helix to coil, but rather corresponds to the change of chain expansion on account of a charge–charge interaction under low ionic strength, which is conceived of by a viscosity measurement.     

The effect of methylmercury (II) binding on the conformation of poly(L-glutamic acid) and poly(L-lysine: a Raman spectroscopic study

The binding of the methylmercury cation CH₃Hg⁺ by poly(L -glutamic acid) (PGA) and by poly(L -lysine) (PLL) has been investigated by Raman spectroscopy. Coordination on the side-chain COO^? and NH groups of these polypeptides gave characteristic ligand–Hg stretching modes at ca. 505 and 450 cm^?1, respectively. Precipitation generally occurred upon formation of the complexes and changes of conformation were common. The solid complex obtained from PGA at pH 4.6 was found to have a mostly disordered conformation, which differed from the respective α-helical and β-sheet structures of the dissolved and precipitated uncomplexed polypeptide in the same conditions. An α-helical structure was generally adopted by the complex formed with PLL, even in pH and temperature conditions where the free polypeptide normally exists in another conformation. The addition of a stronger complexing agent, glutathione, to the PLL/CH₃Hg⁺ complex caused a migration of the bound cations and a restoration of the polypeptide to its original state.     

On the flexibility of poly(γ-benzyl L-glutamate) in helicogenic solvents

The molecular-weight dependence of the rms radius of gyration of poly(γ-benzyl L -glutamate) (PBLG) in helicogenic solvents shows negative and positive deviations from expectations for an intact and rigid α-helix in the higher and lower molecular-weight ranges, respectively. In order to study the reason for both deviations, we compare the extant experimental data of with those computed for wormlike chain, freely jointed rod, and a rigid rod having random-coil portions at both ends. The computation for the freely jointed rod and the rigid rod having frayed ends is carried out by a simulation method of Muroga. From the Zimm and Bragg theory and the above comparisons, it is concluded that both deviations can be self-consistently explained if PBLG in helicogenic solvents has an essentially intact α-helical structure with some flexibility arising from random fluctuations in hydrogen bond length. This flexibility explains the negative deviations in the high molecular weight region. The positive deviations in the low molecular weight region result from the tendency of helices to unwind at the ends. © 1998 John Wiley & Sons, Inc. Biopoly 45: 281–288, 1998     

Solution conformation of poly(L-lysyl-L-glutamic acid) and poly(L-lysyl-L-glutamine)

Solution conformation of poly(L-lysyl-L-glutamic acid) (PLGU) and poly(L-lysyl-L-glutamine) (PLGN) was studied in water as a function of pH, added salt, detergents, methanol and trifluoroethanol (TFE). Both the polypeptides exhibit no ordered conformation in the pH range 1.5-12.5; salts and detergents did not have any marked effect. Replacement of side chain carboxyl by an amide group did not help in inducing PLGN to adopt a helical conformation even at pH as high as 12.0, unlike poly(L-lysine). The helicogenic solvents, methanol and TFE, induce formation of weak helices in PLGU as well as in PLGN. It is not unlikely that H-bonding between the side chains leads to stabilizing an unordered conformations.     

The two beta forms of poly(L-glutamic acid).

β-Helical poly(L -glutamic acid) in a gel state was found to be easily converted to the antiparallel β form by heating. Two β forms were obtained, depending on the temperature of heating. Temperatures between 40° and 85°C produced a β form with a spacing between pleated sheets (d₀₀₁) of 9.03 Å, termed β₁. If the heating was carried out at temperatures higher than 85°C, the β₁ form underwent another conformational transition reducing the d₀₀₁ value from 9.03 to 7.83 Å (termed β₂) without any prominent change in the fiber repeat distance (i.e., the polypeptide backbone conformation). The time course of these two transitions was followed by measuring the infrared spectra of the samples, and it was concluded that the α → β₁ transition in its initial stage obeys a pseudo-first order rate process with activation enthalpy and entropy of 54 kcal/mol and 92 eu, respectively. On the other hand, the typical sigmoidal conversion curves observed for the transition between the two types of β forms (β₁ → β₂) indicate that this transition proceeds via a socalled “nucleation and growth” process. The kinetic theory of phase transitions developed by Avrami can be applied with success to explain this transition. The infrared spectra, in the region from 1800 to 200 cm^?1, were measured for these two β forms and the results showed that the conformation of the side chains and the mode of the hydrogen bonding between the side-chain carboxyl groups undergo appreciable change during the transition. The heat-induced conformational transition of poly(L -Glu⁷⁸ L -Val²²) was also studied. The copolymer was transformed from the α-helical conformation directly to the β₂ form. The reason for this was thought to be due to the fact that the L -valine residues and the L -glutamyl residues near the L -valine residues have a strong tendency to take the more compact β₂ form.     

Molecular dynamics simulation of poly(spiropyran-L-glutamate): Influence of chromophore isomerization

A study of the influence of the spiropyran to merocyanine ring opening on a model of poly(spiropyran-L -glutamate) as implied by the experimental data (T. M. Cooper, K. A. Obermeier, L. V. Natarajan, and R. L. Crane (1992) Photochemistry and Photobiology, 55, 1–7) is presented. The individual chromophore is studied by the AM1 semiempirical approach, while molecular mechanics and dynamics calculations are employed in the analysis of the poly(spiropyran-L -glutamate) model. It is shown that the α-helical secondary structure is less conserved in the polypeptide substituted with the merocyanine form of the chromophore. In particular, larger side-chain flexibility, increased backbone hydrogen-bond lengths, as well as a larger helix bending are calculated. Furthermore, a random conformational minimization calculation finds the intrinsic behavior of the spiropyran molecular system as being more of a helix “maker” than its merocyanine analogue. The interactions of the chromophore substituent with other side chains prove, in part, that an early event in the decay of the α-helical structure is the formation of hydrogen bonds between the carboxylic acid groups and the merocyanine oxygens. The results lend support to the experimental observation that the merocyanine group destabilizes the α-helical framework of the polypeptide, thus possibly allowing the entry of solvent molecules into the α-helical core, while spiropyran in its closed form shields it from the solvent. © 1992 John Wiley & Sons, Inc.     

Helical screw sense of peptide molecules: The pentapeptide system (Aib)4/L-Val[L-(αMe)Val] in the crystal state

The crystal-state preferred conformations of six N^α-blocked pentapeptide esters, each containing four helicogenic, achiral α-aminoisobutyric acid (Aib) residues followed by one chiral L -valine (L -Val) or C^α-methyl-L -valine [(αMe)Val] residue at the C-terminus, have been assessed by x-ray diffraction analysis. In all of the compounds the  (Aib)₄ sequence is folded in a regular 3₁₀-helical conformation. In the four pentapeptides characterized by the L -(αMe)Val residue two conformationally distinct molecules occur in the asymmetric unit. Conversely, only one molecule is observed in the asymmetric unit of two pentapeptides with the C-terminal L -Val residue. In the L -Val based peptides the helical screw sense of the  (Aib)₄ sequence is right-handed, whereas in the L  (αMe)Val analogues both right- and left-handed helical screw senses concomitantly occur in the two crystallographically independent molecules. © 1998 John Wiley & Sons, Inc. Biopoly 46: 433–443, 1998