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.     

Synthesis and conformational study of poly(N delta-carbobenzoxy, N delta-benzyl-L-ornithine) and poly(N delta-benzyl-L-ornithine)

Poly(N^δ-carbobenzoxy, N^δ-benzyl-L -ornithine) (PCBLO) was prepared by the standard NCA method. PCBLO was converted into poly(N^δ-benzyl-L -ornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N^δ-benzyl-L -ornithine was synthesized by reacting L -ornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix–coil transition at 12% (v/v) DCA at 25°C, as compared with 36% for poly(N^δ-carbobenzoxy-L -ornithine) (PCLO). Like PCLO, the helix–coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a “coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pH_tr of about 10 for poly-L -ornithine in the same solvent.     

Ferric complexes of acetoacetyl derivatives of poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid). II. Conformational properties in solution. Evidence for a stereospecific complex formation

The conformational properties of ferric complexes of poly(N^ε-acetoacetyl-L -lysine), poly(N^δ-acetoacetyl-L -ornithine), and poly(N^γ-acetoacetyl-L -diaminobutyric acid) were investigated in 1:1 water/dioxane by CD techniques. Optical activity was found in the visible and in the uv absorption region of the polymeric complexes. The conformation of the peptide backbone was always that of a right-handed α-helix, and was found independent of the degree of complexation, at least up to a degree of binding of 20%. In the absorption region of the side-chain chromophores the optical activity is substantially affected by complex formation. In all three cases a splitting of the ligand π → π* transition centered at 257 nm is observed. These data suggest a stereospecific complex formation. From the signs of the splitting it also appears that the chirality of the poly(N^δ-acetoacetyl-L -ornithine) complex is opposite that of the other two polymers.     

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 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.     

Ferric complexes of acetoacetyl derivatives of poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid). I. Preparation and absorption properties in solution

Poly(N^ε-acetoacetyl-L -lysine), poly(N^δ-acetoacetyl-L -ornithine) and poly(N^γ-acetoacetyl-L -diaminobutyric acid) form colored complexes with ferric ions in water/dioxane solutions. These complexes are soluble at pH values lower than 2.8 and show their maximum absorption at 257 nm in the uv and at 478 nm in the visible region; whereas the ferric complex of the model compound n-hexylacetoacetamide exhibits absorptions centered at 258 and 536 nm, respectively. It is shown that in the complex of the model compound one metal ion is bound per acetoacetamide group, while in the complexes of the three polymers two β-ketoamides side chains are bound per ferric ion under the same solvent, pH, concentration, and ionic strength conditions. The binding constants of ferric ions to the three polymers, and the formation constant of the ferric complex of the model compound are also evaluated.     

Comparison of helix stabilities of poly-L-lysine, poly-L-ornithine, and poly (L-diaminobutyric acid)

The helix–coil transitions of aqueous solutions of poly-α-L -lysine (PLL), poly-α-L -ornithine (PLO), and poly(α,γ-L -diaminobutyric acid) (PLDBA) have been investigated as functions of pH at 25°C and of temperature at pH 11.75, where these polymers are uncharged; in the cases of the latter two polyamino acids, the transitions have also been studied as functions of apparent pH in methanol-water solution (50/50 by volume). The helix stability of the polypeptides is shown to be a direct function of the number of methylene groups on the side chain. From an analysis of potentiometric titration data, we find that the difference between the helix stability of PLL and that of PLO is due to a difference of about 1 e.u. in the ΔS° of the transition. Combining the “melting curves” obtained from optical rotatory dispersion studies with the potentiometric titration data permits evaluation of the initiation parameter Z (or 1/σ^½) of the statistical mechanical theories for these transitions. The value obtained for Z in the case of uncharged aqueous PLO is ca. 35.     

Metal complexs of poly (α-amino acids): Cu(II) chelates of acetoacetylated poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid)

The cupric complexes of poly(N^ε-acetoacetyl-L -lysine), [Lys(Acac)]_n′ poly(N^δ-acetoacetyl-L -ornithine), [Orn(Acac)]_n′ and poly(N^γ-acetoacetyl-L -diaminobutyric acid), [A₂bu-(Acac)]_n, as well as of the model compound n-hexyl acetoacetamide, have been investigated by means of absorption, potentiometric, equilibrium dialysis, and CD measurements. While in the complex of the model compound, one chelating group is bound to one cupric ion, in the polymeric complexes two β-ketoamide groups are bound to Cu(II) under the same experimental conditions. The binding constant of cupric ions to the three polymers and the formation constant of the Cu(II)-nhexylacetoacetamide complex have been evluated. Investigation on the chiroptical properties of the three polymeric complexes shows that the peptide backbone does not undergo conformational transitions, remaining α-helical when up to 20% of the side chains are bound to Cu(II). The optical activity of the β-ketoamide chromophores is substantially affected by complex formation and is discussed in terms of asymmetric induction from the chiral backbone.     

Polymerization of L- and DL-phenylalanine N-carboxyanhydride by poly(N-methyl-L-alanine)

Polymerizations of L - and DL -phenylalanine N-carboxyanhydride in nitrobenzene by poly (N-methyl-L -alanine) of varying degrees of polymerization (n = 1–30) were investigated. Poly(N-methyl-L -alanine) was prepared by the polymerization of N-methyl-L -alanine NCA with N-methyl-L -alanine diethylamide and the degree of polymerization was controlled by the molar ratio [NCA]/[Catalyst] + 1. This polymer was shown to be an asymmetrically selective catalyst which polymerized L -phenylalanine NCA at a faster rate than DL -phenylalanine NCA. With increasing degree of polymerization the stability of the secondary structure of poly(N-methyl-L -alanine) increased. This was confirmed by circular dichroism spectra. However, the degree of asymmetric selection did not increase as the stability of the secondary structure of poly(N-methyl-L -alanine) increased. These findings indicate that the interaction of a growing polypeptide in an ordered structure with NCA molecules prior to the reaction does not lead to an asymmetric selection, and that the mechanism of the asymmetric selection by poly(N-methyl-L -alanine) should be different from those proposed so far.     

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.     

Potentiometric titrations and the helix-coil transition of poly(L-glutamic acid) and poly-L-lysine in aqueous salt solutions

The objective have been to establish if those ions which are known to change the stability of the structure of proteins, have any influence on the properties of ionizable polypeptides. Potentiometric titrations and complementary optical rotation data are presented for aqueous solutions of poly-L -lysine (PLL) in the presence of KSCN, KCl, and KF, and for poly(L -glutamic acid) (PLGA) in the presence of KSCN, KCl, and LiCl. The following measured quantities which are affected by salt concentration were obtained: intrinsic pK (pK₀), slope of pK_app versus degree of ionization (α) curves, the degree of ionization at which the helix to coil transition occurs, and the free energy of this transition for the uncharged molecule (δG°_hel). The effects of nonspecific salts (KCl and LiCl for PLL and KSCN and KCl for PLGA) are small, and about, as expected from general electrostatic considerations. In line with the observations made with isoelectric and cat ionic collagen, specific, effects were noted with KSCN–PLL and with LiCl–PLGA. In the presence of KSCN, the poly-L -lysine helix becomes stabilized at much lower degree of ionization than in the presence of KCl, and the slope of the pK_app versus α plots is greatly reduced. However, ΔG°_hel (for the uncharged molecule) is not affected, and pK₀ is only slightly higher. We interpret these data in terms of binding of SCN^? primarily to the side-chain amino groups (both to R? NH₃⁺ and to R? NH₂) solutions. (L -glutamic acid) in LiCl solution has its transition at the same α value as in KCl solution. However, both the slopes of the pK_app versus α plots and the absolute values of ΔG°_hel are lower than in KCl solution. We interpret these results in terms of binding of Li⁺ to side chains as well as to the peptide bond.     

Interaction of sodium decyl sulfate with poly(L-ornithine) and poly(L-lysine) in aqueous solution

The binding isotherms of sodium decyl sulfate to poly(L -ornithine), poly(D ,L -ornithine), and poly(L -lysine) at neutral pH were determined potentiometrically. The nature of a highly cooperative binding in all three cases suggests a micelle-like clustering of the surfactant ions onto the polypeptide side groups. The hydrophobic interaction between the nonpolar groups overshadows the coulombic interaction between the charged groups. The titration curves can be interpreted well by the Zimm–Bragg theory. The average cluster size of bound surfactant ions is sufficiently large to promote the β-structure of (L -Lys)_n even at a very low binding ratio of surfactant to polypeptide residue, whereas the onset of the helical structure for (L -Orn)_n begins after about 7 surfactant ions are bound to two turns of the helix. The CD results are consistent with this explanation.     

Side-chain effect on conformation of ionizable polypeptides in aqueous solution

In order to study the effect of side-chain length on the conformation of polypeptides, conformational changes of various ionic polypeptides with various lengths of side chain, poly-N^ε-glutaryl-L -lysine (PGL), poly-N^δ-glutaryl-L -ornithine (PGO), poly-N^ε-succinyl-L -lysine (PSL), and poly-N^δ-succinyl-L -ornithine (PGO), were investigated by ORD, potentiometric titration, and dilatometric measurements in aqueous solution. The results of optical rotation and potentiometric titration measurements indicate strongly that the α-helix stability increases in the sequence PSO < PSL ～ PGO < PGL, which corresponds to increased side-chain length. The volume change associated with the helix–coil transition also increased in the above sequence. This series of polymers seems to be more hydrophobic compared with poly-L -glutamic acid or poly-L -lysine, as suggested from the values of enthalpy and entropy changes for coil–helix transitions.     

Calorimetric measurement of enthalpy change in the isothermal helix-coil transition of poly(L-ornithine) in aqueous solution.

The enthalpy change associated with the isothermal pH-induced uncharged coil-to-helix transition ΔH_h° in poly(L -ornithine) in 0.1 N KCl has been determnined calorimetrically to be ?1530 ± 210 and ?1270 ± 530 cal/mol at 10° and 25°C, respectively. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH_h°, the observed calorimetric heat was corrected for the heat of breaking the sample cell, the heat of dilution of HCl, the heat of neutralization of the OH^? ion, and the heat of ionization of the δ-amino group in the random coil. The latter was obtained from similar calorimetric measurements on poly(D ,L -ornithine). Since it was discovered that poly(L -ornithine) undergoes chain cleavage at high pH, the calorimetric measurements were carried out under conditions where no degradation occurred. From the thermally induced uncharged helix–coil transition curve for poly(L -ornithine) at pH 11.68 in 0.1 N KCl in the 0°–40°C region, the transition temperature T_tr and the quantity (?θ_h/?T)_Ttr have been obtained. From these values, together with the measured values of ΔH_h°, the changes in the standard free energy ΔG_h° and entropy ΔG_h°, associated with the uncharged coil-to-helix transition at 10°C have been calculated to be ?33 cal/mol and ?5.3 cal/mol deg, respectively. The value of the Zimm–Bragg helix–coil stability constant σ has been calculated to be 1.4 × 10^?2 and the value of s calculated to be 1.06 at 10°C, and between 0.60 and 0.92 at 25°C.     

Synthesis and conformational properties of polypeptides containing long aliphatic side chains. Poly(Nepsilon-stearyl-L-lysine) and poly(Nepsilon-pelargonyl-L-lysine).

Poly(N^ε-stearyl-L -lysine) and poly(N^ε-pelargonyl-L -lysine) were synthesized both by polymerization of N^ε-pelargonyl and N^ε-stearyl-L -lysine NCA and by acylation of poly(L-lysine) with pelargonyl and stearyl chloride. This second route has proven to be very useful, since completely acylated polymers are obtained in almost quantitative yield, whereas the usual scheme of preparation of ε protected poly(L-lysine) cannot easily be applied due to solubility problems. Poly(N^εpelargonyl and stearyl-L -lysine) are soluble in alcohols containing linear aliphatic chains such as n-butanol and n-octanol and in mixtures of these alcohols with hydrocarbons such as n-hexane and n-heptane. Both polymers show an α-helical conformation in the above solvents, which can be disrupted upon addition of sulfuric acid. Also in the solid state, poly(N^ε-stearyl-L -lysine) and poly(N^ε-pelargonyl-L -lysine) show X-ray diffraction patterns typical of order structure.     

The importance of coulombic interactions for the induction of beta structure in lysine oligomers by sodium dodecyl sulfate.

Circular dichroism spectra have been obtained for tri(L -lysine), tetra(L -lysine), and penta(L -lysine) in aqueous sodium dodecyl sulfate at 25°C. None of the oligomers are affected significantly by sodium dodecyl sulfate at detergent concentrations exceeding 0.01 M. Literature results show that the high-molecular-weight polymer forms a β strucure under these conditions. At detergent concentrations near 3.5 × 10^?4 M the penta(L -lysine), but not the smaller oligomers, undergoes a conformational change. Its circular dichroism under these conditions is essentially identical to that observed with poly(L -lysine) when it forms a β structure in sodium dodecyl sulfate. Solutions of the penta(L -lysine), which exhibit this modified circular dichroism, are also turbid, leading to the conclusion that the oligomer has formed an intermolecular β structure. When these experiments are conducted in the presence of 0.1 M sodium hydroxide, the sodium dodecyl sulfate produces neither turbidity nor a modified circular dichroism spectrum. These observations provide compelling evidence that Coulombic interaction between the anionic detergent head and the cationic lysyl amino groups is essential for the conformational change induced in penta(L -lysine) by sodium dodecyl sulfate.     

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.     

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.     

Polymerization of optical isomers of phenylalanine N-carboxyanhydride by poly(N-methylalanine) diethylamide

In the polymerization of phenylalanine N-carboxyanhydride (NCA) using poly(N-methyl-L -or DL -alanine) diethylamide as initiator, the polymerization rate was L -NCA ? D -NCA > DL -NCA. This is a new type of selective polymerization and indicates the incompleteness of earlier investigations to study the asymmetrically selective polymerization without D -NCA. Neither secondary structure nor optical activity of the polymeric initiator is a reason for the selectivity. Hence the cause for the selectivity was sought in the properties of the NCA's in solution. However, the selectivity was not observed in the polymerization initiated by poly(L -phenylalanine) dimethylamide. The importance of the initiator being a secondary amine type was suggested. The experimental results are discussed on the basis of these considerations.     

Experimental study of the conformation of two stereoregular polymethionines: poly(D-methionyl-L-methionine) and poly(L-methionyl-D-methionyl-L-methionine) (author's transl)]

Infrared spectroscopy, X-ray diffraction, and nuclear magnetic resonance spectroscopy have been used in investigating the conformation of two stereoregular polymethionines, poly(D -methionyl-L -methionine) and poly(L -methionyl-D -methionyl-L -methionine). When dissolved in a helicogenic solvent, such as chloroform and hexafluoroisopropanol, the polytripeptide is in an α-helical conformation. A helix-to-coil transition can then be induced by addition of trifluoroacetic acid. On the other hand, it appears that the most stable conformation of poly(D -Met-L -Met) is a β antiparallel folded structure in which the linear polypeptide segments are near to the planar extension. This structure has been evidenced through X-ray examination of oriented films, casted from solutions in chloroform. It has also been identified in solution in the same solvent, by use of infrared spectroscopy and by measuring the δ_Hα chemical shift which characterizes the H^α proton in the peptide units. This δ_Hα value is found equal to 5.4 ppm and differs significantly from those which are usually attributed to the α-helical conformation (δ_Hα = 4.2 ppm) and to the random coil (δ_Hα = 4.6 ppm). The β folded conformation of the poly(D -Met-L -Met) appears to be comparatively less stable than the α-helical one for the poly(L -Met) macromolecular stereoisomer since hexafluoroisopropanol is a helicogenic solvent for this last solute and a destabilizing one for the poly(D -Met-L -Met) β folded conformer. X-ray examinations carried out with stretched films, casted from a solution of poly(D -Met-L -Met) in chloroform, result in several data concering the cross β structure of this stereoregular polypeptide in the solid state.