Conformational aspects of polypeptides. XVII. Synthesis of oligomers and co-oligomers derived from L-alanine and L-glutamic acid

A series of oligomers was synthesized of the form: Where Z is the benzyloxycarbonyl group and n = 2, 3, 5, 10. We encountered difficulty in dissolving the decamer in any solvent except strong organic acids. We therefore prepared co-oligomers of the type: where (a) w = x = y = 1; (b) w = 0, x = y = 3; and (c) w = x = y = 3. A series of model compounds composed of glycine L -alamine and γ-methyl-L -glutamate were also prepared. We converted manyof the benzyloxycarbonyl oligopeptides into their corresponding acetyl derivatives. Some of these peptides were synthesized by mixed anhydride and active ester techniques while others were prepared via azide coupling techniques using the tert-butoxycarbonylhydrazide blocking group. All reactions proceeded with retention of configuration.     

Circular dichroism studies of isoleucine oligopeptides in solution

A conformational analysis was carried out on a series of L -isoleucine oligomers having the general formula BOC-(Ileu)_n-OMe (n = 2–8). These oligopeptides were examined in trifluoroethanol, trifluoroethanol-acid and mixed organic-water media. The results indicate that these oligomers form β-conformations beginning at the heptamer. The stability of the β-conformations was found to be greater than those formed by oligopeptides derived from L -alanine.     

Sequential oligopeptides. Conformational studies of the oligopeptides and a polypeptide with the repeating sequence L-norvalyl-glycyl-L-proline

Conformational studies of a series of oligopeptides (from the tripeptide to the octadecapeptide) with the repeating sequence L -norvalyl-glycyl-L -proline and a polytripeptide with this sequence are reported. By means of chiroptical techniques, unordered conformations are found for all oligopeptides in water, trifluoroethanol, and ethylene glycol and for the water-insoluble polymer in trifluoroethanol. In ethylene glycol the polymer assumes a collagen-like structure. Infrared studies indicate that all the oligomers, in contrast to the polymer, are unordered in the solid state.     

Critical chain length for helix formation in L-methionine oligopeptides

The circular dichroism of a series of L -methionine oligopeptides [BOC-(Met)_n-OMe] was examined in trifluoroethanol and hexafluoroacetone sesquihydrate. The results indicate that the trimer through the hexamer exists predominantly in disordered conformations in these solvents. An abrupt change in the CD pattern at the heptamer in trifluoroethanol suggests that L -methionine oligopeptides begin forming helices at this chain length.     

Circular dichroism studies of helical oligopeptides. Can 3(10) and alpha-helical conformations be chiroptically distinguished?

Circular dichroism studies of seven helical oligopeptides containing alpha-aminoisobutyric acid (Aib) in methanol and trifluoroethanol (TFE) solutions are reported. Peptides ranging from 10 to 21 residues in length have been examined. In all cases distinct negative CD bands characteristic of helical peptides are obtained at approximately 220 and 205 nm corresponding to the n-pi and pi-pi transitions, respectively. The ratio R = [theta] pi-pi is less than 1.0 for all peptides studied. Using crystal structure and n.m.r. results for a 10 residue 3(10) helical peptide and literature values for an alpha-helical 11-residue peptide, it is shown that both helical conformations yield R values of approximately 0.8 in alcoholic solvents. The CD data are considered in the light of 1H n.m.r. studies on these oligopeptides. The results suggest that 3(10) and alpha-helical conformations cannot be distinguished by CD methods.     

Infrared spectra and conformation of monodisperse Oligo-γ-Benzyl-L-Glutamates in solution

The ir spectra of σ–nitrophenylthio-tetra- and hexa-γ-benzyl-L -glutamate ethylamides were measured at different concentrations in chloroform and ethylene dichloride. The molar extinction coefficients of two bands, each for the amide I and A modes, were observed as indicating the content of β structure and the fraction of hydrogen bonds; they were analyzed for elucidating the hydrogen-bonded state of peptide residues in the σ and β conformations of oligopeptides. While the content of β structure of the tetra-peptide increases with increasing concentration, the hexapeptide is in the β conformation above the critical concentration only. The fraction of hydrogen bonds remains finite even at infinite dilution or below the critical concentration, indicating the intramolecular hydrogen-bonding in the σ—form. As the fundamental structure of folded forms having only intramolecular hydrogen bonds, the 2₇ ribbon is most likely. With increasing concentration or above the critical concentration, the extended forms are stabilized by the intermolecular hydrogen bonds between residues of the β-form. The β-form is present only when intermolecular hydrogen bonds link two residues in an antiparallel way. Possible structures of the oligopeptides in the σ– and β conformations in the two solvents are described briefly.     

Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. II. Ultraviolet absorption and circular dichroism properties of co-oligopeptides of tryptophan and glycine

The uv absorption and circular dichroism (CD) properties in water (pH 5.9) and trifluoroethanol of several co-oligopeptides of glycine and tryptophan have been investigated. These compounds contain one tryptophyl residue, such as H-Gly-Trp-OH, H-Trp-Gly-OH, and H-Gly-Trp-Gly-OH; or two, such as H-Trp-Trp-OH, H-Gly-Trp-Trp-OH, H-Trp-Trp-Gly-OH, and H-Gly-Trp-(Gly)_n-Trp-Gly-OH (I, n = 0,1,2). Furthermore, the case of some protected derivatives, such as Z-Trp-Gly-OMe, Z-Gly-Trp-Gly-OMe, and Z-Trp-Trp-OMe, Z-Trp-Trp-Gly-OMe, Z-Gly-Trp-Trp-Ome were investigated. The extinction coefficients in H₂O in the region of the L bands, referred to one mole of tryptophyl residue, are essentially the same for all compounds within the limit of experimental error, thus indicating that no strong chromophoric interaction takes place in the oligopeptides containing two aromatic residues. However, in the far uv region, anomalies in the absorption properties cannot be excluded. The investigated compounds show marked differences in their CD properties. Whereas in the case of lower molecular-weight peptides (e.g., H-Trp-Gly-OH and H-Gly-Trp-OH), these differences can be ascribed, at least in part, to the influence of the charge end groups on the indole chromophore, in the case of the compounds containing two tryptophyl residues, the differences in CD properties are assumed to reflect the different structures and conformations of the oligopeptides. In particular, we observed a negative dichroic band at ～225 nm in H-Gly-Trp-Trp-OH and I(n = 0) in water. In trifluoroethanol, this band has a much larger intensity, and it is present also in Z-Gly-Trp-Trp-OMe, Z-Trp-Trp-Gly-OMe, and Z-Trp-Trp-OMe. It is argued that such a negative band is characteristic of the sequence -CO-Trp-Trp-, and the possibility that it derives from intramolecular chromophoric interaction is discussed. It is also pointed out, particularly in view of the high ellipticity values found in some cases (up to 100,000 deg cm² dmol^?1), that this feature may reflect a high degree of conformational rigidity connected with the sequence -CO-Trp-Trp-.     

Linear oligopeptides. XLIII. Study of the relationship between conformation and nature of side chain: Homologous series derived from γ-branched amino acid residues

Conformational studies of the three homologous series, from dimer through heptamer, of monodisperse, N-and C-protected oligopeptides derived from the γ-branched α-amino acid residues L -leucine, β-cyclohexyl-L -alanine, and L -phenylalanine are reported. By means of ir absorption and CD in the vacuum-uv (150nm), the occurrence of intermolecular β-conformations in the higher oligomers in the solid state was established. In solvents of low polarity at high dilution, the extent of intramolecularly hydrogen-bonded folded-structure formation was assessed as a function of chain length and the nature of the side chain. Unordered and intermolecular β-conformations were found in alcohols and aqueous alcoholic mixtures. The results obtained indicate that—the position of branching being equal–steric requirements, electronic properties, and hydrophobic character of the amino acid side chains are all important in determining the nature and stability of oligopeptide conformations.     

The effect of homologous amino acid replacement on the conformation of oligopeptides. II. Synthesis of co-oligopeptides containing methionine and glycine

The synthess of 18 co-oligopeptides of L -methionine and glycine is reported. A series of oligomers, Boc-Gly-Met_n-OMe (n = 1–6), and six hexamers-containing five methionyl and one glycyl residue were synthesized using the mixed anhydride procedure. Polarimetric studies give evidence that oligomers in the Boc-Gly-Met_n-Ome series are essentially disordered in hexafluoroacetone sesquihydrate but begin forming secondry structures at n > 4 in trifluorethanol. Difference in the molar rotation values found for the six hexamers in hexafluoroacetone sesquihydrate may indicate that these compounds, while primirily disordered, are present in slightly different conformations.     

Conformational aspect of polypeptide structure. XLIV. Conformational transitions of poly(N-methyl-alanines) induced by trifluoroacetic acid

The synthesis of poly(N-methyl-L -alanine) and poly (N-methyl-DL -alanine) are described. The polymers were examined by 220 MHz high-resolution nuclear magnetic resonance (nmr) and circular dichroism (CD). The results demonstrate that poly(N-methyl-L -alanine) exists as an ordered helical structure with all the amide bonds in the trans configuration in appropriate solvents. As trifluoroacetic acid (TFA) is added to the solutions of the polymer in helix-supporting solvents, resonances corresponding to both trans and cis amide conformations of N-methyl, C-methyl, and α-CH are observed. The presence of both the trans and the cis peptide bonds in a polymer chain disrupts the ordered structures. Our conclusions from CD data are in agreement with the nmr results. Ultracentrifugation shows that degradation of the polymer chain does not occur during the TFA treatment.     

Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins.

Spectroscopic measurement of protein concentration requires knowledge of the value of the relevant extinction coefficient. If the amino acid composition of a protein is known, however, extinction coefficients can be calculated approximately, provided that the values of the molar absorptivities for tryptophan and tyrosine residues in the protein are known. We have applied a matrix linear regression procedure and a mapping of average absolute deviations between experimental and calculated values to find molar extinction coefficients (epsilon M, 1 cm, 280 nm) of 5540 M-1 cm-1 for tryptophan and 1480 M-1 cm-1 for tyrosine residues in an "average" protein, as defined by a set of experimentally determined extinction coefficients for more than 30 proteins. Use of these values provides a significant improvement in extinction coefficient estimation over that obtained with the commonly used values obtained from solutions of model compounds in guanidine-HCl. The consistency of these results when compared to the large deviations often observed between experimentally determined extinction coefficients suggest that this method may offer acceptable accuracy in the initial estimation of molar absorptivities of globular proteins.     

Conformational analysis of polyoxyethylene-bound homo-oligo-L-glutamates in aqueous environment

A detailed conformational analysis of homo-oligo-L -glutamates was carried out in aqueous solution using ¹H-nmr spectroscopy. Three series of side-chain protected (α-OMe) glutamate oligopeptides, attached to polyoxyethylene (POE) to enhance their solubility, were synthesized. The effect of the N-terminal blocking groups—Boc, Ac, and pGlu—on the conformations of these peptides in water is discussed. Unequivocal assignments were obtained for all amide NH resonances through use of selectively α-deuterated oligo-glutamates. Analysis of vicinal coupling constants, temperature dependence of NH chemical shifts, transfer of saturation experiments, and titration studies with a denaturing solvent (DMSO) were used to investigate the peptide structure. These data suggest that the Glu¹ and Glu² NH protons of each heptamer are solvent exposed, while the NH protons of interior glutamate residues chain are solvent sequestered. The nmr data are consistent with the onset of helical structure at the heptamer of each series in aqueous solution. The POE-peptides with pGlu at the N-terminus showed considerably reduced stability of structure than those with the Boc or acetyl blocking groups. Peptide conformations and their stability in water are compared to those in other solvents.     

Conformational investigations on glycosylated asparagine-oligopeptides of increasing chain length.

Stepwise solution syntheses of the homo-oligomers Boc-(Asn)n-NHCH3 (n = 1-5; I1-5), Boc-[[GlcNAc(Ac)3beta]Asn]n-NHCH3 (n = 1-8; II1-8), and Boc-[(GlcNAcbeta)Asn]n-NHCH3 (n = 1-8; III1-8) are described. Members of the series III were obtained by deacetylation of the corresponding members of the series II. The conformational preferences of the N-protected homo-peptides of the three series were investigated by spectroscopic techniques. 1H-NMR measurements were carried out in various solvents; the CD spectra were recorded in water, aqueous SDS and TFE. The poor solubility of the oligomers of the three series prevented FT-IR measurements in solution. NMR and IR measurements indicate the existence of unordered structures containing some gamma-turns in the carbohydrate-free oligomers and the presence of beta-turns in the glycosylated oligopeptides, whether acetylated or not. The CD spectra do not indicate the presence of organized structures. The sugar moieties apparently do not have a structure-inducing effect on the asparagine homo-oligomer main chain.     

Conformational studies of alanine oligopeptides by nuclear magnetic resonance.

We have studied the nmr spectra of the series of alanine oligopeptides containing a methoxyethoxyethoxyacetyl blocking group on the N-terminal residue and a morpholino blocking group on the C-terminal residue. Spectra were measured in chloroform–trifluoroacetic acid solvent systems. For oligomers with chain lengths of five or more, “double peaks” are observed for the α-CH protons. Addition of trifluoroacetic acid causes the peaks to coalesce. The amount of trifluoroacetic acid necessary for coalescence increases from the pentamer to the nonamer. These findings are general since alanine oligomers with different blocking groups exhibit similar “double peak” phenomena. We explain the “double peak” phenomenon in terms of specific folded forms of the oligopeptides which arise from intramolecular hydrogen bonding. Additional evidence for such hydrogen bonding is presented based upon infrared studies. Slight aggregation probably occurs for the pentamer and hexamer which may stabilize the folded forms.     

Dependence of some conformational properties of polyglycine on composition and sequence of secondary structure

The values of four conformational properties, unperturbed dimensions 0, dipole moment , mean squared optical anisotropy and molar Kerr constant have been calculated for polyglycine chains of x = 100 repeat units with varying composition of alpha-helix, beta-sheet and random coil conformations. The influence of the conformational sequence on these properties has been investigated by calculating the four above-mentioned properties together with the end-to-end vector for several polyglycine oligomers.     

Phenylalanine oligopeptides: Circular dichroism studies in solution

The conformations of a series of l-phenylalanine oligomers having the general formula BOC-(Phe)_n-OMe (n = 1–9) were investigated by circular dichroism in a number of solvent systems. These studies indicate that in trifluoroethanol and in hexafluoroisopropanol these oligomers probably form β-associated conformations beginning at the hexamer.     

Nmr studies on linear homo-oligopeptides: A perspective view

The use of high-resolution nmr to determine the structure of linear homo-oligopeptides in solution is described. Complete assignments of NH and α-CH resonances were elucidated based upon a guest–host series of compounds and selective α-deuteration of residues in the chain. The conformations for oligomethionines and oligoglutamates in dimethylsulfoxide, chloroform, and trifluoroethanol are discussed, and molecular models are described. Recent studies of L -glutamate peptides attached to polyoxyethylene are also included. These peptide–polyoxyethylene conjugates are soluble in a broad range of solvents, such as dimethyl-sulfoxide, chloroform, trifluoroethanol, and water. Preliminary conclusions are drawn concerning the effects of amine terminal end groups and the nature of the solvent on the conformations of these peptide derivatives.     

Poly(L-lysyl-L-alanyl-alpha-L-glutamic acid). II. Conformational studies.

The solution characterization of poly(Lys-Ala-Glu) is described. This polytripeptide is zwitterionic at neutral pH and is shown to take on a conformation which is dictated by the state of ionization, molecular weight, temperature, and solvent. The polypeptide is almost entirely α-helical at low pH and temperature for polymers of greater than 25,000 molecular weight. Melting profiles for these conditions show t_m ～ 20°C. Analysis of circular dichroism curves shows the α-helical content to vary in a linear manner with molecular weight in the range 3000–30,000. At neutral pH the charged polypeptide is essentially random, but substantial α-helix could be induced by addition of methanol or trifluoroethanol. At temperatures where the sequential polypeptide is a random coil, addition of trifluoroethanol produces a polymer which is mostly α-helical but also contains an appreciable ammount of β-structure. The infrared spectrum of a low-molecular-weight fraction assumed to be cyclo(Lys-Ala-Glu)₂ was tentatively assigned a β-pleated sheet structure. A comparison of this polytripeptide in various ionization states with other polytripeptides containing L -alanine and L -glutamate or L -lysine shows the α-helix directing properties for the (uncharged) residues to lie in the order Ala > Glu > Lys.     

Molecular extinction coefficients of lead sulfide and polymerized diaminobenzidine as final reaction products of histochemical phosphatase reactions.

Molar extinction coefficients of precipitated lead sulfide (PbS) and polymerized diaminobenzidine (polyDAB) have been determined at wavelengths of 450 nm and 480 nm, respectively, for quantitative histochemical analysis of phosphatase reactions. These values are essential for the conversion of cytophotometric (mean integrated) absorbance values to absolute units of substrate converted per unit time and volume of tissue. This conversion allows direct comparison of histochemical and biochemical data. The molar extinction coefficient of PbS at 450 nm was found to be 3,800 and therefore, per mole phosphate liberated, the molar extinction coefficient is 5,700 because 3 moles phosphate are captured by 2 moles lead at neutral or alkaline pH. Parallel experiments with the cerium-DAB method revealed that the molar extinction coefficient of polyDAB at 480 nm is 5,500 with respect to liberated phosphate. The molar extinction coefficients were applied for comparison of data from biochemical and histochemical assays of glucose-6-phosphatase activity in rat livers. A significant correlation was found between both sets of data. The values were in the same order of magnitude with histochemical values approximately 1.4 times higher than biochemical values.     

Conformational studies of poly-L-alanine in water

The conformational properties of poly-L -alanine have been examined in aqueous solutions in order to investigate the influence of hydrophobic interactions on the helix–random coil transition. Since water is a poor solvent for poly-L -alanine, water-soluble copolymers of the type (D , L -lysine)_m–(L alanine)_n-(D , L -lysine)_m, having 10, 160, 450, and 1000 alanyl residues, respectively, in the central block, were synthezised. The optical rotatory dispersion of the samples was investigated in the range 190–500 mμ, and the rotation at 231 mμ was related to the α-helix content, θ_H, of the alanine section. In salt-free solutions, at neutral pH, the three large polymers show high θ_H values, which are greatly reduced when the temperature is increased from 5 to 80°C. No helicity was observed for the small (n = 10) polymer. By applying the Lifson-Roig theory, the following parameters were obtained for the transition of a residue from a coil to a helical state: ν = 0.012; ΔH = ?190 ± 40 cal./mole; ΔS = ?0.55 ± 0.12 e.u. Since ΔH and ΔS differ from the values expected for a process involving only the formation of a hydrogen bond, and in a manner predicted by theories for the influence of hydrophobic bonding on helix stability, it is concluded that a hydrophobic interaction is also involved. In the presence of salt (0.2M NaCl), or when the ε-amino groups of the lysyl residues are not protonated (pH = 12), the helical form of the two large polymers (n = 450 and n = 1000) is more stable than in water. Since the electrostatic repulsion between the lysine end blocks is greatly reduced under these conditions, the alanine helical sections fold back on themselves, and this conformation is stabilized by interchain hydrophobia bonds. This structure was predicted by the theory for the equilibrium between such interacting helices, non-interacting helices, and the random coil.