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.     

Raman spectra of poly-L-lysines

The Raman spectra of poly-L -lysine hydrochloride and poly-?-carbobenzoxy-L -lysine in the solid state have been obtained and are consistent with the presence of an α-helical structure. The Raman spectrum of poly-L -lysine in aqueous solution suggests the presence of random coil structures.     

Synthesis of poly-L-arginine

Poly-L -arginine was prepared by the guanidization of poly-L -ornithine with 1-guanyl-3,5-dimethylpyrazole. The poly-L -ornithine was derived from poly-δ,N-trifluroacetyl-L -ornithine by removal of the blocking groups under mild basic conditions (1M piper-idine).     

Raman scattering of some synthetic polypeptides: poly(gamma-benzyl L-glutamate), poly-L-leucine, poly-L-valine, and poly-L-serine

The Raman spectra of poly-γ-benzyl-L -glutamate, poly-L -leucine, poly-L -valine, and poly-L -serine are reported. For the α-helical polymers, the conformationally sensitive amide I, II, and III modes are observed in the Raman as, well as the infrared. For the β form, the Raman effect, supplies the infrared inactive inphase motion which is useful for the determination of a parallel or antiparallel chain alignment. Modes characteristic of the specific polypeptide are also observed which are insensitive to conformation.     

Template specific inhibitor of mammalian DNA polymerases.

A study of the inhibition of mouse cellular DNA polymerases by poly-nucleotides and their vinyl analogs is presented. Poly(dT)-directed poly(dA) synthesis by representatives of all three classes of cellular DNA polymerase could be completely inhibited by poly(9-vinyladenine), although higher concentrations were required in the case of the gamma class enzyme. Studies on the mechanism of the inhibition using the alpha class DNA polymerase and different templates showed that the enzyme activity was inhibited in all cases where base-pairing between the vinyl polymer and the template occurred; poly(9-vinyladenine) did not interfere with the replication of templates to which it does not bind. The inhibition occurred shortly after addition of poly(9-vinyladenine) to ongoing reactions, yet the enzyme was not displaced from the template - primer complex.     

Recognition of (d(TTTATT) and d(TTATTT) by capillary affinity gel electrophoresis (CAGE) using poly(9-vinyladenine)-polyacrylamide conjugated gel.

Sequence-specific recognition of oligodeoxynucleotide isomers (TTTATT and TTATTT) was achieved by using polyacrylamide-poly(9-vinyladenine) conjugated gel filled capillary affinity gel electrophoresis. It was found that the interaction between poly(9-vinyladenine) and the isomers was dependent on the sequential thymidylic acids of them.     

Circular dichroism and proton magnetic resonance studies of random chain poly-L-lysine

The 218-nm peak, characteristic of the circular dichroism of randomly coiled poly-α-amino acids can be demonstrated in solutions of penta-L -lysine, α-glycyl-L -lysine, as well as poly-L -lysine. The thermal stability of the particular state that gives rise to this 218-nm band in the CD is similar for all three peptides. These results eliminate the possibility that poly-L -lysine forms a structure with long-range order in acidic aqueous solution since the stability of such a structure would be expected to be greater for a higher molecular weight polymer than for a pentamer. The intrinsic viscosity of poly-L -lysine of molecular weight 180,000 varies only slightly between 25 and 60°C. The proton magnetic resonance spectra of poly-L -lysine and penta-L -lysine are indistinguishable on the basis of the chemical shift of all resonances, their line widths, and the exchange rates of the N? H protons. This demonstrates that poly-L -lysine does not possess a cooperatively formed ordered structure in acidic solutions. A weak band at 238 nm is observed in the circular dichroism of poly-L -lysine and other peptides. It is suggested that the effects of change in temperature, salt concentration, or polymer on both the magnitude and position of the 238-nm band may be explained if it is assumed that it is a shoulder of a lower wavelength peak.     

Synthesis of poly-L-asparagine and poly-L-glutamine

Poly-β-N-diphenylmethyl-L -asparagine and poly-γ-N-diphenylmethyl-L -glutamine were prepared from the corresponding N-carboxyanhydrides. Poly-L -aspuragine and poly-L -glutamine were obtained by removal of the diphenylmethyl protecting groups with liquid anhydrous hydrofluoric acid.     

Conformations and interactions of excited states. II. Polystyrene, polypeptides, and proteins

Previous fluorescence and phosphorescence studies of aromatic model compounds have been extended to polymers: “atactic” and isotactic polystyrene, seven aromatic poly-(amino acids), and two proteins. We have confirmed previous observations that both forms of polystyrene exhibit strong excimer fluorescence emission at room temperature but not at 77°K. Of the poly(amino acids) (all observed in helix-supporting solvents), poly-L -phenylalanine, poly(α-benzyl-L -aspartic acid), and poly-1-benzyl-L -histidine likewise show excimer emission at room temperature but not at 77°K., while poly-L -tyrosine, poly-L -tryptophan, poly(γ-benzyl-L -glutamic acid), and poly-S-benzyl-L -cysteine exhibit no excimer emission at either temperature. The aromatic residues of bovine serum albumin exhibit only “normal” fluorescence, but, lysozyme appears to be unique among proteins in showing excimer-like tryptophan emission in the native state; its luminescence becomes “normal” upon denaturation. It appears very probable that none of these polymers has a ground-state conformation in which the aromatic groups have face-to-face orientations appropriate for excimer interaction. It is concluded that at room temperature absorption of light can cause local “melting” of regular (usually helical) structures and thus, in some polymers, permit the attainment of a face-to-face arrangement of aromatic rings within the radiative lifetime of their excited singlet states. In certain other polymers (for reasons not clear at present), and in all polymers at 77°K., this does not occur. This concept is extended to provide a bettor basis for understanding the mechanism of formation of the photodimer of thy mine in irradiated DNA.     

Proof of the charging of polypeptides and polyamides in organic acids

The electrophoresis of poly-γ-benzyl-L -glutamate (PBLG), poly-L -alanine (PLA) and nylon dissolved in various solvents was studied in a glass cell containing three sintered glass partitions. After the passage of a measured amount of charge the concentration of PBLG remained constant in all four chambers when the helicogenic solvents dimethylformamide and ethylene dichloride were used, but in mixtures of ethylene dichloride and dichloroacetic acid and in trifluoroacetic acid, polypeptide migrated to the cathode. Electrophoresis also occurred with PLA in trifluoroacetic acid and with nylon in formic acid. Although the total charge on the polyion could not be determined, the results show beyond reasonable doubt that polypeptides and polyamides are protonated in the presence of moderately strong organic acids.     

Biomesogenic matrix systems

The bifunctionally reactive nucleoside and distant nucleoside analogs adenosine (Ado), S-[(adenine-9-yl)methoxyethyl]-L-cysteine (Na-salt) (cysA) and 9-vinyladenine (vA) in aqueous solutions assemble on complementary polyuridylic acid templates to form complex lyomesophases. The systems are investigated by polarizing microscopy, differential scanning calorimetry (DSC) and 1H- and 31P-nmr spectroscopies, assisted by molecular modeling studies. The results indicate the importance of biomesogenic (pre)ordering in nucleic acid native and artificial matrix reactions.     

Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin a-chain

8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to that of 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure for characterization of the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome-inactivating proteins. Ricin toxin A-chain (RTA) and pokeweed antiviral protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (GAGA) sequence at the elongation factor (eEF2) binding site of the 28S subunit of eukaryotic ribosomes, thereby arresting translation. RTA does not catalyze the release of 8-vinyladenine from 8vdA-10. Molecular dynamics simulations implicate a role for Arg180 in oxacarbenium ion destabilization and the lack of catalysis. However, 8vdA-10 is an active site analogue and inhibits RTA with a Ki value of 2.4 microM. Adenine is also released from the second adenosine in the modified tetraloop, demonstrating an alternative mode for the binding of this motif in the RTA active site. The 8vdA analogue defines the specificities of RTA for the two adenylate depurination sites in a RNA substrate with a GAGA tetraloop. The rate of nonenzymatic acid-catalyzed solvolysis of 8-vinyladenine from the stem-loop RNA is described. Unlike RTA, PAP catalyzes the slow release of 8-vinyladenine from 8vdA-10. The isolation of 8-vA and its physicochemical characterization is described.     

Interaction between chondroitin-6-sulfate and poly-L-arginine in aqueous solution

The interactions between chondroitin-6-sulfate and poly-L -arginine in aqueous salt solution have been investigated by circular dichroism techniques. In the presence of chondroitin-6-sulfate, at neutral pH, poly-L -arginine adopts the α-helical conformation rather than “charged coil” form observed in the absence of mucopolysaccharide. This interaction is at a maximum when the ratio of arginine to disaccharide residues is 2:1. Elevation of the temperature leads to a sharp melting transition at 76.0 ± 1.0°C. This behavior is in marked contrast to that for poly-L -lysine-chondroitin-6-sulfate interactions, which are at a maximum at a 1:1 residue ratio and have a melting transition at 47.0 ± 1.0°C. These results indicate a stronger interaction for poly-L -arginine than for poly-L -lysine. The positive arginine side chains appear to interact with both the negative sulfate and carboxyl residues, while those of the lysines are involved only with the sulfates. Poly-L -ornithine at neutral pH shows no conformation directing interaction with chondroitin-6-sulfate, although a small proportion of α-helix is formed on dilution of the mixture with methanol. The extent of the interaction of cationic polypeptides with chondroitin-6-sulfate increases in the order poly-L -ornithine, poly-L -lysine, poly-L -arginine, i.e., in the order of increasing side-chain length.     

以聚γ-谷氨酸为材质研制微胶囊的工艺

微胶囊制剂能够利用壁材将囊芯物质包裹起来,减少外界环境的不良因素对其造成的影响,但存在产品残效期和速效性的矛盾、成本过高等问题。聚γ-谷氨酸具有成膜性,可生物降解。本文通过自制的枯草芽胞杆菌聚γ-谷氨酸,对开发聚γ-谷氨酸微胶囊的工艺展开研究。对壁材浓度、搅拌转速、反应温度、聚γ-谷氨酸∶明胶质量比、菌悬液体积和甲醛的用量进行优化,建立了聚γ-谷氨酸微胶囊制备工艺,微胶囊对枯草芽胞杆菌的包埋率达到94.2%。同时考察了微胶囊制剂对热、紫外线和极端pH的抗逆性,结果表明聚γ-谷氨酸-明胶微胶囊能赋予微生物细胞更强的抗紫外能力和耐热性。在极端pH条件下热处理,聚γ-谷氨酸-明胶微胶囊剂中枯草芽胞杆菌的存活率也显著提高。     

Novel 5-vinyl pyrimidine nucleosides with potent anti-hepatitis B virus activity.

Synthesis and antiviral activities of novel N-1 alkyl substituted pyrimidines, 1-[(2-hydroxyethoxy)methyl]-5-vinyluracil (5), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-vinyluracil (6), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-vinyluracil (7) are reported. Compounds 6 and 7 were potent inhibitors of DHBV in cell culture, in contrast, all of the compounds described were devoid of activity against TK(+) HSV-1 and TK(-) HSV-1.     

Biomesogenic Matrix Systems

Abstract

The bifunctionally reactive nucleoside and distant nucleoside analogs adenosine (Ado), S-[(adenine-9-yl)methoxyethyl]-L-cysteine (Na-salt) (cysA) and 9-vinyladenine (vA) in aqueous solutions assemble on complementary polyuridylic acid templates to form complex lyomesophases. The systems are investigated by polarizing microscopy, differential scanning calorimetry (DSC) and ¹H- and ³¹P-nmr spectroscopies, assisted by molecular modeling studies. The results indicate the importance of biomesogenic (pre)ordering in nucleic acid native and artificial matrix reactions.     

The effect of neutral salts on the conformational transition of poly-alpha-amino acids. I. Potentiometric titration and optical rotatory dispersion studies

The effect of salts on the coil-to-helix transition of poly-α-amino acids was investigated by optical rotatory dispersion and potentiometric titration techniques. Both charge-dependent and charge-independent contributions to the free energy were considered. The free energy of formation ΔF° of the uncharged α-helix from the uncharged random coil for poly-L -glutamic acid (PGA) decreases very rapidly in the limit of zero added salt concentration. This effect probably depends on the uncertainty affecting the choice of the extrapolation of the apparent pK for the random coil at low ionic strength. Above 0.1 M salt, where the free energy determination becomes meaningful, the anions and cations investigated do not affect the value of ΔF°, with the exception of Li⁺. Our data support the point of view that this cation binds to the peptide group. A class of salts produces an increase of the helical content of poly-L -ornithine (PO) both at low and high degree of ionization. This effect appears to be anion dependent. It is shown that (1) no change of ΔF° is involved; (2) recent theories of polyelectrolyte solutions cannot account for our results. We suggest that a true site binding of the anions to the charged amino groups occurs. The role of electrostatic binding in determining the conformational stability of proteins in the presence of some anions is stressed, and a general treatment for the electrostatic binding equilibria is outlined.     

Conformation of poly-L-tyrosine in aqueous solution

The conformational transition of poly-L -tyrosine in 0.1M KCl was investigated by ORD and infrared spectroscopy, potentiometric titration, and sedimentation velocity experiments. It is shown that the fully ordered conformer is obtained by slow titration of the random coil with 0.1N HCl at 25°C. The charge-induced transition, at variance with other poly-α-amino acids, is completed in a narrow range of α. An aggregation process was detected both by potentiometric titration and sedimentation velocity. The polyamino acid aggregates around α = 0.7 at 25°C when the conformational transition is almost complete. Infrared spectra, in the region of the amide I band (1650 cm^?1) showed that the transition is a random coil → antiparallel β one. Evidence exists that the form is of the intramolecular type. The foregoing interpretations of ORD and CD spectra in terms of the α-helix conformation are discussed.     

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

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