Conformations of a monodisperse homohexapeptide,Nps-[Glu(OBzl)]6-NHEt,and its critical concentration of β-form formation in solution

Nps-[Glu(OBzl)]₆-NHEt has been prepared by coupling Nps-[Glu(OBzl)]₂-OH with HCl,H-[Glu(OBzl)]₄-NHEt by means of dicyclohexylcarbodiimide. The ir spectra of its nujol mull show that the hexapeptide has the β-structure of antiparallel chains. When it is dissolved in dioxane or ethylene dichloride, the hexapeptide consists of a mixture of the β-form and the solvated σ-form, but the β-form can exist only above a certain critical concentration. The critical concentration is about 0.4g dl^?1 in dioxane and 0.08g dl^?1 in ethylene dichloride, and the content of β-form increases with increasing concentration above it. The CD of the dioxane and ethylene dichloride solutions shows concentration dependence in visible and uv regions.     

Local conformations of ends of α-helical poly[Glu(OBzl)] studied by circular dichroism of terminal chromophores

Conformations of terminal peptide units of α-helical poly(γ-benzyl-L -glutamate), poly-[Glu(OBzl)], were examined by an induced circular dichroism (CD) of chromophores which were covalently attached to both ends of the chain. In chloroform, where the helical poly-[Glu(OBzl)] exists as a head-to-tail-type dimeric associate, the chromophores showed a strong CD induced by an asymmetric perturbation from the helical structure. The induced CD almost disappeared by an addition of a few percent of dichloroacetic acid, which has been reported as a powerful breaker of the associate. These results are explained by an incorporation of terminal peptide units into a helical conformation in the head-to-tail associate and a local unfolding of the terminal portions by the addition of acid. An induced CD of a charge-transfer complex between the two terminal chromophores was also observed and the structure of the helix–helix junction of the head-to-tail dimer is discussed.     

Base tilt of poly[d(A)]-poly[d(T)] and poly[d(AT)]-poly[d(AT)] in solution determined by linear dichroism

We have measured the CD, isotropic absorption, and LD of poly[d(A)]–poly[d(T)] and poly[d(AT)]–poly[d(AT)] in the vacuum-uv spectral region. The reduced dichroism (LD divided by isotropic absorption) varied as a function of wavelength and was independent of shear gradient. Thus, the bases are not perpendicular to the helix axis in solution. Since the directions of the transition dipoles are known, the orientations of the bases in the polymers can be calculated from the reduced dichroism spectra. The results show that the base normals are tilted at angles greater than 25°, with respect to the helix axis, and thymine is tilted more than adenine for both polymers. The tilt axes of adenine and thymine are not parallel, indicating a large propeller twist. Space-filling models of poly[d(A)]–poly[d(T)] and poly[(AT)]–poly[d(AT)] are built based on our results, and the conformations of the two (A + T) polymers in solution are discussed.     

Base tilt of B-form poly[d(G)]-poly[d(C)] and the B- and Z-conformations of poly[d(GC)]-poly[d(GC)] in solution

We have measured the CD, isotropic absorption, and linear dichroism (LD) in the vacuum-uv spectral region for the B-conformations of poly[d(G)]-poly[d(C)] and poly[d(GC)]-poly[d(GC)], and for the Z-conformation of poly[d(GC)]-poly[d(GC)] formed in 70% trifluoroethanol. The reduced dichroism (LD divided by isotropic absorption) for all conformations varied with wavelength, indicating that the bases are not perpendicular to the helix axis. Since the directions of the transition dipoles are known, the inclinations and axes of inclination of each base can be determined from the wavelength dependence of the reduced dichroism spectra. The results indicate that the base normals of the (G + C) polymers in the B- and Z-conformations are tilted at angles greater than 19° with respect to the helix axis. The guanine and cytosine bases have different inclinations, and the tilt axes are not parallel. Therefore, the bases for all the (G + C) polymer conformations studied are buckled and propeller twisted.     

Rhodamine B-labeled thyroid hormone forms high molecular weight aggregates in solution: a possible source of artifacts in binding experiments

The fluorescence intensity of rhodamine B-labeled thyroid hormone is a nonlinear function of probe concentration. The concentration dependence was analyzed in terms of a model with continuous aggregation of the probe molecules to give very high molecular weight species. The apparent association constant was 3.65 microM-1 at 25 degrees C, pH 7.2. Studies at different temperatures showed apparent delta H = 6.8 kcal/mol and delta S = -7.3 cal/mol/degree for association. Based on their absorbance spectra, these aggregates seem to be linear, with the rhodamine chromophores slanted with respect to the long axis.     

Polynucleotide base-pair orientation in solution: linear dichroism and molecular mechanical studies of poly[d(A)]-poly[d(T)

Model helices for poly[d(A)]-poly[d(T)] in solution were constructed based on the base orientations determined by linear dichroism [LD; S. P. Edmondson & W. C. Johnson, Jr. (1985) Biopolymers 24 , 824–841] and refined by molecular mechanical energy minimization. The results demonstrate that poly[d(A)]-poly[d(T)] can form energetically stable conformations with the base orientations measured by LD. Further, only one of the four possible base-pair orientations that are consistent with the LD results is feasible. Models with negative base tilts had large potential energies, indicating that the LD results do not reflect base motions. The LD models of poly[d(A)]-poly[d(T)] are stabilized mainly by intrastrand base-stacking interactions, particularly for the adenine strand.     

In vitro and in vivo opioid activity of [DPro(6)]dermorphin,a new dermorphin analogue

To study the effects of inducing stereo-chemical modifications in the structure of dermorphin (DM) so as to improve its mu-opioid receptor affinity and its resistance to C-terminal enzymatic degradation, in the Institute of Molecular Genetics of Moscow, we synthesized a new DM analogue ([DPro(6)]DM) and analyzed the changes induced in the biological activities of DM by substituting the Pro(6) residue with DPro(6). We compared the activity of the new DM analogue and DM in in vitro assays and in in vivo tests of analgesia, thermoregulation, heart rate recordings, and gastrointestinal motility in rats. In the in vitro tests, guinea pig ileum (GPI) and mouse vas deferens (MVD), although the opioid activities of [DPro(6)]DM indicated that the peptide was always less potent than DM, its lower IC(50) ratios (mu/delta) showed that it had higher mu-opioid receptor selectivity. In the in vivo analgesic test, [DPro(6)]DM, when injected intraperitoneally (i.p.) (0.5-5 and 10mg/kg) in rats, had the same antinociceptive efficacy as DM and when injected intranasally (i.n.) (0.005 and 0.02 mg/kg) it induced a more stable and long-lasting analgesia than DM (the AUC was about 91% higher for [DPro(6)]DM than for DM). Moreover, these data confirm that the intranasal route is advantageous for peripheral drug administration. In the heart rate study, [DPro(6)]DM and DM (0.5mg/kg, i.p.), induced a similar, weak bradycardia. The only difference was that [DPro(6)]DM induced a longer-lasting effect than DM. Conversely, in body temperature regulation [DPro(6)]DM induced weaker inhibitory activity than DM (56% of the DM-induced response); it did so only in a cold environment and at the maximal used dose (0.5mg/kg, i.p.) without inducing vasomotor effects. In the gastrointestinal study, [DPro(6)]DM and DM (0.005, 0.05, and 0.5mg/kg, i.p.) significantly slowed upper gastrointestinal transit of a charcoal meal and inhibited colonic propulsion. Comparison of the ED(50) values of [DPro(6)]DM (0.03 mg/kg) and DM (0.009 mg/kg) showed that the DM analogue was about three times less potent than DM in slowing gastrointestinal and colonic transit. In conclusion, all these data overall suggest that structural maneuvering in the Pro(6)-residue of the DM molecule changes its affinity for mu-opioid receptor subtypes and confirms the usefulness of experimental studies involving structural modifications in obtaining new therapeutic agents.     

Synthesis, characterization, DNA-binding and photocleavage of complexes [Ru(phen)2(6-OH-dppz)]2+ and [Ru(phen)2(6-NO2-dppz)]2+

Two new complexes, ([Ru(phen)(2)(6-OH-dppz)](2+)) (1) and ([Ru(phen)(2)(6-NO(2)-dppz)](2+)) (2) (phen=1,10-phenanthroline; 6-OH-dppz=6-hydroxyl-dipyrido[3,2-a:2',3'-c]phenazine; 6-NO(2)-dppz=6-nitro-dipyrido[3,2-a:2',3'-c]phenazine), have been synthesized and characterized by elemental analysis, ES-MS (electrospray mass spectra), (1)H NMR, UV-Vis (UV-visible) and CV (cyclic voltammetry). The DNA-binding behaviors of both complexes have been studied by spectroscopic methods and viscosity measurements. The results indicate that the two complexes all bind to calf thymus DNA (CT-DNA) in an intercalative mode, and the DNA-binding affinity of complex 2 is greater than that of complex 1. In addition, complex 1 can promote photocleavage of pBR322 DNA upon irradiation, whereas complex 2 can promote cleavage of pBR322 DNA both upon irradiation and in the dark, with more efficient cleavage occurring upon irradiation. Theoretical studies for these complexes have been also carried out with the density functional theory (DFT) method. The difference in the DNA-binding behaviors of the two complexes can be reasonably explained by the DFT calculations.     

Synthesis of a [Glu5, Ala12, Ala18, Ala21]sheep insulin-A-chain by fragment condensation on a polymer support (author's transl)]

The synthesis of a [Glu5, Ala12, Ala18,-Ala21]sheep insulin-A-chain by condensation of 5 fragments on a polymer support is described. The 5 fragments Boc-Gly-Ile-Val-Glu(gammaOBut)-Glu(gammaOBut) (V), Boc-Cys(SiPr) (IV), Boc--Cys(SiPr)-Ala-Gly-Val (III), Boc-Cys(SiPr)-Ala-Leu-Tyr-Gln-Leu (II) and Boc-Glu(gammaOBut)-Ala--Tyr(Bzl)-Cys(SiPr)-N2H3 (I) were synthesized by conventional methods and coupled with dicyclohexylcarbodiimide/1-hydroxybenzotriazol (II, III, IV, V) and by the azide method (I) with coupling yields of 60-98% on an Ala-polymer. The failure sequence peptides were separated by ion exchange chromatography on DEAE-Sephadex and by chromatography on Biogel P4. The A-chain was obtained in 9% yield, which, after combinations with natural B-chain, gave insulin activities comparable to that obtained with natural A-chain. These results demonstrate that fragment condensation by the solid-phase method together with simple techniques for purification can be used for the synthesis of longer peptides.     

NMR solution structure of the glucagon antagonist [desHis1, desPhe6, Glu9]glucagon amide in the presence of perdeuterated dodecylphosphocholine micelles

Glucagon, a 29-residue peptide hormone, plays an important role in glucose homeostasis and in diabetes mellitus. Several glucagon antagonists and agonists have been developed, but limited structural information is available to clarify the basis of their biological activity. The solution structure of the potent glucagon antagonist, [desHis1, desPhe6, Glu9]glucagon amide, was determined by homonuclear 2D NMR spectroscopy at pH 6.0 and 37 degrees C in perdeuterated dodecylphosphocholine micelles. The overall backbone root-mean-square deviation (rmsd) for the structured portion (residues 7-29, glucagon numbering) of the micelle-bound 27-residue peptide is 1.36 A for the 15 lowest-energy structures, after restrained molecular dynamics simulation. The structure consists of four regions (segment backbone rmsd in A): an unstructured N-terminal segment between residues 2 and 5 (1.68), an irregular helix between residues 7 and 14 (0.79), a hinge region between residues 15 and 18 (0.54), and a well-defined alpha-helix between residues 19 and 29 (0.33). The two helices form an L-shaped structure with an angle of about 90 degrees between the helix axes. There is an extended hydrophobic cluster, which runs along the inner surface of the L-structure and incorporates the side chains of the hydrophobic residues of each of the amphipathic helices. The outer surface contains the hydrophilic side chains, with two salt bridges (D15-R18 and R17-D21) implied from close approach of the charged groups. This result is the first clear indication of an overall tertiary fold for a glucagon analogue in the micelle-bound state. The relationship of the two helical structural elements may have important implications for the biological activity of the glucagon antagonist.     

Synthesis and some pharmacological properties of [Glu(OMe)4]oxytocin and [Mpr1, Glu(OMe)4]oxytocin.

[Glu(OMe)4]oxytocin (XVI) and [Mpr1, Glu(OMe)4]oxytocin (XVII) bearing a methyl ester group in place of the carboxamide group in position 4 of oxytocin were synthesized by (3 + 6) segment condensation using the S-trityl group for the protection of the cysteine side chains. Analogue XVI exhibited 10.5 U/mg in vitro uterotonic, and 42 U/mg avian vasodepressor, activity, and analogue XVII 21.4 U/mg and 82 U/mg of the respective activities. Both compounds showed no response in the rat pressor assay.     

超活性胰高血糖素的分泌表达

通过多肽化学合成方法 ,人们对胰高血糖素的结构与功能关系有了比较深刻的了解 ,其中最引人注目的成就之一是发现 [Lys17,18,Glu2 1] 胰高血糖素具有比天然胰高血糖素更高的生物活性 ,称之为超活性胰高血糖素(superactiveglucagon ,下称SA glucagon)。为了通过基因工程途径获得SA glucagon ,用PCR方法从以前构建的胰高血糖素表达载体pAGluT得到SA glucagon的基因 (SAG) ,构建了含PL 启动子 ,phoA信号肽和SAG的分泌表达载体pBLSG7。pBLSG7转化到大肠杆菌BL2 1中 ,进行SAG的分泌表达 ,在摇瓶条件下 ,该菌种能分泌表达SA glucagon达 3.6 5mg/L(A60 0 =1) ,占上清液中蛋白质的 19.5 % ,并进一步研究了诱导温度和菌株对表达的影响。     

Structural and functional significance of association of a low molecular weight chalone from JB-1 ascites tumours with high molecular weight factors

Abstract. The cell-specific inhibitory (chalone) activity of JB-1 ascites tumour cell proliferation has been purified using five different procedures. By combining (1) molecular weight estimations based on ultrafiltration and gel chromatography, and (2) partitioning in organic solvent and ion exchangers, it is concluded that the active factor associates, in a complex manner, with various other components involving both hydrophobic and ionic forces. The active factor appears to be a slightly acidic, hydrophobic peptide (molecular weight 500–1000 D). When assessing the activity in vivo , it appears to be highly dependent on associated serum factors. Thus, the chalone studied appears to interact both structurally and functionally with various associated factors which affect its physicochemical behaviour and biological activity.     

Physicochemical analysis of (Pro-Pro-Gly)n with defined molecular weight--temperature dependence of molecular weight in aqueous solution

Sequenced polytripeptides, (Pro-Pro-Gly)_n, (n = 10, 15, 20), with defined molecular weights were synthesized by the solid-phase method. Conformational changes of these sample as a function of temperature were studied by measurements of optical rotation and sedimentation equilibrium. The temperature dependence of optical rotation was shown similar to thermal transition of collagen molecule. Each of these polymers existed as a timer at lower temperature. (Pro-Pro-Gly)₁₀ existed as a monomer at higher temperature, and the others were expected to behave analogously.     

Contribution of translational and rotational motions to molecular association in aqueous solution

Much uncertainty and controversy exist regarding the estimation of the enthalpy, entropy, and free energy of overall translational and rotational motions of solute molecules in aqueous solutions, quantities that are crucial to the understanding of molecular association/recognition processes and structure-based drug design. A critique of the literature on this topic is given that leads to a classification of the various views. The major stumbling block to experimentally determining the translational/rotational enthalpy and entropy is the elimination of vibrational perturbations from the measured effects. A solution to this problem, based on a combination of energy equi-partition and enthalpy-entropy compensation, is proposed and subjected to verification. This method is then applied to analyze experimental data on the dissociation/unfolding of dimeric proteins. For one translational/rotational unit at 1 M standard state in aqueous solution, the results for enthalpy (H degrees (tr)), entropy (S degrees (tr)), and free energy (G degrees (tr)) are H (degrees) (tr) = 4.5 +/- 1.5RT, S (degrees) (tr) = 5 +/- 4R, and G (degrees) (tr) = 0 +/- 5RT. Therefore, the overall translational and rotational motions make negligible contribution to binding affinity (free energy) in aqueous solutions at 1 M standard state.     

Phosphorescence and optically detected magnetic resonance of 4',6-diamidino-2-phenylindole (DAPI) and its complexes with [d(CGACGTCG)]2 and [d(GGCCAATTGG)]2

Phosphorescence and optical detection of magnetic resonance (ODMR) is used to study the excited triplet state of 4',6-diamidino-2-phenyl indole (DAPI) and its complexes with the oligonucleotides [d(CGACGTCG)](2) and [d(GGCCAATTGG)](2), where binding occurs by intercalation between GC base pairs and by minor groove insertion, respectively. Weaker binding of DAPI to phosphate is also detected, and the triplet state of this complex is characterized. Intercalation with [d(CGACGTCG)](2) produces a phosphorescence redshift, while groove binding with [d(GGCCAATTGG)](2) leads to a blueshift. Both binding modes give rise to a small decrease in the zero-field splitting (zfs) of the DAPI triplet state. The largest redshift and zfs decrease are found for the phosphate complex. The phosphorescence lifetimes are shorter by an order of magnitude than that of indole or tryptophan as expected for the lower triplet state energy, E(00), of DAPI. The lifetimes agree well with a correlation with E(00) introduced by Siebrand [Siebrand, W. (1966) J. Chem. Phys. 44, 4055-4057] except for the [d(GGCCAATTGG)](2) minor groove complex with a lifetime that is about 20% too long. The longer lifetime is attributed to distortion of the amidino groups in this complex, resulting in less efficient intersystem crossing.     

Synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp+ ++-(1----6)] - beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp- (1----6)]-D- GlcpNAc, a core glycoheptaose of a "bisected" complex-type glycan of glycoproteins

A synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp++ +-(1----6)]- beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-( 1----6)]-D- GlcpNAc was achieved by employing benzyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(3,6-di-O-benzyl-2-deoxy-2 - phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O-benzyl-2-deoxy-6-O-p- methoxyphenyl-2-phthalimido-beta-D-glucopyranoside as a key glycosyl acceptor. Highly stereoselective mannosylation was performed by taking advantage of the 2-O-acetyl group in the mannosyl donors. The alpha-L-fucopyranosyl residue was also stereoselectively introduced by copper(II)-mediated activation of methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside.     

Biological activities of des-His[Glu]glucagon amide, a glucagon antagonist

Hyperglycemia in diabetes mellitus is generally associated with elevated levels of glucagon in the blood. A glucagon analog, des-His¹[Glu⁹] glucagon amide, has been designed and synthesized and found to be an antagonist of glucagon in several systems. It has been a useful tool for investigating the mechanisms of glucagon action and for providing evidence that glucagon is a contributing factor in the pathogenesis of diabetes. The in vitro and in vivo activities of the antagonist are reported here. The analog bound 40% as well as glucagon to liver membranes, but did not stimulate the release of cyclic AMP even at 10⁶ higher concentration. However, it did activate a second pathway, with the release of inositol phosphates. In addition, the analog enhanced the glucose-stimulated release of insulin from pancreatic islet cells. Of particular importance were the findings that the antagonist also showed only very low activity (<0.2%) in the in vivo glycogenolysis assay, and that at a ratio of 100:1 the analog almost completely blocked the hyperglycemic effects of added glucagon in normal rabbits. In addition, it reduced the hyperglycemia produced by endogenous glucagon in streptozotocin diabetic rats. Thus, we have an analog that possesses properties that are necessary for a glucagon antagonist to be potentially useful in the study and treatment of diabetes.