Conformation and properties of DNA–(Lys33, Leu67)100–(Orn)20 complex: A nucleohistone model

The synthesis and characterization of the block copolypeptide (Leu⁶⁷, Lys³³)₁₀₀Orn₂₀, a synthetic model of histone, are reported. In neutral aqueous solutions, 80% of the etheropolypeptide block assumes an α-helical conformation, whereas the polyornithine block is in a random-coil conformation. In the association complexes with DNA, melting and titration experiments, as well as CD results, indicate that the polyornithine block interacts with DNA, whereas at least 2/3 of the lysine residues of the (Leu, Lys) moiety are excluded from the direct binding with DNA. CD spectra of the association complexes reveal significant differences from those obtained with DNA–polyornithine and DNA–polylysine complexes but substantial similarities with CD spectra of native and reconstituted nucleohistones. In contrast to DNA–polyornithine complexes, the CD spectra of the ternary complexes, copolypeptide–DNA–ethidium bromide, indicate a strong reduction of the dye intercalation. The low-angle x-ray diffraction pattern, reminiscent of that of chromatin, reveals the presence of a superstructure in these complexes. The results obtained are discussed in connection with the expected structural features of the model.     

Cyclic analogs of substance P. I. Cyclo(11----epsilon 5)-[Lys5] substance P-(5-11)

Two solution syntheses of cyclo(11----5 epsilon)-[Lys5]substance P-(5-11) (CLP) were carried out. The first synthesis involved the stepwise elongation of the peptide chain starting from glycine tert-butyl ester. At the stage of hexapeptide deprotection, the cleavage of Boc and But groups was accompanied by tert-butylation of the Met residue. Cyclization was carried out via a pentafluorophenyl ester intermediate. The benzyloxycarbonyl-cyclopeptide (Z-CLP) formed was deprotected by catalytic transfer hydrogenation. A (3 + 4) block coupling strategy was used in course of the repeated preparation of the linear precursor of CLP. Optimization of the cyclization and subsequent deprotecting stages lead to increased yields and facilitated the synthetic procedure. Z-CLP was found to possess myotropic activity on isolated guinea pig ileum (alpha = 0.55 +/- 0.18; pD2 = 7.97 +/- 0.20), whereas CLP was inactive in these experiments. Z-CLP causes a slight two-phase effect on arterial pressure in rats, CLP being inactive. Similar to substance P, CLP displays an antidepressant-like effect in mice as indicated by the swimming test.     

Natural peptides and their analogs. XXXV. Synthesis and properties of [Ala5, Orn9]somatostatin

A total chemical synthesis of [Ala5, Orn9]somatostatin has been performed. This structural analogue of natural somatostatin inhibits the release of somatotropin, insulin and glucagon, but shows no inhibitory effect on secretion of prolactin.     

Chromatin models. Thermal denaturation studies of (Lysx, Leuy)n-and (Lys)n(Leu)m-DNA complexes.

The structural transitions of (Lysx, Leuy)n-DNA and (Lysx)n(Leuy)m-DNA complexes have been studied by thermal denaturation utilizing simultaneous absorption and circular dichroism (CD) measurements [R. Mandel and G.D. Fasman (1974), Biochem. Biophys. Res. Commun. 59, 672]. These complexes are used as models for nucleohistones. At amino acid/nucleotide ratios r less than 1, the copolymers bind to DNA in a ratio of one amino acid residue per nucleotide, and such binding stabilizes the DNA double helix against thermal denaturation relative to the unbound regions. The leucine residues in the copolymers stabilize the bound portion of the complex against thermal denaturation but to a lesser degree than does poly(L-lysine). This study confirms the hypothesis that absorption melting profiles reflect only the change in secondary structure (helix-coil transition) of DNA. It was found that, in the absence of a higher ordered structure (condensed), the CD melting profile also reflects this same conformational transition, and the melting temperatures, Tm, in CD are equal to those in absorption. However, when a higher ordered structure (tertiary) exists in the complex, then the CD melting profile will be dominated by the structural transitions related to the melting of the higher ordered asymmetric structure in the condensed state, followed by the melting of the secondary structure. Under such circumstances, the Tm obtained from absorption may be slightly different from that of the CD, since only the secondary structural changes are being reflected in absorption. The relevance of these studies to the structure of chromatin is discussed.     

Convenient preparation of [Orn(Tfa)2]- and [Orn(Boc)2, Orn(Tfa)2]gramicidin S, versatile unsymmetrically protected derivatives of gramicidin S.

Treatment of gramicidin S (GS) with trifluoroacetic anhydride afforded a derivative in which only one of the two Orn side chains was trifluoroacetylated in 72% yield, furnishing the first efficient method for the preparation of a monoprotected derivative of GS. The mono(Tfa) derivative [Orn(Tfa)2']GS was treated with di-tert-butyl dicarbonate to yield dually protected derivative [Orn(Boc)2,Orn(Tfa)2']GS from which another monoprotected derivative [Orn(Boc)2]GS was prepared in high yield. These unsymmetrically protected GS derivatives are versatile starting materials for the preparation of various other GS derivatives. As an example of application of the unsymmetrically protected derivatives, a dimeric GS derivative was prepared via a singly p-nitrobenzenesulfonyl(NBS)-activated derivative [Orn(Boc)2,Orn(NBS)2']GS.     

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 2. Tadalafil analogs

In this Letter we describe our ongoing target repurposing efforts focused on discovery of inhibitors of the essential trypanosomal phosphodiesterase TbrPDEB1. This enzyme has been implicated in virulence of Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). We outline the synthesis and biological evaluation of analogs of tadalafil, a human PDE5 inhibitor currently utilized for treatment of erectile dysfunction, and report that these analogs are weak inhibitors of TbrPDEB1.     

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 1. Sildenafil analogs

Parasitic diseases, such as African sleeping sickness, have a significant impact on the health and well-being in the poorest regions of the world. Pragmatic drug discovery efforts are needed to find new therapeutic agents. In this Letter we describe target repurposing efforts focused on trypanosomal phosphodiesterases. We outline the synthesis and biological evaluation of analogs of sildenafil (1), a human PDE5 inhibitor, for activities against trypanosomal PDEB1 (TbrPDEB1). We find that, while low potency analogs can be prepared, this chemical class is a sub-optimal starting point for further development of TbrPDE inhibitors.     

Tryptic hydrolysis of hGH-RH(1-29)-NH2 analogues containing Lys or Orn in positions 12 and 21.

Two analogues of the 29 amino acid sequence of human growth hormone-releasing hormone, namely [Nle27]hGH-RH(1-29)-NH2 and [Orn(12,21),Nle27]hGH-RH(1-29)-NH2, have been synthesized and subjected to digestion by trypsin. The course of degradation was followed using RP-HPLC and ESI-MS. Several intermediates and final products of degradation were identified and conclusions regarding the rate of cleavages at different positions occupied by Lys and Arg residues were drawn. The analogue containing ornithine was found to be less susceptible to hydrolysis by trypsin: the 12-13 and 21-22 peptide bonds were completely resistant to the cleavage. The results show that by replacing Lys with Orn, a possibility exists to design new peptides, which could be more stable in biological fluids.     

Synthesis and kinetic study of transition state analogs for ribonuclease T1.

Based on the proposal that ribonucleases cleave the RNA phosphodiester bond with a mechanism involving pentacovalent phosphorous as transition state, complexes of guanosine and inosine with vanadate-(IV, V), molybdate-(VI), tungstate-(VI), chromate-(VI) and hexacyanochromate-(III) were synthesized and probed as inhibitors of recombinant ribonuclease T1, obtained from an E. coli. overproducing strain. The apparent dissociation constants of these inhibitors and RNase T1, as determined by Michaelis-Menten kinetics, vary between 0.5-0.9 microM and indicate very strong binding, 100- to 1000-fold stronger than the binding of guanosine (Kd = 545 microM) and inosine (Kd = 780 microM), and 50-100-fold stronger than the binding of the product 3' GMP (Kd = 55 microM). Therefore the synthesized inhibitors may be considered as genuine transition state analogs for the enzyme.     

Synthesis of [cyclo(Glu gamma-----epsilon Lys(Gly)]ACTH-(5-14) undecapeptide. Biological and physico-chemical properties of analogs of ACTH-(5-10)- and ACTH-(5-14)-peptides

Modified corticotropin fragment - [Lys11 (Gly)]ACTH-(5-14)- and its cyclic analogue - [cyclo (Glu gamma----epsilon Lys (Gly)] ACTH-(5-14)-undecapeptides have been synthesized by classical approach. The cyclic structure has been fixed by amide bond between gamma-COOH group of glutamic acid and alpha-NH2 group of glycine coupled to the epsilon-NH2 group of lysine. Fragment condensation has been achieved by azide or dicyclohexylcarbodiimide methods. Cyclization has been performed using diphenylphosphorylazide. The melanotropic activity of the cyclicanalogue on isolated frog skin exceeds by two orders of magnitude that of the linear undecapeptide, however the steroidogenic activity in isolated cells of rat adrenal cortex is diminished by an order of magnitude as compared with that of the linear precursor. A similarity of the CD spectra for the cyclic ACTH peptides and their linear counterparts in water and trifluoroethanol points to the similarity and relative rigidity of their structures.     

Conformational studies on sequential polypeptides. Part V. Synthesis and characterization of (Pro-Leu-Gly)10, (Pro-LeuqGly)n and (Leu-Pro-Gly)n.

Syntheses of (Pro-Leu-Gly)n and (Leu-Pro-Gly)n, two synthetic polytripeptide analogues of the non-polar regions of collagen, via the corresponding tripeptide p-nitrophenyl-esters are described. The sequential polypeptide (Pro-Leu-Gly)10 was also obtained by solid-phase synthesis. In the following paper, conformational investigations on these polymers, both in solution and in solid state, will be described.     

Studies on trypsin inhbitors. Part II. Synthesis of the protected tetrapeptide (sequence 11-14) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

Synthesis is described of the protected tetrapeptide corresponding to positions 11-14 of the primary structure of the porcine pancreatic secretory trypsin inhibitor II (Kazal), in the form of free acid as well as protected hydrazide. The tetrapeptide tert-butyloxycarbonylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyl-lysine was prepared by stepwise elongation from the C-terminal Nepsilon-trifluoroacetyllysine using successively 1-succinimidyl benzyloxycarbonylprolinate, p-nitrophenyl N-tert-butyloxycarbonyl-S-acetamidomethylcysteinate and 1-phenyl-3-methyl-4-(tert-butyloxycarbonylglycyl)-oximinyl-5-(benzyloxycarbonylglycyl)-imino-2-pyrazoline as acylating agents. Alternately, the dipeptide benzyloxycarbonylprolyl-Nepsilon-trifluoroacetyllsine was transformed into the corresponding tert-butyloxycarbonylhydrazide which was reacted, after catalytic hydrogenolysis, with tritylglycyl-S-acetamido-methylcysteine to give the tetrapeptide tritylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyllsine tert-butyloxycarbonylhydrazide. The stereochemical homogeneity of the final products was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M, followed by quantitative amino acid analysis.     

Synthesis of M and N active glycopeptides. Part of the N-terminal region of human glycophorin A

The synthesis of two series of glycopeptides, part of the N-terminal region of human glycophorin A, was accomplished starting from derivatives ofO--d-galactopyranosyl-(1–3)-O-(2-acetamido-2-deoxy--d-galactopyranosyl)-l-serine and-l-threonine.     

Synthesis and characterization of 1-(13) C-D X Leu12, 14 gramicidin A

The 13C-D-Leu12, 14 gramicidin A was synthesized by the solid phase method incorporating 13C-D-leucine in positions 12 and 14 with about 25 and 50% enrichment, respectively. The pentadecapeptide was removed from the resin by ethanolamine treatment, with the N-protecting group (Boc) still on. After removal of the protecting group, the peptide was formylated and purified by preparative t.l.c. to obtain 13C-D-Leu12, 14 gramicidin A in a very pure state in an overall yield of about 12.5%. The peptide was then thoroughly characterized by HPLC which gave one single peak with the same retention time as that of Val1-gramicidin A of the natural gramicidin mixture. The CD spectra of the synthetic and the HPLC purified natural Val1-GA were obtained and found to be identical, indicating the optical purity of the sample. The synthetic GA was characterized by 13C n.m.r. spectrum and compared with that of natural GA. Single channel conductance parameters of the synthetic GA were determined and found to be indistinguishable from those of natural Val1-GA in lipid bilayer membranes and the mean channel lifetime was found to be as reported earlier by others.