Azide Procedure Research Articles

Characterization and in vitro release studies of tetracycline and rolitetracycline imobilized on anionic collagen membranes

This work reports the covalent immobilization of tetracycline and rolitetracycline over anionic collagen membranes and the drug release studies as an effort to develop a two stage drug release based on diffusion (fast release) and on the rate of membrane biodegradation (slow release). Independent from casting conditions antibiotics incorporated by dispersion were released in the range from 80 to 100% within 7 hours in concentrations significantly higher than those described for the prevention of bacterial growth. Antibiotic release within this period was predominantly diffusion controlled. Covalent immobilization by a modified azide procedure occurred with preservation of collagen structure independently from pH of casting and reaction conditions. Its expected that anionic collagen membranes with dispersed and covalently bound rolitetracycline or tetracycline, in association with conventional therapy, may significantly reduce membrane induced infections observed post-implantation, one of the major problem associated with periodontal ligaments reconstruction by the Guided Tissue Regeneration procedure.

Application of improved iodine–azide procedure for the detection of thiouracils in blood serum and urine with planar chromatography

The application of iodine–azide reaction for the determination of thiouracils in thin-layer chromatography and high-performance thin-layer chromatography is described. The developed plates were sprayed with a freshly prepared mixture of sodium azide, adjusted to a proper pH, and starch solution, and exposed to iodine vapour for 5 s. The detection limits were established at pmol level. The factors depending on the detection limits were described. A comparison of iodine–azide tests reaction with other procedures is presented. The developed method was applied to detection of thiouracils in blood serum and urine. The possibility of detection of a thiouracils mixture was demonstrated.

Design and Synthesis of N-terminal Cyclic Motilin Partial Peptides: A Novel Pure Motilin Antagonist.

Motilin antagonist was designed and synthesized on the basis of the structure-activity relationship analysis of porcine motilin that we reported recently. The drug design was performed on a specific concept to reduce a flexibility of peptide conformation of porcine motilin partial peptide by its cyclization. The cyclic peptide was synthesized using Boc (tert-butyloxycarbonyl) solid phase methodology, followed by cyclization using the azide procedure, and tested for the binding activity to motilin receptor and smooth muscle contractile activity. The cyclic peptides 3, 4, and 5 showed antagonistic property on contraction assay (pA2 [the negative logarithm of molar concentration of antagonist causing a 2-hold shift to the right of the concentration-response curve for motilin]: 4.5, 4.34, and 4.04, respectively, in rabbit duodenum) and no contractile activity even at high concentration.

Iodine–azide reagent for the detection of biologically oriented thiophosphoryl compounds in thin-layer chromatography systems

The correlation between induction factors ( F i) of phosphorothioates in the iodine–azide reaction and their detection limits (DLs) using the iodine–azide reagent has been established. The iodine–azide reagent has been used for the selective thin-layer chromatographic detection of several sugar phosphorothioates and related compounds. In several cases, hydrolytic treatment of phosphorothioates, prior to their detection on thin-layer chromatography (TLC) plates with the iodine–azide reagent, was accompanied by a dramatic increase in the detectability of the starting compounds. Comparison of TLC detection systems for phosphorothioates using iodine–azide procedures and other representative procedures are presented. The application of the iodine–azide procedure combined with the molybdate procedure for selective TLC detection of phosphates and phosphorothioates is illustrated.

Synthesis and biological activities of head‐to‐tail cyclic bradykinin analogues of varying ring size

Syntheses of cyclic kinin analogues with different backbone atom numbers are described. Cyclization, by either the O-benzotriazolyl-N,N,N',N'-tetramethyluronium tetrafluoroborate/1-hydroxybenzotriazole/diisopropylethyl amine (TBTU-HOBt-DIPEA) or the diphenylphosphoryl azide (DPPA) procedure of linear peptides prepared by the solid-phase method based on the g-fluorenyl methyloxycarbonyl chemistry, was used for preparing cyclo-Gly-Ile-Ile-Gly-bradykinin, cyclo-Lys-kallidin (cyclo-Lys-Lys-bradykinin) and cyclo-des Arg-bradykinin. Peptides were characterized by amino acid analysis, optical rotation, analytical high-performance liquid chromatography and matrix-assisted laser desorption ionization-time flight mass spectrometry. Pharmacological experiments showed that cyclo-Gly-Ile-Ile-Gly-bradykinin (39 backbone atoms) and cyclo-Lys-bradykinin (30 backbone atoms) are about equipotent, when tested on the relaxation of the isolated rat duodenum preparation. The potency of cyclo-des Arg-bradykinin is at least three orders of magnitude lower. The potency of cyclo-Lys-Lys-bradykinin (33 backbone atoms) is one tenth the activity of bradykinin but about 10 times higher than the potency of the above-mentioned cyclokinins and makes the latter analogue the most potent end-to-end cyclic analogue known currently. The present results, in agreement with data from earlier reports, seem to indicate that the enhancement of the number of backbone atoms in the cyclic kinins first increases and subsequently decreases the potency, whereas a reduction in the atom number from 27 to 24 causes a dramatic decrease in potency.

Synthesis of human angiotensinogen (1‐17) containing one of the putative glycosylation binding sites and its hydrolysis by human renin and porcine pepsin

The N-terminal heptadecapeptide of human angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-Glu-Ser-Thr-NH2 ), with the C-terminal carboxyl group amidated, was synthesized in order to study the role of Asn-Glu-Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h-angiotensinogen (1-17)-NH2 with a Km value of 3.4 x 10(-5) M, at pH 7.3 and 37 degrees being similar to h-angiotensinogen (1-13), an analog without the carbohydrate binding site. However, the Vmax value of 4.1 x 10(-9) mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu10-Val11 bond and, surprisingly, His9-Leu10 as well.

Strategy for the chemical synthesis of large peptides; synthesis of angiogenin as an example

Although large peptides are now being synthesized by recombinant DNA technology, chemical synthesis remains important for clarifying their chemical and biological properties. In 1969, two notable reports were published on the chemical synthesis of enzymes: one described solid-phase synthesis of ribonuclease A by Gutte & Merrifield [ 1, 21; and the other, solution synthesis of ribonuclease S-protein by a group at Merck Sharp & Dohme [3]. Gutte & Merrifield demonstrated that their product exhibited ribonuclease activity, but their efforts to isolate a fully active product were unsuccessful. Since then, the techniques for solid-phase synthesis have continuously been improved and many effective purification techniques have been introduced. In 1989, Kent attempted synthesis of HIV-1 acid protease, a 99-residue enzyme, by applying the Boc-strategy to an improved solidphase method and, as a result of which, the product was successfully isolated as crystals [4, 51. In the solution synthesis of ribonuclease S-protein, Mercks group demonstrated that their product exhibited enzymic activity in the presence of excess S-peptide, but the amount of their final product was so small that they could not characterize it. Solution synthesis of ribonuclease A was achieved by Fujii & Yajima in 198 1 [6, 71. They assembled the fully protected peptide from 30 segments by the azide procedure and, after deprotection followed by air-oxidation, they succeeded in isolating the product as crystals via affinity chromatography. Since 197 1, we have been deeply involved in a project for supplying a variety of biologically active peptides to researchers for use as standards. To supply reliable peptides, we concluded that all should be synthesized by the segment condensation method in solution, with standardization of the strategy and techniques. After several trials, we designed a maximum protection strategy as follows [8,9]: ( a ) A water-soluble carbodi-imide, 1 -ethyl-3-( 3-dimethylaminopropy1)-carbodi-imide, is used as a reagent for forming peptide bonds in the presence of HOBt or HOOBt. This procedure is better than the dicyclohexylcarbodi-imide (DCC)/HOBt or DCC/HOOBt method because the urea or acylurea formed, if any, is far more easily removable from the reaction products than when DCC is used. ( h ) Each segment is synthesized by stepwise elongation of Boc-amino acids starting from amino acid phenacyl esters: if the C’-terminus of the objective peptide has a free acid or amide, the C-terminal segment is elongated from the terminal amino acid benzyl ester or amide. The side-chain protected amino acids used are: Asp(cHx), Glu(cHx), Arg(Tos), Ser( Bzl). Thr(Bzl), Cys(MeB) or Cys(Acm), Tyr(BrZ), Lys(CIZ), His(Tos) or His( Bom) and Trp( For). ( c ) When the protected segment, Boc-peptide phenacyl ester, is used as an acid component, the terminal phenacyl ester is removed by treatment with zinc powder in acetic acid. When the segment is used as an amino component, it is treated with trifluoroacetic acid. During these deprotection reactions, all side-chain protecting groups remain intact. ( d ) After completion of all coupling reactions, the final fully protected product is deprotected by the HF method. SHIN-ICHIRO KUMAGAYE, HISAYA KURODA,

Studies on peptides. CXLVIII. Application of a new deprotecting procedure with trimethylsilyl trifluoromethanesulfonate for the syntheses of two porcine spinal cord peptides, neuromedin U-8 and neuromedin U-25.

The usefulness of a new deprotecting procedure was demonstrated in the solution syntheses of two porcine spinal cord peptides, designated neuromedin U-8 and neuromedin U-25. Protected neuromedin U-8 (8-residue peptide), prepared by condensation of two fragments, served as a C-terminal amino component for the synthesis of neuromedin U-25 (25-residue peptide). Onto this fragment, five peptide fragments were successively condensed by the azide procedure to construct the entire amino acid sequence of neuromedin U-25, a possible biosynthetic precursor of neuromedin U-8. All protecting groups were cleaved from protected neuromedin U-8 and neuromedin U-25 by 1 M trimethylsilyl trifluoromethanesulfonate-thioanisole in trifluoroacetic acid. The results were compared with those obtained by trifluoromethanesulfonic acid deprotection. In terms of contractile activity in rat uterus, neuromedin U-25 was twice as active as neuromedin U-8.

Studies on peptides. CXXXIX. Solution synthesis of a 42-residue peptide corresponding to the entire amino acid sequence of human glucose-dependent insulinotropic polypeptide (GIP).

Eight peptide fragments were prepared by known amide-forming reactions as building blocks for the solution synthesis of the dotetracontapeptide corresponding to the entire amino acid sequence of human intestinal GIP (gastric inhibitory polypeptide or glucose-dependent insulinotropic polypeptide). Besides Lys(Z), Trp(Mts) and Gln-OBzl, two new amino acid derivatives, Asp(OChp) and Glu(OChp) [Mts=mesitylenesulfonyl, Chp=cycloheptyl], were employed to suppress various side reactions. These fragments were successively assembled by the azide procedure to minimize racemization and all protecting groups employed were removed from the protected GIP by using 1M trifluoromethanesulfonic acid-thioanisole in trifluoroacetic acid.Synthetic GIP exhibited a significant glucose-dependent insulinotropic activity in dogs, but failed to produce any notable anti-gastric activity in rats.

Amino acids and peptides. VIII. Synthesis of a hexacosapeptide corresponding to the C-terminal sequence 36-61 of human metallothionein II (hMT II) and determination of its heavy metal binding activity.

A C-terminal hexacosapeptide corresponding to residues 36-61 of human liver metallothionein (hMT II) was synthesized by the fragment condensation method using the azide procedure. Protecting groups were removed by the methanesulfonic acid (MSA) method or hydrogen fluoride (HF) method to give the desired peptide. The binding ability of this peptide with Cd and Zn was examined by measuring the absorbance in the ultraviolet region (200-260 nm) as a function of heavy metal concentration and by the gel-filtration method. The heavy metal-binding behavior of this peptide is quite similar to that of thionein.

Synthesis of serum thymic factor (FTS) and two analogs

Serum thymic factor (FTS) and [Gln1]-FTS were synthesized by fragment condensation using the azide procedure. [d-Ala6]-FTS was prepared by stepwise amino acid condensation in combination with fragment condensation on solid-phase resin. Synthetic FTS and [Gln1]-FTS both enhanced the delayed-type hypersensitivity (DTH) level to sheep red blood cells (SRBC) in mice at a dose of 0.1 ng/mouse per day, while a dose of 1.0 ng/mouse per day of [d-Ala6]-FTS was needed for the enhancement reaction.

Human chorionic gonadotropin. VI. Synthesis of a tetratriacontapeptide corresponding to the C-terminal sequence 112-145 of the .BETA.-subunit of human chorionic gonadotropin (hCG).

The tetratriacontapeptide corresponding to sequence 112-145 of the β-subunit of human chorionic gonadotropin (hCG) was synthesized by assembling peptide fragments by the azide procedure, followed by TFA treatment and catalytic hydrogenation. This peptide was conjugated with bovine serum albumin (BSA). New Zealand white rabbits were immunized with the conjugated antigen in Freund's complete adjuvant. Antiserum capable of 30% specific binding of 125I-hCG or 125I-β-subunit of hCG at a dilution of 1 : 5000 was produced.

Synthesis of a tetratriacontapeptide corresponding to sequence 112–145 of the β-subunit of human chorionic gonadotropin

A tetratriacontapeptide corresponding to sequence 112–145 of the β-subunit of human chorionic gonadotropin (hCG), which was synthesized by assembling peptide fragments by the azide procedure, was then conjugated to bovine serum albumin; injection of the conjugated antigen into rabbits produced antisera which exhibited 30% specific binding with [125I]hCG at a 1 : 1000 dilution.

Studies on trypsin inhibitors. Part IX. Synthesis and trypsin inhibitory activity of the duopentacontapeptide corresponding to the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal).

The synthesis of the protected duopentacontapeptide corresponding to the entire amino acid sequence I-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The benzyloxycarbonyltetradecapeptide tert-butyloxycarbonylhydrazide (sequence 1-14) was selectively deblocked with trifluoroacetic acid and used to acylate, by the azide procedure, the peptide free base corresponding to the sequence 15-52. The isolated material was purified by ion exchange chromatography and the protecting groups were removed by successive treatments with anhydrous hydrogen fluoride, 1 M piperidine and mercuric acetate. F02M phosphate buffer, pH8. Determination of the inhibitory capacity indicated that the synthetic material is about 50% effective, at 30:1 inhibitor:trypsin molar ratio in inhibiting the tryptic hydrolysis of Nalpha-benzoyl-DL-arginine-4-nitroanilide. Full inhibition was achieved at a higher inhibitor:trypsin molar ratio. The stability constants and the standard free energy of binding of the complex between trypsin and the synthetic inhibitor have been determined.

STUDIES ON TRYPSIN INHIBITORS

The synthesis by fragment condensation of protected peptides corresponding to the amino acid sequences 15-35, 25-52 and 15-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The Rudinger modification of the azide procedure was used in the fragment coupling steps. The tert-butyloxycarbonylheptapeptide hydrazide (sequence 22-28) was reacted with the heptapeptide methyl ester free base (sequence 29-35) and the resulting tert-butyloxycarbonyltetradecapeptide methyl ester after selective deprotection, coupled with the benzyloxycarbonylheptapeptide hydrazide (sequence 15-21) to give the protected peptide methyl ester corresponding to the 15-35 sequence which was then converted to the corresponding hydrazide. The synthesis of the 25-52 sequence was achieved by assembling the protected peptide hydrazide corresponding to the amino acid residues 25-35, with the C-terminal heptadecapeptide 36-52. The resulting protected octaeicosapeptide (sequence 25-52) was selectively deblocked with trifluoroacetic acid and acylated with the benzyloxycarbonyldecapeptide hydrazide 15-24 to give the desired octatriacontapeptide corresponding to sequence 15-52 of the inhibitor. An attempt to prepare the 15-52 sequence through the condensation of fragments corresponding to 15-35 and 36-52 sequences was unsuccessful. The identity and purity of the synthetized peptide derivatives wre established by elemental analysis (in some cases), amino acid analysis, optical rotation, and thin-layer chromatography in two solvent systems. The final products were also evaluated, after partial deprotection with anhydrous hydrogen fluoride or aqueous 90% trifluoroacetic acid, by paper electrophoresis at different pH values.

STUDIES ON TRYPSIN INHIBITORS

Synthesis is described of the protected undecapeptide tert‐butyloxycarbonylisoleucyl‐threonyltyrosylserylasparaginyl‐γ‐ tert‐butylglutamyl‐S‐acetamidomethylcysteinyl‐valylleucyl‐S‐acetamidomethylcysteinylserine hydrazide corresponding to positions 25–35 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal).The heptapeptide free base methyl asparaginyl‐γ‐tert‐butylglutamyl‐S‐aceta‐midomethylcysteinylvalylleucyl‐S‐acetamidomethylcysteinylserinate (sequence 29–35) was acylated, by the azide procedure, with the tetrapeptide tert‐butyloxycarbonyl‐isoleucylthreonyltyrosylserine hydrazide (sequence 25–28) and the resulting tert‐butyloxycarbonylundecapeptide methyl ester was transformed into the corresponding hydrazide by hydrazinolysis.The stereochemical homogeneity of the final product was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.

STUDIES ON TRYPSIN INHIBITORS

Synthesis is described of the partially protected nonapeptide tert‐butyloxycarbonyl‐valylleucylisoleucylglutaminyl ‐ Nε ‐ trifluoroacetyllsylserylglycylprolyl‐ S‐acetamido ‐ methylcysteine corresponding to positions 44–52 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II. The hexapeptide free base glutaminyl‐Nε ‐ trifluoroacetyllysylserylglycylprolyl‐S‐acetamidomethylcysteine, prepared by two alternative routes, was acylated by the azide procedure, with the tripeptide tert‐butyloxycarbonylvalylleucylisoleucine hydrazide. The stereochemical homogeneity of the resulting nonapeptide was assessed, after partial deprotection by treatment with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.

STUDIES ON TRYPSIN INHIBITORS

Synthesis is described of the partially protected octapeptide tert‐butyloxycarbonyl‐γ‐tert‐butylglutamylasparaginyl‐Nε‐trifluoroacetyllysyl‐Nε‐trifluoroacetyllysylarginyl‐Nγ‐4,4'‐dimethoxybenzhydrylglutaminylthreonylproline corresponding to positions 36–43 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II. The tetrapeptide free base arginyl‐Nγ‐4,4′‐dimethoxybenzhydrylglutaminylthreonyl‐proline was acylated, by the azide procedure, with the tripeptide benzyloxycarbonyl‐asparaginyl‐Nε‐trifluoroacetyllysyl‐Nε‐trifluoroacetyllysine hydrazide. The resulting protected heptapeptide was partially deblocked by catalytic hydrogenation and reacted with α‐1‐succinimidyl‐γ‐tert‐butyl tert‐butyloxycarbonylglutamate. The stereochemical homogeneity of the ensuing octapeptide was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.

STUDIES ON TRYPSIN INHIBITORS

The general strategy for the synthesis, by conventional procedures, of the entire sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal) is discussed. The synthesis of two protected peptides corresponding to positions 2-10 and 1-10 of the proposed primary structure of the inhibitor is described. The heptapeptide free base threonyl-S-acetamidomethylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide (sequence 4-10) was acylated, by the azide procedure, with either the dipeptide benzyloxycarbonyl-gamma-tert-butylglutamylalanine hydrazide (sequence 2-3) or the tripeptide Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylala-nine hydrazide (sequence 1-3). The stereochemical homogeneity of the resulting peptides, benzyloxycarbonyl-gamma-tert-butylglutamylalanylthreonyl-S-acetamidomethyl-cysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide and Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylalanylthreonyl-S-acetamido-methylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonyl-hydrazide, was assessed, after partial deprotection with liquid hydrogen fluoride, by digestion with aminopeptidase M followed by quantitative amino acid analysis.

ChemInform Abstract: ACID DISSOCIATION CONSTANTS OF SOME HISTIDINE‐CONTAINING PEPTIDES AND FORMATION CONSTANTS OF THEIR METAL COMPLEXES

Glycyl-L-histidine, L-histidylglycine, glycylglycyl-L-histidine, glycyl-L-histidylglycine, and L-histidylglycylglycine were synthesized by the carbobenzoxy azide procedure. The acid dissociation constants of these peptides and their related compounds were determined from the potentiometric titration curves of 0.01 M solutions at 21°C. The dissociation of one ionization group is greatly influenced by the charge on the other groups. The formation constants of metal complexes of L-histidylglycine, L-histidylglycylglycine, and L-3-benzylhistidine were calculated by the least-squares method from the titration curves of the solution containing the ligand (0.01 M) and metal in the molar ratio 2: 1, assuming the formation of 1: 1 and 2: 1 normal complexes (ML+ and ML2), and the results were compared with those of L-histidine-metal complexes. For any metal ion, the order of formation constants was found to be histidine≈3-benzylhistidine>histidylglycine>histidylglycylglycine, in agreement with that of the basicity ...