Synthesis and utilization of 13C and 15N backbone-labeled proline: NMR study of synthesized oxytocin with backbone-labeled C-terminal tripeptide amide.

The 13C and 15N backbone-labeled proline was prepared using Oppolzer's method based on application of a sultam as chiral auxiliary. This isotopomer was used in the synthesis of the 13C, 15N backbone-labeled C-terminal tripeptide amide fragment of neurohypophyseal hormone oxytocin. Finally, this tripeptide amide was coupled by segment condensation with N-Boc- or N-Fmoc-tocinoic acid, followed by N-deprotection with TFA or piperidine. The labeled oxytocin exhibited biological activity identical with that of natural oxytocin. A detailed 1H, 13C and 15N NMR study confirmed the assigned oxytocin conformation containing a beta-turn in the cyclic part of the molecule, stabilized by H-bond(s) that can be perturbed by the C-terminal tripeptide amide moiety as indicated by comparison of NMR data for both the tocine ring in oxytocin and tocinoic acid.

Solid phase synthesis of partially protected tocinoic acid: optimization with respect to resin and protecting groups.

A few solid phase and solution approaches of good repute were applied in parallel with the aim to provide optimized routes to Boc- and Fmoc-tocinoic acid (3a and 3c) and the corresponding Tyr(Bu(t)) derivatives (3b and 3d). Boc-tocinoic acid is known to couple with tripeptide amides to give substituted oxytocin precursors in high yields, requiring only Boc-cleavage to furnish the corresponding hormone analogs with minimal loss of material. For comparison, two protected linear hexapeptides (2a and 2b) were prepared on three polystyrene supports, two with acid-labile handles and one a conventional chloromethylated resin, in yields of 62-82 and 58-76%, respectively. The intermediate 2a could be converted to 3a with physical data in agreement with those earlier reported. Similarly, the intermediate 2b was converted to 3b. The highest yields for both 2a and 2b were obtained with a 2-chlorotrityl chloride resin, which in addition provided advantages with respect to overall speed and convenience. Additional syntheses of 3c and 3d on this and of 3c on SASRIN resin, in conjunction with trityl instead of benzyl for side-chain protection of cysteine, were also elaborated.

Enhancing reductive cleavage of aromatic carboxamides.

[reaction: see text] A set of aromatic and especially heteroaromatic N-benzyl carboxamides, derived from naphthalene, pyridine, pyrazine, and quinoline, and the corresponding tert-butyl acylcarbamates have been synthesized and studied by cyclic voltammetry with respect to facilitated reduction. The latter undergo regiospecific cleavage of their C(O)-N bonds under very mild reductive conditions with formation of Boc-protected (benzyl)amine in most cases in nearly quantitative yields. Examples of preparative cleavage by controlled potential electrolysis, activated aluminum, and NaBH(4) are given.

Determination of the optical purity of threonine and hydroxyproline by capillary gas chromatography on a chiral stationary phase.

Experimental conditions for the derivatization and resolution by GLC of all stereoisomers of threonine and 4-hydroxyproline are reported. Threonine was in two steps converted to N,O-bisisobutoxycarbonyl 2,2,2-trifluoroethyl ester derivatives, the second of which was performed under anhydrous conditions. As such the enantiomers could pairwise be separated by capillary gas chromatography on a Chirasil-Val column. Since L- and D-threonine eluted much earlier than the corresponding allo forms, quantitative determination of the allothreonine content in D- or L-threonine down to the one percent level could be simply accomplished but also enantiomeric impurities could be determined. Unlike for threonine, the corresponding 4-hydroxyproline isomers could not all be resolved as N,O-bisisobutoxycarbonyl 2,2,2-trifluoroethyl esters on this column. Although diastereomers could still be separated, the allo pair cochromatographed and the resolution for the L- and D-isomers was low. Complete separation of the 4-hydroxyproline isomers could be accomplished as N,O-bisprotected isobutyl amides, the formation of which required three derivatization steps. These were used for the determination of allohydroxyproline.

Modifications outside the proteinase binding loop in Cucurbita maxima trypsin inhibitor III (CMTI-III) analogues change the binding energy with bovine beta-trypsin.

Five 26-peptide analogues of the trypsin inhibitor [Pro18]CMTI-III containing Leu or Tyr in position 7 and Val or Tyr in position 27: 1 (Leu7, Tyr27), 2 (Tyr7, Val27), 3 (Tyr7, Tyr27), 4 (Leu7, Val27) and 5 (Leu7, Ala18, Tyr27) were synthesized by the solid-phase method. Analogues 1-4 displayed Ka with bovine beta-trypsin of the same order of magnitude as the wild CMTI-III inhibitor, whereas for analogue 5, this value was lower by about 3 orders of magnitude. This indicated that for the analogues with Pro (but not with Ala) in position 18, the side-chain interactions between positions 7 and 27 did not play a critical role for the stabilization of the active structure. In addition, these results also suggest that Tyr7 is involved in an additional aromatic interaction with position 41 of the enzyme.

Distance between the basic group of the amino acid residue's side chain in position P1 of trypsin inhibitor CMTI-III and Asp189 in the substrate pocket of trypsin has an essential influence on the inhibitory activity.

Three new analogues of trypsin inhibitor CMTI-III were synthesized by the solid-phase method: [Lys5]-CMTI-III, [Orn5]CMTI-III and [Dab5]CMTI-III. Only one analogue with L-lysine residue in position P1 showed inhibitory activity of the same order of magnitude as did wild CMTI-III. Two remaining analogues were completely inactive. A conclusion was drawn that the distance between the basic group of the amino acid residue's side chain in position P1 of the trypsin inhibitor CMTI-III and Asp189 in the substrate pocket of trypsin plays an essential role for the trypsin-inhibitor interaction.

Enzymatic synthesis of isotopically labelled serine and tryptophan for application in peptide synthesis.

L-[1.2-13C2, 15N]Serine was prepared from [1,2-13C2, 15N]glycine on a gram scale by the use of the enzyme serine hydroxymethyltransferase. The reaction was monitored by 13C-NMR spectroscopy. This is the first simultaneously 13C- and 15N-labelled serine isotopomer so far reported. Part of the product was directly converted by tryptophan synthase to L-[1,2-13C2, 15N]tryptophan which could conveniently be purified and isolated as Boc-derivative in a yield of 71%. Most of the serine was isolated similarly but to remove remaining starting material in this case purification by column chromatography was required.

Proteinogenic amino acids labelled with 15N and/or 13C for application in peptide synthesis: a short review with a comprehensive list of published derivatives.

A review is given of the literature dealing with the most common protected derivatives of 15N- and/or 13C-labelled amino acids of interest in peptide synthesis. The list contains all such Boc-, Z- and Fmoc-amino acids as well as published methyl, ethyl, t-butyl and benzyl esters.

Synthesis and antiviral activity of three pyrazole analogues of distamycin A.

The synthesis of three new monopyrazole analogues of the antiviral compound distamycin A is reported. Suitably protected 4-amino-1-methylpyrrole-2-carboxylic acid and 3-amino-1-methylpyrazole-5-carboxylic acid derivatives were chosen as starting materials. The construction of the trimeric polyamide framework was accomplished by assembly of the monomeric precursors under condensing conditions by analogy with our previous methodology, although with significant improvements in some pivotal steps. After chromatographic purification and spectroscopic characterisation, the analogues were assayed for antiviral activity. Compounds 7a-c inhibited vaccinia virus at a concentration similar to or lower than distamycin A and the related antibiotic netropsin. Analogues 7b and 7c exhibited an antiviral effect comparable to those of distamycin A and netropsin against HSV-1 and HSV-2, whereas their antiviral activity against several other viruses including HIV-1 and HIV-2 was somewhat lower. The cellular toxicity of 7a-c toward different host cell types proved to be of similar magnitude or lower than those of distamycin A and netropsin.

D-amino acid residues as substrate specificity determinants for casein kinase II.

A set of diastereomeric peptides RRRDDDSDDD each with a corresponding D-amino acid residue successively in every position (except the arginines) was tested as substrates for casein kinase II. It was found that the D-serine containing peptide was not detectably phosphorylated. The replacements at the positions +1, -1 and +2 decreased the kII values more than 60, 30 and 20 times, respectively. The effect of the L/D replacements decreased with increased distance from the serine. The D-amino acid scan used herein seems to be a helpful complementary tool for studies of substrate specificity determinants for different protein kinases.

Analogues of Cucurbita maxima trypsin inhibitor III (CMTI-III) with elastase inhibitory activity.

Three new CMTI-III analogues containing the Val residue in the reactive site (position 5) were synthesized by the solid-phase method. The analogues displayed an elastase inhibitory activity. It is shown that the removal of the N-terminal Arg residue and the introduction of the Gly-Pro-Gln tripeptide in the region 23-25 decreases the antielastase activity by two orders of magnitude. The removal of the disulfide bridge in positions 16-28 and the substitution of Ala for Cys16 and Gly for Cys28 decreases the activity (measured as Ka with HLE) by five orders of magnitude as compared with [Val5]CMTI-III.

New active analogues of Cucurbita maxima trypsin inhibitor III (CMTI-III) modified in the non-contact region.

Four new analogues of trypsin inhibitor CMTI-III(3-28) = [desArg1,desVal2,desGly29]CMTI-III which was recently shown to be fully active, were synthesized by the solid-phase method. The introduction of glycine in position 9 (peptide 1) and Gly-Pro-Gly (peptide 2) and Gly-Pro-Asn (peptide 3) in the regions 17-19 and 23-25, respectively, did not change the antitrypsin activity of all modified peptides. All of these substitutions are presumed to be outside the trypsin-binding loop as judged from the X-ray structure of the complex between beta-trypsin and the related inhibitor CMTI-I. Also the fourth analogue which was substituted in all the positions mentioned, exhibited the full activity.

Glycine-rich analogues of Cucurbita maxima trypsin inhibitor (CMTI-III) substituted by valine in position 27 display relatively low antitrypsin activity.

Five new analogues of the trypsin inhibitor CMTI-III were synthesized by the solid-phase method. All analogues containing a valine residue in position 27 and glycine residues in some or all of the positions 9, 11, 14, 17, 19, 29 as well as in two cases a norleucine residue in position 8 displayed association equilibrium constants by 6-7 orders of magnitude lower than the native CMTI-III inhibitor.

Substrate specificity of protein kinase C studied with peptides containing D-amino acid residues.

A set of stereoisomeric nonapeptides KRPSQRAKY with one, two, or all L-amino acid residues replaced by the corresponding D-amino acids, and two analogs with L- and D-threonine instead of serine, were synthesized and tested as substrates for protein kinase C. All of the peptides were phosphorylated by the enzyme. The maximal rate of the reaction with the all-D peptide was more than one order of magnitude lower than that for all-L peptide with serine. The same applied to the peptides with D-Ser or with D-Arg in position +2 with respect to Ser. The Km values for the peptides containing one D-amino acid were close to that for the prototype peptide (53 microM). On the other hand, when two or more D-amino acids were present, the Km value increased considerably. Replacement of serine by threonine also reduced the phosphorylation rate and increased the Km values. One can conclude that the stereospecificity of protein kinase C is much less pronounced than that of protein kinase A, which is in agreement with the less clearly pronounced substrate specificity of the former enzyme.

Synthesis and application of N,N-bis-(1-adamantyloxycarbonyl) amino acids.

The preparation of novel bis-(1-adamantyloxycarbonyl) amino acid derivatives has been undertaken and their properties studied. Among them, the p-nitrophenyl esters were subsequently applied to the stepwise synthesis of Leu-enkephalin. In the last coupling step, some hydantoin formation was encountered but it could be nearly completely overcome by working with more concentrated solution. The preparation of a tyrosine derivative presented special problems owing to the existence of the phenolic group in the precursor. The relative stability of 1-adamantyloxycarbonyl as N- and O-protecting groups was also studied.

Protein kinase assay using tritiated peptide substrates and ferric adsorbent paper for phosphopeptide binding.

The recently described synthesis of ferric adsorbent paper has made possible the modification of protein kinase assays. The adsorbent contains ferric chelate groups, which are responsible for the binding of phosphopeptide via phosphate group. The selective adsorption of phosphopeptide contra nonphosphorylated peptide allows the use of tritium-labeled peptides and unlabeled ATP as substrates. The binding of the reaction product to the adsorbent was complete and was not affected by the amino acid sequence of the peptide. The conditions required for the separation of the produced phosphopeptide from the initial peptide have been worked out as well. The firmly bound phosphopeptide should be released from the ferric adsorbent paper prior to liquid scintillation counting. Using 0.1 m NH4HCO3 solution (pH 8.0), the elution of phosphopeptides was almost complete. The modified protein kinase assay proposed herein is rapid and allows handling of multiple samples simultaneously. In addition, the ferric paper method avoids the use of 32P-isotope, replacing it with 3H which has lower radiation energy and a much longer half-life.

New analogues of Cucurbita maxima trypsin inhibitor III (CMTI III) with simplified structure.

Seven new analogues of trypsin inhibitor CMTI III were obtained by solid-phase peptide synthesis. Three analogues contained only two, instead of three, disulfide bridges, whereas the molecules of the next four analogues were shortened at the N- and/or C-terminus. The elimination of one disulfide bridge in CMTI III induces a decrease in the association equilibrium constants by 6-7 orders of magnitude, whereas the removal of one, two or three amino-acid residues at the N- and/or C-terminus does not significantly affect the activity.

Peptide fragments of myelin basic protein as substrates of protein kinase C.

A set of peptides derived from myelin basic protein was synthesized and the kinetics of their phosphorylation by protein kinase C was studied. The replacement or the removal of the N-terminal Gln had no effect on the activity of the parent peptide. The removal of the following Lys or Arg led to a systematic decrease in substrate activity. The modifications in the C-terminal part of the peptide had a weaker influence on the parameters Vmax and KM than those in the N-terminal. The rather regular dependence of the activity of substrates upon their structure does not allow the strict definition of a minimum substrate for protein kinase C.

Synthesis and characterization of some collagen sequence analogs.

Some analogs of natural collagen sequences (773-779) were synthesized. The peptides were hydrolyzed at the Gly-Ile bond not only by crude collagenase isolated from normal rat liver, but also by the bacterial Clostridium histolyticum collagenase. The reason for this unusual cleavage site in the latter case may lie in the unordered secondary structure of the substrates measured by CD spectroscopy.