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.

Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1.

The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.

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.

Adverse responses to analgesia, sedation, and neuromuscular blocking agents in infants and children.

Analgesics, sedation, and neuromuscular blocking agents are commonly used in treating critically ill infants and children. Although these medications are beneficial and imperative to the care of the child, their use is not risk free. Adverse responses occur in these children. With improved methods for use and monitoring, adverse responses can be minimized.

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.

Use of a dehydroalanine-containing peptide as an efficient inhibitor of tripeptidyl peptidase II.

Tripeptidyl peptidase II is an intracellular exopeptidase, which has been purified from rat liver and human erythrocytes. An efficient specific inhibitor was obtained through beta-elimination of phosphate from the phosphopeptide Arg-Ala-Ser(P)-Val-Ala. The dehydroalanine-containing peptide formed was a competitive inhibitor with a Ki of 0.02 +/- 0.01 microM. This study demonstrated that replacing a serine residue in a good inhibitor with a dehydroalanine residue reduced the Ki 45 times. It is proposed that dehydroalanine-containing peptides could be of interest in the development of inhibitors for other peptidases as well.

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.

TRH analogs with 4-nitro- and 4-N-butyloxycarbonylamino-1-methyl-2-pyrolylcarboxylic acid in position 1. Synthesis and biological properties.

Two new analogs of thyrotropin-releasing hormone (TRH), obtained by the replacement of the L-pyroglutamic acid residue with 4-nitro- or 4-N-butyloxycarbonylamino-1-methyl-2-pyrolylcarboxylic acid (analogs 1, 3), and three related derivatives, in which also the L-histidine residue was replaced with L-norvaline (analogs 2, 4) or L-norleucine (analog 5), were synthesized and tested for endocrine and central nervous system (CNS) activity. The replacement of the L-pyroglutamic acid residue with 4-nitro-1-methyl-2-pyrolylcarboxylic acid (analogs 1 and 2) resulted in the separation of the endocrine from the direct CNS activity. The effect of these analogs on the sleeping time, rectal temperature and breathing frequency, was either the same or greater than that of TRH. However, neither the correlation between the binding of analogs to TRH receptors in the brain nor their activity on the CNS parameters measured was found. Analogs 3, 4 and 5, containing 4-N-butyloxycarbonylamino-1-methyl-2-pyrolylcarboxylic acid in place of L-pyroglutamic acid, were inactive.

Facile synthesis of monoacetylated spermidines, illustrating selective deacetylation and application of a common precursor.

The synthesis of all three monoacetylated spermidines is reported. N4-Acetylspermidine was obtained in four steps from spermidine via the triacetylated intermediate by selective deacetylation after exhaustive t-butoxycarbonylation as well as directly from a previously described protected precursor. N1-Acetylspermidine and N8-acetylspermidine were both obtained in four simple protection/deprotection steps from a common, selectively protected compound, thus illustrating the versatility of the latter.

Rotational behaviour produced by intranigral injections of bovine and human beta-casomorphins in rats.

The biological activity of beta-casein derived beta-casomorphin peptides was evaluated by injecting bovine beta-casomorphin-5 (Tyr-Pro-Phe-Pro-Gly), the homologous sequence in human beta-casein (Tyr-Pro-Phe-Val-Glu) and the corresponding N-terminal tetrapeptides into the left substantia nigra of rats. Their ability to produce rotational behaviour was compared to that produced by three reference compounds, morphine, D-ala2D-leu5 enkephalin and U50,488H, ligands for mu, delta and kappa types of opioid receptors, respectively. The relative potencies of beta-casomorphins and morphine were compared to those tested in two in vitro assays for opioid activity: (1) inhibition of the electrically induced contraction of the isolated myenteric plexus-longitudinal muscle of the guinea-pig ileum and (2) displacement of 3H-dihydromorphine binding to brain membranes. The same ranking order of potency was found in all three assays, the peptides from human beta-casein being about 10-fold less potent than those from bovine beta-casein. The effects of both morphine and bovine beta-casomorphin-5 in producing rotational behaviour were antagonized by naloxone; however, approximately 10-fold more naloxone was required to antagonize the beta-casomorphin-5 effect than that of morphine. The present data are discussed in the light of the recent observation that high concentrations of beta-casomorphin-like peptides are found in the cerebrospinal fluid and plasma of women with postpartum psychosis.

Structure-activity relationships in distamycin A analogues: effect of alkyl groups on the pyrrole nitrogen at the non-amidine end of the molecule combined with methyl elimination in the following ring.

Distamycin A analogues 5a-f (R = CnH2n+1, n = 0-5) were synthesized using our previous strategy with some improved modifications and screened for their effects on herpes simplex virus (HSV-1). Virus yield assays show that 5a-5d were potent antiviral agents whereas 5e and 5f had lower activity. Considerable cellular toxicity was however observed for 5a-5c. Thus 5d combining significant antiviral activity with moderate cellular toxicity seems to be the most promising derivative in this series.

Synthesis and antiviral activity of distamycin A analogues: substitutions on the different pyrrole nitrogens and in the amidine function.

Several new analogues of the antiviral antibiotic distamycin A were synthesized and assayed for their effects on influenza and herpes simplex virus. The new compounds 5b-j (R1-3 = H, CH3, and C2H5, R4,5 = H and CH3) were obtained via stepwise prepared formylated trimeric benzyl 4-aminopyrrole-2-carboxylates 3a-h, which after catalytic hydrogenolysis were coupled as N-succinimidyl esters directly with the proper beta-aminopropionamidine, unsubstituted or substituted with one or two methyl groups in the amidine function. Most of the new analogues did not exhibit significant effects on the viruses studied, but three compounds (5f-h) displayed activity on herpes virus as demonstrated in plaque formation and virus yield assays. Elevated cytotoxicity was simultaneously observed for 5g and 5h. For compound 5f, a partial separation of antiherpes activity and cytotoxicity was accomplished. The differences in antiherpes activity did not correspond to the differences in the inhibition of herpes virus DNA polymerase.