Stabilized analogs of thymopentin. 1. 4,5-Ketomethylene pseudopeptides.

The pentapeptide, thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5) is known for its activity as an immunomodulating drug, but with limited half-life in plasma. In this first paper of a series of three studies, the synthesis of analogs stabilized at the peptide bond between the C-terminal amino acids via insertion of a ketomethylene moiety is described. N-Blocked pseudopeptides containing Val(k)Phe, Ala(k)Phe, and Val(k)Val units were prepared and attached to chloromethyl Merrifield resin via the carboxy terminal. Removal of the N-BOC group by trifluoroacetic acid was followed by sequential coupling with N-BOC dipeptides of aspartic acid to yield resin-bound N-BOC pseudotetrapeptides. Removal of N-BOC and coupling with N-BOC-r-N-tosylarginine followed by total cleavage of blocking groups and resin by HF afforded the target pseudopentapeptides. The analogs were found to compete favorably with thymopentin for binding to CEM cells, but binding was reduced by about 20-30% on average. All analogs showed significant enhancement of half-life versus thymopentin in mouse serum, but most showed only modest improvement in human serum. Insertion of proline or norleucine at position 2 in the chain caused a substantial increase in half-life (3-4-fold), while N-methylnorleucine conferred complete stability in the analogs.

Stabilized analogs of thymopentin. 2. 1,2- and 3,4-ketomethylene pseudopeptides.

In this second paper in a series of three studies of stable analogs of thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5), the synthesis of analogs stabilized at peptide bonds 1,2 and 3,4 via insertion of ketomethylene units is described. A tris(carbobenzyloxy)arginyl(k)norleucine pseudopeptide was synthesized and coupled to Asp-Val-Phe-resin units followed by HF cleavage to prepare Arg(k)Nle-Asp-Val-Phe analogs. Preparation of N-BOC Asp(k)Val and N-BOC Asp(k)Ala units followed by coupling to Phe- or Tyr-resin units provided resin-bound pseudotripeptide substrates for attachment of various arginyl dipeptides. Cleavage from the resin afforded 3,4-ketomethylene-stabilized pseudopeptide analogs of thymopentin. The Arg-Lys-Asp(k)Val-Phe and Arg-Lys-Asp(k)Val-Tyr analogs were more strongly bound to CEM cells than thymopentin itself. There was significant enhancement of stability in serum for the analogs, especially those containing Arg(k)Nle or Arg-NMeLys moieties at the 1,2-peptide bond.

Inactivation of monoamine oxidase by 3,3-dimethyl analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-2,3-dihydropyridinium ion. Dramatic effect of beta-mercaptoethanol on substrate turnover and enzyme inactivation.

It was previously shown (Sayre, L. M., Arora, P. K., Feke, S. C., and Urbach, F. L. (1986) J. Am. Chem. Soc. 108, 2464-2466) that 1,3,3-trimethyl-4-phenyl-2,3-dihydropyridinium salt (the 3,3-dimethyl analogue of 1-methyl-4-phenyl-2,3-dihydropyridinium ion or MPDP+) is a good model for MPDP+ on the basis of its redox potential and was used to show that MPDP+ is unlikely to possess reactivity characteristics which could contribute to the neurotoxicity observed with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). 3,3-Dimethyl-MPTP and 3,3-dimethyl-MPDP+ are now shown to interact with monoamine oxidase similar to MPTP and MPDP+, but only in the presence of beta-mercaptoethanol (beta-ME). In the absence of beta-ME, mixed competitive-noncompetitive inhibition kinetics are observed for 3,3-dimethyl-MPTP and 3,3-dimethyl-MPDP+, whereas competitive inhibition kinetics are exhibited by MPTP. In the presence of beta-ME, however, 3,3-dimethyl-MPTP also is a competitive inhibitor. 3,3-Dimethyl-MPTP and 3,3-dimethyl-MPDP+ also are time-dependent inactivators of monoamine oxidase, having identical kinetic constants, as is the case with MPTP and MPDP+. In the presence of beta-ME, but not glutathione, the rate of inactivation increases dramatically. When [beta-ME] and [3,3-dimethyl-MPTP] or [3,3-dimethyl-MPDP+] are varied, there is an optimal concentration of 1.0 mM for all three at which maximal inactivation rates are obtained. Another dramatic effect of the beta-ME is to lower the partition ratio for inactivation from greater than 50 to about one. This suggests that the effect of the beta-ME toward inactivation may be to induce a conformational change in the enzyme, which reorients an active site nucleophile for attack on the activated species. Support for involvement of an active site nucleophile is the finding that inactivation does not lead to a flavin adduct. Three possible mechanisms for inactivation of monoamine oxidase by MPTP and MPDP+ are suggested.

Inactivation of monoamine oxidase A by the monoamine oxidase B inactivators 1-phenylcyclopropylamine, 1-benzylcyclopropylamine, and N-cyclopropyl-alpha-methylbenzylamine.

Three known mechanism-based inactivators of beef liver mitochondrial monoamine oxidase (MAO) B are tested as inactivators of human placental mitochondrial MAO A. 1-Phenylcyclopropylamine (1-PCPA), 1-benzylcyclopropylamine (1-BCPA), and N-cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) are time-dependent irreversible inactivators of MAO A. The KI values for 1-PCPA and N-C alpha MBA, analogues of the MAO B substrate benzylamine, are much higher with MAO A than with MAO B. Evidence is presented to show that 1-PCPA inactivates MAO A by attachment to the flavin cofactor, unlike the reaction with MAO B in which 1-PCPA can attach to both a cysteine residue and the flavin [Silverman, R.B., & Zieske, P.A. (1985) Biochemistry 24, 2128-2138]. The reaction of 1-BCPA with MAO A was too slow to study in detail. N-C alpha MBA exhibits the same properties toward inactivation of MAO A that it does for inactivation of MAO B. Attachment in both cases is shown to be to one cysteine residue per enzyme molecule. The results with 1-PCPA indicate that the active site topographies of MAO A and MAO B are different. The ability of N-C alpha MBA to undergo attachment to a cysteine residue in both MAO A and MAO B may lead the way toward peptide mapping of the two isozymes in order to determine differences in their primary structures.

Synthesis of fluorescent muramyl dipeptide congeners. 2.

Muramyl dipeptide (MDP) analogues were prepared and utilized in the synthesis of new fluorescently labeled MDP derivatives for use as biologic probes. Thus, N alpha-(N-acetylmuramyl)-L-lysyl-D-isoglutamine (Lys-MDP, 4) and N alpha-(N-acetylmuramyl)-L-alanyl-D-isoglutaminyl)-L-lysine [MTP, 5] were synthesized and then reacted with 2-(fluoresceinylamino)-4,6-dichloro-s-triazine (DTAF, 2) to yield the fluorescent adducts, DTAF-Lys-MDP (6) and DTAF-MTP (7). The adjuvant activity of the fluorescent MDP derivatives was determined by the ability of the compounds to promote delayed skin test responses in guinea pigs immunized with ovalbumin (OA) and by evaluating the anti-OA activity of these guinea pigs.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine analogues. Inactivation of monoamine oxidase by conformationally rigid analogues of N,N-dimethylcinnamylamine.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin and also an inactivator of monoamine oxidase (MAO). Since MPTP is a conformationally rigid analogue of N,N-dimethylcinnamylamine, other conformationally rigid analogues of N,N-dimethylcinnamylamine were synthesized and tested as inhibitors and inactivators of MAO. (E)-2-(Phenylmethylene)cyclohexanamine (5a), (E)-N,N-dimethyl-2-(phenylmethylene)cyclohexanamine (5b), 3-phenyl-2-cyclohexen-1-amine (6a), N,N-dimethyl-3-phenyl-2-cyclohexen-1-amine (6b), and (E)- and (Z)-N-methyl-3-(phenylmethylene)piperidine (7 and 8) are all inhibitors and time-dependent inactivators of MAO B, but none is as potent as MPTP. alpha-Methylation and methylation of the amino group in all cases increases the Ki value relative to that for the parent compound. Compounds 5a, 5b, 6a and 6b are highly cytotoxic, but cytotoxicity is not prevented by pretreatment of the cells with pargyline. There does not appear to be a correlation between the configuration of the N,N-dimethylcinnamylamine analogue and its potency as a MAO inactivator.

Inactivation of monoamine oxidase by allylamine does not result in flavin attachment.

[1-3H]Allylamine was synthesized by sodium boro[3H]hydride reduction of acrolein followed by direct conversion of the [1-3H]allyl alcohol to N-allylphthalimide with triphenylphosphine, diethylazodicarboxylate, and phthalimide. The protecting group was removed with hydrazine. Inactivation of beef liver mitochondrial monoamine oxidase with [1-3H]allylamine led to incorporation of 1-6 eq of inactivator/active site depending upon the length of incubation time. Inactivation and radioactivity incorporation coincided; however, after 1 eq of tritium was incorporated and 5% enzyme activity remained, additional radioactivity continued to become incorporated into the enzyme. The optical spectrum of the FAD coenzyme changed during inactivation from that of oxidized to reduced flavin. Following dialysis of the inactivated enzyme, the spectrum remained reduced, but denaturation in urea rapidly resulted in reoxidation of the flavin. Under these same denaturing conditions, 96% of the radioactivity associated with the enzyme remained bound, therefore indicating that allylamine attachment is not to the flavin coenzyme but rather to an active site amino acid residue. The adduct also was stable to base and, to a lesser degree, acid treatment. Although allylamine and N-cyclopropylbenzylamine appear to be oxidized by monoamine oxidase to give 3-(amino acid residue) propanal adducts, two different amino acids seem to be involved because of a difference in stability of the adducts. The mechanisms for inactivation of monoamine oxidase by allylamine and reactivation by benzylamine are discussed in relation to previously reported results.

Synthesis of a biologically active fluorescent muramyl dipeptide congener.

A fluorescent-labeled muramyl dipeptide (MDP) has been prepared to probe immunoadjuvant cellular interactions. N-Acetylmuramyl-L-alanyl-D-isoglutamine (1) was synthesized in improved yield and reacted with 2-(fluoresceinylamino)-4,6-dichloro-s-triazine (DTAF, 2) to give the fluorescent adduct DTAF-MDP (3), attached through the 6-position of the sugar moiety. Adjuvant activity was assessed by using two different in vitro assays, macrophage spreading, and inhibition of macrophage migration. Both assays indicated that the apparent adjuvant activity of 3 is comparable to that of 1.