Peptidyl Esters Research Articles

RNA-directed peptide synthesis across a nicked loop.

Ribosomal translation at the origin of life requires controlled aminoacylation to produce mono-aminoacyl esters of tRNAs. Herein, we show that transient annealing of short RNA oligo:amino acid mixed anhydrides to an acceptor strand enables the sequential transfer of aminoacyl residues to the diol of an overhang, first forming aminoacyl esters then peptidyl esters. Using N-protected aminoacyl esters prevents unwanted peptidyl ester formation in this manner. However, N-acyl-aminoacyl transfer is not stereoselective.

Synthesis and evaluation of peptidyl α,β-unsaturated carbonyl derivatives as anti-malarial calpain inhibitors

Malarial calpain is a cysteine protease believed to be a central mediator essential for parasitic activities. N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN), a calpain inhibitor, showed an excellent inhibitory effect on the erythrocytic stages of Plasmodium falciparum. However the aldehyde group of ALLN makes it susceptible to metabolism. Therefore, we designed α,β-unsaturated carbonyl peptides that could serve as electrophiles for cysteine residues in calpain. Among the synthetic analogs based on the structure of ALLN, peptidyl esters 7, 8 and 9 showed the most potent anti-malarial effects, with the same IC50 values of 5.0 μM. Also they showed the high selective toxicity for the malaria versus Hela cell with 40.6, 69.2 and 24.3 fold for 7, 8 and 9, respectively. Dipeptidyl α,β-unsaturated carbonyl derivatives consisting of two amino acids gave better anti-malarial effects than those consisting with one amino acid. The fluctuation in anti-malarial activity with small changes in chemical structure indicates the possibilities of improving synthetic analogs.

Direct crystallographic observation of an acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of a peptidyl ester substrate: Exploiting the “glass transition” in protein dynamics

The crystal structure of the acyl complex of porcine pancreatic elastase with its peptidyl ester substrate N-acetyl-ala-ala-ala-methyl ester (Ac(Ala) 3OMe) has been determined at 2.5 Å resolution. The complex was stabilized by exploiting the “glass transition” in protein dynamics that occurs at around −53 °C (220 K). Substrate was flowed into the crystal in a cryoprotective solvent above this temperature, and then the crystal was rapidly cooled to a temperature below the transition to trap the species that formed. The use of a flow cell makes the experiment a kinetic one and means that the species prior to the rate determining transition state has a chance to accumulate. The resulting crystal structure shows an acyl-enzyme intermediate in which the leaving group is absent and the carbonyl carbon of the C-terminal alanine residue is covalently bound to the gamma oxygen of the active site serine. The ester carbonyl shows no significant distortion from planarity, with the carbonyl oxygen forming one hydrogen bond with the oxyanion hole. The tripeptide is bound in an extended antiparallel β-sheet with main chain residues of the enzyme. The geometry and interactions of this acyl-enzyme suggest that it represents a productive intermediate. To test this hypothesis, the same crystal was then warmed above the glass transition temperature and a second data set was collected. The resulting electron density map shows no sign of the substrate, indicating hydrolysis of the intermediate followed by product release. This experiment provides direct evidence for the importance of dynamic properties in catalysis and also provides a blueprint for the stabilization of other short-lived species for direct crystallographic observation.

Sphingolipids as Coenzymes in Anion Transfer and Tumor Death

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Sphingolipids as coenzymes in anion transfer and tumor death

Many kinds of natural sphingolipids and their analogs stimulate or inhibit a wide assortment of biochemical phenomena and enzymes. The puzzle considered here is: how can these lipids control so many different kinds of processes? In almost every study in which a structural comparison was made, an allylic alcohol moiety [–CHCH–CH(OH)–] was found to be an essential feature of the sphingolipid. Many of those stimulations lead to cell death, emphasizing the importance of allylic sphingolipid structure in the design of chemotherapeutic agents. The proposal offered here is that these lipids function as coenzymes, in which the allylic moiety acts as an anion transferring agent, forming transient phosphate or acyl or peptidyl esters for the synthesis or hydrolysis of phosphoproteins, proteins, and phospholipids. Sphingolipids that inhibit these reactions may simply displace the active sphingolipids from their sites in the enzymes’ active regions, or bind to the enzymes’ allosteric region. This kind of competition could act as a major homeostatic control mechanism. Some of the allylic sphingolipids also generate reactive oxygen, possibly by oxidation of the allylic alcohol group. This explains the need to control redox-controlling metabolites in sphingolipid-controlled processes (e.g., glutathione). Many anticancer drugs that produce apoptosis in tumors possess an allylic alcohol residue, affect protein phosphorylation, and produce reactive oxygen species. They may be therapeutically useful because they control the action of sphingolipids as anion transfer agonists or inhibitors.

Mechanism-based inactivation of α-chymotrypsin

The peptidyl ester derivatives of 2,2-dichlorocyclopropanol and the amide derivative of 2,2-dichlorocyclopropylamine were prepred as novel mechanism-based inactivators of α-chymotrypsin. The esters inactivated α-chymotrypsin irreversibly but the amide did not show any irreversible inhibitory activity toward α-chymotrypsin.

Stereoselective formation of bis(α-aminoacyl) esters of 5′-AMP suggests a primitive peptide synthesizing system with a preference for l-amino acids

In the biosynthesis of proteins, each amino acid passes from the aminoacyl adenylate to become an amino acid ester and finally a 2′ (3′) peptidyl ester of the AMP residue at the end of a tRNA. Consequently, the chemistry of protein synthesis is the chemistry of aminoacyl and peptidyl AMP. Our data has revealed properties of 5′-AMP and its esters which should allow the preferential catalytic synthesis of l-amino acid peptides via a bis(2′, 3′-aminoacyl) ester intermediate. Results in this paper concern one step in the proposed process and show that preexisting Ac- l-Phe monoester reacts about 2.5-times faster to form diester than preexisting Ac- d-Phe monoester.

Reactions of Escherichia coli TEM beta-lactamase with cephalothin and with C10-dipeptidyl cephalosporin esters.

Two novel C10-(dipeptidyl)cephalosporin esters (3-(beta-chloro-L-alanyl-beta-chloro-L-alanyloxymethyl)-7 beta-(2-thienylacetamido)-3-cephem-4-carboxylic acid (7) and sodium 3-(L-alanyl-L-alanyloxymethyl)-7 beta-(2-thienylacetamido)-3-cephem-4-carboxylate, toluene-sulfonic acid salt (18] were synthesized, and their reactions with Escherichia coli TEM beta-lactamase were examined. Kinetic parameters determined for the enzymatic reactions of 7 (Km = 0.32 mM; Vmax = 338 mumol min-1 (mg protein)-1) and of 18 (Km = 0.33 mM, Vmax = 338 mumol min-1 (mg protein)-1) demonstrate that both of the peptidyl esters are good substrates for the lactamase. In fact, the Vmax rates for 7 and 18 are each more than 4-fold greater than that obtained for cephalothin, 1 (Vmax = 78 mumol min-1 (mg protein)-1), a well characterized substrate for the lactamases. Analysis of the enzymatic reactions by high field (500 MHz) 1H NMR revealed similar patterns for fragmentation of the cephem nucleus of 1, 7, and 18. However, while hydrolysis of 1 produces acetate, cleavage of 7 and 18 releases beta Cl-LAla-beta Cl-LAla and LAla-LAla, respectively, from the dipeptidyl cephalosporin esters. Based on these findings, a strategy for co-opting the beta-lactamases of Gram-negative bacteria for "delivery" of bactericidal agents is described, and an explanation for the previously reported (Mobashery, S., Lerner, S.A., and Johnston, M. (1986) J. Am. Chem. Soc. 108, 1685) antibacterial activity of 7 is offered.

New indolyl peptidyl esters as MAO inhibitors

Substituted indole-3-peptidyl esters were synthesized and were characterized. The compounds were studied for their effect on the monoamine oxidase activity both in vitro as well as in vivo and compared with known MAO inhibitors such as tranylcypromine. Approximate lethal dose (ALD 50) of the compounds was also determined.

Amino acid esters of 9-(2?,3?-dihydroxypropyl-1?)-adenine are the specific inhibitors of protein synthesis on ribosomes

Peptide acceptor properties of phenylalanine and glycine esters of 9'-(2',3'-dihydroxypropyl-1')-adenine and 1-(2',3'-dihydroxypropyl-1')-4-thiouracyl were investigated. All these esters appeared to be powerful inhibitors of polyphenylalanine synthesis in E. coli MRE-600 ribosomes charged with poly U. Like puromycin, esters of adenine derivatives accepted the AcPhe residue from Ac-[(14)C] Phe-tRNA in a ribosomal system charged with poly U. However, peptidyl esters of 9-(2',3'-dihydroxypropyl-1')-adenine remained bound with ribosomes. The structure of the peptide esters synthesized was ascertained after dissociation of ribosomes into subparticles by direct comparison with the synthetic specimens.

Renal dipeptidase: Localization and inhibition

The principle locus of porcine renal dipeptidase activity has been found to be the microsomal fraction of kidney cortex. When the microsomal preparation was incubated with the enzymes, chymotrypsin, trypsin, lipase, and ribonuclease, the peptidase was released to no greater degree than other protein components of this fraction. Treatment of the microsomal fraction with deoxycholate and sodium dodecyl sulfate led to the identification of the dipeptidase within the resulting ribosomal fraction. Relatively large-scale isolation methods applied to the preparation of renal dipeptidase resulted in an enzyme of purity comparable to that reported in previous studies. The molecular weight of the purified peptidase was estimated by gel filtration on Sephadex G-200 to be approximately 90 000. The enzyme was shown to be competitively inhibited by phosphate and phosphate esters, and the inhibition demonstrated to be instantaneous and completely reversible upon dilution. A comparison of K i values for the adenine series indicated that the effectiveness of inhibition was in the order ATP > ADP > AMP > inorganic phosphate. It is suggested that the phosphate inhibition occurs as a result of the formation of a ternary (peptidase-Zn-inhibitor) complex at the active site of the enzyme. Inhibition of renal dipeptidase by 1,10-phenanthroline was measurably time dependent and could not be completely reversed by dilution or by the addition of excess zinc ions. However, removal of chelating agent followed by dialysis against zinc containing buffers led to reversal of inhibition. It seems likely that the inhibitory effect of 1,10-phenanthroline is caused by the removal of zinc from the active site of the peptidase to produce an inactive apoenzyme. The native enzyme can be reconstituted with additional zinc to restore full activity. When the rate of peptidase-catalyzed hydrolysis of dipeptides was measured in the presence of the free amino acid components, inhibition of hydrolysis was clearly demonstrated. The amino acids with bulkier side chains were the more effective inhibitors regardless of their positions as N-terminal or C-terminal components of the substrate. There was observed a lack of esterase activity against amino acid and peptidyl esters indicating that the free carboxyl group is required for renal dipeptidase activity.

A ribonucleic acid-iodo-proteinaceous complex from the thyroid

1. RNA-iodo-proteinaceous complexes occur in the tRNA fractions isolated from bovine, ovine, and porcine thyroids. The calf thyroid complex has been partially characterized and contains diiodotyrosine and probably the hormone thyroxine in addition to several other amino acids. 2. Different preparative procedures and techniques capable of disrupting a variety of non-covalent bonds and aminoacyl and peptidyl ester bonds had no obvious effect on the calf thyroid RNA-iodo-proteinaceous complex which remained associated with tRNA. 3. Nucleolytic enzymes can degrade the complex to lower molecular weight products that contain iodine, proteinaceous, and RNA components. However, most of the iodine and proteinaceous material is associated with a relatively high molecular weight, ribonuclease-resistant, RNA-containing core. 4. It is concluded that the tRNA fractions of bovine and probably other mammalian thyroid glands contain a low molecular weight RNA linked to proteinaceous material that contains iodinated amino acids.