Inhibition of Aminopeptidase and Acetylcholinesterase by Puromycin and Puromycin Analogs

Abstract: Puromycin analogs in which the O -methyl-L-tyrosine moiety was substituted by a number of amino acids were examined as inhibitors of the puromycin-sensitive rat brain aminopeptidase and bovine erythrocyte acetyl-cholinesterase. In the case of the aminopeptidase, the structure and stereochemistry of the amino acid substituent were important factors in determining inhibitor effectiveness. In the case of the acetylcholinesterase reaction, the aminonucleoside of puromycin was nearly as effective an inhibitor as puromycin itself, with little effect dependent on the nature or stereochemistry of the amino acid.     

Origin of amino acid homochirality: relationship with the RNA world and origin of tRNA aminoacylation

The origin of homochirality of l-amino acids has long been a mystery. Aminoacylation of tRNA might have provided chiral selectivity, since it is the first process encountered by amino acids and RNA. An RNA minihelix (progenitor of the modern tRNA) was aminoacylated by an aminoacyl phosphate oligonucleotide that exhibited a clear preference for l- as opposed to d-amino acids. A mirror-image RNA system with l-ribose exhibited the opposite selectivity, i.e., it exhibited an apparent preference for the d-amino acid. The selectivity for l-amino acids is based on the stereochemistry of RNA. The side chain of d-amino acids is located much closer to the terminal adenosine of the minihelix, causing them collide and interfere during the amino acid-transfer step. These results suggest that the putative RNA world that preceded the protein theatre determined the homochirality of l-amino acids through tRNA aminoacylation.     

Conformational relationships between amino acids and their anticodons in the primitive decoding system

Detailed calculations of the conformational characteristics of a primitive decoding system are presented. A penta-nucleotide serves as the primitive tRNA (PIT) with a triplet of primitive anticodon (PAC) in a helical conformation. This molecular moiety has a cleft in the middle. An amino acid can comfortably nestle into the cleft. The conformation of this molecular association is stabilised by a few hydrogen bonds. The stereochemistry of the moiety restricts the conformational possibilities and the sidechain of the amino acid gets oriented at a proper position and in the correct direction to interact intimately with the PAC in the middle of the PIT. The model favours L-amino acids for beta-D-ribonucleotides. The location of the sidechain of the amino acid in the PIT gives a raison d'être for the important features of the organisation of nucleotide triplets for amino acids in the Genetic Code. The interaction of a few key amino acids with the different combinations of bases as PAC sequences has been studied and the stereochemical basis for the selection of the anticodons for amino acids is elucidated.     

Vascular activity of polycations and basic amino acids: L-arginine does not specifically elicit endothelium-dependent relaxation

Irrespective of their stereochemistry (D- or L-form), polycations such as poly-lysine, poly-arginine and poly-histidine elicited endothelium dependent relaxation of pre-contracted rat aortic rings in a dose-dependent manner (ED50 less than or equal to 10-7 M). In contrast, the basic amino acids arginine, glutamine, histidine and lysine caused only endothelium-potentiated relaxation at high concentrations (ED 50 greater than 10-3 M). Both heparin (1U/ml) and dextran sulphate (10 microgram/ml) abolished relaxation by the polycations but had no effect on the responses to the basic amino acids or acetylcholine. These results indicate that the vasodilatory property of the polycations is due to an electrostatic interaction with anionic domains on the endothelial surface, whereas the basic amino acids elicit a non-specific relaxation. Therefore, L-arginine per se cannot be the immediate precursor of nitric oxide, the proposed endothelium-derived relaxing factor.     

Stereochemical origins of the genetic code

The origin of the genetic code may be attributed to a postulated prebiological stereochemistry in which amino acid dimers, the trans -R,R'-diketopiperazines, interacted with prototype codon and anticodon nucleotide sequences. An intricately coupled stereochemistry is formulated which displays a binary logic for amino acid-codon recognition. It is shown that the diketopiperazine ring system can be inserted between any terminal pair of base paired nucleotides in a codon-anticodon structure with exact registration of complementary hydrogen bonding functional groups. This yields a codon-dimer-anticodon structure in which each amino acid residue is projected towards and interacts with a particular sequence of vicinal nucleotides on either codon or anticodon. The projection direction and the sequence of nucleotides encountered is a strongly coupled function of the choice of codon terminal nucleotide and the handedness of the amino acid. The reciprocal chemical nature of the complementary base pairs drives the selection of dimers containing quite dissimilar and chirally opposed amino acids. Application of the stereochemical model to the in vivo system leads to a general correlation for amino acid-codon assignments. The genetic code is restated in terms of the dimers selected. The profound symmetry of the code is elucidated and this proves useful for correlative and predictive purposes.     

Synthesis of octahydropyrano[3,2-b]pyrrole-2-carboxylic acid derivatives from D-mannose

Bicyclic amino acids are useful building blocks in synthesizing biologically active molecules and peptidomimetics. 2-Carboxy-6-hydroxyloctahydroindole (Choi) is a novel bicyclic amino acid found in the marine natural products aeruginosins. Many compounds in the aeruginosin family exhibit inhibition activities toward serine proteases including thrombin and trypsin. The unique Choi structure is the common feature of this family of oligopeptides and this motif is important for their observed biological activities. To better understand the influence of the stereochemistry of the Choi core structure on the inhibition activities, we have previously synthesized ring-oxygenated variants from glucose. The preparation of octahydro-pyrano[3,2-b]pyrrole 2-carboxylic acids from d-mannose is reported here. These novel bicyclic amino acids can be used in the preparation of aeruginosin analogs, as well as conformationally constrained peptidomimetics or other biologically active molecules.     

Nakijiquinones G-I, new sesquiterpenoid quinones from marine sponge

Three new sesquiterpenoid quinones, nakijiquinones G-I (1-3), containing a different amino group derived from amino acids have been isolated from Okinawan marine sponges of the family Spongiidae, and the structures and relative stereochemistry of 1-3 were elucidated on the basis of the spectral data. Nakijiquinones G-I (1-3) showed modest cytotoxicity and inhibitory activity against HER2 kinase, while nakijiquinone H (2) exhibited antimicrobial activity.     

Peptido-mimetic Approach in the Design of Syndiotactic Antimicrobial Peptides

Biocompatibility, low toxicity and high selectivity towards bacterial cells has been the hallmark of peptide-based antibiotics. The innate immune system has been employing such molecular systems against invading pathogens as a successful defense strategy. In this study, we attempt to develop topologically constrained antimicrobial peptides with syndiotactic stereochemical arrangement, by incorporating L and D amino acids successively in its amino acid sequence. Acetylated versions of the designed peptides were also examined for its influence on bactericidal potency, against Gram-positive and Gram-negative bacteria. Syndiotactic stereochemical arrangement of the polypeptide main chain mimics stereochemistry of Gramicidin, a naturally occurring antimicrobial peptides. Gramicidin is a class of penta-deca-peptides isolated from soil bacteria Bacillus brevis, but their utility as antibiotic was limited to topical use due to high levels of hemotoxicity. Activity profiles of the four de novo designed peptide variants show higher specificity towards Gram-positive bacteria than Gram-negative variants, matching earlier reports on the therapeutic potential of gramicidin as a broad spectrum antibiotic. Significantly, our hemolytic assay confirms very low (<1%) levels of toxicity for the designed peptides unlike gramicidin. Earlier reports confirm that incorporation of D amino acids effectively negates the possibility of proteolytic degradation, thus pointing to the potential utility of de novo designed peptides with diversified stereochemistry as a promising new approach in the generation of novel antibiotic peptides.     

Solid-phase synthesis of functionalized bis-peptides

We demonstrate the first solid-phase synthesis of highly functionalized bis-peptides. Bis-peptides are ladder oligomers composed of stereochemically pure, cyclic bis-amino acids joined by substituted diketopiperazine linkages. They have a shape-programmable backbone that is controlled by controlling the stereochemistry and sequence of the monomers within each oligomer. Functionalized bis-peptides are assembled using a new amide bond forming reaction (acyl-transfer coupling) that we have previously developed and a novel activation strategy that allows the sequential formation of penta- and hexa-substituted diketopiperazines from extremely hindered N-alkyl-alpha,alpha-disubstituted amino acids. We present mechanistic evidence that acyl-transfer coupling is competitive with direct acylation in the formation of hindered amide bonds. We also detail the synthesis of four functionalized bis-peptides, and that by combining bis-peptides with amino acids through diketopiperazine linkages, bis-peptides can mimic the display of residues i, i+4, i+7 of an alpha-helical peptide.     

Synthesis and activity of analogues of the isoleucyl tRNA synthetase inhibitor SB-203207

Twenty two analogues of SB-203207 have been prepared by total synthesis, and evaluated as inhibitors of a range of tRNA synthetases. Changes to the bicyclic core, removing either the terminal amino substituent or the sulfonyl group from the side chain, and altering either the carbon skeleton or stereochemistry of the isoleucine residue, decreases the potency of inhibition of isoleucyl tRNA synthetase. Substituting the isoleucine residue with other amino acids produces inhibitors of the corresponding synthetases. In particular, a methionine derivative is 50-100 times more potent against methionyl tRNA synthetase than against any of the corresponding isoleucyl, leucyl, valyl, alanyl and prolyl synthetases.     

Paecilopeptin,a new cathepsin S inhibitor produced by Paecilomyces carneus

Paecilopeptin, a novel cathepsin S inhibitor, was produced and isolated from the culture supernatant of the fungal strain, Paecilomyces carneus. A spectroscopic analysis revealed the planar structure of paecilopeptin to be acetyl-Leu-Val-CHO. The stereochemistry of the constituent amino acids was analysed by chiral HPLC after oxidation and 6N HCl hydrolysis of paecilopeptin. The total synthesis of paecilopeptin was completed in six steps. Paecilopeptin inhibited human cathepsin S with an IC50 value of 2.1 nM in vitro.     

Isolation and structure determination of a new lasso peptide specialicin based on genome mining

Based on genome mining, a new lasso peptide specialicin was isolated from the extract of Streptomyces specialis. The structure of specialicin was established by ESI-MS and NMR analyses to be a lasso peptide with the length of 21 amino acids, containing an isopeptide bond and two disulfide bonds in the molecule. The stereochemistries of the constituent amino acids except for Trp were determined to be L and the stereochemistry of Trp at C-terminus was determined to be D. Three dimensional structure of specialicin was determined based on NOE experimental data, which indicated that specialicin possessed the similar conformational structure with siamycin I. Specialicin showed the antibacterial activity against Micrococcus luteus and the moderate anti-HIV activity against HIV-1 NL4-3. The biosynthetic gene cluster of specialicin was proposed from the genome sequence data of S. specialis.     

Mirrors in the PDB: left-handed α-turns guide design with D-amino acids

Background  

Incorporating variable amino acid stereochemistry in molecular design has the potential to improve existing protein stability and create new topologies inaccessible to homochiral molecules. The Protein Data Bank has been a reliable, rich source of information on molecular interactions and their role in protein stability and structure. D-amino acids rarely occur naturally, making it difficult to infer general rules for how they would be tolerated in proteins through an analysis of existing protein structures. However, protein elements containing short left-handed turns and helices turn out to contain useful information. Molecular mechanisms used in proteins to stabilize left-handed elements by L-amino acids are structurally enantiomeric to potential synthetic strategies for stabilizing right-handed elements with D-amino acids.     

Di(1‐naphthyl) methanol ester of carboxylic acids for absolute stereochemical determination

The absolute stereochemistry of chiral carboxylic acids is determined as a di(1‐naphthyl)methanol ester derivative. Computational scoring of conformations favoring either P or M helicity of the naphthyl groups, capable of exciton‐coupled circular dichroic coupling, leads to a predicted stereochemistry for the derivatized carboxylic acids.     

Unguisin C,a GABA-containing cyclic peptide from the fungus Emericella unguis

Besides the known unguisins A and B, a new cyclic heptapeptide, unguisin C, containing a GABA-derived moiety in the ring, was isolated from the fungus Emericella unguis. The structure was determined by 1D and 2D NMR techniques. Marfey's method was used to determine the absolute stereochemistry. Precursor-directed biosynthesis of the unguisins was performed by supplementation of the culture medium with amino acids (L-Ala, L-Ser, L-Phe and L-Leu). A related cyclic heptapeptide, unguisin D, was detected by HPLC and characterized by sequence analysis using LC-QITMS.     

Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to alpha-carbon cross-links: formation and reduction of alpha-thio-alpha-amino acid derivatives

The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled [(13)C,(15)N]medium derived from Anabaena sp. grown on sodium [(13)C]bicarbonate and [(15)N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-(13)C,(15)N]-L-phenylalanine and [U-(13)C,(15)N]-L-threonine using otherwise unlabeled media. The results demonstrate that in addition to having a cyclized peptide backbone (amide between N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively. The stereochemistry of all residues in 1 except for the three modified ones was confirmed to be L by complete desulfurization with nickel boride, acid hydrolysis to the constituent amino acids, and conversion of these to the corresponding pentafluoropropanamide isopropyl esters for chiral GC MS analysis. The stereochemistry at the modified residues was determined by subjecting each of the eight possible stereoisomers of 1 to eight rounds of ARIA structure calculations, starting with the same NMR peak files and assignments. The stereoisomer with the l stereochemistry at Phe22 (alpha-R) and d stereochemistry at Thr28 (alpha-S) and Phe31 (alpha-S) (LDD isomer) fit the NMR data, giving the lowest energy family of structures with the best rmsd. Thus, biochemical formation of the unusual thio links proceeds with net retention of configuration at Phe22, and inversion at Thr28 and Phe31. Model amino acid derivatives bearing a sulfide moiety at the alpha-carbon were synthesized by reaction of the corresponding alpha-alkoxy compounds with benzyl thiol and SnCl(4). Separation of their pure stereoisomers and desulfurization with nickel boride demonstrated that the reduction of such compounds proceeds with epimerization, in contrast to the previously reported retention of stereochemistry for analogous reaction of steroidal sulfides. However, desulfurization of subtilosin A to cyclic peptide 14, which is inactive as an antimicrobial agent, occurs with inversion of stereochemistry at the alpha-carbons of Phe22 and Thr28 and with 4:1 retention at Phe31. This indicates that the desulfurization reaction proceeds via an N-acyl imine and that the structure of the surrounding peptide controls the geometry of reduction. Posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is unprecedented in ribosomally synthesized peptides or proteins, and very rare in secondary metabolites. Subtilosin A (1) represents a new class of bacteriocins.     

Use of Marfey's reagent and analogs for chiral amino acid analysis: assessment and applications to natural products and biological systems

The present paper describes an updated knowledge and status on Marfey's reagent (MR), 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDNP-L-Ala-NH(2)). The reagent is used for pre-column derivatization of amino acids followed by HPLC separation of the diastereomers so formed. Emphasis is put on the design and application of structural variants which are synthesized by introducing different (other than L-Ala-NH(2)) L- and D-amino acid amides and amino acids in the 1,5-difluoro-2,4-dinitro benzene (DFDNB) moiety, as the chiral auxiliary. Advantages, disadvantages, the required precautions and suitability of the approach for the separation of multi component mixtures of DL-amino acids are assessed. Use of two dimensional (2D) techniques, in particular online HPLC in combination with various mass spectrometry techniques is discussed as well as methods designated 'advanced Marfey's method' and 'C(3) Marfey's method'. Application of MR and its variants for the determination of the stereochemistry of protein and non-protein amino acids in bioactive natural products isolated from living organisms (bacteria including blue-green algae, filamentous fungi, plants, marine sponges, invertebrates and vertebrates), in physiological samples including human beings, and in biologically relevant synthetic peptides are presented. In an outlook future applications are envisaged.     

A stereochemical switch in the aDrs model system, a candidate for a functional amyloid

Amyloid fibrils are commonly observed to adopt multiple distinct morphologies, which eventually can have significantly different neurotoxicities, as e.g. demonstrated in case of the Alzheimer peptide. The architecture of amyloid deposits is apparently also determined by the stereochemistry of amino acids. Post-translational changes of the chirality of certain residues may thus be a factor in controlling the formation of functional or disease-related amyloids. Anionic dermaseptin (aDrs), an unusual peptide from the skin secretions of the frog Pachymedusa dacnicolor, assembles to amyloid-like fibrils in a pH-dependent manner, which could play a functional role in defense. aDrs can be enzymatically converted into the diastereomer [d-Leu2]-aDrs by an l/d-isomerase. EM and AFM on fibrils formed by these isomers have shown that their predominant morphology is controlled by the stereochemistry of residue 2, whereas kinetic and thermodynamic parameters of aggregation are barely affected. When fibrils were grown from preformed seeds, backbone stereochemistry rather than templating-effects apparently dominated the superstructural organization of the isomers. Interestingly, MD indicated small differences in the conformational propensities between the isomers. Our results demonstrate how d-amino acid substitutions could take active part in the formation of functional or disease-related amyloid. Moreover, these findings contribute to the development of amyloid-based nanomaterials.     

On the Structural Regularity in Nucleobases and Amino Acids and Relationship to the Origin and Evolution of the Genetic Code

To explore how chemical structures of both nucleobases and amino acids may have played a role in shaping the genetic code, numbers of sp² hybrid nitrogen atoms in nucleobases were taken as a determinative measure for empirical stereo-electronic property to analyze the genetic code. Results revealed that amino acid hydropathy correlates strongly with the sp² nitrogen atom numbers in nucleobases rather than with the overall electronic property such as redox potentials of the bases, reflecting that stereo-electronic property of bases may play a role. In the rearranged code, five simple but stereo-structurally distinctive amino acids (Gly, Pro, Val, Thr and Ala) and their codon quartets form a crossed intersection “core”. Secondly, a re-categorization of the amino acids according to their β-carbon stereochemistry, verified by charge density (at β-carbon) calculation, results in five groups of stereo-structurally distinctive amino acids, the group leaders of which are Gly, Pro, Val, Thr and Ala, remarkably overlapping the above “core”. These two lines of independent observations provide empirical arguments for a contention that a seemingly “frozen” “core” could have formed at a certain evolutionary stage. The possible existence of this codon “core” is in conformity with a previous evolutionary model whereby stereochemical interactions may have shaped the code. Moreover, the genetic code listed in UCGA succession together with this codon “core” has recently facilitated an identification of the unprecedented icosikaioctagon symmetry and bi-pyramidal nature of the genetic code.     

Unguisin C, a GABA-containing cyclic peptide from the fungus Emericella unguis

Besides the known unguisins A and B, a new cyclic heptapeptide, unguisin C, containing a GABA-derived moiety in the ring, was isolated from the fungus Emericella unguis. The structure was determined by 1D and 2D NMR techniques. Marfey's method was used to determine the absolute stereochemistry. Precursor-directed biosynthesis of the unguisins was performed by supplementation of the culture medium with amino acids ( -Ala, -Ser, -Phe and -Leu). A related cyclic heptapeptide, unguisin D, was detected by HPLC and characterized by sequence analysis using LC-QITMS.