The carboxyl methylation of aspartyl residues in eledoisin and tetragastrin by rabbit erythrocyte aspartyl β-carboxyl methyltransferase

Rabbit erythrocytes contain a soluble aspartyl β-carboxyl methyltransferase capable of specifically carboxyl methylating the β-carboxyl group of an internal aspartyl residue in the synthetic polypeptide eledoisin, a hypotensively active peptide from the cephalopodsEledone moschata andE. aldrovandi, and tetragastrin, the biologically active C-terminal tetrapeptide of human gastrin. However, the aspartyl residue in delta sleep-inducing peptide (DSIP) could not be carboxyl methylated, nor could glutamyl residues in any polypeptide tested.     

Potent transglutaminase inhibitors,aryl β-aminoethyl ketones

Aryl β-aminoethyl ketones were discovered as potent inhibitors of tissue transglutaminase. Heteroaryl-like thiophene groups and N-benzyl N-t-butyl aminoethyl group are critical to the strong inhibitory activity of aryl β-aminoethyl ketones.     

Potent transglutaminase inhibitors, dithio β-aminoethyl ketones

Potent transglutaminase inhibitors were obtained from disulfide compounds, cystamine, dimethyl cystine, and dimethyl homocystine. The disulfide bond and thiophene ring play an important role in inhibitory activity of synthesized aryl β-amino ketones.     

HIV aspartyl protease inhibitors as promising compounds against Candida albicans André Luis Souza dos Santos

Cells of Candida albicans (C. albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated mycoses of almost all inner organs, especially in immunocompromised patients. In this context, both the host immune status and the ability of C. albicans to modulate the expression of its virulence factors are relevant aspects that drive the candidal susceptibility or resistance; in this last case, culminating in the establishment of successful infection known as candidiasis. C. albicans possesses a potent armamentarium consisting of several virulence molecules that help the fungal cells to escape of the host immune responses. There is no doubt that the secretion of aspartyl-type proteases, designated as Saps, are one of the major virulence attributes produced by C. albicans cells, since these hydrolytic enzymes participate in a wide range of fungal physiological processes as well as in different facets of the fungal-host interactions. For these reasons, Saps clearly hold promise as new potential drug targets. Corroborating this hypothesis, the introduction of new anti-human immunodeficiency virus drugs of the aspartyl protease inhibitor-type (HIV PIs) have emerged as new agents for the inhibition of Saps. The introduction of HIV PIs has revolutionized the treatment of HIV disease, reducing opportunistic infections, especially candidiasis. The attenuation of candidal infections in HIV-infected individuals might not solely have resulted from improved immunological status, but also as a result of direct inhibition of C. albicans Saps. In this article, we review updates on the beneficial effects of HIV PIs against the human fungal pathogen C. albicans, focusing on the effects of these compounds on Sap activity, growth behavior, morphological architecture, cellular differentiation, fungal adhesion to animal cells and abiotic materials, modulation of virulence factors, experimental candidiasis infection, and their synergistic actions with classical antifungal agents.     

Mutagen production by chlorination of methylated α,β-unsaturated ketones

Mesityl oxide and isophorone, two β-methylated-α,β-unsaturated industrial solvent ketones, were found to be converted to mutagens by aqueous chlorination under conditions of pH and reactant concentration that may be relevant to waste water and drinking water chlorination. Chlorination of millimolar concentrations of isophorone generated mutagens at a pH as low as 8.5, while mutagens were formed from submillimolar concentrations of mesityl oxide at pH 8.5, or millimolar concentrations at pH 7.5. It is suggested that mutagen formation can occur via a haloform reaction at such low pH levels because of extended resonance stabilization of an intermediate carbanion.     

Molecular modelling and comparative structural account of aspartyl β-semialdehyde dehydrogenase of Mycobacterium tuberculosis (H37Rv)

Aspartyl β-semialdehyde dehydrogenase (ASADH) is an important enzyme, occupying the first branch position of the biosynthetic pathway of the aspartate family of amino acids in bacteria, fungi and higher plants. It catalyses reversible dephosphorylation of l-β-aspartyl phosphate (βAP) to l-aspartate-β-semialdehyde (ASA), a key intermediate in the biosynthesis of diaminopimelic acid (DAP)—an essential component of cross linkages in bacterial cell walls. Since the aspartate pathway is unique to plants and bacteria, and ASADH is the key enzyme in this pathway, it becomes an attractive target for antimicrobial agent development. Therefore, with the objective of deducing comparative structural models, we have described a molecular model emphasizing the uniqueness of ASADH from Mycobacterium tuberculosis (H37Rv) that should generate insights into the structural distinctiveness of this protein as compared to structurally resolved ASADH from other bacterial species. We find that mtASADH exhibits structural features common to bacterial ASADH, while other structural motifs are not present. Structural analysis of various domains in mtASADH reveals structural conservation among all bacterial ASADH proteins. The results suggest that the probable mechanism of action of the mtASADH enzyme might be same as that of other bacterial ASADH. Analysis of the structure of mtASADH will shed light on its mechanism of action and may help in designing suitable antagonists against this enzyme that could control the growth of Mycobacterium tuberculosis. Anupama Singh and Hemant R. Kushwaha contributed equally to this work.     

Chemoenzymatic resolution of racemic Wieland–Miescher and Hajos–Parrish ketones

Enantiospecific microbial reduction of bicyclic ketones was described. Racemic Wieland–Miescher (1) and Hajos–Parrish (2) ketones were used as substrates. In a 4-h biotransformation of Hajos–Parrish ketone (2) using the strain of Didymosphaeria igniaria an optically pure ketone (R)-2 was obtained, whereas the (S)-2 ketone underwent reduction to (4aS,5S)-4 alcohol with 100% of enantiomeric excess and with over 60% of diastereoisomeric excess. Jones oxidation of the alcohol obtained in the biotransformation gave an optically pure ketone (S)-2. Enzymatic system of Coryneum betulinum reduced the (R)-2 ketone to (4aR,5S)-4 alcohol with a high enantiomerical purity in a 6-day reaction. Wieland-Miescher (1) ketone was transformed by these microorganisms in an analogous way, but the reaction times were longer.     

Determination of rate constants for β-linkage isomerization of three specific aspartyl residues in recombinant human αA-crystallin protein by reversed-phase HPLC

The major soluble eye lens protein, αA-crystallin, has a very long half-life. Thus, many post-translational modifications, including stereoinversion, have been found in its constituent amino acids. We determine the rates of β-linkage isomerization, which is the main reaction through the formation of a succinimide intermediate, of specific Asp residues of recombinant human αA-crystallin protein by simple RP-HPLC method. Kinetic analyses of the β-linkage isomerization were performed on the three Asp residues of αA-crystallin, (58)Asp, (84)Asp, and (151)Asp, because the d/l ratios of both the (58)Asp and (151)Asp residues were higher than 1.0 in the αA-crystallin isolated from aged human eye lens. The β-linkage isomerizations of both the (58)Asp and (84)Asp residues were suppressed in the recombinant protein by approximately 0.4-0.5 times compared to those in the synthetic peptide below 50 °C, whereas the isomerization of the (151)Asp residue occurred at the same rate for the whole protein and synthetic fragmentary peptide. The suppression of (58)Asp isomerization in the recombinant protein relaxed to some extent when the αA-crystallin protein was incubated at a high temperature. The far-UV CD spectra showed that the secondary structure of the protein was partially disordered at temperatures greater than 60 °C in the recombinant αA-crystallin protein. These results suggest that the (58)Asp residue was restrained from forming the succinimide intermediate by the higher order structure of the αA-crystallin protein, and that the structural environment around the (151)Asp residue of the αA-crystallin was similar to that of the synthetic fragmentary peptide with respect to succinimide formation. The difference in the influence of the secondary structure of the αA-crystallin protein inverts the order of the succinimide formations of the (58)Asp and (151)Asp residues in the recombinant protein as compared with the order in the synthetic fragmentary peptides.     

4-Aminoethylpiperazinyl aryl ketones with 5-HT₁A/5-HT₇ selectivity

The well-known 5-HT(1A)/5-HT(7) selectivity issue was tackled by a new series of 4-aminoethylpiperazinyl aryl ketones (1a-1l) specifically designed to distinguish the two hydrophobic sites centered at the anchoring salt bridge. The 4-aminoethylpiperazinyl aryl ketones showed a wide spectrum of activity and selectivity for the 5-HT receptors depending on the type of the hydrophobic groups attached at the aryl piperazinyl ketone scaffold. Docking study of the most active compounds against 5-HT(7)R and 5-HT(1A)R revealed that both receptors have two hydrophobic pockets around the anchoring salt bridge. These two binding sites are perpendicular to each other in 5-HT(7)R but parallel in 5-HT(1A)R, and this observation is well matched with the previous report which claimed that 5-HT(7)R affinity arises from bent conformation of the bound ligand whereas an extended one is best suited for 5-HT(1A)R selectivity. Also, as these pockets have different size and shape, inhibitory activity as well as selectivity of the 4-aminoethylpiperazinyl aryl ketones against 5-HT(7)R and 5-HT(1A)R seemed to be determined by combination of two hydrophobic substituents attached at both ends of the title compounds.     

UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of Nε-carboxymethyl lysine (CML) in keratin of skin

UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-β-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-β-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-β-Asp and CML, immunohistochemical analysis using anti-D-β-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-β-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50-70 kDa were immunoreactive towards antibodies for both D-β-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14.     

Pyrrolidine based chiral organocatalyst for efficient asymmetric Michael addition of cyclic ketones to β-nitrostyrenes

An efficient asymmetric Michael addition of cyclic ketones to β-nitrostyrenes using secondary diamine as an organocatalyst derived from l-proline and (R)-α-methylbenzyl amine has been described. This pyrrolidine based catalyst 1 was found to be very effective to synthesize various γ-nitrocarbonyl compounds in good yield (up to 81%) with excellent stereoselectivity (up to >99:1 dr and >99% ee).     

On the microbial transformation of α,β-unsaturated aryl ketones by the fungus Beauveria bassiana

α,β-unsaturated aryl ketones, 1a–12, have been submitted to the action of the fungus Beauveria bassiana (ATCC 7159) in growing conditions. The saturation of the double bond strictly depends from the substituent α to the carbonyl group. The saturated ketone is then oxidised in a Baeyer-Villiger type reaction. This new oxidative capacity of the fungus has been studied and the adaptability of the micro organism towards structural modifications has been investigated.     

Studies on the BF3·Et2O catalyzed Baeyer-Villiger reaction of spiroketalic steroidal ketones

During reactions of 23-oxosapogenins and the corresponding isomeric 22-oxo-23-spiroketals with MCPBA in the presence of BF₃·Et₂O, equilibration occurs between the ketones. The Baeyer-Villiger type oxidation is followed by fragmentation to the dinorcholanic lactones and 3-methylbutyrolactone. The mechanistic aspects of these reactions in the 25R and 25S series are discussed.     

A chiral benzoylthiourea–pyrrolidine catalyst for the highly enantioselective Michael addition of ketones to chalcones

A benzoylthiourea–pyrrolidine catalyst was developed for the asymmetric Michael addition of ketones to chalcones. The corresponding products were obtained in high yields with high level of diastereoselectivities (up to 99:1 dr) and high level of enantioselectivities (up to 94% ee) under mild conditions.     

Bioreduction of α,β-unsaturated ketones and aldehydes by non-conventional yeast (NCY) whole-cells

The bioreduction of α,β-unsaturated ketones (ketoisophorone, 2-methyl- and 3-methyl-cyclopentenone) and aldehydes [(S)-(−)-perillaldehyde and α-methyl-cinnamaldehyde] by 23 “non-conventional” yeasts (NCYs) belonging to 21 species of the genera Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Kazachstania, Kluyveromyces, Lindnera, Nakaseomyces, Vanderwaltozyma, and Wickerhamomyces was reported. The results highlight the potential of NCYs as whole-cell biocatalysts for selective biotransformation of electron-poor alkenes. A few NCYs exhibited extremely high (>90%) or even total ketoisophorone and 2-methyl-cyclopentenone bioconversion yields via asymmetric reduction of the conjugated CC bond catalyzed by enoate reductases. Catalytic efficiency declined after switching from ketones to aldehydes. High chemoselectivity due to low competing carbonyl reductases was also sometimes observed.     

Are endophytic microorganisms involved in the stereoselective reduction of ketones by Daucus carota root?

Four strains of endophytic microorganisms isolated from carrot root were able to carry out the reduction of the carbonyl group with diverse degree of enantio-, and diasteroselectivity. Furthermore, biotransformation in the presence of bacterial inhibitor affects the stereochemical outcome of the reaction, and the concomitant addition of a yeast inhibitor results in a large decrease in the conversion percentage. These results indicate that endophytic microorganisms might be involved in the enantioselective reduction of ketones and ketoesters with fresh carrot root pieces.     

TTC-based screening assay for ω-transaminases: a rapid method to detect reduction of 2-hydroxy ketones

A rapid TTC-based screening assay for ω-transaminases was developed to determine the conversion of substrates with a 2-hydroxy ketone motif. Oxidation of the compounds in the presence of 2,3,5-triphenyltetrazolium chloride (TTC) results in a reduction of the colourless TTC to a red-coloured 1,3,5-triphenylformazan. The enzymatic reductive amination of a wide range of various aliphatic, aliphatic-aromatic and aromatic-aromatic 2-hydroxy ketones can be determined by the decrease of the red colouration due to substrate consumption. The conversion can be quantified spectrophotometrically at 510 nm based on reactions, e.g. with crude cell extracts in 96-well plates. Since the assay is independent of the choice of diverse amine donors a panel of ω-transaminases was screened to detect conversion of 2-hydroxy ketones with three different amine donors: l-alanine, (S)-α-methylbenzylamine and benzylamine. The results could be validated using HPLC and GC analyses, showing a deviation of only 5-10%. Using this approach enzymes were identified demonstrating high conversions of acetoin and phenylacetylcarbinol to the corresponding amines. Among these enzymes three novel wild-type ω-transaminases have been identified.     

Metabolically driven equilibrium shift of asymmetric amination of ketones by ω-transaminase using alanine as an amino donor

Removal of a side product to overcome unfavorable equilibrium is a prerequisite for the asymmetric amination of ketones using ω-transaminase (ω-TA). Alanine has been preferred as an amino donor because its deamination product (i.e. pyruvate) is easily removable by several enzymatic methods. Here, we demonstrated that the removal of pyruvate by an innate metabolic pathway could afford equilibrium shift of the ω-TA reactions.     

Purification and characterization of a novel carbonyl reductase involved in oxidoreduction of aromatic β-amino ketones/alcohols

Aromatic β-amino ketones/alcohols such as adrenalone play an important role in some stereoselective synthesis of pharmaceuticals. Unfortunately, the transformation of aromatic β-amino ketones to their chiral alcohols has been carried out chemically as no corresponding biocatalyst has been available. Here, a novel carbonyl reductase responsible for the reduction of adrenalone to (R)-(−)-epinephrine was identified and characterized from Kocuria rhizophila. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by ion-exchange column chromatography, hydrophobic chromatography and gel chromatography. The purified enzyme yielded pure (R)-enantiomer product with high activity and utilized NADH as the cofactor. The enzyme had special significance by showing selectivity for many aromatic β-amino ketones/alcohols such as 2-amino-acetophenone, 2-amino-4′-hydroxyacetophenone, isoproterenol and ephedrine. The maximum reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) for adrenalone and NADH were 14.62 μmol/(min mg) protein and 0.189 mM, 11.66 μmol/(min mg) protein and 0.204 mM respectively. These properties ensure the enzyme a promising future for industrial application as a replacement of chemical synthesis of aromatic β-amino chiral alcohols.