Solid phase synthesis of peptidyl aldehydes from C-terminal thiazolidinyl peptides

Summary Peptidyl aldehydes are potent transition state analogue inhibitors of cysteine and serine proteinases. The aldehyde function has recently been used for chemoselective peptide ligation. The preparation of peptidyl aldehydes on a solid support requires that the aldehyde be masked during peptide elongation and generated in a final step under mild conditions. We report here the preparation of peptidyl aldehydes by copper salt-mediated neutral hydrolysis of the corresponding C-terminal thiazolidinyl peptides which were elongated on a solid support.     

Microbial Engineering for Aldehyde Synthesis

Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity.     

Progress in the preparation of peptide aldehydes via polymer supported IBX oxidation and scavenging by threonyl resin.

Peptide aldehydes are of interest due to their inhibitory properties toward numerous classes of proteolytic enzymes such as caspases or the proteasome. A novel access to peptide aldehydes is described using a combination of solid phase peptide synthesis with polymer-assisted solution phase synthesis based on the oxidation of peptide alcohols with a mild and selective polymer-bound IBX derivative. The oxidation is followed by selective purification via scavenging the peptide aldehyde in a capture-release procedure using threonine attached to an aminomethyl resin. Peptide aldehydes are obtained in excellent purity and satisfying yield. The optical integrity of the C-terminal residue is conserved in a high degree. The procedures are compatible with the use of common side-chain protecting groups. The potential for using the method in parallel approaches is very advantageous. A small collection of new and known peptide aldehydes has been tested for inhibitory activity against caspases 1 and 3.     

Enzymatic Improvement of Food Flavor IV. Oxidation of Aldehydes in Soybean Extracts by Aldehyde Oxidase

Aldehyde oxidase (aldehyde: oxygen oxidoreductase, EC 1.2.3.1) was partially purified from bovine liver. The enzyme irreversibly oxidized various aldehydes to the corresponding acids by using dissolved oxygen as an electron acceptor. Although the Km value for n-hexanal was low (6 µm), that for acetaldehyde was high (20 mm).

Medium-chain aldehydes such as hexanal and pentanal appear to be mainly responsible for green beany odor of soybean products. A great reduction in the beany odor was observed after the soybean extract was incubated with aldehyde oxidase under aerobic conditions. Dissolved oxygen was utilized as the electron acceptor throughout the enzyme-catalyzed oxidation of aldehydes and none of other cofactors were found to be required.

It has been shown that bovine liver mitochondrial aldehyde dehydrogenase oxidizes the soybean protein-bound aldehyde with a rate comparable to that for free n-hexanal (Agric. Biol. Chem., 43, in press). Comparative studies of aldehyde oxidase and aldehyde dehydrogenase with respect to oxidation-rates of free aldehydes and the soybean protein-bound aldehydes indicated that aldehyde oxidase acted on the bound aldehyde with a much slower rate.     

Characterization of rat cornea aldehyde dehydrogenase

Aldehyde dehydrogenase has been purified from rat cornea in a single step. The enzyme is a class 3 aldehyde dehydrogenase. Cornea aldehyde dehydrogenase is a 100-kDa dimer composed of 51-kDa subunits, prefers NADP+ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes as well as medium chain length (4-9 carbons) aliphatic aldehydes. The substrate and coenzyme specificity, immunochemical properties, effect of disulfiram, pH profile, and isoelectric point of cornea aldehyde dehydrogenase are identical to those of tumor-associated aldehyde dehydrogenase, the prototype class 3 enzyme. The substrate and coenzyme preferences are consistent with a role for cornea aldehyde dehydrogenase in the oxidation of a variety of aldehydes generated by lipid metabolism, including lipid peroxidation.     

Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Interactions between aldehyde derivatives and the aldehyde binding site of bacterial luciferase

The interaction of trizine aldehydes with the aldehyde binding site of bacterial luciferases was investigated using a series of triazine aldehydes with different aldehyde chain length, and substituents on the s-triazine ring. Substrate activity was determined using luciferase from Photobacterium fischeri and Vibrio harveyi in a dithionite-based luciferases assay. The chain length optimum was determined for two triazine aldehyde classes to be C-10 and C-11, respectively. Only the substrate activity of 10-(4-chloro-6-methyithio-s-triazine-2-yl)aminodecanal (5) was as high as n-decanal, the reference aldehyde. All other triazine derivatives reduced light emission, probably by hindered binding of the substrates. The degree of activity reduction correlated with the volume of the triazine ring moiety. The triazine moiety volume of compound 5 was estimated to be 200 × 10^?30 m³. Triazine aldehydes which showed reduced light emission had an estimated volume of 228 × 10^?30 m³ or greater. All triazine aldehydes showed approximately 10-fold lower activities for Vibrio harveyi than for Photobacterium fischeri luciferase. Substrate specificity was the same for both luciferases. A schematic superposition of quinone aldehydes and triazine aldehydes which showed substrate activities equivalent to n-decanal, indicated potential interaction sites of aldehyde substrates with the aldehyde binding site of bacterial luciferases. The in vivo relevance of the results is discussed.     

Synthesis of peptide aldehydes.

The functionalization of peptides and proteins by aldehyde groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes towards various enzymes. Furthermore, peptide aldehydes are of great interest for peptide backbone modification or ligation reactions. This review focuses upon their synthesis, which has been developed following two main strategies. The first strategy consists of prior synthesis of the peptide, followed by the introduction of the aldehyde function. The second possible strategy uses alpha-amino aldehydes as starting materials. After protection of the aldehyde, peptide elongation occurs. At the end of the synthesis, the aldehyde function can be unmasked.     

SOME ENZYMIC ASPECTS OF THE PRODUCTION OF OXIDIZED OR REDUCED METABOLITES OF CATECHOLAMINES AND 5-HYDROXYTRYPTAMINE BY BRAIN TISSUES

The Michaelis constants of purified aldehyde dehydrogenase (aldehyde: NAD oxidoreductase, EC 1.2.1.3) and aldehyde reductases (alcohol: NADP oxidoreductase, EC 1.1.1.2) from pig brain have been obtained for a number of biologically important aldehydes. The aldehydes include 3,4-dihydroxyphenylacetaldehyde, D-3,4-dihydroxyphenylglycolaldehyde, and 5-hydroxyindoleacetaldehyde. The relative activities of the aldehyde-catabolizing enzymes in the soluble fractions of the cerebral cortex and caudate nucleus of pig brain have also been obtained. The values are used to show that the metabolic fates of the various aldehydes—and hence of the parent amines—may be explained in terms of the simple kinetics of these enzymes. It is also shown that the metabolic fates of the aldehydes may be influenced by their rates of synthesis. As the rate of aldehyde production increases the proportion of aldehyde reduced may be expected to increase at the expense of the proportion of aldehyde oxidized. It is further concluded from the kinetic constants that selective inhibition of aldehyde dehydrogenase may greatly affect the catabolism of dopamine and 5-hydroxytryptamine by altering the relevant aldehyde concentrations, while the catabolism of norepinephrine is little affected under these circumstances. Conversely, it is concluded that selective inhibition of the aldehyde reductases should scarcely affect the catabolism of dopamine and 5-hydroxytryptamine, but that the catabolism of norepinephrine should be markedly affected. The results also indicate that the concentrations of the various deaminated metabolites of the biogenic amines could be selectively controlled by modulation of the activity of the enzymes of aldehyde catabolism in brain.     

Biogenic Aldehydes in Brain: On Their Preparation and Reactions with Rat Brain Tissue

When 1 mM serotonin, dopamine, or norepinephrine was incubated with a monoamine oxidase preparation (mitochondrial membranes) in the presence of 4 mM sodium bisulfite, 85-95% of the amines were oxidized to the corresponding aldehydes. In the absence of bisulfite, the recoveries were only approximately 30%, and dark colored products were formed during the incubations. The aldehydes derived from tyramine, octopamine, methoxytyramine, and normetanephrine were also prepared by the use of this method. The bisulfite-aldehyde compounds were stable during storage at -20 degrees C. Bisulfite-free aldehyde solutions were made by diethylether extraction. When the aldehydes derived from dopamine or serotonin were incubated with rat brain homogenates, they were found to disappear in an aldehyde dehydrogenase- and aldehyde reductase-independent manner. The disappearance of the latter aldehyde was more pronounced, and the results indicated that this aldehyde may react with both proteins and phospholipids.     

Hepatic microsomal oxygenation of aldehydes to carboxylic acids

Hepatic microsomal oxygenation of aldehydes to carboxylic acids was investigated. Aldehydes (veratrum aldehyde, cinnamic aldehyde, myrtenal, cuminaldehyde, 3-phenylpropionaldehyde, perillaldehyde and 9-anthraldehyde) were incubated with hepatic microsomes of mice in the presence of an NADPH-generating system under 18O2 (97 atom%). The incorporation of oxygen-18 into carboxylic acids formed was determined by gas chromatography-mass spectrometry. Oxygen-18 was incorporated into the carboxylic acids formed from all aldehyde substrates examined. Hepatic microsomal formation of 3,4-dimethoxybenzoic acid and cumic acid from veratrum aldehyde and cuminaldehyde, respectively, was inhibited by CO and SKF 525-A. These results indicate that the oxygenation of aldehydes which may be catalyzed by cytochrome P450 is a common reaction in the biotransformation of xenobiotic aldehydes.     

Removal of mixtures of acetaldehyde and propionaldehyde from waste gas in packed column with immobilized activated sludge gel beads

The removal of mixed acetaldehyde and propionaldehyde as a model of the binary contaminants in waste gas was studied in the packed column containing the immobilized activated sludge gel beads together with the hollow plastic balls developed for the removal of a single aldehyde in the previous work. The rate of each aldehyde biodegradation by the gel beads in the aldehydes mixture was expressed by the Michaelis-Menten type rate equation with an inhibitory term due to the other coexistent aldehyde. The kinetic parameters involved were found to be the same as those determined previously for biodegradation of a single aldehyde. A model for prediction of removal of each aldehyde in the packed column was developed assuming that each aldehyde dissolved in the aqueous phase within the gel bead was biodegraded according to the above rate equation with no mass transfer effect. The packed column was stable and efficient for removal of the binary aldehydes mixture with a very low pressure drop for gas flow due to a reduced gel beads bed compaction by the hollow plastic balls. Removal of each aldehyde decreased with increasing the inlet aldehyde concentrations since each biodegradation rate itself approached asymptotically the maximum one with increase in each aldehyde concentration. The observed removals for each aldehyde in the aldehydes mixture agreed well with those calculated from the design equations developed. The contact efficiency of gel beads with the waste gas stream was estimated to be the same value of 0.24 as in the previous work, supporting that the efficiency was specific to the geometrical and physical properties of the packed column used.     

应用生物技术降解木质纤维素水解液中呋喃醛

木质纤维素是地球上储量最为丰富的可再生有机碳资源,但由于其结构的复杂性,必须经过一系列预处理过程才能被微生物高效利用,这就不可避免地带来了呋喃醛等典型抑制物,严重阻碍了微生物的生长和后续发酵过程。认知微生物的呋喃醛代谢途径,并基于此开发耐受性和转化能力强的微生物菌株是生物炼制领域的重要研究内容。文中综述了呋喃醛抑制物的来源、呋喃醛对微生物的抑制机理以及微生物降解呋喃醛的代谢途径,并重点讨论了基于生物法降解呋喃醛抑制物的研究进展,涉及的主要技术手段包括传统的适应性进化工程和代谢工程,以及近年来新兴的微生物共培养系统和功能化材料辅助微生物脱除呋喃醛等。     

Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004

Aldehyde binding to liver alcohol dehydrogenase in the absence and presence of coenzymes has been characterized by spectrometric equilibrium methods, using auramine O and bipyridine as reporter ligands. Free enzyme shows a significant affinity for aldehydes, and equilibrium constants for dissociation of the binary complexes formed with typical aldehyde substrates are reported. Binary-complex formation does not lead to any detectable inner-sphere coordination of aldehydes to the catalytic zinc ion of the enzyme subunit. Complex formation with NAD+ or NADH increases the affinity of the enzyme for aromatic aldehydes by a factor of 1.8 - 3.5 and 6-17, respectively. Benzaldehyde and dimethylaminocinnamaldehyde binding to the enzyme . NAD+ complex is not detectably associated with inner-sphere coordination of the aldehyde to zinc. It is concluded that binding of NADH is required to induce catalytically adequate bonding interactions between enzyme and aromatic aldehydes. The effect of reduced coenzyme in this respect is attributed to hydrophobic interactions leading to dehydration of the active-site region, which allows aldehyde substrates to compete successfully with water for inner-sphere coordination to the catalytic zinc ion. Oxidized coenzyme is proposed to have a similar promoting effect on metal coordination of aldehydes which function as substrates for the dismutase activity of the enzyme.     

The effect of aldehyde fixation on selected substrates for energy metabolism and amino acids in mouse brain.

The effect of aldehyde fixation on concentrations of low molecular weight constituents was determined by comparing amounts of selected intermediates in brains of mice exposed to aldehyde fixative solutions with those perfused with phosphate buffer solution alone. Aldehyde perfusion resulted in excellent preservation of cerebral cortex ultrastructure in the presence of dramatic declines in adenosine triphosphate, phosphocreatine, glucose and glucose-6-phosphate that occureed before exposure of the tissue to aldehyde fixatives. Decreases in hexose were accompanied by approximately a 4-fold increase in lactate and a 2-fold increase in pyruvate. Glycogen levels decreased by about 60% during the initial operative procedure but remained constant after aldehyde fixation. Glycogen content declined approximately 90% in tissues that were not treated with aldehyde. Concentrations of aspartate and glutamate changed only slightly during the initial period (1-5 min) and remained constant for at least 90 min in cerebral cortices fixed with aldehydes. Alanine levels increased in both fixed and unfixed tissue; however, this increase was much smaller in tissues exposed promptly to aldehydes. Total ninhydrin-positive material in perchloric acid extracts of brain decreased in mice exposed to aldehyde solutions but increased in tissues that were not. These results indicated that several amino acids may be measured reliably in tissues preserved for light and electron microscopy. In addition, determination of glutamate: alanine ratios in tissues perfused with aldehydes may provide an indication of the timing of fixation.     

Oxidation of aldehydic products of lipid peroxidation by rat liver microsomal aldehyde dehydrogenase

Lipid peroxidation in microsomal membranes produces a large number of aldehydes, alcohols, and ketones, some of which have been shown to be cytotoxic. This study has determined the kinetic parameters for the oxidation of aldehyde lipid peroxidation products by purified rat hepatic microsomal aldehyde dehydrogenase (ALDH). Livers were obtained from male Sprague-Dawley rats for preparation of microsomal ALDH which was purified 400-fold. Kinetic parameters, Vmax and V/K, were determined for saturated and unsaturated aldehydes of three to nine carbons in length in the presence of NAD+. Of the aldehydes examined, only acrolein and 4-hydroxynonenal were not oxidized by ALDH. The Vmax values (mumol NADH produced/min/mg protein) increased linearly with carbon chain length and ranged from 6.5 to 23 for the saturated series and 4.0 to 9.0 for the unsaturated aldehydes. The affinity constant V/K (nmol NADH produced/min/mg protein/nmol aldehyde/liter) also increased with carbon chain length and ranged from 12 to 9000 for the saturated aldehydes and 13 to 5300 for the unsaturated aldehydes. These results suggest that microsomal ALDH may serve a biological role for detoxification of reactive aldehydes produced by lipid peroxidation of microsomal membranes.     

Detection of alkanes, alcohols, and aldehydes using bioluminescence

We report a novel method for the rapid, sensitive, and quantitative detection of alkanes, alcohols, and aldehydes that relies on the reaction of bacterial luciferase with an aldehyde, resulting in the emission of light. Primary alcohols with corresponding aldehydes that are within the substrate range of the particular luciferase are detected after conversion to the aldehyde by an alcohol dehydrogenase. In addition, alkanes themselves may be detected by conversion to primary alcohols by an alkane hydroxylase, followed by conversion to the aldehyde by alcohol dehydrogenase. We developed a rapid bioluminescent method by genetically engineering the genes encoding bacterial luciferase, alcohol dehydrogenase, and alkane hydroxylase into a plasmid for simultaneous expression in an E. coli host cell line. Alkanes, alcohols, or aldehydes were detected within seconds, with sensitivity in the micromolar range, by measuring the resulting light emission with a microplate reader. We demonstrate the application of this method for the detection of alkanes, alcohols, and aldehydes and for the detection of alkane hydroxylase and alcohol dehydrogenase activity in vivo. This method is amenable to the high-throughput screening needs required for the identification of novel catalysts.     

An induced aliphatic aldehyde dehydrogenase from the bioluminescent bacterium, Beneckea harveyi. Purification and properties.

A NAD+-dependent aldehyde dehydrogenase, the activity of which induces at the same time as luceriferase, has been purified from the bioluminescent bacterium Beneckea harveyi, and its chemical and physical properties have been investigated. The purification is accomplished in three steps resulting in an enzyme preparation that gives a single protein band on three different gel electrophoresis systems. The molecular weight of the purified enzyme was estimated to be 120,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 59,000 indicating that aldehyde dehydrogenase has a dimeric structure with subunits of similar molecular weight. The purified enzyme has a high specificity for long chain aliphatic aldehydes; the Michaelis constants for aldehydes decrease with increasing chain length as also observed for bacterial aldehyde dehydrogenases involved in the metabolism of hydrocarbons. The aldehyde specificity of the aldehyde dehydrogenase is similar to that of luciferase indicating that the functional role of the enzyme may be linked with the bioluminescent system.     

The heptadecanoic fatty aldehyde--one of the main aldehydes of the far-eastern Bryozoa.

1. The relative content of 16:0, 17:0 and 18:0 fatty aldehydes in the lipids of eight species of the far-eastern Bryozoa was studied. 2. Heptadecanoic aldehyde is one of the main aldehydes in the seven species investigated comprising about 30% of the sum of these main bryozoan aldehydes. 3. We suggest the unusually high relative heptadecanoic aldehyde content in the lipids of Bryozoa may be helpful in settling some problems concerning their system.