Industrial biocatalysis.

The number of industrial processes for the synthesis of fine and commodity chemicals, pharmaceutical and agrochemical intermediates and drug substances utilizing biological catalysts continues to grow. The combination of new molecular biology techniques, such as directed evolution and pathway engineering, with new and efficient high-throughput screening methods is poised to bolster this field and further advance the contribution of biocatalysis to the chemical and the pharmaceutical industries.

Enzymatic kinetic resolution of piperidine atropisomers: synthesis of a key intermediate of the farnesyl protein transferase inhibitor, SCH66336.

The resolution of secondary amines via enzyme-catalyzed acylation is a relatively rare process. The kinetic resolution of a series of intermediates of SCH66336 (1), by either enzymatic acylation of the pendant piperidine (4, 5) or hydrolysis of the corresponding carbamate 3, was investigated. In the case of 4, the molecule exists as a pair of enantiomers due to atropisomerism about the exocyclic double bond. The enzymatic acylation of (+/-)-4 was optimized in terms of acylating agent, solvent, and moisture content. The use of lipase, Toyobo LIP-300, and trifluoroethyl isobutyrate as acylating agent resulted in isobutyrylation of the (+)-enantiomer, which is easily separated from the unwanted (-)-4. Hydrolysis of the isobutyramide 6c yielded the desired (+)-4 in high enantiomeric excess. (-)-4 may be recovered from the resolution step, racemized, and resubjected to enzymatic acylation to increase material throughput.

An enzymatic synthesis of glucuronides of azetidinone-based cholesterol absorption inhibitors.

Two derivatives, 1 and 3, of a novel cholesterol absorption inhibitor, Sch 58235, were glucuronidated with the help of glucuronyl transferases derived from bovine and dog liver microsomes. An efficient procedure for the iodination of 4 was developed on an analytical scale to be used for the preparation of a 125I-labeled radioactive glucuronide 5.

Enzymatic glucuronidation of a novel cholesterol absorption inhibitor, Sch 58235.

A glucuronide of a novel cholesterol absorption inhibitor was synthesized on a 200-mg scale in one step via bovine liver glucuronyltransferase-catalyzed coupling of the glucuronyl moiety of UDP-glucuronic acid with the phenolic hydroxyl of Sch 58235. It was shown that the product yield is limited by the hydrolysis of UDP-glucuronic acid by impurities present in the commercial microsomal preparation of the transferase. This detrimental effect of UDPGluA hydrolysis could be diminished by the presence of high concentration of glucuronlytransferase. Optimization of reaction conditions and purification procedure resulted in a process that proceeded with 95% conversion and 88% isolated product yield. The 13C6-glucuronide of Sch 58235 was prepared with the help of a cascade of eight enzymes operating concurrently in one pot.

Relationship between plasma IGF-I levels, in vitro correlates of immunity, and human senescence.

Insulin-like growth factor-I (IGF-I) is a polypeptide mitogen which is regulated by growth hormone (GH). IGF-I mediates many of the biological functions of GH, including the maintenance of lymphoid mass and functions. Since GH secretion declines with age, we asked whether changes in the availability of IGF-I might contribute to age-associated alterations in immune functions. As a first step, we examined relationships between plasma levels of IGF-I and in vitro correlates of immunity in young and elderly subjects. Heparinized plasma and lymphocytes were collected from the peripheral blood of 34 healthy young (aged 27 +/- 0.9 years, mean +/- SEM) and 41 elderly (79 +/- 1.3 years) volunteers (31 males and 44 females in total). Plasma levels of IGF-I, measured by radioimmunoassay after the removal of IGF-I-binding proteins, were reduced among elders compared to young controls (138 +/- 8.7 ng/mL vs 80.2 +/- 4.7 ng/mL, P < 0.001). The number of circulating lymphocytes did not change with age. The proliferative response ([3H]thymidine uptake into DNA) of T-cells to concanavalin A and B-cells to pokeweed mitogen were reduced among elders (P < 0.05). An increased spontaneous antitumor natural killer (NK) activity (P < 0.001) was accompanied by a higher percentage of CD16(+) NK cells among lymphocytes in older subjects (P < 0.001). The NK cell number was positively related to IGF-I levels in young volunteers but not among elders. Correlation analysis demonstrated a highly significant relationship between plasma IGF-I levels and T-cell (but not B-cell) proliferative response during aging (r = 0.492, P < 0.001). Our results imply that reduced immunocompetence may be one of the consequences of reduced IGF-I levels in human aging. Among the three types of immune cells tested, the T-cells were most sensitive to fluctuations in IGF-I levels. Reduced IGF-I availability may be one of the determinants of the decline in T-cell-mediated immune function in the elderly. To our knowledge, this is the first report presenting correlative data on concurrent changes in IGF-I levels and immune parameters in human aging.

[The participation of the T system and macrophages in the immediate immune response to drug compounds]. / Uchastie T-sistemy i makrofagov v nemedlennom immunnom otvete na lekarstvennye soedineniia.

Experiments on rats and on human lymphocytes have indicated that a prompt immune response to low-molecular-weight biologically active agents is thymus-dependent, calls for cooperative interaction of T and B lymphocytes and macrophages, it is enhanced by T mitogens and implemented through the specific pharmacological receptors of T and B lymphocytes. The findings offer ample scope for controlling the immune mechanisms of habituation to drugs and poisons.

The effect of water on enzyme action in organic media.

Three model, unrelated enzymes (yeast alcohol oxidase, mushroom polyphenol oxidase, and horse liver alcohol dehydrogenase) were found to be catalytically active in a variety of organic solvents. For all enzymes and solvents tested, the enzymatic activity greatly increased upon an increase in the water content in the solvents (which always remained below the solubility limit). Much less water was required to reach the maximal activity in hydrophobic solvents than in their hydrophilic counterparts. However, when the catalytic activity was plotted versus the amount of water bound to the enzymes, a common pattern emerged for different solvents. These data suggest that the effect of organic solvents on an enzyme is primarily due to interactions with the enzyme-bound, essential layer of water rather than with the enzyme itself. At optimal water contents, enzymatic activities in organic solvents were in the range from 20 to 40% of those in aqueous solutions. From experiments on (i) replacement of water with other hydrogen bond-forming additives and (ii) titration of enzyme amino groups in an organic medium, as well as the literature data on dehydrated enzymes, it is concluded that the water required by enzymes in nonaqueous solvents provides them with sufficient conformational flexibility needed for catalysis.

Enzymatic catalysis in nonaqueous solvents.

Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.

Enzyme-catalyzed processes in organic solvents.

Three different lipases (porcine pancreatic, yeast, and mold) can vigorously act as catalysts in a number of nearly anhydrous organic solvents. Various transesterification reactions catalyzed by porcine pancreatic lipase in hexane obey Michaelis-Menten kinetics. The dependence of the catalytic activity of the enzyme in organic media on the pH of the aqueous solution from which it was recovered is bell-shaped, with the maximum coinciding with the pH optimum of the enzymatic activity in water. The catalytic power exhibited by the lipases in organic solvents is comparable to that displayed in water. In addition to transesterification, lipases can catalyze several other processes in organic media including esterification, aminolysis, acyl exchange, thiotransesterification, and oximolysis; some of these reactions proceed to an appreciable extent only in nonaqueous solvents.

[Spasmolytic and anti-inflammatory activity of 8-hydroxyquinolines]. / Spazmoliticheskaia i protivovospalitel'naia aktivnost' 8-oksikhinolinov.

It has been shown that quinozole (aqueous solution), enteroseptol and nitroxoline (suspension with Tween-80) in a concentration of 0.2 X 10(-6)-1.10(-5) decrease the tone of the rat and guinea-pig ileum and diminish their peristalsis. When administered in the same concentrations quinozole removes or prevents the spasmogenic effects of barium chloride (1 X 10(-5)-4.10(-5), of histamine (2 X 10(-5) and -6) but not of acetylcholine (1 X 10(-6)). When administered orally in a dose 50 mg/kg to rats enteroseptol and nitroxoline inhibit the serotonin-, agar- and carrageenin-induced edemas of the rat paws without changing the response to subplantar injection of histamine. The data obtained should be taken into consideration during the use of 8-hydroxyquinolines as antimicrobial substances.

Enzymatic catalysis in organic media at 100 degrees C.

Porcine pancreatic lipase catalyzes the transesterification reaction between tributyrin and various primary and secondary alcohols in a 99 percent organic medium. Upon further dehydration, the enzyme becomes extremely thermostable. Not only can the dry lipase withstand heating at 100 degrees C for many hours, but it exhibits a high catalytic activity at that temperature. Reduction in water content also alters the substrate specificity of the lipase: in contrast to its wet counterpart, the dry enzyme does not react with bulky tertiary alcohols.

[Autoimmune and diabetogenic effects of bucarban]. / Autoimmunnyi i diabetogennyi éffekty bukarbana.

Experiments on animals were made to study the diabetogenous effect of repeated administrations of bucarban. The drug-induced immunization and insulin release were accompanied by the increased immune response of the humoral and cellular types specific for bucarban and insulin as well. The changes in the animals' immune status were accompanied by disorders of carbohydrate and lipid metabolism, characteristic for diabetes mellitus and by a lowering of the hypoglycemic effect of the antidiabetic drug in question. It was shown for the first time that the changes in carbohydrate, lipid and lipoprotein blood indicators seen in the animals given sulfanilamide antidiabetics occur in the presence of intense autoimmune response to the body own insulin and are undoubtedly related to this response.

[Kinetics of NAD-dependent formate dehydrogenase from the methanol-utilizing yeast Candida methylica]. / Kineticheskii mekhanizm deistviia NAD-zavisimoi formiatdegidrogenazy metilotrofnykh drozhzhei Candida methylica.

A kinetic analysis of the mechanism of action of NAD-dependent formate dehydrogenase (EC 1.2.1.2) from the methanol-utilizing yeast Candida methylica has been carried out. The dependence of the initial reaction rate on substrate concentrations and the inhibition by the reaction products and substrate analogs were investigated. The data obtained suggest that the kinetics of the formate dehydrogenase action are consistent with the formation of a ternary enzyme--substrate complex. NAD is the first substrate and NADH is the last product of the reaction, respectively.

[Early triggering of the immune mechanism of chemical homeostasis in response to exo- and endogenous chemical compounds]. / O rannem vkliuchenii immunnogo mekhanizma khimicheskogo gomeostaza v otvet na ékzo- i éndogennye khimicheskie soedineniia.

An early triggering of the immune mechanism of chemical homeostasis occurs in response to intravenous, intraperitoneal or intramuscular injection of drugs affecting the content of endogenous compounds of the mediator, enzymatic and hormonal type. This response is accompanied by morphological and functional changes in lymphoid cells of blood and organs as confirmed by immune rosette-formation and blast transformation, by variation of intercellular interactions that manifest in activation of immunocytoadhesion, in an increase in the titers of antibodies to the drug administered and in those of autoantibodies to the endogenous compounds that realize the drug effect. It has been shown that lymphocyte receptors specific for the endo- and exogenous compounds participate in the cell immune response to a chemical agent that might be pharmacologically monitored by means of the action of antagonists of the endogenous mediators on the receptor structure.

Cellular component of the immune mechanism of maintaining chemical homoeostasis in the internal environment of the organism.

In the study of obtained data on the part of immune mechanism in maintaining chemical homeostasis the authors found, by means of in vitro experiments, certain properties characterizing participation of the lymphatic apparatus of various rabbit and rat organs in the synthesis of antibodies against mediators, their metabolites, enzymes and hormons in comparison with effect of stimulators, [fytohemagglutinin, staphylococcal filtrate, xenogenic mixed lymphocytic cultures], active and passive immunization of potential cell preparations for the production of specific autoantibodies. Formation of the mechanism is shown on ontogenesis. It was ascertained that pathologic processes [adjuvant arthritis, rejection reaction, immobilization stress, sepsis] essentially act upon conditions of the immune mechanism of chemical homeostasis.

[Immune and autoimmune reactions in the mechanism of action of drug substances, their detoxication and the development of habituation to them]. / Immunnye i autoimmunnye reaktsii v mekhanizme deistviia lekarstvennykh veshchestv, ikh detoksikatsii i razvitiia pyrivykaniia k nim.

It has been shown that rausedyl administration is followed by enhanced synthesis of autoantibodies to serotonine and adrenaline. Meanwhile bucarban enhances the synthesis of autoantibodies to endogenous insulin and ACTH to hydrocortisone and testosterone. Activation of the autoimmune system of regulating the balance of endogenous compounds was shown by an increased number of specific antibody-producing cells in the organs and tissues and by the growth of the titer of circulating antibodies in the blood.