A solid-supported phosphine-free ruthenium alkylidene for olefin metathesis in methanol and water

The synthesis and olefin metathesis activity in protic solvents of 7, a phosphine-free ruthenium alkylidene bound to a hydrophilic solid support are reported. This heterogeneous catalyst promotes relatively efficient ring closing- and cross-metathesis reactions in both methanol and water. The potential utility of homogeneous catalyst 2 for olefin metathesis in methanol is also demonstrated.     

Design, synthesis and biological evaluation of aryl-substituted sialyl Lewis X mimetics prepared via cross-metathesis of C-fucopeptides.

Several aryl substituted C-fucopeptides have been developed as sialyl Lewis X mimetics. Although the compounds have a much simpler structure compared to SLe(x), up to 3-times higher binding affinity toward E-selectin and > 1000 times toward P-selectin was observed. Furthermore, a convenient strategy for generating a number of analogues from a SLe(x) mimetic template at a very late stage of the synthesis was introduced, using a ruthenium catalyzed cross olefin metathesis under benchtop conditions.     

Aspects Of Biocatalyst Stability In Organic Solvents

The stability of biocatalysis in systems containing organic solvents is reviewed. Among the examples presented are homogeneous mixtures of water and water-miscible organic solvents, aqueous/organic two-phase systems, solid biocatalysts suspended in organic solvents, enzymes in reverse micelles and modified enzymes soluble in water immiscible solvents. The stability of biocatalysts in organic solvents depends very much on the conditions. The hydrophobicity or the polarity of the solvent is clearly of great importance. More hydrophobic solvents (higher log P values) are less harmful to enzymes than less hydrophobic solvents. The water content of the system is a very important parameter. Some water is essential for enzymatic activity; however, the stability of enzymes decreases with increasing water content. Mechanisms of enzyme inactivation are discussed.     

Rules for optimization of biocatalysis in organic solvents

General rules for the optimization of different biocatalytic systems in various types of media containing organic solvents are derived by combining data from the literature, and the logarithm of the partition coefficient, log P, as a quantitative measure of solvent polarity. (1) Biocatalysis in organic solvents is low in polar solvents having a log P < 2, is moderate in solvents having a log P between 2 and 4, and is high in a polar solvents having a log P > 4. It was found that this correlation between polarity and activity parallels the ability of organic solvents to distort the essential water layer that stabilizes the biocatalysts. (2) Further optimization of biocatalysis in organic solvents is achieved when the polarity of the microenvironment of the biocatalyst (log P(i)) and the continuous organic phase (log P(cph)) is tuned to the polarities of both the substrate (log P(s)) and the product (log P(p)) according to the following rules: |log P(i) - log P(s)| and |log P(cph) - log P(p)| should be minimal and |log P(cph) - log P(s)| and |log P(i) - log P(p)| should be maximal, with the exception that in the case of substrate inhibition log P(i), should be optimized with respect to log P(s) In addition to these simple optimization rules, the future developments of biocatalysis in organic solvents are discussed.     

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.     

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.     

Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents

A comprehensive study of the hydration mechanism of an enzyme in nonaqueous media was done using molecular dynamics simulations in five organic solvents with different polarities, namely, hexane, 3-pentanone, diisopropyl ether, ethanol, and acetonitrile. In these solvents, the serine protease cutinase from Fusarium solani pisi was increasingly hydrated with 12 different hydration levels ranging from 5% to 100% (w/w) (weight of water/weight of protein). The ability of organic solvents to 'strip off' water from the enzyme surface was clearly dependent on the nature of the organic solvent. The rmsd of the enzyme from the crystal structure was shown to be lower at specific hydration levels, depending on the organic solvent used. It was also shown that organic solvents determine the structure and dynamics of water at the enzyme surface. Nonpolar solvents enhance the formation of large clusters of water that are tightly bound to the enzyme, whereas water in polar organic solvents is fragmented in small clusters loosely bound to the enzyme surface. Ions seem to play an important role in the stabilization of exposed charged residues, mainly at low hydration levels. A common feature is found for the preferential localization of water molecules at particular regions of the enzyme surface in all organic solvents: water seems to be localized at equivalent regions of the enzyme surface independently of the organic solvent employed.     

Cross-metathesis mediated synthesis of new acyclic nucleoside phosphonates

With the commercial availability of well-defined ruthenium metathesis catalysts which combine high stability and broad functional group compatibility, olefin metathesis is now routinely integrated in various syntheses. We will report here the overwhelming power and scope of cross-metathesis in the area of new acyclic nucleoside phosphonates. Scope and limitations of this approach, and especially the E/Z stereocontrol, are discussed on selected examples from our drug discovery group.     

Heat Effects of Dehydration of Human Serum Albumin in Hydrophilic Organic Solvents

A thermochemical model for describing the transfer of water from the protein phase to the organic solvent liquid phase and for determining how the solvation ability of organic solvents affects this process was developed. Enthalpy changes on the interaction of dried and hydrated human serum albumin (HSA) with hydrophilic organic solvents (dimethyl sulfoxide, formamide, ethanol, methanol and acetic acid) and water were measured by isothermal calorimetry at 25 °C. The initial hydration level of human serum albumin was varied in the entire water content range from 0–30 % [g water/g HSA]. The dependence of the interaction enthalpies on the initial water content is complex. The interaction enthalpies of the dried HSA with organic solvents are exothermic. At low water contents (less than 0.1 g/g), there is a sharp increase in the interaction enthalpy values. At the highest water contents (more than 0.2 g/g), the interaction enthalpies are endothermic for acetic acid and formamide and exothermic for DMSO, methanol, and ethanol. These thermochemical data were analyzed in conjunction with the results for the water adsorption in organic solvents to calculate the molar enthalpies of dehydration of HSA in organic liquids. It was found that the dehydration enthalpy changes may be endothermic or exothermic depending on the initial water content and the water solvation enthalpy value. From the results obtained, it can be concluded that: (i) only the solvation of water by hydrophilic organic solvent determines the changes in the dehydration enthalpy values, and (ii) the data for the enthalpies of solvation of water by the solvent at infinite dilution reflect this effect.     

Synthesis and characterization of water-soluble two-photon excited blue fluorescent chromophores for bioimaging.

We report here the synthesis and characterization of a new type of non-ionic blue fluorescent water-soluble chromophores specifically designed for two-photon absorption microscopy. The water solubility is induced by introduction of short oligo(ethylene glycol) monomethyl ether moieties. This work has led to low molecular weight dyes with efficient two-photon absorption cross sections and high fluorescence quantum yield in organic solvents as well as in aqueous solutions.     

Reaction rate with suspended lipase catalyst shows similar dependence on water activity in different organic solvents.

We have studied the effect of thermodynamic water activity (a W) on the initial rate of esterification catalysed by an immobilised lipase (Lipozyme) suspended in an organic reaction mixture. The catalyst and the organic phase were separately pre-equilibrated to the same aw value. The rate shows similar dependence on aw in reaction mixtures based on five different organic solvents ranging in polarity from pentan-3-one to hexane, and in a liquid reactant mixture. There is a maximum at aw about 0.5, with a decline to 30-70% at aw of either 0.9 or less than 0.01. When the rates are presented in terms of water concentration in the organic phase (or total water content of the system), the maxima for the various solvents come at very different positions, reflecting the widely varying solubilities of water in the organic phase.     

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

Homogeneous biocatalysis in organic solvents and water-organic mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

Lipase-catalyzed transesterification in several reaction systems: An application of room temperature lonic liquids for bi-phasic production ofn-butyl acetate

Organic solvents are widely used in biotransformation systems. There are many efforts to reduce the consumption of organic solvents because of their toxicity to the environment and human health. In recent years, several groups have started to explore novel organic solvents called room temperature ionic liquids in order to substitute conventional organic solvents. In this work, lipase-catalyzed transesterification in several uni-and bi-phasic systems was studied. Two representative hydrophobic ionic liquids based on 1-butyl-3-methylimidazolum coupled with hexafluorophosphate ([BMIM][PF₆]) and bis[(trifluoromethylsulfonyl) imide] ([BMIM] [Tf₂N]) were employed as reaction media for the transesterification ofn-butanol. The commercial lipase, Novozym 435, was used for the transesterification reaction with vinyl acetate as an acyl donor, The conversion yield was increased around 10% in a water/[BMIM][Tf₂N], bi-phasic system compared with that in a water/hexane system. A higher distribution of substrates into the water phase is believed to enhance the conversion yield in a water/[BMIM][Tf₂N] system. Partion coefficients of the substrates in the water/[BMIM][Tf₂N] bi-phasic system were higher than three times that found in the water/hexane system, while n-butyl acetate showed a similar distribution in both systems. Thus, RTILs appear to be a promising substitute of organic solvents in some biotransformation systems.     

Exfoliated MoS2 in Water without Additives

Many solution processing methods of exfoliation of layered materials have been studied during the last few years; most of them are based on organic solvents or rely on surfactants and other funtionalization agents. Pure water should be an ideal solvent, however, it is generally believed, based on solubility theories that stable dispersions of water could not be achieved and systematic studies are lacking. Here we describe the use of water as a solvent and the stabilization process involved therein. We introduce an exfoliation method of molybdenum disulfide (MoS₂) in pure water at high concentration (i.e., 0.14 ± 0.01 g L⁻¹). This was achieved by thinning the bulk MoS₂ by mechanical exfoliation between sand papers and dispersing it by liquid exfoliation through probe sonication in water. We observed thin MoS₂ nanosheets in water characterized by TEM, AFM and SEM images. The dimensions of the nanosheets were around 200 nm, the same range obtained in organic solvents. Electrophoretic mobility measurements indicated that electrical charges may be responsible for the stabilization of the dispersions. A probability decay equation was proposed to compare the stability of these dispersions with the ones reported in the literature. Water can be used as a solvent to disperse nanosheets and although the stability of the dispersions may not be as high as in organic solvents, the present method could be employed for a number of applications where the dispersions can be produced on site and organic solvents are not desirable.     

Effect of water activity on the lipase catalyzed esterification of geraniol in ionic liquid [bmim]PF6

Enzymatic reactions in non-aqueous media have been shown to be effective in carrying out chemical transformation where the reactants are insoluble in water or water is a byproduct limiting conversion. Ionic liquids, liquid organic salts with infinitesimal vapor pressure, are potentially useful alternatives to organic solvents. It is known that the thermodynamic water activity is an important variable affecting the activity of enzymes in non-aqueous solvents. This study investigated the influence of water activity on the esterification of geraniol with acetic acid in ionic liquid [bmim]PF6 catalyzed by immobilized Candida antarctica lipase B. The conversion of geraniol in [bmim]PF6 was significant although the reaction rate was slower than in organic solvents. The profile of initial reaction rate-water activity was determined experimentally, and differed from the data reported for other non-aqueous solvents. A maximum in the initial reaction rate was found at aw = 0.6. The pseudo reaction equilibrium constant, Kx, was measured experimentally for the reaction. The average value of Kx in [bmim]PF6 was 12, 20-fold lower than the value reported for the same system in hexane.     

Recent advances of enzymatic reactions in ionic liquids

The tremendous potential of room temperature ionic liquids as an alternative to environmentally harmful ordinary organic solvents is well recognized. Ionic liquids, having no measurable vapor pressure, are an interesting class of tunable and designer solvents, and they have been used extensively in a wide range of applications including enzymatic biotransformation. In fact, ionic liquids can be designed with different cation and anion combinations, which allow the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ionic liquids provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. As compared to those observed in conventional organic solvents, the use of enzymes in ionic liquids has presented many advantages such as high conversion rates, high enantioselectivity, better enzyme stability, as well as better recoverability and recyclability. To date, a wide range of pronounced approaches have been taken to further improve the performance of enzymes in ionic liquids. This review presents the recent technological developments in which the advantages of ionic liquids as a medium for enzymes have been gradually realized.     

Application of lipases in kinetic resolution of racemates

Lipases have been well established as valuable catalysts in organic synthesis. This review article focuses on some of the recent developments in the rapidly growing field of lipase-catalyzed kinetic resolution of racemates as a versatile method for the separation of enantiomers. The literature search dates back to the last five years and covers some comprehensive examples. The main emphasis is on the use of lipases in organic solvents.     

Development of a Method for the Solid-Phase Peptide Synthesis in Water

Various organic solvents are routinely used in peptide synthesis, safe disposal of which are now an important environmental problem. To circumvent this problem, during the last few years we focused on developing an organic solvent-free SPPS method using aqueous solvents. For the SPPS in water, we designed protected amino acids that could be used in the aqueous media. Here we described development of several types of water-soluble protected amino acids and their application to the SPPS in water, and a novel technology that uses water-dispersible protected amino acids for in-water peptide synthesis.