Chemically modified "polar patch" mutants of subtilisin in peptide synthesis with remarkably broad substrate acceptance: designing combinatorial biocatalysts

A significant enhancement of the applicability of the serine protease subtilisin Bacillus lentus (SBL) in peptide synthesis was achieved by using the strategy of combined site-directed mutagenesis and chemical modification to create chemically modified mutant (CMM) enzymes. The introduction of polar and/or homochiral auxiliary substituents, such as X=oxazolidinones, alkylammonium groups, and carbohydrates at position 166 at the base of the primary specificity S(1) pocket created SBL CMMs S166C-S-X with strikingly broad structural substrate specificities. These CMMs are capable of catalyzing the coupling reactions of not only L-amino acid esters but also D-amino acid esters as acyl donors with glycinamide to give the corresponding dipeptides in good yields. These powerful enzymes are also applicable to the coupling of L-amino acid acyl donors with alpha-branched acyl acceptor, L-alaninamide. Typical increases in isolated yields of dipeptides of 60-80 % over SBL-WT (e.g. 0 % yield of Z-D-Glu-GlyNH(2) using SBL-WT-->74 % using S166C-S-(CH(2))(2) NMe(3) (+)) demonstrate the remarkable synthetic utility of this "polar patch" strategy. Such wide-ranging systems displaying broadened and therefore similarly high, balanced yields of products (e.g. 91 % Z-L-Ala-GlyNH(2) and 86 % yield of Z-D-Ala-GlyNH(2) using S166C-S-(3R,4S)-indenooxazolidinone) may now allow the use of biocatalysts in parallel library synthesis.     

Initiation of polymer degradation via transfer of infectious species

A novel dual stage chemiluminescence detection system incorporating individually controlled hot stages has been developed and applied to probe for material interaction effects during polymer degradation. Utilization of this system has resulted in experimental confirmation for the first time that in an oxidizing environment a degrading polymer A (in this case polypropylene, PP) is capable of infecting a different polymer B (in this case polybutadiene, HTPB) over a relatively large distance. In the presence of the infectious degrading polymer A, the thermal degradation of polymer B is observed over a significantly shorter time period. Consistent with infectious volatiles from material A initiating the degradation process in material B it was demonstrated that traces (micrograms) of a thermally sensitive peroxide in the vicinity of PP could induce degradation remotely. This observation documents cross-infectious phenomena between different polymers and has major consequences for polymer interactions, understanding fundamental degradation processes and long-term aging effects under combined material exposures.     

Geometric and electronic structure of clusters

The interplay between electronic and geometric structure is investigated for covalently bonded phosphorus clusters. We use a modification of the molecular dynamics/ density functional (MD/DF) method of Car and Parrinello, describing the electronic structure by a simplified linear combination of atomic orbitals (LCAO) approach. The results show clearly the tendency of phosphorus to threefold coordination, and substantial variations in bond angles lead to a large variety of isomers.     

Infrared spectra and rotational isomerism of epifluorohydrin and other propene oxide derivatives

The infrared spectrum for the vapour, liquid and solid states of epifluorohydrin has been studied, and evidence has been obtained for the presence of rotational isomers. The dominant conformer in all phases corresponds to a gauche structure, which was the only form identified in the vapour by microwave spectroscopy [1]. Similar evidence for conformational equilibria has been found for the chloro, bromo and iodo derivatives, and the enthalpy differences between the rotational isomers of these compounds were measured respectively as 4580 ± 970, 3950 ± 400, and 2350 ± 170 J mol^?1 respectively. At low temperatures, epiiodohydrin was found to crystallize in one of two possible crystalline phases. These two phases correspond to the two rotational isomeric conformations of the molecule.     

Reactions of a carbene stabilised indium trihydride complex, [InH3{CN(Mes)C2H2N(Mes)}] Mes = mesityl,with transition metal complexes

The reactivity of the carbene stabilised indium trihydride complex, [InH₃(IMes)] IMes = 1,3-dimesitylimidazol-2-ylidene, toward a variety of transition metal complexes has been investigated. The study has shown that the InH₃ complex can act as a carbene and/or hydride transfer reagent to transition metal centres but does not yield heterobimetallic materials. Two new complexes, [Cp₂Ti(-Cl)₂Zn(IMes)Cl] and [CpNi(H)(IMes)], have resulted from this work, both of which have been spectroscopically and structurally characterised.     

Macroscale high-performance liquid chromatographic separation and instrumental identification of components of diethylaminoethyl murine epidermal growth factor

Murine epidermal growth factor (m-EGF), a polypeptide produced as a chromatographically homogeneous peak on diethylaminoethyl (DEAE) cellulose by the method of Savage and co-workers, and characterised as a single compound, has been shown by ourselves and several other groups to be a mixture. The present contribution extends our previously reported work and discusses the separation of this material, termed DEAE-m-EGF, into its components by preparative ion-pair reversed-phase high-performance liquid chromatography (RP-HPLC) on C18 mu Bondapak in quantities up to 50 mg per run. Isocratic elution was used and the mobile phase was acetonitrile-water (26:74, v/v, 0.04 M in triethylamine acetate); pH was 5.6, temperature 40 degrees C, and detection was by ultraviolet absorption at 254 nm, and (for some runs) by differential refractometry. Seven significant peaks, four major, three minor, were detected. Of the major peaks, two designated alpha- and beta-EGF, constituted 70% of the total mass and were the most important to our work. Each of the eluted peaks was recovered by lyophilisation, and this product checked for homogeneity by ion-pair RP-HPLC on a C18 mu Bondapak analytical column, with ultraviolet detection as before. All recovered peaks were found to be homogeneous by this criterion. These chromatographically homogeneous compounds were investigated by modern physicochemical instrumentation to determine their structure. The molecular weight of each of the species was determined by fast atom bombardment mass spectrometry. High-field proton magnetic resonance at 270 MHz provided structural and conformational information. Polarimetry and ultraviolet absorption were also used to characterise the compound. alpha-EGF, for example, had a molecular weight of 6040 corresponding to the 53 amino acid residue peptide previously designated EGF; beta-EGF had a molecular weight of 5930. This molecular weight differential of 110 suggested the hypothesis that beta-EGF was a 52 residue peptide corresponding to alpha-EGF minus the terminal asparagine at position 1. Proton magnetic resonance difference spectroscopy (beta spectrum subtracted from alpha) provided powerful confirmatory evidence for this hypothesis. All materials recovered from RP-HPLC were tested in the sheep and found to retain their biological activity.