No abstract

The work described herein presents a strategy for the regioselective introduction of organometallic complexes into the active site of the lipase cutinase. Nitrophenol phosphonate esters, well known for their lipase inhibitory activity, are used as anchor functionalities and were found to be ideal tools to develop a single-site-directed immobilization method. A small series of phosphonate esters, covalently attached to ECE "pincer"-type d8-metal complexes through a propyl tether (ECE=[C6H3(CH2E)(2)-2,6]-; E=NR2 or SR), were designed and synthesized. Cutinase was treated with these organometallic phosphonate esters and the new metal-complex/protein hybrids were identified as containing exactly one organometallic unit per protein. The organometallic proteins were purified by membrane dialysis and analyzed by ESI-mass spectrometry. The major advantages of this strategy are: 1) one transition metal can be introduced regioselectively and, hence, the metal environment can potentially be fine-tuned; 2) purification procedures are facile due to the use of pre-synthesized metal complexes; and, most importantly, 3) the covalent attachment of robust organometallic pincer complexes to an enzyme is achieved, which will prevent metal leaching from these hybrids. The approach presented herein can be regarded as a tool in the development of regio- and enantioselective catalyst as well as analytical probes for studying enzyme properties (e.g., structure) and, hence, is a "proof-of-principle design" study in enzyme chemistry.

Granular potato starch and amylopectin potato starch were methylated in aqueous suspension to molar substitutions (MS) up to 0.29. A method was developed to determine the MS of both branched and linear regions. After exhaustive digestion of the methylated starches with -amylase, the highly branched fraction with a degree of polymerisation (DP) > 8 was separated from the linear oligomers by selective precipitation of the former in methanol. The substitution levels of branched and linear regions were determined. It was found that methylation takes place preferably at the branched regions of amylopectin and that amylose is higher substituted than linear regions of amylopectin. The distribution of methyl substituents in trimers and tetramers was determined by FABMS and compared to the outcome of a statistically random distribution. The results provided evidence for heterogeneous substituent distributions. Quanti®cation of the degree of heterogeneity of the branched and linear regions showed a much larger deviation from random distribution in the linear regions. #

Triple bond analogues of polyunsaturated fatty acids irreversibly inactivate lipoxygenases. During the inactivation the inhibitors are converted enzymatically [Kuhn, H., et al. (1984) Eur. J. Biochem. 139, 577-5831. Since the converted inhibitor molecules may hold important information about the inactivation mechanism, we have determined the structure of the product that is formed during the irreversible inactivation of soybean lipoxygenase-1 by octadeca-9,12-diynoic acid (ODYA), the triple bond analogue of linoleic acid. This product is formed only in the presence of Fe(II1)-lipoxygenase-1 and 0 2 . It was purified by C18 solid phase extraction and reversed phase HPLC and was identified with UV, IR, and NMR spectroscopic and mass spectrometric techniques as the novel lipoxygenase product,

Granular potato starch and amylopectin potato starch were methylated in aqueous suspension with dimethyl sulfate to molar substitution (MS) up to 0.29. The percentage of amorphous starch compared with crystalline domains increased with increasing MS. Prolonged treatment of these methylated starches with hydrochloric acid below the swelling temperature resulted in the release of D-glucose and small D-glucose-oligomers from the amorphous domains. The granular structure was maintained during the acidic treatment, indicating that the crystalline lamellae were less affected by acid. The amorphous domains contained on average about twice as many substituents per glucose unit as the remaining crystalline network. The distributions of methyl substituents in trimers and tetramers, released from amorphous domains and prepared from crystalline fractions, were determined by FABMS and compared to the outcome of a statistically random distribution. Quantification of the degree of heterogeneity of the thus-obtained trimers and tetramers showed a much larger deviation from random substitution in the crystalline fractions compared with the amorphous ones. These results are in agreement with our previous study that describes substitution patterns in branched and linear regions of methylated starch granules.