Universiẗat, Diss., 2001--Münster (Westfalen).

Techn. Hochsch., Diss., 2002--Aachen.

This thesis describes the work carried out on the lithiation and rearrangement of N-aryl- and N-vinyl allylic thiocarbamates, with the aim of preparing a wide range of versatile tertiary thiols. The synthesis of the racemic allylic starting materials has been achieved through in situ [3,3]-sigmatropic rearrangement of O-allylthiocarbamates to their S-counterparts (II.A). The enantioselective version relies either on asymmetric metal catalysis (achiral substrates) or stereospecificity (enantiopure substrates) in the [3,3]-sigmatropic rearrangement. Lithiation followed by N to C aryl migration proceeds in generally good to excellent yields, with both electron-poor and electron-rich rings. The scope and the influence of various substituents at Cα or on the allylic double bond are presented in Section II.B.1. Section II.B.2 proposes a mechanistic pathway and details NMR studies carried out to get structural data in the α-thioallyllithium intermediates. Investigations of the stereospecificity of the rearrangement show that most rings migrate without loss of enantioenrichment in substrates bearing an unsubstituted allylic double bond (II.B.3.a). Complete enantiospecificity with all rings has been achieved in thiocarbamates bearing a cyclohexyl-substituted double bond (II.B.3.b). Section II.C reports the results obtained in the N to C transfer of non-aromatic groups. Excellent enantiospecificities have been achieved in the migration of a vinyl substituent, although yields remain moderate. Higher yields can be obtained but they come along with lower enantiomeric ratios. Section II.D details the transformations undertaken in the rearranged tertiary allylic thiocarbamates. A wide range of tertiary thiols has been obtained with good to excellent yields. Functionalisation of the allylic double bond in these hindered substrates was not straightforward. Eventually, ring-closing metathesis in S-allyl sulfides allows the preparation of biologically interesting 2,5-dihydrothiophenes bearing a highly enantioenriched quaternary centre. Finally, evidence for retentive aryl migration in allylic thiocarbamates is outlined in Section II.E. Circular dichroism experiments were carried out in the derivatised 2,5-dihydrothiophenes and compared with predictions obtained via DFT calculations for both enantiomers of a model compound. The absolute configuration of the 2,5-dihydrothiophenes and their tertiary thiocarbamate precursors has been unambiguously established, confirming a retentive pathway in both aryl and vinyl migrations.

The biophysical and spectroscopic characterisation of two novel P450 fusion enzymes is reported. The first of these is CYP102A3, which is a fusion of P450 haem and cytochrome P450 reductase (CPR)-like domains and functions as a catalytically self-sufficient fatty acid hydroxylase in its host organism Bacillus subtilis. The elucidation of structural aspects of the isolated haem domain of CYP102A3 (HDCYP102A3) is described. This reveals a strong homology between HDCYP102A3 and the haem domain of the related, well studied enzyme CYP102A1 (known as BM3). Examination of the substrate binding and redox properties of HDCYP102A3 reveals variations in substrate selectivity and the influence of substrate binding over the haem-iron redox potential compared to BM3. Of particular note is the apparent cooperative binding profile displayed for some branched chain fatty acid substrates with CYP102A3. The second system characterised is CYP116B1 from Cupriavidus metallidurans, a P450 fusion with a reductase domain that resembles phthalate dioxygenase reductase (PDOR). The purification of the intact CYP116B1 enzyme, and also of its isolated haem domain (expressed from the relevant gene section), is optimised and biophysical characterisations are reported. The haem iron redox potential is found to be unusually positive (-85 mV) and the influence of thiocarbamate herbicide substrate binding upon this potential is found to be minimal, unlike the case in CYP102A£ with its fatty acid substrates and likely as a consequence of the relatively small degree of shift in haem-iron spin-state towards the high-spin form. From a panel of eight potential substrates for CYP116B1, six were found to stimulate NADPH oxidation, but only two of these were themselves oxidised by the enzyme, with hydroxylated products observable. The genetically dissected reductase domain of CYP116B1 was also expressed and purified, and kinetic studies of the reductase domain revealed a preference for NADPH over NADH coenzyme, and enables comparisons with kinetic features and coenzyme selectivity in other members of the ferredoxin reductase family of enzymes. Collectively, these studies advance our knowledge of the properties of two distinct types of P450-redox partner fusion enzymes, a growing class of enzymes with potential for biotechnological applications.

Tertiary thiols are a synthetically challenging class of compounds to prepare asymmetrically. The few reported methods for preparing these species require restrictive functionality to be incorporated into the products or are limited to employing simple carbon electrophiles. This thesis details investigations into the lithiation of N-aryl thiocarbamates. A stereoselective intramolecular arylation within lithiated thiocarbamates has been developed allowing the construction of quaternary stereocentres next to sulfur. Simple deprotection allows the isolation of enantiomerically pure tertiary thiols. A procedure for aryl migration within benzylic thiocarbamates has been developed and optimised. Rearrangement occurs in good yield and excellent stereoselectivity in a wide range of thiocarbamate substrates. Various substitution patterns are tolerated on the migrating aryl ring, the benzylic aryl ring and on the benzylic carbon centre. Extension of this methodology has incorporated an asymmetric alkylation of achiral benzylic thiocarbamates as a method of preparing aryl migration substrates. This allows the asymmetric synthesis of tertiary thiols in 2 steps from simple achiral precursors. Aryl migration has also been found to occur in lithiated allylic thiocarbamates with high stereospecificity, allowing preparation of a wider range of tertiary thiols.

No description available.