Thesis (PhD (Chemistry and Polymer Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The use of fluorine in medicinal chemistry has increased dramatically in the last 20 years. The addition of fluorine to a lead compound has various advantages such as the blocking of metabolic active sites, the increase of solubility and lipophilicity of a compound, acting as conformational probes for the active site of an enzyme, and influencing (in most cases increasing) the binding affinity of a compound to a target protein. Their use as mechanism based inhibitors is also well known. In this study we set out to synthesize hydroxyl- and fluorinated-amino acid analogues as potential inhibitors and probes towards the active site of various enzymes. The synthesis of the hydroxylamino acid analogues would precede the fluorinated analogues to serve as precursors with fuorination achieved via a fluoro-dehydroxylation reaction. These aims have successfully been achieved with the synthesis of the two enantiopure isomers of 3-fluoro-aspartic acid. The fluorinated aspartic acid analogues were subsequently used in a conformational analysis, with regards to substrate- and binding activity, which investigated the interaction of these compounds with aspartate decarboxylase (PanD). The synthesis of the 3- hydroxy-analogues of ornithine and diamino pimelic acid was also successfully achieved. These syntheses were done in a stereospecific manner to provide one enantiomer of the L-amino acid analogue. However, our efforts toward the synthesis of the other enantiomer of hydroxy analogues as well as our attempts at the conversion of the hydroxyl group to a fluorine were unsuccessful to date. Nevertheless, these results gave us a new direction towards the synthesis of the desired compounds and have led us to new strategies and ideas. Hopefully, the work done in this study will be part of the ground work towards new methodologies for the synthesis of desired halogenated amino acid analogues as small molecule inhibitors. / AFRIKAANSE OPSOMMING: The use of fluorine in medicinal chemistry has increased dramatically in the last 20 years. The addition of fluorine to a lead compound has various advantages such as the blocking of metabolic active sites, the increase of solubility and lipophilicity of a compound, acting as conformational probes for the active site of an enzyme, and influencing (in most cases increasing) the binding affinity of a compound to a target protein. Their use as mechanism based inhibitors is also well known. In this study we set out to synthesize hydroxyl- and fluorinated-amino acid analogues as potential inhibitors and probes towards the active site of various enzymes. The synthesis of the hydroxylamino acid analogues would precede the fluorinated analogues to serve as precursors with fuorination achieved via a fluoro-dehydroxylation reaction. These aims have successfully been achieved with the synthesis of the two enantiopure isomers of 3-fluoro-aspartic acid. The fluorinated aspartic acid analogues were subsequently used in a conformational analysis, with regards to substrate- and binding activity, which investigated the interaction of these compounds with aspartate decarboxylase (PanD). The synthesis of the 3- hydroxy-analogues of ornithine and diamino pimelic acid was also successfully achieved. These syntheses were done in a stereospecific manner to provide one enantiomer of the L-amino acid analogue. However, our efforts toward the synthesis of the other enantiomer of hydroxy analogues as well as our attempts at the conversion of the hydroxyl group to a fluorine were unsuccessful to date. Nevertheless, these results gave us a new direction towards the synthesis of the desired compounds and have led us to new strategies and ideas. Hopefully, the work done in this study will be part of the ground work towards new methodologies for the synthesis of desired halogenated amino acid analogues as small molecule inhibitors.

It has been discovered during the past few years that demosponges of the order Verongida such as Aplysina cavernicola exhibit chitin-based skeletons. Verongida sponges are well known to produce bioactive brominated tyrosine derivatives. We could recently demonstrate that brominated compounds do not exclusively occur in the cellular matrix but also in the skeletons of the marine sponges Aplysina cavernicola and Ianthella basta. Our measurements imply that these yet unknown compounds are strongly, possibly covalently bound to the sponge skeletons. In the present work, we determined the skeletal amino acid composition of the demosponge A. cavernicola especially with respect to the presence of halogenated amino acids. The investigations of the skeletons before and after MeOH extraction confirmed that only a small amount of the brominated skeleton-bound compounds dissolves in MeOH. The main part of the brominated compounds is strongly attached to the skeletons but can be extracted for example by using Ba(OH)2. Various halogenated tyrosine derivatives were identified by GC-MS and LC-MS in these Ba(OH)2 extracts of the skeletons.

Demosponges of the order Verongida such as Ianthella basta exhibit skeletons containing spongin, a collagenous protein, and chitin. Moreover, Verongida sponges are well known to produce bioactive brominated tyrosine derivatives. We recently demonstrated that brominated compounds do not only occur in the cellular matrix but also in the skeletons of the marine sponges Aplysina cavernicola and I. basta. Further investigations revealed the amino acid composition of the skeletons of A. cavernicola including the presence of several halogenated amino acids. In the present work, we investigated the skeletal amino acid composition of the demosponge I. basta, which belongs to the Ianthellidae family, and compared it with that of A. cavernicola from the Aplysinidae family. Seventeen proteinogenic and five non-proteinogenic amino acids were detected in I. basta. Abundantly occurring amino acids like glycine and hydroxyproline show the similarity of I. basta and A. cavernicola and confirm the collagenous nature of their sponging fibers. We also detected nine halogenated tyrosines as an integral part of I. basta skeletons. Since both sponges contain a broad variety of halogenated amino acids, this seems to be characteristic for Verongida sponges. The observed differences of the amino acid composition confirm that spongin exhibits a certain degree of variability even among the members of the order Verongida.

It has been discovered during the past few years that demosponges of the order Verongida such as Aplysina cavernicola exhibit chitin-based skeletons. Verongida sponges are well known to produce bioactive brominated tyrosine derivatives. We could recently demonstrate that brominated compounds do not exclusively occur in the cellular matrix but also in the skeletons of the marine sponges Aplysina cavernicola and Ianthella basta. Our measurements imply that these yet unknown compounds are strongly, possibly covalently bound to the sponge skeletons. In the present work, we determined the skeletal amino acid composition of the demosponge A. cavernicola especially with respect to the presence of halogenated amino acids. The investigations of the skeletons before and after MeOH extraction confirmed that only a small amount of the brominated skeleton-bound compounds dissolves in MeOH. The main part of the brominated compounds is strongly attached to the skeletons but can be extracted for example by using Ba(OH)2. Various halogenated tyrosine derivatives were identified by GC-MS and LC-MS in these Ba(OH)2 extracts of the skeletons.

Demosponges of the order Verongida such as Ianthella basta exhibit skeletons containing spongin, a collagenous protein, and chitin. Moreover, Verongida sponges are well known to produce bioactive brominated tyrosine derivatives. We recently demonstrated that brominated compounds do not only occur in the cellular matrix but also in the skeletons of the marine sponges Aplysina cavernicola and I. basta. Further investigations revealed the amino acid composition of the skeletons of A. cavernicola including the presence of several halogenated amino acids. In the present work, we investigated the skeletal amino acid composition of the demosponge I. basta, which belongs to the Ianthellidae family, and compared it with that of A. cavernicola from the Aplysinidae family. Seventeen proteinogenic and five non-proteinogenic amino acids were detected in I. basta. Abundantly occurring amino acids like glycine and hydroxyproline show the similarity of I. basta and A. cavernicola and confirm the collagenous nature of their sponging fibers. We also detected nine halogenated tyrosines as an integral part of I. basta skeletons. Since both sponges contain a broad variety of halogenated amino acids, this seems to be characteristic for Verongida sponges. The observed differences of the amino acid composition confirm that spongin exhibits a certain degree of variability even among the members of the order Verongida.