A structural glycoprotein has been extracted from bovine ligamentum nuchae, using 5M guanidine hydrochloride containing a disulphide bond reducing agent, and purified by preparative gel electrophoresis. The isolated material appeared to be monodisperse with a molecular weight of ~ 34 OOO as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by analytical ultracentrifugation. It contains 10% carbohydrate comprising mannose, N-acetyl-glucosamine, galactose and sialic acid in a 6:5:3:3 molar ratio. The glycoprotein has been assayed for peptidyl-lysine oxidase activity using 3H lysine-aortic elastin, prepared from 15- to 17-day-old chick embryos, as a substrate. In the absence of free lysine, the specific activity of the preparation over a 2h incubation was ~ 60 x 10⁴ dpm/mg purified protein. Addition of 10mM lysine resulted in an ~ 50% decrease in the specific activity. Free lysine was shown to act as a substrate for the glycoprotein preparation as indicated by control experiments using 3H lysine in place of the aortic substrate. These results demonstrate that the glycoprotein exhibits a dual amine oxidase activity. In the presence of 0.27mM aminopropionitrile fumarate, a concentration which completely inhibits peptidyl-lysine oxidase activity in other lysyl oxidases, the glycoprotein preparation was inhibited by ~ 14%. In the absence of 5M guanidine hydrochloride and a reducing agent, the glycoprotein undergoes aggregation which in the presence of copper ions results in the formation of cylindrical tactoids, the diameter of which (11nm) corresponds closely to that of the fibrils which in the majority of connective tissue matrices constitute the microfibrillar component mainly associated with elastic fibres.

The work presented within this thesis expands upon the theme within this laboratory, of utilising epitope-labelled recombinant proteins for the construction of multivalent subunit vaccines. Mammalian-cell expression- vectors were constructed which encoded a 14 amino acid epitope-tag, termed Pk-tag. The genes encoding the haemagglutinin (PIN) and fusion (F) glycoproteins (model type II and type I proteins respectively) from the paramyxovirus simian virus 5 (SV5), were inserted into the above vectors such that the sequence encoding the Pk-tag was present at the amino (N) or carboxy (C) terminus of SV5 HN, and the C-terminus of SV5 F. The genes were expressed in mammalian cells by utilising the vaccinia virus/T7 transient-expression system. Encouraging results were obtained which demonstrated that the addition of the Pk-tag to the N or C termini of SV5 HN, or to the C-terminus of SV5 F, did not prevent the production of; full length, N-linked glycosylated, oligomeric, natively folded and cell-surface localised Pk-tagged protein. An attempt was made to produce secretable forms of Pk-tagged SV5 HN and F. For this purpose, a vector was constructed which encoded a truncated version of the SV5 F protein in which the C-terminal transmembrane anchor & cytoplasmic tail were deleted, but which still possessed the sequence encoding the C-terminal Pk-tag at the C-terminus of the ectodomain. Expression of this gene in mammalian cells resulted in the production of a protein which had undergone N-linked glycosylation and partial oligomerisation. No secretion of the truncated Pk-tagged protein into the external milieu was detected. Furthermore, production of potentially secretable forms of N & C- terminally Pk-tagged SV5 HN was achieved by the construction of plasmid vectors in which the non-cleavable native HN signal sequence was replaced by a putative cleavable signal sequence from the Epstein Barr virus gp220/360 glycoprotein. Expression of the modified Pk-tagged HN genes in mammalian cells produced proteins of a lower mwt. than expected and which, apart from a small proportion of the N-terminally Pk-tagged molecules, did not possess N-linked oligosaccharides and were not recognised by conformationally sensitive mAbs. No secretion of the modified Pk-tagged HN into the external milieu was detected. Following the first initial characterisation of the Pk-tagged HN & F, in which very encouraging results were obtained, an attempt was made to isolate cell-lines which constitutively or inducibly expressed Pk-tagged HN. Production of Pk-tagged HN could not be detected from constructs in which expression was driven from constitutive promoters. However, production of N-terminally Pk-tagged HN (but not C-terminally tagged HN) was detected when expression was driven by the tTa inducible expression system. As a further development to the tTa system, a 293 cell-line was isolated which expressed high-levels of functional tTa. The tTa-producing cell-line was subsequently utilised in an attempt to isolate cell-lines which inducibly produce N-terminally Pk-tagged HN. These cells are currently undergoing selection for drug resistance. Further experiments were performed to try and develop a system whereby the low copy number of episomally-maintained EBV-based vectors present in a stable cell-line could be amplified to high copy number, whereby a subsequent increase in protein production would be envisaged. For this purpose, SV40 ori-containing, EBV-based vectors were constructed in which the expression of the non-toxic SV5 P protein was under the control of the hCMV IE promoter/enhancer. Cell-lines were isolated which produced the SV5 P in various amounts. Transient amplification of the episomal copy number was attempted via a transient expression of the SV40 LTAg, using plasmid DNA transfection. No subsequent increase in protein production was observed by way of a Western blot analysis.

L'identification de nouveaux agents anti-infectieux, actifs contre les pathogènes et les micro-organismes multi-résistants, reste un enjeu majeur pour la science. Parmi les molécules développées pour combattre ces infections, les peptides thérapeutiques apparaissent comme un champ prometteur de recherche. Ils se synthétisent rapidement, grâce à la synthèse sur support solide automatisée, et leur structure modulable facilite la découverte et l'amélioration d'activités biologiques. Le principal inconvénient des peptides est leur manque de résistance face à la dégradation protéolytique, et donc leur rapide élimination du corps humain. L'introduction d'aminoacides modifiés, comme les aza-bêta³-aminoacides, au sein de la séquence peptidique, permet de renforcer la biodisponibilité de ces peptides, et peut conduire à une augmentation de l’activité biologique et/ou de la sélectivité. Les aza-bêta3-aminoacides sont des analogues aza des bêta³-aminoacides, où le carbone portant la chaîne latérale est remplacé par un atome d'azote chiral à configuration non-fixée. Introduit au sein d'une séquence peptidique, ces monomères donnent accès à des pseudopeptides dont la biodisponibilité est augmentée, et l’activité et/ou la sélectivité peuvent être améliorée. Ce mémoire de thèse présente la synthèse et la fonctionnalisation d’aza-bêta³-aminoacides, à chaînes latérales protéinogènes ou non, en vue de leur insertion en synthèse peptidique sur support solide. Deux séries de pseudopeptides cycliques de novo ont été développées. La première série cible les vésicules d’endocytose résultant d’une infection adénovirale, et la seconde série mime la séquence RGD, ligand des intégrines alpha-nu-bêta₃, qui est une cible d’intérêt contre la néo-angiogénèse tumorale. / Discovering new anti-pathogenic agent, which are effective against new or multi-drug resistant microorganisms, is still a major challenge for science. Among all the drugs, which are currently developed to fight these infections, therapeutic peptides arise as a promising research field. Their synthesis is fast, due to automated solid phase synthesis, and their adjustable structure makes the discovery and the enhancement of biological activities easier. The main drawback of peptides is their lack of resistance against proteolytic degradation, and therefore their quick elimination from the human body. Modification of peptide sequence, by introduction of aminoacids analogues, such as aza-bêta3-aminoacids, reinforces the peptide bioavailability, and can lead to an increase of the biological activity, and/or of the selectivity. Aza-bêta3-amino acids are aza analogues of bêta3-amino acids, where the side chain is carried by a chiral nitrogen atom, with a non-fixed configuration. Their introduction in a peptide sequence affords pseudopeptides, with a better bioavailability, and with an activity/selectivity which could be increased. This report describes the synthesis of aza-bêta3-aminoacids, with proteinogenic side chains or not, in order to insert them in solid phase peptide synthesis. Two sets of cyclic pseudopeptides de novo have been developed. The first one targets endocytosis vesicles, resulting from an adenoviral infection, and the second one copies the RGD sequence, ligand of alpha-nu- bêta₃ integrins, which is one the main targets against the tumorous neo-angiogenesis.

Les glycoconjugués comme les glycolipides et les glycopeptides sont impliqués dans de nombreux processus biologique, physiologique et pathologique, tels que les interactions cellule-cellule, les infections virales et bactériales, les réponses immunitaires, le cancer, etc. Ces propriétés ont suscité de recherche intensive pour la synthèse de mimes de glycoconjugés pour des applications en biologie et en pharmacie. Cette thèse est consacrée à la synthèse de glycolipides et de glycopeptides liés par la liaison N-oxyamide qui possède une meilleure stabilité métabolique et une facilité de formation de liaison H conduisant à des structures secondaires très intéressantes.Les dérivés de pyranoid glycoaminooxy acides fonctionnés en positions 2 et 6 du sucre sont synthétisés à partir du methyl alpha-D-glucopyranoside, puis transformés en N-oxyamide glycolipides. En tant que nouveaux mimes de glycoglycérolipides et glycosphingolipides, les N-oxyamide beta-glycolipides possédant une ou deux chaines lipidiques sont préparés à partir du glucose ou du galactose penta-acétate et de (S)-1,2-di-O-benzyl-glycérol.De plus, une synthèse stéréosélective de (2R) et de (2S)-3-beta-O-glycosyl aminooxy esters a été réalisée à partir du (2R)-beta-glycoglycérol, avec la réaction de Mitsunobu et l’épimérisation de Lattrell-Dax comme étapes clés. Les N-oxyamide glycopeptides ont pu être préparés à partir de glycosyl aminooxy esters orthogonalement protégés. / As part of glycoconjugate family, glycolipids and glycopeptides are involved in a variety of important biological, physiological and pathological processes, such as cell-cell interactions, viral and bacterial infections, immune response, cancer progression, etc. Synthesis of glycoconjugate mimics has attracted intensive research interest for biological and pharmaceutical applications. This thesis was devoted to the synthesis of N-oxyamide-linked glycolipids and glycopeptides, since N-oxyamide-containing compounds have shown improved metabolic stability, and interesting secondary structures due to the easy H-bond formation property of N-oxyamide compounds.From methyl alpha-D-glucopyranoside, the 2,6-functionalized pyranoid glycoaminooxy acid derivatives have been successfully prepared as a multifunctional building block for further derivatization to new N-oxyamide glycolipids. From glucose or galactose pentaacetate and (S)-1,2-di-O-benzyl-glycerol, we have successfully achieved the first synthesis of N-oxyamide-linked beta-glycolipids with one or two lipids chains, as novel mimics of glycoglycerolipids and glycosphingolipids.In addition, the (2R) and (2S)-3-beta-O-glycosyl aminooxy esters have been stereoselectively synthesized from (2R)-beta-glycoglycerol, with Mitsunobu reaction and Lattrell-Dax epimerization as key steps. N-Oxyamide-linked glycopeptides have been prepared from the orthogonally protected glycosyl aminooxy esters.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains ix, 219 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 212-219).

L’objectif de nos travaux consiste en la préparation de C-glycosides et d’aminoacides glycosylés trifluorométhylés dans le but d'obtenir des structures biologiquement actives stabilisées. L'intérêt des C-glycosides est lié à leur stabilité autorisant une pharmacocinétique plus appropriée pour un usage thérapeutique. La 1ière partie de mon travail a été de créer une liaison C-C en position anomérique d'un sucre par différentes méthodes d’alkylation, d’alcynylation à l’indium et par la réaction de Réformatsky. Ensuite après avoir fonctionnalisé les C-glycosides synthétisés, une étude tournée vers la synthèse d’aminoacides C-glycosylés par le biais d’alkylations énantiosélectives a été réalisée. Ensuite nous avons étudié la synthèse d’aminoacides glycosylés stabilisés par l’introduction d’un groupement trifluorométhylé en une position stratégique. D’abord nous avons étudié la synthèse d’aminoacides N-glycosylés obtenus par la réaction entre un aminoacide trifluorométhylé et un sucre. Le groupe fluoré, en  de l’azote, diminue la basicité de l’amine et empêche sa protonation. Ainsi, l’hydrolyse du lien anomérique est très défavorisée. Plusieurs conditions réactionnelles ont été étudiées. Le milieu acide protonique a montré des résultats encourageant notamment entre le 2-déoxy-glucose et un dipeptide trifluorométhylé. Ensuite nous avons travaillé sur la synthèse de O-glycosides. 2 stratégies ont été développé à partir d’un sucre trifluorométhylé. D’abord l’éthérification de Williamson a été étudié entre un sucre trifluorométhylé et différents dérivés halogénés. Cette voie a donné des résultats satisfaisant avec des dérivés halogénés linéaires uniquements. Puis la réaction de Mitsunobu a été étudié entre un sucre trifluorométhylé et différents alcools. La réaction donne des résultats variés dépendant de l’alcool. Cette voie nous a aussi permis de synthétiser des aminoacides O-glycosylés trifluorométhylé en utilisant la sérine comme alcool. / The aim of our work consists in the preparation of C-glycosides and trifluorinated glycosylated aminoacids in order to obtain biogically active structures. The interest of such C-glycosides, is due to its stability. It allows a better pharmacokinetics to a therapeutic use. The first part of my work was to create a C-C bond at the anomeric position of a carbohydrate by different methods such as alkylation, alcynylation and Reformatsky reaction. Then, after functionalized these C-glycosides, a study on C-glycosylated aminoacids synthetized by an enantioselectiv alkylation way was made. The second part of my work was to synthesized stabilized glycoaminoacidsby the introduction of a trifluoromethylated group at a strategic position: at the anomeric position of the carbohydtare or at  position of the anomeric position of the carbohydtare. We first studied the synthesis of N-glycosides with a trifluoromethylated group at  position of the anomeric position of the carbohydtare. This strategy is based on the reaction of a protected sugar with a trifluoromethylated amine catalysed by an acid. Then we studied the synthesis of O-glycoaminoacids with a trifluoromethylated group at the anomeric position of the carbohydtare. Two strategies have been developed. The first one is the alkylation of Williamson. The second one is the reaction of Mitsunobu.

The synthesis of galactose clusters that are linked to a steroid moiety by a peptide-like spacer unit is described. The galactose cluster is obtained by Koenigs-Knorr glycosylation of TRIS-Gly-Fmoc (2b) under Helferich conditions. Peptide and ester bonds are formed after activation of carboxylic acids as diphenylthiophene dioxide (TDO) esters. 6a is synthesized in a convergent way by coupling of (Ac4Gal)3-TRIS-Gly (3e) with cholesteryl TDO succinate (5b). Coupling of (Ac4Gal)3-TRIS-Gly hydrogen succinate (3f) with Gly-O-Chol (5d) by means of EEDQ yields 6d. Reaction of (Ac4Gal)3-TRIS-Gly-SUCC-O-TDO (3g) with 25-hydroxycholesterol leads in a linear sequence to the oxysterol derivative 6f. Selective cleavage of the acetyl groups from galactose units yields the known compound 6b and the new derivatives 6e and 6g.

Thèse de doctorat : Biochimie : Lille 1 : 2003. / N° d'ordre (Lille) : 3282. Résumé en français et en anglais. Textes en français et en anglais. Pagination multiple pour les annexes. Bibliogr. 10 p.. Notes bibliogr.

Improved procedures for the glycosylation of serine and threonine utilizing Schiff base activation are reported. The procedures are less expensive and more efficient alternatives to previously published methods. The Schiff bases exhibited ring-chain tautomerism in CDCl₃ as shown by ¹H NMR. Acting as glycosyl acceptors, the Schiff bases reacted at RT with simple sugar peracetate donors with BF₃•OEt₂ promotion to provide the corresponding protected amino acid glycosides in good yields. With microwave irradiation, the reactions were complete in 2-5 minutes. Glycosylation with the dipeptide Schiff base shows the potential of this method in the preparation of peptide building blocks. To investigate this reaction further, direct glycosylation of sugar acetates with FMOC-Ser-OH/OBZl under BF₃•OEt₂ promotion in a microwave provided glycosides in high yield. In addition to the expected glycoside products acetylated side products resulting from acetate migration were isolated, suggesting that activation of the anomeric sugar acetates with a Lewis acid such BF₃•OEt₂ led to an oxocarbenium ion, which rearranged to a 1,2-dioxocarbenium ion because of the acetate participating group at C-2. Solvent participation was also illustrated with acetate migration being more pronounced when CH₃CN was used as a solvent and resulted in less product yield and higher amounts of the acetylated product. The acyl transfer products in these reactions where sugar acetates serve as glycosyl donors is reported for the first time, which also implies that ortho-ester like intermediates are important in the reaction mechanism. Keeping the message segment constant in the sequence H-Tyr-DThr-Gly-Phe-Leu- Ser-CO-NH₂ and modification of the address segment with different carbohydrate moieties had little effect on selectivity for binding at the μ, δ, or κ-opiod receptors. However, substitution of D-threonine with D-serine or the less polar D-alanine in the message segment resulted in a loss of κ-receptor affinity. Further replacement of D-threonine with the more hydrophobic D-valine resulted in complete loss of κ-binding affinity generating pure μ-δ agonists. These data suggests that changes in the message segment of the pharmacophore results in the glycopeptide adopting a conformation that is less favorable for 􀀁-binding receptor activity. Finally, the peripheral administration and i.c.v. tests of the drugs suggest that modifications in the message segment of the pharmacophore influences the potency of these compounds.