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Bioconjugation chemistry generally refers to the covalent derivatisation of biomolecules. Derivatisation of cysteine’s thiol of peptides and proteins is a common method in bioconjugation chemistry as the thiolate is an excellent nucleophile in aqueous conditions. The propensity for thiols to oxidise in an aqueous environment necessitates the need for a disulfide reduction step prior to the addition of ligands derivatised with thiol alkylating linkers. Disulfide reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP) are disulfide reducing agents that are often marketed as being non-reactive with thiol alkylating reagents. The reaction of TCEP and THPP with thiol alkylation linkers was therefore investigated. Characterisation of reaction products and mechanistic studies revealed that TCEP and THPP both react with thiol alkylation reagents. A novel protocol was, therefore, developed utilising the Staudinger reaction to oxidise excess TCEP and THPP prior to the addition of thiol alkylating reagents. The protocol offers a simple “one-pot” method for effecting conjugate production via thiol alkylation, without the need for an intermediate purification step for the removal of excess disulfide reducing agents. 4-Vinyl pyridine (4-VP) derivatives were developed and explored as an alternative Michael acceptor class for thiol alkylation of peptides and proteins. The 4-VP derivatives exhibited high reactivity and specificity for thiol alkylation between pH = 7 and pH = 8. A selection of 4-VP linkers were subsequently functionalised with either carbohydrates or polyethylene glycol (PEG) and successfully utilised to produce peptide or protein conjugates via thiol alkylation reactions.

Thiol-ene radical coupling is increasingly used for the biofunctionalisation of biomaterials and the formation of 3D hydrogels enabling cell encapsulation. Indeed, thiol-ene chemistry presents interesting features that are particularly attractive for platforms requiring specific reactions of peptides or proteins, in particular in situ, during cell culture or encapsulation: thiol-ene coupling occurs specifically between a thiol (from cysteine residues for example) and a non-activated alkene (unlike Michael addition); it is relatively tolerant to the presence of oxygen; it can be triggered by light, to trigger dynamic systems or for patterning. Despite such interest, little is known about the factors impacting thiol-ene chemistry in situ, under biologically relevant conditions. Here we explore some of the molecular parameters controlling photo-initiated thiol-ene coupling chemistry with a series of alkenes and thiols, including peptides, in buffered conditions. 1H NMR spectroscopy and HPLC were used to quantify the efficiency of couplings and the impact of the intensity of UV exposure, pH of the buffer, as well as the molecular structure and local microenvironment close to alkenes and thiols to be coupled. Our studies demonstrate that molecular design should be carefully selected in order to achieve high biofunctionalisation levels in biomaterials with peptides.

The kinetics of nitrosation of cysteine, cysteine methyl ester, N-acetylcysteine, penicillamine, N-acetylpenicillamine, glutathione and thioglycolic acid was undertaken. These thiols exhibited identical rate laws which are interpreted as nitrosation at sulphur by H(_2)NO(_2)(^+)/NO(^+). The rate constants determined show the high reactivity of thiols towards the nitrosating agent. The nucleophile catalyzed reactions were also investigated and the order of reactivity NOCl > NOBr > NOSCN was observed. Normally in these nucleophile catalyzed reactions there is a first order dependence on [thiol]. However, for N-acetylcysteine and thioglycolic' acid at high [thiol] the rate of formation of NOX tends to become the rate determining stage. The difference in rate constants between cysteine and penicillamine and their N-acetyl derivatives is explained in terms of internal stabilization. The decomposition of S-nitrosocysteine (S-NOCys) at pH 5.5, 7 and 9.8 in the presence and absence of C1(^-), Br(^-) and SCN(^-), and also alanine and sodium bicarbonate at pH 7, and S-nitrosoglutathione (GS-NO) at pH 7 in the presence and absence of alanine, C1(^-), and sodium bicarbonate was studied. The decomposition profiles were complex, but showed that S-NOCys was least stable at pH 7, and that GS-NO was more stable than S-NOCys. The addition of the aforementioned species did not significantly affect the rate of decomposition of the thionitrites. Finally the potential of S-NOCys, GS-NO and S-nitroso-N-acetyl- penicillamine as nitrosating agents towards amines was investigated at pH 7 and pH 8. These thionitrites nitrosated morpholine to give approximately the same yield of N-nitrosomorpholine (ca -17%) at pH 7, and less at pH 8 for S-NOCys and GS-NO. The addition of sodium acetate, sodium chloride, sodium bicarbonate, alanine and glucose, compounds liable to be present in vivo, did not significantly affect the yield of N-nitrosomorpholine. The transnitrosation reaction was complete before total decomposition of the thionitrite and a direct reaction between the thionitrite and morpholine is proposed.

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Thiol-ene chemistry is versatile and efficient and can be used as a powerful tool in polymer synthesis. In this thesis, the concept of thiol-ene chemistry has been central, where it has been explored as a tool for the synthesis of well-defined hydrogels and dopa-functional materials towards biomedical applications; such as hydrogels, primers for adhesive fixation of bone fractures, self-healing gels, and micelles for drug-delivery. Using thiol-ene chemistry, well-defined hydrogels were realized in order to study how the structure influences properties such as swelling, stiffness and hydrolytic degradation. It was found that all these characteristics are related to each other, as a more loosely crosslinked hydrogel experiences higher swelling, lower stiffness and higher degradation rates. Dopa-functional materials have gained a lot of interest throughout the years due to the remarkable adhesive properties they possess in wet environments. In the pursuit of new primers towards thiol-ene functional crosslinked bone adhesives, compounds with dopa moieties were proposed. Primers derived from dopamine were found to enhance the adhesion towards bone, and it was concluded that addition of NaOH was essential to achieve good adhesion. The strongest adhesion was achieved when thiol and ene-functional primers were used in combination. Most synthetic routes to dopa-functional polymers involve several protection and deprotection steps and a more simplistic synthetic route is therefore desired. The possibility of using UV-initiated thiol-ene chemistry to produce dopa-functional polymers was therefore investigated. The resulting polymers were shown to exhibit self-healing properties upon complexation with Fe3+ ions. Finally, the developed synthetic route was used to produce dopa and allyl-functional triblock-co-polymers. These triblock-co-polymers were then used to form micelles and evaluated as drug-delivery vehicles for the cancer-drug doxorubicin. The micelles were found to have high drug-loading capacities and slow release profiles and showed promising results when evaluated against breast-cancer cells. / Reaktioner mellan tioler och omättade kemiska föreningar utgör ett mångsidigt och effektivt redskap inom polymersyntes. I denna avhandling har begreppet tiol-en kemi varit centralt och kemin har använts för syntes av såväl väldefinierade hydrogeler som dopa-funktionella material. Dessa material har sedan utvärderats mot biomedicinska tillämpningar såsom hydrogeler, primers för fixering av benfrakturer, självläkande geler och kontrollerad läkemedelsleverans. Tiol-en-kemi har i denna avhandling använts för att framställa väldefinierade hydrogeler som sedan utvärderats med avseende på hur strukturen påverkar egenskaper såsom svällningsgrad, styvhet och nedbrytningshastighet. Det visade sig att alla dessa egenskaper är relaterade till varandra och att lösare tvärbundna hydrogeler uppvisar högre svällning, lägre styvhet och högre nedbrytningshastigheter. Marina musslor har en exceptionell förmåga att fästa mot olika ytor och på grund av detta har det visats en hel del intresse för dopa-funktionella material genom åren. På jakt efter en primer för att öka vidhäftningen hos benlim proponerades därför föreningar med dopafunktionella grupper. Det visade sig att dopaminderivat kunde förbättra vidhäftningen mot ben och det visade sig även att tillsats av natriumhydroxid var viktigt för att uppnå god vidhäftningsförmåga. Den starkaste vidhäftning uppnåddes när derivat med tiol och omättade bindningar användes i kombination. Syntes av dopafunktionella material involverar ofta flera reaktionssteg och en förenklad syntesväg är därför att eftersträva. UV-initierad tiol-en-kemi undersöktes därför som en möjlig syntesväg för att framställa dopafunktionella polymerer. Polymererna visade sig ha självläkande egenskaper vid komplexbildning med järnjoner. Slutligen användes denna syntesväg för att framställa blocksampolymerer. Dessa blocksampolymerer användes sedan för att bilda miceller med lovande resultat vid utvärdering för leverans av läkemedel mot bröstcancer. / <p>QC 20160125</p>

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