Development and Application of Chemical Sulfation Methods

Abstract

Sulfation is one of the most important modifications that occurs to a wide range of bioactive small molecules including carbohydrates, proteins, flavonoids, and steroids. In turn, these sulfated molecules have significant biological and pharmacological roles in diverse processes including cell signalling, modulation of immune and inflammation response, anti-coagulation, anti-atherosclerosis, and anti-adhesion. However, methods to incorporate sulfate and related sulfur functionality have drawbacks. This thesis is composed of seven individual chapters and contains the collective works of six separate investigations. Chapter 1 describes the approaches to the sulfation of small molecules: current progress and future directions. This chapter highlights the importance of sulfation in critical biological signalling cascades and a key phase II drug metabolism step. It also summarises the most encountered chemical sulfation approaches of small molecules that have been applied to a wide range of molecules, including carbohydrates, proteins, steroids, amongst many others. Chapter 2 describes the design and synthesis of novel sulfur trioxide complexes for sulfation chemistry. This includes the synthesis of tertiary amine sulfur trioxide complexes such as tripropylamine and bulky tertiary amines like triisobutylamine and isopropyl-N-methyl-tert-butylamine. This chapter also provides a general preparation of novel hydrophilic N-substituted morpholine sulfur trioxide (4-methylmorpholine SO3 and 4-ethylmorpholine SO3) which is suitable for sulfating hydrophilic substrates, including amino acids. These reagents were investigated on a simple benzyl alcohol and benzylamine to test whether they could be used as a sulfation source. The results of this investigation led to the preparation of a series of novel sulfating reagents including tripropylamine sulfur trioxide, isopropyl-N-methyl-N-tert-butylamine sulfur trioxide and 4-methyl and 4-ethylmorpholine sulfur trioxide complexes. Chapter 3 describes the sulfation of selected amino acids using 4-methylmorpholine SO3 and 4-ethylmorpholine SO3. This chapter describes the optimised conditions for the sulfation of selected amino acids using a lower temperature strategy and H2O/MeCN solvent system without the need for column chromatography techniques. Chapter 4 describes a novel exchange method to access sulfated molecules using Py·SO3 and Me3N·SO3 complexes. This chapter reports a low-cost in-situ version of Bu3N•SO3 using Py•SO3 and Me3N•SO3 followed by a lipophilic exchange with tributylamine (Bu3N). This method provides an alternative sulfation method based on a cheap, molecularly efficient and solubilising cation exchanging method. This method is amenable to a range of differentially substituted benzyl alcohols, benzylamines and aniline and can also be performed at low temperature for sensitive substrates in good to excellent isolated yields. Chapter 5 provides a convenient chemoselective conversion of the steroidal alcohol and phenol moieties to their corresponding organosulfate using tributylsulfoammonium betaine (TBSAB). This method can be conducted on a millimolar scale and the corresponding steroid sulfates isolated as their biologically relevant sodium salts without the need for ion-exchange chromatography. Furthermore, this chapter reports a convenient method to install an isotopic label, deuterium (2H), combined with estrone sulfation which could be exploited for mass-spectrometric quantification in biological studies. The results of this investigation have demonstrated a suitable method for the preparation of mono- or di-sulfated steroidal skeletons of importance to the fields of biology and spectroscopy. A simplified deuterium labelling-sulfation strategy for estrone is also reported. This isotopically labelled estrone could be used for the detection of substances of abuse through to cancer diagnosis applications as well as pharmacokinetics studies. Chapter 6 describes the investigation in to whether the biologically active heparan sulfate-glycomimetic could act as a source of the sulfation pathway in the body. This hypothesis of in situ sulfur transfer was tested on a simple benzyl alcohol using a biologically active heparan sulfate-glycomimetic. Unfortunately, this attempt was not successful as confirmed by 1H NMR spectroscopic data. Additionally, several attempts were made to sulfate other benzyl alcohol analogues using synthesised sulfation sources such as sodium 4-methylbenzyl sulfate and sodium 4-chlorobenzyl sulfate but also there was no robust evidence of the formation of the desired molecule as confirmed by 1H NMR spectroscopic data. However, for the first time, robust evidence for the stability of benzylic sulfates was found a major anticipated issue with sulfation chemistry more generally. Chapter 7 discusses the biological role of hydrogen sulfide (H2S) and the synthesis of cysteine trisulfide (Cys-SSS-Cys). This chapter describes the synthesis of cysteine trisulfide and highlights the proposed reaction mechanism as well as purification and isolation strategies. The gram-scale synthesis of cysteine trisulfide was submitted to our biological collaborator; Dr Madhani (Institute of Cardiovascular Sciences, University of Birmingham, UK) and screened for biological activity. Treatment of HEK293T (Human embryonic kidney) cells with the cysteine trisulfide resulted in high intracellular levels of hydropersulfides and thus, protection from electrophilic stress.