A J S C T I G N-L A
D G M S
Submitted in total fulfilment of the degree of Doctor of Philosophy
School of Chemistry University of Melbourne
January 2018
Don’t Panic1
Abstract
Microbes, both pathogenic and commensal, produce a wide range of glycolipids that act as unique molecular signatures. The ability of the human immune system to fight infec- tion as well as to modulate commensal organisms are active areas of research. Microbial glycolipids are known to interact with the immune system though discrete protein fam- ilies including CD1 and Mincle. The main challenge in the study of such systems is the difficulty in, and often impossibility of, obtaining pure, homogeneous material from natural sources. We synthesised four classes of molecules of both natural and unnatural origin to investigate their potential to modulate the human immune system through the CD1 and Mincle axes. Chapter 2 describes the synthesis of a range of cholesteryl α-glucosides that are found in members of the Helicobacter family, including the prominent gut bacterium Helicobacter pylori. As part of this work we investigated the effect of remote protecting groups on the sugar on the stereochemical outcome of glucosylation reactions. In chapter 3 we designed and synthesised a set of purely synthetic glycolipids draw- ing upon the structures of known Mincle agonists. We investigated these compounds for their ability to signal through Mincle as a prelude to the development of improved vaccine adjuvants that promote cellular and humoral immunity. Chapter 4 discloses the total synthesis of α-glucosyl and α-glucuronosyl diglycerides, found in both pathogenic and commensal organisms relevant to human health. Finally, we prepared a set of analogues of the unique, non-lipidic synthetic CD1d- restricted effector, PPBF, to explore structure activity relationships for T cell activation. In collaboration with immunologists, the synthetic glycolipids and non-lipidic anti- gens have been studied for their ability to activate CD1d-restricted natural killer T cells or for their ability to stimulate signalling through Mincle.
i Declaration
This is to certify that:
• This thesis comprises only my original work towards the PhD except where in- dicated in the Preface
• Due acknowledgement has been made in the text to all other material used
• The thesis is less than 100,000 words in length, exclusive of tables, bibliographies and appendices
Dylan Smith January 2018
ii Preface
All work reported herein has been conducted by the candidate Dylan Smith except where indicated. Mincle immuno-assays have been conducted in the laboratory of Prof. Sho Yamasaki at the Division of Molecular Immunology, Medical Institute of Bioregu- lation, Kyushu University, Japan. NKT cell immuno-assays have been conducted in the laboratory of Prof. Dale Godfrey at the Department of Microbiology and Immunology, University of Melbourne.
The work contained in this thesis has been published in part:
Smith, D. G. M. & Williams, S. J. Immune sensing of microbial glycolipids and related conjugates by T cells and the pattern recognition receptors MCL and Mincle. Carbohydr. Res., 2016, 420, 32–45. DOI:10.1016/j.carres.2015.11.009
Richardson, M. B., Smith D. G. M., & Williams, S. J. Quantitation in the regi- oselectivity of acylation of glycosyl diglycerides: total synthesis of a Streptococ- cus pneumoniae α-glucosyl diglyceride. Chem. Commun., 2017, 53, 1100–1103. DOI:10.1039/C6CC09584D
Smith D. G. M., & Williams, S. J. A carbon tetrachloride-free synthesis of N-phenyltrifluoroacetimidoyl chloride. Carbohydr. Res., 2017, 450, 10–11. DOI:10.1016/j.carres.2017.08.004
iii Acknowledgements
First and foremost, I thank my wonderful wife Amy for her unwavering support through- out my PhD. She has always inspired me to do my best, and I’m glad to have her by my side as I enter a new chapter of my life.
To my family, I am also infinitely grateful; their love of science and the world at large encouraged me to question the how of everything from an early age. Without them, I wouldn’t be where I am today.
What doctoral program wouldn’t be complete without a supportive and academ- ically challenging supervisor? So I also thank Prof. Spencer Williams for his support, understanding and assistance. Spencer has always been available to provide that little bit of knowledge that was missing in solving a challenging problem.
The members of the Williams lab, past and present, have been invaluable in my train- ing as a practical chemist and I am forever in their debt. Their friendship and support has managed to make even the most challenging of days achievable.
iv Contents
Abstract ...... i Declaration ...... ii Preface ...... iii Acknowledgements ...... iv Contents ...... v Abbreviations ...... ix
1 Introduction 1 1.1 Overview of the human immune system ...... 1 1.1.1 The innate immune system ...... 2 1.1.2 The adaptive immune system ...... 3 1.2 Select components of the innate immune system ...... 6 1.2.1 Toll-like receptors: prototypical PRRs of the innate immune system 6 1.2.2 Mincle is responsible for the recognition of carbohydrate-containing mo- lecular signatures ...... 8 1.3 Recognition of glycolipids by the CD1 proteins ...... 15 1.3.1 CD1 presents non-classical antigens to T cells ...... 15 1.3.2 CD1 restricted T cells ...... 17 1.4 Harnessing the immune system for the treatment and prevention of disease 23 1.4.1 Adjuvants improve the efficacy of vaccines ...... 24 1.4.2 Stimulation of the immune system to combat disease ...... 27 1.5 Outline of the discussions to follow ...... 28
2 The development of an improved α-glucosylation; Cholesteryl α-glucosides from Helicobacter spp. signal through Mincle 30 2.1 Introduction ...... 30 2.1.1 Helicobacter pylori: a human gut pathogen with an Australian story 30 2.1.2 Helicobacter spp. possess cholesteryl α-glucosides ...... 31 2.1.3 Mincle recognizes a structurally diverse range of carbohydrate and cholesterol containing molecules ...... 35 2.1.4 Are Helicobacter spp. cholesteryl glucosides Mincle agonists? ... 37 2.1.5 Formation of cis-glucosides ...... 37 2.1.6 N-Phenyl-trifluoroacetimidates are better glycosylation leaving groups than trichloroacetimidates ...... 46
v 2.1.7 Research aims ...... 49 2.2 Results and Discussion ...... 50
2.2.1 A new CCl4-free preparation of N-phenyl trifluoroacetimidoyl chlor- ide ...... 50 2.2.2 Donors for the comparative glycosylation of cholesterol ...... 54 2.2.3 Comparative glycosylation of cholesterol to evaluate influence of remote acetate protecting groups ...... 57 2.2.4 Removal of the 2-O-benzyl protecting group ...... 59 2.2.5 Installation of the 6-O acyl chain ...... 62 2.2.6 Helicobacter spp. αCG and αCAG’s as Mincle ligands ...... 65 2.3 Summary ...... 67 2.4 Experimental ...... 68 2.4.1 General Methods ...... 68
3 Rational design of synthetic Mincle agonists. Towards the development of safe vaccine adjuvants 86 3.1 Introduction ...... 86 3.1.1 Structural summary of glycolipids with demonstrated ability to elicit signalling through Mincle ...... 86 3.1.2 Investigations into the Mincle binding site ...... 91 3.1.3 A structural model for Mincle agonism ...... 92 3.1.4 Mincle agonists are a promising class of vaccine adjuvants .... 93 3.1.5 Research aims ...... 95 3.2 Results and discussion ...... 95 3.2.1 Selective acylation of unprotected glucosides ...... 96 3.2.2 Straight chain esters of alkyl β-glucosides ...... 98 3.2.3 Branched chain esters of alkyl β-glucosides ...... 98 3.2.4 Cholesteroloxyacetyl esters of alkyl β-glucosides ...... 100 3.2.5 Acylated alkyl β-glucosides signal potently through Mincle .... 100 3.3 Summary ...... 102 3.3.1 Future work ...... 102 3.4 Experimental ...... 103
4 α-Glucosyl and α-glucuronosyl diglycerides from commensal and pathogenic bacteria 109 4.1 Introduction ...... 109 4.1.1 Nomenclature of glycerides ...... 109
vi 4.1.2 Glucuronosyl diglycerides from Mycobacterium smegmatis and Coryne- bacterium glutamicum ...... 110 4.1.3 αGlcADAGs with undefined regiochemistry have been identified in Aspergillus fumigatus ...... 116 4.1.4 αGlcDAGs from Lactobacillus plantarum agonise immune signalling through Mincle ...... 117 4.1.5 αGlcDAGs from Streptococcus pneumoniae possess immunological activity ...... 118 4.1.6 Synthetic methodologies for the construction of glucosyl digly- cerides ...... 119 4.1.7 Regioisomerization during the synthesis of mixed diglycerides .. 123 4.1.8 Regiochemical determination of diglycerides ...... 125 4.1.9 Research aims ...... 126 4.2 Results and Discussion ...... 126 4.2.1 Quantitative 13C NMR for the measurement of acyl regioselectivity 127 4.2.2 Preparation of S. pneumoniae SPN-s2, a CD1d-restricted immune effector ...... 133 4.2.3 Preparation of Gl-A(18:1) from Mycobacterium smegmatis and Coryne- bacterium glutamicum, and its regioisomer ...... 135 4.2.4 Confirmation of the natural Gl-A(18:1) regioisomer by fragment- ation mass spectrometry ...... 141 4.2.5 Type I, Ia and II NKT TCR activity ...... 142 4.3 Summary ...... 144 4.3.1 Future work ...... 145 4.4 Experimental ...... 146
5 Phenylpentamethylbenzofuranyl sulfonate analogues as type II NKT immune effectors 160 5.1 Introduction ...... 160 5.1.1 Identification of PPBF as the stimulatory molecule ...... 160 5.1.2 A small library of analogues identified a more potent compound . 162 5.1.3 PPBF possesses a structural similarity to many ‘sulfa’ drugs .... 163 5.1.4 Project aims ...... 163 5.2 Results and discussion ...... 164 5.2.1 Expanding the structure-activity relationship of PPBF; resynthesis of PPBF ...... 164 5.2.2 Second generation PPBF analogues ...... 168 5.2.3 Potent activation of ABd NKT cells by 3-halo PPBF derivatives .. 171
vii 5.2.4 Identification of novel TCR clones that recognise ClPPBF in a CD1d- restricted fashion ...... 171 5.2.5 Efforts to discover clinically relevant molecules with structural sim- ilarities to PPBF that can activate NKT cells ...... 172 5.3 Summary ...... 177 5.3.1 Future work ...... 177 5.4 Experimental ...... 179
Bibliography 188
viii Abbreviations
Ab antibody FAME fatty acid methyl ester
αCAG cholesteryl 6-O-acyl α- G-CSF granulocyte colony- glucoside stimulating factor
APC antigen presenting cell GMCM glucose monocorynomy- colate APM antigen presenting mo- lecule GMM glucose monomycolate
BMM bone marrow macrophage HBTU 2-(1H-benzotriazol-1-yl)- 1,1,3,3-tetramethyluronium CD1 cluster of differentiation 1 hexafluorophosphate CD69 cluster of differentiation 69 HKR hydrolytic kinetic resolu- tion CID-MS/MS collision-induced dissoci- ation fragmentation mass HLA human leukocyte antigen spectrometry IFN-γ interferon-γ ClPPBF 3-chlorophenylpenta- methylbenzofuranyl sulf- IL interleukin onate iNOS inducible nitric oxide syn- CRD carbohydrate recognition thase domain MAIT mucosal-associated invari- DAMP damage-associated molecu- ant T cell lar pattern MAMP microbe-associated mo- DCC N,N'-dicyclohexylcarbodi- lecule pattern imide mCPBA m-chloroperoxybenzoic DDA dimethyldioctadecylam- acid monium bromide MePPBC 3-methylphenylpenta- DIPEA N,N-diisopropylethylamine methylbenzochromanyl sulfonate DMAP N,N-dimethylaminopyrid- ine MePPBF 3-methylphenylpenta- methylbenzofuranyl sulf- DDQ 2,3-dichloro-5,6-dicyano- onate 1,4-benzoquinone MHC major histocompatibility ESI electrospray ionization complex FAB-MS/MS fast atom bombardment NFAT-GFP nuclear factor of activated fragmentation mass spec- T cells - green fluorescent trometry protein T cell hybridoma
ix NKT natural killer T cell rt room temperature
NSAID non-steroidal anti-inflam- TBDPS tert-butyldiphenylsilyl matory drug TCA trichloroacetonitrile NOE nuclear Overhauser en- TCAI trichloroacetimidate hancement TCR T cell receptor PBC pentamethylbenzochroman TDB trehalose dibehenate PBF pentamethylbenzofuran TDCM trehalose dicorynomycolate PMB p-methoxybenzyl TDM trehalose dimycolate PPBC phenylpentamethylbenzo- chromanyl sulfonate TEMPO 2,2,6,6-tetramethyl-1- piperidinyloxy PPBF phenylpentamethylbenzo- furanyl sulfonate TFA trifluoroacetic acid
PPARγ peroxisome proliferator- THF tetrahydrofuran activated receptor γ TNF-α tumour necrosis factor-α PRE paramagnetic relaxation TLC thin layer chromatography enhancement TLR Toll-like receptor PRR pattern recognition re- ceptor TMS trimethylsilyl
PTFAI N-phenyltrifluoroacetimidateTsOH p-toluenesulfonic acid
x Chapter 1
Introduction
1.1 Overview of the human immune system
The revelation more than a century ago that microbes are the cause of infectious dis- ease immediately led to new questions; how are they detected by our body? And how does our body respond appropriately? The human immune system possesses multiple lines of defence against threats both external, such as bacteria and fungi, as well as those that originate from the human self, such as cancer and other cellular dysfunctions. Im- mune responses are directed by the actions of immune cells known as leukocytes; these include B cells, phagocytes, natural killer (NK) cells, and T cells. Central to this is the ability of immune cells to recognise unique danger signatures such as peptidic anti- gens, microbe-associated molecular patterns (MAMPs), and damage-associated mo- lecular patterns (DAMPs).5,6 Recognition prompts the immune system to act, generally through the recruitment of cells not responsible for the initial detection, and remove or neutralize the threat.
MAMPs and DAMPs are comprised of sugars, peptides, nucleic acids, lipids, vit- amins and other metabolites.7,8 While the specific molecular structures are diverse they are usually structurally-conserved across the microbiota or disease state and therefore provide good general signatures for immune recognition.
Commonly, the immune system is divided into two parts, each with a diverse set of functions: innate immunity and adaptive immunity; however, this distinction is less clear than it was once thought to be.9 Adaptive immunity relies on the development of an immunological memory resulting in a strong and highly specific response to anti- gens it has encountered before. The term antigen is often incorrectly applied to describe
1 any immune stimulating molecule in contrast to its root, ‘antibody generator’. DAMPs and MAMPs can be true antigens, however, when they play a role in the immune system not resulting directly in the generation of antibodies they are more accurately termed ‘immune effectors’. The innate immune system is involved in rapid recognition and re- sponse to threats, and in the recruitment and activation of the adaptive immune system. While immune cells are often attributed specific positions in this paradigm they are typ- ically involved in multiple, and sometimes opposing, roles in immune function.
1.1.1 The innate immune system
Activation of the innate immune system is typically mediated by the recognition of MAMPs and DAMPs by pattern recognition receptors (PRRs) (Figure 1.1).5 PRRs are expressed on professional antigen presentation cells (APCs) such as macrophages and dendritic cells, are evolutionarily conserved, and can recognise a broad range of danger signals. The recognition of these danger signals by PRRs leads to the release of cytokines into the interstitial space. Cytokines are cell-to-cell signalling molecules used for loc- alized recruitment and activation of innate cells, such as other APCs and NK cells, to deal with the threat. Cytokine production is also crucial for the correct activation of the adaptive immune system. In this respect, the innate immune system acts as a ‘first responder’ that is able to elicit immune responses very rapidly, and prime the adaptive immune system for a robust response; this response can occur even to immune effector species never encountered previously. anti-inflammatory cytokines IL-4 IL-10 MAMP/DAMP IL-13 APC
PRR IFN-γ TNF-α
proinflammatory cytokines IL-1
Figure 1.1. Recognition of MAMPs or DAMPs by the innate immune system, often via a PRR, results in the release of immunoregulatory cytokines.
2 NK cells and APCs, upon activation, can neutralize pathogenic organisms by an im- mune cell specific form of endocytosis known as phagocytosis; this process is known as cell-mediated immunity. In addition to their ability to directly neutralize threats, APCs can present antigens to T cells of the adaptive immune system. This dual role places APCs as a central player for the overall outcome of an immune response. An ‘innate- like’ system where MAMPs and DAMPs including lipids and glycolipids, presented by the CD1 antigen presentation molecules (APM), and vitamin metabolites, presented by MR1, are presented to innate-like T cells also plays a role in the rapid response of the innate immune system.
1.1.2 The adaptive immune system
Antigen-presentation to the adaptive immune system is mediated by the major histo- compatibility complexes (MHC; also known as human leukocyte antigen, HLA) (Fig- ure 1.2).10 MHCs are APMs that present peptide fragments, typically 8-20 amino acids long, to T cells. MHC class I (MHC-I) molecules are found on all cell types (except red blood cells) and present antigens derived from cytosolic proteins.11 Both self and for- eign peptides are presented on MHC-I and both play important roles in immune reg- ulation.10 MHC class II (MHC-II) molecules are typically found only on professional APCs;12 MHC-II presents antigens derived from extracellular sources such as bacteria and fungi.10 Both classes of MHCs are heterodimers that possess a binding cleft with a high degree of genetic polymorphism allowing them to present a wide range of anti- gens.13
T cells recognise MHC-antigen complexes via their T cell receptors (TCRs).15 Like the MHC molecules they are recognising, the TCR binding regions are highly poly- morphic both within and between individuals. It has been estimated that there are greater than two orders of magnitude more unique TCR structures at any one time than there are genes in the human genome.16,17 MHC-I-antigen complexes are recognised by the TCRs of CD8+ cytotoxic T cells responsible for removal of damaged or infected
3 Figure 1.2. A pre-proinsulin related peptide (MVWGPDPLYV) presented in the binding groove of HLA-A02, a human form of MHC-I.14
+ host cells. Alternatively, TCR engagement of a naïve CD4 T helper cell (TH0) with an appropriate MHC-II-antigen complex results in T helper cell maturation. This matura- tion of naïve TH0 cells into cytokine-producing T cells can be described by a simplified
18–20 paradigm: TH0 cells that mature into TH1 cells are pro-inflammatory and assist with clearance of intracellular pathogens, while TH0 cells that mature into TH2 cells are anti- inflammatory and assist with clearance of extra-cellular pathogens (Figure 1.3). TH0 maturation is oriented by the cytokine environment present at the time of TCR en- gagement and can therefore be manipulated by the APCs and other innate cells in the local environment. Because the initial innate cytokine release is typically limited spa- tially and temporally, maturation and clonal expansion of cytokine-releasing TH cells provides amplification of the cytokine signal. Naïve T cells can also mature into long lived T memory cells that can embed within tissues and are able to quickly reactivate in the presence of their cognate antigen, stimulating the production of T helper cells, and thus providing long-lasting immunological memory.
While the TH1/TH2 paradigm serves as a useful conceptual aid, it ignores the pres- ence of many other types of T cells that do not conform to its narrow definition;21,22 these
23 24 25 26 include TH9, TH17, Treg and natural killer T (NKT) cells. Moreover, an optimum immune response requires both pro- and anti-inflammatory actions to maximise the ability to clear the threat while minimizing damage to surrounding tissues.27,28
4 proinflammatory cytokine
MAMP APC TH0 TH1 TCR PRR peptide MHC
APC TH0 TH2
anti-inflammatory cytokine
Figure 1.3. Maturation of naïve TH0 cells into cytokine producing TH cells is driven by recognition, via their TCR, of an appropriate antigen presented by an MHC. The differentiation into different classes of cytokine producing cells is mediated bythe cytokine environment present during TCR engagement.
The maturation of B cells provides the final component of the ability of the adaptive immune system to generate immunological memory. Through their B cell receptor, B cells can quickly recognise and generate antibodies to antigens in solution. However, the antibodies generated by this process have low affinity for the target antigen. Alternat- ively, in a TH2 cell mediated process, antigens are presented by MHC-II on the surface of B cells and engaged by a TCR leading to B cell maturation, immunoglobulin class switching and ultimately the production of high affinity and avidity antibodies that af- ford long lasting immune memory.
In addition to the presentation of peptides by the MHCs, lipids and glycolipids are also presented to the adaptive immune system by the CD1 proteins.29
The adaptive immune system can take days to effect an immune response on first en- counter with an antigen; after the production of antigen specific antibodies and memory T cells the immune response is quick, in the order of minutes, and protection may last a life-time. On the other-hand, innate immune responses are rapid irrespective of prior recognition, typically within seconds or minutes, but the effects rapidly wane, often last-
5 ing only days.
1.2 Select components of the innate immune system
The innate immune system consists of PRRs, as well as APMs responsible for the present- ation of MAMPs and DAMPs to T cells. Due to the prevalence of carbohydrates in the essential molecules of biology, such as DNA, glycolipids and post-translational protein decorations, many danger signatures contain carbohydrates in their structures and the innate immune system is highly tuned to recognise and present these foreign or self danger-associated molecules.30
1.2.1 Toll-like receptors: prototypical PRRs of the innate immune
system
The Toll-like receptors (TLRs) are germline encoded, membrane-spanning receptors in- volved in the detection of PAMPs.31 Expressed on wide array of cellular types, thirteen TLRs (TLR1–13) have been described with the first eleven expressed by humans. The TLRs possess affinities for a wide range of molecular structures. Recognition of anap- propriate ligand by the TLRs, with the exception of TLR3, results in a signalling cascade mediated by the intracellular adaptor molecule MyD88. This signalling cascade leads to activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), up-regulating the production of proinflammatory cytokines (Figure 1.4).
The first TLR to be described was TLR4, which is responsible for the recognition of lipopolysaccharide (LPS; also known as endotoxin).32,33 LPS is a lipidic phospho- polysaccharide that is the major component of the outer membrane of Gram-negative bacteria (Figure 1.5). While LPS was long known to cause significant immune activ- ation, including systemic shock and death in experimental animals, the mechanism of its activity was for many years unknown.34 It was observed by several groups that single point mutations within the Lps locus in mice could completely abolish the re- sponse to LPS; this suggested the response to LPS was mediated though a single, non-
6 OH HO 3PO HN O OH O O OH OH TNF-α HO 3PO HN O OH O O O HN O O O OH O O HO O O HN O O O OPO 3H O IL-1 O HO O O OPO 3H O IL-12
TIRAP IRAK2 MyD88 IRAK1 NF-κB IRAK4 TLR dimer Figure 1.4. Typically, TLRs require homo- or hetero-dimerization to initiate a MyD88-mediated signalling cascade that leads to the production of NF-κB and the production and release of proinflammatory cytokines. redundant receptor.35–38 The Toll receptor was first described in Drosophila and shown to be important in development; it was shown that a homologous human gene (termed the Toll-like receptor) was responsible for the recognition of, and immune stimulation by LPS.39–42 The LPS core, a glycolipid termed Lipid A, anchors LPS to the membrane and is responsible for the TLR4-dependent activity of LPS. LYMErix, a vaccine used for protection against Lyme disease, contains recombin- ant OspA, a lipoprotein from the surface of Borrelia burgdorferi recognised by TLR2.43,44 The vaccine was formulated with aluminium hydroxide (often incorrectly termed alum), a vaccine adjuvant that affords a strong TH2 response through stimulation of the release of the endogenous danger signal uric acid (aluminium hydroxide based adjuvants are the most widely used adjuvants in human vaccines).45 The vaccine was withdrawn in 2002 because of the suggestion that it caused an autoimmune reaction led to a large decrease in sales.46 The FDA approved drugs Imiquimod and Resiquimod, which are used to treat cutaneous warts and cancers, act through activation of TLR7 and TLR8 (Figure 1.5).47
7 n = 4 - 40 NGc
Gal NGa NGa Gal
Gal Gal
NGa
NH2 O Gal NGa PhoEtn = P O O- NGc GlcGal Glc NH2 N Hep N HO3PO NGc Hep N Hep
PhoEtnO3PO Imiquimod KDO KDO KDO OPhoEtn NH2 O N O N O O OPO3H O NH NH N HO3PO HO O O O O O O O O HO O O HO Resiquimod
Figure 1.5. LPS, also known as endotoxin, is an lipidic phosphopolysaccharide found in Gram-negative bacteria that agonises TLR4 signalling. KDO: 3-deoxy-α-D-mannooctulosonic acid; Hep: heptulose; NGa: galactosamine; Gal: galactose; NGc: glucosamine; Glc: glucose. Imiquimod and Resiquimod are TLR7/8 agonists used for the treatment of cutaneous warts and cancers.
1.2.2 Mincle is responsible for the recognition of carbohydrate-
containing molecular signatures
Macrophage inducible C-type lectin (Mincle) is a C-type lectin receptor (CLR) that is a PRR responsible for the recognition of glycolipids by the innate immune system. Mincle has become an important target for investigation with the discovery that it is re- sponsible for recognition of the Mycobacterium tuberculosis lipid cord-factor, trehalose 6,6'-dimycolate (TDM) (Figure 1.6).48 Thought to have arisen from a gene duplication of Mincle, macrophage C-type lectin (MCL) is also capable of recognising TDM.49 Chal-
8 lenge of the immune system with purified TDM can elicit the formation of granulo- mas, a protective immune response to foreign substances that are difficult to elimin- ate, which are characterized by mass localization of macrophages, epithelioid cells and multi-nucleated giant cells.50,51
O OH
O HO O HO HO O
O O HO OH OH O OH
Figure 1.6. Trehalose 6,6'-dimycolate (TDM), an abundant M. tuberculosis lipid, is the cognate Mincle antigen and is responsible for the formation of granulomas in a Mincle-dependent fashion.52
Like the TLRs, CLRs are germline-encoded transmembrane receptors with the abil- ity to recognise a diverse range of MAMPs and DAMPs.53,54 The genes encoding Mincle and MCL are clustered close to those of related CLRs of the Dectin-2 family, and as a result, genetic and molecular analysis of Mincle and MCL preceded an understand- ing of their immune functions.55 Mincle was initially cloned as a target of the nuclear transcription factor NF-IL-6; it was found that Mincle RNA production was strongly upregulated upon exposure to inflammatory signalling including LPS, tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin 6 (IL-6).56
The transmembrane domain of Mincle contains a positively charged arginine that facilitates a crucial interaction with the immuno-receptor tyrosine-based activation mo- tif (ITAM)-containing adaptor molecule Fc receptor γ-chain (FcRγ).57 Binding of an ag- onistic ligand to Mincle results in phosphorylation of the ITAM of FcRγ and promotes recruitment of spleen tyrosine kinase (Syk). Syk recruitment leads to NF-κB activation via a signalling cascade mediated by CARD9-Bcl10-MALT1 (Figure 1.7).58 While MCL also signals through Syk, the nature of the ITAM-containing adaptor is unclear as MCL does not possess a positively charged arginine in its transmembrane domain. Several
9 reports suggest a covalently-linked heterodimer of Mincle and MCL may be involved in MCL signalling.59,60 MCL appears to play a role in recruitment of Mincle to the cell surface in bone marrow-derived dendritic cells.61 MCL also appears to be crucial for robust cytokine production after stimulation with TDM in bone marrow-derived mac- rophages.62 In both humans and rodents, Mincle is predominately expressed on APCs including monocytes, macrophages, neutrophils, and dendritic cells.56,57,63,64 The cy- tokine release mediated by Mincle stimulation directs the maturation of naïve TH0 cells
65 towards the TH1 and TH17 subtypes.
TNF-α
O
O IL-6 O
O O O O IL-10 Mincle G-CSF FcRγ iNOS + --
P CARD9 Syk P MALT1 NF-κB Bcl10 ITAM Figure 1.7. Signalling through Mincle involves an FcRγ-provided ITAM adaptor and ultimately leads to the production of a set of cytokines that promote the maturation of TH0 cells towards the TH1 and TH17 subtypes.
CLRs are named after the presence of a Ca2+ atom often found in their carbohydrate recognition domains (CRD) that are usually required for ligand binding. Screening of a 326-member carbohydrate micro-array against a soluble MCL-Fc fusion protein re- turned no positive binding interactions suggesting that MCL is not directly involved in carbohydrate binding.66 Crystal structures of both human67 and bovine68 Mincle have been solved; both contain two Ca2+ atoms in their CRDs with the latter also containing an Na+ atom distant from the CRD. A crystal structure of bovine Mincle complexed with trehalose has additionally been obtained providing crucial details on the mode of binding in the CRD (Figure 1.8). Studies of the Mincle CRD have identified a typical Ca2+ sugar binding site as well as a novel Ca2+-free secondary carbohydrate binding
10 site.68 Only one of the glucose moieties of trehalose, through O-2 and O-3, is bound to a Ca2+. Adjacent to the Ca2+ bound carbohydrate is a lipophilic cleft that has been proposed to act as a lipid binding channel.69 AB Ca 2+ glucose 2 Na + glucose 1
O3 O4 Ca 2+
Ca 2+
C glucose 2 glucose 1
Figure 1.8. X-ray structures of A bovine Mincle bound to trehalose (PDB: 4KZV); B close-up of Ca2+ site of bovine Mincle; C protein surface near trehalose binding site, with the potential lipid binding groove running diagonally right-down from glucose 1.
Since its initial description, a wide variety of glycolipids, both natural and synthetic, have been identified as Mincle signalling agonists. With the exception of spliceasome- associated protein 130 (SAP130) all reported agonists are insoluble and may be a con- sequence of Mincle detecting intact microbial cells. In vitro studies typically involve plate-bound or crystalline forms of the agonists. The recognition of TDM as the pro- totypical Mincle ligand occurred contemporaneously with the discovery that the long- known simplified analogue, trehalose dibehenate (TDB), also signals through Mincle (Figure 1.9).52 Produced by members of the Mycobacterial taxon, mycolic acids are long chain, α-alkyl-β-hydroxy fatty acids with up to 90 carbons, and can possess un- saturation, cyclopropanation, ketones, esters and methoxy groups in the mero chain.
11 Corynebacteria produce related fatty acids, termed corynomycolic acids, which like mycolic acids are α-alkyl-β-hydroxy fatty acids, but possess shorter chains (30–40 car- bons), and are typically found only in saturated and unsaturated forms. Van der Peet and colleagues showed that synthetic trehalose dicorynomycolate (TDCM), prepared us- ing C32-corynomycolic acid, elicited strong signalling through both human and mouse Mincle.70 Trehalose monomycolate (TMM) and trehalose monocorynomycolate (TMCM) were shown to be potent Mincle agonists.48,70 In both cases the monoesters caused less potent stimulation than the comparable diesters; a finding that has been replicated in other studies.71–74 OR OR HO O HO O HO HO HO HO O O OR OH HO O O OR O OH HO OH HO OH HO OH OH OH HO OH OR R
O OH
TDM TMM GMM GroMM O OH
TDCM TMCM GMCM GroMCM O
TDB TMB GMB (not active) Figure 1.9. A range of trehalose di- and mono-esters, as well as monoesters of glucose and glycerol, signal through Mincle.
In addition to trehalose-based molecules, monoesters of glucose and glycerol are potent Mincle agonists (Figure 1.9). Glucose-6-monomycolate (GMM) has been shown to cause granulomas in mice, a process known to be Mincle-dependent;75 in addition, glucose-6-monocorynomycolate (GMCM) agonist of both human and mouse Mincle.70 In comparison, the mycobacterial glycerol-monomycolate (GroMM) and its coryne-
12 bacterial counterpart, glycerol-monocorynomycolate (GroMCM), are potent agonists of human but not mouse Mincle in GFP-reporter assays.70,76 In the case of glucose- 6-monobehenate very little signalling was seen in contrast to the potent activity of TDB and trehalose monobehenate, highlighting the importance of multiple components of the structure of a potent agonist. Glycosyl diglycerides from several bacterial species have Mincle signalling activity (Figure 1.10). A series of simple β-gentiobiosyl diglycerides from M. tuberculosis bear- ing iso-branched fatty acids as well as straight chain acids showed weak activity towards mouse but not human Mincle, with the most potent compound bearing straight-chain
77 C12 fatty acids. Truncation of the gentiobiose to a single glucose unit resulted in potent activation of mouse and human Mincle for the diglyceride bearing iso-C17:0 fatty acids. Shah and co-workers demonstrated that α-glucosyl diglycerides bearing cyclopropane- containing fatty acids from Lactobacillus plantarum are Mincle agonists.78 A glucosyl diglyceride isolated from Streptococcus pneumoniae was also shown to significantly in- crease cytokine release in mice in a Mincle-dependent fashion; the same report showed that Mincle was required for survival of mice when challenged with serotype 19F S. pneumoniae.79 In addition to a wide range of microbe-derived molecules, Mincle also contributes to sterile (non-infected) inflammatory signalling in response to stimulation by DAMPs. A report showed involvement of Mincle in the damaged cell response via recognition of the soluble protein SAP130.57 Mutation of the Mincle CRD demonstrated that signalling agonism by SAP130 occurs at a site outside of the CRD.
13 R
HO iso-C15:0 HO O HO O iso-C16:0 HO OR iso-C17:0 HO O HO O OR C16:0 HO C12:0 C8:0 C4:0 HO OR HO O iso-C17:0 O OR HO C HO 12:0
HO O 11 18 HO O HO O HO 16 O O
O Streptococcus pneumoniae Glc-DAG-s2 HO O HO O HO O HO O O 9S 10R O Lactobacillus plantarum GL1
Figure 1.10. A range of microbial diglycerides have been identified as Mincle agonists.
Several, self-derived, insoluble Mincle agonists have been described to be involved in sterile inflammation (Figure 1.11). Crystalline cholesterol, present in atheroscler- otic plaques caused by hypercholesterolemia as well as cholesterol granulomas, stim- ulates inflammatory signalling via Mincle.80 Importantly, only crystalline cholesterol, and not solubilised or membrane bound cholesterol, elicited a response. In a screening of endogenous sterol structures only cholesterol, and its biosynthetic precursor des- mosterol, elicited signalling; masking the 3-OH of cholesterol as a range of esters ab- lated activity. Reporter cells expressing human Mincle, but not those expressing rat or mouse Mincle, were able to elicit a response upon stimulation with cholesterol. This specificity is due to the presence of the cholesterol recognition/interaction amino acid consensus (CRAC) motif in the human Mincle sequence but not that of the rat or mouse sequence. In a related finding, subcutaneous injection of cholesterol sulfate resulted in
14 a Mincle-dependent localized inflammatory response characterised by recruitment of neutrophils, monocytes and eosinophils.81
H H O H H O H H S O HO O cholesterol cholesterol sulfate
Figure 1.11. Cholesterol and cholesterol sulfate have been identified as potent human, but not rodent, Mincle agonists.80,81
A β-glucosyl ceramide (β-GlcCer), a glycolipid released upon cellular damage, eli- cits inflammatory signalling through human and mouse Mincle.82 A range of β-GlcCer acyl homologues were identified in dead cells with βGlcCer (C24:1) eliciting the strongest response in a Mincle reporter cell-line (Figure 1.12). β-GlcCer is notably accumulated in Gaucher’s disease, an inherited disease in which the β-glucosyl ceramidase (GBA1) enzyme is defective, which is characterized by systemic inflammation. O HO HN HO O HO O OH OH
Figure 1.12. βGlcCer (C24:1), a compound associated with cellular damage, was identified as the most active of βGlcCer lipid homologues in a Mincle reporter cell assay.82
1.3 Recognition of glycolipids by the CD1 proteins
1.3.1 CD1 presents non-classical antigens to T cells
CD1 is a family of antigen presentation molecules (APM) that presents a diverse range of lipidic MAMPs and DAMPs to T cells of the innate/innate-like immune system.83 Analogous to the function of MHC molecules as surveyors of the protein and peptide environment for antigens, CD1 proteins survey the intra- and extra-cellular lipid en- vironment for MAMPs and DAMPs.84 In humans, the CD1 locus encodes five protein
15 isoforms that can be further categorized into three groups: Group 1 is comprised of CD1a,b,c; Group 2 of CD1d; and Group 3 of CD1e.85 Groups 1 and 2 are directly in- volved in effector molecule presentation, while Group 3 is thought to be involved in the intracellular loading of lipids into the other CD1 molecules.86,87 CD1 molecules are ubiquitous amongst mammals, however, not all isoforms are universal. Humans pos- sess representative members of all three groups,88 while mice only possess the Group 2 CD1d (two isoforms)89,90 and cattle the Group 1 isoforms.91 Structurally, the Group 1 and 2 CD1 isoforms are similar to MHC-I and contain two α-helices forming a ligand binding cleft;92 however, in contrast to MHC-I, the CD1 isoforms are genetically monomorphic. In addition to similarities in the binding re- gion, CD1 proteins possess a single transmembrane region and an extra-cellular, non- covalent, association with β2-microglobulin. Unlike MHC-I, CD1 molecules present lipids with long hydrophobic regions, which are accommodated into deep hydrophobic tunnels under the binding groove. CD1 molecules are typically expressed only on pro- fessional APCs and CD4/8 double positive thymocytes. However, CD1d has been re- ported to be more widely expressed, including on non-hematopoietic cells.93 A common antigen-binding paradigm exists amongst the isoforms whereby the lipophilic chains are accommodated within pockets beneath the binding groove while the polar, typically carbohydrate, head-group projects into the binding cleft (Figure 1.13). A large number of crystal structures have been solved for ligands bound to the CD1 isoforms and differences in these hydrophobic tunnels helps to differentiate the setof lipids presented by each isoform.94–98 The lipophilic pockets of the CD1 molecules range in volume from 1340 Å3 for CD1a to 2200 Å3 for CD1b; with the volumes of CD1c (1780 Å3) and CD1d (1650 Å3) falling in between. The pockets are further differenti- ated by their overall shape and inter-connectivity. These differences, in combination with different intracellular trafficking behaviour, largely explains the differences inlipid repertoire that the CD1 molecules present.84,99 The pockets of CD1d are termed the' A pocket, able to accommodate 26 carbons, and F' pocket (18 carbons).
16 A B
C D
Figure 1.13. Crystal structures showing CD1 ligand binding: A CD1a in complex with dideoxymycobactin; B CD1b in complex with GMM; C CD1c in complex with mannose-1-β-phosphomycoketide (MPM); D CD1d in complex with αGalCer.
1.3.2 CD1 restricted T cells
Like the TCRs that recognise peptidic antigens bound to MHC molecules, the TCRs that recognise lipidic antigens bound to CD1 molecules are typically composed of an α- and β-chain (αβ TCR). The gene coding for the TCR possesses multiple gene segments coding for the variable (V), diversity (D), joining (J) and constant regions; somatic re- combination of these gene segments, a process known as V(D)J combination, results in high variability of the sequence in the binding region of the TCR (Figure 1.14).100 For
αβ TCRs the α-chain is composed of Vα and Jα segments, and the β-chain is composed of Vβ,Dβ and Jβ segments.
Additionally, TCRs possessing γ- and δ-chains (γδ TCR) can recognise immune effectors bound to CD1 molecules.101 The TCRs of γδ T cells undergo somatic recom- bination like their αβ counterparts. γδ T cells have been reported to recognise effector molecules bound to CD1c and CD1d.102,103 Due to a lack of recognition of MHC presen- ted antigens far less is known about γδ TCRs than αβ TCRs.
17 TCR chain segments Variable Diversity Joining Constant
Unique TCR arrangement Figure 1.14. The somatic recombination of different gene segments of the TCR α, β,δ and γ genes enables production of an immense number of unique TCRs that can recognise an astonishingly large repertoire of molecules. Further diversity is generated (not shown) through additional processing events before the transcript is complete.
CD1 Group 1 restricted T cells
The Group 1 CD1 molecules are recognised by T cells possessing the genetically diverse TCRs typically associated with recognition of peptides presented by MHCs. These T cells recognise a range of ligands including squalene104 and dideoxymycobactin (CD1a),105,106 glucose and glycerol monomycolate (CD1b),107 and mannose phosphomycoketide (CD1c).108 Most of the known Group 1 presented effector molecules are mycobacterial in origin.
Initially, it was thought that Group 1 restricted T cells exclusively possessed diverse TCRs. When a T cell clone (LDN5) reactive towards CD1b-presented GMM was isol- ated from a leprosy patient, it was thought that it simply possessed a patient-specific TCR.109 However, sampling of the human T cell population from unrelated donors us- ing CD1b tetramers loaded with GMM enabled isolation of two T cell populations with conserved TCR sequences, including one with similar sequence identity to the original
110–112 LDN5 clone. LDN5-like T cells possess a bias towards Vα17 and Vβ4.1 expression in their α-chains, and the other ‘germline-encoded mycolyl-reactive’ (GEM) T cells pos- sess an invariant TCR containing Vα1.2 and Jα9 in the α-chain and a bias towards Vβ6.2 in the β-chain. These discoveries have highlighted the existence of innate-like T cells recognising CD1 Group 1 presented effector molecules.
18 CD1 Group 2 restricted T cells, the natural killer T cells
Natural killer T (NKT) cells are a sub-population of T cells that possess characteristics of both T cells and NK cells. Initially defined by the presence of NK cell surface marker NK1.1, NKT cells are now more accurately defined by their specificity for the group 2 CD1 molecule, CD1d.26 CD1d, and therefore NKT cells, are the best studied members of the CD1 system owing to two critical coincidences: mice being the ideal research animals for CD1d study, and the early discovery of a potent synthetic CD1d-restricted ligand, αGalCer (vida infra). The discovery of αGalCer was so instrumental in early studies that NKT cells are separated into two distinct groups based primarily on their ability to recognise it. Type I NKT cells recognise αGalCer presented by CD1d while type II do not.
NKT cells are functionally distinct from their MHC-restricted cousins, and for ex- ample possess the ability to produce large amounts of cytokines, including IFNγ, IL-4, IL-13, IL-17 and TNF-α, without requiring maturation; enabling NKT cells to effect general immune regulation without requiring clonal expansion (Figure 1.15).26 Con- sequently, NKT cells are generally considered innate-like, with their TCRs acting like PRRs. TNF-α IFNγ APC NKT IL-4 IL-13 Lipid CD1d TCR IL-17 Figure 1.15. The engagement of the TCR of an NKT cell with a CD1d-presented effector molecule results in the rapid release of large amounts of immunoregulatory cytokines.
Type I NKT cells possess an invariant TCR α-chain and recognise αGalCer
In 1993, a series of glycosphingolipids, termed the agelasphins, were isolated from a sea sponge off the Okinawan coast.113 These compounds shared a common structure of a galactose head-group attached in an α-configuration to a ceramide tail. All the
19 agelasphins showed potent anti-tumour activity in mice. Further work examining the structural basis for this activity, through the synthesis of analogues, led to the develop- ment of KRN7000 (commonly, although somewhat confusingly, termed αGalCer) (Fig- ure 1.16).114 αGalCer is a purely synthetic compound that activates a CD1d-restricted NKT cells. Although αGalCer produces a strong cytokine release from these type I NKT cells, its relevance to the role that glycolipids play in the human immune system, owing to its origin as a synthetic derivative of a marine natural product, remains an active area of inquiry. Recently, a series of α-galactosyl ceramides has been identified in Bacteroides fragilis, a member of the human gut microbiota.115 In retrospect, it seems likely that the agelasphins are not produced by the sponge itself but from a resident microbe.
OH HO OH O O HO NH OH HO O
OH agelasphin-9b HO OH O O HO NH OH HO O
OH KRN 7000 (αGalCer) HO OH O O HO NH OH HO O
OH αGalCerBf Figure 1.16. The α-galactosyl ceramides, Agelasphin 9b and KRN7000 (αGalCer), have limited relevance to human health due to their origins as a marine natural product and synthetic derivative thereof. Potentially more relevant to human health is 115 αGalCerBf an α-galactosyl ceramide identified in Bacteroides fragilis, a member of the human gut microbiota.
In humans, type I NKT cells possess a canonical TCR with a Vα24-Jα18 (Vα14-Jα18 in mice) α-chain.116 It should be noted that while the α-chain is invariant, the β-chain is still variable leading to the term semi-invariant NKT cells (iNKT). Differences in β- chain usage results in minor changes in ligand specificities.117 In addition to αGalCer,
20 type I NKT cells are stimulated by a range of microbial and self-derived lipid effector molecules.118 To date, crystal structures of type I-CD1d-ligand complexes have demon- strated a conserved docking strategy where the TCR sits parallel to the binding cleft, en- gaging in significant contact both with the ligand and with CD1d itself (Figure 1.17).116 Interactions with the lipid are mediated though the TCR α-chain, while CD1d makes intimate contacts with both the α- and β-chains.
TCR α-chain
TCR β-chain αGalCer
CD1d
βM
Figure 1.17. The TCR of a type I NKT cell recognises CD1d-presented αGalCer.119
Recently, type I NKT cells with different ligand specificities, termed atypical type I (type IA), have been described in mice.120–122 These NKTs, while still able to recog- nise αGalCer, also recognise CD1d presented ligands that the canonical type I TCR does not. A type 1a NKT cell that possessed a Vα10-Jα50 TCR α-chain, and displayed a robust response to CD1d-presented αGalCer, demonstrated even greater reactivity to- wards α-glucosyl ceramide (αGlcCer);120 it was also reactive towards an α-glucuronosyl diglyceride from Mycobacterium smegmatis, a compound that does not activate type I NKTs. A crystal structure of this type 1a-CD1d-αGalCer complex showed a similar mode of binding to that of a type 1 NKT-CD1d complex.
21 Type II NKT cells possess diverse TCR arrangements
Type II NKT cells are a group of CD1d-restricted T cells that by definition do not re- cognise CD1d presented αGalCer.26,123 Unlike type I NKT cells there does not appear to be a unifying motif that defines the TCRs expressed by type II NKTs. Although type II NKTs do not express a singular conserved TCR, studies of mouse type II TCRs suggest enrichment towards Vα3 and Vα8 as well as Vβ8.124 Currently, sulfatide, a sulfated β-galactosyl lipid, is the best studied type II NKT ef- fector molecule (Figure 1.18);125 sulfatide is found endogenously in neuronal tissue and has been associated with inhibition of experimental autoimmune encephalomyelitis.126
The sulfatide reactive TCR possesses a Vα1-Jα26 α-chain and Vβ16-Jβ2.1 β-chain. Another prominent, albeit less well studied, type II NKT cell effector is the small molecule phenylpentamethylbenzofuranyl sulfonate (PPBF) (Figure 1.18). Discovered by van Rhijn and co-workers, PPBF is the first non-lipidic species able to elicit a stimu- latory response in a CD1d-restricted fashion and does not stimulate type I NKT cells.127 In comparison to the sulfatide-reactive TCR, the PPBF-reactive TCR possesses a Vα2- Vβ21 TCR. O O HO OH HN O O 24 HO3SO O S 14 O HO O OH Figure 1.18. PPBF and sulfatide both activate type II NKT cells in a CD1d-restricted fashion.
While only a small number of structures have been solved for type II NKT-CD1d complexes, the docking mode for type II NKTs appears strikingly different to that of type I NKTs (Figure 1.19).128 The TCR sits orthogonal to the A' pocket and makes most of its interactions with the ligand via the β-chain.
22 TCR
sulfatide
β
CD1d
βM
Figure 1.19. The docking mode of type II NKT TCRs is different to that of typeI TCRs, adopting an orthogonal configuration above the CD1d binding cleft.
1.4 Harnessing the immune system for the treatment
and prevention of disease
Subunit vaccines provide the adaptive immune system with epitopes matching a par- ticular pathogen, enabling generation of antibodies and other immunological memory without the risk of disease associated with contracting the pathogen. Co-stimulation of the innate and adaptive immune systems can improve the effectiveness of subunit vac- cines. An adjuvant is a material or mixture of materials added to the vaccine to stimulate the innate immune system and assist in generation of a robust adaptive responses to the provided epitopes.129 The most widely used adjuvant in human use is aluminium hy- droxide.45
23 1.4.1 Adjuvants improve the efficacy of vaccines
Freund’s complete adjuvant (FCA) is the gold standard and most widely used adjuvant for experimental antibody production.130 Freund and co-workers administered an water- in-oil emulsion of heat-killed tuberculosis bacilli in paraffin oil to rabbits, and observed increased antibody titres, as well as longer lasting complement effect when compared to those treated with wet or dry killed-tuberculosis bacilli alone.131–133 Although FCA is a potent stimulator of the immune system, it is reported to have severe side-effects including the production of granulomas and lesions both at the site of injection and systemically.134 Infection with live tuberculosis also causes granulomas and is associ- ated with the presence of TDM and TMM in the mycobacterial outer leaflet.51,135 These side effects have prevented FDA approval for the use of FCA in humans and limited its use to research settings. An incomplete Freund’sadjuvant (IFA), lacking the dried myco- bacterial cells and therefore simply a water-in-oil emulsion, is in animal use. While IFA possesses a more favourable side effect profile than CFA it induces a weaker immune response and a less ideal cytokine profile for stimulation of immunological memory.136 More recently, significant research effort has been applied to the development of adjuvants suitable for use in humans.129 Two adjuvants have been developed and are in active use for co-administration with the seasonal and pandemic influenza vaccines. MF59, developed by Novartis, consists of an water-in-oil emulsion of squalene (Fig- ure 1.20).137 The second, AS03, developed by GlaxoSmithKline (GSK), also contains squalene but combined with α-tocopherol and a surfactant, polysorbate 80.138,139 GSK have also developed AS04, which contains aluminium salts in combination with mono- phosphoryl lipid A (MPL), a less toxic derivative of Lipid A.140 AS04 is used in GSK’s hepatitis B and human papillomavirus vaccines.
24 squalene O
O O OH O P O O O O OH O O O N OH O H O HO O OH O O N H OH MPL Figure 1.20. Squalene is the active component of the AS03 and MF49 vaccine adjuvants. MPL, a less toxic derivative of Lipid A, is used in the AS04 combination adjuvant.
Under active study is a saponin adjuvant, a water-soluble terpene glycoside, isolated from the bark of the soap bark tree (Quillaja saponaria).141,142 Forty years ago Dalsgaard and colleagues isolated a mixture of saponins, later named Quil A, from another Quil- laja species and showed that it strongly stimulated both adaptive and innate immune re- sponses as well as induction of differential antibody isotypes.143–146 Quil A was a mixture of up to 23 different saponins and its toxicity precluded use in humans. Fractionation and analysis of the adjuvanticity of the individual components revealed that fraction 21 (QS21), contained a potent adjuvant with reduced toxicity compared to other com- ponents of the mixture (Figure 1.21).147,148 While the toxicity of QS21 is still reasonably high can be significantly reduced though co-administration with liposomes and MPL (AS01).149 David Gin and co-workers took a different approach to the toxicity of QS21 and found that simplification of the QS21 structure retained adjuvant activity while sig- nificantly reducing the toxicity that is associated with the natural QS21.150,151
25 O HO O HO H H O O O HO H O HO CO2 O CH3 OR O HO C OH 2 H OH HO OH O O O HO O HO O HO OH HO O O HO CH3 OH O CHO HO O HO OH O HO OH HO O QS-21Aapi: R = QS-21Axyl: R = HO HO OH OH Figure 1.21. Structures of the apiose and xylose variants of QS-21 which occur in an approx. 2:1 ratio in the naturally isolated material. The Mincle signalling agonist TDB is under investigation in phase 1 clinical trials for use as an adjuvant.152 Incorporation of 11% TDB into dimethyldioctadecylammonium bromide (DDA) liposomes affords an adjuvant named CAF01 that provides robust im- mune stimulation.52,153 The adjuvanticity of CAF01 is thought to be mediated predom- inately via Mincle due to the lack of adjuvant effect when co-administered with the TB sub-unit H1 vaccine to Mincle-/- mice. A human trial of the H1 vaccine in combination with CAF01 found long lasting antigen-specific T cell responses in addition to minimal adverse effects.152
Immune stimulation via TLR activation is used by many currently marketed vac- cine adjuvants. AS04 and other adjuvants containing MPL, as analogues of LPS, are known to cause immune stimulation through TLR4.33 While the immune stimulation caused by CFA was thought to be mediated through both TLR2 and TLR4,130 a recent re- port demonstrated dependence on recognition by a receptor using a CARD9-containing signalling pathway.154 The mechanism of action of squalene in MF59 and AS03 is cur- rently undetermined; however, the ability of CD1a-presented squalene to stimulate T cells presents one likely candidate.104 The QS-21 mechanism of action is under active investigation. Increasingly, agonists of Mincle signalling, such as TDB, are also being explored as adjuvants. These different modes of activation highlight the potential for different types of stimulation for different antigens and that no one adjuvant is optimal for all vaccines.155
26 1.4.2 Stimulation of the immune system to combat disease
In addition to enhancing the efficacy of prophylactic vaccines, adjuvants can also be ap- plied directly to the treatment of disease by way of immune-therapy.156 Danishefsky and colleagues are exploring the treatment of cancer by activation of the immune system to- wards cancer specific signatures and utilizing adjuvants to ensure a robust and effective immune response.157,158 In support of this, it has been observed that in rare instances cancer patients raise an immune response against tumour-associated carbohydrate an- tigens and that doing so promotes significantly improved survival rates.159 GM2, Globo-H, LeY, sialyl Tn (sTN) and Thompson Friedreich (TF) are carbo- hydrate antigens that are over-expressed on the cell surface of multiple cancers.160,161 While monovalent vaccines against these epitopes can deliver immunologic responses, the heterogeneity of cancer cell surface expression mitigates their ability to have broad effect (Figure 1.22).162–165 Using all five of these epitopes, Danishefsky and co-workers prepared a pentavalent carbohydrate structure supported on a peptide backbone, con- jugated to keyhole limpet haemocyanin for use as a vaccine157 In a Phase I clinical trial of the vaccine, which was co-administered with QS21 as an adjuvant, 83% of patients treated developed antibody titres against at least three of the antigens.166 Only mild side- effects were observed and were mostly attributed to the relatively high dose of QS21. HO OH OH CO2H OH OH OH OH OH O O O HO O NHAc OH HO O O O AcHN O OH HO OH HO O AcHN HO O OH OH O AcHN O O HO O O O OH OH HO O O OH HO HO HO
O O H H H N N N NHR AcHN N N OH OH H H O O O O HO HO OH HO O OH OH HO2C OH O HO O O O O O O AcHN AcHN HO HO HO HO O O HO O OH OH OH OH Figure 1.22. A synthetic vaccine consisting of the epitopes for five different cancer-associated oligosaccharide structures. R = KLH.
27 While adjuvants were initially unknown quantities in their ability to stimulate the immune system, it is now widely accepted that most adjuvants are natural MAMPs or DAMPs, or analogues thereof, and simulate a real infection. The activation of the innate immune system by stimulation of PRRs leads to downstream regulation of the adapt- ive immune system and assists in the development of robust immunological memory. The continued development of adjuvants will be crucial in the production of effective prophylactic and therapeutic vaccines.
1.5 Outline of the discussions to follow
Chapter 2: The development of an improved α-glucosylation; Cholesteryl α-glucosides from Helicobacter spp. signal through Mincle
Cholesteryl α-glucosides are produced in abundance in Helicobacter spp. but their im- munological functions are poorly understood. The objectives of this chapter were two- fold. First we sought to compare the stereochemical outcome of glucosylation of choles- terol using glucosyl donors with varying acylation patterns. Second, using the optimum donor developed in the first objective, we synthesised five cholesteryl 6'-O-acyl-α-gluco- sides that have been isolated from members of the Helicobacter family, and in particular from Helicobacter pylori. These compounds, in collaboration with the Yamasaki labor- atory, were investigated for their ability to stimulate signalling through Mincle.
Chapter 3: Rational design of synthetic Mincle agonists. Towards the development of safe vaccine adjuvants
This small chapter describes an initial foray into the rational design of Mincle signalling agonists. A series of alkyl 6-O-acyl-β-glucosides were designed based on an analysis of the currently described Mincle agonists and on their ability to be prepared from readily available materials. The compounds were synthesised and assessed for their ability to stimulate Mincle signalling.
28 Chapter 4: α-Glucosyl and α-glucuronosyl diglycerides from commensal and pathogenic bacteria
This chapter describes the application of the highly regioselective methodology for fra- ternal diglyceride synthesis to the synthesis of several α-glucosyl and α-glucuronosyl diglycerides from pathogenic and commensal bacteria. The primary challenge of this work was the application of existing techniques for late stage C-6 oxidation due to the highly insoluble nature of these compounds. Assessment of the CD1d-restricted activity of these compounds against several different NKT TCRs was conducted in collaboration with the Godfrey laboratory.
Chapter 5: Phenylpentamethylbenzofuranyl sulfonate analogues as potent type II NKT antigens
This chapter discusses the exploration of structure-activity relationships for NKT ac- tivation of the novel, non-lipidic CD1d-restricted ligand PPBF. This work involved the synthesis of approximately 20 novel compounds to optimize their activity. This work resulted in discovery of more potent NKT effectors based on the PPBF structure, which were used to discover several novel TCRs with affinity towards PPBF analogues bound to CD1d. We used structural similarity to PPBF to identify drugs with similar chemical structures and assessed their ability to stimulate PPBF reactive NKT cells in a CD1d- restricted fashion.
29 Chapter 2
The development of an improved α-glucosylation; Cholesteryl α-glucosides from Helicobacter spp. signal through Mincle
2.1 Introduction
Gastric and duodenal ulcers have a lifetime risk of around 10% and accounted for 300,000 deaths in 2013.167,168 Until the late-80s the treatment of ulcers was often ineffective. It is now well-known that gastric ulcers can be treated with antibiotics owing to the dis- covery that infection with the bacterium Helicobacter pylori is the cause — a discovery that led to the Nobel Prize in Physiology or Medicine in 2005, awarded to Australian scientists Barry Marshal and Robin Warren.
2.1.1 Helicobacter pylori: a human gut pathogen with an Australian
story
Before Marshall and Warren published their dogma-breaking findings in The Lancet in 1984, gastric ulcers were thought to have been caused primarily by either stress or excess stomach acid. Controversially, Marshall and Warren proposed that ulcers were instead caused by infection with a pathogenic bacterium.169 When they announced their findings they were met with more than mild scepticism, although the findings were not the first to show an association between an unidentified “gastric spiral bacteria” and ul- cers.170 The pair attempted to infect piglets with H. pylori to show a direct link but were unsuccessful. Later that year, Marshall intentionally ingested broth containing cultured H. pylori, and rapidly developed symptoms like those of gastric ulcer disease. Treatment
30 with antibiotics reversed his symptoms.171 This “experiment”,along with the earlier find- ings allowed Marshall and Warren to fulfil Koch’s postulatesa and demonstrate H. pylori as the cause of gastric ulcers.
Helicobacter are Gram-negative bacteria possessing a characteristic helical shape. The family contains around 35 species, of which H. pylori is the most relevant to human health. Other members of the family while not well characterized in human disease may play a role as opportunistic pathogens. Non-pylori Helicobacter species play an important role in animal models of H. pylori infection in humans.172
The distribution of H. pylori in humans is very wide, infecting as much as 50% of the world’s population with a larger proportion in third world countries.173 Only 20% of those are symptomatic of gastritis, suggesting a tight interplay between the immune system and H. pylori.174
2.1.2 Helicobacter spp. possess cholesteryl α-glucosides
Cholesterol is a component of eukaryotic cell membranes where it is crucial for proper biophysical functioning of the membrane.175 Vaz and Simons demonstrated that cho- lesterol is essential for robust H. pylori growth.175 Multiple reports in the mid-90s by Haque, Hirai and co-workers demonstrated the presence of cholesteryl containing glycol- ipids in several different members of the Helicobacter family.176–178 These included cho- lesteryl α-glucoside (αCG), and various modified glucosides such as cholesteryl 6-O- acyl-α-glucosides (αCAG) and cholesteryl 6-O-phosphatidyl-α-glucosides (αCPG) (Fig- ure 2.1).
The H. pylori cholesteryl glucosides are highly abundant and comprise a signific- ant proportion, 15-30% (>25% in H. pylori), of the total lipid content in the species in which they are present. The acyl chain composition in the αCAG and αCPGs differs
aKoch’s postulates state: 1. The micro-organism must be found in abundance in all organisms suf- fering from the disease, but should not be found in healthy organisms. 2. The micro-organism must be isolated from a diseased organism and grown in pure culture. 3. The cultured micro-organism should cause disease when introduced into a healthy organism. 4. The micro-organism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific caus- ative agent.
31 for the individual Helicobacter species. αCAGs were found in four of the species in- vestigated: H. nemestrinae, H. muridarum, H. pylori and H. rappini (Figure 2.2). The fatty acid repertoire of αCAG from H. rappini was the broadest ranging from C12:0 to
C18:1, whereas H. pylori produced mostly αCAG(C14:0) and H. nemestrinae produced
176–178 only αCAG(C14:0).
HO H HO O HO H H HO O OR1 cholesteryl α-glucoside (αCG) OR2 OH O P O O R3O H H HO O HO O HO H H HO H H HO HO O O cholesteryl 6-O-phosphatidyl-α-glucoside (αCPG) cholesteryl 6-O-acyl-α-glucoside (αCAG)
acyl chains O 3 R C12:0 O 1 2 3 R /R /R C14:0 O 1 2 3 R /R /R C16:0 O 1 2 3 R /R /R C18:0 O 1 2 3 R /R /R C18:1 O 1 2 R /R C18:2 O 1 2 R /R C19c:0
Figure 2.1. Various cholesteryl glucoside structures found in Helicobacter species grown on cholesterol rich media.176–178
None of the Helicobacter species studied have the ability to biosynthesize choles- terol, suggesting that these glycolipids are formed using cholesterol taken from the en- vironment, particularly the human host. Meyer and co-workers showed that H. pylori will actively move along a cholesterol gradient and in vivo it was shown to extract cho- lesterol directly from host cells.179 Fukada and co-workers found that inhibition of a key H. pylori α-glucosyltransferase (αCgT) responsible for glycosylating cholesterol lead to
32 RO H HO O HO H H HO O R acyl chains Helicobacter spp. O
C12:0 H. rappini, H. muridarum O
C14:0 H. pylori, H. rappini, H. nemestrinae, H. felis O
C16:0 H. pylori, H. rappini O
C18:0 H. pylori O
C18:1 H. rappini Figure 2.2. Distribution of acyl chain composition in αCAGs found in Helicobacter spp grown on cholesterol rich media.176–178 a significant decrease in growth potential of the bacteria.180 Ito and co-workers repor- ted a direct correlation between αCgT activity and gastric mucosal atrophy181 and that the glycosylation of cholesterol was essential for immune evasion in the gut epithelium. Fukuda, Nakayama and co-workers demonstrated that the gut mucosa produce α1,4-N- acetylglucosamine (α1,4GlcNAc)-capped core 2-branched O-glycans that competitively inhibit αCgT and thereby suppress H. pylori growth in the gut.180,182
The lipid profile of H. pylori cholesteryl glucosides changes post-infection
Jan and co-workers cultured standard and clinically isolated strains of H. pylori with an azide derivative of cholesterol either in isolation or co-cultured with a human stom- ach cancer (AGS) cell line (Figure 2.3).183 The azide tag was used to attach an ionized fluorophore using click-chemistry that was used to aid the quantification of cholesteryl glucoside lipoforms by HPLC/MS. They found that when standard H. pylori was grown in isolation it produced >95% of the C14:0 lipoform with small amounts (<2%) of each
176,178 of the C16:0 and C18:1 lipoforms, a finding concordant with previous reports. When co-cultured with AGS cells, the standard and four clinically isolated strains of H. pylori produced an expanded pattern of lipoforms, including C18:0,C18:2 (low abundance) and
C22:4 (low abundance), as well as producing much less C14:0 lipoform than in isolated
33 culture. This finding suggests that the repertoire of lipoforms reported for standard H. pylori grown on a plate may not represent those produced upon infection and present for recognition by the immune system.
N 3 N3
O O H. pylori H RO culture H Cu(I) HO O HPLC and MS analysis H H HO H H HO HO O
Isolated R acyl chains Co-cultured O > 94% C14:0 ~17% O < 5% C16:0 ~17% O
C18:0 ~20% O < 5% C18:1 ~35% O
C18:2 ~5% O
C22:4 ~10% Figure 2.3. Using an azide-labelled cholesterol derivative different lipoforms of acylated cholesteryl α-glucoside were observed for H. pylori grown in isolation or co-cultured with AGS cells.183
Reported immunological activity of cholesteryl glucosides from Helicobacter spp.
Ito and co-workers demonstrated a link between αCgT activity and gastric musical at- rophy and found that this effect was mediated by a CD1d-cholesteryl glucoside-type I NKT interaction.181 They found a 5-fold increase in bacterial recovery in Jα18-/- knock- out mice, which lack the ability to develop type I NKT cells, 10 days after inoculation with a H. pylori strain with high αCgT activity. Of the three classes of cholesteryl glycos- ides found in H. pylori, the αCAGs elicited the highest cytokine response when murine type I NKT cells were challenged with glycolipid loaded CD1d-tetramers.
NKT cells are known to be activated by influenza A and also to play a role in asthma.184 As well, infection with H. pylori is inversely correlated with childhood asthma.185 Chang and colleagues showed that influenza-induced protection against airway hyper-reactivity could be mimicked by inoculation with H. pylori lipids.186 Chang found that the pro-
34 tective effect relied on a CD1d-lipid-NKT cell interaction and the production of IFN-γ; this effect was not replicated by αGalCer, the archetypal type I NKT cell ligand, sug- gesting that a type Ia or type II NKT cell was responsible for the recognition of H. pylori glycolipids presented by CD1d.
Devi and colleagues reported molecular modelling findings demonstrating recog- nition of H. pylori LPS by Mincle.187 They proposed that an observed upregulation in Mincle and an associated release of anti-inflammatory cytokines may result from this interaction and aid in immune evasion by H. pylori.
2.1.3 Mincle recognizes a structurally diverse range of carbohydrate
and cholesterol containing molecules
Mincle, and specifically human Mincle, has been demonstrated by Kiyotake and co- workers to recognize the endogenous ligand cholesterol in both crystalline and plate- coated forms.80 This discovery arose from the observation that Mincle-dependent sig- nalling was induced by a fraction of liver-extracted lipids that was subsequently shown to contain cholesterol. This response was shown to be specific to Mincle and not other CLRs, to human Mincle and not rodent Mincle, and specific to cholesterol and not other steroids (Figure 2.4).80 The specificity for human Mincle arises from the presence of the cholesterol recognition/interaction amino acid consensus (CRAC) motif in the human Mincle sequence that is absent in rat and mouse sequences. Cells may be activated in a Mincle-dependent fashion by cholesterol sulfateb, another endogenous human lig- and.81 The agonism of Mincle signalling by cholesterol sulfate is thought to play a role in the sterile (non-infected) inflammatory response. Sterile inflammatory responses are an important step in the process of traumatic wound healing but are also implicated in a multitude of common diseases including allergies and some autoimmune diseases.188 In an allergic dermatitis model, the absence of Mincle correlated with a decrease in the magnitude of the inflammatory response.81
bYamasaki and colleagues have failed to independently reproduce this claim, personal communica- tion.
35 O O
OH O OH H H H
H H H H H H HO O O cholesterol cortisone progesterone
OH HO
H H H CO2H
H H H H H H HO HO OH OSO3 H estradiol cholesterol sulfate cholestanoic acid
O OH
H H H H H H H H H HO O HO dehydroepiandrosterone testosterone desmosterol (DHEA)
O O HO OH H H
H H H H H H HO O HO sitosterol aldosterone ergosterol
Mincle agonists Do not signal through Mincle
Figure 2.4. A range of sterol structures are agonists of human but not mouse Mincle.80,81
In addition to cholesterol and cholesterol sulfate, a wide range of carbohydrate (predominantly glucose derived) moieties also signal through Mincle. The best known Mincle ligand is the mycobacterial 6,6' -O-mycolate trehalose (TDM).48 Other mono- and di-acyl trehaloses can also signal through Mincle including TMM, TDCM, TMCM, TDB and TMB (Figure 2.5).70,189 A single glucose moiety acylated at O-6 is also known to be a ligand, however, in this case only α-branched fatty esters, such as the mycolates, can agonise Mincle signalling.189,190
36 OR HO O HO HO O RO O OR' HO O HO OH HO OH OH OH R = R' = fatty acid esters (TDB, TDM, TDCM) R = branched esters (GMM, GMCM) R' = H (TMM, TMCM)
Figure 2.5. Trehalose mono- and di-esters consisting of branched and straight chain fatty acids are agonists of Mincle signalling; as are glucose derivatives possessing a branched acid ester at O-6.48,189,190
2.1.4 Are Helicobacter spp. cholesteryl glucosides Mincle agonists?
The similarities between known Mincle agonists and the cholesteryl glucosides pro- duced by Helicobacter spp. are unmistakable. Although no Mincle agonists have yet been defined with both a carbohydrate and cholesterol moiety we hypothesise that these two common motifs combined, as in the H. pylori αCAGs, could result in signalling through Mincle (Figure 2.6).
RO H HO O HO H H HO O R acyl chains O
αCAG(C12:0) O
αCAG(C14:0) O
αCAG(C16:0) O
αCAG(C18:0) O
αCAG(C18:1) Figure 2.6. Synthesis of known acyl variants of the αCAGs from the different Helicobacter spp. would allow for evaluation as Mincle agonists.
2.1.5 Formation of cis-glucosides
The formation of cis-glucosides (α-glucosides), such as that between glucose and cho- lesterol in αCAG, has always presented a synthetic challenge and many methods have been devised to provide a solution. While good general strategies, most notably neigh-
37 bouring group participation that proceeds via cis-configured intermediates, have been successfully developed for the synthesis of trans-glucosides, compounds containing cis- glucosides remain a challenging synthetic target. There are four generalised approaches to attaining, with high stereo-selectivity, α-glucosides: intramolecular aglycon delivery, adjacent neighbouring groups able to form trans-configured intermediates, halide-ion catalysed glycosylations and variants thereof, and the use of non-adjacent participating groups.
Intramolecular aglycon delivery
Intramolecular aglycon delivery (IAD) was independently developed in the early nineties by the groups of Hindsgaul and Ogawa/Ito for the synthesis of β- mannosides.191,192 Bols soon after applied this approach to the synthesis of α- glucosides.193 IAD glycosylation works on the principle of tethering the aglycon to C-2 of the carbohydrate to form an adduct in which the aglycon is held close to the face on which it is to react. Upon activation of the glycosyl donor the tether is broken and the aglycon is ‘delivered’ and reacts at the desired face of the carbohydrate (Figure 2.7). A major problem with IAD is the synthesis of the complex adduct, including finding an appropriate linking group.
X OR' X RO RO RO HO XOR' O O RO O RO O RO O RO RO RO OR' LG LG Figure 2.7. β-Mannosylation driven by an IAD glycosylation. The aglycon is initially linked to O-2, and upon activation of the leaving group is selectively transferred to the same face of the carbohydrate.
A 2011 patent outlined the synthesis of αCAG(C14:0), the most abundant αCAG from H. pylori grown in isolation.194 The tethered precursor was synthesised in 5 steps from glucose and the IAD glycosylation delivered an 82% yield of protected cho- lesteryl α-glucoside. Further elaboration of this glycoside yielded αCAG(C14:0) in 4 steps (Scheme 2.1).
38 AcO benzenesulfinylpiperidine, AcO H AcO O Tf2O, di-tert-butylpyridine AcO O SPh AcO AcO H H O 82% HO Si O O Cholesterol
O 4 steps C13H27 O H HO O HO H H HO O
αCAG(C14:0) Scheme 2.1. Synthesis of cholesteryl α-glucoside using IAD with a silyl linker group.194
1,2-Trans-configured intermediates
Both the Boons and Turnbull laboratories have reported glycosylation methodologies involving the formation of ‘1,2-trans-decalin’-like intermediates (Figure 2.8).195,196 These glycosylations generate highly α-selective products in a manner similar to the widely employed cis-configured intermediates in the synthesis of β-glucosides using esters at C-2. Unfortunately, this methodology suffers from the same draw-back as IAD, that is, complex donor synthesis, as well as challenging auxiliary removal. RO RO RO NH Ph RO O RO O RO O RO 'activation' RO S HOR' RO O O CCl3 O O OR' Ph Ph Ph SPh PhS Figure 2.8. 1,2-trans glycosylation developed by the Boons and Turnbull groups.
Halide-ion catalysis
Although substitution of glucosyl halides has long been used in Koenigs-Knorr reac- tion, it was for many years applied only to the formation of β-glucosides due to the ease of formation of peracetylated α-glycosyl halides which provide anchimeric assistance.197 Lemieux and co-workers discovered that the addition of tetraethylammonium bromide to 2-O-benzyl-α-glucosyl bromides resulted in equilibration with the β-glucosyl brom-
39 ide (Figure 2.9A).198 The resulting β-glucosyl bromide is more reactive and rapidly un- dergoes nucleophilic displacement with an alcohol resulting in the α-glucoside with inversion of the stereochemistry (Figure 2.9B). The Curtin-Hammett principle can be invoked whereby the outcome of a reaction with two rapidly inter-converting starting materials that lead irreversibly to two different products is determined by the difference in energy between the two starting materials as well as the difference in free energy of the transition-states leading to each product. These observations led to the development of a glycosidation methodology that Lemieux termed “halide-ion catalysis”. A
O TBABr O HOR O PO PO Br PO Br OR
B H OR Br O O H H Br Br O OR H H Br
Energy O O Br O O OR Br OR
Figure 2.9. A The facile preparation of glycosyl iodide and glycosylation to form the cis-glycoside. B The proposed energetic landscape of a halide-ion catalysed glycosylation demonstrating the facile isomerization between α- and β-glycosyl halide followed by SN2-like substitution of the β-glycosyl halide to form the α-glycoside.
For many years only glycosyl bromides and chlorides were thought to be useful re- agents in carbohydrate chemistry, with glycosyl iodides considered too reactive to syn- thesize and handle. Two developments changed the thinking about glycosyl iodides: first, the finding that they could be easily synthesized from assorted precursors using iodotrimethylsilane (TMSI); and secondly, a report from Gervay-Hague demonstrating their use in halide-ion catalysed glycosylations.199,200 However, glycosyl iodides are not without their issues and represent technically demanding compounds to work with.
40 Gervay-Hague has reported mixed successes with α-glucosylations. A 2010 report applied per-TMS-glucosyl iodide to the synthesis of several glucosyl ceramides and diglycerides with good stereoselectivity but poor to moderate yields.201 More recently, several articles from the same laboratory disclosed the synthesis of αCAG and αCPG glycolipids from H. pylori.202–204 Initially, a microwave assisted glycosylation was com- plicated by partial deprotection of the 6-O-TMS group. This was overcome by first in- stalling the acyl group at O-6; unfortunately, the glycosylation only provided mod- erate α-selectivity (Scheme 2.2).202 More recently performing the glycosylation under non-‘microwave irradiation’ conditions enabled the synthesis of the cholesteryl gluc- oside in 80% yield with 39:1 α/β selectivity (Scheme 2.2).203 The 6-O-acyl group was installed by an enzymatic regioselective acylation using Novozym 435. While this acyl- ation methodology occurs with very high regioselectivity it requires a large excess of the fatty acid vinyl ester, limiting it to commercially available vinyl esters, and precluding its use for more complex substrates.
O O
C13H27 OH O C13H27 O HO O 1. Novozym 435 TMSO O HO TMSO HO OH 2. TMSCl, DMAP TMSO OTMS
1. TMSI 2. Cholesterol, DIPEA, TBAI 3. Dowex 50WX8-200 46% (8:1 α:β) O
C13H27 O H HO O HO H H O HO O O C13H27 Novozym 435, 80% (39:1 α/β) 1. TMSI OTMS 2. Cholesterol, OH H O TBAI, DIPEA TMSO HO O TMSO HO H H TMSO OTMS 3. Dowex 50WX8-200 HO O Scheme 2.2. Glycosyl iodides have been applied to the challenging synthesis of 202,203 α-glucosyl compounds including αCAG(C14:0) with mixed success. Studies in our own group have found that while glucosyl iodides are often effective glycosyl donors, many reactions lack reproducibility or occur in low yields.205–207
41 Non-adjacent participating groups
The final approach to achieving a cis-selective glycosylation is the utilization of remote (i.e. non-O-2) participating groups. Although α-stereoselectivity is sometimes achieved in a system with no participating groups (e.g. a tetrabenzylated donor), often a poor stereochemical outcome is obtained, influenced by the intrinsic facial selectivity in the reaction of the oxocarbenium ion intermediate with a nucleophile (Figure 2.10). O PO O 'activation' O+ OR PO PO HOR O LG PO
OR Figure 2.10. An activated glycosyl donor without anchimeric assistance forms a ’naked’ oxocarbenium species whose facial preference determines the stereochemical outcome of a glycosylation.
Incorporation of a remote group can alter the stereoselectivity of a glycosylation re- action by biasing the facial kinetic preference of the nucleophilic attack through either steric effects, stereo-electronic effects, or a combination thereof. The most commonly used donor of this type is that bearing all non-participating benzyl ether protecting groups. This donor is highly sensitive to all aspects of the reaction conditions includ- ing donor leaving group, nature of the nucleophile, activating reagent, and solvent. Presser and colleagues glucosylated cholesterol by reaction of a tetra-O-benzyl brom- ide donor promoted by AgOTf with cholesterol to achieve a 3:2 α/β ratio of the cho- lesteryl glucoside.208 Shirahata and colleagues reported that the glycosylation of choles- terol with a tetra-O-benzyl trichloroacetylcarbamate donor gave only 21:79 α/β selectiv- ity when activated with TMSOTf in EtCN; yet the same donor activated with TMSClO4 in Et2O gave 94:6 α/β selectivity, demonstrating the tight interplay between these factors.209 Seeberger and co-workers synthesised cholesteryl α-glucoside using a 2-O- tert-butyldimethylsilyl donor that yielded only 75% of a 1:1 α/β mixture (Scheme 2.3).180 These results demonstrate the sensitivity of non-participating donors to all aspects of glycosylation conditions.
42 H Ph O cholesterol, AgOTf Ph O O O O O H H TBSO SEt TBSO O TBSO sym-collidine, CH2Cl2 TBSO 75% (1:1 α/β)
Scheme 2.3. Glucosylation of cholesterol with a non-participating 2-O-TBS group gave the glucoside in 75% yield with a 1:1 α/β ratio.180
In 1972 Fréchet showed that glucosylations using donors bearing an acetate or ben- zoate at O-6 often gave higher α-selectivity than the equivalent O-6 benzyl donors. He proposed that an interaction, termed ‘vertical stabilization’, between an acetyl group at O-6 and a nascent oxocarbenium ion formed during activation of a glycosyl donor blocks the top-face of the donor and promotes α-stereoselectivity (Figure 2.11A).210 In this vein, Shingu and co-workers reported that 6-O-acetyl-2,3,4-O-benzylglucose hemi- acetal under Appel-like glycosylation conditions reacted with cholesterol providing a >99:1 α/β ratio of cholesteryl glucoside (Figure 2.11B); under the same conditions, tetra- O-benzylglucose only yielded a 9:1 α/β ratio of cholesteryl glucosides.211,212 Possibly, these conditions also incorporate aspects of halide-ion catalysed glycosylations. A O O 6 1 O B RO 1. Ph3P, CBr4 RO H BnO O 2. cholesterol BnO O BnO OH BnO H H BnO CH2Cl2 BnO O R = Bn 95% (90:10 α/β) R = Ac 95% (>99:1 α/β)
Figure 2.11. A Vertical stabilization of an oxocarbenium with a 6-O-acetyl group blocks the top face from attack by a nucleophile. B Influence of the 6-O-acetyl group was observed in the glycosylation of cholesterol under Appel-like conditions.211,212
Nifantiev and co-workers investigated the stereodirecting effect of esters at O-3 of glucosyl donors. They proposed a similar concept to vertical stabilization whereby the O-3 carbonyl oxygen stabilizes the transient oxocarbenium ion and partially blocks the
43 top face from attack (Figure 2.12A). In the same report, calculation of the energy dif- ference between the stabilized and non-stabilized structures identified a –11.3 kcal/mol “stabilization energy”.In spite of this, they found that on an otherwise benzylated donor, an acetate at O-3 had mixed effects on promoting α-stereoselectivity.213 however, when paired with an acetate at O-6, 3,6-di-O-acetyl donors provided better α-selectivity than the 6-O-acetyl donor on its own (Figure 2.12B).214 A more recent report from Nifant- ievs’ group explored a wider scope of aglycon structures and supported the earlier find- ings, but exemplified the highly variable nature of glycosylation outcomes when varying substrates, activators and solvents.215
A PO O O O 1 3 OP OBn
B OH OR1 BnO O 1 BzO OMP BnO O OR 2 NPh OBn R O BnO O BnO O R2O MeOTf, AW-300 O CF OBn 3 CH2Cl2 BnO O BzO OMP OBn tetrabenzyl R1 = Bn R2 = Bn 99% (1:3.3 α/β) 6-O-acetyl R1 = Ac R2 = Bn 94% (4:1 α/β) 3-O-acetyl R1 = Bn R2 = Ac 93% (1:2.3 α/β) 3,6-O-acetyl R1 = Ac R2 = Ac 97% (11.2:1 α/β)
Figure 2.12. Nifantiev and co-workers proposed that stabilization of an oxocarbenium ion with a 3-O-acetyl group blocks the top face from attack by a nucleophile.213
The van Boom group have used a 3,4,6-tri-O-acetyl-2-O-benzylglucosyl donor to achieve good α-selectivity when reacted with primary alcohols, which typically react with poor stereoselectivity owing to their high reactivity (Scheme 2.4).216,217 No ra- tionale was provided for the usage of these particular donors; however, the protecting group pattern has a history in older Koenigs-Knorr type glycosylations.218
44 AcO OTBDPS AcO HO AcO O AcO O AcO AcO NIS/TfOH BnO SEt AcO 1,2-DCE/Et2O O 83% (α only) OTBDPS
Scheme 2.4. van Boom used a 2-O-benzyl-3,4,6-O-acetyl glucosyl donor for a highly α-selective glucosylation of tert-butyldiphenylsilylglycol as part of the synthesis of adenophostin A analogues.217
Crich and colleagues investigated the role of remote participation of esters at O-3, O-4, and O-6 on a range of carbohydrates.219 Using a tert-butyloxylcarbonate to probe participation they found that a Boc group on the axial O-3 of an allose-configured donor could form the 1,3-O-cyclic carbonate ester, through loss of a tert-butyl cation (Scheme 2.5). This reaction was not observed for any of the other positions probed, and led them to conclude that under normal glycosylation conditions there is no participa- tion from equatorial O-3, axial/equatorial O-4 and O-6 esters.
BnO BnO BnO BnO O promotor O BnO O SPh BnO OC6H11 OBn C H OH BnO OBn OBoc 6 11 O O OBoc 1:11.2 α/β O Scheme 2.5. Crich and co-workers found that activation of a glycosyl donor bearing an axial 3-O-Boc protecting group resulted in the formation of the 1,3-O-cyclic carbonate ester though remote participation. This effect was not observed for other positions/steroisomeric Boc carbonates.219
Contrary to the findings of Crich, Yu and co-workers reported the formation of
3,6-di-O-acetyl-1,2,4-O-orthoacetyl-α-D-glucopyranose in 12% yield from the gold catalysed activation of a tetra-acetyl glucopyranosyl donor.220 They proposed two pos- sible pathways for its formation (Figure 2.13): the first invoked attack from O-4 on the 1,2-dioxolenium ion that is typically formed during glycosylation with 2-O-acetyl donor. The second involves the reverse, whereby O-2 attacks a 1,4-dioxolenium ion. Only the second pathway would be a demonstration of O-4 ester participation. The two possibilities were distinguished by synthesising donors bearing O-2 or O-4 d3-labelled acetates. Because both pathways involve the loss of acetaldehyde from the attacking es-
45 ter oxygen, survival of the deuterium label in the product would show it was from the ester that initially formed the orthoester. Using these donors, they demonstrated that the 1,4-dioxolenium ion is formed, as the deuterium label attached to O-4 was retained, while the label on O-2 was lost. AcO OAc Pathway 1 O x 2-CD3CO O D3C O O AcO OAc OAc O OAc AcO O AcO O O AcO LG AcO O OAc O OAc AcO O O OAc O CD Pathway 2 O 3 O 3-CD3CO O O
CD3 Figure 2.13. Yu and co-workers demonstrated remote participation of an O-4 acetate using a deuterium label to distinguish two potential participation pathways.220
The survey above highlights that it is possible to obtain high α-stereoselectivity by judicious choice of protecting groups and reaction conditions. However, it is difficult to compare these various examples as each uses different donors and acceptors under dif- ferent reaction conditions. To identify the most promising α-selective glycosyl donors that utilize remote participation, it is important to perform reactions using systematic- ally varied glycosyl donors with a common acceptor under identical reaction conditions.
2.1.6 N-Phenyl-trifluoroacetimidates are better glycosylation leaving
groups than trichloroacetimidates
Most glycosylation methodologies involve the activation of a leaving group on the sugar donor, usually with a Lewis acid promoter, resulting in the formation of a transient ox- ocarbenium ion. This transient intermediate can undergo a variety of intra- and inter- molecular interactions, and as previously discussed, many factors then influence the nucleophilic attack on the anomeric centre and formation of a glycoside. In the early 1980s, Schmidt and colleagues reported the development of a class of glycosyl donor
46 bearing a trichloroacetimidate (TCAI) leaving group.221,222 These donors are easily syn- thesised by reaction of a sugar hemiacetal with trichloroacetonitrile in the presence of inorganic or organic base. Schmidt’s initial report demonstrated activation of these donors with TsOH and BF3 · Et2O. Subsequent developments have seen a wide variety
223 224 225 of activators developed including TMSOTf, Tf2O, TfOH and I2\Et3SiH. TCAI methodology has proven to be a remarkably versatile glycosylation approach that has perfused modern carbohydrate chemistry.
Nonetheless, trichloroacetimidate methodology has some draw-backs. Firstly, after activation, the trichloroacetamide product can compete with the nucleophile in the glycosylation; this is especially problematic when the nucleophile has poor reactiv- ity. In the glycosylation of a model amide, acetamide, with a TCAI donor, 80% of the donor was converted to the adduct of trichloroacetamide rather than that of the amide (Scheme 2.6).226 Secondly, certain TCAI donors are unstable and must be prepared dir- ectly before use. This is especially true for 2-deoxy TCAIs.227
To alleviate these issues, Yu and co-workers developed a more stable leaving group, the N-phenyl trifluoroacetimidate (PTFAI).228,229 Activated under similar conditions to TCAI donors, PTFAI donors of 2-deoxy sugars are usually stable for moderate periods of time at 0 ◦C. Compared to trichloroacetamide the amide formed from a PTFAI donor is less nucleophilic (Scheme 2.6) and does not compete during glycosylation reactions. In addition, N-phenyltrifluoroacetamide is UV active enabling its detection on TLC; this aids in purification of the glycoside products. PFTAI donors are prepared by reac- tion of the sugar hemiacetal with the N-phenyl trifluoroacetimidoyl chloride (PTFAI- Cl, 1) in the presence of a weak base.
Preparation of N-phenyl trifluoroacetimidoyl chloride
The most widely used method for the preparation of 1 involves the reaction of aniline
230 with trifluoroacetic acid,3 Ph P and Et3N in CCl4 (Scheme 2.7). The “Montreal Pro- tocol on Substances that Deplete the Ozone Layer” is an international treaty signed in
1987 designed to protect the ozone layer and which legislated the phasing out of CCl4
47 1 2 TCAI (R = H, R = CCl3): BnO O OBn BnO OBn R2 = CCl (80% bosm) R1 3 O O 2 2 H N CH R = CH3 (42%) BnO O R 2 3 BnO N R2 PTFAI (R1 = Ph, R2 = CF ): BzO TMSOTf BzO 3 N O R2 = CF (0%) 1.5 equiv. R1 CH3NO2 3 2 R = CH3 (71%) Scheme 2.6. The glycosylation of acetamide with a TCAI donor gave predominantly glycosylated trichloroacetamide rather than glycosylated acetamide. The use of a PTFAI donor showed no glycosylation of the leaving group due to its lower nucleophilicity.226
production owing to its highly potent greenhouse gas and ozone depleting properties.
231 In addition, CCl4 is a very potent hepatotoxic agent. Increasingly, CCl4 is difficult to source and while most laboratories maintain legacy stocks, its use into the future is un- tenable. These points led us to investigate alternate synthetic options for the preparation of 1.
NH2 O Ph P, Et N 3 3 N F C OH 3 CCl4 CF3 PSfrag replacements Cl 1 Scheme 2.7. Reaction of aniline and trifluoroacetic acid with triphenylphosphine and 230 Et3N in CCl4 provides a facile synthesis of PTFAI-Cl.
Although many strategies have been developed for the synthesis of acetimidoyl chlorides, most have not been applied to the electron-poor trifluoroacetate system.232 Strategies that have been applied to the synthesis of acetimidoyl chlorides include the
previously mentioned Ph3P/Et3N/CCl4 system, reaction of amides with PCl5, POCl3
232 or SOCl2, and reactions of an alkyl nitro functionality with LiOMe/TiCl4. Still, sev- eral alternative strategies for the synthesis of PTFAI-Cl have been reported. Norris and Jonassen sought to convert N-phenyltrifluoroacetamide into the corresponding acetim-
idoyl chloride using PCl5; low yields were obtained with a diazadiphosphetidine formed as the major product.233 This result was replicated by another group using similar condi- tions.234 Chen and co-workers reported the conversion of this amide to the acetimidoyl
chloride using POCl3 under high temperature and high-pressure autoclave conditions,
48 which are not readily applied in the research laboratory.235 A 2006 patent outlined an approach from the amide using the chlorinating reagent (PhO)2POCl and attaining a 81% yield of the chloride.236
2.1.7 Research aims
We plan on synthesizing acyl variants of the cholesteryl 6-O-acyl α-glucosides (αCAG) from Helicobacter spp., and in particular H. pylori. We plan to prepare the C12:0,C14:0,
C16:0,C18:0 and C18:1 O-6 esters. This set of compounds presents two primary synthetic challenges. First is attaining, with high stereoselectivity, the α-anomer; second is the regioselective installation of the fatty acid esters on O-6. Towards the first challenge, we elected to prepare various glucosyl donors bearing different arrangements of acetyl protecting groups to explore the effect of remote participation on the stereochemical outcome of our glycosylation. These donors will bear a common PTFAI leaving group.
Aim 1: Develop a new approach for the preparation of PTFAI-Cl that avoids the highly toxic greenhouse pollutant CCl4.
Aim 2: Prepare a set of common glucosyl donors bearing a common PTFAI cleaving group and varying arrangements of remote acetyl groups and assess their ability to con- fer high α-selectivity through the glycosylation of a common alcohol, cholesterol. This will allow us to identify the optimal protecting group arrangement for synthesis of the αCAG compounds as well as probe the role of remote acyl groups in controlling or in- fluencing the stereochemical outcome of this glucosylationScheme ( 2.8).
RO Ph O N TfOH, conditions RO H RO H RO RO O BnO O CF3 H H RO H H BnO HO O Scheme 2.8. Glycosylation of cholesterol utilizing differently acetylated donors under the same conditions will allow us to assess the effect of remote acetyl participation on the stereochemical outcome of the glycosylation.
Aim 3: Synthesise known acyl variants of the αCAGs from the various Helicobacter spp.
We propose to undertake the synthesis of the C12:0,C14:0,C16:0,C18:0 and C18:1 αCAGs
49 isomers.
Aim 4: Investigate the ability of αCAGs to stimulate Mincle signalling in collaboration with Yamasaki and colleagues at Kyushu University. This will help to further elucidate the complex immunological interplay between a human host and H. pylori.
2.2 Results and Discussion
2.2.1 A new CCl4-free preparation of N-phenyl trifluoroacetimidoyl chloride
We elected to synthesise PTFAI-Cl (1) from N-phenyltrifluoroacetamide 2. Treatment
237 of aniline with (CF3CO)2O in CH2Cl2 afforded 2 in very good yield (Scheme 2.9). H O O NH2 a N CF3 F C O CF PSfrag replacements 3 3 86% O 2
◦ Scheme 2.9. Preparation of 2. a) CH2Cl2, 0 C -> rt.
We first investigated the treatment of 2 with the common dehydrative chlorinating
238 reagent SOCl2, which has been employed successfully for other imidoyl chlorides. A report from Chandler and co-workers demonstrated the applicability of this system to the non-fluorinated derivative of 2, with clean conversion to the imidoyl chloride in
239 75% yield. Reflux of 2 in neat SOCl2 containing catalytic DMF (Scheme 2.10) did not result in formation of imidoyl chloride (monitored by TLC). To confirm that 2 was
amenable to conversion to the imidoyl chloride, it was treated with Ph3P and Et3N in
CCl4 and saw complete conversion to the chloride (by TLC), affording 1 in 85% yield. a H N CF3 N CF3 PSfrag replacements b O Cl
2 1
Scheme 2.10. Attempted synthesis of 1 using SOCl2 was unsuccessful; we confirmed that 2 was amenable to conversion using the reported CCl4 conditions. a) SOCl2, cat. DMF, reflux b) Ph3P, Et3N, CCl4, reflux, 85%.
50 A detailed literature review of the reported Ph3P/Et3N/CCl4 system found it to be based on conditions originally reported by Lee for the formation of acyl chlorides.240 Appel, well known for his name-sake reaction, had subsequently reported the conver- sion of amides into the corresponding imidoyl chlorides under similar conditions.241 In a 1975 review, Appel reported a wide range of dehydrating and chlorinating applications
242 of the Ph3P/CCl4 system. Appel proposed that these reactions proceed through a mechanism involving the
+ + - 243 abstraction of Cl from CCl4 by Ph3P, which rearranges to afford [Ph3PCCl3] Cl .
+ - While it was initially postulated that [Ph3PCCl3] Cl was the reactive interme- diate responsible for the chlorination of substrates it was subsequently shown that this salt reacts rapidly with a second equivalent of Ph3P to form the ylide
Ph3P – CCl2 and dichlorotriphenylphosphorane Ph3PCl2. The ylide undergos reaction with a third equivilent of Ph3P resulting in an overall transformation of 3 PhP3 +
+ - CCl4 –> [Ph3P – C(Cl) – PPh3] Cl + Ph3PCl2. Both Ph3PCl2 and the intermediate
+ - [Ph3PCCl3] Cl can effect chlorination reactions and both pathways are believed to be involved (Scheme 2.11).
Cl Ph3P CCl3 A + - + - Ph3P CCl4 Ph3P Cl CCl3 [Ph3PCl] CCl3 [Ph3PCCl3] Cl Ph3PCl2
Ph3P Ph3P CCl2 B + + - Ph3P + - [Ph3PCCl3] Cl Ph3P [Ph3C(Cl)PPh3] Cl X Ph PO RX R CHCl3 RCl 3 Cl- X = carbonyl, alcohol, amine Scheme 2.11. A Proposed mechanism for the formation of the active chlorinating species from the reaction of Ph3P and CCl4. B Formation of the favourable P-X (X = O, N) bond followed by substitution by Cl- enables the synthesis of chlorides from alcohols and amines as well as carbonyl containing substrates.
Several groups have reported using alternate ‘Cl’ sources to CCl4 under similar re- action conditions. Most of these reports involve the conversion of alcohols to chlorides
51 or carboxylic acids to their corresponding acyl chlorides. Magid and co-workers repor- ted substitution of CCl4 with hexachloroacetone (HCA) for the conversion of allylic
244 alcohols into chlorides. While they found that HCA was able to substitute for CCl4 with similar yields, they were disappointed to find that their products contained a small
CCl4 impurity; the mechanistic origin of this CCl4 was unclear. Villeneuve and Chan extended this work by employing HCA as a CCl4 substitute in the synthesis of acyl chlor- ides.245 The method was used in a one pot synthesis of N-benzylbenzamide and N-4- methylphenylformamide in very high yield (Scheme 2.12). Unlike Magid, who used HCA in excess as the solvent, Villeneuve and Chan used a stoichiometric quantity of HCA to enact their transformations.
O O O HCA, Ph3P Ph NH2
Ph OH pyridine, THF Ph Cl 99% Ph N Ph H -78 °C
O O TCA, Ph3P OH Cl CH2Cl2 90% Br Br
Scheme 2.12. Both HCA and TCA have been used as substitutes for CCl4 in the Ph3P promoted preparation of acid chlorides.245–247
Kim and co-workers have published multiple articles investigating the use of tri-
246,247 chloroacetonitrile (TCA) as an alternative to CCl4. They reported its use for the preparation of acid chlorides as well as the one pot synthesis of amides and esters (Scheme 2.12). Like Villeneuve, TCA was employed in stoichiometric amounts. In ad- dition, other workers have reported on the use of cyanuric chloride248 or tetramethyl- α-chloroenamine249 for the preparation of acid chlorides. In the latter case, the require- ment of phosgene for the preparation of tetramethyl-α-chloroenamine makes this re- agent incongruous with our aim of avoiding a highly toxic reagent.
We opted to investigate whether TCA, a readily available reagent in a carbohydrate chemistry laboratory, could replace CCl4 in the conversion of 2 to 1. Under identical conditions to those for the CCl4 reaction, that is, with TCA used as reaction solvent,
52 we observed only trace amounts of PTFAI-Cl (Scheme 2.13). The stoichiometric con- ditions established by the work of Kim and co-workers for acid chloride synthesis were also unsuccessful. Attempts at optimizing this reaction through varying stoi- chiometry, as well as reaction temperature and time failed to drive this reaction to com- pletion. In this regard, it is interesting to note that Kim reported the synthesis of N- phenyltrifluoroacetamide by reaction of aniline and TFA under TCA/Et3N/Ph3P con- ditions similar to those reported for the original multicomponent synthesis of PTFAI-Cl using CCl4/Et3N/Ph3P, yet did not report the formation of imidoyl chloride, PTFAI-Cl, suggesting this is a less potent chlorinating system.250 H N CF3 a N CF3 PSfrag replacements O Cl
2 1 Scheme 2.13. Our attempt to synthesis 1 using TCA provided only trace amounts of PTFAI-Cl. a) TCA, Ph3P, Et3N, reflux.
Appel had shown that the phosphorane Ph3PCl2 can replicate the reactivity of the
243 Ph3P/CCl4 system. Ph3PCl2 is a cheap, commercially available reagent synthesised during the recycling of Ph3P – O back to Ph3P after it is generated as part of the Wittig
251 252 reaction. Ph3P – O can be converted to Ph3PCl2 by reaction with Cl2. The phos- phorane can then be reduced back to Ph3P by treatment with elemental P,a process that also produces PCl3 as a valuable by-product.
Appel reported that treatment of a small set of amides with either Ph3P/CCl4 or
Ph3PCl2, both in the presence of Et3N, provided essentially equivalent yields of the im-
241 idoyl chlorides. In our hands treatment of 2 with Ph3PCl2 and Et3N in MeCN at re- flux resulted in almost complete transformation to 1 as determined by TLC. Optimiz- ation of this procedure demonstrated that complete consumption of the amide could be achieved with 2.5 equivalents of Ph3PCl2 (Scheme 2.14). Work-up was hampered by the large excess of Ph3P – O formed, removal of which could be achieved by sequential filtrations following the addition of hexanes. Purification of 1 was achieved by vacuum distillation or by flash chromatography, the latter providing slightly higher yields.
53 H N CF3 a N CF3
O 61% Cl 2 1
H NH2 O N CF3 N CF3 PSfrag replacements a F3C OH O Cl 2 1 Scheme 2.14. Dichlorotriphenylphosphorane was successful in driving the transformation of 2 to 1. a) Ph3PCl2, Et3N, MeCN, reflux.
Our successes with using dichlorophosphorane led us to attempt a two-step/one-pot procedure as we surmised that this reagent should be able to effect the transformation of TFA and aniline into the amide via an acid chloride intermediate and then effect the already demonstrated transformation of amide into PTFAI-Cl. Unfortunately, even with 3 equivalents of phosphorane 1 was not observed; however, aniline and TFA were cleanly transformed into the amide (Scheme 2.14). Additional reagent should enable
this second transformation to occur; however, the large amounts of Ph3P – O generated in doing so make this transformation impractical.
2.2.2 Donors for the comparative glycosylation of cholesterol
Weproposed the synthesis of a set of common glucosyl donors to systematically evaluate the best acetyl/benzyl protecting pattern to confer high α-glucosylation selectivity. All four donors will bear a 2-O-benzyl group and combinations of acetyl/benzyl protecting groups on the other positions. Herein, each of these donors, bar the tetra-benzyl (3), has been named after the major contributor to its design; the Frèchet (6-O-acetyl, 4), the Nifantiev (3,6-di-O-acetyl, 5) and the van Boom (3,4,6-tri-O-acetyl, 6)(Scheme 2.15).
The simplest of our donors is that bearing all benzyl protecting groups. Its prepara-
tion involved reaction of 2,3,4,6-tetra-O-benzyl glucose (7) with 1 and K2CO3 in acet- one, affording tetra-benzyl donor 3 in 95% yield (Scheme 2.16).
The Frèchet donor was accessed through mild acetolysis of 7 using 1 molar equival-
54 BnO Ph AcO Ph BnO O N BnO O N BnO BnO BnO O CF3 BnO O CF3 tetra-benzyl “Frèchet” 3 4
PSfrag replacements AcO Ph AcO Ph BnO O N AcO O N AcO AcO BnO O CF3 BnO O CF3 "Nifantiev" “van Boom" 5 6 Scheme 2.15. Four proposed glycosyl donors for comparison of their ability to perform an α-selective glucosylation of cholesterol.
BnO BnO a Ph BnO O BnO O N PSfrag replacements BnO BnO 95% BnO OH BnO O CF3 7 3
Scheme 2.16. a) 1,K2CO3, MeOH, rt.
253,254 ent of TsOH in Ac2O to afford diacetate 8 in 61% yield (Scheme 2.17). Anomeric deacetylation utilizing benzylamine afforded the hemiacetal 9 in 78% yield. Treatment
of 9 with PTFAI-Cl and K2CO3 in acetone afforded the Frèchet donor 4 in 95% yield.
BnO AcO a BnO O BnO O BnO BnO 61% BnO OH BnO OAc 7 8 PSfrag replacements AcO AcO b c Ph BnO O BnO O N BnO BnO 78% 95% BnO OH BnO O CF3 9 4
Scheme 2.17. a) TsOH, Ac2O, reflux; b) BnNH2, THF, rt; c) 1,K2CO3, MeOH, rt.
A key step to the synthesis of remaining two donors was the selective installation of the 2-O-benzyl group. Several options are reported for this transformation including the use of stannylene-acetals for regioselective benzylation and the selective removal of a 2-O-acetyl group with piperidine.218,255 We opted to conduct the 2-O-selective ben- zylation under phase-transfer conditions, as first reported by Garegg and colleagues.256
55 While the procedure of Garegg et al. occurs in moderate yield, it is operationally simple and starts from easily available starting materials enabling large quantities of product
to be readily prepared. Methyl 4,6-O-benzylidene-α-D-glucoside (10) was prepared in
75% yield on a 20 g scale by reaction of methyl α-D-glucoside with benzaldehyde di- methyl acetal and camphorsulfonic acid in MeCN (Scheme 2.18). Treatment of 10 with
benzyl bromide and tetrabutylammonium hydrogensulfate in a water/CH2Cl2 mixture afforded 11 in 50% yield.
HO Ph O Ph O a O b O HO O O O PSfrag replacements HO HO HO 75% 50% HO HO BnO OMe OMe OMe 10 11
Scheme 2.18. a) PhCH(OMe)2, camphorsulfonic acid, MeCN, reflux; b) BnBr,4 Bu NHSO4, 5% NaOH soln., CH2Cl2, reflux.
With 11 in hand, the Nifantiev donor was synthesised starting with regioselective opening of the benzylidene acetals. Unlike approaches using Lewis acids, metal triflates can be used in genuinely catalytic amounts rather than stoichiometric or excess, and
have improved water stability. Shie reported that Cu(OTf)2 (5 mol%) with BH3.THF
regioselectively cleaved methyl 2,3-di-O-benzyl-4,6-benzylidene-α-D-glucopyranoside
into methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside in 95% yield.257 Treatment of 11
with Cu(OTf)2 in BH3.THF afforded 12 in 94% yield (Scheme 2.19). Acetolysis of 12
with TsOH/Ac2O gave the tri-acetate 13 in 95% yield. Anomeric deacetylation with
benzylamine gave 14 and treatment with PTFAI-Cl and K2CO3 afforded the Nifantiev
PSfrag replacementsdonor 5 in very good yield (92%). Ph O HO AcO AcO O a b d Ph O O O O N HO BnO BnO BnO 94% HO 95% AcO 92% AcO BnO OMe BnO BnO OR OMe BnO O CF3 R = Ac 13 11 12 c 5 67% R = H 14
Scheme 2.19. a) 10% Cu(OTf)2, BH3.THF; b) TsOH (1.5eq), Ac2O; c) BnNH2, THF; d) 1, K2CO3, acetone.
The van Boom donor was prepared from 11 by performing an acetolysis to cleave both the acetals and acetylate the free hydroxyls affording the tetra-acetate 15 in 79%
56 yield (Scheme 2.20). Anomeric deprotection using hydrazine acetate, followed by treat- ment with 1, afforded the van Boom donor 6 in excellent yield.
Ph O AcO AcO a c Ph O O PSfrag replacements O AcO AcO O N HO 91% AcO 94% AcO BnO OR OMe BnO BnO O CF3 11 6 b R = Ac 15 79% R = H 16
Scheme 2.20. a) TsOH (2eq), Ac2O; b) N2H4.AcOH, DMF; c) 1,K2CO3, acetone.
2.2.3 Comparative glycosylation of cholesterol to evaluate influence
of remote acetate protecting groups
We initially envisioned conducting the glycosylation study under similar conditions
215 to those reported by Nifantiev; namely, CH2Cl2 and acid washed molecular sieves (AW-300) at -35 ◦C. To our surprise we saw activation of the tetra-benzyl donor by the AW-300 sieves at rt during setup for the glycosylation. Adinolfi and co-workers have reported the use of AW-300 sieves as activators for trihaloacetimidate donors; typic- ally, long reaction times or elevated temperatures were required.258 This led us to use non-acid washed sieves to preserve donor integrity prior to activation.
We chose to investigate CH2Cl2, Et2O and a 4:1 pet. spirits/toluene mixture as
solvents. Et2O is a widely used glycosylation solvent for performing cis-glycosylations due to a purported solvent participation mechanism involving an equatorial diethylox-
onium ion intermediate; this intermediate is propose to react in an SN2 fashion lead- ing to preferential formation of the α-glucoside. Wu and colleagues reported almost
1 complete α selectivity (trace β by H-NMR) when using Et2O for the glucosylation of
259 isopropylidene-sn-glycerol, while under identical conditions but in CH2Cl2 a 5:1 α/β
ratio was obtained. We also investigated pet. spirits/toluene as an alternative to CH2Cl2 as a non-participating solvent system in which cholesterol exhibited good solubility.
Our first glycosylation experiment using tetra-benzyl donor and TfOH in each
of these solvents highlighted poor solubility of cholesterol in both Et2O and 1:4 pet.
57 O O PO
O 'activation' O PO PO LG OR O+ O PO
HOR
Scheme 2.21. It is proposed that Et2O improves α-selectivity via the formation of a equatorial complex with the activated donor; SN2 substitution leads to the α-glycoside. spirits/toluene at -35 ◦C. Upon warming to 0 ◦C the solubility of cholesterol improved sufficiently for the glycosylation reaction to occur. While2 Et O provided moderate α- selectivity (4:1), both CH2Cl2 and pet. spirits/toluene provided no selectivity (1:1) at
◦ 0 C(Table 2.1). This result led us to pursue the use of2 Et O for our evaluation of remote acetylation on the stereo-outcome of our glucosylation of cholesterol.
BnO cholesterol, TfOH Ph solvent, 0°C BnO H O N BnO BnO O BnO BnO H H BnO O CF 3 BnO O Solvent α/β ratio (approx.)
CH2Cl2 1:1 Pet. spirits/toluene 1:1 Et2O 4:1
Table 2.1. Stereoselectivity of the glucosylation of cholesterol with 3 in different solvents.
We elected to perform the glycosylation reactions using donors 3, 4, 5 and 6 at 0 ◦C, in Et2O, activated by TfOH. The four donors under these conditions demonstrated a trend of improving α-selectivity as the number of acetyl groups on the donor increased (Table 2.2). The tri-O-acetyl van Boom donor 6 provided the highest α-selectivity with a 14:1 α/β ratio. Flash chromatography, followed by repeated crystallization, afforded the pure α anomer of cholesteryl glucoside 17 in 88% yield.
58 BnO Ph AcO Ph BnO O N BnO O N BnO BnO BnO O CF3 BnO O CF3 PSfrag replacements 3 4 cholesterol, TfOH H R1O Et2O, 0°C 2 O R O H H R3O AcO AcO Ph Ph BnO O BnO O N AcO O N AcO AcO BnO O CF3 BnO O CF3 5 6
Donor α/β ratio Yield (%) Per-benzyl 3 4:1 91 Frèchet 4 8:1 97 Nifantiev 5 10:1 93 van Boom 6 14:1 88
Table 2.2. Glucosylation of cholesterol with four glucosyl donors under identical conditions.
We also sought to confirm that Et2O was providing a stereodirecting effect to the outcome of the glycosylation using the best performing van Boom donor, 6. Perform-
ing the glycosylation in CH2Cl2, only a 3:1 α/β ratio was attained. Cooling the reaction to -40 ◦C, provided a marked improvement in selectivity, yet did not surpass that of
◦ the reaction performed in Et2O at 0 C(Table 2.3). This supported our initial observa- tion that the stereochemical outcome of this glucosylation benefits from the presence
of Et2O.
Temperature α/β ratio 0 ◦C 3:1 -40 ◦C 10:1
Table 2.3. Glycosylation of cholesterol with the van Boom donor 6 in CH2Cl2 at different temperatures.
2.2.4 Removal of the 2-O-benzyl protecting group
Benzyl ethers are one of the most common protecting groups used in oligosaccharide and carbohydrate synthesis, and as such, much work has been done on the development of conditions for their removal. The most commonly employed methodology involves
260 the use of H2 and catalytic Pd. However, the unsaturation in cholesterol precludes the
59 use of H2/Pd. The removal of benzyl groups under conditions that retain the alkene was therefore investigated.
The oxidant 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) has been applied to the removal of benzyl ethers. During their synthesis of the polyether antibiotic, sa- linomycin, Oikawa and co-workers attempted to remove a para-methoxybenzyl group with DDQ but discovered concomitant deprotection of a tertiary benzyl ether.261 This unexpected reactivity, which was subsequently extended to secondary benzyl ethers, was exploited by other groups to allow orthogonal protecting group manipulations.262–264 Crich and co-workers reported that DDQ allowed the selective removal of 4-O-benzyl
265 ethers on a range of carbohydrate structures. Crich noted that wet CH2Cl2 was ne- cessary for the reaction to occur. The reaction likely proceeds through a single electron transfer process that involves abstraction of one of the benzylic protons followed by subsequent hydrations and elimination of benzaldehyde.
We investigated the use of DDQ for the debenzylation of 17. Initial attempts follow- ing a protocol similar to that reported by Crich showed only a trace of deprotected 18.265 We suspected that our 2-O-benzyl group was more stable than those reported by Crich
and moved to the higher boiling ClCH2CH2Cl as the solvent allowing us to increase the reflux temperature. After considerable optimization it was found that 7 equiv of DDQ were required to complete the transformation; unfortunately, under these conditions only 10-20% yields of debenzylated product could be isolated (Scheme 2.22).
a AcO H AcO H PSfrag replacements AcO O 10-20% AcO O AcO H H AcO H H BnO HO O O 17 18
Scheme 2.22. a) DDQ, wet ClCH2CH2Cl, reflux.
At this juncture we entertained the possibility of replacing our 2-O-benzyl group with a protecting group with better orthogonality to an alkene. Two possibilities were the para-methoxybenzyl ether or the 2-naphthylmethyl ether, both of which can be
60 cleaved readily under oxidative conditions owing to their lowered oxidation potentials. We were however unsure what effect modification of the substituent at O-2 would have on the stereochemical outcome of our glycosylation. Resynthesis of all of the donors for re-evaluation with a new protecting group seemed counter to the aim of establish- ing the effect of the commonly used acetyl and benzyl protecting groups. Therefore, we returned to investigate other non-reductive methods for benzyl removal.
AlCl3/N,N-dimethylaniline has been reported by Akiyama and colleagues as a mild method to remove benzyl and allyl ethers but this system showed no reactivity towards 17 even with long reaction times (Scheme 2.23); we hypothesise this could be due to the abundance of coordinating oxygens present in the substrate that can sequester the Lewis
266 267 acid. Adinolfi and co-workers reported debenzylation using 3NaBrO /Na2S2O4.
Unfortunately, under NaBrO3/Na2S2O4 conditions we observed significant by-product formation due to the formation of a bromohydrin across the double bond.268,269
AcO a H AcO O NR AcO H H HO O AcO H 18 AcO O AcO H H BnO O b 17 AcO H PSfrag replacements AcO O AcO H H HO O OH Br
Scheme 2.23. a) AlCl3, N,N-dimethylaniline, CH2Cl2, rt; b) NaBrO3, Na2S2O4, EtOAc, water, rt.
61 While traditional hydrogenation with H2 is not selective for debenzylation over al- kene saturation, there are reports of selective debenzylation when using catalytic trans-
fer hydrogenation. Hannesian and co-workers used Pd(OH)2/C with cyclohexene as the hydrogen transfer source to effect the cleavage of benzyl ethers from a range of carbo- hydrates.270 Presser and colleagues used this method for the removal of benzyl groups from cholesteryl glucosides while maintaining the integrity of the cholesterol double bond.208 We applied this methodology to the deprotection of 17 and isolated 18 in 91% yield (Scheme 2.24). Zemplén transesterification of the acetates then afforded us cho- lesteryl α-glucoside 19 in 92% yield.271
AcO RO H a H AcO O RO O AcO RO H H 92% H H BnO HO PSfrag replacements O O 17 R = Ac 18 b 92% R = H 19
Scheme 2.24. a) Pd(OH)2/C, cyclohexene, EtOH; b) NaOMe, MeOH/THF.
2.2.5 Installation of the 6-O acyl chain
Completion of the synthesis of the αCAGs required a means to install the acyl chains on to unprotected 19. Grindley and colleagues have published several reports on O- 6 selective acylations utilizing uronium based peptide coupling reagents.272–274 Other groups have looked at the use of Mitsonobu chemistry to selectively form esters with primary alcohols in the presence of secondary alcohols.275
We chose to investigate a report by Baczko and colleague who demonstrated the use of 3-acyl-thiazolidine-2-thiones and 3-acyl-5-methyl-1,3,5-thiadiazole-2(3H)-thiones as primary selective carbohydrate acylating reagents.276 3-Acyl-thiazolidine-2-thiones have previously been reported as efficient amide bond forming reagents in the con- text of peptide synthesis and for the preparation of amide alkaloids.277,278 Baczko et al. proposed that the selectivity achieved under the reaction conditions (NaH, catalytic
62 DMAP) resulted from an equilibration of an O-2 centred anion, via intermolecular hy- drogen bonds, to the more sterically accessible O-6 anion, which is acylated by the bulky, loose ion-pair 20 formed with DMAP (Scheme 2.25). HO O- H O O O O H O O O- H HO H OMe OMe
SH O N N N S PSfrag replacements Acyl N 20
Scheme 2.25. Baczko proposed that 20 could only effectively acylate O-6 due to steric bulk.276
We took the lead from Nagao and co-workers and reacted myristic acid with 2-mercapto-5-methyl-1,3,4-thiadiazole in the presence of DCC and isolated reagent 21 in 80% yield.279 A model glycoside, methyl α-glucopyranoside, was treated with 21 but afforded a complex mixture of products (Scheme 2.26); as a result, this approach was abandoned. SH O S a N N S 12 S N 80% N
21 O
HO 12 O HO O b HO O PSfrag replacements HO HO HO HO OMe OMe
Scheme 2.26. a) Myristic acid, DCC, CH2Cl2, rt; b) 21, DBU, pyridine
Gervay-Hague’s synthesis of H. pylori αCAG(C14:0) relied on the use of an enzymatic approach for the selective installation of the O-6 myristoyl chain.203 As previously dis- cussed this approached is limited to the available fatty acid vinyl esters, which do not include all of those we are interested in studying. A report from the same lab utilized a selective TMS deprotection methodology to gain access to a free hydroxyl at O-6.204
63 Tetra-O-TMS protected glucoside obtained after iodide glycosylation was subjected to
6-O-TMS selective deprotection using NH4OAc. We investigated whether the method used by Gervay-Hague et al. was optimum for the selective deprotection of the 6-O- TMS verses other methods used for similar transformations. Monitoring side-by-side
deprotections of 22 with NH4OAc, AcOH and K2CO3 by TLC revealed that AcOH pro- moted very rapid deprotection of the 6-position along with partial deprotection of the
◦ secondary alcohols even at 0 C; K2CO3 also demonstrated deprotection of the primary
and secondary positions. NH4OAc, while also fast, provided a clean and selective de- protection of the 6-O-TMS group. Consequently, we applied Gervay-Hague’s approach:
TMSCl/Et3N afforded the tetra-TMS derivative; treatment with NH4OAc effected se- lective removal of the 6-O-TMS affording 23 in 68% yield (Scheme 2.27).
HO HO H a H HO O TMSO O PSfrag replacements HO H H 68% TMSO H H HO TMSO O O 19 23
Scheme 2.27. a) i. TMSCl, Et3N, CH2Cl2 ii. NH4OAc, CH2Cl2/MeOH
Acylation of 23 was achieved by reaction with the appropriate acyl chloride in the presence of pyridine. Due to the poor stability of TMS ethers to handling we imme- diately treated the protected compound with Dowex 50WX8-200 strong acid resin in
methanol to cleave the TMS groups. In doing so we were able to synthesise the C12:0,
C14:0,C16:0,C18:0,C18:1 αCAG’s in good yield (Scheme 2.28).
64 HO 2 H a R O H O TMSO R1O O TMSO 1 H H R O H H TMSO R1O O O 23 R1 = TMS b R1 = H R2 acyl chains O
24 (C12:0) 73% PSfrag replacements O
25 (C14:0) 79% O 26 (C16:0) 67% O
27 (C18:0) 72% O
28 (C18:1) 63%
2 Scheme 2.28. a) R Cl, DMAP, pyridine/CH2Cl2; b) Dowex 50WX8, MeOH/CHCl3.
2.2.6 Helicobacter spp. αCG and αCAG’s as Mincle ligands
The ability of αCG and the αCAGs to induce signalling through Mincle was investigated in the laboratory of Sho Yamasaki (Division of Molecular Immunology, Kyushu Univer- sity, Japan).c Yamasaki and co-workers have engineered a nuclear factor of activated T cells - green fluorescent protein (NFAT-GFP) T cell hybridoma that is activated by sig- nalling through Mincle. NFAT are a group of transcription factors that are involved in immune responses. These cell lines have been developed for both human and mouse Mincle, and express Mincle as a Mincle-FcRγ fusion. The hybridoma produces GFP when Mincle binds an activating ligand, in turn allowing an indirect measure of ligand potency from the intensity of fluorescence as measured by flow cytometry. This cell line has been crucial in identifying the Malassezia Mincle ligands, βGlcCer, and cholesterol as well as confirming TDB and cord-factor as Mincle signalling agonists.49,280
Firstly, we investigated whether non-acylated cholesteryl α-glucoside (αCG) acts to agonise signalling through human Mincle (hMincle) and mouse Mincle (mMincle) NFAT-GFP hybridomas. We observed with interest that plates coated with αCG, which
cNFAT-GFP reporter cell assays were performed by Shojiro Haji and Masahiro Nagata.
65 is also found in the Helicobacter spp., caused cellular activation (Figure 2.14). In this instance mMincle was significantly less stimulated than hMincle. This result is not unex- pected given hMincle is known to be stimulated by crystalline cholesterol while mMincle was unresponsive;80 however, it raises the possibility that this cholesteryl glucoside is not being recognised in the same way as cholesterol (ie. through the CRAC motif). 60 50 40 30 20 10 NFAT-GFP NFAT-GFP (%) 0 0.016 nmol 0.08 nmol 0.4 nmol 2 nmol αCG
FcRγ only mMincle-γ hMincle-γ
Figure 2.14. NFAT-GFP reporter cells expressing either human Mincle/FcRγ or mouse Mincle/FcRγ, as well as those expressing FcRγ alone were tested for their reactivity to plate-bound non-acylated αCG. Assays were performed in duplicate; the mean values and standard deviations are shown.
Of the acylated cholesteryl α-glucosides, H. pylori predominantly produces
176–178 αCAG(C14:0) when grown in isolation, but mixtures of the C14:0,C16:0,C18:0,C18:1,
183 C18:2 and C22:4 lipoforms when co-cultured with AGS cells. We found that all the acyl lipoforms synthesised were able to stimulate cells through both mouse and human Mincle, with similar potency (Figure 2.15). Generally, mMincle reporter cells were less stimulated than hMincle reporter cells; however, this likely represents differences in the sensitivities of the report cell lines than intrinsic differences in recognition. The C14:0–
C18:0 lipoforms produced robust responses even at 16 pmol. This compares favourably with TDM which produces a similar response at 10 pmol.49 The most common Helico- bacter found in rodent species is H. muridarum, which possess a different profile of
αCAGs than H. pylori. αCAG(C12:0), the only αCAG detected in H. muridarum, was an agonist of human and mouse Mincle. H. rappini produces mainly the unsaturated acyl variant, αCAG(C18:1), which elicited a similar response to αCAG(C12:0). H. rappini has been implicated in human disease.281,282
66 100
90
80
70
60
50
40
NFAT-GFP NFAT-GFP (%) 30
20
10
0 0.016 0.08 0.4 2 0.016 0.08 0.4 2 0.016 0.08 0.4 2 0.016 0.08 0.4 2 0.016 0.08 0.4 2 nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol nmol αCAG(C12:0) αCAG(C14:0) αCAG(C16:0) αCAG(C18:0) αCAG(C18:1)
FcRγ only mMincle-γ hMincle-γ Figure 2.15. NFAT-GFP reporter cells expressing either human Mincle/FcRγ or mouse Mincle/FcRγ, as well as those expressing FcRγ alone were tested for their reactivity to plate-bound αCAG(C12:0), αCAG(C14:0), αCAG(C16:0), αCAG(C18:0), αCAG(C18:1). Assays were performed in duplicate; the mean values and standard deviations are shown.
2.3 Summary
The work in this chapter looked at investigated the devlopment of an more enviroment- ally friendly preperation of PTFAI-Cl, the comparison of glyosylation steroselectivy of donors bearing different remote acetylation patterns and the preperation of cholesteryl 6-O-acyl α-glucosides from members of the Helicobacter family. Using the phosphor- ane chlorination reagent Cl2Ph3P, a CCl4-free, we synthesised the leaving group reagent PTFAI-Cl. This in turn, enabled the preparation of four PTFAI glucosyl donors bear- ing different arrangements of acetates at O-3, O-4 and O-6. Glucosylation of cholesterol under identical conditions using each of these donors allowed an assessment of the ste- reodirecting effect afforded by remote acetylation. The optimum donor, bearing acetates at O-3, O-4 and O-6, afforded cholesteryl glucoside in a 14:1 α:β ratio and the highly crystalline nature of this material allowed purification of α-anomer by crystallization.
Deprotection, and subsequent elaboration of cholesteryl α-glucoside to a series of 6-O-acyl derivatives represented the synthesis of known glucosides produced by mem- bers of the Helicobacter family. These compounds were assessed for their ability to stim-
67 ulate Mincle signalling in reporter cell lines where they were found to possess potencies similar to that of TDM.
Future work
Our collaborators at Osaka University are currently performing a H. pylori infection study in Mincle-/- mice to assess what role Mincle plays in disease progression and in- fection clearance.
2.4 Experimental
2.4.1 General Methods
1H and 13C NMR were recorded using an Agilent Inova-400, Inova-500 or Inova-600
◦ 1 at 25 C. All signals were referenced to solvent peaks (CDCl3 7.27 ppm for H and
13 1 13 77.16 ppm for C; d4-MeOH: 3.49 ppm for H and 49.00 ppm for C). TLC analysis used aluminium-backed Merck Silica Gel 60 F254 sheets, detection was achieved using
UV light, 10% H2SO4 in MeOH with charring as necessary. Flash chromatography was
283 performed using Geduran silica gel according to the method of Still et al. Dry CH2Cl2,
Et2O and THF were obtained from a dry solvent apparatus (Glass Contour of SG Wa- ter, Nashua, U.S.A.).284 Pyridine was dried by distillation over KOH. Optical rotations were obtained using a JASCO DIP-1000 polarimeter. Melting points were obtained us- ing a Reichert-Jung hot stage melting point apparatus and are corrected. High resolution mass spectra (HRMS) were acquired at the Bio21 Institute, University of Melbourne on an Agilent ESI-TOF.Acid chlorides were purchased from Sigma-Aldrich and used as-is.
N-Phenyl-2,2,2-trifluoroacetamide (2) O CF3
2,2,2-Trifluoroacetic anhydride (9.50 mL, 68.3 mmol) was NH added drop-wise to a stirred solution of aniline (4.89 g,
◦ 52.5 mmol) in CH2Cl2 (50 mL) at 0 C. The reaction was allowed to warm to rt. After consumption of the aniline (1 h), the reaction was concentrated and the crude crystal-
285 1 lized from EtOH/H2O to afford 2 as colourless needles (8.5 g, 86% yield). H NMR
68 (400 MHz, CDCl3) δ 7.23–7.27 (1 H, m), 7.38–7.42 (2 H, m), 7.55–7.58 (2 H, m), 7.92
13 (1 H, br s); C NMR (101 MHz, CDCl3) δ 115.7 (q, J 288.5 Hz), 120.7, 126.4, 129.3, 135.1, 155.0 (q, J 37.4 Hz); 19F NMR (470 MHz, CDCl3) δ -75.77 (d, J 1.3 Hz);
Cl CF3 N-Phenyl-2,2,2-trifluoroacetimidoylchloride (1)
N Et3N (7.67 mL, 55.0 mmol) was added to a suspension of 2 (4 g, 21.2 mmol) and dichlorotriphenylphosphorane (17.6 g, 52.9 mmol) in MeCN (80 mL). The suspension was refluxed for 3 h and then cooled
◦ to 0 C. Precipitated Et3N.HCl and Ph3O were filtered and the filtrate concentrated. The residue was dissolved in hexanes, filtered and the filtrate concentrated to provide a yel- low oil. Flash chromatography (Et2O/hexanes 1:4) afforded 1 as a colourless liquid (2.68
1 g, 61%). H NMR (400 MHz, CDCl3) δ 7.10–7.14 (2 H, m), 7.30–7.34 (1 H, m), 7.43–
+ + 7.48 (2 H, m); HRMS (ESI ) calcd for C8H6ClF3N [M + H] 208.0135. Found 208.0136.
Methyl 4,6-O-benzylidene-α-D-glucopyranoside (10) Ph O O O HO Methyl α-D-glucopyranoside (20 g, 103 mmol) was suspen- HO OMe ded in a solution of benzaldehyde dimethyl acetal (27.8 mL, 185 mmol) in MeCN (360 mL). Camphorsulfonic acid was added (1.2 g, 5 mmol) and the solution stirred at reflux for 20 min. Upon cooling, the reaction was neutralized with
Et3N (approx. 1 mL) and concentrated. The residue was dissolved in EtOAc and washed with H2O and brine. The crude was purified by crystallization from EtOAc/pet. spirits to afford 10 as white needles (21.7 g, 75% yield). mp 163–168 ◦C (lit.286 166–167 ◦C);
24 286 23 1 [α]D +100.0 (c 1.00, CHCl3) (lit. [α]D +103.4 (c 2.00, CH2Cl2)); H NMR (400 MHz,
CDCl3) δ 2.45 (1 H, s, OH), 2.97 (1 H, s, OH), 3.44 (3 H, s, CH3), 3.48 (1 H, t, J 9.2 Hz, H4), 3.61 (1 H, br. s, H2), 3.68–3.85 (2 H, m, H5, H6b), 3.91 (1 H, t, J 9.3 Hz, H3), 4.28 (1 H, dd, J 4.3, 9.6 Hz, H6a), 4.77 (1 H, d, J 3.9 Hz, H1), 5.52 (1 H, s, PhCH), 7.33–
13 7.41 (3 H, m, Ph), 7.45–7.53 (2 H, m, Ph); C NMR (101 MHz, CDCl3) δ 55.7 (OCH3), 62.5 (C5), 69.1 (C6), 71.8 (C3), 73.0 (C2), 81.1 (C4), 99.9 (C1), 102.1 (PhCH), 126.4,
+ + 128.5, 129.4, 137.2 (Ph); HRMS (ESI ) calcd for C14H18O6 [M + Na] 305.0996. Found 305.0995.
69 Methyl 2-O-benzyl-4,6-O-benzylidene-α-D-glucopyranoside Ph O O O HO (11) BnO OMe 10 (5 g, 17.7 mmol) was added to a solution of tetrabutyl- ammonium sulfate (1.18 g, 3.5 mmol) and benzyl bromide (3.58 mL, 30.1 mmol) in
CH2Cl2 (300 mL). A 5% NaOH solution (25.3 mL) was added and the biphasic mix- ture stirred vigorously at reflux for 2 days. Upon cooling, the mixture was diluted with
H2O and the organic phase separated. The organic phase was washed with2 H O and brine, dried with MgSO4 and concentrated. Flash chromatography (30-50% EtOAc/pet. spirits) afforded 11)as a colourless solid (3.3 g, 50% yield). mp 128–129 ◦C (lit.256
◦ 1 131–132 C); H NMR (600 MHz, CDCl3) δ 2.60 (1 H, s, 3-OH), 3.38 (3 H, s, CH3), 3.47 (1 H, dd, J 3.5, 9.2 Hz, H2), 3.50 (1 H, t, J 9.5 Hz, H4), 3.70 (1 H, t, J 10.3 Hz, H6a), 3.82 (1 H, ddd, J 4.9, 9.9, 9.9 Hz, H5), 4.16 (1 H, t, J 9.3 Hz, H3), 4.26 (1 H, dd, J 4.9,
10.2 Hz, H6b), 4.62 (1 H, d, J 3.6 Hz, H1), 4.71 (1 H, d, J 12.2 Hz, PhCH2), 4.79 (1 H, d,
13 J 12.1 Hz, PhCH2), 5.52 (1 H, s, PhCH), 7.30–7.52 (10 H, m, Ph); C NMR (151 MHz,
CDCl3) δ 55.5 (OCH3), 62.1 (C5), 69.1 (C6), 70.4 (C3), 73.5 (PhCH2), 79.7 (C2), 81.4 (C4), 98.8 (C1), 102.1 (PhCH), 126.4, 128.2, 128.3, 128.4, 128.7, 129.3, 137.2, 138.0 (Ph);
+ + HRMS (ESI ) calcd for C21H24O6 [M + Na] 395.1465. Found 395.1468.
Second to elute was the 3-O-benzyl glucoside as a colourless solid.
1 H NMR (600 MHz, CDCl3) δ 2.35 (1 H, d, J 7.3 Hz, 2-OH), 3.45 (3 H, s, CH3), 3.65 (1 H, t, J 9.1 Hz, H4), 3.70–3.88 (4 H, m, H2, H3, H5, H6a), 4.31 (1 H, dd, J 4.4, 9.8 Hz, H6b),
4.77–4.83 (2 H, m, H1, PhCH2)), 4.97 (1 H, d, J 11.6 Hz, PhCH2)), 5.58 (1 H, s, PhCH)),
13 7.27–7.53 (10 H, m, Ph); C NMR (151 MHz, CDCl3) δ 55.5 (OCH3), 62.1 (C5), 69.1
(C6), 70.4 (C2), 73.5 (PhCH2), 79.7 (C3), 81.4 (C4), 98.8 (C1), 102.1 (PhCH), 126.4,
+ 128.2, 128.3, 128.4, 128.7, 129.3, 137.2, 138.0 (Ph); HRMS (ESI ) calcd for C21H24O6 [M + Na]+ 395.1465. Found 395.1466.
70 AcO 1,3,4,6-Tetra-O-acetyl-2-O-benzyl-D-glucopyranose (15) AcO O AcO p-Toluenesulfonic acid (747 mg, 3.93 mmol) was added to BnO OAc a solution of 11 (2.93 mg, 7.86 mmol) in acetic anhydride (45 mL). After stirring at rt for 2 h a further portion of p-toluenesulfonic acid (2.24 g, 11.8 mmol) was added and the solution refluxed for 2 h. Upon completion, the reaction was cooled, diluted with EtOAc and quenched with icy satd. aq. NaHCO3. The organic phase was separated and washed with satd. aq. NaHCO3 and brine, dried (MgSO4) and concentrated. Flash chromatography (40-60% EtOAc/pet. spirits) afforded an anomeric mixture (4:1 α/β) of 15 as a viscous yellow oil (3.30 g, 96% yield).253
1 α anomer: H NMR (600 MHz, CDCl3) δ 1.98 (3 H, s, CH3(C=O)), 2.00 (3 H, s,
CH3(C=O)), 2.05 (3 H, s, CH3(C=O)), 2.16 (3 H, s, CH3(C=O)), 3.66 (1 H, dd, J 3.6, 9.9 Hz, H2), 3.99–4.09 (2 H, m, H5, H6a), 4.26 (1 H, dd, J 4.1, 12.4 Hz, H6b), 4.50 (1 H, d, J
12.2 Hz, PhCH2), 4.62 (1 H, d, J 12.2 Hz, PhCH2), 5.01 (1 H, t, J 9.9 Hz, H3), 5.38 (1 H, t, J 9.7, H4), 6.33 (1 H, d, J 3.6 Hz, H1), 7.21–7.35 (5 H, m, Ph); 13C NMR (151 MHz,
CDCl3) δ 20.6, 20.74, 20.77, 21.0 (CH3(C=O)), 61.6, 67.9, 69.6, 71.5, 72.9, 75.3, 89.2 (C1), 127.9, 128.1, 128.5, 137.1 (Ph), 169.0, 169.6, 170.1, 170.5 (C=O); HRMS (ESI+)
+ calcd for C21H26O10 [M + Na] 461.1418. Found 461.1397.
1 β anomer: H NMR (600 MHz, CDCl3) δ 1.91 (3 H, s, CH3(C=O)), 1.99 (3 H, s,
CH3(C=O)), 2.08 (3 H, s, CH3(C=O)), 2.15 (3 H, s, CH3(C=O)), 3.60 (1 H, dd, J 8.5, 9.0 Hz, H2), 3.80 (1 H, ddd, J 2.1, 4.5, 10.1 Hz, H5), 3.99–4.09 (1 H, m, H6a), 4.26–4.31
(1 H, m, H6b), 4.60 (1 H, d, J 12.0 Hz, PhCH2), 4.69 (1 H, d, J 11.9 Hz, PhCH2), 5.00 (1 H, t, J 9.7, H4), 5.21 (1 H, t, J 9.4 Hz, H3), 5.64 (1 H, d, J 8.2 Hz, H1), 7.21–7.35 (5 H,
13 m, Ph); C NMR (151 MHz, CDCl3) δ 20.61, 20.66, 20.7, 20.9 (CH3(C=O)), 61.6, 68.0, 72.5, 74.0, 74.6, 77.9, 93.7 (C1), 127.8, 128.0, 128.1, 128.4, 137.1 (Ph), 168.8, 169.7, 170.1,
+ + 170.5 (C=O); HRMS (ESI ) calcd for C21H26O10 [M + Na] 461.1418. Found 461.1394.
2-O-Benzyl-3,4,6-tri-O-acetyl-D-glucopyranose (16) AcO AcO O Hydrazine acetate (330 mg, 3.59 mmol) was added to a solu- AcO BnO OH tion of 15 (1.05 g, 2.39 mmol) in DMF (15 mL) and stirred
71 overnight. The solution was diluted with EtOAc and washed with satd. aq. NaHCO3,
H2O and brine, dried (MgSO4) and concentrated. Flash chromatography (EtOAc/pet. spirits) afforded an anomeric mixture (1:0.85 α/β) of 16 as a colourless oil (750 mg,
216 1 79% yield). H NMR (600 MHz, CDCl3) δ 1.91–2.09 (16.7H, 6 × s, 6 × CH3(C=O)), 3.38–3.43 (0.85 H, m, βH2), 3.57–3.62 (1 H, m, αH2), 3.70 (0.85 H, ddd, J 2.2, 4.7, 9.7 Hz, βH5), 4.05 (1 H, dd, J 1.9, 12.2 Hz, αH6a), 4.12 (0.85 H, dd, J 2.3, 12.3 Hz, βH6a),
4.19–4.29 (2.85 H, m, αH5, αH6b, βH6b), 4.62–4.68 (2.85 H, m, 2 × αPhCH2, βPhCH2),
4.81 (0.85 H, d, J 7.4 Hz, βH1), 4.86 (1 H, d, J 11.8 Hz, βPhCH2), 4.97 (1.85 H, t, J 9.8 Hz, αH4, βH4), 5.14–5.19 (0.85 H, m, βH3), 5.24 (1 H, d, J 3.3 Hz, αH1), 5.43 (1 H, t,
13 J 9.6 Hz, αH3), 7.26–7.37 (9.25 H, m, Ph); C NMR (151 MHz, CDCl3) δ 20.61, 20.65,
20.68, 20.72, 20.77, 20.8 (CH3(C=O)), 62.0, 62.2, 67.3, 68.3, 68.6, 71.7, 73.2, 73.8, 74.3, 76.9, 77.2, 79.6, 90.9 (C1α), 97.4 (C1β), 127.8, 127.9, 128.0, 128.05, 128.2, 128.4, 128.6, 137.2, 137.8 (Ph), 169.7, 169.8, 170.1, 170.2, 170.7, 170.7 (C=O); HRMS (ESI+) calcd for
+ C19H24O9 [M + Na] 419.1313. Found 419.1299.
O-(3,4,6-Tri-O-acetyl-2-O-benzyl-D-glucopyranosyl)-N- AcO Ph AcO O N phenyl-2',2',2'-trifluoroacetimidate (6) AcO BnO O CF3 Hydrazine acetate (330 mg, 3.59 mmol) was added to a solu- tion of 15 (1.05 g, 2.39 mmol) in DMF (15 mL) and stirred overnight. The solution was diluted with EtOAc and washed with satd. aq. NaHCO3,H2O and brine, dried
(MgSO4) and concentrated. Flash chromatography (EtOAc/pet. spirits) afforded an anomeric mixture (1:0.85 α/β) of 16 as a colourless oil (750 mg, 79% yield). 1H NMR
(600 MHz, CDCl3) δ 1.94 (3 H, s, βCH3(C=O)), 2.00 (3 H, s, βCH3(C=O)), 2.01 (1.5H, s, αCH3(C=O)), 2.04 (1.5H, s, αCH3(C=O)), 2.06 (3 H, s, βCH3(C=O)), 2.08 (1.5H, s,
αCH3(C=O)), 3.43–3.87 (2.5H, m, αH2, βH2, βH5), 4.04–4.18 (2 H, m, αH5, αH6b,
βH6b), 4.21–4.33 (1.5H, m, αH6a, βH6a), 4.59–4.71 (2 H, m, αPhCH2, βPhCH2), 4.82
(1 H, d, J 11.7 Hz, βPhCH2), 4.99–5.10 (1.5H, m, αH4, βH4), 5.14–5.26 (1 H, m, βH3), 5.47 (0.5H, t, J 9.7 Hz, αH3), 5.73 (1 H, br s, βH1), 6.49 (0.5H, br s, αH1), 6.74 (1 H, d, J 7.5 Hz, ortho-NPhα), 6.83 (1 H, d, J 7.7 Hz, ortho-NPhβ), 7.07–7.41 (12 H, m, Ph);
72 13 C NMR (151 MHz, CDCl3) δ 20.62, 20.67, 20.71, 20.74, 20.8 (CH3(C=O)), 61.6 (C6α,
βC6), 67.8 (C4α), 68.0 (C4β), 69.9 (C5α), 71.5 (C3α), 72.5 (C5β), 73.3 (PhCH2α), 73.7
(C3β), 74.7 (PhCH2β), 75.8 (C2α), 77.6 (C2β), 92.4 (C1α), 96.7 (C1β), 119.2, 119.3, 124.4 (NPh), 124.5, 127.7, 128.12, 128.14, 128.2, 128.5, 128.6, 128.8, 137.25, 137.29, 143.1, 143.2 (Ph), 169.6, 169.7, 170.0, 170.5, 170.5 (C=O); HRMS (ESI+) calcd for
+ C27H28F3NO9 [M + Na] 590.1608. Found 590.1595.
Methyl 2,4-di-O-benzyl-α-D-glucopyranoside (12) HO
BnO O 10 (372 mg, 1 mmol) was added to BH3·THF (5 mL, 5 mmol) HO BnOOMe and the solution stirred for 10 min. Freshly dried Cu(OTf)2 (18 mg, 0.05 mmol) was added and the mixture stirred for a further 4h. The reaction mixture was cooled to 0°C and quenched with Et3N (0.14 mL) and MeOH (1.8 mL). The mixture was then concentrated followed by co-evaporation with MeOH. Flash chro- matography (EtOAc/pet. spirits) afforded 12 as a white solid (351 mg, 94% yield). mp
◦ 287 ◦ 1 68–71 C (lit. 64–65 C); H NMR (600 MHz, CDCl3) δ 3.30 (3 H, s, CH3), 3.33 (1 H, dd, J 3.5, 9.6 Hz, H2), 3.41–3.47 (1 H, m, H4), 3.58–3.64 (1 H, m, H5), 3.70 (1 H, dd, J 4.0, 11.8 Hz, H6a), 3.77 (1 H, dd, J 2.8, 11.8 Hz, H6b), 4.08 (1 H, t, J 9.2 Hz, H3), 4.58
(1 H, d, J 3.5 Hz, H1), 4.65 (1 H, d, J 12.1 Hz, PhCH2), 4.68 (1 H, d, J 11.4 Hz, PhCH2),
4.69 (1 H, d, J 12.1 Hz, PhCH2), 4.89 (1 H, d, J 11.3 Hz, PhCH2), 7.23–7.38 (10 H, m,
13 Ph); C NMR (151 MHz, CDCl3) δ 55.2 (OCH3), 61.9 (C6), 70.2 (C5), 73.1 (PhCH2),
73.4 (C3), 74.5 (PhCH2), 77.2 (C4), 79.6 (C2), 97.5 (C1), 127.8, 128.0, 128.13, 128.17,
+ + 128.5, 128.6, 137.9, 138.3 (Ph); HRMS (ESI ) calcd for C21H26O6 [M + K] 413.1361. Found 413.1356.
1,3,6-Tri-O-acetyl-2,4-di-O-benzyl-D-glucopyranose (13) AcO BnO O p-Toluenesulfonic acid (171 mg, 0.90 mmol) was added to a AcO BnO OAc solution of 12 (1.12 mg, 3.01 mmol) in Ac2O (18 mL) and stirred at rt for 1 h. Additional p-Toluenesulfonic acid (687 mg, 3.61 mmol) was ad- ded and the solution heated to 70 ◦C for 3 h. Upon disappearance of the di-acetylated intermediate from the mass spectrum (ESI, 150 V) the solution was cooled to 0 ◦C and
73 quenched with the addition of ice and MeOH. The cold solution was poured onto icy EtOAc and the phases separated. The aqueous layer was extracted with EtOAc and the combined organic phases washed with satd. aq. NaHCO3 and H2O, dried (MgSO4) and concentrated. Flash chromatography (EtOAc/pet. spirits) afforded an anomeric mixture
253 1 (4:1 α/β) of 13 as a yellow oil (1.30 g, 99% yield). H NMR (600 MHz, CDCl3) δ 1.89
(0.75 H, s, βCH3(C=O)), 1.98 (3 H, s, αCH3(C=O)), 2.02 (0.75 H, s, βCH3(C=O)), 2.03
(3 H, s, αCH3(C=O)), 2.06 (0.75 H, s, βCH3(C=O)), 2.13 (3 H, s, αCH3(C=O)), 3.47– 3.51 (0.25 H, m, βH2), 3.53–3.59 (2.25 H, m, αH2, αH4, βH4), 3.70 (0.25 H, ddd, J 2.1, 4.4, 9.8 Hz, βH5), 3.98 (1 H, ddd, J 3.1, 3.1, 10.1 Hz, αH5), 4.20–4.30 (2.5H, m, αH6,
βH6), 4.47 (1 H, d, J 12.3 Hz, αPhCH2), 4.50 (0.25 H, d, J 11.1 Hz, βPhCH2), 4.50 (1 H, d, J 11.0 Hz, αPhCH2), 4.54 (0.25 H, d, J 11.2 Hz, βPhCH2), 4.57 (1 H, d, J 11.0 Hz,
αPhCH2), 4.58 (0.25 H, d, J 11.9 Hz, βPhCH2), 4.63 (1 H, d, J 12.3 Hz, αPhCH2), 4.69
(0.25 H, d, J 11.9 Hz, βPhCH2), 5.31 (0.25 H, t, J 9.3 Hz, βH3), 5.50 (1 H, t, J 9.6 Hz, αH3), 5.64 (0.25 H, d, J 8.1 Hz, βH1), 6.32 (1 H, d, J 3.6 Hz, αH1), 7.20–7.36 (12.5H, m,
13 Ph); C NMR (151 MHz, CDCl3) δ 20.83, 20.87, 20.94, 20.98, 21.02, 21.05 (CH3(C=O)), 62.4, 62.5, 70.8, 72.5, 73.2, 73.5, 74.3, 74.4, 74.7, 75.4, 75.51, 75.56, 75.8, 78.3, 89.1 (C1α), 93.8 (C1β), 127.82, 127.85, 127.9, 128.00, 128.04, 128.1, 128.4, 128.52, 128.56, 137.1, 137.3, 137.7 (Ph), 168.7, 169.2, 169.6, 169.8, 170.5, 171.0 (C=O); HRMS (ESI+) calcd
+ for C26H30O9 [M + Na] 509.1782. Found 509.1760.
3,6-Di-O-acetyl-2,4-di-O-benzyl-D-glucopyranose (14) AcO Benzyl amine (0.42 mL, 3.88 mmol) was added to a solution of BnO O AcO 13 (1.26 g, 2.59 mmol) in THF (6 mL) and stirred overnight. BnO OH
The reaction mixture was concentrated and the residue dissolved in CHCl3 and washed with icy water. The aqueous layer was extracted with CHCl3 and the combined organic phases washed with cold dilute HCl, satd. aq. NaHCO3, water and brine, dried (MgSO4) and concentrated. Flash chromatography (EtOAc/pet. spirits) afforded an anomeric
1 mixture (4:3 α/β) of 14 as a clear oil (995 mg, 86% yield). H NMR (600 MHz, CDCl3)
δ 1.89 (3 H, s, αCH3(C=O)), 1.96 (3 H, s, αCH3(C=O)), 2.02 (2.25 H, s, βCH3(C=O)),
74 2.03 (2.25 H, s, βCH3(C=O)), 3.28 (1 H, dd, J 7.7, 9.6 Hz, βH2), 3.45–3.54 (2.75 H, m, αH2, αH5, βH4), 3.59 (0.75 H, ddd, J 2.1, 4.8, 9.8 Hz, βH5), 4.12–4.18 (1.75 H, m, αH4, βH6a), 4.22 (0.75 H, dd, J 4.2, 12.1 Hz, αH6a), 4.29 (1 H, dd, J 2.3, 12.1 Hz, βH6b), 4.34
(0.75 H, dd, J 2.1, 12.0 Hz, αH6b), 4.46–4.58 (4.25 H, 2 × αPhCH2, 3 × βPhCH2), 4.62
(1 H, d, J 11.9 Hz, αPhCH2), 4.63 (0.75 H, d, J 12.2 Hz, βPhCH2), 4.77 (0.75 H, d, J 7.7
Hz, βH1), 4.85 (1 H, d, J 11.9 Hz, αPhCH2), 5.23 (1 H, d, J 3.6 Hz, αH1), 5.24 (0.75 H, t, J 9.3 Hz, βH3), 5.51 (1 H, t, J 9.4 Hz, αH3), 7.20–7.36 (17.5H, m, Ph); 13C NMR
(151 MHz, CDCl3) δ 20.8, 21.06, 21.09, 21.1 (CH3(C=O)), 62.94, 62.98, 68.5, 72.7, 72.8, 73.2, 74.0, 74.3, 74.4, 75.4, 75.8, 75.9, 77.5, 79.9, 90.7 (αC1), 97.3 (βC1), 127.7, 127.8, 128.02, 128.07, 128.03, 128.09, 128.1, 128.15, 128.3, 128.53, 128.55, 128.5, 137.2, 137.3,
+ 137.4, 138.1 (Ph), 169.92, 169.96, 170.7, 170.8 (C=O); HRMS (ESI ) calcd for C24H28O8 [M + Na]+ 467.1676. Found 467.1662.
O-(3,6-Di-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranoyl)-N- AcO Ph BnO O N AcO phenyl-2',2',2'-trifluoroacetimidate (5) BnO O CF3 K2CO3 (297 mg, 2.14 mmol) was added to a solution of 14 (477 mg, 1.07 mmol) and 1 (0.177 mL, 1.61 mmol) in acetone (10 mL). The mixture was stirred for 3 h and then filtered through a bed of Celite and the filtrate concentrated.
Flash chromatography (Et3N/EtOAc/pet. spirits) afforded an anomeric mixture (1:2
215 1 α/β) of 5 as a yellow oil (626 mg, 95% yield). H NMR (600 MHz, CDCl3) δ 1.91 (3 H, s, βCH3(C=O)), 2.01 (1.5H, s, αCH3(C=O)), 2.05 (3 H, s, βCH3(C=O)), 2.07 (1.5H, s,
αCH3(C=O)), 3.39–3.83 (4 H, m, αH2, βH2, αH4, βH4, βH5), 4.02–4.39 (3.5H, m, αH5,
αH6, βH6), 4.47–4.65 (4.5H, m, 3 × αPhCH2, 3 × βPhCH2), 4.70 (0.5H, d, J 12.4 Hz,
αPhCH2), 4.81 (1 H, d, J 11.7 Hz, βPhCH2), 5.26 (1 H, br s, βH3), 5.59 (0.5H, br s, αH3), 5.59 (1 H, t, J 9.6 Hz, βH1), 6.49 (0.5H, br s, αH1), 6.73 (1 H, d, J 7.1 Hz, αortho-NPh)), 6.82 (1 H, d, J 7.6 Hz, βortho-NPh)), 7.06–7.40 (19 H, m, Ph); 13C NMR (151 MHz,
CDCl3) δ 20.8, 20.9, 21.0 (CH3(C=O)), 62.4, 71.2, 72.9, 73.0, 73.4, 74.3, 74.4, 74.6, 75.2, 75.31, 75.34, 76.3, 77.9, (αC1 not visible), 96.8 (βC1), 119.2, 119.3, 124.5 (NPh), 127.7, 128.01, 128.06, 128.12, 128.14, 128.2, 128.55, 128.59, 128.64, 128.69, 128.8, 137.1, 137.4,
75 + 143.2 (Ph), 169.6, 169.7, 170.5, 170.5 (C=O); HRMS (ESI ) calcd for C32H32F3NO8 [M + Na]+ 638.1972. Found 637.1971.
1,6-Di-O-acetyl-2,3,4-tri-O-benzyl-α-D-glucopyranose (8) AcO p-Toluenesulfonic acid (120 mg, 0.63 mmol) was added to a BnO O BnO OAc solution of tetrabenzyl glucopyranose (1.00 g, 1.85 mmol) in BnO
Ac2O (16 mL) and stirred at rt for 2 h. Additional p-Toluenesulfonic acid (457 mg, 2.40 mmol) was added and the mixture heated to 70 ◦C for 2 h. Upon disappearance of the starting material as monitored by TLC the solution was cooled to 0 ◦C and quenched with the addition of ice. The cold solution was poured onto icy EtOAc and the phases separated. The aq. layer was extracted with EtOAc and the combined organic phases washed with satd. aq. NaHCO3 and water, dried (MgSO4) and concentrated. Flash chromatography (EtOAc/pet. spirits) afforded an anomeric mixture of 8 as a yellow oil
1 (600 mg, 61% yield). α anomer: H NMR (600 MHz, CDCl3) δ 2.16, 2.03 (6 H, 2 × s,
CH3(C=O)), 3.57 (1 H, t, J 9.6 Hz, H4), 3.68 (1 H, dd, J 3.5, 9.6 Hz, H2), 3.92 (1 H, t, J 2.9 Hz, H5), 3.97 (1 H, t, J 9.4 Hz, H3), 4.24 (1 H, dd, J 2.2, 12.1 Hz, H6b), 4.29 (1 H, dd,
J 3.9, 12.1 Hz, H6a), 4.57 (1 H, d, J 10.6 Hz, PhCH2), 4.64 (1 H, d, J 11.3 Hz, PhCH2),
4.71 (1 H, d, J 11.5 Hz, PhCH2), 4.83 (1 H, d, J 10.8 Hz, PhCH2), 4.89 (1 H, d, J 10.8
Hz, PhCH2), 5.00 (1 H, d, J 10.8 Hz, PhCH2), 6.32 (1 H, d, J 3.5 Hz, H1), 7.20 – 7.40
13 (15 H, m, Ph); C NMR (151 MHz, CDCl3) δ 20.8, 21.1 (CH3(C=O)), 62.6, 71.0, 73.2, 75.3, 75.7, 76.6, 78.8, 81.6, 89.6 (C1), 127.8, 128.8, 128.0, 128.06, 128.10, 128.18, 128.46, 128.51, 128.53, 137.45, 137.56, 138.4 (Ph), 169.3, 170.6 (C=O); HRMS (ESI+) calcd for
+ C31H34O8 [M + Na] 557.2146. Found 557.2158.
6-O-Acetyl-2,3,4-tri-O-benzyl-D-glucopyranose (9) AcO Benzyl amine (0.18 mL, 1.68 mmol) was added to a solution of BnO O BnO 8 (600 mg, 1.12 mmol) in THF (2.6 mL). The reaction mixture BnO OH was stirred overnight and then concentrated. The residue was redissolved in CHCl3 and washed with icy water. The aq. layer was extracted with CHCl3 and the com- bined organic phases washed with cold dilute HCl, satd. aq. NaHCO3, water and brine,
76 dried (MgSO4) and concentrated. Flash chromatography (EtOAc/pet. spirits) afforded an anomeric mixture (~1:0.6 α/β) of 9 as a clear oil (430 mg, 78% yield). 1H NMR
(600 MHz, CDCl3) δ 2.06 (~1.8H, s, βCH3(C=O)), 2.07 (3 H, s, αCH3(C=O)), 3.50–3.69 (4 H, m), 3.77 (0.6H, t, J 8.7 Hz, βH4), 4.14–4.51 (6.6H, m), 4.64–5.14 (10.6H, m), 5.30
13 (1 H, d, J 3.4 Hz, αH1), 7.29–7.51 (24 H, m, Ph); C NMR (151 MHz, CDCl3) δ 20.9,
20.9 (CH3(C=O)), 63.32, 63.36, 68.7, 72.9, 73.1, 74.8, 75.13, 75.16, 75.81, 75.87, 77.3, 77.4, 77.7, 80.1, 81.8, 83.1, 84.7, 91.0 (αC1), 97.5 (βC1), 127.8, 128.04, 128.06, 128.13, 128.15, 128.26, 128.59, 128.5, 128.60, 128.64, 128.69, 137.8, 138.05, 138.09, 138.5, 138.6
+ + (Ph), 171.1, 171.2 (C=O); HRMS (ESI ) calcd for C29H32O7 [M + Na] 515.2040. Found 515.2039.
O-(6-O-Acetyl-2,3,4-tri-O-benzyl-D-glucopyranosyl)-N- AcO Ph BnO O N BnO phenyl-2',2',2'-trifluoroacetimidate (4) BnO O CF3 K2CO3 (362 mg, 1.75 mmol) was added to a solution of 9 (430 mg, 0.87 mmol) and 1 (0.192 mL, 1.75 mmol) in acetone (8.7 mL). The mixture was stirred for 3 h and then filtered through a bed of Celite and the filtrate concen- trated. Flash chromatography (Et3N/EtOAc/pet. spirits) afforded an anomeric mixture
288 1 of 4 as a yellow oil (550 mg, 95% yield). α anomer: H NMR (600 MHz, CDCl3) δ 2.04
(3 H, s, CH3(C=O)), 3.54–4.37 (6 H, m, H2, H3, H4, H5, H6a, H6b), 4.60 (1 H, d, J 10.7
Hz, PhCH2), 4.71–4.80 (2 H, m, PhCH2), 4.87 (1 H, d, J 10.8 Hz, PhCH2), 4.90 (1 H, d, J 10.8 Hz, PhCH2), 5.02 (1 H, d, J 10.8 Hz, PhCH2), 6.46 (1 H, br s, H1), 6.73 (2 H,
13 d, J 6.5 Hz, ortho-NPh)), 7.05–7.39 (18 H, m, Ph); C NMR (151 MHz, CDCl3) δ 20.8
(CH3(C=O)), 62.6 (C6), 71.4 (C5), 73.3 (PhCH2), 75.26 (PhCH2), 75.81 (PhCH2), 76.40 (C4), 79.2 (C2), 81.4 (C3), 93.0 (C1), 119.3, 124.2 (NPh), 127.63, 127.76, 127.93, 127.95, 128.07, 128.20, 128.44, 128.50, 128.53, 128.69, 137.51, 137.65, 138.33, 143.5 (Ph), 170.6
+ + (C=O); HRMS (ESI ) calcd for C37H36F3NO7 [M + K] 702.2075. Found 702.2047.
O-(2,3,4,6-Tetra-O-benzyl-D-glucopyranosyl)-N-phenyl-2',2',2'- BnO Ph BnO O N BnO trifluoroacetimidate (3) BnO O CF3 K2CO3 (165 mg, 1.20 mmol) was added to a solution of
77 2,3,4,6-tetra-O-benzyl-α-D-glucopyranose (324 mg, 0.60 mmol) and 1 (0.099 mL, 0.90 mmol) in acetone (6 mL). The mixture was stirred for 3 h and then filtered through a bed of Celite and the filtrate concentrated. Flash chromatography3 (Et N/EtOAc/pet. spirits) afforded an anomeric mixture of 3 as a yellow oil (406 mg, 95% yield).289 α
1 anomer: H NMR (600 MHz, CDCl3) δ 4.06–3.65 (6 H, m, H2, H3, H4, H5, H6a, H6b),
4.50 (1 H, d, J 12.1 Hz, PhCH2), 4.52 (1 H, d, J 10.7 Hz, PhCH2), 4.61 (1 H, d, J 12.1 Hz,
PhCH2), 4.75 (2 H, s, PhCH2), 4.86 (2 H, d, J 10.9 Hz, PhCH2), 4.99 (1 H, d, J 10.9 Hz,
PhCH2), 6.52 (1 H, s, H1), 6.72 (2 H, d, J 6.1 Hz, ortho-NPh)), 7.05–7.39 (20 H, m, Ph);
13 C NMR (151 MHz, CDCl3) δ 68.1 (C6), 73.07, 73.29, 73.49, 75.29, 75.74, 76.8, 79.2, 81.5 (C1), 119.4, 124.1 (NPh), 127.62, 127.63, 127.75, 127.82, 127.84, 127.90, 127.92, 127.97, 128.38, 128.39, 128.41, 128.45, 128.66, 137.76, 137.78, 137.96, 138.55, 143.7;
+ + HRMS (ESI ) calcd for C42H40F3NO6 [M + Na] 734.2700. Found 734.2683.
General procedure for glycosylation reactions A solution of a cholesterol (1 equiv) and N-phenyltrifluoroacetimidoyl donor (1.2 equiv) in anhydrous solvent (8 mL/1 mmol of the donor) was stirred with activated 3 Å mo- lecular sieves at rt for 20 min. The mixture was then cooled to 0 ◦C, and TfOH (0.2 equiv) was added. The reaction mixture was stirred at 0 ◦C for 15 min, at which stage
TLC showed that the reaction was complete. The reaction was quenched with3 Et N (2.3
◦ equiv) at 0 C. The resulting mixture was diluted with CH2Cl2, and the solids were re- moved by filtration through a pad of Celite and washed with CH2Cl2. The combined filtrates were washed with aq. sat. NaHCO3 and then H2O. The organic phase was dried
(MgSO4), and the solvent was removed under reduced pressure to give a crude residue. The crude was analysed to 1H NMR to determine the stereo-outcome based on integ- ration of the α/β H1 signals.
Cholesteryl 3,4,6-tri-O-acetyl-2-O-benzyl-α-D-glucopyran- oside (17)
AcO H AcO O AcO H H Cholesterol (69.8 mg, 0.188 mmol), 6 (123 mg, 0.216 mmol) in BnO O dry Et2O (1.5 mL) and powdered 3 Å molecular sieves were treated with TfOH (3.2 µL)
78 according to the General Procedure for Glycosylation Reactions. The crude residue was purified by flash chromatography (EtOAc/pet. spirits) to afford an anomeric mixture (14:1 α/β) of cholesteryl glucoside as a white solid (122 mg, 88% yield). This solid was recrystallised from ethanol to afford the pure α-anomer17 ( ).
◦ 24 1 mp 178–181 C; [α]D +68.96 (c 0.94, CHCl3); H NMR (600 MHz, CDCl3) δ 0.61 (3 H, s), 0.79 (3 H, d, (J) 2.5 Hz), 0.80 (3 H, d, (J) 2.5 Hz), 0.85 (3 H, d, (J) 6.5 Hz), 0.94 (3 H, s), 0.75–1.85 (24 H, m), 1.87–1.98 (2 H, m), 1.99, 1.95, 1.94 (9 H, 3 × s, CH3(C=O)), 2.23 (1 H, ddd, (J) 13.3, 4.7, 1.8 Hz), 2.31–2.40 (1 H, m), 3.30–3.38 (1 H, m, H3), 3.48 (1 H, dd, (J) 3.6, 10.0 Hz, H2), 3.97 (1 H, dd, (J) 2.0, 12.2 Hz, H6b'), 4.03 (1 H, ddd, (J) 2.1, 4.7, 10.3 Hz, H5'), 4.17 (1 H, dd, (J) 4.8, 12.2 Hz, H6a'), 4.51 (1 H, d, (J) 12.4
Hz, PhCH2), 4.56 (1 H, d, (J) 12.4 Hz, PhCH2), 4.84–4.91 (2 H, m, H1', H4'), 5.29 – 5.24 (1 H, m, H6), 5.37 (1 H, t, (J) 9.7 Hz, H3'), 7.18 – 7.29 (5 H, m, Ph); 13C NMR (151 MHz,
CDCl3) δ 11.8, 18.7, 19.4, 20.68 (CH3(C=O)), 20.73 (CH3(C=O)), 20.83 (CH3(C=O)), 21.02, 22.54, 22.80, 23.8, 24.3, 27.64, 27.99, 28.20, 31.82, 31.90, 35.75, 36.15, 36.71, 37.0, 39.48, 39.72, 39.92, 42.3, 50.1, 56.11, 56.71, 62.2 (C6'), 67.2 (C5'), 68.9 (C4'), 71.9 (C3'),
72.7 (PhCH2), 76.5 (C2'), 78.2 (C3), 94.9 (C1'), 121.9 (C6), 127.84 (Ph), 127.99, 128.5,
+ 137.8, 140.5 (C5), 169.90, 170.07, 170.63 (C=O); HRMS (ESI ) calcd for C46H68O9 [M + Na]+ 782.5202. Found 782.5219.
Cholesteryl 3,4,6-tri-O-acetyl-α-D-glucopyranoside (18)
A mixture of Pd(OH)2 (20% on carbon, 100 mg) and 17
AcO H AcO O AcO H H HO (100 mg, 0.13 mmol) in ethanol/cyclohexene (2:1, 1.5 mL) was O stirred at reflux overnight then filtered through Celite and concentrated. Flash chro- matography (EtOAc/pet. spirits) afforded 18 as a white solid (77 mg, 88% yield). mp
◦ 290 ◦ 24 290 24 160–162 C (lit. 160–162 C); [α]D +90.23 (c 0.89, CHCl3) (lit. [α]D +91 (c 0.95,
1 CHCl3)); H NMR (500 MHz, CDCl3) δ 0.67 (3 H, s), 0.85 (3 H, d, J 2.2 Hz), 0.86 (3 H, d, J 2.2 Hz), 0.90 (3 H, d, J 6.5 Hz), 1.01 (3 H, s), 0.82–1.61 (20 H, m), 1.77–2.04 (7 H, m), 2.02 (3 H, s, Ac), 2.07 (6 H, s, Ac), 2.37 (2 H, d, J 7.7 Hz), 3.46–3.54 (1 H, m, H3), 3.64 (1 H, ddd, J 4.0, 9.9, 11.6 Hz, H2'), 4.10–4.03 (2 H, m, H5', H6b'), 4.20–4.27 (1 H, m,
79 H6a'), 4.98 (1 H, t, J 9.7 Hz, H4'), 5.04 (1 H, d, J 4.0 Hz, H1'), 5.21 (1 H, t, J 9.7 Hz, H3'),
13 5.35 (1 H, m, H6); C NMR (126 MHz, CDCl3) δ 11.8, 18.7, 19.3, 20.63 (CH3(C=O)),
20.73 (CH3(C=O)), 20.89 (CH3(C=O)), 21.0, 22.54, 22.80, 23.8, 24.3, 27.99, 28.09, 28.20, 31.84, 31.90, 35.8, 36.16, 36.62, 36.93, 39.49, 39.70, 40.1, 42.3, 50.1, 56.12, 56.69, 62.2 (C6'), 67.7 (C5'), 68.2 (C4'), 70.7 (C2'), 73.6 (C3'), 79.2 (C3), 97.0 (C1'), 122.4 (C6), 140.1
+ + (C5), 169.6, 170.6, 171.0 (C=O); HRMS (ESI ) calcd for C39H62O9 [M + Na] 697.4286. Found 697.4298.
Cholesteryl α-D-glucopyranoside (19) NaOMe solution (1 M, 0.10 mL) was added to a solution of 18
HO H HO O HO H H (100 mg, 0.13 mmol) in MeOH/THF (3:2, 2.5 mL). The mixture HO O was stirred at rt for 2 h and then was quenched with Dowex resin (H+ form), filtered and concentrated to afford 19 as a white solid (66 mg, 92% yield). mp 220–230 ◦C (de-
203 ◦ 24 203 24 comp.) (lit. 220–222 C); [α]D +51.81 (c 0.50, DMSO) (lit. [α]D +30.2 (c 0.54,
1 CHCl3:MeOH)); H NMR (400 MHz, DMSO – d6) δ 0.61 (3 H, s), 1.55–0.73 (34 H, m), 2.00–1.64 (5 H, m), 2.19 (1 H, t, J 11.5 Hz,), 2.35–2.24 (1 H, m), 2.46 (1 H, dt, J 1.8, 3.5 Hz), 3.00 (1 H, td, J 5.5, 9.0 Hz, H4'), 3.14–3.07 (1 H, m, H2'), 3.38 (3 H, m, H3', H5', H3), 3.60–3.51 (1 H, m, H6b'), 4.37 (1 H, t, J 5.7 Hz, OH), 4.42 (1 H, d, J 6.7 Hz, H6a'), 4.65 (1 H, d, J 4.8 Hz, OH), 4.73 (1 H, d, J 3.7 Hz, H1'), 4.78 (1 H, d, J 5.4 Hz, OH), 5.25
13 (1 H, m, H6); C NMR (101 MHz, DMSO – d6) δ 11.7, 18.6, 19.1, 20.6, 22.42, 22.69, 23.23, 23.89, 27.42, 27.81, 31.38, 31.45, 35.23, 35.69, 36.21, 36.63, 38.89, 38.97, 40.14, 40.43, 41.9, 49.6, 55.6, 56.2, 61.1 (C6'), 70.4 (C4'), 71.9 (C2'), 72.9 (C5'), 73.2 (C3'), 76.5
+ + (C3), 96.9 (C1'), 121.1 (C6), 140.7 (C5); HRMS (ESI ) calcd for C33H56O6 [M + Na] 566.4415. Found 566.4442.
3-Myristoyl-5-methyl-1,3,5-thiadiazole-2(3H)-thione (21) O S Myristic acid (250 mg, 1.09 mmol) was added to a solution of N 12 S 2-mercapto-5-methyl-1,3,4-thiadiazole (131 mg, 1 mmol) and N
N,N'-dicyclohexylcarbodiimide (204 mg, 1.1 mmol) in CH2Cl2 (1 mL). The reaction was stirred for 6 h then concentrated and purified by flash chromatography. The partially
80 purified product was recrystallized from MeOH/H2O to afford 21 as yellow needles
276 ◦ 1 (274 mg, 80%). mp 52–53 C H NMR (600 MHz, CDCl3) δ 0.84–0.88 (3 H, t, J 6.9, 6.9 Hz), 1.24–1.44 (20 H, m), 1.70–1.78 (2 H, m), 2.46 (3 H, s), 2.99 (2 H, t, J 7.4 Hz); 13C
NMR (151 MHz, CDCl3) δ 14.1, 16.3, 22.7, 24.1, 28.92, 29.29, 29.34, 29.43, 29.58, 29.63,
+ + 31.9, 37.8, 154.9, 171.6; HRMS (ESI ) calcd for C17H30N2OS2 [M + Na] 365.1692. Found 365.1685.
Cholesteryl 2,3,4-tris-O-trimethylsilyl-α-D-glucopyranoside (23)
HO H TMSO O TMSO H H TMSO . O Trimethylsilyl chloride (266 ul, 1 78 mmol) and Et3N (260 ul,
1.85 mmol) were added to a stirred solution of 19 (195 mg, 0.355 mmol) in CH2Cl2 (5 mL). The reaction was stirred overnight and then diluted with pentane and sat.
NaHCO3. The organic phase was separated and washed with satd. aq. NaHCO3, wa- ter and brine, then dried (Na2SO4) and concentrated. The residue was dissolved in
CH2Cl2/MeOH (1:1, 4ml) and NH4OAc (61 mg, 0.79 mmol) was added. The reaction was allowed to stir for 48h then concentrated and purified by flash chromatography
(pre-treated with 4% Et3N/pet. spirits, EtOAc/pet. spirits) to afford 23 as a clear glass
1 (185 mg, 68%). H NMR (400 MHz, CDCl3) δ 0.10–0.14 (9 H, m, CH3Si), 0.14–0.16
(9 H, m, CH3Si), 0.16–0.20 (9 H, m, CH3Si), 0.67 (3 H, s), 0.81–1.61 (34 H, m), 1.79– 1.88 (3 H, m), 1.93–2.03 (2 H, m), 2.27–2.32 (1 H, m), 2.33–2.41 (1 H, m), 3.34–3.40 (1 H, m, H3), 3.40 (1 H, dd, J 3.6, 9.2 Hz, H2'), 3.43–3.47 (1 H, m, H4'), 3.66–3.73 (2 H, m, H5, H6b'), 3.76 (2 H, m, H3', H6a'), 4.82 (1 H, d, J 3.6 Hz, H1'), 5.31–5.34 (1 H, m,
13 H6); C NMR (101 MHz, CDCl3) δ 0.70 ((C)H3Si), 1.09 ((C)H3Si), 1.47 ((C)H3Si), 12.0, 18.88, 19.6, 21.2, 22.72, 22.98, 23.9, 24.5, 27.9, 28.17, 28.39, 32.05, 32.09, 35.94, 36.35, 36.90, 37.33, 39.68, 39.95, 40.3, 42.5, 50.3, 56.31, 56.93, 62.0, 71.99, 72.18, 73.7, 74.6, 77.9 (C3), 97.7 (C1'), 121.8 (C6), 141.1 (C5).
Cholesteryl 6-O-dodecanoyl-α-D-glucopyranoside (24)
O Lauryl chloride (22 µL, 0.094 mmol) was added to a solution of 10 O H HO O HO H H HO DMAP (5.2 mg, 0.047 mmol) and 23 (36 mg, 0.047 mmol) in O
81 ◦ pyridine/CH2Cl2 (3:97, 1.2 mL) at 0 C. The solution was allowed to stir at rt for 4 h, then MeOH (50 µL) was added and the mixture was to stirred for 30 min. The solvent was evaporated under reduced pressure to afford a colourless residue. The residue was dis-
+ solved in MeOH/CHCl3 (1:1, 2 mL) and Dowex 50WX8-200 (H form, approx. 100 mg) was added. After stirring for 1 h the mixture was filtered and the filtrate concentrated. Flash chromatography (EtOAc/pet. spirits/MeOH) afforded 24 as a white solid (25 mg,
24 1 73% yield). [α]D +32.2 (c 0.95, CHCl3); H NMR (500 MHz, CDCl3) δ 0.67 (3 H, s), 0.81–1.65 (54 H, m), 1.78–2.06 (5 H, m), 2.20–2.41 (5 H, m), 3.31 (2 H, br s, 2 × OH), 3.33 (1 H, t, J 9.5 Hz, H4'), 3.44–3.53 (2 H, m, H2', H3), 3.72 (1 H, t, J 9.2 Hz, H3'), 3.86 (1 H, ddd, J 9.9, 4.8, 2.0 Hz, H5'), 4.26 (1 H, m, H6b'), 4.43–4.50 (1 H, m, H6a'), 5.01 (1 H,
13 d, J 4.0 Hz, H1'), 5.33–5.37 (1 H, m, H6); C NMR (126 MHz, CDCl3) δ 12.0, 14.3, 18.9, 19.5, 21.2, 22.71, 22.85, 22.96, 23.9, 24.4, 25.1, 28.16, 28.37, 29.39, 29.46, 29.51, 29.73, 29.82, 32.02, 32.08, 34.34, 35.94, 36.34, 36.80, 37.14, 39.66, 39.89, 40.26, 42.5, 50.3, 56.31, 56.88, 63.3 (C6'), 70.2 (C5'), 70.2 (C4'), 72.2 (C2'), 74.8 (C3'), 78.6 (C3), 97.0 (C1'), 122.4
+ + (C6), 140.4 (C5), 174.8 (C=O); HRMS (ESI ) calcd for C47H86O7 (M + NH4) 748.6086. Found 748.6077.
Cholesteryl 6-O-tetradecanoyl-α-D-glucopyranoside (25)
Myristoyl chloride (24 µL, 0.088 mmol) was added to a solution O 12 O H HO O HO H H of DMAP (5 mg, 0.044 mmol) and 23 (34 mg, 0.044 mmol) in HO O ◦ pyridine/CH2Cl2 (3:97, 1.2 mL) at 0 C. The solution was allowed to stir at rt for 4 h, then MeOH (50 µL) was added and the mixture was stirred for 30 min. The solvent was evaporated under reduced pressure to afford a colourless residue. The residue was redis-
+ solved in MeOH/CHCl3 (1:1, 2 mL) and Dowex 50WX8-200 (H form, approx. 100 mg) was added. After stirring for 1 h the mixture was filtered and the filtrate concentrated. Flash chromatography (EtOAc/pet. spirits/MeOH) afforded 25 as a white solid (26 mg,
24 24 1 79% yield). [α]D +31.5 (c 1, CHCl3) (lit. [α]D +21.9 (c 0.7, CHCl3)); H NMR (500 MHz,
CDCl3) δ 0.67 (3 H, s), 1.70 – 0.81 (58 H, m), 2.04 – 1.77 (5 H, m), 2.20 (1 H, br s, OH), 2.28–2.42 (4 H, m), 3.04 (1 H, br s, OH), 3.15 (1 H, br s, OH), 3.29–3.36 (1 H, m, H4'),
82 3.44–3.54 (2 H, m, H2', H3), 3.72 (1 H, t, J 9.2 Hz, H3'), 3.86 (1 H, ddd, J 2.1, 4.8, 9.9 Hz, 1H, H5'), 4.25 (1 H, dd, J 2.0, 12.2 Hz, H6b'), 4.48 (1 H, dd, J 4.9, 12.2 Hz, H6a'),
13 5.01 (1 H, d, J 4.0 Hz, H1'), 5.33–5.37 (1 H, m, H6); C NMR (126 MHz, CDCl3) δ 11.8, 14.1, 18.7, 19.3, 21.0, 22.54, 22.68, 22.80, 23.8, 24.27, 24.94, 27.99, 28.01, 28.21, 29.22, 29.28, 29.36, 29.56, 29.65, 29.66, 29.70, 31.85, 31.92, 34.2, 35.8, 36.17, 36.64, 36.97, 39.50, 39.72, 40.1, 42.3, 50.1, 56.15, 56.71, 63.1 (C6'), 70.0 (C4', C5'), 72.1 (C2'), 74.6 (C3'), 78.4
+ (C3), 96.9 (C1'), 122.2 (C6), 140.3 (C5), 174.6 (C=O); HRMS (ESI ) calcd for C49H90O7
+ (M + NH4) 776.6399. Found 776.6370.
Cholesteryl 6-O-hexadecanoyl-α-D-glucopyranoside (26)
O Palmitoyl chloride (27 µL, 0.088 mmol) was added to a solution 14 O H HO O HO H H HO of DMAP (5 mg, 0.044 mmol) and 23 (34 mg, 0.044 mmol) in O ◦ pyridine/CH2Cl2 (3:97, 1.2 mL) at 0 C. The solution was allowed to stir at rt for 4 h, then MeOH (50 µL) was added and the mixture was stirred for 30 min. The solvent was evaporated under reduced pressure to afford a colourless residue. The residue was redis-
+ solved in MeOH/CHCl3 (1:1, 2 mL) and Dowex 50WX8-200 (H form, approx. 100 mg) was added. After stirring for 1 h the mixture was filtered and the filtrate concentrated. Flash chromatography (EtOAc/pet. spirits/MeOH) afforded 26 as a white solid (23 mg,
24 1 67% yield). [α]D +28.9 (c 1, CHCl3); H NMR (500 MHz, CDCl3) δ 0.67 (3 H, s), 0.79– 1.66 (62 H, m), 1.77–2.05 (5 H, m), 2.23–2.41 (5 H, m), 3.27 (2 H, br s, 2 × OH), 3.33 (1 H, td, J 3.1, 9.7 Hz, H4'), 3.43–3.54 (2 H, m, H2', H3), 3.72 (1 H, t, J 9.3 Hz, H3'), 3.86 (1 H, ddd, J 2.0, 4.9, 9.9 Hz, H5'), 4.26 (1 H, d, J 12.0 Hz, H6b'), 4.46 (1 H, dd, J 3.3, 12.1 Hz, H6a'), 5.01 (1 H, d, J 4.0 Hz, H1'), 5.32–5.37 (1 H, m, H6); 13C NMR (126 MHz,
CDCl3) δ 12.0, 14.3, 18.9, 19.5, 21.2, 22.71, 22.85, 22.96, 24.00, 24.43, 25.1, 28.16, 28.38, 29.40, 29.46, 29.53, 29.74, 29.83, 29.88, 32.02, 32.09, 34.4, 35.94, 36.34, 36.80, 37.1, 39.7, 39.9, 40.3, 42.5, 50.3, 56.32, 56.88, 63.3 (C6), 70.15 (C4'), 70.21 (C5'), 72.2 (C2'), 74.8 (C3'), 78.6 (C3), 97.0 (C1'), 122.4 (C6), 140.4 (C5), 174.7 (C=O); HRMS (ESI+) calcd
+ for C51H94O7 (M + NH4) 804.6712. Found 804.6725.
83 Cholesteryl 6-O-octadecanoyl-α-D-glucopyranoside (27)
O
Stearoyl chloride (28 µL, 0.082 mmol) was added to a solution 16 O H HO O HO H H HO of DMAP (4.5 mg, 0.044 mmol) and 23 (31 mg, 0.041 mmol) O
◦ in pyridine/CH2Cl2 (3:97, 1.2 mL) at 0 C. The solution was allowed to stir at rt for 4 h, then MeOH (50 µL) was added and the mixture was stirred for 30 min. The solvent was evaporated under reduced pressure to afford a colourless residue. The residue was redis-
+ solved in MeOH/CHCl3 (1:1, 2 mL) and Dowex 50WX8-200 (H form, approx. 100 mg) was added. After stirring for 1 h the mixture was filtered and the filtrate concentrated. Flash chromatography (EtOAc/pet. spirits/MeOH) afforded 27 as a white solid (24 mg,
24 1 72% yield). [α]D +29.5 (c 1, CHCl3); H NMR (500 MHz, CDCl3) δ 0.67 (s, 3H), 0.80– 1.75 (66 H, m), 1.77–2.06 (5 H, m), 2.16–2.42 (5 H, m), 3.02–3.34 (2 H, br s, 2 × OH), 3.33 (1 H, t, J 9.5 Hz, H4'), 3.43–3.53 (2 H, m, H2', H3), 3.72 (1 H, t, J 9.2 Hz, H3'), 3.86 (1 H, ddd, J 2.0, 4.9, 9.9 Hz, H5'), 4.26 (1 H, d, J 12.2 Hz, H6b'), 4.46 (1 H, dd, J 5.1, 11.4
13 Hz, H6a'), 5.01 (1 H, d, J 3.8 Hz, H1'), 5.35 (1 H, m, H6); C NMR (126 MHz, CDCl3) δ 12.0, 14.3, 18.9, 19.5, 21.2, 22.71, 22.85, 22.96, 24.00, 24.44, 25.1, 28.16, 28.38, 29.40, 29.46, 29.52, 29.75, 29.83, 29.88, 32.02, 32.08, 34.4, 35.94, 36.34, 36.81, 37.1, 39.7, 39.9, 40.3, 42.5, 50.3, 56.32, 56.88, 63.3 (C6'), 70.16 (C4', C5'), 72.2 (C2'), 74.8 (C3'), 78.6 (C3),
+ 97.0 (C1'), 122.4 (C6), 140.4 (C5), 174.7 (C=O); HRMS (ESI ) calcd for C53H94O7 (M
+ + NH4) 832.7025. Found 832.6982.
Cholesteryl 6-O-oleyl-α-D-glucopyranoside (28)
Oleoyl chloride (28 µL, 0.086 mmol) was added to a solution O 7 O 7 H HO O HO H H of DMAP (5 mg, 0.043 mmol) and 23 (33 mg, 0.043 mmol) in HO O ◦ pyridine/CH2Cl2 (3:97, 1.2 mL) at 0 C. The solution was allowed to stir at rt for 4 h, then MeOH (50 µL) was added and the mixture was stirred for 30 min. The solvent was evaporated under reduced pressure to afford a colourless residue. The residue was redis-
+ solved in MeOH/CHCl3 (1:1, 2 mL) and Dowex 50WX8-200 (H form, approx. 100 mg) was added. After stirring for 1 h the mixture was filtered and the filtrate concentrated. Flash chromatography (EtOAc/pet. spirits/MeOH) afforded 28 as a white solid (22 mg,
84 24 1 63% yield). [α]D +31.1 (c 1, CHCl3); H NMR (500 MHz, CDCl3) δ 0.67 (3 H, s), 0.81– 1.66 (60 H, m), 1.77–2.09 (9 H, m), 2.21–2.42 (5 H, m), 3.29 (2 H, br s, 2 × OH), 3.33 (1 H, t, J 9.5 Hz, H4'), 3.43–3.53 (2 H, m, H2', H3), 3.72 (1 H, t, J 9.2 Hz, H3'), 3.86 (1 H, ddd, J 2.0, 4.8, 9.9 Hz, H5'), 4.26 (1 H, d, J 12.1 Hz, H6b'), 4.47 (1 H, dd, J 3.9, 11.8 Hz, H6a'), 5.01 (1 H, d, J 4.0 Hz, H1'), 5.29–5.40 (3 H, m, H6, 9'' , 10'' ); 13C NMR (126 MHz,
CDCl3) δ 12.0, 14.3, 18.9, 19.5, 21.2, 22.71, 22.83, 22.96, 24.0, 24.44, 25.1, 27.35, 27.38, 28.16, 28.18, 28.38, 29.35, 29.47, 29.49, 29.69, 29.89, 29.93, 32.02, 32.06, 32.08, 34.4, 35.94, 36.34, 36.81, 37.1, 39.66, 39.89, 40.3, 42.5, 50.3, 56.31, 56.88, 63.3 (C6'), 70.2 (C4', C5'), 72.2 (C2'), 74.8 (C3'), 78.6 (C3), 97.0 (C1'), 122.4 (C6), 129.8, 130.2 (C9'' , C10''
+ + ), 140.4 (C5), 174.7 (C=O); HRMS (ESI ) calcd for C53H92O7 (M + NH4) 830.6868. Found 830.6900.
85 Chapter 3
Rational design of synthetic Mincle agonists. Towards the development of safe vaccine adjuvants
3.1 Introduction
A wide range of lipidic species have been shown to be Mincle signalling agonists with varying potency. Additionally, a small number of crystal structures have been obtained with ligands bound to Mincle, and binding studies have been performed with simplified non-agonist analogues. Collectively, these data have the potential to be used to develop a model of the structural requirements for Mincle agonism.
3.1.1 Structural summary of glycolipids with demonstrated ability to
elicit signalling through Mincle
Trehalose-derived Mincle agonists
6,6'-Trehalose dimycolate (TDM) is the archetypal Mincle ligand. A range of 6,6'-trehalose mono and diesters have been synthesised and shown to exhibit Mincle signalling agonism. Of those derived or inspired by natural microbial products, a rep- resentative corynebacterial C32-monocorynomycolate (TMCM) and dicorynomycolate (TDCM) were prepared, both of which afforded levels of signalling stimulation similar to that of TDM in both mouse and human Mincle reporter cell lines.70 Consistent with this observation, a Corynebacterium diphtheriae strain lacking the ability to synthes- ise corynomycolic acids failed to stimulate macrophages or bind to a mouse Mincle-Fc fusion while the natural strain stimulated the production of nitrites and G-CSF in a
86 Mincle-dependent fashion.291 These findings suggest intermediate length mycolates are sufficient to induce Mincle signalling.
Straight chain primary-esters of trehalose have also been widely investigated as Mincle agonists. 6,6'-Trehalose dibehenate (TDB) is the best studied of the straight chain TDM analogues and has been demonstrated to signal through Mincle.52 Stocker and colleagues investigated a series of straight chain mono and diesters of trehalose C4:0
(diester only), C7:0 (diester only), C10:0 (diester only), C18:0 (diester only), C20:0 (diester only), C22:0 (mono and diester) and C26:0 (mono and diester) for their ability to stim- ulate Mincle-dependent cytokine production in bone marrow macrophages (BMMs)
72,73 (Scheme 3.1). Short-chain esters (C4:0–C10:0) did not elicit the production of cy- tokines from BMMs while longer chain esters (C18:0–C26:0) elicited production of IL-6 and IL-1b with maximal production seen for TDB. The monoesters were less potent than their equivalent diesters. These results suggest that simple straight chain esters of
C18:0–C26:0 can replace mycolic acids and that esterification on both primary positions is preferred. O
OH OH O 24 HO O 1. BSA, TMSO O hexacosanoic acid, RO O HO TBAF, DMF TMSO EDCl, DMAP RO HO TMSO RO O O O 2. K2CO3, toluene O OH O OH O OH HO OH propanol TMSO OTMS 45% RO OR OH 85% OTMS OR
Dowex-H+ R = TMS
CH2Cl2, MeOH 97% R = H Scheme 3.1. Stocker prepared long-chain trehalose monoesters from selectively deprotected trimethylsilyl trehalose.73
Mycobacteria produce other trehalose-based glycolipids. Decout and co-workers investigated trehalose derivatives acylated at the 2- and 3- positions (Ac2GL), and re- ported that these glycolipids, which bear combinations of straight and methyl-branched fatty esters, elicited poor signalling through Mincle (Figure 3.1).190 Of particular in- terest was the observation that a 2-O-sulfated derivative of Ac2GL (Ac2GSL) did not signal through Mincle at all, suggesting that a free 2-OH is crucial for robust Mincle signalling.
87 OH HO O O OH HO R1O HO O 16 O OH R2O OH hydroxyphthioceranoic acid OR3 1 1 R = H R = SO3H R2 = stearic/palmitic acid R2 = stearic/palmitic acid R3 = hydroxyphthioceranoic acid R3 = hydroxyphthioceranoic acid
Ac2GL (active) Ac2SGL (inactive)
Figure 3.1. Mycobacteria-derived Ac2GL was able to weakly signal through 190 Mincle. The 2-O-sulfated derivative, Ac2SGL, had no activity suggesting a free 2-OH is important for Mincle signalling.
Glucose-derived structures with Mincle activity
An early report noted that TDM treated with porcine trehalase lost its ability to signal through Mincle.48 This was interpreted to mean that glucosyl monomycolate (GMM) did not elicit Mincle signalling; however, more recent reports have demonstrated that authentic GMM signals through Mincle with greater potency than TDM.190 This dis- crepancy was proposed to arise from contamination of the trehalase preparation with an esterase. Van der Peet and co-workers prepared the corresponding glucosyl mono- corynomycolate (GMCM) and demonstrated that it also possessed activity similar in potency to TMCM (Figure 3.2).70 A follow-up report explored variation in the struc- ture of the mycolate ester to include extended (C23) and shortened (C5) α-chains as well as an α-chain possessing a terminal phenyl ring.189 Both the extended and phenyl ring glucosyl esters possessed Mincle stimulation potency similar to that of GMCM, while the activity of the shortened ester was attenuated. A C21 symmetrical β-branched fatty ester was also assessed and found to lack activity. In contrast to the potent activity of trehalose behenate esters the corresponding glucose monobehenate possessed very little activity.70,189,190
88 OH O O 18 O
O n O O 30 O 18 O R HO O HO HO HO HO HO m m HO OH HO OH HO OH (v. low activity) R = CH m = 3 n = 9 m = 1 αC βC Glc 3 (low activity) 4 12 (low activity) O R = CH3 m = 21 (active) n = 9 m = 7 αC9βC12Glc (low activity) α β O R = Ph m = 10 (active) n = 15 m = 12 C14 C18Glc (active) 20 HO O HO HO OH (v. low activity)
Figure 3.2. Van der Peet189 and Decout,190 independently prepared GMM/GMCM analogues and found that while short α-chains resulted in low Mincle stimulation, medium-length chains were well tolerated, as was functionalization of the α-chain terminus. Glucose monobehenate and a β-branched analogue were only weakly active.
Decout and colleagues prepared glucose esters with several α-branched fatty acids
(αC4βC12, αC9βC12 and αC14βC18) and found that the βC12 6-O-glucose esters possessed low activity regardless of the α-chain length (Figure 3.2).190 A 6-O-acyl glucose bearing the αC14βC18 fatty ester possessed Mincle signalling activity with potency similar to that of TDM and GMM and greater than TDB.
α- and β-glucosyl diglycerides, and β-gentiobiosyl diglycerides bearing a variety of fatty acid esters on the glycerol signal through Mincle (Figure 3.3).77,78,280 β-Gentiobiosyl diglycerides have been identified in Malassezia pachydermatis and Malassezia dermatis,280 bearing arrangements of anteiso fatty acids and for which weak signalling was seen through mouse Mincle but not human Mincle. Closely related species were reported from M. tuberculosis H37Ra,292 which bear arrangements of straight-chain and iso fatty acids. Richardson and co-workers synthesised several representative M. tuberculosis β- gentiobiosyl diglycerides, and demonstrated that like the Malassezia compounds, they
77 signal weakly through mouse but not human Mincle. Short-chain (C4/C8) and delipid- ated glycerol derivatives did not signal through Mincle. A β-glucosyl diglyceride bearing iso-C17:0 fatty acids was demonstrated to elicit moderate to strong mouse Mincle sig- nalling, and moderate human Mincle signalling in reporter cell lines. α-Glucosyl digly- cerides from several bacterial species have been identified as Mincle signalling agonists including from Streptococcus pneumoniae79 and Lactobacillus plantarum.78
89 HO HO O O HO OR HO HO O HO O OR HO HO HO OR O O HO HO HO OR HO O OR HO HO O OR
R = Straight, unsaturated and branched-chain esters
Figure 3.3. Diglycerides from M. pachydermatis, M. tuberculosis, S. pneumoniae and L. plantarum have been identified as Mincle agonists.
Non-carbohydrate structures with Mincle activity
Chapter 2 discussed the ability of a variety of sterol structures, including cholesterol, to elicit signalling through Mincle.80,81 Cholesterol elicits signalling through human, but not rodent Mincle.80 In Chapter 2 we showed that cholesteryl α-glucoside and 6-O-acyl derivatives thereof, from Helicobacter ssp. including H. pylori, and can signal through both mouse and human Mincle (Figure 3.4). The 6-O-acyl derivatives were more potent than the parent compound αCG.
H RO H HO O H H HO H H HO HO O
R = H or straight and unsaturated esters (C12:0-C18:1) Figure 3.4. Crystalline cholesterol, as well as cholesteryl α-glucoside and its 6-O-acyl derivatives can signal though human Mincle, and human and mouse Mincle, respectively.
All known active Mincle signalling molecules are insoluble at the concentrations required for signalling. Reporter cell and other in vitro assays rely on plates coated with ligand onto which cells are cultured. The physiochemical properties of the identified agonists may be representative of their role in cellular membranes. As such, Mincle may require multimerization for robust signalling.
90 3.1.2 Investigations into the Mincle binding site
Crystallography and molecular modelling
Crystal structures of bovine Mincle with and without trehalose bound have been solved, identifying the presence of primary and secondary carbohydrate recognition domains (CRD).67,68 Modelling around the CRD has revealed the presence of a lipophilic groove capable of accommodating two lipid chains extending away from O-6 of the primary CRD.68 Co-crystals of trehalose monobutyrate with the Mincle CRD showed the start of the alkyl chain at the ‘mouth’ of the proposed lipophilic groove; however, electron density for the remainder of the alkyl chain was not observed suggesting it may not have a fixed configuration.69 glucose 2 glucose 1
lipophilic groove
Figure 3.5. Mincle possesses two CRDs and a lipophilic groove capable of accommodating two lipid chains.
Soluble derivatives have provided insights into the Mincle binding region
As the known Mincle agonists are insoluble, binding affinity studies have been limited to soluble derivatives that do not typically elicit signalling. Moreover, binding studies have been limited to the soluble, extra-cellular domain of the Mincle receptor. Nonetheless, these studies have provided insightful data regarding the binding affinities of ligands. Feinberg and colleagues using surface plasmon resonance (SPR) reported that Mincle binds trehalose 36-fold stronger than methyl α-D-glucopyranoside, which highlights
68 the importance of the two sugar binding sites. C6:0 fatty acids on a trehalose diester
91 afforded a 250-fold increase in binding over non-acylated trehalose. Additional studies utilizing SPR to measure relative affinities have shown a steady increase in the affinity of Mincle for trehalose ester derivatives with increasing acyl chain length.67,69 Zheng and colleagues used an array bearing the major carbohydrate components of the mycobacterial cell wall to investigate bovine Mincle binding.293 The CRD of bovine Mincle was expressed with a biotin tag which was used to form a streptavidin-based fluorescent tetramer. Binding of this fluorescent tetramer to the array revealed that bovine Mincle would only bind to structures containing trehalose; in addition to the disaccharide, the Mincle CRD showed activity towards complex trehalose-containing glycans.
3.1.3 A structural model for Mincle agonism
The structure-function studies, crystallography, and molecular modelling, taken as a whole, at least for glycolipid ligands, suggest some important and common structural motifs necessary for robust Mincle signalling. Ligands can possess one or two sugars, which bind in a primary and secondary CRD; and possess one or two alkyl chains as part of one or more fatty esters (Figure 3.6). The 2-OH group must be unmodified. While two sugars are well tolerated in acylglycoses, a single sugar is preferred in glycosyl diglycerides. Decout and co-workers proposed that when considering the binding of TDM/GMM analogues, 3 of these 4 structural motifs (two sugars, two alkyl chains) must be present for robust signalling. However, they also noted that with appropriate branching and chain-length, the alkyl chains are more important than a second sugar unit, as high-
190 lighted by the branched αC14βC18Glc possessing greater activity than TDB. In addi- tion, branching at the α-position is preferred to the β-position.189 Cholesterol and derivatives thereof appear to represent an important yet mostly un- explored type of Mincle signalling agonist. The finding that cholesteryl glucosides pos- sessing fatty acid esters at O-6 are Mincle agonists opens a new direction of study.
92 O (di)acyltrehaloses O n HO O HO HO O glycosyl diglycerides O O O O n HO OH HO OH O HO O n O O O HO n HO O n O 1 sugar HO O HO n multiple alkyl chains HO OH acylglucoses
1 or 2 sugars multiple or branched alkyl chains
Figure 3.6. Several structural motifs have been identified for robust Mincle signalling for acyl glucose/trehalose derivatives: a free 2-OH on the primary carbohydrate, one or two alkyl chains as part of one or two fatty esters, one (firmer requirement for two alkyl chains) or two sugars. For glucosyl diglycerides a single sugar with two acyl chains appears to be preferred.
3.1.4 Mincle agonists are a promising class of vaccine adjuvants
There is growing interest in the development of safe and efficient vaccine adjuvants. This is driven by the development of subunit vaccines, which instead of whole cells only possess a subset of the antigenic compounds. This makes them less likely to result in adverse effects and simplifies the process of production; however, at the same time it makes them less immunogenic as they lack a full suite of immune effector molecules. Adjuvants currently in use promote humoral immune responses with little effect on cellular immunity.
TDM analogues, such as TDB, have long been investigated as immunomodulators and adjuvants and are known to be strong stimulators of cellular immunity.294 Oil-in- water emulsions of TDB were typically less effective than TDM or TMCM emulsions for protecting against bacterial infection. However, TDM, and likely TMCM, promote the formation of granulomas in a Mincle-dependent fashion and therefore are unsuitable
93 for human use. The recent development of a two-component adjuvant consisting of TDB and di- methyldioctylammonium bromide (DDA) provides an adjuvant that promotes strong humoral and cell mediated immune responses.52,153 Alone, DDA is a potent immunos- timulator but it lacks the stability required for use as an adjuvant. Co-formulation of DDA with TDB provides stable liposomes that play a dual role of acting as ‘storage ves- sels’ for antigen as well as actively stimulating the immune system.153 Davidsen and col- leagues found that the optimum ratio of TDB to DDA for immune stimulation activity as well as liposome stability to be 11% TDB (a formulation termed CAF01), and formu- lations with 6% or 20% TDB were less effective. The CAF01 formulation was optimal for induction of IFN-γ and IgG2b Ab production and afforded a 2-fold increase in IFN-γ production compared to DDA alone. This immune stimulation was shown to be Mincle- dependent by the lack of adjuvant effect in Mincle-/- mice when administered with the M. tuberculosis H1 (Ag85B-ESAT-6) subunit vaccine. As is typical of Mincle stimula- tion, the profile of cytokines released directs TH0 cell maturation towards the TH1 and
155 TH17 sub-types (Figure 3.7). Due to its novel induction of cellular as well as humoral immunity, CAF01 is currently undergoing trials as a human vaccine adjuvant. A recent phase I clinical study of the H1 vaccine adjuvanted by CAF01 found strong antigen- specific T cell responses 150 weeks after vaccination.152 CAF01 was demonstrated to be a safe and well tolerated adjuvant that promotes strong TH1 immune responses when administered with subunit vaccines in humans. Decout and co-workers have investigated other Mincle agonist/DDA formulations
190 as vaccine adjuvants. αC14βC18Glc, αC14βC18Man and TDB were formulated in a 1:25 ratio with DDA. As this ratio is well below the optimal ratio for TDB/DDA formulation, no statistical increase for TDB or αC14βC18Man above DDA alone was seen for IL-2, IFN-γ or IL-17 production. A significant increase in cytokine production was observed for the αC14βC18Glc/DDA formulation with an 80-fold increase in IgG2b titres over DDA alone.
94 TH1/TH17 directing cytokines
DDA/TDB liposome TH1
APC TH0 FcRy TCR Mincle antigen APM TH17
Figure 3.7. DDA/TDB liposomes, via stimulation of Mincle signalling, are able to direct the maturation of TH0 cells into TH1 and TH17 pro-inflammatory cells.
3.1.5 Research aims
Mincle agonists are a promising avenue of research towards the production of safe and effective vaccine adjuvants that stimulate both cellular and humoral immune responses to afford long lasting vaccine effect. This chapter seeks to exploit developing structure- activity relationships to rationally design novel Mincle agonists.
Aim 1: Using a structural template derived from observed and proposed structural re- quirements for signalling though Mincle we propose to prepare a small set of purely synthetic compounds as potential adjuvants. Tothis end, we will design synthetic targets that can be easily prepared. An ideal compound would be accessible in a few synthetic steps from commercially available materials.
Aim 2: Confirmation of Mincle activity with the potential for further evaluation of these compounds as useful adjuvants in collaboration with Sho Yamasaki and colleagues at Kyoto University.
3.2 Results and discussion
Taking note from the strong Mincle signalling agonism of the acylated cholesteryl α- glucosides from Helicobacter ssp., which possess lipophilic functionality at the 1 and 6 positions, we chose to investigate compounds with an alkyl group at the anomeric position of glucose and acylation at O-6. Compounds possessing this structure would
95 fit the observed structural requirements for Mincle signalling by possessing two alkyl chains as well as a sugar with free 2-OH.
Alkyl glucosides are widely used in consumer products, including cosmetics and
soaps. Medium-chain (C8–C16) alkyl β-glucosides are often used as non-ionic surfact- ants with octyl β-glucoside used for the non-denaturing solubilisation of integral mem- brane proteins.295 Both octyl (29) and lauryl β-glucosides (30) are cheap, commercially available reagents that would enable easy synthetic access to potential Mincle stimu- lating adjuvants (Figure 3.8). Their intermediate length alkyl chains should surffice to occupy the hydrophobic binding site of Mincle. HO HO HO O HO O PSfrag replacements HO O HO O OH OH octyl β-glucoside 29 lauryl β-glucoside 30
Figure 3.8. Octyl (29) and lauryl β-glucosides (30) provide attractive commercially available starting materials for the preparation of rationally-designed Mincle agonists.
Almost all previous work towards the synthesis of both natural and synthetic gluc- osyl and trehalose Mincle agonists has involved numerous protecting group manip- ulations to install the required functionality.70,73,77,78,189,190,296 Acylated trehalose and glucoses are typically prepared using benzyl or TMS protected sugars with either free primary hydroxyls or with appropriate leaving groups on the 6-position to enable se- lective installation of esters. As we sought to develop vaccine adjuvants that could be easily prepared we sought to avoid the use of protecting groups. This prompted us to revisit the regioselective acylation of the glucosides 29 and 30.
3.2.1 Selective acylation of unprotected glucosides
We chose to investigate a method for selective installation of esters on unprotected glycosides developed by Twibanire and colleagues involving uronium-based peptide coupling reagents under basic conditions.272–274 An earlier report from Hakki and co- workers had demonstrated that HBTU in the presence of DMAP and NMM could be applied to the selective preparation of a glucose 1,6-diester.297 Twibanire investigated
96 the benzotriazole-based reagents TBTU and TATU, and the Oxyma-based COMU in the presence of various bases for their ability to promote the formation of primary, sec- ondary and tertiary esters (Scheme 3.2).274 Only COMU in the presence of the strong base 7-methyl-1,5,7-triazabicyclo-[4.4.0]dec-5-ene (MTBD) could catalyse the forma- tion of tertiary esters. TATU, TBTU and COMU catalysed the formation of primary and secondary esters in the presence of DBU, while in the presence of the weaker base DIPEA, the benzotriazole reagents could only form primary esters. Of particular in- terest for our work was the finding that TBTU in the presence of DIEA promoted the chemoselective installation of both aryl and aliphatic esters on the primary alcohol of methyl α-glucopyranoside. BF BF N 4 N 4 N N N N N N O N O N O O R OH N HO R O TBTU, DIEA TBTU TATU HO O HO O HO HO CN HO CH2Cl2 HO PF6 OMe OMe N O N CO Et 2 R = Ph 81% N R = (BnOCH2)3C 78%
O COMU Scheme 3.2. Twibanire and colleagues assessed TBTU, TATU and COMU for the regioselective formation of esters and showed that primary esters formed selectively with TBTU in the presence of DIPEA.274
Subsequent publications from the same lab reporting the synthesis of cholesteryl 6-O-acyl β-galactosides from Borrelia burgdorferi273 and 6,6'-diacyltrehalose maradol- ipids from Caenorhabditis elegans272 detailed further optimizations and demonstrated that when performed in pyridine a high degree of chemoselectivity is maintained without the need for a tertiary amine base (Scheme 3.3).
97 O OH 1. oleic acid (1.1 eq), O HO O HBTU, pyridine 7 7 HO 65% HO O HO HO O HO 2. 13-methyltetradecanoic acid (1.1 eq), O O OH HO OH HBTU, pyridine O O OH 81% HO OH 11 OH O Scheme 3.3. Reaction optimization by Twibanire and co-workers demonstrated regioselective installation of acyl chains on trehalose using pyridine as base and solvent.272,273
3.2.2 Straight chain esters of alkyl β-glucosides
We chose to install three representative straight chain esters, short (octanoyl, C8:0), me-
dium (palmitoyl, C16:0) and long (behenoyl, C22:0), onto each of our alkyl glucosides. Reaction of the appropriate acid with 29 and HBTU in pyridine afforded the esters
C8GlcC8 (31), C16GlcC8 (32) and C22GlcC8 (33) in 61%, 33% and 47% yields, respect-
ively (Scheme 3.4). Similarly, C8GlcC12 (34), C16GlcC12 (35) and C22GlcC12 (36) were prepared from 30 in 56%, 46% and 47% yields, respectively. We did not optimize these reactions as the moderate yields provided sufficient material for Mincle signalling as- PSfrag replacements says.
O 31 61% n = 8, y = 6 32 56% n = 12, y = 6 y = 6, a HO O 33 33% n = 8, y = 14 y = 14, b y O O 34 46% n = 12, y = 14 HO y = 20, c HO HO O HO O 35 47% n = 8, y = 20 n n OH OH 36 47% n = 12, y = 20 29 n = 7 30n = 11
Scheme 3.4. a) octanoic acid, HBTU, pyridine; b) palmitic acid, HBTU, pyridine; c) behenic acid, HBTU, pyridine.
3.2.3 Branched chain esters of alkyl β-glucosides
Decout and colleagues have demonstrated that 6-O-acyl glucoses bearing medium- length α-branched esters signal potently through Mincle.190 The fatty acids used in these studies were prepared by reaction of a straight-chain fatty acid with LDA and NaH to obtain the enolate followed by substitution of an alkyl iodide to afford the α-branched
98 acid (Scheme 3.5). This procedure does not control the stereochemistry at the α-position and therefore creates a racemic mixture of the R and S enantiomers. It is not known if the stereochemistry of the branch is relevant to Mincle activity. Two possible ways to overcome this are a stereo-controlled synthesis, for example using a chiral auxiliary, or the preparation of an acid with equal α- and β-chain lengths, thereby avoiding the formation of enantiomers.
O
OH
LDA, NaH then 1-iodotetradecane 14% O
OH
Scheme 3.5. Utilizing a non-stereoselective approach Decout and colleagues prepared a small set of α-branched fatty acids that were converted to the 6-O-acylglucoses.190 It is unknown whether the stereochemistry plays a role in Mincle activity.
Fortuitously, we found that the symmetric αC16βC18 symmetric acid (1-hexadecyloctadecanoic acid) is commercially available. Reaction of 1-hexadecyloctadecanoic acid with 29 or 30 in the presence of HBTU afforded
αC16βC18GlcC8 (37) and αC16βC18GlcC12 (38) in 15% and 12% yields, respectively (Scheme 3.6). These yields were lower than those obtained with the straight chain acids and may represent a technical limitation of the HBTU-catalysed esterification although
no optimization was performed. An attempt to prepare an ester of C32-corynomycolic acid with 30 was unsuccessful.
O HO PSfrag replacements 15 O HO O O O a HO HO 15 HO O n n OH OH 29 n = 7 37 15% 30 n = 11 38 12% Scheme 3.6. a) 1-hexadecyloctadecanoic acid, HBTU, pyridine.
99 3.2.4 Cholesteroloxyacetyl esters of alkyl β-glucosides
Due to the potent Mincle signalling agonism seen for the cholesteryl 6-O-acyl-α- glucosides from Helicobacter ssp. we investigated incorporation of cholesterol into an ester install at O-6. Preparation of 1-cholesteryloxyacetic acid was undertaken by reac-
tion of ethyl diazoacetate with cholesterol and BF3·etherate in CH2Cl2 to afford 39 in 61% yield (Scheme 3.7).298 Saponification with 2M NaOH in EtOH afforded the acid (40) in quantitative yield. Reaction of 40 with 29 or 30 in the presence of HBTU af-
forded CholAcGlcC8 (41) and CholAcGlcC12 (42) in 47% and 45% yields, respectively (Scheme 3.7).
H a H b H
H H 61% H H quant. H H EtO HO HO O O O 39O 40
H PSfrag replacements H H O O
HO O HO O c HO O HO O HO O n n OH OH 29n = 7 41 47% 30n = 11 42 45%
Scheme 3.7. a) ethyl diazoacetate, BF3·Et2O, CH2Cl2; b) 2 M NaOH, EtOH, H2O; c) 40, HBTU, pyridine.
3.2.5 Acylated alkyl β-glucosides signal potently through Mincle
The set of 6-O-acyl alkyl β-glucosides as well as the parent octyl and lauryl β-glucosides were investigated for their ability to induce signalling through Mincle by Masahiro Nagata in the laboratory of Sho Yamasaki (Division of Molecular Immunology, Kyushu University, Japan).
Assays were performed by coating wells with microgram amounts of the lipids,
100 followed by the culturing of reporter cells and measurement of GFP fluorescence to quantify the degree of Mincle signalling agonism. Octyl and lauryl β-glucosides did not signal through Mincle, a finding that was unsurprising as these compounds do not meet the criteria for Mincle signalling (Figure 3.9). All of the synthesised acylated derivatives signalled through both mouse and human Mincle, with similar potency potency for each compound with the two receptor orthologs. Little difference in activity was ob- served between the equivalent octyl and lauryl glucosides. For the straight-chain acyl- ated compounds those bearing longer acyl chains exhibited greater potency, with the exception of C22GlcC12. The most potent compounds were those possessing branched- chain and cholesteryloxy esters, which elicited a similar degree of activity to that of TDB at the lowest tested concentration. An experiment (data not shown) of the C22:0 com- pounds at 0.01 µg showed lower potency than TDB at the same concentration.
120 100 mMincle-γ 80 60 40
NFAT-GFP NFAT-GFP (%) 20 0
TDB GlcC GlcC GlcC GlcC GlcC CGlcC GlcC GlcC CGlcC C C C C βC 120 βCGlcC αC CholAcGlcCCholAcGlcC 100 hMincle-γ αC 80 60 40
NFAT-GFP NFAT-GFP (%) 20 0
TDB GlcC GlcC GlcC GlcC GlcC GlcC C C C CGlcC CGlcC CGlcC 120 βC βCGlcC CholAcGlcC 100 FcRγ αC αC CholAcGlcC 80 60 40
NFAT-GFP NFAT-GFP (%) 20 0
TDB GlcC GlcC GlcC GlcC GlcC CGlcC C CGlcC CGlcC CGlcC C βCGlcC βC CholAcGlcC αC αC CholAcGlcC Figure 3.9. NFAT-GFP reporter cells expressing either human Mincle/FcRγ or mouse Mincle/FcRγ, as well as those expressing FcRγ alone were tested for their reactivity to plate-bound synthetic potential agonists. Assays were performed in duplicate; the mean values and standard deviations are shown.
101 3.3 Summary
Building upon the known structural features required for robust Mincle signalling ag- onism we rationally-designed synthetic agonists. Intermediate length alkyl β-glucosides were elaborated with straight-chain, branched-chain and cholesteryloxy esters at O-6. These compounds were assessed for their ability to stimulate Mincle signalling and were all found to the potent agonists for mouse and human Mincle. Non-functionalised, crystalline cholesterol has been demonstrated to signal though the human but not the mouse form of Mincle.80,81 Cholesterol esters do not possess Mincle activity at all. The findings presented here for synthetic Mincle agonists possess- ing cholesteryloxy esters and in Chapter 2 for 6-O-acyl cholesteryl glucosides suggest that these compounds bind in a fashion distinct from that of cholesterol itself.
3.3.1 Future work
As these compounds possess the ability to stimulate signalling through Mincle, evalu- ation of their ability to act as vaccine adjuvants should be investigated. Retrogenic mice expressing the human form of Mincle are required for these experiments. We are cur- rently working with our collaborators to determine if such studies are feasible.
102 3.4 Experimental
General procedure for the synthesis of 6-O esters from unprotected glycosides:273 Carboxylic acid (1.2 equiv.) was added to a suspension of HBTU (1.2 equiv.) in pyridine (approx 10 mL/mmol) and the mixture was stirred for 20-30 minutes before addition of the glycoside (1 equiv.). The solution was stirred for 2-3 days and then concentrated. Flash chromatography of the residue (pet. spirits/EtOAc/MeOH) afforded the 6-O-acyl glycoside.
Octyl 6-O-octanoyl-β-D-glucoside (31) O
6 O Octyl β-D-glucoside (50 mg, 0.171 mmol) was reacted with HO O HO O HO 7 octanoic acid (32 µL, 0.205 mmol) according to the general pro-
24 1 cedure to afford 31 as a colourless glass (44 mg, 61%). [α]D -36.5 (c 2.2, CHCl3); H
NMR (400 MHz, CDCl3:CD3OD 95:5) δ 0.81 (6 H, t, J 6.5 Hz, CH2CH2CH3), 1.23 (18 H, m, alkyl), 1.50 – 1.60 (4 H, m, β-CH2), 2.27 (3 H, t, J 7.6 Hz, CO2CH2), 3.19 – 3.32
(2 H, m, H2,4), 3.35 – 3.42 (2 H, m, H3,5), 3.43 – 3.51 (1 H, m, OCH2CH2), 3.73 – 3.84
(1 H, m, OCH2CH2), 4.20 (2 H, m, H1,6a), 4.27 (0.2 H, s, OH), 4.32 (1 H, m, H6b), 4.65
13 (0.2 H, s, OH), 4.87 (0.2 H, s, OH); C NMR (100 MHz, CDCl3:CD3OD 95:5) δ 14.0, 14.0, 22.6, 22.6, 24.9, 25.9, 28.9, 29.1, 29.3, 29.4, 29.6, 31.7, 31.8, 34.2 (alkyl), 63.6 (C6),
70.2 (C4), 70.3 (OCH2CH2), 73.4 (C2), 73.8 (C3), 76.3 (C5), 102.7 (C1), 174.4 (CO2);
+ + HRMS (ESI ) calcd for C22H42O7 [M + H] 419.3003. Found 419.3005.
Octyl 6-O-palmitoyl-β-D-glucoside (32) O
14 O Octyl β-D-glucoside (50 mg, 0.171 mmol) was reacted with HO O HO O HO 7 palmitic acid (53 mg, 0.205 mmol) according to the general pro-
24 1 cedure to afford 32 as a colourless glass (30 mg, 33%). [α]D -33.7 (c 1.5, CHCl3); H
NMR (400 MHz, CDCl3:CD3OD 95:5) δ 0.82 (6 H, t, J 6.7 Hz, CH2CH2CH3), 1.15 – 1.32
(34 H, m, alkyl), 1.51 – 1.62 (4 H, m, β-CH2), 2.28 (2 H, t, J 7.6 Hz, CO2CH2), 3.20 – 3.31
(2 H, m, H2,4), 3.36 – 3.43 (2 H, m, H3,5), 3.46 (1 H, dt, J 9.5, 7.0 Hz, OCH2CH2), 3.80
103 (1 H, dt, J 9.5, 7.0 Hz, OCH2CH2), 4.19 (1 H, d, J 7.8 Hz, H1), 4.22 (1 H, dd, J 12.1, 6.1
13 Hz, H6a), 4.32 (1 H, dd, J 12.1, 2.1 Hz, H6b); C NMR (100 MHz, CDCl3:CD3OD 95:5) δ 14.07, 14.09, 22.67, 22.70, 24.96, 25.93, 29.18, 29.27, 29.33, 29.38, 29.43, 29.52, 29.64,
29.68, 29.69, 29.72, 31.8, 31.9, 34.2 (alkyl), 63.6 (C6), 70.2 (C4), 70.4 (OCH2CH2), 73.5
+ (C2), 73.9 (C3), 76.3 (C5), 102.8 (C1), 174.5 (CO2); HRMS (ESI ) calcd for C30H58O7 [M + H]+ 531.4255. Found 531.4260.
Octyl 6-O-behenoyl-β-D-glucoside (33) O
Octyl β-D-glucoside (50 mg, 0.171 mmol) was reacted with be- 20 O HO O HO O henic acid (70 mg, 0.205 mmol) according to the general pro- HO 7
24 1 cedure to afford 33 as a colourless glass (49 mg, 47%). [α]D -29.3 (c 1.6, CHCl3); H
NMR (400 MHz, CDCl3:CD3OD 95:5) δ 0.82 (6 H, t, J = 6.7 Hz, CH2CH2CH3), 1.00 –
1.32 (46 H, m, alkyl), 1.51 – 1.61 (4 H, m, β-CH2), 2.28 (2 H, t, J = 7.6 Hz, CO2CH2), 3.20 – 3.32 (2 H, m, H2,4), 3.36 – 3.43 (2 H, m, H3,5), 3.47 (1 H, dt, J = 7.0, 9.4 Hz,
OCH2CH2), 3.80 (1 H, dt, J = 7.0, 9.4 Hz, OCH2CH2), 4.19 (1 H, d, J = 8.0 Hz, H1), 4.17 – 4.26 (1 H, m, H6a), 4.32 (1 H, dd, J = 2.1, 11.9 Hz, H6b); 13C NMR (100 MHz,
CDCl3:CD3OD 95:5) δ 14.07, 14.10, 22.67, 22.71, 25.0, 25.9, 29.2, 29.27, 29.34, 29.38, 29.43, 29.5, 29.6, 29.68, 29.71, 29.73, 31.8, 31.9, 34.2 (alkyl), 63.6 (C6), 70.2 (C4), 70.4
+ (OCH2CH2), 73.5 (C2), 73.9 (C3), 76.3 (C5), 102.8 (C1), 174.5 (CO2); HRMS (ESI )
+ calcd for C36H70O7 [M + H] 615.9154. Found 615.9156.
Octyl 6-O-cholestryloxyacetyl-β-D-glucoside (41) O O O H H HO O HO O H Octyl β-D-glucoside (25 mg, 0.086 mmol) was reacted with OH 7 cholesteryloxyacetic acid (46 mg, 0.103 mmol) according to the general procedure to afford 41 as a colourless glass (29 mg, 47%). 1H NMR
(400 MHz, CDCl3:CD3OD 95:5) δ 0.62 (3 H, s), 0.74 – 1.66 (48 H, m, alkyl, choles- terol), 1.71 – 2.00 (5 H, m), 2.20 (1 H, m, H4' ), 2.33 (1 H, m, H4' ), 3.16 – 3.49 (6 H, m, OCH2CH2,H2,3,4,5,3' ), 3.79 (1 H, dt, J = 6.9, 9.5 Hz, OCH2CH2), 4.11 (2 H, s,
104 O=CCH2O), 4.20 (1 H, d, J = 7.7 Hz, H1), 4.30 (1 H, dd, J = 5.5, 11.9 Hz, H6a), 4.39 (1 H, dd, J = 2.2, 11.9 Hz, H6b), 5.28 – 5.32 (1 H, m, HC=H); 13C NMR (100 MHz,
CDCl3:CD3OD 95:5) δ 11.8, 14.0, 18.6, 19.2, 21.0, 22.4, 22.6, 22.7, 23.7, 24.2, 25.8, 27.9, 28.0, 28.1, 29.2, 29.4, 29.5, 31.76, 31.79, 31.84, 35.7, 36.1, 36.7, 37.0, 38.5, 39.4, 39.7,
42.2, 50.1, 56.1, 56.7 (alkyl, cholesterol), 63.8 (C6), 65.3 (O=CCH2O), 69.9 (C4), 70.3
(OCH2CH2), 73.3 (C2), 73.6 (C3), 76.2 (C5), 80.1 (C3' ), 102.7 (C1), 122.0 (HC=C),
+ + 140.2 (C=CH), 171.2 (CO2); HRMS (ESI ) calcd for C43H74O8 [M + H] 719.5457. Found 719.5456.
Octyl 6-O-(1-hexadecyloctadecanoyl)-β-D-glucoside (37) O 14 O Octyl β-D-glucoside (25 mg, 0.086 mmol) was reacted with HO O O 14 HO HO 7 1-hexadecyloctadecanoic acid (52 mg, 0.103 mmol) according to the general procedure to afford 37 as a colourless glass (11 mg, 15%). 1H NMR
(400 MHz, CDCl3:CD3OD 95:5) δ 0.82 (9 H, t, J = 6.8 Hz, CH2CH2CH3), 1.14 – 1.33
(66 H, m, acyl), 1.33 – 1.45 (2 H, m, β-CH2), 1.55 (4 H, m, β-CH2), 2.31 (1 H, tt, J = 5.4,
8.6 Hz, CO2CH), 3.19 – 3.30 (2 H, m, H2,4), 3.35 – 3.50 (3 H, m, OCH2CH2,H3,5), 3.81
(1 H, dt, J = 6.9, 9.6 Hz, OCH2CH2), 4.17 (1 H, dd, J = 6.6, 11.8 Hz, H6a), 4.20 (1 H, d, J =
13 7.8 Hz, H1), 4.39 (1 H, dd, J = 2.0, 11.9 Hz, H6b); C NMR (100 MHz, CDCl3:CD3OD 95:5) δ 13.97, 13.99, 22.57, 22.60, 25.9, 27.3, 29.2, 29.3, 29.36, 29.43, 29.5, 29.56, 29.58, 29.60, 29.63, 31.75, 31.84, 32.26, 32.28 (alkyl), 45.7 (α-acyl), 63.5 (C6), 70.1 (C4), 70.3
+ (OCH2CH2), 73.4 (C2), 73.9 (C3), 76.2 (C5), 102.6 (C1), 177.1 (CO2); HRMS (ESI )
+ calcd for C48H94O7 [M + H] 783.7072. Found 783.7073.
Lauryl 6-O-octanoyl-β-D-glucoside (34) O
6 O Lauryl β-D-glucoside (50 mg, 0.143 mmol) was reacted with HO O HO O HO 11 octanoic acid (27 µL, 0.172 mmol) according to the general pro-
24 1 cedure to afford 34 as a colourless glass (38 mg, 56%). [α]D -24.9 (c 1.3, CHCl3); H
NMR (400 MHz, CDCl3:CD3OD 95:5) δ 0.82 (6 H, t, J = 6.6 Hz, CH2CH2CH3), 1.16 –
105 1.30 (26 H, m, alkyl), 1.51 – 1.61 (4 H, m, β-CH2), 2.29 (2 H, t, J = 7.6 Hz, CO2CH2), 3.21 – 3.31 (2 H, m, H2,4), 3.36 – 3.43 (2 H, m, H3,5), 3.47 (1 H, dt, J = 7.0, 9.5
Hz, OCH2CH2), 3.80 (1 H, dt, J = 7.0, 9.5 Hz, OCH2CH2), 4.20 (1 H, d, J = 7.8 Hz, H1), 4.22 (1 H, dd, J = 6.0, 12.1 Hz, H6a), 4.32 (1 H, dd, J = 2.1, 11.9 Hz, H6b); 13C
NMR (100 MHz, CDCl3:CD3OD 95:5) δ 14.0, 14.1, 22.6, 22.7, 24.9, 25.9, 29.0, 29.1, 29.4, 29.5, 29.63, 29.65, 29.66, 29.70, 31.7, 31.9, 34.2 (alkyl), 63.6 (C6), 70.2 (C4), 70.4
+ (OCH2CH2), 73.5 (C2), 73.9 (C3), 76.3 (C5), 102.8 (C1), 174.5 (CO2); HRMS (ESI )
+ calcd for C26H50O7 [M + H] 475.3629. Found 479.3630.
Lauryl 6-O-palmitoyl-β-D-glucoside (35) O
Lauryl β-D-glucoside (50 mg, 0.143 mmol) was reacted with 14 O HO O HO O palmitic acid (44 mg, 0.172 mmol) according to the general pro- HO 11
24 1 cedure to afford 35 as a colourless glass (39 mg, 46%). [α]D -26.6 (c 1.3, CHCl3); H
NMR (400 MHz, CDCl3:CD3OD 95:5) δ 0.82 (6 H, t, J = 6.7 Hz, CH2CH2CH3), 1.13 –
1.31 (42 H, m, alkyl), 1.50 – 1.62 (4 H, m, β-CH2), 2.28 (2 H, t, J = 7.6 Hz, CO2CH2), 3.21 – 3.31 (2 H, m, H2,4), 3.36 – 3.43 (2 H, m, H3,5), 3.47 (1 H, dt, J = 7.1, 9.5 Hz,
OCH2CH2), 3.80 (1 H, dt, J = 7.0, 9.5 Hz, OCH2CH2), 4.20 (1 H, d, J = 7.8 Hz, H1), 4.22 (1 H, dd, J = 6.1, 12.1 Hz, H6a), 4.32 (1 H, dd, J = 2.1, 12.0 Hz, H6b); 13C NMR
(100 MHz, CDCl3:CD3OD 95:5) δ 14.1, 22.7, 25.0, 25.9, 29.2, 29.3, 29.4, 29.50, 29.52, 29.64, 29.65, 29.67, 29.70, 29.71, 29.71, 29.73, 31.9, 34.2 (alkyl), 63.6 (C6), 70.2 (C4),
+ 70.4 (OCH2CH2), 73.5 (C2), 73.9 (C3), 76.3 (C5), 102.8 (C1), 174.5 (CO2); HRMS (ESI )
+ calcd for C34H66O7 [M + H] 587.4881. Found 587.4880.
Lauryl 6-O-behenoyl-β-D-glucoside (36) O
Lauryl β-D-glucoside (50 mg, 0.143 mmol) was reacted with be- 20 O HO O HO O henic acid (59 mg, 0.172 mmol) according to the general pro- HO 11
24 1 cedure to afford 36 as a colourless glass (45 mg, 47%). [α]D -24.4 (c 1.5, CHCl3); H
NMR (400 MHz, CDCl3:CD3OD 95:5) δ 0.82 (6 H, t, J = 6.7 Hz, CH2CH2CH3), 1.14 –
106 1.31 (54 H, m, alkyl), 1.51 – 1.62 (4 H, m, β-CH2), 2.28 (2 H, t, J = 7.6 Hz, CO2CH2), 3.21 – 3.31 (2 H, m, H2,4), 3.36 – 3.43 (2 H, m, H3,5), 3.47 (1 H, dt, J = 7.0, 9.5 Hz,
OCH2CH2), 3.80 (1 H, dt, J = 7.0, 9.5 Hz, OCH2CH2), 4.20 (1 H, d, J = 7.7 Hz, H1), 4.22 (1 H, dd, J = 6.1, 12.1 Hz, H6a), 4.32 (1 H, dd, J = 2.2, 12.0 Hz, H6b); 13C NMR
(100 MHz, CDCl3:CD3OD 95:5) δ 14.1, 22.7, 25.0, 25.9, 29.2, 29.3, 29.4, 29.50, 29.53, 29.64, 29.65, 29.67, 29.69, 29.71, 29.74, 32.0, 34.2 (alkyl), 63.6 (C6), 70.2 (C4), 70.4
+ (OCH2CH2), 73.5 (C2), 73.9 (C3), 76.3 (C5), 102.8 (C1), 174.5 (CO2); HRMS (ESI )
+ calcd for C40H78O7 [M + H] 671.5820. Found 671.5822.
Lauryl 6-O-cholesteryloxyacetyl-β-D-glucoside (42) O O O H H HO O HO O H OH 11 Lauryl β-D-glucoside (25 mg, 0.072 mmol) was reacted with cholesteryloxyacetic acid (38 mg, 0.086 mmol) according to the general procedure to afford 42 as a colourless glass (25 mg, 45%). 1H NMR (400 MHz,
CDCl3:CD3OD 95:5) δ 0.63 (3 H, s), 0.76 – 1.61 (56 H, m, alkyl, cholesterol), 1.73 – 2.01 (5 H, m), 2.17 – 2.26 (1 H, m, H4' ), 2.30 – 2.37 (1 H, m, H4' ), 3.17 – 3.51
(6 H, m, OCH2CH2,H2,3,4,5,3' ), 3.80 (1 H, dt, J = 6.9, 9.5 Hz, OCH2CH2), 4.12 (2 H,
O=CCH2O), 4.21 (1 H, d, J = 7.7 Hz, H1), 4.32 (1 H, dd, J = 5.7, 11.9 Hz, H6a), 4.40 (1 H,
13 dd, J = 2.3, 11.9 Hz, H6b), 5.31 (1 H, m, HC=H); C NMR (100 MHz, CDCl3:CD3OD 95:5) δ 11.8, 14.0, 18.6, 19.2, 21.0, 22.5, 22.6, 22.7, 23.8, 24.2, 25.9, 27.9, 28.0, 28.2, 29.3, 29.5, 29.57, 29.60, 29.64, 31.8, 31.85, 31.86, 35.7, 36.1, 36.7, 37.0, 38.5, 39.4, 39.7,
42.3, 50.1, 56.1, 56.7 (alkyl, cholesterol), 63.8 (C6), 65.4 (O=CCH2O), 69.9 (C4), 70.3
(OCH2CH2), 73.4 (C2), 73.6 (C3), 76.2 (C5), 80.1 (C3' ), 102.7 (C1), 122.0 (HC=C),
+ + 140.2 (C=CH), 171.2 (CO2); HRMS (ESI ) calcd for C47H82O8 [M + H] 775.6083. Found 775.6080.
Lauryl 6-O-(1-hexadecyloctadecanoyl)-β-D-glucoside (38) O 14 O Lauryl β-D-glucoside (25 mg, 0.072 mmol) was reacted with HO O O 14 HO HO 11 1-hexadecyloctadecanoic acid (44 mg, 0.086 mmol) according
107 to the general procedure to afford 38 as a colourless glass (7 mg, 12%). 1H NMR
(400 MHz, CDCl3:CD3OD 95:5) δ 0.83 (9 H, t, J = 6.7 Hz), 1.10 – 1.33 (74 H, m, alkyl),
1.33 – 1.45 (2 H, m, β-CH2), 1.48 – 1.61 (4 H, m, β-CH2), 2.31 (1 H, tt, J = 5.5, 8.7 Hz,
CO2CH), 3.21 – 3.29 (2 H, m, H2,4), 3.37 – 3.49 (3 H, m, OCH2CH2,H3,5), 3.80 (1 H, dt,
J = 6.9, 9.5 Hz, OCH2CH2), 4.17 (1 H, dd, J = 6.7, 11.9 Hz, H6a), 4.20 (1 H, d, J = 7.8 Hz,
13 H1), 4.39 (1 H, dd, J = 2.1, 12.0 Hz, H6b); C NMR (100 MHz, CDCl3:CD3OD 95:5) δ 14.0, 22.6, 25.9, 27.3, 29.3, 29.4, 29.5, 29.58, 29.61, 29.63, 31.8, 32.3 (alkyl), 45.7 (α-acyl),
63.5 (C6), 70.1 (C4), 70.3 (OCH2CH2), 73.4 (C2), 73.9 (C3), 76.2 (C5), 102.6 (C1), 177.1
+ + (CO2); HRMS (ESI ) calcd for C52H102O7 [M + H] 839.7698. Found 839.7698.
108 Chapter 4
α-Glucosyl and α-glucuronosyl diglycerides from commensal and pathogenic bacteria
4.1 Introduction
4.1.1 Nomenclature of glycerides
Before embarking on a discussion of glycosyl diglycerides, it is necessary to discuss the relevant nomenclature of glycerolipids as it stands somewhat distant to the carbohydrate parlance. The glyceryl (Gro) component utilizes ‘sn’ (stereospecific numbering) nomen- clature to indicate the absolute stereochemistry at C-2. When a glycerol is drawn in a Fischer projection such that its secondary alcohol is to the left, C-1 is considered to be at the top and C-3 at the bottom (Figure 4.1). This allows for absolute configuration to be defined in the absence of a carbonyl group, such as in an aldohexose. Curiously, this notation also means that two glycerol enantiomers can be defined by the numbering of the connections rather than with R/S or D/L notation; for example, 1,2-di-O-palmityl- sn-glycerol is the enantiomer of 2,3-di-O-palmityl-sn-glycerol. 1 OH OH 2 HO H HO OH 2 3 1 3 OH Figure 4.1. Stereochemical numbering (‘sn’) nomenclature for glycerolipids.
The fatty acid components of these molecules also have well established nomen- clature, with an extensive list of widely accepted common names. When we refer to the fatty acids we will do so by one of two ways. Either we will use their most commonly used name, such as stearic acid, or, we may use the shorthand, C18:0 to refer to the same
109 material. For acids with unsaturation, the number after the colon defines the number of double-bonds and can be suffixed with both the configuration and position such as
C18:1(cis-9) otherwise known as oleic acid.
The nomenclature for glycosyl diglycerides is as follows: αGlcGroAc2 (C14:0/C16:0) corresponds to the C14:0 ester at the primary (sn-1) position on the glycerol and the C16:0 ester at the secondary (sn-2) position; D-glucose is connected at sn-3 of the glycerol via an α-linkage (Figure 4.2).
HO O HO O HO O 14 HO O O 12 O
Figure 4.2. Structure of the example αGlcGroAc2 (C14:0/C16:0), 1'-O-myristoyl-2'-O-palmitoyl-sn-glyceryl α-D-glucopyranoside.
4.1.2 Glucuronosyl diglycerides from Mycobacterium smegmatis and
Corynebacterium glutamicum
Many mycobacteria are causative agents of human disease; the best known is Mycobac- terium tuberculosis, the cause of tuberculosis. When the full genome of M. tuberculosis was published it was discovered that it contained more than 250 genes involved in fatty acid metabolism.299 This is perhaps unsurprising as the M. tuberculosis cell envelope lipids constitute approx. 40% of the dry cell mass.300 Mycobacteria and corynebacteria possess an atypical cell wall in which the peptidoglycan layer has an additional waxy layer outside it. Covalently linked to the peptidoglycan is arabinogalactan modified with esters of mycolic acids, which form one part of a bilayer with the other part comprised of lipids, glycolipids, sulfoglycolipids and glycophospholipids.301
The easily cultivated M. smegmatis is generally considered non-pathogenic,302 and is often used as a biochemical and genetic model for the study of M. tuberculosis.303 While investigating the glycolipid repertoire of M. smegmatis MNC strain 13, Wolucka and co-workers came across several new compounds when the bacteria were grown
110 on plates with high glycerol content.304 Upon extraction and isolation of these mater- ials, chemical and mass spectrometric analysis identified them as glycolipids contain- ing D-glucuronic acid, glycerol and a mixture of fatty acid esters derived from palmitic
(C16:0), oleic (C18:1) and R-tuberculostearic acids (C19:0). Additional structural elucida- tion allowed the identification of two distinct compounds differing in their fatty acid compositions; both were α-glucuronosyl diglycerides (αGlcAGroAc2) bearing esters of palmitic acid and either oleic or R-tuberculostearic acids. Both compounds were pro- posed to bear the palmitic ester at the sn-2 position and the other ester at the sn-3 position: αGlcAGroAc2 (C18:1/C16:0) and αGlcAGroAc2 (C19:0/C16:0) respectively (Fig- ure 4.3). No rationale for this proposal was given and the acyl regioisomers were also possible structures: αGlcAGroAc2 (C16:0/C18:1) and αGlcAGroAc2 (C16:0/C19:0). While neither of these compounds have been isolated from M. tuberculosis, the isolated com- pounds were shown to cross-react with anti-M. tuberculosis and anti-Mycobacterium avium antibodies from rabbit sera. This result, in addition to the phylogenetic similar- ity of M. tuberculosis and M. smegmatis, suggest the presence of similar glycolipids in M. tuberculosis that remain to be described.
CO2H O HO O HO OR1 HO 2 2 O OR R O oleic acid Proposed regioisomer stearic acid CO2H O HO O HO 2 OR 1 HO R O OR1 palmitic acid
Figure 4.3. The proposed structure for the two M. smegmatis glucuronosyl diglycerides as well as their regioisomers which represent possible alternatives.304
Glucuronosyl diglycerides from Corynebacterium glutamicum
C. glutamicum is a non-pathogenic bacterium used widely for industrial scale pro- duction of amino acids.305 In 2007, Tatituri and co-workers discovered two glycol- ipids in C. glutamicum that were related to αGlcAGroAc2 (C18:1/C16:0) from M. smeg-
111 matis.306 The first was depicted as the regioisomer of the M. smegmatis compound,
αGlcAGroAc2 (C16:0/C18:1); once again no evidence was provided for the ester regio- chemistry. The second compound depicted was elongated by mannosylation at O-4 to
αManαGlcAGroAc2, formed through the action of the mannosyltransferase MgtA; this compound was termed Gl-X. As Gl-X is derived from the mono-glucosyl compound its regiochemistry is intrinsically the same (Figure 4.4). Although M. tuberculosis and M. smegmatis both possess homologues of MgtA, there is no evidence for the elongation of
307 αGlcAGroAc2 to αManαGlcAGroAc2 in these organisms.
CO2H O HO O HO O HO O O
O Gl-A HO HO (proposed acyl regiochemistry) HO O HO CO2H O O O HO O HO O O
O Gl-X Figure 4.4. Two glucuronosyl diglycerides from C. glutamicum, Gl-A and Gl-X; proposed structures have the opposite acyl regiochemistry to the related M. smegmatis compounds.306
Structural assignment of Gl-A(19:0) from M. smegmatis and Gl-A(18:1) from M. smegmatis and C. glutamicum
Cao and co-workers in the Williams lab undertook the synthesis of αGlcAGroAc2
205 (C19:0/C16:0) and αGlcAGroAc2 (C16:0/C19:0). With these glycolipids they were able to use mass spectrometry to unequivocally assign the regiochemistry of the natural M. smegmatis tuberculostearyl-containing glucuronosyl diglyceride. When the two glycol- ipids underwent CID-MS/MS both compounds revealed identical m/z fragment ions but with different intensities (Figure 4.5). It was observed that the [M - H]– fragment corresponding to the loss of the ketene of the sn-2 attached fatty acid had an intensity approximately 3 times that of [M - H]– fragment corresponding to the loss of the sn-1 attached fatty acid. For synthetic αGlcAGroAc2 (C19:0/C16:0) the m/z 547.3 signal (cor-
112 responding to the loss of the palmitate) had an intensity ~3 times that of the m/z 505.3 signal (corresponding to the loss of the tuberculostearate) and this ratio was reversed in the synthetic regioisomer αGlcAGroAc2 (C16:0/C19:0). This data established some ‘rules’
205 for the fragmentation of other αGlcAGroAc2 glycolipids.
CO 2H O HO O 547 HO O HO 255 O 297 O 505 O 547 505
505 CO 2H O HO O 505 HO O HO 297 O 255 O 547 O 547
Figure 4.5. MS fragmentation for αGlcAGroAc2 (C16:0/C19:0) and αGlcAGroAc2 (C19:0/C16:0) demonstrating a 3:1 ratio of ions representing the fragmentation of the secondary acyl group over the primary acyl group.205
In the original M. smegmatis report, FAB-MS/MS was performed on the natural tuberculostearyl-containing glycolipid (Figure 4.6A) and both of the m/z 547.3 and 505.3 fragmentation ions were observed in a 3:1 ratio,304 identical to the fragmenta- tion spectra of the synthetic αGlcAGroAc2 (C19:0/C16:0). This provided compelling evid- ence for the natural arrangement of the esters as αGlcAGroAc2 (C19:0/C16:0) (hence- forth known as Gl-A(19:0)). Using the FAB-MS/MS data for the oleoyl-containing
αGlcAGroAc2 isolated from M. smegmatis (Figure 4.6B), Cao and co-workers were able to apply the fragmentation rules to assign its acyl regiochemistry.205,304 The natural, oleoyl-containing, glucuronosyl diglyceride possessed peaks at m/z 531.3 and 505.3, corresponding to the loss of the palmityl and oleoyl esters, respectively, with an intens- ity ratio of approximately 3:1. This data supported assignment of the oleoyl group at
113 sn-1 and the palmityl group at sn-2; and as such, identified the natural regioisomer as
αGlcAGroAc2 (C18:1/C16:0) (henceforth known as Gl-A(18:1)). Relative Intensity A Relative Intensity Relative Intensity Relative Intensity
Relative Intensity Relative
B
Relative Intensity Relative
Figure 4.6. FAB-MS/MS for A αGlcAGroAc2 (C19:0/C16:0) B αGlcAGroAc2 (C18:1/C16:0). Isolated from M. smegmatis showing the same 3:1 intensity ratio demonstrated with the synthetic materials.205,304 No fragmentation data had been reported for the proposed Gl-A from C. glutamicum. Using a crude lipid extract from C. glutamicum, CID-MS/MS data for both Gl-A and Gl-X was attained. C. glutamicum Gl-A gave a spectrum with identical ratios for the m/z 531.3 and 505.3 peaks as for the M. smegmatis Gl-A(18:1) (Figure 4.7). This is the opposite of what would be expected if the compound possessed the acyl regio- chemistry that was proposed by Tatituri and co-workers.306 Fragmentation of Gl-X gave a Gl-A associated fragment that upon further fragmentation displayed ions correspond- ing to the loss of the same sn-2 and sn-1 esters as Gl-A in the same 3:1 ratioa. This data allowed the reassignment of the C. glutamicum Gl-A and Gl-X acyl regiochemistry to be the same as the M. smegmatis Gl-A(18:1), αGlcADAG(C18:1/C16:0).
aData not shown.
114 CO H O 2 531 HO O HO O HO 255 O 281 O 505 531 O
505
Figure 4.7. MS fragmentation of αGlcAGroAc2 (C18:1/C16:0) isolated from C. glutamicum.
Immunological activity of Gl-A(19:0)
Access to synthetic Gl-A(19:0) allowed for CD1d-restricted NKT cell activity to be in- vestigated. Uldrich and co-workers reported the discovery of a novel NKT clone from a Jα18-/- mouse possessing a TCR that was capable of recognising Gl-A(19:0).120 This
NKT cell expressed a Vα10-Jα50 TCR α-chain, which differs from the canonical type
I mVα14-Jα18 TCR α-chain, and a Vβ8.3 β-chain (Figure 4.8). Despite this difference, the new TCR also recognised CD1d presented αGalCer and structurally did so in a way similar to the type I-αGalCer-CD1d interaction. This population of NKT cells was the first to be defined as an atypical type I (type Ia); that is, they possess the ability tore- cognise αGalCer but have different antigen specificities conferred through differences in TCR chains. Gl-A(19:0) was not recognised by the canonical type I NKT TCR.
Of particular interest was the finding that only the natural regioisomer of Gl-A(19:0) was able to stimulate the NKT cells.120 The structural basis for this difference in react- ivity is well understood; fatty acids with unsaturation or branching preferentially bind into the A’ pocket regardless of their glycerol position. This results in the sugar head group rotating depending on the position of the A’ binding fatty acid and changing the TCR interaction surface.308 A related compound bearing a non-branched stearate in place of the tuberculostearate was also inactive. Ulrich reported that another type Ia
NKT cell clone with the same Vα10-Jα50 α-chain and a Vβ8.1 β-chain was unresponsive to Gl-A(19:0).120
115 Type 1a TCR
GlcADAG(C 19:0/C 16:0)
CD1d
Figure 4.8. Crystal structure of a novel type Ia NKT TCR recognising CD1d-presented synthetic Gl-A(19:0).
An unpublished report from the Godfrey laboratory has demonstrated that Gl-A(19:0) is recognised by a new type II NKT cell clone. The details of this clone will be discussed later in this chapter.
4.1.3 αGlcADAGs with undefined regiochemistry have been
identified in Aspergillus fumigatus
A. fumigatus, a species of mould, is the most common cause of fungal infection in immunosuppressed individuals, typically invading the lungs.309,310 Fontaine and col- leagues reported the isolation of a glucuronic acid containing glycerolipids from A. fu- migatus, the first time acidic glycerolipids had been identified in a fungal species..311 Spectroscopic analysis, including mass spectrometry and 1H/13C NMR, as well as fatty acid methyl ester (FAME) analysis enabled them to conclude that the compounds were
αGlcADAGs bearing mainly C16:0,C18:0,C18:1 and C18:2 fatty acids (Figure 4.9). Al-
116 though no MS/MS data was reported for intact compounds, meaning the acyl regio- chemistry could not be defined, the high abundance of C16:0 and C18:1 fatty acids sug- gests Gl-A(18:1) or iso-Gl-A(18:1) as reasonable structures for some of the glycolipids identified. O
O CO2H HO O HO OR1 R1/R2 O HO O OR2 O
Figure 4.9. α-Glucuronosyl diglycerides with undefined acyl regiochemistry have been identified in the pathogenic fungus A. fumigatus.
A. fumigatus has been reported to activate NKT cells in a CD1d-restricted man- ner. The βGalCer, asperamide B, has been identified as one molecular component re- sponsible for this activity.312,313 As related αGlcADAGs from M. smegmatis have shown CD1d-restricted activity it is plausible that the αGlcADAGs from A. fumigatus play a role in this observed NKT activity.
4.1.4 αGlcDAGs from Lactobacillus plantarum agonise immune
signalling through Mincle
Lactobacillus plantarum, a Gram positive fermentative bacterium, is used for the indus- trial scale preparation of various fermented foods; it also has relevance as a source of probiotics and dietary supplements.314,315 L. plantarum also forms part of the human intestinal microbiota and can colonize the mucosa of the mouth and nose.316,317 Us- ing FAME, 1- and 2-D NMR and mass spectrometry, Sauvageau and co-workers char- acterized several α-glucosyl diglycerides isolated from L. plantarum differing in their fatty acid composition; the most abundant compound bore a cyclopropyl-containing 9S,10R-dihydrosterculic acid at sn-1 and an oleic acid at sn-2, which they named GL1 (Figure 4.10).318
117 HO O HO O HO O HO O O
O Figure 4.10. GL-1, a α-glucosyl diglyceride bearing a cyclopropane fatty acid isolated from L. plantarum.
Sayali Shah in the Williams laboratory undertook the synthesis of GL1.78 Access to pure, synthetic GL1 enabled the discovery of its activity as a Mincle signalling agonist. At the lowest active concentration it possessed approximately half the activity of TDM and TDB in a NFAT-GFP report cell assay. A range of analogues bearing both linear acyl chains, and regio- and diastereo-isomers of dihydrosterculic acid at sn-1 were also found to stimulate Mincle with equal potency.
4.1.5 αGlcDAGs from Streptococcus pneumoniae possess
immunological activity
Streptococcus pneumoniae is a pathogenic, Gram positive, haemolytic bacterium that is the leading cause of pneumonia, bacterial meningitis and sepsis. Primarily an op- portunistic pathogen, S. pneumoniae is found in 5-10% of healthy adults and 20-40% of healthy children. In the late 1970s two groups described mono- and diglycosyl digly- cerides in S. pneumoniae.319,320 Using FAME analysis, gas chromatography and TLC they identified the most abundant fatty acids asC16:0,C16:1 and vaccenic (C18:1(cis-11)). Chem- ical and enzymatic hydrolysis of the glycosyl diglycerides led them to conclude that they were α-glucopyranosyl-sn-1,2-diglyceride and 3-[O-α-galactopyranosyl-(1->2)- O-α-glucopyranosyl]-sn-1,2-diglyceride; however, the fatty acid regiochemistry was not elucidated. Tatituri and co-workers used an MS/MS fragmentation technique similar to that used to elucidate the acyl regiochemistry of Gl-A from M. smegmatis to show that both the mono- and diglycosyl diglycerides from S. pneumoniae contained a major spe- cies bearing a vaccenic acid at sn-2 of the glycerol and palmitic acid at sn-1.321
118 Kinjo and colleagues reported that the major mono glucosyl glyceride from S. pneu- moniae, acylated as described above and termed SPN-s2, was a CD1d-restricted iNKT cell effector.322 In the same report they also tested the regioisomer of SPN-s2 and a double bond isomer where the vaccenic acid was replaced with oleic acid (C18:1(cis-9)) as potential CD1d-restricted effector molecules. It was observed that neither the regioi- somer nor the double bond isomer could activate NKT cells (Figure 4.11); this result is reminiscent of the similar result for the regioisomer of Gl-A(19:0) and demonstrates that the arrangement of acyl groups is critical for appropriate loading and presentation in CD1d for TCR engagement.
HO HO O double bond O O 11 12 O 9 10 HO positional isomer HO HO O HO O HO HO O O O O
O SPN-s2 O
acyl regioisomer acyl regioisomer
HO O HO O HO O HO O HO O HO O HO HO O O double bond O O 11 12 positional isomer 9 10 O O Figure 4.11. Of a small series of analogues, only SPN-s2, a compound found in S. pneumoniae, demonstrated CD1d-restricted NKT cell activation. Its acyl regioisomer as well as the double bond isomers were inactive.322
Behler-Janbeck and co-workers demonstrated that S. pneumoniae can activate a Mincle reporter cell line in vivo.79 This activity was determined to be mediated via α-glucosyl diglycerides including SPN-s2, and treatment with purified S. pneumoniae glycosyl diglycerides and synthetic analogues bearing dimyristoyl or dioleoyl fatty acids resulted in cytokine release by human and mice alveolar macrophages in a Mincle- dependent fashion. A decrease in survivability was shown for Mincle-/- mice relative to WT when challenged with focal pneumonia inducing S. pneumoniae.
4.1.6 Synthetic methodologies for the construction of glucosyl
diglycerides
There are three challenges associated with the synthesis of high purity α-glucosyl digly- cerides: first, stereoselective construction of the α-linkage; second, installation of the
119 glycerol fragment with correct C-2 stereochemistry; and third, the regioselective in- stallation of the fatty acid esters. The formation of α-glucosyl linkages was discussed in subsection 2.1.5 and as such will be mostly passed-over here. In regard to anomeric linkage, a report from Brennan and co-workers ascribed iNKT cell self reactivity in mammalian systems to an abundant endogenous lipid, β-glucosyl ceramide (βGlcCer); this report was later corrected by the same authors after demonstration that the spe- cies responsible for the observed iNKT cell activation was in fact a minor component, αGlcCer.323,324 More recently, Brennan and co-workers developed a technique for dir- ectly sampling the CD1d-restricted effector molecules from cows milk and found them to be α-linked monohexosyl ceramides; this represents direct biochemical evidence of α-linked lipid antigens from a common dietary source.325 Overall, these findings serve to highlight the necessity for high stereoselectivity during the synthetic preparation of glycolipids for assessment as CD1d-restricted effector molecules. Historically, the main approach for attaining the glycerol fragment for glycolipid synthesis has been to use a chiral pool starting material such as D-mannitol. For ex- ample, treatment of D-mannitol with acetone and ZnCl2 followed by oxidative cleav- age with periodate and reduction with NaBH4 gives two equivalents of the common glyceryl precursor (S)-1,2-isopropylidene glycerol.326,327 Although easily prepared and commercially available, (S)-1,2-isopropylidene glycerol has a significant draw-back: under mildly acidic conditions the isopropylidene group can migrate from a 1,2-O- isopropylidene to a 2,3-O-isopropylidene, in effect forming its enantiomer, (R)-1,2- isopropylidene glycerol (Figure 4.12). This was also shown to occur for other acid-labile protecting groups under typical NIS/TfOH glycosylation conditions.b Enantiopure gly- cidol has also been used as a glyceryl precursor for the synthesis of glycosyl digly- cerides.328,329
bMark Richardson, PhD Thesis, unpublished.
120 O O + O H+ O -H HO O HO O H HO OH HO O natural enantiomer unnatural enantiomer
Figure 4.12. Mechanism for the migration of an isopropylidene leading to glycerol racemization.
There are two general protecting group strategies used for glyceryl precursors de- pending on the need for a simple or mixed acylation pattern. If a simple acylation is de- sired, the same protecting group can be used for both O-1 and O-2 of the glycerol. Con- versely, for the synthesis of the mixed diglyceride Gl-A(19:1), Cao and co-workers pre- pared the differentially protected 1-O-(tert-butyldiphenylsilyl)-2-O-(p-methoxybenzyl)- glycerol from mannitol.205,330 Glycosylation of this glycerol using a glucosyl iodide donor under halide-ion catalysis afforded the protected α-glucosyl glycerol (Scheme 4.1). De- protection and oxidation at O-6 established the glucuronic acid which was then pro- tected as the benzyl ester. The sn-1 TBDPS was removed with HF.pyridine and acylated with (R)-tuberculostearic acid. Removal of the sn-2 PMB and immediate acylation en- abled regioselective installation of the sn-2 palmitic acid.
1. NaOMe, MeOH OPMB 2. TEMPO, BnO BnO HO OTBDPS PhI(OAc)2, BnO2C BnO O BnO O CH2Cl2/H2O BnO O BnO BnO OPMB BnO OPMB Bu NI, TTBP, BnO 4 BnO 3. BnOH, HBTU, BnO I CH Cl O OTBDPS O OR 2 2 DMAP, CH Cl 63% 2 2 55% over 3 steps HF·pyr, R = TBDPS THF 94% R = H
tuberculostearic acid BnO C 2 palmitic acid, RO C O COMU, DIPEA, BnO O 2 COMU, DIPEA, RO O BnO OR RO O cat. DMAP, DMF BnO 14 O O DMAP, DMF RO 63% 8 7 O O 60% over 2 steps 8 7 O O R = PMB CAN, R = Bn MeCN/H O 2 R = H R = H Gl-A(19:0) Scheme 4.1. Cao and co-workers prepared Gl-A(19:1) utilizing a step-wise, deprotection/acylation methodology.205
A major draw-back of the approach applied by Cao and colleagues is the necessity for orthogonal protecting groups on the sugar, and for the sugar and glycerol protecting group deblocking to be compatible with the fatty acid esters and unsaturation therein.
121 Often, this type of strategy necessitates protecting group interchanges to maintain or- thogonality. The choice of protecting groups is challenging when the acyl chains contain unsaturation. While our group has previously attempted the synthesis of Gl-A(18:1) us- ing the approach established by Cao, we were unsuccessful in establishing conditions for the selective removal of the sugar benzyl protecting groups in the presence of the double bond of the oleoyl ester. The prevailing approach for mixed diglyceride synthesis is the use of chemoselect- ive acylation.331–338 This approach relies on the innate difference in reactivity of primary and secondary alcohols and often employs low temperature couplings to selectively in- stall the sn-1 fatty acid. Srikanth and co-workers prepared β-linked analogues of the L. plantarum glycolipid GL1 for evaluation of their in vitro cytotoxicity against several cancer cell lines; the glycosyl glycerol diol was prepared by glycosylation of (R)-1,2-iso- propylidene glycerol with a glycosyl trichloroacetimidate followed by removal of the isopropylidene (Scheme 4.2).334 Selective installation of the sn-1 fatty acid was achieved by treatment of the diol with EDC-HCl and catalytic DMAP at 0 ◦C. Subsequently, es- terification at sn-2 was carried out under the same conditions at rt. AcO
AcO O O AcO Zn(NO3)2 · 6H2O, AcO AcO HO O O OH AcO O AcO O AcO O O OH O CCl AcO O MeCN, 50 °C AcO 3 75% TMSOTf, CH2Cl2 AcO AcO NH 62%
O O O AcO HO OH HO AcO 7 7 AcO O 7 7 O O 7 7 EDC-HCl, DMAP, AcO O O EDC-HCl, DMAP, AcO 7 7 AcO O O AcO 7 7 CH Cl , 0 °C O CH Cl , rt AcO 2 2 2 2 O 88% 98% Scheme 4.2. Srikanth and co-worker utilized regioselective EDC.HCl couplings for preparation of a series of mixed diglyceride analogues of L. plantarum GL-1.334
Another strategy for the formation of glycosyl diglycerides is glycosylation of a pre- formed diacylglycerol.201,339,340 Du and colleagues synthesised BbGL-II, an α-galactosyl diglyceride from Borrelia burgdorferi, by glycosylation of commercially available 1-O- palmityl-2-O-oleoyl-sn-glycerol with a galactosyl iodide in 89% yield with 10:1 α- selectivity (Scheme 4.3). When also undertaking the synthesis of BbGL-II, Pozsgay noted that inspection of the commercially available 1,2-di-O-acylglycerol by 13C-NMR
122 revealed it contained around 25% of a related compound which they speculate to a re- gioisomer of the naturally occurring material.337 It has been demonstrated that the re- giochemistry of the BbGL-II acyl chains is important in its ability to signal in a CD1d- restricted fashion; the C16:0/C18:1 regioisomer elicited signalling via a CD1d-iNKT cell
341 interaction while the C18:1/C16:0 regioisomer did not. O
O 7 7 HO O TMSO OTMS TMSO OTMS 14 O O O O TMSO TMSO O Bu NI, DIPEA, 7 7 TMSO 4 TMSO I CH Cl , rt O O 2 2 14 81% (10:1 α/β) O Scheme 4.3. Du and co-workers used an α-selective halide ion catalysed glycosylation to directly install a mixed diacylglycerol.339
Wang and colleagues also approached the synthesis of a polyunsaturated α-gluc- uronosyl diglyceride using a preformed diacylglycerol. Their mixed diacylglycerol was prepared using consecutive selective acylations of a 3-O-TBS protected glycerol.340 Re- moval of the TBS group enabled a poorly α-selective glycosylation with a glucuronosyl ortho-alkynylbenzoate donor under gold(I) catalysed conditions. They observed sev- eral glycosylation by-products that they proposed were due to loss of protecting group and subsequent involvement of the C-6 uronyl group of their donor.
4.1.7 Regioisomerization during the synthesis of mixed diglycerides
A major problem with existing strategies for the preparation of mixed glycosyl digly- cerides is the involvement of hydroxy-ester intermediates, which are known to facilitate acyl migration. Migration occurs when the vicinal hydroxyl attacks the carbonyl carbon of the fatty acid ester; this can occur under both basic and acid conditions (Figure 4.13). The migration is an equilibrium process with a preference for the shift from a 1,2-di- O-acyl glycerol to a 1,3-di-O-acyl glycerol, likely due to steric effects.342 This migration is facile and even occurs during silica chromatography.343–346 Although quick and care- ful handling is reported to minimize or eliminate these migrations, it can be difficult to
123 quantify the degree of migration as their detection is troublesome, with NMR spectra of the regioisomers often identical, or at least indistinguishable. During the preparation of a mixed diacylphosphatidylethanolamine from M. tuberculosis, Fodran and Minnaard used chiral HPLC to assess the regiopurity of a monoacylglyceride; this technique is limited by its requirement of authentic standards for comparison.343
O R1 -O H B+ R1 O O O B: HO O R2 O R2
O O
base catalysed O OH 1 R O R1 O O R2 HO O R2 O O O acid catalysed -H+ H+ O R1 H+ H O 1 R O -H+ O O HO O R2 O R2
O O Figure 4.13. Diglycerides undergo facile acyl migration under acidic and basic conditions.
In an attempt to solve this problem, Dr Sayali Shah in the Williams group developed an improved method for the preparation of fraternal glycosyl diglycerides like GL-1 (Scheme 4.4).78 Commercially-available, and anomerically pure, allyl α-glucoside was protected with methoxyacetates, a protecting group that provides orthogonality to fatty acid esters. A non-selective epoxidation of the alkene using mCPBA followed by a Jacob- sen’s hydrolytic kinetic resolution allowed separation of the two epoxide diastereomers by furnishing the epoxide of correct stereochemistry and the diol of its diastereomer. A
‘soft’ bromination using Li2NiBr4 was then used to regioselectively open the epoxide at the primary position.
124 RO MAcO (S,S)-OTs HKR, MAcO O RO mCPBA, THF MAcO O THF MAcO O RO MAcO MAcO RO 95% MAcO O MAcO O O O 48% O
MAcCl, R = H pyr R = MAc 98% MAcO Li NiBr THF 2 4, MAcO O MAcO OH 98% MAcO O Br
Scheme 4.4. A novel glycosyl diglyceride synthesis utilised a Jacobsen’s hydrolytic kinetic resolution to attain the correct glycerol diastereomer in high purity.78
The bromohydrin is an important intermediate that allows for a highly selective installation of the two acyl chains by affording different modes of reactivity for their installation and never exposing a hydroxy-ester intermediate (Scheme 4.5). The sn-2 fatty acid can be installed using traditional coupling chemistry while the sn-1 acid is installed using a nucleophilic substitution with the tetrabutylammonium salt of the fatty acid. While mechanistically it seems reasonable that this approach should afford mixed diglycerides with high regioselectivity, this has not been confirmed; nor has the fidelity of the regioisomeric acylation used in the Cao synthesis of Gl-A(19:1). 1 MAcO 1. R COCl MAcO O 2 MAcO O 2. R CO2Bu4N MAcO O MAcO OH MAcO O R1 MAcO MAcO O Br O O R2
O Scheme 4.5. In the second generation route, high fidelity acylation is achieved through the use of orthogonal acylation chemistry for the sn-1 and sn-2 positions.
4.1.8 Regiochemical determination of diglycerides
The equivalence of regioisomeric diglycerides under most analytical techniques presents a pressing problem for assessing the fidelity of mixed diglyceride synthesis. The MSn approach used by Cao, and Tsu and Turk,205,321 while sufficient for determining gross regiochemical structure cannot detect and quantify low levels of regioisomers. Mazur and co-workers used NMR to quantify regioselectivity of esterification of glycerol derivatives with medium chain-length acyl groups by exploiting a little appre-
125 ciated chemical shift difference in the 13C signals of the ω-3 carbon of their esters.347 Unfortunately, in our experience long chain diglycerides have indistinguishable chem- ical shifts. Other reports have used NMR to quantify regioisomers of mixed short/long chain diglycerides; however, the technique can not be extended to long/long chain digly- cerides, owing to their indistinguishable NMR shifts.348,349
4.1.9 Research aims
Aim 1: Develop a methodology for assessing the acyl regioisomeric purity of a synthetic diglyceride. We will apply this method to compare different acylation methodologies commonly employed for the preparation of glycosyl diglycerides.
Aim 2: There exists a body of evidence supporting an important role for α-glucuronosyl diglycerides as immunomodulators though both CD1d and Mincle interactions. We propose to synthesise Gl-A(18:1) from M. smegmatis, C. glutamicum and potentially A. fumigatus. As well, we propose to synthesize iso-Gl-A(18:1), which in addition to representing the regioisomer of the natural diglyceride from M. smegmatis and C. glutamicum may represent a compound from A. fumigatus. We will also synthesise the α-glucosyl diglyceride SPN-s2 from S. pneumoniae, whose synthesis has not been re- ported previously.
Aim 3: In collaboration with Dale Godfrey’s group at The Peter Doherty Institute, Uni- versity of Melbourne, assess whether Gl-A(18:1) possesses similar CD1d-restricted activ- ity to that of Gl-A(19:0); and assess the activity of its regioisomer. We will also study the CD1d-restricted activity of SPN-s2.
4.2 Results and Discussion
Since the synthesis of high quality natural glycolipids relies on the ability to perform high fidelity installation of the fatty esters, we decided to first develop a technique for quantification of acyl regioisomeric purity of synthetic glycosyl diglycerides.
126 4.2.1 Quantitative 13C NMR for the measurement of acyl
regioselectivity
Our approach utilizes 13C-isotopic labelling in conjunction with quantitative 13C NMR to accurately measure the regioisomeric ratio of synthetic diglycerides. This work was inspired by the observation that glycosyl diglycerides, almost universally, exhibit dis- tinct 13C NMR signals for the carbonyl signals of sn-1 and sn-2 attached esters; the sn-1 carbonyl signal is ~0.5 – 1.0 ppm down-field of the sn-2 carbonyl signal. This re- lationship is independent of chain length or functionalisation within the acyl chain. We reasoned that incorporation of a 13C label into the carbonyl of one of the esters would enable evaluation of the success of a regioselective esterification by integration of the sn-1 and sn-2 signals in the 13C NMR spectrum. For example, a diglyceride where only the sn-1 carbonyl is labelled with 13C (with 100% efficiency) will exhibit 13C car- bonyl signals with a 100:1.1 ratio. In contrast, a diglyceride where the label was installed non-regioselectively will exhibit a 1:1 integration ratio of the carbonyl 13C signals (Fig- ure 4.14). These are representative for the extremes for acylation of a glycerol, complete regioselectivity and no regioselectivity respectively; we expect that practical regioselect- ive acylations will fall somewhere between these two.
The typical 13C NMR experiment used for the analysis of small molecules uses a Fourier transformed proton-decoupled experiment. These experiments enable the ob- servation of 13C nuclei as singlet peaks with good sensitivity in a reasonable amount of time. The protons are decoupled to give singlet 13C peaks by simultaneous saturation of the full 1H spectral range during the experiment; in turn, this allows enhancement of the sensitivity by utilizing the nuclear Overhauser effect (NOE) to transfer magnetiza- tion from nearby protons to 13C nuclei. These experiments do not provide quantitative peak intensities and a different experiment is required to obtain quantitative data.
In order to obtain accurate and meaningful signal intensities the 13C nuclei must be allowed to fully relax between each scan. This is complicated by long T1 relaxation
127 A δ 170.4, sn-1 δ 169.8, sn-2
173 172 171 170 169 168 δ (ppm)
B δ 169.8, sn-2
δ 170.4, sn-1
173 172 171 170 169 168 δ (ppm)
Figure 4.14. Relative signal intensities from simulated 13C NMR spectra of diglyceride ester carbonyls: A) non-regioselective incorporation of a 1-13C-labelled fatty ester onto the glycerol diol. B) regioselective incorporation of a 1-13C-labelled fatty ester onto the sn-2 position, and an unlabelled fatty ester onto the sn-1 position.
times (0.1–100 sec) for 13C nuclei, especially for quaternary carbonyl carbons, and the difference in relaxation time for nuclei in different chemical environments. In addition, the low natural abundance of 13C requires averaging over many scans to attain sufficient signal to noise. To minimize measurement error a minimum delay of 5 × T1 relaxation is often used; however, delays of up to 10 × T1 are recommended.350–352 Also, NOEs can distort signal intensities due to unequal magnetization transfer and so presents an ad- ditional complication.353 In combination, these factors suggest that without additional assistance these experiments would take many days to complete sufficient scans for a good signal to noise ratio.
To overcome this issue we implemented two changes to the standard 13C NMR ex- periment. The first is the use of an inverse-gated decoupled-proton experiment.350 This experiment, unlike the standard experiment, only irradiates the proton frequencies dur- ing data acquisition and not during the relaxation delay. This enables decoupling of the protons from the 13C signals, thus generating singlets, but allows NOE magnetization to dissipate and not distort peak intensities. The second is the use of a paramagnetic
128 relaxation enhancement (PRE) agent. PRE agents are additives that can drastically reduce T1 relaxation times and aid in dissipation of NOE magnetization. Although several PRE agents are reported, we chose
chromium(III) acetylacetonate (Cr(Acac)3) as a non-chemical shift altering PRE agent that possesses good solubility in organic solvents and little anisotropy.354 A T1 relaxation
measurement of a diglyceride performed by Dr Mark Richardson both with Cr(Acac)3 (0.05 M) and without found a 10-fold decrease in relaxation time in the presence of the PRE agent. This shortened relaxation time allowed us to perform a quantitative meas- urement (>2000 scans, 10 sec relaxation delay) in a single overnight experiment. Using commercially available 1-13C-palmitic acid Dr Mark Richardson undertook the synthesis of two labelled mixed diglycerides utilizing different acylation method- ologies and a negative control. For each of the labelled compounds both the expec- ted acyl arrangement as well as its regioisomer, occurring from poor regioselectivity or acyl migration, is depicted. A non-regiospecific control was prepared by treatment of 3-(2,3,4,6-tetra-O-benzyl-α-glucopyranosyl)-sn-glycerol (43) with a 1:1 mixture of
13 1- C-palmitic acid and 11-methyldodecanoic acid in the presence of COMU, iPr2NEt and catalytic DMAP to afford a 77% yield of a statistical mixture of diglycerides con- taining both mixed regioisomers and both simple isoforms 44 (Scheme 4.6). O BnO R1 R2 O 1 13 13 BnO O R 1- C-C16:0 1- C-C16:0 BnO a PSfrag replacements OH 2 13 BnO O R 1- C-C16:0/iso-C13:0 O OH 77% iso-C13:0 iso-C 43 44 O 13:0 . Scheme 4.6. Dr Mark Richardson’s PhD: a) 1-13C-palmitic acid, 11-methyldodecanoic acid, COMU, iPr2NEt, DMAP, CH2Cl2
A labelled mixed diglyceride was prepared using the widely used regioselect- ive Steglich coupling. To selectively install the sn-1 ester, 43 was treated with 11-methyldodecanoic acid, DCC and catalytic DMAP at 0 ◦C to afford 45 (Scheme 4.7). Purification of 45 was achieved in <15 min using chilled solvents and silicagel to min- imize the potential for acyl migration. Immediate treatment of 45 with 1-13C-palmitic
129 acid, COMU and catalytic DMAP installed the labelled acid at the sn-2 position and afforded the diglyceride 46 in 79% yield over two steps. O
O R1 BnO OH O R2 BnO O b BnO a O OH 9 46 O BnO PSfrag replacements O OH O 79% R1 R2 43 45 over 2 steps 13 iso-C 1- C-C16:0 13:0 iso-C 13 13:0 1- C-C16:0 Scheme 4.7. Dr Mark Richardson’s PhD: a) DCC, DMAP, 11-methyldodecanoic acid, ◦ 13 CH2Cl2, 0 C, 12 h; b) DMAP, COMU, 1- C-palmitic acid, CH2Cl2, rt.
A labelled diglyceride was also prepared using the methodology established by Cao and colleagues for the synthesis of Gl-A(19:0).205 Treatment of 3-(2,3,4,6-tetra- O-benzyl-α-glucopyranosyl)-2-(p-methoxybenzyl)-sn-glycerol (47) with lauryl chlor- ide and catalytic DMAP in pyridine installed the sn-1 ester affording 48 in 99% yield (Scheme 4.8). The PMB protecting group was removed by treatment of 48 with CAN in
CH3CN/H2O and without purification of the alcohol intermediate was esterified with
13 1- C-palmitic acid using COMU, iPr2NEt and catalytic DMAP to afford diglyceride 49 in 77% yield. O
O R1 BnO O R2 OPMB BnO O a b BnO OPMB O 49 O PSfrag replacements BnO 10 O OH R1 R2 99% O 77% 47 48 13 1- C-C16:0 C12:0 13 C12:0 1- C-C16:0 Scheme 4.8. Dr Mark Richardson’s PhD: a) lauryl chloride, DMAP, pyridine, rt; b) CAN, 13 MeCN, H2O, rt, ; c) COMU, 1- C-palmitic acid, iPr2NEt, DMF, rt.
I prepared a labelled diglyceride under the regioselective esterification conditions established by Shah and co-workers for the preparation of GL-1.78 Commercially- available allyl α-glucopyranoside was protected by treatment with methoxyacetyl chlor- ide in pyridine to afford 50 in 91% yield (Scheme 4.9). Epoxidation of the alkene was
130 carried out by treatment with mCPBA in CH2Cl2 to afford 51 in 95% yield. A Jacobsens HKR utilizing S,S-salen cobalt(II) tosylate enabled separation of the epoxide diastereo- mers by hydrolysing the unwanted S isomer to the diol and afforded a 41% yield of pure R epoxide 52. Epoxide opening and bromination by treatment with a saturated solution
of Li2NiBr4 in THF afforded 53 in 96% yield.
PSfrag replacements RO MAcO MAcO MAcO O RO b MAcO O c MAcO O d O RO MAcO MAcO MAcO MAcO OH RO 95% MAcO O 41% MAcO O 96% MAcO O O O O Br 51 52 53 a R = H 91% R = MAc 50
Scheme 4.9. a) Methoxyacetyl chloride, pyridine; b) mCPBA, CH2Cl2; Co(II)-salen-OTs, H2O (0.55 eq), THF; d) Li2NiBr4, THF.
Selective acylation of 53 was carried out in two steps: the sn-2 ester was installed
by treatment of 53 with palmitoyl chloride in CH2Cl2 to afford 54 in 87% yield (Scheme 4.10). Substitution of the primary bromide to install the sn-1 ester was achieved by treatment of 54 with tetrabutylammonium 1-13C-palmitate (55) to afford the labelled diglyceride 56 in 71% yield; 55 is easily prepared by treatment of 1-13C-palmitic acid
with tetrabutylammonium hydroxide in H2O. O
O R1 MAcO O O R2 MAcO O a b MAcO O OH 14 56 O MAcO PSfrag replacements O Br 87% Br 71% R1 R2 53 54 13 C16:0 1- C-C16:0 13 1- C-C16:0 C16:0
Scheme 4.10. a) Palmitoyl chloride, pyridine, CH2Cl2; b) tetrabutylammonium 1-13C-palmitate, toluene, reflux.
To compare the integrations of our carbonyl signals we need to consider both the natural abundance of 13C in the unlabelled ester as well as the 13C atom efficiency of 1-13C-palmitic acid. Using negative-ion electrospray mass spectrometry the 13C atom efficiency of the labelled palmitic acid was determined to be 99.4% assuming that the other carbons had natural 13C abundances. The percentage ratio between the two iso-
131 mers can then be calculated according to Equation 4.1; this relationship considers the carbonyl 13C NMR peak integrations ( x and y, labelled and unlabelled integrations, respectively) and the 13C abundances (NR andR L, natural and labelled abundances, re- spectively).
x N − L · y R = R · 100 (4.1) x Rx N · y − L · y + N − L R R R R Using inverse-gated proton decoupling and a 10 sec relaxation delay, a quantitative spectrum for each compound (80 90mmol with 50 mmol Cr(Acac)3) or mixture of com- pounds was collected over 2000 scans (Figure 4.15). We used Lorentzian curve fitting bundled with MestreNova NMR software to accurately measure the peak integrations. The negative control, 44, displayed a regioisomeric ratio of 50%, corresponding to indis- criminate incorporation of 13C labelled acid at the sn-1 and sn-2 positions. Analysis of the diglyceride prepared using the Steglich approach revealed a 13C carbonyl signal ra- tio of 10:1 which corresponds to a regioisomeric ratio of 92% of the desired isomer and 8% of its regioisomer. Two possibilities for this are imperfect regioselectivity of the first Steglich esterification or acyl migration. Mattson and colleague noted that the equilib- ration position for a mono glyceride is approximately 9:1 primary and secondary esters respectively,355 and on this basis we propose that acyl migration is the cause of this 10:1 ratio. The diglyceride prepared using the approach of Cao displayed a carbonyl ratio of 25:1, corresponding to a regioselectivity ratio of 97.2% in favour of the desired isomer with 2.7% of the regioisomer. While highly selective, this suggests that a small degree of acyl migration occurs during deprotection of the PMB group. Finally, the labelled digly- ceride prepared using Shah’s approach that avoids hydroxy-ester intermediates revealed a 13C carbonyl ratio of 56:1, corresponding to >99% regioselectivity. This supports our initial proposition that avoidance of hydroxy-ester intermediates allows for high fidelity synthesis of mixed diglycerides.
132 A B
1.02 1.00 1.00 10.22 CD
56.50 1.00 1.00 25.20
δ ppm δ ppm
Figure 4.15. Quantitative 13C NMR spectrum of the carbonyls of A) 44, a statistical mixture bearing 50% labelling at each position; B) 46, a diglyceride prepared using regioselective DCC coupling chemistry; C) 49, a diglyceride prepared using the step-wise approach of Cao; D) 56, a diglyceride prepared using the methodology of Shah.
4.2.2 Preparation of S. pneumoniae SPN-s2, a CD1d-restricted
immune effector
With establishment of the high regioselectivity for the Shah diglyceride route we now sought to synthesise SPN-s2 from S. pneumoniae. SPN-s2 contains a cis-vaccenoate at sn-2 of the glycerol and a palmitate at sn-1. The bromohydrin 53 was treated with vaccenoyl chloride in the presence of pyridine in CH2Cl2 to afford bromide 57 in 87% yield (Scheme 4.11). Initially we attempted this transformation in the presence DMAP to catalyse the acylation through the formation of the activated acyl dimethyl- aminopyridinium species; however, we observed significant formation of a stable qua- ternary ammonium species resulting from the substitution of the primary bromide (53)
133 with the pyridine nitrogen. Fortunately, the reaction proceeded without incident when the less nucleophilic pyridine was employed. Substitution of the bromide (57) with tet- rabutylammonium palmitate afforded protected SPN-s2 (58) in 52% yield. Treatment of 58 with tert-butylamine in a methanol/chloroform mixture effected the selective re- moval of the MAc groups and afforded SPN-s2 (59) in 57% yield.
MAcO RO O RO O MAcO O a RO O b RO O PSfrag replacements MAcO OH RO O RO O MAcO RO 7 7 RO 7 7 O Br 82% O Br 52% O O 14 53 57 O R = MAc 58 c 57% R = H 59
Scheme 4.11. a) cis-Vaccenoyl chloride, pyridine, CH2Cl2; b) tetrabutylammonium palmitate, toluene, reflux; c) tert-butylamine, MeOH, CHCl3.
Confirmation of the natural structure of SPN-s2 by fragmentation mass spectrometry
Although it is presumed that SPN-s2 is the naturally occuring acyl regioisomer, this has not been independently confirmed using authentic synthetic material. Tatituri and co- workers reported the MS/MS spectrum for the natural material isolated from S. pneu- moniae and proposed that it possesses the regiochemistry as drawn for SPN-s2 (Fig- ure 4.16B).321 We therefore sought to compare MS/MS data for the synthetic material with that of the natural material. Comparison of MS/MS spectra obtained for our syn- thetic material with that published for the natural material revealed peaks at m/z 507 and m/z 481 corresponding to loss of the sn-2 and sn-1 acyl groups, respectively (Fig- ure 4.16A). In both spectra, the peaks were in a 5:4 ratio for m/z 507 and m/z 481, respectively, confirming matching acyl regiochemistry in the natural and synthetic ma- terials.
134 Figure 4.16. A) MS/MS fragmentation spectrum of synthetic SPN-s2; B) MS/MS fragmentation spectrum of SPN-s2 and minor related materials isolated from S. pneumoniae.321
4.2.3 Preparation of Gl-A(18:1) from Mycobacterium smegmatis and
Corynebacterium glutamicum, and its regioisomer
Gl-A(18:1) and its regioisomer, iso-Gl-A(18:1), can be synthesised in a fashion similar to that used for SPN-s2 (Scheme 4.12). The palmityl β-bromo-ester 54 from our 13C NMR study was substituted with tetrabutylammonium oleate to afford protected digly- ceride 60 in 63% yield. The protected regioisomer was prepared by treatment of 53 with oleoyl chloride in the presence of pyridine affording 61 in 95% yield, and then substi- tution with tetrabutylammonium palmitate afforded a 55% yield of 62. Both 60 and 62 were deprotected by treatment with tert-butylamine to afford the glucosyl diglycerides GlcDAG(18:1/16:0) (63) and its regioisomer GlcDAG(16:0/18:1) (64), in 59% and 81% yields, respectively.
135 MAcO O RO O a MAcO O RO O MAcO O RO O MAcO 14 63% RO 14 O Br O O 54 7 7 O 60 PSfrag replacements b R = MAc 59% R = H 63
MAcO MAcO O RO O MAcO O c MAcO O d RO O MAcO OH MAcO O RO O MAcO 95% MAcO 7 7 RO 7 7 O Br O Br 55% O O 53 61 14 O 62 b R = MAc 81% R = H 64
Scheme 4.12. a) Tetrabutylammonium oleate, toluene, reflux; b) tert-butylamine, MeOH, CHCl3; c) oleoyl chloride, pyridine, CH2Cl2; d) tert-butylamine, MeOH, CHCl3.
Selective oxidation at C-6: conversion to the glucuronic acid
For the synthesis of Gl-A(19:1), Cao and co-workers utilized an early oxidation to the glucuronic acid performed prior to introduction of the fatty acyl groups using the well
205 known 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)/PhI(OAc)2 reagent system.
Wang and colleagues have also used the TEMPO/PhI(OAc)2 system for preparation of glucuronosyl diglycerides with the oxidation performed prior to installation of the fatty acid esters.340 Thus, in contrast to our need to conduct the oxidation on an unprotected substrate as the final step in our synthesis, both of these studies conducted the oxidation on otherwise protected glycosides early on in their synthetic routes.
Most commonly employed oxidation methodologies, such as the Jones (CrO3), py-
idinium dichromate (PDC) and RuO4 oxidations, exhibit poor selectivity for primary over secondary alcohols resulting in a mixture of carboxylic acids and ketones.356 For selective oxidation of deprotected carbohydrates two methods are generally applied: the aforementioned TEMPO radical derived systems and the Heyns oxidation.
The Heyns oxidation utilizes catalytic platinum and molecular oxygen to selectively oxidise primary alcohols to carboxylic acids in the presence of secondary alcohols. In the
1940s Heyns reported the transformation of L-sorbose to 2-keto-L-gulonic acid upon
357 treatment with Pt and O2 under basic aqueous conditions. Although basic condi-
136 tions are required for useful reaction rates, Bols applied the Heyns’ oxidation to the synthesis of β-glucuronosyl pivalate, thus demonstrating the stability of a pivalate ester under the reported conditions (Scheme 4.13).358 However, the oxidation is primarily carried out in H2O and is highly susceptible to sequestration of the heterogeneous cata- lyst by non-soluble substrates leading to termination of the reaction;359 we suspected that our amphipathic starting material and the soap-like oxidation product would not be conducive to these reaction conditions. HO O2, Pt, NaHCO3 HO2C HO O HO O HO O HO O HO H2O, 90°C HO O 60% O Scheme 4.13. Bols utilized the Heyns oxidation to selectively oxidise glucose at O-6 without of cleavage of an anomeric pivalate.358
We therefore turned our focus towards TEMPO-derived systems. Golubev and col- leagues described the oxidation of ethanol by treatment with a stoichiometric amount of the oxoammonium salt, 2,2,6,6-tetramethyl-4-hydroxy-piperidineoxoammonium chloride.360 Ten years later Cella and co-workers reported on the oxidation of primary alcohols into the corresponding carboxylic acids by treatment with catalytic 2,2,6,6-tetramethyl-1-piperidine in the presence of stoichiometric mCPBA as a co- oxidant.361 mCPBA oxidises the amine, firstly into the stable radical TEMPO and sub- sequently into the oxoammonium cation that operates as the primary oxidant. Although Cella’s procedure employs a peracid as the co-oxidant, making it inappropriate for many complex substrates, it was seminal in showing that these oxo-species could be employed catalytically for the conversion of primary alcohols into carboxylic acids in the presence of an appropriate co-oxidant.
Golubev reported that under acidic conditions TEMPO oxidations proceed slowly and have a preference for oxidation of secondary over primary alcohols and that this preference is reversed under basic conditions with a concurrent increase in reaction rate.360 Semmelhack and co-workers proposed that a cyclic transition state is formed under basic conditions by addition of the substrate alcohol to the nitrogen centre of
137 the oxoammonium species. Intramolecular proton abstraction and elimination of the substrate as a carbonyl completes the reaction.362 For primary alcohols the oxidation to carboxylic acid occurs in two steps: firstly an oxidation to the aldehyde; which, under anhydrous conditions, is where the reaction stops. Hydration of the aldehyde to a gem- inal diol allows a second oxidation to occur to form the carboxylic acid. A comprehens- ive mechanistic study by DeNooy and co-workers supported this mechanism. These workers suggested that the faster oxidation of primary alcohols was due to steric in- teraction between the TEMPO methyl groups and the substrate.363 They demonstrated that secondary alcohols with increasing steric bulk were oxidised more slowly under basic conditions. B: - O H OH OH H O O N pH < 4 pH > 6 N R R' R O R R' R O R H R' 2° > 1° 2° < 1° R'
Scheme 4.14. Golubev observed both slow reaction rates and a preference for secondary alcohols under acidic conditions, and the opposite preference under basic conditions; DeNooy and co-workers proposed this difference was due to the presence ofa cyclic intermediate under basic conditions.360,363
Anelli and co-workers developed an improved TEMPO oxidation methodology that used NaClO and HOBr, generated by oxidation of NaBr, as a stoichiometric and sec- ondary catalytic oxidant respectively; the HOBr is responsible for the regeneration of the active oxoammonium species (Scheme 4.15).364 Subsequently, Zhao and colleagues employed NaClO2 as a second co-oxidant along with catalytic NaClO; this helped to re- duce, although not eliminate, by-product formation caused by chlorination by NaClO.365
Epp and Widlanski reported that the hypervalent iodine species PhI(OAc)2 and TEMPO could oxidise 2',3'-isopropylidene protected nucleosides to their 5'-carboxylic
366 acid derivatives in an aqueous buffer/MeCN solvent system; PhI(OAc)2 acts as the co- oxidant and affords iodobenzene and acetic acid as easily removed reduction products.
While PhI(OAc)2 is responsible for the oxidation of the hydroxylamine back to the TEMPO radical, liberated AcOH catalyses a bismutation of two molecules of radical into the active oxoammonium cation and the hydroxylamine.
138 HO2C HO O HO HO OR
N O O- N HO H OH HO O HO HOCl HO HOBr OR
N N O HO O - OH Cl- Br H HO O HO H2O O HO OR H HO O HO - HOCl HO Br OR N O OH N O O HO N HO - HO O O OR - H H HOBr Cl HO O HO N HO OR OH Scheme 4.15. Mechanism for the Anelli TEMPO methodology.364
While the TEMPO/NaClO system has been used for the regioselective preparation of uronic acids, we suspected that the basic conditions employed, in addition, to the po- tential for chlorination of double bonds would make it sub-optimal to employ on a digly- ceride containing oleic acid esters. We instead chose to apply the TEMPO/PhI(OAc)2 system as PhI(OAc)2 is compatible with the oleoyl double bond and the reaction can be conducted at a lower pH. We attempted oxidation of 64 by treatment with TEMPO and PhI(OAc)2 in a MeCN and NaHCO3 buffer (pH 9, 0.1 M) (Scheme 4.16); although consumption of 64 was evident by TLC the reaction required multiple additions of both
TEMPO and PhI(OAc)2 and did not reach completion after 7 days. In addition, after the long reaction time several by-products with similar TLC mobilities were evident. The product was ultimately isolated in low yield. PhI(OAc)2 is reported to undergo slow hydrolysis under aqueous conditions;367 poor solubility of our substrate leading to low reaction rates could result hydrolysis of PhI(OAc)2 and premature arrest of the reaction.
139 Long reaction times also risk ester hydrolysis due to the basic conditions.
HO O HO2C O HO O HO O HO O a HO O PSfrag replacements HO 7 7 HO 7 7 O O O O 14 14 64 65 O O
Scheme 4.16. a) TEMPO, PhI(OAc)2, MeCN, NaHCO3 buffer (pH 9, 0.1 M).
Most oxoammonium-based oxidation protocols use a catalytic amount of oxo- species with a stoichiometric amount of one or more secondary oxidants. While these have many demonstrated successes, they can be difficult to apply to complex substrates with sensitive functionalities or poor solubility. The early mechanistic work establishing the oxoammonium cation as the oxidatively active species has led to the development of stable oxoammonium salts that are applied stoichiometrically without the need for a co-oxidant. Mercandante and colleagues developed 4-acetamido-2,2,6,6-tetramethyl- N-oxopiperidinium tetrafluoroborate (Bobbitt’s salt, 66), a stable oxoammonium salt for use as a stoichiometric oxidation reagent (Figure 4.17).368 O
HN
- BF4 N PSfrag replacements O 66
Figure 4.17. Bobbitt’s salt, 4-acetamido-2,2,6,6-tetramethyl-N-oxopiperidinium tetrafluoroborate, was developed by Mercandante and colleagues as a stoichiometric oxoammonium oxidation reagent.
We applied a Bobbitt’s salt mediated oxidation to the preparation of Gl-A and its re-
gioisomer. Treatment of 63 with 66 in MeCN and aqueous KHCO3 afforded Gl-A(18:1) (67) in 50% yield (Scheme 4.17). The same conditions also oxidised 64 to afford iso-Gl- A(18:1) (65) in 45% yield. While these reactions are slow (2-5 days), they afforded clean glucuronides with no evidence of by-products (by TLC).
140 HO O HO2C O HO O HO O HO O a HO O HO 7 7 HO 7 7 O O O O 14 45% 14 64 65 O O
PSfrag replacements HO O HO2C O HO O HO O HO O a HO O HO 14 HO 14 O O O O 7 7 50% 7 7 63 67 O O
Scheme 4.17. a) 66, KHCO3,H2O, MeCN.
4.2.4 Confirmation of the natural Gl-A(18:1) regioisomer by
fragmentation mass spectrometry
An MS/MS spectrum for our synthetic Gl-A(18:1) was obtained which allowed com- parison to the data collected by Cao and co-workers for Gl-A(18:1) isolated from C. glutamicum,205 and the FAB-MS/MS spectrum reported for Gl-A(18:1) from M. smeg- matis.304 Our synthetic Gl-A possessed a spectrum that was concordant with the natural materials and with the acyl regiochemistry rules established by the structural elucida- tion of Gl-A(19:0) (Figure 4.18). Peaks at m/z 531.3 and m/z 505.3 possessed a 3:1 ratio corresponding to the loss of the sn-2 and sn-1 acids, respectively, thus reaffirming the more facile loss of the sn-2 acyl group. 770 CO H O 2 531 Relative Intens HO O HO O Relative Intensity HO 255 O 281 Relative Intensity O Relative Intensity 505 531 O Relative Intensityity 281
Relative Intensity Relative 255 505 752
m/z Figure 4.18. An MS/MS spectrum of synthetic Gl-A(18:1) demonstrated consistent fragmentation patterns to those established for natural Gl-A(18:1).
141 4.2.5 Type I, Ia and II NKT TCR activity
In collaboration with the Dale Godfrey’s laboratory at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, both the glucosyl and glucuronosyl Gl-A regioisomers, as well as SPN-s2, were assessed for their ability to stimulate NKT cells in a CD1d-restricted manner. The assay used for the determination of CD1d-NKT cell interactions involves the use of CD1d tetramers. Tetramers were initially developed by Altman and colleagues as a way to detect CD8+ T cells with TCRs reactive to MHC class I presented peptides.369 Matsuda and Benlagha independently developed CD1d tetramers.370,371 Their forma- tion involves the production of recombinant CD1d possessing biotinylation at the C- terminus. The biotinylated CD1d molecules are loaded with an appropriate ligand (ini- tially this was αGalCer), and then incubated with fluorescently labelled avidin, forming stable tetrameric structures. The resulting tetramers can be used to label T cells express- ing TCRs that interact with the presented ligand. Detection and quantification of the labelled T cells can be achieved with flow cytometry. We were interested in assessing the ability of our synthetic glycolipids to be re- cognised by type I, type Ia and type II NKT cells in a CD1d-restricted fashion. First we investigated whether our diglycerides would be recognised by canonical type I NKT cells. We were able to determine that both glucuronides as well as SPN-s2 and GlcDAG(18:1/16:1) were recognised by the type I TCR (Figure 4.19). Natural Gl-A(18:1) possessed the strongest interaction, while the regioisomer, SPN-s2 and GlcDAG(18:1/16:0) were approximately 20% as potent. It was also noted that Gl-A(18:1) provided a response more than twice that of Gl-A(19:0). The artificial ligand αGalCer provides a response more than 20-fold stronger than the most potent natural compound in these assays.
142 250000
50000 20000 18000 16000 14000 12000 10000 8000 6000 4000 Mean Fluorescence IntensityFluorescenceMean 2000 0 0) Gl-A(19:0) Gl-A(18:1) iso-Gl-A(18:1) :1) SPN-s2 Vehicle (tween) αGalCer Endo 1/16: 18: ( (16:0/18
lcDAG GlcDAG G Type I Type Ia Figure 4.19. Jurkat cells expressing canonical type I or a type IA (Vα10) TCR were stained with CD1d tetramers loaded with Gl-A(19:0), Gl-A(18:1), iso-Gl-A(18:1), GlcDAG(18:1/16:0), GlcDAG(16:0/18:1), SPN-s2, αGalCer or endogenous lipids. Staining was measured by flow cytometry. Assays were performed in duplicate.
Type Ia NKT (Vα10) cells, first identified using Gl-A(19:1), stained to similar levels with CD1d tetramers loaded with iso-Gl-A(18:1), Gl-A(19:0) or SPN-s2 (Figure 4.19); however, none of the other compounds were active. This result is surprising as iso-Gl- A(18:1) was not the observed regiochemistry in C. glutamicum or M. smegmatis; how- ever, as previously discussed, MS/MS is insufficient to conclusively reject its presence.
The natural regiochemistry of the18:1 C /C16:0 glucuronosyl diglyceride produced by A. fumigatus has not been reported; it is possible that iso-Gl-A(18:1) is produced. Staining of type II NKT cells (TRAV401c) demonstrated a different profile of tetra- mer staining with only the glucuronosyl diglycerides showing activity (Figure 4.20). The natural regioisomer of Gl-A(18:1) afforded approx. 80% of the staining of Gl-A(19:1), while the regioisomer iso-Gl-A(18:1) was much less active.
cUnpublished.
143 4000
3500
3000
2500
2000
1500
1000
Mean Fluorescence IntensityFluorescenceMean 500
0 Gl-A(19:0) Gl-A(18:1) iso-Gl-A(18:1) SPN-s2 Vehicle (tween) αGalCer Endo
(18:1/16:0) (16:0/18:1) DAG GlcDAG Glc Figure 4.20. Jurkat cells expressing a type II (TRAV401) TCR were stained with CD1d tetramers loaded with Gl-A(19:0), Gl-A(18:1), iso-Gl-A(18:1), GlcDAG(18:1/16:0), GlcDAG(16:0/18:1), SPN-s2, αGalCer or endogenous lipids. Staining was measured by flow cytometry. Assays were performed in duplicate.
These results for Gl-A(18:1) and iso-Gl-A(18:1) stand apart from those of Gl-A(19:0) with differences seen in all three cell lines. The recognition of iso-Gl-A(18:1) bythe type Ia TCR is a highly surprising result and warrants further study. The type 1a TCR has the ability to recognise a wide range of α-glucosyl and α-glucuronosyl diacylgly- cerides. On the other hand, the type II TRAV401 TCR appears to be highly specific for
α-glucuronosyl diacylglycerides bearing natural arrangements of C19:0 and C18:1 found in mycobacteria and corynebacteria.
4.3 Summary
Part of the work in this chapter investigated the use of quantitative 13C NMR to assess the regioselectivity of acylation methodologies for diglyceride synthesis in combina- tion with a 13C labelled fatty acid. Using this technique, we determined that most com- monly used acylation methodologies lead to some degree of regioisomer formation; of the assessed methodologies, a recently reported orthogonal acylation from the Williams group provided the highest selectivity.
Using this highly selective orthogonal acylation, we prepared SPN-s2, a glucosyl diglyceride from S. pneumoniae that had previously been identified as a type I NKT cell effector molecule. MS/MS of the synthetic material confirmed the acylation pattern to be the same as that reported for authentic material.
144 The same acylation technique was also employed in the total synthesis of aC18:1/C16:0 glucuronosyl diglyceride (Gl-A(18:1)), and its regioisomer (iso-Gl-A(18:1)), from C. glutamicum and M. smegmatis. Gl-A(19:0) (also from M. smegmatis), Gl-A(18:1), iso-Gl-A(18:1) and SPN-s2 were assessed for their ability to stimulate Jurkat cells expressing a range of TCRs in a CD1d- restricted fashion. Differences were seen between the natural M. smegmatis glucuronides in stimulating type 1 and type 1a cell lines, however both were able to stimulate a re- cently identified type II cell line.
4.3.1 Future work
Work is underway to attain ternary crystal complexes with both regioisomers of Gl-A(18:1) with various TCRs to gain an improved understanding of the structural basis for their activity.
145 4.4 Experimental
Allyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-glucopyranoside (50) MAcO MAcO O Methoxyacetyl chloride (3.32 mL, 36.3 mmol) was added drop- MAcO MAcO O wise to a stirred solution of allyl α-D-glucopyranoside (1.00 g, 4.54 mmol) in pyridine (30 mL) at 0 ◦C. The solution was gradually warmed to room temperature and stirred overnight. The reaction mixture was diluted with water and
EtOAc, and the organic phase washed with sat. aq. CuSO4, sat. aq. NaHCO3 and wa- ter, dried (MgSO4), filtered and concentrated. Flash chromatography of the residue
24 (EtOAc/pet. spirits) afforded 50 as a yellow oil (2.1 g, 91%). [α]D +90.1 (c 1, CHCl3)
78 26 1 (lit. [α]D +91.7 (c 1.19, CHCl3)); H NMR (500 MHz, CDCl3) δ 3.39, 3.40, 3.42, 3.45
(12 H, 4 × s, 4 × CH3OCH2), 3.95 – 4.14 (2 H, m, H3', 3'), 3.95, 3.99, 4.09 (6 H, 3 × s,
3 × CH3OCH2), 4.02 (2 H, m, CH3OCH2), 4.16 – 4.21 (2 H, m, H5, 6), 4.37 (1 H, dd, J 4.1, 12.5 Hz, H6), 4.98 (1 H, dd, J 3.6, 10.2 Hz, H2), 5.13 – 5.18 (1 H, 2 × d, H1, 4), 5.22 – 5.33 (2 H, m, H1', 1'), 5.58 (1 H, t, J 10 Hz, H3), 5.82 – 5.59 (1 H, m, H2'); 13C NMR
(125 MHz, CDCl3) δ 59.38, 59.47 (4 × OCH3), 61.87, 67.10, 68.62, 69.08, 69.35, 69.40, 69.52, 70.62, 70.82, 94.72 (C1), 118.60, 132.85 (C=C), 169.26, 169.54, 170.02 (4 × C=O);
HRMS (ESI+) calcd for C21H32O14 (M + Na)+ 531.1684. Found 531.1675.
2',3'-Epoxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-gluco- MAcO MAcO O pyranoside (51) MAcO MAcO O A solution of m-CPBA (1.53 g, 8.85 mmol) and 50 (3.00 g, O
5.90 mmol) in dry CH2Cl2 (40 mL) was stirred at room temperature overnight. Ad- ditional m-CPBA (0.51 g, 2.95 mmol) was added and stirring was continued for an- other night. The solution was diluted with dichloromethane, washed successively with aqueous Na2S2O5, sat. aq. NaHCO3 and water, dried (MgSO4) and concentrated. Flash chromatography (EtOAc) afforded 51 (as a mixture of 2'R/2'S isomers in a 1:0.95 ratio)
24 78 26 as a colourless oil (2.94 g, 95%). [α]D +87 (c 1, CHCl3) (lit. [α]D +89.8 (c 2, CHCl3));
146 1 H NMR (500 MHz, CDCl3) δ 2.60 (1 H, dd, J 2.5, 4.6 Hz, H1', 2'R epimer), 2.65 (1 H, dd, J 2.5, 4.8 Hz, H1', 2'S epimer), 2.76 – 2.83 (2 H, m), 3.10 – 3.19 (2 H, m), 3.33 – 3.49 (24 H, m), 3.54 (1 H, dd, J 5.7, 12.0 Hz), 3.87 (1 H, dd, J 2.0, 12.0 Hz), 3.91 – 4.09 (17 H, m), 4.10 – 4.23 (4 H, m), 4.30 – 4.39 (2 H, m), 4.92 – 5.00 (2 H, m), 5.09 – 5.21 (4 H,
13 m), 5.50 – 5.61 (2 H, m); C NMR (125 MHz, CDCl3) δ 44.2, 44.4, 50.2, 50.4, 59.5, 59.58, 59.59, 61.89, 61.94, 67.27, 67.30, 68.58, 68.61, 68.9, 69.4, 69.5, 69.6, 69.8, 70.5,
70.6, 70.8, 70.9, 96.1, 169.4, 169.7, 169.8, 170.1; HRMS (ESI+) calcd for C21H32O15 (M + Na)+ 547.1633. Found 547.1645.
(2'R)-2',3'-Epoxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D- MAcO MAcO O MAcO -glucopyranoside (52) MAcO O O A mixture of (S,S)-salen Co(II) (0.040 g, 0.066 mmol) and p-toluenesulfonic acid (0.012 g, 0.072 mmol) in CH2Cl2 (1 mL) was stirred vigorously at room temperature for 30 min. The solvent was evaporated and the solid was dried under reduced pressure. A solution of the resulting (S,S)-salen Co(III) complex and S (1.72 g,
◦ 3.28 mmol) in dry THF (2 mL) was cooled to 0 C and was treated with H2O (0.033 mL, 1.81 mmol). The mixture was warmed to room temperature and was stirred overnight. The solvent was evaporated and flash chromatography (EtOAc) afforded (2'R)-52 as a
24 78 26 colourless oil (0.77 g, 45%). [α]D +81.2 (c 1.0, CHCl3) (lit. [α]D +84.8 (c 1.51, CHCl3));
1 H NMR (500 MHz, CDCl3) δ 2.59 (1 H, dd, J 2.6, 4.4 Hz, H1'), 2.80 (1 H, t, J 4.8, H1'),
3.12 – 3.19 (1 H, m, H2'), 3.33 – 3.47 (12 H, 4 × s, 4 × CH3OCH2), 3.90 – 4.22 (12 H, m,
4 × CH3OCH2, H5,6,3',3'), 4.34 (1 H, dd, J 4.0, 12.4 Hz, H6), 4.97 (1 H, dd, J 3.6, 10.2 Hz, H2), 5.13 (1 H, t, J 9.8, H4), 5.17 (1 H, d, J 3.5, H1), 5.54 (1 H, t, J 9.7, H3); 13C NMR
(125 MHz, CDCl3) δ 44.4, 50.4, 59.4, 59.53, 59.54, 62.0, 67.3, 68.6, 69.4, 69.5, 69.6, 69.8,
70.5, 70.7, 96.1, 132.9, 169.3, 169.6, 169.7, 170.1; HRMS (ESI+) calcd for C21H32O15 (M + Na)+ 547.1633. Found 547.1626.
147 (2'S)-3'-Bromo-2'-hydroxypropyl 2,3,4,6-tetra-O-methoxy- MAcO MAcO O MAcO OH acetyl-α-D-glucopyranoside (53) MAcO O Br
LiBr (1.00 g, 11.5 mmol) and NiBr2 (1.25 g, 5.72 mmol) were stirred for 48 h in dry THF (13.7 mL) at room temperature. Stirring was stopped and undissolved material was allowed to settle, affording a clear dark blue/green solution of Li2NiBr4 ( 0.4 M). Excess Li2NiBr4 (2.75 mL) in THF was added to a solution of 52 (0.36 g, 0.68 mmol) in dry THF (1 mL) and the solution was stirred overnight. The re- action was treated with phosphate buffer (7 mL, pH 7) and extracted withCH2Cl2. The organic layer was washed with water, dried (Na2SO4), filtered and concentrated. Flash
24 chromatography (EtOAc/pet. spirits) afforded 53 as a colourless oil (0.39 g, 96%) [α]D
78 25 1 +67.5 (c 1, CHCl3) (lit. [α]D +67.4 (c 1.35, CHCl3)); H NMR (500 MHz, CDCl3) δ
3.34 – 3.43 (14 H, 4 × s, 4 × CH3OCH2), 3.45 (1 H, dd, J 5.4, 10.5 Hz, H3'), 3.49 (1 H, dd, J 5.7, 10.4 Hz, H3'), 3.57 (1 H, dd, J 5.9, 10.5 Hz, H1'), 3.83 (1 H, dd, J 4.2, 10.5 Hz,
H1'), 3.90 – 4.08 (9 H, m, 4 × CH3OCH2, H2'), 4.11 – 4.16 (1 H, m, H5), 4.18 (1 H, dd, J 2.2, 12.4 Hz, H6a), 4.33 (1 H, dd, J 4.4, 12.3 Hz, H6b), 4.97 (1 H, dd, J 3.7, 10.1 Hz, H2),
13 5.07 – 5.17 (2 H, m, H1,4), 5.51 (1 H, t, J 9.7, H3); C NMR (125 MHz, CDCl3) δ 34.5, 59.5, 59.59, 59.61, 59.7, 62.0, 67.4, 68.6, 69.5, 69.6, 69.68, 69.71, 70.6, 70.8, 70.9, 96.6,
169.3, 169.5, 169.7, 170.0; HRMS (ESI+) calcd for C21H33BrO15 (M + Na)+ 627.0895. Found 627.0901.
(2'S)-3'-Bromo-2'-palmitoyloxypropyl 2,3,4,6-tetra-O-meth- MAcO O MAcO O MAcO oxyacetyl-α-D-glucopyranoside (54) O 14 MAcO O Br Palmitoyl chloride (0.103 mL, 0.337 mmol) was added to a stirred solution of 53 (102 mg, 0.168 mmol) in dry CH2Cl2 (4 mL) and pyridine (0.270 mL, 3.37 mmol) at 0 ◦C. The reaction mixture was allowed to warm to room temperature and stirring was continued overnight. The reaction mixture was diluted with CH2Cl2, washed with water, sat. aq. CuSO4 and sat. aq. NaHCO3. The organic lay- ers were dried (MgSO4), filtered and concentrated under reduced pressure. Flash chro-
148 matography of the residue (EtOAc/pet. spirits) afforded 54 as a colourless oil (123 mg,
24 1 87%). [α]D +66.6 (c 0.68, CHCl3); H NMR (500 MHz, CDCl3) δ 0.87 (3 H, t, J 7.0 Hz,
CH2CH3), 1.24 – 1.31 (24 H, m, alkyl), 1.59 – 1.65 (2 H, m, β-CH2) 2.33 – 2.37 (2 H, m,
α-CH2), 3.39, 3.40, 3.42, 3.45 (12 H, 4 × s, 4 × CH3OCH2), 3.52 (1 H, dd, J 4.6, 10.7 Hz, H1'), 3.60 (1 H, dd, J 6.3, 10.6 Hz, H1'), 3.72 (1 H, dd, J 4.9, 10.8 Hz, H3'), 3.89 (1 H, dd, J
4.7, 10.8 Hz, H3'), 3.95, 4.00, 4.08 (6 H, 3 × s, 3 × CH3OCH2), 4.02 (2 H, m, CH3OCH2), 4.07 – 4.11 (1 H, m, H5), 4.19 (1 H, dd, J 2.2, 12.4 Hz, H6a), 4.38 (1 H, dd, J 4.2, 12.4 Hz, H6b), 4.99 (1 H, dd, J 3.8, 10.1 Hz, H2), 5.09 – 5.17 (3 H, m, H1,2',4), 5.53 (1 H, t,
13 J 9.7 Hz, H3); C NMR (125 MHz, CDCl3) δ 14.2, 22.8, 25.0, 29.3, 29.41, 29.44, 29.5, 29.6, 29.75, 29.79, 29.8, 30.0, 32.1, 34.3 (fatty acyl), 59.5, 59.57, 59.59, 59.60, 61.8, 67.41, 67.45, 68.5, 69.42, 69.44, 69.5, 69.6, 70.5, 70.6, 70.7, 96.3 (C1), 169.3, 169.6, 169.7, 170.1
(MeOCH2C=O), 172.9 (sn-2-CO2); HRMS (ESI+) calcd for C37H63O16Br (M + NH4)+ 860.3638. Found 860.3646.
(2'S)-3'-Bromo-2'-oleoyloxypropyl 2,3,4,6-tetra-O-methoxy- MAcO O MAcO O MAcO O 7 7 MAcO O Br acetyl-α-D-glucopyranoside (61) Oleoyl chloride (0.27 mL, 0.816 mmol) was added to a stirred solution of 53 (247 mg, 0.408 mmol) in dry CH2Cl2 (9 mL) and pyridine (0.657 mL, 8.16 mmol) at 0 ◦C. The reaction mixture was allowed to warm to room temperature and stirring was continued overnight. The reaction mixture was diluted with CH2Cl2, washed with water, sat. aq. CuSO4 and sat. aq. NaHCO3. The organic layers were dried
(MgSO4), filtered and concentrated under reduced pressure. Flash chromatography of
24 the residue (EtOAc/pet. spirits) afforded 61 as a colourless oil (337 mg, 95%). [α]D +52.3
25 1 (c 1, CHCl3) (lit. [α]D +55.8 (c 1.15, CHCl3)); H NMR (500 MHz, CDCl3) δ 0.87 (3H, t, J 6.9 Hz, CH2CH3), 1.18 – 1.39 (20H, m, alkyl), 1.60 – 1.66 (2H, m, β-CH2), 1.96 –
2.05 (4H, m, CH2CH – CHCH2), 2.29 – 2.38 (2H, m, α-CH2), 3.39, 3.40, 3.42, 3.44 (12H,
4 × s, 4 × CH3OCH2), 3.51 (1H, dd, J 4.6, 10.6 Hz, H1'), 3.59 (1H, dd, J 6.2, 10.7 Hz, H1'), 3.72 (1H, dd, J 4.9, 10.9 Hz, H3'), 3.88 (1H, dd, J 4.8, 10.8 Hz, H3'), 3.95, 3.99, 4.08
149 (6H, 3 × s, 3 × CH3OCH2), 4.01 – 4.11 (3H, m, CH3OCH2, H5), 4.19 (1H, dd, 2.3, 12.4 Hz, H6a), 4.38 (1H, dd, J 4.2, 12.4 Hz, H6b), 4.99 (1H, dd, J 3.8, 10.1 Hz, H2), 5.09 –
5.17 (3H, m, H1,4), 5.29 – 5.37 (2H, m, HC – CH), 5.52 (1H, t, J 9.7 Hz, H3); 13C NMR
(125 MHz, CDCl3) δ 14.2, 22.8, 25.0, 27.3, 27.4, 29.20, 29.24, 29.3, 29.43, 29.44, 29.6, 29.8, 29.9, 30.0, 32.0, 34.3 (fatty acyl), 59.5, 59.56, 59.57, 59.59, 61.8, 67.40, 67.44, 68.5, 69.41, 69.42, 69.5, 69.6, 70.4, 70.5, 70.7, 96.2 (C1), 129.8, 130.2 (HC=CH), 169.3, 169.6,
169.7, 170.1 (MeOCH2C=O), 172.9 (sn-2-CO2); HRMS (ESI+) calcd for C35H65BrO16 (M + Na)+ 891.3348. Found 891.3343.
(2'S)-3'-Bromo-2'-vaccenoyloxypropyl 2,3,4,6-tetra-O-meth- MAcO O MAcO O MAcO O 9 5 oxyacetyl-α-D-glucopyranoside (57) MAcO O Br Vaccenoyl chloride (0.054 mL, 0.162 mmol) was added to a stirred solution of 53 (49 mg, 0.081 mmol) in dry CH2Cl2 (2 mL) and pyridine (0.130 mL, 1.62 mmol) at 0 ◦C. The reaction mixture was allowed to warm to room temperature and stirring was continued overnight. The reaction mixture was diluted with CH2Cl2, washed sequentially with water, sat. aq. CuSO4 and sat. aq. NaHCO3. The organic layers were dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography of the residue (EtOAc/pet. spirits) afforded 57 as colourless oil
24 1 (61 mg, 87%). [α]D +62.3 (c 1.29, CHCl3); H NMR (500 MHz, CDCl3) δ 0.87 (3 H, t,
J 8 Hz, CH2CH3), 1.24 – 1.31 (20 H, m, alkyl), 1.59 – 1.65 (2 H, m, β-CH2) 1.98 – 2.04
(4 H, m, H2CCH=CHCH2), 2.33 – 2.37 (2 H, m, α-CH2), 3.39, 3.41, 3.43, 3.45 (12 H,
4 × s, 4 × CH3OCH2), 3.52 (1 H, dd, J 4.6, 10.7 Hz, H1'), 3.60 (1 H, dd, J 6.2, 10.6 Hz, H1'), 3.72 (1 H, dd, J 4.9, 10.9 Hz, H3'), 3.89 (1 H, dd, J 4.7, 10.8 Hz, H3'), 3.95, 4.00,
4.08 (6 H, 3 × s, 3 × CH3OCH2), 4.02 (2 H, m, CH3OCH2), 4.07 – 4.11 (1 H, m, H5), 4.19 (1 H, dd, J 2.3, 12.3 Hz, H6), 4.38 (1 H, dd, J 4.3, 12.4 Hz, H6), 4.99 (1 H, dd, J 3.8, 10.1 Hz, H2), 5.09 – 5.17 (3 H, m, H1,2',4), 5.30 – 5.38 (2 H, m, HC=CH), 5.53 (1 H,
13 t, J 9.6 Hz, H3); C NMR (125 MHz, CDCl3) δ 14.2, 22.8, 25.0, 27.3, 27.4, 29.1, 29.2, 29.3, 29.4, 29.58, 29.64, 29.8, 29.9, 30.0, 31.9, 34.3 (fatty acyl), 59.48, 59.56, 59.59, 59.60,
150 61.8, 67.41, 67.45, 68.5, 69.42, 69.43, 69.47, 69.6, 70.4, 70.5, 70.7, 96.3 (C1), 129.9, 130.1
(HC=CH), 169.3, 169.6, 169.7, 170.1 (MeOCH2C=O), 172.9 (sn-2-CO2); HRMS (ESI+) calcd for C39H65O16Br (M + NH4)+ 886.3794. Found 886.3830.
1'-O-(1'-13C-Palmitoyl)-2'-O-palmitoyl-sn-glyceryl 2,3,4,6- MAcO O MAcO O MAcO O MAcO 14 tetra-O-methoxyacetyl-α-D-glucopyranoside (56) O O13 C 14 O Tetrabutylammonium hydroxide solution in H2O (1.5 M, 0.089 mL, 0.137 mmol) was added to a suspension of 1-13C-palmitic acid (39 mg,
0.152 mmol) in H2O. The resulting mixture was vigorously stirred at room temperature overnight. The solvent was evaporated and the crude residue was co-evaporated with toluene several times to give the tetrabutylammonium salt of palmitic acid. A mixture of tetrabutylammonium palmitate (69 mg, 0.137 mmol) and 54) (58 mg, 0.069 mmol) in toluene (2 mL) was heated to 85 ◦C and stirred vigorously for 25 min. The solvents were evaporated under high vacuum. Flash chromatography of the residue (EtOAc/pet
24 1 spirits 2:3) afforded 56 as a colourless oil (50 mg, 71%). [α]D +57.8 (c 0.65, CHCl3); H
NMR (500 MHz, CDCl3) δ 0.81 – 0.94 (6 H, m, CH2CH3), 1.25 – 1.42 (48 H, m, alkyl),
1.48 – 1.66 (4 H, m, β-CH2), 2.23 – 2.35 (4 H, m, α-CH2), 3.38, 3.39, 3.41, 3.43 (12 H,
4 × s, 4 × CH3OCH2), 3.62 (1 H, dd, J 5.5, 11.2 Hz, H3'), 3.79 (1 H, dd, J 4.6, 11.2 Hz,
H3'), 3.94, 3.98, 4.07 (6 H, 3 × s, 3 × CH3OCH2), 4.04 (2 H, m, CH3OCH2), 4.06 – 4.08 (1 H, m, H5), 4.12 (1 H, ddd, J 3.2, 6.0, 11.9 Hz, H1'), 4.17 (1 H, dd, J 2.3, 12.4 Hz, H6), 4.32 (1 H, ddd, J 2.8, 4.2, 11.8 Hz, H1'), 4.37 (1 H, dd, J 4.1, 12.4 Hz, H6), 4.96 (1 H, dd, J 4.1, 12.4 Hz, H2), 5.08 – 5.23 (3 H, m, H1,2',4), 5.5 (1 H, t, J 9.7 Hz, H3); 13C
NMR (125 MHz, CDCl3) δ 14.2, 22.8, 25.0, 29.2, 29.40, 29.42, 29.5, 29.61, 29.63, 29.75, 29.77, 29.78, 29.79, 29.81, 32.0, 32.0, 34.3, 34.4 (fatty acyl), 59.4, 59.51, 59.53, 59.54, 61.7, 62.1, 67.0, 67.4, 68.5, 69.32, 69.39, 69.40, 69.6, 70.5, 70.7, 96.2 (C1), 169.3, 169.6,
169.7, 170.1 (MeOCH2C=O), 172.9 (sn-2-CO2), 173.2 (sn-1-CO2); HRMS (ESI+) calcd
13 for C52 CH94O18 (M + NH4)+ 1037.6812. Found 1037.6778.
151 1'-O-Oleoyl-2'-O-palmitoyl-sn-glyceryl 2,3,4,6-tetra-O-me- MAcO O MAcO O MAcO O 14 MAcO O O thoxyacetyl-α-D-glucopyranoside (60) 7 7 O
Tetrabutylammonium hydroxide solution in H2O (1.5 M, 0.056 mL, 0.085 mmol) was added to a suspension of oleic acid (30 µL, 0.095 mmol) in H2O (1 mL). The resulting mixture was vigorously stirred at room temperature overnight. The solvent was evaporated and the crude residue was co-evaporated with toluene several times to give the tetrabutylammonium salt of oleic acid. A mixture of tet- rabutylammonium oleate (45 mg, 0.085 mmol) and (54) (40 mg, 0.047 mmol) in tolu- ene (1 mL) was heated to 85 ◦C and stirred vigorously for 25 min. The solvents were evaporated under high vacuum. Flash chromatography of the residue (EtOAc/pet spir-
24 1 its 2:3) afforded 60 as a colourless oil (31 mg, 63%). [α]D +50.8 (c 1, CHCl3); H NMR
(500 MHz, CDCl3) δ 0.88 (6 H, t, J 6.9 Hz, CH2CH3), 1.18 – 1.36 (44 H, m, alkyl), 1.55
– 1.66 (4 H, m, β-CH2), 1.96 – 2.06 (4 H, m, CH2CH=CHCH2), 2.27 – 2.35 (4 H, m,
α-CH2), 3.39, 3.40, 3.42, 3.45 (12 H, 4 × s, 4 × CH3OCH2), 3.63 (1 H, dd, J 5.5, 11.2
Hz, H3'), 3.81 (1 H, dd, J 4.5, 11.2 Hz, H3'), 3.95 – 4.12 (8 H, m, 4 × CH3OCH2), 4.04 – 4.07 (1 H, m, H5), 4.13 (1 H, dd, J 5.6, 12.3 Hz, H1'), 4.18 (1 H, dd, J 2.3, 12.4 Hz, H6), 4.33 (1 H, dd, J 4.3, 11.8 Hz, H1'), 4.38 (1 H, dd, J 4.1, 12.4 Hz, H6), 4.97 (1 H, dd, J 3.8, 10.1 Hz, H2), 5.09 – 5.23 (3 H, m, H1,2',4), 5.30 – 5.39 (2 H, m, HC=CH),
13 5.49 – 5.55 (1 H, m, H3); C NMR (126 MHz, CDCl3) δ 14.3, 22.83, 22.84, 25.0, 25.1, 27.3, 27.4, 29.27, 29.29, 29.4, 29.46, 29.47, 29.51, 29.7, 29.81, 29.83, 29.85, 29.91, 32.0, 32.1, 34.2, 34.4 (fatty acyl), 59.49, 59.56, 59.58, 59.59, 61.7, 62.1, 67.1, 67.4, 68.5, 69.36, 69.44, 69.5, 69.6, 69.8, 70.6, 70.8, 96.3 (C1), 129.8, 130.2 (HC=CH), 169.3, 169.6, 169.7,
170.1 (MeOCH2C=O), 173.1 (sn-2-CO2), 173.4 (sn-1-CO2); HRMS (ESI+) calcd for
C55H96O18 (M + NH4)+ 1062.6935. Found 1062.6918.
152 MAcO O 1'-O-Palmitoyl-2'-O-oleoyl-sn-glyceryl 2,3,4,6-tetra-O-me- MAcO O MAcO O 7 7 MAcO O O 14 thoxyacetyl-α-D-glucopyranoside (62) O
Tetrabutylammonium hydroxide solution in H2O (1.5 M, 0.478 mL, 0.738 mmol) was added to a suspension of palmitic acid (210 mg,
0.820 mmol) in H2O (8 mL). The resulting mixture was vigorously stirred at room temperature overnight. The solvent was evaporated and the crude residue was co- evaporated with toluene several times to give the tetrabutylammonium salt of palmitic acid. A mixture of tetrabutylammonium palmitate (0.354 g, 0.738 mmol) and (61) (343 mg, 0.382 mmol) in toluene (2.5 mL) was heated to 85 ◦C and stirred vigorously for 25 min. The solvents were evaporated under high vacuum. Flash chromatography of the residue (EtOAc/pet spirits 2:3) afforded 62 as a colourless oil (0.220 mg, 55%).
24 1 [α]D +47.7 (c 0.5, CHCl3); H NMR (500 MHz, CDCl3) δ 0.86 (6 H, t, J 6.9 Hz,
CH2CH3), 1.20 – 1.38 (44 H, m, alkyl), 1.55 – 1.64 (4 H, m, β-CH2), 1.97 – 2.03 (4 H, m,
CH2CH=CHCH2), 2.26 – 2.34 (4 H, m, α-CH2), 3.38, 3.39, 3.41, 3.44 (12 H, 4 × s, 4 ×
CH3OCH2), 3.62 (1 H, dd, J 5.5, 11.2 Hz, H3'), 3.80 (1 H, dd, J 4.6, 11.2 Hz, H3'), 3.93 –
4.08 (8 H, m, 4 × CH3OCH2), 4.03 – 4.07 (1 H, m, H5), 4.12 (1 H, dd, J 6.0, 11.9 Hz, H1'), 4.17 (1 H, dd, J 2.3, 12.4 Hz, H6), 4.32 (1 H, dd, J 4.3, 11.8 Hz, H1'), 4.37 (1 H, dd, J 4.1, 12.4 Hz, H6), 4.96 (1 H, dd, J 3.8, 10.1 Hz, H2), 5.08 – 5.23 (3 H, m, H1,2',4), 5.28 – 5.38
13 (2 H, m, HC=CH), 5.51 (1 H, t, J 9.7 Hz, H3); C NMR (126 MHz, CDCl3) δ 14.2, 22.8, 22.8, 24.99, 25.00, 27.30, 27.34, 29.22, 29.27, 29.34, 29.41, 29.43, 29.44, 29.5, 29.61, 29.64, 29.75, 29.77, 29.78, 29.82, 29.85, 29.88, 32.02, 32.04, 34.1, 34.3 (fatty acyl), 59.45, 59.49, 59.53, 59.54, 61.7, 62.1, 67.0, 67.4, 68.5, 69.3, 69.4, 69.45, 69.6, 69.8, 70.5, 70.7, 96.2 (C1),
129.8, 130.1 (HC=CH), 169.3, 169.6, 169.7, 170.1 (MeOCH2C=O), 173.0 (sn-2-CO2),
173.4 (sn-1-CO2); HRMS (ESI+) calcd for C55H96O18 (M + NH4)+ 1062.6935. Found 1062.6921.
153 1'-O-Oleoyl-2'-O-oleoyl-sn-glyceryl 2,3,4,6-tetra-O-methoxy- MAcO O MAcO O MAcO O 7 7 MAcO O O acetyl-α-D-glucopyranoside (68) 7 7 O
Tetrabutylammonium hydroxide solution in H2O (1.5 M, 247 µL, 0.383 mmol) was added to a suspension of oleic acid (0.134 µL, 0.426 mmol) in H2O (4 mL). The resulting mixture was vigorously stirred at room temperature overnight. The solvent was evaporated and the crude residue was co-evaporated with toluene several times to give the tetrabutylammonium salt of oleic acid. A mixture of tet- rabutylammonium oleate (0.220 g, 0.383 mmol) and (61) (172 mg, 0.192 mmol) in tolu- ene (1.2 mL) was heated to 85 ◦C and stirred vigorously for 25 min. The solvents were evaporated under high vacuum. Flash chromatography of the residue (EtOAc/pet spir-
24 1 its 2:3) afforded 68 as a colourless oil (134 mg, 65%). [α]D +50.2 (c 1, CHCl3); H NMR
(500 MHz, CDCl3) δ 0.85 (6 H, t, J 6.7 Hz, CH2CH3), 1.20 – 1.36 (40 H, m, alkyl), 1.53 –
1.63 (4 H, m, β-CH2), 1.93 – 2.02 (8 H, m, CH2CH=CHCH2), 2.22 – 2.33 (4 H, mα-CH2),
3.37, 3.38, 3.40, 3.42 (12 H, 4 × s, 4 × CH3OCH2), 3.61 (1 H, dd, J 5.4, 11.2 Hz, H3'), 3.79
(1 H, dd, J 4.5, 11.2 Hz, H3'), 3.90 – 4.07 (8 H, m, 4 × CH3OCH2), 4.04 – 4.07 (1 H, m, H5), 4.11 (1 H, dd, J 6.0, 11.8 Hz, H1'), 4.16 (1 H, dd, J 2.3, 12.5 Hz, H6), 4.31 (1 H, dd, J 4.2, 11.9 Hz, H1'), 4.36 (1 H, dd, J 4.1, 12.4 Hz, H6), 4.95 (1 H, dd, J 3.8, 10.1 Hz, H2), 5.07 – 5.21 (3 H, m, H1,2',4), 5.25 – 5.39 (4 H, m, HC=CH), 5.50 (1 H, t, J 9.7 Hz,
13 H3); C NMR (125 MHz, CDCl3) δ 14.2, 22.7, 24.9, 25.0, 27.2, 27.3, 29.18, 29.20, 29.22, 29.27, 29.29, 29.4, 29.6, 29.76, 29.78, 29.80, 29.83, 32.0, 34.1, 34.3 (fatty acyl), 59.40, 59.46, 59.49, 61.7, 62.0, 67.0, 67.3, 68.4, 69.27, 69.35, 69.4, 69.5, 69.7, 70.5, 70.7, 96.2
(C1), 129.72, 129.74, 130.1 (HC=CH), 169.2, 169.56, 169.62, 170.0 (MeOCH2C=O),
173.0 (sn-2-CO2), 173.3 (sn-1-CO2); HRMS (ESI+) calcd for C57H98O18 (M + NH4)+ 1088.7091. Found 1088.7078.
154 MAcO O 1'-O-Palmitoyl-2'-O-vaccenoyl-sn-glyceryl 2,3,4,6-tetra-O- MAcO O MAcO O 9 5 MAcO O O 14 methoxyacetyl-α-D-glucopyranoside (58) O
Tetrabutylammonium hydroxide solution in H2O (1.5 M, 0.219 mL, 0.147 mmol) was added to a suspension of palmitic acid (42 mg, 0.163 mmol) in H2O (1.5 mL). The resulting mixture was vigorously stirred at room temperature overnight. The solvent was evaporated and the crude residue was co-evaporated with toluene several times to give the tetrabutylammonium salt of palmitic acid. A mixture of tetrabutylammonium palmitate (73 mg, 0.147 mmol) and (57) (64 mg, 0.074 mmol) in toluene (2 mL) was heated to 85 ◦C and stirred vigorously for 25 min. The solvents were evaporated under high vacuum. Flash chromatography of the residue (EtOAc/pet
24 1 spirits 2:3) afforded 58 as a colourless oil (40 mg, 52%). [α]D +46.8 (c 1.8, CHCl3); H
NMR (500 MHz, CDCl3) δ 0.81 – 0.94 (6 H, m, CH2CH3), 1.25 – 1.42 (44 H, m, alkyl),
1.54 – 1.67 (4 H, m, β-CH2), 1.97 – 2.03 (4 H, m, CH2CH=CHCH2), 2.27 – 2.35 (4 H, m, α-CH2), 3.39, 3.40, 3.42, 3.44 (12 H, 4 × s, 4 × CH3OCH2), 3.62 (1 H, dd, J 5.5, 11.2
Hz, H3'), 3.80 (1 H, dd, J 4.6, 11.2 Hz, H3'), 3.94, 3.98, 4.07 (6 H, 3 × s, 3 × CH3OCH2),
4.00 – 4.04 (2 H, m, CH3OCH2), 4.04 – 4.08 (1 H, m, H5), 4.13 (1 H, dd, J 6.0, 11.9 Hz, H1'), 4.18 (1 H, dd, J 2.3, 12.4 Hz, H6), 4.33 (1 H, dd, J 4.3, 11.9 Hz, H1'), 4.38 (1 H, dd, J 4.1, 12.4 Hz, H6), 4.97 (1 H, dd, J 3.8, 10.1 Hz, H2), 5.09 – 5.23 (3 H, m, H1,2',4), 5.30
13 – 5.39 (2 H, m, HC=CH), 5.52 (1 H, t, J 9.7 Hz, H3); C NMR (125 MHz, CDCl3) δ 14.2, 14.3, 22.80, 22.83, 25.01, 25.04, 27.4, 29.1, 29.3, 29.4, 29.5, 29.6, 29.7, 29.80, 29.81, 29.84, 29.87, 29.92, 31.9, 32.1, 34.2, 34.4 (fatty acyl), 59.48, 59.55, 59.56, 59.58, 61.7, 62.1, 67.1, 67.4, 68.5, 69.4, 69.5, 69.6, 69.8, 70.6, 70.7, 96.3 (C1), 129.9, 130.1 (2C, HC=CH),
169.3, 169.6, 169.7, 170.1 (MeOCH2C=O), 173.1 (sn-2-CO2), 173.4 (sn-1-CO2); HRMS
(ESI+) calcd for C55H96O18 (M + NH4)+ 1062.6935. Found 1062.6954.
155 1'-O-Oleoyl-2'-O-palmitoyl-sn-glyceryl α-D-glucopyranoside HO O HO O HO O 14 HO O O (63) 7 7 O A solution of tert-butylamine (516 µL, 4.92 mmol) and 60
◦ (257 mg, 0.246 mmol) in CH2Cl2 (1.6 mL) and MeOH (6.5 mL) was stirred at 0 C for 10 min and then at 10 ◦C for 1 h. The solvents were evaporated under high vacuum without heating. Flash chromatography of the residue (MeOH/CHCl3 5:95) afforded
24 1 63 as a white semisolid (110 g, 59%). [α]D +48.1 (c 1, CHCl3); H NMR (500 MHz,
CDCl3:CD3OD) δ 0.66 (6 H, t, J 6.8 Hz, CH2CH3), 0.98 – 1.16 (44 H, m, alkyl), 1.35
– 1.43 (4 H, m, β-CH2), 1.76 – 1.83 (4 H, m, CH2CH=CHCH2), 2.07 – 2.13 (4 H, m,
α-CH2), 3.17 (1 H, t, J 9.4 Hz, H4), 3.21 (1 H, dd, J 3.8, 9.7 Hz, H2), 3.33 (1 H, ddd, J 3.0, 4.3, 9.9 Hz, H5), 3.38 – 3.45 (2 H, m, H3,3'), 3.50 – 3.59 (2 H, m, H6,6), 3.61 (1 H, dd, J 5.6, 10.8 Hz, H3'), 3.95 (1 H, dd, J 6.4, 12.0 Hz, H1'), 4.19 – 4.23 (1 H, m, H1'), 4.60 (1 H, d, J 3.8 Hz, H1), 5.00 – 5.06 (1 H, m, H2'), 5.07 – 5.16 (2 H, m, HC=CH); 13C
NMR (125 MHz, CDCl3:CD3OD) δ 13.6, 22.4, 24.60, 24.62, 26.88, 26.91, 28.83, 28.84, 28.85, 28.93, 29.02, 29.04, 29.1, 29.2, 29.37, 29.39, 29.42, 29.44, 29.47, 31.6, 31.7, 33.8, 34.0 (fatty acyl), 61.2 (C6), 62.4 (C1'), 65.8 (C3'), 69.8, 69.9 (H4, H2'), 71.8 (C2), 72.0
(C5), 73.5 (C3), 99.1 (C1), 129.4, 129.7 (HC=CH), 173.4 (sn-2-CO2), 173.8 (sn-1-CO2);
HRMS (ESI+) calcd for C43H80O10 (M + NH4)+ 774.6090. Found 774.6069.
1'-O-Palmitoyl-2'-O-oleoyl-sn-glyceryl α-D-glucopyranoside HO O HO O HO O 7 7 (64) HO O O 14 A solution of tert-butylamine (0.438 mL, 4.17 mmol) and 62 O
◦ (218 mg, 0.209 mmol) in CH2Cl2 (1.3 mL) and MeOH (5.5 mL) was stirred at 0 C for 10 min and then at 10 ◦C for 1 h. The solvents were evaporated under high vacuum without heating. Flash chromatography of the residue (MeOH/CHCl3 5:95) afforded
24 1 64 as a white semi-solid (128 g, 81%). [α]D +49.3 (c 0.9, CHCl3); H NMR (500 MHz,
CDCl3:CD3OD) δ 0.72 (6 H, t, J 7.0 Hz, CH2CH3), 1.03 – 1.22 (44 H, m, alkyl), 1.39
– 1.50 (4 H, m, β-CH2), 1.81 – 1.89 (4 H, m, CH2CH=CHCH2), 2.12 – 2.20 (4 H, m,
156 α-CH2), 3.24 (1 H, t, J 9.5 Hz, H4), 3.27 (1 H, dd, J 3.7, 9.7 Hz, H2), 3.39 (1 H, ddd, J 3.7, 10.0 Hz, H5), 3.44 – 3.50 (2 H, m, H3,3'), 3.58 – 3.64 (2 H, m, H6,6), 3.67 (1 H, dd, J 5.5, 10.8 Hz, H3'), 3.99 – 4.03 (1 H, m, H1'), 4.26 (1 H, dd, J 3.4, 12.0 Hz, H1'), 4.66 (1 H, d, J 3.7 Hz, H1), 5.06 – 5.13 (1 H, m, H2'), 5.14 – 5.24 (2 H, m, HC=CH);
13 C NMR (125 MHz, CDCl3:CD3OD) δ 13.8, 22.5, 24.7, 27.01, 27.04, 28.9, 28.97, 28.99, 29.1, 29.15, 29.20, 29.35, 29.36, 29.48, 29.50, 29.51, 29.54, 29.57, 29.60, 31.7, 31.8, 34.0, 34.1 (fatty acyl), 61.3 (C6), 62.4 (C1'), 66.0 (C3'), 69.90, 69.95 (H4, H2'), 71.9 (C2), 72.0
(C5), 73.6 (C3), 99.1 (C1), 129.5, 129.8 (HC=CH), 173.5 (sn-2-CO2), 173.9 (sn-1-CO2);
HRMS (ESI+) calcd for C43H80O10 (M + NH4)+ 774.6090. Found 774.6089.
1'-O-Oleoyl-2'-O-oleoyl-sn-glyceryl α-D-glucopyranoside (69) HO O HO O HO O 7 7 HO O O A solution of tert-butylamine (307 µL, 2.93 mmol) and 68 7 7 O (157 mg, 0.147 mmol) in CH2Cl2 (1.0 mL) and MeOH (4.0 mL) was stirred at 0 ◦C for 10 min and then at 10 ◦C for 1 h. The solvents were evaporated un- der high vacuum without heating. Flash chromatography of the residue (MeOH/CHCl3
24 1 5:95) afforded 69 as a white semi-solid (75 mg, 65%). [α]D +46.9 (c 1, CHCl3); H NMR
(500 MHz, CDCl3) δ 0.87 (6 H, t, J 6.7 Hz, CH2CH3), 1.18 – 1.39 (40 H, m, alkyl), 1.54
– 1.65 (4 H, m, β-CH2), 1.93 – 2.08 (8 H, m, CH2CH=CHCH2), 2.26 – 2.35 (4 H, m,
α-CH2), 3.49 – 3.63 (4 H, m, H2,4,5,3'), 3.68 – 3.89 (4 H, m, H3,6,6,3'), 4.15 (1 H, dd, J 6.1, 12.1 Hz, H1'), 4.39 (1 H, dd, J 3.3, 12.1 Hz, H1'), 4.85 (1 H, d, J 3.4 Hz, H1),
13 5.21 – 5.28 (1 H, m, H2'), 5.28 – 5.40 (4 H, m, HC=CH); C NMR (125 MHz, CDCl3) δ 14.3, 22.8, 25.01, 25.04, 27.35, 27.37, 29.26, 29.29, 29.34, 29.40, 29.41, 29.46, 29.47, 29.68, 29.90, 29.91, 32.0, 34.3, 34.4 (fatty acyl), 61.6 (C6), 62.7 (C1'), 66.3 (C3'), 69.8, 69.9 (H4, H2'), 72.06 (C2), 72.09 (C5), 74.2 (C3), 99.3 (C1), 129.8, 130.1 (HC=CH),
173.4 (sn-2-CO2), 173.9 (sn-1-CO2); HRMS (ESI+) calcd for C45H82O10 (M + NH4)+ 800.6246. Found 800.6247.
157 1'-O-Palmitoyl-2'-O-vaccenoyl-sn-glyceryl α-D-glucopyran- HO O HO O HO O 9 5 HO O O oside (59) 14 O A solution of tert-butylamine (0.180 mL, 1.7 mmol) and 58
◦ (37 mg, 0.035 mmol) in CHCl3 (0.2 mL) and MeOH (0.8 mL) was stirred at 0 C for 10 min and then at 10 ◦C for 1 h. The solvents were evaporated under high vacuum without heating. Flash chromatography of the residue (MeOH/CHCl3 5:95) afforded 59
24 1 (Glc-DAG-s2) as a white semi-solid (15 mg, 57%). [α]D +50.0 (c 0.75, CHCl3); H NMR
(500 MHz, CDCl3) δ 0.88 (6 H, m, CH2CH3), 1.10 – 1.42 (46 H, m, alkyl), 1.52 – 1.66
(4 H, m, β-CH2) 1.98 – 2.06 (4 H, m, CH2CH=CHCH2), 2.13 (1 H, br. s, OH), 2.26 – 2.36
(4 H, m, α-CH2), 2.98 (1 H, br. s, OH), 3.48 – 3.65 (4 H, m, H2,4,5,3'), 3.70 – 3.91 (4 H, m, H3,6,6,3'), 4.15 (1 H, dd, J 6.1, 12 Hz, H1'), 4.22 (1 H, br. s, OH), 4.39 (1 H, dd, J 3.3, 12 Hz, H1'), 4.47 (1 H, br. s, OH), 4.85 (1 H, s, H1), 5.20 – 5.29 (1 H, m, H2'), 5.30 – 5.41
13 (2 H, m, HC=CH); C NMR (125 MHz, CDCl3) δ 14.1, 22.64, 22.67, 24.9, 27.2, 29.0, 29.2, 29.3, 29.50, 29.54, 29.6, 29.71, 29.78, 31.8, 31.9, 34.1 (fatty acyl), 34.26, 61.6, 62.5,
66.2, 69.8, 71.8, 72.0, 74.2, 99.1 (C1), 129.8, 129.9 (HC=CH), 173.3 (sn-2-CO2), 173.8
(sn-1-CO2); HRMS (ESI+) calcd for C43H80O10 (M + NH4)+ 774.6089. Found 774.6094.
1'-O-Oleoyl-2'-O-palmitoyl-sn-glyceryl α-D-glucopyranosi- O HO O HO O HO O duronic acid (67) 14 HO O O 7 7 Bobbitt’s salt (30 mg, 0.099 mmol) was added to a vigorously O stirred solution of 63 (25 mg, 0.033 mmol) in MeCN (266 µL) and aq. KHCO3 (1.5 M, 133 µL). The mixture was stirred for 2 days and then concentrated. Sequen- tial flash chromatography of the residue (CHCl3/MeOH/H2O(20:1)/TFA(0.1%) and toluene:EtOAc(2:1):MeOH:H2O(20:1):-
24 TFA(0.1%)) afforded 67 (Gl-A (18:1)) as a colourless glass (13 mg, 50%). [α]D +28.7 (c
1 0.45, CHCl3); H NMR (500 MHz, CDCl3:CD3OD:TFA 95:5:0.1) δ 0.81 (6 H, t, J 6.9 Hz,
CH2CH3), 1.12 – 1.34 (44 H, m, alkyl), 1.48 – 1.60 (4 H, m, β-CH2), 1.91 – 1.99 (4 H, m, CH2CH=CHCH2), 2.22 – 2.30 (4 H, m, α-CH2), 3.47 – 3.67 (4 H, m, H2,3,4,3'), 3.80
158 (1 H, dd, J 5.1, 10.8 Hz, H3'), 4.03 (1 H, d, J 9.4 Hz, H5), 4.10 (1 H, dd, J 6.2, 12.0 Hz, H1'), 4.34 (1 H, dd, J 3.6, 12.0 Hz, H1'), 4.85 (1 H, s, H1), 5.15 – 5.21 (1 H, m, H2'), 5.23
13 – 5.35 (2 H, m, HC=CH); C NMR (125 MHz, CDCl3:CD3OD:TFA 95:5:0.1) δ 14.1, 22.7, 24.85, 24.88, 27.19, 27.22, 29.12, 29.16, 29.22, 29.33, 29.37, 29.53, 29.67, 29.72, 29.74, 29.77, 31.9, 34.1, 34.2 (fatty acyl), 62.4, 66.8, 69.9, 71.4, 71.6, 73.1, 99.3 (C1),
129.7, 130.0 (HC=CH), 172.1 (C6), 173.6 (sn-2-CO2), 174.0 (sn-1-CO2); HRMS (ESI+) calcd for C43H78O11 (M - H)- 769.5471. Found 769.5478.
D O 1'-O-Palmitoyl-2'-O-oleoyl-sn-glyceryl α- -glucopyranosid- HO O HO O HO O uronic acid (65) 7 7 HO O O 14 O Bobbitt’s salt (54 mg, 0.180 mmol) was added to a vigorously stirred solution of 64 (34 mg, 0.045 mmol) in MeCN (420 µL) and aq. KHCO3 (1.5 M, 180 µL). The mixture was stirred for 2 days and then concentrated. Sequen- tial flash chromatography of the residue (CHCl3/MeOH/H2O(20:1)/TFA(0.1%) and toluene:EtOAc(2:1):MeOH:H2O(20:1):-
24 TFA(0.1%)) afforded 65 as a colourless glass (16 mg, 45%). [α]D +33.0 (c 0.8, CHCl3);
1 H NMR (500 MHz, CDCl3:CD3OD:TFA 95:5:0.1) δ 0.82 (6 H, t, J 6.9 Hzz), 1.11 – 1.35
(44 H, m, alkyl), 1.47 – 1.61 (4 H, m, β-CH2), 1.89 – 2.01 (4 H, m, CH2CH=CHCH2),
2.19 – 2.33 (4 H, m, α-CH2), 3.43 – 3.70 (4 H, m, H2,3,4,3'), 3.82 (1 H, s, H3'), 4.04 (1 H, s, H5), 4.11 (1 H, dd, J 5.9, 12.1 Hz, H1'), 4.35 (1 H, dd, J 3.3, 12.1 Hz, H1'), 4.89 (1 H, s, H1), 5.19 (1 H, s, H2'), 5.24 – 5.35 (2 H, m, HC=CH); 13C NMR (125 MHz,
CDCl3:CD3OD:TFA 95:5:0.1) δ 14.0, 22.57, 22.58, 24.75, 24.77, 27.09, 27.12, 28.98, 29.03, 29.06, 29.13, 29.20, 29.22, 29.26, 29.41, 29.43, 29.55, 29.56, 29.58, 29.60, 29.64, 29.67, 31.81, 31.83, 34.0, 34.1 (fatty acyl), 62.3, 66.7, 69.7, 71.3, 71.5, 73.1, 99.3 (C1),
129.6, 129.9 (HC=CH), 171.7 (C6), 173.5 (sn-2-CO2), 173.9 (sn-1-CO2); HRMS (ESI+) calcd for C43H78O11 (M - H)- 769.5471. Found 769.5455.
159 Chapter 5
Phenylpentamethylbenzofuranyl sulfonate analogues as type II NKT immune effectors
5.1 Introduction
In 2004, a serendipitous observation led to the discovery of the first non-lipidic CD1d- restricted effector molecule. A mixture of lipopeptides, made by acylation of a sub- sequence of IL-1α (LKKRLL) with long-chain acids, was co-cultured with APCs and was used to identify a T cell line that was dependent on the mixture of lipopeptides for proliferation.127 This response was determined to be CD1d-dependent, as blocking with an anti-CD1d mAb or using CD1-/- APCs ablated the proliferative effect. Sequencing of the TCR from the reactive cell line revealed conserved α- and β-chains, namely Vα2- Vβ21, and leading them to name this novel T cell line ‘ABd’. ABd was not stimulated when challenged with αGalCer and therefore represents a type II NKT cell line.
5.1.1 Identification of PPBF as the stimulatory molecule
Since ABd had been identified using a mixture of compounds originally designed to have structural similarities to a mycobacterial antigen (DDM-838) presented by CD1a, isolation of the active effector species was necessary. HPLC fractionation of the mixture and stimulation assays identified one fraction with activity against ABd; mass spectro- metry revealed a single compound corresponding to a peak at m/z 347.1, a mass that did not correspond to any of the lipopeptides. A strong isotope peak at M + 2 was observed which suggested the presence of sulfur. They noted that only one reactant used during the preparation of the lipopeptides contained sulfur, 9-fluorenylmethyloxycarbonyl-N-
160 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-L-Arg-N-PBF- OH) (Figure 5.1A). The authors speculated that during deprotection in the presence of phenol, the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group could form phenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonate (PPBF, 70, m/z 347.1). MS/MS fragmentation of the parent ion produced fragment ions at m/z 283.1 and m/z
253.1, corresponding to the loss of SO2 and phenoxy respectively (Figure 5.1B); an ad-
ditional peak at m/z 190.1 corresponded to loss of PhOSO2. A B
O O O S O O m/z 253.1 O O S O S O O NH O HN O O m/z 347.1 HN NH 70 PSfrag replacements Fmoc O
m/z 283.1 m/z 190.1
Figure 5.1. A PBF-protected arginine was used during the preparation of the IL-1α lipopeptide library; B Fragmentation of the isolated active fraction revealed masses consistent with the structure of PPBF.
As there was no precedent for an aromatic small molecule stimulating NKT cells in a CD1d-restricted fashion, independent confirmation was achieved via preparation of
PPBF by reaction of phenol with Fmoc-L-Arg-N-PBF-OH. HPLC/MS of the products identified two compounds with masses of m/z 347, but with different retention times (Figure 5.2). The MS/MS fragmentation pattern of the late eluting isomer was identical to that of the compound isolated from the lipopeptide mixture. This independently syn- thesised compound stimulated the ABd cell line in a CD1d-restricted manner, thus confirming the identity of the stimulatory molecule as PPBF. The early eluting com- pound, representing the minor component, was identified as a sulfone (regiochemistry not defined) and was approx. 100-fold less active than PPBF. This compound presum- ably arises from electrophilic aromatic substitution of the phenol.
161 O
O O phenol O O S OH O S O S CHCl3 O HO O O NH O 70 PSfrag replacements HN HN NH Fmoc Figure 5.2. PPBF was synthesised by reaction of phenol with PBF-protected arginine
5.1.2 A small library of analogues identified a more potent
compound
With two non-lipidic small molecules now identified as CD1d-restricted ABd effectors, Van Rhijn and colleagues prepared a small set of analogues. Reaction of various cresols
with Fmoc-L-Arg-N-PBF-OH in chloroform followed by HPLC purification afforded the ortho-, meta- and para-methyl PPBF derivatives, as well as a sulfone analogue of undefined regiochemistry from the reaction with meta-cresol (Figure 5.3A). These com- pounds, with exception of the meta-methyl derivative, produced reduced proliferation of ABd cells in comparison to PPBF (Figure 5.3B). The meta-methyl compound, hence- forth known as MePPBF (71), stimulated proliferation of ABd cells approx. 20-fold more potently than PPBF.
A B O o -cresol O S O CHCl O 3 O O S O O O PPBF O p-cresol O S OH O S O O CHCl 3 O O NH HN HN NH Fmoc O O m-cresol O O S S CHCl O O 3 O MePPBF HO Figure 5.3. A Van Rhijn and colleagues prepared several PPBF analogues by reaction of different cresols with PBF-protected arginine; B Stimulation of ABd T cells with CD1d-presented PPBF analogues.
162 5.1.3 PPBF possesses a structural similarity to many ‘sulfa’ drugs
Van Rhijn and co-workers argued that PPBF and its analogues possess structural sim- ilarities to a range of clinically-approved drugs such as the antibiotics sulfisomidine, sulfadiazine and sulfasalazine, and the NSAID celecoxib (Figure 5.4). Sulfonamides, and especially antibiotics of this class bearing an arylamine, cause hypersensitivity re- actions in 3% of the population.372 It has been proposed that the hypersensitivity to sulfonamides bearing an arylamine is mediated by oxidation of the amine to the ni- troso compound via the hydroxyamine.373 In addition to its ability to directly stimulate lymphocytes, the nitroso compound can form covalent protein conjugates leading to re- cognition by the adaptive immune system as altered-self. The mode of hypersensitivity of sulfur-containing drugs not possessing an arylamine is less well understood leading to speculation that CD1 presentation may be involved. H O O H N N N S S O N N O N H N 2 H2N sulfisomidine sulfadiazine
O H N O S H2N S O N N O H2N N sulfasalazine OH celecoxib N O OH CF3 Figure 5.4. The antibacterial drugs sulfisomidine, sulfadiazine, sulfasalazine and the NSAID celecoxib possesses structures similar to that of PPBF.
5.1.4 Project aims
While the small set of PPBF derivative studied by Van Rhijn etal. provided some insight into the structural features that underpin the activation of ABd, its limited scope does not provide detailed insight into the structural features required for activity. A broader study of structural variation to PPBF should provide a deeper understanding of the structural requirements for immunological activity.
163 Aim 1: Wewill develop a structure-activity relationship for PPBF and MePPBF as CD1d- restricted immune effectors. We aim to develop synthetic approaches that will allow the construction of analogues with modifications on the phenyl ring, in the linker between the aryl rings, as well as the ring size of the pentamethylbenzofuran ring.
Aim 2: We plan to employ a combination of computational database searching as well as manual structure searches to identify commercially available drugs and vitamins with structural similarities to PPBF.
Aim 3: In collaboration with Prof. Dale Godfrey’s group at The Peter Doherty Institute, University of Melbourne, we will assess the activity of synthetic PPBF analogues and drugs as CD1d-restricted effectors. High potency analogues of PPBF will be used as probes for discovery of CD1d-restricted PPBF reactive T cell populations from human whole blood samples.
5.2 Results and discussion
5.2.1 Expanding the structure-activity relationship of PPBF;
resynthesis of PPBF
Since we were hoping to perform our immunological studies in a laboratory that had not previously worked with the ABd TCR, we initially planned to resynthesise PPBF and MePPBF to confirm their activity and to establish a baseline upon which to com- pare new synthetic analogues. The ability to stimulate ABd TCR+ cells will allow devel- opment of a structure-activity relationship ABd TCR binding.
Resynthesis of PPBF and MePPBF
Van Rhijn and co-workers prepared PPBF by reaction of phenol with PBF-protected arginine.127 We took a more direct approach involving reaction of pentamethbenzofur-
374 anylsulfonyl chloride (PBFCl, 72) with phenol and Et3N in CH2Cl2, which yielded 70 in 55% yield (Scheme 5.1). MePPBF was prepared by reaction of 72 with meta-cresol to afford 71 in 57% yield. In both cases no sulfone regioisomers were observed.
164 R O O a) 70 R = H 55% PSfrag replacements cond. O O b) 71 R = Me 57% S S Cl O O O 72
◦ Scheme 5.1. General conditions: Et3N, CH2Cl2, 0 C rt; a) phenol; b) m-cresol.
→ Modification of ring size: a benzochroman analogue of MePPBF
We initially chose to investigate the effect of enlargement of the benzofuran ring to the corresponding benzochroman. While the sulfonyl chloride 72 is commercially available, the analogous benzochroman 73 is not; like PBFCl it has also previously been used as an acid-labile protecting group.375 Ramage and colleague reported the preparation of 73 starting from 2,3,5-trimethylphenol.375 Treatment of 2,3,5-trimethylphenol with 2-methyl-3-buten-2-ol in TFA afforded 74 in 16% yield (Scheme 5.2). Installation of the sulfonyl chloride by treatment with chlorosulfuric acid afforded 73 in 28% yield.
O OH HO a O b + O 16% 26% S Cl O 74 73
PSfrag replacements OH O c O d O + O 76% 55% S Cl O 75 72
◦ ◦ Scheme 5.2. a) TFA; b) HSO3Cl, CH2Cl2, 0 C; c) H2SO4, toluene, 110 C; d) HSO3Cl, ◦ CH2Cl2, 0 C.
Although commercially available, we suspected that 72 was not particularly stable and moderate yields reported in Scheme 5.1 were a result of this. Carpino reported a synthesis of 72 similar to that utilized for 73 which we decided to follow.374 Treatment
of 2,3,5-trimethylphenol with isobutaldehyde and H2SO4 in toluene afforded 75 in 76% yield on a 15 g scale (Scheme 5.2). Chlorosulfonation with chlorosulfuric acid afforded 72 in 55% yield.
165 Since the m-cresol derivative MePPBF had been shown to stimulate ABd more po- tently than PPBF, we synthesised the m-cresol benzochroman derivative by treatment of 73 with m-cresol affording MePPBC76 ( ) in 68% yield (Scheme 5.3).
O O a O O PSfrag replacements S 68% S Cl O O O 73 76
◦ Scheme 5.3. a) m-cresol, Et3N, CH2Cl2, 0 C rt.
→ Modification of phenyl substituent: 3-substituted phenols
We sought to extend the structure activity relationship initiated by Van Rhijn and col- leagues by modification of the substituent at the 3-position of the phenyl ring. We treated 72 with a range of different phenols to obtain the corresponding PPBF deriv- atives (Scheme 5.4). Substituent bulkiness was investigated in the 3-iPr (iPrPPBF, 77), 3-tBu (tBuPPBF, 78) and 3-phenyl (PhPPBF, 79) series. Different halides were explored PSfrag replacements through the preparation of the 3-F (FPPBF, 80), 3-Cl (ClPPBF, 81) and 3-Br derivat- ives (BrPPBF, 82). Different electron demand was studied using the donating groups
3-OMe (MeOPPBF, 83) and 3-NMe2 (NMePPBF, 84), and the electron withdrawing
groups 3-NO2 (NOPPBF, 85) and 3-CF3 (CF3PPBF, 86).
R a) 80 R=F 46% f) 79 R=Ph 48% O O b) 81 R=Cl 44% g) 86 R=CF3 44% cond. O O c) 82 R=Br 45% h) 85 R=NO2 49% S S d) 77 R=iPr 40% i) 83 R=OMe 30% Cl O O O e) 78 R=tBu 46% j) 84 R=NMe2 50%
◦ Scheme 5.4. Conditions: Et3N, CH2Cl2, 0 C rt; a) 3-fluorophenol; b) 3-chlorophenol; c) 3-bromophenol; d) 3-isopropylphenol; e) 3-tert-butylphenol; f) 3-hydroxybiphenyl; g) 3-(trifluoromethyl)phenol; h) 3-nitrophenol;→ i) 3-methoxyphenol; j) 3-(N,N-dimethyl)phenol.
Stimulation of Jurkat cells expressing the ABd TCR by first generation PPBF analogues
Access to 70 and 71 as well as Jurkat cells expressing the ABd TCR enabled confirmation of the reported CD1d-restricted activity of these compounds (Figure 5.5a). The level of
aCatarina Almeida from the Godfrey laboratory performed all PPBF analogue assays.
166 CD69 expression on the Jurkat cell surface was measured by flow cytometry as a meas- ure of stimulation. Stimulation with MePPBF (71) resulted in greater stimulation than PPBF (70). We assessed the benzochroman derivative 76 as well as the non-sulfonated parent benzofuran (PBF, 75) and benzochroman (PBC, 74) for their ability to stimulate the ABd cell line. We observed no stimulation with the non-sulfonated compounds; however, 76 afforded stimulation greater than that afforded by its 5-membered benzo- furan analogue 71.
3000
2500
2000
1500
1000
intensity intensity 500
0
mean fluorescence mean fluorescence
PBF
PBC
PPBF
FPPBF
ClPPBF
BrPPBF
PhPPBF
iPrPPBF
αGalCer
NOPPBF
MePPBF
DClPPBF
tBuPPBF
MePPBC
NMePPBF
MeOPPBF
neg. control neg.
Size Halogenaon EDG EWG
Figure 5.5. Jurkat cells expressing the ABd TCR were stimulated with first generation ligands bound to CD1d tetramers; level of stimulation was determined by CD69 expression measured by flow cytometry.
Investigation of the 3-substituted phenyl derivatives revealed that bulky substitu- ents are not well tolerated with complete ablation of stimulation observed by substitu- ents larger than methyl. The halide substituted derivatives all possessed potency equal to or greater than that of 71, with ClPPBF (81) being the most potent. Of the derivatives probing the effect of electron demand on the phenyl ring 3-NO2 (85) and 3-OMe (83) possessed similar activity to 71; 3-CF3 (86) possessed similar potency to PPBF while
3-NMe2 (84) was inactive. As OMe is strongly donating and NO2 is strongly withdraw- ing this suggests that size, rather than electron demand is more important for potency.
The low activity of 3-NMe2 (84) may relate to the large size of this group which is similar to iPr.
167 5.2.2 Second generation PPBF analogues
The successful identification of a highly active set of active PPBF derivatives led usto investigate further structural modifications, with an aim to further enhance potency of ABd stimulation.
Modification of phenyl substituent: di-substituted phenols
Van Rhijn et al. noted that 2-methyl PPBF possessed a similar potency to PPBF.127 Our confirmation of the high potency of 3-methyl PPBF led us to investigated whether methyl groups at both the 2- and 3-positions may be beneficial for ABd TCR stimula- tion. Based on the positive result observed for ClPPBF we also investigated whether a 3,5-di-chloro derivative would have similar or greater efficacy than 81, the most active compound discovered thus far. Both the dimethyl (DMEPPBF, 87) and di- chloro (DClPPBF, 88) derivatives were prepared by reaction of 2,3-di-methylphenol or 3,5-di-chlorophenol with 72 to afford 87 and 88 in 95% and 42% yields, respectively (Scheme 5.5).
R2 O 1 O PSfrag replacements R a) 87 R1/R2 = Me R3 = H 95% O cond. O b) 88 R2/R3 = Cl R1 = H 42% S S Cl R3 O O O
◦ Scheme 5.5. Conditions: Et3N, CH2Cl2, 0 C rt; a) 2,3-di-methylphenol; b) 3,5-di-chlorophenol. → It should be noted that while most of the PBF derived analogues were synthesised using commercially sourced 72, 87 was produced in 95% yield when freshly prepared 72 was used, compared to only 44% when prepared with commercial 72. This supported the notion that 72 is not particularly shelf stable and fresh preparation is recommended for high yielding reactions.
Modification of linker: sulfonamide and sulfone derivatives
Our last synthetic investigation focused on the linkage between the two aryl groups. We proposed two modifications: replacement of the S-O bond with an S-N bond to form
168 the sulfonamide and replacement of the S-O bond with an S-C bond to form a sulfone. Since sulfonamides are present in many drugs and since the approach utilized com- mercially available amines, our synthetic effort initially focused on preparing sulfonam- ide derivatives of PPBF.In an analogous way to the sulfonates, we treated 72 with aniline to afford the phenyl sulfonamide (APBF, 89) in 46% yield (Scheme 5.6). Preparation of the N-methyl (MAPBF, 90) and a 3-methyl (mAPBF, 91) derivatives was achieved in the same fashion in 61% and 50% yields, respectively.
R2 PSfrag replacements O O a) 89 R1 = H R2 = H 46% cond. O O b) 90 R1 = Me R2 = H 61% S S 1 2 Cl N c) 91 R = H R = Me 50% O O R1 ◦ Scheme 5.6. Conditions: Et3N, CH2Cl2, 0 C rt; a) aniline; b) N-methylaniline; c) m-toluidine. → We envisioned we could prepare a derivative bearing a sulfone linkage by con- version of the sulfonyl chloride to the sulfinate followed by nucleophilic substitution of benzyl bromide. A well established procedure for the preparation of sulfinate salts from the corresponding sulfonyl chloride involves reduction with sodium sulfite in the presence of base. The preparation of benzyl tosylate via the sodium p-toluenesulfinate is often employed in teaching laboratories due to the highly crystalline nature ofthe product; and indeed, upon reaction of sodium p-toluenesulfinate with benzyl bromide the product crystallized in high purity and excellent yield directly from the reaction mixture (Scheme 5.7). We attempted to prepare the sulfone derivative of PPBF in the same way, however we found that reaction of 72 with sodium sulfite, followed by benzyl bromide led predominantly to the formation of the sulfonic acid and we were unable to isolate any sulfone.
169 b O a O O S S- S Cl + 88% over Na O O two steps O
O O a,b O O S S Cl HO PSfrag replacements O O 72
Scheme 5.7. a) Na2SO3, NaHCO3,H2O; b) benzyl bromide.
Stimulation of Jurkat cells expressing the ABd TCR by second generation PPBF analogues
We assessed the ability of the sulfonamide derivatives 89, 90 and 91 to stimulate an ABd TCR+ cell line and observed no increase in CD69 expression above endo (Figure 5.6b). We note that the failure of the sulfonamide PPBF derivatives to stimulate ABd does not preclude the possibility that other sulfonamide structures, such as sulfa drugs and NSAIDs, may afford stimulation. Of the di-substituted derivatives, only 3,5-di-chloro (88) was able to stimulate ABd TCR+ cells, however, only weakly. This supports the earlier observation that the binding region for the phenyl ring prefers small derivatives.
3000
2500
2000
1500
1000
500
intensity intensity 0
mean fluorescence mean fluorescence
PPBF
APBF
ClPPBF
mAPBF
MAPBF
MePPBF
DClPPBF
DMePPBF
neg. control neg.
Di-substuted Sulfonamide
Figure 5.6. ABd TCR+ cells were stimulated with ligands bound to CD1d tetramers; level of stimulation was determined by CD69 expression measured by flow cytometry.
bCatarina Almeida from the Godfrey laboratory performed all PPBF analogue assays.
170 5.2.3 Potent activation of ABd NKT cells by 3-halo PPBF derivatives
Our structure activity study revealed that 3-halo derivatives and especially 3-chloro PPBF (ClPPBF) possess improved activity when compared to the most potent com- pound from the original report, MePPBF.127 We also observed that the activity of the ABd TCR+ cell line is tolerant of small modifications of the substitution on the phenyl ring as well as increasing the size of heterocyclic core but not to the modifications we made to the sulfonate linkage. Identification of ClPPBF as a more potent analogue of PPBF and MePPBF will support elucidation of the structure and function of the CD1d- ClPPBF-NKT interaction.
5.2.4 Identification of novel TCR clones that recognise ClPPBF ina
CD1d-restricted fashion
With the development of ClPPBF as a potent CD1d-restricted effector molecule our collaborators sought to discover novel ClPPBF reactive CD1d-restricted T cell popula- tions. Using whole human blood, ClPPBF-loaded CD1d-tetramers were used to identify reactive T cells. Isolation and TCR sequencing of these T cell identified several new TCR clones with different chain usage to the ABd TCR. Of these, two of the clones possess- ing αβ TCRs (Vα26-Vβ13 and Vα14-Vβ19) and two possessing γδ TCRs (Vγ9-Vδ1 and Vγ8-Vδ3) were expressed in Jurkat cells and their reactivity towards both ClPPBF and αGalCer determined (Table 5.1c). The Vα26-Vβ13 clone was highly auto-reactive as well as reactive to both ClPPBF and αGalCer; in contrast, the Vα14-Vβ19 clone, while still auto-reactive, showed no reactivity towards αGalCer and greater reactivity to ClPPBF. Neither of the γδ TCR clones possessed reactivity towards CD1d-presented αGalCer; the Vγ8-Vδ3 clone was highly auto-reactive and the Vγ9-Vδ1 clone only reactive to ClPPBF. These results support the discovery of at least two novel type II TCRs with reactivity towards PPBF-based compounds.
cCatarina Almeida from the Godfrey laboratory performed TCR screen assays.
171 TCR clone Reactivity Auto αGalCer ClPPBF Vα26-Vβ13 High High High Vα14-Vβ19 Moderate None High Vδ1-Vγ9 Low None High Vδ3-Vγ8 High None High
Table 5.1. Four novel TCR clones were identified and sequenced using ClPPBF loaded tetramers. These clones were assessed for their ability to be stimulated by CD1d-tetramers loaded with endogenous lipids (auto), the archetypal type I NKT effector molecule αGalCer or ClPPBF.
5.2.5 Efforts to discover clinically relevant molecules with structural
similarities to PPBF that can activate NKT cells
In Van Rhijn’sinitial report disclosing PPBF they noted the structural similarity between PPBF and drug-like structures.127 There is precedent for small molecules modulating T cell responses with drug-specific T cell populations being identified for a wide range of drugs.376,377 T cells reactive towards sulfonamide drugs presented by HLA have also been identified.378 The antiviral drug abacavir was demonstrated to bind to HLA and modify the repertoire of presented self-peptides resulting in a hypersensitivity reac- tion.379 Our observation of ABd TCR reactivity in a CD1d-restricted fashion of an ex- panded array of structures supported the possibility for discovery of a drug-like inter- action; we envisioned expanding our library to include a small set of clinically available drugs and assessing their ability to stimulate the ABd TCR+ cell line.
Keller and co-workers investigated the ability of drug-like molecules to modulate the function of mucosal-associated invariant T (MAIT) cells, which recognise vitamin B metabolites presented by the MHC-I-like molecule MR1.380 81 drug-like structures were discovered using three different approaches. The first set of compounds were iden- tified using an in silico fragment docking approach to identify compounds with func- tionality that would likely interact with residues in the binding cleft of MR1. The second approach screened drugs for structural similarity with the bound conformation of the
172 known ligand 6-formylpterin. The third approach utilized in silico docking of drugs into the binding cleft of MR1 to predict their ability to bind. Their candidate structures had a range of activities ranging from non-active to strongly stimulatory with most of the compounds falling in the former category.
A crystal structure of PPBF bound to CD1d has yet to be obtained, preventing use of in silico docking techniques for virtual screening of candidate structures. However, we noted that the final step of many of these computational processes involves visual inspection and ‘picking’ by a trained chemist. We reasoned that in silico screening of a library of drugs based on their structural similarity to PPBF could provide a subset of drugs from which selection of candidates by visual inspection would enable us to obtain a library of PPBF-like candidates without requiring a crystal structure.
Using structural similarity and manual selection to develop a library of PPBF-like drug molecules
The structural comparison of two compounds relies on the ability to accurately con- struct and compare fingerprints for each compound. While the graphical representation of chemicals commonly used in organic chemistry is ideal for human brains, it is not computationally efficient. A chemical fingerprint is a computationally efficient repres- entation of a given compound.381 Typically, a fingerprint breaks down the structure into a graph that contains information about connectivity and/or an array of sub-structural features (Figure 5.7). Fingerprints enable comparison and ranking of a reference com- pound against a library by enabling computation of the ‘distance’ or similarity between the reference and each library compound.382 The most common metric used for simil- arity of chemical structures is the Tanimoto coefficient, which compares (1) the number of features or substructures common to both fingerprints to (2) the sum of the features present in each fingerprint but not in both. This affords a number between 0, represent- ing no common features and 1, where all structural features are shared.
Using the databases available at ChEMBL,383 DrugBank384 and PubChem we iden- tified pre-candidate structures using a Tanimoto similarity coefficient of 0.5 compared
173 O
O
O OH
Substructures in fingerprint shared by both compounds A = 1 0 0 1 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 Aspirin fingerprint 2 0.66 Tanimoto = 3 = 0 0 0 0 0 0 0 0 0 1 coefficient
OH 0 0 0 0 1 0 0 1 0 0 O Substructures in fingerprint present in only one compound
B = 1 0 0 1 1 0 0 1 0 0 Ibuprofen fingerprint
Figure 5.7. Simplified example whereby the structure of two drugs, Aspirin and ibuprofen, are mapped onto a 10-bit substructure fingerprint, which is used for determination of their Tanimoto coefficient. to MePPBF. Final identification of candidate structures involved visual inspection and agreement of two chemists. Using this approach, we identified 14 structures across a diverse range of drug classes (Figure 5.8): sulfonamide antibiotics (sulfadimidine, sulfamethoxazole, sulfadiazine and sulfapyridine), NSAIDs (celecoxib, valdecoxib, rofe- coxib and etoricoxib), anti-tumor agents (belinostat), erectile dysfunction (sildenafil), insomnia treatment (ramelteon), β-blockers (tamsulosin) and anti-diabetics (troglit- azone).
H C NH2 3 O H2N H N S N O N N O S O N N O O CF H N 3 Cl O N Sulfadimidine Tamsulosin O H CO S S 3 2 NH H N Etoricoxib 2 2 O S O N O Celecoxib O S H O HN O N N O O O HO S H N O Sulfamethoxazole N O H Troglitazone OH O Belinostat NH2 N O H2N O S S N N O O N H O O N O Valdecoxib O N Sulfadiazine S N N H O O NH2 O N N N O O H S N Sildenafil Ramelteon H O
Sulfapyridine H3CO2S Rofecoxib Figure 5.8. 14 drugs were selected as candidates for ABd reactivity based on their structural similarity to MePPBF.
174 Vitamin E possesses structural similarities to PPBF
While under-taking unrelated work we noted that the heterocyclic core structure of vit- amin E is similar to that of PPBC (Figure 5.9). The term vitamin E describes a group of compounds composed of tocopherols and tocotrienols; these two classes of compounds possess structurally distinct aliphatic tails and conserved heterocyclic cores. Specific- ally, α-tocopherol and α-tocotrienol bear the highest degree of structural similarity to PPBC; they each bear three methyl groups on the phenyl ring with a hydroxyl para to the endocyclic oxygen of the chroman. While there are no reports of immune or allergy re- sponses to vitamin E, its biological function is still not well understood.385,386 We chose to investigate α-tocopherol as it possesses the highest degree of biological activity and the best structural resemblance to PPBC.
3 R HO R4 R2 O O O
HO HO R1 α R1 = Me R2 = Me R3 = Me trolox β R1 = Me R2 = H R3 = Me δ R1 = H R2 = Me R3 = Me γ R1 = H R2 = H R3 = Me
R4 = O tocotrienol O S O R4 = O MePPBC tocopherol
Figure 5.9. The structures of the tocopherols and tocotrienols that make up vitamin E and the water soluble vitamin E analogue trolox.
Due to its low water solubility α-tocopherol is problematic in biological assays per- formed in water. For this reason the water soluble vitamin E analogue trolox was de- veloped. In trolox aliphatic tail of α-tocopherol is replaced with a carboxylic acid. This change increases the structural similarity to PPBC.
175 Neither PPBF-like drugs nor vitamin E possess CD1d-restricted ABd activity
The activity of our PPBF-like drugs was assessed by cell-mediated presentation onCD1d by the THP-1 cell line. Unsurprisingly, α-tocopherol proved too insoluble for the re- quired experiments. Its water soluble analogue trolox, while soluble, did not elicit in- creased CD69 expression (Figure 5.10).
700 600 500 400 300
intensity 200 100 meanfluorescence 0
Figure 5.10. ABd TCR+ cells were stimulated with drugs identified as being MePPBF-like bound to CD1d tetramers; level of stimulation was determined by CD69 expression measured by flow cytometry.
Both troglitazone and belinostat demonstrated elevated CD69 expression, a result that was replicated with the novel PPBF-reactive TCR clones. However, a control ex- periment uncovered that both of these compounds stimulated an increase in CD69 ex- pression when presented to the archetypal type I NKT TCR;d an unexpected result if the drugs were being presented or recognised in a similar manner to the PPBFs. Tiper and Webb have shown that histone deacetylase inhibitors, such as belinostat, cause an upregulation of CD1d expression.387 In addition, several reports detail the in- fluence of the peroxisome proliferator-activated receptor γ (PPARγ) on CD1d expres- sion;388–390 troglitazone’s mode of action involves the activation of PPARγ.391 Additional experiments performed by our collaborators investigating upregulation of the Class I CD1 molecules revealed that belinostat caused upregulation of CD1a, CD1b and CD1d by THP cells while troglitazone caused upregulation of CD1a, CD1b, CD1c and CD1d.d Two patents were discovered covering the use of belinostat392 and troglitazone393 for novel immune stimulation, specifically targeting the upregulation of CD1 molecules.
dData not shown.
176 In combination with the experimental data, these findings suggest that neither of these PPBF-like drugs cause upregulation of CD69 in a manner consistent with CD1d-restricted TCR recognition.
5.3 Summary
The work in this chapter unveils a structure-activity relationship extending that ini- tially reported by Van Rhijn et al.127 that led to the discovery of PPBF and the develop- ment of MePPBF. Further structural modification led us to ClPPBF which represents the most potent derivative yet discovered for the ABd TCR. We also found that the 6-membered PPBC (92) was more potent than the 5-membered PPBF (70) suggesting that the 3-chloro derivative of PPBC would likely be more potent than ClPPBF.
Using ClPPBF-loaded tetramers we discovered new TCR clones with reactivity to- wards the PPBF-class of immune effectors. These novel TCRs were of both the αβ and γδ types.
We pursued efforts to discover clinically relevant drugs capable of stimulating Tcells expressing the ABd TCR, however, were unsuccessful.
5.3.1 Future work
Due to the way that the ABd and our novel TCRs were selected (screening with PPBF and ClPPBF-loaded tetramers, respectively) it would be feasible to employ the same technique to identify novel TCR populations with reactivity towards PPBF-like drugs by loading individual drugs or cocktails thereof into CD1d tetramers.
The molecular mechanism of the CD1d loading of PPBF-class immune effectors, and recognition by TCRs is unknown. Structural studies are underway in Prof. Jamie Rossjohn’s laboratory to obtain structures of CD1d and PPBF-type molecules. In this regard BrPPBf is especially promising due to the presence of the heavy Br atom.
We currently speculate that it is unlikely that PPBF-like molecules are loaded into CD1d in the typical glycolipid binding cleft; however, it is possible that they act allos-
177 terically to alter binding of typically non-antigenic lipid species. In order to probe this,
CD1d-ClPPBF-TCRABd ternary complexes have been generated in the Godfrey labor- atory and purified by size exclusion chromatography. The intact complexes are analysed by nanospray ESI-MS in Prof. Branch Moody’s laboratory to identify the composition of bound endogenous lipids. Preliminary results have identified an enrichment of sphin- gomyelin in these ternary complexes. These studies hope to elucidate the precise role that PPBF-like molecules play in immune stimulation. In addition, synthesis of the likely more potent ClPPBC may afford more stable ternary complexes for analyses.
178 5.4 Experimental
2,2,4,6,7-Pentamethyldihydrobenzofuran (75)
Isobutylaldehyde (12.1 mL, 132 mmol) and H2SO4 (250 µL)
O were added to a stirred solution of 2,3,5-trimethylphenol (15 g, 110 mmol) in toluene (25 mL). The reaction mixture was refluxed for 4 h. The reaction mixture was concentrated and flash chromatography of the residue (EtOAc/pet. spir- its) afforded 75 as a white solid (15.9 g, 76%). mp 43–44 ◦C (lit.394 44–46 ◦C); 1H NMR
(400 MHz, CDCl3) δ 1.48 (6H, s), 2.09 (3H, s), 2.16 (3H, s), 2.21 (3H, s), 2.92 (2H, s),
6.50 (1H, s); HRMS (ESI+) calcd for C13H18O (M + Na)+ 213.1250. Found 213.1261.
2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl chlor- O Cl S ide (72) O O Chlorosulfuric acid (1.09 g, 9.31 mmol) was added dropwise to
◦ a solution of 75 (0.5 g, 2.63 mmol) in CH2Cl2 (5 mL) at 0 C. The reaction was stirred at rt for 1.5 h. The mixture was quenched with ice, then the organic phase was separated and washed successively with 5% Na2CO3 solution, saturated NaHCO3, and water, then dried (MgSO4) and concentrated. The crude was recrystallized (pet. spirits) to afford 72 as a grey solid (0.418 g, 55%) which was used without further purification.
2,2,5,7,8-Pentamethylbenzochromane (74) 2-Methyl-3-buten-2-ol (348 mg, 4.04 mmol) was added drop-
O wise to a solution of 2,3,5-trimethylphenol (500 mg, 3.67 mmol) in TFA (2 mL). The solution was stirred at rt for 2 h then concentrated. The residue dis- solved in Et2O and washed with saturated NaHCO3, water and brine, dried (MGSO4) and concentrated. Flash chromatography of the residue (CH2Cl2/pet. spirits) afforded
1 74 as a yellow oil (123 g, 16%). H NMR (400 MHz, CDCl3) δ 1.45 (6 H, s), 1.88 – 1.97 (2 H, m), 2.23 (3 H, s), 2.31 (3 H, s), 2.35 (3 H, s), 2.68 – 2.80 (2 H, m), 6.69 (1 H, s); 13C
179 NMR (100 MHz, CDCl3) δ 11.45, 18.93, 19.87, 20.55, 26.99, 32.85, 73.09, 116.66, 122.05,
122.36, 133.41, 134.69, 151.76; HRMS (ESI+) calcd for C14H20O (M + Na)+ 227.1406. Found 227.1405.
2,2,5,7,8-Pentamethylbenzochromane-6-sulfonyl chloride (73) O Cl Chlorosulfuric acid (150 µL, 2.08 mmol) was added dropwise to S O ◦ a solution of 74 (120 mg, 0.587 mmol) in CH2Cl2 (2 mL) at 0 C. O The reaction was stirred at rt for 1.5 h. The mixture was quenched with ice, then the or- ganic phase was separated and washed with 5% Na2CO3 solution, saturated NaHCO3, and water, then dried (MgSO4) and concentrated to afford 73 as a yellow solid (50 mg, 28%) which was used without further purification.
General procedure for the synthesis of benzofuran and benzochroman sulfonates and sulfonamides: An appropriate phenol or aniline (0.58 mmol) was added to a solution of sulfonyl chlor-
◦ ide (72 or 73, 0.69 mmol) and Et3N (100 µL) in CH2Cl2 (5 mL) at 0 C. The reaction was stirred for 24 h and then concentrated. The residue was purified by flash chromato- graphy (ethyl ether/pet. sprits) to afford the sulfonate or sulfamide.
Phenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfon- O O S ate (70) O O Phenol (55 mg, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 70 (105 mg, 55%). 1H NMR
(400 MHz, CDCl3) δ 1.48 (6 H, s), 2.13 (3 H, s), 2.33 (3 H, s), 2.56 (3 H, s), 2.95 (2 H, s), 7.00 – 7.06 (2 H, m), 7.19 – 7.25 (1 H, m), 7.28 (2 H, t, J 7.8 Hz); 13C NMR (100 MHz,
CDCl3) δ 12.6, 18.0, 19.2, 28.6, 43.0, 87.4, 118.3, 122.5, 125.3, 126.8, 129.6, 135.2, 141.0,
149.8, 160.8; HRMS (ESI+) calcd for C19H22O4S (M + Na)+ 369.1131. Found 369.1139.
180 O O 3-Methylphenyl 2,2,4,6,7-pentamethyldihydrobenzofuran- S O O 5-sulfonate (71) m-Cresol (60 ul, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 71 (120 mg, 57%). 1H NMR
(400 MHz, CDCl3) δ 1.49 (6 H, s), 2.14 (3 H, s), 2.30 (3 H, s), 2.35 (3 H, s), 2.57 (3 H, s), 2.96 (2 H, s), 6.75 – 6.81 (1 H, m), 6.89 – 6.93 (1 H, m), 7.00 – 7.06 (1 H, m), 7.14 (1 H,
13 t, J 7.9 Hz); C NMR (100 MHz, CDCl3) δ 12.6, 18.1, 19.3, 21.4, 28.6, 43.1, 87.4, 118.3, 119.1, 123.1, 125.3, 125.4, 127.6, 129.3, 135.2, 139.9, 141.0, 149.7, 160.8; HRMS (ESI+) calcd for C20H24O4S (M + Na)+ 383.1288. Found 383.1277.
3-Fluorophenyl 2,2,4,6,7-pentamethyldihydrobenzofuran- O F O S O 5-sulfonate (80) O 3-Fluorophenol (60 ul, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 80 (124 mg, 46%). 1H
NMR (400 MHz, CDCl3) δ 1.49 (6 H, s), 2.14 (3 H, s), 2.35 (3 H, s), 2.56 (3 H, s), 2.96 (2 H, s), 6.78 (1 H, dt, J 9.4, 2.4 Hz), 6.86 (1 H, dd, J 8.3, 2.2 Hz), 6.95 (1 H, td, J 8.4, 2.5
13 Hz), 7.22 – 7.31 (1 H, m); C NMR (100 MHz, CDCl3) δ 12.6, 18.1, 19.3, 28.6, 43.0, 87.6, 110.4, 110.6, 113.9, 114.1, 118.3, 118.6, 124.8, 125.5, 130.3, 130.4, 135.3, 141.1, 161.1.;
HRMS (ESI+) calcd for C19H21FO4S (M + Na)+ 387.1037. Found 387.1030.
O 3-Chlorophenyl 2,2,4,6,7-pentamethyldihydrobenzofuran- Cl O S O 5-sulfonate (81) O 3-Chlorophenol (82 ul, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 81 (97 mg, 44%). 1H NMR
(400 MHz, CDCl3) δ 1.49 (6 H, s), 2.14 (3 H, s), 2.35 (3 H, s), 2.56 (3 H, s), 2.97 (2 H, s), 6.92 – 7.00 (1 H, m), 7.02 – 7.07 (1 H, m), 7.19 – 7.24 (2 H, m); 13C NMR (100 MHz,
CDCl3) δ 12.5, 17.9, 19.1, 28.5, 42.9, 87.4, 118.4, 120.7, 122.9, 124.6, 125.3, 130.2, 134.7,
135.1, 150.0, 160.9; HRMS (ESI+) calcd for C19H21ClO4S (M + Na)+ 403.0741. Found
181 403.0753.
3-Bromophenyl 2,2,4,6,7-pentamethyldihydrobenzofuran- O Br O S 5-sulfonate (82) O O 3-Bromophenol (61 ul, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 82 (111 mg, 45%). 1H
NMR (400 MHz, CDCl3) δ 1.49 (6 H, s), 2.14 (3 H, s), 2.35 (3 H, s), 2.56 (3 H, s), 2.97 (2 H, s), 6.96 – 7.02 (1 H, m), 7.16 (1 H, t, J 8.1 Hz), 7.19 (1 H, t, J 2.0 Hz), 7.33 – 7.38
13 (1 H, m); C NMR (100 MHz, CDCl3) δ 12.6, 18.0, 19.3, 28.6, 42.9, 87.6, 118.5, 121.2, 122.4, 124.6, 125.5, 125.8, 130.0, 130.6, 135.2, 141.1, 150.1, 161.1; HRMS (ESI+) calcd for C19H21BrO4S (M + Na)+ 447.0236. Found 447.0237.
3-Isopropylphenyl 2,2,4,6,7-pentamethyldihydrobenzofur- O iPr O S an-5-sulfonate (77) O O 3-Isopropylphenol (48 ul, 0.35 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 77 (54 mg, 40%). 1H NMR
(400 MHz, CDCl3) δ 1.12 (6 H, d, J 6.9 Hz), 1.48 (6 H, s), 2.13 (3 H, s), 2.31 (3 H, s), 2.55 (3 H, s), 2.81 (1 H, p, J 6.9 Hz), 2.94 (2 H, s), 6.72 – 6.79 (1 H, m), 6.86 – 6.91 (1 H, m),
13 7.05 – 7.10 (1 H, m), 7.19 (1 H, t, J 7.9 Hz); C NMR (100 MHz, CDCl3) δ 12.6, 18.0, 19.2, 23.8, 28.6, 33.9, 43.1, 87.3, 118.3, 119.9, 120.2, 125.0, 125.22, 125.25, 129.4, 135.3,
141.1, 149.9, 150.7, 160.8; HRMS (ESI+) calcd for C22H28O4S (M + NH4)+ 406.2047. Found 406.2051.
182 O tBu O 3-tert-Butylphenyl 2,2,4,6,7-pentamethyldihydrobenzofur- S O O an-5-sulfonate (78) 3-tert-Butylphenol (48 ul, 0.35 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 78 (65 mg, 46%). 1H NMR
(400 MHz, CDCl3) δ 1.17 (9 H, s), 1.48 (6 H, s), 2.13 (3 H, s), 2.29 (3 H, s), 2.54 (3 H, s), 2.94 (2 H, s), 6.78 – 6.81 (1 H, m), 6.89 – 6.93 (1 H, m), 7.19 – 7.24 (2 H, m); 13C
NMR (100 MHz, CDCl3) δ 12.6, 18.0, 19.2, 28.6, 31.1, 34.8, 43.1, 87.3, 118.3, 119.4, 119.7, 123.6, 125.2, 125.2, 129.1, 135.3, 141.1, 149.8, 153.1, 160.8; HRMS (ESI+) calcd for C23H30O4S (M + Na)+ 425.1757. Found 425.1745.
O [1,3']-Biphenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5- Ph O S O sulfonate (79) O 3-Hydroxybiphenyl (99 mg, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 79 (118 mg, 48%).
1 H NMR (400 MHz, CDCl3) δ 1.50 (6 H, s), 2.17 (3 H, s), 2.38 (3 H, s), 2.60 (3 H, s), 2.97 (2 H, s), 7.06 – 7.09 (1 H, m), 7.14 – 7.16 (1 H, m), 7.32 – 7.49 (7 H, m); 13C NMR
(100 MHz, CDCl3) δ 12.5, 18.0, 19.3, 28.6, 43.0, 87.4, 118.4, 121.0, 121.3, 125.1, 125.4, 125.5, 127.0, 127.9, 128.9, 129.9, 135.3, 139.8, 141.1, 142.8, 150.1, 160.9; HRMS (ESI+) calcd for C25H26O4S (M + Na)+ 445.1444. Found 445.1462.
O 3-Trifluoromethylphenyl 2,2,4,6,7-pentamethyldihydroben- F3C O S O zofuran-5-sulfonate (86) O 3-Trifluoromethylphenol (71 ul, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 86 (106 mg, 44%).
1 H NMR (400 MHz, CDCl3) δ 1.48 (6 H, s), 2.14 (3 H, s), 2.33 (3 H, s), 2.56 (3 H, s), 2.96 (2 H, s), 7.17 – 7.19 (1 H, m), 7.26 – 7.30 (1 H, m), 7.41 – 7.51 (2 H, m); 13C NMR
(100 MHz, CDCl3) δ 12.4, 17.9, 19.1, 28.4, 42.8, 87.5, 118.5, 119.46 (q, J 3.9 Hz), 121.9, 123.42 (q, J 3.8 Hz), 124.3, 124.6, 125.4, 126.00, 126.01, 130.2, 131.92 (q, J 33.1 Hz),
183 135.2, 141.0, 149.7, 161.0; HRMS (ESI+) calcd for C20H21F3O4S (M + Na)+ 437.1005. Found 437.0092.
3-Nitrophenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5- O O2N O S sulfonate (85) O O 3-Nitrophenol (81 mg, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 85 (111 mg, 49%). 1H
NMR (400 MHz, CDCl3) δ 1.49 (7 H, s), 2.14 (3 H, s), 2.36 (3 H, s), 2.56 (3 H, s), 2.97 (2 H, s), 7.43 – 7.48 (1 H, m), 7.51 (1 H, t, J 8.2 Hz), 7.81 (1 H, t, J 2.2 Hz), 8.08 – 8.13
13 (1 H, m); C NMR (100 MHz, CDCl3) δ 12.6, 18.0, 19.3, 28.6, 42.9, 87.8, 117.8, 118.8, 121.6, 124.2, 125.8, 128.9, 130.4, 135.3, 141.2, 148.8, 150.0, 161.4; HRMS (ESI+) calcd for C19H21NO6S (M + Na)+ 414.0981. Found 414.0996.
3-Methoxyphenyl 2,2,4,6,7-pentamethyldihydrobenzofuran- O MeO O S 5-sulfonate (83) O O 3-Methoxyphenol (72 mg, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 83 (66 mg, 30%). 1H NMR
(400 MHz, CDCl3) δ 1.48 (6 H, s), 2.13 (3 H, s), 2.36 (3 H, s), 2.57 (3 H, s), 2.96 (2 H, s), 3.72 (3 H, s), 6.58 – 6.63 (2 H, m), 6.74 – 6.80 (1 H, m), 7.13 – 7.20 (1 H, m); 13C NMR
(100 MHz, CDCl3) δ 12.4, 17.9, 19.1, 28.5, 42.9, 55.4, 87.3, 108.1, 112.7, 114.2, 118.2,
125.2, 129.8, 135.1, 140.9, 150.6, 160.3, 160.7; HRMS (ESI+) calcd for C20H24O5S (M + Na)+ 399.1237. Found 399.1220.
2,3-Di-methylphenyl 2,2,4,6,7-pentamethyldihydrobenzofur- O O S an-5-sulfonate (87) O O 2,3-di-Methylphenol (71 mg, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 87 (206 mg, 95%). 1H
NMR (400 MHz, CDCl3) δ 1.50 (6 H, s), 2.15 (3 H, s), 2.23 (3 H, s), 2.28 (3 H, s), 2.36
184 (3 H, s), 2.57 (3 H, s), 2.99 (2 H, s), 6.59 (1 H, d, J 8.1 Hz), 6.93 (1 H, t, J 7.8 Hz), 7.02 (1 H,
13 d, J 7.4 Hz); C NMR (100 MHz, CDCl3) δ 12.5, 13.1, 17.9, 19.1, 20.2, 28.5, 42.9, 87.2, 118.2, 119.0, 125.2, 125.7, 126.4, 128.0, 130.8, 134.7, 139.0, 140.6, 148.4, 160.6; HRMS
(ESI+) calcd for C21H26O4S (M + Na)+ 397.1444. Found 397.1462.
3,5-Di-chlorophenyl 2,2,4,6,7-pentamethyldihydrobenzofur- Cl O O S O an5-sulfonate (88) Cl O 3,5-di-Chlorophenol (95 mg, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 88 (101 mg, 42%). 1H
NMR (400 MHz, CDCl3) δ 1.78 (15 H, s), 2.43 (3 H, s), 2.66 (3 H, s), 2.85 (3 H, s), 3.27
13 (2 H, s), 7.50 – 7.52 (2 H, m), 7.55 (1 H, s); C NMR (100 MHz, CDCl3) δ 12.6, 18.0, 19.3, 28.5, 42.9, 87.7, 118.7, 121.4, 124.3, 125.6, 127.2, 135.3, 135.3, 141.1, 150.2, 161.3;
HRMS (ESI+) calcd for C19H20Cl2O4S (M + Na)+ 437.0352. Found 437.0368.
N,N-Di-methyl-aminophenyl 2,2,4,6,7-pentamethyldihydro- O N O S O benzofuran-5-sulfonate (84) O N,N-di-Methyl-aminophenol (80 mg, 0.58 mmol) was reacted with 72 (200 mg, 0.69 mmol) according to the general procedure to afford 84 (113 mg,
1 50%). H NMR (400 MHz, CDCl3) δ 1.48 (6 H, s), 2.13 (3 H, s), 2.38 (3 H, s), 2.57 (3 H, s), 2.85 (6 H, s), 2.96 (2 H, s), 6.29 – 6.34 (2 H, m), 6.50 – 6.56 (1 H, m), 7.05 – 7.11 (1 H,
13 m); C NMR (100 MHz, CDCl3) δ 12.6, 18.0, 19.3, 28.6, 40.4, 43.0, 87.3, 106.1, 109.5, 110.6, 118.2, 125.2, 125.8, 129.7, 135.1, 140.9, 151.0, 151.6, 160.6; HRMS (ESI+) calcd for C21H27NO4S (M + H)+ 390.1734. Found 390.1720.
O 3-Methylphenyl 2,2,5,7,8-pentamethylbenzochromane-6- O S O sulfonate (76) O m-Cresol (13 mg, 0.121 mmol) was reacted with 73 (50 mg, 0.181 mmol) according to the general procedure to afford 76 (31 mg, 68%). 1H NMR
185 (400 MHz, CDCl3) δ 1.34 (6 H, s), 1.84 (2 H, t, J 6.9 Hz), 2.14 (3 H, s), 2.30 (3 H, s), 2.47 (3 H, s), 2.53 (3 H, s), 2.65 (2 H, t, J 6.8 Hz), 6.78 – 6.81 (1 H, m), 6.91 – 6.93 (1 H, m), 7.02
13 (1 H, d, J 7.6 Hz), 7.14 (1 H, t, J 7.9 Hz); C NMR (100 MHz, CDCl3) δ 12.2, 17.3, 18.4, 21.3, 21.4, 26.7, 32.5, 74.3, 118.4, 119.0, 123.0, 124.8, 125.9, 127.4, 129.1, 137.5, 137.6,
139.8, 149.5, 155.6; HRMS (ESI+) calcd for C20H24O4S (M + Na)+ 383.1288. Found 383.1302.
Phenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5- H O N S sulfamide (89) O O Freshly distilled aniline (76 ul, 0.83 mmol) was reacted with 73 (200 mg, 0.69 mmol) according to the general procedure to afford 89 (110 mg, 46%).
1 H NMR (400 MHz, CDCl3) δ 1.46 (6 H, s), 2.09 (3 H, s), 2.43 (3 H, s), 2.56 (3 H, s), 2.94 (2 H, s), 6.62 (1 H, s), 6.93 – 7.00 (2 H, m), 7.04 – 7.10 (1 H, m), 7.19 – 7.25 (2 H,
13 m); C NMR (100 MHz, CDCl3) δ 12.7, 17.9, 19.6, 28.7, 43.2, 87.1, 118.2, 121.2, 124.9,
125.4, 129.4, 134.5, 137.1, 139.6, 160.1; HRMS (ESI+) calcd for C19H23NO3S (M + H)+ 346.1471. Found 346.1466.
3-Methylphenyl 2,2,4,6,7-pentamethyldihydrobenzofur- H O N S an-5-sulfamide (91) O O Freshly distilled m-toluidine (62 ul, 0.58 mmol) was reacted with 73 (200 mg, 0.69 mmol) according to the general procedure to afford 91 (104 mg,
1 50%). H NMR (400 MHz, CDCl3) δ 1.46 (6 H, s), 2.09 (3 H, s), 2.25 (3 H, s), 2.44 (3 H, s), 2.56 (3 H, s), 2.95 (2 H, s), 6.42 (1 H, s), 6.69 – 6.77 (1 H, m), 6.76 – 6.81 (1 H, m),
13 6.84 – 6.92 (1 H, m), 7.09 (1 H, t, J 7.8 Hz); C NMR (100 MHz, CDCl3) δ 12.5, 17.7, 19.4, 21.3, 28.5, 43.1, 86.9, 117.8, 121.6, 124.8, 125.6, 129.0, 135.1, 137.6, 139.4, 159.9.;
HRMS (ESI+) calcd for C20H25NO3S (M + H)+ 360.1628. Found 360.1609.
186 O Phenyl N-methyl-2,2,4,6,7-pentamethyldihydrobenzofuran- N S O 5-sulfamide (90) O Freshly distilled N-methylaniline (89 ul, 0.83 mmol) was re- acted with 73 (200 mg, 0.69 mmol) according to the general procedure to afford 90 (152
1 mg, 61%). H NMR (400 MHz, CDCl3) δ 1.45 (6 H, s), 2.08 (3 H, s), 2.27 (3 H, s), 2.42
13 (3 H, s), 2.91 (2 H, s), 3.20 (3 H, s), 7.19 – 7.29 (5 H, m); C NMR (100 MHz, CDCl3) δ 12.6, 17.8, 19.3, 28.6, 37.7, 43.1, 86.9, 118.0, 125.1, 126.6, 127.2, 127.41, 129.01, 135.5,
140.9, 142.1, 160.0; HRMS (ESI+) calcd for C20H25NO3S (M + Na)+ 382.1447. Found 382.1461.
187 Bibliography
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Minerva Access is the Institutional Repository of The University of Melbourne
Author/s: Smith, Dylan Glendon Martin
Title: A journey of synthetic chemistry towards immunogenic glycolipids and non-lipidic antigens
Date: 2018
Persistent Link: http://hdl.handle.net/11343/212092
File Description: A journey of synthetic chemistry towards immunogenic glycolipids and non-lipidic antigens
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