The Synthesis of Branched Trehalose Glycolipids and their Mincle

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The Synthesis of Branched Trehalose Glycolipids and their Mincle Agonist Activity Jessie H. Bird, Ashna A Khan, Naoya Nishimura, Sho Yamasaki, Mattie S. M. Timmer, and Bridget L. Stocker J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b03269 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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The Journal of Organic Chemistry

The Synthesis of Branched Trehalose Glycolipids and their Mincle Agonist Activity

Jessie H. Bird,1 Ashna A. Khan,1 Naoya Nishimura,2,3 Sho Yamasaki,2,3 Mattie S. M. Timmer,1* Bridget L. Stocker1*

1

School of Chemical and Physical Sciences, Victoria University of Wellington, PO

Box 600, Wellington, New Zealand 2

Division of Molecular Immunology, Medical Institute of Bioregulation, Kyushu

University, Fukuoka 812-8582, Japan 3

Department of Molecular Immunology, Research Institute for Microbial Diseases,

Osaka University, Suita, 565-0871, Japan

Email: [email protected]; [email protected]

Abstract The macrophage inducible C-type lectin (Mincle) is a pattern recognition receptor that recognises trehalose dimycolate (TDM), and trehalose dibehenate (TDB) and related trehalose diesters, and thus represents a promising target for the development of vaccine adjuvants based on the trehalose glycolipid scaffold. To this end, we report on the synthesis of a series of long-chain α-branched, β-modified trehalose monoesters and diesters to explore how glycolipid structure affects signalling through Mincle. Key steps in our synthetic strategy include a Fráter-Seebach α-alkylation to install the

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C20 aliphatic lipid on a malic acid derivative, and the formation of a β,γ-epoxide as an intermediate from which modifications to the β-position of the lipid can be made. Biological evaluation of the derivatives using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cell lines expressing mMincle or hMincle revealed that the hMincle agonist activity of all diesters was superior to that of the current lead trehalose glycolipid adjuvant TDB, while the activity of several monoesters was similar to that of their diester counterparts for mMincle, but all showed reduced hMincle agonist activity. Taken together, diesters 2d-g are thus potent Mincle agonists and promising vaccine adjuvants.

Graphical Abstract


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Introduction Trehalose glycolipids (Figure 1) are a diverse family of long-chain fatty acid monoesters (1) and diesters (2) of trehalose (α,α′-1,1′-di-D-glucose).1 Representative members include the more complex trehalose dimycolates (TDMs, 2a), which are composed of a functionalised meromycolate branch with a linear side chain at the αposition,1,2 and the simple trehalose glycolipids (TGLs) such as the trehalose diesters (TDEs, e.g. 2b and c) and the trehalose monoesters (TMEs, e.g. 1b and c).1 TDM was originally isolated from the cell wall of M. tuberculosis and has been found to contribute to the survival and virulence of the bacterium.3 However, it is the ability of TDM and TDE to stimulate the innate and early adaptive immune response that has led to interest in the potential of these molecules as adjuvants.4,5,6,7 In particular, the C22 acyl chain derivative, trehalose dibehenate, (TDB, 2b) has shown much promise as a vaccine adjuvant for both tuberculosis (TB)8 and HIV9 when formulated in the CAF01 liposome system.

Figure 1: Representative Trehalose glycolipids (TGLs)



With regard to their mode of action, TDM and representative TDEs and TMEs have been shown to bind to the macrophage-inducible C-type lectin (Mincle),4,5,10,11,12 thereby leading to the activation of the FcRγ-Syk-Card9 dependent pathway and NFκB-mediated gene expression.13,14,15,16 In the case of TDM (2a) and TDB (2b), further studies have demonstrated that the glycolipids favour the development of a Th1/Th17 immune response.14,17 It has also been demonstrated that changes to the glycolipid structure can affect the immune response to the glycolipid, which is important for the development of optimal vaccine adjuvants.18 For example, we demonstrated that trehalose diesters with a linear carbon chain of ≥ C18 were required to activate macrophages in a Mincle-dependent manner,19 while elegant syntheses by the group of Baird20 and subsequent immunological testing21,22 revealed that changing the functional groups along the mycolic acid branch of TDM altered the cytokine response of macrophages to these glycolipids.

In 2013, the crystal structures of human23 and bovine24 Mincle (hMincle and bMincle, respectively) were determined, thereby shedding light on the ability of different ligands to bind this receptor. From these and related studies,25,26 it was determined that Mincle uses a carbohydrate recognition domain (CRD) comprising a Ca2+ ion in an EPN motif (residues 169-171) to bind the 3- and 4-OH groups of one glucose residue in trehalose. One side of the EPN motif contains a binding site for the second glucose residue with an adjacent minor hydrophobic groove, while the other side of the EPN motif contains a major hydrophobic groove capable of binding both linear and branched trehalose glycolipids.


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In an effort to better understand how trehalose glycolipid structure influences Minclebinding and signalling through the receptor, we sought to synthesise several TDEs and TDMs with systematic structural variations to the lipid backbone. Mincle ligand structure activity studies have been the subject of some excellent reviews,18,27 and it has been speculated that branched but less highly functionalised glycolipids may have superior adjuvant activity.28,29 Moreover, it has been suggested that trehalose glycolipids containing longer acyl chains bind more strongly to Mincle23,30 and are better able to induce cytokine production by macrophages.10,19 Accordingly, we sought to prepare a series of long-chain branched trehalose monoesters 1d-g and diesters 2d-g (Figure 1), whose activity could be compared to TDB (2b) using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mMincle or hMincle.31 Moreover, our derivatives would allow us to explore the effect of the β-hydroxyl substituent on macrophage activation. The amino acid residue Thr196 is found at the entrance to the major hydrophobic groove in Mincle and thus has the potential to form hydrogen bonds with the TGL β-hydroxyl or ether groups. We also envisioned that any of the Glu, Asp, Thr or Tyr residues in the vicinity of the Mincle CRD might be able to act as a nucleophile and form a covalent bond with electrophilic traps, and form a covalent bond with Mincle-ligands 1f and 2f, which contain epoxides as potential electrophilic traps, as well as ligands 1g and 2g, containing an α,β-unsaturated esters. To this end, we envisioned that TGLs with hydroxyl (1d and 2d), methoxy (1e and 2e), and epoxy (1f and 2f) substituents at the β-position, or TGLs with α,β-unsaturated esters (1g and 2g) might show enhanced Mincle signaling. Finally, determining the activity of monoester derivatives 1d-g will build on our earlier studies where we demonstrated that TMEs 1b and 1c activate



macrophages in a Mincle-dependent manner.10 The improved solubility of TMEs can make them more attractive synthetic targets.10

Results and Discussion: To synthesise the target trehalose glycolipids we proposed a retrosynthetic strategy whereby TMEs 1d-g and TDEs 2d-g are accessible via the coupling of TMSprotected trehalose 3 to one or two of the mycolic acid analogues 4-7 (Scheme 1).10,19,32 TMS-protected trehalose 3, in turn, is available from α,α´-trehalose (8) in two steps via persilylation followed by selective cleavage of the primary TMS ethers.33,34 The mycolic acid derivatives 4-7 can be obtained via the regioselective ring opening of the common epoxide precursor 9 using H2 with Pd(OH)2/C, followed by TBS protection or O-methylation and subsequent ester hydrolysis to give 4 and 5, respectively. In the case of epoxy derivative 6, only ester hydrolysis of epoxide 9 is required. α,β-Unsaturated acid 7, could be formed through base mediated epoxide opening and ester hydrolysis of intermediate 9, followed by protection of the primary hydroxyl with a TBS group. Epoxide 9 can itself be prepared from allylic iodide 10 and diethyl L-malate (11) via a Fráter-Seebach alkylation,35 subsequent reduction of the α-alkyl-β-hydroxy diester,36 tosylation of the primary alcohol, and base-mediated cyclisation. In previous studies in our laboratory, we demonstrated that the FráterSeebach alkylation gave an unsatisfactory (

The Synthesis of Branched Trehalose Glycolipids and their Mincle

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