Synthetic Î²â1,2-Mannosyloxymannitol Glycolipid ... - ACS Publications

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A synthetic #-1,2-mannosyloxymannitol glycolipid from the fungus Malassezia pachydermatis signals through human Mincle Phillip L. van der Peet, Christian Gunawan, Miyuki Watanabe, Sho Yamasaki, and Spencer J. Williams J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00544 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 3, 2019

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The Journal of Organic Chemistry

A synthetic -1,2-mannosyloxymannitol glycolipid from the fungus Malassezia pachydermatis signals through human Mincle Phillip L. van der Peet,† Christian Gunawan,† Miyuki Watanabe,‡ Sho Yamasaki,‡,¶ Spencer J. Williams*,† †School of Chemistry and Bio21 Institute, University of Melbourne, Parkville, Australia 3010 ‡Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, 565-0871, Japan ¶Department of Molecular Immunology, Immunology Frontier Research Center, Osaka University, Suita, 565-0871, Japan KEYWORDS glycosylation, immunology, natural product, total synthesis.

ABSTRACT: Mincle is a C-type lectin receptor of the innate immune system with the ability to sense pathogens and commensals through lipidic metabolites. While a growing number of bacterial glycolipids have been discovered that can signal through human Mincle, no fungal metabolites are known that can signal through the human form of this receptor. We report the total synthesis of a complex -1,2-mannosyloxymannitol glycolipid from Malassezia pachydermatis 44-2, which was reported to signal through the murine Mincle receptor. Assembly of 44-2 was achieved through a highly convergent route that exploits symmetry elements inherent within this molecule, and delineation of conditions that maintain the delicate Lmannitol triester-triol array. We show that 44-2 is a potent agonist of human Mincle signalling and constitutes the first fungal metabolite identified that can signal through the human Mincle receptor, providing new insights into antifungal immunity.

1. INTRODUCTION The macrophage inducible C-type lectin (Mincle) receptor provides the capacity for cells to sense a wide range of bacterial and fungal pathogens.1-5 Sensing is initiated when insoluble (glyco)lipidic species bind to the extracellular carbohydrate recognition domain6-7 recruiting the adaptor molecule adaptor molecule Fc receptor -chain (FcR) and leading to signal transduction, transcriptional activation and the expression of cytokines and other defence proteins and enzymes of the immune system.8-10 An important role for Mincle as a glycolipid sensor for a range of bacteria has emerged and the specific factors responsible for signalling through Mincle have been identified in a growing number of pathogenic and commensal bacteria.1 For example, acyl trehaloses, glucoses and glycerols have been identified as Mincle signalling agonists from mycobacteria11-14 (and the closely related corynobacteria15); and glycosyl diglycerides have been identified as Mincle signalling agonists from mycobacteria,16 streptococci17-18 and commensal lactobacilli.19 While roles for Mincle in antifungal immunity have been identified in Candida albicans,20 Pneumocystis carinii,21 Fonsecaea monophora22-23 and Malassezia spp.,24 the specific small molecule ligands have been identified only in the case of Malassezia pachydermatis. M. pachydermatis is a lipiddependent fungus that causes skin diseases and otitis in humans and carnivorous pets, and can result in serious infections in susceptible humans.25 M. pachydermatis produces a series of gentiobiosyl diacylglycerides and the

unprecedented -1,2-mannosyloxymannitol glycolipid 442 (Figure 1).24 The initial report by Ishikawa et al. only reported signalling of these compounds through mouse Mincle; a subsequent study of one of the M. pachydermatis gentiobiosides failed to show signalling though human Mincle.16 Indeed this observation extends others identifying significant differences in the specificity of human and rodent Mincle – crystalline cholesterol26 and glycerol monomycolate14 are known to signal only through the former. However, it has not been reported whether 44-2 can signal though human Mincle. O 10

18

O O HO O

10

18 HO

O

HO

-D-Man

O O

L-mannitol O

HO HO

3

4 OH

OH

-D-Man

HO

O

-D-Man

OH HO

HO OH

OH

OH 1

O

-D-Man O O

OH

HO

OH O

OH 18

10 O

Figure 1. Structure of -1,2-mannosyloxymannitol glycolipid 44-2 from Malassezia pachydermatis. The stereochemistry at the 10-positions of the stearoyl chains is unknown and was chosen arbitrarily.

Compound 44-2 was isolated by bioassay guided fractionation of a chloroform/methanol extract of M. pachydermatis using a reporter cell line expressing murine Mincle.24 Structural assignment of 44-2 was supported by mass spectrometry, extensive NMR analysis, and chemical

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detected for the methyl esters of the fatty acids derived from cleavage from the L-mannitol core, showing that the intact structure is required for full activity. A recent study reported Mincle signalling agonists developed from the side-chains of 44-2, but linked to 12-hydroxystearic acid.27

degradation studies as well as chiral hplc. Ultimately, the complete structure of 44-2 could be assigned with the exception of the stereochemistry at the 10-positions of the stearoyl chains. Compound 44-2 signalled through mouse Mincle with a potency similar to that of the archetypal agonist, trehalose dimycolate. Only weak activity was BnO BnO BnO

OBn O

BnO

11

BnO BnO

O

CH3(CH2)7

+

2

Compound 44-2

BnO BnO

OBn O

BnO BnO

O

OH O

10 O

+

CH3(CH2)7

15

O

1

O

(CH2)8CO2H

+

O HO

OP OP

PO PO

OMe

CH3(CH2)7

O

CH3(CH2)7

HO

OH

(CH2)8CO2Me

BnO BnO BnO

O AcO OC(NH)CCl3

(CH2)8CO2H BnO

BnO

Page 2 of 12

OH

HO HO

OH

OH

OMe

5

O

OH

L-mannitol

Scheme 1. Blueprint for the synthesis of compound 44-2.

Mannolipid 44-2 possesses a remarkable, trioltriester architecture comprised of an L-mannitol core adorned with unusual -mannosylated lipids. The mannosyl-1,2--mannoside motif found within one of these is especially rare in nature; aside from within this mannolipid it has been observed in the cell wall of fungi (eg. Candida albicans)28-29 and the soluble intracellular carbohydrate reserve of Leishmania.30-31 In this work we present a total synthesis of 44-2 that takes advantage of the symmetry inherent in this molecule. As the stereochemistry of the 10-hydroxystearic acid group is unknown, we arbitrarily chose to prepare the illustrated isomer. We confirm that the NMR data for synthetic material matches that of the natural material, and that its activity conforms with that reported previously for murine Mincle. Finally, we show that synthetic 44-2 is a potent signalling agonist of human Mincle. 2. RESULTS AND DISCUSSION Our synthetic analysis of 44-2 pivoted on the observation that the -mannosyloxystearate groups at positions 3 and 4 of the L-mannitol chain are identical; and that the -mannosyl-1,2--mannosyloxystearate at position 1 of L-mannitol is the -1,2-mannosylated version of the mannosyllipids at positions 3 and 4. Our synthetic blueprint identified methyl R-10-hydroxystearate 1 as a suitable starting point (Scheme 1). Installation of the -mannosyl groups would be achieved using the 2-O-acyl glucosyl donor 2, which benefits from neighboring group participation to provide exclusively the -anomer, followed by a sequence involving cleavage of the ester protecting group, and 2position epimerization by an oxidation/reduction process via the ulosyl sugar to give a -mannoside. We elected to use this stepwise -glucosylation/inversion approach over

direct -mannosylation owing to the minimization of the number of protecting groups and the high diastereoselectivity in both the glycosylation and reduction steps. Benzylation of the 2-position provides mannoside fragment 3, whereas a second glucosylation, oxidation/reduction epimerization and finally benzylation delivers dimannoside fragment 4. Assembly of the complete glycolipid 44-2 requires the double esterification of the 3and 4-positions of a protected L-mannitol with 2 equiv of the monomannoside acid derived from saponification of 3, deprotection of the mannitol core (removal of 'P'), selective primary esterification of the mannitol core with 1 equiv of the dimannoside acid derived from 4, and finally deprotection of the benzyl ethers. Only one asymmetric synthesis of 10-hydroxystearic acid has been reported, which utilized a three-step route involving a titanium-mediated asymmetric catalytic addition of dioctylzinc to an aldehyde in the presence of a chiral ligand.32 We developed a new approach to access methyl (R)-10-hydroxystearate 1 from methyl undecylenate, involving epoxidation and then Jacobsen's hydrolytic kinetic resolution with (S,S)-Co(II)-salen-OTs, as reported by Shair and co-workers, to obtain the epoxide 5.33 Alkylation of 5 using the organocuprate derived from heptylmagnesium bromide and Li2CuCl4 in THF afforded 1 (Scheme 2). Previous work described the ability of Oacetylmandelic acid esters of methyl 10-hydroxystearate to perturb the chemical shift of the methyl ester group in the 1H NMR spectrum to allow spectral resolution of the two resultant diastereoisomers.32, 34 Instead, we used DCC/pyr/DMAP and the R- and S-Mosher acids to convert the alcohol 1 to the corresponding Mosher esters 6 and 7. Upon screening several solvents, almost baseline resolution of the methyl ester signals of the diastereoisomers in the 1H NMR spectrum could be obtained in CD3OD (Figure S1),


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allowing determination of de >95% for the Mosher diastereoisomers, and thus an ee >95% for 1.

with Dess-Martin periodinane and then stereoselective reduction with sodium borohydride to provide exclusively the D-manno alcohol 10, with the high stereoselectivity most likely a result of the benzyl ether at O3.35 Benzylation using benzyl trichloroacetimidate (BnOTCA) and TfOH36 afforded 3, and the methyl ester was saponified with K2CO3 in MeOH/H2O, furnishing acid 11. To construct the -1,2mannobiosyl fragment, 10 was glucosylated with trichloroacetimidate 2, again under the agency of TfOH, to afford the disaccharide 12. This was subjected to the same sequence of deacetylation (13), and then oxidation/reduction with NaBH4 to provide a 7:10 mixture of the D-gluco alcohol 13 and D-manno alcohol 14. We also examined L-selectride37 and while this afforded the Dmanno isomer 14 exclusively, this reagent led to decomposition of the product providing 14 in a similar yield as obtained using NaBH4, but without the potential for recovery and recycling of the D-gluco isomer. Benzylation of the dimannoside alcohol 14 with BnOTCA/TfOH, as before, gave the -mannobioside 4, and finally saponification (K2CO3 in MeOH/H2O) afforded the dimannoside acid 15.

O (CH2)8CO2Me

5

1

C7H15MgBr, Li2CuCl4 THF, -78 to 0 °C 94%

R-Mosher acid DCC, pyr, DMAP

6

HO CH3(CH2)7

H NMR  3.649 ppm

MeO O (d4-methanol) F3C O R Ph CH3(CH2)7 (CH2)8CO2Me

(CH2)8CO2Me

1

H NMR  3.654 ppm

F3C O (d4-methanol) MeO O S Ph CH3(CH2)7 (CH2)8CO2Me

1 S-Mosher acid DCC, pyr, DMAP

7

Scheme 2. Enantioselective synthesis of methyl R-10hydroxystearate.

Assembly of the monomannoside acid commenced with glucosylation of the alcohol 1 with 2 under the agency of TfOH, to give the -glucoside 8 (Scheme 3). Selective transesterification (NaOMe/MeOH) of the acetyl group afforded the alcohol 9, which was epimerized by oxidation A

BnO O

BnO BnO

HO

+

AcO OC(NH)CCl3

CH3(CH2)7

2

BnO

0.05 eq TfOH (CH2)8CO2Me

O

OR

CH2Cl2, -10 °C 89%

1

O

BnO BnO

CH3(CH2)7

(CH2)8CO2Me 8 R = Ac (-only) 9R=H

NaOMe, MeOH 93% BnO

1. Dess-Martin, CH2Cl2 2. NaBH4, MeOH 93% over 2 steps

BnO

OR O

BnO BnO

CH3(CH2)7

(CH2)8CO2Me

BnOTCA, TfOH Et2O, 91%

B

K2CO3

O

OBn O

BnO BnO

MeOH, H2O 91%

O

CH3(CH2)7

(CH2)8CO2H

11

10 R = H 3 R = Bn BnO

BnO

BnO O

BnO BnO

+

AcO OC(NH)CCl3

BnO BnO

2

BnO

2. NaBH4, MeOH, CH2Cl2 39% over 2 steps

O

CH3(CH2)7 10

BnO BnO

1. Dess-Martin, CH2Cl2

1.1 eq TfOH

OH O

O

BnO BnO

O

OR BnO

CH2Cl2, -10 °C 97% (CH2)8CO2Me

O

BnO BnO

CH3(CH2)7

BnO

O

K2CO3

BnO BnO BnO

O

CH3(CH2)7 BnOTCA, TfOH Et2O, 97%

O

BnO BnO

MeOH, H2O 92% (CH2)8CO2Me

OBn O

(CH2)8CO2Me 12 R = Ac (-only) 13 R = H

NaOMe, MeOH 97% OR O

O

O

BnO BnO BnO

O

CH3(CH2)7 15

14 R = H 4 R = Bn

O

(CH2)8CO2H

Scheme 3. Synthesis of -mannosyloxy-stearic acid fragments A) Mannoside 10 and B) dimannoside 14.

We next turned our attention to a suitable Lmannitol fragment. Initially, we explored the use of 2,5-di-O-benzyl-1,6-di-O-(t-butyldimethylsilyl)-Lmannitol; however, this diol proved poorly reactive and we could not achieve a double acylation at both 3and 4-positions with 11 (or even stearic acid) under a range of conditions.38 Presumably, the congested

nature of the vicinal diol crowded by large adjacent protecting groups precludes formation of a diester on this substrate. We therefore investigated the use of 1,2;5,6-di-O-isopropylidene-L-mannitol 16,39 in which the constrained nature of the acetonide groups were expected to present less hindrance to acylation. After extensive screening, we were able to achieve a high-



yielding double-esterification of 16 by mannoside acid 11 using the Yamaguchi mixed anhydride method promoted by 2,4,6-trichlorobenzoyl chloride, Et3N, and DMAP (Scheme 4). O O HO

11 (3 eq) Cl3H2C5COCl

O O

O O

DMAP, CH2Cl2 96%

OH

BnO

O

O

O BnO

O

BnO

O

O OBn BnO

17

16

O

OBn OBn OBn

O

O O TFA/H2O, CH2Cl2 0 C, 94% O O BnO OH

HO

O

BnO

OBn BnO

OH

HO

O OBn

BnO

18

O

OBn O

OBn

O

Compound 44-2

O

15 (1.2 eq) HBTU, iPr2NEt MeCN, 23%

O

Pd(OH)2/C, AcOH iPrOH, 97%

O O

BnO O

BnO

O

BnO

OH

O O

O

BnO

OBn BnO

O

BnO BnO

OBn OBn

OBn

OH

HO

19

O OBn

BnO O

OBn O

Page 4 of 12

acquisition of 13C NMR data in this solvent. 44-2 was also unstable in methanol but proved more stable over a period of 1 week in the secondary alcohol isopropanol, particularly under acidic conditions; satisfactory 13C NMR, HMBC and HMQC NMR data were obtained in d8-isopropanol + 1% d4acetic acid. Ishikawa et al. noted HMBC correlations between the mannitol H3 and H4 with the corresponding attached fatty acyl carbonyl groups, which were also evident in our spectra. A more detailed comparison of the published spectral data for natural 44-2 with our data for synthetic material can be found in SI Table S2. Compound 44-2 was originally reported to activate reporter cell lines comprised of murine Mincle coupled to a nuclear factor activator of T cells (NFAT) promoter that drives expression of green fluorescent protein. In this assay, the synthetic ligand is coated onto plates upon which the reporter cells are cultured for 18 h, and activated cells quantified by flow cytometry. Synthetic 44-2 agonized signalling through mouse Mincle with high potency, similar to that reported by Ishikawa et al. for natural 44-2 (Figure 2).24 Extending those earlier observations we tested that ability of plate-coated synthetic 44-2 to activate an equivalent reporter cell line expressing human Mincle, thereby showing that this remarkable fungal glycolipid can signal through the human C-type lectin receptor.

OBn

O O

Scheme 4. Assembly and deprotection of compound 44-2.

The final stages required careful optimization owing to the propensity of the ester groups to migrate under acidic and basic conditions. The acetonide groups of 16 could be removed to give 18 with a brief treatment with TFA/H2O, but the acyl groups underwent positional scrambling if the solvent was directly removed under reduced pressure. Scrambling was suppressed when the reaction was quenched with aqueous sodium bicarbonate, followed by extraction into an organic phase. The resulting unstable tetraol 18 was treated with one equivalent of the dimannoside acid 15 and HBTU/iPr2NEt, providing the triester 19 in a modest yield of 23%. The low yield here reflects the challenges in achieving a selective esterification of one hydroxyl within the tetraol array of 18, the complex structures of the two coupling partners, and the small scale upon which this reaction was conducted. Finally, deprotection required considerable optimization owing to the propensity for acyl migration and solvolysis of the esters, and was ultimately achieved using H2, Pd(OH)2/C in iPrOH with 1% AcOH, affording 44-2 in 75% yield. The 1H NMR spectrum of synthetic 44-2 in 20:1 d5pyridine/D2O was broadly consistent with that published for the natural material by Ishikawa et al., in the same solvent.24 In particular the diagnostic chemical shifts and coupling patterns of the mannitol H3/H4 protons at  6.22 (1 H, d, J = 8.3 Hz) and 6.16 (1 H, d, J = 8.0 Hz) ppm, respectively. However, a direct comparison with the published work is complicated as it was acquired at two temperatures, and is incompletely reported at each temperature. We observed that compound 44-2 decomposed overnight in d5-pyridine/D2O, preventing

Figure 2. Synthetic mannolipid 44-2 signals through mouse and human Mincle. NFAT-GFP reporter cells expressing either human Mincle/FcR or mouse Mincle/FcR, as well as those expressing FcR alone were tested for their reactivity to platebound trehalose dimycolate (TDM) and glycolipid 44-2. Quantities denote amount in nmol; assays were performed in triplicate; the mean values and standard deviations are shown. This data is representative of two independent experiments.

This work divulges a convergent and remarkably concise approach for the assembly of the unprecedented architecture of the mannolipid 44-2, taking strategic advantage of symmetry elements embedded within the molecule. The work highlights the need for careful experimentation to overcome challenges in the assembly and handling of the sensitive ester groups situated within this congested molecular framework. Access to synthetic 44-2 allowed confirmation of its ability to signal through murine Mincle and for the first-time identification of its ability to signal through human Mincle. Compound 44-2 constitutes the first example of a fungal metabolite with the ability to signal through this human C-type lectin receptor


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hinting at a role for this glycolipid in antifungal immunity in humans.

3. EXPERIMENTAL SECTION 3.1 General. Pyridine was distilled over KOH before use. Dichloromethane and THF were dried over alumina according to the method of Pangborn et al.40 Reactions were monitored using TLC, performed with silica gel 60 F254. Detection was effected by charring in a mixture of 5% sulfuric acid in methanol, 10% phosphomolybdic acid in EtOH, and/or visualizing with UV light. Flash chromatography was performed according to the method of Still et al.41 using silica gel 60. [α]D values are given in deg 10−1 cm2 g−1. NMR experiments were conducted on 400, 500 or 600 MHz instruments, with chemical shifts referenced relative to residual protiated solvent and are in ppm. 1H−1H COSY spectra were used to confirm proton assignments and HMQC and HMBC spectra used for carbon assignments. Mass spectra were acquired in the ESI-QTOF or ESI-FTMS modes. Atom numbering: A, B, C, D refers to L-mannitol, the 1-O-diglycosylstearoyl chain, the 3-O- and 4-O-glycosyl stearoyl chains, respectively. The first attached sugar on each lipid is denoted with '; the second attached sugar with ''. Methyl (R)-10-hydroxystearate (1). A stirred solution of LiCuCl4 in THF (0.1 M, 40.0 mL, 4.00 mmol) was cooled to 35 °C in an N2 atmosphere and heptylmagnesium bromide42 in THF (100 mL, 1.0 M, 100 mmol) was added slowly by cannula. After 20 min the mixture was slowly cannulated into a flask containing a solution of methyl (S)-9-(oxiran-2yl)nonanoate33 5 (6.06 g, 28.3 mmol) in THF (50 mL) at -78 °C with stirring. The mixture was warmed to 0 °C and stirred for 10 min then 20% aq NH4OAc (250 mL) was added. The mixture was extracted with EtOAc (3 × 100 mL) and the combined organic extracts were washed sequentially with 20% aq NH4OAc, H2O then dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography of the residue gave compound 5 (8.34 g, 94%). The characterization data was in accordance with Behrouzian et al.43 The enantiomeric excess was determined to be >95% by 1H NMR analysis of the Mosher ester 7 in CD3OD. [α]D18 +0.33 ° (c 4.00, CHCl3). Methyl (R)-10-(((R)-3,3,3-trifluoro-2-methoxy-2phenylpropanoyl)oxy)stearate (6). Synthesized from 1 and the R-Mosher acid according to Hoye et al.44 [α]D25 +22.6 ° (c 1.00, CHCl3); 1H NMR (500 MHz, CD3OD) δ 0.90 (3 H, t, J = 7.1 Hz, CH2CH3), 1.07–1.41 (22 H, m), 1.51– 1.72 (6 H, m), 2.32 (2 H, t, J = 7.4 Hz, CH2CO2), 3.55 (3 H, s, OMe), 3.649 (3 H, s, OMe), 5.09 (1 H, p, J = 6.0 Hz, H10), 7.40– 7.47 (3 H, m, Ph), 7.51–7.56 (2 H, m, Ph); 13C{1H} NMR (125 MHz, CD3OD) δ 14.5 (1 C, CH2CH3), 23.8, 25.96, 26.01, 26.3, 30.1, 30.26, 30.33, 30.36, 30.41, 30.6, 33.1, 34.6, 34.8, 34.9 (15 C, CH2), 52.0 (1 C, q, J = 4.3 Hz, CF3COCH3), 56.0 (1 C, OCH3), 78.6 (1 C, C10), 85.7 (1 C, q, J = 27.8 Hz, CCF3), 124.9 (1 C, q, J = 288.7 Hz, CF3), 128.4, 129.4, 130.8, 133.8 (6 C, Ph), 167.5, 176.0 (2 C, C=O); HRMS (ESI-QTOF) m/z: [M+H]+ Calcd for C48H69F3O9 531.3292; Found 531.3295. Partial 1H NMR (400 MHz, d4-methanol)  3.649 (3 H, s, CO2Me) ppm.

Methyl (R)-10-(((S)-3,3,3-trifluoro-2-methoxy-2phenylpropanoyl)oxy)stearate (7). Synthesised as for the diastereoisomer above but using 6 and the S-Mosher acid. The 1H NMR spectral data of 6 and 7 were identical in CDCl3. Partial 1H NMR (400 MHz, d4-methanol)  3.654 (3 H, s, CO2Me) ppm. Methyl 10-(2-O-acetyl-3,4,6-tri-O-benzyl-β-Dglucopyranosyloxy)stearate (8). Triflic acid (10 L, 0.11 mmol) was added to a stirred mixture of 2-O-acetyl-3,4,6tri-O-benzyl-β-D-glucopyranosyl trichloroacetimidate45 2 (1.63 g, 2.56 mmol), methyl (R)-10-hydroxystearate 1 (671 mg, 2.13 mmol) and dry CH2Cl2 (10 mL) at -10 ° C under N2 atmosphere. After 15 min the mixture was poured into sat. aq. NaHCO3 (50 mL) and extracted with CH2Cl2 (3 ×10 mL). The combined organic extracts were dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by flash chromatography (10-30% EtOAc in petroleum spirits) to afford compound 8 as an oil (1.49 g, 89%). [α]D25 +4.6 ° (c 4.00, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.89 (3 H, t, J = 6.8 Hz, H18), 1.17–1.64 (28 H, m, CH2), 1.95 (3 H, s, J = 4.9 Hz, Ac), 2.28 (2 H, t, J = 7.6 Hz, CH2CO2), 3.46 (1 H, m, H5'), 3.49– 3.56 (1 H, m, H10), 3.62–3.76 (7 H, m, H3',4',6a',6b',CO2Me), 4.37 (1 H, d, J = 8.0 Hz, H1'), 4.55–4.69 (4 H, m, 4  CH2Ph), 4.80 (2 H, overlapping d, J = 11.2 Hz, 2  CH2Ph), 4.92–5.04 (1 H, t, J = 8.5 Hz, H2), 7.19–7.24 (2 H, m, Ph), 7.25–7.35 (13 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.2 (C18), 21.0, 22.8, 25.0, 25.1, 25.4, 29.2, 29.38, 29.43, 29.75, 29.83, 30.0, 32.0, 34.2, 34.3, 35.0 (15 C, CH2), 51.5 (CO2Me), 69.2 (C6'), 73.6, 73.7 (2 C, C2',CH2Ph), 75.0, 75.1, 75.3 (3 C, C5',2 × CH2Ph), 78.3 (1 C, C3' or C4'), 80.8 (C10), 83.3 (1C, C3' or C4'), 100.9 (C1'), 127.6, 127.7, 127.8, 127.9, 128.1, 128.4, 128.49, 128.51, 138.1, 138.39, 138.43 (18 C, Ph), 169.4, 174.4 (2 C, C=O); HRMS (ESI-QTOF) m/z: [M+NH4]+ Calcd for C48H72NO9 806.5202; Found 806.5212. Methyl (S)-10-(3,4,6-tri-O-benzyl-β-Dglucopyranosyloxy)stearate (9). The glycoside 8 (2.23 g, 2.83 mmol) was dissolved in MeOH (200 mL) and Na(s) (200 mg) was added with stirring. After 24 h the mixture was neutralized with Amberlite IR-120 resin (H+ form), filtered and concentrated in vacuo. Flash chromatography of the residue (20-30% EtOAc/petroleum spirits) gave the alcohol 9 as an oil (1.96 g, 93%). [α]D24 -8.9 ° (c 1.50, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (3 H, t, J = 6.8 Hz, H18), 1.16–1.64 (28 H, m, CH2), 2.28 (3 H, overlapping br s and t, J = 7.6 Hz, CH2CO2,OH), 3.45–3.47 (1 H, m, H5'), 3.50–3.68 (7 H, m, H2',3',4',10,CO2Me), 3.57–3.74 (2H, m, H6a',6b') 4.28 (1 H, d, J = 7.5 Hz, H1'), 4.53–4.64 (3 H, m, CH2Ph), 4.82 (1 H, d, J = 7.8 Hz, CHPh), 4.84 (1 H, d, J = 7.4 Hz, CHPh), 4.96 (1 H, d, J = 11.3 Hz, CHPh), 7.17–7.41 (15 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.3 (C18), 22.8, 25.1, 25.4, 29.3, 29.4, 29.6, 29.7, 29.9, 30.0, 32.0, 34.2, 34.3, 35.0 (15 C, CH2), 51.6 (CO2Me), 69.3 (C6'), 73.7 (CH2Ph), 75.1, 75.23, 75.24, 75.4 (4 C, 2 × CH2Ph,C2',5'), 77.8 (1 C, C3' or C4'), 80.2 (C10), 84.8 (1 C, C3' or C4'), 102.1 (C1'), 127.71, 127.73, 127.9, 128.0, 128.1, 128.5, 128.55, 128.57, 138.3, 138.5, 138.9 (18 C, Ph), 174.4 (C=O); HRMS (ESI-FTMS) m/z: [M+NH4]+ Calcd for C46H70NO8 764.5096; Found 764.5086. Methyl (S)-10-(3,4,6-tri-O-benzyl-β-Dmannopyranosyloxy)stearate (10). Dess-Martin periodinane (4.53 g, 10.7 mmol) was added to a stirred



mixture of 9 (3.99 g, 5.34 mmol) in dry CH2Cl2 (50 mL) under N2. After 3 h the mixture was poured into 5% aq. Na2S2O3 (200 mL) and the organic layer separated. The organic layer was washed with 5% aq. Na2S2O3 (200 mL), and the combined aqueous layers back-extracted with CH2Cl2 (4 × 50 mL). The combined organic extracts were washed with H2O (100 mL), dried (MgSO4), filtered and concentrated in vacuo. The residue was dissolved in MeOH (100 mL) and NaBH4 (2.02 g, 53.5 mmol) was added slowly with vigorous stirring. After 60 min silica was added and the mixture concentrated under reduced pressure to a freeflowing powder. Flash chromatography (30-50% EtOAc/petroleum spirits) afforded compound 10 as an oil (3.72 g, 93%). [α]D21 -13.7 ° (c 7.38, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.89 (3 H, t, J = 6.8 Hz, H18), 1.19–1.70 (28 H, m, CH2), 2.28 (2 H, t, J = 7.6 Hz, CH2CO2), 2.41 (1H, br s, 2’OH), 3.40 (1 H, ddd, J = 9.6, 5.0, 1.8 Hz, H5'), 3.56 (1 H, dd, J = 9.1, 3.0 Hz, H3'), 3.63–3.79 (6 H, m, H6a',6b',10,CO2Me), 3.86 (1 H, t, J = 9.4 Hz, H4'), 4.06 (1 H, br d, J = 2.1 Hz, H2'), 4.45 (1 H, d, J = 2.0 Hz, H1'), 4.54–4.72 (4 H, m, CH2Ph), 4.79 (1 H, d, J = 10.9 Hz, CH2Ph), 4.90 (1 H, d, J = 10.9 Hz, CH2Ph), 7.17–7.43 (15 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.3 (C18), 22.8, 25.1, 25.3, 25.5, 29.3, 29.4, 29.6, 29.7, 29.9, 30.0, 32.0, 34.0, 34.3, 35.0 (15 C, CH2), 51.6 (CO2Me), 69.0 (C2'), 69.7 (C6'), 71.4 (CH2Ph), 73.7 (CH2Ph), 74.5 (C4'), 75.3 (CH2Ph), 75.6 (C5'), 79.6 (C10), 82.0 (C3'), 98.8 (C1'), 127.6, 127.77, 127.82, 127.9, 128.0, 128.2, 128.4, 128.5, 128.6, 138.1, 138.5, 138.6 (18 C, Ph), 174.4 (C=O); HRMS (ESIQTOF) m/z: [M+Na]+ Calcd for C46H66O8Na 769.4650; Found 769.4644. Methyl (S)-10-(2,3,4,6-tetra-O-benzyl-β-Dmannopyranosyloxy)stearate (3). Triflic acid (18 µL, 0.20 mmol) was added to a stirred mixture of the alcohol 10 (1.00 g, 1.34 mmol), benzyl trichloroacetimidate (380 µL, 2.02 mmol) and dry Et2O (30 mL) under N2 atmosphere. After 16 h additional benzyl trichloroacetimidate (380 µL, 2.02 mmol) was added followed by additional triflic acid (18 µL, 0.20 mmol) and the mixture left to stir for 22 h. The mixture was poured into sat. aq. NaHCO3 (100 mL), the organic layer was separated, and the aq. phase extracted with Et2O (3 × 20 mL). The combined organic extracts were washed with H2O (100 mL), dried (MgSO4), filtered and concentrated in vacuo. Flash chromatography of the residue (10-20% EtOAc/petroleum spirits) afforded compound 3 as an oil (1.02 g, 91%). [α]D21 -33.9° (c 0.50, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.89 (3 H, t, J = 6.6 Hz, H18), 1.21–1.64 (28 H, m, CH2), 2.28 (2 H, t, J = 7.6 Hz, CH2CO2), 3.40–3.46 (1 H, m, H5'), 3.51 (1 H, dd, J = 9.3, 2.9 Hz, H3'), 3.61–3.68 (4 H, m, H10,CO2Me), 3.74–3.80 (2 H, m, H6a',6b'), 3.84–3.90 (2 H, m, H2',4'), 4.42 (1 H, s, H1'), 4.44–4.72 (5 H, m, CH2Ph), 4.86 (1 H, d, J = 12.5 Hz, CH2Ph), 4.91 (1 H, d, J = 10.9 Hz, CH2Ph), 5.00 (1 H, d, J = 12.5 Hz, CH2Ph), 7.14–7.56 (20 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.3 (C18), 22.8, 25.1, 25.3, 25.4, 29.33, 29.5, 29.6, 29.8, 30.00, 30.1, 32.1, 33.8, 34.3, 35.1 (15 C, CH2), 51.6 (CO2Me), 70.0 (C6'), 71.5 (CH2Ph), 73.7, 73.8, 74.4, 75.15, 75.24 (5 C, 3 × CH2Ph,C2',4'), 76.2 (C5'), 79.6 (C10), 82.8 (C3'), 100.8 (C1'), 127.1, 127.4, 127.5, 127.6, 127.68, 127.72, 127.9, 128.15, 128.17, 128.38, 128.45, 128.7, 138.4, 138.6, 138.8, 139.2 (24 C, Ph), 174.4 (C=O); HRMS (ESI-QTOF) m/z: [M+Na]+ Calcd for C53H72O8Na 859.5119; Found 859.5120.

Page 6 of 12

(S)-10-(2,3,4,6-Tetra-O-benzyl-β-Dmannopyranosyloxy)stearic acid (11). A mixture of K2CO3 (248 mg, 1.79 mmol) and the ester 3 (150 mg, 0.179 mmol) in water (0.50 mL) and MeOH (5 mL) was stirred with heating in an oil bath at 65 ° C for 16 h. The mixture was cooled to r.t. and poured into potassium hydrogen phthalate buffer (pH 4, 50 mM, 100 mL). The resultant mixture was extracted with Et2O (3 ×15 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography (20% EtOAc/petroleum spirits with 1% AcOH) to give compound 11 as an oil (134 mg, 91%). [α]D21 -43.1 ° (c 3.37, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.91 (3 H, t, J = 6.8 Hz, H18), 1.14– 1.73 (28 H, m, CH2), 2.31 (2 H, t, J = 7.5 Hz, CH2CO2), 3.45 (1 H, ddd, J = 9.5, 5.3, 2.2 Hz, H5'), 3.53 (1 H, dd, J = 9.4, 3.0 Hz, H3'), 3.66 (1 H, m, H10), 3.75–3.84 (2 H, m, H6a',6b'), 3.85– 3.95 (2 H, m, H2',4'), 4.44 (1 H, s, H1'), 4.45–4.62 (4 H, m, CH2Ph), 4.69 (1 H, d, J = 12.0 Hz, CH2Ph), 4.89 (1 H, d, J = 12.5 Hz, CH2Ph), 4.92 (1 H, d, J = 12.5, CH2Ph), 5.02 (1 H, d, J = 12.5 Hz, CH2Ph), 7.12–7.55 (20 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.3 (C18), 22.8, 24.8, 25.3, 25.4, 29.2, 29.3, 29.4, 29.5, 29.8, 29.9, 30.1, 32.0, 33.8, 34.1, 35.1 (15 C, CH2), 70.0 (C6'), 71.4, 73.7, 73.8 (3 C, 3 × CH2Ph), 74.4, 75.1, 75.2 (CH2Ph,C2',4'), 76.2 (C5'), 79.6 (C10), 82.7 (C3'), 100.8 (C1'), 127.1, 127.4, 127.5, 127.65, 127.68, 127.72, 127.9, 128.15, 128.17, 128.38, 128.45, 128.71, 138.4, 138.6, 138.8, 139.1 (24 C, Ph), 179.8 (C=O); HRMS (ESI-QTOF) m/z: [M+H]+ Calcd for C52H71O8 823.5143; Found 823.5155. Methyl (S)-10-(2-O-(2-O-acetyl-3,4,6-tri-O-benzyl-β-Dglucopyranosyl)-3,4,6-tri-O-benzyl-β-Dmannopyranosyloxy)stearate (12). Triflic acid (50 L) was added to a stirred mixture of the alcohol 10 (390 mg, 0.523 mmol), the trichloroacetimidate45 2 (355 mg, 0.557 mmol) and dry CH2Cl2 (5 mL) at -15 ° C. After stirring for 1 h, Et3N (100 L) was added. Celite (5 g) was added and the mixture was concentrated to a powder. The powder was applied to a silica gel column and purified by flash chromatography (30% EtOAc/petroleum spirits) to give compound 12 as an oil (450 mg, 95%). [α]D21 -27.9 ° (c 1.50, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (3 H, t, J = 6.6 Hz, H18), 1.16–1.61 (28 H, m, CH2), 1.94 (3 H, Ac), 2.24 (2 H, t, J = 7.5 Hz, CH2CO2), 3.36–3.44 (1 H, m, H5'), 3.47 (1 H, dd, J = 9.2, 2.6 Hz, H3'), 3.51–3.79 (12 H, m, H4',6a',6b',3'',4'',5'',6a'',6b'',10,CO2Me), 4.22 (1 H, d, J = 2.0 Hz, H2'), 4.36 (1 H, s, H1'), 4.43–4.60 (7 H, m, CH2Ph), 4.70– 4.95 (5 H, m, CH2Ph), 4.98 (1 H, d, J = 8.1 Hz, H1''), 5.12 (1 H, t, J = 8.7 Hz, H2''), 7.08–7.44 (30 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.2 (C18), 21.2 (COCH3), 22.8, 25.0, 25.1, 25.2, 29.3, 29.4, 29.5, 29.70, 29.73, 29.8, 30.0, 30.1, 32.0, 32.9, 34.1, 34.7 (15 C, CH2), 51.5 (CO2Me), 69.7 (CH2Ph), 69.9, 70.5 (C6',6''), 72.1 (C2), 73.2 (CH2Ph), 73.4 (C2''), 73.6, 74.8, 74.97, 75.0, 75.15, 75.23, 75.6, 78.2, 78.4, 80.4, 83.5 (4 × CH2Ph,C10,3',3'',4',4'',5',5''), 99.2 (C1'), 100.7 (C1''), 127.5, 127.5, 127.6, 127.67, 127.68, 127.74, 127.8, 127.9, 128.1, 128.2, 128.32, 128.33, 128.36, 128.43, 128.5, 138.0, 138.1, 138.4, 138.5, 138.6, 138.7 (36 C, Ph), 169.9, 174.2 (2 C, 2 × C=O); HRMS (ESI-QTOF) m/z: [M+NH4]+ Calcd for C75H10NO14 1238.7138; Found 1238.7165. Methyl (S)-10-(2-O-(3,4,6-tri-O-benzyl-β-Dglucopyranosyl)-3,4,6-tri-O-benzyl-β-Dmannopyranosyloxy)stearate (13). Na(s) (25.0 mg, 1.09


Page 7 of 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mmol) was added to a solution of 12 (17.0 mg, 13.9 µmol) dissolved in dry MeOH (4 mL). The mixture was stirred under N2 for 24 h. The mixture was neutralized with AcOH and evaporated to dryness. The residue was purified by flash chromatography to give the alcohol 13 as an oil (15.9 mg, 97%). [α]D22 -31.3° (c 0.50, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.89 (3 H, t, J = 6.7 Hz, H18), 1.18–1.65 (28 H, m, CH2), 2.24 (2 H, t, J = 7.6 Hz, CH2CO2), 3.34–3.45 (2 H, H5',5''), 3.48–3.78 (12 H, m, H3',6a',6b',2'',3'',4'',6a'',6b'',10,Me), 3.88 (1 H, t, J = 9.5 Hz, H4'), 4.19 (1 H, d, J = 3.0 Hz, H2'), 4.43 (1 H, s, H1'), 4.46–4.61 (6 H, m, CH2Ph), 4.62–4.71 (2 H, m, H1'',CH2Ph), 4.79 (1 H, d, J = 11.2 Hz, CH2Ph), 4.84–4.98 (3 H, m, CH2Ph), 5.08 (1 H, d, J = 11.2 Hz), 7.15–7.45 (30 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.3 (C18), 22.8, 25.1, 25.2, 25.4, 29.3, 29.47, 29.53, 29.67, 29.73, 29.98, 30.01, 32.0, 33.8, 34.2, 34.7 (15 C, CH2), 51.5 (CO2Me), 69.6, 70.0 (2 C, C6',6''), 70.4, 73.6, 73.7 (3 C, 3 × CH2Ph), 74.6, 74.8, 75.2, 75.4, 75.5, 75.6, 75.9, 77.3, 80.0, 80.7, 85.5 (C2',3',4',5',2'',3'',4'',5'',C10,3 × CH2Ph), 99.4 (C1'), 104.5 (C1''), 127.57, 127.60, 127.65, 127.7, 127.8, 127.9, 128.16, 128.20, 128.21, 128.3, 128.38, 128.42, 128.45, 128.5, 138.2, 138.3, 138.4, 138.6, 138.7, 139.2, (36 C, Ph), 174.4 (C=O); HRMS (ESI-QTOF) m/z: [M+Na]+ Calcd for C73H94O13Na 1201.6587; Found 1201.6594. Methyl (S)-10-(2-O-(3,4,6-tri-O-benzyl-β-Dmannopyranosyl)-3,4,6-tri-O-benzyl-β-Dmannopyranosyloxy)stearate (14). Dess-Martin periodinane (507 mg, 1.20 mmol) was added to a stirred mixture of the alcohol 13 (470 mg, 0.398 mmol), NaHCO3 (247 mg, 2.95 mmol) and CH2Cl2 (as received, 10 mL) in a flask open to the atmosphere. The mixture was stirred at r.t. for 3 h, then was poured into a 2:1:1 mixture of H2O/sat. aq. Na2S2O3/sat. aq. NaHCO3 (50 mL) and shaken vigorously. The organic layer was separated, and the aqueous phase extracted with CH2Cl2 (4 × 25 mL). The combined organic extracts were dried (MgSO4), filtered and concentrated under reduced pressure. The residue was dissolved in 1:1 MeOH/CH2Cl2 (20 mL) and NaBH4 (301 mg, 7.96 mmol) was added slowly with stirring. After 1 h the mixture was poured into 50% sat. aq. NaHCO3 and extracted with CH2Cl2 (5 × 20 mL). The combined organic extracts were dried (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to afford compound 14 as an oil (261 mg, 55%) as well as recovered starting material 13 (182 mg, 39%). Data for 14: [α]D26 -43.0 ° (c 1.43, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (1 H, t, J = 6.8 Hz, H18), 1.63–1.10 (28 H, m, CH2), 2.25 (2 H, t, J = 7.6 Hz, CH2CO2), 3.36–3.44, 3.44–3.52 (2 H, 2 × m, H5',5''), 3.53–3.60 (2 H, m, H3',3''`), 3.60–3.69 (4 H, m, H10,OMe), 3.67–3.82 (5 H, m, H4'/4'',6a',6b',6a'',6b''), 3.94 (1 H, t, J = 9.3 Hz, H4'/4''), 4.34 (1 H, d, J = 2.7 Hz, H2''), 4.41 (1 H, d, J = 3.2 Hz, H2'), 4.43 (1 H, s, H1'), 4.45–4.69 (8 H, m, CH2Ph), 4.80–4.99 (5 H, m, H1'',CH2Ph), 7.06–7.56 (30 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.3 (C18), 22.8, 25.1, 25.3, 25.4, 29.3, 29.47, 29.49, 29.69, 29.73, 29.9, 30.0, 32.0, 33.8, 34.2, 35.0 (15 C, CH2), 51.6 (CO2Me), 67.8, 69.8, 70.0, 70.2, 70.9, 71.9, 73.5, 73.7, 74.3, 74.5, 75.2, 75.3, 75.8, 79.6, 80.8, 81.9 (C2',3',4',5',6',2'',3'',4'',5'',6'',10,CH2Ph), 99.7, 100.0 (2 C, C1',1''), 127.6, 127.7, 127.78, 127.80, 127.83, 128.0, 128.23, 128.24, 128.3, 128.39, 128.42, 128.44, 128.45, 128.5, 138.2,

138.3, 138.4, 138.5, 138.57, 138.65 (36 C, Ph), 174.4 (C=O); HRMS (ESI-QTOF) m/z: [M+Na]+ Calcd for C73H94O13 1201.6587; Found 1201.6596. Methyl (S)-10-(2-O-(2,3,4,6-Tetra-O-benzyl-β-Dmannopyranosyl)-3,4,6-tri-O-benzyl-β-Dmannopyranosyloxy)stearate (4). A solution of 1% triflic acid (v/v) in dry Et2O (400 µL, 0.044 mmol) was added to a stirred mixture of the alcohol 14 (260 mg, 0.220 mmol), benzyl trichloroacetimidate (204 µL, 1.10 mmol) and dry Et2O (7 mL) at 0 ° C under N2 atmosphere and then kept at 20 °C for 16 h. The mixture was then poured into sat. aq. NaHCO3 (100 mL), the organic layer separated, and the aq. phase extracted with Et2O (3 × 20 mL). The combined organic extracts were dried (MgSO4), filtered and concentrated in vacuo. Flash chromatography of the residue (10-20% EtOAc/petroleum spirits) afforded compound 4 as an oil (273 mg, 97%). [α]D26 -49.7 ° (c 2.74, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.90 (3 H, t, J = 6.7 Hz, H18), 1.16–1.66 (28 H, m, CH2), 2.26 (2 H, t, J = 7.5 Hz, CH2CO2), 3.44 (1 H, m, H5'), 3.50–3.56 (2 H, m, H3'',5''), 3.66 (1 H, dd, J = 9.1, 2.8 Hz, H3'), 3.64–3.72 (5 H, m, H6a',10,OMe), 3.84 (4 H, m, H4',6b',6a'',6b''), 3.88 (1 H, t, J = 9.5 Hz, H4''), 4.21 (1 H, d, J = 2.9 Hz, H2''), 4.32–4.39 (2 H, m, H2',CH2Ph), 4.41–4.62 (9 H, m, H1',CH2Ph), 4.85–5.14 (6 H, m, H1'',CH2Ph), 7.11–7.40 (31 H, m, Ph), 7.44–7.60 (4 H, m Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.2 (C18), 22.8, 25.1, 25.2, 25.5, 29.3, 29.4, 29.5, 29.71, 29.72, 29.96, 29.99, 32.0, 33.7, 34.2, 35.0 (15 C, CH2), 51.5 (CO2Me), 69.7, 69.9, 70.4, 70.8, 73.2, 73.3, 73.48, 73.50, 74.0, 74.4, 75.0, 75.2, 75.3, 75.6, 75.8, 79.2, 80.5, 82.4 (C2',3',4',5',6',2'',3'',4'',5'',6'',10,7 × CH2Ph), 100.1, 102.4 (2 C, C1',1''), 127.2, 127.4, 127.49, 127.51, 127.56, 127.63, 127.7, 127.8, 128.0, 128.17, 128.25, 128.33, 128.34, 128.4, 129.0, 138.4, 138.5, 138.60, 138.61, 138.8, 139.3 (42 C, Ph), 174.3 (C=O); HRMS (ESI-QTOF) m/z: [M+NH4]+ Calcd for C80H104O13N 1286.7502; Found 1286.7521. 10-(2-O-(2,3,4,6-Tetra-O-benzyl-β-Dmannopyranosyl)-3,4,6-tri-O-benzyl-β-Dmannopyranosyloxy)stearic acid (15). A solution of K2CO3 (134 mg, 0.97 mmol) in H2O (1.00 mL) was added to stirred mixture of the ester 4 (123 mg, 0.0969 mmol) and methanol (20 mL). The resulting mixture was heated at 75 °C for 4 h then 90 °C for 90 min. The mixture was cooled to r.t. and poured into 0.37 M aq. HCl and extracted with Et2O (4 × 20 mL). The combined organic extracts were dried (MgSO4), filtered and concentrated. The residue was purified by flash chromatography (10-30% EtOAc/petroleum spirits with 1% AcOH) to give the acid 15 as an oil (112 mg, 92%). [α]D26 -61.4 ° (c 2.06, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.91 (1 H, t, J = 6.7 Hz, H18), 1.21– 1.64 (28 H, m, CH2), 2.29 (2 H, t, J = 7.5 Hz, CH2CO2), 3.45 (1 H, dd, J = 9.4, 5.0 Hz, H5'), 3.51–3.57 (2 H, m, H3'',5''), 3.62 (1 H, dd, J = 9.2, 3.0 Hz, H3'), 3.66–3.74 (2 H, m, H6a',10), 3.75–3.85 (4 H, m, H4',6b',6a'',6b''), 3.89 (1 H, t, J = 9.5 Hz, H4''), 4.22 (1 H, d, J = 2.9 Hz, H2''), 4.33–4.39 (2 H, m, H2',CH2Ph), 4.42–4.63 (9 H, m, CH2Ph,H1'), 4.86–5.02 (4 H, m, H1'',CH2Ph), 5.08 (1 H, d, J = 11.5 Hz, CH2Ph), 5.12 (1 H, d, J = 12.1 Hz, CH2Ph), 7.15–7.37 (31 H, m, Ph), 7.43–7.61 (4 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.2 (C18), 22.8, 24.8, 25.2, 25.4, 29.2, 29.4, 29.5, 29.66, 29.70, 30.0, 32.0, 33.7, 34.1, 35.0 (15 C, CH2), 69.7, 69.9, 70.4, 70.8, 73.2, 73.3, 73.46, 73.50, 74.0, 74.4, 75.0, 75.2, 75.3, 75.5, 75.8, 79.1,



80.5, 82.4 (18 C, C2',3',4',5',6',2'',3'',4'',5'',6'',10,CH2Ph), 100.0, 102.4 (2 C, C1',1''), 127.2, 127.4, 127.51, 127.52, 127.58, 127.64, 127.7, 127.9, 128.1, 128.2, 128.26, 128.33, 128.35, 128.39, 129.0, 138.3, 138.4, 138.5, 138.6, 138.7, 139.2 (42 C, Ph), 179.4 (C=O); HRMS (ESI-QTOF) m/z: [M+NH4]+ Calcd for C79H102NO13 1273.7346; Found 1273.7361. 1,2;5,6-Di-O-isopropylidene-3,4-di-O-((S)-10-(2,3,4,6tetra-O-benzyl-β-D-mannopyranosyloxy)stearoyl)-Lmannitol (17). 2,4,6-Trichlorobenzoyl chloride (38.0 µL, 0.243 mmol) was added to a stirred mixture of DMAP (14.8 mg, 0.122 mmol), Et3N (67.7 µL, 0.486 mmol), 1,2;5,6-di-Oisopropylidene-L-mannitol39 16 (10.6 mg, 0.0405 mmol), the acid 11 (100 mg, 0.122 mmol) and dry CH2Cl2 under N2. After 22 h the mixture was poured into sat. aq. NaHCO3 (25 mL) the organic layer separated and the aqueous phase extracted with CH2Cl2 (2 × 5 mL). The combined organic extracts were dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (10-50% EtOAc/petroleum spirits with 1% AcOH) to give the diester 17 as a glass (73.0 mg, 96%). [α]D24 -42.5 ° (c 2.00, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.91 (6 H, t, J = 6.7 Hz, (H18)C,D), 1.16–1.75 (68 H, m, CH2,C(CH3)2), 2.33 (4 H, t, J = 7.5 Hz, (H2)C,D), 3.45 (2 H, ddd, J = 9.2, 5.3, 1.8 Hz, (H5')C,D), 3.53 (2 H, dd, J = 9.4, 2.9 Hz, (H3')C,D), 3.67 (2 H, p, J = 5.5 Hz, (H10)C,D), 3.75–3.97 (12 H, m, (H2',4',6a',6b')C,D,(1a,1b,6a,6b)A), 4.15 (2 H, q, J = 6.0 Hz, (H2,5)A), 4.44 (2 H, s, (H1')C,D), 4.46–4.63 (8 H, m, CH2Ph), 4.69 (2 H, d, J = 12.0 Hz, CH2Ph), 4.89 (2 H, d, J = 11.5 Hz, CH2Ph), 4.93 (2 H, d, J = 11.5 Hz, CH2Ph), 5.02 (2 H, d, J = 11.5 Hz, CH2Ph), 5.35 (2 H, d, J = 5.6 Hz, (H3,4)A), 7.15–7.43 (36 H, m, Ph), 7.39–7.60 (4 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.3 (2 C, (C18)C,D), 22.8 (2 C, CH2), 25.0, 25.3, 25.38, 25.43 (8 C, C(CH3),CH2), 26.6 (C(CH3)), 29.35, 29.44, 29.5, 29.67, 29.74, 30.03, 30.04, 32.0, 33.8, 34.3, 35.1 (22 C, CH2), 66.0 (2 C, (C1,6)A), 70.0 (2 C, (C6')C,D), 71.38 (2 C, (C3,4)A), 71.45 (2 C, CH2Ph), 73.7, 73.8, 74.4, 74.6), 75.1, 75.2 (12 C, 6 × CH2Ph,(C2,5)A,(C2',4')C,D), 76.2 (2 C, (C5')C,D), 79.6 (2 C, (C10)C,D), 82.7 (2 C, (C3')C,D), 100.8 (2 C, (C1')C,D), 109.4 (2 C, C(CH3)2), 127.4, 127.5, 127.63, 127.65, 127.7, 127.8, 128.13, 128.14, 128.36, 128.42, 128.43, 138.4, 138.6, 138.8, 139.1 (48 C, Ph), 172.6 (2 C, C=O); HRMS (ESI-QTOF) m/z: [M+Na]+ Calcd for C116H158O20 1894.1239; Found 1894.1235. 3,4-Di-O-((S)-10-(2,3,4,6-tetra-O-benzyl-β-Dmannopyranosyloxy)stearoyl)-L-mannitol (18). TFA/H2O (3:1, 2.00 mL) was added to a vigorously stirred mixture of 17 (94.4 mg, 0.504 mmol) in CH2Cl2 (8 mL) at 0 °C. After 30 min sat. aq. NaHCO3 (25 mL) was added. The organic layer was separated, and the aq. phase extracted with CH2Cl2 (3 × 5 mL). The combined organic extracts were dried (MgSO4), filtered and concentrated under reduced pressure to give the tetraol 18 as a glass (85.4 mg, 94%). [α]D21 -39.0 ° (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.90 (6 H, t, J = 6.7 Hz, (H18)C,D), 1.17–1.71 (56 H, m, CH2), 2.37 (4 H, dt, J = 7.3, 1.2 Hz, (H2)C,D), 3.44 (2 H, ddd, J = 9.3, 5.3, 1.9 Hz, (H5')C,D), 3.47–3.57 (6 H, m, (H3')C,D,(H1a,6a,2,5)A), 3.61–3.72 (4 H, m, (H10)C,D,(H1b,6b)A), 3.75–3.83 (4 H, m, (H6a',6b')C,D), 3.84– 3.91 (4 H, m, (H2',4')C,D), 4.43 (2 H, s, (H1')C,D), 4.44–4.62 (8 H, m, CH2Ph), 4.67 (2 H, d, J 12.0 Hz, CH2Ph), 4.88 (2 H, d, J =

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12.5 Hz, CH2Ph), 4.92 (2 H, d, J = 10.9 Hz, CH2Ph), 5.00 (2 H, d, J = 12.5 Hz, CH2Ph), 5.22 (2 H, d, J = 8.8 Hz, (H3,4)A), 7.19– 7.37 (36 H, m, Ph), 7.44–7.51 (4 H, m, Ph); 13C{1H} NMR (100 MHz, CDCl3) δ 14.27 (2 C, CH3), 22.8, 25.0, 25.3, 25.4, 29.2, 29.37, 29.44, 29.6, 29.7, 29.8, 29.9, 30.0, 32.0, 33.8, 34.3, 35.1 (30 C, CH2), 62.8 (2 C, (C1,6)A), 68.8 (2 C, (C2,5)A), 70.0 (2 C, (C6')C,D), 70.8 (2 C, (C3,4)A), 71.5, 73.6, 73.8, 74.4, 75.1, 75.2 (12 C, (C2',4')C,D,CH2Ph), 76.1 (2 C, (C5')C,D), 79.6 (2 C, (C10)C,D), 82.7 (2 C, (C3')C,D), 100.8 (2 C, (C1')C,D), 127.4, 127.5, 127.66, 127.71, 127.9, 128.2, 128.38, 128.43, 128.5, 138.4, 138.6, 138.7, 139.1 (48 C, Ph), 175.1 (2 C, C=O); HRMS (ESI-QTOF) m/z: [M+Na]+ Calcd for C110H150O20Na 1814.0613; Found 1814.0637. 1-O-((S)-10-(2-O-(2,3,4,6-Tetra-O-benzyl-β-Dmannopyranosyl)-3,4,6-tri-O-benzyl-β-Dmannopyranosyloxy)stearoyl)-3,4-di-O-((S)-10(2,3,4,6-tetra-O-benzyl-β-Dmannopyranosyloxy)stearoyl)-L-mannitol (19). A solution of HBTU (4.6 mg, 12.1 µmol), the acid 15 (12.2 mg, 9.71 µmol), iPr2NEt (4.2 µL, 24 µmol) and dry MeCN (1 mL) was stirred under an N2 atmosphere for 40 min. Then a solution of 18 (14.5 mg, 8.09 µmol) in MeCN (2 mL) was added and the mixture allowed to stir for 22 h. The mixture was diluted with Et2O (20 mL) and washed with aq. HCl (0.1 M, 20 mL). The aqueous layer was extracted with Et2O (4 × 10 mL). The combined organic extracts were dried (MgSO4). filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (2:9:9 Et2O/PhMe/hexanes with 1% AcOH) to afford the triester 19 as a glass (5.7 mg, 23%). [α]D21 -31.9 ° (c 0.0428, CHCl3); 1H NMR (400 MHz, CDCl ) δ 0.87 (9 H, m, (H18)B,C,D), 1.12– 3 1.61 (84 H, m, CH2), 2.25 (2 H, t, J = 7.5 Hz, (H2)B), 2.35 (4 H, t, J = 7.5 Hz, (H2)C,D), 3.35–3.53 (7 H, m, (H5,6a,6b)A, (H5')B, (H3',5')C,D), 3.54–3.59 (2 H, m, (H3'',5'')B), 3.60–3.90 (19 H, m, (H2)A, (H10,3',4',6a',6b',4'',6a'',6b'')B, C,D (H10,2',4',6a',6b') ), 4.16 (3 H, m, (H1a,1b)A,(H2'')B), 4.27– 4.35 (2 H, m. (H2')B,CHPh), 4.36–4.61 (19 H, m, (H1')B,(H1')C,D,16×CHPh), 4.66 (2 H, d, J = 12.1 Hz, CHPh), 4.80–5.11 (12 H, m, (H1'')B,11 × CHPh), 5.16–5.23 (2 H, m, (H3,4)A), 7.35–7.01, 7.44–7.48 (75 H, 2 × m, Ph); 13C{1H} NMR (176 MHz, CDCl3) δ 14.1 (3 C, (C18)B,C,D), 22.64, 22.65, 24.79, 24.83, 25.10, 25.15, 25.21, 25.26, 25.4, 29.09, 29.13, 29.16, 29.24, 29.28, 29.3, 29.4, 29.50, 29.57, 29.58, 29.63, 29.67, 29.79, 29.82, 29.88, 31.84, 31.87, 31.89, 33.57, 33.64, 34.0, 34.1, 34.88, 34.92, 34.94 (45 C, CH2), 62.8, 64.2, 67.2, 68.5, 69.5, 69.7, 69.9, 70.3, 70.4, 70.58, 70.61, 71.3, 73.0, 73.2, 73.33, 73.35, 73.5, 73.7, 73.9, 74.17, 74.19, 74.3, 74.86, 74.96, 75.02, 75.06, 75.1, 75.4, 75.7, 76.0, 79.2, 79.5, 80.4, 82.2, 82.6 (44 C, (C1,2,3,4,5,6)A, B (C10,2',3',4',5',6',2'',3'',4'',5'',6'') , (C10,2',3',4',5',6')C,D, 15×CH2Ph), 100.0, 100.7, 102.2 (4 C, (C1',1'')B,(C1')C,D), 127.1, 127.25, 127.35, 127.42, 127.50, 127.54, 127.56, 127.69, 127.71, 127.91, 127.99, 128.0, 128.1, 128.20, 128.21, 128.25, 128.28, 128.3, 128.9, 138.23, 138.25, 138.3, 138.42, 138.47, 138.6, 138.9, 139.1 (90 C, Ph), 173.8, 174.8, 174.9 (3 C, C=O); HRMS (ESI-QTOF) m/z: [M+Na]+ Calcd for C189H246O32Na 3050.7514; Found 3050.7524. 1-O-((S)-10-(2-O-β-D-Mannopyranosyl-β-Dmannopyranosyloxy)stearoyl)-3,4-di-O-((S)-10-β-Dmannopyranosyloxystearoyl)-L-mannitol, 44-2. A mixture of the triester 19 (6.0 mg, 1.98 µmol), Pd(OH)2/C


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(13.0 mg), AcOH (30 µL) and isopropanol (3.0 mL) was stirred under H2 atmosphere (100 psi) for 4 h. The mixture was sparged with N2, filtered through a 0.22 µm syringe cartridge and concentrated in vacuo to give near pure triester 44-2 as a glass (2.9 mg, 97%). Attempts to further purify by HPLC and silica gel chromatography gave material of reduced purity owing to acyl migration and ester cleavage. [α]D21 -35.4 ° (c 0.0653, 1% AcOH/isopropanol). Selected 1H NMR data for 44-2 in d5-pyridine/D2O 20:1 (400 MHz, 25 °C) δ 0.70–0.78 (9 H, m, (H18)B,C,D), 0.95–1.75 (84H, m, CH2), 2.32 (6 H, m, (H2)B,C,D), 3.75 (4 H, m, (H5',5'')B,(H5')C,D), 3.82–3.91 (3H, m, (H10)B,C,D), 4.00–4.66 (26 H, m, (H1a,1b,2,5,6a,6b)A, (H2',2'',3',3'', B 4',4'',6a',6b',6a'',6b'') , (H2',3',4',6'a,6'b)C,D), 4.85–4.93 (3 H, m, (H1')B,C,D), 5.45 (1 H, s, H1''B), 6.16 (1 H, d, J = 8.0 Hz, H4A), 6.22 (1 H, d, J = 8.3 Hz, H3A). 1H NMR (600 MHz, 1% d -AcOD/CD OD) δ 0.88–0.93 (9 H, 4 3 m, (H18)B,C,D), 1.23–1.67 (84 H, m, CH2), 2.32–2.41 (6 H, m, (H2)B,C,D), 3.14–3.22 (4 H, m, (H5',5'')B,(H5')C,D), 3.38 (1 H, dd, J = 9.5, 3.2 Hz, (H3'')B), 3.41–3.49 (4 H, m, (H6a)A, (H3')B,C,D), 3.50–3.65 (6 H, m, (H5,6b)A,(H4,4')B,(H4')C,D), 3.66–3.78 (7 H, m, (H10,6a',6a'')B, (H10,6a')C,D), 3.82 (2 H, m, (H2')C,D), 3.83–3.89 (5 H, m, (H2)A,(H6b',6b'')B,(H6b')C,D), 4.00 (1 H, d, J = 3.2 Hz, (H2'')B), 4.04–4.13 (3 H, m, (H1a,1b)A,(H2')B), 4.56 (2 H, s, (H1')C,D), 4.65 (1 H, s, (H1')B), 4.81 (1 H, s, (H1'')B), 5.29 (1 H, dd, J = 8.2, 2.4 Hz, (H4)A), 5.36 (1 H, dd, J = 8.5, 2.5 Hz, (H3)A). Compound 44-2 exhibited better stability over a period of one week in 1% d4-AcOD/d8-isopropanol. 13C{1H} NMR (176 MHz, 1% d -AcOD/d -isopropanol, 4 8 referenced to AcOD, 20.31 ppm) δ 14.3 (3 C, (C18)B,C,D), 21.0, 23.1, 24.5, 24.6, 25.2, 25.5, 25.7, 29.6, 29.7, 29.8, 30.2, 30.4, 32.4, 33.9, 34.4, 35.2, 35.4 (45 C, CH2), 61.6, 67.2, 67.7, 67.8, 68.3, 70.4, 71.0, 71.2, 71.7, 72.0, 72.3, 73.6, 74.4, 74.6, 77.0, 77.2, 77.7, 78.1, 79.3 (28 C, (C1,2,3,4,5,6)A, (C10,2',3',4',5',6')B,C,D, (C2'',3'',4'',5'',6'')B, 99.4 ((C1')B), 100.1 ((C1')C,D), 101.1 ((C1'')B), 173.3, 173.6, 174.1 (3 C, C=O, HMBC); HRMS (ESI-QTOF) m/z: [M+Na]+ Calcd for C84H156O32Na 1700.0472; Found 1700.0477. Mincle reporter cell assays. 2B4-NFAT-GFP reporter cells expressing mouse or human Mincle together with FcRγ, as well as an FcRγ control, were prepared as previously described.11, 15 Each glycolipid dissolved in chloroform methanol (2 : 1) at 1 mg ml − 1 was diluted in isopropanol, then added to 96-well plates at 20 μl per well, followed by the evaporation of the solvent as described previously. Reporter cells were stimulated for 16 to 20 h, and activation of NFAT-GFP was monitored by flow cytometry.

■ ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/xxx NMR spectra for new compounds

■ AUTHOR INFORMATION Corresponding Author * [email protected]

ORCID Spencer J. Williams: 0000-0001-6341-4364

Sho Yamasaki: 0000-0002-5184-691 Phillip van der Peet: 0000-0002-9790-2427

Author Contributions PvdP and CG conducted all synthetic chemistry. MW performed immunological assays. SJW, PvdP and SY designed the experiments. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS We thank the Australian Research Council (DP160100597) and the Japan Agency for Medical Research and Development (JP17gm0910010) for funding support.

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Immune System from a Novel Array of Synthetic Mycobacterial Glycans, ACS Chem. Biol. 2017, 2990-3002. (13) Decout, A.; Silva-Gomes, S.; Drocourt, D.; Barbe, S.; Andre, I.; Cueto, F. J.; Lioux, T.; Sancho, D.; Perouzel, E.; Vercellone, A.; Prandi, J.; Gilleron, M.; Tiraby, G.; Nigou, J., Rational Design of Adjuvants Targeting the C-Type Lectin Mincle, Proc. Natl. Acad. Sci. USA 2017, 114, 2675-2680. (14) Hattori, Y.; Morita, D.; Fujiwara, N.; Mori, D.; Nakamura, T.; Harashima, H.; Yamasaki, S.; Sugita, M., Glycerol Monomycolate is a Novel Ligand for the Human, but not Mouse Macrophage Inducible C-Type Lectin, Mincle, J. Biol. Chem. 2014, 289, 15405-15412. (15) van der Peet, P. L.; Gunawan, C.; Torigoe, S.; Yamasaki, S.; Williams, S. J., Corynomycolic Acid-Containing Glycolipids Signal Through the Pattern Recognition Receptor Mincle, Chem. Commun. 2015, 51, 5100-3. (16) Richardson, M. B.; Torigoe, S.; Yamasaki, S.; Williams, S. J., Mycobacterium tuberculosis β-Gentiobiosyl Diacylglycerides Signal Through the Pattern Recognition Receptor Mincle: Total Synthesis and Structure Activity Relationships, Chem. Commun. 2015, 51, 15027-30. (17) Imai, T.; Matsumura, T.; Mayer-Lambertz, S.; Wells, C. A.; Ishikawa, E.; Butcher, S. K.; Barnett, T. C.; Walker, M. J.; Imamura, A.; Ishida, H.; Ikebe, T.; Miyamoto, T.; Ato, M.; Ohga, S.; Lepenies, B.; van Sorge, N. M.; Yamasaki, S., Lipoteichoic Acid Anchor Triggers Mincle to Drive Protective Immunity Against Invasive Group A Streptococcus Infection, Proc. Natl. Acad. Sci. USA 2018, 115, E10662-e10671. (18) Behler-Janbeck, F.; Takano, T.; Maus, R.; Stolper, J.; Jonigk, D.; Tort Tarres, M.; Fuehner, T.; Prasse, A.; Welte, T.; Timmer, M. S.; Stocker, B. L.; Nakanishi, Y.; Miyamoto, T.; Yamasaki, S.; Maus, U. A., C-Type Lectin Mincle Recognizes GlucosylDiacylglycerol of Streptococcus pneumoniae and Plays a Protective Role in Pneumococcal Pneumonia, PLoS Pathog. 2016, 12, e1006038. (19) Shah, S.; Nagata, M.; Yamasaki, S.; Williams, S. J., Total Synthesis of a Cyclopropane-Fatty Acid α-Glucosyl Diglyceride from Lactobacillus plantarum and Identification of its Ability to Signal Through Mincle, Chem. Commun. 2016, 52, 10902-5. (20) Bugarcic, A.; Hitchens, K.; Beckhouse, A. G.; Wells, C. A.; Ashman, R. B.; Blanchard, H., Human and Mouse MacrophageInducible C-Type Lectin (Mincle) Bind Candida albicans, Glycobiology 2008, 18, 679-685. (21) Kottom, T. J.; Hebrink, D. M.; Jenson, P. E.; Nandakumar, V.; Wüthrich, M.; Wang, H.; Klein, B.; Yamasaki, S.; Lepenies, B.; Limper, A. H., The Interaction of Pneumocystis with the C-Type Lectin Receptor Mincle Exerts a Significant Role in Host Defense Against Infection, J. Immunol. 2017, 198, 3515-3525. (22) Wevers, B. A.; Kaptein, T. M.; Zijlstra-Willems, E. M.; Theelen, B.; Boekhout, T.; Geijtenbeek, T. B.; Gringhuis, S. I., Fungal Engagement of the C-Type Lectin Mincle Suppresses Dectin-1-Induced Antifungal Immunity, Cell Host Microbe 2014, 15, 494-505. (23) da Glória Sousa, M.; Reid, D. M.; Schweighoffer, E.; Tybulewicz, V.; Ruland, J.; Langhorne, J.; Yamasaki, S.; Taylor, P. R.; Almeida, S. R.; Brown, G. D., Restoration of Pattern Recognition Receptor Costimulation to Treat Chromoblastomycosis, a Chronic Fungal Infection of the Skin, Cell Host Microbe 2011, 9, 436-43. (24) Ishikawa, T.; Itoh, F.; Yoshida, S.; Saijo, S.; Matsuzawa, T.; Gonoi, T.; Saito, T.; Okawa, Y.; Shibata, N.; Miyamoto, T.; Yamasaki, S., Identification of Distinct Ligands for the C-Type Lectin Receptors Mincle and Dectin-2 in the Pathogenic Fungus Malassezia, Cell Host Microbe 2013, 13, 477-488.

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(25) Grice, E. A.; Dawson, T. L., Jr., Host-Microbe Interactions: Malassezia and Human Skin, Curr. Opin. Microbiol. 2017, 40, 8187. (26) Kiyotake, R.; Oh-Hora, M.; Ishikawa, E.; Miyamoto, T.; Ishibashi, T.; Yamasaki, S., Human Mincle Binds to Cholesterol Crystals and Triggers Innate Immune Responses, J. Biol. Chem. 2015, 290, 25322-25332. (27) Van Huy, L.; Tanaka, C.; Imai, T.; Yamasaki, S.; Miyamoto, T., Synthesis of 12-O-Mono- and Diglycosyl-oxystearates, a New Class of Agonists for the C-type Lectin Receptor Mincle, ACS Med. Chem. Lett. 2019, 10, 44-49. (28) Nitz, M.; Ling, C. C.; Otter, A.; Cutler, J. E.; Bundle, D. R., The Unique Solution Structure and Immunochemistry of the Candida albicans beta-1,2-Mannopyranan Cell Wall Antigens, J. Biol. Chem. 2002, 277, 3440-3446. (29) Crich, D.; Li, H.; Yao, Q.; Wink, D. J.; Sommer, R. D.; Rheingold, A. L., Direct Synthesis of beta-Mannans. A Hexameric [-->3)-beta-D-Man-(1](3) Subunit of the Antigenic Polysaccharides from Leptospira biflexa and the Octameric (1->2)-linked beta-D-Mannan of the Candida albicans Phospholipomannan. X-ray Crystal Structure of a Protected Tetramer, J. Am. Chem. Soc. 2001, 123, 5826-5828. (30) Ralton, J. E.; Naderer, T.; Piraino, H. L.; Bashtannyk, T. A.; Callaghan, J. M.; McConville, M. J., Evidence that Intracellular β1,2-Mannan is a Virulence Factor in Leishmania Parasites, J. Biol. Chem. 2003, 278, 40757-40763. (31) Sernee, F. M.; Ralton, J. E.; Dinev, Z.; Khairallah, G. N.; O'Hair, R. A. J.; Williams, S. J.; McConville, M. J., Biosynthesis of Leishmania Mannan is Primed by a Novel Mannose-Cyclic Phosphate, Proc. Natl. Acad. Sci. USA 2006, 103, 9458-9463. (32) Brunner, A.; Hintermann, L., Configurational Assignment of ‘Cryptochiral’ 10-Hydroxystearic Acid through an Asymmetric Catalytic Synthesis, Helv. Chim. Acta 2016, 99, 928943. (33) Magdziak, D.; Lalic, G.; Lee, H. M.; Fortner, K. C.; Aloise, A. D.; Shair, M. D., Catalytic Enantioselective Thioester Aldol Reactions that are Compatible with Protic Functional Groups, J. Am. Chem. Soc. 2005, 127, 7284-7285. (34) Yang, W.; Dostal, L.; Rosazza, J. P., Stereospecificity of Microbial Hydrations of Oleic Acid to 10-Hydroxystearic acid, Appl. Environ. Microbiol. 1993, 59, 281-4. (35) Lichtenthaler, F. W.; Schneider-Adams, T., 3,4,6-Tri-Obenzyl-alpha-D-arabino-hexopyranos-2-ulosyl Bromide: A Versatile Glycosyl Donor for the Efficient Generation of beta-DMannopyranosidic Linkages, J. Org. Chem. 1994, 59, 67286734. (36) Iversen, T.; Bundle, D. R., Benzyl Trichloroacetimidate, a Versatile Reagent for Acid-Catalyzed Benzylation of HydroxyGroups, J. Chem. Soc. Chem. Commun. 1981, 1240-1241. (37) Nitz, M.; Purse, B. W.; Bundle, D. R., Synthesis of a β1,2Mannopyranosyl Tetrasaccharide Found in the Phosphomannan Antigen of Candida albicans, Org. Lett. 2000, 2, 2939-2942. (38) Using either 11 or stearic acid, the following reagents were explored: DIC/DCC, DMAP; HBTU/HATU/COMU with and without HOAt; DIC/DMAP; Ph3P/CCl3CN, pyr; HBTU or HATU or COMU; and 2,4,6-trichlorobenzoyl chloride, Et3N, DMAP. Typically, monoacylation products were obtained and only traces of diesters were detectable. Attempts using stearoyl chloride and fluoride with pyridine/DMAP were also unsuccessful, and with stronger base (Et3N) yielded only the diketene. (39) Baer, E.; Fischer, H. O. L., Studies on AcetoneGlyceraldehyde. VII. Preparation of l-Glyceraldehyde and l-(-)Acetone Glycerol, J. Am. Chem. Soc. 1939, 61, 761-765.


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(40) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J., Safe and Convenient Procedure for Solvent Purification, Organometallics 1996, 15, 1518-1520. (41) Still, W. C.; Kahn, M.; Mitra, A. M., Rapid Chromatographic Technique for Preparative Separations with Moderate Resolution, J. Org. Chem. 1978, 43, 2923-2925. (42) Holm, T., Thermochemical Bond Dissociation Energies of Carbon–Magnesium Bonds, J. Chem. Soc., Perkin Trans. 2 1981, 464-467. (43) Behrouzian, B.; Savile, C. K.; Dawson, B.; Buist, P. H.; Shanklin, J., Exploring the Hydroxylation−Dehydrogenation Connection: Novel Catalytic Activity of Castor Stearoyl-ACP Δ9 Desaturase, J. Am. Chem. Soc. 2002, 124, 3277-3283.

(44) Hoye, T. R.; Jeffrey, C. S.; Shao, F., Mosher Ester Analysis for the Determination of Absolute Configuration of Stereogenic (Chiral) Carbinol Carbons, Nat. Protoc. 2007, 2, 2451-2458. (45) Marino-Albernas, J. R.; Bittman, R.; Peters, A.; Mayhew, E., Synthesis and Growth Inhibitory Properties of Glycosides of 1O-Hexadecyl-2-O-Methyl-sn-Glycerol, Analogs of the Antitumor Ether Lipid ET-18-OCH3 (Edelfosine), J. Med. Chem. 1996, 39, 3241-3247.



Page 12 of 12

Table of Contents artwork O

Compound 44-2 from Malassezia pachydermatis

O O HO O

HO

O

HO

OH

O O

O

HO

OH HO

HO

O

OH

OH

HO HO

OH

O OH

HO O

OH O

OH

O

OH O


12

Synthetic Î²â1,2-Mannosyloxymannitol Glycolipid ... - ACS Publications

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