design of potent mincle signalling agonists based on an

doi.org/10.26434/chemrxiv.10567097.v1

Design of Potent Mincle Signalling Agonists Based on an Alkyl b-Glucoside TemplateDYLAN SMITH, Yuki Hosono, Masahiro Nagata, Sho Yamasaki, Spencer Williams

Submitted date: 15/01/2020 • Posted date: 17/01/2020Licence: CC BY-NC-ND 4.0Citation information: SMITH, DYLAN; Hosono, Yuki; Nagata, Masahiro; Yamasaki, Sho; Williams, Spencer(2020): Design of Potent Mincle Signalling Agonists Based on an Alkyl b- Glucoside Template. ChemRxiv.Preprint. https://doi.org/10.26434/chemrxiv.10567097.v1

The innate immune receptor Mincle senses lipid-based molecules derived from pathogens, commensals andaltered self. Based on emerging structure-activity relationships we design simple alkyl6-O-acyl-b-D-glucosides that are effective agonists of Mincle and signal with potency on par with theprototypical ligand trehalose dimycolate.

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Received 00th January 20xx,Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x Design of potent Mincle signalling agonists based on an alkyl -glucoside template Dylan G.M. Smith,a Yuki Hosono,b,c Masahiro Nagata,b,c Sho Yamasakib,c and Spencer J. Williams*a

a. School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Vic 3010.

b. Department of Molecular Immunology, Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan.

c. Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan

† Footnotes relating to the title and/or authors should appear here. Electronic Supplementary Information (ESI) available: [details of any supplementaryinformation available should be included here]. See DOI: 10.1039/x0xx00000x

Please do not adjust margins


COMMUNICATION Journal.The innate immune receptor Mincle senses lipid-basedmolecules derived from pathogens, commensals and alteredself. Based on emerging structure-activity relationships wedesign simple alkyl 6-O-acyl--D-glucosides that are effectiveagonists of Mincle and signal with potency on par with theprototypical ligand trehalose dimycolate.

Macrophage inducible C-type lectin (Mincle) receptor is anFcRγ-associated pattern recognition receptor involved ininnate immunity.1 Mincle senses a range of self and foreign(glyco)lipids2 and has become an important target for studysince the discovery that it is responsible for recognition ofmycobacterial cord factor (trehalose dimycolate; TDM)3 andthe synthetic adjuvant trehalose dibehenate (TDB) (Fig.1a).4 Ligand binding to Mincle results in phosphorylation ofthe immunoreceptor tyrosine activation motif of FcRγ5, 6

and activation of NF-B via Card9–Bcl10–Malt1signalosomes.7 Mincle is required for the classicgranulomatous response to TDM3 and the adjuvant activityof TDB.4 TDB was developed as an optimized analogue ofTDM, and both compounds induce strong Th1 and Th17cellular immune responses.2 Mincle possesses anextracellular C-type lectin domain that appears to be theprimary site for ligand binding. X-ray structures of human8

and bovine Mincle (the latter bound to trehalose andtrehalose monobutyrate)9, 10 revealed two monosaccharidebinding subsites, with the primary site involvingcoordination of O3 and O4 to Ca2+, and a shallowhydrophobic groove that can accommodate lipid chains(Fig. 1b).

Fig. 1. a) Examples of potent agonists of Mincle signalling. b) X-raystructure of trehalose binding to the Ca2+ ion in the carbohydratebinding domain of Mincle (PDB 4KZV). c) Summary of structure-activitymodel for Mincle signalling.

A growing appreciation of the features necessary foragonism of Mincle signalling have emerged throughidentification of other naturally-occurring Mincle agonists,and through structure-activity relationship studies(reviewed in Refs2, 11). Broadly, signalling through Mincleincreases as lipid chain length increases for both mono- anddiacyl-trehaloses, with activity maximized for a C22

(behenate) chain,12, 13 or slightly shorter for a series ofalkylated brartemicin analogues (Fig. 1a).14 A single glucoseresidue is sufficient for Mincle signalling: both glucosemonomycolate (GMM)15 and monocorynomycolate(GMCM)16 are potent Mincle signalling agonists. Glucose 6-monobehenate (GMB)16 is a weak Mincle agonist, yetglucose analogues bearing long, branched fatty acids linkedoff the 6-position (such as C14C18)15 are potent agonists.Collectively, these results suggest that at least two lipidchains off a monosaccharide are necessary for optimumactivity. Separately, various - and -glucosyldiacylglycerides17-19 and -glucosyl ceramide20 can signalthrough Mincle, showing that the lipid group can be sited atthe anomeric or C6-positions of glucose. Consistent withthe requirement for at least two lipid chains for activity ofmonosaccharide glycolipids, a lyso -glucosyldiacylglyceride did not signal through Mincle.18 Throughstudies of tetraacylated trehalose glycolipids it wasconcluded that a free 2-hydroxyl group on a glucose residuewas essential for activity.15

A good understanding now exists of structure-activityrelationships for glucolipid signalling through Mincle (Fig.1c). Decout and co-workers have argued that at least threeof four key structural motifs (two sugars, two alkyl chains)must be present for Mincle agonism, and when combinedwith appropriate branching and length, the alkyl chains aremore important than the second sugar unit.15 A separatestudy showed that branching at the -position of a 6-O-acyl-glucose analogue provided only weak Mincleagonism.21 Based on these results Decout and co-workersdeveloped C14C18Glc, which possessed greater activitythan TDB in a Mincle reporter assay.15 However, C14C18Glcis comprised of complex mixture of 8 compounds: epimersat the -branched stereocentre of the lipid, the sugarhemiacetal that exists as both - and -anomers, as well asfuranose and pyranose ring isomers; the contribution ofeach of these forms to activity is unclear.

In this work we report new, simplified agonists ofMincle signalling based on a -glucoside template. Inparticular, we focussed on synthetic targets that can beeasily prepared in just a few steps from commercialmaterials and which exist in a single isomeric form tosimplify characterization, purification and interpretation ofresults. Cognizant of the potency of GMM and analogues aswell as -glucosyl diglycerides, we investigated compoundsthat possess lipophilic functionality at the 1- and 6-positions. Compounds of this general structure fit with thestructure-activity relationships for Mincle signalling by

2 | Journal ., 20xx, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx



Journal. COMMUNICATIONpossessing at least two alkyl chains, a single sugar residueand maintaining the free sugar 2-, 3- and 4-hydroxyl groups.

Alkyl glucosides are widely used in consumer productsincluding cosmetics and soaps and both octyl and lauryl -glucoside are commercially available reagents that couldprovide easy synthetic access to potential Mincle agonists.We avoided the use of protecting groups and insteadperformed direct acylation. Uronium-based peptidecoupling reagents allow primary-selective acylation ofsimple glucosides through the choice of appropriate aminebases.22-24 We chose to install three representative straightchain esters: a short octanoyl (C8), medium palmitoyl (C16)and long behenoyl (C22) group (Scheme 1). Reaction of thecorresponding acids with octyl -glucoside afforded C8GlcC8

3, C16GlcC8 4 and C22GlcC8 5 in 61, 33 and 47% yields,respectively. Similarly, the same acids and lauryl -glucoside afforded C8GlcC12 6, C16GlcC12 7 and C22GlcC12 8 in56, 46 and 47% yields, respectively.

Scheme 1. Synthesis of lauryl and octyl 6-O-acyl--D-glucopyranosides.Reagents and conditions: a) octanoic acid, HBTU, pyr, b) palmitic acid,HBTU, pyr, c) behenic acid, HBTU, pyr, d) HO2CCH[(CH2)15CH3]2, HBTU,pyr.

Racemic 2-tetradecyloctanoic acid was used by Decoutet al.15 for preparation of C14C18Glc, and thus thiscompound is a mixture of isomers epimeric at the -position. We instead utilized commercially-available achiral2-hexadecyloctanoic acid. Reaction of octyl and lauryl -glucosides with this acid and HBTU in pyridine afforded themonoesters C18C16GlcC8 9 and C18C16GlcC12 10 in 15 and 12%yields, respectively. These yields were lower than that ofthe straight chain acids and may reflect reduced reactivityarising from the -branch.

Cholesterol can signal through Mincle,25 and is alipophilic group that we speculated could occupy the lipid

binding pocket. We synthesized 1-cholesteryloxyacetic acid13 by reaction of ethyl diazoacetate26 with cholesterolpromoted by BF3.Et2O in CH2Cl2 to afford 12 in 61% yield(Scheme 2). Saponification with 2 M NaOH in EtOH afforded13. Reaction of octyl and lauryl -glucosides with 13 andHBTU/pyridine afforded the monoesters CholGlcC8 14 andCholGlcC12 15 in 47 and 45% yields, respectively.

Scheme 2. Synthesis of lauryl and octyl 6-O-(cholesteryloxyacetyl)--D-glucopyranosides. Reagents and conditions: a) ethyl diazoacetate,BF3.Et2O, CH2Cl2, b) 2 M NaOH, EtOH, H2O, c) 1 or 2, HBTU, pyr.

The panel of alkyl 6-O-acyl--D-glucopyranosides aswell as the parent octyl and lauryl glucosides wereinvestigated for their ability to induce signalling throughMincle. Assays were performed by culturing reporter cellsexpressing mouse and human Mincle on plate-boundglycolipids, and measurement of green fluorescent proteinby flow cytometry to quantify the degree of Minclesignalling agonism. Octyl and lauryl -D-glucopyranosidesdid not signal through Mincle, which is unsurprising as theyare soluble detergents, and do not fulfil the expectedstructural criteria for Mincle agonists. All of the remainingcompounds signalled to varying degrees through the tworeceptor orthologs.

For the straight-chain 6-O-acyl derivatives, a smallincrease in signalling potency was seen in changing fromoctyl to lauryl, except in the case of C22GlcC8 and C22GlcC12,where the potency was lower in the latter. Also, theintensity of signalling through the mouse and humanorthologs was similar for most analogues, except the C22

derivatives, where signalling through human Mincle wasreduced relative to the mouse ortholog. Similarly, withinthe individual series of octyl or lauryl glycosides, anincrease in 6-O-acyl chain length generally led to anincrease in potency, with the exception of C22GlcC12, wherethe potency was reduced. In all cases, signalling potencywas less than that of TDB. This trend in activity isreminiscent of the ability of trehalose mono-13 anddiesters12 to activate macrophages, wherein the C22

compounds were more potent than the C26 analogues, anda homologous series of acyl glycerols, where potencypeaked at C28.27

This journal is © The Royal Society of Chemistry 20xx Journal. , 20xx, 00, 1-3 | 3



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(5)

neg

nmol0.010.1

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(8)

Fig. 2 Agonism of Mincle signalling by unsubstituted octyl or lauryl -D-glucopyranosides or straight-chain 6-O-acyl variants. NFAT-GFP reporter cellsexpressing either human Mincle/FcRγ or mouse Mincle/FcRγ, as well as those expressing FcRγ alone were tested for their reactivity to plate-boundTDB and analogues 1-8. Assays were performed in duplicate; the mean values and standard errors are shown. neg = isopropanol vehicle control.

We next evaluated the activities of the branched-chain6-O-acyl -D-glucopyranosides and the cholesteryloxyacetylanalogues (Fig. 3). The branched-chain analogues weremore potent than any of the straight-chain derivatives, andagain, little difference was seen between the octyl andlauryl glycosides. As for the C22 analogues, signallingintensity was lower for human Mincle than for mouseMincle. The most potent signalling was seen for thecholesterol analogues, which provided signalling withsimilar intensities and potencies as TDB and comprise themost potent agonists identified in this study.

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(9)

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Fig. 3 Agonism of Mincle signalling by octyl or lauryl 6-O-acyl--D-glucopyranosides bearing a branched lipid chain or acholesteryloxyacetyl group. NFAT-GFP reporter cells expressing eitherhuman Mincle/FcRγ or mouse Mincle/FcRγ, as well as those expressingFcRγ alone were tested for their reactivity to plate-bound TDB andanalogues 9, 10, 14 and 15. Assays were performed in duplicate; themean values and standard errors are shown. neg = isopropanol vehiclecontrol. Results for TDB and neg were obtained in the same experimentas for Fig. 2.

In conclusion this work reports development of simplealkyl glucosides as highly potent Mincle agonists based onemerging structure-activity relationships. Long-chain lipidicesters at O6 provided potent agonists of Mincle signalling,with even greater activity seen for an -branched fatty acylchain. These results are broadly in concordance with thestructure-activity relationship articulated by Decout et al.15

but extend that work by showing that a lipid chain at C1

can contribute to potency and simplify their structures byboth blocking the anomeric position and locking itsstereochemistry. Our results also show that alkyl -glucosides esterified with a cholesteryl group at O6 arepotent Mincle antagonists. Cholesterol, in its crystallineinsoluble form, signals through human, but not rodentMincle.25 Signalling by cholesterol involves interaction withthe cholesterol recognition site (CRAC) of human Mincle,which is absent in rodent Mincle, and alkylation ofcholesterol leads to loss of ability to signal throughMincle.25 As our analogues are alkylated cholesterolderivatives, and signal through both mouse and humanMincle, it seems unlikely that the cholesteryl moiety bindsat the CRAC site of Mincle and is likely simply a surrogatefor a lipid group.

This work builds on related efforts exploring structureactivity relationships of acyl-hexoses (as analogues ofglucose monomycolate),15, 16 glycosyloxy-stearates (asanalogues of the mannosyloxymannitol glycolipid fromMalassezia pachydermatis),28 glycerolipids (as analogues ofglycerol monomycolate),27, 29 mono-13 and diacyltrehaloses,12, 30 and lipidated diaroyltrehaloses (asanalogues of brartemicin).14, 31 The present work is notablefor the simplicity of the resulting agonists and the fact thatunlike acyl-hexoses they exist as single stereoisomers.

We thank the Australian Research Council(DP160100597) and AMED (JP19gm0910010,JP19ak0101070 and JP19fk0108075) for grant support.

Conflicts of interestThere are no conflicts to declare.

Notes and references1 M. B. Richardson and S. J. Williams, Front. Immunol.,

2014, 5, 288.2 S. J. Williams, Front. Immunol., 2017, 8, 1662.




Journal. COMMUNICATION3 E. Ishikawa, T. Ishikawa, Y. S. Morita, K. Toyonaga, H.

Yamada, O. Takeuchi, T. Kinoshita, S. Akira, Y. Yoshikaiand S. Yamasaki, J. Exp. Med., 2009, 206, 2879.

4 H. Schoenen, B. Bodendorfer, K. Hitchens, S.Manzanero, K. Werninghaus, F. Nimmerjahn, E. M.Agger, S. Stenger, P. Andersen, J. Ruland, G. D. Brown,C. Wells and R. Lang, J. Immunol., 2010, 184, 2756.

5 S. Yamasaki, E. Ishikawa, M. Sakuma, H. Hara, K. Ogataand T. Saito, Nat. Immunol., 2008, 9, 1179.

6 A. Lobato-Pascual, P. C. Saether, S. Fossum, E. Dissenand M. R. Daws, Eur. J. Immunol., 2013, 43, 3167.

7 K. Werninghaus, A. Babiak, O. Gross, C. Holscher, H.Dietrich, E. M. Agger, J. Mages, A. Mocsai, H.Schoenen, K. Finger, F. Nimmerjahn, G. D. Brown, C.Kirschning, A. Heit, P. Andersen, H. Wagner, J. Rulandand R. Lang, J. Exp. Med., 2009, 206, 89.

8 A. Furukawa, J. Kamishikiryo, D. Mori, K. Toyonaga, Y.Okabe, A. Toji, R. Kanda, Y. Miyake, T. Ose, S. Yamasakiand K. Maenaka, Proc. Natl. Acad. Sci. USA, 2013, 110,17438.

9 H. Feinberg, S. A. Jegouzo, T. J. Rowntree, Y. Guan, M.A. Brash, M. E. Taylor, W. I. Weis and K. Drickamer, J.Biol. Chem., 2013, 288, 28457.

10 H. Feinberg, N. D. Rambaruth, S. A. Jegouzo, K. M.Jacobsen, R. Djurhuus, T. B. Poulsen, W. I. Weis, M. E.Taylor and K. Drickamer, J. Biol. Chem., 2016, 291,21222.

11 X. Lu, M. Nagata and S. Yamasaki, Int. Immunol., 2018,30, 233.

12 A. A. Khan, S. H. Chee, R. J. McLaughlin, J. L. Harper, F.Kamena, M. S. M. Timmer and B. L. Stocker,Chembiochem, 2011, 12, 2572.

13 B. L. Stocker, A. A. Khan, S. H. Chee, F. Kamena and M.S. Timmer, Chembiochem, 2014, 15, 382.

14 A. J. Foster, M. Nagata, X. Lu, A. T. Lynch, Z. Omahdi, E.Ishikawa, S. Yamasaki, M. S. M. Timmer and B. L.Stocker, J. Med. Chem., 2018, 61, 1045.

15 A. Decout, S. Silva-Gomes, D. Drocourt, S. Barbe, I.Andre, F. J. Cueto, T. Lioux, D. Sancho, E. Perouzel, A.Vercellone, J. Prandi, M. Gilleron, G. Tiraby and J.Nigou, Proc. Natl. Acad. Sci. USA, 2017, 114, 2675.

16 P. L. van der Peet, C. Gunawan, S. Torigoe, S. Yamasakiand S. J. Williams, Chem. Commun., 2015, 51, 5100.

17 S. Shah, M. Nagata, S. Yamasaki and S. J. Williams,Chem. Commun., 2016, 52, 10902.

18 M. B. Richardson, S. Torigoe, S. Yamasaki and S. J.Williams, Chem. Commun., 2015, 51, 15027.

19 F. Behler-Janbeck, T. Takano, R. Maus, J. Stolper, D.Jonigk, M. Tort Tarres, T. Fuehner, A. Prasse, T. Welte,M. S. Timmer, B. L. Stocker, Y. Nakanishi, T. Miyamoto,S. Yamasaki and U. A. Maus, PLoS Pathog., 2016, 12,e1006038.

20 M. Nagata, Y. Izumi, E. Ishikawa, R. Kiyotake, R. Doi, S.Iwai, Z. Omahdi, T. Yamaji, T. Miyamoto, T. Bamba andS. Yamasaki, Proc. Natl. Acad. Sci. USA, 2017, 114,E3285.

21 P. L. van der Peet, M. Nagata, S. Shah, J. M. White, S.Yamasaki and S. J. Williams, Org. Biomol. Chem., 2016,14, 9267.

22 Z. Hakki, B. Cao, A. M. Heskes, J. Q. Goodger, I. E.Woodrow and S. J. Williams, Carbohydr. Res., 2010,345, 2079.

23 J. D. Twibanire and T. B. Grindley, Org. Lett., 2011, 13,2988.

24 N. K. Paul, J. D. Twibanire and T. B. Grindley, J. Org.Chem., 2013, 78, 363.

25 R. Kiyotake, M. Oh-Hora, E. Ishikawa, T. Miyamoto, T.Ishibashi and S. Yamasaki, J. Biol. Chem., 2015, 290,25322.

26 D. Lafont, P. Boullanger and A. Gambetta, J. LabelledCompds. Radiopharmaceut., 2012, 55, 88.

27 T. Matsumaru, R. Ikeno, Y. Shuchi, T. Iwamatsu, T.Tadokoro, S. Yamasaki, Y. Fujimoto, A. Furukawa and K.Maenaka, Chem. Commun., 2019, 55, 711.

28 L. Van Huy, C. Tanaka, T. Imai, S. Yamasaki and T.Miyamoto, ACS Med. Chem. Lett., 2019, 10, 44.

29 A. Khan, C. D. Braganza, K. Kodar, M. S. M. Timmer andB. L. Stocker, Org. Biomol. Chem., 2020, DOI:10.1039/C9OB02302J.

30 J. H. Bird, A. A. Khan, N. Nishimura, S. Yamasaki, M. S.M. Timmer and B. L. Stocker, J. Org. Chem., 2018, 83,7593.

31 A. J. Foster, K. Kodar, M. S. M. Timmer and B. L.Stocker, Org. Biomol. Chem., 2020, DOI:10.1039/C9OB02397F.

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Received 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

Design of potent Mincle signalling agonists based on an alkyl -glucoside template

Dylan G.M. Smith,a Yuki Hosono,b,c Masahiro Nagata,b,c Sho Yamasakib,c and Spencer J. Williams*a

The innate immune receptor Mincle senses lipid-based molecules

derived from pathogens, commensals and altered self. Based on

emerging structure-activity relationships we design simple alkyl 6-

O-acyl--D-glucosides that are effective agonists of Mincle and

signal with potency on par with the prototypical ligand trehalose

dimycolate.

Macrophage inducible C-type lectin (Mincle) receptor is an

FcRγ-associated pattern recognition receptor involved in innate

immunity.1 Mincle senses a range of self and foreign

(glyco)lipids2 and has become an important target for study

since the discovery that it is responsible for recognition of

mycobacterial cord factor (trehalose dimycolate; TDM)3 and the

synthetic adjuvant trehalose dibehenate (TDB) (Fig. 1a).4 Ligand

binding to Mincle results in phosphorylation of the

immunoreceptor tyrosine activation motif of FcRγ5, 6 and

activation of NF-B via Card9–Bcl10–Malt1 signalosomes.7

Mincle is required for the classic granulomatous response to

TDM3 and the adjuvant activity of TDB.4 TDB was developed as

an optimized analogue of TDM, and both compounds induce

strong Th1 and Th17 cellular immune responses.2 Mincle

possesses an extracellular C-type lectin domain that appears to

be the primary site for ligand binding. X-ray structures of

human8 and bovine Mincle (the latter bound to trehalose and

trehalose monobutyrate)9, 10 revealed two monosaccharide

binding subsites, with the primary site involving coordination of

O3 and O4 to Ca2+, and a shallow hydrophobic groove that can

accommodate lipid chains (Fig. 1b).

Fig. 1. a) Examples of potent agonists of Mincle signalling. b) X-ray structure

of trehalose binding to the Ca2+ ion in the carbohydrate binding domain of

Mincle (PDB 4KZV). c) Summary of structure-activity model for Mincle

signalling.

A growing appreciation of the features necessary for

agonism of Mincle signalling have emerged through

identification of other naturally-occurring Mincle agonists, and

through structure-activity relationship studies (reviewed in

Refs2, 11). Broadly, signalling through Mincle increases as lipid

chain length increases for both mono- and diacyl-trehaloses,

with activity maximized for a C22 (behenate) chain,12, 13 or

slightly shorter for a series of alkylated brartemicin analogues

(Fig. 1a).14 A single glucose residue is sufficient for Mincle

signalling: both glucose monomycolate (GMM)15 and

monocorynomycolate (GMCM)16 are potent Mincle signalling

a. School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Vic 3010.

b. Department of Molecular Immunology, Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan.

c. Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan

† Footnotes relating to the title and/or authors should appear here. Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x





agonists. Glucose 6-monobehenate (GMB)16 is a weak Mincle

agonist, yet glucose analogues bearing long, branched fatty

acids linked off the 6-position (such as C14C18)15 are potent

agonists. Collectively, these results suggest that at least two

lipid chains off a monosaccharide are necessary for optimum

activity. Separately, various - and -glucosyl diacylglycerides17-

19 and -glucosyl ceramide20 can signal through Mincle, showing

that the lipid group can be sited at the anomeric or C6-positions

of glucose. Consistent with the requirement for at least two

lipid chains for activity of monosaccharide glycolipids, a lyso -

glucosyl diacylglyceride did not signal through Mincle.18

Through studies of tetraacylated trehalose glycolipids it was

concluded that a free 2-hydroxyl group on a glucose residue was

essential for activity.15

A good understanding now exists of structure-activity

relationships for glucolipid signalling through Mincle (Fig. 1c).

Decout and co-workers have argued that at least three of four

key structural motifs (two sugars, two alkyl chains) must be

present for Mincle agonism, and when combined with

appropriate branching and length, the alkyl chains are more

important than the second sugar unit.15 A separate study

showed that branching at the -position of a 6-O-acyl-glucose

analogue provided only weak Mincle agonism.21 Based on these

results Decout and co-workers developed C14C18Glc, which

possessed greater activity than TDB in a Mincle reporter assay.15

However, C14C18Glc is comprised of complex mixture of 8

compounds: epimers at the -branched stereocentre of the

lipid, the sugar hemiacetal that exists as both - and -anomers,

as well as furanose and pyranose ring isomers; the contribution

of each of these forms to activity is unclear.

In this work we report new, simplified agonists of Mincle

signalling based on a -glucoside template. In particular, we

focussed on synthetic targets that can be easily prepared in just

a few steps from commercial materials and which exist in a

single isomeric form to simplify characterization, purification

and interpretation of results. Cognizant of the potency of GMM

and analogues as well as -glucosyl diglycerides, we

investigated compounds that possess lipophilic functionality at

the 1- and 6-positions. Compounds of this general structure fit

with the structure-activity relationships for Mincle signalling by

possessing at least two alkyl chains, a single sugar residue and

maintaining the free sugar 2-, 3- and 4-hydroxyl groups.

Alkyl glucosides are widely used in consumer products

including cosmetics and soaps and both octyl and lauryl -

glucoside are commercially available reagents that could

provide easy synthetic access to potential Mincle agonists. We

avoided the use of protecting groups and instead performed

direct acylation. Uronium-based peptide coupling reagents

allow primary-selective acylation of simple glucosides through

the choice of appropriate amine bases.22-24 We chose to install

three representative straight chain esters: a short octanoyl (C8),

medium palmitoyl (C16) and long behenoyl (C22) group (Scheme

1). Reaction of the corresponding acids with octyl -glucoside

afforded C8GlcC8 3, C16GlcC8 4 and C22GlcC8 5 in 61, 33 and 47%

yields, respectively. Similarly, the same acids and lauryl -

glucoside afforded C8GlcC12 6, C16GlcC12 7 and C22GlcC12 8 in 56,

46 and 47% yields, respectively.

Scheme 1. Synthesis of lauryl and octyl 6-O-acyl--D-glucopyranosides.

Reagents and conditions: a) octanoic acid, HBTU, pyr, b) palmitic acid, HBTU,

pyr, c) behenic acid, HBTU, pyr, d) HO2CCH[(CH2)15CH3]2, HBTU, pyr.

Racemic 2-tetradecyloctanoic acid was used by Decout et

al.15 for preparation of C14C18Glc, and thus this compound is

a mixture of isomers epimeric at the -position. We instead

utilized commercially-available achiral 2-hexadecyloctanoic

acid. Reaction of octyl and lauryl -glucosides with this acid and

HBTU in pyridine afforded the monoesters C18C16GlcC8 9 and

C18C16GlcC12 10 in 15 and 12% yields, respectively. These yields

were lower than that of the straight chain acids and may reflect

reduced reactivity arising from the -branch.

Cholesterol can signal through Mincle,25 and is a lipophilic

group that we speculated could occupy the lipid binding pocket.

We synthesized 1-cholesteryloxyacetic acid 13 by reaction of

ethyl diazoacetate26 with cholesterol promoted by BF3.Et2O in

CH2Cl2 to afford 12 in 61% yield (Scheme 2). Saponification with

2 M NaOH in EtOH afforded 13. Reaction of octyl and lauryl -

glucosides with 13 and HBTU/pyridine afforded the monoesters

CholGlcC8 14 and CholGlcC12 15 in 47 and 45% yields,

respectively.

Scheme 2. Synthesis of lauryl and octyl 6-O-(cholesteryloxyacetyl)--D-

glucopyranosides. Reagents and conditions: a) ethyl diazoacetate, BF3.Et2O,

CH2Cl2, b) 2 M NaOH, EtOH, H2O, c) 1 or 2, HBTU, pyr.

The panel of alkyl 6-O-acyl--D-glucopyranosides as well as

the parent octyl and lauryl glucosides were investigated for

their ability to induce signalling through Mincle. Assays were

performed by culturing reporter cells expressing mouse and

Journal. COMMUNICATION

This journal is © The Royal Society of Chemistry 20xx Journal. , 20xx, 00 , 1-3 | 3



human Mincle on plate-bound glycolipids, and measurement of

green fluorescent protein by flow cytometry to quantify the

degree of Mincle signalling agonism. Octyl and lauryl -D-

glucopyranosides did not signal through Mincle, which is

unsurprising as they are soluble detergents, and do not fulfil the

expected structural criteria for Mincle agonists. All of the

remaining compounds signalled to varying degrees through the

two receptor orthologs.

For the straight-chain 6-O-acyl derivatives, a small increase

in signalling potency was seen in changing from octyl to lauryl,

except in the case of C22GlcC8 and C22GlcC12, where the potency

was lower in the latter. Also, the intensity of signalling through

the mouse and human orthologs was similar for most

analogues, except the C22 derivatives, where signalling through

human Mincle was reduced relative to the mouse ortholog.

Similarly, within the individual series of octyl or lauryl

glycosides, an increase in 6-O-acyl chain length generally led to

an increase in potency, with the exception of C22GlcC12, where

the potency was reduced. In all cases, signalling potency was

less than that of TDB. This trend in activity is reminiscent of the

ability of trehalose mono-13 and diesters12 to activate

macrophages, wherein the C22 compounds were more potent

than the C26 analogues, and a homologous series of acyl

glycerols, where potency peaked at C28.27

Fig. 2 Agonism of Mincle signalling by unsubstituted octyl or lauryl -D-glucopyranosides or straight-chain 6-O-acyl variants. 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 TDB and analogues 1-

8. Assays were performed in duplicate; the mean values and standard errors are shown. neg = isopropanol vehicle control.

We next evaluated the activities of the branched-chain 6-O-

acyl -D-glucopyranosides and the cholesteryloxyacetyl

analogues (Fig. 3). The branched-chain analogues were more

potent than any of the straight-chain derivatives, and again,

little difference was seen between the octyl and lauryl

glycosides. As for the C22 analogues, signalling intensity was

lower for human Mincle than for mouse Mincle. The most

potent signalling was seen for the cholesterol analogues, which

provided signalling with similar intensities and potencies as TDB

and comprise the most potent agonists identified in this study.

Fig. 3 Agonism of Mincle signalling by octyl or lauryl 6-O-acyl--D-

glucopyranosides bearing a branched lipid chain or a cholesteryloxyacetyl

group. 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 TDB and analogues 9, 10, 14 and 15. Assays

were performed in duplicate; the mean values and standard errors are

shown. neg = isopropanol vehicle control. Results for TDB and neg were

obtained in the same experiment as for Fig. 2.

In conclusion this work reports development of simple alkyl

glucosides as highly potent Mincle agonists based on emerging

structure-activity relationships. Long-chain lipidic esters at O6

provided potent agonists of Mincle signalling, with even greater

activity seen for an -branched fatty acyl chain. These results

are broadly in concordance with the structure-activity

relationship articulated by Decout et al.15 but extend that work

by showing that a lipid chain at C1 can contribute to potency

and simplify their structures by both blocking the anomeric

position and locking its stereochemistry. Our results also show

that alkyl -glucosides esterified with a cholesteryl group at O6

are potent Mincle antagonists. Cholesterol, in its crystalline

insoluble form, signals through human, but not rodent Mincle.25

Signalling by cholesterol involves interaction with the

cholesterol recognition site (CRAC) of human Mincle, which is

absent in rodent Mincle, and alkylation of cholesterol leads to

loss of ability to signal through Mincle.25 As our analogues are

alkylated cholesterol derivatives, and signal through both

mouse and human Mincle, it seems unlikely that the cholesteryl

0

10

20

30

40

50

60

70

80

ー 0.1 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1

NFA

T-G

FP

(%

)

TDB GlcC8

(1)GlcC12

(2)C8GlcC8

(3)C8GlcC12

(6)C16GlcC8

(4)C16GlcC12

(7)C22GlcC8

(5)

neg

nmol0.010.1

1mMincle-g

hMincle-g

FcRg only

0.1 1

C22GlcC12

(8)

mMincle-g

hMincle-g

FcRg only

0

10

20

30

40

50

60

70

80

90

ー 0.1 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1

NFA

T-G

FP

(%

)

neg

TDB C18C16GlcC8

(9)

0.1 1

C18C16GlcC12

(10)CholGlcC8

(14)CholGlcC12

(15)

nmol0.010.1

1





moiety binds at the CRAC site of Mincle and is likely simply a

surrogate for a lipid group.

This work builds on related efforts exploring structure

activity relationships of acyl-hexoses (as analogues of glucose

monomycolate),15, 16 glycosyloxy-stearates (as analogues of the

mannosyloxymannitol glycolipid from Malassezia

pachydermatis),28 glycerolipids (as analogues of glycerol

monomycolate),27, 29 mono-13 and diacyl trehaloses,12, 30 and

lipidated diaroyltrehaloses (as analogues of brartemicin).14, 31

The present work is notable for the simplicity of the resulting

agonists and the fact that unlike acyl-hexoses they exist as

single stereoisomers.

We thank the Australian Research Council (DP160100597)

and AMED (JP19gm0910010, JP19ak0101070 and

JP19fk0108075) for grant support.

Conflicts of interest

There are no conflicts to declare.

Notes and references

1 M. B. Richardson and S. J. Williams, Front. Immunol., 2014, 5, 288.

2 S. J. Williams, Front. Immunol., 2017, 8, 1662. 3 E. Ishikawa, T. Ishikawa, Y. S. Morita, K. Toyonaga, H.

Yamada, O. Takeuchi, T. Kinoshita, S. Akira, Y. Yoshikai and S. Yamasaki, J. Exp. Med., 2009, 206, 2879.

4 H. Schoenen, B. Bodendorfer, K. Hitchens, S. Manzanero, K. Werninghaus, F. Nimmerjahn, E. M. Agger, S. Stenger, P. Andersen, J. Ruland, G. D. Brown, C. Wells and R. Lang, J. Immunol., 2010, 184, 2756.

5 S. Yamasaki, E. Ishikawa, M. Sakuma, H. Hara, K. Ogata and T. Saito, Nat. Immunol., 2008, 9, 1179.

6 A. Lobato-Pascual, P. C. Saether, S. Fossum, E. Dissen and M. R. Daws, Eur. J. Immunol., 2013, 43, 3167.

7 K. Werninghaus, A. Babiak, O. Gross, C. Holscher, H. Dietrich, E. M. Agger, J. Mages, A. Mocsai, H. Schoenen, K. Finger, F. Nimmerjahn, G. D. Brown, C. Kirschning, A. Heit, P. Andersen, H. Wagner, J. Ruland and R. Lang, J. Exp. Med., 2009, 206, 89.

8 A. Furukawa, J. Kamishikiryo, D. Mori, K. Toyonaga, Y. Okabe, A. Toji, R. Kanda, Y. Miyake, T. Ose, S. Yamasaki and K. Maenaka, Proc. Natl. Acad. Sci. USA, 2013, 110, 17438.

9 H. Feinberg, S. A. Jegouzo, T. J. Rowntree, Y. Guan, M. A. Brash, M. E. Taylor, W. I. Weis and K. Drickamer, J. Biol. Chem., 2013, 288, 28457.

10 H. Feinberg, N. D. Rambaruth, S. A. Jegouzo, K. M. Jacobsen, R. Djurhuus, T. B. Poulsen, W. I. Weis, M. E. Taylor and K. Drickamer, J. Biol. Chem., 2016, 291, 21222.

11 X. Lu, M. Nagata and S. Yamasaki, Int. Immunol., 2018, 30, 233.

12 A. A. Khan, S. H. Chee, R. J. McLaughlin, J. L. Harper, F. Kamena, M. S. M. Timmer and B. L. Stocker, Chembiochem, 2011, 12, 2572.

13 B. L. Stocker, A. A. Khan, S. H. Chee, F. Kamena and M. S. Timmer, Chembiochem, 2014, 15, 382.

14 A. J. Foster, M. Nagata, X. Lu, A. T. Lynch, Z. Omahdi, E. Ishikawa, S. Yamasaki, M. S. M. Timmer and B. L. Stocker, J. Med. Chem., 2018, 61, 1045.

15 A. Decout, S. Silva-Gomes, D. Drocourt, S. Barbe, I. Andre, F. J. Cueto, T. Lioux, D. Sancho, E. Perouzel, A. Vercellone, J. Prandi, M. Gilleron, G. Tiraby and J. Nigou, Proc. Natl. Acad. Sci. USA, 2017, 114, 2675.

16 P. L. van der Peet, C. Gunawan, S. Torigoe, S. Yamasaki and S. J. Williams, Chem. Commun., 2015, 51, 5100.

17 S. Shah, M. Nagata, S. Yamasaki and S. J. Williams, Chem. Commun., 2016, 52, 10902.

18 M. B. Richardson, S. Torigoe, S. Yamasaki and S. J. Williams, Chem. Commun., 2015, 51, 15027.

19 F. Behler-Janbeck, T. Takano, R. Maus, J. Stolper, D. Jonigk, M. Tort Tarres, T. Fuehner, A. Prasse, T. Welte, M. S. Timmer, B. L. Stocker, Y. Nakanishi, T. Miyamoto, S. Yamasaki and U. A. Maus, PLoS Pathog., 2016, 12, e1006038.

20 M. Nagata, Y. Izumi, E. Ishikawa, R. Kiyotake, R. Doi, S. Iwai, Z. Omahdi, T. Yamaji, T. Miyamoto, T. Bamba and S. Yamasaki, Proc. Natl. Acad. Sci. USA, 2017, 114, E3285.

21 P. L. van der Peet, M. Nagata, S. Shah, J. M. White, S. Yamasaki and S. J. Williams, Org. Biomol. Chem., 2016, 14, 9267.

22 Z. Hakki, B. Cao, A. M. Heskes, J. Q. Goodger, I. E. Woodrow and S. J. Williams, Carbohydr. Res., 2010, 345, 2079.

23 J. D. Twibanire and T. B. Grindley, Org. Lett., 2011, 13, 2988.

24 N. K. Paul, J. D. Twibanire and T. B. Grindley, J. Org. Chem., 2013, 78, 363.

25 R. Kiyotake, M. Oh-Hora, E. Ishikawa, T. Miyamoto, T. Ishibashi and S. Yamasaki, J. Biol. Chem., 2015, 290, 25322.

26 D. Lafont, P. Boullanger and A. Gambetta, J. Labelled Compds. Radiopharmaceut., 2012, 55, 88.

27 T. Matsumaru, R. Ikeno, Y. Shuchi, T. Iwamatsu, T. Tadokoro, S. Yamasaki, Y. Fujimoto, A. Furukawa and K. Maenaka, Chem. Commun., 2019, 55, 711.

28 L. Van Huy, C. Tanaka, T. Imai, S. Yamasaki and T. Miyamoto, ACS Med. Chem. Lett., 2019, 10, 44.

29 A. Khan, C. D. Braganza, K. Kodar, M. S. M. Timmer and B. L. Stocker, Org. Biomol. Chem., 2020, DOI: 10.1039/C9OB02302J.

30 J. H. Bird, A. A. Khan, N. Nishimura, S. Yamasaki, M. S. M. Timmer and B. L. Stocker, J. Org. Chem., 2018, 83, 7593.

31 A. J. Foster, K. Kodar, M. S. M. Timmer and B. L. Stocker, Org. Biomol. Chem., 2020, DOI: 10.1039/C9OB02397F.

download fileview on ChemRxivSpencer Williams - Design of potent Mincle.pdf (1.22 MiB)



Design of potent Mincle signalling agonists based on an alkyl -glucoside template

Dylan G.M. Smith, Yuki Hosono, Masahiro Nagata, Sho Yamasaki and Spencer J. Williams*

ContentsExperimental..............................................................................................................................3

General...................................................................................................................................3

Ethyl 2-(cholesteryloxy)-ethanoate (12)................................................................................3

2-(Cholesteryloxy)-ethanoic acid (13)...................................................................................3

General procedure for the synthesis of 6-O-acyl-β-D-glucosides:.........................................3

Octyl 6-O-octanoyl-β-D-glucoside (3)...................................................................................4

Octyl 6-O-palmitoyl-β-D-glucoside (4).................................................................................4

Octyl 6-O-behenoyl-β-D-glucoside (5)..................................................................................4

Octyl 6-O-cholestryloxyacetyl-β-D-glucoside (14)...............................................................5

Octyl 6-O-(1-hexadecyloctadecanoyl)-β-D-glucoside (9).....................................................5

Lauryl 6-O-octanoyl-β-D-glucoside (6).................................................................................5

Lauryl 6-O-palmitoyl-β-D-glucoside (7)...............................................................................6

Lauryl 6-O-behenoyl-β-D-glucoside (8)................................................................................6

Lauryl 6-O-cholesteryloxyacetyl-β-D-glucoside (15)............................................................6

Lauryl 6-O-(1-hexadecyloctadecanoyl)-β-D-glucoside (10).................................................7

NMR spectra...............................................................................................................................8

Ethyl 2-(cholesteryloxy)-ethanoate (12)................................................................................8

2-(Cholesteryloxy)-ethanoic acid (13).................................................................................10

Octyl 6-O-octanoyl-β-D-glucoside (3).................................................................................11

Octyl 6-O-palmitoyl-β-D-glucoside (4)...............................................................................13

Octyl 6-O-behenoyl-β-D-glucoside (5)................................................................................14

Octyl 6-O-cholesteryloxyacetyl-β-D-glucoside (14)...........................................................15

S1

Octyl 6-O-(1-hexadecyloctadecanoyl)-β-D-glucoside (9)...................................................16

Lauryl 6-O-octanoyl-β-D-glucoside (6)...............................................................................17

Lauryl 6-O-palmitoyl-β-D-glucoside (7).............................................................................18

Lauryl 6-O-behenoyl-β-D-glucoside (8)..............................................................................19

Lauryl 6-O-cholesteryloxyacetyl-β-D-glucoside (15)..........................................................20

Lauryl 6-O-(1-hexadecyloctadecanoyl)-β-D-glucoside (10)...............................................21

S2

Experimental

General

Pyridine was distilled over KOH before use. Dichloromethane and THF were dried over alumina

according to the method of Pangborn et al.1 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.2 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 mode.

Ethyl 2-(cholesteryloxy)-ethanoate (12)

Boron trifluoride etherate (31 μL, 0.25 mmol) was added to a cold solution of cholesterol (1.00 g,

2.59 mmol) and ethyl diazoacetate (2.42 mL, 2.85 mmol) in dry CH2Cl2 (10 mL). The solution was

stirred overnight at 20 °C and then poured into saturated aqueous NaHCO3 (30 mL), and the product

was extracted with CH2Cl2 (225 mL). The organic phase was dried (Na2SO4) and concentrated under

reduced pressure. The crude product was purified by flash chromatography (ethyl acetate/petroleum

ether) afforded 12 as an amorphous solid (760 mg, 62%). 1H NMR (400 MHz, CDCl3) δ 1.28 (3 H,

t), 0.65–2.41 (43 H, m), 4.11 (2 H, s), 3.24 (1 H, m), 4.21 (2 H, q, J 7.2 Hz), 5.35 (1 H, m); 13C

NMR (100 MHz , CDCl3) δ 11.9, 14.2, 18.7, 19.3, 21.1, 22.6, 22.8, 23.8, 24.3, 28.0, 28.1, 28.2,

31.9, 31.9, 35.8, 36.2, 36.8, 37.1, 38.7, 39.5, 39.8, 42.3, 50.2, 56.1, 56.8, 60.8, 65.8, 80.0, 121.9,

140.5, 170.9. HRMS (ESI+) calcd for C31H52O3 [M + H]+

2-(Cholesteryloxy)-ethanoic acid (13)

Sodium hydroxide (2M, 320 μl) was added to 12 (200 mg, 0.42 mmol) in ethanol (2.5 ml) and

stirred at rt overnight. The solution was neutralized with Dowex 50WX8-200, then filtered and the

filtrate concentrated under reduced pressure to give 13 as a glass that was used without further

purification. 1H NMR (400 MHz, CDCl3) δ 0.62–2.43 (44 H, m), 3.31 (1 H, m), 4.14 (2 H, s), 5.37

(1 H, m); 13C NMR (100 MHz, CDCl3) δ 11.8, 18.7, 19.3, 21.1, 22.5, 22.8, 23.8, 24.3, 28.0, 28.1,

28.2, 31.8, 31.9, 35.8, 36.2, 36.7, 36.9, 38.7, 39.5, 39.7, 42.3, 50.1, 56.1, 56.7, 65.2, 80.5, 122.5,

139.8. HRMS (ESI+) calcd for C31H52O3 [M + H]+

General procedure for the synthesis of 6-O-acyl-β-D-glucosides:

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 min before addition of the glycoside (1 equiv.).

The solution was stirred for 2-3 d and then concentrated. Flash chromatography of the residue (pet.

spirits/EtOAc/MeOH) afforded the 6-O-acyl-β-D-glucoside.

Octyl 6-O-octanoyl-β-D-glucoside (3)

Octyl β-D-glucoside (50 mg, 0.171 mmol) and octanoic acid (32 μL, 0.205 mmol) according to the

General procedure afforded 3 as a colourless glass (44 mg, 61%). [α]D25 -36.5 (c 2.2, CHCl3); 1H

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, H1,6),

4.27 (0.2 H, s, OH), 4.32 (1 H, m, H6), 4.65 (0:2 H, s, OH), 4.87 (0.2 H, s, OH); 13C 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 (4)

Octyl β-D-glucoside (50 mg, 0.171 mmol) and palmitic acid (53 mg, 0.205mol) according to the


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 (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 Hz, H6), 4.32 (1 H, dd, J 12.1, 2.1

Hz, H6); 13C 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 (5)

Octyl β-D-glucoside (50 mg, 0.171 mmol) and behenic acid (70 mg, 0.205 mmol) according to the


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,



OCH2CH2), 4.19 (1 H, d, J 8.0 Hz, H1), 4.17–4.26 (1 H, m, H6), 4.32 (1 H, dd, J 2.1, 11.9 Hz, H6);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 (14)

Octyl β-D-glucoside (25 mg, 0.086 mmol) and 13 (46 mg, 0.103 mmol) according to the General

procedure afforded 14 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, cholesterol), 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, O=CCH2O), 4.20 (1 H, d, J 7.7 Hz, H1), 4.30 (1 H, dd, J 5.5, 11.9 Hz,

H6), 4.39 (1 H, dd, J 2.2, 11.9 Hz, H6), 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 (9)

Octyl β-D-glucoside (25 mg, 0.086 mmol) and 1-hexadecyloctadecanoic acid (52 mg, 0.103 mmol)

according to the General procedure afforded 9 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, H6), 4.20 (1 H, d, J 7.8 Hz, H1), 4.39 (1 H, dd, J 2.0, 11.9 Hz, H6); 13C 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 (6)

Lauryl β-D-glucoside (50 mg, 0.143 mmol) and octanoic acid (27 μL, 0.172 mmol) according to the






Hz, H6); 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 (7)

Lauryl β-D-glucoside (50 mg, 0.143 mmol) and palmitic acid (44 mg, 0.172 mmol) according to the


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,





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


C34H66O7 [M + H]+ 587.4881. Found 587.4880.

Lauryl 6-O-behenoyl-β-D-glucoside (8)

Lauryl β-D-glucoside (50 mg, 0.143 mmol) and behenic acid (59 mg, 0.172 mmol) according to the







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


C40H78O7 [M + H]+ 671.5820. Found 671.5822.

Lauryl 6-O-cholesteryloxyacetyl-β-D-glucoside (15)

Lauryl β-D-glucoside (25 mg, 0.072 mmol) and 13 (38 mg, 0:086 mmol) according to the General

procedure afforded 15 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, H6), 4.40 (1 H, dd, J 2.3, 11.9 Hz, H6), 5.31 (1 H, m, HC=H); 13C NMR (100 MHz,

CDCl3:CD3OD 95:5) δ 11.8, 14.0, 18.6, 19.2, 21.0, 22.5, 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 (10)

Lauryl β-D-glucoside (25 mg, 0.072 mmol) and 1-hexadecyloctadecanoic acid (44 mg, 0.086 mmol)

according to the General procedure afforded 10 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, H6), 4.20 (1 H, d, J 7.8 Hz, H1), 4.39 (1 H, dd, J 2.1, 12.0 Hz, H6); 13C 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.

References

1 A. B. Pangborn, M. A. Giardello, R. H. Grubbs, R. K. Rosen and F. J. Timmers,Organometallics, 1996, 15, 1518.

2 W. C. Still, M. Kahn and A. M. Mitra, J. Org. Chem., 1978, 43, 2923.

NMR spectra

Ethyl 2-(cholesteryloxy)-ethanoate (12)1H NMR

13C NMR

2-(Cholesteryloxy)-ethanoic acid (13)1H NMR

13C NMR

Octyl 6-O-octanoyl-β-D-glucoside (3)1H NMR

13C NMR

Octyl 6-O-palmitoyl-β-D-glucoside (4)1H NMR

13C NMR

Octyl 6-O-behenoyl-β-D-glucoside (5)1H NMR

13C NMR

Octyl 6-O-cholesteryloxyacetyl-β-D-glucoside (14)1H NMR

13C NMR

Octyl 6-O-(1-hexadecyloctadecanoyl)-β-D-glucoside (9)1H NMR

13C NMR

Lauryl 6-O-octanoyl-β-D-glucoside (6)1H NMR

13C NMR

Lauryl 6-O-palmitoyl-β-D-glucoside (7)1H NMR

13C NMR

Lauryl 6-O-behenoyl-β-D-glucoside (8)1H NMR

13C NMR

Lauryl 6-O-cholesteryloxyacetyl-β-D-glucoside (15)1H NMR

13C NMR

Lauryl 6-O-(1-hexadecyloctadecanoyl)-β-D-glucoside (10)1H NMR

13C NMR

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