Total synthesis of tetraacylated phosphatidylinositol hexamannoside and evaluation of its immunomodulatory activity

Abstract

Tuberculosis, aggravated by drug-resistant strains and HIV co-infection of the causative agent Mycobacterium tuberculosis, is a global problem that affects millions of people. With essential immunoregulatory roles, phosphatidylinositol mannosides are among the cell-envelope components critical to the pathogenesis and survival of M. tuberculosis inside its host. Here we report the first synthesis of the highly complex tetraacylated phosphatidylinositol hexamannoside (Ac₂ PIM₆ ), having stearic and tuberculostearic acids as lipid components. Our effort makes use of stereoelectronic and steric effects to control the regioselective and stereoselective outcomes and minimize the synthetic steps, particularly in the key desymmetrization and functionalization of myo-inositol. A short synthesis of tuberculostearic acid in six steps from the Roche ester is also described. Mice exposed to the synthesized Ac₂ PIM₆ exhibit increased production of interleukin-4 and interferon-γ, and the corresponding adjuvant effect is shown by the induction of ovalbumin- and tetanus toxoid-specific antibodies.

ARTICLE

Received 26 Jan 2015 | Accepted 20 Apr 2015 | Published 3 Jun 2015

Tuberculosis, aggravated by drug-resistant strains and HIV co-infection of the causative agent Mycobacterium tuberculosis, is a global problem that affects millions of people. With essential immunoregulatory roles, phosphatidylinositol mannosides are among the cell-envelope components critical to the pathogenesis and survival of M. tuberculosis inside its host. Here we report the rst synthesis of the highly complex tetraacylated phosphatidylinositol hexamannoside (Ac2PIM6), having stearic and tuberculostearic acids as lipid components.

Our effort makes use of stereoelectronic and steric effects to control the regioselective and stereoselective outcomes and minimize the synthetic steps, particularly in the key desymmetrization and functionalization of myo-inositol. A short synthesis of tuberculostearic acid in six steps from the Roche ester is also described. Mice exposed to the synthesized Ac2PIM6 exhibit increased production of interleukin-4 and interferon-g, and the corresponding adjuvant effect is shown by the induction of ovalbumin- and tetanus toxoid-specic antibodies.

DOI: 10.1038/ncomms8239 OPEN

Total synthesis of tetraacylated phosphatidylinositol hexamannoside and evaluation of its immunomodulatory activity

Pratap S. Patil1, Ting-Jen Rachel Cheng1, Medel Manuel L. Zulueta1, Shih-Ting Yang1, Larry S. Lico1 & Shang-Cheng Hung1

1 Genomics Research Center, Academia Sinica, No. 128, Section 2, Academia Road, Taipei 115, Taiwan. Correspondence and requests for materials should be addressed to S.-C.H. (email: mailto:[email protected]

Web End [email protected] ).

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Mycobacterium tuberculosis is a dreaded pathogen that causes tuberculosis, one of the leading causes of death in the world. Although the disease becomes active for

only 510% of infected individuals, 1.5 million people died of tuberculosis in 2013 alone despite progress in the global effort for diagnosis, treatment and prevention1. Moreover, it is estimated that one-third of the human population is latently infected withM. tuberculosis and is highly vulnerable if immunocompromised. The antituberculosis vaccine bacillus Calmette-Gurin, made by using an attenuated strain of M. bovis, only gives protection to children but is highly variable in adults2. Co-infection with HIV and the rising cases of multidrug and extensive drug resistance also add to the high morbidity and mortality of the disease3. Clearly, fresh insights on the character of the causative agent and its pathogenesis are needed to help alleviate this human condition4.

The thick glycolipid-containing cell envelope5 of M. tuberculosis is critical for bacterial survival and growth. It is involved in sabotaging immunoregulatory responses68 and it forms a protective barrier for various drugs9,10. Among the vital cell-envelope components, phosphatidylinositol mannosides (PIMs) and their hypermannosylated structural relatives (lipomannans and lipoarabinomannans) are found noncovalently anchored to the plasma membrane and the outer capsule through palmitate, stearate and tuberculostearate lipid chains11. PIMs, in particular, dictate the intercellular fate of mycobacteria by binding to macrophages12, regulate cytokines and reactive radical species and stimulate early endosomal fusion by acting as ligands to Toll-like receptors, C-type lectins and DC-SIGN13. PIMs can also act as CD1d antigen to activate natural killer T cells for the production of interferon-g (ref. 14), indicating their potential as vaccine or adjuvant candidates. In addition, PIMs interact with a5b1 integrin on CD4 lymphocytes, which can either promote granuloma formation and enhance host immune response or help in bacterial survival15.

Structurally, myo-inositol is the central support unit of PIMs with a diacylated glycerophospholipid moiety at O1 and a-mannosylation sites at O2 and O6 (ref. 16). Additional lipid chains may be linked at the primary hydroxyl of the 2-O-mannosyl unit and at the O3 position of myo-inositol to form triacylated PIMs (AcPIMs) and tetraacylated PIMs (Ac2PIMs), respectively.

Higher PIMs (for example, AcnPIM3 AcnPIM6) are formed by

elongation at the mannose residue linked at O6 of myo-inositol. The number of mannose residues and the degree and type of the fatty acyl groups present in the PIM molecules determine their unique role in immunoregulation11. As a result, elegant synthetic strategies have been developed for PIMs and their related compounds1734. Nevertheless, the synthesis of a tetraacylated phosphatidylinositol hexamannoside (Ac2PIM6), the most complex among this class of compounds, is yet to be reported. Thus far, previous disclosures explored the synthesis of Ac2PIM2 (ref. 34), PIM4 (ref. 24), PIM6 (ref. 32) and AcPIM6 (ref. 25) to name a few.

We describe herein the rst synthesis of Ac2PIM6, using stearic and tuberculostearic acids as the lipid components. The immunomodulatory activity of the synthesized Ac2PIM6 was also evaluated.

ResultsSynthetic strategy. Compound 1 possesses multiple components and functionalizations. To arrive at this molecule, one rational synthetic design would be to fragment this sizeable structure into separate segments, which could later be assembled in a convergent manner. For this purpose, we conceived the pseudo-trisaccharide 2, tetramannoside donor 3 and phosphonate 4 as the primary targets (Fig. 1). The readily perceptible synthetic issues include the transformation of the ordinarily meso myoinositol into the unsymmetrical counterpart in 2 as well as the regioselective protection to afford the mannosyl-building blocks useful enough to deliver the necessary a1-2 and a1-6 linkages and the acylation of one mannosyl unit. Accordingly, along with benzyl groups for the global protection of hydroxyls that would be free in the desired product, we selected two additional orthogonal protecting groups for the primary positions of the mannosyl residues in intermediates leading to compound 2. The tert-butyldiphenylsilyl (TBDPS) group should allow, on deprotection, the subsequent coupling with the tetramannoside 3, whereas the 2-naphthylmethyl (2-NAP) group protects the position that would later be acylated. Being a core constituent of inositol phosphates and other phosphatidyl lipoglycans, various methods have been published for the myo-inositol resolution and

O OO

HO

OH HO

HO

OBn

BnO

OBn BnO

BnO

BnO

HO

O

HO

O HO

HO

OBn BnO

BnO

O-2-NAP

O

O BnO

BnO

BnO

O

OOBn

BnO

HO

O

O HO

HO

O O

HO

OBn

OH

O

BnO

O

2

O BnO

BnO

O

O

OH HO

HO

O

OBn BnO

BnO

O

OH

HO

O

O

O

O

STol

OH HO

HO

O

3

OOH

HO

Et3HN+

OH

O

O

O O

H

Na+

O

O O

1 2 3

P

P

O

O

O

O

O

O

O

O

O

4

Ac2PIM6 1

Figure 1 | Our target tetraacylated phosphatidylinositol hexamannoside (Ac2PIM6) and the main blocks designed to represent each segment. 2-NAP, 2-naphthylmethyl; Bn, benzyl; Tol, p-tolyl.

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desymmetrization35,36. However, the suitably protected chiral myo-inositol derivative required for PIMs and glycosyl phosphatidylinositide synthesis were mainly achieved through multistep synthesis from D-glucose via the Ferrier reaction37. Recently, we have shown that a mannosyl donor can directly act as chiral auxiliary to differentiate the enantiotopic hydroxyls of meso myo-inositol derivatives30,31. This direct approach bypasses many transformation steps in affording an appropriately mannosylated and desymmetrized myo-inositol. We intend to apply this capacity towards diol 2 while also relying on the steric hindrance created by the installed mannosyl residues to strategically position the 4,5-di-O-benzyl moieties on the myoinositol unit and sufciently favour regioselective acylation at the free 3-OH. An extra temporary protecting group and participating moiety is also needed to permit the construction of the tetramannoside 3, in which case we chose a 2-O-benzoyl group. We planned to assemble 3 through linear glycosylation from the reducing end to the nonreducing end using a single elongation unit. For the phosphonate 4, access to the rare tuberculostearic acid is the main concern, and this fatty acid should be synthesized to complete the desired phospholipid moiety.

Mannosyl-building blocks. Considering the stability of the thiotolyl leaving group on various functional group

interconversions, we selected the thiomannoside 5 (ref. 38) as a starting point in our transformations towards several mannosyl building blocks (Fig. 2). In general, the building blocks needed for the assembly of our target structure required differentiation at either O6 or O2. With a bulky functionality such as the TBDPS group, the protection sequence aimed regioselectively at the primary O6 position seems clear-cut. Thus, the 6-O-silylation of 5 using tert-butyldiphenylchlorosilane, triethylamine and 4-(N,N-dimethylamino)pyridine gave compound 6 in excellent yield. Subsequent benzylation under Williamson condition supplied the necessary thioglycoside 7. The 6-alcohol 8, intended as the starting acceptor in generating the tetramannoside 3, was also readily acquired from 7 by acidic desilylation.

In contrast to the route above, traditional approaches concerning the effective acquisitions of the 6-O-naphthylmethylated thiomannoside 13 and the 2-benzoate 14 do not appear to be straightforward. The complexity arises from the desire to carry out the fully regioselective installations of the vital ether groups. Apparently, the regioselective one-pot protection strategy that we introduced39,40 and further expanded to other sugars4144 could simplify such preparations. Our recent work on the stereoselective dioxolane-type benzylidene formation on thiomannosides43 should provide a convenient gateway to the 2,6-diol 11, a potential common intermediate towards compounds 13 and 14. It was envisioned that, with benzyl groups permanently protecting O3 and O4, the

O

HO

TMSO

OH HO

HO

O

ref. 40 O

OTMS TMSO

TMSO

STol

STol

5 9

a

d

TBDPSO

Ph

OH HO

HO

O

O O

Ph

O

STol

O

6

7

8

STol

10

11

12

b

e

OBn BnO

BnO

TBDPSO

HO

O

OH BnO

BnO

O

OBn BnO

BnO

2-NAPO

O

STol

STol

g

STol

c

f

13

14

OBn BnO

BnO

HO

TMSO

BnO

h i

O

OTMS BnO

BnO

O

OBz BnO

BnO

O

STol

STol

STol

Figure 2 | Preparations of the mannosyl-building blocks. Reagents and conditions: (a) tert-butyldiphenylchlorosilane, Et3N, DMAP, 93%; (b) NaH, BnBr, DMF, 94%; (c) PTSA, MeOH, CH2Cl2, 92%; (d) benzaldehyde (2.1 equivalents), TMSOTf, MeCN, 0 C, 30 min; (e) BH3 THF, Cu(OTf)2, CH2Cl2, 87% (one

pot from 9); (f) trimethylchlorosilane, Et3N, quantitative; (g) 2-naphthaldehyde, Et3SiH, TMSOTf, CH2Cl2, 78 to 40 C, 2 h, then, NaH, BnBr, DMF, 81%

(one pot); (h) 2-naphthaldehyde, Et3SiH, TMSOTf, CH2Cl2, 78 to 40 C, 2 h, then, NaH, BnBr, DMF, then, DDQ, H2O, 73% (one pot); (i) benzaldehyde,

Et3SiH, TMSOTf, 78 C, 1.5 h, then, BF3 Et2O, MeCN, 78 to 20 C, 30 min, then, Bz2O, Et3N, 93% (one pot). Bz, benzoyl; Bz2O, benzoic anhydride;

Cu(OTf)2, copper(II) triuoromethanesulfonate; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; Ph, phenyl; PTSA, p-toluenesulfonic acid; TBDPS, tert-butyldiphenylsilyl; THF, tetrahydrofuran; TMS, trimethylsilyl, TMSOTf, trimethylsilyl triuoromethanesulfonate.

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primary 6-hydroxyl could be easily differentiated from the secondary and axial 2-hydroxyl. We were also keen to check whether stereoselective dibenzylidenation and simultaneous regioselective ring opening could be achieved in one pot.

Starting from the tetrakis-trimethylsilyl ether 9 acquired in one step from 5 (ref. 43), treatment with 2.1 equivalents of benzaldehyde along with catalytic trimethylsilyl triuoromethanesulfonate (TMSOTf) in acetonitrile at 0 C exclusively delivered the exo-product 10 as evidenced by NMR spectroscopy and X-ray crystallography (Supplementary Fig. 1, Supplementary Data 1). This fully stereoselective transformation is benecial because unlike the regioselectivity in the 4,6-O-benzylidene ring opening, which is determined by the choice of reducing agent45, the opening of the dioxolane-type 2,3-O-benzylidine moiety is guided by the orientation of the phenyl group (that is, exo-isomers generally open at the axial position). Delightfully, the subsequent exposure of 10 to BH3 tetrahydrofuran in the same vessel provided the diol 11

in a two-stage one-pot yield of 87%. Anticipating a smooth regioselective reductive etherication at O6 (ref. 46), we subjected the diol 11 to trimethylsilylation. Consequently, treatment of the so-formed 12 with 2-naphthaldehyde, triethylsilane and TMSOTf in subzero temperature supplied the intermediate that was benzylated in one pot using the typical etherication method to afford compound 13. An X-ray single crystal analysis fully supported the desired structure (Supplementary Fig. 1,

Supplementary Data 2). Continuing further, addition of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to the in situ-generated 13 successfully delivered the same acceptor 8, thus providing an alternative pathway for its acquisition. Similarly, the corresponding reductive 6-O-benzylation of 12 was carried out, followed by desilylation at O2 with tetrabutylammonium uoride (TBAF) and basic benzoylation in the same ask. Unfortunately, the yield for compound 14 was less than satisfactory even when benzoic acid and TBAF were used together41 to perform the desilylation, probably due to the interference of TBAF to the benzoylation stage. Desilylation with BF3 Et2O apparently solved

this issue and furnished 14 in an excellent 93% yield from 12.

Synthesis of the pseudotrisaccharide 2. For the desymmetrization of myo-inositol, we evaluated the coupling of the mannosyl donor 7 with the meso diol 16 (Fig. 3), which can be easily prepared in one step30 from the commercially available Kishis triol. The asymmetric nature of the mannosyl donor itself should provide certain preferences between the axial hydroxyls of 16 as we have demonstrated previously30 but without the TBDPS group on the sugar. Because of the wider opening available for a nucleophilic attack on the half-chair mannosyl oxocarbenium ion intermediate by O6 as compared with O4 (Supplementary Fig. 2), it is expected that the required 6-O-mannosylation would be more favoured. Our attempts at coupling of 7 and 16 using

OO

O

OO

6

O

OO

O

BzO

4

HO OH

RO

BzO

OBn BnO

BnO

TBDPSO

HO

O

O

O

O

O OH

BnO

+

b

BnO

R

TBDPSO BnO

OBn

OBn

a

7: R = -STol

15: R = OC(=NH)CCl3

16

TBDPSO BnO

18

c

17: R = Bz

19: R = H

OBn BnO

BnO

2-NAPO

OO O

O

O

OBn BnO

BnO

2-NAPO

O

d

R

O O

BnO

OH

a

13: R = -STol

20: R = OC(=NH)CCl3

OBn

TBDPSO BnO

21

e

BnO

OBn O-2-NAP

BnO OBn O-2-NAP

OH

OTBDPS

f

OBn BnO

BnO

OOBn

O

OBn BnO

BnO

OOBn

O

BnO

O

HO

O

O

HO

OBn OH

O

HO

OH OH

2

22

Figure 3 | Preparation of the pseudotrisaccharide 2. Reagents and conditions: (a) (1) NBS, acetone, H2O; (2) K2CO3, CCl3CN, 15: 94% (two steps), 20: 90% (two steps); (b) silver triuoromethanesulfonate, 1,4-dioxane, CH2Cl2, 17: 68%, 18: 20%; (c) NaOMe, MeOH, CH2Cl2, quantitative; (d) BF3 Et2O, CH2Cl2, 60 to 20 C, 72%; (e) (1) PTSA, MeOH, CH2Cl2, 84%; (2) tert-butyldiphenylchlorosilane, Et3N, DMAP, 82%; (f) (1)

trimethylchlorosilane, Et3N, quantitative; (2) benzaldehyde (3 equivalent), Et3SiH, TMSOTf, CH2Cl2, 40 C, then, tetrabutylammonium uoride, 72%.

NBS, N-bromosuccinimide.

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N-iodosuccinimide and TMSOTf in CH2Cl2 and 1,4-dioxane to improve the solubility of diol 16, unfortunately, led only to donor hydrolysis and full recovery of the acceptor. We suspected that the axial hydroxyls are too unreactive for mannosyl thioglycoside to foster productive couplings. With strong activators avoided to maintain the acid-sensitive orthoformate group, focus was shifted to the imidate versions of the donor. After some optimization (see the Supplementary Table 1), silver triuoromethanesulfonate promoted the glycosylation step at room temperature, supplying the desired pseudodisaccharide 17 at 68% yield, along with its regioisomer 18 (20%). Here and in the succeeding glycosylations, we veried the a-orientations of the mannosidic bonds through the coupling constants of the anomeric carbons and protons (B170 Hz, see Supplementary Methods)47,48. Distinguishing the structures of 17 and 18 with condence is not possible with NMR analysis alone. We, therefore, resorted to exchange the primary TBDPS with benzyl group (Supplementary Fig. 3) and compare agreement with the NMR spectra from previously published data30.

For the preparation of the key intermediate 21, the pseudodisaccharide 17 was subjected to Zempln deacylation to generate the diol 19. Regioselective mannosylation at the equatorial hydroxy group should be more likely because of steric reasons. A thorough evaluation of the subsequent coupling also made us consider the application of the imidate 20 over the thioglycoside 13. Under BF3 Et2O promotion, compound 21

was, therefore, acquired in 72% yield with complete regioselectivity and stereoselectivity. The orthoformate group was cleaved using p-toluenesulfonic acid, which also removed the TBDPS group. The tetraol 22 was obtained after re-installation of the silyl group. With 22 in hand, regioselective benzylation at O4 and O5 of the inositol unit is the next challenge. Williamson condition and acidic benzylation using benzyl imidate are not sufciently selective, whereas the reductive benzylation of the trimethylsilylated substrate showed greater promise. True enough, excellent selectivity was achieved by using 3 equivalents of benzaldehyde, furnishing, after further full desilylation with TBAF, the desired compound 2 in 72% yield from 22.

It should be stated that other less successful means in acquiring the pseudotrisaccharide backbone have been studied. Our effort at condensation of the imidate donor 20 with the diol 16 led to mixtures of inseparable regioisomers and stereoisomers, a demonstration of the known potential of the bulky 6-O-TBDPS group at enhancing a-selectivity49. A participating moiety at

O2 of the mannosyl donor was ruled out to avoid complications that may be encountered in later reactions. Sequential dimannosylation of Kishis triol also seemed feasible under our synthetic design, with glycosylation at the more reactive O2 using donor 20 followed by asymmetric 6-O-mannosylation with donor 15. Unfortunately, poor yields for both couplings were observed. Another procedure we have tried included the 2-O-mannosylation of 19 with a donor already carrying the fatty acyl functionality at the primary position. While the glycosylation step worked as intended, the acyl moiety was also removed along with the TBDPS group on acid treatment intended to cleave the orthoformate function.

Synthesis of tuberculostearic acid and the H-phosphonate 4. Tuberculostearic acid was rst isolated from M. tuberculosis in 1927 (ref. 50) and several methods for its synthesis have been reported25,26,5153. Nevertheless, an updated, shorter and more effective method for accessing this important fatty acid is still desirable. We decided to acquire the chiral carbon of tuberculostearic acid from the commercially available Roche ester (23). Tosylation of 23 to afford compound 24, followed by reduction with diisobutylaluminium hydride and methylene insertion by Wittig reaction furnished the olen 25 (ref. 54; Fig. 4). The rst long-chain elongation of 25 towards compound 26 was achieved by Grignard reaction under catalytic Li2CuCl2.

Grubbs metathesis of olen 26 with the olenic acid 27 provided the E/Z olen mixture, which was exposed to palladium-catalysed hydrogenation to nally secure tuberculostearic acid (28). Accomplished in just six steps, this acquisition is the shortest synthetic preparation reported, thus far, for this compound.

Elaborations of the commercially available 3-O-benzyl-snglycerol were performed next. Under dicyclohexylcarbodiimide and 4-(N,N-dimethylamino)pyridine, the fatty acid 28 was rst condensed with the primary hydroxyl followed by stearic acid esterication at the secondary position in good yields. Cleavage of the benzyl group was achieved through hydrogenolysis and the generated alcohol was phosphorylated using PCl3, imidazole and

Et3N to afford the H-phosphonate 2.

Ac2PIM6 assembly and nal transformations. Our planned sugar assembly towards the tetramannoside 3 hinges on the chemoselective activation of a trichloroacetimidate donor in the presence of a thioglycoside acceptor55 (Fig. 5). Accordingly,

O

HO

6

MeO2C OR23: R = H

24: R = Ts

b c

OTs

d

6

a

25 26

27

Et3HN+

BnO

O

O

P

H

O

O

e

O

O

O

C17H35

C18H37

f

O

O C17H35 C18H37

HO

O

7

6

O

O

28 29 4

Figure 4 | Preparation of tuberculostearic acid (28) and the H-phosphonate 4. Reagents and conditions: (a) TsCl, Et3N, DMAP, 94%;(b) (1) diisobutylaluminium hydride, 78 C; (2) Ph3P CH2, 72% (two steps); (c) C7H15MgBr, Li2CuCl4, 78 to 0 C, 92%; (d) (1) Grubbs second-

generation catalyst, CH2Cl2, reux; (2) H2, Pd/C, 75% (two steps); (e) (1) 3-O-benzyl-sn-glycerol, DCC, DMAP, 75%; (2) stearic acid, DCC, DMAP, 88%; (f) (1) H2, Pd/C, 92%; (2) PCl3, imidazole, Et3N, 10 C, 69%. DCC, dicyclohexylcarbodiimide; Ts, Tosyl.

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BnO

BnO

OR BnO

BnO

OBn BnO

BnO

O BnO

BnO

BnO

O

BnO

O

BnO

O BnO

BnO

BnO

R

14: R = -STol

30: R = OC(=NH)CCl3

OH BnO

BnO

O

OBz BnO

BnO

O BnO

BnO

O

b

c

BnO

O

a e

BnO

O

O

O

OBn BnO

BnO

O BnO

BnO

O

O

O

a

O

STol

OBn BnO

BnO

O

OBn BnO

BnO

O

NH

31

STol

O

CCl3

d

33

32: R = Bz

3: R = Bn

RO

OR RO

RO

OBn BnO

BnO

BnO

O RO

RO

RO

O

BnO

O

RO

O

O BnO

BnO

O RO

RO

O

O BnO

BnO

g

BnO

O

O

O

OR RO

RO

O

OR

RO

O

OBn BnO

BnO

O

O

O

OR RO

RO

O

OBn

BnO OO

O

O

C17H35

OOR

O

RO

OBn BnO

BnO

OR

OOBn

O

OR

O

O

O

Na+

C17H35

O

O OHO

OBn OR'

BnO

P

O

O

O

O C17H35

C18H37

f

34: R = 2-NAP, R' = H

35: R = R' = C(=O)C17H35

h

36: R = Bn

1: R = H

O

Figure 5 | Synthesis of compound 1. Reagents and conditions: (a) (1) NBS, acetone, H2O; (2) CCl3CN, 1,8-diazabicyclo[5.4.0]undec-7-ene, 30: 96% (two steps), 33: 86% (two steps); (b) 8, TMSOTf, CH2Cl2, 78 C, then, NaOMe, MeOH, 87% (one pot); (c) (1) 30, TfOH, CH2Cl2, 60 to 40 C, then,

NaOMe, MeOH, 70% (one pot); (2) 30, TfOH, CH2Cl2, 60 to 40 C, 74%; (d) BnBr, NaH, 98%; (e) 2, TMSOTf, Et2O, 40 C, 52% (89% yield

based on the recovered 2); (f) (1) DDQ, CH2Cl2, H2O, 71%; (2) stearic acid, DCC, DMAP, 86%; (g) (1) 4, pivaloyl chloride, pyridine; (2) I2, pyridine, H2O;(3) DOWEX 50WX8 Na form, 77% from 35; (h) H2, Pd/C, 88%. TfOH, triuoromethanesulfonic acid.

the elongation unit is formed by converting compound 14 to 30 under the usual procedures. Glycosylation of the thioglycoside 8 with 30 followed by debenzoylation in the same ask supplied the disaccharide acceptor 31 in 87% yield. Two more elongation cycles easily formed the tetramannoside 32. Knowing that the benzoate group is base-sensitive, the benzoyl-to-benzyl exchange was achieved in one step by NaH and benzyl bromide treatment, smoothly offering the target compound 3 in 98% yield.

With all segment backbones available, we moved forward in putting all these pieces together. The glycosylation of the pseudotrisaccharide acceptor 2 by the thioglycoside 3, however, produced only a meager 10% yield for compound 34 despite our best efforts, prompting us to use the more reactive imidate counterpart 33 instead (see Supplementary Table 2). With Et2O as solvent and TMSOTf as activator, we eventually obtained the desired 34 in 52% yield (89%, if the recovered acceptor is considered). The 2-NAP ether was then cleaved, which paved the way for the concurrent installation of two stearate esters at the mannosyl and the inositol units, leading to the alcohol 35. This reaction exhibited no regioselectivity issues, with O1 of the inositol unit spared because it experiences the highest steric hindrance among the three free hydroxyls. The attachment of the H-phosphonate 4 and the pseudo-heptasaccharide 35 was carried out by using pivaloyl chloride,

followed by iodine-mediated in situ oxidation and cation exchange, delivering the derivative 36. Global hydrogenolysis of the benzyl ethers provided the Ac2PIM6 construct 1 in 82%

yield.

Evaluation of immunomodulatory activity. The adjuvant effects of compound 1 were examined through co-administration with ovalbumin (Fig. 6a) or tetanus toxoid (Fig. 6b) antigen in BALB/c mice. PIMs isolated from M. tuberculosis strain H37Rv (iPIM1,2

and iPIM6) and alum were also investigated in parallel for comparison. It was observed that compound 1 induced an approximately two to fourfold increase in the level of antigen-specic antibodies. The adjuvant activity of 1 is similar to the bacteria-derived PIMs and slightly lower than alum.

Furthermore, we evaluated the cytokine-producing activity of compound 1 as well as iPIM1,2 and iPIM6 (Fig. 6c,d). The level of interleukin-4 and interferon-g was not detectable in mouse sera at 1 h after injection of Ac2PIM 1 and the bacteria-derived PIMs. At 18 h after injection, the cytokine levels increased. Lipid and glycolipid molecules derived from M. tuberculosis are presented to T cells by CD1 antigen-presenting molecules, specically CD1d14,56. Compared with the well-known CD1d-targeting a-galactosylceramide, which can activate the invariant natural killer T cells and induce high levels of interleukin-4 and

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a

51,200

b

51,200

*

Ag (ovalbumin)

25,600

25,600

*

12,800

*

*

*

12,800

* *

End point titre of

Ag-specific antibodies

End point titre of

Ag-specific antibodies

6,400

6,400

*

3,200

3,200

1,600

1,600

800

800

400

400

Background

<100

<100

No adjuvant

+ Alum

+ iPIM 6

+ iPIM 1,2

+ Ac 2PIM 6 1

Background

No adjuvant

+ Alum

+ iPIM 6

+ iPIM 1,2

+ Ac 2PIM 6 1

Ag (tetanus toxoid)

c

60

0 Control

d

300

Control

Interleukin-4 (pg ml1 )

Interferon-(pg ml1 )

40

200

20

100

0

iPIM 6

iPIM 1,2

Ac 2PIM 6 1

Control

1 h 18 h

iPIM 6

iPIM 1,2

Ac 2PIM 6 1

iPIM 6

iPIM 1,2

Ac 2PIM 6 1

Control

iPIM 6

iPIM 1,2

Ac 2PIM 6 1

1 h 18 h

Figure 6 | Immunological evaluation in BALB/c mice. (a,b) Induction of antigen (Ag)-specic antibodies in mice immunized with ovalbumin or tetanus toxoid adjuvanted with alum or various PIMs; (c,d) secreted cytokines (interleukin-4 and interferon-g) in mice 1 and 18 h after injection with various PIMs (control represents injection only with PBS). Both end point antibody titres and the cytokine levels were measured by using enzyme-linked immunosorbent assay. The results displayed represent the mean s.d.s (n 5). Data were analysed by using one-way analysis of variance, and differences were

considered signicant at *Po0.05. iPIM6 and iPIM1,2, isolated PIM6 and mixture of PIM1 and PIM2, respectively, from M. tuberculosis strain H37Rv.

interferon-g within 24 h (ref. 57), Ac2PIM6 1 appeared to have moderate effects.

DiscussionWe have successfully developed a convenient route to synthesize an Ac2PIM6 construct in the form of compound 1 containing tuberculostearic acid and stearic acid as the fatty acid components. This is the rst time that an Ac2PIM6 molecule was synthesized. Further, a novel and short synthetic route towards tuberculostearic acid was developed, with only six synthetic steps from the commercially available Roche ester and four purication stages. Our synthetic approach benetted from the use of shared mannoside-building blocks, the carefully chosen orthogonal protecting groups and the features of the regioselective one-pot transformations from trimethylsilylated starting materials previously developed by us. The trichloroacetimidate donor types58 are vital factors in achieving the successful assembly processes. Regioselectivity and stereoselectivity were achieved through the aid of steric and stereoelectronic effects. Steric effects were also exploited in the direct desymmetrization of myo-inositol by mannosyl donors and in minimizing the number of protecting groups used in the synthesis. With practical access and functional group exibility, the key intermediates such as the pseudotrisaccharide 2 possess good potential in supplying PIMs of different mannosylation and lipidation patterns as well. Our synthesized Ac2PIM6 has comparable adjuvant activity with the natural PIMs against ovalbumin and tetanus toxoid antigens and induced the production of interleukin-4 and interferon-g, thus,

validating the immunological qualities of PIM molecules and its value in vaccine research.

Methods

Chemical synthesis. The complete experimental details and compound characterization data can be found in the Supplementary Methods. For the NMR spectra of the compounds in this article, see Supplementary Figs 4120. The mass spectrum of the synthesized Ac2PIM6 1 is shown in Supplementary Fig. 121.

Materials for immunological evaluation. All BALB/c mice were housed at the animal facility in the Institute of Cell Biology, Academia Sinica, Taiwan in accordance with the Institutional Animal Care Committee guidelines. Puried iPIM1,2 (NR-14846) and iPIM6 (NR-14847) were obtained through BEI Resources,

National Institute of Allergy and Infectious Diseases, National Institutes of Health (USA). Ovalbumin and tetanus toxoid were purchased from InvivoGen (San Diego, CA, USA) and Adimmune Inc. (Taichung, Taiwan), respectively.

Evaluation of adjuvant activity. Five- to six-week-old female BALB/c mice were immunized with ovalbumin (100 mg) or tetanus toxoid (2 mg) adjuvanted with 10 mg of PIM compounds (Ac2PIM6 1, iPIM1,2 or iPIM6) or alum in PBS for three times at 2-week intervals by intramuscular injection. Two weeks after the third immunization, the immunized mice were bled for antigen-specic antibody analysis.

Ovalbumin- and tetanus toxoid-specic antibodies in heat-inactivated serum were monitored with direct enzyme-linked immunosorbent assay (ELISA). The ovalbumin- or tetanus toxoid-coated plates were incubated with mouse serum in twofold serial dilutions for 1 h. Antigen-specic IgG was monitored by using horseradish peroxidase-conjugated anti-mouse antibodies and 3,30,5,50-tetramethylbenzidine substrate (Thermo Scientic Inc). After colour development, absorbance at 450 nm was recorded by using a plate reader (SpectraMax M5, Molecular Device). The end point antibody titre was dened as the highest dilution of serum to produce an absorbance 2.5 times higher than the optical absorbance produced by the pre-immune serum. The background end point antibody titre was assigned as o1:100.

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Evaluation of cytokine-producing activity. Five- to six-week-old female BALB/c mice were intramuscularly injected with 10 mg of the PIM compounds (Ac2PIM6 1, iPIM1,2 or iPIM6) in PBS and bled at 1 or 18 h after injection (ve mice per group). The cytokines in the sera were measured with sandwich ELISA using paired anti-interleukin-4 and anti-interferon-g monoclonal antibodies (R&D Systems).

Statistical analysis. The response of each mouse was counted as an individual data point for statistical analysis. Data obtained from animal studies were analysed by using one-way analysis of variance from Graphpad and differences were considered signicant at Po0.05.

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Acknowledgements

This work was supported by the Ministry of Science and Technology (MOST 100-2113-M-001-019-MY3 and MOST 102-2628-M-001-001), National Health Research Institute (NHRI-EX103-10146NI) and Academia Sinica.

Author contributions

S.-C.H. designed the study, supervised students and staffs, and nalized the manuscript preparation. P.S.P. performed the synthesis of Ac2PIM6 1. T.-J.R.C. and S.-T.Y. carried out the evaluation of immunological activity. M.M.L.Z. prepared the gures and wrote

the manuscript. L.S.L. is involved in the early stages of manuscript preparation and assisted in compiling the Supplementary Information. All authors discussed the results and commented on the manuscript.

Additional information

Accession codes: The X-ray crystallographic coordinates for compounds 10 and 13 in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition numbers CCDC 1040371 and CCDC 1040372, respectively. These data can be obtained free of charge from the CCDC via http://www.ccdc.cam.ac.uk/data_request/cif

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How to cite this article: Patil, P. S. et al. Total synthesis of tetraacylated phosphatidylinositol hexamannoside and evaluation of its immunomodulatory activity. Nat. Commun. 6:7239 doi: 10.1038/ncomms8239 (2015).

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