Synthesis of a pentasaccharide and neoglycoconjugates related to fungal α-(1→3)-glucan and their use in the generation of antibodies to trace Aspergillus fumigatus cell wall.

DOI: 10.1002/chem.201404770

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Synthesis of a Pentasaccharide and Neoglycoconjugates Related to Fungal a-(1!3)-Glucan and Their Use in the Generation of Antibodies to Trace Aspergillus fumigatus Cell Wall Bozhena S. Komarova,[a] Maria V. Orekhova,[a] Yury E. Tsvetkov,[a] Remi Beau,[b] Vishukumar Aimanianda,[b] Jean-Paul Latg,[b] and Nikolay E. Nifantiev*[a]

synthesis of pentasaccharides. Coupling of free a-(1!3)pentaglucoside with biotin and bovine serum albumin (BSA) gave glycoconjugate tools for mycological studies. Immunization of mice with the BSA conjugate induced the generation of antibodies that recognize a-(1!3)-glucan on A. fumigatus cell wall and distinguish its morphotypes. This discovery represents a first step to the development of a diagnostic test system and a vaccine to detect and fight this life-threatening pathogen.

Abstract: 3-Aminopropyl a-(1!3)-pentaglucoside, a fragment of a-(1!3)-glucan of the cell wall of Aspergillus fumigatus, has been synthesized in a blockwise approach. The application of mono- and disaccharide N-phenyltrifluoroacetimidates bearing a stereodirecting 6-O-benzoyl group was essential for stereoselective a-glucosylations. In the products, p-methoxyphenyl and levulinoyl groups served as orthogonal protecting groups for the anomeric position and 3-OH group, respectively. Their removal from shared blocks led to donors and acceptors that were used for the

Introduction

as macrophages and neutrophils). Treg cells function to maintain self-tolerance and immune homeostasis. Th2 inhibition suppresses the humoral immune system. Although the a-(1!3)-glucans have been shown to be essential in the pathogenic life of A. fumigatus,[1] their immunological role has not been properly analyzed because the insolubility of the a-(1!3)-glucans in water makes such investigations difficult. To date, the phagocyte receptor for a-(1!3)glucans is unknown and has not been investigated because of the lack of soluble a-(1!3)-glucans that could be used as a ligand. Moreover, although the association of T cell immunity with the protective function of a-(1!3)-glucans has been demonstrated, the putative protective role of anti-a-(1!3)-glucans antibodies has not been investigated. On the other hand, passive administration of monoclonal antibodies (mAb) to bglucans has been shown to be protective.[7] This could also be the case for anti-a-(1!3)-glucan antibodies because it was shown that a-(1!3)-glucans play an essential role in conidial swelling, which is the first step of vegetative growth. A combination of mAb directed against A. fumigatus cell wall polysaccharides (including a-(1!3)-glucans) has also been shown to reduce the severity of allergic airways diseases in mice.[8] Anti-a-(1!3)-glucan antibodies have been described, but the immunization protocols leading to B cell activation remains empirical. For example, polyclonal antibodies were raised in rabbits inoculated with a-(1!3)-glucans coupled to BSA.[9] Kearney et al.[10] generated MAbs after inoculation of bacteria of the Enteobacteriacae family. A commercial antibody, MOPC104e (Sigma), was shown to bind specifically to a-(1!3)-

a-(1!3)-Glucan is a major cell wall component of most ascomycete and basidiomycete fungal pathogens,[1, 2] which establish their disease upon inhalation of their infective morphotypes. In A. fumigatus, the most ubiquitous human pathogen, a-(1!3)-glucan accounts for 19–40 % of the conidial and mycelial cell wall polysaccharides, respectively.[3] a(1!3)-Glucan is the major cell wall polysaccharide involved in the aggregation of germinating conidia and biofilm formation.[4, 5] Moreover, it has been shown in experimental murine aspergillosis models that a-(1!3)-glucans have a prominent immunological role.[6] Repeated inoculations of a-(1!3)-glucan before immunosuppression confer a long-term survival of mice following intranasal inoculation of the fungus. Immune protection was associated with a reduced inflammatory pathology,[6] which is linked to Th1/Treg activation and concomitant Th2 inhibition. Th1 activation results in stimulation of the cellular immune system, maximizing killing efficiency of the phagocytes (such [a] Dr. B. S. Komarova, M. V. Orekhova, Dr. Y. E. Tsvetkov, Prof. N. E. Nifantiev Laboratory of Glycoconjugate Chemistry N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences Leninsky Prospect 47, 119991 Moscow (Russia) E-mail: [email protected] [b] R. Beau, Dr. V. Aimanianda, Prof. J.-P. Latg Unit des Aspergillus, Institut Pasteur 25 rue du Docteur Roux, 75724 Paris Cedex 15 (France) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404770. Chem. Eur. J. 2014, 20, 1 – 8

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Full Paper glucans; it is an IgM produced in the BALB/c mice ascites upon intraperitoneal injection of the MOPC-104e tumor line.[11] Accordingly, the availability of soluble oligo-a-(1!3)-glucan conjugates produced by chemical synthesis would allow better control of antibody production with better definition of the epitope recognized and is expected to help in the identification of the human receptor for this polysaccharide. However, the synthesis of a-(1!3)-glucooligosaccharides of predetermined lengths is not a simple task. Preparation of homologous a-(1!3)-oligoglucosides from di- up to the nonasaccharides by partial acid hydrolysis of natural polysaccharides followed by laborious chromatographic isolation of the individual oligomers has been described.[12] a-(1!3)-Linked di- and trisaccharides were also obtained by acetolysis of glucan.[13] Only a few chemical syntheses of a-(1!3)-glucooligosaccharides have been reported. Some works described the formation of a-(1! 3)-linked disaccharide derivatives as a result of model glycosylations,[14] whereas the targeted synthesis of homologous a(1!3)-glucooligosaccharides composed of two to five monosaccharides was reported in only a single publication.[15] It should be noted that all oligosaccharides of this type, both synthetic and those isolated from polysaccharide hydrolysates, contain a free reducing end. To our knowledge, the synthesis of a-(1!3)-glucooligosaccharides functionalized at the reducing end as well as their further application for the preparation of glycoconjugates has not been reported. In this communication, we describe an efficient blockwise approach to the preparation of 3-aminopropyl glycosides of a-(1!3)glucooligosaccharides illustrated by the synthesis of a pentasaccharide, its transformation into conjugates with biotin and BSA, and we present our first results on the use of these conjugates in immunizing mice to obtain antibodies against a-(1!3)-glucans.

group as a temporary protection of 3-OH because published data[18] as well as our own recent experience[16b,c, 19] indicated that this group can be selectively removed by mild acidic methanolysis in the presence of benzoyl groups. Thus, the glucosyl bromide obtained in situ from hemiacetal 4[16d] was coupled with protected 3-aminopropanol under the conditions of Lemieux glycosylation (Scheme 1). As a result, a-glucoside 5 was obtained in good yield. However, the 3-O-acetyl group in 5 proved to be unusually stable; its complete removal required prolonged reaction time and was accompanied by competitive splitting of the 6-O-benzoyl and N-trifluoroacetyl groups. Therefore, additional steps of 6-O-benzoylation and N-trifluoroacetylation were necessary to obtain the deacetylation product 6, which was isolated in modest yield (28 %). Further glycosylation of 6 with disaccharide N-phenyltrifluoroacetimidate 3 (prepared from p-methoxyphenyl glycoside 1[16d] via hemiacetal 2) afforded trisaccharide 7, but its yield was unsatisfactory. These results clearly indicated that the acetyl group was not suitable for the temporary protection of the 3-OH group, and that the reactivity of the latter in the glycosyl acceptor 6 was too low to employ it in the assembly of the target oligoglucosides. We then examined the use of a levulinoyl group as an alternative to the acetyl group as the temporary 3-O-protecting group. A benzylidene group was used for protection of O-4 and O-6 in the hope of achieving higher reactivity of the 3-OH group in the corresponding glycosyl acceptor 13. The latter was prepared as depicted in Scheme 2. p-Methoxyphenyl glucoside 8[16d] was treated with levulinic acid in the presence

Results and Discussion The prerequisite for effective preparation of sufficiently long a(1!3)-glucooligosaccharides is high a-stereoselectivity of each glycosylation step. Recently, we have shown[16] that 3,6-di-Oacyl-2,4-di-O-benzyl-d-glucopyranosyl N-phenyltrifluoroacetimidates as glucosyl donors meet this requirement, providing high stereoselectivity of glycosylation presumably due to remote anchimeric assistance of the acyl groups.[17] In addition, model experiments showed that the presence of an acyl group only at O-6 of the donor molecule was sufficient to achieve high a-stereoselectivity of (1!3)-glycosylation.[16d] This observation enabled us to use disaccharide and potentially higher oligosaccharide donor blocks, containing a 6-O-acyl group in the glycosylating glucose unit, for a-(1!3) chain elongation without loss of a-stereoselectivity. Such a convergent approach is clearly more effective for the synthesis of higher glucose oligomers than chain elongation by one monosaccharide residue. We intended to synthesize the target oligomers as 3-aminopropyl glycosides, therefore the coupling of 3-trifluoroacetamidopropanol with an appropriate glucosyl donor had to be carried out in the first step. Initially we planned to use an acetyl &

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Scheme 1. First approach to a-(1!3)-linked trisaccharide acceptor. Reagents and conditions: a) CAN, CH3CN (aq. 80 %); b) ClC(= NPh)CF3, K2CO3, acetone; c) CBr4, Ph3P, CH2Cl2 ; d) Bu4NBr, CH2Cl2 ; e) 1.6 m anhydrous HCl in MeOH (76 %); f) CF3CO2Et, Et3N (50 %); g) BzCl, Py (74 %); h) AgOTf, CH2Cl2, 35! 15 8C.

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Scheme 2. Assembly of the pentasaccharide. Reagents and conditions: a) LevOH, DCC, DMAP, CH2Cl2 ; b) CAN, CH3CN (aq. 80 %) (benzene was added for preparation of 15 and 18) (78–81 %); c) CBr4, Ph3P, CH2Cl2 ; d) HO(CH2)3NHTFA, Bu4NBr, CH2Cl2 ; e) NH2NH2·H2O, AcOH, Py; f) ClC(= NPh)CF3, K2CO3, acetone (94– 95 %); g) MeOTf, CH2Cl2, 35!15 8C.

solvent, and temperature, was carried out before the preparative synthesis of disaccharide 17 (Table 1). The conditions developed (Table 1, entry 6) provided excellent efficiency and a-stereoselectivity of glycosylation and these were used for the preparative synthesis of 17, which was thus obtained in 90 % isolated yield. Further conversion of 17 into disaccharide donor 19 was performed as described above for the transformation of 14 into 16. Glycosylation of the 4,6O-benzylidene protected acceptor 12 with imidate 19 provided much higher yield of the trisaccharide 20 than the yield of similar trisaccharide 7 achieved on glycosylation of 6 with 3. As expected, the presence of a single stereodirecting 6-O-benzoyl group in the glycosylating unit of donor 19 was sufficient to ensure very high a-selectivity of glycosylation (a/b = 14:1). Disaccharide imidate 19 afforded exclusively a-linked pentasaccharide 22; no formation of the corresponding b-anomer was detected.

of N,N-dicyclohexylcarbodiimide (DCC) and 4-(N,N-dimethylamino)pyridine (DMAP) to provide ester 9 in high yield. Removal of the anomeric group in 9 with cerium(IV) ammonium nitrate (CAN) afforded hemiacetal 10, which was converted into the corresponding bromide by treatment with CBr4 and Ph3P. Halide-catalyzed coupling of the bromide with protected 3aminopropanol gave a-glucoside 11 in good yield. Removal of the levulinoyl group in 11 with hydrazine acetate in pyridine smoothly gave the required acceptor 12 in high yield, thus demonstrating the applicability of this group for temporary protection of 3-OH. Disaccharide donor 19 was obtained from the single precursor 13.[16d] Introduction of the levulinoyl group into 13 provided ester 14, which had to be converted into the corresponding hemiacetal. The yield of the anomeric deprotection of 14 under published conditions, that is, by treatment with CAN in a two-phase solvent mixture of acetonitrile/water/toluene, did not exceed 70 %. This yield seemed to be insufficiently high, particularly in the case of the valuable disaccharide block. An attempt to improve the yield by performing the reaction in aqueous acetonitrile, that is, under homogeneous conditions, failed because of the poor solubility of 14. Application of Nbromosuccinimide (NBS) as an oxidant did not lead to any improvement. Surprisingly, the use of benzene instead of toluene in the above two-phase solvent mixture resulted in increased yield of the hemiacetal 15 to 80 %. Treatment of the hemiacetal with N-phenyltrifluoroacetimidoyl chloride provided imidate 16. A series of model couplings of 13 with 16, aimed at optimization of the reaction conditions with respect to the promoter, Chem. Eur. J. 2014, 20, 1 – 8

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Table 1. Optimization of the reaction conditions of glycosylation of 13 with 16 Entry

Solvent

Promoter[a]

T [8C]

Yield [%] (a/b)

1 2 3 4 5 6

CH2Cl2 ClCH2CH2Cl Et2O CH2Cl2 CH2Cl2 CH2Cl2

MeOTf MeOTf MeOTf AgOTf TfOH[b] MeOTf

35 30 35 20 35 35!20

78 82 22 58 92 96

(27:1) (16:1) (25:1) (5:1) (26:1) (27:1)

[a] Except for entry 5, donor excess was 20 %. [b] Donor excess was 85 %.

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Full Paper 100.6 ppm) in the 13C NMR spectrum were in agreement with the published data for a-(1!3)-glucooligosaccharides.[12, 15, 20] Low-field location of signals for C-3 (d = 81.1–81.3 ppm) compared with that of unsubstituted a-glucopyranosides (d 74 ppm) confirmed the position of glycoside bonds. Acylation of the amino group in the aglycon of 25 with active ester 26,[19c] which derived from biotin equipped with a hexaethylene glycol linker, afforded conjugate 27. The attachment of 25 to BSA was carried out by using the squarate procedure.[22] In the first step, 25 was acylated with diethyl squarate at pH 7 to produce monoamide 28. Further conjugation of 28 with BSA at pH 9 provided conjugate 29. According to MALDI TOF data, the latter compound contained 18 pentasaccharide residues per BSA molecule on average. The obtained conjugate 29 (penta-a-(1!3)-gluco-BSA) was used to immunize mice. The BALB/c mice immunization with penta-a-(1!3)-gluco-BSA resulted in the induction of antibody production (Figure 1). In contrast to the production of polyclonal antibodies upon immunization with penta-a-(1!3)-glucoBSA, repeated immunization (five times with 15 day intervals) with native water-insoluble a-(1!3)-glucan (extracted from the A. fumigatus cell wall) suspension or penta-a-(1!3)gluco-Biotin did not induce the production of antibodies. Furthermore, a series of A. fumigatus morphotypes were immunolabelled with the anti-a-(1!3)-glucan mouse hyperimmune serum (Figure 2, Row A). In these testes, the p-formaldehyde fixed-permeabilized swollen and germinating conidia were positive whereas the dormant conidia were negative.

Five signals for anomeric protons were observed in the H NMR spectrum of 22; each of them appeared as a doublet with J1,2 < 4 Hz, thus proving the a-configuration of all glucose residues. In conclusion to this part of the work, we have elaborated an efficient blockwise approach to the assembly of a-(1!3)glucooligosaccharides as well as a set of mono- and oligosaccharide building blocks for its implementation. Compound 22 can be easily converted into a pentasaccharide acceptor 23 (Scheme 3) and subjected to further iterative chain elongation with the donor 19, thus opening access to longer oligomers. Alternatively, the protecting group pattern in the disaccharide block 17 allows its simple transformation into tri- or tetrasaccharide donor blocks by removal of the levulinoyl group followed by coupling with imidates 16 or 19 and modification of the anomeric position of the glucose unit at the reducing end. The transformation of protected pentasaccharide 22 into free pentamer 25 (Scheme 3) included removal of the levulinoyl group (!23), catalytic hydrogenolysis of the benzylidene and benzyl groups (!24), and one-pot debenzoylation and splitting of the N-trifluoroacetyl group by alkaline hydrolysis. The structure of 25 was unambiguously confirmed by 1H and 13 C NMR data. Although coupling constant values J1,2 in the 1H NMR spectrum of 25 could not be measured because of the overlap of individual signals for H-1, their chemical shifts (d = 5.37–5.40) were characteristic for the a-(1!3)-linkage[20] and strongly differed from those for the b-(1!3)-linkage (d 4.75).[21] Chemical shifts of signals for C-1 (d = 99.7– 1

Scheme 3. Preparation of the free pentasaccharide and its conjugation with biotin and BSA. Reagents and conditions: a) NH2NH2·H2O, AcOH, Py; b) H2, Pd(OH)2/C, MeOH; c) NaOH, MeOH; d) NaOH, H2O; e) diethyl squarate, aq EtOH, Et3N, pH 7; f) borate buffer, pH 9.

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Full Paper germinating conidia, which lacks a-(1!3)-glucan in their cell wall, confirmed the specificity of the polyclonal antibodies (Figure 2, Row B). In addition, preincubation of the serum containing polyclonal antibodies with a-(1!3)-glucan extracted from the A. fumigatus cell wall or a-(1!3)-gluco-oligosaccharide mixture (degree of polymerization, 8–14) obtained from the A. fumigatus cell wall a-(1!3)-glucan induced the loss of labeling by the serum polyclonal antibodies (Figure 2, Row C).

Conclusion The 3-aminopropyl glycoside of a pentasaccharide fragment of a-(1!3)-glucan of A. fumigatus cell wall has been synthesized in a blockwise manner. Highly efficient and stereoselective aglucosylation was ensured because of the use of the glucosyl donors with the N-phenyltrifluoroacetimidoyl leaving group and the 6-O-benzoyl protecting group, which is capable of the remote anomeric stereocontrol. The 3-O-levulinoyl group proved to be much more effective than the acetyl group for temporary protection of the glycosylation site. The BSA conjugate of the synthetic pentasaccharide was used for raising antibodies in mice. These polyclonal antibodies were shown to be effective for immunolabeling a-(1!3)glucan positive morphotypes of A. fumigatus. These data show that synthetic glycoconjugates of the described types represent very useful tools for the investigation of fungal cell-wall a-(1!3)-glucan. Further synthesis of longer a-(1!3)-gluco-oligosaccharides and glycoconjugates thereof is in progress to understand the immunogenicity of this molecule and to evaluate their vaccine potential as well as their suitability for diagnostics development. From the therapeutic viewpoint, adjunctive combination therapy to maximize antifungal efficacy is known. Accordingly, in the case of invasive aspergillosis, treatment with antifungal drugs in combination with a glycoconjugate vaccine based on synthetic ligands related to fungal cell wall polysaccharides or monoclonal antibodies raised against such carbohydrate chains are worth testing.

Figure 1. Raising the polyclonal antibodies against a-(1!3)-pentamer-BSA in mice. Immunization details are described in the Experimental Section.

Experimental Section Conjugate of the pentasaccharide with biotin (27)

Figure 2. Immunofluorescence labeling (IFL) of the wild-type and Dags mutant A. fumigatus conidial morphotypes with polyclonal antibodies: Row A: IFL of the p-formaldehyde fixed-permeabilized wild-type swollen conidia and germ tubes with penta-a-(1!3)-gluco-BSA antiserum from mice; Bright field (BF; visible light) and fluorescence microscopy studies: Row B: Triple Dags mutant swollen conidium (lacking cell wall a-(1!3)-glucan) was negative for immunolabeling by mouse serum containing polyclonal antibodies raised against penta-a-(1!3)-gluco-BSA; Row C: Labeling of wild-type germinating conidia was abolished upon prior incubation of the polyclonal antibodies with a-(1!3)-glucan extracted and purified from A. fumigatus cell wall (scale bar 2 mm).

A solution of 26 (0.062 m in DMF, 46.8 mL) and anhydrous triethylamine (11.6 mL) were added to a solution of 25 (2 mg, 0.0023 mmol) in anhydrous DMF (100 mL). After 12 h, the solvent was removed under reduced pressure and the product was purified by gel-permeation chromatography (TSK HW-40(S), 0.1 m AcOH). The appropriate fractions were freeze-dried to give 27 (2.5 mg, 76 %) as a fluffy solid; Rf = 0.44 (CHCl3/MeOH/H2O, 5:5:1); 1H NMR (600 MHz, D2O): d = 5.40–5.36 (m, 4 H; H-1b, H-1c, H-1d, H-1e), 4.93 (d, J(1a,2a) = 3.8 Hz, 1 H; H-1a), 4.63 (dd, J(6a,6A) = 4.9 Hz, J(6a,3a) = 8.0 Hz, 1 H; H-6a), 4.44 (dd, J(3a,4) = 4.5 Hz, J(3a,6a) = 8.0 Hz, 1 H; H3a), 4.08–4.01 (m, 4 H; H-5b, H-5c, H-5d, H-5e), 3.93–3.89 (m, 3 H; H3b, H-3c, H-3d), 3.89–3.83 (m, 6 H; H-3a, H-6Aa, H-6Ab, H-6 Ac, H-6Ad, H-6Ae), 3.83–3.74 (m, 9 H; H-6Ba, H-6Bb, H-6Bc, H-6Bd, H-6Be, H-3e, NC(O)CH2CH2O, OCH2CH2CH2NA), 3.73–3.63 (m, 30 H; H-4a, H-4b, H-

The specificity of the polyclonal antibodies raised was verified by using A. fumigatus lacking a-(1!3)-glucan (triple Dags mutant[1, 23]). The negative labeling of the triple Dags mutant Chem. Eur. J. 2014, 20, 1 – 8

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Full Paper 4c, H-4d, H-2a, H-2b, H-2c, H-2d, H-5a, 5 OCH2CH2O), 3.65 (t, J = 5.4 Hz, 2 H; OCH2CH2N), 3.60–3.54 (m, 2 H; H-2e, OCH2CH2CH2NB), 3.45 (t, J(4e,3e) = J(4e,2e) = 9.7 Hz, 1 H; H-4e), 3.41 (t, J = 5.4 Hz, 2 H; OCH2CH2N), 3.38–3.31 (m, 3 H; H-4, OCH2CH2CH2N), 3.02 (dd, J(6A,6a) = 5.0 Hz, J(6A,6B) = 13.0 Hz, 1 H; H-6A), 2.80 (d, J(6B,6A) = 13.0 Hz, 1 H; H-6B), 2.54 (t, J = 6.2 Hz, 2 H; NC(O)CH2CH2O), 2.29 (t, J = 7.3 Hz, 2 H; C(O)CH2CH2CH2CH2), 1.89 (m, 2 H; OCH2CH2CH2N), 1.75 (m, 1 H; C(O)CH2CH2CH2CH2A), 1.71–1.58 (m, 3 H; C(O)CH2CH2CH2CH2, C(O)CH2CH2CH2CH2A), 1.47–1.41 ppm (m, 2 H; C(O)CH2CH2CH2CH2); 13C NMR (150.9 MHz, D2O): d = 177.7, 174.7 (C(O)), 100.11, 100.02, 99.95, 99.04 (C-1b, C-1c, C-1d, C-1e), 99.0 (C1a), 80.9, 80.8, 80.7 (C-3b, C-3d, C-3e, C-3a), 73.6 (C-3e), 72.6, 72.5, 72.4 (C-5a, C-5b, C-5c, C-5d, C-5e, C-2e), 70.7, 70.63, 70.58, 70.4, 70.3, 70.2 (C-2a, C-2b, C-2c, C-2d, C-4a, C-4b, C-4c, C-4d, C-4e,), 69.6 (NC(O)CH2CH2O), 67,6 (C(O)CH2CH2O), 66.2 (OCH2CH2CH2N), 62.8 (C3a), 61.3, 61.13, 61.05 (C-6a, C-6b, C-6c, C-6d, C-6e, C-6a, C-6a), 56.1 (C-4), 40.4 (C-6), 39.7 (NC(O)CH2CH2O), 37.3 (OCH2CH2CH2N), 36.9 (NC(O)CH2CH2O), 36.2 (C(O)CH2CH2CH2CH2), 29.0 (OCH2CH2CH2N), 28.6, 28.5 (C(O)CH2CH2CH2CH2, C(O)CH2CH2CH2CH2), 25.9 ppm (C(O)CH2CH2CH2CH2); HRMS (ESI): m/z calcd for C58H102N4O35S + Na: 1469.5938 [M+Na] + ; found: 1469.5926.

Mouse immunization with the conjugate of the pentasaccharide with BSA (29) Two female BALB/c mice were injected with 20 mg of a-(1!3)-pentamer-BSA at 15 day intervals (first injection with Freund’s complete adjuvant and the booster doses with Freund’s incomplete adjuvant). After five boosts with 15 day intervals, the serum titer was estimated by ELISA. For that purpose, microtiter plates were coated with 10 mg mL1 penta-m-(1!3)-gluco-BSA as described earlier.[24] When mice serum samples diluted at 1:5000 were positive (for which serum samples were collected from the orbital vein), then mice were sacrificed and serum samples were collected. Control wells coated with BSA (10 mg mL1, 100 mL per well) were included in the assay to verify the lack of cross-reactivity of the polyclonal antibodies with BSA. Experiments on mice were approved by the Institut Pasteur Institutional Ethics Committee (C2EA n889) under the approval statement #2013-0055 issued on March 13, 2013.

Immunolabeling assays The A. fumigatus conidial (dormant, swollen and germinating) morphotypes were fixed with 2.5 % p-formaldehyde and subjected to permeabilization as described earlier (Harris et al., 1994).[25] Assays showed that permeabilization was a pre-requisite for positive labeling. After fixation and permeabilization A. fumigatus morphotypes were immunolabeled with anti-penta-a-(1!3)-gluco-BSA hyperimmune mouse serum diluted 1:100 in PBS containing 1 % BSA using mouse whole IgG conjugated to FITC as the secondary antibodies. Labeling of the triple Dags deletion mutant, devoid of cell wall a-(1!3)-glucan,[23] was also undertaken as described for the wild type A. fumigatus morphotypes. Inhibition of the immunolabeling was undertaken upon prior incubation of the hyper-immune serum (diluted 1:100) with a-(1!3)-glucan (10 mg) or a-(1!3)gluco-oligosaccharide mixture (10 mg) of degree of polymerization 8–14 produced as described earlier.[5]

3-(3,4-Dioxo-2-ethoxycyclobut-1-enylamino)propyl a-dglucopyranosyl-(1!3)-a-d-glucopyranosyl-(1!3)-a-d-glucopyranosyl-(1!3)-a-d-glucopyranosyl-(1!3)-a-d-glucopyranoside (28) Diethyl squarate (2.6 mL, 0.018 mmol) and Et3N (2 mL) were added to a solution of 25 (11.5 mg, 0.013 mmol) in aq 50 % ethanol (0.8 mL). When TLC showed the disappearance of starting material (ca. 30 min), the mixture was concentrated, the residue was dissolved in water (1 mL) and applied on a Sep-Pak C-18 cartridge. The cartridge was washed with water, then with aqueous methanol in 2 mL portions increasing the concentration of methanol from 5 to 20 %. The appropriate fractions were concentrated, the residue was dissolved in water, and the solution was freeze-dried to give 28 (11 mg, 84 %); Rf = 0.55 (n-butanol/ethanol/H2O/15 % NH4OH, 1 0.5:1:0.8:0.4); [a]24 D = + 170.0 (c = 1, H2O); H NMR (600 MHz, D2O): d = 5.39–5.34 (m, 4 H; H-1b, H-1c, H-1d, H-1e), 4.93 (d, J(1a,2a) = 3.7 Hz, 1 H; H-1a), 4.07–4.00 (m, 4 H; H-5b, H-5c, H-5d, H-5e), 3.94– 3.58 (m; 2H-6a, 2H-6b, 2H-6c, 2H-6d, 2H-6e, H-5a, H-3a, H-3b, H-3c, H3d, H-3e, H-2a, H-2b, H-2c, H-2d, H-2e, H-4a, H-4b, H-4c, H-4d), 3.44 (t, J(4e,3e) = J(4e,5e) = 9.6 Hz, 1 H; H-4e), 2.05–1.94 (m, 2 H; OCH2CH3), 1.95 ppm (m, 3 H; OCH2CH3); 13C NMR (150.9 MHz, D2O): d = 100.6, 100.4 (C-1b, C-1c, C-1d, C-1e), 99.6 (C-1a), 81.3, 81.2 (C-3b, C-3d, C-3e, C-3a), 74.1 (C-3e), 73.1, 73.06, 72.95, 72.8 (C-5a, C-5b, C-5c, C-5d, C-5e, C-2e), 71.60, 71.00, 70.7 (C-2a, C-2b, C-2c, C-2d, C-4a, C-4b, C-4c, C-4d, C-4e), 66.4, 66.1 (OCH2CH2CH2N), 61.7, 61.5 (C-6a, C-6b, C-6c, C-6d, C6e), 43.2, 42.9 (OCH2CH2CH2N), 27.8 (OCH2CH2CH2N), 16.3 ppm (OCH2CH3); HRMS (ESI): m/z calcd for C39H63NO29 + Na: 1032.3383 [M+Na] + ; found: 1032.3371.

Acknowledgements This work was supported RSF grant 14-23-00199 (NEN). Keywords: glycoconjugates · carbohydrates · imaging agents · protecting groups · glycosylation [1] A. Beauvais, S. Bozza, O. Kniemeyer, C. Formosa, V. Balloy, C. Henry, R. W. Roberson, E. Dague, M. Chignard, A. A. Brakhage, L. Romani, J. P. Latg, PLoS Pathog. 2013, doi: 10.1371/journal.ppat.1003712. [2] J. P. Latg, A. Beauvais, Curr. Opin. Microbiol. 2014, 17, 111 – 117. [3] D. Maubon, S. Park, M. Tanguy, M. Huerre, C. Schmitt, M. C. Prevost, D. S. Perlin, J. P. Latge, A. Beauvais, Fungal Genet. Biol. 2006, 43, 366 – 375. [4] A. Beauvais, C. Schmidt, S. Guadagnini, P. Roux, E. Perret, C. Henry, S. Paris, A. Mallet, M. C. Prvost, J. P. Latg, Cell Microbiol. 2007, 9, 1588 – 1600. [5] T. Fontaine, A. Beauvais, C. Loussert, B. Thevenard, C. C. Fulgsang, N. Ohno, C. Clavaud, M. C. Prevost, J. P. Latg, Fungal Genet. Biol. 2010, 47, 707 – 712. [6] S. Bozza, C. Clavaud, G. Giovannini, T. Fontaine, A. Beauvais, J. Sarfati, C. D’Angelo, K. Perruccio, P. Bonifazi, S. Zagarella, S. Moretti, F. Bistoni, J. P. Latg, L. Romani, J. Immunol. 2009, 183, 2407 – 2414. [7] A. Torosantucci, C. Bromuro, P. Chiani, F. De Bernardis, F. Berti, C. Galli, F. Norelli, C. Bellucci, L. Polonelli, P. Costantino, R. Rappuoli, A. Cassone, J. Exp. Med. 2005, 202, 597 – 606.

Conjugate of the pentasaccharide with BSA (29) A solution of 28 (11 mg, 0.011 mmol) and BSA (36.6 mg, 0.00055 mmol) in buffer solution (280 mL, 350 mm KHCO3 and 70 mm Na2B4O7·10 H2O, pH 9.0) was kept at RT for 5 days. The reaction mixture was applied on a Sephadex G-15 column and eluted with water. Appropriate fractions were pooled and lyophilized to yield conjugate 29 (42 mg, 90 %). MALDI-TOF MS showed a broad peak with a maximum at m/z 85 000 (ca. 18 pentasaccharide units per BSA molecule).

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Received: August 7, 2014 Published online on && &&, 0000

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Full Paper

FULL PAPER & Glycoconjugates B. S. Komarova, M. V. Orekhova, Y. E. Tsvetkov, R. Beau, V. Aimanianda, J.-P. Latg, N. E. Nifantiev* && – && Synthesis of a Pentasaccharide and Neoglycoconjugates Related to Fungal a-(1!3)-Glucan and Their Use in the Generation of Antibodies to Trace Aspergillus fumigatus Cell Wall

Directed disaccharides: Adhering to a concept of remote anchimeric assistance, a disaccharide block bearing a stereodirecting benzoyl group at O-6 was constructed. A combination of orthogonal levulinic and methoxyphenyl protecting groups in shared blocks ensured straightforward chain exten-

sion. A couple of a-selective glycosylations lead to spaced a-pentasaccharide. The a-(1!3)-glucan conjugates derived from the pentasaccharide allowed specific anti-a-(1!3)-glucan serum to be obtained for further immunolabeling of Aspergillus fumigatus cell wall.

The synthetic oligosaccharide conjugate… …representing a fragment of surface a-(1!3)-glucan of Aspergillus fumigatus, is depicted on the cover. The conjugate was obtained by a convergent astereoselective scheme and used to generate polyclonal antibodies capable to trace this harmful pathogen. This discovery made a first step to the development of a diagnostic test system and a vaccine to detect and fight this life-threatening pathogen. Details are discussed in the Full Paper by N. E. Nifantiev et al. on p. && ff.

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