Synthesis of oligosaccharides related to galactomannans from Aspergillus fumigatus and their NMR spectral data.

The synthesis of model oligosaccharides related to antigenic galactomannans of the dangerous fungal pathogen Aspergillus fumigatus has been performed employing pyranoside-into-furanoside (PIF) rearrangement and controlled O(5) → O(6) benzoyl migration as key synthetic methods. The prepared compounds along with some previously synthesized oligosaccharides were studied by NMR spectroscopy with the full assignment of 1H and 13C signals and the determination of 13C NMR glycosylation effects. The obtained NMR database on 13C NMR chemical shifts for oligosaccharides representing galactomannan fragments forms the basis for further structural analysis of galactomannan related polysaccharides by a non-destructive approach based on the calculation of the 13C NMR spectra of polysaccharides by additive schemes.


Introduction
Aspergillus fumigatus is an opportunistic fungal pathogen that can cause allergic bronchopulmonary aspergillosis, chronic aspergillosis and especially invasive aspergillosis (IA)a life threatening disease with high morbidity and mortality rates among immunocompromised patients. 1-3 A major carbohydrate antigen produced by A. fumigatus is the polysaccharide galactomannan. 4 Its identification in a patient's serum employing a commercial sandwich enzyme immunoassay is used for the diagnosis of IA. 5 Galactomannan is a complex heteropolysaccharide whose samples can be highly structurally diverse depending on the conditions of A. fumigatus cultivation. Despite numerous structural studies, some inconsistencies can be seen among the published data. Latgé et al. proposed a structure for two forms 4,6 of galactomannan representing in both cases an α-(1→2)and α-(1→6)-linked poly-D-mannoside backbone bearing β-(1→5)-linked oligogalactofuranoside side chains attached to some of the mannose units via either β-(1→3)or β-(1→6)-bonds (Fig. 1A). Recently, Shibata et al. 7 also revealed the presence of the β-(1→6)-linkage within the galactofurano-side chain 8 and the β-(1→2)but not the β-(1→3)-attachment of the galactofuranoside side chain to the mannan backbone (Fig. 1B). Significant variations in the polysaccharide structure have been reported under different culture growth conditions. 7 Polysaccharides structurally related to galactomannans from A. fumigatus have been reported in other fungal species in particular Aspergillus ochraceus, 9 Malassezia furfur, 10 Malassezia pachydermatis, 10 Trichophyton rubrum, 11 Trichophyton mentagrophytes, 11 Paracoccidioides brasiliensis 12 and others. 13 This stimulates the development of sensitive methods for the structural analysis of galactomannans. NMR spectroscopy is the most potent non-destructive method used to accomplish these tasks.
The chemical syntheses of galactofuranoside-containing oligosaccharides, related to mycobacterial arabinogalactan [14][15][16] or Aspergillus galactomannan [17][18][19] and others, 20 were previously reported. Surprisingly, there are well visible contradictions among the published 13 C NMR data for very similar Galfresidues present in these oligosaccharides. For example, the 13 C NMR chemical shifts of Galf-residues at the non-reducing end linked via (1→5)-bonds to the next Galf-ring reported by Lowary et al., 14 Reynolds et al. 15 and Gallo-Rodriguez et al. 17 (see compounds A-C in Table 1) are quite different. Similar deviations are found in data published for the terminal unit in Galf-(1→6)-Galf-fragments in oligosaccharides D and E. These examples of spectral discrepancy cannot be attributed to the differences of the used spectra recording conditions (temperature and concentration) or the remote influence of structural fragments along the oligosaccharide chains and probably are the results of the tentative assignment of NMR signals. Thus, a careful revision of the previous data is required for their further use in the NMR analysis of fungal galactomannans.
Herein, we report an NMR spectral study of a series of synthetic galactomannan related oligosaccharides 1-13 (Fig. 2). This work is aimed towards the development of NMR database suitable for further structural analysis of Aspergillus galactomannan and structurally related oligo-and polysaccharides. The synthesis of compounds 1-6, 8 and 13 is also reported, while the preparation of oligosaccharides 7 and 9-12 was previously published. 21,22 Results and discussion

Synthesis of oligosaccharides
The investigation of fine correlation between a galactomannan structure and NMR chemical shift values requires a representative series of model oligosaccharides with strictly defined composition containing different fragments of studied polysaccharide. Previously, 21 we reported the synthesis of two galactomannan related pentasaccharides 7 and 10 and oligosaccharides 9, 11 and 12. 22 In the present work, additionally required models such as homo-galactofuranosides 1-4 containing both β-(1→5)and β-(1→6)-linked galactofuranosyl units and hetero-  Table 1 Assigned signals in the 13  a Not described.  saccharides 5, 6, 8 and 13 have been obtained (Fig. 2). For their synthesis, glycosyl donor 16 22 was chosen as a key galactofuranosyl building block. Its main feature was the presence of Fmoc-protection at O(6) which could be selectively removed by deblocking either a 6-OH or 5-OH group depending on the reaction conditions. 22 This made building block 16 suitable for assembling both types of linkages in the oligogalactofuranosyl chains. The synthesis of glycosyl donor 16 was performed according to the procedure using pyranoside-into-furanoside (PIF) rearrangement, 23,24 first described in 2014. Starting pyranoside 14 was initially transformed into the corresponding furanoside 15 and then subjected to subsequent per-O-benzoylation, deallylation and imidation (15 → 16). The glycosylation of 3-trifluoroacetamidopropanol with donor 16 in the presence of TMSOTf gave 17, a precursor to disaccharides 1 and 3 with β-(1→6)and β-(1→5)-bonds, respectively (Scheme 1).
The removal of Fmoc-protection in monosaccharide 17 by treatment with morpholine in DMF gave product 18 with the free OH-group at C-6, which was further glycosylated with imidate 22 25 yielding β-linked disaccharide 23. The configuration of the newly formed bond was confirmed by the singlet shape of the H(1)′-signal ( J H1′,H2′ < 1.0 Hz) and the characteristic low-field chemical shift of C(1)′ (105.86 ppm) in the NMR spectra. One-step deblocking of the obtained disaccharide 23 gave target compound 1.
Alternatively, the removal of Fmoc-protection in 17 under conditions activating O(5) → O(6) benzoyl migration 22 22 in the presence of TMSOTf gave β-linked di-24 and trisaccharide 21, respectively. Following the deblocking of the obtained oligosaccharides resulted in the formation of target compounds 3 and 4.
Disaccharide 24 was also used for the preparation of trisaccharide 2 with alternating (1→6)-and (1→5)-linkages. For this purpose, Fmoc-protection in 24 was removed under conditions preventing benzoate migration (morpholine in DMF) to give acceptor 25 with the free hydroxyl group at O′(6) which was further glycosylated with imidate 22. The deblocking of the obtained protected derivative 26 afforded target trisaccharide 2.

NMR analysis of oligosaccharides
A complete signal assignment in the 1 H and 13 C NMR spectra of oligosaccharides 1-13 was successfully performed employing 2D NMR experiments: COSY, TOCSY, ROESY, HSQC, and HMBC. The chemical shifts were referenced to CH 3 CN (δ 1 H -2.06; 13 C -1.47 ppm) used as an internal standard. The 13 C chemical shifts of the studied compounds are summarized in Table 2 (for 1 H chemical shifts see Table S1 in the ESI †). The 13 C chemical shifts were measured using 1 H-decoupled 1D 13 C NMR spectra, and the 1 H chemical shifts of overlapping signals were established from 2D 1 H- 13 C HSQC spectra using the centres of the corresponding correlation signals.
It can be noted that the assignment of signals summarized in Table 2 has certain, often quite substantial deviations from the previously reported chemical shifts (see Introduction), in particular for the signals of C(1), C(6) and some other carbons. Typically, in the previous papers dedicated to the synthesis of Galf-containing oligosaccharides, NMR signal assignment procedures were described only briefly and the resulting attribution was probably tentative or based on possibly erroneous data from older studies.
The studied series of oligosaccharides (1-13) contained 10 types of galactofuranosyl residues which differed in (1) the type of the glycoside linkage in them and (2) the type of the carbohydrate unit as the aglycon (Fig. 3). The variation in chemical shifts among different compounds for the same type of residue did not exceed ±0.1 ppm. The analysis of chemical shifts did not reveal any chain length influence or influence from distant residues which allowed the use of these data for the spectral investigation of different types of oligo-and polysaccharides.
An important feature for the analysis of the NMR spectra of large carbohydrate chains is the determination of 13 C NMR glycosylation effects. 27 They are the differences in chemical shifts for the corresponding carbon atoms within a residue taken as a part of an oligo-or polysaccharide and for the same residue in the parent mono-or oligosaccharide, non-glycosylated at the position under consideration. The differences in chemical shifts for atoms involved directly in the glycosidic linkage are called the α-effects of glycosylation.
The differences in chemical shifts for atoms bonded to the substituted carbon are the β-effects of glycosylation. To calculate the glycosylation effects, the 13  used. For example, Fig. 4 shows two monosaccharides used for the calculation of the glycosylation effects in the spectrum of disaccharide 5.
The α-effects for anomeric carbon atoms are most informative for the elucidation of monosaccharide sequences in oligoor polysaccharide chains. They are dependent on the direction of the linkage and the stereochemical configuration of the anomeric center involved, and are not influenced by the type of substitution in the glycosylating residue under consideration. The exceptions to this rule are 2-O-substituted residues due to the interference of the β-effects of the substitution with the α-effects of glycosylation.
The C(1) glycosylation effects for differently linked β-Galf residues are shown in Table 3. It can be seen that their values are different for 2-(4.8 ppm, entry 1), 3-(3.4 ppm, entry 2), 5-(6.1 ppm, entry 4) and 6-linked (6.8 ppm, entries 3 and 5) glycosylating furanoses, but the two latter disacchar-ides are not distinguishable on the basis of the glycosylation effects for C (1). Unfortunately, H(1) chemical shifts for the glycosylating Galf residues in these two disaccharide fragments are also very close (5.02-5.04 ppm, Table S1 †). In this case, more information can be given by the α-effects of substitution at C(6) of the glycosylated sugar residues, α-D-Manp (5.8 ppm) and β-D-Galf (6.5 ppm). Additionally, the positions and integral intensities of H(6a,b)/C(6) correlation peaks in the HSQC spectra can also be used for the quantitative assessment of the relative content of the two disaccharide fragments in the polymer. However, this two-dimensional approach requires full analysis and certain assignment of NMR spectra.

NMR analysis of natural polysaccharides
All types of differently substituted Galf residues were characterized by specific 13 C chemical shift patterns and 13 C NMR glycosylation effects which permitted their unambiguous identification in the 13 C-NMR spectra of natural polysaccharides. These signals could be regarded as "structure reporting" signals 29,30 suitable for the identification of the characteristic fragments of galactomannans and related polysaccharides which are usually available in very limited amounts and thus produce spectra with low signal intensities.
For example, the C(1) resonances of β-galactofuranoside residues represented the highest interest as structure report-  ing signals because they are located in a specific range (105-109 ppm) different from the anomeric signals of mannopyranoside units. The chemical shifts of β-galactofuranoside residues were strongly influenced by the nature of monosaccharide substituents at the anomeric site connected via β-(1→2)-, β-(1→3)-, β-(1→5)-, or β-(1→6)linkages as illustrated by 13 C NMR data for model oligosaccharides (Table 4). They coincided well with the corresponding chemical shifts in the spectrum of galactomannan. 7,11 A small deviation (of 0.9 ppm) was observed only in the case of anomeric carbon in the β-galactofuranoside unit connected via the (1→3)-linkage to the (1→2)mannosylated D-Man residue of the mannan backbone (see entry 2 in Table 4) and could be attributed to 2,3-vicinal branching.
To illustrate the applicability of the above mentioned structure reporting signals for the investigation of fungal galactomannans, we analysed the spectrum of galactomannan reported by Latgé et al. 4,6 Despite the fact that the presence of the β-D-Galf-(1→3)-D-Man fragment was claimed by the authors, the signal of the anomeric carbon of the corresponding β-D-Galf-unit was not observed in the spectrum ( Fig. 1 in ref. 4). Hence, in the present study, the 13 C NMR spectrum was recorded for the galactomannan obtained under the reported conditions 4 and it contained the discussed signal. Due to its low intensity in the 1D 13 C NMR spectrum, the presence of the β-(1→3)-linked galactofuranoside unit was detected by a 2D HSQC spectral protocol (Fig. 5A).
The full assignment of the signals in the HSQC spectrum (Fig. 5A) revealed the presence of all the types of carbohydrate residues reported by Latgé et al. 4,6 However, the careful analysis of low intensity signals (Fig. 5B) suggested the presence of a very minor portion of structural elements reported by Shibata et al.: 7 the (1→2)-linkage between β-D-Galf and α-D-Man residues and O(6)-glycosylated β-D-Galf residues (see Fig. 1B). Their presence was confirmed by characteristic 1 H and 13 C chemical shifts that perfectly matched the data obtained for the corresponding model compounds 13 and 12.      Conclusions A series of model oligosaccharides representing key structural fragments of Aspergillus galactomannans was synthesised and studied by NMR spectroscopy. The obtained NMR database on 13 C NMR chemical shifts for different galactomannan fragments is shown to be useful for the verification of the previously established structures and further development of a non-destructive approach towards the structural analysis of galactomannan related polysaccharides based on the calculation of their 13   ments were performed in a positive ion mode (interface capillary voltage −4500 V); mass range from m/z 50 to m/z 3000 Da; external or internal calibration was made with Electrospray Calibrant Solution (Fluka). A syringe injection was used for solutions in a mixture of acetonitrile and water (50 : 50 v/v, flow rate 3 μL min −1 ). Nitrogen was applied as a dry gas; the interface temperature was set at 180°C. Preparation of the galactomannan sample. Aspergillus galactomannan was obtained as described. 4,6 The NMR spectra of galactomannan (solution of 4 mg samples in 0.3 mL of D 2 O) were recorded at 333 K.
Glycosylation with imidate donors (general procedure). A carefully dried mixture of imidate donor and glycosyl acceptor was dissolved in CH 2 Cl 2 and molecular sieves MS300 AW (100 mg per 1 mL of the reaction mixture) were added. After 10 min of stirring, the temperature was decreased to −80°C and TMSOTf (0.40 eq. to imidate donor) was added. The mixture was stirred for 50 min and the temperature was gradually raised to −10°C and then the mixture was quenched with one drop of Et 3 N. The reaction mixture was purified by column chromatography on silica gel (toluene-EtOAc, gradient 8 : 1 → 3 : 1) to give the glycosylation product.  4 mL), morpholine (20 μL) was added. After 35 min, the reaction mixture was poured into 1 M HCl (aq., 50 mL) and extracted with CH 2 Cl 2 (50 mL × 2), and the combined organic phase was concentrated in vacuo. The residue was purified by column chromatography on silica gel (toluene-EtOAc, gradient 6 : 1 → 3 : 1) to afford the 6-OH product.
3-Trifluoroacetamidopropyl 2,3,5-tri-O-benzoyl-β-D-galactofuranoside 18. The removal of Fmoc-protection without Bzmigration in monosaccharide 17 (68 mg, 0.078 mmol) as described in the general procedure gave the product 18 CH 2 N), 1.90-1.79 (m, 2H, OCH 2 CH 2 CH 2 N). 13 (1 mL) was added to the protected oligosaccharide (0.014 mmol) and the mixture was stirred for 1 h. Then, 1 drop of water was added and the reaction mixture was left overnight. Base was neutralized with AcOH (10 μL), and the reaction mixture was diluted with water and concentrated in vacuo. Gel chromatography on TSK-40 HW(S) followed by lyophilization afforded the unprotected compound as a white foam.