Synthesis of a unique mannose α-1-phosphate side chain moiety found in Candida auris cell wall mannan

Abstract

Candida auris is an emerging fungal pathogen that has become a world-wide public health threat. While there have been numerous studies into the nature, composition and structure of the cell wall of Candida albicans and other Candida species, much less is known about the C. auris cell wall. We have shown that C. auris cell wall mannan contains a unique phosphomannan structure which distinguishes C. auris mannan from the mannans found in other fungal species. Specifically, C. auris exhibits two unique acid-labile mannose α-1-phosphate (Manα1PO₄) sidechains that are absent in other fungal mannans and fungal pathogens. This unique mannan structural feature presents an opportunity for the development of vaccines, therapeutics, diagnostic tools and/or research reagents that target C. auris. Herein, we describe the successful synthesis and structural characterization of a Manα1PO₄-containing disaccharide moiety that mimics the phosphomannan found in C. auris. Additionally, we present evidence that the synthetic Manα1PO₄ glycomimetic is specifically recognized and bound by cell surface pattern recognition receptors, i.e. rhDectin-2, rhMannose receptor and rhMincle, that are known to play important roles in the innate immune response to C. auris as well as other fungal pathogens. The synthesis of the Manα1PO₄ glycomimetic may represent and important starting point in the development of vaccines, therapeutics, diagnostics and research reagents which target a number of C. auris clinical strains. In addition, these data provide new insights and understanding into the structural biology of this unique fungal pathogen.

Graphical Abstract

Introduction

Candida auris is an emerging fungal pathogen that has become a world-wide public health threat. Candida auris is notable for its resistance to antifungal therapy, its transmissibility, its high mortality rate as well as its ability to colonize patients, healthcare personnel and healthcare environments[1]. C. auris strains are typically resistant to fluconazole and approximately half of C. auris strains are resistant to two or more anti-fungal drugs[2]. C. auris causes nosocomial outbreaks of invasive candidiasis with mortality rates of ~60%[3]. In addition to its drug resistance, C. auris can colonize skin, spread from person-to-person and survive in healthcare environments for long periods of time[4]. These features are unique to C. auris as other Candida species do not show the same colonization, transmissibility or persistence in the environment[5]. C. auris is also resistant to many standard decontamination reagents and protocols, which has resulted in the rapid spread of this pathogen to hospitals throughout the world[6]. This makes C. auris a particularly dangerous fungal pathogen. Thus, it is not surprising that it is the first fungal pathogen that has been identified as a public health threat[7].

There have been numerous investigations into the nature, composition and structure of the cell wall of Candida albicans and other Candida species[8–10]. From these studies a general model has evolved, describing the Candida cell wall predominantly composed of a network of chitin, glucan and mannan[10]. Mannan fibrils are located at the exterior of the cell wall where they interact with the environment and ultimately the human host. It is now accepted that mannan is a major fungal pathogen associated molecular pattern (PAMP). Recognition of mannan and other fungal cell wall PAMPs by cell surface pattern recognition receptors (PRRs) plays an important role in determining or “shaping” the innate immune response to the fungal pathogen[11].

While the cell wall of C. albicans has been extensively characterized, much less is known about the cell wall of Candida auris. In 2020, we reported that C. auris mannan exhibits a unique phosphomannan structure that distinguishes it from other fungi[12]. We found that mannan derived from eight different clinical strains of C. auris exhibited this unique phosphomannan structure. More specifically, C. auris clinical strains exhibit two unique acid labile Manα1PO₄ side chains that are not found in mannans derived from other fungal pathogens[12]. Thus, the mannan which coats the outermost surface of C. auris clinical strains distinguishes C. auris from other fungal pathogens, such as C. albicans, C. haemulonii and C. glabrata (see reference 12 and Fig. 1). The presence of two distinctive Manα1PO₄ epitopes, linked via a more flexible phosphodiester linkage rather than a glycosidic linkage, renders this unique phosphomannan a promising target for the development of C. auris specific vaccines, diagnostics and/or reagents. However, it is not practical to isolate natural product C. auris phosphomannan for large scale production of vaccines, reagents and/or diagnostics. Therefore, it becomes imperative to develop a synthetic phosphomannan that mimics the structural and biological characteristics of naturally occurring C. auris phosphomannan.

Figure 1.

General structures of C. auris mannan with two unique Manα1PO₄ side chains. Backbone and side chain length, side chain composition and position vary among different strains.

Previous reports from our group and others have described the synthesis of the 1,6-α-linked backbone[13, 14], the 1,2- and 1,3-α-linked side chain, the 1,2-α-linked side chain with and without phosphate[15]. However, the Manα1PO₄ side chain moiety, which has only been found in C. auris phosphomannan and is attached to the acid stable side chain at a C4 position (shown in red in Fig. 1), has not been synthesized previously. Herein, we describe the de novo synthesis of a glycomimetic C. auris Manα1PO₄ side chain moiety. The successful synthesis of this compound provides a foundation for research into the development of vaccines, therapeutics, diagnostics and research reagents that may target a number of C. auris clinical strains.

2. Results and discussion

Synthesis of mannose α 1-phosphate glycomimetic.

The synthesis of disaccharide 7 involves the preparation of hydrogen phosphonate donor 2 and acceptor 5 with C4 being the only unprotected reaction site (Scheme 1). Donor 2 was conveniently prepared from the readily available hemiacetal 1 in good yield (64%) under modified Dang’s conditions[15]. The preparation of acceptor 5 began with the benzoylation of the C2 position of compound 3, followed by debenzylidenation, resulting in the diol 4 in high yield (83% for two steps). Selective benzoylation of 4 at the C6 position yielded acceptor 5 (65%). Following the procedure described in Dang’s paper[15], donor 2 and acceptor 5 were coupled to generate the Manα1PO₄-4Manα1OAll disaccharide 6 in good yield (74%). Deprotection of disaccharide 6 was carried out using 7N NH₃/MeOH and subsequently purified by C18 column chromatography, resulting in the final product 7 in the form of a sodium salt in good yield (75%).

Scheme 1.

Synthesis of disaccharide 7 containing ManαPO₄ side chain moiety. Reagents and Conditions: a). i. imidazole, PCl₃, Et₃N, CH₃CN, 0°C - rt, 1h; ii. Et₃NHHCO₃, H₂O (pH 8), −20°C - 0°C, 30min, 64%; b). i. BzCl/Py, rt, 2h; ii. 80%HOAc, 80°C, 3h, 83%; c). BzCl, Py/DCM, −20°C - rt, 16h, 65%; d). i. PivCl, Py, rt, 1h; ii. I₂, H₂O, −70C - 0°C, 1h, 74%; e). i. 7N NH₃/MeOH, 4°C, 3d; ii. Sat. aq. Na₂CO₃, rt, 10min, 75%.

In the initial attempts to prepare the hydrogenphosphonate 2, an issue arose concerning the proton attached to phosphorus (H-P), which posed challenges in interpreting the ¹H NMR results (Fig. 2A). According to the previous ¹H NMR assignment of a hydrogenphosphonate compound[15], the 6.16 ppm peak was attributed to the H-P. However, in the NMR analysis of sample 2, we observed a peak at 6.16 ppm with an integration value of only 0.5 instead of the expected 1. Initially, we considered factors like the relaxation rate of H-P or the presence of α/β isomers might be influencing this discrepancy. However, the splitting pattern and integrals of other protons indicated that a mixture was unlikely. Upon closer examination of the spectrum, we identified an additional unassigned singlet resonance at 7.78 ppm, with an integral value of approximately 0.5. This signal had not been documented in the earlier paper[15] due to its overlap with multiple aromatic protons. We suspected these two singlet resonances (6.16 ppm, 7.78 ppm) represent the proton attached to phosphorus with a coupling constant J_HP = 652 Hz. A phosphorus-decoupled ¹H NMR of this compound revealed that the two peaks merged into a single peak (6.97 ppm), with an integral value of 1 (Fig. 2B). Furthermore, the proton resonating at 7.78 and 6.16 ppm as well as the H-1 proton at 5.59 ppm correlated to phosphorus in a 2D ¹H-³¹P HMBC spectrum (Fig. 2C). These results collectively confirmed that the two singlet resonances were indeed a doublet resonance of the proton attached to phosphorus, providing further confirmation that hydrogen phosphonate 2 was successfully prepared as an α-isomer.

Figure 2.

NMR analysis of the hydrogenphosphonate 2: A. ¹H NMR; B. ¹H(P) NMR; C. ¹H-³¹P HMBC.

With the aim of preserving the relatively sensitive phosphodiester, a mild deprotection method (7N NH₃/MeOH) was employed for the disaccharide 6. However, the purification of product 7 proved to be a challenging task due to its relatively small molecular weight, which ruled out any methods of separation by size, such as dialysis and size exclusion chromatography typically used for larger oligosaccharides. Standard silica gel chromatography yielded a product mixture comprising Et₃HN⁺ and H₄N⁺ salts, complicating the ¹H NMR analysis due to the presence of multiple Et₃N resonances, exhibiting variations in chemical shift, integration and ratio among different fractions (Fig. 3A). To resolve this issue, sodium carbonate (Na₂CO₃) was utilized to convert the Et₃HN⁺ and H₄N⁺ salts into sodium salt. The reaction mixture was dissolved in water with ten equivalents of Na₂CO3, followed by loading onto a reversed-phase (C18) column using water as the eluent. After the carbonate salts fractions, disaccharide 7 was eluted exclusively as the sodium salt, as indicated in the ¹H NMR (Fig. 3B).

Figure 3.

NMR analysis of the disaccharide: A. ¹H NMR of the disaccharide N⁺H₄/N⁺HEt₃ salts; B. ¹H NMR of the disaccharide sodium salt (7); C. ¹H-³¹P HMBC of 7.

As outlined in the introduction section, one of the research objectives was to offer additional information to facilitate further structural analysis of the complex fungal phosphomannans. Consequently, comprehensive NMR analyses were conducted on disaccharide 7, including ¹H-³¹P HMBC (Fig. 3C), as well as other 1D and 2D NMR experiments (¹³C NMR, ³¹P NMR, ¹H-¹H COSY, ¹H-¹³C HSQC, (Supplemental Fig. S5). With the assistance of 2D NMR results, the distinctive proton signals in ¹H NMR were readily assigned, particularly the protons H-1’ and H-4, located three bonds away from the phosphorus. The splitting patterns of these two protons were found to differ from their counterparts in mannose units linked through glycosidic bond rather than phosphodiester bond. Both H-1’ and H-4 exhibited coupling constants of 7.5 Hz and 9.8 Hz with ³¹P, respectively, resulting in the doubling of the proton resonances for H-1’ and H-4. This information is particularly valuable when analyzing natural products containing this disaccharide moiety.

Recognition and binding of synthetic Manα1PO₄ glycomimetic by recombinant human Dectin-2, mannose receptor and Mincle.

Mannan is the outermost layer of Candida auris and many other fungal species[16]. Recognition of mannan by cell surface pattern recognition receptors (PRRs) is a first step in anti-fungal innate immune host defense[17–19]. The interaction of fungal PAMPs with PRR in the innate immune system plays an important role in determining or “shaping” the innate immune response to the fungal pathogen[12, 19]. We examined the interaction of the synthetic Manα1PO₄ glycomimetic with three PRRs that are known to play a role in anti-fungal host defense. We observed that the synthetic Manα1PO₄ glycomimetic was recognized and bound by recombinant human (rh) rhDectin-2, rhMannose receptor and rhMincle in a dose dependent and saturable manner (Table 1 and Fig. S6). rhMincle showed the highest affinity interaction with Manα1PO₄ glycomimetic, i.e. K_D of 1.59 μM. rhMannose receptor and rhDectin-2 bound the synthetic Manα1PO₄ glycomimetic with affinities of 2.87 and 13.57 μM. We also compared and contrasted the binding interactions of synthetic Manα1PO₄ glycomimetic with natural product mannan isolated from C. auris KCTC17810 as previously described by our group[12]. Not surprisingly, all three receptors showed significantly higher binding affinities (K_D = ~3 nM) for natural product C. auris mannan when compared to the Manα1PO₄ glycomimetic (See supplemental data Fig. S6). This is due in large part to the fact that natural product C. auris mannan has a much more complex structure with many more available receptor binding sites than does the Manα1PO₄ glycomimetic.

Table 1.

Receptor binding interactions of synthetic Manα1PO₄ glycomimetic with rhMannose receptor, rhDectin-2 and rhMincle.¹

Receptor	Synthetic Manα1PO4 glycomimetic KD (μM)2	C. auris mannan KD (μM)
rhDectin-2	13.57 (11.05 to 16.10)3	0.0033 (0.001080 to 0.0009884)
rhMannose receptor	2.87 (0.99 to 8.35)	0.0031 (0.001752 to 0.005661)
rhMincle	1.59 (1.165 to 2.016)	0.0031 (0.001602 to 0.006125)

¹A minimum of N = 6/ligand receptor was employed.

²K_D = dissociation constant

³95% confidence intervals

In conclusion, the synthesis of the unique phosphomannan disaccharide present in C. auris mannan was successfully accomplished. In addition, we incorporated an allyl group at the reducing end as an attachment point that may be useful when formulating the Manα1PO₄ glycomimetic for in vitro, ex vivo or in vivo studies. The structure of this disaccharide was verified and thoroughly examined through 1D and 2D NMR techniques, providing valuable insights for further investigations into natural products containing this specific moiety. Additionally, we confirmed that the synthetic Manα1PO₄ glycomimetic was recognized and bound by cell surface pattern recognition receptors that are known to play important roles in the innate immune response to C. auris as well as other fungal pathogens.

3. Experimental

3.1. General methods.

All chemicals were purchased from commercial suppliers and used as received. Molecular sieves were activated by heating with heating mantle under reduced pressure. Thin layer chromatography (TLC) was carried out on silica gel F₂₅₄. Carbohydrate compounds were visualized by UV light or/and by charring with 5% H₂SO₄ in ethanol. Flash chromatography was performed with silica gel or C18 functionalized silica gel, P60, (43–60mm, 230–400 mesh). Column chromatography was conducted by elution of a column (16 × 240 mm, 18 × 300 mm, 35 × 400 mm) with EA/Hex, EA/MeOH/Et₃N or water as the eluents. Solutions were concentrated at < 60 °C under reduced pressure.¹H NMR and ¹³C NMR spectra were recorded with Bruker Avance III NMR spectrometer (400 MHz for ¹H, 100 MHz for ¹³C, 162 MHz for ³¹P) at 22°C for solutions in CDCl₃ or D₂O as indicated. Chemical shift referencing was accomplished relative the ¹H resonance of residual protonated chloroform (7.28 ppm) for ¹H NMR and the ¹³C resonance for deuterated chloroform (77.1 ppm) for ¹³C NMR. Assignments of 1D NMR were achieved with additional information from 2D NMR (¹H-¹H COSY, ¹H-¹³C HSQC) (Supplemental data Figs. S1–S5). High-resolution mass spectra (HRMS) for the synthetic compounds were recorded by electron spray ionization mass spectroscopy (time-of-flight analyzer) at the mass spectrometry facility, University of Guelph, Ontario.

3.2. Isolation of Candida auris mannan.

C. auris mannan was cultivated, extracted and structurally characterized from strain KCTC17810 as described previously by our group[12].

3.3. Synthetic procedure

3.3.1. Triethylammonium 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl hydrogenphosphonate (2).

To a solution of imidazole (1.6 g, 23 mmol), and triethylamine (Et₃N) (3.2 mL, 23 mmol) in dichloromethane (DCM) (30 mL), phosphorus trichloride (1.66 mL, 19.2 mmol) was added at 0 °C. The resulting cloudy mixture was stirred at rt for 15 min. The solution of 1 (0.7 g, 1.9 mmol) in DCM (15 mL) was added dropwise at 0 °C over 25 min, then stirred at rt for further 30 min. Triethylammonium bicarbonate buffer (TEAB) (1.0 M, pH 8.0–8.5) (30 mL) was added slowly at −20 °C, and stirred for 15 min. Pyr/Et₃N (4mL/1mL) was added, then the reaction mixture was stirred at rt for 30 min. The organic layer was collected, the aqueous layer was extracted with DCM (50mL), then the organic layers were combined and dried, followed by silica gel chromatography (10:1:0.1 to 5:1:0.06 EA/MeOH/Et₃N) to yield the product 2 as a semi solid (0.66 g, 64%). Knerr et al. previously reported this compound[20], but spectroscopic data were not available. In this report, we present our findings. Rf = 0.3 (10:1:0.1 EA/MeOH/Et₃N). ¹H NMR (400 MHz, CDCl₃): δ 12.11 (s, 1H, NH), 7.78 (s, 0.5H, PH), 6.16 (s, 0.5H, PH), 5.59 (dd, 1H, J_1,2 = 3.5 Hz, J_POCH = 10 Hz, H-1), 5.39 (dd, 1H, J_2,3 = 4 Hz, J_3,4 = 12 Hz, H-3), 5.34–5.26 (m, 2H, H-2, H-3), 4.30–4.22 (m, 2H, H-5, H-6a), 4.12–4.05 (m, 1H, H-6b), 3.12–3.02 (m, 6H, CH₂CH₃ × 3), 2.12 (s, 3H, CH₃CO), 2.08 (s, 3H, CH₃CO), 2.03 (s, 3H, CH₃CO), 1.96 (s, 3H, CH₃CO), 1.34 (t, 9H, J = 10 Hz, CH₂CH₃ × 3). ¹³C NMR (100 MHz, CDCl3): δ 170.74, 169.93, 169.87, 169.73 (CH₃CO), 92.94, 92.90 (C-1), 69.88, 69.81, 69.41, 69.08, 65.82, 62.26 (C-2, C-3, C-4, C-5, C-6), 45.64 (CH₂CH₃), 20.86, 20.76, 20.71, 20.67 (CH₃CO), 8.56 (CH₂CH₃). ³¹P NMR (162 MHz, CDCl₃): δ 0.25. HRMS (ESI): Calcd for C₁₄H₂₀O₁₂P [M+H]⁺ : 514.2048, found: 514.2014

3.3.2. Allyl 2,3-di-O-benzoyl-α-D-mannopyranoside (4).

Benzoyl chloride (5 mL, 43 mmol) was added slowly to a solution of 3 (5.0 g, 12.1 mmol) in DCM/Pyr (6:1, 35 mL) at rt. The reaction mixture was stirred at rt for 2 hr, at which time TLC (3:1 Hex/EA) indicated that the reaction was complete. Methanol (3 mL) was added slowly to quench the reaction, and the reaction solution was concentrated to dryness. The resulting residue was dissolved in 80% HOAc (50 mL), then heated at 80 °C for 3 hr, at which time TLC (2:1 Hex/EA) indicated that the debenzylidenation was complete. The mixture was diluted with toluene (40 mL) and concentrated in vacuo directly. The residue was passed through a short silica gel column using 2:1 Hex/EA as the eluent to give 4 as a syrup (4.3 g, 83% for two steps). Rf = 0.35 (1:1 Hex/EA). ¹H NMR (400 MHz, CDCl₃): δ 8.12–7.42 (m, 10H, Ph), 6.01–5.91 (m, 1H, –CH₂–CH=CH₂), 5.66–5.61 (m, 2H, H-2, H-3), 5.39–5.25 (m, 2H, –CH₂–CH=CH₂), 5.07 (s, 1H, H-1), 4.37 (dd, 1H, J_3,4 = J_4,5 = 10 Hz, H-4), 4.32–4.06 (m, 2H, –CH₂–CH=CH₂), 4.03–3.97 (m, 2H, H-6a, H-6b), 3.95–3.87 (m, 1H, H-5), 3.02 (br s, 2H, OH). ¹³C NMR (100 MHz, CDCl3): δ 166.83, 165.51 (PhCO), 133.53, 133.39, 133.19, 129.84, 129.45, 129.30, 129.61, 128.40 (Ar, –CH₂–CH=CH₂), 118.20 (–CH₂–CH=CH₂), 96.73 (C-1), 73.07, 72.52, 70.69, 68.54, 66.85, 62.25 (C-2, C-3, C-4, C-5, C-6, –CH₂–CH=CH₂). HRMS (ESI): Calcd for C₂₃H₂₄NaO₈ [M+H]⁺ : 429.1544, found: 429.1527

3.3.3. Allyl 2,3,6-tri-O-benzoyl-α-D-mannopyranoside (5).

Benzoyl chloride (1.2 mL, 10.3 mmol) was added slowly to a solution of 4 (4.0 g, 9.4 mmol) in DCM/Pyr (10:1, 33 mL), at −20 °C, then the reaction solution was warmed to rt and stirred for 16 hr. The reaction mixture was washed with water (30 mL) and the organic layer was dried with Na₂SO₄, then concentrated and passed through a short silica gel column (4:1 Hex/EA) to separate the product, 5, as a syrup (3.3 g, 65%) and recovered starting material 4 (1.2 g, 30%). Wang et al. previously published the synthesis of this compound[21], but only included a portion of the ¹H NMR data, which aligns with our findings. In this paper, we present complete set of findings. Rf = 0.3 (3:1 Hex/EA). ¹H NMR (400 MHz, CDCl₃): δ 8.17–7.31 (m, 15H, Ph), 6.02–5.92 (m, 1H, –CH₂–CH=CH₂), 5.72–5.63 (m, 2H, H-2, H-3), 5.40–5.24 (m, 2H, –CH₂–CH=CH₂), 5.09 (d, 1H, J_1,2 = 2.2 Hz, H-1), 4.91 (dd, 1H, J_5,6a = 3.8 Hz, J_6a,6b = 12.5 Hz, H-6a), 4.66 (dd, 1H, J_5,6b = 2.5 Hz, J_6a,6b = 12.5 Hz, H-6b), 4.36–4.09 (m, 4H, H-4, H-5, –CH₂–CH=CH₂), 3.58 (br s, 1H, OH). ¹³C NMR (100 MHz, CDCl3): δ 166.92, 166.72, 165.36 (PhCO), 133.41, 133.36, 133.26, 133.17, 129.95, 129.89, 129.83, 129.76, 129.70, 129.48, 129.33, 128.57, 128. 51, 128.44, 128.37 (Ar, –CH₂–CH=CH₂), 118.27 (–CH₂–CH=CH₂), 96.77 (C-1), 72.69, 71.40, 70.62, 68.66, 66.35, 63.42 (C-2, C-3, C-4, C-5, C-6, –CH₂–CH=CH₂). HRMS (ESI): Calcd for C₃₀H₂₈NaO₉ [M+Na]⁺ : 533.1806, found: 533.1905

3.3.4. Allyl 2,3,6-tri-O-benzoyl-α-D-mannopyranoside [2,3,4,6-tetraacetyl-α-D-mannopyranosyl]-4-(triethylammonium phosphate (6).

Hydrogenphosphonate donor 2 (65 mg, 127 μmol) and acceptor 5 (90 mg, 170 μmol) were dissolved in DCM/Pyr (4:1, 1 mL), followed by addition of pivaloyl chloride (63 μL, 510 μmol) at rt. The reaction mixture was stirred for 1 hr, then cooled to −70 °C, followed by addition of the freshly prepared solution of iodine (46 mg, 180 μmol) in Pyr/water(95:5, 5 mL). The resulting yellow reaction mixture was stirred at −70 °C to 0 °C for 1 hr. The reaction mixture was diluted with DCM (35 mL) and washed with 1M Na₂S₂O₃ (15 mL), then water (10 mL × 2). The organic layer was dried over Na₂SO₄, then concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (10:1:0.11 to 5:1:0.06 EA/MeOH/Et₃N) to yield the disaccharide 6 as a colorless solid (98 mg, 74%). Rf = 0.3 (5:1:0.06 EA/MeOH/Et₃N). ¹H NMR (400 MHz, CDCl₃): δ 11.78 (s, 1H, NH), 8.18–7.24 (m, 15H, Ph), 5.98–5.88 (m, 1H, –CH₂–CH=CH₂), 5.73–5.64 (m, 2H, H-2, H-3), 5.54 (dd, 1H, J_1,2 = 1.5 Hz, J_POCH = 6.5 Hz, H-1’), 5.37–5.15 (m, 5H, H-2’, H-4, H-4’, –CH₂–CH=CH₂), 5.11 (dd, 1H, J_2,3 = 2.1 Hz, J_3,4 = 10 Hz, H-3), 5.05–4.95 (m, 2H, H-1, H-6b), 4.77–4.68 (dd, 1H, J_5,6b = 3.1 Hz, J_6a,6b = 13.2 Hz, H-6b), 4.32–4.03 (m, 4H, H-5, H-6’a, –CH₂–CH=CH₂), 4.03–3.81 (m, 2H, H-5’, H-6’b), 2.87–2.73 (m, 6H, CH₂CH₃ × 3), 2.05 (s, 3H, CH₃CO), 2.03 (s, 3H, CH₃CO), 1.90 (s, 3H, CH₃CO), 1.88 (s, 3H, CH₃CO), 1.10 (t, 9H, J = 10 Hz, CH₂CH₃ × 3). ¹³C NMR (100 MHz, CDCl3): δ 181.64, 170.73, 169.93, 169.71, 169.60, 166.27, 165.62, 165.52 (PhCO, CH₃CO), 133.27, 133.12, 132.75, 130.43, 130.25, 130.10, 129.83, 129.79, 129.45, 128.40, 128.37, 128.26 (Ar, –CH₂–CH=CH₂), 118.08 (–CH₂–CH=CH₂), 96.48 (C-1), 94.06, 94.01 (C-1’), 71.55, 70.50, 70.34, 70.29, 69.80, 69.75, 69.62, 69.53, 69.19, 69.17, 68.51, 65.30, 63.42, 61.93 (C-2, C-3, C-4, C-5, C-6, C-2’, C-3’, C-4’, C-5’, C-6’, –CH₂–CH=CH₂), 45.34 (CH₂CH₃), 20.81, 20.79, 20.64, 20.62 (CH₃CO), 8.31 (CH₂CH₃). ³¹P NMR (162 MHz, CDCl₃): δ −4.63. HRMS (ESI): Calcd for C₄₄H₄₆O₂₁P [M+H]⁺ : 1044.3625, found: 1044.3529

3.3.5. Allyl α-D-mannopyranoside α-D-mannopyranosyl-4-sodium phosphate (7).

Disaccharide 6 (40 mg, 38.4 μmol) was dissolved in 7N NH₃/MeOH (20 mL) and kept at 4 °C for 3 days. The solvent was removed with airflow to give a crude product (41 mg, 384 μmol), into which sat. aq. Na₂CO₃ (0.5 mL) was added, followed by purification with reversed phase (H₂O) chromatography to give the product 7 as a colorless solid (14mg, 75%). ¹H NMR (400 MHz, D₂O): δ 6.06–5.93 (m, 1H, –CH₂–CH=CH₂), 5.73–5.64 (m, 2H, H-2, H-3), 5.51 (d, 1H, J_POCH = 7.5 Hz, H-1’), 4.94 (s, 1H, H-1), 4.31–4.07 (m, 3H, H-4, –CH₂–CH=CH₂), 4.06–3.68 (m, 11H, H-2, H-3, H-5, H-6a, H-6b, H-2’, H-3’, H-4’, H-5’, H6’a, H-6’b). ¹³C NMR (100 MHz, CDCl3): δ 133.12 (–CH₂–CH=CH₂), 118.50 (–CH₂–CH=CH₂), 98.71 (C-1), 96.42, 96,36 (C-1’), 73.73, 72.52, 72.46, 71.7, 71.64, 70.35, 70.26, 70.04, 69.93, 69.82, 68.22, 66.29, 60.71, 60.67. (C-2, C-3, C-4, C-5, C-6, C-2’, C-3’, C-4’, C-5’, C-6’, –CH₂–CH=CH₂). ³¹P NMR (162 MHz, CDCl₃): δ −2.18. HRMS (ESI): Calcd for C₁₅H₂₆O₁₄P [M+Na]⁺ : 507.0850, found: 507.0834

3.4. Binding interaction of Manα1PO₄ glycomimetic with PRRs rhDectin-1, rhMannose and rhMincle.

Assessment of mannan binding interactions to the recombinant human Dectin-1, Mannose receptors and Mincle (R&D systems) were carried out on an Octet K2 BLI instrument (ForteBio) in 10X Kinetics Buffer (pH7.4) at 30°C and 1000 rpm. Increasing concentrations of the Manα1PO₄ glycomimetic (3.125–400 μg/mL) were used to generate respective saturation curves, after which the binding affinities were calculated for mannans isolated from C. albicans and C. auris clinical strains as previously described[12]. The Ni-NTA biosensor was subjected to a 3-minute equilibration prior to 10 minutes of exposure 0.1 ug/mL HIS-tagged rhDectin-2 or rhMannose receptor proteins and final a 10-minute dissociation in 10X kinetics buffer for measuring the BLI signal, consistently 20 seconds after transferring. Subsequently followed by a series of similar 5-minute exposures to an increasing concentration (2-fold) of carbohydrate. To control for receptor dissociation during the experiment, a parallel biosensor with the immobilized receptors was placed in the 10X kinetics buffer without respective carbohydrate exposure. Data analysis was performed using the GraphPad Prism 9.0 software and the dissociation constant K_D is presented as mean value with 95% confidence intervals.

High Lights.

• Candida auris is an emerging fungal pathogen that is a public health threat

• Synthesis of a Manα1PO₄ disaccharide found in Candida auris phosphomannan

• Structural characterization of the Manα1PO₄ glycomimetic using 1D and 2D NMR

• Manα1PO₄ glycomimetic is recognized by rhDectin-2, rhMincle and rhMannose receptors

• Manα1PO₄ glycomimetic may be useful in developing drugs/vaccines against C. auris

Data statement

All data are available in the manuscript or supplementary materials. All data reported in this manuscript will be made available through the lead contact.