Facile synthesis of nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranosides from ManNAc-oxazoline

The synthetic procedures for a large-scale preparation of o- and p-nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranoside are described. The synthetic pathway employs the glycosylation of phenol with ManNAc oxazoline, followed by nitration of the aromatic moiety yielding a separable mixture of the o- and p-nitrophenyl derivative in a 2:3 ratio.


Introduction
Hexosamines are fundamental structural elements and precursors of the peptidoglycan and membrane lipopolysaccharide layer as well as of capsular polysaccharides in Gram-negative bacteria. N-Acetyl-D-mannosamine (ManNAc) has been found to be, presumably, the strongest monosaccharidic ligand for the natural killer cells (NK-cells) activating protein NKR-P1 [1], and some ManNAc-containing oligosaccharides (e.g., GlcpNAc-β-(1→4)ManpNAc) have been identified to be strong immunoactivators [2]. Detection of ManNAc by the immune system is probably very important for recognizing bacterial infection, as this carbohydrate is an unambiguous signal of a microbial invader [3]. In the somatic cells (vertebrates) there are no structures composed of ManNAc. This carbohydrate is a precursor of sialic acid(s). The pathogenicity of some bacterial strains occurring in their R-forms (R stands for rough: virulent, containing ManNAc in the capsular structures) and S-forms (S stands for smooth: nonvirulent, lower level of ManNAc in the capsules) is related partly to the content of ManNAc. Thus, ManNAc units play a significant role in bacterial pathogenicity and virulence (e.g., Streptococcus pneumoniae) [4,5]. Surprisingly, glycosidases active upon β-ManpNAc and α-ManpNAc glycosides are not known so far. Therefore, the building blocks for the chemical synthesis of ManNAc-containing compounds, as well as the substrates for the hypothetical α-N-acetylmannopyranosidase, are required. This paper describes new robust and effective methods for the synthesis of both o-nitrophenyl 2-acetamido-2-deoxy-α-D-Scheme 1: Synthetic pathway. mannopyranoside (7) and p-nitrophenyl 2-acetamido-2-deoxyα-D-mannopyranoside (8) usable as practical chromogenic substrates for the screening of such enzymes.
Oxazolines, such as 3, have been used as glycosylation agents in the preparation of glycoproteins [9,10], various alcohol glycosides [11,12], phosphates [13] and oligosaccharides [12]. To the best of our knowledge, glycosylation of phenolic OH with oxazoline has not been accomplished so far. Therefore, we decided to test oxazoline 3 [14] glycosylation in our synthetic approach.
The glycosylation of phenol and p-nitrophenol with oxazoline 3 was extensively tested. We tested a large array of reaction conditions, including variation of catalyst (copper(II) chloride [12,15], 2,2-diphenyl-1-picrylhydrazyl, zinc chloride, tin(IV) chloride) and solvent (CH 2 Cl 2 , THF, toluene, benzene), all of which were not successful. Finally, we found that trimethylsilyl trifluoromethanesulfonate in dichloromethane [11] was capable of catalyzing the oxazoline ring opening with phenol to afford α-phenyl glycoside 4 in a reasonable 56% yield. This reaction was also tested with p-nitrophenol; however, the required p-nitrophenyl glycoside 6 was produced under these conditions only in trace amounts (observed by TLC). Our and previously published results [11] indicate that the deactivation of phenol by the electron-withdrawing nitro group substantially decreases its reactivity in the glycosylation reaction with oxazoline and, on the other hand, the presence of electron-donating groups increases the yields.
The resulting phenyl glycoside 4 was treated with a solution of red fuming nitric acid in acetic acid [16], producing a mixture of o-nitrophenyl glycoside 5 (22% yield) and p-nitrophenyl glycoside 6 (34% yield) in approximately 2:3 ratio, which was separated by flash chromatography. Zemplén deacetylation of 4 and 5 afforded the title compounds 7 and 8 in almost quantitative yields.

Conclusion
In conclusion, a simple and robust procedure for the synthesis of oand p-nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranoside (7 and 8) from commercially available ManNAc, via its oxazoline, is described affording an overall total yield of both 7 and 8 of over 21%, including the purification steps.

Experimental General methods
All chemicals were purchased from Sigma-Aldrich, except for 2-acetamido-2-deoxy-D-mannose, which was purchased from BIOSYNTH AG, Staad, CH. The reactions were monitored by TLC with precoated silica gel 60 F 254 aluminium sheets from Merck, detected with UV light and/or charred with sulfuric acid (5% in EtOH). The compounds were purified either by column flash chromatography with silica gel 60 (230-240 mesh, Merck) or by gel permeation chromatography with Sephadex LH20 from Sigma Aldrich. The solvents were distilled and dried according to the standard procedures before use.

NMR spectroscopy
NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer (400. 13 13 C NMR, gCOSY, gHSQC, and gHMBC were performed using the manufacturer's software. 1 H NMR and 13 C NMR spectra were zero filled to fourfold data points and multiplied by a window function before Fourier transformation. A two-parameter double-exponential Lorentz-Gauss function was applied for 1 H to improve resolution, and line broadening (1 Hz) was applied to get a better 13 C signal-to-noise ratio. Chemical shifts are given on a δ-scale with the digital resolution justifying the reported values to three (δ H ) or two (δ C ) decimal places.
The anomeric configuration of manno-structures was determined based on the value of 1 J (C-1, H-1) [17], which was measured by using gHMQC or gHSQC with retained direct coupling constants between directly bonded carbon and hydrogen.

Mass spectrometry
Mass spectra were measured on MALDI-TOF/TOF ultraFLEX III mass spectrometers (Bruker-Daltonics). Positive spectra were calibrated externally by using the monoisotopic [M + H] + ions of PepMixII calibrant (Bruker-Daltonics). For the MALDI experiment 0.4 μL of sample dissolved in 50% acetonitrile was allowed to dry at ambient temperature on the target and overlaid with matrix solution (either 2,5-dihydroxybenzoic acid, DHB or α-cyano-4-hydroxycinnamic acid, CCA). The spectra were collected in reflectron mode.