Nonenzymatic synthesis of anomerically pure, mannosyl-based molecular probes for scramblase identification studies

The chemical synthesis of molecular probes to identify and study membrane proteins involved in the biological pathway of protein glycosylation is described. Two short-chain glycolipid analogs that mimic the naturally occurring substrate mannosyl phosphoryl dolichol exhibit either photoreactive and clickable properties or allow the use of a fluorescence readout. Both probes consist of a hydrophilic mannose headgroup that is linked to a citronellol derivative via a phosphodiester bridge. Moreover, a novel phosphoramidite chemistry-based method offers a straightforward approach for the non-enzymatic incorporation of the saccharide moiety in an anomerically pure form.


Overview Synthesis
The synthetic steps are outlined in Schemes 1 and 2:
The synthesis of α-4Ac-Man-CEP is described in the literature. 7 Its analytical data ( 1 H-, 13 C and 31 P-NMR), however, are published herein for the first time (see below), making the comparison with β-4Ac-Man-CEP easier.
NMR spectra were recorded on a Bruker Avance III HD 300 or Avance II 400 / Avance III HD 400. MS data were obtained from a ThermoScientific LTQ Orbitrap XL, equipped with a Nanoelectrospray Ion Source (NSI

Compound DMT-Cit-OH
DMT-Cit (1.02 g, 2.22 mmol) was dissolved in a mixture of pyridine/EtOH (10:1, 40 mL) under argon at rt. The mixture was stirred for 20 min. Then, SeO2 (245 mg, 2.22 mmol, 1 eq) was added, the mixture was heated to 80°C and stirred overnight. Afterwards, the solvents were removed under vacuum, the resultant oil was dissolved in ethyl acetate, washed with brine and dried with Na2SO4. The solvent was removed and the crude product was re-dissolved in EtOH (30 mL) at 0°C. NaBH4 (168 mg, 2 eq) was added and the mixture was stirred for 1 h. Then, H2O was added, the crude product was extracted with DCM, dried with Na2SO4 and purified by column chromatography on silica gel (Rf = 0.2; hexane/EtOAc 8:2). DMT-Cit-OH (580 mg, 55% yield) was obtained as a yellow oil. at 0°C under argon. Et3N (1.65 mL, 11.85 mmol, 5 eq) was added, followed by the addition of methanesulfonyl chloride (Ms-Cl) (0.37 mL, 4.74 mmol, 2 eq). The reaction mixture was allowed to slowly reach rt, and the mixture was stirred for 1 h. The mixture was washed with brine, the organic phase was dried with Na2SO4 and the volatile components were removed under reduced pressure. The resultant oil (Ms-Dod-TBDMS) was used in the next reaction step without any further purification.

HRMS (ESI
(HR-MS analysis, see below) Note: Due to the apparent presence of a substantial amount of byproduct(s), estimated to be ⅓ (based on NMR data) and most likely resulting from degradation of the NBD-tag (when treated with alkaline conditions), further purification via RP-HPLC was necessary (see below).

RP-HPLC purification:
Ca. 20 mg of the obtained material (see above) were dissolved in 4 mL Milli-Q H2O, to perform 4 runs (1 mL each