Protocol for the purification of protected carbohydrates: toward coupling automated synthesis to alternate-pump recycling high-performance liquid chromatography

Abstract

Analytical techniques that can be coupled to automated oligosaccharide synthesis platforms are needed to purify to homogeneity protected carbohydrates at levels of ≥99.5% purity.

Recent liquid chromatographic approaches and developments for the separation and purification of carbohydrates

“…As compared to alkyl-linked supports that separate analytes primarily based on hydrophobicity, phenyl-alkyl supports can also separate analytes based on π-π interactions 40–42 . Despite the prevalence in synthetic carbohydrate chemistry of benzyl and acyl protecting groups containing π-bonds, phenyl supports have seen surprisingly little exploration for the purification of protected carbohydrates; however, these phenyl columns can provide increased separation as compared to their alkyl-linked counterparts 43 . The use of methanol rather than the more common acetonitrile as the organic modifier proves particularly valuable when these phenyl-modified columns are operated in reversed-phase mode.…”

“…As with phenyl supports, these perfluoro-phenyl rings also separate analytes based on π-π interactions; therefore, methanol again proves to be a superior organic modifier as compared to acetonitrile, especially for the purification of protected/partially protected carbohydrates. Recently, these phenyl/perfluoro-phenyl supports proved far superior to alkyl-linked supports in the purification of various globally protected carbohydrates 43 . The development of additional modified-phenyl and other hydrophobic supports could further tune separations capabilities compared to existing column packings.…”

“…Detailed information on valve actuation as well as the ease of implementation of alternate-pump R-HPLC has been discussed previously 43,95 . With the growing need for carbohydrates as authentic analytical standards or as components of bioassay screens, high-level purification of synthetic compounds remains of the utmost important.…”

“…After 53 effective columns, mannose-7 N -glycan isomers were successfully separated and purified from one another 95 . In another study that employed alternate-pump R-HPLC, it was determined that synthesized protected carbohydrates could be successfully purified to ≥99.5% purity 43 with a reversed-phase approach. It was observed that pentafluorophenyl supports were ideal for the purification of protected monosaccharides, whereas phenyl hexyl supports were well suited for the purification of protected di-/tri-saccharides 43 .…”

“…In another study that employed alternate-pump R-HPLC, it was determined that synthesized protected carbohydrates could be successfully purified to ≥99.5% purity 43 with a reversed-phase approach. It was observed that pentafluorophenyl supports were ideal for the purification of protected monosaccharides, whereas phenyl hexyl supports were well suited for the purification of protected di-/tri-saccharides 43 . Interestingly, both phenyl/fluoro-phenyl stationary phases were far superior in the purification of protected carbohydrates, especially when methanol was used as the organic modifier instead of acetonitrile to increase π-π interactions between analyte and stationary phase, as compared to the more commonly employed alkyl-linked, C5/C18, supports 43 .…”