Automated Glycan Assembly in a Variable-Bed Flow Reactor Provides Insights into Oligosaccharide–Resin Interactions

A pressure-based variable-bed flow reactor built for peptide synthesis and capable of real-time monitoring of resin swelling was adapted for automated glycan assembly. In the context of the solid-phase synthesis of several oligosaccharides, the coupling efficiencies, resin growth patterns, and saccharide solvation during the synthesis were determined. The presented work provides the first estimation of on-resin oligosaccharide solvation and an alternative technique to UV–vis monitoring.


General Information
All reagents and solvents were acquired from commercial sources, unless stated otherwise. Thioglycoside monosaccharide building block was purchased from GlycoUniverse. Conversion to mannosyl phosphate donor 1 were prepared according to literature procedures. 1 Pentaacetatyled mannosyl phosphate donor matched that of previously reported. 2 HPLC grade solvents were used. Resin was prepared according to literature. 3 ESI-HRMS were performed with a Xevo G2-XS Q-Tof (Waters). Anhydrous solvents were prepared by drying over activated molecular sieves. NMR spectra were obtained using Ascend 400 (Bruker) and Agilent 400 MHz NMR Magnet (Agilent Technologies) spectrometers at 400 MHz ( 1 H) and 100 MHz ( 13 C). CDCl 3 was used as solvent and chemical shifts (δ) referenced to internal standards (CDCl 3 : 7.26 ppm 1 H, 77.16 ppm 13 C) unless stated otherwise. Assignments were supported by COSY and HSQC experiments. Only diagnostic signals are listed. For monitoring reactions by mass spectrometry, an Agilent 1100 Series LC/MSD mass spectrometer was used. HPLCs were performed on Agilent 1200 Series systems. A YMC-Diol-300-NP column (150 mm x 4.60 mm I.D.) was used for analytical NP-HPLC, with a flow rate of 1.00 mL/min and hexanes:EtOAc as eluent. A YMC-Pack Diol-300-NP column (150 mm x 20.0 mm I.D.) was used for preparative NP-HPLC, with a flow rate of 15.0 mL/min and hexanes:EtOAc as eluent. Unless stated otherwise, the gradient program detailed hereafter was used: 1. Isocratic 20% EtOAc in hexanes (5 min). 2. Linear gradient 20 to 55% EtOAc in hexanes (35 min). 3. Linear gradient to 100% EtOAc (10 min). UV-vis (312 nm) and VBFR data were processed using Origin software.

Oligosaccharide Synthesis
Resin Preparation: Desired resin (0.1 g, 0.33 mmol g -1 ) was loaded into the Omnifit column and the pistons were put into place. The VBFR was set to a resin bed height of 1.5 cm and resin was allowed to swell by flowing CHCl 3 from pumps A and B at a flow rate of 0.3 mL min -1 . After 10 min with the pumps still flowing, the cooling coils were then cooled to -10 °C for an additional 5 min.
Once at temperature (-10 °C) the VBFR was set to achieve a differential pressure of 8 bar to pack the resin. Pumps A and B continued to pass CHCl 3 (0.3 mL/min) over until synthesis commenced.
Building Block Preparation: Mannosyl donor 1 was separated into individual 20 mL vials with each containing enough material for one cycle (270 mg). Each vial was then co-evaporated with toluene three times and placed under high vacuum overnight. Building block stock solutions (2 mL, 0.15 M) in anhydrous CHCl 3 were prepared and transferred into individual 10 mL microwave reactor vessels with a crimped septum under argon. Stock activator solutions were made by addition of 2 mL of anhydrous CHCl 3 into individual 10 mL microwave reactor vessels with a crimped septum under argon. The appropriate amount of TMSOTf was then added (60 µL, 0.15 M) to each activator vessel (one for every glycosylation cycle). Solutions were placed in proper autosampler position.
Modules for Automated Synthesis:

Oligosaccharide Resin Cleavage and Purification
After completion of AGA, the resin was transferred into a 10 mL fritted syringe and washed with CH 2 Cl 2 and dried in vacuo. The Vapourtec E-Series UV-150 Photoreactor Flow Chemistry System was employed. The medium pressure metal halide lamp was filtered using the commercial longpass filter (No. 3, red). The resin, suspended in CH 2 Cl 2 , was loaded into a 20 mL plastic syringe. The suspension was pumped using a syringe pump (PHD2000, Harvard Apparatus) at 0.7 mL min -1 through a 10 mL reactor, constructed of 1/8inch o.d. FEP tubing. The temperature of the photoreactor was maintained at 20 °C and the lamp power was adjusted to 80%. The exiting flow was filtered and the filtrate was collected into a collection flask. The crude oligosaccharide was dried in vacuo, then dissolved in 50:50 hexanes:ethyl acetate and analyzed by analytical HPLC (Agilent 1200). Crude mixture was purified with preparative NP-HPLC.