Synthesis of a heparan sulfate tetrasaccharide using automated glycan assembly

Herein we utilise automated glycan assembly to complete solid-phase synthesis of defined heparan sulfate oligosaccharides, employing challenging d-glucuronate disaccharide donors. Using an orthogonally protected d-GlcN-α-d-GlcA donor, milligram-scale synthesis of a heparan sulfate tetrasaccharide is completed in 18% yield over five steps. Furthermore, orthogonal protecting groups enabled regiospecific on-resin 6-O-sulfation. This methodology provides an important benchmark for the rapid assembly of biologically relevant heparan sulfate sequences.


General Experimental
The reagents and solvents used in the following experiments were bought commercially and used without further purification.Anhydrous solvents were obtained using equipment based on Grubb's design 1 and stored under N2 in Young's flask over 4 Å molecular sieves.Anhydrous DMF and Pyridine were purchased from Acros.For air-sensitive reactions, solvents were added via syringe through rubber septa.Reactions were monitored by thin layer chromatography using Merck silica-coated 60F254 aluminium plates and the eluents are outlined in the respective experiments; spots were detected under 254 nm UV light and 10% H2SO4/EtOH staining followed by heat.Flash column chromatography was performed using silica gel [Davisil,].Analysis and purification by normal and reverse phase HPLC were performed using the Agilent 1260 series equipped with a Multiple Wavelength Detector (MWD) and an Evaporative Light Scattering Detector (ELSD). 1 H NMR, 13 C NMR and 2D NMR were carried out at 400 MHz on a Bruker AVIII400 or on a Varian 600-MR (600 MHz) spectrometer using deuterated chloroform (CDCl3).Chemical shifts are reported in parts per million (ppm), coupling constants (J) are reported in Hertz (Hz) and multiplicities are abbreviated as; s (singlet), d (doublet), t (triplet) or m (multiplet) or combinations thereof.
Chemical shifts were referenced to the residual proton of TMS for 1 H NMR spectra and to the 13 C chemical shift of deuterated chloroform (CDCl3) for 13 C NMR spectra.For compounds not reported in literature, NMR assignments have been made using COSY, HSQC and HMBC.
HRMS were recorded on a ThermoScientific LTQ Orbitrap XL at the ESPRC National Mass Spectrometry Facility at Swansea University, 6210 ESI-TOF mass spectrometer (Agilent) and MALDI-TOF autoflexTM (Bruker) instruments.The automated syntheses were performed on an automated solid phase synthesiser developed at the Max Planck Institute of Colloids and Interfaces.

Synthesis of Building Blocks for AGA
Synthesis and characterisation of S1, 1 and 3 have been reported previously.A solution of disaccharide S1 (757 mg, 0.788 mmol) in Tol/MeCN/H2O (1:1.5:1;16 mL) was cooled to 0 °C and treated with CAN (2.16 g,3.94 mmol).The reaction mixture was then stirred at room temperature for 1 h.The reaction was diluted with EtOAc (25 mL) and washed with H2O (50 mL).The layers were separated, and the product was extracted with EtOAc (3 x 25 mL).The combined organic layers were dried over anhydrous MgSO4, filtered and concentrated in vacuo to give a brown paste.Purification by column chromatography (Rf = 0.58, 1:1; hex/EtOAc) gave hemiacetal 2 as a light brown foam (622 mg, 92% yield, a/b = 88:12).

Materials and Measurements
Solvents for dissolving all building blocks and making of solutions were taken from Solvent Dispensing System (J.C.Meyer).Wash solvents were HPLC grade.Prior to automated synthesis, the building blocks were co-evaporated three times with toluene and dried for at least 1 hour under high vacuum.All solutions were freshly prepared and kept under argon during the automation process.Isolated yields of products were calculated on the basis of resin loading.Functionalized resin 2 was synthesized and resin loading (0.45 mmol/g) was determined as previously reported. 3

Preparation of Stock Solutions
Building Block Solution: Glycosyl phosphate building block (0.0653 mmol, 4.5 eq per cycle) was dissolved in anhydrous CH 2 Cl 2 (1 mL per cycle).
Pre-capping Solution: Pyridine (10 mL) was added to 90 mL of DMF.

Modules for Automated Synthesis
Initiation: The resin loaded in the reaction vessel was washed with DMF, THF, and CH2Cl2 (3 x 2 mL for 25 s, respectively).The resin was then swollen in CH2Cl2 (2 mL) for 20 minutes while the temperature of the reaction vessel was cooled to -20 °C.

Module A: Acidic Washing
Once the temperature of the reaction vessel was adjusted -20 °C, Acidic Wash Solution (1 mL) was delivered to the reaction vessel.After bubbling for 3 minutes, the solution was drained.Finally, the resin was washed with CH2Cl2 (25 mL) for 25 s and drained.

Module B: Glycosylation for glycosyl phosphate
Upon draining the CH2Cl2 in the reaction vessel, the Building Block Solution (1 mL) was delivered to the reaction vessel.After the temperature reached the desired temperature (T 1 ), Activator Solution (1 mL) was delivered dropwise to the reaction vessel.The glycosylation mixture was incubated for the selected duration (t 1 ) at the desired T 1 , then the reaction temperature was linearly ramped to T 2 .Once T 2 was reached, the reaction mixture was incubated for an additional time (t 2 ).Once the incubation time was finished, the reaction mixture was drained to fraction collector and the resin was washed with CH2Cl2 (6 x 2 mL for 25 s).

Module C: Capping
The resin was washed with DMF (2 x 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 30 °C.Pre-capping Solution (2 mL) was delivered to the reaction vessel and drained after 1 minute.The resin was then washed with CH2Cl2 (3 x 2 mL for 25 s).Upon washing, Capping Solution (2 mL) was delivered and the resin and the reagents are incubated for 10 minutes.The solution was drained from the reaction vessel and the pre-capping and capping steps were repeated once more.Finally, the solution was drained and the resin was washed with CH2Cl2 (3 x 2 mL for 25 s).

Module D: Fmoc Deprotection
The resin was washed with DMF (3 x 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25 °C.Fmoc Deprotection Solution (2 mL) was delivered to the reaction vessel and the reaction was incubated for 5 minutes.The reaction solution was drained and the resin was washed with DMF (3 x 2 mL for 25s).Then, fresh Fmoc Deprotection Solution (2 mL) was delivered and the process was repeated twice.Finally, the resin was washed with DMF (3 x 2 mL for 25 s) and CH2Cl2 (3 x 2 mL for 25 s).The temperature of the reaction vessel was decreased to -20 °C for the next module.

Module E: Lev Deprotection
The resin was washed with CH2Cl2 (3 x 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 40 °C.Lev Deprotection Solution (2 mL) was delivered to the reaction vessel and the reaction was incubated for five minutes.The reaction solution was drained from the reactor vessel and the resin was washed with CH2Cl2 (3 x 2 mL for 25 s).Then, fresh Lev Deprotection Solution (2 mL) was delivered and the process was repeated twice.Finally, the resin was washed with DMF, THF, and CH2Cl2 (3 x 2 mL for 25 s, respectively).

Module F: AcCl Deprotection
The resin was first washed with CH2Cl2 (3 x 2 mL for 25 s) then ClAc Deprotection Solution (2mL) was delivered to the reaction vessel.The temperature of the reaction vessel was then adjusted to and maintained at 80 °C.After 22 min, the reaction solution was drained from the reactor vessel.The resin was washed with DMF (3 x 2 mL for 15 s).Then, fresh ClAc Deprotection Solution (2 mL) was delivered and the process was repeated once more.Finally, the resin was washed with DMF (3 x 2 mL for 25 s) and CH2Cl2 (5 x 2 mL for 25 s).

Module G: Sulfation
The resin was first washed with CH2Cl2 (3 x 2 mL for 15 s) then Sulfation Solution (2 mL) was delivered to the reaction vessel.The temperature of the reaction vessel was then adjusted to and maintained at 90 °C.After 20 minutes the reaction solution was drained from the reaction vessel.Then, fresh Sulfation Solution (2 mL) was added and the process was repeated once more.Upon completion, the resin was washed with DMF (3 x 2 mL for 25 s).