Traceless Photolabile Linker Expedites the Chemical Synthesis of Complex Oligosaccharides by Automated Glycan Assembly

Automated glycan assembly (AGA) aims at accelerating access to synthetic oligosaccharides to meet the demand for defined glycans as tools for molecular glycobiology. The linkers used to connect the growing glycan chain to the solid support play a pivotal role in the synthesis strategy as they determine all chemical conditions used during the synthesis and the form of the glycan obtained at the end of it. Here, we describe a traceless photolabile linker used to prepare carbohydrates with a free reducing end. Modification of the o-nitrobenzyl scaffold of the linker is key to high yields and compatibility with the AGA workflow. The assembly of an asymmetrical biantennary N-glycan from oligosaccharide fragments prepared by AGA and linear as well as branched β-oligoglucans is described to illustrate the power of the method. These substrates will serve as standards and biomarkers to examine the unique specificity of glycosyl hydrolases.


General Information
All chemicals were reagent grade and were used as supplied unless otherwise noted. The automated syntheses were performed on a home-built synthesizer developed at the Max Planck Institute of Colloids and Interfaces. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates (0.25mm). Compounds were visualized by UV irradiation or dipping the plate in a panisaldehyde (PAA) solution. Flash column chromatography was carried out by using forced flow of the indicated solvent on Fluka Kieselgel 60 M (0.04-0.063 mmm). Analysis and purification by normal and reverse phase HPLC was performed by using an Agilent 1200 series. Products were lyophilized using a Christ Alpha 2-4 LD plus freeze dryer. 1 H, 13 C, 19 F, and HSQC NMR spectra were recorded in parts per million () relative to the resonance of the solvent on a Varian 400-MR (400 MHz), Varian 600-MR (600MHz), or Bruker Biospin AVANCE700 (700 MHz) spectrometer. High resolution mass spectra were obtained using 6210 ESI-TOF mass spectrometer (Agilent) and MALDI-TOF autoflex TM (Bruker) instruments. IR spectra were recorded on a Perkin-Elmer 1600 FTIR spectrometer. Optical rotations were measured by using a Perkin-Elmer 241 and Unipol L1000 polarimter, with concentrations expressed in g per 100ml. The loading determination of functionalized resins was obtained using a Shimadzu UV-MINI-1240 spectrometer.

Materials and measurements
Solvents used for dissolving all building blocks and making of various solutions were taken from an anhydrous solvent system (jcmeyer phoenix solvent drying system). Wash solvents were HPLC grade. The building blocks were purchased from GlycoUniverse GmbH & CO KGaA, co-evaporated three times with anhydrous toluene and dried for 1 hour under high vacuum prior to use. All solution bottles were freshly prepared and kept under Argon during the automation process. Isolated yields of products were calculated on the basis of resin loading. Resin loading was determined following a reported protocol. 1 Briefly, functionalized resin (40 mg) was treated with one glycosylation cycle using 65 mg of building block A, followed by DBU-promoted Fmoc cleavage and determination of dibenzofulvene concentration by measuring its UV absorbance. All automated syntheses were performed on 0.0125 mmol scale. Resin was placed in the reaction vessel and was swollen in CH2Cl2 for 20 min at room temperature before starting the first module. During this time, all reagent lines involved in the synthesis were washed and primed.

Preparation of stock solutions
 Building Block: 0.08 mmol of building block was dissolved in 1 mL of CH2Cl2.
 Acidic Wash: 0.45 mL of TMSOTf was added to 40 mL of CH2Cl2.
 Activator Solution 1: 1.35 g of recrystallized NIS was dissolved in 40 mL of a 2:1 mixture of anhydrous CH2Cl2 and dioxane, followed by addition of 55 L of triflic acid. The solution was kept under ice-bath cooling for the duration of the automated run.
 Activator Solution 2: 0.9 mL of TMSOTf was added to 40 mL of CH2Cl2.
 Pre-capping Solution: 10 mL of pyridine was added to 90 mL of anhydrous DMF.
 Capping Solution: 1.2 mL of methansulfonic acid, 6 mL of acetic anhydride were added to 50 mL of anhydrous CH2Cl2.
 Fmoc Deprotection Solution 1: 20 mL of piperidine was added to 80 mL of anhydrous DMF.
 Fmoc Deprotection Solution 2: 20 mL of Et3N was added to 80 mL of anhydrous DMF.

Modules for automated synthesis
 Module I -Acidic Wash (ca. 20 min): CH2Cl2 (2 mL) was added and the temperature was adjusted to -20 C. Next, Acidic Wash solution (1 mL) was added dropwise to the reaction vessel. After bubbling for 3 min, the solution was drained and the resin was washed with CH2Cl2 for 25 s. The building block solution (0.08 mmol of BB in 1mL of CH2Cl2) was delivered to the reaction vessel. After initiation temperature (T1) was reached, Activator Solution 1 (1 mL) was added dropwise to the reaction vessel. After initiation time (t1), an incubation temperature (T2) was set and the incubation duration (t2) was adjusted depending on the BB. The values for building block A were shown in the table below. The solution was drained and the resin was washed with CH2Cl2, CH2Cl2/dioxane (1:2, 3 mL for 20 s) and CH2Cl2 (twice, each with 2 mL for 25 s). The temperature was increased to 25 C.

Modules for post-automated synthesis
 Module V -Photocleavage: 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 CH2Cl2, was loaded into a plastic syringe. The suspension was pumped using a syringe pump (PHD2000, Harvard Aparatus) at 1 mL/min through a 10 mL reactor, constructed of 1/8 inch o.d. FEP tubing. The temperature of the photoreactor was maintained at 20 C and the lamp power was adjusted to 80%. Irradiation was set to 30 min, after which the solution was pushed out of the reactor. The existing flow was filtered and the filtrate was collected into a collection flask.
 Module VI -Methanolysis: the resin was loaded into a filter syringe with a capped tip and was suspended in 4 mL of anhydrous THF. A solution of NaOMe in MeOH (0.4 mL, c 5.4M) was then added. The filter syringe was wrapped in aluminium foil and was attached to a rotavap. Spinning was continued for 1 hour, after which the solution was drained. The resin was then washed successively with MeOH, H2O, 1M citric acid solution, DMF, THF, CH2Cl2.
 Module VII -Hydrogenation: the compound was dissolved in 4 mL of EA/tBuOH/H2O (2:1:1). Pd-C (10%) was added to the solution (100% by weight) and the suspension was stirred in a H2 bomb with 60 psi pressure for 1 hour. The insoluble material was removed by a CHROMAFIL®Xtra, RC 0.45 syringe filter. The solid was washed once with tBuOH and several times with H2O. The filtrate was collected, dried and purified by reverse-phase HPLC.

Modules for HPLC purification
Crude products were analyzed and purified using analytical or preparative HPLC (Agilent 1200 Series System). All unprotected products were isolated as formate salt.

O-linked oNB-type resin 8
Merrifield resin LL (100-200 mesh, initial loading 0.5 mmol/g, 2.00 g, 1 mmol) was pre-washed three times with CH2Cl2 and was swollen in anhydrous DMF before 3-(hydroxymethyl)-4-nitrophenol 10 (0.68 g, 4 mmol), Cs2CO3 (1.63 g, 5 mmol) and TBAI (0.370 g, 1 mmol) were added. The suspension was gently shaken on a rotavap at 60 C for 24 hours at 600 mbar. The mixture was filtered and the resin was washed successively with 20 mL each of THF, H2O, THF, DMF, MeOH, CH2Cl2. The resin was suspended in anhydrous DMF and CsOAc (0.96 g, 5 mmol) was added. The suspension was gently shaken on a rotavap at 60 C for 24 hours at 600 mbar. The mixture was filtered and the resin was washed successively with 20 mL each of THF, H2O, THF, DMF, MeOH, CH2Cl2. The resin 8 was dried under high vacuum overnight and protected from light with aluminum foil. Loading (ca. 0.34 mmol/g) was determined according to reported protocol. 1

S11
To a solution of compound S2 (18.3 g, 88 mmol) in trifluoroacetiac acid (60 mL) was added KNO3 (9.86 g, 98 mmol) portion-wise at 10 C. The resulting black solution was heated at 60 C for 4 hours under argon. Upon complete consumption of starting material was indicated by TLC spotting, CH2Cl2 and cold water were added and the organic layer was extracted. The aqueous layer was extracted once with CH2Cl2. The combined organic layer was neutralized with saturated NaHCO3 solution until off-gassing ceased. The organic layer was dried over Na2SO4, filtered, concentrated and purified by flash column chromatography (Hex/EA 3:1) to provide compound 13 (13.4 g, 60%, yellow solid). IR (thin film) νmax = 3675,2982,2310,1706,1576,1519,1335,1282,1212,1037,814

S13
To a stirred solution of compound 14 (3.79 g, 15 mmol) in anhydrous MeOH was added a catalytic amount of Pd(PPh3)4 (346 mg, 0.30 mmol) under a nitrogen atmosphere. The solution was stirred for 5 min, and K2CO3 (6.25 g, 45 mmol) was added. The reaction was run overnight. The reaction mixture was dried, and the residue was treated with citric acid (1M). The aqueous phase was extracted with CH2Cl2. The combined organic layer was dried over Na2SO4, filtered, concentrated and purified by flash column chromatography (CH2Cl2/EA = 2:1) to give compound 15 (

S14
The N-linked oNB-type resin 5 was prepared following the protocol of resin 8. Loading (ca. 0.35 mmol/g) was measured.

N-linked MeNV-type resin 6
To a solution of compound 13 (7.50 g, 30 mmol) and 5-aminopentanol (9.23 g, 90 mmol) in absolute ethanol (70 mL) was added titanium(IV)-isopropoxide(13.25ml, 45 mmol). The reaction was stirred under argon at room temperature overnight. Sodium borohydride (6.59g, 180 mmol) was then added at 0 C and the resulting mixture was stirred for an additional 2 hours. The reaction was quenched by addition of water and stirring was continued for additional 20 minutes. The solvent was removed under reduced pressure, and 1M citric acid (50 mL) was added. The crude mixture was filtered over Celite and extracted with CH2Cl2. The acidic aqueous phase was neutralized with sodium hydroxide NaOH to pH 10-12 and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to afford the crude amine S3, which was used immediately in the next step.

OBn-linked MeNV-type resin 7
To a solution of 4-allyloxymethylbenzyl alcohol 2 (1.14 g, 6.3 mmol) in anhydrous CH2Cl2 was added dropwise phosphorus tribromide (1.2 mL, 12.6 mmol). The solution was stirred at room temperature for an hour. The reaction mixture was diluted with CH2Cl2 (40 mL) and washed twice with saturated aqueous NaHCO3. The combined organic layer was dried over Na2SO4, filtered, concentrated and purified by flash column chromatography (CH2Cl2) to give 4-allyloxymethylbenzyl bromide S4, which was used immediately for the next step.

S20
The OBn-linked MeNV-type resin 7 was prepared following the protocol of resin 8. Loading (ca. 0.36 mmol/g) was measured.

Synthesis of monosaccharide 18
Automation sequence: A