Automated Glycan Assembly of Oligogalactofuranosides Reveals the Influence of Protecting Groups on Oligosaccharide Stability

Galactofurans are an important structural constituent of arabinogalactan and lipopolysaccharides (LPS) ubiquitously present on the envelopes of all Mycobacteria. Key to the automated glycan assembly (AGA) of linear galactofuranosides as long as 20-mers was the identification of thioglycoside building blocks with a fine balance of stereoelectronic and steric effects to ensure the stability of oligogalactofuranoside during the synthesis. A benzoylated galactofuranose thioglycoside building block proved most efficient for oligosaccharide construction.

T uberculosis caused by Mycobacterium tuberculosis (M. tb.), kills more people than any other infectious disease. 1 M. tb. bacteria are surrounded by an intricate network of mycoyl chains that form a dense outer hydrophobic framework that is critical for survival and pathogenicity of the organism. 2 The TB cell wall consists of two major structural components, arabinogalactan (AG) and lipoarabinomannan (LAM) that are both composed of D-galactose and D-arabinose furanoses. Arabinogalactan consists of a linear galactan backbone of approximately 30 alternating β-(1 → 5)-and β-(1 → 6)-linked galactofuranose (Galf) residues. 3 Furanose-containing oligosaccharides are important for microorganisms, but rarely found in humans and other primates. Therefore, the enzymes that are necessary for the construction of galactofuranosyl motifs in microorganisms are attractive targets for the development of new antituberculosis drugs. 4 The low abundance of bifunctional galactofuranosyltransferase (GlfT2) and the structural heterogeneity of oligogalactofuranosides limits the access to probes for cell-wall biosynthesis and to determine substrate specificities. 5 Well-defined synthetic galactofuranosides that resemble the interior portion of AG are necessary to establish structure− activity relationships for these carbohydrates. 6 Solution phase syntheses of galctofuranosyl oligomers ranging from 4 to 12 Galf residues have been reported. 7 A stepwise synthesis of a galactan tetramer revealed structural constraints in the trisaccharide nucleophile that resulted in drastically reduced reactivity. Therefore, a "nonreducing to reducing end" strategy relying on monosaccharide nucleophiles was employed, to prepare a tetrasaccharide galactan. 7a The synthesis of longer galctofuranosyl oligomers relied on an iterative glycosylation approach. 7b, 8 A range of galactofuranosides were synthesized to probe substrate specificities in biological systems. 9 Most oligosaccharide sequences were prepared via stepwise syntheses that require many discrete operations and multiple purifications.
Automated glycan assembly was developed to accelerate oligosaccharide synthesis. 10 Over the past two decades, it has been improved to access more complex glycans. 11 However, oligofuranosides were not prepared by AGA beyond short arabinofuranosides. 12 To explore the utility of AGA 11 to prepare oligogalactofuranosides, we wanted to test the limits of preparing linear galactans found on the surface of M. tb. Here, we disclose the automated synthesis of linear oligogalactofuranoside 20-mer 1 using building blocks with judiciously selected orthogonal protecting groups ( Figure 1).
The power of automated synthesis relies on the section of differentially protected monosaccharide building blocks that result in high yielding and completely selective glycosylations. Galactofuranose thioglycoside building blocks were designed to carry a temporary 9-fluorenylmethoxycarbonyl (Fmoc) protecting group at C-5 (3) or C-6 (4) respectively. A C-2 benzoate provides anchimeric assistance to ensure stereoselectivity for trans-glycosidic linkages. Regioselective benzoylation of thioglycoside 7 13 was followed by 3-O-benzylation and subsequent aqueous acetic acid mediated hydrolysis of acetonide protection afforded diol 8 (Scheme 1). Transacetalation of 8 with benzylaldehyde dimethyl acetal under acidic conditions preceded the regioselective opening of the benzylidene acetal using triethyl silane under acidic conditions before the C-5 hydroxyl was protected with Fmoc to furnish building block 3 in excellent yield. Building block 4 was prepared from 8 by selective protection as the corresponding TBDPS ether and 5-O-benzylation to access thiofuranoside 9. Selective cleavage of the silyl ether using acetic acid buffered TBAF, followed by installation of the C-6 Fmoc provided building block 4 (Scheme 1).
With thioglycoside building blocks 3 and 4 in hand, photocleavable aminopentanol linker immobilized on polystyrene resin 2 was placed in the reaction vessel of the automated synthesizer to prepare galactan heptamer 10 (Scheme 2). A four-step AGA process consisting of acidic wash, glycosylation, capping to mask unreacted nucleophiles, and removal of the temporary protecting group to expose the nucleophile for the next glycosylation was executed. UV irradiation using a continuous flow device released the protected oligosaccharide products from the polymer support that were analyzed using analytical HPLC and MALDI. In addition to desired galactan heptamer 10, a host of deletion sequences were obtained. A careful analysis of the deletion sequences revealed that the temporary Fmoc protecting groups remained intact even after treatment with 20% piperidine in DMF. Changing the deprotection solution on the synthesizer to triethylamine (20% in DMF), or DBU (5% in DMF) and a higher reaction temperature (60°C) failed to cleave Fmoc. The very hydrophobic Fmoc group may interact with hydrophobic regions of the sugar scaffold during oligosaccharide assembly to result in aggregation and poor reactivity.
To counteract aggregation and improve resin swelling, dichloromethane was used as solvent and the use of DBU (5% in CH 2 Cl 2 ) resulted in complete Fmoc cleavage. However, AGA of galactan heptamer 10 using the improved deprotection step revealed unwanted deletion sequences with exposed hydroxyl groups. Apparently, the arming benzyl ethers at C-3, C-6 in building block 3 and C-3, C-5 positions in 4 have profound impact on the stability of the growing galactofuranoside due to intrinsic steric and stereoelectronic effects. 14 On the basis of previous observations, we speculated that thiofuranosides 5 and 6 containing disarming benzoate esters may facilitate the assembly of linear oligogalactofuranoses. Building blocks 5 and 6 were prepared from thiofuranoside 7 13 by benzoylation and isopropylidene cleavage to afford 11. Regioselective benzoylation of 11 at low temperature and placement of Fmoc on the remaining secondary hydroxyl furnished 5. Selective silylation of the 6-hydroxyl in 11 with TBDPSCl and benzoylation gave 12. Desilylation of 12 by HF/pyridine followed by Fmoc protection yielded thioglycoside 6 (Scheme 3).
AGA of galactan heptamer 13 using thiofuranosides 5 and 6 produced a single product according to the HPLC trace of the crude product (Scheme 4 and Figures S1 and S2). This encouraging result prompted us to prepare longer galactofuranose oligomers and to evaluate the influence of the protecting groups on the building blocks (Bn vs Bz) on the stability of growing oligogalactofuranoside. Therefore, using the AGA process developed for shorter sequences, linear galactan 20mer 1 was assembled using building blocks 5 and 6. HPLC and MALDI analysis of the crude mixture revealed that per-Obenzoylated furanoside glycosides 5 and 6 performed well. The desired product was purified by preparative HPLC and the structural integrity of protected galactofuranoside 20-mer 14 was confirmed by 1 H, 13 C NMR, as well as MALDI mass spectrometry (Scheme 5). Fully protected galactan 14 (17 mg) was treated with sodium methoxide to cleave all benzoate ester groups, followed by Pd(OH) 2 /C-catalyzed hydrogenolysis in the presence of hydrogen to cleave the Cbz group furnishing linear galactan 20-mer 1 (2 mg).
In conclusion, we disclose the first automated glycan assembly of oligogalactofuranosides. The identification of differentially protected benzoyl substituted galactofuranose thioglycoside building blocks was key to the successful automated synthesis of the glycans as long as 20-mers found on the cell surface of bacteria. The building blocks will be useful for the construction of many other oligofuranosides.
■ EXPERIMENTAL SECTION General Information. All chemicals used were reagent grade and used as supplied unless otherwise noted. Automated syntheses were performed on a home-built synthesizer developed at the Max Planck Institute of Colloids and Interfaces. 15 Merrifield resin LL (100−200 mesh, Novabiochem) was modified and used as solid support. 16 Analytical thin-layer chromatography (TLC) was performed on Merck silica gel 60 F 254 plates (0.25 mm). Compounds were visualized by UV irradiation or dipping the plate in a p-anisaldehyde (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 mm). Analysis and purification by normal and reverse phase HPLC was performed using an Agilent 1200 series. Products were lyophilized using a Christ Alpha 2−4 LD plus freeze-dryer. 1 H, 13 C, and HSQC NMR spectra were recorded on a Varian 400-MR (400 MHz), Varian 600-MR (600 MHz), or Bruker Biospin AVANCE700 (700 MHz) spectrometer. Spectra were recorded in CDCl 3 by using the solvent residual peak chemical shift as the internal standard (CDCl 3 : 7.26 ppm 1 H, 77.16 ppm 13 C) or in D 2 O using the solvent as the internal standard in 1 H NMR (D 2 O: 4.79 ppm 1 H) unless otherwise stated. High resolution mass spectra were obtained using a 6210 ESI-TOF mass spectrometer (Agilent) and a MALDI-TOF Autoflex (Bruker). MALDI and ESI mass spectra were run on IonSpec Ultima instruments.
Automated Synthesis. Solvents used for dissolving building blocks and preparing the activator, TMSOTf, and capping solutions were taken from an anhydrous solvent system (jcmeyer-solvent systems). Other solvents used were HPLC grade. The building blocks were coevaporated three times with toluene and dried 2 h under a high vacuum before use. Activator, deprotection, acidic wash, capping, and building block solutions were freshly prepared and kept under argon during the automation run. All yields of products obtained by AGA were calculated based on resin loading. Resin loading was determined by performing one glycosylation (Module C) with ten equivalents of building block followed by DBU promoted Fmoccleavage and determination of dibenzofulvene production by measuring its UV absorbance.
Preparation of Stock Solutions. 17 Building Block. Building block was dissolved in 1 mL dichloromethane (DCM).
Activator Solution. Recrystallized NIS (1.56 g) was dissolved in 60 mL of a 2:1 mixture of anhydrous CH 2 Cl 2 and anhydrous dioxane. Then triflic acid (67 μL) was added. The solution was kept at 0°C for the duration of the automation run.
Modules for Automated Synthesis. Module A: Resin Preparation for Synthesis (20 min). All automated syntheses were performed on 140 μmol scale (40 mg). Resin was placed in the reaction vessel and swollen in DCM for 20 min at room temperature prior to synthesis. During this time, all reagent lines required for the synthesis were washed and primed. Before the first glycosylation, the resin was washed with the DMF, tetrahydrofuran (THF), and CH 2 Cl 2 (three times each with 2 mL for 25 s). This step is conducted as the first step for every synthesis.
Module B: Acidic Wash with TMSOTf Solution (20 min). The resin was swollen in CH 2 Cl 2 (2 mL) and the temperature of the reaction vessel was adjusted to −20°C. Upon reaching the temperature, TMSOTf solution (1 mL) was added dropwise to the reaction vessel. After bubbling for argon 3 min, the acidic solution was drained and the resin was washed with 2 mL CH 2 Cl 2 for 25 s.
Module C: Thioglycoside Glycosylation (20−60 min). The building block solution (0.095−0.123 mmol (5−6.5 equiv) of BB in 1 mL of CH 2 Cl 2 per glycosylation) was delivered to the reaction vessel. After the set temperature (−20°C) was reached, the reaction was started by dropwise addition of the activator solution (1.0 mL, excess). The glycosylation was performed by increasing the temperature to 0°C for 20−60 min (depending on oligosaccharide length). After completion of the reaction, the solution is drained and the resin was washed with CH 2 Cl 2 , CH 2 Cl 2 :dioxane (1:2, 3 mL for 20 s) and CH 2 Cl 2 (twice, each with 2 mL for 25 s). The temperature of the reaction vessel is increased to 25°C for the next module.
Module D: Capping (30 min). The resin was washed with DMF (twice with 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25°C. Pyridine solution (2 mL, 10% in DMF) was delivered into the reaction vessel. After 1 min, the reaction solution was drained and the resin washed with CH 2 Cl 2 (three times with 3 mL for 25 s). The capping solution (4 mL) was delivered into the reaction vessel. After 20 min, the reaction solution was drained and the resin washed with CH 2 Cl 2 (three times with 3 mL for 25 s).
Module E: Fmoc Deprotection (14 min). The resin was washed with DMF (three times with 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25°C. Fmoc deprotection solution (2 mL) was delivered into the reaction vessel. After 5 min, the reaction solution was drained and the resin washed with DMF (three times with 3 mL for 25 s) and CH 2 Cl 2 (five times each with 2 mL for 25 s). The temperature of the reaction vessel is decreased to −20°C for the next module.