Towards a Systematic Understanding of the Influence of Temperature on Glycosylation Reactions

Abstract Glycosidic bond formation is a continual challenge for practitioners. Aiming to enhance the reproducibility and efficiency of oligosaccharide synthesis, we studied the relationship between glycosyl donor activation and reaction temperature. A novel semi‐automated assay revealed diverse responses of members of a panel of thioglycosides to activation at various temperatures. The patterns of protecting groups and the thiol aglycon combine to cause remarkable differences in temperature sensitivity among glycosyl donor building blocks. We introduce the concept of donor activation temperature to capture experimental insights, reasoning that glycosylations performed below this reference temperature evade deleterious side reactions. Activation temperatures enable a simplified temperature treatment and facilitate optimization of glycosyl donor usage. Isothermal glycosylation below the activation temperature halved the equivalents of building block required in comparison to the standard “ramp” regime used in solution‐ and solid‐phase oligosaccharide synthesis to‐date.


Modules
Initiation: For tetramer syntheses, resin loaded in the reaction vessel is washed with DMF, THF, and CH2Cl2 (3 x 3 mL for 15 s, respectively). The resin is then swollen in 2 mL CH2Cl2 for 20 minutes while the temperature of the reaction vessel is cooled to the lowest temperature required throughout the synthesis.
Acidic Washing: Once the temperature of the reaction vessel has adjusted to the desired temperature of the subsequent glycosylation by the cooling device, 1 mL of the Acidic Wash Solution is delivered to the reaction vessel through the precooling device (set at -20 °C). After three minutes, the solution is drained. Finally, the resin is washed with 3 mL CH2Cl2 (bubbling = 15 s) and drained.
Glycosylation: Upon draining the CH2Cl2 in the reaction vessel, 1 mL of Building Block Solution containing the appropriate building block is delivered from the building block storing component to the reaction vessel through the precooling device (set at -20 °C). After the temperature reaches the desired temperature (T1), Activator Solution (1 mL) is delivered to the reaction vessel from the respective activator storing component to the reaction vessel through the precooling device (set at -20 °C). The glycosylation mixture is incubated for the selected duration (t1) at the desired T1. In control cases SUPPORTING INFORMATION 5 microwave irradiation (max power = 120 W) is employed to ramp the reaction temperature to T2 (rate = 4 °C/min). Once T2 is reached, it is maintained by microwave irradiation and the reaction mixture is incubated for an additional time (t2). Once the incubation time is finished, the reaction mixture is drained and the resin is washed with CH2Cl2 (1 x 2 mL for 15 s), then dioxane (1 x 2 mL for 15 s), and finally CH2Cl2 (2 x 2 mL for 15 s). During the module, the active cooling element is maintained at the lowest temperature required throughout the synthesis.
Capping: The resin is initially washed with DMF (2 x 3 mL for 15 s). Then Pre-capping Solution (2 mL) is delivered and under microwave irradiation the reaction temperature is adjusted to and maintained at 50 °C for one minute (max power = 5 W). The resin is then washed with CH2Cl2 (3 x 2 mL for 15). Upon washing, Capping Solution (4 mL) is delivered and the temperature is adjusted to and maintained at 25 °C by microwave irradiation (max power = 100 W). The resin and reagents are incubated for 8 min. The solution is then drained from the reactor vessel and the resin is washed with CH2Cl2 (2 x 3 mL for 15 s).
Then, DMF (4 mL) is delivered and irradiated with microwaves (max power = 5 W) for 10 s and the solution is allowed to incubate for an additional 50 s before draining. During the module, the active cooling element is maintained at the lowest temperature required throughout the synthesis.

Fmoc Deprotection:
The resin is first washed with DMF (3 x 3 mL for 15 s), and then Fmoc Deprotection Solution (2 mL) is delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is then adjusted to and maintained at 60 °C by microwave irradiation (max power = 60 W). After 1 min the reaction solution is drained and the resin is washed with DMF (3 x 3 mL for 15 s) and CH2Cl2 (5 x 3 mL for 15 s). During the module, the active cooling element is maintained at the lowest temperature required throughout the synthesis. After this module the resin is ready for the next glycosylation cycle.
Cleavage from Solid Support: After automated synthesis, the resin was removed from the reaction vessel, suspended in CH2Cl2 (20 mL), and photocleaved in a continuous-flow photoreactor. A Vapourtec E-Series easy-MedCHem, equipped with a UV-150 photochemical reactor having a UV-150 mediumpressure mercury lamp (arc length 27.9 cm, 450 W) surrounded by a long-pass UV filter (Pyrex, 50% transmittance at 305 nm). A Pump 11 Elite Series (Harvard Apparatus syringe pump at a flow rate of 0.7 mL/min was used to pump the mixture through a FEP tubing (i.d. 3.0 inch, volume: 12 mL) at 20 °C. The reactor was washed with 20 mL CH2Cl2 at a flow rate of 2.0 mL/min. The output solution was filtered to remove the resin and the solvent was evaporated in vacuo. The crude product was then analyzed by MALDI and analytical HPLC.

Temperature of activation experiments
To assay the temperature of activation for various thioglycoside building blocks, the glycosylation module above was adapted, with t1 = 5 min and T1 corresponding to the desired temperature for activation assay. Upon completion of 5 minutes of the modified glycosylation module, pre-capping solution is added, and the reaction mixture is ejected to an external fraction containing 10% aqueous Na2S2O3. The reaction vessel is washed with CH2Cl2 (1 x 2 mL for 15 s), then dioxane (1 x 2 mL for 15 s), and finally CH2Cl2 (2 x 2 mL for 15 s). After washing, the adapted glycosylation protocol is repeated for the next desired temperature. Collected fractions are extracted twice with 2 ml CH2Cl2, then the combined organic phase is dried over Na2SO4, filtered, and concentrated, coevaporating thrice with anhydrous toluene to remove residual solvent. The residue is placed under high vacuum for at least eight hours before NMR analysis. References are included in select overlays to highlight donor differences. Regions marked red are time periods where modified glycosylation module occurred.

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7 Activation experiment spectra and temperature profiles

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8

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4-Methylphenyl 2-O-benzoyl-4,6-di-O-benzyl-3-O-(9-fluorenylmethoxycarbonyl)-1-thio-β-Dgalactopyranoside (16)
-31 --35 °C with aq. sat. NaHCO3. The organic layer is washed twice with Na2S2O3 and once with water, followed by drying with Na2SO4, filtration, and evaporation to dryness. Purification by silica gel chromatography (hexanes/EtOAc 70:30) affords the glycosyl phosphate in yields from 72 to 86% as a colorless oil and α/β mixture. Phosphate compounds matched previously described spectra. 1 The glycosyl phosphate is combined with the desired thiol (1.5 equiv.), then the mixture is coevaporated thrice to dryness with anhydrous toluene. The residue is dissolved in anhydrous CH2Cl2 (0.1 M). The solution is cooled to 0 °C, then TMSOTf (1.1 equiv.) is added dropwise during vigorous stirring. The reaction is removed from ice and allowed to warm to room temperature or is gently heated until completion as judged by disappearance of the starting material spot in TLC, 30 min to 1 h. The reaction mixture is diluted with CH2Cl2 and quenched with aq. sat. NaHCO3. The organic layer is washed twice with Na2S2O3 and once with water, followed by drying with Na2SO4, filtration, and evaporation to dryness.

Relative reactivity value (RRV) experiments
To determine the relative reactivity of thioglycosides, 0.03 mmol each of an experimental donor (Dexp) and reference donor (Dref) and 50 mg of an inert internal standard (S), 4-methylphenyl 2,3,4,6-tetra-Oacetyl-1-thio-α-D-mannopyranoside, are combined and coevaporated thrice with anhydrous toluene before placement over high vacuum overnight. The donor mixture is then dissolved in anhydrous CH2Cl2 (3 mL To establish absolute RRVs, each building block is compared to another with known reactivity. The relative reactivity of 4-Methylphenyl 2,3,4,6-O-acetyl-1-thio-α-D-mannopyranoside. 3 To avoid peak integration issues, reactivities are included only if the relative difference in RRV is less than 20-fold.
Values reflect agreement of at least two independent experiments.

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43 Figure

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45 Figure S5. Comparison of activation temperature with natural logarithms of RRVs. Red dashes represent Tdecomposition, green dashes are Tactivation, and yellow points are the geometric mean of these values. A light trend could be seen between RRV and activation temperature for mannosides and glucosides, where less temperature sensitive donors reacted were more reactive as measured by competitive experiments.