Simple and Practical Real-Time Analysis of Solid-Phase Reactions by Thin-Layer Chromatography

Abstract

Solid-phase synthesis is a practical approach for simplifying the time-consuming and routine purification steps in the preparation of numerous naturally occurring molecules; however, studying such reactions is difficult due to the lack of a convenient monitoring method. By using thin-layer chromatography in conjunction with a photolabile linker on a resin, we developed a convenient and simple method for monitoring solid-phase reactions in real time by thin-layer chromatography. This method provides a user-friendly protocol for examining reaction conditions for solid-state syntheses.

Key words

solid-phase reactions - real-time analysis - thin-layer chromatography - photolabile linkers - glycosylation - click chemistry

• References and Notes

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• 13 Solid-Phase Glycosylation Procedure for the Synthesis of Glycoside 4aResin 1 (500 mg, 0.1 mmol/g) was swollen in CH₂Cl₂ (4 mL) for 2 h. Swollen 1 in CH₂Cl₂ was mixed with sugar 2a (139 mg, 0.3 mmol) and TolSCl (38 μL, 0.3 mmol), then AgOTf (64 mg, 0.3 mmol) was added and the mixture was kept at r.t. for 3 h. Unreacted reagents were removed by sequential washing with CH₂Cl₂ and MeOH (×3). The resins in CH₂Cl₂ were exposed to UV radiation for 1 h, then filtered. The products in the filtrate were purified by flash column chromatography [silica gel, hexane–EtOAc (3:1 for 4a; 1:2 for 5)] to give 4a as a white solid; yield: 19 mg (56%). Linker 5 was also obtained as a white solid: yield: 5 mg (40%). Eluting solvent for TLC: hexane–EtOAc (1:1).
• 14 Real-Time Analyses of Solid-Phase Reactions by TLC; General ProcedureA minute sample of the reaction mixture in the reaction vessel was captured by capillary attraction in a capillary tube (see Supporting Information, Figure S1). The liquid solution in the capillary was absorbed by using a TLC plate, while the resin beads were retained in the capillary (Figure S2). The beads were then washed sequentially with CH₂Cl₂ and MeOH three times to remove excess reactants. Both CH₂Cl₂ and MeOH were able to flow into the capillary through capillary attraction, and could be subsequently removed by absorption onto the TLC plate. After the repeated washing steps, the capillary loaded with the beads and CH₂Cl₂ was irradiated with UV radiation for 10–15 minutes, and the resulting reaction mixture from the capillary was spotted onto another TLC plate. After eluting the sample, the TLC plate was stained with Hanessian’s reagent and heated on a hotplate.
• 15 Determination of the Reaction Time for PhotocleavageResin 1 (500 mg, 0.1 mmol/g) was immersed in CH₂Cl₂ (7.8 mL) for 1 h. Nine 0.1 mL aliquots were extracted from the resin solution and exposed to UV radiation (254 nm) for various times (5, 10, 15, 20, 30, 40, 60, 90, or 120 min). Linker 5, obtained after irradiation and the removal of CH₂Cl₂, was dissolved in CD₂Cl₂ (0.4 mL) containing (5 × 10^–6)% TMS (v/v) as an internal standard. The results are given in the Supporting Information (Figure S3).
• 16 Solid-phase Glycosylation to Give Products 4b–d; General ProcedureSwollen resin 1 (500 mg, 0.1 mmol/g) in CH₂Cl₂ was mixed with the appropriate sugar derivate 2b–d (0.3 mmol) and NIS (56 mg, 0.3 mmol). TfOH (22 μL, 0.3 mmol) was added at –40 °C, and the mixture was maintained at –40 °C for 2–3 h. All unreacted reagents were washed out three times with CH₂Cl₂ and MeOH. The resins in CH₂Cl₂ were exposed to UV radiation for 1 h then filtered. The products in the filtrate were purified by flash column chromatography [silica gel, hexane–EtOAc (3:1 for 4b, 4:1 for 4c, 1:2 for 5)]. 4b was obtained as a colorless oil; yield: 34 mg (90%). 4c was also obtained as a colorless oil; yield: 12 mg (31%). 4d was not obtained. Eluting solvent for TLC: hexane–EtOAc (1:1 for 4b, 2:1 for 4c and 4d).
• 17 Solid-Phase Syntheses of Aminopentyl Esters 7a–cA solution of the appropriate amino acid 6a–c (0.5 mmol) and HOBt (68 mg, 0.5 mmol) in DMF (200 μL) was injected into CH₂Cl₂ (4 mL) containing swollen resin 1 (500 mg, 0.1 mmol/g). A solution of DIC (63 mg, 0.5 mmol) in DMF (200 μL) was dropped into the reaction mixture, followed by the addition of a catalytic amount of DMAP. The microwave-assisted reactions were conducted at 90 °C for 20 min. Unreacted reagents were washed out five times with CH₂Cl₂ and MeOH. The resin in CH₂Cl₂ were exposed to UV radiation for 1 h, then filtered. The products in the filtrate were purified by flash column chromatography [silica gel, hexane–EtOAc (2:1 for 7a, 3:1 for 7b, 4:1 for 7c)]. 7a was obtained as white solid; yield: 19 mg (60%). 7b was obtained as a colorless oil; yield: 32 mg (93%). 7c was obtained as a colorless oil; yield 29 mg (82%). Eluting solvent for TLC: hexane–EtOAc (1:2 for 7a, 1:1 for 7b and 7c).
• 18 Pathigoolla A. Pola RP. Sureshan KM. Appl. Catal., A 2013; 453: 151
• 19 Triazole 11 by Solid-Phase Click ReactionA mixture of resin 1 (500 mg, 0.1 mmol/g) swollen in DMF (2 mL) was slowly added to a 60% dispersion of NaH (50 mg, 0.6 mmol) in mineral oil at 0 °C, and the mixture was stirred at 0 °C for 6 h. Propargyl bromide (54 μL, 0.6 mmol) was then slowly added at 0 °C, and the mixture was kept at r.t. for 6 h. Unreacted reagents were washed out five times with 1:1 v/v MeOH–H₂O and CH₂Cl₂. The resulting resin 8 was swollen in CH₂Cl for 2 h. A mixture of resin 8 in 2:2:1 CH₂Cl₂–MeCN–H₂O (2 mL) was treated with azide 9 (79 μL, 0.6 mmol) and CuSO₄·5H₂O (154 mg, 0.6 mmol), and the mixture was kept at r.t. for 3 h. N₂H₄·H₂O (30 μL, 0.6 mmol) was added, and the mixture was allowed to react for 4 h. Unreacted reagents were washed out five times with CH₂Cl₂ and MeOH. A mixture of the resin and CH₂Cl₂ was exposed to a UV lamp for 1 h then filtered. The filtrate was purified by flash column chromatography [silica gel, hexane–EtOAc (5:1)] to give 11 as a brown solid; yield: 9 mg (43% over three steps). Eluting solvent for TLC: hexane–EtOAc (2:1).
• 20 Products 15a and 15b by Sequential Solid-Phase ReactionsA 1 M soln of TBAF in THF (500 μL, 0.5 mmol) was added dropwise to a solution of swollen resin 12 (500 mg, 0.1 mmol/g) in CH₂Cl₂, and the mixture was stirred at r.t for 4 h. Unreacted reagents were washed out with CH₂Cl₂ and MeOH to give resin 13. Resin 13 and a solution of glucopyranoside 2c (323 mg, 0.5 mmol) in CH₂Cl₂ were mixed with 1-(phenylsulfinyl)piperidine (105 mg, 0.5 mmol) and Tf₂O (82 μL, 0.5 mmol), and the mixture was stirred at –78 °C for 2–3 h. Unreacted reagents were washed out with CH₂Cl₂ and MeOH to give resin 14, which was exposed to a UV lamp for 1 h. The filtrate was purified by flash column chromatography [silica gel, hexane–EtOAc (7:1 for 15a, 6:1 for 15b)]. 15a was obtained as a colorless oil; yield 25 mg (50% overall). 15b was also obtained as a colorless oil; yield 33 mg (66%). Eluting solvent for TLC: hexane–EtOAc (1:1).