Synthesis of Next-Generation Maleimide Radical Labels

Abstract

The synthesis and characterization of four new nitroxide-radical-containing next-generation maleimides are presented. Each new label has a single leaving group which is either a phenoxyl or bromide. The linker between the maleimide and the nitroxide-containing framework is either a racemic mixture of a short chain or an achiral longer chain. These molecules have been designed to site-specifically label vicinal cysteines in proteins for magnetic resonance studies. The characterization of the final products includes crystallography and the labeling of sperm whale myoglobin protein.

Key words

next-generation maleimide - succinimide - nitroxide spin label - radical - EPR spectroscopy - PRE NMR - cysteine labeling - site-directed spin labeling

• References and Notes

• 1 New address: PharmaCytics Operations BV, Novio Tech Campus, Office 3.12, Transistorweg 5v, 6534 AT Nijmegen, NL.
• 2 New address: Almac Group, 5 Fleming Building, Edinburgh Technopole, Milton Bridge, EH26 0BE, UK.
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• 18 The magnetic resonance data supporting this publication can be accessed at http://dx.doi.org/10.17630/19ac1929-f829-449b-adee-323baeedf0e5. CCDC 1468783-1468786 contains the supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/getstructures. The primary data for the LC–MS analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003824.²¹
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• 20 3-{[(Ethoxycarbonyl)oxy]carbonyl}-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-1-yloxy (6) Ethylchloroformate (2.9 g, 26.7 mmol) was added dropwise to a stirred cold (–10 °C) solution of 5 (3.6 g, 19.5 mmol) in a mixture of dry toluene (155.0 mL) and Et₃N (3.5 mL, 35.5 mmol). After stirring for 50 min, the solvent was evaporated, and the residue was triturated with Et₂O. The precipitate was filtered off, washed with Et₂O. The organic layer was concentrated in vacuo, and the residue was recrystallized from hexane yielding 6 (5.0 g, quant.) as a yellow solid. ESI-HRMS: m/z calcd for C₁₂H₁₈NO₅ ⁺: 256.1179; found: 256.1176. 3-Hydroxymethyl-2,2,5,5-tetramethylpyrroline-N-oxyl (7) A solution of NaBH₄ (1.5 g, 39.0 mmol) in EtOH was cooled down with an ice bath and 6 (5.0 g, 19.5 mmol) was added portionwise upon stirring. After stirring for 2 h, the solvent was evaporated under reduced pressure, and the residue was diluted with water and extracted with Et₂O. The extract was dried and concentrated to give 7 (2.8 g, 83%) as a yellow powder. ¹H NMR (400 MHz, acetone d ₆/D₂O + 1.5 equiv Na₂S₂O₄): δ = 1.14 (s, 6 H), 1.16 (s, 6 H), 4.02 (d, J = 1.7 Hz, 2 H), 5.50 (s, 1 H).²²,²³ ESI-HRMS: m/z calcd for C₉H₁₆NO₂Na⁺: 193.1073; found: 193.1070. 2,5-Dihydro-2,2,5,5-tetramethyl-3-({[(4-methylphenyl)sulfonyl]oxy}methyl)-1H-pyrrol-1-yloxy (8) A solution of 7 (2.5 g, 14.5 mmol) and triethylamine (1.9 mL, 19.3 mmol) in dry CH₂Cl₂ (40.0 mL) was cooled at –10 °C, and then TsCl (2.8 g, 14.5 mmol) was added portionwise upon vigorous stirring. The solution was stirred for 3 h at r.t., washed with water and with a sat. solution of NaHCO₃, dried, and concentrated under vacuo. The crude was purified by column chromatography (cyclohexane–EtOAc) yielding 8 (3.5 g, 67%) as a yellow solid. ESI-HRMS: m/z calcd for C₁₆H₂₂O₄NNaS⁺: 347.1162; found: 347.1154. 3-(Aminomethyl)-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-1-yloxy (9) A solution of 8 (1.5 g, 4.6 mmol) in anhydrous MeOH was added dropwise into NH₃ solution (75.0 mL, 7 N in MeOH). The mixture was stirred for 2 h at r.t., then left to stand overnight. The solvent was evaporated under reduced pressure. The residue was treated with a buffer solution (60.0 mL; mixture of citric acid and Na₂HPO₄) at pH 5 and extracted with Et₂O. The aqueous layer was saturated with NaOH and extracted with Et₂O. The extract was dried and concentrated yielding 9 (706.0 mg, 80%) as an orange oil. ¹H NMR (400 MHz, CDCl₃ + phenylhydrazine to reduce the nitroxide radical to a diamagnetic N-hydroxylamine,²⁴ since we found that our product was degraded by Na₂S₂O₄): δ = 1.24 (s, 6 H), 1.25 (s, 6 H), 3.31 (d, J = 1.8 Hz, 2 H), 5.42 (s, 1 H). ESI-HRMS: m/z calcd for C₉H₁₈ON₂ ⁺: 170.1414; found: 170.1408. 3-[(3-Bromo-2,5-dihydro-1H-pyrrole-2,5-dione-1-yl)-methyl]-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-1-yloxy (1) Bromomaleic anhydride (388.2 μL, 4.2 mmol) was dissolved in AcOH (7.0 mL). Nitroxyl 9 (707.9 mg, 4.2 mmol) in AcOH (7.0 mL) was added, and the reaction was heated at 80 °C for 3 h. The solvent was removed under vacuo, and the mixture was purified by column chromatography (cyclohexane–EtOAc) to give the bromomaleimides 1 (644.7 mg, 47%) as an orange powder; mp 146–147 °C. ¹H NMR (400 MHz, CDCl₃ + phenylhydrazine): δ = 1.38 (s, 6 H), 1.45 (s, 6 H), 4.17 (d, J = 1.4 Hz, 2 H), 5.40 (s, 1 H), 6.94 (s, 1 H). NSI-HRMS: m/z calcd for C₁₃H₁₇O₃N₂Br⁺: 328.0417; found: 328.0417. 3-(3-Bromo-2,5-dihydro-1H-pyrrole-2,5-dione-1-yl)-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-1-yloxy (3) To a stirred solution of amine 10 (489.0 mg, 3.1 mmol) in dry Et₂O (24.7 mL) bromomaleic anhydride (286.7 μL, 3.1 mmol) was added. The reaction was left stirring at r.t. for 3 h. The precipitate was filtered and washed with Et₂O yielding a mixture of 11 and 11′ (910.0 mg, 87%) as a yellow solid. The mixture of 11 and 11′ (910 mg, 2.7 mmol) and NaOAc (222.2 mg, 2.7 mmol) was dissolved in Ac₂O (13.5 mL) and heated at 60–70 °C for 3 h. The reaction mixture was then concentrated, dissolved in CH₂Cl₂ and filtered. The filtrate was concentrated and purified by column chromatography (cyclohexane–EtOAc) to give 3 (853 mg, 64%) as an orange solid; mp 101–102 °C. ¹H NMR (400 MHz, CDCl₃ + phenylhydrazine): δ = 1.08 (s, 3 H), 1.25 (s, 3 H), 1.26 (s, 3 H), 1.36 (s, 3 H), 1.82 (dd, J = 12.5, 8.8 Hz, 1 H), 2.93 (dd, J = 12.5, 11.0 Hz, 1 H), 4.47 (dd, J = 11.0, 8.8 Hz, 1 H), 6.89 (s, 1 H). ESI-HRMS: m/z calcd for C₁₂H₁₆O₃N₂BrNa⁺: 338.0237; found: 338.0223. Phenoxymaleimide 2 and 4 – General Procedure To molten phenol (13.2 mmol), t-BuOK (1.1 mmol) in dry dioxane (0.8 mL) was added dropwise, and the solution was left stirring for 10 min at 40 °C. Then a solution of bromomaleimide 1 or 3 (0.8 mmol) in dry dioxane (0.8 mL) was added dropwise, and the resulting mixture was stirred at 40 °C for 30 min. After this time, the solvent was evaporated under reduced pressure. The mixture was purified by column chromatography (cyclohexane–EtOAc) to give the corresponding phenoxymaleimide. 3-[(3-Phenoxy-2,5-dihydro-1H-pyrrole-2,5-dione-1-yl)-methyl]-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-1-yloxy (2) Yield 279 mg (92%); light yellow powder; mp 92–94 °C. ¹H NMR (400 MHz, CDCl₃ + phenylhydrazine): δ = 1.32 (s, 6 H), 1.40 (s, 6 H), 4.14 (d, J = 1.4 Hz, 2 H), 5.34 (s, 1 H), 5.48 (s, 1 H), 7.36–7.29 (m, 2 H), 7.51–7.43 (m, 3 H). NSI-HRMS: m/z calcd for C₁₉H₂₂O₄N₂ ⁺: 342.1574; found: 342.1570. 3-(3-Phenoxy-2,5-dihydro-1H-pyrrole-2,5-dione-1-yl)-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-1-yloxy (4) Yield 345 mg (92%); yellow solid; mp 120–122 °C. ¹H NMR (400 MHz, CDCl₃ + phenylhydrazine): δ = 1.13 (s, 3 H), 1.24 (s, 3 H), 1.27 (s, 3 H), 1.36 (s, 3 H), 1.80 (dd, J = 12.4, 8.7 Hz, 1 H), 2.98 (dd, J = 11.2, 12.4 Hz, 1 H), 4.46 (dd, J = 11.2, 8.7 Hz, 1 H), 5.29 (s, 1 H), 7.37–7.30 (m, 2 H), 7.54–7.43 (m, 3 H). ESI-HRMS: m/z calcd for C₁₈H₂₁N₂O₄Na⁺: 352.1394; found: 352.1371.
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