Synlett 2016; 27(16): 2357-2361
DOI: 10.1055/s-0035-1562451
letter
© Georg Thieme Verlag Stuttgart · New York

Synthesis of Next-Generation Maleimide Radical Labels

Bouchra Hajjaj
a   SUPA School of Physics and Astronomy, University of St Andrews, St Andrews, KY16 9SS, UK, eMail: jel20@st-andrews.ac.uk
b   BSRC, University of St Andrews, St Andrews, KY16 9ST, UK
,
Anokhi Shah
a   SUPA School of Physics and Astronomy, University of St Andrews, St Andrews, KY16 9SS, UK, eMail: jel20@st-andrews.ac.uk
b   BSRC, University of St Andrews, St Andrews, KY16 9ST, UK
,
Stacey Bell
a   SUPA School of Physics and Astronomy, University of St Andrews, St Andrews, KY16 9SS, UK, eMail: jel20@st-andrews.ac.uk
b   BSRC, University of St Andrews, St Andrews, KY16 9ST, UK
,
Sally L. Shirran
b   BSRC, University of St Andrews, St Andrews, KY16 9ST, UK
,
Catherine H. Botting
b   BSRC, University of St Andrews, St Andrews, KY16 9ST, UK
c   EaStChem School of Chemistry, University of St Andrews, St Andrews, KY16 9ST, UK
,
Alexandra M. Z. Slawin
c   EaStChem School of Chemistry, University of St Andrews, St Andrews, KY16 9ST, UK
,
Alison N. Hulme
d   EaStChem School of Chemistry, University of Edinburgh, David Brewster Road, Edinburgh, EH9 3FJ, UK
,
Janet E. Lovett*
a   SUPA School of Physics and Astronomy, University of St Andrews, St Andrews, KY16 9SS, UK, eMail: jel20@st-andrews.ac.uk
b   BSRC, University of St Andrews, St Andrews, KY16 9ST, UK
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Weitere Informationen

Publikationsverlauf

Received: 01. April 2016

Accepted after revision: 28. Mai 2016

Publikationsdatum:
05. Juli 2016 (online)


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.

Supporting Information

 
  • References and Notes

  • 1 New address: PharmaCytics Operations BV, Novio Tech Campus, Office 3.12, Transistorweg 5v, 6534 AT Nijmegen, NL.
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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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