Ten mg hydrocortisone
3-(O-carboxymethyl)oxime (Toronto Research Chemicals) and 10.4 mg
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU, Fluorochem) were dissolved in 2 mL DMF. Subsequently, 44 μL
diisopropylethylamine (DIPEA, Sigma) was added together with 16.5
mg 1-(2 aminoethyl)maleimide hydrochloride (Sigma) before the mixture
was stirred for 4 h at room temperature. DMF was removed using a Buchi
rotary evaporator and a diaphragm pump. The remaining compound was
dissolved in Milli-Q water with 25% HPLC-grade acetonitrile and 0.1%
formic acid. Cortisol-3-CMO-maleimide was purified with a preparative
LC-MS system comprising of an LCQ Deca XP Max (Thermo Finnigan) ion-trap
mass spectrometer equipped with a Surveyor photodiode detector array
(PDA) detector (Thermo Finnigan). The mixture was purified with a
Phenomex kinetex 2.6 μm EVO C18 50 × 2.1 mm column and
an isocratic acetonitrile gradient of 34%. Fractions with the correct
mass (E/Z isomers were not separated) were collected using a PrepFC
fraction collector (Gilson Inc.) and subsequently freeze-dried before
being dissolved in DMSO and stored at −30 °C (Figure S4 ).
3-(O-carboxymethyl)oxime (Toronto Research Chemicals) and 10.4 mg
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU, Fluorochem) were dissolved in 2 mL DMF. Subsequently, 44 μL
diisopropylethylamine (DIPEA, Sigma) was added together with 16.5
mg 1-(2 aminoethyl)maleimide hydrochloride (Sigma) before the mixture
was stirred for 4 h at room temperature. DMF was removed using a Buchi
rotary evaporator and a diaphragm pump. The remaining compound was
dissolved in Milli-Q water with 25% HPLC-grade acetonitrile and 0.1%
formic acid. Cortisol-3-CMO-maleimide was purified with a preparative
LC-MS system comprising of an LCQ Deca XP Max (Thermo Finnigan) ion-trap
mass spectrometer equipped with a Surveyor photodiode detector array
(PDA) detector (Thermo Finnigan). The mixture was purified with a
Phenomex kinetex 2.6 μm EVO C18 50 × 2.1 mm column and
an isocratic acetonitrile gradient of 34%. Fractions with the correct
mass (E/Z isomers were not separated) were collected using a PrepFC
fraction collector (Gilson Inc.) and subsequently freeze-dried before
being dissolved in DMSO and stored at −30 °C (
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