Diisopropylethylamine → HCID 86706042, compound

Temperature

0 °C

Cyclooctyne free acid 8 (4.8 mg, 0.018 mmol) was dissolved in CH3CN (1 mL, anhyd) and cooled to 0° C. N,N-Diisopropylethylamine (10 μL, 0.06, 3 equiv) was added and this solution was stirred under N2 for 10 min at which point, pentafluorophenyl trifluoroacetate (10 μL, 0.058 mmol, 3 equiv) was added dropwise and the reaction was allowed to warm to rt. The r×n was monitored by TLC (1:1 toluene/acetone) for the disappearance of 8 (Rf=0.3-0.4). Upon reaction completion (approx 1 h), the mixture was filtered, and the filtrate was evaporated to dryness. The pentafluorophenyl activated cyclooctyne was purified on silica gel using anhyd toluene and anhyd ether in a gradient solvent system of 10:1 toluene/ether to 4:1 toluene/ether. This product was dried and immediately used for the coupling to biotin. N-(13-amino-4,7,10-trioxamidecanyl)biotinamide3 (7.8 mg, 0.018 mmol, 1 equiv) was dissolved in DMF (0.5 mL, anhyd) and cooled to 0° C. N,N-Diisopropylethylamine (2 drops) was added. The pentafluorophenyl activated cyclooctyne was dissolved in DMF (0.5 mL, anhyd) and this solution was added dropwise to the biotin solution at 0° C. Upon addition of all activated cyclooctyne, the reaction was warmed to rt and monitored by ESI-LCMS for the formation of 9 ([M+H]+=698, [M+Na]+=720). Upon reaction completion (approx 6 h), the mixture was evaporated to dryness and purified by flash chromatography on silica gel. A gradient solvent system was used beginning at 50:3:1 EtOAc/MeOH/H2O and ending with 8:3:1 EtOAc/MeOH/H2O. This procedure resulted in pure 9 (5.0 mg, 0.0072 mmol, 40%). Rf=0.4 in 5:3:1 EtOAc/MeOH/H2O. 1H NMR (500 MHz, D2O): δ 4.58 (dd, J=7.9, 4.9 Hz, 1H), 4.40 (dd, J=7.9, 4.5 Hz, 1H), 4.36 (dt, J=7.8, 2.0 Hz, 0.1H), 4.22 (dt, J=8.6, 2.5 Hz, 1H), 4.12 (dd, 14.9, 5.4 Hz, 0.9H), 4.05 (d, J=14.2 Hz, 0.9H), 4.00 (d, J=16.2 Hz, 0.1H), 3.82 (t, J=8.4 Hz, 0.1H), 3.71-3.65 (m, 8.9H), 3.57-3.45 (m, 7.1H), 3.37-3.19 (m, 8.9H), 3.03 (dd, J=14.3, 9.0 Hz, 1H), 2.97 (dd, J=13.1, 5.0 Hz, 1H), 2.92-2.88 (m, 0.1H), 2.79-2.75 (m, 2.9H), 2.65-2.62 (m, 1H), 2.58-2.49 (m, 2H), 2.32 (dt, J=16.8, 3.0 Hz, 0.9H), 2.24 (t, J=7.2 Hz, 2.1H), 1.79-1.53 (m, 8H), 1.44-1.33 (m, 2H). 13C NMR (125 MHz, D2O): δ 176.7, 174.7, 174.5, 165.2, 98.9, 89.9, 84.3, 76.1, 69.5, 69.3, 68.4, 68.3, 62.0, 60.2, 58.0, 56.5, 55.3, 54.2, 52.0, 39.6, 36.3, 36.2, 35.4, 30.7, 28.24, 28.18, 27.8, 27.6, 25.1, 20.8. HRMS (ESI) calcd for C33H55N5O9SNa [M+Na]+: 720.3618. found: 720.3593.

1. additionwas added dropwise
2. temperatureto warm to rt
3. customUpon reaction completion (approx 1 h)
4. filtrationthe mixture was filtered
5. customthe filtrate was evaporated to dryness
6. customwas purified on silica gel using anhyd toluene
7. customThis product was dried
8. temperatureanhyd) and cooled to 0° C
9. temperaturethe reaction was warmed to rt
10. customUpon reaction completion (approx 6 h)
11. customthe mixture was evaporated to dryness
12. custompurified by flash chromatography on silica gel

ORD record

ord-4e953e80247342cfb2dc12872a04714e

Source dataset

uspto-grants-2016_02

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US09260371B2