3-Mercaptopropionic Acid → Succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate

HRID 526284

Reaktionsgleichung

Reaktionsgleichung von HRID 526284
[SH:1][CH2:2][CH2:3][C:4]([OH:6])=[O:5].[CH2:7]1[CH:12]([CH2:13][N:14]2[C:19](=[O:20])[CH:18]=[CH:17][C:15]2=[O:16])[CH2:11][CH2:10][CH:9]([C:21]([O:23][N:24]2[C:29](=[O:30])[CH2:28][CH2:27][C:25]2=[O:26])=[O:22])[CH2:8]1.CCN(C(C)C)C(C)C>>[CH2:7]1[CH:12]([CH2:13][N:14]2[C:19](=[O:20])[CH:18]=[CH:17][C:15]2=[O:16])[CH2:11][CH2:10][CH:9]([C:21]([O:23][N:24]2[C:25](=[O:26])[CH2:27][CH2:28][C:29]2=[O:30])=[O:22])[CH2:8]1.[SH:1][CH2:2][CH2:3][C:4]([OH:6])=[O:5].[CH2:7]1[CH:12]([CH2:13][N:14]2[C:19](=[O:20])[CH:18]=[CH:17][C:15]2=[O:16])[CH2:11][CH2:10][CH:9]([C:21]([O:23][N:24]2[C:25](=[O:26])[CH2:27][CH2:28][C:29]2=[O:30])=[O:22])[CH2:8]1

Komponenten

5

Edukt 3

HCID 65143-Mercaptopropionic AcidHCID 81531DiisopropylethylamineHCID 125175Succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate

Produkt 2

HCID 125175Succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylateHCID 131731991MPr SMCC

Versuchsvorschrift

The drug-linker SMCC-MDC was prepared in the following reactions: (1) 3-mercaptopropanoic acid (MPr) was reacted with N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC) in the presence of DIEA, giving the MPr-SMCC at a yield of over 95%; Secondly, condensation of N-Me-L-Ala-MDC, which was prepared by deprotection of Fmoc-N-Me-Ala-MDC under a base piperidine in CH3CN, with MPr-SMCC under a coupling reagent EDC, giving the desired coupled product SMCC-MDC in 60-70% yield over two steps. Antibody Bat0206 (Abu) was diluted to 2.5 mg/mL in solution A (50 mM potassium phosphate, 50 mM NaCl, and 2 mM EDTA, pH 6.5). SMCC-MDC was added to give a ratio of SMCC-MDC to antibody of 7:1 mole equivalent. Then DMA was added to 15% (v/v) to the reaction and reaction was mixed by stirring for 4 h at ambient temperature. D-Lmcc-Bat0206 conjugate was purified from excess unreacted or hydrolyzed reagent and excess SMCC-MDC using a G25 gel filtration column equilibrated in pH 7.4 phosphate buffer (aqueous). The conjugate was then dialyzed overnight into pH 7.4 phosphate buffer (aqueous) and then filtered through a 0.22 μm filter for final storage. The number of SMCC-MDC molecule per Abu molecule in the final conjugate was measured by determining absorbance of the conjugate at 252 and 280 nm and using known extinction coefficients for SMCC-MDC and antibody at these two wavelengths. A ratio of maytansinoid compound to antibody of 3.5:1.0 was normally obtained.

Quellen und Nachweise

ORD-Eintrag
ord-8fc8471c73e945fbbcbfc45cfbcdcb85
Quelldatensatz
uspto-grants-2016_05
Patentnummer
US09345786B2

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