m-Maleimidobenzoyl-N-hydroxysuccinimide → 7-Mercapto-2-propylheptanoic acid

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Reaction Steps

A solution of 7-mercapto-2-propylheptanoic acid(prepared in Example 2, 150 mg) in 0.1 M phosphate buffer (pH 7.0,10 ml) was added to a solution of BSA(350 mg) in 0.1 M phosphate buffer (pH 7.0,30 ml) in an argon atmosphere. To the solution was added slowly a solution of N-(m-maleimidobenzoyloxy)succinimide (hereinafter referred to as m-MBS, 150 mg) in dioxane (10 ml). The mixture was stirred for 3 hours at ambient temperature, and dialyzed overnight against running water. The dialized solution was adjusted to pH 8.5 with 1 N sodium hydroxide solution and concentrated to about 10 ml by ultrafiltration using PM30 membrane (Amicon Corp.). The concentrate was adjusted to pH 4.5 with diluted hydrochloric acid and left in a cold place for 20 hours. The resulting precipitates were collected by centrifuge (10000 r.p.m., 15 minutes), suspended in a small amount of water, dissolved by adjusting to pH 8.5 with diluted alkali solution and dialyzed for 48 hours against 0.9% sodium chloride solution (2 liters), while the outer solution was renewed four times. The dialyzed solution was lyophilized to give the conjugate (330 mg).

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