Preparation of Protein Conjugates via Intermolecular Disulfide Bond Formation (1)
Protein A containing thiol groups reacts with protein B containing 4-dithiopyridyl groups by a disulfide interchange reaction with the release of 4-thiopyridone. The required thiol and 4-dithiopyridyl groups can be introduced upon amidination of the lysyl side chains of proteins with the appropriate imidate ester, 2-iminothiolane, or a mixture of 2-iminothiolane and 4,4′- dithiopyridine.
4-Dithiopyridyl containing proteins can be prepared by treatment of proteins with methyl 3-(4′-dithiopyridy1)pro- pionimidate ester, or with a mixture of 2-iminothiolane and 4,4′-dithiopyridine, reactions 3 and 4 of Scheme I.
Methyl 3-mercaptopro- pionimidate and 4-mercaptobutyrimidate ester hydrochloride are two known reagents for preparing thiolated proteins upon amidination of the e-amino groups of the lysyl residues. They can be synthesized on methanolysis of the appropriate mercaptonitrile, as shown for 4-mercaptobuty- ronitrile in reaction 3 of Scheme II.
The required imidate ester can be prepared on treatment of methyl 3-mercaptopropionimidate ester with excess 4,4′-dithiodipyridine. These reactions are depicted in Scheme III.
The extent of amidination of proteins with the two propionimidates and 2-iminothiolane was higher at pH 9 than those at pH values of less than 9. This is in accord with the known reactivity of methyl acetimidate, the amidination reaction being optimal at pH 9-10. The thiol groups of the amidinated proteins are moderately stable to air oxidation in 0.1 M phosphate buffer (pH 6.6) containing 1 mM EDTA. For example, the thiol content of a solution of 3-mercaptopropionimidinyl bovine plasma albumin ( 11 thiol groups per mol) in pH 6.6 buffer decreased by 9% and 45%, respectively, after 1 – and 5-day storage at room temperature.
The dithiopyridyl groups are fairly stable at pH values more acidic than 6.6, but they are labile at pH values more alkaline than 6.6. 4-(4’-Dithiopyridyl)butyriminidyl group appears to be more stable than 3- (4’-dithiopyridyl)propionimidinyl group. With the reactants at 50-100 pM concentration range and the pH of the medium in the range of 6.6 to 8.0, the reaction was near com- pletion after 7 h room temperature. Therefore, the coupling reaction in this pH range is a much faster reaction than the decomposition of 4-dithiopyridyl group.
The extent of coupling was followed spectropho- tometrically for the release of 4-thiopyridone, and chromato- graphically for the formation of oligomeric protein conjugates. In all cases studied, coupling of proteins occurred readily in the concentration range of 20-80 uM at room temperature and neutral pH to give 60-90% yields of conjugates after a reaction time of 3 h or more. The coupling reaction is faster at pH 8 than that of pH 6.6 as shown in experiments 3 and 4 for the coupling of 3-mercaptopropionimidinyl albumin with 4-(4’-dithiopyridyl)butyrimidinyl albumin. Cuts 1 and 2 contained at least three more compo- nents than did cut 3,and the relative mobilities of these slower components as compared with that of the major component in cut 3 are suggestive of their dimer, trimer, and tetramer nature. On treatment with 0.11 M mercaptoethanol at 25 OC for IO min, the slower migrating components of cuts I and 2 were converted to the fast migrating major component present in cut 3.
The coupling mixture of albumin with ribonuclease indicates that the conjugates are well resolved from ribonuclease but not from albumin. It was rechromatographed on a column of Sepharose 6B. The conjugates were still not well resolved from albumin, but the results do show that the conjugates formed are relatively small in size, mainly dimers, trimers, and tetramers
The coupling mixture of albumin with D-GLwas separated on a column of Sepharose CL-6B. The conjugates were eluted as a broad zone extending from the column breakthrough volume to that of albumin. Cuts 1,2, 3, and 4 contained 14, 37,36, and 14%, respectively, of the albumin used.
The coupling mixture of sheep antibody specific for human IgE with horseradish peroxidase was separated on a column of Sepharose CL-6B. About 75% of the enzyme activity used in the reaction mixture was recovered in the chromatogram. The first peak of the chromatogram contained the enzyme-antibody conjugate as indicated by its elution position and enzyme activity. Its elution position indicates the conjugates to be comprised of mainly dimers and trimers. The indicated cut, comprising 75% of the first peak, was pooled and concentrated by ultrafiltration.
