5-[[AMINO(IMINO)METHYL]AMINO]-2-(BENZOYLAMINO)PENTANOIC ACID synthesis

Yield:-

Reaction Conditions:

with trypsin from bovine pancreas in aq. buffer at 22; pH=7.7;Microwave irradiation;Enzymatic reaction;Temperature;

Steps:

Trypsin digestion of Nα(±)-benzoyl-DL-arginine-4-nitroanilide hydrochloride

Trypsin stocksolution (20 μM) and BAPNAstock solution (1 mM) were freshly prepared prior to reactions.For each digestion experiment, the trypsin stock solution (296 μl) was added to a solution of BAPNA (740 μl), 2200 μl Tris-HCl(40 mM, pH 7.7) and 740 μl CaCl₂(10 mM). Aliquots (270 μl) were withdrawn fromthe reaction mixture every 10 minutes, and added to a 96 well quartz plate.Absorbance was measured at 410 nm using a Biotek Powerwave xs plate reader. Control experimentswere carried out in the microwave reaction vessel without microwave irradiationat 25°C. Using the same reaction vessel is important asdifferent reaction vessels/containers could lead to varying degrees of adsorptionof trypsin on the surface, and thus change the reaction kinetics. Microwave experiments were carried out using a CEM Coolmate reactor. Temperatureof the reaction mixture was kept constant (at 25°C), as monitored by anoptic fibre temperature probethat was inserted into the reaction mixture, by flowing coolant through thecooling jacket around the reaction vessel. In these experiments, the maximal microwavepower was set at 20 W, however, the actual microwave power was adjusted by the Synergysoftware that controls the microwave reactor, based on the temperature feedbackfrom the optic fibre temperature probe.

References:

Mazinani, Sina Atrin;Delong, Benjamin;Yan, Hongbin [Tetrahedron Letters,2015,vol. 56,# 42,p. 5804 - 5807] Location in patent:supporting information