Photodimerization of maleic anhydride (MA) gives insoluble precipitated products that can be a trigger to clog a conventional microreactor. To avoid this problem, we devised a microreactor that uses liquid/gas slug flow and ultrasonication. Inert N 2 gas introduced into the reaction solution swept through the reactor tube and transported precipitated products in the liquid segments.Ultrasound vibrations inhibited the adhesion and sedimentation of precipitate in the reactor tube. The combination of gas and ultrasound prevented the tube from clogging. Fluorinated ethylene propylene (FEP) tubes of various sizes were investigated to use as a tube reactor. The tubes were wound around a high-pressure Hg lamp with a Pyrex immersion well which has been using generally as a light source of photoreaction, and the reaction solution was then passed through the tube and irradiated through the tube wall. The slug flow microreactor could be operated for more than 16 h continuously without clogging. Compared to using a batch reactor, this method achieves better product quality, improved conversion, and reduced waste.

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.

Atomically flat, sputter-grown Au(111) films allowed well-ordered alkanethiol (exemplified by octanethiol) monolayers to be self-assembled from solution markedly faster and in larger domain sizes than previously reported. An X-ray photoelectron spectroscopy analysis showed that complete monolayer coverage was reached by 0.2−60 min of incubation in 0.1−0.001 mM ethanolic solution at room temperature (∼17 °C), with single-step (0.1 and 0.01 mM) or two-step (0.001 mM) adsorption kinetics. Increasing the temperature to 35 °C was enough to cause a single-step, diffusion-controlled adsorption also from the 0.001 mM solution, yielding the full monolayer coverage in approximately 10 min. Scanning tunneling microscopy (STM) imaging proved that well-ordered islands, with the (√3 × √3)R30° structure more-or-less strongly modulated by the c(4 × 2) superlattice, begin to form at 0.6−0.7 monolayer coverage most likely by homogeneous nucleation and grow rapidly thereafter. This kinetics of ordering requiring the considerably high threshold coverage for the nucleation, but allowing the fast growth of the nuclei was independently confirmed by the infrared reflection absorption spectroscopy. A typical c(4 × 2) domain size at the saturation coverage was estimated to be no less than 10−15 nm, and the structural identity often seemed not to be disrupted even across the etch pits. This superior structural order is reflected on the highest level of molecular resolution achieved by the in-air STM imaging. The expected registry of the (√3 × √3)R30° or c(4 × 2) lattice with that of Au(111) was also confirmed. On Au films that were also sputter-grown but no longer atomically flat, we observed at least by 1 order of magnitude slower self-assembly.

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The extended substrate binding site of cathepsin G from human leukocytes has been mapped by using a series of peptide 4-nitroanilide substrates. The enzyme has a significant preference for substrates with a P1 Phe over those with the other aromatic amino acids Tyr and Trp. The S2 subsite was mapped with the substrates Suc-Phe-AA-Phe-NA where AA was 13 of the 20 amino acid residues commonly found in proteins. The best residues were Pro and Met. The S3 subsite was mapped with the sequence Suc-AA-Pro-Phe-NA by using 14 different amino acid residues for AA. The two best residues were the isosteric Val and Thr. No significant improvement in reactivity was obtained by extending the substrate to include seven different P4 residues. The kinetic parameters for cathepsin G are significantly slower than those for many other serine proteases. Changes in the reaction conditions and addition of possible cofactors or ligands were in general found to have little effect on the enzymatic activity, while chemical modifications and proteolysis destroyed the activity of cathepsin G. Cathepsin G hydrolyzed peptides containing model desmosine residues and prefers the hydrophobic picolinoyllysine derivative over lysine by substantial margins at both the S4 and S2 subsites but will not tolerate it at S3. Substrates with sequences related to the cathepsin G cleavage site in angiotensin I and angiotensinogen, and the reactive site of alpha 1-antichymotrypsin, were hydrolyzed effectively by enzyme, but with unexceptional rates. Our results indicate that the natural substrate(s) and function(s) of cathepsin G still remain to be discovered.

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A series of 14 tripeptide 4-nitroanilide substrates of the type Z-AA-Gly-Arg-NA and Z-AA-Phe-Arg-NA where AA = Ala, Asn, Glu, Lys, Phe, Pro, or Ser were used to map the S3 subsite of several serine proteases involved in blood coagulation. The enzymes studied included bovine thrombin, factor IXa, factor Xa, factor XIa, human beta-factor XIIa (factor XIIa fragment), and activated bovine and human protein C. Kinetic constants (kcat, KM, and kcat/KM) for the enzymatic hydrolysis of the substrates by each enzyme were determined and used to compare the relative reactivities of the individual enzymes. Most of the enzymes reacted with all the substrates, although a few showed considerable specificity. Human beta-factor XIIa showed the highest reactivity of all the coagulation proteases studied and was also very substrate specific (kcat/KM ranged over 470-fold). The best substrate was Z-Lys-Phe-Arg-NA with kcat/KM = 140 000 M-1 s-1. Activated bovine protein C (best substrate = Z-Ser-Phe-Arg-NA), factor Xa (best substrate = Z-Glu-Gly-Arg-NA), and thrombin (best substrate = Z-Lys-Gly-Arg-NA) were the group of enzymes that showed next highest reactivity toward the substrates. Activated bovine protein C, thrombin, and factor Xa displayed relatively little substrate specificity. Activated human protein C (best substrate = Z-Ser-Phe-Arg-NA) and factor XIa (best substrate = Z-Glu-Gly-Arg-NA) are moderately reactive enzymes. Activated human protein C is an extremely specific enzyme since it has such a large range of kcat/KM values.(ABSTRACT TRUNCATED AT 250 WORDS)