The synthetic potential of phthalimide SET photochemistry.

The authors' studies in the area of phthalimide photochemistry are discussed in the context of the development of new methods for N-heterocycle synthesis. Emphasis is given to reactions which are initiated by both intermolecular and intramolecular SET from silicon-containing electron donors to excited states of phthalimides and related maleimides and conjugated imides. The photoaddition and photocyclization processes which ensue follow mechanistic pathways, in which efficient desilylation of initially formed radical cation occurs to generate radical pair and biradical intermediates that serve as precursors of the products. Several examples that demonstrate the preparative potential of these reactions are presented. These are taken from the authors' investigations of (1) phthalimido-alkylsilane photocyclization reactions, (2) azomethine ylide-forming excited-state processes of N-(trimethylsilylmethyl)phthalimide, and (3) photoaddition and photocyclization reactions of phthalimide alpha-silyl ether, thioether, amine, and amide systems.

Applications of phthalimide photochemistry to macrocyclic polyether, polythioether, and polyamide synthesis.

Irradiation of phthalimides which contain N-linked omega-trimethylsilylmethyl-substituted polyether, polythioether, and polysulfonamide chains results in efficient production of the corresponding macrocyclic polyether, polythioether, and polysulfonamide products. These photocyclization reactions follow sequential single electron transfer (SET)-desilylation pathways. Only in the cases of phthalimides, bearing mixed ether-thioether N-substituents, do these excited-state cyclization reactions proceed with lower degrees of regioselectivity. This is a result of competitive desilylation and alpha-to-sulfur deprotonation reactions of the zwitterionic diradical intermediates formed by initial SET.

Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium symbiosum pyruvate phosphate dikinase. 1. Identification of an essential cysteine by chemical modification with [1-14C]bromopyruvate and site-directed mutagenesis.

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PPi), and phosphoenolpyruvate (PEP). The reaction takes place according to the following steps: (1) E+ATP+P(i)<-->E-PP.AMP.P(i), (2) E-PP.AMP.P(i)<-->E-P+AMP+PP(i), and (3) E-P+pyruvate<-->E+PEP, where E represents free enzyme; E-PP, pyrophosphorylenzyme; and E-P, phosphorylenzyme. Steps 1 and 2 comprise the nucleotide partial reaction, and step 3 comprises the pyruvate partial reaction. The present studies were carried out to locate amino acid residues within the primary structure of Clostridium symbiosum PPDK participating in the catalysis of the pyruvate partial reaction. The enzyme was treated with the affinity label [1-14C]bromopyruvate, reduced with NaBH4, proteolyzed with trypsin, and chromatographed on an HPLC column. The radiolabeled tryptic peptide isolate was sequenced to reveal Cys 831 as the site of alkylation. Using PCR techniques Cys 831 was replaced by Ala, and the C831A PPDK mutant formed was then subjected to kinetic analysis. Rapid quench studies of single turnover reactions on the enzyme showed that the mutant is as efficient as wild-type PPDK in catalyzing the nucleotide partial reaction while it is unable to catalyze the pyruvate partial reaction. These results were interpreted as evidence for a role of Cys 831 in pyruvate/PEP binding and/or catalysis.

Investigation of the substrate binding and catalytic groups of the P-C bond cleaving enzyme, phosphonoacetaldehyde hydrolase.

Kinetic studies with substrate analogs and group-directed chemical modification agents were carried out for the purpose of identifying the enzyme-substrate interactions required for phosphonoacetaldehyde (P-Ald) binding and catalyzed hydrolysis by P-Ald hydrolase (phosphonatase). Malonic semialdehyde (Ki = 1.6 mM), phosphonoacetate (Ki = 10 mM), phosphonoethanol (Ki = 10 mM), and fluorophosphate (Ki = 20 mM) were found to be competitive inhibitors of the enzyme but not substrates. Thiophosphonoacetaldehyde and acetonyl phosphonate underwent phosphonatase-catalyzed hydrolysis but at 20-fold and 140-fold slower rates, respectively, than did P-Ald. In the presence of NaBH4, acetonyl-phosphonate inactivated phosphonatase at a rate exceeding that of its turnover. Sequence analysis of the radiolabeled tryptic peptide generated from [3-3H]acetonylphosphonate/NaBH4-treated phosphonatase revealed that Schiff base formation had occurred with the catalytic lysine. From the Vm/Km and Vm pH profiles for phosphonatase-catalyzed P-Ald hydrolysis, an optimal pH range of 6-8 was defined for substrate binding and catalysis. The pH dependence of inactivation by acetylation of the active site lysine with acetic anhydride and 2,4-dinitrophenyl acetate evidenced protonation of the active site lysine residue as the cause for activity loss below pH 6. The pH dependence of inactivation of an active site cysteine residue with methyl methanethiol-sulfonate indicated that deprotonation of this residue may be the cause for the loss of enzyme activity above pH 8.

Investigation of the Bacillus cereus phosphonoacetaldehyde hydrolase. Evidence for a Schiff base mechanism and sequence analysis of an active-site peptide containing the catalytic lysine residue.

Reaction of Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) with phosphonoacetaldehyde or acetaldehyde in the presence of NaBH4 resulted in complete loss of enzymatic activity. Treatment of phosphonatase with NaBH4 in the absence of substrate or product had no effect on catalysis. Inactivation of phosphonatase with [3H]NaBH4 and phosphonoacetaldehyde, NaBH4 and [14C]acetaldehyde, or NaBH4 and [2-3H]phosphonoacetaldehyde produced in each instance radiolabeled enzyme. The nature of the covalent modification was investigated by digesting the radiolabeled enzyme preparations with trypsin and by separating the tryptic peptides with HPLC. Analysis of the peptide fractions revealed that incorporation of the 3H- or 14C-radiolabel into the protein was reasonably selective for an amino acid residue found in a peptide fragment observed in each of the three trypsin digests. Sequence analysis of the 3H-labeled peptide fragment isolated from the digest of the [2-3H]phosphonoacetaldehyde/NaBH4-treated enzyme identified N epsilon-ethyllysine as the radiolabeled amino acid. The ability of the phosphonatase competitive inhibitor (Ki = 230 +/- 20 microM) acetonylphosphonate to protect the enzyme from phosphonoacetaldehyde/NaBH4-induced inactivation suggested that the reactive lysine residue is located in the enzyme active site. Comparison of the relative effectiveness of phosphonoacetaldehyde and acetaldehyde as phosphonatase inactivators showed that the N-ethyllysine imine that is reduced by the NaBH4 is derived from the corresponding N-(phosphonoethyl) imine. On the basis of these findings, a catalytic mechanism for for phosphonatase is proposed in which phosphonoacetaldehyde is activated for P-C bond cleavage by formation of a Schiff base with an active-site lysine. Accordingly, an N-ethyllsysine enamine rather than the high-energy acetaldehyde enolate anion is displaced from the phosphorus.

Evidence for a pH-dependent irreversible formation of a stable conformation of phenacyl-alpha-chymotrypsin.

A reinvestigation of the modification reactions of alpha-chymotrypsin with phenacyl bromide was carried out. Results conclusively demonstrate that the chemically and physically different modified enzymes prepared at pH 4 and at pH 7 both contain the phenacyl group at methionine-192 in the sulphonium salt form. Evidence to suppoort this conclusion derives from 13C nuclear-magnetic-resonance spectroscopic observations on [methylene-13C]phenacyl-enriched enzymes. More conclusively, the methionine-192-containing C-chain, derived by performic acid oxidative cleavage of radioactively-labelled enzyme prepared at pH 7, was shown to contain the phenacyl moiety and to undergo dealkylation by 2-mercaptoethanol with loss of this moiety. In addition, thermolytic cleavage of the high-pH enzyme results in fragmentation of the polypeptide chain in a fashion analogous to model reactions of phenacylmethionyl dipeptides and other methionine-192 sulphonium salts. A rationalization of the unusual nature of the high-pH phenacyl-modified enzyme based on the irreversible formation of stable conformation in which the phenacyl moiety is rigidly located in interior regions of the enzyme is presented and discussed.

Reinvestigation of the phenacyl bromide modification of alpha-chymotrypsin.

The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.