9-(5,5-Difluoro-5-phosphonopentyl)guanine ( 3) was synthesized as a potential multisubstrate analogue inhibitor of purine nucleoside phosphorylase (EC 2.4.2.1, PNP). At pH 7.4, 3 has a K, value 18-, 26-, 25-, and 5.5-fold lower than that of the nonfluorinated analogue 9-(5-phosphonopentyl)guanine (2) regarding PNP from human erythrocyte, rat erythrocyte, calf spleen, and Escherichia coli, respectively. Further studies with human erythrocytic PNP show that at pH 6.2 the difference in K¡ value is more pronounced { / is 96), and at pH 8.8, where 2 and 3 are both essentially present in the unprotonated form, the ratio is 8. The superiority of the difluorophosphonate 3 over the phosphonate 2 is explained by electronic as well as by steric effects.
Formation of the 4-kDa peptides, which are essential constituents of the extracellular plaques in Alzheimer's disease, involves the sequential cleavage of the amyloid precursor protein (APP) by p-and y-secretases. The carboxy-terminal 99-amino-acid peptide which is liberated from APP by p-secretase was used as a potential native substrate of the y-secretase(s). With the addition of an initiator Met and a FLAG sequence at the C-terminus (PAPP100-FLAG), it was expressed in Escherichia coli under the control of the T7 promotor. The preferred site(s) of cleavage in the N-terminal 40-amino-acid P-amyloid peptide and PAPPI 00-FLAG by potential y-secretase(s) were rapidly identified using matrix-assisted Iaser-desorptionlionization time-of-flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis. Since y-secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing. Studies using different detergents indicated that the cleavage preference of cathepsin D for the PAPP100-FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins were found to be capable of catabolizing both p-amyloid peptides and the PAPPIOO-FLAG. As cathepsin D was found to cleave the PAPP100-FLAG in the vicinity of the C-terminus of the /l-aniyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the possibility cannot be excluded that cathepsins D and B are involved in the amyloidogenic processing of APP.
There is presently an upsurge of interest in glycosidase inhibitors. Such compounds have been shown to be important tools in mechanistic studies on glycohydrolases,1 as well as having promising therapeutic applications.2 However, only a few examples of enzyme-activated irreversible inhibitors of glycosidases have been described. They include conduritol epoxides,3 glycosylmethyltriazenes,4 and the aziridine triol derivatives of piperidine.5 These compounds are activated in the first protonation step of the enzyme-assisted glycoside hydrolysis. Due to their high intrinsic chemical reactivity, insufficient specificity could limit their use as drugs. Inhibitors activated only after or during enzymic cleavage of the glycosidic bond should be more selective.The first examples of this new class are 2-deoxy-2-fluoroglycosides.6 These inhibitors are believed to be cleaved before inactivating the enzyme by the formation of a covalent glycosyl-enzyme complex. We now wish to report a novel enzymeactivated irreversible inhibitor of yeast -glucosidase based on the activation, following hydrolytic cleavage, of the aglycon moiety of the molecule.-Glucosidases catalyze the hydrolysis reaction shown in Scheme I where R can be either a glycosidic residue or an aglycon leaving group. Our approach toward irreversible inhibition of -glucosidase was to design R in such a way that the glucosidase 1 would be a stable substrate, while the alcohol 2 would be spontaneously and rapidly converted into a reactive alkylating agent.1,1-Difluoroalkyl glucosides 3 (Scheme II) were chosen to illustrate this concept. The , -difluoro alcohols 4 (products of the -glucosidase-catalyzed hydrolysis of 3) are known7 to rapidly lose HF and to be transformed into acid fluorides 5. These acylating agents are then expected to form a covalent adduct 6 with a nucleophilic residue of the enzyme-active site.8
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