SummaryGaucher disease, resulting from deficient lysosomal glucocerebrosidase (GC) activity, is the most common lysosomal storage disorder. Clinically important GC mutant enzymes typically have reduced specific activity and reduced lysosomal concentration, the latter due to compromised folding and trafficking. We and others have demonstrated that pharmacological chaperones assist GC folding by binding to the variants active site, stabilizing the native conformation of GC in the neutral pH environment of the endoplasmic reticulum (ER), enabling their trafficking from the ER to the Golgi and on to the lysosome. The mutated GC fold is generally stable in the lysosome after pharmacological chaperone dissociation, owing to the low pH environment for which the fold was evolutionarily optimized and the high substrate concentration, enabling GC to hydrolyze glucosylceramide to glucose and ceramide. The hypothesis of this study was that we could combine GC pharmacological chaperone structure activity relationships from distinct chemical series to afford potent novel chaperones comprising a carbohydrate-like substructure that binds in the active site with a hydrophobic substructure that binds in a nearby pocket. We combined isofagomine and 2,5-anhydro-2,5-imino-D-Glucitol active site binding substructures with hydrophobic alkyl adamantyl amides to afford novel small molecules with enhanced ability to increase GC activity in patientderived fibroblasts. The cellular activity of N370S and G202R GC in fibroblasts is increased by 2.5 and 7.2 fold with isofagmine-based pharmacological chaperones N-Adamantanyl-4-((3R,4R, 5R)-3,4-dihydroxy-5-(hydroxymethyl)piperidin-1-yl)-butanamide (3) and N-Adamantanyl-4-((3R, 4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)piperidin-1-yl)pentanamide (4), respectively, the best enhancements observed to date.

A one-pot chemoenzymatic method for the synthesis of a variety of new iminocyclitols from readily available, non-phosphorylated donor substrates has been developed. The method utilizes the recently discovered fructose-6-phosphate aldolase (FSA), which is functionally distinct from known aldolases in its tolerance of different donor substrates as well as acceptor substrates. Kinetic studies were performed with dihydroxyacetone (DHA), the presumed endogenous substrate for FSA, as well as hydroxy acetone (HA) and 1-hydroxy-2-butanone (HB) as donor substrates, in each case using glyceraldehyde-3-phosphate as acceptor substrate. Remarkably, FSA used the three donor substrates with equal efficiency, with kcat/KMvalues of 33, 75, and 20 M-1 s-1, respectively. This level of donor substrate tolerance is unprecedented for an aldolase. Furthermore, DHA, HA, and HB were accepted as donors in FSA-catalyzed aldol reactions with a variety of azido- and Cbz-amino aldehyde acceptors. The broad substrate tolerance of FSA and the ability to circumvent the need for phosphorylated substrates allowed for one-pot synthesis of a number of known and novel iminocyclitols in good yields, and in a very concise fashion. New iminocyclitols were assayed as inhibitors against a panel of glycosidases. Compounds 15 and 16 were specific alpha-mannosidase inhibitors, and 24 and 26 were potent and selective inhibitors of beta-N-acetylglucosaminidases in the submicromolar range. Facile access to these compounds makes them attractive core structures for further inhibitor optimization.

Dihydroxyacetone phosphate (DHAP)dependent aldolases have been widely used for the organic synthesis of unnatural sugars or derivatives. The practicality of using DHAP-dependent aldolases is limited by their strict substrate specificity and the high cost and instability of DHAP. Here we report that the DHAP-dependent aldolase l-rhamnulose 1-phosphate aldolase (RhaD) accepts dihydroxyacetone (DHA) as a donor substrate in the presence of borate buffer, presumably by reversible in situ formation of DHA borate ester. The reaction appears to be irreversible, with the products thermodynamically trapped as borate complexes. We have applied this discovery to develop a practical one-step synthesis of the non-caloric sweetener lfructose. l-Fructose was synthesized from racemic glyceraldehyde and DHA in the presence of RhaD and borate in 92 % yield on a gram scale. We also synthesized a series of l-iminocyclitols, which are potential glycosidase inhibitors, in only two steps.

Several model mechanism-based inhibitors (MbIs) were designed and evaluated for their ability to inhibit sulfatases. The MbI motifs were based on simple aromatic sulfates, which are known to be commonly accepted substrates across this highly conserved enzyme class, so that they might be generally useful for sulfatase labeling studies. (Difluoro)methyl phenol sulfate analogs, constructed to release a reactive quinone methide trap, were not capable of irreversibly inactivating the sulfatase active site. On the other hand, the cyclic sulfamates (CySAs) demonstrated inhibition profiles consistent with an active-site directed mode of action. These molecules represent a novel scaffold for labeling sulfatases and for probing their catalytic mechanism.

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