Urszula Grabowska

The resorptive apparatus of osteoclasts supports lysosomotropism and increases potency of basic versus non-basic inhibitors of cathepsin K

In mice and humans, the effect of genetic deficiency of cathepsin K (catK) is impaired bone resorption, or osteopetrosis. Inhibition of catK is therefore a promising strategy for the treatment of osteoporosis. The enzyme acts in an acid environment. This provides a further potential opportunity: if the inhibitor is basic it is more likely to accumulate in membrane-bound acidic compartments (lysosomotropism), so minimizing off-target effects. However, the resorptive hemivacuole is not membrane-bound, and so might not retain lysosomotropic compounds. We therefore elected to determine whether the osteoclastic resorptive apparatus supports such accumulation. First, we attempted to compare the persistence of a lysosomotropic dye in the hemivacuole versus intracellular vesicles. To our surprise the dye could not be detected in the ruffled border region by confocal microscopy. We found that this could be explained by the tight packing of the folds of the ruffled border, and their close apposition to the bone surface. We also found that the dye persisted similarly in resorbing osteoclasts and macrophages, consistent with the notion that resorbing osteoclasts support lysosomotropism. Next, we compared the ability of basic and non-basic inhibitors of catK to suppress bone resorption by human osteoclasts. We found that basic compounds were considerably more potent than non-basic compounds at suppression of osteoclastic resorption than would be anticipated from their potency as enzyme inhibitors. Also consistent with osteoclastic lysosomotropism, basic inhibitors suppressed resorption for substantially longer than a non-basic inhibitor after washout from cell cultures. Furthermore, selectivity of basic inhibitors for inhibition of catK versus other cathepsins persisted: concentrations that inhibited catK in osteoclasts had no detectable effect on cathepsin S (catS) in a cell-based assay. This data is consistent with accumulation and enrichment of such basic inhibitors in the resorptive apparatus of the osteoclast, allowing for prolonged efficacy at the intended site of action. Our results suggest a major advantage for lysosomotropic compounds as inhibitors of bone resorption by osteoclasts in osteoporosis and other diseases caused by excessive osteoclastic activity.

New routes to β-cycloalkylalanine derivatives using serine-derived organozinc reagents

Two distinct routes to beta-cycloalkylalanine derivatives have been developed. The first route employs the reaction of the iodoalanine-derived zinc-copper reagent 2 with cycloalk-1-en-3-yl phosphates, and the second uses the palladium-catalysed coupling of the iodoalanine-derived zinc reagent 1 with cycloalkenyl triflates; in each case, catalytic hydrogenation of the unsaturated product leads to the protected beta-cycloalkylalanine. The latter route allows access to a range of cycloalkyl derivatives, with ring sizes of 5-8. beta-(1-Methyl-1-cyclohexyl)alanine may be prepared using reaction of the zinc-copper reagent 2 with 3-methyl-2-cyclohexenyl chloride, followed by hydrogenation. The corresponding cyclopentyl derivative may be prepared by reaction of the same zinc-copper reagent 2 with diethyl geranylphosphate, followed by ring-closing metathesis and hydrogenation.

A new route to hydrophobic amino acids using copper-promoted reactions of serine-derived organozinc reagents

Copper-catalysed reaction of the serine-derived zinc reagent 1 with allylic electrophiles gives products arising formally from both SN2 and SN2′ pathways. These constitutional isomers can be separated, either directly, or in the case of (2S)-2-tert-butoxycarbonylamino-6-methylhept-5-enoic acid methyl ester (11) and (2S)-2-tert-butoxycarbonylamino-4,4-dimethylhex-5-enoic acid methyl ester (12) by selective epoxidation of 11. Hydrogenation of the double bond, followed by protecting group manipulation, allows the synthesis of the Fmoc-protected amino acids 3–7 ready for automated peptide synthesis.