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It is conceivable that the irreversible inhibition by LG analogues could be the result of a stable covalent bond between the inhibitor and the proteasome borne from direct alkylation via the chlorethyl group (as an alternative to or in addition to the covalent ester bond to Thr1Oγ, However, there was no evidence of any direct alkylation product in the crystal structure of 1 with the 20S proteasome; only the intramolecular chloride displacement product (cyclic ether end product Ib′) was observed.26 Thus, there is little evidence to support the hypothesis that the longer duration of proteasome inhibition in the case of 1 (or other analogues in the LG family) results from direct alkylation of proteasome amino acid(s) by the chloroethyl group. Moreover, direct reactions of 1 or closely related compounds with nucleophiles that may mimic amino acid side chains, including primary alcohols, water or hydroxide, thiols
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