A general procedure which selectively introduced a nucleophilic group at a particular location in the active site of lysozyme has been developed. The coupling of hen egg white lysozyme with amine nucleophiles by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was studied at pH 5 and room temperature. In the presence of an amine nucleophile, such as ethanolamine, ethylenediamine, methylamine, or 4(5)-(aminomethyl)imidazole, the carboxyl side chain of aspartic acid-101 in lysozyme was selectively modified by using a small excess of EDC. The reactivity of Asp-101 is probably due to the specific binding of EDC to the substrate binding site close to Asp-101. With histamine or D-glucosamine, the selectively of Asp-101 was somewhat decreased. This may be due to the specific binding of these amines to lysozyme in competition with EDC, causing a decrease of the selective activation of Asp-101 by EDC. Depending on the amine employed, the lysozyme derivatives obtained exhibited decreased activity (83-52% of native enzyme), suggesting that the modification of Asp-101 weakened substrate binding.

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Three types of reactions of 2,3-anhydro-beta-cyclodextrins, namely nucleophilic ring-opening, reduction to 2-enopyranose, and reduction to 3-deoxypyranose, have been investigated to regio- and stereoselectively functionalize the secondary face of beta-cyclodextrin. Upon treatment with various nucleophiles, both 2,3-mannoepoxy and 2,3-alloepoxy-beta-cyclodextrins are found to undergo nucleophilic ring-opening reaction generating 3- and 2-modified cyclodextrin derivatives. In each case, the 3-position is more easily accessible than the 2-position. By using these ring-opening reactions, imidazolyl, iodo, azido, and benzylmercapto groups are selectively introduced to the secondary face of beta-cyclodextrin in place of the 2- or 3-hydroxyl groups. The functionalized cyclodextrins have either modified glucosidic subunits or modified altrosidic subunits that make the hydrophobic cavity slightly distorted from that of native beta-cyclodextrin. Thiourea also reacts with the cyclodextrin epoxides. In this case, thiirane and olefin species are generated instead of any ring-opening products. By ameliorating the reaction condition, cyclodextrin olefin, diene, and triene derivatives are prepared in moderate to good yields. Reduction of per[6-(tert-butyldimethyl)silyl]-beta-cyclodextrin permannoepoxide with lithium aluminum hydride produces the per(3-deoxy)-beta-cyclomannin. All these chemically modified cyclodextrins are structurally well characterized and most of them are expected to serve as versatile scaffolds for diverse purposes such as the construction of catalysts and development of synthetic receptors and molecular containers.

Specific sulfonation on a secondary hydroxyl group (C-2-OH or C-3-OH) of -cyclodextrin was elucidated. A reaction of -cyclodextrin with m-nitrobenzenesulfonyl chloride exclusively gave the C-2-sulfonate, while /3-naphthalenesulfonyl chloride selectively afforded the C-3-sulfonate. The former reaction also gave selectively 2A2B-, 2A2C-, and 2A2D-disulfonates as the products of disulfonation. They were well separated by reversed-phase column chromatography, isolated, and converted to corresponding di-manno-epoxides. Structural determinations of the disulfonates and the diepoxides were made from their