A combinatorial library composed from nine alcohols and six isocyanates to formally generate 54 carbamates has been designed, prepared, and screened against acetylcholinesterase from the electric eel. In order to deduce the most active member of the library, it was prepared as 15 sublibraries in which one of the reacting components was fixed and the other reactants were used as an equimolar mixture. The product mixtures were tested and their activities used as "indices" to the rows or columns of a two-dimensional matrix reflecting the activities of individual carbamates. A number of carbamates in the most active row and column were synthesized and assayed, demonstrating that the most active cell in the matrix could be identified by the sublibrary synthesis procedure. Other methods for generating large libraries of molecules for biological screening that have recently been developed have relied on a covalent attachment between library members and a label to identify the active components. Indexed libraries offer the advantage that they can be prepared from any class of compounds composed from multiple subunits and that any type of assay (binding, enzyme inhibition, agonism/antagonism, cell-based, or even whole organism assays for biological activity) can be used because all compounds are generated in a free form.

No abstract

The enzyme that oxidatively converts 1-aminocyclopropanecarboxylic acid (ACC) to ethylene, a key plant growth hormone, has been classified, on the basis of a comparison of homologous protein sequences (derived from the cDNA sequences), as a member of a family of non-heme iron proteins that includes plant and bacterial oxidative enzymes. This knowledge has facilitated the purification of the relatively abundant ethylene-forming enzyme to homogeneity from apple tissue. The properties of the enzyme are consistent with two other recent reports that describes its purification by different protocols, lending credence to the assertion that the key protein has been isolated. New characterizations of the protein have been conducted. Electrospray mass spectrometry shows that its molecular weight (35 331.8 +/- 5 amu) is approximately 50 amu higher than that predicted from the cDNA sequence, identifying the blocking group at the N-terminus as acetyl. The enzyme is activated by bicarbonate at low concentration but is inhibited at high concentration, with the maximum activation occurring at 5 mM. The iron concentration leading to half-maximal activity is 1 microM. The enzyme self-inactivates during turnover. The availability of the purified enzyme will permit definitive studies of the mechanism by which ethylene is produced and provide opportunities to discover molecules that inhibit the process.

Our results provide support for a radical mechanism in the final step of ethylene biosynthesis and refute an alternative N-hydroxylation mechanism. This work extends the idea that the intrinsic chemical reactivity of a high energy iron-oxo intermediate can account for the observed products in ethylene biosynthesis.

In this work, we have provided a relatively rare example of carbon-carbon bond formation in a pool synthesis and have discovered a potentially useful acetylcholinesterase inhibitor.