A general approach was developed for the regio-and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by posttranslationally modifying the cysteine residue in a CaaX recognition motif with functional groups suitable for "click" chemistry or a Staudinger ligation. Farnesyl diphosphate analogs bearing ω-azide or ω-alkyne moieties were attached to the cysteine residue in Cys-Val-Ile-Ala motifs at the C-termini of engineered versions of green fluorescent protein (GFP) and glutathione S-transferase (GST) by protein farnesyltransferase. The derivatized proteins were attached to glass slides bearing linkers containing azide ("click" chemistry) or phosphine (Staudinger ligation) groups. "Click" immobilized proteins were detected by fluorescently labeled antibodies and remained attached to the slide through two cycles of stripping under stringent conditions at 80 °C. GFP immobilized by a Staudinger ligation was detected by directly imagining the GFP fluorophore over a period of 6 days. These methods for covalent immobilization of proteins should be generally applicable. CaaX recognition motifs can easily be appended to the C-terminus of a cloned protein by a simple modification of the corresponding gene, and virtually any soluble protein or peptide bearing a CaaX motif is a substrate for protein farnesyltransferase.Protein "chips" are useful for studying protein-ligand and protein-protein interactions, i including the detection of antibody-antigen interactions, ii and permit high-throughput screening of limited quantities of analytes in a microarray format. iii Devices with covalently attached proteins 1,iv are more robust than their non-covalent counterparts. v In addition, substantial enhancements in sensitivity are seen when proteins are attached in a uniform manner. 4,5,vi Typically covalent immobilization is accomplished by reactions that rely on nucleophilic moieties found in the side chains of naturally occurring amino acids. i,vii We now report a general approach for the regio-and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by posttranslationally modifying a cysteine residue with functional groups suitable for "click" chemistry viii or a Staudinger ligation. ix Protein farnesyltransferase (PFTase) catalyzes the alkylation of the sulfhydryl moiety in the cysteine located in C-terminal CaaX motifs, where X = A, S, M, or Q, by farnesyl diphosphate (1) (see Scheme 1). x The reaction is general for any soluble protein bearing a CaaX motif. We synthesized farnesyl analogs 2 and 3, both of which are excellent alternate substrates for yeast PFTase with catalytic efficiencies (1.6 and 0.58 µM −1 s −1 ) similar to that of 1 (0.76 µM −1 s −1 ). xi As model proteins for immobilization, we engineered C-terminal CVIA motifs into green fluorescent protein (GFP) and glutathione S-transferase (GST) proteins with azide analog 2 or alkyne analog 3 gave GFP-N 3 , GFP-C 2 , GST-N 3 , or G...

More than 1000 yields and ee determinations are possible per day using an immunoassay. This highly efficient screening method is based on the remarkable binding specificity of antibodies. It has been employed in the development of straightforward procedures for the direct conversion of α‐keto acids into chiral α‐hydroxy acids, for which the enantioselective reduction of benzoyl formic acid to mandelic acid serves as a model reaction (see scheme).

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