Kruithof_2005_Chemistry_11_6869

The work described herein presents a strategy for the regioselective introduction of organometallic complexes into the active site of the lipase cutinase. Nitrophenol phosphonate esters, well known for their lipase inhibitory activity, are used as anchor functionalities and were found to be ideal tools to develop a single-site-directed immobilization method. A small series of phosphonate esters, covalently attached to ECE "pincer"-type d8-metal complexes through a propyl tether (ECE=[C6H3(CH2E)(2)-2,6]-; E=NR2 or SR), were designed and synthesized. Cutinase was treated with these organometallic phosphonate esters and the new metal-complex/protein hybrids were identified as containing exactly one organometallic unit per protein. The organometallic proteins were purified by membrane dialysis and analyzed by ESI-mass spectrometry. The major advantages of this strategy are: 1) one transition metal can be introduced regioselectively and, hence, the metal environment can potentially be fine-tuned; 2) purification procedures are facile due to the use of pre-synthesized metal complexes; and, most importantly, 3) the covalent attachment of robust organometallic pincer complexes to an enzyme is achieved, which will prevent metal leaching from these hybrids. The approach presented herein can be regarded as a tool in the development of regio- and enantioselective catalyst as well as analytical probes for studying enzyme properties (e.g., structure) and, hence, is a "proof-of-principle design" study in enzyme chemistry.