DOI: 10.1002/cbic.201000159 Artificial Metalloenzymes through Cysteine-Selective Conjugation of Phosphines to Photoactive Yellow Protein Wouter Laan, [a] Bianca K. MuÇoz, [a] RenØ den Heeten, [b] and Paul C. J. Kamer* [a] The embedding of organometallic catalysts into the chiral envi- ronment of proteins and DNA to develop enantioselective hybrid transition metal catalysts is attracting increasing atten- tion. [1] Phosphine ligands are ubiquitous in transition metal chemistry and can afford extremely reactive and versatile ho- mogeneous catalysts. Consequently, various efforts have been made to create hybrid catalysts with this attractive class of ligands. The covalent embedding of phosphine ligands into DNA [2] and the application of phosphine-functionalized DNA in asymmetric catalysis has recently been described. [3] So far, pro- tein-based artificial metalloenzymes containing phosphine li- gands have mainly been developed by using noncovalent an- choring strategies. The introduction of biotinylated phosphines to (strept)avidin has been a particularly successful approach for the development of enantioselective artificial metalloen- zymes. [4] Furthermore, the potential of using antibodies for the development of hybrid catalysts through supramolecular an- choring of phosphine catalysts has recently been demonstrat- ed. [5] In contrast, no examples of artificial metalloenzymes based on robust covalent phosphine conjugation to a protein have been reported to date, and so the protein structure space combined with this class of ligand remains limited. Neverthe- less, Reetz has modified the active-site serine of a number of lipases with a diphosphine coupled to a phosphonate inhibitor, but unfortunately the resulting hybrids turned out to be hy- drolytically unstable, which hampered application in catalysis. [6] The unique reactivity of the nucleophilic thiol side chain of cysteine makes it a very attractive target for site-selective bio- conjugation to proteins, which has previously been used for the covalent anchoring of synthetic catalysts. However, be- cause of the nucleophilic character of phosphines, the most common methods for cysteine-selective bioconjugation, such as, disulfide bridge formation or alkylation by using haloaceta- mides and maleimides are incompatible with phosphines. Thus, alternative strategies need to be explored for this class of ligand. De Vries et al. turned to less-nucleophilic phosphites that could be covalently attached to papain. This resulted in an active, but nonselective hydrogenation catalyst. [7] Following a different approach, we have developed for the first time the site-selective covalent conjugation of phosphine ligands and phosphine–transition-metal complexes to a protein, and we report the application of some of the hybrids in catalysis. Photoactive yellow protein (PYP) is a small (15 kDa) water- soluble photoreceptor protein from the bacterium Halorhodo- spira halophila (Figure 1). [8] The protein has a strong absorb- ance peak at 446 nm due to its chromophore, p-hydroxycin- namic acid, which is located in a small hydrophobic binding pocket and covalently linked by a thioester linkage to the unique cysteine, Cys69, of the protein. [9] The (recombinant) PYP apo-protein can be reconstituted in vitro with activated forms of the p-hydroxycinnamic acid chromophore or other chromophore derivatives: the use of the thiophenyl ester, the anhydride and the imidazolide of p-hydroxycinnamic acid can all lead to highly efficient and selective formation of the de- sired thioester linkage with the protein. [10] We decided to explore whether this reconstitution approach could be adopted for the site-selective coupling of phosphine ligands to the cysteine of PYP by using phosphino-carboxylic acids. The only by-products of the activation of a carboxylic acid with N,N-carbonyldiimidazole (CDI) to form the reactive imidazolide and subsequent coupling reaction are imidazole and CO 2 . Because they are easily removed from the protein after coupling, we chose to use CDI-activated phosphine ligands for the protein functionalization. The imidazolides of phosphino-carboxylic acids 1–7 were synthesised by treating them with an excess of N,N-carbonyldiimidazole (CDI) in DMF (Scheme 1). For all ligands, a shift of the signal in the 31 P NMR spectrum occurs upon imidazolide formation, thus allowing the extent of activation to be monitored by NMR. Treatment of the PYP with imidazolides of 1–7 afforded in all cases the desired conjugate in high yield and with excellent chemoselectivity. The predominant LC-MS signal found for each conjugate corresponds to PYP containing free phosphine (Table 1 and Figures S1–S3 in the Supporting Information). While the bidentate ligands 4 and 7 were the least reactive, the use of a larger excess still afforded excellent conversion. The lower reactivity might be due to the increased steric hin- drance encountered by these bulkier bidentate ligands or a faster rate of hydrolysis of the imidazolide. Due to their nucleo- philicity, free phosphines react similarly to free thiols with re- agents used for the colorimetric detection of thiol groups. This prohibited the use of such assays to determine the specificity and efficiency of the coupling reactions. Instead, we relied on mass spectrometry to determine the extent of modification. Al- though in most cases a signal corresponding to the unmodi- fied protein was still observed, the ionization efficiency of each hybrid was found to be about 80–100-fold less than that of the parent-protein, therefore revealing that all ligands coupled with more than 90 % efficiency (see the Supporting Informa- [a] Dr. W. Laan, Dr. B. K. MuÇoz, Prof. Dr. P.C. J. Kamer School of Chemistry, University of St Andrews North Haugh, KY16 9ST, St. Andrews (UK) Fax: (+ 44) 1334463808 E-mail : pcjk@st-andrews.ac.uk [b] Dr. R. den Heeten Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam Nieuwe Achtergracht 166, 1018 WV Amsterdam (NL) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000159. 1236 # 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2010, 11, 1236 – 1239