Metal-Conjugated Affinity Labels: A New Concept to Create Enantioselective Artificial Metalloenzymes

Invited for this month′s cover is the group of Prof. Jorg Eppinger. The cover picture illustrates the concept of using metal-conjugated affinity labels (m-ALs) to convert proteases into well-defined and catalytically active artificial metalloenzymes. For more details, see the Communication on p. 50 ff.

S-1 Chromogenic assays for the determination of residual enzyme activity of organometallic enzyme hybrids pg. 7 3 Hydrogenation of ketones using organometallic enzyme hybrids pg. 9 3.1 General procedure for hydrogenation of ketones pg. 9 3.2 Prochiral substrates for asymmetric hydrogenation used in this work pg. 9 3. 3 Variation of different parameters pg. 10

1.3
General procedure for the synthesis of metal-conjugated affinity labels Dichloromethane (10 ml) was added to polystyrene bound cyclohexyl carbodiimide (0.24 mmol, 1.3 mmol/g) at 0° C and the resin was allowed to swell for several minutes. The corresponding carboxylic acid (1 or 2; 0.08 mmol) was added to the suspension. After 15 min, pentafluorophenol (0.12 mmol) in dichloromethane was added slowly. The resulting suspension was stirred at 0° C for 1 h, warmed to RT and stirred for further 8 h. After filtration, the metal complex (0.08 mmol) and triethylamine (0.36 mmol) were added at RT. After 30 min the reaction mixture was extracted with water and brine (each 3 x 10 ml), dried over MgSO 4 and the solvent was removed under reduced pressure. The resulting red solid was washed with diethylether and hexanes (each 3 x 10 ml) to yield the analytically pure metal-conjugated affinity label.

2.1
Preparation of organometallic enzyme hybrids for MALDI-TOF/TOF analysis 2.1.1 MALDI TOF/TOF analysis of papain based organometallic enzyme hybrids: 50 L of a papain suspension were dissolved in 2950 L of phosphate buffer (40 mM, pH 7.0), containing 1 mM DTT. To 50 L of this solution 5 L of metalla-affinity label (0.342 mM in DMSO) were added and incubated for 2 h at RT. Prior to measurement, the samples were filtered using Millipore ZipTips C4 to remove buffer salts. The samples were analyzed with a Bruker Ultraflex TOF/TOF with a SOUT-MTP ion source, samples were prepared in cyanohydroxycinnamic acid, laser pulse at 337 nm, ~100 mJ, 1 ns pulse width. Spectra were calibrated to the residual papain peak.     General procedure for asymmetric hydrogenation of ketones catalyzed by organometallic enzyme hybrids: A 1 mL glass vial, equipped with a stirring bar was charged with 200 μL of protease suspension (c = 22 mg/mL) and 260 μL aqueous buffered solution (120 mM PO 4 2-) containing DTT (0 to 7.7 mM). After incubating for 1 h, 50 μL DMSO and 5 μL of the m-AL (30 mM in DMSO) were added and the solution was stirred gently for 3 h. Subsequently 5μL 1-OH (20 mM in DMSO) were added and the reaction solution was stirred for another 3 h, before 5 μL of ketone (2 M in DMSO) was added. The vial was placed in a standard multivial autoclave and pressurized with H 2 (10 to 75 bar) at the desired temperature (20 to 40 °C). After the desired amount of time, pressure was released and the reaction solution was extracted with dichloromethane (600 μL, 2.5 h). Yields were determined either with 19 F NMR spectroscopy or gas chromatography. Enantiomer enrichment was determined by chiral GC-FID (Chirasil-Dex).
Substoichometric addition of the catalyst motive ensures that m-ALs bind to the protease and that metal centers are embedded in a chiral environment. However, after addition and binding of the m-AL, residual protease activity can be detected. If quenching of the residual protease activity by addition of 1-OH is neglected, selfcleavage of the protease results in slow digestion of the OMEH catalyst leading to irreproducible results.
Overall, efficient covalent anchoring of the m-AL in the protease's binding site is a key-factor to achieve good catalytic activities and stereoinduction. This was also reflected in a pronounced dependence of the OMEH catalyst's performance on the composition of the protease batch used, which we encountered during our studies. Hence, it is mandatory to control the quality of the enzyme batches used. We realized that the quality of commercially available papain and bromelain varies from batch to batch and even more from vendor to vendor. Low quality batches lead to reduced yields and lower enantiomer enrichment. Usually, batches of lower quality can already be identified by a substantial amount of impurities visible by the MALDI MS. To ensure comparability of the results presented in this study, all reactions were conducted with enzymes of the highest quality level (twice recrystallized) available from Sigma Aldrich. For any new batch we recorded a MALDI-MS spectrum of the protease and tested its proteolytic activity using the chromogenic assay described above. Further a test run with m-AL 1 Rh and substrate S-4 was carried out under standard conditions (see below) to identify potential changes in performance. This way we achieved good reproducibility of our results.

3.2
Prochiral substrates for asymmetric hydrogenation used in this work