DOI: 10.1002/asia.201403005
Full Paper
Metalloproteins
Hybrid Ruthenium ROMP Catalysts Based on an Engineered Variant of b-Barrel Protein FhuA DCVFtev : Effect of Spacer Length Daniel F. Sauer,[a] Marco Bocola,[b] Claudio Broglia,[a] Marcus Arlt,[b] Lei-Lei Zhu,[b] Melanie Brocker,[c] Ulrich Schwaneberg,[b] and Jun Okuda*[a]
tween the N-heterocyclic carbene ligand and the cysteine site 545 increased the ROMP activity toward a water-soluble 7-oxanorbornene derivative. The cis/trans ratio of the double bond in the polymer was influenced by the hybrid catalyst.
Abstract: A biohybrid ring-opening olefin metathesis polymerization catalyst based on the reengineered b-barrel protein FhuA DCVFtev was chemically modified with respect to the covalently anchored Grubbs–Hoveyda type catalyst. Shortening of the spacer (1,3-propanediyl to methylene) be-
Introduction
terms of coupling efficiency and catalytic activity, the maleimide linker appeared to be superior, offering the possibility of varying spacer length in order to bring the catalytic active center closer to the protein scaffold. In an additional step, the protein matrix will be modified in order to affect the activity or selectivity of the target benchmark ring-opening metathesis polymerization (ROMP) reaction. We report here the spacer modification based on the recently reported biohybrid system with the covalently attached Grubbs–Hoveyda type catalyst.[2h] These unique biohybrid metathesis catalysts with shortened spacer length incorporated in a membrane protein were employed in ring-opening metathesis polymerization (ROMP) reaction of a water-soluble 7-oxanorbornene derivative. The objective of this work was to explore the catalytic site before directed mutagenesis will be applied to modify the second ligand sphere.
There is growing recognition that the second ligand sphere influences the activity and selectivity of organometallic catalysts.[1] Biohybrid catalysts composed of an active organometallic catalyst site and a protein scaffold offer unprecedented opportunities for designing new catalysts for transformations beyond those in metabolic reactions (“artificial metalloenzymes”) by combining principles of chemocatalysis with those of biocatalysis containing non-natural metal centers.[2] We have recently reported a modified Grubbs–Hoveyda type catalyst bearing an alcohol function in the backbone of the NHC ligand.[2h] The advantage of the Grubbs–Hoveyda type catalyst is the relatively high robustness against air, moisture, and functional groups, making this type of catalyst suitable to perform metathesis reactions in an exceptional environment.[2a–c, h] This catalyst was anchored covalently to the rationally designed variant of the transmembrane b-barrel protein ferric hydroxamate uptake protein component A (FhuA), extracted from E. coli.[2h] Covalent anchoring to the protein is achieved through an a-bromoacetyl group or, alternatively, through a maleimide group with a 1,3-propanediyl spacer. In
Results and Discussion Grubbs–Hoveyda type catalysts with varied spacer length were prepared. Three maleimide derivatives containing carboxylic acids were synthesized following a procedure described by Christmann and co-workers.[3] After transforming the carboxylic group into an acetyl chloride group, the linking units were attached to a Grubbs–Hoveyda type catalyst by esterification with an alcohol function in the backbone of the NHC ligand. The influence of the FhuA DCVFtev ligand sphere was determined by comparing the performance of the biohybrid catalysts with that of the protein-free catalysts and a water-soluble Grubbs–Hoveyda type catalyst containing an alcohol function in the backbone of the NHC-ligand and a tertiary ammonium group on the benzylidene part. This water-soluble catalyst was obtained by reacting the Grubbs 2nd generation catalyst 3 with 1-(4-isopropyloxy-3-vinylphenyl)-N,N,N-trimethylmethanamonium chloride 4 (Scheme 1). The reactivity of the maleimide group towards the thiol group was explored in a model reac-
[a] D. F. Sauer, C. Broglia, Prof. Dr. J. Okuda Institut fr Anorganische Chemie RWTH Aachen University Landoltweg 1, 52056 Aachen (Germany) E-mail: [email protected] [b] Dr. M. Bocola, M. Arlt, Dr. L.-L. Zhu, Prof. Dr. U. Schwaneberg Lehrstuhl fr Biotechnologie RWTH Aachen University Worringer Weg 1, 52074 Aachen (Germany) [c] Dr. M. Brocker Institut fr Bio- und Geowissenschaften, IBG-1: Biotechnologie Forschungszentrum Jlich 52425 Jlich (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201403005. Chem. Asian J. 2015, 10, 177 – 182
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Full Paper bond. Performing the reaction under slightly basic conditions was necessary for complete conjugation. In aqueous solutions the reaction was slightly slower. In pure water the reaction could not be performed due to low solubility of the corresponding Grubbs–Hoveyda type complexes 13–15. By adding 20 % (v/ v) of THF to water, the catalysts were dissolved homogeneously. The pH value was adjusted to 7.4 with either NaOH or with a sodium phosphate buffer (NaPi, 0.1 m). In the case of sodium phosphate buffer, formation of precipitates was observed and the obtained yield was significantly lower. The best Scheme 1. Syntheses of Grubbs–Hoveyda type catalysts bearing a maleimide linking unit and the water-soluble conditions in aqueous solutions Grubbs–Hoveyda type catalyst 6. are in presence of 20 % (v/v) THF and NaOH at a pH value of around 7.5. These conditions are Table 1. Conditions for conjugation with cysteine. applicable to the host protein FhuA DCVFtev due to its high tolerance against organic solvents such as THF.[2h, 4] Under these optimized conditions, the anchoring with all catalysts was performed under oxygen-free conditions, and an excess of the corresponding catalyst (10 equiv) was employed. Complete reaction at the cysteine residue 545 was ensured by stirring the reaction mixture overnight (16 h). The work-up procedure was conducted as previously reported (Scheme 2).[2h] To prove the successful anchoring and refolding, several analyses were undertaken. To determine the degree of conjugation, a fluorescence titration with the fluorescence dye ThioGlo was perEntry Solvent Base/Buffer Reaction Isolated t [h] Yield [%] formed.[5] The results reproduced a nearly quantitative reaction at the cysteine 545 as reported recently.[2h] The conjugation 1 THF NMM 1 95 2 Water NaOH 24 – yield was above 95 % for all three Grubbs–Hoveyda type cata3 Water + 20 % (v/v) THF NaOH 2 94 lysts 13–15. The final protein concentration was determined by 4 51 4 Water + 20 % (v/v) THF NaPi (0.1 M) using the BCA quantification method.[6]
tion of the catalysts 13, 14, and 15 with a protected l-cysteine. Results of varying reaction conditions are shown in Table 1. The addition of the cysteine to the catalyst was easily followed by monitoring the olefinic signal of the maleimide moiety at d 6.78 ppm in the 1H NMR spectra. In THF and with N-methylmorpholine (NMM) present, the reaction was completed within 1 h leading to quantitative conversion into the cysteine adduct at the maleimide double Chem. Asian J. 2015, 10, 177 – 182
Scheme 2. Synthesis of the biohybrid conjugates FhuA-Cn.
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Full Paper reagent polyethylene-polyethylene glycol copolymer (PE-PEG, 0.125 mm). To confirm correct refolding, circular dichroism (CD) spectra were recorded (Figure 2).[2h, 4] Absorption effects below 200 nm occurred because of absorptions of the chlorine, leading to peak variations in the CD spectra below 200 nm; additionally, the change of the dielectric constant affected by the hydrophobic surrounding led to spectra slightly different from that of the standard b-sheet.[8] Nevertheless, the characteristic features of a b-barrel structure (minimum at 215 nm, maximum at 195 nm) can be observed in the CD spectra.[8, 9] The structure of the folded apo form FhuA DCVFtev in the refolding buffer is represented by the light blue line. In comparison, the biohybrid conjugates FhuA-Cn show the same behavior in the CD spectra. Additionally, a shortening of the linker length does not interfere with the FhuA DCVFtev secondary structure. Figure 1. MALDI TOF MS analysis of digested biohybrid catalyst FhuA-C2 (matrix: 2,5-dihydroxybenzoic acid, Molecular modeling of hybrid T = 25 8C). catalysts was performed based on the X-ray crystal structure of FhuA (PDB 1by3) and the X-ray structure of the Grubbs–Hoveyda catalyst (CCDC 620588) by In addition to the cysteine titration, MALDI TOF MS analysis using the YASARA software package (details can be found in was performed. The high molecular mass of the biohybrid catthe Supporting Information).[10] We focused on the FhuA alyst (MW > 65 kDa) together with attached detergent mole[2 h] DCVFtev ligand sphere surrounding of the Ru catalyst attached The cules caused problems for mass spectrometric analysis. latter were solved by introducing two specific protease cleato the cysteine at position 545. Two possible orientations of vages sites intro the FhuA DCVFtev. As a result (Figure 1) it was the catalyst within the FhuA DCVFtev were determined. The cattev possible to specifically digest FhuA DCVF into three smaller fragments, whereby the smallest one with an approximately molecular weight of 5.9 kDa contains the cysteine at position 545 with the attached metal catalyst. Digestion was performed with a TEV-protease under basic conditions at slightly elevated temperatures for 24 h.[2h, 7] This led to saponification of the ester bond which attaches the catalyst to the protein. Additionally, a nucleophlic attack of the hydroxyl anion on the maleimide group occurred. The resulting masses of digested FhuA-C2 (saponification product 6077.2 Da (calc. 6077.1 Da) and saponified and ring-opened product 6095.5 Da (calc. 6095.4 Da)) in addition to the very low intensity of the apo form signal clearly indicated the successful anchoring on the cysteine residue at position 545 (MALDI TOF MS spectra in Figure 1). Figure 2. CD spectra of the biohybrid conjugates at 25 8C in sodium phosRefolding of FhuA DCVFtev to the b-barrel structure was achphate buffer (pH 7.4, 0.1 m) with PE-PEG (0.125 mm). Teal: FhuA DCVFtev ; ieved by dialysis with a buffer solution containing the refolding Red: FhuA-C3; Green: FhuA-C2; Purple: FhuA-C1. Chem. Asian J. 2015, 10, 177 – 182
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Full Paper alytic active site points toward the rim with the loops or may orientate to the interior of the barrel structure. In both orientations, interactions with the hydrophilic amino acid residues containing -OH, -COO or -CONH2 cannot be excluded. To avoid these interactions, one possibility would be the removal of these coordinating residues generating a more hydrophobic protein environment. On the other hand, with shortening the spacer length, it should be possible to elongate the distance to these coordinating groups, and therefore the competition of coordinating an amino acid residue or a substrate molecule could be favored for substrates. Figure 3 shows catalyst FhuAC3 with the orientation toward the interior barrel structure.
Scheme 3. ROMP of water-soluble oxanorbornene derivative 23.
due to diffusion, the reaction time was set to 68 h.[2h] THF was chosen as the solvent because it can be used as co-solvent for the biohybrid conjugates.[2h, 4] As catalyst 6 did not dissolve in THF, catalyst 9 was used as reference in organic solvents. Catalyst 9 reached full conversion in THF within 68 h, producing polymer 24 with a cis-content of 71 %. The attached linker of catalysts 13–15 and the additionally conjugated cysteine of catalysts 16–18 did not influence the activity and the selectivity compared to that of catalyst 9 (see Table S1 in the Supporting Information). Catalysts 9–18 are not soluble in water or in aqueous 1 % (w/w) sodium dodecyl sulfate (SDS) and therefore inactive without any co-solvent. Catalyst 6 polymerized substrate 23 in water without requiring co-solvents. The activity of Grubbs–Hoveyda catalysts in water is known to be pH-dependent. Under acidic conditions, metathesis reactions are accelerated.[11] The initiation is faster due to protonation of the isopropoxy styrene moiety, generating a vacant coordination site for the substrates in the first step of the catalytic cycle. Coordinating groups or molecules may also be protonated, facilitating the olefin coordination.[2c, 11, 14] At pH 7.4 (NaPi, 0.1 m) catalyst 6 showed 38 % conversion, and in more acidic conditions (pH 5.8, NaPi, 0.1 m) 57 % conversion was achieved. Conversion was increased by the addition of 10 % (v/v) THF. At pH 7.4 the conversion amounted to 49 %, and at pH 5.8 conversion reached 78 %. The cis/trans ratio of the double bond in the polymer chain is about 1:1 (Table S3, Supporting Information). The higher conversion in the presence of THF can be explained with a higher solubility of the produced polymer. In pure water, precipitation of the polymer was observed at 50 % conversion. In an aqueous mixture with 10 % (v/v) THF, catalysts 9–18 are soluble and polymerized 7-oxanorbornene 23 and, for instance, compound 9 reached > 90 % conversion without affecting the cis/trans ratio. The following findings were made: 1) Catalysts 9–18 were not water-soluble. Addition of 10 % (v/v) THF was necessary to promote ROMP in aqueous solutions; 2) The cis/trans ratio was not affected by the nature of the linker or the first amino acid unit attached to the catalysts. Additionally, the presence of 10 % THF (v/v) increased conversion but did not influence selectivity; and 3) Conversion in aqueous solutions was pH-dependent, and higher conversions were achieved under more acidic conditions. The activity of the unfolded and refolded biohybrid conjugates was evaluated in water containing a sodium phosphate buffer (0.1 m) and the detergent (either 1 % SDS (w/w) or 0.125 mm PE-PEG), using 10 % (v/v) THF as co-solvent. The reaction time was set to 68 h. After this time, ethyl vinyl ether (0.5 mL) was added to quench the ruthenium carbene cata-
Figure 3. Biohybrid catalyst FhuA-C3 with distances to E529, Y548, and E585 marked by arrows.
The arrows mark the distances to the closest, potentially coordinating residues E529 (7.93 ), Y548 (5.72 ), and E585 (6.39 ). By shortening the spacer by two methylene units, the distances to residues E529 and Y548 in the biohybrid catalyst FhuA-C1 are increased significantly (E529: 8.89 ; Y548: 6.37 ) (Figure 4). The catalytic activity of the biohybrid catalysts was investigated using the ROMP reaction of water-soluble 7-oxanorbornene derivative 23 as a benchmark (Scheme 3). In order to account for slower catalysis by the biohybrid catalysts FhuA-Cn
Figure 4. Biohybrid catalyst FhuA-C1 with distances to E529, Y548, and E585 marked by arrows. Chem. Asian J. 2015, 10, 177 – 182
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Full Paper lysts.[12] The unfolded as well as the refolded biohybrid catalyst catalyzed the ring-opening polymerization of 7-oxanorbornene 23. The structure of the unfolded catalyst is unknown and cannot be predicted by CD spectroscopy (catalytic experiments can be found in the Supporting Information, Table S2). Therefore the discussion focuses on the folded biohybrid catalysts. Under more acidic conditions at pH 5.8, the reactions progressed faster. By shortening the linker length, the ruthenium center may be repositioned further away from the coordinating amino acid residues within the b-barrel structure, as shown in Figure 2. This leads to higher conversion compared to the system with a longer linker. Table 2 shows results for
with benchmark RCM substrates are on-going. Additionally, the chemogenetic approach will be completed by mutagenesis of the protein. The medium-term target of generating a whole cell system might be achieved with this combination of the Grubbs–Hoveyda type catalyst and the FhuA protein scaffold, making the system applicable in pure organic solvents and atmospheric conditions, due to the robustness of a whole cell.
Experimental Section General All operations were performed under an inert atmosphere of argon or nitrogen using standard Schlenk or glovebox techniques. Water was thoroughly degassed with argon or nitrogen prior to use (> 1 h). Other solvents were degassed by using “freeze-pumpthaw” cycles. Diethyl ether, dichloromethane, toluene, tetrahydrofuran, and pentane for complex synthesis were obtained dry and degassed from a SPS 800 solvent purification system (MBraun). [D6]Dimethylsulfoxide, CD2Cl2, and CDCl3 were dried over calcium hydride, distilled, degassed, and stored in a glovebox. [D6]Benzene was dried over sodium/benzophenone, distilled, degassed and stored in a glovebox. NMR spectra were recorded on a Bruker DRX 400 spectrometer (1H, 400.1 MHz; 13C{1H}, 100.6 MHz). Chemical shifts were referenced internally by using the residual solvent resonances. Compounds 2,[2h, 13] 4,[14] 7–12,[2h, 3] 15[2h] , and 23[2h] were synthesized according to literature procedures. The analytical data and detailed synthesis of compounds 5, 6, 13, and 14 are given in the Supporting Information. FhuA DCVFtev was prepared as previously reported.[2h] The Grubbs 1 st generation catalyst was a generous gift by Umicore and used without further purification. All other chemicals were used as received if not mentioned otherwise.
Table 2. ROMP of 23 with refolded biohybrid catalysts. Entry[a]
Catalyst
Conversion[b] [%]
Selectivity[b] cis/trans
TON[c]
1 2 3 4 5[2h]
6 9 FhuA-C1 FhuA-C2 FhuA-C3
78 99 41 24 37
48/52 50/50 58/42 56/44 56/44
780 990 555 325 365
[a] Reactions were carried out in degassed water containing PE-PEG (0.125 mm), sodium phosphate buffer (0.1 m, pH 5.8) and THF (10 % (v/v)); c(substrate) = 0.1 m. [b] Determined by 1H NMR spectroscopy in CDCl3 ; formation of polymer was confirmed by gel permeation chromatography measurements.[16] [c] c(6) and c(9) = 0.1 mm; c(biohybrid catalyst) determined with BCA assay and consideration of ThioGlo titration.
ROMP of 23 using the folded biohybrid catalysts in an acidic reaction medium. Catalysis under other reaction conditions and the results of the unfolded catalyst are given in Table S4 in the Supporting Information. The cis/trans selectivity in the polymer chain was slightly affected with catalysts FhuA-Cn when compared to the catalysts 6 and 9. Higher cis content was observed for hybrid catalysts FhuA-C2 and FhuA-C3. Additionally, with shorter linker length the cis content of the 7-oxanorbornene polymer increased when compared with the longer linkers (catalyst FhuA-C1). Differences in cis content may therefore be attributed to interactions with the surrounding FhuA DCVFtev.
Equipment of 9 with maleimide linking units In a glovebox in an oven dried Schlenk tube, Grubbs–Hoveyda type catalyst 9 (30 mg, 0.045 mmol) was dissolved in anhydrous, degassed THF (1 mL). Dry NaHCO3 (30 mg, 0.36 mmol) was suspended in the solution. Acetyl chloride 10/11/12 (0.05 mmol) in THF (0.2 mL) was slowly added to the mixture. The solution was vigorously stirred at 25 8C for 16 h. The solid was filtered off, and diethyl ether (25 mL) was added. The organic solution was washed with water (2 25 mL) and brine (1 25 mL). The organic layer was dried over MgSO4, and the solvent removed in vacuo to yield complexes 13/14/15 as a green powder.
Conclusion and Outlook In summary, the influence of the spacer length on the activity and selectivity of a biohybrid catalyst consisting of the transmembrane protein FhuA DCVFtev and a Grubbs–Hoveyda type catalyst was investigated. By shortening the linker length, it was possible to increase the activity (TON). Additionally, a slightly increased cis content of the double bond in the synthesized polymers was observed with biohybrid catalysts FhuA-Cn. Compared to water-soluble catalysts 6 and 9, a significant change in the cis/trans ratio was observed (increase of the cis content from 50 % (catalysts 6 and 9) to 58 % (FhuAC1). These results of the generated biohybrid catalysts will be applied in the ring-closing metathesis (RCM) reaction. Studies Chem. Asian J. 2015, 10, 177 – 182
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Conjugation of Grubbs–Hoveyda type catalysts 13/14/15 with l-cysteine In a glovebox, Grubbs–Hoveyda type catalysts 13/14/15 (0.036 mmol) were dissolved in dry THF (1 mL). To this solution, a solution of l-cysteine (0.040 mmol) in THF (0.5 mL) was added dropwise. To this mixture, N-methyl morpholine (0.040 mmol) was added via a microsyringe. The reaction mixture was stirred for 2 h at 25 8C and then transferred into a separatory funnel. After addition of water (20 mL), the aqueous phase was extracted with dichloromethane (2 20 mL). The combined organic layer was concentrated and filtered over aluminium oxide. The organic layer was dried over MgSO4, and the solvent was removed in vacuo to give the cysteine-conjugated complexes as a dark green solid.
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Full Paper Synthesis of complex 6
(SeleCa), the Cluster of Excellence “Tailor-Made Fuels from Biomass” (TMFB), and Umicore, Frankfurt (Dr. A. Doppiu), for a generous gift of Grubbs 1st Generation catalyst.
Complex 5 (385 mg, 0.45 mmol) was dissolved in EtOH (20 mL), and hydrochloric acid (5 mL 1 m) was added dropwise to the solution. The flask was capped with a septum, and the mixture was purged with argon for 20 min. The seal was reinforced with parafilm, and the mixture was allowed to stir for 20 h at 25 8C. After filtering through celite, the solvent was concentrated in vacuo. The residue was precipitated in diethyl ether (30 mL) and centrifuged. The green solid was washed with THF (2 20 mL) and pentane (2 20 mL) and dried in vacuo to yield water-soluble Grubbs–Hoveyda type catalyst 6 (312 mg, 0.41 mmol, 90 %) as a green solid. 1H NMR (D2O/MeOD; 25 8C): d = 16.62 (s, 1 H, Ru = CH), 7.68–7.66 (m, 1 H, aryl H), 7.07–6.96 (m, 6 H, aryl H), 4.91 (sept, 3JHH = 6.4 Hz, 1 H, CH(CH3)2), 4.50–4.41 (m, 3 H, CH, CH2NMe3), 4.28–4.23 (m, 1 H, CH2), 4.05–4.00 (m, 1 H, CH2) 3.67–3.61 (m, 2 H, CH2), 2.96 (br. s, 9 H, NMe3), 2.35 (br s, 18 H, aryl Me), 1.17–1.14 ppm (m, 6 H, CH(CH3)2); 13 C NMR (D2O/MeOD; 25 8C): d = 296.67 (br, Ru = CH), 211.75 (br, Ru-C), 155.23, 147.03, 140.52, 135.39, 131.19, 130.77, 130.53, 126.69, 123.31, 115.37, 77.90, 76.84, 69.77, 66.87, 63.24, 62.92, 62.82, 53.07, 21.84, 21.46, 21.38 ppm; ESI HR MS (positive mode): m/z calcd. for C36H50Cl2N3O2Ru [M Cl] + : 728.2313; found: 728.2279.
Keywords: biohybrid catalysis · chemogenetic approach · metalloenzymes · ring-opening polymerization · ruthenium [1] a) P. J. Deuss, R. den Heeten, W. Laan, P. C. J. Kamer, Chem. Eur. J. 2011, 17, 4680 – 4698; b) Y. Lu, N. Yeung, N. Sieracki, N. M. Marshall, Nature 2009, 460, 855 – 862; c) C. M. Thomas, T. R. Ward, Chem. Soc. Rev. 2005, 34, 337 – 346; d) T. R. Ward, Acc. Chem. Res. 2011, 44, 47 – 57; e) J. C. Lewis, ACS Catal. 2013, 3, 2954 – 2975; f) J. Steinreiber, T. R. Ward, Coord. Chem. Rev. 2008, 252, 751 – 766. [2] a) C. Lo, M. R. Ringenberg, D. Gnandt, Y. Wilson, T. R. Ward, Chem. Commun. 2011, 47, 12065 – 12067; b) T. Matsuo, C. Imai, T. Yoshida, T. Saito, T. Hayashi, S. Hirota, Chem. Commun. 2012, 48, 1662 – 1664; c) C. Mayer, D. G. Gillingham, T. R. Ward, D. Hilvert, Chem. Commun. 2011, 47, 12068 – 12070; d) J. M. Zimbron, T. Heinisch, M. Schmid, D. Hamels, E. S. Nogueira, T. Schirmer, T. R. Ward, J. Am. Chem. Soc. 2013, 135, 5384 – 5388; e) K. Fukumoto, A. Onoda, E. Mizohata, M. Bocola, T. Inoue, U. Schwaneberg, T. Hayashi, ChemCatChem 2014, 6, 1229 – 1235; f) A. Onoda, K. Fukumoto, M. Arlt, M. Bocola, U. Schwaneberg, T. Hayashi, Chem. Commun. 2012, 48, 9756 – 9758; g) C. Zhang, P. Srivastava, K. Ellis-Guardiola, J. C. Lewis, Tetrahedron 2014, 70, 4245 – 4249; h) F. Philippart, M. Arlt, S. Gotzen, S.-J. Tenne, M. Bocola, H.-H. Chen, L. Zhu, U. Schwaneberg, J. Okuda, Chem. Eur. J. 2013, 19, 13865 – 13871; i) M. E. Wilson, G. M. Whitesides, J. Am. Chem. Soc. 1978, 100, 306 – 307. [3] R. M. de Figueiredo, P. Oczipka, R. Frçhlich, M. Christmann, Synthesis 2008, 1316 – 1318. [4] S.-J. Tenne, U. Schwaneberg, Int. J. Mol. Sci. 2012, 13, 2459 – 2471. [5] S. K. Wright, R. E. Viola, Anal. Biochem. 1998, 265, 8 – 14. [6] P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, D. C. Klenk, Anal. Biochem. 1985, 150, 76 – 85. [7] J. Phan, A. Zdanov, A. G. Evdokimov, J. E. Tropea, H. K. Peters, R. B. Kapust, M. Li, A. Wlodawer, D. S. Waugh, J. Biol. Chem. 2002, 277, 50564 – 50572. [8] B. A. Wallace, J. G. Lees, A. J. W. Orry, A. Lobley, R. W. Janes, Protein Sci. 2003, 12, 875 – 884. [9] a) N. J. Greenfield, G. D. Fasman, Biochemistry 1969, 8, 4108 – 4116; b) N. Sreerama, R. W. Woody, Anal. Biochem. 2000, 287, 252 – 260. [10] E. Krieger, T. Darden, S. B. Nabuurs, A. Finkelstein, G. Vriend, Proteins Struct. Funct. Bioinf. 2004, 57, 678 – 683. [11] a) D. M. Lynn, B. Mohr, R. H. Grubbs, L. M. Henling, M. W. Day, J. Am. Chem. Soc. 2000, 122, 6601 – 6609; b) S. J. P’Poo, H.-J. Schanz, J. Am. Chem. Soc. 2007, 129, 14200 – 14212; c) T. M. Trnka, R. H. Grubbs, Acc. Chem. Res. 2001, 34, 18 – 29. [12] a) T.-L. Choi, R. H. Grubbs, Angew. Chem. Int. Ed. 2003, 42, 1743 – 1746; Angew. Chem. 2003, 115, 1785 – 1788. [13] M. Mayr, M. R. Buchmeiser, K. Wurst, Adv. Synth. Catal. 2002, 344, 712 – 719. [14] a) J. P. Jordan, R. H. Grubbs, Angew. Chem. Int. Ed. 2007, 46, 5152 – 5155; Angew. Chem. 2007, 119, 5244 – 5247. [15] W. J. Feast, D. B. Harrison, Polym. Bull. 1991, 25, 343 – 350. [16] For FhuA-C1, average values Mn of 181 000 g mol 1 and Mw/Mn of 2.7 were measured. For FhuA-C2, average values Mn of 244 000 g mol 1 and Mw/Mn of 2.8 were measured.
Typical ring-opening metathesis polymerization (ROMP) using Grubbs–Hoveyda type catalyst To a solution containing the Grubbs–Hoveyda type catalyst in either pure THF (1 mL THF, 0.1 mm (1 mL H2O, 0.1 mm) or 1 % SDS (NaPi = 0.1 m, pH 7.4 or 5.8) or PE-PEG (0.125 mm, NaPi = 0.1 m, pH 7.4 or 5.8) was added degassed THF (0.1 mL). The 7-oxanorbornene derivative 23 (18 mg, 15 mL, 0.1 m) was added dropwise via a microsyringe. The reaction mixture was allowed to stir for 68 h at 25 8C. Subsequently, ethyl vinyl ether was added to quench the catalyst, and the residual monomer and the polymer were extracted with CDCl3. The conversion and the cis/trans ratio were determined by 1H NMR spectroscopy as reported by Feast and Harrison.[15]
Typical ROMP using the biohybrid catalysts To a solution containing the biohybrid catalyst (1 mL, 5 mg mL 1, 0.1 mm) in either 1 % SDS (NaPi = 0.1 m, pH 7.4 or 5.8) or PE-PEG (0.125 mm, NaPi = 0.1 m, pH 7.4 or 5.8) was added degassed THF (0.1 mL). The solution was stirred for 10 min at 25 8C. The 7-oxanorbornene derivative 23 (18 mg, 15 mL, 0.1 m) was added dropwise via a microsyringe. The reaction mixture was allowed to stir for 68 h at 25 8C. Subsequently, ethyl vinyl ether was added to quench the catalyst, and the residual monomer and the polymer were extracted with CDCl3. The conversion and the cis/trans ratio were determined by 1H NMR spectroscopy as reported by Feast and Harrison.[15]
Acknowledgements We gratefully acknowledge the financial support by the Deutsche Forschungsgemeinschaft through the International Research Training Group “Selectivity in Chemo- and Biocatalysis”
Chem. Asian J. 2015, 10, 177 – 182
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Received: August 27, 2014 Revised: November 10, 2014 Published online on November 25, 2014
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Full Paper
Metalloproteins
Hybrid Ruthenium ROMP Catalysts Based on an Engineered Variant of b-Barrel Protein FhuA DCVFtev : Effect of Spacer Length Daniel F. Sauer,[a] Marco Bocola,[b] Claudio Broglia,[a] Marcus Arlt,[b] Lei-Lei Zhu,[b] Melanie Brocker,[c] Ulrich Schwaneberg,[b] and Jun Okuda*[a]
tween the N-heterocyclic carbene ligand and the cysteine site 545 increased the ROMP activity toward a water-soluble 7-oxanorbornene derivative. The cis/trans ratio of the double bond in the polymer was influenced by the hybrid catalyst.
Abstract: A biohybrid ring-opening olefin metathesis polymerization catalyst based on the reengineered b-barrel protein FhuA DCVFtev was chemically modified with respect to the covalently anchored Grubbs–Hoveyda type catalyst. Shortening of the spacer (1,3-propanediyl to methylene) be-
Introduction
terms of coupling efficiency and catalytic activity, the maleimide linker appeared to be superior, offering the possibility of varying spacer length in order to bring the catalytic active center closer to the protein scaffold. In an additional step, the protein matrix will be modified in order to affect the activity or selectivity of the target benchmark ring-opening metathesis polymerization (ROMP) reaction. We report here the spacer modification based on the recently reported biohybrid system with the covalently attached Grubbs–Hoveyda type catalyst.[2h] These unique biohybrid metathesis catalysts with shortened spacer length incorporated in a membrane protein were employed in ring-opening metathesis polymerization (ROMP) reaction of a water-soluble 7-oxanorbornene derivative. The objective of this work was to explore the catalytic site before directed mutagenesis will be applied to modify the second ligand sphere.
There is growing recognition that the second ligand sphere influences the activity and selectivity of organometallic catalysts.[1] Biohybrid catalysts composed of an active organometallic catalyst site and a protein scaffold offer unprecedented opportunities for designing new catalysts for transformations beyond those in metabolic reactions (“artificial metalloenzymes”) by combining principles of chemocatalysis with those of biocatalysis containing non-natural metal centers.[2] We have recently reported a modified Grubbs–Hoveyda type catalyst bearing an alcohol function in the backbone of the NHC ligand.[2h] The advantage of the Grubbs–Hoveyda type catalyst is the relatively high robustness against air, moisture, and functional groups, making this type of catalyst suitable to perform metathesis reactions in an exceptional environment.[2a–c, h] This catalyst was anchored covalently to the rationally designed variant of the transmembrane b-barrel protein ferric hydroxamate uptake protein component A (FhuA), extracted from E. coli.[2h] Covalent anchoring to the protein is achieved through an a-bromoacetyl group or, alternatively, through a maleimide group with a 1,3-propanediyl spacer. In
Results and Discussion Grubbs–Hoveyda type catalysts with varied spacer length were prepared. Three maleimide derivatives containing carboxylic acids were synthesized following a procedure described by Christmann and co-workers.[3] After transforming the carboxylic group into an acetyl chloride group, the linking units were attached to a Grubbs–Hoveyda type catalyst by esterification with an alcohol function in the backbone of the NHC ligand. The influence of the FhuA DCVFtev ligand sphere was determined by comparing the performance of the biohybrid catalysts with that of the protein-free catalysts and a water-soluble Grubbs–Hoveyda type catalyst containing an alcohol function in the backbone of the NHC-ligand and a tertiary ammonium group on the benzylidene part. This water-soluble catalyst was obtained by reacting the Grubbs 2nd generation catalyst 3 with 1-(4-isopropyloxy-3-vinylphenyl)-N,N,N-trimethylmethanamonium chloride 4 (Scheme 1). The reactivity of the maleimide group towards the thiol group was explored in a model reac-
[a] D. F. Sauer, C. Broglia, Prof. Dr. J. Okuda Institut fr Anorganische Chemie RWTH Aachen University Landoltweg 1, 52056 Aachen (Germany) E-mail: [email protected] [b] Dr. M. Bocola, M. Arlt, Dr. L.-L. Zhu, Prof. Dr. U. Schwaneberg Lehrstuhl fr Biotechnologie RWTH Aachen University Worringer Weg 1, 52074 Aachen (Germany) [c] Dr. M. Brocker Institut fr Bio- und Geowissenschaften, IBG-1: Biotechnologie Forschungszentrum Jlich 52425 Jlich (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201403005. Chem. Asian J. 2015, 10, 177 – 182
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Full Paper bond. Performing the reaction under slightly basic conditions was necessary for complete conjugation. In aqueous solutions the reaction was slightly slower. In pure water the reaction could not be performed due to low solubility of the corresponding Grubbs–Hoveyda type complexes 13–15. By adding 20 % (v/ v) of THF to water, the catalysts were dissolved homogeneously. The pH value was adjusted to 7.4 with either NaOH or with a sodium phosphate buffer (NaPi, 0.1 m). In the case of sodium phosphate buffer, formation of precipitates was observed and the obtained yield was significantly lower. The best Scheme 1. Syntheses of Grubbs–Hoveyda type catalysts bearing a maleimide linking unit and the water-soluble conditions in aqueous solutions Grubbs–Hoveyda type catalyst 6. are in presence of 20 % (v/v) THF and NaOH at a pH value of around 7.5. These conditions are Table 1. Conditions for conjugation with cysteine. applicable to the host protein FhuA DCVFtev due to its high tolerance against organic solvents such as THF.[2h, 4] Under these optimized conditions, the anchoring with all catalysts was performed under oxygen-free conditions, and an excess of the corresponding catalyst (10 equiv) was employed. Complete reaction at the cysteine residue 545 was ensured by stirring the reaction mixture overnight (16 h). The work-up procedure was conducted as previously reported (Scheme 2).[2h] To prove the successful anchoring and refolding, several analyses were undertaken. To determine the degree of conjugation, a fluorescence titration with the fluorescence dye ThioGlo was perEntry Solvent Base/Buffer Reaction Isolated t [h] Yield [%] formed.[5] The results reproduced a nearly quantitative reaction at the cysteine 545 as reported recently.[2h] The conjugation 1 THF NMM 1 95 2 Water NaOH 24 – yield was above 95 % for all three Grubbs–Hoveyda type cata3 Water + 20 % (v/v) THF NaOH 2 94 lysts 13–15. The final protein concentration was determined by 4 51 4 Water + 20 % (v/v) THF NaPi (0.1 M) using the BCA quantification method.[6]
tion of the catalysts 13, 14, and 15 with a protected l-cysteine. Results of varying reaction conditions are shown in Table 1. The addition of the cysteine to the catalyst was easily followed by monitoring the olefinic signal of the maleimide moiety at d 6.78 ppm in the 1H NMR spectra. In THF and with N-methylmorpholine (NMM) present, the reaction was completed within 1 h leading to quantitative conversion into the cysteine adduct at the maleimide double Chem. Asian J. 2015, 10, 177 – 182
Scheme 2. Synthesis of the biohybrid conjugates FhuA-Cn.
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Full Paper reagent polyethylene-polyethylene glycol copolymer (PE-PEG, 0.125 mm). To confirm correct refolding, circular dichroism (CD) spectra were recorded (Figure 2).[2h, 4] Absorption effects below 200 nm occurred because of absorptions of the chlorine, leading to peak variations in the CD spectra below 200 nm; additionally, the change of the dielectric constant affected by the hydrophobic surrounding led to spectra slightly different from that of the standard b-sheet.[8] Nevertheless, the characteristic features of a b-barrel structure (minimum at 215 nm, maximum at 195 nm) can be observed in the CD spectra.[8, 9] The structure of the folded apo form FhuA DCVFtev in the refolding buffer is represented by the light blue line. In comparison, the biohybrid conjugates FhuA-Cn show the same behavior in the CD spectra. Additionally, a shortening of the linker length does not interfere with the FhuA DCVFtev secondary structure. Figure 1. MALDI TOF MS analysis of digested biohybrid catalyst FhuA-C2 (matrix: 2,5-dihydroxybenzoic acid, Molecular modeling of hybrid T = 25 8C). catalysts was performed based on the X-ray crystal structure of FhuA (PDB 1by3) and the X-ray structure of the Grubbs–Hoveyda catalyst (CCDC 620588) by In addition to the cysteine titration, MALDI TOF MS analysis using the YASARA software package (details can be found in was performed. The high molecular mass of the biohybrid catthe Supporting Information).[10] We focused on the FhuA alyst (MW > 65 kDa) together with attached detergent mole[2 h] DCVFtev ligand sphere surrounding of the Ru catalyst attached The cules caused problems for mass spectrometric analysis. latter were solved by introducing two specific protease cleato the cysteine at position 545. Two possible orientations of vages sites intro the FhuA DCVFtev. As a result (Figure 1) it was the catalyst within the FhuA DCVFtev were determined. The cattev possible to specifically digest FhuA DCVF into three smaller fragments, whereby the smallest one with an approximately molecular weight of 5.9 kDa contains the cysteine at position 545 with the attached metal catalyst. Digestion was performed with a TEV-protease under basic conditions at slightly elevated temperatures for 24 h.[2h, 7] This led to saponification of the ester bond which attaches the catalyst to the protein. Additionally, a nucleophlic attack of the hydroxyl anion on the maleimide group occurred. The resulting masses of digested FhuA-C2 (saponification product 6077.2 Da (calc. 6077.1 Da) and saponified and ring-opened product 6095.5 Da (calc. 6095.4 Da)) in addition to the very low intensity of the apo form signal clearly indicated the successful anchoring on the cysteine residue at position 545 (MALDI TOF MS spectra in Figure 1). Figure 2. CD spectra of the biohybrid conjugates at 25 8C in sodium phosRefolding of FhuA DCVFtev to the b-barrel structure was achphate buffer (pH 7.4, 0.1 m) with PE-PEG (0.125 mm). Teal: FhuA DCVFtev ; ieved by dialysis with a buffer solution containing the refolding Red: FhuA-C3; Green: FhuA-C2; Purple: FhuA-C1. Chem. Asian J. 2015, 10, 177 – 182
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Full Paper alytic active site points toward the rim with the loops or may orientate to the interior of the barrel structure. In both orientations, interactions with the hydrophilic amino acid residues containing -OH, -COO or -CONH2 cannot be excluded. To avoid these interactions, one possibility would be the removal of these coordinating residues generating a more hydrophobic protein environment. On the other hand, with shortening the spacer length, it should be possible to elongate the distance to these coordinating groups, and therefore the competition of coordinating an amino acid residue or a substrate molecule could be favored for substrates. Figure 3 shows catalyst FhuAC3 with the orientation toward the interior barrel structure.
Scheme 3. ROMP of water-soluble oxanorbornene derivative 23.
due to diffusion, the reaction time was set to 68 h.[2h] THF was chosen as the solvent because it can be used as co-solvent for the biohybrid conjugates.[2h, 4] As catalyst 6 did not dissolve in THF, catalyst 9 was used as reference in organic solvents. Catalyst 9 reached full conversion in THF within 68 h, producing polymer 24 with a cis-content of 71 %. The attached linker of catalysts 13–15 and the additionally conjugated cysteine of catalysts 16–18 did not influence the activity and the selectivity compared to that of catalyst 9 (see Table S1 in the Supporting Information). Catalysts 9–18 are not soluble in water or in aqueous 1 % (w/w) sodium dodecyl sulfate (SDS) and therefore inactive without any co-solvent. Catalyst 6 polymerized substrate 23 in water without requiring co-solvents. The activity of Grubbs–Hoveyda catalysts in water is known to be pH-dependent. Under acidic conditions, metathesis reactions are accelerated.[11] The initiation is faster due to protonation of the isopropoxy styrene moiety, generating a vacant coordination site for the substrates in the first step of the catalytic cycle. Coordinating groups or molecules may also be protonated, facilitating the olefin coordination.[2c, 11, 14] At pH 7.4 (NaPi, 0.1 m) catalyst 6 showed 38 % conversion, and in more acidic conditions (pH 5.8, NaPi, 0.1 m) 57 % conversion was achieved. Conversion was increased by the addition of 10 % (v/v) THF. At pH 7.4 the conversion amounted to 49 %, and at pH 5.8 conversion reached 78 %. The cis/trans ratio of the double bond in the polymer chain is about 1:1 (Table S3, Supporting Information). The higher conversion in the presence of THF can be explained with a higher solubility of the produced polymer. In pure water, precipitation of the polymer was observed at 50 % conversion. In an aqueous mixture with 10 % (v/v) THF, catalysts 9–18 are soluble and polymerized 7-oxanorbornene 23 and, for instance, compound 9 reached > 90 % conversion without affecting the cis/trans ratio. The following findings were made: 1) Catalysts 9–18 were not water-soluble. Addition of 10 % (v/v) THF was necessary to promote ROMP in aqueous solutions; 2) The cis/trans ratio was not affected by the nature of the linker or the first amino acid unit attached to the catalysts. Additionally, the presence of 10 % THF (v/v) increased conversion but did not influence selectivity; and 3) Conversion in aqueous solutions was pH-dependent, and higher conversions were achieved under more acidic conditions. The activity of the unfolded and refolded biohybrid conjugates was evaluated in water containing a sodium phosphate buffer (0.1 m) and the detergent (either 1 % SDS (w/w) or 0.125 mm PE-PEG), using 10 % (v/v) THF as co-solvent. The reaction time was set to 68 h. After this time, ethyl vinyl ether (0.5 mL) was added to quench the ruthenium carbene cata-
Figure 3. Biohybrid catalyst FhuA-C3 with distances to E529, Y548, and E585 marked by arrows.
The arrows mark the distances to the closest, potentially coordinating residues E529 (7.93 ), Y548 (5.72 ), and E585 (6.39 ). By shortening the spacer by two methylene units, the distances to residues E529 and Y548 in the biohybrid catalyst FhuA-C1 are increased significantly (E529: 8.89 ; Y548: 6.37 ) (Figure 4). The catalytic activity of the biohybrid catalysts was investigated using the ROMP reaction of water-soluble 7-oxanorbornene derivative 23 as a benchmark (Scheme 3). In order to account for slower catalysis by the biohybrid catalysts FhuA-Cn
Figure 4. Biohybrid catalyst FhuA-C1 with distances to E529, Y548, and E585 marked by arrows. Chem. Asian J. 2015, 10, 177 – 182
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Full Paper lysts.[12] The unfolded as well as the refolded biohybrid catalyst catalyzed the ring-opening polymerization of 7-oxanorbornene 23. The structure of the unfolded catalyst is unknown and cannot be predicted by CD spectroscopy (catalytic experiments can be found in the Supporting Information, Table S2). Therefore the discussion focuses on the folded biohybrid catalysts. Under more acidic conditions at pH 5.8, the reactions progressed faster. By shortening the linker length, the ruthenium center may be repositioned further away from the coordinating amino acid residues within the b-barrel structure, as shown in Figure 2. This leads to higher conversion compared to the system with a longer linker. Table 2 shows results for
with benchmark RCM substrates are on-going. Additionally, the chemogenetic approach will be completed by mutagenesis of the protein. The medium-term target of generating a whole cell system might be achieved with this combination of the Grubbs–Hoveyda type catalyst and the FhuA protein scaffold, making the system applicable in pure organic solvents and atmospheric conditions, due to the robustness of a whole cell.
Experimental Section General All operations were performed under an inert atmosphere of argon or nitrogen using standard Schlenk or glovebox techniques. Water was thoroughly degassed with argon or nitrogen prior to use (> 1 h). Other solvents were degassed by using “freeze-pumpthaw” cycles. Diethyl ether, dichloromethane, toluene, tetrahydrofuran, and pentane for complex synthesis were obtained dry and degassed from a SPS 800 solvent purification system (MBraun). [D6]Dimethylsulfoxide, CD2Cl2, and CDCl3 were dried over calcium hydride, distilled, degassed, and stored in a glovebox. [D6]Benzene was dried over sodium/benzophenone, distilled, degassed and stored in a glovebox. NMR spectra were recorded on a Bruker DRX 400 spectrometer (1H, 400.1 MHz; 13C{1H}, 100.6 MHz). Chemical shifts were referenced internally by using the residual solvent resonances. Compounds 2,[2h, 13] 4,[14] 7–12,[2h, 3] 15[2h] , and 23[2h] were synthesized according to literature procedures. The analytical data and detailed synthesis of compounds 5, 6, 13, and 14 are given in the Supporting Information. FhuA DCVFtev was prepared as previously reported.[2h] The Grubbs 1 st generation catalyst was a generous gift by Umicore and used without further purification. All other chemicals were used as received if not mentioned otherwise.
Table 2. ROMP of 23 with refolded biohybrid catalysts. Entry[a]
Catalyst
Conversion[b] [%]
Selectivity[b] cis/trans
TON[c]
1 2 3 4 5[2h]
6 9 FhuA-C1 FhuA-C2 FhuA-C3
78 99 41 24 37
48/52 50/50 58/42 56/44 56/44
780 990 555 325 365
[a] Reactions were carried out in degassed water containing PE-PEG (0.125 mm), sodium phosphate buffer (0.1 m, pH 5.8) and THF (10 % (v/v)); c(substrate) = 0.1 m. [b] Determined by 1H NMR spectroscopy in CDCl3 ; formation of polymer was confirmed by gel permeation chromatography measurements.[16] [c] c(6) and c(9) = 0.1 mm; c(biohybrid catalyst) determined with BCA assay and consideration of ThioGlo titration.
ROMP of 23 using the folded biohybrid catalysts in an acidic reaction medium. Catalysis under other reaction conditions and the results of the unfolded catalyst are given in Table S4 in the Supporting Information. The cis/trans selectivity in the polymer chain was slightly affected with catalysts FhuA-Cn when compared to the catalysts 6 and 9. Higher cis content was observed for hybrid catalysts FhuA-C2 and FhuA-C3. Additionally, with shorter linker length the cis content of the 7-oxanorbornene polymer increased when compared with the longer linkers (catalyst FhuA-C1). Differences in cis content may therefore be attributed to interactions with the surrounding FhuA DCVFtev.
Equipment of 9 with maleimide linking units In a glovebox in an oven dried Schlenk tube, Grubbs–Hoveyda type catalyst 9 (30 mg, 0.045 mmol) was dissolved in anhydrous, degassed THF (1 mL). Dry NaHCO3 (30 mg, 0.36 mmol) was suspended in the solution. Acetyl chloride 10/11/12 (0.05 mmol) in THF (0.2 mL) was slowly added to the mixture. The solution was vigorously stirred at 25 8C for 16 h. The solid was filtered off, and diethyl ether (25 mL) was added. The organic solution was washed with water (2 25 mL) and brine (1 25 mL). The organic layer was dried over MgSO4, and the solvent removed in vacuo to yield complexes 13/14/15 as a green powder.
Conclusion and Outlook In summary, the influence of the spacer length on the activity and selectivity of a biohybrid catalyst consisting of the transmembrane protein FhuA DCVFtev and a Grubbs–Hoveyda type catalyst was investigated. By shortening the linker length, it was possible to increase the activity (TON). Additionally, a slightly increased cis content of the double bond in the synthesized polymers was observed with biohybrid catalysts FhuA-Cn. Compared to water-soluble catalysts 6 and 9, a significant change in the cis/trans ratio was observed (increase of the cis content from 50 % (catalysts 6 and 9) to 58 % (FhuAC1). These results of the generated biohybrid catalysts will be applied in the ring-closing metathesis (RCM) reaction. Studies Chem. Asian J. 2015, 10, 177 – 182
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Conjugation of Grubbs–Hoveyda type catalysts 13/14/15 with l-cysteine In a glovebox, Grubbs–Hoveyda type catalysts 13/14/15 (0.036 mmol) were dissolved in dry THF (1 mL). To this solution, a solution of l-cysteine (0.040 mmol) in THF (0.5 mL) was added dropwise. To this mixture, N-methyl morpholine (0.040 mmol) was added via a microsyringe. The reaction mixture was stirred for 2 h at 25 8C and then transferred into a separatory funnel. After addition of water (20 mL), the aqueous phase was extracted with dichloromethane (2 20 mL). The combined organic layer was concentrated and filtered over aluminium oxide. The organic layer was dried over MgSO4, and the solvent was removed in vacuo to give the cysteine-conjugated complexes as a dark green solid.
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Full Paper Synthesis of complex 6
(SeleCa), the Cluster of Excellence “Tailor-Made Fuels from Biomass” (TMFB), and Umicore, Frankfurt (Dr. A. Doppiu), for a generous gift of Grubbs 1st Generation catalyst.
Complex 5 (385 mg, 0.45 mmol) was dissolved in EtOH (20 mL), and hydrochloric acid (5 mL 1 m) was added dropwise to the solution. The flask was capped with a septum, and the mixture was purged with argon for 20 min. The seal was reinforced with parafilm, and the mixture was allowed to stir for 20 h at 25 8C. After filtering through celite, the solvent was concentrated in vacuo. The residue was precipitated in diethyl ether (30 mL) and centrifuged. The green solid was washed with THF (2 20 mL) and pentane (2 20 mL) and dried in vacuo to yield water-soluble Grubbs–Hoveyda type catalyst 6 (312 mg, 0.41 mmol, 90 %) as a green solid. 1H NMR (D2O/MeOD; 25 8C): d = 16.62 (s, 1 H, Ru = CH), 7.68–7.66 (m, 1 H, aryl H), 7.07–6.96 (m, 6 H, aryl H), 4.91 (sept, 3JHH = 6.4 Hz, 1 H, CH(CH3)2), 4.50–4.41 (m, 3 H, CH, CH2NMe3), 4.28–4.23 (m, 1 H, CH2), 4.05–4.00 (m, 1 H, CH2) 3.67–3.61 (m, 2 H, CH2), 2.96 (br. s, 9 H, NMe3), 2.35 (br s, 18 H, aryl Me), 1.17–1.14 ppm (m, 6 H, CH(CH3)2); 13 C NMR (D2O/MeOD; 25 8C): d = 296.67 (br, Ru = CH), 211.75 (br, Ru-C), 155.23, 147.03, 140.52, 135.39, 131.19, 130.77, 130.53, 126.69, 123.31, 115.37, 77.90, 76.84, 69.77, 66.87, 63.24, 62.92, 62.82, 53.07, 21.84, 21.46, 21.38 ppm; ESI HR MS (positive mode): m/z calcd. for C36H50Cl2N3O2Ru [M Cl] + : 728.2313; found: 728.2279.
Keywords: biohybrid catalysis · chemogenetic approach · metalloenzymes · ring-opening polymerization · ruthenium [1] a) P. J. Deuss, R. den Heeten, W. Laan, P. C. J. Kamer, Chem. Eur. J. 2011, 17, 4680 – 4698; b) Y. Lu, N. Yeung, N. Sieracki, N. M. Marshall, Nature 2009, 460, 855 – 862; c) C. M. Thomas, T. R. Ward, Chem. Soc. Rev. 2005, 34, 337 – 346; d) T. R. Ward, Acc. Chem. Res. 2011, 44, 47 – 57; e) J. C. Lewis, ACS Catal. 2013, 3, 2954 – 2975; f) J. Steinreiber, T. R. Ward, Coord. Chem. Rev. 2008, 252, 751 – 766. [2] a) C. Lo, M. R. Ringenberg, D. Gnandt, Y. Wilson, T. R. Ward, Chem. Commun. 2011, 47, 12065 – 12067; b) T. Matsuo, C. Imai, T. Yoshida, T. Saito, T. Hayashi, S. Hirota, Chem. Commun. 2012, 48, 1662 – 1664; c) C. Mayer, D. G. Gillingham, T. R. Ward, D. Hilvert, Chem. Commun. 2011, 47, 12068 – 12070; d) J. M. Zimbron, T. Heinisch, M. Schmid, D. Hamels, E. S. Nogueira, T. Schirmer, T. R. Ward, J. Am. Chem. Soc. 2013, 135, 5384 – 5388; e) K. Fukumoto, A. Onoda, E. Mizohata, M. Bocola, T. Inoue, U. Schwaneberg, T. Hayashi, ChemCatChem 2014, 6, 1229 – 1235; f) A. Onoda, K. Fukumoto, M. Arlt, M. Bocola, U. Schwaneberg, T. Hayashi, Chem. Commun. 2012, 48, 9756 – 9758; g) C. Zhang, P. Srivastava, K. Ellis-Guardiola, J. C. Lewis, Tetrahedron 2014, 70, 4245 – 4249; h) F. Philippart, M. Arlt, S. Gotzen, S.-J. Tenne, M. Bocola, H.-H. Chen, L. Zhu, U. Schwaneberg, J. Okuda, Chem. Eur. J. 2013, 19, 13865 – 13871; i) M. E. Wilson, G. M. Whitesides, J. Am. Chem. Soc. 1978, 100, 306 – 307. [3] R. M. de Figueiredo, P. Oczipka, R. Frçhlich, M. Christmann, Synthesis 2008, 1316 – 1318. [4] S.-J. Tenne, U. Schwaneberg, Int. J. Mol. Sci. 2012, 13, 2459 – 2471. [5] S. K. Wright, R. E. Viola, Anal. Biochem. 1998, 265, 8 – 14. [6] P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, D. C. Klenk, Anal. Biochem. 1985, 150, 76 – 85. [7] J. Phan, A. Zdanov, A. G. Evdokimov, J. E. Tropea, H. K. Peters, R. B. Kapust, M. Li, A. Wlodawer, D. S. Waugh, J. Biol. Chem. 2002, 277, 50564 – 50572. [8] B. A. Wallace, J. G. Lees, A. J. W. Orry, A. Lobley, R. W. Janes, Protein Sci. 2003, 12, 875 – 884. [9] a) N. J. Greenfield, G. D. Fasman, Biochemistry 1969, 8, 4108 – 4116; b) N. Sreerama, R. W. Woody, Anal. Biochem. 2000, 287, 252 – 260. [10] E. Krieger, T. Darden, S. B. Nabuurs, A. Finkelstein, G. Vriend, Proteins Struct. Funct. Bioinf. 2004, 57, 678 – 683. [11] a) D. M. Lynn, B. Mohr, R. H. Grubbs, L. M. Henling, M. W. Day, J. Am. Chem. Soc. 2000, 122, 6601 – 6609; b) S. J. P’Poo, H.-J. Schanz, J. Am. Chem. Soc. 2007, 129, 14200 – 14212; c) T. M. Trnka, R. H. Grubbs, Acc. Chem. Res. 2001, 34, 18 – 29. [12] a) T.-L. Choi, R. H. Grubbs, Angew. Chem. Int. Ed. 2003, 42, 1743 – 1746; Angew. Chem. 2003, 115, 1785 – 1788. [13] M. Mayr, M. R. Buchmeiser, K. Wurst, Adv. Synth. Catal. 2002, 344, 712 – 719. [14] a) J. P. Jordan, R. H. Grubbs, Angew. Chem. Int. Ed. 2007, 46, 5152 – 5155; Angew. Chem. 2007, 119, 5244 – 5247. [15] W. J. Feast, D. B. Harrison, Polym. Bull. 1991, 25, 343 – 350. [16] For FhuA-C1, average values Mn of 181 000 g mol 1 and Mw/Mn of 2.7 were measured. For FhuA-C2, average values Mn of 244 000 g mol 1 and Mw/Mn of 2.8 were measured.
Typical ring-opening metathesis polymerization (ROMP) using Grubbs–Hoveyda type catalyst To a solution containing the Grubbs–Hoveyda type catalyst in either pure THF (1 mL THF, 0.1 mm (1 mL H2O, 0.1 mm) or 1 % SDS (NaPi = 0.1 m, pH 7.4 or 5.8) or PE-PEG (0.125 mm, NaPi = 0.1 m, pH 7.4 or 5.8) was added degassed THF (0.1 mL). The 7-oxanorbornene derivative 23 (18 mg, 15 mL, 0.1 m) was added dropwise via a microsyringe. The reaction mixture was allowed to stir for 68 h at 25 8C. Subsequently, ethyl vinyl ether was added to quench the catalyst, and the residual monomer and the polymer were extracted with CDCl3. The conversion and the cis/trans ratio were determined by 1H NMR spectroscopy as reported by Feast and Harrison.[15]
Typical ROMP using the biohybrid catalysts To a solution containing the biohybrid catalyst (1 mL, 5 mg mL 1, 0.1 mm) in either 1 % SDS (NaPi = 0.1 m, pH 7.4 or 5.8) or PE-PEG (0.125 mm, NaPi = 0.1 m, pH 7.4 or 5.8) was added degassed THF (0.1 mL). The solution was stirred for 10 min at 25 8C. The 7-oxanorbornene derivative 23 (18 mg, 15 mL, 0.1 m) was added dropwise via a microsyringe. The reaction mixture was allowed to stir for 68 h at 25 8C. Subsequently, ethyl vinyl ether was added to quench the catalyst, and the residual monomer and the polymer were extracted with CDCl3. The conversion and the cis/trans ratio were determined by 1H NMR spectroscopy as reported by Feast and Harrison.[15]
Acknowledgements We gratefully acknowledge the financial support by the Deutsche Forschungsgemeinschaft through the International Research Training Group “Selectivity in Chemo- and Biocatalysis”
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Received: August 27, 2014 Revised: November 10, 2014 Published online on November 25, 2014
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