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A Highly Active Biohybrid Catalyst for Olefin Metathesis in Water: The Impact of a Hydrophobic Cavity in a #-Barrel Protein Daniel F. Sauer, Tomoki Himiyama, Kengo Tachikawa, Kazuki Fukumoto, Akira Onoda, Eiichi Mizohata, Tsuyoshi Inoue, Marco Bocola, Ulrich Schwaneberg, Takashi Hayashi, and Jun Okuda ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.5b01792 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 20, 2015
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A Highly Active Biohybrid Catalyst for Olefin Metathesis in Water: The Impact of a Hydrophobic Cavity in a β-Barrel Protein Daniel F. Sauer,† Tomoki Himiyama,‡ Kengo Tachikawa,‡ Kazuki Fukumoto,‡ Akira Onoda,‡ Eiichi Mizohata,‡ Tsuyoshi Inoue,‡ Marco Bocola,§ Ulrich Schwaneberg,§ Takashi Hayashi,*,‡ Jun Okuda*,† †
Institute of Inorganic Chemistry, RWTH Aachen University, Landoltweg 1, D-52056 Aachen, Germany Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan § Institute of Biotechnology, RWTH Aachen University, Worringer Weg 1, D-52056 Aachen, Germany ‡
Keywords: Biohybrid catalysis, chemogenetic approach, metalloenzymes, ruthenium, metathesis ABSTRACT: A series of Grubbs-Hoveyda type catalyst precursors for olefin metathesis containing a maleimide moiety in the backbone of the NHC-ligand was covalently incorporated in the cavity of the β-barrel protein nitrobindin. By using two protein mutants with different cavity sizes and choosing the suitable spacer length, an artificial metalloenzyme for olefin metathesis reactions in water in the absence of any organic co-solvents was obtained. High efficiencies reaching TON > 9000 in the ROMP of a water-soluble 7-oxanorbornene derivative and TON > 100 in ring closing metathesis (RCM) of 4,4bis(hydroxymethyl)-1,6-heptadiene in water under relatively mild conditions (pH 6, T = 25 to 40 °C) were observed.
Artificial metalloenzymes with catalytically active metal sites integrated into a protein matrix could extend the reaction scope of natural enzymes and the performance of organometallic catalysts. Moreover, fine-tuning substrate recognition and stereoselectivity by virtue of the second ligand sphere provided by the protein offers new 1 possibilities of catalyst control. To construct biohybrid catalysts, the metal catalyst precursor is either incorporated in the cavity of a protein or attached to the protein surface 2 3 4 by supramolecular, dative, or covalent anchoring. The chemogenetic approach is a useful tool to fine-tune the activity and/or selectivity of metalloenzymes. For example, Hayashi et al. accomplished protein modification with the easily tunable and robust protein matrix 4c,4d nitrobindin. By mutations of selected amino acids in the 5 vicinity of an (η -cyclopentadienyl)rhodium site, the cis/trans selectivity of poly(phenylacetylene) was shifted from 9% to 4d 82% trans. Furthermore, synergy in catalyst optimization can be expected by modifying both the metal site and the protein environment. Grubbs-Hoveyda (GH) type catalysts are known to catalyze olefin metathesis in organic solvents at relatively 5 low catalyst loadings. Performing olefin metathesis in water 6 as solvent is of interest for various synthetic applications. The main challenge is to achieve a highly active and longlived catalyst requiring low catalyst loadings. We have recently reported the incorporation of a GH type catalyst in a variant of the transmembrane protein Ferric hydroxamate 7 uptake protein component A (FhuA). By changing the spacer length on the first coordination sphere of the
catalyst, the ring-opening metathesis polymerization (ROMP) activity was doubled. So far, the efficiency of the artificial olefin metatheases reported in the literature still 2e,3c,4a,4b,7 We need to be improved for practical applications. report here the incorporation of a GH type catalyst in the βbarrel protein nitrobindin (NB) and a significant increase in the activity in water for olefin metathesis. O O O
n
NMes
MesN
S
O N
O
O
Ru O
n = 1 - C1 n = 2 - C2 n = 3 - C3
NB
O
O NB-SH H 2O, DMSO (1-10% (v/v))
NMes
MesN
Cl Cl
N n
Tris-HCl buffer (pH = 7.5; 10 mM) 50 mM NaCl 60 min., 25 °C
Cl Ru Cl O
n = 3; NB n = 1; NB n = 2; NB n = 3; NB
= NB4 - NB4_C3 = NB11 - NB11_C1 = NB11 - NB11_C2 = NB11 - NB11_C3
Scheme 1. Conjugation of the Grubbs-Hoveyda type catalysts. A series of GH type catalyst precursors C1–C3 containing a maleimide moiety in the backbone of the NHC ligand was covalently anchored via thioether formation with an NB variant carrying a cysteine in position C96 (Scheme 1). The hydrophobic cavity of NB has a suitable size for anchoring 4c,4d mononuclear organometallics. The mostly hydrophobic residues inside the cavity are likely to protect the catalyst from coordination of polar solvent molecules or coordinating
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residues such as amino or carboxylic groups to the active center. Starting with a variant of NB having a relatively small cavity volume (M75L/H76L/Q96C/M148L/H158L (NB4), Table S2), GH type catalysts C1–C3 (Figure S1) were anchored in NB4. Only the complex C3 was anchored with NB4 in a moderate conjugation yield (25%); coupling with C1 and C2 was unsuccessful. Computational studies using the YASARA software package suggested that the cavity of NB4 is too small to incorporate the sterically demanding NHC ligand of the GH type catalyst (Table S2, entry 3 and 4c,4d,7-8 4). Although the maleimide moiety with an elongated spacer is able to be covalently linked with the thiol of C96, the catalyst is apparently still too bulky to yield higher coupling efficiency. We thus prepared the variant L75A/H76L/Q96C/M148L/H158A (NB11) with a larger cavity 4d (Table S2, entry 5). The cavity depth of 15-20 Å for both mutants is similar (Table S2). Conjugating the catalysts C1– C3 (Figure 1), resulted in higher coupling efficiency of up to 89% (Supporting Information). C96
L75
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indicating successful anchoring. The amount of attached ruthenium ion was further confirmed by ICP-AES, suggesting that almost one (0.85~0.90) ruthenium ion per NB protein had been incorporated. The β-barrel structure of the biohybrid catalysts was determined by CD spectroscopy. All biohybrid catalysts containing the GH type catalyst showed the characteristic minimum at 215 nm in a Tris-HCl buffer solution (10 mM, pH 7.5; 200 mM NaCl) (Figure S5). The biohybrid catalysts retain the proper folding structure under the acidic conditions of a MES buffer solution (5 mM, pH 6.0; 200 mM NaCl). This medium was preferred for metathesis catalysis at temperatures up to 50 °C (Figure 2).
NB4_C3
Figure 2. CD spectra of of NB11_C3 at 25 °C (black), 40 °C (red), 50 °C (blue), and 60 °C (purple).
L158 C96
A75
NB11_C3
A158
Figure 1. Illustration of the cavity size in the monomeric structures of NB4_C3 and NB11_C3 with selected amino acid residues. After the metal catalysts C1–C3 were anchored in the protein, the biohybrid catalysts were purified by centrifugation and size exclusion chromatography to remove the unreacted catalyst precursors (See Supporting Information, page S3) and characterized by UV/vis, ICP-AES, MALDITOF MS and ESI-TOF MS (Figure S2, S3, and S4). The calculated mass for catalyst NB11_C3 (m/z 20153), in which one equivalent of the catalyst is attached to the protein, was observed and the signal for the apo-form (m/z 19332) was not detected in the MALDI-TOF MS spectra,
The crystal structure of NB11 confirmed the changes of the size in the interior of the cavity. The space group is 9 identical to that of wild-type NB (PDB code 3EMM, space group: P21212) with a dimeric assembly, as seen in the wild 4c,4d,9 This type and all NB mutants (Figure S6 and S7). dimeric assembly was also indicated in solution by size exclusion chromatography (Figure S12). The cavity of each of the two monomers is twisted against each other around 140°, making the catalysts’ active site pointing away from each other (Figure 3). The contact between the two proteins is formed mainly by hydrophobic contact containing the aliphatic amino acids such as valine (V93, V101, V103) and leucine (L121). Despite the dimeric structure, the cavities of NB4 and NB11 are not blocked by other amino acid residues or the second NB. The activities of the hybrid catalysts and protein-free catalyst (COH, Figure S1) were evaluated in the RCM 10 reaction of 4,4-bis(hydroxymethyl)-1,6-heptadiene 1 (Table 1) and in the ROMP reaction of the water-soluble 7oxanorbornene derivative 3 (Table 2). The conversions of all catalytic reactions were low in Tris-HCl buffer (pH 7.5, 10 mM, 200 mM NaCl), probably because a Tris molecule might be involved in coordination, competiting with substrate binding. As metathesis in aqueous solutions is 4a,11 accelerated under acidic conditions, the reaction was performed in MES buffer (pH 6.0, 5 mM, 200 mM NaCl). Under these conditions, the biohybrid catalysts were active at low catalyst loadings in both the RCM and ROMP reactions. NB11_C2 and NB11_C3 performed the RCM reaction with high activity with a catalyst loading of 1 mol %, reaching full conversion within 24 h in the best case (Table 1, entry 10). Additionally, catalyst variant NB11_C2 showed significant activity in the RCM reaction, supporting the
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assumption that during ROMP the growing polymer blocks the cavity, as explained in the next paragraph.
Table 1. RCM of Substrate 1.
conversion occurred (Table 2, entry 4). By attachment of the catalyst C3 to NB4, the activity significantly increased, leading to 10% conversion with a total TON of 1100 (Table 2, entry 5).
Table 2. ROMP of substrate 3. O OMe
n
#
Catalyst (mol %)
a
%-Conv.
b
OMe
TON
1
c
2
c
3
c
COH (5)
>99
20
1
COH
4
d
COH (0.01)
99
100
3
COH
6
d
COH (5)
>99
20
4
7
e
COH (5)
n.d.
n.d.
8
NB11_C2 (1)
89
9
NB11_C3 (0.008)
