Enzyme Activity by Design: An Artificial Rhodium Hydroformylase for Linear Aldehydes

Abstract Artificial metalloenzymes (ArMs) are hybrid catalysts that offer a unique opportunity to combine the superior performance of natural protein structures with the unnatural reactivity of transition‐metal catalytic centers. Therefore, they provide the prospect of highly selective and active catalytic chemical conversions for which natural enzymes are unavailable. Herein, we show how by rationally combining robust site‐specific phosphine bioconjugation methods and a lipid‐binding protein (SCP‐2L), an artificial rhodium hydroformylase was developed that displays remarkable activities and selectivities for the biphasic production of long‐chain linear aldehydes under benign aqueous conditions. Overall, this study demonstrates that judiciously chosen protein‐binding scaffolds can be adapted to obtain metalloenzymes that provide the reactivity of the introduced metal center combined with specifically intended product selectivity.

Abstract: Artificial metalloenzymes (ArMs) are hybrid catalysts that offer au nique opportunity to combine the superior performance of natural protein structures with the unnatural reactivity of transition-metal catalytic centers.T herefore,t hey providet he prospect of highly selective and active catalytic chemical conversions for whichn atural enzymes are unavailable.Herein, we show how by rationally combining robust sitespecific phosphine bioconjugation methods and al ipid-binding protein (SCP-2L), an artificial rhodium hydroformylase was developed that displays remarkable activities and selectivities for the biphasic production of long-chain linear aldehydes under benign aqueous conditions.O verall, this study demonstrates that judiciously chosen protein-binding scaffolds can be adapted to obtain metalloenzymes that provide the reactivity of the introduced metal center combined with specifically intended product selectivity.
The development of substrate-and product-specific catalytic processes that operate efficiently at mild reaction temperatures is am ajor challenge for the synthetic chemistry community. [1] Enzymes are naturesm ain catalysts and catalyze numerous chemical transformations,t ypically under benign conditions.However,many desired chemical reactions are not performed by nature,a nd therefore,t here are no suitable natural enzymes.A rtificial metalloenzymes (ArMs) provide aw ay to bridge that gap between natural and chemical synthesis,p roviding enzymes for unnatural reactions. [2] Despite these successes,m ost ArMs do not meet the rates and performances achieved by natural enzymes. [3] In addition, the molecular recognition and shape selectivity of proteins have typically not been exploited. Themost successful approach to create ArMs has been the use of non-covalent anchoring strategies utilizing protein scaffolds with strong supramolecular recognition motifs such as avidin. [4] In these ArMs,the binding site is used to carry the active metal center or bind metal-containing cofactors,r estricting the possible applications of the binding properties of the protein. An alternative approach utilizes site-selective protein modification methods [5] to incorporate transition metals into aw ide range of protein scaffolds whilst leaving the proteinsi nnate binding capabilities largely intact. Any protein scaffold can be used, allowing the exploitation of the almost unlimited range of highly specific substrate-binding capabilities of proteins. Therefore,v irtually any organometallic non-natural catalytic reaction can be merged with the sophisticated biological performance of enzymes,and this approach offers significant opportunities for the design of ArMs aiming at high selectivity by shape-selective product formation. Herein, we demonstrate the potential of such ArMs through the development of artificial rhodium enzymes derived from ap rotein scaffold that was selected for its apolar substrate-binding properties.T hese ArMs enable selective aldehyde formation in the biphasic hydroformylation of long-chain linear alkenes, ar eaction for which no natural enzymes are known, and which is challenged in current industrial applications by the low solubility of the substrates in the aqueous phase. [6] We have previously reported amethod that enables such an approach for the synthesis of ArMs containing metalbinding phosphine ligands (Scheme 1A). [5a, 7] Rhodium-phosphine complexes are known to be highly active and robust hydroformylation catalysts,and thus our strategy provides the prospect of enzymatic hydroformylation reactions.Rhodiumprotein hybrids tested to date in hydroformylation have utilized dative protein-rhodium interactions with limited success. [8] Although this has led to unprecedented linear selectivities for the hydroformylation of styrene, [9] thee xact nature of the active species,a nd thus the origin of the selectivity,isstill unclear. [10] Rhodium-catalyzed hydroformylation is used on a800 000 ton scale to produce butyraldehyde from propene under biphasic conditions, [11] allowing for the recovery of the expensive rhodium 3,3',3''-phosphanetriyltris(benzenesulfonic acid) trisodium salt catalyst (Rh/ TPPTS). Long-chain aldehydes are desired by industry as they are important precursors for the production of detergents and plasticizers. [12] This process is not feasible for longchain alkenes (> 5Catoms) owing to their low solubility in water. [6] Fatty acid transporter proteins contain apolar tunnels and clefts to bind their hydrophobic cargo.T he steroid carrier protein type 2l ike (SCP-2L) domain of the human multifunctional enzyme 2( MFE-2) was identified as as uitable linear alkene binding protein scaffold as it is known to bind avariety of linear aliphatic substrates [13] and can be obtained in high yields. [5a] Our hypothesis was that the apolar tunnel present in SCP-2L ( Figure 1B)would be able to facilitate the transport of alkenes to the aqueous environment and orient the starting alkene along the tunnel towards the rhodium center, enabling the shape-selective production of the desired linear product ( Figure 1A). To introduce the catalytic rhodium-phosphine complexes,t wo mutants containing unique cysteines at either end of the tunnel were prepared (SCP-2L V83C and SCP-2L A100C;F igure 1B). [5a, 14] These two mutants,o btained in excellent yields,s howed little structural permutations and similar aliphatic substrate binding capabilities as the wild-type (WT) protein (see the Supporting Information, Table S3). Both SCP-2L mutants were successfully modified with aldehyde phosphines P1-P3 through ac ysteine modification strategy (Scheme 1A;f or characterization data for SCP-2L V83C-1-(P1-P3), see Ref.
Therhodium proteins (SCP-2LV83C/A100C-1-(P1-P3)-Rh) were obtained by the addition of Rh(acac)(CO) 2 .O ther Rh precursors did not selectively bind to the phosphine (see Table S4 for MS and metal loading analysis) . Their catalytic activity was investigated in the hydroformylation of 1-octene at 35 8 8Ca nd 80 bar synthesis gas (Figure 2, Scheme 1B,a nd Table S5). To minimize rhodium leaching and therefore false results from "free Rh" (leading to low selectivity (ca. 55 % linearity) and high turnover numbers (TONs > 500;T able 1, entry 2)), as light excess of protein (2 equiv) was used. Even though these reactions were performed at ar elatively low temperature (typical industrial conditions are 125 8 8C), significant hydroformylation activity was detected over 48 hfor several of the rhodium-phosphine ArMs (Figure 2A). Reac-   [13] the tunnel dimensions, and the positions of A100 and V83.
tions over time showed that the enzyme was active over the whole 48 h ( Table S6). Control reactions with the ArM phosphine selenide or ArM phosphine gold complexes showed that the rhodium was required for the hydroformylation reaction to occur (Table S5).
Thestructure of the phosphine cofactor was found to have al arge effect on the TON,w ith the activity increasing by factors of 30 and 70, respectively,a st he phosphine moved from the ortho (P1)t ot he meta position (P2)a nd from the ortho (P1)t ot he para position (P3)f or SCP-2L V83C.T he protein scaffold also influenced the reactivity,with amarked improvement in turnover found when using SCP-2L A100C-1-P3,achieving TONs of > 400, versus 75 for SCP-2L V83C-1-P3.The selectivity of the reaction was also found to depend on the combination of protein scaffold and phosphine cofactor applied. It ranged from 69 to 79 %f or the linear product (nonanal), matching typical selectivities for doublephosphine-ligated rhodium-catalyzed hydroformylation. [15] Selectivities of 80 %a re rarely seen with monoligated P/Rh systems.C ontrol reactions with P1-P3 or Rh(acac)(CO)-(PPh 3 )i nn eat alkene showed that with monophosphines in the organic phase of our reaction, selectivities of up to 74 % can be achieved (Tables S8 and S9). Catalyst degradation and leaching of the phosphine and rhodium to the organic phase were not responsible for the observed selectivities as only minimal rhodium leaching and degradation were observed (Table S11 and Figures S12 and S13). It should be noted that our ArM system has al ow P/Rh ratio,w hich, when using abenchmark biphasic Rh/TPPTS catalytic system, only gives activities and selectivities that correspond to rhodium leaching into the organic phase (Table 1, entries 2, 4, and 5). At the same concentration as in our ArM reactions,over 300 equivalents of the TPPTS ligand are needed to prevent metal leaching (Table 1, entries 4-6) and obtain high selectivities. Thesame effect of rhodium leaching was observed when the protein was simply mixed with Rh(acac)(CO) 2 (Table 1,  entry 3). This,a longside the differences between the two protein mutants,s hows that it is the hybrid catalysts that are responsible for the hydroformylation results.SCP-2L A100C-1-P3 gave the best performance overall (79 %n onanal, TON = 409;T able 1, entry 1). These conversions and selectivities are remarkable as ab enchmark catalyst (Rh/TPPTS) gives negligible conversion approaching the detection limit (TON % 1) when the TPPTS/Rh ratio is optimized to give similar selectivities to the metalloenzyme (TPPTS/Rh = 30:1 at ca. 10 times the Rh concentration of the ArM reaction gives 72 % linearity;see Table S7). Following the successful hydroformylation of 1-octene,t hese artificial metalloenzymes were tested in the hydroformylation of 1decene,1 -dodecene,a nd 1-octadecene ( Figure 2B). [6] When TPPTS was used, at enfold rate decrease was observed on the addition of two carbon atoms to the chain owing to the reduced water solubility of the alkene. [6] Using the ArM, aless than fourfold

Angewandte Chemie
Communications decrease in activity was observed upon going from 1-octene to 1-decene,a nd an only tenfold decrease when going to octadecene.U nder selectivity-optimized conditions (high ligand concentrations to give adequate linear selectivity while preventing Rh leaching), Rh/TPPTS displayed no significant activity (Figure 2A,T able 1, entry 6, and Table S7). Control experiments using the Rh/TPPTS system in the presence of the protein scaffold showed no significant difference in turnover for 1-octene (Table 1, entry 6vs. 7and 8), providing evidence that the increase in activity for the ArMs cannot solely be explained by the protein acting as ap hase-transfer reagent. We therefore attribute the higherthan-expected activity to the presence of the lipid-binding tunnel in the protein scaffold in direct proximity of the Rh center.
Overall, the selectivities for the linear hydroformylation products were remarkably high for monophosphine-ligated rhodium in water, indicating that the protein scaffold counterbalances the lack of phosphine ligands.I na ddition, both the phosphine cofactor and the protein mutant affect the activity of the reaction. To better understand the observed selectivity of our hydroformylase,weinvestigated the local environment of the Rh center in the protein scaffold. Both the X-ray absorption near-edge structure (XANES) and extended Xray absorption fine structure (EXAFS) of SCP-2L A100C-1-P3-Rh at the Rh Kedge were assessed. Comparing the XANES of SCP-2L A100C-1-P3-Rh to model Rh complexes ( Figure S10) suggested the loss of carbonyl functionalities from the Rh(acac)(CO) 2 precursor.T his was further supported by the lack of CO stretches in the IR spectrum and fitting the EXAFS data of SCP-2L A100C-1-P3-Rh (Table S1).
Thef itting model applied used characteristic scattering paths from both acac and PPh 2 Ar ligands,with aderived Rh-Pc oordination number of two.E XAFS is unable to distinguish between scattering neighbors of like atomic number, especially where Z =AE 1. Thep rospect of two phosphorus atoms coordinated to the rhodium center appeared unlikely owing to the steric congestion resulting from placing two protein scaffolds around the metal. Thus we postulated the possibility of one of the observed Rh-P neighbors arising from aR h-S interaction ( Figure 3A); the protein scaffold contains functionalized sulfur in the form of methionine,a t the N-terminus and on the flexible a-helices,a nd the introduced cysteine ( Figure 3B).
To probe the possibility of an earby methionine coordinating to the Rh center, the selenomethionine derivatives of the protein scaffold were expressed and purified. Rhodium complexation of the phosphine-modified SeMet protein with Rh(acac)(CO) 2 gave similar results as for SCP-2L A100C-1-P3,showing the RhCO adducts in the mass spectra, and also resulted in as mall shift in the 77 Se NMR spectrum upon Rh addition ( Figure S11). There was aprofound change in the Rh Kedge EXAFS data of SeMet-A100C-1-P3-Rh compared to SCP-2L A100C-1-P3-Rh ( Figure 3C). Thed ifferences observed can be rationalized by coordination to aneighbor of higher atomic number with alarger backscattering amplitude; between the analogous systems,Rh-S interactions have been replaced by Rh-Se interactions.T he EXAFS analysis sup-ports our hypothesis that there is monophosphine coordination, coupled with further interaction with sulfur from am ethionine residue.M oreover,t hrough observation of this Rh-S/Se interaction, we have direct evidence for the coordination of the protein scaffold to the Rh center.
We were intrigued if the methionine coordination would have an effect on the catalytic performance of the ArM, or if the methionine would just decoordinate under the reaction conditions to give the same active catalyst, and thus the same catalytic results in all cases.T op robe this further, four mutants of SCP-2L A100C were prepared in which each Met residue was replaced by alanine,a nd the proteins were then modified as above.T he results of the initial catalytic experiments are ambiguous;the activity of SCP-2L A100C M105A-1-P3 was on average higher than that of A100C or the other mutants (TON = 112(AE 33) and TON = 60-86 for the others, see Table S10), which could indicate that M105 is indeed involved in rhodium coordination. However,this can also be due to other factors,s uch as structural changes or decreased protein stability.M ore detailed studies are required before firm conclusions can be drawn on the role of the methionine residues in the hydroformylation by these ArMs.
In summary,w eh ave shown that rhodium phosphine modified SCP-2Ls are linear-selective catalysts in the hydroformylation of long-chain alkenes.S CP-2L A100C-1-P3-Rh showed ar ate enhancement of at least 10 3 compared to the traditional Rh/TPPTS system in the biphasic hydroformylation of 1-octene and 1-decene.T his demonstrates that ap rotein chosen for its specific binding properties can be converted into an enzyme in which these properties are used to transmit product selectivity.C ombining this technology with the recent advances in chemical biology will allow us to rapidly engineer highly selective catalysts that operate under benign conditions.M oving forward, we believe that this approach has the potential to be used for aw hole range of reactions that traditionally use phosphines as ligands and to convert these into biocatalytic processes.Inthe long term, as chemogenetic optimization is used to improve activity,t his could open the door to an ew era of biocatalytic chemical production.