DOI: 10.1002/cctc.201801755 Full Papers 1 2 3 Mechanistic Investigation of Biocatalytic Heme Carbenoid 4 À 5 Si H Insertions 6 [a] [b] [b] [a] 7 Rahul L. Khade, Ajay L. Chandgude, Rudi Fasan,* and Yong Zhang* 8 9 10 Recent studies reported the development of biocatalytic heme heme catalyzed SiÀ H insertion pathways were provided to help 11 carbenoid SiÀ H insertions for the selective formation of carbon- understand the origin of experimental reactivity trends. Quanti- 12 silicon bonds, but many mechanistic questions remain unad- tative relationships between reaction barriers and some proper- 13 dressed. To this end, a DFT mechanistic investigation was ties such as charge transfer from substrate to heme carbene II 14 performed which reveals an Fe -based concerted hydride trans- and SiÀ H bond length change from reactant to transition state 15 fer mechanism with early transition state feature. The results were found. Results suggest catalyst modifications to facilitate 16 from these computational analyses are consistent with exper- the charge transfer from the silane substrate to the carbene, 17 imental data of radical trapping, kinetic isotope effects, and which was determined to be a major electronic driving force of 18 structure-reactivity data using engineered variants of hemopro- this reaction, should enable the development of improved 19 teins. Detailed geometric and electronic profiles along the biocatalysts for SiÀ H carbene insertion reactions. 20 21 Introduction roles do conformation and spin state play on the transition 22 state (TS)? What factors are critical to catalyst design? Computa- 23 Engineered hemoproteins have been proven useful for promot- tional work on SiÀ H insertion is scarce[8] and detailed analyses 24 ing a broad range of carbene-mediated transformations, of geometric or electronic property changes along these metal- 25 including carbene insertion into XÀ H bonds (X=N, S, Si, C, B),[1] carbenoid SiÀ H insertion pathways have been missing. 26 cyclopropanations,[2] aldehyde olefinations,[3] and sigmatropic To address these mechanistic questions, a quantum chem- 27 rearrangements.[4] Among these reactions, the synthesis of ical investigation of heme-carbenoid SiÀ H insertions was 28 organosilicon compounds find important applications in the performed here, using basically the same computational 29 area of material science[5] and medicinal chemistry.[6] While approach in recent accurate predictions of iron porphyrin 30 synthetic methodologies for the transition metal-catalyzed carbenes’ (IPCs) experimental X-ray crystal structures, Mössba- 31 carbene insertion into SiÀ H bonds have been reported,[7] these uer and NMR properties, and their experimental formation, 32 protocols require organic solvents and are typically character- cyclopropanation, and CÀ H insertion reactivity and selectivity 33 ized by limited catalytic turnovers (<100). Recently, biocatalytic results,[9] see details in Experimental section. Reproducing 34 methods for accomplishing this type of transformations in experimentally observed structure-activity trends and kinetic 35 aqueous solvents and under mild reaction conditions were isotope effects (KIEs), our computational analyses provide key 36 reported using engineered variants of cytochrome c[1c] and insights into the basic mechanism of heme-catalyzed SiÀ H 37 myoglobin.[1h] These systems offer several attractive features for carbene insertion and into the impact of structural changes at 38 sustainable chemistry due to the accessibility and biocompati- the level of the substrate, carbene, and heme catalyst on this 39 bility of these iron-based metalloprotein biocatalysts, along reactivity. 40 with their promising catalytic activity in these transformations 41 (>1,500–8,000 catalytic turnovers).[1c,h] 42 Despite this progress, many mechanistic questions remain Results and Discussion 43 unanswered. For instance, what is the major electronic driving 44 force for this reaction? What is the origin of the experimentally Basic Mechanism 45 observed substrate and carbene reactivity trends? What kind of 46 Since the previously reported active biocatalysts for this 47 reaction (i.e., cytochrome c and myoglobin) both contain a 48 [a] Dr. R. L. Khade, Prof. Dr. Y. Zhang histidine-ligated heme cofactor,[1c,h] [Fe(Por)(His)(C(Me)CO Et)] 49 Department of Chemistry and Chemical Biology 2 Stevens Institute of Technology was first investigated to model the core part of these 50 1 Castle Point on Hudson, Hoboken, NJ 07030 (USA) biocatalysts in the SiÀ H carbene insertion reaction 1 of 51 E-mail: [email protected] Scheme 1. Por is a non-substituted porphyrin, His is modeled as 52 [b] Dr. A. L. Chandgude, Prof. Dr. R. Fasan 5-methylimidazole as done previously,[9bÀ d] and C(Me)CO Et is 53 Department of Chemistry 2 University of Rochester the carbene group derived from the corresponding diazo ester 54 120 Trustee Road, Rochester, NY 14627 (USA) reagent.[1c] This carbene structure was also confirmed in a recent 55 E-mail: [email protected] X-ray crystallographic study.[8c] Since there is no prior computa- 56 Supporting information for this article is available on the WWW under https://doi.org/10.1002/cctc.201801755 tional study of effects of substrates, carbenes, and catalysts on 57 ChemCatChem 2019, 11, 3101–3108 3101 © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Wiley VCH Mittwoch, 19.06.2019 1913 / 138593 [S. 3101/3108] 1 Full Papers concerted TS(1) described above. Similarly the initial broken- 1 symmetry setup of FeIII (S=5/2), carbene (S =À 1/2), H (S =1/2), 2 and the remaining substrate (S=À 1/2) for the overall quintet 3 radical-based transition state was also optimized to the same 4 quintet concerted TS(1) mentioned above. In contrast with 5 these results at singlet and quintet levels, the triplet TS(1) has 6 obvious radical feature with 1.092, 0.558, and 0.377 e spin 7 densities for Fe, the carbene α-carbon atom, and Si, respec- 8 tively. However, it has the highest Gibbs free energy of 9 activation, with ΔΔG� of 12.81 kcal/mol larger than that of the 10 lowest energy TS(1), see Table S4. 11 In previous work, an engineered active site variant of sperm 12 whale myoglobin, Mb(H64V,V68A), was determined to catalyze 13 Scheme 1. SiÀ H insertion pathways in reactions 1–5. a SiÀ H carbene insertion reaction between dimethyl(phenyl) 14 silane (1a) and ethyl α-diazoacetate (2a) with up to 1,545 total 15 turnovers.[1h] To probe experimentally the occurrence of a 16 heme carbenoid SiÀ H insertions, this work focuses on the SiÀ H radical mechanism in this hemoprotein-catalyzed SiÀ H insertion 17 insertion reactivities of the core part of the protein due to these process, the reaction was performed in the absence and in the 18 effects. presence of a large excess (100 mM) of the spin trap reagent 19 Both a concerted mechanism as proposed for other metal 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). As shown in Sche- 20 carbenoid SiÀ H insertions[7b,c,8b,10] and a stepwise radical mecha- me 2a, the addition of DMPO did not have a significant impact 21 nism reminiscent of heme-enzyme catalyzed oxidation 22 reactions[11] were investigated for reaction 1 to explore the basic 23 mechanism, with details in Supporting Information (SI) and key 24 results discussed here. 25 Comparisons of different mechanisms, conformations, 26 and spin states. We first studied the concerted transition state. 27 Because electronic state and conformation may influence the 28 mechanism, such effects were examined first to identify the 29 most favorable conformations and spin states, see SI for details. 30 Overall, the conformation effect was found to be small. For 31 example, the Gibbs free energy differences for different 32 conformations of TS(1) (the reaction number is in parenthesis) 33 (see Figure S1) are within ~0.3 kcal/mol, indicating a weak steric 34 interaction due to the long C…Si distance (~3.1 Å). 35 In contrast, the spin state effect on mechanism is much 36 larger. Here, we focused on both singlet and quintet transition 37 states because the iron-containing product is of quintet ground 38 state,[12] which is different from the reactant’s singlet ground Scheme 2. Radical trap (a) and kinetic isotope effect experiments (b) for Mb 39 state for various IPCs.[13] These experimental ground states of (H64 V,V68 A)-catalyzed SiÀ H carbene insertion reaction. 40 reactants and products were reproduced in our calculations.[9] 41 Regarding concerted TS(1), the FeII-based quintet transition 42 state has Fe spin density of 3.885 e and carbene’s carbon spin on the yield of the Mb(H64V,V68A)-catalyzed SiÀ H insertion 43 density of only À 0.165 e. It has significantly higher energy reaction (52�5% vs. 45�7%), suggesting the absence of a 44 (ΔΔG� of 10.27 kcal/mol) than the FeII-based closed-shell singlet radical intermediate. To gain further insights into the mecha- 45 (Table S4). In contrast, the third TS spin state with S=1 is of the nism of this reaction, kinetic isotope effect (KIE) experiments 46 highest energy, by ΔΔG� of 12.81 kcal/mol (Table S4). These were performed using a deuterated form of the dimethyl 47 results support the singlet TS(1) as the most favorable spin (phenyl)silane substrate (d–1a). Competition experiments with 48 state, and therefore there is no spin crossover from the singlet equimolar amount of 1a and d–1a yielded a positive KIE (k /k ) 49 H D reactant. The results obtained for the other reactions consid- corresponding to 1.19�0.05 (Scheme 2b and Figure S4). A 50 ered here (Scheme 1) also indicated that the singlet state TS is similar KIE value of 1.3� 0.2 was also obtained by comparing 51 the most favorable transition state (see SI for details).