Chemoenzymatic synthesis of optically active α-cyclopropyl-pyruvates and cyclobutenoates via enzyme-catalyzed carbene transfer with diazopyruvate

Abstract

Cyclopropanes are recurrent structural motifs in natural products and bioactive molecules. Recently, biocatalytic cyclopropanations have emerged as a powerful approach to access enantioenriched cyclopropanes, complementing chemocatalytic approaches developed over the last several decades. Here, we report the development of a first biocatalytic strategy for cyclopropanation using ethyl α-diazopyruvate as a novel enzyme-compatible carbene precursor. Using myoglobin variant Mb(H64V,V68G) as the biocatalyst, this method afforded the efficient synthesis of α-cyclopropylpyruvates in high diastereomeric ratios and enantiomeric excess (up to 99% ee). The ketoester moiety in the cyclopropane products can be used to synthesize diverse optically pure cyclopropane derivatives. Furthermore, the enzymatically obtained α-cyclopropylpyruvate products could be converted into enantiopure cyclobutenoates via a metal-free photochemical ring expansion without loss of optical activity.

INTRODUCTION

Cyclopropane is a recurrent structure in natural products^1–3 and bioactive molecules,^4–6 and the third most represented carbocycle, after benzene and cyclohexane, within the structure of FDA-approved drugs.⁷ In recent years, motivated by their high prevalence in drug molecules and drug candidates, a variety of powerful and selective approaches have been developed to synthesize optically pure cyclopropanes.^8–11 Complementing chemocatalytic cyclopropanation protocols, significant progress has been made over the past decade in the development of biocatalytic strategies for the stereoselective cyclopropanation using engineered hemoproteins such as myoglobin and cytochrome P450s.^12–18 These strategies have also proven to be synthetically relevant for the preparation of cyclopropane containing drugs and drug precursors.^14–17

In this context, there is an ongoing interest in broadening the accessible chemical space of optically active cyclopropanes through biocatalysis. Efforts in this direction have included the use of readily available diazo carbene precursors, which have resulted in different examples of acceptor only diazo compounds for biocatalytic cyclopropanation^13,19,20 as well as the use of new carbene precursors like diaziridines²¹. This progress has resulted in newly functionalized enantiopure cyclopropanes, which can be readily manipulated through further chemical modifications to achieve greater diversification of the cyclopropane scaffolds.

Recently, some of us reported biocatalytic methods for olefin cyclopropanation in the presence of α-diazoacetonitrile and α-cyclopropylketones,^22,23 leading to enantioenriched cyclopropanes in up to >99% ee (scheme 1). Both the cyano and the keto-groups in these products can be converted into various new moieties, with a complete stereoretention of the cyclopropane chiral centers. This work showcased the importance of introducing new densely functionalized biocatalyst-compatible diazo compounds that ultimately allow further manipulations to increase the molecular complexity of the cyclopropane. On the other hand, the Arnold group, demonstrated the diasterodivergent cyclopropanation of vinyl boronic acid pinacol ester with ethyldiazoacetate and diversification of the resulting cyclopropanes by Suzuki coupling (Scheme 1).²⁴

Scheme 1.

Biocatalytic synthesis of functionalized cyclopropanes and library extension.

As part of our ongoing program dedicated to the development of straightforward access to functionalized cyclopropanes,²⁵ here we report the first example of biocatalytic enantioselective cyclopropanation using ethyl diazopyruvate (EDPv) as a new biocatalyst-compatible carbene source. The resulting α-cyclopropylpyruvates can be produced with high enantioselectivity (up to >99% ee). To date, the catalytic asymmetric synthesis of α-cyclopropylpyruvates remained elusive, and only racemic chiral derivatives have been reported.^26,27 We also showcased the α-cyclopropylpyruvate motif as a privilege precursor of enantiopure cyclobutenoates and other enantioenriched compounds.

RESULTS AND DISCUSSION

To initiate our investigations dealing with the biocatalytic synthesis of chiral α-cyclopropylpyruvates, different myoglobin variants were tested on the model reaction between EDPv 1 and styrene in whole cell transformation. Variant Mb(H64V,V68A), which previously showed activity for different carbene transfer reactions with ethyl diazoacetate (EDA),¹² afforded only traces of the desired product (Table 1, entry 4), whereas no activity was shown by wild-type Mb or hemin alone (Table 1, entries 2–3).

Table 1.

Catalyst Screening for Cyclopropanation of Styrene with the EDPv.

Entry	Catalyst	YieldC
1	Hemina	0 %
2	Mb (WT)	0 %
3	Mb (H64V, V68A)	<2%
4	Mb (H64G, V68A)	8 %
5	Mb (H64V, V68G)	34 %
6	Mb (H64A, V68G)	<2 %
7	Mb (H64G, V68G)	4%
8	Mb (H64V, V68G)b	5%

Reaction Conditions: 20 mM olefin, 10 mM of EDPv 1, OD₆₀₀ = 20; in 50 mM potassium phosphate (pH 7) buffer (KPi) with 10% ethanol, room temperature under anaerobic conditions.

^a)60 μM of hemin, 10 mM sodium dithionite in 50 mM potassium phosphate buffer (pH 7) containing 10% DMF, room temp.

^b)20 μM of purified Mb (H64V, V68G), 10 mM sodium dithionite in 50 mM potassium phosphate buffer (pH 7) containing 10 % Ethanol.

^c)yield is determined by GC compared with authentic standards.

Decreasing the size of the amino acid at the level of the distal histidine 64 by switching from valine to glycine, Mb(H64G,V68A), increased the yield of the cyclopropane 2a to 8% as shown in entry 5 of Table 1. A similar modification of the amino acid residue at the position 68 (from valine to glycine) resulted in the improved biocatalyst Mb(H64V,V68G), which provides 2a in 34% yield (Table 1, entry 6). Other modifications in position 64 did not further improve the yield (Table 1, entries 7–8). Similarly, additional mutations at positions 111, 28, and 43 were also tested but none of them resulted in improved reactivity (Table S3). Finally the use of Mb(H64V,V68G) as purified enzyme led to an important decrease of the yield of 2a from 34% to 5% (Table 1, entry 9), demonstrating a beneficial effect of conducting the reaction in whole cells compared to the isolated enzyme.

Reaction optimization studies showed that an excess of styrene (5-fold) over the diazo reagent was beneficial to obtain quantitative yields of 2a in the Mb(H64V,V68G)-catalyzed reaction (Table 2, entry 3 and 5) with up to 210 TON (Scheme 2 product 2a). A similar trend was previously observed in Mb-catalyzed cyclopropanation with the α-diazo ketone reagent as the carbene precursor.²³ More recent studies indicated that once the metal carbene is formed, it can either react with the olefin to produce the desired cyclopropane product or inserts into the heme cofactor in a non-productive pathway to produce a green colored adduct.²⁸ A similar non-productive reactivity can occur in the case of the diazopyruvate 1, as supported by the observation of a green coloration of the reaction mixture after the addition of the diazo compound in the absence of the olefin (Figure S3). For synthetic purposes, a scaled-up reaction was performed and product 2a was obtained in 61% isolated yield and an excellent enantiomeric excess (99% ee) (Table 2, entry 6). Interestingly, we observed that extended reaction times (>12–18 hrs) led to only traces of the cyclopropane product. Time course experiments revealed that optimal yields were achieved after 3 hours of reaction (see SI for details, figure S1), whereas a rapid decrease in yield was noted at longer reaction times, likely due to degradation of 2a in the reaction mixture or the intracellular milieu.

Table 2.

Concentrations screening for Cyclopropanation of Styrene with the EDPv.

Entry	Mb (H64V, V68G) (OD600)	Styrene (mM)	EDPv (mM)	Yielda (%)	de (%)
1	20	10	5	43	>99
2	10	50	10	39	>99
3	20	50	10	>99	>99
4	20	25	5	85	>99
5	20	50	5	>99	>99
6b	20	50	10	61%	>99

Reaction Conditions: 50 mM potassium phosphate (pH 7) buffer (KPi) with 10% ethanol, room temperature under anaerobic conditions.

^a)yield is determined by GC compared with authentic standards.

^b)reaction performed on 50 mL scale (volume of the enzymatic scale-up reaction).

Scheme 2.

Substrate scope of Mb(H64V,V68G)-catalyzed olefin cyclopropanation with EDPv. Reaction Conditions: reaction performed in 400 μL KPi buffer 50 mM (pH 7) with 10 mM of EDPv 1 and 50 mM of olefin during 3h. yield is determined by GC compared with authentic standards. (In parenthesis, isolated yield obtained using slow addition (see SI, general procedure A) of the diazo compound 1)

Having defined optimal conditions for this reaction, its scope was further examined. As summarized in Scheme 2, several styrene derivatives could be transformed through this biocatalytic process with excellent enantioselectivity (>99% ee) being obtained in all cases. Different functionalities are tolerated at the para position of the aryl moiety, as exemplified by the para-methyl- and para-bromostyrenes affording the desired cyclopropane 2g and 2e²⁹ in good to high yields (45–80%) and with excellent ee. Electron-withdrawing CF₃ group significantly decreased the yield of cyclopropane (2d: 15% yield). Interestingly, para-methoxy cyclopropane 2b was obtained in low yield, which can result from a combination of the steric effects and the formation of a side product deriving from the undesired Clock-Wilson rearrangement,³⁰ which was detected by GC-MS. A similar phenomenon was observed during the cyclopropanation of electron rich olefins such as 2-vinyl furan and 2-vinyl thiophene, whose transformation primarily led to the corresponding Clock-Wilson rearrangement products. Methyl substitution at the meta-, ortho- or α-position were all tolerated, yielding the desired cyclopropanes in 42 %, 28 % and 79 % yield and 98 %, 90 % and 99 % ee. As fluorinated compounds are of high interest in medicinal chemistry,³¹ this biocatalytic approach was tested also with styrene derivatives bearing a fluorinated group (CF₃, CHF₂, CH₂F) at the α-position. Using the optimized conditions, the corresponding cyclopropanes 2j-l were obtained in excellent de and ee, albeit in modest yields (5–23%). The 2-pyridyl cyclopropane 2m, which is not accessible via known alternative methods using transition metal chiral catalyst, was also synthesized in good yield (66%) and excellent diastereoselectivity.³² Non-styrenyl substrates such as vinylbenzoate, N-vinyl phtalimide, 3-phenylpropene and 4-phenylpropene were tested but no activity was observed.

With these enantiopure cyclopropanes in hand, we investigated their transformations into cyclobutenoates. Indeed, four-membered rings are present in the structure of multiple natural products^33–35 and biologically active compounds.^36–40 Cyclobutenes, in particular have been prepared via photochemical^41–43 or metal-catalyzed^44–46 [2+2]-cycloaddition, as well as from ring expansion reactions, starting from a large variety of cyclopropane.^47–50 After establishing an efficient route to optically active α-cyclopropylpyruvates, we aimed at developing a sustainable metal-free approach to cyclobutenoates via a photochemical ring expansion of α-cyclopropyl diazo derivatives of these enzymatic products. Indeed, in analogy to the work reported by Tang⁴⁷ using transition metal carbene, the photoinduced formation of the free carbene might allow its rearrangement into the corresponding cyclobutene.

To this end, we chose racemic cyclopropane (±)-2a as model substrate for this transformation. Ketone 2a was efficiently converted into the corresponding diazo derivative 3a in 87% yield over two steps. Thanks to the UV spectra analysis (see SI for details, Figure S2) of this diazo compound (λ_abs = 423 nm), we selected a 425 nm irradiation to perform a photoinduced ring-expansion reaction. The reaction parameters were evaluated and among the various solvents tested (see SI for details, Table S2), and optimal yield for 4 (64%) was obtained using diethyl ether as solvent. Note that 4 was isolated as a 50:50 mixture of regioisomers. Performing the photochemical rearrangement in acetonitrile led to an improved regioselectivity of 70:30 in favour of product 4a (Scheme 3). Using these optimized conditions, the reaction was then carried out the rearrangement with enantiopure cyclopropane 2a. To our delight the expected cyclobutenoates 4a and 4b were obtained in identical global yield (46%) and regioisomeric ratio, along with a complete stereoretention of the absolute configuration for each regioisomer. Similarly, the sequence was then applied to the enantiopure cyclopropanes 2b and 2c, which were converted into cyclobutenoates 5 and 6 with identical efficiency (Scheme 3).

Scheme 3.

Stereoselectivity retention through photochemical ring expansion reaction. Reaction conditions: a) TsNHNH2, PhMe, reflux, 5h; b) DBU, DCM, 16h; c) MeCN, blue light 425 nm (Hepatochem), 3h 30min.

Regioisomeric ratios were determined on ¹H NMR crude.

In addition to this unprecedented photochemical rearrangement of α-diazocyclopropanes, we examined further functionalization of the pyruvate unit to gain access to other functionalized cyclopropanes. First, the ketoester residue of 2a was reduced in the presence of LiAlH₄ in high yield (97%), giving the diol 7 as a 2:1 mixture of diasteroisomers without any degradation of the cyclopropane ring. Then, 7 was converted into the α-cyclopropyl epoxide 9a, via the selective activation of the primary hydroxyl group into a tosyl one, followed by epoxide ring formation under basic conditions. The product was obtained in 90% yield, without erosion of the optical purity (Scheme 4). On the other hand, the α-keto group of 2a could be reduced selectively in the presence of NaBH₄ to give the α-hydroxyester 10 as a 60:40 mixture of diastereoisomers. This product was successfully used in a Mitsunobu and Staudinger reactions sequence to afford the corresponding amino-ester 12 as a nearly 1:1 mixture of diastereoisomers, both in an enantiopure form.

Scheme 4.

Synthetic pathway for enantiopure alpha-cyclopropyl epoxide and non-natural amino-ester.

Reaction conditions: a) LiAlH₄, THF; 0 °C, 3 h; b) TsCl, Bu₂SnO (2 eq), DCM, r.t., 16h; c) nBuLi (1 eq), −78 °C, 1 h; d) NaBH₄, EtOH, −10 °C, 30 min; e) PPH₃, DIAD, DPPA, pyridine, 0 °C to r.t., 16h; f) PPh₃, PhMe, 60 °C then H₂O, 6 h

Conclusions

In summary, we have developed an efficient and stereoselective biocatalytic strategy for the synthesis of pyruvate containing cyclopropanes, using engineered myoglobin as the catalyst and ethyl α-diazopyruvate as carbene precursor. This method provides a scalable access to these enantioenriched cyclopropanes by chemoenzymatic routes, which can be used as valuable building blocks to access different cyclopropane derivatives. Interestingly, the enzymatic cyclopropanation sequence was coupled to the photoinduced ring expansion to access optically active cyclobutenes in high enantiomeric excess and good yields.