Synthesis of β-Quaternary Lactams Using Photoenzymatic Catalysis

Abstract

Quaternary carbons are useful motifs in chemical synthesis but can be challenging to prepare using many chemical methods. Here, we report a stereoselective synthesis of β-quaternary lactams using flavin-dependent ‘ene’-reductases via a 5-exo-trig radical cyclization. The products are formed in moderate to good levels of enantioselectivity using an ‘ene’-reductase variant from Zymomonas mobilis. This method highlights the opportunity for biocatalysis to form quaternary centers using non-natural reactions.

Graphical Abstract

This manuscript describes the use of photoenzymatic catalysis to prepare lactams bearing quaternary stereocenters. This type of motif can be challenge to prepare stereoselectively, highlighting the opportunity for an enzyme to solve difficult challenges in chemical synthesis.

Introduction

Quaternary carbons are carbon centers fully substituted by four other carbon substituents. The sterically crowded nature around these sites confers conformational rigidity and metabolic stability desirable in bioactive small molecule natural products and pharmacophores.¹ As such, quaternary centers are present in a variety of small molecule drugs (Figure 1a).² Despite their bioactivity, synthesis of quaternary carbons is challenging.³ The same steric factors which promote their bioactivity makes substitution reactions challenging. Moreover, the tertiary radicals or cations formed through dissociative processes are susceptible to undesired side reactions, making them less synthetically attractive. The most well studied catalytic asymmetric methods for preparing quaternary stereocenters involve enolate alkylation or arylation, conjugate additions, asymmetric Tsuji-Trost and Heck reactions, cyclopropanations, and desymmetrizations reactions.⁴ Radical additions to 1,1,2-trisubstituted alkenes are a potentially attractive methods for synthesizing quaternary stereocenters because of their early transitions states which make them amenable to forming congested stereocenters.

Figure 1.

Photoenzymatic Synthesis of Quaternary Stereocenters via Radical Cyclization. (a) Examples of quaternary stereocenter in natural products. (b) Previous studies engineering NCR to prepare α-tertiary amines stereoselectively. (c) Model reaction using NCR and NCR-Y343W-D294D to prepare β-quaternary lactams.

Over the past five years, our group has focused on developing biocatalytic methods for render non-natural radical transformations asymmetric. During these studies, we found that flavin-dependent ‘ene’-reductases (EREDs) were effective catalysts for an array of inter- and intramolecular radical reactions. Mechanistically, these reactions occur via photoexcitation of a charge transfer complex involving the flavin cofactor in its hydroquinone oxidation state (FMN_hq), the alkyl halide, and the π-system with which the resulting radical will react. This approach was originally demonstrated on a 5-exo-trig radical cyclization of α-chloroamides to afford π-lactams in high yield and excellent enantioselectivity.⁴ Building on these results, we found that the alkene can be replaced with an oxime ether to afford γ-lactams with an α-tertiary amines.⁵ While the wild-type enzymes were low yielding with modest selectivity, rounds of iterative site saturation mutagenesis result in variants of the EREDs from Gluconabacter (GluER) and Zymomonas mobilis (NCR) which provide synthetically useful yields and enantioselectivities (Figure 1b).

We hypothesized the steric similarities between a ketoxime and a 1,1,2-trisubstituted alkene could enable the variants engineering for radical addition to oxime ethers might also be effective for stereoselective additions to 1,1,2-trisubstituted alkenes to form γ-lactam products bearing a β-quaternary stereocenter.

We began by looking at the cyclization of the α-chloroamide bearing a 1,1,2-trisubstituted alkene 1a. While many of the EREDs tested failed to catalyze the desired cyclization, wild-type NCR formed the desired product in 16% yield with low levels of enantioselectivity (41:59 e.r.) (Figure 1c). In contrast, the structurally related ketoxime ether formed product with higher enantioselectivity, underlining the subtle differences between these substrates. Next, we tested the engineering NCR variant that provided the highest yields and enantioselectivities for the ketoxime ester (NCR-D294W-Y343W) on substate 1a and found the desired product formed in 68% yield with good levels of enantioselectivity (87:13 e.r.), highlighting the ability to use existing variants on new substrates (Figure 1c).

With this initial result in hand, we examined a collection of structurally related substrates (Figure 2). NCR-D294W-Y343W is tolerant of electron-donating (4-methoxy 2a and 4-methyl 4a) and electron-withdrawing (4-bromo 3a) substituents on the styrene with diminished yields and enantioselectivity. This variant will also accept alkyl substitution at the 2, and 3 position on the arene with modest yield and enantioselectivity. Gratifyingly, we found that replacing the aryl group with a tert-butyl substituent 7a was tolerated, forming product in 91% yield, unfortunately as a racemate.

Figure 2.

Photoenzymatic Radical Cyclization. ^aSubstrate (15 mM), Enzyme (2 mol %), NADP+ (1 mol %), GDH-105 (20% w/w), Glucose (6 equiv.), KPi (100 mM, pH 7.0), 25 °C, 18 h

Pleased with these results, we tested whether this system would be effective for preparing spirocyclic compounds. These [4.5.0] spirocyclic systems are well represented in bioactive natural products and pharmaceutical molecules (Figure 3).⁶ We prepare a tetrasubstituted cyclohexene derivative 8a and subjected it to the reaction conditions found the spirocyclic compound prepare in 91% yield with 1.4:1 d.r. and 91:9 e.r. for the major enantiomer.

Figure 3.

Radical Cyclization to Afford a Spirocyclic Compound. (a) Examples of spirocyclic lactam natural products and drugs. (b) A model for the synthesis of spirocyclic radical cyclization.

Conclusion

In conclusion, we found flavin-dependent ‘ene’-reductases evolved to form α-tertiary amines with high yield and enantioselectivity were also competent at forming sterically related quaternary centers. These chiral quaternary centers are difficult to form, and as such methods for their asymmetric synthesis are underexplored. This work expands the growing body of literature developing methods for photoenzymatic carbon-carbon bond formations enabled by photoexcited EREDs. In a larger sense, the expansion of EREDs selected for formation of α-tertiary amines to quaternary carbons highlights the ability of directed evolution to remodel enzyme active sites to enable a suite of reactivities on related substrates inaccessible to their wild-type precursors.