Use of a Robust Dehydrogenase from an Archael Hyperthermophile in Asymmetric Catalysis−Dynamic Reductive Kinetic Resolution Entry into (S)-Profens

Abstract

Described is an efficient heterologous expression system for Sulfolobus solfataricus ADH-10 (Alcohol Dehydrogenase isozyme 10) and its use in the dynamic reductive kinetic resolution (DYRKR) of 2-arylpropanal (Profen-type) substrates. Importantly, among the 12 aldehydes tested, a general preference for the (S)-antipode was observed, with high ee’s for substrates corresponding to the NSAIDs (nonsteroidal anti-inflammatory drugs) naproxen, ibuprofen, flurbiprofen, ketoprofen, and fenoprofen. To our knowledge, this is the first application of a dehydrogenase from this Sulfolobus hyperthermophile to asymmetric synthesis and the first example of a DYRKR with such an enzyme. The requisite aldehydes are generated by Buchwald−Hartwig-type Pd(0)-mediated α-arylation of tert-butyl propionate. This is followed by reduction to the aldehyde in one [lithium diisobutyl tert-butoxyaluminum hydride (LDBBA)] or two steps [LAH/Dess−Martin periodinane]. Treatment of the profenal substrates with SsADH in 5% EtOH/phosphate buffer, pH 9, with catalytic NADH at 80 °C leads to efficient DYRKR, with ee’s exceeding 90% for 9 aryl side chains, including those of the aforementioned NSAIDs. An in silico model, consistent with the observed broad side chain tolerance, is presented. Importantly, the SsADH-10 enzyme could be conveniently recycled by exploiting the differential solubility of the organic substrate/product at 80 °C and at rt. Pleasingly, SsADH-10 could be taken through several “thermal cycles,” without erosion of ee, suggesting this as a generalizable approach to enzyme recycling for hyperthermophilic enzymes. Moreover, the robustness of this hyperthermophilic DH, in terms of both catalytic activity and stereochemical fidelity, speaks for greater examination of such archaeal enzymes in asymmetric synthesis.

Hyperthermophilic archaea are of great interest in evolutionary microbiology, owing to their ability to withstand high temperatures and often extremes of pressure, pH, and salinity. Enzymes from these organisms¹ may offer particular opportunities for asymmetric synthesis, complementary to approaches with mesophilic enzymes,² or those involving enzyme³ and pathway⁴ reengineering. However, perhaps due to a bias that hyperthermophilic enzymes have “narrow substrate specificities,”⁵ archaeal extremophiles remain a largely untapped resource in asymmetric synthesis.⁶

Herein, we disclose a remarkably general Dynamic Reductive Kinetic Resolution (DYRKR) entry into (S)-profens, including several important NSAIDs. The enzyme employed is alcohol dehydrogenase (ADH)-10, one of 13 annotated ADHs in the hyperthermophile Sulfolobus solfataricus. Protein phylogenetic analysis of this paralogous family indicates SsADH-10 is most closely related to homologues in distant taxa (Figure 1). The highest identity between SsADH-10 and any other SsADHs is only 34%, suggesting that the SsADH family was established prior to the emergence of other archaeal lineages. Though not described as such, the SsADH-10 appears to be the only SsADH isozyme for which structural information is available in the pdb.⁷

Figure 1.

Protein phylogeny of the SsADH proteins. A consensus neighbor joining distance tree is shown of all SsADHs and homologues of highest sequence identity in related taxa. Distances are indicated by the bar (lower left corner) and represent 10 substitutions per 100 residues. Percent occurrence among 100 trees was greater than 50% for all nodes except those indicated with an asterisk.

The requisite 2-arylpropionaldehydes were readily assembled via Pd(0)-catalyzed arylation of tert-butyl propionate under Buchwald−Hartwig-type⁸ conditions, followed by reduction to the aldehyde (LDBBA⁹ or LAH/DMP oxid; see Supporting Information (SI)). Optimal DYRKR conditions (Table 1, 80 °C, pH 9) led to efficient throughput of rac-aldehyde to the (S)-2-arylpropionaldehyde, particularly with m- and p-substitution. Notably, (S)-profenols corresponding to the NSAIDs naproxen (3b, scaled to 1 g @ 98% yield and 95% ee), ibuprofen (3d, IP), flurbiprofen (3h, FlP), fenoprofen (3j, FP), and ketoprofen (3l, KP) were obtained in excellent yields (up to 96%) and high enantioselectivity (up to 99%).

Table 1. SsADH10-Mediated DYRKR Entry into Profenols.

^aDYRKR performed on a 1 mmol scale (1 mol % NADH; 5 vol% EtOH).

^bIsolated yields.

^cee’s by chiral LC or GC. Blue - Profen drug precursor.

Naproxen is FDA-approved as the active (S)-antipode. While most individuals can invert (R)-ibuprofen to the (S)-antipode, the pathway is inefficient for KP^10a and FlP.^10b Moreover, the recent observation that the Profen-CoA thioester intermediates in this pathway inhibit G6PDH^10c argues for “chiral switching” to single (S)-antipodes.^10d Entries into (S)-profens^11,12 include asymmetric hydrogenation (NP 98% ee, IP 97% ee)^11g and hydroformylation (IP, 92% ee).^11f DKR processes include enantioselective crystallization (NP >99% ee),^11b DYRKR with H₂ as reductant under Ru(II) catalysis (IP 92% ee),^11d and lipase/Ru(II)-mediated DKR of allylic acetates, followed by Cu-mediated Grignard arylation (FlP 97% ee;^11c Knochel arylation:^11e IP 97% ee). The hydrovinylation/oxidation approach is impressive (IP, FP, FlP, NP >96% ee),^11a but access to KP requires late stage arylation. Thus, the broad side chain tolerance of SsADH-10 makes the method presented here among the most generally (S)-selective.

To explore how these extended hydrophobic substrates bind to SsADH-10, docking was carried out (Figure 2) for the (S)-antipodes of flurbiprofenal, naproxenal, ketoprofenal, and fenoprofenal. A detailed discussion of the approach and results is provided in the SI. Briefly, W95 is seen as enforcing (S)-selectivity, with ligands clustering into two distinct distal ring binding modes. “Channel-gating” L272 and L295 appear to form a hydrophobic pocket for naproxenal and flurbiprofenal. For the more flexible ketoprofenal and fenoprofenal, edge-to-face π-π-interactions with W117 and F49 are proposed.

Figure 2.

Structures of thermally relaxed (GROMACS 4.07) SsADH-10 (from 1R37) to which has been docked (Autodock Vina, left to right): (i) (S)-flurbiprofenal, (ii) (S)-naproxenal, (iii) (S)-ketoprofenal, and (iv) (S)-fenoprofenal (Zn ligation sphere: H68, C38, C154, and substrate carbonyl).

From a practical viewpoint, we have also found that SsADH-10 may be engaged in a “thermal recycling” approach that may be generalizable to other hyperthermophilic enzymes. Namely, while 30 vol% cosolvent is often needed to dissolve hydrophobic DH substrates,¹³ we use a higher T (80 °C) @ just 5% EtOH (solvent and biorenewable reductant). Importantly, upon completion of the reaction, cooling to rt allows the product to precipitate and be collected by filtration (see TOC graphic and SI). Reclaimed SsADH may be recycled (5 cycles @ 94−96% ee). Given the growing interest in thermophilic enzymes in synthesis,^1,14 and in engineering thermostability into mesophilic enzymes,¹⁵ this “thermal switching” approach is likely to find broad application, well beyond the domain of geothermal dehydrogenases.

Supporting Information Available

Details of SsADH-10 expression, synthesis, spectra, DYRKR, and modeling. This material is available free of charge via the Internet at http://pubs.acs.org.

Funding Statement

National Institutes of Health, United States