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

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 (Fig. 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.

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

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 (Fig. 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.

Leveraging enzymes for asymmetric synthesis and new in situ combinatorial screening methods

The Berkowitz group has developed an in situ enzymatic screening (ISES) method that allows for real time kinetic readout on a reaction of interest under biphasic conditions. In the newest iteration of this ISES method, a colorimetric variant has been developed. This approach employs two reporting enzymes, alcohol oxidase and peroxidase, the latter of which utilizes a dye cofactor (ABTS = 2,2\u27-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) giving rise to a green radical cation for reactions releasing an alcoholic (by)product. Using this method, 1152 nucleophile/metal/substrate combinations were screened for a targeted halometalation/carbocyclization transformation. Two new reaction manifolds were identified in this manner; a formal bromorhodiation/carbocyclization reaction and a formal thiocyanopalladation/carbocyclization. In the former case, significant diastereoselectivity was observed for closure to 5- and 6-ring systems in the carbocyclization. A post-cyclization ring-closing metathesis (RCM) step then allows for the synthesis of 5,7-fused xanthanolide core scaffolds, bearing a halovinyl moiety that can be functionalized/extended to decorate the core. The 6-ring manifold of this bromorhodiation/carbocyclization transformation is then exploited for a streamlined entry into the oxabicyclo[4.3.1]decyl exomethylene-δ- lactone cores of linearifolin and zaluzanin A. In this case, the absolute stereochemistry derives from kinetic resolution of 5-benzyloxypentene-1,2-oxide, utilizing a β-pinene-derived-Co(III)-salen catalyst discovered by ISES screening. Post-carbocyclization RCM with the Grubbs-II catalyst yields oxabicyclo[4.3.1]decyl exomethylene-δ-lactone cores. RCM with the Grubbs-I catalyst provides the ring-contracted oxabicyclo[3.3.1]nonyl exomethylene-δ-lactone cores of xerophilusin R and zinagrandinolide. In related work, collaboratively with the Blum group in the NU School of Biological Sciences, new synthetic/screening applications have been discovered for a dehydrogenase from an archaeal hyperthermophile. SsADH-10 ( Sulfolobus solfataricus alcohol dehydrogenase, isozyme-10) was found to be a useful enzyme in asymmetric synthesis; namely for a dynamic reductive kinetic resolution (DYRKR) entry into the (S)-profen family of non-steroidal anti-inflammatory drugs. Interestingly, this enzyme permits for thermal switching, allowing for DYRKR at elevated temperatures and product recovery by simple filtration at room temperature. For screening applications, SsADH-10 is being leveraged for a thermal -variant of ISES. This thermal-ISES has been applied to the exploration of intramolecular allylic aminations at elevated temperatures (50-90 °C), with the aim of developing new, catalytic asymmetric synthetic entries into quaternary, &agr;-vinyl amino acids

Combinatorial Catalysis Assisted by a Visible Enzymatic Beacon in Real Time: New Synthetically Versatile (Pseudo)Halometalation/Carbocyclizations

Combinatorial approaches to catalysis have made an impact in targeted transformation development, including Ag-mediated carbene insertion,[1] Sc-pybox-based asymmetric cyclopropanation,[2] and Rh/Ir-based asymmetric hydrogenation.[3] Useful design elements have emerged from these studies, e.g. the value of ligand self assembly,[4] or of the inclusion of peptide-like structural elements[5],[6],[7] in building ligand arrays. Efficient screening methods are of paramount importance for such efforts. Methods based on fluorescence,[8] REMPI,[9] MS,[10] NMR,[11] and IR thermography[12] have appeared. A chromophore may be installed into the substrate[13] or product[14] of the reaction under study. Alternatively, one can exploit chromophores inherent in proteins[15] or enzyme-associated reactions,[16] and use these sensors to report back on product formation and composition

Halocarbocyclization Entry into the Oxabicyclo[4.3.1]decyl Exomethylene-δ-Lactone Cores of Linearifolin and Zaluzanin A: Exploiting Combinatorial Catalysis

A streamlined entry into the sesquiterpene lactone (SQL) cores of linearifolin and zaluzanin A is described. Stereochemistry is controlled through transformations uncovered by ISES (In Situ Enzymatic Screening). Absolute stereochemistry derives from kinetic resolution of 5-benzyloxypentene-1,2-oxide, utilizing a β-pinene-derived-Co(III)-salen. Relative stereochemistry (1,3-cis-fusion) is set via formal halometalation/carbocyclization, mediated by [Rh(O₂CC₃F₇)₂]₂/LiBr. Subsequent ring-closing metathesis (RCM-Grubbs II) yields the title exomethylene-δ-lactone SQL cores. In complementary fashion, RCM with Grubbs-I catalyst provides the oxabicyclo[3.3.1]nonyl core of xerophilusin R and zinagrandinolide

Halocarbocyclization Entry into the Oxabicyclo[4.3.1]decyl Exomethylene-δ-Lactone Cores of Linearifolin and Zaluzanin A - Exploiting Combinatorial Catalysis

A streamlined entry into the sesquiterpene lactones (SQL) cores of linearifolin and zaluzanin A is described. Stereochemistry is controlled through transformations uncovered by ISES (In-Situ- Enzymatic-Screening). Absolute stereochemistry derives from kinetic resolution of 5- benzyloxypentene-1,2-oxide, utilizing a β-pinene-derived-Co(III)-salen. Relative stereochemistry (1,3-cis-fusion)is set via formal halometalation/carbocyclization, mediated by [Rh(O2CC3F7)2]2/ LiBr. Subsequent ring-closing metathesis (RCM-Grubbs II) yields the title exomethylene-δ- lactone SQL-cores. In complementary fashion, RCM with Grubbs-I catalyst provides the oxabicyclo[3.3.1]nonyl-core of xerophilusin R and zinagrandinolide