P450 Fingerprinting Method for Rapid Discovery of Terpene Hydroxylating P450 Catalysts with Diversified Regioselectivity

Engineered P450 enzymes constitute attractive catalysts for the selective oxidation of unactivated C−H bonds in complex molecules. A current bottleneck in the use of P450 catalysis for chemical synthesis is the time and effort required to identify the P450 variant(s) with the desired level of activity and selectivity. In this report, we describe a method to map the active site configuration of engineered P450 variants in high throughput using a set of semisynthetic chromogenic probes. Through analysis of the resulting ‘fingerprints’, reliable predictions can be made regarding the reactivity of these enzymes toward complex substrates structurally related to the fingerprint probes. In addition, fingerprint analysis offers a convenient and time-effective means to assess the regioselectivity properties of the fingerprinted P450s. The described approach can represent a valuable tool to expedite the discovery of P450 oxidation catalysts for the functionalization of relevant natural products such as members of the terpene family

Intramolecular Hydrogen Bond-Controlled Prolyl Amide Isomerization in Glucosyl 3′(<i>S</i>)-Hydroxy-5′-hydroxymethylproline Hybrids: Influence of a <i>C</i>-5′-Hydroxymethyl Substituent on the Thermodynamics and Kinetics of Prolyl Amide <i>Cis</i>/<i>Trans</i> Isomerization

Peptide mimics containing spirocyclic glucosyl-(3′-hydroxy-5′-hydroxymethyl)proline hybrids (Glc3′(S)-5′(CH2OH)HypHs) with a polar hydroxymethyl substituent at the C-5′ position, such as C-terminal ester Ac-Glc3′(S)-5′(CH2OH)Hyp-OMe and C-terminal amide Ac-Glc3′(S)-5′(CH2OH)Hyp-N′-CH3, were synthesized. C-Terminal esters exhibit increased cis population (23−53%) relative to Ac-3(S)HyPro-OMe (17%) or Ac-Pro-OMe (14%) in D2O. The prolyl amide cis population is further increased to 38−74% in the C-terminal amide form in D2O. Our study shows that the stereochemistry of the hydroxymethyl substituent at the C-5′ position of proline permits tuning of the prolyl amide cis/trans isomer ratio. Inversion−magnetization transfer NMR experiments indicate that the stereochemistry of the hydroxymethyl substituent has a dramatic effect on the kinetics of prolyl amide cis/trans isomerization. A 200-fold difference in the trans-to-cis (ktc) isomerization and a 90-fold rate difference in the cis-to-trans (kct) isomerization is observed between epimeric C-5′ 3 and 4. When compared to reference peptide mimics Ac-Pro-OMe and Ac-3(S)Hyp-OMe, our study demonstrates that a (13−16)-fold decrease in ktc and kct is observed for the C-5′(S), while a (5−24)-fold acceleration is observed for the C-5′(R) epimer. DFT calculations indicate that the pyrrolidine ring prefers a Cβ exo pucker in both Ac-Glc3′(S)-5′(CH2OH)Hyp-OMe diastereoisomers. Computational calculations and chemical shift temperature coefficient (Δδ/ΔT) experiments indicate that the hydroxymethyl group at C-5′ in Ac-Glc3′(S)-5′(CH2OH)Hyp-OMe forms a stabilizing intramolecular hydrogen bond to the carbonyl of the N-acetyl group in both epimeric cis isomers. However, a competing intramolecular hydrogen bond between the hydroxymethyl groups in the pyrrolidine ring and pyran ring stabilizes the trans isomer in the C-5′(S) diastereoisomer. The dramatic differences in the kinetic properties of the diastereoisomeric peptide mimics are rationalized by the presence or absence of an intramolecular hydrogen bond between the hydroxymethyl substituent located at C-5′ and the developing lone pair on the nitrogen atom of the N-acetyl group in the transition state

Intramolecular Hydrogen Bond-Controlled Prolyl Amide Isomerization in Glucosyl 3(<i>S</i>)-Hydroxy-5-hydroxymethylproline Hybrids: A Computational Study

Peptide mimics containing spirocyclic glucosyl-(3(S)-hydroxy-5(S)-hydroxymethyl)proline (1) and glucosyl-(3(S)-hydroxy-5(R)-hydroxymethyl)proline (2) hybrids differing in the stereochemistry of the polar hydroxymethyl substitutent at the C-5- or (Cδ)-position have been investigated computationally. A computational “build and search” protocol of molecular mechanics systematic search/Monte Carlo search, followed by density functional theory (DFT), has been developed to ensure complete coverage of the large conformational space. Gas-phase DFT optimizations at the B3LYP level of theory lead to a strong preference for the cis conformation in the prolyl amide bond for both compounds 1 and 2. However, inclusion of the solvent water by means of continuum solvation (PCM) results in a reduction of the prolyl amide cis population in both compounds, leading to good agreement with previous experimental observations. Intramolecular hydrogen bonding involving the C-5-hydroxymethyl substitutent is seen to play a crucial role to tune the thermodynamics of prolyl amide cis/trans isomerization and is responsible for the high cis prolyl amide population in compound 2. Our results indicate that H-bond-forming substituents like the hydroxymethyl group at the C-5-position in proline can be used to control cis/trans prolyl amide isomerization. High cis prolyl amide conformer populations can be achieved by proper choice of the stereochemistry at the C-5-position

Controlled Oxidation of Remote sp³ C–H Bonds in Artemisinin via P450 Catalysts with Fine-Tuned Regio- and Stereoselectivity

The selective oxyfunctionalization of isolated sp³ C–H bonds in complex molecules represents a formidable challenge in organic chemistry. Here, we describe a rational, systematic strategy to expedite the development of P450 oxidation catalysts with refined regio- and stereoselectivity for the hydroxylation of remote, unactivated C–H sites in a complex scaffold. Using artemisinin as model substrate, we demonstrate how a three-tier strategy involving first-sphere active site mutagenesis, high-throughput P450 fingerprinting, and fingerprint-driven P450 reactivity predictions enabled the rapid evolution of three efficient biocatalysts for the selective hydroxylation of a primary and a secondary C–H site (with both <i>S</i> and <i>R</i> stereoselectivity) in a relevant yet previously inaccessible region of this complex natural product. The evolved P450 variants could be applied to provide direct access to the desired hydroxylated derivatives at preparative scales (0.4 g) and in high isolated yields (>90%), thereby enabling further elaboration of this molecule. As an example, enantiopure C7-fluorinated derivatives of the clinical antimalarial drugs artesunate and artemether, in which a major metabolically sensitive site is protected by means of a C–H to C–F substitution, were afforded via P450-mediated chemoenzymatic synthesis

Development of a Nitric Oxide-Releasing Analogue of the Muscle Relaxant Guaifenesin for Skeletal Muscle Satellite Cell Myogenesis

Nitric oxide (NO) mediates activation of satellite precursor cells to enter the cell cycle. This provides new precursor cells for skeletal muscle growth and muscle repair from injury or disease. Targeting a new drug that specifically delivers NO to muscle has the potential to promote normal function and treat neuromuscular disease, and would also help to avoid side effects of NO from other treatment modalities. In this research, we examined the effectiveness of the NO donor, iosorbide dinitrate (ISDN), and a muscle relaxant, methocarbamol, in promoting satellite cell activation assayed by muscle cell DNA synthesis in normal adult mice. The work led to the development of guaifenesin dinitrate (GDN) as a new NO donor for delivering nitric oxide to muscle. The results revealed that there was a strong increase in muscle satellite cell activation and proliferation, demonstrated by a significant 38% rise in DNA synthesis after a single transdermal treatment with the new compound for 24 h. Western blot and immunohistochemistry analyses showed that the markers of satellite cell myogenesis, expression of myf5, myogenin, and follistatin, were increased after 24 h oral administration of the compound in adult mice. This research extends our understanding of the outcomes of NO-based treatments aimed at promoting muscle regeneration in normal tissue. The potential use of such treatment for conditions such as muscle atrophy in disuse and aging, and for the promotion of muscle tissue repair as required after injury or in neuromuscular diseases such as muscular dystrophy, is highlighted