Divergent Cascade Ring-Expansion Reactions of Acryloyl Imides.

Macrocyclic and medium-sized ring ketones, lactones and lactams can all be made from common acryloyl imide starting materials through divergent, one-pot cascade ring-expansion reactions. Following either conjugate addition with an amine or nitromethane, or osmium(VIII)-catalysed dihydoxylation, rearrangement through a four-atom ring expansion takes place spontaneously to form the ring expanded products. A second ring expansion can also be performed following a second iteration of imide formation and alkene functionalisation/ring expansion. In the dihydroxylation series, three- or four-atom ring expansion can be performed selectively, depending on whether the reaction is under kinetic or thermodynamic control.

Preparative scale Achmatowicz and aza-Achmatowicz rearrangements catalyzed by Agrocybe aegerita unspecific peroxygenase.

The unspecific peroxygenase (UPO) from Agrocybe aegerita (rAaeUPO-PaDa-I-H) is an effective and practical biocatalyst for the oxidative expansion of furfuryl alcohols/amines on a preparative scale, using the Achmatowicz and aza-Achmatowicz reaction. The high activity and stability of the enzyme, which can be produced on a large scale as an air-stable lyophilised powder, renders it a versatile and scalable biocatalyst for the preparation of synthetically valuable 6-hydroxypyranones and dihydropiperidinones. In several cases, the biotransformation out-performed the analogous chemo-catalysed process, and operates under milder and greener reaction conditions.

Preparative-Scale Biocatalytic Oxygenation of N-Heterocycles with a Lyophilized Peroxygenase Catalyst.

A lyophilized preparation of an unspecific peroxygenase variant from Agrocybe aegerita (rAaeUPO-PaDa-I-H) is a highly effective catalyst for the oxygenation of a diverse range of N-heterocyclic compounds. Scalable biocatalytic oxygenations (27 preparative examples, ca. 100 mg scale) have been developed across a wide range of substrates, including alkyl pyridines, bicyclic N-heterocycles and indoles. H2 O2 is the only stoichiometric oxidant needed, without auxiliary electron transport proteins, which is key to the practicality of the method. Reaction outcomes can be altered depending on whether hydrogen peroxide was delivered by syringe pump or through in situ generation using an alcohol oxidase from Pichia pastoris (PpAOX) and methanol as a co-substrate. Good synthetic yields (up to 84 %), regioselectivity and enantioselectivity (up to 99 % ee) were observed in some cases, highlighting the promise of UPOs as practical, versatile and scalable oxygenation biocatalysts.

Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT)*.

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (Ki =38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.

A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate.

Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structure-guided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.

Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT).

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (K i=38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.

(±)-trans-Dihydronarciclasine and (±)-trans-dihydrolycoricidine analogues modified in their ring A: Evaluation of their anticancer activity and a SAR study.

A series of (±)-trans-dihydronarciclasine and (±)-trans-dihydrolycoricidine derivatives with variously substituted ring A was synthesised and evaluated for their antiproliferative activity against 60 human tumour cell lines (NCI60), representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate, as well as kidney in vitro. Among the 13 alkaloids screened, (±)-trans-dihydronarciclasine showed the highest potency as a cytotoxic molecule. A structure-activity relationship (SAR) study indicated that the presence of a hydroxy group at position 7 and a rigid, 1,3-benzodioxole scaffold were essential for the antiproliferative activity.

Stereoselective synthesis of trans-dihydronarciclasine derivatives containing a 1,4-benzodioxane moiety.

ABSTRACT: Some new trans-dihydronarciclasine derivatives containing a 1,4-benzodioxane moiety were stereoselectively synthesised using our feasible and efficient method developed recently. These new phenanthridone alkaloid analogues were obtained in both racemic and optically active forms. High enantioselectivities (up to 99% ee) were achieved by applying (8S,9S)-9-amino(9-deoxy)epiquinine as an organocatalyst. Due to a side reaction, various methoxyphenanthridine regioisomers were also prepared which afforded further synthetic trans-dihydronarciclasine analogues modified in the ring A of the phenanthridone scaffold.