Biomacromolecule-Assisted Screening for Reaction Discovery and Catalyst Optimization.

Reaction discovery and catalyst screening lie at the heart of synthetic organic chemistry. While there are efforts at de novo catalyst design using computation/artificial intelligence, at its core, synthetic chemistry is an experimental science. This review overviews biomacromolecule-assisted screening methods and the follow-on elaboration of chemistry so discovered. All three types of biomacromolecules discussed─enzymes, antibodies, and nucleic acids─have been used as "sensors" to provide a readout on product chirality exploiting their native chirality. Enzymatic sensing methods yield both UV-spectrophotometric and visible, colorimetric readouts. Antibody sensors provide direct fluorescent readout upon analyte binding in some cases or provide for cat-ELISA (Enzyme-Linked ImmunoSorbent Assay)-type readouts. DNA biomacromolecule-assisted screening allows for templation to facilitate reaction discovery, driving bimolecular reactions into a pseudo-unimolecular format. In addition, the ability to use DNA-encoded libraries permits the barcoding of reactants. All three types of biomacromolecule-based screens afford high sensitivity and selectivity. Among the chemical transformations discovered by enzymatic screening methods are the first Ni(0)-mediated asymmetric allylic amination and a new thiocyanopalladation/carbocyclization transformation in which both C-SCN and C-C bonds are fashioned sequentially. Cat-ELISA screening has identified new classes of sydnone-alkyne cycloadditions, and DNA-encoded screening has been exploited to uncover interesting oxidative Pd-mediated amido-alkyne/alkene coupling reactions.

Crystal Structures of Cystathionine ß-Synthase from Saccharomyces cerevisiae: One Enzymatic Step at a Time.

Cystathionine ß-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of H2S biogenesis. Roles for CBS have been reported for neuronal death pursuant to cerebral ischemia, promoting ovarian tumor growth, and maintaining drug-resistant phenotype by controlling redox behavior and regulating mitochondrial bioenergetics. The trans-sulfuration pathway is well-conserved in eukaryotes, but the analogous enzymes have different enzymatic behavior in different organisms. CBSs from the higher organisms contain a heme in an N-terminal domain. Though the presence of the heme, whose functions in CBSs have yet to be elucidated, is biochemically interesting, it hampers UV-vis absorption spectroscopy investigations of pyridoxal 5'-phosphate (PLP) species. CBS from Saccharomyces cerevisiae (yCBS) naturally lacks the heme-containing N-terminal domain, which makes it an ideal model for spectroscopic studies of the enzymological reaction catalyzed and allows structural studies of the basic yCBS catalytic core (yCBS-cc). Here we present the crystal structure of yCBS-cc, solved to 1.5 Å. Crystal structures of yCBS-cc in complex with enzymatic reaction intermediates have been captured, providing a structural basis for residues involved in catalysis. Finally, the structure of the yCBS-cc cofactor complex generated by incubation with an inhibitor shows apparent off-pathway chemistry not normally seen with CBS.

Synthesis and Deployment of an Elusive Fluorovinyl Cation Equivalent: Access to Quaternary α-(1'-Fluoro)vinyl Amino Acids as Potential PLP Enzyme Inactivators.

Developing specific chemical functionalities to deploy in biological environments for targeted enzyme inactivation lies at the heart of mechanism-based inhibitor development but also is central to other protein-tagging methods in modern chemical biology including activity-based protein profiling and proteolysis-targeting chimeras. We describe here a previously unknown class of potential PLP enzyme inactivators; namely, a family of quaternary, α-(1'-fluoro)vinyl amino acids, bearing the side chains of the cognate amino acids. These are obtained by the capture of suitably protected amino acid enolates with ß,ß-difluorovinyl phenyl sulfone, a new (1'-fluoro)vinyl cation equivalent, and an electrophile that previously eluded synthesis, capture and characterization. A significant variety of biologically relevant AA side chains are tolerated including those for alanine, valine, leucine, methionine, lysine, phenylalanine, tyrosine, and tryptophan. Following addition/elimination, the resulting transoid α-(1'-fluoro)-ß-(phenylsulfonyl)vinyl AA-esters undergo smooth sulfone-stannane interchange to stereoselectively give the corresponding transoid α-(1'-fluoro)-ß-(tributylstannyl)vinyl AA-esters. Protodestannylation and global deprotection then yield these sterically encumbered and densely functionalized quaternary amino acids. The α-(1'-fluoro)vinyl trigger, a potential allene-generating functionality originally proposed by Abeles, is now available in a quaternary AA context for the first time. In an initial test of this new inhibitor class, α-(1'-fluoro)vinyllysine is seen to act as a time-dependent, irreversible inactivator of lysine decarboxylase from Hafnia alvei. The enantiomers of the inhibitor could be resolved, and each is seen to give time-dependent inactivation with this enzyme. Kitz-Wilson analysis reveals similar inactivation parameters for the two antipodes, L-α-(1'-fluoro)vinyllysine (Ki = 630 ± 20 µM; t1/2 = 2.8 min) and D-α-(1'-fluoro)vinyllysine (Ki = 470 ± 30 µM; t1/2 = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.

"Zipped Synthesis" by Cross-Metathesis Provides a Cystathionine ß-Synthase Inhibitor that Attenuates Cellular H2S Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model.

The gaseous neuromodulator H2S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine ß-synthase (CBS) is the primary mediator of H2S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a "zipped" approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-C 2-symmetric CBS product (l,l)-cystathionine. The "zipped" concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine-imine interchange. It is demonstrated that the most potent "zipped" inhibitor 6S reduces H2S production in SH-SY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor 6S dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia.

A useful methoxyvinyl cation equivalent: α-t-butyldimethylsilyl-α-methoxyacetaldehyde.

Described are the synthesis and application of α-t-butyldimethylsilyl-α-methoxyacetaldehyde as a formal methoxyvinyl cation equivalent. Addition of Grignard reagents to the title aldehyde, followed by treatment of the intermediate ß-hydroxysilanes with KH, gives good yields of large Z-methoxyvinylated products. Assuming a Peterson-like elimination mechanism, one can infer that the Grignard addition proceeds with high syn selectivity. These results are consistent with a chelation control model involving coordination to the α-methoxy group in the title aldehyde rather than an alternative stereoelectronic Felkin-Anh-type model. It must be noted that a steric Felkin-Anh model also accounts for the observed stereochemistry. All told, the title reagent can be employed to efficiently append a Z-configured methoxyvinyl group to an appropriate R-M species, in two steps.

Use of Fluorinated Functionality in Enzyme Inhibitor Development: Mechanistic and Analytical Advantages.

On the one hand, owing to its electronegativity, relatively small size, and notable leaving group ability from anionic intermediates, fluorine offers unique opportunities for mechanism-based enzyme inhibitor design. On the other, the "bio-orthogonal" and NMR-active 19-fluorine nucleus allows the bioorganic chemist to follow the mechanistic fate of fluorinated substrate analogues or inhibitors as they are enzymatically processed. This article takes an overview of the field, highlighting key developments along these lines. It begins by highlighting new screening methodologies for drug discovery that involve appropriate tagging of either substrate or the target protein itself with (19)F-markers, that then report back on turnover and binding, respectively, via an the NMR screen. Taking this one step further, substrate-tagging with fluorine can be done is such a manner as to provide stereochemical information on enzyme mechanism. For example, substitution of one of the terminal hydrogens in phosphoenolpyruvate, provides insight into the, otherwise latent, facial selectivity of C-C bond formation in KDO synthase. Perhaps, most importantly, from the point of view of this discussion, appropriately tailored fluorinated functionality can be used to form to stabilized "transition state analogue" complexes with a target enzymes. Thus, 5-fluorinated pyrimidines, alpha-fluorinated ketones, and 2-fluoro-2-deoxysugars each lead to covalent adduction of catalytic active site residues in thymidylate synthase, serine protease and glycosidase enzymes, respectively. In all such cases, (19)F NMR allows the bioorganic chemist to spectrally follow "transition state analogue" formation. Finally, the use of specific fluorinated functionality to engineer "suicide substrates" is highlighted in a discussion of the development of the alpha-(2'Z-fluoro)vinyl trigger for amino acid decarboxylase inactivation. Here (19)F NMR allows the bioorganic chemist to glean useful partition ratio data directly out of the NMR tube.