N–O Tethered Carbenoid Cyclopropanation Facilitates the Synthesis of a Functionalized Cyclopropyl-Fused Pyrrolidine

We report a facile approach to a cyclopropyl-fused pyrrolidine, which contains four stereogenic centers, by employing the N–O tethered carbenoid methodology. The synthesis was facilitated by the development of a direct Mitsunobu reaction of alcohols with <i>N</i>-alkyl-<i>N</i>-hydroxyl amides to give diazo precursors, which upon intramolecular cyclopropanation yielded a library of N–O containing cyclopropyl-fused bicyclic intermediates. Elaboration of the N–O moiety of one member of this library resulted in the formation of the desired pyrrolidine ring demonstrating the potential of this methodology for making cyclopropyl-fused heterocycles

Unexpected Complex Formation between Coralyne and Cyclic Diadenosine Monophosphate Providing a Simple Fluorescent Turn-on Assay to Detect This Bacterial Second Messenger

Cyclic diadenosine monophosphate (c-di-AMP) has emerged as an important dinucleotide that is involved in several processes in bacteria, including cell wall remodeling (and therefore resistance to antibiotics that target bacterial cell wall). Small molecules that target c-di-AMP metabolism enzymes have the potential to be used as antibiotics. Coralyne is known to form strong complexes with polyadenine containing eight or more adenine stretches but not with short polyadenine oligonucleotides. Using a panel of techniques (UV, both steady state fluorescence and fluorescence lifetime measurements, circular dichroism (CD), NMR, and Job plots), we demonstrate that c-di-AMP, which contains only two adenine bases is an exception to this rule and that it can form complexes with coralyne, even at low micromolar concentrations. Interestingly, pApA (the linear analog of c-di-AMP that also contains two adenines) or cyclic diguanylate (c-di-GMP, another nucleotide second messenger in bacteria) did not form any complex with coralyne. Unlike polyadenine, which forms a 2:1 complex with coralyne, c-di-AMP forms a higher order complex with coralyne (≥6:1). Additionally, whereas polyadenine reduces the fluorescence of coralyne when bound, c-di-AMP enhances the fluorescence of coralyne. We use the quenching property of halides to selectively quench the fluorescence of unbound coralyne but not that of coralyne bound to c-di-AMP. Using this simple selective quenching strategy, the assay could be used to monitor the synthesis of c-di-AMP by DisA or the degradation of c-di-AMP by YybT. Apart from the practical utility of this assay for c-di-AMP research, this work also demonstrates that, when administered to cells, intercalators might not only associate with polynucleotides, such as DNA or RNA, but also could associate with cyclic dinucleotides to disrupt or modulate signal transduction processes mediated by these nucleotides

Synthesis of (−)-6,7-Dideoxysqualestatin H5 by Carbonyl Ylide Cycloaddition–Rearrangement and Cross-electrophile Coupling

An asymmetric synthesis of (−)-6,7-dideoxysqualestatin H5 is reported. Key features of the synthesis include the following: (1) highly diastereoselective <i>n</i>-alkylation of a tartrate acetonide enolate and subsequent oxidation–hydrolysis to provide an asymmetric entry to a β-hydroxy-α-ketoester motif; (2) facilitation of Rh­(II)-catalyzed cyclic carbonyl ylide formation–cycloaddition by co-generation of keto and diazo functionality through ozonolysis of an unsaturated hydrazone; and (3) stereoretentive Ni-catalyzed Csp³–Csp² cross-electrophile coupling between tricarboxylate core and unsaturated side chain to complete the natural product

Evolved Quorum Sensing Regulator, LsrR, for Altered Switching Functions

In order to carry out innovative complex, multistep synthetic biology functions, members of a cell population often must communicate with one another to coordinate processes in a programmed manner. It therefore follows that native microbial communication systems are a conspicuous target for developing engineered populations and networks. Quorum sensing (QS) is a highly conserved mechanism of bacterial cell–cell communication and QS-based synthetic signal transduction pathways represent a new generation of biotechnology toolbox members. Specifically, the <i>E. coli</i> QS master regulator, LsrR, is uniquely positioned to actuate gene expression in response to a QS signal. In order to expand the use of LsrR in synthetic biology, two novel LsrR switches were generated through directed evolution: an “enhanced” repression and derepression eLsrR and a reversed repression/derepression function “activator” aLsrR. Protein modeling and docking studies are presented to gain insight into the QS signal binding to these two evolved proteins and their newly acquired functionality. We demonstrated the use of the aLsrR switch using a coculture system in which a QS signal, produced by one bacterial strain, is used to inhibit gene expression via aLsrR in a different strain. These first ever AI-2 controlled synthetic switches allow gene expression from the <i>lsr</i> promoter to be tuned simultaneously in two distinct cell populations. This work expands the tools available to create engineered microbial populations capable of carrying out complex functions necessary for the development of advanced synthetic products

Altering the Communication Networks of Multispecies Microbial Systems Using a Diverse Toolbox of AI-2 Analogues

There have been intensive efforts to find small molecule antagonists for bacterial quorum sensing (QS) mediated by the “universal” QS autoinducer, AI-2. Previous work has shown that linear and branched acyl analogues of AI-2 can selectively modulate AI-2 signaling in bacteria. Additionally, LsrK-dependent phosphorylated analogues have been implicated as the active inhibitory form against AI-2 signaling. We used these observations to synthesize an expanded and diverse array of AI-2 analogues, which included aromatic as well as cyclic C-1-alkyl analogues. Species-specific analogues that disrupted AI-2 signaling in <i>Escherichia coli</i> and <i>Salmonella typhimurium</i> were identified. Similarly, analogues that disrupted QS behaviors in <i>Pseudomonas aeruginosa</i> were found. Moreover, we observed a strong correlation between LsrK-dependent phosphorylation of these acyl analogues and their ability to suppress QS. Significantly, we demonstrate that these analogues can selectively antagonize QS in single bacterial strains in a physiologically relevant polymicrobial culture