Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis.

Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues.

Fluorescent Probes for Monitoring Serine Ubiquitination.

In a radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of effectors utilize NAD to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism.

Enzymatic Syntheses and Applications of Fluorescent Cyclic Dinucleotides.

Bacterial cyclic dinucleotides (CDNs) play important roles in regulating biofilm formation, motility and virulence. In eukaryotic cells, theses bacterial CDNs are recognized as pathogen-associated molecular patterns (PAMPs) and trigger an innate immune response. We report the photophysical analyses of a novel group of enzymatically synthesized emissive CDN analogues comprised of two families of isomorphic ribonucleotides.

Bio-inspired synthesis of xishacorenes A, B, and C, and a new congener from fuscol.

The xishacorene natural products are structurally unique apolar diterpenoids that feature a bicyclo[3.3.1] framework.

Fluorescing Isofunctional Ribonucleosides: Assessing Adenosine Deaminase Activity and Inhibition.

The enzymatic conversion of isothiazolo[4,3-d]pyrimidine-based adenosine ( A) and 2-aminoadenosine ( 2-AA) analogues to the corresponding isothiazolo[4,3-d]pyrimidine-based inosine ( I) and guanosine ( G) derivatives is evaluated and compared to the conversion of native adenosine to inosine. Henri-Michaelis-Menten analyses provides the foundation for a high-throughput screening assay, and the efficacy of the assay is showcased by fluorescence-based analysis of A conversion to I in the presence of known and newly synthesized inhibitors.

Total Synthesis of (-)-Xishacorene B from ( R)-Carvone Using a C-C Activation Strategy.

The activation of C-C bonds that are traditionally viewed as unreactive, when coupled with other bond-forming processes, can offer new approaches to the synthesis of complex molecular scaffolds. In this Communication, we demonstrate the conversion of carvone to unusual bicyclo[3.3.

Emissive Synthetic Cofactors: Enzymatic Interconversions of A Analogues of ATP, NAD , NADH, NADP , and NADPH.

A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3-d]pyrimidine analogue of adenosine ( A), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and ATP to yield N AD , which can be enzymatically phosphorylated by NAD kinase and ATP or ATP to the corresponding N ADP . The latter can be engaged in NADP-specific coupled enzymatic transformations involving conversion to N ADPH by glucose-6-phosphate dehydrogenase and reoxidation to N ADP by glutathione reductase.

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD Analogue.

The synthesis, photophysics, and biochemical utility of a fluorescent NAD analogue based on an isothiazolo[4,3-d]pyrimidine core (NAD) are described. Enzymatic reactions, photophysically monitored in real time, show NAD and NADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as NAD is converted to NADH, reflecting a complementary photophysical behavior to that of the native NAD/NADH.

Synthesis of unique spirocyclic orthoester-type derivatives of isothiazolo[4,3-d]pyrimidine nucleosides.

A set of unique nucleoside analogs, containing 'spirocyclic orthoester-type' scaffolds, were synthesized from a common isothiazolo[4,3-d]pyrimidine-riboside precursor. The key reaction, using 1,2-di-heteroatomic nucleophiles (e.g.

Expanding a fluorescent RNA alphabet: synthesis, photophysics and utility of isothiazole-derived purine nucleoside surrogates.

A series of emissive ribonucleoside purine mimics, all comprised of an isothiazolo[4,3-]pyrimidine core, was prepared using a divergent pathway involving a key Thorpe-Ziegler cyclization. In addition to an adenosine and a guanosine mimic, analogues of the noncanonical xanthosine, isoguanosine, and 2-aminoadenosine were also synthesized and found to be emissive. Isothiazolo 2-aminoadenosine, an adenosine surrogate, was found to be particularly emissive and effectively deaminated by adenosine deaminase.

Chemical Mutagenesis of an Emissive RNA Alphabet.

An evolved fluorescent ribonucleoside alphabet comprising isomorphic purine ((tz)A, (tz)G) and pyrimidine ((tz)U, (tz)C) analogues, all derived from isothiazolo[4,3-d]pyrimidine as a common heterocyclic core, is described. Structural and biochemical analyses illustrate that the nucleosides, particularly the C-nucleosidic purine analogues, are faithful isomorphic and isofunctional surrogates of their natural counterparts and show improved features when compared to an RNA alphabet derived from thieno[3,4-d]-pyrimidine. The restoration of the nitrogen in a position equivalent to the purines' N7 leads to "isofunctional" behavior, as illustrated by the ability of adenosine deaminase to deaminate (tz)A as effectively as adenosine, the native substrate.