Search

The construction of hydrindane ring system of coronafacic acid is examined in order to show that the canonical polyketide chain elongation and functionalization encoded by type I modular polyketide synthases (PKSs) has taken place along with cascade reactions during assembly line-like biosynthesis. The latent reactivity within the substrate is disclosed upon elongation in order for cascade reactions to proceed at a rate faster than the functionalization reactions encoded by the catalytic domains within the type I PKS.

CmaE is a commonly used transferase, which helps in moving the aminoacyl groups between various carrier protein domains in the coronamic acid biosynthetic pathway. The analysis shows that CmaE can also be used for generating chemical diversity within a nonribosomalpeptide (NRPS).

A study of the identity of the BINAP (1,1'-binaphthalene-2,2'-diylbis(diphenylphosphine))-ligated palladium complexes present in reactions of amines with aryl halides and rate measurements of these catalytic reactions initiated with pure precatalysts and precatalysts generated in situ from [Pd2(dba)(sub 3)] and BINAP is presented. This mechanism has helped the amine and base to react with [Pd(BINAP)(Ar)(Br)] to form an arylpalladium amido complex and reductive elimination from this amido complex to form the arylamine.

A mechanistic study identifying the role of the diamine in preventing multiple ligation of the amide is described. A copper (I) amidate that can serve as a potential intermediate during N-arylation process is also demonstrated.

A relationship between the steady-state concentration of active Pd and the ligand size of monophosphinobiaryl ligands used in the amination of ary1 chlorides is demonstrated. The catalyst derived from the bulkiest ligand in the series, the tri-i-propyl ligand, exhibits both accelerated rate and the increased stability required for practical application of this reaction.

Kinetic studies under synthetically relevant conditions are carried out to study the mechanism of the amination of bromobenzene with primary and secondary amines using Pd2(dba)(sub 3)/BINAP mixtures. The results show that the reaction mechanism is dependent on the relative concentrations of substrates and also depends upon the catalyst resting state.

Protein motion is intimately linked to enzymatic catalysis, yet the stereoelectronic changes that accompany different conformational states of a substrate are poorly defined. Here we investigate the relationship between conformation and stereoelectronic effects of a scissile amide bond. Structural studies have revealed that the C-terminal glycine of ubiquitin and ubiquitin-like proteins adopts a syn (ψ ∼ 0°) or gauche (ψ ∼ ±60°) conformation upon interacting with deubiquitinases/ubiquitin-like proteases. We used hybrid density functional theory and natural bond orbital analysis to understand how the stereoelectronic effects of the scissile bond change as a function of φ and ψ torsion angles. This led to the discovery that when ψ is between 30° and −30° the scissile bond becomes geometrically and electronically deformed. Geometric distortion occurs through pyramidalization of the carbonyl carbon and amide nitrogen. Electronic distortion is manifested by a decrease in the strength of the donor-acceptor interaction between the amide nitrogen and antibonding orbital (π*) of the carbonyl. Concomitant with the reduction in nN → π* delocalization energy, the sp² hybrid orbital of the carbonyl carbon becomes richer in p-character, suggesting the syn configuration causes the carbonyl carbon hybrid orbitals to adopt a geometry reminiscent of a tetrahedral-like intermediate. Our work reveals important insights into the role of substrate conformation in activating the reactive carbonyl of a scissile bond. These findings have implications for designing potent active site inhibitors based on the concept of transition state analogues. [ABSTRACT FROM AUTHOR]

The diversity of ubiquitin modifications calls for methods to better characterize ubiquitin chain linkage, length, and morphology. Here, we use multiple linear regression analysis coupled with ion mobility mass spectrometry (IM-MS) to quantify the relative abundance of different ubiquitin dimer isomers. We demonstrate the utility and robustness of this approach by quantifying the relative abundance of different ubiquitin dimers in complex mixtures and comparing the results to the standard, bottom-up ubiquitin AQUA method. Our results provide a foundation for using multiple linear regression analysis and IM-MS to characterize more complex ubiquitin chain architectures. [ABSTRACT FROM AUTHOR]

The copper-catalyzed N-arylation of amides, i.e., the Goldberg reaction, is an efficient method for the construction of products relevant to both industry and academic settings. Herein, we present mechanistic details concerning the catalytic and stoichiometric N-arylation of amides. In the context of the catalytic reaction, our findings reveal the importance of chelating diamine ligands in controlling the concentration of the active catalytic species. The consistency between the catalytic and stoichiometric results suggests that the activation of aryl halides occurs through a 1,2-diamine-ligated copper(l) amidate complex. Kinetic studies on the stoichiometric N-arylation of aryl iodides using 1,2-diamine ligated Cu(l) amidates also provide insights into the mechanism of aryl halide activation. [ABSTRACT FROM AUTHOR]

To produce next-generation, shelf-stable biosensors for point-of-care diagnostics, a combination of rugged biomolecular recognition elements, efficient encapsulants, and innocuous deposition approaches is needed. Furthermore, to ensure that the sensitivity and specificity that are inherent to biological recognition elements are maintained in solid-state biosensing systems, site-specific immobilization chemistries must be invoked such that the function of the biomolecule remains unperturbed. In this work, we present a widely applicable strategy to develop robust solid-state biosensors using emergent nanobody (Nb) recognition elements coupled with a vapor-deposited polymer encapsulation layer. As compared to conventional immunoglobulin G antibodies, Nbs are smaller (12-15 kDa as opposed to ~150 kDa), have higher thermal stability and pH tolerance, boast greater ease of recombinant production, and are capable of binding antigens with high affinity and specificity. Photoinitiated chemical vapor deposition affords thin, protective polymer barrier layers over immobilized Nb arrays that allow for retention of Nb activity and specificity after both storage under ambient conditions and complete desiccation. Most importantly, we also demonstrate that vapor-deposited polymer encapsulation of Nb arrays enables specific detection of target proteins in complex heterogeneous samples, such as unpurified cell lysate, which is otherwise challenging to achieve with bare Nb arrays., Competing Interests: The authors declare no competing financial interest.

Active-site directed probes are powerful tools for studying the ubiquitin conjugation and deconjugation machinery. Branched ubiquitin chains have emerged as important proteasome-targeting signals for aggregation-prone proteins and cell cycle regulators. By implementing a new synthetic strategy for the electrophilic warhead, we herein report on the generation and reactivity of a series of branched triubiquitin active-site directed probes. These new tools can be used to dissect the molecular basis of branched chain assembly and disassembly.

Access to well-defined ubiquitin conjugates has been key to elucidating the biochemical functions of proteins in the ubiquitin signaling network. Yet, we have a poor understanding of how deubiquitinases and ubiquitin-binding proteins respond to ubiquitin modifications when anchored to a protein other than ubiquitin or a ubiquitin-like protein. This is due to the difficulty of synthesizing ubiquitinated proteins comprised of native isopeptide bonds. Here we report on the evolution of a deubiquitinase capable of site-specifically modifying itself with defined ubiquitin chains. Following mutagenesis and yeast display screening, we identify a variant of the yeast ubiquitin C-terminal hydrolase Yuh1 that has a 28-fold improvement in the transamidation to hydrolysis ratio relative to the wild type enzyme. The switch in activity enables robust autoubiquitination of a lysine in the crossover loop to form an isopeptide bond. We demonstrate the utility of autoubiquitinating the evolved Yuh1 variant by investigating the consequences of ubiquitin chain anchoring on the activities of other deubiquitinases. Much to our surprise, we find that certain deubiquitinases are exquisitely sensitive to chain anchoring. These results highlight the importance of investigating the biochemical activities of deubiquitinases with both substrate-anchored and unanchored ubiquitin chains.

Small-angle X-ray scattering (SAXS) has become an increasingly popular technique for characterizing the solution ensemble of flexible biomolecules. However, data resulting from SAXS is typically low-dimensional and is therefore difficult to interpret without additional structural knowledge. In theory, molecular dynamics (MD) trajectories can provide this information, but conventional simulations rarely sample the complete ensemble. Here, we demonstrate that accelerated MD simulations can be used to produce higher quality models in shorter time scales than standard simulations, and we present an iterative Bayesian Monte Carlo method that is able to identify multistate ensembles without overfitting. This methodology is applied to several ubiquitin trimers to demonstrate the effect of linkage type on the solution states of the signaling protein. We observe that the linkage site directly affects the solution flexibility of the trimer and theorize that this difference in plasticity contributes to their disparate roles in vivo.

Protein ubiquitination, the covalent attachment of ubiquitin to target proteins, has emerged as one of the most prevalent posttranslational modifications (PTMs), regulating nearly every cellular pathway. The diversity of signaling associated with this particular PTM stems from the myriad ways in which a target protein can be modified by ubiquitin, e.g., monoubiquitin, multi-monoubiquitin, and polyubiquitin linkages. In this Review, we focus on developments in both enzymatic and chemical methods that engender ubiquitin with new chemical and physical properties. Moreover, we highlight how these methods have enabled studies directed toward (i) characterizing enzymes responsible for reversing the ubiquitin modification, (ii) understanding the influence of ubiquitin on protein function and crosstalk with other PTMs, and (iii) uncovering the impact of polyubiquitin chain linkage and length on downstream signaling events.

Several examples of 1,7-dioxaspiro[5.5]undecane spiroketal systems have been synthesized from the common bicyclic intermediate 1 via acid-catalyzed rearrangement. Intermolecular ketalization of C(2) symmetric diene diol 3 with ketone 9 and then desymmetrization by ring-closing metathesis rapidly constructs bicyclic acetal 1. The locked conformation and steric bias of 1 allow stereoselective functionalization of one or both double bonds before spiroketalization.