ortho-Boronic Acid Carbonyl Compounds and Their Applications in Chemical Biology.

Iminoboronates and diazaborines are related classes of compounds that feature an imine ortho to an arylboronic acid (iminoboronate) or a hydrazone that cyclizes with an ortho arylboronic acid (diazaborine). Rather than acting as independent chemical motifs, the arylboronic acid impacts the rate of imine/hydrazone formation, hydrolysis, and exchange with competing nucleophiles. Increasing evidence has shown that the imine/hydrazone functionality also impacts arylboronic acid reactivity toward diols and reactive oxygen and nitrogen species (ROS/RNS). Untangling the communication between C=N linked functionalities and arylboronic acids has revealed a powerful and tunable motif for bioconjugation chemistries and other applications in chemical biology. Here, we survey the applications of iminoboronates and diazaborines in these fields with an eye toward understanding their utility as a function of neighboring group effects.

Interfacing Whole Cell Biocatalysis with a Biocompatible Pictet-Spengler Reaction for One-Pot Syntheses of Tetrahydroisoquinolines and Tryptolines.

Biocatalytic processes are highly selective and specific. However, their utility is limited by the comparatively narrow scope of enzyme-catalysed transformations. To expand product scope, we are developing biocompatible processes that combine biocatalytic reactions with chemo-catalysis in single-flask processes. Here, we show that a chemocatalysed Pictet-Spengler annulation can be interfaced with biocatalysed alcohol oxidation. This two-step, one-pot cascade reaction converts tyramine and aliphatic alcohols to tetrahydroisoquinoline alkaloids in aqueous buffer at mild pH. Tryptamine derivatives are also efficiently converted to tryptolines. Optimization of stoichiometry, pH, reaction time, and whole-cell catalyst deliver the tetrahydroisouinolines and tryptolines in >90 % and >40 % isolated yield, respectively, with excellent regioselectivity.

Connecting the dynamics and reactivity of arylboronic acids to emergent and stimuli-responsive material properties.

Over the past two decades, arylboronic acid-functionalized materials have been used in a variety of sensing and stimuli-responsive scaffolds. Their diverse applications result from the various modes of reactivities of arylboronic acids. Arylboronate ester-crosslinked hydrogels are self-healing because the boronate ester bond is dynamic covalent. The hydrogels degrade in acidic environments because of pH-sensitive boronate ester degradation, in the presence of diols because of reversible boronate ester formation, and in the presence of reactive oxygen species (ROS) because of arylboronic acid oxidation. Connecting small-molecule reactivities and dynamics to mechanical and stimuli-responsive properties enables a better understanding of material properties and informs next-generation material design. Here, we highlight recent advances in arylboronic acid-based networks and nanomaterials and how the fundamental chemistry of arylboronic acids can enhance an understanding of the emergent material properties and improve the rational design of stimuli-responsive materials.

Diazaborines oxidize slowly with H2O2 but rapidly with peroxynitrite in aqueous buffer.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) oxidize arylboronic acids to their corresponding phenols. When used in molecular imaging probes and in ROS-responsive molecules, however, simple arylboronic acids struggle to discriminate between H2O2 and ONOO- because of their fast rate of reaction with both ROS. Here, we show that diazaborines (DABs) react slowly with H2O2 but rapidly with peroxynitrite in an aqueous buffer. In addition to their slow reaction with H2O2, the immediate product of DAB oxidation with H2O2 and ONOO- can yield a kinetically trapped CN Z-isomer that slowly equilibrates with its E-isomer. Taken together, our work shows that diazaborines exhibit enhanced kinetic discrimination between H2O2 and ONOO- compared to arylboronic acids, opening up new opportunities for diazaborine-based tools in chemical biology.

Tuning the exchange dynamics of boronic acid hydrazones and oximes with pH and redox control.

Dynamic bonds continually form and dissociate at equilibrium. Carbonyl compounds with proximal boronic acids, including 2-formylphenylboronic acid (2-FPBA), have been reported to form highly dynamic covalent hydrazone and oxime bonds in physiological conditions, but strategies to tune the dynamics have not yet been reported. Here, we characterize the dynamics of 2-FPBA-derived hydrazones and oximes and account for both the rapid rate of formation (∼102-103 M-1 s-1) and the relatively fast rate of hydrolysis (∼10-4 s-1) at physiological pH. We further show that these substrates undergo exchange with α-nucleophiles, which can be reversibly paused and restarted with pH control. Finally, we show that oxidation of the arylboronic acid effectively abolishes the rapid dynamics, which slows the forward reaction by more than 30 000 times and increases the hydrolytic half-life from 50 minutes to 6 months at physiological pH. These results set the stage to explore these linkages in dynamic combinatorial libraries, reversible bioconjugation, and self-healing materials.

Enhancing Biosynthesis and Manipulating Flux in Whole Cells with Abiotic Catalysis.

Metabolic engineering uses genetic strategies to drive flux through desired pathways. Recent work with electrochemical, photochemical, and chemocatalytic setups has revealed that these systems can also expand metabolic pathways and manipulate flux in whole cells. Electrochemical systems add or remove electrons from metabolic pathways to direct flux to more- or less-reduced products. Photochemical systems act as synthetic light-harvesting complexes and yield artificial photosynthetic organisms. Biocompatible chemocatalysis increases product scope, streamlines syntheses, and yields single-flask processes to deliver products that would be challenging to synthesize through biosynthetic means alone. Here, we exclusively highlight systems that combine abiotic systems with living whole cells, taking particular note of strategies that enable the merger of these typically disparate systems.

Catalyst-free, aza-Michael polymerization of hydrazides: polymerizability, kinetics, and mechanistic origin of an α-effect.

Despite the powerful nature of the aza-Michael reaction for generating C-N linkages and bioactive moieties, the bis-Michael addition of 1° amines remains ineffective for the synthesis of functional, step-growth polymers due to the drastic reduction in reactivity of the resulting 2° amine mono-addition adduct. In this study, a wide range of commercial hydrazides are shown to effectively undergo the bis-Michael reaction with divinyl sulfone (DVS) and 1,6-hexanediol diacrylate (HDA) under catalyst-free, thermal conditions to afford moderate to high molecular weight polymers with M n = 3.8-34.5 kg mol-1. The hydrazide-Michael reactions exhibit two distinctive, conversion-dependent kinetic regimes that are 2nd-order overall, in contrast to the 3rd-order nature of amines previously reported. The mono-addition rate constant was found to be 37-fold greater than that of the bis-addition at 80 °C for the reaction between benzhydrazide and DVS. A significant majority (12 of 15) of the hydrazide derivatives used here show excellent bis-Michael reactivity and achieve >97% conversions after 5 days. This behavior is consistent with calculations that show minimal variance of electron density on the N-nucleophile among the derivatives studied. Reactivity differences between hydrazides and hexylamine are also explored. Overall, the difference in reactivity between hydrazides and amines is attributed to the adjacent nitrogen atom in hydrazides that acts as an efficient hydrogen-bond donor that facilitates intramolecular proton-transfer following the formation of the zwitterion intermediate. This effect not only activates the Michael acceptor but also coordinates with additional Michael acceptors to form an intermolecular reactant complex.

Dynamic and Responsive DNA-like Polymers.

The synthesis of thiolactone monomers that mimic natural nucleosides and engage in robust ring opening polymerizations (ROP) is herein described. As each repeat unit contains a thioester functional group, dynamic rearrangement of the polymer is feasible via thiol-thioester exchange, demonstrated here by depolymerization of the polymers and coalescing of two polymers of different molecular weight or chemical composition. This approach constitutes the first step toward a platform that enables for the routine synthesis of sequence controlled polymers via dynamic template directed synthesis.

New Generation of Clickable Nucleic Acids: Synthesis and Active Hybridization with DNA.

Due to the ability to generate oligomers of precise sequence, sequential and stepwise solid-phase synthesis has been the dominant method of producing DNA and other oligonucleotide analogues. The requirement for a solid support, however, and the physical restrictions of limited surface area thereon significantly diminish the efficiency and scalability of these syntheses, thus, negatively affecting the practical applications of synthetic polynucleotides and other similarly created molecules. By employing the robust photoinitiated thiol-ene click reaction, we developed a new generation of clickable nucleic acids (CNAs) with a polythioether backbone containing repeat units of six atoms, matching the spacing of the phosphodiester backbone of natural DNA. A simple, inexpensive, and scalable route was utilized to produce CNA monomers in gram-scale, which indicates the potential to dramatically lower the cost of these DNA mimics and thereby expand the scope of these materials. The efficiency of this approach was demonstrated by the completion of CNA polymerization in 30 seconds, as characterized by size-exclusive chromatography (SEC) and infrared (IR) spectroscopy. CNA/DNA hybridization was demonstrated by gel electrophoresis and used in CdS nanoparticle assembly.

Synthesis and Assembly of Click-Nucleic-Acid-Containing PEG-PLGA Nanoparticles for DNA Delivery.

Co-delivery of both chemotherapy drugs and siRNA from a single delivery vehicle can have a significant impact on cancer therapy due to the potential for overcoming issues such as drug resistance. However, the inherent chemical differences between charged nucleic acids and hydrophobic drugs have hindered entrapment of both components within a single carrier. While poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (PEG-PLGA) copolymers have been used successfully for targeted delivery of chemotherapy drugs, loading of DNA or RNA has been poor. It is demonstrated that significant amounts of DNA can be encapsulated within PLGA-containing nanoparticles through the use of a new synthetic DNA analog, click nucleic acids (CNAs). First, triblock copolymers of PEG-CNA-PLGA are synthesized and then formulated into polymer nanoparticles from oil-in-water emulsions. The CNA-containing particles show high encapsulation of DNA complementary to the CNA sequence, whereas PEG-PLGA alone shows minimal DNA loading, and non-complementary DNA strands do not get encapsulated within the PEG-CNA-PLGA nanoparticles. Furthermore, the dye pyrene can be successfully co-loaded with DNA and lastly, a complex, larger DNA sequence that contains an overhang complementary to the CNA can also be encapsulated, demonstrating the potential utility of the CNA-containing particles as carriers for chemotherapy agents and gene silencers.

Synthesis of Small-Molecule/DNA Hybrids through On-Bead Amide-Coupling Approach.

Small molecule/DNA hybrids (SMDHs) have been considered as nanoscale building blocks for engineering 2D and 3D supramolecular DNA assembly. Herein, we report an efficient on-bead amide-coupling approach to prepare SMDHs with multiple oligodeoxynucleotide (ODN) strands. Our method is high yielding under mild and user-friendly conditions with various organic substrates and homo- or mixed-sequenced ODNs. Metal catalysts and moisture- and air-free conditions are not required. The products can be easily analyzed by LC-MS with accurate mass resolution. We also explored nanometer-sized shape-persistent macrocycles as novel multitopic organic linkers to prepare SMDHs. SMDHs bearing up to six ODNs were successfully prepared through the coupling of arylenethynylene macrocycles with ODNs, which were used to mediate the assembly of gold nanoparticles.

Multicatalytic, Light-Driven Upgrading of Butanol to 2-Ethylhexenal and Hydrogen under Mild Aqueous Conditions.

Microbes produce low-molecular-weight alcohols from sugar, but these metabolites are difficult to separate from water and possess relatively low heating values. A combination of photo-, organo-, and enzyme catalysis is shown here to convert C4 butanol (BuOH) to C8 2-ethylhexenal (2-EH) using only solar energy to drive the process. First, alcohol dehydrogenase (ADH) catalyzed the oxidation of BuOH to butyraldehyde (BA), using NAD+ as a cofactor. To prevent back reaction, NAD+ was regenerated using a platinum-seeded cadmium sulfide (Pt@CdS) photocatalyst. An amine-based organocatalyst then upgraded BA to 2-EH under mild aqueous conditions rather than harsh basic conditions in order to preserve enzyme and photocatalyst stability. The process also simultaneously increased total BuOH conversion. Thus, three disparate types of catalysts synergistically generated C8 products from C4 alcohols under green chemistry conditions of neutral pH, low temperature, and pressure.

Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli.

Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E. coli genome as a stable testing platform.

Catalytic Upgrading in Bacteria-Compatible Conditions via a Biocompatible Aldol Condensation.

Integrating non-enzymatic chemistry with living systems has the potential to greatly expand the types and yields of chemicals that can be sourced from renewable feedstocks. The in situ conversion of microbial metabolites to higher order products will ensure their continuous generation starting from a given cellular reaction mixture. We present here a systematic study of different organocatalysts that enable aldol condensation in biological media under physiological conditions of neutral pH, moderate temperature, and ambient pressure. The relative toxicities of each catalyst were tested against bacteria, and the catalysts were found to provide good yields of homoaldol products in bacterial cultures containing aldehydes. Lastly, we demonstrate that a biocompatible oil can be used to selectively extract the upgraded products, which enabes facile isolation and decreases the product toxicity to microbes.

Enhanced Hydrogen Production from DNA-Assembled Z-Scheme TiO2-CdS Photocatalyst Systems.

A wide range of inorganic nanostructures have been used as photocatalysts for generating H2. To increase activity, Z-scheme photocatalytic systems have been implemented that use multiple types of photoactive materials and electron mediators. Optimal catalysis has previously been obtained by interfacing different materials through aggregation or epitaxial nucleation, all of which lowers the accessible active surface area. DNA has now been used as a structure-directing agent to organize TiO2 and CdS nanocrystals. A significant increase in H2 production compared to CdS or TiO2 alone was thus observed directly in solution with no sacrificial donors or applied bias. The inclusion of benzoquinone (BQ) equidistant between the TiO2 and CdS through DNA assembly further increased H2 production. While the use of a second quinone in conjunction with BQ showed no more improvement, its location within the Z-scheme was found to strongly influence catalysis.

Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

High-yielding and photolabile approaches to the covalent attachment of biomolecules to surfaces via hydrazone chemistry.

The development of strategies to couple biomolecules covalently to surfaces is necessary for constructing sensing arrays for biological and biomedical applications. One attractive conjugation reaction is hydrazone formation--the reaction of a hydrazine with an aldehyde or ketone--as both hydrazines and aldehydes/ketones are largely bioorthogonal, which makes this particular reaction suitable for conjugating biomolecules to a variety of substrates. We show that the mild reaction conditions afforded by hydrazone conjugation enable the conjugation of DNA and proteins to the substrate surface in significantly higher yields than can be achieved with traditional bioconjugation techniques, such as maleimide chemistry. Next, we designed and synthesized a photocaged aryl ketone that can be conjugated to a surface and photochemically activated to provide a suitable partner for subsequent hydrazone formation between the surface-anchored ketone and DNA- or protein-hydrazines. Finally, we exploit the latent functionality of the photocaged ketone and pattern multiple biomolecules on the same substrate, effectively demonstrating a strategy for designing substrates with well-defined domains of different biomolecules. We expect that this approach can be extended to the production of multiplexed assays by using an appropriate mask with sequential photoexposure and biomolecule conjugation steps.

Aniline-terminated DNA catalyzes rapid DNA-hydrazone formation at physiological pH.

We report the effect of DNA-templation on aniline-catalyzed N-acylhydrazone formation. The reaction occurs in both two-component and three-component systems. Through systematic catalyst modifications, we are able to increase the efficiency of the DNA-templated variant to 85-fold above that of the uncatalyzed reaction at physiological pH.