Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.

Thermally Triggered Triazolinedione-Tyrosine Bioconjugation with Improved Chemo- and Site-Selectivity.

A bioconjugation strategy is reported that allows the derivatization of tyrosine side chains through triazolinedione-based "Y-clicking". Blocked triazolinedione reagents were developed that, in contrast to classical triazolinedione reagents, can be purified before use, can be stored for a long time, and allow functionalization with a wider range of cargoes and labels. These reagents are bench-stable at room temperature but steadily release highly reactive triazolinediones upon heating to 40 °C in buffered media at physiological pH, showing a sharp temperature response over the 0 to 40 °C range. This conceptually interesting strategy, which is complementary to existing photo- or electrochemical bioorthogonal bond-forming methods, not only avoids the classical synthesis and handling difficulties of these highly reactive click-like reagents but also markedly improves the selectivity profile of the tyrosine conjugation reaction itself. It avoids oxidative damage and "off-target" tryptophan labeling, and it even improves site-selectivity in discriminating between different tyrosine side chains on the same protein or different polypeptide chains. In this research article, we describe the stepwise development of these reagents, from their short and modular synthesis to small-molecule model bioconjugation studies and proof-of-principle bioorthogonal chemistry on peptides and proteins.

A UPLC-MS/MS method for quantification of metabolites in the ethylene biosynthesis pathway and its biological validation in Arabidopsis.

The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that 1-aminocyclopropane-1-carboxylic acid (ACC) plays a role beyond its function as an ethylene precursor. However, the absence of reliable methods to quantify ACC and its conjugates malonyl-ACC (MACC), glutamyl-ACC (GACC), and jasmonyl-ACC (JA-ACC) hinders related research. Combining synthetic and analytical chemistry, we present the first, validated methodology to rapidly extract and quantify ACC and its conjugates using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Its relevance was confirmed by application to Arabidopsis mutants with altered ACC metabolism and wild-type plants under stress. Pharmacological and genetic suppression of ACC synthesis resulted in decreased ACC and MACC content, whereas induction led to elevated levels. Salt, wounding, and submergence stress enhanced ACC and MACC production. GACC and JA-ACC were undetectable in vivo; however, GACC was identified in vitro, underscoring the broad applicability of the method. This method provides an efficient tool to study individual functions of ACC and its conjugates, paving the road toward exploration of novel avenues in ACC and ethylene metabolism, and revisiting ethylene literature in view of the recent discovery of an ethylene-independent role of ACC.

Arinole, a novel auxin-stimulating benzoxazole, affects root growth and promotes adventitious root formation.

The triple response phenotype is characteristic for seedlings treated with the phytohormone ethylene or its direct precursor 1-aminocyclopropane-carboxylic acid and is often employed to find novel chemical tools to probe ethylene responses. We identified a benzoxazole-urea derivative (B2) partially mimicking ethylene effects in a triple response bioassay. A thorough phenotypic analysis demonstrated that B2 and its closest analogue arinole (ARI) induced phenotypic responses reminiscent of seedlings with elevated levels of auxin, including impaired hook development and inhibition of seedling growth. Specifically, ARI reduced longitudinal cell elongation in roots, while promoting cell division. In contrast to other natural or synthetic auxins, ARI mostly acts as an inducer of adventitious root development, with only limited effects on lateral root development. Quantification of free auxins and auxin biosynthetic precursors as well as auxin-related gene expression demonstrated that ARI boosts global auxin levels. In addition, analyses of auxin reporter lines and mutants, besides pharmacological assays with auxin-related inhibitors, confirmed that ARI effects are facilitated by TRYPTOPHAN AMINOTRANSFERASE1 (TAA1)-mediated auxin synthesis. ARI treatment resulted in AR formation in an array of species, including Arabidopsis, pea, tomato, poplar, and lavender, a desirable trait in both agriculture and horticulture.

Vinylogous Urea-Urethane Vitrimers: Accelerating and Inhibiting Network Dynamics through Hydrogen Bonding.

Vinylogous urethane (VUO ) based polymer networks are widely used as catalyst-free vitrimers that show rapid covalent bond exchange at elevated temperatures. In solution, vinylogous ureas (VUN ) undergo much faster bond exchange than VUO and are highly dynamic at room temperature. However, this difference in reactivity is not observed in their respective dynamic polymer networks, as VUO and VUN vitrimers prepared herein with very similar macromolecular architectures show comparable stress relaxation and creep behavior. However, by using mixtures of VUO and VUN linkages within the same network, the dynamic reactions can be accelerated by an order of magnitude. The results can be rationalized by the effect of intermolecular hydrogen bonding, which is absent in VUO vitrimers, but is very pronounced for vinylogous urea moieties. At low concentrations of VUN , these hydrogen bonds act as catalysts for covalent bond exchange, while at high concentration, they provide a pervasive vinylogous urea - urethane (VU) network of strong non-covalent interactions, giving rise to phase separation and inhibiting polymer chain dynamics. This offers a straightforward design principle for dynamic polymer materials, showing at the same time the possible additive and synergistic effects of supramolecular and dynamic covalent polymer networks.

Dithioallyl Cations in Stereoselective Dearomative (3 + 2) Cycloadditions of Benzofurans: Mechanism and Synthetic Applications.

A stereoselective dearomative cyclopentannulation of benzofurans is reported. A previously reported dearomative (3 + 2) cycloaddition of indoles with 1,4-dithiane-fused allyl cations was found to lack stereoselectivity when more substituted cyclopentene rings are targeted. However, for benzofuran substrates, excellent levels of stereoselectivity were observed for the same allyl cation reagents under very similar reaction conditions. In this full account, we provide a mechanistic rationale and some design principles that govern the stereoselectivity of the intriguing dearomative transformations using dithioallyl cations and demonstrate how the stereoselectivity depends on electronic factors of the starting materials. The stereoselective methodology is also applied in a straightforward dearomative synthesis of the tricyclic sesquiterpenoid natural product aplysin and its analogues, starting from a simple benzofuran.

Dithioallyl cation (3 + 2) cycloadditions under aprotic reaction conditions: rapid access to spiro-fused cyclopentane scaffolds.

We report a general method to effect all-carbon (3 + 2) cycloadditions that can elaborate cyclopentenes from a range of olefins. The required dithioallyl cation reagents can be generated in a newly developed mild protocol starting from 2-allyloxypyridine precursors, thus avoiding the use of strong Brønsted acids. The novel method significantly expands the substrate scope, which now also includes acid-sensitive olefins, and thus enables the preparation of previously inaccessible spiro-fused scaffold types from simple and readily available starting materials.

1,4-Dithianes: attractive C2-building blocks for the synthesis of complex molecular architectures.

This review covers the synthetic applications of 1,4-dithianes, as well as derivatives thereof at various oxidation states. The selected examples show how the specific heterocyclic reactivity can be harnessed for the controlled synthesis of carbon-carbon bonds. The reactivity is compared to and put into context with more common synthetic building blocks, such as 1,3-dithianes and (hetero)aromatic building blocks. 1,4-Dithianes have as yet not been investigated to the same extent as their well-known 1,3-dithiane counterparts, but they do offer attractive transformations that can find good use in the assembly of a wide array of complex molecular architectures, ranging from lipids and carbohydrates to various carbocyclic scaffolds. This versatility arises from the possibility to chemoselectively cleave or reduce the sulfur-heterocycle to reveal a versatile C2-synthon.

A Highly Dynamic Covalent Polymer Network without Creep: Mission Impossible?

Dynamic covalent polymer networks provide an interesting solution to the challenging recyclability of thermosets and elastomers. One of the remaining design constraints, however, is balancing thermal reprocessability in the form of material flow with dimensional stability during use. As a result, many chemistries are being investigated in order to improve bond reactivity control and material robustness. This Minireview highlights a number of promising concepts, with a particular emphasis on disconnecting chemical reactivity in low and high temperature regimes to obtain creep resistant, yet highly dynamic polymer networks. In addition, we will highlight the impact of sharp reactivity changes when applying extrapolation-based approaches during rheological analysis. As a result, we are confident that abandoning the myth of "permanent" reactivity will aid in the development of sustainable polymeric materials that can truly combine the benefits of thermoplastic and thermoset behaviour.

Suppressing Creep and Promoting Fast Reprocessing of Vitrimers with Reversibly Trapped Amines.

We report a straightforward chemical strategy to tackle current challenges of irreversible deformation in low Tg vitrimers at operating temperature. In particular, vinylogous urethane (VU) vitrimers were prepared where reactive free amines, necessary for material flow, were temporarily shielded inside the network backbone, by adding a small amount of dibasic ester to the curing mixture. The amines could be released as reactive chain ends from the resulting dicarboxamide bonds via thermally reversible cyclisation to an imide moiety. Indeed, (re)generation of the required nucleophilic amines as network defects ensured reprocessing and rapid material flow at higher temperature, where exchange dynamics are (re)activated. As a result, VU vitrimers were obtained with limited creep at service temperature, yet with good reprocessability at elevated temperatures. Thus, by exerting strong control on the molecular level over the availability of exchangeable functional groups, a remarkable improvement of VU properties was obtained.

Reprocessing of Covalent Adaptable Polyamide Networks through Internal Catalysis and Ring-Size Effects.

Here, we report the introduction of internally catalyzed amide bonds to obtain covalent adaptable polyamide networks that rely on the dissociation equilibrium between dicarboxamides and imides. While amide bonds are usually considered to be robust and thermally stable, the present study shows that their dynamic character can be activated by a smart choice of available building blocks without the addition of any external catalyst or other additives. Hence, a range of polyamide-based dynamic networks with variable mechanical and viscoelastic properties have been obtained in a systematic study, using a straightforward curing process of dibasic ester and amine compounds. Since the dissociation process involves a cyclic imide formation, the correlation between ring size and the thermomechanical viscosity profile was studied for five- to seven-membered ring intermediates, depending on the chosen dibasic ester monomer. This resulted in a marked temperature response with activation energies in the range of 116-197 kJ mol-1, yielding a sharp transition between elastic and viscous behavior. Moreover, the ease and versatility of this chemistry platform were demonstrated by selecting a variety of amines, resulting in densely cross-linked dynamic networks with Tg values ranging from -20 to 110 °C. With this approach, it is possible to design amorphous polyamide networks with an acute temperature response, allowing for good reprocessability and, simultaneously, high resistance to irreversible deformation at elevated temperatures.

Nonselective Chemical Inhibition of Sec7 Domain-Containing ARF GTPase Exchange Factors.

Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function.

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.

Stereodivergent Synthesis of Biologically Active Spironucleoside Scaffolds via Catalytic Cyclopropanation of 4-exo-Methylene Furanosides.

Cyclopropane fusion of the only rotatable carbon-carbon bond in furanosyl nucleosides (i.e., exocyclic 4'-5') is a powerful design strategy to arrive at conformationally constrained analogues. Herein, we report a direct stereodivergent route toward the synthesis of the four possible configurations of 4-spirocyclopropane furanoses, which have been transformed into the corresponding 4'-spirocyclic adenosine analogues. The latter showed differential inhibition of the protein methyltransferase PRMT5-MEP50 complex, with one analogue inhibiting more effectively than adenosine itself, demonstrating the utility of rationally probing 4'-5' side chain orientations.

Internal catalysis for dynamic covalent chemistry applications and polymer science.

Strong covalent chemical bonds that can also be reversed, cleaved or exchanged are the subject of so-called dynamic covalent chemistry (DCC). Applications range from classical protective groups in organic chemistry and cleavable linkers for solid phase synthesis, to more modern applications in dynamic compound libraries and adaptive materials. Interest in dynamic, reversible or responsive chemistries has risen in particular in the last few decades for the design and synthesis of new DCC-based polymer materials. Implementation of DCC in polymers yields materials with unique combinations of properties and in some cases even unprecedented properties for covalent materials, such as self-healing materials, covalent adaptable networks (CANs) and vitrimers. In particular, the incorporation of DCC in polymer materials aims to find a balance between a swift and triggerable reactivity, combined with a high degree of intrinsic robustness and stability. Applying harsh conditions, highly active catalysts or highly reactive bonding groups, as is done in classical DCC, is often not feasible or desirable, as it can damage the polymer's integrity, leading to loss of function and properties. In this context, so-called internally catalysed DCC platforms have started to receive more interest in this area. This approach relies on the relative proximity and orientation of common functional groups, which can influence a chemical exchange reaction in a subtle but significant way. This approach mimicks the strategies found in enzymic reactions, and is known in classical organic chemistry as neighbouring group participation (NGP). The use of internal catalysis or NGP within polymer material science has proven to be a highly attractive strategy. This tutorial review will outline examples showing the scope, advantages and pitfalls of using internal catalysis within different DCC applications, ranging from small molecules to dynamic polymer materials.

Stereoselective Gold(I)-Catalyzed Vinylcyclopropanation via Generation of a Sulfur-Substituted Vinyl Carbene Equivalent.

A stereoselective gold(I)-catalyzed vinylcyclopropanation of alkenes has been developed. A gold-coordinated cationic vinyl carbene species, readily generated via a rearrangement of the ethylenedithioacetal of propargyl aldehyde, reacts with a wide range of alkenes to afford thio-substituted vinylcyclopropanes. The gold-catalyzed vinyl cyclopropanation proceeds under mild conditions at room temperature and is generally selective for the formation of cis-substituted cyclopropanes. The reaction allows the formal introduction of a "naked" vinyl carbene, by subsequent chemoselective hydrodesulfurisation of the ethylenedithio-bridge. The synthetic utility of the new method is demonstrated by a short, racemic formal synthesis of the alkaloid cephalotaxin, hinging on a key vinyl cyclopropane-cyclopentene rearrangement.

Covalent Adaptable Networks with Tunable Exchange Rates Based on Reversible Thiol-yne Cross-Linking.

The design of covalent adaptable networks (CANs) relies on the ability to trigger the rearrangement of bonds within a polymer network. Simple activated alkynes are now used as versatile reversible cross-linkers for thiols. The click-like thiol-yne cross-linking reaction readily enables network synthesis from polythiols through a double Michael addition with a reversible and tunable second addition step. The resulting thioacetal cross-linking moieties are robust but dynamic linkages. A series of different activated alkynes have been synthesized and systematically probed for their ability to produce dynamic thioacetal linkages, both in kinetic studies of small molecule models, as well as in stress relaxation and creep measurements on thiol-yne-based CANs. The results are further rationalized by DFT calculations, showing that the bond exchange rates can be significantly influenced by the choice of the activated alkyne cross-linker.

Internal Catalysis in Covalent Adaptable Networks: Phthalate Monoester Transesterification As a Versatile Dynamic Cross-Linking Chemistry.

Covalent adaptable networks (CANs) often make use of highly active external catalysts to provide swift exchange of the dynamic chemical bonds. Alternatively, milder species can act as internal catalysts when covalently attached to the matrix and in close proximity to the dynamic bonds. In this context, we introduce the dynamic exchange of phthalate monoesters as a novel chemistry platform for covalent adaptable networks. A low-molecular-weight (MW) model study shows that these monoesters undergo fast transesterification via a dissociative mechanism, caused by internal catalysis of the free carboxylic acid, which reversibly forms an activated phthalic anhydride intermediate. Using this dynamic chemistry, a wide series of CANs with a broad range of properties have been prepared by simply curing a mixture of diols and triols with bifunctional phthalic anhydrides. The dynamic nature of the networks was confirmed via recycling experiments for multiple cycles and via stress relaxation using rheology. The networks proved to be resistant to deformation but showed a marked temperature response in their rheological behavior, related to the swift exchange reactions that have a high activation energy (120 kJ/mol). While densely cross-linked and hydrolytically stable polyester networks with low soluble fractions can be obtained, we found that, by swelling the networks in a hot solvent, a gel-to-sol transition happened, which resulted in the full dissolution of the network.

Fluorinated Vitrimer Elastomers with a Dual Temperature Response.

Vitrimers are an emerging new class of permanently cross-linked polymeric materials that show a liquid behavior upon heating wherein the macroscopic deformation is controlled by the rate of internal chemical bond exchange reactions. Thus, quite uniquely among polymeric materials, flow rates and material viscosities can be enhanced or controlled by the addition of catalysts and additives. We now report a catalyst-free vitrimer system, prepared from mixing two simple components, wherein two competing bond exchange mechanisms coexist, each showing a strikingly different temperature dependence, related to the large difference in activation energy for the different exchange pathways (60 vs 130-170 kJ/mol). The low barrier process is predominant at lower temperatures, but is outcompeted by the high barrier process that becomes dominant at higher temperatures because of its much more pronounced temperature dependence. The result is an interesting and highly unusual dual viscosity profile for this new class of vitrimer materials: a very gradual decrease in viscosity at lower temperatures, intercepted by a much sharper drop in viscosity at higher temperatures. The highly counterintuitive effect where a higher barrier pathway is dominant over a much lower barrier process can be rationalized by the exchange mechanisms that involve different reactive species, but lead to the overall same exchange. We observed this unusual but highly promising behavior first for fluorinated vitrimer elastomers, aimed at high performance materials, but the effect was also shown to hold in related nonfluorinated elastomers. A new way to control and design the rheological behavior of vitrimers toward finely tuned and precisely controlled processing applications has thus been provided.

The Elusive Seven-Membered Cyclic Imino Ether Tetrahydrooxazepine.

Cyclic imino ether heterocycles are used as ligands in transition metal catalysis, in various drugs and as reactive monomers in living cationic ring-opening polymerization (CROP). While five- and six-membered cyclic imino ethers, i.e. 2-oxazolines and 4,5-dihydro-1,3-oxazines, have extensively been studied in these areas, their seven-membered ring counterparts have remained unexplored. Herein, we report the synthesis of 2-phenyl-4,5,6,7-tetrahydro-1,3-oxazepine allowing reassignment of the earlier, incorrectly reported 4,5,6,7-tetrahydro-1,3-oxazepines as their N-acylated pyrrolidine isomers. Finally, we also report a comparison of the CROP reactivity of a homologous series of cyclic imino ethers with a 2-carbon, 3-carbon, and 4-carbon methylene bridge, revealing a remarkable ring size effect.