Photocatalytic Decarboxylative Functionalization of Cyclopropenes via Cyclopropenium Cation Intermediates.

A photocatalytic decarboxylative functionalization of cyclopropenes is reported. Starting from a broad range of redox-active ester-substituted cyclopropenes, cyclopropenylphthalimides can be synthesized in the absence of a nucleophile. Alternatively, different carbon and heteroatom nucleophiles can be introduced. The transformation proceeds most probably through the formation of an aromatic cyclopropenium cation, followed by trapping with the nucleophiles.

Heavy-Atom Tunneling in Bicyclo[4.1.0]hepta-2,4,6-trienes.

Heavy-atom tunneling limits the lifetime and observability of bicyclo[4.1.0]hepta-2,4,6-triene, a key intermediate in the rearrangement of phenylcarbene. Bicyclo[4.1.0]hepta-2,4,6-triene had been proposed as the primary intermediate of the rearrangement of phenylcarbene, but despite many efforts evaded its characterization even in cryogenic matrices. By introducing fluorine substituents into the ortho-positions of the phenyl ring of phenylcarbene, the highly strained cyclopropene 1,5-difluorobicyclo[4.1.0]hepta-2,4,6-triene becomes stable enough to be characterized in argon matrices. However, even at 3 K this cyclopropene is only metastable and rearranges via heavy-atom tunneling to the corresponding cycloheptatetraene. Calculations suggest that fluorination is necessary to slow down the tunneling rearrangement of the bicycloheptatriene. The parent bicycloheptatriene rapidly rearranges via heavy-atom tunneling and therefore cannot be detected under matrix isolation conditions.

Cobalt-Catalyzed Facial-Selective Hydroalkylation of Cyclopropenes.

Cyclopropene hydrofunctionalization has been a promising strategy for accessing multi-substituted cyclopropanes; however, cyclopropene hydroalkylation remains underdeveloped. Herein, we report a low-valent CoH-catalyzed facial-selective cyclopropene hydroalkylation to access multi-substituted cyclopropanes. This reaction exhibits a broad substrate scope of alkyl halides and cyclopropenes and tolerates many functional groups. Moderate-to-good facial-selectivity is obtained without any directing groups. Mechanism studies provide evidence that alkyl radicals are generated from alkyl halides and irreversible CoH insertion is responsible for the facial-selectivity. Our preliminary exploration demonstrates that asymmetric cyclopropene hydroalkylation can be realized without conspicuous auxiliary groups.

Live-Cell Imaging of Sterculic Acid-a Naturally Occurring 1,2-Cyclopropene Fatty Acid-by Bioorthogonal Reaction with Turn-On Tetrazine-Fluorophore Conjugates.

In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene-containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron-demand Diels-Alder reaction with quenched tetrazine-fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

Metal-Free Ring-Opening Metathesis Polymerization with Hydrazonium Initiators.

The ring-opening metathesis polymerization (ROMP) of cyclopropenes using hydrazonium initiators is described. The initiators, which are formed by the condensation of 2,3-diazabicyclo[2.2.2]octane and an aldehyde, polymerize cyclopropene monomers by a sequence of [3+2] cycloaddition and cycloreversion reactions. This process generates short chain polyolefins (Mn ≤9.4 kg mol-1 ) with relatively low dispersities (D≤1.4). The optimized conditions showed efficiency comparable to that achieved with Grubbs' 2nd generation catalyst for the polymerization of 3-methyl-3-phenylcyclopropene. A positive correlation between monomer to initiator ratio and degree of polymerization was revealed through NMR spectroscopy.

Selective Alkynylallylation of the C-C σ Bond of Cyclopropenes.

A Pd-catalyzed regio- and stereoselective alkynylallylation of a specific C-C σ bond in cyclopropenes, using allyl propiolates as both allylation and alkynylation reagents, has been achieved for the first time. By merging selective C(sp2 )-C(sp3 ) bond scission with conjunctive cross-couplings, this decarboxylative reorganization reaction features fascinating atom and step economy and provides an efficient approach to highly functionalized dienynes from readily available substrates. Without further optimization, gram-scale products can be easily obtained by such a simple, neutral, and low-cost catalytic system with high TONs. DFT calculations afford a rationale toward the formation of the products and indicate that the selective insertion of the double bond of cyclopropenes into the C-Pd bond of ambidentate Pd complex and the subsequent nonclassical ß-C elimination promoted by 1,4-palladium migration are critical for the success of the reaction.

Catalytic Synthesis of Trifluoromethyl Cyclopropenes and Oligo-Cyclopropenes.

The synthesis of trifluoromethylated cyclopropenes is often associated with important applications in drug discovery and functional materials. In this report, we describe the use of readily available chiral rhodium(II) catalysts for a highly efficient asymmetric cyclopropenation reaction of fluorinated donor-acceptor diazoalkanes with a broad variety of aliphatic and aromatic alkynes. Further studies highlight the unique reactivity of fluorinated donor-acceptor diazoalkanes in the synthesis of oligo-cyclopropenes. Subsequent C-H functionalization of trifluoromethyl cyclopropenes furnishes densely substituted cyclopropene frameworks and also allows the alternative synthesis of bis-cyclopropenes.

Modular Enzyme- and Light-Based Activation of Cyclopropene-Tetrazine Ligation.

We describe a modular activation strategy for cyclopropene-tetrazine ligation. This activation strategy uses chemically diverse enzyme- or photolabile protecting groups as cyclopropene reactivity cages. The linkages between the caging groups and cyclopropene are through carbamates, thus permitting the application of diverse cages to allow bioorthogonal reactivity by administering enzymes or light.

A Cyclopropene Electrophile that Targets Glutathione S-Transferase Omega-1 in Cells.

Cyclopropenes are an important new addition to the portfolio of functional groups that can be used for bioorthogonal couplings. The inert nature of these highly strained compounds in complex biological systems is almost counterintuitive given their established electrophilic properties in organic synthesis. Here we provide the first demonstration of a cyclopropene that is capable of direct conjugation to protein targets in cells and show that this compound preferentially alkylates the active site cysteine of glutathione S-transferase omega-1 (GSTO1).

Efficient Phage Display with Multiple Distinct Non-Canonical Amino Acids Using Orthogonal Ribosome-Mediated Genetic Code Expansion.

Phage display is a powerful approach for evolving proteins and peptides with new functions, but the properties of the molecules that can be evolved are limited by the chemical diversity encoded. Herein, we report a system for incorporating non-canonical amino acids (ncAAs) into proteins displayed on phage using the pyrrolysyl-tRNA synthetase/tRNA pair. We improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode a cyclopropene-containing ncAA (CypK) at diverse sites on a displayed single-chain antibody variable fragment (ScFv), in response to amber and quadruplet codons. CypK and an alkyne-containing ncAA are incorporated at distinct sites, enabling the double labeling of ScFv with distinct probes, through mutually orthogonal reactions, in a one-pot procedure. These advances expand the number of functionalities that can be encoded on phage-displayed proteins and provide a foundation to further expand the scope of phage display applications.

Diastereo- and enantioselective preparation of cyclopropanol derivatives.

The diastereoselective carbocupration reaction of alkoxy-functionalized cyclopropene derivatives, followed by a subsequent trapping of the resulting cyclopropylmetal species with an electrophilic source of oxygen (oxenoid) afforded various tetrasubstituted cyclopropanol derivatives in high diastereo- and enantiomeric ratios. Similarly, the enantioselective copper-catalyzed carbomagnesiation/oxidation (or amination) sequence on achiral nonfunctionalized cyclopropenes provided the desired cyclopropanol (and cyclopropylamine) derivatives in excellent diastereo- and enantiomeric excesses.

RhI -Catalyzed Carbonylative [3+1] Construction of Cyclobutenones via C-C σ-Bond Activation of Cyclopropenes.

With a catalytic amount of Rh(cod)2 BF4 and dppm, cyclopropenes undergo a direct carbonylative [3+1] cycloaddition reaction under an atmosphere of CO to produce the cyclobutenones in excellent yields, in which the regio- and diastereoselectivities can be controlled in certain cases with the help of chelating groups. Cyclobutenone with a chiral 4-position was prepared by diastereoselective induction. Rhodacyclopentenone has been determined as the key intermediate, as it was synthesized and applied to the reductive elimination step.

Lipidated cyclopropenes via a stable 3-N spirocyclopropene scaffold.

Lipidated cyclopropenes serve as useful bioorthogonal reagents for imaging cell membranes due to the cyclopropene's small size and ability to ligate with pro-fluorescent tetrazines. Previously, the lipidation of cyclopropenes required modification at the C3 position because methods to append lipids at C1/C2 were not available. Herein, we describe C1/C2 lipidation with the biologically active lipid ceramide and a common phospholipid using a cyclopropene scaffold whose reactivity with 1,2,4,5-tetrazines has been caged.

Rapid and Efficient Generation of Stable Antibody-Drug Conjugates via an Encoded Cyclopropene and an Inverse-Electron-Demand Diels-Alder Reaction.

Homogeneous antibody-drug conjugates (ADCs), generated by site-specific toxin linkage, show improved therapeutic indices with respect to traditional ADCs. However, current methods to produce site-specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene derivative of lysine (CypK) into antibodies through genetic-code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivatives to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid production of stable therapeutic protein conjugates.

Efficient Synthesis of Arylated Furans by a Sequential Rh-Catalyzed Arylation and Cycloisomerization of Cyclopropenes.

A novel and efficient strategy for the synthesis of arylated furans was successfully developed by a RhIII -catalyzed coupling of N-phenoxyacetamides and cyclopropenyl esters. Mechanistic investigation reveals that the arylated furans are formed via arylation of the cyclopropenyl esters followed by cycloisomerization.

Coupling and Dearomatization of Pyridines at a Transient η2 -Cyclopropene/Bicyclobutane Zirconocene Complex.

This paper reports on stereospecific coupling reactions between an η2 -cyclopropene ligand and pyridine derivatives, which are preferred to alternative C-H bond activation reactions. The dicyclopropylzirconocene complex [Cp2 Zr(c-C3 H5 )2 ] (1) eliminates cyclopropane to generate the η2 -cyclopropene/bicyclobutane intermediate [Cp2 Zr(η2 -c-C3 H4 )] (A). A does not activate any pyridine C-H bonds, but rather pyridine inserts into a Zr-C bond of A, yielding an azazirconacycle with a dearomatized pyridyl group [Cp2 Zr{κ2 -N,C8 -(2-c-C3 H4 )-C5 H5 N}] (2). Kinetic data, isotopelabelling experiments, and DFT calculations indicate that the rate-determining step of this stereospecific reaction is cyclopropane elimination, and that the stability of the intermediate [Cp2 Zr(η2 -c-C3 H4 )(NC5 H5 )] (A-py) governs the selectivity of the reaction. Complex 2 tautomerizes to [Cp2 Zr{κ2 -N,C8 -(2-C3 H5 )-C5 H4 N}] (6) through a base-catalyzed proton migration accompanied by cyclopropyl opening and restoration of conjugation within the zirconacycle.

Modulable and highly diastereoselective carbometalations of cyclopropenes.

The copper-catalyzed carbomagnesiation reaction of cyclopropenyl esters 1 leads to various substituted cyclopropanes species 3 in good yields with very high diastereoselectivities. The reaction proceeds through a syn-chelated carbomagnesiation reaction and could be extended to various cyclopropenylmethyl ester derivatives 5. The potential of this approach was illustrated by the preparation of two consecutive all-carbon quaternary stereocenters. However, the carbometalation reaction needs to be performed at temperature ranging from -35 to -20 °C to avoid subsequent fragmentation reaction into stereodefined ß,γ-nonconjugated unsaturated esters 4. Alternatively, the carbocupration reaction with organocopper species could also be performed to leads to configurationally stable cyclopropyl copper species 2[Cu]. Additionally, when the Lewis acid character of the copper center is decreased (i.e., RCuCNLi), the reaction proceed with an anti-selectivity. The diastereodivergent behavior of these organometallic species is of synthetic interest, since both diastereomers syn-3 and anti-3 can be obtained, at will, from the same precursor cyclopropenyl esters 1.

Synthesis and reactivity comparisons of 1-methyl-3-substituted cyclopropene mini-tags for tetrazine bioorthogonal reactions.

Substituted cyclopropenes have recently attracted attention as stable "mini-tags" that are highly reactive dienophiles with the bioorthogonal tetrazine functional group. Despite this interest, the synthesis of stable cyclopropenes is not trivial and their reactivity patterns are poorly understood. Here, the synthesis and comparison of the reactivity of a series of 1-methyl-3-substituted cyclopropenes with different functional handles is described. The rates at which the various substituted cyclopropenes undergo Diels-Alder cycloadditions with 1,2,4,5-tetrazines were measured. Depending on the substituents, the rates of cycloadditions vary by over two orders of magnitude. The substituents also have a dramatic effect on aqueous stability. An outcome of these studies is the discovery of a novel 3-amidomethyl substituted methylcyclopropene tag that reacts twice as fast as the fastest previously disclosed 1-methyl-3-substituted cyclopropene while retaining excellent aqueous stability. Furthermore, this new cyclopropene is better suited for bioconjugation applications and this is demonstrated through using DNA templated tetrazine ligations. The effect of tetrazine structure on cyclopropene reaction rate was also studied. Surprisingly, 3-amidomethyl substituted methylcyclopropene reacts faster than trans-cyclooctenol with a sterically hindered and extremely stable tert-butyl substituted tetrazine. Density functional theory calculations and the distortion/interaction analysis of activation energies provide insights into the origins of these reactivity differences and a guide to the development of future tetrazine coupling partners. The newly disclosed cyclopropenes have kinetic and stability advantages compared to previously reported dienophiles and will be highly useful for applications in organic synthesis, bioorthogonal reactions, and materials science.

Novel aromatic and antiaromatic systems.

We contributed to the field of non-benzenoid aromatic compounds by creating the cyclopropenyl cation and various of its derivatives, including cyclopropenone; it was the first aromatic system with other than six pi electrons in a single ring, and the simplest aromatic system. The pioneering work of Tetsuo Nozoe in tropolone chemistry was celebrated with the founding of ISNA, the International Symposium on Non-Benzenoid Aromatic Compounds, where I described our work in the field. It fit the prediction that aromaticity would be found in systems with 4n + 2 pi electrons, where n is an integer. I was also concerned with the properties of monocyclic systems with 4n cyclically conjugated pi electrons. They were expected not to be aromatic, but the interesting question was whether they were actually antiaromatic, especially destabilized by the cyclic conjugation in such 4n species as the cyclopropenyl anion, cyclobutadiene, and cyclopentadienyl cation. The evidence supports antiaromaticity in these cases. We also examined compounds where 4n cyclic pi systems were fused with aromatic systems, and most interestingly systems in which two 4n pi systems were fused. In these cases the periphery of the molecules had 4n + 2 pi electrons, for aromaticity, but the components were antiaromatic. Recently we have studied electrical conductivities in aromatic molecules such as thiophene and saw that aromaticity added resistance to the systems, so non-aromatic compounds are better conductors and antiaromatic compounds are predicted to be the best of all.

Encoding Noncanonical Amino Acids into Phage Displayed Proteins.

Phage display facilitates the evolution of peptides and proteins for affinity selection against targets, but it is mostly limited to the chemical diversity provided by the naturally encoded amino acids. The combination of phage display with genetic code expansion allows the incorporation of noncanonical amino acids (ncAAs) into proteins expressed on the phage. In this method, we describe incorporation of one or two ncAAs in a single-chain fragment variable (scFv) antibody in response to amber or quadruplet codon. We take advantage of the pyrrolysyl-tRNA synthetase/tRNA pair to incorporate a lysine derivative and an orthogonal tyrosyl-tRNA synthetase/tRNA pair to incorporate a phenylalanine derivative. The encoding of novel chemical functionalities and building blocks in proteins displayed on phage provides the foundation for further phage display applications in fields such as imaging, protein targeting, and the production of new materials.