Development of Novel Immobilized Copper-Ligand Complex for Click Chemistry of Biomolecules.

Copper-catalyzed azide-alkyne cycloaddition click (CuAAC) reaction is widely used to synthesize drug candidates and other biomolecule classes. Homogeneous catalysts, which consist of copper coordinated to a ligand framework, have been optimized for high yield and specificity of the CuAAC reaction, but CuAAC reaction with these catalysts requires the addition of a reducing agent and basic conditions, which can complicate some of the desired syntheses. Additionally, removing copper from the synthesized CuAAC-containing biomolecule is necessary for biological applications but inconvenient and requires additional purification steps. We describe here the design and synthesis of a PNN-type pincer ligand complex with copper (I) that stabilizes the copper (I) and, therefore, can act as a CuAAC catalyst without a reducing agent and base under physiologically relevant conditions. This complex was immobilized on two types of resin, and one of the immobilized catalyst forms worked well under aqueous physiological conditions. Minimal copper leaching was observed from the immobilized catalyst, which allowed its use in multiple reaction cycles without the addition of any reducing agent or base and without recharging with copper ion. The mechanism of the catalytic cycle was rationalized by density functional theory (DFT). This catalyst's utility was demonstrated by synthesizing coumarin derivatives of small molecules such as ferrocene and sugar.

Facile de Novo Sequencing of Tetrazine-Cyclized Peptides through UV-Induced Ring-Opening and Cleavage from the Solid Phase.

While most FDA-approved peptide drugs are cyclic, the robust cyclization chemistry of peptides and the deconvolution of cyclic peptide sequences by using tandem mass spectrometry render cyclic peptide drug discovery difficult. Here we present the successful design of cyclic peptides on solid phase that addresses both of these problems. We demonstrate that this peptide cyclization method using dichloro-s-tetrazine on solid phase allows successful cyclization of a panel of random peptide sequences with various charges and hydrophobicities. The cyclic peptides can be linearized and cleaved from the solid phase by simple UV light irradiation, and we demonstrate that accurate sequence information can be obtained for the UV-cleaved linearized peptides by using tandem mass spectrometry. The tetrazine linker used in the cyclic peptides can further be explored for inverse electron-demand Diels-Alder (IEDDA) reactions for screening or bioconjugation applications in the future.

Copper-ligand clusters dictate size of cyclized peptide formed during alkyne-azide cycloaddition on solid support.

Peptide and peptidomimetic cyclization by copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction have been used to mimic disulfide bonds, alpha helices, amide bonds, and for one-bead-one-compound (OBOC) library development. A limited number of solid-supported CuAAC cyclization methods resulting in monomeric cyclic peptide formation have been reported for specific peptide sequences, but there exists no general study on monocyclic peptide formation using CuAAC cyclization. Since several cyclic peptides identified from an OBOC CuAAC cyclized library has been shown to have important biological applications, we discuss here an efficient method of alkyne-azide 'click' catalyzed monomeric cyclic peptide formation on a solid support. The reason behind the efficiency of the method is explored. CuAAC cyclization of a peptide sequence with azidolysine and propargylglycine is performed under various reaction conditions, with different catalysts, in the presence or absence of an organic base. The results indicate that piperidine plays a critical role in the reaction yield and monomeric cycle formation by coordinating to Cu and forming Cu-ligand clusters. A previously synthesized copper compound containing piperidine, [Cu4I4(pip)4], is found to catalyze the CuAAC cyclization of monomeric peptide effectively. The use of 1.5 equivalents of CuI and the use of DMF as solvent is found to give optimal CuAAC cyclized monomer yields. The effect of the peptide sequence and peptide length on monomer formation are also investigated by varying either parameter systemically. Peptide length is identified as the determining factor for whether the monomeric or dimeric cyclic peptide is the major product. For peptides with six, seven, or eight amino acids, the monomer is the major product from CuAAC cyclization. Longer and shorter peptides on cyclization show less monomer formation. CuAAC peptide cyclization of non-optimal peptide lengths such as pentamers is affected significantly by the amino acid sequence and give lower yields.

Beyond azide-alkyne click reaction: easy access to 18F-labelled compounds via nitrile oxide cycloadditions.

Radiofluorinated 4-fluorobenzonitrile oxide and N-hydroxy-4-fluorobenzimidoyl chloride rapidly react with different alkenes and alkynes under mild conditions. These cycloadditions are suitable for the preparation of low-molecular weight radiopharmaceuticals and, in a strain-promoted variant, can enable easy labelling of sensitive biopolymers.

C-(4-[18F]fluorophenyl)-N-phenyl nitrone: A novel 18F-labeled building block for metal free [3+2]cycloaddition.

Radiofluorination via [3+2]-nitrone-alkene cycloaddition was studied using the model reaction between (18)F-labeled C-(4-fluorophenyl)-N-phenyl nitrone ([(18)F]1) and substituted maleimides 2a-c. [(18)F]1 was prepared in RCY of 73.6±5.8% and radiochemical purity of >95%. Cycloaddition of [(18)F]1 to 2a in toluene at 80°C and in EtOH at 110°C gave the respective isoxazolidine [(18)F]5a in >80% RCY at 10min reaction time. Reaction between [(18)F]1 and 2b, c also went smoothly to afford the respective cycloaddition products in high radiochemical yields.