An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA.

As the applications of CRISPR-Cas9 technology diversify and spread beyond the laboratory to diagnostic and therapeutic use, the demands of gRNA synthesis have increased and access to tailored gRNAs is now restrictive. Enzymatic routes are time-consuming, difficult to scale-up and suffer from polymerase-bias while existing chemical routes are inefficient. Here, we describe a split-and-click convergent chemical route to individual or pools of sgRNAs. The synthetic burden is reduced by splitting the sgRNA into a variable DNA/genome-targeting 20-mer, produced on-demand and in high purity, and a fixed Cas9-binding chemically-modified 79-mer, produced cost-effectively on large-scale, a strategy that provides access to site-specific modifications that enhance sgRNA activity and in vivo stability. Click ligation of the two components generates an artificial triazole linkage that is tolerated in functionally critical regions of the sgRNA and allows efficient DNA cleavage in vitro as well as gene-editing in cells with no unexpected off-target effects.

CuAAC: An Efficient Click Chemistry Reaction on Solid Phase.

Click chemistry is an approach that uses efficient and reliable reactions, such as Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), to bind two molecular building blocks. CuAAC has broad applications in medicinal chemistry and other fields of chemistry. This review describes the general features and applications of CuAAC in solid-phase synthesis (CuAAC-SP), highlighting the suitability of this kind of reaction for peptides, nucleotides, small molecules, supramolecular structures, and polymers, among others. This versatile reaction is expected to become pivotal for meeting future challenges in solid-phase chemistry.

The facile assembly of bis-, tris- and poly(triazaphosphole) systems using "click" chemistry.

Uncatalysed 1,3-dipolar cycloaddition reactions between two phosphaalkynes, P≡CR (R = Bu(t) or Me), and a series of di-, tri- and poly-azido precursor compounds have given very high yields of a range of triazaphosphole substituted systems. These comprise the 1,1'-bis(triazaphosphole)ferrocenes, [Fe{C5H4(N3PCR)}2], the tris(triazaphosphole)cyclohexane, cis-1,3,5-C6H9(N3PCBu(t))3, and the poly(allyltriazaphosphole)s, {C3H5(N3PCR)}∞. Electrochemical studies on the 1,1'-bis(triazaphosphole)ferrocenes reveal the compounds to undergo reversible 1-electron oxidation processes, at significantly more positive potentials than ferrocene itself. Attempts to chemically oxidise one 1,1'-bis(triazaphosphole)ferrocene with a silver salt, Ag[Al{OC(CF3)3}4] were not successful, and led to the formation of a silver coordination complex, [{Fe[µ-C5H4(N3PCBu(t))]2(µ-Ag)}2][Al{OC(CF3)3}4]2, thereby demonstrating the potential the reported triazaphosphole substituted systems possess as novel ligands in coordination chemistry.

Rapid and versatile construction of diverse and functional nanostructures derived from a polyphosphoester-based biomimetic block copolymer system.

A rapid and efficient approach for the preparation and modification of a versatile class of functional polymer nanoparticles has been developed, for which the entire engineering process from small molecules to polymers to nanoparticles bypasses typical slow and inefficient procedures and rather employs a series of steps that capture fully the "click" chemistry concepts that have greatly facilitated the preparation of complex polymer materials over the past decade. The construction of various nanoparticles with functional complexity from a versatile platform is a challenging aim to provide materials for fundamental studies and also optimization toward a diverse range of applications. In this paper, we demonstrate the rapid and facile preparation of a family of nanoparticles with different surface charges and functionalities based on a biodegradable polyphosphoester block copolymer system. From a retrosynthetic point of view, the nonionic, anionic, cationic, and zwitterionic micelles with hydrodynamic diameters between 13 and 21 nm and great size uniformity were quickly formed by suspending, independently, four amphiphilic diblock polyphosphoesters into water, which were functionalized from the same parental hydrophobic-functional AB diblock polyphosphoester by click-type thiol-yne reactions. The well-defined (PDI < 1.2) hydrophobic-functional AB diblock polyphosphoester was synthesized by an ultrafast (<5 min) organocatalyzed ring-opening polymerization in a two-step, one-pot manner with the quantitative conversions of two kinds of cyclic phospholane monomers. The whole programmable process starting from small molecules to nanoparticles could be completed within 6 h, as the most rapid approach for the anionic and nonionic nanoparticles, although the cationic and zwitterionic nanoparticles required ca. 2 days due to purification by dialysis. The micelles showed high biocompatibility, with even the cationic micelles exhibiting a 6-fold lower cytotoxicity toward RAW 264.7 mouse macrophage cells, as compared to the commercial transfection agent Lipofectamine.

Fast RNA conjugations on solid phase by strain-promoted cycloadditions.

Strain promoted cycloaddition is presented as a tool for RNA conjugation on the solid phase; RNA-cyclooctyne conjugates are prepared by cycloaddition to both azide (strain-promoted azide-alkyne cycloaddition, SPAAC) and nitrile oxide dipoles (strain-promoted nitrile oxide-alkyne cycloaddition, SPNOAC). The conjugation is compatible with 2'-OMe blocks and with 2'-O-TBDMS protection on the ribose moieties of the sugar. Nitrile oxide dipoles are found to be more reactive click partners than azides. The conjugation proceeds within 10 min in aqueous solvents, at room temperature without any metal catalyst and tolerates dipoles of varying steric bulk and electronic demands, including pyrenyl, coumarin and dabcyl derivatives.

DNA or RNA oligonucleotide 2'-hydrazides for chemoselective click-type ligation with carbonyl compounds.

An efficient method for the synthesis of DNA or RNA oligonucleotide 2'-hydrazides is described. Fully deprotected oligonucleotides containing a hydrazide group at the 2'-position of a uridine residue were obtained by a novel two-step procedure: periodate cleavage of an oligonucleotide with 1,2-diol group followed by conversion of the aldehyde to hydrazide with an extended linker arm using a homobifunctional reagent succinic dihydrazide and NaBH(3)CN. The resulting oligonucleotide 2'-hydrazides were efficiently conjugated by a click-type reaction at acidic pH to aliphatic or aromatic aldehydes with or without NaBH(3)CN reduction to afford novel 2'-conjugates.

Fluorophore-labeled, peptide-based glycoclusters: synthesis, binding properties for lectins, and detection of carbohydrate-binding proteins in cells.

A facile and efficient solid-phase synthesis of linear peptide-based glycoclusters with various valences and different spatial arrangements of the sugar ligands is described. The synthetic strategy includes 1) solid-phase synthesis of fluorophore-labeled, alkyne-containing peptides, 2) coupling of azide-linked, unprotected mono-, di-, and trisaccharides to the alkyne-conjugated peptides on a solid support by click chemistry, and 3) release of the fluorophore-labeled glycoclusters from the solid support. By using this methodology, 32 fluorescent glycoclusters with a valence ranging from 1 to 4 and different spatial arrangements of the sugar ligands were prepared. Lectin-binding properties of the glycoclusters were initially examined by using microarrays immobilized by various lectins. These glycoclusters were then employed to detect the cell-surface carbohydrate-binding proteins in bacteria. Finally, the uptake of glycoclusters by mammalian cells through receptor-mediated endocytosis was evaluated. The results, obtained from the in vitro and in vivo studies, indicate that the binding affinities toward immobilized and cell-surface proteins are highly dependent on the valence and spatial arrangements of the sugar ligands in glycoclusters.

Simple and stereoselective preparation of an 4-(aminomethyl)-1,2,3-triazolyl nucleoside phosphoramidite.

A simple and stereoselective synthesis of a protected 4-(aminomethyl)-1-(2-deoxy-ß-D-ribofuranosyl)-1,2,3-triazole cyanoethyl phosphoramidite was developed for the modification of synthetic oligonucleotides. The configuration of the 1,2,3-triazolyl moiety with respect to the deoxyribose was unambiguously determined in ROESY experiments. The aminomethyl group of the triazolyl nucleotide was fully functional in labelling reactions. Furthermore, the hybridization behavior of 5' triazole-terminated oligonucleotide was similar to that of 5' aminohexyl-terminated oligomer with the same sequence. Internal modifications of the oligonucleotide strands resulted in significant decrease of duplex stability.