Click to Join Peptides/Proteins Together

We report here that the efficiency of the click chemistry between the terminal azide and alkyne groups of different polymer chains could be drastically increased with the addition of an optimum amount of water into a reaction system. That is, the efficiency was only slightly promoted by the addition of a small amount of water into the reaction mixture. However, the reaction efficiency was increased dramatically near the water volume fraction to lead the reaction mixture into nanosized phase separation. Further increasing in water content caused the polymer(s) to undergo macroscopic phase separation and the click reaction efficiency was decreased once again. The enhanced efficiency of click coupling reaction including conversion and rate was also demonstrated via in-situ 1H-NMR. The reaction kinetics as well as reaction rate constant for these reaction system with typical water content were also evaluated. This finding on enhanced click reaction is of practical value, because click reactions in polymer synthesis are generally more difficult to be carried out and proceed relative slowly at low yield in most case because of the strong steric hindrance effect from “large and long” polymer chains, as compared to “click” reactions which are employed for preparation of the low molecular weight organic compounds.

Cyclic peptides have higher stability and better properties as therapeutic agents than their linear peptide analogues. Consequently, intramolecular click chemistry is becoming an increasingly popular method for the synthesis of cyclic peptides from their isomeric linear peptides. However, assessing the purity of these cyclic peptides by mass spectrometry is a significant challenge, as the linear and cyclic peptides have identical masses. In this paper, we have evaluated the analytical capabilities of energy-resolved mass spectrometry (ER MS) and mid-infrared microscopy (IR) to address this challenge. On the one hand, mixtures of both peptides were subjected to collision-induced dissociation tandem mass spectrometry (CID MS/MS) experiments in an ion trap mass spectrometer at several excitation energies. Two different calibration models were used: a univariate model (at a single excitation voltage) and a multivariate model (using multiple excitation voltages). The multivariate model demonstrated slightly enhanced analytical performance, which can be attributed to more effective signal averaging when multiple excitation voltages are considered. On the other hand, IR microscopy was used for the quantification of the relative amount of linear peptide. This was achieved through univariate calibration, based on the absorbance of an alkyne band specific to the linear peptide, and through Partial Least Squares (PLS) multivariate calibration. The PLS calibration model demonstrated superior performance in comparison to univariate calibration, indicating that consideration of the full IR spectrum is preferable to focusing on the specific peak of the linear peptide. The advantage of IR microscopy is that it is linear across the entire working interval, from linear peptide molar ratios of 0 (equivalent to pure cyclic peptide) up to 1 (pure linear peptide). In contrast, the ER MS calibration models exhibited linearity only up to 0.3 linear peptide molar ratio. However, ER MS showed better performances in terms of the limit of detection, intermediate precision and the root-mean-square-error of calibration. Therefore, ER MS is the optimal choice for the detection and quantification of the lowest relative amounts of linear peptides.

The copper(I)-catalyzed 1,2,3-triazole-forming reaction between azides and terminal alkynes has become the gold standard of 'click chemistry' due to its reliability, specificity, and biocompatibility. Applications of click chemistry are increasingly found in all aspects of drug discovery; they range from lead finding through combinatorial chemistry and target-templated in vitro chemistry, to proteomics and DNA research by using bioconjugation reactions. The triazole products are more than just passive linkers; they readily associate with biological targets, through hydrogen-bonding and dipole interactions. The present review will focus mainly on the recent literature for applications of this reaction in the field of medicinal chemistry, in particular on use of the 1,2,3-triazole moiety as pharmacophore.

Application of azide–alkyne cycloaddition ‘click chemistry’ for the synthesis of Grb2 SH2 domain-binding macrocycles

The copper catalytic azide and terminal alkyne cycloaddition reaction, namely “click chemistry”, gives a new and convenient way to create l,4-disubstitutd-l,2,3-triazoles. In this work, 2-pyrrolecarbaldiminato–Cu(II) complexes were established as efficient catalysts for the three-component 1,3-dipolar cycloaddition reaction of arylboronic acid and sodium azide (NaN3) with terminal alkynes in ethanol at room temperature to 50 °C, 1,4-disubstituted 1,2,3-triazoles were synthesized. Following the optimized protocol, two series of new aryl-1,2,3-triazole-β-carboline hybrids have been designed and synthesized, and the chemical structures were characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS). All of the target compounds were evaluated in vitro for their antifungal activity against Rhizoctorzia solani, Fusarium oxysporum, Botrytis cinerea Pers., sunflower sclerotinia rot, and rape sclerotinia rot by mycelia growth inhibition assay at 50 μg/mL. The antifungal evaluation of the novel hybrids showed that, among the tested compounds, 5a, 5b, 5c, and 9b showed good antifungal activity against sunflower sclerotinia rot. Specifically, compound 9b also exhibited high broad-spectrum fungicidal against all the tested fungi with inhibition rates of 58.3%, 18.52%, 63.07%, 84.47%, and 81.23%. However, for F. oxysporum, all the target compounds showed no in vitro antifungal activities with an inhibition rate lower than 20%. These results provide an encouraging framework that could lead to the development of potent novel antifungal agents.

This thesis consists of two main parts, Part I and Part II. The six chapters in Part I cover various multicomponent coupling reactions of arynes generated via the hexadehydro Diels-Alder (HDDA) reactions of multiynes, whereas Part II is devoted to describe an unprecedented click reaction of terminal 1,3-diynes. Below is the brief outline of each chapter. Chapter 1 provides a brief overview of the origins, developments and synthetic applications of HDDA reactions. Aryne-based transformations developed under thermal and metal-catalyzed conditions to generate highly functionalized aromatic systems constitute the major portion of this chapter. In addition, herein are summarized recently developed multicomponent reactions of arynes derived from multiynes. In Chapter 2, Ritter-type transformations of arynes with nitriles are discussed. Nitriles, usually unreactive towards aryne, could be used as nucleophilic component upon activating arynes with a silver catalyst. Trapping of the initially formed aryne-nitrile adduct, a nitrilium species, with water contained in wet solvent provided amides. When nitrilium intermediate was trapped by carboxylic acid, arylimide was generated. In Chapter 3, an unprecedented [2+2+2] annulation reaction of arynes with nitriles has been highlighted. It was found that, in the absence of water or carboxylic acid, the nitrilium species generated from an aryne and a nitrile molecule could react further with another nitrile molecule to generate quinazoline derivatives. The generality of this transformation has been studied with a broad array of multiyne systems and with different sets of aliphatic and aromatic nitriles. Chapter 4 describes a novel and efficient [2+1+1] annulation reaction of arynes with isonitriles. In the presence of silver catalyst, arynes react with two molecules of isonitrile to generate benzocyclobutene-1,2-diimines. However, this reaction is limited to aromatic isonitriles. The mechanisms of these annulation reactions are investigated by DFT calculations. Synthetic utilities of benzocyclobutene-1,2-diimines are demonstrated in the latter part. Continued with silver catalyzed transformations of arynes with nitriles or isonitriles, a cross condensation involving arynes, nitriles, and isonitriles has been presented in Chapter 5. Initial screenings showed the impact of the stoichiometry of nitrile and isonitrile on the formation of aryne-nitrile-isonitrile adduct. A sequential addition of nitrile and isonitrile to aryne was observed to deliver 3-iminoindolin-2-ol and 3H-indol-3-imine. Because of the higher reactivity of isonitrile as a nucleophile, the incorporation of less reactive nitrile was feasible only with its significantly higher concentration compared to that of isonitrile and activated aryne with a silver catalyst. In Chapter 6, a variety of aryne-based multicomponent coupling reactions for the formation of functionalized aromatic compounds are presented. Arynes readily reacted with isonitriles to generate a nitrilium intermediate, which was intercepted with various weak nucleophiles including carboxylic acids, alcohols, sulfonamides, and water to generate the corresponding imides, imidates, amidines, and amides. An unprecedented click reaction of terminal 1,3 diynes is presented in Chapter 7. Cu( catalyzed [3+2] cycloaddition between terminal alkynes and tosyl azide generated N sulfonyl triazolyl copper species, which subsequently released N2 to generate ketenimide intermediates, which could be trapped with amines or alcohols to generate 3 alkynyl or allenyl imidamides and imidates. The selectivity for the formation of these products critically depended on the substituents of the diynes and the nucleophiles that reacted with the ketenimide intermediate. Depending on the type of nucleophiles, base additives, and the substituents patterns of the 1,3 diynes, this click reaction could promote the formation of alternative products such as heterocycles, [3]cumulenes and triple bond migrated products.

Malaria is nowadays one of the most serious health concerns in a global scale and, although there is an evident increase in research studies in this area, pointed by the vast number of hits and leads, it still appears as a recurrent topic every year due to the drug resistance shown by the parasite exposing the urgent need to develop new antimalarial medications. In this work, 38 molecules were synthesized via copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) or “click” chemistry, following different routes to produce 2 different organic azides, obtained from a 4,7 dicholoquinoline, reacted with 19 different commercially available terminal alkynes. All those new compounds were evaluated for their in vitro activity against the chloroquine resistant malaria parasite Plasmodium falciparum (W2). The cytotoxicity evaluation was accomplished using Hep G2 cells and SI index was calculated for every molecule. Some of the quinoline derivatives have shown high antimalarial activity, with IC50 values in the range of 1.72-8.66 µM, low cytotoxicity, with CC50>1000 µM and selectivity index (SI) in the range of 20-100, with some compounds showing SI>800. Therefore, the quinolinotriazole hybrids could be considered a very important step on the development of new antimalarial drugs.

A novel fluorescent sensor for mutational p53 DNA sequence detection based on click chemistry

First described by the groups of Sharpless (Rostovtsev et al., Angew. Chem., Int. Ed., 2002, 41, 2596) and Meldal (Tornoe et al., J. Org. Chem., 2002, 67, 3057) in 2002, Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) became one of the most effective and reliable synthetic tools in diverse areas of modern chemistry, such as organic, medicinal and polymer chemistry, material science and chemical biology. The advantages of this reaction are high selectivity, simple reaction conditions, wide scope and excellent functional group tolerance. This efficient transformation can be used for the synthesis of various 1,2,3-triazoles. On the other hand, CuAAC can be efficiently used for bioconjugation of two units bearing alkyne and azide functions via the triazole moiety. Moreover, the use of azides and alkynes bearing orthogonal functional groups in this reaction opens broad opportunities for the synthesis of valuable polyfunctionalized triazole-containg molecules. Thus, the initial discussion is focused on synthetic applications of functionally substituted azides and terminal alkynes in the CuAAC, combined with other transformations. The prominent part of this review is devoted to an application of these bi (or tri)-functional reagents for the design, synthesis or labeling of biologically active molecules.

In recent years, there has been a lot of interest in the progress of polymers through modification into graft copolymers. Click chemistry was used to synthesize graft copolymers, mostly through the Copper catalyzed Azide-Alkyne cycloaddition process, in order to get a high yield of product quickly. For postmodification, the clickable group on the polymer backbone was grafted by different polymer or chains. This succinct review emphasizes the most recent research on grafted copolymers produced by copper catalyzed azide alkyne click chemistry.

We have used the quantitative polymerase chain reaction (qPCR) to measure the extent of oxidative DNA damage under varying reaction conditions used for copper(I)-catalyzed click chemistry. We systematically studied how the damage depends on a number of key reaction parameters, including the amounts of copper, ascorbate, and ligand used, and found that the damage is significant under nearly all conditions tested, including those commonly used for bioconjugation. Furthermore, we discovered that the addition of dimethyl sulfoxide, a known radical scavenger, into the aqueous mixture dramatically suppresses DNA damage during the reaction. We also measured the efficiency of cross-linking two short synthetic oligonucleotides via click chemistry, and found that the reaction could proceed reasonably efficiently even with DMSO present. This approach for screening both DNA damage and reactivity under a range of reaction conditions will be valuable for improving the biocompatibility of click chemistry, and should help to extend this powerful synthetic tool for both in vitro and in vivo applications.

The fixation of ligands onto molecules, surfaces and materials by use of reactions using a simple and unified chemistry is among the everlasting desires of chemists. Besides the general insensitivity with respect to the chemical structures of the ligand, the completeness of the reaction as well as the insensitivity from external reaction parameters (i.e.: solvents, ambient temperature) is wished. The copper(I)-catalysed azide/alkyne “click”-reaction (also termed Sharpless “click”-reaction, a variation of the Huisgen 1,3-dipolar cycloaddition reaction between terminal acetylenes and azides) is a recent re-discovery of a reaction fulfilling these requirements. Extremely high yields (usually above 95%) are combined with a high tolerance of functional groups and reactions running at moderate temperatures (25°C - 70 °C). The present review assembles recent literature for applications of these reactions in the field of material science, in particular on surfaces, polymers, and for the ligation of ligands to larger biomolecules, including own publications in this field. Since this is an extremely fast developing area, this review offers important knowledge to the interested reader. A number of > 64 references are included. Keywords: cycloaddition reaction, regioselectivity, Cu-acetylide, Sharpless-type-click-reaction, self assembled monolayers (SAMs)

The synthesis of novel acyclic pyrimidine nucleoside analogues with 1,2,3-triazole ring bound via ethylene spacer or directly to C-5 of pyrimidine ring has been reported. 1,4-Disubstituted 1,2,3-triazoles were synthesized by click chemistry approach using regioselective copper(I)-catalyzed 1,3-dipolar cycloaddition reaction between terminal alkyne and azido pyrimidines under both conventional (method A) and microwave (method B) conditions. N-Alkylation of triazolyl pyrimidines afforded 5-[2-(1,2,3- triazolyl)ethyl]pyrimidine derivatives with penciclovir-, ganciclovir-like and 2,3-dihydroxypropyl side chain, as well as 5-(1,2,3- triazolyl)pyrimidine derivatives with penciclovir- and ganciclovir-like side chain. The stereostructures of 2-hydroxyethyl, 2- tosyloxyethyl, 2-azidoethyl and 4-acetoxymethyl-1,2,3-triazolyl C-5 substituted pyrimidine derivatives were unambiguously confirmed by their X-ray crystal structure analysis. Keywords: Acyclic pyrimidine nucleoside, Azide-alkyne cycloaddition, Click chemistry, 1, 4-disubstituted 1, 2, 3-triazoles, X-ray diffraction, supramolecular assembling.

Click chemistry with fullerene derivatives

The synthesis and polymerization of representative acrylic‐type esters containing a terminal acetylene group, CH2C(R)COO(CHR′)mCCH, where R and R′ are H and CH3 and m = 1 or 2, by anionic initiation to linear polymers are described. In contrast, crosslinked polymers were formed when radical and cationic initiators were used. Crosslinked polymers were also obtained with organolithium compounds but not with sodium naphthalene and sodium benzalaniline; this observation is discussed and compared to the behavior of the acetylenic acrylic esters which do not contain a terminal acetylenic hydrogen. The unpolymerized acetylenic bonds in the resulting linear polymers were shown to be present by infrared spectroscopic methods and by the following post‐reactions of these bonds: (1) the heat‐ and radical‐initiated crosslinking of the polymers through the acetylenic bonds; (2) the post‐bromination of the acetylenic bonds; and (3) the reaction of decaborane with the acetylenic bonds. The anionic copolymerization of acrylonitrile and styrene with these acetylenic monomers were performed and compared to the copolymerizations with 1‐acryloxy‐2‐butyne and 1‐methacryloxy‐2‐butyne. Dibromination of the linear polymers affords self‐extinguishing polymers, while decaboronation yields soluble polymers which do not soften up to 300°C. The linear polymers may be classified as “self‐reactive” polymers which yield thermosetting polymers.