The Diels-Alder-Reaction with inverse-Electron-Demand, a very efficient versatile Click-Reaction Concept for proper Ligation of variable molecular Partners

Abstract

The ligation of active pharmaceutical ingredients (API) for working with image processing systems in diagnostics (MRT) attracts increasing notice and scientific interest. The Diels-Alder ligation Reaction with inverse electron demand (DAR inv ) turns out to be an appropriate candidate. The DAR inv is characterized by a specific distribution of electrons of the diene and the corresponding dienophile counterpart. Whereas the reactants in the classical Diels-Alder Reaction feature electron-rich diene and electron-poor dienophile compounds, the DAR inv exhibits exactly the opposite distribution of electrons. Substituents with pushing electrones increase and, with pulling electrons reduce the electron density of the dienes as used in the DAR inv . We report here that the DAR inv is an efficient route for coupling of multifunctional molecules like active peptides, re-formulated drugs or small molecules like the alkyalting agent temozolomide (TMZ). This is an example of our contribution to the "Click chemistry" technology. In this case TMZ is ligated by DAR inv as a cargo to transporter molecules facilitating the passage across the cell membranes into cells and subsequently into subcellular components like the cell nucleus by using address molecules. With such constructs we achieved high local concentrations at the desired target site of pharmacological action. The DAR inv ligation was carried out using the combination of several technologies, namely: the organic chemistry and the solid phase peptide synthesis which can produce 'tailored' solutions for questions not solely restricted to the medical diagnostics or therapy, but also result in functionalizations of various surfaces qualified amongst others also for array development. We like to acquaint you with the DAR inv and we like to exemplify that all ligation products were generated after a rapid and complete reaction in organic solutions at room temperature, in high purity, but also, hurdles and difficulties on the way to the TMZ-BioShuttle conjugate should be mentioned. With this report we would like to stimulate scientists working with the focus on "Click chemistry" to intensify research with this expanding DAR inv able to open the door for new solutions inconceivable so far.

A cyclic-RGD-BioShuttle functionalized with TMZ by DARinv “Click Chemistry” targeted to αvβ3 integrin for therapy

“…Chemoselective reaction-conditions in aqueous solution and at room temperature were obtained by chemical ligation of functional peptides via the DAR inv . The “click chemistry” based on the DAR inv with “inverse electron demand” resulted in an impressive efficiency as illustrated in scheme 3/Figure S3 and is well documented 12. This reaction enhanced the economics of the chemical reaction by the following parameters: ① increase of the reaction rate, ② gentle reaction conditions at room temperature, and ③ resulting in reaction kinetics with stoichiometric reaction partners without excess of the educts.…”

“…This could be explained with a pharmacologic effect of the cRGD, as well as of the TMZ alone. The BioShuttle construct containing the cRGD as targeting modul connected to the TMZ via the Diels-Alder Reaction with inverse electron demand 12 targets the α v β 3 and α v β 5 integrin MCF-7 cell surface proteins as shown in the Figure 3. According to CLSM-pictures of the MCF-7 cells (Figure 3), an increased cytoplasmic coloring is indicative for a strongly increased amount of granularity in cells (R2) in the cytometric results.…”

“…shows the mass of the N -(2-Aminopropyl)-4-(6-(pyrimidine-2-yl)-1,2,4,5-tetrazine-3-yl)benzamide ( 6 in scheme 1/Figure S1), as discussed by Wiessler 12.…”

A genetically encoded cyclobutene probe for labelling of live cells

“…23–27 Curiously, cyclobutenes, a class of small and strained alkenes known to be reactive towards IEDDA reactions, 28–30 have received much less attention, with most efforts involving derivatives of the Reppe anhydride, a bicyclic derivative of cyclooctatetraene. 31 Monocyclic cyclobutenes, such as those discussed here, have surprisingly good thermal stability, 29–30 and, in contrast to cyclopropenes, 25 have good chemical stability even in the absence of additional substituents on the alkene. 29–30 Preliminary work in one of our labs had demonstrated that the rate of IEDDA cycloaddition of a cyclobutene-containing fatty acid (3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine; k = 1.21±0.12 M −1 s −1 ) 30 is faster than that observed with the majority of cyclopropenes 24 and comparable to the most reactive cyclopropenes ( k = 2.78±0.37 M −1 s −1 ).…”

Live‐Cell Imaging of Cyclopropene Tags with Fluorogenic Tetrazine Cycloadditions

“…[1][2][3] Fluorogenic tetrazines that increase in fluorescence after reaction with dienophiles are particularly useful for live-cell imaging applications. [4] Fluorogenic tetrazines have been recently used for live-cell imaging of small molecules, biomolecules tagged enzymatically with dienophiles, and proteins modified by reactive unnatural amino acids.…”