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.

Keywords: Adaptor Systems; Click-Chemistry; Cycloaddition; Diagnostics; Ligation chemistry; Linker Systems; Tetrazines; Therapy; Triazines; inverse Diels Alder Reaction.

Figure 1

Simplified mechanistic illustration of the electron rearrangement during DAR_inv. While the first step of association between diene and dienophile is reversible, the release of nitrogen during rearrangement of the intermediate product is irreversible and switches the reaction to the product side.

Figure 2

¹H-NMR-Spectrum of the 9 in D₆-DMSO_. The structure illustrates the shift calculation for protons of the compound with ChemDraw Ultra 2004. (Numbers indicate the predicted shift of the signals in ppm; quality of estimation is indicated in colour: blue = good, red = rough)

Figure 3

¹H-NMR-Spectrum of the oxidised 9 in D₆-DMSO_. The structure illustrates the shift calculation for protons of the compound with ChemDraw Ultra 2004. (Numbers indicate the predicted shift of the signals in ppm; quality of estimation is indicated in colour: blue = good, red = rough). Insert indicates the detailed peak analysis for the signals found together with the coupling constants measured.

Figure 4

¹H-NMR-Spectrum of the ammonium salt of 9 in D₆-DMSO_. The structure illustrates the shift calculation for protons of the compound with ChemDraw Ultra 2004. (Numbers indicate the predicted shift of the signals in ppm; quality of estimation is indicated in colour: blue = good, red = rough).

Figure 5

¹H-NMR-Spectrum of 9 in CDCl_3. The structure illustrates the shift calculation for protons of the compound with ChemDraw Ultra 2004. (Numbers indicate the predicted shift of the signals in ppm; quality of estimation is indicated in colour: blue = good, red = rough)

Figure 6

¹H-NMR-Spectrum of the Reppe Anhydride 15 in CDCl₃. The structure illustrates the shift calculation for protons of the compound with ChemDraw Ultra 2004. (Numbers indicate the predicted shift of the signals in ppm; quality of estimation is indicated in colour: blue = good, red = rough)