Kinetically-tunable self-immolative aromatic spacers – KITUSE

The idea is to synthesize molecules based on three moieties: a photosensitizer group, a self-immolative spacer, and a fluorescent reporter. Irradiation of the compound will trigger the cleavage of the photosensitizer group under controlled conditions. The system will evolve spontaneously and we can measure its progress through fluorescence of reporters. These measurements were used to calculate the various kinetic parameters of the self-immolation step. By varying the structure of the self-immolative spacer, we have determined the relationship between the structure of the spacer and its self-immolation properties. We could obtain a library of spacers adapted to either a slow-release or a fast release depending of the considered biological or therapeutic applications.

We synthesized model compounds able of releasing one or two fluorophores. A self-immolative spacer library was obtained. Relationships between chemical structure and kinetic properties were studied and were then used to understand the influence of various parameters. These in vitro results have been extended to cells.

Potential applications go from the most fundamental aspects to applications in imaging and therapy. In biology, deliver one or more active compounds in a controlled manner at a given time within a cell will open new horizons. In therapy, the release of an bioactive compound from its prodrug is an important strategy that could benefit from kinetic data provided by our studies.

(1) Labruère R, Alouane A, Le Saux T, Aujard I, Pelupessy P, Gautier A, Dubruille S, Schmidt F, Jullien L. «Self-immolative« spacer for uncaging with fluorescence reporting. Angew Chem Int Ed Engl. 2012 Sep 10;51(37):9344-7.
doi: 10.1002/anie.201204032.

(2) Alouane A, Labruère R, Le Saux T, Aujard I, Dubruille S, Schmidt F, Jullien L. Light activation for the versatile and accurate kinetic analysis of disassembly of self-immolative spacers. Chemistry. 2013 Aug 26;19(35):11717-24.
doi: 10.1002/chem.201301298.

(3) Alouane A, Labruère R, Silvestre KJ, Le Saux T, Schmidt F, Jullien L. Disassembly kinetics of quinone-methide-based self-immolative spacers that contain aromatic nitrogen heterocycles. Chem Asian J. 2014 May;9(5):1334-40.
doi: 10.1002/asia.201400051.

3 additional publications (2 articles and 1 review) in preparation

Self-immolative spacers are covalent chemical assemblies used to increase the diversity of enzyme substrates for therapeutic or bio-analytical aims: the enzymatic reaction triggers the release of a drug or a probe. In this project we wish to develop a library of kinetically tunable self-immolative spacers, which will further broaden their scope. We also want to use them to introduce powerful tools for quantitative biology.
The most commonly used self-immolative spacers rely on an elimination reaction involving electron reorganization on an aromatic ring. This project develops a rational approach designed to provide reliable structure-properties, which are currently insufficiently known. Thanks to the synthesis and the detailed kinetic study of several model series, it will become possible, from its structure, to choose a spacer exhibiting a self-immolation kinetics, which is suited for a given use of an enzyme-drug/probe combination. More specifically, we will also seek to maximize the elimination rate of the drug/probe after the enzymatic activation step.
In this project, light will be used both to initiate the activation of spacer self-immolation and to observe the progress of the elimination leading to the release of a fluorescent reporter. Kinetic studies will be primarily based on the use of optical methods (absorption and steady-state emission spectroscopies, fluorescence correlation spectroscopy). In addition to its implementation for physicochemical analysis, the use of light to initiate product release and to report on its occurrence will allow us to propose new photo-labile protecting groups with fluorescent reporting. Thus we will use fast self-immolative spacers, which we will equip with ramifications. After synthesis and extensive photochemical studies in vitro, these systems will be evaluated in cultured cells.
Self immolative spacers can even reveal themselves more than passive connectors undergoing an elimination reaction. We propose to synthesize spacers incorporating a photochromic moiety, which will make possible to use light for piloting the release kinetics of the fluorescent elimination product. This feature will be used in a bio-analytics context to detect and quantify enzyme activity in experimentally adverse conditions (presence of interfering compounds, noise level) such as those encountered in vivo. We will use an original observation protocol. Upon modulating light excitation, the enzyme activity will give rise to a modulation of the fluorescence emission whose analysis by Fourier transform or by synchronous detection will permit to selectively extract the sole contribution of the fluorophore reporting on enzyme activity.