New water-soluble porphyrins for oxygen sensing in protein-based materials

Oxygen is a vital molecule for life and its precise quantification is pivotal to ensure correct functioning of cells. Instruments able to quantify accurately the oxygen levels are extremely important to understand cells behaviour especially in a three-dimension (3D) environment, where uneven distribution of oxygen can occur leading to zones of low cell viability. Optical oxygen sensing is a relatively recent method to monitor oxygen concentration. This approach exploits the emissive behaviour of a broad family of molecules (organic species and organometallic complexes), for which the luminescence emission is proportional to the quantity of oxygen present in the environment. Unlike methods based on the use of electrodes, optical oxygen sensing allows monitoring of oxygen levels without causing oxygen depletion in the observed sample and can be more easily applied in miniaturised devices. Metalloporphyrins are widely employed as optical oxygen sensors thanks to their favourable physico-chemical properties. The aim of this project was to synthesise a library of oxygen-sensing porphyrins to monitor variation of oxygen in 3D cell cultures. For this specific application, the chromophore needed to (1) be a platinum or palladium complex to ensure oxygen response, (2) present a suitable functional group to allow conjugation to a support (e.g., a biomacromolecule supporting the 3D cell construct) to avoid leaching and cell uptake, and (3) water-solubility to allow conjugation in aqueous solutions. We first focused our attention on the synthesis of tetra meso-substituted porphyrins bearing a carboxylic group as the conjugatable function on one of the beta-positions of the macrocycle. This approach was overall successful in terms of yield and reaction conditions, but the side-chain proved insufficiently stable to allow further manipulation of the species. Attention was then directed to the synthesis of asymmetric porphyrin A3B, bearing the conjugable group on one of the meso-substituted aryl ring. Novel positively and negatively charged water-soluble conjugatable porphyrins were developed during this work, obtained from a single high-value intermediate. The new species presented overall good yields employing relatively mild reaction conditions, via a synthetic procedure with gram-scale applicability. In addition, in this work the synthesis of two known water-soluble conjugatable porphyrin was improved and scaled-up. Platinum (II) and palladium (II) insertion in the water-soluble macrocycles was achieved employing a microwave-assisted reaction, which ensured reaction completion in short times. Finally, a high potential purification approach based on ion exchange was developed for cationic and anionic water-soluble derivatives. This method allows obtaining highly pure compounds employing cheap reagents and without the use of expensive purification instruments. The conjugation of the species to the proteins chosen as matrices for hydrogels (silk fibroin and collagen) was performed via N-hydroxysuccinimide chemistry. Oxygen response of the complexes synthesised was tested both in aqueous solution and supported on protein-based hydrogels. Most species showed oxygen-dependent fluorescence and linear Stern-Volmer plot supported on solid support, indicating their suitability to monitor the oxygen levels in a heterogeneous system such as a hydrogel. To the best of our knowledge, this work represents the first example of optical oxygen measurements in silk fibroin hydrogels.