Grant TEAM funded by The Foundation for Polish Science. POIR.04.04.00-00-3CF4/16-00
from 01.11.2017 to 30.06.2023
The new super-resolution microscopy techniques enable studying cells on the level unthinkable even a decade ago and have already advanced our understanding of key cellular processes. At the same time they require novel, brighter fluorophores exhibiting ideal combination of photophysical properties. The overall research objective of this project is to design, synthesize and apply advanced fluorescent dyes in bio-oriented research, with the use of stimulated emission depletion super-resolution microscopy. This will open the route to the deciphering of as yet inaccessible key biological events in a wide range of cellbiological questions. As a result of this project, new families of dyes will be developed, following three breakthrough strategies. More importantly still, these dyes will be a platform for functionalization with scaffolds able to recognize various molecules of interest in the cell. We will achieve this goal by combining organic chemists with specialists in molecular biology.
In 2019 the expansion of the project has been initiated. The new project entitled ‘New generation of fluorophores for construction of proteases activity-based probes’ was written in collaboration with prof. Marcin Drąg (Wrocław University of Technology). An additional funding was granted by Foundation for Polish Science.
In the aftermath of the coronavirus pandemic, and in response to Foundation for Polish Science call, a new expansion of the project has been initiated in 2020 also in collaboration with prof. Marcin Drąg. The title of the new project was “New detection methods for SARS-CoV-2-Mpro and SARS-CoV-2-PLpro proteases involved in COVID-19 development”.
Read more about expansionTetraaryl-pyrrolo[3,2-b]pyrroles (TAPPs) discovered in our research group in 2012, are chromophores with significant potential in optoelectronic applications. Despite recent advances in the synthesis of 1,4-dihydro-pyrrolo[3,2-b]pyrroles, many heterocycles and key functional groups have proved incompatible with the multicomponent reaction leading to these heterocycles. Although salts of such metals as vanadium, niobium, cerium and manganese were found to facilitate the TAPP formation, we found that iron (III) salts are the most efficient catalysts, resulted in the formation of TAPPs in 6-69% yield. Moreover, modified reaction conditions allows the synthetic access towards TAPPs with very electron-rich, sterically hindered substituents and TAPPs bearing and ordered arrangement of N-substituents and C-substituents ranging from coumarin, quinoline, phthalimide to truxene, which was not possible to synthesize before (Scheme 1). The advances in the reaction scope enable a fine-tuning the photophysical properties including the Stokes shift value and emission color ranging from violet-blue to red. Furthermore, the optimized conditions allow the synthesis of TAPPs in amounts of more than 10 grams per run without concomitant decrease in yield or product contamination.
On the basis of better functional group compatibility, previously unavailable functional groups for TAPPs have been introduced that allowed novel derivatization strategies such as a double Knoevenagel reaction with various CH-acids and also to obtain the corresponding dyes possessing to C-C triple bonds, precursors for acceptor-donor-acceptor dyes, although showing a weak fluorescence, the dyes are expected to possess large two-photon absorption cross-section (currently under investigation in Japan). (Scheme 2).
The particularly modified TAPPs allow the reactions such as intramolecular direct arylation, alkyne benzannulation with the formation of 7-membered ring, addition of tetracyanoethylene and dimesitylboron fluoride, leading to previously unknown heterocyclic cores. In particular formation of fused derivative possessing two azaborole moieties turned out to be very interesting showing the for the first time red emission (Scheme 3).
Based on the strong electron-donor properties of the TAPP chromophore we developed quadrupolar dyes by the addition of the electron-withdrawing heterocyclic moieties such as benzofurazan, its nitro derivatives, benzothiadiazole and water soluble pyridinium (Figure 4). This approach enabled the emission shift to 690 nm.
Research into this class of substances is still ongoing.
A high versatility of pyrrolo[3,2-b]pyrrole architecture at many positions allows a π-expansion of the basic chromophore. Initially we have discovered the possibility to obtain an entirely new type of π-expanded pyrrolo[3,2-b]pyrrole with a geodesic geometry only via surface-assisted synthesis in scanning tunneling microscope. It took us three years of work to discover the method of the synthesis of this molecule in classical way in a 50 mg scale of final compound, which has been fully characterized (Scheme 1). The curving architecture causes significant bathochromic shift of absorption and emission, decrease in fluorescence efficiency and numerous other interesting effects.
Further developing of this synthetic method allowed the synthesis of an analogous dye with different geometry. It turned out, however, that if the substrate shows more hindered terminal substituents the final ring closure occurs only on the one side of the molecule to form helicene-like distortion (Scheme 2).
The unsymmetrical diketopyrrolopyrroles enable the fine-tuning of various physicochemical properties (emission maxima, solubility, affinity towards given ion/biomolecule/organelle) in parallel. We utilized entirely new synthetic methodology to synthesize unsymmetrically substituted diketopyrrolopyrroles for the preparation of highly complex derivatives (Figure 1). These dyes were found to show very good combination of photophysical properties. In particular some of them have weak emission which is recovered in the presence of potassium cations.
For the selective localization in mitochondrion we modified DPPs possessing azacrown ether with a moiety bearing a phosphonium site. A particularly modified fluorescent probe appeared to be highly sensitive towards K+ and localize selectively in mitochondria of cardiac H9C2 cells. We discovered that fast efflux/influx of mitochondrial K+ can be observed upon stimulation with nigericin (Figure 2).
Based on our methodology >10 DPPs possessing Zn2+-selective binding sites were synthesized and characterized (Figure 3). The binding event is accompanied by changes of absorption and emission. The extension of this studies into the cells is now in progress.
Cyanine type dyes such as rhodamines or rhodols possess very good cell permeability, however they suffer from the lack of stability. To overcome this we designed the new type of rhodols bearing additional electron-withdrawing groups group. Two synthetic methods have been discovered to achieve this goal. For the first time the direct transformation of coumarins into rhodols has been performed (Scheme 1). In the final step the double Knoevenagel condensation of 3-formyl-4-arylcoumarins with dimethyl acetonedicarboxylate led to rhodols. After the thorough optimization attempts we found that the best synthetic results were obtained with pyrrolidine as a catalyst. Reaction was extended to lead to the whole library of dyes possessing various aryl groups.
As a development of rhodol dyes with the increased optical stability we designed and synthesized a series of core-modified benzo[g]coumarins as a new generation of fluorophores for construction of proteases activity-based probes The synthetic path started from 2,6-dihydroxynaphthalene and 5- or 8-amino-2-hydroxynaphthalene and comprised 3-4 steps altogether (Scheme 2). Photophysical measurements reveal the strongest fluorescence response in nonpolar solvents.
As the second approach towards the electron deficient rhodol chromophore we discovered that rhodols can be directly prepared from 3-dialkylaminophenols and 4-hydroxy-2,3,5,6-tetrafluorobenzaldehyde (Scheme 3). This one step-procedure furnished rhodol possessing three fluorine atoms and significantly enhanced stability.
The optimization of the tandem reaction allowed preparation the family of diverse analogs of rhodols including heretofore unknown skeletons (Figure 4).
These new dyes absorb at ≈ 530-585 nm and show a strong emission from orange to near-infrared region, with a brightness up to 40,000 M-1 cm-1. Moreover, the new dyes feature an excellent photostability
The π-expansion of diketopyrrolopyrroles (DPPs) is a fantastic tool enabling assembling the fluorophore possessing the right combination of photophysical properties. A fine-tuning the reaction condition and substrates’ structure allowed obtaining a library of these dyes. The strategy consists of double N-arylation of DPPs, followed by double intramolecular direct arylation (Scheme 1). Thanks to the new design we were able to extend emission beyond 700 nm. Moreover it was found that NO2 group present in para position versus the heterocyclic core has no negative effect on the fluorescence, in contrast to the nitro group in ortho position which totally quenches the fluorescence.
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