Nemkovalens kölcsönhatások szerepe biológiai fontosságú vegyületek fényelnyelés hatására végbemenő folyamataiban = Effect of noncovalent interactions on the photoinduced processes of biologically important compounds

Feltártuk, hogy molekulák spontán önszerveződése komplexekké vagy jól definiált sztöchiometriájú aggregátumokká miként befolyásolja a fényelnyelést követő folyamatok kinetikáját és mechanizmusát. Biokompatibilis makrociklusokkal és polielektrolitokkal történő asszociációt tanulmányoztuk, hogy megismerjük a molekulaszerkezet hatását a kötődés erősségére és a fluoreszcenciás sajátságokra. Kukurbiturilok üregébe ékelődéssel módosítani tudtuk fotokróm anyagok tulajdonságait, limikróm tautomerizációját idéztük elő, alkaloid fotooxidációját és nukleofil addiciós átalakulását sikerült gátolnunk, lassítottuk egy merocianin hidrolízisét, megváltoztattuk a saverősséget és a gerjesztett állapotból kiinduló energiavesztés sebességét. Érzékeny fluoreszcenciás módszereket fejlesztettünk ki különféle komplexek kimutatására. Vizsgáltuk a triplett gerjesztett állapot és gyökképződés szerepét alkaloidok és benzoilftálimid származékok fotooxidációs és fotoredukciós reakcióiban. Megállapítottuk, hogy 4-hidroxi-4’-nitrosztilbének fotoiniciált folyamatainak sebessége jelentősen függ az oldószer polaritásától és hidrogen-híd kötő képességétől. 1-Alkil-3-metilimidazólium típusú ionos folyadékok kölcsönhatását vizsgálva kukurbiturilokkal és szulfonátokalixarénekkel összefüggést találtunk a molekulaszerkezet és a keletkezett beékelődési komplexek termodinamikai sajátságai között. | This project revealed how the spontaneous assembly of molecules into complexes or aggregates of well-defined stoichiometry influences the kinetics and mechanism of processes following light absorption. Association with biocompatible macrocycles and polyelectrolytes was studied to understand the effect of molecular structure on the binding strength and the fluorescent properties. It was demonstrated that the inclusion in cucurbiturils modifies photochromic behavior, induces tautomerization of lumichrome, protects alkaloid against photooxidation or nucleophilic addition, hinders the hydrolysis of a merocyanine, alters the acidity, and changes the rate of radiationless energy dissipation from the excited state. Sensitive fluorescence spectroscopic methods were developed to detect the formation of various complexes. The role of the triplet excited state and short-lived radicals in photooxidation, photoreduction of alkaloids and benzoylphthalimide derivatives was clarified. Effect of solvent polarity and hydrogen bonding on the competition among the excited state deactivation pathways of 4-hydroxy-4’-nitrostilbenes was unraveled. The results of systematic studies on the encapsulation of 1-alkyl-3-methylimidazolium type of ionic liquids in cucurbiturils and sulfonatocalixarenes showed how the length of the aliphatic chain of the guest compound and the properties of the macrocycles affect the thermodynamics of inclusion

Kinetics and Mechanism of Cation‐Induced Guest Release from Cucurbit[7]uril

Abstract: The release of two organic guests from cucurbit[7]uril (CB7) was selectively monitored by the stoppedflow method in aqueous solutions of inorganic salts to reveal the mechanistic picture in detail. Two contrasting mechanisms were identified: The symmetric dicationic 2,7- dimethyldiazapyrenium shows a cation-independent complex dissociation mechanism coupled to deceleration of the ingression in the presence of alkali and alkaline earth cations (M$^{+n}$) due to competitive formation of CB7–M$^{+n}$ complexes. A much richer, unprecedented kinetic behaviour was observed for the ingression and egression of the monocationic and non-symmetric berberine (B$^{+}$). The formation of ternary complex B+–CB7–M$^{+n}$ was unambiguously revealed. A difference of more than two orders of magnitude was found in the equilibrium constants of Mn+ binding to B$^{+}$–CB7 inclusion complex. Large cations, such as K$^{+}$ and Ba$^{2+}$, also promoted B$^{+}$ expulsion from the ternary complex in a bimolecular process. This study reveals a previously hidden mechanistic picture and motivates systematic kinetic investigations of other host–guest systems

Kinetics and Mechanism of Cation-Induced Guest Release from Cucurbit[7]uril

The release of two organic guests from cucurbit[7]uril (CB7) was selectively monitored by the stopped-flow method in aqueous solutions of inorganic salts to reveal the mechanistic picture in detail. Two contrasting mechanisms were identified: The symmetric dicationic 2,7-dimethyldiazapyrenium shows a cation-independent complex dissociation mechanism coupled to deceleration of the ingression in the presence of alkali and alkaline earth cations (Mn+) due to competitive formation of CB7-Mn+ complexes. A much richer, unprecedented kinetic behaviour was observed for the ingression and egression of the monocationic and non-symmetric berberine (B+). The formation of ternary complex B+-CB7-Mn+ was unambiguously revealed. A difference of more than two orders of magnitude was found in the equilibrium constants of Mn+ binding to B+-CB7 inclusion complex. Large cations, such as K+ and Ba2+, also promoted B+ expulsion from the ternary complex in a bimolecular process. This study reveals a previously hidden mechanistic picture and motivates systematic kinetic investigations of other host-guest systems

Simultaneous analyte indicator binding assay (SBA) for the monitoring of reversible host–guest complexation kinetics

Very little information is available on the kinetics of the self-assembly and dissociation of optically silent building blocks despite the importance of such data in the rational design of tailor-made host–guest systems. We introduce here a novel time-resolved method that enables the simultaneous determination of complex formation and complex dissociation rate constants for inclusion-type host–guest complexes. The simultaneous analyte indicator binding assay (SBA) gives also direct access to binding affinities, thus largely simplifying the experimental procedure for a full kinetic and thermodynamic characterisation of host–guest systems

Change of the kinetics of inclusion in cucurbit[7]uril upon hydrogenation and methylation of palmatine

The inclusion of protonated (-)-tetrahydropalmatine (THP+) and dehydrocorydaline (DHC+), natural alkaloids, in the cavity of cucurbit[7]uril was monitored in real time by a spectrofluorimetric method in water at various temperatures. Both guests produced 1:1 complexes in enthalpy controlled processes without any detectable intermediates. The tight entrance of CB7 imposed substantial steric hindrance for encapsulation making the entry into the host several orders of magnitude slower than diffusion. Despite the approximate to 6 kJ mol(-1) lower activation enthalpy, the rate constant of THP+ ingression into CB7 was about 44-fold smaller at 298 K than that of DHC+ as a consequence of the considerably negative activation entropy of the former binding. The egression rates of the two studied alkaloids differed to a much lesser extent because the lower energy barrier of THP+ release was almost compensated by the unfavourable activation entropy. In comparison with the kinetics of the reversible confinement of the palmatine parent compound, the presence of the methyl substituent on the aromatic heterocyclic ring in DHC+ barely modified the rate constant of entry into CB7 but caused about 10-fold increase in the dissociation rate at 298 K

In Silico Model Estimates the Clinical Trial Outcome of Cancer Vaccines

Over 30 years after the first cancer vaccine clinical trial (CT), scientists still search the missing link between immunogenicity and clinical responses. A predictor able to estimate the outcome of cancer vaccine CTs would greatly benefit vaccine development. Published results of 94 CTs with 64 therapeutic vaccines were collected. We found that preselection of CT subjects based on a single matching HLA allele does not increase immune response rates (IRR) compared with non-preselected CTs (median 60% vs. 57%, p = 0.4490). A representative in silico model population (MP) comprising HLA-genotyped subjects was used to retrospectively calculate in silico IRRs of CTs based on the percentage of MP-subjects having epitope(s) predicted to bind ≥ 1–4 autologous HLA allele(s). We found that in vitro measured IRRs correlated with the frequency of predicted multiple autologous allele-binding epitopes (AUC 0.63–0.79). Subgroup analysis of multi-antigen targeting vaccine CTs revealed correlation between clinical response rates (CRRs) and predicted multi-epitope IRRs when HLA threshold was ≥ 3 (r = 0.7463, p = 0.0004) but not for single HLA allele-binding epitopes (r = 0.2865, p = 0.2491). Our results suggest that CRR depends on the induction of broad T-cell responses and both IRR and CRR can be predicted when epitopes binding to multiple autologous HLAs are considered

Dimer-Promoted Fluorescence Quenching of Coralyne by Binding to Anionic Polysaccharides

The effects of anionic chondroitin or dextran sulfates on the absorption and fluorescence properties of coralyne, a cationic benzo[c]phenanthridine type alkaloid, were studied in aqueous solution. The remarkably strong binding to both polysaccharides promotes the coralyne dimer formation, which was evidenced by the changes in the absorption and fluorescence spectra and the fluorescence decay. The extent of dimerization, induced by chondroitin, shows a significant pH dependence because the competitive protonation of the carboxylate moieties of the polymer chain decreases the number of binding sites. A larger molecular weight of dextran sulfate stabilizes the coralyne dimer more efficiently

Fluorescent properties of berberine alkaloid in self-assembled systems

Az alkaloidok nagy orvosi, biológiai jelentősége ellenére, nagyon kevés információ áll rendelkezésre a fényelnyelést követő reakcióikról és fotofizikai sajátságaikról. Ez különösen meglepő, hiszen több alkaloidról ismert, hogy fény hatására bomlik és fototoxikus. E farmakológiailag aktív vegyületcsalád sikeres, biztonságos alkalmazásához alapvető fontosságú a fotokémiai átalakulásaik feltárása, és annak megértése, hogy fény hatására milyen kedvezőtlen folyamatokat indítanak az élő szervezetben. Vizsgálataimhoz modellvegyületként egy rákellenes hatású, természetes izokinolin-vázas alkaloidot, a berberint (9,10-dimetoxi-5,6-dihidro-[1,3]dioxolo[4,5-g]izokino[3,2-a]izokinolinilium-klorid) válasz-tottam, mert kimutatására fontos lenne kidolgozni egy érzékeny analitikai módszert, ugyanis vízben elhanyagolható mértékben fluoreszkál. A szupramolekuláris komplexképződést széleskörűen alkalmazzák különböző analitikai eljárások, optikai szenzorok kifejlesztésére, valamint a hidrofób vegyületek, gyógyszerek oldékonyságának a növelésére. A világszerte tapasztalható széleskörű érdeklődés és intenzív kutatómunka ellenére az önszerveződő rendszereket jórészt alapállapotban vizsgálták, kevésbé ismertek a fény hatására lejátszódó átalakulásaik. Munkám során különös figyelmet kívánok fordítani a berberin (B+) szupramolekuláris komplexeiben, aggregátumokban lejátszódó fotoindukált folyamatok megismerésére. Az asszociáció megváltoztatja a gerjesztett állapotok energiáját, ami jelentős változást okozhat az energiavesztési folyamatok sebességében. Szupramolekuláris komplex képzés akadályozhatja a bimolekuláris reakciókat, ezáltal védelmet nyújt a fotokémiai átalakulások ellen. Feltártam a makrociklus gyűrűméretének hatását a berberin4-szulfonáto-kalix[n]arén komplexek sajátságaira. Vizsgálataimat kiterjesztettem, egy viszonylag új, hét glikoluril egységből felépülő makrociklusba, a kukurbit[7]urilba ékelődött B+ vizsgálatára is. A rákellenes és számos egyéb kedvező biológiai hatást mutató B+ iontranszport gátló, kálium-ioncsatorna blokkoló sajátságokkal is rendelkezik, és csökkenti a kloridion kiválasztást. Ezért tanulmányoztam a berberin kation és különféle anionok kölcsönhatását is. Munkámat kiterjesztettem természetes felületaktív anyagok, epesavak aggregációjának tanulmányozására is. Annak ellenére, hogy e vegyületek alapvető szerepet játszanak az élő szervezetben apoláros anyagok vízoldhatóságának növelésében és szállításában, nem ismert olyan fluoreszcenciás jelzőanyag, amely különböző jellegű epesav aggregátumokhoz egyaránt képes kapcsolódni. Olyan fluoreszcenciás jelzőanyagot kerestem, amely detektálni tudja az epesav-aggregátumok szerkezetének és nagyságának változását. Despite the large biological and pharmaceutical importance of alkaloids, very few systematic studies are available in the literature on their light initiated processes. This is particularly surprising because some of them are phototoxic, and it would be desirable to know the photostability and photochemical reactions of this class of compounds before their utilization. The topical application of alkaloids to the skin or eyes raises the possibility that light absorption by these substances may induce adverse reactions in living organism. My dissertation focuses on the fluorescence properties of a clinically important natural isoquinoline alkaloid, berberine (B+), which is a promising drug for the treatment of Alzheimer’s disease, exhibits anticancer, antiviral, antibacterial activity, reduces fever and lowers cholesterol level. It would be important to develop a sensitive analytical method for the quantitative determination of B+, because it has a negligible fluorescence in water. Despite of the biological importance of B+ very few information is available in the literature about its photophysical properties. Supramolecular complex formation of macrocyclic compounds is widely utilized in nanotechnology to develop molecular-scale devices, sensors, and fluorescent probes and to increase the solubility of hydrophobic compounds. Despite the widespread interest in self-organized systems, they have been studied mainly in the ground state, and less attention has been devoted to their excited state. The main goal of the dissertation has been to unveil the most important factors, which should be taken into account when B+ is used as a fluorescent probe. Since spontaneous assembly into aggregates of well-defined structure can induce new energy dissipation pathways or modify the kinetics of the existing excited state relaxation processes, I also intend to reveal how the fluorescence properties of B+ changes upon encapsulation in macromolecules and bile salt aggregates. The change of the fluorescent behavior upon binding to a receptor serves as a basis for biological assays and other highly sensitive analytical procedures. Moreover, supramolecular complex formation may hinder bimolecular reactions. Hereby, the encapsulated compound is protected against photochemical transformation. Therefore, I studied how complex formation with sulfonatocalixarenes (SCXn) of different cavity size and cucurbit[7]uril (CB7) affect the fluorescent behavior of B+ cation. This alkaloid inhibits intestinal ion transport, reduces Clsecretion and can serve as a K+ channel-blocking agent in cells. For this reason, we examined the effect of anions on the fluorescence properties of B+. The medium polarity dependence of the fluorescence characteristics of B+ can be exploited to examine the aggregation of a simple bile salt, sodium cholate (NaC). Despite the fact that the bile salts are the most important natural surfactants, which are responsible for the solubilization of lipids, cholesterol, bilirubin, lecithin, and fat-soluble vitamins in living organisms, no probe has been reported in the literature, which can be encapsulated into both types of bile salt aggregates. Since B+ contains both hydrophilic positively charged and hydrophobic aromatic moieties, we anticipated that B+ might be incorporated in both primary and secondary NaC agglomerates.d