Entropy-Driven Inclusion of Natural Protoberberine Alkaloids in Sulfobutylether-ß-Cyclodextrin.

The understanding of the relationship between molecular structure and the thermodynamics of host-guest binding is essential for the rational design of the applications of inclusion complexes. To obtain insight into the factors governing the driving force of complex formation in aqueous solutions, the encapsulation of five pharmaceutically important protoberberine alkaloids was studied in sulfobutylether-ß-cyclodextrin having on average 6.4 degrees of substitution (SBE6.4ßCD). Spectrophotometric, fluorescence spectroscopic, and isothermal calorimetric measurements showed 1:1 complexation in dilute solutions. From 1.92 × 104 M−1, about an eight-fold decrease of the association constant was observed in the series of berberine ≈ coptisine >> palmatine > epiberberine > dehydrocorydaline. The embedment of these alkaloids in the SBE6.4ßCD cavity was entropy-controlled with mildly negative enthalpy contributions. These findings suggest that the stabilization of the examined complexes arises primarily from the hydrophobic interaction between the constituents. The more than three orders of magnitude smaller association constants of protoberberine alkaloids with SBE6.4ßCD than with cucurbit[7]uril, a host having similar cavity size, originates from the much smaller exothermicity of the confinement in the former macrocycle.

Encapsulation of Metronidazole in Biocompatible Macrocycles and Structural Characterization of Its Nano Spray-Dried Nanostructured Composite.

Due to the great potential of biocompatible cucurbit[7]uril (CB7) and 4-sulfonatocalix[4]arene (SCX4) macrocycles in drug delivery, the confinement of the pharmaceutically important metronidazole as an ionizable model drug has been systematically studied in these cavitands. Absorption and fluorescence spectroscopic measurements gave 1.9 × 105 M-1 and 1.0 × 104 M-1 as the association constants of the protonated metronidazole inclusion in CB7 and SCX4, whereas the unprotonated guests had values more than one order of magnitude lower, respectively. The preferential binding of the protonated metronidazole resulted in 1.91 pH unit pKa diminution upon encapsulation in CB7, but the complexation with SCX4 led to a pKa decrease of only 0.82 pH unit. The produced protonated metronidazole-SCX4 complex induced nanoparticle formation with protonated chitosan by supramolecular crosslinking of the polysaccharide chains. The properties of the aqueous nanoparticle solutions and the micron-sized solid composite produced therefrom by nano spray drying were unraveled. The results of the present work may find application in the rational design of tailor-made self-assembled drug carrier 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.

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.

Teaching indicators to unravel the kinetic features of host-guest inclusion complexes.

Both thermodynamic and kinetic insights are needed for a proper analysis of association and dissociation processes of host-guest interactions. However, kinetic descriptions of supramolecular systems are scarce in the literature because suitable experimental protocols are lacking. We introduce here three time-resolved methods that allow for convenient determination of kinetic rate constants of spectroscopically silent or even insoluble guests with the macrocyclic cucurbit[n]uril family and human serum albumin (HSA) protein as representative hosts.

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.

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 ∼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.

Photooxidation of alkaloids: considerable quantum yield enhancement by rose bengal-sensitized singlet molecular oxygen generation.

The photooxidation of sanguinarine, coralyne and berberine was studied in oxygenated alkaline methanol solutions. Rose bengal as photosensitizer significantly accelerates the process, indicating the importance of singlet molecular oxygen in the reaction mechanism. The quantum yield of sensitized oxidation was found to increase significantly with pH and reaches 0.4 for berberine at pH 13.8. The direct oxidation of alkaloids is less efficient, the quantum yield does not exceed 0.01 even in oxygen-saturated solutions. The photoinduced electron ejection does not play a role in the oxidation. The uncharged pseudobase forms, which are present in alkaline medium, are oxidized much more easily than the alkaloid cations.

Effect of electrolytes, nucleotides and DNA on the fluorescence of flavopereirine natural alkaloid.

The absorption and fluorescence characteristics of flavopereirine, a pharmaceutically important natural alkaloid, were studied to reveal how the complex formation and the change of the microenvironment affect the deactivation kinetics from the singlet-excited state. The fluorescence lifetime was not influenced by the ionic strength, but a significant deuterium effect was observed showing that hydrogen bonding in the singlet-excited state promoted energy dissipation. Nucleotides caused both static and dynamic quenching. The rate constant of the latter process increased when the nucleobase was capable of donating electron to the excited flavopereirine. The spectrophotometric measurements provided evidence for non-cooperative binding to double-stranded DNA with an equilibrium constant of 4.6 × 10(5) M(-1). Time-resolved fluorescence signals showed that three kinds of complexes are formed with distinct fluorescence lifetimes. Flavopereirine binding to chondroitin sulfate was also found, which led to different fluorescence characteristics at pH 2 and 6.

Inclusion complex formation of sanguinarine alkaloid with cucurbit[7]uril: inhibition of nucleophilic attack and photooxidation.

The inclusion of sanguinarine, a biologically active natural benzophenanthridine alkaloid, in cucurbit[7]uril (CB7) was studied by NMR and ground-state absorption spectroscopy, as well as steady-state and time-resolved fluorescence measurements in aqueous solution. The iminium form of sanguinarine (SA(+)) produces very stable 1 : 1 inclusion complex with CB7 (K = 1.0 × 10(6) M(-1)), whereas the equilibrium constant for the binding of the second CB7 is about 3 orders of magnitude smaller. Marked fluorescence quantum yield and fluorescence lifetime enhancements are found upon encapsulation of SA(+) due to the deceleration of the radiationless deactivation from the single-excited state, but the fluorescent properties of 1 : 1 and 1 : 2 complexes barely differ. The equilibrium between the iminium and alkanolamine forms is shifted 3.69 pK unit upon addition of CB7 as a consequence of the preferential encapsulation of the iminium form and the protection of the 6 position of sanguinarine against the nucleophilic attack by hydroxide anion. On the basis of thermodynamic cycle, about 225 M(-1) is estimated for the equilibrium constant of the complexation between the alkanolamine form of sanguinarine (SAOH) and CB7. The confinement in the CB7 macrocycle can be used to impede the nucleophilic addition of OH(-) to SA(+) and to hinder the photooxidation of SAOH.

Photoreduction and ketone-sensitized reduction of alkaloids.

The photoprocesses of berberine, palmatine, coralyne, sanguinarine, flavopereirine and ellipticine were studied in several solvents. The quantum yields Φ(Δ) of singlet molecular oxygen formation of berberine, palmatine and sanguinarine are moderate in dichloromethane (0.2-0.6) and much smaller in acetonitrile or trifluoroethanol. For the other alkaloids examined, Φ(Δ) is rather independent of solvent polarity. The direct and ketone-sensitized photolysis, using steady-state irradiation at 313 nm or 248/308 nm laser pulses, was studied by absorption and fluorescence spectroscopy. Thereby, radicals were observed yielding eventually dihydro derivatives as major products, which are thermally back-converted on admission of oxygen. The quantum yield of conversion of alkaloids to dihydroalkaloids is enhanced in the presence of triethylamine. The reaction in the presence of ketones and electron or H-atom donors has a quantum yield of close to unity.

Considerable change of fluorescence properties upon multiple binding of coralyne to 4-sulfonatocalixarenes.

The interaction of coralyne, an analogue of natural protoberberine alkaloids, with 4-sulfonatocalixarenes (SCXn) was studied in aqueous solution at pH 2 to reveal the major factors determining the stability, stoichiometry, and fluorescent properties of the species formed. Addition of SCXn to coralyne solution brought about remarkable fluorescence intensity diminution and hypochromism in the 300-440 nm absorption domain. SCXn hosts were capable of binding as many coralyne molecules as the number of their hydroxybenzenesulfonate units. The SCXn-promoted interaction among coralyne molecules was evidenced by the appearance of a long-lived fluorescence component. In dilute alkaloid solution, 1:1 and 1:2 coralyne/4-sulfonatocalix[4]arene complexes were formed, but only 1:1 association occurred with 4-sulfonatocalix[8]arene. Time-resolved fluorescence measurements demonstrated that photoinduced electron transfer from a hydroxybenzenesulfonate moiety to the singlet-excited coralyne can compete efficiently with the other deactivation processes.

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.

Inclusion complex formation of ionic liquids and other cationic organic compounds with cucurbit[7]uril studied by 4',6-diamidino-2-phenylindole fluorescent probe.

The encapsulation of 4',6-diamidino-2-phenylindole (DAPI) in the cucurbit[7]uril (CB7) cavity was studied by absorption, fluorescence, and NMR spectroscopic methods in aqueous solution. The profound change in the fluorescence characteristics was attributed to the formation of a very stable 1:1 inclusion complex. Three independent methods provided (1.1+/-0.1)x10(7) M(-1) value for the binding constant. DAPI proved to be an excellent fluorescent probe for the investigation of the competitive binding of ionic liquids, surfactants, and biologically important compounds to CB7. The equilibrium constant of 1-alkyl-3-methylimidazolium inclusion was found to go through a maximum as the aliphatic chain length was increased, reaching the highest value for the hexyl derivative. The variation of the anion had a small effect. Among cationic surfactants containing a dodecyl tail, the stability of CB7 complex diminished with the growing hydrophobicity of the head group.

Berberine alkaloid as a sensitive fluorescent probe for bile salt aggregates.

Effect of sodium cholate (NaC) bile salt on the absorption and fluorescence properties of berberine cation was studied in aqueous solution and water-cosolvent mixtures. The alteration of the fluorescent behavior with increasing NaC concentration showed an entirely different trend from that found previously in the presence of sodium dodecylsulfate. Binding to bile salt agglomerates led to significant fluorescence intensity enhancement, and the fluorescence lifetime of berberine proved to be highly sensitive to the structure and size of the aggregates. The dual exponential decay kinetics above 10 mM NaC concentration showed that the probe resided in two totally different binding sites. At 2-10 mM NaC concentrations, only primary aggregates were detected. The aggregate disrupting power of cosolvents decreased in the series of dimethylformamide, acetonitrile, formamide, and methanol. These compounds enhanced the water accessibility of berberine bound to aggregates and diminished the number of secondary aggregates.