Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10^?5–10^?9 M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10^?4 M. The stimulatory effect of 1,10-phenanthroline manifets itself after 6 h inhubation and increased with time up to 48 h. 2,2′-dipyridyl and 5,6-dimethyl-1-1,10-phemamthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect.Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells.While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyxed controls.These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.     

DNA binding and cleavage activity of [Ru(NH3)4(diimine)]Cl2 complexes

The interaction of a series of mixed ligand complexes of the type [Ru(NH₃)₄(diimine)]Cl₂, where diimine=2,2^′-bipyridine (bipy), 1,10-phenanthroline (phen), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp), 4,7-dimethyl-1,10-phenanthroline (4,7-dmp), 2,9-dimethyl-1,10-phenanthroline (2,9-dmp), 3,4,7,8-tetra-methyl-1,10-phenanthroline (Me₄phen), with calf thymus DNA has been studied using absorption, emission and circular dichroic spectral measurements and viscometry and electrochemical techniques. On interaction with DNA the complexes show hypochromism and red-shift in their MLCT band suggesting that the complexes bind to DNA. The magnitude of the binding constant (K_b) obtained from absorption spectral titration varies depending upon the nature of the diimine ligand: Me₄phen > 5,6-dmp > 4,7-dmp > phen suggesting the use of diimine ‘face’ of the octahedral complexes in binding to DNA. The interaction of phen complex possibly involves phen ring partially inserted into the DNA base pairs. In contrast, the methyl-substituted phen complexes would involve hydrophobic interaction of the phen ring in the grooves of DNA, which is supported by hydrogen bonding interactions of the ammonia ligands with the intrastrand nucleobases. Also the shape and size of the phen ligand as modified by the methyl substituents determine the DNA binding site sizes (0.12-0.45 base pairs). The relative emission intensities (I/I₀) of the DNA-bound complexes parallel the variation in K_b values. Almost all the metal complexes exhibit induced CD bands on binding to B DNA, with the 4,7-dmp and Me₄phen complexes inducing certain structural modifications on the biopolymer. DNA melting curves obtained in the presence of metal complexes reveal a monophasic melting of the DNA strands, the Me₄phen complex exhibiting a slightly enhanced tendency to stabilize the double-stranded DNA. There were slight to appreciable changes in the relative viscosities of DNA, which are consistent with enhanced hydrophobic interaction of the methyl-substituted phen rings. Upon interaction with CT DNA, the Me₄phen, 4,7-dmp and 5,6-dmp complexes, in contrast to bipy, phen and 2,9-dmp complexes, show a decrease in anodic peak current in their cyclic voltammograms suggesting that they exhibit enhanced DNA binding. DNA cleavage experiments show that all the complexes induce cleavage of pBR322 plasmid DNA, the Me₄phen and 5,6-dmp complexes being remarkably more efficient than other complexes.     

Anionic PAMAM dendrimers as drug delivery vehicles for transition metal-based anticancer drugs

The use of anionic half-generation poly(amidoamine) dendrimers as drug delivery vehicles for [Pt(S,S-dach)(5,6-Me₂phen)]²⁺ (56MESS) (where S,S-dach = 1S,2S-diaminocyclohexane; 5,6-Me₂phen = 5,6-dimethyl-1,10-phenanthroline) and [{Δ,Δ-Ru(phen)₂}₂(μ-bb7)]⁴⁺ (Rubb₇) (where phen = 1,10-phenanthroline; bb7 = 1,7-bis[4-(4′-methyl-2,2′-bipyridyl)heptane]) has been studied by nuclear magnetic resonance spectroscopy. From one- and two-dimensional ¹H NMR spectra both 56MESS and Rubb₇ were found to bind to the surface of generation 3.5, 4.5, 5.5 and 6.5 dendrimers through electrostatic interactions. The higher charge and larger size of Rubb₇ resulted in stronger binding to all dendrimer generations (K_b ? 2 × 10⁵ M⁻¹) compared with 56MESS (K_b ? 1 × 10⁴ M⁻¹). Interestingly, there appeared to be no observable trend between dendrimer size and binding constant strength. The size of the free and 56MESS-bound dendrimers were examined using pulsed-gradient spin-echo NMR. The dendrimers ranged in hydrodynamic diameter from 11 to 20 nm and in all cases were larger than their corresponding full-generation dendrimer. Upon the addition of 56MESS the diameter of the dendrimers increased, consistent with surface binding.     

Biotransformation of citronellol by the basidiomycete Cystoderma carcharias in an aerated-membrane bioreactor

The basidiomycete Cystoderma carcharias transformed citronellol into 3,7-dimethyl-1,6,7-octanetriol as the main product. 3,7-Dimethyl-6,7-epoxy-1-octanol was identified as important intermediary product of the biotransformation, and the allylic diols 2,6-dimethyl-2-octene-1,8-diol, 3,7-dimethyl-5-octene-1,7-diol and 3,7-dimethyl-7-octene-1,6-diol were found to be minor products. Microbial formation of rose oxide, a flavour-impact component, was observed for the first time. The formation of the main products was inhibited by 70% after addition of 0.1 mmol l⁻¹ cytochrome monooxygenase inhibitors. Formation of 3,7-dimethyl-1,6,7-octanetriol was effective in a bioreactor with aeration over a coil of a hydrophobic microporous polypropene capillary membrane. Production rates of up to 150 mg l⁻¹ day⁻¹ were reached and led to a product concentration of 866 mg l⁻¹ (conversion rate: 52%). The total loss of the added volatile substrate via the exhaust air was 4.5% when this aeration method was used. Received: 30 July 1998 / Received revision: 2 November 1998 / Accepted: 7 November 1998     

Mixed ligand ruthenium(II) complexes of 5,6-dimethyl-1,10-phenanthroline: The role of ligand hydrophobicity on DNA binding of the complexes

A series of mixed ligand Ru(II) complexes of 5,6-dimethyl-1,10-phenanthroline (5,6-dmp) as primary ligand and 1,10-phenanthroline (phen), 2,2′-bipyridine (bpy), pyridine (py) and NH₃ as co-ligands have been prepared and characterized by X-ray crystallography, elemental analysis and ¹H NMR and electronic absorption spectroscopy. The X-ray crystal structure of the complex [Ru(phen)₂(bpy)]Cl₂ reveals a distorted octahedral coordination geometry for the RuN₆ coordination sphere. The DNA binding constants obtained from the absorption spectral titrations decrease in the order, tris(5,6-dmp)Ru(II) > bis(5,6-dmp)Ru(II) > mono(5,6-dmp)Ru(II), which is consistent with the trend in apparent emission enhancement of the complexes on binding to DNA. These observations reveal that the DNA binding affinity of the complexes depend upon the number of 5,6-dmp ligands and hence the hydrophobic interaction of 5,6-dimethyl groups on the DNA surface, which is critical in determining the DNA binding affinity and the solvent accessibility of the exciplex. Among the bis(5,6-dmp)Ru(II) complexes, those with monodentate py (4) or NH₃ (5) co-ligands show DNA binding affinities slightly higher than the bpy and phen analogues. This reveals that they interact with DNA through the co-ligands while both the 5,6-dmp ligands interact with the exterior of the DNA surface. All these observations are supported by thermal denaturation and viscosity measurements. Two DNA binding modes - surface/electrostatic and strong hydrophobic/partial intercalative DNA interaction - are suggested for the mixed ligand complexes on the basis of time-resolved emission measurements. Interestingly, the 5,6-dmp ligands promote aggregation of the complexes on the DNA helix as a helical nanotemplate, as evidenced by induced CD signals in the UV region. The ionic strength variation experiments and competitive DNA binding studies on bis(5,6-dmp)Ru(II) complexes reveal that EthBr and the partially intercalated and kinetically inert [Ru(phen)₂(dppz)]²⁺ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine) complexes revert the CD signals induced by exciton coupling of the DNA-bound complexes with the free complexes in solution.     

Synthesis, spectroscopic, and cytotoxicity studies of some diamine and diimine platinum(II) complexes of diethyldithiocarbamate

Four new water soluble complexes of the formula [Pt(DA)(DDTC)]NO3 (where DA is 2,2'-bipyridine, 1,10-phenanthroline, 1,2-diaminopropane, or 1,2-diaminocyclohexane, and DDTC is diethyldithiocarbamate anion) have been synthesized by reaction of platinum-diamine/diimine diaqua complex with sodium diethyldithiocarbamate in molar ratio of 1:1. These complexes have been characterized by the chemical analysis, and ultraviolet-visible, infra-red and 1H NMR spectroscopy. The infrared and 1H NMR spectral studies of these complexes have ascertained the modes of binding of diamine/diimine and diethyldithiocarbamate to platinum. The molar conductance values of these platinum complexes in conductivity water suggest them to be 1:1 electrolytes. These four complexes and two other complexes containing ethylenediamine and 1,3-diaminopropane ligands have been tested against P-388 lymphocytic leukemic cells. Out of them only 2,2'-bipyridine and 1,10-phenanthroline complexes show 1.D.50 values less than cisplatin.     

Influence of steric and electronic factors in the stabilization of five-coordinate ethylene complexes of platinum(II): X-ray crystal structure of [PtCl2(2,9-dimethyl-1,10-phenanthroline-5,6-dione)]

Reactions of Zeise’s salt (K[Pt(η²-C₂H₄)Cl₃]) with oxidized phenanthroline ligands (1,10-phenanthroline-5,6-dione, phedon, and 2,9-dimethyl-1,10-phenanthroline-5,6-dione, Me₂phedon) are reported. Comparison with analogous reactions involving unoxidized phen (1,10-phenanthroline) and Me₂phen (2,9-dimethyl-1,10-phenanthroline) ligands indicates that these latter ligands are less capable to stabilize the five-coordinate species [PtCl₂(η²-C₂H₄)(phenanthroline)] in which the phenanthroline and the olefin share the trigonal plane and two chlorines are in the axial positions. The X-ray structure of the four-coordinate species [PtCl₂(Me₂phedon)] indicates that the major difference between oxidized and unoxidized phenanthrolines is the loss of aromaticity of the central ring of phenanthroline. As a consequence, the oxidized phenanthroline becomes more flexible and can undergo a bow-like distortion so to reduce steric interaction between ortho substituents of phenanthroline and cis chlorine ligands. The increase in stability of the four-coordinate species with Me₂phedon is concomitant with an increase in stability of the five-coordinate precursor complex with ethylene. In the latter case the stabilization is not of sterical origin but stems from reduced electron-donor properties of oxidized phenanthrolines. The balance of the two effects is such that the equilibrium between five- and four-coordinate species is more shifted in favour of the former species in the case of Me₂phedon than in the case of Me₂phen.     

A polarographic, oxygen-selective, vibrating-microelectrode system for the spatial and temporal characterisation of transmembrane oxygen fluxes in plants

 A simple procedure is described for the fabrication of micrometer to nanometer-scale platinum electrodes to be used in a vibrating oxygen-selective system. The electrode was prepared by etching a fine platinum wire and insulating it with an electrophoretic paint. The dimensions allowed this electrode to be used with the “vibrating probe technique” in exploratory studies aimed at mapping and measuring the patterns of net influxes as well as effluxes of oxygen in Olea europaea L. leaves and roots with spatial and temporal resolutions of a few microns and a few seconds, respectively. The magnitude and spatial localisation of O₂ influxes in roots was characterised by two distinct peaks. The first, in the division zone, averaged 38 ± 5 nmol m⁻² s⁻¹; the second, in the elongation region, averaged 68 ± 6 nmol m⁻² s⁻¹. Long-term records of oxygen influx in the elongation region of the root showed an oscillatory regime characterised by a fast oscillation with periods of about 8–9 min. In leaves, the system allowed the measurement of real-time changes in O₂ evolution following changes in light. Furthermore, it was possible to obtain “topographical” images of the photosynthetically generated oxygen diffusing through different stomata from a region of the leaf of 120 μm × 120 μm. The combination of topographic and electrochemical information at the micrometer scale makes the system an efficient tool for studying biological phenomena involving oxygen diffusion. Received: 12 November 1999 / Accepted: 1 February 2000     

A Series of Novel Rare Earth Molybdotungstosilicate Heteropolyoxometalates Binding to Bovine Serum Albumin: Spectroscopic Approach

Heteropolyoxometalate complexes have been widely applied in many fields. In this paper, the interaction between a series of novel rare earth molybdotungstosilicate heteropolyoxometalates, K₁₀H₃[Ln(SiMo₆W₅O₃₉)₂]·xH₂O (abbr. LnW₅, Ln = Pr (x = 30), Gd (x = 29), Dy (x = 28), and Yb (x = 31)), and bovine serum albumin (BSA) was investigated by spectroscopic approach under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by LnW₅ is a result of the formation of LnW₅–BSA complex. Fluorescence quenching constants were determined using the Stern–Volmer equation to provide a measure of the binding affinity between LnW₅ and BSA. The binding affinity ranked in the order GdW₅ > DyW₅ > PrW₅ > YbW₅. The results of thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures indicate that van der Waals interactions and hydrogen bonds play a major role for LnW₅–BSA association. Furthermore, the distance r between donor (BSA) and acceptor (LnW₅) was obtained according to fluorescence resonance energy transfer.     

Synthesis, spectroscopic and structural characterization of the reaction products of quaternary cationic 2,2′-bipyridylium ligand bromide salts with metal halides

The synthesis and spectroscopic characterization of quaternary salts containing 2,2,-bipyridine, 1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline and 3,4,7,8-tetramethyl-1,10-phenanthroline is reported. The coordinating behaviour of the bipyridylium salts toward palladium, copper and dimethyltin(IV)halides is investigated. The reaction of N-R,2,2′-bipyridylium (= L¹) bromide (R = 4-Cl-C₆H₄ · CO · CH₂) with palladium(II) chloride in acetonitrile solution yields a crystalline product, shown by a single crystal X-ray study to be L¹PdX₃, where X is a Cl/Br (1:1) composite.     

Metal-binding and active-site structure of di-zinc Streptomyces griseus aminopeptidase

 Streptomyces griseus aminopeptidase has been characterized to have a dinuclear active site and to follow a dinuclear hydrolytic mechanism by means of activity assay, optical, and NMR spectroscopy. A sequential binding of Co²⁺ to the dinuclear sites in 20 mM Mes buffer at pH 6.1 has also been established. The results from these studies suggest that the two metal sites have a five-coordination sphere, with at least one coordinated His each. A di-Cu²⁺-substituted derivative of the enzyme has been prepared which exhibits a ¹H NMR spectrum with sharp hyperfine-shifted signals, again indicating the presence of a dinuclear active site. This ¹H NMR spectrum with sharp hyperfine-shifted features represents a first of its kind for a di-Cu²⁺ center in metalloproteins. Received: 19 May 1997 / Accepted: 4 September 1997     

Molecular dynamics simulations of nafion and sulfonated poly ether sulfone membranes II. Dynamic properties of water and hydronium

We measured the self-diffusion coefficients of water in a Nafion membrane and two sulfonated polyethersulfone (SPES) membranes with varying ion-exchange capacities (IEC) in terms of relative humidity using the pulse field gradient NMR (PFG-NMR) technique. The self-diffusion coefficients were plotted against the number of water molecules per sulfonic acid group, λ, and compare these values with the results of molecular dynamics (MD) simulations. Classical MD simulations for all membranes were carried out using a consistent force field at λ = 3, 6, 9, 12, and 15. The dynamic properties of water (H₂O) and hydronium (H₃O⁺) on a molecular level were estimated as self-diffusion coefficients and residence times around a sulfonate group ( \textSO₃^- {\text{SO}}_3^{-} ). The diffusion coefficients of H₂O and H₃O⁺ followed the order, Nafion > SPES with IEC = 1.4 > SPES with IEC = 1.0 > SPES with IEC = 0.75, which agreed with the experimental data. The residence time distribution of H₂O around \textSO₃^- {\text{SO}}_3^{-} in Nafion was in the range of 1–6 ps, whereas H₂O in the SPES exhibited a residence time of greater than 20 ps.     

Internation of zinc ions with DNA-dependent RNA polymerases A,B and C isolated from calf thymus

Purified DNA-dependent RNA polymerases A, B and C isolated from calf thymus contain a significant amount of zinc. Atomic absorption spectroscopy revealed the presence of 6.7, 5.35 and 2.6–4.1 g-atoms of zinc per mole of polymerase A, B and C, respectively. These enzymes are inhibited by treatment with 1,10-phenanthroline at concentrations varying from 10^-5 to 10^-4 M. However, the addition of zinc ions does not restore fully the activity of 1,10-phenanthroline treated enzymes. Exogenous zinc ions reducein vitro an overall RNA synthesis catalysed by RNA polymerases from calf thymus. In addition to the sites which bind zinc in a specific and stoichiometric way these enzymes possess other classes of binding sites with high and low affinity. Occupancy by exogenous zinc of these additional binding sites inhibits polymerase activity.     

Synthesis,Characterization, Cellular Uptake,Apoptosis, Cytotoxicity,Dna-Binding,and Antioxidant Activity Studies of Ruthenium(II) Complexes

Two new ruthenium(II) polypyridyl complexes [Ru(dmb)₂(HECIP)](ClO₄)₂ (1) (HECIP = N-ethyl-4-[(1,10)-phenanthroline(5,6-f)imidazol-2-yl]carbazole, dmb = 4,4’-dimethyl-2,2’-bipyridine) and [Ru(dmp)₂(HECIP)](ClO₄)₂ (2) (dmp = 2,9-dimethyl-1,10-phenanthroline) have been synthesized and characterized. The DNA-binding behaviors of the two complexes were investigated by absorption spectra, viscosity measurements, and photoactivated cleavage. The DNA-binding constants for complexes 1 and 2 were determined to be 8.03 (± 0.12) × 10⁴ M^?1 (s = 1.62) and 2.97 (± 0.15) × 10⁴ M^?1 (s = 1.82), respectively. The results suggest that these complexes interact with DNA through intercalative mode. The photocleavage of pBR322 DNA by Ru(II) complexes was investigated. The cytotoxicity of complexes 1 and 2 has been evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)] method. Complex 1 shows higher anticancer potency than 2 against the four tumor cell lines. Apoptosis and cellular uptake were investigated. The antioxidant activities of the ligand and these complexes were also performed.     

The TpG chelate of cis(diammineplatinum) forms two head-to-head rotamers in H2O solution

 d(TpG)^– reacts with cis-[Pt(NH₃)₂(H₂O)₂]²⁺ in two steps to yield the platinum chelate cis-[Pt(NH₃)₂{d(TpG)-N3(1),N7(2)}]. In the latter, hindered rotation of the bases leads to an equilibrium between two rotamers interconverting slowly on the NMR time scale. The structure of the two rotameric chelates was studied by means of ¹H NMR and molecular modeling techniques. The major and minor rotamers could be assigned unambiguously to the two head-to-head conformational domains which are characterized by syn/anti and anti/anti sugar-base orientations, respectively. Molecular models derived for both rotamers show that the orientations of the bases are mutually quasi-enantiomeric. The interconversion between the two rotamers (k ≈ 1 s^–1 at 293 K) is approximately 10⁴ times faster than the analogous rotamer interconversion observed in cis-[Pt(NH₃)₂{r(CpG)-N3(1),N7(2)}]⁺ [Girault J-P, Chottard G, Lallemand J-Y, Huguenin F, Chottard J-C (1984) J Am Chem Soc 106 : 7227–7232], suggesting that the steric clash of the exocyclic amino group of the platinum-bound cytosine with the ligands in cis position is more severe than that of the two thymine oxo groups. Received: 23 June 1997 / Accepted: 30 September 1997     

Selectivity at a three-base bulge site in the DNA binding of ΔΔ-[{Ru(phen)2}2(μ-dppm)]4+ [dppm is 4,6-bis(2-pyridyl)pyrimidine; phen is 1,10-phenanthroline]

The binding of the stereoisomers of [{Ru(phen)₂}₂(μ-bpm)]⁴⁺, [{Ru(phen)₂}₂(μ-dppm)]⁴⁺ and [{Ru(phen)₂}₂(μ-bb)]⁴⁺ {phen is 1,10-phenanthroline; bpm is 2,2′-bipyrimidine, dppm is 4,6-bis(2-pyridyl)pyrimidine, bb is 1,2-bis[4-(4′-methyl-2,2′-bipyridyl)]ethane} to an oligonucleotide duplex [d(GCATCGAAAGCTACG)•d(CGTAGCCGATGC)] containing a three-base bulge has been studied using a fluorescence intercalator displacement assay. Of the dinuclear ruthenium complexes, the dppm-linked species showed the strongest binding to the oligonucleotide, with the ΔΔ isomer binding slightly more strongly than the meso isomer and the ΛΛ isomer exhibiting the weakest binding. In order to determine whether the ΔΔ-[{Ru(phen)₂}₂(μ-dppm)]⁴⁺ metal complex specifically bound at the three-base bulge site, a ¹H NMR study of the binding of the metal complex to the oligonucleotide duplex d(GCATCGAAAGCTACG)•d(CGTAGCCGATGC) was carried out. Although a detailed picture of the metal complex–oligonucleotide association could not be determined from the NMR results owing to the broadening of the resonances from the metal complex and nucleotide residues at the bulge site, the NMR results do indicate that the metal complex specifically binds at the three-base bulge site. The combined results of this study suggest that the dppm-bridged dinuclear ruthenium complexes have considerable potential as probes for the unusual secondary structure obtained by the insertion of a three-base bulge within duplex DNA.     

Analysis of the DNA Interaction of Copper Compounds Belonging to the Casiopeínas® Antitumoral Series

Casiopeínas® are mixed-chelate copper complexes with antitumor tested potential. Their activity, both in vitro and in vivo, as antiproliferative, cytotoxic, and genotoxic drugs has been assessed. Biological results of these copper compounds have deserved some of them entering clinical trials. Significant efforts have been devoted to the in-depth identification of their mechanism of action. Using gel electrophoresis analysis, we have previously shown that the interaction of the Casiopeínas® Cas II-gly, [Cu(4,7-dimethyl-1,10-phenanthroline)(glycinate)]NO₃ with DNA, triggers the cleavage of the biomolecule by a free radical mechanism. In this work, we further study the behavior of different complexes of the same Casiopeínas® series also including glycinate as co-ligand {Cas VI-gly (5,6 dimethyl-1,10-phenanthroline glycinato copper(II) nitrate), Cas VII-gly (1,10-phenanthroline glycinato copper(II) nitrate), and Cas IX-gly (2,2′-bipyridine glycinato copper(II) nitrate)} and of a Casiopeínas® with a different co-ligand (Cas III-Cs; 4,7-dimethyl-1,10-phenanthroline salicylaldehydato-copper(II) nitrate). While all of them produce DNA degradation, the performance in the presence of a radical scavenger suggests the existence of differences in their mechanism of interaction with DNA.     

NMR Reinvestigation of the Caffeine–Chlorogenate Complex in Aqueous Solution and in Coffee Brews

Caffeine complexation by chlorogenic acid (3-caffeoylquinic acid, CAS Number [327-97-9]) in aqueous solution as well as caffeine–chlorogenate complex in freshly prepared coffee brews have been investigated by high-resolution ¹H-NMR. Caffeine and chlorogenic acid self-associations have also been studied and self-association constants have been determined resorting to both classical isodesmic model and a recently introduced method of data analysis able to provide also the critical aggregation concentration (cac). Furthermore, caffeine–chlorogenate association constant was measured. For the caffeine, the average value of the self-association constant determined by isodesmic model (K _i = 7.6 ± 0.5 M⁻¹) is in good agreement with the average value (K _a = 10 ± 1.8 M⁻¹) determined with the method which permits the determination of the cac (8.43 ± 0.05 mM). Chlorogenic acid shows a slight decreased tendency to aggregation with a lower average value of association constants (K _i = 2.8 ± 0.6 M⁻¹; K _a = 3.4 ± 0.6 M⁻¹) and a critical concentration equal to 24 ± 1 mM. The value of the association constant of the caffeine–chlorogenate complex (30 ± 4 M⁻¹) is compatible with previous studies and within the typical range of reported association constants for other caffeine–polyphenol complexes. Structural features of the complex have also been investigated, and the complex conformation has been rediscussed. Caffeine chemical shifts comparison (monomeric, complexed, coffee brews) clearly indicates a significant amount of caffeine is complexed in beverage real system, being chlorogenate ions the main complexing agents.