Tautomeric Switching and Metal‐Cation Sensing of Ligand‐Equipped 4‐Hydroxy‐/4‐oxo‐1,4‐dihydroquinolines

Novel 4‐hydroxyquinoline (4HQ) based tautomeric switches are reported. 4HQs equipped with coordinative side arms (8‐arylimino and 3‐piperidin‐1‐ylmethyl groups) were synthesized to access O‐ or N‐selective chelation of Zn²⁺ and Cd²⁺ ions by 4HQ. In the case of the monodentate arylimino group, O chelation of metal ions induces concomitant switching of phenol tautomer to the keto form in nonpolar or aprotic media. This change is accompanied by selective and highly sensitive fluorometric sensing of Zn²⁺ ions. In the case of the bidentate 8‐(quinolin‐8‐ylimino)methyl side arm, NMR studies in CD₃OD indicated that both Cd²⁺ and Zn²⁺ ions afford N chelation for 4HQ, coexisting with tautomeric switching from quinolin‐4(1H)‐one to quinolin‐4‐olate. In corroboration, UV/Vis‐monitored metal‐ion titrations in toluene and methanol implied similar structural changes. Additionally, fluorescence measurements indicated that the metal‐triggered tautomeric switching is associated with compound signaling properties. The results are supported by DFT calculations at the B3LYP 6‐31G* level. Several X‐ray structures of metal‐free and metal‐chelating 4HQ are presented to support the solution studies.     

Modulating affinities of di-2-picolylamine (DPA)-substituted quinoline sensors for zinc ions by varying pendant ligands

We have developed a series of di-2-picolylamine (DPA)-substituted quinoline sensors, HQ1- 4, bearing a pendant ligand at the 8 position of quinoline. UV-vis spectra of HQ1- 4 showed similar variations to that of HQ5 but with different varying extents upon the titration of zinc ions. Fluorescence intensities of HQ1, HQ3, and HQ4 were enhanced 4-6 times upon the addition of 1 equiv of zinc ions under an aqueous buffer. Somewhat unexpectedly, HQ2 is nonfluorescent in the presence of metal ions, including zinc ions. The affinities of HQ sensors are distributed in a broad range from nanomolarity to femtomolarity by varying the pendant ligands near the coordination unit. More importantly, these new sensors exhibited very high selectivity for Zn(2+) over Na(+), K(+), Mg(2+), and Ca(2+) at the millimolar level and over other transition metal ions at the micromolar level, except for Cd(2+). These findings indicated that the incorporations of the pendant groups exerted no effect on the spectroscopic properties and selectivity of the parent fluorescent sensor, with the exception of HQ2. Finally, X-ray crystal structures of ZnHQ's revealed that the auxiliary pendant groups at the 8 position participated in zinc coordination and were able to tune the affinities of HQ sensors.     

A highly selective fluorescent sensor for distinguishing cadmium from zinc ions based on a quinoline platform

A fluorescent sensor, N-(quinolin-8-yl)-2-(quinolin-8-yloxy)acetamide (HL), based on 8-aminoquinoline and 8-hydroxyquinoline platforms has been synthesized. This sensor displays high selectivity and sensitive fluorescence enhancement to Cd(2+) in ethanol. Moreover, sensor HL can distinguish Cd(2+) from Zn(2+) via two different sensing mechanisms (photoinduced electron transfer for Cd(2+); internal charge transfer for Zn(2+)). The composition of the complex Cd(2+)/HL or Zn(2+)/L(-) has been found to be 1:1, based on the fluorescence/absorption titration and further confirmed by X-ray crystallography.     

Coumarin-derived transformable fluorescent sensor for Zn2+

We report a coumarin-derived fluorescent sensor for Zn(2+) termed CTS. CTS shows excellent binding selectivity for Zn(2+) over competing metal ions due to the transformable ability of CTS, that is the displacement of other metal ions by Zn(2+), which induces transformation of chelation from an amide to an imidic acid tautomeric form.     

A pyrenyl-appended triazole-based calix[4]arene as a fluorescent sensor for Cd2+ and Zn2+

The synthesis and evaluation of a novel calix[4]arene-based fluorescent chemosensor 8 for the detection of Cd(2+) and Zn(2+) is described. The fluorescent spectra changes observed upon addition of various metal ions show that 8 is highly selective for Cd(2+) and Zn(2+) over other metal ions. Addition of Cd(2+) and Zn(2+) to the solution of 8 results in ratiometric measurement.     

An 2-(2'-aminophenyl)benzoxazole-based OFF-ON fluorescent chemosensor for Zn2+ in aqueous solution

A water-soluble fluorescent sensor, 1, based on the "receptor-spacer-fluorophore" [2-(2'-aminophenyl)benzoxazole-amide-2-picolylamine] sensor platform, demonstrates the high sensitivity for Zn(2+) with a 25-fold fluorescence enhancement upon chelation to Zn(2+) and also exhibits high selectivity to Zn(2+) over other metal ions. X-ray crystal structure of Zn(2+) complex reveals that the amide oxygen (O2) cooperates with 2-picolylamine unit (N3, N4) as a receptor bind Zn(2+).     

Single domain metallothioneins: supermetalation of human MT 1a

Metallothioneins are a family of small, cysteine rich proteins that have been implicated in a range of roles including toxic metal detoxification, protection against oxidative stress, and as metallochaperones involved in the homeostasis of both essential zinc and copper. We report that human metallothionein 1a, well-known to coordinate 7 Zn(2+) or Cd(2+) ions with 20 cysteinyl thiols, will bind 8 structurally significant Cd(2+) ions, leading to the formation of the supermetalated Cd(8)-βα-rhMT 1a species, for which the structure is a novel single domain. ESI-mass spectrometry was used to determine the exact metalation status of the βα-rhMT. The derivative-shaped CD envelope of Cd(7)-βα-rhMT [peak extrema (+) 260 and (-) 239 nm] changed drastically upon formation of the Cd(8)-βα-rhMT with the appearance of a sharp monophasic CD band centered on 252 nm, a feature indicative of the loss of cluster symmetry. The structural significance of the eighth Cd(2+) ion was determined from a combination of direct and indirect (113)Cd nuclear magnetic resonance (NMR) spectra. In the case of Cd(8)-βα-rhMT, only four peaks were observed in the direct (113)Cd NMR spectrum. Significantly, while both of the isolated domains can be supermetalated forming Cd(4)-β-rhMT and Cd(5)-α-rhMT, Cd(8)-βα-rhMT and not Cd(9)-βα-rhMT was observed following addition of excess Cd(2+). We propose that both domains act in concert to coordinate the eighth Cd(2+) atom, and furthermore that this interaction results in a coalescence of the two domains leading to collapse of the two-domain structure. This is the first report of a possible single-"superdomain" metallothionein structure for Zn(2+) and Cd(2+) binding mammalian proteins. A computational model of a possible single-domain structure of Cd(8)-βα-rhMT is described.     

The -Cys-Cys- motif in Helicobacter pylori's Hpn and HspA proteins is an essential anchoring site for metal ions

The Hpn and HspA proteins from H. pylori are significant for nickel homeostasis and protect the cells from higher concentrations of external metal ions. Both proteins have a unique histidine- and cysteine-rich domain at the C terminus. The interactions of Ni(2+), Bi(3+), Zn(2+) and Cd(2+) ions with C-terminal Ac-CCSTSDSHHQ-NH(2) and Ac-EEGCCHGHHE-NH(2) fragments from Hpn and the Ac-GSCCHTGNHD-NH(2) sequence from HspA were studied by potentiometry, mass spectrometry, circular dichroism and UV-Vis spectroscopy. Ac-CC-NH(2) was used as a reference peptide. The studies have shown that nickel ions form planar complexes with a {2S(-),N(-)} binding mode. The thiol sulfurs of the -Cys-Cys- motif are also the anchoring sites for Bi(3+), Zn(2+) and Cd(2+) ions. The studied protein fragments have the highest affinity for Bi(3+) ions. The thermodynamic stability of Ni(2+) is much higher then that of Zn(2+).     

A chelation enhanced selective fluorescence sensing of Hg2+ by a simple quinoline substituted tripodal amide receptor

Two tris(2-aminoethyl)amine (tren) based tripodal amide fluoroionophores, 1 and 2, functionalized with quinoline (chelating fluorophore) and naphthalene (non-chelating fluorophore) respectively, are synthesized in good yields. Fluoroionophore 1 shows a selective UV-Vis spectral shift in the case of Hg(2+) in acetonitrile among different metal ions like Li(+), Na(+), Ca(2+), Mg(2+), Cr(2+), Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ag(+). On the other hand, fluoroionophore 2 shows no selectivity towards any of the above metal ions in the UV-Vis study. Furthermore, 1 shows a selective chelation induced fluorescence enhancement in the presence of Hg(2+) whereas 2 shows the enhancement of fluorescence with most of the metal ions via a photoinduced charge transfer mechanism. The naked eye detection of Hg(2+) in an acetonitrile solution of 1 shows a greenish fluorescence upon UV light irradiation. The isolated Hg(2+) complex of 1, 3, shows a similar UV-Vis and fluorescence spectral output as observed from in situ spectroscopic studies of 1 in the presence of Hg(2+). Infra-red (IR) and (1)H- NMR studies also reveal the interaction of Hg(2+) with the quinoline nitrogen atoms as well as with the amide functionality.     

Coordination networks from a bifunctional molecule containing carboxyl and thioether groups

The bifunctional molecule tetrakis(methylthio)-1,4-benzenedicarboxylic acid (TMBD) interacts with the increasingly harder metal ions of Cu (I), Cd (II), and Zn (II) to form the coordination networks of Cu 2TMBD, CdTMBD, and Zn 4O(H 2O) 3(TMBD) 3, where the carboxyl group consistently bonds to metal ions, while the softer methylthio group binds with preference to the softer metal ions (i.e., chelation to Cu (+), single-fold coordination to Cd (2+), and nonbonding to Zn (2+)). Diffuse-reflectance spectra show that the metal-thioether interaction is associated with smaller electronic band gaps of the solid-state networks.     

Ratiometric fluorescent receptors for both Zn2+ and H2PO4(-) ions based on a pyrenyl-linked triazole-modified homooxacalix[3]arene: a potential molecular traffic signal with an R-S latch logic circuit

A ratiometric fluorescent receptor with a C(3) symmetric structure based on a pyrene-linked triazole-modified homooxacalix[3]arene (L) was synthesized and characterized. This system exhibited an interesting ratiometric detection signal output for targeting cations and anions through switching the excimer emission of pyrene from the "on-off" to the "off-on" type in neutral solution. (1)H NMR titration results suggested that the Zn(2+) center of receptor L·Zn(2+) provided an excellent pathway of organizing anion binding groups for optimal host-guest interactions. It is thus believed that this receptor has potential application in sensing, detection, and recognition of both Zn(2+) and H(2)PO(4)(-) ions with different optical signals. In addition, the fluorescence emission changes by the inputs of Zn(2+) and H(2)PO(4)(-) ions can be viewed as a combinational R-S latch logic circuit at the molecular level.     

Development of a novel rhodamine-type fluorescent probe to determine peroxynitrite

A flow injection (FI) method with on-line preconcentration using a mini-column loaded with 8-hydroxyquinoline immobilized on controlled pore glass (CPG-8HQ) is described for the determination of trace metals by ion chromatography (IC) with pyridine-2-6-dicarboxylic acid (PDCA) as the eluent. Copper, cadmium, lead, zinc, nickel and iron were determined at ppb level after post-column derivatization with 4-(2-pyridylazo)-resorcinol (PAR). The detection limits (3sigma) for the FI/IC system were 8.27, 0.89, 0.09, 0.06, 0.09 and 0.07 g l(-1) for Pb(2+), Cd(2+), Cu(2+) Ni(2+), Zn(2+) and Fe(3+), respectively, using 5 ml sample volume. The method was applied to the analysis of Malaysian natural waters.     

Separation and gravimetric determination of cerium and lanthanum with N-m-tolyl-m-nitrobenzohydroxamic acid

Cerium and lanthanum were determined gravimetrically by selective precipitation with N-m-tolyl-m-nitrobenzohydroxamic acid and separated from several metal ions such as Ag(+), Be(2+) , Pb(2+) , Mn(2+) , Cu(2+), Zn(2+) , Cd(2+) , Hg(2+) , Pd(2+) , Ga(3+) A1(3+) , Bi(3+) , Sb(3+), Sn(4+), Ce(3+) , Pr(3+) , Nd(3+) , Ti(4+), Zr(4+), Th(4+), V(5+) , Mo(6+) and U(6+) . The precipitates were weighted directly after drying at 110 degrees . The analytical results indicated the composition of the complexes to be (C(14)H(11)N(2)O(4))(n)M.     

Binding and chemical fractionation of heavy metals in typical peat matter

Laboratory batch studies were conducted to evaluate the binding capacity and the mobility of metal species bound to typical humus peat matter. The identification of phase composition of mineral fractions and functional groups in the organic matter was assessed. The results showed generally high, but different retention capacity and binding strength, suggesting distinct diversity in binding mechanisms, phases and chemical nature of binding sites, depending on the metal species and their input concentrations. In general, the binding capacity of peat for the metal ions studied follows the order: Cr(3+) > Cu(2+) > Zn(2+) > Cd(2+) and results in the decrease of pH in the same order, due to displacement of H(3)O(+) from the peat by metal ions. The highest metal enrichment occurs in fractions F1(EXC), F2(CARB), F4(MRO) and F5(OM) of different binding strength adequate to exchangeable, carbonatic, moderately reducible amorphous Fe-oxide and organic/ sulphidic fractions in soils and sediments. In relation to species distribution in peats, the prevailing part of Cr(3+) is strongly bound in oxidizable organic substrate, while Cu(2+) is highly enriched in the moderately reducible F4(MRO) and the most labile F2(EXC) fractions. Cd(2+) and Zn(2+) are predominantly bound in the labile F1(EXC) and F2(CARB) fractions. Diversity of the predominant binding phases for the studied metals suggests rather weak competition for binding sites between chromium and copper ions; the strongest competition between the sorbed metal ions is anticipated for F1(EXC) and F2(CARB) fractions.     

Metal complexes of N-benzamidoporphyrin: (N-benzimido-meso-tetraphenylporphyrinato)(methanol)zinc(II) methanol solvate and (acetato)(N-benzamido-meso-tetraphenylporphyrinato)cadmium(II) benzene solvate

The crystal structures of N-benzamido-meso-tetraphenylporphyrin (NHCOC(6)H(5)-Htpp; 1), (N-benzimido-meso-tetraphenylporphyrinato)(methanol)zinc(II) [Zn(N-NCOC(6)H(5)-tpp)(MeOH); 2(MeOH)], and (acetato)(N-benzamido-meso-tetraphenylporphyrinato)cadmium(II) [Cd(N-NHCOC(6)H(5)-tpp)(OAc); 3] were established. The coordination sphere around Zn(2+) ion in 2(MeOH) is a distorted trigonal bipyramid with N(2), N(5), and O(2) lying in the equatorial plane, whereas, for Cd(2+) ion in 3, it is a sitting-atop derivative with a distorted trigonal bipyramidal geometry in which the apical site is occupied by atoms N(2) and O(2). Cd in 3 acquires five-coordination with five strong bonds [Cd(1)-N(1) = 2.319(5) A, Cd(1)-N(2) = 2.252(5) A, Cd(1)-N(3) = 2.332(5) A, Cd(1)-O(2) = 2.292(5) A, and Cd(1)-O(3) = 2.317(5) A] and with one secondary intramolecular interaction [Cd(1)...N(4)]. The porphyrin ring in these two complexes is distorted to a large extent. The plane of the three pyrrole nitrogen atoms [i.e., N(1)-N(3)] strongly bonded to Zn(2+) in 2(MeOH) and to Cd(2+) in 3 is adopted as a reference plane 3N. For the Zn(2+) complex, the pyrrole nitrogen bonded to the benzamido (BA) ligand lies in a plane with a dihedral angle of 33.8 degrees with respect to the 3N plane, but for the Cd(2+) complex, this dihedral angle is found to be 31.4 degrees. In the former complex, Zn(2+) and N(5) are located on the different side at -0.08 and 1.39 A from its 3N plane, and in the latter one, Cd(2+) and N(5) are also located on the different side at 1.08 and -1.51 A from its 3N plane. VT NMR ((1)H and (13)C) studies of 3 show that the acetate acts as a bidentate ligand and the OAc(-) exchange does not occur in CD(2)Cl(2). Moreover, the NH proton [i.e., H(5)] of 3 in CD(2)Cl(2) is observed as a sharp singlet at delta = -1.13 ppm with Delta nu(1/2) = 4 Hz at 20 degrees C indicating that the intermolecular proton exchange between water and NH proton is rapid.     

1H and (113)Cd NMR Investigations of Cd(2+) and Zn(2+) Binding Sites on Serum Albumin: Competition with Ca(2+), Ni(2+), Cu(2+), and Zn(2+)

1H and (113)Cd NMR studies are used to investigate the Cd(2+) binding sites on serum albumin (67 kDa) in competition with other metal ions. A wide range of mammalian serum albumins possess two similar strong Cd(2+) binding sites (site A 113-124 ppm; site B 24-28 ppm). The two strong sites are shown not to involve the free thiol at Cys34. Ca(2+) influences the binding of Cd(2+) to isolated human albumin, and similar effects due to endogenous Ca(2+) are observed for intact human blood serum. (1)H NMR studies show that the same two His residues of human serum albumin are perturbed by Zn(2+) and Cd(2+) binding alike. Zn(2+) displaces Cd(2+) from site A which leads to Cd(2+) occupation of a third site (C, 45 ppm). The N-terminus of HSA is not the locus of the two strong Cd(2+) binding sites, in contrast to Cu(2+) and Ni(2+). After saturation of the N-terminal binding site, Cu(2+) or Ni(2+) also displaces Cd(2+) from site A to site C. The effect of pH on Cd(2+) binding is described. A common Cd(2+)/Zn(2+) binding site (site A) involving interdomain His residues is discussed.     

Adsorption of Pb2+, Zn2+, and Cd2+ from waters by amorphous titanium phosphate

In the current study, amorphous titanium phosphate (TiP) was prepared as an adsorbent for heavy metals from waters. Uptake of Pb(2+), Zn(2+), and Cd(2+) onto TiP was assayed by batch tests; a polystyrene-sulfonic acid exchanger D-001 was selected for comparison and Ca(2+) was chosen as a competing cation due to its ubiquitous occurrence in waters. The pH-titration curve of TiP implied that uptake of heavy metals onto TiP is essentially an ion-exchange process. Compared to D-001, TiP exhibits more preferable adsorption toward Pb(2+) over Zn(2+) and Cd(2+) even in the presence of Ca(2+) at different levels. FT-IR analysis of the TiP samples laden with heavy metals indicated that the uptake of Zn(2+) and Cd(2+) ions onto TiP is mainly driven by electrostatic interaction, while that of Pb(2+) ions is possibly dependent upon inner-sphere complex formation, except for the electrostatic interaction. Moreover, uptake of heavy metals onto TiP approaches equilibrium quickly and the exhausted TiP particles could be readily regenerated by HCl solution.     

Coordination geometry of cadmium at the zinc and copper sites of superoxide dismutases: a study using perturbed angular correlation of gamma-rays from excited 111Cd.

111Cd time-differential perturbed gamma-gamma angular correlation (PAC) has been used to investigate the Zn site in yeast and bovine copper and zinc-containing superoxide dismutases by substitution of the zinc ions with excited 111Cd(2+) ions. The PAC spectra obtained from the enzymes in aqueous solution reveal a single coordination geometry of 111Cd(2+) showing that the coordination of 111Cd(2+) to the Zn site in the two subunits is identical. Furthermore, the PAC spectra of the yeast and bovine enzymes show that the Zn sites are very similar in the two enzymes. The PAC experiments show a clear difference depending on whether the copper ion is in the oxidized or the reduced state. In the latter case the results resemble those obtained for derivatives with no metal ion at the Cu site. Hence the coordination geometry of the Zn site in these two situations must be similar, and it is very unlikely that the imidazole ring of His61 bridges the two metal ions in the reduced enzyme. The PAC spectrum of 111Cd(2+) ions at the Zn site with copper(II) ions at the Cu site is in agreement with that predicted by applying the angular overlap model (AOM) to the known crystal structure of the bovine enzyme, with known nuclear quadrupole interactions for the ligands involved. Furthermore results from experiments with copper in the reduced state show that reduction of the copper ion causes a significant change at the Zn site. An explanation for this conformational change has been proposed by computer modelling. The PAC experiments also show that it is possible to incorporate cadmium ions into the Cu site in the absence of copper ions, and the result has also been interpreted in terms of the AOM.     

Discrimination in metal-ion binding to RNA dinucleotides with a non-bridging oxygen or sulfur in the phosphate diester link

Replacement of a non-bridging oxygen in the phosphate diester bond by a sulfur has become quite popular in nucleic acid research and is often used as a probe, for example, in ribozymes, where the normally essential Mg(2+) is partly replaced by a thiophilic metal ion to reactivate the system. Despite these widely applied rescue experiments no detailed studies exist quantifying the affinity of metal ions to such terminal sulfur atoms. Therefore, we performed potentiometric pH titrations to determine the binding properties of pUp((S))U(3-) towards Mg(2+), Mn(2+), Zn(2+), Cd(2+), and Pb(2+), and compared these data with those previously obtained for the corresponding pUpU(3-) complexes. The primary binding site in both dinucleotides is the terminal phosphate group. Theoretically, also the formation of 10-membered chelates involving the terminal oxygen or sulfur atoms of the (thio)phosphate bridge is possible with both ligands. The results show that Mg(2+) and Mn(2+) exist as open (op) isomers binding to both dinucleotides only at the terminal phosphate group. Whereas Cd(pUpU)(-) only exists as Cd(pUpU)(-)(op), Cd(pUp((S))U)(-) is present to about 64 % as the S-coordinated macrochelate, Cd(pUp((S))U)(-)(cl/PS). Zn(2+) forms with pUp((S))U(3-) three isomeric species, that is, Zn(pUp((S))U)(-)(op), Zn(pUp((S))U)(-)(cl/PO), and Zn(pUp((S))U)(-)(cl/PS), which occur to about 33, 12 (O-bound), and 55 %, respectively. Pb(2+) forms the 10-membered chelate with both nucleotides involving only the terminal oxygen atoms of the (thio)phosphate bridge, that is, no indication of S binding was discovered in this case. Hence, Zn(2+) and Cd(2+) show pronounced thiophilic properties, whereas Mg(2+), Mn(2+), and Pb(2+) coordinate to the oxygen, macrochelate formation being of relevance with Pb(2+) only.     

Isomorphic coordination polymers of cobalt(II), zinc(II) with a flexible ligand of cis,cis,cis-1,2,3,4-cyclopentanetetracarboxylic acid and their molecular alloys: crystal structures, thermal decomposition mechanisms and magnetic properties

Three new isomorphic coordination polymers of Co(2+), Zn(2+) ions with flexible multicarboxylic acid ligand of the cis,cis,cis-1,2,3,4-cyclopentanetetracarboxylic acid (H(4)L), [Co(4)L(2)(H(2)O)(8)]·3H(2)O (1), [Zn(4)L(2)(H(2)O)(8)]·3H(2)O (2) and [Co(0.8)Zn(3.2)L(2)(H(2)O)(8)]·3H(2)O (3), have been synthesized under hydrothermal conditions and by means of controlling the pH of the reaction mixtures (with an initial pH of 6.0 for 1, 4.0 for 2, and 5.0 for 3, respectively). In the crystal of 1, two crystallographically different Co(2+) ions (Co1 and Co2) form a negatively-charged coordination polymeric chain, which contains a centrosymmetric, linear, trinuclear Co(2+) cluster (Co(3)L(2)) subunit; another crystallographically independent Co(2+) ion (Co3) coordinated to six water molecules acts as a counter ions to link the neighboring coordination polymeric chains via intermolecular H-bond interactions. The Co(2+) ions in 1 were completely and partially replaced by Zn(2+) ions to give 2 and 3, respectively. Complex 3 shows a novel molecular alloy nature, due to the random distributions of the Co(2+) and Zn(2+) ions. Three isomorphic complexes exhibit distinct thermal decomposition mechanisms. The deprotonated cis,cis,cis-1,2,3,4-cyclopentanetetracarboxylic acid ligands decompose at 420-750 °C to give the residue CoO in 1, ZnO + C in 2 and CoO + ZnO in 3. Complex 1 shows a complicated magnetic behavior with co-existence of antiferromagnetic exchange interactions between neighboring Co(2+) ions as well as strong spin-orbital coupling interactions for each Co(2+) ion; complex 3 exhibits a magnetically isolated high-spin Co(2+) ion behavior with strong spin-orbital coupling interactions.