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© 2021 Elsevier B.V. All rights reserved., Staphylococcus aureus medical devices related-infections, such as blood stream catheter are of major concern. Their prevention is compulsory and strategies, not prone to the development of resistance, to prevent S. aureus biofilms on catheter surfaces (e.g. silicone) are needed. In this work two different approaches using sophorolipids were studied to prevent S. aureus biofilm formation on medical grade silicone: i) an antiadhesive strategy through covalent bond of sophorolipids to the surface; ii) and a release strategy using isolated most active sophorolipids. Sophorolipids produced by Starmerella bombicola, were characterized by UHPLC-MS and RMN, purified by automatic flash chromatography and tested for their antimicrobial activity towards S. aureus. Highest antimicrobial activity was observed for C18:0 and C18:1 diacetylated lactonic sophorolipids showing a MIC of 50 μg mL-1. Surface modification with acidic or lactonic sophorolipids when evaluating the anti-adhesive or release strategy, respectively, was confirmed by contact angle, FTIR-ATR and AFM analysis. When using a mixture of acidic sophorolipids covalently bonded to silicone surface as antiadhesive strategy cytocompatible surfaces were obtained and a reduction of 90 % on biofilm formation was observed. Nevertheless, if a release strategy is adopted with purified lactonic sophorolipids a higher effect is achieved. Most promising compound was C18:1 diacateylated lactonic sophorolipid that showed no cellular viability reduction when a concentration of 1.5 mg mL-1 was selected and a reduction on biofilm around 5 log units. Results reinforce the applicability of these antimicrobial biosurfactants on preventing biofilms and disclose that their antimicrobial effect is imperative when comparing to their antiadhesive properties., The authors thank Fundação para a Ciência e Tecnologia (FCT) for the financial support under Projects PTDC/BTM-SAL/29335/2017, UIDB/04138/2020, UIDP/04138/2020 (iMed.ULisboa) and Portugal 2020 for the Portuguese Mass Spectrometry Network (Rede Nacional de Espectrometria de Massa RNEM; LISBOA 01 0145 FEDER 402 022125).

Biosurfactants have been investigated as potential alternatives for synthetic surfactants in several areas, for example, in environmental and pharmaceutical fields. In that regard, extensive research has been carried out with sophorolipids and rhamnolipids that also present various biological properties with therapeutic significance. These biosurfactants are obtained as complex mixtures of slightly different molecules, and thus when studying these microbial glycolipids, the ability to identify and purify the produced compounds is of extreme importance. This study aimed to develop improved methodologies for the identification, separation, and purification of sophorolipids and rhamnolipids. Therefore, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was modified to ensure faster characterization of both sophorolipids and rhamnolipids, enabling the identification and fragmentation pattern description of 10 and 13 congeners, respectively. The separation and purification of these biosurfactants was achieved with novel reversed-phase solid-phase extraction methods guaranteeing the isolation of different glycolipids, including those considered for their significant biological activity (e.g. antimicrobial, anticancer). It was possible to isolate sophorolipids and rhamnolipids with purity of 94% and 99%, respectively. The methods presented herein can be easily implemented and are expected to make purification of these biosurfactants easier, facilitating the study of their individual properties in further works.

© 2021 by the authors., The common bean (Phaseolus vulgaris L.) represents a sustainable and affordable source of protein, namely, to populations with vegetarian dietary habits. Despite the national germplasm genetic diversity, little is known about the Portuguese accessions’ nutritional and protein quality, leading to their underuse in breeding programs. To fill this gap, a representative collection (106 accessions) was cropped under two contrasting environments (traditional versus heat stress) and evaluated in terms of nutritional quality by near-infrared spectroscopy. Protein quality was assessed, under the stressful environment, considering the individual amino acid contents and the activity of trypsin inhibitors through mass spectrometry (LC-MS/MS) and spectrophotometry, respectively. On top of strong genotypic control, the nutritional composition (protein, fat, fiber, moisture and ash) was also highly influenced by the environment and by genotype × environment interaction, with a clear nutritional quality ranking change for the accessions in heat stress conditions. Classified into three clusters, the accessions from the cluster with the highest individual amino acid and protein contents also showed higher trypsin inhibitor activity (TIA). Since different levels of TIA had no translation into contrasting protein digestibility, breeders focusing on common beans’ protein quality improvement, especially under challenging warming climate conditions, may take advantage of this group of accessions., This research was funded by FCT, Portugal, through BEGEQA project (PTDC/AGRTEC/3555/2012), E.M. PhD fellowship (SFRH/BD/89287/2012 and R&D unit, UIDB/04551/2020 (GREEN-IT–Bioresources for sustainability), PORTUGAL 2020, grant number LISBOA-01-0145- FEDER-402-022125 and PDR2020 Operação 7.8.4, Recursos genéticos (PDR2020-784-042734).

OBJECTIVE: Dental device is a very broad term that can be used to include any foreign material or product that is introduced in the host oral cavity to replace missing tissues. These devices are subjected to different environments which include dental hard tissues, tissue fluids, blood and saliva. All dental devices are continuously challenged microbiologically and a number of failures in clinical management are related to microbial colonization. Thus, the assessment of the antimicrobial properties of dental devices are extremely important. In this paper, a classification of dental devices is being proposed. This classification distinguishes the devices based on whether they are implantable or not, and also sub-classified based on their specific application and the substrate receiving the device. METHODS AND RESULTS: A literature search was conducted to identify how dental devices have been tested with relation to the microbial strains used and whether the testing has been performed in isolation or reported with other relevant tests such as material characterization and biological activity. The results of the literature review were analyzed and recommendations for antimicrobial testing of dental devices are proposed. These recommendations include the need for the setting up of pre-testing parameters such as ageing and the details of the pre-testing sterilization procedures, as these may affect the material chemistry and the specification for antimicrobial testing to be done with specific single strains or polymicrobial that are native to the region where the device is located are also suggested. Testing can be undertaken in vitro, ex vivo and in vivo. Since the antimicrobial and biological activities influence/condition one another and the material chemistry may affect both the antimicrobial and biological testing this document also makes recommendations regarding biological assessment which can be carried out in isolation or integrated with the microbiological testing and also material testing methods including chemical and physical characterization of bulk, surface, eluted and degraded materials as well as physical characterization methods. SIGNIFICANCE: The level of standardization of antimicrobial testing for the dental devices needs to be based on the device location and host interaction in order to increase the clinical applicability of the mentioned tests.

International audience; A symmetrical squaraine-based ligand, sbdpa, bearing two dipicolylamine (dpa) units, was synthesized for the first time, using an environment friendly procedure, such as one-pot and solvent-free reaction. The sbdpa ligand was found to have a small blue-green emission, in aqueous solution, due to a photoinduced electron transfer (PET) mechanism. Metal complexes of sbdpa with a large panel of cations, Na+, K+, Ag+, Mg2+, Ca2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, and Pb2+, were prepared in aqueous buffered solution and screened, in search of a selective response for one of them. A turn-on fluorescence response was then found only for Hg2+, as a consequence of a chelation-enhanced fluorescence (CHEF) effect that disrupts the PET mechanism in sbdpa. Subsequently, the complexation behaviour of Hg2+ with sbdpa was investigated in aqueous solution by potentiometric titrations and other spectroscopic techniques. The acid-base reactions of the ligand were also studied by potentiometry, as well as by 1H-NMR and UV-vis titrations. For comparison purposes, the complexation of two other divalent cations, Cu2+ and Zn2+, was also assessed by potentiometry. Additionally, results are compared with dpa all along the study. Both mono- and dinuclear complexes of sbdpa were found for the three metal cations studied having the mercury(II) complex the largest thermodynamic stability. These findings support a stronger uptake of Hg2+ cation by sbdpa, possibly involving at least one oxygen atom from the squaraine moiety on its coordination sphere.

Methylmercury (MeHg) is a highly neurotoxic compound to which human populations are exposed via fish consumption. Once in cells, MeHg actively binds thiols and selenols, interfering with the activity of redox enzymes such as thioredoxin (Trx) and the selenoenzyme thioredoxin reductase (TrxR) which integrate the thioredoxin system. In fact, it has been shown that inhibition of this system by MeHg is a critical step in the unfolding of cell death. Current clinical approaches to mitigate the toxicity of MeHg rely on the use of chelators, such as meso-2,3-dimercaptosuccinic acid (DMSA) which largely replaced British anti-Lewisite or 2,3-dimercapto-1-propanol (BAL) as the prime choice. However, therapeutic efficacy is limited and therefore new therapeutic options are necessary. In this work, we evaluated the efficacy of a macrocyclic chelator, 1-thia-4,7,10,13-tetraazacyclopentadecane ([15]aneN4S), in preventing MeHg toxicity, namely by looking at the effects over relevant molecular targets, i.e., the thioredoxin system, using both purified enzyme solutions and cell experiments with human neuroblastoma cells (SH-SY5Y). Results showed that [15]aneN4S had a similar efficacy to DMSA and BAL in reversing the inhibition of MeHg over purified TrxR and Trx by looking at both the 5,5&prime, dithiobis(2-nitrobenzoic acid) (DTNB) reduction assay and insulin reduction capability. In experiments with cells, none of the chelating agents could reverse the inhibition of TrxR by MeHg, which corroborates the high affinity of MeHg to the selenol in TrxR active site. [15]aneN4S and BAL, unlike DMSA, could prevent inhibition of Trx, which allows the maintenance of downstream functions, although BAL showed higher toxicity to cells. Overall these findings highlight the potential of using [15]aneN4S in the treatment of MeHg poisoning and encourage further studies, namely in vivo.

The role of metalloproteinases (MMPs) on the migration and invasion of cancer cells has been correlated with tumor aggressiveness, namely with the up-regulation of MMP-2 and 9. Herein, two pyridine-containing macrocyclic compounds, [15]pyN5 and [16]pyN5, were synthesized, chemically characterized and evaluated as potential MMP inhibitors for breast cancer therapy using 3D and 2D cellular models. [15]pyN5 and [16]pyN5 (5&ndash, 20 &micro, M) showed a marked inhibition of MMPs activity (100% at concentrations &ge, 7.5 &mu, M) when compared to ARP-100, a known MMP inhibitor. The inhibitory activity of [15]pyN5 and [16]pyN5 was further supported through in silico docking studies using Goldscore and ChemPLP scoring functions. Moreover, although no significant differences were observed in the invasion studies in the presence of all MMPs inhibitors, cell migration was significantly inhibited by both pyridine-containing macrocycles at concentrations above 5 &mu, M in 2D cells (p &lt, 0.05). In spheroids, the same effect was observed, but only with [16]pyN5 at 20 &mu, M and ARP-100 at 40 &mu, M. Overall, [15]pyN5 and [16]pyN5 led to impaired breast cancer cell migration and revealed to be potential inhibitors of MMPs 2 and 9.

The nucleus is connected to the cytoskeleton, and these connections are involved in multiple functions such as nuclear positioning, shape and stiffness, cytoskeleton organization, mechanotransduction, gene expression, chromosome positioning, DNA repair, and cell migration.

A novel gene, csi2+ (chromosome segregation impaired 2), is reported. It localizes to the spindle pole body, and its deletion leads to a transient monopolar spindle and subsequent chromosome lagging. It is proposed that csi2p regulates mitotic microtubule length, defects in which may cause kinetochore–microtubule attachment problems., Proper chromosome segregation is of paramount importance for proper genetic inheritance. Defects in chromosome segregation can lead to aneuploidy, which is a hallmark of cancer cells. Eukaryotic chromosome segregation is accomplished by the bipolar spindle. Additional mechanisms, such as the spindle assembly checkpoint and centromere positioning, further help to ensure complete segregation fidelity. Here we present the fission yeast csi2+. csi2p localizes to the spindle poles, where it regulates mitotic microtubule dynamics, bipolar spindle formation, and subsequent chromosome segregation. csi2 deletion (csi2Δ) results in abnormally long mitotic microtubules, high rate of transient monopolar spindles, and subsequent high rate of chromosome segregation defects. Because csi2Δ has multiple phenotypes, it enables estimates of the relative contribution of the different mechanisms to the overall chromosome segregation process. Centromere positioning, microtubule dynamics, and bipolar spindle formation can all contribute to chromosome segregation. However, the major determinant of chromosome segregation defects in fission yeast may be microtubule dynamic defects.

The purpose of this work was to synthesize and characterize the thiatetraaza macrocycle 1-thia-4,7,10,13-tetraazacyclopentadecane ([15]aneN4S). Its acid-base behaviour was studied by potentiometry at 25 °C and ionic strength 0.10 M in KNO3. The protonation sequence of this ligand was investigated by 1H-NMR titration that also allowed the determination of protonation constants in D2O. Binding studies of [15]aneN4S with Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+ and Pb2+ metal ions were further performed under the same experimental conditions. The results demonstrated that this compound has a higher selectivity and thermodynamic stability for Hg2+ and Cu2+, followed by Ni2+. The UV-visible-near IR spectroscopies and magnetic moment data for the Co(II) and Ni(II) complexes indicated a tetragonal distorted coordination geometry for both metal centres. The value of magnetic moment and the X-band EPR spectra of the Cu(II) complex are consistent with a distorted square pyramidal geometry.

The unique redox and catalytic chemistry of Cu has justified the development of novel Cu complexes for different therapeutic uses including cancer therapy. In this work, four pyridine-containing aza-macrocyclic copper(II) complexes were prepared (CuL1–CuL4) varying in ring size and/or substituents and their superoxide scavenging activity evaluated. CuL3, the most active superoxide scavenger, was further studied as a modulator of the cytotoxicity of oxaliplatin in epithelial breast MCF10A cells and in MCF7 breast cancer cells. Our results show that CuL3 enhances the therapeutic window of oxaliplatin, by both protecting non-tumour cells and increasing its cytotoxic effect in breast carcinoma cells. CuL3 is thus a promising complex to be further studied and to be used as a lead compound for the optimization of novel chemotherapy sensitizers.

Human exposure to cadmium (Cd) occurs via different routes, including diet. The increasing amount of data linking Cd with different cellular effects in the mammary gland justifies additional toxicological assessments using human mammary epithelial cells. This work aimed therefore to assess the cytotoxic effects of Cd in MCF10A cells and to characterize the cytoprotective role of the macrocycle [15]pyN 5 in the form of calcium salt. Cadmium chloride revealed to be cytotoxic to MCF10A cells, decreasing cell viability and proliferation in a concentration-dependent manner. Comparable dose–response curves and IC50 values (57–63 μM, 24 h treatment) were obtained using the MTT reduction, crystal violet and BrdU assays. In terms of reactive oxygen species formation, only a slight increase in superoxide radical anion was observed at very high Cd concentrations (⩾100 μM). Chelation should thus constitute the primary strategy to mitigate the cytotoxic effects induced by Cd in mammary cells. In this context, [15]pyN 5 which presents appropriate chemical and thermodynamic features was studied as a Cd chelator. This macrocycle (25 and 50 μM) significantly reduced or even abolished Cd-induced cytotoxicity. Protective effects were observed in terms of cell viability, cell proliferation and morphological alterations, being the protection mostly attributed to a chelating-based mechanism.

A Pb2+-selective sensor was fabricated from polyvinyl chloride (PVC) matrix membranes containing the ionophore 3,7,11-tris (2-pyridylmethyl)-3,7,11,17-tetraazabicyclo [11.3.1] heptadeca-1(17),13,15-triene (I). The effects of anion excluders (KTpClPB, NaTPB) and plasticizers (o-NPOE, DBP, DOP) on the performance of the membrane sensor were studied. The membrane with the composition of I:PVC:o-NPOE:KTpClPB in the percentage ratio (wt.%) of 2:32:60:6 exhibited the best performance, having a slope of 28.5 ± 0.2 mV/decade in the concentration range 10− 6–10− 1 M, a response time of 20 s and a lifetime longer than four months. The sensor was selective for Pb2+ ions over other monovalent, divalent and trivalent interfering cations and could be used in the pH range of 5–8. The high selectivity in the presence of Cd2+ may be important for further studies. The efficiency of the proposed sensor was demonstrated by its application in Pb2+ ions determination in water samples.

The rigid [6]ferrocenophane, L-1, was synthesised by condensation of 1,1'-ferrocene dicarbaldehyde with trans-1,2-diaminocyclohexane in high dilution at r.t. followed by reduction. When other experimental conditions were employed, the [6,6,6]ferrocenephane (L-2) was also obtained. Both compounds were characterised by single crystal X-ray crystallography. The protonation of L-1 and its metal complexation were evaluated by the effect on the electron-transfer process of the ferrocene (fc) unit of L-1 using cyclic voltammetry (CV) and square wave voltammetry (SWV) in anhydrous CH3CN solution and in 0.1 M (Bu4NPF6)-Bu-n as the supporting electrolyte. The electrochemical process of L-1 between 300 and 900 mV is complicated by amine oxidation. On the other hand, an anodic shift from the fc/fc(+) wave of L-1 of 249, 225, 81 and 61 mV was observed by formation of Zn2+, Ni2+, Pd2+ and Cu2+ complexes, respectively. Whereas Mg2+ and Ca2+ only have with L-1 weak interactions and they promote the acid-base equilibrium of L-1. This reveals that L-1 is an interesting molecular redox sensor for detection of Zn2+ and Ni2+, although the kinetics of the Zn2+ complex formation is much faster than that of the Ni2+ one. The X-ray crystal structure of [(PdLCl2)-Cl-1] was determined and showed a square-planar environment with Pd(II) and Fe(II) centres separated by 3.781(1) angstrom. The experimental anodic shifts were elucidated by DFT calculations on the [(MLCl2)-Cl-1] series and they are related to the nature of the HOMO of these complexes and a four-electron, two-orbital interaction.

Synthetic superoxide dismutase mimetics have emerged as a potential novel class of drugs for the treatment of oxidative stress related diseases. Among these agents, metal complexes with macrocyclic ligands constitute an important group. In this work we synthesized five macrocyclic copper(II) complexes and evaluated their ability to scavenge the superoxide anions generated by the xanthine-xanthine oxidase system. Two different endpoints were used, the nitro blue tetrazolium (NBT) reduction assay (colorimetric method) and the dihydroethidium (DHE) oxidation assay (fluorimetric method). IC(50) values in the low micromolar range were found in four out of five macrocyclic complexes studied, demonstrating their effective ability to scavenge the superoxide anion. The IC(50) values obtained with the NBT assay for the macrocyclic copper(II) complexes, were consistently higher, approximately threefold, than those obtained with the DHE assay. Spectroscopic and electrochemical studies were performed in order to correlate the structural features of the complexes with their superoxide scavenger activity. Cytotoxicity assays were also performed using the MTT method in V79 mammalian cells and we found that the complexes, in the range of concentrations tested in the superoxide scavenging assays were not considerably toxic. In summary, some of the presented macrocyclic copper(II) complexes, specially those with a high stability constant and low IC(50), appear to be promising superoxide scavenger agents, and should be considered for further biological assays.

The stability constants of La 3+ , Sm 3+ and Ho 3+ complexes with 13- and 14-membered macrocycles having methylcarboxylate (trita and teta) or methylphosphonate (tritp and tetp) arms were determined. All the ligands were labelled with 153 Sm and 166 Ho in order to evaluate the effect of the macrocyclic cavity size and type of appended arms on their in vitro and in vivo behaviour. The radiolabelling efficiency was found to be higher than 98% for all the complexes, except for those of tetp. All radiocomplexes studied are hydrophilic with an overall negative charge and low plasmatic protein binding. Good in vitro stability in physiological media and human serum was found for all complexes, except the 153 Sm/ 166 Ho–teta, which are unstable in phosphate buffer (pH 7.4). In vitro hydroxyapatite (HA) adsorption studies indicated that 153 Sm/ 166 Ho–tritp complexes bind to HA having the 166 Ho complex the highest degree of adsorption (>80%, 10 mg). Biodistribution studies in mice demonstrated that 153 Sm/ 166 Ho–trita complexes have a fast tissue clearance with more than 95% of the injected activity excreted after 2 h, value that is comparable to the corresponding dota complexes. In contrast, the 153 Sm–teta complex has a significantly lower total excretion. 153 Sm/ 166 Ho–tritp complexes are retained by the bone, particularly 166 Ho–tritp that has 5–6% (% I.D./g) bone uptake and also a high rate of total excretion. Thus, these studies support the potential interest of 153 Sm/ 166 Ho–trita complexes for therapy when conjugated to a biomolecule and the potential usefulness of the 166 Ho–tritp complex in bone pain palliation.

The protonation constants of two series of tetraazamacrocyclic ligands with acetate and methylphosphonate pendant arms, as well as their stability constants with Cu2+, La3+, Sm3+, and Ho3+, were determined. All the values were determined in aqueous solution at 298.0 K and 0.10 mol dm–3 in N(CH3)4NO3. In the first series, the effect of both types of pendant arms was observed by appending them in the same macrocyclic backbone, a 14-membered tetraazamacrocycle containing pyridine (ac3py14, p2py14, and p3py14). In the second series, two effects were taken into account, the increase of the cavity size of the macrocycle, from 12- to 14-membered, and the appending of acetate (dota, trita, and teta) or methylphosphonate (dotp, tritp, and tetp) arms. The ligands containing methylphosphonate arms have higher thermodynamic stability compared to the corresponding ones with acetate arms, especially in the series of compounds containing pyridine, even upon correction of the different basicity values of the ligands. On the other hand, the ligands with smaller macrocyclic cavity size, namely, dota and dotp, exhibit the largest values of stability constants. In contrast, ac3py14 presents low stability constants with lanthanides. An interpretation of these features based on the known adopted arrangement of dota and teta when free or coordinated with lanthanides is evaluated.

A set of tetraaza macrocycles containing pyridine and methylcarboxylate (ac(3)py14) or methylphosphonate (MeP(2)py14 and P(3)py14) pendant arms were prepared and their stability constants with La(3+), Sm(3+), Gd(3+) and Ho(3+) determined by potentiometry at 25 degrees C and 0.10 M ionic strength in NMe(4)NO(3). The metal:ligand ratio for (153)Sm and (166)Ho and for ac(3)py14, MeP(2)py14 and P(3)py14, as well as the pH of the reaction mixtures, were optimized to achieve a chelation efficiency higher than 98%. These radiocomplexes are hydrophilic and have a significant plasmatic protein binding. In vitro stability was studied in physiological solutions and in human serum. All complexes are stable in saline and PBS, but 20% of radiochemical impurities were detected after 24 h of incubation in serum. Biodistribution studies in mice indicated a slow rate of clearance from blood and muscle, a high and rapid liver uptake and a very slow rate of total radioactivity excretion. Some bone uptake was observed for complexes with MeP(2)py14 and P(3)py14, which was enhanced with time and the number of methylphosphonate groups. This biological profile supports the in vitro instability found in serum and is consistent with the thermodynamic stability constants found for these complexes.

Multiple mechanisms related to metastases undergo redox regulation. Cu[15]pyN5 is a redox-active copper(II) complex previously studied as a chemotherapy sensitizer in mammary cells. The effects of a cotreatment with Cu[15]pyN5 and doxorubicin (dox) were evaluated in two human breast cancer cell lines: MCF7 (low aggressiveness) and MDA-MB-231 (highly aggressive). Cu[15]pyN5 decreased MCF7-directed cell migration. In addition, a cotreatment with dox and Cu[15]pyN5 reduced the proteolytic invasion of MDA-MB-231 cells. Cell detachment was not affected by exposure to these agents. Cu[15]pyN5 and dox significantly increased intracellular ROS in both cell lines. This increase could be at least partially due to H2 O2 accumulation. The combination of Cu[15]pyN5 with dox may be beneficial in breast cancer treatment as it could help reduce cancer cell migration and invasion. Moreover, the ligand [15]pyN5 has a high affinity for copper(II) and displays potential anti-angiogenic properties. Overall, we present a potential drug that might arrest the progression of breast cancer by different and complementary mechanisms.

The novel tetraaza-macrocyclic compound 3,7,11-tris(carboxymethyl)-3,7,11,17-tetraaza-bicyclo[11.3.1]hep tadeca-1(17),13,15-triene, abbreviated as ac 3 py14, was investigated for its activity against Mycobacterium tuberculosis and for induction of protective cellular immune responses. Perspective results show that ac 3 py14 and its Fe 3+ 1:1 complex, [Fe(ac 3 py14)], inhibited radiometric growth of several strains of M. tuberculosis . Inhibition with 25 μg/mL varied from 99% for H37Rv to 80% and above for multiple drug-resistant clinical isolates. The capacity of ac 3 py14 to elicit a beneficial immune response without cellular apoptosis was assessed and compared to the effects of virulent M. tuberculosis . The present study produces evidence that after stimulation with ac 3 py14 there was significant production of interferon gamma (IFN- γ ), whereas the production of interleukin-5 (IL-5) remained low, and there was development of a memory population (CD45RO). The level of binding of Annexin V, a marker of apoptosis, was not sufficient to result in toxic effects toward αβ and γδ T cells and CD14 + macrophages. This preliminary study is the first report of a compound that simultaneously exerts an inhibitory effect against M. tuberculosis and induces factors associated with protective immune responses.

A new 14-membered oxatriaza macrocycle containing pyridine has been synthesized, 7-oxa-3,11,17-triazabicyclo[11.3.1]heptadeca-1(17),13,15-triene, L1. The protonation constants of this compound and stability constants of its complexes with the Mn2+ to Zn2+, Cd2+, and Pb2+ were determined by potentiometric methods at 25°C and ionic strength 0.10 Mol dm−3 in KNO3. L1 presents two high values of protonation constants, and the third is very low. Its overall basicity is low because the three basic centres are in close proximity. Only mononuclear complexes were found, and the stability constants with all the metal ions studied are of the same order as those of the corresponding complexes of the oxatriaza macrocycle L2 (1-oxa-4,8,12-triazacyclotetradecane), but lower than those of tetraaza macrocycles of similar or lower cavity size. The electronic spectra together with the values of magnetic moments of the cobalt(II) and nickel(II) complexes of L1 suggest that five co-ordinate species are formed in aque...

A new redox-responsive receptor, 3,11-bis(ferrocenylmethyl)-7-methyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene, L-1, has been synthesized. The protonation constants of this compound and the stability constants of its complexes with Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ were determined at 25.0 degrees C, in methanol-water (1:1, v/v), and at ionic strength 0.10 mol dm(-3) in KNO3. The values of the protonation constants of L-1 are similar to those of the parent macrocycle, 7-methyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene L-2, except for K-2 which is 1.82 log units lower than that of L-2. Structural reasons are proposed to explain this behaviour. The stability constants of the metal complexes of L-1 are lower than those of L-2 as expected on the basis of its lower overall basicity, but the Cd2+ complex is exceptional exhibiting a higher stability for L-1. The complexation of L-1 with different metals shifts anodically the ferrocene-ferrocenium half-wave potential in relation to that of the free compound, the largest shift being observed for Cu2+, followed by Ni2+, and then Zn2+ and Cd2+. No shift was observed for Pb2+. It was verified that L-1 is a copper-selective sensor in the presence of Ni2+, Zn2+, Cd2+ and Pb2+ and although it is not the first compound for which this property is claimed it is probably the easiest to synthesize. The electronic spectra of [CuL1](2+) reveal that the four nitrogen atoms of the macrocycle form a square-planar arrangement with a pronounced tetrahedral distortion. The single crystal structures of [(CuLCl)-Cl-1][(CuLI)-I-1]Cl-2. 1.25H(2)O 1 and [(ZnLI)-I-1]Cl . 2H(2)O 2 have shown that these complex cations have distorted square pyramidal co-ordination spheres, the basal being planes formed by the four nitrogen atoms of the macrocyclic framework in 1a(+) and 1b(+) while in 2(+) it is defined by three nitrogen atoms of L-1 and one iodine atom. The apical positions are occupied by a chlorine atom in 1a(+) and by an iodine atom in 1b(+), and by the nitrogen donor atom of the macrocycle trans to the pyridine ring in complex 2(+). To achieve the geometric arrangement described for 2(+) the macrocycle folds considerably through the line defined by the two nitrogen atoms contiguous to the pyridine group. It was also found that the intramolecular distances between the ferrocene groups and transition metal receptor centres studied play an important role in the redox behaviour of these complexes.

Microtubules exhibit dynamic instability, stochastically switching between infrequent phases of growth and shrinkage. In the cell, microtubule dynamic instability is further modulated by microtubule-associated proteins and motors, which are specifically tuned to cell cycle stages. For example, mitotic microtubules are more dynamic than interphase microtubules. The different parameters of microtubule dynamics can be measured from length versus time data, which are generally obtained from time-lapse acquisition using the optical microscope. The typical maximum resolution of the optical microscope is ~λ/2 or ~300 nm. This scale represents a challenge for imaging fission yeast microtubule dynamics specifically during early mitosis, where the bipolar mitotic spindle contains many short dynamic microtubules of ~1-μm scale. Here, we present a novel method to image short fission yeast mitotic microtubules. The method uses the thermosensitive reversible kinesin-5 cut7.24(ts) to create monopolar spindles, where asters of individual mitotic microtubules are presented for imaging and subsequent analysis.

The behaviour in solution of nickel(II) complexes of the macrocycles L1{3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene} and L2(7-methyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene} containing pyridine rings was investigated spectroscopically (UV/VIS/near IR and 1H NMR) and several species were found. In order to understand these and previous experimental data concerning stability constants, some crystals were prepared. The crystal structures of L1 and the complexes [CuL1][PF6]21 and [NiL2(ClO4)][ClO4]2 were determined, the copper complex adopting a square-planar geometry and the nickel one a square-pyramidal geometry, where the four nitrogen atoms are planar and one perchlorate occupies the axial co-ordination position. Molecular mechanics calculations were performed in order to find the preferred conformations for square-planar and square-pyramidal complexes of both L1 and L2, and the related macrocycles which do not contain a pyridine ring. The electronic preferences were analysed with extended-Huckel calculations. The introduction of pyridine into macrocycle does not significantly affect its behaviour upon co-ordination in square-planar complexes. For square-pyramidal complexes, the structures where a fifth donor occupies the axial position are usually more stable, though the introduction of methyl groups as substituents on the nitrogen may favour the presence of this donor in an equatorial site and lead to folding of the macrocycle.

Microtubules exhibit dynamic instability, stochastically switching between infrequent phases of growth and shrinkage. In the cell, microtubule dynamic instability is further modulated by microtubule-associated proteins and motors, which are specifically tuned to cell cycle stages. For example, mitotic microtubules are more dynamic than interphase microtubules. The different parameters of microtubule dynamics can be measured from length versus time data, which are generally obtained from time-lapse acquisition using the optical microscope. The typical maximum resolution of the optical microscope is ~λ/2 or ~300 nm. This scale represents a challenge for imaging fission yeast microtubule dynamics specifically during early mitosis, where the bipolar mitotic spindle contains many short dynamic microtubules of ~1-μm scale. Here, we present a novel method to image short fission yeast mitotic microtubules. The method uses the thermosensitive reversible kinesin-5 cut7.24(ts) to create monopolar spindles, where asters of individual mitotic microtubules are presented for imaging and subsequent analysis.

Potentiometric titrations showed that the sum of the first three protonation constants of diethylene-triaminepentaacetate N,N″-bis(butylamide)(L1) decreases by units when compared to the diethylenetriaminepentaacetate (dtpa), and that the complexes of Ga3+ and In3+ of L1 have stability constants 6.1–6.3 long units lower than those of dtpa. The decreased basicity of the bis (amide) derivative thus correlates with the lower stability of its complexes. However, at pH 7.4 the lower overall basicity of L1 compared to dtpa partly compensates the lower stability of its complexes. thus causing their conditional stability constants to be comparable to those of the dtpa complexes. The efficacy of L1 in binding Ga3+ and In3+ under physiological conditions is discussed by comparing pM values at pH 7.4, showing That this compound can withstand the competition of transferring for biological media relevant to medical applications. The 13C and 1H N M R shifts were measured for the complexes of Al,3+, Ga3+, and Y3+ with L1and compared with 13C and are hexadentate, possibly of octahedral geometry, and form various structuraly isomers, whereas those of In3+ and Y3+, with a single structural isomer of eight-co-ordination, are very similar to the lanthanide complexes. The complexes of Al3+ and Ga3+ of L1 have considerable populations of isomers with bound amide groups, whereas the single structural isomer of the complexes of In3+ and Y3+ with both amide groups co-ordinated yield a variety of enantimoers in solution.

The synthesis of the cross-bridged cyclen CRpy(2) {4,10-bis((pyridin-2-yl)methyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane}, a constrained analogue of the previously described trans-methylpyridine cyclen Cpy(2) is reported. The additional ethylene bridge confers to CRpy(2) proton-sponge type behaviour which was explored by NMR and potentiometric studies. Transition metal complexes have been synthesized (by complexation of both ligands with Co(2+), Cu(2+) and Zn(2+)) and characterized in solution and in the solid state. The single crystal X-ray structures of [CoCpy(2)](2+), [CuCpy(2)](2+) and [ZnCpy(2)](2+) complexes were determined. Stability constants of the complexes, including those of the cross-bridged derivative, were determined using potentiometric titration data and the kinetic inertness of the [CuCRpy(2)](2+) complex in an acidic medium (half-life values) was evaluated by spectrophotometry. The pre-organized structure of the cross-bridged ligand imposes an additional strain for the complexation leading to complexes with smaller thermodynamic stability in comparison with the related non-bridged ligand. The electrochemical study involving cyclic voltammetry underlines the importance of the ethylene cross-bridge on the redox properties of the transition metal complexes.

Some macrocyclic compounds containing pyridinc have been synthetized: 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene(1), 3,7,10,16-tetraazabicyclo[10.3.1]hexadeca-1(16),12,14-triene(2), 3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene (3), and 7-methyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15 trienc (4). The protonation reactions of these ligands were studied by potentiometric and 1 H NMR techniques

Summary Background Mitochondria form a dynamic tubular network within the cell. Proper mitochondria movement and distribution are critical for their localized function in cell metabolism, growth, and survival. In mammalian cells, mechanisms of mitochondria positioning appear dependent on the microtubule cytoskeleton, with kinesin or dynein motors carrying mitochondria as cargos and distributing them throughout the microtubule network. Interestingly, the timescale of microtubule dynamics occurs in seconds, and the timescale of mitochondria distribution occurs in minutes. How does the cell couple these two time constants? Results Fission yeast also relies on microtubules for mitochondria distribution. We report here a new microtubule-dependent but motor-independent mechanism for proper mitochondria positioning in fission yeast. We identify the protein mmb1p, which binds to mitochondria and microtubules. mmb1p attaches the tubular mitochondria to the microtubule lattice at multiple discrete interaction sites. mmb1 deletion causes mitochondria to aggregate, with the long-term consequence of defective mitochondria distribution and cell death. mmb1p decreases microtubule dynamicity. Conclusions mmb1p is a new microtubule-mitochondria binding protein. We propose that mmb1p acts to couple long-term mitochondria distribution to short-term microtubule dynamics by attenuating microtubule dynamics, thus enhancing the mitochondria-microtubule interaction time.

Two pentaaza macrocycles containing pyridine in the backbone, namely 3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene ([15]pyN(5)), and 3,6,10,13,19-pentaazabicyclo[13.3.1]nonadeca-1(19),15,17-triene ([16]pyN(5)), were synthesized in good yields. The acid-base behaviour of these compounds was studied by potentiometry at 298.2K in aqueous solution and ionic strength 0.10 M in KNO(3). The protonation sequence of [15]pyN(5) was investigated by (1)H NMR titration that also allowed the determination of protonation constants in D(2)O. Binding studies of the two ligands with Ca(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Pb(2+) metal ions were performed under the same experimental conditions. The results showed that all the complexes formed with the 15-membered ligand, particularly those of Cu(2+) and especially Ni(2+), are thermodynamically more stable than with the larger macrocycle. Cyclic voltammetric data showed that the copper(II) complexes of the two macrocycles exhibited analogous behaviour, with a single quasi-reversible one-electron transfer reduction process assigned to the Cu(II)/Cu(I) couple. The UV-visible-near IR spectroscopic and magnetic moment data of the nickel(II) complexes in solution indicated a tetragonal distorted coordination geometry for the metal centre. X-band EPR spectra of the copper(II) complexes are consistent with distorted square pyramidal geometries. The crystal structure of [Cu([15]pyN(5))](2+) determined by X-ray diffraction showed the copper(II) centre coordinated to all five macrocyclic nitrogen donors in a distorted square pyramidal environment.

The present work addresses the role of two ortho-substituted Mn(III) N-alkylpyridylporphyrins, alkyl being ethyl in MnTE-2-PyP(5+) and n-hexyl in MnTnHex-2-PyP(5+), on the protection against the oxidant tert-butylhydroperoxide (TBHP). Their protective role was studied in V79 cells using endpoints of cell viability (MTT and crystal violet assays), intracellular O(2)*- generation (dihydroethidium assay) and glutathione status (DTNB and monochlorobimane assays). MnPs per se did not show cytotoxicity (up to 25 microM, 24 h). The exposure to TBHP resulted in a significant decrease in cell viability and in an increase in the intracellular O(2)(*-) levels. Also, TBHP depleted total and reduced glutathione and increased GSSG. The two MnPs counteracted remarkably the effects of TBHP. Even at low concentrations, both MnPs were protective in terms of cell viability and abrogated the intracellular O(2)(*-) increase in a significant way. Also, they augmented markedly the total and reduced glutathione contents in TBHP-treated cells, highlighting the multiple mechanisms of protection of these SOD mimics, which at least in part may be ascribed to their electron-donating ability.

Recent development in soft lithography and microfluidics enables biologists to create tools to control the cellular microenvironment. One such control is the ability to quickly change the temperature of the cells. Genetic model organism such as fission yeast has been useful for studies of the cell cytoskeleton. In particular, the dynamic microtubule cytoskeleton responds to changes in temperature. In addition, there are temperature-sensitive mutations of cytoskeletal proteins. We describe here the fabrication and use of a microfluidic device to quickly and reversibly change cellular temperature between 2 degrees C and 50 degrees C. We demonstrate the use of this device while imaging at high-resolution microtubule dynamics in fission yeast.

The kinetics of acid-catalyzed dissociation of the copper(II) complex with 7-methyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene-3,11-diacetic acid (ac2Me[14]pyN4) at [H+] = 0.05-0.25 mol l-1, I = 0.25 mol l-1 (Na, H)ClO4, and T = 298.16 K was studied with conventional and stopped-flow UV/VIS spectroscopy. Three steps of consecutive complex reaction were observed. The very fast first and second steps characterized by k1 = 70 ± 10 and k2 = 0.23 ± 0.01 l mol-1 s-1 depend on the H+ concentration. The third step is very slow, k3 = (1.08 ± 0.03) × 10-3 s-1, and does not depend on the H+ concentration. Latter rate-determining step involves an isomerisation process forcing the copper(II) ion to leave rapidly the macrocyclic cavity. The reaction mechanism of the complex dissociation has been proposed, taking into account the results obtained for related systems by independent methods: potentiometry, UV/VIS and EPR spectroscopies, X-ray diffraction analysis, and molecular mechanics calculations.

A new bis-N,N'-(5-methylimidazol-4-ylmethyl) derivative of a 14-membered tetraazamacrocycle, L-1, has been synthesized. The protonation constants of this compound and the stability constants of its complexes with divalent first-row transition metal ions and Fe3+ were determined at 298.2 K in aqueous 0.10 mol dm(-3) KNO3. Compound L-1 exhibits high overall basicity, which is mainly conferred by theimidazolyl groups. The complexes of the divalent first row-transition metal ions of L-1 follow the Irving-Williams order of stability with the maximum for Cu2+ as expected, but a steep fall of constants is verified for the Mn2+, Fe2+ and Co2+, in one side, and for the Zn2+ complexes, in the other side. Additionally, L-1 shows a large affinity for Fe3+, and the relative stability constants for its Cd2+ and Pb2+ complexes indicate that L-1 may be useful for the complexometric determination of these two toxic metal ions in solutions containing both metal ions. These studies together with NMR, UV-vis and EPR spectroscopic data indicated the presence of mononuclear complexes, which adopt distorted pyramidal or octahedral geometries depending on the metal centre. The X-ray crystal structure of [ Cu(HL1)](PF6)(2)(NO3) center dot H2O showed that the coordination sphere of the copper centre can be described as a distorted square pyramid with the basal plane defined by three nitrogen donors of the macrocycle backbone and one nitrogen atom from one imidazolyl pendant arm. The apical position is occupied by the nitrogen atom of the macrocycle trans to the pyridine ring. To achieve this coordination environment, the macrocycle is folded along the axis defined by the two N atoms contiguous to the pyridine ring. The free methylimidazolyl arm points away from the metal centre leading to an intramolecular Cu center dot center dot center dot N distance of 5.155( 1) angstrom.

Structural studies of metal complexes of five ditopic hexaazamacrocycles containing two pyridine rings ([n]py2N4 n= 18, 20, 22, 24 and 26) have been carried out. The synthesis of macrocycles [22]- to [26]-py2N4 are also reported. The protonation constants of the last three compounds and the stability constants of their complexes with Ni2+, Cu2+, Zn2+, and Pb2+ were determined at 25 degrees C in 0.10 mol dm(-3) KNO3 in aqueous solution. Our results with [22]py2N4 show significant differences from those described previously, while [24]py2N4 has not been studied before and [26]py2N4 is a new compound. Mononuclear and dinuclear complexes of the divalent metal ions studied with [22]- to [26]-py2N4 were found in solution. The stability constants for the ML complexes of the three ligands follow the Irving-Williams order: NiL2+ > ZnL2+ > PbL2+, however for the dinuclear complexes the values for Pb2+ complexes are higher than the corresponding values for the Ni2+ and the Zn2+ complexes. The X-ray single crystal structures of the supramolecular aggregates [Cu2([20]py2N4)(H2O)4][Cu(H2O)6](SO4)3 x 3H2O and [Cu(2)([20]py(2)N4)(CH3CN)4][Ni([20]py2N4)]2(ClO4)8 x H2O, which are composed of homodinuclear [Cu2([20]py2N4])(H2O)4]4+ and [Cu2([20]py2N4])(CH3CN))4]4+, and mononuclear species, [Cu(H2O)6]2+ and [Ni([20]py2N4)]2+, respectively, assembled by an extensive network of hydrogen bonds, are also reported. In both homodinuclear complexes the copper centres are located at the end of the macrocycle and display distorted square pyramidal coordination environments with the basal plane defined by three consecutive nitrogen donors and one solvent molecule, water in and acetonitrile in . The macrocycle adopts a concertina-type conformation leading to the formation of macrocyclic cavities with the two copper centres separated by intramolecular distances of 5.526(1) and 5.508(7) A in 1a and 2a, respectively. The mononuclear complex [Ni([20]py2N4])]2+ displays a distorted octahedral co-ordination environment with the macrocycle wrapping the metal centre in a helical shape. EPR spectroscopy of the copper complexes indicated the presence of mono- and dinuclear species.

A new tetraazamacrocycle containing two 5-chloro-8-hydroxyquinoline side arms, L 1 , was synthesized. The protonationconstants of L 1 and the stability constants of its complexes with Cu 2 ,Zn 2 ,Cd and Pb were determined by potentiometric orspectrophotometric methods, at 20 8C in methanol:water (5:1, v/v) and ionic strength 0.10 mol dm 3 in tetrabutylammonium nitrate(TBAN). Compound L 1 has three high protonation constants, the most basic centres being both phenolates of the arms. Mono- anddinuclear complexes were found in solution for all the metal ions, the complexes of copper are thermodynamically very stable, andthe dinuclear complexes of lead(II) are the predominant species even in 1:1 mixtures. UV / Vis spectra of some complexes and EPRof the copper ones confirmed the presence of dinuclear complexes.# 2003 Elsevier B.V. All rights reserved. Keywords: Tetraazamacrocycle; Stability constants; Dinuclear complexes; EPR copper complexes; 8-Hydroxyquinolinyl groups 1. IntroductionMacrocycles containing quinoline side arms havefound many applications as analytical reagents inabsorption spectrophotometry, fluorometry, solvent ex-traction, and partition chromatography [1,2]. Thesecompounds are also used as pesticides [2].The 8-hydroxyquinoline (HQ) forms a stable five-membered chelate ring with metal centres and verystable complexes with divalent transition and post-transition metal ions of the type M(HQ) , M(HQ)

The hexaaza macrocycles 3,6,14,17,23,24-hexaazatricyclo[ 17.3.1.1(8,12)]tetracosa-1(23), 8,10,12(24), 19,21-hexaene ([18]py(2)N(4)) and 3,7,15,19,25,26-hexaazatricyclo[19.3.1.1(9,13)]hexacosa-1(25), 9,11,13(26), 21,23-hexaene ([20]py(2)N(4)) were synthesised. The protonation constants of both compounds and the stability constants of their complexes with a wide range of metal ions (Mn2+, Ni2+, Cu2+, Zn2+, Cd2+, Pb2+, Mg2+, Ca2+, Ba2+, La3+ and Gd3+) were determined at 25degreesC with ionic strengths of 0.10 mol dm(-3) in KNO3. The overall basicity of [20] py(2)N(4) is 3.20 log units larger than that of [18]py(2)N(4) due to the weaker repulsion between the contiguous protonated ammonium sites, which are separated by propyl chains in [20]py(2)N(4) rather than ethyl chains in [18]py(2)N(4). The stability constants of complexes of each metal decrease as the cavity size of the macrocycle is increased, taking into account the difference in basicity of the ligands; the values for the Cu2+, Ni2+ and Zn2+ complexes of both ligands are exceptionally high. Single crystal structures of complexes [Ni([18]py(2)N(4))](ClO4)(2).2CH(3)CN (1), [Cu([18]py(2)N(4))](ClO4)(2).2CH(3)CN (2), [Co([20]py(2)N(4))]-[Co(H2O)(6)](0.5)(SO4)(2).CH3OH.4H(2)O (3), [Ni([20]py(2)N(4))](ClO4)(2)(4) and [Cu([20]py(2)N(4))](ClO4)(2) (5) were determined. In all complexes, the metal centre exhibit a hexaco-ordinate environment and the macrocycle adopts a twisted helical topology. The effect of the cage sizes of [18]py(2)N(4) and [20]py(2)N(4) on the molecular dimensions of metal complexes of both macrocycles is evaluated and a significant decrease in the helicity is observed in complexes of the 18-membered macrocycle compared to complexes of the 20-membered ring. The X-ray structural results, together with molecular mechanics calculations and NMR studies performed for metal complexes of both macrocycles, indicate that both ligands have enough flexibility to encapsulate smaller and larger metal ions, although it is clear that [20]py(2)N(4) is more flexible than [18]py(2)N(4).

Two N-(2-pyridylmethyl) derivatives of a 14-membered tetraaza macrocycle containing pyridine have been synthesized, L-1 and L-2. The protonation constants of these compounds and the stability constants of complexes of both ligands with Co2+ to Zn2+, Cd2+, Pb2+, Fe3+ and In3+ were determined by potentiometric methods at 25 degrees C and ionic strength 0.10 mol dm(-3) in KNO3. The values of the protonation constants for L-1 and L-2 are quite different. The metal complexes of both ligands present lower values of stability constants than expected. The only exception is the case of the Cd2+ complex with L-2 which exhibits a particularly high stability constant, indicating that this ligand has a remarkable selectivity for this metal ion relative to zinc. The Cd2+ complex seems to be the only one to bond to all the donor atoms of the ligand or at least to more than the other metal ions studied. The pM values determined at physiological pH, which take into account the competition of the protons for the ligand, have shown that L-2 is the most selective compound for cadmium relative to zinc. The single crystal structure of [CuL2][ClO4](2) determined by X-ray diffraction has shown that the co-ordination geometry around the copper atom can be described approximately as a distorted square pyramid. To achieve this geometric arrangement the macrocycle adopts a folded conformation. Furthermore the nitrogen atom of one free pyridylmethyl pendant arm is directed towards the copper centre leading to a distance between these two atoms of 3.304(11) Angstrom, which suggests a weak bonding interaction consistent with a [5+1] co-ordination.

The N,N'-bis(butanamide) derivative of TTHA (TTHA = triethylenetetramine-N,N,N',N",N"',N"'-hexaacetic acid), and its Ga(3+) and In(3+) complexes were synthesized and characterized. The crystal X-ray diffraction structure of [Ga(2)(OH)(2)(TTHA)][Na(2)(H(2)O)(6)].2H(2)O was determined. The complex crystallizes in the monoclinic space group P2(1)/n with a = 7.179(2) Å, b = 20.334(3) Å, c = 10.902(5) Å, beta = 101.90(2) degrees, and Z = 2. Each gallium atom is bonded to six donor atoms (N(2)O(4)) in a slightly distorted octahedral geometry. The values of the protonation constants and the protonation sequence were determined by potentiometry and NMR. The stability constants of the Al(3+), Ga(3+), Fe(3+), and In(3+) complexes of TTHA-(BuA)(2) and of the Ga(3+) complex of TTHA were determined by potentiometry. The structures, in solution, of the Al(3+), Ga(3+), and In(3+) complexes of TTHA-(BuA)(2) and TTHA were analyzed by (1)H, (13)C, (27)Al, (71)Ga, and (115)In NMR techniques. Derivatization of two terminal carboxylates by butanamide substituents leads to a significant decrease of the total ligand basicity (5.77 log units) and to a change of the solubility of the resulting complexes. The stability constant of the ML complexes of TTHA-(BuA)(2) with Fe(3+) exhibits the highest value of the series (10(23.92)). The In(3+) complex is more stable than that of Ga(3+) and almost as stable as that of the Fe(3+). However, the decrease in indium and iron complex stability is less drastic going from TTHA to TTHA-(BuA)(2) (about 3 log units) than for Al(3+) or Ga(3+) (about 6 log units). pM values calculated under physiological conditions for DTPA, TTHA, and the bis(butanamide) derivatives have shown that while DTPA remains a ligand of choice to chelate Fe(3+) and In(3+) ions in vivo compared to transferrin as competitor ligand, TTHA, surprisingly, appears to be the best of these four ligands (pM = 22.71) to chelate Ga(3+).

The protonation constants of H2L1, 3,11-bis(carboxymethyl)-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene and H3L2, 3,7,11-tris(carboxymethyl)-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene, and stability constants of complexes formed by these macrocycles with Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Pb2+, Ga3+, Fe3+ and In3+ were determined at 25 °C and ionic strength 0.10 mol dm–3 in NMe4NO3. Both compounds are very selective for the divalent first-row transition-metal ions, exhibiting very high stability constants for Cu2+, fairly high values for Ni2+, but sharply decreasing ones for the remaining metal ions of this row. Their complexes with the alkaline-earth or larger metal ions, such as Pb2+, have low stability constants. The crystal structure of [CuL1]·4H2O was determined. The copper atom is encapsulated by the macrocycle in a distorted octahedral environment. The equatorial plane contains the four nitrogen atoms of the tetraaza ring and six-co-ordination is completed via two oxygen atoms of the appended carboxylate groups. The angles at the metal centre are close to the expected values of 90 and 180° for octahedral geometry. Molecular mechanics studies carried out for the cis and the trans octahedral [ML1] complexes were consistent with the structure found in the solid state. For a mean CuII–N distance of 2.01 A the experimentally observed trans isomer is 6.5 kcal mol–1 more stable than the cis one. On the other hand these calculations suggest that larger ions such as Pb2+, Ca2+ or Mn2+ can be accommodated by the macrocycle in a cis-octahedral environment. However, these ions allow co-ordination numbers higher than six and so other structures ought to be also considered. The low stability constants for metal complexes of Co2+ and Zn2+ indicate that these complexes do not have a trans-octahedral structure, while the molecular mechanics calculations reveal that the cis isomer is not the most stable form. Therefore, other structures with co-ordination numbers lower than six should be considered, implying that one or more donor atoms are not co-ordinated. Stability constants of metal complexes of (L2)3– and EPR studies suggest that not all the donor atoms in this macrocycle are co-ordinated when complexes are formed with first-row-transition divalent metal ions.

Using data on a series of polyaza and polyoxa-polyaza macrocyclic compounds, a satisfactory linear correlation was established between the dissociation constants of their acid forms obtained in deuterium oxide (p K D ) and in water (p K H ). The isotopic effect, Δp K =p K D - p K H , increases with increasing p K , being larger for the weaker acids. The p K D vs. p K H correlation may be used to determine p K D or p K H values when one of the constants is known; the statistical errors of the estimates are not significatively higher than those of the experimental determinations. By including data available in the literature for a series of different acids (mineral and carboxylic acids, phenols, protonated amines, amino acids, etc.; a total of 138 pairs of data for 70 different compounds), a good general p K D vs. p K H correlation was also obtained (correlation coefficient 0.998), showing that the dependence of the isotopic effect on the nature of the acid is not as important as previous studies, based on limited and not always satisfactory Δp k vs. p K H correlations, suggested.