High-phasing-power lanthanide derivatives: taking advantage of ytterbium and lutetium for optimized anomalous diffraction experiments using synchrotron radiation.

Ytterbium and lutetium are well suited for optimized anomalous diffraction experiments using synchrotron radiation. Therefore, two lanthanide complexes Yb-HPDO3A and Lu-HPDO3A have been produced that are similar to the Gd-HPDO3A complex already known to give good derivative crystals. Derivative crystals of hen egg-white lysozyme were obtained by co-crystallization using 100 mM solutions of each lanthanide complex. De novo phasing has been carried out using single-wavelength anomalous diffraction on data sets collected on each derivative crystal at the L(III) absorption edge of the corresponding lanthanide (ff" = 28 e(-)). A third data set was collected on a Lu-HPDO3A derivative crystal at the Se K absorption edge with f"(Lu) = 10 e(-). The structures were refined and compared with the known structure of the Gd-HPDO3A lysozyme derivative. The quality of the experimental electron-density maps allows easy model building. With L(III) absorption edges at shorter wavelengths than the gadolinium absorption edge, lutetium and ytterbium, when chelated by a ligand such as HPDO3A, form lanthanide complexes that are especially interesting for synchrotron-radiation experiments in structural biology.

A new class of gadolinium complexes employed to obtain high-phasing-power heavy-atom derivatives: results from SAD experiments with hen egg-white lysozyme and urate oxidase from Aspergillus flavus.

Seven gadolinium complexes are shown to be excellent compounds for the preparation of heavy-atom derivatives for macromolecular crystallography projects. De novo phasing has been carried out using single-wavelength anomalous diffraction (SAD) on a series of gadolinium-derivative crystals of two proteins: hen egg-white lysozyme and urate oxidase from Aspergillus flavus. Lysozyme derivative crystals were obtained by co-crystallizing the protein with the corresponding gadolinium complex at a concentration of 100 mM. Diffraction data were collected to a resolution of 1.7 A using Cu K(alpha) radiation from a rotating-anode generator, making use of the high anomalous signal of gadolinium at this wavelength. Urate oxidase derivative crystals were obtained by soaking native crystals in 100 mM gadolinium complex solutions. Diffraction data were collected to a resolution close to 3 A using X-rays at the Gd L(III) absorption edge, taking advantage of the sharp white line on that edge. For all urate oxidase derivative crystals and three of the lysozyme crystals, SAD phasing led to electron-density maps of very high quality, allowing unambiguous chain tracing. From this study, the binding effectiveness of the gadolinium complexes seems to be related to the nature of the precipitant used for crystallization. These gadolinium complexes represent a new class of high-phasing-power heavy-atom derivatives that may be used for high-throughput structure-determination projects.

DOTA tris(phenylmethyl) ester: a new useful synthon for the synthesis of DOTA monoamides containing acid-labile bonds.

The synthesis of DOTA tris(phenylmethyl) ester 2, a new monoreactive derivative of DOTA, is described. This versatile synthon can be easily coupled to compounds bearing an amino group and then deprotected to DOTA monoamide under mild and neutral conditions by catalytic hydrogenolysis. Accordingly, compound 2 has been used in the synthesis of a DOTA monoamide gadolinium complex containing two palmitic esters, which is a component of mixed micelles as MRI contrast agents.

Mixed micelles containing lipophilic gadolinium complexes as MRA contrast agents.

Mixed micelles for MRA are multicomponent systems containing a phospholipid, a biocompatible non-ionic surfactant (e.g. Synperonic(R) F-108) and a lipophilic gadolinium complex. A variety of lipophilic gadolinium complexes were designed taking into account features such as: (i) nature of ligand (cyclic versus acyclic); (ii) lipophilic moiety; (iii) global charge of the complex; and (iv) nature of bond connecting the complex to the lipophilic moiety. All the lipophilic gadolinium complexes after formulation as mixed micelles show high relaxivities in water and in blood (rat). Mixed micelles containing gadolinium complexes bearing only one aliphatic chain cannot be used as MRA contrast agents because they have a high haemolytic effect. Furthermore, in rats they are quickly eliminated from the blood stream. These drawbacks are completely circumvented using gadolinium complexes bearing two aliphatic chains. Mixed micelles containing such complexes show high relaxivities, no haemolytic effect and long blood permanence. This makes them promising candidates as MRA contrast agents. However, elimination, which occurs exclusively through the liver, is not complete, even after 7 days. Complexes containing labile (e.g. ester) bonds between the lipophilic moieties and the chelate subunit are eliminated through both the liver and the kidneys. However, elimination is still not complete after 7 days.

Abc protein transport of MRI contrast agents in canalicular rat liver plasma vesicles and yeast vacuoles.

The mechanism of excretion into bile of hepatospecific magnetic resonance imaging (MRI) contrast media employed labeled Gd-reagents EOB.DTPA, BOPTA, B 20790 (iopanoate-linked), and B 21690 (glycocholate-linked) for measurement in rat liver canalicular plasma membrane vesicles and yeast vacuoles. The presence of ATP gave threefold greater transport of B 20790 and B 21690 than of EOB.DTPA and BOPTA. In yeast vacuoles the ATP stimulatory effect was eightfold with B 20790 and fivefold greater for B 21690, whereas in YCF1- or YLLO115w-deleted yeast cells the transport was significantly reduced and absent from double mutants, YCF1 and YLLO15w. The transport was similar in wild-type and deletant cells for B 21690; taurocholate gave 85% inhibition. These data suggest that bilary secretion of structurally related MRI agents depend on molecular structure. The findings are suggestive as of possible value for clinical diagnosis of inherited hyperbilirubinemias and other liver disorders.

Molecular mechanisms for the hepatic uptake of magnetic resonance imaging contrast agents.

The mechanisms were investigated for the hepatic transport of 4 different gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI). In basolateral rat hepatocyte plasma membrane vesicles, Gd-DTPA uptake was indistinguishable from non-specific binding to vesicles; Gd-BOPTA and Gd-EOB-DTPA entered plasma membrane vesicles following a linear, concentration-dependent mechanism up to 1.5 mM of substrate. By contrast, Gd-B 20790 uptake followed a saturative kinetic with an apparent Km of 92 +/- 15 microM and a Vmax of 143 +/- 42 pmol/mg prot/15 sec, and it occurred into an osmotic-sensitive space. Sulfobromophthalein ant taurocholate, but not unconjugated bilirubin inhibited the uptake rate of Gd-B 20790 but not that of the other three compounds. Injection into Xenopus laevis oocytes of 5 ng of human OATP cRNA resulted, after 3 days, in a >/=2-fold stimulation (p < 0.001) of transport of Gd-B 20790 but not of Gd-BOPTA or Gd-EOB-DTPA. Collectively, these data indicate that the hepatic uptake of the MRI contrast agent Gd-B 20790 is a carrier-mediated mechanism operated by OATP while MRI compounds with other chemical structures enter the hepatocyte by other mechanisms.

L-Glutamic acid and L-lysine as useful building blocks for the preparation of bifunctional DTPA-like ligands.

Bisalkylation of suitably protected L-glutamic acid and L-lysine derivatives with tert-butyl N-(2-bromoethyl)iminodiacetate 2, followed by deprotection of the omega functional group affords N, N-bis[2-[bis[2-(1, 1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamic acid 1-(1, 1-dimethylethyl) ester 4 and N2,N2-bis[2-[bis[2-(1, 1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine 1,1-dimethylethyl ester 7. Such compounds feature a carboxylic or an amino group, respectively, which are available for conjugation with a suitable partner via formation of an amide bond. The conjugates, which can be prepared in this way, contain a chelating subunit in which all five acetic residues of DTPA are available for the complexation of metal ions. Direct bisalkylation of glycine with 2 promptly gives N, N-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]glycine 11. The latter allows to achieve conjugates in which the central acetic group of DTPA is selectively converted into an acetamide.

Hepatocyte-directed MR contrast agents. Can we take advantage of bile acids?

A series of gadolinium complexes conjugated to bile acids was prepared and investigated as possible hepatospecific MR imaging contrast agents. In the design of such compounds, features such as the nature of the bile acid, the site of conjugation on the bile acid skeleton, and the global charge of the conjugate were taken into account. Relaxivity measurements carried out in human serum indicate interaction of the conjugates with human serum proteins; even small structural variations significantly affect relaxivity in human serum. Pharmacokinetic data (biliary elimination in the range of 18.4-45.6%) show that bile acids can be used as address moieties to transport gadolinium complexes through hepatocytes. For a homogeneous series of compounds, differing only in the bile acid residue conjugated, it was unexpectedly found that cholic acid is twice as efficient an address moiety as cholylglycine or cholyltaurine. Preliminary results show that none of the conjugates is transported through the basolateral membrane of hepatocytes by the Na+/taurocholate carrier.

Gd(DOTP)5-outer-sphere relaxation enhancement promoted by nitrogen bases.

The relaxation properties of Gd(DOTP)5- (1, 4, 7, 10-tetra-azacyclododecane- N,N',N'',N'''-tetrakis(methylenephosphonic acid)) have been investigated as a function of pH, temperature, concentration, and magnetic field strength. We have found that the complex has one exchangeable water molecule in its inner coordination sphere, at a distance of 3.26 A from the metal ion, and it does not form oligomers in solution in the concentration range 0.2 to 10 mM. The possible presence of two species in solution with an average fractional hydration number is also taken into accounts. The NMRD profiles were recorded at 5 degrees C, 25 degrees C, and 35 degrees C and quantitatively analyzed in terms of the paramagnetic relaxation equations. Interestingly the addition to a solution of the Gd(III)-complex of nitrogen bases results in a marked relaxation enhancement, which shows a strong pH dependence with a maximum around pH = 9. The relaxivity gain has been shown to depend on outer-sphere effects originating from multiple electrostatic interactions between the anionic complex and the organic cations that bring the exchangeable protons of the substrate molecules in to close proximity with the paramagnetic center. High resolution NMR relaxation data for N-methyl-D(-)-glucamine suggest that the hydroxyl group on the beta-carbon plays a role in stabilizing the interaction, presumably through a hydrogen bond with an uncoordinated oxygen atom of the complex.