Lanthanide DO3A-Complexes Bearing Peptide Substrates: The Effect of Peptidic Side Chains on Metal Coordination and Relaxivity

Two DO3A-type ligands conjugated to substrates of urokinase (L3) and caspase-3 (L4) via a propyl-amide linker were synthesized and their lanthanide(III) (Ln³⁺) complexes studied. A model compound without peptide substrate (L2) and an amine derivative ligand mimicking the state after enzymatic cleavage (L1) were also prepared. Proton Nuclear Magnetic Relaxation Dispersion (NMRD) profiles recorded on the gadolinium(III) (Gd³⁺) complexes, complemented with the assessment of hydration numbers via luminescence lifetime measurements on the Eu³⁺ analogues, allowed us to characterize the lanthanide coordination sphere in the chelates. These data suggest that the potential donor groups of the peptide side chains (carboxylate, amine) interfere in metal coordination, leading to non-hydrated LnL3 and LnL4 complexes. Nevertheless, GdL3 and GdL4 retain a relatively high relaxivity due to an important second-sphere contribution generated by the strongly hydrophilic peptide chain. Weak PARACEST effects are detected for the amine-derivative EuL1 and NdL1 chelates. Unfortunately, the GdL3 and GdL4 complexes are not significantly converted by the enzymes. The lack of enzymatic recognition of these complexes can likely be explained by the participation of donor groups from the peptide side chain in metal coordination.     

Isoquinoline-based lanthanide complexes: bright NIR optical probes and efficient MRI agents

In the objective of developing ligands that simultaneously satisfy the requirements for MRI contrast agents and near-infrared emitting optical probes that are suitable for imaging, three isoquinoline-based polyaminocarboxylate ligands, L1, L2 and L3, have been synthesized and the corresponding Gd(3+), Nd(3+) and Yb(3+) complexes investigated. The specific challenge of the present work was to create NIR emitting agents which (i) have excitation wavelengths compatible with biological applications and (ii) are able to emit a sufficient number of photons to ensure sensitive NIR detection for microscopic imaging. Here we report the first observation of a NIR signal arising from a Ln(3+) complex in aqueous solution in a microscopy setup. The lanthanide complexes have high thermodynamic stability (log K(LnL) =17.7-18.7) and good selectivity for lanthanide ions versus the endogenous cations Zn(2+), Cu(2+), and Ca(2+) thus preventing transmetalation. A variable temperature and pressure (17)O NMR study combined with nuclear magnetic relaxation dispersion measurements yielded the microscopic parameters characterizing water exchange and rotation. Bishydration of the lanthanide cation in the complexes, an important advantage to obtain high relaxivity for the Gd(3+) chelates, has been demonstrated by (17)O chemical shifts for the Gd(3+) complexes and by luminescence lifetime measurements for the Yb(3+) analogues. The water exchange on the three Gd(3+) complexes is considerably faster (k(ex)(298) = (13.9-15.4) × 10(6) s(-1)) than on commercial Gd(3+)-based contrast agents and proceeds via a dissociative mechanism, as evidenced by the large positive activation volumes for GdL1 and GdL2 (+10.3 ± 0.9 and +10.6 ± 0.9 cm(3) mol(-1), respectively). The relaxivity of GdL1 is doubled at 40 MHz and 298 K in fetal bovine serum (r(1) = 16.1 vs 8.5 mM(-1) s(-1) in HEPES buffer), due to hydrophobic interactions between the chelate and serum proteins. The isoquinoline core allows for the optimization of the optical properties of the luminescent lanthanide complexes in comparison to the pyridinic analogues and provides significant shifts of the excitation energies toward lower values which therefore become more adapted for biological applications. L2 and L3 bear two methoxy substituents on the aromatic core in ortho and para positions, respectively, that further modulate their electronic structure. The Nd(3+) and Yb(3+) complexes of the ligand L3, which incorporates the p-dimethoxyisoquinoline moiety, can be excited up to 420 nm. This wavelength is shifted over 100 nm toward lower energy in comparison to the pyridine-based analogue. The luminescence quantum yields of the Nd(3+) (0.013-0.016%) and Yb(3+) chelates (0.028-0.040%) are in the range of the best nonhydrated complexes, despite the presence of two inner sphere water molecules. More importantly, the 980 nm NIR emission band of YbL3 was detected with a good sensitivity in a proof of concept microscopy experiment at a concentration of 10 μM in fetal bovine serum. Our results demonstrate that even bishydrated NIR lanthanide complexes can emit a sufficient number of photons to ensure sensitive detection in practical applications. In particular, these ligands containing an aromatic core with coordinating pyridine nitrogen can be easily modified to tune the optical properties of the NIR luminescent lanthanide complexes while retaining good complex stability and MRI characteristics for the Gd(3+) analogues. They constitute a highly versatile platform for the development of bimodal MR and optical imaging probes based on a simple mixture of Gd(3+) and Yb(3+)/Nd(3+) complexes using an identical chelator. Given the presence of two inner sphere water molecules, important for MRI applications of the corresponding Gd(3+) analogues, this result is particularly exciting and opens wide perspectives not only for NIR imaging based on Ln(3+) ions but also for the design of combined NIR optical and MRI probes.     

Synthesis neutral rare earth complexes of diethylenetriamine-N,N'-bis(acetyl-isoniazid)-N,N',N'-triacetic acid as potential contrast enhancement agents for magnetic resonance imaging

A novel ligand, diethylenetriamine-N,N'-bis(acetyl-isoniazid)-N,N',N'-triacetic acid (H(3)L) has been synthesized from diethylene triamine pentaacetic acid (DTPA) and isoniazid. Ligand and its five neutral rare earth (RE=La, Sm, Eu, Gd, Tb) complexes holding promise of magnetic resonance imaging (MRI) were characterized on the basis of elemental analysis, molar conductivity, (1)H-NMR spectrum, FAB-MS, TG-DTA analysis and IR spectrum. The relaxivity (R(1)) of complexes and Gd(DTPA)(2-) used as a control were determined. The relaxivity of LaL, SmL, EuL, GdL, TbL and Gd(DTPA)(2-) were 0.14, 1.66, 3.14, 6.08, 2.79 and 4.34 l.mmol(-1).s(-1), respectively. The spin-lattice relaxivity of GdL was larger than that of Gd(DTPA)(2-). The relaxivity of GdL had also been investigated in human serum albumin (HSA) solution, the relaxivity of GdL was enhanced from 6.08 l.mmol(-1).s(-1) in water solution to 9.09 l.mmol(-1).s(-1) in HSA solution. In addition, thermodynamics stability constant of GdL complex was determined, the thermodynamic stability constant of GdL complex (K(GdL)=10(20.84)) was a few larger than that of Gd(DTPA)(2-) (K(Gd-DTPA)=10(20.73)). The results showed that complex of GdL may be a prospective MRI contrast agent with low osmotic pressure due to non-ion complex, high spin-lattice relaxivity, good stability and binding affinity for the serum protein.     

Influence of calcium-induced aggregation on the sensitivity of aminobis(methylenephosphonate)-containing potential MRI contrast agents

A novel class of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(methylenecarboxylic) acid (DO3A)-based lanthanide complexes with relaxometric response to Ca(2+) was synthesized, and their physicochemical properties were investigated. Four macrocyclic ligands containing an alkyl-aminobis(methylenephosphonate) side chain for Ca(2+)-chelation have been studied (alkyl is propyl, butyl, pentyl, and hexyl for L(1), L(2), L(3), and L(4), respectively). Upon addition of Ca(2+), the r(1) relaxivity of their Gd(3+) complexes decreased up to 61% of the initial value for the best compounds GdL(3) and GdL(4). The relaxivity of the complexes was concentration dependent (it decreases with increasing concentration). Diffusion NMR studies on the Y(3+) analogues evidenced the formation of agglomerates at higher concentrations; the aggregation becomes even more important in the presence of Ca(2+). (31)P NMR experiments on EuL(1) and EuL(4) indicated the coordination of a phosphonate to the Ln(3+) for the ligand with a propyl chain, while phosphonate coordination was not observed for the analogue bearing a hexyl linker. Potentiometric titrations yielded protonation constants of the Gd(3+) complexes. log K(H1) values for all complexes lie between 6.12 and 7.11 whereas log K(H2) values are between 4.61 and 5.87. Luminescence emission spectra recorded on the Eu(3+) complexes confirmed the coordination of a phosphonate group to the Ln(3+) center in EuL(1). Luminescence lifetime measurements showed that Ca-induced agglomeration reduces the hydration number which is the main cause for the change in r(1). Variable temperature (17)O NMR experiments evidenced high water exchange rates on GdL(1), GdL(2), and GdL(3) comparable to that of the aqua ion.     

Structure-related variable responses of calcium sensitive MRI probes

A new series of Gd(3+) complexes based on DO3A (GdL(1)-GdL(4)) was synthesized and investigated. They possess side chains with different structures which determine their varying binding properties and response towards endogenous metal ions, measured by changes in the longitudinal relaxivity (r(1)). GdL(4) exhibits the highest selectivity toward Ca(2+) in comparison to the other complexes, with up to a 63% increase of the r(1). GdL(2) and GdL(3) also respond to different Ca(2+) concentration ranges, however with a lower selectivity since the r(1) changes are also observed in the presence of other cations such as Mg(2+), Zn(2+) or Cu(2+). Assessment of the hydration number (q) via luminescence lifetime measurements confirmed that the change in q is responsible for the r(1) response for all the complexes.     

Synthesis, thermodynamics stability constant and relaxation properties of neutral Gd(III) complex with derivative from diethylene triamine pentaacetic acid and p-hydroxybenzoyl hydrazine

A novel ligand, diethylenetriamine-N,N'-bis(acetyl-p-hydroxybenzoyl hydrazine)-N,N',N'-triacetic acid (H3L) was synthesized and characterized on the basis of elemental analysis, molar conductivity, 1H-NMR spectrum, FAB-MS, TG-DTA analysis and IR spectrum. Its complex of Gd(III) holding promise of magnetic resonance imaging (MRI) was synthesized, and relaxivity (R1) of complex and Gd(DTPA)2- used as a control was determined in water solution, respectively. The relaxivity of GdL (R1 = 6.39 l.mmol(-1).s(-1)) was larger than that of Gd(DTPA)2- (R1 = 4.34 l.mmol(-1).s(-1)). The relaxivity of GdL has also been investigated in human serum albumin (HSA) solution, the relaxivity of GdL was enhanced from 6.39 l.mmol(-1).s(-1) in water solution to 7.69 l.mmol(-1).s(-1) in HSA solution. In addition, thermodynamics stability constant of GdL was determined. The results showed that complex of GdL is a prospective MRI contrast agent, although the thermodynamic stability constant of GdL complex (K(GdL) = 10(19.56)) was a little less than that of Gd(DTPA)2- (K(Gd-DTPA) = 10(20.73)).     

The effect of ligand scaffold size on the stability of tripodal hydroxypyridonate gadolinium complexes

The variation of the size of the capping scaffold which connects the hydroxypyridonate (HOPO) binding units in a series of tripodal chelators for gadolinium (Gd) complexes has been investigated. A new analogue of TREN-1-Me-3,2-HOPO (1) (TREN = tri(ethylamine)amine) was synthesized: TREN-Gly-1-Me-3,2-HOPO (2) features a glycine spacer between the TREN cap and HOPO binding unit. TRPN-1-Me-3,2-HOPO (3) has a propylene-bridged cap, as compared to the ethylene bridges within the TREN cap of the parent complex. Thermodynamic equilibrium constants for the acid-base properties of 2 and the Gd(3+) complexation strength of 2 and 3 were measured and are compared with that of the parent ligand. The most basic ligand is 2 while 3 is the most acidic. Both 2 and 3 form Gd(3+) complexes of similar stability (pGd = 16.7 and 15.6, respectively) and are less stable than the parent complex Gd-1 (pGd = 19.2). Two of the three complexes are more stable than the bis(methylamide)diethylenetriamine pentaacetate complex Gd(DTPA-BMA) (pGd = 15.7) while the other is of comparable stability. Enlargement of the ligand scaffold decreases the stability of the Gd(3+) complexes and indicates that the TREN scaffold is superior to the TRPN and TREN-Gly scaffolds. The proton relaxivity of Gd-2 is 6.6 mM(-)(1) s(-)(1) (20 MHz, 25 degrees C, pH 7.3), somewhat lower than the parent Gd-1 but higher than that of the MRI contrast agents in clinical practice. The pH-independent relaxivity of Gd-2 is uncharacteristic of this family of complexes and is discussed.     

Syntheses,crystal structures,and luminescent properties of lanthanide complexes with tripodal ligands bearing benzimidazole and pyridine groups

Two tripodal ligands, bis(2-benzimidazolylmethyl)(2-pyridylmethyl)amine (L(1)) and bis(2-pyridylmethyl)(2-benzimidazolylmethyl)amine (L(2)), were synthesized. With the third chromophoric ligand antipyrine (Antipy), three series of lanthanide(III) complexes were prepared: [LnL(1)(Antipy)(3)](ClO(4))(3) (series A), [LnL(1)(Antipy)Cl(H(2)O)(2)]Cl(2)(H(2)O)(2) (series B), and [LnL(2)(NO(3))(3)] (series C). The nitrate salt of the free ligand H(2)L(1).(NO(3))(2) and six complexes were structurally characterized: Pr(3+)A, Y(3+)A, Eu(3+)B, Eu(3+)C, Gd(3+)C and Tb(3+)C, in which the two A and three C complexes are isomorphous. Crystallographic studies showed that tripodal ligands L(1) and L(2) exhibited a tripodal coordination mode and formed 1:1 complexes with all lanthanide metal ions. The coordination numbers of the lanthanide metal ions for the A, B, and C complexes were 7, 8, and 10, respectively. Conductivity studies on the B and C complexes in methanol showed that, in the former, the coordinated Cl(-) dissociated to give 3:1 electrolytes and, in the latter, two coordinated NO(3)(-) ions dissociated to give 2:1 electrolytes. Detailed photophysical studies have been performed on the free ligands and their Gd(III), Eu(III), and Tb(III) complexes in several solvents. The results show a wide range in the emission properties of the complexes, which could be rationalized in terms of the coordination situation, the (3)LC level of the complexes, and the subtle variations in the steric properties of the ligands. In particular the Eu(3+)A and Tb(3+)A complexes, in which the central metal ions were wholly coordinated by chromophoric ligands of one L(1) and three antipyrine molecules, had relatively higher emission quantum yields than their corresponding B and C complexes.     

Design and synthesis of a novel magnetic resonance imaging contrast agent for selective sensing of zinc ion

A series of new diethylenetriaminepentaacetic acid (DTPA)-bisamide chelators has been prepared and characterized for application as zinc sensors. We have designed and synthesized (GdL(a))(2-), which contains a DTPA-bisamide moiety. The R(1) relaxivity of (GdL(a))(2-) solution decreased monotonically on the addition of Zn(2+). Moreover, (GdL(a))(2-) showed high selectivity for Zn(2+) against Ca(2+) and Mg(2+). We also measured the UV-visible spectra and the coldspray ionization (CSI) MS spectra and concluded that the 1-to-1 Zn(2+) complex of (GdL(a))(2-) is stable at higher concentrations of Zn(2+). These complexes should provide the basis for creating a superior Zn(2+)-sensitive MRI contrast agent and are excellent candidates for incorporation into sensors designed for selective detection of Zn(2+) in biological applications.     

Physicochemical properties of the high-field MRI-relevant [Gd(DTTA-Me)(H2O)2]- complex

To study the physicochemical properties of the DTTA chelating moiety (H4DTTA = diethylenetriaminetetraacetic acid = N,N'-[iminobis(ethane-2,1-diyl)]bis[N-(carboxymethyl)glycine]), used in several compounds proposed as magnetic resonance imaging (MRI) contrast agents, the methylated derivative H4DTTA-Me (N,N'-[(methylimino)bis(ethane-2,1-diyl)]bis[N-(carboxymethyl)glycine]) was synthesized. Protonation constants of the ligand were determined in an aqueous solution by potentimetry and (1)H NMR pH titration and compared to various DTTA derivatives. Stability constants were measured for the chelates formed with Gd(3+) (log K(GdL) = 18.60 +/- 0.10) and Zn(2+) (log K(ZnL) = 17.69 +/- 0.10). A novel approach of determining the relative conditional stability constant of two paramagnetic complexes in a direct way by (1)H NMR relaxometry is presented and was used for the Gd(3+) complexes [Gd(DTTA-Me)(H2O)2](-) (L1) and [Gd(DTPA-BMA)(H2O)] (L2) [K(L1/L2)*(at pH 8.3, 25 degrees C) = 6.4 +/- 0.3]. The transmetalation reaction of the Gd(3+) complex with Zn(2+) in a phosphate buffer solution (pH 7.0) was measured to be twice as fast for [Gd(DTTA-Me)(H2O)2](-) in comparison to that for [Gd(DTPA-BMA)(H2O)]. This can be rationalized by the higher affinity of Zn(2+) toward DTTA-Me(4-) if compared to DTPA-BMA(3-). The formation of a ternary complex with L-lactate, which is common for DO3A-based heptadentate complexes, has not been observed for [Gd(DTTA-Me)(H2O)2](-) as monitored by (1)H NMR relaxometric titrations. From the results, it was concluded that the heptadentate DTTA-Me(4-) behaves similarly to the commercial octadentate DTPA-BMA(3-) with respect to stability. The use of [Gd(DTTA-Me)(H2O)2](-) as an MRI contrast agent in vitro and in animal studies is conceivable, mainly at high magnetic fields, where an increase of the inner-sphere-coordination water actually seems to be the most certain way to increase the relaxivity.     

Structural and photophysical properties of visible- and near-IR-emitting tris lanthanide(III) complexes formed with the enantiomers of N,N'-bis(1-phenylethyl)-2,6-pyridinedicarboxamide

The enantiomers of N,N'-bis(1-phenylethyl)-2,6-pyridinedicarboxamide (L), namely, (R,R)-1, and (S,S)-1, react with Ln(III) ions to give stable [LnL(3)](3+) complexes in an anhydrous acetonitrile solution and in the solid state, as evidenced by electrospray ionization mass spectrometry, NMR, luminescence titrations, and their X-ray crystal structures, respectively. All [LnL(3)](3+) complexes [Ln(III) = Eu, Gd, Tb, and Yb; L = (R,R)-1 and (S,S)-1] are isostructural and crystallize in the cubic space group I23. Although the small quantum yields of the Ln(III)-centered luminescence clearly point to the poor efficiency of the luminescence sensitization by the ligand and the intersystem crossing and ligand-to-metal energy transfers, the ligand triplet-excited-state energy seems relatively well suited to sensitize many Ln(III) ion's emission for instance, in the visible (Eu and Tb), near-IR (Nd and Yb), or both regions (Pr, Sm, Dy, Er, and Tm).     

Pyridine-based lanthanide complexes combining MRI and NIR luminescence activities

A series of novel triazole derivative pyridine-based polyamino-polycarboxylate ligands has been synthesized for lanthanide complexation. This versatile platform of chelating agents combines advantageous properties for both magnetic resonance (MR) and optical imaging applications of the corresponding Gd(3+) and near-infrared luminescent lanthanide complexes. The thermodynamic stability constants of the Ln(3+) complexes, as assessed by pH potentiometric measurements, are in the range log K(LnL)=17-19, with a high selectivity for lanthanides over Ca(2+), Cu(2+), and Zn(2+). The complexes are bishydrated, an important advantage to obtain high relaxivities for the Gd(3+) chelates. The water exchange of the Gd(3+) complexes (k(ex)(298)=7.7-9.3×10(6) s(-1)) is faster than that of clinically used magnetic resonance imaging (MRI) contrast agents and proceeds through a dissociatively activated mechanism, as evidenced by the positive activation volumes (ΔV(≠)=7.2-8.8 cm(3) mol(-1)). The new triazole ligands allow a considerable shift towards lower excitation energies of the luminescent lanthanide complexes as compared to the parent pyridinic complex, which is a significant advantage in the perspective of biological applications. In addition, they provide increased epsilon values resulting in a larger number of emitted photons and better detection sensitivity. The most conjugated system PheTPy, bearing a phenyl-triazole pendant on the pyridine ring, is particularly promising as it displays the lowest excitation and triplet-state energies associated with good quantum yields for both Nd(3+) and Yb(3+) complexes. Cellular and in vivo toxicity studies in mice evidenced the non-toxicity and the safe use of such bishydrated complexes in animal experiments. Overall, these pyridinic ligands constitute a highly versatile platform for the simultaneous optimization of both MRI and optical properties of the Gd(3+) and the luminescent lanthanide complexes, respectively.     

Zn(2+) Responsive Bimodal Magnetic Resonance Imaging and Fluorescent Imaging Probe Based on a Gadolinium(III) Complex

A Zn(2+)-responsive bimodal magnetic resonance imaging (MRI) and luminescence imaging probe GdL was synthesized. The relaxivity and luminescence properties were examined. In the presence of 0.5 equiv of Zn(2+), the longitudinal relaxivity is increased from 3.8 mM(-1) s(-1) to 5.9 mM(-1) s(-1) at 23 MHz and 25 °C with 55% enhancement, whereas the fluorescence exhibits a 7-fold increase. The Zn(2+) responsive imaging probe shows favorable selectivity and tolerance over a variety of biologically relevant anions and metal ions in physiological pH range for both relaxivity and luminescence. In vitro phantom images and confocal fluorescence images in living cells show that the bimodal Zn(2+) probe can effectively enhance T(1)-weighted imaging contrast and luminescence imaging effect through Zn(2+) coordination with excellent cellmembrane permeability and biocompatibility. Spectral and electrospray ionization mass spectrometry (ESI-MS) studies indicate that two different Zn(2+)-bound species, (GdL)(2)Zn and GdLZn, are formed when 0.5 and 1 equiv of Zn(2+) are bound to GdL complex, respectively. Crystal structural determination and dysprosium-induced (17)O NMR shift (DIS) experiment demonstrate that the increased molecular weight and the improved molecular rigidity upon complexation of Zn(2+) with GdL is the primary factor for relaxivity enhancement. Significant enhancement of the luminescence is due to a heavy atom effect and much increased molecular rigidity upon Zn(2+) binding to 8-sulfonamidoquinoline chromophore.     

Mononuclear Rare Earth Metal Complexes based on 1,10-phenanthroline Derivatives as Catalysts for Hydrolysis of p-nitrophenyl Phosphate (NPP)

Two multidentate ligands: N,N′-di-(propionic acid-2′-yl-)-2,9-diaminomethyl-1,10-phenanthroline (L1) and N,N′-di-(3′-methylbutyric acid-2′-yl-)-2,9-diaminomethyl-1,10-phenanthroline (L2) were synthesized. The hydrolytic kinetics of p-nitrophenyl phosphate (NPP) catalyzed by complexes of L1 and L2 with La(III), Gd(III) have been studied. Both LnL and LnLH₋₁ have been examined as catalysis for the hydrolysis of NPP in aqueous solution at 298 K, I = 0.10 mol dm⁻³ KNO₃ at the pH range 7.4–9.1, respectively. Kinetic studies show that both LnL and LnLH₋₁ have catalytic activity, but LnLH₋₁ is more active than LnL in the hydrolysis of NPP. The second-order rate constants for the hydrolysis of NPP are k_GdL1H−1 = 0.01399 mol⁻¹ dm³ s⁻¹, k_GdL1 = 0.0000110 mol⁻¹ dm³ s⁻¹ for complexes GdL1H₋₁ and GdL1, respectively. A new mechanism was proposed for the hydrolysis of NPP catalyzed by LnL and LnLH₋₁.     

Facile synthesis and relaxation properties of novel bispolyazamacrocyclic Gd3+ complexes: an attempt towards calcium-sensitive MRI contrast agents

Three novel GdDO3A-type bismacrocyclic complexes, conjugated to Ca (2+) chelating moieties like ethylenediaminetetraacetic acid and diethylenetriamine pentaacetic acid bisamides, were synthesized as potential "smart" magnetic resonance imaging contrast agents. Their sensitivity toward Ca (2+) was studied by relaxometric titrations. A maximum relaxivity increase of 15, 6, and 32% was observed upon Ca (2+) binding for Gd 2L (1), Gd 2L (2), and Gd 2L (3), respectively (L (1) = N, N-bis{1-[{[({1-[1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-10-yl]eth-2-yl}amino)carbonyl]methyl}-(carboxymethyl)amino]eth-2-yl}aminoacetic acid; L (2) = N, N-bis[1-({[({alpha-[1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-10-yl]- p-tolylamino}carbonyl)methyl]-(carboxymethyl)}amino)eth-2-yl]aminoacetic acid; L (3) = 1,2-bis[{[({1-[1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-10-yl]eth-2-yl}amino)carbonyl]methyl}(carboxymethyl)amino]ethane). The apparent association constants are log K A = 3.6 +/- 0.1 for Gd 2L (1) and log K A = 3.4 +/- 0.1 for Gd 2L (3). For the interaction between Mg (2+) and Gd 2L (1), log K A = 2.7 +/- 0.1 has been determined, while no relaxivity change was detected with Gd 2L (3). Luminescence lifetime measurements on the Eu (3+) complexes in the absence of Ca (2+) gave hydration numbers of q = 0.9 (Eu 2L (1)), 0.7 (Eu 2L (2)), and 1.3 (Eu 2L (3)). The parameters influencing proton relaxivity of the Gd (3+) complexes were assessed by a combined nuclear magnetic relaxation dispersion (NMRD) and (17)O NMR study. Water exchange is relatively slow on Gd 2L (1) and Gd 2L (2) ( k ex (298) = 0.5 and 0.8 x 10 (6) s (-1)), while it is faster on Gd 2L (3) (k ex (298) = 80 x 10 (6) s (-1)); in any case, it is not sensitive to the presence of Ca (2+). The rotational correlation time, tau R (298), differs for the three complexes and reflects their rigidity. Due to the benzene linker, the Gd 2L (2) complex is remarkably rigid, with a correspondingly high relaxivity despite the low hydration number ( r 1 = 10.2 mM (-1)s (-1) at 60 MHz, 298 K). On the basis of all available experimental data from luminescence, (17)O NMR, and NMRD studies on the Eu (3+) and Gd (3+) complexes of L (1) and L (3) in the absence and in the presence of Ca (2+), we conclude that the relaxivity increase observed upon Ca (2+) addition can be mainly ascribed to the increase in the hydration number, and, to a smaller extent, to the Ca (2+)-induced rigidification of the complex.     

Towards targeted MRI: new MRI contrast agents for sialic acid detection

The detection of sialic acid in living systems is of importance for the diagnosis of several types of malignancy. We have designed and synthesized two new lanthanide ion ligands (L1 and L2) that are capable of molecular recognition of sialic acid residues. The basic structure of these ligands consists of a DTPA-bisamide (DTPA, diethylenetriamine pentaacetic acid) whose amide moieties each bear both a boronic function for interaction with the diol groups in the side chain of sialic acid, and a functional group that is positively charged at physiologic pH values and is designed to interact with the carboxylate anion of sialic acid. The relaxometric properties of the Gd3+ complexes of these two ligands were evaluated. The relaxivity of the GdL1 complex has a significant second-sphere contribution at pH values above the pKa of its phenylboronic acid moiety. The interaction of the Gd3+ complexes of L1 and L2 with each of several saccharides was investigated by means of a competitive fluorescent assay. The results show that both complexes recognize sialic acid with good selectivity in the presence of other sugars. The adduct formed by GdL2 with sialic acid has the higher conditional formation constant (50.43+/-4.61 M(-1) at pH 7.4). The ability of such complexes to recognize sialic acid was confirmed by the results of a study on the interaction of corresponding radiolabeled complexes (153SmL1 and 153SmL2) with C6 glioma rat cells. 153SmL2 in particular is retained on the cell surface in significant amounts.     

A gadolinium chelate for detection of beta-glucuronidase: a self-immolative approach

New classes of physiologically responsive magnetic resonance (MR) contrast agents are being developed that are activated by enzymes, secondary messengers, pH, and temperature. To this end, we have prepared a new class of enzyme-activated MR contrast agents using a self-immolative mechanism and investigated the properties of these agents using novel in vitro assays. We have synthesized in nine steps a Gd(III) agent 1 that is activated by the oncologically significant beta-glucuronidase. 1 consists of Gd(III)DO3A (DO3A = 1,4,7-tricarboxymethylene-1,4,7,10-tetraazacyclododecane) bearing a pendant beta-glucuronic acid moiety connected by a self-immolative linker to the macrocycle. LC-MS analysis reveals that 1 is enzymatically processed as predicted by bovine liver beta-glucuronidase, generating 2-aminoethylGdDO3A, 2. Compound 2 was prepared independently in a four-step synthetic procedure. Complex 1 displays a decrease in relaxivity upon titration with bicarbonate anion. The relaxivity increases when 1 is converted to 2 in a buffer mimicking in vivo anion concentrations (Parker, D. In Crown Compounds: Towards Future Applications; Cooper, S. R., Ed.; VCH: New York, 1992; pp 51-67) by 17%, while the relaxivity decreases by 27% for the same experiment in human blood serum. Hydrolytic kinetics catalyzed by bovine liver beta-glucuronidase at interstitial pH = 7.4 fit the Michaelis-Menten model with k cat/Km = 74.9 +/- 10.9 M(-1) s(-1). Monitoring of bulk water proton T1 during incubation with enzyme shows an increase in T1 that mirrors results obtained through the relaxivity measurements of compounds 1 and 2.     

Modifying LnHPDO3A Chelates for Improved T1 and CEST MRI Applications

The new ligand HPDO3MA [(R,R,R,R)-10-(2-hydroxypropyl)-α,α′,α′′-trimethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid] was designed to combine and optimize the chemical properties of the macrocyclic ligands HPDO3A and DOTMA. The presence of the methyl groups on the acetic pendant arms of HPDO3A is expected to rigidify the structure of the ligand and favor an increase of the kinetic inertness of the Ln complexes. ¹H NMR spectra of Eu(HPDO3MA) displayed the presence of two pairs of diastereoisomers: SAP (square antiprismatic) and TSAP (twisted square antiprismatic) isomers (56 and 44 %, respectively). In addition, ¹H and ¹⁷O relaxometric NMR studies of Gd(HPDO3MA) showed approximately a 10 % increase in relaxivity and a faster water exchange rate with respect to Gd(HPDO3A). Moreover, a detailed chemical exchange saturation transfer (CEST) characterization of Yb(HPDO3MA) displayed a sensitivity about two times larger than that of Yb(HPDO3A) both in phantom and in cell labeling experiments. Finally, the kinetic inertness of Yb(HPDO3MA) was measured to be twice as high as that of Yb(HPDO3A), with a dissociation half-life at physiological pH of about 2500 years.     

Synthesis, relaxometric and photophysical properties of a new pH-responsive MRI contrast agent: the effect of other ligating groups on dissociation of a p-nitrophenolic pendant arm

Two gadolinium(III) chelates, GdNP-DO3A (1-methlyene-(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triAcetate) and GdNP-DO3AM (1-methlyene(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide), containing a single nitrophenolic pendant arm plus either three acetate or three amide pendant arms were synthesized and characterized. The properties of the gadolinium, terbium, and dysprosium complexes of these ligands were examined as a function of pH. The extent and mechanism of the changes in water relaxivity with pH of each gadolinium complex was found to differ substantially for the two complexes. The water relaxivity of Gd(NP-DO3A) increases from 4.1 mM(-1) s(-1) at pH 9 to 7.0 mM(-1) s(-1) at pH 5 as a result of acid-catalyzed dissociation of the nitrophenol from the lanthanide. The nitrophenol group in Gd(NP-DO3AM) does not dissociate from the metal center even at pH 5; therefore, the very modest increase in relaxivity in this complex must be ascribed to an increase in prototropic exchange rate of the bound water and/or phenolic protons.     

Equilibrium and NMR studies on GdIII, YIII, CuII and ZnII complexes of various DTPA-N,N'-bis(amide) ligands. Kinetic stabilities of the gadolinium(III) complexes

Three DTPA-derivative ligands, the non-substituted DTPA-bis(amide) (L(0)), the mono-substituted DTPA-bis(n-butylamide) (L(1)) and the di-substituted DTPA-bis[bis(n-butylamide)] (L(2)) were synthesized. The stability constants of their Gd3+ complexes (GdL) have been determined by pH-potentiometry with the use of EDTA or DTPA as competing ligands. The endogenous Cu2+ and Zn2+ ions form ML, MHL and M(2)L species. For the complexes CuL(0) and CuL(1) the dissociation of the amide hydrogens (CuLH(-1)) has also been detected. The stability constants of complexes formed with Gd3+, Cu2+ and Zn2+ increase with an increase in the number of butyl substituents in the order ML(0) < ML(1) < ML(2). NMR studies of the diamagnetic YL(0) show the presence of four diastereomers formed by changing the chirality of the terminal nitrogens of their enantiomers. At 323 K, the enantiomerization process, involving the racemization of central nitrogen, falls into the fast exchange range. By the assignment and interpretation of 1H and 13C NMR spectra, the fractions of the diastereomers were found to be equal at pH = 5.8 for YL(0). The kinetic stabilities of GdL(0), GdL(1) and GdL(2) have been characterized by the rates of the exchange reactions occurring between the complexes and Eu3+, Cu2+ or Zn2+. The rates of reaction with Eu3+ are independent of the [Eu3+] and increase with increasing [H+], indicating the rate determining role of the proton assisted dissociation of complexes. The rates of reaction with Cu2+ and Zn2+ increase with rising metal ion concentration, which shows that the exchange can take place with direct attack of Cu2+ or Zn2+ on the complex, via the formation of a dinuclear intermediate. The rates of the proton, Cu2+ and Zn2+ assisted dissociation of Gd3+ complexes decrease with increasing number of the n-butyl substituents, which is presumably the result of steric hindrance hampering the formation or dissociation of the intermediates. The kinetic stabilities of GdL(0) and GdL(1) at pH = 7.4, [Cu2+] = 1 x 10(-6) M and [Zn(2+)] = 1 x 10(-5) M are similar to that of Gd(DTPA)2-, while the complex GdL2 possesses a much higher kinetic stability.