Chemical Communications

Synthesis of luminescent heterometallic bis-lanthanide complexes via selective, sequential metallation

Matthew S. Tremblay and Dalibor Sames*
Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA. E-mail: sames@chem.columbia.edu; Fax: +1 212-932-1289; Tel: +1 212-854-7108

A modular synthetic method for the differential incorporation of two lanthanide ions into a single molecular scaffold is reported; the mixed bimetallic Tb/Eu complex displays an interesting solvent polarity-dependent ratiometric luminescence.

The utility of lanthanide chelates as luminescent probes and magnetic resonance (MR) contrast agents in biological systems is well established.¹ The sharp emission bands of Tb³⁺ and Eu³⁺ occur in a useful wavelength domain (500–700 nm) and often have lifetimes in the order of a few milliseconds. Although direct excitation of the metal center leads to forbidden transitions, sensitization via energy transfer from an appended antenna occurs readily with a variety of organic chromophores, including the aromatic amino acids tyrosine and tryptophan.² The highly paramagnetic Gd³⁺ ion is used frequently in MR imaging applications owing to its ability to provide image contrast by decreasing the T₁ relaxation time of nearby water molecules.³ Responsive lanthanide probes capable of sensing analyte binding or enzymatic activity have been developed for both the luminescent⁴ and magnetic modes.⁵

The toxicity of lanthanide aqua ions makes their residence in a soluble chelate obligatory for biological applications. In addition, inner sphere water molecules can be detrimental to Tb³⁺ and Eu³⁺ luminescence quantum efficiency due to the strong coupling of O–H vibrations. Fulfilment of the high coordination requirements (7–9) of the lanthanides is typically accomplished using the water-soluble polyaminocarboxylates 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid (DOTA) and 1,1,4,7,7-diethylenetriamine pentaacetic acid (DTPA),⁶ although several other motifs have been studied.⁷ As a result of their popularity, several methods for the preparation of DOTA and DTPA bioconjugates are available.⁸

The incorporation of two different lanthanide ions into a single probe molecule may afford interesting properties, in either a dual-emissive (Tb³⁺ and Eu³⁺) or bimodal (Eu³⁺/Tb³⁺ and Gd³⁺) context.^7b Although several lanthanide-containing heterometallic complexes have been prepared by exploiting differences in ligand preference between lanthanides and other transition metals,^7b,9 the minute differences in coordination behavior¹⁰ across the lanthanide period—particularly the immediate neighbors Eu³⁺, Gd³⁺ and Tb³⁺—severely limit the synthesis of heterometallic bis-lanthanide complexes. To the best of our knowledge, there is only one example of a discretely synthesized heterometallic complex with two different lanthanide ions,¹¹ though other complexes containing two different lanthanide ions have been prepared and studied as components of statistical mixtures.^7b,12

As part of a broad program aimed at sensing chemical and enzymatic events with fluorescent¹³ and luminescent¹⁴ probes, we wanted to develop a modular synthetic strategy for the differential incorporation of two lanthanide ions into a single molecular scaffold, preferably using the stable, soluble, and readily bioconjugatable DOTA and DTPA chelates. Although the two chelates are almost indistinguishable thermodynamically,^10,15 only the acyclic DTPA chelate is prone to kinetic dissociation.^7a,10,16 We postulated that the sequential, differential complexation of a molecule containing both DOTA and DTPA chelates could be accomplished by capitalizing on this difference in decomplexation rates (Fig. 1). Specifically, treatment of DOTA/DTPA bis-chelate 1 with an excess of Ln_A³⁺ should result in homobimetallic complex 2, which could be prompted to undergo selective kinetic dissociation (e.g. acid-promoted demetallation) to give the corresponding monometallic species 3. Further complexation of this complex with Ln_B³⁺ should yield the pure heterobimetallic complex 4. A conceptually similar approach has been used elegantly by Horrocks, Jr. et al. to study the distances between calcium binding sites in proteins.¹⁷ The proposed sequential, differential metallation may represent a more general approach than a previously reported method,¹¹ since it obviates the need to couple metallated fragments.

	Fig. 1 Schematic representation of the sequential, differential metallation of a DOTA/DPTA bis-chelate with two different lanthanide ions (LnA3+ and LnB3+).

The branched tetrapeptide-based DOTA/DTPA bis-chelate 5 was prepared via standard solid phase peptide synthesis on Rink Amide AM resin using the orthogonally-protected diamino acid Fmoc-Dpr(Mtt)-OH for the selective introduction of the DOTA(^tBu)₃ and DTPA(^tBu)₄ units (Scheme 1). After cleavage and global deprotection, the crude peptide 5 was purified by reverse phase HPLC (RP-HPLC) and complexed with excess Tb³⁺ in pH 5 triethylammonium acetate buffer. When analyzed by RP-HPLC using a H₂O/MeCN eluent system buffered with 0.1% TFA, the crude mixture contained a single major peak, which was isolated by preparative RP-HPLC and identified by mass spectrometry as the mono-Tb³⁺ species 6, containing only a trace amount of the corresponding bis-Tb³⁺ species. The TFA-promoted dissociation of Tb:DTPA complexes during preparative HPLC has been noted by others^4b and found by us to be nearly quantitative. Following the isolation of 6, complexation with Eu³⁺ or Gd³⁺ took place quantitatively in unbuffered aqueous media; the resulting heterobimetallic complexes 7 and 8 could be used directly for photophysical measurements.⁶ This simple protocol permits the rational design and synthesis of heterobimetallic complexes containing any pair of lanthanide metals, making available new probes and bioconjugates with potentially interesting properties and applications.

	Scheme 1 Synthesis of the branched tetrapeptide ligand DOTA-Dpr(DTPA)-TrpNH2 (5) and its sequential, differential metallation, producing the heterometallated bis-lanthanide complexes 7 and 8.

The steady state emission spectra of 6, 7 and 8 in water are shown in Fig. 2a. As expected, a portion of the energy absorbed by the tryptophan residue is emitted as fluorescence (_max = 390 nm) and a portion is transferred to the ⁵D₄ state of Tb³⁺ (E = 20400 cm^–1), resulting in sensitized luminescence (_max = 487, 544, 585 and 620 nm) from the Tb:DOTA moiety. Although the Tb:DOTA and Eu:DTPA chelates are most likely at similar distances from the tryptophan sensitizer in 7, the triplet energy of tryptophan is too high to efficiently populate the emissive ⁵D₀ state of Eu³⁺ (E = 17200 cm^–1) and no sensitized Eu³⁺ luminescence is expected to be observed.^1,17 However, careful comparison of 6 and 7 reveals a small shoulder at 613 nm in the spectrum of 7 but not in the spectrum of 6. This emission corresponds to the ⁵D₀⁷F₂ transition of Eu³⁺. This shoulder was more pronounced in less polar solvents, such as tert-butanol (Fig. 2b). The luminescence properties of 6 and 7 were compared in a series of alcohol solvents, and the Eu³⁺ emission band increased at the expense of Tb³⁺ emission as the polarity of the solvent was decreased, allowing ratiometric measurement of the relative solvent polarity (Fig. 2c).

	Fig. 2 (a) Emission spectra of compounds 6, 7 and 8 in H2O. (b) Emission spectra of 7 in H2O and in tert-butanol. (c) The Eu/Tb emission ratio dependence on solvent polarity of 6 and 7.

This polarity-sensitive emission could be the result of several factors. The first is deactivation of the Tb³⁺ excited state via energy transfer to Eu³⁺, which, although it has limited precedent, is known to be efficient.^7b The second scenario involves ratiometric proportioning of the tryptophan excited state energy directly to Eu³⁺ in addition to Tb³⁺; a control compound containing only an Eu³⁺ chelate and tryptophan exhibited similar solvent-dependent sensitized luminescence, albeit significantly weaker.§ A third possibility is direct excitation of the Eu³⁺ chelate, but this has been ruled out by control studies.§ The precise nature of this ratiometric emission and its potential applications to polarity sensing are under investigation. Since both Tb³⁺ and Eu³⁺ have long radiative lifetimes and luminesce at relatively long wavelengths, this is the first system that would provide a ratiometric measurement where both components are outside of the frequency as well as the time domain of cellular autofluorescence.

In summary, a novel synthetic route to heterometallic bis-lanthanide complexes via sequential, differential metallation has been presented. Complex 7, containing Tb³⁺ and Eu³⁺, exhibits ratiometric luminescence as a function of solvent polarity and may provide a useful platform for time-resolved polarity sensing.

Financial support for this work was provided by the G. Harold and Leila Y. Mathers Foundation. Marlin Halim is gratefully acknowledged for her intellectual and editorial support.

Footnotes

Electronic supplementary information (ESI) available: Synthetic protocols and characterization; experimental details and additional data for photophysical experiments.

A long pass filter, used to remove diffracted excitation light, truncates the tryptophan emission and shifts its maximum.

§ For additional details, see the ESI.