Synthesis and characterization of glucose-functionalized Fe₄ tetrahedron

With the goal of building a new class of carbohydrate-functionalized systems capable of binding to protein targets, aqueous compatibility and solubility considerations were imperative. Incorporation of water-soluble building blocks has been shown to enable the subcomponent self-assembly of metal-anchored cages that are both soluble and stable in aqueous media.^27,40,41 Specifically, the use of organic building blocks bearing polar⁴² or charged^27,43 functional groups combined with the use of water-soluble metal salts and counterions^44,45 has permitted the successful synthesis of water-stable cages. Metal-ion complexation has also been shown to stabilize the typically water reactive imine bond against hydrolysis.⁴⁶

With these considerations in mind, we initially selected benzidinedisulfonate as a polar, charged, ditopic amine subcomponent that has been shown to form tetrahedral assemblies in aqueous solution.^47–49 Accordingly, treatment of glucose-substituted subcomponent A with commercial benzidinedisulfonic acid and Fe(BF₄)₂·6H₂O in the presence of [NMe₄][OH] in water (Scheme 1) resulted in the formation of one major species as assayed by ¹H NMR spectroscopy. Notably, these reaction conditions were optimized to include an excess of hydroxide ion, which was used to both deprotonate benzidinedisulfonic acid, and to achieve de-O-acetylation of acetate-protected subcomponent Ain situ. The one-pot self-assembly and ester hydrolysis of A conveniently and cleanly enabled rapid access to the desired deprotected product and simplified the synthetic procedure by eliminating an independent de-O-acetylation step. After precipitation from the crude reaction mixture, the product was easily purified away from the low molecular weight by-products by size-exclusion chromatography.

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Scheme 1

Synthetic scheme for the preparation of tetrahedral ([NMe₄]₄[1]), helical ([NMe₄]₂[2]), and mononuclear ([3][SO₄]) glycosylated self-assemblies containing β-d-glucose (glc), α-d-mannose (man), β-d-galactose (gal), and β-d-maltose (mal) appendages.

The [NMe₄]₄[1-glc] product contains Fe(ii) in the low-spin, S = 0, state as indicated by resonances exclusively located in the diamagnetic region of the ¹H NMR spectrum (Fig. 2a). The intense magenta colour (λ = 505 nm [ε 20000 M⁻¹ cm⁻¹], 545 nm [ε 24000 M⁻¹ cm⁻¹], Fig. 2b) of [NMe₄]₄[1-glc] is also ascribed to strong metal-to-ligand charge transfer excitations characteristic of low-spin Fe(ii) in an iminopyridine coordination environment.^47,50,51 Only one set of aromatic signals were observed by ¹H and ¹³C NMR spectroscopy, which is in good agreement with the isolation of a single, highly symmetric species in solution. The ¹H NMR aromatic resonances of [NMe₄]₄[1-glc] are slightly broadened (Δν = 7–18 Hz) when compared with the signals of the A precursor, which is attributed to slow molecular tumbling of the rigid cage core in solution, and typical of large molecular-weight molecules (MW of [1-glc]^4– = 6009 g mol⁻¹).^18,52 Imine bond formation during self-assembly was supported by the disappearance of the aldehyde ¹H NMR signal of subcomponent A (Fig. 2a, δ 9.95 ppm, CDCl₃), and the appearance of a new up-field shifted signal attributed to the imine proton located at δ 9.09 ppm in the ¹H NMR spectrum of the product (Fig. 2a, D₂O). Infrared spectroscopy also confirmed the formation of an imine bond and coordination of the nitrogen donor atom to the Fe centre as evidenced by the absence of an aldehyde vibration in the IR spectrum of [NMe₄]₄[1-glc], and the appearance of a strong vibration ascribed to the imine CN stretch (ν_CN 1558 cm⁻¹, ESI Fig. S33^†).^53,54 Formation of the twelve-fold glucose-substituted assembly was unequivocally confirmed by high-resolution electrospray mass spectrometry (HR-ESI-MS(–)) by the presence of the tetraanionic molecular ion peak (1502.2307 m/z, Fig. 2c), which established both the charge and the intact Fe₄L₆ metal–ligand stoichiometry of [1-glc]^4–.

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Fig. 2

(a) ¹H NMR spectra of subcomponent A (CDCl₃ (*), 25 °C, 400 MHz), [NMe₄]₄[1-glc], [NMe₄]₂[2-glc], and [3-glc][SO₄] (D₂O, 25 °C, 400 MHz); (b) UV-vis spectra of [NMe₄]₄[1-glc], [NMe₄]₂[2-glc], and [3-glc][SO₄] (50 μM, H₂O); (c) HR-ESI-MS of [1-glc]⁴⁻, [2-glc]²⁻, and [3-glc]²⁺; (d) solid-state molecular structure of [2-glc]²⁻ (Fe, orange; N, blue; O, red; S, yellow; C, grey; H, white).

With a protocol for the clean synthesis of [NMe₄]₄[1-glc] in place, we next conducted an analogous self-assembly procedure employing either NaOH or KOH as the hydroxide source to prepare the sodium and potassium salts of [1-glc]^4–. The facile preparation of Na₄[1-glc] and K₄[1-glc] (ESI Section S2.3.2 and S2.3.3^†) showcases the versatility of this methodology and the ease of which salts of various countercations can be prepared. Ready access to alkali metal salts of [1-glc]^4– also provides an excellent opportunity to study this system and others with higher potential biological compatibility when compared with the [NMe₄]⁺ analogues.

Scope of saccharide-functionalized assemblies

Applying the modularity of this synthetic approach, we next sought out other simple amine building blocks that would offer the ability to easily program the size, shape, and valency of resulting assemblies. Tetrahedral cage [1-glc]⁴⁻ bears twelve peripheral glucose groups as a consequence of coordination at each of the four Fe(ii) centres by three glycosylated iminopyridine fragments. Therefore, the preparation of analogously-substituted mono- and bimetallic Fe(ii) complexes would offer structures tethered with three and six glucose groups, respectively, thereby providing rational control over saccharide substitution. We selected 4,4′-diaminodiphenyl ether-2,2′-disulfonic acid and tris(2-aminoethyl)amine (tren) as di- and tritopic amine subcomponents to pair with subcomponent A on account of the reported ability of these building blocks to direct assemblies into di- and mononuclear structures, respectively.^55,56

Previous reports have demonstrated that the reaction between 2-formylpyridine and 4,4′-diaminodiphenyl ether-based subcomponents in the presence of divalent transition metal ions results in the formation of bimetallic triple-stranded helicates as opposed to tetrahedral assemblies on account of the enhanced flexibility in the ligand backbone.^56,57 With this precedent, we employed a protocol analogous to the one described for the synthesis of [NMe₄]₄[1-glc] to prepare a bimetallic glucose-functionalized helicate. Treatment of 4,4′-diaminodiphenyl ether-2,2′-disulfonic acid with A in the presence of Fe(BF₄)₂·6H₂O and [NMe₄][OH]·5H₂O resulted in clean conversion to the triple-stranded helix, [NMe₄]₂[2-glc], as judged by ¹H NMR spectroscopic characterization of the crude product. Complex [NMe₄]₂[2-glc] was purified via size-exclusion chromatography, and the purified product was characterized by ¹H (Fig. 2a) and ¹³C NMR spectroscopy (ESI, Fig. S37^†). NMR characterization revealed two sets of resonances that are likely due to the presence of the right-handed, ΔΔ, and left-handed, ΛΛ, configurations (one set of resonances),⁵⁸ and the presence of the ΛΔ isomer (second set of resonances), which contains two different configurations at the metal centres (Fig. 2a). Indeed, mixtures of ΔΔ, ΛΛ, and ΛΔ configurations are reported for other dinuclear M₂L₃ helicates in the literature.^59,60 The presence of both ΔΔ and ΛΛ configurations in this product was confirmed by an X-ray diffraction study conducted on a single crystal of the salt (vide infra and ESI Section S7^†). Complex [NMe₄]₂[2-glc] was also characterized by UV-vis (Fig. 2b) and infrared (ESI Fig. S41^†) spectroscopy, with spectroscopic signatures consistent with those observed for the tetrametallic [NMe₄]₄[1-glc] analogue. The identity of [2-glc]^2– was confirmed as the sixfold-glucose substituted bimetallic complex by HR-ESI-MS(−) (Fig. 2c).

To establish the structures of this new class of glycosylated molecules, we focused efforts on growing single crystals of the isolated assemblies for X-ray diffraction analysis. Crystals of [NMe₄]₂[2-glc] of sufficient quality for structural determination were grown by vapor diffusion of acetone into a saturated aqueous solution of the complex at 25 °C over the course of 48 h. An X-ray diffraction study of these crystals permitted structural determination of the [2-glc]²⁻ anion, and the solid-state structure is displayed in Fig. 2d. As shown in the molecular structure, the framework of [2-glc]²⁻ spans a 3 nm distance from end-to-end, in which each end of the helix is capped by three glucose fragments. The solid-state structure also displays all six sulfonate groups directed towards the exterior of the helicate, which enforces the C₃ symmetry of the molecular core that is reflected in the high symmetry observed by NMR spectroscopy. Such crystallographic characterization is critical to this work by enabling a more complete understanding of the molecular structure of this class of assemblies.

We next expanded the series of glycosylated assemblies to include a monometallic Fe(ii) complex that would enforce a trivalent saccharide framework. Treatment of tris(2-aminoethyl)amine with subcomponent A in the presence of stoichiometric amounts of [NBu₄][OH] and FeSO₄ in H₂O at 70 °C resulted in the formation of mononuclear, [3-glc][SO₄] (Scheme 1), as evidenced by ¹H NMR spectroscopic analysis of the product. This self-assembly reaction is straightforward and proceeds cleanly to generate C₃-symmetric [3-glc]²⁺ as supported by the high symmetry observed in the ¹H and ¹³C NMR spectra of the product. Monometallic [3-glc][SO₄] was also characterized by UV-vis and infrared spectroscopy, and the identity of this product as the triply-substituted glucose complex was confirmed by HR-ESI-MS(+) (Fig. 2c).

With a successful self-assembly procedure established, we extended this synthetic approach to the preparation of analogous glycosylated complexes employing subcomponents B (α-d-mannose), C (β-d-galactose), and D (β-d-maltose) as shown in Scheme 1. The mannose-, galactose- and maltose-substituted tetra-, bi-, and monometallic analogues were prepared following similar synthetic procedures described for the glucose-derivatives. This versatile synthetic strategy allowed us to easily and rapidly establish a molecular library of well-defined saccharide-appended assemblies that includes twelve unique examples. All complexes were characterized by NMR spectroscopy (¹H and ¹³C), IR and UV-vis spectroscopy, and the identities of all products were confirmed by HR-ESI-MS (ESI Section 2.3^†).

Stability studies

Employing [NMe₄]₄[1-glc] as a model complex, the stability of the present glycosylated assemblies in biologically-relevant media was evaluated. Complex [NMe₄]₄[1-glc] was exposed to fetal bovine serum cell-culture media, solutions of various buffers and pH environments (4–10), as well as the common biological nucleophile, glutathione,⁶¹ for extended periods of time (72 h) with minimal observable signs of degradation as assayed by UV-vis spectroscopy (ESI Section S4.2^†). The excellent stability profile of [NMe₄]₄[1-glc] under these conditions is a direct consequence of the robust framework of the tetrahedral cage and the metal–ligand bonding strength. The high water solubility of [NMe₄]₄[1-glc] (302 g L⁻¹, ESI Section S3^†) also plays a critical role in the overall biological compatibility of the complex and its ability to remain intact in solution.⁴⁶ The [NMe₄]₄[1-glc] complex also has an excellent shelf-life and can be stored on the benchtop as a solid under open-atmosphere conditions and in aqueous solution for extended periods of time. No decomposition of [NMe₄]₄[1-glc] in D₂O (5 mM, 25 °C) was detected even after nine months as monitored by ¹H NMR spectroscopic analysis (ESI Section S4.1^†).

We are grateful for financial support from the Department of Chemistry and Biochemistry at UCSD. We thank Professor Jerry Yang at UCSD for generously allowing us to use his ITC instrument and laboratory space, and Aashish Shivkumar is thanked for assistance with ITC experiments. Professor Fleur Ferguson (UCSD) is thanked for helpful discussions and Professor Arnold Rheingold (UCSD) is thanked for assistance with crystallographic characterization.