Carbohydrates, as the carriers of energy in life, are essential mediators of a wide range of biological processes including protein folding, cell-cell recognition, infection by pathogens or tumor metastasis [1, 2]. The recognition of carbohydrates with high specificity and sensitivity is biologically important but intrinsically challenging, for both nature and host-guest chemists [3-5]. Saccharides are high hydrophilic, non-charged, subtle variation of the three-dimensional structures, and the only one kind of the recognition units are the hydrogen bonding groups. Even the most common class of natural receptors, lectins, show notably low affinities and modest selectivity [6, 7]. Biomimetic synthetic organic-box-like lectins equipped with multi-hydrogen bonding groups in their inner cavities have been devoted to sensor saccharides operating through non-covalent interactions [8-16]. However, these purely organic hosts [17-21] commonly required lengthy and low-yielding synthetic process. Noted that complexation of saccharides with an organic host receptor was often detected by the ¹H NMR chemical-shift changes [22-28], but this method showed a limited sensitivity. Alternatively, the using of fluorescence spectroscopic method to recognize target carbohydrates is highly anticipant. This method possesses excellent signaling properties to modulate the binding process and thus might leave an improved detection sensitivity [29, 30].

On the other hand, self-assembled metal-organic cages (MOCs) with unique hollow cavity environment and rich chemical/physical properties enable them as promising receptors for various compounds recognition [31-33]. The abundant structural configurations and fruitful functionalities can be successfully achieved by rational selection of different metal centers and well-designed ligands. Especially, when amide groups as multiple hydrogen bonding triggers used in nature protein–carbohydrate complexes were incorporated into MOCs, the novel synthetic hosts could be used as efficient receptors for mimicking biological carbohydrate recognition processes [34-38]. The key issues remain how to subtly modulate the distribution of recognition sites, as well as to form appropriate shape and size to encapsulate specific saccharides. Furthermore, fluorescent properties are also desired by integrating emissive ligands and/or coordination complexes into the supramolecular architectures [39] to possess improved signal output properties.

Herein, the design and synthesis of a metal-organic tetrahedron (Zn₄L₄) equipped with four phenoxazinean-based signaling components and a dozen of amide-based hydrogen binding sites as biomimetic lectin for selectively recognition of glucosamine (GlcN) over other related natural mono- and disaccharides was reported (Scheme 1). By contrast, free phenoxazine-based ligand (L) showed no obvious enhancement of the fluorescent intensity towards GlcN. Fluorescence titration and ESI-MS studies demonstrated that the newly obtained biomimetic lectin host could encapsulate one GlcN in the cavity environment of Zn₄L₄ with high association constant. ¹H NMR spectroscopic studies confirmed that the selectivity resulted from the complementary multiple hydrogen bonding interactions between amine, hydroxy groups of GlcN and the amide moieties of Zn₄L₄. The present results suggested that rational arrangement of recognition sites in the confined space of metal-organic cage is crucial for the selectivity toward target guests, and the supramolecular host-guest species formation between the cage containing hydrogen-bond recognition sites and the encapsulated carbohydrates is responsible for the effective detection process.

As shown in Fig. 1a, ligand L was obtained by connecting phenoxazine-based core (1) and 2,2′-bipyridine chelators through amine-linkages in two steps [40-42]. Acylating of 1 with thionyl chloride, and subsequently reacted with 2 to afford ligand L in 78% yield. The successfully obtained ligand L was confirmed by ¹H and ¹³C NMR spectra (Fig. 1b and Section S2 in Supporting information). The reaction of ligand L (1.0 equiv.) and Zn(OTf)₂ (1.0 equiv.) (OTf=trifluoromethanesulfonate) in DMF at 80 ℃ for 12 h resulted in the formation of Zn₄L₄ as the unique product. The ¹H NMR spectra of tetrahedron Zn₄L₄ displayed only one set of ligand resonances in solution, suggesting the exclusive formation of a discrete structure to be highly symmetrical (Fig. 1c). Diffusion-ordered NMR spectroscopy (DOSY) of the product also showed that the multiple aromatic signals possessed similar diffusion coefficients (D=6.31×10⁻¹¹ m²/s, Fig. S8 in Supporting information).

The formation of a discrete supramolecular species containing a Zn₄L₄⁸⁺ cation was confirmed by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). As showed in Fig. 1d, a series of intense peaks at m/z=458.0999, 544.8200, 660.4491, 822.3308, 1065.1522 and 1469.8522 were observed. Comparison of the experimental peaks with these simulated results obtained on the basis of natural isotopic abundance, these peaks were safely assigned to the species [Zn₄(L)₄]⁸⁺ (calcd. 458.1022), [Zn₄(L)₄+OTf]⁷⁺ (calcd. 544.8243), [Zn₄(L)₄+2OTf]⁶⁺ (calcd. 660.4538), [Zn₄(L)₄+3OTf]⁵⁺ (calcd. 822.3351), [Zn₄(L)₄+4OTf]⁴⁺ (calcd. 1065.1570) and [Zn₄(L)₄+5OTf]³⁺ (calcd. 1469.8602), respectively. This result demonstrated the Zn-based tetrahedral architecture was successfully assembled with substantially stability in solution.

To confirm the formation of a [4+4] metal-organic cage, UV–vis titration of ligand L (20.0 μmol/L) upon the addition of Zn²⁺ was further well conducted. As showed in Fig. 2, the ligand L exhibited two broad bands at λ = 310 nm and 390 nm, which assignable to the π–π* and n–π* charge transfer bands. Upon the addition of Zn²⁺ ions, the band at λ = 310 nm was decreased gradually, while the intensity of new peaks appeared at λ=324 and λ = 432 nm were increasing significantly. These bands remained constant after adding approximately 1.0 equiv. of Zn²⁺ ions. The presence of sharp isosbestic point at λ = 322 nm indicated that only two species coexisted in the equilibrium system. The almost linear relations between the absorbance at both bands and the concentration of Zn²⁺ added revealed that the formation of the cage complex was quantitative and the complex exhibited 1:1 stoichiometry. This result demonstrated that the M₄L₄ cage compound was the only one complexation specie and the association constant of the complexation specie is relatively high.

Numerous efforts for crystallization of Zn₄L₄ to obtain suitable crystals for the high-quality X-ray diffraction have been unsuccessful. Therefore, in order to understand the three-dimensional structure of Zn₄L₄ better, we use a MM2 energy-minimized model to simulate the structure of Zn₄L₄ (Fig. 3). Through the optimized structures, we can more intuitively insight that the Zn₄L₄ was a twisted tetrahedron structure with a hollow cavity. The four metal atoms occupied the vertices and each of them was coordinated with three ligands. Each ligand was positioned on one of the four faces of the tetrahedron which defined by four metal ions. The Zn⋯Zn distance in the long edges of Zn₄L₄ was about 19.38(1) Å, and in the short edges was about 18.34(1) Å. The average dihedral angle between the phenyl groups and phenoxazine planes in Zn₄L₄ was about 74.41(1)°. The structure possessed a confined cavity and contains 12 amide groups, which provides a great possibility for carbohydrate recognition.

The fluorescent nature of ligand L was preserved and transferred to the final cage, which enable the cage Zn₄L₄ with luminescent recognition process of relative guests (Fig. S10 in Supporting information). Fluorescent emission spectrum of Zn₄L₄ exhibited a ligand-based broad emission band at λ_em = 448 nm when excited at λ_ex = 396 nm (5.0 μmol/L in DMF). Subsequently, the potential application of as a fluorescent detector for natural saccharides recognition was explored. Impressively, upon the addition of GlcN (0–10 equiv.) into the DMF solution of Zn₄L₄ (5 μmol/L), the wavelength of the emission maximum at λ_em = 448 nm did not change but the luminescence intensity enhanced gradually with the increasing concentration of the guest. When adding of 10.0 equiv. GlcN, the fluorescence intensity of Zn₄L₄ was dramatically increased around 82%. The hill-plot profile of the fluorescence titration curves at 448 nm demonstrated a 1:1 stoichiometric host-guest complex was formed with an association constant about 4.03×10⁴ L/mol (Fig. 4). The formation of donor-type hydrogen bonds between the amide groups of Zn₄L₄ and the guest molecules could alter the electronic distribution of the ligand backbone, which suppress the process of photo-induced electron transfer (PET) process, thus leading to the significant luminescence enhancement [36]. Interestingly, the glucose (Glu) only induced negligible emission enhancement of Zn₄L₄ under the same condition (Fig. 5). The only difference between these two saccharides is that there is one amine group in the skeleton of GlcN. However, the very different recognition ability of Zn₄L₄ toward them suggested that the amine might played an important role. Under the same condition, when 10.0 equiv. of cyclohexanamine and aniline were added, the fluorescent emission intensity of Zn₄L₄ was increased around 25% and 29%, respectively (Fig. S13 in Supporting information). Noted that other mono-saccharides including mannose (Man), ribose (Rib) and xylose (Xyl) showed negligible emission enhancement less than 10%. Furthermore, the addition of excess disaccharides including the sucrose (Suc), lactose (Lac), melibiose (Mel), maltose (Mal), and trehalose (Tre) did not cause any obvious spectroscopic changes of Zn₄L₄ as well (Fig. 5). Combined with the result that free ligand L showed no recognition ability to GlcN, these results suggested that the specific recognition process mainly occurs in the cavity of the cage, and the larger disaccharide could not be encapsulated into the cavity.

The excellent selectivity of Zn₄L₄ toward GlcN encouraged us to further investigate the interaction detail of the host–guest complexation. As shown in Fig. 6, ¹H NMR spectroscopic titration studies on the GlcN confirmed that, as expected, when increasing the concentrations of GlcN (0–10.0 equiv.), the hydrogen signal of the amide groups in Zn₄L₄ showed upfield shifts (Δδ_Ha = 0.16 ppm, Δδ_Hb = 0.16 ppm) that indicated the amide group in Zn₄L₄ acts as the hydrogen bond receptor, and the hydrogen signal of the GlcN showed downfield shifts (especially, Δδ_H1α= 1.50 ppm, Δδ_H1β=1.24 ppm), which indicated that the GlcN was encased in the cavity of Zn₄L₄ and the Zn₄L₄ and GlcN formed a host–guest complex. In addition, when GlcN (10.0 equiv.) were added into the solution of Zn₄L₄ in DMF, the electrospray ionization mass spectrometry (ESI-MS) of the mixture exhibited two new peaks at m/z 554.8465 and 672.1405. Compared with the simulation on the basis of natural isotopic abundance, these two peaks can assign to the [GlcN⊂Zn₄(L)₄-H+CH₃CN]⁷⁺ and [GlcN⊂Zn₄(L)₄-H+OTf+CH₃CN]⁶⁺ (Fig. S21 in Supporting information). The NOESY spectra of the mixture of and GlcN (10.0 equiv.) also gave clear evidence that intermolecular contacts between them were formed (Fig. S22 in Supporting information). Based on the above results, we speculated that the main reason of the specific selectivity was that Zn₄L₄ with three-dimensional configuration and inner confined environment could encapsulate one GlcN into the cavity, which lead to the formation of complementary multiple hydrogen binding between amine, hydroxy groups of GlcN and the amide moieties of Zn₄L₄.

In summary, we reported a supramolecular tetrahedron as biomimetic lectin for GlcN recognition with high specificity and sensitivity over other related natural mono- and disaccharides. The Zn₄L₄ displayed phenoxazine-based broad fluorescent emission property, and equipment of 12 amide groups in the skeleton. The formation of a 1:1 host-guest complexes has been characterized by fluorescence titration spectroscopy and ESI-MS. ¹H NMR spectroscopic titration studies and NOESY spectra confirmed that the hydrogen bonding interactions between GlcN and receptor Zn₄L₄, resulting a stronger host-guest binding affinity. The present results demonstrated that the rational distribution of amide groups and luminescent ligands into the 3D metal-organic cage structure not only provides a recognizable hydrogen-bonding functional site for the guest molecule, which facilitates the formation of the host-guest complex, but also provides a way to convert identification information into effective communicator for optical signals. Accordingly, changing the topology and the arrangement of hydrogen binding sites in the confined space of host, the selectivity toward other target guests could be expected.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We appreciate the National Natural Science Foundation of China (Nos. 21701019 and 21861132004) and Doctoral Scientific Research Launching Fund Project of Liaoning province (No. 2019-BS-050).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.cclet.2020.07.028.