In Situ Ligation of High‐ and Low‐Affinity Ligands to Cell Surface Receptors Enables Highly Selective Recognition

Abstract This paper reports an entirely unexplored concept of simultaneously recognizing two receptors using high‐ and low‐affinity ligands through ligating them in situ on the target cell surface. This de novo approach is inspired by the pretargeting strategy frequently applied in molecular imaging, and has now evolved as the basis of a new paradigm for visualizing target cells with a high imaging contrast. A distinct advantage of using a labeled low‐affinity ligand such as glycan is that the excess labeled ligand can be washed away from the cells, whereas the ligand bound to the cell, even at the milli molar affinity level, can be anchored by a bioorthogonal reaction with a pretargeted high‐affinity ligand on the surface. Consequently, nonspecific background is minimized, leading to improved imaging contrast. Importantly, despite previously unexplored for molecular imaging, a notoriously weak glycan/lectin interaction can now be utilized as a highly selective ligand to the targets.


Synthesis of 8.
To a solution of 7 obtained above (8.9 mg, 9.0 µmol) in DMF (200 µL) was added N-hydroxysuccinimide (2.1 mg, 18 µmol) and EDC・HCl (10.3 mg, 53.7 µmol). The solution was stirred for 1.5 h at room temperature, and the mixture was directly purified by RP-HPLC using the semi-preparative column at a flow rate of 3 mL/min with a linear gradient of MeCN/0.1%TFA (30-60% for 80 min) to obtain 8 (6.7 mg, 68%) as a mixture of regioisomers. HRMS calcd for C 60 H 63 N 6 O 14 ([M+H] + ) 1091.4397, found 1091.4356 for the isomer t R : 15.7 min, 1091.4424 for the isomer t R : 16.7 min (flow rate of 1 mL/min with a linear gradient from 30 to 80% MeCN/0.1%TFA for 25 min using the analytical column).

Synthesis of 2a.
To a solution of 9a 2 (3.0 mg, 1.2 µmol) in MeOH/H 2 O (2 : 1, v/v, 1 mL) was added 10% palladium on carbon (wet with 55% H 2 O, 320 µg). After the reaction mixture was stirred under H 2 atmosphere for 4.5 h, the catalyst was removed by filtration, and concentrated in vacuo. The residue was dissolved in H 2 O, and lyophilized to obtain the amine 10a (3.4 mg, quant), which was used for the reaction with 8 without further purification. HRMS calcd for C 95 H 159 N 9 O 65 ([M+2H] 2+ ) 1232.9707, found 1232.9750.
To a solution of 8 (1.1 mg, 970 nmol) in DMF (50 µL) was added a solution of 10a (1.2 mg, 480 nmol) in 0.1 M sodium phosphate buffer (pH 7.0, 100 µL). The reaction mixture was stirred for 25 h at room temperature, quenched with 50% AcOH, and purified by RP-HPLC using the semi-preparative column at a flow rate of 3 mL/min with a linear gradient of MeCN/0.1%TFA (30-60% for 80 min) to obtain 2a (
To a solution of 2a (0.77 mg, 0.33 µmol) in PBS (50 µL) was added 1a (0.22 mg, 0.26 µmol. The reaction mixture was stirred for 1.5 h at room temperature, and purified by RP-HPLC using the semi-preparative column at a flow rate of 3 mL/min with a linear gradient of MeCN/0.1%TFA (20-50% for 80 min) to obtain the clicked product (0.4 mg for the isomer t R : 17.8 min, 0.6 mg for the isomer t R : 17.

LC-MS/MS analysis of clicked products (1a+2a) from cell surface
HUVECs, which were treated with 1a and 2a according to the established procedure, were washed with PBS twice, and treated with KCl-HCl buffer (pH 2.0, 1.0 mL) on ice, three times for 1 min to dissociate the clicked products from the cell surface. The collected KCl-HCl buffer solution was combined and centrifuged. The supernatant was then lyophilized for MS analysis. The sample was subjected to nano-liquid chromatography-mass spectrometry. The clicked product were purified using nano ESI spray column (100 mm length ×75 µm inside diameter, 3 µm, NTCC analytical column C18, Nikkyo Technos Co., Ltd., Tokyo, Japan) that was equilibrated with A buffer (0.1% aqueous formic acid) and eluted with a linear gradient of 35% buffer B (0.1% formic acid in 100% acetonitrile) over 10 min at a flow rate of 300 nl/min (Easy nLC, Thermo Fisher Scientific). The mass spectrometer (Q-Exactive, Thermo Fisher Scientific) was operated in the positive-ion mode, and the MS/MS spectra were acquired in a data-dependent TOP10 method or a targeted MS/MS (m/z = 1425.58) method. Figure S17. Importance of using low-affinity ligand of the surface receptor on the target cell A for highly selective imaging. In comparison with the Figure 1b using the low-affinity ligand "D", when applying the high-affinity ligand, e.g., ligand "E", could bind and stick to the other cells ( Figure S17d), e.g., "cell C" expressing this receptor, so that the imaging selectivity is reduced.    HUVECs (1.2 × 10 5 cells, 1 mL) were plated onto a 24-well microplate (Iwaki) and incubated in the EBM TM -2 cell culture medium for 24 h at 37°C under 5% CO 2 . After complete adhesion, the cells were washed with the cell culture medium and treated with the cyclic RGD peptide 1a (50 µM, 600 µl/well) for 15 min at room temperature (25°C) before washing with the EBM TM -2 cell culture medium (triple washing, 200 µl). The cells were treated with the TAMRA-glycan derivative 2a (50 µM, 600 µl/well) for 30 min at 4°C. After washing with PBS (triple washing, 200 µl), the cells were treated with 2 mM EDTA (200 µl/well) at 37°C for 10 min and then centrifuged at 3,000 rpm (800 × g) for 5 min at 4°C. After removal of the supernatant, the cells were washed with PBS (400 µl) and centrifuged at 3,000 rpm for 5 min at 4°C. This washing cycle was repeated, and the cells were suspended in PBS (400 µl) and subjected to fluorescence analysis with a guava easyCyte (Merck Millipore) flow cytometer using 488 nm laser excitation and a 583 nm emission filter. Live cells (10,000 cells/sample) for the detection of cellular fluorescence intensity were quantified based on forward-scattering and side-scattering analyses.   The RGD peptide 1a and the glycan ligand 2a were mixed at a final concentration of 50 µM each. The reaction mixture was allowed to stand at 4 o C for 30 min, and monitored by RP-HPLC. (flow rate of 1 mL/min with a linear gradient from 10 to 80% MeCN/0.1%TFA for 25 min using the analytical column). Figure S24. Effects of linker length in 1a-1d for SPAAC reaction on HUVECs. a, HUVECs were labeled using the ligand combination of the RGDyK peptides 1a-1d with the glycan 2a (red), fixed, stained with DAPI (blue), and observed by confocal microscopy under the identical conditions described in Figure 3. The scale bar indicates 20 µm, b, Comparison of the fluorescent intensities measured in Figure S23a. Data are presented as the means ± S.E. [n = 10 (10,000 cells x 10), one way ANOVA post hoc Tukey-Kramer's test, *p<0.01, **p<0.05]. Figure S25. Imaging of HUVECs using both 1a and 2a and SPAAC reaction under cell fixing conditions. a, HUVECs were fixed with 4% paraformaldehyde (PFA) for 30 min at rt, and then labeled with the glycan ligand 2a alone (red). b, HUVECs were treated with the RGDyK peptide 1a, then fixed with PFA followed by treated with 2a. After treatment with the ligands, the cells were stained with DAPI (blue). The scale bar indicates 20 µm. c, Comparison of the fluorescent intensities. Data are presented as the means ±S.E. [n = 10 (10,000 cells x 10), Student's t test, *p < 0.0001].