biotin  (Alomone Labs)

Journal: EMBO Molecular Medicine

Article Title: Selective inhibition of mitochondrial Kv1.3 prevents and alleviates multiple sclerosis in vivo

doi: 10.1038/s44321-025-00307-2

Figure Lengend Snippet: ( A ) Upper panel: Chemical Structure of 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled PAPTP (PAPTP-NBD). Lower panel: Synthesis of PAPTP-NBD. Reagents and Conditions (i) TsCl, DMAP, pyridine, DCM, r.t., 18 h; (ii) NaN 3 , DMF, 90 °C, 3 h; (iii) Pd/C, H2, MeOH, r.t., 16 h; (iv) NBD-Cl, DIPEA, MeOH, r.t., 16 h; (v) TsCl, DMAP, pyridine, DCM, r.t., 5 h; (vi) NaI, acetone, 40 °C, 16 h; (vii) PAPTP-OH, K 2 CO 3 , DMF, r.t., 16 h. Synthesis of 2-(2-(2-hydroxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (1). To a solution of triethylene glycol (PEG3-OH, 77.28 g, 514.6 mmol, 8.0 equiv) in dichloromethane (DCM, 255 mL) at 0 °C were added 4-dimethylaminopyridine (DMAP, 15.7 g, 128.6 mmol, 2.0 equiv) and pyridine (10.18 g, 128.6 mmol, 2.0 equiv). After 10 min of stirring, p-toluenesulfonyl chloride (TsCl, 12.3 g, 64.3 mmol, 1.0 equiv), previously dissolved in DCM (165 mL), was added dropwise. The reaction mixture was stirred at room temperature for 18 h. The reaction was then quenched by dilution with 300 mL of 1.0 M HCl, the organic layer separated and the aqueous phase was extracted with DCM (2 × 250 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using a DCM/acetone mixture (8:2) to afford 1 as a pale yellow oil (13.2 g, 43.4 mmol, yield: 67%). 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 4.18–4.15 (m, 2H), 3.72–3.69 (m, 4H), 3.61 (s, 4H), 3.58–3.56 (m, 2H), 2.44 (s, 3H).13 C NMR (101 MHz, CDCl3) δ 145.01, 133.07, 129.98, 128.11, 72.59, 70.92, 70.43, 69.29, 68.85, 61.89, 21.78. ESI-MS (ion trap): m /z 305 [M + H]+. Synthesis of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (2). To a solution of compound 1 (13.2 g, 43.4 mmol, 1.0 equiv) in anhydrous N,N-dimethylformamide (DMF, 100 mL), sodium azide (NaN₃, 8.5 g, 130.2 mmol, 3.0 equiv) was added. The reaction mixture was stirred at 90 °C for 3 h until thin-layer chromatography (TLC) analysis (EtOAc/PE, 6:4) indicated complete consumption of the starting material. The reaction mixture was then diluted with ethyl acetate (EtOAc, 300 mL) and washed with brine/water 1:1 (5 × 100 mL). The organic layer was dried, and the solvent was removed under reduced pressure. The flask was left under high vacuum overnight to remove residual DMF, yielding compound 2 as a pale yellow oil (6.8 g, 38.7 mmol, yield: 89%). 1H NMR (400 MHz, CDCl3) δ 3.73–3.71 (m, 2H), 3.68–3.64 (m, 6H), 3.61–3.59 (m, 2H), 3.38 (t, J = 5.0 Hz, 2H), 2.45 (s, 1H).13 C NMR (101 MHz, CDCl3) δ 72.59, 70.74, 70.48, 70.13, 61.84, 50.74. ESI-MS (ion trap): m / z 176 [M + H]+. Synthesis of 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol (3). Palladium on carbon (Pd/C, 10% w/w, 0.70 g) was suspended in methanol (30 mL) in a round-bottom flask under nitrogen atmosphere. Compound 2 (6.8 g, 38.7 mmol, 1.0 equiv), previously dissolved in methanol (10 mL), was added to the suspension. The reaction atmosphere was then replaced with hydrogen, and the mixture was stirred at room temperature for 16 h, until thin-layer chromatography (TLC) analysis (DCM/acetone, 8:2) confirmed complete consumption of the starting material. Hydrogen was removed by nitrogen stream and the reaction mixture was filtered through a celite pad to remove the catalyst. The solvent was evaporated under reduced pressure and the crude product was purified by flash column chromatography on silica gel using a DCM/MeOH/NH3(aq) (87:12:1) as the eluent, affording compound 3 as a colorless oil (2.6 g, 17.4 mmol, yield: 45%). 1H NMR (400 MHz, CDCl3) δ 3.73–3.70 (m, 2H), 3.68–3.62 (m, 4H), 3.61–3.58 (m, 2H), 3.56–3.53 (m, 2H), 2.88 (t, J = 5.1 Hz, 2H), 2.55 (s, 3H).13 C NMR (101 MHz, CDCl3) δ 72.81, 72.79, 70.47, 70.24, 61.63, 41.53. ESI-MS (ion trap): m / z 150 [M + H]+. Synthesis of 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethan-1-ol (4). To a solution of NBD-Cl (1.0 g, 5.0 mmol, 1.0 equiv) in methanol (25 mL) at 0 °C were added N,N-diisopropylethylamine (DIPEA, 2.6 g, 20 mmol, 4.0 equiv) and compound 3 (0.82 g, 5.5 mmol, 1.1 equiv). The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was then diluted with ethyl acetate (150 mL) and washed with saturated NH4Cl solution (3 × 50 mL). The organic layers were combined, dried, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using an EtOAc/MeOH mixture (99:1) as the eluent, affording compound 4 as a brown powder (1.0 g, 3.2 mmol, yield: 64%). 1H NMR (400 MHz, MeOD) δ 8.51 (d, J = 8.9 Hz, 1H), 6.43 (d, J = 8.9 Hz, 1H), 3.84–3.80 (m, 2H), 3.76 (s, 2H, broad signal), 3.71–3.66 (m, 2H), 3.66–3.62 (m, 4H), 3.56–3.53 (m, 2H).13 C NMR (101 MHz, DMSO) δ 154.82, 153.90, 153.60, 147.35, 130.29, 109.01, 81.83, 79.36, 79.22, 77.44, 69.66, 52.88. ESI-MS (ion trap): m / z 313 [M + H]+. Synthesis of 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (5). To a solution of compound 4 (100 mg, 0.32 mmol, 1.0 equiv) in dichloromethane (DCM, 1.5 mL) at 0 °C were added pyridine (76 mg, 0.96 mmol, 3.0 equiv), 4-dimethylaminopyridine (DMAP, 78 mg, 0.64 mmol, 2.0 equiv), and p-toluenesulfonyl chloride (TsCl, 122 mg, 0.64 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature for 5 h. The mixture was then diluted with brine (50 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using DCM/MeOH (99:1) as the eluent, affording compound 5 as a brown solid (94 mg, 0.20 mmol, yield: 63%). 1H NMR (400 MHz, (CD3)2CO) δ 8.50 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 4.19–4.11 (m, 2H), 3.84–3.80 (m, 4H), 3.71–3.66 (m, 2H), 3.64 – 3.60 (m, 2H), 3.60–3.53 (m, 2H), 2.86 (s, 1H), 2.42 (s, 3H).13 C NMR (101 MHz, (CD3)2CO) δ 145.78, 145.45, 145.08, 137.77, 134.22, 130.77, 128.63, 123.50, 99.90, 71.24, 71.07, 70.61, 69.34, 44.68, 21.49. ESI-MS (ion trap): m/z 467 [M + H]+. Synthesis of N-(2-(2-(2-iodoethoxy)ethoxy)ethyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (6). To a solution of compound 5 (30 mg, 0.064 mmol, 1.0 equiv) in acetone (0.8 mL) was added sodium iodide (NaI, 39 mg, 0.257 mmol, 4.0 equiv). The reaction mixture was stirred at 40 °C for 16 h in a sealed vial. After completion, the mixture was diluted with ethyl acetate (EtOAc, 40 mL) and washed with brine (3 × 10 mL). The organic layer was dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether (1:1) as the eluent, affording compound 6 as a brown solid (14.5 mg, 0.034 mmol, yield: 54%). 1H NMR (400 MHz, (CD3)2CO) δ 8.54 (d, J = 8.8 Hz, 1H), 8.13 (s, 1H, broad signal), 6.55 (d, J = 8.8 Hz, 1H), 3.93–3.79 (m, 4H), 3.73–3.58 (m, 6H), 3.29 (t, J = 6.5 Hz, 2H).13 C NMR (101 MHz, (CD3)2CO) δ 145.81, 145.41, 145.02, 137.67, 123.43, 99.98, 72.35, 71.08, 70.67, 69.32, 44.59, 4.26. ESI-MS (ion trap): m/z 423 [M + H]+ Synthesis of (4-(2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethoxy)phenyl) (3-(4-(4-((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)butoxy)phenyl)propyl)diphenylphosphonium (PAPTP-NBD) A solution of compound 6 (14.5 mg, 0.034 mmol, 1.1 equiv) in DMF (0.5 mL) was cooled to 0 °C, and PAPTP-OH (25 mg, 0.031 mmol, 1.0 equiv, synthesized as previously reported, 10.3390/ph14020129) and potassium carbonate (K₂CO₃, 4 mg, 0.031 mmol, 1.0 equiv) were added. The reaction mixture was stirred at room temperature for 16 h. After completion, the reaction was diluted with EtOAc (30 mL) and washed with 0.5 M HCl (2 × 10 mL) followed by brine (1 × 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by preparative HPLC and lyophilized to afford PAPTP-NBD as an orange solid (9.6 mg, 0.009 mmol, yield: 29%, purity UPLC > 95%). 1H NMR (400 MHz, (CD3)2CO) δ 8.42 (s, 1H, broad signal), 8.21 (dd, J = 9.8, 0.7 Hz, 1H), 7.93–7.68 (m, 13H), 7.26 (dd, J = 2.4, 1.0 Hz, 1H), 7.22 (dd, J = 9.0, 2.6 Hz, 2H), 7.16–7.08 (m, 3H), 6.82 (d, J = 8.6 Hz, 2H), 6.55 (s, 1H, broad signal), 6.19 (d, J = 9.8 Hz, 1H), 4.67 (t, J = 5.8 Hz, 2H), 4.24–4.21 (m, 2H), 4.09 (t, J = 5.8 Hz, 2H), 3.90–3.82 (m, 6H), 3.68 (s, 4H), 3.60–3.48 (m, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.12–1.98 (m, 6H). 13 C NMR (101 MHz, (CD3)2CO) δ 165.18, 159.24, 158.66, 153.86, 150.18, 146.37, 140.10, 136.81 (d, J = 11.5 Hz), 135.87 (d, J = 3.0 Hz), 134.61 (d, J = 10.0 Hz), 133.12, 131.25 (d, J = 12.6 Hz), 130.52, 120.83, 119.97, 117.48, 117.35, 115.48, 113.33, 109.39, 108.46, 107.37, 106.47, 94.02, 73.61, 71.46, 71.36, 70.02, 69.73, 69.23, 68.23, 49.07, 36.03 (d, J = 16.9 Hz), 27.59, 26.72, 25.49 (d, J = 3.7 Hz), 22.25 (d, J = 52.6 Hz). ESI-MS (ion trap): m / z 363 [M]+. ( B ) Confocal microscopy images showing the fluorescent signal in untreated cells and in those treated with fluorescent PAPTP (PAPTP-fluor) in mitochondria of CD4 + CD25 - Tconv cells isolated from healthy mice before and after treatment with 100 nM PAPTP-Fluor for 30 min. The scale bar is 10 μm. Control experiment for Fig. . The same cells shown in this representative image are also shown in Fig. at higher magnification. ( C ) Left panels: Upper panel: Downregulation of Kv1.3 in cells treated with CRIPSR/Cas9 and Kv1.3 staining in sorted cells. Yellow: CRISPR/Cas transfected, unstained. Blue: CRISPR/Cas transfected, cells positively sorted for Kv1.3. This is the fraction, which was positively sorted and then stained with FITC-anti-Kv1.3 antibodies.For sorting, cells were collected, washed with H/S, incubated with Fc receptor blocking reagent (BioLegend, #101302; 1:50 dilution) for 15 min at 4 °C, washed, and labeled with biotin-conjugated anti-Kv1.3 antibody (Alomone Labs, #APC-101B) for 30 min at 4 °C. Following a second wash, cells were incubated with streptavidin-conjugated microbeads (Miltenyi Biotec, #130-048-101) for 30 min at 4 °C. Kv1.3-positive and -negative populations were isolated using LS columns (Miltenyi Biotec, 130-042-401). For the flow cytometry, Fc-receptors were blocked with True stain (1:50 dilution, cells were collected, washed with H/S, incubated with Fc receptor blocking reagent (Clone S17011E, BioLegend, #156604) and then an aliquot of the samples was stained with a FITC-coupled anti-rabbit IgG (1:500, Jackson Immunoresearch 711-096-152) to detect the anti-Kv1.3, which was already bound to the cells. Red: CRISPR/Cas transfected, cells negatively sorted for Kv1.3. This is the fraction, which was negatively sorted and then stained exactly as the blue fraction. The staining was done on aliquots just before retransfection of the mito-Kv1.3 construct to confirm downregulation. Lower panel: Same as above, but with aliquots that were fixed for 10 min at room temperature in 1% buffered PFA, then washed and permeabilized for 8 min with 0.1% Triton X-100 at room temperature, washed again and then stained. This confirms downregulation of extra- and intracellular Kv1.3. Right upper panel: Flow cytometry of sorted Kv1.3-negative cells that were re-transfected. Blue: Control-transfected (empty vector). Aliquots were fixed for 10 min at room temperature in 1% buffered PFA, then washed and permeabilized for 8 min with 0.1% Triton X-100 at room temperature, washed again, Fc-receptors were blocked with True stain (1:50, Biolegend, #156604) and then stained with FITC-coupled anti-Kv1.3 (Alamone, #APC-101-F). Red: EYFP-Mito-Kv1.3 transfected cells, as above. Right lower panel: Representative dot plot showing the gating strategy for the identification of EYFP-mitoKv1.3 + Annexin + and EYFP-mitoKv1.3 - Annexin + cells after PAPTP treatment. Kv1.3 - cells were transfected with EYFP-mitoKv1.3 construct and subsequently treated with PAPTP for 48 h. Cells were then analyzed for apoptosis using flow cytometry. ( D ) Representative images of longitudinal spinal-cord sections stained with luxol fast blue from mice receiving MOG 35–55 activated untreated or 1 μM PAPTP-treated lymphocytes. Scale bar, 100 μm. Demyelinated are indicated with white arrows. Right: Average ± SEM of myelin area per field ( n = 3 for mice receiving untreated lymphocytes, n = 5 for mice receiving 1 μM PAPTP-treated lymphocytes). p -value of Student’s t test. ( E ) Representative images of longitudinal spinal-cord sections stained with Bielschowsky staining from mice receiving MOG 35–55 activated untreated or 1 μM PAPTP-treated lymphocytes. Scale bar, 200 μm. Right upper panel: enlarged image from the lower EAE AT untreated sample shown on the left. Right lower panel: enlarged image from the upper EAE AT + PAPTP sample shown on the left. Please note damaged axon fibers in the EAE. ( F ) Quantification of axon damage from longitudinal sections of Blieschowsky-stained spinal cords from EAE AT and EAE AT + PAPTP animals. Quantification was performed following the method used for Fig. (left panel) and according to (Theotokis et al, ). ( G ) Representative images of a brain section of a wild-type mouse injected with MOG 35–55 activated untreated lymphocytes, stained with the indicated antibodies. The sections are from the same experiment shown in Fig. . ( H ) Average ± SEM of the number of GFAP + cells per field in brain slices of healthy animals (Ctrl) and mice receiving untreated or 1 μM PAPTP-treated lymphocytes ( n = 3 for each group). On the right, representative immunohistochemical images of GFAP + in brain slices from mice of the indicated groups. The images were taken from the same region for each animal. The scale bar corresponds to 100 μm. p -value from one-way ANOVA. ( I ) Additional examples of Klüver-Barrera dual staining performed as in Fig. . Red arrows indicate infiltrated/demyelinated zones. Please note also vacuolation, as e.g., in (Morales et al, ) in the enlarged image.

Article Snippet: This is the fraction, which was positively sorted and then stained with FITC-anti-Kv1.3 antibodies.For sorting, cells were collected, washed with H/S, incubated with Fc receptor blocking reagent (BioLegend, #101302; 1:50 dilution) for 15 min at 4 °C, washed, and labeled with biotin-conjugated anti-Kv1.3 antibody (Alomone Labs, #APC-101B) for 30 min at 4 °C.

Techniques: Labeling, Purification, Column Chromatography, Thin Layer Chromatography, Solvent, Suspension, Synthesized, Confocal Microscopy, Isolation, Control, Staining, CRISPR, Transfection, Incubation, Blocking Assay, Flow Cytometry, Construct, Plasmid Preparation, Injection, Immunohistochemical staining

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Summary of anti-Kv1.3 antibody identification.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Enzyme-linked Immunosorbent Assay, Functional Assay, Magnetic Beads

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Summary of anti-Kv1.3 antibody functional activity.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Functional Assay, Activity Assay, Blocking Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Kv1.3 ortholog sequence alignment analysis.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Sequencing