Recyclable Cu(i)/melanin dots for cycloaddition, bioconjugation and cell labelling

We successfully transferred melanin into a novel catalytic platform. Ligand-free, water-soluble, recyclable and biocompatible Cu(i)-loaded melanin dots [Cu(i)/M-dots] was easily prepared and demonstrate excellent properties for classic CuAAC, bioconjugation and cell labelling.

Supplementary Figures S1-S11 S3-13 2 Table S1-S3 S14-16 3 General method S17     Figure S6. (a) The pathway of ascorbate-mediated, Cu(I)-induced hydroxyl radical production was scavenged by M-dots; (b) A fluorogenic ROS reporter 3-CCA (3-coumarin carboxylic acid, 100 µM) was used for the ROS detection in vitro. The HPLC spectra monitored this radical reaction after 12 h. The condition of CuSO 4 (100 µM) and ascorbate (5 mM) could produce ROS product 7-OH CCA (black line), while with M-dots (100µM), no ROS product generates in the presence of CuSO 4 /ascorbate (blue line). As a control, no ROS signal generate in the presence of ascorbate (red line).    Table S1. The data of hydrodynamic sizes and zeta potentials of M-dots and Cu(I)/M-dots in aqueous solution. The hydrodynamic sizes of M-dots and Cu(I)/M-dots were measured by dynamic light scattering (DLS) instrument using a 90 Plus particle size analyzer (Malvern, Zetasizer Nano ZS90). Zeta potential was measured using a zeta potential analyzer (Malvern, Zetasizer Nano ZS90).

Preparation of M-dots
Tyrosine-derived synthetic melanin (10 mg) was first dissolved in 5 mL of 0.

Preparation of Cu(I)/M-dots catalyst
The M-dots solubilized in 1 mL of degassed water (1 mg in 1 mL H 2 O) and 20 µL of fresh

Inductively coupled plasma-mass spectrometry (ICP-MS) analysis
Thermo Scientific Xseries 2 Quadrupole was used to measure the concentration of Cu(I) in Cu(I)/M-dots. Standards (0.2 ppm, 0.5 ppm, 1 ppm, 4 ppm and 10 ppm of copper) were prepared in dilute nitric acid (2% w/w) solution and 2% nitric acid solution was used as the blank.
For freshly prepared Cu(I)/M-dots catalyst: 100 µL of detected samples were firstly heated to evaporate the water solvent and then digested with 0.5 mL of trace metal grade concentrated nitric acid (HNO 3 , 70% w/w) under heating until completely dissolved, then the residue was dissolved in 7 mL of dilute nitric acid (2% w/w) for final ICP-MS analysis.
For recycled Cu(I)/M-dots catalyst: after each click reaction, Cu(I)/M-dots were separated immediately from the reaction mixture (30 kDa centrifugal-filter, washed with water for three times) and then stored in water. Recycled samples (100 µL) were prepared firstly and heated to evaporate the water solvent and then digested with 0.5 mL of trace metal grade concentrated nitric acid (HNO 3 , 70% w/w) under heating until completely dissolved. Then the residue was dissolved in 7 mL of dilute nitric acid (2% w/w) for final ICP-MS analysis.

The stability of Cu(I)/M-dots catalyst
The  DMEM. Addition of 10 μL of MTT (0.5 mg/mL) solution to each well and incubation for 4 h at 37 °C was followed to produce formazan crystals. Then, the supernatant was removed and the products were lysed with 100 μL of DMSO. The absorbance value was recorded at 490 nm using a microplate reader. The absorbance of the untreated cells was used as a control and its absorbance was as the reference value for calculating 100% cellular viability.

DNA bioconjugation
Oligonucleotide synthesized by Protein and Nucleic Acid (PAN) facility at Stanford. The 3'position modified oligonucleotide (S1, 5 nmol, MW=6317.2) in 20 μL of water was reacted with an excess of NHS ester V1 (50 eq) in 30 μL of water at room temperature for 12 h.

DNA denaturing PAGE Gel for Electrophoresis
The product S3 was analyzed on a 16% PAGE gel (PAGE gel was prepared by following the standard protocol

Chemical synthesis and characterization
General procedure for the two-componets [3+2] cyclo-addition reaction.

The free radical quenching ability of M-dots in CuAAC conditions.
The ascorbate-driven, metal-induced hydroxyl radical production was monitored by HPLC using the hydroxyl radical scavenging compound 3-CCA (sigma-aldrich). The hydroxyl radical reaction was carried out with or without M-dots (100 µM) in the presence of 100 µM 3-CCA and ascorbate (5 mM) after 12h. General conditions: To a solution of 1f (5.6 mg, 0.1 mmol), Compound 4a (20.4 mg, 0.12 mmol, 1.2 equiv) and sodium azide (7.8 mg, 0.12 mmol) in PBS at room temperature was added 2 nmol Cu(I)/M-dots (containing 0.1% mol Cu + ). The reaction mixture was stirred at this temperature for 1.5 h. The catalyst was separated by a centrifugal-filter (MWCO = 30 kDa), and washed with water for three times. Then the recycle catalyst was reused directly in the next reaction.