## 3 Discrete Cu(I/II) Complexes
### Cross-Linked Synthetic Organic Polymer Supports
The preparation of the material (Scheme 18) began with the dissolution of synthetic melanine granules into NaOH, followed by US-assisted re-precipitation of the polymer on lowering the pH. After this step, aimed to disentangle the macromolecular chains, the resulting water-insoluble melanine nanoparticles were subjected to chemical modification with NH\({}_{2}\)-PEG\({}_{000}\)-NH\({}_{2}\)[170]. This second stage afforded nanoparticles **37** (M-dots) that, probably thanks to their surface charge (\(-2.2\) mV zeta potential), resulted well dispersible in water but could be recovered by centrifugal separation through a 30 kDa cutoff filter. After loading with an aqueous solution of CuSO\({}_{4}\) and reducing with sodium ascorbate, the same technique was applied for the purification of the corresponding supported Cu(I) complex **38**. Characterization of the material by ICP-MS revealed that, on the average, each M-dot particle was loaded with 50 \(\pm\) 2 copper centers, whose +I oxidation state was confirmed by XPS. Specific tests on the M-dots/Cu(I) system evidenced just a modest metal leaching in phosphate PBS buffer at pH = 7.4 and no toxicity against two cellular lines. In addition, M-dots displayed the ability to quench the production of hydroxyl radicals in the Cu(II)-ascorbate reactive system, thus circumventing one of the main issues when this chemistry is applied for bio-conjugation purposes. The appealing features of **38** prompted its study in CuAAC reactions between low molecular weight reactants (Scheme 1) and for the click-derivatisation of biomolecules. In the former scenario, very good product yields (90%-95%) were obtained in 30 min at r.t., by stirring the azide and alkyne reactants in PBS buffer in the presence of 0.1 mol % of the copper catalyst. Similar, albeit slightly worse results (67%-90% yield) were obtained in the corresponding three-component process with halides in PBS-DMSO (Scheme 2a). Reusability of the catalytic system was studied in the reaction of phenylacetylene and a tetra(ethylene glycol)-derived azide. With this choice, **38** could be separated by centrifugal filtration from the solution containing the triazole product, without leaving behind measurable amounts of copper. The subsequent direct reuse of the recovered catalyst, i.e., without adding any reducing agent, afford then reasonably constant results in the course of seven reaction cycles (88% \(\rightarrow\) 82% yield). In a second set of applications, **38** was used for click-modifying biotin and AE105 or RGDyK polypeptide derivatives, provided with either alkyne or azide side chains. Moreover, the same protocol could be applied to the fluorescent tagging of a 3'-alkyne DNA strand with the cyanine 5.5-azide dye, in this case using a 10 kDa cut-off centrifugal filter for separating the polynucleotide product  
Figure 18: Preparation of the M-dots/Cu(I) nanoparticles and likely structure of M-dots (Note: The formula shown for the latter is the dihydroxylonde tautomer of the dihydroindolequinone one depicted in the original publication [171]). from **38**. In a final noteworthy example, the in situ imaging of the integrin \(\alpha_{\text{v}}\beta_{3}\) receptor in human glioblastoma U87 MG cells was realized. In this case, the procedure involved preliminary labelling the receptor sites with RGD-alkyne, followed by exposure of the whole cells to Cyanine 5.5-azide dye and **38**. As for the other biomolecules mentioned above, the possibility of using the melanine-based Cu(I) catalytic system, without the need of any added ascorbate, was undoubtedly a major advantage in order to prevent unwanted side reactions in the CuAAC process. As anticipated in the Introduction, the necessity of handling potentially explosive azides under safe conditions prompted several studies on continuous-flow CuAAC [31]. In this context, the combination of flow-chemistry with another enabling technology [172], viz.