Research Article Chemistry—A European Journal doi.org/10.1002/chem.202301945 www.chemeurj.org Hot Paper (Bio)Functionalisation of Metal–Organic Polyhedra by Using Click Chemistry Laura Hernández-López,[a, b] Cornelia von Baeckmann,[a, b] Jordi Martínez-Esaín,[a] Alba Cortés-Martínez,[a, b] Jordi Faraudo,[c] Caterina Caules,[a, b] Teodor Parella,[d] Daniel Maspoch,*[a, b, e] and Arnau Carné-Sánchez*[a, b] The surface chemistry of Metal–Organic Polyhedra (MOPs) is Rh(II)-based MOP with 24 available surface alkyne groups was crucial to their physicochemical properties because it governs synthesised. Each of the 24 alkyne groups on the surface of the how they interact with external substances such as solvents, “clickable” Rh-MOP can react with azide-containing molecules synthetic organic molecules, metal ions, and even biomolecules. at room temperature, without compromising the integrity of Consequently, the advancement of synthetic methods that the MOP. The wide substrate catalogue and orthogonal nature facilitate the incorporation of diverse functional groups onto of CuAAC click chemistry was exploited to densely functionalise MOP surfaces will significantly broaden the range of properties MOPs with diverse functional groups, including polymers, and potential applications for MOPs. This study describes the carboxylic and phosphonic acids, and even biotin moieties, use of copper(I)-catalysed, azide–alkyne cycloaddition (CuAAC) which retained their recognition capabilities once anchored click reactions to post-synthetically modify the surface of onto the surface of the MOP. alkyne-functionalised cuboctahedral MOPs. To this end, a novel Introduction This is especially true for the archetypical cuboctahedral [M2(m- bdc)2]12 MOP (where m-bdc is isophthalate), which resembles a As a subclass of molecular cages assembled from metal-ions spherical ~2.5 nm-in-diameter molecular nanoparticle.[1] As and organic linkers, Metal–Organic Polyhedra (MOPs) exhibit such, as in inorganic nanoparticles, most of its physicochemical unique porosity capabilities: for example, they can host properties (e. g. solubility, recognition, and assembly) rely on its molecules in solution and are permanently porous in the solid surface chemistry.[2–11] For instance, on its outer surface this state. Importantly, most of these materials fall within the cuboctahedral MOP contains up to 84 potential sites that can nanoscale regime, having outer diameters of ~2 nm to 5 nm. be used to introduce new functionalities: 72 that stem from positions 4, 5, and 6 of the aromatic organic linkers, and 12 that [a] L. Hernández-López, Dr. C. von Baeckmann, Dr. J. Martínez-Esaín, A. Cortés- belong to the axial metal sites. Martínez, C. Caules, Prof. Dr. D. Maspoch, Dr. A. Carné-Sánchez Among the existing functionalisation strategies, copper(I)- Catalan Institute of Nanoscience and Nanotechnology (ICN2) catalysed azide–alkyne cycloaddition (CuAAC), commonly CSIC and The Barcelona Institute of Science and Technology known as click chemistry, is probably the most popular, Campus UAB, Bellaterra, 08193 Barcelona (Spain) E-mail: arnau.carne@icn2.cat powerful, and versatile chemical tool to post-synthetically daniel.maspoch@icn2.cat modify molecules and materials.[12–14] This is mainly due to its [b] L. Hernández-López, Dr. C. von Baeckmann, A. Cortés-Martínez, C. Caules, quantitative and orthogonal reactivity, which makes it compat- Prof. Dr. D. Maspoch, Dr. A. Carné-Sánchez ible with myriad functional groups and even biomolecules.[15,16] Departament de Química, Facultat de Ciencies However, the straightforward use of CuAAC for the post- Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain) synthetic modification of MOPs presents several challenges due [c] Dr. J. Faraudo Institut de Ciència de Materials de Barcelona (ICMAB-CSIC) to the instability of coordination bonds to common click 08193 Bellaterra (Spain) chemistry reagents (e. g., reducing agents and metal salts).[17] [d] Dr. T. Parella Therefore, researchers have developed alternative reaction Servei de Ressonància Magnètica Nuclear conditions for the CuAAC reaction to avoid the use of reducing Universitat Autònoma de Barcelona agents. These conditions involve the use of Cu(I) complexes as Campus UAB, Bellaterra, 08193 Barcelona (Spain) catalysts,[6] or of the strain-promoted version of the azide– [e] Prof. Dr. D. Maspoch ICREA alkyne cycloaddition, the latter of which has been employed for Pg. Lluís Companys 23, 08010 Barcelona (Spain) most partial modifications of parent cages.[18,19] Only very Supporting information for this article is available on the WWW under recently did Bloch and co-workers show that robust rectangular https://doi.org/10.1002/chem.202301945 prismatic MOPs assembled from Co(II) calixarene-capped clus- © 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH ters and 5-azido isophthalic acid (5-N3-bdc) or 5-propargyl GmbH. This is an open access article under the terms of the Creative isophthalic acid can withstand the reaction conditions of the Commons Attribution Non-Commercial License, which permits use, dis- tribution and reproduction in any medium, provided the original work is classical CuAAC reaction.[20] Indeed, they reported the quantita- properly cited and is not used for commercial purposes. Chem. Eur. J. 2023, e202301945 (1 of 8) © 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH 15213765, 0, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202301945 by Readcube (Labtiva Inc.), Wiley Online Library on [25/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article Chemistry—A European Journal doi.org/10.1002/chem.202301945 tive conversion of 4 pendant alkyne or azide groups of the MOP required to make Rh-MOPs, including the use of high temper- through this reaction run at high temperature. atures and basic conditions. Thus, after reacting dimethyl 5- Herein we have adapted and optimised CuAAC to enable iodoisophtalate with (triisopropylsilyl)acetylene and subse- covalent functionalisation of up to 24 positions of cuboctahe- quently deprotecting the carboxylic groups, the protected dral Rh-MOPs[21] with a diverse repertoire of molecules. To this version of ethynyl-bdc, 5-((triisopropylsilyl)ethynyl))isophthalic end, we initially introduced an alkynyl group onto position 5 of acid (TIPS-bdc), was obtained. Next, TIPS-bdc was reacted with the aromatic organic linker of a 24-functionalized cuboctahedral Rh2Ac4 in DMA at 100 °C in the presence of Na2CO3 for 48 h to Rh-MOP through a protection/deprotection method,[22] forming yield a MOP with formula [Rh24(TIPS-bdc)24] (yield: 80 %) (Fig- a “clickable” Rh-MOP with formula [Rh24(ethynyl-bdc)24] (here- ure 1a). The formation of the alkyne-protected Rh-MOP was after named BCN-23; where ethynyl-bdc is 5-ethynyl-1,3- confirmed by matrix-assisted laser desorption/ionisation-time- benzenedicarboxylate). Afterwards, we demonstrated that each of-flight (MALDI-TOF) spectroscopy, which showed a signal at of the 24 alkyne groups located on the outer surface of the 11024.5 m/z, which closely agrees with the targeted cuboctahe- cuboctahedron could be reacted at room temperature with dral Rh(II)-MOP ([Rh24(TIPS-bdc)24 + H + ] + · 2DMA · 2MeOH, ex- azide-containing molecules without compromising the integrity pected m/z = 11024.9) (Figure S4). The 1H NMR spectrum of the of the MOP. Thus, we used click chemistry to quantitively and alkyne-protected MOP in CDCl3-d1 further confirmed the syn- densely functionalise the surface of BCN-23 with a wide range thesis of a discrete metal–organic assembly with 24 triisopro- of functionalities that could not otherwise be easily introduced pylsilyl protecting groups on its surface (δ = 1.05 ppm; Fig- (e. g. by direct synthesis or current post-synthetic approaches). ure S1). Furthermore, diffusion-ordered spectroscopy (DOSY) These functionalities include polymers, free carboxylic acid NMR analysis showed the same diffusion coefficient of groups, free phosphonic acid groups, and bioactive biotin 3.7 · 10 10 m2 s 1 for the aromatic and protecting group protons, molecules. Furthermore, we proved that addition of these which further corroborated that both moieties belong to the functionalities modulates the properties (e. g. solubility) of BCN- same metal–organic structure (Figure S2). The integrity of the 23, and that the biorecognition capabilities of biotin moieties Rh Rh paddlewheel was confirmed through UV-Vis spectro- grafted onto the surface of BCN-23 to avidin are preserved. scopy in chloroform, which showed the characteristic band centred at 630 nm, which corresponds to Band I of the Rh Rh paddlewheel (Figure S4). Results and Discussion The alkyne-available Rh-MOP (BCN-23) was synthesised by treating a THF solution of the alkyne-protected MOP with Synthesis of the “clickable” alkyne-terminated Rh-MOP 36 mol. equiv. of tetrabutylammonium fluoride (TBAF) solution (1.0 M in THF) (Figure 1b). Upon addition of the TBAF solution, a Traditionally, cuboctahedral Rh-MOPs are synthesised through a blue precipitate was immediately formed. This solid was solvothermal reaction between Rh2Ac4 and the corresponding collected by centrifugation, washed with THF and finally, linker. However, BCN-23 cannot be synthesised this way dissolved in DMSO for further analysis (yield: > 95 %). The 1 because the linker ethynyl-bdc is not stable at high temper- H NMR spectrum of BCN-23 in DMSO-d6 confirmed the atures or under basic conditions. Thus, we developed a quantitative fading of the peaks ascribed to the TIPS protecting protection-deprotection strategy that entailed the synthesis of group, together with the appearance of a new signal at the alkyne-protected Rh-MOP and its subsequent deprotection 4.3 ppm (Figure S5), which corresponds to the alkyne proton. to finally yield the targeted clickable MOP (Figure 1). Triisopro- The aromatic and alkyne protons showed the same diffusion pylsilyl was selected as a protecting group because of its coefficient of 6.5 · 10 11 m2 s 1 in the DOSY 1H NMR data (Fig- compatibility with the linker and the synthetic conditions ure S6). The hydrodynamic radius of BCN-23 was calculated to Figure 1. (a) Schematic of the cuboctahedral Rh-MOP, highlighting the positions at which the CuAAC click reaction is performed. (b) Schematic showing the synthesis of the “clickable” BCN-23 MOP, and its subsequent post-synthetic modification with azidobenzene through a CuAAC (top), with the corresponding 1 H-NMR spectra and DOSY NMR representation for each synthetic step (bottom). NMR data for the alkyne-protected Rh-MOP was obtained in CDCl3-d1 whereas the DMSO-d6 was used for BCN-23 and Bz-BCN-23. Chem. Eur. J. 2023, e202301945 (2 of 8) © 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH 15213765, 0, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202301945 by Readcube (Labtiva Inc.), Wiley Online Library on [25/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article Chemistry—A European Journal doi.org/10.1002/chem.202301945 be approximately 1.7 nm, which agrees with the value obtained Bz-BCN-23 revealed complete disappearance of the stretching from the computer simulation of this structure (Figure S7).[18] band corresponding to the alkyne group at 3284 cm 1 (Fig- MALDI-TOF analysis of BCN-23 was characterised by a single ure S20). DOSY NMR data of Bz-BCN-23 showed the same broad peak centred at 7061.6 m/z, which closely agrees with diffusion coefficient (5.3 · 10 11 m2 s 1) for all aromatic protons the expected mass of 7064.1 m/z for the targeted formula (Figure 1b and S12). The calculated hydrodynamic radius was [Rh24(ethynyl-bdc)24 + H + ] + · DMSO (Figure S9). Additionally, the found to be 2.1 nm, in agreement with the equilibrium integrity of the Rh Rh paddlewheel after the deprotection configuration of the simulated structure (Figure S21). reaction was further confirmed by UV-Vis spectroscopy in DMF, Further evidence of the quantitative conversion of the which showed the characteristic band centred at 589 nm that alkyne groups was provided by the 1H NMR analysis of the acid- corresponds to Band I of the Rh Rh paddlewheel (Figure S9). digested Bz-BCN-23. The spectrum of the digested Bz-BCN-23 Altogether, these results corroborate the successful synthesis of showed only the signals corresponding to 5-(1-phenyl-1H-1,2,3- BCN-23 as a cuboctahedral Rh-MOP functionalised with 24 triazol-4-yl)-bdc, which is the linker expected to be created available alkyne groups on its outer surface. upon coupling of the initial 5-ethynyl-bdc linker of BCN-23 to azidobenzene via CuAAC (Figure S13). Finally, as expected for this type of click reaction, only the 1,4-substituted triazole ring CuAAC click reactions on BCN-23: functionalisation with small was formed, as confirmed by the ROESY NMR data of the and polymeric azide molecules as model substrates digested Bz-BCN-23 (Figure S18). From the above model reaction, we corroborated that all 24 We initially assessed the reactivity and functionalisation of BCN- alkyne groups on the outer surface of BCN-23 can be reacted 23 through CuAAC click chemistry using azidobenzene as our through CuAAC click chemistry with an azide group without model azide compound. To this end, 36 mol. equiv. of compromising the integrity of the Rh-MOP. To further confirm azidobenzene per MOP unit (i. e. 1.5 mol. equiv. of azidoben- this quantitative functionalisation, we next targeted the zene per alkyne group) were added to a DMSO solution of BCN- functionalisation of BCN-23 with a polymer, to form a polymer 23 followed by the stepwise addition of CuSO4 (1.5 mol. equiv. grafted Rh-MOP.[6,7,25,26] To this end, BCN-23 was reacted with per alkynyl group) and sodium ascorbate (4.5 mol. equiv. per 36 mol. equiv. (i. e. 1.5 mol. equiv. per alkynyl group) of an azide alkynyl group, 3 mol. equiv. per Cu(II)). The reaction mixture terminated polyethylene glycol polymer (mPEG42-N3) with 42 was left to react overnight at room temperature. The reaction repeating units (average molecular weight = 2000 � 300 g/mol) proceeded under homogenous conditions: no precipitate was overnight at room temperature. As for the previous click observed throughout the reaction. The product of the reaction reaction, this reaction also proceeded homogenously in DMSO. (hereafter named Bz-BCN-23) was precipitated out by adding The resulting PEGylated Rh-MOP (hereafter named mPEG42- ethyl acetate as counter solvent, and further purified through BCN-23) was isolated by precipitation from the reaction mixture successive washing with 1 : 2 mixtures of DMF/HCl (0.3 M) and with diethyl ether, collected by centrifugation, and then purified of DMF/water (yield: 95 %). (yield: > 95 %). The UV-Vis spectrum of Bz-BCN-23 in DMF showed a λmax = The 1H NMR spectrum of the PEGylated Rh-MOP in MeOD-d4 590 nm, which confirmed the integrity of the Rh(II) paddlewheel showed a broad signal in the aromatic region at ca. δ = unit through the catalytic reaction (Figure S19). This result 8.54 ppm, which corresponds to the bdc and to the triazole corroborated that Rh(II) is not reduced upon addition of moieties (Figure S22). Additionally, it showed peaks at δ = ascorbic acid or exchanged in the presence of Cu(II). Further- 3.64 ppm, corresponding to the aliphatic ethylene glycol units. more, UV-Vis spectrum of Bz-BCN-23 also confirmed that the Both sets of signals belong to the same molecule, as confirmed newly formed triazole ring does not coordinate to the exposed by DOSY experiments on mPEG42-BCN-23 that revealed a Rh(II) axial sites due to the strong steric hindrance around the diffusion coefficient of 8.1 · 10 11 m2 s 1 (Figure S23). The calcu- coordinating N atoms of the triazole ring.[24] MALDI-TOF analysis lated hydrodynamic size of mPEG42-BCN-23 was 4.9 nm. These revealed a single broad peak centred at 10045 m/z, which results reflected a considerable increase in the hydrodynamic closely agrees with the expected molecular mass for [Rh24(5-(1- radius relative to the previously found values, supporting the phenyl-1H-1,2,3-triazol-4-yl)-bdc)24 + H + ] + · DMF · 3H2O (ex- notion that the PEG chains were indeed attached to the surface pected = 10045 g/mol) (Figure S19). 1H NMR analysis of a of BCN-23. DMSO-d6 solution of Bz-BCN-23 showed a downfield shift of the Next, to elucidate the degree of PEGylation, we analysed aromatic protons of the BDC moiety, and the appearance of a the digested mPEG42-BCN-23 by 1H NMR. The spectrum of the new broad aromatic signal at δ = 7.55 ppm, which corresponds digested mPEG42-BCN-23 in DMSO-d6 revealed that all 24 5- to the incorporated phenyl moiety on the surface of the MOP ethynyl-bdc linkers of the parent BCN-23 had been clicked with (Figure S11). Furthermore, the formation of the triazole ring the polymer chains (Figure S24). The quantitative PEGylation of linking the MOP unit to the phenyl ring was first evidenced by BCN-23 was further confirmed by MALDI-TOF analysis of the appearance of a signal at δ = 9.35 ppm, which corresponds mPEG42-BCN-23, which showed a broad peak at ca. 53470.5 g/ to the olefinic proton of the triazole ring, and by the mol, which corresponds to a PEGylated Rh-MOP with formula quantitative fading of the signals of the alkyne protons groups ([Rh24(5-(mPEG42-1H-1,2,3-triazol-4-yl)-bdc)24 + H + ] + · 20MeOH (δ = 4.30 ppm, Figure S10). Supporting this later observation, (expected m/z = 53468.4) (Figure 2 and Figure S28). Also, FTIR the Fourier transform infrared spectroscopy (FTIR) spectrum of analysis of mPEG42-BCN-23 revealed the complete disappear- Chem. Eur. J. 2023, e202301945 (3 of 8) © 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH 15213765, 0, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202301945 by Readcube (Labtiva Inc.), Wiley Online Library on [25/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article Chemistry—A European Journal doi.org/10.1002/chem.202301945 methanol, chloroform, DMF and DMSO and water in a wide range of pH values (Figure S27). Functionalisation of BCN-23 with carboxylic and phosphonic acid groups Having demonstrated the regioselective and quantitative nature of click chemistry on the alkyne surface groups of BCN-23, we next sought to take advantage of the orthogonal reactivity of this chemistry to decorate the surface of BCN-23 with coordi- nating groups such as carboxylic or phosphonic acid groups. The formation of MOPs with surface-available carboxylic or phosphonic acid groups is very challenging, as these groups tend to react with metal ions, thus precluding formation of the desired MOP. Indeed, there is only one reported example of a covalent COOH-functionalised MOP, which was prepared through a post-synthetic protection/deprotection strategy, and there are no reports of any phosphonic acid-functionalised MOPs.[22] To prove that the CuAAC reaction could be used to post-synthetically functionalise MOPs with available carboxylic or phosphonic acid groups, we ran experiments with four azide Figure 2. Schematic showing the structure of mPEG42-BCN-23 (top) and the substrates: three terminated with carboxylic acid groups corresponding MALDI-TOF spectrum (bottom). The molecular weight (azidobenzoic acid, 6-azidohexanoic acid, and COOH-PEG3-N3) corresponding to the formula ([Rh24(5-(mPEG42-1H-1,2,3-triazol-4-yl)-bdc)24 + H + ] + · 20MeOH has been highlighted: expected m/z = 53468.4; found and one terminated with a phosphonic acid group (H2PO3-PEG3- m/z = 53470.5. N3). The reaction of BCN-23 with azidobenzoic acid (36 mol. equiv.), 6-azidohexanoic acid (36 mol. equiv.) or COOH-PEG3-N3 (36 mol. equiv.) in the presence of CuSO4 (1.5 mol. equiv.), ance of the stretching band at 3284 cm 1, further confirming sodium ascorbate (2.25 mol. equiv.) and HCl (0.0031 mol. equiv.) that all the alkyne groups on the surface of the MOP had been in DMSO yielded three new COOH-functionalised MOPs: reacted (Figure S29). Importantly, due to the quantitative COOH Bz-BCN-23 (yield: > 95 %), COOH C5-BCN-23 (yield: PEGylation, the physicochemical properties of the product were > 95 %), and COOH-PEG3-BCN-23 (yield: > 95 %) (Figure 3). It is drastically different from those of the initial BCN-23. For important to note here that the addition of HCl to the catalytic example, whereas the initial BCN-23 is only soluble in DMSO reaction was required to prevent coordination of the free and DMF, the densely PEGylated mPEG42-BCN-23 exhibits a carboxylic acid groups to the copper catalyst. In all cases, the broad solubility profile, covering most organic solvents such as click reaction was quantitative, as evidenced by the 1H NMR spectra of the corresponding acid-digested samples (Figur- Figure 3. Schematic composition (top) and simulated structure (bottom) of COOH Bz-BCN-23 (a), COOH-C5-BCN-23 (b), COOH-PEG3-BCN-23 (c) and H2PO3- PEG3-BCN-23 (d). The structure shown corresponds to snapshots of the equilibrium configuration obtained in the computer simulations (snapshots made with VMD).[23] Color code: Rhodium (green); carbon (black); hydrogen (white); oxygen (red); nitrogen (blue); phosphor (gold). Rh atoms are shown in their Van der Waals size while linker is represented with Licorice drawing method. For simplicity, the MOP core is displayed as translucent. Chem. Eur. J. 2023, e202301945 (4 of 8) © 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH 15213765, 0, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202301945 by Readcube (Labtiva Inc.), Wiley Online Library on [25/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article Chemistry—A European Journal doi.org/10.1002/chem.202301945 es S32, S39, and S46, respectively). Moreover, DOSY NMR same diffusion coefficient of 7.2 · 10 11 m2 s 1, as determined by analysis of the three COOH-functionalised MOPs in DMSO-d6 DOSY NMR (Figures S51 and S52, respectively). The calculated corroborated the successful coupling of the COOH moiety onto hydrodynamic radius was 2.6 nm, in agreement with the the surface of the BCN-23 unit (Figures S31, S38, and S45 simulated structure. As in the case of COOH-PEG3-BCN-23 the respectively). The estimated hydrodynamic radii values derived PEG chains are wrapped around the MOP core (Figure 3d). from the DOSY NMR representation were found to be 2.2 nm The MALDI-TOF spectrum of COOH-PEG3-BCN-23 revealed a (COOH Bz-BCN-23), 1.9 nm (COOH-C5-BCN-23), and 2.5 nm broad peak with a centred m/z value of 13855.5 g/mol, in good (COOH-PEG3-BCN-23). These values are in agreement with the agreement with a Rh-MOP of formula [Rh24(5-phosphono-PEG3- simulated structures (Figure 3a, 3b and 3c). Furthermore, the ( 1H-1,2,3-triazol-4-yl)-bdc)24 + H + ] + · 4H2O (expected m/z for simulations also revealed the orientation of the three types of this formula = 13855.0) (Figure S54). The presence of the free carboxylic acids on the surface of the MOP core. Whereas the phosphonic acid group on H2PO3-PEG3-BCN-23 was further carboxylic acids of COOH Bz-BCN-23 and COOH C5-BCN-23 are confirmed by FTIR, which exhibited the characteristic band stretched out from the MOP core, the PEG chains of the COOH- centred at 1044–1046 cm 1 (Figure S57). Altogether, these PEG3-BCN-23 are wrapped around the MOP core, in what is results demonstrate that BCN-23 is stable under homogenous called “mushroom” configuration in nanoparticles.[27] conditions in the presence of free phosphonic acids and MALDI-TOF analysis also proved that, upon reaction of BCN- chelating agents. This outstanding stability enabled quantitative 23 with each of the three types of carboxylic acid substrates functionalisation of the surface of BCN-23 with 24 available separately, each functionalised product has a higher molecular phosphonic acid groups. weight than the starting MOP. In all cases, a broad signal The porosity of the new carboxylic or phosphonic acid- containing the expected molecular formula for each acid functionalised MOPs was assessed in CO2-adsorption experi- derivative of BCN-23 was observed (Figures S33, S40, and S47, ments at 200 K, which revealed that all the tested MOPs were respectively). Free carboxylic acid groups were also evident in porous to CO2. Specifically, the maximum CO2-uptake capacities the FTIR spectrum of all three COOH-functionalised MOPs, at 1 bar were: 2.13 mmol/g (COOH Bz-BCN-23); 1.61 mmol/g which showed a new stretching C=O band centred at ca. (COOH-C5-BCN-23); 3.53 mmol/g (COOH-PEG3-BCN-23); and 1728 cm 1 that corresponds to the presence of free carboxylic 2.23 mmol/g (H2PO3-PEG3-BCN-23) (Figure S64). Therefore, syn- acid groups (Figures S35, S42, and S49). Therefore, CuAACs thesised carboxylic or phosphonic acid-functionalised MOPs enable not only the synthesis of COOH-functionalised MOPs but combine permanent porosity with on-surface coordination and also enable control over the type (i. e. aliphatic or aromatic) and covalent reactivity, making them ideal candidates as porous length of the spacer between the MOP core and the surface monomers in subsequent self-assembly processes. carboxylic acid groups. Finally, we studied the pH-dependent solubility of all the Next, we challenged our approach by attempting the synthesised carboxylic or phosphonic-acid functionalised MOPs. synthesis of the first ever MOP functionalised with available As expected, both types of MOPs were soluble in aqueous phosphonic acid groups. Free phosphonic acids are highly solution upon the stoichiometric deprotonation of the periph- coordinating groups that are generally incompatible with eral carboxylic or phosphonic acids, to obtain the correspond- metal–organic structures, although they have been used to ing carboxylate or phosphonate groups, respectively, affording build up extended structures as an alternative to the metal- negatively charged MOPs (Figure S36, S43, S50 and S58). carboxylate bond.[28,29] However, we found BCN-23 to be stable However, the pH range in which each MOP was found to be against free phosphonic acids such as phenylphosphonic acid, water-soluble varied accordingly to its respective functionalisa- which encouraged us to pursue its post-synthetic functionalisa- tion (Figures S34, S41, S48 and S55). Thus, whereas carboxylic tion with pendant phosphonic acid groups (Figure S56). The acid-functionalised MOPs precipitated out at pH 4.5 to 5.5, due click reaction between BCN-23 and H2PO3-PEG3-N3 (36 mol. to protonation of the surface COOH group, the phosphonic equiv.) was performed in the presence of the previously acid-functionalised one was soluble in water up to pH 2.7, employed catalytic mixture comprising CuSO4 (0.25 mol. equiv. which reflects the higher pKa of carboxylic acids relative to per alkynyl group) and sodium ascorbate (7.5 mol. equiv. per phosphonic acid. alkynyl group, 30 mol. equiv. per Cu(II) ion). Moreover, in this case, we also added the chelating agent tris- hydroxypropyltriazolylmethylamine (THPTA) to the mixture to Synthesis and bio-recognition capabilities of biotinylated prevent coordination of the phosphonate groups to the copper MOPs catalyst. After 1 h of reaction at room temperature, a precipitate (hereafter named H2PO3-PEG3-BCN-23) was obtained, isolated Biotinylation of organic or inorganic nanoparticles, which by centrifugation, and subsequently washed with 0.3 M HCl and involves attaching biotin molecules to their surface, has proven water (yield: 87 %). The integrity of the Rh(II) paddlewheel in to be an effective method for enhancing their ability to interact H2PO3-PEG3-BCN-23 was first confirmed by the presence of the with biological systems through the widely known biotin- characteristic λmax = 582 nm in the UV-Vis spectrum (Figure S54). streptavidin/avidin interaction.[30] For example, biotinylated Furthermore, the 1H NMR spectrum of H2PO3-PEG3-BCN-23 in nanoparticles have proven invaluable in the development of D2O revealed the disappearance of the alkyne protons and the innovative biosensors and targeted delivery systems.[31,32] Con- appearance of broad PEG and aromatic signals, which had the sequently, as a first step towards the development of MOP- Chem. Eur. J. 2023, e202301945 (5 of 8) © 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH 15213765, 0, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202301945 by Readcube (Labtiva Inc.), Wiley Online Library on [25/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article Chemistry—A European Journal doi.org/10.1002/chem.202301945 based bioconjugates, we employed CuAAC for the biotinylation aliquots that remained in solution revealed that biotin-PEG5- of MOPs, and then studied the resultant products for their BCN-23 does indeed recognise avidin. Furthermore, the molec- interactions with avidin. ular nature of the biotin-PEG5-BCN-23 enabled calculation of BCN-23 was biotinylated by reacting it with biotin-PEG5- the stoichiometry of these interactions. Thus, the average azide under the same standardised click conditions previously number of avidin groups attached to the MOP was found to be used for the model compounds. The structure of the product, 1, irrespective of the excess amount of avidin in solution hereafter named biotin-PEG5-BCN-23, was confirmed by 1H NMR (Figure 4c and Figure S66-S68). This result implies that, once in DMSO-d6, which showed new broad signals that correspond one avidin protein has attached to the MOP surface, there is to PEGylated biotin (Figure S59 and S60). DOSY NMR revealed strong steric hindrance that inhibits the recognition by the the same diffusion coefficient (D = 7.6 · 10 11 m2 s 1) for these remaining 23 available biotin moieties on the surface. This steric signals and for the aromatic peaks assigned to the MOP core hindrance might arise from the relative size of avidin (maximum (Figure S61). The calculated hydrodynamic radius was 2.5 nm in cross section of ca. 8 nm) relative to the biotin-PEG5-BCN-23 agreement with equilibrium configuration of the simulated (diameter of ca. 5 nm), and/or to the mutual steric hindrance of structure (Figure 4a and S63). The incorporation of the PEG- surface-bound biotin groups, given their high surface density biotin moiety onto the surface of BCN-23 was further confirmed (ca. 0.5 biotin molecules per nm2). by mass spectrometry, which revealed a broad peak that included the expected mass for [Rh24(5-biotin-PEG5-( 1H-1,2,3- triazol-4-yl)-bdc)24 + H + ] + of 19770 g/mol (Figure S62). Conclusions Next, we aimed to test the biorecognition capabilities of biotin-PEG5-BCN-23. To this end, we employed the competitive We have reported the synthesis of a novel alkyne-functionalised HABA-avidin binding assay. In this assay, 2-(4- MOP that can be post-synthetically modified through CuAAC hydroxyphenylazo)benzoic acid (HABA) initially occupies the reactions. The CuAAC reaction proceeds quantitatively without four possible binding sites of avidin (Figure 4b). When this compromising the structure of the parent MOP structure, which interaction occurs, λmax is centred at 500 nm, in contrast to the enables the synthesis of densely functionalised MOPs. We free form of HABA, whose λmax is at 350 nm. The addition of free validated the scope of our approach by decorating the MOP biotin to this complex induces the quantitative replacement of surface with a wide range of chemically diverse substrates. For HABA due to the greater affinity of avidin for biotin (Kd = 10 15) example, we synthesised novel MOPs with unprecedented than for HABA (Kd = 10 6). Therefore, the stoichiometric ex- functionalities, including carboxylic or phosphonic acid groups, change between HABA and biotin can be followed through UV- or bioactive biotins. We envisage the use of CuAACs on Vis spectroscopy, by monitoring the changes in absorbance of “clickable” MOPs to further expand the repertoire of functional the band centred at 500 nm (Figure S65). Thus, to study the MOPs to guide the development of novel catalysts, sensors, and biorecognition capabilities of MOP-bounded biotin moieties, we building blocks for hierarchical bio-mediated self-assembly performed a competitive test in which increasing amounts of processes. biotin-PEG5-BCN-23 were added to separated aliquots of HABA- avidin at a constant concentration. The reaction mixture was maintained in solution up to the addition of one biotin-PEG5- BCN-23 per avidin (i. e. 24 biotin molecules per avidin), after which partial precipitation was observed. The analysis of the Figure 4. (a) Snapshot of the equilibrium structure of biotin-PEG5-BCN-23 as obtained from molecular dynamics simulations in water solvent. Snapshots made with VMD.[17] Color code: Rhodium (green); carbon (black); hydrogen (white); oxygen (red); nitrogen (blue); Sulphur (yellow). Rh atoms are shown in their van der Waals size while linker is represented with Licorice drawing method. For simplicity, the MOP core is displayed as translucent. (b) Structure of avidin. (c) Plot of the average number of avidin proteins interacting with biotin-PEG5-BCN-23 at different molar ratios of biotin-BCN-23/avidin. Chem. Eur. J. 2023, e202301945 (6 of 8) © 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH 15213765, 0, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202301945 by Readcube (Labtiva Inc.), Wiley Online Library on [25/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article Chemistry—A European Journal doi.org/10.1002/chem.202301945 Experimental Section Acknowledgements Synthesis of alkyne-protected Rh(II)-MOP: Rh2Ac4 · 2MeOH (20 mg, 0.04 mmol), H2bdc-TIPS (74.4 mg 0.2 mmol) and Na2CO3 (25 mg, This work was supported by the Spanish MINECO (project 0.2 mmol) were dispersed in 4 mL of DMA. The mixture was then RTI2018-095622-B I00) and the Catalan AGAUR (project 2021 transferred to a scintillation vial and heated at 100 °C for 48 h. A SGR 00458). It was also funded by the CERCA Programme deep green solution was obtained and separated from the residual /Generalitat de Catalunya and through a fellowship (LCF/BQ/ solids by centrifugation. The crude product was obtained by PR20/11770011) from the “la Caixa” Foundation (ID 100010434). precipitation with water (20 mL) and then, separated by centrifuga- tion. The solid was washed with water and dried by lyophilization. ICN2 is supported by the Severo Ochoa Centres of Excellence The resulting green solid was washed two times with methanol programme, Grant CEX2021-001214-S, funded by MCIN/AEI/ (20 mL) for further purification and dried in a vacuum oven at 85 °C 10.13039.501100011033. A.C.S. is indebted to the Ramón y Cajal overnight (93.9 mg, yield: 80 %). Program (RYC2020-029749-I Fellowship) and the Europa Exce- Synthesis of BCN-23: The alkyne-protected Rh(II)-MOP (10 mg, lencia grant (EUR2021-121997) L.H.L acknowledges the support 0.96 μmol) was dissolved in THF (1 mL). Then, tetrabutylammonium from the Spanish State Research Agency (PRE2019-088056). fluoride (34.7 μL, 34.7 μmol, 1 M solution in THF) was added to this C.v.B. thanks the Austrian Science Fund (FWF), Erwin Schrö- solution under stirring, from which a blue precipitate appeared. dinger fellowship for supporting the project J 4637. J. F This solid was rapidly centrifuged and subsequently washed three acknowledges the financial support from MCIN/AEI/10.13039/ times with THF (2 mL) and dried under air. 501100011033 agency through Grant PID2021-124297NB-C33 General procedure to perform CuAAC reactions on BCN-23: BCN- and the “Severo Ochoa” Programme for Centres of Excellence in 23 (6.72 mg, 0.96 μmol) and the desired azide-functionalized ligand R\&D (CEX2019-000917-S) awarded to ICMAB. We also thank the (34.56 μmol) were dissolved in DMSO (10 mL). Then, two aqueous Government of Catalonia (AGAUR) for Grant 2021SGR01519. solutions of sodium ascorbate (1.04 M) and CuSO4 (0.7 M) were prepared. The addition of the required amount of catalyst (100 μL of CuSO4 and 200 μL of sodium ascorbate) to complete the reaction was added stepwise in five additions over a period of 1.25 h under Conflict of Interests stirring. Once the additions were completed, the mixture was reacted at room temperature overnight. Finally, the obtained The authors declare no conflict of interest. reaction products were separated and purified according to their solubility. Synthesis of acid-functionalized Rh(II)-MOPs through CuAAC Data Availability Statement post-synthetic reactions: BCN-23 (6.72 mg, 0.96 μmol) and 34.56 μmol of 5-azidohexanoic acid (5.1 μL), 4-azidobenzoic acid (172.8 μL from a 0.2 M solution in tert-butyl ether) or COOH-PEG3- The data that support the findings of this study are available N3 (8.6 mg) were dissolved in DMSO (10 mL). The presence of free from the corresponding author upon reasonable request. carboxylic acids in the reaction required the addition of acid to avoid precipitation during the reaction (20 μL, HCl 0.3 M). The CuAAC reaction were performed following the above-described Keywords: biofunctionalization · click chemistry · metal– general procedure. After the reaction, ethyl acetate (40 mL) was organic polyhedra · recognition · surface functionalisation used to precipitate the product obtaining an orange coloured solid. These solids were washed once with a mixture of DMF: 0.3 M HCl (40 mL, 1 : 2) and twice with a DMF/H2O mixture (40 mL, 1 : 2). The [1] M. Eddaoudi, J. Kim, J. B. Wachter, H. K. Chae, M. O’Keeffe, O. M. Yaghi, J. Am. Chem. Soc. 2001, 123, 4368–4369. final blue solids were dried in a vacuum oven at 85 °C for 2 h. [2] A. Carné-Sánchez, J. Albalad, T. Grancha, I. Imaz, J. Juanhuix, P. Larpent, Synthesis of phosphonic acid-functionalized Rh(II)-MOPs through S. Furukawa, D. 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Chemistry - A European Journal published by Wiley-VCH GmbH 15213765, 0, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202301945 by Readcube (Labtiva Inc.), Wiley Online Library on [25/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License RESEARCH ARTICLE Copper(I)-catalysed alkyne-azide cy- L. Hernández-López, Dr. C. von Baeck- cloaddition click chemistry is a mann, Dr. J. Martínez-Esaín, A. Cortés- versatile post-synthetic tool to func- Martínez, Dr. J. Faraudo, C. Caules, tionalize the surface chemistry of Dr. T. Parella, Prof. Dr. D. Maspoch*, alkyne-functionalized cuboctahedral Dr. A. Carné-Sánchez* Metal–Organic Polyhedra (MOPs). This 1–9 methodology enables quantitative functionalization of MOPs with a wide (Bio)Functionalisation of Metal– range of functional groups, including Organic Polyhedra by Using Click polymers, carboxylic and phosphonic Chemistry acids and biomolecules.