Nanocopper-Doped Cross-Linked Lipoic Acid Nanoparticles for

May 24, 2018 - ... gold nanoparticle-embedded ruthenium and palladium catalysts for .... (22) By monitoring the real-time fluorescence change of the s...
2 downloads 0 Views 1MB Size
Download PDF
Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

Letter

Nano-Copper Dopped Cross-Linked Lipoic Acid Nanoparticles for Morphology-Dependent Intracellular Catalysis Jingsheng Huang, Liang Wang, Pengxiang ZHAO, Fuqing Xiang, Jie Liu, and Shiyong Zhang ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b01337 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Nano-Copper Dopped Cross-Linked Lipoic Acid Nanoparticles for Morphology-Dependent Intracellular Catalysis Jingsheng Huang,† Liang Wang,†,‡ Pengxiang Zhao,§ Fuqing Xiang,† Jie Liu,∥ and Shiyong Zhang*,†,‡ †

National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, 610064, China. College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China. § Science and Technology on Surface Physics and Chemistry Laboratory, Mianyang 621907, China. ∥ State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China. ‡

ABSTRACT: The metal catalysts encapsulated in nanomaterials have recently been applied successfully in bioorthogonal chemistry for intracellular generation of bioactive compounds. However, the nanomaterial involved intracellular catalysis is intrinsically different from that in solution or in extracellular fluid. Except for the reactivity of metal catalyst itself, the supporting material’s morphology and biocompatibility are essential factors for building optimal nanocatalysts. We herein present a new nanocopper dopped cross-linked lipoic acid nanoparticle (Cu@cLANP), which meets the stringent requirements for the intracellular nanocatalyst. It comes from endogenous lipoic acid and can achieve easily the morphology change by different reduction methods (Scheme 1). The optimal rugby-like Cu@cLANPs I did show much better catalytic efficiency for intracellular azide–alkyne cycloaddition than that of the other two spherical nanocatalysts Cu@cLANPs II and III, hinting the importance of nanoparticle morphology for the intracellular bioorthogonal transformation. KEYWORDS: nanocatalyst, intracellular catalysis, lipoic acid, morphology, biocompatibility Benefited from high catalytic efficiency, good water solubility, reduced toxicity, and structural similarity with metalloenzymes, the metal catalysts encapsulated in various nanomaterials, which have been widely applied over decades in synthetic chemistry,1-4 has recently been successfully expanded in bioorthogonal chemistry in intracellular generation of bioactive compounds for therapeutic and labelling applications. For example, Bradley and coworkers developed a series of nano-palladium catalysts supported by polystyrenes for intracellular drug synthesis;5-7 Rotello and colleagues reported the supramolecular regulation of bioorthogonal catalysis in cells with gold nanoparticle-embedded ruthenium and palladium catalysts for imaging and therapeutic applications;8 Zimmerman and co-workers synthesized a copper-containing metal-polyolefin nanoparticle for catalyzing cellular azide–alkyne cycloaddition at parts-per-million catalyst levels;9 and Weissleder et al developed poly(lactic-co-glycolic acid)-bpolyethyleneglycol supported palladium catalysts for intracellular activation of prodrugs.10 However, the nanomaterial involved intracellular catalysis is intrinsically different from that in solution or in extracellular fluid.11-14 Except for the reactivity of catalyst itself, the endocytosis of nanoparticles would be taken into account for efficient transformations. This requires that the nanocatalyst would be better prepared in morphology controlled for achieving the optimal catalyst formula. Yet as far as we know, there is no report regarding the effect of nanoparticle morphology on intracellular catalysis. Another important consideration is the biocompatibility of supporting materials. Although the reported nanomaterials are claimed to be biologically benign at their work conditions, they are after all exogenous matters, and would no doubt have hazard to some extent. Taking popu-

lar supporting material polystyrene as example, it has been listed as Class 3 carcinogen by World Health Organization on 27 Oct., 2017.15 We present herein a new nano-copper dopped cross-linked lipoic acid nanoparticle (Cu@cLANP, Scheme 1), which meets the stringent requirements mentioned above for the nanocatalyst of intracellular catalysis. On one hand, the supporting material consists of endogenous lipoic acid, a coenzyme existing in the mitochondria,16 and thus owns extremely good biocompatibility. On the other hand, the copper dopped cross-linked lipoic acid nanoparticles, by virtue of a large number of interlaced disulfide groups, could reassemble under different reduction conditions and achieve the construction of nanocatalysts with different morphologies. The experimental results showed that the resulting three morphological nanocatalysts Cu@cLANPs I-III demonstrated similar parts-permillion catalytic efficiency in solution for azide–alkyne cycloaddition, but gave distinct catalytic activity in creating the same compounds in living cells, hinting the importance of nanoparticle morphology for the intracellular bioorthogonal transformation. The supporting material, namely cross-linked lipoic acid nanoparticles (cLANPs), was prepared simply by dissolving lipoic acid in water above the critical aggregation concentration (CAC) of 17.3 µg/mL (Figure S1), followed by reduction with 10 mol% dithiothreitol (DTT) and dialysis against deionized water (see Supporting Information for details).17 The successful synthesis of the cLANPs was directly proven by the broadened proton signals in 1H NMR spectra (Figure S2), the great increase in absolute weight-average molecular weight determined by static light scattering (SLS) measurement [Mw

ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Schematic illustration of the preparation of nanocatalysts Cu@cLANPs I-III.

Figure 1. TEM and DLS characterization of the supporting material cLANPs and nanocatalysts Cu@cLANPs I-III. (a) cLANPs, (b) Cu@cLANP I, (c) Cu@cLANP II, and (d) Cu@cLANP III.

= (3.79 ± 0.13) ×104 g mol-1 (Figure S3)], and the enhanced stability both in water and in 10% fetal bovine serum (Figure S4). The transmission electron microscopy (TEM) image disclosed the formation of spherical nanoparticles with an average size of ~93 nm by dynamic light scattering (DLS) measurement (Figure 1a). The resulting aqueous solution of cLANPs was treated with 1.0 eq. NaBH4 and 20 mol% CuCl2•2H2O (relative to LA) to generate the nano-copper dopped cross-linked lipoic acid nanoparticle I (Cu@cLANP I), which gave the similar hydrophobic size with cLANPs, but changed a lot the morphology from sphere- to rugby-like structure with aspect ratio of ~2.8:1 determined by TEM measurement (Figure 1b). The high-resolution TEM (HRTEM) confirmed the formation of nano-copper(0) with the clear lattice stripe of the particles (Figure S5). We rationalize that the rugby-like Cu@cLANP I would be attributed to partial cleaved disulfide bonds by NaBH4. The resulting thiol intermediates, which were detected by ellmann’s reagent (Figure S6),18 recombined and/or anchored to the newly formed nano-

copper(0) to achieve the establishment of observed rugby-like structure (Scheme 1). Notably, in spite of the absence of metal particles, the cLANPs could still be partially reduced by NaBH4, the morphology of the obtained nanoparticles was basically the same as that of the original cLANPs, indicating the synergistic effect of metal nanoparticles in the morphological change of cLANPs. More intriguingly, the morphology of Cu@cLANP I can be further changed by different reducing agent doses or reducing methods. As exhibited in Scheme 1, under the condition of 10 eq. NaBH4, the spherical Cu@cLANP II with the size of only ~11 nm were obtained (Figure 1c), while exposed under a handheld UV lamp (365 nm, 8 W), the rugby-like Cu@cLANP I changed to spherical Cu@cLANP III with the size of ~47 nm (Figure 1d). The nanocatalysts Cu@cLANPs I-III were further characterized via energy dispersive spectroscopy (EDS) analyses, which indicated that the copper was successfully encapsulated (Figure 2a). Moreover, X-ray photoelectron spectroscopy (XPS) analysis at Cu 2p region showed two distinct peaks at 932.6 eV (Cu 2p3/2) and 952.6 eV (Cu 2p1/2), implying the successfully fabrication of copper(0) nanoparticles (Figure 2b).19 It should be mentioned that the Cu content of all above three nanocatalyts were determined by ICP-AES, and the leaching levels were also estimated and shown less than 1.6% after a 48 h treatment in 10 mM PBS at 37 oC (Figure S7), suggesting the robustness of the Cu@cLANP nanocatalysts.

Figure 2. Physicochemical properties of the nanocatalysts. (a) EDS spectra of Cu@cLANPs I-III. (b) XPS spectrum of Cu@cLANP I.

ACS Paragon Plus Environment

Page 2 of 6

Page 3 of 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Figure 3. Investigation of the catalytic activity of Cu@cLANPs I-III in water and in living cells. (a) The Cu@cLANPs mediated “click” reaction between 3-azido-7-coumarin and phenyl acetylene. (b) The emission intensity at 460 nm (I460) of the reaction system in water as a function of time. Data are expressed as mean ± standard deviation of three separate experiments. (c) CLSM images of HeLa cells preincubated with Cu@cLANPs I-III ([Cu] = 15 ppm) and with no catalyst for 2 h, washed with PBS, and subsequently incubated with 1:1 (mol/mol) 3-azido-7-coumarin/phenyl acetylene for 48 h at 37 oC for 0.5, 4, and 6 h. The scale bars are 10 µm in all images. (d) Relative pixel intensity of the reaction system in cells as a function of time. The pixel intensity from the 6 h image of cells pre-incubated with Cu@cLANP I is defined as 1.0. Data are expressed as mean ± standard deviation of three separate experiments. (e) Flow cytometry analysis of HeLa cells incubated with the Cu@cLANPs I-III at 37 °C for 2 h. The untreated cells were used as a control.

The catalytic efficiency of Cu@cLANPs I-III was first tested by the classic “click” reaction between benzyl azide and phenyl acetylene with various amounts of catalyst in neat water at 35 oC, and the results are shown in Table S1. One can find that the traditional catalysts CuSO4/sodium ascorbate and Cu powder with the amount of 50 ppm gave only 43% and 7% yields due to the low contact between the substrates and catalysts in neat water condition (Table S1, entries 1 and 3). By contrast, the catalysts Cu@cLANPs I-III with the amount of only 25 ppm can reach almost the quantitative isolated yields in 24 h (Table S1, entries 7-9). The parts-per-million (ppm) catalyst level is comparable to the lowest catalyst dosage reported so far for “click” reaction in pure water.9, 20, 21 To further compare the catalytic efficiency of the three morphological nanocatalysts, the substrate 3-azido-7-coumarin was employed to “click” with phenyl to generate the well-known fluorogenic coumarin derivative 1 with high fluorescence (Figures 3a and S8).22 By monitoring the real-time fluorescence change of the system, the reaction process can be easily tracked. Figure 3b shows the emission intensity at 460 nm (I460) of the reaction system as a function of reaction time, which disclosed that the three nanocatalysts Cu@cLANPs I-III have almost uniform conversion efficiency at each given time point, suggesting that the morphology has little effect on the solution reaction rate. This result is reasonable in consideration of the homology of the copper nanoparticles buried in the three nanocatalysts. Moreover, the three nanocatalysts Cu@cLANPs I-III showed similar reaction activity with a wide scope of substrates (6 h, Table S2) and gave the corresponding 1,2,3-triazoles with final yields ranging from 83% to 97% (24 h, Table S2).

After confirmation of the solution reactivity of the three nanocatalysts, their potential for intracellular catalysis was tested in living cells using confocal microscopy. Briefly, human cervical cancer (HeLa) cells were pre-incubated with Cu@cLANPs I-III (0.5 µM in Dulbecco’s modified Eagle medium, DMEM) for 2 h. The DMEM was removed and the cells were washed three times to eliminate extracellular Cu@cLANPs, and subsequently incubated with substrates 3azido-7-coumarin and phenyl acetylene (15 µM) at 37 °C for 0.5, 4, and 6 h (See Supporting Information for details). As seen in Figure 3c, only very weak fluorescence, likely from unreacted 3-azido-7-coumarin, could be observed in the absence of Cu@cLANPs, whereas cells were clearly fluorescent in the presence of nanocatalysts, suggesting the generation of fluorogenic molecule 1. The high-resolution MS confirmed the intracellular formation of compound 1 with the molecular weight of 306.0875 ([M + H]+, Figure S9). Quantitative data showed that at every identical incubation time, the fluorescence signals of the cells treated by Cu@cLANP I were more intense than that of the other two types of nanocatalysts (Figure 3d). Especially for 4 h incubation, the relative fluorescence signals of Cu@cLANP I are 2.66 and 1.78 times higher than that of Cu@cLANP II and Cu@cLANP III, respectively, hinting more fluorescent molecules produced. We rationalize that the nanoparticle morphology plays an important role in the intracellular catalytic performance of the three catalysts. The rugby-like Cu@cLANP I looks like entering cells faster so as to achieve higher catalytic efficiency, while the cell uptake of spherical Cu@cLANP II and III is relatively slow, and so get lower conversion efficiency. To

ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

testify the hypothesis, the cell uptakes of the three nanocatalysts were compared quantitatively using the flow cytometry technique (See Supporting Information for details). As exhibited in Figure 3e, one can find that the intracellular fluorescence of HeLa cells pre-incubated with Cu@cLANP I was much stronger than that incubated with the other two nanocatalysts. The corresponding mean fluorescence intensity (MFI) of the Cu@cLANP I (∼6752) was 1.54-fold higher than that of Cu@cLANP II (∼4367) and 1.21-fold higher than that of Cu@cLANP III (∼5535) (Figure S10a), demonstrating that the rugby-like structure can indeed efficiently improve the cell uptake of the nanocatalysts. Furthermore, the cellular Cu contents after the three catalysts incubated with cells for 1 h were measured by ICP-MS, which shows that the cells treated by Cu@cLANP I own up to 9.0 ppm Cu content whereas those treated by Cu@cLANP II and Cu@cLANP III are only 6.0 and 7.0 ppm, respectively (Figure S10b). This result verifies again our hypothesis that the rugby- like Cu@cLANP I owns better cell uptake efficiency. Up to this point, it is clear that the rugby-like Cu@cLANP I is an optimal catalyst for intracellular azide–alkyne cycloaddition. To further demonstrate the application advantage of the morphology-dependent intracellular catalysis, the in situ generation of bioactive compounds for tumor therapy was conducted. Here triazole-containing anticancer agent 4, which is known to inhibition of tubulin polymerization to interfere with the mitotic spindle assembly during cell division,23,24 was synthesized from two benign segments 2 and 3 (Figure 4a). As expected, the three catalysts showed consistent catalytic activities in solution (Figure S11), but gave distinct catalytic activities in living cells. As shown in Figure 4b, under the same concentration of pre-incubated catalysts, the cells treated by Cu@cLANP I demonstrated a lower viability at every given substrate concentration compared to the other two catalysts, suggesting that the intracellular formation of cytotoxic 4 mediated by the Cu@cLANP I (Figure S12) was more efficient so as to give stronger cell growth inhibition. Since the amount of intracellularly generated product 4 was hard to be measured, we utilized the substrate 2 or 3 to indirectly determine the half-maximal inhibitory concentration (IC50) of cell proliferation. It was found that the IC50 of sample mediated by Cu@cLANP I could be as low as 10.18 µM, a value 0.73 and 0.62-time lower than that from Cu@cLANP II (13.96 µM) and Cu@cLANP III (16.38 µM), respectively. Notably, the IC50 of 10.18 µM from Cu@cLANP I is just 1.19 µM higher than that of the antitumor product 4 (8.99 µM, Figure S13), which means that the overwhelming majority of the substrates were converted to 4 for cell proliferation inhibition. Considering the complex intracellular environment, this in situ synthesis is really highly efficient. By the way, the control experiments exhibited either 2/3, 2/Cu@cLANP I or 3/Cu@cLANP I showing any inhibitory effect even up to 50 µM (Figure S14). The biocompatibility of the nanocatalysts was finally evaluated. Derived from the endogenous lipoic acid, and decomposed into dihydrolipoic acid participating in a variety of biochemical transformations,25,26 the supporting material cLANPs demonstrated high biocompatibility, and showed noncytotoxicity at the concentration of even 750 µg/mL (Figure S15). Benefited from the biocompatible cLANPs, the nanocopper dopped cLANPs (Cu@cLANPs I-III) were also noncytotoxic with full cell viability at up to 100 ppm of Cu (Figure S16). The conventional homogeneous catalyst CuSO4,

Page 4 of 6

however, showed significant toxicity with the survival rate of treated cells less than 50% at only the concentration of 50 ppm, indicating the great potential of the Cu@cLANPs for biologically compatible in vivo applications.

Figure 4. In situ synthesis of anticancer bioactive compound 4. (a) The Cu@cLANPs mediated “click” reaction between 2 and 3. (b) Cell viability of HeLa cells pre-incubated with Cu@cLANPs I-III ([Cu] = 15 ppm) for 2 h, washed with PBS, and subsequently incubated with 1:1 (mol/mol) 2/3 for 48 h at 37 oC with a series of concentrations. HeLa cells incubated without any materials were used as the control. Data are expressed as mean ± standard deviation of five separate measurements (mean ± SD, n = 5, P1* < 0.05, P2* < 0.05).

In summary, this work gave us a clue that except for the reactivity of metal catalyst itself, two key elements of the supporting materials, i.e. morphology and biocompatibility, should be taken into account to build optimal nanocatalysts for intracellular transformations. The cross-linked lipoic acid nanoparticles (cLANPs) represent a leading example that meets simultaneously the two stringent requirements for the supporting materials of intracellular nanocatalysts. It comes from endogenous lipoic acid and can achieve easily the morphology change by different reduction methods. The optimal rugby-like Cu@cLANPs I did show much better catalytic efficiency for intracellular azide–alkyne cycloaddition than that of the other two spherical nanocatalysts Cu@cLANPs II and III. Notably, although only nano-copper dopped cLANPs was exampled here, this new cLANPs is generally applicable and may extend to encapsulate other nano-metals to generate nanocatalysts with diverse morphologies, which would give great opportunities to search for novel bioorthogonal reactions. In fact, some interesting metal@cLANPs have been screened out in our lab, and the results will be reported later.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Notes The authors have no conflicts of interest to declare for this article.

ASSOCIATED CONTENT

ACS Paragon Plus Environment

Page 5 of 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedure, NMR, reaction monitoring, and cell test (PDF)

sis in Living Systems and In Situ Drug Synthesis. Angew. Chem. Int. Ed. 2016, 55, 15662-15666. (15) International Agency for Research on Cancer, World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 2017, volume 1–121, date accessed on 27-10-2017:

http://monographs.iarc.fr/ENG/Classification/index.php.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21372170 and 51703145), the Excellent Young Foundation of Sichuan Province (2016JQ0028), the Applied Basic Research Project of Sichuan Province (15JC0440), and the Discipline Development Foundation of Science and Technology on Surface Physics and Chemistry Laboratory (Nos. XKFZ201505, XKFZ201506). We thank the Center of Testing and Analysis, Sichuan University, for TEM, EDS and XPS measurements.

REFERENCES (1) Crooks, R. M.; Zhao, M. Q.; Sun, L.; Chechik, V.; Yeung, L. K. Dendrimer-encapsulated metal nanoparticles: synthesis, characterization, and applications to catalysis. Acc. Chem. Res. 2001, 34, 181-190. (2) Wang, C.; Ciganda, R.; Salmon, L.; Gregurec, D.; Irigoyen, J.; Moya, S.; Ruiz, J.; Astruc, D. Highly Efficient Transition Metal Nanoparticle Catalysts in Aqueous Solutions. Angew. Chem. Int. Ed. Engl. 2016, 55, 3091-3095. (3) Zhu, Q.; Xu, Q. Immobilization of ultrafine metal nanoparticles to high-surface-area materials and their catalytic applications. Chem 2016, 1, 220-245. (4) Navalon, S.; Dhakshinamoorthy, A.; Alvaro, M.; Garcia, H. Metal nanoparticles supported on two-dimensional graphenes as heterogeneous catalysts. Coord. Chem. Rev. 2016, 312, 99-148. (5) Yusop, R. M.; Unciti-Broceta, A.; Johansson, E. M.; SanchezMartin, R. M.; Bradley, M. Palladium-mediated intracellular chemistry. Nat. Chem. 2011, 3, 239-243. (6) Unciti-Broceta, A.; Johansson, E. M.; Yusop, R. M.; SanchezMartin, R. M.; Bradley, M. Synthesis of polystyrene microspheres and functionalization with Pd(0) nanoparticles to perform bioorthogonal organometallic chemistry in living cells. Nat. Protoc. 2012, 7, 1207-1218. (7) Clavadetscher, J.; Indrigo, E.; Chankeshwara, S. V.; Lilienkampf, A.; Bradley, M. In-Cell Dual Drug Synthesis by Cancer-Targeting Palladium Catalysts. Angew. Chem. Int. Ed. 2017, 56, 6864-6868. (8) Tonga, G. Y.; Jeong, Y.; Duncan, B.; Mizuhara, T.; Mout, R.; Das, R.; Kim, S. T.; Yeh, Y. C.; Yan, B.; Hou, S.; Rotello, V. M. Supramolecular regulation of bioorthogonal catalysis in cells using nanoparticle-embedded transition metal catalysts. Nat. Chem. 2015, 7, 597-603. (9) Bai, Y.; Feng, X.; Xing, H.; Xu, Y.; Kim, B. K.; Baig, N.; Zhou, T.; Gewirth, A. A.; Lu, Y.; Oldfield, E.; Zimmerman, S. C. A Highly Efficient Single-Chain Metal-Organic Nanoparticle Catalyst for Alkyne-Azide "Click" Reactions in Water and in Cells. J. Am. Chem. Soc. 2016, 138, 11077-11080. (10) Miller, M. A.; Askevold, B.; Mikula, H.; Kohler, R. H.; Pirovich, D.; Weissleder, R. Nano-palladium is a cellular catalyst for in vivo chemistry. Nat. Commun. 2017, 8, 15906-15918. (11) Weiss, J. T.; Dawson, J. C.; Macleod, K. G.; Rybski, W.; Fraser, C.; Torres-Sanchez, C.; Patton, E. E.; Bradley, M.; Carragher, N. O.; Unciti-Broceta, A. Extracellular palladium-catalysed dealkylation of 5-fluoro-1-propargyl-uracil as a bioorthogonally activated prodrug approach. Nat. Commun. 2014, 5, 3277-3285. (12) Martínez-Calvo, M.; Mascareñas, J. L. Organometallic catalysis in biological media and living settings. Coord. Chem. Rev. 2018, 359, 57-79. (13) Indrigo, E.; Clavadetscher, J.; Chankeshwara, S. V.; Lilienkampf, A.; Bradley, M. Palladium-mediated in situ synthesis of an anticancer agent. Chem. Commun. 2016, 52, 14212-14214. (14) Clavadetscher, J.; Hoffmann, S.; Lilienkampf, A.; Mackay, L.; Yusop, R. M.; Rider, S. A.; Mullins, J. J.; Bradley, M. Copper Cataly-

(16) Novotny, L.; Rauko, P.; Cojocel, C. alpha-lipoic acid - the potential for use in cancer therapy. Neoplasma 2008, 55, 81-86. (17) Liao, C. Y.; Chen, Y.; Yao, Y. C.; Zhang, S. Y.; Gu, Z. W.; Yu, X. Q. Cross-Linked Small-Molecule Micelle-Based Drug Delivery System: Concept, Synthesis, and Biological Evaluation. Chem. Mater. 2016, 28, 7757-7764. (18) Inal, B. B.; Emre, H. O.; Baran, O.; Ahmedov, M.; Ozdemir, A. F.; Kemerdere, R.; Ates, S.; Tanriverdi, T. Dynamic thiol-disulphide homeostasis in low-grade gliomas: Preliminary results in serum. Clin. Neurol. Neurosurg. 2017, 161, 17-21. (19) Irie, H.; Kamiya, K.; Shibanuma, T.; Miura, S.; Tryk, D. A.; Yokoyama, T.; Hashimoto, K. Visible Light-Sensitive Cu(II)-Grafted TiO2 Photocatalysts: Activities and X-ray Absorption Fine Structure Analyses. J. Phys. Chem. C 2009, 113, 10761-10766. (20) Deraedt, C.; Pinaud, N.; Astruc, D. Recyclable Catalytic Dendrimer Nanoreactor for Part-Per-Million CuI Catalysis of “Click” Chemistry in Water. J. Am. Chem. Soc. 2014, 136, 12092-12098. (21) Wang, C.; Wang, D.; Yu, S.; Cornilleau, T.; Ruiz, J.; Salmon, L.; Astruc, D. Design and Applications of an Efficient Amphiphilic “Click” CuI Catalyst in Water. Acs Catal. 2016, 6, 5424-5431. (22) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.; Wang, Q. A fluorogenic 1,3-dipolar cycloaddition reaction of 3azidocoumarins and acetylenes. Org. Lett. 2004, 6, 4603-4606. (23) Odlo, K.; Fournier-Dit-Chabert, J.; Ducki, S.; Gani, O. A.; Sylte, I.; Hansen, T. V. 1,2,3-triazole analogs of combretastatin A-4 as potential microtubule-binding agents. Bioorg. Med. Chem. 2010, 18, 6874-6885. (24) Akselsen, O. W.; Odlo, K.; Cheng, J. J.; Maccari, G.; Botta, M.; Hansen, T. V. Synthesis, biological evaluation and molecular modeling of 1,2,3-triazole analogs of combretastatin A-1. Bioorg. Med. Chem. 2012, 20, 234-242. (25) Fujiwara, K.; Okamura-Ikeda, K.; Motokawa, Y. Chicken liver H-protein, a component of the glycine cleavage system. Amino acid sequence and identification of the Nepsilon-lipoyllysine residue. J. Biol. Chem. 1986, 261, 8836-8841. (26) Milne, J. L.; Wu, X.; Borgnia, M. J.; Lengyel, J. S.; Brooks, B. R.; Shi, D.; Subramaniam, S. Molecular structure of a 9-MDa icosahedral pyruvate dehydrogenase subcomplex containing the E2 and E3 enzymes using cryoelectron microscopy. J. Biol. Chem. 2006, 281, 4364–4370.

ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Paragon Plus Environment

Page 6 of 6