Analyte-Driven Disassembly and Turn-On ... - ACS Publications

Feb 20, 2018 - MCx-1 provides a real-time, turn-on fluorescence signal in response to .... These data show that host–guest recognition within the ca...
0 downloads 3 Views 743KB Size
Subscriber access provided by UNIV OF DURHAM

Communication

Analyte-driven disassembly and turn-on fluorescent sensing in competitive biological media Meagan A. Beatty, Jorge Borges-González, Nicholas J. Sinclair, Aidan T. Pye, and Fraser Hof J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b13298 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 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.

Journal of the American Chemical Society 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 4 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

Journal of the American Chemical Society

Analyte-driven disassembly and turn-on fluorescent sensing in competitive biological media Meagan A. Beatty, Jorge Borges-González, Nicholas J. Sinclair, Aidan T. Pye, Fraser Hof* Department of Chemistry, University of Victoria, PO Box 3065, STN CSC, Victoria, V8W 3V6, Canada. E-mail: [email protected] Supporting Information ABSTRACT: Many indicator displacement assays can detect biological analytes in water, but these often have reduced performance in the presence of an unavoidable component: NaCl. We report here a new self-assembled sensor—DimerDye—that uses a novel photochemical guest-sensing mechanism and that is intrinsically tolerant of co-solutes. We synthetically integrated a dye into a calixarene macrocycle, forming two new merocyanine calixarenes (MCx-1 and MCx-2). Both compounds self-assemble into non-emissive dimers in water. The addition of good guests like trimethyllysine induces a turn-on fluorescence response of MCx-1 due to simultaneous dimer dissociation and formation of an emissive host-guest complex. DimerDyes remain functional in solutions containing the various salts, metal ions, cofactors that are needed for enzymatic reactions. MCx-1 provides a real-time, turn-on fluorescence signal in response to the lysine methyltransferase reaction of PRDM9.

Indicator displacement assays (IDAs) are the central mechanism by which supramolecular hosts are converted into sensors.1 This approach has been demonstrated with many different dyes complexed with macrocycles2 or clefts3 that target analytes of many classes. IDAs that are intended to detect analytes in real biological solutions often need extensive optimization. The Kd of host-dye and host-analyte equilibria must be compatible with each other, and both equilibria are sensitive to co-solutes. We report here a supramolecular sensing scheme that involves a new kind of disassembly-triggered emission, and that is intrinsically tolerant of competitive, biologically relevant co-solutes. Real-world solutions contain salts, organic molecules, complex buffers, and biological components that can effect binding equilibria and/or the dye’s emission in ways that diminish signal or render the IDA inoperative. Sulfonatocalix[n]arene hosts (e.g. PSC) are often paired with the dye lucigenin (LCG) (Figure 1a), creating IDAs for neurotransmitters, post-translationally modified peptides, and other analytes.4 Quinolinium dyes like LCG are quenched by Cl–, which is ubiquitous in biology.5 In a groundbreaking example of IDA conducted inside of living cells, the problem was circumvented by use of an unnatural Cl–-free growth medium.6 Cucurbiturils (e.g. CB7, Figure 1b) have been paired with a variety of dyes to detect many different amines. 7 However, cucurbiturils operate best in low-Na+ solutions because Na+ binds to the oxygen-lined portals and decreases binding of both analytes and indicators.8

Figure 1. Responses of common assemblies to NaCl. a) Lucigenin is often used in IDA, but is quenched by halides. b) Cucurbiturils have weakened affinity for dyes and guests in the presence of Na+. c) Compound 1 forms a homodimer in water that becomes stronger with NaCl. We recently reported the discovery of a new family of selfassembling hosts (e.g. 1, Figure 1c). This motif is notable for the fact that the dimers get stronger in the presence of added salt and remain assembled in real biological fluids.9 The salt tolerance of these dimers arises intrinsically from their architecture. The assembly is driven by hydrophobic attraction in tension with mutual repulsions between like-charged monomers. Increasing salt concentrations reduce the repulsion between anionic monomers, and dimerization is preserved or strengthened.

Figure 2. Cartoon depiction of a) an Indicator Displacement Assay and b) a DimerDye Disassembly Assay which involves an integrated host-dye sensor that disassembles in the presence of an analyte to produce a turn-on fluorescence response. 1 Environment ACS Paragon Plus

Journal of the American Chemical Society 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

We sought to exploit this robust architecture to develop a new class of reagents that can sense analytes in biological solutions. The new sensors integrate both host and dye within a single molecular species (a “DimerDye”) that homodimerizes in water through a combination of hydrophobic attraction and electrostatic repulsion, and therefore would preserve the salt tolerance of 1. A DimerDye Disassembly Assay (DDA) would achieve analyte sensing because 1) the self-assembly forces two copies of fluorophore to stack upon each other, and 2) guest binding causes programmed disassembly of the dimers into a host-guest complex, thereby changing the photophysical response (Figure 2).

macrocycle (Figure S1). All protons on the merocyanine arm show upfield shifts in D2O relative to values in DMSO, consistent with the existence of an assembled state in water and free monomer in DMSO (Table S1). The N-ethyl derivative, MCx-2, showed similar upfield chemical shifts (Figure 4a and Table S2) while the N-ethyl triplet and quartet provided easily assigned protons for structural analysis. 2-D NOESY correlations between Et protons (−1.01 ppm) and upper-rim protons (7.64 ppm) are diagnostic of the assembled state (Figure 4b). The rigidity of the Ealkene ensures that intramolecular self-inclusion of pyridinium arms is impossible. DOSY proves homodimeric assemblies for both MCx-1 and MCx-2 (Tables S3–S5). We used quantitative NMR to show that MCx-1 homodimerizes with single-digit micromolar Kd values in phosphate buffers (Tables S6 and S7).

Figure 3. Brooker’s Merocyanine (2) is integrated into the calix[4]arene macrocycle to form MCx-1, which dimerizes in a similar fashion to 1. We designed the integrated host-dye merocyanine calixarene (MCx-1) to test the DDA concept. Brooker’s merocyanine (2) is a solvatochromic styryl dye.10 The design of MCx-1 includes the phenolic ring of 2 directly within the macrocyclic core of calix[4]arene11 (Figure 3). We anticipated that the merocyanine arm would serve as a hydrophobic element to bind within the pocket of a second monomer to drive a dimeric self-assembly, wherein each monomer bears a net –3 charge.12 MCx-1 was synthesized by site-specific functionalization of the calix[4]arene skeleton. The triprotected calix[4]arene 313 was selectively formylated by treatment with hexamethylenetetramine (HMTA) to produce 4. Subsequent ester removal (5, not shown) and sulfonation produced the aldehyde trisulfonate 6 as a key intermediate. Condensation reactions with picolinium derivatives produced the targeted merocyanine calixarenes, MCx-1 (Nmethyl) and MCx-2 (N-ethyl), with 37% and 30% yield, respectively, after HPLC purification.

Scheme 1. Synthesis of MCx-1 and MCx-2. NMR studies provide a detailed picture of the self-assembled homodimers in water. The pyridinium aryl protons of MCx-1 shift upfield by ≥1 ppm relative to chemical shifts observed for free 2, while the N-methyl resonance moves >3.5 ppm upfield, which is diagnostic of encapsulation deep within the calixarene

Figure 4. 1-D and 2-D NMR data support the formation of the expected dimers. a) N-ethylpyridinium protons are upfield-shifted in D2O (dimer) relative to their normal positions in d6-DMSO (monomer). See Supp. Info. for comparisons to chemical shifts of the free parent dye (2). b) The upfield-shifted MCx-2 ethyl group shows an NOE with calix[4]arene upper rim protons. The self-assembly of MCx-1 gives it photophysical properties that are distinct from those of 2. Compound 2 is fluorescent in water and DMSO (Figure S5), while MCx-1 is fluorescent in DMSO (Figure 5b) but is non-emissive in neutral water (Figure 5a). Compound 2 remained fluorescent when mixed with PSC (Figure S4), showing that the dark state of MCx-1 does not simply arise from the binding of a merocyanine dye to a calixarene pocket. Fluorescence and NMR data together demonstrate the structural principles of the DimerDye design: 1) NMR data show that MCx-1 is monomeric in DMSO, and dimeric in water. 2) MCx-1 is emissive as a monomer, but not emissive as a dimer due to quenching of the fluorophore’s excited state by the second adjacent fluorophore.

Figure 5. Absorbance (dotted line) and fluorescence (solid line) spectra of MCx-1 (4 µM) in a) Na2HPO4/NaH2PO4 buffer (10 mM, pH 7.4, λex. 382 nm) and b) DMSO (λex. 482 nm, λem 585 nm). Pictures of MCx-1 show the lack of fluorescence in water (left vials) and visible emission in DMSO (right vials) when irradiated by a handheld UV lamp at 365 nm. All solutions are homogeneous.

2 Environment ACS Paragon Plus

Page 2 of 4

Page 3 of 4 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

Journal of the American Chemical Society MCx-1 is a turn-on fluorescent sensor for guests in water. A panel of amino acids were studied by fluorescence (Figures 6, S13) and NMR. Sulfonated calixarenes bind Kme3 and other amino acids with Kd values between 30 and 2000 µM, meaning that concentrations of these analytes in this range are needed to disrupt the homodimers.14a The greatest fluorescence response was observed for Kme3, and a negligible response was seen for K (Figure 6b and 6c). Added salts are tolerated, causing slightly decreased signal intensity but better Kme3/K selectivity (Figure S14). NMR titrations of Kme3 into MCx-1 provided insight into the interactions between analyte and sensor. Most MCx-1 resonances broadened with the increase of Kme3 concentration indicating intermediate exchange of [MCx-1]2 and [MCx-1]-Kme3 on the NMR timescale. The resonances that were the most broadened were protons that experience the largest chemical shift change during the transition from dimer to analyte-sensor complex, notably, the N-methyl, and pyridinium protons (Figure 7). Titrating the weaker guest lysine into MCx-1 produced only slight broadening even at higher lysine concentrations (Figure S9). These data show that host-guest recognition within the calixarene pocket causes disassembly of the dimer, which is accompanied by a turn-on fluorescence response.

Figure 6. Absorbance (dotted line) and fluorescence (solid line) spectra of MCx-1 (10 µM) in Na2HPO4/NaH2PO4 buffer (10 mM, pH 7.4, λex. 382 nm) a) without guest c) with Kme3 (1 mM, λex. 382 nm, λem. 575 nm). b) Comparison of fluorescence intensities observed with various amino acids (250 µM, λex. 382 nm, λem 575 nm).

Figure 7. Complexation of Kme3 with MCx-1 (250 µM) is observed with 1H NMR titration in Na2HPO4/NaH2PO4 buffer (100 mM, pD 7.8). Blue boxes highlight resonances that support the formation of a host-guest complex undergoing intermediate exchange relative to NMR timescale. MCx-1 operates in complex media and on complex analytes. Histone peptide tails are home to epigenetic methyllysine marks that are under the control of methyltransferases and demethylases. Calixarenes bearing three sulfonates routinely bind cationic Kme3 peptides with Kd values of 0.17–5 µM, while retaining selectivity over unmethylated peptides.14b-c We tested MCx-1 with histone tails in typical reaction buffers for Fe2+- and cofactor-dependent

demethylases (Figure S17), and for S-adenyl-methionine (SAM)dependent methyl transferases (Figure 8a). In each case, the methylated histone tail gave a significant turn-on fluorescent signal. We conducted an enzymatic methylation of a 21-mer histone tail peptide with methyltransferase PRDM9 in 96-well format. MCx-1 produced a real-time, turn-on fluorescence signal as the reaction produced methylated product (Figure 8b, c, S18). Demethylation of a 21-mer H3K9me3 peptide by JMJD2D was also measurable, in this case by real-time decrease in MCx-1 emission (Figure S19).

Figure 8. a) Emission spectra of MCx-1 (8 µM, λex. 384 nm) in the reaction conditions (solid gray line), with H3K4me3 (40 µM, solid black line), and H3K4 (40 µM, dotted line). b) Fluorescence increases as methyltransferase, PRDM9 (460 nM), methylates H3K4 (40 µM) with MCx-1 (10 µM, λex. 384 nm, λem 585 nm) in the reaction conditions: Tris (50 mM, pH 8.5), NaCl (30 mM), DTT (1 mM), SAM (300 µM). c) Reaction scheme of PRDM9catalysed conversion of H3K4 to H3K4me3, which complexes with MCx-1 inducing a turn-on fluorescence response. The sensing of enzymatic reaction by MCx-1 is a rare example of real-time analysis of methyltransferase activity. All commercial assays for methyltransferases and demethylases require the reaction to be stopped before undergoing separate steps to develop signal through a coupled colorimetric/fluorometric reaction,15 isolation and detection of an incorporated radioisotope,16 or by some antibody-mediated binding event.17 Only a few continuous assays that use supramolecular approaches have been reported. In one example, a conventional indicator displacement approach works when using a Cl–-free reaction buffer.4b, 18 In another elegant example, the disagreggation of a micellar aggregate containing cavitand, indicator guest, and lipids is triggered by binding a methylated peptide.2d, 19 Our approach incorporates features of other supramolecular sensing schemes in unique ways. Previously published alternatives to IDA fall broadly into three categories: intramolecular indicator displacement assays (IIDAs)20 and receptor-spacerreporter (RSR) sensors21 involve hosts covalently linked to chromophores; another collection of host-type sensors rely on aggregation-induced emission (AIE) arising in various ways from analyte binding.22 DimerDyes relate structurally to IIDAs, but differ in that the dye is included into a recognition pocket in an assembled, intermolecular complex. This dimer assembly is similar to AIE-based sensors in that a change in supramolecular environment induces a photophysical response. But it differs by involving a programmed dimeric assembly with tight control over chromophore-chromophore interactions instead of extended aggregates, and by producing a turn-on response to sensor disassembly that is

3 Environment ACS Paragon Plus

Journal of the American Chemical Society 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

the opposite of AIE. Unlike RSRs, DimerDyes don’t need the analyte to have any direct influence on the excited state of the fluorophore. This design is general and adaptable, and we anticipate that this mode of disassembly-induced turn-on sensing will operate for many different dimeric species that include a host and an integrated dye in an appropriate architecture. The DimerDyes reported here have an intrinsic tolerance to diverse biological solutions that we will aim to preserve and exploit as we expand the concept to more hosts, dyes, and analytes.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis of novel compounds, NMR/UV-Vis/fluorescence characterization of assembly, enzyme assay optimization and procedure and fluorescence analyte detection (PDF).

AUTHOR INFORMATION Corresponding Author *[email protected].

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank Christopher Barr for establishing the DOSY experiments, Shaun MacLean for assistance with molecular modeling, and the National Science and Engineering Research Council (Discovery Grant #05382) and a Canada Research Chair for support.

REFERENCES (1) (a) Dsouza, R. N.; Pischel, U.; Nau, W. M., Chem. Rev. 2011, 111, 7941-80. (b) You, L.; Zha, D.; Anslyn, E. V., Chem. Rev. 2015, 115, 7840-92. (c) Ghale, G.; Nau, W. M., Acc. Chem. Res. 2014, 47, 21502159. (2) (a) Dsouza, R. N.; Nau, W. M., J. Org. Chem. 2008, 73, 5305-10. (b) Praetorius, A.; Bailey, D. M.; Schwarzlose, T.; Nau, W. M., Org. Lett. 2008, 10, 4089-92. (c) Koh, K. N.; Araki, K.; Ikeda, A.; Otsuka, H.; Shinkai, S., J. Am. Chem. Soc. 1996, 118, 755-758. (d) Liu, Y.; Perez, L.; Mettry, M.; Easley, C. J.; Hooley, R. J.; Zhong, W., J. Am. Chem. Soc. 2016, 138, 10746-9. (3) (a) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V., Acc. Chem. Res. 2001, 34, 963-72. (b) Kumar, V.; Anslyn, E. V., J. Am. Chem. Soc. 2013, 135, 6338-44. (c) Shcherbakova, E. G.; Zhang, B.; Gozem, S.; Minami, T.; Zavalij, P. Y.; Pushina, M.; Isaacs, L. D.; Anzenbacher, P., Jr., J. Am. Chem. Soc. 2017, 139, 14954-14960. (4) (a) Ghale, G.; Lanctôt, A. G.; Kreissl, H. T.; Jacob, M. H.; Weingart, H.; Winterhalter, M.; Nau, W. M., Angew. Chem. Int. Ed. 2014, 53, 27622765. (b) Guo, D.-S.; Uzunova, V. D.; Su, X.; Liu, Y.; Nau, W. M., Chem. Sci. 2011, 2, 1722-1734. (c) Florea, M.; Kudithipudi, S.; Rei, A.; Gonzalez-Alvarez, M. J.; Jeltsch, A.; Nau, W. M., Chem. Eur. J. 2012, 18,

3521-8. (d) Florea, M.; Kudithipudi, S.; Rei, A.; González-Álvarez, M. J.; Jeltsch, A.; Nau, W. M., Chem. Eur. J. 2012, 18, 3521-3528. (5) (a) Legg, K. D.; Hercules, D. M., J. Phys. Chem. 1970, 74, 2114-2118. (b) Lakowicz, J. R.; Principles of Fluorescence Spectroscopy; Springer, New York, NY, 2006. (6) Norouzy, A.; Azizi, Z.; Nau, W. M., Angew. Chem. Int. Ed. 2015, 54, 792-795. (7) (a) Miskolczy, Z.; Biczók, L.; Megyesi, M.; Jablonkai, I., J. Phys. Chem. B 2009, 113, 1645-1651. (b) Zhang, H.-M.; Yang, J.-Y.; Du, L.-M.; Li, C.-F.; Wu, H., Anal. Methods 2011, 3, 1156-1162. (c) Hennig, A.; Bakirci, H.; Nau, W. M., Nat. Methods. 2007, 4, 629-632. (8) (a) Liu, Y.; Li, C.-J.; Guo, D.-S.; Pan, Z.-H.; Li, Z., Supramol. Chem. 2007, 19, 517-523. (b) Ong, W.; Kaifer, A. E., J. Org. Chem. 2004, 69, 1383-1385. (9) Garnett, G. A.; Daze, K. D.; Pena Diaz, J. A.; Fagen, N.; Shaurya, A.; Ma, M. C.; Collins, M. S.; Johnson, D. W.; Zakharov, L. N.; Hof, F., Chem. Commun. 2016, 52, 2768-71. (10) (a) Linn, M. M.; Poncio, D. C.; Machado, V. G., Tetrahedron Lett. 2007, 48, 4547-4551. (b) Miskolczy, Z.; Biczok, L., J. Phys. Chem. B. 2013, 117, 648-53. (11) Shinkai, S.; Araki, K.; Shibata, J.; Tsugawa, D.; Manabe, O., Chem. Lett. 1989, 18, 931-934. (12) (a) Guo, D.-S.; Wang, K.; Liu, Y., J. Inclusion Phenom.Macrocyclic Chem. 2008, 62, 1-21. (b) Lhotak, P.; Nakamura, R.; Shinkai, S., Supramol. Chem. 1997, 8, 333-344. (13) Arora, V.; Chawla, H.; Santra, A., Tetrahedron Lett. 2002, 58, 55915597. (14) (a) Beshara, C. S.; Jones, C. E.; Daze, K. D.; Lilgert, B. J.; Hof, F., ChemBioChem 2010, 11, 63-66. (b) Tabet, S.; Douglas, S. F.; Daze, K. D.; Garnett, G. A. E.; Allen, K. J. H.; Abrioux, E. M. M.; Quon, T. T. H.; Wulff, J. E.; Hof, F. Bioorg. Med. Chem. 2013, 21, 7004. (c) Allen, H. F.; Daze, K. D.; Shimbo, T.; Lai, A.; Musselman, C. A.; Sims, J. K.; Wade, P. A.; Hof, F.; Kutateladze, T. G. Biochem. J. 2014, 459, 505. (15) Koh-Stenta, X.; Joy, J.; Poulsen, A.; Li, R.; Tan, Y.; Shim, Y.; Min, J.-H.; Wu, L.; Ngo, A.; Peng, J., Biochem. J 2014, 461, 323-334. (16) Eram, M. S.; Bustos, S. P.; Lima-Fernandes, E.; Siarheyeva, A.; Senisterra, G.; Hajian, T.; Chau, I.; Duan, S.; Wu, H.; Dombrovski, L., J. Biol. Chem. 2014, 289, 12177-12188. (17) Jacob, Y.; Voigt, P. Plant Chromatin Dynamics: Methods and Protocols, Humana Press, New York, NY, 2018. (18) Dsouza, R. N.; Hennig, A.; Nau, W. M., Chem. Eur. J. 2012, 18, 3444-59. (19) Liu, Y.; Perez, L.; Gill, A. D.; Mettry, M.; Li, L.; Wang, Y.; Hooley R.; Zhong, W. J. Am. Chem. Soc. 2017, 139, 10964–10967. (20) Minami, T.; Liu, Y.; Akdeniz, A.; Koutnik, P.; Esipenko, N. A.; Nishiyabu, R.; Kubo, Y.; Anzenbacher, P., Jr., J. Am. Chem. Soc. 2014, 136, 11396-401. (21) Liu, Y.; Minami, T.; Nishiyabu, R.; Wang, Z.; Anzenbacher, P., J. Am. Chem. Soc. 2013, 135, 7705-7712. (22) (a) Chapin, B. M.; Metola, P.; Vankayala, S. L.; Woodcock, H. L.; Mooibroek, T. J.; Lynch, V. M.; Larkin, J. D.; Anslyn, E. V., J. Am. Chem. Soc. 2017, 139, 5568-5578. (b) Watt, M. M.; Engle, J. M.; Fairley, K. C.; Robitshek, T. E.; Haley, M. M.; Johnson, D. W., Org. Biomol. Chem 2015, 13, 4266-4270. (c) Wang, J.; Gu, X.; Zhang, P.; Huang, X.; Zheng, X.; Chen, M.; Feng, H.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z., J. Am. Chem. Soc. 2017, 139, 16974-16979.

Graphic entry for the Table of Contents (TOC)

4 Environment ACS Paragon Plus

Page 4 of 4