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Micelles Protect Intact Metallo-supramolecular Block Copolymer Complexes from Solution to Gas Phase during Electrospray Ionization Kai Hung Huang, Tsung-Han Tu, Shi-Cheng Wang, Yi-Tsu Chan, and Cheng-Chih Hsu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01576 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Analytical Chemistry

Micelles Protect Intact Metallo-supramolecular Block Copolymer Complexes from Solution to Gas Phase during Electrospray Ionization Kai‐Hung Huang, Tsung‐Han Tu, Shi‐Cheng Wang, Yi‐Tsu Chan,* and Cheng‐Chih Hsu* Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan Supporting Information Placeholder ABSTRACT: Supramolecular diblock copolymers using metal‐ligand coordination can be synthesized under ambient conditions by delicate design of the end groups of the homopolymer chains. However, mass spectrometric analysis of such metallo‐supramo‐ lecular copolymers is challenging. One of the reasons is the nonpolarity of the polymer chains, making it hard to disperse the complexes in electrospray ionization (ESI)‐friendly environments. The other difficulty is the noncovalent nature of such copoly‐ mers, which is easily disrupted during the ionization. Here, we demonstrate that the intact metallo‐supramolecular diblock co‐ polymers can be maintained sufficiently during the ESI process in aqueous solution within micelles. The high resolution mass spectrometric evidence revealed that the surfactant molecules effectively protect the noncovalent binding of the complexes into gaseous ions. Intriguingly, surfactant molecules were sufficiently detached away from the copolymer complexes, giving unambig‐ uous mass spectra which were predominated by intact diblock copolymers. This ESI‐based approach allowed us to investigate the relative bond strengths of metal‐to‐ligand complexation using collision‐induced dissociation (CID) in the ion trap mass spectrom‐ etry. Conformational features and collision cross‐sections of the copolymers were thus obtained using subsequent ion mobility spectrometry mass spectrometry (IMS‐MS). Remarkable environment‐dependent conformations of the denoted diblock copoly‐ mers were found using this mass spectrometric platform.

Rod‐coil diblock copolymers, comprising rigid macro‐ molecules covalently connected with flexible counterparts, have drawn tremendous research interest because of their unique self‐assembled morphologies both in solution and in the solid state.1‐4 In the midst of various rod segments,5 poly(3‐hexylthiophene) (P3HT) has aroused particular at‐ tention due to its wide applicability,6 but tedious synthetic procedures have impeded development of diversified pol‐ ymer architectures. In contrast to irreversible covalent binding, supramolecular polymerization and coupling methods through reversible noncovalent interactions such as metal−coordination bonds,7‐15 aromatic interactions,16‐19 host−guest interactions,20‐23 and hydrogen bonding,24‐28 not only offer a facile access to preparation of sophisticated to‐ pologies, but also endow the materials with dynamic func‐ tions.29‐33 Nevertheless, the structural characterization of supramolecular polymers by size exclusion chromatog‐ raphy (SEC) or mass spectrometry (MS) commonly en‐ counters difficulties because the weak interactions are un‐ sustainable in such harsh analytical environments.13, 34 Hence, developing new characterization techniques for molecular weight measurements of intact supramolecular polymers is still an urgent and challenging task. Mass spectrometric interrogation of supramolecular pol‐ ymers containing such noncovalent interactions provides not only structural but also dynamic information,35,36 which is essential to the rational design and synthesis of

macromolecules.37 Electrospray ionization (ESI) coupled with high resolution MS has become a powerful method to the study of noncovalent biomacromolecular com‐ plexes,38,39 including protein‐protein,40‐43 protein‐metal ion,44,45 protein‐lipids,46‐47 and protein‐nucleotide48‐50 in‐ teractions in their native environments. However, mass spectrometric analysis of supramolecular hydrophobic or amphiphilic copolymers remains challenging in several as‐ pects. First, these supramolecular copolymers containing hydrophobic blocks are not readily dispersed in regular ESI conditions, e.g. 50% methanol aqueous solution. Second, the other ionization methods amenable to nonpolar com‐ pounds are too harsh to retain their noncovalent nature. Third, control over supramolecular conformation to a con‐ fined morphology from the native environment at con‐ dense phases into gas phase during ionization is difficult. The abovementioned aspects put a limit on the usefulness of mass spectrometry away from the structural characteri‐ zation of environment‐sensitive multi‐functional copoly‐ mers. Furthermore, it is not hard to imagine mass spec‐ trometry‐based methodology that could probe the supra‐ molecular chemistry at heterogeneous ambience, e.g., wa‐ ter‐lipids bilayer interface, will be of great potential in ap‐ plication.51‐52 Recently, Robinson group reported a series of mass spec‐ trometry platforms, that by applying micelle aqueous solu‐ tion of membrane protein complexes to regular ESI mass

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spectrometry, the subunit stoichiometry, structural infor‐ mation and interaction networks of hydrophobic macro‐ molecules with small molecules/lipid and their assemblies are thus determined in well‐defined morphological states.47,53‐59 Intriguingly, such methodology has not been applied to the structural characterization of supramolecu‐ lar polymers, motivating us to implement similar ap‐ proaches to a series of amphiphilic metallo‐supramolecu‐ lar PEO‐b‐P3HT diblock copolymers bearing Zn2+ and Cd2+ ions.10 These copolymers were originally designed by an‐ choring complementary terpyridine (tpy) ligand pairs to the polymer chain ends such that the spontaneous key‐ lock complexations take place in the presence of metal ions at room temperature in CHCl3/MeOH mixed solvents. Re‐ markably, in this study, the exact mass of the intact diblock copolymers embedded in nonionic micelles dispersed in water were clearly resolved by ESI coupled with high reso‐ lution mass spectrometers. This led us to the subsequent tandem mass spectrometric analysis (MS/MS) and ion mo‐ bility spectrometry mass spectrometry (IMS‐MS). More specifically, the relative metal‐ligand bond strengths of Zn2+/Cd2+ polymeric complexes were determined using col‐ lision‐induced dissociation (CID), and their collision cross‐ sections (CCSs) were given by the drift times obtained with IMS‐MS. Details of the sample preparation, instrumental settings, mass spectrometric characterization, and inter‐ pretations, are elaborated in the following sections. ■ MATERIALS AND METHODS Materials. Five different metallo‐supramolecular PEO‐ b‐P3HT diblock copolymers, including PEO1‐Zn2+‐P3HT1, PEO2‐Zn2+‐P3HT1, PEO1‐Cd2+‐P3HT1, PEO2‐Cd2+‐P3HT1, and PEO1‐Cd2+‐P3HT2, were constructed using the same strategy described in the literature (Scheme 1).10 The num‐ ber‐average degree of polymerization (m and n in Scheme 1) of the terpyridine‐functionalized homopolymers was es‐ timated based on 1H NMR spectroscopy. Details of the syn‐ theses and 1H NMR analyses were elaborated in the Sup‐ porting Information (Figure S1‐S15). To minimize the influ‐ ence to our mass spectrometers, the non‐ionic surfactant, decyl β‐D‐maltopyranoside (DM), was used in this study. DM was purchased from Sigma‐Aldrich and used to pre‐ pare the micelle aqueous solution. The critical micelle con‐ centration (CMC) in water is 0.087% (1.6 mM). For decom‐ plexation experiments, a MeOH solution (50 μL, 50 mg/mL) of tetrakis(triethyammonium) EDTA was added into 1 mL of PEO1‐Zn2+‐P3HT1 (1.5 mg/mL) in a DM solution (1.5x CMC) and CHCl3/MeOH (1/1, v/v), respectively. Tetrakis(triethylammonium) EDTA was synthesized fol‐ lowing the reported protocol.60 Sample Preparation. DM (54.6 mg, 0.113 mmol) was dissolved in water (20.14 g) to yield 0.270% DM solution (3.11x CMC), and 10 mL of 0.270% DM solution was mixed with 10 mL of water to yield 20 mL of 0.135% DM aqueous solution (1.55x CMC).

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In the mass spectrometric measurements using ESI, 0.135% DM solution was used to dissolve PEO1‐Zn2+‐ P3HT1. In the concentration‐dependent experiments, pure water, 0.30x CMC (192.5 mg of 1.55x CMC DM solution mixed with 0.79 mL of water), 0.77x CMC (488.6 mg of 1.55x CMC DM solution mixed with 0.48 mL of water), and 1.55x CMC DM solutions were prepared and used to dis‐ solve PEO1‐Zn2+‐P3HT1 (Figure S16). PEO1‐Zn2+‐P3HT1, PEO1‐Cd2+‐P3HT1, PEO2‐Zn2+‐P3HT1, PEO2‐Cd2+‐P3HT1, and PEO1‐Cd2+‐P3HT2 were dissolved in 1.55x CMC DM solution for subsequent ESI‐MS and IMS‐MS analyses. To make sure that the copolymers were fully dispersed and embedded in micelles, all the DM solutions containing copolymers were subjected to ultrasonicator (QSonica Q125). Dried powder of the copolymers and aqueous DM solutions were mixed in a 1.5 mL Eppendorf tube, and the mixture was sonicated using QSonica Q125 probe sonicator (sonication time : pause time = 10 sec : 2 sec) for 45 minutes at 65% full power (125W) under an ice bath. Mass Spectrometry Analysis. A homemade ESI source (Figure S17) was used to gener‐ ate the ions for subsequent high resolution mass spectro‐ metric measurements. The copolymers in DM solutions were injected via a fused silica capillary continuously pumped with a flowrate of 5 　L/min. The size of the ca‐ pillary for the source was 250 μm/350 μm (I.D./O.D.). A high voltage was applied to the capillary via an alligator clip onto the needle of the syringe. The distance between the capillary and the ion inlet of the mass spectrometer was kept at about 1‐2 cm. Pressure of the nebulizing gas (nitro‐ gen) was set at 120 psi throughout the experiment. Details of MALDI‐TOF and high resolution mass spec‐ trometry, IMS‐MS, and molecular modeling are described in the Supporting Information. Details of data processing are described in the Supporting Information and Figure S18. Scheme 1. Construction of Metallo‐supramolecular Diblock Copolymers

■ RESULTS AND DISCUSSION As Matrix‐assisted laser desorption ionization time‐of‐ flight mass spectrometry (MALDI‐TOF MS) has been pro‐ foundly used to determine the molecular weights of mac‐ romolecules,68‐72 we first subjected the metallo‐supramo‐ lecular copolymers to the common MALDI‐TOF MS inter‐ rogation.


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Figure 1. Mass spectra of PEO1‐Zn2+‐P3HT1 obtained using different ionization methods. Mass spectrum of pure DM in aqueous solution (b) was shown as the blank (MS annotation in Table S1). Mass spectra of PEO1‐Zn2+‐P3HT1 in DM aqueous solution (1.55x CMC) using (a) MALDI‐TOF MS and (c) ESI‐orbitrap mass spectrometers reveal that the micelle protects the metal‐ligand coor‐ dination during ESI. Annotations of intact PEO1‐Zn2+‐P3HT1 in the range of m/z 1745‐1985 are exhibited in (d), where copolymers with the same number of P3HT monomers are shown as the same colors. A 　m/z of ~22 (z=2) between each pair of isotope clusters represents the mass of PEO monomer (~44 Da). The annotation of each peak in (c) is shown in Tables S2‐S4.

Using PEO1‐Zn2+‐P3HT1 as a paradigm, Figure 1a re‐ vealed two groups of polymeric species at m/z around 1600 to 2800 in the MALDI‐TOF mass spectrum. Based on the

prior knowledge to the materials used for the synthesis in this study, the mass of PEO1‐Zn2+‐P3HT1 is expected to be about 3000 to 5000 Dalton (Da), rendering a distribution



of ions at m/z about 1500 to 2500, with charges equal to 2 and consecutive polymeric peaks of Δm/z equal to 22 or 84 between adjacent ion species. However, these charac‐ teristic patterns were not observed in the MALDI‐TOF MS analysis with all experimental settings, even when the laser excitation power was tuned to the minimal value at which level a distinguishable ion signal could be detected. Instead of the intact molecular ions, dissociated homopolymers ‧ were largely found in forms of [PEO+Na]+ and [P3HT] + ions, indicating that the metal‐to‐ligand coordinative in‐ teraction was not retained upon laser desorption/ioniza‐ tion. This was not surprising. Even though that MALDI is usually categorized as “soft ionization,” direct detection of noncovalent assemblies is often challenging without care‐ fully optimized instrumental parameters or chemical cross‐linking. 73,74 Figure 1c shows the high resolution ion spectrum of di‐ block PEO1‐Zn2+‐P3HT1 dissolved in DM micellar aqueous solution using the homemade ESI source. In contrast to the MALDI‐TOF results, a much more informative ion spec‐ trum was observed at the range of m/z 1600‐2400. In order to assign the peaks of copolymer ions, a background spec‐ trum was acquired using a blank micellar aqueous solution (Figure 1b). The characteristic 241.14 m/z shift (z=2) of the serial peaks at m/z 1600‐2800 unequivocally indicates that the oligo‐DM surfactant ions (heptamer to undecamer with Ca2+ adduct) were dominant in the spectrum. Such feature was also observed in the solution of PEO1‐Zn2+‐ P3HT1, whereas the surfactant ions were Zn2+‐bound in‐ stead (Figure 1c). We then made attempts to annotate the rest of the doubly charged ions contributing to the major‐ ity of the spectrum in the m/z range. As shown in Figure 1d, a series of intact diblock PEO‐Zn2+‐P3HT copolymers with different chain lengths were resolved using high res‐ olution mass spectrometry. Increases in the number of pol‐ ymerized monomers were reflected as the characteristic 　m/z of 22.013 (PEO monomer, 44.026 Da) and 83.041 (P3HT monomer, 166.082 Da) between each pair of iso‐ topic clusters. The assignments of the intact copolymers are unambiguous, considering the observed exact masses and isotope patterns for most of the ion clusters are highly consistent with the theoretical predictions (Figure S19). For example, the molecular weight of PEO(12)‐Zn2+‐ P3HT(15) (shown in the inset of Figure 1c) obtained in our experiments (4083.8568 Da) possesses less than 1‐ppm de‐ viation opposed to the calculated value (4083.8584 Da), and its isotope abundances match undoubtedly with the simulated levels (Figure S20). Orbitrap mass spectrometer provides us an ability to resolve these supramolecular ions in a greater detail, enabling us to identify most of the ion species at the denoted m/z range. More importantly, we can thus conclude that the non‐ covalent nature of the diblock PEO1‐Zn2+‐P3HT1 survived during the ESI process in this platform. The sharp contrast between the mass spectral behaviors of the DM‐

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incorporated PEO1‐Zn2+‐P3HT1 complex in ESI MS from that in MALDI‐TOF MS emphasized the importance of mi‐ celle involvement in protecting the noncovalent interac‐ tions from harsh environmental stress during ionization. To gain more insight into the role of micelles, we investi‐ gated how the mass spectral behavior of PEO1‐Zn2+‐P3HT1 evolves with the concentration of DM. As shown in Figure 2, only negligible copolymer ions were observed in solu‐ tions without micelles, including those surfactant concen‐ trations made below or close to CMC. Most decisively, the ion intensity of oligo‐DM increased proportionally from 0.77x to 1.55x CMC, whereas that of the intact PEO‐Zn2+‐ P3HT enhanced for more than 2 order‐of‐magnitude. It is obvious that the formation of micelles is crucial to ions of intact PEO1‐Zn2+‐P3HT1 in ESI MS.

Figure 2. DM concentration‐dependent ESI‐Orbitrap high resolution mass spectra of PEO1‐Zn2+‐P3HT1 revealing a stark improvement to the intensity of intact diblock copolymers when micelles are generated. ESI‐MS spectra of (a) pure water, (b) 0.30x, (c) 0.77x, and (d) 1.55x CMC DM solutions with PEO1‐Zn2+‐P3HT1 after ultrasonication. (e) Intensity of PEO(11)‐Zn2+‐P3HT(13) at m/z 1855.850.

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The above‐mentioned finding was serendipitous but not surprising. In fact, the use of micelles has been well‐docu‐ mented for mass spectrometric analysis of hydrophobic protein assemblies, e.g. membrane proteins.47, 53,55‐56,59,61 It is noticeable that for membrane proteins in these reports, surfactant molecules were bundled with the analyte ions into the mass spectrometers. Thus, a subsequent imple‐ mentation of collision‐induced dissociation (CID) was re‐ quired to remove the surfactants from the target com‐ pounds. Without the prerequisite CID enhancement, the spectra would barely be interpretable. This behavior was in stark contrast to what was observed in our case, in which PEO1‐Zn2+‐P3HT1 complex was fully desorbed from sur‐ rounding DM layers during ESI before it reached mass spectrometer, enabling us to resolve the intact copolymer complex ions easily without spectral deconvolution. To ra‐ tionalize the above results, we postulated that the micelle may eventually erupt into smaller spheres containing only few to a dozen of DM in the electrostatic microdroplets. When the size of ESI microdroplets keeps shrinking, the micelle becomes more and more exposed to the atmos‐ phere (in the most extreme scenario, it turns into a micel‐ lar aerosol), making individual DM more prone to ther‐ mally evaporate, as the stabilizing force in aqueous envi‐ ronment that drives micelle formation disappears. Bearing the hypothesized mechanism in mind, we then continued to see if the ambient temperature is decisive to the formation of free PEO1‐Zn2+‐P3HT1 ions. Figure S21 demonstrates that the ion intensity increased significantly when the temperature of the ion inlet rose above 150 oC. Such temperature‐dependency suggests that a thermally‐ activated detachment of surfactants took place during the ESI of the DM‐incorporated copolymer and it has a consid‐ erable effect in facilitating the ion complexes escaping from DM micelles. In addition, the ambient gas molecules at higher inlet temperature also possess higher kinetic en‐ ergy and collision frequency, assisting desorption of DM from the micelle. To explore a wider usefulness of DM micelles in ESI MS analysis to metallo‐supramolecular diblock copolymers, a series of PEO‐b‐P3HT copolymers with different metal centers and various block chain lengths were investigated. These copolymers with heteroleptic bis(terpyridine) com‐ plexes, including (a) PEO1‐Zn2+‐P3HT1, (b) PEO2‐Zn2+‐ P3HT1, (c) PEO1‐Cd2+‐P3HT1, (d) PEO2‐Cd2+‐P3HT1, and (e) PEO1‐Cd2+‐P3HT2, were originally constructed using the predesigned complementary ligand pairs.10 As shown in Figure 3, doubly charged intact diblock copolymers (a‐ d) were repeatedly discovered at the denoted m/z range. These copolymers of the same P3HT block chain exhibited similar spectral profiles, except that copolymers with longer PEO chains (b and d) showed an ~200 m/z shift of the entire ion packets, revealing the contribution from the additional ten PEO monomers to their molecular weights. However for the copolymer with longer P3HT chains (e), a

much more complicated spectrum was observed as shown in Figure S22. Interestingly, quadruply charged ions were largely found in the m/z range of 2500 to 3000, implying that the dimer [PEO1‐Cd2+‐P3HT2]2 was measured. This was further evidenced by the presence of doubly charged intact PEO1‐Cd2+‐P3HT2 copolymer when a narrower m/z window was preselected by the ion trap before high reso‐ lution MS measurements (Figure S22b). This was not sur‐ prising as dimerization of hydrophobic compounds was commonly found in ESI MS.38,62 Details of the peak assign‐ ments are shown in the supporting information. The high resolving power of Orbitrap allowed us to specify the dis‐ tinct isotope distribution of Zn2+ and Cd2+ copolymers.

Figure 3. High resolution mass spectra of diblock copolymers (a) PEO1‐Zn2+‐P3HT1, (b) PEO2‐Zn2+‐P3HT1, (c) PEO1‐Cd2+‐ P3HT1, and (d) PEO2‐Cd2+‐P3HT1 in micellar environment using ESI‐Orbitrap mass spectrometer. Insets: zoomed‐in spectra of each complex; isotopic clusters of the same ion spe‐ cies are shown in the same colors. Details of peak m/z were shown in Tables S5‐S12.

Tandem Mass Spectrometric Analysis. Encouraged by these results, we proceeded to perform the MS/MS analysis of these copolymers to further validate

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our peak assignments using CID. Figure 4 shows the MS/MS spectra of the representative diblock copolymers (a‐d). In general, the terpyridine‐modified P3HT radical ‧ cations (for example, m/z 2137, [P3HT(11)] +, the exact number of monomer is denoted in parenthesis and the fol‐ lowings) were commonly found, presumably due to the loss of a valence electron from the electron‐rich P3HT.63 In addition to the free P3HT ions, the other free ions, such as ‧ PEO‐M2+ and their fragmented radical cations ([PEO] + ‧+ and [PEO‐M] in Figure 4), were largely found, indicating that decomplexation of the metal‐to‐ligand coordination occurred upon CID. Notably, in contrast to P3HT ions, metal ion‐bound terpyridine‐PEO fragments were con‐ stantly found. This result is in agreement with the fact of the stronger binding affinity of the functional PEO end‐ capped with 6,6’’‐anthracenyl‐substituted tpys to the metal ions, possibly resulting from the additional cation‐π interactions. The MS/MS spectrometry using CID not only helps us to verify our structural identification but also provided us an ability to gain more insights into the binding strengths of the metal‐ligand coordination. As shown in Figure S23, a considerable amount of fragmented terpyridine‐PEO ions were bound with Zn2+ at ~1000 m/z, whereas none of those species were observed in copolymers with Cd2+, suggesting that the coordination bond of Cd2+ is not as stable as that of Zn2+ to survive the collisions. In this contribution, we meticulously mapped the frag‐ mentation profiles against the CID energy as demonstrated in Figure 5. Specifically, intensities of ion species associ‐ ated with the decomplexation between PEO/P3HT end‐ capped ligands and center metal ions were probed as a function of the normalized CID energy. In general, as sup‐ ported by the fragmentation profiles in Figure 5, a precur‐ sor‐successor relation holds firmly for parent and daughter ions in both Zn2+ and Cd2+ copolymer complexes. However, a higher normalized CID energy is required to dissociate the homopolymer from the metal center in PEO1‐Zn2+‐ P3HT1 than that in PEO1‐Cd2+‐P3HT1. Moreover, the in‐ ‧ tensity of Cd2+‐bound ion ([PEO(13)‐Cd] +) was found to decrease when normalized CID energy kept turned on above 40% where the parent PEO(13)‐Cd2+‐P3HT(11) com‐ plex ion has vanished completely (Figure 5c), while the ‧ Zn2+‐bound product ([PEO(13)‐Zn] +) levels stayed un‐ changed (Figure 5a). These results indicate that for the co‐ polymers bearing about 10 to 20 PEO monomers (PEO1), Zn2+ metal center possesses a more robust coordination than Cd2+ does, which are consistent with the slightly larger binding constant for Zn2+ ions.64 On the other hand, such phenomenon was not observed in the copolymers with longer PEO chains, e.g. PEO2 (Figures 5b and 5d). This may be rationalized by the fact that a greater steric hindrance in the larger copolymer complexes, leading the energy obtained from the collision of gas molecules in CID experiments accumulated onto the long block chains and

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redistributed into vibrationally excited manifolds, rather than a direct breakdown at the metal centers. Thus a less center metal‐dependent CID character was observed. The aforementioned statement was supported by a greater amount of intra‐ligand fragment products (e.g. [PEO*]+, PEO**, and PEO**‐Zn) in PEO(26)‐Zn2+‐P3HT(11) com‐ plexes (Figure S23).

Figure 4. MS/MS spectra of (a) PEO(13)‐Zn2+‐P3HT(11) (m/z 1733.789), (b) PEO(26)‐Zn2+‐P3HT(11) (m/z 2019.949), (c) PEO(13)‐Cd2+‐P3HT(11) (m/z 1757.264), (d) PEO(26)‐Cd2+‐ P3HT(11) (m/z 2043.952) measured with normalized CID en‐ ergy 45%, and (e) PEO(17)‐Cd2+‐P3HT(29) (m/z 2588.672) with normalized CID energy 40%. Details of intra‐ligand fragment products (highlighted in yellow) are listed in the Supporting Information Figure S23.

Ion Mobility Mass Spectrometry This ESI‐compatible approach allows us to probe the na‐ tive structures of diblock copolymer complexes from a mi‐ cellar environment into gas phase using IMS‐MS. Figure 6 shows the IMS‐MS spectra of copolymers a‐d. The intact copolymer ions of various block chain lengths constitute a staggered formation laddering up to the right, indicating that the CCS of the copolymer complex ions is proportional to their molecular weights. Each species of different m/z was separated from the others into distinct ion packets and could be explicitly annotated (Figure S24 and S25). Upon monitoring at every single diblock copolymer chain one at

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whereas the CCS of PEO(17)‐Zn2+‐P3HT(10) (M.W. 3473.5799 Da) raises to 627.9 Å2 (Table S17). This result im‐ plies that the rigid rod‐like P3HT block may adopt a more extended conformation in comparison to the flexible coil‐ like PEO segment for PEO‐Zn2+‐P3HT embedded in mi‐ celles. The experimental result was also compared with MD simulation (Figure S27). The calculated CCSs of PEO(13)‐Zn2+‐P3HT(11) ranged from 500 to 830 Å2, with an average value of 654.7 Å2, which agrees well with the exper‐ imental one. To gain further insight into whether this conformational feature is unique to micellar environment, we performed a separated IMS‐MS study of diblock PEO1‐Zn2+‐P3HT1 dis‐ solved in a DM‐free MeOH/CHCl3 (1/1, v/v) system. Intri‐ guingly, in addition to the drift time distribution (DTD) originally found in diblock copolymers embedded in mi‐ celles, i.e., the drift time peak maximum at 14.47 ms (CCS 641.2 Å2) for PEO(13)‐Zn2+‐P3HT(11), a minor DTD with the peak top at 13.24 ms (CCS 606.0 Å2) was found for the co‐ polymer suspended in the organic solvent (Figures 7 and S26).

a time, the drift time of each ion species was revealed in the subsequent extracted ion chromatograms (Figures 6 and S26), allowing us to determine the CCSs of intact co‐ polymer ions individually as shown in Table 1. In this re‐ gard, the contributions of every additional monomer to the CCSs for each complex species were solicitously calculated and are exhibited in the Supporting Information.

Figure 5. MS/MS fragmentation plots of (a) PEO(13)‐Zn2+‐ P3HT(11), (b) PEO(26)‐Zn2+‐P3HT(11), (c) PEO(13)‐Cd2+‐ P3HT(11), and (d) PEO(26)‐Cd2+‐P3HT(11). The proposed structures and the corresponding exact masses of each frag‐ menting products are shown in Tables S13‐S16.

Notably, we found a nonequivalent contribution from PEO and P3HT block chains. For example, the CCS raises from 609.2 Å2 (PEO(13)‐Zn2+‐P3HT(10), M.W. 3297.4750 Da) to 635.9 Å2 (PEO(13)‐Zn2+‐P3HT(11), M.W. 3463.5566 Da),

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Figure 7. Ion mobility spectrometry analysis of PEO(13)‐Zn2+‐ P3HT(11) in micelle aqueous solution (red dots) and MeOH/CHCl3 (blue dots) and their Gaussian fittings (red and blue lines).

Figure 6. IMS‐MS analysis of (a) PEO1‐Zn2+‐P3HT1, (b) PEO2‐ Zn2+‐P3HT1, (c) PEO1‐Cd2+‐P3HT1, and (d) PEO2‐Cd2+‐ P3HT1 at m/z 1600‐2500 and drift time 12‐18 ms. The mass spectra and chromatograms of the denoted copolymer species in Table 1 are shown on the right panel. The chromatogram were fitted by Gaussian normal distribution (red) from the ex‐ perimental data (black dots).

This suggested that a more compact conformation (Fig‐ ure S27) of the copolymer exists in MeOH/CHCl3, while disappears in the hydrophobic micellar environment, pre‐ sumably due to a further P3HT chain folding induced by the poor solvent MeOH. Moreover, the most striking fea‐ ture is that the bandwidths for both DTDs were notably larger than what was obtained in micelles (see Figures 7, S25 and S26). This unprecedented phenomenon might be reflected to the limited degrees of freedom of copolymers in micelles, rendering a well‐defined conformation during ESI.

Table 1. Cross‐sections of denoted PEO‐M2+‐P3HT copolymers drift CCS species m/z time (Å2) (ms) PEO(13)‐ 1733.2528 14.28 635.9 Zn2+‐P3HT(11) PEO(26)‐ 2019.9689 15.69 674.2 Zn2+‐P3HT(11) PEO(13)‐ 1757.2813 14.32 636.8 Cd2+‐P3HT(11) PEO(26)‐ 2043.6484 16.10 685.3 Cd2+‐P3HT(11) Bearing this mechanism in mind, we then made at‐ tempts to gain an in‐depth insight into the behavioral dif‐ ferences of PEO2‐Zn2+‐P3HT1 in organic solvent (MeOH/CHCl3) opposed to in the micellar environment by investigating the dynamical changes of its decomplexation induced by EDTA (Figures S28‐32). After adding EDTA for 24 hours, a great deal of precipitation was observed in the organic solution, and the corresponding mass spectrum of the supernatant was dominant with free PEO2 ligands (Figure S28b). As what has been discussed previously,10 this was unambiguously explained by the decomplexation of the copolymer complex due to the stronger competition from EDTA. However, the major species were still intact PEO2‐Zn2+‐P3HT1 in micelle aqueous solution after EDTA treatment (Figure S28c), with less than 10% of the polymer ligands were dissociated (Table S18). This was also evi‐ denced from the absence of precipitation in micelle solu‐ tion. Moreover, if the micelle‐protected copolymers were

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subjected to ultrasonication, a slight precipitation was ob‐ served (Figure S30), whereas the extent of decomplexation remained below 15% (Table S18), implying that the metal center was mostly immersed into the DM layer. Obviously, the micelle worked as a vesicular shell to carry the metallo‐supramolecular copolymers away from the EDTA chelation. Most importantly, had it not been for the unconventional ESI approach involving the use of sur‐ factant, the stoichiometry, stability, and conformational characters of the multi‐functional supramolecules in het‐ erogeneous systems would be difficult to measure. In spite of that micelle‐protected ESI‐MS has been used to investi‐ gate the noncovalent interactions of hydrophobic mem‐ brane proteins,47, 53‐59 we believe that this work is the first comprehensive exploration of the effectiveness to am‐ phiphilic supramolecules, which incorporates tandem mass spectrometric analysis, ion mobility spectrometry, and theoretical calculation. Moreover, the environment‐ dependent IMS‐MS behavior of metallo‐supramolecular diblock copolymers was found for the first time. ■ CONCLUSIONS Herein, using a series of newly designed PEO‐Zn2+‐P3HT and PEO‐Cd2+‐P3HT diblock copolymers as the paradigm, we demonstrated that noncovalent nature of metallo‐su‐ pramolecular copolymers could be successfully preserved during ESI via a simple micellar protection. This ESI‐ friendly approach allowed subsequent MS analysis, includ‐ ing MS/MS analysis and IMS‐MS. Intriguingly, the surfac‐ tant molecules were substantially desorbed from the ion complex prior to the mass spectrometric interrogation, dis‐ regarding the use of CID.47, 53‐59 Coupling with high resolu‐ tion orbitrap mass spectrometer, our result unambigu‐ ously revealed that the abovementioned diblock copoly‐ mers kept predominantly at intact molecular states in gas phase. Relative metal‐ligand bond strengths among differ‐ ent copolymer species were thus sketched by probing the product fragments upon CID. Moreover, CCSs of individ‐ ual copolymers could be determined by IMS‐MS, allowing us to probe the environment‐sensitive conformation of the metallo‐supramolecular copolymers. In summary, micelle plays a crucial role in protecting labile copolymers from harsh environments, rendering a well‐defined confor‐ mation which is the key to the study of multi‐functional supramolecular macromolecules. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected]

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was supported by Ministry of Science and Technology (MOST), R.O.C. (Grant #: MOST105‐2113‐M‐ 002‐004‐MY2, MOST105‐2923‐M‐002‐008‐MY2, MOST 106‐2113‐M‐002 ‐013‐MY2, and MOST106‐2628‐M‐002‐007‐ MY3) and Center for Emerging Materials and Advanced Devices, National Taiwan University (Grant #: NTU‐ERP‐ 106R880211, 106R880219, and 106R880204). REFERENCES 1. Lee, M.; Cho, B. K.; Zin, W. C., Chem. Rev. 2001, 101, 3869‐92. 2. Olsen, B. D.; Segalman, R. A., Mat. Sci. Eng. R. 2008, 62, 37‐66. 3. Hadjichristidis, N.; Hirao, A.; Tezuka, Y.; Du Prez, F., Complex macromolecular architectures: synthesis, char‐ acterization, and self‐assembly. John Wiley & Sons: 2011. 4. Zhang, J.; Chen, X. F.; Wei, H. B.; Wan, X. H., Chem. Soc. Rev. 2013, 42, 9127‐9154. 5. Liu, C.‐L.; Lin, C.‐H.; Kuo, C.‐C.; Lin, S.‐T.; Chen, W.‐C., Prog. Polym. Sci. 2011, 36, 603‐637. 6. Ludwigs, S., P3ht Revisited‐from Molecular Scale to Solar Cell Devices. Springer: 2015. 7. Kumpfer, J. R.; Jin, J. Z.; Rowan, S. J., J. Mater. Chem. 2010, 20, 145‐151. 8. Burnworth, M.; Tang, L. M.; Kumpfer, J. R.; Dun‐ can, A. J.; Beyer, F. L.; Fiore, G. L.; Rowan, S. J.; Weder, C., Nature 2011, 472, 334‐U230. 9. Kumpfer, J. R.; Rowan, S. J., J. Am. Chem. Soc. 2011, 133, 12866‐12874. 10. He, Y. J.; Tu, T. H.; Su, M. K.; Yang, C. W.; Kong, K. V.; Chan, Y. T., J. Am. Chem. Soc. 2017, 139, 4218‐4224. 11. Kelch, S.; Rehahn, M., Macromolecules 1998, 31, 4102‐4106. 12. Yount, W. C.; Loveless, D. M.; Craig, S. L., J. Am. Chem. Soc. 2005, 127, 14488‐14496. 13. Winter, A.; Schubert, U. S., Chem. Soc. Rev. 2016, 45, 5311‐5357. 14. Burnworth, M.; Knapton, D.; Rowan, S. J.; Weder, C., J. Inorg. Organomet. Polym. Mater. 2007, 17, 91‐103. 15. Moughton, A. O.; O’Reilly, R. K., J. Am. Chem. Soc. 2008, 130, 8714‐8725. 16. Burattini, S.; Greenland, B. W.; Merino, D. H.; Weng, W. G.; Seppala, J.; Colquhoun, H. M.; Hayes, W.; Mackay, M. E.; Hamley, I. W.; Rowan, S. J., J. Am. Chem. Soc. 2010, 132, 12051‐12058. 17. Burattini, S.; Greenland, B. W.; Hayes, W.; Mackay, M. E.; Rowan, S. J.; Colquhoun, H. M., Chem. Mater. 2011, 23, 6‐8. 18. Tayi, A. S.; Shveyd, A. K.; Sue, A. C. H.; Szarko, J. M.; Rolczynski, B. S.; Cao, D.; Kennedy, T. J.; Sarjeant, A.

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