Mapping the Compaction of Discrete Polymer Chains by Size

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

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Mapping the Compaction of Discrete Polymer Chains by Size Exclusion Chromatography Coupled to High-Resolution Mass Spectrometry Tobias Nitsche,† Jan Steinkoenig,∥ Kevin De Bruycker,† Fabian R. Bloesser,† Stephen J. Blanksby,‡ James P. Blinco,*,† and Christopher Barner-Kowollik*,†,§

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School of Chemistry, Physics and Mechanical Engineering, and ‡Central Analytical Research Facility, Institute for Future Environments, Queensland University of Technology (QUT), 2 George Street, Brisbane, QLD 4000, Australia § Macromolecular Architectures, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstrasse 18, 76131 Karlsruhe, Germany ∥ Department of Organic and Macromolecular Chemistry, Polymer Chemistry Research Group, Center of Macromolecular Chemistry (CMaC), Ghent University, Krijgslaan 281 S4bis, 9000 Ghent, Belgium S Supporting Information *

ABSTRACT: We introduce a powerful approach based on the combination of size exclusion chromatography with highresolution mass spectrometry to selectively follow the compaction of discrete polymer chains that have uniform elemental composition. Single-chain nanoparticles (SCNP) have attracted considerable interest for a wide range of applications associated with their adjustable morphology. However, the precise characterization of morphological changes during the compaction is still challenging using existing analysis techniques. We employ a polystyrene backbone functionalized with tetrazole and fumarate moieties to utilize the nitrile imine-mediated tetrazole−ene cycloaddition for compaction. As every compaction step is associated with an elimination of one nitrogen molecule, it can be monitored via high-resolution electrospray ionization mass spectrometry. The combination with size exclusion chromatography enables the direct correlation of changes in mass with changes in morphology associated with the compaction. By establishing a calibration between the retention time and the hydrodynamic radius, ion chromatograms of discrete chains can be directly applied to determine the reduction in hydrodynamic radius associated with each cross-linking event. Therefore, accessing the compaction of discrete polymer chains becomes possible for the first time.

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INTRODUCTION Single-chain nanoparticles (SCNPs) have emerged as a highly interesting folded macromolecular materials class1−6 with wide application fields as varied as synthetic proteins,7−13 transition metal catalysis,8,14−19 and imaging agents.20−26 In addition, SCNPs are discussed as potent drug delivery agents.12,26−30 We have recently explored the future of SCNPs in a wide range of application fields and projected their potential functions, including the ability to change their morphology reversibly, ideally in response to a photonic or electric field enabling perfect external stimulus control over catalytic activity.16 However, these developments will only be possible if powerful characterization methods for SCNP analysis are available, particularly those that provide information about the size and morphological changes occurring during collapse. Significant progress has been made in this realm, e.g., advanced triple detection methods coupled to size exclusion chromatography (SEC), enabling the mapping of changes in hydrodynamic diameter upon chain collapse in arguably the most reliable variant of SCNPs analysis.31 Dynamic light scattering (DLS) is an alternative © XXXX American Chemical Society

method that has been frequently used to determine hydrodynamic radii both before and after folding.32−35 However, the method is challenging on a routine basis with polymeric materials in the sub-10 nm size regime due to their very low scattering intensity. In addition, colored and fluorescent particles can exhibit incorrectly deduced particle sizes as a result of light absorption by the fluorophores.36,37 These difficulties lead to a considerable error and scatter as recently summarized by our team, suggesting that SCNP analysis via DLS is a nontrivial task affiliated with large uncertainties.32 Further methods employed for following chain collapse include diffusion ordered spectroscopy (DOSY) NMR8,38−41 as well as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) imaging techniques.42,43 Alternative methods that yield information regarding the chemical changes that occur during collapse are NMR spectroscopy andrecently Received: January 29, 2019 Revised: February 26, 2019

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DOI: 10.1021/acs.macromol.9b00203 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. (A) Synthesis of functional polymer chains via nucleophilic substitution under mild basic conditions. (B) General scheme of the NITEC compaction reaction involving nitrile imine formation and subsequent cycloaddition forming a pyrazoline product. (C) Monitoring the folding of single chains via the expulsion of a nitrogen molecule for every nitrile imine formation. (D) Schematic depiction of the classical analytic approach as well as the XIC extraction utilized to determine the elution behavior of discrete chains.

occur upon folding, it does not provide information about folding-induced changes in hydrodynamic volume. Ideally, single-chain collapse should be studied on foldable monodisperse and sequence-defined macromolecules.46 In such a system, the observed change in hydrodynamic volume is

introduced by ushigh-resolution electrospray ionization (ESI) mass spectrometry using an Orbitrap mass analyzer.44,45 While mass spectrometry is a powerful method to identify chemical transformations, associated with a change in mass that B


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technique enables a slice-by-slice mass spectrometric analysis of the eluted macromolecules. The determination of the signal intensity of a specific m/z value corresponding to a discrete chain in every elution volume element creates an XIC, which can be determined for both the folded and nonfolded species. The power of our approach rests on its ability to follow the compaction of discrete polymer chains individually within a disperse sample rather than observing the overall size reduction of the entire polymer distribution. We therefore submit that the results presented herein represent the only instance to date of a direct observation of the compaction of discrete synthetic polymer chains.

dependent only on the folding of a perfect chain, similar to the folding of naturally occurring paragons such as proteins. Inspection of the literature, however, indicates that the structural collapse of defined single synthetic macromolecules has not previously been observed. This comes as no surprise, since the synthesis of sequence-defined polymers in sufficient quantities, length, and functionality is still in its infancy, despite the remarkable progress that has been made over the past few years.47 In lieu of direct synthetic access to uniform polymers for subsequent folding via the formation of covalent bonds, we sought to exploit high-resolution mass spectrometry to track the structural collapse of selected polymer compositions within a disperse mixture. Herein, we introduce a method to measure changes in hydrodynamic volume for individual molecular masses at high mass accuracy that correspond to direct observation of the covalent folding behavior of discrete polymer chains uniform in composition and molecular weight within a disperse polymer sample. Every discrete mass value corresponds to only one specific monomer and end-group composition; however, it does not contain information about the sequence of incorporation. By isolating elution chromatograms of these discrete polymer chains and subsequently determining their specific elution time shift caused by their compaction, the hydrodynamic size reduction during SCNP formation can be established. These elution traces of MS systems hyphenated with chromatographic setups have been termed single oligomer profiles (SOPs) in relation to polymeric systems or more broadly in other application fields of mass spectrometry extracted ion chromatograms (XICs).48−50 Our study shows that XICs of single molecular masses are readily accessible by combination of SEC with high-resolution ESI mass spectrometry, resulting in access to the elution behavior of defined and discrete polymer chains. So far, the isolation of XICs from SEC-ESI-MS data has not been exploited for mapping single-chain collapses. Herein, we close this capability gap, which opens a powerful access route to following the morphological changes of defined polymer segments as they fold. By establishing a calibration between SEC elution volumes and hydrodynamic radii (RH), changes in RH as a function of the number of compaction events can be readily established. The only prerequisite of the herein established mapping process is that each compaction event, i.e., each new intramolecular covalent bond, must be associated with a defined mass change. Fortunately, a number of the established folding chemistries currently employed in SCNP preparation are associated with a mass change and therefore lend themselves to this novel analytical method.51−53 We demonstrate the compaction of specific macromolecular chains into folded entities by employing a photochemical reaction sequence that expels a unit of nitrogen for each group that is activated. Specifically, tetrazoles were employed that afford nitrile imines under UV irradiation, which readily react with activated enes such as fumarates or maleimides in a so-called NITEC (nitrile imine-mediated tetrazole−ene cycloaddition) process to generate fluorescent pyrazoline adducts.39,54 We construct a polystyrene chain prepared by nitroxide-mediated radical polymerization (NMP) decorated with tetrazole as well as fumarate moieties following established procedures.54 Upon irradiation in dilute solution, nitrile imines are generated on the polymer chain that react with the fumarate moieties, leading to multiple intramolecular cross-links and folding of the precursor chain. Figure 1 summarizes the analytical approach employed to assess discrete polymer chains. Our hyphenated SEC-ESI-MS

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RESULTS AND DISCUSSION Initially, the synthesis of the utilized polymers and the reaction to yield SCNPs are showcased. Subsequently, the conventional SCNP analysis via SEC including the advantages and disadvantages of different calibration methods is discussed, in particular, the determination of hydrodynamic radii (RH) to attain a compaction value that expresses the size change of the macromolecule more accurately. Following that, a mass spectrometric folding analysis is carried out. Finally, the separation of single-chain chromatograms via the hyphenated characterization setup is described providing a direct link of RH to the number of folding steps. The analysis of copolymers comprising three or more monomers leads to challenging mass spectra due to the sheer number of possible monomer combinations and the resulting quantity of signals. In addition, the formation of multiply charged ions during ESI can increase the signal overlap and complicate the analysis. Therefore, singly charged ions are preferred for the evaluation of complex polymers. Matrixassisted laser desorption/ionization (MALDI), which results mainly in singly charged ions, is thus often preferred for polymer analysis. MALDI, however, is not readily incorporated with chromatography, and as a result ESI is preferred for coupling with SEC. We have recently shown that polystyrenes can be readily ionized via ESI by doping the spray solution with halide salts.55 To take advantage of this approach, a narrow dispersity statistical copolymer was synthesized via nitroxide-mediated radical polymerization (NMP) utilizing styrene and 4-(chloromethyl)styrene (CMS) as building blocks. The CMS units are convenient synthons for introducing functional groups via a simple substitution by carboxylic acids under mild basic conditions (Figure 1A). The purity and structure of the functional polymer were confirmed by 1H NMR, SEC, and SEC-ESI-MS (refer to the Supporting Information, Figures S3− S5). As noted, the NITEC reaction was selected as the compaction reaction due to the possibility of tracing the change in mass resulting from nitrogen expulsion. The formation of the nitrile imine during UV irradiation, as well as the formation of the pyrazoline adduct with a suitable dipolarophile, is schematically shown in Figure 1B. The folding reaction was performed under UV irradiation (λmax = 313 nm) in dichloromethane at low concentration (0.0175 mg mL−1) to avoid intermolecular crosslinking. Because of the fluorescent nature of the folded units, the reaction can be conveniently monitored via UV−vis spectroscopy, showing a quantitative reaction within few a minutes (refer to Figure S5). SEC Analysis and Hydrodynamic Radius Calibration. Most commonly, SCNP formation is traced via SEC, which confirms the successful compaction of the precursor through the reduction of the hydrodynamic radius visualized by a shift of the C


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Figure 2. Depiction of the resulting RH calibration curves depending on different calibration methods, as well as SEC traces of the functional PS precursor (black) and the formed SCNP (red) exhibiting a lower elution volume and hydrodynamic radius due to the reduction in the hydrodynamic radius.

employed MHS constants for PS in THF at 30 °C (K = 0.0128 mL g−1, α = 0.712). However, while these values are well established for PS of higher molecular mass, they might exhibit a greater deviation for molecular weights smaller than 10 kg mol−1. A more specific set of MHS values (K = 0.17 mL g−1, α = 0.428) specific to low mass PS was used to compare the absolute variation and to estimate the error of possibly mismatched MHS values. Furthermore, the data were compared to a power law from the literature,56 utilizing the established mass calibration. The limited availability of MHS parameters, especially for uncommon solvents and column conditions, hampers the applicability to all SEC conditions. To address this issue and to eliminate the uncertain error stemming from MHS values, the hydrodynamic radius of a set of narrow single PS standards (Mp = 682−34800 g mol−1) was determined via DOSY NMR to establish a calibration method that can be used for every SEC system and does not rely on assumptions or literature values. The resulting calibration curves (refer to the Supporting Information Section 4 for DOSY measurements and viscosity correction) are in excellent agreement with the MHS calibrations and underpin the relatively low error of the MHS method in the calibrated region as well as the applicability of the DOSY approach to rarer SEC systems lacking suitable MHS literature values. The resulting RH calibration curves as well as the application on SCNP data are depicted in Figure 2. The relationship between RH and elution time is critical for the below discussion of the changes in elution time for different species. Conventional SEC-ESI-MS Analysis. Initially, we analyze specific retention time slices before and after the compaction reaction to determine characteristic mass changes.44,45 In general, the coupling of SEC to a mass spectrometer has several advantages. First, the separation from any possible lower molecular weight impurities results in the prevention of

polymer distribution (Figure 2, top left). Typically, the shift to higher retention volume and the resulting molecular weight values are reported in the SCNP literature. However, the retention volume is influenced by the instrument configuration, such as the column number, material, length, and dead volume. Consequently, SEC data cannot be compared and published without reference values. Therefore, polymer specific calibrations with PMMA or PS using narrow polymer standards as a reference are required to obtain comparable values regarding the actual shift of the polymer distribution, eliminating the dependence on the experimental setup. Molecular weights obtained via such a calibration are comparable between different systems; nonetheless, they are not applicable to SCNPs due to their complex morphology. To enable a mass comparison, the same polymer standard has to be used as a reference for the polymer pre- and postreaction, resulting in an incorrect reduction in molecular weight for the compacted particle. Thus, while the retention behavior of the formed particle changes, the difference in retention volume is not caused by a variation in molecular weight, which is implied by these values. Therefore, the calibration of the SEC should compare the hydrodynamic volume of the precursor and folded particle instead. For the narrowly distributed, commercially available PS or PMMA samples used for the SEC standard calibration, the Mark−Houwink−Sakurada (MHS) parameters are readily available allowing direct access to a hydrodynamic radius calibration. Therefore, hydrodynamic radii should be reported as reference values for SCNP formation, while molecular weights and retention volumes should be reported to retain the comparability to older data. To assess the error margin, the hydrodynamic radius calibration was performed with two sets of MHS parameters. The first data set represents the commonly D


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of the monomer units, including some residual chloromethylstyrene units from the synthetic precursor. Despite the complexity, the simulation of different monomer combinations can successfully recreate the experimentally observed spectrum apart from one unidentified signal (marked red in Figure 3). The same mass range and retention time window were analyzed after irradiation, resulting in significant changes to the spectrum (refer to Figures S12−S13 and Table S2 for full assignments). A detailed analysis of the differences between the spectra obtained before and after irradiation was performed for the region m/z 2615−2685 (Figure 4). The peaks in this region correspond to ionized polymer chains containing either 0, 1, or 2 tetrazole units. The top panel (A) shows the averaged ion abundance profile of the precursor material at the retention time t1 (18.0− 18.3 min). The most intense signals arising from the abundant isotopes of each polymer chain are colored based on their tetrazole content (no tetrazole = red, one tetrazole unit = light blue, two tetrazole units = dark blue). Mass spectrum (B) depicts the ion intensity summation at the same retention time t1 after the NITEC reaction. Comparison to (A) shows that the red signals remain unchanged. In contrast, the blue signals have disappeared, and new signals appear at masses corresponding to one (light blue) or two (dark blue) nitrogen losses depending on their initial tetrazole content due to the nitrogen loss caused by the nitrile imine formation. The expanded view of ions at m/z 2618 (left side of Figure 4) demonstrates the necessity of highresolution analysis to distinguish the ions observed in (A) from the new signal caused by the nitrogen loss observed in (B) and (C). The ESI-MS data thus confirm the loss of nitrogen from the tetrazole side chains, but on its own, it cannot be confirmed if the formed nitrile imines have subsequently undergone intramolecular cross-linking with fumarate double bonds. As previously mentioned, the compaction via intramolecular bonds leads to a reduction in the hydrodynamic radius. Therefore, the ions relating to species that expelled nitrogen (light and dark blue) should shift to higher elution times in the

potential ion suppression effects. Second, the fractionation of the polymer minimizes the effects of ionization bias toward smaller polymer chains.57 In addition, the mass separation avoids overlap between different chain sizes with the same m/z values due to different charge states that further complicate the spectrum. The precursor was measured utilizing the ionization via attachment of halide ions reported previously.55 Sodium iodide was used because of its better solubility and lower tendency to form clusters compared to sodium chloride. The analyzed mass region was selected due to the comparably high signal intensity. The resulting spectrum demonstrates excellent end-group fidelity and confirms the successful functionalization (Figure 3; for a full list of all signals including

Figure 3. An example mass spectrum of the precursor copolymer showcasing one styrene repeating unit measured over the SEC retention time from 18.0 to 18.3 min including the simulation of the assigned species. For a full list of signal assignments refer to Table S1.

relative intensities and deviations refer to Table S1). The high signal density in this region results from different combinations

Figure 4. Example mass spectra depicting the region m/z 2615−2685: (A) Precursor polymer at the SEC retention time t1 (18.0−18.3 min) with the most prominent signals colored depending on the tetrazole content (0 = red, 1 = light blue, 2 = dark blue). (B) Compacted polymer at the same retention time t1 with remaining red signals as well as signals marked with a single nitrogen loss (light blue) compared to (C) the same mass region at a higher SEC retention time t2 (18.5−18.7 min) showing the remaining signals relating to polymer chains with one (light blue) or two (dark blue) nitrogen losses. E


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To determine the changes in the elution behavior, the XIC curves of the unfolded state are determined from the precursor sample, and subsequently, an SCNP sample was used to determine the elution times of the compacted SCNP species. Because of their consistent elution behavior, the nonphotoactive, tetrazole-free polymer chains can effectively be used as an internal standard for the correction of the small internal shift of the retention time between measurements. Four nonreactive signals were compared pre- and postirradiation; they exhibited a constant, slight deviation in retention time (Δt = 0.05 ± 0.01 min). This retention time shift was corrected, resulting in identical peak positions for these species. Because of the lower overall signal intensity of the folded nanoparticle compared to the precursor, the XICs exhibit less tailing resulting in slightly varying fit curves. The resulting XIC curves for one tetrazole-free polymer chain are plotted in Figure 6 to illustrate the resulting identical peak positions pre- and

case of SCNP formation, while tetrazole-free signals (red) should exhibit the same elution behavior. To confirm this behavior, the same mass region was investigated at a higher retention time t2 in (C) to underpin the selective SEC shift of compacted polymer chains. Mass spectrum (C) shows that in this mass region only ions that underwent nitrogen loss remain at t2, while ions of tetrazole-free polymer chains are absent. In conclusion, the mass analysis qualitatively describes the selective shift in retention times for polymer chains containing tetrazole molecules, which further underpins that, apart from the nitrile imine formation, the pyrazoline adduct was formed intramolecularly. Determination of the Size Reduction of Discrete Numbers of Compaction Events via XIC Extraction. In the critical next step, we exploit the two-dimensional nature of SEC-ESI-MS to follow the compaction of single chains. Individual retention time shifts depend on the number of compaction events and connect these values to the previously established RH calibration. Postacquisition SEC-ESI-MS data sets can be interrogated to extract the ion abundance profile of a specific mass range as a function of retention time, resulting in an extracted ion chromatogram (XIC). The XIC provides information comparable to an SEC curve. The difference lies in the possibility to isolate a specific isotopic pattern or single ion signal and therefore reveals the retention behavior of a defined polymer chain. The XICs for specific polymer chain compositions were determined by summing a region of 0.025 m/z (10 ppm at 2500 m/z) around the simulated theoretical m/z of every wellresolved peak of the isotope distribution. The data points were subsequently fitted with exponentially modified Gaussian (EMG) functions, which are well suited for chromatographic peaks exhibiting slight tailing.58 The XICs of the most intensive signals in the mass range of 2580−2850 were determined and fitted (refer to Figures S14−S17). Figure 5 shows the raw data

Figure 6. XIC comparison of a nonphotoactive monomer combination (i.e., void of tetrazole) before and after irradiation exhibiting a consistent XIC curve apart from stronger signal tailing with stronger overall signal intensity.

postirradiation. After establishing a consistent elution behavior between samples, the selective shift in elution volume per folding step of discrete single chains can be determined. The XICs of 17 polymer compositions containing 0−3 tetrazole units were analyzed preirradiation. Subsequently, the sample was irradiated, and the XICs of the fully compacted species were determined. The XICs of partially compacted single polymer chains were determined for the signals exhibiting sufficiently strong intensities and little overlap with other distributions. The peak position of the EMG fits was used for the calculation of the hydrodynamic radii based on the previously established hydrodynamic radius calibration curve. Alternatively, the entire EMG fit curve could be employed; however, the peak positions are less susceptible to the tailing effects as observed in Figure 6. A representative set of XICs of one discrete single polymer chain is shown in Figure 7, demonstrating the selective SEC retention time shift depending on the number of nitrogen losses, which corresponds to a compaction step each (refer to the Figures S14−S17 for all XICs). The resulting elution time values were converted to hydrodynamic radii using the calibration with the MHS parameter set specific to small polymer chains (MHS2). The removal of a nitrogen molecule for every folding point introduces a small error through the SEC retention time shift

Figure 5. Raw XIC data points and EMG fits of two polymer chains with different polymer compositions a mass difference of 4 m/z showcasing consistent elution behavior.

points and the resulting XIC EMG fit of two polymer chains with a mass difference of 4 m/z and slightly overlapping isotopic distributions. The XICs show that polymer chains with nearly the same molecular mass exhibit consistent and similar elution behavior independent from the type of monomers that were incorporated. F


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Figure 7. Example XIC raw data points and EMG fits of the polymer composition depicted in (A) showcasing the gradual elution time shift depending on the amount of folding steps during irradiation, illustrated in (B).

abundancies of singly charged high molecular weight analytes. The restriction of the analysis was caused by the resolution of the MS analysis which resulted in overlapping signals for higher charge states. However, the usage of simpler polymer systems with less and smaller monomer units could increase the mass range by accessing higher charge states, therefore giving access to a higher number of compaction steps. Comparison to Previous Work. Here we compare the determined compaction values for SCNPs to previous theoretical and experimental studies. Lemcoff et al. investigated the size reduction by internally cross-linking dendrimers via ring-closing metathesis (RCM).59 The mass change of the RCM enabled the conversion determination via MALDI-TOF, which revealed a linear decrease in the radius of gyration with the number of cross-links. The difference to our SCNP system which exhibits a diminished size reduction with consecutive stepsmay be explained by the regular conformation of the monodisperse dendrimer compared to the random polymer coil conformation during SCNP compaction. The difference showcases the influence of the initial geometry on the size reduction of every compaction step. Macrocycle formations are another synthetic approach with defined folding points that can be compared to our work. Schmidt et al. investigated a synthetic strategy to yield differently compacted macrocycles by utilizing polymer chains with azide and maleimide functionalities in predefined chain positions (Figure 9).60 The different polymers

caused by the molecular mass difference itself. To remove this influence on the overall elution behavior for every nitrile imine formation, a correction of the retention time shift can be established. The linear correlation between retention time and molecular mass in the investigated mass region leads to a simple correction factor depending on the amount of folding steps (Δt = 0.017 × nN2). To ensure the comparability of different sizes, the hydrodynamic radius change was plotted in percent with and without nitrogen loss in Figure 8. The data points represent the

Figure 8. Determined mean average RH reduction as a function of compaction steps with and without correction for nitrogen loss with error bars for the RH values with the highest deviation.

mean average of all determined peak XIC values with the error representing the values with the highest deviation from the mean in both directions resulting in asymmetric error bars. The determined numbers underpin the assumption that the first compaction step leads to the highest relative size reduction, with every subsequent compaction step further reducing the size to a lesser extent. In summary, the XIC extraction could be utilized to determine the elution behavior of defined single chains. In combination with a bimolecular reaction that results in a mass change, the XIC extraction could be directly utilized to determine hydrodynamic volume changes depending on the number of compaction steps. The analysis of the SCNPs in this study was limited to three compaction steps due to the low ion

Figure 9. Different shapes synthesized by Schmidt et al. via introduction of azide and alkene functionalities in predefined positions (adapted from ref 60). G


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free species exhibit values consistent with the expanded coil exponent. After the first compaction step, the reduced hydrodynamic radii match the expected reduced values for IDP resembling structures very closely. In the case of a second compaction step a size reduction resulting in compact globular particles occurs. However, the comparison has to be reviewed critically due to the assumptions made in the theoretical framework by Pomposo and co-workers resulting in the intersection of all curves at 1 nm. Therefore, the difference between the predicted morphologies is relatively small in the investigated mass region. The three-step compaction was observed with slightly larger particles (Figure 10, violet dots) showcasing a higher deviation from the compact, globular state although the linear precursor underwent one additional compaction step. Overall, the comparison to the literature shows that the hydrodynamic size reductions determined via XIC extraction give numbers in agreement with both experimental and theoretical literature.

were compacted, which resulted in different hydrodynamic size reductions that were compared regarding their molecular weights determined at the SEC curve peak position (Mp). The size reductions were described through the compaction parameter ⟨G⟩ = Mp,f/Mp,l (f = folded, l = linear). The ⟨G⟩ values for every compaction step were determined likewise for our SCNPs by determining the peak molecular weight of every compaction state for all analyzed chain compositions. The mean average compaction parameters ⟨G⟩ of all compositions are shown in Table 1 on the right side. The α- and P-shape result in ⟨G⟩ values comparable to a random single compaction in our system. Table 1. Compaction Parameter ⟨G⟩ Comparison between Different Literature Shapes60 (Left Side) and the Herein Determined Mean Average Values of Different Numbers of Folding Points (Right Side) folded shape

⟨G⟩

⟨G⟩

no. of folding points

α-shape P-shape Q-shape 8-shape

0.93 0.92 0.81 0.76

0.91 0.84 0.80

1 2 3

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CONCLUSION We establish a powerful analytical protocol based on ion chromatograms extracted from size exclusion chromatography (SEC) coupled with electrospray ionization mass spectrometry (ESI-MS) to map the compaction of discrete polymer chains. The single-chain collapse was performed via nitrile iminemediated tetrazole-ene cycloaddition (NITEC) under UV irradiation utilizing a tetrazole and fumarate decorated polystyrene (PS) backbone. Because of the expulsion of one nitrogen (28.01 Da) during the NITEC process, the number of compaction steps for every molecular mass can be determined, and the elution behavior can be established by plotting the ion chromatogram as a function of retention time using the SEC coupling. These extracted ion chromatograms (XIC) can be interpreted as SEC analogues of separate polymer chains with defined compositions. As a result, compaction values per intermolecular cross-link can be obtained by introducing a RH calibration. The determined RH changes show that the first compaction step results in the highest size reduction, with every subsequent compaction step further reducing the size to a lesser extent. Importantly, the obtained RH values correlate with theoretically predicted morphologies depending on the number of folding steps. Thus, our work constitutes an unprecedented data evaluation protocol for discrete single-chain compaction allowing for the extraction of RH values and their corresponding SCNP shape.

The pseudocyclic Q-shape results in a denser structure with a size reduction comparable to the structures achieved by several random compactions. The 8-shape, achieved by connecting both end groups to functionalities in the middle of the chain, resulted in a slightly stronger compaction than three random compactions in our study. To connect our data to a theoretical framework, we compared it to the work of Pomposo and co-workers on the theoretical size reduction during chain collapse depending on the nature of the cross-link and the reaction conditions.61−65 The SCNP morphologies are described resembling either intrinsically disordered proteins (IDPs) with locally compact segments connected by flexible segments or truly resembling compact globular particles. The hydrodynamic radii determined with MHS parameters (MHS2 for small PS chains) exhibit excellent accordance with the theoretical work (Figure 10). The tetrazole-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.9b00203. Further experimental procedures for syntheses, 1H NMR and SEC characterizations, UV−vis spectrum of SCNP formation, detailed DOSY measurements including viscosity correction, full SEC-ESI-MS assignments including deviations and simulated spectra and XIC raw data with EMG fits (PDF)

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Figure 10. Comparison between experimentally determined size reductions in this study depending on the resulting SCNP morphology (dots) and expected theoretical values as proposed by Pomposo and coworkers61 (dashed lines).

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], christopher. [email protected]. H


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ORCID

Jan Steinkoenig: 0000-0002-6365-7179 Stephen J. Blanksby: 0000-0002-8560-756X James P. Blinco: 0000-0003-0092-2040 Christopher Barner-Kowollik: 0000-0002-6745-0570 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.B.-K. acknowledges funding from the Australian Research Council (ARC) in the form of a Laureate Fellowship (FL170100014) enabling his photochemical research program as well as continued key support from the Queensland University of Technology (QUT). S.J.B. acknowledges generous financial assistance provided by the ARC Discovery grant (DP1701011596). The Central Analytical Research Facility (CARF) operated by the Institute for Future Environments (IFE) and supported by the Faculty of Science and Engineering at QUT is gratefully acknowledged for access to analytical instrumentation.

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Mapping the Compaction of Discrete Polymer Chains by Size

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