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Imaging Single-Chain Nanoparticle Folding via High-Resolution Mass Spectrometry Jan Steinkoenig, Hannah Rothfuss, Andrea Lauer, Bryan T. Tuten, and Christopher Barner-Kowollik J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b10952 • Publication Date (Web): 14 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016
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Journal of the American Chemical Society
Imaging Single-Chain Nanoparticle Folding via High-Resolution Mass Spectrometry Jan Steinkoenig,a Hannah Rothfuss,a Andrea Lauer,a Bryan T. Tuten,a Christopher Barner-Kowollik*a,b a
Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76128 Karlsruhe, Germany and Institut für Biologische Grenzflächen, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany. b School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology (QUT), 2 George St, QLD 4000, Brisbane, Australia.
ABSTRACT: Herein, we introduce the first approach to map single-chain nanoparticle (SCNP) folding via high-resolution electrospray ionization mass spectrometry coupled with size exclusion chromatography (SEC-ESI MS). For the first time, the successful collapse of polymeric chains into SCNPs is imaged by characteristic mass changes, allowing to draw detailed mechanistic information regarding the folding mechanism. As SCNP system, methyl methacrylate (MMA) statistically copolymerized with glycidyl methacrylate (GMA) resulting in p(MMA-stat-GMA) subsequently collapsed by using B(C6F5)3 as catalyst is employed. Both the precursor polymer and the SCNPs can be well-ionized via ESI MS and the strong covalent crosslinks are stable during ionization. Our high resolution mass spectrometric approach can unambiguously differentiate between two mechanistic modes of chain collapse for every chain constituting the SCNP sample.
Precisely folded single-chain polymer nanoparticles (SCNPs) have emerged as a major research area in the field of macromolecular architectures.1–7 SCNPs generated through various motifs such as Diels–Alder cycloadditions,8 metal ligation,9–11 hydrogen bonding,3,12,13 a variety of click and modular ligation chemistries,14,15 and several reversible folding chemistries,16 have found interesting applications in drug delivery,17 imaging,18 nano-containers and catalysis.19 Beyond the standard techniques utilized for characterizing these SCNPs (nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy, size exclusion chromatography (SEC), dynamic light scattering (DLS) and diffusion ordered NMR spectroscopy (DOSY)), more advanced techniques such as triple-detection SEC,20 atomic force microscopy,18 tunneling electron microscopy,20 small angle X-ray scattering,21 and small angle neutron scattering21 have proven to be powerful tools in the morphological characterization of SCNPs. However, a short-coming of these methods lies in the fact that they are not capable of imaging single chains and the individual chemical transformations they undergo. Herein, we introduce an advanced high-resolution ESI mass spectrometric protocol for SCNP imaging. ESI MS is a powerful characterization tool employed for the structural elucidation of homoand copolymers,22 polyelectrolytes,23 and various reaction pathways24 keeping the structural integrity of the chains intact based on the soft ionization process.25 The structural elucidation can be further underpinned by employing collision-induced dissociation (CID) techniques such as higher-energy collision dissociation (HCD).
Taking inspiration from the work of Pomposo and co-workers,26 a statistical copolymerization of methyl methacrylate (MMA) and glycidyl methacrylate (GMA) as precursor polymer p(MMA-statGMA) (1) was prepared, benefitting from the reversible additionfragmentation chain transfer (RAFT) process affording excellent end group fidelity. Subsequently, a Lewis acid catalyst B(C6F5)327,28 initiates an intrachain cationic ring-opening polymer-
Scheme 1 Preparation of p(MMA-stat-GMA) (1) (Mn = 13100 g∙mol-1, Ð = 1.26) under RAFT conditions (bulk, 80 °C, 1 h) and the single-chain collapse using B(C6F5)3 as catalyst for intrachain ROP (a.t., 72 h). ization (ROP)29,30 collapsing the linear chain (1) into a nanoparticle (2) (Scheme 1).26 We decided to utilize a methacrylate-type system for imaging SCNPs via mass spectrometry for three reasons: (i) pMMA – due to its complexation to Na+ – ionizes excellently; (ii) the ethylene glycol motifs resulting from the intrachain crosslinking event do not negatively affect the ionization,31 and (iii) the intrachain ROP of glycidyl moieties allows for the elucidation of two possible collapse avenues: via a bimolecular coupling (i.e. a single glycidyl unit finds another glycidyl unit and reacts only once, terminating in a single ether bridge) or via propagation (i.e. one glycidyl unit ringopens to find another glycidyl unit, which then ring-opens again to find another glycidyl unit, and so on). The solubility of precursor (1) and SCNP (2) allows for the characterization using SEC coupled with high-resolution ESI MS (SEC-ESI MS). As we reported
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Figure 1 (a) SEC traces of p(MMA-stat-GMA) (1, purple line) and SCNP (2, blue line) illustrating the decrease of hydrodynamic radius; (b) SEC-ESI Orbitrap spectrum of p(MMA-stat-GMA) (1) from m/z 1500 to m/z 2750 depicting the single oligomer profiles of the double charged copolymer species; (c) SEC-ESI Orbitrap spectrum of SCNP (2) from m/z 1500 to m/z 3000 depicting the single oligomer profiles of the double charged collapsed species. previously,32 the single oligomer profiles obtained by SEC-ESI MS are useful to assess the retention volume dependence of the single, double or triple charged species of the polymer. In the present study, we focused on the double charged oligomer profiles (rather than the additionally observed single, triple and quadrupole charged oligomer profiles) covering a broad mass range and thus high molar masses of p(MMA-stat-GMA) (1) and the SCNP (2). More precisely, we performed the following experiments and mapped the resulting polymers: (i) direct addition of the catalyst B(C6F5)3; (ii) slow addition of the catalyst with a syringe pump at a flow rate of 1 mL·h-1; (iii) GMA feed ratios of 15% and 10% (with direct addition of the catalyst); (iv) multi-dimensional SECESI MS2 experiments to evidence the folding; and (v) aqueous quenching of all GMA units for a study in negative mode. To date, the limitation of the method is dictated by ionization ability of the polymers (e.g. polar macromolecules), the accessible mass range (e.g. for Orbitrap
