Interfacing multistage mass spectrometry with liquid chromatography or ion mobility separation for synthetic polymer analysis

Synthetic polymers are naturally mixtures of homologs, even in pure form. More complexity is introduced by the presence of different comonomers, end groups and/or macromolecular architectures. The analysis of such systems is substantially facilitated by interfacing mass spectrometry (MS), which disperses based on mass, with an additional level of separation involving either interactive liquid chromatography (LC) or ion mobility (IM) spectrometry, both of which are readily coupled online with electrospray ionization and MS detection. IM-MS separates in the gas phase, post-ionization and, therefore, is ideally suitable for labile and reactive polymers. Its usefulness is illustrated with the characterization of non-covalent siloxane-saccharide complexes, metallosupramolecular assemblies and an air- and moisture-sensitive inorganic polymer, poly(dichlorophosphazene). Conversely, LC-MS which separates in solution phase, before ionization, is most effective for the analysis of polymeric mixtures whose components differ in polarity. Interactive LC conditions can be optimized to disperse by the content of hydrophobic units, as is demonstrated for amphiphilic polyether copolymers and sugar-based nonionic surfactant blends. Both LC-MS and IM-MS can be extended into a third dimension by tandem mass spectrometry (MS(2)) studies on select oligomers, in order to obtain insight into individual end groups and isomeric architectures, comonomer sequences and degree of substitution, for example, by hydrophobic functionalities.     

Ion‐Mobility Mass Spectrometry for the Rapid Determination of the Topology of Interlocked and Knotted Molecules

A rapid screening method based on traveling‐wave ion‐mobility spectrometry (TWIMS) combined with tandem mass spectrometry provides insight into the topology of interlocked and knotted molecules, even when they exist in complex mixtures, such as interconverting dynamic combinatorial libraries. A TWIMS characterization of structure‐indicative fragments generated by collision‐induced dissociation (CID) together with a floppiness parameter defined based on parent‐ and fragment‐ion arrival times provide a straightforward topology identification. To demonstrate its broad applicability, this approach is applied here to six Hopf and two Solomon links, a trefoil knot, and a [3]catenate.     

Distinguishing Protein Chemical Topologies Using Supercharging Ion Mobility Spectrometry-Mass Spectrometry

A technique combining ion mobility spectrometry-mass spectrometry (IMS-MS) and supercharging electrospray ionization (ESI) has been demonstrated to differentiate protein chemical topology effectively. Incorporating as many charges as possible into proteins via supercharging ESI allows the protein chains to be largely unfolded and stretched, revealing their hidden chemical topology. Different chemical topologies result in differing geometrical sizes of the unfolded proteins due to constraints in torsional rotations in cyclic domains. By introducing new topological indices, such as the chain-length-normalized collision cross-section (CCS) and the maximum charge state (z_M) in the extensively unfolded state, we were able to successfully differentiate various protein chemical topologies, including linear chains, ring-containing topologies (lasso, tadpole, multicyclics, etc.), and mechanically interlocked rings, like catenanes.     

Weighing synthetic polymers of ultra‐high molar mass and polymeric nanomaterials: What can we learn from charge detection mass spectrometry?

Advances in soft ionization techniques for mass spectrometry (MS) of polymeric materials make it possible to determine the masses of intact molecular ions exceeding megadaltons. Interfacing MS with separation and fragmentation methods has additionally led to impressive advances in the ability to structurally characterize polymers. Even if the gap to the megadalton range has been bridged by MS for polymers standards, the MS‐based analysis for more complex polymeric materials is still challenging. Charge detection mass spectrometry (CDMS) is a single‐molecule method where the mass and the charge of each ion are directly determined from individual measurements. The entire molecular mass distribution of a polymer sample can be thus accurately measured. Described in this perspective paper is how molecular weight distribution as well as charge distribution can provide new insights into the structural and compositional studies of synthetic polymers and polymeric nanomaterials in the megadalton to gigadalton range of molecular weight. The recent multidimensional CDMS studies involving couplings with separation and dissociation techniques will be presented. And, finally, an outlook for the future avenues of the CDMS technique in the field of synthetic polymers of ultra‐high molar mass and polymeric nanomaterials will be provided.     

A Solomon Link through an Interwoven Molecular Grid

A molecular Solomon link was synthesized through the assembly of an interwoven molecular grid consisting of four bis(benzimidazolepyridyl)benzthiazolo[5,4‐d]thiazole ligands and four zinc(II), iron(II), or cobalt(II) cations, followed by ring‐closing olefin metathesis. NMR spectroscopy, mass spectrometry, and X‐ray crystallography confirmed the doubly interlocked topology, and subsequent demetalation afforded the wholly organic Solomon link. The synthesis, in which each metal ion defines the crossing point of two ligand strands, suggests that interwoven molecular grids should be useful scaffolds for the rational construction of other topologically complex structures.     

Native mass spectrometry—A valuable tool in structural biology

Proteins and the complexes they form with their ligands are the players of cellular action. Their function is directly linked with their structure making the structural analysis of protein‐ligand complexes essential. Classical techniques of structural biology include X‐ray crystallography, nuclear magnetic resonance spectroscopy and recently distinguished cryo‐electron microscopy. However, protein‐ligand complexes are often dynamic and heterogeneous and consequently challenging for these techniques. Alternative approaches are therefore needed and gained importance during the last decades. One alternative is native mass spectrometry, which is the analysis of intact protein complexes in the gas phase. To achieve this, sample preparation and instrument conditions have to be optimised. Native mass spectrometry then reveals stoichiometry, protein interactions and topology of protein assemblies. Advanced techniques such as ion mobility and high‐resolution mass spectrometry further add to the range of applications and deliver information on shape and microheterogeneity of the complexes. In this tutorial, we explain the basics of native mass spectrometry including sample requirements, instrument modifications and interpretation of native mass spectra. We further discuss the developments of native mass spectrometry and provide example spectra and applications.     

Signatures of Mechanically Interlocked Topology of Lasso Peptides by Ion Mobility–Mass Spectrometry: Lessons from a Collection of Representatives

Lasso peptides are characterized by a mechanically interlocked structure, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Their compact and stable structures have a significant impact on their biological and physical properties and make them highly interesting for drug development. Ion mobility - mass spectrometry (IM-MS) has shown to be effective to discriminate the lasso topology from their corresponding branched-cyclic topoisomers in which the C-terminal tail is unthreaded. In fact, previous comparison of the IM-MS data of the two topologies has yielded three trends that allow differentiation of the lasso fold from the branched-cyclic structure: (1) the low abundance of highly charged ions, (2) the low change in collision cross sections (CCS) with increasing charge state and (3) a narrow ion mobility peak width. In this study, a three-dimensional plot was generated using three indicators based on these three trends: (1) mean charge divided by mass (ζ), (2) relative range of CCS covered by all protonated molecules (ΔΩ/Ω) and (3) mean ion mobility peak width (δΩ). The data were first collected on a set of twenty one lasso peptides and eight branched-cyclic peptides. The indicators were obtained also for eight variants of the well-known lasso peptide MccJ25 obtained by site-directed mutagenesis and further extended to five linear peptides, two macrocyclic peptides and one disulfide constrained peptide. In all cases, a clear clustering was observed between constrained and unconstrained structures, thus providing a new strategy to discriminate mechanically interlocked topologies.

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Anion templated assembly of mechanically interlocked structures

This tutorial review describes the evolution of the field of chemical templation, in particular, emphasising the impact its application has made to the synthesis of mechanically interlocked structures. Recent advances in the use of negatively charged template species for the synthesis of interlocked structures are detailed, with the main focus of this review describing the development of a general anion templation strategy that combines anion recognition with ion-pairing. The versatility of this methodology is demonstrated by the chloride anion templated synthesis of a series of interpenetrated pseudorotaxane, rotaxane and catenane structures. Upon template removal, the mechanically interlocked rotaxanes and catenanes are shown to bind anions within their topologically unique anion binding clefts by virtue of electrostatic and hydrogen bonding interactions, exhibiting a strong selectivity for the chloride halide anion template. The incorporation of the photo-active rhenium(I) bipyridyl signalling group into the rotaxane structural framework highlights the potential of these interlocked systems in future chemical sensor design.     

Arborescent polymers and other dendrigraft polymers: A journey into structural diversity

Arborescent polymers are characterized by a dendritic, multilevel branched architecture derived from successive grafting reactions. In spite of their much larger size, these materials display properties analogous to dendrimers and hyperbranched polymers, the two other dendritic polymer families. The distinguishing features of arborescent polymers are their assembly from polymeric building blocks of uniform size and their very high molecular weights attained in few synthetic steps. This article offers an overview of the historical aspects of the development of dendrigraft polymers, starting from our initial efforts on the synthesis of arborescent polystyrenes. Major subsequent developments in the synthetic techniques from our and other research groups allowing the synthesis of dendrigraft copolymers, tailoring of the structural characteristics of the molecules, and further simplifications to their synthesis are also reviewed, with emphasis over the broad range of architectures attainable in these systems. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 3803–3810, 2007     

Modern mass spectrometry in the characterization and degradation of biodegradable polymers

In the last decades, the solid-waste management related to the extensively growing production of plastic materials, in concert with their durability, have stimulated increasing interest in biodegradable polymers. At present, a variety of biodegradable polymers has already been introduced onto the market and can now be competitive with non biodegradable thermoplastics in different fields (packaging, biomedical, textile, etc.). However, a significant economical effort is still directed in tailoring structural properties in order to further broaden the range of applications without impairing biodegradation. Improving the performance of biodegradable materials requires a good characterization of both physico-chemical and mechanical parameters. Polymer analysis can involve many different features including detailed characterization of chemical structures and compositions as well as average molecular mass determination. It is of outstanding importance in troubleshooting of a polymer manufacturing process and for quality control, especially in biomedical applications. This review describes recent trends in the structural characterization of biodegradable materials by modern mass spectrometry (MS). It provides an overview of the analytical tools used to evaluate their degradation. Several successful applications of MALDI-TOF MS (matrix assisted laser desorption ionization time of flight) and ESI MS (electrospray mass spectrometry) for the determination of the structural architecture of biodegradable macromolecules, including their topology, composition, chemical structure of the end groups have been reported. However, MS methodologies have been recently applied to evaluate the biodegradation of polymeric materials. ESI MS represents the most useful technique for characterizing water-soluble polymers possessing different end group structures, with the advantage of being easily interfaced with solution-based separation techniques such as high-performance liquid chromatography (HPLC).     

Mass spectrometric imaging of synthetic polymers

The analysis of synthetic polymers represents today an important part of polymer science to determine their physical properties and to optimize the performance of polymeric materials for block copolymers as well as blend systems. The characterization can easily and rapidly be performed by mass spectrometry. In particular, the film formation of a synthetic polymer is of interest in material research and quality control, which can be determined by employing mass spectrometric imaging (MSI) using secondary ion mass spectrometry (SIMS) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. MALDI-MSI has been rapidly improved for the analysis of tissue cross-sections due to its soft ionization and accessible m/z range, which both also play an important role in polymer science. On the other hand, SIMS-MSI enables a sub-micrometer molecular spatial resolution, which is limited in MALDI-MSI due to the spatial resolution capabilities of the laser desorption process. The aim of the present contribution is to summarize recent advances in both imaging techniques for the analysis of synthetic polymers and to highlight their capabilities to correlate several imaging modalities in future applications.     

Gradient polymers and copolymers

Gradient polymers are two component polymeric systems in which the concentration of one component varies in a continuous way from one side to the other in systems with plane-parallel geometry. Such systems can be obtained from an amorphous polymeric matrix by diffusing into another monomer creating a gradient of concentration, which is fixed by, for example, photo-polymerization. Properties of such systems with plane-parallel geometry are discussed. Paricular attention is given to the systems with cylindrical geometry in which the gradient of the second polymer varies from the center to outside. This class of gradient polymers has a great practical application as gradient optical polymeric fibers and multifocal lenses. An interesting and new class of gradient polymers are systems systems consisting of semicrystalline polymeric matrices in which a gradient of structure is created by appropriate thermal treatment and an amorphous polymer gradient is formed by diffusion of a monomer and its subsequent polymerization. The structural, thermal and mechanical properties are discussed mainly for a model system consisting of polyethylene and polystyrene. The polymeric gradient systems, consisting of an oriented semicrystalline polymer and amorphous gradient polymer, are discussed showing that the structurally gradient matrices and amorphous polymer offer a great variety of factors which can influence the properties of multicomponent gradient polymers. Recently obtained gradient copolymers in which the chemical composition varies from one end to the other a macromolecule are presented. It is shown how such macromolecules can be obtained with different type of changes of the composition. The unusual properties of gradient copolymers are discussed considering their mechanical and thermal properties as well their specific behaviour as compatibilizers. © 1998 John Wiley & Sons, Ltd.     

Cooperative self-assembly: producing synthetic polymers with precise and concise primary structures

The quest to construct mechanically interlocked polymers, which present precise monodisperse primary structures that are produced both consistently and with high efficiencies, has been a daunting goal for synthetic chemists for many years. Our ability to realise this goal has been limited, until recently, by the need to develop synthetic strategies that can direct the formation of the desired covalent bonds in a precise and concise fashion while avoiding the formation of unwanted kinetic by-products. The challenge, however, is a timely and welcome one, as a consequence of, primarily, the potential for mechanically interlocked polymers to act as dynamic (noncovalent) yet robust (covalent) new materials for a wide array of applications. One such strategy which has been employed widely in recent years to address this issue, known as Dynamic Covalent Chemistry (DCC), is a strategy in which reactions operate under equilibrium and so offer elements of "proof-reading" and "error-checking" to the bond forming and breaking processes such that the final product distribution always reflects the thermodynamically most favourable compound. By coupling DCC with template-directed protocols, which utilise multiple weak noncovalent interactions to pre-organise and self-assemble simpler small molecular precursors into their desired geometries prior to covalent bond formation, we are able to produce compounds with highly symmetric, robust and complex topologies that are otherwise simply unobtainable by more traditional methods. Harnessing these strategies in an iterative, step-wise fashion brings us ever so much closer towards perfecting the controlled synthesis of high order main-chain mechanically interlocked polymers. This tutorial review focuses (i) on the development of DCC-namely, the formation of dynamic imine bonds-used in conjunction with template-directed protocols to afford a variety of mechanically interlocked molecules (MIMs) and ultimately (ii) on the synthesis of highly ordered poly[n]rotaxanes with high conversion efficiencies.     

A Dynamic Hydrogen‐Bonded Azo‐Macrocycle for Precisely Photo‐Controlled Molecular Encapsulation and Release

A light‐responsive system constructed from hydrogen‐bonded azo‐macrocycles demonstrates precisely controlled propensity in molecular encapsulation and release process. A significant decrease in the size of the cavity is observed in the course of the E→Z photoisomerization based on the results from DFT calculations and traveling wave ion mobility mass spectrometry. These macrocyclic hosts exhibit a rare 2:1 host–guest stoichiometry and guest‐dependent slow or fast exchange on the NMR timescale. With the slow host–guest exchange and switchable shape change of the cavity, quantitative release and capture of bipyridinium guests is achieved with the maximum release of 68 %. This work underscores the importance of slow host–guest exchange on realizing accurate release of organic cations in a stepwise manner under light irradiation. The light‐responsive system established here could advance further design of novel photoresponsive molecular switches and mechanically interlocked molecules.     

Current limitations in native mass spectrometry based structural biology

Nowadays, mass spectrometry plays an important role in structural biology. At one end it can be used to investigate intact protein complexes, providing details about the complex composition, topology, stability, and dynamics, whereas at the other end the protein’s identity and possible modifications can be visualized using proteomics approaches. Combining all this information allows the generation of detailed models for functional biological assemblies. Here, a perspective on the application of native mass spectrometry in structural biology is presented. The potential of this technique and some important current limitations are discussed. This includes issues regarding the quality/homogeneity of the sample, the dissociation efficiency of protein complexes during tandem mass spectrometric analysis, and some boundaries of ion mobility mass spectrometry.     

π‐Conjugated [2]Catenanes Based on Oligothiophenes and Phenanthrolines: Efficient Synthesis and Electronic Properties

Novel π‐conjugated topologies based on oligothiophenes and phenanthroline have been assembled by combining their outstanding electronic and structural benefits with the specific properties of the topological structure. Macrocycles and catenanes are prepared by using an optimized protocol of transition metal‐templated macrocyclization followed by efficient Pd‐catalyzed cross‐coupling reaction steps. By using this method, [2]catenanes comprising two interlocked π‐conjugated macrocycles with different ring sizes have been synthesized. The structures of the [2]catenanes and corresponding macrocycles are confirmed by detailed ¹H NMR spectroscopy and high resolution mass spectrometry. Single crystal X‐ray structural analysis of the quaterthiophene–diyne macrocycle affords important insight into the packing features and intermolecular interaction of the new systems. The fully conjugated interlocked [2]catenanes are fully characterized by spectroscopic and electrochemical measurements.     

Polymer architectures via mass spectrometry and hyphenated techniques: A review

This review covers the application of mass spectrometry (MS) and its hyphenated techniques to synthetic polymers of varying architectural complexities. The synthetic polymers are discussed as according to their architectural complexity from linear homopolymers and copolymers to stars, dendrimers, cyclic copolymers and other polymers. MS and tandem MS (MS/MS) has been extensively used for the analysis of synthetic polymers. However, the increase in structural or architectural complexity can result in analytical challenges that MS or MS/MS cannot overcome alone. Hyphenation to MS with different chromatographic techniques (2D × LC, SEC, HPLC etc.), utilization of other ionization methods (APCI, DESI etc.) and various mass analyzers (FT-ICR, quadrupole, time-of-flight, ion trap etc.) are applied to overcome these challenges and achieve more detailed structural characterizations of complex polymeric systems. In addition, computational methods (software: MassChrom2D, COCONUT, 2D maps etc.) have also reached polymer science to facilitate and accelerate data interpretation. Developments in technology and the comprehension of different polymer classes with diverse architectures have significantly improved, which allow for smart polymer designs to be examined and advanced. We present specific examples covering diverse analytical aspects as well as forthcoming prospects in polymer science.     

Ring Shuttling Controls Macroscopic Motion in a Three‐Dimensional Printed Polyrotaxane Monolith

Amplification of molecular motions into the macroscopic world has great potential in the development of smart materials. Demonstrated here is an approach that integrates mechanically interlocked molecules into complex three‐dimensional (3D) architectures by direct‐write 3D printing. The design and synthesis of polypseudorotaxane hydrogels, which are composed of α‐cyclodextrins and poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO‐PPO‐PEO) triblock copolymers, and their subsequent fabrication into polyrotaxane‐based lattice cubes by 3D printing followed by post‐printing polymerization are reported. By switching the motion of the α‐cyclodextrin rings between random shuttling and stationary states through solvent exchange, the polyrotaxane monolith not only exhibits macroscopic shape‐memory properties but is also capable of converting the chemical energy input into mechanical work by lifting objects against gravity.     

A mechanically interlocked bundle

The prototype of an artificial molecular machine consisting of a trisammonium tricationic component interlocked with a tris(crown ether) component to form a molecular bundle with averaged C(3v) symmetry has been designed and synthesized. The system is based on noncovalent interactions, which include 1) N(+)-H...O hydrogen bonds; 2) C-H...O interactions between the CH(2)NH(2) (+)CH(2) protons on three dibenzylammonium-ion-containing arms, which are attached symmetrically to a benzenoid core, and three dibenzo[24]crown-8 macrorings fused onto a triphenylene core; and 3) pi...pi stacking interactions between the aromatic cores. The template-directed synthesis of the mechanically interlocked, triply threaded bundle involves post-assembly covalent modification, that is, the efficient conversion of three azide functions at the ends of the arms of the bound and threaded trication into bulky triazole stoppers, after 1,3-dipolar cycloaddition with di-tert-butylacetylenedicarboxylate to the extremely strong 1:1 adduct that is formed in dichloromethane/acetonitrile (3:2), on account of a cluster effect associated with the paucivalent adduct. Evidence for the averaged C(3v) symmetry of the molecular bundle comes from absorption and luminescence data, as well as from electrochemical experiments, (1)H NMR spectroscopy, and mass spectrometry. The photophysical properties of the mechanically interlocked bundle are very similar to those of the superbundle that precedes the formation of the bundle in the process of supramolecular assistance to covalent synthesis. Although weak non-nucleophilic bases (e.g., nBu(3)N and iPr(2)NEt) fail to deprotonate the bundle, the strong tBuOK does, as indicated by both luminescence and (1)H NMR spectroscopy. While deprotonation undoubtedly loosens up the interlocked structure of the molecular bundle by replacing relatively strong N(+)-H...O hydrogen bonds by much weaker N-H...O ones, the pi...pi stacking interactions ensure that any structural changes are inconsequential, particularly when the temperature of the solution of the neutral molecular bundle in dichloromethane is cooled down to considerably below room temperature.     

Rapid and sensitive differentiation of anomers,linkage, and position isomers of disaccharides using High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS)

A challenging aspect of structural elucidation of carbohydrates is gaining unambiguous information for anomers, linkage, and position isomers. Such isomers with identical mass can't be easily distinguished in mass spectrometry and a separation step is required prior to mass spectrometry identification. In our laboratory, gas-phase separation and differentiation of anomers, linkage, and position isomers of disaccharides was achieved using High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). The FAIMS method responds to changes in ion mobility at high field rather than absolute values of ion mobility, and was shown to provide efficient separation and identification of disaccharide isomers at high sensitivity. Separation of analyzed disaccharide isomers can be accomplished at low nM level in a matter of seconds without sample purification or fractionation. Capability for examining a large population of ionic species of disaccharides by this method allowed for correlating structural details of disaccharide isomers with their separation properties in FAIMS. Results for disaccharide isomers indicate that this method could be applied to a larger group of carbohydrates.