Mass spectrometry of polymers: A tutorial review.

Ever since the inception of synthetic polymeric materials in the late 19th century, the number of studies on polymers as well as the complexity of their structures have only increased. The development and commercialization of new polymers with properties fine-tuned for specific technological, environmental, consumer, or biomedical applications requires powerful analytical techniques that permit the in-depth characterization of these materials. One such method with the ability to provide chemical composition and structure information with high sensitivity, selectivity, specificity, and speed is mass spectrometry (MS). This tutorial review presents and exemplifies the various MS techniques available for the elucidation of specific structural features in a synthetic polymer, including compositional complexity, primary structure, architecture, topology, and surface properties. Key to every MS analysis is sample conversion to gas-phase ions. This review describes the fundamentals of the most suitable ionization methods for synthetic materials and provides relevant sample preparation protocols. Most importantly, structural characterizations via one-step as well as hyphenated or multidimensional approaches are introduced and demonstrated with specific applications, including surface sensitive and imaging techniques. The aim of this tutorial review is to illustrate the capabilities of MS for the characterization of large, complex polymers and emphasize its potential as a powerful compositional and structural elucidation tool in polymer chemistry.

Separation of Perfluorooctanoic Acid from Water Using Meso- and Macroporous Syndiotactic Polystyrene Gels.

Per- and polyfluoroalkyl substances are an emerging class of contaminants that are environmentally persistent, bioaccumulative, and noxious to human health. Among these, perfluorooctanoic acid (PFOA) molecules are widely found in ground and surface water sources. A novel high surface area, meso- and macroporous syndiotactic polystyrene (sPS) wet gel is used in this work as the adsorbent of PFOA molecules from water at environmentally relevant PFOA concentrations (≤1 µg/L) and cleanse water to below the U.S. EPA's 2023 health advisory limit of 4 parts per trillion (ppt). The sigmoidal shape of the PFOA adsorption isotherm indicates a two-step adsorption mechanism attributed to the strong affinity of PFOA molecules for the sPS surface and molecular aggregation at solid-liquid interfaces or within the pores of the sPS wet gel. The adsorption kinetics and the effects of sPS wet gel porosity, pore size, and pore volume on the removal efficiency are reported. The adsorption kinetics is seen to be strongly dependent on pore size and pore volume.

Native mass spectrometry analysis of conjugated HSA and BSA complexes with various flavonoids.

Mass spectrometry was used to study the binding interaction between serum albumin proteins (BSA and HSA) and flavone dyes, which is known to induce large fluorescence signals for protein detection. By electrospray ionization mass spectrometry (ESI-MS), multiple charged species/states could be produced in ammonium acetate buffer, while preserving the native structures of the proteins. Subsequent introduction of a flavone dye into the buffered solution resulted in an immediate interaction, forming the respective protein-dye conjugates associated by non-covalent interactions. Formation of protein-dye conjugates induced a notable response in the ESI-MS spectra, including changes in both the charge states and molecular mass of the protein species. The resulting data pointed out that the protein-flavone dye maintained a 1 : 1 ratio in the conjugate, although multiple binding sites for drug molecules are present in albumin proteins.

Rapid and simple determination of average molecular weight and composition of synthetic polymers via electrospray ionization-mass spectrometry and a Bayesian universal charge deconvolution.

RATIONALE: Simple, affordable, and rapid methods for identifying the molecular weight (MW) distribution and macromolecular composition of polymeric materials are limited. Current tools require extensive solvent consumption, linear calibrations, and expensive consumables. A simple method for the determination of average MW (Mn , Mw ) and chain end groups is demonstrated for synthetic homopolymer standards using direct injection electrospray ionization-mass spectrometry (ESI-MS) and an open-sourced charge deconvolution (CDC) algorithm. METHODS: Five homopolymer standards in the 1-7 kDa MW range were analyzed using direct-injection ESI-MS on a quadrupole/time-of-flight mass spectrometer. The samples investigated, viz. two poly(ethylene oxide) (PEO) and two poly(styrene sulfonic acid) (PSS) standards with narrow polydispersity and one poly(d,l-alanine) (pAla) standard with undefined polydispersity, were chosen to illustrate challenges with ESI-MS quantitation. Using the UniDec program, weight average MWs (Mw ) obtained from the charge-deconvoluted spectra were compared to the reported Mw data of the standards from size exclusion chromatography (SEC) measurements. RESULTS: The MW data derived for the PSS, PEO, and pAla standards agreed well with the corresponding reported Mw or MW range values. The method was able to provide MW, degree of polymerization (DP), and polydispersity index (PDI) information for polymers with narrow (PSS, PEO) as well as broader (pAla) molecular weight distribution; this feature provides an advantage over MW analysis via matrix-assisted laser desorption/ionization (MALDI) for ESI-compatible materials. PSS standards differing in average MW by only a few repeat units could be confidently distinguished. Additionally, the oligomeric resolution observed for all samples studied unveiled chain-end information not available through chromatographic analysis. CONCLUSIONS: Overall, the free and easy-to-use UniDec CDC algorithm provides a simple, alternative method to measuring MW and DP for polymeric materials without high solvent consumption, expensive ionization sources, or calibration curves. Information about the masses of individual oligomers and the possibility to further characterize these oligomers using tandem mass spectrometry and/or ion mobility techniques constitutes additional benefits of this approach vis-à-vis traditional MW and PDI elucidation through SEC.

Analysis of Thermoplastic Copolymers by Mild Thermal Degradation Coupled to Ion Mobility Mass Spectrometry.

Thermal desorption/degradation with an atmospheric solids analysis probe (ASAP) and ion mobility (IM) separation are coupled with mass spectrometry (MS) analysis and tandem mass spectrometry (MS/MS) fragmentation to characterize thermoplastic elastomers. The compounds investigated, which are used in the manufacture of a wide variety of packaging materials, are mainly composed of thermoplastic copolymers, but also contain additional chemicals ("additives"), like antioxidants and UV stabilizers, for enhancement of their properties or protection from degradation. The traditional method for analyzing such complex mixtures is vacuum pyrolysis followed by electron or chemical ionization mass spectrometry, often after gas chromatography separation. Here, an alternative, faster approach, involving mild degradation at atmospheric pressure (ASAP) and subsequent characterization of the desorbates and pyrolyzates by IM-MS, and if needed, MS/MS is presented. Such multidimensional dispersion considerably simplifies the resulting spectra, permitting the conclusive separation, characterization, and classification of the multicomponent materials examined.

Molecular Geometry-Directed Self-Recognition in the Self-Assembly of Giant Amphiphiles.

Three sets of polyoxometalate (POM)-based amphiphilic hybrid macromolecules with different rigidity in their organic tails are used as models to understand the effect of molecular rigidity on their possible self-recognition feature during self-assembly processes. Self-recognition is achieved in the mixed solution of two structurally similar, sphere-rigid T-shape-linked oligofluorene(TOF4 ) rod amphiphiles, with the hydrophilic clusters being Anderson (Anderson-TOF4 ) and Dawson (Dawson-TOF4 ), respectively. Anderson-TOF4 is observed to self-assemble into onion-like multilayer structures and Dawson-TOF4 forms multilayer vesicles. The self-assembly is controlled by the interdigitation of hydrophobic rods and the counterion-mediated attraction among charged hydrophilic inorganic clusters. When the hydrophobic blocks are less rigid, e.g., partially rigid polystyrene and fully flexible alkyl chains, self-recognition is not observed, attributing to the flexible conformation of hydrophobic molecules in the solvophobic domain. This study reveals that the self-recognition among amphiphiles can be achieved by the geometrical limitation of the supramolecular structure due to the rigidity of solvophobic domains.

Separation, identification, and confirmation of cyclic and tadpole macromolecules via UPLC-MS/MS.

Macrocyclic poly(glycidyl phenyl ether) (pGPE) synthesized via zwitterionic ring opening polymerization is typically contaminated by chains with linear and tadpole architecture. Although mass spectrometry (MS) analysis can readily confirm the presence of the linear byproduct, due to its unique mass, it is unable to differentiate between the cyclic and tadpole structures, which are constitutional isomers produced by backbiting reactions in monomeric or dimeric chains, respectively. To overcome this problem, ultraperformance reversed-phase liquid chromatography interfaced with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) was employed. The separation achieved by UPLC revealed that the tadpole isomer elutes before the cyclic structure because of the increased polarity afforded by its distinctive substituents. The ratio of tadpole to cyclic species increased with the degree of polymerization, in agreement with the synthetic method used, as the potential for forming tadpole structures by backbiting is entropically favored in longer polymer chains. Once separated, the two isomers could be independently characterized by tandem mass spectrometry. The macrocyclic and tadpole species exhibit unique fragmentation patterns, including structurally diagnostic fragments for each structure.

Multidimensional Mass Spectrometry of Multicomponent Nonionic Surfactant Blends.

Ultraperformance liquid chromatography (UPLC) and ion mobility (IM) spectrometry were interfaced with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) to characterize a complex nonionic surfactant mixture. The surfactant was composed of a glycerol core, functionalized with poly(ethylene oxide) units (PEOn) that were partially esterified by caprylic and/or capric acid. Reversed-phase UPLC classified the blend based on polarity into four groups of eluates, corresponding to compounds with zero, one, two, or three fatty acid residues. Additional separation within each eluate group was achieved according to the length of the fatty acid chains. Coeluting molecules of similar polarity were dispersed in the gas phase by their collision cross section in the IM dimension. Performed in series, UPLC and IM allowed for the separation and detection of several isomeric and isobaric blend constituents, thereby enabling their isolation for conclusive MS/MS analysis to confirm or elucidate their primary structures and architectures (overall four-dimensional, 4D, characterization).

Synthesis, characterization, in vitro SAR study, and preliminary in vivo toxicity evaluation of naphthylmethyl substituted bis-imidazolium salts.

A series of novel bis-imidazolium salts was synthesized, characterized, and evaluated in vitro against a panel of non-small cell lung cancer (NSCLC) cells. Two imidazolium cores were connected with alkyl chains of varying lengths to develop a structure activity relationship (SAR). Increasing the length of the connecting alkyl chain was shown to correlate to an increase in the anti-proliferative activity. The National Cancer Institute's NCI-60 human tumor cell line screen confirmed this trend. The compound containing a decyl linker chain, 10, was chosen for further in vivo toxicity studies with C578BL/6 mice. The compound was well tolerated by the mice and all of the animals survived and gained weight over the course of the study.

Sierpinski Pyramids by Molecular Entanglement.

Planar, terpyridine-based metal complexes with the Sierpinski triangular motif and alkylated corners undergo a second self-assembly event to give megastructural Sierpinski pyramids; assembly is driven by the facile lipophilic-lipophilic association of the alkyl moieties and complementary perfect fit of the triangular building blocks. Confirmation of the 3D, pyramidal structures was verified and supported by a combination of TEM, AFM, and multiscale simulation techniques.

Unraveling Chiral Selection in the Self-assembly of Chiral Fullerene Macroions: Effects of Small Chiral Components Including Counterions, Co-ions, or Neutral Molecules.

Lactic acid-functionalized chiral fullerene (C60) molecules are used as models to understand chiral selection in macroionic solutions involving chiral macroions, chiral counterions, and/or chiral co-ions. With the addition of Zn2+ cations, the C60 macroions exhibit slow self-assembly behavior into hollow, spherical, blackberry-type structures, as confirmed by laser light scattering (LLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques. Chiral counterions with high charge density show no selection to the chirality of AC60 macroions (LAC60 and DAC60) during their self-assembly process, while obvious chiral discrimination between the assemblies of LAC60 and DAC60 is observed when chiral counterions with low charge density are present. Compared with chiral counterions, chiral co-ions show weaker effects on chiral selection with larger amounts needed to trigger the chiral discrimination between LAC60 and DAC60. However, they can induce a higher degree of discrimination when abundant chiral co-ions are present in solution. Furthermore, the self-assembly of chiral AC60 macroions is fully suppressed by adding significant amounts of neutral molecules with opposite chirality. Thermodynamic parameters from isothermal titration calorimetry (ITC) reveal that chiral selection is controlled by the ion pairing and the destruction of solvent shells between ions, and meanwhile originates from the delicate balance between electrostatic interaction and molecular chirality.

Collision cross-section analysis of self-assembled metallomacrocycle isomers and isobars via ion mobility mass spectrometry.

RATIONALE: Coordinatively driven self-assembly of transition metal ions and bidentate ligands gives rise to organometallic complexes that usually contain superimposed isobars, isomers, and conformers. In this study, the double dispersion ability of ion mobility mass spectrometry (IM-MS) was used to provide a comprehensive structural characterization of the self-assembled supramolecular complexes by their mass and charge, revealed by the MS event, and their shape and collision cross-section (Ω), revealed by the IM event. METHODS: Self-assembled complexes were synthesized by reacting a bis(terpyridine) ligand exhibiting a 60o dihedral angle between the two ligating terpyridine sites (T) with divalent Zn, Ni, Cd, or Fe. The products were isolated as (Metal2+ [T])n (PF6 )2n salts and analyzed using IM-MS after electrospray ionization (ESI) which produced several charge states from each n-mer, depending on the number of PF6 - anions lost upon ESI. Experimental Ω data, derived using IM-MS, and computational Ω predictions were used to elucidate the size and architecture of the complexes. RESULTS: Only macrocyclic dimers, trimers, and tetramers were observed with Cd2+ , whereas Zn2+ formed the same plus hexameric complexes. These two metals led to the simplest product distributions and no linear isomers. In sharp contrast, Ni2+ and Fe2+ formed all possible ring sizes from dimer to hexamer as well as various linear isomers. The experimental and theoretical Ω data indicated rather planar macrocyclic geometries for the dimers and trimers, twisted 3D architectures for the larger rings, and substantially larger sizes with spiral conformation for the linear congeners. Adding PF6 - to the same complex was found to mainly cause size contraction due to new stabilizing anion-cation interactions. CONCLUSIONS: Complete structural identification could be accomplished using ESI-IM-MS. Our results affirm that self-assembly with Cd2+ and Zn2+ proceeds through reversible equilibria that generate the thermodynamically most stable structures, encompassing exclusively macrocyclic architectures that readily accommodate the 60o ligand used. In contrast, complexation with Ni2+ and Fe2+ , which form stronger coordinative bonds, proceeds through kinetic control, leading to more complex mixtures and kinetically trapped less stable architectures, such as macrocyclic pentamers and linear isomers.

Mechanism of 6-Hydroxynicotinate 3-Monooxygenase, a Flavin-Dependent Decarboxylative Hydroxylase Involved in Bacterial Nicotinic Acid Degradation.

6-Hydroxynicotinate 3-monooxygenase (NicC) is a Group A FAD-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) with concomitant oxidation of NADH in nicotinic acid degradation by aerobic bacteria. Two mechanisms for the decarboxylative hydroxylation half-reaction have been proposed [Hicks, K., et al. (2016) Biochemistry 55, 3432-3446]. Results with Bordetella bronchiseptica RB50 NicC here show that a homocyclic analogue of 6-HNA, 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420-fold lower catalytic efficiency than is 6-HNA. The 13( V/ K), measured with wild-type NicC by isotope ratio mass spectrometry following the natural abundance of 13C in the CO2 product, is inverse for both 6-HNA (0.9989 ± 0.0002) and 4-HBA (0.9942 ± 0.0004) and becomes negligible (0.9999 ± 0.0004) for 5-chloro-6-HNA, an analogue that is 10-fold more catalytically efficient than 6-HNA. Covalently bound 6-HNA complexes of NicC are not observed by mass spectrometry. Comparative steady-state kinetic and Kd6HNA analyses of active site NicC variants (C202A, H211A, H302A, H47E, Y215F, and Y225F) identify Tyr215 and His47 as critical determinants both of 6-HNA binding ( KdY215F/ KdWT > 240; KdH47E/ KdWT > 350) and in coupling rates of 2,5-DHP and NAD+ product formation ([2,5-DHP]/[NAD+] = 1.00 (WT), 0.005 (Y215F), and 0.07 (H47E)]. Results of these functional analyses are in accord with an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 may serve as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC.

Monitoring Metallo-Macromolecular Assembly Equilibria by Ion Mobility-Mass Spectrometry.

Ion mobility-mass spectrometry (IM-MS) allows the separation of isomeric and isobaric species on the basis of their size, shape, and charge. The fast separation timescale (ms) and high sensitivity of these measurements make IM-MS an ideally suitable method for monitoring changes in macromolecular structure, such as those occurring in interconverting terpyridine-based metallosupramolecular self-assemblies. IM-MS is used to verify the elemental composition (size) and architecture (shape) of the self-assembled products. Additionally, this article demonstrates its applicability to the elucidation of concentration-driven association-dissociation (fusion-fission) equilibria between isobaric structures. IM-MS enables both quantitative separation and identification of the interconverting complexes as well as derivation of the corresponding equilibrium constants (i.e., thermodynamic information) from extracted IM-MS abundance data.

Self-Assembly of Polyoxometalate-Peptide Hybrids in Solution: Elucidating the Contributions of Multiple Possible Driving Forces.

Incorporating the building blocks of nature (e.g., peptides and DNA) into inorganic polyoxometalate (POM) clusters is a promising approach to improve the compatibilities of POMs in biological fields. To extend their biological applications, it is necessary to understand the importance of different non-covalent interactions during self-organization. A series of Anderson POM-peptide hybrids have been used as a simple model to demonstrate the role of different interactions in POM-peptide (biomolecules) systems. Regardless of peptide chain length, these hybrids follow similar solution behaviors, forming hollow, spherical supramolecular structures in acetonitrile/water mixed solvents. The incorporation of peptide tails introduces interesting stimuli-responsive properties to temperature, hybrid concentration, solvent polarity and ionic strength. Unlike the typical bilayer amphiphilic vesicles, they are found to follow the blackberry-type assemblies of hydrophilic macroions, which are regulated by electrostatic interaction and hydrogen bonding. The formation of electrostatic assemblies before the supramolecular formation is confirmed by ion-mobility mass spectrometry (IMS-MS).

Analysis of monodisperse, sequence-defined, and POSS-functionalized polyester copolymers by MALDI tandem mass spectrometry.

Monodisperse, sequence-defined polymers can be potentially used for digital data storage. This study reports the sequence analysis and differentiation of monodisperse polyester copolymers carrying side chains functionalized in a specific order by polyhedral oligomeric silsesquioxane (POSS) nanoparticles. Steglich esterification and succinic anhydride ring-opening chemistries were utilized iteratively to synthesize the intended sequences, which were characterized by matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS2). Isomeric oligomers were readily distinguished based on their different fragmentation patterns. The sequences embedded in the oligomers were decrypted by their specific backbone dissociation pathways. The robustness of using MALDI-MS2 as a sequencing method for monodisperse synthetic macromolecules was assessed and validated by the characterization of longer oligomers.

Geometry induced sequence of nanoscale Frank-Kasper and quasicrystal mesophases in giant surfactants.

Frank-Kasper (F-K) and quasicrystal phases were originally identified in metal alloys and only sporadically reported in soft materials. These unconventional sphere-packing schemes open up possibilities to design materials with different properties. The challenge in soft materials is how to correlate complex phases built from spheres with the tunable parameters of chemical composition and molecular architecture. Here, we report a complete sequence of various highly ordered mesophases by the self-assembly of specifically designed and synthesized giant surfactants, which are conjugates of hydrophilic polyhedral oligomeric silsesquioxane cages tethered with hydrophobic polystyrene tails. We show that the occurrence of these mesophases results from nanophase separation between the heads and tails and thus is critically dependent on molecular geometry. Variations in molecular geometry achieved by changing the number of tails from one to four not only shift compositional phase boundaries but also stabilize F-K and quasicrystal phases in regions where simple phases of spheroidal micelles are typically observed. These complex self-assembled nanostructures have been identified by combining X-ray scattering techniques and real-space electron microscopy images. Brownian dynamics simulations based on a simplified molecular model confirm the architecture-induced sequence of phases. Our results demonstrate the critical role of molecular architecture in dictating the formation of supramolecular crystals with "soft" spheroidal motifs and provide guidelines to the design of unconventional self-assembled nanostructures.

Synthesis, Self-Assembly and Characterization of Tandem Triblock BPOSS-PDI-X Shape Amphiphiles.

In this article, we report the facile synthesis, self-assembly, and characterization of shape amphiphiles (BPOSS-PDI-X) based on isobutyl-functionalized polyhedral oligomeric silsesquioxane (BPOSS), perylene tetracarboxylic diimide (PDI), and (60)fullerene (C60) moieties. Firstly, an asymmetrically functionalized diblock shape amphiphile precursor (BPOSS-PDI-OH) was obtained through the one-pot reaction between perylene-3,4,9,10-tetracarboxylic dianhydride and two different amines, namely BPOSS-NH2 and 3-amino-1-propanol. It was further conjugated with C60-COOH to give a tri-block shape amphiphile (BPOSS-PDI-C60). Their chemical structures were thoroughly characterized by NMR, IR and MALDI-TOF MS spectrometry. In order to gain insights on the structure-property relationship, their self-assembly in gas phase, in solution, and in solid state were characterized using traveling wave ion mobility mass spectrometry (TWIM-MS), UV/Vis absorption, fluorescence emission spectrophotometer, and transmission electron microscopy, respectively. It was found that BPOSS-PDI-OH formed more complicated dimers than BPOSS-PDI-C60. Both samples showed unique aggregation behaviors in solution with increasing concentration, which could be attributed neither to H- nor to J-type and might be related to the discrete dimers. While BPOSS-PDI-C60 could hardly crystalize into ordered structures, BPOSS-PDI-OH could form nanobelt-shaped single crystals, which may hold potential applications in microelectronics.

Breaking Parallel Orientation of Rods via a Dendritic Architecture toward Diverse Supramolecular Structures.

Self-assembled nanostructures of rod-like molecules are commonly limited to nematic or layered smectic structures dominated by the parallel arrangement of the rod-like components. Distinct self-assembly behavior of four categories of dendritic rods constructed by placing a tri(hydroxy) group at the apex of dendritic oligo-fluorenes is observed. Designed hydrogen bonding and dendritic architecture break the parallel arrangement of the rods, resulting in molecules with specific (fan-like or cone-like) shapes. While the fan-shaped molecules tend to form hexagonal packing cylindrical phases, the cone-shaped molecules could form spherical motifs to pack into various ordered structures, including the Frank-Kasper A15 phase and dodecagonal quasicrystal. This study provides a model system to engineer diverse supramolecular structures by rod-like molecules and sheds new light into the mechanisms of the formation of unconventional spherical packing structures in soft matter.

Magnesium Catalyzed Polymerization of End Functionalized Poly(propylene maleate) and Poly(propylene fumarate) for 3D Printing of Bioactive Scaffolds.

The ring-opening copolymerization of maleic anhydride and propylene oxide, using a functionalized primary alcohol initiator and magnesium 2,6-di-tert-butyl phenoxide as a catalyst, was investigated in order to produce high end-group fidelity poly(propylene maleate). Subsequent isomerization of the material into 3D printable poly(propylene fumarate) was utilized to produce thin films and scaffolds possessing groups that can be modified with bioactive groups postpolymerization and postprinting. The surface concentration of these modifiable groups was determined to be 30.0 ± 3.3 pmol·cm-2, and copper-mediated azide-alkyne cycloaddition was used to attach a small molecule dye and cell adhesive GRGDS peptides to the surface as a model system. The films were then studied for cytotoxicity and found to have high cell viability before and after surface modification.