Tandem mass spectrometry and ion mobility mass spectrometry for the analysis of molecular sequence and architecture of hyperbranched glycopolymers

Abstract

Multidimensional mass spectrometry techniques, combining matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) with tandem mass spectrometry (MS(2)), multistage mass spectrometry (MS(n)) or ion mobility mass spectrometry (IM-MS), have been employed to gain precise structural insight on the compositions, sequences and architectures of small oligomers of a hyperbranched glycopolymer, prepared by atom transfer radical copolymerization of an acrylate monomer (A) and an acrylate inimer (B), both carrying mannose ester pendants. The MS data confirmed the incorporation of multiple inimer repeat units, which ultimately lead to the hyperbranched material. The various possible structures of n-mers with the same composition were subsequently elucidated based on MS(2) and MS(n) studies. The characteristic elimination of bromomethane molecule provided definitive information about the comonomer connectivity in the copolymeric AB2 trimer and A2B2 tetramer, identifying as present only one of the three possible trimeric isomers (viz. sequence BBA) and only two of the six possible tetrameric isomers (viz. sequences BBA2 and BABA). Complementary IM-MS studies confirmed that only one of the tetrameric structures is formed. Comparison of the experimentally determined collision cross-section of the detected isomer with those predicted by molecular simulations for the two possible sequences ascertained BBA2 as the predominant tetrameric architecture. The multidimensional MS approaches presented provide connectivity information at the atomic level without requiring high product purity (due to the dispersive nature of MS) and, hence, should be particularly useful for the microstructure characterization of novel glycopolymers and other types of complex copolymers.

Fig. 1

Monomer and inimer used for the synthesis of the glycopolymer studied (top); the inimer includes both an initiating moiety and a polymerizable group. The monomer and inimer can copolymerize to yield linear and branched chain segments (bottom).

Fig. 2

MALDI mass spectrum of the glycopolymer studied. Four [M + Na]⁺ ion distributions of A_nB_m oligomers, m = 1–4, are clearly discerned. Each oligomer observed includes m Br atoms. The insets show the structures of A and B (with their pendants shadowed by different colors) and the measured and calculated isotope pattern of one oligomer (A₃B₂), which corroborates the detection of brominated acrylates upon MALDI. Monoisotopic m/z ratios are given on top of the peaks. R = CH₃CO.

Fig. 3

(a) ESI-MS² (CAD) mass spectrum of sodiated A₁B₁ (m/z 949.1); (b) MS³ (CAD) mass spectrum of m/z 889.1, formed by acetic acid (AcOH) loss from sodiated A₁B₁; (c) MS⁴ (CAD) mass spectrum of m/z 829.1, formed by AcOH loss from m/z 889.1. The numbers on top of the peaks give the monoisotopic m/z ratio (in black) and the mass of the neutral loss(es) in Da (in color); −60 and −42 indicate losses of acetic acid and ketene, respectively; −80 and −94 indicate losses of HBr and CH₃Br, respectively; −330, −270 and −210 indicate losses of a mannose pendant or a mannose pendant that lost one or two AcOH molecules, respectively. The subscripts indicate the number of losses.

Fig. 4

MALDI-MS² mass spectra of sodiated (a) A₂B₁ (m/z 1351.3) and (b) A₃B₁ (m/z 1753.4); the numbers on top of the peaks give the monoisotopic m/z ratio (in black) and the mass of the neutral loss(es) in Da (in color). The insets show the structures of A₂B₁ and A₃B₁.

Fig. 5

ESI-MS² (CAD) mass spectra of sodiated A₁B₂ (m/z 1473.3); the numbers on top of the peaks give the monoisotopic m/z ratio (in black) and the mass of the neutral loss(es) in Da (in color). The inset shows schematic representations of the three isomers possible with the A₁B₂ stoichiometry and the actual structure of the A₁B₂ architecture from which CH₃Br can be eliminated to form a five-membered ring lactone.

Fig. 6

(a) ESI-MS² and (b) MALDI-MS² mass spectra of sodiated A₂B₂ (m/z 1875.3); the numbers on top of the peaks give the monoisotopic m/z ratio (in black) and the mass of the neutral loss(es) in Da (in color). The insets show (a) the isomers possible with the A₂B₂ stoichiometry and (b) the A₂B₂ architectures from which CH₃Br can be eliminated to form a five-membered ring lactone. The low-mass fragments are not detected in the ESI-MS² spectrum due to the low-mass cutoff in CAD experiments with QIT instrumentation.

Fig. 7

ESI-IM-MS drift time distributions of sodiated (a) A₂B₁ (m/z 1351.3), (b) A₃B₁ (m/z 1753.4) and (c) A₂B₂ (m/z 1875.3); at each m/z value, three peaks are observed, corresponding to ions with +1 to +3 sodium charges. (d) Charge states were determined from the isotope patterns in the mass spectra extracted from each IM-separated peak, as exemplified for the +2 and +1 peaks of A₂B₁.

Scheme 1

Charge-remote 1,5-H rearrangements in sodiated A₁B₁, leading to (a) AcOH and CH₂CO losses from the mannose ring and (b) expulsion of the sugar pendant from monomer unit A; the Na⁺ has been omitted for brevity. Note that AcOH and CH₂CO losses can occur at the mannose group of A (as shown) as well as the mannose group of B. (c) Intramolecular nucleophilic displacement of Br by the adjacent ester group to form a five-membered ring lactone with concomitant elimination of CH₃Br. The latter reaction is possible only at a terminal unit (A or B) attached at the branching site of an inimer unit B (see text).