Modern Electrospray Ionization Mass Spectrometry Techniques for the Characterization of Supramolecules and Coordination Compounds

Mass spectrometry is routinely used for myriad applications in clinical, industrial, and research laboratories worldwide. Developments in the areas of ionization sources, high-resolution mass analyzers, tandem mass spectrometry, and ion mobility have significantly extended the repertoire of mass spectrometrists; however, for coordination compounds and supramolecules, mass spectrometry remains underexplored and arguably underappreciated. Here, the reader is guided through different tools of modern electrospray ionization mass spectrometry that are suitable for larger inorganic complexes. All steps, from sample preparation and technical details to data analysis and interpretation are discussed. The main target audience of this tutorial is synthetic chemists as well as technicians/mass spectrometrists with little experience in characterizing labile inorganic compounds.


Case Example of Am Mn
The above discussed [Ring M ] -are able to encapsulate ammonium cations, and in this detailed case example the data of a polymetallic hybrid inorganic-organic rotaxane with the formula  S1). 2,3Previous crystal structure analysis suggested that the tert-butyl groups of the cation are too bulky to allow the slipping of the thread through the ring, indicating a kinetically trapped rotaxane structure. 3,4The monoisotopic mass (sum of the accurate masses of most abundant isotopes) was calculated as 2571.8Da.The sample of Am Mn was prepared in 7:3 in a solution of Toluene:MeOH and with 500 µM NaI.

Table S1:
The solvent mixture was empirically determined to include a solvent that can dissolve the analyte Am Mn (toluene) and one that is able to dissolve NaI (methanol), which was used to enhance the signal of the sodiated ion.Sample concentration was set at 10 µM based on standard concentrations used on the instrument of choice (Select Series Cyclic IMS by Waters Corp.). 5ere are two main regions of interest in the mass spectrum, one between 1240 and 1340 m/z and one around 2600 m/z (Figure S3a).The most obvious place to look for our analyte with the monoisotopic mass of 2571.8Da is the latter region, where only +1 ions are present.
As Am Mn is neutral and NaI was added for signal enhancement, [Am Mn + Na] + is the most likely occurring ion.The isotopic pattern was calculated, and showed high agreement with the most intense peak in that region with a maximum at 2595.8 Da (comparable to the agreement in Figure 2a).The second most intense peak is at 2611.8, which is difference of 16 Da.This could correspond to the oxidation with a single oxygen atom, but is also typical for the difference between the alkali metal adducts of Na + (M = 23 Da) and K + (M = 39 Da).The former would likely require the presence of a neutral oxygen, as z remains +1, which suggests that the latter is more likely and the ion at 2611.8 Da corresponds to [Am Mn + K] + .This was confirmed with the simulation of the isotopic distribution and accurate mass, and similarly for the protonated ion [Am Mn + H] + at 2573.9 Da.
The most intense peaks are in the region between 1240 and 1340 m/z, and these are all doubly charged (Figure S3a Zoom).Their masses are therefore in the region of the molecule Am Mn , and the assignments [Am Mn + 2 Na] 2+ (1309.4Da), [Am Mn + Na + K] 2+ (1317.9Da) and [Am Mn + 2 K] 2+ (1325.9Da) are hence relatively obvious, confirmed with the simulated isotopic patterns.The ions at 1247.4 Da and 1255.9Da are also doubly charged, and most likely correspond to the loss of negative charged fragments from Am Mn .Scientifically, and from mass considerations, the most likely explanation is the loss of one pivalate ligand (M = 101 Da) and the simultaneous addition of an alkali metal cation.Once again, based on comparisons of isotopic patterns, the two ions were identified as [(Am Mn -Piv) + Na] 2+ and [(Am Mn -Piv) + K] 2+ .
The mass spectrum of Am Mn in positive mode shows no global patterns of repeating units, except for some polymeric units between 750 and 950 m/z (Figure S3a).As they are singly charged cations, the distance of 14 m/z between the peaks corresponds to 14 Da, which is likely due to the subsequent addition of -CH 2 -groups.The masses of these species are significantly below the mass of Am Mn , and are hence not further discussed.Similarly anything in the region below 750 m/z only involves singly charged cations, which are most likely contaminations and not relevant for our studies.For example, the most prominent peaks of these regions at 685 and 701 m/z were identified as contaminations in plastic ware, as found in the blank mass spectrum.While all the above peaks do not necessarily have to occur, this spectrum highlights how unideal, but real data looks like.The ions [Am Mn + H] + and [Am Mn + Na] + were investigated using CID, and the protonated species fragments showed the loss of a pivalic acid (HPiv) unit, whereas the sodiated ion loses the secondary ammonium cation TAm + and a pivalate ligand as the main channel, leading to [(Ring Mn -Piv) + Na] + (Figure S3b for sodiated species).The E 50 values were quantified under comparable instrument conditions, yielding values of E 50 = 0.25 eV ([Am Mn + H] + ) and E 50 = 1.10 eV ([Am Mn + Na] + ), showing a significantly higher stability of the sodiated ion.
IM allowed to understand the structure of both rotaxane ions [Am Mn + A] + (A = H + , Na + ), and their collision cross section distribution revealed that the sodiated species is slightly larger than the protomer. 2The combination of CID and IM was applied to investigate how a) the structures of the rotaxane ions change upon collisional activation, and b) what structures the fragments exhibit.The sodiated species [Am Mn + Na] + loses the ammonium cation (Figure S3b), and the mechanism of this disassembly is critical in understanding whether these architectures are pseudorotaxanes, where the ring can slip of the thread, or rotaxanes, where this is not possible and the ring has to break in order to release the thread.IM is a great tool to investigate these questions, however, these discussions are beyond the scope of this article and details can be found in the corresponding publications. 2,3Overall, this case example illustrates how the mass spectrum of the polymetallic supramolecule Am Mn can be analysed, and how the combination of high-resolution mass spectrometry, tandem mass spectrometry including stability analysis, and ion mobility can characterise its different properties.

Figure S2 :
Figure S2: Crystal structure of the ternary complex involving Z 2 with one endo-complexed CHCl 3 (no figure was available for endo-complexed acetone), and two exo-coordinated PF 6 -.Hydrogen bonds are shown as dashed orange lines.Reproduced from Ref. 1 with permission, © 2017 Wiley-VCH Verlag GmbH & Co. KGaA.
Overview of the discussed polymetallic rings [Ring M ] -, the thread TAm + and the rotaxanes Am M .Reproduced from ref. 3, © 2022 The Authors.

Figure S3 :
Figure S3: a) Mass spectrum of 10 µM Am Mn in 7:3 Toluene:MeOH with 500 µM NaI including Zoom.Occurring peaks are labelled, including possible small molecule and polymer contaminations.*Ions are assigned as [(Am Mn -Piv) + Na] 2+ and [(Am Mn -Piv) + K] 2+ .b) MS 2 data from [Am Mn + Na] + (m/z = 2596) at a collision energy of E lab = 100 eV.Fragmentation pathways are labelled, and the dominant primary channel includes the loss of the secondary ammonium thread.Schematic structures of precursor ion and primary fragment are shown (Cr: green, Mn: black, N: blue, C: gray).Reproduced from ref. 3, © 2022 The Authors.