Synthesis of model humic substances: a mechanistic study using controllable H/D exchange and Fourier transform ion cyclotron resonance mass spectrometry

Abstract

The products of the oxidative coupling of phenols are frequently used as synthetic analogues to natural humic substances (HS) for biomedical research. However, their molecular compositions and exact structures remain largely unknown. The objective of this study was to develop a novel approach for the molecular-level analysis of phenolic polymerisates that is capable of inventorying molecular constituents and resolving their distinct structural formulas. For this purpose, we have synthesized the model HS using the oxidative coupling of a specifically designed phenylpropanoic monomer, 3-(4-hydroxy-3-methoxyphenyl)-3-oxopropionic acid, to hydroquinone. We have characterized the synthesized model HS using high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS), (1)H NMR spectroscopy, and controllable hydrogen/deuterium (H/D) exchange. We succeeded in the molecular inventory of the model HS. The assigned molecular formulas occupied the substantial space of CHO compositions in the Van Krevelen diagram with a maximum density found in the regions of tannins and lignins, resembling those of natural HS. To identify the exact structural formulas of the individual constituents in the model HS, we have applied selective H/D exchange of non-labile backbone protons by a choice of basic or acidic catalytic conditions followed by FTICR MS. The determined formulas allowed us to verify the proposed pathways of hydroxylation and carboxylation in the course of the phenolic coupling and to identify the acetylation of aromatic rings as an important side reaction. We conclude that the proposed analytical approach may be used to identify the molecular carriers of biological activity within the phenolic polymerisates and eventually within natural HS.

Hydrogen/deuterium exchange in mass spectrometry

“…The exchange of the labile amide hydrogens for deuterium in the solution is used for the studying of the protein conformation dynamic. The exchange of labile hydrogens from −OH groups and exchange of non‐labile −CH hydrogens can be used for the obtaining structural information about individual compounds of complex organic mixtures such as humic substances . Gas phase exchange can be applied for fundamental studies of the folding and unfolding of macromolecular ions in the vacuum as well as for determination of particular reaction center in organic molecules .…”

“…The enumeration of carboxylic groups can be performed based on deuteromethylation . Preliminary information about the position of substituents on the aromatic ring can be obtained using the acid and base catalyzed exchange of the −CH hygrogens (see Figure ). By the simultaneous analysis of the isotopic exchange data the structure of the individual compounds of the humic substances can be predicted.…”

“…Now the performing of the H/D exchange reaction in the ion source has become the reliable analytical technique. This approach enables enumeration of the functional groups in the individual molecules of the natural complex mixtures such as petroleum, dissolved organic matter, bio‐oil . The in‐ESI source H/D exchange allows to observe different conformations of oligosaccharides, investigate a rearrangement of subunits in protein‐protein complexes .…”

CID fragmentation, H/D exchange and supermetallization of Barnase-Barstar complex

“…Recently it was suggested to perform the H/D exchange reaction directly in the ESI source 28, 29 . This approach was applied to study proteins 30–32 , oligosaccharides 33 , natural complex mixtures 34–37 and small organic molecules 38 .…”

“…One of the advantageous features of the gas phase H/D exchange reaction is the use of elevated temperatures in the region of droplet evaporation, when several unusual gas phase reactions can take place. One of such reactions is the supermetallization 30 , which is also described in the paper. The investigation of the pathways of fragmentation of supermetallized complexes may provide structural information whic cannot be obtained in the H/D exchange experiments because metal atoms are less labile than protons, so the effect of scrambling would be minimized 39–42 .…”