Oleg V. Boyarkin

Controlling the stereoselective outcome of glycosidic bond formation remains a major challenge in chemical synthesis. This process typically proceeds along a complex SN1-SN2 mechanistic continuum. In solution, the key contributions to these mechanisms are difficult to determine due to influence by structural and environmental variables, such as solvents, counterions or temperature. To isolate the intrinsic factors driving steroselectivity in glycosylations, we investigate the reactions for the monosaccharide galactose in the gas phase. We perform ion–molecule galactosylation reactions in complete isolation inside an ion trap of a mass spectrometer and directly probe the stereochemistry of the formed products using mass-resolved ultraviolet cold ion spectroscopy. The results show that α-selectivity is significantly weaker in the gas phase than in solution, and that the product stereochemistry might be influenced by the nucleophile’s electron density rather than its overall nucleophilic strength.

UV-induced phosphorescence from the lowest triplet state T₁ of tryptophan (Trp) residues is widely used to monitor the structural dynamics of host proteins on long time scales. Probing the intrinsic properties of this optically "dark" state requires studies of Trp isolated in the gas phase, which are challenging due to low sample concentrations and the need to monitor the triplet over extended time scales. We discovered that excitation of cold, protonated noncovalent TrpH⁺-(H₂O)_n complexes (n ≥ 6) by UV light of specific wavelengths induces evaporation of two water molecules and promotes tryptophan to the triplet state. Subsequent photofragmentation dynamic, monitored by mass spectrometry and ion spectroscopy, yields rate constants for intrinsic triplet-state quenching via phosphorescence and reverse intersystem crossing. The T₁ lifetime is approximately 1 s at 7 K and is dominated by phosphorescence; it decreases to tens of milliseconds at ∼40 K and is estimated to be ∼10 ms at room temperature.

The identification of chemical structures of metabolites available in trace-level quantities remains a challenging task in analytical chemistry. Although cold ion spectroscopy (CIS) has considerable potential to assist in such identification, the widespread use of the method is currently hindered by the lack of suitable commercial instrumentation. In this study, we present a compact CIS add-on module, which was noninvasively interfaced to an Orbitrap-based host mass spectrometer (MS). This instrument was employed to demonstrate how, in certain cases, high-resolution UV-MS photofragmentation CIS allows for the identification of the chemical structure of a metabolite from a pool of rationally predicted candidates. We show that MS detection of specific "prompt" photofragments indicates the attachment of the associated labile functional group to the chromophores of a metabolite, while a comparison of the UV spectrum of the metabolite with the spectra of chromophore standards determines the exact location of the group. As a proof of principle, we successfully applied this workflow to the blind structural identification of a metabolite of a prospective drug.

Cold ion spectroscopy coupled with mass spectrometry and quantum chemical calculations enhanced by neural network search reveal 3D structures of Phe ₂ oligomers.

Enantiomers of a lipid erythro-sphingosine have been quantified with ≈4% accuracy by UV cold ion spectroscopy of their non-covalent complexes with a chiral aromatic molecule. The diastereomeric configuration of such complexes enables the quantification using just a single enantiomeric lipid standard and the identification of non-racemic solutions with no standards at all.

Protein ubiquitin in its +7 charge state microhydrated by 5 and 10 water molecules has been interrogated in the gas phase by cold ion UV/IR spectroscopy. The complexes were formed either by condensing water onto the unfolded bare proteins in a temperature-controlled ion trap or by incomplete dehydration of the folded proteins. In the case of cryogenic condensation, the UV spectra of the complexes exhibit a resolved vibrational structure, which looks similar to the spectrum of bare unfolded ubiquitin. The spectra become, however, broad-band with no structure when complexes of the same size are produced by incomplete dehydration under soft conditions of electrospray ionization. We attribute this spectroscopic dissimilarity to the structural difference of the protein: condensing a few water molecules cannot refold the gas-phase structure of the bare ubiquitin, while the retained water preserves its solution-like folded motif through evaporative cooling. This assessment is firmly confirmed by IR spectroscopy, which reveals the presence of free NH and carboxylic OH stretching vibrations only in the complexes with condensed water.

The geometry of biomolecules isolated in the gas phaseusuallydiffers substantially from their native structures in aqueous solution,which are the only ones truly relevant to life science. To connectthe high resolution of cold ion spectroscopy that can be achievedin the gas phase and the key role of intermolecular hydrogen bondsthat shape biomolecules in water, we study protonated tryptophan microhydratedby 1-6 water molecules. IR/UV spectra measured with the sameinstrument under similar conditions appear to be identical for thecomplexes of the same size produced by soft dehydration and cryogeniccondensation methods. This observation points to the lack of kinetictrapping in the dehydration/rehydration processes. Quantum chemistrycomputations allow for the unambiguous assignment of the measuredIR spectra to the most stable conformers of the complexes. The calculationsreveal that retaining as few as four water molecules still conservesmost of the TrpH(+) native structural features.

Aminoacids and peptides generally exhibit zwitterionic forms withsalt bridge (SB) structures in solution but charge-solvated (CS) motifsin the gas phase. Here, we report a study of non-covalent complexesof the protonated amino acid arginine, ArgH(+)(H2O)( n ) (n = 1-5),produced in the gas phase from an aqueous solution with a controllednumber of retained water molecules. These complexes were probed bycold ion spectroscopy and treated by quantum chemistry. The spectroscopicchanges induced upon gradual dehydration of arginine were assignedby structural calculations to the transition from SB to CS geometries.SB conformers appear to be present for the complexes with as few as3 retained water molecules, although energetically CS structures shouldbecome prevailing already for ArgH(+) with 7-8 watermolecules. We attribute the revealed kinetic trapping of argininein native-like zwitterionic forms to evaporative cooling of the hydratedcomplexes to as low as below 200 K.

We present herein the design of a nano-electrospray ion source capable of reliable generation of large quantities of microsolvated ions. The source is based on a triple molecular skimmer scheme and can be quickly tuned to generate bare ions or their ionic complexes with up to more than 100 solvent molecules retained from solution. The performance of this source is illustrated by recording the mass spectra of distributions of ionic complexes of protonated water, amino acids, and a small protein ubiquitin. Protonated water complexes with more than 110 molecules and amino acids with more than 45 water molecules could be generated. Although the commercial ion source based on the double ion funnel design with orthogonal injection, which we used in our laboratory, is more efficient in generating ions than our triple skimmer ion source, they both exhibit comparable short-term stability in generating bare ions. In return, only the new source is capable of generating microsolvated ions.

The difference in functionality of many isomeric biomolecules requires their analytical identification for life science studies. We present a universal approach for quantitative identification of different small- to medium-sized isomeric biomolecules that can be brought to the gas phase from solution by electrospray ionization (ESI). The method involves infrared (IR) fragment cold ion spectroscopy of analyte molecules that are incompletely desolvated by soft ESI. The use of solvent molecules as natural tags removes a need for adding to solutions any special compounds, which may interfere with liquid chromatography or mass spectrometric measurements. The tested peptides and especially monosaccharides and lipids exhibit highly isomer-specific IR fragment spectra of such noncovalent complexes, which were produced from water, methanol, acetonitrile, and 2-butanol solutions. The relative concentrations in solution mixtures of, for instance, two isomeric dipeptides can be quantified with the accuracy of 1.6% and 2.9% for the acquisition time of 25 min and, potentially, 5 s, respectively; for three isomeric phospho-octapeptides, the accuracy becomes 4.1% and 11% for 17 min and, potentially, 10 s measurements, respectively.