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. 2026 Aug 5;148(32):34045–34050. doi: 10.1021/jacs.6c09131

Stereoselectivity of Galactosylation Reactions In Vacuo

Chun-Wei Chang †,‡, Andrei Zviagin §, Dana Wehner †,‡, Niklas Geue †,‡, Jacob S Jordan †,‡, Ana Zidar †,‡, Ariane R Wieseke †,‡, Kim Greis †,‡, Gaël M Vos †,‡, Michael Götze †,‡, Oleg Boyarkin §,*, Kevin Pagel †,‡,*
PMCID: PMC13495751  PMID: 42619095

Abstract

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.

graphic file with name ja6c09131_0004.webp

Glycosylation is a fundamental reaction for building complex oligosaccharides in nature. , It links glycosylating agents and nucleophiles to form new glycosidic bonds. The reaction forms a new chiral center at the anomeric position. This in turn, generates two possible stereoisomeric products: α- and β-glycosides. Traditionally, the reaction does not proceed via a single, discrete mechanism. Instead, the mechanism lies along a continuum between two pathways: the unimolecular SN1 and bimolecular SN2 pathways (Figure a). , While kinetic isotope effect experiments are often employed to probe these pathways, the boundary between these two extremes remains elusive. − This uncertainty arises because changes in temperature, solvent, or the structure of the building blocks easily shift the reaction pathway. ,, Consequently, quantifying the individual contributions of SN1 and SN2 throughout the reaction remains a significant analytical hurdle.

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Challenges in the stereoselective installation of glycosidic bonds. (a) The glycosylation mechanism exists on an SN1-SN2 continuum. The reaction often results in a mixture of α- and β-anomers. (b) The SN2 pathway proceeds with stereoinversion at the anomeric center. It allows for predictable product formation. (c) In the SN1 pathway, a cationic intermediate is formed. Nucleophiles can attack the intermediate from either the top or bottom, creating α- or β-glycosides as respective products. (d) Various factors influence the stereochemical outcome of SN1-type glycosylations, ranging from external environmental conditions (solvent, temperature) to the intrinsic properties of the building blocks.

SN2 glycosylation involves covalent glycosyl intermediates and leads to stereoinverted products (Figure b). Nuclear magnetic resonance (NMR) spectroscopy has provided detailed characterization of this process. − Specifically, exchange NMR has enabled the observation of dynamic exchange between glycosyl triflate and the counterions in solution. , Under this framework, the final stereochemical outcome was found to be governed by the Curtin-Hammett kinetic principle, ,, with the product ratio depending on the relative energy barriers of the competing transition states.

In contrast, the understanding of the SN1 mechanism remains limited. The reaction is governed by highly reactive species, known as glycosyl cations (Figure c). The supposed oxocarbenium ions feature an sp 2-hybridized anomeric carbon. , Their planar geometry allows the nucleophile to attack from either the top (β) or bottom (α) face, ultimately dictating the product stereoselectivity. To understand the origin of stereoselectivity, it is essential to characterize the three-dimensional structure of glycosyl cations. However, these cations are highly transient with extremely short lifetimes, posing a significant analytical challenge. ,, Various spectroscopic techniques have been developed to address this. ,− For instance, NMR-based methods analyze these cations by stabilizing them with superacids. , Alternatively, gas-phase infrared (IR) spectroscopy has emerged as a powerful tool for the direct structural analysis of glycosyl cations. − It provides unique vibrational fingerprints that allow the assignment of ion structures, when compared with theoretical spectra simulated from density functional theory (DFT) calculations.

Despite effective methods for characterizing intermediates, the coupling step in glycosylation reactions remains elusive. Its complexity stems from the fact that it involves a bimolecular reaction with a nucleophile. Besides its role as a reactant, the nucleophile alters the sugar conformation to minimize steric hindrance, thereby ensuring an approach along the lowest energy pathway. − In solution, this process is further complicated by external factors (Figure d). , The solvent, for example, orients the cationic intermediate relative to the nucleophile and dictates the reactivity of solvent-separated ion pairs (SSIPs). , Ultimately, the dynamic equilibria between these intermediates and the solvent govern the stereoselectivity of the glycosylation. Another key parameter for tuning sugar reactivity and controlling donor activation is temperature, which dictates both the overall yield and the stereoselective outcome of the glycosylation.

Because these intrinsic and external factors are strongly interconnected in solution, it remains a challenge to determine the true driver of stereoselectivity. It is often unclear whether the outcome depends primarily on the sugar building blocks, the nucleophile, or external solvent effects. This is particularly relevant for galactose, whose building blocks are often highly reactive and exceptionally sensitive to their environment. Even small changes in temperature or solvent can completely alter the stereochemical outcome. Therefore, without observing the glycosyl cations and nucleophiles together in a controlled environment, these individual contributions cannot be easily distinguished. This lack of clarity represents a major hurdle in predicting the formation of challenging linkages, such as 1,2-cis-glycosidic bonds.

To obtain this controlled environment for SN1-glycosylations, we performed galactosylation reactions in complete isolation inside a mass spectrometer (MS). This allows us to study the reactions free from the complicating factors in solution. Technically, this is achieved by trapping the galactosyl cations in an ion trap, where they are brought into reaction with volatile nucleophiles to form a new glycosidic bond. The resulting products are monitored in situ using gas-phase cold ion UV fragmentation spectroscopy (Figure S1). The results reveal that the inherent chemical properties of the reactants have a considerably smaller impact on the SN1-glycosylation than was previously expected from solution-phase experiments.

To conduct galactosylation reactions in the gas phase (Figure a), a benzyl-protected thiogalactoside building block was chosen as precursor. Thiols are commonly used as leaving groups in carbohydrate synthesis and the benzyl-protecting groups prevent unwanted side reactions. Thiogalactoside precursor ions were generated from an organic solution using nanoelectrospray ionization (n-ESI). On the way into MS, the ions undergo controlled in-source dissociation via collisions with ambient air molecules to produce galactosyl cations. Finally, the ions are transferred into MS and stored in a dedicated reaction ion trap at room temperature and a residual gas pressure of 10–4 mbar. While these intermediates are highly reactive in solution, they remain stable in the gas phase under this low pressure due to the inherent lack of nucleophiles in vacuo. Bleeding low-pressure vapor of a nucleophile into the trap initiates a gas-phase galactosylation reaction (Figure b and Figure S2).

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Galactosylation reaction in the gas phase. (a) A galactosyl cation is generated through in-source collision induced dissociation. Methanol vapor reacts with the galactosyl cation producing an α/β mixture of the protonated GalOMe via an SN1 mechanism. (b) Mass spectrometry is used to monitor the gas-phase galactosylation reaction. Electrospray ionization of the thiogalactoside precursor yields protonated, ammonium and sodium adducts. In-source fragmentation of the protonated precursor at m/z 647 yields galactosyl cations at m/z 523. The gas-phase reaction with methanol vapor results in GalOMe product ions at m/z 555. (c) Reference UV fragmentation spectra of the anomerically pure standards of β-GalOMe (blue), α-GalOMe (red), and of the gas-phase galactosylation product (black). The α/β ratio of the in situ product is derived from the best fit of the black trace by a linear combination of the blue and red ones. This analysis relies on distinct spectral fingerprints. A broad feature between 37100 cm–1 and 37300 cm–1 characterizes the α-anomer, whereas distinct bands from 37300 cm–1 and 37600 cm–1 are related to the β-anomer.

A galactosylation reaction yields either α- or β-galactoside anomers. The α/β anomeric ratio of the products formed in solution was determined using NMR and liquid chromatography. To determine the anomeric ratio for the products of the reaction in the gas phase, we employed UV photofragmentation cold ion spectroscopy, which is a well-established method for the analysis of isomeric biomolecules. − It involves measuring UV spectra of the anomerically mixed products and of two anomerically pure α/β-galactoside standards. The mixed spectrum is then numerically decomposed into a linear combination of the two standard reference spectra (Supporting Information, section 3), yielding relative concentrations of the individual anomers in the product mixture. To measure a UV spectrum of the product ions, they are m/z-isolated and transferred from the reaction ion trap into a cryogenic ion trap, where they are collisionally cooled to ∼ 10 K. Next, the ions undergo photofragmentation by a UV laser pulse. Spectra are recorded by monitoring the abundance of fragment ions as a function of laser wavelength. UV spectra of the anomerically pure synthetic α/β-galactosides were recorded similarly, except that the ESI-generated ions were not subjected to in-source fragmentation and no nucleophile vapor was bled into the reaction ion trap.

Isolation of the reactants in the gas phase at low background pressure essentially removes all external interferences present in solution. This allows a precise examination of the stereoselective contribution of the reactants in SN1-glycosylation reactions (Figure c). Spectral differences arise because the orientation of the anomeric O-methyl substituent alters the inductive polarization of the sugar, which in turn shifts the electronic and vibrational energy levels in the UV spectra.

The obtained spectral difference reveals a relative concentration of α-GalOMe produced in the gas-phase of 21 ± 6% (Figures S3 and S4). This is nearly half of the 42% obtained in solution, as determined by NMR (section 8 in Supporting Information). The product ratio reflects the stereoselectivity that arises in a pure SN1-type glycosylation reaction. To assess the stability of the glycosyl products, NMR spectroscopy (in solution) and ion mobility mass spectrometry (in the gas phase) were employed (Figures S5 and S6). These data and literature precedents indicate that the anomeric center is locked, which suppresses low-energy mutarotation pathways. − This suggests that both solution- and gas-phase ratios are meaningful and can be analyzed quantitatively.

To systematically assess the effect of different steric and electronic factors in SN1-glycosylation, we investigated gas-phase galactosylations with different nucleophiles under otherwise identical conditions (Figures S7–S10). The anomeric composition of the gas-phase products at 298 K was compared to that of the solution reactions (Figure ). In solution, the nonparticipating solvent dichloromethane was used to generate conditions that facilitate the SN1 mechanism. Consistently with previous reports, , the solution galactosylation reactions show an inverse relationship between α-selectivity and nucleophile strength (gray bars), such that a higher abundance of the α-product is observed for the weaker nucleophiles (Figure S11).

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Stereoselectivity in solution and gas-phase galactosylation reactions at 25 °C. In the solution phase, α-product formation correlates with the strength of the nucleophile. In contrast, the α-content is significantly reduced in the gas phase and appears to be largely independent of nucleophilic strength.

This general trend stands in a stark contrast to what is observed when the influence of the solution is removed. First, for all the studied nucleophiles, α-selectivity is significantly reduced in gas-phase galactosylation reactions, with a minimum reduction of 1.9 times measured for the methanol substituent. The reaction with the weakest tested nucleophile, trifluoroethanol, exhibits a particularly striking difference between the abundance of the α-product in solution (76%) and in the gas phase (0%). Interestingly, the relative selectivity in the gas phase might be influenced by electronic effects. Electron-rich nucleophiles, such as benzyl alcohol, show a slightly higher α-content than electron-poor nucleophiles such as trifluoroethanol. This data aligns with earlier theoretical calculations suggesting that α-galactoside formation is predominantly driven by electrostatic interactions in vacuo. , Analogous in vacuo glycosylation reactions were also attempted with glucose and mannose (Figures S12–S17); however, these yielded inconclusive results. Future studies will investigate how general the observed effects are for other monosaccharides.

Another fundamental question is how the structure of the intermediate correlates with the observed herein stereoselectivities, and how these correlations differ between solution and gas phase. Previously, we characterized the structure of the perbenzylated galactosyl cation using cryogenic gas-phase IR spectroscopy, suggesting interactions of the C6-OBn group with the positively charged anomeric center (C1). Here, we extend this study with a detailed conformational analysis of the galactosyl cation (Figure S20). The computational results show that, besides previously reported conformations that facilitate C6–C1 interactions and hence α-selectivity, other structures occur at marginally higher energies that would prefereably lead to β-products. , This might rationalize our experimentally observed gas-phase β-selectivity, however this warrants further investigations.

In conclusion, we present herein the first direct evaluation of galactosylation reactions by removing interference from external factors, such as solvent and counterions. This is achieved by performing ion–molecule reactions of glycosyl cations in the gas phase and subsequent in situ sensing of the formed products with mass-selective UV cold ion spectroscopy. Compared to galactosylation reactions in solution, the intrinsic gas-phase α-selectivity is significantly reduced and appears to be independent of the nucleophilic strength. Instead, α-glycosylation appears to be enhanced for electron-donating nucleophiles. These results illustrate how glycosylation reactions are heavily influenced by the surrounding environment. Ultimately, isolating these intrinsic reactivity factors will provide datasets that can be used to train machine learning models in the future to predict solution-phase stereoselectivities. Combining in vacuo insights with solution-phase data and predictive AI could enable the rational design of glycosylation strategies.

Supplementary Material

ja6c09131_si_001.zip (287.1KB, zip)
ja6c09131_si_002.pdf (6MB, pdf)

Acknowledgments

O.B. thanks Swiss National Science Foundation (grant 200020_204072) for supporting this work. K.P. acknowledges generous funding by the European Research Council, ERC-2019-CoG-863934-GlycoSpec. K.G. thanks the Fonds National de la Recherche (FNR) Luxembourg, for funding the project GlycoCat (13549747). C.-W.C. and N.G. thank Lisa Hietzig for assisting with the ion mobility mass spectrometry measurements. G.M.V. acknowledges generous funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under the CRC 1449-Project ID 431232613. N.G. acknowledges the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for funding through a Walter Benjamin fellowship (Project ID: 559720072). J.S.J. acknowledges generous funding by the DFG under the CRC 1340-Project C02. We thank the NMR and Mass Spectrometry core facility team at Freie Universität Berlin for their support with structural characterization.

The data reported in this paper are available in the main text or the Supporting Information. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.6c09131.

  • Deconvolution (ZIP)

  • Additional information as detailed in the text (PDF)

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C.-W.C. and A.Z. contributed equally to this work.

The authors declare no competing financial interest.

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