Fluorinated Man9 as a High Mannose Mimetic to Unravel Its Recognition by DC-SIGN Using NMR

Fluorinated Man9as a High Mannose Mimetic to Unravel Its Recognition by DC-SIGN Using NMR Adrián Silva-Díaz, ∥ Jonathan Ramírez-Cárdenas, ∥ Juan C. Munoz-García, M. Carmen de la Fuente, Michel Thépaut, Franck Fieschi, Javier Ramos-Soriano,*Jesus Angulo,*and Javier Rojo* Cite This: J. Am. Chem. Soc. 2023, 145, 26009−26015 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Lectins are capable of reading out the structural information contained in carbohydrates through specific recognition processes. Determining the binding epitope of the sugar is fundamental to understanding this recognition event. Nuclear magnetic resonance (NMR) is a powerful tool to obtain this structural information in solution; however, when the sugar involved is a complex oligosaccharide, such as high mannose, the signal overlap found in the NMR spectra precludes an accurate analysis of the interaction. The introduction of tags into these complex oligosaccharides could overcome these problems and facilitate NMR studies. Here, we show the preparation of the Man9of high mannose with some fluorine tags and the study of the interaction with its receptor, dendritic cellspecific intercellular adhesion molecule-3-grabbing nonintegrin (DCSIGN). This fluorinated ligand has allowed us to apply heteronuclear two-dimensional (2D) 1H,19F STD-TOCSYreF NMR experiments, using the initial slope approach, which has facilitated the analysis of the Man9/DC-SIGN interaction, unequivocally providing the binding epitope. ■INTRODUCTION Dendritic cell-specific intercellular adhesion molecule-3-grab- bing nonintegrin (DC-SIGN) 1 is a C-type lectin that has attracted the interest of the scientific community during the last two decades due to its key role in pathogen infection and immunomodulation processes. 2 This lectin recognizes carbohydrates, mainly high mannose (Man9GlcNAc2) and fucosylated oligosaccharides, in a Ca2+-dependent way. Man9GlcNAc2decorates several pathogen envelope glycoproteins, such as HIV gp120 or Ebola virus GP1. 3,4 Understanding how DC-SIGN recognizes Man9GlcNAc2is a topic of enormous interest, but at the same time, it remains a big challenge. Although some X-ray crystal structures of complexes of DC-SIGN with oligosaccharides, from Manα1,2Man disaccharide up to Man6hexasaccharide, 5−9 have been solved, providing relevant structural features at the molecular level of this recognition, the structure of the DC-SIGN/Man9complex has remained elusive despite all of the efforts devoted to its resolution. The size, flexibility, and nature of the Man9 oligosaccharide make this a challenging goal. Furthermore, the fixed picture obtained in the solid state for the multiple binding modes exhibited typically by carbohydrates/lectin recognition processes provides only partial information about the event. NMR has been successfully used to analyze protein/ carbohydrate complexes in solution at the molecular level. 10 Different techniques such as STD NMR, tr-NOE, etc., in combination with CORCEMA-ST, can provide accurate information on the different binding modes that carbohydrates can show in the recognition process by lectins and facilitate the identification of the binding epitope. 11 For complex oligosaccharides such as Man9with strong NMR signal overlap, isotopic labeling (15N for the protein partner and/or 13C for the carbohydrate ligand) is required. Recently, the recognition of 13C-labeled Man9by DC-SIGN and microvirin has been studied by NMR spectroscopy. 12 These studies have shown that DC-SIGN recognizes an epitope of Man9that does not agree with the information previously published 7 based on the X-ray crystallographic analysis of its complex with Man6. Therefore, more information is necessary to have a better picture of how Man9is recognized by DC-SIGN. In this context, new strategies are demanded to develop useful chemical tools for gaining structural information on complex carbohydrate recognition by lectins in solution. Fluorinated mannosyl di- and trisaccharides have been Received: June 13, 2023 Revised: November 7, 2023 Accepted: November 7, 2023 Published: November 18, 2023 Articlepubs.acs.org/JACS © 2023 American Chemical Society 26009 https://doi.org/10.1021/jacs.3c06204 J. Am. Chem. Soc. 2023, 145, 26009−26015 This article is licensed under CC-BY 4.0 Downloaded via UNIV DE SEVILLA on April 29, 2025 at 10:42:52 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. synthesized and have proven to be valuable tools to diminish the signal overlap in 1H NMR and also to allow the use of 19F NMR. 13 These fluorinated sugars have provided structural information about their interaction with DC-SIGN by NMR. 14 For instance, using 19F NMR relaxation filter experiments allowed to evaluate the relative affinities and binding modes of several fluorinated monosaccharides (fucose, mannose, glucose, and galactose) toward DC-SIGN. 15 Based on these precedents, we have synthesized a fluorine-tagged Man9(F- Man9) with the aim of gaining fundamental information about the binding epitope of this Man9oligosaccharide with DCSIGN. The new picture of this recognition event will provide the basis for the design of better and more selective ligands to target this receptor with higher specificity. ■RESULTS AND DISCUSSION To this end, and considering that the configuration of the reducing end plays only a minor role in DC-SIGN recognition, 16 F-Man91with αconfiguration at the reducing end, synthetically more accessible than the β-epimer, 19F- labeled at C-2 or C-6 positions, was rationally designed (Scheme 1). While the disaccharides (yellow) of the pentasaccharide building block were 19F-labeled at the C-2 positions for synthetic simplicity, the internal branching mannosyl unit (green) supporting the D2- and D3-arms was fluorinated at the same position because the C-6 position was glycosylated. Similarly, the reducing end terminal residue (red) was 19F-labeled at the C-2 position. C-6 fluorination in the trisaccharide (blue) was selected in order to avoid 19F-signal overlap, with the rest of the fluorinated mannosyl units complicating the NMR analysis. Based on our previous convergent straightforward synthetic strategy to obtain Man9oligosaccharides, 16,17 we envisioned the synthesis of F-Man9(1) in a similar way (Scheme 1). The synthesis of building blocks 5,7, and 14 was afforded from 1,3,4,6-tetra-O-acetyl-2-deoxy-2-fluoro-D-mannopyranose 2. 18 Briefly, the synthesis of acceptor 5started with the formation of glycosyl thiol 3 18 followed by acetyl group removal to yield thioglycoside 4. After that, four sequential synthetic steps with a final chromatography purification on silica gel afforded acceptor 5in a 41% yield. This consecutive approach involved the chemoselective protection of C-6 and C-3 hydroxyl groups as silyl ethers, benzoylation at the C-4 position, and, finally, the selective removal of silyl groups. Second, a Zemplen deprotection−benzoylation sequence of 2followed by the selective removal of the anomeric benzoyl ester and treatment with trichloroacetonitrile and DBU afforded the corresponding trichloroacetimidate 7. The glycosylation reaction of this compound with acceptor 8 19 in the presence of a catalytic amount of TMSOTf as a promoter afforded disaccharide 9in a 76% yield. Disaccharide 9was converted to the corresponding trichloroacetimidate 10 by the selective removal of the S-Tolyl group with NBS/water and subsequent reaction of the C-1 hydroxyl group with trichloroacetonitrile and DBU. Then, a 2 + 1 glycosylation of donor 10 with acceptor 5yielded in a single step (90% yield) the pentasaccharide 11 using TMSOTf as the glycosylation promoter. The synthesis of the terminal mannoside 14 started with the glycosylation of trichloroacetimidate 12 20 with 2-bromoetha- Scheme 1. Synthesis of Fluorinated Man9Oligosaccharide 1 (F-Man9) Journal of the American Chemical Society pubs.acs.org/JACS Article https://doi.org/10.1021/jacs.3c06204 J. Am. Chem. Soc. 2023, 145, 26009−26015 26010 nol. Next, the substitution of bromine with an azido group gave compound 13 in excellent yield. Zemplen deprotection and subsequent protection of C-4 and C-6 hydroxyl groups as benzylidene acetal provided the desired acceptor 14 in a very good yield. The 6-deoxy-6-fluoro compound 15 was obtained from D- mannose via one-pot tritylation and benzoylation, followed by trityl removal under acidic conditions. Fluorination at C-6 with DAST was performed as previously described elsewhere to give compound 15. 21 This intermediate was subjected to anomeric deprotection with hydrazine acetate and treatment with trichloroacetonitrile in basic media to give trichloroacetimidate 16, which was glycosylated with disaccharide acceptor 17 17 to afford trisaccharide 18 in a 77% yield. Glycosylation of the S-tolyl donor 18 with the hydroxyl group at C-3 of the acceptor 14 using NIS and TfOH at −20 °C gave tetrasaccharide 19 bearing the benzylidene group spanning C-4 and C-6 positions, which was removed in acidic media, with p-TsOH in acetonitrile affording tetrasaccharide 20 in high yield. With the two required building blocks 11 and 20 in hand, the protected nonasaccharide 21 was assembled by glycosylation of the tetrasaccharide acceptor 20, with pentasaccharide donor 11 in a 41% yield. The final step to obtain F-Man91 was the global deprotection of nonasaccharide 21 (82% yield) using NaOMe in methanol. The interaction of F-Man91with DC-SIGN was analyzed in solution using STD NMR experiments. First, standard onedimensional (1D) 1H STD NMR experiments were carried out. Due to a strong 1H signal overlap, only STD signals from the anomeric protons could be accurately integrated (Figure 1). This provided very limited structural information about the oligosaccharide contacts with the protein (binding epitope mapping). This first analysis supported that the nonreducing Man residues at D1 and D2 branches make the most intimate contacts with DC-SIGN (Figure 1). However, to be able to answer the key question of whether there is a preferential recognition of any branch of the nonamannoside by DC-SIGN, a more detailed comparison of the total saturation transferred to each terminal mannose residue, measured as an average of different ring protons, was necessary, which was not possible by standard 1D 1H STD NMR. We demonstrate here that the introduction of fluorine atoms into the F-Man9oligosaccharide, indeed, is instrumental to access a more reliable description of the determinants for the molecular recognition by DC-SIGN. The presence of the spectroscopically orthogonal 19F spins allowed us to take advantage of the recently developed 2D 1H,19F STD- TOCSYreF NMR experiment. 13 Thanks to the TOCSY mixing element, the information on saturation transfer is extended to further protons within the same spin system for each fluorinated Man residue of F-Man9. Besides, the dispersion of signals along the 1H and 19F frequency axes allowed an accurate integration of STD intensities beyond the limited set of well-dispersed 1H signals of the anomeric protons in the 1D 1H STD NMR approach (Figure S1). Any potential disadvantage in terms of an intrinsic loss of sensitivity for the heteronuclear transfer in this experiment is well compensated for by the much better spectral resolution, allowing more detailed epitope information to be obtained. 13 The analysis of the 2D 1H,19F STD-TOCSYreF NMR experiments led to a more complete and accurate binding epitope mapping of the oligosaccharide upon interaction with DC-SIGN (Figure 2). In fact, the results show a more detailed and significantly different picture of the epitope than in the previous 1D 1H STD NMR experiments (Figure 1). We demonstrate here, for the first time, that an initial slope analysis of full 2D 1H,19F STD-TOCSYreF build-up curves is necessary to obtain a reliable binding epitope mapping of the interaction to avoid T1relaxation bias dominating measurements at one single saturation time (Table S1). Indeed, a simplified 2D 1H,19F STD-TOCSYreF single saturation time analysis led to an incorrect determination of the binding epitope mapping of the F-Man9oligosaccharide for its binding to DC-SIGN (Figure S4). Figure 2 shows the 2D 1H,19F STD-TOCSYreF NMR initial slope analysis, which demonstrates that DC-SIGN indeed preferentially recognizes the D2-arm in the context of the whole nonamannoside in solution. What is more, the recognition of the D3-arm is preferred over that of the D1- arm, in contrast to the results from the 1D 1H STD NMR analysis (Figure 1). The results show that both ramifications of the nonamannoside pending from the sugar ring ManV (D2- and D3-arms, from C-3 and C-6 positions of ManV, respectively) are better recognized by DC-SIGN compared to the D1-arm pending from ManI. We further investigated whether the presence of the fluorine atoms in F-Man9could induce conformational differences relative to natural Man9. An extensive conformational study of both ligands was carried out by NOESY and transferred- NOESY experiments. 22 We analyzed 2D NOESY and tr- NOESY spectra of F-Man9and Man9in the absence and Figure 1. Binding epitope mapping of the F-Man9oligosaccharide from 1D 1H STD NMR experiments. Selective protein 1H saturation was achieved by irradiation at 0.3 ppm at different saturation times. Colored spheres represent normalized STD NMR intensities, and the values are indicated by the colored ellipses. STD responses are shown only for protons that could accurately be measured (colored spheres). Asterisks indicate averaged STD values due to 1H overlap. Journal of the American Chemical Society pubs.acs.org/JACS Article https://doi.org/10.1021/jacs.3c06204 J. Am. Chem. Soc. 2023, 145, 26009−26015 26011 presence of DC-SIGN (Figure S5). No major changes in the conformations of the ligands were observed upon binding to the protein. Besides, for both ligands, an analysis of key strong NOEs from 2D tr-NOESY spectra concluded that there are no conformational differences between F-Man9and the natural Man9(Figure S6). We also investigated if the presence of fluorine atoms could impact the affinity of the oligosaccharide for DC-SIGN. Competition studies between fluorinated and nonfluorinated Man9were carried out using STD NMR experiments (Figure S7), clearly demonstrating that both F-Man9and Man9have the same affinity for DC-SIGN. All of these results support that fluorine atoms do not impact neither the conformation of the F-Man9ligand nor the affinity of the interaction, validating the use of the fluorinated Man9as a high mannose mimetic to unravel its recognition by DC-SIGN using NMR. Finally, we used molecular dynamics (MD) simulations to correlate the experimental 2D 1H,19F STD-TOCSYreF NMR to three-dimensional (3D) molecular models of the complex of the nonamannoside with DC-SIGN. Starting from previous 3D models based on X-ray data, 7 three molecular models of the F- Man9and three of the natural Man9oligosaccharides were built bound to DC-SIGN through each of the different arms (D1-, D2-, or D3-bound). The six starting protein−ligand 3D models for the D1, D2, and D3 Ca2+-coordinated orientations were built by superimposition of the nonreducing and adjacent mannose residues of each branch to the alike residues of the Man6ligand bound to DC-SIGN CRD in the PDB structure 2IT5. 7 It should be noted that this PDB structure shows two possible orientations for Man6, which are the so-called major and minor orientations. For ligand superimposition, we resorted to the experimental 1H STD NMR data (Figure 1), which clearly indicated that for the nonamannoside, only a bound conformation with a “minor” orientation of the nonreducing dimannoside on each arm is in agreement with the experimental binding epitope. In particular, 1H STD NMR data showed that the anomeric protons of both the nonreducing and adjacent residues point toward the protein surface, whereas they would be solvent-exposed in the “major” orientation. As a result, in the three starting structures, the residue adjacent to the nonreducing terminal of each of the nonamannoside branches (D1, D2, and D3, corresponding to ManIII, ManVI, and ManVIII, respectively) coordinated the Ca2+ ion of DC-SIGN CRD. No significant steric clashes were observed between the oligosaccharides and the protein in any of the orientations. A normalized protein−ligand contact analysis along the MD simulations revealed that, globally, the nonamannoside oligosaccharide binds preferentially through the D2-arm, in excellent agreement with the experimental NMR data (Figure Figure 2. Structural details of the molecular recognition of the F-Man9oligosaccharide by DC-SIGN from 2D 1H,19F STD-TOCSYreF NMR experiments. Selective protein saturation was achieved by irradiation at 0.3 ppm. Binding epitope mapping of F-Man9from 2D 1H,19F STD- TOCSYreF NMR experiments, using an initial slope analysis of full STD build-up curves (shown as insets). A one-saturation time analysis was demonstrated to be inaccurate for determining the binding epitope of the oligosaccharide (Figure S4). Colored spheres represent normalized STD NMR intensities, only determined for protons that could accurately be measured. Colored ellipses show the total relative STD per mannose residue (average of initial slopes), using the same color code. Journal of the American Chemical Society pubs.acs.org/JACS Article https://doi.org/10.1021/jacs.3c06204 J. Am. Chem. Soc. 2023, 145, 26009−26015 26012 3). The MD simulations showed a well-defined binding mode of the terminal ManVII−ManVI ending disaccharide within the binding pocket, with OH-3 and OH-4 hydroxyl groups of ManVI coordinating the Ca2+ ion, whereas the two remaining arms, D1 and D3, where highly flexible (Figure 4). Overall, our data show that in the context of the whole nonamannoside, Man9, each branch is recognized by DCSIGN CRD at the Ca2+ binding site through the residue adjacent to the nonreducing mannose, with a preference for the D2-arm. In the context of previous data in the literature, this mode of binding corresponds to the so-called “minor” orientation. 7,23 Our experimental evidence, and theoretical data, are consistent with the existence of only one orientation (Figure 4) for each nonreducing dimannoside terminal in Man9, excluding the presence of the so-called “major” orientation. 7 This highly relevant result indicates that, compared to the binding of smaller oligosaccharides mimicking different segments of Man9, 14,23 the whole nonamannoside does not show multiple binding modes for the recognition of its nonreducing terminal mannose residues by DC-SIGN. This is the first time that such a detailed study of the preferential branch recognition of Man9by DC-SIGN has been performed in solution, and the results further underscore the importance of using 19F labeling, along with an initial slope analysis of the heteronuclear 19F-edited STD NMR experiments for such branched large oligosaccharides to elucidate the populations of each branch in the protein-bound state. ■ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c06204. Experimental procedures, characterization data (1H, 13C, and 19F NMR and HRMS), copies of NMR spectra of new compounds, and NMR and computational details (PDF) ■AUTHOR INFORMATION Corresponding Authors Javier Ramos-Soriano −Instituto de Investigaciones Químicas (IIQ), CSIC −Universidad de Sevilla, Seville 41092, Spain; orcid.org/0000-0002-3054-0679; Email: fj.ramos@ iiq.csic.es. Jesus Angulo −Instituto de Investigaciones Químicas (IIQ), CSIC −Universidad de Sevilla, Seville 41092, Spain; orcid.org/0000-0001-7250-5639; Email: j.angulo@ iiq.csic.es Javier Rojo −Instituto de Investigaciones Químicas (IIQ), CSIC −Universidad de Sevilla, Seville 41092, Spain; Figure 3. Bar graph comparing the experimental binding epitope mapping of F-Man9(center) with the theoretical time-average contact-based epitopes derived from the MD simulations of F-Man9 (left) and Man9(right). The epitope values are individually shown for all three branches: D1 (blue), D2 (orange), and D3 (green). Overall, the pattern D2 > D3 > D1, in terms of the preference of contacts with the protein, is observed in the MD simulations for both the natural and the fluorinated Man9, hence in agreement with the experimental epitope. Figure 4. Superposition of four frames of the MD simulation of DC-SIGN CRD bound to F-Man9with the D2 branch coordinating the Ca2+ ion. Protein and Ca2+ atoms are shown as light gray cartoon and pale-yellow spheres, respectively. The ligand is shown as cyan (carbon atoms), red (oxygen atoms), and green (fluorine atoms) sticks. Key protein residues involved in the interaction with the ligand are shown as light gray sticks and with labels next to them. The three F-Man9branches are labeled as well as the two residues of the D2 branch occupying most of the binding pocket. Journal of the American Chemical Society pubs.acs.org/JACS Article https://doi.org/10.1021/jacs.3c06204 J. Am. Chem. Soc. 2023, 145, 26009−26015 26013 orcid.org/0000-0003-3173-3437; Email: javier.rojo@ iiq.csic.es. Authors Adrián Silva-Díaz −Instituto de Investigaciones Químicas (IIQ), CSIC −Universidad de Sevilla, Seville 41092, Spain Jonathan Ramírez-Cárdenas −Instituto de Investigaciones Químicas (IIQ), CSIC −Universidad de Sevilla, Seville 41092, Spain Juan C. Munoz-García −Instituto de Investigaciones Químicas (IIQ), CSIC −Universidad de Sevilla, Seville 41092, Spain; orcid.org/0000-0003-2246-3236 M. Carmen de la Fuente −Instituto de Investigaciones Químicas (IIQ), CSIC −Universidad de Sevilla, Seville 41092, Spain Michel Thépaut −Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble F-38044, France Franck Fieschi −Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble F-38044, France; Institut Universitaire de France (IUF), Paris 75231, France Complete contact information is available at: https://pubs.acs.org/10.1021/jacs.3c06204 Author Contributions ∥ A.S.-D. and J.R.-C. contributed equally to this work. Notes The authors declare no competing financial interest. ■ACKNOWLEDGMENTS This research was funded by grants from MCIN/AEI/ 10.13039/501100011033 (PID2020-118403GB-I00 and PID2019-109395GB-I00) cofunded by the European Regional Development Fund (ERDF) “A way of making Europe”. J.R.-S. acknowledges the support of Juan de la Cierva-Incorporación (IJC2020-044072-I) and A.S.-D. and J.R.-C. FPI scholarships (PRE2018-083270 and PRE2020-092754, respectively) financed by MCIN/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRT. The authors also acknowledge helpful and useful discussions with Dr Javier López from the NMR facility at the Biomolecular Interactions Platform (cicCartuja) and Dr Tammo Diercks from the NMR Platform at cicBioGUNE. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRSCEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10- INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). F.F. also acknowledges the French Agence Nationale de la Recherche (ANR) PIA for Glyco@Alps (ANR-15-IDEX-02). ■REFERENCES (1) Geijtenbeek, T. B. H.; Torensma, R.; van Vliet, S. 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ChemBioChem 2008,9(14), 2225−2227. ■NOTE ADDED AFTER ASAP PUBLICATION This paper was published on November 18, 2023. Footnotes have been added to Scheme 1. The revised version was reposted on November 21, 2023. Journal of the American Chemical Society pubs.acs.org/JACS Article https://doi.org/10.1021/jacs.3c06204 J. Am. Chem. Soc. 2023, 145, 26009−26015 26015