Journal of Medicinal Chemistry pubs.acs.org/jmc Article added to the supernatant and the mixture was kept overnight at −20 ■ AUTHORINFORMATION °C and centrifuged at 10,000 rpm for 10 min to precipitate glycoproteins. The new supernatant (containing free saccharides) Corresponding Authors was dried under a nitrogen flow, while the new pellet was pooled with the previous one to extract protein- and lipid-bound saccharides. All Isabelle McCort-Tranchepain − Laboratoire de Chimie et samples were subsequently treated with 100 μL of trifluoroacetic acid Biochimie Pharmacologiques et Toxicologiques, CNRS, UMR ((TFA) 0.1 M, 2 h, 80 °C) for selective release of bound sialic acids 8601, Université de Paris, F-75006 Paris, France; orcid.org/ and dried overnight into a vacuum concentrator (Concentrator 5301, 0000-0001-7447-8806; Email: isabelle.mccort@ Eppendorf). Dried samples were incubated with 50 μL of the derivatization solution containing 1,2-diamino-4,5-methylenedioxy- parisdescartes.fr benzene dihydrochloride (DMB, 7 mM), 2-mercaptoethanol (1 M), ̀ Bruno Gasnier − SPPIN - Saints-Peres Paris Institute for the Na S O (18 mM), and TFA (20 mM) for 2 h at 50 °C in the dark. Neurosciences, CNRS, Université de Paris, F-75006 Paris, 2 2 4 Samples were kept at −20 °C before analysis. Quantitative analyses France; orcid.org/0000-0003-4458-6296; 3 were performed by micro-LC/ESI MRM-MS in the positive ion Email: [email protected] mode on an amaZon speed ETD ion trap mass spectrometer (Bruker ̀ Daltonics) equipped with a standard ESI source and controlled by Christine Anne − SPPIN - Saints-Peres Paris Institute for the 2 fragment ions Neurosciences, CNRS, Université de Paris, F-75006 Paris, Hystar software (ver. 3.2). The identification of MS France; Email: [email protected] was based on previous papers.64,65 DMB-coupled sialic acid separation was achieved on Prominence LC-20AB micro LC system (Shimadzu). Samples were diluted 10-fold in formic acid (0.1%), and dilutions Authors were applied (5 μL) to a Luna 3 μm analytical column (C18, 100 Å, Lilian Dubois − Laboratoire de Chimie et Biochimie 150 × 1 mm, Phenomenex) with isocratic elution in acetonitrile/ methanol/water (4:6:90, v/v/v) at 60 μL/min. Multiple reaction Pharmacologiques et Toxicologiques, CNRS, UMR 8601, 3 was used for DMB-coupled sialic acid Université de Paris, F-75006 Paris, France monitoring (MRM) of MS quantification (ion spray voltage of 4500 V; dry gas slow rate of 8 L/ ́ Nicolas Pietrancosta − Laboratoire des Biomolecules, LBM, min). Absolute quantifications were calculated by comparing ion ́ ́ ́ Sorbonne Universite,Ecole Normale Superieure, PSL University, intensities to a standard curve established for DMB-coupled sialic CNRS, F-75005 Paris, France; Neurosciences Paris Seine - acids (Neu5Ac, Neu5Gc, KDN). Results were normalized for the total Institut de Biologie Paris Seine (NPS - IBPS), Sorbonne protein amount (nmol of sialic acids/mg of protein). ́ In another protocol, 250 μLofa85μmol/L solution of Neu5Ac Universite, INSERM, CNRS, F-75005 Paris, France 13 Alexandre Cabaye − Laboratoire de Chimie et Biochimie 1,2,3- C3 (internal standard, IS; Sigma-Aldrich, ref no. 649694) was Pharmacologiques et Toxicologiques, CNRS, UMR 8601, added to each fibroblast pellet. Samples were sonicated (3 × 10 s with Université de Paris, F-75006 Paris, France; BIOVIA, Dassault 5sresting intervals in ice) and assayed for protein concentrations. For ̀ free sialic acid, 100 μL of this homogenate was mixed with 150 μLof Systemes, F-78140 Velizy-Villacoublay, France ̀ acetonitrile (ACN), homogenized, and centrifuged. For the total sialic Isabelle Fanget − SPPIN - Saints-Peres Paris Institute for the acid, 100 μL of the homogenate was mixed with 200 μL of sulfuric Neurosciences, CNRS, Université de Paris, F-75006 Paris, acid (63 mM) and incubated for 1 h at 80 °C for hydrolysis. Samples France were supplemented with 450 μL of ACN, homogenized, and ́ ̀ Cecile Debacker − SPPIN - Saints-Peres Paris Institute for the centrifuged. The two supernatants (free and bound sialic acid) were Neurosciences, CNRS, Université de Paris, F-75006 Paris, 2 66 quantitated by LC/MS as described. Transitions 308.1 > 86.9 (for France Neu5Ac) and 311.1 > 89.9 (for IS) were monitored with a Pierre-André Gilormini − UMR 8576, UGSF, Unité de declustering potential of −50 V and a collision energy of −16 V. Glycobiologie et Fonctionnelle, Université de Lille, CNRS, F- Peak integration was performed with Analyst software (version 1.6.2, 59650 Lille, France Applied Biosystems; smoothing width: 3 points). Neu5Ac concen- Patrick M. Dansette − Laboratoire de Chimie et Biochimie trations were calculated from the Neu5Ac area/IS area ratio and the calibration curve (linear through zero) and normalized to protein Pharmacologiques et Toxicologiques, CNRS, UMR 8601, concentrations. Université de Paris, F-75006 Paris, France; orcid.org/0000- 0002-3694-3348 ■ ASSOCIATED CONTENT Julien Dairou − Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS, UMR 8601, sı *Supporting Information Université de Paris, F-75006 Paris, France The Supporting Information is available free of charge at Christophe Biot − UMR 8576, UGSF, Unité de Glycobiologie https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b02119. et Fonctionnelle, Université de Lille, CNRS, F-59650 Lille, Compound structure, abbreviations, and code numbers; France; orcid.org/0000-0002-7396-1959 synthesis scheme of Fmoc-amino acid 11, 12, 16, 22, 24, Roseline Froissart − Service de Biochimie et Biologie ́ and 43; HPLC conditions and retention time for Moleculaire Grand Est, Centre de Biologie et de Pathologie Est, commercially available compounds; HPLC and HPLC- Hospices Civils de Lyon, F-69677 Bron, France ̀ MS spectra for compounds 3, 13, 45, 46, and 47; Anne Goupil-Lamy − BIOVIA, Dassault Systemes, F-78140 experimental details and analytical data for compounds Velizy-Villacoublay, France ̀ 11, 12, 16, 22, 24−28, and 43; NMR spectra for Hugues-Olivier Bertrand − BIOVIA, Dassault Systemes, F- compounds 45−47; Figures S1 to S5; Supplementary 78140 Velizy-Villacoublay, France Methods; Supplementary References (PDF) Francine C. Acher − Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS, UMR 8601, Molecular formula strings (SMILES) and associated Université de Paris, F-75006 Paris, France; orcid.org/0000- data (CSV). 0002-5413-4181 Sialin homology model docked with 45 (PDB) Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.9b02119 8246 https://dx.doi.org/10.1021/acs.jmedchem.9b02119 J. Med. Chem. 2020, 63, 8231−8249