Influence of the reducing-end anomeric configuration of the Man9 epitope on DC-SIGN recognition

O ganic & Biomolecula Chemis y PAPER Ci e his: O g. Biomol. Chem., 2020, 18, 6086 Recei ed 6 h July 2020, Accep ed 27 h July 2020 DOI: 10.1039/d0ob01380c sc.li/obc Influence o he educing-end anome ic configu a ion o he Man 9 epi ope on DC-SIGN ecogni ion† Noelia de la C uz, a Ja ie Ramos-So iano,* a José J. Reina, a José L. de Paz, a Michel Thépau , b F anck Fieschi, b Ana Sousa-He es a and Ja ie Rojo * a High-mannose (Man 9 GlcNAc 2 ) is he main ca bohyd a e uni p esen in i al en elope glycop o eins such as gp120 o HIV and he GP1 o Ebola i us. This oligosaccha ide comp ises he Man 9 epi ope conjuga ed o wo e minal N-ace ylglucosamines by o he wise a ely-encoun e ed β-mannose glycosidic bond. Fo ma ion o his challenging linkage is he bo leneck o he ew syn he ic app oaches desc ibed o p epa e high mannose. He ein, we epo he syn hesis o he Man 9 epi ope wi h bo h alpha and be a configu a ions a he educing end, and subsequen e alua ion o he impac o his configu a ion on binding o na u al ecep o o high-mannose, DC-SIGN. Using fluo escence pola iza ion assays, we demons a e ha bo h anome s bind o DC-SIGN wi h compa able affini y. These ele an esul s he e- o e indica e ha he mo e syn he ically-accesible Man 9 alpha epi ope may be deployed as ligand o DC-SIGN in bo h in i o and in i o biological assays. In oduc ion Ca bohyd a es a e ubiqui ous in na u e and pa icipa e in many biological e en s ele an o heal h and disease, such as cell g ow h and diffe en ia ion, e iliza ion, in lamma ion, umou p og ession and me as asis, i al in ec ion, and many o he s. 1 The unc ion o ca bohyd a es in hese p ocesses esul s om in e ac ions wi h speci ic ecep o s, mainly p o- eins known as lec ins. 2 Ca bohyd a e–p o ein in e ac ions a e cha ac e ised by a high selec i i y and a low affini y, he la e o which is compensa ed by mul i alen in e ac ions esul ing om he p esen a ion o mul iple copies o he ca bohyd a e epi opes and ecep o s in na u e. 3 Unde s anding ca bo- hyd a e-media ed p ocesses he e o e equi es he accessibili y o mul i alen ca bohyd a e pla o ms capable o in e ening wi h hese p ocesses. 4–6 High-mannose (Man 9 GlcNAc 2 ) is a complex ele an N-glycan p esen in se e al i al en elope glycop o eins (Fig. 1). This suga plays a c ucial ole du ing he a achmen o pa hogens o cells in he i s s ages o he in ec ion p ocess h ough he in e ac ion wi h he DC-SIGN (Dend i ic Cell- Speci ic ICAM-3 G abbing Non-in eg in) ecep o , ound in lipid pa ches a he dend i ic cell (DC) su ace. 7 In pa icula , gp120 o HIV-1 has an a e age o 24 N-linked glycans clus e ed on he p o ein su ace, o which 53–76% a e high-mannose de i a i es. 8 This clus e p esen a ion is undamen al o he efficien in e ac ion wi h DC-SIGN. Fig. 1 Chemical s uc u e o high-mannose and i s cons i u i e pa s, he be a Man9 and he GlcNAc 2 epi opes. †Elec onic supplemen a y in o ma ion (ESI) a ailable: 1 H and 13 C NMR spec a and selec ed HSQC and MS spec a. See DOI: 10.1039/D0OB01380C a Glycosys ems Labo a o y, Ins i u o de In es igaciones Químicas (IIQ), CSIC –Uni e sidad de Se illa, A . Amé ico Vespucio 49, Se ille 41092, Spain. E-mail: ja i [email p o ec ed]m, ja[email p o ec ed] b Uni . G enoble Alpes, CNRS, CEA, Ins i u de Biologie S uc u ale, 38000 G enoble, F ance 6086 |O g. Biomol. Chem.,2020,18,6086–6094 This jou nal is © The Royal Socie y o Chemis y 2020 Open Access A icle. Published on 27 July 2020. Downloaded on 9/24/2020 5:20:41 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online View Jou nal | View Issue Towa ds be e unde s anding he ecogni ion p ocess be ween high-mannose and DC-SIGN, se e al mul i alen sys ems ha e been en isaged. Howe e , he s uc u al complex- i y o he cons i uen suga s and he difficul y o ob aining sufficien quan i ies o pu e ma e ial om na u al sou ces ende s he p epa a ion o mul i alen sys ems ca ying he na u al be a epi ope a he challenging. In ac , e y ew examples a e epo ed in he li e a u e desc ibing he syn hesis o Man 9 and high-mannose mul i alen sys ems. 9–11 In his con ex , diffe en app oaches ha e been de eloped o he p epa a ion o simple compounds ha mimic he mul i alen p esen a ion o he suga ound in na u e. Small agmen s o he Man 9 epi ope ha e been syn hesised and e alua ed in diffe en glycomime ic sys ems in pu sui o suga s simple han Man 9 bu wi h easonable binding affini ies o DC-SIGN. 12–15 Recen ly, we ha e de eloped a s aigh o wa d syn he ic s a egy o he p epa a ion o he Man 9 epi ope wi h he na u al be a con igu a ion a he educing end. 16 Al hough he syn hesis ha we ha e desc ibed is compe i i e wi h hose published p e iously by o he labo a o ies, he p epa a ion o he β-mannose uni emains he limi ing s ep. The syn hesis o he app op ia ely- unc ionalised be a-mannose monosaccha - ide building block, equi ed o pe o m he necessa y glycosy- la ion s eps owa ds he p epa a ion o he nonasaccha ide, demands ele en syn he ic s eps wi h a global yield o 43% om S- olyl-α-D-mannopy anoside. 16 As a possible solu ion, we conside ed whe he he mo e syn he ically-accessible α-mannose analogue would p o ide a simila affini y o DC-SIGN han he be a anome , he e o e ci cum en ing he equi emen o he complica ed β-mannose building block, in he p epa a ion o he co esponding ca bohyd a e mul i alen sys ems. To e alua e ou p oposed s a egy, we ha e syn hesised he alpha and be a anome s o he Man 9 epi ope and he co es- ponding i alen glycoclus e o examine he diffe ences in binding affini ies o he DC-SIGN ecep o using a luo escence pola iza ion assay. Resul s and discussion P epa a ion o Man 9 epi opes and glycoclus e s The syn hesis o be a Man 9 using a con e gen s aigh o wa d syn he ic s a egy was p e iously desc ibed by ou labo a o y. 16 In his wo k, we used he same s a egy o p epa e he co es- ponding alpha anome . The di-, pen a-, and isaccha ides building blocks we e common in e media es o bo h anome s, (Fig. 2) being he mannose o he educing end wi h he alpha con igu a ion in he anome ic posi ion p epa ed as desc ibed in Scheme 1. The syn hesis o he app op ia ely-p o ec ed educing end mannose was achie ed as depic ed in Scheme 1, s a ing om he p e iously desc ibed mannosyl de i a i e 5. 17 Mannose 5 was o hogonally p o ec ed using a consecu i e app oach, pe - o ming pu i ica ion only a he inal s ep. Posi ions 4 and 6 we e p o ec ed ia he o ma ion o he benzylidene ace al by subjec ing 5 o benzaldehyde dime hyl ace al and campho sul- onic acid (CSA). Subsequen ly, posi ion 3 was p o ec ed as he p-me hoxybenzy e he , ia he ini ial o ma ion o a in ace al be ween posi ion 2 and 3, ollowed by eac ion wi h p-me hoxy- benzyl chlo ide (PMBCl) and e abu yl ammonium iodine (TBAI). The ee OH in posi ion 2 was hen p o ec ed by ben- zoyla ion wi h benzoyl anhyd ide in he p esence o ie hyl amine and a ca aly ic amoun o dime hylamino py idine (DMAP). Finally, he posi ion 3 was selec i ely dep o ec ed wi h 2,3-dichlo o-5,6-dicyano-1,4-benzoquinone (DDQ) o yield he a ge mannose de i a i e 6in 26% o e all yield om mannose 5 ollowing inal ch oma og aphic pu i ica ion. Wi h he monosaccha ide 6in hand, glycosyla ion wi h he p e iously syn hesised isaccha ide 7and pen asaccha ide 8, 16 enabled he o ma ion o he p o ec ed Man 9 nonasaccha - ide 11 (Scheme 2). The glycosyla ion be ween isaccha ide 7and mannose de i a i e 6was pe o med using N-iodo succinimide and i- luo ome hanesul onic acid as he glycosyla ion p omo o o ob ain e asaccha ide 9in 88% yield. Nex , he benzylidene ace al was emo ed in acidic media using p-TsOH o affo d he e asaccha ide 10 (bea ing unp o ec ed hyd oxyl g oups a posi ions 4 and 6 o he e minal mannose a he educing end) in 78% yield. The inal glycosyla ion be ween e asac- cha ide 10 and pen asaccha ide 8 ook place only a he posi- ion 6 hyd oxyl g oup o he accep o , since his is mo e eac- i e han he s e ically hinde ed hyd oxyl g oup a posi ion 4. This glycosyla ion was ca ied ou using he same condi ions Fig. 2 Re osyn he ic scheme o he p epa a ion o αMan 9 and βMan 9 . Scheme 1 Consecu i e syn hesis o mannose de i a i e 6. O ganic & Biomolecula Chemis y Pape This jou nal is © The Royal Socie y o Chemis y 2020 O g. Biomol. Chem.,2020,18,6086–6094 | 6087 Open Access A icle. Published on 27 July 2020. Downloaded on 9/24/2020 5:20:41 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online as abo e o yield he p o ec ed nonasaccha ide 11 in 73% yield. The nonasaccha ide 11 was cha ac e ised by 1 H and 13 C NMR and ESI-MS. Finally, global dep o ec ion s ep wi h NaOMe and 2 M NaOH in MeOH and oluene clea ed all O-benzoyl g oups affo ding he αMan 9 12 in excellen yield (Scheme 2). In his way, he syn hesis o he alpha anome o Man 9 was achie ed in an expedien manne h ough a con e - gen s a egy. Bo h Man 9 epi opes (alpha and be a) we e p e- pa ed wi h a sho space a he anome ic posi ion unc iona- lised wi h a e minal azido g oup. This g oup pe mi s conju- ga ion o mul i alen scaffolds using he Cu(I) azide–alkyne cycloaddi ion (CuAAC) eac ion. 20,21 To examine he effec ha he mul i alen p esen a ion has on he binding DC-SIGN o bo h anome s, we p epa ed he i- alen glycoclus e s wi h he alpha and be a Man 9 epi opes (Scheme 3). Fo his pu pose, we employed a ialkynyla ed pen ae y h i- ol scaffold, equen ly used o he p epa a ion o ca bo- hyd a e mul i alen sys ems by ou g oup. This scaffold 14 was p epa ed in wo s eps om pen ae y h i ol as p e iously epo ed. 18 The coupling o αMan 9 and βMan 9 ligands was hen ca ied ou ia a click chemis y 19 CuAAC eac ion p o- mo ed by CuSO 4 , sodium asco ba e and is[(1-benzyl-1H-1,2,3- iazol-4-yl)me hyl]amine (TBTA) unde mild condi ions o ob ain he co esponding i alen glycoclus e s. These mul i- alen sys ems we e pu i ied by ea men wi h Quad asil MP esin o emo e he coppe ca alys and by G50 Sephadex ch oma og aphy o affo d he glycoclus e s 15 and 16 in good yields (Scheme 3). The inal s ep was he subs i u ion o he chlo ine a om o an azido g oup a he end o he linke a he ocal posi ion o he scaffold. This eac ion was pe o med using an excess o NaN 3 in DMF a 70 °C o wo days u nish azido- unc ionalised glycoclus e s 17 and 18 in excellen yields. Finally, i was necessa y o in oduce a ch omopho e o he luo escence pola iza ion assays. An alkynyl de i a i e o luo - escein, he comme cially a ailable FAM-alkyne 6-isome 19, was he e o e conjuga ed o he αMan 9 12, he βMan 9 13, and hei co esponding i alen glycoclus e s 17 and 18, espec - i ely, by a CuAAC click eac ion p omo ed by CuB and TBTA in DMSO a oom empe a u e (Scheme 4). The compounds we e ea ed wi h Quad asil MP esin and hen, submi ed o a LH20 Sephadex ch oma og aphy o deli e he luo escen ly-labelled compounds 20,21,22 and 23 in excellen yields. Wi h he luo escen ool compounds p epa ed, we e alu- a ed hei capaci y o in e ac wi h DC-SIGN, he na u al ecep- o o his Man 9 ligand. Fluo escence pola iza ion assays Fluo escence pola iza ion is widely used o s udy binding e en s owing o se e al ad an ages o his echnique. 22,23 The assay equi es only small quan i ies o ligands and ecep o s, is well sui ed o high- h oughpu sc eening and does no equi e he a achmen o any o he pa ne s o a su ace, allowing ligands and ecep o s diffuse eely in solu ion. In pa icula , luo escence pola iza ion expe imen s ha e been success ully employed in he analysis o ca bohyd a e–p o ein in e ac ions. 24–27 Thus, we selec ed his assay o e alua e he binding affini y o ou alpha and be a epime s o Man 9 (20 and 21) and hei co esponding mul i alen sys ems (22 and 23) o DC-SIGN using he e a alen Ex acellula Domain (ECD) o he lec in. Fo his, we ha e used a mic o i e pla e whe e diffe en concen a ions o he p o ein (DC-SIGN ECD), anging om 75 nM o 28 µM in T is buffe , we e added o he Scheme 2 Syn hesis o he αMan 9 12. Scheme 3 Syn hesis o he i alen glycoclus e s 17 and 18. Pape O ganic & Biomolecula Chemis y 6088 |O g. Biomol. Chem.,2020,18,6086–6094 This jou nal is © The Royal Socie y o Chemis y 2020 Open Access A icle. Published on 27 July 2020. Downloaded on 9/24/2020 5:20:41 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online wells con aining a ixed inal 10 nM concen a ion o app op i- a e luo escen ligands (20–23). We obse ed an inc ease in he luo escence pola iza ion alue wi h inc easing p o ein con- cen a ions, demons a ing ha he luo escence ligands bound o DC-SIGN ECD. The esul ing Langmui iso he m cu es a e ep esen ed in Fig. 3. F om hese cu es, he K D o he binding p ocess o each ligand was calcula ed. Fo he α and βanome s o Man 9 , simila alues o K D , (5.2 ± 1.2 and 4.6 ± 1.3 μM, espec i ely) we e ound demons a ing ha he con- igu a ion o he anome in he mannosyl uni a he educing end o he oligosaccha ide does no play a c i ical ole in he binding p ocess. In o de o e alua e he in luence o he anome con igu - a ion when he Man 9 ligand is p esen ed in a mul i alen scaffold, luo opho e labelled glycoclus e s 22 and 23 we e es ed in he luo escence pola iza ion assays (Fig. 3). In his case, he K D we e one o de o magni ude lowe han he da a ound o he mono alen sys ems, indica ing a clea mul i- alen effec (0.53 ± 0.09 and 0.37 ± 0.04 μM o he i alen αMan 9 and βMan 9 , espec i ely). Again, no signi ican diffe - ences be ween he dissocia ion cons an s o he wo anome s we e ound as in he case o he mono alen ligands. Conclusions The s e eochemical con igu a ion o suga s is undamen al o hei selec i e ecogni ion by lec ins. Indeed, jus a simple diffe ence in he con igu a ion o one posi ion can d i e he ecogni ion by one speci ic lec in, e.g. glucose (wi h all OH g oups in equa o ial disposi ion) e sus galac ose (which bea s an axial OH a C4). Fu he mo e, he con igu a ion o he gly- cosidic bonds be ween he monosaccha idic uni s ha o m an oligosaccha ide a e ele an o hei ecogni ion and unc- ion. In he case o high mannose, all he mannoses o he Man 9 epi ope display he alpha con igu a ion o he glycosidic bonds, as usual o his ype o suga . Howe e , he linkage be ween he Man 9 epi ope and he GlcNAc disaccha ide exhi- bi s a be a con igu a ion. This glycosidic bond con igu a ion is a e o mannoses and p esen s a signi ican hu dle owa ds add essing his bond con igu a ion ia chemical syn hesis. All N-glycans ha e his moie y as connec ion wi h he co es- ponding Asn in he N-glycan p o eins. This speci ic a ange- men is c i ical o he p ope o ien a ion o he mannosyl clus e , p o iding a igid s uc u e on he p o ein su ace o op imize he binding o DC-SIGN. Howe e , when new glyco- mul i alen sys ems a e designed, he glycan epi ope is no - mally conjuga ed o he mul i alen scaffold wi h a lexible Scheme 4 Syn hesis o fluo escence labelled compounds 20,21,22 and 23. Fig. 3 Langmui iso he mal cu es o binding be ween DC-SIGN ECD and fluo escence compounds: (a) αMan 9 (20); (b) βMan 9 (21); (c) i alen glycoclus e 22; and (d) i alen glycoclus e 23. O ganic & Biomolecula Chemis y Pape This jou nal is © The Royal Socie y o Chemis y 2020 O g. Biomol. Chem., 2020, 18,6086–6094 | 6089 Open Access A icle. Published on 27 July 2020. Downloaded on 9/24/2020 5:20:41 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online linke and he e o e he speci ic o ien a ion p o ided by he be a-mannose linkage is likely no necessa y. To demons a e his hypo hesis, we ha e syn hesised he Man 9 epi ope o high mannose wi h alpha and be a con igu a ion in he e minal mannose a he educing end (20 and 21) and he co es- ponding i alen glycoclus e s (22 and 23). These ligands we e labelled wi h a luo escein de i a i e and hei binding o DC-SIGN e alua ed by luo escence pola iza ion assays. Ou indings ha e shown ha he con igu a ion, alpha o be a, o he anome ic posi ion o he e minal mannose does no in lu- ence in he binding affini y o he na u al ecep o DC-SIGN and he e o e hey can be used in e changeably, meaning he mo e syn he ically-accessible alpha-linked epime s can be deployed ins ead o he mo e challenging na u al be a ana- logues. We an icipa e his knowledge is o key ele ance in he design o new glycosys ems o high mannose, since no ably educes he complexi y, he ime and he cos o he glycan syn hesis by ci cum en ing he syn he ic bo leneck o he be a mannose p epa a ion. Expe imen al Ma e ials and me hods Sol en s we e HPLC g ade and used as ecei ed unless o he - wise s a ed. Size exclusion ch oma og aphy was pe o med wi h Sephadex LH20, G-25 and G-50 (GE Heal hca e). Thin- laye ch oma og aphy (TLC) was pe o med on silica pla es (Me ck). Reagen s and Quad aSil® MP we e pu chased om Sigma Ald ich. 2-Azidoe hyl α-D-mannopy anoside 17 and ialk- ynyla ed scaffold 14 18 we e syn hesised as p e iously desc ibed. NMR expe imen s we e pe o med using a B uke Ad ance DRX 400 ins umen . NMR chemical shi s a e epo ed in ppm (δuni s) down ield om he CDCl 3 signal o he HOD peak (D 2 O). 2D expe imen s (COSY and HSQC) we e pe o med when necessa y. NMR spec a we e analysed wi h Mes eNo a so wa e. Syn hesis o ligands 2-Azidoe hyl 2-O-benzoyl-4,6-O-benzylidene-α-D-mannopy a- noside (6). To a solu ion o 2-azidoe hoxy-α-D-mannopy anose (5) (800 mg, 3.21 mmol) in CH 3 CN (8 mL), CSA (186 mg, 0.8 mmol) and benzaldehyde dime hyl ace al (0.5 mL, 3.55 mmol) we e added a . The eac ion mix u e was s i ed o e nigh and he mixing became solidi ied du ing addi ion o he eagen , which indica es he p og ess o he eac ion. The eac ion mix u e was quenched wi h E 3 N (250 μL) and dis- sol ed in excess o E OAc and wa e . The eac ion mix u e was ex ac ed wice wi h E OAc and he combined o ganic laye s we e d ied o e anh. MgSO 4 , il e ed and concen a ed unde acuum. A e being dissol ed in anh. oluene (29 mL), dibu yl- in oxide (879 mg, 3.55 mmol) was added o he esul ing mix u e con aining he 4,6-O-benzylidene de i a i e. The eac- ion mix u e was kep unde e lux a 110 °C o 4 h, cooled o and PMBCl (0.5 mL, 3.55 mmol) and TBAI (370 mg, 3.55 mmol) we e added o i unde A a mosphe e. The eac- ion mix u e was kep unde e lux a 110 °C o 1 h. A e emo al he sol en , he c ude was dilu ed wi h CH 2 Cl 2 and washed wi h wa e . The o ganic laye was d ied o e anh. MgSO 4 , il e ed and concen a ed unde acuum. To he esul - ing mix u e con aining 4,6-O-benzylidene-3-O-p-me hoxybenzyl de i a i e in CH 2 Cl 2 (15 mL), Bz 2 O (1.5 g, 6.42 mmol), E 3 N (1.3 mL, 9.63 mmol) and a ca aly ic amoun o DMAP we e subsequen ly added and he eac ion was s i ed a o 1 h. The eac ion mix u e was washed wi h NaHCO 3 sa . aq. soln. The o ganic phase was d ied o e anh. MgSO 4 , il e ed and concen a ed unde acuum. Finally, o a s i ed solu ion o he α-D-mannoside de i a i e in CH 2 Cl 2 (70 mL) and wa e (2.7 mL), DDQ (2.23 g, 9.63 mmol) was added a . A e 1 h, NaHCO 3 sa . aq. soln. was added, and he mix u e was ex ac ed wi h CH 2 Cl 2 . The ex ac was washed se e al imes wi h NaHCO 3 sa . aq. soln. and hen d ied o e anh. MgSO 4 , il e ed and concen a ed unde acuum. The c ude was pu i- ied by column ch oma og aphy on silica gel (E OAc/n-hexane 1 : 3) o gi e he compound 6(320 mg, 0.72 mmol, 26%) as a colou less oil. [α] D =−37 (c1.00, CHCl 3 ). 1 H-NMR (400 MHz, CDCl 3 )δ: 8.11 (m, 2H, H–A ), 7.60 (m, 1H, H–A ), 7.56–7.45 (m, 4H, H–A ), 7.43–7.35 (m, 3H, H–A ), 5.67 (s, 1H, H ace al ), 5.52 (dd, J 2,3 = 3.6, J 2,1 = 1.6 Hz, 1H, H-2), 5.01 (d, J 1,2 = 1.6 Hz, 1H, H-1), 4.41 (d , J 3,4 = 9.6, J 3,2 = 3.9 Hz, 1H, H-3), 4.33 (dd, J 6a,6b = 9.9, J 6a,5 = 4.4 Hz, 1H, H-6a), 4.06 ( , J 4,3 =J 4,5 = 9.5 Hz, 1H, H-4), 4.03–3.84 (m, 3H, H-5, H-6b, H-1′a), 3.73–3.63 (m, 1H, H-1′b), 3.55–3.40 (m, 2H, H-2′), 2.32 (d, J OH,3 = 4.2 Hz, 1H, OH). 13 C-NMR (100 MHz, CDCl 3 )δ: 166.0, 137.1, 133.5, 129.9, 129.5, 129.3, 128.5, 128.3, 126.3, 102.2, 98.7, 79.2, 72.5, 68.7, 67.1, 66.9, 63.8, 50.4. ESI-MS m/zcalcd o C 22 H 23 N 3 O 7 : 441.2; ound: 464.2 [M + Na] + . Te asaccha ide 9. To a solu ion o accep o 6(49 mg, 0.11 mmol), dono 7(275 mg, 0.17 mmol) and 4 Å molecula sie es in anh. CH 2 Cl 2 (5.8 mL) we e added and he mix u e was s i ed a −20 °C o 30 min. Then, NIS (39 mg, 0.17 mmol) and T OH (2.9 μL, 0.03 mmol) we e added and he eac ion was s i ed a −20 °C o 15 min. The eac ion was quenched wi h NaHCO 3 sa . aq. soln. The eac ion mix u e was il e ed h ough Celi e and washed se e al imes wi h CH 2 Cl 2 . The o ganic laye was washed wi h Na 2 S 2 O 3 sa . aq. soln. and hen d ied o e anh. MgSO 4 , il e ed and concen a ed unde acuum. The c ude was pu i ied by column ch oma og aphy on silica gel (E OAc/n-hexane 1 : 2 o 1 : 1.25) o gi e he e a- saccha ide 9(193 mg, 0.10 mmol, 88%) as a whi e solid. [α] D = −22 (c1.00, CHCl 3 ). 1 H-NMR (400 MHz, CDCl 3 )δ: 8.22 (m, 1H, H–A ), 8.19–8.12 (m, 2H, H–A ), 8.09–7.92 (m, 7H, H–A ), 7.92–7.81 (m, 7H, H–A ), 7.76–7.68 (m, 2H, H–A ), 7.64–7.19 unde CDCl 3 (m, 36H, H–A ), 7.14–6.95 (m, 4H), 6.78 (m, 1H), 6.05–5.81 (m, 5H), 5.79–5.69 (m, 2H), 5.64 (dd, J= 9.2, J= 3.1 Hz, 1H), 5.57 (s, 1H, H-1), 5.49 (b s, 1H, H ace al ), 5.35 (s, 1H, H-1), 5.05 (d, J= 1.5 Hz, 1H, H-1), 4.91 (s, 1H, H-1), 4.72–4.50 (m, 4H), 4.47 ( , J= 2.8 Hz, 1H), 4.38 (m, 1H), 4.35–4.13 (m, 4H), 4.07–3.94 (m, 3H), 3.94–3.79 (m, 3H), 3.65 (m, 1H), 3.35 (m, 2H). 13 C-NMR (100 MHz, CDCl 3 )δ: 166.4, 166.0, 165.9, 165.8, 165.5, 165.3, 165.3, 165.2, 165.2, 165.0, 164.8, 137.0, 133.7, 133.5, 133.3, 133.2, 133.1, 133.1, 133.1, 133.0, 132.8, Pape O ganic & Biomolecula Chemis y 6090 |O g. Biomol. Chem.,2020,18,6086–6094 This jou nal is © The Royal Socie y o Chemis y 2020 Open Access A icle. Published on 27 July 2020. Downloaded on 9/24/2020 5:20:41 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online 130.1–129.6, 129.3, 129.2, 129.2, 129.1, 128.9–128.3 101.9, 99.8, 90.1, 79.1, 71.9, 71.4, 70.4, 70.1, 69.8, 69.7, 69.5, 68.8, 67.6, 67.2, 67.0, 66.3, 64.1, 63.6, 63.5, 62.9, 62.5, 50.4. ESI-MS m/zcalcd o C 110 H 93 N 3 O 32 : 1967.6; ound: 1990.4 [M + Na] + . ESI-HRMS m/zcalcd o C 110 H 93 N 3 O 32 Na [M + Na] + : 1990.5634; ound: 1990.5609 Te asaccha ide 10. p-Toluenesul onic acid monohyd a e (25 mg, 0.13 mmol) was added o a s i ed solu ion o e asac- cha ide 9(173 mg, 0.09 mmol) in CH 3 CN (4.4 mL) a . A e 24 h, he eac ion mix u e was quenched wi h E 3 N (30 μL) and concen a ed unde acuum. The esidue was pu i ied by column ch oma og aphy on silica gel (ace one/ oluene 1 : 5) o gi e he e asaccha ide 10 (130 mg, 0.07 mmol, 78%) as a whi e amo phous solid. [α] D =−15 (c1.00, CHCl 3 ). 1 H-NMR (400 MHz, CDCl 3 )δ: 8.14 (m, 2H, H–Bz), 8.07–7.93 (m, 13H, H–Bz), 7.90–7.82 (m, 4H, H–Bz), 7.70 (m, 2H, H–Bz), 7.61–7.18 unde CDCl 3 (m, 34H, H–Bz), 6.13 ( , J= 10.2 Hz, 1H), 6.03–5.90 (m, 3H), 5.84–5.72 (m, 2H), 5.67–5.57 (m, 3H, 1H-1), 5.52 (d, J= 1.6 Hz, 1H, H-1), 5.09 (b s, 1H, H-1), 5.00 (d, J= 1.7 Hz, 1H, H-1), 4.73–4.31 (m, 12H), 4.31–4.15 (m, 2H), 4.01 (m, 2H), 3.95–3.78 (m, 3H), 3.59 (d , J= 10.4, 4.8 Hz, 1H), 2.34 (m, 2H). 13 C-NMR (100 MHz, CDCl 3 )δ: 166.4, 166.3, 166.0, 165.9, 165.7, 165.5, 165.2, 164.9, 133.6, 133.5, 133.4, 133.3, 133.2, 133.0, 130.2–129.8, 129.6, 129.5, 129.4, 129.3, 129.1, 129.0, 128.9, 128.6–128.4, 100.8, 99.4, 98.1, 76.4, 73.3, 72.4, 70.5, 70.3, 69.9, 69.7–69.6, 69.4, 68.2, 67.8, 67.0, 66.6, 63.9–63.8, 62.6, 62.3, 50.4 (C-2′). ESI-MS m/zcalcd o C 103 H 89 N 3 O 32 : 1879.5; ound: 962.5 [M + Na] + . ESI-HRMS m/zcalcd o C 103 H 89 N 3 O 32 Na [M + Na] + : 1902.5321; ound: 1902.5307. αMan 9 p o ec ed (11). To a solu ion o accep o 10 (110 mg, 0.06 mmol), dono 8(228 mg, 0.09 mmol) and 4 Å molecula sie es in anh. CH 2 Cl 2 (4.1 mL) we e added and he mix u e was s i ed a −20 °C o 30 min. Then, NIS (42 mg, 0.09 mmol) and T OH (3.1 μL, 17.55 μmol) we e added and he eac ion was s i ed a −20 °C o 15 min. The eac ion was quenched wi h NaHCO 3 sa . aq. soln. The eac ion mix u e was il e ed h ough Celi e and washed se e al imes wi h CH 2 Cl 2 . The o ganic laye was washed wi h Na 2 S 2 O 3 sa . aq. soln. and hen d ied o e anh. MgSO 4 , il e ed and concen a ed unde acuum. The c ude was pu i ied by column ch oma og aphy on silica gel (E OAc/n-hexane 4 : 5 →1 : 1) o gi e he nonasac- cha ide 11 (185 mg, 0.04 mmol, 73%) as a whi e solid. [α] D = −19 (c1.00, CHCl 3 ). 1 H-NMR (400 MHz, CDCl 3 )δ: 8.30–7.67 (m, 52H, HBz), 7.56–7.18 (m, 80H, HBz), 6.99 (m, 2H, HBz), 6.81 ( , J= 7.5, 1H, HBz), 6.22–5.80 (m, 16H), 5.55 (s, 1H, H-1), 5.46 (m, 2H, 2H-1), 5.32 (s, 1H, H-1), 5.18 (s, 1H, H-1), 5.05 (m, 2H, 2H-1), 4.74–4.02 (m, 36H, H-1, H-1), 3.84–3.72 (m, 2H), 3.49–3.32 (m, 3H). 13 C-NMR (100 MHz, CDCl 3 )δ: 166.5, 166.4, 166.4, 166.3, 166.1, 166.0, 165.8, 165.6, 165.6, 165.4, 165.4, 165.3, 165.0, 165.0, 164.8, 164.8, 164.6, 133.5, 133.4, 133.4, 133.2, 133.2, 133.1, 130.2–129.5, 129.5, 129.5, 129.3, 129.2, 129.2, 129.0, 129.0, 128.9, 128.9, 128.7–128.3, 101.1, 100.9, 100.3, 99.9, 99.5, 98.5, 97.7, 97.5, 78.2, 77.9, 75.6, 72.4, 72.2, 71.9, 71.1, 70.8, 70.5, 70.1–69.4, 68.9, 68.1, 68.0, 67.7, 67.3–67.0, 66.6, 66.4, 66.1, 63.7, 63.5, 63.1, 62.6, 62.4, 50.4. ESI-MS m/zcalcd o C 245 H 203 N 3 O 73 : 4354.2; ound: 2200.1 [M + 2Na] 2+ and 1473.5 [M + 3Na] 3+ . ESI-HRMS m/zcalcd o C 245 H 203 N 3 O 73 Na [M + 2Na] 2+ : 2189.1115; ound: 2189.1088. αMan 9 (12). To a solu ion o compound 11 (166 mg, 0.04 mmol) in MeOH/ oluene (4 : 1, 1.7 mL), NaOMe (11 mg, 0.20 mmol) and a NaOH 2 M solu ion (0.7 mL) we e added and he eac ion was s i ed a 50 °C o 5 h. A e neu aliz- a ion wi h Ambe li e IR-120H + , he solu ion was il e ed and concen a ed. The c ude was pu i ied by size-exclusion ch om- a og aphy (Sephadex G-25, H 2 O/MeOH 9/1), gi ing Man 9 (12) (55 mg, 0.04 mmol, 93%) as a whi e amo phous solid. 1 H-NMR (400 MHz, D 2 O) δ: 5.40 (s, 1H, H-1), 5.33 (s, 1H, H-1), 5.30 (s, 1H, H-1), 5.15 (s, 1H, H-1), 5.08–5.01 (m, 3H, 3 × H-1), 4.88 (s, 1H, H-1), unde D 2 O (1H, H-1), 4.19–3.60 (m, 58H). 13 C-NMR (100 MHz, D 2 O) δ: 102.2, 100.8, 100.6, 100.0, 99.5, 98.0, 78.8–78.5, 73.2, 73.2, 72.7, 71.1, 71.1, 70.3–70.0, 69.6, 66.9, 66.8, 66.6, 65.7, 65.5, 65.2, 61.2–61.0, 50.2. ESI-MS m/z calcd o C 56 H 95 N 3 O 46 : 1545.5; ound: 1568.2 [M + Na] + , 795.5 [M + 2Na] 2+ . D3-αMan 9 -Cl (15). Nonasaccha ide 12 (22.0 mg, 14.23 μmol) and [2-[2-(2-chlo oe hoxy)e hoxy]e hoxy]e hoxyme hyl ikis(2- p opyniloxyme hyl)-me hane (14) (1.6 mg, 3.59 μmol) we e dis- sol ed in H 2 O/DMSO (1 : 1, 0.5 mL). F esh solu ions o CuSO 4 ·5H 2 O (1.80 μmol), is[(1-benzyl-1H-1,2,3- iazol-4-yl) me hyl]amine (TBTA) (3.59 μmol) and sodium asco ba e (5.39 μmol) we e added o a sealed mic owa e ial. The solu- ion was hea ed a 60 °C in a mic owa e o en o 30 min. A me al sca enge esin, Quad asilMP, was added o he eac ion solu ion and s i ed o 20 min a . A e ha , he mix u e was il e ed and he esul ing solu ion was pu i ied by size- exclusion ch oma og aphy (Sephadex G-50, H 2 O/MeOH 9 : 1) yielding he glycoclus e 15 (12 mg, 2.37 μmol, 66%) as a whi e amo phous solid. 1 H-NMR (400 MHz, D 2 O) δ: 8.06 (s, 3H, H iazole ), 5.41 (b s, 3H, 3H-1), 5.37–5.27 (m, 6H, 6H-1), 5.15 (b s, 3H, 3H-1), 5.09–5.02 (m, 9H, 9H-1), unde D 2 O (6H, 6H-1), 4.67 (m, 3H, O CH 2 CHHN), 4.57 (b s, 3H, OCHHC iazole ), 4.14–3.62 (m, 198H). 13 C-NMR (100 MHz, D 2 O) δ: 144.2, 125.4, 102.2, 100.7, 99.8, 99.5, 98.0, 78.8–78.4, 73.3–73.2, 72.7, 72.0, 71.1–71.0, 70.8, 70.3, 70.0, 70.0, 69.7, 69.6, 69.5, 66.9, 66.8, 65.9, 65.8, 65.6, 65.4, 65.2, 64.8, 63.4, 62.5, 61.0, 50.1, 43.2. ESI-MS m/zcalcd o C 191 H 320 N 9 O 145 Cl: 5080.7; ound: 2562.9 [M + 2Na] 2+ , 1715.5 [M + 3Na] 3+ , and 1295.7 [M + 4Na] 4+ . D3-αMan 9 -N 3 (17). To a solu ion o glycoclus e 15 (9.0 mg, 1.77 μmol) in DMF (1 mL) sodium azide (1.2 mg, 17.71 μmol) was added. The mix u e was s i ed a 70 °C o 2 days. A e ha ime, he sol en was e apo a ed unde acuum and he c ude was pu i ied using Amicon Ul a-15 cen i ugal il e s (MWCO 3 kDa), yielding he glycoclus e 17 (9 mg, 1.77 μmol, quan .) as a whi e amo phous solid. 1 H-NMR (400 MHz, D 2 O) δ: 8.06 (s, 3H, H iazole ), 5.41 (b s, 3H, 3H-1), 5.35–5.29 (m, 6H, 6H-1), 5.15 (b s, 3H, 3H-1), 5.07–5.03 (m, 9H, 9H-1), unde D 2 O (6H, 6H-1), 4.67 (m, 3H, OCHHCH 2 N), 4.58 (b s, 3H, OCHHC iazole ), 4.14–3.60 (m, 198H). 13 C-NMR (100 MHz, D 2 O) δ: 144.2, 125.5, 102.3, 100.7, 99.8, 99.5, 98.0, 78.8–78.4, 73.3–73.2, 72.0, 71.1–71.0, 70.3–69.9, 69.7–69.5, 66.9–66.8, 65.9, 65.6, 65.4, 65.2, 63.4, 62.5, 61.1–61.0, 50.1, 50.1, 44.6. O ganic & Biomolecula Chemis y Pape This jou nal is © The Royal Socie y o Chemis y 2020 O g. Biomol. Chem.,2020,18,6086–6094 | 6091 Open Access A icle. Published on 27 July 2020. Downloaded on 9/24/2020 5:20:41 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online ESI-MS m/zcalcd o C 191 H 320 N 12 O 145 Cl: 5087.2; ound: 2567.0 [M + 2Na] 2+ , 1781.1 [M + 3Na] 3+ , and 1294.6 [M + 4Na] 4+ . D3-βMan 9 -Cl (16). Nonasaccha ide 13 (22.0 mg, 14.23 μmol) and 14 (1.6 mg, 3.59 μmol) we e dissol ed in H 2 O/DMSO (1 : 1, 0.5 mL). F esh solu ions o CuSO 4 ·5H 2 O (1.80 μmol), TBTA (3.59 μmol) and sodium asco ba e (5.39 μmol) we e added o a sealed mic owa e ial. The solu ion was hea ed a 60 °C in a mic owa e o en o 30 min. A me al sca enge esin, Quad asilMP, was added o he eac ion solu ion and s i ed o 20 min a . A e ha , he mix u e was il e ed and he esul ing solu ion was pu i ied by size-exclusion ch oma o- g aphy (Sephadex G-50, H 2 O/MeOH 9 : 1) yielding he glyco- clus e 16 (14.8 mg, 2.91 μmol, 81%) as a whi e amo phous solid. 1 H-NMR (400 MHz, D 2 O) δ: 8.05 (s, 3H, 3H iazole ), 5.42 (b s, 1H, 3H-1), 5.34–5.28 (m, 6H, 6H-1), 5.15 (b s, 3H, 3H-1), 5.09–5.03 (m, 9H, 9H-1), 4.87 (b s, 3H, 3H-1), 4.67 (m, 3H, OCH 2 CHHN), 4.61–4.56 (b s, 6H, H-1, OCHHC iazole ), 4.18–3.56 (m, 198H). 13 C-NMR (100 MHz, D 2 O) δ: 144.1, 125.5, 102.3, 102.2, 100.8, 100.7, 100.6, 99.9, 99.5, 98.0, 80.9, 78.9–78.5, 74.0, 73.3–73.2, 72.7, 71.1, 70.8, 70.3–69.9, 69.7–69.5, 68.3–67.7, 66.9–66.8, 65.6–65.2, 63.5, 62.5, 61.1–61.0, 50.3, 44.7, 43.2. ESI-MS m/zcalcd o C 191 H 320 N 9 O 145 Cl: 5080.7; ound: 2561.1 [M + 2Na] 2+ , 1715.2 [M + 3Na] 3+ , and 1293.5 [M + 4Na] 4+ . D3-βMan 9 -N 3 (18). To a solu ion o glycoclus e 16 (20.0 mg, 3.94 μmol) in DMF (1 mL) sodium azide (2.6 mg, 39.36 μmol) was added. The mix u e was s i ed a 70 °C o 2 days. A e ha ime, he sol en was e apo a ed unde acuum and he c ude was pu i ied using Amicon Ul a-15 cen i ugal il e s (MWCO 3 kDa), yielding he glycoclus e 18 (20 mg, 3.94 μmol, quan .) as a whi e amo phous solid. 1 H-NMR (400 MHz, D 2 O) δ: 8.05 (s, 3H, 3H iazole ), 5.41 (b s, 1H, 3H-1), 5.34–5.29 (m, 6H, 6H-1), 5.15 (b s, 3H, 3H-1), 5.07–5.03 (m, 9H, 9H-1), unde D 2 O (3H, 3H-1), 4.68 (m, 3H, OCHHC iazole ), 4.60–4.55 (m, 6H, H-1, OCH 2 CHHN), 4.12–3.46 (m, 198H). 13 C-NMR (100 MHz, D 2 O) δ: 144.1, 125.5, 102.3, 102.2, 100.8, 100.7, 100.6, 99.9, 99.5, 98.0, 80.9, 78.9–78.4, 74.0, 73.3–73.2, 72.7, 72.0, 71.1, 70.3–69.9, 69.7–69.5, 69.2, 68.3, 67.7, 66.9–66.8, 65.6–65.2, 63.5, 62.4, 61.1–61.0, 50.2, 50.1, 44.7. ESI-MS m/z calcd o C 191 H 320 N 12 O 145 : 5087.2; ound: 2566.5 [M + 2Na] 2+ , 1717.9 [M + 3Na] 3+ , and 1293.9 [M + 4Na] 4+ . αMan 9 - luo escein (20). To a solu ion o he FAM-alkyne 6-isome 19 (2.67 mg, 6.48 μmol) and he epi ope αMan 9 12 (5 mg, 3.24 μmol) in DMSO (300 μL), ano he solu ion o CuB (3.24 μmol) and TBTA (6.48 μmol) in he same sol en (150 μL) was added. The eac ion mix u e was s i ed on a . The ea e , a me al sca enge esin, Quad asilMP, was added o he eac ion solu ion and s i ed o 20 min a . A e ha , he mix u e was il e ed and he esul ing solu ion was pu i ied by size-exclusion ch oma og aphy (Sephadex LH-20, H 2 O/ MeOH 9 : 1), yielding he luo escen p obe 20 (6.3 mg, quan .) as an o ange amo phous solid. 1 H-NMR (400 MHz, D 2 O) δ: 8.22–7.87 (m, 3H), 7.68 (b s, 1H), 7.30–7.07 (m, 2H), 6.87–6.54 (m, 4H), 5.40–5.24 (m, 3H, 3H-1), 5.11 (b s, 1H, 1H-1), 5.04 (b s, 3H, 3H-1), unde D 2 O (2H, 2H-1), 4.64 (m, 2H), 4.12–3.57 (m, 58H). 13 C-NMR (100 MHz, selec ed da a ob ained om HSQC, D 2 O) δ: 131.2, 128.2, 128.2, 127.9, 121.7, 103.0, 102.2, 100.8, 100.6, 100.0, 99.5, 98.0, 78.8–78.5, 73.2, 73.2, 72.7, 71.1, 71.1, 70.3–70.0, 69.6, 66.9, 66.8, 66.6, 65.7, 65.5, 65.2, 61.2–61.0, 50.2. ESI-MS: m/zcalcd o C 80 H 110 N 4 O 52 : 1958.6, ound: 978.1 [M −2H] 2− . βMan 9 - luo escein (21). To a solu ion o he FAM-alkyne 6-isome 19 (6.6 mg, 10.36 μmol) and he epi ope β-Man 9 13 (8 mg, 5.18 μmol) in DMSO (300 μL), ano he solu ion o CuB (5.18 μmol) and TBTA (10.36 μmol) in he same sol en (150 μL) was added. The eac ion mix u e was s i ed on a . The ea e , a me al sca enge esin, Quad asilMP, was added o he eac ion solu ion and s i ed o 20 min a . A e ha , he mix u e was il e ed and he esul ing solu ion was pu i ied by size-exclusion ch oma og aphy (Sephadex LH-20, H 2 O/ MeOH 9 : 1), yielding he luo escen p obe 21 (10.1 mg, quan .) as an o ange amo phous solid. 1 H-NMR (400 MHz, D 2 O) δ: 8.05–7.98 (m, 2H), 7.94 (m, 1H), 7.57 (b s, 1H), 7.15–7.07 (m, 2H), 6.69–6.63 (m, 4H), 5.37 (b s, 1H, H-1), 5.31–5.26 (m, 2H, 2H-1), 5.10 (b s, 1H, H-1), 5.06–5.00 (m, 4H, 4H-1), 4.65 (m, 2H, CH 2 C iazole ), 4.59 (m, 2H, CH 2 CH 2 N), 4.49 (b s, 1H, H-1), 4.21 (m, 1H), 4.12–4.05 (m, 6H), 4.03–3.92 (m, 6H), 3.90–3.60 (m, 42H), 3.41 (m, 1H). 13 C-NMR (100 MHz, selec ed da a ob ained om HSQC, D 2 O) δ: 131.0, 128.2, 128.2, 127.9, 121.7, 103.0, 102.3, 102.1, 100.7, 100.5, 99.9, 99.5, 97.9, 81.0, 78.9, 78.6, 78.5, 78.4, 74.0, 73.2–73.1, 72.6, 71.1, 70.3–69.9, 69.4, 68.4, 66.9–66.8, 65.6–65.4, 61.1–60.9, 50.3. ESI-MS: m/zcalcd o C 80 H 110 N 4 O 52 : 1958.6, ound: 1982.3 [M −H] − , and 1002.1 [M −2H] 2− . D3-αMan 9 - luo escein (22). To a solu ion o he FAM-alkyne 6-isome 19 (1.3 mg, 3.14 μmol) and he i alen glycoclus e o αMan 9 17 (8 mg, 1.57 μmol) in DMSO (400 μL), ano he solu ion o CuB (1.57 μmol) and TBTA (3.14 μmol) in he same sol en (200 μL) was added. The eac ion mix u e was s i ed on a . The ea e , a me al sca enge esin, Quad asilMP, was added o he eac ion solu ion and s i ed o 20 min a . A e ha , he mix u e was il e ed and he esul ing solu ion was pu i ied by size-exclusion ch oma o- g aphy (Sephadex LH-20, H 2 O/MeOH 9 : 1), yielding he luo- escen p obe 22 (8.7 mg, quan .) as an o ange amo phous solid. 1 H-NMR (400 MHz, D 2 O) δ: 8.06–7.78 (m, 6H), 7.57 (b s, 1H), 7.07–6.88 (m, 2H), 6.62–6.42 (m, 4H), 5.35–5.16 (m, 9H, 9H-1), 5.06 (b s, 3H, 3H-1), 4.96 (b s, 9H, 9H-1), unde D 2 O (6H, 6H-1), 4.55–4.29 (m, 6H), 4.07–3.11 (m, 213H). 13 C-NMR (100 MHz, selec ed da a ob ained om HSQC, D 2 O) δ: 126.8, 122.9, 102.9, 102.3, 100.7, 99.8, 99.5, 98.0, 78.8–78.4, 73.3–73.2, 72.0, 71.1–71.0, 70.3–69.9, 69.7–69.5, 66.9–66.8, 65.9, 65.6, 65.4, 65.2, 63.4, 62.5, 61.1–61.0, 50.1. ESI-MS: m/z calcd o C 215 H 335 N 13 O 151 : 5514.9, ound: 2749.4 [M −2H] 2− , and 1833.1 [M −3H] 3− . D3-βMan 9 - luo escein (23). To a solu ion o he FAM-alkyne 6-isome 19 (2.3 mg, 5.5 μmol) and he i alen glycoclus e s o βMan 9 18 (14 mg, 2.75 μmol) in DMSO (180 μL), ano he solu ion o CuB (2.75 μmol) and TBTA (5.5 μmol) in he same sol en (90 μL) was added. The eac ion mix u e was s i ed on a . The ea e , a me al sca enge esin, Quad asilMP, was added o he eac ion solu ion and s i ed o 20 min a . Pape O ganic & Biomolecula Chemis y 6092 |O g. Biomol. Chem.,2020,18,6086–6094 This jou nal is © The Royal Socie y o Chemis y 2020 Open Access A icle. Published on 27 July 2020. Downloaded on 9/24/2020 5:20:41 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online A e ha , he mix u e was il e ed and he esul ing solu ion was pu i ied by size-exclusion ch oma og aphy (Sephadex LH-20, H 2 O/MeOH 9 : 1), yielding he luo escen p obe 23 (15.0 mg, quan .) as an o ange amo phous solid. 1 H-NMR (400 MHz, D 2 O) δ: 8.02–7.80 (m, 6H), 7.54 (b s, 1H), 7.01–6.87 (m, 2H), 6.58–6.46 (m, 4H), 5.40–5.30 (m, 6H, 6H-1), 5.21 (m, 6H, 6H-1), 5.06 (b s, 3H, 3H-1), 4.96 (m, 9H, 9H-1), 4.53–4.41 (m, 12H, 3H-1), 4.29 (b s, 6H), 4.07–3.05 (m, 204H). 13 C-NMR (100 MHz, selec ed da a ob ained om HSQC, D 2 O) δ: 131.0, 129.6, 128.5, 124.0, 122.4, 103.8, 102.3, 102.2, 100.8, 100.7, 100.6, 99.9, 99.5, 98.0, 80.9, 78.9–78.4, 74.0, 73.3–73.2, 72.7, 72.0, 71.1, 70.3–69.9, 69.7–69.5, 69.2, 68.3, 67.7, 66.9–66.8, 65.6–65.2, 63.5, 62.4, 61.1–61.0, 50.1 (OCH 2 CH 2 N). ESI-MS: m/z calcd o C 215 H 335 N 13 O 151 : 5514.9, ound: 2748.5 [M −2H] 2− , and 1830.8 [M −3H] 3− . DC-SIGN ECD exp ession DC-SIGN ECD exp ession and p oduc ion was pe o med as p e iously desc ibed. B ie ly, he DC-SIGN ECD p o ein is exp essed as inclusion bodies in E. coli, e olded and pu i ied using a mannose affini y ch oma og aphy s ep ollowed by a size exclusion. 28,29 Bo h s eps ensu e espec i ely he selec ion o co ec ly olded and oligome ized p o ein. DC-SIGN binding assays by luo escence pola iza ion The luo escence pola iza ion measu emen s we e pe o med in 384-well mic opla es (black polys y ene, non- ea ed, Co ning), using a TRIAD mul imode mic opla e eade ( om Dynex) wi h exci a ion and emission wa eleng hs o 485 and 535 nm, espec i ely. Fluo escen compounds 20–23 and DC-SIGN-ECD we e dissol ed in T is buffe (25 mM, pH 8, 150 mM NaCl, 4 mM CaCl 2 ). 15 μL o a 20 nM luo escen ligand solu ion we e ans e ed o each well. Then, 15 μLo p o ein solu ions, wi h concen a ions anging om 75 nM o 28 μM, we e added o he mic opla e wells. The e o e, he o al sample olume in each well was 30 μL. The mic opla e was shaken in he da k o 5 min, be o e eading. Blank wells con- ained 15 μL o he DC-SIGN-ECD solu ion and 15 μL o T is buffe , and hei measu emen s we e sub ac ed om all alues. All expe imen s on samples we e pe o med in epli- ca es o h ee. Wells con aining 15 μL o he 20 nM luo escence com- pound solu ion and 15 μL o T is buffe affo ded he back- g ound pola iza ion o he luo escen molecule, in he absence o p o ein. This alue was sub ac ed om he pola iz- a ion alues o all he samples, gi ing he inc emen in he luo escence pola iza ion (ΔP). The a e age ΔP alues o h ee eplica e wells we e plo ed agains he concen a ion o DC-SIGN-ECD, and he esul ing cu e was i ed o he equa ion o a one-si e binding model: y=ΔP max x/[K D +x] whe e ΔP max is he maximal alue o ΔPand K D is he dis- socia ion cons an o he in e ac ion. Conflic s o in e es The e a e no con lic s o decla e. Acknowledgemen s This wo k was inancially suppo ed by MINECO (CTQ2017- 86265-P, PGC2018-099497-B-100 and IJCI-2015-23272), and ISCIII RETICS ARADyAL (RD16/0006/0011). G an s we e co- unded by he Eu opean Regional De elopmen Fund (ERDF). A. S.-H. and N. d. l. C hank MINECO o hei Juan de la Cie a-Inco po acion and FPI con ac s, espec i ely. This wo k used he Mul is ep P o ein Pu i ica ion Pla o m o human CLRs p oduc ion o he G enoble Ins uc -ERIC Cen e (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) wi hin he G enoble Pa ne ship o S uc u al Biology (PSB), suppo ed by FRISBI (ANR-10-INBS-05-02) and GRAL, inanced wi hin he Uni e si y G enoble Alpes g adua e school (Ecoles Uni e si ai es de Reche che) CBH-EUR-GS (ANR-17-EURE-0003). F. F. also acknowledge he F ench Agence Na ionale de la Reche che (ANR) PIA o Glyco@Alps (ANR-15-IDEX-02). We acknowledge D Michael P. O’Hagan o he e ision o he English e sion o he manusc ip . Re e ences 1 A. Va ki, Glycobiology, 2017, 27,3–49. 2 H. Kal ne , J. 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