Conformational preferences in diglycosyl disulfides: NMR and molecular modeling studies

Accep ed Manusc ip Con o ma ional P e e ences in Diglycosyl Disul ides: Nm and Molecula Mod‐ elling S udies K isz ina Fehé , Richa d P. Ma hews, Ka alin E. Kö é , Ke in J. Naidoo, László Szilágyi PII: S0008-6215(11)00371-5 DOI: 10.1016/j.ca es.2011.07.013 Re e ence: CAR 5864 To appea in: Ca bohyd a e Resea ch Recei ed Da e: 13 May 2011 Re ised Da e: 5 July 2011 Accep ed Da e: 12 July 2011 Please ci e his a icle as: Fehé , K., Ma hews, R.P., Kö é , K.E., Naidoo, K.J., Szilágyi, L., Con o ma ional P e e ences in Diglycosyl Disul ides: Nm and Molecula Modelling S udies, Ca bohyd a e Resea ch (2011), doi: 10.1016/j.ca es.2011.07.013 This is a PDF ile o an unedi ed manusc ip ha has been accep ed o publica ion. As a se ice o ou cus ome s we a e p o iding his ea ly e sion o he manusc ip . The manusc ip will unde go copyedi ing, ypese ing, and e iew o he esul ing p oo be o e i is published in i s inal o m. Please no e ha du ing he p oduc ion p ocess e o s may be disco e ed which could a ec he con en , and all legal disclaime s ha apply o he jou nal pe ain. CONFORMATIONAL PREFERENCES IN DIGLYCOSYL DISULFIDES: NMR AND MOLECULAR MODELLING STUDIES K isz ina Fehé #a,b, Richa d P. Ma hews#c,d, Ka alin E. Kö é b, Ke in J. Naidoo*c,d, László Szilágyi*a Depa men s o O ganica and Ino ganicb Chemis y, Uni e si y o Deb ecen , H-4010 Deb ecen, P . 20., Hunga y and Scien i ic Compu ing Resea ch Uni c and Depa men o Chemis yd, Uni e si y o Cape Town, Rondebosch 7701, Sou h A ica Abs ac The con o ma ions o se e al 1 1’ diglycosyl disul ides we e in es iga ed by NMR and compu a ional me hods. Expe imen al da a such as NOEs, p o on-p o on and p o on-ca bon-13 coupling cons an s, measu ed o solu ions in DMSO, a e in good ag eemen wi h alues ob ained by MD simula ions in explici DMSO. The disul ide o sion angles (C1-S-S-C1’) p e e en ially sample alues close o ei he +90o o –90o (+g o –g) and appea as he main me ic ha de e mines he con o ma ional beha io o hese glycomime ics. The e is mo e con o ma ional eedom a ound he C1-S and C1’-S’ bonds (Φ and Ω o sions, espec i ely) and popula ion clus e analysis allowed o iden i y up o ou allowed con o ma ional egions o each o he +g o –g o ms. Popula ion analysis o he hyd oxylic g oup o ame s, based on p o on- p o on and p o on-ca bon-13 couplings as well as on calcula ed hyd ogen bonding s a is ics, did no e eal any signi ican in amolecula hyd ogen bonds in DMSO solu ion. *Au ho s o whom co espondence should be add essed e-mail: [email p o ec ed] e-mail: lszilagyi@ ig is.unideb.hu # These au ho s con ibu ed equally o compu a ional analyses. Keywo ds: diglycosyl disul ides, con o ma ion, MD simula ion, hyd oxylic o ame popula ions, NMR coupling cons an s, NOEs 1. In oduc ion Making and b eaking o he glycosidic bond is undamen al o ca bohyd a e chemis y and biology. Na u al glycosides a e hyd olyzed by glycosidase enzymes, a eac ion o en implica ed in pa hological p ocesses. Resis ance o glycoside hyd olases is mos e icien ly achie ed by manipula ing he glycosidic linkage; i is he e o e o p ime impo ance o in es iga e he physicochemical p ope ies o his bond. Na u al glycosidic linkages usually comp ise wo bonds (C-X-C) be ween he suga ing and an aglycone, o be ween wo suga ings, wi h he b idging a om being an oxygen (X=O) in he majo i y o cases. Th ee-bond in e glycosidic linkages (3BIGLs, C-X-Y-C) wi h X=C and Y=O a e no uncommon ei he bu 3BIGLs wi h wo he e oa oms (X,Y = O, N, S) we e unknown in Na u e un il he disco e y o he espe amicin-calicheamicin g oup o an i umo an ibio ics. A s iking ea u e o hese s uc u es is he unusual –N-O- glycosidic bond in he oligosaccha ide pa s o he molecules ( o a e iew, see:1) NMR2 and X- ay3 da a oge he wi h o ce ield calcula ions4 highligh ed he impo ance o he con o ma ion a ound he –N-O- linkage o ensu e op imal binding o DNA o subsequen clea age.5,6 In oduc ion o a disul ide mo i (X,Y = S) o connec wo di e en monosaccha ide uni s led o a no el class o disaccha ide mimics7-10 cha ac e ized by ano he wo he e oa om-3BIGL ( o a e iew, see Szilágyi e . al11). Some neoglycop o eins ep esen u he examples o in e es ing hyb id s uc u es in which glycosyl uni s a e a ached o p o eins h ough S-S linkages.12-14 Symme ic diglycosyl disul ides we e shown o bind speci ically o he plan lec in Concana alin A15, u he mo e, inhibi o y ac i i ies agains an endogenous lec in we e de ec ed in i o on human umo cell lines.16 Based on hese esul s disul ide-linked suga de i a i es we e sugges ed “as new subs ance pla o m o lec in-di ec ed d ug design”.16 We ha e ecen ly desc ibed speci ic binding o oligo alen a oma ic mannosyl disul ide de i a i es o Concana alin A.17 In o de o gain insigh in o biomolecula in e ac ions, s uc u al cha ac e iza ion o he molecules in ol ed is necessa y. Expe imen al and heo e ical s udies on suga disul ides ha e been limi ed hus a . The c ys al and molecula s uc u es o symme ic18,19 and nonsymme ic diglycosyl disul ide de i a i es20,21 and some alkyl-glycosyl disul ide s uc u es22,19 ha e been published. Recen ly we in es iga ed he chi op ical p ope ies o diglycosyl disul ides and - selenides in solu ion and in he solid s a e.23 P e iously he con o ma ions o a 1Æ4 disul ide disaccha ide we e es ima ed in solu ion on he basis o quali a i e NOEs and simple MO conside a ions,7 while molecula dynamics simula ions wi hou expe imen al es ain s we e epo ed o he symme ic β(1Æ1)digalac osyl-disul ide.16 High esolu ion NMR spec oscopy holds he mos p omise o expe imen al s udies o ca bohyd a e con o ma ions and dynamic p ope ies in solu ion ( o a ecen e iew, see:24). Vicinal coupling cons an s p o ed aluable o con ey in o ma ion on he con o ma ion a he glycosidic dihed als25-27 o in de e mining he dis ibu ion o hyd oxyme hyl o ame s.28-30 Nuclea O e hause e ec s (NOEs) a e use ul o es ima e in e nuclea dis ances bu in e p e a ion o he da a is complica ed by con o ma ional a e aging on he NMR imescale and he limi ed numbe o obse able NOEs be ween monosaccha ide uni s.31,32 Supplemen ing expe imen al da a wi h molecula dynamics (MD) simula ions p o ides an e icien app oach o handle mul iple con o ma ions33,34,27,35,36,31 and o explo e he lexibili y o glycosidic linkages. He e we use an in eg a ed expe imen al and compu a ional app oach o gain insigh in o he con o ma ional beha io o a se ies o diglycosyl-disul ides. Speci ically we epo ou esul s on he disaccha ide mimics D-Glcp-βS(1Æ1’)Sβ-D-Glcp, D-Glcp-βS(1Æ1’)Sβ-D-Galp, D-Glcp- βS(1Æ1’)Sβ-D-Manp, and D-Glcp-βS(1Æ1’)Sβ-D-GlcNAcp o solu ions in DMSO. These a e e e ed o as GSSG, GSSGa and GSSM and GSSGN (Figu e 1), espec i ely o simpli y he discussion below. R 1 R 2 R 3 R 4 GSSG H OH H OH GSSGa H OH OH H GSSM OH H H OH GSSGN H NHAc H OH Figu e 1. Schema ic o he disaccha ides s udied and de ini ion o he o sion angles abou he disul ide in e glycosidic linkage. 2. Ma e ials and me hods The compounds in es iga ed in he p esen s udy ha e been syn hesized acco ding o published p ocedu es.10 2.1 NMR measu emen s NMR expe imen s we e un on a B uke A ance DRX-500 spec ome e . 10-15 mg o he samples we e dissol ed in DMSO-d6 and he p obe empe a u e was se o 300K. 2D NOE (NOESY) and o a ing ame ROE (ROESY) spec a in phase sensi i e mode (TPPI) we e ob ained using s anda d B uke pulse p og ams; he mixing ime being 300 ms in bo h expe imen s o each compound. A CW spinlock ield o 3.57 kHz s eng h was used o he ROESY expe imen s. HOHAHA e ec s37,38 and J- elayed c oss peaks39,40 we e iden i ied by epea ing he expe imen using a di e en o se equency o he spinlock ield.41 The aw da ase s ypically consis ed o 2K 512 complex da a poin s. The c oss peak in ensi ies we e de e mined by olume in eg a ion om he baseplane co ec ed spec a. The indi idually assigned c oss peaks we e checked o he absence o he a e ac s abo e be o e con e ing hem in o dis ances using he Isola ed Spin Pai App oxima ion (ISPA).42 The dis ances be ween 1,3 and 1,5 diaxial p o ons wi hin he glycopy anosyl ings wi h 4C1 chai geome y43,44 we e used o calib a ion o he in eg als. Long- ange p o on-ca bon coupling cons an s we e de e mined using a sensi i i y enhanced, g adien selec ed 13C- il e ed TOCSY pulse sequence called gs-HETLOC45 and HSQMBC expe imen s.46,47 Theo e ical alues o homo- and he e onuclea icinal couplings in ol ing hyd oxyl p o ons we e calcula ed om MD-simula ed o sion angles using he Ka plus ype equa ions below,48,49 2.0cos5.1cos4.10 2 , 3+−= ϕϕ OHH J (1) 102.0cos585.0cos494.5 23 +−= ϑϑ HOCC J (2) whe e φ is de ined as Hi-Ci-O-Hi and ϑ as Ci-1-Ci-O-Hi. Popula ions o he hyd oxyl o ame s a ound he C-O bond we e es ima ed om he measu ed 3JH,OH and 3JHOCC coupling cons an s49 and compa ed wi h calcula ed alues (see Resul s and Discussion). 2.2 Compu a ional 2.2.1 Adiaba ic maps The ene gy landscapes o each disaccha ide we e explo ed as a unc ion o he h ee o sion angles ha desc ibe he con o ma ion abou he disul ide linkage. They a e de ined as: Φ = H1 – C1 – S1 – S1’, Ψ = C1– S1 – S1’ – C1’, and Ω = S1 – S1’ – C1’ – H1’ (see Fig.1). Adiaba ic maps we e cons uc ed in h ee dimensional ( , , ) space using he simula ed annealing algo i hm50 as desc ibed p e iously o he h ee-bond glycosidic linkage in isomal ose36 and panose.35 The disaccha ides we e modeled using he CSFF o ce ield51 wi h he addi ion o CSFF consis en pa ame e s o he SS-linkage.52 2.2.2 Molecula Dynamics simula ions Based on he h ee-dimensional adiaba ic maps, low lying minima we e chosen as s a ing poin s o simula ions in explici DMSO. The molecula dynamics p og am CHARMM33b53 was used o all he simula ions. DMSO MD simula ions we e conduc ed using he S ade e al. model o DMSO.54 The dynamics we e un in a cubic box wi h sides 41.7262 Å applying pe iodic bounda y condi ions. The disaccha ides we e sol a ed wi h 612 DMSO molecules. The o e lapping DMSO molecules wi hin a hea y a om dis ance o 3.5 Å om he solu e we e subsequen ly emo ed. A e an ini ial 1 ns equilib a ion he MD simula ions we e ex ended o a u he 10 ns o he con o ma ions ha we e mos consis en wi h expe imen al NOE da a, while sho e , 5 ns simula ions we e un o he emaining con o ma ions. The simula ions we e ca ied ou using he leap og Ve le in eg a o and implemen a ion o he iso he mal-isoba ic ensemble (NPT) whe e he p essu e and empe a u e a e kep cons an (P = 1 ba , T = 300 K) by making use o he Lange in pis on me hod.55 Da a o all espec i e con igu a ions we e s o ed a in e als o 0.05 ps and 0.5 ps o he 10 ns and 5 ns long simula ions, espec i ely. 2.2.3 Compu a ional Analysis Popula ion clus e analysis (PCA) was pe o med using he ART2’ adap i e analogue pa e n ecogni ion scheme.56,57 A clus e adius alue58 was used o p e en alse clus e s om a ising. In o de o measu e he occu ence o in a- and in e molecula hyd ogen bonds du ing he MD simula ion hyd ogen bonding s a is ics we e calcula ed. We used a geome ic de ini ion o a maximum dis ance c i e ion o 2.4 Å be ween he accep o oxygen and he hyd ogen along wi h an angle o no less han 100o o (dono oxygen)-hyd ogen-(accep o oxygen) a angemen . 3. Resul s and discussion 3.1 Con o ma ional analysis o he in e glycosidic o sion angles based on adiaba ic maps and MD simula ions in explici DMSO The h ee dimensional adiaba ic ene gy su ace e ealed local minima p ima ily in wo main a eas cha ac e ized by −90o and +90o along he Ψ angle o all disul ide disaccha ides. The e o e he con o ma ional space is discussed in e ms o hese wo low ene gy egions sepa a ely. Con o ma ions close o Ψ = −90o and +90o minima a e designa ed as –g and +g con o ma ions, espec i ely. The po en ial ene gy su aces shown in Figs. 2 and 3 we e ob ained by slicing along he Ψ = −90° and Ψ = +90° plane o he h ee dimensional adiaba ic su aces and con ou ing a ene gies o 2 kcal.mol-1 om he lowes minimum and up o 12 kcal.mol-1. Wi hin hese egions se e al local minima exis o each o he disaccha ides, hese a e iden i ied by le e s A o D in he –g con o ma ion (Fig. 2) and E o G in he +g con o ma ion (Fig. 3; o a de ailed lis ing o he da a, see Supplemen a y Table S1). Figu e 2. Rep esen a i e wo-dimensional sec ions om he (a) GSSG, (b) GSSGa, (c) GSSM and (d) GSSGN adiaba ic maps o Ψ = −90°. The ene gy is con ou ed in inc emen s o 2 kcal.mol-1 abo e he local minimum. Figu e 3. Rep esen a i e wo-dimensional sec ions om he (a) GSSG, (b) GSSGa, (c) GSSM and (d) GSSGN adiaba ic maps o Ψ = +90°. The ene gy is con ou ed in inc emen s o 2 kcal.mol-1 abo e he local minimum. The esul s o he MD simula ions in explici DMSO a e displayed in Figs. 4 and 5 by depic ing clus e dis ibu ions in he Φ/Ω space. Popula ion a e aged angles o he clus e s o he MD simula ion a e summa ized in Table 1 along wi h he o sion angles o hei s a ing s uc u es. Figu e 4. Rep esen a ions o he clus e s a ising om MD ajec o ies calcula ed in DMSO and s a ed in he −g con o ma ion. Each symbol iden i ies a clus e ob ained by PCA o he MD un s a ed om he adiaba ic minima A-D labeled wi h symbols ,, S and { espec i ely. Con o ma ional egions a e deno ed by oman nume als in acco dance wi h Table 1. Da a a e shown o he –g con o ma ions o GSSG (a), GSSGa (b), GSSM (c), and GSSGN (d). Figu e 5. Rep esen a ions o he clus e s a ising om MD ajec o ies calcula ed in DMSO and s a ed in he +g con o ma ion. Each symbol iden i ies a clus e ob ained by PCA o he MD un s a ed om he adiaba ic minima A-D labeled wi h symbols U, , and { espec i ely. Con o ma ional egions a e deno ed by oman nume als in acco dance wi h Table 1. Da a a e shown o he +g con o ma ions o GSSG (a), GSSGa (b), GSSM (c), and GSSGN (d). Fo GSSG he lowes ene gy minimum is A in he –g con o ma ion ollowed closely by minimum E wi h ela i e po en ial ene gy o 0.256 kcal.mol-1 in he +g con o ma ion as shown in Table S1. All o he minima ha e ene gies mo e han 1 kcal.mol-1 highe han he global minimum. MD simula ions s a ed om each o he adiaba ic minima esul ed in ou con o ma ional egions lis ed in Table 1 (las column). Inspec ion o he da a shows ha o GSSG in he –g con o ma ion minima A and D we e quasi conse ed in he MD simula ions yielding clus e g oups II and III, while minimum C shi ed along he angle o a egion, designa ed as I as shown in Fig. 4a. In con as , all simula ions in he +g con o ma ion esul ed in a single egion cen ed close o minimum G, named as egion V as depic ed in Fig. 5a. The popula ion-a e aged Ψ alues a e sligh ly less han he ideal +/−90o o all clus e s excep o egion I, which is cha ac e ized by Ψ = −100o (Table 1). Fo GSSGa he global ene gy minimum is s a e A in he -g con o ma ion as shown in Table S1, while he second lowes ene gy minimum is well B wi h 0.98 kcal.mol-1, also ound in he –g con o ma ion. The MD simula ions s a ed om he di e en adiaba ic minima yielded al oge he six egions lis ed in Table 1. Fou egions o he con o ma ional space we e sampled in he –g con o ma ion, co esponding o he a eas a ound minima A, C, D yielding con o ma ional g oups III, II, I and an addi ional new egion, con o ma ional a ea IV was ound, which eme ged om he simula ion s a ed om minimum B as shown in Fig. 4b. In he +g con o ma ional s a e he MD simula ions sampled con o ma ions in one b oad egion encompassing all minima E, F and G. By compa ison, +g con o ma ions o he o he de i a i es could be clus e ed along Ω in o he wo egions designa ed as V and VI, as seen in Fig. 5b. The popula ion a e aged Ψ angles a e sligh ly less han he ideal +/−90o o all clus e s, while egion IV displays a dis inc ly di e en Ψ angle (−107o). Fo GSSM he global ene gy minimum is in he +g con o ma ion E, hough i is closely ollowed by minimum A in he -g con o ma ion, which has only 0.2 kcal.mol-1 highe ene gy han E as shown in Table S1. The e a e ye ano he wo minima which a e ela i ely close o he global minimum: s a e B wi h 0.775 kcal.mol-1 and F wi h 0.997 kcal.mol-1. The MD simula ions s a ed om he a ious minima explo ed only ou egions o he con o ma ional space lis ed in Table 1. In he –g con o ma ion only a eas a ound minima D and C we e e i ied by he MD simula ion esul ing in con o ma ional egions I and II as shown in Fig. 4c. In he +g con o ma ion a egion a ound minimum E designa ed as a ea VI and clus e s a ising om he simula ion s a ed om minimum G, named as con o ma ional g oup VII, we e iden i ied as shown in Fig. 5c. All esul ing egions ha e simila Ψ alues o somewha smalle han +/−90o wi h he excep ion o he clus e s a ing om G (105o). Fo GSSGN he global ene gy minimum is A in he -g con o ma ion wi h all o he con o ma ions ha ing signi ican ly highe po en ial ene gy as shown in Table S1. The MD simula ions o GSSGN p oduced h ee dis inc egions in bo h o he –g (I, II and III, Fig. 4d) and +g ( V, VI and VIII, Fig. 4b) con o ma ional s a es as lis ed in Table 1. All six egions displayed e y simila Ψ alues o somewha less han he ideal +/−90o. Table 1 To sion angles o minimum ene gy s a ing s uc u es o MD simula ions yielding con o ma ional egions wi h popula ion a e aged o sion angles. MD popula ion a e aged Adiaba ic map minima Φ Ψ Ω Φ Ψ Ω Con o ma ional egions GSSG A -170 -90 0 -166 -82 38 III B 60 -90 -40 182 -85 19 III C 50 -90 50 -2 -100 -21 I D 30 -90 -180 30 -86 179 II E 60 90 -50 -5 84 46 V F 60 90 -50 -22 86 55 V G -40 90 40 -22 85 46 V GSSGa A 180 -90 20 -171 -83 -4 III B -140 -90 -160 -50 -107 171 IV C 30 -90 170 39 -78 164 II D 30 -90 30 35 -88 38 I D -178 -90 35 III E -40 90 40 -8 81 33 V E 21 86 -22 VI F 80 90 -20 -35 90 44 V G 40 90 -30 -1 87 -39 VI GSSM A -150 -90 170 30 -81 181 II B 180 -90 -10 21 -91 59 I C 30 -90 150 -3 -86 194 II D 50 -90 30 45 -86 43 I E 60 90 -30 28 82 -56 VI F -170 90 20 42 79 -62 VI G -20 90 50 174 105 52 VII GSSGN A 180 -90 30 -164 -85 13 III B 180 -90 0 -27 -86 -20 I C -30 -90 20 13 -90 28 I D 40 -90 170 50 -89 178 II E 70 90 -20 37 85 -53 VI F -20 90 50 18 84 27 V G -40 90 170 12 90 164 VIII The adiaba ic maps ga e an o e all imp ession o he minimum ene gy con o ma ions, bu some o hese egions disappea ed o shi ed o a sligh ly di e en a ea o he po en ial ene gy su ace du ing he MD simula ions. The di e ences be ween he adiaba ic maps and he space sampled by MD may be a ibu ed o sol a ion e ec s in he MD simula ion. O e all he numbe o inal con o ma ional egions we e educed in compa ison wi h he numbe o adiaba ic minima. The MD simula ions con i med ha he Ψ angle p e e ences we e ca. +/−90o. The dis ibu ions o Ψ alues in he MD simula ions we e es ic ed o a compa a i ely na ow ange Good co ela ion was obse ed be ween expe imen al and calcula ed alues o all 1H-1H coupling cons an s. The majo i y o he calcula ed 3JHOCC couplings also show easonable ag eemen wi h measu ed alues. Di e ences exceeding +/- 1Hz may be due o pa ial exchange o OH-p o ons on he expe imen al side and/o subs i uen e ec s no p ope ly aken in o accoun in eq. (2) in he calcula ions. 3JHC coupling cons an s a e known o be no o iously sensi i e o subs i uen e ec s (see63 and e e ences ci ed he ein). A g aphical ep esen a ion (using GSSGa as an illus a ion) o he co ela ion be ween expe imen al and calcula ed alues is shown in Figu e 8. Fig. 8. Co ela ion be ween expe imen al and calcula ed coupling cons an s o GSSGa (c . Tables 4 & 5). Colo coding and symbols: *, blue: 3JHOCH measu ed; o, ed: 3JHOCH calcula ed; •, g een: 3JHOCC measu ed; o, black: 3JHOCC calcula ed. E o ba s a e added in ma ching colou s. Acco ding o he Ka plus equa ions (1) & (2) coupling cons an s o he o de o ~ 5.4 Hz and ~ 2.8 Hz, o 3JHOCH and 3JHOCC, espec i ely, ep esen o a ion-a e aged alues. The da a in Tables 4 and 5 a e close o hese alues and he e o e indica e quasi ee o a ion a ound he C-O bond o all hyd oxyl g oups in es iga ed. When bo h couplings a e a ailable o a pa icula OH u he in o ma ion can be deduced, namely, popula ions o each o he OH o ame ic s a es can be es ima ed in e ms o an ipe iplana (ap) and synclinal (sc) con o ma ions a ound he C-O bond.49 Popula ion analyses o he con o ma ional p e e ences o he same hyd oxyl g oups we e pe o med o bo h he +g and –g simula ions by binning he sampled o ame s in inc emen s o 5o. Ro ame s wi hin 5o o any ansi ions, i.e. +sc o -sc, e c. we e excluded om he inal summa ion. OH o ame ic popula ions ha we e es ima ed om NMR coupling cons an s show good co ela ion wi h alues ob ained om MD simula ions (Table 6). Ro ame ic s abiliza ion may be p omo ed by s e eo-elec onic e ec s and/o hyd ogen bonding. Calcula ed H-bonding s a is ics p esen ed abo e a e, howe e , uling ou he occu ence o pe sis en hyd ogen bonds in DMSO solu ion. This is (also) in line wi h expe imen al and heo e ical esul s demons a ing he dis up ion o in e nal hyd ogen bonds in sol en s wi h inc eased pola i y such as wa e o DMSO.60-62 The da a in Table 6 indica e no p e e ed o ame ic s a e o he majo i y o he OH g oups in GSSGa, GSSM and GSSGN. Low pe cen age o he ap o ame s o Glu-OH3 and Gal-OH4 in GSSGa and one o he sc o ame s o GlcNAc-OH3 in GSSGN we e ound, howe e , by expe imen and calcula ion alike (Table 6). Acco ding o he conside a ions abo e his es ic ion o he o ame ic eedom may be due s e eo-elec onic e ec s a he han engagemen in o in e nal hyd ogen bonds o hese pa icula OH g oups. Table 6. NMR and MD o ame popula ion dis ibu ions o OH g oups Compound G oup NMR measu ed o ame s (%)* MD calcula ed o ame s (%)Ŧ P(-sc) P(ap) P(+sc) P(-sc) P(ap) P(+sc) GSSGa Glu-OH2 36 37 17 32 40 21 Glu-OH3 17 9 28 26 4 49 Glu-OH4 33 23 37 35 10 45 Glu-OH6 36 29 n.a. 45 40 13 Gal-OH2 40 43 28 40 40 18 Gal-OH3 37 17 37 36 19 40 Gal-OH4 60 9 29 60 18 20 GSSM Glu-OH2 38 48 20 37 42 20 Glu-OH3 35 27 28 28 6 47 Glu-OH4 34 27 44 24 22 50 Glu-OH6 37 23 n.a. 46 42 8 Man-OH2 13 n.a. 34 11 53 34 Man-OH3 23 33 34 11 25 44 Man-OH4 30 23 45 18 14 50 Man-OH6 37 23 n.a. 45 38 15 GSSGN Glu-OH2 38 35 n.a. 40 40 17 Glu-OH3 n.a. n.a. n.a. 55 42 1 Glu-OH4 29 30 41 30 15 50 Glu-OH6 31 23 n.a. 44 41 5 GlcNAc- OH3 0 45 33 3 37 57 *As de e mined om h ee-bond p o on-p o on- (3JH,OH) and p o on-ca bon (3JHOCC) coupling cons an s (see ex ). The +sc, -sc and ap no a ions e e o he synclinal o an ipe iplana o ien a ion o p o on OH(i) wi h espec o C(i-1). Popula ions P(n) a e app oxima e (+/−10%) alues. ŦMD o ame popula ions calcula ed o he +g/-g a e age con o ma ion. 4. Conclusion In summa y, expe imen al NMR da a such as NOEs, p o on-p o on and p o on-ca bon-13 coupling cons an s supplemen ed wi h MD calcula ions in explici DMSO ha e clea ly indica ed ha he main me ic o de e mine he con o ma ions in 1 1’ diglycosyl disul ides is he disul ide o sion angle (C1-S-S-C1’) which p e e en ially samples alues close o ei he +90o o –90o (+g o –g). Signi ican ly mo e con o ma ional eedom was obse ed a ound he C1-S and C1’-S’ bonds (Φ and Ω o sions, espec i ely) and popula ion clus e analysis (PCA) allowed o iden i y up o ou allowed con o ma ional egions o each o he +g o –g o ms. Rega ding con o ma ional simila i ies s. di e ences be ween he membe s o he cu en panel o diglycosyl disul ide s uc u es i can be s a ed in gene al ha subs i uen e ec s (s e ic o elec onic) o g oups close o he disul ide b idge ha e la ge impac on he con o ma ional dis ibu ions han changes a emo e posi ions. Visual compa ison o adiaba ic maps and MD simula ions bo h show he highes con o ma ional simila i y o GSSG o GSSGa, less simila i y o GSSGN and e en less o GSSM. In pa icula , in he –g o m con o ma ional g oups I, II and III a e sampled by GSSG, GSSGa and GSSGN whe eas only g oups I and II a e p esen o GSSM. In he +g o m con o ma ional g oup V, which is dominan o GSSG, is sha ed only by GSSGa and GSSGN, bu is no p esen o GSSM. This is in line wi h he gene al end s a ed abo e. The change o he 4-OH g oup om equa o ial (GSSG) o axial (GSSGa) posi ion is a away om he disul ide b idge and he e o e does no esul in la ge con o ma ional di e ences. The 2-NAc g oup in GSSGN is close o disul ide b idge, howe e , he subs i u ion does no signi ican ly a ec he s e eoelec onic cha a e isi ics o GSSGN compa ed o GSSG. The GSSM de i a i e ea u es he la ges di e ence compa ed o GSSG because he equa o ial 2-OH g oup, nex o he disul ide linkage, in he la e is changed o an axial one in he mannosyl uni o he o me . Es ima ion o he hyd oxylic g oup o ame popula ions, based on calcula ed hyd ogen bonding s a is ics and 1H-1H / 1H-13C J-coupling analysis, did no e eal any signi ican in amolecula hyd ogen bonds in DMSO solu ion. 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Ma hews, Ka alin E. Kö é , Ke in J. Naidoo, László Szilágyi* The disul ide o sion angles (Ψ) sample alues close o +90o o –90o (+g o –g) and up o ou allowed con o ma ional egions a ound Φ and Ω we e iden i ied o each o he Ψ alues. HIGHLIGHTS Con o ma ions o diglycosyl disul ides we e s udied by NMR and compu a ions The disul ide o sion angles (Ψ) a e close o +90o o –90o (+g o –g) Up o ou con o ma ional egions a ound Φ and Ω a e allowed o each Ψ alues No signi ican in amolecula hyd ogen bonds we e e ealed in DMSO solu ion.