Chirality-Puckering correlation and intermolecular interactions in Sphingosines: Rotational spectroscopy of jaspine B3 and its monohydrate

Chi ali y-Pucke ing co ela ion and in e molecula in e ac ions in Sphingosines: Ro a ional spec oscopy o jaspine B3 and i s monohyd a e Rizalina T. Sa agi a , Ma cos Juanes a , José L. Abad b , Ru h Pinacho c , José E. Rubio c , Albe o Lesa i a, ⇑ a Depa amen o de Química Física y Química Ino gánica, Facul ad de Ciencias - I.U. CINQUIMA, Uni e sidad de Valladolid, Paseo de Belén 7, Valladolid 47011, Spain b Resea ch Uni on BioAc i e Molecules (RUBAM), Depa amen de Química Biològica, Ins i u de Química A ançada de Ca alunya (IQAC-CSIC), Jo di Gi ona 18-26, Ba celona 08034, Spain c Depa amen o de Elec ónica, ETSIT, Uni e sidad de Valladolid, Paseo de Belén 11 Valladolid 47011, Spain highligh s In a-/in e molecula in e ac ions a he head g oup o Jaspine B analyzed o a ionally. Obse a ion o six isome s o jaspine B and he monohyd a e in he gas phase. In e molecula in e ac ions linked o ing pucke ing and chi ali y, plausibly connec ed o biological ac i i y. g aphical abs ac a icle in o A icle his o y: Recei ed 19 Augus 2021 Recei ed in e ised o m 9 Oc obe 2021 Accep ed 21 Oc obe 2021 A ailable online 16 No embe 2021 Keywo ds: Nonco alen in e ac ions Sphingosines Mic osol a ion Ring-pucke ing Ro a ional Spec oscopy Je spec oscopy abs ac Chi ali y is de e minan o sphingosine bio unc ions and pha macological ac i i y, ye he easons o he biological chi al selec ion a e no well unde s ood. He e, we cha ac e ized he in a- and in e molec- ula in e ac ions a he headg oup o he cy o oxic anhyd ophy osphingosine jaspine B, e ealing chi ali y-dependen co ela ions be ween he pucke ing o he ing co e and he o ma ion o amino- alcohol hyd ogen bond ne wo ks, bo h in he monome and he monohyd a e. Following he speci ic syn- hesis o a sho ened 3-ca bon side-chain molecule, deno ed jaspine B3, six di e en isome s we e obse ed in a je expansion using b oadband (chi ped-pulsed) o a ional spec oscopy. Addi ionally, a single isome o he jaspine B3 monohyd a e was obse ed, e ealing he inse ion o wa e in be ween he hyd oxy and amino g oups and he o ma ion o a ne wo k o O-HN-HO ing hyd ogen bonds. The speci ic jaspine B3 s e eochemis y hus c ea es a double- aced molecule whe e he exposed lone-pai elec ons may easily ca alyze he o ma ion o in e molecula agg ega es and de e mine he sphingosine biological p ope ies. Ó2021 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). 1. In oduc ion In e es in sphingolipids (SLs) is wo- old. F om a s uc u al poin o iew SLs a e key componen s o euka yo ic cell mem- b anes and lipid signaling messange s [1,2]. As an example, SLs h ps://doi.o g/10.1016/j.saa.2021.120531 1386-1425/Ó2021 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). ⇑ Co esponding au ho . Spec ochimica Ac a Pa A: Molecula and Biomolecula Spec oscopy 267 (2022) 120531 Con en s lis s a ailable a ScienceDi ec Spec ochimica Ac a Pa A: Molecula and Biomolecula Spec oscopy jou nal homepage: www.else ie .com/loca e/saa pa icipa e wi h s e ols in he o ma ion o memb ane mic odo- mains o lipid a s, which a e c i ically dependen on he sphin- golipid chi ali y [3]. F om he pha macological side, some sphingosines display s ong chi ali y-dependen an ip oli e a i e p ope ies and can be used as an i umo o cy o oxic compounds, exploi ing hei ole in cell p oli e a ion, mig a ion and apop osis [4]. Phy osphingosines o m a sub-class o sphingoid bases (1, Fig. 1), mos commonly consis ing o a 1,3,4- ihyd oxy-2-amino head and an 18-ca bon alipha ic chain, like D- ibo- phy osphingosine (2). Some unusual phy osphingosines de i a- i es om ma ine o ganisms, like jaspine A (3) and jaspine B (4), ha e impo an pha macological p ope ies. Jaspine B (pachas is- samine), isola ed om he sponges Pachas issa sp.[5] and Jaspis sp. [6] in 2002, is biosyn hesized om D- ibo-phy osphingosine 2by in amolecula nucleophilic displacemen and possesses a unique all-syn (2S,3S,4S)- isubs i u ed e ahyd o u an ing co e. Jaspine B inhibi s he ce amide and sphingomyelin syn hases and shows ema kable an i umo p ope ies on se e al human cance cell lines [7–9]. Since sphingoid bases acqui e biological unc ions wi h speci ic s e eochemical con igu a ions, in e es has g own in he syn hesis and biological assessmen o jaspine s e eoisome s [10,11] and de i a i es [12]. He e we add ess he co ela ion be ween s e eo- chemis y, ing-pucke ing and he in a- and in e -molecula in e - ac ions o jaspine B and i s monohyd a es om a molecula poin o iew, p obing he isola ed molecule in he cooled and collision- less en i onmen o a supe sonic je expansion. Gas-phase s udies bene i om disagg ega ion [13], ende ing in insic single- molecule p ope ies di ec ly compa able o he in acuo molecula o bi al compu a ions. This in o ma ion may help he a ional design o new d ugs based on he jaspine skele on. Howe e , he 14-ca bon alipha ic chain o jaspine B would c ea e a my iad o compe ing con o ma ions, impeding a high- esolu ion spec o- scopic s udy. Fo his eason, we i s p epa ed a specially un- ca ed jaspine B wi h a sho ened side chain, whe e we can cap u e he con o ma ional and s uc u al p ope ies o he bioac- i e pola headg oup and i s ela ion o he side chain using high- esolu ion o a ional spec oscopy. Following chemical and spec- oscopic conside a ions, we syn hesized he 3-ca bon alipha ic chain jaspine analogue o (2S,3S,4S)-4-amino-2-p opyl e ahyd o u an-3-ol, deno ed jaspine B3 (5). Jaspine B3 is expec ed o display a ich h ee-le el-deep iso- me ism a ising om he lexible e ahyd o u an ing, he p esence o wo pola amino and alcohol g oups in adjacen ing a oms and he alipha ic side chain. The e ahyd o u an i e-membe ed ing is a common sca old in biological compounds like nucleic acids, dis- playing unique o sional cha ac e is ics. The acile in e con e sion be ween wis ed and en elope o ms may be desc ibed as a monodimensional la ge-ampli ude mo ion o pseudo o a ion [14], in which ben / wis pucke ing o a es a ound he ing a oid- ing plana con igu a ions. Pucke ing can be e y sensi i e o ing subs i uen s o hyd a ion and may de e mine mac omolecula a angemen s [15–17], as in he di e en pucke ing o DNA/RNA A o B chains. P ecise de e mina ion o he pucke ing cha ac e is- ics is hus necessa y o speci y he pseudo o a ion pa hways, o compa e wi h ela ed biomolecules o clus e s and o assess he in luence o he pola and alipha ic g oups in he p e e ed s uc- u es o jaspine B. In addi ion, he amino and alcohol adjacen g oups o e se e al op ions o in a and in e -molecula hyd ogen bonding, such as O-HN o N-HO[15,18]. The alipha ic side chain, e en unca ed he e o 3 ca bons, adds se e al h ee- old o o s which may engage wi h he ing o he pola g oups c ea ing mul iple isome s. The mic osol a ion o jaspine B uses one wa e molecule o p obe he s eng h o he in amolecula o ces in he monome and he possibili y o wa e o modula e molecula con o ma ions o hyd ogen bonding in he isola ed molecule [13,15,19]. Speci i- cally, wa e may ei he add o dis up he monome in amolecula hyd ogen bonding o pucke ing, o e ing clues on he s eng h and na u e o he in e molecula in e ac ions [20]. All hese ac o s make jaspine B3 a es bed o examine he ela ion be ween in a- and in e -molecula in e ac ions and con o ma ion in sphin- gosine headg oups and o ela e o biomolecula in e ac ions. The p esen wo k ex ends p e ious gas-phase s uc u al s udies on mimics o sphingoid headg oups, es ic ed o small 3- o 4- ca bon open-chain amino-alcohols like se inol [21] o he h eoni- nol [22] and allo- h eoninol [23] dias e eoisome s. These s udies ha e de ec ed mul iple low-ene gy isome s whe e in a- molecula hyd ogen bonds may p oduce linea o cyclic ne wo ks o hyd ogen bonds, e ealing ini ial connec ions be ween chi ali y and in amolecula in e ac ions. The in es iga ion o jaspine B3 ep esen s a quali a i e leap in he p og ess owa d molecula s udies o la ge SLs. The spec oscopic wo k used ad anced chi ped-pulse b oad- band mic owa e spec oscopy [24], o e ing much highe esolu- Fig. 1. Uppe panel: Schema ics o he inse ion o sphingosine in o sphingomyelin and in o a (simpli ied) lipidic bilaye . Lowe panel: Molecula o mulas o sphingosine (1), D- ibo-phy osphingosine (2), jaspine A (3), jaspine B (4) and jaspine B3 (5). R.T. Sa agi, M. Juanes, José L. Abad e al. Spec ochimica Ac a Pa A: Molecula and Biomolecula Spec oscopy 267 (2022) 120531 2 ion (10 -7 cm 1 ) han ib a ional echniques and unambiguously disc imina ing o ame s and e en iso opologues h ough hei momen s o ine ia. Compu a ional ools p o ided a ionaliza ion o he expe imen . 2. Expe imen al me hods 2.1. Chemical syn hesis and spec oscopy The molecule o jaspine B3 was ini ially p epa ed by hyd o- gena ion and dep o ec ion o he p ope isop opylydene-Boc p o- ec ed alkene in e media e used o he o al syn hesis o Jaspine B[25], as de ailed in he Suppo ing In o ma ion. Jaspine B3 was in es iga ed in a supe sonic je expansion wi h a chi ped-pulse b oadband Fou ie - ans o m mic owa e spec ome e [24] wo k- ing in he egion 2–8 GHz, desc ibed in he Suppo ing In o ma ion. B ie ly, he sample was apo ized (95–110 °C) in si u a he spec- ome e injec o , dilu ed in a s eam o neon and co-expanded nea -adiaba ically o o m a pulsed supe sonic je in he e acua ed expansion chambe . The es ima ed o a ional empe a u e is 2 K. The je was hen p obed wi h chi ped mic owa e pulses (4 l s), exci ing he ull bandwid h by a as -passage elec ic-dipole mech- anism [26]. The subsequen ee-induc ion decay is egis e ed in ime scales o 40 l s and Fou ie ans o med o he equency domain. 2.2. Compu a ional me hods The compu a ional me hods included a con o ma ional sea ch and molecula o bi al calcula ions, which a e desc ibed in he Sup- po ing In o ma ion. B ie ly, he compu a ional sea ch s a ed wi h molecula mechanics and he MMFFs o ce ield [27], which p o- ided an ini ial se o molecula geome ies. These con o me s we e ully e-op imized using D3 dispe sion-co ec ed hyb id den- si y unc ional heo y (DFT) me hods [28] and a iple- basis se (B3LYP-D3(BJ)/de 2-TZVP), limi ing he inal sea ch o isome s wi hin ee ene gies below 5 kJ mol 1 . The mos s able con o me s o he monome and he monohyd a es we e addi ionally ecalcu- la ed using a double-hyb id [29] B2PLYP-D3(BJ) unc ional and Fig. 2. Con o ma ional landscape o jaspine B3 and p edic ed ee ene gies a 1 a m and 298 K (B3LYP-D3(BJ)/de 2-TZVP, kJ mol 1 ; see Suppo ing In o ma ion o B2PLYP and MP2 alues). The species obse ed expe imen ally a e enci cled (Ring pucke ing no a ion: T = wis , E = en elope, see Fig. 3; Side chain o ien a ions gi en by he wo dihed als s 1 and s 2 :A=an i, G+/G-=gauche +/-). The oxygen and ni ogen a oms a e in ed and blue colo s, espec i ely (Fo in e p e a ion o he e e ences o colou in he igu e legends, he eade is e e ed o he web e sion o his a icle.) Fig. 3. Ring-pucke ing diag am compa ing he ing con o ma ion o he six isome s o jaspine B3 ( ed ci cles: 1, 2, ...6) and i s obse ed monohyd a e (blue squa e W2). The c osses ep esen p edic ed unobse ed monome con o ma ions. The diag am shows he pucke ing ampli ude (q) and phase (/, see Suppo ing In o ma ion) and e eals a clus e ing o con o ma ions a ound he 3 T 4 wis and E 5 en elope s uc u es ( ed and yellow egions, espec i ely), wi h speci ically associa ed in amolecula in e ac ions. (Fo in e p e a ion o he e e ences o colou in his igu e legend, he eade is e e ed o he web e sion o his a icle.) R.T. Sa agi, M. Juanes, José L. Abad e al. Spec ochimica Ac a Pa A: Molecula and Biomolecula Spec oscopy 267 (2022) 120531 3 second-o de ab ini io pe u ba ion heo y [30] (MP2) and he same basis se . Vib a ional equency calcula ions p o ided a ha - monic o ce ield, cen i ugal dis o ion and ze o-poin ene gy co - ec ions. Fo he dime , complexa ion ene gies we e co ec ed o basis-se supe posi ion e o s wi h he coun e poise me hod [31]. 3. Resul s and discussion 3.1. Po en ial ene gy su ace The in es iga ion o jaspine B3 s a ed wi h a desc ip ion o he po en ial ene gy su ace (PES). The compu a ional esul s a e sum- ma ized in Tables S1-S3 (monome ) and S4-S5 (monohyd a e, Sup- po ing In o ma ion). The calcula ions sugges ed a ich con o ma ional landscape, gene a ing up o 52 con o me s o he monome and 212 isome s o he monohyd a e wi hin 25 kJ mol 1 . The con o ma ional landscape o jaspine B3 can be ca ego ized using he ing con o ma ion and he side chain o ien a ion (dihe- d als s 1 = O1C2–C a -Cb, s 2 = C2C a –CbC c in Scheme S1). The con o - ma ional maps in Fig. 2 and S1-S2 (Suppo ing In o ma ion) and he pucke ing diag am o Fig. 3 speci y ing pucke ing using he C eme -Pople (CP) cu ilinea coo dina es [32], allowing p ecise compa isons wi h ela ed i e-membe ed dominan ing geome- ies, ei he wis ed 3 T 4 (con o me s 1, 3, 5, 7,10, 13) o en elope E 5 (con o me s 2, 4, 6, 9, 11, 15), bo h wi h he alcohol poin ing o he amino g oup. Th ee highe -ene gy isome s show a di e en 3 T 2 (8, 12) o 3 T 4 (14) wis con o ma ion, wi h he amino g oup Fig. 4. PES scans o he sidechain o he 3 T 4 and E 5 ing con o ma ions, showing he loca ion o he mos s able con o me s. The sidechain beha io is simila o bo h 3 T 4 and E 5 pucke ing con o ma ions, wi h p e e ence o G- and A o ien a ions (nume ical alues in Suppo ing In o ma ion). In his blue- o- ed colo scheme he smalle ene gies a e da k blue. The isome loca ions a e ma ked 1-11. Fig. 5. A 4 GHz sec ion o he je -cooled mic owa e spec um o Jaspine B3. Uppe ace: Expe imen al spec um; Lowe ace: Fi o he di e en con o me s 1–6 using he o a ional pa ame e s o Table 1 and a o a ional empe a u e o 2 K. R.T. Sa agi, M. Juanes, José L. Abad e al. Spec ochimica Ac a Pa A: Molecula and Biomolecula Spec oscopy 267 (2022) 120531 4 owa ds he alcohol. Fo each geome y se e al side-chain o ien a- ions a e possible. Addi ional bidimensional PES scans o he 3 T 4 and E 5 ing con o ma ions in Fig. 4 used B3LYP-D3(BJ) and show p e e ence o a oms Cband C c o be o ien ed ei he an ipe iplana (A, s 1 180°)o gauche– (G–, s 1 300°) espec o he ing he - e oa om, wi h ela i ely small ene gy di e ences be ween he lowes -lying con o me s and close simila i y be ween he 3 T 4 and E 5 ing con o ma ions. 3.2. Jaspine B3 monome The compu a ional p edic ions we e assessed in ou spec- oscopy expe imen . The dense o a ional spec um in Fig. 5 immedia ely sugges ed he p esence o mul iple compe ing iso- me s. The spec um was hen sea ched i e a i ely using simula- ions wi h he ial o a ional cons an s in Tables S1 (B3LYP), S2 (B2PLYP) and S3 (MP2) in he Suppo ing In o ma ion. Mos ansi- ions exhibi ed small (<1 MHz) nuclea quad upole coupling e ec s (Figu es S3-S4, Suppo ing In o ma ion), so he spec um was analyzed wi h a Wa son’s semi igid- o o Hamil onian wi h quad upole e ms app op ia e o he 14 N nucleus (nuclea spin I=1)[33]. Bo h he global spec al pa e ns and he indi idual hype ine e ec s we e ex emely sensi i e o molecula geome y, leading o an unambiguous s uc u al iden i ica ion. The p ocess was epea ed i e a i ely and inally a o al o six di e en con o m- e s we e posi i ely iden i ied, wi h he expe imen al pa ame e s and measu ed ansi ions p esen ed in Table 1 and S6-S12 (Sup- po ing In o ma ion). The compa ison o expe imen and heo y con i med he de ec ion o all six jaspine B3 isome s wi h con o - ma ional ene gies below 3 kJ mol 1 (ou lined in Fig. 2). Di e ences be ween he expe imen al and heo e ical o a ional cons an s we e below 0.8% o B3LYP and B2PLYP, while MP2 o e ed wo se esul s (<2.8%). Rela i e in ensi ies on a common se o l a ansi- ions ga e es ima ions o he je popula ions o 2:1:3:4:6:5 = 1.0 > 0.69(6) > 0.34(3) 0.34(3) > 0.09(1) > 0.06(1), indica ing ha iso- me 2 is he mos popula ed. Howe e , popula ion a ios canno be ansla ed in o ene gy di e ences as con o ma ional elaxa ion is o en obse ed in he je in case o low (<5–10 kJ mol 1 ) in e con- e sion ba ie s [34,35]. We explo ed plausible con e sions be ween non-obse ed and de ec ed species in Figu es S5-S8 (Sup- po ing In o ma ion), ob aining ba ie s in he ange 6.9–17.3 kJ m ol 1 e en o changes limi ed o he side chain o ien a ion. The smalle ba ie s exchanging con o me s 10 and 3 o 9 and 2 (6.9 and 7.1 kJ mol 1 , espec i ely) may be su moun able in he je , explaining he absence o addi ional speci ic isome s. The expe imen ally obse ed isome s o jaspine B3 comp ise h ee 3 T 4 wis (1, 3, 5) and h ee E 5 en elope (2, 4, 6) con o ma- ions. Topological analyses o he educed elec onic densi y wi h NCI plo s [36] in Figu es S9-S10 (Suppo ing In o ma ion) and he s uc u al da a in Tables S13-S18 (Suppo ing In o ma ion) con i med he p esence o in amolecula hyd ogen bonds in all Table 1 Expe imen al o a ional pa ame e s o he six obse ed isome s o jaspine B3 and he dime (jaspine B3)-wa e . See Suppo ing In o ma ion o heo e ical alues. Sys em Jaspine B3 Jaspine B3- wa e Isome 1 2 3 4 5 6 W2 Pucke ing 3 T 4 E 53 T 4 E 53 T 4 E 53 T 4 Side chain AA G - AG - AAAG - G - G - G - AA A/ MHz a 2795.4709(11) c 2243.8144(10) 3174.6108(18) 2462.18(14) 2901.8114(71) 2298.2034(14) 1382.331(28) B/ MHz 741.10679(29) 801.63564(35) 686.67666(33) 804.3406(12) 750.5540(14) 857.91008(53) 667.2511(10) C/ MHz 645.70568(22) 704.55488(32) 616.73418(28) 696.1520(11) 677.8354(14) 743.21068(50) 485.6647(10) 3/2 aa 1.569(16) 2.2950(97) 3.090(17) 0.392(52) 4.04(13) 2.897(35) 3.355(47) 1/4 ( bb - cc ) 0.3322(51) 1.7099(27) 0.2650(49) 1.314(13) 0.585(32) 1.6560(90) 0.893(40) D J / kHz [ 0.0] d 0.1626(66) [ 0.0] [ 0.0] [0.0] 0.3314(62) [0.] D JK / kHz [ 0.0] 0.754(11) [ 0.0] [ 0.0] [0.0] [ 0.0] [0.] D K / kHz [ 0.0] 3.24(13) [ 0.0] [ 0.0] [0.0] [ 0.0] [0.] | l a | / D +++ e +++ +++ +++ ++ + +++ | l b |/D + ++ + – – ++ – | l c |/D ++ + ++ – – – – N b 161 168 103 62 36 56 45 / kHz 15.2 10.2 13.4 12.5 13.5 15.7 12.4 a Ro a ional cons an s (A,B,C), nuclea quad upole coupling pa ame e s ( a , b , c ), Wa son’s S- educ ion cen i ugal dis o ion cons an s (D J ,D JK ,D K ; he cons an s d 1 and d 2 we e ixed o ze o) and elec ic dipole momen s ( l a , a =a,b,c). b Numbe o i ed ansi ions (N) and ms de ia ion ( ) o he i . c S anda d e o s in pa en heses uni s o he las digi . d Values in squa e b acke s we e ixed o ze o. e One o mo e posi i e signs indica es he obse a ion and in ensi y o he co esponding ansi ions. Fig. 6. Con o ma ional landscape o he mos s able (jaspine B3)-wa e monohy- d a es W1 o W5 (T = wis , E = en elope), wi h p edic ed ee ene gies a 1 a m and 298 K (B3LYP-D3, kJ mol 1 ; see Suppo ing In o ma ion o B2PLYP and MP2 alues). The obse ed species is enci cled. R.T. Sa agi, M. Juanes, José L. Abad e al. Spec ochimica Ac a Pa A: Molecula and Biomolecula Spec oscopy 267 (2022) 120531 5 isome s. The wis ed 3 T 4 isome s display a single O-HN hyd ogen bond, as his ing con o ma ion inc eases he dis ance be ween he amino g oup and he ing oxygen (B3LYP: (O-HN) = 2.07 Å). Con- e sely, he E 5 con o me s may es ablish a ne wo k o wo consec- u i e O-HN-HO ing hyd ogen bonds, since he ing pe mi s a close app oach o one o he amino N-H bonds owa ds he ing he e oa om. These isome s combine a ela i ely sho O-HN hyd ogen bond ( (O-HN) = 1.94–1.99 Å) wi h a long N-HO ing con ac ( (N-H O ing ) = 2.59–2.61 Å). All in amolecula in e ac- ions a e cons ained by he ing geome y bu all wi hin he s a- is ical bounds o hese hyd ogen bonds [15,18]. 3.3. Jaspine B3 monohyd a e The addi ion o wa e o he ca ie gas line pe mi ed he obse a ion o new ansi ions om he monohyd a ed dime (Table S19, Suppo ing In o ma ion). Figu e S11 (Suppo ing In o - ma ion) shows he nuclea quad upole coupling hype ine e ec s o some ypical o a ional ansi ions, which we e well ep oduced wi h a semi igid- o o model. The expe imen al esul s in Table 1 and Table S20 (Suppo ing In o ma ion) can be compa ed wi h cal- cula ions o he con o ma ional landscape in Tables S4 (B3LYP) and S5 (B2PLYP and MP2, Suppo ing In o ma ion), which p edic ed Fig. 7. Uppe panel: NCI plo mapping he in e - and in amolecula in e ac ions in he obse ed monohyd a e o jaspine B3. The blue and g een shades indica e s ongly and weakly a ac i e in e ac ions, espec i ely (see Suppo ing In o ma ion), cha ac e izing he hyd ogen bonds. Lowe panel: Plo o he educed elec onic densi y g adien s ¼ 1 23 p 2 ðÞ 1=3 q jj q 4=3  s. he signed elec onic densi y compa ing con o me 1 (= 3 T 4 AA, ed ace) and he wa e dime W2 (= 3 T 4 AA, blue ace). A ac i e c i ical poin s a e shown as nega i e minima o he cu e (see Suppo ing In o ma ion). The compa ison shows he o ma ion o new in e ac ions in he dime . (Fo in e p e a ion o he e e ences o colou in his igu e legend, he eade is e e ed o he web e sion o his a icle.) R.T. Sa agi, M. Juanes, José L. Abad e al. Spec ochimica Ac a Pa A: Molecula and Biomolecula Spec oscopy 267 (2022) 120531 6 hi een di e en isome s below 5 kJ mol 1 . The expe imen al da a unequi ocally co espond o isome W2 o Fig. 6-7 and Figu e S12 (Suppo ing In o ma ion), associa ed o a 3 T 4 ing pucke ing con- o ma ion. A ew ansi ions we e en a i ely assigned o isome W1, bu he small da ase did no pe mi a de ini i e con i ma ion. Consis en ly, he W2 monohyd a e shows he la ges complexa ion ene gy (B2PLYP: 49.5 kJ mol 1 ). The obse ed W2 monohyd a e can be a ionalized as esul o he inse ion o a wa e molecule in be ween he alcohol and amino g oups o he monome 3 T 4 glo- bal minimum, also p edic ed o isome W3 (E 4 ). Con e sely, iso- me s W1, W4 and W5 would co espond o a common pa e n o addi ion o he ing oxygen o h ee di e en pucke ings. The pucke ing plo o Fig. 3 e idences small ing dis o ions o isome W2 espec o he monome , so he wa e ole is o ein o ce he cha ac e is ic ne wo k o hyd ogen bonds in which wa e always pa icipa es simul aneously as p o on dono and accep o . NCI plo s in Fig. 7 and s uc u al da a in Table S21 (Suppo ing In o - ma ion) con i med he complemen a i y and o ma ion o new in a- and in e molecula in e ac ions in he dime . The 3 T 4 dime exhibi s wo cha ac e is ic O-HO w -HN-H hyd ogen bonds, while o he hyd a es may show up o h ee consecu i e O-HN- HO w -HO hyd ogen bonds. The addi ion o wa e o he ing oxygen gi es he sho es hyd ogen bond (B3LYP: (O w -HO )= 1.83–1.85 Å). Wa e also beha es as p o on dono o he amino g oup when inse ing in be ween he pola g oups in he ing ( (O w -HN) = 1.90 Å), con i ming he amino g oup as be e hyd o- gen bond accep o and poo e dono , as obse ed in se inol [21] and h eoninol [22,23]. 4. Conclusion The obse a ion o six isome s and he mos s able monohyd a e o jaspine B3 p o ides an accu a e desc ip ion o he con o ma- ional landscape and mic osol a ion p ope ies. As obse ed in o he biomolecules wi h a e ahyd o u an co e, in pa icula nucleo ides, ing pucke ing u ns undamen al, as ela i ely small pucke ing di e ences a e ampli ied by he ing subs i uen s, condi- ioning in e molecula in e ac ions and la ge-scale molecula beha iou [15]. Pucke ing p e e ences a e ela i ely unchanged o he monome and hyd a es and speci ically dependen on he ing subs i uen s, as obse ed in u anosides [37]. This esul con- i ms p e ious obse a ions ha molecula s abili y a ises om an in e play o in insic hype conjuga i e elec onic e ec s and in amolecula in e ac ions [16,17]. Simila i ies in he side chain o di e en pucke ing con o ma ions sugges a seconda y ole o he alipha ic g oup, as in amino acids [38]. As a esul , he speci ic jaspine s e eochemis y, which loca es he pola g oups on he same side o he ing, is de e minan o i s in a- and in e molecula in e ac ions, c ea ing a double- aced molecule whe e he exposed lone-pai elec ons on he subs i uen s ace may easily ca alyze he o ma ion o in e molecula agg ega es. The mic osol a ion o jaspine B3 shows mul iple hyd a ion pa e ns by p o on dona ion o he oxygen ing o he amino g oup, c ea ing chi ali y- dependen hyd ogen bond ne wo ks amilia in suga hyd a ion [39,40], which may be ins umen al o he jaspine biological ole. Finally, he impo ance o gas-phase molecula s udies o comple- men c ys al di ac ion o liquid NMR in o ma ion o biochemical building blocks should be ou lined, ma king u u e ou es o in e- g a ed s udies co e ing om he ba e molecule o he in eg a ion o biomolecula uni s in he hyd a ed biological en i onmen . CRediT au ho ship con ibu ion s a emen Rizalina T. Sa agi: Valida ion, Fo mal analysis, In es iga ion, Da a cu a ion, W i ing – o iginal d a , W i ing – e iew & edi ing, Visualiza ion. Ma cos Juanes: Valida ion, In es iga ion, W i ing – e iew & edi ing. José L. Abad: Concep ualiza ion, Me hodology, Resou ces, In es iga ion, W i ing – e iew & edi ing. Ru h Pina- cho: So wa e, Resou ces, W i ing – e iew & edi ing. José E. Rubio: So wa e, Resou ces, W i ing – e iew & edi ing. Albe o Lesa i: Concep ualiza ion, Me hodology, Resou ces, Da a cu a ion, W i ing – o iginal d a , W i ing – e iew & edi ing, Supe ision, P ojec adminis a ion, Funding acquisi ion. Decla a ion o Compe ing In e es The au ho s decla e ha hey ha e no known compe ing inan- cial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape . Acknowledgemen s Funding suppo om he Spanish MICINN-FEDER (g an PGC2018-098561-B-C22) is g a e ully acknowledged. M.J. and R. T.S. a e hank ul o p edoc o al con ac s om he MICINN and UVa, espec i ely. Appendix A. Supplemen a y ma e ial Supplemen a y da a o his a icle can be ound online a h ps://doi.o g/10.1016/j.saa.2021.120531. Re e ences [1] B. Cinque, L. Di Ma zio, C. Cen i, C. Di Rocco, C. Ricca di, M.G. Ci one, Sphingolipids and he immune sys em, Pha macol. Res. 47 (2003) 421–437, h ps://doi.o g/10.1016/S1043-6618(03)00051-3. [2] T. A iga, W.D. Ja is, R.K. Yu, Role o sphingolipid-media ed cell dea h in neu odegene a i e diseases, J. Lipid Res. 39 (1) (1998) 1–16, h ps://doi.o g/ 10.1016/S0022-2275(20)34198-5. [3] K. Simons, E. 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