Biomime ic Tweeze s o N-Glycans: Selec i e Recogni ion
o he Co e GlcNAc2Disaccha ide o he Sialylglycopep ide
SGP
F ancesco Milanesi,[a, b] Luca Unione,[c, d] Ana A dá,[c, d] C is ina Na i i,[a]
Jesús Jiménez-Ba be o,[c, d, e, ] S e ano Roelens,[a] and Osca F ancesconi*[a]
Abs ac : In ecen yea s, glycomics ha e shown how
pe asi e he ole o ca bohyd a es in biological sys ems is
and how chemical ools a e essen ial o in es iga e glycan
unc ion and modula e ca bohyd a e-media ed p ocesses.
Biomime ic ecep o s o ca bohyd a es can ca y ou his ask
bu , al hough signi ican a ini ies and selec i i ies owa d
simple saccha ides ha e been achie ed, a ge ing complex
glycoconjuga es emains a goal ye una ained. In his wo k
we epo he unp eceden ed ecogni ion o a complex
bian enna y sialylglycopep ide (SGP) by a weeze s-shaped
biomime ic ecep o , which selec i ely binds o he co e
GlcNAc2disaccha ide o he N-glycan wi h an a ini y o
170 μM. Because o he simple s uc u e and he ema kable
binding abili y, his biomime ic ecep o can ep esen a
e sa ile ool o glycoscience, opening he way o use ul
applica ions.
In oduc ion
Oligosaccha ides (glycans, ca bohyd a es) a e widely exp essed
on he su ace o euka yo ic cells as glycoconjuga es o p o eins
and lipids.[1,2] Thei selec i e ecogni ion by di e en classes o
p o eins, such as lec ins, igge s essen ial physio- and pa ho-
logical p ocesses, like cell adhesion and in ec ion, cell di e -
en ia ion, in lamma ion, and immune esponse.[3–5] Glycosyla ion
is an ubiqui ous pos - ansla ional modi ica ion o p o eins ha
leads o conjuga ion wi h O- and N-linked oligosaccha ides. In
pa icula , N-glycans, ha is, N-linked oligosaccha ides, all sha e
he common pen asaccha ide s uc u e Man3GlcNAc2[Manα1-
3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ] N-linked o he aspa -
agine esidue o he Asn-X-Th /Se (X is any amino acid excep
P o) sequence in he p o ein backbone.[6] Depending on he
s uc u al di e si y o he oligosaccha ides ex ending beyond
he common s uc u e, N-glycans a e classi ied as high-
mannose ype, complex- ype, and hyb id ype. N-glycans play
essen ial oles in he chemical and biological beha iou o
p o eins, including p o ein olding and s abili y,[7,8] solubili y,[9]
esis ance o p o eolysis,[10] in acellula a ic,[11] an igenici y,[12]
ac i i y, and ecogni ion by ca bohyd a e-binding p o eins.[13]
Because o glycan ubiqui y, molecula agen s a ge ing N-
glycans can lead o a deepe unde s anding o hei unc ion
and o he de elopmen o new diagnos ic and p ognos ic
s a egies.[14] Typically, ecogni ion o speci ic saccha ides o
elici unc ion is achie ed in na u e by lec ins, which a e glycan
binding p o eins specialized in ecognizing speci ic suga
epi opes.[15] Howe e , hei pha maceu ical de elopmen has
emained la gely un ul illed, due o hei suscep ibili y o
p o eolysis, hei abili y o elici immune esponse, and o he
ea u es ela ed o hei p o ein na u e.
Syn he ic biomime ic ecep o s o ca bohyd a es a e a
he e ogeneous class o molecules de eloped o selec i e
ecogni ion o suga s o biological in e es .[16–21] To mimic he
ecogni ion unc ion o lec ins, he binding abili y o biomime ic
ecep o s elies exclusi ely on non-co alen in e ac ions. How-
e e , achie ing e ec i e ecogni ion o pola species like
saccha ides in wa e le e aging a biomime ic app oach is a
[a] D . F. Milanesi, P o . C. Na i i, D . S. Roelens, D . O. F ancesconi
Depa men o Chemis y “Ugo Schi ” DICUS and INSTM
Uni e si y o Flo ence
Polo Scien i ico e Tecnologico, I–50019 Ses o Fio en ino, Fi enze (I aly)
E-mail: [email p o ec ed]
[b] D . F. Milanesi
Magne ic Resonance Cen e CERM
Uni e si y o Flo ence
Via L. Sacconi 6, I-50019
Ses o Fio en ino, Fi enze (I aly)
[c] D . L. Unione, D . A. A dá, P o . J. Jiménez-Ba be o
CICbioGUNE,
Basque Resea ch & Technology Alliance (BRTA)
Bizkaia Technology Pa k, Building 800,
48160 De io, Bizkaia (Spain)
[d] D . L. Unione, D . A. A dá, P o . J. Jiménez-Ba be o
Ike basque, Basque Founda ion o Science
Ma ia Diaz de Ha o 3, 48013 Bilbao, Bizkaia (Spain)
[e] P o . J. Jiménez-Ba be o
Depa men o O ganic Chemis y,
II Facul y o Science and Technology
Uni e si y o he Basque Coun y, EHU-UPV
48940 Leioa (Spain)
[ ] P o . J. Jiménez-Ba be o
Cen o de In es igación Biomédica En Red de En e medades Respi a o ias
Mad id (Spain)
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non- i ial ask.[22,23] Indeed, miles ones in his ield ha e been
achie ed only ecen ly, mainly by means o sophis ica ed
mac ocyclic a chi ec u es.[24–30] In con as , acyclic ecep o s
ha e been la gely unexplo ed,[31–34] al hough hey may p esen
dis inc ad an ages o e mac ocyclic s uc u es in e ms o
simple syn hesis, mo e easily modi iable s uc u e, easy adap -
abili y o di e en gues s and, mos o all, he possibili y o
binding non- e minal saccha ide en i ies.
We ha e ecen ly epo ed design, syn hesis, and applica-
ions o he acyclic, weeze s-shaped ecep o 1(Figu e 1),[35,36]
which e ec i ely ecognizes s anda d disaccha ides in wa e a
pH 7.4. Recep o 1is based on a e y simple s uc u e,
consis ing o a diaminoca bazole hyd ogen-bonding uni [25,37]
and wo an h acene ings, es ablishing CH-πin e ac ions[38] wi h
he non-pola egion o ca bohyd a es. Recep o 1selec i ely
ecognized he me hyl βglycoside o N,N’-diace ylchi obiose
(MeβGlcNAc2), he co e disaccha ide o he conse ed pen asac-
cha ide s uc u e o N-glycans, o e a wide ange o mono- and
disaccha ides, showing a ema kable a ini y o 160 μM. This
a ini y alue no only exceeds ha exhibi ed by mo e complex
mac ocyclic ecep o s, bu also ha shown by he lec in he ein
(isola ed om He ea b asiliensis) by one o de o magni ude
(Kd=1.61 mM, logβ=2.79).[39] The e o e, ecep o 1s ands as
he ideal sca old o ecognize complex- ype N-glycans o
biological in e es by binding o he co e GlcNAc2disaccha ide.
Among N-glycans, sialyla ed N-glycans a e o pa icula
in e es , because a e he a ge o a class o lec ins named
Siglecs (sialic acid binding immunoglobulin ype lec ins).[40–42]
Siglecs a e cell su ace immunomodula o y ecep o s, which
selec i ely ecognize speci ic sialyla ed glycans exposed on all
mammalian cells and help immune cells o dis inguish be ween
sel and non-sel epi opes. One o he mos ubiqui ous
sialyla ed N-glycans in mammals is he complex- ype bian en-
na y sialylundecasaccha ide SGP, which can be ob ained om
egg yolk as he glycopep ide[4,43] (Figu e 1). Recogni ion o
glycans by lec ins, including siglecs, usually occu s by binding
o he epi ope loca ed a he end o he oligosaccha ide chain;
weeze s-shaped ecep o s may o e he oppo uni y o
le e age a di e en binding si e, loca ed in he conse ed uni
o he glycan co e, hus p o iding a new and po en ially e y
use ul ecogni ion ool.
Following his al e na i e ecogni ion app oach, in his wo k
we epo on he unp eceden ed ea u es o a weeze s-shaped
ecep o e ec i ely ecognizing he na u al N-glycan SGP by
selec i ely binding o he GlcNAc2co e disaccha ide.
Resul s and Discussion
E alua ion o he ecogni ion p ope ies o ecep o 1 owa d
complex- ype N-glycans is, un o una ely, p e en ed by s ong
sel -associa ion in wa e (logβdim =2.65�0.07),[36] due o he
p esence o he ex ended a oma ic su aces o he an h acene
moie ies, s acking in e molecula ly unde he p essu e o hyd o-
phobic e ec s.[44] In addi ion, such a s ong dime iza ion
phenomenon p e en s no only a quan i a i e de e mina ion o
binding a ini ies, bu also a s uc u al de ini ion o he binding
modes when binding o complex glycans. To o e come he sel -
associa ion issue, we ha e unc ionalized he pa a- posi ions o
he an h acene ings o 1wi h wo addi ional phosphona e
g oups, gi ing ise o he e aphosphona e ecep o 2(Fig-
u e 1).
Recep o 2was p epa ed om comme cially a ailable 9,10-
dib omoan h acene (3) in six s eps (Figu e 2a). A e o myla ion
o 3using n-BuLi ollowed by DMF, he ca baldehyde 4was
p o ec ed as he dime hylp opylene ace al 5by he pTsOH
ca alyzed condensa ion wi h neopen yl glycol. The b omo-
subs i uen o he ace al in e media e was hen eplaced by a
die hylphosphona e g oup using n-BuLi ollowed by PO(OE )2Cl.
The ace al 6was hen hyd olyzed by HCl in ace one, o gi e he
aldehyde 7, which was condensed wi h he diaminoca bazole 8,
p epa ed acco ding o a p e iously epo ed me hod,[25] o
p o ide he co esponding bis-imino de i a i e. Subsequen
educ ion wi h NaBH4ga e he liposoluble ecep o 9. Hyd ol-
ysis o he phosphona e es e s wi h TMSB , ollowed by
ea men wi h me hanol ga e he desi ed ecep o 2.
The concen a ion dependen oligome iza ion s a e o
ecep o 2was in es iga ed by NMR. In con as o i s
p ogeni o , dilu ion expe imen s ca ied ou on 2a pH 7.4
e ealed sha p NMR signals in a wide concen a ion ange, om
24 o 1 mM, oge he wi h a modes a ia ion o 1H NMR
chemical shi s, sugges ing a weak sel -associa ion beha iou
Figu e 1. Concep o he wo k: con e sion o he biomime ic ecep o 1 o 2(wi h p o on labelling) o be es ed s. he sialylglycopep ide SGP.
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(Figu e S17). Nonlinea eg ession o he co esponding da a
ga e a dime iza ion cons an (logβdim =0.98�0.05) ha ,
al hough s ill de ec able, is mo e han one o de o magni ude
smalle han ha obse ed wi h he pa en ecep o 1. As
expec ed, he p esence o addi ional hyd osolubilising g oups
on he an h acene moie ies p e en ed π-s acking in e ac ions
and, consequen ly, sel -associa ion phenomena.
To asce ain whe he he in oduc ion o phosphona e
g oups on an h acene ings may pe u b he binding p ope ies
o ecep o 1, we es ed ecep o 2 owa d he same se o
mono- and disaccha ides used o ecep o 1. A sc eening o
he binding p ope ies o ecep o 2was hus ca ied ou by 1H
NMR spec oscopy (Figu es S14-S16) owa d he mono- and
disaccha ides shown in Figu e 2b and 2c, espec i ely. The pool
included se e al monosaccha ides [glucose (Glc), N-ace ylgalac-
osamine (GalNAc), hamnose (Rha), ucose (Fuc), xylose (Xyl),
sialic acid (Sia), and αand βme hyl de i a i es o Glc, Gal, Man,
and GlcNAc], oge he wi h a ew disaccha ides [suc ose (Suc),
ehalose (T e), cellobiose (CeB), mal ose (Mal), lac ose (Lac) and
he me hyl glycoside o N,N’-diace ylchi obiose (MeβGlcNAc2)].
In all cases, he chemical shi pe u ba ions (CSPs) o he 1H
NMR signals o he suga s upon addi ion o equimola amoun s
o 2we e moni o ed. Excep o me hyl-β-glucoside (MeβGlc),
o which an app eciable a ia ion was measu ed (ΔδH-1 =
0.05 ppm), a he small (Δδ <0.04 ppm) o no a ia ions we e
de ec ed o o he monosaccha ides. Conce ning disaccha ides,
MeβGlcNAc2expe ienced a massi e pe u ba ion o he p o on
signals, showing a ma ked line b oadening ha sp ead mos
signals in o he spec um baseline, which indica ed chemical
exchange p ocesses be ween ee and bound species in he
medium/ as egime in he NMR ime scale. While no changes
we e obse ed o Suc and T e, a ma ked shi was ins ead
obse ed o he βanome o CeB and, o a smalle ex en , o
hose o Lac and Mal, analogous o ha p e iously epo ed o
he pa en ecep o 1. Mo eo e , o he CeB and Lac
disaccha ides, he addi ion o ecep o 2induced a signi ican
line b oadening o he NMR signals. This e idence s ongly
sugges s a simila binding p o ile o bo h ecep o s.
Figu e 2. (a) Syn he ic scheme o he biomime ic ecep o 2. In es iga ed monosaccha ides (b) and disaccha ides (c) and hei abb e ia ions.
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A quan i a i e in es iga ion was hus ca ied ou by NMR
spec oscopy on he in e ac ion o 2wi h MeβGlc, MeβCeB,
MeβMal, MeβLac, and MeβGlcNAc2. Me hyl glycosides we e
used o a oid in e con e sion equilib ia be ween anome s. The
cumula i e associa ion cons an s epo ed in Table 1 we e
measu ed by 1H NMR i a ions in D2O (pD 7.4) a 298 K, by
simul aneously i ing he complexa ion induced shi s o all he
a ailable signals o he app op ia e associa ion model by
nonlinea eg ession analysis. Because mul iple binding con-
s an s we e de ec ed, a ini ies we e assessed hough he
in insic median binding concen a ion pa ame e BC500,[45]
calcula ed om he measu ed binding cons an s and epo ed
in Table 1.
A i s glance, da a con i m a binding p o ile closely simila
o ha obse ed o he p ogeni o 1. Indeed, ecep o 2
e ec i ely disc imina es disaccha ides om monosaccha ides,
showing a signi ican ly lowe a ini y o MeβGlc han o
MeβCeB. Like seen o 1, ecep o 2shows a dis inc p e e ence
o all-equa o ial disaccha ides, and o MeβCeB and MeβGlc-
NAc2wi h espec o MeβLac and MeβMal. Mos impo an ly,
he la ges a ini y was obse ed o MeβGlcNAc2(270 μM), e y
simila o ha measu ed o 1(160 μM), showing ha addi ional
phosphona e g oups a ec binding p ope ies only ma ginally.
To shed ligh on he ene ge ics o binding and o suppo
he NMR-based a ini ies measu ed o he 1,4-linked disaccha -
ides, binding was also in es iga ed by ITC. Because he
dime iza ion cons an o he ecep o 2is negligible in H2O a
pH 7.4, dime iza ion was no included in he nonlinea
eg ession analysis o ITC da a. Cumula i e binding cons an s,
oge he wi h a ini y alues a e ga he ed in Table 1 o p o ide
a di ec compa ison wi h he NMR esul s. The good ag eemen
be ween he wo independen echniques con i med he
eliabili y o he obse ed a ini ies. In con as o ecep o 1, o
which s ong dime iza ion p e en ed he de e mina ion o
he modynamic pa ame e s, eliable da a o he o ma ion o
1:1 complexes could be ob ained o 2(Table 2), om which i
can be app ecia ed ha en halpy p o ides he impe us o he
o ma ion o all 1:1 complexes, whe eas an ad e se en opic
con ibu ion is consis en wi h a p ominen ole o in e molec-
ula o ces o e sol ophobic e ec s.
Based on he selec i i y shown by ecep o 2 o MeβGlc-
NAc2, he de e mina ion o binding a ini y owa d he SGP N-
glycan was a emp ed by NMR echniques. Because o s ong
signal o e lapping, use ul chemical shi da a could no be
collec ed o a quan i a i e analysis (Figu e S23). Thus, he
binding abili y o 2 owa d SGP was measu ed by ITC i a ions
(Figu e 3a), which we e ca ied ou in H2O a pH 7.4 (Table 1),
gi ing an a ini y alue o 170 μM, sligh ly la ge han ha
measu ed o MeβGlcNAc2. In con as , he he modynamic
pa ame e s we e unexpec edly di e en (Table 2): while he ee
ene gy o binding is essen ially he same compa ed o
MeβGlcNAc2, he en halpic con ibu ion o SGP ecogni ion is
signi ican ly smalle , whe eas a posi i e en opy is esponsible
o binding. Resul s sugges a d as ic change in he sol a ion
ea u es o SGP wi h espec o MeβGlcNAc2.
The a ini y o 2 o SGP measu ed by ITC was u he
con i med by Ci cula Dich oism (CD) as an independen
echnique. Because bo h, he UV ch omopho e 2and he chi al
SGP, a e silen on CD, he echnique is pa icula ly con enien ,
because i moni o s he unambiguous o ma ion o he complex
be ween he wo pa ne s. CD i a ions we e ca ied ou on
p e-neu alized eac an s in H2O a pH 7.4. Upon addi ion o
SGP o ecep o 2, he CD in ensi y inc eased a 254.5, 260 and
265.5 nm, ea u ing nega i e sign o he i s wo wa eleng h
and posi i e o he la e (Figu e 3b). Nonlinea leas -squa e
eg ession analysis o CD da a i ed a 1:1 s oichiome y model,
gi ing logβ=3.737�0.003, co esponding o BC500=183�
Table 1. Cumula i e o ma ion cons an s (logβn)[a] and in insic median binding concen a ion (BC500, mM)[b] o ecep o o glycoside (R:G) complexes o 2
wi h me hyl glycosides and SGP, measu ed a 298 K om NMR da a in D2O a pD 7.4 and om ITC da a in H2O a pH 7.4.[c] BC500(mM) o ecep o 1a e
epo ed o compa ison.
Recep o 2(NMR) 2(ITC) 1(NMR)
Glycoside R:G log βBC500log βBC500BC500
MeβGlc 1:1 1.59�0.01 38.4�1.7
MeβCeB 1:1 2.40�0.01 1.04�0.06 2.49�0.07 1.32�0.43 0.94�0.10
2:1 5.79�0.07 5.53�0.38
2:2 8.00�0.09
MeβMal 1:1 2.00�0.01 11.9�0.3 1.78�0.02 16.6�0.7 31.0�4.4
MeβLac 1:1 2.11�0.01 9.01�016 1.97�0.01 10.5�0.3 30.7�4.7
MeβGlcNAc21:1 3.47�0.02 0.27�0.01 3.45�0.10 0.18�0.04 0.16�0.01
2:1 6.48�0.05 7.16�0.23
SGP 1:1 3.44�0.26 0.17�0.05
2:1 7.30�0.26
[a] Fo ma ion cons an s we e ob ained by nonlinea leas -squa e eg ession analysis o NMR da a. [b] Calcula ed om he log β alues using he “BC50
Calcula o ” p og am.[45] [c] The ecep o dime iza ion cons an s a pH 7.4 (2: log βdim =0.98�0.05) was se in a ian in he nonlinea eg ession analysis o
NMR.
Table 2. The modynamic pa ame e s (kJ mol1) o he o ma ion o
ecep o 2 o glycoside 1:1 complexes o MeβCeB. MeβMal. MeβLac,
MeβGlcNAc2, and SGP in H2O. pH 7.4 a 298 K.
Glycoside -ΔG0-ΔH0TΔS0
MeβCeB 14.2�0.4 26.1�1.4 11.9�1.0
MeβMal 10.2�0.1 17.9�0.5 7.7�0.6
MeβLac 11.3�0.1 23.4�0.5 12.1�0.6
MeβGlcNAc219.7�0.6 47.3�2.2 27.7�2.7
SGP 19.6�1.5 14.5�5.0 5.2�6.5
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1μM, closely compa able o he a ini y ob ained by ITC. The
highly dilu ed condi ions used o he CD i a ion (63 μM)
accoun o he simple model, in which highe s oichiome y
complexes a e unde ec able.
I should be emphasized ha , o he bes o ou knowledge,
he molecula ecogni ion o N-glycans by biomime ic a i icial
ecep o s has no been p e iously epo ed. Indeed, he na u al
p oduc s belonging o he an ibio ic p adimicin amily is he
only known non-pep idic amily binding o N-glycans .[46]
Mo eo e , i is no ewo hy ha he a ini y achie ed by ecep o
2is ully compa able o ha o na u al lec ins.[39]
P omp ed by he abo e ecogni ion p ope ies, an in es-
iga ion on he h ee-dimensional s uc u e o he complex
be ween 2and SGP was a emp ed using NMR expe imen al
da a assis ed by molecula modelling calcula ions.[47] 1D and 2D
NMR spec a (Figu es S24–S31) we e ca ied ou o assign all
he a ailable p o on signals o SGP.
In o ma ion on he geome y o he majo complex was
ex ac ed om he analysis o 2D NMR NOESY spec a eco ded
o a 1:1 mix u e o 2and SGP. Se e al in e molecula NOE
con ac s we e unambiguously de ec ed be ween he ecep o
and esidues a, b, c, and d o he undecasaccha ide, indica ing
ha molecula ecogni ion akes place on he N-glycan co e
(Figu e 4a). As obse ed o he in e ac ion o 2wi h he simple
disaccha ide MeβGlcNAc2, he me hyl o he N-ace yl g oup
(CH3- a) displayed a NOE con ac wi h he ca bazole HB
p o on. Mo eo e , H-2-b o GlcNAc2showed a NOE c oss peak
wi h he an h acene HF, clea ly indica ing ha he disaccha -
ide is loca ed be ween he wo an h acene ings, inside he
ecep o cle . Addi ional in e molecula con ac s we e in e ed
by he p esence o NOE c oss peaks in ol ing HD and HE o
he an h acene and H-5-c o he β-mannose esidue, and wi h
H-6-d and H-6-d’ o he α6-linked mannose esidue, sugges ing
ha he glycan ex ends ou side he binding cle o 2beyond
he an h acene moie ies. E en ually, he ca bazole HB shows a
con ac wi h H-5-c, while HC shows a con ac wi h he H-6’ o
esidue d.
We nex ca ied ou a de ailed analysis o he CSPs obse ed
in he NMR spec a o SGP. 1H-13C HSQC NMR expe imen s
(Figu e 4c) showed signi ican changes in speci ic N-glycan
signals upon addi ion o an equimola amoun o ecep o 2. A
d ama ic line b oadening e ec , sp eading he signal below he
limi o signal de ec ion, was expe ienced by he c oss-peaks
co esponding o he co e isaccha ide ( esidues a, b, and c),
u he con i ming ha ecogni ion akes place on he N-glycan
co e. Conce ning he wo a ms, he H-1 and H-2 o he α6Man
( esidue d) also disappea ed, whe eas o H-3, H-4, H-5, and H-
6,6’ signi ican CSPs (>0.05 ppm) we e de ec ed. Pe u ba ions
obse ed o he α3Man ( esidue d’) we e di e en , as H-1 and
H-2 did no disappea , a he hey showed CSPs abo e
0.05 ppm, while he H-3, H-4, H-5 and H-6,6’ showed modes
CSPs (<0.05 ppm). Beyond he mannose esidues, only ma gin-
al CSPs we e de ec ed o some o he esonances om he
GlcNAc esidues e and e’, whe eas no a ia ions we e obse ed
o Gal ( and ’) and Neu5Ac (g and g’). Simila ly, he amino
acids N, A, and K disappea , he amino acid V shows CSP,
whe eas he absence o CSP o N and C e minal amino acids K’
and T indica es ha hese esidues a e loca ed a om he
ecep o cle .
F om he abo e e idence, we conclude ha he ecogni ion
selec i ely occu s on he co e GlcNAc2disaccha ide, lea ing
e minal saccha ide esidues and dis an amino acids unpe -
u bed. Howe e , he abo e di e ences in CSPs and line
b oadening o he wo Man esidues sugges ha he wo
a ms o he SGP span di e en con o ma ional spaces wi h
espec o he a oma ic sca old o he biomime ic ecep o .
In e es ingly, con o ma ional s udies on bian enna y N-glycans
s uc u ally ela ed o SGP ha e shown ha , beyond he
“ex ended” con o ma ion, o he geome ies o he α6 a m a e
also possible, including he so-called “back olded”
o ien a ion,[48] in which he α6-linked Man is o a ed owa d he
GlcNAc2co e, so ha con ac s wi h he ecep o a e also
expec ed. Thus, he obse ed di e ences in CSPs o he wo
Man esidues poin o he exis ence o an equilib ium be ween
he ex ended and he back olded con o ma ions.
To p o ide a h ee-dimensional desc ip ion o he majo
binding modes, he expe imen al da a we e suppo ed by
Figu e 3. Ti a ions o 2wi h SGP in H2O (pH 7.4) a 298 K: a) ITC plo o
expe imen al da a poin s, [black ci cles - o al hea (TQ) s. 2/SGP mole a io]
and i ed cu e [solid line] calcula ed by non-linea eg ession analysis; b)
CD i a ion spec a o 2(63 μM) wi h SGP.
Chemis y—A Eu opean Jou nal
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molecula modelling calcula ions. A con o ma ional sea ch was
ca ied ou on he 1:1 complex o med be ween 2and SGP. A
amily o minimum ene gy con o me s was ound ha ag ees
wi h all he NOE con ac s expe imen ally obse ed. A ep esen-
a i e geome y wi hin his minimum ene gy amily is depic ed
in Figu e 5a. I can be app ecia ed ha he weeze s-shaped
a chi ec u e o 2is pe ec ly sui ed o bind he co e GlcNAc2
disaccha ide in he N-glycan s em. In e es ingly, all he NOE
con ac s ound, including hose be ween H-6-d and he HD
and HE p o ons o an h acene, ag ee wi h an ex ended
con o ma ion, in which he α6-linked Man (d) is close o one o
he an h acene moie ies. The di e en chemical shi pe u ba-
ion and chemical exchange beha iou obse ed o he wo
a ms, which a e mo e signi ican o he α6- han o he α3-
linked Man, may be due o a back olded con o ma ion, which
was indeed ound in he con o ma ional sea ch (Figu e 5b), and
ha may be p esen o a non-negligible ex en in solu ion.
Finally, se e al hyd ogen-bonding, wi h O···H in e a omic dis-
ances sho e han he sum o he an de Waals adii, and CH-
πin e ac ions ha e been de ec ed in he calcula ed models
in ol ing he co e GlcNAc2disaccha ide (Figu e 5c). As p e i-
ously obse ed in he complex o 1wi h MeβGlcNAc2, while he
iden a e diaminoca bazole o 2binds he OH-3a, he N-ace yl
g oup es ablishes CH-πin e ac ions wi h he ca bazole uni .
Addi ional hyd ogen bonds in ol ing he phosphona e g oup
and se e al CH-πin e ac ions wi h bo h he an h acenes
con ibu e o he complex s abiliza ion.
Conclusion
In conclusion, a second-gene a ion biomime ic ecep o 2is
p esen ed, o which he sel -associa ion issues o he p ogeni-
o ha e been add essed by in oducing addi ional phospho-
na e subs i uen s, lea ing he binding p ope ies owa d
MeβGlcNAc2essen ially una ec ed. The weeze s-shaped bio-
mime ic acyclic a chi ec u e o 2shows he unp eceden ed
ea u e o ecognizing wi h high selec i i y he GlcNAc2
Figu e 4. S udies on he binding mode be ween 2and SGP: a) sec ions o he 800 MHz NOESY (500 ms) spec um eco ded o an equimola mix u e o SGP
and 2(2 mM each) in D2O a 298 K. Unambiguous in e molecula NOE c oss peaks a e indica ed by squa es; b) A schema ic ep esen a ion o he
in e molecula NOEs ound be ween 2and SGP is depic ed a he igh hand side; c) Supe imposi ion o he 1H-13C HSQC spec a eco ded o a 2 mM solu ion
o SGP be o e (blue peaks) and a e ( ed peaks) he addi ion o an equimola amoun o 2. Selec ed peaks a e squa ed and labelled: peaks ha p esen
chemical shi a ia ion a e squa ed using a dashed line; peaks ha disappea a e addi ion o 2a e squa ed using a do ed line.
Chemis y—A Eu opean Jou nal
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disaccha ide uni in he co e o a complex- ype N-glycan. Due
o he simple and easily accessible/modi iable s uc u e,
ecep o 2s ands as a new and e ec i e ool o glycoscience
esea ch. Indeed, because ecogni ion occu s a a si e di e en
om he sialic e minal usually ecognized by biological
ecep o s, ecep o 2sugges s po en ial applica ions in analysis,
sepa a ion, and imaging o N-glycans, as well as modula o o
N-glycan ecogni ion p ocesses.
Acknowledgemen s
We hank MIUR-I aly “P oge o Dipa imen i di Eccellenza 2018–
2022” alloca ed o Depa men o Chemis y Ugo Schi , COST
Ac ion (CA18132), MIUR-I aly PRIN2017 (2017XZ2ZBK) o g an -
ing a ellowship o F.M. and En e Cassa di Rispa mio di Fi enze
(I aly) is acknowledged o g an ing an ITC nanocalo ime e and
a high- ield NMR spec ome e . Open Access unding p o ided
by Uni e si à degli S udi di Fi enze wi hin he CRUI-CARE
Ag eemen .
Con lic o In e es
The au ho s decla e no con lic o in e es .
Da a A ailabili y S a emen
The da a ha suppo he indings o his s udy a e a ailable in
he supplemen a y ma e ial o his a icle.
Keywo ds: biomime ic a i icial ecep o s ·ca bohyd a es ·
chi obiose ·glycan ·molecula ecogni ion
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