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
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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.
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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.
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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.
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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
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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.
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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 .
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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 .
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Pape O ganic & Biomolecula Chemis y
6094 |O g. Biomol. Chem.,2020,18,6086–6094 This jou nal is © The Royal Socie y o Chemis y 2020
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