Oligosaccha ides
Syn he ic Zwi e ionic S ep ococcus pneumoniae Type 1
Oligosaccha ides Ca ying Labile O-Ace yl Es e s
Zhen Wang, Ana Gimeno, Ma a G. Le e, He man S. O e klee , Gijsbe A. an de Ma el,
Fab izio Chiodo, Jesús Jiménez-Ba be o, and Je oen D. C. Codée*
Abs ac : We he ein epo he i s o al syn hesis o
he S ep ococcus pneumoniae se o ype 1 (Sp1) oligosac-
cha ide, a unique zwi e ionic capsula polysaccha ide
ca ying labile O-ace yl es e s. The a ge oligosaccha -
ides, ea u ing a e α-2,4-diamino-2,4,6- ideoxy galac-
ose (AAT) and α-galac u onic acids, we e assembled
up o he 9-me le el, in a highly s e eoselec i e manne
using isaccha ide building blocks. The labili y o he
O-ace yl es e s imposed a ca e ul dep o ec ion scheme
o p e en mig a ion and hyd olysis. The mig a ion was
in es iga ed in de ail a a ious pD alues using NMR
spec oscopy, o show ha mig a ion and hyd olysis o
he C-3-O-ace yl es e s eadily akes place unde neu al
condi ions. S uc u al in es iga ion showed he oligom-
e s o adop a igh -handed helical s uc u e wi h he
ace yl es e s exposed on he pe iphe y o he helix in
close p oximi y o he neighbo ing AAT esidues, he e-
by imposing con o ma ional es ic ions on he AATα1-
4GalA(3OAc) glycosidic linkages, suppo ing he helical
shape o he polysaccha ide, ha has been p oposed o
be c i ical o i s unique biological ac i i y.
In oduc ion
Bac e ial cell-su ace ca bohyd a es play a signi ican ole in
binding e en s wi h componen s o he hos immune
sys em,[1,2] and bac e ial capsula polysaccha ides (CPS) can
e ec i ely be used in he gene a ion o ca bohyd a e-based
an ibac e ial accines. S ep ococcus pneumoniae (o pneu-
mococcus) is a d eaded alpha-hemoly ic G am-posi i e
pa hogen ha can cause a ious ypes o po en ially le hal
in ec ions, including pneumonia, sep icemia, meningi is,
leading o high mo bidi y and mo ali y a es wo ldwide.[3–5]
Al hough an ibio ic ea men o mos pneumococcal
in ec ions is e ec i e, i does induce he e olu ion o d ug-
esis an pneumococcal bac e ia. As ea ly as 1946, he i s
pneumococcal polysaccha ide accine was licensed[6] and
cu en ly a 23- alen CPS accine and 20- alen glycoconju-
ga e accine a e a ailable o o e p o ec ion agains he
mos p e alen se o ypes.[7] Howe e , e en hough hese
accines ha e been e y success ul, he immunological
mechanism o he di e en se o ypes is no well
unde s ood.[8–12] S. pneumoniae ype 1 is a highly i ulen
s ain, and i is cha ac e ized by a unique CPS, Sp1, ha is
buil up om isaccha ide epea ing uni s, ha in u n a e
composed o he a e α-2,4-di-amino-2,4,6- i-deoxygalac ose
(D-AAT) and wo α-D-galac u onic acid esidues (Fig-
u e 1).[13–15] I has been epo ed ha he polysaccha ide can
ca y O-ace yl g oups a he C-2 o C-3 posi ions o he 4-
linked galac u onic acid esidues and ha app oxima ely
wo hi d o he epea ing uni s ca y an ace yl es e .[14] O-
ace yla ion is commonly encoun e ed in bac e ial polysac-
cha ides, and he ole o his modi ica ion can a y.[15,16] I
may be an impo an s uc u al elemen o ecogni ion by
opsonic an ibodies bu can also shield immunogenic epi o-
pes. I has been p oposed ha ace yla ion o Sp1 can impac
he con o ma ion and lexibili y o he polysaccha ide chains
al hough p e ious models ha e allen sho in explaining he
s uc u al ole o hese labile unc ional g oups.[17–21] The
labili y o he O-ace yl g oups complica es he isola ion o
well-de ined and pu e CPS agmen s o s uc u al s udies
[*] Z. Wang, H. S. O e klee , G. A. an de Ma el, J. D. C. Codée
Leiden Ins i u e o Chemis y, Leiden Uni e si y
Eins einweg 55, 2333 CC Leiden (The Ne he lands)
E-mail: [email p o ec ed].nl
A. Gimeno, M. G. Le e, J. Jiménez-Ba be o
CIC bioGUNE
Bizkaia Technology Pa k, Building 801A, 48170 De io (Spain)
F. Chiodo
Ins i u e o Biomolecula Chemis y, Na ional Resea ch Council
(CNR)
Pozzuoli, Napoli (I aly)
and
Ams e dam In ec ion and Immuni y Ins i u e, Depa men o
Molecula Cell Biology and Immunology
Ams e dam UMC, Loca ion VUmc, 1007 MB Ams e dam (The
Ne he lands)
J. Jiménez-Ba be o
Ike basque, Basque Founda ion o Science, Plaza Euskadi 5, 48009
Bilbao, Bizkaia, Spain
J. Jiménez-Ba be o
Depa men o O ganic Chemis y, II Facul y o Science and
Technology, EHU-UPV, 48940 Leioa, Spain
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
© 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion
published by Wiley-VCH GmbH. This is an open access a icle unde
he e ms o he C ea i e Commons A ibu ion License, which
pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided
he o iginal wo k is p ope ly ci ed.
Angewand e
Chemie
Resea ch A icles www.angewand e.o g
How o ci e: Angew. Chem. In . Ed. 2023, 62, e202211940
In e na ional Edi ion: doi.o g/10.1002/anie.202211940
Ge man Edi ion: doi.o g/10.1002/ange.202211940
Angew. Chem. In . Ed. 2023,62, e202211940 (1 o 11) © 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202211940 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [06/03/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
and he es ablishmen o s uc u e-ac i i y ela ionships a
he molecula le el. Mig a ion o O-ace yl g oups has been
obse ed be ween di e en hyd oxyl g oups wi hin ca bohy-
d a e esidues o polysaccha ides bu also be ween neighbo -
ing ca bohyd a e ings.[22–24] While biosyn hesis enzymes can
ans e ace yl es e s in a highly egioselec i e manne ,
spon aneous mig a ion e en s can lead o highly he e oge-
neous sequences.
In con as o na u ally sou ced oligo/polysaccha ides,
syn he ic oligosaccha ides a e well de ined, uni o m mole-
cules, o a single leng h wi h p ede ined subs i u ion
pa e ns, making hem expe ly sui ed o s uc u al s udies
and de ailed in e ac ion s udies wi h binding pa ne s.[25–27]
Mul iple syn he ic e o s ha e been di ec ed a he assembly
o Sp1 oligosaccha ides, bu none ha e add essed he
challenging inco po a ion o he labile O-ace yl es e s.[28,29]
The assembly o he Sp1 isaccha ide epea ing uni has
been epo ed by mul iple g oups[30–32] and Bundle and co-
wo ke s we e he i s o epo on he syn hesis o Sp1
oligome s comp ising wo isaccha ide epea s.[33] We ha e
epo ed on he assembly o Sp1-oligosaccha ides, up o he
dodecasaccha ide le el and we showed h ough molecula
dynamics (MD) simula ions and NMR spec oscopy s udies
ha he oligome s adop a igh -handed helical s uc u e
wi h he nonasaccha ide comple ing a ull u n.[34] Kaspe
and co-wo ke s ha e linked he helical s uc u e o zwi e -
ionic polysaccha ides o hei unique capaci y o bind o
MHC-II molecules, elici ing a T-cell media ed immune
esponse.[35]
The Sp1 polysaccha ide ep esen s majo syn he ic
challenges, amongs which a e he p esence o he a e 2-
ace amido-4-amino-2,4,6- ideoxy-D-galac ose (D-AAT),
he galac u onic acids, which a e well known o be signi i-
can ly less eac i e in glycosyla ion eac ions han hei non-
oxidized galac ose coun e pa s, and he cis-glycosidic link-
ages ha connec all monosaccha ides. Al hough he p es-
ence o he O-ace yl es e s ep esen s only a small s uc u al
modi ica ion, he inco po a ion o hese labile es e s
p esen s an addi ional majo hu dle, because o he
mig a o y ap i ude o hese g oups[22–24] and because hei
p esence signi ican ly limi s p o ec ing g oup manipula ions
as well as op ions o inal dep o ec ion chemis ies.
We he e desc ibe he de elopmen o a syn he ic ou e
o gene a e ace yla ed Sp1 agmen s anging in leng h om
a isaccha ide o a nonasaccha ide, which ha e been
syn hesized in mul i-millig am amoun s o allow o de ailed
s uc u al s udies. Molecula dynamics (MD) simula ions
and NMR spec oscopy we e used o p obe he 3-dimen-
sional s uc u e o he Sp1 oligosaccha ides. These e ealed
ha he oligome s adop a s able helical s uc u e wi h he
C-3-O-ace yl g oups being posi ioned on he ou side o he
helix and close enough o he neighbo ing AAT- esidues o
p o ide an addi ional ba ie o es ic ing he o a ion o
he AATα1-4GalA(3OAc) glycosidic linkages. The s abili y
o he O-ace yl g oups has been assessed by NMR spec o-
scopy a di e en pH alues o show ha hey mig a e
eadily a neu al and sligh ly basic pH, while being s able
o p olonged pe iod o ime a sligh ly acidic pH (>1 yea ).
Resul s and Discussion
The ace yla ed Sp1- agmen s a ge ed in his s udy ( ime
1, hexame 2and noname 3) a e shown in Scheme 1. We
p e iously epo ed a ou e owa ds he de-OAc-Sp1
oligosaccha ides, combining a p e-glycosyla ion oxida ion
s a egy wi h a pos -glycosyla ion oxida ion app oach, o
minimize he di icul oxida ion e en s equi ed on la ge
oligosaccha ides while enabling a obus and highly s e eo-
selec i e glycosyla ion p o ocol.[34] A Bi ch- ype educ ion
was employed a he end o he syn hesis o unmask all
benzyl- ype p o ec ing g oups. Taking he ace yl g oups in
he a ge compounds 1–3in o conside a ion, his global
dep o ec ion scheme can ob iously no be used and he e-
o e a hyd ogena ion s ep will be equi ed a he end o he
syn hesis. We decided o inco po a e a icinal diol e mi-
na ed space o enable u u e conjuga ion chemis y a e a
selec i e oxida ion by a Malap ade eac ion.[36,37] Building
on ou p e iously syn he ic s a egy, we se ou o use
isaccha ide 7as he key building block (Scheme 1). This
building block ea u es a silylidene p o ec ed galac ose
dono moie y, ha allows o he eliable, s e eoselec i e
o ma ion o he equi ed cis-glycosidic linkages. To mini-
mize oxida ion e en s in he oligosaccha ide s age he
building block con ains a galac u onic acid in he middle.
The galac u onic acid also ca ies a C-3-O-ace yl g oup ha
has o be main ained h ough he assembly. T isaccha ide 7
can be ob ained om he monome ic building blocks 8–10.
We s a ed ou syn hesis wi h he assembly o he
equi ed monome ic building blocks 8–10 as depic ed in
Scheme 2. The syn he ic ou e o p o ide he a e 2-
ace amido-4-amino-2,4,6- ideoxy-D-galac ose (D-AAT)
building block 8,[38,39] was op imized and s a ed om 6-
deoxy mannose 11[34,39] (Scheme 2A). We ins alled a bulky
TIPS g oup a he C-3-OH, a e which he diol was i la ed
and he C-2- and C-4-O- i la es we e subsequen ly sub-
s i u ed wi h an azide and ammonia espec i ely. A e Cbz-
p o ec ion o he C-4-amine, AAT building block 13 was
ob ained in 62% om diol 12. Fo compa ison, ou old
ou e using a C-3-O-ace yl and po assium ph halimide
ins ead o ammonia, p o ide he co esponding C-3-O-ace yl
AAT building block in only 21% yield.[34] Desilyla ion,
le ulinoyla ion and ans o ma ion o he hiophenol g oup
Figu e 1. The s uc u e o Sp1.
Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2023,62, e202211940 (2 o 11) © 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202211940 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [06/03/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
in o he co esponding N-phenyl i luo oace imida e hen
p o ided building block 8.
The equi ed galac u onic acid syn hon 9was ob ained
om known C-2-O-benzyl galac ose 15[34] by silyla ion o he
p ima y alcohol, a e which he C-3-OH was egioselec-
i ely ace yla ed by ea ing he diol wi h a ca aly ic amoun
o dime hyl in dichlo ide, di-iso-p opyl e hylamine (DiPEA)
and ace ylchlo ide (Scheme 2B).[40,41] A e ea men o he
esul ing alcohol wi h HF-py idine, diol 16 was ob ained
ha was egio- and chemoselec i ely oxidized using he
TEMPO/BAIB combina ion. Benzyla ion o he so- o med
ca boxylic acid deli e ed galac u onic acid 17 o which he
C-4-OH was masked wi h a le ulinoyl es e . T ans o ma ion
o he hiophenol 18 in o he N-phenyl i luo oace imida e
comple ed he syn hesis o he cen al galac u onic acid
building block 9. The silylidene-p o ec ed galac ose building
block 10 was assembled as p e iously desc ibed.[34]
Wi h all building blocks in hand, he syn hesis o he
oligosaccha ides commenced wi h he assembly o isac-
cha ide a ge 1(Scheme 2C). Fi s , he glycosyla ion
be ween imida e galac u onic acid dono 9and galac ose
accep o 10 was pe o med in he p esence o TBSOT o
p o ide disaccha ide 19 in 75% yield wi h excellen s e eo-
selec i i y. Subsequen ly, selec i e dep o ec ion o he
le ulinoyl p o ec ing g oup was e ec ed by ea men o he
disaccha ide wi h hyd azine ace a e unde acidic condi ions
o p e en mig a ion o he C-3’-ace a e, o deli e he C4’-
OH disaccha ide 20 in 95% yield. Nex , he glycosyla ion
be ween accep o 20 and AAT building block 8was ca ied
ou unde he p omo ion o TBSOT o p o ide 21 in 82%
yield and a 5/1 α/β- a io. The anome s could be sepa a ed a
his s age and he syn hesis was con inued wi h he
hyd olysis o he hioglycoside moie y using NIS-TFA,[42] o
p o ide he co esponding hemiace al 22, which was ol-
lowed by ins alla ion o he imida e moie y deli e ing he
pi o al isaccha ide imida e dono 7. Nex , he space , R-
5,6-bis(benzyloxy)hexan-1-ol 23, p epa ed acco ding o li e -
a u e p ocedu es,[43,44] was a ached o he isaccha ide.
Unde con ol o he bulky silylidene, he glycosyla ion
p oceeded smoo hly o u nish 24 in 85% yield wi h
comple e s e eoselec i i y. Subsequen ly, he le ulinoyl
g oup was clea ed by using hyd azine monohyd a e o
p o ide he isaccha ide 25 in excellen yield. Reduc ion o
he azide g oup using a S audinge eac ion, was ollowed by
selec i e N-ace yla ion o p o ide ace amide 26 in quan i a-
i e yield. T iol in e media e 4could hen be p epa ed in
95% yield om 26 by ea men wi h hyd ogen luo ide in
py idine. The egioselec i e oxida ion o p o ide he ca box-
ylic moie y p oceeded une en ully and was achie ed using
he TEMPO-BAIB oxida ion sys em in a e -bu anol-DCM-
wa e mix u e a 4°C, which was ollowed by benzyla ion
using BnB in DMF o p o ide isaccha ide 27 in 81%
yield. To comple e he syn hesis o he isaccha ide a ge 1,
all benzyl e he s, he benzyl es e s and he benzyl ca bama e
we e emo ed h ough a hyd ogena ion eac ion using
Pd(OH)2as a ca alys . A e pu i ica ion by gel il a ion
column, howe e , he p oduc p o ed o be impu e and
NMR analysis indica ed he p esence o side p oduc s, in
which he ace yl g oup had mig a ed (1a) o was hyd olyzed
(1b) (see Scheme 2D). The labile ace yl g oup in he
isaccha ide appa en ly canno wi hs and he sligh ly basic
condi ions used o he size exclusion ch oma og aphy o
which an aqueous NH4OAc solu ion was used as eluen .[22–24]
The e o e, we checked he in eg i y and pu i y o he
isaccha ide immedia ely a e he hyd ogena ion eac ion.
A e il a ion o he Pd-ca alys and concen a ion, he
isaccha ide p o ed o be pu e and no sign o ace yl
mig a ion was de ec ed by NMR spec oscopy.
Ha ing success ully assembled he isaccha ide, we
mo ed o he p epa a ion o longe oligome s (See Sche-
me 3A). Hexasaccha ide 28 was syn hesized using 26 and 7
in a TBSOT media ed [3+3] glycosyla ion in 83% yield as a
Scheme 1. Re osyn he ic analysis o he assembly o a ge compounds 1–3. Bn=benzyl, Cbz=ca bobenzyloxy.
Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2023,62, e202211940 (3 o 11) © 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202211940 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [06/03/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
Scheme 2. A) Syn hesis o D-AAT building block 8. B) Syn hesis o he cen al C-3-O-ace yl galac u onic acid building block 9. C) Assembly o
isaccha ide 1. D) Mig a ion and hyd olysis o he C-3’-O-ace yl es e . Reagen s and condi ions: a) TIPSCl, imidazole, DMF, 0°C o , 88%; b) i,
T 2O, Py, DMAP, DCM, 10°C o 10°C; ii, TBAN3, MeCN, 30°C! 20°C; iii, 7 N NH3in MeOH; i , CbzCl, Na2CO3, THF, H2O, 0°C o ; ou
s eps 62%; c) TBAF, AcOH, THF, quan .; d) Le OH, EDCI, DMAP, DCM, 14, 91%; 18, 99%; e) NIS, TFA, DCM, 0°C, 8b, quan .; 9a, 96%; 22,
95%; ) N-phenyl i luo oace imidoyl chlo ide, Cs2CO3, ace one, 8, 79%; 9, 91%; 7, 94%; g) TBDPSCl, imidazole, DMF, 96%; h) AcCl, Me2SnCl2,
DIPEA, ThF; i) HFPy, THF, Py, 0°C, 85% ( wo s eps); j) i, TEMPO, BAIB, DCM, -BuOH, H2O; ii, BnB , Cs2CO3, DMF, 17, 77%; 27, 81% (o e wo
s eps); k) TBSOT , DCM, 5Å MS, 0°C, 19, 75%; 21, 82% (αanome , 68%; βanome , 14%); 24, 85%; l) N2H4·AcOH, AcOH, THF, MeOH, 0°C,
95%; m) N2H4·H2O, py idine, AcOH, 0°C!RT, 95%; n) i, PPh3, py idine, H2O, THF, 70°C, 7 h; ii, Ac2O, NaHCO3, THF, H2O, quan i a i e;
o) Pd(OH)2/C, H2, -BuOH, H2O, 3 days, quan i a i e. BAIB=bis(ace oxy)iodobenzene, DCM=dichlo ome hane, DIPEA=N,N-diisop opyle hyl-
amine, DMAP=4-dime hylaminopy idine, DMF=N,N-dime hyl o mamide, EDCI=1-(3-dime hylaminop opyl)-3-e hylca bodiimide, NIS=N-iodo-
succinimide, Py=py idine, TBAF= e abu ylammonium luo ide, TBAN3= e abu ylammonium azide, TBDPS= e -bu yldiphenylsilyl, TBS= e -
bu yldime hylsilyl, TEMPO=2,2,6,6- e ame hylpipe idin-1-oxyl, T = i luo ome hanesul onyl, TFA= i luo oace ic acid, TIPS= iisop opylsilyl.
Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2023,62, e202211940 (4 o 11) © 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202211940 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [06/03/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
single dias e eoisome . Remo al o he le ulinoyl g oup
using hyd azine monohyd a e, as desc ibed o he syn hesis
o 25, u nished hexasaccha ide 29. Subsequen ly, a [3+6]
glycosyla ion and dele ulina ion cycle was ca ied ou o
une en ully p o ide he nonasaccha ide 31 in excellen
yield. Following he syn he ic app oach o isaccha ide
a ge 1, a simila unc ionaliza ion and dep o ec ion
sequence was pe o med wi h hexasaccha ide 29 and non-
asaccha ide 31 o p o ide he pa ially p o ec ed hexasac-
cha ide 5and nonasaccha ide 6, espec i ely. P e iously we
no iced ha he egioselec i e oxida ion o mul iple p ima y
alcohols in he oligosaccha ides became inc easingly di icul
wi h inc easing subs a e leng h. When he oxida ion con-
di ions, as p e iously op imized,[34] we e applied on hexasac-
cha ide 5, con aining i e ee hyd oxyls, he equi ed
p oduc was o med in mode a e yield and a majo side
p oduc was isola ed. The s uc u e o his compound was
elucida ed using NMR and HRMS analysis and p o ed o be
unca ed disaccha ide 36e. A possible mechanism o he
o ma ion o his isaccha ide is shown in Scheme 3B.[45]
Appa en ly, he oxida i e condi ions led o clea age o he
glycosidic bond a he junc ion o he isaccha ide epea ing
uni s. This would lead o wo isaccha ide agmen s 36a,
and 36b. The hemiace al 36b can unde go a u he
oxida ion o he di-acid 36c, he diol o which can be
oxida i ely clea ed o p o ide, a e ano he oxida ion, di-
acid 36d, which upon benzyla ion wi h phenyldiazome hane
hen p o ides 36e. The o ma ion o his side p oduc
indica ed ha he long eac ion ime (3 days) and he la ge
excess o oxidan s used o he oxida ion we e oo ha sh o
Scheme 3. A) Assembly o a ge s 2and 3. B) Oxida i e clea age leading o agmen a ion o he oligosaccha ides. Reagen s and condi ions:
a) TBSOT , DCM, 5Å MS, 0°C, 28, 83%; 30, 85%; b) N2H4·H2O, py idine, AcOH, 0°C!RT, 29, 94%; 31, 89%; c) i, PPh3, py idine, H2O, THF, 70°C,
7 h; ii, Ac2O, NaHCO3, THF, H2O, 32, 88%; 33, 99%; d) HF·Py, THF, py idine, 0°C, 5, 92%; 6, 96%; e) i, TEMPO, BAIB, -BuOH, H2O, NaHCO3,
E OAc o MeCN, 4°C, 1 day; ii, PhCHN2, DCM, 34, 58%; 35, 66% (o e wo s eps); ) Pd(OH)2/C, H2, -BuOH, 0.1% AcOH in H2O, 3 days, 2,
91%; 3, 90%.
Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2023,62, e202211940 (5 o 11) © 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202211940 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [06/03/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
he subs a e. The e o e, sho e eac ion imes we e ex-
plo ed and he eac ion was ca e ully moni o ed by hin-
laye ch oma og aphy om 10 h o 3 days. This indica ed a
eac ion o 24 h o be op imal o he con e sion o 5in o
36a. This way, hexasaccha ide 34 was ob ained in 58% yield
a e a eac ion wi h he TEMPO/BAIB eagen combina-
ion o 24 h a 4°C in a -BuOH-wa e -E OAc sol en
sys em, ollowed by a benzyla ion using phenyldiazome-
hane. Simila ly, he h ee p ima y alcohols o he non-
asaccha ide 6we e oxidized and benzyla ed o p o ide he
co esponding noname 35 in 66% yield o e wo s eps.
Clea age o he nonasaccha ide unde he oxida i e con-
di ions could no be comple ely p e en ed as e ealed by
LC–MS analysis o he eac ion mix u e, which showed he
o ma ion o i- and hexasaccha ide agmen s, including 27
and 34. The o ma ion o hese side p oduc s con i ms he
egioselec i i y o he clea age eac ions, aking place a he
anome ic cen e o he galac ose esidues ha ha e o be
oxidized. The mechanism o he oxida i e glycosidic bond
clea age emains o be es ablished.
Un o una ely, he dep o ec ion p ocedu e used o
p epa e isaccha ide 1 om 27 was no sui able o he
hexasaccha ide, and ace yl mig a ion and hyd olysis we e
de ec ed by NMR and LC–MS analysis. A e ca e ul
op imiza ion o he sol en sys ems used and closely
moni o ing he pH o he eac ion mix u e, we managed o
achie e he global dep o ec ion by pe o ming he hyd o-
gena ion unde mild acidic condi ions o p o ide he a ge
hexasaccha ide 2in 91% and nonasaccha ide 3in 90% yield
(Scheme 3A).
The syn heses o he ace yla ed oligome s clea ly
indica ed he C-3-O-ace yl o be e y labile. To e alua e he
s abili y o he ace yl g oup mo e accu a ely, a se o NMR
expe imen s was se up. NMR analyses we e pe o med on
samples o 1in deu e a ed phospha e bu e s a di e en pD
alues (pD=pH+0.4), anging om pD 5.0 o pD 8.0 using
he diagnos ic peaks o H3 and H2 o he ace yla ed GalA
esidue (1:δ=5.33 ppm H3(OAc); 1a:δ=5.01 ppm H2-
(OAc); 1b:δ=4.10 H3(OH)). T isaccha ide 1a was o med
as an in e media e du ing hese expe imen s and was no
isola ed. The assignmen o his s uc u e was based on he
cha ac e is ic chemical shi s o he men ioned p o ons and
compa ison o li e a u e alues[14] (See Suppo ing In o ma-
ion o ull de ails). As shown in Figu e 2 and Scheme 2D,
he ace yl g oup can mig a e and hyd olyze om 1 o 1a and
1b unde sligh ly basic condi ions (pD=8.0), and mig a ion
was also possible, albei e y slowly, a pD 7.0, indica ing
he ace yl o be labile unde basic, neu al and sligh ly acidic
(pH 6.6) condi ions. O no e, we did no obse e any
mig a ion o he AAT ni ogen a om. A pD=8.0 he 3-
OAc
$
2-OAc mig a ion was as (Figu e 2), wi h mo e han
50% o he 3-OAc mig a ing in 15 days.[23,24] A e 380 days
he a io o 1,1a and 1b was app oxima ely 5:5:90. A
lowe pD (pD=5.0 and 6.0) no mig a ion o hyd olysis was
obse ed o e a pe iod as long as 380 days. These mig a ion
and clea age s udies unde pin he equi emen o sligh ly
acidic condi ions du ing he dep o ec ion o he oligosac-
cha ides. They also indica e ha ca e should be aken when
in e ac ion s udies a e pe o med wi h hese syn he ic ag-
men s o du ing isola ion and manipula ion o na u ally
sou ced Sp1-polysaccha ides.
S uc u al s udies
Wi h he h ee a ge oligosaccha ides in hand, we se ou o
p obe hei s uc u e. All 1H NMR esonances o ime 1
we e assigned h ough s anda d TOCSY, NOESY and
Figu e 2. O-Ace yl mig a ion and hyd olysis in isaccha ide 1a pD=5.0, 6.0 (A), 7.0 (B), o 8.0 (C).
Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2023,62, e202211940 (6 o 11) © 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202211940 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [06/03/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
HSQC expe imen s. No ably, all ing p o ons o ace yla ed
GalA esidue showed down ield-shi ed esonances com-
pa ed wi h he de-O-ace yla ed Sp1 oligome s we p e iously
assembled. In addi ion, he NOESY spec um o he O-
ace yla ed ime showed key in e - esidue c oss-peaks
which allowed us o unequi ocally de ine he con o ma ion
o he ime (Figu e 3A). The NOEs o he H1AAT-H4GalA-
(3OAc), H1GalA(3OAc)-H3GalA, and H1GalA(3OAc)-H4GalA p o on
pai s de ined exo-syn-Φ/syn-Ψcon o ma ions a ound he
glycosidic linkages. Fi ingly, MD simula ions p edic ed exo-
syn-Φ/syn(+)-Ψand exo-syn-Φ/syn()-Ψcon o ma ions o
he AAT-(α1–4)-GalA(3OAc) and GalA(3OAc)-(α1–3)-
GalA glycosidic linkages, espec i ely, and hey emained
conside ably s able along all he ajec o y (Figu e 3B and
C). The p o on signal o H5 o AAT shi ed up ield due o
he p esence o he OAc g oup, while he AAT C6-CH3
g oup appea ed o be mo e deshielded appea ing a a highe
chemical shi . The MD simula ion e ealed ha he AAT-
(α1-4)-GalA(3OAc) glycosidic linkage showed a he low
lexibili y while he GalA(3OAc)–(α1-3)-GalA linkage was
mo e lexible, wi h con inuous ansi ions be ween syn()-Ψ
and syn(+)-Ψcon o me s occu ing along he en i e simu-
la ion. The NOE-es ima ed in e esidual p o on-p o on
dis ances ma ched well wi h he a e age dis ances de i ed
om he MD simula ion.
Nex , a simila analysis was pe o med o he hexasac-
cha ide 2and nonasaccha ide 3using NOESY expe imen s
in combina ion wi h MD simula ions o analyze hei
con o ma ional ea u es. No ewo hy, he H1-H4 p o on
signals o all GalA(3OAc) esidues we e shi ed down ield,
highligh ing he pe u ba ion upon he in oduc ion o he 3-
OAc g oups. As obse ed o he ime , he AAT H5
p o ons we e shi ed o highe ield, while all AAT C6-CH3
signals appea ed a a highe chemical shi as compa ed o
he non-ace yla ed s uc u es. NOESY expe imen s indi-
ca ed ha he exo-syn-Φ/syn-Ψcon o ma ions a ound AAT-
(α1-4)-Gal(3OAc), Gala(3OAc)-(α1-3)-GalA, and GalA-
(α1-3)-AAT glycosidic linkages we e he mos popula ed
(Figu e 4A,B). 500 ns MD simula ions suppo ed hese
esul s and desc ibed a he simila con o ma ional ea u es
o he longe oligosaccha ides wi h exo-syn-Φ/syn(+)-Ψ,
exo-syn-Φ/syn(�)-Ψ, and exo-syn-Φ/syn()-Ψcon o ma ions
o he AAT-(α1-4)-GalA(3OAc), Gala(3OAc)-(α1-3)-
GalA, and GalA-(α1–3)-AAT linkages, espec i ely (Fig-
Figu e 3. A) Supe imposi ion o he HSQC spec a o he O-ace yla ed Sp1 ime (in black) and he de-O-ace yla ed homologue (in ed).
B) Expansion o he NOESYspec um o isaccha ide 1. Key c oss-peaks de ining he con o ma ion a ound he glycosidic linkages a e indica ed.
C) 10 snapsho supe imposi ion and Φ/Ψmaps o he con o ma ions explo ed along he 500 ns MD simula ion o isaccha ide 1.
Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2023,62, e202211940 (7 o 11) © 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202211940 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [06/03/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
u e 4C, 5A). Thus, as obse ed o he de-O-ace yla ed Sp1
agmen s, he addi ion o epea ing uni s does no al e he
con o ma ional lexibili y a ound each glycosidic linkage.
The MD simula ions allowed us o analyze he global
s uc u e o he oligosaccha ides e ealing hem o adop an
ex ended con o ma ion, whe e on a e age he noname is
h ee imes as long as he ime as de ined by he in e -
esidue dis ances (Figu e 5C). In e es ingly, noname 3
displayed a igh -handed helical s uc u e, wi h eigh
esidues comple ing a ull u n (spanning ca. 25 Å,
igu e 5B). This s uc u e is almos iden ical o he one
desc ibed o i s non-ace yla ed coun e pa and no majo
con o ma ional changes could be obse ed. The GalA-
(3OAc) ace yl g oups a e sol en -exposed and in close
p oximi y o he C6-CH3g oups o he neighbo ing AAT
esidues, explaining he deshielding e ec s obse ed by
Figu e 4. A) Expansion o NOESYspec um o hexame 2. B) Expansion o he NOESYspec um o noname 3. Key c oss-peaks de ining he
con o ma ion a ound he glycosidic linkages we e indica ed. C) Φ/Ψmaps and supe imposi ion o ep esen a i e s uc u es explo ed along he
500 ns MD simula ion o hexame 2.
Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2023,62, e202211940 (8 o 11) © 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202211940 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [06/03/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
NMR. Indeed, he icini y o he O-ace yl g oups o he
AAT me hyl g oup may p o ide an addi ional ba ie o
he ee o a ion a ound he AAT-(α1-4)-Gal(3OAc) glyco-
sidic linkage, hen hampe ing he access o o he (highe -
ene gy) con o ma ions and hus s abilizing he helix. The
close p oximi y o he me hyl g oups o he GalA(3OAc)
ace yl and C6-CH3g oups o he neighbo ing AAT esidues
c ea es hyd ophobic pa ches on he pe iphe y o he OAc-
Sp1 helix, which may play a ole in he in e ac ion wi h an i-
Sp1 an ibodies.
Conclusion
In conclusion, h ee O-ace yla ed Sp1 agmen s, a isac-
cha ide, hexasaccha ide and nonasaccha ide we e success-
Figu e 5. A) Φ/Ψmaps and supe imposi ion o ep esen a i e s uc u es explo ed along he 500 ns MD simula ion o and noname 3.
B) Molecula model o noname 3whe e he ace yl g oups we e highligh ed as balls. C) A e age alues o RoG and in e - esidue dis ances Rn-R1 o
he h ee oligome s, measu ed om he cen e o mass o e e y esidue. The s anda d de ia ion is indica ed in pa en heses. R1is he esidue a he
educing end.
Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2023,62, e202211940 (9 o 11) © 2022 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202211940 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [06/03/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
