Ci a ion: Clemen e, F.;
Ma ínez-Bailén, M.; Ma assini, C.;
Mo one, A.; Falliano, S.; Cacio i, A.;
Paoli, P.; Go i, A.; Ca dona, F.
Syn hesis o a New β-Galac osidase
Inhibi o Displaying Pha macological
Chape one P ope ies o GM1
Gangliosidosis. Molecules 2022,27,
4008. h ps://doi.o g/10.3390/
molecules27134008
Academic Edi o : Ramón J. Es é ez
Cabanas
Recei ed: 1 June 2022
Accep ed: 17 June 2022
Published: 22 June 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
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Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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dis ibu ed unde he e ms and
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A ibu ion (CC BY) license (h ps://
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molecules
A icle
Syn hesis o a New β-Galac osidase Inhibi o Displaying
Pha macological Chape one P ope ies o GM1 Gangliosidosis
F ancesca Clemen e 1,* , Maca ena Ma ínez-Bailén1,2, Camilla Ma assini 1, Amelia Mo one 3,4 ,
Sil ia Falliano 4, Anna Cacio i 4, Paolo Paoli 5, And ea Go i 1and F ancesca Ca dona 1,*
1Dipa imen o di Chimica “Ugo Schi ” (DICUS), Uni e si àdi Fi enze, Via Della Las uccia 3-13,
50019 Ses o Fio en ino, I aly; maca [email p o ec ed] (M.M.-B.); [email p o ec ed] (C.M.);
[email p o ec ed] (A.G.)
2Glycosys 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, 41092 Se illa, Spain
3Depa men o Neu osciences, Pha macology and Child Heal h (NEUROFARBA), Uni e si y o Flo ence,
Viale Pie accini n. 24, 50139 Fi enze, I aly; amelia.mo [email p o ec ed]
4Labo a o y o Molecula Biology o Neu ome abolic Diseases, Neu oscience Depa men ,
Meye Child en’s Hospi al, Viale Pie accini n. 24, 50139 Fi enze, I aly; sil ia. alliano@meye .i (S.F.);
anna.cacio i@meye .i (A.C.)
5Dipa imen o di Scienze Biomediche Spe imen ali e Cliniche “Ma io Se io” (DSBSC), Uni e si y o Flo ence,
Viale Mo gagni 50, 50134 Flo ence, I aly; [email p o ec ed]
*Co espondence: [email p o ec ed] (F.C.); [email p o ec ed] (F.C.);
Tel.: +39-055-4573453 (F.C.); +39-055-4573504 (F.C.)
Abs ac :
GM1 gangliosidosis is a a e lysosomal disease caused by he de iciency o he enzyme
β
-galac osidase (
β
-Gal; GLB1; E.C. 3.2.1.23), esponsible o he hyd olysis o e minal
β
-galac osyl
esidues om GM1 ganglioside, glycop o eins, and glycosaminoglycans, such as ke a an-sul a e.
Wi h he aim o iden i ying new pha macological chape ones o GM1 gangliosidosis, he syn hesis
o i e new ihyd oxypipe idine iminosuga s is epo ed in his wo k. The a ge compounds ea u e
a pen yl alkyl chain in di e en posi ions o he pipe idine ing and di e en absolu e con igu a ions
o he alkyl chain a C-2 and he hyd oxy g oup a C-3. The o ganome allic addi ion o a G igna d
eagen on o a ca bohyd a e-de i ed ni one in he p esence o absence o a sui able Lewis Acid was
exploi ed, p o iding s uc u al di e si y a C-2, ollowed by he ing-closu e educ i e amina ion
s ep. An oxida ion- educ ion p ocess allowed access o a di e en con igu a ion a C-3. The N-pen yl
ihyd oxypipe idine iminosuga was also syn hesized o he pu pose o compa ison. The biological
e alua ion o he newly syn hesized compounds was pe o med on leucocy e ex ac s om heal hy
dono s and iden i ied wo sui able
β
-Gal inhibi o s, namely compounds
10
and
12
. Among hese,
compound
12
showed chape oning p ope ies since i enhanced
β
-Gal ac i i y by 40% when es ed
on GM1 pa ien s bea ing he p.Ile51Asn/p.A g201His mu a ions.
Keywo ds:
iminosuga s;
β
-galac osidase inhibi o s; GM1 gangliosidosis; GLB1; pha macological
chape ones; ni ones; G igna d eagen s; educ i e amina ion
1. In oduc ion
GM1 gangliosidosis (MIM# 230500) is a lysosomal s o age diso de (LSD) caused by
he de iciency o lysosomal
β
-galac osidase (
β
-Gal; GLB1; E.C. 3.2.1.23), an enzyme depu ed
o he hyd olysis o he e minal
β
-galac osyl esidues om GM1 ganglioside, glycop o-
eins, and glycosaminoglycans, such as ke a an-sul a e [
1
]. GM1 gangliosidosis is consid-
e ed a neu odegene a i e diso de classi ied in o h ee clinical sub ypes: se e e in an ile
(
ype I; OMIM #230500
), la e-in an ile/ju enile ( ype II; OMIM #230600), and milde adul
( ype III; OMIM #230650) o ms [
2
]. The se e e in an ile o m ( ype I) is a al by 1–2 yea s
old and associa ed wi h se e e p og essi e neu ological symp oms ha mani es du ing
ea ly in ancy. Pa ien s wi h he ju enile o m ( ype II) mani es i s symp oms a 2–3 yea s,
Molecules 2022,27, 4008. h ps://doi.o g/10.3390/molecules27134008 h ps://www.mdpi.com/jou nal/molecules
Molecules 2022,27, 4008 2 o 21
show he slowe p og ession o he disease and ha e a ela i ely highe li e expec ancy (la e
childhood o ea ly adolescence). Type III GM1 gangliosidosis is he mildes o m, wi h la e
onse o symp oms (ea ly o mid-adolescence) and highe li e expec ancy. The es ima ed
incidence o GM1 gangliosidosis is in he ange o
1:100.000–200.000
li e bi hs [
2
,
3
], wi h
some isola ed communi ies being pa icula ly a ec ed (e.g., Mal a, 1:3.700) [4].
A p esen , he e is no cu e a ailable o GM1 gangliosidosis. Symp oma ic ea -
men s o some o he neu ologic symp oms a e a ailable bu do no signi ican ly al e he
condi ion’s p og ession [
2
]. The apies elying on pha macological chape ones (PCs) may
cons i u e a u u e op ion o he ea men o his disease. PCs a e e e sible inhibi o s
o he enzyme used in sub-inhibi o y amoun s, which in e ac wi h he mu a ed p o ein,
a o ing i s p ope con o ma ion, imp o ing i s s abili y in he endoplasmic e iculum,
p e en ing i s agg ega ion and p ema u e deg ada ion, hus esul ing in he escue o
enzyma ic ac i i y [
5
–
9
]. In he pas wen y yea s, se e al glycomime ic small molecules
ha e been in es iga ed as PCs o GM1 gangliosidosis, such as 1-deoxygalac onoji imycin
(DGJ (
1
); Figu e 1) [
10
,
11
]. This compound was la e app o ed o Fab y Disease, ano he
LSD, unde he ade name o Gala oldTM [12].
Molecules 2022, 27, x FOR PEER REVIEW 2 o 21
by 1–2 yea s old and associa ed wi h se e e p og essi e neu ological symp oms ha
mani es du ing ea ly in ancy. Pa ien s wi h he ju enile o m ( ype II) mani es i s
symp oms a 2–3 yea s, show he slowe p og ession o he disease and ha e a ela i ely
highe li e expec ancy (la e childhood o ea ly adolescence). Type III GM1 gangliosidosis
is he mildes o m, wi h la e onse o symp oms (ea ly o mid-adolescence) and highe
li e expec ancy. The es ima ed incidence o GM1 gangliosidosis is in he ange o
1:100.000–200.000 li e bi hs [2,3], wi h some isola ed communi ies being pa icula ly
a ec ed (e.g., Mal a, 1:3.700) [4].
A p esen , he e is no cu e a ailable o GM1 gangliosidosis. Symp oma ic
ea men s o some o he neu ologic symp oms a e a ailable bu do no signi ican ly
al e he condi ion's p og ession [2]. The apies elying on pha macological chape ones
(PCs) may cons i u e a u u e op ion o he ea men o his disease. PCs a e e e sible
inhibi o s o he enzyme used in sub-inhibi o y amoun s, which in e ac wi h he mu a ed
p o ein, a o ing i s p ope con o ma ion, imp o ing i s s abili y in he endoplasmic
e iculum, p e en ing i s agg ega ion and p ema u e deg ada ion, hus esul ing in he
escue o enzyma ic ac i i y [5–9]. In he pas wen y yea s, se e al glycomime ic small
molecules ha e been in es iga ed as PCs o GM1 gangliosidosis, such as 1-
deoxygalac onoji imycin (DGJ (1); Figu e 1) [10,11]. This compound was la e app o ed
o Fab y Disease, ano he LSD, unde he ade name o Gala oldTM [12].
Figu e 1. S uc u es o some imino- and azasuga s inhibi o s and chape ones o β-Gal.
The abili y o 1 o escue he ac i i y o mu an β-Gal up o 7- old in human ib oblas s
om pa ien s wi h GM1 gangliosidosis was demons a ed in 2001 [13]. Since hen, g ea
syn he ic e o s ha e iden i ied mo e po en PCs owa ds GM1 gangliosidosis (Figu e 1)
[11]. Se e al N-alkyla ed DGJ de i a i es we e syn hesized wi h enhanced speci ici y and
a ini y o galac osidases, such as N-bu yl-DGJ (NB-DGJ (2); Figu e 1) and N-nonyl-DGJ
(NN-DGJ (3); Figu e 1) [13,14]. The bicyclic sp2-iminosuga iso hiou ea de i a i e 6S-NBI-
DGJ (5N,6S-[N’-bu yliminome hylidene)-6- hio-1-deoxygalac onoji imycin (4); Figu e 1)
esul ed in a six old β-Gal ac i i y enhancemen in he ib oblas s om pa ien s
homozygous o he ju enile GM1 mu a ion p.A g201Cys [9,15,16] and showed p omising
p ope ies as PC in a GM1 gangliosidosis mouse model [17]. A signi ican con ibu ion
was also p o ided by he G az g oup, s a ing wi h he compound coined as DLHEX-DGJ
(5), which showed signi ican ac i i y enhancemen (18- old) wi h chape one-sensi i e
p.A g201Cys and p.A g201His cell lines a 500 µM [18]. In addi ion, se e al C-alkyla ed
azasuga s (C-pen yl-4-epi-iso agomine 6 and i s nonyl analogous 7, Figu e 1) we e ound
o be po en and selec i e inhibi o s o human lysosomal β-Gal able o he hal -maximal
eco e y o β-Gal ac i i y. Howe e , compound 7 also s ongly inhibi ed lysosomal β-
glucosidase, which may cause undesi able side e ec s [19,20]. Mo eo e , he “all-cis”
ihyd oxypipe idines 8 and 9 (Figu e 1) we e sui able inhibi o s o lysosomal β-Gal and
Figu e 1. S uc u es o some imino- and azasuga s inhibi o s and chape ones o β-Gal.
The abili y o
1
o escue he ac i i y o mu an
β
-Gal up o 7- old in human i-
b oblas s om pa ien s wi h GM1 gangliosidosis was demons a ed in 2001 [
13
]. Since
hen, g ea syn he ic e o s ha e iden i ied mo e po en PCs owa ds GM1 gangliosido-
sis (
Figu e 1
) [
11
]. Se e al N-alkyla ed DGJ de i a i es we e syn hesized wi h enhanced
speci ici y and a ini y o galac osidases, such as N-bu yl-DGJ (NB-DGJ (
2
); Figu e 1) and N-
nonyl-DGJ
(NN-DGJ (3)
; Figu e 1) [
13
,
14
]. The bicyclic sp
2
-iminosuga iso hiou ea de i a-
i e 6S-NBI-DGJ (5N,6S-[N
0
-bu yliminome hylidene)-6- hio-1-deoxygalac onoji imycin (
4
);
Figu e 1) esul ed in a six old
β
-Gal ac i i y enhancemen in he ib oblas s om pa-
ien s homozygous o he ju enile GM1 mu a ion p.A g201Cys [
9
,
15
,
16
] and showed
p omising p ope ies as PC in a GM1 gangliosidosis mouse model [
17
]. A signi ican
con ibu ion was also p o ided by he G az g oup, s a ing wi h he compound coined as
DLHEX-DGJ (5)
, which showed signi ican ac i i y enhancemen (18- old) wi h chape one-
sensi i e p.A g201Cys and p.A g201His cell lines a 500
µ
M [
18
]. In addi ion, se e al
C-alkyla ed azasuga s (C-pen yl-4-epi-iso agomine
6
and i s nonyl analogous
7
, Figu e 1)
we e ound o be po en and selec i e inhibi o s o human lysosomal
β
-Gal able o he
hal -maximal eco e y o
β
-Gal ac i i y. Howe e , compound
7
also s ongly inhibi ed
lysosomal
β
-glucosidase, which may cause undesi able side e ec s [
19
,
20
]. Mo eo e ,
he “all-cis”
ihyd oxypipe idines 8
and
9
(Figu e 1) we e sui able inhibi o s o lysosomal
β
-Gal and we e able o inc ease
β
-Gal ac i i y in GM1 gangliosidosis pa ien ib oblas s up
o wo–six old (a <100 µM concen a ion) [21].
Based on he obse a ion ha he con igu a ion o he ca bon a oms bea ing he
hyd oxy g oups and he posi ion o he alkyl chain play a sub le ole in he biological
Molecules 2022,27, 4008 3 o 21
ac i i y o
β
-Gal, he s aigh o wa d s e eoselec i e syn hesis o ou no el C-2 pen yl
ihyd oxypipe idines
10
,
11
,
12
and
13
(Scheme 1) was unde aken. The new compounds
a e C-2 alkyla ed ihyd oxypipe idines wi h wo di e en s e eochemical pa e ns a he
hyd oxy g oups and he opposi e absolu e con igu a ion a C-2. The choice o a pen yl
alkyl chain (ins ead o a longe one) was made o a oid undesi able side e ec s due o
lysosomal β-glucosidase inhibi ion [22,23].
Molecules 2022, 27, x FOR PEER REVIEW 3 o 21
we e able o inc ease β-Gal ac i i y in GM1 gangliosidosis pa ien ib oblas s up o wo–
six old (a <100 µM concen a ion) [21].
Based on he obse a ion ha he con igu a ion o he ca bon a oms bea ing he
hyd oxy g oups and he posi ion o he alkyl chain play a sub le ole in he biological
ac i i y o β-Gal, he s aigh o wa d s e eoselec i e syn hesis o ou no el C-2 pen yl
ihyd oxypipe idines 10, 11, 12 and 13 (Scheme 1) was unde aken. The new compounds
a e C-2 alkyla ed ihyd oxypipe idines wi h wo di e en s e eochemical pa e ns a he
hyd oxy g oups and he opposi e absolu e con igu a ion a C-2. The choice o a pen yl
alkyl chain (ins ead o a longe one) was made o a oid undesi able side e ec s due o
lysosomal β-glucosidase inhibi ion [22,23].
Scheme 1. Re osyn he ic s e eodi e gen s a egy o yield C-2 pen yl ihyd oxypipe idines 10, 11,
12 and 13, s a ing om he wo common in e media es 14 and 15, in u n, ob ained om D-
mannose-de i ed ni one 18.
The a ge compounds we e ob ained s a ing om wo common in e media es 14
and 15, which could a o d, espec i ely, bo h he ihyd oxypipe idines 10 and 12
h ough simple O- and N-dep o ec ion, and he “all-cis” ihyd oxypipe idines 11 and 13,
epime ic a C-3, h ough an oxida ion- educ ion sequence a e empo a y ni ogen
p o ec ion. The pipe idine in e media es 14 and 15 could be ob ained ia in amolecula
educ i e amina ion (RA) [24] o hyd oxylamines 16 and 17 wi h an S o R absolu e
con igu a ion a he newly o med s e eocen e , in u n, de i ed om G igna d eagen
addi ions on o ni one 18 in he p esence o absence o a sui able Lewis Acid (Scheme 1).
Ni one 18 was eadily accessed om 19 wi h an 85% yield by eac ion wi h N-benzyl
hyd oxylamine in d y CH2Cl2 [25]. Aldehyde 19 was syn hesized in ou s eps om D-
mannose on a g am scale [26]. To con i m he ole o he chain posi ion on ac i i y,
compound 21 was syn hesized s a ing om he pipe idine in e media e 20 h ough N-
Alkyla ion. The pipe idine in e media e 20 was ob ained om 18 by RA [23].
In his wo k, he syn hesis o he a ge compounds 10–13 and 21 is desc ibed,
oge he wi h he biological e alua ion o hei e ec on he human lysosomal β-Gal
enzyme and an in i o s udy on ib oblas cell lines bea ing he p.Ile51Asn/p.A g201His
and he p.A g201His/Ty 83Leu sX8 mu a ions om ju enile GM1 gangliosidosis pa ien s.
Scheme 1.
Re osyn he ic s e eodi e gen s a egy o yield C-2 pen yl ihyd oxypipe idines
10,11,12 and 13
, s a ing om he wo common in e media es
14
and
15
, in u n, ob ained om
D-mannose-de i ed ni one 18.
The a ge compounds we e ob ained s a ing om wo common in e media es
14
and
15
, which could a o d, espec i ely, bo h he ihyd oxypipe idines
10
and
12
h ough
simple O- and N-dep o ec ion, and he “all-cis” ihyd oxypipe idines
11
and
13
, epime ic
a C-3, h ough an oxida ion- educ ion sequence a e empo a y ni ogen p o ec ion.
The pipe idine in e media es
14
and
15
could be ob ained ia in amolecula educ i e
amina ion (RA) [
24
] o hyd oxylamines
16
and
17
wi h an So Rabsolu e con igu a ion
a he newly o med s e eocen e , in u n, de i ed om G igna d eagen addi ions on o
ni one
18
in he p esence o absence o a sui able Lewis Acid (Scheme 1). Ni one
18
was
eadily accessed om
19
wi h an 85% yield by eac ion wi h N-benzyl hyd oxylamine
in d y CH
2
Cl
2
[
25
]. Aldehyde
19
was syn hesized in ou s eps om D-mannose on
a g am scale [
26
]. To con i m he ole o he chain posi ion on ac i i y, compound
21
was syn hesized s a ing om he pipe idine in e media e
20
h ough N-Alkyla ion. The
pipe idine in e media e 20 was ob ained om 18 by RA [23].
In his wo k, he syn hesis o he a ge compounds
10–13
and
21
is desc ibed, oge he
wi h he biological e alua ion o hei e ec on he human lysosomal
β
-Gal enzyme and
an
in i o
s udy on ib oblas cell lines bea ing he p.Ile51Asn/p.A g201His and he
p.A g201His/Ty 83Leu sX8 mu a ions om ju enile GM1 gangliosidosis pa ien s.
2. Resul s and Discussion
2.1. Chemis y: Syn hesis and S uc u al Assignmen
The addi ion o pen ylmagnesium b omide o ni one
18
in d y THF a
−
78
◦
C o
3 h a o ded a good yield (70%, en y 1, Table 1) o he co esponding hyd oxylamines
16
and
17
in a 3.5:1 a io in a o o he hyd oxylamine
16
wi h he (S) absolu e con igu-
Molecules 2022,27, 4008 4 o 21
a ion a he newly o med s e eocen e . The addi ion o he same G igna d eagen in
he p esence o BF
3·
E
2
O (1.0 equi .) esul ed in a e e sal o s e eoselec i i y, and he
hyd oxylamine
17
wi h an (R) absolu e con igu a ion a he newly o med s e eocen e
was o med wi h a
d = 5.0:1
(en y 2, Table 1). In keeping wi h he indings o p e ious
expe imen s wi h di e en G igna d eagen s, he wo hyd oxylamines
16
and
17,
which
we e eadily sepa able by lash column ch oma og aphy, we e no s able in ai and spon-
aneously pa ially oxidized o he co esponding ni ones
22
and
23
(Scheme 2) [
22
,
23
].
Thei o ma ion was a es ed by
1
H-NMR and MS analyses immedia ely a e pu i ica ion
by column ch oma og aphy; comple e cha ac e iza ion was ca ied ou a e oxida ion o
he hyd oxylamines
16
and
17
o ni ones
22
and
23
wi h he hype alen iodine eagen
IBX in d y CH2Cl2(Scheme 2) [27].
Table 1. Addi ion o pen yl magnesium b omide o ni one 18.
Molecules 2022, 27, x FOR PEER REVIEW 4 o 21
2. Resul s and Discussion
2.1. Chemis y: Syn hesis and S uc u al Assignmen
The addi ion o pen ylmagnesium b omide o ni one 18 in d y THF a −78 °C o 3 h
a o ded a good yield (70%, en y 1, Table 1) o he co esponding hyd oxylamines 16 and
17 in a 3.5:1 a io in a o o he hyd oxylamine 16 wi h he (S) absolu e con igu a ion a
he newly o med s e eocen e . The addi ion o he same G igna d eagen in he p esence
o BF3·E 2O (1.0 equi .) esul ed in a e e sal o s e eoselec i i y, and he hyd oxylamine
17 wi h an (R) absolu e con igu a ion a he newly o med s e eocen e was o med wi h
a d = 5.0:1 (en y 2, Table 1). In keeping wi h he indings o p e ious expe imen s wi h
di e en G igna d eagen s, he wo hyd oxylamines 16 and 17, which we e eadily sep-
a able by lash column ch oma og aphy, we e no s able in ai and spon aneously pa -
ially oxidized o he co esponding ni ones 22 and 23 (Scheme 2) [22,23]. Thei o ma ion
was a es ed by 1H-NMR and MS analyses immedia ely a e pu i ica ion by column ch o-
ma og aphy; comple e cha ac e iza ion was ca ied ou a e oxida ion o he hyd oxyla-
mines 16 and 17 o ni ones 22 and 23 wi h he hype alen iodine eagen IBX in d y
CH2Cl2 (Scheme 2) [27].
Table 1. Addi ion o pen yl magnesium b omide o ni one 18.
En y C5H11MgB
Equi .
Lewis Acid
(1 Equi .)
Temp
(°C)
Time
(h)
16:17
Ra io a Yield b
1 1.8 none −78 3 3.5:1 70%
2 1.8 BF3·E 2O −30 2 1:5 75%
a De e mined by he in eg a ion o signals in he 1H-NMR spec a o he c ude eac ion mix u e. b
De e mined based on he o al amoun o R and S adduc s eco e ed a e pu i ica ion by column
ch oma og aphy.
Scheme 2. Oxida ion o hyd oxylamines 16 and 17 wi h IBX: syn hesis o ni ones 22 and 23.
The hyd oxylamine/ni one mix u es we e employed in he ing-closu e RA s ep
wi h H2 as a educing agen (balloon), Pd/C as a ca alys and wo equi alen s o ace ic acid
in MeOH, a o ding he pipe idines 14 and 15, (S)- and (R)-con igu ed a C-2, espec i ely,
En y C5H11MgB
Equi .
Lewis
Acid
(1 Equi .)
Temp
(◦C)
Time
(h)
16:17
Ra io aYield b
1 1.8 none −78 3 3.5:1 70%
2 1.8 BF3·E 2O−30 2 1:5 75%
a
De e mined by he in eg a ion o signals in he
1
H-NMR spec a o he c ude eac ion mix u e.
b
De e mined
based on he o al amoun o Rand Sadduc s eco e ed a e pu i ica ion by column ch oma og aphy.
Molecules 2022, 27, x FOR PEER REVIEW 4 o 21
2. Resul s and Discussion
2.1. Chemis y: Syn hesis and S uc u al Assignmen
The addi ion o pen ylmagnesium b omide o ni one 18 in d y THF a −78 °C o 3 h
a o ded a good yield (70%, en y 1, Table 1) o he co esponding hyd oxylamines 16 and
17 in a 3.5:1 a io in a o o he hyd oxylamine 16 wi h he (S) absolu e con igu a ion a
he newly o med s e eocen e . The addi ion o he same G igna d eagen in he p esence
o BF3·E 2O (1.0 equi .) esul ed in a e e sal o s e eoselec i i y, and he hyd oxylamine
17 wi h an (R) absolu e con igu a ion a he newly o med s e eocen e was o med wi h
a d = 5.0:1 (en y 2, Table 1). In keeping wi h he indings o p e ious expe imen s wi h
di e en G igna d eagen s, he wo hyd oxylamines 16 and 17, which we e eadily sep-
a able by lash column ch oma og aphy, we e no s able in ai and spon aneously pa -
ially oxidized o he co esponding ni ones 22 and 23 (Scheme 2) [22,23]. Thei o ma ion
was a es ed by 1H-NMR and MS analyses immedia ely a e pu i ica ion by column ch o-
ma og aphy; comple e cha ac e iza ion was ca ied ou a e oxida ion o he hyd oxyla-
mines 16 and 17 o ni ones 22 and 23 wi h he hype alen iodine eagen IBX in d y
CH2Cl2 (Scheme 2) [27].
Table 1. Addi ion o pen yl magnesium b omide o ni one 18.
En y C5H11MgB
Equi .
Lewis Acid
(1 Equi .)
Temp
(°C)
Time
(h)
16:17
Ra io a Yield b
1 1.8 none −78 3 3.5:1 70%
2 1.8 BF3·E 2O −30 2 1:5 75%
a De e mined by he in eg a ion o signals in he 1H-NMR spec a o he c ude eac ion mix u e. b
De e mined based on he o al amoun o R and S adduc s eco e ed a e pu i ica ion by column
ch oma og aphy.
Scheme 2. Oxida ion o hyd oxylamines 16 and 17 wi h IBX: syn hesis o ni ones 22 and 23.
The hyd oxylamine/ni one mix u es we e employed in he ing-closu e RA s ep
wi h H2 as a educing agen (balloon), Pd/C as a ca alys and wo equi alen s o ace ic acid
in MeOH, a o ding he pipe idines 14 and 15, (S)- and (R)-con igu ed a C-2, espec i ely,
Scheme 2. Oxida ion o hyd oxylamines 16 and 17 wi h IBX: syn hesis o ni ones 22 and 23.
The hyd oxylamine/ni one mix u es we e employed in he ing-closu e RA s ep
wi h H
2
as a educing agen (balloon), Pd/C as a ca alys and wo equi alen s o ace ic
acid in MeOH, a o ding he pipe idines
14
and
15
, (S)- and (R)-con igu ed a C-2, espec-
i ely, in 2 days and excellen yields a e ea men wi h a s ongly basic anion exchange
esin (Scheme 3).
Molecules 2022,27, 4008 5 o 21
Molecules 2022, 27, x FOR PEER REVIEW 5 o 21
in 2 days and excellen yields a e ea men wi h a s ongly basic anion exchange esin
(Scheme 3).
Scheme 3. The ing-closu e educ i e amina ion sequence.
Ca e ul analysis o he 1H-NMR, 2D-NMR and 1D-NOESY spec a ca ied ou on pi-
pe idines 14 and 15 con i med he s e eochemical ou come o he G igna d addi ion. The
1H-NMR signals o he azasuga po ion o pipe idines 14 and 15, oge he wi h hei
coupling cons an s, a e shown in Figu e 2. Fo compound 14, 1D-NOESY spec a did no
help o elucida e he s uc u al assignmen . Howe e , i s 1H-NMR spec a showed small
couplings cons an s o 3J2–3, 3J3–4 and 3J4–5 (2.0 Hz, 3.2 Hz and 5.2 Hz, espec i ely). This
pa e n is in ag eemen wi h an (S) absolu e con igu a ion a C-2, wi h he pipe idine dis-
playing a p e e ed 1C4 con o ma ion (sligh ly dis o ed due o he used dioxolane ing)
in which he bulky chain lies in he equa o ial posi ion and H-3 and H-4 a e in an equa o-
ial o ien a ion (Figu e 2). In he 1D-NOESY spec a o compound 15, s ong NOE co e-
la ion peaks we e obse ed be ween H-2 and H-4, H-4 and Hb-6, and H-2 and Hb-6, which
es i y o hei mu ual 1,3-diaxial posi ion and allow o con i m he (R) con igu a ion a C-
2 (see Supplemen a y Ma e ials). Indeed, he axial o ien a ion o H-2 de i es om a p e-
e ed 4C1 con o ma ion, which accommoda es he bulky chain again in an equa o ial po-
si ion (Figu e 2).
Ha
NH
OH
H
H
R
H
O56
2(S)
O
43
1C4
Hb
H
R = pen yl NH
HO
R
H
O
H
H
3
6
54
2
H
O
H
H
(S)
4C1
H
NH
OH
H
R
H
H
O56
(R)
O
43
1C4
H
H
R = pen yl NH
HO
H
R
O
H
Ha
3
6
54
2
H
O
Hb
H
(R)
4C1
2
14
15
1H-NMR (selec ed
signals)
H-2 = 2.73 d (7.0, 2.0)
H-3 = 3.80 (2.6)
H-4 = 4.14 dd (5.2, 3.2)
H-5 = 4.19 q (6.3)
Ha-6 = 2.66 dd (13.2,
7.6)
Hb-6 = 3.05 dd (13.2,
5.6)
1H-NMR (selec ed
signals)
H-2 = 2.27-2.21 m
H-3 = 3.32-3.24 m
H-4 = 3.84 (6.2)
H-5 = 4.21-4.19 m
Ha-6 = 3.32-3.24 m
Hb-6 = 2.93 d (13.2)
Scheme 3. The ing-closu e educ i e amina ion sequence.
Ca e ul analysis o he
1
H-NMR, 2D-NMR and 1D-NOESY spec a ca ied ou on
pipe idines
14
and
15
con i med he s e eochemical ou come o he G igna d addi ion. The
1
H-NMR signals o he azasuga po ion o pipe idines
14
and
15
, oge he wi h hei
coupling cons an s, a e shown in Figu e 2. Fo compound
14
, 1D-NOESY spec a did
no help o elucida e he s uc u al assignmen . Howe e , i s
1
H-NMR spec a showed
small couplings cons an s o
3
J
2–3
,
3
J
3–4
and
3
J
4–5
(2.0 Hz, 3.2 Hz and 5.2 Hz, espec i ely).
This pa e n is in ag eemen wi h an (S) absolu e con igu a ion a C-2, wi h he pipe idine
displaying a p e e ed
1
C
4
con o ma ion (sligh ly dis o ed due o he used dioxolane
ing) in which he bulky chain lies in he equa o ial posi ion and H-3 and H-4 a e in an
equa o ial o ien a ion (Figu e 2). In he 1D-NOESY spec a o compound
15
, s ong NOE
co ela ion peaks we e obse ed be ween H-2 and H-4, H-4 and H
b
-6, and H-2 and H
b
-6,
which es i y o hei mu ual 1,3-diaxial posi ion and allow o con i m he (R) con igu a ion
a C-2 (see Supplemen a y Ma e ials). Indeed, he axial o ien a ion o H-2 de i es om a
p e e ed
4
C
1
con o ma ion, which accommoda es he bulky chain again in an equa o ial
posi ion (Figu e 2).
Molecules 2022, 27, x FOR PEER REVIEW 5 o 21
in 2 days and excellen yields a e ea men wi h a s ongly basic anion exchange esin
(Scheme 3).
Scheme 3. The ing-closu e educ i e amina ion sequence.
Ca e ul analysis o he 1H-NMR, 2D-NMR and 1D-NOESY spec a ca ied ou on pi-
pe idines 14 and 15 con i med he s e eochemical ou come o he G igna d addi ion. The
1H-NMR signals o he azasuga po ion o pipe idines 14 and 15, oge he wi h hei
coupling cons an s, a e shown in Figu e 2. Fo compound 14, 1D-NOESY spec a did no
help o elucida e he s uc u al assignmen . Howe e , i s 1H-NMR spec a showed small
couplings cons an s o 3J2–3, 3J3–4 and 3J4–5 (2.0 Hz, 3.2 Hz and 5.2 Hz, espec i ely). This
pa e n is in ag eemen wi h an (S) absolu e con igu a ion a C-2, wi h he pipe idine dis-
playing a p e e ed 1C4 con o ma ion (sligh ly dis o ed due o he used dioxolane ing)
in which he bulky chain lies in he equa o ial posi ion and H-3 and H-4 a e in an equa o-
ial o ien a ion (Figu e 2). In he 1D-NOESY spec a o compound 15, s ong NOE co e-
la ion peaks we e obse ed be ween H-2 and H-4, H-4 and Hb-6, and H-2 and Hb-6, which
es i y o hei mu ual 1,3-diaxial posi ion and allow o con i m he (R) con igu a ion a C-
2 (see Supplemen a y Ma e ials). Indeed, he axial o ien a ion o H-2 de i es om a p e-
e ed 4C1 con o ma ion, which accommoda es he bulky chain again in an equa o ial po-
si ion (Figu e 2).
Ha
NH
OH
H
H
R
H
O56
2(S)
O
43
1C4
Hb
H
R = pen yl NH
HO
R
H
O
H
H
3
6
54
2
H
O
H
H
(S)
4C1
H
NH
OH
H
R
H
H
O56
(R)
O
43
1C4
H
H
R = pen yl NH
HO
H
R
O
H
Ha
3
6
54
2
H
O
Hb
H
(R)
4C1
2
14
15
1H-NMR (selec ed
signals)
H-2 = 2.73 d (7.0, 2.0)
H-3 = 3.80 (2.6)
H-4 = 4.14 dd (5.2, 3.2)
H-5 = 4.19 q (6.3)
Ha-6 = 2.66 dd (13.2,
7.6)
Hb-6 = 3.05 dd (13.2,
5.6)
1H-NMR (selec ed
signals)
H-2 = 2.27-2.21 m
H-3 = 3.32-3.24 m
H-4 = 3.84 (6.2)
H-5 = 4.21-4.19 m
Ha-6 = 3.32-3.24 m
Hb-6 = 2.93 d (13.2)
Figu e 2.
The wo possible chai con o ma ions o pipe idines
14
and
15
. Dihed al angles be ween
bonds analysed o he de e mina ion o he p e e ed con o ma ion a e shown in blue. Red double-
ended a ows show he obse ed diagnos ic NOE co ela ion peaks in he 1D-NOESY NMR spec a.
1
H-NMR chemical shi s o selec ed signals a e epo ed, wi h he co esponding coupling cons an s
in pa en hesis (in Hz).

Molecules 2022,27, 4008 6 o 21
Final emo al o he ace onide p o ec ing g oups unde acidic condi ions (aqueous
HCl in MeOH) ollowed by basic ea men a o ded he a ge ihyd oxypipe idines
10
and 12 as ee amines in good yields (Scheme 4).
Molecules 2022, 27, x FOR PEER REVIEW 6 o 21
Figu e 2. The wo possible chai con o ma ions o pipe idines 14 and 15. Dihed al angles be ween
bonds analysed o he de e mina ion o he p e e ed con o ma ion a e shown in blue. Red double-
ended a ows show he obse ed diagnos ic NOE co ela ion peaks in he 1D-NOESY NMR spec a.
1H-NMR chemical shi s o selec ed signals a e epo ed, wi h he co esponding coupling cons an s
in pa en hesis (in Hz).
Final emo al o he ace onide p o ec ing g oups unde acidic condi ions (aqueous
HCl in MeOH) ollowed by basic ea men a o ded he a ge ihyd oxypipe idines 10
and 12 as ee amines in good yields (Scheme 4).
Scheme 4. The inal dep o ec ion s ep.
The in e sion o con igu a ion a C-3 in compounds 11 and 13 was achie ed h ough
an oxida ion– educ ion sequence ca ied ou on empo a ily N-p o ec ed pipe idines
(Schemes 5 and 6).
Scheme 5. P o ec ion–oxida ion sequence.
Scheme 4. The inal dep o ec ion s ep.
The in e sion o con igu a ion a C-3 in compounds
11
and
13
was achie ed h ough
an oxida ion– educ ion sequence ca ied ou on empo a ily N-p o ec ed pipe idines
(Schemes 5and 6).
Molecules 2022, 27, x FOR PEER REVIEW 6 o 21
Figu e 2. The wo possible chai con o ma ions o pipe idines 14 and 15. Dihed al angles be ween
bonds analysed o he de e mina ion o he p e e ed con o ma ion a e shown in blue. Red double-
ended a ows show he obse ed diagnos ic NOE co ela ion peaks in he 1D-NOESY NMR spec a.
1H-NMR chemical shi s o selec ed signals a e epo ed, wi h he co esponding coupling cons an s
in pa en hesis (in Hz).
Final emo al o he ace onide p o ec ing g oups unde acidic condi ions (aqueous
HCl in MeOH) ollowed by basic ea men a o ded he a ge ihyd oxypipe idines 10
and 12 as ee amines in good yields (Scheme 4).
Scheme 4. The inal dep o ec ion s ep.
The in e sion o con igu a ion a C-3 in compounds 11 and 13 was achie ed h ough
an oxida ion– educ ion sequence ca ied ou on empo a ily N-p o ec ed pipe idines
(Schemes 5 and 6).
Scheme 5. P o ec ion–oxida ion sequence.
Scheme 5. P o ec ion–oxida ion sequence.
Molecules 2022, 27, x FOR PEER REVIEW 7 o 21
Scheme 6. Reduc ion-dep o ec ion sequence.
The p o ec ion o pipe idines 14 and 15 wi h he e -bu yloxyca bonyl g oup o ob-
ain compounds 24 and 25, ollowed by oxida ion wi h Dess Ma in pe iodinane, p o ided
he key ke one in e media es 26 and 27 in good yields o e wo s eps (Scheme 5). Bo h
ke ones unde wen sodium bo ohyd ide educ ion in good yields and high selec i i y,
showing he same p e e ence o an a ack on he Re ace, which p oduced he all-cis ela-
i e con igu a ion in he esul ing ihyd oxypipe idines (Scheme 6). The high s e eose-
lec i i y obse ed in he educ ion o 26 and 27 o 28 and 29, espec i ely, can be asc ibed
o a s ongly a ou ed axial a ack o hyd ide a he C-3 ca bonyl in he 1C4 chai con o -
ma ion in bo h cases. Indeed, his con o ma ion allows a kine ically a ou ed app oach o
hyd ide an i o he icinal C-O bond, hus enjoying TS s abiliza ion by he low-lying en-
e gy σ* o C-O bond acco ding o he Felkin–Anh model (Figu e 3) [28,29].
Figu e 3. Fa o ed axial a ack o hyd ide o C=O bond.
Final emo al o he ace onide p o ec ing g oups o 28 and 29 unde acidic condi ions
(aqueous HCl in MeOH), ollowed by basic ea men a o ded he a ge ihyd oxypi-
pe idines 11 and 13 as ee amines in excellen yields (92% and 98%, Scheme 6).
The occu ed in e sion o con igu a ion a C-3 in compounds 11 and 13 and hei
p e e ed con o ma ion we e es ablished on he basis o ca e ul analysis o hei 1H-NMR
and 1D-NOESY spec a. In pa icula , o compound 11, a s ong NOE co ela ion peak
be ween H-3 and H-5 was obse ed (Figu e 4). Toge he wi h he high coupling cons an s
obse ed o he signal o H-3, his inding con i med an axial o ien a ion o his p o on
wi h an ax-ax ela ionship wi h H-2 (J = 8.0 Hz) and a 1,3-diaxial in e ac ion wi h H-5. This
pa e n ag ees wi h an (S) absolu e con igu a ion a C-3, wi h he pipe idine displaying a
p e e ed 1C4 con o ma ion in which he bulky chain lies in he equa o ial posi ion. 1D-
NOESY s udies o compound 13 showed s ong NOE peaks co ela ing p o ons H-2, Hb-
6 and H-4 (Figu e 4) (see Supplemen a y Ma e ials). Mo eo e , he 1H-NMR spec um
showed b oad single s o p o ons H-3 and H-5, consis en wi h hei equa o ial posi ion.
These e idences con i m he (S) con igu a ion a C-3 and a 4C1 con o ma ion in which he
bulky chain is equa o ial.
Scheme 6. Reduc ion-dep o ec ion sequence.
Molecules 2022,27, 4008 7 o 21
The p o ec ion o pipe idines
14
and
15
wi h he e -bu yloxyca bonyl g oup o ob ain
compounds
24
and
25
, ollowed by oxida ion wi h Dess Ma in pe iodinane, p o ided
he key ke one in e media es
26
and
27
in good yields o e wo s eps (Scheme 5). Bo h
ke ones unde wen sodium bo ohyd ide educ ion in good yields and high selec i i y,
showing he same p e e ence o an a ack on he Re ace, which p oduced he all-cis ela i e
con igu a ion in he esul ing ihyd oxypipe idines (Scheme 6). The high s e eoselec i i y
obse ed in he educ ion o
26
and
27
o
28
and
29
, espec i ely, can be asc ibed o a
s ongly a ou ed axial a ack o hyd ide a he C-3 ca bonyl in he
1
C
4
chai con o ma ion
in bo h cases. Indeed, his con o ma ion allows a kine ically a ou ed app oach o hyd ide
an i o he icinal C-O bond, hus enjoying TS s abiliza ion by he low-lying ene gy
σ
* o
C-O bond acco ding o he Felkin–Anh model (Figu e 3) [28,29].
Molecules 2022, 27, x FOR PEER REVIEW 7 o 21
Scheme 6. Reduc ion-dep o ec ion sequence.
The p o ec ion o pipe idines 14 and 15 wi h he e -bu yloxyca bonyl g oup o ob-
ain compounds 24 and 25, ollowed by oxida ion wi h Dess Ma in pe iodinane, p o ided
he key ke one in e media es 26 and 27 in good yields o e wo s eps (Scheme 5). Bo h
ke ones unde wen sodium bo ohyd ide educ ion in good yields and high selec i i y,
showing he same p e e ence o an a ack on he Re ace, which p oduced he all-cis ela-
i e con igu a ion in he esul ing ihyd oxypipe idines (Scheme 6). The high s e eose-
lec i i y obse ed in he educ ion o 26 and 27 o 28 and 29, espec i ely, can be asc ibed
o a s ongly a ou ed axial a ack o hyd ide a he C-3 ca bonyl in he 1C4 chai con o -
ma ion in bo h cases. Indeed, his con o ma ion allows a kine ically a ou ed app oach o
hyd ide an i o he icinal C-O bond, hus enjoying TS s abiliza ion by he low-lying en-
e gy σ* o C-O bond acco ding o he Felkin–Anh model (Figu e 3) [28,29].
Figu e 3. Fa o ed axial a ack o hyd ide o C=O bond.
Final emo al o he ace onide p o ec ing g oups o 28 and 29 unde acidic condi ions
(aqueous HCl in MeOH), ollowed by basic ea men a o ded he a ge ihyd oxypi-
pe idines 11 and 13 as ee amines in excellen yields (92% and 98%, Scheme 6).
The occu ed in e sion o con igu a ion a C-3 in compounds 11 and 13 and hei
p e e ed con o ma ion we e es ablished on he basis o ca e ul analysis o hei 1H-NMR
and 1D-NOESY spec a. In pa icula , o compound 11, a s ong NOE co ela ion peak
be ween H-3 and H-5 was obse ed (Figu e 4). Toge he wi h he high coupling cons an s
obse ed o he signal o H-3, his inding con i med an axial o ien a ion o his p o on
wi h an ax-ax ela ionship wi h H-2 (J = 8.0 Hz) and a 1,3-diaxial in e ac ion wi h H-5. This
pa e n ag ees wi h an (S) absolu e con igu a ion a C-3, wi h he pipe idine displaying a
p e e ed 1C4 con o ma ion in which he bulky chain lies in he equa o ial posi ion. 1D-
NOESY s udies o compound 13 showed s ong NOE peaks co ela ing p o ons H-2, Hb-
6 and H-4 (Figu e 4) (see Supplemen a y Ma e ials). Mo eo e , he 1H-NMR spec um
showed b oad single s o p o ons H-3 and H-5, consis en wi h hei equa o ial posi ion.
These e idences con i m he (S) con igu a ion a C-3 and a 4C1 con o ma ion in which he
bulky chain is equa o ial.
Figu e 3. Fa o ed axial a ack o hyd ide o C=O bond.
Final emo al o he ace onide p o ec ing g oups o
28
and
29
unde acidic condi ions
(aqueous HCl in MeOH), ollowed by basic ea men a o ded he a ge ihyd oxyp-
ipe idines 11 and 13 as ee amines in excellen yields (92% and 98%, Scheme 6).
The occu ed in e sion o con igu a ion a C-3 in compounds
11
and
13
and hei
p e e ed con o ma ion we e es ablished on he basis o ca e ul analysis o hei
1
H-NMR
and 1D-NOESY spec a. In pa icula , o compound
11
, a s ong NOE co ela ion peak
be ween H-3 and H-5 was obse ed (Figu e 4). Toge he wi h he high coupling cons an s
obse ed o he signal o H-3, his inding con i med an axial o ien a ion o his p o on
wi h an ax-ax ela ionship wi h H-2 (J = 8.0 Hz) and a 1,3-diaxial in e ac ion wi h H-5. This
pa e n ag ees wi h an (S) absolu e con igu a ion a C-3, wi h he pipe idine displaying
a p e e ed
1
C
4
con o ma ion in which he bulky chain lies in he equa o ial posi ion.
1D-NOESY s udies o compound
13
showed s ong NOE peaks co ela ing p o ons H-2,
H
b
-6 and H-4 (Figu e 4) (see Supplemen a y Ma e ials). Mo eo e , he
1
H-NMR spec um
showed b oad single s o p o ons H-3 and H-5, consis en wi h hei equa o ial posi ion.
These e idences con i m he (S) con igu a ion a C-3 and a
4
C
1
con o ma ion in which he
bulky chain is equa o ial.
To in es iga e he ole played by he posi ion o he alkyl chain on he biological
ac i i y, he ihyd oxypipe idine
21
alkyla ed a ni ogen wi h a pen yl chain was also
syn hesized. This aim was achie ed by alkyla ion o pipe idine
20
ollowed by dep o ec ion
o he ace onide g oup o 30 (40% yield o e wo s eps, Scheme 7).
Molecules 2022,27, 4008 8 o 21
Molecules 2022, 27, x FOR PEER REVIEW 8 o 21
Figu e 4. The wo possible chai con o ma ions o pipe idines 11 and 13. Dihed al angles be ween
bonds analysed o he de e mina ion o he p e e ed con o ma ion a e shown in blue. Red double-
ended a ows show he obse ed diagnos ic NOE co ela ion peaks in he 1D-NOESY NMR spec a.
1H-NMR chemical shi s o selec ed signals a e epo ed, wi h he co esponding coupling cons an s
in pa en hesis (in Hz).
To in es iga e he ole played by he posi ion o he alkyl chain on he biological ac-
i i y, he ihyd oxypipe idine 21 alkyla ed a ni ogen wi h a pen yl chain was also syn-
hesized. This aim was achie ed by alkyla ion o pipe idine 20 ollowed by dep o ec ion
o he ace onide g oup o 30 (40% yield o e wo s eps, Scheme 7).
Scheme 7. Alkyla ion and dep o ec ion o a o d compound 21.
2.2. P elimina y Biological Sc eening owa ds Human β-Galac osidase and β-Glucosidase
I is known ha 3,4,5- ihyd oxipipe idine display i s biological p ope ies in i s ully
dep o ec ed o m [30]. The e o e, he a ge compounds 10–13 and 21 we e i s e alua ed
as human lysosomal β-Gal inhibi o s a 1 mM in human leukocy e homogena es, and as
human lysosomal β-Glu (β-glucosidase) inhibi o s a 1 mM in o de o e alua e he selec-
i i y o he new compounds. The esul s a e shown in Table 2. Only compounds 10 and
12 showed a good selec i i y and conside able 90% and 42% inhibi o y ac i i y owa ds
β-Gal, wi h a mode a e IC50 (400 ± 15 µM and 1.15 ± 0.1 mM, espec i ely). The ihyd ox-
ypipe idine 10 wi h he (S) con igu a ion a C-2 (Table 2, En y 1) was mo e ac i e han
he co esponding epime ic ihyd oxypipe idine 12 wi h he (R) con igu a ion (Table 2,
En y 2). The p esence o a pen yl chain in ihyd oxypipe idines 10 and 12 appea s o
bene i he inhibi o y ac i i y, in ag eemen wi h p e ious esul s [19,20].
Figu e 4.
The wo possible chai con o ma ions o pipe idines
11
and
13
. Dihed al angles be ween
bonds analysed o he de e mina ion o he p e e ed con o ma ion a e shown in blue. Red double-
ended a ows show he obse ed diagnos ic NOE co ela ion peaks in he 1D-NOESY NMR spec a.
1
H-NMR chemical shi s o selec ed signals a e epo ed, wi h he co esponding coupling cons an s
in pa en hesis (in Hz).
Molecules 2022, 27, x FOR PEER REVIEW 8 o 21
Figu e 4. The wo possible chai con o ma ions o pipe idines 11 and 13. Dihed al angles be ween
bonds analysed o he de e mina ion o he p e e ed con o ma ion a e shown in blue. Red double-
ended a ows show he obse ed diagnos ic NOE co ela ion peaks in he 1D-NOESY NMR spec a.
1H-NMR chemical shi s o selec ed signals a e epo ed, wi h he co esponding coupling cons an s
in pa en hesis (in Hz).
To in es iga e he ole played by he posi ion o he alkyl chain on he biological ac-
i i y, he ihyd oxypipe idine 21 alkyla ed a ni ogen wi h a pen yl chain was also syn-
hesized. This aim was achie ed by alkyla ion o pipe idine 20 ollowed by dep o ec ion
o he ace onide g oup o 30 (40% yield o e wo s eps, Scheme 7).
Scheme 7. Alkyla ion and dep o ec ion o a o d compound 21.
2.2. P elimina y Biological Sc eening owa ds Human β-Galac osidase and β-Glucosidase
I is known ha 3,4,5- ihyd oxipipe idine display i s biological p ope ies in i s ully
dep o ec ed o m [30]. The e o e, he a ge compounds 10–13 and 21 we e i s e alua ed
as human lysosomal β-Gal inhibi o s a 1 mM in human leukocy e homogena es, and as
human lysosomal β-Glu (β-glucosidase) inhibi o s a 1 mM in o de o e alua e he selec-
i i y o he new compounds. The esul s a e shown in Table 2. Only compounds 10 and
12 showed a good selec i i y and conside able 90% and 42% inhibi o y ac i i y owa ds
β-Gal, wi h a mode a e IC50 (400 ± 15 µM and 1.15 ± 0.1 mM, espec i ely). The ihyd ox-
ypipe idine 10 wi h he (S) con igu a ion a C-2 (Table 2, En y 1) was mo e ac i e han
he co esponding epime ic ihyd oxypipe idine 12 wi h he (R) con igu a ion (Table 2,
En y 2). The p esence o a pen yl chain in ihyd oxypipe idines 10 and 12 appea s o
bene i he inhibi o y ac i i y, in ag eemen wi h p e ious esul s [19,20].
Scheme 7. Alkyla ion and dep o ec ion o a o d compound 21.
2.2. P elimina y Biological Sc eening owa ds Human β-Galac osidase and β-Glucosidase
I is known ha 3,4,5- ihyd oxipipe idine display i s biological p ope ies in i s ully
dep o ec ed o m [
30
]. The e o e, he a ge compounds
10
–
13
and
21
we e i s e alua ed
as human lysosomal
β
-Gal inhibi o s a 1 mM in human leukocy e homogena es, and
as human lysosomal
β
-Glu (
β
-glucosidase) inhibi o s a 1 mM in o de o e alua e he
selec i i y o he new compounds. The esul s a e shown in Table 2. Only compounds
10
and
12
showed a good selec i i y and conside able 90% and 42% inhibi o y ac i i y
owa ds
β
-Gal, wi h a mode a e IC
50
(400
±
15
µ
M and 1.15
±
0.1 mM, espec i ely).
The ihyd oxypipe idine
10
wi h he (S) con igu a ion a C-2 (Table 2, En y 1) was mo e
ac i e han he co esponding epime ic ihyd oxypipe idine
12
wi h he (R) con igu a ion
(Table 2, En y 2). The p esence o a pen yl chain in ihyd oxypipe idines
10
and
12
appea s o bene i he inhibi o y ac i i y, in ag eemen wi h p e ious esul s [19,20].
Con e sely, he opposi e con igu a ion a C-3 as in compounds
11
and
13
esul ed in a
d ama ic dec ease o
β
-Gal inhibi ion (down o 36% and 35%, espec i ely, ega dless o he
con igu a ion a C-2) (Table 2, en ies 3 and 4 s. en ies 1 and 2) in line wi h ou p e ious
esul s on alkyla ed azasuga s wi h a ela i e “all-cis” con igu a ion a he hyd oxy/amine-
subs i u ed s e eocen e s [
31
]. These esul s con u ed ou expec a ions ha inhibi ion o
β
-Gal migh bene i om he “all-cis” con igu a ion o he h ee hyd oxy g oups. Mo eo e ,
he “all-cis” con igu a ion made compounds
11
and
13
be e inhibi o s o
β
-Glu, e oding
any selec i i y in inhibi ion o he wo enzymes. N-Alkyla ion in compound
21
was also
dele e ious o inhibi ion agains
β
-Gal (Table 2, En y 5). None o he newly syn hesized
Molecules 2022,27, 4008 9 o 21
compounds showed a signi ican inhibi ion owa ds
β
-glucosidase a 1 mM ( om 1% o
45%). Once again, he esul s highligh ha he p esence o a longe alkyl chain is essen ial
o p o iding a ini y owa ds β-glucosidase [22,23,31–34].
Table 2. β-Gal and GCase (β-Glu) inhibi ion in human leukocy es om heal hy dono s.
En y Compounds
β-Gal β-Glu
Inhibi ion
[%] a
Inhibi ion
[%] a
110 90
400 ±15 µMb1
212 42
1.15 ±0.1 mM b5
311 36 34
413 35 44
521 20 45
a
Pe cen age inhibi ion o
β
-Gal o
β
-Glu in human leukocy e ex ac s om heal hy dono s incuba ed wi h
compounds (1 mM).
b
IC
50
alues we e de e mined by measu ing
β
-Gal ac i i y a di e en concen a ions o
each inhibi o o compounds showing inhibi o y ac i i y highe han 40% a 1 mM.
Since li e a u e da a show ha po en PCs o
β
-Gal can also be ound among mode a e
and ela i ely good inhibi o s [
21
], he abili y o compounds
10
and
12
o escue he
enzyma ic ac i i y o β-Gal on cell lines bea ing selec ed mu a ions was es ed.
Kine ic analyses we e also pe o med o de e mine he mechanism o ac ion o
12
(see Supplemen a y Ma e ials), which showed chape oning p ope ies (see Sec ion 2.3).
The esul s indica e ha i ac s as a noncompe i i e
β
-Gal inhibi o , wi h a K
i
alue o
1.4 ±0.7 mM.
2.3. Pha macological Chape oning Ac i i y
In his expe imen , he inhibi o s o
β
-Gal
10
and
12
we e incuba ed o ou days wi h
ib oblas s om wo ju enile GM1 gangliosidosis pa ien s bea ing p.Ile51Asn/p.A g201His
and p.A g201His/Ty 83Leu sX8 mu a ions, espec i ely, ollowed by assays o cell lysa es
o lysosomal
β
-Gal ac i i y (see Supplemen a y Ma e ials). The expe imen s showed
ha all compounds a e non- oxic a high concen a ions in bo h cell lines. Compound
12
showed a
β
-Gal ac i i y escue o 1.40- old a 600
µ
M on GM1 pa ien ib oblas s bea ing
he p.Ile51Asn/p.A g201His mu a ions.
The esul s ob ained sugges ha only he p.Ile51Asn/p.A g201His mu a ions a e
esponsi e o ou compound. In e es ingly, he s onge inhibi o
10
did no show any
enzyme ac i i y escue.
To he bes o ou knowledge, compound
12
ep esen s he i s example o a non-
compe i i e
β
-Gal inhibi o ac ing as a pha macological chape one on GM1 pa ien s’ cells.
A chape oning beha iou o o he noncompe i i e inhibi o s o lysosomal enzymes has
been p e iously obse ed, in pa icula o gene mu a ions leading o Gauche , Fab y,
Pompe, and Tay–Sachs diseases [35].
3. Ma e ials and Me hods
3.1. Gene al Expe imen al P ocedu es o he Syn heses
Comme cial eagen s we e used as ecei ed. All eac ions we e ca ied ou unde mag-
ne ic s i ing and moni o ed by TLC on 0.25 mm silica gel pla es (Me ck F254). Column ch o-
ma og aphies we e ca ied ou on Silica Gel 60 (32–63
µ
m) o on silica gel (
230–400 mesh
,
Me ck, Da ms ad , Ge many). Yields e e o spec oscopically and analy ically pu e com-
pounds unless o he wise s a ed.
1
H-NMR spec a we e eco ded on a Va ian Gemini
200 MHz,
a Va ian Me cu y 400 MHz, o on a Va ian INOVA 400 MHz ins umen a 25
◦
C.
13
C-NMR spec a we e eco ded a 50 MHz o a 100 MHz. Chemical shi s a e epo ed
ela i e o CDCl
3
(
1
H:
δ
= 7.27 ppm,
13
C:
δ
= 77.0 ppm). In eg als a e in acco dance wi h
assignmen s; coupling cons an s a e gi en in Hz. Fo de ailed peak assignmen s, 2D spec-
a we e measu ed (g-COSY, g-HSQC) and 1D-NOESY. Small-scale mic owa e-assis ed
Molecules 2022,27, 4008 16 o 21
a H-3, and i adia ion o H-3 ga e a NOE a H-5. MS (ESI): m/z(%) = 226.10 (100) [M + H]
+
.
C10H21NO3(203.28): calcd. C, 59.09; H, 10.41; N, 6.89; ound C, 59.12; H, 10.23; N, 6.51.
3.1.15. Syn hesis o (2R,3S,4R,5R)-2-Pen ylpipe idine-3,4,5- iol (13)
A solu ion o
29
(24 mg, 0.07 mmol) in MeOH (3 mL) was le s i ing wi h 12 M
HCl (15
µ
L) a oom empe a u e o 16 h. The c ude mix u e was concen a ed o yield
he hyd ochlo ide sal o
29
. The co esponding ee amine was ob ained by dissol ing
he esidue in MeOH, hen he s ongly basic esin Ambe sep 900-OH was added, and
he mix u e was s i ed o 40 min. The esin was emo ed by il a ion o a o d 14 mg
(0.069 mmol, 98%) o 13 as ee base, as a pale-yellow oil.
13
: pale yellow oil.
[α]25
D
=
−
40.4 (c = 0.50, CH
3
OH).
1
H-NMR (400 MHz, CD
3
OD)
δ
= 3.79 (b s, 1H, H-5), 3.73 (b s, 1H, H-3), 3.46 (b s, 1H, H-4), 3.02 (d, J = 15.0 Hz, 1H,
H
a
-6), 2.69 (d, J = 14.0 Hz, 1H, H
b
-6), 2.44 ( , J = 6.8 Hz, 1H, H-2), 1.58–1.53 (m, 1H, H
a
-1
0
),
1.51–1.44 (m, 1H, H
a
-2
0
), 1.43–1.29 (m, 6H, H
b
-1
0
, H
b
-2
0
, H-3
0
–H-4
0
), 0.92 ( , J = 6.0 Hz, 3H,
H-5
0
) ppm.
13
C-NMR (100 MHz, CD
3
OD) = 72.6 (C-3), 71.3 (C-4), 71.7 (C-5), 60.0 (C-2),
51.6 (C-6)
, 33.2, 32.7, 26.7, 23.7 (4C, C-1
0
–C-4
0
), 14.4 (C-5
0
) ppm. 1D-NOESY: I adia ion
o H-2 ga e a NOE a H-4 and H
b
-6, and i adia ion o H-4 ga e a NOE a H-2 and H
b
-6.
MS (ESI): m/z (%) = 204.12 (100) [M + H]
+
. C
10
H
21
NO
3
(203.28): calcd. C, 59.09; H, 10.41;
N, 6.89; ound C, 59.17; H, 10.11; N, 6.70.
3.1.16. Syn hesis o (3R,4S,5R)-1-Pen yl-5-hyd oxy-3,4-O-(1-me hyle hylidene)-pipe idine (30)
To a solu ion o compound
20
(96 mg, 0.55 mmol) in ace oni ile (2 mL) and Milli-
Q wa e (0.6 mL), pen ylb omide (87
µ
L, 0.83 mmol) and po assium ca bona e (115 mg,
0.83 mmol
) we e added. The mix u e was s i ed unde mic owa e i adia ion a
120 ◦C
o
3 h, un il a TLC con ol a es ed o he disappea ance o he s a ing ma e ial
(CH
2
Cl
2
/MeOH/NH
4
OH (6%) 15:1:0.1). The mix u e was il e ed h ough Celi e
®,
and he
sol en was emo ed unde educed p essu e. The c ude eac ion was pu i ied by silica gel
lash column ch oma og aphy (eluen CH
2
Cl
2
/MeOH/NH
4
OH (6%) 15:1:0.1) ob aining
compound 30 (62 mg, 0.25 mmol, 45%, R = 0.34) as a waxy solid.
30:
waxy solid.
[α]24
D
= +15.0 (c = 0.50, CHCl
3
).
1
H-NMR (400 MHz, CDCl
3
)
δ
= 4.27
(q, J = 6.0 Hz, 1H, H-3), 4.03 ( , J = 4.6 Hz, 1H, H-4), 3.93–3.92 (m, 1H, H-5), 2.87 (b s, 1H,
OH), 2.72 (dd, J = 6.0, 12.0 Hz, 1H, H
a
-2), 2.55 (d, J = 12.0 Hz, 1H, H
a
-6), 2.46 (dd, J = 5.2,
11.6 Hz, 1H, H
b
-6), 2.40–2.33 (m, 3H, H
b
-2 and H-1
0
), 1.49 (s, 3H, C(CH
3
)
2
), 1.47–1.41 (m,
2H, H-2
0
), 1.34 (s, 3H, C(CH
3
)
2
), 1.31–1.20 (m, 4H, H-3
0
–H-4
0
), 0.87 ( , J = 7.0 Hz, 1H, H-5
0
)
ppm.
13
C-NMR (100 MHz, CDCl
3
)
δ
= 109.4 (C(CH
3
)
2
), 77.0 (C-4), 72.2 (C-3), 67.7 (C-5),
57.9 (C-1
0
), 56.0 (C-2 o C-6), 55.6 (C-2 o C-6), 29.7, 28.4, 26.6, 26.5, 22.7 (5C, C-2
0
–C-4
0
and
C(CH
3
)
2
), 14.1 (C-5
0
) ppm. MS (ESI): m/z(%) = 244.20 (100) [M + H]
+
. IR (CDCl
3
):
ν
= 1059,
1381, 1468, 2247, 2826, 2862, 2935, 2989, 3478 cm
−1
. C
13
H
25
NO
3
(243.35): calcd. C, 64.16; H,
10.36; N, 5.76; ound C, 64.34; H, 10.11; N, 5.70.
3.1.17. Syn hesis o (3R,5R)-1-Pen yl-3,4,5- ihyd oxy-pipe idine (21)
A solu ion o
30
(42 mg, 0.17 mmol) in MeOH (5 mL) was le s i ing wi h 12 M
HCl (25
µ
L) a oom empe a u e o 16 h. The c ude mix u e was concen a ed o yield
he hyd ochlo ide sal o
21
. The co esponding ee amine was ob ained by dissol ing
he esidue in MeOH, hen he s ongly basic esin Ambe sep 900-OH was added, and
he mix u e was s i ed o 40 min. The esin was emo ed by il a ion o a o d 30 mg
(0.15 mmol, 88%) o 21 [36] as he ee base, as a whi e solid.
21
: whi e solid m.p. 63–66
◦
C.
[α]24
D
=
−
39.7 (c = 0.61, CH
3
OH).
1
H-NMR (400 MHz,
CD
3
OD)
δ
= 3.91–3.90 (m, 1H, H-3), 3.83–3.78 (m, 1H, H-5), 3.42 (b s, 1H, H-4), 2.84–2.81
(m, 2H, H
a
-2 and H
a
-6), 2.43- 2.36 (m, 2H, H-1
0
), 2.34–2.30 (m, 1H, H
b
-6), 2.14 (b s, 1H,
H
b
-2), 1.56–1.51 (m, 2H, H-2
0
), 1.39–1.26 (m, 4H, H-3
0
–H-4
0
), 0.92 ( , J = 6.0 Hz, 3H, H-5
0
)
ppm.
13
C-NMR (100 MHz, CD
3
OD) = 75.1 (C-4), 69.4 (C-5), 69.0 (C-3), 59.3 (C-1
0
), 58.1
(C-2 o C-6), 57.4 (C-2 o C-6), 30.8, 27.1, 23.6, (3C, C-2
0
–C-4
0
), 14.4 (C-5
0
) ppm. MS (ESI):

Molecules 2022,27, 4008 17 o 21
m/z(%) = 204.08
(100) [M + H]
+
. C
10
H
21
NO
3
(203.15): calcd. C, 59.09; H, 10.41; N, 6.89;
ound C, 59.22; H, 10.33; N, 6.81.
3.2. Biological Sc eening owa ds Human Lysosomal β-Galac osidase (β-Gal) and
β-Glucosidase (GCase)
All expe imen s on biological ma e ials we e pe o med in acco dance wi h he e hical
s anda ds o he ins i u ional esea ch commi ee and wi h he 1964 Helsinki Decla a ion
and i s la e amendmen s. In keeping wi h e hical guidelines, all blood and cell samples
we e ob ained o s o age and analysed only a e w i en in o med consen o he pa ien s
(and/o hei amily membe s) was ob ained, using a o m app o ed by he local E hics
Commi ee (Codice P o ocollo: Lysola e “La e onse Lysosomal S o age Diso de s (LSDs)
in he di e en ial diagnosis o neu odegene a i e diseases: de elopmen o new diagnos ic
p ocedu es and ocus on po en ial pha macological chape ones (PCs). P ojec ID code:
16774_bio, 5 May 2020, Comi a o E ico Regionale pe la Spe imen azione Clinica della
Regione Toscana, A ea Vas a Cen o, Flo ence, I aly).
Con ols and pa ien s’ samples we e anonymized and used only o esea ch pu poses.
The new compounds we e sc eened a 1 mM concen a ion owa ds
β
-Galac osidase
(β-Gal) and β-Glucosidase (GCase) in leukocy es isola ed om heal hy dono s (con ols).
Isola ed leukocy es we e dis up ed by sonica ion, and a Mic o BCA P o ein Assay Ki
(Sigma–Ald ich, S . Louis, MO, USA) was used o de e mine he o al p o ein amoun o
he enzyma ic assay, acco ding o he manu ac u e ’s ins uc ions.
3.2.1. Human Lysosomal β-Galac osidase (β-Gal) Ac i i y
β
-Gal ac i i y was measu ed in a la -bo omed 96-well pla e. Compounds
10
–
13
and
21
solu ion (3
µ
L), 4.29
µ
g/
µ
L leukocy es homogena e 1:10 (7
µ
L), and subs a e
4-me hylumbelli e yl β
-D-galac opy anoside (1.47 mM, 20
µ
L, Sigma–Ald ich) in ace a e
bu e (0.1 M, pH 4.3) con aining NaCl (0.1 M) and sodium azide (0.02%) we e incuba ed a
37
◦
C o 1 h. The eac ion was s opped by he addi ion o sodium ca bona e (200
µ
L;
0.5 M
,
pH 10.7) con aining T i on X-100 (0.0025%), and he luo escence 4-me hylumbelli e one
eleased by
β
-galac osidase ac i i y was measu ed in Spec aMax M2 mic opla e eade
(
λ
ex = 365 nm,
λ
em = 435 nm; Molecula De ices). Inhibi ion is gi en wi h espec o
he con ol (wi hou compound). Pe cen age
β
-Gal inhibi ion is gi en wi h espec o he
con ol (wi hou compound). Da a a e mean SD (n = 3). Fo compounds showing
β
-Gal
inhibi o y ac i i y highe han 40% a 1 mM concen a ion, he IC
50
alues we e de e mined
by measu ing he ini ial hyd olysis a e wi h 4-me hylumbelli e yl
β
-D-galac opy anoside
(1.47 mM). Da a ob ained we e i ed by using he app op ia e equa ion ( o mo e de ails,
see he Supplemen a y Ma e ials).
3.2.2. Human Lysosomal β-Glucosidase (GCase) Ac i i y
GCase ac i i y was measu ed in a la -bo omed 96-well pla e. Compound solu ion
(
3µL
), 4.29
µ
g/
µ
L leukocy es homogena e (7
µ
L), and subs a e 4-me hylumbelli e yl-
β
-
D-glucoside (3.33 mM, 20
µ
L, Sigma–Ald ich) in ci a e/phospha e bu e (0.1:0.2, M/M,
pH 5.8
) con aining sodium au ochola e (0.3%) and T i on X-100 (0.15%) a 37
◦
C we e incu-
ba ed o 1 h. The eac ion was s opped by he addi ion o sodium ca bona e (200
µ
L; 0.5 M,
pH 10.7) con aining T i on X-100 (0.0025%), and he luo escence o 4-me hylumbelli e one
eleased by
β
-glucosidase ac i i y was measu ed in Spec aMax M2 mic opla e eade
(
λex = 365 nm
,
λ
em = 435 nm; Molecula De ices). A pe cen age GCase inhibi ion is gi en
wi h espec o he con ol (wi hou compound). Da a a e mean SD (n = 3).
3.3. Kine ic Analysis o Compound 12 s. β-Gal
The ac ion mechanism o compound
12
was de e mined s udying he dependence o he
main kine ic pa ame e s (Km and Vmax) on he inhibi o concen a ion. Kine ic da a we e
analysed using he Linewea e -Bu k plo ( o mo e de ails, see Supplemen a y Ma e ials).
Molecules 2022,27, 4008 18 o 21
3.4. Pha macological Chape oning Ac i i y
Fib oblas s wi h he p.Ile51Asn/p.A g201His and he p.A g201His/Ty 83Leu sX8
mu a ions om Ju enile GM1 pa ien s we e ob ained om Meye Child en’s Hospi al
(50139 Fi enze, I aly).
Fib oblas cells (15.0
×
10
4
) we e seeded in T25 lasks wi h DMEM supplemen ed wi h
e al bo ine se um (10%), penicillin/s ep omycin (1%), and glu amine (1%) and incuba ed
a 37
◦
C wi h 5% CO
2
o 24 h. The medium was emo ed, and esh medium con aining
he compounds was added o he cells and incuba ed o ou days. The medium was
emo ed, and he cells we e washed wi h PBS and de ached wi h ypsin o ob ain cell
pelle s, which we e washed ou imes wi h PBS, ozen, and lysed by sonica ion in wa e .
Enzyme ac i i y was measu ed as epo ed abo e. Repo ed da a a e mean S.D. (n = 2).
4. Conclusions
Fou new ihyd oxypipe idines wi h a C-2 pen yl chain wi h bo h con igu a ions
we e syn hesized, oge he wi h hei “all-cis” hyd oxy epime s, wi h he aim o inding
new human lysosomal β-Gal inhibi o s and po en ial PCs o GM1-gangliosidosis.
The syn hesis exploi ed he addi ion o pen ylmagnesium b omide o ni one
18
,
de i ed om aldehyde
19
, in he p esence o absence o Lewis acid ollowed by RA, o
yield he wo 2-pen yl 3,4,5- ihyd oxypipe idines. The in e sion o con igu a ion a C-3
was achie ed h ough an oxida ion– educ ion sequence. To in es iga e he ole o he chain
posi ion on he ac i i y, compound
21
was syn hesized o compa ison, s a ing om he
pipe idine in e media e 20 ia N-alkyla ion.
Biologic es s o he new compounds showed ha
10
and
12
a e
β
-Gal inhibi o s
wi h a mode a e IC
50
(400
±
15
µ
M and 1.15
±
0.1 mM). Kine ic analyses e ealed a
noncompe i i e mode o inhibi ion o
12
, which also showed chape oning p ope ies, wi h
K
i
alue o 1.4
±
0.7 mM. Mo eo e , good selec i i y owa ds
β
-Gal wi h espec o
β
-Glu
was obse ed. The poo
β
-Glu inhibi o y ac i i y o all compounds con i med ha he
p esence o a longe linea alkyl chain (a leas eigh ca bon a oms) is essen ial o impa
s ong β-Glu inhibi o y ac i i y.
Tes ing compounds
10
and
12
as po en ial PCs in ib oblas s om ju enile GM1 gan-
gliosidosis pa ien s bea ing he p.Ile51Asn/p.A g201His and he p.A g201His/Ty 83Leu sX8
mu a ions highligh ed ha only
12
allows an ac i i y escue o
β
-Gal (40% a 600
µ
M) on
GM1 pa ien s bea ing he p.Ile51Asn/p.A g201His mu a ions, hus ep esen ing, o he bes
o ou knowledge, a unique example o a non-compe i i e inhibi o wi h chape oning abil-
i y o GM1 gangliosidosis. The he e epo ed ib oblas s, which de i e om ju enile GM1
gangliosidosis pa ien s, sha e he p.A g201His mu a ion in one allele. Thus, he escue o
he
in i o
sys em bea ing he bi-allelic composi ion p.Ile51Asn/A g201His can be asc ibed
o he chape one ac i i y o compound 12 on he p.Ile51Asn mu a ion-bea ing allele.
I has been p e iously epo ed ha he p.Ile51Asn mu a ion, eplacing a nonpola
esidue in a hyd ophobic pocke in o a pola esidue, is likely o ad e sely a ec he old
o he
β
-Gal p o ein [
3
]. This biochemical cha ac e is ic makes he p.Ile51Asn mu a ion
pa icula ly p one o an enzyma ic s abilisa ion and a escue o
β
-Gal ac i i y induced by a
chape one, as demons a ed by he use o compound 12.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/molecules27134008/s1: 1H-NMR, 13C-NMR and selec ed 1D
NOESY spec a o new compounds (Figu es S1–S40); enzyma ic sc eening and kine ic analysis
(Figu es S41–S48); in i o assays on cell lines (Figu es S49 and S50).
Au ho Con ibu ions:
F.C. (F ancesca Clemen e), C.M., A.G. and F.C. (F ancesca Ca dona) planned
he syn he ic s a egy. F.C. (F ancesca Clemen e) pe o med he syn heses. F.C. (F ancesca Clemen e),
S.F. and M.M.-B. pe o med he biological expe imen s. A.C., P.P. and A.M. supe ised he biological
expe imen s. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
The esea ch was unded by Regione Toscana (Bando Salu e 2018) o he p ojec : “La e
onse Lysosomal S o age Diso de s (LSDs) in he di e en ial diagnosis o neu odegene a i e diseases:
Molecules 2022,27, 4008 19 o 21
de elopmen o new diagnos ic p ocedu es and ocus on po en ial pha macological chape ones
(PCs), Ac onym: Lysola e) and by Uni e si àdi Fi enze and Fondazione CR Fi enze (“Bando
congiun o pe il inanziamen o di p oge i compe i i i sulle mala ie neu odegene a i e 2018”.
P ojec : A mul idisciplina y app oach o a ge Pa kinson’s disease in Gauche - ela ed popula ion,
Ac onym: MuTaPa Ga).
Ins i u ional Re iew Boa d S a emen :
This s udy was conduc ed in acco dance wi h he e hical
s anda ds o he ins i u ional esea ch commi ee and wi h he 1964 Helsinki Decla a ion and i s
la e amendmen s. and app o ed by he Comi a o E ico Regionale pe la Spe imen azione Clinica
della Regione Toscana, A ea Vas a Cen o, Flo ence, I aly (Codice P o ocollo: Lysola e “La e onse
Lysosomal S o age Diso de s (LSDs) in he di e en ial diagnosis o neu odegene a i e diseases:
de elopmen o new diagnos ic p ocedu es and ocus on po en ial pha macological chape ones (PCs).
P ojec ID code: 16774_bio, 5 May 2020).
In o med Consen S a emen :
In o med consen was ob ained om all subjec s in ol ed in he
s udy. Da a we e anonymized.
Da a A ailabili y S a emen :
The au ho s con i m ha he da a suppo ing he indings o his s udy
a e a ailable wi hin he a icle and/o i s Suppo ing In o ma ion ile.
Acknowledgmen s:
We hank MIUR-I aly (“P oge o Dipa imen i di Eccellenza 2018
−
2022”) allo-
ca ed o he Depa men o Chemis y “Ugo Schi ”, Uni e si àdi Fi enze and he AMMeC (Associ-
azione Mala ie Me aboliche e Congeni e, I alia).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Sample A ailabili y:
Samples o he compounds epo ed in he manusc ip a e no a ailable om
he au ho s.
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