O ganic & Biomolecula Chemis y RSCPublishing
ARTICLE
This jou nal is © The Royal Socie y o Chemis y 2015 O g. Biomol. Chem., 2015, 00, 1-3 | 1
Ci e his: DOI: 10.1039/x0xx00000x
Recei ed 00 h Feb ua y 2015,
Accep ed 00 h Feb ua y 2015
DOI: 10.1039/x0xx00000x
www. sc.o g/
Con o ma ionally-Locked C-Glycosides: Tuning
Aglycone In e ac ions o Op imal Chepe one
Beha iou in Gauche Fib oblas s
C. D. Na o,a F. Co zana,a E. M. Sánchez-Fe nández,b J. H. Bus o,a A. A enoza,a
E. Namba,c K. Higaki,c C. O iz Melle ,b* J. M. Ga cía Fe nándezd* and J. M.
Pe eg inaa*
A se ies o con o ma ionally locked C-glycosides based on he 3-aminopy ano[3,2-b]py ol-
2(1H)-one (APP) sca old has been syn hesized. The key s ep in ol ed a o ally
s e eocon olled C-Michael addi ion o a se ine-equi alen C-nucleophile o i-O-benzyl-2-
ni o-
D
-galac al. S e eoselec i e ans o ma ions o he co esponding Michael adduc
allowed us he syn hesis o compounds wi h mono- o dian enna ed aglycone moie ies and
di e en opologies. In i o sc eening showed highly selec i e inhibi ion o bo ine li e β-
glucosidase/-galac osidase and speci ic inhibi ion o human -glucoce eb osidase among
lysosomal glycosidases o compounds bea ing palmi oyl chains in he aglycone, wi h a
ma ked dependence o he inhibi ion po ency upon hei numbe and loca ion. Molecula
dynamics simula ions highligh ed he pa amoun impo ance o an op imal o ien a ion o he
hyd ophobic subs i uen o wa an e icien non-glycone in e ac ions, which a e c i ical o
he binding a ini y. The esul s u he p o ide a a ional o he s ong dec ease o he
inhibi on po ency o APP compounds on going om neu al o acidic pH. The bes candida e
was ound o beha e as a chape one in Gacueh ib oblas s wi h homozygous N370S and
F213I mu a ions, wi h enzyme ac i i y enhancemen s simila o hose encoun e ed o he
e e ence compound Amb oxol®.
In oduc ion
Glycosidases a e enzymes ha ca alyze he hyd olysis o
glycosidic bonds, he eby playing a key ole in biochemical
ou es ha a e essen ial o li e such as he p ocessing o
glycop o eins and he ca abolism o polysaccha ides and
glycoconjuga es. De egula ion o any o hese enzymes
gene ally ansla es in o signi ican biological and pa hological
consequences, which has uelled esea ch in compounds
in e e ing wi h hei ac i i y as po en ial d ug candida es.
1
The
ca bohyd a e mimics (glycomime ics) o he iminosuga ype,
such as he pipe idine de i a i e 1-deoxynoji imicyn (DNJ) o
he indolizidine analogue cas anospe mine (CS, Figu e 1),
ep esen by a he mos s udied amily o molecules owa ds
hese channels.
2
Thei polyhyd oxyla ed s uc u e emula es ha
o he na u al monosaccha ides, enabling hem o compe e wi h
he subs a e o he glycone pocke in he ac i e si e o
glycosidases, beha ing as compe i i e inhibi o s..Howe e ,
classical iminosuga s canno ully ep oduce he s uc u e o he
pu a i e glycoside subs a es due o he ins abili y o
aminoace al unc ional g oups, which o en ansla es in o poo
selec i i y. E en hough igidi ica ion o he azahe e ocyclic
co e in bicyclic de i a i es can be exploi ed o imp o e he
disc imina ion capaci y be ween isoenzymes, he absence o a
de ined anome ic con igu a ion gene ally esul s in
simul aneous inhibi ion o glycosidases ac ing on anome ic
subs a es, i.e. - and -glucosidases in he case o DNJ and
CS, which ep esen s a se ious handicap o clinical
applica ions.
3
The u gen need o mo e speci ic glycosidase inhibi o s has
uelled esea ch on syn he ic glycomime ics a ge ing medically
ele an enzymes.
4,5.
In p inciple, molecula p o o ypes
mi o ing he ull s uc u e o glycosides, ha is inco po a ing
bo h glycone- and aglycone-like po ions and bene i ing om a
de ined pseudoanome ic con igu a ion, a e be e sui ed o ha
pu pose.
6
Fo example, sp
2
-iminosuga co es,
7
in which he
endocyclic ni ogen is pa o a pseudoamide unc ionali y, a e
compa ible wi h he inco po a ion o axially-o ien ed
pseudoanome ic subs i uen s h ough s able O-, S- o N-
ARTICLE O ganic & Biomolecula Chemis y
2 | O g. Biomol. Chem., 2015, 00, 1-3 This jou nal is © The Royal Socie y o Chemis y 2015
glycosidic linkages (Figu e 1), which has been exploi ed in he
design o speci ic -glucosidase inhibi o s exhibi ing an icance
ad an ileishmanial ac i i ies.
8
N-alkyla ed bicyclic isou eas
de i ed om aminocycli ol sca olds (e.g.,
benzo[d]oxazolimine de i a i es, Figu e 1) beha ed ins ead as
e y selec i e inhibi o s o human lysosomal -glucosidase,
exhibi ing s ong pha macological chape one po en ial o he
ea men o Gauche disease.
9
In e es ingly, some examples o
glycosidase inhibi o s keeping he py anose ing as he glycone
cons i uen ha e also been epo ed, which p esen s ad an ages
in e m o syn he ic p ocedu es. An example is hiame G
(Figu e 1), a used py anose- hiazoline de i a i e, ha ac s as a
po en inhibi o o O-linked 2-ace amino-2-deoxy-β-
D
-
glucopy anoside hyd olysis cu en ly in p eclinical e alua ion
as a po en ial he apeu ic o he ea men o Alzheime ’s
disease.
10
The con o ma ionally-locked bicyclic N-glycoside
de i a i es o he py anose-sul anyl-1,3-oxazoline amily
(PSO, Figu e 1) exhibi β-glucosidase inhibi o y ac i i y ha is
s ongly dependen on he na u e o he psudoaglycone S-
subs i uen , some ep esen a i es beha ing as pha macological
chape ones o Gauche disease.
11
Figu e 1.
Some iminosuga s and bicyclic de i a i es glycosidase
inhibi o s.
The py ano[3,2-b]py ole amewo k (Figu e 1) has also been
sugges ed as a sui able sca old o he p epa a ion o
glycosidase inhibi o s.
12
The lack o e ec i e syn he ic
me hods compa ible wi h he inco po a ion o aglycone- ype
susb i uen s has bu dened op imiza ion o he a ini y and
selec i i y owa ds biomedically ele an enzyme a ge s,
howe e .
13
The de elopmen o e icien app oaches owa ds
his amewo k h ough syn he ic ou es compa ible wi h
s uc u ally di e si y-o ien ed s a egy is he e o e highly
wan ed. The possibili y o accessing con o ma ionally-
es ic ed mul ian enna ed de i a i es wi h C-glycoside
s uc u e is pa icula ly appealing
14
gi en ha many medically
ele an glycosidases, such as hose ha a e dys unc ional in
glycosphingolipid- ela ed me abolic diso de s, ac on subs a es
wi h b anched aglycone segmen s.
15
Wi h his backg ound, we ha e now en isioned he syn hesis o
3-
a
mino-6,7-dihyd oxy-3,5-
bis(hyd oxyme hyl)hexahyd o
p
y ano[3,2-b]
p
y ol-2(1H)-one
de i a i es (APP, Figu e 1) as a new amily o non-iminosuga -
ype glycosidase inhibi o s. The disubs i u ion pa e n a he
exocyclic qua e na y anome ic ca bon, which bea s
simul aneously a hyd oxyme hyl and an amino g oup
suscep ible o chemical modi ica ion, o e b oad oppo uni ies
o he inco po a ion o non-glycone moie ies. The
op imiza ion o he syn hesis o a key syn he ic p ecu so o
APP syn hesis, he subsequen ans o ma ion in o a b oad
ba e y o mono- and dian enna ed APP de i a i es and he
assessmen o he a ini y and selec i i y o he inal
compounds agains a panel o comme cial and human
glycosidase enzymes is p esen ed.
Resul s and discussion
Recen ly, a o ally s e eocon olled C-Michael addi ion o a
se ine-equi alen C-nucleophile o i-O-benzyl-2-ni o-
D
-
galac al (ni ogalac al) was used as he key s ep o syn hesize
se e al py ano[3,2-b]py ole s uc u es, e.g. compound
1
,
ollowing educ ion o he ni o g oup in he co esponding
Michael adduc .
16
In e es ingly, spi ocycle
1
can be ega ded as
a p o ec ed o m o he a ge APP de i a i es (Scheme 1). To
explo e his channel, an op imized p epa a ion o
1
in g am
scale was a p e equisi e. This goal was con enien ly achie ed
by using pla inized Raney Ni unde an a mosphe e o
hyd ogen
17
in he educ ion s ep. The ansien amino g oup
eac ed spon aneously wi h he me hyl es e g oup o close he
co esponding i e-membe ed lac am ing wi h e en ion o he
con igu a ion a bo h s e eocen e s.
Scheme 1.
Re osyn he ic analysis o APP de i a i es.
Compound
1
was nex used as a p ecu so o he syn hesis o a
ba e y o APP de i a i es. In a i s s ep, concomi an
hyd olysis o he ace al and cyclic ca bama e g oups, by
Jou nal Name ARTICLE
This jou nal is © The Royal Socie y o Chemis y 2012 O g. Biomol. Chem., 2015, 00, 1-3 | 3
ea men wi h 4
M
aqueous HCl a 40 °C o 12 h and libe a ion
o he esul ing ammonium hyd ochlo ide sal by he ac ion o
p opylene oxide in e hanol a e lux, p o ided aminoalcohol
2
in excellen yield (96%). Acyla ion o
2
wi h palmi oyl chlo ide
in he p esence o ime hylamine a oom empe a u e o 12 h
a o ded he dian enna ed de i a i e
3
(70% yield), which was
subsequen ly de-O-benzyla ed by hyd ogenolysis in acidi ied
me hanol, using palladium on ca bon as a ca alys , o gi e he
unp o ec ed APP
4
in only h ee s eps and 62% o e all yield
(Scheme 2).
Scheme 2.
Syn hesis o a ge compounds
4
,
6
,
8
and
11
inco po a ing palmi oyl sub i uen s.
Compound
3
was also selec i ely de-O-acyla ed wi h sodium
me hoxide in me hanol a pH 9 o gi e he co esponding
alcohol
5
, which a e debenzyla ion ollowing he abo e
p ocedu e a o ded he co esponding APP de i a i e
6
, bea ing
a single-chain N-palmi oyl aglycone moie y (Scheme 2). The
homologous O-palmi oyl monan enna ed adduc
8
, keeping a
ee amino g oup in he aglycone, was ob ained om he
pi o al p ecu so
1
a e acid hyd olysis, in si u egioselec i e
O-acyla ion wi h palmi oyl chlo ide, neu aliza ion wi h
p opylene oxide in e hanol and hyd ogenolysis o he ibenzyl
e he
7
(62% o e all yield; Scheme 2). The possibili y o
conduc ing chemoselec i e acyla ion o he masked hyd oxy
and amino g oups in
1
is u he compa ible wi h he
inco po a ion o di e en subs i uen a each posi ion. Thus,
palmi oyla ion o he p e iously epo ed N-ace yla ed
compound
9
16
ollowed by hyd ogenolysis o he esul ing es e
10
led o he new APP de i a i e
11
(Scheme 2). Finally,
compounds
13
-
15
, ea u ing O-ace yl and a ee amino g oups,
ee hyd oxyl and amino g oups, o ee hyd oxy and N-ace yl
g oups in he aglycone segmen , espec i ely we e p epa ed as
con ols o assess he in luence o he p esence o he long
hyd ophobic segmen s in he glycosidase inhibi o y p ope ies
o he PAA (Scheme 3).
ARTICLE O ganic & Biomolecula Chemis y
4 | O g. Biomol. Chem., 2015, 00, 1-3 This jou nal is © The Royal Socie y o Chemis y 2015
Scheme 3.
Syn hesis o a ge compounds
13
-
15
wi hou la ge alkyl
chains.
Compounds
4
,
6
,
8
,
11
,
13
,
14
and
15
we e i s e alua ed
agains a panel o comme cially a ailable glycosidase enzymes
including -glucosidase (bake yeas ), isomal ase (bake yeas ),
amyloglucosidase (A. nige ), -glucosidase/-galac osidase
(bo ine li e ), -galac osidase (E. coli), -galac osidase (g een
co ee), -glucosidase (almonds), -mannosidase (Jack bean),
-mannosidase (H. poma ia) and -N-ace ylglucosaminidase
(human placen a, bo ine kidney and Jack bean). Inhibi ion
cons an s (K
i
, 
M
) we e de e mined om he slope o
Linewea e -Bu k plo s (see Figu e 2 o a ep esen a i e
example) and a e collec ed in Table 1. The da a con i m ha he
p esence o a hyd ophobic segmen in he aglycone (
4
,
6
,
8
and
11
) is necessa y o elici a signi ican ac i i y, isomal ase (K
i
,
13-80 
M
) and -glucosidase/-galac osidase (K
i
, 5.8-86 
M
)
being he mo e sensi i e enzymes o APP inhibi ion. The
p esence o a bulky subs i uen a he ni ogen a om (
4
and
6
) is
de imen al o isomal ase inhibi ion, whe eas i has he
opposi e e ec in he case o he mammalian -glucosidase/-
galac osidase enzyme. Compound
8
, keeping a ee amino
g oup in he aglycone, addi ionally beha es as a 
M
inhibi o o
-glucosidase (K
i
, 27 
M
) and -galac osidase (K
i
, 67 
M
)
Table 1.
Glycosidase inhibi o y ac i i ies (K
i
, 
M
) o APP de i a i es agains comme cial glycosidases.
[a]
[a]
Inhibi ion, when de ec ed, was compe i i e in all cases. No inhibi ion was de ec ed o any o he compounds a concen a ions up o 2 mM
agains amyloglucosidase (A. nige ),

-glucosidase (almonds),

-galac osidase (E. coli),

-mannosidase (Jack bean),

-mannosidase (H. poma ia)
and

-N-ace ylglucosaminidase (human placen a, bo ine kidney and Jack bean).
[b]
De e mined om he slope o Linewea e _Bu k Plo s and
Double Recip ocal Analysis. n.i. no inhibi ion de ec ed a concen a ions up o 2 mM.
Inhibi ion o bo ine li e β-glucosidase/-galac osidase has
been o en used as a p elimina y pa ame e o selec candida es
as pha macological chape ones o mu an o ms o human β-
glucoce eb osidase (GCase) associa ed wi h Gauche disease.
18
Al hough he p edic i e cha ac e o he da a mus be aken
wi h ca e,
19
he signi ican inhibi o y po en ial and high
selec i i y encoun e ed o some o he new APP compounds in
Table 1 agains he comme cial enzyme wa an ed u he
e alua ion in his sense. The concen a ions o he di- (
4
) and
monoan enna ed de i a i es (
6
,
8
and
11
) gi en 50% inhibi ion
o human GCase a e collec ed in Table 2. The co esponding
da a o he non-glycomime ic ype pha macological chape one
Amb oxol® (ABX), cu en ly in p eclinical s udies o he
ea men o Gauche disease,
20
a e also included o
compa a i e pu poses.
Enzymes
4
6
8
11
13
14
15

-Glucosidase
(Bake yeas )
225±25
124±13
27±3
244±28
n.i.
532±58
n.i.
Isomal ase
(Bake yeas )
80±9
34±4
13±1
19±2
97±11
283±31
n.i.

-Glucosidase/

-Galac osidase
(Bo ine li e )
10±1
[b]
5.8±0.6
[b]
86±9
9.2±8
[b]
n.i.
n.i.
n.i.

-Galac osidase
(G een co ee)
657±72
515±56
67±7
n.i.
n.i.
n.i.
n.i.
Jou nal Name ARTICLE
This jou nal is © The Royal Socie y o Chemis y 2012 O g. Biomol. Chem., 2015, 00, 1-3 | 5
Figu e 2.
Linewea e -Bu k plo o Ki de e mina ion (5.8

M
) o APP
6
agains

-galac osidase (bo ine li e ) (pH 7.3).
Table 2.
IC
50
alues (M) agains human GCase o APP
de i a i es
4
,
6
,
8
and
11
.
Compound
GCase pH 7
GCase pH 5
41±1
470±20
23±1
272±10
49±2
606±10
67±5
707±20
7.6±0.5
90±5
The inhibi ion po ency agains GCase ollowed a sligh ly
di e en end as compa ed o ha encoun e ed o he
comme cial mammalian β-glucosidase/-galac osidase. The
p esence o he O-acyl subs i uen in he aglycone seems o be
de imen al o he human enzyme. Thus, compound
6
(IC
50
23
M a pH 7), missing his g oup, is an abou 2- o 3- old
s onge inhibi o han he h ee o he APP de i a i es assayed
(IC
50
41-67 M a pH 7), hough i is s ill 3- old weake han
he e e ence compound ABX (7.6 M). No inhibi ion o o he
lysosomal enzymes, such as -glucosidase, -galac osidase, -
galac osidase, and -hexosaminidase, was obse ed. Mos
in e es ingly, an abou one-o de -o -magni ude dec ease in he
GCase inhibi ion s eng h was obse ed a pH 5, a a o able
ea u e o chape one candida es.
21
The ensemble o da a in Tables 1 and 2 highligh he pa amoun
impo ance o non-glycone in e ac ions in he a ini y and
selec i i y owa ds glycosidases. They also unde line he need
o implemen ing s a egies allowing s uc u al modi ica ions
wi h a ela i ely low syn he ic cos o op imize such
in e ac ions. The supe io i y o compound
6
as GCase inhibi o
wi hin he APP se ies is ema kable. To ge u he s uc u al
in o ma ion, we pe o med 50 ns MD simula ions
22
on
6
in
complex wi h he human enzyme (pdb code: 2WCG).
23
The
esul s suppo ha he py anoid glycone moie y is engaged in
hyd ogen bond ne wo king in a simila manne o ha
p e iously encoun e ed o o he glycomime ic- ype
compe i i e inhibi o s (Figu e 3). Indeed, al hough he pu a i e
subs a e o GCase is D-glucosylce amide, i has been shown
ha D-galac o con igu ed glycomime ics do also i well in he
ca aly ic si e. No iceably, he calcula ions p edic ha he
exocyclic amide ni ogen is in ol ed in a hyd ogen bond wi h
he ca aly ic glu amic acid nucleophile Glu235. P o ona ion o
his esidue may be esponsible o he la ge di e ence
obse ed in he inhibi o y po ency o APP compounds a
neu al and acidic pH. Mo eo e , his hyd ogen bond o ien s
he alipha ic N-subs i uen owa ds he hyd ophobic egion a
he en ance o he ac i e si e o he enzyme, ein o cing he
s abili y o he complex.
Figu e 3.
Rep esen a i e ame ob ained om he 50 ns MD simula ion
on human GCase:APP
6
complex showing he hyd ogen bonds be ween
he glycone uni o compound
6
and he esidues in he binding si e (le
panel). The side chain o compound APP
6
is engaged in hyd ophobic
con ac s wi h he su ace o he enzyme ( igh panel). The hyd ophobic
esidues o he enzyme a e shown in o ange.

ARTICLE O ganic & Biomolecula Chemis y
6 | O g. Biomol. Chem., 2015, 00, 1-3 This jou nal is © The Royal Socie y o Chemis y 2015
All he ou amphiphilic APP compounds we e u he assayed
o hei chape oning capabili ies using heal hy and Gauche
ib oblas s om pa ien s ha ing he N370S/N370S,
F213I/F213I o L444P/L444P GCase mu a ions. These a ian s
a e associa ed o he non-neu onopa hic ( ype 1) and
neu onopa hic ( ypes 2 and 3) pheno ypes o Gauche disease,
espec i ely.
24
Only he i s one, wi h he highes p e alence
and loca ed in he ca aly ic domain o GCase, is esponsi e o
he enzyme eplacemen o subs a e educ ion he apies
cu en ly a ailable, The F213I/F213I mu a ion is also loca ed in
he ca aly ic domain o he enzyme, while he L444P/L444P
mu a ion is loca ed in a nonca aly ic domain, which makes i
less p one o escuing by pha macological chape ones.
25
The
cells we e cul u ed o 5 days in he absence and in he p esence
o 2 o 20 M concen a ions o he APP compounds, hen
lysed and he GCase ac i i y de e mined using 4-
me hylumbelli e yl -D-glucopy anoside as subs a e. Enzyme
ac i i y a ia ions ela i e o he con ol in he absence o any
compound we e hus moni o ed. S a is ically signi ican ac i i y
enhancemen s we e obse ed only o he N370S/N370S and
F213I/F213I GCase mu an s a e ea men wi h compound
6
a 20 M concen a ion, wi h ela i e inc eases o 1.3- and 1.5-
olds, espec i ely, which app oach hose achie ed wi h he
e e ence compound ABX (1.5- and 1.8- old, espec i ely).
None o he APP de i a i es did exhibi oxic e ec on any o
he no mal o mu an cell lines assayed o 5 days incuba ion.
Conclusions
In summa y, we ha e de ised an e icien me hodology o he
syn hesis o con o ma ionally locked C-glycosides based on he
3-amino-3-hyd oxyme hylpy ano[3,2-b]py ol-2(1H)-one
(APP) sca old compa ible wi h he inco po a ion o mono and
mul ib anched aglycone moie ies. E alua ion agains a a ie y
o glycosidase enzymes showed ha amphiphilic de i a i es
bea ing long hyd ophobic subs i uen s beha ed as M
compe i i e inhibi o s o bo ine li e -glucosidase/-
galac osidase. Mos in e es ingly, hey also beha ed as selec i e
inhibi o s o human lysosomal -glucosidase (-
glucoce eb osidase) a neu al pH, wi h a ema kable dec ease
in he inhibi o y po ency upon acidi ica ion (pH 5). The pH
dependency o glucoce eb osidase binding makes his ype o
compounds good candida es as pha macological chape ones o
Gauche disease. Indeed, compound
6
, ha ing a
palmi oylamido segmen in he aglycone, was able o inc ease
he ac i i y o N370S/N370S and F213I/F213I
glucoce eb osidase mu an s in ib oblas s o Gauche pa ien s
wi h an e iciency simila o ha o he e e ence compound
Amb oxol®. MD simula ions suppo he exis ence o a
hyd ogen bond in ol ing he amide p o on o he chape one
and he ca boxyla e g oup o he ca aly ic glu amic acid esidue
Glu235 h a a o ably o ien s he palmi oyl chain owa ds a
hyd ophobic pocke in heenzyme. Since he p o ona ion s a e
o Glu235 changes in he pH 7-5, his s uc u al ea u e o e
u he in e es ing oppo uni ies o pH-dependen chape one
design. Resea ch in ha di ec ion is cu en ly sough in ou
labo a o ies.
Expe imen al
Gene al In o ma ion
Sol en s we e pu i ied acco ding o s anda d p ocedu es. All
eac ions we e ollowed by hin laye ch oma og aphy (TLC)
whe e p ac ical, using silica gel 60 F254 luo escence ea ed
silica gel pla es, which we e isualized unde UV ligh (250
nm). Column ch oma og aphy was pe o med using silica gel
60 (230−400 mesh).
1
H and
13
C NMR spec a we e eco ded on
a 400 MHz spec ome e using CDCl
3
, CD
3
OD, o D
2
O as he
sol en ; chemical shi s a e epo ed in pa s pe million on he
δ scale, and coupling cons an s a e epo ed in He z. All o he
esol ed signals in he
1
H NMR spec a we e assigned on he
basis o coupling cons an s and ge-COSY and ge-HSQC
expe imen s pe o med on he 400 MHz spec ome e . The
esul s o hese expe imen s we e p ocessed wi h Mes ReC and
Mes eNo a so wa e. Mel ing poin s we e de e mined on a
mel ing-poin appa a us and a e unco ec ed. Op ical o a ions
we e measu ed on a pola ime e om solu ions in 1.0 dm cells
o capaci y 1.0 o 0.3 mL. Elec osp ay mass spec a we e
eco ded on a mic OTOF spec ome e ; accu a e mass
measu emen s we e achie ed by using sodium o ma e as an
ex e nal e e ence. Copies o NMR spec a o all new
compounds a e p o ided in he Suppo ing In o ma ion.
P epa a ion o (3R,3aS,5R,6R,7R,7aS,7′R,7′aS)-6,7-dibenzyloxy-
5-(benzyloxyme hyl)-7′-me hoxy-7′,7′a-
dime hylspi o[1,3a,5,6,7,7a-hexahyd opy an[3,2-b]py ol-3,3′-
2H-oxazolo[4,3-b]oxazol]- 2,5′-dione 1
Raney Ni (2.00 g) was suspended in H
2
O (12 mL), and
hexachlo opla inic acid (50 mg) and sodium hyd oxide 20%
(400 μL) we e added unde s i ing. The mix u e was hea ed a
50 °C. A e he mix u e was s i ed o 2.5 h, sodium
hyd oxide 40% (6 mL) was added, keeping he s i ing and he
hea ing. A e 1.5 h o s i ing, a whi e cloud in he op o he
lask appea ed, which was emo ed by decan a ion; he
esul ing solu ion was hen washed wi h wa m wa e (3 × 15
mL) and e hanol (3 × 15 mL). The ca alys ob ained was
suspended in e hanol (10 mL) and p ehyd ogena ed o 10 min.
An e hanol/e hyl ace a e solu ion (5:2, 7 mL) o Michael adduc
(200 mg, 0.28 mmol) was added, and he mix u e was s i ed
unde molecula hyd ogen a oom empe a u e and
a mosphe ic p essu e o 5 h. The c ude p oduc was il e ed,
and he liquid phase was concen a ed and pu i ied by silica gel
column ch oma og aphy (e hyl ace a e/hexane, 7:3), o a o d
compound
1
(105 mg, 0.16 mmol, 57%) as a colo less oil.
Physical da a a e ag ee wi h hose epo ed in he li e a u e.
e
P epa a ion o (3R,3aS,5R,6R,7R,7aS)-3-Amino-6,7-
bis(benzyloxy)-5-(benzyloxyme hyl)-3-(hyd oxyme hyl)-2-oxo-
1,3a,5,6,7,7a-hexahyd opy ano[3,2-b]py ol 2
Jou nal Name ARTICLE
This jou nal is © The Royal Socie y o Chemis y 2012 O g. Biomol. Chem., 2015, 00, 1-3 | 7
Compound
1
(105 mg, 0.163 mmol) was dissol ed in THF (10
ml). A 4
M
solu ion o HCl (3.6 mL) was hen added and he
mix u e was hea ed up o 40 ºC o 12 h unde s i ing. The
c ude ob ained a e concen a ion was dissol ed in absolu e
e hanol (5 mL). P opylene oxide (5 mL) was added and he
mix u e was hea ed o e lux o 2 h unde s i ing.
Concen a ion and pu i ica ion o he c ude p oduc by silica
gel column ch oma og aphy (dichlo ome hane/me hanol, 15:1)
a o ded compound
2
(60 mg, 0.116 mmol, 71%) as a
colou less oil.
[𝛼]
𝐷
20
= +64.8 (c 1.0 in CHCl
3
). HRMS ESI+
(m/z) = 519.2495 [M+H]
+
; calcula ed o C
30
H
35
N
2
O
6+
=
519.2490.
1
H NMR (400 MHz, CDCl
3
)  = 3.52-3.61 (m, 2H,
H
7
, BnOCH
a
H
b
), 3.67 (d, 1H, J = 12.0 Hz, CH
c
H
d
OH), 3.71-
3.83 (m, 2H, CH
c
H
d
OH, BnOCH
a
H
b
), 4.03-4.15 (m, 3H, H
5
,
H
6
, H
7a
), 4.24 (d, 1H, J = 4.8 Hz, H
3a
), 4.48-4.59 (m, 4H,
PhCH
2
O, PhCH
e
H
O, PhCH
g
H
h
O), 4.68 (d, 1H, J = 11.6 Hz,
PhCH
e
H
O, 4.84 (d, 1H, J = 11.6 Hz, PhCH
g
H
h
O), 6.84 (s, 1H,
NH), 7.20-7.40 (m, 15H, Ph).
13
C NMR (100 MHz, CDCl
3
)  =
56.2 (C
7a
), 62.3 (C
3
), 65.1 (CH
2
OH), 67.2 (BnOCH
2
), 71.9
(PhCH
e
H
O), 72.5 (C
6
), 73.6 (PhCH
2
O), 74.5 (PhCH
g
H
h
O),
76.2 (C
5
), 76.7 (C
3a
), 81.3 (C
7
), 127.8, 127.9, 128.0, 128.3,
128.5, 128.6, 128.8, 137.6, 137.9, 138.1 (Ph), 177.8 (CO).
P epa a ion o [(3R,3aS,5R,6R,7R,7aS)-6,7-Bis(benzyloxy)-5-
(benzyloxyme hyl)-2-oxo-3-palmi amido-1,3a,5,6,7,7a-
hexahyd opy ano[3,2-b]py ol-3-yl]me hyl palmi a e 3
Compound
2
(45 mg, 0.086 mmol) was dissol ed in
dichlo ome hane (2 mL) and palmi oyl chlo ide (80 L, 0.26
mmol) was added. E
3
N (50 L, 0.36 mmol) was hen added
and he mix u e was s i ed a oom empe a u e o 12 h. A 0.5
M
solu ion o HCl (2 mL) was added. The o ganic laye was
sepa a ed and he aqueous one was washed wi h
dichlo ome hane (3 × 2 mL). The o ganic phases we e collec ed
and d ied wi h anhyd ous Na
2
SO
4
. Concen a ion and
pu i ica ion o he c ude p oduc by silica gel column
ch oma og aphy (hexane/e hyl ace a e, 65:35) a o ded
compound
3
(60 mg, 0.06 mmol, 70%) as a colou less oil.
[𝛼]
𝐷
20
= +34.5 (c 1.0 in CHCl
3
). HRMS ESI+ (m/z) = 995.7071
[M+H]
+
; calcula ed o C
62
H
95
N
2
O
8+
= 995.7083.
1
H NMR (400
MHz, CDCl
3
)  = 0.88 ( , 6H, J = 6.5 Hz, CH
3
), 1.19-1.35 (m,
48H, (CH
2
)
12
CH
3
), 1.51-1.64 (m, 4H, CH
2
(CH
2
)
12
CH
3
), 2.18 ( ,
2H, J = 7.6 Hz, NHCOCH
2
), 2.24 ( , 2H, J = 7.6 Hz,
OCOCH
2
), 3.52-3.60 (m, 2H, H
7
, BnOCH
a
H
b
), 3.63-3.70 (m,
1H, BnOCH
a
H
b
), 4.03-4.10 (m, 1H, H
5
), 4.11-4.15 (m, 1H, H
6
),
4.29-4.36 (m, 2H, H7a, CH
c
H
d
OCO), 4.42-4.54 (m, 4H,
CH
c
H
d
OCO, PhCH
2
O, PhCH
e
H
O), 4.58 (d, 1H, J = 11.6 Hz,
PhCH
g
H
h
O), 4.66-4.72 (m, 2H, H
3a
, PhCH
e
H
O), 4.88 (d, 1H, J
= 11.6 Hz, PhCH
g
H
h
O), 6.06 (NH), 6.32 (NH), 7.22-7.40 (m,
15H, Ph).
13
C NMR (100 MHz, CDCl
3
)  = 14.2 (CH
3
), 22.8,
24.9, 25.3, 29.3, 29.5, 29.6, 29.7, 29.8, 32.1 ((CH
2
)
13
CH
3
), 34.2
(OCOCH
2
), 36.2 (NHCOCH
2
), 53.5 (C
3
), 56.0 (C
7a
), 63.1
(CH
2
OCO), 67.8 (BnOCH
2
), 71.5 (PhCH
e
H
O), 71.7 (C
6
), 73.6
(PhCH
2
O), 74.4 (PhCH
g
H
h
O), 75.9 (C
3a
), 76.5 (C
5
), 81.4 (C
7
),
127.7, 127.8, 127.9, 128.2, 128.4, 128.6, 128.8, 137.6, 138.1,
138.4 (Ph), 172.4 (OCO), 173.2 (NHCO), 173.7 (NHCO).
P epa a ion o [(3R,3aS,5R,6R,7R,7aR)-6,7-Dihyd oxy-5-
(hyd oxyme hyl)-2-oxo-3-palmi amido-1,3a,5,6,7,7a-
hexahyd opy ano[3,2-b]py ol-3-yl]me hyl palmi a e 4
A hyd ogenolysis o a me hanol solu ion (5 mL) o he
compound
3
(37 mg, 0.037 mmol) was held unde ambien
p essu e and empe a u e, using Pd-C (35 mg) and HCl conc. (3
d ops) as ca alys . The eac ion was pe o med o 12 h and he
ca alys was il e ed o e dia omaceous ea h. The liquid phase
was concen ed a o ding compound
4
(25 mg, 0,034 mmol,
93%) as a yellow oil.
[𝛼]
𝐷
20
= +20.6 (c 0.99 in me hanol).
HRMS ESI+ (m/z) = 725.5669 [M+H]
+
; calcula ed o
C
41
H
77
N
2
O
8+
= 725.5674.
1
H NMR (400 MHz, CD
3
OD)  =
0.89 ( , 6H, J = 6.5 Hz, CH
3
), 1.24-1.35 (m, 48H, (CH
2
)
12
CH
3
),
1.54-1.67 (m, 4H, CH
2
(CH
2
)
12
CH
3
), 2.23 ( , 2H, J = 6.7 Hz,
NHCOCH
2
), 2.36 ( , 2H, J = 7.3 Hz, OCOCH
2
), 3.70-3.78 (m,
3H, CH
2
OH, H
7
), 3.79-3.85 (m,1H, H
5
), 3.91-4.02 (m, 2H, H
6
,
H
7a
), 4.31 (d, 1H, J = 10.7 Hz, CH
a
H
b
OCO), 4.45-4.52 (m, 1H,
CH
a
H
b
OCO), 4.83 (m, 1H, H
3a
).
13
C NMR (100 MHz, CD
3
OD)
 = 14.4 (CH
3
), 23.7, 25.8, 26.8, 30.2, 30.4, 30.5, 30.6, 30.7,
30.8, 33.0 ((CH
2
)
13
CH
3
), 34.9 (OCOCH
2
), 36.7 (NHCOCH
2
),
56.9 (C
7a
), 61.6 (CH
2
OH), 62.6 (CH
2
OCO), 63.1 (C
3
), 69.4
(C
6
), 73.3 (C
7
), 76.0 (C
3a
), 78.8 (C
5
), 174.7 (OCO), 175.7
(NHCO), 176.0 (NHCO).
P epa a ion o N-[(3R,3aS,5R,6R,7R,7aS)-6,7-Bis(benzyloxy)-5-
(benzyloxyme hyl)-3-hyd oxyme hyl-2-oxo -1,3a,5,6,7,7a-
hexahyd opy ano[3,2-b]py ol-3-yl]palmi amide 5
A me hanol solu ion o sodium me hoxide 0.5
M
(2 mL) was
added o a me hanol solu ion (4 mL) o he compound
3
(76
mg, 0.076 mmol). The mix u e was s i ed o 1 h and sul onic
acid esine Dowex
®
was hen added. The liquid phase was
il e ed, concen a ed and he esidue was pu i ied by silica gel
column ch oma og aphy (hexane/e hyl ace a e, 15:85) o a o d
compound
5
(40 mg, 0.053 mmol, 70%) as a colou less oil.
[𝛼]
𝐷
20
= +66.0 (c 1.0 in CHCl
3
). HRMS ESI+ (m/z) = 757.4788
[M+H]
+
; calcula ed o C
46
H
65
N
2
O
7+
= 757.4786.
1
H NMR (400
MHz, CDCl
3
)  = 0.88 ( , 3H, J = 6.7 Hz, CH
3
), 1.22-1.33 (m,
24H, (CH
2
)
12
CH
3
), 1.55-1.65 (m, 2H, CH
2
(CH
2
)
12
CH
3
), 2.20 ( ,
2H, J = 7.6 Hz, NHCOCH
2
), 3.48 (dd, 1H, J = 10.3, 4.7 Hz,
BnOCH
a
H
b
), 3.56 (d, 1H, J = 8.1 Hz, H
7
) , 3.67-3.75 (m, 2H,
BnOCH
a
H
b
, CH
c
H
d
OH), 3.98 (d, 1H, J = 12.0 Hz, CH
c
H
d
OH),
4.09 (d, 1H, J = 4.9 Hz, H
6
), 4.11-4.18 (m, 1H, H
5
), 4.38 ( , 1H,
J = 7.2 Hz, H
7a
), 4.47 (d, 1H, J =11.9 Hz, PhCH
e
H
O), 4.49 (s,
2H, PhCH
2
O), 4.55 (d, 1H, J = 11.8 Hz, PhCH
g
H
h
O), 4.61 (d,
1H, J = 6.3, H
3a
) 4.68 (d, 1H, J = 11.8 Hz, PhCH
e
H
O), 4.87 (d,
1H, J = 11.8 Hz, PhCH
g
H
h
O), 6.22 (NH), 6.56 (NH), 7.20-7.39
(m, 15H, Ph).
13
C NMR (100 MHz, CDCl
3
)  = 14.1 (CH
3
),
22.7, 25.3, 29.2, 29.4, 29.5, 29.7, 32.0 ((CH
2
)
13
CH
3
), 35.8
(NHCOCH
2
), 56.3 (C
7a
), 63.5 (C
3
), 64.0 (CH
2
OH), 67.4
(BnOCH
2
), 71.5 (C
6
), 71.6 (PhCH
e
H
O), 73.5 (PhCH
2
O), 74.2
(PhCH
g
H
h
O), 76.7 (C
5
, C
3a
), 81.4 (C
7
), 127.6, 127.8, 127.9,
128.2, 128.4, 128.5, 128.7, 137.5, 137.8, 138.0 (Ph), 173.5
(NHCO), 173.9 (NHCO).
P epa a ion o N-[(3R,3aS,5R,6R,7R,7aR)-6,7-Dihyd oxy-3,5-
bis(hyd oxyme hyl)-2-oxo -1,3a,5,6,7,7a-hexahyd opy ano[3,2-
b]py ol-3-yl]palmi amide 6
A
hyd ogenolysis o a me hanol solu ion (5 mL) o he
compound
5
(35 mg, 0.046 mmol) was held unde ambien
p essu e and empe a u e, using Pd-C (35 mg) and HCl conc. (3
d ops) as ca alys . The eac ion was pe o med o 12 h and he
ca alys was il e ed o e dia omaceous ea h. The liquid phase
ARTICLE O ganic & Biomolecula Chemis y
8 | O g. Biomol. Chem., 2015, 00, 1-3 This jou nal is © The Royal Socie y o Chemis y 2015
was concen ed a o ding compound
6
(22 mg, 0,045 mmol,
98%) as a yellow oil.
[𝛼]
𝐷
20
= +15.1 (c 1.0 in me hanol). HRMS
ESI+ (m/z) = 487.3386 [M+H]
+
; calcula ed o C
25
H
47
N
2
O
7+
=
487.3378.
1
H NMR (400 MHz, CD
3
OD)  = 0.83 ( , 3H, J = 5.8
Hz, CH
3
), 1.13-1.36 (m, 24H, (CH
2
)
12
CH
3
), 1.49-1.62 (m, 2H,
CH
2
(CH
2
)
12
CH
3
), 2.38 ( , 2H, J = 6.7 Hz, NHCOCH
2
), 3.65-
4.02 (m, 6H, CH
2
OH, H
5
, H
6
, H
7
, H
7a
), 4.39 (d, 1H, J = 11.3
Hz, C
3
CH
a
H
b
OH), 4.48 (d, 1H, J = 11.7 Hz, C
3
CH
a
H
b
OH),
4.74 (d, 1H, J = 5.3 Hz, H
3a
).
13
C NMR (100 MHz, CD
3
OD) 
= 14.3 (CH
3
), 23.5, 25.5, 30.0, 30.3, 30.4, 30.5, 30.6, 32.9
((CH
2
)
13
CH
3
), 34.4 (NHCOCH
2
), 55.8 (C
7a
), 61.6 (C
3
CH
2
OH),
61.9 (CH
2
OH), 63.0 (C
3
), 69.2 (C
6
), 73.3 (C
7
), 74.6 (C
3a
), 79.0
(C
5
), 174.1 (2 NHCO).
P epa a ion o [(3R,3aS,5R,6R,7R,7aS)-3-Amino-6,7-
bis(benzyloxy)-5-(benzyloxyme hyl)-2-oxo-1,3a,5,6,7,7a-
hexahyd opy ano[3,2-b]py ol-3-yl]me hyl palmi a e 7
Compound
1
(82 mg, 0.127 mmol) was dissol ed in THF (8
mL). A 4
M
solu ion o HCl (2.8 mL) was hen added and he
mix u e was hea ed up o 40 ºC o 12 h unde s i ing. The
c ude ob ained a e concen a ion was dissol ed in TFA (4
mL) and palmi oyl chlo ide was added (385 L, 1.27 mmol).
The mix u e was s i ed a oom empe a u e o 15 min and
absolu e e hanol (10 mL) was added o quench he eac ion.
A e concen a ion, he c ude was dissol ed in absolu e e hanol
(5 mL). P opylene oxide (5 mL) was added and he mix u e was
hea ed o e lux o 2 h unde s i ing. Concen a ion and
pu i ica ion o he c ude p oduc by silica gel column
ch oma og aphy (hexane/e hyl ace a e, 65:35) a o ded
compound
7
(64 mg, 0.085 mmol, 67%) as a colou less oil.
[𝛼]
𝐷
20
= +52.9 (c 1.0 in CHCl
3
). HRMS ESI+ (m/z) = 757.4788
[M+H]
+
; calcula ed o C
46
H
65
N
2
O
7+
= 757.4786.
1
H NMR (400
MHz, CDCl
3
)  = 0.88 ( , 3H, J = 6.7 Hz, CH
3
), 1.20-1.36 (m,
24H, (CH
2
)
12
CH
3
), 1.54-1.64 (m, 2H, CH
2
(CH
2
)
12
CH
3
), 2.27 ( ,
2H, J = 7.8 Hz, OCOCH
2
), 3.55 (d, 1H, J = 5.3 Hz, H
7
), 3.70
(d, 1H, J = 5.2 Hz, BnOCH
2
) , 4.05-4.12 (m, 2H, H
3a
, H
5
), 4.14-
4.20 (m, 2H, H
7a
, H
6
), 4.47 (d, 1H, J = 2.7 Hz, CH
2
OCO), 4.49-
4.61 (m, 4H, PhCH
a
H
b
O, PhCH
c
H
d
O, PhCH
2
O), 4.69 (d, 1H, J
= 11.8 Hz, PhCH
a
H
b
O), 4.81 (d, 1H, J = 11.5 Hz, PhCH
c
H
d
O),
6.57 (NH), 7.24-7.38 (m, 15H, Ph).
13
C NMR (100 MHz,
CDCl
3
)  = 14.2 (CH
3
), 22.8, 26.1, 29.3, 29.5, 29.6, 29.7, 29.8,
32.0 ((CH
2
)
13
CH
3
), 28.2 (NHCOCH
2
), 57.3 (C
7a
), 67.2
(BnOCH
2
), 68.3 (CH
2
OCO), 72.0 (PhCH
a
H
b
O), 73.0 (C
3a
), 73.5
(PhCH
2
O), 74.2 (PhCH
c
H
d
O), 74.6 (C
6
), 76.2 (C
5
), 78.7 (C
3
),
80.1 (C
7
), 127.8, 127.9, 128.2, 128.5, 128.6, 128.7, 137.7,
138.1, 138.2 (Ph), 170.9 (OCO), 175.5 (NHCO).
P epa a ion o [(3R,3aS,5R,6R,7R,7aR)-3-Amino-6,7-
bis(hyd oxy)-5-(hyd oxyme hyl)-2-oxo-1,3a,5,6,7,7a-
hexahyd opy ano[3,2-b]py ol-3-yl]me hyl palmi a e 8
A hyd ogenolysis o a me hanol solu ion (5 mL) o he
compound
7
(50 mg, 0.065 mmol) was held unde ambien
p essu e and empe a u e, using Pd-C (50 mg) and HCl conc. (3
d ops) as ca alys . The eac ion was pe o med o 12 h and he
ca alys was il e ed o e dia omaceous ea h. The liquid phase
was concen ed a o ding compound
8
(30 mg, 0,062 mmol, 94
%) as a yellow oil.
[𝛼]
𝐷
20
= -3.9 (c 1.0 in me hanol). HRMS
ESI+ (m/z) = 487.3388 [M+H]
+
; calcula ed o C
25
H
47
N
2
O
7+
=
487.3378.
1
H NMR (400 MHz, CD
3
OD)  = 0.89 ( , 3H, J = 6.4
Hz, CH
3
), 1.22-1.39 (m, 24H, (CH
2
)
12
CH
3
), 1.58-1.67 (m, 2H,
CH
2
(CH
2
)
12
CH
3
), 2.43 ( , 2H, J = 7.4 Hz, OCOCH
2
), 3.70-3.78
(m, 3H, CH
2
OH, H
7
), 3.79-3.85 (m, 1H, H
5
), 3.93 ( , 1H, J =
7.6 Hz, H
7a
), 3.99 (s, 1H, H
6
), 4.44 (d, 1H, J = 12.1 Hz,
CH
a
H
b
OCO), 4.53 (d, 1H, J = 11.9 Hz, CH
a
H
b
OCO), 4.77 (d,
1H, J = 7.2 Hz, H
3a
).
13
C NMR (100 MHz, CD
3
OD)  = 14.4
(CH
3
), 23.7, 25.7, 30.1, 30.4, 30.6, 30.7, 30.8, 33.0
((CH
2
)
13
CH
3
), 34.5 (OCOCH
2
), 55.9 (C
7a
), 61.7 (CH
2
OCO),
62.0 (CH
2
OH), 63.1 (C
3
), 69.3 (C
6
), 73.5 (C
7
), 74.8 (C
3a
), 79.2
(C
5
), 170.3 (OCO), 174.1 (NHCO).
P epa a ion o [(3R,3aS,5R,6R,7R,7aS)-3-Ace amido-6,7-
bis(benzyloxy)-5-(benzyloxyme hyl)-2-oxo-1,3a,5,6,7,7a-
hexahyd opy ano[3,2-b]py ol-3-yl]me hyl palmi a e 10
Compound
9
(60 mg, 0.107 mmol) was dissol ed in
dichlo ome hane (2 mL) and palmi oyl chlo ide (70 L, 0.23
mmol) was added. E
3
N (44 L, 0.32 mmol) was hen added
and he mix u e was s i ed a oom empe a u e o 12 h. A 0.5
M
solu ion o HCl (2 mL) was added. The o ganic laye was
sepa a ed and he aqueous one was washed wi h
dichlo ome hane (3 × 2 mL). The o ganic phases we e collec ed
and d ied wi h anhyd ous Na
2
SO
4
. Concen a ion and
pu i ica ion o he c ude p oduc by silica gel column
ch oma og aphy (hexane/e hyl ace a e, 35:65) a o ded
compound
10
(63 mg, 0.079 mmol, 74%) as a colou less oil.
[𝛼]
𝐷
20
= +41.0 (c 1.0 in CHCl
3
). HRMS ESI+ (m/z) = 799.4853
[M+H]
+
; calcula ed o C
48
H
67
N
2
O
8+
= 799.4892.
1
H NMR (400
MHz, CDCl
3
)  = 0.88 ( , 3H, J = 6.7 Hz, CH
3
), 1.21-1.30 (m,
24H, (CH
2
)
12
CH
3
), 1.51-1.58 (m, 2H, CH
2
(CH
2
)
12
CH
3
), 1.98 (s,
3H, NHAc), 2.23 ( , 2H, J = 7.5 Hz, OCOCH
2
), 3.53-3.59 (m,
2H, BnOCH
a
H
b
, H
7
), 3.66 (dd, 1H, J = 9.5, 6.9 Hz,
BnOCH
a
H
b
), 4.07 (dd, 1H, J = 11.1, 6.3 Hz, H
5
), 4.13 (d, 1H, J
= 4.2 Hz, H
6
), 4.27-4.34 (m, 2H, H
7a
, CH
c
H
d
OCO), 4.42-4.52
(m, 4H, CH
c
H
d
OCO, PhCH
2
O, PhCH
e
H
O), 4.58 (d, 1H, J =
11.6, PhCH
g
H
h
O), 4.66-4.72 (m, 2H, PhCH
e
H
O, H
3a
), 4.87 (d,
1H, J = 11.6 Hz, PhCH
g
H
h
O), 6.07-6.18 (m, 1H, NHAc), 6.38
(s, 1H, NHCO), 7.22-7.40 (m, 15H, Ph).
13
C NMR (100 MHz,
CDCl
3
)  = 14.1 (CH
3
), 23.1 (NHCOCH
3
), 22.7, 24.8, 29.2,
29.3, 29.4, 29.5, 29.7, 31.9, 34.1 (CO(CH
2
)
14
CH
3
), 55.9 (C
7a
),
63.0 (CH
2
OCO), 63.2 (C
3
) 67.7 (BnOCH
2
), 71.4 (PhCH
e
H
O),
71.6 (C
6
), 73.5 (PhCH
2
O), 74.3 (PhCH
g
H
h
O), 75.7 (C
3a
), 76.4
(C
5
), 81.2 (C
7
), 127.7, 127.8, 127.9, 128.2, 128.3, 128.5, 128.7,
137.5, 138.0, 138.2 (Ph), 170.0 (OCO), 172.3 (NHCOCH
3
),
173.6 (NHCO).
P epa a ion o [(3R,3aS,5R,6R,7R,7aR)-3-Ace amido-6,7-
bis(hyd oxy)-5-(hyd oxyme hyl)-2-oxo-1,3a,5,6,7,7a-
hexahyd opy ano[3,2-b]py ol-3-yl]me hyl palmi a e 11
A hyd ogenolysis o a me hanol solu ion (5 mL) o he
compound
10
(63 mg, 0.079 mmol) is held unde ambien
p essu e and empe a u e, using Pd-C (60 mg) and HCl conc. (3
d ops) as ca alys . The eac ion is pe o med o 12 h and he
ca alys is il e ed o e dia omaceous ea h. The liquid phase is
concen ed a o ding he compound
11
(35 mg, 0,066 mmol, 84
%) as a yellow oil.
[𝛼]
𝐷
20
= +22.7 (c 1.0 in H
2
O). HRMS ESI+
(m/z) = 529.3493 [M+H]
+
; calcula ed o C
27
H
49
N
2
O
8+
=
529.3483.
1
H NMR (400 MHz, CD
3
OD)  = 0.88 ( , 3H, J = 6.4
Hz, CH
3
), 1.19-1.37 (m, 24H, (CH
2
)
12
CH
3
), 1.53-1.66 (m, 2H,
CH
2
(CH
2
)
12
CH
3
), 2.26-2.39 (m, 2H, OCOCH
2
), 3.68-4.03 (m,
6H, CH
2
OH, H
5
, H
6
, H
7
, H
7a
), 4.25-4.35 (m, 1H, CH
a
H
b
OCO),
4.46-4.54 (m, 1H, CH
a
H
b
OCO), 4.83 (m, 1H, H
3a
).
13
C NMR
(100 MHz, CD
3
OD)  = 14.3 (CH
3
), 23.6, 25.8, 25.9, 30.1,
30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 32.9 ((CH
2
)
13
CH
3
), 34.7
(OCOCH
2
), 56.9 (C
7a
), 61.6 (CH
2
OH), 62.2 (CH
2
OCO), 64.9
Jou nal Name ARTICLE
This jou nal is © The Royal Socie y o Chemis y 2012 O g. Biomol. Chem., 2015, 00, 1-3 | 9
(C
3
), 69.3 (C
6
), 73.1 (C
7
), 75.9 (C
3a
), 78.7 (C
5
), 171.4 (OCO),
174.6 (NHCO), 176.0 (NHCO).
P epa a ion o [(3R,3aS,5R,6R,7R,7aS)-3-Amino-6,7-
bis(benzyloxy)-5-(benzyloxyme hyl)-2-oxo-1,3a,5,6,7,7a-
hexahyd opy ano[3,2-b]py ol-3-yl]me hyl ace a e 12
Compound
1
(73 mg, 0.113 mmol) was dissol ed in THF (7
mL). A 4
M
solu ion o HCl (2.5 mL) was hen added and he
mix u e was hea ed up o 40 ºC o 12 h unde s i ing. The
c ude ob ained a e concen a ion was dissol ed in TFA (4
mL) and ace yl chlo ide was added (80 L, 1.13 mmol). The
mix u e was s i ed a oom empe a u e o 15 min and
absolu e e hanol (10 mL) was added o quench he eac ion.
A e concen a ion, he c ude was dissol ed in absolu e e hanol
(5 mL). P opylene oxide (5 mL) was added and he mix u e was
hea ed o e lux o 2 h unde s i ing. Concen a ion and
pu i ica ion o he c ude p oduc by silica gel column
ch oma og aphy using e hyl ace a e as eluen a o ded
compound
12
(27 mg, 0.048 mmol, 42%) as a colou less oil.
[𝛼]
𝐷
20
= +76.7 (c 1.0 in CHCl
3
). HRMS ESI+ (m/z) = 561.2590
[M+H]
+
; calcula ed o C
32
H
37
N
2
O
7+
= 561.2595.
1
H NMR (400
MHz, CDCl
3
)  = 1.99 (s, 3H, CH
3
), 3.54 (s, 1H, H
7
), 3.67-3.73
(m, 2H, BnOCH
2
), 4.06-4.12 (m, 2H, H
3a
, H
5
), 4.15-4.20 (m,
2H, H
6
, H
7a
), 4.41-4.49 (m, 2H, CH
2
OAc), 4.49-4.61 (m, 4H,
PhCH
a
H
b
O, PhCH
c
H
d
O, PhCH
2
O), 4.70 (d, 1H, J = 11.8 Hz,
PhCH
a
H
b
O), 4.82 (d, 1H, J = 11.4 Hz, PhCH
c
H
d
O), 6.51 (s, 1H,
NH), 7.24-7.39 (m, 15H, Ph).
13
C NMR (100 MHz, CDCl
3
)  =
14.0 (CH
3
), 57.4 (C
7a
), 67.2 (BnOCH
2
), 68.6 (CH
2
OAc), 71.9
(PhCH
a
H
b
O), 72.9 (C
3a
), 73.5 (PhCH
2
O), 74.3 (PhCH
c
H
d
O),
74.6 (C
6
), 76.2 (C
5
), 78.8 (C
3
), 80.2 (C
7
), 127.8, 127.9, 128.3,
128.5, 128.6, 128.8, 137.7, 138.1, 138.2 (Ph), 167.8 (COCH
3
),
175.5 (NHCO).
P epa a ion o [(3R,3aS,5R,6R,7R,7aR)-3-Amino-6,7-
bis(hyd oxy)-5-(hyd oxyme hyl)-2-oxo-1,3a,5,6,7,7a-
hexahyd opy ano[3,2-b]py ol-3-yl]me hyl ace a e 13
A hyd ogenolysis o a me hanol solu ion (5 mL) o he
compound
12
(27 mg, 0.048 mmol) was held unde ambien
p essu e and empe a u e, using Pd-C (25 mg) and HCl conc. (3
d ops) as ca alys . The eac ion was pe o med o 12 h, he
ca alys was hen il e ed o e dia omaceous ea h and he
liquid phase is concen ed. The esidue was dissol ed in H
2
O (5
mL) and ex ac ed wi h e hyl ace a e (2 × 5 mL). The combined
aqueous phases we e concen a ed and he esidue was
dissol ed in H
2
O (2 mL) and elu ed h ough a e e se-phase
Sep-pak C18 ca idge a o ding, a e e apo a ion o wa e ,
compound
13
(13 mg, 0,044 mmol, 94%) as a yellow oil.
[𝛼]
𝐷
20
= +42.3 (c 1.0 in H
2
O). HRMS ESI+ (m/z) = 291.1188 [M+H]
+
;
calcula ed o C
11
H
19
N
2
O
7+
= 291.1187.
1
H NMR (400 MHz,
D
2
O)  = 2.17 (s, 3H, CH
3
), 3.69-3.84 (m, 2H, CH
2
OH), 3.84-
4.08 (m, 4H, H
5
, H
6
, H
7
, H
7a
), 4.51 (d, 1H, J = 12.3 Hz,
CH
a
H
b
OAc), 4.66 (d, 1H, J = 12.3 Hz, CH
a
H
b
OAc), 4.96 (d,
1H, J = 8.1 Hz, H
3a
).
13
C NMR (100 MHz, D
2
O)  = 20.1
(CH
3
), 53.1 (C
7a
), 61.1 (CH
2
OAc), 61.4 (CH
2
OH), 61.8 (C
3
),
67.4 (C
6
), 71.8 (C
7
), 74.3 (C
3a
), 77.4 (C
5
), 169.7 (COAc), 172.6
(NHCO).
P epa a ion o (3R,3aS,5R,6R,7R,7aR)-3-Amino-6,7-dihyd oxy-
3,5-bis(hyd oxyme hyl)- 1,3a,5,6,7,7a-hexahyd opy ano[3,2-
b]py ol-2-one 14
A hyd ogenolysis o a me hanol solu ion (5 mL) o he
compound
2
(35 mg, 0.067 mmol) was held unde ambien
p essu e and empe a u e, using Pd-C (35 mg) and HCl conc. (3
d ops) as ca alys . The eac ion was pe o med o 12 h, he
ca alys was hen il e ed o e dia omaceous ea h and he
liquid phase was concen ed. The esidue was dissol ed in H
2
O
(5 mL) and ex ac ed wi h e hyl ace a e (2 × 5 mL). The
combined aqueous phases we e concen a ed a o ding
compound
14
(16 mg, 0,064 mmol, 96 %) as a yellow oil.
[𝛼]
𝐷
20
= +32.6 (c 1.0 in H
2
O). HRMS ESI+ (m/z) = 249.1087 [M+H]
+
;
calcula ed o C
9
H
17
N
2
O
6+
= 249.1081.
1
H NMR (400 MHz,
D
2
O)  = 3.71-3.84 (m, 2H, CH
2
OH), 3.90-4.16 (m, 6H, H
5
, H
6
,
H
7
, H
7a
, C
3
CH
2
OH), 4.96 (d, 1H, J = 5.6 Hz, H
3a
).
13
C NMR
(100 MHz, D
2
O)  = 53.0 (C
7a
), 59.0 (C
3
CH
2
OH), 61.4
(CH
2
OH), 63.6 (C
3
), 67.4 (C
6
), 71.8 (C
7
), 73.8 (C
3a
), 77.3 (C
5
),
170.7 (NHCO).
Inhibi ion S udies wi h Comme cial Enzymes
. Inhibi ion
cons an (K
i
) alues we e de e mined by spec opho ome ically
measu ing he esidual hyd oly ic ac i i ies o he glycosidases
agains he espec i e p-ni ophenyl α- o β-
D
-glycopy anoside,
o o-ni ophenyl β-
D
-galac opy anoside ( o β-galac osidases),
in he p esence o compounds
4
,
6
,
8
,
11
,
13
,
14
and
15
. Each
essay was pe o med in phospha e bu e a he op imal pH o
each enzyme. The eac ions we e ini ia ed by addi ion o
enzyme o a solu ion o he subs a e in he absence o p esence
o a ious concen a ions o inhibi o . The mix u e was
incuba ed o 10-30 min a 37 °C, and he eac ion was
quenched by addi ion o 1
M
Na
2
CO
3
. Reac ion imes we e
app opia e o ob ain 10
−
20% con e sion o he subs a e in
o de o achie e linea a es. The abso bance o he esul ing
mix u e was de e mined a 405. App oxima e alues o K
i
we e
de e mined using a ixed concen a ion o subs a e (a ound he
K
m
alue o he di e en glycosidases) and a ious
concen a ions o inhibi o . Full K
i
de e mina ions and enzyme
inhibi ion mode we e de e mined om he slope o Linewea e
−
Bu k plo s and double ecip ocal analysis.
Lysosomal enzyme ac i i y assay
. Lysosomal enzyme
ac i i ies in cell lysa es we e de e mined as desc ibed
p e iously.
26
B ie ly, cells we e sc aped in ice-cold 0.1% T i on
X-100 in wa e . A e cen i uga ion (6,000 pm o 15 min a 4
ºC) o emo e insoluble ma e ials, p o ein concen a ions we e
de e mined using P o ein Assay Rapid Ki (Wako, Tokyo,
Japan). The lysa es we e incuba ed a 37 ºC wi h he
co esponding 4-me hylumbelli e yl -D-glycopy anoside
solu ion in 0.1 M ci a e bu e (pH 4). The lib a ed 4-
me hylumbelli e one was measu ed wi h a luo escence pla e
eade (exi a ion 340 nm; emission 460 nm; In ini e F500,
TECAN Japan, Kawasaki, Japan). Fo enzyme inhibi ion assay,
cell lysa es om no mal skin ib oblas s we e mixed wi h he 4-
me hylumbelli e yl -D-glycopy anoside subs a es in he
absence o p esence o inc easing concen a ions o he es ed
de i a i es.
Cell cul u e and GCase ac i i y enhancemen assay.
Human
skin ib oblas s om a heal hy and h ee Gauche disease
pa ien s (wi h N370S/ N370S, F213I/ F213I and L444P/L444P
mu a ions) we e main ained in ou labo a o y wi h DMEM
supplemen ed wi h 10% FBS as he cul u e medium. Fo
enzyme ac i i y enhancemen assay, cells we e cul u ed in he
p esence o di e en concen a ions o he candida es o DMSO
alone (as a con ol) o 5 days and ha es ed by sc aping.
26
Cy o oxici y o he compounds was moni o ed by measu ing
he lac a e dehyd ogenase ac i i ies in he cul u ed supe na an s
(LDH assay ki , Wako, Tokyo, Japan).