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Volume 11 | Numbe 16 | 21 Augus 2024
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ORGANIC CHEMISTRY
FRONTIERS
RESEARCH ARTICLE
Ci e his: O g. Chem. F on ., 2024,
11, 4356
Recei ed 17 h May 2024,
Accep ed 22nd June 2024
DOI: 10.1039/d4qo00886c
sc.li/ on ie s-o ganic
Chemoselec i e eac ion o me hoxyaminome hyl
BODIPYs wi h unp o ec ed ca bohyd a es:
a powe ul ool o accessing BODIPY
neoglycosides†
Ana M. Gómez, *
a
Luis Ga cía-Fe nández,
b,c
And és G. San ana,
a
Cla a U iel,
a
Lei e Ga zia-Ri e o,
d
Jo ge Bañuelos, *
d
Inmaculada Ga cia-Mo eno,
e
Lou des In an es,
Ma ía Rosa Aguila
b,c
and
J. C is obal Lopez *
a
The neoglycosyla ion o me hoxyaminome hyl-appended BODIPYs wi h unp o ec ed educing mono-,
di-, and isaccha ides p oduces, in a egio- and s e eoselec i e manne , cyclic N-glycosyl-N-me hoxy–
BODIPY conjuga es, as a ele an class o neoglycosides ha display excellen pho ophysical cha ac e -
is ics in pu e wa e , e en a high dye concen a ions. In addi ion, he cellula up ake o some o he neo-
glycosyla ed BODIPYs has been confi med ia fluo escence mic oscopy, and a BODIPY–aca bose conju-
ga e showed compa able enzyma ic inhibi o y ac i i y o aca bose o wo diffe en α-amylases: A. o yzae
α-amylase (AOA) and human sali a y α-amylase (HSA).
In oduc ion
Ca bohyd a es, ubiqui ous in Na u e, play signi ican oles in
many biological p ocesses anging om in ec ion ( i al and
bac e ial), cell ecogni ion, igge ing o immune esponses,
and cance me as asis.
1
In ecen yea s, i has also become
clea ha ca bohyd a e–p o ein in e ac ions in ol ing cell
su ace p o eins,
2
o cell su ace ca bohyd a es,
3
a e key o
heal h and disease mechanisms.
4,5
The in es iga ion o hese
p ocesses, which alls unde he umb ella o Glycobiology
6
is,
he e o e, a ield a ac ing inc easing in e es . In his con ex ,
luo escence imaging echniques ha e become powe ul ools
o he isualiza ion o biomolecules, and he assessmen o
hese phenomena. Such s udies o en equi e he de i a iza-
ion o glycans by labeling wi h luo opho es
7
o by a achmen
o su aces.
8
In his con ex , di luo obo on dipy ome hene
(4,4-di luo o-4-bo a-3a,4a-diaza-s-indacene) o BODIPY dyes,
e.g.,1(Fig. 1A),
9
ha e a guably become one o he mos
popula luo opho es o saccha ide agging.
7b,10
BODIPYs
display ela i ely high pho os abili y, neu al o al cha ge,
sha p abso p ion and emission spec a, no able chemical
obus ness, and high luo escence quan um yield (Φ
F
).
9
Mo e
in e es ingly, all o he abo e p ope ies can be modula ed by
sub le pos unc ional modi ica ions o he dipy ome hene
co e.
11,12
Thus, inco po a ing di e se unc ional g oups o he
BODIPY co e can ine- une he abso p ion and emission wa e-
leng hs o hese dyes. This unabili y enables he design o
BODIPY de i a i es wi h ailo ed luo escence p ope ies,
making hem sui able o a ious applica ions.
13
BODIPY
de i a i es possess many ideal pho osensi ize (PS) ea u es,
which makes hem use ul agen s in pho odynamic he apy
(PDT),
14
and, mo e ecen ly, in pho o he mal he apy (PTT).
15
They ha e also been used in op oelec onic de ices, including
o ganic ligh -emi ing diodes (OLEDs)
16
and o ganic pho o ol-
aics (OPVs).
17
De i a i es o BODIPY a e equen ly used in
biological imaging.
18
Thei luo escence makes hem excellen
o obse ing cellula s uc u es, biomolecule dis ibu ion
wi hin cells, and dynamics.
19
In his ega d, because o hei wide ange o uses, adjus a-
ble luo escence cha ac e is ics, and pho os abili y, lumines-
cen BODIPY–suga p obes ha e gained he a en ion o
†Elec onic supplemen a y in o ma ion (ESI) a ailable. CCDC 2351271. Fo ESI
and c ys allog aphic da a in CIF o o he elec onic o ma see DOI: h ps://doi.
o g/10.1039/d4qo00886c
a
Ins i u o de Química O gánica Gene al (IQOG-CSIC), Juan de la Cie a 3,
28006 Mad id, Spain. E-mail: ana.[email p o ec ed], [email p o ec ed]
b
Ins i u o de Ciencia y Tecnología de Políme os (ICTP-CSIC), Juan de la Cie a 3,
28006 Mad id, Spain
c
Cen o de In es igación Biomédica en Red de Bioingenie ía, Bioma e iales y
Nanomedicina (CIBER-BBN), Ins i u o de Salud Ca los III, Mon o e de Lemos 3-5,
28029 Mad id, Spain
d
Depa amen o de Química Física, Uni e sidad del País Vasco (UPV/EHU),
Apa ado 644, 48080 Bilbao, Spain. E-mail: jo [email protected]
e
Depa amen o de Química-Física de Ma e iales, Ins i u o de Química-Física
“Blas Cab e a”(IQF-CSIC), Se ano 119, 28006 Mad id, Spain
Depa amen o de C is alog a ía y Biología Es uc u al, Ins i u o de Química-Física
“Blas Cab e a”(IQF-CSIC), Se ano 119, 28006 Mad id, Spain
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esea che s o he po en ial applica ions o such molecula
sys ems in bio-imaging.
20
The ca bohyd a e componen o
BODIPY–ca bohyd a e conjuga es plays an impo an ole
since i con e s ema kable p ope ies o he BODIPY glycop-
obes. Thus, he suga componen p o ides enhanced solubi-
li y in pola sol en s (including wa e ),
21
biocompa ibili y,
22
a ge ing abili y,
23
cell endocy osis,
24
and, in some ins ances,
educed oxici y
25
o he glyco- luo opho es.
7,8
In a b oad sense, BODIPY–ca bohyd a e conjuga es can be
di ided in o linke - ee o e he ed de i a i es. Classically, syn-
he ic ou es o he o me class ha e been less
exploi ed,
10,21,26
and a en ion has been mainly de o ed o he
syn hesis o BODIPY–ca bohyd a e conjuga es connec ed
h ough a “linke ”. Among he la e , syn he ic app oaches o
e he ed BODIPY–ca bohyd a e hyb ids, e.g.,2(Fig. 1A), o en
ely on he use o click eac ions, in pa icula he coppe (I)-
ca alyzed azide–alkyne cycloaddi ion (CuAAC)
27
on azido- o
alkynyl BODIPYs.
28,29
On he con a y, me hods based on
ca bohyd a e ans o ma ions, o ins ance, he glycosyla ion
o hyd oxyl-appended BODIPYs, e.g.,4, wi h glycosyl dono s
3a, leading o glycosyl BODIPYs 5(Fig. 1B),
30
ha e been sca -
cely employed.
31
Seeking a powe ul me hod o co alen ly link BODIPYs and
ca bohyd a es, we we e mind ul o he seminal con ibu ion by
Pe i, Dumy and Mu e ,
32
which epo ed he egio- chemo-
and s e eoselec i e o ma ion o glycosidic bonds be ween
unp o ec ed educing suga s, e.g. 3b (Fig. 1C), and seconda y
me hoxyamine-con aining aglycons. This ans o ma ion p o-
ides access o cyclic N-glycosyl-N,O-dialkyl neoglycosides in
he he modynamically mos s able con igu a ions.
33
These
de i a i es display con o ma ional beha io simila o na u al
O-glycosides.
34
This liga ion me hod, u he alida ed by
Langenhan and Tho son,
35
has p o en use ul in he p epa-
a ion o bioac i e p obes and ea ly-s age leads in d ug
disco e y.
36
Along hese lines, we epo in his manusc ip he p epa-
a ion o me hoxyaminome hyl BODIPY dyes, i.e.,6, and hei
eac ion wi h unp o ec ed educing suga de i a i es e.g.,3b,
o affo d BODIPY-neoglycosides, i.e.,7(Fig. 1C). Speci ically,
he me hod is applied o diffe en me hoxyaminome hyl
BODIPY compounds as well as o saccha ides o diffe en
chain leng hs. The ensuing BODIPY conjuga es displayed
excellen pho ophysical p ope ies in wa e and a high dye
concen a ions. Fu he mo e, some o hese conjuga es we e
submi ed o biological s udies including cellula up ake,
in acellula localiza ion, and cy o oxici y in heal hy and
umo al cells. One o he suga de i a i es employed in hese
s udies has been aca bose. Aca bose, an α-amylase and
α-glucosidase inhibi o , exe s a well-de ined gluco egula o y
effec . Cance cells a e known o exhibi a heigh ened depen-
dence on glucose o ATP p oduc ion compa ed o hei non-
malignan coun e pa s.
37
Consequen ly, a ge ing his me a-
bolic pa hway by es ic ing glucose a ailabili y ep esen s a
well-es ablished s a egy in cance he apy.
38–40
Finally, he
aca bose–BODIPY conjuga e was also e alua ed as a ch omo-
genic inhibi o o α-amylases.
Resul s and discussion
To e alua e he easibili y, scope and limi a ions o his
app oach, we ini ia ed ou s udies wi h he p epa a ion o
me hoxyaminome hyl BODIPYs 6a–c(Fig. 2). These luo escen
dyes a e de i ed om 8-a yl, 1,3,5,7 e ame hyl BODIPYs. In
hese compounds he me hyl g oups a C-1 and C-7 o ce he
8-a yl g oup o adop an o hogonal o ien a ion ela i e o he
BODIPY co e.
31a
This a angemen p e en s unwan ed agg ega-
ion and es ic s he o a ion o he a yl subs i uen , he eby
p ese ing he ch omopho e’s emission p ope ies. In
addi ion, he p esence o he a yl and me hyl g oups in he
BODIPY amewo k has a bene icial effec on i s pho os abili y
and chemical obus ness.
31b
Ou syn he ic s a egy began by eac ing p e iously
desc ibed o myl BODIPYs 8a,
41
8b,
42
and 8c,
31a
wi h me hox-
yamine hyd ochlo ide sal using py idine as a media o
(Fig. 2). This p oduced he co esponding BODIPY-O-me hyl
oximes 9a–cin good yields (81%, 84%, and 74%, espec -
i ely).
43
Compounds 9a and 9b we e ob ained as single s e eoi-
some s while 9c exhibi ed a 6 : 1 mix u e o isome s a he
oxime double bond, as de e mined by
1
H-NMR spec oscopy.
Fig. 1 (A) BODIPY (1) and e he ed BODIPY–ca bohyd a e conjuga es
(2). (B) Glycosyla ion o hyd oxyl-con aining BODIPYs (4) wi h glycosyl
dono s 3a, leading o BODIPY glycosides 5. (C) Reac ion o me hoxyami-
nome hyl BODIPYs 6, wi h unp o ec ed educing suga s 3b, leading o
BODIPY-neoglycosides 7.
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Reduc ion o he O-me hyl oximes 9a–cwi h NaCNBH
3
in
glacial ace ic acid hen ga e he expec ed me hoxyaminome hyl
BODIPYs 6a,6b and 6c in ai o good yields (74%, 73% and
38%, espec i ely, Fig. 2). In he la e case, he zwi e ionic
N-cyanobo ona ed-N-alkoxyamine de i a i e 6d was also iso-
la ed (32% yield) as a c ys alline de i a i e (Fig. 2).
44
This bo o-
na ed species was cha ac e ized by NMR spec oscopy, mass
spec ome y, and single c ys al X- ay c ys allog aphy. The
1
H-NMR spec um o compound 6d showed benzylic p o ons
ha appea ed as dias e eo opic signals, due o he chi al ni o-
gen a om a he benzylic posi ion. X-Ray diff ac ion con i med
ha compound 6d c ys allized as a acemic compound
(Fig. S84 in ESI†).
Nex , we es ed he compa ed eac i i y o me hoxyamino-
me hyl BODIPY de i a i es 6a–din he neoglycosyla ion eac-
ion using D-glucose as he glycosyl dono .
45
Thus, ea men
o me hoxyaminome hyl BODIPYs 6a–c, unde he eac ion
condi ions ini ially ecommended by Pe i e al. (DMF/AcOH, .
.),
32
esul ed in he syn hesis o glucosyl de i a i es 10a,10b,
and 10c in 37%, 25% and 40% yields, espec i ely (Fig. 3).
Simila ly, neoglycosyla ion o N-cyanobo ona ed-N-me hoxya-
mine 6d, wi h D-glucose yielded de i a i e 10c in a sligh ly
lowe yield (25%, Fig. 3).
In ag eemen wi h li e a u e p eceden s,
32
he glycosyla ion
o N,O-disubs i u ed seconda y hyd oxylamino BODIPYs (6)
wi h D-glucose ook place in a comple ely egio- and s e eocon-
olled manne , leading o he co esponding β-D-glucopy ano-
syl de i a i es (10a–c), wi h he expec ed 1,2- ans s e eo-
selec i i y on he ca bohyd a e moie y (Fig. 3).
The β-con igu a ion a he BODIPY-a ached anome ic
ca bon was igo ously es ablished o compounds 10a–c,on
he basis o hei obse ed J
1′,2′
coupling cons an s in hei
1
H-NMR spec a. In he case o compound 10b, wi h no o e -
lapping wi h o he p o on signals, he obse ed diagnos ic
coupling cons an (4.16 ppm, J
1′,2′
= 9.0 Hz) could be de e -
mined om i s
1
H-NMR spec um. On he con a y, he s e eo-
chemical assignmen o he C-1′con igu a ion in gluco-
BODIPYs 10a and 10c had o be ca ied ou in hei co es-
ponding pe -O-ace yl de i a i es, 10a-OAc and 10c-OAc (see
ESI† o de ails), whe e he imp o ed spli ing o he p o on
signals allowed he unequi ocal assignmen o hei anome ic
p o ons.
The s e eoselec ion o he p ocess has been asc ibed o a
he modynamic equilib ium be ween he open iminium in e -
media e, i.e.,11, and he closed ing isome , i.e.,10, in i s
mos s able o m (Fig. 3).
32,36
Ha ing es ablished ha all h ee me hoxyamino BODIPYs
(6a–c) could be used as aglycons in he neoglycosyla ion eac-
ion, we hen se up o op imize he eac ion condi ions o gly-
cosyl coupling using BODIPY 6a and D-glucose as pa ne s.
Compound 6a was selec ed owing o he easiness o i s p epa-
a ion and i s obse ed imp o ed eac i i y owa d D-glucose,
unde he assump ion ha he esul s ob ained wi h his com-
pound could be ex ended o isome ic BODIPYs 6b and 6c.
Acco dingly, we examined a a ie y o eac ion condi ions
o he ans o ma ion 6a →10a (Table 1).
36
In gene al, he
diffe en me hods e alua ed in ol ed changes in he sol en
sys em, empe a u e (T), and ca alys . Thus, he use o DMF/
AcOH (1 : 1) sol en mix u es led o modes yields o 10a,
which could be sligh ly imp o ed by inc easing he eac ion
empe a u e (compa e en ies iand ii, . . s. 40 °C, Table 1).
The use o MeOH/CH
2
Cl
2
(6 : 1) as a sol en sys em, in he
absence o acid, p oduced a modes 25% yield o 10a (Table 1,
en y iii). The use o a AcONa/AcOH buffe , as he eac ion
media, did no esul in he o ma ion o 10a (Table 1, en y
Fig. 2 Me hoxyaminome hyl BODIPY de i a i es 6a–d, ob ained om
o myl-BODIPYs 8a–c, ia BODIPY oximes 9a–c.
Fig. 3 S e eoselec i e syn hesis o BODIPY neoglucosides 10a–c, om
he eac ion o me hoxyaminome hyl BODIPYs 6a–dwi h D-glucose
(DMF/AcOH, . .).
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i ). Be e esul s we e ob ained when he neoglycosyla ion
eac ion was ca ied ou in MeOH/AcOH (1 : 1) sol en mix-
u es. Thus hea ing (60 °C) o he eac ion o 48 h p oduced a
40% yield o 10a (Table 1, en y ). In e es ingly, he use mic o-
wa e i adia ion (MW) allowed he eac ion ime o be educed
o 1 h, and he yield inc eased o 73% (Table 1, en y i).
Finally, bes yields we e ob ained unde mic owa e i adia ion
(MW, 60 °C, 1 h, 87% yield) in he p esence o 5-me hoxyan-
h anilic acid as nucleophilic ca alys , as sugges ed by
Langenhan and cowo ke s (Table 1, en y ii).
46
Likewise, he applica ion o he op imized eac ion con-
di ions (MeOH/AcOH, 5-me hoxyan h anilic acid ca ., MW,
60 °C, 1 h) o me hoxyamino BODIPYs 6b and 6c, allowed he
p epa a ion o glucoconjuga es 10b and 10c in 76% and 84%
yields, espec i ely (compa e wi h yields displayed in Fig. 3).
To e alua e he scope o he me hod ega ding he suga
dono , we s udied he eac ion o BODIPY 6a wi h a a ie y o
comme cially a ailable unp o ec ed educing suga s, includ-
ing: (i) cellobiose, lac ose, and mal ose disaccha ides, which
diffe in he con igu a ion o hei in e glycosidic bond (β-
e sus α-, e.g., cellobiose and lac ose e sus mal ose); (ii) mal o-
iose as an example o a isaccha ide, and (iii) aca bose as a
pseudo e asaccha ide. In each neoglycosyla ion eac ion, he
expec ed BODIPY-saccha ides 12,13,14,15, and 16, espec -
i ely, wi h β-anome ic con igu a ion a he linking posi ion,
could be isola ed (Fig. 4). The β-con igu a ion a he BODIPY-
a ached anome ic ca bon in compounds 15 and 16 was igo -
ously es ablished om hei co esponding pe -O-ace yl de i a-
i es, 15-OAc and 16-OAc (see ESI† o de ails) on he basis o
hei obse ed J
1′,2′
coupling cons an s in hei
1
H-NMR
spec a. On he o he hand, he β-con igu a ion a he BODIPY-
a ached anome ic ca bon in compounds 12–14 was pos ula ed
in acco dance wi h he li e a u e p eceden s and he simila i y
wi h he ela ed BODIPY glycosides p epa ed in his s udy,
since only one isome was isola ed in each case.
Pho ophysical s udies
The pho ophysical ea u es o he no el BODIPY glycoconju-
ga es we e nex s udied. The a achmen o p og essi ely
complex ca bohyd a e uni s a C-2 o he BODIPY co e, acili-
a ed by he me hoxyaminome hyl space , enhanced he hyd o-
philici y o he dye signi ican ly, ul ima ely ende ing i eady
soluble in wa e . Indeed, he in insic pho ophysical p ope ies
o ep esen a i e BODIPY glycoconjuga es 10a and 14–16 in
dilu ed wa e solu ions (Table 2) closely esembled hose o
hei hyd ophobic 8-phenyl BODIPY p ecu so s (which lead o
o myl dyes 8).
32
Rega dless o he numbe o ca bohyd a e
Table 1 Op imiza ion o eac ion condi ions
a
o he p epa a ion o neoglucoside 10a by eac ion o 6a wi h D-glucose
En y Sol en (s) T(°C)/ Ca alys Yield
b
(%)
iDMF/AcOH (1 : 1) . ./20 h —37
ii DMF/AcOH (1 : 1) 40 °C/20 h —40
iii MeOH/CH
2
Cl
2
(6 : 1) 60 °C/48 h —25
i AcONa/AcOH pH = 5 . ./24 h ——
MeOH/AcOH (1 : 1) 60 °C/48 h —40
i MeOH/AcOH (1 : 1) 60 °C/1 h (MW) —73
ii MeOH/AcOH (1 : 1) 60 °C/1 h (MW) 5-Me hoxy-an h anilic acid 87
a
Reac ion condi ions: 6a (1.0 mmol), D-glucose (3.0 mmol).
b
Isola ed yields.
Fig. 4 Sc eening o ca bohyd a e subs a es: s e eoselec i e syn hesis
o BODIPY saccha ides 12–16 by eac ion o unp o ec ed educing
suga s wi h BODIPY 6a.
Table 2 Pho ophysical p ope ies o ep esen a i e BODIPY neoglyco-
sides in wa e (dye concen a ion 2 μM)
Dye λ
aba
(nm) ε
maxb
×10
4
(M
−1
cm
−1
)λ
lc
(nm) ϕ
d
τ
e
(ns)
10a 502.5 2.7 513.5 0.52 3.80
14 502.5 2.8 513.5 0.53 3.60
15 502.5 4.2 513.5 0.49 3.70
16 502.5 5.0 513.5 0.50 3.70
a
Maximum abso p ion wa eleng h.
b
Maximum mola abso p ion.
c
Maximum luo escence wa eleng h.
d
Fluo escence quan um yield.
e
Fluo escence li e ime.
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uni s appended ( anging om one o ou ), hese wa e -soluble
dyes exhibi ed s ong abso p ion and luo escence bands in
pu e wa e (peaked a 502 nm and 513 nm, espec i ely, wi h
mola ex inc ion coefficien s up o 50 000 M
−1
cm
−1
and 53%
espec i ely, Table 2). No e ha e en a his low dye concen-
a ion, he pa en 8-phenyl BODIPY was en i ely insoluble and
was p one o agg ega e in aqueous solu ions, esul ing in he
comple e quenching o i s luo escen signal.
47
To ensu e he efficiency o hese dyes as luo escen biop-
obes in physiological media, i is c ucial o main ain hei
solubili y and luo escence esponse a highe concen a ions.
Thus, o assess he solubili y and luo escence pe o mance o
he BODIPY glycoconjuga es a ele a ed concen a ions in
wa e , we s udied he impac o dye concen a ion on hei
pho ophysical p ope ies.
The inco po a ion o D-glucose o D-mal ose o he BODIPY
co e, as in 10a o 14, espec i ely, acili a ed he a ainmen o
homogeneous wa e solu ions up o 0.1 mM (Fig. 5). This
wa e solubili y was signi ican ly enhanced by inc easing he
numbe o appended ca bohyd a e uni s, enabling he a ain-
men o aqueous solu ion up o 1.9 mM in 15 wi h h ee suga
uni s (D-mal o iose), and e en 2.4 mM g a ing ou suga
uni s (aca bose) in 16 (Fig. 5 and Fig. S85 in ESI†). Howe e , a
he highes concen a ion, he abso p ion p o ile o 15 and 16
became b oade wi h a no able inc ease in he abso bance a
sho e wa eleng hs (Fig. 5 and Fig. S85 in ESI†). Acco ding o
he exci on model,
48
his spec al end is indica i e o H- ype
agg ega ion. Such agg ega ion should be a consequence o a
weak exci on coupling, since i s spec oscopic con ibu ion
a ises as a shoulde o he main abso p ion band, e en a he
highes op ical densi y (dye concen a ion a ound 2 mM o
BODIPY conjuga es 15 and 16) he ein es ed in pu e wa e .
H-Agg ega es a e usually no luo escen , as e idenced by bo h,
a d as ic dec ease o he luo escence efficiency (Fig. S87 in
ESI†) and he absence o new emission bands in he luo-
escence spec um (Fig. 5). I is no ewo hy ha he obse ed
inc ease o he long-wa eleng h shoulde in he luo escence
p o ile a his concen a ion was likely a ibu ed o he eab-
so p ion/ eemission phenomena, which we e no ully co -
ec ed solely by educing he op ical pa hway in such highly
concen a ed media.
49
To quan i y he impac o he agg ega ion and/o eabso p-
ion/ eemission phenomena on he luo escence esponse, he
absolu e luo escence quan um yields we e es ima ed as a
unc ion o dye concen a ion (Fig. S87 in ESI†). As expec ed,
an inc ease in dye concen a ion led o a dec ease in luo-
escence efficiency due o eabso p ion and eemission effec s
( he op ical pa h leng h was main ained a 1 mm o all dye
concen a ions) and he p omo ion o H-agg ega es.
Rema kably, all he BODIPY glycoconjuga es e ained a sub-
s an ial luo escence esponse (highe han 20%) a
0.1–0.2 mM concen a ions, which a e ypical o he biological
assays. Beyond his dye concen a ion, he luo escence
efficiency sha ply dec eased due o an inc ease in eabso p-
ion/ eemission and agg ega ion p obabili ies, which we e
enhanced a high op ical densi ies, as shown in Fig. 5. Despi e
hese ad e se condi ions, dyes 15 and 16 (bea ing mal o iose
and aca bose esidues, espec i ely) s ill we e able o e ain a
measu able luo escence signal. Fu he mo e, hei mola
abso p ion coefficien s we e he highes ones eco ded (almos
double han hose eco ded o 10a and 14, bea ing glucose
and mal ose espec i ely, Table 2). Consequen ly, an enhance-
men in he hyd ophilici y o he BODIPY de i a i e is also
e iden in a mo e efficien ha es ingo incomingexci a ion
ligh , likely because he dye is be e sol a ed and s abilized in
he aqueous en i onmen . These pho ophysical p ope ies a e
expec ed o enhance he po en ial o BODIPYs 15 and 16 as biop-
obes. In pa icula , he good solubili y in pu e wa e , wi hou
any hin o agg ega ion, and he b igh luo escence signal up o
0.1 mM, a concen a ion high enough o bioimaging essays in
he aqueous cellula media, suppo his no ion.
Toxici y on mammalian cells (heal hy and umo )
To analyze he oxici y o he diffe en compounds on heal hy
and umo cells, we conduc ed a oxici y es using heal hy
human b eas epi helial cells (HMEpiC) and human b eas
adenoca cinoma epi helial cells (MCF-7, ECACC) (Fig. 6A and
B, espec i ely). We obse ed ha he oxici y o BODIPY-sac-
cha ides inc eased wi h he numbe o suga uni s in bo h
heal hy and umo cells. Rega ding cy o oxic de i a i es 15
and 16, he o me (mal o iose–BODIPY conjuga e) displayed
highe cy o oxici y agains umo cells (MCF-7) han agains
heal hy human b eas epi helial cells (HMEpiC) (Table 3). On
he con a y, BODIPY–aca bose glycoconjuga e (16), showed a
diffe en beha io , displaying simila LC
50
agains HMEpiC
and MCF-7 cells (Fig. 7 and Table 3).
Fig. 5 No malized abso p ion (solid line) and fluo escence (dashed
line) spec a o BODIPY glycoconjuga es 10a and 15 bea ing one and
h ee ca bohyd a e uni s, espec i ely, as a unc ion o he dye concen-
a ion in wa e using op ically ma ched solu ions (see ESI† o de ails).
The spec a o BODIPY conjuga es 14 and 16 a e collec ed in Fig. S85 in
ESI.†The eco ded abso p ion spec a scaled by he mola abso p ion
coefficien a e collec ed in Fig. S86 in ESI.†
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Cance cells exhibi heigh ened glucose abso p ion and ely
on an ae obic glycoly ic pa hway o ul ill hei me abolic
equi emen s o g ow h and p oli e a ion. Ta ge ing he inhi-
bi ion o ae obic glycolysis p esen s a s a egic he apeu ic
a enue o impede cance cell p og ession.
40
In his sense, he
use o aca bose o mal o iose could inhibi glucose up ake
and p omo e glucose dep i a ion. In his way, bo h cell ypes
showed he capaci y o up ake he diffe en BODIPYs de i a-
i es (Fig. 7), which accumula ed a ound he nucleus o he
cell.
Cell in e naliza ion o BODIPY–aca bose conjuga e 16
To e alua e cell in e naliza ion, BODIPY–aca bose conjuga e
16 was selec ed due o i s capaci y o p oduce glucose dep i-
a ion.
40
We conduc ed se e al s aining expe imen s.
Lysosomes and mi ochond ia s ains we e pe o med oge he
wi h ac in and nucleus (Fig. 8, and Fig. S88–S90† o ull-size
pic u es).
Acco ding o Fig. 8A, aca bose–BODIPY 16 is loca ed nea
he nucleus bu ou side he mi ochond ia. The in e naliza ion
p ocess o 16 appea s o be h ough he lysosomes as can be
obse ed in Fig. 8B and C, and no accumula ion was obse ed
in he mi ochond ia. Ligh ning con ocal mic oscopy (Fig. 8C)
p o ides images o s udy spa io empo al localiza ion. The
esul s showed a colocaliza ion o he BODIPYs wi h he lyso-
somes and co obo a e he capaci y o cells o in e nalize aca -
bose–BODIPY 16.
Enzyma ic s udies
To alida e he neoglycosyla ion agging p o ocol om an enzy-
ma ic pe spec i e, we decided o s udy how he newly inco po -
a ed BODIPY appendage could affec he binding affini y o a
Fig. 6 Toxici y assays o BODIPY conjuga es 10a (D-glucose), 14
(D-mal ose), 15 (D-mal o iose), and 16 (aca bose), on HMEpiC (A) and
MCF-7 (B) cells.
Table 3 Obse ed LC
50
alue on heal hy human b eas epi helial cells
(HMEpiC) and human b eas adenoca cinoma epi helial cells (MCF-7,
ECACC) o BODIPY–saccha ide conjuga es 15 and 16
LC
50
(mM) 15 (D-mal o iose) 16 (aca bose)
HMEpiC 1.8 0.32
MCF7 0.75 0.24
Fig. 7 Epifluo escence imaging o HMEpiC and MCF-7 cells showing
BODIPYs in e naliza ion in g een. Ac in is ma ked in ed and he nucleus
in blue. Scale ba : 50 µm.
Fig. 8 (A) Con ocal imaging o mi ochond ia (ligh blue), nucleus (blue),
ac in ( ed) s ain and BODIPY in e naliza ion (g een). Scale ba : 30 µm (B)
con ocal imaging o lysosomes (ligh blue), nucleus (blue), ac in ( ed)
s ain and BODIPY in e naliza ion (g een). Scale ba : 30 µm (C) ligh ning
image and colocaliza ion analysis o he lysosomes ( ed) and BODIPY
(g een). Scale ba : 20 µm.
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well-known amylase inhibi o , such as aca bose.
50
The la e is
a pseudo- e asaccha ide composed o an aca osine uni ,
esponsible o he inhibi o y ac i i y, which has been de i a-
ized wi h D-mal ose a he educing end. Wi h his pu pose in
mind, we ackled enzyma ic inhibi ion s udies o wo diffe en
α-amylases: A. o yzae α-amylase (AOA) and human sali a y
α-amylase (HSA). The ob ained IC
50
alues (Table 4) a e in
acco dance wi h epo ed da a,
51
con i m a dose-dependen
compe i i e inhibi ion mode and e eal ha he inco po a ion
o he me hoxyamino-BODIPY aglycon o aca bose, no only
does no in e e e wi h he inhibi o y ac i i y o AOA, bu
imp o es by wo- old he binding affini y o HSA. O e all,
hese esul s indica e ha he neoglycosyla ion o selec i e
enzyma ic ligands wi h BODIPY dyes could be a con enien
way o u n hem in o ch omogenic p obes wi h biological
applica ions wi hou affec ing hei inhibi o y po ency.
Conclusions
The neoglycosyla ion p o ocol used o he conjuga ion o
BODIPYs o unp o ec ed ca bohyd a e de i a i es is a e sa ile
and highly selec i e coupling me hod, which occu s unde mild
eac ion condi ions. The me hod can be applied o a a ie y o
ca bohyd a e de i a i es, including hose wi h complex s uc-
u es. The a achmen o a sufficien numbe o saccha ide
uni s o he BODIPY co e led o comple ely wa e -soluble dyes,
e aining a high luo escence signal e en a high concen a ions
and hence sui able o use as luo escen p obes in physiologi-
cal media. The biological s udies o he BODIPY-neoglycosides
showed excellen biocompa ibili ies and no cy o oxici y up o
0.1 mM o he p obes. The cell’s abili y o up ake a ious
BODIPY de i a i es wi hin a non- oxic ange could ha e appli-
ca ions in a ious biological ields including bio-imaging.
Speci ically, he luo escen aca bose–BODIPY conjuga e 16
demons a ed he capabili y o be aken up by cells and loca-
lized wi hin lysosomes, as e idenced by ligh ing con ocal
mic oscopy. This de i a i e also showed a binding affini y o
α-amylases ha is compa able o o be e han ha o aca bose
alone, acco ding o enzyma ic ac i i y s udies. Acco dingly, he
up ake capaci y combined wi h he PS ea u es o BODIPY con-
juga es show p omise in u u e biological applica ions in pho o-
dynamic and pho o he mal he apies.
Au ho con ibu ions
A. M. G.: concep ualiza ion, p ojec adminis a ion, unding
acquisi ion, w i ing –o iginal d a , w i ing – e iew &
edi ing. L. G. F.: in es iga ion and w i ing –o iginal
d a . A. G. S.: in es iga ion. C. U.: in es iga ion and w i ing –
o iginal d a . L. G.-R.: in es iga ion. J. B.: unding acquisi ion,
supe ising and w i ing. I. G.-M.: unding acquisi ion, supe -
ising and w i ing. L. I.: in es iga ion and unding acquisi ion.
M. R. A.: in es iga ion and unding acquisi ion. J. C. L.: con-
cep ualiza ion, unding acquisi ion, w i ing –o iginal d a ,
w i ing – e iew & edi ing. All au ho s ha e ead and ag eed o
he published e sion o he manusc ip .
Conflic s o in e es
The e a e no con lic s o decla e.
Acknowledgemen s
This esea ch ecei ed inancial suppo om he Spanish
Minis e io de Ciencia e Inno ación (MCIN)/Agencia Es a al de
In es igación (AEI) G an s: PID2020-114755GB-C31 and -C33,
and PID2021-122504NB-I00 unded by MCIN/AEI/10.13039/
501100011033 and by ERDF A way o making Eu ope. LGR and
JB acknowledge Gobie no Vasco o inancial suppo (IT1639-
22). MRA and LGF acknowledge inancial suppo om MCIN
(PID2020-114086RB-I00) and Comunidad de Mad id (P2022/
BMD-7406). MRA and LGF a e membe s o he SusPlas pla -
o m om Consejo Supe io de In es igaciones Cien í icas
(CSIC). This esea ch wo k was pe o med in he amewo k o
he Nanomedicine CSIC HUB ( e 202180E048). The au ho s
hank Ms. Rosa Ana Ramí ez (ICTP-CSIC) o assis ance in he
biological expe imen s. We a e also indeb ed o Ms. Ma ina
Rod íguez and M Diego Raúl Pozas (IQOG-CSIC) o skil ul
echnical suppo .
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