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a.
Depa amen o de Química O gánica, Fac. CC. Químicas, Uni e sidad Complu ense
de Mad id, A . Complu ense s/n, 28040 Mad id (Spain).
b.
Glycosys ems Labo a o y, Ins i u o de In es igaciones Químicas (IIQ), CSIC-
Uni e sidad de Se illa, A . Amé ico Vespucio 49, 41092 Se ille (Spain).
c.
IMDEA-Nanoscience, Campus Can oblanco, 28049 Mad id (Spain).
d.
These au ho s con ibu ed equally o his wo k.
† Foo no es ela ing o he i le and/o au ho s should appea he e.
Elec onic Supplemen a y In o ma ion (ESI) a ailable: [de ails o any
supplemen a y in o ma ion a ailable should be included he e]. See
DOI: 10.1039/x0xx00000x
Recei ed 00 h Janua y 20xx,
Accep ed 00 h Janua y 20xx
DOI: 10.1039/x0xx00000x
www. sc.o g/
Cyclooc yne [60]Fulle ene Hexakis Adduc s: a Globula Sca old
o Coppe -F ee Click Chemis y
Ja ie Ramos-So iano,a,b,d José J. Reina,b,d Al onso Pé ez-Sánchez,a Bea iz M. Illescas,*a Ja ie
Rojo*b and Naza io Ma ín*a,c
The syn hesis o a new highly symme ic hexakis adduc o C60
appended wi h 12 cyclooc yne moie ies has been ca ied ou . This
compound has been used o he coppe - ee s ain-p omo ed
cycloaddi ion eac ion o a se ies o azides wi h excellen yields.
This s a egy o he ob en ion o clicked adduc s o [60] ulle ene
is o special in e es o biological applica ions.
[60]Fulle ene hexakis adduc s wi h Th symme y cons i u e an
a ac i e class o compounds ha allow he globula
disposi ion o subs i uen s a ound he C60 co e.1 These
compounds ha e a ac ed much a en ion in he las ecen
yea s, bo h in he a eas o ma e ials science and biomedicine.
Thus, some o hem p esen liquid c ys al beha io ,2 ha e been
employed o he s udy o elec onic and ene gy ans e
p ocesses,3 as o ganic connec i i y cen e s o he syn hesis o
Me al O ganic F amewo ks (MOFs),4 o as ca alys s.5 On he
o he hand, in he a ea o biological applica ions, hei ac i i y
has been es ed in di e en ields showing in e es ing
p ope ies and good biocompa ibili y. Hexakis adduc s o
[60] ulle ene ha e been es ed as gene ans ec ion ec o s,6
mul iplying uni s o pho odynamic he apy,7 glycosidase and
glycosyl ans e ase inhibi o s,8 o e icien an ibac e ial9 o
an i i al sys ems.10
The syn hesis o hese adduc s was i s s udied by Hi sch by
he one-po addi ion o malona es empla ed by 9,10-
dime hylan h acene11 and la e modi ied by Sun.12 Howe e ,
hese p ocedu es a e o en limi ed by he size o he
malona es, as he s e ic aspec s limi he yield o he eac ion.
To o e come his d awback, Nie enga en p oposed he
employmen o an azide appended malona e o ob ain a
clickable hexakis adduc o C60 o e ec i ely allow he co alen
unc ionaliza ion o [60] ulle ene by Cu(I) alkyne-azide
cycloaddi ion (CuAAC) eac ion.13 La ely, Nie enga en and we
desc ibed he 12-alkyne modi ied hexakis adduc o
[60] ulle ene and i s use in he click chemis y addi ion o
azides and alkynes.14 This e ec i e eac ion equi es, howe e ,
he use o coppe (I) as ca alys , which in ol es a subsequen
pu i ica ion s ep o he emo al o coppe , especially wi h
hose ma e ials o bio-medical applica ions, owing o i s high
cy o oxici y. This s ep can be hampe ed by he p esence in he
inal p oduc s o unc ional g oups, and e en he iazole ings
hemsel es, capable o binding coppe , hus limi ing hei
biological applica ions.15
A s a egy o a oid his pu i ica ion s ep is o employ o s ain
p omo ed alkyne-azide cycloaddi ion (SPAAC) click eac ion.
This eac ion, de eloped by Be ozzi and cowo ke s,16 consis s
o he eac ion o s ained cyclooc ynes wi h azides and has
ecei ed conside able a en ion owing o i s simplici y and
as e eac ion a es, a oiding he use o a me al as ca alys . As
i does no equi e he use o coppe , i has especial ele ance
o biological applica ions.
In he p esen communica ion we epo he syn hesis o a
new hexakis adduc o [60] ulle ene subs i u ed wi h wel e
cyclooc yne moie ies o u he ca y ou SPAAC eac ions. The
e sa ili y o his new de i a i e has been es ed wi h he
addi ion o di e en azides appended wi h pola and non-pola
chains, na u al p oduc s such as bio in, amino acids such as
phenylalanine and pep ide nucleic acid (PNAs) monome s such
as hymine. All compounds we e ob ained wi h high yields and
sho eac ion imes unde mild condi ions, and he comple e
cha ac e iza ion o all new de i a i es is also epo ed.
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Scheme 1 Syn hesis o compound 5. Reagen s and condi ions: (i) C60, DBU, CB 4, ODCB,
, 72h (50%); (ii) H2, Pd-C, DCM/MeOH, , o e nigh (100%); (iii) DPTS, DCC,
DCM/DMF, , o e nigh (99%).
The syn hesis o compound
5
by di ec Bingel addi ion o he
co esponding cyclooc yne subs i u ed malona e unde he
condi ions epo ed by Sun12 o he p epa a ion o hexakis
adduc s o C60 yielded a complex mix u e o compounds whe e
he b omina ion o he alkyne moie y was obse ed.
The e o e, we decided o ollow a syn he ic s a egy in h ee
s eps, as depic ed in Scheme 1. Malona e
1
(12 equi .) was
added o C60 (1 equi .) in he p esence o DBU (20 equi .) and
CB 4 (100 equi .) in ODCB a oom empe a u e. A e 72 h,
hexakis adduc
2
was ob ained in 50% yield a e column
ch oma og aphy. Dep o ec ion o he hyd oxyl g oups was
ca ied ou by hyd ogena ion a a mosphe ic p essu e, yielding
compound
3
in quan i a i e yield. Finally, he es e i ica ion o
cyclooc yne ca boxylic acid
4
(see SI o syn he ic de ails) wi h
hexakis adduc
3
using DCC/DPTS led o compound
5
in a
quan i a i e yield, which was pu i ied by size-exclusion
ch oma og aphy employing Sephadex.
Compound
5
was comple ely cha ac e ized by he usual
analy ical and spec oscopic echniques. The 13C NMR
spec um con ains only h ee signals o he C60 ca bons, wo
o he Csp2 a δ

145.8, 141.05 and one o he Csp3 a δ

69.1 (see ESI). Two di e en ca bonylic signals o he succinic
and he malona e appea espec i ely a δ

172.2 and 163.6
and he Csp o he alkyne a e obse ed a 110.1 and 92.8 o
he ca bons d and c o he cyclooc yne moie y, espec i ely
(see Figu e 1).
The abili y o compound
5
o be clicked o di e en azides was
es ed wi h compounds
6a-e
(Scheme 2). The addi ion
eac ions we e ca ied ou in DMSO unde mic owa e
i adia ion a 50ºC du ing 30 min. Compounds
7a-e
(ob ained
espec i ely om
6a-e
) we e pu i ied by size-exclusion
ch oma og aphy using Sephadex (see ESI). The yields ob ained
we e o e 90% in all cases.
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Scheme 2. Syn hesis o clicked adduc s 7a-e. Reagen s and condi ions: (i) DMSO, 50°C
unde MW, 30 min (93-99%). The wo possible egioisome s a e ep esen ed a he
bo om.
Cycloadduc s
7a-e
we e ully cha ac e ized by FTIR, 1H and 13C
NMR and MS and he assignmen o he signals in NMR was
elucida ed by COSY and HSQC NMR spec oscopies (see
suppo ing in o ma ion). The lack o he ypical band o he
azide g oup in he FTIR spec a (

2100 cm-1) o he clicked
adduc s was indica i e o he absence o un eac ed azide a e
pu i ica ion. 1H NMR spec a show wo di e en signals o he
CH o he cyclooc yne adjacen o iazole moie y (i.e. δ

4.82
and 4.69 o
7a
). Se e al o he signals appea duplica ed,
indica ing he p esence o he wo expec ed egioisome s (see
Scheme 2). Once assigned he signals o he 1H and 13C spec a
using COSY and HSQC expe imen s (see ESI), he a io o
egioisome s was calcula ed by in eg a ion o he signals in he
1H NMR spec um. Fo all he compounds, almos equal
p opo ion o he wo isome s was ob ained (
7a
: 57% (A), 43%
(B);
7b
: 54% (A), 46% (B);
7c
: 53% (A), 47% (B);
7d
: 55% (A),
45% (B);
7e
: 57% (A), 43% (B)), wi h a sligh excess o isome A,
wi h less s e ic hind ance han isome B (see Figu e 1). This
esul is compa able o p e ious s udies on monome ic
compounds, indica ing ha he globula p esen a ion o
cyclooc ynes p o ided by he hexakis adduc o C60
5
does no
a ou he o ma ion o one egioisome o e he o he .17
13C NMR also shows he p esence o he wo possible
egioisome s (see Figu e 1). Thus, while wo signals a e
obse ed o he Csp2 o C60 o
7a-e
(i.e.: 145.8 and 141.1 o
7a
), ou signals appea in he 13C NMR spec um o each
compound co esponding o he wo ca bons o he iazole
ing o he wo egioisome s (i.e.: 144.9, 144.8, 133.5 and
132.4 o
7a
). The es o he signals o he cyclooc yne moie y
also appea duplica ed (see ESI).
MS da a ob ained by MALDI-TOF spec ome y con i med he
p esence o he molecula ion peak o
7a,b,e
. Fo
7c-d
,
howe e , high le el o occu ing agmen a ion a oided he
obse a ion o he expec ed molecula ion peaks.
In conclusion, we ha e ca ied ou he syn hesis o a new
building block o he syn hesis o hexakis adduc s o
[60] ulle ene by using SPAAC. This compound is ob ained in
h ee s eps by cyclop opana ion (Bingel eac ion),
dep o ec ion and es e i ica ion, wi h e y good yields. The use
o his building block was es ed in eac ions wi h a se ies o
azides, leading o he clicked adduc s wi h yields o e 90%.
Mos impo an ly, his me hodology a oids he use o coppe
as a ca alys in he click cycloaddi ion eac ion, being o special
in e es o he p epa a ion o globula ulle ene de i a i es
o biological s udies.
Figu e 1.
13
C NMR spec a o compounds
5
(up) and
7a
(down) (CDCl
3
, 125.8
MHz).
We hank inancial suppo by he Eu opean Resea ch Council (ERC-
320441-Chi alca bon), he Minis e io de Economía y Compe i i idad
(MINECO) o Spain (p ojec s CTQ2014-52045-R and CTQ2014-
52328-P) and he Comunidad Au ónoma de Mad id
(PHOTOCARBON p ojec S2013/MIT-2841). JRS hanks MINECO o
a FPI ellowship and JJR acknowledges o CSIC o a JAEdoc con ac .
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