C60 pyrrolidine bis-carboxylic acid derivative as a versatile precursor for biocompatible fullerenes

Article abstract of DOI:10.1021/ol500363r

A C₆₀ Prato derivative with bis-^tBu ester was prepared as a stable and convenient scaffold for the development of fullerene derivatives such as water-soluble C₆₀-PEG conjugates, fulleropeptides via solid phase synthesis, and bis-functionalized C₆₀.

Full text of DOI:10.1021/ol500363r

Letter
pubs.acs.org/OrgLett
C60 Pyrrolidine Bis-carboxylic Acid Derivative as a Versatile Precursor
for Biocompatible Fullerenes
Safwan Aroua, W. Bernd Schweizer, and Yoko Yamakoshi*
Laboratorium fur Organische Chemie, ETH-Zurich, Vladimir-Prelog-Weg 3, CH-8093 Zurich, Switzerland
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* Supporting Information
ABSTRACT: A C60 Prato derivative with bis-tBu ester was
prepared as a stable and convenient scaffold for the development
of fullerene derivatives such as water-soluble C60−PEG
conjugates, fulleropeptides via solid phase synthesis, and bis-
functionalized C60.
ince the initial discovery of buckminsterfullerene,1
Senormous numbers of studies on fullerene-based materials
have been reported.2−10 For the development of fullerene
biomaterials, chemical functionalization of fullerenes is often
used to enhance their properties by the addition of moieties
that increase their water solubility or to promote interactions
with biomolecules such as DNA. Such chemical functionaliza-
tions can also be employed for the development of new classes
of materials including donor−acceptor dyads and supra-
molecular assemblies. For the preparation of those materials,
simple fullerene derivatives that can be used as a versatile
platform for further elaboration are in high demand.
The Prato reaction,11 a 1,3-dipolar cycloaddition of full-
erenes, has been widely used in the derivatization of fullerenes.
The features of this reaction include easy access to the starting
materials (aldehydes and amino acids), good yields, and
chemical stability of the fulleropyrrolidine adducts. By taking
advantage of the photosensitivity and metal encapsulation of
fullerenes, we are working on the preparation of biocompatible
fullerene materials in combination with a nontoxic polymer,
PVP (poly(vinylpyrrolidone)), by complexation12 or chemical
attachment.13,14 For the attachment of water-soluble or
biorelevant moieties to the fullerene core, it is important to
develop a versatile and easily handled C60 derivative as a
scaffold molecule. In the present study, we report the synthesis
of a C60 fulleropyrrolidine bis-carboxylic acid derivative and its
applications to a variety of C60 derivatives.
Figure 1. Synthesis of glycine derivative 4 and Prato reaction with C60
(inset: 1H NMR of Prato adduct 5 in CDCl3). Reagents and conditions:
(i) NaBH3CN, MeOH, rt, 48 h, 88%; (ii) H2, Pd/C, EtOAc−MeOH,
rt, 24 h, 98%; (iii) C60, HCOH, toluene, refl., 1 h, 38%; (iv) TFA,
1
CHCl3, rt, 18 h, >99%. In the H NMR, diastereotopic Hc and Hd
appeared as magnetically nonequivalent protons. Due to the presence
of two symmetry mirrors of [6,6]-adduct, four methylene protons in
pyrrolidine appeared as one singlet (Ha).
Although a small amount of polyadducts was observed, the
[6,6]-monoadduct was produced as a major product. The
structure of adduct 5 was determined by 1H and 13C NMR. On
the basis of the symmetry of the molecule (Figures 1 and S12),
it was confirmed to be a [6,6]-adduct, the same as the other C60
fulleropyrrolidine derivatives reported previously. Adduct 5 was
A glycine derivative 4 with two tert-butyl ester groups was
synthesized by reductive amination of 1 and 2 with NaBH3CN,
followed by deprotection of the benzyl group (Figure 1). The
Prato reaction of 4 and formaldehyde with C60 was complete
within 1 h, as monitored by HPLC (Figures 2 and S9), to
provide fulleropyrrolidine 5 in a sufficient yield (38%).
Received: February 4, 2014
Published: March 7, 2014
© 2014 American Chemical Society
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Letter
Figure 2. HPLC traces of Prato reaction of C60 to provide
fulleropyrrolidine 5 ([6,6]-adduct) (Buckyprep-M 4.6 mm × 250
mm, toluene, 390 nm, 1 mL·min−1).
highly soluble in most organic solvents and very stable against
heating (at 180 °C for 4 h) without observing products from
retro-cycloaddition (often observed in Diels−Alder adducts of
C60),15,16 decarboxylation (can be observed in Hirsch−Bingel
derivatives of C60),17,18 or rearrangement (often observed in
19
azafulleroids of C60 or Prato adducts of TNT-EMF).20−22
Figure 4. Production of functional C60 derivatives via Prato adduct 5
as a key intermediate.
Figure 5. Addition of mPEG2000−NH2 to C60 bis-carboxylic acid 6 to
provide water-soluble C60−(mPEG2000)2 7a (a) and 1 mM aqueous
solution of 7a (b).
solvents such as pyridine and DMF and could be subjected to
subsequent reactions (Figure 4, Path 1−2 and Figure 5) to
provide water-soluble C60 derivative 7 (Path 1) or C60−peptide
conjugates (fulleropeptide) 8 (Figure 4, Path 2 and Figure 6).
Alternatively, compound 5 lead to heterofunctionalized C60
derivative 10 through an acid anhydride intermediate 9 (Figure
4, Path 3 and Figure 7).
Bis-carboxylic acid 6 was subjected to an amide conjugation
reaction with mPEG2000−NH2 using HBTU as a coupling
reagent (Path 1 in Figure 4 and Figure 5a). Using 3 equiv (1.5
equiv for each carboxylic acid) of PEG2000−NH2 provided 7a in
a good yield (78%). The structure of the C60−PEG 7a was
Figure 3. Crystal structure of Prato adduct 5.
X-ray crystal structure analysis was employed to confirm the
structure of 5 (Figure 3). In the crystal lattice, the hydrophobic
C60 parts were located in line parallel to the axes a and c (Figure
3b−d; the shortest distance between C atoms of neighboring
1
confirmed by MALDI-TOF-MS and H, 13C NMR (Figures
S28, S29, and S31). Compound 7a was highly water-soluble
(Figure 5b), forming particles with a diameter of about 10 nm
in water (DLS; see Figure S27). The highly water-soluble 7a
(>3 mM) can potentially be a biocompatible fullerene material.
Bis-carboxylic acid 6 was subjected to the solid-phase
coupling with a resin-supported peptide (Path 2 in Figure 4
and Figure 6) to provide a fulleropeptide. Previous studies
reported fulleropeptides with interesting bioactivities such as a
higher cell penetration property than the corresponding
peptides with even higher water solubility23 and antimicrobial
C
60 all related by an inversion center are 3.03, 3.17, and 3.40 Å,
Figure 3e). The distance of 3.03 Å is the shortest contact found
between C60 moieties in known crystal structures (CSD
database search version 5.34).
Adduct 5 was subjected to acidic deprotection of the two tert-
butyl groups. In contrast to basic deprotection, which could
often cause hydroxylation of the C60 core, the deprotection with
TFA provided 6 in an excellent yield (99%) without affecting
the C60 cage (Figure 4). Compound 6 was soluble in polar
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Letter
nearly colorless suggesting that conjugation of 6 to amino
groups of GABA of the peptide on solid phase was successfully
proceeding. The resulting resin was subjected to conditions for
peptide cleavage from the solid support (TFA/H2O), and the
fulleropeptide 8a was obtained without many byproducts, as
shown in the HPLC trace of a crude mixture (Figure 6b top
diagram). By further HPLC purification (Figure 6b bottom
diagram) fulleropeptide 8a was obtained in 64% yield and
confirmed by MALDI-TOF-MS analysis (Figure 6c).
Bis-carboxylic acid ester 5 was converted to an acid
anhydride 9 by simultaneous deprotection and dehydration of
5 in the presence of TFA and TFAA for the preparation of
heterofunctionalized C60 derivatives (Path 3 in Figure 4 and
Figure 7a). The in situ generation of 9 was monitored by NMR,
with a decrease of peak Hb in 5 and a simultaneous increase of
Hb′ in 9 observed (Figure 7b). Although compound 9 was
somewhat water-sensitive, it was stable enough for isolation and
full characterization (stable at room temperature at least for 5 h
under ambient conditions, Figures S37−S44). Compound 9
could not be prepared via acid 6, since 6 was not soluble in
toluene or chloroform. The addition of 5 equiv of tridecylamine
to 9 allowed selective single amide conjugation without the
need for coupling reagents to provide 10a with one substituted
moiety (tridecyl group) and one unsubstituted carboxylic acid.
This remaining carboxylic acid was converted to the amide with
benzylamine in the presence of a coupling reagent (HBTU) to
provide a heterofunctionalized C60 derivative 11a with two
different side chains.
Figure 6. (a) Solid phase reaction of bis-carboxylic acid 6 to peptide-
resin to provide a fulleropeptide 8a (solid phase: chlorotrityl resin,
peptide sequence: NH2-GABA-GPLGVRGA-COO-resin). (b) HPLC
trace of crude mixture of fulleropeptide cleaved from resin (top) and
purified one (bottom) (column: C4, 4.6 mm × 250 mm, Vydac 214
MS protein, solvents: MeCN−H2O (5:95 (0−5 min), gradient to 95:5
(5−15 min), 95:5 (15−27 min), gradient to 10:90 (27−29 min),
detection: 280 nm). (c) MALDI-TOF-MS spectra of purified
fulleropeptide (matrix: HCCA, m/z calcd for C137H132N25O22:
2479.00, found: 2479.00 M+).
In this study, we reported a new, simple, and stable Prato
derivative bearing two carboxylic acid groups. The Prato
reaction of C60 with formaldehyde and a glycine derivative 4,
which were easily accessible using commercially available
chemicals, proceeded in good yield. Under acidic conditions,
tBu deprotection proceeds smoothly without affecting the C60
core and efficiently provided a bis-acid derivative 6. The Prato
derivative 6 was stable compared to other C60 carboxylic acid
derivatives, such as those derived by Bingel reactions.17,18 By
addition of an amine derivative or peptide, this Prato derivative
was easily functionalized even under solid phase conditions and
was shown potentially to provide various other biocompatible
derivatives. In addition, the anhydride intermediate 9 was used
to provide C60 derivatives bearing multiple functional moieties.
These results demonstrate the versatility of the Prato derivative
5 for easy access to a variety of C60 functional derivatives
including biocompatible ones.
ASSOCIATED CONTENT
* Supporting Information
■
S
Figure 7. (a) Conjugation of two different amines to C60 via an acid
anhydride 9. Two distinct amines (ntridecyl and benzyl groups were
used as examples) were attached successfully to the C60 core by a
stepwise procedure to provide heterofunctionalized C60 derivative 11a.
(b) In situ generation of acid anhydride 9 from the reaction of 5 and
Spectral data of all new compounds and crystallographic data of
5. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
1
TFA/TFAA observed by H NMR (in TFA-d1, CDCl3, and toluene-
d8).
■
Corresponding Author
*E-mail: yamakoshi@org.chem.ethz.ch.
Notes
activity to Gram-positive bacteria.24 Previously, there were a
few examples of fullerene derivatives that can be applied in
solid-phase synthesis.24−27 In this study, a typical peptide
GABA-GPLGVRGA, prepared on a 2-chlorotrityl resin (3 equiv
to 6), was subjected to coupling with 6 using HBTU. After 1 h
of reaction subsequent to the addition of 6 to a suspension of
peptide resin, the solution, which was initially very dark, turned
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Authors thank Ms. Sean Oriana (ETH-Zurich) for her help in
solid phase peptide preparation. Authors thank Dr. Aaron M.
̈
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Dumas (ETH-Zurich) for his discussion. Authors thank Profs.
Letter
̈
Morbidelli and Gruzmacher (ETH-Zurich) for the access to
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DLS and CV machines. This research was supported in part by
the Swiss National Foundation (200021_140451), ETH
Individual Investigator’s Research Award (ETH-25 11-1),
PRESTO program from JST, CEET pilot grant from the
University of Pennsylvania, and American Heart Association
Researcher’s Developing Grant (0930140N).
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