Interlaced capsules by self-assembly of cavitands substituted with tripeptides and tetrapeptides

Article abstract of DOI:10.1080/10610278.2017.1406603

A strategy that utilizes macrocyclic resorcin[4]arenes for the pre-positioning of peptides to form cavitands that subsequently self-assemble through hydrogen bonds was used for the formation of molecular capsules. Hydrophobic tri- and tetrapeptides were attached via their C-termini to tetraformylresorcin[4]arene using acylhydrazone linkers. The resulting cavitands self-assemble in relatively non-polar environments (chloroform or chloroform-methanol) forming non-covalent dimers through hydrogen bonding motifs resembling beta-sheets. NMR studies (ROESY and DOSY) indicate that the binding motif involves C-terminal amino acids from the peptide strands, while the N-terminal parts are positioned outside of the cavity. The capsules possess stable porous structures and they are able to quantitatively complex fullerenes C₆₀ and C₇₀.

Full text of DOI:10.1080/10610278.2017.1406603

Supramolecular Chemistry
ISSN: 1061-0278 (Print) 1029-0478 (Online) Journal homepage: http://www.tandfonline.com/loi/gsch20
Interlaced capsules by self-assembly of cavitands
substituted with tripeptides and tetrapeptides
Marek P. Szymański, Jakub S. Czajka, Piotr Cmoch, Waldemar Iwanek &
Agnieszka Szumna
To cite this article: Marek P. Szymański, Jakub S. Czajka, Piotr Cmoch, Waldemar Iwanek &
Agnieszka Szumna (2017): Interlaced capsules by self-assembly of cavitands substituted with
tripeptides and tetrapeptides, Supramolecular Chemistry, DOI: 10.1080/10610278.2017.1406603
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Date: 29 November 2017, At: 23:13

Supramolecular chemiStry, 2017
https://doi.org/10.1080/10610278.2017.1406603
Interlaced capsules by self-assembly of cavitands substituted with tripeptides
and tetrapeptides
Marek P. Szymańskia , Jakub S. Czajkab, Piotr Cmocha, Waldemar Iwanekc and Agnieszka Szumnaa
ainstitute of organic chemistry, polish academy of Sciences, Warsaw, poland; bFaculty of chemistry, Warsaw university of technology, Warsaw,
poland; cFaculty of chemistry, adam mickiewicz university in poznań, poznań, poland
ABSTRACT
ARTICLE HISTORY
received 7 September 2017
accepted 7 November 2017
A strategy that utilizes macrocyclic resorcin[4]arenes for the pre-positioning of peptides to form
cavitands that subsequently self-assemble through hydrogen bonds was used for the formation
of molecular capsules. Hydrophobic tri- and tetrapeptides were attached via their C-termini to
tetraformylresorcin[4]arene using acylhydrazone linkers. The resulting cavitands self-assemble in
relatively non-polar environments (chloroform or chloroform-methanol) forming non-covalent
dimers through hydrogen bonding motifs resembling beta-sheets. NMR studies (ROESY and DOSY)
indicate that the binding motif involves C-terminal amino acids from the peptide strands, while the
N-terminal parts are positioned outside of the cavity. The capsules possess stable porous structures
and they are able to quantitatively complex fullerenes C60 and C70.
KEYWORDS
peptides; artificial
beta barrels; fullerene;
encapsulation
Introduction
Combination of inherent features of peptides with porosity
offers additional prospective applications, e.g. in recog-
nition, separation, delivery or catalysis. However, exam-
ples of porous peptidic systems are scarce (10–12). This is
mainly caused by high conformational lability of peptide
strands and presence of numerous non-covalent inter-
acting sites that imply low predictability of the resulting
structures and their high tendency to collapse. Therefore,
The advantageous features of peptides that include func-
tionality, chirality, biocompatibility, availability and syn-
thetic tunability have been exploited in various chemical
and biological applications (1–6). Particularly promising
applications have been found in the field of biocompat-
ible functional materials, where self-assembled peptides
serve as fibrous scaffolds for regenerative medicine (7–9).
CONTACT agnieszka Szumna
agnieszka.szumna@icho.edu.pl
the supplemental data for this article can be accessed at https://doi.org/10.1080/10610278.2017.1406603
© 2017 informa uK limited, trading as taylor & Francis Group

2
M. P. SZYMAŃSKI ET AL.
various strategies that aim at ordering and rigidifying of
the peptide-based compounds have to be employed.
Among them application of concave-shaped macrocyclic
scaffolds have been tested by the group of Sherman (13),
Ungaro (14), Feigel (15) and, more recently by our group.
that further self-assemble through hydrogen bonds to
form dimeric molecular capsules with cavity sizes up to
835 Å3 (16–18). In this tactic peptides are attached to tetr-
aformylresorcin[4]arene 1 using imine chemistry, that
implies attachment of the peptide through its N-terminus
(type A, Figure 1(a)) (13) or acylhydrazone chemistry that
allows peptide attachment through its C-terminus (type B,
Figure 1(b)) (14). Capsules A are barrel-shaped and their
binding motifs, involving peptides’ backbones, are fully
complementary. The length of the barrels systematically
Results and discussion
We have recently presented an ordering strategy that uti-
lizes macrocyclic scaffolds for pre-positioning of peptides
Figure 1. (colour online) comparison of structures, shapes and binding motifs of self-assembled capsules formed by attachment of
peptides (orange) toresorcinareneskeleton (blue) by: (a) iminelinkers (typeA); (b) acylhydrazonelinkers (typeB, red – solventmolecules).

SUPRAMOLECULAR CHEMISTRY
3
increases with the elongation of peptides (studied up to
tetrapeptides). Sidechains of peptides in capsules A are
positioned outside the capsule. Attempts to introduce
functional sidechains into interiors lead to disruption of
the self-assembled dimers. This may be caused by rela-
tively small internal diameters of the barrels of capsules
of type A. Hydrazone based capsules (type B) are wider
because they have longer semi-rigid linkers. As a conse-
quence, peptides’backbones are positioned further from
each other and they are not able to form a fully comple-
mentary binding motif. For single amino acid derivatives
the gaps in the binding motif are filled by solvent mole-
cules, while for dipeptides the second amino acid (glycine)
forms a turn and complements the binding motif (14).
Even though the binding motif is not fully complemen-
tary, capsules of type B form spontaneously in non-polar
environments (14), while in polar environment they form
upon templation (15). Additionally, they are chemically
more stable than capsules A (due to higher chemical sta-
bility of acylhydrazones compared to imines). Based on the
structures of mono- and dipeptide-based acylhydrazone
capsules B it is non-trivial to predict structures formed by
longer peptides. In this work we address the question of
the self-assembly abilities and geometric preferences of
resorcin[4]arenes substituted with tri- and tetrapeptides.
The work is motivated by the possibility of modulation of
Figure 2. Synthesis of cavitands 4 and 5. coupling conditions: eDci∙hcl, oXyma, et3 N, DmF. Deprotection conditions: tFa, Dcm for Boc
removal, or pd/c, meoh/h2o for cbz and Bn removal.

4
M. P. SZYMAŃSKI ET AL.
the cavity size and/or the internal or external functionali-
zation (for catalytic functions and solubility modifications).
Tripeptide hydrazide 2 and tetrapeptide hydrazide 3
have sequences consisting of alternating phenylalanine and
glycine residues. Phenylalanine ensures solubility in non-po-
lar solvents while glycine provides conformational lability
for plausible adaptation. The hydrazides were synthesized in
solution using coupling procedures (EDCI/OXYMA) and sub-
sequent removal of the Cbz/Boc protecting groups (Figure
2). In the final step hydrazide 2 was attached to tetraformyl-
resorcin[4]arene 1 in chloroform at 70 °C and product 4 was
formed in quantitative yield. Hydrazide 3 gave product 5 in
quantitative yield in an analogous reaction with 1 carried
out in chloroform/methanol mixture (95: 5, v: v).
formation of (4)2 capsule (Figure 3(b)). These signals also
suggest that the binding motif involves the amino acid
that is positioned next to the linker. In such a structure
the remaining part of the peptide should be positioned
outside of a resorcinarene bowl. This conclusion is sup-
ported by the presence of correlation signals between
protons of the N-terminal amino acid and protons of the
lower rim of resorcinarene (a + a′↔j) in the ROESY spec-
trum. DOSY measurements reveal a diffusion coefficient
of 2.8 × 10−10 m2 s−1 (in CDCl3 at 298 K), corresponding to
an average diameter of molecule of ~2.8 nm (calculated
using the Stokes-Einstein equation). The averaged diam-
eter of the capsule (4)2 of the suggested interlaced shape
is ~2.9 nm, that is in fair agreement with the experimental
1
Tripeptide product 4 was characterized by 2D NMR in
CDCl3 and all signals were assigned. The most significant
feature in the ROESY spectrum is the presence of f↔h + h′
correlation signals indicative of a close contact between
hemispheres in a head-to-head arrangement and the
value. The number of signals in H and 13C NMR spectra
indicates that chiral capsule (4)2 has a dynamically-aver-
aged D4 symmetry
Product 5 is not soluble in pure chloroform, but addition
of a small amount of methanol results in a homogenous
Figure 3. (colour online) Nmr spectra of 4: (a) 1h Nmr spectrum with signals’ assignments; (b) parts of roeSy spectrum; (c) suggested
binding motif with marked short contacts based on roeSy spectrum (cDcl3, 298 K, 600 mhz).

SUPRAMOLECULAR CHEMISTRY
5
solution (Figure 4). 2D NMR spectra were recorded in CDCl3:
CH3OH (95: 5, v: v). ROESY spectrum show the presence of
f↔h + h’correlation signals. This observation suggests that
tetrapeptide 5 forms a head-to head dimer (5)2 in CDCl3:
CH3OH (95: 5, v: v) solution. Additionally, these correlation
signals suggest that the binding motif in both dimers is
similar and involves the amino acid positioned next to
the linker. The ROESY spectrum of (5)2 also shows correla-
tion signals between N-terminal amino acid protons and
resorcinarene’s lower rim protons. DOSY measurements
reveal a diffusion coefficient of 2.6 × 10−10 m2 s−1 (in CDCl3:
CH3OH, 95: 5, v: v at 298 K). Since the viscosity coefficients
for both solvents are similar (0.534 mPa·s for CDCl3 and
0.543 mPa·s for methanol) and the amount of methanol
is small we used the viscosity coefficient of CDCl3 for cal-
culations and estimated the molecules’ sizes as ~3.1 nm.
The averaged diameter of the capsule based on a model
structure is ~2.9 nm, which is in agreement with the exper-
imental value.
formation, complexation experiments were carried out.
We have previously demonstrated that for acylhydrazone
capsules based on single amino acids the size of the cav-
ities is appropriate for complexation of fullerenes C60 and
C70 (14). However, further experiments showed that the
complexation processes are accompanied with a high
kinetic barrier, that precludes complexation of fullerenes
by already-formed capsules. Therefore, the complexes
can be obtained either at the reaction step or by mech-
anochemical activation (19). Here fullerenes were added
during the synthetic reactions. Complexes (4)2⊃C60 or
(4)2⊃C70 were obtained in the reaction between 1, 2
with stoichiometric amounts of C60 or C70 and isolated in
quantitative yields (as sole products, without formation
of free capsules). Complexes (5)2⊃C60 and (5)2⊃C70 were
obtained in the reaction between 1, 3 and stoichiomet-
ric amounts of C60 or C70 (also as sole products, without
formation of free capsules). The complexes were charac-
terized by NMR, mass spectrometry and circular dichro-
ism (Figures 5–7, Table 1). In the ESI MS spectra signals of
doubly and triply negatively charged ions for complexes
In order to probe the porosity and presence of cavities
in capsules (4)2 and (5)2 and to further confirm capsules’
Figure 4. (colour online) Nmr spectra of 5: (a) 1h Nmr spectrum with signals’ assignments; (b) parts of roeSy spectrum; (c) suggested
binding motif with marked short contacts based on roeSy spectrum (cDcl3: ch3oh, 95: 5, v: v, 298 K, 600 mhz, solvent suppression).

6
M. P. SZYMAŃSKI ET AL.
are observed (Figure 5(m)–(p)) without presence of signals
corresponding to free capsules. 13C NMR spectra show sin-
gle signals of complexed C60 at 141.6 ppm for (4)2⊃C60 and
141.3 ppm for (5)2⊃C60. The signals of C60 are onefold and
narrow indicating free rotation of a guest molecule in the
cavity. For complexes (4)2⊃C70 and (5)2⊃C70 there are seven
13C NMR signals for complexed C70. This may seem surpris-
ing, since in case of free C70 there are five magnetically
non-equivalent carbon atoms. The presence of seven sig-
nals for complexed C70 originates from symmetry lowering
in chiral environment – the symmetry of C70 in the chiral
cavity changes from D5 h to D5, that justifies the presence
of 7 non-equivalent carbon atoms. Considering that the
symmetry of the capsule is D4 and all signals are narrow, it
can be concluded that rotation of C70 in the cavity is fast (at
least along the main axis) and the fullerene does not expe-
rience significant constraints. DOSY measurements show
similar diffusion coefficients for complexes as for free cap-
sules indicating that the size of capsules does not change
during complexation (Table 1). The ROESY spectrum and
complexation-induced changes in H NMR spectra also
1
support this conclusion. The trough-space ROE correla-
tion signals for complexes are analogous to free capsules
(Figure 6). Complexation induced shifts in 1H NMR spectra
are the largest for the backbone protons of the first and the
second amino acid (counting from the attachment point,
Figure 5(a)–(f)). These spectral features indicate that com-
plexation of fullerenes in capsules (4)2 and (5)2 proceeds
very effectively (quantitatively) without substantial struc-
tural rearrangement of their skeletons (involving binding
motifs and shapes). This conclusion corresponds well to
the previous results for capsules consisting of single amino
acid chains, that also were able to accommodate C60 and
C70 and allows us to assume that the cavity sizes for cap-
sules (4)2 and (5)2 are similar (~800 Å3) (14). Considering
the molecular volumes of fullerenes (549 Å3 for C60 and
646 Å3 for C70 with 1.76 Å as a radius of C atom) (20), pack-
ing coefficients of 67% for C60 complexes and 81% for C70
1
Figure 5. (colour online) comparison of h Nmr (a-f) and 13c Nmr (g-l) spectra of free capsules and their complexes with fullerenes:
(a) (4)2; (b) (4)2⊃c60; (c) (4)2⊃c70 (cDcl3, 298 K, 600 mhz); (d) (5)2; (e) (5)2⊃c60; (f) (5)2⊃c70 (cDcl3: ch3oh, 95: 5, v: v, 298 K, 600 mhz,
solvent suppression); (g) (4)2; (h) (4)2⊃c60; (i) (4)2⊃c70 (cDcl3, 298 K, 150 mhz); (j) (5)2; (k) (5)2⊃c60; (l) (5)2⊃c70 (cDcl3: ch3oh, 95: 5, v: v,
298 K, 150 mhz, solvent suppression), – indicates signals of encapsulated fullerenes; eSi-mS (m-p) spectra of capsules complexes with
fullerenes: (m) (4)2⊃c60; (n) (4)2⊃c70; (o) (5)2⊃c60; (p) (5)2⊃c70.

SUPRAMOLECULAR CHEMISTRY
7
Figure 6. (colour online) (a) part of the roeSy spectrum of (5)2⊃c70; (b) suggested binding motif with marked short contacts based on
roeSy spectrum (cDcl3: ch3oh, 95: 5, v: v, 298 K, 600 mhz, solvent suppression).
Figure 7. uV/Vis (a, b) and ecD spectra (c, d) of capsules and their complexes (298 K, chcl3).
complexes are calculated. For comparison, the packing
coefficients observed for pure crystalline fullerenes are
around 74%, while for their molecular complexes they are
usually lower. One can expect that for such high packing
coefficients expansion of the cavities of (4)2 and (5)2 is
structurally justified and feasible or complexation should
be disfavoured, especially for C70. However, current

8
M. P. SZYMAŃSKI ET AL.
Table 1. Diffusion coefficients for capsules and their complexes
measured by DoSy.
Funding
This work was supported by Ministerstwo Nauki i Szkolnictwa
Wyższego [grant number 0206/DIA/2016/45]; and Narodowe
Centrum Nauki [grant number 2016/20/W/ST5/00478].
D/m2 s−1
Conditions
cDcl3, 298 K, 600 mhz
cDcl3, 298 K, 600 mhz
(4)2
2.8 × 10−10
2.7 × 10−10
3.0 × 10−10
2.6 × 10−10
2.2 × 10−10
2.3 × 10−10
(4)2⊃c60
(4)2⊃c70
(5)2
cDcl3, 298 K, 600 mhz
cDcl3: ch3oh (95: 5, v: v), 298 K, 600 mhz
cDcl3: ch3oh (95: 5, v: v), 298 K, 600 mhz
cDcl3: ch3oh (95: 5, v: v), 298 K, 600 mhz
ORCID
(5)2⊃c60
(5)2⊃c70
Marek P. Szymański
Agnieszka Szumna
http://orcid.org/0000-0003-0916-1575
http://orcid.org/0000-0003-3869-1321
experimental evidences indicate that this is not the case,
and such densely packed complexes are favoured.
Highly ordered structures of capsules and their com-
plexes are also reflected in their electronic circular dichro-
ism spectra (ECD). All spectra exhibit intensive effects
at lowest energy bands (280–350 nm, Figure 7). These
bands are the most sensitive to conformational inherent
chirality and symmetry of these molecules, as they come
from HOMO→LUMO transition involving an inherently
chiral chromphore (21). For cavitands and capsules that
are conformationally labile or disordered intensities of
these bands are substantially lower (even 100 times) (15).
Therefore, high intensities of CD bands and their similar
values for free capsules and complexes and indicate that
all compounds (capsules and complexes) exhibit ordered
conformations, which is in agreement with their stabiliza-
tion by hydrogen bonding.
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In conclusions we have synthesized new cavitands sub-
stituted with tripeptides and tetrapeptides and we have
shown that in relatively non-polar environment (chloro-
form or chloroform: methanol) the cavitands form dimeric
capsules. These capsules retain their porous structure and
they are able to quantitatively complex fullerenes C60 and
C70. Detailed structural studies indicate that the binding
motif involves the C-terminal amino acid from the pep-
tides’strands, while the remaining N-terminal part is posi-
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Acknowledgements
M.P.S. acknowledges the financial support of the Ministry of
Science and Higher Education, Poland (Diamond Grant 0206/
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Disclosure statement
No potential conflict of interest was reported by the authors.