Synthesis of cyclic peptide hemicryptophanes: Enantioselective recognition of a chiral zwitterionic guest

Article abstract of DOI:10.1039/c3cc44784g

The synthesis of the first members of a new class of cyclic peptide-containing hemicryptophanes is described. Synthesis was achieved through attachment of veratryl groups to the l-tyrosine side chains of a cyclic hexapeptide, c(YG)₃, followed by intramolecular cyclodehydration to generate the CTV unit. The diastereomeric P- and M-hemicryptophanes were generated in a 2 : 1 ratio and were separated by chromatography. The enantioselective binding properties of the hemicryptophanes were investigated by complexation with carnitine. Both isomers were found to have significant selectivity for binding (R)-carnitine. The Royal Society of Chemistry 2013.

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Synthesis of cyclic peptide hemicryptophanes: enantioselective
recognition of a chiral zwitterionic guest
James R. Cochrane, Aline Schmitt, Uta Wille and Craig A. Hutton*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
yield after 1 h. Cyclisation of the unprotected linear peptide 4
The synthesis of the first members of a new class of cyclic
peptide-containing hemicryptophanes is described. Synthesis
was achieved through attachment of veratryl groups to the
proceeded much faster than that of the corresponding protected
50 peptide 3 and without the formation of byproducts and poor
solubility that plagued purification of 6 generated from 3.
L-tyrosine side chains of
a cyclic hexapeptide, c(YG)3,
10 followed by intramolecular cyclodehydration to generate the
CTV unit. The diastereomeric P- and M-hemicryptophanes
were generated in a 2:1 ratio and were separated by
chromatography. The enantioselective binding properties of
the hemicryptophanes were investigated by complexation
15 with carnitine. Both isomers were found to have significant
selectivity for binding (R)-carnitine.
MeO
OMe
O
MeO
OMe
O
O
O
OMe
O
OMe
O
O
O
O
O
O
O
O
O
H
O
N
HO
OH
The use of molecular containers as supramolecular host
molecules that mimic enzymes and receptors has received
significant recent interest.1-3 Cryptophane hosts, which are
20 comprised of two cyclotriveratrylene (CTV) units, have been
investigated for their binding properties towards neutral and
charged guests,3 and for potential applications in chiral
recognition.3,4 Hemicryptophanes are related molecular
containers that consist of a single CTV moiety connected to a
25 capping moiety of C3-symmetry by linkers whose nature and
length can be varied (e.g., 1, Fig. 1).5-9 The dissymmetry of the
hemicryptophanes leads to heteroditopic host molecules that can
encapsulate two different guest molecules,6a or bifunctional
molecules.6b,c The heteroditopicity of hemicryptophanes has been
30 exploited for catalytic7 and recognition properties.6,8-9
Functionalised cyclic peptide scaffolds have been investigated
as host molecules capable of selective binding of anions and
cations,1,10-11 yet their incorporation into heteroditopic hosts is
rare.11 We report the synthesis of a new class of hemicryptophane
35 that incorporates a CTV unit connected to a cyclic hexapeptide
scaffold (e.g. 2, Fig. 1). The cyclic peptide–hemicryptophane
design incorporates a cyclic hexapeptide such that the three-fold
symmetry matches that of the CTV unit. Three tyrosine residues
provide both hydrophobic ‘walls’ to the cavity and functional
40 groups to link to the CTV unit. Glycine was chosen for the
alternate residues to facilitate the synthesis of the cyclic peptide
and minimise steric bulk within the cage.
N
H
N
H
O
O
H
N
H
N
O
HO
O
N
H
O
N
O
1- Dutasta
2 - this work
Fig. 1 Chiral hemicryptophanes
Hemicryptophanes are commonly assembled by appending
55 three linkers to a preformed CTV unit and attaching the linkers to
central ‘cap’;5a,6b,7b,8b however, such an approach was
a
considered implausible to link a CTV to a cyclic peptide. A
template approach was therefore chosen in which the three
veratryl units were attached to the cyclic peptide ‘cap’, with
60 intramolecular cyclodehydration generating the CTV unit as the
final step. Accordingly, the cyclic hexapeptide was triply
alkylated with bromoethyl vanillyl alcohol 75b,12 to generate
hemicryptophane precursor 8 in 55% yield. The CTV unit was
generated by treatment of 8 with formic acid under high dilution
65 conditions, providing hemicryptophane 2 in good yield (64%) as
a mixture of two diasteromers (Scheme 1). The P- and M-isomers
at the CTV unit were formed in a 2:1 ratio, and were readily
separated by column chromatography. The inherent chirality of
the cyclic peptide presumably induces a modest degree of
70 selectivity for one configuration of the CTV unit.
The absolute configuration of the CTV units of the
hemicryptophane isomers was determined by CD spectroscopy.
The spectrum of the major isomer exhibits a characteristic
negative–positive bisignate curve from 230–250 nm
75 corresponding to the P-configuration (ESI Fig. 10).13 The minor
isomer shows a similar but inverted signal, thus allowing the
assignment the M-configuration to the minor diastereomer.
The linear hexapeptide was prepared by standard microwave-
assisted Fmoc solid phase peptide synthesis. Cleavage of the
45 hexapeptide from the resin with 5% TFA in CH3CN:H2O (4:1)
gave the unprotected linear peptide [L-Tyr–Gly]3 4 in 85% yield.
Cyclisation of 4 generated the cyclic peptide 6 in quantitative
The 1H NMR spectra of hemicryptophanes PL-2 and ML-2
indicate that both molecules exhibit C3-symmetry, with only one
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set of resonances for each of the three tyrosyl, glycyl, veratryl
and linker units. ESI-MS experiments demonstrated the formation
of host–guest complexes with simple cations such as sodium,
cesium, ammonium and tetrabutyl ammonium, as well as with
zwitterions taurine and lysine (see ESI Figs 2,4–9).
OR
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DOI: 10.1039/C3CC44784G
5
a) 1% TFA
CH3CN:H2O (4:1)
[Tyr(tBu)-Gly]3
O
H
N
OR b) 5% TFA,
CH3CN:H2O (4:1)
OH
H
N
H
O
3
3 R = tBu, condition a), 91%
4 R = H, condition b), 85%
THPO
PyBOP, DMF
OR
THPO
THPO
OTHP
O
MeO
MeO
MeO
O
MeO
Br
O
N
H
7
O
O
O
O
O
NH
NH
HN
HN
O
Cs2CO3, DMF
55%
O
30 Fig. 2 1H NMR spectra (500 MHz, 298 K) from the titration of PL-2 with
(R)-carnitine R-9 in CD3CN.  Gly-NH; ‚ Tyr-NH; ® OMe; l NMe; ê
Tyr-βHs.
O
H
N
O
O
O
O
H
N
N
H
N
RO
O
H
N
H
H
N
O
N
H
experiments, which indicated the presence of a 1:1 complex ([PL-
2+R-9+H]+; m/z 1308.57, ESI Fig. 5). Association constants (Ka)
35 were determined from plots of Δδ vs. [guest 9]/[host 2],15 and
ranged from 690–4100 (Fig. 3 and Table 1). In all titration
experiments only a single set of peaks were observed indicating
fast exchange on the NMR time scale between the free and
complexed species. The complexes retain the C3 symmetry of the
40 host. Significant downfield shifts were observed for both the
glycyl and tyrosyl NH protons of the cyclic peptide scaffold, with
these induced shifts indicating encapsulation of the guest within
the cavity.16
5 R = tBu
6 R = H,
OR
TFA
O
O
quant., 1 step from 4, OR
24%, 2 steps from 3
8
HCO2H
MeO
OMe
O
O
OMe
MeO
O
MeO
O
O
O
OMe
O
O
O
O
O
+
O
O
O
O
H
N
O
H
N
N
H
N
H
H
N
H
N
H
O
O
H
N
H
N
H
N
N
O
O
N
N
H
O
H
O
O
O
2:1
64%
ML-2
PL-2
Scheme 1 Synthesis of hemicryptophanes PL-2 and ML-2.
The use of inherently chiral hemicryptophanes in
enantioselective recognition shows much promise, but has yet to
10 widely explored.9 The presence of fixed asymmetric centres
introduces additional chiral recognition elements into the host
structure and also avoids difficulties in the separation of
enantiomeric hemicryptophanes by chiral HPLC or derivatisation
of the hemicrytophanes into diastereomers.4,13 The binding
15 properties of PL-2 and ML-2 were investigated employing
carnitine 9 as the guest molecule. L-Carnitine (R-9) is involved in
fatty acid transport through the formation of fatty acid–carnitine
esters, while the unnatural D- or (S)-isomer has no known
biological role.14 Carnitine 9 was chosen for investigation as it
20 allows exploration of enantioselective binding properties in
addition to exploitation of the ditopic nature of the hosts in
association with the zwitterionic character of the guest.6b,c
Binding studies of the hemicryptophane isomers PL-2 and ML-
2 with the (R)- and (S)-enantiomers of carnitine 9 were
25 undertaken through 1H NMR spectroscopic analysis upon
titration in CD3CN (Fig. 2). Job plot analysis of the titration of
(R)-carnitine with PL-2 suggested a 1:1 binding model (ESI Fig.
10). Further evidence for this 1:1 ratio was obtained by ESI-MS
45
Fig. 3 Binding-induced change in 1H NMR δ of glycyl-NH protons of
host 2 as a function of [guest 9]:[host 2] ratio.
Table 1 Association constants (Ka) of hosts PL-2 and ML-2 with guests
(R)- and (S)-carnitine 9
a
Host
PL-2
PL-2
ML-2
ML-2
Guest
R-9
S-9
Δδmax /ppm
–0.8945
–0.5937
–0.7194
–0.4791
Ka b /M–1
4.1 ± 0.3 × 103
2.7 ± 0.2 × 103
9.1 ± 0.1 × 102
6.9 ± 0.5 × 102
R-9
S-9
a
δ Gly-NH of host 2.
50 b determined using WinEQNMR2 (ref. 15).
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Binding of carnitine within the cavity of PL-2 in acetonitrile
Notes and references
was found to be enantioselective with R-9 bound with a 1.5-fold
greater Ka than S-9. Binding of carnitine within the cavity of ML-
2 was also found to be enantioselective, with R-9 having a 1.3-
School of Chemistry and Bio21 Molecular Science and Biotechnology
View Article Online
50 Institute, University of Melbourne, Parkville, Victoria, 3010, Australia.
DOI: 10.1039/C3CC44784G
Fax: +613 9347 8124; Tel: +613 8344 2393; E-mail:
chutton@unimelb.edu.au
5
fold
greater
Ka.
Although
the
D-tyrosine-derived
† Electronic Supplementary Information (ESI) available: Synthetic
procedures, NMR and mass spectra of PL-2 and ML-2 and complexes,
55 NMR titration data for Ka determinations. See DOI: 10.1039/b000000x/
1. (a) M. J. Hardie, “Cyclotriveratrylene and Cryptophanes”, in
Supramolecular Chemistry: from Molecules to Nanomaterials, P. A.
Gale, & J. W. Steed (eds), John Wiley & Sons Ltd., Chichester, UK,
2012, 3, 895-916. (b) J. W. Steed, D. R. Turner, K. J. Wallace, Core
hemicryptophanes were not synthesised, association constants for
the PDR-complex, for example, can be determined from the
enantiomeric MLS-complex. Accordingly, a 5.9-fold difference in
association constants of R-9 with the PL-2 and PD-2 hosts was
10 calculated. In contrast, switching from PL-2 to ML-2 results in a
4.5-fold drop in binding affinity for R-9. These results suggest
that of all four stereoisomers of the hemicryptophane host 2, the
PL-2 host is matched to bind with (R)-carnitine R-9.
Modelling of the inclusion complex was performed with the
15 Gaussian 03 program17 using a two-layer ONIOM method, where
the three ‘walls’ comprising the tyrosine side-chain and
ethyleneglycol units were computed at the B3LYP/6-31G* level
of theory, with the remainder treated with the semiempirical PM3
method. Carnitine binds to the host in a ditopic fashion exploiting
20 hydrogen-bonding interactions between the cyclic peptide moiety
and the carnitine carboxylate group, together with cation-π/C–H-
π interactions between the CTV unit and the carnitine quaternary
ammonium group (Fig. 4). The amide groups are all twisted such
that the N–H groups are directed into the cavity to make H-bonds
25 with the carnitine carboxylate. The glycyl NHs, which point
‘down’ (away from the CTV unit), are significantly more
distorted than the ‘upwards’ pointing tyrosyl NHs, consistent
with a greater shift of the glycyl NH protons in the 1H NMR
spectrum. No H-bond interactions are observed between the host
30 and the carnitine OH group; instead the OH group is involved in
an intramolecular H-bond with the carnitine carboxylate.
60
Concepts in Supramolecular Chemistry and Noanochemistry, John
Wiley & Sons Ltd., Chichester, UK, 2007.
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70 5. (a) B. Chatelet, E. Payet, O. Perraud, P. Dimitrov-Raytchev, L.-L.
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Fig. 4: Modelled PL-2:S-9 complex.
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In conclusion, we have designed and synthesised a new class of
35 hemicryptophane cage, where the CTV unit is attached to a cyclic
peptide scaffold. The efficient synthesis yielded hemicryptophane
2 as a mixture of diastereomers in an overall yield of 30% over 4
steps. The inherent chirality of the L-tyrosine-containing cyclic
peptide scaffold induces moderate selectivity for formation of the
40 P-configuration of the CTV unit. Host–guest interactions of the
hemicryptophanes with the chiral, biologically relevant zwitterion
carnitine were investigated, with the hemicryptophanes exhibiting
modest chiral discrimination behaviour. The modular design of
the cyclic peptide-based hemicryptophane framework should
45 enable the synthesis of a range of analogues of these host
molecules, with binding properties tunable through substitution of
the amino acid residues.
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16. As a control, the Ka of R-9 with cyclic peptide 6 (in d6-DMSO) was
determined and shown to be 2.6-fold less than with PL-2, indicating
that both the cyclic peptide and CTV moieties of the
hemicryptophane are required for optimum binding (see ESI).
17. M. J. Frisch, et al., Gaussian 09, Revision A.01, Gaussian,
Wallingford, CT, 2009.
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