Synthesis and host-guest studies of chiral N-linked peptidoresorc[4]arenes

Article abstract of DOI:10.1021/jo7016636

(Figure Presented) Four cone resorc[4]arene octamethyl ethers (10, 11, ent-10, and ent-11) tetrafunctionalized at the feet with valyl-leucine [LL- (6); DD- (ent-6)] and leucyl-valine [LL- (9); DD- (ent-9)] methyl esters have been synthesized. These compounds, obtained by conjugation of macrocycle tetracarboxylic acid chlorides with the appropriate terminal amino groups of the above dipeptides, are N-linked peptidoresorc[4]arenes. We found that these macrocycles (M) are capable of recognizing the homologue dipeptides as guests (G), both in solution and in the gas phase, by forming relatively stable host-guest complexes ([M·G]), resistant to chromatographic purification but not to heating. Complexation phenomena between M and G in solution were investigated by NMR methods, including NMR DOSY experiments, for the detection of translational diffusion. Heteroassociation constants of 2030 and 186 M ^-1 were obtained by the Foster-Fyfe method for the complexes [10·6] and [10·ent-6], respectively, the latter being comparable to the self-association constant of dipeptide itself. Conversely, the structural features of the proton-bound complexes [M·H·G_n] ⁺ (n = 1, 2), generated in the gas phase by electrospray ionization mass spectrometry (ESI-MS), were investigated by collision-induced dissociation (CID) experiments. In both cases, the four N-linked peptidoresorc[4]arenes were shown to act as synthetic receptors and to recognize the homologue dipeptide by means of hydrogen bonds.

Full text of DOI:10.1021/jo7016636

Synthesis and Host-Guest Studies of Chiral N-Linked
Peptidoresorc[4]arenes†
Bruno Botta,*,‡ Ilaria D’Acquarica,‡ Giuliano Delle Monache,‡ Deborah Subissati,‡
Gloria Uccello-Barretta,*,§ Massimo Mastrini,§ Samuele Nazzi,§ and Maurizio Speranza*,‡
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente AttiVe, UniVersita` “La
Sapienza”, P.le Aldo Moro 5, 00185 Roma, Italy, and Dipartimento di Chimica e Chimica Industriale,
UniVersita` di Pisa, Via Risorgimento 35, 56125 Pisa, Italy
bruno.botta@uniroma1.it; gub@dcci.unipi.it; maurizio.speranza@uniroma1.it
ReceiVed July 30, 2007
Four cone resorc[4]arene octamethyl ethers (10, 11, ent-10, and ent-11) tetrafunctionalized at the feet
with valyl-leucine [LL- (6); DD- (ent-6)] and leucyl-valine [LL- (9); DD- (ent-9)] methyl esters have been
synthesized. These compounds, obtained by conjugation of macrocycle tetracarboxylic acid chlorides
with the appropriate terminal amino groups of the above dipeptides, are N-linked peptidoresorc[4]arenes.
We found that these macrocycles (M) are capable of recognizing the homologue dipeptides as guests
(G), both in solution and in the gas phase, by forming relatively stable host-guest complexes ([M‚G]),
resistant to chromatographic purification but not to heating. Complexation phenomena between M and
G in solution were investigated by NMR methods, including NMR DOSY experiments, for the detection
of translational diffusion. Heteroassociation constants of 2030 and 186 M-1 were obtained by the Foster-
Fyfe method for the complexes [10‚6] and [10‚ent-6], respectively, the latter being comparable to the
self-association constant of dipeptide itself. Conversely, the structural features of the proton-bound
complexes [M‚H‚Gn]+ (n ) 1, 2), generated in the gas phase by electrospray ionization mass spectrometry
(ESI-MS), were investigated by collision-induced dissociation (CID) experiments. In both cases, the four
N-linked peptidoresorc[4]arenes were shown to act as synthetic receptors and to recognize the homologue
dipeptide by means of hydrogen bonds.
Introduction
self-assembling capsules.5 A number of calix[4]arenes with
either R-amino acids or peptides attached to the upper rim are
Calixarenes have had an enormous impact in supramolecular
chemistry,1 as building blocks in the field of molecular
recognition. The modulation of shape and size of their cavities
led to the preparation of cavitands,2,3 (hemi)carcerands,4 and
(2) (a) Starnes, S. D.; Rudkevich, D. M.; Rebek, J., Jr. J. Am. Chem.
Soc. 2001, 123, 4659. (b) Lu¨cking, U.; Tucci, F. C.; Rudkevich, D. M.;
Rebek, J., Jr. J. Am. Chem. Soc. 2000, 122, 8880.
(3) (a) Rudkevich, D. M.; Rebek, J., Jr. Eur. J. Org. Chem. 1999, 1991.
(b) Rudkevich, D. M. Bull. Chem. Soc. Jpn. 2002, 75, 393.
(4) Cram, D. J.; Cram, J. M. Container Molecules and their Guests; The
Royal Society of Chemistry: Cambridge, UK, 1994.
(5) (a) Heinz, T.; Rudkevich, D. M.; Rebek, J., Jr. Nature 1998, 394,
764. (b) Rudkevich, D. M. In Calixarenes 2001; Asfari, Z., Bo¨hmer, V.,
Harrowfield, J., Vicens, J., Eds.; Kluwer: Dordrecht, The Netherlands, 2001;
pp 155-180. (c) Hof, F.; Craig, S. L.; Nuckolls, C.; Rebek, J., Jr. Angew.
Chem., Int. Ed. 2002, 41, 1488.
† Dedicated to the memory of Dmitry Rudkevich, an enthusiastic, exceptional
scientist, and friend who passed away too early.
‡ Universita` degli Studi di Roma “La Sapienza”.
§ Universita` di Pisa.
(1) (a) Gutsche, C. D. Calixarenes ReVisited; The Royal Society of
Chemistry: Cambridge, UK, 1998. (b) Calixarenes 2001; Asfari, Z.,
Bo¨hmer, V., Harrowfield, J., Vicens, J., Eds.; Kluwer Academic: Dordrecht,
The Netherlands, 2001.
10.1021/jo7016636 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/25/2007
J. Org. Chem. 2007, 72, 9283-9290
9283

Botta et al.
a
known,6 and the first generation of calix[4]arene peptide
dendrimers has recently been synthesized.7 Mostly, the presence
of a N- or C-linked peptide endows the macrocycle with novel
properties, such as the recognition of protein surfaces8 and
carbohydrates,9 the formation of stable inclusion complexes,10
and self-assembly phenomena.11 A four-armed hydrophilic
peptidocalix[4]arene library consisting of 1000 members, suit-
able for peptide recognition in aqueous media, has recently been
developed.12 Only a few papers reported on cavitands equipped
with dipeptide residues. For example, either peptides of helical
conformation have been connected to a cavitand platform, using
a thioether bridge at the external carbon of the aromatic rings,13
or the C-termini of four short peptide chains have been linked
to the aromatic amino methyl groups.14
We have previously functionalized resorc[4]arene octamethyl
ethers at the feet with L- and D-valine ethyl ester units,15 and
later exploited their capability of enantiodiscriminating amino
acidic guests16 in the gas phase by mass spectrometry (MS).
The limited body of work addressed to investigate by MS
techniques the gas-phase stability of host-guest complexes
formed by calixarenes and resorcarenes with organic molecular
guests has been discussed in detail in a recent comprehensive
review.17 In this paper we report on the synthesis and host-
guest properties of N-linked peptidoresorc[4]arene octamethyl
SCHEME 1
a Reagents and conditions: (i) 2 N NaOH, EtOH, reflux, 4 h; glacial
AcOH; (ii) thionyl chloride, THF, reflux, 4 h.
ethers, obtained by conjugation of macrocycle tetracarboxylic
acid chlorides in the cone conformation with the appropriate
terminal amino groups of valyl-leucine and leucyl-valine methyl
esters. Complexation phenomena between peptidoresorc[4]-
arenes and the homologue dipeptides as guests were investigated
in solution by NMR methods and in the gas phase by ESI-MS.
Results and Discussion
Synthesis of the N-Linked Peptidoresorc[4]arenes. We
chose for the synthesis resorc[4]arene octamethyl ether tetraester
1 (Scheme 1) in the cone conformation for the presence of four
all-cis substituents that could preorganize the peptide chain on
the same side of the scaffold. Resorcarene 1, obtained as
previously described,18 was hydrolyzed to tetracarboxylic acid
2 (Scheme 1) and quantitatively converted into the correspond-
ing acid chloride 3 by reaction with thionyl chloride in
tetrahydrofuran (THF).
(6) (a) Sansone, F.; Barboso, S.; Casnati, A.; Fabbi, M.; Pochini, A.;
Ugozzoli, F.; Ungaro, R. Eur. J. Org. Chem. 1998, 897. (b) Sansone, F.;
Barboso, S.; Casnati, A.; Sciotto, D.; Ungaro, R. Tetrahedron Lett. 1999,
40, 6821. (c) Lazzarotto, M.; Sansone, F.; Baldini, L.; Casnati, A.; Cozzini,
P.; Ungaro, R. Eur. J. Org. Chem. 2001, 595. (d) Casnati, A.; Sansone, F.;
Ungaro, R. Acc. Chem. Res. 2003, 36, 246 and references cited therein
from the authors laboratory. (e) Hioki, H.; Yamada, T.; Fujioka, C.; Kodama,
M. Tetrahedron Lett. 1999, 40, 6821. (f) Hioki, H.; Kubo, M.; Yoshida,
H.; Bando, M.; Ohnishi, Y.; Kodama, M. Tetrahedron Lett. 2002, 43, 7949.
(g) Frkanec, L.; Visnjevac, A.; Kojic-Prodic, B.; Zinic, M. Chem. Eur. J.
2000, 6, 442. (h) Shuker, S. B.; Esterbrook, J.; Gonzalez, J. Synlett 2001,
210.
Standard peptide coupling was employed to gather the four
dipeptide chains 6, 9, ent-6, and ent-9. Commercially available
N-BOC-L-valine 4 (Figure 1) was coupled with L-leucine methyl
ester hydrochloride 5 in the presence of HOBT and triethylamine
to afford, after deprotection with TFA-DCM, dipeptide L-valyl-
L-leucine methyl ester 6, as TFA salt, in a 93% overall yield.
Analogously, dipeptide L-leucyl-L-valine methyl ester 9 (TFA
salt, 95% overall yield) was prepared from commercially
available N-BOC-L-leucine 7 and L-valine methyl ester hydro-
chloride 8. The same procedure was applied to the DD-dipeptides
series, to afford the TFA salts of dipeptides ent-6 and ent-9
with comparable yields, as summarized in Figure 1. The
conjugation of acid chloride 3 with the single dipeptides gave
the final compounds. In a general reaction, diisopropylethy-
lamine (DIPEA) was added, under nitrogen, to a dry THF
solution of 3 and the reaction mixture was stirred at room
temperature for 20 min. After the slow addition of the proper
dipeptide (in a 1.5 excess for each acid chloride group), the
mixture was held at reflux for 3 h. Chromatographic purification
afforded the N-linked peptidoresorc[4]arenes 10, 11, ent-10, and
ent-11 in 48-51% yield, starting from dipeptides 6, 9, ent-6,
and ent-9, respectively (Figure 2). The structures of compounds
10, 11, ent-10, and ent-11 were confirmed by 1H and 13C NMR
spectroscopy and by electrospray ionization mass spectrometry
(ESI-MS).
(7) Xu, H.; Kinsel, G. R.; Zhang, J.; Rudkevich, D. M. Tetrahedron 2003,
59, 5837.
(8) (a) Hamuro, Y.; Calama, M. C.; Park, H. S.; Hamilton, A. D. Angew.
Chem. 1997, 109, 2797; Angew. Chem., Int. Ed. 1997, 36, 2680. (b) Park,
H. S.; Lin, Q.; Hamilton, A. D. Biopolymers (Peptide Science) 1998, 47,
285. (c) Park, H. S.; Lin, Q.; Hamilton, A. D. J. Am. Chem. Soc. 1999,
121, 8. (d) Lin, Q.; Hamilton, A. D. Compt. Rend. 2002, 5, 441. (e) Francese,
S.; Cozzolino, A.; Caputo, I.; Esposito, C.; Martino, M.; Gaeta, C.; Troisi,
F.; Neri, P. Tetrahedron Lett. 2005, 46, 1611.
(9) Sansone, F.; Baldini, L.; Casnati, A.; Lazzarotto, M.; Faimani, G.;
Ugozzoli, F.; Ungaro, R. PNAS 2002, 99, 4842.
(10) Sansone, F.; Baldini, L.; Casnati, A.; Chierici, E.; Ugozzoli, F.;
Ungaro, R. J. Am. Chem. Soc. 2004, 126, 6204.
(11) Segura, M.; Bricoli, B.; Casnati, A.; Munoz, E. M.; Sansone, F.;
Ungaro, R.; Vicent, C. J. Org. Chem. 2003, 68, 6296.
(12) Kubo, M.; Nashimoto, E.; Tokiyo, T.; Morisaki, Y.; Kodama, M.;
Hioki, H. Tetrahedron Lett. 2006, 47, 1927.
(13) (a) Gibb, B. C.; Mezo, A. R.; Causton, A. S.; Fraser, J. R.; Tsai, F.
C. S.; Sherman, J. C. Tetrahedron 1995, 51, 8719. (b) Causton, A. S.;
Sherman, J. C. Bioorg. Med. Chem. 1999, 7, 23. (c) Mezo, A. R.; Sherman,
J. C. J. Am. Chem. Soc. 1999, 121, 8983. (d) Causton, A. S.; Sherman, J.
C. J. Peptide Sci. 2002, 8, 275.
(14) Berghaus, C.; Feigel, M. Eur. J. Org. Chem. 2003, 3200.
(15) Botta, B.; Delle Monache, G.; Salvatore, P.; Gasparrini, F.; Villani,
C.; Botta, M.; Corelli, F.; Tafi, A.; Gacs-Baitz, E.; Santini, A.; Carvalho,
C. F.; Misiti, D. J. Org. Chem. 1997, 62, 932.
(16) (a) Botta, B.; Botta, M.; Filippi, A.; Tafi, A.; Delle Monache, G.;
Speranza, M. J. Am. Chem. Soc. 2002, 124, 7658. (b) Tafi, A.; Botta, B.;
Botta, M.; Delle Monache, G.; Filippi, A.; Speranza, M. Chem. Eur. J.
2004, 10, 4126. (c) Botta, B.; Subissati, D.; Tafi, A.; Delle Monache, G.;
Filippi, A.; Speranza, M. Angew. Chem., Int. Ed. 2004, 43, 4767. (d) Botta,
B.; Caporuscio, F.; Subissati, D.; Tafi, A.; Botta, M.; Filippi, A.; Speranza,
M. Angew. Chem., Int. Ed. 2006, 45, 2717. (e) Botta, B.; Caporuscio, F.;
D’Acquarica, I.; Delle Monache, G.; Subissati, D.; Tafi, A.; Botta, M.;
Filippi, A.; Speranza, M. Chem. Eur. J. 2006, 12, 8096.
The 1H and 13C NMR spectral data were consistent with the
distribution pattern of a resorc[4]arene containing four identical
side chains. The inner protons (i.e., H-25, H-26, H-27, and H-28)
gave only one signal, as well as the outer ones (H-5, H-11,
(18) Botta, B.; Di Giovanni, M. C.; Delle Monache, G.; De Rosa, M.
C.; Gacs-Baitz, E.; Botta, M.; Corelli, F.; Tafi, A.; Santini, A.; Benedetti,
E.; Pedone, C.; Misiti, D. J. Org. Chem. 1994, 59, 1532.
(17) Vincenti, M.; Irico, A. Int. J. Mass Spectrom. 2002, 214, 23 and
references cited therein.
9284 J. Org. Chem., Vol. 72, No. 24, 2007

Chiral N-Linked Peptidoresorc[4]arenes
FIGURE 1. Building blocks for N-linked peptidoresorc[4]arenes.
TABLE 1. Chemical Shifts (δ, ppm) and Chemical Shift Variation
(∆δ, ppm) of ent-6 (600 MHz, CDCl3, 298 K)
concn
0.1 mM
concn
10 mM
∆δ (ppm)
δ10mM - δ0.1mM
protons
NH
6.98
4.59
3.76
3.74
2.27
1.06
1.05
0.93
0.92
7.50
4.45
3.93
3.72
2.21
1.07
1.03
0.91
0.89
0.52
0.14
0.17
0.02
0.06
0.01
0.02
0.02
0.03
CH-N (leucine)
CH-N (valine)
OMe
CH (valine)
Me (valine)
Me (valine)
Me (leucine)
Me (leucine)
FIGURE 2. N-linked peptidoresorc[4]arene octamethyl ethers.
H-17, and H-23). The same was observed for the methine
protons (H-2, H-8, H-14, and H-20) and for the elements of the
four side chains. The methoxyl groups as well as those for
CAr-C and CAr-O carbons are instead doubled, because, for
instance, the 4-OMe is equivalent to the 22-OMe, but not to
the 24-OMe. These findings are consistent with the introduction
of stereogenic centers in the side chains, which destroyed all
the symmetry planes of the resorc[4]arene skeleton. The above
data are in agreement with a C4 group of symmetry; in fact the
molecule is superimposable to itself after a clockwise rotation
of 90°. Finally, the broadening (sensitive to temperature
increase) of the two methoxyl group signals was attributed to
the influence of the peptide chain19 on the interchange time
between the two flattened cone forms, which becomes compa-
rable to the NMR time scale. Surprisingly, all the synthesized
N-linked peptidoresorc[4]arenes showed in the NMR spectra
the presence of additional signals for each proton and carbon
of the dipeptide chains. These minor peaks, still present after
purification on Sephadex LH-20, were attributed to host-guest
[M‚G] complexes of peptidoresorc[4]arenes 10 and 11 (M), the
guests (G) being the dipeptide reactants themselves (6 and 9,
respectively). Only after heating at reflux in MeOH for 4 h and
a further purification on Sephadex did we observe clean NMR
spectra for compounds 10, 11, ent-10, and ent-11, the spectra
of ent-10 and ent-11 being superimposable with those of the
corresponding LL-forms (10 and 11, respectively). As expected,
optical rotations with coincident absolute value and opposite
sign were observed for each couple of enantiomers.
the interaction between peptidoresorc[4]arene 10 and dipeptide
6 or ent-6 in CDCl3 solutions. Because both resorcarene and
dipeptide contain several hydrogen bond donor and acceptor
groups, the analysis of self-association processes concerning the
single 10 or 6/ent-6 compounds was mandatory prior to the
analysis of heteroassociation processes. The self-association
1
processes of 10 and 6 were thus investigated by the H NMR
analysis of chemical shifts of progressively diluted CDCl3
solutions. The low solubility in CDCl3 of dipeptides 6/ent-6
allowed the use of a concentration range of 10.0-0.1 mM only.
Within this concentration gradient, the signals for the amide
NH proton and the two methine nuclei bonded to the two
stereogenic centers of ent-6 underwent remarkable chemical shift
variations, whereas those for the methyl groups almost did not
move (Table 1, Figure 3). The above trend strongly supports
the self-aggregation of ent-6 by hydrogen bond interactions.
On the hypothesis that dimerization would be more favored
than the formation of higher order self-aggregates (at least within
the low concentration ranges investigated), a self-association
constant of 328 M-1 was calculated by fitting chemical shifts
versus concentration (Table 1) on the basis of eq 1 (see also
Figure 4).
(δobs - δm)(δd - δm)
C )
(1)
2
Complexation Studies in Solution by NMR Spectroscopy.
For a deeper insight, we investigated by NMR spectroscopy
2K(δd - δobs
)
The presence of self-association equilibria was fully confirmed
by NMR DOSY20 measurement of diffusion coefficients (D)
(19) (a) Ho¨gberg, A. G. S. J. Org. Chem. 1980, 45, 4498. (b) Ho¨gberg,
A. G. S. J. Am. Chem. Soc. 1980, 102, 6046.
J. Org. Chem, Vol. 72, No. 24, 2007 9285

Botta et al.
FIGURE 5. DOSY maps (600 MHz, CDCl3, 298 K) of 0.1 (D ) 8.1
× 10-6 cm2 s-1) and 4 mM (D ) 5.7 × 10-6 cm2 s-1) solutions of
dipeptide ent-6.
TABLE 2. Diffusion Coefficients (D) of ent-6 at Variable
FIGURE 3. 1H NMR spectra (600 MHz, CDCl3, 298 K) of a 0.1 mM
(a) and a 10 mM (b) solution of dipeptide ent-6.
Concentrations (600 MHz, CDCl3, 298 K)
concn (mM)
Xd
D (×10-6 cm2 s-1
)
10.0
4.0
2.0
0.1
0.68
0.54
0.43
0.06
4.9
5.7
6.1
8.1
in the dimer (Dd) and monomer (Dm), diffusion coefficients of
3.4 × 10-6 and 8.3 × 10-6 cm2 s-1, respectively, were
calculated by eq 3, where Xm and Xd are the molar fractions of
the monomer and the dimer forms of ent-6.
Dobs ) DmXm + DdXd
(3)
The analogous NMR analysis of peptidoresorc[4]arene 10 in
the concentration range 10.0-0.1 mM did not show significant
variations both of the chemical shifts and of the diffusion
coefficient which did not change from the initial value of 4.8
× 10-6 cm2 s-1. This behavior can be attributed to the very
close arrangement of the four dipeptide side chains that favors
intramolecular interactions in place of intermolecular associative
processes. The heteroassociation between 10 and 6/ent-6 was
investigated in conditions where the self-aggregation of 6 was
minimized, i.e., using very low concentrations of 6 (0.1 mM)
versus a high molar excess of 10 (from 4 to 16 mM). By the
Foster-Fyfe method,22 the heteroassociation constant for the
homochiral complex [10‚6] was shown to be 2030 M-1 (Figure
6 and Table 1S in the Supporting Information). For the
heterochiral complex [10‚ent-6], a significantly lower heteroas-
sociation constant was obtained by the same method (186 M-1),
which is comparable to the self-association constant of dipeptide
itself (see Table 2S in the Supporting Information).
FIGURE 4. Self-association constant determination: δobs is the
observed chemical shift variation of the NH proton of ent-6.
in solution, which are correlated by the hydrodynamic radius
(RH) to the molecular sizes by means of eq 2:
kT
cπηRH
D )
(2)
where k is the Boltzmann constant, T is the absolute temperature,
η is the solvent viscosity, and c is a numerical factor21
substantially dependent on the size of diffusing species.
The resort to NMR DOSY in the analysis of homo- and
heteroassociation phenomena is justified by the fact that
complexation processes originate species with increased sizes,
and hence with lower diffusion coefficients. Notably, DOSY
maps of compound ent-6 (Figure 5, Table 2) at 0.1 mM revealed
a diffusion coefficient of 8.1 × 10-6 cm2 s-1, which decreased
to 4.9 × 10-6 cm2 s-1 at 10 mM, in agreement with an increase
of molecular size due to self-aggregation processes.
Once having calculated the self-association constant of
dipeptide ent-6, we obtained the dimer versus monomer molar
fractions (Table 2) at each concentration value. Taking into
account that in a fast exchange condition the observed diffusion
coefficients (Dobs) represent the weighted average of their values
The analysis of the diffusion coefficients of mixtures 10‚6
gave further support to the tight interaction between guest 6
and host 10. Because we could not obtain accurate diffusion
coefficient measurements of 6 in the same experimental condi-
tions employed for the determination of the stability constant,
we analyzed the DOSY map of an equimolar mixture of 10‚6
(0.1 mM), in which the molar fraction of 6 as dimer was quite
low (about 6%). The diffusion coefficient of 6 in the mixture
was shown to be 7.5 × 10-6 cm2 s-1, which was lower than
the value found for the pure dipeptide 6 (8.1 × 10-6 cm2 s-1
)
(20) (a) Johnson, C. S., Jr. Prog. NMR Spectrosc. 1999, 34, 203. (b)
Special Issue, NMR and Diffusion. Magn. Reson. Chem. 2002, 40, S1-
S152.
at the same concentration. The diffusion coefficient of 10
(21) Zuccaccia, D.; Macchioni, A. Organometallics 2005, 24, 3476.
(22) Foster, R.; Fyfe, C. A. Trans. Faraday Soc. 1965, 61, 1626.
9286 J. Org. Chem., Vol. 72, No. 24, 2007

Chiral N-Linked Peptidoresorc[4]arenes
FIGURE 6. Heteroassociation constant determination by the Foster-
Fyfe method: ∆δobs is the observed chemical shift variation (δmix
-
δf, Hz) for the NH proton of 6 (0.1 mM) in the presence of an increasing
molar excess of 10 (from 4 to 16 mM) and [10] is the total concentration
of the peptidoresorc[4]arene.
remained 4.8 × 10-6 cm2 s-1. The small difference of diffusion
coefficients of 6 was unambiguously attributed to heteroasso-
ciation processes rather than to the viscosity gradient of the
analyzed solution (see eq 2), as was confirmed by using
tetramethylsilane (TMS) as the internal standard;23 the TMS
diffusion coefficient was the same in both a solution of pure 6
(0.1 mM) and in an equimolar mixture of 10‚6 (0.1 mM).
In the rapid exchange conditions, the diffusion coefficient of
6 in the mixture 10‚6 is the weighed average of the value in
free (Df) and complexed (Dc) forms (see eq 4):
FIGURE 7. 1D ROESY spectrum (600 MHz, CDCl3, 298 K, mixing
time ranging 0.5 s) showing the perturbation of the leucine methine
proton of 6 in the [10‚6] complex (molar ratio 1:10).
interactions with valine and leucine methine protons of resor-
carene dipeptide chains, in conjunction with an effect on the
aromatic proton at δ 6.29 ppm that is located between the two
methoxy groups (Figure 7). Therefore, we concluded that
interaction between resorcarene 10 and its homologous dipeptide
occurs at the external surface of resorcarene by means of
attractive hydrogen bond interactions involving polar groups
of both host and guest.
Complexation Studies in the Gas Phase by ESI-MS. We
first run ESI-MS spectra of free peptidoresorc[4]arenes 10-11
and dipeptides 6-9 to investigate their mass spectral features.
As already observed for tetraethylresorc[4]arenes,25 compounds
10 and 11 (MW ) 1737 amu) show a strong tendency to form
complexes with sodium (Na+) (m/z 1760), which is a ubiquitous
contaminant present in glassy vessels (see Figure 8 for
compound 10). The most intense peak in the spectrum is that
of the doubly charged disodium adduct [M‚Na2]2+, at m/z 891.5.
The protonated molecule [M‚H]+ at m/z 1738 was barely
observable. The change of solvent from methanol to acetonitrile
did not help in increasing the [M‚H]+ signal, since essentially
the same, though weaker spectrum was observed. Addition of
acetic acid did not help either, because only extensive decom-
position of the sample took place.
The ESI-MS spectrum of dipeptide 6 (L-valyl-L-leucine
methyl ester, MW ) 244 amu) is shown in Figure 9. The most
abundant signal is due to the protonated molecule [G‚H]+, at
m/z 245, and to the proton-bound dimer [G2‚H]+, at m/z 489,
which is accompanied by the sodium-bound analogue [G2‚Na]+,
at m/z 511. Figure 10 presents the ESI-MS spectrum of the
[resorcarene 10]:[dipeptide 6] ) 1:7 mixture. Besides the above
signals typical of free M and G, we observed the appearance of
new signals corresponding to the complexes [M‚H‚G]+ (m/z
1982), [M‚H2‚G]2+ (m/z 991.5), and [M‚H‚Na‚G]2+ (m/z
1002.5). In spite of the sevenfold excess of G relative to M,
the minor abundance of the m/z 489 ([G2‚H]+) and 511 ([G2‚
Dobs ) XfDf + XcDc
(4)
where Xf and Xc are the molar fractions of the free and the
complexed forms of 6, respectively.
Thus, by using the NMR approach proposed for the analysis
of the diffusion of cyclodextrin inclusion complexes,24 we
applied the approximation that the diffusion coefficient of 6 in
the bound state was equal to that of the complexing agent
(resorcarene 10), and we calculated by eq 5 the molar fractions
of bound 6 (Xc) by a single-point measurement:
Dobs - Df
Dc - Df
Xc )
(5)
A bound fraction value of 0.18 was obtained, in very good
agreement with the value (0.15) expected (on the basis of the
association constant) by linear fitting of chemical shift data.
Because the heteroassociation constant of the heterochiral
complex [10‚ent-6] was lower than the self-association of
dipeptide ent-6, diffusion measurements did not allow heteroas-
sociation phenomena to be shown unambiguously aside from
self-aggregation processes for ent-6.
Information on the nature of heteroassociation processes
involving the formation of the highly stable [10‚6] complex was
achieved by measuring intermolecular dipolar interactions by
1D ROESY of 6 (1 mM solutions) in the presence of resorcarene
10 (10 mM), thus providing a high bound molar fraction of the
[10‚6] complex, in spite of the autoaggregation tendency of
dipeptide. In particular, perturbation of the leucine methine
proton at δ 4.55 ppm of 6 produced detectable dipolar
(23) Cabrita, E. J.; Berger, S. Magn. Reson. Chem. 2001, 39, S142.
(24) Wimmer, R.; Aachmann, F. L.; Larsen, K. L.; Petersen, S. B.
Carbohydr. Res. 2002, 337, 841.
(25) (a) Ma¨kinen, M.; Vainiotalo, P.; Rissanen, K. J. Am. Soc. Mass
Spectrom. 2002, 13, 851. (b) Ma¨kinen, M.; Vainiotalo, P.; Nissinen, M.;
Rissanen, K. J. Am. Soc. Mass Spectrom. 2003, 14, 143.
J. Org. Chem, Vol. 72, No. 24, 2007 9287

Botta et al.
intense peaks at m/z 869.5 ([M‚H2]2+), 880.5 ([M‚H‚Na]2+),
and 888.5 ([M‚H‚K]2+). The formation of the [M‚H2‚G2]+ (m/z
2227) complex could not be directly observed in the ESI-MS
spectrum because of mass range limitation of the instrument
used (e2000 amu). However, its formation is witnessed by the
presence of a peak at m/z 1113.5, corresponding to the [M‚H2‚
G2]2+ ion. Notably, the ESI-MS of the M/G mixture revealed
also the presence of small signals, absent in the spectrum of
the pure host, at m/z 797 ([(M - LeuOMe)‚H2]2+) and 1593
([(M - LeuOMe)‚H]+), clearly deriving from the loss of the
external amino acid of the dipeptide chain (leucine methyl ester,
MW ) 145 amu).
The much larger affinity of the resorc[4]arene host M for
the H+ cation, as compared with the dipeptide guest G, is further
testified by collision-induced dissociation (CID) experiments
on the [M‚H‚G]+ (m/z 1982) and [M‚H2‚G2]2+ (m/z 1113.5)
complexes. CID of the isolated [M‚H‚G]+ complex (isolation
width (10 amu, collision energy 20%) led to the exclusive loss
of G to form the m/z 1738 ([M‚H]+) ion. Similarly, CID of the
isolated [M‚H2‚G2]2+ complex (isolation width (10 amu,
collision energy 12%) gave rise exclusively to the [M‚H2‚G]2+
(m/z 991.5) fragment through the loss of a neutral G molecule.
Formation of the [M‚H2]2+ fragment (m/z 869.5) by loss of the
second neutral G required a significantly higher collision energy
(35%). Under no condition did CID of the [M‚H2‚G2]2+ and
[M‚H2‚G]2+ complexes lead to the loss of a charged fragment
in spite of the intense electrostatic repulsive forces operating
in doubly charged species.
FIGURE 8. ESI mass spectrum of N-linked peptidoresorc[4]arene 10
(positive ion mode).
Similar ESI-MS spectral features were obtained with the
[resorcarene 11]:[dipeptide 9] ) 1:7 mixture, the only difference
being the presence of the ions at m/z 804 ([(M - ValOMe)‚
H2]2+) and 1607 ([(M - ValOMe)‚H]+), due to the loss of valine
methyl ester (MW ) 131 amu), i.e., the external amino acid of
the dipeptide chain. The heterologue M‚G complexes (i.e., M
) 10 and G ) 9; M ) 11 and G ) 6) provided similar mass
spectral patterns as well. Again, no differences were found in
the ESI-MS spectra of the heterochiral complexes (i.e., M )
10 and G ) ent-6; M ) 11 and G ) ent-9). On the ground of
these observations as well as on the CID experiments, we
concluded that (i) in all the examined cases, the dipeptide guest
G is noncovalently bound to the peptidoresorc[4]arene M and
(ii) the stereochemistry (LL versus DD) or the regioisomerism
(leucyl-valine versus valyl-leucine sequences) of the dipeptide
chain does not have any significant influence on the formation
of the host-guest noncovalent complexes with the peptidoresorc-
[4]arenes.
FIGURE 9. ESI mass spectrum of dipeptide 6 (positive ion mode).
Conclusions
Four dipeptides, differing for composition and stereochem-
istry, have been attached to the feet of cone resorc[4]arene
octamethyl ethers through their terminal amino groups to give
N-linked peptidoresorc[4]arenes, which may represent a new
class of synthetic receptors endowed with the ability of
recognizing the homologue dipeptides, both in solution and in
the gas phase. The resulting complexes appeared to be quite
stable, since hosts and guests could not be separated by column
chromatography. The NMR spectra revealed that hydrogen
bonding interaction is at the basis of the guest recognition by
the peptidoresorc[4]arenes. In particular, resorcarene 10 and its
homologue and homochiral dipeptide 6 interact at the external
surface of resorcarene by means of attractive hydrogen bond
interactions involving polar groups of both host and guest, as
FIGURE 10. ESI mass spectrum of a 1:7 molar ratio solution of resorc-
[4]arene 10 and dipeptide 6.
Na]+) signals can only be attributed to the much larger affinity
of the resorc[4]arene host M for the H+, Na+, and K+ ions, as
compared with the dipeptide guest G. This hypothesis is
corroborated by the presence in the spectrum of Figure 10 of
9288 J. Org. Chem., Vol. 72, No. 24, 2007

Chiral N-Linked Peptidoresorc[4]arenes
solution (30 mL) of 6 (322 mg, 0.9 mmol) was added dropwise in
a 4 h period. The reaction mixture was held at reflux under nitrogen
for 3 h. Evaporation of the solvent and purification by column
chromatography (CHCl3/MeOH mixtures) gave a crude fraction as
a pale yellow powder, identified by ESI-MS as the complex [10‚
6]. This fraction was heated at reflux in MeOH for 4 h, evaporated
to dryness, and afterward processed on Sephadex LH-20, to yield
peptidoresorc[4]arene 10 as a white powder (127 mg, 49%). Mp:
220-221 °C.
judged by intermolecular dipolar interactions measured by 1D
ROESY. Heteroassociation constants of 2030 and 186 M-1 were
obtained for [10‚6] and [10‚ent-6] complexes, respectively, the
latter being comparable to the self-association constant of
dipeptide itself.
ESI mass spectrometry allowed detection of resorcarene/
dipeptide complexes with 1:1 and 1:2 stoichiometry, which were
shown to have different stabilities by the different collisional
energies required to undergo dissociation. In the gas phase, no
significant stereochemical (LL versus DD) or regioisomeric
(leucyl-valine versus valyl-leucine sequences) influence was
evidenced in the formation of host-guest complexes. The
present results represent a very promising starting point for
future studies on the intrinsic enantioselectivity of the new
receptors toward small polyfunctional molecules or more
complex frames such as dipeptide chains.
Experimental Section
2,8,14,20-Tetrakis(carboethoxymethyl)resorc[4]arene, Octam-
ethyl Ether (1). 1 was synthesized as previously described.18
2,8,14,20-Tetrakis(carboxymethyl)resorc[4]arene, Octamethyl
Ether (2). Compound 1 (1.6 g, 1.7 mmol) was dissolved in
EtOH (10 mL), and 2 N NaOH (1.2 g, 30 mmol) was added
(5 mL). The reaction mixture was stirred for 4 h at reflux. EtOH
was removed under vacuo and the aqueous solution was acidified
with glacial acetic acid. The precipitate was filtered, rinsed several
1H NMR (600 MHz, CD3CN, 298 K): δ 0.60 (d, J ) 6.6 Hz, 3H,
Me-h), 0.78 (d, J ) 6.6 Hz, 3H, Me-h′), 0.84 (d, J ) 6.5 Hz, 3H,
Me-o), 0.88 (d, J ) 6.5 Hz, 3H, Me-o′), 1.50 (m, 2H, H-m), 1.59
(m, 1H, H-n), 1.83 (m, 1H, H-g), 2.55 (dd, J ) 14.2, 7.3 Hz, 1H,
H-d), 2.77 (dd, J ) 14.2, 7.3 Hz, 1H, H-d′), 3.63 (s, 3H, COOMe),
3.66 (s, 3H, OMe), 3.71 (s, 3H, OMe), 4.20 (t, J ) 8.1 Hz, 1H,
H-f), 4.27 (dt, J ) 8.4, 6.9 Hz, 1H, H-l), 4.84 (t, J ) 7.3 Hz, 1H,
H-c), 6.44 (s, 1H, H-b), 6.84 (s, 1H, H-a), 6.85 (d, J ) 8.1 Hz, 1H,
H-e), 7.34 (d, J ) 6.9 Hz, 1H, H-i). 13C NMR (150 MHz, CD3CN,
298 K): δ 173.8 (CO), 173.2 (CO), 172.6 (CO), 157.2 (quaternary
C), 156.9 (quaternary C), 128.7 (CH, Ar), 124.4 (quaternary C),
123.9 (quaternary C), 97.4 (CH, Ar), 59.1 (CH), 56.7 (OMe), 56.3
(OMe), 52.5 (OOMe), 52.1 (CH), 42.1 (CH2), 40.9 (CH2), 34.4
(CH), 31.9 (CH), 25.5 (CH), 22.9 (Me), 22.2 (Me), 19.3 (Me), 18.7
(Me). ESI-MS (pos.): m/z found 1760.7 ([M + Na]+), C92H136N8O24-
Na requires 1761.12. Anal. Calcd for C92H136N8O24 (1738.10): C,
1
times with water, and dried, giving 2 in a quantitative yield. H
NMR (400 MHz, CDCl3, 298 K, TMS): δ 9.57 (br s, 1H, COOH),
6.52 (s, 4H, H-i), 6.30 (s, 4H, H-e), 4.95 (t, 4H, CH, J ) 7.0 Hz),
3.63 (s, 24H, OMe), 2.78 (d, 8H, CH2, J ) 7.0 Hz). 13C NMR
(75 MHz, CDCl3, 298 K): δ 175.9 (COOH), 156.0 (C-O), 125.9
(CHi), 124.0 (C-C), 96.3 (CHe), 55.9 (OMe), 36.3 (CH2), 33.0
(CH).
2,8,14,20-Tetrakis(chlorocarboxymethyl)resorc[4]arene, Oc-
tamethyl Ether (3). SOCl2 (2.4 mL, 33 mmol) was added under
nitrogen to a solution of 2 (150 mg, 0.18 mmol) in dry THF (15
mL). The reaction mixture was stirred for 4 h at reflux and allowed
to stand at rt overnight. Final evaporation under vacuo gave 3 in a
quantitative yield.
General Procedure for the Preparation of Methyl Esters 5,
8, ent-5, and ent-8 as HCl Salts. CH3COCl (5 mL) was added
slowly to cooled dry MeOH (50 mL), in which the proper amino
acid (1.7 mmol) has been dissolved. The mixture was heated at
reflux for about 2 h, and then evaporated to dryness.
General Procedure for the Preparation of Dipeptide Methyl
Esters 6, 9, ent-6, and ent-9 as TFA Salts. The proper amino
acid methyl ester hydrochloride (5.5 mmol), HOBT (0.75 g, 5.5
mmol), Et3N (0.96 mL, 7.1 mmol), and the proper commercially
available N-BOC protected amino acid (5.5 mmol) were dissolved
in CH2Cl2 (250 mL). DCC (1.14 g, 5.5 mmol) was added to the
solution cooled in an ice bath. After 4 h at rt, the solvent was
removed under vacuo and the residue was purified by silica gel
column chromatography (hexane-EtOAc, 7.5:2.5) to yield the
N-BOC protected dipeptide methyl esters as a white powder (yields
93-96%). The dipeptides (0.9 mmol) were deprotected by treatment
with TFA-DCM (1:1, 25 mL). The reaction mixture was stirred
for 1 h at rt, and then evaporated to dryness; the addition of Et2O
(50 mL) under vigorous stirring yielded white solids that were
filtered, washed several times with Et2O, and dried under vacuo,
to afford the pure salts 6, 9, ent-6, and ent-9 in quantitative yields.
For the characterization of the above compounds, see the Supporting
Information.
63.57; H, 7.89; N, 6.45. Found: C, 63.59; H, 7.91; N, 6.47. [R]20
D
-91.8 (c 0.66, MeOH).
2,8,14,20-Tetrakis(L-leucyl-L-valinamido)resorc[4]arene (11).
11 was obtained as described for 10, starting from tetracarboxylic
acid chloride 3 and dipeptide methyl ester 9. White powder, 49%
1
overall yield. Mp: 248-249 °C. H and 13C NMR signals are
reported in the Supporting Information. ESI-MS (pos.): m/z found
1760.7 ([M + Na]+), C92H136N8O24Na requires 1761.12. Anal. Calcd
for C92H136N8O24 (1738.10): C, 63.57; H, 7.89; N, 6.45. Found:
C, 63.53; H, 7.85; N, 6.41. [R]20 -86.8 (c 0.68, MeOH).
D
2,8,14,20-Tetrakis(D-valyl-D-leucinamido)resorc[4]arene (ent-
10). ent-10 was obtained as described for 10, starting from
tetracarboxylic acid chloride 3 and dipeptide methyl ester ent-6.
1
White powder, 48% overall yield. H NMR, 13C NMR, ESI-MS
data, and elemental analyses are coincident with those reported for
10. [R]20 + 91.9 (c 0.20, MeOH).
D
2,8,14,20-Tetrakis(D-leucyl-D-valinamido)resorc[4]arene (ent-
11). ent-11 was obtained as described for 10, starting from
tetracarboxylic acid chloride 3 and dipeptide methyl ester ent-9.
1
White powder, 51% overall yield. H NMR, 13C NMR, ESI-MS
data, and elemental analyses are coincident with those reported for
11. [R]20 +87.8 (c 1.00, MeOH).
D
NMR Experiments. CDCl3 solution ranges of 0.1-0.3 and 4.0-
16 mM for dipeptides and resorcarenes, respectively, were prepared
for the Foster-Fyfe22 determination of the association constants.
ESI-MS Experiments. Stock solutions of peptidoresorc[4]arenes
10, 11, ent-10, and ent-11 (0.6 × 10-5 M) and dipeptides 6, 9,
ent-6, and ent-9 (4 × 10-5 M) were in MeOH. To achieve complex
formation, all sample solutions contained a 7-fold excess of
guest.
2,8,14,20-Tetrakis(L-valyl-L-leucinamido)resorc[4]arene (10).
DIPEA (0.42 mL, 2.5 mmol) was added under nitrogen to a dry
THF solution (45 mL) of tetracarboxylic acid chloride 3 (150 mg,
0.15 mmol). The mixture was stirred 20 min at rt and a dry THF
J. Org. Chem, Vol. 72, No. 24, 2007 9289

Botta et al.
Supporting Information Available: Data used for the deter-
mination of heteroassociation constants by the Foster-Fyfe method
for complexes [10‚6] and [10‚ent-6] (Tables 1S and 2S); general
experimental methods; characterization of dipeptide methyl esters
Acknowledgment. Financial support by Universita` “La
Sapienza”, Roma, Italy (Funds for selected research topics
2003-2005), and by FIRB 2003 is acknowledged. This work
was partially supported by the Istituto Pasteur-Fondazione
Cenci Bolognetti, Universita` “La Sapienza”, Roma, Italy. G.U-
B. is grateful to MIUR (Project “High performance separation
systems based on chemo- and stereoselective molecular recogni-
tion”, contract no. 2005037725), to University of Pisa, and to
ICCOM-CNR (Italy).
1
6, 9, ent-6, ent-9; and H and 13C NMR signals of peptidoresorc-
[4]arene 11 and 1H NMR spectra of peptidoresorc[4]arenes 10 and
11. This material is available free of charge via the Internet at
http://pubs.acs.org.
JO7016636
9290 J. Org. Chem., Vol. 72, No. 24, 2007