1,3-diamido-calix[4]arene conjugates of amino acids: Recognition of -COOH side chain present in amino acids, peptides, and proteins by experimental and computational studies

Article abstract of DOI:10.1021/jo101759f

Lower rim 1,3-diamido conjugates of calix[4]arene have been synthesized and characterized, and the structures of some of these have been established by single crystal XRD. The amido-calix conjugates possessing a terminal -COOH moiety have been shown to exhibit recognition toward guest molecules possessing -COOH moiety, viz., Asp, Glu, and reduced and oxidized glutathione (GSH, GSSG), by switch-on fluorescence in aqueous acetonitrile and methanol solutions when compared to the control molecules via forming a 1:1 complex. The complex formed has been shown by mass spectrometry, and the structural features of the complexes were derived on the basis of DFT computations. The association constants observed for the recognition of Asp/Glu by Phe-calix conjugate, viz., 532/676 M^-1, are higher than that reported for the recognition of Val, Leu, Phe, His, and Trp (16-63 M^-1) by a water-soluble calixarene (Arena, G., et al. Tetrahedron Lett. 1999, 40, 1597). For this recognition, there should be a free -COOH moiety from the guest molecule. AFM, SEM, and DLS data exhibited spherical particles with a hundred-fold reduction in the size of the complexes when compared to the particles of the precursors. These spherical particles have been computationally modeled to possess hexameric species reminiscent of the hexameric micellar structures shown for a Ag⁺ complex of a calix[6]arene reported in the literature (Houmadi, S., et al. Langmuir 2007, 23, 4849). Both AFM and TEM studies demonstrated the formation of nanospheres in the case of GSH-capped Ag nanoparticles in interaction with the amido-calix conjugate that possesses terminal -COOH moiety. The AFM studies demonstrated in this paper have been very well applied to albumin proteins to differentiate the aggregational behavior and nanostructural features exhibited by the complexes of proteins from those of the uncomplexed ones. To our knowledge, this is the first report wherein a amido-calix[4]arene conjugate and its amino acid/peptide/protein complexes have been differentiated on the basis of spectroscopy and microscopy studies followed by species modeling by computations.

Full text of DOI:10.1021/jo101759f

pubs.acs.org/joc
1,3-Diamido-calix[4]arene Conjugates of Amino Acids: Recognition
of -COOH Side Chain Present in Amino Acids, Peptides, and Proteins
by Experimental and Computational Studies
Amitabha Acharya, Balaji Ramanujam, Jugun Prakash Chinta, and Chebrolu P. Rao*
Bioinorganic Lab, Department of Chemistry, Indian Institute of Technology Bombay, Powai,
Mumbai 400 076, India
cprao@iitb.ac.in
Received September 10, 2010
Lower rim 1,3-diamido conjugates of calix[4]arene have been synthesized and characterized, and the
structures of some of these have been established by single crystal XRD. The amido-calix conjugates
possessing a terminal -COOH moiety have been shown to exhibit recognition toward guest mole-
cules possessing -COOH moiety, viz., Asp, Glu, and reduced and oxidized glutathione (GSH,
GSSG), by switch-on fluorescence in aqueous acetonitrile and methanol solutions when compared to
the control molecules via forming a 1:1 complex. The complex formed has been shown by mass
spectrometry, and the structural features of the complexes were derived on the basis of DFT com-
putations. The association constants observed for the recognition of Asp/Glu by Phe-calix conjugate,
viz., 532/676 M-1, are higher than that reported for the recognition of Val, Leu, Phe, His, and Trp
(16-63 M-1) by a water-soluble calixarene (Arena, G., et al. Tetrahedron Lett. 1999, 40, 1597). For
this recognition, there should be a free -COOH moiety from the guest molecule. AFM, SEM, and
DLS data exhibited spherical particles with a hundred-fold reduction in the size of the complexes
when compared to the particles of the precursors. These spherical particles have been computation-
ally modeled to possess hexameric species reminiscent of the hexameric micellar structures shown for
a Agþ complex of a calix[6]arene reported in the literature (Houmadi, S., et al. Langmuir 2007, 23, 4849).
Both AFM and TEM studies demonstrated the formation of nanospheres in the case of GSH-capped
Ag nanoparticles in interaction with the amido-calix conjugate that possesses terminal -COOH
moiety. The AFM studies demonstrated in this paper have been very well applied to albumin proteins
to differentiate the aggregational behavior and nanostructural features exhibited by the complexes of
proteins from those of the uncomplexed ones. To our knowledge, this is the first report wherein a
amido-calix[4]arene conjugate and its amino acid/peptide/protein complexes have been differentiated
on the basis of spectroscopy and microscopy studies followed by species modeling by computations.
Introduction
proteins containing these are of prime interest in biological
systems.1-3 Asp and Glu are known to play important roles
as excitatory amino acid neurotransmitters in the central
nervous system of mammalians, whereas GSH is known to
protect intracellular components against oxidative damage
Amino acids possessing side chain -COOH functionality,
viz., aspartic acid and glutamic acid, and peptides, GSH
(reduced glutathione) GSSG (oxidized glutathione), and
(1) Marc, R. E.; Lam, D. M. Proc. Natl Acad. Sci. U.S.A. 1981, 78, 7185.
(2) Mizrahi, V.; Brooksbank, R. L.; Nkabinde, N. C. J. Biol. Chem. 1994,
269, 19245.
(3) Voet, D.; Voet, J. G. Biochemistry; John Wiley & Sons: New York,
1990.
DOI: 10.1021/jo101759f
r
Published on Web 12/01/2010
J. Org. Chem. 2011, 76, 127–137 127
2010 American Chemical Society

Acharya et al.
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SCHEME 1. Synthesis of the Precursors and Amido-calix Conjugatesa
aReagents and conditions: (a) acetone, K2CO3, BrCH2COOEt, reflux for 15 h; (b) EtOH, aq NaOH, reflux for 24 h; (c) HCl salt of the ester of the
corresponding amino acid, dry THF, Et3N, HOBT, DCC; (d) THF, LiOH. Similar method has been used for the synthesis of NC-Phe-OH (1e), where p-
tert-butyl phenol has been used instead of C4A (Supporting Information). R = tert-butyl. Compounds 1a-1d are given in Scheme 3.
and also in detoxifying heavy metal ions by using its mer-
capto groups.4 Concentration changes of Asp and Glu levels
in certain regions of the brain are closely related to Parkinson’s
disease.5-7 Therefore, the recognition of such amino acids,
peptides, and proteins possessing -COOH moieties is a
challenging task. Molecular systems capable of recognizing
these by eliciting easily detectable spectroscopic signals and
microscopic structures are of paramount importance. Recently,
a galactosyl based naphthyl-imine derivative has been shown
to recognize Glu by fluorescence spectroscopy.8 A set of
organic and inorganic molecular species have been employed
in combination as receptor units for the identification of
naturally occurring amino acids by indicator displacement
assays controlled by pH.9 Calix[4]arene provides a congenial
platform for bringing appropriate functionalization and
hence acts as useful molecular framework. Such systems
are expected to yield interesting nanostructural features in
interaction with the guest species. Water-soluble calixarenes
have also been shown to complex Val, Leu, Phe, His, and Trp
noted in the literature, though the examples of such systems
were limited.10-12,16-21 Recently our group reported the
selective recognition of amino acids by the metal ion com-
plexes of calix[4]arene conjugates.22-24
To our knowledge, none of the calixarene derivative have
been reported for the detection of Asp, Glu, or GSH. There-
fore, the present paper demonstrates for the first time
the selective recognition of Asp, Glu, and GSH by lower
rim 1,3-diamido conjugates of calix[4]arene possessing ter-
minal -COOH moieties by fluorescence spectroscopy wherein
the complex species formed have been further studied by
DFT calculations. The nanoscopic aggregate formation of
these amido-calix[4]arene conjugates with Asp or Glu has
been shown by scanning electron microscopy (SEM) and
atomic force microscopy (AFM), and the nanospheres
formed with the GSH-coated Ag nanoparticles by transmis-
sion electron microscopy (TEM) as well as by AFM. The
utility of the amido-calix conjugates has been further ex-
tended even to proteins to show their novel nanostructural
features by microscopy.
with Ka values in the range of 16-63 M-1 10
Sulfonato-
.
calix[6]arene has been shown to complex basic amino acids,
viz., Lys and Arg, and their peptides as studied by 1H NMR
and microcalorimetry.11-13 Some were shown to exhibit
enantioselective differentiation14,15 as well as amino acid
transport.16 Thus, the importance of calix[4]arene conju-
gates as biosensors for amino acids and peptides has been
Results and Discussion
The amido-calix[4]arene conjugates have been synthe-
sized in four steps starting from simple p-tert-butyl-calix[4]-
arene (C4A) as given in Scheme 1.25 All synthetic compounds
(Scheme 1) were checked for their purity and authenticity
and were characterized by NMR, FTIR, and ESI MS as
given in the Experimental Section.26 Since the terminals of
(4) Mehra, R. K.; Tran, K.; Scott, G. W.; Mulchandani, P.; Saini, S. S.
J. Inorg. Biochem. 1996, 61, 125.
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(7) Fornai, F.; Vaglini, F.; Maggio, R.; Bonuccelli, U.; Corsini, G. U.
Neurosci. Biobehav. Rev. 1997, 21, 401.
(8) Ahuja, R.; Singhal, N. K.; Ramanujam, B.; Ravikumar, M.; Rao,
C. P. J. Org. Chem. 2007, 72, 3430.
(9) Buryak, A.; Severin, K. A. J. Am. Chem. Soc. 2005, 127, 3700.
(10) Arena, G.; Contino, A.; Gulino, F. G.; Magri, A.; Sansone, F.;
Sciotto, D.; Ungaro, R. Tetrahedron Lett. 1999, 40, 1597.
(11) Douteau-Guevel, N.; Coleman, A. W.; Morel, J.-P.; Morel-Desrosiers,
N. J. Chem. Soc., Perkin Trans. 2 1999, 3, 629.
(17) Selkti, M.; Tomas, A.; Coleman, A. W.; Douteau-Guevel, N.;
Nicolis, I.; Villain, F.; de Rango, C. Chem. Commun. 2000, 2, 161.
(18) Buschmann, H.-J.; Mutihac, L.; Jansen, K. J. Inclusion Phenom.
Macrocyclic Chem. 2001, 39, 1.
(19) Hassen, W. M.; Martelet, C.; Davis, F.; Higson, S. P. J.; Abdelghani,
A.; Helali, S.; Jaffrezic-Renault, N. Sens. Actuators, B 2007, 124, 38.
(20) Lu, J. Q.; Zhang, L.; Sun, T. Q.; Wang, G. X.; Wu, L. Y. Chin. Chem.
Lett. 2006, 17, 575.
(21) Lazar, A. N.; Danylyuk, O.; Suwinska, K.; Coleman, A. W. J. Mol.
Struct. 2006, 825, 20.
(12) Douteau-Guevel, N.; Coleman, A. W.; Morel, J.-P.; Morel-Desrosiers,
N. J. Phys. Org. Chem. 1998, 11, 693.
(22) Joseph, R.; Ramanujam, B.; Acharya, A.; Rao, C. P. J. Org. Chem.
2009, 74, 8181.
ꢀ
(13) Douteau-Guevel, N.; Perret, F.; Coleman, A. W.; Morel, J.-P.;
(23) Jugun, P. C.; Acharya, A.; Kumar, A.; Rao, C. P. J. Phys. Chem. B
2009, 113, 12075.
(24) Joseph, R.; Jugun, P. C; Rao, C. P. J. Org. Chem. 2010, 75, 3387.
(25) Collins, E. M.; McKervey, M. A.; Madigan, E.; Moran, M. B.;
Owens, M.; Ferguson, G.; Harris, S. J. J. Chem. Soc., Perkin Trans.1 1991,
12, 3137.
Morel-Desrosiers, N. J. Chem. Soc., Perkin Trans. 2 2002, 3, 524.
(14) Liu, S.; He, Y.; Qing, G.; Xu, K.; Qin, H. Tetrahedron: Asymmetry
2005, 16, 1527.
(15) Liu, F.; Lu, G.-Y.; He, W.-J.; Liu, M.-H; Zhu, L.-G. Thin Solid Films
2004, 468, 244.
(16) Antipin, I. S.; Stoikov, I. I.; Pinkhassik, E. M.; Fitseva, N. A.; Stibor,
I.; Konovalov, A. I. Tetrahedron Lett. 1997, 38, 5865.
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Chem. Soc. 1981, 103, 3782.
128 J. Org. Chem. Vol. 76, No. 1, 2011

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FIGURE 1. ORTEP diagrams of the molecular structures of (a)
C4A-Phe-OMe, (b) C4A-Phe-OH, and (c) NC-Phe-OH as deter-
mined by single crystal XRD. Encircled portion possesses sites for
interaction.
FIGURE 2. Lattice structures of (a) C4A-Phe-OMe and (b) NC-
Phe-OH, as viewed down “c” and “a”, respectively.
TABLE 1. Crystallographic Parameters for the Structure Determina-
tion and Refinement
species. Similar structural features were observed even with
C4A-Phe-OH where the terminal -COOH is poised suitable
for interaction (Figure 1b).28 Comparison of the amido arm
present in the structures of C4A-Phe-OMe and C4A-Phe-
OH with that present in the noncalixarene conjugate, NC-
Phe-OH, exhibits considerable changes in the dihedral angles
(Supporting Information) of the arm, and hence the side
chain phenyl moiety is more exposed. This difference is
expected to reflect on their ability to interact with the incom-
ing moiety. This has indeed been seen from the titrations
carried out by fluorescence spectroscopy. In the crystal
lattice, the C4A-Phe-OMe stacks in the form of columns
wherein the adjacent pair is arranged in a head-to-tail
fashion (Figure 2a). The water of crystallization indeed
connects the amido-calix conjugates through hydrogen bond
interactions and thus forms a supramolecular structure.
However, the lattice of the single strand derivative, namely,
NC-Phe-OH, does not show such supramolecular formation
(Figure 2b).
C4A-Phe-OMe
NC-Phe-OH
empirical formula
temperature (K)
crystal system
space group
2(C68H82N2O10) H2O C21H25NO4
120
150
monoclinic
P21 (No. 4)
14.4663(4)
26.1325(7)
17.0631(5)
96.133(3)
6413.6(3)
2
orthorhombic
P212121 (No. 19)
7.594(3)
9.670(3)
27.818(8)
ꢀ
˚
a/A
ꢀ
˚
b/A
ꢀ
˚
c/A
β/deg
volume/A
3
ꢀ
˚
2042.8(12)
4
Z
reflections collected
independent reflections
Rint
66808
22341
0.103
16584
3593
0.092
1328
250
reflections used [I>2.0σ(I)] 13689
parameters
final R
1478
0.0863
0.1814
0.0448
0.1033
a
R0w
aw = 1/[s2(Fo2) þ (0.0553P)2 þ 4.9051P] in the case of C4A-Phe-
OMe; w = 1/[ s2(Fo2) þ (0.0440P)2] in the case of NC-Phe-OH; where
2
P = (Fo þ 2Fc2)/3.
Necessity of the Amido-calix[4]arenes for the Selective
Recognition of Asp, Glu, GSH, and GSSG. All fluorescence
spectral studies were carried out using 5 μM solutions of the
amido-calix[4]arene conjugate. When C4A-Phe-OH is ex-
cited at 280 nm, it exhibits a strong emission around 313 nm.
All titrations of C4A-Phe-OH with 20 naturally occurring
amino acids revealed that only the Asp and Glu exhibits an
increase in fluorescence intensity, while the others exhibit no
significant change (Figure 3a,c). The enhancement is 3.5-
to 4.0-fold in methanol (Supporting Information), and it is
5.5- to 6.0-fold in 1:1 aqueous acetonitrile (Supporting
Information). The fluorescence intensity data fit well with
a 1:1 complex formed between C4A-Phe-OH and Asp/Glu
by exhibiting Ka values of 532/676 M-1. These association
constants are much higher than that observed for Val, Leu,
Phe, His, and Trp (16-63 M-1) in the literature in the case of
a water-soluble calix[4]arene.10 Titration of C4A-Phe-OH
with the totally esterified Asp/Glu, viz., Asp-(OBz)2 or
Glu-(OMe)2, exhibited no change in the fluorescence inten-
sity during the titration (Supporting Information). Combin-
ing this result with that observed in the case of other natu-
rally occurring amino acids suggests the involvement of the
side chain carboxylic group in the interaction and not the CR
bound groups. Thus Asp and Glu elicit fluorescence re-
sponse by switch-on mechanism, and hence only these two
amino acids are amenable for sensing by C4A-Phe-OH,
both pendants of these conjugates possess a -COOH moi-
ety, it is of interest to study their recognition abilities toward
biologically important molecules, such as amino acids and
peptides. To elicit the selectivity of these conjugates (C4A-
Phe-OH, C4A-Gly-OH, and C4A-Ala-OH) as well as the
role of the two amido arms, appropriate control molecular
systems were generated by converting the -COOH terminal
into -COOR {e.g., C4A-Phe-OMe, C4A-Gly-OMe, and
C4A-Ala-OMe}, and a single strand version of these, namely,
NC-Phe-OH, and a nonamido version of these, namely,
{C4A-OEt, C4A-OH},27 were synthesized and characterized
(Schemes 1 and 3; Experimental Section) and were used in
the titration studies (Supporting Information).
Crystal Structures. Single crystals of C4A-Phe-OMe and
NC-Phe-OH were obtained by slow evaporation of their
ethanol solutions. These crystals yielded satisfactory dif-
fraction data suitable to determine their 3-D structures.
(Figure 1a,c). The corresponding details of the structure deter-
mination and refinement are given in Table 1 (Supporting
Information). The crystal structure of C4A-Phe-OMe ex-
hibited cone conformation for the calix[4]arene and is in
conformity with the results obtained on the basis of NMR.
The structure exhibits rather exposed amide and ester moi-
eties that are amenable for interaction with the incoming
(27) Joseph, R.; Ramanujam, B.; Acharya, A.; Khutia, A.; Rao, C. P.
J. Org. Chem. 2008, 73, 5745.
(28) Dey, M.; Rao, C. P. Unpublished results.
J. Org. Chem. Vol. 76, No. 1, 2011 129

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FIGURE 3. Fluorescence titration of C4A-Phe-OH with amino acids: (a) spectral traces obtained in the titration with Asp in 1:1 H2O/CH3CN;
(b) plots of relative fluorescence intensity (I/I0) of the titrations of Asp (black 9), Glu (red b), Asp ester (blue 2), Glu ester (green 1), GSH (pink
left-facing triangle), and GSSG (gray right-facing triangle) (G=guest species); (c) histogram showing the number of times of enhancement
(positive axis) or quenching (negative axis) of fluorescence intensity of C4A-Phe-OH when titrated with amino acids. Filled bars are for
methanol (at 200 equiv), and unfilled ones are for aqueous acetonitrile (at 100 equiv) solutions. Error bars are given based on three different
measurements.
the C4A-Phe-OH (Figure 4), and it was found that there is no
role of side chain in the recognition process.
Involvement of the Terminal -COOH Moiety on the Amido
Arms in the Recognition. The esters of these, namely, C4A-
Gly-OMe and C4A-Ala-OMe, do not show any change in the
fluorescence, suggesting the necessity of the terminal -COOH
moiety on the arm (Supporting Information) (Figure 4).
All of the fluorescence titration results together clearly
suggest that the calix[4]arene platform, the amido arm as
well as the terminal -COOH group of the host, and the side
chain -COOH of the guest species are essential for the
recognition. Thus the amido-calix conjugates selectively
recognize Asp, Glu, GSH, and GSSG.
ESI MS Studies. The stoichiometry of the complexes
FIGURE 4. Histogram showing the number of times of enhance-
formed between Glu/GSH and amido-calix[4]arene con-
ment (positive axis) or quenching (negative axis) of fluorescence
jugates has been established to be 1:1 by the molecular
intensity in the titration of amido-calix conjugates and other control
ion peaks observed in ESI MS spectrometry (Supporting
Information). These are found at 1206.7/1366, 1026.5/
1186.6, and 1054.6/1214.7, respectively, for the amido con-
jugates, namely, C4A-Phe-OH, C4A-Gly-OH, and C4A-
Ala-OH, indicating the formation of their 1:1 complexes
with Glu/GSH. To our knowledge, there is only one paper
in the literature in which the complex formed between
O-ethylated calix[6]arene and His/Lys or Arg has been
shown by mass spectrometry.29 The structural features of
the complexes formed in the present case have been com-
puted on the basis of DFT calculations.
Computational Modeling of the Complex Formed between
Amido-calix Conjugate and Asp, Glu, and GSH. To address
the structural features of the complexes formed between the
amido-calix conjugates, C4A-Phe-OH0/C4A-Gly-OH0 and
Asp/Glu/GSH, computational modeling studies have been
carried out by geometry optimizations using the Gaussian 03
package30 as per the details given in the Experimental Section.
The final structure of the optimized complex obtained through
DFT calculations exhibits the presence of three hydrogen
bonds between the side chain -COOH of Asp/Glu/GSH and
the two arms of C4A-Phe-OH0/C4A-Gly-OH0 (Figure 5a-c;
Supporting Information). Such 1:1 complexation leads to
a stabilization of -24 to -27 kcal/mol in the case of all
the three complexes. This is comparable with that obtained
molecules with Asp (open bar), Glu (black bar), GSH (lined bar),
and GSSG (cross-hatched bar) in aqueous acetonitrile (at 100 equiv).
Similar results have also been obtained in methanol (Supporting
Information).
among the 20 naturally occurring amino acids. Similar
results were observed when a tripeptide, namely, GSH,
possessing γ-glutamyl moiety or its oxidized form,
namely, GSSG, was used in the titration (Figure 3b). This
further supports the involvement of -COOH moiety in the
recognition, besides ruling out the involvement of the -SH
moiety. The latter has indeed been supported by the
titrations carried out with Cys. Thus C4A-Phe-OH has
been clearly shown to be selective toward Asp and Glu
among the 20 amino acids studied in addition to the
tripeptides, GSH and GSSG.
Involvement of the Amido Arms in the Recognition. Com-
parison of the fluorescence titration results of the C4A-
OH and C4A-OEt with those of the amido conjugates
clearly suggests the necessity of the amido arms in the
recognition process. On the other hand, the comparison of
the fluorescence titration results of NC-Phe-OH with that
of the C4A-Phe-OH supports the necessity of the calix-
[4]arene platform in the recognition process (Supporting
Information) (Figure 4).
Involvement of the Side Chain Moiety in the Recognition.
To see the involvement of the side chain of the amido arm of
the conjugate in the recognition process, similar fluorescence
titrations were carried out using C4A-Gly-OH and C4A-
Ala-OH. The corresponding data was compared with that of
(29) Stone, M. M.; Franz, A. H.; Lebrilla, C. B. J. Am. Soc. Mass
Spectrom. 2002, 13, 964.
(30) Frisch, M. J. et al. Gaussian 03, Revision C.02; Gaussian, Inc.:
Wallingford, CT, 2004.
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FIGURE 5. Structure of the DFT optimized amino acid complexes of C4A-Phe-OH0 and NC-Phe-OH: (a) {C4A-Phe-OH0 þ Asp}, (b) {C4A-
Phe-OH0 þ Glu}, (c) {C4A-Phe-OH0 þ GSH}, and (d) {NC-Phe-OH þ Glu}. Encircled portions show the region of interaction. Visualization
has been performed by using Chemcraft.32
FIGURE 6. Scanning electron micrographs of (a) C4A-Phe-OH, (b) {C4A-Phe-OH þ Asp}, and (c) {C4A-Phe-OH þ Glu}.
from the literature (i.e., -21 to -28 kcal/mol) by plugging
in the H A distances of the optimized structure (Supporting
3 3 3
Information).31 On the other hand, the computations carried
out with the single strand version, viz., NC-Phe-OH showed
only one hydrogen bond (Figure 5d).
Spherical Clusters Formed between C4A-Phe-OH and Asp
or Glu. Because calixarene systems are known to exhibit
supramolecular structures, it is of interest to see whether
there are any changes in the microscopic architectural fea-
tures of the amido conjugate and its complexes by micro-
scopy studies.
FIGURE 7. Atomic force microscopy images of (a) C4A-Phe-OH,
(b) {C4A-Phe-OH þ Asp}, and (c) {C4A-Phe-OH þ Glu}.
same in the precursor amido conjugate (93-159 nm), in-
dicating a drastic shrinking of the particle size in the complex
and thereby bringing a volume reduction of ∼100-fold. Thus
the volume and shape of the particles differ drastically between
the precursor conjugate and its Asp complex. Similar results
have also been obtained for the Glu complex, {C4A-Phe-
OH þ Glu} (Figure 7c) (Supporting Information).
SEM Studies. While the SEM micrographs of C4A-Phe-
OH exhibit crystalline rods of (0.4-1.4) ꢀ (2.8-11.1) μm2
(Figure 6a), its complex {C4A-Phe-OH þ Asp} exhibits
almost uniformly distributed spherical surfaces with a size
distribution of 200-300 nm (Figure 6b). Similar results were
obtained even for the complex, {C4A-Phe-OH þ Glu}, but
the size of particles range from 400 to 600 nm (Figure 6c).
The SEM features of C4A-Phe-OH and its complex {C4A-
Phe-OH þ Asp} are quite different from that of the simple
Asp (Supporting Information). Thus the microscopic fea-
tures of the amido conjugate differ substantially from its
complexes.
Dynamic Light Scattering (DLS) Studies. DLS experi-
ments carried out with C4A-Phe-OH, {C4A-Phe-OH þ Asp},
and {C4A-Phe-OH þ Glu} exhibited average aggregates of
405, 66, and 110 nm, respectively, indicating a volume reduc-
tion of at least 100-fold in the aggregates upon complexation
when compared to the uncomplexed C4A-Phe-OH.
AFM Studies. The shape and size of the particles observed
in the AFM image of C4A-Phe-OH (Figure 7a) differ largely
from that of its complex, {C4A-Phe-OH þ Asp} (Figure 7b),
suggesting that the complex can be easily differentiated from
its precursor amido-calix conjugate. While C4A-Phe-OH
exhibits uniformly distributed particles of 225-250 nm, its
Asp complex exhibits marginally elongated spherical ones
75-110 nm in size that were also distributed uniformly.
However, both exhibited clusters that were formed from
basic units, where the ratio of the smaller to the cluster
particles was found to be ∼1:2. Height of these particles is
much smaller in the complex (7-16 nm) as compared to the
Thus all three techniques, viz., AFM, SEM, and DLS, sug-
gest a reduction in the surface area of the particles by about
100-fold upon complexation when compared to the particles
of C4A-Phe-OH owing to the nature of the growth of the
particles in each of this case. Thus, to our knowledge, this is
the first calixarene conjugate that has been demonstrated to
recognize amino acids and peptides based on microscopy
studies.
Computational Modeling of the Spherical Cluster. The
spherical clusters observed from microscopy for the Asp or
Glu complex of C4A-Phe-OH may result from the aggrega-
tion of the originally formed 1:1 complexes already given in
this paper. Because Glu exhibits dimers to hexamers in
solution to an extent of >90% of the species as established
(31) Grabowski, S. J. Annu. Rep. Prog. Chem., Sect. C 2006, 102, 131.
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FIGURE 8. (a) A PM3 optimized hexamer of Glu. (b) Proposed model for the species formed from 1:1 Asp/Glu complexes of C4A-Phe-OH.
Inner core represents the hexameric closed cluster formed by the Asp/Glu units. The amino end (blue) of one of the amino acids interacts
with the carboxylic end (red) of the neighbor. The green cups represent calixarene units. (c) Space-filling model of the hexameric aggregate of
{C4A-Phe-OH0 þ Glu} obtained from geometry minimization using HYPERCHEM program, shown as the dimer formed by two such
aggregates.
by mass spectrometry,33 it might be appropriate to start the
modeling from one such species. The oligomers of Ser and
Pro have been computationally modeled.34,35 Therefore, in
order to emerge with the spherical nature of the particles, a
hexamer of simple Glu has been modeled by semiempirical
PM3 computations that results in a cyclic hexamer where the
amino terminal of one of the Glu interacts with the car-
boxylic end of the neighbor and so on, through hydrogen
bonds (Figure 8a) (Supporting Information). In turn, the
Glu-hexamer has been developed starting from dimer and
then going through trimer, tetramer, and pentamer (Supporting
Information) in a cascade fashion. The Glu-hexamer has a
diameter of ∼2.2 nm. The stabilization energy of the hex-
amer is commensurate with the H-bond interactions formed
between them. The Glu-hexamer has its side-chain -COOH
moiety projecting out and is suitable for further complexa-
tion with the amido-calix conjugate.
Thus the complexation of the side chain -COOH unit of
each residue of the Glu-hexamer with one C4A-Phe-OH
results in the hexameric aggregate shown in Figure 8b, and
this has been computationally modeled by molecular me-
chanics. The structures of the Asp and Glu complexes of the
amido conjugate as obtained from B3LYP/6-31G computa-
tions were taken in order to generate the hexameric clusters
using the SymmDock web server.36,37 Among the top ranked
solutions generated, the best one was subjected to molecu-
lar mechanics calculations using the HYPERCHEM pro-
gram.38 The minimizations were done by using Polak-qRi-
biere conjugate gradient method by considering rms gradient
of 0.1 kcal/mol. The resulting structures exhibit a bowl-shaped
arrangement with a rim diameter of ∼3.2 and ∼3.1 nm, and a
bowl depth of ∼1.6 and ∼1.5 nm, respectively, for Glu and
Asp complexes. The diameter of the channel observed in this
structure is found to be 0.55 ( 0.05 nm. Such structures are
amenable for the formation of a dimer through the hydro-
phobic interaction between the rim portions of two bowls
resulting in an almost spherical particle as shown in Figure 8c.
About 50% of the surface area of such spherical particles is
hydrophilic groups, while the rest is hydrophobic, and hence
these can lead to the formation of higher size aggregates in
3 dimensions through hydrophobic and hydrophilic interac-
tions to result in supramolecular structures already demon-
strated through microscopy. These hexameric aggregates are
reminiscent of the hexameric micellar structures shown for
1:1 complexes of Agþ and calix[6]arene reported in the liter-
ature,39 wherein the amino acid in the present case can be con-
sidered to be replaced by Agþ, and the C4A-Phe-OH is being
replaced by calix[6]arene.
MALDI Studies. The aggregational behavior of the Asp/
Glu complex of C4A-Phe-OH has been further revealed based
on MALDI mass spectrum (Supporting Information) which
exhibited peaks at 5698.3 and 5696.5 respectively for Asp and
Glu and are assignable to the oligomers, viz., {4 ꢀ C4A-Phe-OH
þ 11 ꢀ Asp þ H}þ (Figure 9) and {4 ꢀ C4A-Phe-OH þ 10 ꢀ
Glu þ7H}þ. No such oligomers were observed with the indivi-
dual precursors, viz., C4A-Phe-OH or Asp or Glu.
Formation of Nanospheres by GSH-AgNP with C4A-Phe-
OH. As the amido-calix conjugates were shown to recognize
GSH through its side chain -COOH function and the Ag
nanoparticles can be coated using -SH moiety of the GSH,
the interaction between the GSH-coated Ag nanoparticles
(GSH-AgNP), and these conjugates were studied by TEM
and AFM. The GSH-AgNP have been characterized by ab-
sorption, TEM, and powder XRD techniques (Supporting
Information), and the data was found to be comparable with
that reported in the literature.40-42
(32) Zhurko, G. A.; Zhurko D. A. ChemCraft; http://www.chemcraftprog.
com.
(33) Nemes, P.; Schlosser, G.; Vekey, K. J. Mass spectrom. 2005, 40, 43.
(34) Cooks, R. G.; Zhang, D.; Koch, K. J. Anal. Chem. 2001, 73, 3646.
(35) Nanita, S. C.; Cooks, R. G. Angew. Chem., Int. Ed. 2006, 45, 554.
(36) Duhovny, D.; Nussinov, R. Wolfson, H. J. In Algorithms in Bioinfor-
matics: Second International Workshop; Guigo, R., Gusfield, D., Eds.; WABI
2002, Rome, Italy, Lecture Notes in Computer Science 2452; Springer
Verlag: New York, 2002; p 185.
(37) Zhang, C.; Vasmatzis, G.; Cornette, J. L.; DeLisi, C. J. Mol. Biol.
1997, 267, 707.
(38) HyperChem 8.0.4; Hypercube, Inc.: Gainesville, FL, 2007.
(39) Houmadi, S.; Coquire, D.; Legrand, L.; Faur, M. C.; Goldmann, M.;
Reinaud, O.; Rmita, S. Langmuir 2007, 23, 4849.
(40) Jiang, X.; Xie, Y.; Lu, J.; Zhu, L.; He, W.; Qian, Y. Langmuir 2001,
17, 3795.
132 J. Org. Chem. Vol. 76, No. 1, 2011

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TEM Studies. In TEM, the GSH-AgNP (see Experimental
Section) were mainly found to be of ∼7 nm in size, as can
be seen from Figure 10a (Supporting Information). When
C4A-Phe-OH was added to GSH-AgNP, the nanospecies
were further aggregated into spherical clusters resulting in
nanospheres with diameters ranging from 60 to 120 nm
(Figure 10b-d). Similar studies carried out with an amido
conjugate-ester, namely, C4A-Phe-OMe, resulted in no nano-
spheres, suggesting that the terminal arm -COOH moiety is
necessary for the formation of nanospheres. Further studies
carried out with amido-calix conjugates that do not possess a
phenyl side chain, viz., C4A-Gly-OH and C4A-Ala-OH, also
resulted in no nanospheres, suggesting the necessity of the
phenyl moiety in forming these (Figure 10e-g). The TEM
results clearly support the role of hydrophobic interactions
in the formation of nanospheres. Thus, it may be visualized
that the nanospheres observed in TEM result from the com-
plexation of GSH-AgNP with C4A-Phe-OH as 1:1 complexes
followed by the hydrophobic interactions extended between
the side chain phenyl moieties of the neighbor silver nano-
species.
AFM Studies. Experiments similar to that reported under
TEM were performed even by AFM, and the formation of
nanospheres was found only in the case of C4A-Phe-OH
(Figure 11) and not with C4A-Phe-OMe or C4A-Gly-OH or
C4A-Ala-OH (Figure 12), suggesting the importance of the
terminal -COOH and the side chain phenyl moiety present
on the calix arms in the formation of these spheres. The size
of GSH-AgNP was found to be in the range of 23-36 nm
FIGURE 9. MALDI-TOF spectra for [C4A-Phe-OH þ Asp].
FIGURE 10. Transmission electron micrographs of (a) GSH-capped silver nanoparticles (GSH-AgNP), (b) nanospheres formed by {GSH-
AgNP þ C4A-Phe-OH} [(c, d) are the magnified version of two different nanospheres seen in (b)], (e) {GSH-AgNP þ C4A-Phe-OMe},
(f) {GSH-AgNP þ C4A-Gly-OH}, and (g) {GSH-AgNP þ C4A-Ala-OH}.
FIGURE 11. Atomic force microscopy images of (a) GSH-capped silver nanoparticles (GSH-AgNP), (b) 3D view of that given in (a),
(c) nanospheres formed by {(GSH-AgNP) þ C4A-Phe-OH}, and (d) 3D view of that given in (c).
J. Org. Chem. Vol. 76, No. 1, 2011 133

Acharya et al.
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when deposited on a mica surface. When C4A-Phe-OH was
added to GSH-AgNP, nanospheres of sizes ∼480-520 nm
were observed. Thus the nanospheres formed were about
10-20 times bigger than the simple GSH-AgNP in both
TEM as well as in AFM.
Studies Extended to Proteins. Albumins are most abun-
dantly found and well studied circulatory proteins possessing
greater R-helical content. Bovine serum albumin (BSA),
human serum albumin (HSA), and R-lactalbumin possess
∼14-16% Asp and Glu residues. Since several of these amino
acids are present on the surface of the protein, the side
chain -COOH groups can complex with amido-calix conjugates
through the interactions analogous to that observed between the
amido conjugate and Asp/Glu. However, in the case of proteins,
such interactions may further lead to specific changes in their
aggregational behavior, and the resulting structures may be
distinguishable from the nanostructures of the simple pro-
tein. Thus it is of interest to monitor the changes observed in
microscopy features of the proteins and their complexes.
SEM Studies. To support the interactions present between
C4A-Phe-OH and BSA/HSA, SEM studies have been car-
ried out. SEM of the corresponding complex of {HSA þ
C4A-Phe-OH} exhibited almost uniformly spherical parti-
cles (Figure 13a,b). The sizes of these protein-bound particles
are larger by at least about 10 times compared to those
observed with simple aspartic acid complex (Figure 6b,c).
AFM Studies. When C4A-Phe-OH was added to BSA/
HSA, the proteins tend to agglomerate (Figure 14b,f ) and
result in particles with distorted spherical shape, as can be
seen from the micrographs. Though the size of these particles
does not seem to change much (∼90-150 nm in free protein
compared to ∼100-180 nm in complexed species), the height
of the particles has been found to change considerably
(∼4-20 nm in free protein compared to 18-60 nm in
complexed species). On the other hand, the aggregation that
results from the interaction of C4A-Gly-OH and BSA/HSA
results in almost spherical particles (Figure 14c,g). This
suggests that the nanospheres formed between the amido-
calix conjugates and BSA/HSA seem to be dependent only
on the presence of the amido arm and not the side chain. This
may be attributable to the arene basket present in these
conjugates. Similar studies carried out with the -COOMe
terminal, namely, C4A-Phe-OMe, resulted in much less
FIGURE 12. Atomic force microscopy images of (a) {(GSH-
AgNP) þ C4A-Gly-OH}, (b) {(GSH-AgNP) þ C4A-Ala-OH},
and (c) {(GSH-AgNP) þ C4A-Phe-OMe}.
FIGURE 13. Scanning electron micrographs of (a) HSA and
(b) {C4A-Phe-OH þ HSA}.
FIGURE 14. Atomic force microscopy images of (a) BSA, (b) {C4A-Phe-OH þ BSA}, (c) {C4A-Gly-OH þ BSA} (d) {C4A-Phe-OMe þ
BSA}, (e) HSA, (f) {C4A-Phe-OH þ HSA}, (g) {C4A-Gly-OH þ HSA}, (h) {C4A-Phe-OMe þ HSA}, (i) R-lactalbumin, (j) {C4A-Phe-OH þ
R-lactalbumin}, and (k) {C4A-Phe-OMe þ R-lactalbumin}.
134 J. Org. Chem. Vol. 76, No. 1, 2011

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SCHEME 2. Microscopy Features of the Recognition of Glu, GSH-AgNP, and R-Lactalbumin by C4A-Phe-OH
aggregation than that observed in the case of C4A-Phe-OH
(Figure 14d,h), suggesting the positive role played by the
terminal -COOH of the arms of the conjugates. Similar
results were observed even with the R-lactalbumin, which is
also an R-helical protein (Figure 14j,k)
present in the tripeptide, viz., GSH/GSSG. Studies based
on AFM and SEM resulted in the formation of spherical
particles followed by reduction in the particle size by about
100-fold upon complexation when compared to the particles
of uncomplexed conjugate, namely, C4A-Phe-OH. These
results were supported by DLS studies. The spherical particle
has been modeled by semiempirical (PM3) followed by molec-
ular mechanics (MMþ) computations as hexameric aggre-
gates that are reminiscent of the hexameric micellar struc-
tures shown for 1:1 complexes of Agþ and calix[6]arene
reported in the literature.39 The aggregation of such nano-
scopic species through interspecies interactions can lead to
the formation of the type of particles observed in the micro-
scopy studies. This has been further supported by studying
the microscopy of the structures formed by the complexes of
the amido-calix conjugates with GSH-AgNP. The TEM and
AFM studies clearly demonstrate the necessity and the role
of terminal -COOH moiety of the arm and the side chain of
the amido unit in the formation of the nanospheres.
Since the recognition and the formation of the nano-
spheres is through the side chain -COOH function, the
studies were extended to R-helical proteins, viz., BSA,
HSA, and R-lactalbumin, and it was found that the principle
observed in the amino acid studies can indeed be applied to
these proteins. The aggregational behavior of these proteins
as studied by AFM clearly differentiates the nanostructural
features of the simple proteins from their complexes. The
present paper clearly provided the nanostructural differences
in the recognition of amido-calix conjugates toward Asp/Glu
and albumin proteins as can be seen from the Scheme 2 given
in the case of C4A-Phe-OH and C4A-Phe-OMe.
Conclusions and Correlations
To our knowledge, this is the first report wherein an
amido-calix[4]arene conjugate and its amino acid/peptide/
protein complexes have been differentiated on the basis of
spectroscopy and microscopy studies followed by modeling
of the species by computational studies. In the present paper,
several amido-calix[4]arene conjugates were synthesized and
characterized, and structures of a few of these were estab-
lished by single crystal XRD. All these were studied for their
selective recognition toward amino acids Asp and Glu and
the peptides GSH and GSSG and were compared with some
appropriate control molecular systems. The fluorescence
data obtained in the present case certainly exhibit higher
association constants (i.e., 532/676 M-1) as compared to that
reported in the literature (16-63 M-1 10
by exhibiting a
)
switch on fluorescence. It has been shown as 1:1 complex
by mass spectrometry. The studies clearly supported the
need for the calix[4]arene platform, amido arm, and term-
inal -COOH moiety in the recognition toward amino
acids and peptides possessing side chain -COOH moiety.
The structural features of the 1:1 complex formed between
amido-calix conjugatge and Asp/Glu/GSH have been derived
on the basis of DFT computational calculations, wherein the
complex is stabilized through three intermolecular hydrogen
bond interactions.
Thus the present study throws light on the manner in
which protein aggregations are induced in the biological
systems. Hence the results reported in this paper may
find implications in medical diagnosis and drug delivery
when the studies are extended appropriately, which can
indeed be monitored through spectroscopy and micro-
scopy methods.
Thus, amido-calix conjugates reported in this paper are
potential receptors that can bind to Asp/Glu residues by
sensing their side chain -COOH moiety as well as those
(41) Slocik, J. M.; Wright, D. W. Biomacromolecules 2003, 4, 1135.
(42) Lieberman, I.; Shemer, G.; Fried, T.; Kosower, E. M.; Markovich,
G. Angew. Chem., Int. Ed. 2008, 47, 4855.
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Experimental Section
SCHEME 3. Synthesis of NC-Phe-OHa
Synthesis and Characterization Data. The p-tert-butyl-calix-
[4]arene (C4A) has been synthesized by the condensation of
p-tert-butyl-phenol with formaldehyde in presence of NaOH as
per the procedure given by Gutsche and co-workers.26 Synthesis
for C4A-OEt, C4A-OH and C4A-Phe-OMe have already been
reported by us earlier,27 and hence only the characterization
data for these is given in this section
C4A-Phe-OMe. C68H82N2O10 (1086): Anal. (% found) C
74.75, H 7.20, N 2.75; (% required) C 75.14, H 7.55, N 2.58.
FTIR (KBr, cm-1): 3458, 3304 (νNH/OH), 1752 (νCdO, COOMe),
1670 (νCdO, CONH). 1H NMR (CDCl3, δ ppm): 1.02, 1.30 (s,
36H, C(CH3)3), 3.02-3.15 (m, 6H, Ar-CH2-Ar, and CβH2-Ph),
3.47 (d, 2H, Ar-CH2-Ar, J = 13.76 Hz), 3.64 (s, 6H, OCH3), 4.06
(d, 2H, Ar-CH2-Ar, J=12.9 Hz), 4.10 (d, 2H, Ar-CH2-Ar, J =
14.2 Hz), 4.14 (d, 2H, O-CH2-CO, J = 15.2 Hz), 5.03 (d, 2H,
O-CH2-CO, J=15.0 Hz), 5.10 (q, 2H, CRH, J = 7.8, 7.0 Hz),
6.87 (d, 4H, Ar-H), 7.03 (m, 14H, Ar-H, Ph-H), 7.73 (s, 2H,
OH), 9.51 (d, 2H, NH, J = 8.3). 13C NMR (CDCl3): δ 30.9,
31.7 (C(CH3)3), 32.0, 32.5 (Ar-CH2-Ar), 33.9, 34.0 (tert-C),
39.0 (CH2-Ph), 52.1 (OCH3), 52.8 (CH), 74.9 (O-CH2-CO),
124.7, 125.3, 125.8, 126.4, 126.5, 126.7, 127.6, 128.1, 128.9,
132.6, 136.1, 142.3, 147.8, 149.7, 150.0 (aromatic carbons),
168.8 (CONH), 171.8 (COOMe) ppm. ES-MS: m/z = 1087
([M þ H]þ, 100%).
a(a) Acetone, K2CO3, BrCH2COOEt, reflux for 15 h; (b) EtOH, aq
NaOH, reflux for 24 h; (c) Phe-OMe.HCl, dry THF, Et3N, HOBT,
DCC; (d) THF, LiOH. R = tert-butyl.
(d, 2H, Ar-CH2-Ar, J = 12.9 Hz), 3.54 (d, 2H, Ar-CH2-Ar, J =
13.1 Hz), 4.15 (d, 2H, Ar-CH2-Ar, J=13.1 Hz), 4.31 (d, 2H,
OCH2CO, J = 15.1 Hz), 4.41-4.36 (m, 2H each, Ar-CH2-Ar þ
CRH), 4.77 (d, 2H, OCH2CO, J = 15.1 Hz), 7.22-7.17 (m, 8H,
Ar-H), 8.30 (s, 2H, OH), 9.09 (d, 2H, HN, J = 7.6 Hz) ppm.
13C NMR (DMSO-d6): δ 17.0(CβH3), 30.8, 31.1 (C(CH3)3), 30.9
(Ar-CH2-Ar), 31.9 (Ar-CH2-Ar), 33.6, 34.0 (C(CH3)3), 47.8
(CRH), 74.5 (OCH2CO), 125.1, 125.6, 125.8, 126.4, 126.7,
127.4, 132.6, 132.9, 142.0, 147.7, 149.3, 150.0 (aromatic carbons),
167.7 (CdO CONH), 173.7 (CdO COOH) ppm. FABMS:
m/z 913 ([MþLi]þ, 100%). Anal. Calcd for C54H70N2O10
3
C4A-Phe-OH. To THF (15 mL) was added C4A-Phe-OMe
(1.086 g, 1 mmol), and the mixture was stirred at 0 °C. LiOH
(0.126 g, 3 mmol) in water (2 mL) was added to this at 0 °C, and
the mixture was brought to room temperature and was stirred
for 6 h. The solvent was removed under reduced pressure to give
a gel-like yellow substance. To this was added chilled water, and
the mixture was acidified with 1 N HCl (pH ∼1) to give a white
solid. The product was filtered, washed with water, and then
dried to yield a white solid. Yield: 0.952 g (90%); mp 180-182 °C.
FTIR (KBr): 3424 (νOH), 3336, 1741 (νCdO, COOH), 1654
4H2O (978): C, 66.23; H, 8.03; N, 2.86. Found: C, 66.40; H,
7.73; N, 2.67.
NC-Phe-OH (1e). Control molecule 1e has been synthesized
as shown in Scheme 3, and the characterization data has been
given under Supporting Information. Compounds 1b and 1c
have already been reported by us.27
1d. To a solution of 1c (0.5 g, 2.40 mmol) in CH2Cl2 (70 mL)
were added Et3N (1.7 mL, 12 mmol), 1-ethyl-(3-dimethylamino-
propyl)-3-carbodiimide hydrochloride (EDCI. HCl) (0.70 g,
3.6 mmol), and a catalytic amount of 1-hydroxybenzotriazole
(HOBT), and the mixture was stirred at 0 °C for 30 min under N2
atmosphere. L-Phenylalaninemethylester hydrochloride (0.73 g,
3.36 mmol) was added to this reaction mixture and stirred at
room temperature for overnight. The resulting mixture was
washed with water followed by saturated NaHCO3 and brine.
The organic residue was dried using sodium sulfate. Organic
solvent was removed under reduced pressure to result in a highly
viscous light yellowish oily liquid. This product was used for the
next step without further purification. Yield (0.55 g, 65%).
1
(νCdO, CONH) cm-1. H NMR (DMSO-d6): δ 1.10 (s, 18H,
C(CH3)3), 1.21 (s, 18H, C(CH3)3), 3.09-3.16 (m, 4H, CH2-Ph),
3.33 (d, 2H, Ar-CH2-Ar, J = 12.9 Hz), 3.38 (d, 2H, Ar-CH2-Ar,
J = 12.9), 4.16 (d, 2H, Ar-CH2-Ar J = 12.8 Hz), 4.25 (d, 2H,
Ar-CH2-Ar J = 12.9 Hz), 4.48 (m, 4H, OCH2CO), 4.64 (q, 2H,
CRH, J = 6.22 Hz), 7.08 (s, 4H, Ar-H), 7.12-7.21 (m, 14H
Ar-H þ phe-H), 8.16 (s, 2H, OH), 8.72 (d, 2H, NH, J = 7.63 Hz)
ppm. 13C NMR (DMSO-d6): δ 30.8, 31.4 (C(CH3)3), 33.6, 33.9
(tert-C), 36.8 (CβH2Ph), 53.6 (CRH), 74.1 (OCH2CO), 125.4,
125.8, 126.3, 127.0, 127.2, 128.1, 129.1, 132.7, 132.8, 137.3,
141.5, 147.2, 149.8, 150.5, (aromatic carbon resonances), 168.0
(CdO CONH), 172.4 (CdO COOH) ppm. ES-MS: m/z 1059
([M þ H]þ, 100%). Anal. Calcd for C66H78N2O10.H2O (1076):
C, 73.58; H, 7.48; N, 2.60. Found: C, 73.37; H, 7.29; N, 2.70
(Supporting Information).
1
C22H27NO4 (369.45). H NMR (CDCl3, δ ppm): 1.24 (s, 9H,
C(CH3)3), 3.06 (t, 2H, J = 6 Hz, Ph-CH2), 3.65 (s, 3H, OCH3),
4.4 (s, 2H, OCH2), 4.89 (q, 1H, J = 14 Hz, CH), 6.72 (d, 2H, J =
10 Hz, Ar-H), 6.97 (m, 1H, J = 10 Hz, Ar-H), 7.13 (d, 2H, J =
2 Hz, Ar-H), 7.16 (d, 2H, J = 2 Hz, Ar-H), 7.23 (m, 2H, Ar-H).
13C NMR: (CDCl3, δ ppm): 31.6, 38.1, 41.2, 52.5, 52.7, 67.5,
114.4, 126.6, 126.9, 127.3, 128.7, 129.3, 135.6, 145.1, 155.1,
168.3, 171.6. ESI- MS: m/z (intensity (%), fragment) 370.04.
(100, [M þ H] þ).
C4A-Gly-OH. Yield 0.781 g (89%); mp 210 °C (decomposes).
FTIR (KBr): 3440 (νOH), 1747 (νCdO, COOH); 1662 (νCdO
,
CONH) cm-1. 1H NMR (DMSO-d6): δ 1.13 (s, 18H, C(CH3)3),
1.20 (s, 18H, C(CH3)3), 3.47 (d, 4H, Ar-CH2-Ar, J = 13.2 Hz),
4.03 (m, 4H, CRH2), 4.23 (d, 4H, Ar-CH2-Ar, J = 12.8 Hz), 4.53
(s, 4H, OCH2CO), 7.17 (s, 8H, Ar-H), 8.41 (s, 2H, OH), 8.89
(t, 2H, HN) ppm. 13C NMR (DMSO-d6): δ 31.1, 31.6 (C(CH3)3),
33.8, 34.2 (C(CH3)3), 40.6 (CRH2), 72.1 (tert C), 74.3 (OCH2CO),
125.5, 125.9, 127.1, 133.0, 141.6, 147.5, 149.8, 150.1 (aromatic
carbons), 168.7 (CdO, CONH), 170.8 (CdO, COOH) ppm.
1e. To THF (50 mL) was added 1d (1.086 g, 1 mmol), and the
mixture was stirred at 0 °C. LiOH (0.126 g, 3 mmol) in water
(2 mL) was added to this at 0 °C, and the mixture was brought to
the room temperature and was stirred for 6 h. The solvent was
removed under reduced pressure to give a gel-like yellow sub-
stance. To this, chilled water was added and acidified with 1 N
HCl (pH ∼1). The compound was separated by organic layer. It
was dried using sodium sulfate and concentrated under reduced
pressure to give a yellowish liquid. To this was added diethyl
ether, and the mixture was kept overnight. A white solid was
formed next day. Yield: (0.42 g, 75%); C21H25NO4 (355.42). 1H
NMR (CDCl3, δ ppm): 1.22 (s, 9H, C(CH3)3), 3.10 (t, 2H, J =
13 Hz, Ph-CH2), 4.4 (s, 2H, OCH2), 4.90 (q, 1H, J = 8 Hz, CH),
6.70 (d, 2H, J = 9 Hz, Ar-H), 7.01 (m, 1H, J = 9 Hz, Ar-H), 7.15
ESI-MS: m/z901 ([MþNa]þ, 100%). Anal. Calcd for C52H66N2O10
3
3H2O (932): C, 66.93; H, 7.78; N, 3.00. Found: C, 66.71; H,
7.72; N, 2.86.
C4A-Ala-OH. Yield 0.843 g (93%); mp 196-198 °C. FTIR
(KBr): 3443, 3329, 1745 (νCdO, COOH); 1661 (νCdO, CONH)
1
cm-1. H NMR (DMSO-d6): δ 1.12 (s, 18H, C(CH3)3), 1.20
(s, 18H, C(CH3)3), 1.40 (d, 6H, CH3-ala, J = 7.27 Hz), 3.45
136 J. Org. Chem. Vol. 76, No. 1, 2011

Acharya et al.
JOCArticle
(d, 2H, J = 2 Hz, Ar-H), 7.17 (d, 2H, J = 2 Hz, Ar-H), 7.23 (m,
2H, Ar-H). 13C NMR (CDCl3, δ ppm): 31.6, 34.3, 37.5, 52.6,
67.4, 114.4, 126.6, 127.4, 128.8, 129.4, 135.4, 145.1, 155.0, 169.2,
174.5. ESI- MS: m/z (intensity (%), fragment) 356.03. (100,
[M þ H]þ) (Supporting Information).
prepared by sonicating the corresponding solutions for approxi-
mately 30 min. About 5-10 μL of the sonicated solutions was
spread on mica sheets as substrate and were allowed to air-dry.
The dried substrates were taken for AFM measurements.
For AFM studies with GSH-AgNP, 50 μL of GSH-AgNP
was added to 50 μL of 3 ꢀ 10-4 M solution of C4A-Phe-OH/
C4A-Gly-OH/C4A-Ala-OH/C4A-Phe-OMe.
For AFM studies with proteins, 100 μL of protein (1 mg/
10 mL) was added to 100 μL of C4A-Phe-OH/C4A-Gly-OH/
C4A-Ala-OH/C4A-Phe-OMe.
Dynamic Light Scattering Studies. The hydrodynamic dia-
meter of all the samples were measured in methanol (concen-
tration were kept same as in the case of fluorescence studies) at
25 °C. The incident laser (Coherent Inc. Santa Clara, CA)
radiation used was 633 nm, wavelength of 90°. The scattered
light was filtered through a vertical polarization filter. The
experiments were carried out using standard cylindrical BI-RC
12 glass cuvettes. The concentrations of the samples were kept
same as in fluorescence studies and the solvo-dynamic diame-
ters have been measured for 1:4 mol ratio of Asp/Glu and C4A-
Phe-OH.
SEM Studies. The powder samples were isolated from C4A-
Phe-OH, Asp, Glu, and mixtures of {C4A-Phe-OH þ Asp} or
{C4A-Phe-OH þ Glu} or corresponding protein samples. The
surface of these powder samples were coated with gold and used
for SEM.
Fluorescence Studies. Fluorescence emission spectra were
measured by exciting the solutions of C4A-Phe-OH/C4A-Gly-
OH/C4A-Ala-OH and the control molecules at 280 nm, and
the emission spectra were recorded in the 290-450 nm range.
The bulk solutions of C4A-Phe-OH/C4A-Gly-OH/C4A-Ala-
OH and other control molecules were prepared in CH3OH
except in the case of the aqueous CH3CN solvent system in
which CH3CN was used for bulk solution preparation. All the
measurements were made in 3 cm quartz cells, and a final
ligand concentration of 5 μM was maintained. During the
titration, the concentration of amino acids/peptides was var-
ied accordingly in order to result in requisite mole ratios of
amino acids/peptides, and the total volume of the solution was
maintained constant at 3 mL in each case by adding appro-
priate solvent or solvent mixtures. Normalized emission
(relative fluorescence) intensities (I/I0) (where I0 is the inten-
sity with no amino acid addition; I is the intensity at different
mole ratios of amino acid to ligand) were plotted against the
mole ratio of amino acid to ligand. The association constant of
the amino acid formed in the solution has been estimated using
the standard Benesi-Hildebrand equation, viz.,
Preparation of GSH-AgNP. Ag(0) nanoparticles have been
prepared by a reported procedure with required modifica-
tions.40-42 Sixty-eight milliliters of AgNO3 (2.5 ꢀ 10-2 M) was
taken in a flask and stirred vigorously for 5 min. Fifty milliliters
of GSH (0.3 ꢀ 10-2 M) was slowly added from a dropping
funnel at a constant rate. The solution was stirred for another
30 min. To this solution was added 16 mL of NaBH4 (10-1 M). The
solution immediately changes its color from yellow to brown.
Vigorous stirring was continued for 8 h in dark. Precipitation of
the product was done by adding absolute ethanol. The product was
isolated by repeated centrifugation at 10,000 rpm.
TEM Sample Preparation. Approximately 5-10 μL of an
aqueous solution of GSH-AgNP (after sonication) was used for
TEM studies. For studies with GSH-AgNP, the concentration
of C4A-Phe-OH/C4A-Gly-OH/C4A-Ala-OH/C4A-Phe-OMe was
kept 3 ꢀ 10-4 M. Fifty microliters of C4A-Phe-OH/C4A-Gly-
OH/C4A-Ala-OH/C4A-Phe-OMe was mixed with 50 μL of
GSH-AgNP and sonicated for 30 min. Approximately 5-10 μL
of this solution was placed in the carbon-coated copper grid and
allowed to air-dry.
1
1
1
¼
þ
I - I0
I1 - I0 ðI1 - I0ÞKa½AAꢁ
where I0 is the intensity of ligand, I is the intensity in the
presence of amino acid, I1 is intensity upon saturation with
amino acid, [AA] is the concentration of amino acid, and Ka is
the association constant of the complex formed.
Computational Optimization of the Complex Formed. The
starting geometry of C4A-Phe-OH has been taken from the
crystallographic coordinates.28 In view of the large computa-
tional times involved, C4A-Phe-OH has been truncated to give
precursor C4A-Phe-OH0 by replacing each tert-butyl group
present at the upper rim of the calix[4]arene with a hydrogen
without affecting the conformation of the calix[4]arene ring. The
geometry optimizations of the precursors (viz., C4A-Phe-OH0
and Asp/Glu/GSH) and the complex have been carried out in a
cascade fashion by starting from a semiempirical method and
ending with DFT as shown in this sequence, viz., AM1 f HF/
3-21G f HF/6-31G f B3LYP/3-21G f B3LYP/6-31G. The
initial structured for the complexes were modeled by taking the
DFT optimized structure of C4A-Phe-OH0 and Asp or Glu or
GSH, wherein the precursors were placed at a noninteracting
distance and further optimized in the cascade fashion. Similar
computations were carried out even with the tert-butyl trun-
cated version of C4A-Gly-OH (C4A-Gly-OH0).
AFM Studies. Tapping mode with a phosphorus-doped Si
probe having sharp fine tip at the end was used for all of the
studies. The sample of C4A-Phe-OH/C4A-Gly-OH/C4A-Ala-
OH for AFM was prepared from a 3 ꢀ 10-4 M solution in
methanol. The solution for the aspartic acid/glutamic acid
complex of C4A-Phe-OH were prepared by mixing 50 μL of
C4A-Phe-OH and 200 μL of aspartic acid/glutamic acid (3 ꢀ
10-3 M) in methanol. All samples for AFM studies were
Acknowledgment. C.P.R. acknowledges financial support
from DST, CSIR, and DAE-BRNS. A.A. and J.P.C.
acknowledge CSIR for SRF. We thank IIT Bombay for AFM
(DST-FIST, Department of Physics), SEM (at MEMS), and
DLS and TEM (at SAIF) facilities.
Supporting Information Available: Characterization data,
crystallographic data, fluorescence data, ESI MS data, compu-
tational data, AFM and SEM data, MALDI TOF data, and
UV-vis, powder XRD, and TEM data for GSH-AgNP. This
material is available free of charge via the Internet at http://
pubs.acs.org.
J. Org. Chem. Vol. 76, No. 1, 2011 137