Calixarene amino acids; building blocks for calixarene peptides and peptide-dendrimers

Article abstract of DOI:10.1016/S0040-4020(03)00947-5

A modular strategy towards receptor macromolecules is presented, which combines synthetically diverse peptide synthesis with highly functional calixarene chemistry. The design and synthesis of calix[4]arene amino acids 1a-f, calix-lysines, is described, which were used as construction blocks to assemble nanoscale, multivalent entities - calix-peptides 2 and calix-peptide-dendrimers 3.

Full text of DOI:10.1016/S0040-4020(03)00947-5

Tetrahedron 59 (2003) 5837–5848
Calixarene amino acids; building blocks for calixarene peptides
and peptide-dendrimers
*
Heng Xu, Gary R. Kinsel, Jiang Zhang, Meiling Li and Dmitry M. Rudkevich
Department of Chemistry and Biochemistry, University of Texas at Arlington, Box 19065, Arlington, TX 76019-0065, USA
Received 28 April 2003; revised 12 June 2003; accepted 16 June 2003
Abstract—A modular strategy towards receptor macromolecules is presented, which combines synthetically diverse peptide synthesis with
highly functional calixarene chemistry. The design and synthesis of calix[4]arene amino acids 1a–f, calix-lysines, is described, which were
used as construction blocks to assemble nanoscale, multivalent entities—calix–peptides 2 and calix–peptide-dendrimers 3.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
calixarenes. Calixarenes are extremely popular building
blocks in molecular recognition, and they have had a great
impact in the history of supramolecular chemistry.4 The
three-dimensional surface, commercial availability and
conformationally rigid structures make calixarenes most
convenient for synthetic elaboration. Calixarene-based
receptors are among the most effective and selective for
cations; they are widely used to transport and extract various
inorganic ions such as Naþ, Kþ, and Csþ,5–7 lanthanides
and actinides,8 as well as organic cationic species.9
Calixarene-based anion receptors show record thermodyn-
amics and selectivities for phosphate, sulfate and chlor-
ide.10,11 Calixarenes are also extremely important cavity-
forming modules and have been employed for the
construction of cavitands,12,13 (hemi)carcerands14 and
self-assembling capsules.15
Among the new challenges of chemistry are macromolecu-
lar entities composed of many identical components,
arranged to serve as receptors for given binding units or
ligands.1 Multifunctionality of receptor molecules reflects a
current trend of chemical sciences going towards ‘smart’
materials, informationally rich molecular devices, and
nanofabrication.2
Through multiple, multivalent interactions macromolecular
receptors display an increased affinity towards substrates,
including biologically relevant ones. Such increase is due to
either purely statistical reasons or positive cooperative
effects. It is important, therefore, to harness the properties of
compounds of intermediate size (e.g. 1–100 nm), somewhat
between the molecular and solid state, and to create
molecules that self-assemble into supramolecular structures,
including solids, the properties of which surpass those of the
molecular collection. This requires understanding the
structure and dynamics of intra- and intermolecular
interactions so that the properties of such molecular
collections can be predicted and controlled. For some
time, polymers have been offering effective routes towards
such multifunctionality and multivalency, and more
recently, dendrimers have entered the field.3 While poly-
mers, used for molecular recognition purposes, are often
heterogeneous, linear, flexible, and not well defined,
dendrimers tend to be monodisperse, globular, with defined
size and shape. To build multivalent, functional nanos-
tructures, polymers and dendrimers may be combined with
Existing syntheses of macromolecules that contain several
identical or different binding sites typically require multiple
steps for the preparation of monomers, thorough protec-
tion–deprotection strategy and are always time-consuming.
Another issue is the potential application, which requires
further synthetic planning and more experimental efforts.
On the other hand, nature employs a limited number of
construction blocks, modules, for instance amino acids, to
assemble—quickly and effectively—a huge variety of
proteins and enzymes. In this paper, we take advantage of
this and introduce a general modular strategy towards
multifunctional receptor macromolecules—calix–peptides
and peptide-dendrimers (Fig. 1). This combines highly
functional, receptor-oriented calixarene chemistry with
synthetically diverse peptide synthesis. Namely, the design
and synthesis of modules—calixarene amino acids is
presented. Further, modular assembly of nanostructures—
calixarene–peptides and calixarene–peptide-dendrimers is
demonstrated. In general, presented here ‘receptor–amino
Keywords: calixarenes; dendrimers; host compounds; supramolecular
chemistry.
*
Corresponding author. Tel.: þ1-817-272-5245; fax: þ1-817-272-3808;
e-mail: rudkevich@uta.edu
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00947-5

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H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
Figure 1. Calixarene amino acids as building blocks for multifunctional nanostructures—calix–peptides and calix–peptide-dendrimers. A modular approach.
acid’ based modular approach may be useful for the
construction of wide variety of multifunctional
nanostructures.
carboxylic group and two distant NH2 groups of dis-
tinguishable reactivity. The 1-NH2 group was attached to
the calixarene fragment, while the a-NH2 group was used in
the coupling reactions with the other lysine C(O)OH group
to form a peptide bond. This is an important feature of the
proposed modular approach: both ends of amino acids are
readily available for further peptide growth (Fig. 1).
Notably, while a number of calix[4]arene–amino acid
conjugates are known,16 they are attached either via N- or
C(O)O-terminus and, therefore, cannot be involved in the
repetitive, multivalent peptide chain elongation.
2. Results and discussion
2.1. Design
The proposed strategy is demonstrated for representative
preparation of calix[4]arene amino acids 1, calix[4]arene
dipeptides 2, and first generation of calix[4]arene peptide
dendrimers 3 (Fig. 2).
The choice of the calixarene component for this project was
justified by its strong affinity towards Naþ cation.5 It has
been known for years that calix[4]arenes, functionalized
with either ester or amide groups (or both) at the lower rim,
In the synthesis of calix[4]arene amino acids, we took
advantage of trifunctional lysine, which possesses a
Figure 2. Calixarene lysines 1a–f, dipeptides 2a,b, and tripeptides 3a,b.

H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
5839
demonstrate a unique Naþ selectivity, with the Kassq106
M21 in apolar solvents. Moreover, the calixarene lower rim
is relatively easy to functionalize. In our studies the
calixarene Naþ receptors were readily converted into the
corresponding acids for the coupling with lysine derivatives.
BOC deprotection with TFA, amine salts 11 and 12 were
isolated in a quantitative yield.
For N-a-BOC-N-1-Cbz-l-lysine 13, the carboxylic group
was similarly methylated (Cs2CO3, CH3I, DMF) to form the
corresponding ester 14 in 65% yield.20 The Cbz protection
was then quantitatively cleaved with 10% Pd/C in CH3OH
to yield pure lysine 15.21 After the a-BOC protection in 14
was cleaved with TFA in THF, lysine derivative 16 was
quantitatively isolated as a TFA salt.
2.2. Calixarene amino acids
First, we prepared a series of regioselectively protected
lysine derivatives (Fig. 3). Thus, commercially available
N-1-BOC-l-lysine 4 was coupled with n-octanoyl chloride
in the two-phase system EtOAc–H2O, 1:1 in the presence of
K2CO3 to afford N-a-acylated derivative 5 in 77% yield.
The long aliphatic chain was used for solubility reasons. As
followed from the absence of optical activity (see Section 4)
and subsequent 1H NMR analysis, compound 5 was
obtained as a racemate. Apparently, base-catalyzed racemi-
zation17 occurred as a result of rather basic conditions for
acylation of enantiomerically pure 4. The carboxylic group
in 5 was then protected through benzylation. Namely, acid 5
was treated with benzyl alcohol and 1,3-dicyclohexylcar-
bodiimide (DCC) in CH2Cl2, containing catalytic quantities
of 4-dimethylaminopyridine (DMAP) under nitrogen with
the formation of benzyl ester 6 in 61% yield. Subsequently,
the BOC protecting group in 6 was cleaved with TFA–THF,
1:4 mixture to afford the TFA salt of free amine 7 in a
quantitative yield.
N-a-BOC-N-1-BOC-l-lysine 1722 was methylated (CH3I,
Cs2CO3, DMF, 65%) and also benzylated (benzyl bromide,
Cs2CO3, DMF, 68%) to afford O-benzyl esters 18 and 19,
respectively. Both the a- and 1-BOC groups in these were
quantitatively cleaved with TFA in THF, yielding lysines 20
and 21 as TFA salts.
Finally, N-1-Cbz-l-lysine 2223 was acylated with n-octanoyl
chloride (K2CO3, EtOAc–H2O, 1:1) to yield lysine acid 23
in 71% yield. The a-Cbz protection in 9 was quantitatively
cleaved with 10% Pd/C in CH3OH to yield pure 24.24
In the coupling experiments between calixarenes and
lysines, calix[4]arene acid chloride 25 was employed,
which was prepared from the corresponding triester
monoacid calix[4]arene and SOCl2.25 An equimolar amount
of 25 in EtOAc was added to a solution of 1-deprotected
lysines 7, 11, 12, or 15 in EtOAc–H2O, 1:1 and excess
K2CO3. The reaction was complete in ,3 h and afforded
calixarene lysines 1a–d in 62–67% yield after column
chromatography.
In another series of experiments, N-1-BOC-l-lysine 4 was
protected by a Cbz group (Cbz–Cl, Na2CO3) with the
formation of N-a-Cbz-N-1-BOC-l-lysine 8 in 65%.18
Derivative 8 is optically active and, as will follow from
the NMR analysis, enantiomerically pure. The carboxylic
group in 8 was methylated (Cs2CO3, CH3I, DMF) to form
the corresponding ester 9 in 58% yield.19 Reaction between
8, 4-t-butylphenol, DCC and a catalytic amount of DMAP in
CH2Cl2 afforded phenyl ester 10 in 62% yield. After the
The structure of compounds 1a–d was confirmed by high-
resolution 1H NMR spectroscopy, matrix-assisted laser
desorption/ionization (MALDI) mass spectrometry and
CHN elemental analysis. Typical 1H NMR spectra are
Figure 3. Building blocks for calixarene amino acids, dipeptides and tripeptides.

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H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
lysine 1a was quantitatively accomplished by catalytic
hydrogenolysis with 10% Pd/C in CH3OH and afforded free
acid 1e. Cleavage of the BOC protection group in derivative
1d was carried out with TFA in THF to give lysine 1f with
free amino group.
2.3. Calix peptides
Peptides have already been functionalized with binding and
catalytic sites.28–30 For example, metalloporphyrin-con-
taining de novo designed proteins effectively mimic natural
photosynthetic centers.29 Peptide-based fluorescent metal
ion sensors comprise a metal recognition domain and a
signal transduction moiety that is triggered upon metal ion
binding.30 A number of selective sensors have been
constructed which are based on naturally occurring zinc
fingers, serum albumin proteins and siderophores. Another
important area of application is based on the DNA binding
ability of proteins and their assemblies.
In principle, any modified amino acid can be incorporated
within the polymeric peptide sequence. Preparative organic
chemistry of amino acids and peptide bond formation is well
developed. Secondary and even higher order structures of
peptides largely depend on the solvent, temperature, etc. and
can be studied by standard spectroscopic techniques and
also somewhat predicted by molecular modeling. As
follows from our own molecular modeling, the calix[4]ar-
˚
ene platform is #10 A in its dimensions, so when
incorporated within the peptide network it can easily fit
there without disrupting important hydrogen bonding
processes.
In the synthesis of calixarene peptides, standard peptide
coupling was employed. Lysines 1f and 1e, possessing free
amino and carboxylic groups, respectively, were mixed with
equimolar amounts of DCC and HOBT in DMF and stirred
at rt for 36 h. Standard workup and chromatography
afforded calix dipepide 2a in 46% yield. In the alternative
procedure, 2.4 equiv. of calixarene acid chloride 25 were
coupled with the 1-NH2 groups of preformed bis-lysine
derivative 26 (K2CO3, EtOAc–H2O, 1:1) with the for-
mation of 2a in 49% yield after column chromatography.
Bis-lysine 26 was prepared from bis-BOC derivative 27,
which itself was synthesized from amino acids 24 and 5
(DCC, HOBT, DMF, 64%). Both protocols gave compar-
able quantities of calyx dipeptide 2a.
Figure 4. 1H NMR spectra (500 MHz, CDCl3, 295^1 K) of calixarene
lysines: (a) 1b, (b) 1a, (c) 1d, (d) 1d·NaþClO42. The residual CHCl3 signals
are marked ‘·’.
consistent with the monosubstituted calix[4]arene pattern,
and contain in particular three calixarene aromatic (appar-
ent) singlets in 1:2:1 ratio and three calixarene t-Bu singlets
in 1:2:1 ratio (CDCl3, 295 K) (Fig. 4). The 1-NH–C(O)
amide proton is seen far down field as a triplet at ,8.4 ppm
and apparently involved in the CvO· · ·H–N hydrogen
bonding with the calixarene lower rim carbonyl oxygens in
apolar CDCl3.26
Similarly, bis-lysine 2831 was prepared from amino acids 13
and 16 (DCC, HOBT, DMF, 53%). This was then
deprotected with Pd/C in CH3OH resulting in 2932 in 94%
yield. Bis-lysine 29 reacted with 2.4 equiv. of monoacid
chloride 25 in presence of K2CO3 (EtOAc–H2O, 1:1) with
the formation of calixarene dipeptide 2b in 45% yield after
column chromatography.
This was additionally confirmed by molecular modeling.27
The a-NH–C(O) proton is recorded as doublet and seen at
,5.5 ppm for carbamate derivatives 1b–d, and at 6.4 ppm
for amide derivative 1a. As follows from molecular
modeling (Fig. 5), the calix[4]arene fragment is positioned
˚
,5–7 A away from the amino acid fragment and should not
sterically interfere with the peptide bond formation. It can
also easily fit within the peptide/dendritic superstructures
without disrupting hydrogen bonding and intramolecular
folding processes.
The structure of calixarene dipeptides 2a,b was confirmed
by FTIR, 1H and COSY NMR spectroscopy, MALDI mass
spectrometry and CHN elemental analysis. Although the
compounds were reasonably soluble in CDCl3, the corre-
sponding H NMR spectra appeared to be rather broad,
especially the amide/peptide NH signals, most probably due
to noncovalent aggregation. Molecular modeling (MM2 and
1
Standard manipulation with protecting groups afforded
calixarene amino acids with free either NH2 or C(O)OH
ends. For example, removal the O-benzyl group in calix-

H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
5841
Figure 5. MacroModel 7.1 (Amberp ForceField) representation of calix[4]arene lysine 1 (A), calixarene dipeptide 2 (B), and calixarene dendritic structure 3
(C). Possible CvO· · ·H–N hydrogen bonding is marked by arrows. For dipeptide 2, both folded and unfolded structures are shown. The CH hydrogen and long
alkyl chains are omitted for viewing clarity.
Amber Force Field)27 suggests that not only the 1-NH–
C(O) amide protons participate in the CvO· · ·H–N
hydrogen bonding with the calixarene lower rim carbonyl
oxygens, but also the a-NH–C(O) proton is now involved in
the intramolecular folding process (Fig. 5). The FTIR
spectra of 2a,b in KBr contain mostly associated NH
used as a core to which photochromic dyes and carbo-
hydrate dendrons were attached.33 This was relatively easy
to achieve through standard, symmetrical tetrafunctional-
ization of calixarene. However, in two published
examples,34 involving calixarenes as branches and/or sur-
face elements, the repetitive branching strategy for the
preparation of higher dendritic generations was not clearly
visible and had not been demonstrated. In a single report,
stretching at ,3300 cm21
.
¨
In contrast, competitive and hydrogen bonding DMSO-d6
produces sharp spectra. Typically, the 1H NMR spectra are
consistent with the monosubstituted calix[4]arene pattern.
Dipeptide 2a and also 26 and 27 exhibit two ,1:1 sets of
signals for all groups of protons, indicating that pairs of
diastereomeric products are formed in these cases. We
attribute this to the base-catalyzed racemization of the
amino acid precursor 5, which was subsequently used in the
preparation of racemic calixarene lysine 1e, and also
resulted in pairs of diastereomers for dipeptides 26 and
27. This is not the case for dipeptides 2b, 28, and 29, which
Bohmer and co-workers postulated the divergent approach
towards dendrimers, based on the multiple amide bond
formation between appropriately functionalized calix[4]ar-
ene amines and calix[4]arene acid chlorides.35 Experimen-
tally, this had not been accomplished. Proposed here is the
convergent36 approach towards calixarene-containing den-
drimers, which employs peptide chemistry. In such
dendrimers, calixarene fragments are situated on the
surface, thus providing the multivalency, and peptide
groups serve as branching units.
1
were obtained enantiomerically pure (optical rotation, H
NMR analysis in different solvents).
In principle, peptide dendrimers are broadly defined as any
dendrimer containing peptide fragments.37 They are well
suited for various biochemical and biotechnological appli-
cations, including diagnostic reagents, protein mimetics,
anticancer and antiviral agents, vaccines, and drug
delivery systems. Synthetically, amino acids are appealing
building blocks because of well-developed peptide-
coupling techniques. Polyamino acids consisting of
branches of a trifunctional acid, especially lysine, represent
the largest and most popular group of branching units being
used today. The diamino nature of lysine creates a unique
situation where each additional level of lysines effectively
doubles the number of sites to which monomers are
attached.
2.4. Calix peptide dendrimers
Dendrimers have revolutionary entered supramolecular
chemistry.3 The layered, nanoscale architecture, globular
shape, controlled nanomolar dimensions and easy modifi-
cations have made dendrimers unique carriers of supramo-
lecular properties. Multivalent surfaces of dendrimers offer
a unique opportunity for the substrate binding, providing
strength and, often, cooperativity, with minimalized energy
loses for reorganization and diffusion.
Surprising, that till now, only a limited progress has been
reported in the literature on the preparation of calixarene
dendrimers. For example, our recent search with SciFinder
Scholar produced ,6000 references on dendrimers and
,5000 references on calixarenes, but less than 10 papers on
calixarene-based dendrimers. A calix[4]arene platform was
Two equivalents of calix amino acid 1e were submitted to
the coupling reaction with lysine methyl ester 20 in the
presence of equimolar amounts of DCC and HOBT in DMF
producing, after purification, 41% of dendritic bis-calixar-
ene 3a.

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H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
In the alternative procedure, triester monoacid chloride 25
was coupled with the preformed, first-generation lysine
dendrimer 30 in presence of K2CO3 (EtOAc–H2O, 1:1).
This yields 47% of 3a after column chromatography on
silica gel. Bis-lysine 30 was prepared by the Pd/C
hydrogenation of bis-Cbz derivative 31 in CH3OH, which
in its turn was obtained from amino acids 23 and O-methyl
ester of l-lysine 20. Accordingly, both procedures success-
fully yielded the same dendrimer. Since racemic lysine 5
was employed, dendrimer 3a in our experiments was
obtained as a mixture of diastereomers, which is highly
difficult to separate. Similarly, calixarene acid chloride 25
was coupled with the preformed, first-generation lysine
dendrimer 32 (K2CO3, EtOAc–H2O) to afford optically
pure dendrimer 3b in 42% after column chromatography.
Precursor dendrimer 32 was prepared by the Pd/C
hydrogenation of bis-Cbz derivative 33, which was obtained
from amino acids 13 and 20.
showed perfect Naþ coordination within the lower rim
pocket, consisting of eight basic, ether and carbonyl oxygen
atoms. Such complexes are kinetically stable on the NMR
time scale (e.g. Kassq106 M21), and the exchange between
free and complexes species is slow.
We found that calix amino acids 1a–d, calix peptides 2a,b
and dendrimer 3a,b strongly complex Naþ cations within
their binding pockets. In the preliminary experiments,
extraction was studied from aqueous solution of NaClO4
to CH2Cl2. Specifically, 1a–d, 2a,b, and 3a,b were
dissolved in CH2Cl2 and stirred overnight with equal
volumes of saturated aqueous solution of NaClO4. Organic
layers were then separated, evaporated under reduced
pressure, dried in vacuum and analyzed by high-resolution
1H NMR spectroscopy in CDCl3. The corresponding Naþ
complexes formed quantitatively. The NMR spectra chan-
ged dramatically and exhibited completely new sets of
signals for all protons. Especially notable are ,0.3 ppm
down field shift of the calixarene aromatic protons, and
,0.2 ppm down field shift of the OCH2 ethyl ester protons
(Fig. 4). This could be attributed to the electron-with-
drawing nature of Naþ cation. The methylene CH2 protons
next to the carbonyl at the lower rim are shifted up field,
which has been observed for simpler calixarene–Naþ
complexes and is caused by complexation-induced fixation
of the carbonyls. Also of interest, the ,1 ppm up field shift
of the lower rim C(O)NH proton. Apparently, Naþ cation
disrupts the intramolecular CvO· · ·H–N hydrogen bonding
at the lower rim upon complexation. No residual uncom-
plexed receptors were detected (Fig. 7).
The structure of compounds 3a,b was confirmed by high-
1
resolution H and COSY NMR spectroscopy and MALDI
mass spectrometry. With their nanoscale dimensions and
masses of .2500 Da, the structural assignment of 3a,b was
heavily relayed on the spectral features of simpler
precursors such as tripeptides 30–33 and also calix lysines
1a–f. Although being soluble in CDCl3, 3a,b exhibit broad
1H NMR spectra, probably due to noncovalent aggregation.
As expected, DMSO-d6 produces sharp spectra. Similar to
dipeptides 2a,b, molecular modeling suggests that not only
the 1-NH–C(O) amide protons are involved in the
CvO· · ·H–N hydrogen bonding with the calixarene lower
rim carbonyl oxygens, but also the a-NH–C(O) proton
participates in the intramolecular folding process (Fig. 5). In
the folded conformation, the calix[4]arene fragments are
The efficiency of compounds 1a–d, 2a,b, and 3a,b as Naþ
receptors was also evident in the MALDI mass spectra:
exclusively [MþNa]þ parent ions were observed. This
behavior is in contrast to most MALDI mass spectra of
peptides which yield predominantly [MþH]þ parent ions.
At the same time, decomplexation of Naþ cation was readily
achieved by an excessive washing with water.
˚
positioned ,15 A away from each other. Such a long
distance is important for building next generations of calix
dendrimers.
Dendritic structures 3a,b may be regarded as a first
generation of more expanded peptide dendrimers with
calixarene surfaces. Indeed, in the proposed here convergent
strategy, the C(O)OMe group of the core may be
deprotected,38 activated (DCC, HOBT) and used in the
coupling step with lysine ester 21. This will result in calix
peptide dendrimer 34 of a second generation (Fig. 6).
Subsequently, the core Bn-ester group can be further
deprotected (H2, Pd/C), activated and coupled with 21.
This will result in the third generation dendrimer 35. These
steps can, in principle, be repeated. Molecular modeling,
however, suggests that further generations of 35 may be
rather sterically crowded. Accordingly, proposed here
repetitive branching strategy would allow for the prep-
aration of higher dendritic generations of calixarenes.
While cooperativity in Naþ binding is not expected for 2a,b,
and 3a,b, the presence of (a) multiple binding sites on the
periphery/surface of such nanoscale receptors and (b)
unique, intramolecular hydrogen bonding within their
peptide scaffolds may offer strong and specific affinity
towards guests. In contrast to simple collections of small
receptor-molecules, which might require prior assembly/
reorganization for transport and delivery, nanostructures
2a,b, and 3a,b and especially their larger relatives can use
intramolecular forces to arrange their multiple and inter-
connected components in ways that minimize free energy.
Such intramolecular processes may lead to the shape changes,
specific internal microenvironments, and cooperative organ-
ization of ion binding surfaces. We are currently exploring
ion-complexing properties of calix–peptides 2a,b and calix
peptide dendrimers 3a,b and report on this in the sequel.
2.5. Preliminary complexation experiments
Calix[4]arene functionalized with C(O)OAlk ester and/or
C(O)NHAlk/C(O)NAlk2 amide groups at the lower rim
demonstrate a unique affinity and selectivity towards Naþ
cation not only in single solvents, but also in extraction
experiments from water to organic solvent and in the
transport experiments through bulk liquid and supported
liquid membranes.5 Solid-state and NMR-derived structures
3. Conclusions and outlook
A modular strategy towards multivalent, nanoscale
receptor macromolecules—calix–peptides and peptide-
dendrimers—has been introduced. This combines unique

H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
5843
Figure 6. Towards higher generations of calix peptide dendrimers.
host–guest capabilities of calixarene chemistry with
synthetically universal peptide synthesis. Calixarene
amino acids are now available to be incorporated into
peptide networks and nanostructured biological materials.
Extended peptide dendrimers and dendritic polymers with
calixarene surfaces can also be constructed. Through
multiple, multivalent interactions such nanoscale receptors
display a strong affinity towards substrates. Unique self-
folding features of the incorporated peptide scaffolds offer
an attractive opportunity to properly arrange and expose the
multiple and interconnected receptor sites. This opens novel
perspectives for the modular design of multifunctional
receptors and sensors, multiply attached cavities and
capsules, macromolecular devices and smart polymeric
materials. We are currently working in these directions.
4. Experimental
4.1. General
Figure 7. Complexation of Naþ cation at the lower rims of multiple
calþix[4]arene fragments in amino acids 1 and peptides 2 and 3. Addition of
Na disrupts intramolecular CvO· · ·H–N hydrogen bonding. The ester
CvO groups turn around to coordinate the cation.
Melting points were determined on a Mel-Temp apparatus
(Laboratory Devices, Inc.) and a Buchi apparatus and are
uncorrected. 1H, 13C and COSY NMR spectra were

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H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
recorded at 295^18C on JEOL Eclipse 500 MHz spec-
trometer. Chemical shifts were measured relative to residual
non-deuterated solvent resonances. FTIR spectra were
recorded on a Bruker Vector 22 FTIR spectrometer.
337 nm MALDI mass spectrometry was performed on a
Bruker BiFLEX I linear time-of-flight mass spectrometer
operated in delayed extraction mode. Elemental analysis
was performed on a Perkin–Elmer 2400 CHN analyzer. For
DCC (0.27 g, 1.3 mmol), catalytic amount of DMAP, and 4-
t-butylphenol (0.24 g, 1.6 mmol) at rt overnight. The
reaction mixture was then filtered and concentrated in
vacuo. Column chromatography with EtOAc–CH2Cl2, 2:8
as an eluent afforded phenyl ester 10 (0.41 g, 62%) as an oil:
1H NMR (CDCl3): d 7.35 (m, 7H), 7.0 (d, J¼8.7 Hz, 2H),
5.54 (d, J¼7.3 Hz, 1H), 5.13 (s, 2H), 4.58 (m, 2H), 3.12 (m,
2H), 1.42 (s, 9H), 1.25 (s, 9H), 2.1–1.9, 1.9–1.8, 1.6–1.2
(3£m, 6H).
˚
column chromatography, Silica Gel 60 A (Sorbent Tech-
nologies, Inc.; 200–425 mesh) was used. All experiments
with moisture- or air-sensitive compounds were run in
freshly distilled, anhydrous solvents under a dried nitrogen
atmosphere. Molecular modeling was performed using
MacroModel 7.1.27
4.1.5. N-a-Cbz-l-lysine, O-methyl ester, TFA salt 11. A
solution of 9 (0.39 g, 1.0 mmol) in THF (20 mL) was treated
with TFA (5 mL) at rt for 2 h. The reaction mixture was
concentrated in vacuo to afford pure salt 11 (0.38 g, 92%).
1H NMR (CDCl3): d 7.30 (m, 5H), 5.74 (d, J¼7.3 Hz, 1H),
5.06, 5.03 (2£d, J¼11.9 Hz, 2H), 4.26 (m, 1H), 3.69 (s, 3H),
2.89 (m, 2H), 1.9–1.7, 1.7–1.5, 1.5–1.2 (3£m, 6H); 13C
NMR (CDCl3): d 173.0, 162.0 (q, JC–F¼37.9 Hz), 156.4,
136.2, 128.6, 128.3, 128.0, 127.8, 116.9 (q, JC–
4.1.1. N-a-(n-Octanoyl)-N-1-BOC-(6)-lysine 5. A sol-
ution of N-1-BOC-l-lysine 4 (0.50 g, 2 mmol) in mixture of
H2O (20 mL) and EtOAc (20 mL) was treated with n-
octanoyl chloride (3.30 g, 20.3 mmol) and then stirred at rt
for 3 h. The reaction mixture was diluted with aq HCl (5%
vol, 50 mL) and CH2Cl2 (80 mL). The formed layers were
separated. The aqueous layer was extracted by CH2Cl2
(3£30 mL), and the combined organic layer was then dried
over anhydrous Na2SO4 and evaporated. The residue was
solidified with hexane, yielding pure 5 (0.57 g, 77%) as a
colorless solid: mp 115–1168C; [a]2D3¼0.0 (c¼0.02, EtOH);
1H NMR (DMSO-d6): d 7.99 (d, J¼7.8 Hz, 1H), 6.77 (t,
J¼7.1 Hz, 1H), 4.12 (dt, J¼8.5, 7.8 Hz, 1H), 2.87 (dt,
J¼7.6, 7.1 Hz, 2H), 2.09 (dt, J¼7.3, 2.5 Hz, 2H), 1.36 (s,
9H), 1.71–1.60, 1.60–1.40, 1.30–1.11 (3£m, 16H), 0.85 (t,
J¼6.6 Hz, 3H); 13C NMR (DMSO-d6): d 174.5, 172.9,
156.1, 77.9, 52.2, 35.6, 31.8, 31.3, 29.6, 29.1, 29.0, 28.8,
25.8, 23.4, 22.6, 14.5; MS-EI m/z 371.9 (Mþ, calcd for
C19H36N2O5 372.50).
¼308.1 Hz), 67.1, 53.7, 52.6, 39.5, 31.7, 26.8, 22.1.
F
4.1.6. N-a-Cbz-l-lysine, O-(4-tert-butyl)phenyl ester,
TFA salt 12. Prepared analogously to compound 11 in a
1
95% yield. H NMR (CDCl3): d 7.35 (m, 7H), 6.96 (d,
J¼8.3 Hz, 2H), 5.71 (d, J¼7.3 Hz, 1H), 5.12, 5.07 (2£d,
J¼12.4 Hz, 2H), 4.50 (dt, J¼8.3, 4.1 Hz, 1H), 2.92 (m, 2H),
1.27 (s, 9H), 2.0–1.0 (m, 6H).
4.1.7. N-1-Cbz-l-lysine, O-methyl ester, TFA salt 16. A
solution of 14 (0.2 g, 0.51 mmol) in THF (15 mL) was
treated with TFA (4 mL) and then stirred at rt for 2 h. The
reaction mixture was concentrated in vacuo to afford pure
1
salt 16 (0.15 g, 95%). H NMR (CDCl3) d 7.30 (m, 5H),
5.16 (br s, 1H), 5.05 (s, 2H), 3.98 (m, 1H), 3.76 (s, 3H), 3.13
(m, 2H), 2.0–1.8 (m, 2H), 1.6–1.3 (m, 4H); 13C NMR
(CDCl3): d 170.2, 162.0 (q, JC–F¼36.0 Hz), 157.1, 136.6,
128.6, 128.2, 128.0, 127.9, 127.8, 116.4 (q, JC–
4.1.2. N-a-(n-Octanoyl)-N-1-BOC-(6)-lysine, O-benzyl
ester 6. A solution of free acid 5 (0.37 g, 1.0 mmol) in THF
(20 mL) was mixed with DCC (0.21 g. 1.0 mmol), catalytic
amount of DMAP, and benzyl alcohol (0.13 g, 1.2 mmol).
The solution was stirred at rt overnight, filtered, and
concentrated in vacuo. Column chromatography on silica
gel with EtOAc–CH2Cl2, 3:7 as an eluent afforded 6
¼291.7 Hz), 62.7, 53.2, 53.1, 40.4, 29.8, 29.1, 21.8.
F
4.1.8. N,N-a,1-Bis-BOC-l-lysine, O-methyl ester 18. To a
solution of l-lysine (2.0 g, 13.7 mmol) in water–dioxane,
1:1 (40 mL) were added BOC-anhydride (7.5 g, 34.3 mmol)
and 1N NaOH (14 mL). The reaction mixture was stirred for
6 h at rt, then concentrated till 15 mL. The pH was adjusted
to 2.4 by adding aqueous NaHSO4, and the product was
extracted with EtOAc (2£40 mL). The solvent was
1
(0.28 g, 61%) as a colorless oil: H NMR (CDCl3): d 7.36
(m, 5H), 6.03 (d, J¼7.8 Hz, 1H), 5.16, 5.12 (2£d,
J¼12.4 Hz, 2H), 4.64 (dt, J¼7.8, 5.0 Hz, 1H), 4.51 (br s,
1H), 3.04 (dt, J¼7.3, 6.0 Hz, 2H), 2.21 (t, J¼7.3 Hz, 2H),
1.43 (s, 9H), 1.90–1.80, 1.72–1.58, 1.50–1.37, 1.37–1.18
(4£m, 16H), 0.87 (t, J¼6.9 Hz, 3H); MS-EI m/z 461.8 (Mþ,
calcd for C26H42N2O5 462.6).
1
evaporated to give 17 (3.37 g, 71%) as an oil: H NMR
(DMSO-d6): d 6.98 (br s, 1H), 6.74 (br s, 1H), 3.83 (m, 1H),
2.86 (m, 2H), 1.35 (s, 18H), 1.7–1.1 (m, 6H). Diprotected
derivative 17 (1.0 g, 2.9 mmol) was dissolved in THF
(30 mL) and H2O (6 mL), and the solution was neutralized
till pH 7 with 20% aqueous Cs2CO3 and evaporated to
dryness. The cesium salt was then stirred CH3I (0.49 g,
3.5 mmol) in DMF (20 mL) for 2 h. Upon removal of the
solvent by evaporation and treatment with H2O (80 mL), the
product was extracted with EtOAc (3£50 mL). The organic
layer was dried over anhydrous Na2SO4 and evaporated to
4.1.3. N-a-(n-Octanoyl)-(6)-lysine, O-benzyl ester, TFA
salt 7. A solution of 6 (0.46 g, 1.0 mmol) in THF (20 mL)
was stirred with TFA (5 mL) at rt for 2 h. The reaction
mixture was concentrated in vacuo to afford salt 7 (0.45 g,
94%), which was used without further purification. 1H NMR
(CDCl3): d 7.33 (m, 5H), 6.71 (d, J¼7.8 Hz, 1H), 5.16, 5.12
(2£d, J¼12.4 Hz, 2H), 4.55 (dt, J¼8.3, 5.0 Hz, 1H), 2.93
(m, 2H), 2.23 (t, J¼7.3 Hz, 2H), 2.0–1.0 (m, 16H), 0.87 (t,
J¼6.9 Hz, 3H).
1
afford methyl ester 18 (0.68 g, 65%) as an oil: H NMR
(CDCl3) d 5.10 (br s, 1H), 4.59 (br s, 1H), 4.26 (m, 1H), 3.72
(s, 3H), 3.08 (m, 2H), 1.42 (s, 18H), 2.0–1.2 (m, 6H).
4.1.4. N-a-Cbz-N-1-BOC-l-lysine, O-(4-tert-butyl)phenyl
ester 10. A solution of 8 (0.50 g, 1.3 mmol, [a]2D3¼23.6
(c¼0.03, EtOH) in dry CH2Cl2 (20 mL) was stirred with
4.1.9. N,N-a,1-Bis-BOC-l-lysine, O-benzyl ester 19. Free
acid 17 (1.0 g, 2.9 mmol) was dissolved in CH3OH (30 mL)

H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
5845
and H2O (6 mL), and the solution was neutralized till pH 7
with 20% aqueous Cs2CO3 and evaporated to dryness. The
resulting cesium salt was then stirred with benzyl bromide
(0.60 g, 3.5 mmol) in DMF (20 mL) for 2 h. The solution
was evaporated, and the product was partitioned between
H2O (60 mL) and EtOAc (120 mL). The organic layer was
dried over anhydrous Na2SO4 and evaporated to afford ester
4H), 2.10 (m, 2H), 1.36 (s, 18H), 2.0–1.0 (m, 22H), 0.85 (t,
J¼6.6 Hz, 3H).
A solution of 27 (0.50 g, 0.81 mmol) in THF (20 mL) was
treated with TFA (5 mL) and stirred at rt for 2 h. The
reaction mixture was concentrated to afford pure 26 (0.50 g,
96%). 1H NMR (DMSO-d6, only one diastereomer is
shown): d 8.30 (d, J¼7.6 Hz, 1H), 7.95 (d, J¼7.8 Hz, 1H),
4.22 (m, 1H), 3.62 (s, 3H), 2.75 (m, 4H), 2.10 (m, 2H), 2.0–
1.0 (m, 22H), 0.85 (t, J¼6.6 Hz, 3H).
1
19 (0.86 g, 68%): H NMR (CDCl3) d 7.32 (m, 5H), 5.16,
5.10 (2£d, J¼11.9 Hz, 2H), 5.10 (br s, 1H), 4.57 (br s, 1H),
4.28 (dt, J¼7.8, 5.0 Hz, 1H), 3.04 (dt, J¼6.9, 6.4 Hz, 2H),
1.43 (s, 18H), 2.0–1.0 (m, 6H); 13C NMR (CDCl3): d 172.8,
156.2, 155.6, 135.5, 128.6, 128.5, 128.3, 79.8, 79.1, 67.2,
53.3, 40.1, 32.1, 29.6, 28.5, 28.4, 22.5.
4.1.13. Tripeptide 30, a mixture of diastereomers. To a
stirred and ice cooled solution of 20 (0.19 g, 0.50 mmol) in
DMF (15 mL) was added Et3N (0.14 mL, 1 mmol) and then
acid 23 (0.42 g, 1 mmol), HOBT (0.28 g, 2 mmol), and
DCC (0.43 g, 2 mmol). The reaction mixture was allowed to
stir for 30 min at 08C and for 36 h at rt, filtered,
concentrated, diluted with EtOAc (200 mL), and washed
successively with 1N NaHSO4 (4£50 mL), water
(3£50 mL), 1N NaHCO3 (4£50 mL), and again water
(3£50 mL). The organic layer was then dried over
anhydrous Na2SO4 and evaporated. The residue was
chromatographed on silica gel eluting with CHCl3–
CH3OH, 9:1 to afford bis-Cbz-protected trilysine 31
(0.21 g, 45%) as an oil: 1H NMR(DMSO-d6, only one
diastereomer is shown): d 8.20 (br s, 1H), 7.84 (m, 3H), 7.33
(m, 10H), 7.20 (t, J¼5.5 Hz, 2H), 4.98 (s, 4H), 4.25 (m, 1H),
4.15 (m, 2H), 3.60 (s, 3H), 2.94 (m, 6H), 2.10 (m, 4H), 1.8–
1.1 (m, 38H), 0.84 (t, J¼6.4 Hz, 3H).
4.1.10. l-Lysine, O-methyl ester, bis-TFA salt 20. A
solution of 18 (0.5 g, 1.4 mmol) in THF (20 mL) was treated
with TFA (5 mL) and stirred at rt for 2 h. The reaction
mixture was concentrated to afford pure 20 (0.51 g, 95%).
1H NMR (DMSO-d6): d 4.03 (br s, 1H), 3.75 (s, 3H), 2.75
(m, 2H), 1.77 (m, 3H), 1.6–1.3 (m, 3H); 13C NMR (DMSO-
d6): d 170.6, 159.3 (q, JC–F¼31.7 Hz), 117.6 (q, JC–
¼298.5 Hz), 53.2, 52.3, 38.9, 30.0, 26.9, 21.8. Benzyl ester
F
21 was prepared analogously in 82% yield: 1H NMR
(DMSO-d6): d 7.37 (m, 5H), 5.20 (s, 2H), 4.06 (m, 1H), 2.70
(dt, J¼8.3, 6.4 Hz, 2H), 1.78 (m, 2H), 1.50 (m, 2H), 1.37
(m, 1H), 1.26 (m, 1H); 13C NMR (DMSO-d6): d 169.9,
159.3 (q, JC–F¼32.2 Hz), 135.7, 129.0, 128.8, 128.8, 117.6
(q, JC–F¼298.0 Hz), 67.6, 52.2, 38.9, 30.1, 26.9, 21.7.
4.1.11. N-a-(n-Octanoyl)-N-1-Cbz-(6)-lysine 23. A sol-
ution of N-1-Cbz-l-lysine 22 (0.50 g, 1.78 mmol) in water
(20 mL) and EtOAc (20 mL) was treated with n-octanoyl
chloride (2.46 g, 17.8 mmol) and stirred at rt for 3 h, after
which 5% HCl (50 mL) and CH2Cl2 (80 mL) were added.
After separation, the aqueous layer was extracted with
CH2Cl2 (3£50 mL) times, and the combined organic
layers were dried over anhydrous Na2SO4 and evapor-
ated. The residue was solidified in hexane to yield 23
(0.51 g, 71%) as a colorless solid: mp 119–1208C;
[a]2D3¼0.0 (c¼0.02, EtOH); 1H NMR (DMSO-d6) d
12.44 (s, 1H), 7.99 (d, J¼7.8 Hz, 1H), 7.34 (m, 5H),
7.20 (t, J¼5.3 Hz, 1H), 4.99 (s, 2H), 4.12 (m, 1H), 2.96
(m, 2H), 2.08 (m, 2H), 1.8–1.1 (m, 16H), 0.84 (t,
J¼6.6 Hz, 3H); 13C NMR (DMSO-d6) d 174.5, 172.9,
156.6, 137.8, 128.9, 128.3, 128.2, 65.7, 52.2, 35.6, 31.8,
31.2, 29.6, 29.1, 29.0, 25.8, 23.4, 22.6, 14.5.
A solution of tripeptide 31 (0.15 g, 0.16 mmol) in CH3OH
(10 mL) was treated with 10% Pd/C (15 mg) and stirred
under a hydrogen atmosphere for 6 h. The mixture was
filtered through Celite and concentrated. The residue was
dried under high vacuum to give 30 (0.10 g, 93%) as an oil:
1H NMR (DMSO-d6, only one diastereomer is shown) d
8.20 (br s, 1H), 7.84 (m, 3H), 4.25 (m, 1H), 4.14 (m, 2H),
3.56 (s, 3H), 3.42 (m, 4H), 3.02 (m, 2H), 2.06 (m, 4H), 1.8–
1.0 (m, 38H), 0.81 (t, J¼6.9 Hz, 6H).
4.1.14. Tripeptide 32. To a stirred and ice cooled solution
of 20 (0.17 g, 0.43 mmol) in DMF (20 mL) was added Et3N
(0.12 mL, 0.86 mmol) and then after 15 min, lysine 13
(0.33 g, 0.86 mmol), HOBT (0.24 g, 1.72 mmol) and DCC
(0.35 g, 1.72 mmol).The mixture was allowed to stir for
30 min at 08C and for 36 h at rt, then filtered, concentrated in
vacuo, diluted with EtOAc (200 mL), and washed succes-
sively with 1N NaHSO4 (4£50 mL), water (3£50 mL), 1N
NaHCO3 (4£50 mL), and again water (3£50 mL).The
organic layer was then dried over anhydrous Na2SO4 and
evaporated. The residue was chromatographed on silica gel
eluting with CHCl3–CH3OH, 9:1 to afford 33 (0.19 g,
50%): [a]2D3¼212.4 (c¼0.02, EtOH); 1H NMR (DMSO-d6):
d 8.13 (br s, 1H), 7.75 (br s, 1H), 7.34 (m, 10H), 7.21 (t,
J¼6.0 Hz, 2H), 6.78 (d, J¼6.4 Hz, 1H), 6.73 (d, J¼7.8 Hz,
1H), 5.01 (s, 4H), 4.20 (m, 1H), 3.91 (m, 1H), 3.79 (m, 1H),
3.59 (s, 3H), 3.15 (m, 4H), 3.04 (m, 2H), 2.0–1.0 (m, 18H),
1.36 (s, 18H); MALDI-TOF MS, m/z 908 ([MþNaþ], calcd
for C45H68N6O12 908).
4.1.12. Dipeptide 26, two diastereomers. To a stirred and
ice cooled solution of amine 24 (0.10 g, 0.38 mmol) in DMF
(15 mL) was added carboxylic acid 5 (0.14 g, 0.38 mmol),
HOBT (0.10 mg, 0.76 mmol), and DCC (0.16 g,
0.76 mmol). The reaction mixture was stirred for 30 min
at 08C and for 36 h at rt. The mixture was filtered,
concentrated, diluted with EtOAc (200 mL) and washed
successively with 1N NaHSO4 (4£50 mL), water
(3£50 mL), 1N NaHCO3 (4£50 mL), and again water
(3£50 mL). The organic layer was then dried over
anhydrous Na2SO4 and evaporated. The residue was
chromatographed on silica gel eluting with THF–hexanes,
1:1 to afford dipeptide 27 (0.15 g, 64%) as an oil: 1H NMR
(DMSO-d6, only one diastereomer is shown): d 8.23 (d,
J¼7.80 Hz, 1H), 7.84 (d, J¼8.0 Hz, 1H), 6.73 (m, 2H), 4.29
(m, 1H), 4.18 (dt, J¼8.5, 5.5 Hz, 1H), 3.61 (s, 3H), 2.87 (m,
A solution of 33 (0.15 g, 0.17 mmol) in CH3OH (10 mL)
was treated with 10% Pd/C (15 mg) and stirred under a
hydrogen atmosphere for 4 h. The mixture was filtered

5846
H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
through Celite and concentrated. The residue was dried
1
under high vacuum to give tripeptide 32 (0.1 g, 95%): H
NMR (DMSO-d6): d 8.16 (br s, 1H), 7.80 (br s, 1H), 6.81 (d,
J¼8.3 Hz, 1H), 6.75 (d, J¼7.1 Hz , 1H), 4.21 (m, 1H), 3.91
(m, 1H), 3.80 (m, 1H), 3.60 (s, 1H), 3.06 (m, 4H), 2.96 (m,
2H), 1.35 (s, 18H), 2.0–1.0 (m, 18H).
(m, 6H), 1.28 (m, 9H), 1.11 (s, 9H), 1.07 (s, 18H), 1.01
(s, 9H); MALDI-TOF MS, m/z 1229.9 ([MþNaþ], calcd
for C70H98N2O15 1230.5). Anal. calcd for C70H98N2O15:
C, 69.63; H, 8.18; N, 2.32. Found: C, 69.60, H, 8.28, N,
2.48.
4.1.16. Calixarene lysine 1e. A solution of benzyl ester 1a
(0.2 g, 0.15 mmol) in CH3OH (10 L) was mixed with 10%
Pd/C (20 mg) and stirred under a hydrogen atmosphere for
4 h. The reaction mixture was filtered through Celite and
concentrated in vacuo to give pure 1e (0.18 g, 98%) as a
colorless solid: 1H NMR (CDCl3) d 8.62 (t, J¼6.4 Hz, 1H),
6.99 (d, J¼5.0 Hz, 1H), 6.82 (s, 2H), 6.78 (s, 4H), 6.72 (s,
2H), 5.0–4.8 (m, 2H), 4.80–4.62 (m, 8H), 4.62–4.40 (m,
2H), 4.18 (m, 6H), 3.50 (dt, J¼6.9, 6.4 Hz, 1H), 3.34 (m,
2H), 3.24, 3.22 (2£d, J¼13.2 Hz, 4H), 2.26 (m, 2H), 2.0–
1.8, 1.8–1.5, 1.5–1.15 (3£m, 25H), 1.11 (s, 9H), 1.07 (s,
18H), 1.04 (s, 9H), 0.85 (t, J¼6.9 Hz, 3H).
4.1.15. Preparation of calix[4]arene amino acids 1a–d. A
typical protocol. A solution of calix[4]arene triester
monoacid chloride 25 (0.49 g, 0.5 mmol) in EtOAc
(10 mL) was added under vigorous stirring to a solution of
TFA salt 7 (0.2 g, 0.42 mmol) and K2CO3 (0.58 g,
4.2 mmol) in EtOAc (10 mL) and H2O (20 mL). The
reaction mixture was stirred at rt for 3 h. The organic
layer was separated and evaporated under reduced pressure.
The residue was chromatographed on silica gel with THF–
hexanes, 3:2 as eluents to afford 1a (0.36 g, 65%) as a
colorless solid: mp 69–708C; IR (KBr): n 3377, 2954, 2868,
1
1754, 1651, 1547, 1480, 1193, 1070; H NMR (CDCl3):
d 8.42 (t, J¼6.4 Hz, 1H), 7.34 (m, 5H), 6.82 (s, 2H), 6.79
(s, 4H), 6.73 (s, 2H), 6.39 (d, J¼7.3 Hz, 1H), 5.15 (s, 2H),
4.89 (d, J¼13 Hz, 2H), 4.74 (d, J¼13 Hz, 2H), 4.69 (s, 2H),
4.65–4.6 (m, 4H), 4.55 (m, 1H), 4.52 (m, 2H), 4.16 (m, 6H),
3.39 (m, 2H), 3.25, 3.21 (2£d, J¼13 Hz, 4H), 2.20
(t, J¼7.3 Hz, 2H), 1.85–1.50 (m, 6H), 1.25 (m, 19H),
1.11 (s, 9H), 1.07 (s, 18H), 1.03 (s, 9H), 0.85 (t, J¼7.5 Hz,
3H); MALDI-TOF MS, m/z 1331.9 ([MþNaþ], calcd for
C79H108N2O14 1332.7). Anal. calcd for C79H108N2O14: C,
72.45; H, 8.31; N, 2.14. Found: C, 72.15, H, 8.67, N, 2.40.
Calixarene 1b: The product was purified by column eluting
with THF–hexanes, 1:1. Yield 67%, mp 71–728C; 1H NMR
(CDCl3): d 8.41 (t, J¼6.0 Hz, 1H), 7.33 (m, 5H), 6.82 (s,
2H), 6.78 (s, 4H), 6.73 (s, 2H), 5.52 (d, J¼8.3 Hz, 1H), 5.11,
5.06 (2£d, J¼12.4 Hz, 2H), 4.90, 4.87 (2£d, J¼16.0 Hz,
2H), 4.75, 4.73 (2£d, J¼12.8 Hz, 2H), 4.70 (s, 2H), 4.63 (m,
4H), 4.50 (br s, 2H), 4.35 (m, 1H), 4.18 (m, 6H), 3.73 (s,
3H), 3.35 (m, 2H), 3.22 (m, 4H), 2.0–1.5 (m, 6H), 1.24 (m,
9H), 1.11 (s, 9H), 1.07 (s, 18H), 1.04 (s, 9H); MALDI-TOF
MS, m/z 1265.2 ([MþNaþ], calcd for C73H96N2O15
1264.6). Anal. calcd for C73H96N2O15: C, 70.62; H, 7.79;
N, 2.26. Found: C, 70.81, H, 7.69, N, 2.53. Calixarene 1c
was chromatographed with THF–hexanes, 3:2. Yield 64%);
mp 70–728C; 1H NMR (CDCl3): d 8.43 (t, J¼6.0 Hz, 1H),
7.34 (m, 7H), 7.01 (d, J¼8.7 Hz, 2H), 6.82 (s, 2H), 6.77 (s,
4H), 6.73 (s, 2H), 5.62 (d, J¼8.3 Hz, 1H), 5.13, 5.09 (2£d,
J¼12.3 Hz, 2H), 4.91, 4.88 (2£d, J¼12.4 Hz, 2H), 4.75,
4.73 (2£d, J¼12.9 Hz, 2H), 4.70 (s, 2H), 4.62 (m, 4H), 4.55
(m, 1H), 4.52 (d, J¼2.8 Hz, 2H), 4.17 (m, 6H), 3.44 (m,
2H), 3.24, 3.22 (2£d, J¼13.2 Hz, 4H), 2.1–1.5 (m, 6H),
1.30 (s, 9H), 1.24 (m, 9H), 1.11 (s, 9H), 1.07 (s, 18H), 1.04
(s, 9H); MALDI-TOF MS, m/z 1382.9 ([MþNaþ], calcd for
C82H106N2O15 1382.7). Anal. calcd for C82H106N2O15: C,
72.43; H, 7.86; N, 2.06. Found: C, 72.12, H, 7.74, N, 1.94.
Calixarene 1d was chromatographed on silica gel eluting
with THF–hexanes, 1:1. Yield 62%); mp 74–758C;
[a]2D3¼22.8 (c¼0.02, EtOH); IR (KBr): n 3382, 2961,
2869, 1755, 1720, 1673, 1480, 1363, 1194, 1128, 1069; 1H
NMR (CDCl3): d 8.40 (t, J¼6.0 Hz, 1H), 6.82 (s, 2H), 6.78
(s, 4H), 6.73 (s, 2H), 5.13 (d, J¼8.3 Hz, 1H), 4.89
(d, J¼16.5 Hz, 2H), 4.74 (d, J¼12.8 Hz, 2H), 4.70 (s,
2H), 4.63 (d, J¼12.8 Hz, 2H), 4.62 (d, J¼16.5 Hz, 2H), 4.53
(m, 2H), 4.28 (m, 1H), 4.19 (m, 6H),3.72 (s, 3H), 3.37 (m,
2H), 3.24, 3.21 (2£d, J¼12.8 Hz, 4H), 1.42 (s, 9H), 2.0–1.3
4.1.17. Calixarene lysine 1f. A solution of 1b (0.2 g,
0.16 mmol) in CH3OH (10 mL) was treated with 10% Pd/C
(20 mg) and stirred under a hydrogen atmosphere for 4 h.
The mixture was filtered through Celite and concentrated in
vacuo. The residue was dried under high vacuum to give
1
0.17 g (96%) of amine 1f. H NMR (CDCl3): d 8.47 (t,
J¼6.0 Hz, 1H), 6.77 (m, 8H), 4.95–4.80 (m, 2H), 4.80–
4.65 (m, 4H), 4.65–4.52 (m, 4H), 4.52–4.45 (m, 2H), 4.15
(m, 6H), 3.72 (s, 3H), 3.60 (m, 1H), 3.35 (m, 2H), 3.22 (t,
J¼12.8 Hz, 4H), 2.0–1.5 (m, 6H), 1.24 (m, 9H), 1.12 (s,
9H), 1.07 (s, 9H), 1.04 (s, 9H), 1.02 (s, 9H).
4.1.18. Calixarene dipeptide 2a. Procedure 1. To a stirred
and ice cooled solution of calix amino acid derivative 1e
(0.15 g, 0.14 mmol) in DMF (15 mL) was added succes-
sively 1f (0.17 g, 0.14 mmol), HOBT (38 mg, 0.28 mmol),
and DCC (58 mg, 0.28 mmol). The mixture was stirred for
30 min at 08C and then for 36 h at rt. The mixture was
filtered, concentrated, diluted with EtOAc (200 mL), and
washed successively with 1N NaHSO4 (4£50 mL), water
(3£50 mL), 1N NaHCO3 (4£50 mL), and again water
(3£50 mL). The organic layer was then dried over
anhydrous Na2SO4 and evaporated. The residue was
chromatographed on silica gel eluting with THF–hexanes,
7:3 to afford calix–peptide 2a (two diastereomers, 0.15 g,
46%) as a colorless solid: mp 97–988C; IR (KBr) nmax 3378,
2959, 2868, 1752, 1671, 1540, 1474, 1369, 1297, 1191,
1124, 1066; 1H NMR (DMSO-d6, only one diastereomer is
shown): d 8.30 (d, J¼7.8 Hz, 1H), 8.10 (m, 2H), 7.89 (d,
J¼8.0 Hz, 1H), 6.84 (m, 16H), 4.85–4.75 (m, 4H), 4.70–
4.50 (m, 16H), 4.36 (m, 4H), 4.22 (m, 1H), 4.13 (m, 12H),
3.62 (s, 3H), 3.40–3.15 (m, 12H), 2.11 (m, 2H), 2.0–1.1 (m,
40H), 1.05 (m, 72H), 0.85 (t, J¼7.7 Hz, 3H); MALDI-TOF
MS, m/z 2332.6 ([MþNaþ], calcd for C137H190N4O26
2332.0).
Procedure 2. A solution of acid chloride 25 (0.37 g,
0.38 mmol) in EtOAc (10 mL) was added to a vigorously
stirred solution of dipeptide 26 (0.1 g, 0.16 mmol) and
K2CO3 (0.53 g, 3.8 mmol) in EtOAc (10 mL) and H2O
(20 mL). The reaction mixture was stirred at rt for 6 h, and
the organic layer was separated and evaporated. The residue
was chromatographed on silica gel eluting with THF–
hexanes, 7:3 to afford 2a (0.18 g, 49%).

H. Xu et al. / Tetrahedron 59 (2003) 5837–5848
5847
4.1.19. Calixarene dipeptide 2b. A solution of acid
chloride 25 (0.61 g, 0.62 mmol) in EtOAc (10 mL) was
added under vigorously stirring to a solution of dipeptide 29
(0.10 g, 0.26 mmol) and K2CO3 (0.86 g, 6.2 mmol) in
EtOAc (10 mL) and H2O (20 mL). The reaction mixture
was stirred for 6 h at rt. The organic layer was separated and
evaporated. The residue was chromatographed on silica gel
eluting with THF–hexanes, 7:3 to afford calix dipeptide 2b
(0.27 g, 45%) as a colorless solid: mp 109–1108C;
[a]2D3¼25.1 (c¼0.02, EtOH); IR (KBr) nmax 3333, 2959,
2864, 2358, 1752, 1673, 1540, 1475, 1368, 1300, 1190,
4.05 (m, 12H), 3.93 (m, 1H), 3.83 (m, 1H), 3.65 (s, 3H),
3.3–3.1 (m, 12H), 3.0 (m, 2H), 1.8–1.4 (m, 18H), 1.36 (s,
18H), 1.7–1.25 (m, 18H), 1.06 (s, 18H), 1.03 (s, 27H), 1.02
(s, 27H); MALDI-TOF MS, m/z 2532 ([MþNaþ], calcd for
C145H204N6O30 2534).
4.2. Liquid–liquid extraction experiments
Compounds 1a–d, 2a,b, and 3a,b were dissolved in CH2Cl2
(,5£1023 M, 10 mL) and vigorously stirred overnight with
saturated aqueous solution of NaClO4 (10 mL) at rt. Organic
layers were then separated, evaporated under reduced
pressure, dried in vacuo and analyzed by high-resolution
1H NMR spectroscopy in CDCl3.
1
1125, 1056; H NMR (DMSO-d6): d 8.16 (d, J¼7.3 Hz,
1H), 8.11 (m, 2H), 6.96 (d, J¼6.9 Hz, 1H), 6.85 (m, 16H),
4.78 (d, J¼15.8 Hz, 4H), 4.7–4.5 (m, 16H), 4.37 (m, 4H),
4.25 (m, 1H), 4.2–4.0 (m, 12H), 3.96 (m, 1H), 3.60 (s, 3H),
3.22 (m, 12H), 1.38 (s, 9H), 2.0–1.1 (m, 30H), 1.1–0.9 (m,
72H); MALDI-TOF MS, m/z 2306.8 ([MþNaþ], calcd for
C134H185N4O27 2305.9). Anal. calcd for C135H184N4O27: C,
70.50; H, 8.12; N, 2.45. Found: C, 70.63, H, 8.19, N, 2.61.
4.3. MALDI mass spectrometry measurements
Samples were initially reconstituted in acetone. A 2 mL
aliquot of a given sample was deposited on the MALDI
sample probe and the solvent was allowed to evaporate. A
2 mL aliquot of the MALDI matrix 2,5-dihydroxybenzoic
acid (10 mg/mL) in MeOH (for compounds 1a–d and 2a,b)
or a 2 mL aliquot of a-cyano-4-hydroxycinnamic acid
(10 mg/mL) in MeOH–formic acid, 1:1 (for compounds
3a,b) was deposited on the sample coated probe. Internal
calibration was achieved by adding the peptide bradykinin
([MþH]þ 1060.1 Da).
4.1.20. Dendrimer 3a. Procedure 1. To a stirred and ice
cooled solution of salt 20 (80 mg, 0.21 mmol) DMF (8 mL)
was added Et3N (0.06 mL, 0.42 mmol) and then after
15 min, calyx amino acid 1e (0.51 g, 0.42 mmol), HOBT
(0.11 g, 0.84 mmol), and DCC (0.17 g, 0.84 mmol). The
mixture was allowed to stir for 30 min at 08C and for 48 h at
rt, then filtered, concentrated in vacuo, diluted with EtOAc
(200 mL), and washed successively with 1N NaHSO4
(4£50 mL), water (3£50 mL), 1N NaHCO3 (4£50 mL),
and again water (3£50 mL). The organic layer was then
dried over anhydrous Na2SO4 and evaporated. The residue
was chromatographed on silica gel eluting with CHCl3–
CH3OH, 9:1 to afford 3a (0.22 g, 41%). 1H NMR (DMSO-
d6, only one diastereomer is shown): d¼8.23 (br s, 1H), 8.10
(br s, 2H), 7.87 (m, 3H), 6.81 (m, 16H), 4.80 (m, 4H), 4.70–
4.50 (m, 16H), 4.34 (s, 4H), 4.30 (m, 1H), 4.2–4.0 (m, 13H),
3.95 (m, 1H), 3.57 (s, 3H), 3.25–3.05 (m, 12H), 3.02 (m,
2H), 2.08 (m, 4H), 2.0–1.1 (m, 66H), 1.1–0.9 (m, 72H),
0.83 (m, 6H); MALDI-TOF MS, m/z 2588.7 ([MþNaþ],
calcd for C151H216N6O28 2586.4).
Acknowledgements
Financial support is acknowledged from the University of
Texas at Arlington (D. M. R), the Alfred P. Sloan
Foundation (D. M. R.), and NSF CHE-9876249 (G. R. K.).
We are also grateful to Stephen P. Stampp for valuable
assistance with molecular modeling. Professor Wim Ver-
boom and Dr Grigory V. Zyryanov are acknowledged for
advice on the calix[4]arene preparation.
Procedure 2. A solution of acid chloride 25 (0.30 g,
0.30 mmol) in EtOAc (20 mL) was added to a vigorously
stirring solution of tripeptide 30 (0.1 g, 0.15 mmol) and
K2CO3 (0.41 g, 3.0 mmol) in EtOAc (10 mL) and H2O
(20 mL). The reaction mixture was stirred for 6 h at rt, the
organic layer was separated and evaporated. The residue
was chromatographed on silica gel eluting with CHCl3–
CH3OH, 9:1 to afford 3a (0.18 g, 47%).
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