Mass spectroscopic investigation of bis-1,3-urea calix[4]arenes and their ability to complex N-protected α-amino acids

Article abstract of DOI:10.1007/s10847-009-9687-6

We report the ability of urea's appended onto the upper-rim of conformationally locked 'cone' calix[4]arenes to show a preference for binding specific N-protected α-amino acids. Superior complexation (as judged by mass spectroscopy) between N-protected α-

Full text of DOI:10.1007/s10847-009-9687-6

J Incl Phenom Macrocycl Chem (2010) 66:195–208
DOI 10.1007/s10847-009-9687-6
ORIGINAL ARTICLE
Mass spectroscopic investigation of bis-1,3-urea calix[4]arenes
and their ability to complex N-protected a-amino acids
•
Sean P. Bew Adam W. J. Barter
•
Sunil V. Sharma
Received: 3 August 2009 / Accepted: 2 October 2009 / Published online: 20 October 2009
Ó Springer Science+Business Media B.V. 2009
Abstract We report the ability of urea’s appended onto the
upper-rim of conformationally locked ‘cone’ calix[4]arenes
to show a preference for binding specific N-protected
a-amino acids. Superior complexation (as judged by mass
spectroscopy) between N-protected a-amino results and
bis-1,3-N-benzylureas calix[4]arenes was observed when
methylene bridges were present between the calix[4]arene
‘host’ and the urea motif. Interestingly we also demonstrate
that subjecting mixtures of structurally diverse N-Fmoc-
a-amino acids to a single bis-1,3-N-benzylurea derived
calix[4]arene allows, in some cases, the calix[4]arene ‘host’
to selectively ‘pick out’ and complex a specific N-Fmoc
amino acid from the mixture.
Developing the idea further Lehn et al. reported in 1982
a polyammonium species that was capable of binding
carboxylic acid anions. Synthesizing and employing spe-
cies based on 3 (Scheme 2) Lehn et al. was able to show
that these macrocycles encapsulated and bound (with
varying strengths) dicarboxylic acids with different lengths
of ‘spacer’ such as adipate, glutarate, oxalate, succinate,
malonate and pimelate as well as N-acetyl-(L)-aspartate,
N-acetyl-(L)-glutamate and N-acetyl-(L)-glutamyl anions [3].
Further work by Lehn et al. resulted in the synthesis of
anion receptors based on guanidinium species 5 and quat-
ernized nitrogen atoms that proved to be highly effective
receptors for carboxylate or phosphate anions (Scheme 2) [4].
Beer et al. has pioneered the synthesis and applications
of metallocene based calix[4]arenes as anion receptors [5].
Thus calix[4]arenes adorned with two, i.e. 7 or one, i.e. 8
cobaltocenium amide derivative (Fig. 1) are able to bind in
the case of 7 dicarboxylate anions e.g. adipate and for 8 an
acetate anion highly effectively via electrostatic and
hydrogen bonding interactions.
Keywords bis-1,3-Urea calix[4]arenes Á Amino acids Á
Hydrogen bonding Á Mass spectroscopy Á Anion binding Á
Carboxylic acids
Introduction to anion receptors
Atwood et al. synthesized a series of electron-poor
calix[4]arene cavities that have the capacity to bind anionic
guests [6, 7]. Thus attaching cationic metal centers (9,
Fig. 1) to the outer face of a calix[4]arene allowed size-
and shape-selective complexation of anions such as tetra-
fluoroborate, triflate or hexafluorophosphate. It was noted
that the tetrafluoroborate anion was held ‘deep’ within the
cavity of 9.
In 1968 Simmons and Park [1, 2] reported the first anion
receptor, i.e. 1 for the chloride anion. It was configured on
a polyammonium cryptand that employed electrostatic and
hydrogen bonding interactions to ‘capture’ the chloride
anion within its cavity (Scheme 1).
Electronic supplementary material The online version of this
article (doi:10.1007/s10847-009-9687-6) contains supplementary
material, which is available to authorized users.
In addition to electrochemical methods for determining
if a binding event has taken place (cf. [8]), fluorescence
emission spectroscopy has also been utilized using ruthe-
nium complexes bound to the lower rim of a calix[4]qui-
none, i.e. 10 (Fig. 2). Anion binding receptors based on 10
display selectivity for acetate over chloride and dihydrogen
phosphate, with a 500% increase in emission for acetate
S. P. Bew (&) Á A. W. J. Barter Á S. V. Sharma
School of Chemistry, University of East Anglia,
Norwich NR4 7TJ, UK
e-mail: s.bew@uea.ac.uk
URL: https://intranet.uea.ac.uk:443/cap/people/faculty/spb
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J Incl Phenom Macrocycl Chem (2010) 66:195–208
and a 60% increase in emission for chloride binding. The
increased emission was attributed to the anionic complex
overcoming intramolecular quenching of the luminescence.
In 1993 Reinhoudt and co-workers reported the syn-
thesis and application of an upper-rim derived tetra-
sulfonamide calix[4]arene, i.e. 11 (Fig. 2) that displayed
selective binding for a tetrahedral sulfate anion, i.e. 102
over and above a dihydrogen phosphate, chloride, nitrate or
perchlorate anion [9]. Interestingly the authors described
Cl-
N
(CH2)8
N
N
D
DCl
(CH2)8
(CH2)8
(CH2)8
(CH2)8 (CH2)8
50% DTFA
D
N
1
2
Scheme 1 Encapsulation of DCl within 2
H
N
H
N
H
N
H
N
NH2
(CH2)7
(CH2)7
O
O
O
O
H
NH2
NH2
H
H
NH
NH
H
H
N
N
H
H
N
N
H
H
H
N
H
N
H
N
H
N
H
(CH2)7
(CH2)7
NH2
4
5
3
Scheme 2 Encapsulation of dicarboxylate anion in 4
6+
Co+
Co+
Co+
O
O
NH
Re
HN
Re
Re
Re
PF6
(PF6)2
OCH3
OH
OH
OH
OH
OTosyl
OCH3
OH
OTosyl
OH
O
O
7
8
9
Fig. 1 Metallocene derived calix[4]arenes for anion recognition
O
O
O
O
O
NH
O
HN
Cl
Cl
Cl
Cl
NH
H
NH
H
H
N
O
N
O
O
O
N
O2S
SO2
O
HN
O2S
NH
SO2
NH
HN
O
O
NH
O
NH
HN
OPr
OPr
O
OPr
O
OPr
O
O
O
O
10
N
N
O
O
O
11
O
12
M = Ru(Bpy)22+(PF6 )2
-
M
Fig. 2 Anion binding calix[4]arenes
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J Incl Phenom Macrocycl Chem (2010) 66:195–208
197
how using FAB mass spectroscopy analysis on the solid
complexes they were able to identify the signals corre-
sponding to the tetra-sulfonamide calix[4]arene complex
with the sulfate anion bound.
reported here (vida infra) Ungaro et al. was the first to
report the use of a qualitative, competitive pre-screening
technique for anion binding that employed mass spectros-
copy [12–14].
Loeb and Cameron have reported the synthesis of a 1,3-bis
amide derived calix[4]arene 12 (equipped with electron-
withdrawing groups that enhanced the acidity of the amide
proton, Fig. 2) and its ability to be an excellent 1:1 binder
ofcarboxylate anionssuchasacetate andbenzoate(Kass 5160)
[10].
The synthesis of upper-rim or lower-rim appended urea
or thiourea derived calix[4]arenes and their application in
anion sensing has been extensive. Early work focused on
appending substituents to the lower rim, with calix[4]arene
16 showing selectivity for chloride over bromide and
iodide (Fig. 4) [15]. The use of FAB mass spectroscopy
1
A number of calix[4]arene derived peptide based anion
sensing systems have been developed. Cyclic peptide
derived calix[4]arene 13 (Fig. 3) displayed a strong bind-
and H-NMR was critical to determining the effectiveness
of the binding event and the stoichiometry of the host–
guest complex, with bis-1,3-N-substituted urea calix[4]-
arenes exhibiting higher binding and enhanced selectivity
over tetra-N-substituted urea calix[4]arenes. The cyclic
amide derived calix[4]arene 17 (Fig. 4) proved to be an
effective receptor for binding carboxylates, with 17 showing
increased selectivity for acetate over phosphate, sulfate or
chloride anions [16]. The synthesis of hybrid calix[4]arene
18 equipped with two thiourea or urea motifs was reported
by Yang et al. they demonstrated that these systems
had significant potential and application in binding dicar-
boxylate anions such as adipate (Kass 4,640 M-1) and
ing potential for 4-nitrophenyl phosphate (Kass 3,900 M-1
)
[11]. Whilst the flexible peptide derived calix[4]arene 14
displayed weak binding for anions, subsequent synthesis of
the constrained cyclic peptide derived calix[4]arene 15
afforded much higher binding affinities for anions such as
acetate or benzoate. Thus the Kass values for the binding of
benzoate using 14 and 15 were 680 M-1 and 44,000 M-1
,
respectively. With the selectivity displayed by 15 attributed
to additional p–p stacking interactions with the aromatic
capping moiety. Of particular interest to the research
O
O
O
O
HN
HN
NH
NH
O
O
O
O
HN
HN
NH
NH
OH
OH
O
O
NH
O
HN
O
S S
13
OPr
OPr
OPr
OPr
OPr
OPr
OPr
OPr
H CO C
CO CH
2 3
3
2
14
15
Fig. 3 Peptide based calix[4]arenes for anion recognition
Fig. 4 Halide and carboxylate
binding calix[4]arenes
OH
OH
O
O
O
O
O
O
OPr
OPr
O
O
O
O
O
HN
NH
HN
HN
HN
HN
O
HN
HN
NH
NH
NH
NH
HN
HN
O
O
S
S
18
16
17
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J Incl Phenom Macrocycl Chem (2010) 66:195–208
succinate which were bound significantly stronger than
chloride or dihydrogen phosphate anions [17].
the iso phthalate dicarboxylic acid (Kass 1100 M-1) over
benzoic acid (Kass 43 M-1) [19].
Binding of anions within upper-rim derived calix[4]-
arenes especially those equipped with four urea species is
complicated by the fact that these ‘calixureas’ are prone to
dimerisation and capsule formation. Monourea calix[4]ar-
ene 19 (Fig. 5) has been synthesized and shown to be an
efficient carboxylate binding species for benzoate, butyrate
and para-nitrobenzoate, a fact that contrasts sharply with
its inability to bind spherical anions such as chloride,
bromide or iodide [18]. Similar to 19 bis-1,3-N-phenylurea
calix[4]arene 20 did not bind spherical anions such as
chloride, bromide or iodide it did however bind a variety of
mono and dicarboxylate species, i.e. butyrate (Kass
133 M-1), para-phthalate (Kass 200 M-1), meta-phthalate
(Kass 230 M-1), benzoate (Kass 290 M-1), ortho-phthalate
(Kass 300 M-1) and acetate (Kass 2200 M-1). Extending
the cavity on 21 via the inclusion of two aryl groups
(Fig. 5) and rigidifying it via cyclisation of the two 1,3-
diurea motifs was recently undertaken by Nakamura et al.
The resulting system had the potential to selectively bind
A consequence of the highly sensitive nature of fluo-
rescence spectroscopy it is widely employed for studying
host–guest binding. Employing titration studies it is often
possible to gain a valuable insight into the location and
nature of the anion-binding event. By way of example a
recent report by Shuang et al. demonstrated that ‘deep’
calix[4]arene 23 (Fig. 6) was able to selectively bind a
carboxylic acid species such as acetate or benzoate in
preference to fluoride or dihydrogen phosphate via hydro-
gen bonding to the upper-rim carbamate species [20]. The
anion binding and sensing properties of a series of calix[4]-
arene derivatives were investigated using fluorescence
titration (emission quenching) where a N-phenylcarbamate
species was switched to
a N-phenylamide species
(24, Fig. 6). The bis-1,3-N-phenylamide (appended onto
the upper-rim) derived calix[4]arene still displayed supe-
rior binding selectivities for acetate over fluoride but
overall of 23 and 24 bis-1,3-N-phenylcarbamate calix[4]-
arene 23 was still the most efficient carboxylic acid binder.
Fig. 5 Carboxylate binding
calix[4]arenes
NH
NH
NH
O
O
HN
HN
HN
NH
O
NH
NH
O
O
NH
O
O
O
O
O
O
O
O
O
O
O
O
20
19
21
Fig. 6 Carboxylate, chloride
and sulfate binding
calix[4]arenes
NH
O
HN
O
HN
NH
O
O
O
O
O
O
O
O
O
O
O
NH
O
HN
NH
O
O
O
O
O
O
O
O
25
24
23
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J Incl Phenom Macrocycl Chem (2010) 66:195–208
199
Beer et al. have demonstrated that lower-rim appended
ferrocene calix[4]arene 25 (Fig. 6) is capable of ‘sensing’
chloride, sulfate and dihydrogen phosphate with the latter
anion producing the largest shift of the ferrocene/ferrocenium
redox couple (160 mV) when titrated into a solution of 25.
dimer in a non-racemic fashion with rotation solely in one
direction. The result being that an asymmetric microenvi-
ronment within the calix[4]arene was generated. Rebek
was able to show that the chiral heterodimers have a
defined urea hydrogen bonding direction that upon addition
of an enantiomeric guest produces two diasteromeric
capsules.
Results and discussion
Shuker et al. synthesized an upper-rim appended tetra-
(S)-alanine calix[4]arene and was able to demonstrate that
these species were able to perform a molecular recognition
event between two calix[4]arenes species thus generating a
dimer via hydrogen bonding. The ability to generate dimers
in methanol, a solvent normally expected to disrupt hydro-
gen bonding, is remarkable. Interestingly the authors uti-
lized mass spectroscopy to verify dimer formation and
were able to demonstrate that they had been formed with an
association constant of Kass 29,000 M-1 in 24:1 CD3OD/
D2O [29]. The authors report this as the first example of a
calix[4]arene dimer that is thermodynamically stable in a
polar solvent.
Even though the potential of amino acid molecular recog-
nition via the calixarene scaffold has long been recognized,
the synthesis of functionalized calix[4]arenes [21–23] or
resorcinarenes [24] and their subsequent investigation/
application as amino acid molecular recognizing agents is
not particularly common. Indeed a search of the literature1
[25] afforded relatively few examples of amino acid
recognition via binding to upper-rim functional groups
appended onto calix[4]arenes [26]. Thus by way of
example Nishimura et al. demonstrated that a conforma-
tionally constrained (on the upper-rim) calix[4]arene spe-
cies that had been appended on the lower-rim with four
pendant chiral esters derived from (S)-1-phenylethanol was
able to selectively extract (from a biphasic system) certain
a-amino acid esters and N-protected-a-amino esters from
an aqueous solution into dichloromethane [27]. Nishimura
demonstrated that glycine, L-alanine and b-alanine were
not extracted, but that on the other hand L-phenylalanine
and L-tryptophan were effectively and efficiently extracted
with a preference for L-phenylalanine over L-tryptophan.
This order of extraction efficiency can be explained by
invoking a hydrophobicity effect, that follows the order:
Phe [ Trp [ Ala [ Gly. Furthermore the constrained
calix[4]arene developed by Nishimura was able to mediate
a chiral molecular recognition event if presented with a
racemic mixture of Z-protected a-amino acids. The con-
formationally constrained calix[4]arene interacting with
and ‘selecting’ L-amino acids over D-amino acids [11.0 to
73.1% excess (L - D/D 9 100)]. The use, in any form, of
mass spectroscopy to determine the ability of the a-amino
acids to bind the calix[4]arenes was not discussed.
In 2003, Parisi et al. reported the synthesis of an upper-
rim ureidocalix[5]arene that behaved as a remarkably
efficient abiotic receptor of x-amino acids and biogenic
amines. Part of the x-amino acids and biogenic amines
were bound within the p-basic cavity and part bound via
secondary hydrogen bonding to urea motifs [30]. The
authors determined the binding affinities of the amine/
1
calix[5]arene complexes using H-NMR which indicated a
1 : 1 stoichiometry between the host and guest. The authors
make no mention of the use of mass spectroscopy to
investigate the host–guest relationship.
Using a convergent synthesis protocol Ito et al. syn-
thesized a series of homocalix[4]arene species, each one
incorporating a different a-amino acid, i.e. L-valine, L-
phenylalanine, L-tyrosine, L-typtophan and L-alanine. Using
these ‘chiral cavity’ calix[4]arenes a quaternary ammo-
nium species was able to bind inside the p-basic cavity and
undergo an interaction.
In an ongoing project focused on generating a new
protocol towards the synthesis of upper-rim derived urea
calix[4]arenes [26] we wanted to extend our interests and
investigate the possibility that mass spectroscopy (LCMS)
be used to detect, in a qualitative sense, host–guest com-
plexes between N-protected a-amino acids and bis-1,3-N-
substituted urea calix[4]arenes that have been fixed in the
cone conformation. Thus in a recent publication we
reported an uncomplicated protocol for synthesizing hybrid
calix[4]arene urea and carbamate motifs via an experi-
mentally straightforward ionic hydrogenation procedure
(Scheme 3). The method is fully amenable to generating
structurally diverse bis-1,3-N-substituted urea calix[4]are-
nes, some of which were able to undergo further elabora-
tion via transition-metal-mediated transformations.
In 1999 Rebek et al. reported [28] on the ability of
upper-rim derived tetra-urea calix[4]arenes to dimerise and
within the resulting interior cavity accommodate a guest.
Thus the synthesis of a series of homochiral a-amino acid
derived calix[4]arene ureas appended with L-norleucine, L-
leucine, L-isoleucine, L-valine, L-O-tBu-serine, L-phenylal-
anine, L-phenylglycine and L-alanine groups allowed Rebek
et al. to show that the resulting calix[4]arene heterodimers
generated via a ‘seam of hydrogen bonds’ generated the
1
We searched SciFinder Scholar and Scopus for the key words
‘calixarene’ and ‘amino acid recognition’ returned 56 and 55 ‘hits’
respectively.
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Scheme 3 Ionic hydrogenation
for synthesis of bis-1,3-urea
calixarenes
H
H
H
O
O
N
N
N
NH
2
O
H
TFA, Et3SiH
toluene, Ar
2
2
OPr
OPr
OPr
Br
OPr
=
I
39% yield
N
63% yield
47% yield
57% yield
Cl
N
N
H
76% yield
57% yield
69% yield
α-amino
acid 1
α-amino
+
+
O
O
acid 2
NHR
1
NHR
1
R HN
O
R HN
O
1
1
NH
NH
HN
α-amino
acid 4
HN
+
+
α-amino
acid 3
α-amino
acid 4
RO
OR
OR
OR
RO
OR
OR
OR
Molecular weight X
Host-guest molecular weight is Z from X + Y
determined via mass spectroscopy
Molecular weight Y
Scheme 4 Selective molecular recognition of amino acids using urea appended calix[4]arenes
For the purposes of the research programme outlined
here, our hypothesis was straightforward and could be
divided into two parts: (1) is it possible that N-protected
a-amino acids have the ability to ‘bind’ to bespoke upper-
rim appended bis-1,3-N-substituted urea calix[4]arene
scaffolds such that subsequent ESI mass spectroscopy
allows the ‘supramolecular complex’ to be detected and
(2) if this proves possible, would it be possible if a
mixture of N-protected a-amino acids were presented to a
single bis-1,3-N-substituted urea calix[4]arene hybrid for
that calix[4]arene to specifically ‘pick out’ one N-protected
a-amino acid and generate a supramolecular host–guest
complex that can be identified from its unique host–guest
mass to be comprised of the N-protected a-amino acid
and bis-1,3-N-substituted urea calix[4]arene. The idea of
comparison to NMR and fluorescence techniques. Thus
although mass spectroscopy has proved itself invaluable to
chemists across all aspects of chemistry, its application as a
sensitive tool for investigating calixarene based molecular
recognition events have been relatively few.
With our hypothesis in mind (vida supra) we initiated
our investigation using two simple bis-1,3-N-phenylurea
calix[4]arenes, i.e. 26 and 27 (Fig. 7). Both afforded strong
‘control’ mass peaks when subjected to ESI (negative
mode) mass spectroscopy, with 26 generating a Mwt of
860.4 and 27 a Mwt of 1140.3 (See ESM, Fig. 3).
Subsequent addition of a molar equivalent of N-Boc
glycine (28, Mwt 175) to the sample of 26 and re-running
the ESI afforded a new mass peak at 1035, i.e. correct mass
for 26 and N-Boc-glycine, although the intensity of this
peak was relatively low,2 furthermore it is interesting to
note that mass spectral evidence for non-complexed 26 was
selective complexation (using calix[4]arene with
a
molecular weight X) of an a-amino acid with molecular
mass Y with concomitant formation of a supramolecular
species with molecular weight Z is shown schematically
in Scheme 4.
2
From a qualitative point of view we have attempted to ‘standard-
ized’ the mass spectroscopic data of the ‘host–guest’ calix[4]arenes
complexes by taking the observed mass intensity of the bis-1,3-N-urea
calix[4]arene amino acid complex and dividing it by the observed
mass intensity of the bis-1,3-N-urea calix[4]arene amino acid, this is a
crude but relatively effect way of allowing us to compare all the data
sets generated.
The application of mass spectroscopy techniques for
investigating anion-binding events within macrocyclic
systems, and for the purposes of this research we have
focused on calixarenes, has received scant attention in
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201
I
I
O
O
O
H
N
O
OH
OH
HN
30
O
O
O
NH
NH
HN
HN
O
HN
HN
O
28
NH
NH
O
O
OH
N
OH
O
O
HN
O
O
PrO
OPr
OPr
PrO
OPr
OPr
OPr
OPr
26, molecular weight = 860.4
31
27, molecular weight = 1140.3
29
Fig. 7 Ureas and amino acids used in molecular recognition study
Cl
Cl
O
O
NH
NH
NH
HN
HN
O
HN
HN
O
NH
O
NH
NH
HN
HN
O
PrO
PrO
OPr
OPr
OPr
OPr
OPr
OPr
33, molecular weight = 916.5
34, molecular weight = 984.4
PrO
OPr
OPr
OPr
32, molecular weight = 1088.5
Fig. 8 Ureas used in molecular recognition study
also present in the spectrum. Performing the same proce-
dure3 with 26 but switching to N-Cbz-proline (29, Mwt 249) a
more intense mass peak at 1110.1 was observed (see ESM,
Fig. 4), which again correlates to the formation of a host–
guest adduct between 26 and 29. Encouraged by these results
we pursued the application of aromatic a-amino acid 30, i.e.
N-acetyl-(S)-phenylalanine, anticipating that the aromatic
group may aid formation of the calix[4]arene host–guest
complex via possible inclusion of the phenyl ring of the
a-amino acid within the calix[4]arene cavity. Gratifyingly
when an equimolar quantity of 30 (Mwt 207) was added to 26
a strong mass peak correlating to the formation of the
anticipated host–guest complex was observed, i.e. 1067 (see
ESM, Fig. 5), finally repeating this process with N-Fmoc-
(S)-valine 31 afforded a strong mass peak that correlated well
with the expected mass for the formation of a host–guest
complex, i.e. 1199 (see ESM, Fig. 6). It seemed from these
preliminary results that ‘strong’ complex formation between
26 and a few select N-protected a-amino acids was observed,
this appeared to be particularly so when aromatic groups
were present on the a-amino acid component.
bis-1,3-N-para-iodophenylurea calix[4]arene to undergo
host–guest complexation. We repeated the process outlined
for 26 with para-iodophenylurea calix[4]arene 27. Inter-
estingly from the mass spectroscopy results it seemed all
four N-protected a-amino acids 28–31 (See Fig. 7), were
only very poorly accommodated or complexed by the para-
iodo substituted calix[4]arene. Seemingly the presence of
the two sterically encumbered large iodine atoms inhibited
host–guest formation (Chart 1).
Synthesising [26] ‘deep’ calix[4]arene 32 (Fig. 8) we
investigated formation of the corresponding host–guest
complexes with 28–31 (See ESM Figs. 11–14), similar to 27
only weak interactions were detected with the uncomplexed
host (32) also present. The application of hosts with ‘deep’
and extended sides did not seem to offer any increased
potential for generating urea appended calix[4]arenes with
superior molecular recognition properties.
Changing tact slightly we considered the possibility that
introducing a methylene spacer between the urea motif and
the aryl group may afford an N-benzylurea calix[4]arene
species that has greater capacity due to increased flexibility
for binding an N-protected a-amino acid. Synthesizing 33
we investigated its potential to bind, individually, the
a-amino acids 28–31 using mass spectroscopy as our
investigative tool. Interestingly incorporating a bis-1,3-
N-benzyl urea system onto the calix[4]arene seemingly
Utilising 27 we set about investigating the effect that
halogens, namely iodine, would have on the ability of the
3
Undertaking the mass spectra experiments was straightforward and
quick, see ESM for details.
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restored (compared to 27 and 32) and increased (judged
against 26) the capacity of the calix[4]arene to bind the
N-protected a-amino acids 28–31 (See ESM, Figs. 15–18).
Again similar to 26, 33 had a preference for aromatic
containing a-amino acids, i.e. 30 and N-Fmoc derived
a-amino acids, i.e. 31 (See Chart 1).
Investigating what effect incorporating a meta-chloro-
benzyl group would have on the ability of the resulting
calix[4]arene to bind 28–31 we synthesized 34. From
Chart 1 it is evident that both 33 and 34 were significantly
better at binding 28–31 than the N-phenylureas 26 and 27.
Furthermore and similar to 33 the chlorine containing 34
displayed broadly comparable and impressive selectivity
for cyclic peptide 29, aromatic a-amino acid 30 and
N-Fmoc derived a-amino acid 31, this was countered by a
decreased for 33 and 34 to bind N-Boc glycine 28.
steric bulk around the carbon adjacent to the two proximal
ureas on 37 serves only to inhibit binding of the N-pro-
tected a-amino acids (See ESM, Figs 31–34).
Intrigued by the fact that N-Fmoc-(S)-valine 31 dis-
played what appeared to be enhanced binding towards
calix[4]arenes 33–35 (Chart 1) we opted to investigate the
possibility that it was the Fmoc group that was critical to the
calix[4]arene binding event. With this in mind we tested
(individually) a further three N-Fmoc protected a-amino
acids, i.e. glycine, (S)-proline and (S)-phenylalanine (38–40
respectively) for their ability to bind to bis-1,3-N-substi-
tuted urea calix[4]arenes 26, 32–37. Employing our stan-
dard mass spectroscopy analysis protocol (see Experimental
section) N-phenyl derivatives 26 and 32 behaved in a
manner similar to previously tested N-protected a-amino
acids 28–31, the results suggested that there was not much
evidence of a strong host–guest relationship (see Chart 2).
On the contrary, screening 38–40 for their ability to undergo
host–guest relationships with 33–35 afforded interesting
and generally positive results (See ESM, Figs. 35–43), with
the aromatic N-Fmoc-(S)-phenylalanine 40 displaying a
particularly strong propensity to bind to para-pyridine
derived urea 35. Interestingly as seen in the previous study,
substituting N-Cbz-(S)-proline (29) for N-Fmoc-(S)-proline
(39) demonstrated a significant improvement in the ability
of the N-Fmoc derivative to bind to the same two
calix[4]arenes 34 and 35 (Chart 2). N-Fmoc-glycine 38 also
appeared to have a greater affinity for 34 and 35 than the
corresponding N-Boc glycine derivative 28 (compare
Charts 1, 2), overall these results tend to confirm that
incorporating an N-Fmoc group did have a positive influ-
ence on amino acid binding and complex formation.
Delighted that N-benzyl derivatives 33 and 34 did indeed
seem to be able to bind to specific N-protected a-amino
acids we synthesized three more calix[4]arene variants, i.e.
35–37 (Fig. 9). Using these substrates we repeated the mass
spectroscopy process outlined above using the same
a-amino acids 28–31; an interesting and striking result was
obtained (Chart 1). Of the four a-amino acids employed
N-acetyl-(S)-phenylalanine and Fmoc-(S)-valine were of
particular interest. Focusing on 35 and these two amino
acids particularly strong mass peaks for complexation were
observed with commensurate very small mass peaks for the
host, i.e. 35 (See ESM, Figs. 23–26). This intriguing result
suggests that with suitable N-substituted urea motifs
appended onto conformationally fixed calix[4]arene scaf-
folds their ability to form host–guest complexes that are
stable and detectable via mass spectroscopy is viable.
Anticipating that benzimidazole 36 may form extended
hydrogen bonding arrays with 28–31 we were disappointed
that only weak binding was observed in all of the four
amino acids investigated (Chart 1). Changing tact slightly
we synthesizing 37 a sterically encumbered secondary
benzylamine derived bis-1,3-urea calix[4]arene. Investi-
gating its potential to bind 28–31 we were disappointed
that only weak binding of 28–31 (See ESM, Figs 27–30)
was observed (Chart 1), it would seem that increasing the
Similar to 28–31 N-benzimidazole 36 displayed no real
binding affinity for 38–40 (See ESM, Figs. 44–46), on the
contrary and for reasons not yet fully understood N-Fmoc a-
amino acids 38–40 do have a reasonable affinity for 37, with
the cyclic a-amino acid 39 standing out (Chart 2) amongst
the three tested cf. N-Fmoc-(S)-valine 31 (Chart 1).
The interesting results obtained with the N-Fmoc
protected derivatives 38–40 prompted us to investigate
the effect of changing the a-amino acid component and
N
N
N
N
N
N
O
O
H
NH
NH
NH
NH
H
O
HN
HN
O
HN
HN
O
NH
NH
HN
HN
O
PrO
PrO
OPr
OPr
OPr
OPr
OPr
OPr
PrO
OPr
OPr
OPr
35, molecular weight = 918.5
37, molecular weight = 944.5
36, molecular weight = 996.5
Fig. 9 Ureas used in molecular recognition study
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J Incl Phenom Macrocycl Chem (2010) 66:195–208
203
Chart 1 Mass spectroscopic
interaction of bis-1,3-urea
calix[4]arenes 26, 27, 32–37
with amino acids 28–31
screened on an individual basis
Chart 2 Mass spectroscopic
interaction of bis-1,3-urea
calix[4]arenes 26, 32–37 with
amino acids 38–40 screened on
an individual basis
determining their ability to generate host–guest complexes
that are detectable via ESI mass spectroscopy with 26,
32–37.
With this in mind we screened structurally diverse
N-Fmoc protected a-amino acids 41–47 (Chart 3). It is clear
from Chart 3 that (S)-arginine derivative 42 (containing the
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204
J Incl Phenom Macrocycl Chem (2010) 66:195–208
Chart 3 Mass spectroscopic
interaction of bis-1,3-urea
calix[4]arenes 26, 32–37 with
amino acids 41–47 screened on
an individual basis
pentamethylchroman sulfonyl N-protecting group or PMC),
(S)-histidine 43 and 44 did not generally form complexes
that were detectable or stable enough to be identified
via ESI mass spectroscopy. N-Fmoc-(S)-tryptophan 41
displayed improved complexation properties with 34–35
and similar to previous N-Fmoc a-amino acids tested, i.e.
28–31 and 38–40 was poor at binding to N-phenyl derived
calix[4]arenes 26 and 32. Switching to (S)-tyrosine deriv-
atives 46 and 47 there was limited evidence, the only
exception being between pyridine derivative 34 and 46, of
any binding to the calix[4]arenes. This indicates that the
addition of the sterically bulky tert-butyl group to the
phenolic hydroxyl in 46 essentially shuts down the formation
of complexes with all but one of the hybrid calix[4]arenes.
A similar situation was observed with N-Fmoc protected
3,5-diiodo-(S)-tyrosine derivative 47, with essentially no
complexation observed with 33–37 (orange bars). The sit-
uation changes somewhat however when N-Fmoc-(S)-
phenylglycine (45) is employed where what appears to be
quite strong and selective complexation (purple bars,
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J Incl Phenom Macrocycl Chem (2010) 66:195–208
205
Chart 3) between 33–35 and N-Fmoc-(S)-phenylglycine is
observed and specifically between phenylglycine derivative
45 and bis-1,3-N-benzylurea calix[4]arene 33 (See ESM,
Figs. 47–53).
mixture of a-amino acids 28–31 is surprising. A rational
explanation that accounts for these observations is difficult
to establish. Needless to say these preliminary results
spurred us onto try alternative a-amino acid mixtures.
Earlier work indicated that individual N-Fmoc derived
a-amino acids such as 28–31 displayed higher affinity for
bis-1,3-N-substituted urea calix[4]arenes such as 33–35
and 37 than N-Boc or N-Cbz cf. Charts 1 and 2. With this
and the preliminary, interesting results from the mixture of
a-amino acids in mind we elected to investigate the pos-
sibility that individual calix[4]arenes 26, 32–37 may when
presented with a mixture comprising four N-Fmoc a-amino
acids (31, 38–40) selectively bind to one of them.
Employing a procedure essentially identical to that outlined
for a-amino acids 28–31 an interesting observation resul-
ted, see Chart 5. Similar to individually tested amino acids
(Chart 2) calix[4]arenes 34 and 35 had a propensity to
complex N-Fmoc-(S)-phenylalanine 40 but at the expense
of forming host–guest complexes with 38, 39 and 31 (See
ESM, Figs 68–69).
From results attained thus far it seemed, that at least from
a qualitative point of view, specific bis-1,3-N-substituted
urea’s did have the ability to selectively bind N-protected
a-amino acids. Introducing a further level of complexity we
considered the possibility of presenting to single bis-1,3-
N-substituted urea calix[4]arenes mixtures of N-protected
a-amino acids, thus allowing us to probe the possibility that
given competition for binding to the bis-1,3-substituted
urea calixarenes the host molecule does indeed demon-
strate a selective binding process.
In the first instance we screened the original N-protected
a-amino acids, i.e. 28–31 against calix[4]arenes 26, 32–37.
If Charts 1 and 4 are compared it is clear that employing a
mixture of N-Fmoc-a-amino acids results in a considerable
change in Fmoc amino acid selectivity, furthermore it is
evident that a number of striking similarities are observed
but also that anomalies become apparent. From Chart 4 the
most obvious feature is the seemingly very high propensity
for 26, 33, 34 and 37 to complex with N-Fmoc-(S)-valine
31 even if they have competition from amino acids 28–30.
Interestingly complexation of amino acids 28–30 with 26,
27, 32–37 seems to have undergone a global reduction if
compared with the individual screenings outlined in
Chart 1. The first and last calix[4]arenes, i.e. 26 and 37 are
interesting. Comparing the individual screenings of 28–31
outlined in Chart 1 with 26 there is a considerable differ-
ence between the mixtures 28–31 (Chart 4) and the
observation in Chart 1 for the individual amino acids,
furthermore the substantial increase in 37 binding to 31
(Chart 4) (See ESM, Figs. 61–67) when presented with the
Comparing the individual a-amino acid study reported
(Chart 3) with a mixture study utilizing 41–42 and 43–44
we screened these a-amino acid mixtures against bis-1,3-
N-substituted urea calix[4]arenes 26, 32–37. The only
significant difference between the ‘single’ versus ‘mix-
ture’ study was the slight drop in the affinity of 41 for
34 and 35 and the increase in complexation between 41
and 37 (compare Chart 3 with Chart 6) (See ESM,
Fig. 70). Focusing on the individual a-amino acids 43
and 44 in Chart 3 with the results from the mixtures
outlined in Chart 7 for the same two N-Fmoc-(S)-histi-
dines there is relatively little difference between
using single amino acids versus a mixture (See ESM,
Figs. 71–72).
Chart 4 Investigating via mass
spectroscopy the interaction of
bis-1,3-urea calix[4]arenes 26,
27, 32–37 with mixtures of
amino acids, i.e. 28–31
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J Incl Phenom Macrocycl Chem (2010) 66:195–208
Chart 8 Investigating the mass spectroscopic interaction between
bis-1,3-urea calix[4]arenes 26, 32–37 and mixtures of amino acids,
i.e. 45 and 47
Chart 5 Investigating the mass spectroscopic interaction between
bis-1,3-urea calix[4]arenes 26, 32–37 and mixtures of amino acids,
i.e. 31 and 38–40
occurred, with the intensity of the majority of the peaks in
Chart 8 greatly reduced to the many of those observed in
Chart 3.
Interestingly, calix[4]arene 26 appears to have generated
approximately equal quantities of host–guest complexes
between all three a-amino-acids (including 47) that com-
prise the mixture, comparing this to the single amino acids
screen in which 47 did not appear to have any great pro-
pensity to form a host–guest complex with 26. Similarly
calix[4]arene 34 when presented with a mixture of 45–47
(See ESM, Figs. 73–74) seems to have an increased affinity
for 47 (Chart 8).
In summary, preliminary evidence for the ability of
M-protected a-amino acids to form host–guest relationships
with bis-1,3-N-substituted urea calix[4]arenes fixed in the
cone conformation has been accumulated. Although spe-
cific, single a-amino acids seem to associate quite well, the
situation with hybrid calix[4]arenes and their ability to
generate bespoke host–guest complexes with specific
a-amino acids ‘extracted’ from within a mixture is consid-
erably more complicated and requires further investigation.
Chart 6 Investigating the mass spectroscopic interaction between
bis-1,3-urea calix[4]arenes 26, 32–37 and mixtures of amino acids,
i.e. 41 and 42
Experimental section
General protocols
All reactions requiring anhydrous conditions were con-
ducted in flame-dried glass apparatus under an atmosphere
of nitrogen or argon. Water refers to distilled water. All
commercially available chemicals, reagents and salts were
used as supplied. Melting points were recorded using open
capillary tubes on a Gallenkamp melting point apparatus
and are uncorrected. Infrared spectra were recorded on a
Perkin-Elmer Paragon 1000 FT infrared spectrometer.
1H- and 13C-NMR spectra were recorded in Fourier
Chart 7 Investigating the mass spectroscopic interaction between
bis-1,3-urea calix[4]arenes 26, 32–37 and mixtures of amino acids,
i.e. 43 and 44
Switching our attention to a mixture comprising Fmoc
derivatives 45–47 a number of differences are apparent,
again a global reduction in binding affinity seems to have
123

J Incl Phenom Macrocycl Chem (2010) 66:195–208
207
ESI–MS experiments
transform mode at the field strength specified either on a
Varian Gemini 300, 400 or 500 spectrometer. Unless
otherwise stated, deuterated chloroform was used as sol-
vent. The 1H-spectra were recorded in ppm and referenced
to the residual CHCl3 signal located at d 7.24 ppm.
13C-NMR spectra were recorded in ppm and referenced to
the residual CHCl3 signal found at d 77.00. Multiplicities
in the NMR spectra are described as: s singlet, d doublet,
t triplet, q quartet, m multiplet, br broad; coupling con-
stants are reported in Hz. Mass spectra were run on either a
Shimadzu LCMS-2010A, Micromass Quattro II, Finnigan
MAT95 or MAT 900 spectrometer. Ion mass/charge (m/z)
ratios are reported as values in atomic mass units. Thin
layer chromatography was performed on Merck aluminium
plates coated with 0.2 mm silica gel-60 F254. Flash column
chromatography was performed on silica gel (Kieselgel
60). All the commercially available amino acids were used
without any further purification. All the sample solutions
were prepared using HPLC grade dichloromethane or
methanol as required. The synthesis of 27, 32–36 has been
reported [26].
The ESI–MS experiments were performed on a Shimadzu
LCMS-2010A instrument (mass range 10–2000 amu)
equipped with Q-array ion optical-quadrupole detector
system, SIL 20A autosampler and LCMS 2010EV detector.
The calix[4]arene stock solutions were prepared by dis-
solving appropriate amounts of sample in a mixture of
dichloromethane-methanol (5% v/v) to give a final con-
centration of 1 mM. Individual amino acid solutions
(1 mM) were prepared in methanol. Calix[4]arene-amino
acid mixture samples were prepared by mixing 20 lL each
of the required stock solution and diluting up to 1.2 mL
with methanol. The samples were introduced as 10 lL
injection using the autosampler and at a flow rate of
0.5 mL/min eluting with methanol. For guest competition
experiments 20 lL solutions of calix[4]arene with 1
equivalent of each guest amino acid were prepared and
analysed as detailed above. A ratio of the intensity of mass
ion peaks of the host–guest complex to the molecular ion
peak of host was used as a qualitative indicator of the
relative binding ability.
Typical synthesis of bis-1,3-N-substituted ureas
is outlined below for 37 [26]
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