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Highly Sensitive Recognition of Substrates of Adrenergic Receptors at the AirWater Interface.

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Artificial Adrenergic Receptors
Highly Sensitive Recognition of Substrates of
Adrenergic Receptors at the Air/Water
Oliver Molt and Thomas Schrader*
One of the major classes of receptors in the human body are
the G-protein-coupled receptors (GPCRs).[1] Their signals
arise from a special binding site incorporated in the phospholipid bilayer: seven membrane-spanning helices create a
binding pocket for the incoming hormones that has a largely
unpolar environment.[2] One of the major subclasses is the
family of adrenergic receptors. Amino alcohols of varying
length and polarity are distinguished and lead to different
transduction processes. In light of the enormous biological
and pharmaceutical importance of this recognition site[3] the
question arises whether it is possible to imitate this microenvironment in a lipid layer and use it for highly efficient and
selective recognition.[4]
Recent developments in the field of artificial catecholamine receptor molecules include bipyridinium/gold nanoparticle arrays,[5] phenyl boronates,[6] and pyrazole-containing
cryptands.[7] Bioorganic alternatives have also been presented
featuring RNA aptamers[8] and copper-containing redox
enzymes.[9] In keeping with the natural image,[10] we have
recently designed a new type of synthetic adrenaline receptor
with the following features: 1) a xylylene bisphosphonate unit
serves as an established amino alcohol binder,[11] with enough
flexibility for an induced fit; 2) the core unit is linked to rigid
building blocks, assuring a high level of preorganization.[12]
Thus, stiff aromatic tolan side walls[13] create a deep hydrophobic cleft, which is flanked by inwards-oriented amide NH
groups that can form hydrogen bonds with the hydroxy groups
of catechol derivatives. Monte Carlo simulations of this host
molecule in its complex with noradrenaline indicate that there
is enough room at the bottom part of the receptor for
accomodating the amino alcohol, and all minimum-energy
conformations find the guest inside the cavity. Subsequent
molecular dynamics calculations suggest a high mobility of
host and guest within the complex—an entropically most
favorable situation.[14]
A modular retrosynthesis leads to three building blocks: a
bisphosphonate-modified p-xylylene dibromide, a tolandicarboxylic acid, and m-xylylene diamine. These can be prepared
by a very direct synthetic route. Arbuzov reaction and
[*] Prof. T. Schrader, Dr. O. Molt
Fachbereich Chemie
Universitt Marburg
Hans-Meerwein-Strasse, 35032 Marburg (Germany)
Fax: (+ 49) 6421-28-25544
[**] We gratefully acknowledge support by the Deutsche Forschungsgemeinschaft (DFG SCHR 604/1-3).
Supporting information for this article is available on the WWW
under or from the author.
Angew. Chem. Int. Ed. 2003, 42, 5509 –5513
DOI: 10.1002/anie.200352186
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
subsequent NBS bromination give bisphosphonate 6, while
two successive Sonogashira couplings furnish the mono-tertbutyl ester component 3 of the tolan spacer.[15] After the ester
function is converted into first the carboxylic acid and then
the acid chloride, this unit is connected to m-xylylene diamine
to give compound 4 after cleavage of the ester. Subsequent
macrocyclization with bisphosphonate 6 via the cesium
carboxylate relied on the intramolecular directing effect of
cesium ions.[16] Final mild monodealkylation of the phosphonates with LiBr completes the high-yielding synthesis of the
new receptor molecule.[17] Host 1 is well soluble in polar
solvents such as DMSO, methanol, and water.
The 1H NMR spectrum of the 1 displays remarkably broad
signals for the P-CH2 and the O-CH2 methylene groups.
Variable-temperature experiments in methanol reveal a clean
and large splitting for the diastereotopic protons at 45 8C.
This is explained by a ten-membered chelate ring in which the
lithium cation bridges the phosphonate and the ester carbonyl
oxygen atoms of the neighboring carboxylate group
(Scheme 1). The phosphonate arm is thus directed inwards
ready to receive the amino alcohol guest.[18] No sharp NMR
signals were observed for 1 in D2O. In a dilution titration
experiment large shifts were found for the unpolar aromatic
parts of 1, and a self-association constant in the order of 102–
103 m 1 was determined.[19] Considering the structural similarity of 1 to amphiphilic phospholipids, an incorporation of the
new host into lipid monolayers seemed feasible in imitation of
the natural adrenergic receptor.
For a quantitative evaluation of the new receptor's
binding profile we performed NMR titrations in methanol
and calculated the association constants by standard non-
linear regression methods.[20] The assumed 1:1 stoichiometry
was confirmed by a representative Job plot.[21] In most cases
all signals of the guest shifted and produced similar binding
constants. However, the results of a systematic variation of
the amino alcohol were disappointing: from small ethanolamine to propranolol the Ka values ranged only between 700
and 1600 m 1 (Table 1). Only amino acids with an additional a
substituent were bound less effectively by one order of
magnitude. Considering the relatively small NMR shifts for
the aromatic protons of the host and guest, we must conclude
that in polar solution the macrocyclic host 1 probably does not
Table 1: A0 and DA0 values for stearic acid monolayers with embedded
receptor 1 over different subphases.
[F2 molecule 1]
l-tyrosine methyl ester
[F2 molecule 1]
[a] S = stearic acid. [b] A0 = apparent total area of molecule of stearic acid
in the liquid-condensed phase. [c] Net influence of the guest: DA0 =
A0 (monolayer with 1 over subphase with guest) A0 monolayer with 1
over water).
Scheme 1. Synthesis of macrocycle 1. a) SOCl2, MeOH, 0 8C (92 %); b) 2-methyl-but-3-yn-2-ol, [Pd(PPh3)2Cl2], CuI, PPh3, NEt3, pyridine, D (98 %);
c) NaOH, 1-butanol, D (99 %); d) SOCl2, cat. DMF, CHCl3, 0 8C (99 %); e) phenol, NEt3, THF (45 %); f) 4-iodobenzoic acid tert-butyl ester,
[PdCl2(PPh3)2], CuI, PPh3, NEt3, 80 8C (93 %); g) 1. Na2CO3, H2O2, dioxane, H2O, D; 2. KHSO4 (86 %); h) (COCl)2, cat. DMF, CH2Cl2 (99 %);
i) m-xylylenediamine, NEt3, CH2Cl2 (89 %); j) TFA, CH2Cl2 (99 %); k) P(OMe)3, D (99 %); l) NBS, AIBN, CCl4, D (64 %); m) 6, Cs2CO3, DMF, high
dilution (28 %); n) LiBr, CH3CN, D (81 %). AIBN = azoisobutyronitrile, DMF = dimethylformamide, NBS = N-bromosuccinimide, TFA = trifluoroacetic acid.
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2003, 42, 5509 –5513
nature of the guest dissolved in the aqueous subphase. Since
the host molecules present their amino alcohol binding site
towards the water phase, the guest molecules are bound by
their bisphosphonate moieties and drawn into the monolayer.
Now a new binding mode becomes operative which distinguishes between minute structural variations with high
sensitivity. Figure 3 shows the p–A diagrams for noradrenaline, adrenaline, and isoproterenol, which differ
only in the size of their N-alkyl substituent
(Scheme 2). Comparison of these curves to those
of b-blockers propranolol and atenolol reveals the
exquisite selectivity of the new binding mode:
although the only structural difference between
both antihypertensives consists of the replacement
of an a-naphthyl moiety by a phenylacetamide, the
receptor–ligand interaction produces in one case
the strongest of all positive substrate shifts, in the
other case even a drastically negative p–A shift.
The affinity of the embedded receptor molecules within the monolayer for their guests is so
high that noradrenaline can be detected even at
micromolar concentrations.[26] A rough estimation
of the binding constants at the air/water interface
leads to Ka values that must clearly surpass 105 m 1;
Figure 1. a) ESI mass spectrum (negative-ion detection) of a solution of receptor 1
thus 1 might be used in new adrenaline-sensing
and noradrenaline hydrochloride (NA) in methanol. Host: m/z = 631 [4 H+] , 949
devices. All the other catecholamines did not show
[1 Li +H ] , 955 [1 Li ] ; 1:1 complex: m/z = 1118 [1 2 Li +NA-Cl ] , 1130
any appreciable shifts in the p–A diagram at
[1 2 H +NA-Cl ] . b) Schematic drawing of the planned binding mode, featuring
concentrations between 10 5 and 10 6 m. Their
inclusion inside the rigid cavity of 1, accompanied by an induced fit of the
association constants must therefore be at least
one order of magnitude lower than that of noradrenaline. A complete list of changes in the p–A
The situation changed profoundly when we embedded the
new amphiphilic receptor molecule in a monomolecular layer
of stearic acid at the air/water interface.[22] A marked increase
in the pressure–area (p–A) diagram of the Langmuir film
balance always indicated the incorporation of additional
material into the monolayer.[23] This was accompanied by the
development of new stable, large patches in the Brewster
angle microscope (BAM) images (Figure 2), which we
attribute to the formation of domains within the monolayer
consisting mainly of self-aggregated receptor molecules.[24]
Recent bioanalytical results show that even the natural
adrenergic receptors form dimeric structures.[25]
Subsequent subinjection of noradrenaline into the subphase led to a drastic increase in the p–A isotherm, but the
patches in the BAM picture remained (Figure 2). These
effects were reproducible and strongly dependent on the
embrace adrenaline-type guests but merely lets them dock
onto its hydrophobic region with their catechol moiety. Other
spectroscopic methods indicate the same: a clean ESI mass
spectrum was obtained from methanol showing peaks for host
and 1:1 complex exclusively (Figure 1), but no change of the
receptor's extinction maxima could be detected in a UV/Vis
experiment in the same solvent.
Figure 2. BAM images (4.8 I 6.4 mm2) of a stearic acid monolayer
(left) and a monolayer with embedded receptor 1 (dark gray areas,
right) in the condensed liquid phase.
Angew. Chem. Int. Ed. 2003, 42, 5509 –5513
Figure 3. Pressure–area (p–A) isotherms of stearic acid (S) and
receptor 1 in a monolayer over water: with a) isoproterenol (IPT),
adrenaline (Adr), and noradrenaline (NA); and b) propranolol (Propr)
and atenolol (Aten.).
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Scheme 2. Guest molecules with structures similar to that of adrenaline. NA = noradrenaline, Adr = adrenaline, IPT = isoproterenol, Dop =
dopamine, Tyr = tyrosine methyl ester, Aten = atenolol, Propr = propranolol. (The hydrochloride salts employed in the experiments are
diagrams induced by a range of guest molecules is summarized in Table 1.
With Langmuir–Blodgett (LB) techniques it was possible
to immobilize 160 (2 G 80) layers of the receptor-containing
stearic acid monolayer on quartz slides. These immobilized
multilayers were observed directly with UV/Vis spectroscopy,
which revealed a drastic blue shift of the receptor's extinction
maximum.[27] The multilayers were subsequently washed off
the glass carrier with methanol, which leads to a complete
reversal of the blue shift and restores the UV/Vis spectrum of
the free receptor molecule (Figure 4). Since solvatochromic
effects with the lipid layer would result in a red shift,[28] this
effect must be attributed to the self-aggregation of the
receptor molecule within the monolayer, as already indicated
in the BAM picture. If noradrenaline was present in the
subphase, the LB deposition on quartz led to a markedly
decreased blue shift, which was shown to be dependent on the
analyte concentration and could again be completely reverted
on washing with methanol. This effect sheds light on the
binding mechanism inside the monolayer. We propose that
the red shift originates from the insertion of the electron-rich
catechol ring into the ensemble of stacked electron-poor
aromatic rings of the surrounding receptor molecules.[29] We
could detect this bathochromic shift down to a noradrenaline
concentration in the subphase of 5 G 10 5 m, indicating again a
similar lowest limit for the dissociation constant of the
complex as judged from the Langmuir p–A diagrams
(Figure 3).
From all the experimental data we infer the following
tentative mechanism of noradrenaline binding by the monolayer. When guest molecules are subinjected into the aqueous
phase, they are bound by solvated receptor molecules close to
the monolayer. The host molecules' negative charges become
neutralized in part and their lipophilicity increases. This in
turn leads to reincorporation of the whole complex into the
monolayer (Figure 5). However, the catechol moiety is not
inserted into a single host's cavity, but rather into the
ensemble of host molecules in receptor domains within the
stearic acid monolayer. The new microenvironment leads to
high selectivity for minute structural changes in the analytes.
Figure 5. Schematic representation of the proposed binding mode
within the monolayer.
We conclude that the incorporation of an artificial
receptor molecule for substrates of the adrenergic receptor
into a monolayer leads to a dramatic increase in both the
recognition efficiency and selectivity for a whole range of
substrates. Thus, noradrenaline can be detected quantitatively
at micromolar concentrations. The basis for this enormous
progress is the altered microenvironment created in the
monolayer.[30] Not only is the dielectric constant lowered
dramatically compared to that of the free aqueous phase,[27]
but the receptor molecules also self-aggregate into assemblies
that are highly sensitive towards the structural features of
amino alcohol guests. In the future we intend to incorporate
the new macrocyclic receptor molecules into vesicles made
from lipids and diacetylenes for colorimetric detection of
adrenaline derivatives.[31]
Received: June 23, 2003 [Z52186]
Figure 4. UV/Vis spectra of LB layers of stearic acid (alone, with
embedded 1, and with attached guest) on quartz slides and in
methanol solution.
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Keywords: adrenergic receptors · b-blockers · interfaces ·
Langmuir–Blodgett films · molecular recognition · monolayers
Angew. Chem. Int. Ed. 2003, 42, 5509 –5513
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Other sensors for adrenaline derivatives reach comparable
detection limits of ~ 10 6 m.[5–9]
K. Ariga, T. Kunitake, Acc. Chem. Res. 1998, 31, 371.
Solvatochromic red shifts with unpolar solvents originate from
the stabilization of their more polarizable LUMO: C. Reichardt,
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A CT band does not develop, as judged from the colorless
monolayer. However, a sandwich arrangement would be conceivable for the guest's catechol ring within the host aggregates.
A simple lipid-modified bisphosphonate embedded in the stearic
acid monolayer did not produce any changes in the p–A diagram
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2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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adrenergic, substrate, receptors, recognition, sensitive, airwater, interface, highly
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