Binding of a hemoregulatory tetrapeptide by a bis-guanidinium crown ether

Article abstract of DOI:10.1016/j.tet.2010.06.024

A synthetic receptor for the molecular recognition of a tetrapeptide in aqueous buffer was obtained by combining a luminescent crown ether with two pyrrole-guanidinium moieties. The compound interacts with ammonium carboxylates of complementary geometry a

Full text of DOI:10.1016/j.tet.2010.06.024

Tetrahedron 66 (2010) 6019e6025
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Binding of a hemoregulatory tetrapeptide by a bis-guanidinium crown ether
*
€
€
Andreas Spath, Burkhard Konig
€
Institut für Organische Chemie, Universitat Regensburg, D-93040 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A synthetic receptor for the molecular recognition of a tetrapeptide in aqueous buffer was obtained by
combining a luminescent crown ether with two pyrrole-guanidinium moieties. The compound interacts
with ammonium carboxylates of complementary geometry and binds the hemoregulatory peptide
Ac-Ser-Asp-Lys-Pro with K¼7ꢀ103 Mꢁ1 at physiological pH. Shorter fragments and other tetrapeptides
show no or significant reduced affinity. The binding of the target peptide to the functionalized crown
ether is signalled by an increase of its emission intensity.
Received 11 March 2010
Received in revised form 7 June 2010
Accepted 8 June 2010
Available online 12 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
The crown ether moiety8,9 of compound 2 is a luminescent
ammonium ion indicator based on photoinduced electron transfer
The naturally occurring hemoregulatory peptide Ac-Ser-Asp-
Lys-Pro (1) shows anti-inflammatory and antifibrotic properties,
thus decreasing hypertension-induced target organ damage.1 It is
known to inhibit the proliferation of hematopoietic stem cells2 and
has recently been reported to inhibit cardiac fibrosis.3 It has been
shown to reduce the damage to specific compartments in the bone
marrow resulting from treatment with chemotherapeutic agents,
ionizing radiations, hyperthermy, or phototherapy.4 The peptide
acts as a specific inhibitor for the N-terminal site of the angiotensin
I-converting enzymes (ACE). The effect is characterized by the
conversion activity of ACE.5 Fluorescent substrates like coumaryl-
acetyl-Ser-Asp-Lys-Pro were used for the determination of the ki-
netic constants and to develop a sensitive assay for ACE activity in
human plasma.6
(PET),10 while the guanidinium ion group strongly interacts with
anions through charge pairing and hydrogen bonding.11 It is used
as recognition motif for carboxylates12 and its interactions are
well-studied.13
A pyrrole substituent improves the binding
strength of the recognition motif further.14 Several interesting
examples employing this carboxylate binding site have been
published by the Schmuck group, recently.15 Supramolecular
studies in water16 are challenging, because solvent hydrogen
bonds disturb the binding process and diminish or even prevent
molecular recognition. Multiple intermolecular interactions can
stabilize host/guest aggregates. The spacer units between the
binding sites of compound 2 were therefore designed to comple-
ment the hydrogen-bonding pattern of the target peptide zwit-
terion (Fig. 1).
We report a synthetic receptor capable of indicating the pres-
ence of the peptide by change of its luminescence. The emission
increase of the host depends on the concentration of the target
peptide, which allows the determination of its concentration
without further functionalisation with a fluorophore or radiolabel.
The structure of the peptide Ac-Ser-Asp-Lys-Pro (1) contains
two carboxylate groups and the amino acid lysine bearing an am-
monium ion side chain. Recently, we presented different receptors
for zwitterionic amino acids, recognizing combinations of ammo-
nium and carboxylate ions, based on luminescent crown ethers
with guanidinium ion binding sites.7 Here we extended this mod-
ular approach to a tridentate receptor 2 with complementary
binding sites for the target peptide 1.
2. Results and discussion
2.1. Synthesis
The synthesis of the substructures of receptor 2, the crown ether
amino acid ester 69 and pyrrole substituted isothiourea 47 were
published previously. 3,5-Bis-aminomethyl-benzoic acid methyl
ester (3)17 was reacted with methyl-isothiourea 4 yielding the
protected pyrrole-guanidine 5a (Scheme 1). The ester group was
hydrolyzed giving 5b, which was coupled to the crown ether amino
acid ester 6a using standard peptide coupling methods to yield
compound 7. Compound 6b was prepared for comparison by acyl-
ation under standard conditions (Scheme 2). The guanidilation
reaction can also be achieved by mercury(II) catalysis in DMF in
6 h.18 The EDC promoted reaction is slower, but the use of toxic
metal ions is avoided, the overall yield is higher and less side
products are formed.19 A small amount of methanol has to be added
* Corresponding author. Fax: þ49 943 941 1717; e-mail address: Burkhard.Koe-
€
nig@chemie.uni-regensburg.de (B. Konig).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.06.024

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A. Spath, B. Konig / Tetrahedron 66 (2010) 6019e6025
H
N
H
N
NH+2
NH+3
O
HN
O
OH
N
H
O
O
H
N
H
N
O
H
NH
N
N
O
N
N
H
N
H
_
NH+2
O
_
COO
O
O
O
COO
O
O
O
O
O
O
O
2
Figure 1. The hemoregulatory peptide 1 and synthetic receptor 2 with complementary ammonium and carboxylate ion binding sites.
O
O
S
O
O
O
O
O
N
N
H
N
O
NH2
O
R
N
H
O
O
N
H
NH
4
a
H
N
H2N
HN
O
3
R = Me
R = H
5a
5b
N
H
N
O
b
NH
Scheme 1. Synthesis of the protected pyrrole-guanidine 5b; conditions: (a) DCM, MeOH, NEt3, EDC$HCl, rt, then 40 ꢂC over night, 77%; (b) NaOH, MeOH, H2O, rt, 20 h, quant.
O
O
O
O
O
O
O
O
O
O
c
O
O
O
R´
N
O
O
O
5b
a
O
O
O
N
O
+
N
O
H
R
H
+
N
H
N
H
N
H
NH
H
N
O
O
HN
R´ = Boc
R´ = H
7
2
6a (R = H)
R´
N
H
N
b
O
NH
6b
R =
Scheme 2. Peptide coupling of compound 5b with 6a; conditions: (a) EDC$HCl, HOBt, CHCl3, DMF, N2, rt, then 40 ꢂC, over night, 75%; (b) DCM, HCl in Et2O, 5 h, rt, quant.; (c) DCM,
NEt3, Ac2O, 3 h, rt, quant.
to ensure a homogeneous reaction mixture.20 The subsequent
1,2
Compound 6b
deprotection step gives quantitative yields of 2. Peptides for the
1,1
Compound 2
determination of binding selectivity were prepared in solution and
1,0
by standard solid phase methods.
0,9
0,8
0,7
2.2. Physical properties
0,6
Compound 2 shows absorption maxima in methanol at 205 nm,
0,5
with a shoulder at 220 nm, and at 290 nm, corresponding to the
0,4
crown ether and pyrrole moieties, respectively. Upon excitation at
0,3
310 nm, the system emits at 390 nm. The quantum yield is
(Fig. 2)
4
¼0.1.21
0,2
0,1
0,0
The compounds emission intensity is sensitive to pH: pro-
tonation of the crown ether nitrogen atom interrupts PET fluores-
cence quenching.22 Solutions of the compound 2 in methanol were
mixed with aqueous buffer at defined pH values and the emission
of the crown ether was recorded (Fig. 3). At pH values smaller than
200
225
250
275
300
325
350
375
400
wavelength [nm]
Figure 2. Absorption spectra of compounds 2 and 6b (c¼3.4ꢀ10ꢁ5 and 3.2ꢀ10ꢁ5 mol/L).

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A. Spath, B. Konig / Tetrahedron 66 (2010) 6019e6025
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340
320
300
280
260
240
220
200
180
160
140
350
2 at pH 6.5
2 at pH 9
2 at pH 4
Compound 2
300
pK value ~ 5.6
a
max.
250
200
150
100
50
min.
0
2
3
4
5
6
7
8
9
10
11
320 340 360 380 400 420 440 460 480 500
pH
wavelength λ [nm]
Figure 3. Emission intensity of compound 2 in methanol/aqueous buffer 4:1 at different pH values of the buffer; lex¼300 nm, lem¼390 nm; (c¼3.0ꢀ10ꢁ5 mol/L).
6 an emission intensity increase is observed by protonation of the
aza-crown nitrogen atom.
ether, the aza-crownnitrogen atom quenching the emission is mainly
deprotonated at this pH. The acyl guanidines are protonated under
these conditions and interact with carboxylate ions. First, the in-
teraction of 2 with ammonium and carboxylate ions was investigated
separately: the addition of up to 1000 equiv guanidine hydrochloride
or tert-butylammonium acetate in methanol/aqueous buffer 4:1
(10 mM HEPES adjusted to pH 6.3 with dilute hydrochloric acid) did
not affect the emission at 390 nm. Butylammonium chloride addition
2.3. Binding studies
Peptide binding to compound 2 was investigated in methanol/
aqueous buffer mixtures adjusted to pH 6.3. This ensures a large gain
in emission intensity upon ammonium ion binding to the crown
NH+3
NH+3
NH+3
OH
O
O
O
H
N
H
N
H
NH2
N
N
H2N
N
H
N
H
N
H
_
_
O
COO
_
COO
O
O
O
O
COO
_
COO
8
1
9
O
O
H
N
_
H3N+
H3N+
COO
11a n = 1
11b n = 3
11c n = 5
NH2
10a
N
H
N
H
n
O
O
O
O
_
COO
H
N
H
N
_
H
N
H3N+
10b
H3N+
COO
12a
N
H
NH2
NH2
O
O
O
O
O
O
_
COO
H
N
_
H3N+
H
N
H3N+
COO
10c
N
H
N
H
N
H
O
O
O
O
O
12b
_
COO
O
H
N
H
N
_
H
N
H
H3N+
10d
H3N+
N
COO
12c
N
H
NH2
_
N
H
O
O
O
O
COO
Figure 4. Peptides investigated for binding to compound 2 in methanol/aqueous buffer 4:1 solution.

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A. Spath, B. Konig / Tetrahedron 66 (2010) 6019e6025
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increases the emission intensity only negligible (F/F0<1.05).23 The
possible effect of ionic strength on the emission intensity was probed
by addition of sodium perchlorate in large excess (1000 equiv), which
induced no observable emission changes. Sodium acetate24 was
added to investigate the pyrrole guanidinium carboxylate binding.
Analysis of changes in the UV/vis spectrum revealed a binding con-
stant of <103 Mꢁ1 with 1:1 stoichiometry, but no changes of the
emission intensity at 390 nm are observed.25
intensity changes (F/F0z1.3) with the receptor 2. Addition of the
other peptides under these conditions caused no emission intensity
change.
For the peptides showing a significant emission enhancement
the binding affinities stoichiometries were determined by emission
titration of solutions of 2 (c¼3ꢀ10ꢁ5 M) in 4:1 methanol/aqueous
buffer-mixture (10 mM HEPES, pH 6.3). For comparison, compound
6b was titrated with peptide 1 under identical conditions. The in-
teraction of Ac-Ser-Asp-Lys-Pro (1) with 2 was additionally in-
vestigated in 1:1 methanol/aqueous buffer. Emission titration data
were analyzed using the Hill equation.28 Figure 6 shows the
emission titration data and Job’s plot analyses of compound 2 ti-
trated with Ac-Ser-Asp-Lys-Pro (1) in methanol/aqueous buffer 4:1
(left) and 1:1 (right). Table 1 summarizes the results of all titrations
with receptor 2. The investigated peptides Lys-Gly (8), Gly-Glu-Gly-
Gly-NH2 (10b), Gly-Gly-Glu-Gly-NH2 (10c), (Gly)3 (12b) and (Gly)4
(12c), bind to 2 with affinities in the range of 220e930 Mꢁ1, which
is significantly weaker than the binding of 2 to 1.
The enhanced binding affinity of 2 for peptide 1 of log K¼3.8 can
be rationalized by the additional electrostatic interactions due to
the carboxylate groups in 1. The dipeptide guest Lys-Gly (8), which
reflects a fragment of the structure of 1, is missing this additional
carboxylate/guanidinium interaction and therefore shows an af-
finity to 2 of log K<3. All other zwitterion guests, which can un-
dergo ditopic binding, show even weaker binding to 2. It was not
possible to derive a binding constant for 1 versus 6b. Even if a large
excess of the guest (>2000 equiv) is added, no saturation of titra-
tion curve is reached.29 This illustrates the importance of the gua-
nidinium/carboxylate interaction for the formation of stable
aggregates.
The binding of compound 2 to peptide 1 and related peptide
structures was investigated by emission titration. Ac-Glu-Lys-am-
ide (8),26 Cbz-Lys-Gly and Lys-Gly (9) are partial structures of the
target peptide. For comparison glycine (11a),
g-aminobutyric acid
(11b), -aminohexanoic acid (11c), Gly-Gly-OH (12a), Gly-Gly-Gly-
3
OH (12b), Gly-Gly-Gly-Gly-OH (12c) and the isomeric peptides Glu-
Gly-Gly-Gly-NH2 (10a), Gly-Glu-Gly-Gly-NH2 (10b), Gly-Gly-Glu-
Gly-NH2 (10c) and Gly-Gly-Gly-Glu-NH2 (10d) were used (Fig. 4). To
quickly identify the strongest interactions, 200 and 500 equiv of the
peptides were added to a solution of 2 (0.5 mL, 3ꢀ10ꢁ5 M) in
methanol/buffer 4:1 (10 mM HEPES; pH 6.3). All peptides were
soluble in the used concentration range.27 Compound 2 shows
a significant 1.7-fold fluorescence enhancement with 1 in 20%
aqueous, buffered methanol (Fig. 5, left). Addition of glycine,
g-aminobutyric acid (GABA) and 3-aminohexanoic acid (AHX) gives
no response, while (Gly)3, (Gly)4, (Cbz, H)-Lys-Gly, Gly-Glu-Gly-Gly-
amide and Gly-Gly-Glu-Gly-amide induce a weak response of the
emission intensity (F/F0¼1.1e1.3). In 50% aqueous buffer/methanol
mixture the emission change upon addition of 1 is still evident (F/
F0¼1.5), while all other peptides induce only small emission
changes (F/F0¼<1.1; Fig. 5, right). In aqueous buffer without added
methanol only the addition of 1 leads to detectable emission
Figure 5. Comparison of the emission response of compound 2 (c¼3ꢀ10ꢁ5 M) and of crown ether 6b (c¼3ꢀ10ꢁ5 M) upon addition of 200 equiv of different peptides (c¼0.006 M) in
MeOH/buffer (10 mM HEPES) 4:1 (left) and 1:1 (right) at pH 6.3 adjusted with Et4NOH and HCl.
Figure 6. Titration of compound 2 with the tetrapeptide 1 in methanol/aqueous buffer 4:1 (left) and 1:1 (right) at pH 6.3; Small inserts: Job plots.

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A. Spath, B. Konig / Tetrahedron 66 (2010) 6019e6025
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Table 1
4.1.1. Methyl
3,5-bis-(1-(tert-butyloxycarbonyl)-3-(pyrrol-2-car-
bonyl)-guanidino-methyl)benzoate (5a). To a cooled solution (2 ꢂC)
of compound 4 (312 mg, 1.1 mmol) and triethylamine (0.66 mL,
500 mg, 5.0 mmol) in 3.0 ml of DCM and methanol (19:1), 3,5-bis-
aminomethyl-benzoic acid methyl ester (3)32 (105 mg, 0.5 mmol)
and EDC$HCl (272 mg, 1.5 mmol) were added. The reaction was
stirred for 3 h, allowed to warm up to room temperature, refluxed
for another 2 h and cooled to room temperature. A second portion
EDC$HCl (272 mg, 1.5 mmol) was added, the reaction mixture was
stirred for 30 min at room temperature and then refluxed for 2 h.
The reaction mixture was diluted with 30 mL of DCM and filtered;
washed with 10% citric acid (10 mL), water (10 mL), dried over
MgSO4 and the solvent was evaporated. The residue was purified by
column chromatography with ethyl acetate/ethanol, 6:1, to give the
product as an orange solid (256 mg, 77%).
Absolute and relative binding constants of compound 2 to different peptides 1
methanol/aqueous buffer 4:1 at pH 6.3
Peptide
Binding
Relative
binding
strengths
K/K (1)
Fluorescence
enhancement
F/F0
constant
K [Mꢁ1
]
Ac-Ser-Asp-Lys-Pro (1)
6800ꢃ360
(3540ꢃ190)a
930ꢃ50
1
1.62
(1.69)a
1.41
1.32
1.34
1.21
1.17
Lys-Gly (8)
0.137
0.032
0.117
0.075
0.057
Gly-Glu-Gly-Gly-NH2 (10b)
Gly-Gly-Glu-Gly-NH2 (10c)
(Gly)3 (12b)
220ꢃ30
800ꢃ60
510ꢃ60
(Gly)4 (12c)
390ꢃ40
a
Values for 1:1 methanol/aqueous buffer.
3. Conclusions
Mp: (uncorrected)¼127e128 ꢂC; 1H NMR (300 MHz, CDCl3):
d
[ppm]¼1.44e1.54 (m, 18H), 3.90 (s, 3H), 4.68e4.81 (m, 4H), 6.22
Compound 2 is a synthetic receptor for the hemoregulatory
peptide 1 presumably binding the lysine ammonium side chain, the
terminal and the aspartic acid side chain carboxylate of the pep-
tide.30 The binding of the peptide is signalled by an increase in
emission intensity of the crown ether fluorophor, as the ammo-
nium ion binding intercepts the photoinduced electron transfer
(PET) quenching mechanism. The combination of the crown ether/
ammonium ion interaction with the guanidinium/carboxylate ion
interaction is essential for sensing the target peptides: The crown
ether/ammonium ion interaction is too weak to be effective in the
aqueous buffer, while the acyl-guanidinium/carboxylate binding
itself will not affect the emission intensity. Small peptides, which
were used for comparison, revealed that the additional carboxylate
group in 1 and the ammonium/carboxylate distance complemen-
tary to 2 are responsible for the observed increased affinity. Figure 7
shows the proposed structure of the peptideereceptor aggregate.
The results provide another example that the combination of sev-
eral weak reversible interactions may lead to synthetic receptors
with affinity and selectivity of biological targets. Although the
binding and emission properties of compound 2 are not sufficient
for analytical applications in complex matrices, its use as an in-
dicator is already possible.
(m, 2H), 6.71e7.01 (m, 4H), 7.59e7.72 (m, 1H), 7.85e8.01 (m, 2H),
8.80 (m, 2H), 9.23 (m, 1H), 9.53 (br s, 1H), 9.76 (br s, 1H), 9.98 (br s,
1H); 13C NMR (75 MHz, CDCl3):33
d
[ppm]¼28.1 and 28.3 (þ, 6C),
44.5 (ꢁ, 2C), 52.3 (þ, 1C), 79.8 and 83.3 (Cquat, 2C), 110.4 (þ, 2C),
114.5 (þ, 2C), 122.1 (þ, 2C), 128.2 (þ, 2C), 131.0 (Cquat, 2C), 131.6 (þ,
1C), 139.1 (Cquat, 2C), 153.3 (Cquat, 2C), 156.0 and 156.3 (Cquat, 2C),
160.6 (Cquat, 1C), 164.0 (Cquat, 1C), 166.5 (Cquat, 1C), 171.1 (Cquat, 1C);
MS (ESI-MS, CH2Cl2/MeOHþ10 mmol NH4OAc): m/z (%)¼665.2 (90,
MHþ), 333.0 (100, (Mþ2Hþ)2þ); HRMS (FAB LSI-MS Glycerine):
calcd for C32H41N8Oþ8 : 665.3047, found: 665.3033; MF:
C33H43N8O8dFW: 679.76 g/mol.
4.1.2. Sodium 3,5-bis-(1- (tert-butyloxycarbonyl)-3-(pyrrol-2-car-
bonyl)-guanidino-methyl)benzoate (5b). Compound 5a (166 mg,
0.25 mmol) in methanol (1.0 mL) was treated with 1 M aqueous
sodium hydroxide solution (0.25 mL) by vigorous stirring at room
temperature over night. Methanol was evaporated and the aqueous
solution was lyophilised to give the product as a yellowish solid
(164 mg, 98%).
Mp: (uncorrected)¼145e146 ꢂC (decomp.); 1H NMR (300 MHz,
CDCl3):33
d
[ppm]¼1.47e1.53 (s,18H), 4.69e4.78 (m, 4H), 6.20e6.31
(m, 2H), 6.83e6.89 (m, 4H), 7.64 (m, 1H), 7.90e8.05 (m, 2H), 8.84
(m, 2H), 9.41 (m, 2H), 9.86 (br s, 1H), 12.43 (br s, 1H); MS (ESI-MS,
CH2Cl2/MeOHþ10 mmol NH4OAc): m/z (%)¼651.2 (94, MHþ), 326.0
(100, (Mþ2Hþ)2þ); HRMS (FAB LSI-MS Glycerine): calcd for
C31H39N8Oþ8 : 651.2891, found: 651.2897; MF: C31H37N8O8dFW:
672.68 g/mol.
H
O
N
N
H
O
N
+
N
H
O
H
-
4.1.3. Dimethyl 14-[2-[3,5-bis-(1-(tert-butyloxycarbonyl)-3-(pyrrol-
2 - c a r b o n y l ) - g u a n i d i n o - m e t h y l ) b e n z a m i d o ] - e t h y l ] -
6,7,9,10,13,14,15,16,18,19,21,22-dodecahydro-12H-5,8,11,17,20,23-hex-
HO
O
H
N
N
H
H
H
N
N
O
O
N
+
aoxa-14-aza-benzocycloheneicosene-2,3-dicarboxylate
(7). Com-
H
O
O
N
H
-
O
O
O
pound 5b (135 mg, 0.20 mmol) was dissolved in 1.0 ml of DMF and
DCM (1:1) at 5 ꢂC, triethylamine (0.06 mL, 0.05 g, 0.5 mmol), HOBt
(29 mg, 0.22 mmol) and EDC hydrochloride (36 mg, 0.21 mmol)
were added subsequently and the mixture was stirred for 30 min in
the ice bath. The TFA salt of compound 6a (163 mg, 0.22 mmol)
dissolved in 1.0 mL of DCM containing 0.06 mL triethylamine
(0.05 g, 0.5 mmol) was added slowly and the reaction mixture was
stirred for 2 h at room temperature. Another portion of triethyl-
amine (0.06 mL, 0.05 g, 0.5 mmol), HOBt (29 mg, 0.22 mmol) and
EDC hydrochloride (36 mg, 0.21 mmol) was added, stirring was
continued for 1 h, the mixture was warmed to 40 ꢂC and stirred for
additional 8 h under nitrogen. The solvents were removed and the
residue was re-dissolved in 10 mL of ethyl acetate. The solution was
filtered and the filter cake was washed with a small portion of ice
cold ethyl acetate. The clear filtrate was washed with 5% aqueous
ammonium chloride solution and with water (3ꢀ3 mL). The or-
ganic phase was dried over MgSO4, evaporated to dryness and the
N
H
H
H
N
O
O
O
O
+
NH3
O
O
N
N
O
O
O
O
2 - Ac-Ser-Asp-Lys-Pro
Figure 7. Proposed structure of the aggregate of 2 and the tetrapeptide 1.
4. Experimental
4.1. Syntheses
Compounds 6,9 431 and 317 were synthesized in solution
according to published procedures. Ac-Ser-Asp-Lys-Pro (1) was
purchased from GENSCRIPT in a purity >95% and used as is.

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6024
remaining solid was purified by column chromatography with ethyl
acetate/ethanol 10:1 to yield 173 mg of a pale yellow glass (75%).
solution, 5e40
m
L for peptide 1 (1ꢀ10ꢁ3 mol/L) or 60e200
mL for G-
E-G-G-NH2, G-E-G-G-NH2, K-G-OH, G-G-G-OH and G-G-G-G-OH
(1ꢀ10ꢁ2 mol/L) were successively added. After each addition the
solution was allowed to equilibrate for 5 min before the fluores-
cence intensity (lex¼310 nm) and the UV spectrum were recorded
at 25 ꢂC. The stoichiometry was determined by Job plot analysis.34
Binding constants were determined from volume corrected emis-
sion intensities by non linear fitting. The pH of the buffer/methanol
mixtures was determined before and after titrations. Measure-
ments with deviations of more than 0.5 pH units were not used for
binding constant determination.
1H NMR (600 MHz, CDCl3):
d
[ppm]¼1.42e1.54 (m, 18H), 2.56
(br m, 4H), 3.49e3.68 (br m, 6H), 3.59 (m, 4H), 3.65e3.70 (m, 4H),
3.81 (m, 4H), 3.82e3.88 (m, 8H), 4.62e4.78 (m, 8H), 6.15e6.33 (m,
2H), 6.81e6.95 (m, 4H), 7.04 (m, 1H), 7.12 (m, 2H), 7.46 (m, 1H), 7.70
(m, 1H), 7.82 (br s, 1H); 13C NMR (150 MHz, CDCl3):33
d
[ppm]¼28.0
and 28.3 and 30.9 (þ, 6C), 40.9 (ꢁ, 1C), 44.2 and 44.4 (ꢁ, 2C), 46.3
(ꢁ, 1C), 52.6 (þ, 2C), 54.5 (ꢁ, 2C), 68.8 (ꢁ, 2C), 69.4 (ꢁ, 2C), 70.2 (ꢁ,
4C), 70.8 (ꢁ, 2C), 79.6 and 82.2 (Cquat, 2C), 110.3 and 110.8 (þ, 2C),
113.1 and 113.6 (þ, 2C), 114.3 (þ, 2C), 121.9 (þ, 2C), 124.2 (þ, 1C),
125.3 (Cquat, 2C), 126.1 (þ, 2C), 130.1 (Cquat, 2C), 138.9 (Cquat, 1C),
150.0 and 153.1 (Cquat, 2C), 155.7 and 155.8 (Cquat, 2C), 165.1 (Cquat
,
Acknowledgements
1C), 167.8 (Cquat, 2C), 170.9 (Cquat, 2C); IR (KBr):
n
(cmꢁ1)¼3300 (br
m), 2948 (m), 2929 (m), 2876 (m), 2356 (w), 2247 (w), 1718 (m),
1607 (s), 1578 (s), 1546 (s), 1406 (s), 1349 (s), 1289 (s), 1256 (m),
1229 (m), 1187 (m), 1124 (s), 1027 (m), 980 (m), 910 (m), 843 (m),
780 (m), 729 (s), 646 (m); MS (ESI-MS, CH2Cl2/MeOHþ10 mmol
NH4OAc): m/z (%)¼1147.6 (21, MHþ), 574.2 (100, (Mþ2Hþ)2þ), 383.2
(22, (Mþ3Hþ)3þ); HRMS (FAB LSI-MS Glycerine): calcd for
We thank the Deutsche Forschungsgemeinschaft (GRK 640) and
the University of Regensburg for support of this work.
Supplementary data
Supplementary data associated with this article can be found in
online version at 10.1016/j.tet.2010.06.024. These data include MOL
files and InChIKeys of the most important compounds described in
this article.
C56H75N10Oþ17: 1147.5312, found: 1147.5307; UV (MeOH):
l
(3
)¼298
(18,900), 205 (30,500); MF: C55H74N10O17dFW: 1147.26 g/mol.
4.1.4. Dimethyl
14-[2-[3,5-bis-(1-(pyrrol-2-carbonyl)-guanidino-
methyl)benzamido]-ethyl]-6,7,9,10,13,14,15,16,18,19,21,22-dodecahy-
dro-12H-5,8,11,17,20,23-hexaoxa-14-aza-benzocycloheneicosene-2,3-
dicarboxylate hydrochloride (2). Compound 7 (115 mg, 0.10 mmol)
was dissolved in 1.0 ml of dry DCM, 0.3 mL of HCl saturated
diethylether was added and the mixture was stirred for 3 h at room
temperature under moisture protection. The product was fully
precipitated by slow addition of diethylether. The solvent was
decanted off, the solid was suspended in a small volume of dieth-
ylether, allowed to precipitate completely and the ether was dec-
anted off. This step was repeated once. After drying in vacuo the
product was isolated as a fine yellow powder (96 mg, 91%).
References and notes
1. Sharma, U.; Rhaleb, N.-E.; Pokharel, S.; Harding, P.; Rasoul, S.; Peng, H.; Carre-
tero, O. A. Am. J. Physiol. Heart Circ. Physiol. 2008, 294, H1226eH1232.
2. Lombard, M.-N.; Sotty, D.; Wdzieczak-Bakala, J.; Lenfant, M. Cell Prolif. 1989, 23,
99e103.
3. Pokharel, S.; Rasoul, S.; Roks, A.; van Leeuwen, R.; van Luyn, M.; Deelman, L. E.;
Smits, J. F.; Carretero, O.; van Gilst, W. H.; Pinto, Y. M. Hypertension 2002, 40,
155e161.
4. Massé, A.; Ramirez, L. H.; Bindoula, G.; Grillon, C.; Wdzieczak-Bakala, J.; Rad-
dassi, K.; Deschamps de Paillette, E.; Mencia-Huerta, J. M.; Koscielny, S.; Potier,
P.; Sainteny, F.; Carde, P. Blood 1998, 91, 441e449.
5. Grillon, C.; Lenfant, M.; Wdzieczak-Bakala, J. Growth Factors 1993, 9, 133e138.
6. Cheviron, N.; Rousseau-Plasse, A.; Lenfant, M.; Adeline, M.-T.; Potier, P.;
Thierry1, J. Anal. Biochem. 2000, 280, 58e64.
1H NMR (400 MHz, CD3OD):
d
[ppm]¼3.48e3.59 (m, 6H),
€
€
3.65e3.71 (m, 8H), 3.75 (t, 2H, 6.4 Hz), 3.82 (s, 6H), 3.84 (m, 4H), 3.91
7. Spath, A.; Konig, B. Tetrahedron 2010, 66, 1859e1873.
€
€
8. Stadlbauer, S.; Riechers, A.; Spath, A.; Konig, B. Chem.dEur. J. 2008, 14,
2536e2541; Kruppa, M.; Mandl, C. P.; Miltschitzky, S.; Konig, B. J. Am. Chem. Soc.
(m, 4H), 4.21 (m, 4H), 6.30 (m, 2H), 7.16 (m, 2H), 7.21 (s, 2H), 7.26 (m,
€
2H), 7.61 (s, 1H), 7.82 (s, 2H); 13C NMR (100 MHz, CDCl3):
d
[ppm]¼
2005, 127, 3362e3365.
45.6 (ꢁ, 2C), 51.7 (ꢁ, 1C), 53.2 (þ, 2C), 55.3 (ꢁ, 2C), 64.1 (ꢁ, 1C), 70.2
(ꢁ, 4C), 70.8 (ꢁ, 2C), 71.4 (ꢁ, 2C), 71.7 (ꢁ, 2C), 111.8 (þ, 2C), 114.3 (þ,
2C), 115.9 (Cquat, 1C), 116.6 (þ, 2C), 126.5 (Cquat, 2C), 127.0 (þ, 2C),
130.9 (Cquat, 2C), 151.7 (Cquat, 2C), 161.7 (Cquat, 2C), 169.4 (Cquat, 2C),
€
9. Mandl, C. . P.; Konig, B. J. Org. Chem. 2005, 70, 670e674.
10. de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C.
P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515e1566; de Silva, A. P.;
Fox, D. B.; Moody, T. S.; Weir, S. M. Trends Biotechnol. 2001, 19, 29e34.
11. Best, M. D.; Tobey, S. L.; Anslyn, E. V. Coord. Chem. Rev. 2003, 240, 3e15.
12. Fitzmaurice, R. J.; Kyne, G. M.; Douheret, D.; Kilburn, J. D. J. Chem. Soc., Perkin
Trans. 1 2002, 841e864; Brooks, S. J.; Edwards, P. R.; Gale, P. A.; Light, M. E. New
J. Chem. 2006, 30, 65e70.
13. Czekalla, M.; Stephan, H.; Habermann, B.; Trepte, J.; Gloe, K.; Schmidtchen, F. P.
Thermochim. Acta 1998, 313, 137e144.
14. Schmuck, C. Coord. Chem. Rev. 2006, 250, 3053e3067.
15. Schmuck, C. Chem.dEur. J. 2000, 6, 709e718; Hernandez-Folgado, L.; Schmuck,
C.; Tomic, S.; Piantanida, I. Bioorg. Med. Chem. Lett. 2008, 18, 2977e2981 and
literature therein.
16. Oshovsky, G. V.; Reinhoudt, D. N.; Verboom, W. Angew. Chem., Int. Ed. 2007, 46,
2366e2393.
17. Maeda, D. Y.; Mahajan, S. S.; Atkins, W. M.; Zebala, J. A. Bioorg. Med. Chem. Lett.
2006, 16, 3780e3783; Ryu, E.-H.; Yan, J.; Zhong, Z.; Zhao, Y. J. Org. Chem. 2006,
71, 7205e7213.
18. Chand, P.; Elliot, A. J.; Montgomery, J. A. J. Med. Chem. 2001, 44 (25), 4379e4392;
Kent, D. R.; Cody, W. L.; Doherty, A. M. Tetrahedron Lett. 1996, 37, 8711e8714.
19. The cyclised by-product 1-oxo-1,2-dihydro-pyrrolo[1,2-c]imidazol-3-ylidene is
formed using Hg(II) as catalyst in DMF.
further signals were not detectable; IR (KBr):
n
(cmꢁ1)¼3200 (br m),
2946 (m), 2883 (m), 2361 (m), 2347 (m),1678 (s),1632 (m),1546 (m),
1434 (m),1293 (s),1186 (m),1120 (s),1051 (m), 974 (m), 948 (m), 887
(m), 746 (m), 667 (m); MS (ESI-MS, CH2Cl2/MeOHþ10 mmol
NH4OAc): m/z (%)¼947.7 (6, MHþ), 531.2 (40, (Mþ2Hþ)2þþTFA),
474.2 (100, (Mþ2Hþ)2þ), 330.1 (98, (Mþ3Hþ)3þþMeCN), 316.4 (14,
(Mþ3Hþ)3þþTFA); HRMS (FAB LSI-MS Glycerine): calcd for
C45H59N10Oþ13: 947.4263, found: 947.4241; UV (MeOH):
l
(3)¼291
(22,300), 206 (29,400); MF: C45H61N10O13Cl3dFW: 1056.40 g/mol.
4.2. Binding investigations
4.2.1. Screening of amino acid and peptide binding affinities. The
screening was performed in UV star 96 well plates (400 mL volume
per cell) in a 4:1 mixture of methanol and aqueous salt free HEPES
buffer (10 mM) at pH 6.3. To a 3ꢀ10ꢁ5 M solution of the receptor
compound 200 equiv of peptide or amino acid (1.5ꢀ10ꢁ2 M) were
added (1:1 v/v). The fluorescence spectrum was recorded
20. Heterogeneous reaction leads to increased amounts of the side product 1-oxo-
1,2-dihydro-pyrrolo[1,2-c]imidazol-3-ylidene.
21. All quantum yields were determined with quinine disulfate in 1 N H2SO4 as the
reference compound (
F
¼0.546).
22. The pKa value for the crown ether nitrogen atom of the protected derivative 7
was determined to be 5.6 by titration with perchloric acid; the pKa value of 5.8
for compound 6b is comparable. The pKa value of the pyrrole guanidinium
groups is 6.5. See Supplementary data.
(lex¼310 nm); all measurements were repeated twice.
23. In pure methanol the crown ether binds this ammonium guest weakly (log K
(2)¼2.2 and log K (6b)¼2.3).
4.2.2. Binding affinity titrations. To a cuvette with 1.0 mL of the
receptor (3ꢀ10ꢁ5 mol/L) in a 4:1 or a 1:1 mixture of methanol and
aqueous HEPES buffer (10 mM, pH 6.3) small aliquots of the peptide
24. Sodium is not bound by the crown ether (21-crown-7). Even if a 2000 fold excess
is added in aqueous methanol no change in luminescence can be observed.

€
€
A. Spath, B. Konig / Tetrahedron 66 (2010) 6019e6025
6025
€ €
31. Spath, A.; Konig, B. Tetrahedron 2009, 65, 690e695.
25. Schmuck, C. Chem.dEur. J. 2006, 6, 709e718.
26. The amino acid Glu is used instead of Asp due to synthetic reasons. The
structural difference should not influence the selectivity and binding strength
to a large extent.
32. The free base was prepared from the hydrochloride by shaking its chloroform
solution several times with an aqueous solution of potassium carbonate (2 M).
The combined aqueous phases were re-extracted three times with chloroform.
33. The splitting of some signals in the spectrum indicates the presence of unequal
populations of two conformers at room temperature. This observation caused
by hindered rotation in the molecule is literature known: Dalton, D. R.; Ramey,
K. E.; Gisler, H. J., Jr.; Lendvay, L. J.; Abraham, A. J. Am. Chem. Soc. 1969, 91,
6367e6370 and literature therein.
27. Fauchere, J. L.; Pliska, V. Eur. J. Med. Chem. 1983, 18, 369e375; Narita, M.; Chen,
J. Y.; Sato, H.; Kim, Y. Bull. Chem. Soc. Japan 1985, 58, 2494e2501.
28. Connors, K. A. Binding Constants; Wiley: New York, NY, 1987.
29. The crown ether binding affinity for ammonium ions in aqueous solution is
known to be small (log K<2) Rüdiger, V.; Schneider, H.-J.; Solov’ev, V. P.;
Kazachenko, V. P.; Raevsky, O. A. Eur. J. Org. Chem. 1999, 8, 1847e1856.
34. MacCarthy, P. Anal. Chem. 1978, 50, 2165; Schmuck, C.; Wich, P. Angew. Chem.,
Int. Ed. 2006, 45, 4277e4281.
€
30. Suhs, T.; Konig, B. Mini-Rev. Org. Chem. 2006, 3, 315e331.