Modular synthesis of di- and tripeptides of luminescent crown ether aminocarboxylic acids

Article abstract of DOI:10.1016/j.tet.2008.10.086

Luminescent benzo crown ether aminocarboxylic acids with ammonium ion affinity were prepared and converted into linear bis- and tris benzo crown ether amides using standard peptide coupling protocols. The affinities of the new crown ethers to ammonium ion

Full text of DOI:10.1016/j.tet.2008.10.086

Tetrahedron 65 (2009) 690–695
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Modular synthesis of di- and tripeptides of luminescent crown ether
aminocarboxylic acids
*
Andreas Spa¨th, Burkhard Ko¨nig
Institut fu¨r Organische Chemie, Universita¨t 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:
Received 14 July 2008
Received in revised form 19 October 2008
Accepted 27 October 2008
Available online 31 October 2008
Luminescent benzo crown ether aminocarboxylic acids with ammonium ion affinity were prepared and
converted into linear bis- and tris benzo crown ether amides using standard peptide coupling protocols.
The affinities of the new crown ethers to ammonium ions and di- and tetrapeptides bearing ammonium
ion moieties were determined by emission titration in methanol and buffered water.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
through a columnar crown ether stack. Gokel16 developed a family
of tris-macrocycles that permits the transport of sodium ions
Many biologically important molecules have ammonium ions
as functional groups. Typical examples are neutrotransmitters,1,2
growth factors,3 histamine2,4 or peptides.5 The development of
synthetic receptors that allow the specific recognition of organic
ammonium ions is therefore an area of ongoing interest and re-
sults have potential applications in medicinal diagnostics or che-
mosensors. Typical artificial binding sites for ammonium ions are
crown ethers,6 calixarenes,7 phosphonate8dand oxazoline-based9
receptors and porphyrine metal complexes; most examples have
been reported for crown ether systems.10 However, for many cases
the binding strengths of a single ammonium ion–receptor site
interactions are too weak in an aqueous environment to give
stable and defined aggregates. The combination of several binding
sites in one synthetic receptor can improve the situation, if their
individual contributions are additive or even show positive
cooperativity.
Several examples of oligovalent artificial cation receptors based
on crown ethers have been reported: bis- and tris-crown ethers as
host compounds for alkaline metal ions have been published by
Huang,11 and a carrier molecule composed of three crown ethers
was used to mediate the transport of mercury ions through
a chloroform phase.12 Nolte13 polymerized an isocyanide derivative
of benzo-18-crown-6 and applied the polymer to liposome medi-
ated copper-ion transport. Oligo- or poly14 crown ethers have been
used as chemical model systems for ion channels. A poly(crown)
channel was constructed using the benzo-21-crown-7 derivative of
phenylalanine and leucine.15 Incorporation of the resulting helical
peptide into a lipid bilayer leads to proton and sodium ion transport
through membranes, using crown ethers, connected by hydro-
phobic alkane spacers, as channel head groups and cation entry
portals.
For ammonium ion binding, only ditopic receptors have been
reported so far. Phenolphtaleine bis-crown ethers have been used
for the binding of diamines and dipeptides with a C-terminal Lys.17
Two crown ether aminocarboxylic acids, which were incorporated
into an oligo Ala peptide were used by Voyer18 to bind
a,u-di-
ammonium alkanes. We have recently reported the synthesis and
dimerization of ammonium ion binding phthalimide crown
ethers.19 We now extend the approach and present here a modular
synthetic route to functionalized, luminescent bis- and tris-crown
ethers with ammonium ion affinity.
2. Results and discussion
2.1. Synthesis
The twofold ring closing substitution reaction of di-tosylate 119
with primary amines 2 under basic conditions yields crown ether
aminocarboxylic acids 3 in good yields (Scheme 1, Table 1). The
presence of potassium ions favours the ring closing reaction by its
template effect. No by-products from intermolecular reactions
were observed under the experimental conditions (c¼6ꢀ10ꢁ2 mol/
L). Alkyne or azide functional groups (entries g, h) are tolerated and
the resulting products are suitable substrates for Huisgen cyclo-
addition reactions. The reaction tolerates cbz- or Boc-protected
amines (entries a–d).
The Boc-deprotection of compound 3a and the ester hydrolysis
of compound 3a or 3e allows the preparation of crown ether ami-
nocarboxylic acid dipeptides 620 in good yield using standard
peptide coupling conditions (Scheme 2). A further extension to
* Corresponding author. Fax: þ49 943 941 1717.
E-mail address: burkhard.koenig@chemie.uni-regensburg.de (B. Ko¨nig).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.10.086

A. Spa¨th, B. Ko¨nig / Tetrahedron 65 (2009) 690–695
691
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Tos
Tos
O
O
O
O
a
O
O
N
R
O
3
1
Scheme 1. Synthesis of a luminescent benzo azacrown ethers 3: (a) H2NCH2R (2, see Table 1), K2CO3, KI, MeCN, H2O, 80 ꢂC, 63–86%.
Table 1
Synthesis of compounds 3a–i
conditions (Scheme 3). The purification of tripeptides 7 requires
repeated column chromatography to remove by-products and
reagents.
Entry
R
Yield 3 [%]
a19
79
68
72
63
NHBoc
NHCbz
NHBoc
NHBoc
b
2.2. Photophysical properties
c
Compounds 3 show absorption maxima in methanol at 220 nm
and 270 nm, and emit upon excitation at 390 nm with a quantum
d
yield of about
4
¼0.1.21 The absorption and emission properties are
e
f
86
76
only marginally affected by the nature of the substituent R. Upon
addition of KSCN or n-butyl ammonium chloride the emission in-
tensity increases significantly and binding affinities were derived
from emission titration experiments. Table 2 summarizes the re-
sults. The affinity for potassium ions is typically one order of
magnitude larger than the ammonium ion binding, which is in
accordance with earlier reports.19,22 The cation affinity for all crown
ethers 3 is very similar in methanol solution. In buffered aqueous
solution (50 mM HEPES, pH 7.5; c¼2ꢀ10ꢁ5 mol/L) no change in the
emission intensity was observed, even if a large excess of KSCN or
butyl ammonium chloride was used.
NHBoc
ga
ha
i
71
69
78
H
N3
O
O
a
In the dark.
compounds 7, bearing three crown ether units, is possible by re-
moval of the Boc-protecting group of 6a yielding amine 6c, and
coupling with 5a or 5e, again, using standard peptide coupling
Upon the addition of the second crown ether moiety, as in
compound 6, the phthalic amide fluorophore is created and a new
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
H
N
N
R
O
O
O
O
O
O
3a
3e or 3a
a
b
O
O
O
O
O
O
O
O
O-
O-
O
O
O
O
O
N+
N
NH3+
R
O
O
O
O
O
5a (R = CH2NHBoc)
5b (R = Ph)
4
c
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
O
R
O
O
O
O
6a (R = CH2NHBoc)
6b (R = Ph)
Scheme 2. Preparation of crown ether aminocarboxylic acid dipeptides 6a and 6b: (a) HCl in Et2O, DCM, 95%; (b) NaOH 1 M, MeOH, H2O, 40 ꢂC, quant.; (c) EDC, HOBt, DIPEA, DMF,
CHCl3, 70 ꢂC, 79% (6a);19 76% (6b).

692
A. Spa¨th, B. Ko¨nig / Tetrahedron 65 (2009) 690–695
6a
a
O
O
O
O
O
O
O
O
O
O
O
O
O
NH3+
N+
O
O
N+
N
O
O
O
6c
b
5a or 5b
O
O
O
O
O
O
R
O
O
O
O
O
O
O
N
N
O
O
O
O
O
O
N
N
O
O
O
O
O
N
O
O
7a (R = CH2NHBoc)
7b (R = Ph)
Scheme 3. Synthesis of crown ether aminocarboxylic acid tripeptides 7a and 7b: (a) HCl in Et2O, DCM, 92%; (b) EDC, HOBt, DIPEA, DMF, CHCl3, 70 ꢂC, 35% (7a); 37% (7b).
Table 2
properties of the phthalic ester and the phthalimide moiety of 6b
allow the individual monitoring of binding to the two-crown ether
cation binding sites. Ammonium ions bind to both crown ethers in
methanol with a stoichiometry of 1:1 and similar affinity in the
order of log K¼2. In aqueous solution no interaction between 6b
and ammonium ions can be detected. The affinity of lysine methyl
ester hydrochloride reaches log K w4 in methanol, due to the si-
multaneous interaction of both ammonium ions with the bis-
crown compound. In buffered aqueous solution the interaction of
Lys–HCl–6b is detectable via the phthalic ester emission change,
but drops to log K<2. The emission of the phthalimide moiety is
quenched in aqueous solution (Table 3).
Binding affinities and emission intensity changes of compounds 3 and n-butyl
ammonium chloride or KSCN in methanol
Compound
n-Butyl ammonium chloride
KSCN
log K
IN/I0
log K
IN/I0
3a
3b
3e
3d
3f
2.8ꢃ0.3
2.8ꢃ0.2
2.8ꢃ0.1
2.7ꢃ0.2
2.7ꢃ0.3
2.6ꢃ0.2
3.2ꢃ0.3
1.7
1.7
1.9
1.8
1.7
1.6
2.1
3.9ꢃ0.4
3.8ꢃ0.3
3.9ꢃ0.3
3.8ꢃ0.4
3.6ꢃ0.3
d
3.5
3.5
3.6
3.4
3.4
d
3g
3i
4.0ꢃ0.4
3.7
Next, the trimeric crown ethers 7 were investigated for am-
monium ion binding (Table 4). As expected, the affinity for am-
monium ions in methanol solution of the crown ether moieties
remain unchanged if compared to 6. Lysyllysine methyl ester tri-
hydrochloride was chosen as a guest with three ammonium groups
to determine the binding to compound 7. Although the aggregate of
7 and lysyllysine is potentially stabilized by three simultaneous
ammonium ions–crown ether interactions, the determined affinity
constants in methanol and buffered water only slightly exceed the
values for the interaction of bis-crown ether 6 with a diammonium
absorption band at 247 nm appears. The tris-crown ether com-
pounds 7 bear two phthalimide chromophores, which results in
a significant increase of the UV absorption intensity at 247 nm if
compared to 6. Excitation of the fluorophores at 309 nm stimulates
emission at 386 nm with a shoulder at 488 nm (Fig. 1).
The binding properties of bis-crown ether 6b to n-butyl am-
monium chloride and lysine methyl ester hydrochloride were
evaluated by fluorescence titration in methanol and buffered
aqueous solution (50 mM HEPES, pH 7.5). The different optical
300
60000
50000
40000
30000
20000
10000
0
3e
6b
7b
7a
7b
250
200
150
100
50
0
250
300
wavelength [nm]
350
400
300
400
wavelength [nm]
500
600
Figure 1. Absorption spectra of compounds 3e, 6b, and 7b (in methanol, left) and emission spectra of compounds 7a and 7b (methanol, c¼2ꢀ10ꢁ5 mol/L, excitation at 309 nm,
right).

A. Spa¨th, B. Ko¨nig / Tetrahedron 65 (2009) 690–695
693
Table 3
ethers does not lead to a significant increase of binding affinities.
The flexible structure of the extended crown ethers and their
peptidic guest molecules is a likely rational for the observation: the
limited preorganization of the extended receptors binding sites
prohibits an additive or cooperative action of the intermolecular
interactions, and illustrates the importance of well balanced en-
tropy and enthalpy contributions in the design of synthetic
receptors.
Binding affinity and emission enhancement of compound 6b in the presence of
n-butyl ammonium chloride and lysine methyl ester hydrochloride in methanol
and buffered aqueous solution
Solvent
Guest
Phthalic ester
moiety of 6b
Phthalimide
moiety of 6b
log K
IN/I0
log K
IN/I0
Methanol
Butyl ammonium
chloride
2.2ꢃ0.4
1.3
2.6ꢃ0.3
4.7
Lysine methyl ester
hydrochloride
4.3ꢃ0.3
1.8ꢃ0.2
1.6
4.6ꢃ0.2
8.1
3. Experimental
H2O, HEPES
(50 mM, pH 7.5,
2% MeOH)
Lysine methyl ester
hydrochloride
1.7
d
d
3.1. General method for the preparation of aza-benzo-21-
crown-7-ethers
Compound 1 (2 g, 2.5 mmol)19 was dissolved in 40 mL of ace-
tonitrile and 0.3 mL of H2O. The corresponding amine or mono Boc-
protected diamine (2.5 mmol), 610 mg (3.6 mmol) KI and 3.45 g
(25 mmol) K2CO3 were added successively and the mixture was
refluxed over night. After cooling to room temperature, the mixture
was filtered over Celite and the solid residue washed with aceto-
nitrile and dichloromethane. The solvent was evaporated and the
crude product was purified by column chromatography. If not
stated differently, silica gel, and ethyl acetate/ethanol; 3:1, as eluent
was used.
compound. The differences of lysyllysine binding affinities of 7a
and 7b are within the error margins of the measurements. For both
interactions a stoichiometry of 1:1 is indicated by Job’s plot analysis
(see supporting information).
Although the extension of the ammonium ion receptor by one
crown ether from 6 to 7 had little impact on the ammonium
binding affinity, the additional chromophore leads to a stronger
emission in the presence of ammonium ions, which becomes visi-
ble by the naked eye. In addition, the ability of compounds 7 to bind
ammonium ions within small peptides was investigated using
isomeric tetrapeptides bearing two lysines (K) and two glycines (G)
in their sequence. H-K-K-G-G–NH2 carrying two lysines at the
N-terminus has the same ammonium ion pattern as H-K-K–OMe. In
H-K-G-K-G–NH2 one glycine and in H-K-G-G-K–NH2 two glycines
separate the lysines. All peptides were prepared on Rink amide
resin (see Supplementary data) and their binding affinities as tri-
hydrotriflates to compounds 7 were determined by titration in
methanol and buffered water (see Supplementary data for data).
The derived affinities are, within the errors of the experiments,
identical to the values obtained for H-K-K–OMe.
In summary, luminescent aza benzo crown ethers and crown
ether aminocarboxylic acids are available by an efficient ring clos-
ing reaction of bis-tosylates and amines. Using standard peptide
coupling procedures, linear bis- and tris-crown ether peptides were
obtained. Emission titrations in methanol or buffered aqueous so-
lution revealed affinities of ammonium ions to the benzo crown
ether moieties of approx. log K¼2 in methanol, but no affinity in
buffered water. The interaction of bis-crown ethers 6 and tris-
crown ethers 7 with bis- or tris-ammonium ions, respectively, is of
similar strengths in methanol (log K w4.5) and in buffered water
(log K w2.5). This shows that the extension of bis- to tris-crown
The molecular structures for the assigned NMR data sets are
given in the Supplementary data to this article.
3.2. 14-[3-(tert-Butyl)oxycarbonylamino-propyl]-
6,7,9,10,13,14,15,16,18,19,21,22-dodecahydro-12H-
5,8,11,17,20,23-hexaoxa-14-aza-benzocycloheneicosene-
2,3-dicarboxylic acid dimethyl ester (3c)
3-(tert-Butyl)oxycarbonylamino-propyl-amine (435 mg) was
reacted according to the general procedure to yield product 3c
(910 mg, 58%) as a clear, yellow oil (Rf¼0.08). 1H NMR (300 MHz,
CDCl3):
d
¼1.33 (s, 9H, s), 1.57 (m, 2H, n), 2.51 (m, 2H, m), 2.65 (br s,
4H, l), 3.12 (m, 2H, o), 3.55–3.57 (m, 4H, k), 3.61–3.65 (m, 4H, i),
3.70–3.76 (m, 4H, h), 3.81 (s, 6H, a), 3.84–3.90 (m, 4H, g), 4.12–4.19
(m, 4H, f), 5.43 (br s, 1H, p), 7.15 (s, 2H, d); 13C NMR (75 MHz,
CDCl3):
d
¼26.6 (ꢁ, 1C, o), 28.5 (þ, 3C, s), 38.3 (ꢁ, 1C, n), 52.5 (þ, 2C,
a), 53.2 (ꢁ, 1C, m), 53.9 (ꢁ, 2C, l), 68.9 (ꢁ, 2C, k), 69.4 (ꢁ, 4C, 6, g),
70.3 (ꢁ, 2C, i), 70.7 (ꢁ, 4C, h), 78.8 (Cquat,1C, r),114.0 (þ, 2C, d),125.7
(Cquat, 2C, c), 150.4 (Cquat, 2C, e), 156.3 (Cquat, 1C, q), 167.7 (Cquat, 2C,
b); IR (KBr):
n
(cmꢁ1)¼3393 (br m), 2939 (m), 2874 (m), 1708 (m),
1593 (m), 1517 (m), 1435 (m), 1352 (m), 1286 (m), 1250 (m),
1186 (m), 1126 (m), 1050 (m), 978 (m), 940 (m), 785 (m); UV
(MeOH):
l
(
3
)¼267 (7700), 224 (24,700); MS (ESI-MS, CH2Cl2/
MeOHþ10 mmol NH4OAc): e/z (%)¼629.6 (100, MHþ); C30H48N2O12
:
calcd C 57.3, H 7.7, N 4.5, found: C 57.1, H 7.5, H 4.2.
Table 4
Affinity constants and emission enhancement of compounds 7 in the presence of
n-butyl ammonium chloride and lysyllysine methyl ester in protic solvents
3.3. 14-[4-(tert-Butyl)oxycarbonylamino-butyl]-
6,7,9,10,13,14,15,16,18,19,21,22-dodecahydro-12H-
5,8,11,17,20,23-hexaoxa-14-aza-benzocycloheneicosene-
2,3-dicarboxylic acid dimethyl ester (3d)
Solvent
Compound Guest
Phthalic ester Phthalimide
log K IN/I0 log K IN/I0
Methanol
Methanol
7a
7a
7a
Butyl ammonium 2.2ꢃ0.4 1.2 2.1ꢃ0.2 4.5
chloride
4-(tert-Butyl)oxycarbonylamino-butyl-amine (470 mg) was
Lysyllysine methyl 4.6ꢃ0.5 2.6 4.4ꢃ0.1 6.8
used as reactant in the procedure. A clear, yellow oil (847 mg, 53%)
ester hydrochloride
is obtained (Rf¼0.05). 1H NMR (300 MHz, CDCl3):
¼1.40 (s, 9H, t),
d
H2O, HEPES
(50 mM,
pH 7.5, 2% MeOH)
Methanol
Lysyllysine methyl 2.6ꢃ0.3 1.4
d
d
ester hydrochloride
1.44 (br s, 4H, o, p), 2.50 (m, 2H, m), 2.74 (m, 4H, l), 3.06 (m, 2H, n),
3.56–3.58 (m, 4H, k), 3.61–3.63 (m, 4H, i), 3.72–3.74 (m, 4H, h), 3.84
(s, 6H, a), 3.88–3.89 (m, 4H, g), 4.18–4.19 (m, 4H, f), 4.90 (br s, 1H, q),
7b
7b
7b
Butyl ammonium 2.0ꢃ0.3 1.3
1.9ꢃ0.2 4.6
chloride
7.17 (s, 2H, d); 13C NMR (75 MHz, CDCl3):
d
¼24.1 (ꢁ, 1C, o), 27.7 (ꢁ,
Methanol
Lysyllysine methyl 4.2ꢃ0.3 2.0 4.3ꢃ0.1 6.8
1C, p), 28.4 (þ, 3C, t), 40.3 (ꢁ, 1C, n), 52.5 (þ, 2C, a), 53.8 (ꢁ, 1C, m),
55.0 (ꢁ, 2C, l), 69.3 (ꢁ, 2C, k), 69.5 (ꢁ, 2C, f), 69.5 (ꢁ, 2C, g), 70.6 (ꢁ,
ester hydrochloride
H2O, HEPES
(50 mM,
pH 7.5, 2% MeOH)
Lysyllysine methyl 2.8ꢃ0.3 1.4
d
d
ester hydrochloride
4C, i), 71.1 (ꢁ, 4C, h), 78.8 (Cquat, 1C, s), 113.7 (þ, 2C, d), 125.4 (Cquat
,
2C, c), 150.5 (Cquat, 2C, e), 156.0 (Cquat, 1C, r), 167.7 (Cquat, 2C, b); IR

694
A. Spa¨th, B. Ko¨nig / Tetrahedron 65 (2009) 690–695
(KBr):
n
(cmꢁ1)¼3375 (br m), 2936 (m), 2870 (m), 1715 (m), 1589
l0), 3.55 (t, 4H, J¼5.30 Hz, k0), 3.59–3.64 (m, 8H, i, k), 3.65–3.75 (m,
10H, h, n, i0), 3.78 (t, 4H, J¼4.80 Hz, h0), 3.86 (s, 6H, a), 3.90 (t, 4H,
J¼4.56 Hz, g), 3.95 (t, 4H, J¼4.56 Hz, g0), 4.19 (t, 4H, J¼4.56 Hz, f),
4.23 (t, 4H, J¼4.56 Hz, f0), 7.17 (s, 4H, d), 7.21–7.28 (m, 5H, o0, p0, q0),
(m), 1519 (m), 1436 (m), 1353 (m), 1284 (m), 1185 (m), 1126 (m),
1053 (m), 979 (m), 945 (m), 785 (m); UV (MeOH):
224 (25,200); MS (ESI-MS, CH2Cl2/MeOHþ10 mmol NH4OAc): e/z
(%)¼643.6 (100, MHþ); C31H50N2O12: calcd C 57.9, H 7.8, N 4.4,
found: C 57.7, H 7.6, H 4.2.
l
(3
)¼267 (8000),
7.33 (br s, 2H, d0); 13C NMR (150 MHz, CDCl3):
d¼36.1 (ꢁ, 1C, n), 52.5
(þ, 2C, a), 52.9 (ꢁ, 1C, m), 53.8 (ꢁ, 2C, l0), 54.3 (ꢁ, 2C, l), 59.8 (ꢁ, 1C,
m0), 69.3 (ꢁ, 2C, f), 69.4 (ꢁ, 2C, g0), 69.5 (ꢁ, 2C, g), 69.6 (ꢁ, 2C, f0),
70.2 (ꢁ, 2C, k), 70.7 (ꢁ, 6C, i, i0, k0), 71.1 (ꢁ, 2C, h), 71.2 (ꢁ, 2C, h0),
107.0 (þ, 2C, d0), 113.7 (þ, 2C, d), 125.3 (Cquat, 2C, c0), 125.6 (Cquat, 2C,
c), 126.9 (Cquat, 1C, n0), 128.2 (þ, 3C, p0, q0), 128.9 (þ, 2C, o0), 150.5
(Cquat, 2C, e), 153.4 (Cquat, 2C, e0), 167.8 (Cquat, 2C, b0), 168.4 (Cquat, 2C,
3.4. 14-Propargyl-6,7,9,10,13,14,15,16,18,19,21,22-dodecahydro-
12H-5,8,11,17,20,23-hexaoxa-14-aza-benzocycloheneicosene-
2,3-dicarboxylic acid dimethyl ester (3g)
Propargylamine (140 mg) was allowed to react according to the
general procedure. The reaction was performed under nitrogen
atmosphere and protection from light. The product 3g was
obtained as a clear, yellow oil (910 mg, 71%, Rf¼0.17). 1H NMR
b); IR (NaCl):
n
(cmꢁ1)¼3400 (br w), 2930 (m), 2880 (m), 1707 (s),
1599 (m), 1510 (m), 1436 (m), 1393 (m), 1352 (m), 1291 (s), 1196 (m),
1129 (s), 1056 (m), 980 (w), 945 (w); MS (ESI-MS, CH2Cl2/
MeOHþ10 mmol NH4OAc): e/z (%)¼506.8 (100, (Mþ2Hþ)2þ),1012.6
(300 MHz, CDCl3):
d
¼2.09 (m, 1H, o), 2.75 (m, 4H, l), 3.45 (m, 2H,
(8, MHþ); UV (MeOH):
l
(3
)¼340 (1200), 248 (43,100), 227 (37,400);
m), 3.55–3.58 (m, 4H, k), 3.61–3.65 (m, 4H, i), 3.68–3.74 (m, 4H, h),
3.80 (s, 6H, a), 3.84–3.88 (m, 4H, g), 4.12–4.17 (m, 4H, f), 7.12 (s, 2H,
HRMS (PI-LSIMS, MeOH/glycerin): calcd for C51H68N3O18$Hþ:
1011.4576, found: 1011.4558.
d); 13C NMR (75 MHz, CDCl3):
d¼31.1 (þ, 1C, o), 43.3 (ꢁ, 1C, m), 52.5
(þ, 2C, a), 53.3 (ꢁ, 1C, l), 69.4 (ꢁ, 2C, k), 69.5 (ꢁ, 2C, f), 70.6 (ꢁ, 4C, 9,
3.7. Compound 7a
g), 71.2 (ꢁ, 4C, h), 73.0 (Cquat, 1C, n), 113.5 (þ, 2C, d), 125.3 (Cquat, 2C,
c), 150.5 (Cquat, 2C, e), 167.8 (Cquat, 2C, b); IR (KBr): (cmꢁ1)¼3310
A solution of 5a (132 mg, 0.24 mmol) in chloroform (10 mL)
containing N-hydroxybenzotriazol (HOBt) (55 mg, 0.40 mmol) and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC) (63 mg, 0.40 mmol) was stirred under nitrogen atmosphere
for 30 min at 0 ꢂC. A mixture of 6c (215 mg, 0.2 mmol) and N,N-
diisopropylethylamine (DIPEA) (156 mg, 1.20 mmol) in chloroform
(10 mL) was added dropwise under cooling. The mixture was
stirred 2 h under nitrogen at ambient temperature and refluxed
over night. After cooling to room temperature the solution was
washed with brine (3ꢀ10 mL) and dried over MgSO4. The solvent
was removed under reduced pressure and the crude product was
purified by repeated column chromatography (chloroform/meth-
anol, 6:1; 8:1/5:1, 6:1/5:1) yielding a yellow glass (106 mg,
35%). [Rf (CHCl3/MeOH, 6:1)¼0.4]. 1H NMR (600 MHz, CDCl3):
n
(s), 2976 (m), 2880 (m), 1721 (s), 1599 (m), 1516 (m), 1438 (m), 1348
(m), 1282 (s), 1194 (m), 1123 (m), 1053 (m), 981 (m), 943 (m); UV
(MeOH):
l
(3
)¼267 (7400), 224 (26,800); MS (ESI-MS, CH2Cl2/
MeOHþ10 mmol NH4OAc): e/z (%)¼510.3 (100, MHþ); C25H35NO10
:
calcd C 58.9, H 6.9, N 2.8, found: C 58.6, H 6.6, N 2.6.
3.5. 14-[2-Azido-ethyl]-6,7,9,10,13,14,15,16,18,19,21,22-
dodecahydro-12H-5,8,11,17,20,23-hexaoxa-14-aza-
benzocycloheneicosene-2,3-dicarboxylic acid dimethyl
ester (3h)
2-Azido-ethyl-amine hydrobromide (250 mg) was added as
amine component, nitrogen atmosphere was used. Column chro-
matography was performed with chloroform and methanol in
a 10:1 ratio. A clear, yellow glass (934 mg, 69%) was obtained
d
¼1.41 (s, 9H, r00), 2.68–2.93 (m, 14H, l, l0, l00, m00), 3.25 (m, 2H, n00),
3.48–3.80 (m, 40H, k, m, n, o, m0, k00), 3.82–3.96 (m, 14H, g, g0, n0,
g00), 3.85 (s, 6H, a), 4.12–4.28 (m, 12H, f, f0, f00), 5.64 (br s, 1H), 7.15
(s, 2H, d), 7.19 (m, 2H, d0), 7.24 (br s, 2H, d00); 13C NMR (150 MHz,
(Rf¼0.42). 1H NMR (300 MHz, CDCl3):
¼2.76 (t, 2H, J¼6.8 Hz, m),
d
2.80 (m, 4H, l), 3.26 (t, 2H, J¼6.8 Hz, n), 3.59 (t, 4H, J¼5.6 Hz, k),
3.66–3.69 (m, 4H, i), 3.72–3.74 (m, 4H, h), 3.86 (s, 6H, a), 3.91 (t, 4H,
J¼5.6 Hz, g), 4.20 (t, 4H, J¼5.6 Hz, 6), 7.19 (s, 2H, d); 13C NMR
CDCl3):
d
¼28.5 (þ, 3C, r00), 35.9 (ꢁ, 2C, n, n0), 52.5 (þ, 2C, a), 52.8
(ꢁ, 1C, m), 53.0 (ꢁ, 1C, m00), 54.1 (ꢁ, 1C, m0), 54.2 (ꢁ, 2C, l0), 54.4 (ꢁ,
2C, l), 54.6 (ꢁ, 2C, l00), 69.1–69.5 (ꢁ, 12C, f, g, f0, g0, f00, g00), 70.0 (ꢁ,
2C, k), 70.6–70.8 (ꢁ, 10C, i, i0, k0, i00, k00), 71.0–71.2 (ꢁ, 6C, h, h0, h00),
80.0 (Cquat, 1C, q00), 106.7 (þ, 4C, d0, d00), 113.7 (þ, 2C, d), 125.3
(75 MHz, CDCl3):
d¼49.4 (ꢁ, 1C, n), 52.6 (þ, 2C, a), 54.5 (ꢁ, 2C, l),
54.7 (ꢁ,1C, m), 69.3 (ꢁ, 2C, k), 69.5 (ꢁ, 2C, f), 70.0 (ꢁ, 2C, g), 70.7 (ꢁ,
4C, i), 71.2 (ꢁ, 4C, h), 113.6 (þ, 2C, d), 125.3 (Cquat, 2C, c), 150.5 (Cquat
,
2C, e), 167.8 (Cquat, 2C, b); IR (KBr):
n
(cmꢁ1)¼2971 (m), 2876 (m),
(Cquat, 2C, c), 125.4 (Cquat, 2C, c0), 125.5 (Cquat, 2C, c00), 150.5 (Cquat
,
1719 (s), 1596 (m), 1521 (m), 1438 (m), 1348 (m), 1282 (s), 1195 (m),
1123 (m), 1049 (m), 983 (m), 942 (m); UV (MeOH):
(7800), 224 (27,300); MS (ESI-MS, CH2Cl2/MeOHþ10 mmol
NH4OAc): e/z (%)¼541.4 (100, MHþ), 513.4 (11, (M-N2)Hþ);
C24H36N4O10: calcd C 53.3, H 6.7, N 10.4, found: C 52.8, H 6.3, N 9.9.
2C, e), 153.2 (Cquat, 2C, e0), 153.3 (Cquat, 2C, e00), 156.2 (Cquat, 1C, p00),
167.7 (Cquat, 4C, b0, b00), 163.1 (Cquat, 2C, b); IR (KBr): (cmꢁ1)¼3458
(br m), 3018 (m), 2952 (m), 2881 (w), 1677 (s), 1599 (m), 1501 (m),
1438 (s), 1391 (s), 1299 (s), 1178 (s), 1116 (s), 1052 (m), 946 (m), 799
(m), 719 (m), 659 (m); MS (ESI-MS, CH2Cl2/MeOHþ10 mmol
NH4OAc): e/z (%)¼487.3 (63, (Mꢁt-Buþ3Hþ)3þ), 506.0 (80,
(Mþ3Hþ)3þ), 758.6 (100, (Mþ2Hþ)2þ), 1516.1 (3, MHþ)þ; UV
l
(
3)¼267
3.6. Compound 6b
(MeOH):
l
(
3)¼247 (59,100), 224 (42,600).
A solution of 4 (144 mg, 0.25 mmol) in chloroform (10 mL)
containing N-hydroxybenzotriazol (HOBt) (68 mg, 0.5 mmol) and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC) (79 mg, 0.5 mmol) was stirred for 15 min at 0 ꢂC. A mixture of
5b (141 mg, 0.25 mmol) and N,N-diisopropylethylamine (DIPEA)
(104 mg, 0.80 mmol) in chloroform (10 mL) was added dropwise
under cooling. The mixture was stirred 3 h at ambient temperature
and then refluxed over night. After cooling, the suspension was
filtered over Celite and the filter cake was washed with chloroform.
The solvent was removed under reduced pressure and the residue
was purified by column chromatography (chloroform/methanol,
6:1) to give a yellow glassy solid (193 mg, 76%, Rf (CHCl3/MeOH,
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft (GRK 760) and
the University of Regensburg for support of this work.
Supplementary data
General experimental procedures, experimental procedures,
and spectral data for the synthesis of compounds 3b, 3e, 3f, 3i, 5b,
6c, 7a, 7b, of the peptide sequences for testing, as well as 1H and 13
C
NMR spectra for all new compounds; comparison of n-butyl am-
monium binding to 6b, 7a and 7b; selected fluorescence titration
6:1)¼0.44). 1H NMR (600 MHz, CDCl3):
¼2.76–2.84 (m, 10H, l, m,
d

A. Spa¨th, B. Ko¨nig / Tetrahedron 65 (2009) 690–695
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