Synthesis and characterization of DMAP-modified NPY Y1 receptor antagonists as acyl-transfer catalysts

Article abstract of DOI:10.1135/cccc2011036

Starting from the working hypothesis that specific chemical labelling may be an attractive approach to detect and study G protein-coupled receptors (GPCRs) we synthesized catalytically active antagonists of the neuropeptide Y₁ receptor (Y₁R). An argininamide-type Y₁R antagonist scaffold was combined with a DMAP moiety via spacers of different length and chemical nature. These hybrid compounds have Y₁R affinities in the two-digit nanomolar range and are capable of catalysing acyl-transfer reaction to surrogates of bionucleophiles, as demonstrated in the absence of cells by using esters of fluorescent dyes as substrates in buffer. By contrast, selective staining of Y₁Rs on living MCF-7 cells was not achieved due to significant non-catalysed (Y₁R ligand independent) reaction with biomolecules and the limited density of Y₁R on the cell surface. Although this may also depend on insufficient selectivity of the staining reagents, the results of this study suggest that the general applicability of catalytic staining to GPCRs has to be reconsidered, as this approach is hampered by a very low portion of receptor of interest compared to the total amount of membrane proteins.

Full text of DOI:10.1135/cccc2011036

Towards Catalytic Staining
763
SYNTHESIS AND CHARACTERIZATION OF DMAP-MODIFIED NPY Y1
RECEPTOR ANTAGONISTS AS ACYL-TRANSFER CATALYSTS
b2
Stefan WEISSa1, Günther BERNHARDTb1, Armin BUSCHAUER and
a2
Burkhard KÖNIG
*
a
Institut für Organische Chemie, Universität Regensburg, D-93040 Regensburg, Germany;
1
2
e-mail: stefan.weiss@chemie.uni-regensburg.de, burkhard.koenig@chemie.uni-regensburg.de
Institut für Pharmazie, Universität Regensburg, D-93040 Regensburg, Germany;
b
e-mail: 1 guenther.bernhardt@chemie.uni-regensburg.de, 2 armin.buschauer@chemie.uni-regensburg.de
Received February 4, 2011
Accepted March 29, 2011
Published online May 31, 2011
Dedicated to Professor Pavel Kočovský on the occasion of his 60th birthday.
Starting from the working hypothesis that specific chemical labelling may be an attractive
approach to detect and study G protein-coupled receptors (GPCRs) we synthesized catalyti-
cally active antagonists of the neuropeptide Y1 receptor (Y1R). An argininamide-type Y1R an-
tagonist scaffold was combined with a DMAP moiety via spacers of different length and
chemical nature. These hybrid compounds have Y1R affinities in the two-digit nanomolar
range and are capable of catalysing acyl-transfer reaction to surrogates of bionucleophiles, as
demonstrated in the absence of cells by using esters of fluorescent dyes as substrates in
buffer. By contrast, selective staining of Y1Rs on living MCF-7 cells was not achieved due to
significant non-catalysed (Y1R ligand independent) reaction with biomolecules and the lim-
ited density of Y1R on the cell surface. Although this may also depend on insufficient selec-
tivity of the staining reagents, the results of this study suggest that the general applicability
of catalytic staining to GPCRs has to be reconsidered, as this approach is hampered by a very
low portion of receptor of interest compared to the total amount of membrane proteins.
Keywords: Medicinal chemistry; Acylation; Molecular recognition; Neuropeptide Y; Antago-
nist.
G protein-coupled receptors (GPCR) represent one of the major classes of
biological targets in drug research1. Therefore, there is a need not only for
selective agonists and antagonists of special GPCRs but also for novel strate-
gies to detect and study such receptors. Apart from eliciting intracellular re-
sponses, stimulation of a GPCR with an agonist results in desensitization.
After phosphorylation of serine and threonine residues, arrestin binding,
internalisation and down-regulation of receptors may occur2. Thus, sus-
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 763–780
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011036

764
Weiss, Bernhardt, Buschauer, König:
tained drug-induced receptor activation can result in a significant decrease
or complete loss of the cellular response. Fluorescent antibodies against
extracellular epitopes of the receptor protein were applied to visualize
internalisation3. However, appropriate antibodies are not always available,
and these macromolecules can interfere with ligand binding and receptor
functionality3. Expression of fusion proteins comprising the receptor of in-
terest and, e.g., the yellow fluorescent protein may alter structure and func-
tion of the protein significantly4. The introduction of specific tags that are
recognized with high specificity by functionalized dyes leading to non-
covalent labelling is another successfully applied strategy, which again re-
quires protein modification5–7
.
A chemical reaction in close proximity to the active site of a receptor
leading to labelling of the protein with a fluorescent dye would be a conve-
nient approach, provided that such a reaction takes places with high selec-
tivity and efficiency. Recently, Hamachi et al. reported the chemical label-
ling of lectins using a catalytically active carbohydrate ligand8. In an at-
tempt to apply such an approach to membrane receptors, we selected
neuropeptide Y (NPY) receptors as a model system of a class A GPCR. Four
subtypes of NPY receptors (Y1, Y2, Y4 and Y5) are functionally expressed in
humans. Here we report the synthesis of antagonists for the NPY Y1 recep-
tor that catalyse acylation reactions of nucleophiles in buffers.
4-Dimethylaminopyridine (DMAP) is a well known nucleophilic acyl-
transfer catalyst that was successfully applied for target-specific acylations8,9
.
In our investigations, DMAP was connected to the potent argininamide-
type Y1R antagonist BIBP 3226 10 (Table I, compound 1) to obtain catalyti-
FIG. 1
Illustration of the working hypothesis aiming at a catalytic staining of the Y1R. The Y1R struc-
ture is simplified in this scheme; it is a heptahelical GPCR with a transmembrane binding
pocket for nonpeptidic antagonists such as BIBP 3226. In a, the DMAP moiety of a catalytic an-
tagonist activates a fluorescent active ester. A nucleophilic amino acid (e.g. lysine) of the re-
ceptor is acylated in step b
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Towards Catalytic Staining
765
cally active Y1R ligands. The BIBP 3226 skeleton tolerates acylation at the
terminal guanidine nitrogen (NG) with moderate decrease or even gain of
affinity11–14. Catalytic Y1R specific staining should be achieved by addition
of a fluorescent active ester (Fig. 1)8.
TABLE I
Structures and Y1R binding data of compounds 1a–1e. Ki values were determined from the
displacement of [3H]-UR-MK114 16 (1.5 nM) using Y1R expressing SK-N-MC cells; Ki values
represent mean values SEM from two or three independent experiments
No.
R
Ki, nM
1
H
1.3 0.2
1a
1b
1c
1d
1e
53 13
96 20
90
6
78 0.4
400 19
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Weiss, Bernhardt, Buschauer, König:
RESULTS AND DISCUSSION
Synthesis and Binding Affinity of Putative Catalytically Active
Y1R Antagonists
Previously, we demonstrated that NG-acylation of BIBP 3226 (1) is tolerated
in terms of Y1R affinity12. In addition, the catalytic activity of N-substituted
DMAP derivatives is comparable to that of the parent compound15. There-
fore, hybrid compounds (Table I) combining Y1R antagonism with catalytic
activity were constructed by connecting the guanidine group in 1 with
DMAP by linkers of different length and chemical nature. Synthesis started
from 4-aminopyridine 2 which was converted to the acid 5 in two steps
(Scheme 1). Acid 5 was converted to a guanidinylation reagent (7) or cou-
SCHEME 1
Synthesis of the precursor 5 of the catalytic substructure: methyl acrylate, reflux (a); HCl, 76%
(b); NaOH, MeOH (c); HCl, 30% (d)
pled to amino-functionalized acylated argininamides via different spacers
as depicted in Scheme 2. Compound 1a was synthesized from N-Boc-
S-methylisothiourea 6 via acylation affording 7, followed by guanidinyl-
ation of the ornithine derivative 8. The synthesis was performed in analogy
to a previously described procedure13,14. Compounds 1b and 1c were ob-
tained from the precursor amines 9 and 10 by coupling with the acid 5 in
moderate yield. The compound 1d was also obtained from precursor amine
9, which was coupled in a one pot reaction with the dicarboxylic acid 13
and amine 12. Amine 12 was synthesized from acid 5 and the mono-Boc
protected propane-1,3-diamine 11. The conformationally constrained
ligand 1e was synthesized starting from 1,4-phenylenediamine, which was
converted to the diazide 15 and subjected to a copper catalysed “click-
reaction” yielding amine 16b. Coupling to N-Boc-S-methylisothiourea and
guanidinylation of compound 8 resulted in azide 18. The latter was allowed
to react with the propargyl derivative 20 to give the conformationally con-
strained Y1R antagonist 1e. The copper(I) ligand 21 was used to form the
active catalyst for the reaction.
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Towards Catalytic Staining
767
SCHEME 2
Synthesis of Y1R antagonists 1a–1e: EDC, HOBt, DIPEA, DCM, 75% (a); DMF, HgCl2, NEt3,
36% (b); TFA/DCM 1:1, quantitative yield (c); DMF, DIPEA, TBTU, 67% (1b), 47% (1c) (d);
MeCN, DIPEA, DCC (e); MeOH/HCl 3:1, 36% (f); DMF, DIPEA, TBTU (g); MeCN, TFA, 16% (h);
NaNO2, H2SO4, NaN3, 66% (i); Boc-propargylamine, CHCl3/MeOH/H2O, ascorbate, copper sul-
fate pentahydrate, 79% (j); DCM/TFA, quantitative (k); triphosgene, DCM, DIPEA;
N-Boc-S-methyl-isothiourea, DCM, 46% (l); Compound 8, DMF, NEt3, HgCl2, 57% (m);
DCM/TFA, quantitative (n); THF, n-BuLi, propargylbromide, 10% (o); DMSO, MeOH, H2O,
ascorbate, copper sulfate pentahydrate, 51% (p). Compound 21 is added as a ligand for the
copper-catalysed cycloaddition reaction
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 763–780

768
Weiss, Bernhardt, Buschauer, König:
Synthesis and Hydrolytic Stability of Active Esters
For the catalytic staining reaction of the receptors we prepared active esters
that are easily activated with DMAP. Carboxyfluorescein and cyanine dyes
were modified for their application as acyl-donors (Fig. 2). Compound 23
was prepared according to the procedure of Hamachi and coworkers8. The
phenyl thioester 26 and the p-nitrophenyl ester 27 were synthesized from
the commercially available cyanine dye 24 in one coupling step.
The hydrolysis kinetics of the active esters 23, 25 and 27 and the ability
of the Y1R ligands to catalyse acyl-transfer to simple nucleophiles was in-
vestigated in buffered aqueous solution at pH 7.4. For monitoring of the
hydrolysis kinetics the dyes were incubated at 50 µM for 3 h in phosphate
buffer (30 mM) at pH 7.4. In a second series they were incubated with addi-
tional 50 µM of DMAP. Samples were taken RF 30, 90 and 150 min. The
amount of decomposition was determined with HPLC (Fig. 3).
The acceleration of the hydrolysis in the presence of DMAP was most sig-
nificant for phenyl thioester 23 (about 400% RF 150 min) and only moder-
ate for p-nitrophenyl ester 27 (60%) and succinimidyl ester 25 (48%).
Although hydrolysis rates are not directly comparable with acyl-transfer re-
action rates to other nucleophiles, the results indicate that the thioester is
more suitable than the p-nitrophenol and the NHS ester. The active ester re-
activity correlates with the acidity of the corresponding acids: pKa(NHS):
6.0 17, pKa(p-nitrophenol): 7.4 17, and pKa(thiophenol): 8.3 18
.
Acyl Donors:
Carboxyfluorescein scaffold
Cyanine dye S0436 scaffold
22 R = OH
24 R = OH
23 R = phenylsulfanyl
25 R = succinimidyloxy
26 R = phenylsulfanyl
27 R = p-nitrophenyloxy
FIG. 2
Structures of the carboxyfluorescein and cyanine dyes used as carboxylic acid derivatives for
catalytic staining
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Towards Catalytic Staining
769
Acyl-Transfer Studies
Intermolecular interactions at the cell surface are rather complex. Therefore
we studied first the catalysed acyl-transfer to surrogates of bionucleophiles
in buffered solution to optimize the reaction conditions. Incubation of cat-
alyst 1a and active ester 25 in aqueous solution and subsequent HPLC anal-
ysis showed the complete decomposition of 1a to compound 30 RF an
incubation time of 3 h. As described in literature certain acyl-guanidines are
labile against intra-molecular cleavage14,19. We assume the formation of
4.0
A
3.0
2.0
1.0
0.0
0
38
75
t, min
113
150
50.0
B
37.5
25.0
12.5
0.0
0
38
75
t, min
113
150
FIG. 3
Diagram A: Hydrolysis of phenyl thioester 23 (red) and p-nitrophenyl ester 27 (blue), both
50 µM in presence (solid line) and absence (dashed line) of 50 µM DMAP. Diagram B: Hydroly-
sis graph of succinimidyl ester 25 (50 µM) in presence (solid line) and absence (dashed line) of
DMAP
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770
Weiss, Bernhardt, Buschauer, König:
diacylguanidine 29 (Scheme 3) and fast hydrolysis of this labile intermedi-
ate20. In contrast, compound 1b was not degraded during incubation with
the active ester. The carbamoylguanidine substructure is more resistant
against cleavage, because of the electron donating effect of the amide
N-atom. After an incubation period of 3 h, compounds 1b and 32 were de-
tected by LC-MS. Thus acyl-guanidine 1a was not applicable for the stain-
ing reaction, but the more stable carbamoylguanidines 1b–1e are promising
compounds. However, the fast intramolecular self-acylation of these com-
pounds competes with the acyl-transfer to other nucleophiles and increases
the rate of hydrolysis of the active ester. The catalytic Y1R antagonist 1e
was designed to circumvent this problem. The conformationally con-
strained spacer connecting the DMAP moiety and the BIBP 3226 scaffold
prevents intramolecular self-acylation.
Catalytic acyl-transfer to small nucleophiles was investigated for the Y1R
antagonists 1b–1e. 2-(Dimethylamino)ethylamine (DMAEA), butanol and
BIBP 3226 (BIBP) were used as mimics of nucleophilic residues in proteins
(DMAEA for lysine, butanol for serine and threonine, BIBP 3226 for
arginine and tyrosine). As an example of an acyl-donor, the succinimidyl
ester 25, was incubated with catalyst and nucleophile for 30 min. The
acylation products were detected by LC-MS coupling. The acyl-transfer ex-
periments were carried out with the succinimidyl ester 25, because the
SCHEME 3
Acylation of the Y1R antagonists 1a and 1b with succinimidyl ester 25
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 763–780

Towards Catalytic Staining
771
thioesters were not suitable for the subsequent receptor staining investiga-
tions on cells in section Catalytic Staining of MCF-7 Cells.
The rate of hydrolysis of active ester 25 (cf. entries 1–15 in Table II) was
accelerated when catalysed (entries 2–6) compared the uncatalysed reaction
(entry 1). Antagonists 1b–1e were even more active than DMAP and con-
sumed succinimidyl ester 25 completely (entries 3–5) or almost completely
(entry 6). Acyl-transfer to BIBP 3226 was increased by adding the catalysts
(entries 8–11) compared to the uncatalysed reaction (entry 7). The sterically
hindered catalyst 1e still forms an appreciable amount of the acylated cata-
TABLE II
Catalysed and non-catalysed acyl-transfer of succinimidyl ester 25 to small nucleophiles
(product yields were not quantified, the peak heights show only detected products and reac-
tion tendencies)
% rel. peak height after 30 min incubation timea
Active
Entry
Catalyst Nucleophile
ester
free acid
(hydrolysis
product)
acylated nucleophile-dye
catalyst (product)
active
ester
(32)
structure 33
1
2
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
–
water
79
92
78
83
83
75
57
55
47
45
42
58
74
77
82
21
8
–
–
DMAP
1b
1c
1d
1e
–
water
–
–
3
water
0
22
17
17
21
–
–
4
water
0
–
5
water
0
–
6
water
4
–
7
BIBP
19
0
24
34
40
40
50
29
13
–
8
1b
1c
1d
1e
–
BIBP
11
13
15
8
9
BIBP
0
10
11
12
13
14
15
BIBP
0
BIBP
0
DMAEA
DMAEA
n-butanol
n-butanol
13
0
–
1e
–
13
–
23
4
1e
14
–
a
Relative fraction of the compounds in percent (the sum of all four peak heights is 100).
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 763–780

772
Weiss, Bernhardt, Buschauer, König:
lyst (entries 6, 11, 13, 15). The primary alcohol n-butanol was not attacked
by the active ester (entry 14, 15) due to its weakly nucleophilic properties.
The catalysts 1b–1e are promising prototypical ligands for the catalytic
staining of GPCRs despite BIBP acylation in the absence of the biological
target, as the BIBP 3226 scaffold binds tightly to the Y1R binding pocket,
which should prevent this process.
Catalytic Staining of MCF-7 Cells
In principle, the active esters 23, 25–27 can react with cellular nucleophiles
in the absence of a DMAP moiety. Therefore, we first determined the extent
of non-catalysed staining of MCF-7 cells. The cells were treated with 10 µM
of active ester for 15–60 min in PBS (pH 7.4). The thioesters 23 and 26 were
not applicable for the staining of the Y1R of MCF-7 cells under these condi-
tions, because they pass the cell membrane and accumulate inside the cell.
The p-nitrophenyl ester 27 penetrated the membrane to a significantly
lower extent, and the succinimidyl ester 25 proved to be the best suited ac-
tive ester, as intracellular accumulation was not observed.
For the catalytic staining reaction the cells were pre-incubated with the
corresponding Y1R antagonist, washed with buffer and incubated with the
succinimidyl ester 25. The Y1R retained their functional activity as con-
firmed by a FURA-2 functional assay (data not shown). However, compared
to the uncatalysed staining reaction, there was no significant difference
detectable by confocal microscopy (data not shown). Obviously, Y1R-
independent unselective, i.e. uncatalysed, staining prevails due to a very
high abundance of nucleophilic groups other than those of Y1Rs at the cell
membrane.
CONCLUSION
The results of this study suggest that more selective staining reagents are re-
quired for further investigations. Although MCF-7 cells express a rather
high number of Y1Rs (approximately 300,000 sites/cell), the portion of Y1Rs
related to the total amount of membrane proteins is very low (<0.1% of the
total membrane protein). Therefore, a very high ratio of the catalysed
acylation reactions compared to the non-catalysed background reaction
must be achieved to allow for the envisaged selective staining. The cur-
rently available DMAP-modified receptor antagonists do not provide the re-
quired selectivity.
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Towards Catalytic Staining
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EXPERIMENTAL
General Experimental Conditions
Unless otherwise noted, solvents (analytical grade) were purchased from commercial suppli-
ers and used without further purification. Ethyl acetate (EA), petrol ether (PE; 60–70 °C),
methanol and dichloromethane were obtained in technical grade and distilled before appli-
cation. Acetonitrile (MeCN) for HPLC was obtained from Merck (Darmstadt, Germany).
Cyanine dye S0436 (24) and NHS ester S0536 (25, FEW Chemicals, Bitterfeld–Wolfen, Ger-
many), 4-(methylamino)pyridine (Alfa Aesar, Karlsruhe, Germany), methyl acrylate (Merck,
Darmstadt, Germany), 3,6,9-trioxaundecanedioic acid (Fluka, Sigma–Aldrich Chemie GmbH,
Munich, Germany), 1,4-diaminobenzene (Fluka, Sigma–Aldrich Chemie GmbH, Munich,
Germany), butyl lithium (Fluka, Sigma–Aldrich Chemie GmbH, Munich, Germany) and
propargyl bromide (Fluka, Sigma–Aldrich Chemie GmbH, Munich, Germany) were pur-
chased. Preparative HPLC was performed with a system from Knauer (Berlin, Germany) con-
sisting of two K-1800 pumps and a K-2001 detector. A Nucleodur 100-5 C18 (250 × 21 mm,
5 µm; Macherey–Nagel, Germany) and a Eurospher-100 C18 (250 × 32 mm, 5 µm; Knauer,
Germany) served as RP-columns at flow rates of 20 and 38 ml/min, respectively. Mixtures of
MeCN and diluted aqueous TFA (0.1%) were used as mobile phase. 1H NMR spectra were
recorded at 300 MHz on a Bruker Avance 300 spectrometer or at 600 MHz on a Bruker
Avance III 600 with cryogenic probehead (Bruker, Karlsruhe, Germany). 13C NMR spectra
were recorded at 75 MHz on a Bruker Avance 300 spectrometer. All chemical shifts values
are reported in ppm (δ-scale), coupling constants J in Hz. UV/VIS spectra were recorded with
a Varian Cary BIO 50 UV/VIS/NIR spectrophotometer (Varian Inc., CA, USA). Mass spectra:
Finnigan SSQ 710A (EI), Finnigan MAT 95 (CI), Finnigan MAT TSQ 7000 (Thermo
FINNIGAN, USA) (ES/LC-MS). LC-system for LC-MS: Agilent 1100 (Palo Alto, USA). LC-MS
method I (LC-MS-I): Column: Phenomex Luna C18, 3.0 µm, 100 × 2 mm HST (Phenomenex,
Aschaffenburg, Germany); flow: 0.30 ml/min; solvent A (water + 0.1% TFA), solvent B
(MeCN); gradient: 0 min [A/B 95/5], 1 min [A/B 95/5], 11 min [A/B 2/98], 18 min [A/B
2/98], 19 min [A/B 95/5], 24 min [A/B 95/5]. LC-MS method II: Column: Phenomex Luna
C18, 2.5 µm, 50 × 2 mm HST (Phenomenex, Aschaffenburg, Germany); flow: 0.40 ml/min;
solvent A (water + 0.1% TFA), solvent B (MeCN); gradient: 0 min [A/B 95/5], 8 min [A/B
2/98], 11 min [A/B 2/98], 12 min [A/B 95/5], 15 min [A/B 95/5]. Melting points were deter-
mined with a Lambda Photometrics Optimelt MPA100 apparatus (Lambda photometrics,
Harpenden, UK), they are not corrected. Thin layer chromatography (TLC) was performed
on alumina plates coated with silica gel (Merck silica gel 60 F245, thickness 0.2 mm). Col-
umn chromatography (CC) was performed with Merck Geduran SI 60 silica gel as the sta-
tionary phase.
Compounds 4 15, 5 15, 6 13, 8 16, 9 20, 10 20, 11 21, 21 22,23, 22 8, 23 8 and Boc-propargylamine24
were prepared according to literature procedures. The synthesis of literature known com-
pound 15 25 was improved.
tert-Butyl-{3-[methyl(pyridin-4-yl)amino]propanamido}(methylthio)-
methylenecarbamate (7)
Acid 5 (216 mg, 1.00 mmol), DIPEA (258 mg, 2.00 mmol) and HOBt (149 mg, 1.10 mmol)
were dissolved in 10 ml cold DCM and EDC (171 mg, 1.10 mmol) was added. After 15 min,
compound 6 (190 mg, 1.00 mmol) was added. The mixture was stirred overnight. Next day
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 763–780

774
Weiss, Bernhardt, Buschauer, König:
the reaction mixture was concentrated and the crude product was purified with column
chromatography (EA/EtOH 1:1, RF 0.1). Compound 7 (323 mg, 75%) was obtained as a col-
orless oil. 1H NMR (300 MHz, CDCl3): 1.48 s, 9 H; 2.36 s, 3 H; 2.79 t, 2 H, J = 6.87; 3.14 s,
3 H; 3.83 t, 2 H, J = 6.88; 6.65–6.75 m, 2 H; 8.10–8.20 m, 2 H; 9.00–13.00 m, 2 H. 13C NMR
(75 MHz, CDCl3): 14.7, 27.9, 31.7, 38.6, 48.0, 79.9, 106.8, 141.5, 141.8, 143.5, 156.0, 170.8.
C
16H24N4O3S: MS (LC-MS-I): m/z (%) [tr = 8.1 min]: 353 (100, MH+), 705 (5).
(R)-4-((3-((4-(2,2-Diphenylacetamido)-5-(4-hydroxybenzylamino)-5-oxopentyl)guanidino)-
3-oxopropyl)(methyl)amino)pyridinium 2,2,2-Trifluoroacetate (1a)
Compound 7 (123 mg, 0.35 mmol) was dissolved in 4 ml DMF and mixed with compound 8
(170 mg, 0.35 mmol). NEt3 (0.5 ml) was added and then a solution of HgCl2 (95 mg,
0.35 mmol) in 1 ml DMF was dropped into the reaction mixture. The mixture was stirred
overnight. The next day DMF was evaporated under high vacuum and DCM (5 ml) was
added. The insoluble mercury salts were filtered off and the solution was concentrated. The
crude material was purified with column chromatography (EA/EtOH 1:1).
The product was deprotected without further characterisation: a mixture of DCM/TFA
(1:1; 4 ml) was added and the solution was stirred for 2 h. Then it was concentrated and
purified with HPLC. A white solid was obtained (109 mg, 36%), m.p. > 190 °C (decomp.).
1H NMR (300 MHz, CD3CN): 1.40–1.90 m, 4 H; 2.78 t, 2 H, J = 7.05; 3.15 s, 3 H;
3.15–3.30 m, 2 H; 3.85 t, 2 H, J = 7.06; 4.10–4.30 m, 2 H; 4.30–4.40 m, 1 H; 5.02 s, 1 H;
6.60–7.20 m, 4 H; 6.71 d, 2 H, J = 8.54; 7.03 d, 2 H, J = 8.50; 7.20–7.35 m, 10 H; 7.96 bs,
2 H; 9.47 bs, 1 H; 9.95 bs, 1 H; 12.13 bs, 1 H; 13.37 bs, 1 H. C36H43N7O4: MS (LC-MS-I): m/z
(%) [tr = 7.6 min]: 318 (100), 339 (35), 636 (50, MH+).
(R)-4-((4-(2,2-Diphenylacetamido)-1-(4-hydroxyphenyl)-9-imino-3,11,18-trioxo-
2,8,10,12,17-pentaazaicosan-20-yl)(methyl)amino)pyridinium 2,2,2-Trifluoroacetate (1b)
The acid 5 (5.6 mg, 26 µmol) was dissolved in DMF (2 ml) and DIPEA (28 mg, 0.21 mmol)
and TBTU (8.3 mg, 26 µmol) were added. After 10 min, amine 9 (20 mg, 22 µmol) was
added. The mixture was stirred overnight. The next day DMF was removed under high
vacuum and the oily residue was purified with HPLC. A white solid was obtained (14 mg,
67%), m.p.
> 8 H;
190 °C (decomp.). 1H NMR (300 MHz, CD3CN): 1.30–1.80 m,
2.00–2.80 m, 2 H; 3.10 s, 3 H; 3.10–3.30 m, 6 H; 3.80 t, 2 H, J = 6.45; 4.10–4.40 m, 3 H;
5.03 s, 1 H; 6.50–7.90 m, 8 H; 6.67 d, 2 H, J = 8.45, 2 H; 7.00 d, 2 H, J = 8.45; 7.20–7.35 m,
10 H; 7.90–8.05 m, 2 H; 9.00–9.20 bs, 1 H; 11.90–13.00 m, 1 H. C41H51N9O5: MS (LC-MS-II):
m/z (%) [tr = 4.9 min]: 375 (100), 750 (30, MH+).
(R)-4-((4-(2,2-Diphenylacetamido)-1-(4-hydroxyphenyl)-9-imino-3,11,22-trioxo-
15,18-dioxa-2,8,10,12,21-pentaazatetracosan-24-yl)(methyl)amino)pyridinium
2,2,2-Trifluoroacetate (1c)
Acid 5 (3.0 mg, 13.7 µmol) was dissolved in DMF (2 ml) and DIPEA (14.7 mg, 114 µmol)
and TBTU (4.4 mg, 13.7 µmol; dissolved in 0.2 ml MeCN) were added. After 10 min,
amine 10 (10 mg, 11.4 µmol) was added. The solution was stirred overnight. Next day DMF
was removed completely and the oily residue was suspended in some MeCN (1 ml). TFA was
added until pH < 2. MeCN was evaporated and the crude material was purified with HPLC.
A white solid was obtained (5.5 mg, 47%), m.p. > 190 °C (decomp.). 1H NMR (300 MHz,
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775
D2O): 1.20–1.80 m, 4 H; 2.38 t, 2 H, J = 6.59; 2.93 s, 3 H; 3.03 t, 2 H, J = 7.28; 3.13 t, 2 H,
J = 5.41; 3.25 t, 2 H, J = 5.16; 3.34 t, 2 H, J = 5.39; 3.40–3.55 m, 6 H; 3.64 t, 2 H, J = 6.61;
4.00–4.10 m, 1 H; 4.10–4.25 m, 2 H; 5.01 s, 1 H; 6.55–6.85 m, 6 H; 6.92–7.00 d, 2 H, J =
8.53; 7.00–7.10 m, 2 H; 7.10–7.30 m, 8 H; 7.82 d, 2 H, J = 7.29. C43H55N9O7: MS (LC-MS-II):
m/z (%) [tr = 4.98 min]: 405 (100), 810 (10, MH+).
4-((3-(3-Ammoniopropylamino)-3-oxopropyl)(methyl)amino)pyridinium Chloride (12)
Acid 5 (216 mg, 1.00 mmol) was dissolved in MeCN (10 ml) and DIPEA (271 mg, 2.10 mmol)
was added. The mixture was cooled in an icebath and DCC (227 mg, 1.10 mmol) was added.
After 15 min, amine 11 (174 mg, 1.00 mmol) was added. The turbid reaction mixture was
stirred at room temperature for 5 h and then the mixture was heated to 50 °C for 30 min.
After that time, MeCN was evaporated completely and the residue (viscous oil) was
redissolved in DCM. The precipitate (DCU) was filtered off and the crude material was puri-
fied with column chromatography (EA/MeOH 9:1 + 1% NEt3, RF 0.05). The Boc-protected,
purified white solid (134 mg, 40%) was deprotected with a 3:1 mixture of methanol and
concentrated hydrochloric acid (4 ml). After complete deprotection (10 min, room tempera-
ture), the solvent was evaporated, water was added (2–3 ml) and the solution was
lyophilized overnight. A white solid was obtained (110 mg, 36% overall), m.p. > 190 °C
(decomp.). 1H NMR (300 MHz, D2O): 1.60–1.75 m, 2 H; 2.51 t, 2 H, J = 6.61; 2.82 t, 2 H, J =
7.62; 3.00–3.15 m, 5 H; 3.78 t, 2 H, J = 6.61; 6.70–7.00 bs, 2 H; 7.93 d, 2 H, J = 7.48.
C
12H20N4O: MS (LC-MS-II): m/z (%) [tr = 0.31 min]: 119 (5), 139 (65), 160 (100), 237 (65,
MH+).
(R)-4-((4-(2,2-Diphenylacetamido)-1-(4-hydroxyphenyl)-9-imino-
3,11,18,28,34-pentaoxo-20,23,26-trioxa-2,8,10,12,17,29,33-hepta-
azahexatriacontan-36-yl)(methyl)amino)pyridinium 2,2,2-Trifluoroacetate (1d)
Acid 13 (6.6 mg, 30 µmol) was dissolved in DMF (2 ml) and DIPEA (35 mg, 270 µmol) and
TBTU (18.9 mg, 59 µmol) were added. After 10 min, the reaction was started with the addi-
tion of the compounds 9 (25.0 mg, 27 µmol) and 12 (8.3 mg, 27 µmol). The reaction mix-
ture was stirred overnight. Next day DMF was removed completely and the oily residue was
suspended in some MeCN (1 ml). TFA was added until pH < 2. MeCN was evaporated and
the crude mixture was purified with HPLC. A white solid was obtained (5.5 mg, 16%),
m.p. > 190 °C (decomp.). 1H NMR (300 MHz, D2O): 1.25–1.90 m, 10 H; 2.44 t, 2 H, J = 6.48;
2.90–3.20 m, 13 H; 3.50–3.60 m, 8 H; 3.72 t, 2 H, J = 6.48; 3.89 d, 4 H, J = 1.70;
4.05–4.15 m, 1 H; 4.15–4.30 m, 2 H; 5.05 s, 1 H; 6.60–6.90 m, 4 H; 6.95–7.30 m, 12 H;
7.87 d, 2 H, J = 7.52. C52H71N11O10: MS (LC-MS-II): m/z (%) [tr = 4.99 min]: 505 (100), 1010
(5, MH+).
1,4-Diazidobenzene (15)
NaNO2 (1.45 g, 21 mmol) was suspended in 10 ml concentrated sulfuric acid at 10 °C in
small amounts. The mixture was stirred 10 min at 20 °C and then heated to 70 °C until
a clear solution was obtained. The mixture was cooled to 10 °C and 1,4-diaminobenzene
(1.08 g, 10 mmol) dissolved in 10 ml glacial acetic acid was dropped slowly into the mix-
ture. A yellow solid precipitated. The mixture was stirred one hour at 15 °C. After that time,
the mixture was dropped in a 500 ml flask containing 150 ml ice-cold water (with ice).
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A clear, yellow solution was obtained. NaN3 (1.43 g, 22 mmol) dissolved in 10 ml water was
added dropwise at 5 °C under vigorous stirring (caution: HN3 gas can develop). Nitrogen was
formed and a yellow solid precipitated. After 2 h, the product was extracted with EA (2 ×
75 ml). The organic layer was separated and dried over MgSO4. The solvent was concen-
trated and the crude material was recrystallized in PE. Yellow crystalls were obtained (1.06 g,
66%), m.p. 155 °C (decomp.).
tert-Butyl-[(1-(4-azidophenyl)-1H-1,2,3-triazol-4-yl)methyl]carbamate (16a)
1,4-Diazidobenzene (500 mg, 3.13 mmol) was dissolved in 5 ml of CHCl3 and was added to
a solution of Boc-propargylamine (484 mg, 3.13 mmol) in 5 ml of MeOH. Then ascorbic
acid (55 mg, 0.31 mmol, dissolved in 0.5 ml of H2O) and CuSO4·5H2O (40 mg, 0.16 mmol;
dissolved in 0.5 ml of H2O) were added. The mixture was stirred at 60 °C for 1 h. EA (50 ml)
and water (50 ml) were added and the organic layer was separated. The mixture was concen-
trated and a white solid precipitated (double coupled side product). The precipitate was fil-
tered off and the remaining solution was concentrated and purified with column chroma-
tography (PE/EA 2:3, RF 0.5). The product was obtained as a white solid (782 mg, 79%), m.p.
155 °C (160 °C decomp.). 1H NMR (300 MHz, CDCl3): 1.45 s, 9 H; 4.47 d, 2 H, J = 6.03;
5.20 bs, 1 H; 7.10–7.20 m, 2 H; 7.65–7.75 m, 2 H; 7.94 s, 1 H. 13C NMR (75 MHz, CDCl3):
28.4, 36.1, 79.9, 120.2, 122.0, 132.1, 133.8, 140.7, 146.4, 155.9. C14H17N7O2: MS (LC-MS-II):
m/z (%) [tr = 3.57 min]: 216 (100, M*H+), 257 (20), 431 (25). The compound was completely
Boc-deprotected during LC-MS.
(1-(4-Azidophenyl)-1H-1,2,3-triazol-4-yl)methanaminium 2,2,2-Trifluoroacetate (16b)
Compound 16a (287 mg, 0.91 mmol) was deprotected with a 2:1 mixture of DCM and TFA
(6 ml). The mixture was stirred for 1 h and then the solvent was removed. Water was added
(5 ml) and the solution was lyophilized. A white solid was obtained (300 mg, quantitative),
m.p. 157 °C (decomp.). 1H NMR (300 MHz, D2O): 4.36 s, 2 H; 7.14–7.17 m, 2 H;
7.63–7.66 m, 2 H; 8.44 s, 1 H. 13C NMR (75 MHz, D2O): 33.9, 120.0, 122.1, 123.4, 132.6,
140.3, 140.9. C9H9N7: MS (LC-MS-II): m/z (%) [tr = 3.57 min]: 216 (100, MH+), 257 (20), 431
(25). IR (ν, cm–1): 3166, 2757, 2136 (N3), 2095 (N3), 1711, 1618, 1555, 1514.
tert-Butyl-(3-((1-(4-azidophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-
(methylthio)methylenecarbamate (17)
Triphosgene (71 mg, 0.24 mmol) was dissolved in icecold DCM (10 ml) and a solution of
compound 16b (200 mg, 0.61 mmol) and DIPEA (285 mg, 2.21 mmol) was dropped slowly
(20 min) at 0 °C into the triphosgene solution. After complete addition, the reaction was al-
lowed to warm up to ambient temperature and a solution of Boc-S-methyl-isothiourea 6
(116 mg, 0.61 mmol) in DCM (10 ml) was added over a period of 10 min. The mixture was
stirred overnight at ambient temperature. The next day, the reaction mixture was concen-
trated and the crude material was purified with column chromatography (PE/EA 3:1 → 2:3)
[RF 0.3 (PE/EA 3:2)]. A light yellow solid was obtained (122 mg, 46%), m.p. 126 °C (162 °C
decomp.). 1H NMR (300 MHz, CDCl3): 1.48 s, 9 H; 2.29 s, 3 H; 4.58 d, 2 H, J = 6.19; 6.23 t,
1 H, J = 5.49; 7.10–7.20 m, 2 H; 7.75–7.75 m, 2 H; 7.97 s, 1 H; 12.19 bs, 1 H. C17H21N9O3S:
MS (LC-MS-II): m/z (%) [tr = 7.83 min]: 432 (100, MH+), 863 (40).
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777
(R)-1-(1-(4-Azidophenyl)-1H-1,2,3-triazol-4-yl)-10-(4-hydroxybenzylcarbamoyl)-
3,12-dioxo-13,13-diphenyl-2,4,6,11-tetraazatridecan-5-iminium 2,2,2-Trifluoroacetate (18)
Compound 17 (101 mg, 0.23 mmol), compound 8 (114 mg, 0.23 mmol) and NEt3 (230 mg,
2.3 mmol) were dissolved in 3 ml DMF. HgCl2 (64 mg, 0.23 mmol) was dissolved in 0.5 ml
of DMF and added to the mixture. The mixture was stirred overnight. Next day DMF was re-
moved completely and the residue was dissolved in 5 ml DCM. Hg-salts were filtered off and
the crude material was purified with column chromatography (PE/EA 7:3, RF 0.4). The com-
pound was deprotected without further characterization. DCM/TFA was added (1:1 mixture,
4 ml) and the mixture was stirred for 2 h. Then it was concentrated and the crude material
was purified with HPLC. A white solid was obtained (112 mg, 57%), m.p. > 190 °C
(decomp.). 1H NMR (600 MHz, DMSO-d6): 1.35–1.48 m, 2 H; 1.48–1.70 m, 2 H; 3.10–3.25 m,
2 H; 4.05–4.20 m, 2 H; 4.30–4.35 m, 1 H; 4.46 d, 2 H, J = 5.29; 5.12 s, 1 H; 6.67 d, 2 H, J =
8.45; 7.00 d, 2 H, J = 8.45; 7.15–7.30 m, 10 H; 7.30–7.35 m, 2 H; 7.88–7.92 m, 2 H; 8.13 bs,
1 H; 8.36 t, 1 H, J = 5.80; 8.37–8.46 bs, 1 H; 8.47 d, 1 H, J = 8.11; 8.67 s, 1 H; 8.89 bs, 1 H;
9.28 s, 1 H; 9.73 bs, 1 H. 13C NMR (HSQC, HMBC, ROESY) (151 MHz, DMSO-d6): 24.55,
29.42, 34.82, 41.58, 52.23, 55.88, 114.97, 120.47, 121.10, 121.59, 126.56, 128.13, 128.17,
128.38, 128.46, 128.48, 129.11, 133.46, 139.70, 140.24, 140.42, 156.23, 170.93, 170.98.
C
37H38N12O4: MS (LC-MS-II): m/z (%) [tr = 6.01 min]: 715 (100, MH+). UV (MeCN): λ (ε) 272
(20 × 103). IR (ν, cm–1): 3277, 2130 (N3), 2100 (N3), 1686, 1638, 1542, 1512, 1446, 1364.
4-[Methyl(prop-2-ynyl)amino]pyridinium Chloride (20)
N-Methylpyridin-4-amine 19 (200 mg, 1.85 mmol) was dissolved in 5 ml of dry THF. Then a
BuLi solution (1.6 M in hexanes; 1.4 ml, 2.22 mmol) was added at room temperature under
nitrogen. After 10 min, when no more gas was formed, a propargylbromide solution
(80 wt.% in toluene; 330 mg, 2.22 mmol) was added slowly at –5 °C over a period of 1 h.
The mixture was stirred overnight. The next day water was added (5 ml) and then THF was
removed completely under reduced pressure. NaOH (1 M, 20 ml) and EA (20 ml) were added
and the organic layer was collected. Organic phase was acidified with NaHSO4 solution
(20 ml, 5 wt.%) and the aqueous layer was collected. NaOH (1 M, 40 ml) and EA (40 ml)
were added and the organic layer was collected. The organic layer was washed several times
with water until not converted N-methylpyridin-4-amine 19 was removed completely. The
organic layer was dried over MgSO4 and the solvent was removed. A brown oil was ob-
tained. Diluted hydrochloric acid (10%) was added dropwise until pH < 2. A yellow precipi-
tate was formed. It was collected and recrystallized with CHCl3/PE. Light yellow-brown
crystals were obtained (33 mg, 10%), m.p. 193 °C (decomp.). 1H NMR (300 MHz, D2O):
2.63 t, 1 H, J = 2.47; 3.15 s, 3 H; 4.26 d, 2 H, J = 2.47; 6.92 d, 2 H, J = 7.26; 8.00 d, 2 H, J =
7.67. 13C NMR (75 MHz, D2O): 38.0, 41.5, 74.1, 77.0, 107.8, 138.9, 157.3. C9H10N2:
MS (LC-MS-II): m/z (%) [tr = 0.56 min]: 147 (100, MH+), 188 (10), 293 (10).
(R)-4-(((1-(4-(4-(10-(4-Hydroxybenzylcarbamoyl)-5-imino-3,12-dioxo-13,13-diphenyl-
2,4,6,11-tetraazatridecyl)-1H-1,2,3-triazol-1-yl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-
(methyl)amino)pyridinium 2,2,2-Trifluoroacetate (1e)
Compound 18 (10 mg, 12.1 µmol) was dissolved in 0.5 ml of DMSO and alkyne 20 (2.2 mg,
12.1 µmol) was added. The solution was diluted with a of 3:1 mixture of MeOH and H2O
(0.5 ml). Then ascorbic acid (1.1 mg, 6.1 µmol [stock solution 100 mg/ml in 1 M NaOH])
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and CuSO4·5H2O (0.91 mg, 3.6 µmol [stock solution 100 mg/ml in H2O]) were dropped into
the mixture. Copper(I) stabilizing ligand 21 (0.8 mg, 1.21 µmol) was added and the mixture
was heated to 65 °C for 1 h. After that time, TLC control demonstrated complete conver-
sion. TFA was added until pH < 2 and the crude material was purified with HPLC. A white
solid was obtained (6.7 mg, 51%), m.p. > 190 °C (decomp.). 1H NMR (300 MHz, DMSO-d6):
1.35–1.75 m, 4 H; 3.15–3.25 m, 2 H; 3.32 s, 3 H; 4.05–4.25 m, 2 H; 4.25–4.40 m, 1 H;
4.40–4.55 m, 2 H; 5.02 s, 2 H; 5.12 s, 1 H; 6.67 d, 2 H, J = 8.45; 7.00 d, 2 H, J = 8.45;
7.05–7.40 m, 12 H; 8.05–8.20 m, 5 H; 8.25–8.60 m, 6 H; 8.81 s, 1 H; 8.91 bs, 1 H; 8.96 s,
1 H; 9.30 s, 1 H; 9.86 bs, 1 H; 13.44 bs, 1 H. C46H58N14O4: MS (LC-MS-II): m/z (%) [tr
=
5.18 min]: 431 (100), 861 (5, MH+), 975 (2).
Phenyl Thioester 26
Compound 24 (3.00 mg, 4.96 µmol) was mixed with DIPEA (1.92 mg, 14.9 µmol), DMAP
(0.06 mg, 0.49 µmol) and TBTU (1.91 mg, 5.95 µmol). After 10 min, the reaction was started
with the addition of thiophenol (0.65 mg, 5.95 µmol). Next day, DMF was removed com-
pletely and the crude material was purified with HPLC. A blue solid was obtained (2.40 mg,
69%). C41H49N2O4S2: MS (LC-MS-II): m/z (%) [tr = 7.87]: 697 (100, M+), 1395 (15).
p-Nitrophenyl Ester 27
Compound 24 (2.70 mg, 4.46 µmol) was dissolved in in 1 ml of DMF and DIPEA (2.7 mg,
21 µmol) was added. Then bis(4-nitrophenyl) carbonate (3.1 mg, 10.20 µmol) was added.
The mixture was stirred overnight at room temperature. The next day, DMF was removed
completely and the residue was purified with HPLC. A blue solid was obtained (2.90 mg,
91%). C41H48N3O7S: MS (LC-MS-II): m/z (%) [tr = 7.61]: 726 (100, M+).
Radioligand Competition Binding Assay
Radioligand competition experiments at SK-N-MC neuroblastoma cells using the radioligand
[3H]UR-MK114 (1.5 nM) were performed as described elsewhere16
.
Fura-2 Assay on HEL Cells
The Fura assay was performed with HEL cells as previously described using a Perkin–Elmer
LS50 B spectrofluorimeter (Perkin–Elmer, Überlingen, Germany)26
.
Hydrolytic Stability of the Active Esters
The studies were performed in 2 ml Eppendorf reaction vessels using 285 µl phosphate
buffer (30 mM, pH 7.4) per vessel (cup). Stock solutions of the active esters (1 mM) and
DMAP (10 mM) in DMSO were prepared. Then 1.5 µl of the DMAP solution was added to the
corresponding cups. Reaction was started with the addition of 15 µl of the corresponding
active ester solution (vortexed). Active ester concentration was 50 µM. After 30, 90 and
150 min, 75 µl aliquots were taken and put into 75 µl quenching solution [25 vol.% MeCN,
62.5 vol.% H2O, 37.5 vol.% TFAaq (1%)]. 5 µl of internal standard (1 mM in MeCN) was
added to every cup and the samples were analyzed with HPLC. The integrals of active ester
and free acid were determined and the total amount of active ester was calculated (calibra-
tion with different concentrations of the free acids from 0.5 to 25 µM).
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Towards Catalytic Staining
779
Acyl-Transfer Studies
The acyl-transfer studies were performed in 2 ml Eppendorf reaction vessels using 200 µl
phosphate buffer (30 mM, pH 7.4) per vessel. Stock solutions of the catalysts, nucleophiles
and active esters in DMSO were prepared (10 mM). According to the particular experiment
1 µl of catalyst and 1 µl of nucleophile were added to the buffer and the mixture was
vortexed. The acyl-transfer was started with the addition of 1 µl of the active ester
(vortexed). Then it was incubated for 30 min. After that time, the reaction was quenched
with the addition of 10 µl AcOH. 1 µl of a 10 mM solution of an internal standard was
added and the samples were analyzed with LC-MS. The internal standard peak height was
set as 100% and the relative peak intensities were determined.
Cell Culture and Confocal Microscopy
MCF-7 cells were cultivated in EMEM medium (Sigma, Deisenhofen, Germany), containing
5% FCS (Biochrom AG, Berlin, Germany), in 8 well µ-slides (Ibidi GmbH, Munich) at 37 °C
for two days. Estradiol (1 nM) was added to the medium to stimulate Y1R expression27. Be-
fore the experiment the medium was replaced by Leibovitz L15 medium (LM). Cells were in-
cubated with 200 nM catalyst (in LM, 200 µl per well) at room temperature for 15 min. Then
the medium was removed and the cells were washed once with PBS. Cells without catalyst
were also washed with PBS. Thereafter, a 10 µM solution of NHS ester 25 in PBS was added
(150 µl per well), and the cells were incubated in the dark at room temperature for 30 min.
The cells were washed carefully three times with LM to remove non-covalently bound NHS
ester and the free acid 24 from the membranes. To determine non-specific binding 100 nM
pNPY (in LM, 200 µl) were added, and cells were incubated at room temperature. Images
were taken 90 min after pNPY incubation.
Confocal microscopy was performed with a Zeiss Axiovert 200M microscope, equipped
with a LSM 510 laser scanner. The dye S0436 was excited with a 633 nm laser, carboxy-
fluorescein was excitated at 488 nm. The emission of S0436 dyes was detected >650 nm
(long pass filter) and the emission of carboxyfluorescein >505 nm (long pass filter). An im-
mersion objective (Plan-Neofluar 40×/1.3) was used.
Data Processing
Data from radioligand competition experiments were analyzed by 4 parameter sigmoidal fits
(SigmaPlot 9.0, Systat Software). IC50 values from radioligand competition studies were con-
verted to Ki values according to the Cheng–Prusoff equation28 using the respective KD value
of the radioligand.
For the determination of Y1R antagonistic activities in fura-2 assays on HEL cells, three
data points (between 20 and 80% inhibition) served for the calculation of IC50 values after
logit-log transformation. IC50 values were converted to Kb values according to the Cheng–
Prusoff equation using an EC50 value of 1.8 nM for pNPY (mean value from 4 independently
determined concentration-effect curves on HEL cells).
This work was supported by the Graduate Training Program (Graduiertenkolleg) GRK 760,
“Medicinal Chemistry: Molecular Recognition – Ligand-Receptor Interactions”, of the Deutsche
Forschungsgemeinschaft.
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REFERENCES
1. Filmore D.: Mod. Drug Discovery 2004, 7, 24.
2. Tobin A. B., Butcher A. J., Kong K. C.: Trends Pharmacol. Sci. 2008, 29, 413.
3. Bourne H., Horuk R. J. K., Michel H.: Ernst Schering Foundation Symposium Proceedings,
Vol. 2, p. 229. Springer, 2006.
4. Hoffmann C., Gaietta G., Bünemann M., Adams S. R., Oberdorff-Maass S., Behr B.,
Vilardaga J., Tsien R. Y., Ellisman M. H., Lohse M. J.: Nat. Methods 2005, 2, 171.
5. Johnsson K.: Nat. Chem. Biol. 2009, 5, 63.
6. Griffin B. A., Adams S. R., Tsien R. Y.: Science 1998, 281, 269.
7. Estévez J. M., Somerville C.: Biotechniques 2006, 41, 569.
8. Koshi Y., Nakata E., Miyagawa M., Tsukiji S., Ogawa T., Hamachi I.: J. Am. Chem. Soc.
2008, 130, 245.
9. Höfle G., Steglich W., Vorbrüggen H.: Angew. Chem. Int. Ed. 1978, 17, 569.
10. Rudolf K., Eberlein W., Engel W., Wieland H. A., Willim K. D., Entzeroth M.,
Wienen W., Beck-Sickinger A. G., Doods H. N.: Eur. J. Pharmacol. 1994, 271, R11.
11. Hutzler C., Kracht J., Mayer M., Graichen F., Bauer B., Schreiber E., Bollwein S.,
Bernhardt G., Dove S., Fricker G., Buschauer A.: Arch. Pharm. (Weinheim) 2001, 334, 17.
12. Keller M., Teng S., Bernhardt G., Buschauer A.: ChemMedChem 2009, 4, 1733.
13. Weiss S., Keller M., Bernhardt G., Buschauer A., König B.: Bioorg. Med. Chem. 2008, 16,
9858.
14. Weiss S., Keller M., Bernhardt G., Buschauer A., König B.: Bioorg. Med. Chem. 2010, 18,
6292.
15. Delaney E. J., Wood L. E., Klotz I. M.: J. Am. Chem. Soc. 1982, 104, 799.
16. Keller M., Pop N., Hutzler C., Beck-Sickinger A. G., Bernhardt G., Buschauer A.: J. Med.
Chem. 2008, 51, 8168.
17. Cline G. W., Hanna S. B.: J. Org. Chem. 1988, 53, 3583.
18. Kleeberg H.: J. Mol. Struct. 1988, 177, 157.
19. Brennauer A., Keller M., Freund M., Bernhardt G., Buschauer A.: Tetrahedron Lett. 2007,
48, 6996.
20. Keller M.: Ph. D. Thesis. University of Regensburg, Regensburg 2008.
21. Cinelli M. A., Cordero B., Dexheimer T. S., Pommier Y., Cushman M.: Bioorg. Med. Chem.
2009, 17, 7145.
22. Ritter S., König B.: Chem. Commun. 2006, 45, 4694.
23. Chan T. R., Hildegraf R., Sharpless K. B., Fokin V. V.: Org. Lett. 2004, 6, 2853.
24. Qiao C., Jeon H., Sayre L. M.: J. Am. Chem. Soc. 2004, 126, 8038.
25. Herring D. L.: J. Org. Chem. 1961, 26, 3998.
26. Müller M., Knieps S., Gessele K., Dove S., Bernhardt G., Buschauer A.: Arch. Pharm.
(Weinheim) 1997, 330, 333.
27. Memminger M.: Ph. D. Thesis. University of Regensburg, Regensburg 2009.
28. Cheng Y., Prusoff W. H.: Biochem. Pharmacol. 1973, 22, 3099.
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