Fluorescently Labeled Amino Acids as Building Blocks for Bioactive Molecules

Article abstract of DOI:10.1055/s-0035-1560361

A series of twelve fluorescently labeled amino acids were designed by assembling different coumarin, fluorescein, or nitrobenzo-furazan fluorophores with N-protected lysine or 2-aminopropionic acid. The synthesized amino acids were evaluated with regard to their spectroscopic properties. The easy introduction of the amino acids into peptides and peptidomimetics was exemplarily shown for one coumarin-labeled- amino acid.

Full text of DOI:10.1055/s-0035-1560361

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
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Georg Thieme Verlag Stuttgart · New York
2016, 48, 245–255
245
paper
Synthesis
D. Häußler, M. Gütschow
Paper
Fluorescently Labeled Amino Acids as Building Blocks for Bioactive
Molecules
Daniela Häußler
Boc-HN
CO2H
Michael Gütschow*
(
)n n = 1, 4
HN
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn,
An der Immenburg 4, 53121 Bonn, Germany
guetschow@uni-bonn.de
Y = O, Cl, CH2, H
X = O, N
Y
X
O
O
O
Received: 16.09.2015
Accepted after revision: 05.10.2015
Published online: 03.11.2015
tives, coumarins represent a widely used class of fluores-
cent dyes and are distinguished by their small molecular
size, low bleaching, and large Stokes shifts.3 Whereas the
unsubstituted coumarin does not possess fluorescent prop-
erties, introduction of an electron-donating group at the 7-
position provokes fluorescence caused by an intramolecu-
lar charge transfer, arising from an electron transfer from
the donor in the 7-position to the α,β-unsaturated lactone
system. A carbonyl group at the 3-position enhances the
fluorescence outcome.
–7
DOI: 10.1055/s-0035-1560361; Art ID: ss-2015-t0540-op
Abstract A series of twelve fluorescently labeled amino acids were de-
signed by assembling different coumarin, fluorescein, or nitrobenzo-
furazan fluorophores with N-protected lysine or 2-aminopropionic acid.
The synthesized amino acids were evaluated with regard to their spec-
troscopic properties. The easy introduction of the amino acids into pep-
tides and peptidomimetics was exemplarily shown for one coumarin-
labeled amino acid.
The introduction of an additional electron-donating sub-
stituent at the 6-position further contributes to the charge
Key words amino acids, coumarins, fluorescence, guanidines,
peptidomimetics
8
transfer. Methoxy groups have been commonly applied as
8,9
electron-donating groups at the 7- and 6-positions, and
7-amino substituents produce a typical red shift to longer
wavelengths of absorption and emission, a favorable feature
with regard to background fluorescence.4
Fluorescence labeling of biologically active peptides is
of vital importance in clinical and biomedical research.
There is a broad field of application of such compounds, for
example, as peptidic enzyme substrates, environmental in-
dicators, and chemical probes to study biological structures
and functions. Fluorescently labeled peptides are useful for
visualizing intracellular processes, or investigating protein
folding, localization of proteins, and interactions between
,5,7,10,11
In this study, we synthesized a series of fluorescently
labeled amino acids and evaluated their potential as fluo-
rescent building blocks by examining their spectroscopic
properties. In addition to a fluorescein and a nitrobenzo-
furazan-labeled amino acid, ten coumarin-labeled deriva-
tives were prepared covering the blue to green region of the
visible light spectrum. Exemplarily, the applicability of one
labeled amino acid was shown by incorporation into a pep-
tidomimetic bis-benzguanidine and demonstration of the
biological activity.
To prepare coumarin building blocks, we followed a lit-
erature procedure,9 involving a Knoevenagel reaction of
ortho-hydroxy aldehydes 1–5 (Figure 1) with Meldrum’s
acid and catalytic amounts of piperidinium acetate to form
the coumarin-3-carboxylic acids 8–12 (Scheme 1). Whereas
aldehydes 1, 2, 3, and 4 were commercially available, 5 and
6 were obtained by multi-step syntheses involving a
Vilsmeier formylation as depicted in the Supporting Infor-
mation (Schemes S1 and S2). The coumarin acids 8–12
1–5
peptides and biological membranes. Imaging by fluores-
cence provides many advantages, such as high sensitivity
and selectivity, as well as a safe and ready detection. The
probes and fluorescent substrates can be provided by facile
syntheses with opportunities for manifold structural varia-
tions. Their photophysical properties can be adjusted by the
selection of an appropriate fluorophore. Several fluorescent
labels are valued for their high chemical and biological sta-
1
bility. To achieve adequate detection of the peptide fluo-
rescence and to maintain its native structure, the peptide
should contain either natural fluorescent amino acids,
namely tryptophan or tyrosine, or non-natural fluorescent
analogues. Various types of fluorophores have been em-
ployed. Besides fluorescein and nitrobenzofurazan deriva-
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Synthesis
D. Häußler, M. Gütschow
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were coupled either to the N-protected lysine 15 or to 3-
amino-2-(tert-butoxycarbonylamino)propanoic acid (14)
using NHS and EDC.
synthesis of the labeled amino acid 22 using NHS and N,N′-
dicyclohexylcarbodiimide (DCC) as coupling reagents has
already been described.13 However, the authors used race-
mic lysine, whereas our compounds exhibit the natural L-
configuration. The ethyl esters of 21 and 23 and their spec-
O
O
O
4
O
O
troscopic properties have been reported by Murase et al.
H
H
H
Compound 28 (Scheme 2) was prepared in a different man-
ner by performing the Knoevenagel condensation of the
salicylaldehyde 6 with dimethyl malonate (25),7 yielding
coumarin 26 with two orthogonally protected carboxyl
groups. The tert-butyl ester was cleaved under acidic condi-
tions, followed by the NHS and EDC-mediated amide cou-
O
OH
OH
N
N
OH
O
1
2
3
O
O
H
OH
H
Cl
H
N
OH
α
pling with N -Boc-L-lysine (15) to synthesize the labeled
HO
OH
O
amino acid 28. Furthermore, two new fluorescent amino
4
5
6
α
O
acids, 29 and 30 (Figure 2), were assembled from N -Boc-L-
lysine (15) and the established fluorophores fluorescein
and nitrobenzofurazan, respectively; similar labeled amino
acids have been described.14 Synthetic details for 29 and 30
are given in the Supporting Information (Schemes S3 and
S4). The final fluorescently labeled amino acids 16–24 and
28–30 can easily be introduced into bioactive molecules, as
they exhibit the free carboxyl group. In a subsequent step,
the amino functionality can be readily deprotected to allow
for a further extension of the structure.
Figure 1 Salicylaldehydes for coumarin syntheses
To access the required compound 14, Boc-asparagine
13) was subjected to a Hofmann rearrangement of the ter-
minal carboxamide moiety utilizing PIDA as the oxidant.
(
12
This route provided the nine coumarin-labeled amino acids
6–24 as final compounds (Scheme 1 and Figure 2). The
1
O
O
O
O
piperidine, toluene,
reflux, 2 h
H
R2
H
O
O
N
OH
O
H
N
OH
O
O
N
O
O
O
R1
OH
O
O
O
O
R3
13
NH2
6
26
O
25
O
1–5
O
O
PIDA, EtOAc, MeCN,
H2O, 16 °C, 30 min,
then r.t., 4 h
piperidinium acetate,
EtOH, r.t., 20 min,
then reflux, 2 h
O
O
O
H
N
O
TFA, CH2Cl2,
r.t., 1 h
OH
NH
7
O
O
O
H
R2
O
N
OH
OH
O
O
O
R1
O
N
n
O
H2N
R3
O
1
1
4
5
n = 1
1. NHS, EDC·HCl,
DMF, 0 °C to r.t., 3 h
n = 4
8-12
N
O
O
2
0
. 15, DIPEA, DMF,
°C to r.t., overnight
O
O
O
OH
1
. NHS, EDC·HCl,
28
27
DMF, 0 °C to r.t., 3 h
O
O
O
O
2
0
. DIPEA, DMF,
O
n
°C to r.t., overnight
HN
HN
O
OH
Scheme 2 Synthesis of the fluorescently labeled amino acid 28
R2
R1
O
O
The fluorescently labeled amino acids 16–24 and 28–30
were analyzed with regard to their spectroscopic proper-
ties. Absorption and emission spectra were recorded in buf-
fer pH 8; maxima are outlined in Table 1, and representa-
tive spectra covering all different fluorophores incorporat-
ed are depicted in Figure 3. A comparison of compounds
R3
1
6–24
Scheme 1 Synthesis of the fluorescently labeled amino acids 16–24.
PIDA: Phenyliodine(III) diacetate; NHS: N-hydroxysuccinimide; EDC:
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; DIPEA: diisopropyl-
ethylamine.
1
6/17 with 18/19 revealed that the introduction of the
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Synthesis
D. Häußler, M. Gütschow
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second methoxy group in 6-position led to a bathochromic
shift, caused by an enhanced push-pull system that contrib-
utes to the charge transfer. Furthermore, 18 and 19 exhib-
clearly quenched in an aqueous environment.4,10 By rigidifi-
cation of the amino group into a ring structure as in 22 and
23 containing coumarin 343, as well as in 28 with a tricyclic
coumarin moiety, fluorescence returned in the aqueous
medium and an additional bathochromic shift occurred.
This expected effect of rigidified 7-aminocoumarins relies
8
ited the largest Stokes shift (79 nm) of all final compounds.
The amino acids bearing 7-aminocoumarins 20–23, 28 pos-
sessed a typical red shift to longer wavelengths of absorp-
tion and emission, an advantageous attribute to facilitate
fluorescence measurements and to overcome background
fluorescence. A further well established fluorophore, 7-di-
on a maximal interaction of the nitrogen lone pair with the
aromatic system.4
,7,16
These amino acids possessed the
highest fluorescence maxima of the coumarin-labeled de-
rivatives of this series.
4,10,15
ethylaminocoumarin-3-carboxylic acid,
was used for
the preparation of 20 and 21. However, its fluorescence is
O
H
O
H
O
N
O
N
OH
NH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
HN
HN
HN
HN
OH
O
OH
O
O
O
O
O
O
O
O
O
O
1
6
17
18
19
O
H
N
O
O
OH
NH
H
N
O
O
OH
NH
O
O
O
O
O
HN
HN
O
O
OH
O
O
O
HN
HN
OH
O
O
O
N
O
O
O
N
N
O
O
O
N
20
21
22
23
H
N
O
O
OH
NH
H
N
O
O
O
OH
NH
H
N
O
O
OH
NH
O
O
O
H
N
O
S
OH
N
O
O
HO
NH
O
O
O
O
NH
O
COOH
O
Cl
O2N
N
O
OH
N
O
O
30
24
28
29
Figure 2 Fluorescently labeled amino acids 16–24, 28–30
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D. Häußler, M. Gütschow
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Table 1 Absorption (UV λmax) and Fluorescence Emission (F λmax) Maxi-
ma of Compounds 16–24, 28–30
into a dibasic molecule of a chemotype known to exhibit
inhibitory properties against several serine proteases of the
trypsin family. Benzamidine and benzguanidine moieties
represent prominent arginine mimetics. They have
frequently been used to address the S1 and S3/S4 binding
a
Compound
UV λmax (nm)
F λmax (nm)
Stokes shift (nm)
1
1
1
1
2
2
2
2
2
2
2
6
7
8
9
0
1
2
3
4
8
9
0
351
350
369
371
428
429
444
447
404
423
481
493
404
406
448
450
472
478
492
492
448
490
552
516
53
56
79
79
44
49
48
45
44
67
71
23
site of trypsin-like serine proteases, such as matriptase,
matriptase-2, thrombin, factor Xa, and trypsin.1
7–19
More-
over, such compounds possess antiprotozoal and antifungal
activities. Their mode of action has been attributed to the
insertion into the minor groove of adenine/thymine-rich
sites of double-strand DNA.20 In addition, it has been shown
that certain bis-benzamidines exhibit DNA-dependent flu-
orescent properties.21
The synthesis of such a compound comprising two
benzguanidine substructures and a central coumarin-
labeled amino acid is shown in Scheme 3. Using HATU as
coupling reagent and DIPEA as base, the amino acid 19 and
4
-nitrobenzylamine (31), the first amide bond was formed.
3
After trifluoroacetic acid-promoted removal of the Boc-
protecting group of 32, the resulting free amine was react-
ed with 4-nitrobenzoic acid (33) reapplying the same cou-
pling protocol. The following simultaneous conversion of
both nitro groups into primary amine moieties occurred
a
Recorded in buffer, pH 8, at concentrations of 10 μM for the absorption
spectra and 1 μM for the emission spectra.
The fluorescein derivative 30 showed a maximum of
516 nm, 24 nm more shifted to longer wavelengths than
after treatment with SnCl acting as the reductive agent.
2
that of the coumarin-labeled compounds 22/23, but it also
possessed the smallest Stokes shift. The 7-nitrobenzo-
furazan fluorophore present in compound 29 provided an
emission maximum of 552 nm, which was the most red-
shifted within this series. However, this fluorophore has
the drawback that its fluorescence is quenched in aqueous
media; for comparison, see the non-normalized spectra in
the Supporting Information (Figure S1).
The bis-aniline 35 was converted in a HgCl -catalyzed reaction
2
with N,N′-di-Boc-S-methylisothiourea (36)22 and triethyl-
amine to produce an intermediate with two protected
guanidine groups. After the subsequent removal of the Boc-
protecting groups with trifluoroacetic acid, the desired bis-
benzguanidine 37 was obtained and purified by means of
preparative HPLC. The hydrochloride salt was obtained by
adding few drops of HCl to a solution of the guanidinium
trifluoroacetate in water, followed by lyophilization.
To demonstrate the applicability of such coumarin-
labeled amino acids, compound 19 was chosen for insertion
Figure 3 Normalized absorption (10 μM, 1% DMSO, dashed lines) and emission spectra (1 μM, 1% DMSO, continuous lines) in buffer (50 mM Tris-HCl,
pH 8.0, 150 mM NaCl). Dark blue: 16; light blue: 18; dark green: 24; yellow: 28; light green: 20; orange: 22; red: 29; brown: 30.
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D. Häußler, M. Gütschow
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O2N
1
. CH2Cl2/TFA, r.t., 1 h
O
H
O
2. HATU, DIPEA, DMF,
r.t., overnight
H
N
HATU, DIPEA, DMF, r.t.,
overnight
O
N
N
N
19
H
H
O
O
NO2
O N
NO2
HN
2
HN
Cl
H3N
O
O
O
O
OH
O
NO2
31
O
O
O
O
O
O
32
33
34
1. HgCl2, Et3N, CH2Cl2, r.t., 24 h
H
H2N
O
S
O
H2N
N
O
O
H
H
N
O
N
N
O
NH2
N
SnCl2, H2O,
EtOAc, reflux, 4 h
N
H
Cl
N
NH2 Cl
N NH2
H
H
36
O
O
HN
NH2
HN
H
2
. CH2Cl2/TFA, r.t., 2 h
O
O
O
O
O
O
O
O
O
O
35
37
Scheme 3 Synthesis of the bis-benzguanidine 37. HATU: N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanamini-
um hexafluorophosphate N-oxide; TFA: trifluoroacetic acid.
Compound 37 was evaluated as an inhibitor of the
serine proteases matriptase-2, thrombin, factor Xa, and
trypsin and with respect to its binding to DNA. The results
of duplicate measurements are depicted in Table 2. The en-
In conclusion, a small library of fluorescently labeled
amino acids were synthesized. The labels comprise differ-
ent coumarin moieties, as well as fluorescein and nitro-
benzofurazan, connected to either a C-1 or C-4 linker via an
amide bond. In aqueous medium, the products show fluo-
rescence in the blue to green region of the visible light
18,19
zymes were assayed as described,
using fluorogenic
peptide substrates. The progress curves were analyzed and
IC50 values were obtained from plots of the rates versus
spectrum.
A 6,7-dimethoxycoumarin-containing amino
inhibitor concentrations. K values were calculated with the
acid was exemplarily introduced into a peptidomimetic
compound, whose bioactivity was evaluated. Accordingly,
the labeled amino acids reported herein are expected to be
useful building blocks for the assembly of various biologi-
cally active, fluorescent compounds.
i
Cheng–Prusoff equation. Whereas 37 did not inhibit factor
Xa, it exhibited a moderate inhibition of thrombin, and Ki
values of less than 10 μM were obtained for matriptase-2
and trypsin. DNA binding of 37 was determined by
anisotropy titration, using short hairpin oligonucleotides,
21
as described. Compound 37 did not bind to a guanine/
cytosine-rich oligonucleotide, but showed a concentration
Solvents and reagents were obtained from Acros (Geel, Belgium),
Aldrich (Steinheim, Germany), Alfa Aesar (Karlsruhe, Germany),
Bachem (Bubendorf, Switzerland), Carbolution (Saarbrücken, Germa-
ny), and Fluorochem (Hadfield, UK). TLC was carried out on alumi-
num sheets, coated with silica gel 60 F254 (Merck, Darmstadt, Germa-
ny). Compounds were visualized under UV light (254 nm). Prepara-
tive column chromatography was performed using Merck silica gel
60, 0.060–0.200 mm. Preparative HPLC was performed on a Euro-
spher 100 column with reversed phase silica gel C-18, and a spectro-
photometer Wellchrome K-2600 (Knauer, Berlin, Germany) was
applied.
dependent, albeit weak affinity (K > 20 μM) to a short hair-
D
pin oligonucleotide featuring the adenine/thymine-rich
binding site AAATTT.
Table 2 Kinetic Parameters of Inhibition (K ) of Compound 37 towards
i
Four Serine Proteases and Dissociation Constant (K ) for the DNA Bind-
D
ing of 37
Assay
i D
K or K value
Mass spectra were recorded on an API 2000 mass spectrometer (elec-
tron spray ion source, Applied Biosystems, Darmstadt, Germany) cou-
pled with an Agilent 1100 HPLC system using a Phenomenex Luna
HPLC C18 column (50 × 2.00 mm, particle size 3 μm). The purity of
the tested compounds was determined by HPLC-UV obtained on an
LC-MS instrument (Applied Biosystems API 2000 LC-MS/MS, HPLC
Agilent 1100). HRMS was recorded on a microTOF-Q mass spectrome-
ter (Bruker, Köln, Germany) with ESI-source coupled with a HPLC
Dionex Ultimate 3000 (Thermo Scientific, Braunschweig, Germany)
using a EC50/2 Nucleodur C18 Gracity 3 μm column (Macherey-Nagel,
Düren, Germany). Elemental analyses were performed with a Vario
human matriptase-2
human thrombin
bovine factor Xa
bovine trypsin
K
K
K
K
K
i
i
i
i
= 6.3 ± 0.8 μM
= 28 ± 2 μM
> 40 μM
= 8.4 ± 0.4 μM
DNA binding
D
> 20 μM
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D. Häußler, M. Gütschow
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EL apparatus. 1H and 13C NMR spectra were recorded on a Bruker
Avance 500 MHz NMR spectrometer. DMSO-d6 was used as the sol-
vent as indicated below. NMR spectra were recorded at 30 °C. Chemi-
cal shifts are given in parts per million (ppm) referring to the signal
Anal. Calcd for C14H15NO : C, 64.36; H, 5.79; N, 5.36. Found: C, 64.03;
H, 5.75; N, 5.72.
4
2,3,6,7-Tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano-[6,7,8-
center using the solvent peaks for reference: DMSO-d 2.49/39.7 ppm.
6
ij]quinolizine-10-carboxylic Acid (11)
Melting points were determined on a Büchi 510 oil bath apparatus
and are uncorrected.
From 8-hydroxyjulolidine-9-carboxaldehyde (4; 2.17 g, 10 mmol), 7
(1.44 g, 10 mmol), and piperidinium acetate (0.029 g, 0.2 mmol) in
anhydrous EtOH (10 mL); yield: 0.69 g (24%, 2.42 mmol); orange sol-
id; mp 248–251 °C (Lit. mp 253 °C).
Coumarin Acids of 8–12; General Prodecure
24
The respective aldehyde 1–5 (1.0 equiv) was dissolved in absolute
EtOH (7–60 mL). Isopropylidene malonate (1.0 equiv) and piperidini-
um acetate (0.02 equiv) were added to the solution. The mixture was
stirred for 20 min at r.t. and refluxed for 2 h. The mixture was allowed
to cool down to r.t. and kept in an ice bath for 30 min. The product
was collected by filtration and washed with EtOH (50 mL).
1
H NMR (500 MHz, DMSO-d ): δ = 1.84–1.90 [m, 4 H, N(CH CH ) ],
6
2
2 2
2
.69–2.73 {m, 4 H, N[(CH ) CH ] }, 3.33–3.36 [m, 4 H, N(CH ) ], 7.23
2 2 2 2 2 2
(s, 1 H, 5-H), 8.44 (s, 1 H, 4-H), 12.38 (s, 1 H, CO H).
2
13
C NMR (125 MHz, DMSO-d ): δ = 19.7, 19.7, 20.6, 26.9, 49.3, 49.8,
6
1
04.9, 105.2, 107.4, 119.7, 127.6, 148.9, 149.3, 152.8, 160.7, 164.8.
LC-MS (ESI): 99% purity; m/z = 286.0 ([M + H]+).
7-Methoxy-2-oxo-2H-chromene-3-carboxylic Acid (8)
Anal. Calcd for C16H15NO : C, 67.36; H, 5.30; N, 4.91. Found: C, 67.69;
4
H, 5.74; N, 5.11.
From 2-hydroxy-4-methoxybenzaldehyde (1; 2.28 g, 15 mmol), 2,2-
dimethyl-1,3-dioxane-4,6-dione (7; 2.16 g, 15 mmol), and piperidini-
um acetate (0.044 g, 0.3 mmol) in anhydrous EtOH (60 mL); yield:
6-Chloro-7-hydroxy-2-oxo-2H-chromene-3-carboxylic Acid (12)
From 5-chloro-2,4-dihydroxybenzaldehyde (5; 0.30 g, 1.73 mmol), 7
2
1
1
.38 g (72%, 10.81 mmol); colorless solid; mp 196–198 °C (Lit.9 mp
92–194 °C).
(0.25 g, 1.73 mmol), and piperidinium acetate (0.005 g, 0.035 mmol)
4
H NMR (500 MHz, DMSO-d ): δ = 3.88 (s, 3 H, OCH ), 6.99 (dd, J = 2.6
in anhydrous EtOH (7 mL), yield: 0.12 g (29%, 0.50 mmol); yellow sol-
id; mp >250 °C.
6
3
3
4
3
Hz, J = 8.6 Hz, 1 H, 6-H), 7.02 (d, J = 2.6 Hz, 1 H, 8-H), 7.81 (d, J = 8.6
Hz, 1 H, 5-H), 8.70 (s, 1 H, 4-H), 12.93 (s, 1 H, CO H).
1
2
H NMR (500 MHz, DMSO-d ): δ = 6.88 (s, 1 H, 8-H), 7.97 (s, 1 H, 5-H),
6
13
C NMR (125 MHz, DMSO-d ): δ = 56.4, 100.4, 111.8, 113.4, 114.0,
8.63 (s, 1 H, 4-H), 11.99 (br s, 1 H, OH), 12.81 (br s, 1 H, OH).
6
1
31.7, 149.1, 157.0, 157.4, 164.2, 164.8.
13
C NMR (125 MHz, DMSO-d ): δ = 103.0, 111.4, 114.2, 117.8, 130.9,
6
+
–
LC-MS (ESI): 100% purity; m/z = 238.23 ([M + NH ] ), 219.03 ([M – H] ).
148.4, 155.3, 157.0, 158.9, 164.1.
4
LC-MS (ESI): 97% purity; m/z = 241.1 ([M + H]+).
Anal. Calcd for C11H O : C, 60.00; H, 3.66; N, 0.00. Found: C, 59.80; H,
8
5
3.62; N, 0.25.
Anal. Calcd for C10H ClO : C, 49.92; H, 2.09; N, 0.00. Found: C, 49.83;
5
5
H, 2.14; N, 0.30.
7,6-Dimethoxy-2-oxo-2H-chromene-3-carboxylic Acid (9)
From 2-hydroxy-4,5-dimethoxybenzaldehyde (2; 2.73 g, 15 mmol), 7
2.16 g, 15 mmol), and piperidinium acetate (0.044 g, 0.3 mmol) in
anhydrous EtOH (60 mL); yield: 2.63 g (70%, 10.51 mmol); light yel-
(S)-3-Amino-2-(tert-butoxycarbonylamino)propanoic Acid (14)
N-Boc-L-asparagine (13; 3.48 g, 15 mmol) and PIDA (5.81 g, 18 mmol)
(
were dissolved in a mixture of EtOAc, MeCN, and H O (2:2:1, 62.5 mL)
2
23
low solid; mp 265–269 °C (Lit. mp 266 °C).
and stirred at 16 °C for 30 min. Afterwards, the reaction mixture was
allowed to warm up to r.t. and stirred for 4 h whereupon a precipitate
was formed, which was collected by filtration and dried; yield: 2.02 g
1
H NMR (500 MHz, DMSO-d ): δ = 3.80 (s, 3 H, OCH ), 3.89 (s, 3 H,
6
3
OCH ), 7.10 (s, 1 H, 8-H), 7.43 (s, 1 H, 5-H), 8.67 (s, 1 H, 4-H), 12.87 (s,
3
25
1
H, CO H).
(66%, 9.89 mmol); colorless solid; mp 211–213 °C (Lit. mp 216 °C).
2
1
3
1
C NMR (125 MHz, DMSO-d ): δ = 56.1, 56.6, 99.8, 110.2, 110.7,
H NMR (500 MHz, DMSO-d
6
): δ = 1.40 [s, 9 H, C(CH
3
2
)
3
], 2.76 (dd,
6
3
2
3
1
13.8, 146.4, 149.2, 151.6, 155.2, 157.9, 164.3.
J = 9.2 Hz, J = 11.8 Hz, 1 H, CH
2
), 3.06 (dd, J = 5.1 Hz, J = 11.8 Hz, 1
LC-MS (ESI): 100% purity; m/z = 249.11 ([M – H]–).
H, CH
2
), 3.65–3.69 (m, 1 H, COCH), 6.08 (s, 1 H, OCONH). Protons of
CO H and the NH group were not detected.
2
2
Anal. Calcd for C12H10O : C, 57.60; H, 4.03; N, 0.00. Found: C, 57.36; H,
6
13
C NMR (125 MHz, DMSO-d ): δ = 28.3, 40.8, 51.1, 78.2, 155.2, 171.2.
6
4.08; N, 0.00.
+
–
LC-MS (ESI): 100% purity; m/z = 205.19 ([M + H] ), 203.09 ([M – H] ).
+
+
7-(Diethylamino)-2-oxo-2H-chromene-3-carboxylic Acid (10)
HRMS (ESI ): m/z calcd for C H N O ([M + H] ): 205.1183; found:
8 16 2 4
2
05.1177.
From 4-(diethylamino)-2-hydroxybenzaldehyde (3; 1.93 g, 10 mmol),
(1.44 g, 10 mmol), and piperidinium acetate (0.029 g, 0.2 mmol) in
anhydrous EtOH (40 mL); yield: 1.65 g (63%, 6.32 mmol); yellow sol-
7
Fluorescently Labeled Amino Acids 16–24; General Procedure
10
id; mp 220–223 °C (Lit. mp 222 °C).
The appropriate coumarin-3-carboxylic acid 8–12 (1.0 equiv) and N-
hydroxysuccinimide (1.5 equiv) were dissolved in DMF (15–60 mL)
and the solution was cooled to 0 °C. EDC·HCl (1.5 equiv) was added
and the mixture was stirred for 3 h. During this time, r.t. was reached
and the mixture was diluted with EtOAc (50–300 mL) and washed
1
3
H NMR (500 MHz, DMSO-d ): δ = 1.13 (t, J = 7.1 Hz, 6 H, CH CH ),
6
2
3
3
4
3
.47 (q, J = 7.1 Hz, 4 H, CH CH ), 6.55 (t, J = 2.3 Hz, 1 H, 8-H), 6.78
2 3
3
3
(
dd, 4J = 2.3 Hz, J = 8.9 Hz, 1 H, 6-H), 7.62 (d, J = 8.9 Hz, 1 H, 5-H),
8
.57 (s, 1 H, 4-H), 12.47 (s, 1 H, CO H).
2
1
3
with aq 10% citric acid (50–150 mL), aq sat. NaHCO
brine (50–150 mL). The organic phase was dried (Na SO ), filtered,
3
(50–150 mL), and
C NMR (125 MHz, DMSO-d ): δ = 12.5, 44.5, 96.1, 107.3, 107.5,
6
2
4
1
10.2, 132.0, 149.6, 153.1, 158.0, 159.7, 164.6.
and evaporated. The NHS-ester was dissolved in DMF (25–50 mL) and
LC-MS (ESI): 98% purity; m/z = 262.1 ([M + H]+).
α
cooled to 0 °C. DIPEA (2.4 equiv) and N -Boc-L-Lys-OH (15) or com-
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 245–255

2
51
Synthesis
D. Häußler, M. Gütschow
Paper
3
pound 14 (1.2 equiv) were added and the solution was stirred at r.t.
overnight. The mixture was diluted with EtOAc (50–300 mL) and
washed with aq 10% citric acid (3 × 50–150 mL) and brine (50–150
mL), dried (Na SO ), filtered, and evaporated.
(m, 1 H, COCH), 3.90 (s, 3 H, OCH ), 6.99 (d, J = 7.9 Hz, 1 H, OCONH),
3
3
7.17 (s, 1 H, 8-H), 7.50 (s, 1 H, 5-H), 8.67 (t, J = 5.7 Hz, 1 H, CONH),
8.78 (s, 1 H, 4-H), 12.37 (s, 1 H, CO H).
2
2
4
13
C NMR (125 MHz, DMSO-d ): δ = 23.3, 28.3, 28.8, 30.6, 39.0, 53.5,
6
5
6.2, 56.6, 78.1, 99.1, 110.1, 111.3, 114.9, 146.7, 147.8, 150.8, 154.2,
2-(tert-Butoxycarbonylamino)-6-(7-methoxy-2-oxo-2H-
155.7, 161.2, 161.5, 174.3.
chromene-3-carboxamido)hexanoic Acid (16)
+
–
LC-MS (ESI): 97% purity; m/z = 479.28 ([M + H] ), 477.18 ([M – H] ).
From 8 (1.10 g, 5 mmol), activated with N-hydroxysuccinimide (0.86
g, 7.5 mmol) and EDC·HCl (1.44 g, 7.5 mmol) in DMF (15 mL); and
subsequent coupling with 15 (1.48 g, 6 mmol) in the presence of
DIPEA (1.55 g, 2.04 mL, 12 mmol) in DMF (25 mL). The product was
crystallized from EtOAc; yield: 0.89 g (40%, 1.98 mmol); colorless sol-
id; mp 104–105 °C.
Anal. Calcd for C23H30N O : C, 57.73; H, 6.32; N, 5.85. Found: C, 56.67;
H, 6.37; N, 6.01.
2
9
2-(tert-Butoxycarbonylamino)-3-(6,7-dimethoxy-2-oxo-2H-
chromene-3-carboxamido)propanoic Acid (19)
1H NMR (500 MHz, DMSO-d ):
From 9 (1.00 g, 4 mmol), activated using N-hydroxysuccinimide (0.69
g, 6 mmol) and EDC·HCl (1.14 g, 6 mmol) in DMF (15 mL); and subse-
quent coupling with 14 (0.98 g, 4.8 mmol) in the presence of DIPEA
δ
=
1.35 [s, 11 H, C(CH ) ,
6
3 3
CH CH CH CH NH], 1.45–1.54 (m, 2 H, CH CH CH CH NH), 1.55–1.62
2
2
2
2
2
2
2
2
(
m, 1 H, CH CH CH CH NH), 1.63–1.71 (m, 1 H, CH CH CH CH NH),
2 2 2 2 2 2 2 2
(1.24 g, 1.63 mL, 9.6 mmol) in DMF (30 mL). The product was crystal-
3
.29–3.31 (m, 2 H, CH CH CH CH NH), 3.89 (s, 3 H, OCH ), 4.02 (q,
2 2 2 2 3
3
3
lized from EtOAc; yield: 0.78 g (45%, 1.78 mmol); yellow solid; mp
199–200 °C.
J = 7.3 Hz, 1 H, COCH), 6.99 (d, J = 7.3 Hz, 1 H, OCONH), 7.03 (dd,
4
3
3
4
J = 2.4 Hz, J = 8.7 Hz, 1 H, 6-H), 7.09 (d, J = 2.4 Hz, 1 H, 8-H), 7.88 (d,
3
1H NMR (500 MHz, DMSO-d
2 H, CH ), 3.81 (s, 3 H, OCH
J = 8.7 Hz, 1 H, 5-H), 8.61 (t, J = 5.8 Hz, 1 H, CONH), 8.79 (s, 1 H, 4-H),
6
): δ = 1.36 [s, 9 H, C(CH
) ], 3.34–3.40 (m,
3 3
1
2.36 (s, 1 H, CO H).
2
3
), 3.90 (s, 3 H, OCH ), 4.09–4.13 (m, 1 H,
3
2
1
3
COCH), 7.17 (s, 1 H, OCONH), 7.18 (s, 1 H, 8-H), 7.53 (s, 1 H, 5-H), 8.82
C NMR (500 MHz, DMSO-d ): δ = 20.9, 23.2, 28.3, 30.6, 39.0, 53.5,
6
3
(
s, 1 H, 4-H), 8.89 (t, J = 5.7 Hz, 1 H, CONH), 12.73 (s, 1 H, CO H).
2
5
1
6.4, 78.1, 100.4, 112.3, 113.7, 115.1, 131.6, 147.8, 155.7, 156.3, 161.0,
61.5, 164.5, 174.3.
13C NMR (125 MHz, DMSO-d
110.2, 111.2, 114.4, 146.7, 148.3, 150.9, 155.1, 155.6, 161.1, 161.9,
): δ = 28.3, 53.5, 56.2, 56.6, 78.4, 99.8,
6
+
–
LC-MS (ESI): 99% purity; m/z = 449.28 ([M + H] ), 447.15 [M – H] .
1
72.3. The CH and DMSO signals were overlapped.
2
Anal. Calcd for C22H28N O : C, 58.92; H, 6.29; N, 6.25. Found: C, 58.46;
H, 6.45; N, 5.72.
2
8
+
–
LC-MS (ESI): 99% purity; m/z = 437.37 ([M + H] ), 435.38 [M – H] .
+
+
HRMS (ESI ): m/z calcd for C20H24N O ([M + H] ): 437.1555; found:
2
9
437.1556.
2-(tert-Butoxycarbonylamino)-3-(7-methoxy-2-oxo-2H-
chromene-3-carboxamido)propanoic Acid (17)
(
S)-2-(tert-Butoxycarbonylamino)-6-[7-(diethylamino)-2-oxo-2H-
From 8 (0.88 g, 4 mmol), activated using N-hydroxysuccinimide (0.86
g, 7.5 mmol) and EDC·HCl (1.14 g, 6 mmol) in DMF (15 mL); and sub-
sequent coupling with 14 (0.98 g, 4.8 mmol) in the presence of DIPEA
chromene-3-carboxamido]hexanoic Acid (20)
From 10 (0.131 g, 0.5 mmol), activated using N-hydroxysuccinimide
(0.086 g, 0.75 mmol) and EDC·HCl (0.14 g, 0.75 mmol) in DMF (10
mL); and subsequent coupling with 15 (0.18 g, 0.6 mmol) in the pres-
ence of DIPEA (0.16 g, 0.20 mL, 1.2 mmol) in DMF (20 mL); yield: 0.20
g (82%, 0.41 mmol); yellow solid; mp 80–82 °C.
(1.24 g, 1.63 mL, 9.6 mmol) in DMF (30 mL). The product was crystal-
lized from EtOAc; yield: 0.58 g (36%, 1.43 mmol); colorless solid; mp
190–191 °C.
1
H NMR (500 MHz, DMSO-d ): δ = 1.36 [s, 9 H, C(CH ) ], 3.35–3.40 (m,
6
3 3
1H NMR (500 MHz, DMSO-d
1.36 [s, 11 H, C(CH CH
CH CH CH CH NH), 1.54–1.61 (m, 1 H, CH
CH CH CH NH), 3.25–3.27 (m, 2 H, CH
): δ = 1.13 (t, 3J = 7.0 Hz, 6 H, CH
CH CH CH NH], 1.44–1.53 (m,
CH CH CH
CH
2
),
H,
1
H, CH ), 3.80–3.85 (m, 1 H, CH ), 3.89 (s, 3 H, OCH ), 4.10–4.14 (m, 1
6
2
3
2
2
3
3
4
4
H, COCH), 7.37 (dd, J = 2.5 Hz, J = 8.8 Hz, 1 H, 6-H), 7.10 (d, J = 2.5
Hz, 1 H, 8-H), 7.20 (d, J = 7.9 Hz, 1 H, OCONH), 7.90 (d, J = 8.8 Hz, 1 H,
3
)
3
,
2
2
2
2
3
3
NH), 1.62–1.71
CH CH CH NH),
2
2
2
2
2
2
2
2
5-H), 8.82–8.84 (m, 2 H, CONH, 4-H), 12.72 (s, 1 H, CO H).
(m, 1 H, CH
2
2
2
2
2
2
2
2
2
3
13
3.47 (q, J = 7.0 Hz, 4 H, CH
2
CH
3
4
), 3.82–3.86 (m, 1H, COCH), 6.59 (d,
C NMR (125 MHz, DMSO-d ): δ = 28.3, 53.5, 56.4, 78.4, 100.4, 112.2,
6
4
3
J = 2.3 Hz, 1 H, 8-H), 6.79 (dd, J = 2.3 Hz, J = 9.1 Hz, 1 H, 6-H), 6.99
113.8, 114.5, 131.8, 148.3, 155.6, 156.4, 160.9, 161.9, 164.7, 172.3.
3
3
(
d, J = 7.6 Hz, 1 H, OCONH), 7.66 (d, J = 9.1 Hz, 1 H, 5-H), 8.61 (t,
The CH and DMSO signals overlapped.
2
3
J = 6.0 Hz, 1 H, CONH), 8.63 (s, 1 H, 4-H), 12.35 (s, 1 H, CO H).
2
+
–
LC-MS (ESI): 99% purity; m/z = 407.32 ([M + H] ), 405.26 ([M – H] ).
1
3
C NMR (125 MHz, DMSO-d ): δ = 12.5, 23.3, 28.3, 28.3, 30.6, 38.8,
6
Anal. Calcd for C19H22N O : C, 56.15; H, 5.46; N, 6.89. Found: C, 56.15;
2
8
44.5, 53.5, 78.1, 96.0, 107.8, 109.7, 110.3, 131.7, 147.7, 152.5, 155.7,
57.3, 161.9, 162.2, 174.3.
H, 5.24; N, 6.82.
1
LC-MS (ESI): 97% purity; m/z = 490.4 ([M + H]+).
2
-(tert-Butoxycarbonylamino)-6-(6,7-methoxy-2-oxo-2H-
+
+
HRMS (ESI ): m/z calcd for C25H N O ([M + H] ): 490.2548; found:
chromene-3-carboxamido)hexanoic Acid (18)
35
3
7
490.2542.
From 9 (1.25 g, 5 mmol), activated using N-hydroxysuccinimide (0.86
g, 7.5 mmol) and EDC·HCl (1.44 g, 7.5 mmol) in DMF (60 mL); and
subsequent coupling with 15 (1.48 g, 6 mmol) in the presence of
DIPEA (1.55 g, 2.04 mL, 12 mmol) in DMF (25 mL); yield: 0.52 g (22%,
(S)-2-(tert-Butoxycarbonylamino)-3-[7-(diethylamino)-2-oxo-2H-
chromene-3-carboxamido]propanoic Acid (21)
1
.09 mmol); yellow solid; mp 178–182 °C.
1H NMR (500 MHz, DMSO-d ):
= 1.36 [s, 11 H, C(CH ) ,
3 3
From 10 (1.05 g, 4 mmol), activated using N-hydroxysuccinimide
(0.69 g, 6 mmol) and EDC·HCl (1.14 g, 6 mmol) in DMF (20 mL); and
subsequent coupling with 14 (0.98 g, 4.8 mmol) in the presence of
DIPEA (1.24 g, 1.63 mL, 9.6 mmol) in DMF (30 mL); yield: 1.31 g (73%,
δ
6
CH CH CH CH NH], 1.47–1.54 (m, 2 H, CH CH CH CH NH), 1.55–1.62
2
2
2
2
2
2
2
2
(m, 1 H, CH CH CH CH NH), 1.63–1.71 (m, 1 H, CH CH CH CH NH),
2 2 2 2 2 2 2 2
2.93 mmol); bright yellow solid; mp 207–210 °C.
3
.27–3.31 (m, 2 H, CH CH CH CH NH), 3.81 (s, 3 H, OCH ), 3.82–3.87
2 2 2 2 3
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 245–255

2
52
Synthesis
D. Häußler, M. Gütschow
Paper
1
H NMR (500 MHz, DMSO-d ): δ = 1.13 (t, 3J = 7.0 Hz, 6 H, CH CH ),
(S)-2-(tert-Butoxycarbonylamino)-6-(6-chloro-7-hydroxy-2-oxo-
2H-chromene-3-carboxamido)hexanoic Acid (24)
6
2
3
3
1
.35 [s, 9 H, C(CH ) ], 3.26–3.37 (m, 1 H, CH ), 3.47 (q, J = 7.0 Hz, 4 H,
3
3
2
CH CH ), 3.78–3.85 (m, 1 H, CH ), 4.08–4.12 (m, 1 H, COCH), 6.59 (s, 1
2
3
2
From 12 (0.13 g, 0.53 mmol), activated using N-hydroxysuccinimide
4
3
3
H, 8-H), 6.79 (dd, J = 2.4 Hz, J = 9.3 Hz, 1 H, 6-H), 7.19 (d, J = 7.9 Hz,
(0.10 g, 0.80 mmol) and EDC·HCl (0.15 g, 0.80 mmol) in DMF (50 mL);
3
1
H, OCONH), 7.67 (d, J = 9.3 Hz, 1 H, 5-H), 8.65 (s, 1 H, 4-H), 8.82 (t,
and subsequent coupling with 15 (0.16 g, 0.64 mmol) in the presence
of DIPEA (0.16 g, 0.22 mL, 1.27 mmol) in DMF (25 mL); yield: 0.19 g
(76%, 0.41 mmol); yellow solid; mp 125–128 °C.
3
J = 5.7 Hz, 1 H, CONH), 12.69 (s, 1 H, CO H).
2
13
C NMR (125 MHz, DMSO-d ): δ = 12.4, 28.3, 44.5, 53.7, 78.4, 96.0,
6
1
1
07.8, 109.1, 110.3, 131.8, 148.1, 152.7, 155.6, 157.4, 161.7, 162.7,
72.3. The CH and DMSO signals overlapped.
1
H NMR (500 MHz, DMSO-d ): δ = 1.32–1.38 [s, 11 H, C(CH ) ,
6
3 3
2
CH CH CH CH NH], 1.44–1.54 (m, 2 H, CH CH CH CH NH), 1.54–1.60
(m, 1 H, CH
3
6
8
1
2
2
2
2
2
2
2
2
LC-MS (ESI): 99% purity; m/z = 448.0 ([M + H]+).
CH
CH
CH
NH), 1.62–1.70 (m, 1 H, CH
CH CH CH NH),
2
2
2
2
2
2 2 2
3
.30 (q, J = 5.6 Hz, 2 H, CH CH CH CH NH), 3.81–3.85 (m, 1 H, COCH),
.95 (s, 1 H, 8-H), 7.00 (d, J = 6.6 Hz, 1 H, OCONH), 8.05 (s, 1 H, 5-H),
.58 (t, J = 4.8 Hz, 1 H, CONH), 8.74 (s, 1 H, 4-H), 11.83 (br s, 1 H, OH),
2
3
2
2
2
Anal. Calcd for C22H29N O : C, 59.05; H, 6.53; N, 9.39. Found: C, 58.98;
H, 6.74; N, 9.28.
3
7
3
2.36 (br s, 1 H, CO H).
2
(S)-2-(tert-Butoxycarbonylamino)-6-(2,3,6,7-tetrahydro-11-oxo-
13
C NMR (125 MHz, DMSO-d ): δ = 23.3, 28.4, 28.1, 30.6, 39.0, 53.6,
1
H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carbox-
6
7
1
8.1, 103.0, 111.9, 115.4, 118.2, 130.9, 147.1, 154.5, 155.8, 158.6,
60.7, 161.3, 174.3.
amido)hexanoic Acid (22)
From 11 (0.54 g, 1.88 mmol), activated using N-hydroxysuccinimide
LC-MS (ESI): 97% purity; m/z = 469.1 ([M + H]+).
(0.33 g, 2.82 mmol) and EDC·HCl (0.54 g, 2.82 mmol) in DMF (30 mL);
+
+
and subsequent coupling with 15 (0.56 g, 2.26 mmol) in the presence
of DIPEA (0.77 g, 1.01 mL, 4.51 mmol) in DMF (30 mL). The product
was chromatographed over silica gel using CH Cl –MeOH–AcOH
HRMS (ESI ): m/z calcd for C21H25ClN O ([M + H] ): 469.1372; found:
469.1360.
2
8
2
2
(
29:1:0.03) as eluent; yield: 0.59 g (61%, 1.16 mmol); orange resin.
1H NMR (500 MHz, DMSO-d ):
1.30–1.34 (m,
H, C(CH ) ], 1.43–1.51 (m,
Methyl 9-(2-tert-Butoxy-2-oxoethyl)-2-oxo-(6,7,8,9-tetrahydro-
2H-pyrano[3,2-g]quinoline-3-carboxylate (26)
δ
=
2
2
H,
H,
6
CH CH CH CH NH), 1.35 [s,
CH CH CH CH NH), 1.53–1.60 (m, 1 H, CH CH CH CH NH), 1.62–1.69
9
2
2
2
2
3
3
tert-Butyl 2-(6-formyl-7-hydroxy-3,4-dihydroquinolin-1(2H)-yl)ace-
tate (6; 0.39 g, 1.33 mmol) was dissolved in toluene (10 mL). Dimethyl
malonate (25; 0.19 g, 0.17 mL, 1.46 mmol) and piperidine (0.13 mL,
1.33 mmol) were added and the mixture was refluxed for 2 h. After
evaporation in vacuo, the residue was purified by column chromatog-
raphy using petroleum ether–EtOAc (3:1 to 1:1); yield: 0.44 g (88%,
1.18 mmol); green solid; mp 143–146 °C.
2
2
2
2
2
2
2
2
(m, 1 H, CH CH CH CH NH), 1.84–1.89 [m, 4 H, N(CH CH ) ], 2.69–
2 2 2 2 2 2 2
3
2
.73 {m, 4 H, N[(CH ) CH ] }, 3.24–3.28 (q, J = 5.5 Hz, 2 H,
2 2 2 2
CH CH CH CH NH), 3.30–3.32 [m, 4 H, N(CH ) ], 3.80–3.84 (m, 1 H,
COCH), 6.94 (d, J = 6.6 Hz, 1 H, OCONH), 7.22 (s, 1 H, 5-H), 8.48 (s, 1
H, 4-H), 8.63 (t, J = 4.8 Hz, 1 H, CONH), 12.10 (s, 1 H, CO H).
1
2
2
2
2
2 2
3
3
2
3
C NMR (125 MHz, DMSO-d ): δ = 19.8, 21.2, 23.3, 27.0, 28.4, 29.0,
1
6
H NMR (500 MHz, DMSO-d ): δ = 1.41 [s, 9 H, C(CH ) ], 1.87 (quint,
6 3 3
3
1
0.7, 38.8, 49.2, 49.7, 53.6, 78.0, 104.8, 107.5, 108.3, 119.6, 127.2,
47.6, 148.1, 152.2, 155.7, 162.1, 162.5, 172.1.
3
3
J = 4.9 Hz, 2 H, NCH CH ), 2.72 [t, J = 4.9 Hz, 2 H, N(CH ) CH ], 3.40
2
2
2
2
2
3
(t, J = 4.9 Hz, 2 H, NCH ), 3.75 (s, 3 H, CH ), 4.20 (s, 2 H, CH CO), 6.63
2
3
2
LC-MS (ESI): 93% purity; m/z = 514.4 ([M + H]+).
(s, 1 H, 8-H), 7.36 (s, 1 H, 5-H), 8.49 (s, 1 H, 4-H).
13C NMR (125 MHz, DMSO-d
): δ = 20.9, 26.8, 27.9, 50.1, 51.9, 53.2,
81.5, 95.6, 107.7, 108.0, 120.7, 129.5, 149.3, 151.5, 156.8, 157.1, 164.0,
68.4.
+
+
HRMS (ESI ): m/z calcd for C27H35N O ([M + H] ): 514.2548; found:
5
6
3
7
14.2548.
1
+
+
(S)-2-(tert-Butoxycarbonylamino)-3-(2,3,6,7-tetrahydro-11-oxo-
LC-MS (ESI): 99% purity; m/z = 374.1 ([M + H] ), 764.3 ([2 M + NH ] ).
4
1
H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carbox-
Anal. Calcd for C20H23NO : C, 64.33; H, 6.21; N, 3.75. Found: C, 64.43;
H, 6.34; N, 3.76.
6
amido)propanoic Acid (23)
From 11 (0.66 g, 2.31 mmol), activated using N-hydroxysuccinimide
(
0.40 g, 3.47 mmol) and EDC·HCl (0.66 g, 3.47 mmol) in DMF (15 mL);
(
S)-2-(tert-Butoxycarbonylamino)-6-{2-[3-(methoxycarbonyl)-2-
and subsequent coupling with 14 (0.56 mg, 2.77 mmol) in the pres-
ence of DIPEA (0.72 g, 0.94 mL, 5.54 mmol) in DMF (30 mL); yield:
0
1
oxo-7,8-dihydro-2H-pyrano[3,2-g]quinolin-9(6H)-yl]acet-
amido}hexanoic Acid (28)
.66 g (61%, 1.40 mmol); orange oil.
Compound 26 (0.69 g, 1.85 mmol) was dissolved in a mixture of CH2-
Cl –TFA (1:1, 20 mL) and stirred for 1 h at r.t. Afterwards, the mixture
was evaporated to obtain 27, which was used in the next step without
further purification. Amide coupling was performed by applying the
aforementioned protocol. Compound 27 (0.59 g, 1.85 mmol) was acti-
vated with N-hydroxysuccinimide (0.32 g, 2.78 mmol) and EDC·HCl
H NMR (500 MHz, DMSO-d ): δ = 1.35 [s, 9 H, C(CH ) ], 1.83–1.89 [m,
6
3
3
2
4
H, N(CH CH ) ], 2.67–2.73 {m, 4 H, N[(CH )CH ] }, 3.29–3.36 [m, 5
2 2 2 2 2 2
3
H, N(CH ) , CH ], 3.77–3.82 (m, 1 H, CH ), 4.06 (q, J = 7.0 Hz, 1 H, CO-
CH), 7.13 (d, J = 7.6 Hz, 1 H, OCONH), 7.22 (s, 1 H, 5-H), 8.49 (s, 1 H, 4-
H), 8.83 (t, J = 5.7 Hz, 1 H, CONH). CO H was not detected.
1
2
2
2
2
3
3
2
3
α
C NMR (125 MHz, DMSO-d ): δ = 19.7, 20.7, 26.9, 28.3, 49.2, 49.7,
(0.53 g, 2.78 mmol) in DMF (50 mL) and coupled with N -Boc-L-Lys-
6
5
3.9, 78.4, 104.8, 107.5, 107.7, 119.6, 127.3, 147.8, 148.3, 152.3, 155.1,
OH (15; 0.55 g, 2.22 mmol) in the presence of DIPEA (0.57 g, 0.76 mL,
4.44 mmol) in DMF (50 mL) to afford compound 28; yield: 0.53 g
(53%, 0.97 mmol); orange solid; mp 194–197 °C.
161.8, 163.0, 172.4. The CH and DMSO signals overlapped.
2
LC-MS (ESI): 97% purity; m/z = 472.2 ([M + H]+).
+
1H NMR (500 MHz, DMSO-d
CH CH CH CH NH), 1.36 [s, H, C(CH ) ], 1.38–1.43 (m,
):
δ
=
1.32–1.38 (m,
2
2
H,
H,
+
6
HRMS (ESI ): m/z calcd for C24H29N O ([M + H] ): 472.2078; found:
3
7
9
2
2
2
2
3 3
472.2073.
CH CH CH CH NH), 1.49–1.58 (m, 1 H, CH CH CH CH NH), 1.59–1.67
2
2
2
2
2
3
2
2
2
(
m, 1 H, CH CH CH CH NH), 1.88 (quint, J = 5.7 Hz, 2 H, NCH CH ),
2
2
2
2
2
2
2.70–2.73 [m,
2
H, N(CH ) CH ], 3.02–3.10 (m,
2
H,
2
2
2
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 245–255

2
53
Synthesis
D. Häußler, M. Gütschow
Paper
3
CH CH CH CH NH), 3.44 (t, J = 5.7 Hz, 2 H, NCH ), 3.74 (s, 3 H, CH ),
(S)-tert-Butyl 3-(6,7-Dimethoxy-2-oxo-2H-chromene-3-carbox-
2
2
2
2
2
3
3
.79–3.85 (m, 1 H, COCH), 4.02 (s, 2 H, CH ), 6.29 (s, 1 H, 8-H), 6.96 (d,
amido)-1-(4-nitrobenzylamino)-1-oxopropan-2-ylcarbamate (32)
2
3
3
J = 8.7 Hz, 1 H, OCONH), 7.33 (s, 1 H, 5-H), 8.07 (t, J = 5.5 Hz, 1 H,
CH CONH), 8.47 (s, 1 H, 4-H), 12.36 (s, 1 H, CO H).
Compound 19 (0.80 g, 1.83 mmol) was activated using HATU (0.70 g,
1.83 mmol) and DIPEA (0.47 g, 0.62 mL, 3.66 mmol) in anhydrous
DMF (20 mL) for 15 min at r.t. Afterwards, 4-nitrobenzylamine hydro-
chloride (31; 0.35 g, 1.83 mmol) was added and the mixture was
stirred for 16 h overnight. Solvents were then removed in vacuo. The
2
2
13
C NMR (125 MHz, DMSO-d ): δ = 20.8, 23.2, 26.8, 28.3, 28.8, 30.5,
6
3
1
8.5, 50.6, 51.9, 54.2, 53.2, 78.1, 95.4, 107.5, 107.5, 120.8, 129.3,
49.3, 151.8, 155.7, 156.9, 157.1, 164.1, 167.7, 174.3.
LC-MS (ESI): 94% purity; m/z = 546.3 ([M + H]+).
+
reaction mixture was diluted with CH Cl (100 mL) and washed with
2 2
aq 10% citric acid (100 mL), aq 10% NaHCO3 (100 mL), H O (100 mL)
2
+
HRMS (ESI ): m/z calcd for C 7H N O ([M + H] ): 546.2446; found:
2
35
3
9
and brine (100 mL), dried (Na SO ), filtered, and the solvent was re-
2
4
546.2441.
moved under reduced pressure. The product was crystallized from
EtOAc; yield: 0.93 g (89%, 1.64 mmol); yellow solid; mp 222 °C (dec.).
(
S)-2-(tert-Butoxycarbonylamino)-6-(7-nitrobenzo[c][1,2,5]oxadi-
1
H NMR (500 MHz, DMSO-d ): δ = 1.57 [s, 9 H, C(CH ) ], 3.40–3.46 (m,
6
3 3
azol-4-ylamino)hexanoic Acid (29)
1
H, CH ), 3.73–3.80 (m, 1 H, CH ), 3.82 (s, 3 H, OCH ), 3.90 (s, 3 H,
2
2
3
α
NaHCO3 (1.19 g, 14.13 mmol), N -Boc-L-Lys-OH (15; 0.70 g, 2.83
mmol), and 4-chloro-7-nitrobenzofurazan (0.49 g, 2.43 mmol) were
dissolved in a mixture of H O and MeCN (1:1; 20 mL). The solution
was stirred at 75 °C for 1 h. The solvents were removed in vacuo, and
the remaining crude product was dissolved in CH Cl2 (100 mL) and
washed with aq 10% KHSO4 (2 × 100 mL) and H O (100 mL), dried
3
OCH ), 4.17–4.21 (q, J = 8.2 Hz, 1 H, COCH), 4.35–4.44 (m, 2 H,
CHCONHCH ), 7.18 (s, 1 H, 8-H), 7.19 (d, J = 7.9 Hz, 1 H, OCONH), 7.51
(d, J = 8.7 Hz, 1 H
8
H, CONH).
13
3
3
2
3
3
2
), 7.53 (s, 1 H, 5-H), 8.10 (d, J = 8.7 Hz, 2 Harom),
arom
3
3
.69 (t, J = 5.5 Hz, 1 H, CONH), 8.81 (s, 1 H, 4-H), 8.86 (t, J = 5.5 Hz, 1
2
2
C NMR (125 MHz, DMSO-d ): δ = 28.3, 40.7, 41.9, 54.5, 56.2, 56.7,
6
(Na SO ), filtered, and evaporated; yield: 0.83 g (83%, 2.03 mmol);
2 4
7
1
8.6, 99.8, 110.2, 111.2, 114.4, 123.3, 128.3, 146.5, 146.7, 147.7, 148.2,
50.9, 155.1, 155.6, 161.1, 162.0, 170.7.
brown solid; mp 99–102 °C.
1
H NMR (500 MHz, DMSO-d ): δ = 1.34 [s, 9 H, C(CH ) ], 1.37–1.46 (m,
6
3
3
LC-MS (ESI): 97% purity; m/z = 571.5 ([M + H]+).
+
2
H, CH CH CH CH NH), 1.55–1.72 (m, 4 H, CH CH CH CH NH), 3.44–
2 2 2 2 2 2 2 2
+
HRMS (ESI ): m/z calcd for C27H N O ([M + H] ): 571.2035; found:
3
.45 (m, 2 H, CH CH CH CH NH), 3.82–3.86 (m, 1 H, COCH), 6.39 (d,
30
4
10
2
2
2
2
3
3
3
571.2032.
J = 8.9 Hz, 1 Harom), 7.04 (d, J = 7.9 Hz, 1 H, OCONH), 8.49 (d, J = 8.9
3
Hz, 1 Harom), 9.54 (t, J = 5.2 Hz, 1 H, NH), 12.42 (s, 1 H, CO H).
2
1
3
(S)-N-[2-(4-Guanidinobenzamido)-3-(4-guanidinobenzylamino)-
-oxopropyl]-6,7-dimethoxy-2-oxo-2H-chromene-3-carboxamide
Dihydrochloride (37)
C NMR (125 MHz, DMSO-d ): δ = 23.3, 27.3, 28.4, 30.6, 43.3, 53.5,
6
3
7
8.2, 99.3, 120.7, 138.2, 144.4, 144.7, 145.4, 155.8, 174.4.
LC-MS (ESI): 95% purity; m/z = 410.4 ([M + H]+).
The N-protecting group was cleaved by dissolving 32 (0.93 g, 1.64
+
+
HRMS (ESI ): m/z calcd for C 7H N O ([M + H] ): 410.1670; found:
4
1
23
5
7
mmol) in a solution of CH Cl and TFA (1:1, 20 mL) and stirring the
2
2
10.1672.
mixture for 1 h at r.t. The corresponding ammonium trifluoroacetate
was obtained after removing the solvent in vacuo. 4-Nitrobenzoic
acid (33; 0.27 g, 1.64 mmol) was dissolved in anhydrous DMF (20 mL)
and activated for 15 min at r.t. using HATU (0.62 g, 1.64 mmol) and
DIPEA (0.64 g, 0.84 mL, 4.92 mmol). Afterwards, the ammonium tri-
fluoroacetate (0.959 g, 1.64 mmol) was added and the mixture was
stirred overnight at r.t. The solvent was removed and the residue was
suspended in EtOAc (100 mL). The insoluble material, compound 34,
was collected by filtration and directly subjected to the next reaction
step. To a stirred suspension 34 (0.73 g, 1.18 mmol) in EtOAc (100 mL)
(S)-5-{3-[5-(tert-Butoxycarbonylamino)-5-carboxypentyl]thio-
ureido}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic Acid (30)
Fluorescein isothiocyanate (0.39 g, 1.0 mmol) was added to a solution
of N -Boc-L-Lys-OH (15; 0.25 g, 1.0 mmol) and Et N (0.15 g, 0.21 mL,
1
h. Afterwards, the mixture was evaporated, and the residue was puri-
fied by column chromatography using EtOAc to EtOAc–MeOH–AcOH
α
3
.5 mmol) in EtOH (15 mL) and the mixture was stirred at 60 °C for 6
(
1
9:1:0.01) as eluent; yield: 0.33 g (52%, 0.52 mmol); red solid; mp
10–112 °C.
1H NMR (500 MHz, DMSO-d ):
were added SnCl ·2 H O (2.66 g, 11.8 mmol) and H O (0.85 g, 47.20
2
2
2
mmol). The solution was stirred under reflux for 4 h. The solvent was
removed in vacuo and the residue was suspended in aq 2 M NaOH
δ
=
1.36 [s, 11 H, C(CH ) ,
6
3 3
CH CH CH CH NH], 1.52–1.63 (m, 3 H, CH CH CH CH NH), 1.65–1.73
2
2
2
2
2
2
2
2
(
200 mL) and extracted with CH Cl (3 × 100 mL). The combined or-
2 2
(
m, 1 H, CH CH CH CH NH), 3.48 (br s, 2 H, CH CH CH CH NH), 3.84–
2 2 2 2 2 2 2 2
ganic extracts were washed with H O (150 mL) and brine (150 mL),
2
3
.89 (m, 1 H, COCH), 6.54–6.57 (m, 2 H
.66 (s, 1 Harom), 6.67 (s, 1 H
J = 8.5 Hz, 1 Harom), 7.73 (d, J = 6.6 Hz, 1 H, OCONH), 8.07 (s, 1H, NH
), 6.59–6.60 (m, 2 Harom),
), 7.01 (d, J = 8.2 Hz, 1 Harom), 7.16 (d,
arom
3
dried (Na
derivative 35 (98 mg, 0.18 mmol), N,N′-di-Boc-S-methylisothiourea
36; 94 mg, 0.32 mmol), HgCl (93 mg, 0.34 mmol), Et N (55 mg, 0.07
SO ), and evaporated. A mixture of the obtained bis-aniline
2
4
6
arom
3
3
(
2
3
or OH), 8.21 (s, 1 Harom), 9.83 (s, 1 H, NH or OH), 10.07 (s, 1 H, NH or
OH), 12.07 (s, 2 H, CO H).
mL, 0.54 mmol), and CH Cl (20 mL) was stirred at r.t. for 24 h. The
2
2
2
solution was passed through a pad of Celite and the pad was washed
with CH Cl (50 mL) and MeOH (50 mL). The solvents were removed
13
C NMR (125 MHz, DMSO-d ): δ = 21.2, 23.3, 28.4, 30.7, 43.8, 53.6,
8.1, 83.1, 102.4, 109.9, 112.7, 116.5, 124.1, 126.7, 129.1, 141.6, 147.2,
52.0, 155.8, 159.6, 168.7, 172.1, 174.3, 180.5.
6
2
2
7
1
in vacuo. The resulting Boc-protected bis-guanidine was purified by
column chromatography using EtOAc–MeOH–Et N (9:1:0.01) as elu-
3
+
–
LC-MS (ESI): 99% purity; m/z = 636.44 ([M + H] ), 634.39 ([M – H] ).
ent. The material (148 mg, 0.14 mmol) was dissolved in CH Cl (5 mL)
2
2
+
+
and a 50% solution of TFA in CH Cl (10 mL) was added. After stirring
at r.t. for 2 h, the solvent was evaporated. The resulting oil was puri-
2 2
HRMS (ESI ): m/z calcd for C32H33N O S ([M + H] ): 636.2010; found:
3
9
636.2008.
fied by preparative HPLC. The HPLC eluent was MeOH–H O (30:70)
2
for 8 min. To the product, few drops of aq 1 M HCl were added to form
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 245–255

2
54
Synthesis
D. Häußler, M. Gütschow
Paper
the hydrochloride salt. The final product 37 was obtained after lyo-
philization; yield: 23 mg (2% over five steps, 0.033 mmol); yellow sol-
id; mp 173–176 °C.
G.; Jan, L. Y. Science 2002, 296, 1700. (b) Vázquez, M. E.;
Rothman, D. M.; Imperiali, B. Org. Biomol. Chem. 2004, 2, 1965.
(c) Kajihara, D.; Abe, R.; Iijima, I.; Komiyama, C.; Sisido, M.;
Hohsaka, T. Nat. Methods 2006, 3, 923. (d) Loving, G.; Imperiali,
B. J. Am. Soc. Chem. 2008, 130, 13630. (e) Lee, H. S.; Guo, J.;
Lemke, E. A.; Dimla, R. D.; Schultz, P. G. J. Am. Chem. Soc. 2009,
131, 12921. (f) Li, H. W.; Li, Y.; Dang, Y. Q.; Ma, L. J.; Wu, Y.; Hou,
G.; Wu, L. Chem. Commun. 2009, 4453. (g) Li, Y.; Li, H. W.; Ma, L.
J.; Dang, Y. Q.; Wu, Y. Chem. Commun. 2010, 46, 3768. (h) Li, C.;
Henry, E.; Mani, N. K.; Tang, J.; Brochon, J. C.; Deprez, E.; Xie, J.
Eur. J. Org. Chem. 2010, 2395. (i) Frizler, M.; Yampolsky, I.;
Baranov, M. S.; Stirnberg, M.; Gütschow, M. Org. Biomol. Chem.
1
H NMR (500 MHz, DMSO-d ): δ = 3.60–3.67 (m, 1 H, CH ), 3.81 (s, 3
6
2
H, OCH ), 3.89–3.98 (m, 1 H, CH ), 3.90 (s, 3 H, OCH ), 4.27–4.35 (m, 2
3
2
3
3
H, CHCONHCH ), 4.66–4.71 (m, 1 H, COCH), 7.14 (d, J = 8.6 Hz, 2
Harom), 7.17 (s, 1 H, 8-H), 7.32 (d, J = 8.6 Hz, 4 Harom), 7.46 (s, 4 H,
NH2guanidine), 7.54 (s, 1 H, 5-H), 7.70 (s, 4 H, NH
Hz, 2 Harom), 8.70 (t, J = 6.2 Hz, 1 H, CONH), 8.79 (d, J = 7.6 Hz, 1 H,
2
3
3
), 7.96 (d, J = 8.6
2guanidine
3
3
3
CONH), 8.84 (s, 1 H, 4-H), 8.99 (t, J = 6.0 Hz, 1 H, CONH), 9.97 (s, 1 H,
NHguanidine), 10.35 (s, 1 H, NHguanidine).
13
C NMR (125 MHz, DMSO-d ): δ = 41.8, 45.5, 54.0, 56.2, 56.7, 99.8,
6
2013, 11, 5913. (j) Speight, L. C.; Muthusamy, A. K.; Goldberg, J.
110.2, 111.2, 114.3, 123.0, 124.3, 128.4, 129.2, 131.2, 133.9, 137.8,
138.6, 146.7, 148.3, 150.9, 155.1, 155.9, 156.2, 161.1, 162.2, 166.0,
170.0.
M.; Warner, J. B.; Wissner, R. F.; Willi, T. S.; Woodman, B. F.;
Mehl, R. A.; Petersson, E. J. J. Am. Chem. Soc. 2013, 18, 18806.
3) For coumarin-labeled amino acids, see, for example: (a) Brun,
M. P.; Bischoff, L.; Garbay, C. Angew. Chem. Int. Ed. 2004, 43,
3432. (b) Paschalidou, K.; Neumann, U.; Gerhartz, B.; Tzougraki,
C. Biochem. J. 2004, 382, 1031. (c) Kitamatsu, M.; Futami, M.;
Sisido, M. Chem. Commun. 2010, 46, 761. (d) Agnes, R. S.;
Jernigan, F.; Shell, J. R.; Sharma, V.; Lawrence, D. S. J. Am. Soc.
Chem. 2010, 132, 6075. (e) Nizamov, S.; Willig, K. I.; Sednev, M.
V.; Belov, V. N.; Hell, S. W. Chem. Eur. J. 2012, 18, 16339.
(
LC-MS (ESI): 98% purity; m/z = 644.5 ([M + H]+).
+
+
HRMS (ESI ): m/z calcd for C31H33N O ([M + AcOH] ): 351.6357;
9
7
found: 351.6369.
Biochemical Investigations
Kinetic parameters of inhibition of the serine proteases human
matriptase-2, human thrombin, bovine factor Xa, and bovine trypsin
were determined in duplicate measurements by using literature pro-
(f) Gehrig, S.; Mall, M. A.; Schultz, C. Angew. Chem. Int. Ed. 2012,
51, 6258. (g) Wysocka, M.; Gruba, N.; Miecznikowska, A.;
18,19
tocols.
Assays were performed in the presence of five different
Popow-Stellmaszyk, J.; Gütschow, M.; Stirnberg, M.; Furtmann,
N.; Bajorath, J.; Lesner, A.; Rolka, K. Biochimie 2014, 97, 121.
concentrations of inhibitor 37 at pH 8, either at 25 °C or 37 °C. Reac-
tions were followed over 15 min.
(h) Meimetis, L. G.; Carlson, J. C.; Giedt, R. J.; Kohler, R. H.;
Affinity to DNA of 37 was measured by anisotropy titration, following
Weissleder, R. Angew. Chem. Int. Ed. 2014, 53, 7531. (i) Mertens,
M. D.; Gütschow, M. Synthesis 2014, 46, 2191.
21
a
literature protocol.
The short hairpin oligonucleotide (GG-
CAAATTTCAGTTTTTCTGAAATTTGCC) was used containing the hairpin
loop TTTTT and the adenine/thymine-rich binding site AAATTT. The
experiments were performed in duplicate at pH 7.5 and 25 °C.
(4) Murase, T.; Yoshihara, T.; Yamada, K.; Tobita, S. Bull. Chem. Soc.
Jpn. 2013, 86, 510.
(5) Wirtz, L.; Auerbach, D.; Jung, G.; Kazmaier, U. Synthesis 2012,
44, 2005.
(
6) For examples, see: (a) Katritzky, A. R.; Narindoshvili, T.;
Angrish, P. Synthesis 2008, 2013. (b) Shilin, S. V.; Garazd, M. M.;
Khilya, V. P. Chem. Nat. Compd. 2008, 44, 301. (c) Vázquez, O.;
Sánchez, M. I.; Mascareñas, J. L.; Vázquez, M. E. Chem. Commun.
2010, 46, 5518. (d) Veselovskaya, M. V.; Shilin, S. V.; Khilya, V. P.
Chem. Nat. Compd. 2012, 48, 757. (e) Wirtz, L.; Kazmeier, U. Eur.
J. Org. Chem. 2011, 7062. (f) Kohl, F.; Schmitz, J.; Furtmann, N.;
Schulz-Fincke, A. C.; Mertens, M. D.; Küppers, J.; Benkhoff, M.;
Tobiasch, E.; Bartz, U.; Bajorath, J.; Stirnberg, M.; Gütschow, M.
Org. Biomol. Chem. 2015, 13, 10310.
Acknowledgment
D.H. holds a fellowship from the Bonn International Graduate School
of Drug Sciences (BIGS DrugS). D.H. was also supported by a fellow-
ship from the NRW International Graduate Research School Biotech-
Pharma. The authors thank Dr. Matthias D. Mertens, Anna Pecht, and
Jessica Geller (all University of Bonn, Germany) for assistance. We
thank Prof. M. Eugenio Vázquez (Departamento de Química Orgánica
and Departamento de Bioquímica y Biología Molecular, Universidade
de Santiago de Compostela, Santiago de Compostela, Spain) for per-
forming the DNA binding experiments.
(7) Mertens, M. D.; Schmitz, J.; Horn, M.; Furtmann, N.; Bajorath, J.;
Mareš, M.; Gütschow, M. ChemBioChem 2014, 15, 955.
(8) Takadate, A.; Masuda, T.; Murata, C.; Tanaka, T.; Irikura, M.;
Goya, S. Anal. Sci. 1995, 11, 97.
Supporting Information
(9) Song, A.; Wang, X.; Lam, K. S. Tetrahedron Lett. 2003, 44, 1755.
10) Frizler, M.; Mertens, M. D.; Gütschow, M. Bioorg. Med. Chem.
Lett. 2012, 22, 7715.
(
(
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1560361.
S
u
p
p
ortioIgnfrm oaitn
S
u
p
p
ortioIgnfrm oaitn
11) (a) Besson, T.; Coudert, G.; Guillaumet, G. J. Heterocycl. Chem.
1991, 28, 1517. (b) Kitamura, N.; Fukagawa, T.; Kohtani, S.;
Kitoh, S.; Kunimoto, K. K.; Nakagaki, R. J. Photochem. Photobiol. A
Chem. 2007, 188, 378.
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(
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(
2) For non-coumarin-labeled amino acids, see, for example:
a) Cohen, B. E.; McAnaney, T. B.; Park, E. S.; Jan, Y. N.; Boxer, S.
(
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 245–255

2
55
Synthesis
D. Häußler, M. Gütschow
Paper
(
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