Chiral N-(Coumarin-3-ylcarbonyl)-α-amino acids: Fluorescent markers for amino acids and dipeptides

Article abstract of DOI:10.1055/s-2008-1067078

N-(Coumarin-3-ylcarbonyl)benzotriazole has been coupled with free amino acids, N-terminal-protected lysines and dipeptides to afford fluorescent amino acids and dipeptides (76-89% yield) with retention of original chirality. N ^α- and N^ε

Full text of DOI:10.1055/s-2008-1067078

PAPER
2013
Chiral N-(Coumarin-3-ylcarbonyl)-a-amino Acids: Fluorescent Markers for
Amino Acids and Dipeptides
Fluorescent
M
arke
l
rs for
A
a
minoAcid
n
s
andDipeptides R. Katritzky,* Tamari Narindoshvili, Parul Angrish
Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
Fax +1(352)3929199; E-mail: katritzky@chem.ufl.edu
Received 17 January 2008; revised 27 March 2008
of an extended spectral range, high emission quantum
yield, photostability and good solubility in many solvents.
Abstract: N-(Coumarin-3-ylcarbonyl)benzotriazole has been cou-
pled with free amino acids, N-terminal-protected lysines and dipep-
tides to afford fluorescent amino acids and dipeptides (76–89%
yield) with retention of original chirality. Na- and Ne-Coumarin-
labeled lysines are obtained by simple, two-step procedures. Na-Cbz-
Ne-(Coumarin-3-ylcarbonyl)-L-lysine is demonstrated to be an opti-
cally active fluorescent marker for labeling amino acids in solution-
phase syntheses.
The many bioanalytical applications of fluorescently la-
beled proteins include in vitro and in vivo imaging,6,23
high-throughput screening,24 proteomics,24–26 diagnos-
tics,9,27,28 single biomolecule spectroscopy,26,29 HPLC
control30 and exploring protein conformations.7
Probes for studying protein dynamics and electrostatics
should be (i) sensitive to the state of hydrogen bonding in
the solvent environment31 and (ii) readily incorporated re-
giospecifically at the C- or the N-terminus of a protein.
The use of an amino acid as a fluorescent chromophore of-
fers a good possibility to synthesize fluorescent peptides
by solid-phase peptide synthesis (SPPS).
Key words: amino acids, peptides, coupling, coumarin fluoro-
phore, benzotriazole methodology
The imaging of living cells is a big challenge in cell biol-
ogy. Understanding the nature and dynamics of cellular
events in living organisms is assisted by imaging cell
components such as the nucleic acids, proteins and metab-
olites. Fluorescent markers are the visualization tools
most commonly used, particularly fluorescent tagging
with organic fluorophores1 or green fluorescent protein
(GFP).2 Highly sensitive fluorescence derivatization
techniques3 are gaining an increasing share of the analyt-
ical world market, becoming competitive with, for exam-
ple, radioimmunoassay.4
Previously, enantioselective syntheses of coumarin-
containing amino acids have been carried out by the dia-
stereoselective alkylation of chiral glycine equiva-
lents.13,20,32 For example, the chiral auxiliaries of
Oppolzer and co-workers and of Williams were used for
the synthesis of L-(6,7-dimethoxycoumarin-4-yl)alanine
(1) and L-(7-methoxycoumarin-4-yl)alanine, respectively.
These methods produced optically pure amino acids;
however, they required multiple steps (Scheme 1) and the
products were obtained in poor overall yields. Recently, a
synthesis of coumarin-containing amino acids was
reported33 utilizing protected aspartic and glutamic acids
as chiral starting materials, which led to coumarinylala-
nines and coumarinylethylglycines in a two- or a three-
step process.
Radioactive labeling of peptides usually involves the in-
troduction of a radiolabeled amino acid as part of the nat-
ural structure of the peptide. In contrast, fluorescent tags
are introduced as an additional moiety to the molecule, by
a variety of strategies.5–10 Derivatives of rhodamine,
fluorescein11,12 and coumarin13,14 are widely used as fluo-
rescent markers for peptides and other biomolecules. Xan-
thenes such as rhodamines, fluoresceins and Alexa dyes
are advantageously bright; however, drawbacks of exist-
ing derivatives in reporting protein conformational chang-
es include (i) relatively long, flexible linkers between the
probe and protein, which questions whether the probe mo-
tions faithfully mirror the motions of the residue to which
it is attached, and (ii) multiple charges and relatively large
surface areas, which can perturb local structure or motion,
and inhibit labeling at certain positions.7,13
Coumarin-labeled lysines are of considerable interest for
the design and synthesis of fluorogenic triple-helical sub-
strates for the analysis of matrix metalloproteinase family
members. However, the attempted preparation of Na-
Fmoc-Ne-[(7-methoxycoumarin-4-yl)acetyl]lysine (10),18
by reaction of commercially available Na-Fmoc-lysine in
N,N-dimethylformamide with the preformed N-hydroxy-
benzotriazole ester of 7-methoxycoumarin-4-acetic acid,
gave problems in (i) separation of 10 from unreacted
fluorophore, (ii) side reactions and (iii) crystallization of
the product. An alternative four-step synthetic approach
(Scheme 2) using a copper-complexed intermediate18
gave 10 in only 17% overall yield.
Coumarins (benzopyran-2-ones), the largest class of laser
dyes for the blue-green region,14–22 are highly sensitive.
They have provided the most commercially acceptable
categories of fluorescent derivatives with the advantages
Berthelot and co-workers19 labeled Na-Fmoc-lysine (11)
in three steps using the active coumarincarboxylic acid N-
hydroxysuccinimide esters 13 (Scheme 3); the target
compounds 14a and 14b were obtained in 64% and 50%
yield, respectively.
SYNTHESIS 2008, No. 13, pp 2013–2022
Advanced online publication: 16.05.2008
DOI: 10.1055/s-2008-1067078; Art ID: M00108SS
© Georg Thieme Verlag Stuttgart · New York
x
x.
x
x
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0
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A. R. Katritzky et al.
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EtO2C
SMe
SMe
O
N
N
SMe
SMe
NH
SO2
N
Me3Al
SO2
3
2
n-BuLi, THF,
4-chloromethyl-6,7-
dimethoxy-
NH2
COOH
O
N
coumarin
SO2
N
MeO
SMe
MeO
MeO
ii) 1 M LiOH, THF
MeS
O
MeO
O
O
1
O
4
overall yield 12%
Scheme 1 Synthesis of L-(6,7-dimethoxycoumarin-4-yl)alanine (1)13
O
O
O
NH2
OH
O
CuCO3, Cu(OH)2
Cu)2
OH
H2N
reflux, 4 h
H2N
NH2
O
MeO
O
6
7
5
O
O
O
Cu)2
Chelex 100 (H+ form)
H2O–MeOH
DCC, HOBt, DIPEA
DMF–MeCN
NH2
NH
O
MeO
O
8
O
OH
O
OH
O
O
NH2
NH
NHFmoc
NH
O
FmocOSu, NaHCO3
H2O–acetone
MeO
O
O
MeO
O
9
10
DCC = N,N-dicyclohexylcarbodiimide
DIPEA = N,N-diisopropylethylamine
FmocOSu = N-(fluoren-9-ylmethoxycarbonyloxy)succinimide
HOBt = N-hydroxybenzotriazole
Scheme 2 Synthesis of Na-Fmoc-Ne-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine (10)18
NH3
NH2
O
HN
O
X
O
O
O
b, c
a
X
O
O
O
N
Fmoc
Fmoc
N
H
O
COO
N
COOH
H
Fmoc
N
H
COOH
11
12
13
14a,b
(a) DIPEA, DMF, 0 °C, 30 min
(b) r.t., 2 h
14a, X = OMe (64%)
14b, X = NEt2 (50%)
(c) 2 M HCl, 4 °C, 12 h
Scheme 3 Synthesis of coumarin-labeled lysines 14a and 14b19
Synthesis 2008, No. 13, 2013–2022 © Thieme Stuttgart · New York

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Fluorescent Markers for Amino Acids and Dipeptides
2015
The quantitative analysis of peptides and proteins in com- lysine (18g) in aqueous acetonitrile at 20°C (Scheme 5) to
plex biological matrixes is hindered by structural similar- give the N-(coumarin-3-ylcarbonyl)-L-a-amino acids
ities between analyte, degradation products, impurities 19a–g and racemate [19c + 19c¢] in 76–89% yield
and endogenous components.34 The development of de- (Table 1). Products 19a–g retained the original chirality;
rivatization strategies for the synthesis of suitable, optical- each showed a single retention-time peak upon analytical
ly pure fluorescent labeling probes remains a considerable HPLC. The racemate [19c + 19c¢] displayed the expected
challenge.
two peaks.
We now report straightforward syntheses of coumarin-
labeled amino acids and dipeptides which afford enantio-
merically pure fluorescent building blocks suitable for
SPPS. Our simple, two-step synthetic route (i) converts
coumarin-3-carboxylic acid (15) into its active benzotria-
zolide 17 and (ii) couples fluorophore with Cbz- and
Fmoc-protected lysines and with the N-terminus of free
amino acids, as well as with dipeptides, providing diverse
optically pure fluorescent probes in 76–89% yield.
R
O
O
O
O
Et3N
OH
H
17
+
H2N
N
OH
MeCN–H2O
r.t., 1 h
O
R
18a–g, [18c + 18c']
19a–g, [19c + 19c']
Scheme 5 Synthesis of coumarin-labeled amino acids
N-Terminus-protected lysines are of great current
interest12,18,19 as versatile fluorescent building blocks for
SPPS. In addition to Na-coumarin-labeled Ne-Cbz-L-
lysine 19g, with the free Ne-amino group suitable for
SPPS, we successfully synthesized the isomeric Ne-cou-
marin-labeled Na-Cbz-L-lysine 21a (89% yield) and the
Fmoc-protected analogue, Ne-coumarin-labeled Na-
Fmoc-L-lysine 21b (87% yield) (Scheme 6) utilizing the
convenient two-step general procedure, as described
above for the preparation of N-(coumarin-3-ylcarbonyl)-
a-amino acids 19. The enantiomeric purity of 21a and of
21b was demonstrated by NMR and HPLC analysis.
In the first step (Scheme 4), coumarin-3-carboxylic acid
(15) was treated with 1H-benzotriazole (16) and thionyl
chloride in tetrahydrofuran at 20 °C to give the corre-
sponding
3-(1H-benzotriazol-1-ylcarbonyl)-2H-
chromen-2-one (17) as a stable compound in 87% yield
(all yields indicated in this work are calculated for pure
products).
O
O
O
O
O
SOCl2
THF
N
N
N
+
N
N
H
COOH
O
N
O
NH2
17
15
16
O
NH
Scheme 4 Preparation of 3-(1H-benzotriazol-1-ylcarbonyl)-2H-
chromen-2-one (17)
Et3N
OH
PG
17
+
Reaction of the coumarinoylbenzotriazole 17 with the
amino group of peptides or proteins should allow the la-
beling of such biomolecules under the gentle conditions
used in recent research in our laboratory.35 Benzotriazole
17 was thus coupled with diverse L-amino acids 18a–f,
with the DL-amino acid [18c + 18c¢]36 and with Ne-Cbz-L-
N
MeCN–H2O
H
OH
PG
N
O
H
O
21a,b
20a,b
20a, 21a: PG = Cbz; 20b, 21b: PG = Fmoc
Scheme 6 Synthesis of coumarin-labeled lysines 21a and 21b
Table 1 Yields and HPLC Analysis of N-(Coumarin-3-ylcarbonyl)-a-amino Acids 19a–g and Racemic Mixture [19c + 19c¢]
Reactant 18
R of coumarin-3-ylcarbonyl product 19
Me (19a)
Yielda (%)
tRb (min)
3.96
L-Ala-OH (18a)
L-Ser-OH (18b)
L-Met-OH (18c)
L-Phe-OH (18d)
L-Trp-OH (18e)
L-Val-OH (18f)
82
76
88
88
78
81
77
89
CH2OH (19b)
4.33
(CH2)2SMe (19c)
3.85
CH2Ph (19d)
4.03
1H-indol-3-ylmethyl (19e)
i-Pr (19f)
4.04
3.64
Ne-Cbz-L-Lys-OH (18g)
DL-Met-OH ([18c + 18c¢])
a Isolated yield.
(CH2)4NHCbz (19g)
(CH2)2SMe ([19c + 19c¢])
3.93
3.69, 4.13
b tR = retention time. For conditions, see the experimental section.
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A. R. Katritzky et al.
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O
O
O
O
Cbz
NH
O
NH
O
NH
O
O
O
O
O
OH
O
OH
N
N
H
OH
Fmoc
H
N
O
O
H
O
19g
22
21b
Figure 1
Coupling 2 equivalents of benzotriazole 17 with 1 equiv- yield, respectively (Table 3). The 13C NMR spectrum of
alent of free lysine gave the bis(coumarin)-substituted [25b + 25b¢] showed duplication of almost all the aliphat-
lysine 22 (Figure 1) in 86% yield.
ic and carbonyl carbon signals. As anticipated, dipeptide
25a showed no duplication of carbon signals and the H
NMR spectrum in DMSO-d6 displayed a single prominent
doublet at 1.30 ppm for the methyl group of the L-alanine
fragment. These data, along with HPLC analysis, demon-
strate the enantiomeric purity of 25a.
1
We synthesized dipeptides containing a coumarin fluoro-
phore by two routes: (i) stepwise elongation of N-cou-
marinoyl-a-amino acids from the C-terminus and (ii)
direct introduction of the coumarin-containing fluoro-
phore into free dipeptides at the N-terminus.
Ne-Coumarin-labeled Na-Cbz-L-lysine 21a proved to be a
convenient starting material in the stepwise elongation
route (i) for the preparation of fluorescent dipeptides in
solution. In future work we plan to use coumarin-labeled
lysines 21a and 21b as fluorescent building blocks for la-
beling peptides in SPPS. The benzotriazole derivative 26
(Figure 2) was synthesized from 21a using a methodology
similar to that described in Scheme 7 for the formation of
derivative 23a. Benzotriazole 26 is stable and able to per-
form efficient acylation of amino groups in aqueous ace-
tonitrile at ambient temperature without side reactions.
Coupling 26 with L-aspartic acid gave the multifunctional
In the stepwise elongation procedure [route (i)], the key
step was the synthesis of the stable N-acylbenzotriazole
intermediates 23a and [23b + 23b¢] (Table 2). These were
prepared from 19a and [19c + 19c¢] by a procedure
(Scheme 7) similar to that described in Scheme 4 for the
formation of coumarinoylbenzotriazole 17.
Subsequent coupling of N-(coumarin-3-ylcarbonyl-a-
aminoacyl)benzotriazoles 23a and [23b + 23b¢] with un-
protected L-phenylalanine (24) in aqueous acetonitrile
(Scheme 8) afforded enantiomerically pure 25a in 74%
yield and the diastereomeric mixture [25b + 25b¢] in 68%
Table 2 Yields of N-(Coumarin-3-ylcarbonyl-a-aminoacyl)benzotriazoles 23a and [23b + 23b¢]
R of coumarin-3-ylcarbonyl reactant 19
Me (19a)
Benzotriazole product 23
Yielda (%)
N-(coumarin-3-ylcarbonyl)-L-Ala-Bt (23a)
N-(coumarin-3-ylcarbonyl)-DL-Met-Bt ([23b + 23b¢])
78
74
(CH2)2SMe ([19c + 19c¢])
a Isolated yield.
O
O
O
O
O
O
O
O
BtH, SOCl2
CH2Cl2
H
H
N
N
OH
N
N
R
R
N
19a, [19c + 19c']
23a, [23b + 23b']
Scheme 7 Synthesis of the N-acylbenzotriazole intermediates 23a and [23b + 23b¢]
O
O
O
O
O
O
O
L-Phe-OH (24)
O
H
H
N
N
H
N
OH
Et3N
N
MeCN–H2O
R
N
N
O
R
23a, [23b + 23b']
25a, [25b + 25b']
Scheme 8 Synthesis of coumarin-labeled dipeptides by stepwise elongation
Synthesis 2008, No. 13, 2013–2022 © Thieme Stuttgart · New York

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Fluorescent Markers for Amino Acids and Dipeptides
2017
Table 3 Yields and HPLC Analysis of Coumarin-Labeled Dipeptides 25a and [25b + 25b¢]
Reactant 23
23a
Coumarin-labeled dipeptide product 25
Yielda (%)
tRb (min)
4.13
N-(coumarin-3-ylcarbonyl)-L-Ala-L-Phe-OH (25a)
N-(coumarin-3-ylcarbonyl)-DL-Met-L-Phe-OH ([25b + 25b¢])
74
68
[23b + 23b¢]
a Isolated yield.
5.67, 6.77
b tR = retention time. For conditions, see the experimental section.
O
O
O
O
O
O
O
NH
O
NH
O
NH
N
O
O
O
N
N
H
H
Cbz
N
N
Cbz
N
Cbz
N
OH
OH
OH
N
H
H
H
R
O
O
O
26
27
29a,b, [29a + 29a']
Figure 2
Table 4 Yields and HPLC Analysis of Coumarin-Labeled, Lysine-Containing Dipeptides 29a,b and [29a + 29a¢] Derived from 26
Amino acid reactant
L-Ala-OH (28a)
Coumarin-labeled dipeptide product 29
Yielda (%)
tRb (min)
Ne-(coumarin-3-ylcarbonyl)-Na-Cbz-L-Lys-L-Ala-OH (29a)
Ne-(coumarin-3-ylcarbonyl)-Na-Cbz-L-Lys-L-Phe-OH (29b)
Ne-(coumarin-3-ylcarbonyl)-Na-Cbz-L-Lys-DL-Ala-OH ([29a + 29a¢])
91
89
94
5.57
L-Phe-OH (28b)
7.43
DL-Ala-OH ([28a + 28a¢])
a Isolated yield.
5.51, 6.92
b tR = retention time. For conditions, see the experimental section.
fluorescent building block 27 in 85% yield; L- and DL- tention-time peak (4.17 min for 27), whereas the diaste-
amino acids 28a,b, [28a + 28a¢] gave targets 29a,b, [29a reomeric mixture [29a + 29a¢] showed two peaks
+ 29a¢] in 89–94% yield (Table 4).
(Table 4).
All couplings proceeded with complete retention of In route (ii), involving direct introduction of the cou-
1
chirality: comparison of the H and 13C NMR spectra of marin-containing fluorophore, the free dipeptides Gly-
29a with those of [29a + 29a¢] demonstrated that 29a is a Gly and Gly-L-Leu were directly labeled with benzotriaz-
1
single stereoisomer. Thus, the H NMR spectrum of 29a ole 17 to give the coumarin-labeled dipeptides 30 and 31
in DMSO-d6 showed a doublet at 1.28 ppm (J = 7.3 Hz) (Figure 3) in 81 and 85% yield, respectively, by a proce-
characteristic of the methyl group of the L-alanine part of dure similar to that adopted for the preparation of N-(cou-
the molecule, while the corresponding signal for the dia- marin-3-ylcarbonyl)-a-amino acids 19 (Scheme 5). This
stereomeric mixture [29a + 29a¢] gave a multiplet at 1.21– direct approach [route (ii)] is preferred for the synthesis of
1.31 ppm. The 13C NMR spectrum showed a duplication fluorescent dipeptides compared to the stepwise elonga-
of carbon peaks corresponding to the carbonyl groups and tion approach [route (i)] described above.
the aliphatic portion of the molecule for [29a + 29a¢],
Absorption (lAbs.) and fluorescence (lEm.) wavelength
while single peaks were observed for each carbon of enan-
maxima and extinction coefficients were measured for
tiomerically pure 29a. The enantiopurity of dipeptides 27
most of the labeled compounds (Figure 4, Table 5). The
and 29a,b was confirmed by HPLC analysis using a chiral
coumarin-labeled a-amino acids and dipeptides (except
column: dipeptides 27, 29a and 29b each gave a single re-
for 21b) had higher emission wavelength values and high-
O
O
O
O
O
O
H
O
N
H
NH
N
N
COOH
O
H
HOOC
30
31
Figure 3
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A. R. Katritzky et al.
PAPER
23a, [23b + 23b¢] and 26 are efficient acylation reagents
for the fluorescent labeling of amino acids and dipeptides
in solution phase because of their stability and ability to
react with amino groups in aqueous acetonitrile at ambient
temperature without side reactions. They also have the po-
tential to be applied for labeling using solid-phase syn-
thetic methods. The protocols which have been employed
should be adaptable for the syntheses of many similar flu-
orescent markers.
Melting points were determined on a Mel-Temp II capillary point
apparatus equipped with a digital thermometer. 1H (300 MHz) and
13C (75 MHz) NMR spectra were recorded on an Oxford 300 spec-
trometer in CDCl3 or DMSO-d6 with TMS as an internal reference.
Free amino acids and N-Cbz- and N-Fmoc-amino acids were pur-
chased from Fluka and Acros and were used without further purifi-
cation. Elemental analyses were performed on a Carlo Erba-1106
instrument. Optical rotations were measured on a Perkin-Elmer 241
polarimeter using the sodium D line. HPLC analyses were per-
formed on a Beckman System Gold® programmable solvent module
126, using a Chirobiotic™ T chiral column (4.6 × 250 mm), detec-
tion at 254 nm, flow rate of 1.0 mL/min and MeOH as the eluting
solvent. UV and fluorescence measurements were recorded on Cary
100 UV–Vis and FluoroMax spectrophotometers, respectively.
Figure 4 Fluorescence emission spectra of Na-(coumarin-3-ylcar-
bonyl)-Ne-Cbz-L-lysine (19g), lEx. = 295 nm, and Na-Cbz-Ne-(cou-
marin-3-ylcarbonyl)-L-lysine (21a), lEx. = 293 nm, in methanol
Table 5 Absorption and Fluorescence Data for Coumarin-Labeled
a-Amino Acids and Dipeptides
Compound
19a
19b
19c
19d
19e
19f
lAbs.a (nm)
295
lEm.a (nm)
408
eb (cm–1M–1)
c
–
294
407
–
3-(1H-Benzotriazol-1-ylcarbonyl)-2H-chromen-2-one (17)
Thionyl chloride (7.5 mmol) was added to a soln of 1H-benzotriaz-
ole (16; 25 mmol) in anhyd THF (30 mL) at r.t. and the reaction
mixture was stirred for 20 min. Coumarin-3-carboxylic acid (15; 5
mmol) was added to the reaction mixture which was stirred for 4 h
at 25 °C. The white precipitate which formed during the reaction
was filtered off, and the filtrate was concentrated under reduced
pressure. The residue was diluted with EtOAc (150 mL) and the so-
lution was washed with sat. Na2CO3 soln (3 × 50 mL) and sat. NaCl
soln (50 mL), and dried (MgSO4). Removal of the solvent under re-
duced pressure gave 17, which was recrystallized from CH2Cl2–
hexanes for elemental analysis.
294
409
–
295
408
–
d
290
–
–
295
408
392
407
405
409
408
408
409
407
406
408
–
19g
21a
21b
22
295
17600
293
17900
265
27500
Yield: 87%; white microcrystals; mp 186–187 °C.
1H NMR (CDCl3): d = 7.41 (t, J = 7.6 Hz, 1 H), 7.46 (d, J = 8.2 Hz,
1 H), 7.58 (t, J = 8.2 Hz, 1 H), 7.64–7.80 (m, 3 H), 8.16 (d, J = 8.2
Hz, 1 H), 8.34 (s, 1 H), 8.36 (d, J = 8.4 Hz, 1 H).
13C NMR (CDCl3): d = 114.4, 117.2, 117.6, 120.5, 121.9, 125.3,
126.8, 129.6, 130.9, 131.2, 134.5, 146.2, 147.0, 154.9, 157.4, 162.6.
293
–
–
–
–
–
–
–
25a
27
297
294
29a
29b
30
294
Anal. Calcd for C16H9N3O3: C, 65.98; H, 3.11; N, 14.43. Found: C,
65.67; H, 3.10; N, 14.22.
294
N-(Coumarin-3-ylcarbonyl)-a-amino Acids 19a–g, 21a,b, 22
and Racemate [19c + 19c¢]; General Procedure
294
31
294
3-(1H-Benzotriazol-1-ylcarbonyl)-2H-chromen-2-one (17; 1 mmol)
was added to a soln of the appropriate a-amino acid 18a–g, 20a,b,
[18c + 18c¢] (1 mmol) in MeCN–H2O (10 mL:5 mL) in the presence
of Et3N (1.1 mmol) [17 (2 mmol) and L-lysine (1 mmol) were used
to obtain 22]. The reaction mixture was stirred at 20 °C for about 1
h (until TLC showed the absence of 17). Aq 4 N HCl (1 mL) was
then added and the MeCN was removed under reduced pressure.
The residue obtained was dissolved in EtOAc (150 mL), washed
with 4 N HCl (3 × 50 mL) and sat. NaCl soln (50 mL), and dried
(MgSO4). After evaporation of the solvent, the residue was crystal-
lized from EtOAc–hexanes or CH2Cl2–hexanes.
a Determined in HPLC-grade methanol.
b Extinction coefficient.
c Not determined.
d Not detected.
er absorption values than tryptophan (lAbs. = 278 nm,
l
Em. = 352 nm) or tyrosine (lAbs. = 274 nm, lEm. = 303
nm).
In conclusion, Na- and Ne-coumarin-labeled Cbz- and
Fmoc-L-lysines 19g, 21a and 21b, chiral fluorescent
probes for amino acids and peptides, have been readily
synthesized in high yields. The N-acylbenzotriazoles 17,
(S)-2-[(2-Oxo-2H-chromen-3-ylcarbonyl)amino]propanoic
Acid [N-(Coumarin-3-ylcarbonyl)-L-Ala-OH, 19a]
23
Yield: 82%; white microcrystals; mp 203–204 °C; [a]D +12.0 (c
1.92, DMF).
Synthesis 2008, No. 13, 2013–2022 © Thieme Stuttgart · New York

PAPER
Fluorescent Markers for Amino Acids and Dipeptides
2019
1H NMR (DMSO-d6):37 d = 1.43 (d, J = 7.1 Hz, 3 H), 4.49 (quin,
J = 7.0 Hz, 1 H), 7.46 (t, J = 7.5 Hz, 1 H), 7.53 (d, J = 8.4 Hz, 1 H),
7.77 (td, J = 8.5, 1.5 Hz, 1 H), 8.01 (dd, J = 7.8, 1.4 Hz, 1 H), 8.91
(s, 1 H), 9.11 (d, J = 6.9 Hz, 1 H).
13C NMR (DMSO-d6): d = 17.9, 48.2, 116.2, 118.3, 118.4, 125.2,
130.4, 134.3, 148.0, 154.0, 160.5, 160.6, 173.6.
13C NMR (DMSO-d6): d = 27.1, 54.9, 108.8, 111.4, 116.2, 118.0,
118.3, 118.4 (2 C), 121.0, 123.9, 125.2, 127.3, 130.4, 134.3, 136.1,
148.2, 154.0, 160.5, 160.6, 172.6.
Anal. Calcd for C21H16N2O5: C, 67.02; H, 4.28; N, 7.44. Found: C,
66.79; H, 4.13; N, 7.25.
(S)-3-Methyl-2-[(2-oxo-2H-chromen-3-ylcarbonyl)amino]bu-
tanoic Acid [N-(Coumarin-3-ylcarbonyl)-L-Val-OH, 19f]
Yield: 81%; white microcrystals; mp 211–212 °C; [a]D23 + 5.58 (c
2.04, DMF).
1H NMR (DMSO-d6): d = 0.97 (d, J = 6.7 Hz, 6 H), 2.15–2.31 (m,
1 H), 4.47 (dd, J = 8.3, 4.7 Hz, 1 H), 7.47 (t, J = 7.5 Hz, 1 H), 7.54
(d, J = 8.2 Hz, 1 H), 7.78 (t, J = 7.8 Hz, 1 H), 8.02 (d, J = 7.6 Hz, 1
H), 8.93 (s, 1 H), 9.09 (d, J = 8.2 Hz, 1 H).
Anal. Calcd for C13H11NO5: C, 59.77; H, 4.24; N, 5.36. Found: C,
59.66; H, 4.40; N, 5.49.
(S)-3-Hydroxy-2-[(2-oxo-2H-chromen-3-ylcarbonyl)ami-
no]propanoic Acid [N-(Coumarin-3-ylcarbonyl)-L-Ser-OH,
19b]
Yield: 76%; white microcrystals; mp 225–226 °C; [a]D23 +20.65 (c
1.85, DMF).
1H NMR (DMSO-d6): d = 3.76 (dd, J = 10.8, 3.2 Hz, 1 H), 3.89 (dd,
J = 10.8, 3.2 Hz, 1 H), 4.48–4.58 (m, 1 H), 5.29 (br s, 1 H), 7.46 (t,
J = 7.5 Hz, 1 H), 7.54 (d, J = 8.4 Hz, 1 H), 7.78 (t, J = 7.5 Hz, 1 H),
8.03 (d, J = 7.5 Hz, 1 H), 8.96 (s, 1 H), 9.27 (d, J = 7.5 Hz, 1 H).
13C NMR (DMSO-d6): d = 54.9, 61.1, 116.2, 118.1, 118.4, 125.3,
130.5, 134.4, 148.3, 154.0, 160.6, 160.7, 171.6.
13C NMR (DMSO-d6): d = 17.6, 19.1, 30.5, 57.3, 116.2, 118.2,
118.5, 125.3, 130.4, 134.4, 148.1, 154.0, 160.9, 161.0, 172.4.
Anal. Calcd for C15H15NO5: C, 62.28; H, 5.23; N, 4.84. Found: C,
62.20; H, 5.11; N, 4.74.
(S)-6-(Benzyloxycarbonylamino)-2-[(2-oxo-2H-chromen-3-yl-
carbonyl)amino]hexanoic Acid [Na-(Coumarin-3-ylcarbonyl)-
Ne-Cbz-L-Lys-OH, 19g]
Anal. Calcd for C13H11NO6·H2O: C, 52.89; H, 4.44; N, 4.74. Found:
C, 53.16; H, 4.31; N, 4.67.
Yield: 77%; white microcrystals; mp 149–150 °C; [a]D23 + 6.27 (c
2.08, DMF).
(S)-4-(Methylsulfanyl)-2-[(2-oxo-2H-chromen-3-ylcarbon-
yl)amino]butanoic Acid [N-(Coumarin-3-ylcarbonyl)-L-Met-
OH, 19c]
1H NMR (DMSO-d6): d = 1.25–1.51 (m, 4 H), 1.68–1.95 (m, 2 H),
2.94–3.06 (m, 2 H), 4.43–4.58 (m, 1 H), 4.98 (s, 2 H), 7.22–7.40 (m,
6 H), 7.46 (t, J = 7.6 Hz, 1 H), 7.53 (d, J = 8.4 Hz, 1 H), 7.77 (t,
J = 7.5 Hz, 1 H), 8.01 (d, J = 7.3 Hz, 1 H), 8.91 (s, 1 H), 9.08 (d,
J = 7.4 Hz, 1 H).
13C NMR (DMSO-d6): d = 22.2, 29.1, 31.2, 52.3, 65.1, 116.2,
118.3, 118.4, 125.2 (2 C), 127.7 (2 C), 128.3 (2 C), 130.4, 134.3,
137.3, 148.0, 154.0, 156.1, 160.6, 160.8, 173.0 (one aliphatic car-
bon is merged in the DMSO-d6 peak).
23
Yield: 88%; white microcrystals; mp 115–116 °C; [a]D +1.33 (c
1.73, DMF).
1H NMR (CDCl3): d = 2.13 (s, 3 H), 2.16–2.26 (m, 1 H), 2.27–2.42
(m, 1 H), 2.64 (t, J = 7.5 Hz, 2 H), 4.86–4.98 (m, 1 H), 7.37–7.47
(m, 2 H), 7.65–7.75 (m, 2 H), 8.93 (s, 1 H), 9.35 (d, J = 7.3 Hz, 1 H).
13C NMR (CDCl3): d = 15.3, 30.0, 31.1, 52.0, 116.6, 117.5, 118.4,
125.4, 130.0, 134.4, 149.2, 154.5, 161.2, 162.0, 175.1.
Anal. Calcd for C24H24N2O7: C, 63.71; H, 5.35; N, 6.19. Found: C,
63.82; H, 5.32; N, 6.11.
Anal. Calcd for C15H15NO5S: C, 56.07; H, 4.70; N, 4.36. Found: C,
55.76; H, 4.75; N, 4.25.
4-(Methylsulfanyl)-2-[(2-oxo-2H-chromen-3-ylcarbonyl)ami-
no]butanoic Acid [N-(Coumarin-3-ylcarbonyl)-DL-Met-OH,
[19c + 19c¢]]
(S)-2-[(2-Oxo-2H-chromen-3-ylcarbonyl)amino]-3-phenylpro-
panoic Acid [N-(Coumarin-3-ylcarbonyl)-L-Phe-OH, 19d]
Yield: 88%; white microcrystals; mp 178–179 °C; [a]D23 +41.53 (c
1.78, DMF).
1H NMR (DMSO-d6): d = 3.12 (dd, J = 13.8, 6.7 Hz, 1 H, B part of
AB system), 3.21 (dd, J = 13.7, 5.3 Hz, 1 H, A part of AB system),
4.73–4.83 (m, 1 H), 7.18–7.34 (m, 5 H), 7.45 (t, J = 7.6 Hz, 1 H),
7.51 (d, J = 8.4 Hz, 1 H), 7.77 (t, J = 7.8 Hz, 1 H), 8.00 (d, J = 7.4
Hz, 1 H), 8.91 (s, 1 H), 9.05 (d, J = 7.4 Hz, 1 H).
Yield: 89%; white microcrystals; mp 136–137 °C.
1H NMR (CDCl3): d = 2.13 (s, 3 H), 2.20 (quin, J = 7.3 Hz, 1 H),
2.27–2.42 (m, 1 H), 2.65 (t, J = 7.4 Hz, 2 H), 4.87–4.98 (m, 1 H),
7.36–7.47 (m, 2 H), 7.65–7.75 (m, 2 H), 8.94 (s, 1 H), 9.36 (d,
J = 7.4 Hz, 1 H).
13C NMR (CDCl3): d = 15.4, 30.0, 31.1, 52.0, 116.7, 117.5, 118.4,
125.4, 130.0, 134.5, 149.2, 154.5, 161.3, 162.1, 175.4.
13C NMR (DMSO-d6): d = 36.8, 53.7, 116.2, 117.9, 118.4, 125.2,
126.7, 128.3 (2 C), 129.3 (2 C), 130.4, 134.4, 136.6, 148.3, 154.0,
160.5, 160.6, 172.1.
Anal. Calcd for C15H15NO5S: C, 56.07; H, 4.70; N, 4.36. Found: C,
55.74; H, 4.70; N, 4.27.
Anal. Calcd for C19H15NO5: C, 67.65; H, 4.48; N, 4.15. Found: C,
67.34; H, 4.34; N, 4.10.
(S)-2-(Benzyloxycarbonylamino)-6-[(2-oxo-2H-chromen-3-yl-
carbonyl)amino]hexanoic Acid [Na-Cbz-Ne-(Coumarin-3-ylcar-
bonyl)-L-Lys-OH, 21a]
Yield: 89%; white microcrystals; mp 144–145 °C; [a]D –8.54 (c
1.68, DMF).
1H NMR (CDCl3): d = 1.37–1.56 (m, 2 H), 1.58–1.74 (m, 2 H),
1.75–2.40 (m, 2 H), 3.33–3.58 (m, 2 H), 4.34–4.45 (m, 1 H), 5.09
(s, 2 H), 5.75 (d, J = 8.0 Hz, 1 H), 7.27–7.42 (m, 7 H), 7.52–7.72
(m, 2 H), 8.92 (s, 1 H), 8.95–9.04 (m, 1 H).
13C NMR (CDCl3): d = 22.3, 28.9, 31.5, 39.3, 53.6, 66.9, 116.5 (2
C), 117.9, 118.5, 125.3, 128.0, 128.1, 128.4 (2 C), 130.0, 134.1,
136.2, 148.8, 154.3, 156.3, 161.3, 162.1, 175.3.
(S)-3-(1H-Indol-3-yl)-2-[(2-oxo-2H-chromen-3-ylcarbonyl)ami-
no]propanoic Acid [N-(Coumarin-3-ylcarbonyl)-L-Trp-OH,
23
19e]
Yield: 78%; yellow microcrystals; mp 201–202 °C; [a]D23 +33.14 (c
1.91, DMF).
1H NMR (DMSO-d6): d = 3.23–3.41 (m, 2 H), 4.80 (q, J = 6.1 Hz,
1 H), 6.95 (t, J = 7.4 Hz, 1 H), 7.06 (t, J = 7.5 Hz, 1 H), 7.19 (d,
J = 2.2 Hz, 1 H), 7.33 (d, J = 8.1 Hz, 1 H), 7.44 (t, J = 7.5 Hz, 1 H),
7.51 (t, J = 8.7 Hz, 2 H), 7.75 (td, J = 7.1, 1.5 Hz, 1 H), 7.99 (dd,
J = 7.5, 1.4 Hz, 1 H), 8.93 (s, 1 H), 9.10 (d, J = 7.3 Hz, 1 H), 10.94
(s, 1 H).
Synthesis 2008, No. 13, 2013–2022 © Thieme Stuttgart · New York

2020
A. R. Katritzky et al.
PAPER
N-[1-(1H-1,2,3-Benzotriazol-1-ylcarbonyl)-3-(methylsulfa-
nyl)propyl]-2-oxo-2H-chromene-3-carboxamide [N-(Cou-
marin-3-ylcarbonyl)-DL-Met-Bt, [23b + 23b¢]]
Yield: 74%; white microcrystals; mp 123–124 °C.
1H NMR (CDCl3): d = 2.13 (s, 3 H), 2.30–2.45 (m, 1 H), 2.52–2.69
(m, 1 H), 2.77 (t, J = 7.0 Hz, 2 H), 6.23–6.35 (m, 1 H), 7.40 (t,
J = 7.6 Hz, 1 H), 7.50 (d, J = 8.7 Hz, 1 H), 7.55 (t, J = 7.6 Hz, 1 H),
7.64–7.78 (m, 3 H), 8.16 (d, J = 8.2 Hz, 1 H), 8.30 (d, J = 8.2 Hz, 1
H), 8.91 (s, 1 H), 9.60 (d, J = 7.1 Hz, 1 H).
13C NMR (CDCl3): d = 15.3, 30.2, 31.8, 52.9, 114.4, 116.7, 117.6,
118.4, 120.3, 125.4, 126.5, 130.0, 130.8, 131.2, 134.5, 146.0, 149.1,
154.6, 161.4, 161.8, 170.5.
Anal. Calcd for C24H24N2O7: C, 63.71; H, 5.35; N, 6.19. Found: C,
63.82; H, 5.09; N, 6.04.
(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-6-[(2-oxo-2H-
chromen-3-ylcarbonyl)amino]hexanoic Acid [Na-Fmoc-Ne-
(Coumarin-3-ylcarbonyl)-L-Lys-OH, 21b]
Yield: 87%; white microcrystals; mp 110–111 °C; [a]D –1.62 (c
1.85, DMF).
1H NMR (DMSO-d6): d = 1.32–1.50 (m, 2 H), 1.50–1.62 (m, 2 H),
1.62–1.85 (m, 2 H), 3.26–3.38 (m, 2 H), 3.92–4.01 (m, 1 H), 4.17–
4.36 (m, 3 H), 7.22–7.54 (m, 6 H), 7.60–7.80 (m, 4 H), 7.87 (d,
J = 7.4 Hz, 2 H), 7.96 (d, J = 7.4 Hz, 1 H), 8.73 (t, J = 5.5 Hz, 1 H),
8.84 (s, 1 H), 12.62 (s, 1 H).
13C NMR (DMSO-d6): d = 23.2, 28.6, 30.5, 46.7, 53.8, 65.6, 116.1,
118.5, 119.0, 120.1, 125.1, 125.3 (2 C), 127.1 (2 C), 127.7 (2 C),
130.2, 134.0, 140.7 (2 C), 143.8 (2 C), 147.3, 153.8, 156.2, 160.4,
161.0 (2 C), 174.0 (one aliphatic carbon is merged in the DMSO-d6
peak).
23
Anal. Calcd for C21H18N4O4S: C, 59.70; H, 4.29; N, 13.26. Found:
C, 59.93; H, 4.17; N, 12.88.
Benzyl{(S)-1-(1H-1,2,3-Benzotriazol-1-ylcarbonyl)-5-[(2-oxo-
2H-chromen-3-ylcarbonyl)amino]pentyl}carbamate [Na-Cbz-
Ne-(Coumarin-3-ylcarbonyl)-L-Lys-Bt, 26]
Anal. Calcd for C31H28N2O7: C, 68.88; H, 5.22; N, 5.18. Found: C,
68.59; H, 5.11; N, 5.16.
Yield: 79%; white microcrystals; mp 156–157 °C.
1H NMR (CDCl3): d = 1.50–1.80 (m, 4 H), 1.96–2.12 (m, 1 H),
2.13–2.28 (m, 1 H), 3.36–3.48 (m, 1 H), 3.48–3.64 (m, 1 H), 5.13
(s, 2 H), 5.69–5.83 (m, 1 H), 6.12 (d, J = 7.8 Hz, 1 H), 7.26–7.47
(m, 8 H), 7.52 (t, J = 7.6 Hz, 1 H), 7.60–7.74 (m, 2 H), 8.13 (d,
J = 8.2 Hz, 1 H), 8.27 (d, J = 8.2 Hz, 1 H), 8.86 (s, 1 H), 8.82–8.97
(m, 1 H).
13C NMR (CDCl3): d = 22.3, 28.9, 31.6, 38.5, 54.6, 67.1, 114.4,
116.5, 118.2, 118.6, 120.3, 125.2, 126.4, 128.0 (2 C), 128.1, 128.5
(2 C), 129.8, 130.6, 131.1, 134.0, 136.2, 145.9, 148.6, 154.3, 156.2,
161.4, 162.0, 171.7.
(S)-2,6-Bis[(2-oxo-2H-chromen-3-ylcarbonyl)amino]hexanoic
Acid [Na,Ne-Bis(coumarin-3-ylcarbonyl)-L-Lys-OH, 22]
Yield: 86%; white microcrystals; mp 220–221 °C; [a]D23 +14.13 (c
1.86, DMF).
1H NMR (DMSO-d6): d = 1.30–1.48 (m, 2 H), 1.50–1.65 (m, 2 H),
1.72–2.00 (m, 2 H), 3.25–3.40 (m, 2 H), 4.47–4.57 (m, 1 H), 7.37–
7.52 (m, 4 H), 7.68–7.80 (m, 2 H), 7.95 (t, J = 8.0 Hz, 2 H), 8.68 (t,
J = 5.6 Hz, 1 H), 8.81 (s, 1 H), 8.88 (s, 1 H), 9.06 (d, J = 7.4 Hz, 1
H).
Anal. Calcd for C30H27N5O6: C, 65.09; H, 4.92; N, 12.65. Found: C,
64.91; H, 4.76; N, 12.59.
13C NMR (DMSO-d6): d = 18.0, 22.1, 28.4, 31.0, 52.1, 116.1,
116.2, 118.3 (2 C), 118.4, 119.0, 125.1, 125.2, 130.2, 130.4, 134.0,
134.3, 147.3, 148.0, 153.8, 153.9, 160.4, 160.6, 160.7, 161.0, 173.0.
Coumarin-Labeled Dipeptides 25a, [25b + 25b¢], 27, 29a,b and
[29a + 29a¢]; General Procedure for Route (i)
Anal. Calcd for C26H22N2O8: C, 63.67; H, 4.52; N, 5.71. Found: C,
63.43; H, 4.42; N, 5.69.
A N-(coumarin-3-ylcarbonyl-a-aminoacyl)benzotriazole 23a, [23b
+ 23b¢], 26 (0.5 mmol) was added to a soln of an a-amino acid 24,
28a,b, [28a + 28a¢] (0.5 mmol), or L-aspartic acid (to obtain 27), in
MeCN–H2O (10 mL:5 mL) in the presence of Et3N (1 mmol). The
reaction mixture was stirred at 20 °C for 1 h. Aq 4 N HCl (1 mL)
was then added and the MeCN was removed under reduced pres-
sure. The obtained residue was dissolved in EtOAc (100 mL),
washed with 4 N HCl (3 × 50 mL) and sat. NaCl soln (50 mL), and
dried (MgSO4). Evaporation of the solvent gave the desired product,
which was recrystallized from CH2Cl2–hexanes.
N-(Coumarin-3-ylcarbonyl-a-aminoacyl)benzotriazoles 23a,
[23b + 23b¢] and 26; General Procedure
Thionyl chloride (1.2 mmol) was added to a soln of 1H-benzotriaz-
ole (16; 4 mmol) in anhyd CH2Cl2 (15 mL) at 20 °C and the reaction
mixture was stirred for 20 min. To the reaction mixture was added
the N-(coumarin-3-ylcarbonyl)-a-amino acid 19a, [19c + 19c¢], 21a
(1 mmol) and the relevant mixture was stirred for 1 h at r.t. to obtain
23a, 26 and for 15 min to obtain the diastereomeric mixture [23b +
23b¢]. The white precipitate which formed during the reaction was
filtered off, the filtrate was diluted with additional CH2Cl2 (80 mL),
and the solution was washed with 4 N HCl (3 × 50 mL) [sat. Na2CO3
soln (3 × 50 mL) to obtain 26] and sat. NaCl soln (50 mL), and dried
(MgSO4). Removal of the solvent under reduced pressure gave the
desired product, which was recrystallized from CH2Cl2–hexanes.
(S)-2-{(S)-2-[(2-Oxo-2H-chromen-3-ylcarbonyl)amino]propio-
nylamino}-3-phenylpropanoic Acid [N-(Coumarin-3-ylcarbon-
yl)-L-Ala-L-Phe-OH, 25a]
Yield: 74%; white microcrystals; mp 208–209 °C; [a]D23 +19.46 (c
1.67, DMF).
1H NMR (DMSO-d6): d = 1.30 (d, J = 6.9 Hz, 3 H), 2.91 (dd,
J = 13.7, 9.5 Hz, 1 H), 3.10 (dd, J = 13.9, 4.8 Hz, 1 H), 4.44–4.50
(m, 1 H), 4.55 (quin, J = 7.0 Hz, 1 H), 7.13–7.32 (m, 5 H), 7.45 (t,
J = 7.6 Hz, 1 H), 7.52 (d, J = 8.2 Hz, 1 H), 7.76 (t, J = 7.3 Hz, 1 H),
8.00 (d, J = 7.4 Hz, 1 H), 8.51 (d, J = 8.0 Hz, 1 H), 8.90 (s, 1 H),
9.13 (d, J = 7.3 Hz, 1 H).
13C NMR (DMSO-d6): d = 19.3, 36.5, 48.3, 53.6, 116.2, 118.3,
118.5, 125.2, 126.5, 128.2 (2 C), 129.2 (2 C), 130.4, 134.3, 137.6,
147.9, 154.0, 160.1, 160.5, 171.5, 172.7.
N-[(1S)-2-(1H-1,2,3-Benzotriazol-1-yl)-1-methyl-2-oxoethyl]-2-
oxo-2H-chromene-3-carboxamide [N-(Coumarin-3-ylcarbon-
yl)-L-Ala-Bt, 23a]
Yield: 78%; white microcrystals; mp 178–179 °C.
1H NMR (CDCl3): d = 1.85 (d, J = 7.3 Hz, 3 H), 6.13 (quin, J = 7.0
Hz, 1 H), 7.39 (t, J = 7.4 Hz, 1 H), 7.45 (d, J = 8.7 Hz, 1 H), 7.54 (t,
J = 7.5 Hz, 1 H), 7.64–7.76 (m, 3 H), 8.16 (d, J = 8.2 Hz, 1 H), 8.31
(d, J = 8.2 Hz, 1 H), 8.90 (s, 1 H), 9.49 (d, J = 6.2 Hz, 1 H).
13C NMR (CDCl3): d = 18.3, 49.6, 114.5, 116.7, 117.7, 118.4,
120.3, 125.4, 126.4, 129.9, 130.7, 131.2, 134.4, 146.0, 148.9, 154.6,
161.3, 161.5, 171.4.
Anal. Calcd for C22H20N2O6: C, 64.70; H, 4.94; N, 6.86. Found: C,
64.66; H, 4.96; N, 6.74.
Anal. Calcd for C19H14N4O4: C, 62.98; H, 3.89; N, 15.46. Found: C,
62.65; H, 3.81; N, 15.26.
Synthesis 2008, No. 13, 2013–2022 © Thieme Stuttgart · New York

PAPER
Fluorescent Markers for Amino Acids and Dipeptides
2021
(S)-2-{4-(Methylsulfanyl)-2-[(2-oxo-2H-chromen-3-ylcarbon-
yl)amino]butyrylamino}-3-phenylpropanoic Acid [N-(Cou-
marin-3-ylcarbonyl)-DL-Met-L-Phe-OH, [25b + 25b¢]]
Yield: 68%; white microcrystals; mp 184–185 °C; [a]D –1.02 (c
1.96, DMF).
1H NMR (DMSO-d6): d = 1.60–2.05 (m, 2 H), 1.92 (s, 1.5 H), 2.01
(s, 1.5 H), 2.03–2.20 (m, 1 H), 2.43 (t, J = 8.0 Hz, 1 H), 2.78–2.98
(m, 1 H), 3.13 (td, J = 13.6, 4.4 Hz, 1 H), 4.42–4.58 (m, 1 H), 4.59–
4.70 (m, 1 H), 7.12–7.32 (m, 5 H), 7.44 (t, J = 7.4 Hz, 1 H), 7.51 (d,
J = 8.4 Hz, 1 H), 7.75 (t, J = 7.8 Hz, 1 H), 7.98 (d, J = 7.6 Hz, 1 H),
8.54 (d, J = 7.8 Hz, 0.5 H), 8.64 (d, J = 8.4 Hz, 0.5 H), 8.88 (s, 1 H),
9.09 (d, J = 7.8 Hz, 1 H).
13C NMR (DMSO-d6): d = 14.5, 14.6, 28.7, 28.8, 32.9, 36.4, 36.7,
51.9, 52.0, 53.3, 53.6, 116.2, 118.4, 125.2, 126.5, 128.2, 128.2,
129.1, 130.3, 134.3, 137.5, 137.5, 147.8, 153.9, 160.5, 160.5, 170.1,
170.2, 172.7, 172.8.
1H NMR (DMSO-d6): d = 1.20–1.80 (m, 6 H), 2.92 (dd, J = 13.7,
8.8 Hz, 1 H), 3.06 (dd, J = 13.7, 4.8 Hz, 1 H), 3.22–3.40 (m, 2 H),
3.90–4.10 (m, 1 H), 4.38–4.51 (m, 1 H), 5.02 (s, 2 H), 7.16–7.48 (m,
12 H), 7.51 (d, J = 8.2 Hz, 1 H), 7.75 (t, J = 7.7 Hz, 1 H), 7.98 (d,
J = 7.6 Hz, 1 H), 8.15 (d, J = 7.7 Hz, 1 H), 8.66–8.78 (m, 1 H), 8.87
(s, 1 H).
13C NMR (DMSO-d6): d = 22.9, 28.7, 31.7, 36.7, 39.1, 53.4, 54.5,
65.4, 116.2, 118.5, 119.1, 125.2, 126.5, 127.7 (3 C), 127.8, 127.8,
128.2, 128.4 (2 C), 129.2, 130.3, 134.1, 137.1, 137.4, 147.4, 153.9,
155.9, 160.5, 161.0, 172.0, 172.9.
23
Anal. Calcd for C33H33N3O8: C, 64.10; H, 5.55; N, 7.01. Found: C,
64.37; H, 5.51; N, 6.61.
2-{(S)-2-(Benzyloxycarbonylamino)-6-[(2-oxo-2H-chromen-3-
ylcarbonyl)amino]hexanoylamino}propanoic Acid [Ne-(Cou-
marin-3-ylcarbonyl)-Na-Cbz-L-Lys-DL-Ala-OH, [29a + 29a¢]]
Yield: 94%; white microcrystals; mp 131–132 °C.
1H NMR (DMSO-d6): d = 1.21–1.31 (m, 3 H), 1.31–1.80 (m, 6 H),
3.27–3.34 (m, 2 H), 3.85–4.08 (m, 1 H), 4.09–4.30 (m, 1 H), 5.02
(s, 2 H), 7.24–7.48 (m, 7 H), 7.51 (d, J = 8.6 Hz, 1 H), 7.76 (t,
J = 7.7 Hz, 1 H), 7.97 (d, J = 8.0 Hz, 1 H), 8.21 (d, J = 6.9 Hz, 1 H),
8.65–8.76 (m, 1 H), 8.86 (s, 1 H), 12.58 (br s, 1 H).
13C NMR (DMSO-d6): d = 17.1, 17.5, 22.9, 28.6, 28.7, 31.6, 32.0,
39.1, 47.4, 54.2, 65.3, 116.1, 118.5, 119.1, 125.2, 127.7, 127.7,
127.8, 128.3, 130.3, 134.1, 137.1, 147.4, 153.9, 155.9, 156.0, 160.4,
161.0, 171.7, 171.8, 174.0, 174.1.
Anal. Calcd for C24H24N2O6S: C, 61.53; H, 5.16; N, 5.98. Found: C,
61.27; H, 5.20; N, 6.00.
(S)-2-{(S)-2-(Benzyloxycarbonylamino)-6-[(2-oxo-2H-
chromen-3-ylcarbonyl)amino]hexanoylamino}succinic Acid
[Ne-(Coumarin-3-ylcarbonyl)-Na-Cbz-L-Lys-L-Asp-OH, 27]
Yield: 85%; white microcrystals; mp 206–207 °C; [a]D23 –10.48 (c
0.83, DMF).
1H NMR (DMSO-d6): d = 1.27–1.42 (m, 2 H), 1.43–1.73 (m, 4 H),
2.60 (dd, J = 16.5, 6.6 Hz, 1 H), 2.68 (dd, J = 16.8, 5.6 Hz, 1 H),
3.20–3.38 (m, 2 H), 3.94–4.09 (m, 1 H), 4.48–4.58 (m, 1 H), 5.00
(s, 2 H), 7.25–7.38 (m, 5 H), 7.40–4.47 (m, 2 H), 7.50 (d, J = 8.2 Hz,
1 H), 7.75 (td, J = 7.8, 1.4 Hz, 1 H), 7.97 (d, J = 7.7 Hz, 1 H), 8.23
(d, J = 8.0 Hz, 1 H), 8.70 (t, J = 5.6 Hz, 1 H), 8.86 (s, 1 H), 12.58
(br s, 2 H).
Anal. Calcd for C27H29N3O8: C, 61.94; H, 5.58; N, 8.03. Found: C,
61.64; H, 5.59; N, 7.65.
Coumarin-Labeled Dipeptides 30 and 31; General Procedure
for Route (ii)
13C NMR (DMSO-d6): d = 22.9, 28.7, 31.7, 36.0, 48.5, 54.3, 65.4,
116.2, 118.5, 119.1, 125.2, 127.7 (2 C), 127.8, 128.4 (2 C), 130.3,
134.1, 134.3, 137.0, 147.4, 153.9, 156.0, 160.4, 161.0, 171.7, 171.9,
172.4.
Benzotriazole 17 (1 mmol) was added to a soln of a dipeptide in
MeCN–H2O (10 mL:5 mL) in the presence of Et3N (1.1 mmol). The
reaction mixture was stirred at 20 °C for 1 h. Aq 4 N HCl (1 mL)
was then added and the solvent was removed under reduced pres-
sure. The residue obtained was dissolved in CH2Cl2 (80 mL), and
the organic extract was washed with 4 N HCl (3 × 40 mL) and sat.
NaCl soln (30 mL), and dried (MgSO4). Evaporation of the solvent
gave the pure product.
Anal. Calcd for C28H29N3O10: C, 59.26; H, 5.15; N, 7.40. Found: C,
59.44; H, 5.14; N, 7.24.
(S)-2-{(S)-2-(Benzyloxycarbonylamino)-6-[(2-oxo-2H-
chromen-3-ylcarbonyl)amino]hexanoylamino}propanoic Acid
[Ne-(Coumarin-3-ylcarbonyl)-Na-Cbz-L-Lys-L-Ala-OH, 29a]
2-{2-[(2-Oxo-2H-chromen-3-ylcarbonyl)amino]acetylami-
no}acetic Acid [N-(Coumarin-3-ylcarbonyl)-Gly-Gly-OH, 30]
Yield: 81%; white microcrystals; mp 243–244 °C.
1H NMR (DMSO-d6): d = 3.82 (d, J = 5.8 Hz, 2 H), 4.06 (d, J = 5.2
Hz, 2 H), 7.46 (t, J = 7.5 Hz, 1 H), 7.52 (d, J = 8.2 Hz, 1 H), 7.77
(td, J = 7.5, 1.2 Hz, 1 H), 8.01 (d, J = 6.9 Hz, 1 H), 8.44 (t, J = 5.8
Hz, 1 H), 8.92 (s, 1 H), 9.11 (t, J = 5.2 Hz, 1 H).
23
Yield: 91%; white microcrystals; mp 156–157 °C; [a]D –8.43 (c
0.83, DMF).
1H NMR (DMSO-d6): d = 1.28 (d, J = 7.3 Hz, 3 H), 1.32–1.76 (m,
6 H), 3.25–3.48 (m, 2 H), 3.95–4.10 (m, 1 H), 4.20 (quin, J = 7.2
Hz, 1 H), 5.01 (s, 2 H), 7.26–7.38 (m, 5 H), 7.41 (d, J = 8.4 Hz, 1
H), 7.46 (d, J = 7.6 Hz, 1 H), 7.51 (d, J = 8.4 Hz, 1 H), 7.70–7.82
(m, 1 H), 7.98 (d, J = 7.7 Hz, 1 H), 8.21 (d, J = 7.1 Hz, 1 H), 8.71
(t, J = 5.5 Hz, 1 H), 8.86 (s, 1 H).
13C NMR (DMSO-d6): d = 40.7, 42.5, 116.2, 118.4, 125.2, 130.4 (2
C), 134.3, 147.8, 154.0, 160.4, 161.1, 168.6, 171.1.
13C NMR (DMSO-d6): d = 17.1, 22.9, 28.7, 31.6, 47.4, 54.2, 65.4,
116.2, 118.5, 119.1, 125.2, 127.7 (2 C), 127.8, 128.3 (2 C), 130.3,
134.1, 137.1, 147.4, 153.9, 156.0, 160.4, 161.0, 171.8, 174.1 (one
aliphatic carbon is merged in the DMSO-d6 peak).
Anal. Calcd for C14H12N2O6·H2O: C, 52.18; H, 3.75; N, 8.69.
Found: C, 52.25; H, 3.86; N, 8.36.
(S)-4-Methyl-2-{2-[(2-oxo-2H-chromen-3-ylcarbonyl)ami-
no]acetylamino}pentanoic Acid [N-(Coumarin-3-ylcarbonyl)-
Gly-L-Leu-OH, 31]
Anal. Calcd for C27H29N3O8: C, 61.94; H, 5.58; N, 8.03. Found: C,
61.64; H, 5.65; N, 7.66.
23
Yield: 85%; white microcrystals; mp 194–195 °C; [a]D –1.02 (c
1.96, DMF).
(S)-2-{(S)-2-(Benzyloxycarbonylamino)-6-[(2-oxo-2H-
chromen-3-ylcarbonyl)amino]hexanoylamino}-3-phenylpro-
panoic Acid [Ne-(Coumarin-3-ylcarbonyl)-Na-Cbz-L-Lys-L-
Phe-OH, 29b]
1H NMR (DMSO-d6): d = 0.86 (d, J = 6.3 Hz, 3 H), 0.91 (d, J = 6.5
Hz, 3 H), 1.48–1.58 (m, 2 H), 1.58–1.74 (m, 1 H), 4.06 (d, J = 5.1
Hz, 2 H), 4.28 (q, J = 7.6 Hz, 1 H), 7.46 (t, J = 7.5 Hz, 1 H), 7.52 (d,
J = 8.2 Hz, 1 H), 7.77 (t, J = 7.8 Hz, 1 H), 8.01 (d, J = 7.7 Hz, 1 H),
8.37 (d, J = 7.8 Hz, 1 H), 8.92 (s, 1 H), 9.09 (t, J = 4.9 Hz, 1 H).
23
Yield: 89%; white microcrystals; mp 170–171 °C; [a]D –5.71 (c
1.75, DMF).
Synthesis 2008, No. 13, 2013–2022 © Thieme Stuttgart · New York

2022
A. R. Katritzky et al.
PAPER
13C NMR (DMSO-d6): d = 21.4, 22.9, 24.3, 42.4, 50.3, 116.2,
118.4, 118.5, 125.2, 130.4 (2 C), 134.3, 147.8, 154.0, 160.4, 161.0,
168.1, 174.0.
(20) Bennett, F. A.; Barlow, D. J.; Dodoo, A. N. O.; Hider, R. C.;
Lansley, A. B.; Lawrence, M. J.; Marriott, C.; Bansal, S. S.
Tetrahedron Lett. 1997, 38, 7449.
(21) Wang, J.; Xie, J.; Schultz, P. G. J. Am. Chem. Soc. 2006,
Anal. Calcd for C18H20N2O6: C, 59.99; H, 5.59; N, 7.77. Found: C,
59.59; H, 5.77; N, 7.52.
128, 8738.
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Acknowledgment
We thank Dr. Kirk Shanze and Dr. Katsu Ogawa (University of
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(36) Two compound numbers given as a combination within
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(37) No signal was seen in the proton NMR spectra for the acid
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Synthesis 2008, No. 13, 2013–2022 © Thieme Stuttgart · New York