Synthesis and evaluation of two coumarin-type derivatization reagents for fluorescence detection of chiral amines and chiral carboxylic acids

Article abstract of DOI:10.1002/chir.22240

The synthesis of two fluorescent coumarin-type chiral derivatization agents (4 and 11) is reported. A chiral side chain was introduced at position 7 of the coumarin via Mitsunobu reaction. The two coumarins bear in this side chain either a free amino group or a carboxyl group, making them useful for further transformations. Conjugates of chiral prototype drugs with 4 or 11 were prepared by amide coupling of the analyte's carboxyl group to the reagent's amine group, or vice versa. The separation of seven diastereomeric conjugates through achiral high-performance liquid chromatography (HPLC) on a common C18 column is demonstrated. Chirality 25:957-964, 2013. 2013 Wiley Periodicals, Inc.

Full text of DOI:10.1002/chir.22240

CHIRALITY 25:957–964 (2013)
Synthesis and Evaluation of Two Coumarin-Type Derivatization
Reagents for Fluorescence Detection of Chiral Amines and
Chiral Carboxylic Acids
*
MATTHIAS D. MERTENS AND MICHAEL GÜTSCHOW
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany
ABSTRACT
The synthesis of two fluorescent coumarin-type chiral derivatization agents
(4 and 11) is reported. A chiral side chain was introduced at position 7 of the coumarin via
Mitsunobu reaction. The two coumarins bear in this side chain either a free amino group or a car-
boxyl group, making them useful for further transformations. Conjugates of chiral prototype drugs
with 4 or 11 were prepared by amide coupling of the analyte’s carboxyl group to the reagent’s
amine group, or vice versa. The separation of seven diastereomeric conjugates through achiral
high-performance liquid chromatography (HPLC) on a common C18 column is demonstrated.
Chirality 25:957–964, 2013.
© 2013 Wiley Periodicals, Inc.
KEY WORDS: reversed-phase high-performance liquid chromatography; diastereomer separation;
chiral coumarins; fluorescence detection; precolumn derivatization; Mitsunobu reaction
Chirality is strikingly important for biological recognition, in-
cluding drug–target interactions. It is well known that the two
enantiomers of a drug can differ in their pharmacological activ-
ity, side effects, stability, and pharmacokinetics.1 Numerous
drugs on the market have a chiral center, such as β-blockers,
2-arylpropionic acid-type nonsteroidal antiinflammatory drugs
(NSAIDs), antidepressants, e.g., fluoxetine and citalopram,
and proton pump inhibitors, e.g., omeprazole. Although some
of them are still used as racemates, the number of enantio-
meric pure drugs is continuously increasing.2 Hence, monitor-
ing of the enantiopurity of drugs is crucial for quality control in
the production of pharmaceuticals. Sensitive methods for the
determination of the optical purity are required and numerous
publications report on the analytical separation of drugs.3,4
Chiral resolution by high-performance liquid chromatography
(HPLC) can be employed to determine the enantiomeric purity.
While the use of a chiral stationary phase (CSP) represents a
direct and accurate method, the formation of diastereomers by
means of a chiral derivatization agent (CDA) is an indirect
approach, which can be performed on an achiral stationary phase.
Both methods possess advantages and disadvantages.5
Derivatization reagents are well established tools for
analytical chemistry. Among them, the fluorescent derivatiza-
tion reagents are valued for their detection, which is far more
sensitive than the corresponding UV detection. Accordingly,
the precolumn fluorescence derivatization is a common
method to improve the limit of detection. For chiral derivatiza-
tion, several reagents have been developed; some of them
make use of their fluorescent properties.6–10 Moreover, a cou-
ple of fluorescence derivatization reagents were reported as
bearing a coumarin moiety as a fluorophore, one of the most
sensitive, generally accepted, and commercially available class
of fluorophors.11–17 Coumarin represents the parent substance
of a broad class of naturally occurring benzopyranes; many of
its derivatives are highly fluorescent. The structure–fluores-
cence relationships are well understood and depend on the
coumarin’s substitution pattern. The excitation wavelengths
range from the near-UV to the blue spectral region and are
often characterized by an acceptable large Stokes shift.18
Besides their use as derivatization reagents, coumarins have
© 2013 Wiley Periodicals, Inc.
also been applied to label bioactive molecules, e.g., as
inhibitors of disease-related proteins or internally quenched
substrates.19–24
Coumarin derivatives with an ester moiety or an aryl group in
position 3 are known to be highly fluorescent. The fluorescence
of the former compounds emerges especially in polar solvents
such as water and methanol, and 3-chloroformyl-
7-methoxycoumarin (MC3C), by forming the corresponding
esters, was useful for the determination of 17-oxosteroids in
urine11 or platelet-activating factors in lysates of human
polymorphonuclear leukocytes.13 Among the 3-aryl coumarins,25
7-diethylamino substituted derivatives such as 7-diethylamino-
3-[(4-(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA)12
or 3-(4-bromomethylphenyl)-7-diethylaminocoumarin (MPAC-
Br)15 are applicable to separate fatty or bile acids. However,
none of these coumarin-type reagents bear a chiral atom,
which would make them suitable for chiral derivatization.
In this work, the preparation and characterization of two
coumarin molecules featuring a chiral carbon atom for
derivatization as well as the resolution of representative
drug-coumarin conjugates is presented. The separation
efficiency of the derivatization reagents was evaluated in
terms of the parameters α and RS.
MATERIALS AND METHODS
Chemicals and Instruments
Thin-layer chromatography was carried out on Merck (Darmstadt,
Germany) aluminum sheets, silica gel 60 F254. Detection was performed
with UV light at 254 nm. Preparative column chromatography was
performed on Merck silica gel 60 (70–230 mesh). Melting points were
determined by a Boëtius melting point apparatus from VEB Wägetechnik
Rapido PHMK and are uncorrected. 1H NMR (500 MHz) and 13C NMR
*Correspondence to: Michael Gütschow, Pharmaceutical Institute, Pharma-
ceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn,
Germany. E-mail: guetschow@uni-bonn.de
Received for publication 18 July 2013; Accepted 6 August 2013
DOI: 10.1002/chir.22240
Published online 23 October 2013 in Wiley Online Library
(wileyonlinelibrary.com).

958
MERTENS AND GÜTSCHOW
spectra (125 MHz) were recorded on a Bruker Avance (Billerica, MA)
DRX 500. Chemical shifts δ are given in ppm referring to the signal center
using the solvent peaks for reference: CDCl3 7.26 / 77.0 ppm and DMSO-
d6 2.49 / 39.7 ppm. Optical rotations were determined on a Perkin-Elmer
(Norwalk, CT) 241 Polarimeter. LC-DAD chromatograms and ESI-MS
spectra were recorded on an Agilent (Palo Alto, CA) 1100 HPLC system
with Applied Biosystems (Foster City, CA) API-2000 mass spectrometer.
Solvents and reagents were obtained from Acros (Geel, Belgium), Alfa
Aesar (Karlsruhe, Germany), or Sigma-Aldrich (Steinheim, Germany).
HPLC water was obtained from a Milli-Q Water Purification System from
Millipore (Darmstadt, Germany) and HPLC acetonitrile was obtained
from VWR BDH Prolabo (Darmstadt, Germany).
8.75 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6, 25 °C) δ 15.1, 46.0,
52.4, 69.6, 101.3, 112.1, 113.7, 113.8, 131.9, 149.5, 156.3, 156.9, 163.4,
163.5. MS ESI+: m/z 278 ([C14H16NO5]+); HPLC purity: 99.6%
(λ = 220–400 nm).
Synthesis of the Conjugate of Reagent 4 and (S)-
(À)-Ibuprofen (5b)
(S)-(À)-Ibuprofen (62 mg, 0.3 mmol) and 4 (94 mg, 0.3 mmol) were
dissolved in DMF (5 mL). DIPEA (125 μl, 0.72 mmol) was added, followed
by O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluoroph-
osphate (HATU) (137 mg, 0.36 mmol ). After 2 h, the mixture was diluted
with EtOAc (50 mL) and washed with citric acid solution (2 × 50 mL),
saturated NaHCO3 (2 × 50 mL), and brine (50 mL). The product was
purified by column chromatography to remove polar byproducts using
petroleum ether/EtOAc (1/1) as eluent, which yielded the conjugate
5b as a white solid (57 mg, 0.12 mmol, 40%): mp 172–174 °C; [α]2D0 = À36.8
(c = 1.03, MeOH); UV (H2O) λmax nm (log ε): 355 (4.44), UV (MeCN) λmax
nm (log ε): 347 (4.35), UV (MeOH) λmax nm (log ε): 350 (4.40); 1H NMR
(500 MHz, DMSO-d6, 25 °C) δ 0.78 (d, 6H, 3 J = 6.6 Hz, CH(CH3)2), 1.16
(d, 3H, 3 J = 6.7 Hz, CH3CHAr), 1.28 (d, 3H, 3 J = 6.9 Hz, CH3CHNH), 1.71
(sept, 1H, 3 J = 6.7 Hz, CH(CH3)2), 2.33 (d, 2H, 3 J = 7.3 Hz, CHCH2Ar),
3.56 (q, 1H, 3 J=6.9Hz, ArCHCH3), 3.80 (s, 3H, OCH3), 3.95-3.96 (m, 2H,
CHCH2O), 4.14 (app sept, 1H, 3 J=6.6 Hz, NHCH), 6.86 (dd, 1H, 3 J=8.7Hz,
4 J= 2.5 Hz, 6-H), 6.91 (d, 1H, 4 J= 2.5 Hz, 8-H), 6.96 (d, 2H, 3 J= 8.9 Hz, 3´-H,
5´-H), 7.15 (d, 2H, 3 J= 8.2 Hz, 2´-H, 6´-H), 7.77 (d, 1H, 3 J=8.8Hz, 5-H), 7.97
(d, 1H, 3 J = 7.9 Hz, NH), 8.72 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-
d6, 25 °C) δ 17.0, 18.6, 22.2, 29.7, 43.7, 44.4, 44.7, 52.3, 71.2, 101.0,
111.6, 113.2, 113.7, 127.0, 128.7, 131.8, 139.1, 139.6, 149.5, 156.3, 157.0,
163.2, 164.2, 173.3. MS ESI+: m/z 466 ([C27H32NO6]+); HPLC purity:
96.9% (λ = 220–400 nm).
Synthesis of Methyl 7-Hydroxy-2-oxo-2H-chromene-
3-carboxylate (2)
2,4-Dihydroxybenzaldehyde (1, 5.52 g, 40.0 mmol) was dissolved in
MeOH (60 mL). Dimethyl malonate (5.81 g, 5.0 mL, 44.0 mmol, 1.1 eq.)
and piperidine (426 mg, 0.50 mL, 5.0 mmol) were added. The solution was
refluxed for 2 h. The stirred reaction mixture was cooled to room tempera-
ture and kept on an ice bath for 30min. The product was filtered off and
washed with cold MeOH (20mL) to yield a white solid (5.74 g, 26.1 mmol,
65%): mp 264-265 °C (lit.26 mp 264-267 °C); 1H NMR (500MHz, DMSO-d6,
30 °C) δ 3.78 (s, 3H, CH3), 6.71 (d, 1H, 4 J = 2.5 Hz, 8-H), 6.82 (dd, 1H,
3 J = 8.5 Hz, 4 J = 2.3 Hz, 8-H), 7.73 (d, 1H, 3 J = 8.8Hz, 5-H), 8.67 (s, 1H, 4-H),
11.04 (br s, 1H, OH); 13C NMR (125 MHz, CDCl3, 25°C) δ 52.2, 101.9,
110.6, 112.0, 114.2, 132.3, 149.8, 156.5, 157.3, 163.6, 164.3. MS ESI+: m/z
221 ([C11H9O5]+); HPLC purity: 99.7% (λ = 220–450 nm).
Synthesis of (S)-(À)-Methyl 7-(2-(tert-
butoxycarbonylamino)propoxy)-2-oxo-2H-chromone-3-
carboxylate (3)
Synthesis of (R)-(+)-Methyl 7-(1-(benzyloxy)-1-oxopropan-
2-yloxy)-2-oxo-2H-chromene-3-carboxylate (6)
Methyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (2, 440 mg, 2.0 mmol),
triphenylphosphine (1.04 g, 4.0 mmol), (S)-(À)-tert-butyl 1-hydroxypropan-2-yl
carbamate (525 mg, 3.0 mmol) were dissolved in THF (30 mL) and cooled to
0 °C. Diisopropyl azodicarboxylate (DIAD) (0.61 g, 0.59 mL, 3.0 mmol) was
added dropwise and the solution was stirred overnight at room temperature.
After evaporation in vacuo the residue was dissolved in EtOAc (100 mL) and
the organic phase was washed with 1 N NaOH (3 × 100 mL) and brine
(100 mL). The organic phase was evaporated and the residue was purified
by column chromatography using petroleum ether/EtOAc (1/1) as eluent.
The product was obtained as pale yellow solid (683 mg, 1.8 mmol, 90%): mp
128–129 °C; [α]2D0 =À38.9 (c= 0.95, MeOH); UV (H2O) λmax nm (log ε): 353
(4.41), UV (MeCN) λmax nm (log ε): 347 (4.29), UV (MeOH) λmax nm (log
ε): 349 (4.42); 1H NMR (500 MHz, DMSO-d6, 25°C) δ 1.12 (d, 3H,
3 J= 7.0 Hz, CHCH3), 1.37 (s, 9H, C(CH3)3), 3.80 (s, 3H, OCH3), 3.82-3.88
(m, 1H, CH), 3.94-3.97 (dd, 2 J=9.8Hz, 3 J= 5.7 Hz, 1H, OCHH), 4.01-4.04
(dd, 1H, 2 J=9.8Hz, 3 J=6.4Hz, OCHH), 6.89 (d, 1H, 3 J= 7.3 Hz, NH), 6.99
(dd, 1H, 3 J=8.5Hz, 4 J= 2.2 Hz, 6-H), 7.01 (d, 1H, 4 J= 2.3 Hz, 8-H), 7.82
(d, 1H, 3 J= 8.5 Hz, 5-H), 8.73 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6,
25 °C) δ 17.3, 28.3, 45.2, 52.3, 71.5, 77.9, 100.9, 111.6, 113.2, 113.8, 131.8,
149.5, 155.2, 156.3, 157.1, 163.5, 164.2. MS ESI+: m/z 378 ([C19H24NO7]+);
HPLC purity: 95.7% (λ = 220–400 nm).
Methyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (2, 440 mg, 2.0 mmol),
triphenylphosphine (1.04 g, 4.0 mmol) and benzyl (S)-(À)-lactate (540 mg,
3.0mmol) were dissolved in THF (30mL) and cooled to 0°C. DIAD (0.61g,
0.59 mL, 3.0 mmol) was added dropwise and the solution was stirred for 2 h
at room temperature. After evaporation in vacuo the residue was diluted
with EtOAc (50 mL), washed with 1 N NaOH (3 × 50 mL), brine (50 mL),
and dried over Na2SO4. After evaporation the residue was purified by col-
umn chromatography using petroleum ether/EtOAc (1/1) to yield a col-
orless oil (540 mg, 1.4 mmol, 71%): [α]2D0 = +123 (c = 1.00, MeOH); 1H
NMR (500 MHz, DMSO-d6, 25 °C) δ 1.57 (d, 3H, 3 J = 6.6 Hz, CHCH3),
3.81 (s, 3H, OCH3), 5.16 (d, 1H, 2 J = 12.3 Hz, CHH), 5.20 (d, 1H,
2 J = 12.3 Hz, CHH), 5.33 (q, 1H, 3 J = 7.0 Hz, CH), 6.94 (d, 1H,
4 J = 2.5 Hz, 8-H), 6.99 (dd, 1H, (d, 1H, 3 J = 8.8 Hz, 4 J = 2.5 Hz, 6-H), 7.28-
7.35 (m, 5H, arom. H), 7.82 (d, 1H, 3 J = 8.5 Hz, 5-H), 8.73 (s, 1H, 4-H);
13C NMR (125 MHz, DMSO-d6, 25 °C) δ 18.0, 52.4, 66.6, 72.4, 101.6,
112.1, 113.7, 113.8, 128.0, 128.3, 128.5, 131.9, 135.7, 149.4, 156.2, 156.8,
162.7, 163.4, 170.6. MS ESI+: m/z 383 ([C21H19O7]+); HPLC purity:
96.5% (λ = 220–400 nm).
Hydrogenolysis of Compound 6
Synthesis of (S)-(À)-1-(3-(Methoxycarbonyl)-2-oxo-2H-
(R)-(+)-Methyl 7-(1-(benzyloxy)-1-oxopropan-2-yloxy)-2-oxo-2H-chromene-
3-carboxylate (501 mg, 1.3 mmol) was hydrogenated with Pd/C (50 mg) as
catalyst. After 2 h at room temperature, the mixture was filtered through a
pad of celite and evaporated in vacuo. The residue was purified by column
chromatography using EtOAc + 1% AcOH as eluent. The product, (R)-2-(3-
(methoxycarbonyl)-2-oxochroman-7yloxy)propanoic acid (7), was obtained
as a white solid (310 mg, 1.05 mmol, 81%): mp 117-118 °C; 1H NMR
(500 MHz, DMSO-d6, 25°C) δ 1.47 (d, 3H, 3 J=6.7 Hz, CHCH3), 3.17-3.19
(m, 2H, CH2), 3.65 and 3.66 (s, 3H, OCH3), 4.09-4.13 (m, 1H, CH2CH), 4.83
and 4.84 (q, 1H, 3 J=6.9Hz, CH), 6.60-6.61 (m, 1H, 8-H), 6.65-6.68 (m, 1H,
6-H), 7.20 (d, 1H, 3 J= 8.2 Hz, 5-H), 13.00 (br s, 1H, OH); 13C NMR
(125 MHz, DMSO-d6, 25°C) δ 18.26 and 18.28, 25.8, 45.73 and 45.75, 52.8,
72.0, 103.0 and 103.2, 111.2 and 111.5, 113.67 and 113.72, 129.2, 151.67 and
151.69, 157.6, 164.77 and 164.79, 168.4, 172.9. MS ESI+: m/z 312
([C14H18NO7]+), 295 ([C14H15O7]+); HPLC purity: 96.2% (λ = 220–400 nm).
chromone-7-yloxy)propan-2-aminium Chloride (4)
(S)-(À)-Methyl 7-(2-(tert-butoxycarbonylamino)propoxy)-2-oxo-2H-c-
hromone-3-carboxylate (3, 1.53 g, 4.0 mmol) was dissolved in EtOAc
(10 mL) and cooled to 0 °C. A solution of HCl in EtOAc (1 N, 20 mL,
20.0 mmol, 5.0 eq.) was added via syringe and the solution was stirred
for 2 h at room temperature. The precipitate was filtered off, washed with
diethyl ether (20 mL) to give the product as a white solid (991 mg,
3.2 mmol, 80%): mp 267–268 °C; [α]2D0 = À31.3 (c = 1.07, H2O); UV (H2O)
λmax nm (log ε): 349 (4.38), UV (MeCN) λmax nm (log ε): 342 (4.41),
UV (MeOH) λmax nm (log ε): 344 (4.39); 1H NMR (500 MHz, DMSO-
d6, 25 °C) δ 1.30 (d, 3H, 3 J = 6.7 Hz, CHCH3), 3.60-3.64 (m, 1H, CHNH3),
3.80 (s, 3H, OCH3), 4.17 (dd, 1H, 2 J = 10.6 Hz, 3 J = 7.1 Hz, OCHH), 4.29
(dd, 1H, 2 J = 10.4 Hz, 3 J = 3.8 Hz, OCHH), 7.04 (d, 1H, 4 J = 2.6 Hz, 8-H),
7.07 (s, 1H, 6-H), 7.87 (d, 1H, 3 J = 8.8 Hz, 5-H), 8.36 (br s, 3H, NH3),
Chirality DOI 10.1002/chir

TWO COUMARIN-TYPE DERIVATIZATION REAGENTS
959
3 J = 8.7 Hz, 4 J = 2.5 Hz, 6-H), 6.98 (d, 2H, 3 J = 8.9 Hz, 3´-H, 5´-H), 7.64-7.67
(m, 3H, 2´-H, 6´-H, 5-H), 8.11 (s, 1H, 4-H). The CO2H proton could not be
detected. 13C NMR (125 MHz, DMSO-d6, 25°C) δ 18.2, 55.4, 72.1, 101.3,
113.1, 113.7, 113.8, 123.3, 127.2, 129.6, 129.7, 139.4, 154.4, 159.5, 160.2,
160.1, 172.6. MS ESI+: m/z 341 ([C19H17O6]+); HPLC purity: 99.5%
(λ = 220–400 nm).
Synthesis of 3-(4-Methoxyphenyl)-2-oxo-2H-chromen-7-yl
Acetate (8)
2,4-Dihydroxybenzaldehyde (1, 2.76 g, 20.0 mmol) and p-
methoxyphenylacetic acid (3.32 g, 20.0 mmol) were refluxed in a mixture
of pyridine (30 mL) and acetic anhydride (30 mL) for 9 h. After cooling to
room temperature, the precipitate was filtered off and washed with
MeOH (30 mL) to give the product as a white solid (1.92 g, 6.2 mmol,
31%): mp 168–170 °C (lit.27 mp 181–182 °C); 1H NMR (500 MHz,
DMSO-d6, 25 °C) δ 2.31 (s, 3H, H3CCO), 3.80 (s, 3H, OCH3), 7.02 (d,
2H, 3 J = 8.9 Hz, 3´-H, 5´-H), 7.16 (dd, 1H, 3 J = 8.5 Hz, 4 J = 2.2 Hz, 6-H),
7.28 (d, 1H, 4 J = 2.2 Hz, 8-H), 7.69 (d, 2H, 3 J = 8.9 Hz, 2´-H, 6´-H), 7.79 (d,
1H, 3 J = 8.5 Hz, 5-H), 8.19 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6,
25 °C) δ 21.0, 55.4, 109.7, 113.8, 117.6, 118.8, 125.9, 126.9, 129.3, 129.9,
138.8, 152.5, 153.3, 159.8, 168.9. MS ESI+: m/z 311 ([C18H15O5]+); HPLC
purity: 93.8% (λ = 220–400 nm).
Synthesis of the Conjugate of Reagent 11 and (S)-(À)-
α-Methylbenzylamine (12e)
(S)-(À)-α-Methylbenzylamine (37 mg, 0.3 mmol) and 11 (106 mg,
0.3 mmol) were dissolved in DMF (5 mL). DIPEA (125 μL, 0.72 mmol) was
added, followed by HATU (137 mg, 0.36 mmol). After 2 h, the mixture was di-
luted with EtOAc (50 mL) and washed with citric acid solution (2 × 50 mL),
saturated NaHCO3 (2 × 50 mL), and brine (50 mL). The product was purified
by column chromatography to remove polar byproducts using petroleum
ether/EtOAc (1/1) as eluent, which yielded the conjugate 12e as a white
solid (99 mg, 0.22 mmol, 74%): mp 152–154 °C; [α]2D0 = +71.8 (c= 1.04,
MeOH); UV (H2O) λmax nm (log ε): 347 (4.42), UV (MeCN) λmax nm (log
ε): 344 (4.49), UV (MeOH) λmax nm (log ε): 346 (4.49); 1H NMR (500 MHz,
DMSO-d6, 25°C) δ 1.35 (d, 3H, 3 J=7.0Hz, CH3CHAr), 1.49 (d, 3H,
3 J=6.6Hz, CH3CHO), 3.80 (s, 3H, OCH3), 4.89-4.94 (m, 2H, CH(CH3)Ar
and CH3CHO), 6.91-6.94 (m, 2H, arom.H), 7.00 (d, 2H, 3 J= 8.9 Hz, 3´-H, 5´-
H), 7.20-7.17 (m, 1H, arom.H), 7.28-7.34 (m, 4H, arom.H), 7.63 (d, 1H,
3 J=8.5Hz, 5-H), 7.67 (d, 2H, 3 J= 9.2 Hz, 2´-H, 6´-H), 8.11 (s, 1H, 4-H), 8.66
(d, 1H, 3 J= 8.2 Hz, NH); 13C NMR (125 MHz, DMSO-d6, 25°C) δ 18.6, 22.3,
48.0, 55.4, 73.9, 101.4, 113.56, 113.63, 113.8, 123.3, 126.0, 126.8, 127.2, 128.3,
129.5, 129.7, 139.4, 144.5, 154.4, 159.5, 160.09, 160.14, 169.6. MS ESI+: m/z
444 ([C27H26NO5]+); HPLC purity: 98.8% (λ =220–400 nm).
Synthesis of 7-Hydroxy-3-(4-methoxyphenyl)-2H-chromen-
2-one (9)
3-(4-Methoxyphenyl)-2-oxo-2H-chromen-7-yl acetate (8, 1.55 g, 5.0 mmol)
and K2CO3 (1.38g, 10.0 mmol) were refluxed for 1 h in MeOH (30mL).
After evaporation in vacuo the residue was dissolved in H2O (50mL). The
pH was adjusted to 2 with 2 N HCl. The precipitate was filtered off and
recrystallized from MeOH (105mL) to give the product as yellow needles
(973 mg, 3.6 mmol, 73%): mp 228 °C (lit.27 mp 215–216); 1H NMR
(500 MHz, DMSO-d6, 25 °C) δ 3.78 (s, 3H, CH3), 6.73 (d, 1H, 4 J = 2.3 Hz,
8-H), 6.80 (dd, 1H, 3 J= 8.5Hz, 4 J = 2.6 Hz, 6-H), 6.98 (d, 2H, 3 J = 8.9 Hz,
3´-H, 5´-H), 7.56 (d, 1H, 3 J = 8.5 Hz, 5-H), 7.64 (d, 2H, 3 J= 8.8Hz, 2´-H, 6´-
H), 8.05 (s, 1H, 4-H), 10.51 (br s, 1H, OH); 13C NMR (125MHz, DMSO-
d6, 25 °C) δ 55.3, 101.8, 112.2, 113.5, 113.8, 122.0, 127.5, 129.6, 129.8,
139.9, 154.8, 159.3, 160.3, 161.1. MS ESI+: m/z 269 ([C16H13O4]+); HPLC
purity: 96.3% (λ = 220–400 nm).
Derivatization Procedure
One equivalent (0.1–0.3 mmol) of the sample (chiral carboxylic acid or
chiral amine) and one equivalent of the reagent (4 or 11) were dissolved
in an appropriate solvent (e.g., DMF or MeCN) and 1.2 equivalents N,
N-diisopropylethylamine (DIPEA) were added, followed by 1.2 equivalents
HATU. The reaction mixture was stirred for 2 h at room temperature. The
solution was diluted with EtOAc (50mL) and washed with 10% citric acid
solution (2 × 50mL), saturated NaHCO3 (2 × 50mL), and brine (50 mL).
Products were purified by column chromatography to remove polar
byproducts using petroleum ether/EtOAc as eluent. The structures were
confirmed by liquid chromatography / mass spectroscopy (LC/MS) and
nuclear magnetic resonance (NMR) analytics (see Supporting Material,
Table S1).
Synthesis of (R)-(+)-Methyl 2-(3-(4-(4-Methoxyphenyl)-2-
oxo-2H-chromen-7-yloxy)propanoate (10)
7-Hydroxy-3-(4-methoxyphenyl)-2H-chromen-2-one (9, 537 mg, 2.0 mmol),
triphenylphosphine (1.04 g, 4.0 mmol) and methyl (S)-(À)-lactate (312 mg,
3.0mmol) were dissolved in THF (30mL) and cooled to 0 °C. Di-tert-butyl
azodicarboxylate (690 mg, 3.0 mmol) was added portionwise and the solution
was stirred for 2 h at room temperature. After evaporation in vacuo the resi-
due was diluted with EtOAc (50 mL), washed with 1 N NaOH (3 × 50 mL),
brine (50 mL), and dried over Na2SO4. After evaporation the residue was
purified by column chromatography petroleum ether/EtOAc (3/1) to yield
a light green solid (545 mg, 1.54 mmol, 77%): mp 172–173 °C; [α]2D0 = +74.7
(c=1.07, MeOH); UV (H2O) λmax nm (log ε): 341 (4.45), UV (MeCN) λmax
UV and Fluorescence Spectra
1
UV spectra were recorded on a Cary 50 Bio device from Varian, fluores-
cence spectra on a Monaco Safas spectrofluorometer flx. The compounds
were solved in DMSO in a concentration of 10 mM, 1.0 mM, and 100 μM.
All UV and fluorescence spectra were recorded in water, MeOH, and
acetonitrile containing 1% DMSO. The final concentration of the
compound was 10 μM for the UV determination and 1.0 μM for the
fluorescence spectra. Determination of the extinction coefficients was
done according to the Beer Lambert law (ε (λ) = A / (c × b)), where b is
the cuvette length (1.0 cm), c the used concentration (10 μM), and A
the measured absorption value.
nm (log ε): 342 (4.54), UV (MeOH) λmax nm (log ε): 345 (4.44); H NMR
(500 MHz, DMSO-d6, 25°C) δ 1.55 (d, 3H, 3 J= 7.0 Hz, CHCH3), 3.70 (s, 3H,
CO2CH3), 3.79 (s, 3H, OCH3), 5.20 (q, 1H, 3 J=7.0Hz, CH), 6.94-6.96
(m, 2H, arom. H), 7.00 (d, 2H, 3 J= 8.8 Hz, 3´-H, 5’-H), 7.65-7.68 (m, 3H, arom.
H), 8.11 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6, 25°C) δ 18.1, 52.4, 55.4,
72.1, 101.5, 113.1, 113.8, 113.9, 123.5, 127.2, 129.70, 129.71, 139.3, 154.4,
159.5, 160.0, 160.1, 171.5. MS ESI+: m/z 355 ([C20H19O6]+); HPLC purity:
99.4% (λ =220–400 nm).
Synthesis of (R)-(+)-2-(3-(4-methoxyphenyl)-2-oxo-2H-
chromen-7-yloxy)propanoic Acid (11)
Chromatographic Conditions
(R)-Methyl 2-(3-(4-(4-methoxyphenyl)-2-oxo-2H-chromen-7-yloxy)propanoate
(10, 469 mg, 1.3 mmol) was dissolved in THF (10 mL) and water
(20 mL). LiOH × H2O (334 mg, 7.8 mmol, 6.0 eq.) was added. The
mixture was stirred overnight at room temperature. After filtration, the
filtrate was diluted with water (40 mL) and acidified with 2 N HCl to pH
~2. The solution was extracted with CH2Cl2 (3 × 60 mL), the combined
organic layers were dried over Na2SO4. After evaporation the residue was
recrystallized from EtOAc and petroleum ether to yield a light gray solid
(321 mg, 0.94 mmol, 73%): mp 145–147 °C; [α]2D0 = +68.3 (c = 1.00, MeOH)
UV (H2O) λmax nm (log ε): 345 (4.40), UV (MeCN) λmax nm (log ε): 342
(4.62), UV (MeOH) λmax nm (log ε): 349 (4.46); 1H NMR (500 MHz,
DMSO-d6, 25°C) δ 1.54 (d, 3H, 3 J= 6.7Hz, CHCH3), 3.79 (s, 3H, OCH3),
5.04 (q, 1H, 3 J = 6.6 Hz, CH), 6.90 (d, 1H, 4 J= 2.5 Hz, 8-H), 6.93 (dd, 1H,
HPLC experiments were performed on a Jasco HPLC 2000 instrument.
Chromatograms were obtained using ChromPass (v. 1.8.6.1, Jasco) chro-
matography software. Chromatographic analyses were performed with a
LiChroCART, LiChrosorb column (250–4, RP-18, 7 μm particle size) from
Merck. Stock solutions (2.5 mM) of the samples were prepared in aceto-
nitrile and diluted with acetonitrile to 25 μM, 1.0 μM, or 250 nM, respec-
tively, with acetonitrile. A volume of 20 μL was injected into the HPLC
device. All mobile phases were degassed with a Jasco DG-2080-53
degasser. A column temperature of 30 °C was applied with a JASCO
CO-2060 column oven. UV detection wavelength was fixed at 350 nm
using a Jasco UV-2075. For derivatives 5, the excitation and emission
wavelengths were adjusted to 360 nm and 410 nm, respectively, for
Chirality DOI 10.1002/chir

960
MERTENS AND GÜTSCHOW
derivatives 10 to 350 nm and 450 nm, respectively. Analytical parameters
were calculated according to the following equations. Capacity factor k
was calculated k = tR´/tM, where tR´ is the reduced retention time tR´ = tR
– tM and tM is the experimental void time. Separation factor α was calcu-
lated from the observed α = 2 k/1 k, where 2 k and 1 k were the calculated
capacity factors of the two diastereomers. Resolution Rs was calculated
from RS = 1.18 (tR2 – tR1)/(2wh + 1wh), where wh is the peak width in 50%
height. Values tM were obtained after injection of a thiourea solution
(0.1 mg/mL) and UV detection at 240 nm and the respective flow rate
(1.0 mL/min or 1.3 mL/min). For the flow rates, the tM values were
2.59 min and 2.03 min, respectively.
analyte with the one, or an amine-type analyte with the other
reagent. The resulting conjugates partake in the coumarins’
fluorescence, thus allowing for sensitive detection.
The preparation of the reagent for chiral carboxylic acids is
shown in Scheme 1. Starting from 2,4-dihydroxybenzaldehyde,
Knoevenagel condensation with dimethyl malonate yielded the
7-hydroxy-coumarin derivative 2. This intermediate represents
a fluorophore, and the 7-alkoxy relatives show fluorescence
without pH dependency. Mitsunobu reaction of 2 with
Boc-(S)-(À)-alaninol produced the protected precursor 3. The
Boc group was removed with HCl to yield the derivatization re-
agent 4 in an overall yield of 47%. The fluorescence properties
of coumarin 4 were evaluated in water, acetonitrile, and
methanol. As exemplified in Figure 1, it has an absorption max-
imum at 342 nm and an emission maximum at 402 nm, when
the excitation wavelength was 350 nm. Such a notable Stokes
shift of 60 nm was similarly observed for 4 in the other
solvents (data not shown). These spectroscopic properties
are comparable to those of the known MC3C products.11 An
amine functionality for the derivatization has been also used
in luminarin 4, a achiral coumarin labeling reagent for
carboxylic acids.14 Compound 4 can be considered as a
candidate for further investigation on enantioselective
inhibition of cholinesterases.28,29
RESULTS AND DISCUSSION
A chiral derivatization reagent has to fulfill the following
requirements: (i) a chiral center in close proximity to its
reactive functional group, (ii) sufficient reactivity towards
the functional moiety of the analyte, (iii) stability of the
diastereomeric products, and (iv) the opportunity to exert a
sensitive detection technique such as fluorescence.5,10 We
have designed two reagents that meet these requirements,
i.e., coumarins 4 (Scheme 1) and 11 (Scheme 2). In both
cases, the chiral center is brought as near as possible to the
reactive group. To form a stable amide bond, only a coupling
additive is needed for the reaction of a carboxylic acid-type
Scheme 1. Synthesis of reagent 4 and reaction with chiral carboxylic acids. Reagents and conditions: (a) dimethyl malonate, piperidine, MeOH; (b) Boc-(S)-
alaninol, PPh3, DIAD, THF; (c) HCl, EtOAc; (d) RCO2H, DIPEA, HATU, DMF or MeCN.
Scheme 2. Synthesis of reagent 11 and reaction with chiral amines. Reagents and conditions: (a) dimethyl malonate, piperidine, MeOH; (b) benzyl (S)-(–)-lactate,
PPh3, DIAD, THF; (c) H2, Pd/C, MeOH; (d) 4-methoxyphenylacetic acid, Ac2O, pyridine; (e) K2CO3, MeOH; (f) methyl-(S)-(–)-lactate, PPh3, di-tert-butyl
azodicarboxylate, THF; (g) LiOH, THF/water; (h) R1R2NH, DIPEA, HATU, DMF or MeCN.
Chirality DOI 10.1002/chir

TWO COUMARIN-TYPE DERIVATIZATION REAGENTS
961
For the design of the other reagent, lactate was chosen as
the chiral recognition site to be introduced into the coumarin
core. A similar strategy was followed to construct chiral
derivatizing agents for 1H NMR inspection.30 We used benzyl
(S)-(À)-lactate in a first attempt to obtain a reagent for chiral
amines (Scheme 2). After Mitsunobu coupling with 2, the
resulting benzyl ester 6 was removed hydrogenolytically.
However, cleavage of the benzyl ester yielded a mixture of
nonfluorescent diastereomers (7), whose C3-C4 single bonds
resulted from a catalytic hydrogenation of the pyran double
bond.31 Therefore, methyl (S)-(À)-lactate was employed in
an alternative approach which was not compatible with the
methyl ester group of 2. It was exchanged for a 4-
methoxyphenyl group as follows. Perkin condensation of
2,4-dihydroxybenzaldehyde and 4-methoxyphenylacetic acid
in a mixture of pyridine and acetic anhydride gave derivative
8, which was deacetylated under basic conditions resulting in
9. Methyl (S)-(À)-lactate was now coupled to 9 using the
Mitsunobu protocol to retain the chiral information via
Mitsunobu inversion.32 In contrast to the two aforementioned
Mitsunobu reactions, di-tert-butyl azodicarboxylate was used,
because the previously used DIAD furnished diisopropyl hy-
drazine-1,2-dicarboxylate, which could not be separated from
the product of this reaction. Alkaline hydrolysis of 10 gave
the free carboxylic acid 11, suitable for coupling to different
chiral amines after activation. The overall yield for the new
3-phenylcoumarin 11 was 13%. The fluorescence properties
of reagent 11 were found to be in accordance with related
3-phenylcoumarins;25 it shows an absorption maximum at
342 nm and an emission maximum at 436 nm, after excitation
at 350 nm. Again, a remarkable Stokes shift of 94 nm was
obtained (Fig. 2).
Fig. 1. Absorption (10 μM, solid line) and emission (1.0 μM, dashed line,
excitation wavelength 350 nm) spectra of 4 in acetonitrile.
Next, we investigated the suitability of 4 and 11 as chiral
derivatizing agents by preparing and analyzing prototype
drug conjugates. Both agents need an activation step prior
Fig. 2. Absorption (10 μM, solid line) and emission (1.0 μM, dashed line,
excitation wavelength 350 nm) spectra of 11 in acetonitrile.
TABLE 1. Chromatographic parameters for different conjugates of 4 and 2-arylpropionic acids*
2-Arylpropionic acid Structure
(+/À)-ibuprofen
Conjugate
5a
k
α
RS
9.50 and 7.13
(6.92 and 6.63)
1.33 (1.04)
5.26 (2.61)
5b
5c
(À)-ibuprofen
6.73
(6.71)
—
—
(+/À)-flurbiprofen
6.74 and 4.58
(6.49 and 6.14)
1.47 (1.06)
5.69 (2.92)
5d
(+/À)-ketoprofen
3.64 and 2.71
(5.89 and 5.58)
1.34 (1.06)
3.47 (2.52)
*For chromatographic conditions, see Figs. 3, 4, and Supporting Material. Parameters from measurements under isocratic conditions and (in parentheses) gradient
elution are given.
Chirality DOI 10.1002/chir

962
MERTENS AND GÜTSCHOW
Fig. 3. Representative chromatogram of 5c (25 μM), indicating the existence of two diastereomers. Top: UV detection (350 nm), 1st peak area 7.5 mV × min (60%),
2nd peak area 5.0 mV × min (40%). Bottom: fluorescence detection (λex = 360 nm and λem = 410 nm), 1st peak area 431.9 mV × min (49.8%), 2nd peak area 436.1 mV × min
(50.2%). Conditions: Mobile phase isocratic H2O (A) : MeCN (B), 0 min/ 50% B, 25 min/ 50% B, 27 min/ 100% B, 28 min/ 50%, 30 min/ 50%; flow rate: 1.0 ml/min.
Fig. 4. Representative chromatogram of 5a (1.0 μM), the conjugate of racemic ibuprofen and 4, indicating the existence of two diastereomers. Top: UV detection
(350 nm), 1st peak area 0.19 mV × min (48%), 2nd peak area 0.21 mV × min (52%). Bottom: fluorescence detection (λex = 360 nm and λem = 410 nm), 1st peak area
19.0 mV × min (51.6%), 2nd peak area 17.8 mV × min (48.4%). Conditions: mobile phase gradient H2O (A) : MeCN (B), gradient table: 0 min/ 10% B, 25 min/ 100%
B, 27 min/ 100% B, 28 min/ 10%, 30 min/ 10%; flow rate: 1.0 ml/min.
to amide coupling. In the case of 4, the carboxylic acid
analyte has to be activated, in the case of 11, the reagent’s
carboxyl group. When demonstrating the facile coupling of
our reagents, HATU was used for the activation and the reac-
tions proceeded smoothly. We applied HATU in order to min-
imize racemization. The reaction courses were monitored by
thin-layer chromatography (TLC) and found to be completed
within 2 h. As a matter of fact, when coupled to a chiral agent,
racemic analytes would differ in the reactivity of their enantio-
mers. This was not further investigated in the course of the
product analysis. However, it should be noted that a deter-
mined optical purity (of the conjugate) would probably differ
from the real optical purity (of the analyte) due to a preferen-
tial loss of one diastereomer in the course of coupling and
subsequent purification by column chromatography. The
spectral properties of the conjugates were similar to those of
the reagents, with respect to UV absorption and fluorescence
emission (see Supporting Material, Figs. S1 and S2).
and enantiopure drug, as well as flurbiprofen and ketoprofen
(Table 1). The HPLC analysis was performed under reversed
phase conditions using a common achiral C18 column with
acetonitrile/water mixtures as eluent. The separation of the
2-arylpropionic acid conjugates was achieved either with
isocratic elution (for an example, see Fig. 3) or by means of
a simple linear gradient (for an example, see Fig. 4). The sep-
aration factor α was greater than 1, indicating the successful
separation of the three diastereomeric conjugates 5a, 5c,
and 5d, and baseline separation was accomplished under
isocratic conditions as the resolution values RS clearly exceed
1.5 (Table 1). This also holds true for gradient elution, with
somewhat weaker, but still sufficient separation (Table 1,
values in parentheses). As expected, conjugate 5b produced
a single HPLC peak. A minor impurity was visible under
isocratic conditions and fluorescence detection, when a
100 μM solution of 5b was injected (see Supporting Material,
S19). This new fluorescent enantiopure ibuprofen conjugate
was fully characterized (see Materials and Methods).
As depicted in Scheme 1, agent 4 was coupled to carboxylic
acids to produce conjugates 5a-d. Three 2-arylpropionic acids
were selected that are frequently used as NSAIDs for the
treatment of inflammation and pain, i.e., ibuprofen, as racemic
Chirality DOI 10.1002/chir
From the pool of chiral amines, three β-blockers and
α-methylbenzylamine were chosen as model analytes to be
coupled with 11 (Scheme 2, Table 2). The separation of the

TWO COUMARIN-TYPE DERIVATIZATION REAGENTS
963
TABLE 2. Chromatographic parameters for different conjugates of 11 and chiral amines*
Conjugate
12a
Chiral amine
(+/À)-atenolol
Structure
k
α
RS
3.97 and 4.76
1.20
1.62
12b
12c
(+/À)-propanolol
(+/À)-metoprolol
9.22 and 10.0
7.70 and 8.76
1.08
1.14
1.41
2.05
12d
12e
(+/À)-α-methyl-benzylamine
(S)-(À)-α-methyl-benzylamine
19.68 and 21.02
1.07
1.18
21.02
—
—
*For chromatographic conditions, see Fig. 5 and Supporting Material. Parameters from measurements under isocratic conditions are given.
Fig. 5. Representative chromatogram of 12c (2.5 μM), the conjugate of racemic metoprolol and 11, indicating the existence of two diastereomers. Top: UV de-
tection (350 nm), 1st peak area 0.8 mV × min (64%), 2nd peak area 0.4 mV × min (36%). Bottom: fluorescence detection (λex = 350 nm and λem = 450 nm), 1st peak area
112.3 mV × min (64.1%), 2nd peak area 62.9 mV × min (35.9%). Conditions: Mobile phase isocratic H2O (A) : MeCN (B): 0 min/ 50% B, 30 min/ 50% B, 32 min/ 100% B,
33 min/ 50%, 35 min/ 50%; flow rate: 1.0 ml/min.
β-blocker conjugates 12a-c was carried out by isocratic elu-
tion, and for atenolol and metoprolol conjugates (12a and
12c), baseline separation was accomplished with RS values
(1.62 and 2.05) greater than 1.5. The discrimination of the
two diastereomeric metoprolol conjugates by HPLC is illus-
trated in Figure 5. For propanolol and α-methylbenzylamine,
an effective enantiomeric separation was attained with some-
what lower resolution. Conjugate 12e, synthesized from 11
and (S)-(À)-α-methylbenzylamine, completes the series of
new compounds of this study (see Materials and Methods).
As expected, fluorescence detection resulted in a much
better detectability of the conjugates than UV detection. We
Chirality DOI 10.1002/chir

964
MERTENS AND GÜTSCHOW
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obtained usable chromatograms with analyte quantities of 5
pmol when, for example, injecting 20 μL of a 250 nM solution
of 5c or 5d and monitoring the fluorescence (see Supporting
Material S21 and S22). To apply both agents (4 and 11) for
the determination of optical purity, the reaction conditions
and sample work-up should be optimized. As can be seen in
Figures 3 and 5, the chromatograms showed different peak
areas for both diastereomers, respectively. The resolution of
conjugate 5c reveals this deviation only in the UV channel,
obviously a result of the poor UV detectability. Besides their
suitability as CDAs for HPLC analysis, the coumarin reagents
can be applied as CDAs for NMR spectroscopy. This is exem-
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conjugates 5a and 12d as diastereomers (see Supporting
Material S5 and S13).
In conclusion, the synthesis of two chiral, coumarin-type de-
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and the chiral center were introduced in a side chain, connected
via an ether bridge to coumarin C-7. The reagents 4 and 11 dif-
fer with respect to their reactive group to allow for the amide
coupling of carboxylic acids or amines, respectively. They bear
different moieties at 3-position resulting in slightly different
spectroscopic properties. The application of 4 and 11 as CDAs
was successfully demonstrated by producing representative
drug conjugates and analyzing their chromatographic separa-
tion under isocratic and gradient conditions.
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