Synthesis of the fluorescent amino acid rac-(7-hydroxycoumarin-4-yl) ethylglycine

Article abstract of DOI:10.3762/bjoc.6.69

The hydrochloride of the racemic amino acid (7-hydroxycoumarin-4-yl) ethylglycine, a versatile fluorescent probe in proteins, has been synthesized in five steps from commercially available (7-hydroxycoumarin-4-yl)acetic acid. The key step involves the alkylation of a glycine-enolate equivalent.

Full text of DOI:10.3762/bjoc.6.69

Synthesis of the fluorescent amino acid
rac-(7-hydroxycoumarin-4-yl)ethylglycine
Manfred Braun* and Torsten Dittrich
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2010, 6, No. 69. doi:10.3762/bjoc.6.69
Institute of Organic and Macromolecular Chemistry, University of
Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany
Received: 27 April 2010
Accepted: 07 June 2010
Published: 24 June 2010
Email:
Manfred Braun* - braunm@uni-duesseldorf.de; Torsten Dittrich -
Torsten.Dittrich@uni-duesseldorf.de
Associate Editor: N. Sewald
* Corresponding author
© 2010 Braun and Dittrich; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
alkylation; coumarin; fluorescent probe; glycine; protecting group
Abstract
The hydrochloride of the racemic amino acid (7-hydroxycoumarin-4-yl)ethylglycine, a versatile fluorescent probe in proteins, has
been synthesized in five steps from commercially available (7-hydroxycoumarin-4-yl)acetic acid. The key step involves the alkyla-
tion of a glycine–enolate equivalent.
Introduction
The incorporation of non-natural fluorescent amino acids into reversed-phase HPLC. Unfortunately, this procedure, despite
proteins has developed into a potent tool for the investigation of the elegant concept of the synthetic route, is not applicable for
proteins with regard to their structure, function and interaction the provision of larger quantities of product. As a result we
with substrates [1-3]. Once in vitro and in vivo biochemical were interested in developing an alternative route to racemic
methods for the inclusion of non-natural amino acids had been amino acid 1.
successfully developed [4-9], an easy synthetic access to amino
acids with suitable fluorophoric groups that provide tailor-made
spectroscopic properties became the next crucial step. The
requirements of a suitable fluorescent probe are largely met by
(7-hydroxycoumarin-4-yl)ethylglycine (1) (Figure 1), which not
only displays a relatively large Stokes shift but also a fluores-
cence that is sensitive to both pH and solvent polarity [10,11].
Although a straightforward, short approach to the L-amino acid
1 has been reported [10], its isolation and purification was
Figure 1: Fluorescent amino acid (7-hydroxycoumarin-4-yl)ethylgly-
cine (1).
found to be difficult and tedious: According to the described
protocol, the isolation of final product 1 required preparative
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Beilstein J. Org. Chem. 2010, 6, No. 69.
Results and Discussion
silyl ether, were removed in a single step by hydrolysis with
The synthesis outlined in Scheme 1 started from coumarinyl hydrochloric acid. After repeated extractions with diethyl ether,
acetic acid 2 which is commercially available or, alternatively, lyophylization of the acidic aqueous solution gave the hydro-
readily accessible [12] through a Pechmann condensation [13] chloride of the racemic amino acid 1 as a colorless crystalline
from resorcinol and diethyl acetonedicarboxylate. First, the acid material, whose spectroscopic data were identical to those
2 was reduced to the alcohol 3 with borane. The protocol reported in the literature [10].
described in the literature [12] was modified, and the reducing
agent borane–tetrahydrofuran replaced by the less expensive In summary, a straightforward synthetic route has been devised
borane–dimethyl sulfide. This also led to a slight increase in that leads to the colorless, crystalline hydrochloride of the
yield (59%). For the planned selective protection of the phen- racemic amino acid 1 in 11% and 19% overall yield starting
olic hydroxyl group, the tert-butyl(dimethyl)silyl group [14] from commercially available coumarinyl acetic acid 2 or the
was chosen. Thus, coumarin 3 was treated with one equivalent known alcohol 3, respectively. The fact that the product
of sodium hydride to generate the phenolate by selective depro- obtained is a racemic mixture might be considered a drawback:
tonation without abstraction of the proton from the primary However, it has been proven for a related non-natural amino
alcohol moiety. Subsequent treatment with tert-butyl(dimethyl)- acid [8], that in the incorporation into a protein during transla-
silyl chloride gave alcohol 4 in 59% yield after removal of the tion on the ribosome, the L-enantiomer is accepted exclusively
doubly silylated side product the formation of which could not from the racemic mixture. Thus, one can assume that, for in
be completely avoided. By means of an Appel reaction [15], the vivo incorporation, the amino acid 1 does not have to be enan-
alcohol 4 was converted into the bromide 5 in 79% yield. In the tiomerically pure. Nevertheless, the elaboration of an enantiose-
following key-step, alkylation of a glycine enolate with the lective route is ongoing in our laboratory.
primary bromide 5 was anticipated. Indeed, its use as the elec-
trophilic component in the coupling with the benzophenone- Experimental
derived imine of tert-butyl glycine 6, which functioned, after General: Melting points (uncorrected) were determined with a
deprotonation, as a synthetic equivalent of a glycine enolate Büchi 540 melting point apparatus. NMR spectra were recorded
synthon, led to the imine 7 in 67% yield. Under the aprotic with a Bruker DXR 500 spectrometer. Mass spectra were
conditions of the alkylation protocol, the silyl protecting group recorded on an ion-trap API mass spectrometer Finnigan LCQ
turned out to be stable, a fact which facilitated the purification Deca (ESI), triple-quadrupole-mass spectrometer Finnigan TSQ
of the imine 7. It is an obvious idea to apply the established 7000, and sector field mass spectrometer Finnigan MAT 8200
protocols for the enantioselective alkylation of ester 6 under (EI, 70 eV). Elemental analyses (C, H, N) were performed on
phase transfer catalysis [16]. However, only an insignificant Perkin-Elmer 2400 series II at the Institute of Pharmaceutical
enantiomeric excess was observed in the alkylation product 7 Chemistry (University of Düsseldorf). High resolution mass
when a representative protocol was applied [17]. Finally, the spectra were obtained with a Bruker FT-ICR APEX III (7.0 T)
three protecting groups, the tert-butyl ester, the imine, and the (ESI) at the University of Bielefeld. Column chromatography
Scheme 1: Synthetic route to racemic amino acid 1·HCl. Reagents and conditions: a) BH3·SMe2, THF, 0 °C to r. t., 59%; b) t-BuMe2SiCl, NaH, THF,
r. t., 59%; c) CBr4, PPh3, CH2Cl2, 0 °C to r. t., 79%; d) Ph2C=N-CH2-CO2t-Bu 6, n-BuLi, THF, −78 °C to r. t., 67%; e) HCl, H2O, r. t., 60%.
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Beilstein J. Org. Chem. 2010, 6, No. 69.
was performed with Fluka silica gel 60 (230–400 mesh) and lized. Rf 0.72; m. p. 100.5 °C. IR (KBr): = 3423, 2958, 2928,
thin layer chromatography was carried out by using Merck TLC 2894, 2856, 1679, 1613, 1410, 1292, 1192, 1143, 997, 846,
Silicagel 60F254 aluminium sheets. Tetrahydrofuran (THF) was 782 cm−1. 1H NMR (CDCl3, 500 MHz): δ = 0.29 [s, 6H,
freshly distilled from sodium/benzophenone under a nitrogen Si(CH3)2], 0.98 [s, 9H, C(CH3)3], 2.99 (t, J = 6.3 Hz, 2H,
atmosphere, and dichloromethane was freshly distilled from CH2CH2OH), 3.98 (t, J = 6.31 Hz, 2H, CH2CH2OH), 6.20 (s,
calcium hydride.
1H, 3-H), 6.78 (m, 2H, 6-H and 8-H), 7.51 (d, J = 9.5 Hz, 1H,
5-H). 13C NMR (CDCl3, 125 MHz): δ = −4.0 [Si(CH3)2], 18.66
7-Hydroxy-4-(2-hydroxyethyl)-2H-chromen-2-one (3): A [SiC(CH3)3], 26.0 [SiC(CH3)3], 35.22 (CH2CH2OH), 61.16
500 mL flask was equipped with a magnetic stirrer, a pressure- (CH2CH2OH), 108.38 (C-8), 112.53 (C-4a), 113.94 (C-3),
equalizing dropping funnel closed with a septum, and a connec- 117.74 (C-6), 125.81 (C-5), 153.78 (C-8a), 155.62 (C-4),
tion to a combined nitrogen/vacuum line. The flask was charged 159.66 (C-7), 161.86 (C-2). MS (EI, 70 eV): m/z (%) = 320
with carboxylic acid 2 (8.95 g, 40.68 mmol), and the air in the (M+, 45), 292 (20), 263 (100), 245 (70), 233 (35), 189 (35).
flask was replaced by nitrogen. Dry THF (150 mL) was added Elemental anal. calcd. for C17H24O4Si: C, 63.72; H 7.55.
and the solution was cooled to 0 °C. Through the dropping Found: C, 63.60; H, 7.71.
funnel, a 10 M solution of borane–dimethyl sulfide (12 mL,
120 mmol) in THF, diluted with 100 mL of dry THF was added 4-(2-Bromoethyl)-7-(t-butyldimethylsilyloxy)-2H-chromen-
dropwise over 60 min at 0 °C. Stirring was continued at r. t. for 2-one (5): A solution of 4 (320 mg, 1.00 mmol) and tetrabro-
24 h. After cooling to 0 °C, water (10 mL) was cautiously momethane (730 mg, 2.20 mmol) in dry dichloromethane
added. The mixture was concentrated in a rotary evaporator and (10 mL) was stirred at 0 °C under a nitrogen atmosphere. A
the residue treated with ethyl acetate (200 mL) and water solution of triphenylphosphane (525 mg, 2.00 mmol) in dry
(200 mL). The organic layer was separated and the aqueous dichloromethane (2 mL) was added slowly. After removing the
phase extracted with two 150 mL portions of ethyl acetate. The ice bath, stirring was continued for 60 min. The mixture was
combined organic layers were dried with sodium sulfate. After concentrated under reduced pressure and the residue purified by
the solvent had been removed, the viscous residue was column chromatography (dichloromethane/n-hexane, 8:1) to
dissolved in acetone and purified by column chromatography give 301 mg (79%) of solid product 5; Rf 0.64; mp
with acetone/ethyl acetate (1:4) to give 5.03 g (59%) of a 88.5–89.4°C. IR (KBr):
= 3428, 3065, 1590, 1438, 1184,
yellowish solid product that was identical with compound 3 1120, 721, 696, 538 cm−1. 1H NMR (CDCl3, 500 MHz): δ =
according to its 1H-NMR spectrum [12]. 13C NMR (DMSO-d6, 0.25 [s, 6H, Si(CH3)2], 0.99 [s, 9H, C(CH3)3], 3.29 (t, J = 7.25
125 MHz): 34.53 (CH2CH2OH), 59.41 (CH2CH2OH), 102.28 Hz, 2H, CH2CH2Br), 3.46 (t, J = 7.25 Hz, 2H, CH2CH2Br),
(C-8), 110.31 (C-4a), 111.46 (C-3), 112.78 (C-6), 126.44 (C-5), 6.19 (s, 1H, 3-H), 6.80 (m, 2H, 6-H and 7-H), 7.44 (d, J = 9.14
154.58 (C-8a), 155.02 (C-4), 160.26 (C-7), 160.92 (C-2).
Hz, 1H, 5-H). 13C NMR (CDCl3, 125 MHz): δ = −3.98
[Si(CH3)2], 13.68 [SiC(CH3)3], 25.97 [Si(CH3)3], 28.77
4-(2-Hydroxyethyl)-7-(t-butyldimethylsilyloxy)-2H- (CH2CH2Br), 35.21 (CH2CH2Br), 108.61 (C-8), 112.74 (C-4a),
chromen-2-one (4): A 50 mL two-necked flask equipped with 113.13 (C-3), 117.83 (C-6, 125.15 (C-5), 152.48 (C-8a), 155.75
a magnetic stirrer and a connection to a combined nitrogen/ (C-4), 159.86 (C-7), 161.29 (C-2). MS (EI, 70 eV): m/z (%) =
vacuum line was charged with alcohol 3 (385 mg, 1.87 mmol) 384 (M+, 25), 327 (95), 302 (25), 245 (100), 217 (25), 189 (40),
and closed with a septum. The air in the flask was replaced by 115 (40). HRMS: calcd. for C17H23O3SiBr [M++H+Na]:
nitrogen, and dry THF (10 mL) was injected by syringe. The 405.0929; Found: 405.0847.
septum was removed for a short time, and a 60% suspension of
sodium hydride in mineral oil (74.8 mg, 1.87 mmol) added in t-Butyl 4-{7-[(t-Butyldimethylsilyl)oxy]-2-oxo-2H-chromen-
one portion. The flask was closed again, and the mixture was 4-yl}-2-[diphenyl(methylidene)amino]butanoate (7): A
stirred for 30 min at r. t. t-Butyl(dimethyl)chlorosilane (281 mg, 25-mL two-necked flask equipped with a magnetic stirrer and a
1.87 mmol) was added and stirring continued at r. t. until TLC connection to a combined nitrogen/vacuum line was charged
showed the absence of alcohol 3 (approximately 1 h). The mix- with the ester 6 (806 mg, 2.73 mmol) and closed with a septum.
ture was poured into ice and water and extracted three times The air in the flask was replaced by nitrogen and dry THF
with ethyl acetate (total volume 100 mL). The combined (10 mL) was added. The mixture was cooled to −78 °C and a
organic layers were washed with 2 N hydrochloric acid (20 mL) 1.6 M solution of n-BuLi in n-hexane (1.7 mL, 2.7 mmol) was
and dried with sodium sulfate. After the solvent had been injected by syringe. In a second flask, bromide 5 (615 mg,
removed in a rotary evaporator, the oily residue was submitted 1.61 mmol) dissolved in dry THF (2 mL) was added dropwise
to column chromatography with ethyl acetate to give 353 mg to the above-mentioned mixture with stirring at −78 °C. Stir-
(59%) of an oily, yellowish product 4 that subsequently crystal- ring was continued at the same temperature for 2 h and then the
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mixture was allowed to warm up to r. t. over 1 h. A saturated
aqueous solution of ammonium chloride (5 mL) was added.
After three extractions with ethyl acetate (20 mL each) the
combined organic layers were dried with sodium sulfate and
concentrated under reduced pressure. The residue was purified
by column chromatography (dichloromethane/ethyl acetate,
20:1) to give solid product 7 (645 mg, 67%); Rf 0.51; mp
191.8–192.4 °C. IR (KBr): = 2929, 2856, 1714, 1616, 1393,
1286, 1245, 1154, 1088, 1000, 885, 701 cm−1. 1H NMR
(CDCl3, 500 MHz): δ =0.25 [s, 6H, Si(CH3)2], 0.99 [s, 9H,
SiC(CH3)3], 1.46 [s, 9H, OC(CH3)3], 2.23 (m, 2H, NCHCH2),
2.77 (m, 2H, NCHCH2CH2), 4.05 (m, 1H, NCH), 6.07 (s, 1H,
3-H), 6.75 (dd, J = 8.51 Hz, J = 2.21 Hz, 1H, 6-H), 6.78 (d, J =
2.52, 1H, 8-H), 7.56 (d, J = 8.5 Hz, 1H, 5-H), 7.14 (m, 2H),
7.35 (t, J = 7.57, 2H), 7.40 (m, 4H), and 7.69 (d, J = 7.57 Hz,
2H) (other aromatic H). 13C NMR (CDCl3, 125 MHz): δ =
−3.98 [Si(CH3)3], 18.69 [SiC(CH3)3], 26.0 [SiC(CH3)3], 28.48
[OC(CH3)3], 32.81 (NCHCH2CH2), 65.31 (NCH), 81.93
[OC(CH3)3], 108.33 (C-8), 111.69 (C-4a), 113.77 (C-3), 125.96
(C-6), 128.53 (C-5), 139.67 (C-8a), 155.65 (C-4), 156.29 (C-7),
159.49 (C-2), 161.75 (C=N), 171.12 (C=O), 128.97, 129.19,
130.94, and 136.74 (other aromatic C). MS (EI, 70 eV): m/z (%)
= 620 ([M+Na]+, 18), 598 ([M+H]+, 25), 542 (100), 208 (26).
Elemental anal. calcd. for C36H43NO5Si: C, 72.33; H, 7.25; N,
2.34. Found: C, 72.18; H, 7.44; N, 2.17.
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7 (300 mg, 0.50 mmol) in 17% hydrochloric acid (10 mL) was
stirred at r. t. for 24 h. The mixture was then repeatedly
extracted with diethyl ether, until TLC of the extracts was found
to be negative. The acid aqueous phase was lyophylized to give
colorless, crystalline product 1 (79 mg, 60%) whose 1H NMR
data were identical with those described in the literature [10].
13C NMR (DMSO-d6, 125 MHz): δ = 28.95 (NCHCH2CH2),
31.07 (NCHCH2CH2), 51.88 (NCH), 102.86 (C-8), 109.91
(C-4a), 111.16 (C-3), 113.41 (C-6), 126.57 (C-5), 155.46
(C-8a), 160.67 (C-4), 161.72 (C-7), 169.27 (C-2), 170.93
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Acknowledgements
This work was supported by the VW Foundation (I/82 605). We
thank Mats Dietrich for the preparation of several compounds,
and we are grateful to Professor Dr. Lutz Schmitt and Dr. Nils
Hanekop for helpful and stimulating discussions.
The license is subject to the Beilstein Journal of Organic
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doi:10.3762/bjoc.6.69
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