Highly enantioselective synthesis of a fluorescent amino acid

Article abstract of DOI:10.1016/S0957-4166(00)00459-6

A high enantiomeric excess (gt;99.5%) synthesis of L-2-amino-3-(7-methoxy-4-coumaryl) propionic acid (L-Amp) is described. The two step synthesis route of this non-proteinogenic amino acid includes an oxazinone derivative as glycine enolate, which is alky

Full text of DOI:10.1016/S0957-4166(00)00459-6

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 4959–4963
Highly enantioselective synthesis of a fluorescent amino acid
P e´ ter Kele, Guodong Sui, Qun Huo and Roger M. Leblanc*
Center for Supramolecular Science and Department of Chemistry, University of Miami, 1301 Memorial Drive,
Coral Gables, FL 33124, USA
Received 2 November 2000; accepted 6 November 2000
Abstract
A high enantiomeric excess (>99.5%) synthesis of L-2-amino-3-(7-methoxy-4-coumaryl) propionic acid
(L-Amp) is described. The two step synthesis route of this non-proteinogenic amino acid includes an
oxazinone derivative as glycine enolate, which is alkylated with the fluorogenic group. © 2001 Published
by Elsevier Science Ltd.
1
. Introduction
Amp, a non-proteinogenic fluorescent amino acid is a fluorophore with a high quantum yield
that can easily be built into peptides. Together with the 2,4-dinitrophenyl group it is often used
1
2
as a fluorophore-quencher pair or by itself, to label bioactive peptides. In our studies on helical
peptides we plan to label peptides with this fluorogenic amino acid. To keep the helical structure
intact, however, a high enantiomeric excess of the label is needed. The previously reported
3,4
method to synthesize Amp in enantiopure L-form afforded 94% enantiomeric excess (ee). Our
goal was to develop a synthetic approach that results in a higher enantiomeric excess of the
L-form.
Chiral glycine equivalents serve as useful a-amino acid templates undergoing homologation5
via carbon–carbon bond formation at the a-position through nucleophilic carbanion alkylation.
6
,7
Diphenyloxazinones are versatile templates from which proteinogenic and non-proteinogenic
8
amino acids can be synthesized in very high enantiomeric excess.
2
. Results and discussion
The synthesis of 3 (Scheme 1) started with the alkylation of benzyl (2R,3S)-(−)-6-oxo-2,3-
diphenyl-4-morpholine-carboxylate, 1: Sodium bis(trimethylsilyl)amide easily deprotonates 1 at
*
Corresponding author. Tel: +1(305)284-2282, fax: +1(305)284-4571; e-mail: rml@umiami.ir.miami.edu
0
957-4166/00/$ - see front matter © 2001 Published by Elsevier Science Ltd.
PII: S0957-4166(00)00459-6

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P. Kele et al. / Tetrahedron: Asymmetry 11 (2000) 4959–4963
position 3. The enolate is then alkylated with 4-bromomethyl-7-methoxycoumarin, which yields
. Compound 2 is poorly soluble in THF at −78°C, and as the reaction mixture was cooled it
3
precipitated. The enolate derived from 1, however, was reactive enough to afford a satisfactory
yield of 3. As the reaction proceeded the precipitate disappeared, implying the formation of 3,
which was readily soluble in THF even at low temperature. The purification of the resulting
compound, 3, had some difficulties, since the originally followed procedure failed for this
molecule. Finally, the method we developed gave the compound in pure form (Scheme 1). This
intermediate was then hydrogenated to remove the benzyloxycarbonyl (Cbz) protecting group
and set the amino acid 4 free from the oxazinone (Scheme 2). The conditions during this second
reaction step were crucial. Not only should they remove the Cbz group and liberate the amino
acid but they should leave the coumarin double bond intact. When THF/MeOH was used as a
solvent during the hydrogenolysis with 0.7 equiv. of the catalyst, the double bond was found to
be saturated and no fluorescence could be detected. Pure THF, however, with 0.5 equiv. catalyst
gave the right product in good yield. The probable explanation for this result is that the Cbz
group removal and the amino acid liberation occurs first then the amino acid precipitates in
THF, which protects the double bond from saturation. For enantiomeric excess determination,
racemic Amp and L-Amp, respectively, were condensed with N-(2,4-dinitro-5-fluorophenyl)-L-
9
alaninamide. The adducts were then checked by analytical HPLC. Within the detection limit of
the instrument (0.5%) a single peak was detected that comigrated with one of the two adducts
formed during the reaction with DL-Amp (Fig. 1).
Scheme 1. Reagents: (a) Sodium bis(trimethylsilyl)amide, THF, −78°C, 2 h; purified on PTLC (CH Cl /EtOAc 20:1
2
2
v/v%)
In summary, we have completed a synthesis of L-Amp in enantiopure form (>99.5% ee).
Conformational studies on peptides labeled with this non-proteinogenic fluorescent amino acid
and also synthesis of other fluorogenic amino acids in enantiopure form are in progress and will
be reported in the near future.

P. Kele et al. / Tetrahedron: Asymmetry 11 (2000) 4959–4963
4961
Scheme 2. Reagents: (b) PtCl (0.5 equiv.), H (50 psi), THF, 24 h; filtered, concentrated, then triturated with Et O
2
2
2
3
. Experimental
3
.1. General
1
13
Compounds 1 and 2 were commercially available. H NMR, and C NMR: Bruker AC 300.
IR: Perkin–Elmer 2000 FT-IR. HRMS: University of Illinois at Urbana-Champaign Mass
Spectrometry Laboratory. Optical rotation: Perkin–Elmer 241 polarimeter. Analytical HPLC
®
was carried out on a Waters Alliance 2690 instrument equipped with a Symmetry C18 3.5 mm
4
.6×75 mm column.
3
2
.2. (3S,5S,6R)-4-(Benzyloxycarbonyl)-5,6-diphenyl-3-[(7-methoxy-4-coumaryl)methyl]-
,3,5,6-tetrahydro-4H-1,4-oxazin-2-one 3
Compounds 1 (216 mg, 0.56 mmol, 1.5 equiv.) and 2 (100 mg, 0.37 mmol, 1 equiv.) were
dissolved in THF (20 ml) and cooled to the temperature of dry ice–acetone. To this stirred
solution sodium bis(trimethylsilyl)amide (280 ml, 2 M solution in THF, 0.56 mmol, 1.5 equiv.)
was added dropwise via syringe. After the addition of the base the mixture was stirred for two
hours then the solution was allowed to warm up to room temperature and poured onto
methylene chloride. The solution was washed with water and brine, dried over anhydrous
Na SO , filtered and concentrated. The yellowish solid was then purified on PTLC (eluted with
2
4
1
CH Cl /EtOAc 20:1) allowing 86 mg of 3, as a white powder (40%). H NMR (CDCl , 393 K
2
2
3
vs TMS): l 3.29 (1H, dd, J=9.64 Hz, J=13.37 Hz), 3.79 (1H, dd, J=4.82 Hz, J=13.17 Hz),
3
5
.88 (3H, s), 4.96 (1H, 1/2AB q, J=12.34 Hz), 5.05 (1H, 1/2AB q, J=12.36 Hz), 5.13 (2H, s),
.41 (1H, dd, J=4.86 Hz, J=9.52 Hz), 5.83 (1H, d, J=2.75 Hz), 6.16 (1H, s), 6.5 (2H, d,
J=7.45 Hz), 6.74 (2H, d, J=7.1 Hz), 6.86–6.95 (4H, m), 7.04–7.4 (8H, m), 8.05 (1H, d, J=8.89
1
3
Hz). C NMR (CDCl ): l 36.65, 55.78, 56.93, 60.98, 68.02, 79.28, 79.46, 101.40, 112.36, 112.80,
3
1
1
2
5
14.12, 125.91, 126.41, 127.40, 127.46, 128.06, 128.22, 128.34, 128.88, 128.97, 133.50, 134.95,
35.18, 149.97, 154.64, 155.72, 160.61, 162.98, 166.65. IR (KBr) 1761.67, 1711.54, 1689.50; mp
2
5
+
15–216°C; [h] −25 (c 0.13, CH Cl ); HRMS: calcd for C H NO : 576.2022 (HM ), found:
D
2
2
35 30
7
+
76.2022 (HM ).

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P. Kele et al. / Tetrahedron: Asymmetry 11 (2000) 4959–4963
Figure 1. Analytical HPLC chromatograms of the products formed by reaction of N-(2,4-dinitro-5-fluorophenyl)-L-
alaninamide (bottom, used as reference), L-Amp (center), DL-Amp (top)
3
.3. Hydrogenolysis
The oxazinone intermediate 3 was dissolved in THF and the solution was hydrogenated for
2
4 hours in the presence of 0.5 equiv. of PtCl catalyst. Methanol was then added to dissolve the
2
precipitated amino acid and the solution was filtered through Celite, then concentrated. The
precipitate was triturated with Et O and the product was collected by centrifugation.
2

P. Kele et al. / Tetrahedron: Asymmetry 11 (2000) 4959–4963
4963
3
.4. Formation of diastereomeric adducts
L-Amp and DL-Amp (0.5 mol), respectively, in 1 M NaHCO (40 ml) and N-(2,4-dinitro-5-
3
fluorophenyl)-L-alaninamide (0.7 mol) in acetone (20 ml) were allowed to react for 1 hour at
4
supernatants were analyzed on analytical HPLC (solvents used: A, 0.1% trifluoroacetic acid in
water; B, 0.08% trifluoroacetic acid in acetonitrile. A linear gradient of 5–95% B over 20 min at
0°C. After that 2 M HCl (20 ml) was added and the solutions were centrifuged. The
1
.25 ml/min. Elute monitored at 300–400 nm).
Acknowledgements
These experiments were supported by the Department of the Army under Contract 669870.
References
1
2
3
4
5
6
7
8
9
. Knight, C. G. Biochem. J. 1991, 274, 45.
. Kele, P.; Illy e´ s, E.; Mezo, G.; D o´ da, M.; M a´ k, M.; Kaposi, A. D.; Sebesty e´ n, F. Lett. Pept. Sci. 1999, 6, 235.
. Knight, C. G. Lett. Pept. Sci. 1998, 5, 1.
. Oppolzer, W.; Moretti, R.; Zhou, C. Helv. Chim. Acta 1994, 77, 2363.
. Izumi, Y.; Chibata, I.; Itoh, T. Angew. Chem., Int. Ed. Engl. 1978, 17, 176.
. Williams, R. M.; Inn, M.-N. Tetrahedron Lett. 1988, 29, 6075.
. Reno, D. S.; Lotz, B. T.; Miller, M. J. Tetrahedron. Lett. 1990, 31, 827.
. Williams, R. M.; Inn, M.-N. J. Am. Chem. Soc. 1991, 113, 9276.
. Aberhardt, D. J.; Cotting, J.-A.; Lin, H.-J. Anal. Biochem. 1985, 151, 88.
.
.