Synthesis of N-[(tert-Butoxy)carbonyl]-3-(9,10-dihydro-9-oxoacridin-2-yl)-L-alanine, a New Fluorescent Amino Acid Derivative

Article abstract of DOI:10.1002/hlca.200390275

A simple synthesis of a new, highly fluorescent amino acid and of its protected derivative useful in peptide studies is described. The obtained derivative, N-[(tert-butoxy)carbonyl]-3-(9,10-dihydro-9-oxoacridin-2-yl)-L-alanine (6), shows intense long-wave absorption (above 360 nm) and emission (above 400 nm). The quantum yield of fluorescence of the investigated compound is very high, so it can serve as a sensitive analytical probe useful, e.g., in analysis of peptide conformations.

Full text of DOI:10.1002/hlca.200390275

3326
Helvetica Chimica Acta Vol. 86 (2003)
Synthesis of N-[(tert-Butoxy)carbonyl]-3-(9,10-dihydro-9-oxoacridin-2-yl)-l-
alanine, a NewFluorescent Amino Acid Derivative
by Aneta Szyman¬ska*, Katarzyna Wegner, and Leszek Ëankiewicz
¬
¬
University of Gdansk, Faculty of Chemistry, Sobieskiego 18, 80-952 Gdansk, Poland
A simple synthesis of a new, highly fluorescent amino acid and of its protected derivative useful in peptide
studies is described. The obtained derivative, N-[(tert-butoxy)carbonyl]-3-(9,10-dihydro-9-oxoacridin-2-yl)-l-
alanine (6), shows intense long-wave absorption (above 360 nm) and emission (above 400 nm). The quantum
yield of fluorescence of the investigated compound is very high, so it can serve as a sensitive analytical probe
useful, e.g., in analysis of peptide conformations.
Introduction. Labelling of peptides with designed fluorescent groups is one of the
most effective and widely used analytical tool, useful for probing their structures,
functions, and interactions [1 4]. Peptides can be derivatized with the fluorescent
probes following their total synthesis, at the N- or C-terminus or at functional groups
(NH2, SH, or COOH) present within the sequence. However, the presence of several
reactive sites can complicate this procedure. Direct incorporation of the chromophore
into the peptide sequence by use of an unnatural amino acid, bearing a fluorescent
function at its side chain, allows to avoid the problems mentioned. The number of
available fluorescence probes is considerable [5]. Among them, special attention is
paid to photostable, efficient dyes, which can be easily incorporated into a side chain of
an amino acid.
Acridine and many of its derivatives (e.g., 9-acridin-9(10H)-one) belong to the
group of very effective fluorescent probes. Furthermore, the acridinone moiety is stable
to light, oxidation, and heat; so it is used as a very sensitive tag in studies involving
prolonged light exposure (e.g., antibody catalysis [6], peptideÀligand interaction
[7][8], and mass-spectrometry-based sequencing of peptides [9]). Additionally,
photophysical profiles of acridinone derivatives (high quantum yield of fluorescence
and long-wave shift of absorption and emission spectra [10][11]) are very beneficial
from a point of view of their application as energy donors in peptide studies by means
of fluorescence resonance energy transfer (FRET) [2][3][12]. These methods, based
on measurements of the efficiency of the donor×s electronic-excitation trapping by an
appropriate acceptor, have found numerous applications in studies of macromolecules.
FRET is widely used in conformational studies of biopolymers since it is capable to
supply spatial data about the investigated system with a precision comparable with low-
resolution X-ray-diffraction or NMR studies [12 14]. Exploring mechanisms of
enzyme action and inhibition is another field of FRET applications [15 17]. Although
there are many donorÀacceptor pairs that can be applied in peptide studies, there is a
steady need for new, rationally designed ones. In this paper, we present the synthesis of
a fluorescent amino acid and its derivatives that can be used in solid-phase peptide

Helvetica Chimica Acta Vol. 86 (2003)
3327
synthesis. On the ground of our investigation on acridine chromophores, useful in
peptide studies [10][18], we decided to prepare a nonnatural amino acid bearing the
acridin-9(10H)-one moiety, to determine its photophysical properties, and to design the
appropriate donorÀacceptor pairs that can be applied for conformational and
enzymatic studies of peptides and proteins.
Results and Discussion. Synthesis. N-[(tert-Butoxy)carbonyl]-3-(9,10-dihydro-9-
oxoacridin-2-yl)-l-alanine (6; Boc-Aka-OH) was obtained from 4-nitro-l-phenyl-
alanine (1) in a multi-step reaction. Thus, reduction of the 4-nitro group of the
protected derivative 2 was carried out in MeOH (Scheme) although the solubility of the
obtained 3 in MeOH is slightly lower than in THF. However, the decomposition of 3
was slower in MeOH than in THF. Due to this decomposition, the subsequent
UllmannÀJourdan condensation of 3 with 2-chlorobenzoic acid [10][19] was carried
out under Ar to give 4 in good yield. Cyclodehydration of 4 to the acridinone derivative
5 with hot polyphosphoric acid (PPA) [20] caused concomitant removal of the Boc
protecting group. To obtain an orthogonally protected amino acid suitable for peptide
synthesis, we incorporated the Boc group again; under the applied basic conditions
(pH 9 10, maintained by use of 1m NaOH), saponification of the ester function of 5
took place simultanously with the N-acylation ( ! 6). Despite of a longer action of
base, no racemization of the final l-alanine derivative 6 (Boc-Aka-OH) was observed.
Scheme

3328
Helvetica Chimica Acta Vol. 86 (2003)
We determined the optical purity of 6 by reversed-phase HPLC using Marfey×s reagent
((2S)-2-[(5-fluoro-2,4-dinitrophenyl)amino]propanamide) as a differentiation agent
[21]. Marfey×s reagent is known to form diastereoisomeric products with amino acids,
which can be analyzed either by TLC or reversed-phase HPLC. A single peak
corresponding to a single diastereoisomer was observed in the chromatogram of the
product obtained from 6 and Marfey×s reagent.
Photophysical Properties. The absorption (Fig. 1) and fluorescence (Fig. 2) spectra
of Boc-Aka-OH (6) are typical of an acridin-9(10H)-one substituted by a carboxyalkyl
group at position 2 [10][18]. The long-wave absorption band (p ! p* transitions),
interesting from a point of view of further application of the chromophore in peptide
studies, is located at 360 420 nm (lmax 383 and 402 nm in MeOH, lmax 388 and 407 nm
in H2O). This allows to excite the acridine chromophore selectively and to avoid an
interference with other natural chromophores present in an investigated peptide.
Interestingly, a strong influence of the solvent on the fluorescence spectra was
observed. In the case of THF, quenching of the acridinone fluorescence occurred. At
this stage of the investigations, it is difficult to elucidate the mechanism of the process;
we suppose that it is caused by a non-specific interaction of the chromophore with the
solvent. In other solvents, the quantum yield of the fluorescence was very high.
Fig. 1. Absorption spectra of Boc-Aka-OH (6) in various solvents
Since Boc-Aka-OH (6) was designed as an energy donor, we had to find an
appropriate acceptor. Taking into account the rules for the donorÀacceptor pairs
engaged in Fˆrster-type interactions [2], we chose two compounds: the well-known and
widely used Dabcyl (4-[4-(dimethylamino)phenylazo]benzoyl) and a lysine derivative
modified in the side chain with an anthraquinone moiety (H-Lys(AQN)-OH [22]). In

Helvetica Chimica Acta Vol. 86 (2003)
3329
Fig. 2. Emission spectra of Boc-Aka-OH (6) in various solvents. QYˆ quantum yield.
Table. Calculated Values of the Fˆrster Distance (R0) for Designed DonorÀAcceptor Pairs
Donor
Acceptor
R0 [ä]
H-Aka-OH
H-Aka-OH
Dabcyl
H-Lys(AQN)-OH
44.3
36.4
both cases, good overlapping of the acceptor×s absorption and donor×s emission spectra
were observed. Calculated values of the Fˆrster distance are given in the Table.
Conclusions. We elaborated a simple and effective synthesis of a highly fluorescent
amino acid in enantiomerically pure form. Excellent photophysical properties (very
high quantum yield of fluorescence, photostability, long-wave absorption and emission)
make this compound a very promising analytical tool, useful in many kinds of peptide
investigations.
This work is supported by the State Committee for Scientific Research (KBN, Poland; grant BW 8000-5-
0303-2 and grant DS/8350-4-0131-3).
Experimental Part
General. M.p.: uncorrected. TLC: aluminium sheets precoated with SiO2 60 F-254 (Merck); detection by
UV light (l 254 nm) or by the ninhydrin reagent. Anal. HPLC: Kromasil column (4.6 Â 250 mm, C-8, 5 mm);
gradient 0 100% B (B ˆ 80% MeCN ‡ 0.08% CF3COOH, A ˆ H2O ‡ 0.1% CF3COOH); tR in min. Optical
rotations: Perkin-Elmer-343 spectropolarimeter. IR Spectra: Bruker-IFS-66 spectrometer, KBr pellets; in cmÀ1
.

3330
Helvetica Chimica Acta Vol. 86 (2003)
NMR Spectra: CD3OD or (D6)DMSO solns.; Varian-Mercury (400-MHz) and Tesla-BF-567A (100 MHz)
spectrometers;d in ppm, J in Hz. Mass spectra: VG-Masslab-Tri-3 spectrometer with quadrupole filter.
N-[(tert-Butoxy)carbonyl]-4-nitro-l-phenylalanine (Boc-Phe(4-NO2)-OH). To the chilled (ice-bath) soln.
of Phe(4-NO2)-OH (1; 16.5 g, 62.6 mmol) in 1m NaOH (63 ml) and t-BuOH (63 ml), Boc2O (15 g, 68.8 mmol)
was added in portions during 30 min, while the pH was maintained at 8 9by addition of 1m NaOH. The mixture
was stirred for 30 min, an additional portion of t-BuOH (60 ml) was added, and the mixture was stirred
overnight at r.t. The soln. was concentrated by evaporation of t-BuOH, and the alkaline residue was extracted
with petroleum ether (2 Â 30 ml), then acidified to pH ca. 2 with 1m HCl, and extracted with AcOEt (4 Â 50 ml).
The combined extract was washed with 1m KHSO4 and brine, dried (MgSO4), and evaporated: Boc-Phe(4-
NO2)-OH (15.8 g, 81.4%).Yellow oil. Crystallization from AcOEt/petroleum ether gave a crystalline solid (first
crop of the crystals; 9.66 g). M.p. 108 1108 ([23]: 110 1128). HPLC (reversed phase): tR 40.97. [a]2D0 ˆ ‡8.3
(c ˆ 1.08, MeOH) ([23]: [a]2D0 ˆ ‡8 (c ˆ 1.0, MeOH). IR (KBr): 3362, 2985, 2930, 1716, 1685, 1520, 1350, 1166.
1H-NMR (100 MHz, (D6)DMSO): 1.37 (s, tBu); 3.01 3.37 (m, CH2(b)); 4.05 4.22 (m,HÀC(a)); 7.28
(br. d, J ˆ 8.0, NH); 7.60 (AA'BB', J ˆ 10.0, HÀC(2), HÀC(6)); 8.22 (AA'BB', J ˆ 10.0, HÀC(3), HÀC(5)).
N-[(tert-Butoxy)carbonyl]-4-nitro-l-phenylalanine Methyl Ester (2). To the chilled soln. of Boc-Phe(4-
¾
NO2)-OH (4.65 g, 15 mmol) in Et2O (50 ml), CH2N2 soln. in Et2O (prepared from Diazogene , Janssen
Chemicals, according to [24]) was added in small portions until N2 evolution ceased. The mixture was left in an
ice-bath for 30 min, warmed to r.t., and evaporated. The residue was recrystallized from Et2O/petroleum ether:
3.07 g (63.2%) of 2. Yellowish solid. M.p. 93 968 ([25]: 95 978). HPLC (reversed phase): tR 46.51. [a]2D0 ˆ À8.5
(c ˆ 1.03, MeOH) ([25]: [a]2D0 ˆ ‡30.4 (c ˆ 1.055, CH2Cl2). IR (KBr): 3359, 2987, 2953, 1733, 1690, 1676, 1524,
1346, 1273, 1163. 1H-NMR (100 MHz, CD3OD): 1.37 (s, tBu); 3.05 3.25 (m, CH2(b)); 3.77 (s, MeO); 4.37 4.62
(m, HÀC(a)); 7.57 (AA'BB', J ˆ 10.0, HÀC(2), HÀC(6)); 8.28 (AA'BB', J ˆ 10.0, HÀC(3), HÀC(5)).
4-Amino-N-[(tert-butoxy)carbonyl]-l-phenylalanine Methyl Ester (3). A soln. of 2 (4.5 g, 13.9mmol) in
MeOH (65 ml) was subjected to heterogeneous reduction with 10% Pd/C as catalyst. After completion of the
reaction (2 h, TLC), the catalyst was filtered off and the filtrate evaporated: 3.86 g (94.4%) of 3. M.p. 85 878.
[a]2D0 ˆ ‡10.8 (c ˆ 1.02, MeOH). IR (KBr): 3398, 3375, 3235, 2984, 2951, 1742, 1691, 1517, 1170. 1H-NMR
(100 MHz, CD3OD): 1.40 (s, tBu); 2.75 3.05 (m, CH2(b)); 3.70 (s, MeO); 4.13 4.45 (m, HÀC(a)); 6.75 (d, J ˆ
9.0, HÀC(2), HÀC(6)); 7.0 (d, J ˆ 9.0, HÀC(3), HÀC(5)).
N-[(tert-Butoxy)carbonyl]-4-[2-carboxyphenyl)amino]-l-phenylalanine Methyl Ester (4). A mixture of 3
(3.32 g, 11.3 mmol), 2-chlorobenzoic acid (0.88 g, 5.65 mmol), K2CO3 (0.39g, 2.8 mmol), freshly prepared,
activated Cu powder [17] (0.225 g), and DMF (4 ml) was heated under reflux for 2 h. After cooling to r.t., the
mixture was filtered, the filtrate slowly added to H2O (30 ml), and the suspension refrigerated overnight. The
oily, brownish precipitate was washed with H2O and dried: 1.845 g (78%). The compound was dissolved in
AcOEt, the soln. washed with 1m Na2CO3, 1m KHSO4, and brine, dried (MgSO4), and evaporated. The residue
was purified by CC (SiO2, AcOEt): 1.54 g (65.7%). HPLC (reversed phase): tR 48.85. IR (KBr): 3307, 2978,
2931, 1746, 1684, 1590, 1519, 1454, 1252, 1163. 1H-NMR (400 MHz, CD3OD): 1.39( s, tBu); 2.85 2.91
(m, CH2(b)); 3.70 (s, MeO); 4.35 4.38 (m, HÀC(a)); 6.69 6.73 ( m, 1 arom. H); 7.14 7.19( m, 5 arom. H);
7.27 7.32 (m, 1 arom. H); 7.94 7.97 (m, 1 arom. H). 13C-NMR: 27.49; 37.06; 51.43; 55.42; 113.67; 116.92;
122.04; 130.45; 132.07; 133.88; 170.65; 173.05.
(2S)-2-Amino-3-(9,10-dihydro-9-oxoacridin-2-yl)propanoic Acid Methyl Ester (5). Polyphosphoric acid
(13 ml) was preheated to 808 and added to 4 (1.44 g, 3.74 mmol). The mixture was stirred mechanically at 80
858 for 1 h, cooled to 508, and poured into ice-water (50 ml). The resulting precipitate was collected, washed
with cold H2O, and dried: 0.82 g (73.9%) of crude 5 as a yellow solid. Recrystallization from DMF/H2O yielded
pure 5 (0.48 g). M.p. 195 1978 (dec.). HPLC (reversed phase): tR 24.50. [a]2D0 ˆ ‡62.9( c ˆ 1.01, THF). IR
(KBr): 3328, 2928, 2851, 1744, 1627, 1597, 1574, 1526, 1477, 1245, 1160. 1H-NMR (400 MHz, (D6)DMSO): 3.17
3.27 (m, CH2(b)); 3.59( s, MeO); 4.35 4.38 (m, HÀC(a)); 7.02 (t, J ˆ 8.0, HÀC(4)); 7.53 7.55 (m, HÀC(5),
HÀC(8)); 7.65 7.69( m, HÀC(6), HÀC(7)); 8.00 (s, HÀC(1)); 8.17 8.19( d, J ˆ 8.0, HÀC(3)). 13C-NMR:
36.24; 52.17; 53.60; 117.39; 117.80; 120.19; 120.34; 120.77; 125.86; 126.21; 133.98; 134.89; 139.96; 140.79; 170.34;
‡
176.52. FAB-MS: 297.3 ([M ‡ 1] ).
(2S)-2-{[(tert-Butoxy)carbonyl]amino}-3-(9,10-dihydro-9-oxoacridin-2-yl)propanoic Acid (6). As de-
scribed above for N-[(tert-butoxy)carbonyl]-4-nitro-l-phenylalanine, from 5 (0.76 mmol; reaction time 24 h).
Crystallization from THF/cyclohexane gave 0.17 g (59%) of 6. M.p. 248 2508 (dec.). HPLC: tR 34.58. [a]D20
ˆ
‡114.3 (c ˆ 0.50, THF). IR (KBr): 3274, 3174, 3145, 3000, 2975, 1746, 1682, 1635, 1590, 1552, 1525, 1181.
1H-NMR (400 MHz, (D6)DMSO): 1.29( s, tBu); 2.92 2.98 (m, 1 HÀC(b)); 3.11 3.16 (m, 1 HÀC(b)); 4.12
4.18 (m, HÀC(a)); 7.13 (d, J ˆ 8.4, NH); 7.22 7.26 (m, HÀC(4)); 7.46 7.54 (m, HÀC(5), HÀC(8)); 7.62 7.65
(m, HÀC(6)); 7.69 7.73 ( m, HÀC(7)); 8.12 (s, HÀC(1)); 8.22 8.24 (d, J ˆ 8.0, HÀC(3)). 13C-NMR: 36.28;

Helvetica Chimica Acta Vol. 86 (2003)
3331
37.76; 58.46; 87.99; 127.18; 127.22; 130.24; 130.39; 130.77; 135.98; 136.05; 140.75; 143.23; 144.62; 149.57; 150.74;
165.40; 183.38; 186.59.
REFERENCES
[1] J. B. A. Ross, W. R. Laws, K. W. Roussland, H. R. Wyssbrod, in −Topics in Fluorescence Spectroscopy×, Ed.
J. R. Lakowicz, Plenum Press, New York, 1992, Vol. 3, −Biochemical Applications×.
[2] P. Wu, L. Brand, Anal. Biochem. 1994, 218, 1.
[3] R. M. Clegg, Curr. Opin. Biotechnol. 1995, 6, 103.
[4] M.-Ch. Chirio-Lebrun, M. Prats, Biochem. Educ. 1998, 26, 320.
[5] R. P. Haughland, − Handbook of Fluorescent Probes and Research Chemicals×, 9th edn., Molecular Probes,
2002.
[6] J.-L. Reymond, T. Koch, J. Schrˆer, E. Tierney, Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4251.
[7] N. Bahr, E. Tierney, J.-L. Reymond, Tetrahedron Lett. 1997, 38, 1489.
[8] J. H. Rothman, W. C. Still, Bioorg. Med. Chem. Lett. 1999, 9, 509.
[9] T. Faller, K. Hutton, G. Okafo, A. Gribble, P. Camilleri, D. E. Games, Chem. Commun. 1997, 1529.
¬
[10] A. Szymanska, L. Ëankiewicz, W. Wiczk, Chem. Heterocycl. Compd. 2000, 7, 914.
¬
¬
[11] J. J. Aaron, M. Maafi, C. Parkanyi, Ch. Boniface, Spectrochim. Acta, Part A 1995, 51, 603.
[12] L. Ëankiewicz, J. Malicka, W. Wiczk, Acta Biochem. Pol. 1997, 44, 477.
[13] C. G. Dos Remedios, P. D. Moens, J. Struct. Biol. 1995, 115, 175.
[14] E. S. de Souza, I. Y. Hirata, L. Juliano, A. S. Ito, Biochim. Biophys. Acta 2000, 1474, 251.
[15] A. A. Gershkovich, V. V. Kholodovych, J. Biochem. Biophys. Methods 1996, 33, 135.
[16] D. Dwojakowska, A. DaÀbrowska, L. Ëankiewicz, W. Wiczk, K. Stachowiak, −Proceedings of the 25th
¬
European Peptide Symposium× Budapest, 1998, in −Peptides 1998×, Ed. S. Bajusz, F. Hudecz, Akademiai
¬
Kiado, Budapest, 1999, p. 632.
[17] S. Grahn, T. Kurth, D. Ullmann, H.-D. Jakubke, Biochim. Biophys. Acta 1999, 1431, 329.
¬
[18] A. Szymanska, W. Wiczk, J. Karolczak, L. Ëankiewicz, −Proceedings of the 27th European Peptide
Symposium× Sorrento, 2002, in −Peptides 2002×, Ed. E. Benedetti, C. Pedone, Edizioni Ziino, 2002, p. 412.
[19] R. F. Pellon, R. Carrasco, T. Marquez, T. Mamposo, Tetrahedron Lett. 1997, 38, 5107.
[20] I. B. Taraporewala, J. M. Kauffman, J. Pharm. Sci. 1990, 79, 173.
[21] P. Marfey, Carlsberg Res. Commun. 1984, 49, 59 1.
¬
[22] A. Szymanska, T. Ossowski, L. Ëankiewicz, Lett. Pept. Sci. 2003, 9, 193.
[23] G. Landis, G. Liu, J. E. Shook, H. I. Yamamura, T. F. Burks, V. J. Hruby, J. Med. Chem. 1989, 32, 638.
[24] −Vogel×s Textbook of Practical Chemistry×, 4th edn. (polish transl.), Ed. U. Sianko, Wydawnictwa
Naukowo-Techniczne, Warszawa, 1984.
[25] R. F. W. Jackson, R. J. Moore, Ch. S. Dexter, J. Org. Chem. 1998, 63, 7875.
Received June 2, 2003