You are on page 1of 6

3326

Helvetica Chimica Acta ± Vol. 86 (2003)

Synthesis of N-[(tert-Butoxy)carbonyl]-3-(9,10-dihydro-9-oxoacridin-2-yl)-lalanine, a New Fluorescent Amino Acid Derivative
by Aneta Szyman¬ska*, Katarzyna Wegner, and Leszek Ëankiewicz
University of Gdan¬sk, Faculty of Chemistry, Sobieskiego 18, 80-952 Gdan¬sk, 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)-lalanine (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], peptide ligand 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 lowresolution 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 donor acceptor 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

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

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

detection by UV light (l 254 nm) or by the ninhydrin reagent.3 36. useful in many kinds of peptide investigations. good overlapping of the acceptor×s absorption and donor×s emission spectra were observed. C-8. gradient 0 ± 100% B (B ˆ 80% MeCN ‡ 0. TLC: aluminium sheets precoated with SiO2 60 F-254 (Merck). IR Spectra: Bruker-IFS-66 spectrometer.6  250 mm. Anal.1% CF 3COOH). A ˆ H2O ‡ 0. 86 (2003) 3329 Fig.: uncorrected. Conclusions.p. long-wave absorption and emission) make this compound a very promising analytical tool. QY ˆ quantum yield.Helvetica Chimica Acta ± Vol. Emission spectra of Boc-Aka-OH (6) in various solvents. M. photostability. HPLC: Kromasil column (4. . 5 mm). We elaborated a simple and effective synthesis of a highly fluorescent amino acid in enantiomerically pure form. grant BW 8000-50303-2 and grant DS/8350-4-0131-3). This work is supported by the State Committee for Scientific Research (KBN. in cm 1.08% CF 3COOH.4 both cases. Table. 2. Optical rotations: Perkin-Elmer-343 spectropolarimeter. Calculated Values of the Fˆrster Distance ( R0 ) for Designed Donor Acceptor Pairs Donor Acceptor R0 [ä] H-Aka-OH H-Aka-OH Dabcyl H-Lys( AQN )-OH 44. Experimental Part General. Calculated values of the Fˆrster distance are given in the Table. tR in min. Poland. Excellent photophysical properties (very high quantum yield of fluorescence. KBr pellets.

17 g (59%) of 6. 9. 13C-NMR: 27. and poured into ice-water (50 ml).25 (m. activated Cu powder [17] (0.77. 3. 3174. 1H-NMR (400 MHz. 1m KHSO4 .70 (s. dried (MgSO4 ).32 g.). 2978.6 mmol) in 1m NaOH (63 ml) and t-BuOH (63 ml). 5. (2S)-2-Amino-3-(9. 7. [a] 20 D ˆ ‡ 8. H C(a)). The mixture was left in an ice-bath for 30 min. CH2N2 soln.02 (t. then acidified to pH ca. 3.67. 1 H C(b)).d in ppm.22 (m. tBu ).p. H). (D6 )DMSO): 1.22 ± 7. CH2(b)). MeOH).86. H C(5). 1552. 4. Boc2O (15 g.12 ± 4.53 ± 7. CH2(b)). and the suspension refrigerated overnight. 1574. H C(1)). d. The combined extract was washed with 1m KHSO4 and brine.73 (m. [a] 20 D ˆ ‡ 114. H C(6)). H C(8)).5 g.845 g (78%). FAB-MS: 297.8 mmol). according to [24]) was added in small portions until N2 evolution ceased. The resulting precipitate was collected.05.06. MeO). 4. 7.52. tBu).16 (m. tBu). 7. M. 2975. 2-chlorobenzoic acid (0.69 (m. M.3 (c ˆ 1. H C(2).39 (s. H C(a)). CH2(b)).0. H C(6)). and evaporated.46 ± 7. J ˆ 8.39. while the pH was maintained at 8 ± 9 by addition of 1m NaOH. 1 arom. 1682.0.01 ± 3.29 (s.35 ± 4.65 (m. 1 arom. 7.42.01. J ˆ 8.17 ± 3. H C(a)). 86 (2003) NMR Spectra: CD3OD or (D6 )DMSO solns. 195 ± 1978 (dec.11 ± 3. H C(2).37 (s.21. 2.27 ± 7.28 (br. 1685.24. J ˆ 8. the soln.17 ± 8.0. 1166. MeOH). 8. 37. 1742. H C(4))..40 (s. Polyphosphoric acid (13 ml) was preheated to 808 and added to 4 (1. H C(a)). from 5 (0. IR (KBr): 3274.0. H C(3)). 85 ± 878. 1163. 1273.54 (m. N-[(tert-Butoxy)carbonyl]-4-nitro-l-phenylalanine Methyl Ester (2). 15 mmol) in Et2O (50 ml). cooled to 508. MeOH) ([25]: [a] 20 D ˆ ‡ 30. 1H-NMR (100 MHz.0.35 ± 4. 8. 8.13 ± 4. CH2Cl2 ). Mass spectra: VG-Masslab-Tri-3 spectrometer with quadrupole filter. (2S)-2-{[(tert-Butoxy)carbonyl]amino}-3-(9.92 ± 2.14 ± 7.0.19.65 g. H C(3). 1519.82 g (73.89.76 mmol. 3.37 (m. 1 arom. H C(8)). 51. 53. 11.39 g. of Boc-Phe(4NO2 )-OH (4.62 ± 7. 1716.0 (d.77 (s.). Janssen Chemicals.75 (d. 117.60 (AA'BB'. 7. J ˆ 10.05 (m.4%). 126. MeO). HPLC (reversed phase): tR 40. IR (KBr): 3398.3 ([M ‡ 1]‡ ). IR (KBr): 3362. 7.97.28 (AA'BB'. 7. 2931.055.22 ± 8. 5 arom. 1525. J ˆ 8. M. 3. 122. 1746.12 (s.18 (m. 1597. J ˆ 10. H C(3)).45 (m. H C(7)). 1691.85. H C(7)). MeO). 1181. and evaporated: Boc-Phe(4NO2 )-OH (15.44 g. reaction time 24 h). THF).0.9 (c ˆ 1. 4-Amino-N-[(tert-butoxy)carbonyl]-l-phenylalanine Methyl Ester (3).2%) of 2. 2928.79.p.75 ± 3. HPLC (reversed phase): tR 46. 1690.38 (m. 113.225 g). H).02.07 g (63.05 ± 3. J ˆ 8. 1170.3 (c ˆ 0. Crystallization from AcOEt/petroleum ether gave a crystalline solid (first crop of the crystals. MeOH) ([23]: [a] 20 D ˆ ‡ 8 (c ˆ 1. 1520. H C(4)).0.8 (c ˆ 1. freshly prepared. 3375. THF).69 ± 7. tBu). 3. 3. The soln. After cooling to r.98 (m. HPLC: tR 34.28. 3145.10-dihydro-9-oxoacridin-2-yl)propanoic Acid Methyl Ester (5). The residue was recrystallized from Et2O/petroleum ether: 8. 1 H-NMR (100 MHz.0. 116. 1350.19 (m. CD3OD): 1. 1 H C(b)).4. H C(a)). 1684. 16. J in Hz. 93 ± 968 ([25]: 95 ± 978). H). H C(5)).65 mmol).85 ± 2.51.9 mmol) in MeOH (65 ml) was subjected to heterogeneous reduction with 10% Pd/C as catalyst. dried (MgSO4 ). To the chilled soln.p.60.32 (m. 3235. and the alkaline residue was extracted with petroleum ether (2  30 ml).7%). 1590. 133.0.4 (c ˆ 1. 1733. of Phe(4-NO2 )-OH (1. Recrystallization from DMF/H2O yielded pure 5 (0.. 170. 55.98.94 ± 7. 7. 2851. of 2 (4. 120. and brine. 176.92.p.66 g).. H).50. 2985. 7.5 g. 7. 1526. H C(1)). H C(6)).9%) of crude 5 as a yellow solid.05 ± 4. H C(5)).t.10-dihydro-9-oxoacridin-2-yl)propanoic Acid (6).65 ± 7. H C(6). To the chilled (ice-bath) soln. 3. 125.74 mmol).t.96.88. 8.34. After completion of the reaction (2 h. [a] 20 D ˆ (c ˆ 1.07.8 g. CH2(b)).5 3. Yellowish solid.34. 81. IR (KBr): 3359. 1H-NMR (400 MHz. NH). 3000. N-[(tert-Butoxy)carbonyl]-4-nitro-l-phenylalanine (Boc-Phe(4-NO2 )-OH). 4. 7. H C(5). 117. the mixture was filtered. K2CO3 (0. 68. MeO). H C(a)). H C(2). 1163. 140. (D6 )DMSO): 3. HPLC (reversed phase): tR 48. 1524.p.13 (d.17. CD3OD): 1.8 mmol) was added in portions during 30 min. 4. TLC).55 (m. 2984. 120. A soln. 173.54 g (65. 248 ± 2508 (dec.00 (s.69 ± 6. and extracted with AcOEt (4  50 ml). As described above for N-[(tert-butoxy)carbonyl]-4-nitro-l-phenylalanine. 1676. 1635. The compound was dissolved in AcOEt. H-NMR (100 MHz. 1746. J ˆ 9.22 (AA'BB'. A mixture of 3 (3. 1454. 2.65.4%) of 3. 8. 1744.08.88 g. warmed to r. (D6 )DMSO): 1. . M.37 (s. 7. 7.86 g (94.3330 Helvetica Chimica Acta ± Vol. N-[(tert-Butoxy)carbonyl]-4-[2-carboxyphenyl)amino]-l-phenylalanine Methyl Ester (4). Varian-Mercury (400-MHz) and Tesla-BF-567A (100 MHz) spectrometers. The residue was purified by CC (SiO2 . 1590. 130. washed with cold H2O.48 g). 4. 133.38 (m. IR (KBr): 3307. 2930. and DMF (4 ml) was heated under reflux for 2 h. 2987. 13. HPLC (reversed phase): tR 24. [a] 20 D ˆ ‡ 62. H C(6)). 139. H C(3). 2. 6.80. washed with 1m Na2CO3 . tBu ). the filtrate slowly added to H2O (30 ml).0.04.57 (AA'BB'. an additional portion of t-BuOH (60 ml) was added. H C(3). The oily. 2951. CH2(b)).37 ± 4. 62. in Et2O (prepared from Diazogene¾. 3. CD3OD): 1. 134.t.26 (m. Crystallization from THF/cyclohexane gave 0. H C(5)). 1517.73 (m.3 mmol). 6. 170.24 (d.Yellow oil.03. 2. The mixture was stirred mechanically at 80 ± 858 for 1 h. 13C-NMR: 36.59 (s.70 (s. 7. the catalyst was filtered off and the filtrate evaporated: 3. The mixture was stirred for 30 min.27 (m.19 (d. 1627. 1 [a] 20 D ˆ ‡ 10. 1252.58. 108 ± 1108 ([23]: 110 ± 1128).91 (m. 1477. J ˆ 10. 2953. 2 with 1m HCl. and evaporated. 4. M. brownish precipitate was washed with H2O and dried: 1. and dried: 0.43. and the mixture was stirred overnight at r.62 (m. 120. J ˆ 9. 1346. J ˆ 10.45.50. 8. IR (KBr): 3328. 1 H-NMR (400 MHz.49. 132. 1245. 7. was concentrated by evaporation of t-BuOH.97 (m. 13C-NMR: 36. NH). 1160. AcOEt): 1. 52.

[13] C. J. 914. Hudecz. 127. Ch.74.98. Reymond. 2002. J. R. Vol. E. Org.05. Taraporewala. Still. 1989. J. Curr. Juliano. Brand. 412. E. 150. 9th edn. 140. K. 51.76. Biochim. F. 186. Ossowski. REFERENCES [1] J. Stachowiak. Anal.59. [17] S. A. Sci. 173. Plenum Press. 1992. Jakubke. Gribble. 7. J. 632. 2003. Ëankiewicz. [24] −Vogel×s Textbook of Practical Chemistry×. [22] A.99. V. 1997. 58. Commun. P. Methods 1996. Sci. J. 329. W. W. Med. U. J. A. Chem. Wyssbrod.62. L. [12] L. 2000.38. 143. 1999. Biophys. L. 49. Wiczk. Burks. 103.Helvetica Chimica Acta ± Vol. Acad. Chem. Ed. L. Part A 1995. Marfey. Biophys. 1998. 44. Landis. Roussland. 135. 509. 1990. A. 1995. Camilleri. 251. (polish transl. Mamposo. Opin. 149. H.40. Tierney. Compd. 3. Biochem. G. S. Chem.46. V. Ullmann. Wiczk. −Proceedings of the 25th European Peptide Symposium× ± Budapest. J. R. in −Peptides 1998×. Biochim. Szyman¬ska. 1997. Wiczk. L. T. Warszawa. Grahn. B. Tierney. Y. [14] E. Med. Biotechnol. Rothman. 2003 . R. G. 130. Kauffman. 32. Pol. 63. 9. Hirata. Dexter. Faller. J.-D. [8] J.24. Reymond. C. p. 4th edn. M. Bahr. 33. Biol. Spectrochim.-L. 9. Commun. Ed. B. D. Acta 2000. T. Akade¬miai Kiado¬. Koch.57. [15] A. [19] R. Heterocycl. 38. T. Sci. Wu. R. 1998. F. E. 130. Acta Biochem.18. Wydawnictwa Naukowo-Techniczne. [7] N. [2] P. W. P. 603. − Handbook of Fluorescent Probes and Research Chemicals×. 135. Malicka. Pa¬rka¬nyi.-L. Haughland. 193.). [9] T. J. [18] A. 4251. Pept. T.39. [3] R. 93. [10] A. U. 1994. K. Carlsberg Res. Carrasco. 1999. S. J. in −Peptides 2002×. 218. [16] D. Acta 1999. [5] R.77. S. Kurth. Karolczak. 638. 1431. Hutton.. M. −Biochemical Applications×. Chirio-Lebrun. Szyman¬ska. A. de Souza. 87. [4] M. R. Budapest. 127. [21] P. Jackson. p. −Proceedings of the 27th European Peptide Symposium× ± Sorrento. Shook. Maafi. Gershkovich. V. Pedone. 86 (2003) 3331 37. Ëankiewicz. Ëankiewicz. Aaron. Biophys. J. Lett. Wiczk. Clegg. F. DaÀbrowska. 136. G. New York. 1. 2002. Schrˆer. L. 1474. H. Okafo. 2002. 38. 165. 1998. Sianko. Hruby. Lakowicz. 183. Ëankiewicz. W. Edizioni Ziino. Games. M. C. Pellon. Ch. J. 1996. 1984. Lett. 26. 1997. 130. Ross. 1984. 175. 144. W. Liu. K. P. T.75. [11] J. Ed. C. Ito. Kholodovych. Bajusz. W. Struct. [23] G. Marquez. 79.S. 5107. Benedetti. H.A. Szyman¬ska. J. 1995. Boniface.23.-Ch. W. F. Tetrahedron Lett. Received June 2. 320. 1997. Chem. in −Topics in Fluorescence Spectroscopy×. Laws. Proc. [25] R. M. Prats. D. Biochem. Educ. Moore. L. [20] I. 115. 477. E. [6] J. D. S. Tetrahedron Lett. Pharm. Ëankiewicz. Yamamura. Moens. W. I. Molecular Probes. 1489. J. Ed. I. T. 6. 7875. Chem. J. Biochem. Dos Remedios. Bioorg. H. 1529.22. 591. Acta. A. E. Dwojakowska. Natl.