Synthesis of Photoactive p-Azidotetrafluorophenylalanine Containing Peptide by Solid-Phase Fmoc Methodology

Article abstract of DOI:10.1021/ol026998f

(Matrix Presented) N-Fmoc-L-p-azidotetrafluorophenylalanine was prepared from achiral starting materials using an acetamidomalonate synthesis and enzymatic resolution. A photoactive peptide containing this fluorinated residue could be assembled using soli

Full text of DOI:10.1021/ol026998f

ORGANIC
LETTERS
2002
Vol. 4, No. 25
4467-4469
Synthesis of Photoactive
p-Azidotetrafluorophenylalanine
Containing Peptide by Solid-Phase
Fmoc Methodology
James E. Redman and M. Reza Ghadiri*
Departments of Chemistry and Molecular Biology and the Skaggs Institute for
Chemical Biology, The Scripps Research Institute, La Jolla, California 92037
ghadiri@scripps.edu
Received September 30, 2002
ABSTRACT
N-Fmoc-L-p-azidotetrafluorophenylalanine was prepared from achiral starting materials using an acetamidomalonate synthesis and enzymatic
resolution. A photoactive peptide containing this fluorinated residue could be assembled using solid-phase Fmoc chemistry.
The technique of photoaffinity labeling,1 in which a reactive
photogenerated intermediate cross-links to a protein, has been
popular as a means for identification of ligand binding sites,
regions of protein-protein interaction, and for affinity
purification of receptors. Phenyl azides have been extensively
employed as photoaffinity reagents, despite the disadvantage
that on photolysis rapid ring expansion occurs to afford a
comparatively long-lived ketenimine azepine intermediate2
that will preferentially react with nucleophiles and conse-
quentially may diffuse until it encounters a suitably reactive
residue or solvent.
subsequently undergo insertion reactions even with unacti-
vated CH bonds.4 This offers the possibility for labeling of
arbitrary residues at a binding site, and minimizes the
opportunity for diffusion of the reactive intermediate. A
number of reagents for convenient derivatization of proteins
or their ligands,5 and nucleic acids6 with fluoroaryl azides
have been described. However, in contrast to p-azidophe-
(4) (a) Pandurangi, R. S.; Katti, K. V.; Barnes, C. L.; Volkert, W. A.;
Kuntz, R. R. J. Chem. Soc., Chem. Commun. 1994, 1841. (b) Schnapp, K.
A.; Poe, R.; Leyva, E.; Soundararajan, N.; Platz, M. S. Bioconj. Chem.
1993, 4, 172. (c) Schnapp, K. A.; Platz, M. S. Bioconj. Chem. 1993, 4,
178. (d) Poe, R.; Schnapp, K.; Young, M. J. T.; Grayzar, J.; Platz, M. S. J.
Am. Chem. Soc. 1992, 114, 5054. (e) Young, M. J. T.; Platz, M. S. J. Org.
Chem. 1991, 56, 6403. (f) Soundararajan, N.; Platz, M. S. J. Org. Chem.
1990, 55, 2034.
(5) (a) Chehade, K. A. H.; Spielmann, H. P. J. Org. Chem. 2000, 65,
4949. (b) Pandurangi, R. S.; Lusiak, P.; Desai, S.; Kuntz, R. R. Bioorg.
Chem. 1998, 26, 201. (c) Pandurangi, R. S.; Karra, S. R.; Katti, K. V.;
Kuntz, R. R.; Volkert, W. A. J. Org. Chem. 1997, 62, 2798. (d) Cai, S. X.;
Keana, J. F. W. Synth. Commun. 1996, 26, 809. (e) Soundararajan, N.; Liu,
S. H.; Soundararajan, S.; Platz, M. S. Bioconj. Chem. 1993, 4, 256. (f) Cai,
S. X.; Glenn, D. J.; Keana, J. F. W. J. Org. Chem. 1992, 57, 1299. (g)
Crocker, P. J.; Imai, N.; Rajagopalan, K.; Boggess, M. A.; Kwiatkowski,
S.; Dwyer, L. D.; Vanaman, T. C.; Watt, D. S. Bioconj. Chem. 1990, 1,
419. (h) Keana, J. F. W.; Cai, S. X. J. Org. Chem. 1990, 55, 3640. (i)
Keana, J. F. W.; Cai, S. X. J. Fluorine Chem. 1989, 43, 151. (j) Cai, S. X.;
Keana, J. F. W. Tetrahedron Lett. 1989, 30, 5409.
Fluorinated aryl azides have been proposed as new
photoaffinity reagents3 which offer the advantage of sup-
pressing the ring expansion of the initially liberated nitrene
intermediate, which in a hydrophobic environment may
(1) (a) Singh, A.; Thornton, E. R.; Westheimer, F. H. J. Biol. Chem.
1962, 237, 3006. (b) Kotzyba-Hibert, F.; Kapfer, I.; Goeldner, M. Angew.
Chem., Int. Ed. Engl. 1995, 34, 1296. (c) Fleming, S. A. Tetrahedron 1995,
51, 12479.
(2) Leyva, E.; Platz, M. S.; Persy, G.; Wirz, J. J. Am. Chem. Soc. 1986,
108, 3783.
(3) (a) Young, M. J. T.; Platz, M. S. Tetrahedron Lett. 1989, 30, 2199.
(b) Leyva, E.; Young, M. J. T.; Platz, M. S. J. Am. Chem. Soc. 1986, 108,
8307.
10.1021/ol026998f CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/09/2002

nylalanine (Pap), p-azidotetrafluorophenylalanine 4 (Fpap)
had only been reported in racemic form5e and its incorpora-
tion into peptide affinity labels appears to be unexplored.
To circumvent the shortcomings associated with the photo-
chemistry of Pap and to take advantage of the preferential
reactivity of fluorinated aryl azides we sought to develop a
means for incorporation of 4 into peptides using conventional
solid-phase synthesis. A diethylacetamidomalonate synthesis7
subtilisin in buffered DMSO/water were not fruitful,10 we
opted for a nonenzymatic basic hydrolysis of this group to
acid 3, followed by kinetic resolution via proteolytic cleavage
of the acetamide. Compound 3 was unreactive to carboxy-
peptidase A catalyzed hydrolysis,11 however Aspergillus
melleus acylase-I treatment12 while maintaining the pH over
the range 7-8 by titration afforded L-p-azidotetrafluorophe-
nylalanine 4 as a white precipitate with good ee (>95%, vide
infra). Conversion to the Fmoc derivative 5 was effected by
reaction with Fmoc-OSu.13
The enantiomeric purity of 4 was assessed by comparison
of the 1H NMR spectra of the Mosher amide derivatives, 6,
of the methyl ester of 4 with R-(+) and S-(-) R-methoxy-
R-trifluoromethylphenylacetic acid (MTPA).14 Both diaster-
eomers of 6 gave distinct NMR spectra, but were indistin-
guishable by TLC. The chemical shifts of the methyl ether
protons of the R-(+) and S-(-) MTPA derivatives of 4 were
3.34 and 3.49 ppm, respectively, consistent with the assign-
ment of an L stereochemistry at the R-carbon, as would be
predicted for this enzymatic resolution.15 Contamination of
4 with the isomer with a D configuration at the R-carbon
was not detectable in the NMR spectra of these derivatives.
Scheme 1a
a Conditions: (a) NaN3, 0.1 equiv of Bu4NN3, DMF, 80 °C; (b)
NaOH, H2O, MeOH; (c) Acylase-I, H2O.
was used for preparation of protected racemic pentafluo-
rophenylalanine derivative 1 with a view to introducing the
azide group by nucleophilic substitution. Although fluori-
nated aromatics are activated toward nucleophilic attack, it
was observed that 1 required comparatively forcing condi-
tions to bring the reaction to completion. Treatment of 1 with
NaN3 in refluxing acetone for 7 h resulted in no reaction.
Treatment under literature conditions5e of NaN3 in DMF at
55 °C for 20 h afforded mostly starting materials, as judged
by 19F NMR. By increasing the temperature to 80 °C and
addition of catalytic Bu4NN3 the reaction could be driven
essentially to completion,8 without excessive accumulation
of impurities arising from thermolysis of the product 2.
Reaction of commercially available FmocPhe(F5) under
similar conditions resulted in significant loss of the Fmoc
group, whereas unprotected Phe(F5) afforded an intractable
mixture of penta- and tetrafluoro species.
To test the ease with which p-azidotetrafluorophenylala-
nine could be incorporated, via 5, into peptides using
conventional solid-phase methodology,16 we prepared a 33
residue peptide, 7, based on the GCN4 leucine zipper
sequence where this residue was substituted at position 16.
This sequence was chosen as a representative peptide to test
the stability of the fluoroaryl azide group to a number of
coupling/Fmoc deprotection cycles, and to the TFA depro-
tection byproducts of a range of side chain protecting groups
commonly employed in Fmoc chemistry. Automated syn-
thesis was performed using diisopropylcarbodiimide/hy-
droxybenzotriazole for couplings and 50% piperidine in
(9) (a) Berger, A.; Smolarsky, M.; Kurn, N.; Bosshard, H. R. J. Org.
Chem. 1973, 38, 457. (b) Tong, J. H.; Petitclerc, C.; D’Iorio, A.; Benoiton,
N. L. Can. J. Biochem. 1971, 49, 877.
(10) Behrens C.; Nielsen J. N.; Fan, X.-J.; Doisy, X.; Kim, K.-H.;
Praetorius-Ibba, M.; Nielsen, P. E.; Ibba, M. Tetrahedron 2000, 56, 9443.
(11) (a) Rivier, J. E.; Porter, J.; Rivier, C. L.; Perrin, M.; Corrigan, A.;
Hook, W. A.; Siraganian, R. P.; Vale, W. W. J. Med. Chem. 1986, 29,
1846. (b) Fones, W. S. J. Biol. Chem. 1953, 204, 323.
(12) Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J. Am. Chem. Soc.
1989, 111, 6354.
As attempts at removal of the ethyl ester group of 2 using
either R-chymotrypsin in water or DMF/water mixture,9 or
(6) (a) Godovikova, T. S.; Kolpashchikov, D. M.; Orlova, T. N.; Richter,
V. A.; Ivanova, T. M.; Grochovsky, S. L.; Nasedkina, T. V.; Victorova, L.
S.; Poletaev, A. I. Bioconj. Chem. 1999, 10, 529. (b) Wlassoff, W. A.;
Dobrikov, M. I.; Safronov, I. V.; Dudko, R. Y.; Bogachev, V. S.;
Kandaurova, V. V.; Shishkin, G. V.; Dymshits, G. M.; Lavrik, O. I. Bioconj.
Chem. 1995, 6, 352. (c) Venyaminova, A. G.; Repkova, M. N.; Ivanova,
T. M.; Dobrikov, M. I.; Bulygin, K. N.; Graifer, D. M.; Karpova, G. G.;
Zarytova, V. F Nucleosides Nucleotides 1995, 14, 1069.
(13) Ten Kortenaar, P. B. W.; Van Dijk, B. G.; Peeters, J. M.; Raaben,
B. J.; Adams, P. J. H. M.; Tesser, G. I. Int. J. Pept. Protein Res. 1986, 27,
398.
(7) (a) Leanna, M. R.; Morton, H. E. Tetrahedron Lett. 1993, 34, 4485.
(b) Krapcho, A. P. Synthesis 1982, 805. (c) Filler, R.; Ayyangar, N. R.;
Gustowski, W.; Kang, H. H. J. Org. Chem. 1969, 34, 534.
(8) Bra¨ndstro¨m, A.; Lamm, B.; Palmertz, I. Acta Chem. Scand. B 1974,
28, 699.
(14) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34,
2543.
(15) Yamaguchi, S. In Asymmetric Synthesis; Morrison, J. D., Ed.;
Academic Press: San Diego, CA., 1983, Chapter 7.
(16) Wellings, D. A.; Atherton, E. Methods Enzymol. 1997, 289, 44.
4468
Org. Lett., Vol. 4, No. 25, 2002

N-methylpyrrolidone for Fmoc deprotection. Although there
was a concern that scavengers used in the final TFA cleavage
and deprotection of the peptide would reduce the azide group,
in practice it was found that this was not a significant
problem using a cleavage mixture with minimal quantities
of thiol and avoiding the use of silanes.
without extensive photolysis, as judged by HPLC. Repeated
acquisition of UV spectra of 7 in phosphate buffer also
resulted in negligible photolysis. However, inclusion of 1
mM dithiothreitol (DTT) in a pH 8.0 solution of 10 µM 7
led to approximately 50% reduction of the azide moiety over
10 min as judged from the decreasing UV absorbance. At
pH 7.0 the reaction proceeded at a lower rate, leading to
approximately 20% depletion of the azide over the same time
period, highlighting the need to avoid reducing buffers to
maintain the integrity of the azide moiety.
In summary, we have developed a synthesis of Fmoc-L-
p-azidotetrafluorophenylalanine and demonstrated its in-
corporation into a peptide using automated Fmoc synthesis
methodology. The azide group is stable to the conditions
required for peptide synthesis and deprotection, although it
was rapidly reduced by dithiothreitol in aqueous buffer.
Peptide 7 was analyzed by sonic spray ionization mass
spectrometry,17 a mild ionization technique similar to elec-
trospray, which revealed an ion at m/z 1384.02, in good
agreement with the calculated value of 1384.08 for [7 +
3H]3+. In contrast, both 7 and a photoproduct arising from
laser induced loss of N2 were observed by MALDI TOF
spectrometry. Peptide 7 displayed the characteristic UV
absorption of the fluoroaryl azide group at 253 nm, and an
IR absorption at 2126 cm-1 corresponding to the N3 stretch.
On exposure to 254-nm light from a handheld lamp, 7 was
photolyzed in minutes, as monitored by the UV absorbance.
Despite photolability, a CD spectrum of 7 could be measured
Acknowledgment. J.E.R. thanks the Wellcome Trust for
a fellowship (061454/Z/00/Z).
Supporting Information Available: Experimental pro-
cedures and characterization for compounds 1-6. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(17) Hirabayashi, A.; Sakairi, M.; Koizumi, H. Anal. Chem. 1994, 66,
4557.
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