Synthesis and evaluation of photoreactive tetrazole amino acids

Article abstract of DOI:10.1021/ol901300h

Image Persented Six photoreactive tetrazole amino acids were efficiently synthesized either by the de novo Kakehi tetrazole synthesis method or by alkylation of a glycine Schiff base with tetrazole-containing alkyl halides, and four of them showed excellent reactivity toward a simple alkene in the photoinduced 1,3-dipolar cycloaddition reaction in acetonitrile/PBS buffer (1:1) mixture.

Full text of DOI:10.1021/ol901300h

ORGANIC
LETTERS
2009
Vol. 11, No. 16
3570-3573
Synthesis and Evaluation of
Photoreactive Tetrazole Amino Acids
Yizhong Wang and Qing Lin*
Department of Chemistry, State UniVersity of New York at Buffalo,
Buffalo, New York 14260-3000
qinglin@buffalo.edu
Received June 10, 2009
ABSTRACT
Six photoreactive tetrazole amino acids were efficiently synthesized either by the de novo Kakehi tetrazole synthesis method or by alkylation
of a glycine Schiff base with tetrazole-containing alkyl halides, and four of them showed excellent reactivity toward a simple alkene in the
photoinduced 1,3-dipolar cycloaddition reaction in acetonitrile/PBS buffer (1:1) mixture.
Photoreactive reagents have been widely used in biochem-
istry and molecular biology in mapping the interfaces of
ligand-receptor and protein-protein interactions.1 Since
photoreactive reagents are “silent” under normal conditions
and become chemically reactive only upon UV irradiation,
they offer a spatial and temporal control of the ligand or
protein function.2 One major approach in exerting photo-
chemical control in protein systems is to incorporate pho-
toreactive amino acids into proteins site-selectively using
either the Amber codon suppression strategy or the metabolic
incorporation strategy.3 To this end, a number of photore-
active amino acids have been successfully developed, e.g.,
p-benzoyl-L-Phe (pBPA),4 p-azido-L-Phe (pAzpa),5 p-3-
(trifluoromethyl)-3H-diazirin-3-yl-L-Phe (pTmdPhe),6 o-ni-
trophenylalanine (oNPA),7 photoleucine, and photomethion-
ine8 (Figure 1). Although these reagents have provided
invaluable photochemical tools for protein functional studies,
additional chemical moieties with novel photoreactivity are
still needed in order to further expand the capability of
photoregulation.
We have recently reported a new class of photoreactive
reagents, 2,5-diaryltetrazoles,9 with exquisite reactivity to-
ward alkenes both in Vitro and in living cells.10 Upon
photoirradiation, unlike previously known photoreactive
(5) (a) Fleet, G. W. J.; Porter, R. R.; Knowles, J. R. Nature 1969, 224,
511–512. (b) Krieg, U. C.; Walter, P.; Johnson, A. E. Proc. Natl. Acad.
Sci. U.S.A. 1986, 83, 8604–8608. (c) Chen, Y.; Ebright, Y. W.; Ebright,
R. H. Science 1994, 265, 90–92. (d) Chin, J. W.; Santoro, S. W.; Martin,
A. B.; King, D. S.; Wang, L.; Schultz, P. G. J. Am. Chem. Soc. 2002, 124,
9026–9027.
(1) (a) Bayley, H. Photogenerated Reagents in Biochemistry and
Molecular Biology; Elsevier: Amsterdam, 1983. (b) Dorm´ , G.; Prestwich,
G. D. Trends Biotechnol. 2000, 18, 64–77.
(6) (a) Nassal, M. J. Am. Chem. Soc. 1984, 106, 7540–7545. (b) Ploug,
M.; Ostergaard, S.; Hansen, L. B. L.; Holm, A.; Dano, K. Biochemistry
1998, 37, 3612–3622. (c) Nakashima, H.; Hashimoto, M.; Sadakane, Y.;
Tomohiro, T.; Hatanaka, Y. J. Am. Chem. Soc. 2006, 128, 15092–15093.
(d) Tippmann, E. M.; Liu, W.; Summerer, D.; Mack, A. V.; Schultz, P. G.
ChemBioChem 2007, 8, 2210–2214.
(2) For a recent review, see: Young, D. D.; Deiters, A. Org. Biomol.
Chem. 2007, 5, 999–1005.
(3) (a) Xie, J.; Schultz, P. G. Nat. ReV. Mol. Cell Biol. 2006, 7, 775–
782. (b) Link, A. J.; Mock, M. L.; Tirrell, D. A. Curr. Opin. Biotechnol.
2003, 14, 603–609.
(7) Peters, F. B.; Brock, A.; Wang, J.; Schultz, P. G. Chem. Biol. 2009,
16, 148–152.
(4) (a) Kauer, J. C.; Erickson-Viitanen, S.; Wolfe, H. R., Jr.; DeGrado,
W. F. J. Biol. Chem. 1986, 261, 10695–10700. (b) Chin, J. W.; Martin,
A. B.; King, D. S.; Wang, L.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 11020–11024. (c) Mori, H.; Ito, K. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 16159–16164. (d) Ye, S.; Ko¨hrer, C.; Huber, T.; Kazmi, M.;
Sachdev, P.; Yan, E. C.; Bhagat, A.; RajBhandary, U. L.; Sakmar, T. P.
J. Biol. Chem. 2008, 283, 1525–1533.
(8) (a) Suchanek, M.; Radzikowska, A.; Thiele, C. Nat. Methods 2005,
2, 261–268. (b) MacKinnon, A. L.; Garrison, J. L.; Hegde, R. S.; Taunton,
J. J. Am. Chem. Soc. 2007, 129, 14560–14561.
(9) (a) Wang, Y.; Vera, C. I; Lin, Q. Org. Lett. 2007, 9, 4155–4158.
(b) Wang, Y.; Hu, W. J.; Song, W.; Lim, R. K.; Lin, Q. Org. Lett. 2008,
10, 3725–3728.
10.1021/ol901300h CCC: $40.75
Published on Web 07/28/2009
 2009 American Chemical Society

ryltetrazoles exhibited excellent photoreactivity in aqueous
buffers in our previous studies,9 and (ii) Kakehi tetrazole
synthesis offers a facile route for the preparation of
diaryltetrazoles from aryl aldehydes and arene diazonium
salts.12 Two types of diaryltetrazole amino acids were
designed: type I, with the amino acid motif attached to
the C5-aryl ring (1, 2), and type II, with the motif attached
to the N2-aryl ring (3) (Scheme 1). In a convergent
manner, type I tetrazole amino acids can be synthesized
from arene diazonium salts and p-formyl-L-phenylalanine,
which was readily prepared via the palladium-catalyzed
hydroformylation of a protected tyrosine triflate.13 When
simple phenyldiazonium chloride was used, an optically
pure diphenyltetrazole amino acid 1 was obtained with
an overall 41% yield in three steps (Scheme 2). Since our
previous studies indicated that the amino substitution at
the para position of the N-aryl ring of 2,5-diaryltetrazoles
generates long-wavelength photoactivatable tetrazoles,9b
we also prepared p-amino-diphenyltetrazole amino acid
2 in four steps with an overall yield of 50% from p-formyl-
L-phenylalanine and acetamidophenyl diazonium chloride
(Scheme 2).
Figure 1. Representative photoreactive amino acids.
motifs (Figure 1) that produce highly reactive intermediates
(radical, nitrene, or carbene)11 that undergo nonselective
C-H (or X-H) insertion, tetrazoles generate significantly
more stable nitrile imine dipoles that selectively react with
suitable alkenes in solution via 1,3-dipolar cycloaddition. In
essence, the tetrazole moiety provides a new photoregulation
modality, i.e., photo modification, by which a multitude of
alkene-tethered biophysical and biochemical probes can be
attached to the tetrazole site through a photoinduced 1,3-
dipolar cycloaddition reaction. To enable rapid access of this
novel photoregulation modality, herein we report the first
synthesis of six tetrazole amino acids (Scheme 1) and the
evaluation of their reactivities toward an alkene dipolarophile
under a mild photoinduced 1,3-dipolar cycloaddition condi-
tion in acetonitrile/PBS buffer (1:1) mixture.
Scheme 2. Synthesis of Diaryltetrazole Amino Acids 1-3
Scheme 1. Structures of Tetrazole Amino Acids 1-6
On the other hand, type II tetrazole amino acid 3 was
synthesized in a similar manner except that the amino acid
moiety was attached to phenyl diazonium chloride, which
was formed smoothly from a protected 4-amino-phenylala-
nine derivative. Kakehi tetrazole synthesis with benzaldehyde
followed by deprotection gave the tetrazole amino acid 3
with an excellent yield of 65% over four steps (Scheme 2).
We initially focused on the synthesis of 2,5-diaryl tetra-
zole-based amino acids 1-3 (Scheme 1) because (i) dia-
Although a number of unnatural amino acids carrying
(10) (a) Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew.
Chem., Int. Ed. 2008, 47, 2832–2835. (b) Song, W.; Wang, Y.; Qu, J.; Lin,
Q. J. Am. Chem. Soc. 2008, 130, 9654–9655. (c) Wang, Y.; Song, W.; Hu,
W. J.; Lin, Q. Angew. Chem., Int. Ed. 2009, 48, 5330–5333.
(12) Ito, S.; Tanaka, Y.; Kakehi, A.; Kondo, K. Bull. Chem. Soc. Jpn.
1976, 49, 1920–1923.
(13) Morera, E.; Ortar, G.; Varani, A. Synth. Commun. 1998, 28, 4279–
(11) Fleming, S. A. Tetrahedron 1995, 51, 12479–12520.
4285.
Org. Lett., Vol. 11, No. 16, 2009
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large aromatic side chains, including benzophenone,
naphthyl, dansyl, 7-amino-coumarin, azobenzene, and
bipyridyl groups, have been genetically incorporated into
proteins,14 the coplanar geometry of 2,5-diaryltetrazole9b
in 1-3 may present a challenge to the biosynthetic
enzymes involved in genetic incorporation of unnatural
amino acids. To this end, we further designed several
sterically less demanding monoaryltetrazole amino acids
4-6 with only two aryl rings (one phenyl and one
tetrazole) at the side chain (Scheme 1). Our preliminary
studies revealed that the N-aryl ring is absolutely required
for the tetrazole photoreactivity.15 As a result, we designed
both type I (4) and type II (5, 6) monoaryltetrazole amino
acids and envisioned that all three of them can be readily
accessed in racemic forms by alkylation of a glycine Schiff
base with the requisite tetrazole-containing alkyl halides
(Schemes 3 and 4).
Scheme 4. Synthesis of Monoaryl Tetrazole Amino Acids 4-6
Scheme 3. Synthesis of Tetrazole-Containing Alkyl Halides
reducing the ester to alcohol followed by iodination with
iodine and triphenylphosphine in the presence of imidazole
for a combined 99% yield. For the type II tetrazole amino
acid side chains, benzyl bromide 5b was derived in 91%
yield by treating 5a with N-bromo succinimide (NBS) and
benzoyl peroxide (BPO) in a straightfoward manner.
Because simple 2-phenyltetrazole was shown to be pho-
toreactive toward methyl acrylate in benzene,17 we decided
to remove the ester group in 5a through decarboxylation16
and obtained the second benzyl bromide 6b in 49% yield
by following an identical procedure as 5b (Scheme 3).
The installation of the photoreactive tetrazole side
chains to glycine Schiff base proceeded smoothly
(Scheme 4). The Boc-protected methyl (or ethyl) esters
of monoaryl tetrazole amino acids (4c, 5c, and 6c) were
obtained in four steps with the key step involving the
alkylation of glycine Schiff base with an excess amount
of the appropriate tetrazole-containing alkyl halide
(4b, 5b, or 6b) with overall yields of 63%, 68%, and 69%,
respectively (Scheme 4). Simple removal of the protecting
groups afforded monoaryl tetrazole amino acids 4 and 6
in excellent yields. It is noteworthy that amino acids 4
and 6 are structural isomers but potentially have discrete
photoreactivities. To reduce side chain bulkiness and
improve water solubility, we subjected the ethyl ester 5c
Since the synthesis of 2-phenyl-5-carboxylatetetrazole
has been reported in the literature,16 we decided to employ
this type of monoaryltetrazole to derive three tetrazole-
containing alkyl halides (Scheme 3). Starting from ethyl
glyoxalate, Kakehi’s method rapidly gave rise to ethyl
2-phenyltetrazole-5-carboxylate (4a) and ethyl 2-tolyl-
tetrazole-5-carboxylate (5a) in 60% and 41% yield,
respectively. The type I tetrazole amino acid side chain,
N-phenyltetrazole-5-methyl iodide (4b), was obtained by
(14) (a) Wang, L.; Schultz, P. G. Angew. Chem., Int. Ed. 2005, 44, 34–
66. (b) Xie, J.; Liu, W.; Schultz, P. G. Angew. Chem., Int. Ed. 2007, 46,
9239–9242.
(15) In our initial study, 2-phenyl-5-ethyltetrazole reacted with methyl
methacrylate to give rise to the pyrazoline adduct in 60% yield upon
photoirradiation at 302 nm in ethyl acetate for 2 h, whereas analogous
2-ethyl-5-phenyltetrazole did not react under identical conditions: Wang,
Y., Lin, Q. Unpublished results.
(16) Lippmann, E.; Konnecke, A.; Beyer, G. Monatsh. Chem. 1975, 106,
437–442.
(17) Huisgen, R.; Fliege, W.; Winfried, K. Chem. Ber. 1983, 116, 3027–
3038.
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Org. Lett., Vol. 11, No. 16, 2009

to aminolysis and obtained 5-amido-2-phenyltetrazole
amino acid 5 in 68% yield after deprotection (Scheme 4).
Table 1. Photoreactivity of Tetrazole Amino Acids 1-6 toward
Methyl Methacrylate in Photoinduced 1,3-Dipolar Cycloadditiona
Having obtained these six tetrazole amino acids, we then
examined their photoreactivities toward methyl methacrylate
in a mixed ACN/PBS buffer (1:1) upon photoirradiation at 302
nm using a hand-held UV lamp (UVP, 302 nm, 0.16 A) (Table
1). We found that all three diaryltetrazole amino acids 1-3
reacted quantitatively with methyl methacrylate to afford the
pyrazoline cycloadducts 7-9 based on the HPLC analysis. The
reactions were highly regioselective9 as the other regioisomers
were not detected by either HPLC or NMR. However, among
three monoaryltetrazole amino acids (4-6), only 5 gave rise
to the pyrazoline cycloadduct in 93% yield with exclusive
regioselectivity. While both 4 and 6 underwent the photoinduced
tetrazole ring rupture (based on the disappearance of the starting
materials), no pyrazoline cycloadducts were detected, presum-
ably because of rapid water-quenching of the nonstabilized
nitrile imine intermediates. By contrast, because of the resonance
effect, tetrazole amino acids 1-3 and 5 generate much more
stable nitrile imine dipoles in the aqueous buffer, e.g., a
diaryltetrazole-derived nitrile imine intermediate was detected
by reverse-phase HPLC and showed a half-life of 5 s.10a
In summary, we report the first synthesis of six unique
tetrazole amino acids, four of which showed excellent
reactivity toward a simple alkene in the photoinduced
cycloaddition reaction in biological buffer. The incorporation
of these photoreactive amino acids site-specifically into
proteins to serve as chemical models for protein post-
translational modifications, such as lipidation and phospho-
rylation, is currently underway.
Acknowledgment. We gratefully acknowledge the Na-
tional Institute of General Medical Sciences, the National
Institutes of Health (GM85092) and New York State Center
of Excellence in Bioinformatics and Life Sciences for
financial support.
a Reactions were conducted by irradiating 0.06 mmol of 1-6 and 6
mmol of methyl methacrylate in 200 mL of ACN/PBS buffer (1:1) in quartz
flasks for 3 h using a hand-held 302-nm UV lamp. pyr ) pyrazoline. b Yields
were determined by HPLC analysis. The standard deviation in yields should
be less than 5% as the purified products at the indicated retention times
(see Supporting Information) were greater than 95% pure on the basis of
the 1H NMR analysis. c 2 h reaction time. d No pyrazoline cycloadduct was
detected.
Supporting Information Available: Experimental details
and characterization of all new compounds. This material
isavailablefreeofchargeviatheInternetathttp://pubs.acs.org.
OL901300H
Org. Lett., Vol. 11, No. 16, 2009
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