J. Am. Chem. Soc. 1998, 120, 8543-8544
8543
A Bifunctional Photoaffinity Probe for Ligand/
Receptor Interaction Studies
Kan Fang, Masaru Hashimoto, Steffen Jockusch,
Nicholas J. Turro, and Koji Nakanishi*
Department of Chemistry, Columbia UniVersity
New York, New York 10027
ReceiVed May 18, 1998
Figure 1. Conceptual outline of BPP.
Photoaffinity labeling has become a powerful tool for inves-
tigating ligand/receptor interactions since its introduction by
Westheimer.1,2 However, despite the progress in the design of
photolabile groups,3 and methods for separating cross-linked
peptide fragments and identifying cross-linking sites,4,5 the cases
in which cross-linked amino acids have been identified are
relatively limited.4,6 This is particularly true with membrane-
bound proteins, including the philanthotoxin/nicotinic acetylcho-
line (nACh) receptor complex.7 With retinal/rhodopsin and
retinal/bacteriorhodopsin, it was possible to characterize cross-
linked amino acids, but with considerable difficulty.8-14
A bifunctional photoaffinity probe (BPP) with a photoaffinity
label (site A) and a photocleavable moiety (site B) has been
prepared to streamline the tedious photoaffinity labeling process.
The concept is shown in Figure 1: (i) The ligand-BPP molecule
is bound to the receptor and photolyzed, and the receptor is
cleaved enzymatically or chemically; (ii) the cross-linked and non-
cross-linked peptide fragments are separated (a biotin tag linked
to the ligand may facilitate separation);15 (iii) the ligand (with
the biotin tag) is detached from the cross-linked peptides by site
B photocleavage; (iv) the mixture of peptides with the nitrophe-
nolic marker is sequenced by tandem MS.
from the peptides before MS sequencing to avoid complications
in MS analysis (Figure 1). This led to the design of the probe
shown in Figure 1, which carries photolabeling (site A) and
photocleavable sites (site B), and which can derivatize various
ligands.
For site A, the 3-(trifluoromethyl)-3-phenyldiazirine moiety19
was chosen as the photolabel because of its outstanding photo-
cross-linking results13,20 and because irradiation at its 350 nm
absorption band would not destroy the protein, λmax 280 nm; it is
also stable to mild reductive, acidic, and basic conditions.3,4 With
respect to site B, the linkage should withstand the manipulations
associated with photoaffinity labeling experiments, but should also
be readily cleavable for subsequent MS measurements.
A
comparison of various linkages and cleavage conditions in model
systems21 led to the “aromatic photosubstitution” system studied
by Havinga.22 Namely, as shown in Scheme 1 (I), irradiation in
mild base displaces the OCH3 group of m-nitroanisole 1 by 18OH-
to yield 2; however, in neutral media no photochemistry occurs.23
Thus, irradiation of 3-nitroanisole 1 and 3-(4-methylphenyl)-3-
(trifluoromethyl)-1,2-diazirine 3 in CD3OD at 365 nm for 180
1
min gave adduct 4, characterized by H NMR, 19F NMR, and
GC-MS (Scheme 1, II). Whereas diazirine 3 reacted completely,
anisole 1 remained intact. It is therefore possible to selectively
excite the photolabeling site A in the presence of the phenoxyl
ether site B.
Tandem MS can directly sequence the respective peptide
fragments, including those from membrane-bound proteins,16 at
the femtomole level,17 without separation of the mixture; more-
over, no radioisotope is required.18 However, it is desirable that
the bulk of the ligand moiety (including biotin tag) be removed
The bifunctional photoaffinity probe should be compact and
carry a m-nitrophenoxyl ether function and a trifluoromethyl
diazirine side chain, and both positions ortho to the diazirine side
chain should be unsubstituted to avoid intramolecular cross-
linking. This led to BPP 11, prepared as in Scheme 2. Aldehyde
524 was converted into trifluoroacetyl ketone 6 by trifluoro-
methylation with Ruppert’s reagent25 and oxidation of the resulting
alcohol with Dess-Martin periodinane.26 Heating of 6 with
NH2OH‚HCl in pyridine at 60 °C gave a mixture of syn- and
anti-oximes 7. Saponification of methyl ester 7 with aqueous
base followed by Curtius rearrangement with DPPA in t-BuOH27
(1) Singh, A.; Thorton, E. R.; Westheimer, F. H. J. Biol. Chem. 1962, 237,
3006-3008.
(2) Bayley, H. Photogenerated Reagents in Biochemistry and Molecular
Biology; Elsevier: Amsterdam, 1983.
(3) Fleming, S. A. Tetrahedron 1995, 51, 12479-12520 (review).
(4) Kotzyba-Hibert, F.; Kapfer, I.; Goeldner, M. Angew. Chem., Int. Ed.
Engl. 1995, 34, 1296-1312 (review).
(5) Hatanaka, Y.; Hashimoto, M.; Kanaoka, Y. J. Am. Chem. Soc. 1998,
120, 453-454.
(6) Brunner, J. Annu. ReV. Biochem. 1993, 62, 483-514 (review).
(7) Choi, S.-K.; Kalivretenos, A. G.; Usherwood, P. N. R.; Nakanishi, K.
Chem. Biol. 1995, 2, 23-32.
(8) Nakanishi, K.; Zhang, H.; Lerro, K. A.; Takekuma, S.; Yamamoto, T.;
Lien, T. H.; Sastry, L.; Baek, D.-J.; Moquin-Pattey, C.; Boehm, M.; Derguini,
F.; Gawinowicz, M. Biophys. Chem. 1995, 56, 13-22.
(9) Zhang, H.; Lerro, K. A.; Takekuma, S.; Baek, D.-J.; Moquin-Pattey,
C.; Boehm, M. F.; Nakanishi, K. J. Am. Chem. Soc. 1994, 116, 6823-6831.
(10) Zhang, H.; Lerro, K. A.; Yamamoto, T.; Lien, T. H.; Sastry, L.;
Gawinowicz, M. A.; Nakanishi, K. J. Am. Chem. Soc. 1994, 116, 10165-
10173.
(18) Tull, D.; Miao, S.; Withers, S.; Aebersold, R. Anal. Biochem. 1995,
224, 509-514.
(19) Brunner, J.; Senn, H.; Richards, F. M. J. Biol. Chem. 1980, 255, 3313-
3318.
(20) Tate, J. J.; Persinger, J.; Bartholomew, B. Nucleic Acids Res. 1998,
26, 1421-1426.
(21) The following linkages and cleavage conditions were not satisfac-
tory: (i) o-nitrobenzyl ether(amine)/photoinduced internal oxidation, the
oxidation process competed with excitation of the diazirine function resulting
in poor selection; (ii) benzyl amine(ether)/hydrogenolysis, Pd catalyst was
poisoned by Cys and Met; (iii) double bond/ozonolysis, Trp damage. Also
see ref 6.
(11) Boehm, M. F.; Gawinowicz, M. A.; Foucault, A.; Derguini, F.;
Nakanishi, K. J. Am. Chem. Soc. 1990, 112, 7779-7782.
(12) Ding, W. D.; Tsipouras, A.; Ok, H.; Yamamoto, T.; Gawinowicz, M.
A.; Nakanishi, K. Biochemistry 1990, 29, 4898-4904.
(13) Nakayama, T.; Khorana, H. G. J. Biol. Chem. 1990, 265, 15762-
15769.
(22) Havinga, E.; Kronenberg, M. E. Pure Applied Chem. 1968, 16, 137-
152.
(14) Huang, K.-S.; Radhakrishnan, R.; Bayley, H.; Khorana, H. G. J. Biol.
Chem. 1982, 257, 13616-13623.
(23) Jongh, R. O. D.; Havinga, E. Recl. TraV. Chim. Pays-Bas 1966, 85,
275-283.
(15) Passage of the peptide mixture obtained upon cleavage of the receptor
through an avidin affinity column should retain all fragments cross-linked to
the ligand; the BPP linked fragments are submitted to tandem MS after removal
of the ligand through photocleavage.
(24) Aldehyde 5 was readily prepared from dimethyl 5-hydroxyisophthalate
in four steps with 55% overall yield: (i) allyl bromide, K2CO3, acetone, reflux;
(ii) NaOH, H2O/MeOH; (iii) isopropyl chloroformate; NaBH4, THF/H2O; (iv)
Dess-Martin periodinane, CH2Cl2. For details, see Supporting Information.
(25) Prakash, G. K. S.; Krishnamurti, R.; Olah, G. A. J. Am. Chem. Soc.
1989, 111, 393-395.
(16) Orlando, R.; Kenny, P. T. M.; Moquin-Pattey, C.; Lerro, K.; Nakanishi,
K. Org. Mass Spectrom. 1993, 28, 1395-1402.
(17) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996,
239, 180-192.
(26) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155-4156.
(27) Shioiri, T.; Yamada, S. J. Am. Chem. Soc. 1972, 94, 6203-6205.
S0002-7863(98)01718-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/06/1998