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The present simple protocol, which provides an alternative to
the BPP approach, is currently being applied to an actual
ligand–receptor system.
This work was supported by NIH Grant AI 100187. We are
grateful to Drs Yasuhiro Itagaki and Masaru Hashimoto, and
Mr Jinsong Guo, for discussions.
O
O
N
N
Ligand
O
Ligand
O
CH N2
CF3
9
10
O
CF3
OMe
O
O
O
Me
O
Et
11
12
Notes and references
Fig. 3 Model compounds for the aminolysis reaction.
† Pyren-1-ylmethylamine hydrochloride and 4-bromobenzylamine hydro-
chloride are commercially available (Aldrich). The free pyren-1-ylmethyl-
amine and 4-bromobenzylamine were obtained by passing their hydro-
chlorides through an ion exchange column (OH2 type). If
pyren-1-ylmethylamine were brominated, the fluorophore will serve as a
MS tag as well.
‡ Although the amino group in Lys in the peptide would be deactivated by
protonation or hydrogen bonding, in principle, it can aminolyze the ester.
Therefore, excess amine is required, which also accelerates the reaction.
§ Amine 5 was made by the coupling of cystamine and (+)-biotin
4-nitrophenyl ester.
B cleavage occurs to detach the bulk of the ligand from the
crosslinked peptide fragment; the ligand moiety and non-
crosslinked fragments are separated from crosslinked fragment
2, which is sequenced by tandem MS. This protocol is currently
being applied to a particular ligand–receptor pair.
In the following, we introduce an alternative to further
facilitate the photoaffinity crosslinking and sequencing proc-
esses. In the case of BPP, the ligand moiety was removed by a
second photolysis performed under mild base conditions. In this
revised protocol which does not use BPP, the ligand moiety is
removed by aminolysis of a labile ester bond, as summarized in
Fig. 2: (i) esterification of the hydroxy group with the
appropriate photolabel, e.g. diazoacetate; (ii) incubation of
ligand with its receptor followed by photolysis; (iii) enzymatic
and/or chemical cleavage of the receptor; (iv) ester aminolysis
and removal of the ligand moiety using amines 3–5†‡ with
functionalities for identification and/or purification purposes,
e.g. bromine or chlorine atoms as tags for MS identification (3),
fluorescence tag for HPLC detection (4), biotin tag for
purification with immobilized avidin (5); (v) sequencing of
tagged peptide fragments by tandem MS (MS/MS).4–6
Here, aminolysis performs the dual function of removing the
undesired ligand moiety7–9 and introducing the tag for MS
sequencing, as well as purification. Namely, benzylamines 3
carry the isotopic halogen atoms10 for facile MS identification,
pyren-1-ylmethylamine 4 introduces a fluorescent tag11 for
chromatographic detection, if necessary, prior to MS, while
biotinylated amine 5 with a cleavable disulfide bond is useful
for separation of cross-linked peptide fragment(s) by avidin
chromatography.12,13
A wide variety of photolabeling probes can be adapted to the
protocol either directly or via short linkers, e.g. ethanolamine
and glycolic acid, to modify either the ligand or the photolabile
probes before linking. The aminolysis was checked with model
compounds 11 and 12, the products that might be produced
from photolabeled ligands 9 and 10 (Fig. 3), respectively.
Ester 11 was converted into the amide upon treatment with
150 equiv.‡ of amines 3a, 3b, 4, and 5,§ 50° C, 1 day, in yields
of 75, 61, 64 and 52%, respectively. Aminolysis of ester 11 with
base 3a, performed in the presence of four peptides Gly-Gly-
Phe, Lys-Phe, Gly-Leu-Tyr and Phe-Tyr, resulted in a 62%
transformation, demonstrating that peptide bonds are not
affected; the sluggish aminolysis of aromatic esters was
accelerated upon addition of NaCN,¶ in which case 70% of
ester 12∑ was aminolized overnight by 100 equiv. of amine 3a in
pyridine.** The disulfide bond in the biotinylated amide
derived from 5 and 11 was readily cleaved by EtSH to give
product 8 (P = phenoxymethyl) without biotin, suited for
tandem MS analysis, in quantitative yield.††
¶ NaCN is a very mild catalyst. It is likely that the attack of cyanide anion
occurs only on the ester bond without damage of the peptide fragment
(ref. 14).
∑
Ester 12 was readily prepared from the photoaffinity labeling probe
4-(1-azi-2,2,2-trifluoroethyl)benzoic acid (ref. 15) by photolysis and then
methylation.
** The aminolysis works in a wide spectrum of polar solvents, such as
water, EtOH, MeOH, pyridine, etc.
†† Selected data for amides: Amide from 3a and 11: m/z (EI) found:
275.0721 [(M)+ C15H14O2N335Cl], calc.: 275.0713. Amide from 3a and 12:
m/z (EI) found: 357.0739 [(M)+ C17H15O2N35ClF3], calc.: 357.0743. Amide
from 3b and 11: m/z (FAB) found: 319.0210 [(M + 1)+ C15H14O2NBr79],
calc.: 319.0208. Amide from 4 and 11: m/z (EI) found: 365.1422 [(M)+
C25H19O2N], calc.: 365.1416. Amide from 5 and 11: m/z (FAB) found:
513.1688 [(M + 1)+ C22H33O4N4S3], calc.: 513.1664.
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