Simple and versatile method for tagging phenyldiazirine photophores

Article abstract of DOI:10.1021/ja066479y

The first effective method for the introduction of a versatile substituent on 3-phenyl-3-trifluoromethyldiazirine has been developed. The simple preparation of a useful aldehyde intermediate allows easy access to various elaborated photoaffinity ligands, including a l-phenylalanine analog bearing a diazirine ring (TmdPhe). The asymmetric synthesis of TmdPhe was easily accomplished in gram quantities. Site-directed incorporation of this compound into the structure of a calmodulin-binding peptide using automated peptide synthesis afforded a photoreactive peptide that was successfully used for the specific labeling of calmodulin. Copyright

Full text of DOI:10.1021/ja066479y

Published on Web 11/04/2006
Simple and Versatile Method for Tagging Phenyldiazirine Photophores
Hiroyuki Nakashima,† Makoto Hashimoto,‡ Yutaka Sadakane,§ Takenori Tomohiro,† and
Yasumaru Hatanaka*,†
Graduate School of Medicine and Pharmaceutical Sciences, UniVersity of Toyama, 2630 Sugitani,
Toyama 930-0194, Japan, Department of Agricultural and Life Science, Obihiro UniVersity of Agriculture and
Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan, and School of Pharmaceutical Sciences,
Kyushu UniVersity of Health and Welfare, 1714-1 Yoshino-cho, Nobeoka, Miyazaki 882-8505, Japan
Received September 7, 2006; E-mail: yasu@pha.u-toyama.ac.jp
Scheme 1. Synthesis of Phenyl Diazirine Derivatives
Efficient access to useful diazirine photophores has been
developed. Although 3-phenyl-3-trifluoromethyl diazirine deriva-
tives satisfy most of the chemical criteria required for photoaffinity
labeling,1 the limited availability of conveniently modified deriva-
tives represents a major drawback. Alkoxy-substituted phenyl-
diazirines are currently the only useful synthetic units for evading
the repetition of time-consuming multistep diazirine synthesis.2,3
3-Phenyl-3-trifluoromethyl-3H-diazirine (1), the simplest analog,
can also be prepared easily on a large scale. However, the use of
this building block has been hampered by the low reactivity of its
simple benzene ring3 and the instability of the diazirine ring to the
acidic conditions of conventional Friedel-Crafts reactions.4,5 The
carboxylation of 1, mediated by a toxic thallium salt, has been
reported,6 but only moderate yields of an isomeric mixture were
obtained after 24 h of reaction. A breakthrough in the direct
modification of 1 would provide access to a range of valuable
photoaffinity probes. Here we report the efficient preparation of
synthetically useful aldehyde 2 from 1, which has enabled a variety
of applications, including the catalytic asymmetric synthesis of the
photoreactive phenylalanine analog L-4-[3-(trifluoromethyl)-3H-
diazirin-3-yl]-phenylalanine (TmdPhe, 3). The straightforward
synthesis of 3 provides sufficient TmdPhe for convenient automated
peptide synthesis. Thus, a TmdPhe-containing calmodulin-binding
peptide (CaMBP) was easily obtained, which specifically photo-
labeled calmodulin (CaM).
Although C-C bond formation on the benzene ring of 1 is crucial
for effective derivatization, only a limited number of reactions can
be used for this purpose because of the rather labile diazirine ring
and the low reactivity of the benzene ring. We previously found
that the Friedel-Crafts reaction of 1 with GaCl3 and dichloromethyl
methyl ether (a suspected carcinogen) produced the desired aldehyde
2 in the presence of TFA, albeit in low yield.5 Under these
conditions, the diazirine group was stable, whereas the aldehyde
source, dichloromethyl methyl ether, rapidly decomposed in the
acidic medium, leaving large amounts of unreacted 1. Dichloro-
methyl methyl ether was reported to be stable in trifluoromethane-
sulfonic acid (TfOH) and can be used successfully for Friedel-
Crafts acylation of inert benzene rings.7 Indeed, we find that if we
use SbF5 in TfOH at 0 °C, formylation of 1 proceeds in 80% yield
to give the desired aldehyde 2, a compound that was previously
obtained only in small quantities after many synthetic steps.5
Triflates of Ag, Pr, Y, Yb, Gd, or Sm in TfOH were ineffective,
whereas GaCl3 and FeCl3 afforded 2 in moderate yields (31% and
47%, respectively), and TiCl4 gave a sufficient yield. The results
are summarized in Scheme 1.
In the presence of TiCl4, the reaction is endothermic, allowing
large-scale preparation without external cooling. Thus, 2 can be
obtained in multigram quantities. Further derivatization to give ester
4, alcohol 5,8 and bromide 6, as shown in Scheme 1, is straight-
forward. Compound 6 could also be obtained directly from 1 with
BrCH2OCH3 in TfOH at 0 °C in a moderate yield.
Photoreactive peptides and proteins are powerful tools to identify
interactions in protein networks. Photoreactive R-amino acids have
been investigated because these analogs can be directly incorporated
at specific positions in the polypeptide chain. For this reason,
phenylalanine derivatives bearing a photophore, such as azide,9
benzophenone,10 or diazirine,11 have found wide use; practical
synthesis of the diazirine-bearing phenylalanine analog 3, which
has been successfully incorporated into peptides12 and proteins,13
is difficult though. The classical method via malonate gives a
racemate of TmdPhe and requires a subsequent enzymatic resolution
step to give the desired enantiomer.11 The first asymmetric synthesis
of TmdPhe was achieved in excellent yield14 but required 1 equiv
of a chiral Ni complex that is prepared by a rather complicated
manner.15 We adopted a cinchonidine-based asymmetric catalyst16
for the alkylation of tert-butylglycinate benzophenone imine with
bromide 6 in the presence of 2-tert-butylimino-2-diethylamino-1,3-
dimethylperhydro-1,3,2-diazaphosphorine (BEMP) as a base.17
Gram quantities of TmdPhe 3 were easily obtained in excellent
yield (86%) after the deprotection of 7 (Scheme 2). The optical
purity of the Fmoc derivative 8 corresponded to an ee of 97%, as
judged by HPLC analysis using a chiral support.
A photoreactive calmodulin-binding peptide (TmdPhe-CaMBP,
9) was easily prepared using 8 via conventional solid-phase peptide
synthesis. A biotin tag was incorporated at the N-terminus for
detecting photolabeled proteins. The tryptophan residue in the
original sequence of CaMBP10 was replaced by TmdPhe to give
the desired photoprobe 9 (MALDI-TOF MS m/z calcd for [MH -
2N]+ 2330.51, found 2332.19). The site of modification was chosen
on the basis of our earlier work in which a diazirine was successfully
† University of Toyama.
‡ Obihiro University of Agriculture and Veterinary Medicine.
§ Kyushu University of Health and Welfare.
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J. AM. CHEM. SOC. 2006, 128, 15092-15093
10.1021/ja066479y CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Preparation of TmdPhe-CaMBP
Photoreactive analogs of peptides, peptide-derived molecules,
and proteins can provide structural information about transient
interactions of proteins and facilitate tracking of signaling processes,
transport/metabolism pathways, immune responses, and so on.
Recently, this approach has received much attention in molecular
biology for proteomic analysis owing to the success of in vitro13,20
and in vivo21 site-specific incorporation of a photoreactive moiety
into polypeptides or proteins. Our method has the potential to make
diazirine photophores useful building blocks for the preparation of
diverse photoaffinity probes. It can therefore extend the potential
of photoaffinity labeling as a sensitive means of rapidly elucidating
protein structures and proteomic profiling.22
Acknowledgment. This research was financially supported by
Japan Science and Technology Agency (JST), Cooperative Link
of Unique Science and Technology for Economy Revitalization
(CLUSTER) (to Y.H. and T.T.), and Ministry of Education, Culture,
Sports, Science and Technology Grants-in-Aid for Scientific
Research (18390036 to Y.H. and T.T., 17035006 to M.H.), for
Encouragement of Young Scientists (16710151 to M.H.).
introduced into the peptide via disulfide linkage by substituting the
Trp3 with cysteine.18,19
Figure 1 shows the photocross-linking of CaM with 9 visualized
by chemiluminescence utilizing the interaction of HRP-conjugated
avidin and biotin attached to CaMBP after blotting onto a PVDF
membrane. Photolysis rapidly proceeded upon UV-A irradiation
with 30 W/m2 of 360 nm light at 0 °C and was complete in a few
minutes (see Supporting Information). Labeled CaM was not
detected in the absence of calcium (lane 2 in Figure 1a), as the
benzophenone-tagged CaMBP was.10,18 Moreover, when trifluo-
perazine was added to the solution as an inhibitor, the amount of
labeled CaM decreased in response to the concentration of
trifluoperazine (Figure 1b). These data indicate that the affinity of
synthetic peptide 9 for CaM varies in a calcium-dependent manner.
Supporting Information Available: Experimental details and
analytical data for new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Figure 1. (a) Calcium dependence of the photolabeling of CaM with
TmdPhe-CaMBP. Samples of 50 µL of Tris-HCl (50 mM, pH 7.4)
containing CaM (5 µM), TmdPhe-CaMBP (9, 10 µM), CaCl2 (10 mM),
and NaCl (0.15 M) were irradiated at 0 °C for 2 min with a 30-W long-
wavelength UV lamp after incubation at 37 °C for 10 min in the dark.
Lane 1: in the presence of 10 mM CaCl2. Lane 2: in the presence of 5
mM EGTA without CaCl2. Lane 3: in the absence of CaM. (b) Competitive
inhibition of photolabeling with trifluoperazine. Lane 4: same condition
as Lane 1 in panel a. Lane 5: the concentration of trifluoperazine was 10
µM. Lane 6: 30 µM. Lane 7: 100 µM. Lane 8: 300 µM.
Although diazirine photophores have been recognized as useful
photocross-linkers to probe the interfacial structure of protein-
protein or protein-peptide complexes, the preparation of diazirine-
based probes has not been convenient. As a consequence, progress
in diazirine-based photoaffinity labeling has been limited. In this
work, we dramatically improved the direct formylation of 1, a
practical diazirine source, to give compound 2 as a versatile
diazirine unit for developing various elaborated diazirine analogs.
The method enabled the practical synthesis of the useful photo-
reactive amino acid analog L-TmdPhe (3) on a gram scale suitable
for standard solid-phase synthesis. The photoreactive peptide 9
containing this amino acid bound CaM and specifically labeled it
in a calcium-dependent manner upon irradiation.
JA066479Y
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