Simple method for the introduction of iodo-label on (3-trifluoromethyl) phenyldiazirine for photoaffinity labeling

Article abstract of DOI:10.1016/j.tetlet.2006.03.068

A simple and mild method was developed for the introduction of iodo-label on (3-trifluoromethyl) phenyldiazirine (TPD) aromatic ring in the presence of three membered diazirine ring. An iodination protocol, I₂-BTI in CH₃CN, was found

Full text of DOI:10.1016/j.tetlet.2006.03.068

Tetrahedron Letters 47 (2006) 3391–3394
Simple method for the introduction of iodo-label on
(3-trifluoromethyl) phenyldiazirine for photoaffinity labeling
Makoto Hashimoto,a,* Yuhi Katoa and Yasumaru Hatanakab
aDepartment of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho,
Obihiro 080-8555, Hokkaido, Japan
bFaculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2360 Sugitani, Toyama 930-0194, Japan
Received 6 February 2006; revised 7 March 2006; accepted 13 March 2006
Available online 31 March 2006
Abstract—A simple and mild method was developed for the introduction of iodo-label on (3-trifluoromethyl) phenyldiazirine (TPD)
aromatic ring in the presence of three membered diazirine ring. An iodination protocol, I2–BTI in CH3CN, was found to be effective
even though affinity ligands are pre-installed.
Ó 2006 Elsevier Ltd. All rights reserved.
Photoaffinity labeling is a powerful method in the study
of biological structures and functions.1–3 It is suitable
for the analysis of biological interactions because it is
based on the affinity of the ligand moiety. Various
photophores, such as phenyldiazirine, arylazide, and
benzophenone, have been used. Comparative irradiation
studies of these three photophors in living cells suggest
that a carbene precursor, (3-trifluoromethyl) phenyl-
diazirine (TPD), is the most promising, especially con-
cerning photolabeling of living cells.4 This is because
other photophors promote cell death by long time irradi-
ation in attempts to generate active species for cross-
links. However, synthesis of TPD analogues is hampered
when trying to repeat constructions of the TPD photo-
phor for each derivative. Approaches for the direct
modification of diazirine photophor bypassed the time
consuming diazirine ring construction steps.5–7 Iodinated
TPD derivatives are widely used for photoaffinity label-
ing. One method is where iodinated compounds are
utilized as a tag to detect labeled components by radioiso-
tope.8 The other one is where iodinated compounds are
used as precursors for chemoselective hydrogenolysis to
introduce hydrogen isotopes.9,10 Furthermore, it was
reported that iodine introduction sometimes caused a
higher affinity for membrane protein than unsubstituted
TPD compound.11 The iodination routine, however,
has still remained as inefficient. There are several reports
on the preparation of iodinated diazirinyl compounds.
Iodination with Chloramine T and NaI could be applied
when the compounds contained OH12 or isothiocya-
nate,13 which promote very strong activation for electro-
philic substitution on the benzene ring. However, this
method does not apply to alkoxy TPD, because the
alkoxy group is less activated for electrophilic substitu-
tion than the hydroxyl group. Several other iodination
methods were examined for alkoxy TPD analogues: (1)
Iodination was performed before construction of the
diazirinyl three-membered ring. The method should in-
volve repeated constructions of a diazirine moiety (over
five steps) for each derivative;14,15 (2) Iodine was ex-
changed for a pre-installed aryl-tin group on TPD with
NaI in the presence of peracetic acid16 or Chloramine
T.17 This required additional steps to introduce the
alkyl-tin group on TPD, and that the yield of iodination
is not sufficient (ꢀ60%) for the applications; (3) Direct
iodination was performed with Tl(OCOCH3)3–NaI18
for alkoxy TPD analogues. This method is capable of
introducing iodine without pre-installed substituent
groups, but thallic compounds were well known as being
very toxic. The last two methods require a long reaction
time for the iodination. Therefore, the direct iodination
of TPD still remains to be improved with mild and effec-
tive conditions. Combination of molecular iodine (I2) and
bis(trifluoroacetyl)phenyl iodinate (BTI) in CH3CN was
reported as one of the electrophilic iodination reagents
for aromatics under mild conditions.19–23 In this report,
we will describe selective and mild iodination of alkoxy
TPD analogues in which affinity ligands have also already
been installed.
*
Corresponding author. Tel.: +81 155 495542; fax: +81 155 495577;
e-mail: hasimoto@obihiro.ac.jp
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.068

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M. Hashimoto et al. / Tetrahedron Letters 47 (2006) 3391–3394
Iodinations of 3- and 4-methoxy TPD (1, 2) were not
efficient with 0.6 equiv of I2 and 1.2 equiv of BTI, which
is consistent with a previous report. Large amounts of
both reagents, 2.4 equiv of I2 and 4.8 equiv of BTI, were
needed for moderate yields (86% and 74% for 3 and 4,
respectively) and no decomposition of the diazirinyl ring
was observed.24 The iodinated position of 3-methoxy
diazirine 1 was defined via the NOESY spectrum from
the methoxy group. Excess reagents seem to prompt
poly-iodinations for the aromatics. Comparative studies
of iodination for 3- and 4-methoxytoluene (5 and 6)
were carried out (Scheme 1), and the results are summa-
rized in Table 1. With the same conditions as in the pre-
vious report, 0.6 equiv of I2 and 1.2 equiv of BTI
produced mono-iodinated products 7 and 10, but the ex-
cess reagents promoted further iodination to mono-
iodinated compounds (8, 9, 11, entries 4–6 and 10–12).
These results indicated that the (3-trifluoromethyl) diazi-
rinyl group deactivated the electrophilic aromatic substi-
tution reactivity of the benzene ring compared with
alkane substituents. This deactivation power by the
(3-trifluoromethyl) diazirinyl moiety was also observed
in Friedel–Crafts reaction for TPD analogues.25,26
The alkoxy moiety will be useful for the introduction of
ligand skeletons in TPD. We elucidated the mild direct
iodination methods for photoreactive diazirinyl ana-
logues, which already contained a ligand moiety. Photo-
affinity fatty acid analogues are useful reagents to
investigate related biomolecules, because many fatty
acids are recognized by membrane proteins, where it is
difficult to utilize other functional analysis methods.
Previous studies have indicated that phenoxy TPD fatty
acid analogues12,27 are very useful. Several researchers
attempted the synthesis of iodinated fatty acid TPD ana-
logues, but they performed iodination before construc-
tion of the (3-trifluoromethyl) diazirinyl ring or used
pre-installed iodinated precursors. The I2–BTI method
was applied for several alkoxy TPD fatty acid deriva-
tives (Scheme 2). The results are summarized in Table 2.
The diazirinyl fatty acid methyl ester (12a) is easily
mono-iodinated with I2 (1.2 equiv) and BTI (2.4 equiv)
in a moderate yield (73%, entry 1). Larger amounts of
both reagents (two times) improved the yield (90%,
entry 2). Excess reagents did not promote further
iodination of the mono-iodinated compound (12b).
The carboxylic acid analogue (13a) showed optimal
iodination with 1.2 equiv of I2 and 2.4 equiv of BTI
(84%, entry 3) for mono-iodination. A larger excess of
reagents caused further iodination, but the di-iodinated
compound was less than 25% (entries 4 and 5). The
results indicated that the acidity of the reaction mixture
caused secondary iodination. The succinimide ester
(14a), which is one of the highly reactive groups for
the condensation of amino groups, was also iodinated
with 1.2 equiv of I2 and 2.4 equiv of BTI (86%, entry
7) without hydrolysis.
F3C
N
N
R1
R2
R1
R2
I2, BTI
1
2
OCH3
H
H
OCH3
3
4
OCH3
I
I
OCH3
CH3CN, rt
R1
R2
R5
R3
I2, BTI
F3C
N
N
F3C
N
N
F3C
N
N
CH3CN, rt
R4
R1
R4
R1
I
I2, BTI
R2
R2
CH3CN, rt
R1
R2
R1
R2
R3
R5
5
6
OCH3
H
7
8
9
OCH3
OCH3
OCH3
I
I
I
H
H
I
H
H
H
H
I
R1
R1
R1
12-14a
H
R1
12-14b
12-14c
I
I
12 O(CH2)10COOMe
13 O(CH2)10COOH
14 O(CH2)10COOSu
H
OCH3
10
11
I
I
OCH3
OCH3
H
H
H
I
H
H
Scheme 1.
Scheme 2.
Table 1. Iodination of alkoxyphenyl compounds with various amounts of I2 and BTI
Entry
Substratea
I2 (equiv)
BTI (equiv)
Mono-iodinated (%)
Di-iodinated (%)
1
2
3
4
5
6
7
8
9
1
1
1
5
5
5
2
2
2
6
6
6
0.6
1.2
2.4
0.6
1.2
2.4
0.6
1.2
2.4
0.6
1.2
2.4
1.2
2.4
4.8
1.2
2.4
4.8
1.2
2.4
4.8
1.2
2.4
4.8
29 (3)
77 (3)
86 (3)
69 (7)
n.d.
n.d.
n.d.
n.d.
26, 5
75 (8), 15 (9)
79 (8), 13 (9)
n.d.
n.d.
n.d.
n.d.
45 (4)
70 (4)
74 (4)
80 (10)
33 (10)
n.d.
10
11
12
n.d
42 (11)
81 (11)
a Each substrates are used 0.2 mmol.

M. Hashimoto et al. / Tetrahedron Letters 47 (2006) 3391–3394
3393
Table 2. Iodination of diazirinyl fatty acid analogues with various amounts of I2 and BTI
Entry
Substratea
I2 (equiv)
BTI (equiv)
Mono-iodinated (%)
Di-iodinated (%)
1
2
3
4
5
6
7
12a
12a
13a
13a
13a
14a
14a
1.2
2.4
1.2
2.4
6.0
1.2
2.4
2.4
4.8
2.4
4.8
12.0
2.4
73 (12b)
90 (12b)
84 (13b)
68 (13b)
70 (13b)
12 (12b)
86 (12b)
n.d.
n.d.
n.d.
25 (13c)
24 (13c)
n.d.
4.8
n.d.
a Each substrate 0.2 mmol.
We have demonstrated that photoaffinity labeling in
combination with avidin–biotin systems (photoaffinity
biotinylation)28–32 is very useful for the manipulation
of photolabeled biomolecules. The iodinated diazirinyl
biotin analogue could be useful for photoaffinity biotin-
ylation. Results for the direct iodination of biotinylated
diazirine 15a to 15b were not sufficient, because the bio-
tin moiety was not stable under the conditions. The Boc
protected analogue 16a was iodinated with a 23% yield
for 16b, but de-Boc reaction of iodinated and uniodi-
nated intermediates occurred because trifluoroacetic
acid was contaminated in BTI and liberated during the
reaction. Trifluoroacetyl derivative 17a was stable under
the iodination conditions (1.2 equiv of I2 and 2.4 equiv
BTI) with a moderate yield (67%). The iodinated com-
pound 17b was de-trifluoroacetyl and biotinylated,
affording the iodinated diazirinyl biotin analogue 15b
(Scheme 3).
Acknowledgments
This research was partially supported by the Ministry of
Education, Science, Sports and Culture, Grant-in-Aid
for Scientific Research on a Priority Area, 17035006,
and for Encouragement of Young Scientists, 16710151
(M.H.). M.H. also thanks the Mitsubishi Chemical
Foundation for financial support for the studies.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2006.03.068.
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(OCH2CH2)3NHR1
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temperature. The reaction mixture was stirred at room
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