Enzyme cleavable and biotinylated photoaffinity ligand with diazirine

Article abstract of DOI:10.1016/j.bmcl.2004.03.011

The efficient synthesis of an enzyme cleavable biotinylated diazirinyl photoaffinity ligand is described to allow the effective manipulation of the photolabeled biocomponents. The compound contains a glutamic acid γ-methyl ester, which is a precursor of the substrate for V8 protease, between the diazirinyl photophor and biotin moiety. After alkaline hydrolysis of the ester, the compound can be proteolyzed at the Glu moiety by V8 protease. The photophore was introduced to L-Phe p-nitroanilide and the ligand was applied to photolabel of chymotrypsin to manipulate the application of the concept.

Full text of DOI:10.1016/j.bmcl.2004.03.011

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2447–2450
Enzyme cleavable and biotinylated photoaffinity ligand with
diazirine
Makoto Hashimoto,a,* Shun’ji Okamoto,a Kensuke Nabetaa and Yasumaru Hatanakab
aDepartment of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine,
Inada-cho, Obihiro, Hokkaido 080-8555, Japan
bFaculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2360 Sugitani, Toyama 930-0194, Japan
Received 3 February 2004; revised 3 March 2004; accepted 4 March 2004
Abstract—The efficient synthesis of an enzyme cleavable biotinylated diazirinyl photoaffinity ligand is described to allow the effective
manipulation of the photolabeled biocomponents. The compound contains a glutamic acid c-methyl ester, which is a precursor of
the substrate for V8 protease, between the diazirinyl photophor and biotin moiety. After alkaline hydrolysis of the ester, the
compound can be proteolyzed at the Glu moiety by V8 protease. The photophore was introduced to
ligand was applied to photolabel of chymotrypsin to manipulate the application of the concept.
Ó 2004 Elsevier Ltd. All rights reserved.
L-Phe p-nitroanilide and the
Photoaffinity labeling is a powerful method in the study
of biological structures and functions.1–5 It will be suit-
able for the analysis of biological interactions in vivo
because it is based on the affinity of the ligand moiety.
Various photophores, such as phenyldiazirine, arylaz-
ide, and benzophenone, were used. Comparative irra-
diation studies of these three photophors in living cells
suggested that a carbene precursor (3-trifluoromethyl)
phenyldiazirine is the most promising.6 Recently,
photoreactive amino acid derivatives containing benzo-
phenone units were utilized, as they are commercially
available. However, comparative incorporation studies
of a photoreactive plasminogen activator peptide having
benzophenone or (3-trifluoromethyl) phenyldiazirine at
the same position revealed that the latter peptide gave
higher labeling efficiency.7 Low photolabeling yield
hampers purification and isolation of the labeled com-
ponents.3;8 We have attempted to resolve these difficul-
tinyl tag to the target molecule by photoaffinity labeling
followed by the use of avidin–biotin interaction, facili-
tated the structural analyses of photolabeled compo-
nents. The combination enabled us to detect and isolate
photolabeled proteins and peptides without the use of
radioisotopes. However, it is difficult to retrieve the
biotinylated labeled components from the avidin–biotin
complex quantitatively.10 This is due to the higher
affinity between the avidin–biotin complex (Kd ¼
10ꢀ15 M).17 Two techniques were considered to solve this
problem. One method was the utilization of monomeric
avidin to retrieve the biotinylated components under
mild conditions.11;12 Monomeric avidin has less affinity
to biotin (Kd ¼ 5 ꢁ 10ꢀ8 M) than native tetrameric avi-
din18, however, a very high concentration and large
amount of this protein is sometimes required. The other
method is a chemical cleavage between the photophore
and biotin. It includes photocleaving,19 periodinate-
cleaving,14 and utilizing thiol-cleavable15 biotinylated
diazirinyl derivatives. However, these chemical cleav-
ages sometimes damage biomolecules due to their reac-
tion with functional groups in biomolecules under
cleavage conditions. In this paper, we describe a new
method by which the enzyme cleavable component is
inserted between the diazirinyl photophore and biotin
moiety and subsequent use of it to remove biotin from
labeled biomolecules. Figure 1 shows the new enzyme
cleavable biotinylated photoaffinity ligand for chymo-
trypsin (Fig. 2).
ties by
a combination of avidin–biotin systems
(photoaffinity biotinylation)9–15 or immune-interaction
for photolabeling reagents.16 We developed a series of
biotinyl probes for the photoaffinity labeling of bio-
functional proteins. The covalent introduction of a bio-
Keywords: Photoaffinity labeling; Diazirine; Avidin–biotin; V8 prote-
ase; Chymotrypsin.
* Corresponding author. Tel.: +81-155-49-5542; fax: +81-155-49-5577;
e-mail: hasimoto@obihiro.ac.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.011

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M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2447–2450
Glu(OMe)
Photoreactive group
Biotin
O
F3C
N
N
O
S
O
HN
NH
O
H
N
O
N
O
O
O
H
N
H
O
NO2
N
H
1
Cleavable by V8 protease after
deprotection of methyl group
O
Phe p-NA
substrate for chymotrypsin
Figure 1. Structure of enzyme cleavable biotinylated photolabel reagent for chymotrypsin.
O
O
O
O
O
O
O
O
O
O
HN NH
HN NH
ii)
O
O
i)
H
OH
OH
O N
N
N
H
H N
2
S
N
N
S
H
O
O
4
O
F C
2
3
3
O
F C
3
N
N
O
H N
2
O
5
O
O
O
iv)
COOH HN NH
O
H
1
N
O
iii)
N
O
O
S
H
6
O
COOH
Figure 2. Synthesis of compound 1. (i) Biotin N-hydroxysuccinimide ester, DMF, rt, 5 h, 81%, (ii) N-hydroxysuccinimide, DCC, DMF, rt, 2 h, 90%,
(iii) TEA, DMF, rt, 5 h, 91%, (iv) N-hydroxysuccinimide, DCC, DMF, rt, then L-Phe p-NA, TEA, DMF, rt, 4 h, 60%.
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
The compound consisted of (1) photoreactive diazirinyl
photophor, (2)
c-Me) spacer, (3) biotin moiety, and (4)
p-nitroanilide ( -Phe p-NA) attached to diazirine, a
substrate for chymotrypsin. Enzymatic cleavage was
controlled with alkaline hydrolysis of the -Glu c-Me.
L
-glutamic acid c-methyl ester (
L-Glu
L
-phenylalanine
L
µ
L
After hydrolysis, the labeled component became the
substrate for V8 protease and released the biotin moiety
from the photolabeled site during enzymatic cleavage.
The compound 1 was synthesized from the Glu c-methyl
ester 2. Biotin was introduced to the N-terminal 3, and
the C-terminal was converted to succinimide ester 4. The
diazirine derivative 515 was condensed to afford com-
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
1 / [compound] (1/µM)
Figure 3. Lineweaver–Burk plots for photoprobe 1 (open square) and
-Phe p-NA (closed circle) in a 96-well titer plate. The assay mixture
(0.1 mL), consisting of various concentrations of the photoprobe or
-Phe p-NA, 8.4 mM chymotrypsin and 0.1 M sodium phosphate
pound 6. L-Phe p-NA was introduced to the carboxylic
acid of the diazirinyl moiety to obtain compound 1.20
Each step proceeded with a moderate yield.
L
L
buffer (pH 7.0), was incubated at 37 °C for 24 h. Released p-NA was
measured at 405 nm.
The biological activity of compound 1 as a chymotryp-
sin substrate was tested by spectrometric assay.21 We
performed the assay in a 96-well titer plate. There were
no differences between this and a normal assay in a tube.
Compound 1 and
The obtained Km value of compound 1 (175 lM) indi-
cated that 1 is better substrate of chymotrypsin than
L
-Phe p-NA were applied to the assay.
irradiated with black light to attach the samples to the
membrane covalently.11 The irradiated membrane was
subjected to chemiluminescence detection with a strep-
tavidin–HRP conjugate.10 The results indicated that a
chemiluminescence signal was observed in the sample
without treatment, alkaline hydrolysis, and V8 digestion
without alkaline hydrolysis (Fig. 3, A–C). In contrast,
no signal was observed in the sample of V8 digestion
after alkaline hydrolysis (Fig. 3, D). These results indi-
cated that effective cleavage of the synthetic compounds
with V8 protease was regulated by alkaline hydrolysis of
methyl ester (Fig. 4).
L
-Phe p-NA (750 lM) (Fig. 3). Cleavage of the photo-
phore and biotin by V8 protease22 was then inspected.
Compound 1 was subjected to several condition as fol-
lows: (i) alkaline hydrolysis with aqueous NaOH at
room temperature for 2 h, (ii) enzymatic digestion with
V8 protease in phosphate buffer (pH 7.8) at 37 °C for
24 h and (iii) alkaline hydrolysis and V8 digestion in an
identical manner for (i) and (ii). After each treatment,
the mixtures were blotted on a PVDF membrane, then

M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2447–2450
2449
resistance of chymotrypsin to digestion under denatur-
ation conditions. However, no signal was observed when
the sample was treated with both alkaline hydrolysis and
V8 digestion (D). These results indicated that photo-
biotinylated chymotrypsin released the biotinyl moiety
selectively and quantitatively with V8 treatment after
alkaline hydrolysis.
Figure 4. Dot–blot analysis of compound 1 under various conditions.
Compound 1 (1 pmol) was subjected to the conditions indicated: (A)
no treatment, (B) alkaline hydrolysis with 10 mM NaOH at room
temperature for 2 h, (C) digestion with V8 protease in phosphate buffer
(pH 7.8) at 37 °C for 24 h, and (D) alkaline hydrolysis then digestion
with V8 protease. Each sample was blotted on a PVDF membrane,
then dried. The membrane was irradiated with black light for 40 min
for immobilization, then chemiluminescence detection was applied
with streptavidin–horseradish peroxidase conjugate in the same man-
ner as described previously.10;11
In conclusion, new diazirinyl photoprobe containing an
amino acid linker is useful in overcoming the problem to
remove photobiotinylated components from an avidin–
biotin complex. The results indicated that the reagent
may be useful for the functional analysis of the proteins
that are stable V8 protease digestion.
Photoaffinity labeling of chymotrypsin with compound
1 was performed. After incubation of a stoichiometric
amount of protein and 1 at 30 °C for 10 min, the sample
was irradiated with black light (15 W) at 0 °C for 40 min.
Acknowledgements
Competitive inhibition with L-Phe p-NA was also per-
formed. The irradiated samples were subjected to SDS–
PAGE, and the gel was electro-transferred to a PVDF
membrane. The membrane was subjected to chemilu-
minescence detection in an identical manner as for dot–
blot analysis. A chemiluminescence signal was detected
on 26 Kda, which corresponds to chymotrypsin (Fig. 5,
lane A). The signal was reduced to 63% or less than 5%
This research was partly supported by the Akiyama
Foundation and the Research and Development Pro-
gram for New Bio-industry of the Bio-oriented Tech-
nology Research Advancement Institution.
References and notes
in the presence of 7.5 or 15 mM of
L-Phe p-NA,
respectively, (lanes B and C) with densitometer analysis.
The results of inhibition clearly showed that compound
1 competed with Phe p-NA at the substrate-binding site
of chymotrypsin.
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hydrolysis (Fig. 6C) afforded identical chemilumines-
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Figure 5. Chemiluminescence detection of photoaffinity labeled chy-
motrypsin with compound 1. A mixture of chymotrypsin and com-
pound 1 (3 nmol each) in phosphate buffer (pH 7.8) was incubated at
37 °C for 30 min, and irradiated with black light at 0 °C for 40 min.
L
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The samples contained 0, 30, and 60 nmol inhibitor for (A), (B), and
(C), respectively. Each mixture (1 lg protein) underwent SDS–PAGE
(12.5%) and chemiluminescence detection in the manner described
previously.
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Figure 6. Chemiluminescence detection of photoaffinity labeled chy-
motrypsin under various conditions. Labeled mixtures of chymotryp-
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alkaline hydrolysis with 10 mM NaOH at room temperature for 2 h,
(C) digestion with V8 protease in phosphate buffer (pH 7.8) at 37 °C for
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20. Compound 3: H NMR (CD3OD) 4.54 (dd, 1H, J ¼ 5:0,
7.6 Hz), 4.47 (dd, 1H, J ¼ 5:0, 9.2 Hz), 4.36 (dd, 1H,

2450
M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2447–2450
J ¼ 4:6, 7.6 Hz), 3.71 (s, 3H), 3.34 (m, 1H), 2.97 (dd, 1H,
Compound 1: 1H NMR (CDCl3 ) 8.15 (d, 2H, J ¼ 8:9 Hz),
8.07 (d, 1H, J ¼ 8:3 Hz), 7.70 (d, 2H, J ¼ 8:9 Hz), 7.20 (m,
5H), 6.89 (d, 1H, J ¼ 8:3 Hz), 6.70 (s, 1H), 4.53 (m, 1H),
4.35 (m, 1H), 4.28 (m, 2H), 3.83 (m, 2H), 3.70–3.20 (m,
13H), 3.18 (m, 1H), 2.96–2.62 (m, 4H), 2.32 (m, 2H), 2.20
(m, 2H), 2.0 (m, 2H), 1.80 (m, 4H), 1.43 (m, 2H), FAB-MS
m=z 1015 (MHþ).
J ¼ 5:0, ꢀ12.9 Hz), 2.97 (d, 1H, J ¼ ꢀ12:9 Hz), 2.48 (t,
2H, J ¼ 7:6 Hz), 2.31 (m, 2H), 2.10 (m, 2H), 1.74 (m, 4H),
1.51 (m, 2H), FAB-MS m=z 388 (MHþ).
Compound 6: 1H NMR (CDCl3) 7.98 (d, 1H, J ¼ 8:3 Hz),
6.85 (d, 1H, J ¼ 8:3 Hz), 6.70 (s, 1H), 4.54 (m, 1H), 4.34 (m,
1H), 4.25 (m, 2H), 3.90 (m, 2H), 3.72 (m, 2H), 3.68 (m, 1H),
3.64 (s, 3H), 3.54 (m, 2H), 3.42 (m, 2H), 3.13 (m, 1H), 2.90
(m, 1H), 2.74 (m, 1H), 2.41 (m, 2H), 2.20 (m, 2H), 2.10 (m,
2H), 1.74 (m, 4H), 1.51 (m, 2H), FAB-MS m=z 747 (MHþ).
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