Post-biotinylation of photocrosslinking by staudinger-bertozzi ligation of preinstalled alkylazide tag

Article abstract of DOI:10.1248/cpb.53.1510

Post-biotinylation of the alkyl azide derivative of trifluoromethyl phenyldiazirine (TPD) was elucidated to apply a photoaffinity biotinylation technique. A photo-modified polyvinilidene difluoride (PVDF) membrane was used as a photolabeled component and

Full text of DOI:10.1248/cpb.53.1510

1510
Notes
Chem. Pharm. Bull. 53(11) 1510—1512 (2005)
Vol. 53, No. 11
Post-biotinylation of Photocrosslinking by Staudinger–Bertozzi Ligation of
Preinstalled Alkylazide Tag
b
Makoto HASHIMOTO*,a and Yasumaru HATANAKA
a Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine; Inada-cho,
Obihiro, Hokkaido 080–8555, Japan: and b Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical
University; 2360 Sugitani, Toyama 930–0194, Japan.
Received July 21, 2005; accepted September 5, 2005; published online September 9, 2005
Post-biotinylation of the alkyl azide derivative of trifluoromethyl phenyldiazirine (TPD) was elucidated to
apply a photoaffinity biotinylation technique. A photo-modified polyvinilidene difluoride (PVDF) membrane was
used as a photolabeled component and we introduced biotin by Staudinger–Bertozzi ligation. The 15 pmol
amount of biotinylated reagent was still effective for the visualization of cross-linked product on the matrix. The
results show the potential utility of alkyl azide carrying TPD derivatives in the application of photoaffinity
biotinylation, which could be useful for the ligands with tight structural requirements.
Key words diazirine; photoaffinity label; chemical biology; Staudinger reaction; avidin-biotin
ing.23) The TPD photophore can be photolyzed much faster
Photoaffinity labeling is a powerful method in the chemi-
cal biology of protein functions.1,2) This method is especially
than aryl azide at a longer wavelength (350 nm), where alkyl
azide decomposition is negligible.3) We report here on the
useful for protein binding site identification where the appli-
photolysis of alkyl azide carrying TPD following the incor-
poration of a biotin tag for the possible application of di-
azirine based photoaffinity biotinylation.
cation of crystallography or NMR is difficult. Taking advan-
tage of the chemically stable cross-link from (3-trifluoro-
methyl) phenyldiazirine (TPD) based photolabeling,3) we
have developed a series of biotinyl TPD derivatives as a fish-
out approach to improve laborious experimental routines
used in binding site identification.4—10) The application of a
N-acetylglucosamine photoprobe carrying biotinylated di-
azirine gave the first information on acceptor site peptides on
b1,4-galactosyltransferase.6,7) We also developed a novel
method for the one-step introduction of a biotinylated di-
azirine photophore into unprotected carbohydrate ligands.11)
Alternatively, biotinylated reagents with a scissile function
have been considered for separation of the labeled compo-
nents from a biotin–avidin complex because the complex is
essentially irreversible (Kdꢀ10ꢁ15 M).12) For displacing the la-
bile disulfide group, photoreactive,13,14) fluoride sensitive,15)
alkali sensitive,16) oxidative17,18) and acylsulfonamide19) link-
ages have been developed.
Benzyl bromide derivative 125) having a diazirinyl group
was easily converted to alkyl azide analogue 2 with sodium
azide (Fig. 1). The half-life of the compound with black light
irradiation in methanol was calculated as 3.5 min (Fig. 2).
The IR spectrum of the photoirradiated sample showed a
strong peak corresponding to the azide moiety at 2100 cmꢁ1,
Large alternations within the ligand structure, such as
through the introduction of a biotinyl group, were reported to
decrease the affinity of some ligands.20) In such cases, the in-
troduction of biotin after photolabeling is one of the rational
approaches to overcome the problem, because the process in-
cludes minimal chemical modifications of the ligand skeleton
to avoid the reduction of ligand activity due to the pre-in-
stalled biotin moiety.21,22) Alkyl azide derivatives were em-
ployed as useful post modifiable tags by application of the
Staudinger–Bertozzi ligation or azide-alkyne [3ꢂ2] cyclo-
addition (click chemistry).23,24) The application of the
Staudinger–Bertozzi ligation and click chemistry in pho-
toaffinity labeling was examined with aryl azide and di-
azirine photophores.23) The results clearly showed that aryl
azide and diazirine photophores are selectively photoacti-
vated to leave the alkyl azide as intact for the possible appli-
cation of the Staudinger–Bertozzi ligation. Although post
fluorescent labeling with the Staudinger–Bertozzi ligation
was shown for the aryl azide photophore, a TPD derivative
carrying an alkyl azide tag was not evaluated for post label-
Fig. 1. Synthesis of the Alkylazide Derivative of the (3-Trifluoromethyl)
Phenyldiazirine Derivative and Its Application to the Staudinger–Bertozzi
Ligation
Fig. 2. The Decay in Absorbance at 360 nm of Compound 2 with Black
Light Photolysis as a Function of the Time of Photolysis in a Semi-Log Rep-
resentation
∗ To whom correspondence should be addressed. e-mail: hasimoto@obihiro.ac.jp
© 2005 Pharmaceutical Society of Japan

November 2005
1511
(25.1 mg, 68.9 mmol) were dissolved in THF–H2O (4 : 1, 0.5 ml). The reac-
tion mixture was stirred for 24 h, and then concentrated. The residue was pu-
rified with silica column chromatography (CHCl3 : CH3OHꢀ8 : 1) to afford 4
as a yellow compound (18.0 mg, 54%). 1H-NMR (CDCl3) 8.629 (1H, t,
Jꢀ5.6 Hz, NH), 8.177 (1H, d, Jꢀ7.9 Hz), 7.922 (1H, dd, Jꢀ7.9, 3.6 Hz),
7.814 (1H, dd, Jꢀ14.5, 1.6 Hz), 7.65—7.43 (m, 10H), 7.115 (1H, d,
Jꢀ7.9 Hz), 6.662 (1H, dd, Jꢀ7.9 Hz), 6.523 (1H, s), 4.075 (2H, d,
Jꢀ5.6 Hz), 3.793 (3H, s). FAB-MS m/z 594 (MꢂHꢂ) HR-FAB-MS Calcd
for C30H24F3N3O5P 594.1406, Found 594.1420.
Half Life of Compound 4 Assessed by UV Spectrophotometry
A
methanolic solution of compound 4 (0.5 mM) was irradiated with black light
at 1 cm at 0 °C. The spectrum was measured at 0.5, 1, 2, 3, 5, 7.5 and
10 min. The broad peak at 360 nm decreased over time.
Irradiation of Compound 4 in Methanol-d3 (5) and 3-N-[2-Methoxy-4-
(3-trifluoromethyl-2-d3-methoxy-2-d-3-yl)benzyl]aminocarbonyl-4-
(diphenylphosphinyl)-benzoic Acid (6) Compound 2 (3.4 mg, 12.5 mmol)
was dissolved in CD3OD (2.5 ml). The solution was irradiated with black
light for 20 min. 1H-NMR (CD3OD) 7.322 (1H, d, Jꢀ7.9 Hz), 7.106 (1H, s),
7.052 (1H, d, Jꢀ8.0 Hz), 4.357 (2H, s), 3.881 (3H, s), IR (neat) 2100 cmꢁ1
.
Fig. 3. Chemiluminescence Detection of the Post-Functionalized PVDF
Membrane with Compound 7
The irradiated mixture was subjected to the Staudinger reaction with
triphenylphosphine derivative 3 (16.5 mg, 45.3 mmol) in THF–H2O (4 : 1,
0.5 ml). The product was purified with PLC (CHCl3 : CH3OHꢀ8 : 1ꢃ3 times
developments) and eluted with CHCl3 : CH3OHꢀ4 : 1 to afford a colorless
amorphous mass (2.9 mg, 39%), 1H-NMR (CDCl3) d: 8.652 (1H, t,
Jꢀ5.7 Hz), 8.237 (1H, d, Jꢀ8.0 Hz), 7.977 (1H, q, Jꢀ7.9, 3.6 Hz), 7.900
(1H, d, Jꢀ13.2 Hz), 7.716—7.490 (10H, m), 7.144 (1H, d, Jꢀ8.0 Hz), 6.895
(1H, s), 6.883 (1H, d, Jꢀ8.0 Hz), 4.101 (2H, d, Jꢀ5.7 Hz), 3.856 (3H, s). IR
(neat) 2100 cmꢁ1. FAB-MS m/z 602 (MꢂHꢂ), HR-FAB-MS Calcd for
C31H24D4F3NO6P 602.1853, Found 602.1837.
(A) Scheme of the post-functional modified PVDF membrane. (B) Chemilumines-
cence detection of blotted compound 7. Amounts blotted were, 35, 25, 15 and 7.5 pmol
for (I) to (IV), respectively.
1
and H-NMR confirmed the presence of a methylene vicinal
to the azide at 4.10 ppm. The results indicate that the azide
moiety remained after photolysis, as reported previously.23,24)
The Staudinger reaction with triphenylphosphine deriva-
Photoimmobilization of Compound 2 on PVDF Membrane PVDF
tive27) 3 with compound 2 afforded the desired ligated prod- membrane (10 cm square) was wetted with 0.5 mM of compound 2 in MeOH,
dried and irradiated by black light for 20 min on one side. After washing
uct 4 in a moderate yield. Compound 2 in methanol-d3 was
with MeOH, the membrane was dried completely. The biotinylated triph-
photoirradiated with black light to afford 5. The photolyzed
enylphosphine derivatives27) in PBS were blotted on the dried modified
membrane and heated to 50 °C for 8 h. The membrane was well washed with
mixture was subjected to the Staudinger–Bertozzi ligation
with compound 3 to afford ligated compound 6. The method
was then examined at the pmol scale, in which many pho-
toaffinity labeling experiments are usually performed. Com-
pound 2 was coated on the surface of a polyvinilidene difluo-
ride (PVDF) membrane followed by photoirradiation6) with
black light to introduce a crosslink (Fig. 3A). The membrane
was then subjected to the post biotinylation reaction with bi-
otinylated triphenylphosphine derivative 7 and the ligated bi-
otin was detected with a Streptavidin–horseradish peroxidase
(HRP) conjugate.5) The 15 pmol amount of reagent 7 was
still effective for visualization of cross-linked product on the
matrix (Fig. 3B).
MeOH, Tween-phosphate buffered saline (T-PBS) and treated with 10%-
skimmed milk/T-PBS for 1 h. After washing with T-PBS three times, the
membrane was immersed in 2000 times diluted Streptavidin–HRP with T-
PBS for 1 h, and then washed with T-PBS five times. The treated membrane
was subjected to chemiluminescence detection.
Acknowledgments The authors wish to thank Dr. Noriyuki Nakajima
(Toyama Prefectual University) for the MS spectral measurements. This re-
search was partially supported by a Ministry of Education, Culture, Sports,
Science and Technology Grant-in-Aid for Scientific Research on a Priority
Area, 17035006, and for the Encouragement of Young Scientists, 16710151
(M.H.), and also by Exploratory Research, 14658185, and CLUSTER (Co-
operative Link of Unique Science and Technology for Economy Revitaliza-
tion) (Y.H.).
The results show the potential utility of alkyl azide carry-
ing TPD derivatives in the application of photoaffinity bi-
otinylation, which could be useful for the ligands with tight
structural requirements.
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All H-NMR spectra were measured using JEOL JNM-FX270 and ECA-
500 spectrometers. MS spectra were obtained using JEOL JNM-LA400
spectrometers. IR spectra were obtained using JASCO FT-IR 420. All sol-
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Vol. 53, No. 11
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