Synthesis and evaluation of novel photoreactive α-amino acid analog carrying acidic and cleavable functions

Article abstract of DOI:10.1016/j.bmcl.2008.11.013

A novel photoreactive α-amino acid bearing an acidic residue and a cleavable diazirine was developed. To mimic common acidic α-amino acids, the residue was designed to be N-acylsulfonamide that possesses an acidic proton and is able to dissociate under th

Full text of DOI:10.1016/j.bmcl.2008.11.013

Bioorganic & Medicinal Chemistry Letters 19 (2009) 80–82
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of novel photoreactive a-amino acid analog
carrying acidic and cleavable functions
*
Nlandu B. Bongo, Takenori Tomohiro, Yasumaru Hatanaka
Laboratory of Biorecognition Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel photoreactive
mimic common acidic
a
-amino acid bearing an acidic residue and a cleavable diazirine was developed. To
Received 28 August 2008
Revised 30 October 2008
Accepted 5 November 2008
Available online 8 November 2008
a-amino acids, the residue was designed to be N-acylsulfonamide that possesses
an acidic proton and is able to dissociate under the physiological conditions. The inhibition assay of its
biotin-tagged derivative with glutamyl endopeptidase from Staphylococcus aureus V8 strain revealed a
Kiapp value of 162
diation, this derivative specifically photolabeled glutamyl endopeptidase,
glutamic oxalacetic transaminase, and -glutamine synthetase, all the enzymes exhibit high affinity
toward acidic -amino acids. In addition, N-acylsulfonamide group functioned as a cleavable linker in
mild basic solution after a brief N-alkylation. Either the multifunctional nature or the simple structure
l
M, which is slightly higher than the Km value of a common substrate. Upon UV irra-
L-glutamate dehydrogenase,
Keywords:
Acidic a-amino acid
Acylsulfonamide
Diazirine
L
a
of this acidic
a
-amino acid surrogate would be useful as versatile photoreactive building block.
Cleavable
Ó 2008 Elsevier Ltd. All rights reserved.
Photoaffinity labeling
Since photoaffinity labeling is a powerful method for the analy-
sis of biological macromolecules, photoreactive -amino acids
a
have become increasingly important tools for identification and
mapping of peptide–protein interactions as well as protein–pro-
tein interactions.1 Introduction of photophores such as aryl azide,
benzophenone, and phenyldiazirine into peptide or proteins has
been mainly achieved by following two ways: (1) selective chem-
ical modification on the amino acid residues of proteins or pep-
tides, and (2) site-directed incorporation of photoreactive amino
acids by using chemical peptide synthesis or transcriptional sys-
tem in vitro1a,2 and in vivo.1b,3 The former method can easily intro-
duce a photophore by the modification of nucleophiles such as
NH2, CO2H, or SH groups, that often causes the loss of the original
nature of the respective residues. The application of latter method
is currently limited for the introduction of hydrophobic amino acid
analogs.
Herein we report a novel photoreactive and cleavable a-amino
acid analog 3 having an anionic residue, which is designed on the
basis of N-acylsulfonamide chemistry (Fig. 1). The pKa value of gen-
eral N-acylsulfonamide is close to that of general carboxylic acid,
which results in the formation of an anion under the physiological
conditions. In fact, N-acylsulfonamide is well known as a carbox-
ylic acid bioisostere in medicinal chemistry.4 It is one of small link-
ages which exhibits useful features including anionic center
generated by deprotonation, possibility to introduce various func-
tions to the acidic residue via acylation, and cleavability by hydro-
Figure 1. Multifunctional nature of N-acylsulfonamide-containing
analog.
a-amino acid
lysis under mild basic condition upon the N-alkylation.5 After
cross-linking the interacting molecules, the cleavability would be
useful for purification of labeled products as reported before.6
The cleavability is also essential in the application of photoreactive
probes for analyzing protein–protein interactions by label transfer
methods.7
* Corresponding author. Tel.: +81 76 434 7515; fax: +81 76 434 5063.
E-mail address: yasu@pha.u-toyama.ac.jp (Y. Hatanaka).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.013

N. B. Bongo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 80–82
81
The formation of N-acylsulfonamide is usually performed by
condensation of a corresponding carboxylic acid with a sulfon-
amide derivative, or by a reaction of sulfonyl chloride and amide.
Other alternatives have been carried out by thio acid/azide amida-
tion,8 or by reaction of carboxylic acid and p-toluenesulfonyl isocy-
beled V8 protease (lane 2) whereas no labeled products were ob-
served when the sample was not irradiated (lane 1). The average
labeling yield was ca. 5% calculated from the dot-blot analysis
using the chemiluminescence detection method compared with
the biotinylated albumin (Sigma). The similar value was obtained
from density of the band of V8 protease after SDS–PAGE separa-
tion. Lanes 3–5 (panel B) and lanes 7–10 (panel C) show the results
anate.9 We firstly synthesized an
sulfonamide group, which was easily prepared from
a
-amino acid derivative 1 with a
L
-cystine,10
and then coupled with 4-(3-trifluoromethyl-3H-diazirin-3-yl)-
benzoic acid 2 to give the compound 3 in good yield (Scheme 1).
The 3-phenyl-3-trifluoromethyl diazirine derivatives have been
recognized to satisfy most of the chemical criteria required for
photoaffinity labeling. We also developed an efficient modification
on the benzene ring to give a variety of the diazirine derivatives.1e
of label inhibition with N-Boc-L-glutamic acid a-amide 5 as a spe-
cific glutamyl endopeptidase substrate15 as well as an analogous
derivative of compound 3 without a diazirine moiety 6, respec-
tively. Depending on the concentration of the competitors, the
amount of labeled proteins clearly decreased in both cases. The
same inhibition profile was observed when using N-Boc-
L
-aspartic
-amide (data not shown) whereas a neutral derivative, N-
-glutamic acid -benzyl ester -amide 7, did not affect the
A biotin was finally introduced to give the
a
-amino acid-based
acid
Boc-
a
L
photoaffinity probe 411 for the radioisotope-free detection of the
labeled protein by chemiluminescence method based on biotin–
avidin interactions without any purification of the labeled prod-
c
a
photolabeling (panel D).
To confirm the specificity of the probe-protein binding, the biot-
inyl probe 4 was examined in an enzyme assay of V8 protease. The
biological activity of compound 4 as a V8 protease inhibitor was
spectrophotometrically determined by tracing the production of
ucts.12 Besides Boc group for N-protection, other N-protected
a-
amino acid derivatives (Z and Fmoc) were easily obtained in simi-
lar way.
The photoaffinity labeling of glutamyl endopeptidase from
Staphylococcus aureus V8 strain (V8 protease, 29 kDa, E.C.
3.4.21.19) has been performed with the biotin-tagged photoaffinity
probe 4. This enzyme is a well-known serine protease exhibiting
phenol from a specific substrate, N-Boc-
ester as described.15 The obtained Kiapp value (161.7 2.9
was slightly higher than the Km value of this substrate (86 M).
The results of photolabeling and kinetic study described above
obviously showed that the photoaffinity probe 4 was specifically
recognized by V8 protease, and indicate that the acylsulfonamide
could mimic the desired acidic residue. In fact, the probe 4 was
L
-glutamic acid
a
-phenyl
lM)
l
unique specificity to acidic
a-amino acid residues (Glu and
Asp),13 and some acylsulfonamide-containing compounds have
been used as protease inhibitors.4b After incubation of the enzyme
with a stoichiometric amount of compound 4 (125
l
M) in Tris–
confirmed to label other proteins,
(GDH, 50 kDa, EC 1.4.1.2-3),16 glutamic oxalacetic transaminase
(GOT, 65 kDa, EC 2.6.1.1),17 and
-glutamine synthetase (GSyn,
52 kDa, EC 6.3.1.2).18 All these enzymes exhibit specificity toward
acidic -amino acids, and the label incorporation was specifically
L-glutamate dehydrogenase
phosphate buffer (0.2 M, pH 7.8) at 37 °C for 15 min, the sample
solution was irradiated with UV light (360 nm, 15 W Â2) for
40 min on ice and was then subjected to 12% SDS–PAGE after the
treatment with SDS sample buffer at room temperature. The pro-
teins were electrophoretically transferred onto a poly(vinylidene
difluoride) (PVDF) membrane, and the visualization of the blots
on the membrane by chemiluminescence detection was carried
out as previously reported.14 In Figure 2, a single band of protein
was detected around 29 kDa, which corresponds to the photola-
L
a
inhibited in the presence of compound 5 (Fig. 3).
To evaluate another attractive characteristics of the N-acylsulf-
onamide-containing photoprobe, its cleavability was examined by
the application of our previous scissile biotin protocole.6 A dot-blot
analysis (Fig. 4, panel A) shows the cleavability of the labeled V8
Scheme 1. Synthesis of photoreactive acidic a-amino acid analogs. Reagents and conditions: (a) CDI, DBU in THF, 71%; (b) 1 M aq. NaOH–MeOH, 96%; (c) (+)-biotinyl-3,6-
dioxaoctanediamine, HATU, HOAt, DIEA in DMF, 72%.
Figure 2. Chemiluminescence detection of V8 protease photolabeled with the biotinyl probe 4. (A) Lane 1: sample not irradiated, lane 2: sample irradiated. (B) Competitive
inhibition of photolabeling of V8 protease with N-Boc-glutamic acid
a-amide 5, lanes 3–5: irradiated sample in the presence of 0, 10, and 20 equiv of 5, respectively. (C)
Competitive inhibition with compound 6, lanes 6–10: samples irradiated in the presence of 0, 0.5, 5, 50, and 100 equiv of 6, respectively. (D) Competitive inhibition with
compound 7, lanes 11–15: samples irradiated in the presence of 0, 10, 50, 100, and 200 equiv of 7. M: molecular size marker. Arrow indicates labeled V8 protease.

82
N. B. Bongo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 80–82
recognized as a ligand for V8 protease, and has successfully labeled
other proteins possessing specificity toward acidic -amino acid
a
residues. The advantage of N-acylsulfonamide group also demon-
strated the expected cleavability after a brief N-alkylation in mild
basic solution. The cleavability should facilitate the fishing-out of
biotin-tagged proteins as well as peptides from avidin matrices.6
Either the simple synthesis or the multifunctional nature of this
acidic a-amino acid surrogate would be useful as a versatile photo-
reactive building block.
Figure 3. Chemiluminescence detection of proteins photolabeled with the biotinyl
probe 4. The samples containing the enzyme and 4 (1 nmol) were incubated in a
0.1 M potassium phosphate buffer (pH 7.4) with or without the compound 5, and
were subjected to 10% SDS–PAGE. (A) GDH; lanes 1–3: samples irradiated in the
presence of 0, 20, and 200 equiv of 5, respectively; (B) GOT; lanes 4–6: samples
irradiated in the presence of 0, 50, and 100 equiv of 5, respectively; (C) GSyn; lane
7–9: samples irradiated in the presence of 0, 20, and 200 equiv of 5, respectively. M:
molecular size marker. Arrows indicate labeled GDH, GOT, and GSyn, respectively.
Acknowledgments
This research was financially supported by the Ministry of Edu-
cation, Culture, Sports, Science and Technology through Special
Coordination Funds for Promoting Science and Technology, and
also Grants-in-Aid for Scientific Research (18390036 and
20390032). We thank Dr. J.-j. Park for his valuable advices on
cleavage protocol.
References and notes
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Figure 4. (A) Dot-blot analysis of the cleavage of the biotin-tagged photoaffinity
probe 4: (a) photolabeled sample without any treatment, (b) photolabeled sample
after treatment with ICH2COONa (0.4 M) in a borate buffer (0.1 M, pH 9) for 10 min
at room temperature following incubation with NH4OH (0.6 M) for 60 min at room
temperature. (B) Chemiluminescence detection of the photolabeled samples. (C)
Silver staining of the samples in (B): lanes 1, 3: photolabeled sample before
cleavage; lanes 2, 4: photolabeled sample after treatment with ICH2COONa and
NH4OH. M: molecular size marker. Arrows indicate labeled V8 protease (B) and
non-labeled one (C), respectively.
4. (a) Yan, S.; Appleby, T.; Larson, G.; Wu, J. Z.; Hamatake, R. K.; Hong, Z.; Yao, N.
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protease within 30 min at pH 9. Dot (b) is the photolabeled product
after the N-alkylation using sodium iodoacetate following treat-
ment with ammonium hydroxide solution for 60 min at room tem-
perature. The significant decrease of signal indicates the loss of the
biotinyl amino acid moiety from the labeled protein. One of impor-
tant point is to define the undesired effects of alkylation on pro-
teins. The use of iodoacetic acid sodium salt has the advantage to
share the well-established S-carboxymethylation conditions19 for
minimizing possible damages on proteins during N-alkylation of
acylsulfonamide group. To confirm this observation, the labeled
samples ‘before’ and ‘after’ the cleavage were subjected to SDS–
PAGE (lanes 1, 3 and lanes 2, 4, respectively) and chemilumines-
cence detection (panel B) was performed as well as the silver stain-
ing of the gel (panel C). The densitometry of the emission band due
to the labeled V8 protease revealed that 10-min alkylation with
iodoacetic acid sodium salt was enough for the completion of the
cleavage. The silver staining of the gel indicates that no aggrega-
tion or degradation of the protein occurred during the cleavage
process. The cleavability of acylsulfonamide was already revealed
within proteins captured on avidin-matrix.6
11. Compound 4 as a white solid. 1H NMR (400 MHz, CD3OD, TMS): d 7.90 (2H, d,
J = 7.2 Hz), 7.33 (2H, d, J = 8 Hz), 4.69 (1H, t, J = 7.4 Hz), 4.48 (1H, dd, J = 4.2,
7.8 Hz), 4.28 (1H, dd, J = 4.6, 7.8 Hz), 4.09 (1H, dd, J = 7.2, 14.4 Hz), 3.50–3.76
(13H, m), 3.15–3.25 (1H, m), 2.91 (1H, dd, J = 5.2, 10.2 Hz), 2.69 (1H, d,
J = 12.8 Hz), 2.18 (2H, t, J = 7.4 Hz), 1.50–1.80 (4H, m), 1.43 (9H, s), 1.21–
1.30 ppm (2H, m); 13C NMR (400 MHz, CD3OD, TMS): d 176.0, 168.8, 165.9,
150.7, 137.0, 133.0, 129.5, 129.0, 127.6, 121.7, 80.9, 79.4, 71.3, 70.5, 63.3, 61.6,
56.9, 56.6, 54.8, 41.0, 40.3, 36.7, 30.7, 29.7, 29.4, 28.6, 26.8, 20.8, 14.4 ppm.
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In summary, a diazirinyl photophore was easily introduced into
the sulfonamide side chain of
a-amino acid to yield the acidic and
cleavable photoprobes. The biotinyl probe has been specifically
19. Hirs, C. H. W. Methods Enzymol. 1967, 11, 199.