Chemical tagging of a drug target using 5-sulfonyl tetrazole

Article abstract of DOI:10.1016/j.bmcl.2013.01.092

Irreversible modification is one of the most promising strategies to identify cellular receptors of bioactive small molecules. Here we report that receptor proteins can be chemically tagged using a 5-sulfonyl tetrazole probe. 5-Sulfonyl tetrazole easily a

Full text of DOI:10.1016/j.bmcl.2013.01.092

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1608–1611
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Bioorganic & Medicinal Chemistry Letters
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Chemical tagging of a drug target using 5-sulfonyl tetrazole
Satsuki Otsuki, Shinichi Nishimura, Hisae Takabatake, Kozue Nakajima, Yasuaki Takasu, Toru Yagura,
⇑
Yuki Sakai, Akira Hattori, Hideaki Kakeya
Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University,
Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Irreversible modification is one of the most promising strategies to identify cellular receptors of bioactive
small molecules. Here we report that receptor proteins can be chemically tagged using a 5-sulfonyl tet-
razole probe. 5-Sulfonyl tetrazole easily accepted nucleophilic attack of thiol groups, while 5-sulfinyl tet-
razole did not. These functional groups were introduced into probe molecules of a natural product.
Cyclosporine A, an immunosuppressant produced by a microbe, was derivatized to possess 5-sulfonyl tet-
razole and a tag group, which enabled chemical tagging of cyclophilin A, the cellular receptor of cyclo-
sporine A. Cyclosporine A derivative possessing 5-sulfinyl tetrazole could not tag cyclophilin A. This
technique will allow efficient identification of cellular receptors of bioactive small molecules.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 10 November 2012
Revised 19 January 2013
Accepted 22 January 2013
Available online 30 January 2013
Keywords:
5-Sulfonyl tetrazole
Chemical tagging
Bioactive small molecule
Receptor protein
Target identification
Small molecules often exhibit potent biological activities and
sometimes they are used as pharmaceuticals. To develop effective
and safer drugs, we have to understand modes of action of cur-
rently-used drugs and newly discovered bioactive small molecules
at a molecular level. Taking advantage of genome-wide genetic
materials and advances in informatics analysis, pathways targeted
by small molecules can often be predicted.1,2 However, identifica-
tion of the receptor molecules to which small molecules directly
bind remains to be challenging, especially when the binding affin-
ity is low or the amount of receptor molecules is not abundant in a
cell.
Irreversible modification is a powerful strategy to detect bind-
ing partners of a molecule of interest. In fact, small molecules nat-
urally possessing reactive functional groups often exhibit potent
biological activities and their cellular receptors have been success-
fully identified.3–5 Reactive groups are usually electrophilic,
accepting nucleophilic attack by amino acids or nucleotides to
form covalent bonds in the active site of the receptors. On the other
hand, technologies based on multifunctional probe molecules con-
taining reactive groups and bioactive small molecules have been
developed, for example, affinity-based protein profiling6 and li-
gand-directed tosyl chemistry.7 Through this technology, specific
tagging of cellular receptors can be achieved in the proteome and
the activity of the receptor proteins can be detected. For these pur-
poses, probe molecules are made to contain reactive groups, for
example, fluorophosphonate, vinylsulfone, sulfonate ester, epox-
ide, and so on.8,9 We can choose the functionality from this reper-
toire. However, the chemical tagging technology is not mature; we
do not fully understand the potential and limitation of this tech-
nology. It is still necessary to explore other functions with distinct
chemical properties for providing opportunities to analyze mode of
action of small molecules and develop new pharmaceuticals. Here,
we demonstrate that 5-sulfonyl tetrazole is an alternative to chem-
ically tag receptor proteins using a bioactive small molecule as a li-
gand (Fig. 1).
5-Sulfonyl tetrazole is known to be reactive toward nucleophilic
attack by an addition-elimination pathway.10–12 First we evaluated
the reactivity of 5-sulfonyl tetrazole toward thiol, one of the most
abundant nucleophiles in biological systems, using three model
compounds 1, 2 and 3 possessing 5-sulfanyl tetrazole, 5-sulfinyl
tetrazole and 5-sulfonyl tetrazole, respectively (Fig. 2a). All these
molecules were designed to possess a tosylate group to compare
the reactivity. When incubated with n-pentanethiol (1 equiv) in
the presence of base, 5-sulfanyl tetrazole in 1 remained intact,
while 5-sulfonyl tetrazole in 3 was completely converted to 5-sul-
fanyl tetrazole (Fig. 2b). A part of 1 was converted to 5, indicating
that tosylate was replaced with n-pentanethiol. In contrast, 3 was
converted to 4, where only 5-sulfonyl tetrazole was attacked by the
thiol. Compound 2 possessing 5-sulfinyl tetrazole exhibited mod-
erate reactivity to produce 4. If only the tosylate group in 1 or 2
was attacked by n-pentanethiol, compounds 6 and 7 would be pro-
duced, respectively. However, we could not detect any peaks other
than the starting compound and the major product in which only
5-sulfonyl/sulfinyl tetrazole was attacked by n-pentanethiol. These
⇑
Corresponding author. Tel.: +81 75 753 4524; fax: +81 75 753 4591.
E-mail address: scseigyo-hisyo@pharm.kyoto-u.ac.jp (H. Kakeya).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.092

S. Otsuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1608–1611
1609
Figure 1. Chemical tagging of a receptor protein using a 5-sulfonyl tetrazole probe. The probe molecule consists of three functions: a ligand for the receptor protein (red), a
tagging function that reacts with nucleophiles in the receptor protein (orange), and a tag group for detection (blue). When the probe molecule is directed to the receptor
protein and the tagging function, that is, 5-sulfonyl tetrazole, is attacked by a nucleophile (green), chemical tagging of the receptor protein will be achieved.
Figure 2. Reactivity of 5-sulfonyl tetrazole. (a) Reaction scheme of model compounds 1, 2 and 3 with pentanethiol. (b) Reaction properties of 1–3. Model compounds were
mixed with pentanethiol in the presence of base, and the reaction mixture was analyzed on ODS-HPLC. Arrowheads with white interior indicate the model compounds 1, 2, or
3. Black arrowheads indicate products. Compounds 4, 5 and 8, synthesized authentic samples, were also analyzed in the same condition.
results indicated that the oxidation state of the sulfur atom was
important for the reactivity and that 5-sulfonyl tetrazole was the
most reactive. It is likely that the reactivity depends on the kind
of electron withdrawing groups substituted at C5 of the tetrazole
ring. In addition, we found that tosyl ester, one of the well-utilized
leaving groups in organic synthesis, was not replaced by alkyl thiol
in the presence of 5-sulfonyl tetrazole when 1 equiv of n-pentane-
thiol was used, indicating the higher reactivity of 5-sulfonyl tetra-
zole. Next, we designed and synthesized probe molecules that
enable chemical tagging of receptor proteins.
structure of a tagging part (step 2); probe synthesis is accom-
plished by conjugation of a ligand and a tag group to the tagging
part (step 3). In this study, the tagging function was 5-sulfonyl tet-
razole. We chose cyclosporine A (CsA) as a ligand and biotin as a
tag. CsA, a peptidic fungal metabolite,14 is an immunosuppressant
drug widely used in organ transplantation to prevent rejection. CsA
binds to cyclophilin A (CyPA) with high affinity, and the heterodi-
mer binds to calcineulin to inhibit its phosphatase activity, leading
to suppression of the expression of inflammatory cytokine
genes.15–17 We expected that a linker chain can be elongated from
Probe molecules for chemical tagging consist of three functions
(Fig. 1); a ligand that recognizes specific receptor proteins, a tag-
ging function that links the probe to the receptor protein, and a
tag group that makes detection or concentration of the receptor
molecules easy. 5-Sulfonyl tetrazole probes were prepared as fol-
lows (Fig. 3); first, simple heating of neat toluenesulfonyl cyanide
with an alkyl azide gave 1-alkyl-5-sulfonyl tetrazole through
[2+3] cycloaddition (step 1);13 second, substitution of the p-tolu-
enesulfonate with a thiol followed by oxidation furnished the core
the side chain of the (4R)-4-[(E)-2-butenyl]-4,N-dimethyl-L-threo-
nine (MeBmt) residue since this side chain seems not to be in-
volved in the binding of CsA to CyPA.18 In fact, fluorescein-
labeled CsA analog in which MeBmt residue was modified, was re-
ported to bind CyPA.19 Conjugation of CsA, 5-sulfonyl tetrazole and
biotin was carried out as shown in Scheme 1. Briefly, tosyl cyanide
9 and alkyl azide 10 was heated to obtain 11. The tosyl group in 11
was exchanged with thiopropionate in the presence of base, fol-
lowed by oxidation to obtain 12. A linker part was condensed with

1610
S. Otsuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1608–1611
Figure 3. Schematic drawing of preparation of 5-sulfonyl tetrazole probes.
N
N
N
N
N
O
N
a
b, c
d-h
N
N
CN
Fmoc
+
S
N3
N
H
S
O
HO
S
O
O
O
O O
9
10
11
12
NH
Fmoc
NH
Fmoc
N
N
N
N
O
N
N
H
N
N
O
O S
Boc
N
O
N
H
N
S
O
H
S
O
O
N
H
H
HN
H
NH
13
i, j
HN
O
NH
O
O
O
HN
H
NH
H
O
O
NH
S
HN
O
N
HO
O
H
N
O
N
N
N
N
O
O
O
O
O
O
N
N
O
H
N
H
N
N
H
N
O
O
14
Scheme 1. Preparation of cyclosporine probe 14. Reagents and conditions: (a) 100 °C, 4 h (quant); (b) HSCH2CH2COOH, K2CO3, CH2Cl2, rt, 10 h (quant); (c) mCPBA, CH2Cl2, 0 °C
to rt, 4 h; (d) BocNH(CH2)4NH2, DIEA, HBTU, DMF, rt, 1 h (45% in two steps); (e) TFA, CH2Cl2, 0 °C to rt, 1 h; (f) BocNH(CH2)3COOH, DIEA, HBTU, DMF, rt, 1 h (67% in two steps);
(g) DBU, DMF, rt, 3 d; (h) biotin, DIEA, HBTU, DMF, rt, 1 h (48% in two steps); (i) TFA, CH2Cl2, 0 °C to rt, 1 h; (j) CsA-COOH,20 DIEA, HBTU, DMF, rt, 1 h (66% in two steps).
Figure 4. Chemical tagging of cyclophilin A using cyclosporine A probe. (a) Chemical structures of CsA probes 14 and 15, and control probes 13 and 16. (b) Binding of CsA
probes to CyPA and tagging of CyPA by biotin. The arrowhead indicates the protein bands specifically detected for the CsA probes. (c and d) Specificity of the tagging. Chemical
tagging was examined in the presence of CsA (c) or NEM (d). Images around the 18 kDa bands are shown. WCL, whole cell lysate.

S. Otsuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1608–1611
1611
the carboxylic acid of 12, while biotin was conjugated to the amino
group, yielding 13. Finally, deprotection and condensation with a
CsA derivative20 furnished CsA-5-sulfonyl tetrazole probe 14. The
sulfanyl tetrazole probe 15 was prepared in the same manner
(Fig. 4a).
is released after 5-sulfonyl tetrazole is attacked by a nucleophile, in
a similar way to that of the tosyl-chemistry probes.24 Enrichment
of chemical tagging methodologies that are complimentary to tra-
ditional genetic tagging methods will be beneficial for functional
analysis of biological macromolecules.25
We first examined if CsA probes 14 and 15 can interact with
CyPA. Probe molecules were incubated with a lysate of Jurkat cells
(human leukemia T cell line), which was mixed with streptavidin
beads to pull down proteins that have affinity to CsA probes, and
proteins captured on the beads were subjected to SDS–PAGE anal-
ysis. Protein bands with 18 kDa were detected when cell lysate was
incubated with CsA probes 14 and 15 (Fig. 4b). In the case of probe
molecules lacking CsA, that is, 13 and 16, no protein signal was de-
tected around 18 kDa. These 18 kDa bands were confirmed to be
CyPA by Western blotting analysis (Fig. 4b), indicating that the pre-
pared CsA-probes bound to CyPA. The binding did not depend on
the oxidation state of the sulfur atom adjacent to the tetrazole ring.
Next we tested if CyPA was tagged by biotin. As we expected,
although the signal was weak, we could repeatedly observe the
biotin signal when incubating with the probe molecule 14
(Fig. 4b).22 As in the case of the model experiment examined in or-
ganic solvent (Fig. 2), 5-sulfonyl tetrazole in 14 was likely attacked
by some nucleophiles in CyPA to form covalent bonds between the
tag group and CyPA (Fig. 1). In contrast, probe molecule 15 could
not tag CyPA. This indicated that the reactivity of 5-sulfanyl tetra-
zole was not enough to be attacked by nucleophiles in proteins
although it is known that nucleophilic substitution can occur in
harder conditions.23 The biotin and CyPA signals were not detected
in the presence of excess amount of CsA, confirming that the biotin
modification occurred on CyPA molecules (Fig. 4c). When cell ly-
sate was treated with N-ethylmaleimide (NEM) before addition
of the CsA probes, the biotin signal of CyPA disappeared while
the affinity between 14 and CyPA was not lost (Fig. 4d). As NEM
preferentially reacts and inactivates thiol groups, Cys residues in
CyPA seem to attack 5-sulfonyl tetrazole in the probe 14. Alterna-
tively, nucleophiles other than thiol group that reacted with NEM
could be tagged by the CsA probe. 5-Sulfonyl tetrazole has been re-
ported to react with various nucleophiles including imidazole and
alcohol in the presence of base.11,12 Currently, however, the modi-
fied residue remains to be identified.
Acknowledgments
This work was supported in part by research Grants from the Ja-
pan Society for the Promotion of Sciences, the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan, the
Ministry of Health Labor and Welfare of Japan, and SRF.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.01.
092.
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In summary, we have demonstrated that chemical tagging of
cellular receptors of a bioactive small molecule can be achieved
using probe molecules containing 5-sulfonyl tetrazole. When 5-
sulfonyl tetrazole was conjugated with CsA and biotin, and it was
incubated with a Jurkat cell lysate, the cellular receptor of CsA,
CyPA, was tagged with biotin. This technology will offer advanta-
ges in the drug target identification process. First, preparation of
the probe molecule is straightforward and can be carried out quan-
titatively.13 Second, 5-sulfonyl tetrazole exhibited high reactivity
toward thiol. Even in the presence of tosyl ester, 5-sulfonyl tetra-
zole preferentially reacted with thiol. Different reactivity will make
opportunities to gain new insights into the interaction between
drugs and receptor molecules. In addition, 5-sulfonyl tetrazole
probes may not kill the protein function. It is likely that the ligand
20. Introduction of
a carboxylic acid to CsA was carried out as reported
previously.19,21
21. Park, S. B.; Meier, G. P. Tetrahedron Lett. 1989, 30, 4215.
22. The efficiency of the tagging by the CsA probe was not high enough to detect
the biotin signal without purification of CsA binding proteins with streptavidin
beads in this case. See the Supplementary material.
23. Koldobskii, G. I.; Hrabalek, A.; Esikov, K. A. Russ. J. Org. Chem. 2004, 40, 447.
24. Hayashi, T.; Hamachi, I. Acc. Chem. Res. 2012, 45, 1460.
25. Wang, H.; Nakata, E.; Hamachi, I. ChemBioChem 2009, 10, 2560.