Optimization of the azobenzene scaffold for reductive cleavage by dithionite; development of an azobenzene cleavable linker for proteomic applications

Article abstract of DOI:10.1002/ejoc.201000546

In this paper we conducted an extensive reactivity study to determine the key structural features that favour the dithionite-triggered reductive cleavage of the azo-arene group. Our stepwise investigation allowed identification of a highly reactive azo-arene structure 25 bearing a carboxylic acid, at the ortho position of the electron-poor arene and an ortho-Oalkyl-resorcinol as the electron-rich arene. Based on this 2(2′-alkoxy-4′-hydroxyphenylazo) benzoic acid (HAZA) scaffold, the orthogonally protected difunctional azo-arene cleavable linker 26 was designed and synthesized. Selective linker deprotection and derivatization was performed by introducing an alkyne reactive group and a biotin affinity tag. This optimized azo-arene cleavable linker led to a total cleavage in less than 10 s with only 1 mM dithionite. Similar results were obtained in biological media.

Full text of DOI:10.1002/ejoc.201000546

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201000546
Optimization of the Azobenzene Scaffold for Reductive Cleavage by Dithionite;
Development of an Azobenzene Cleavable Linker for Proteomic Applications
Geoffray Leriche,[a] Ghyslain Budin,[a] Laurent Brino,[b] and Alain Wagner*[a]
Keywords: Affinity purification / Azo compounds / Proteomics / Cleavable linkers / Reduction
In this paper we conducted an extensive reactivity study to
determine the key structural features that favour the dithio-
nite-triggered reductive cleavage of the azo–arene group.
Our stepwise investigation allowed identification of a highly
reactive azo–arene structure 25 bearing a carboxylic acid at
the ortho position of the electron-poor arene and an ortho-O-
alkyl-resorcinol as the electron-rich arene. Based on this 2-
(2Ј-alkoxy-4Ј-hydroxyphenylazo)benzoic acid (HAZA) scaf-
fold, the orthogonally protected difunctional azo–arene
cleavable linker 26 was designed and synthesized. Selective
linker deprotection and derivatization was performed by in-
troducing an alkyne reactive group and a biotin affinity tag.
This optimized azo–arene cleavable linker led to a total
cleavage in less than 10 s with only 1 m
M dithionite. Similar
results were obtained in biological media.
Introduction
Since the discovery of “aniline yellow” in 1861 by
Mene,[1] azo–arenes have become the largest class of syn-
thetic dyes. More recently, these compounds attracted much
attention for the development of molecular switches and
cleavable linkers in biological applications. The specific and
reversible photo-induced (Z)/(E) isomerization of the N=N
motif enabled the control of peptide conformations,[2] re-
mote control of ion channels in excitable cells[3] and the
switch-cell adhesion on surfaces coated with arginine–gly-
cine–aspartic acid (RGD) peptides.[4] Because of its ability
to undergo smooth cleavage upon treatment with dithionite,
a mild and potentially bio-orthogonal reducing agent,[5]
azo–arene compounds are used as cleavable linkers. Azo–
arene reduction found innovative applications in protein
cross linking[6] and very recently in functional proteom-
ics.[7a] In the latter application, the incorporation of an azo–
arene cleavable linker between a protein’s affinity probe and
a purification tag, allows specific elution of captured pro-
teins (Scheme 1).[7] Noteworthy, azo–arene reductions re-
quire multiple washings and high concentrations of the re-
ducing agent, which can involve denaturation of the pro-
teins and presence of strong background signals.
Scheme 1. Cleavage of the azo–arene probe with sodium dithionite.
With the continuous need for improved tools for modern
proteomics, the search of mild cleavable linkers is a very
active field of research. Vinyl sulfide,[8] acylhydrazone,[9] di-
aryl hydrazone[10] or azo–arene[7] based cleavable linkers are
therefore object of recent research. In this paper we report
our study to fine-tune the structure of azo–arenes toward
dithionite reduction. This study led to the design of a new
orthogonally diprotected cleavable linker, which can be deri-
vatized by many tags.
Results and Discussion
Many studies have been published dealing with the sensi-
tivity of azo–arene dyes under reducing biological condi-
tions.[11] However, only scarce data concerning the reactiv-
ity of these compounds toward dithionite were reported.[12]
In order to optimize the sensitivity of the azo–arene motif
toward dithionite reduction, we therefore decided to carry
out an extensive structure/reactivity study. A set of substi-
tuted azo–arene compounds was synthesized by using clas-
sical procedures.[13] Cleavage assays were performed under
standardized condition at room temperature with 47.5 µ
azo–arene and 6 m sodium dithionite in phosphate buffer
[a] Laboratory of Functional Chemo-Systems UMR 7199,
74 Route du Rhin, 67401 Illkirch-Graffenstaden, France
Fax: +33-3-68854306
E-mail: wagner@bioorga.u-strasbg.fr
[b] Laboratory of Structural Biology and Genomics, Institute of
Genetics and Molecular and Cellular Biology, Inserm U964
UMR7104 CNRS UdS,
B. P. 10142, 67404 Illkirch-Graffenstaden, France
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201000546.
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Azobenzene Cleavable Linker for Proteomic Applications
(pH = 7.4). Azo bond cleavage was monitored by UV/Vis Table 2. Half-lives of azo–arenes substituted with different EWGs
at the R3 position (R1, R2, R4, R6, R7 = H; R5 = OH).
spectrophotometry at 376 nm (measurement every second)
R3
Azo–arene
Half-life [s]
and the half-life was calculated for each compound
(Tables 1, 2, 3, 4, and 5). This wavelength was chosen, be-
cause below 376 nm dithionite perturbations were observed
in the UV spectra.[14]
Entry
1
2
3
4
5
COOH
PO(OEt)2
CN
SO3H
NO2
6
7b
8
20
9
12
16
576
9
As a first set, substrates bearing combinations of elec-
tron-withdrawing and electron-donating groups (EWGs
and EDGs) at the para position of each aromatic ring were
assayed (Table 1). Introducing a hydroxy group on one aro-
matic ring (2) resulted in a clear acceleration of the re-
duction compared to unsubstituted compound 1 (Table 1,
Entries 1 and 2). Introducing a carboxylic acid (3) de-
creased the kinetics of cleavage compared to the phenol de-
rivative (Table 1, Entry 3). The reduction kinetics of sym-
metric compounds 4 and 5 were less effective than those of
2, and the best result was obtained with 4-(4Ј-hydroxyphen-
ylazo)benzoic acid (6) (Table 1, Entries 4–6). These obser-
vations confirm the literature results stating that azo–arenes
substituted with both EDGs and EWGs are reduced faster
than their analogues bearing only EDGs.[11a] Due to their
reduction efficiency, azo compounds possessing an electron-
poor arene (A) and an electron-rich arene (B) will therefore
be used for further investigations (Figure 1).
10
tron-rich azo–arene substrate.[15] A carboxylic acid group
was chosen as the most convenient EWG to ease post-func-
tionalizations of the scaffold in order to obtain our difunc-
tional cleavable linker. We next investigated the influence of
carboxylic acid positions on the reduction kinetic (Table 3).
Table 3. Half-lives of azo–arenes substituted with carboxylic acid
in different positions of ring A (R4, R6, R7 = H; R5 = OH).
Entry
R1
R2
R3
Azo–arene
Half-life [s]
1
2
3
H
H
COOH
H
COOH
H
COOH
H
H
6
11
12
20
70
Ͻ1
As anticipated, due to mesomeric effects, substrate 11
with the COOH group in position R2 showed a longer half-
life than the previous compound 6 bearing the acid group
in position R3 (Table 3, Entries 1 and 2). With the acid
group in position R1, a significant decrease in half-life was
observed with a cleavage in less than 1 s (Table 3, Entry 3).
Assuming that the carboxylic acid might be used as a
straightforward conjugation point to introduce a reactive
linker, we decided to study the effect of acid modifications
on the half-life (Table 4).
Table 1. Half-lives of azo–arenes substituted by a combination of
EDGs and EWGs at the 4- and 4Ј-positions.
Entry
R1
R2
Azo–arene
Half-life [s]
Table 4. Half-lives of azo–arenes substituted with carboxylic acid
1
2
3
4
5
6
H
H
COOH
COOH
OH
–H
–OH
–H
–COOH
–OH
–OH
1
2
3
4
5
6
455[a]
39
121
110
185
20
analogues at the R1 position (R2, R3, R4, R6, R7 = H; R5 = OH).
Entry
R1
Azo–arene Half-life [s][a] Half-life [s][b]
1
2
3
COOH
COOMe
CONH(CH2CϵCH)
12
13
14
Ͻ1
Ͻ1
40
Ͻ1
13
193
COOH
[a] Cleavage in a mixture of acetonitrile/phosphate buffer (2:8).
[a] 6 m dithionite. [b] 1 m dithionite.
With a 6 m dithionite solution, ester analogue 13 and
free acid 12 were cleaved in less Ͻ 1 s, whereas amide ana-
logue 14 displayed a longer half-life (Table 4, Entries 1–3).
The dithionite concentration was lowered to 1 m in order
to discriminate compounds 12 and 13. Under these condi-
tions, the free acid proved to undergo a significantly faster
reduction. It thus appeared that for linker development pur-
poses the carboxylic acid had to remain free. The derivati-
Figure 1. General structure of azo–arene compounds synthesized
for the structure/reactivity study.
The influence of the structure of ring A was first evalu- zation point, enabling conjugation with the molecular
ated. In the following set of experiments, the phenol group probe or the affinity tag, will be introduced at the R3 posi-
was fixed in position R5 and different EWGs in position R3 tion of ring A through C–C linkage.
were introduced (Table 2).
We then investigated the effect of the substitution pattern
For this series, comparable half-lives were observed of electron-rich arene ring B to further improve the cleavage
(Table 2, Entries 1–4) except for the nitro compound 10, efficiency. In order to enable accurate half-life measure-
which cleaved in more than 500 s (Table 2, Entry 5). This ments, kinetics were slowed down by using unsubstituted
surprising result could be ascribed to a possible competitive ring A. Several azo–arenes with different substituents on
reduction of the nitro group, giving an unfavorable elec- ring B were synthesized and assayed (Table 5).
Eur. J. Org. Chem. 2010, 4360–4364
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4361

G. Leriche, G. Budin, L. Brino, A. Wagner
SHORT COMMUNICATION
Table 5. Half-lives of azo–arenes substituted on ring B by different
EDGs (R1, R2, R3 = H).
Entry
R4
R5
R6
R7
Azo–arene Half-life [s]
1
2
3
4
5
6
7
8
9
H
H
H
H
H
OH
OH
OMe
OH
OMe
OMe
NH2
NEt2
NHAc
OH
OMe
H
OH
OH
OMe
OH
OH
H
H
H
H
H
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
15
16
17
2
18
19
20
21
22
23
24
94
890
1839
39
2694
46
121
4
1540
3354
7
Figure 2. Structure of optimal compound 25 for azo–arene re-
duction by sodium dithionite and deduced orthogonally protected
azo–arene cleavable linker 26.
10
11
OH
OMe
allow the subsequent introduction of a bioorthogonal
Of all R5-monosubstituted arenes, 4-(hydroxyphenylazo)- chemical hook (e.g. alkyne, phosphane, azide, ...) at one end
benzene (2) remained the most efficient (Table 5, Entry 4). and the introduction of different tags such as biotin or a
In fact, aniline 15, N-alkylaniline 16 and N-acylaniline 17 fluorescent dye at the other end. In addition, the carboxylic
were found to undergo much slower cleavages (Table 5, En- acid will be kept protected to avoid side-reaction and purifi-
tries 1–3). The reduction of the O-methylated analogue 18 cation problems. On the phenol side, three units of an ethyl-
showed a dramatic decrease of the cleavage kinetics com- ene glycol spacer will be introduced to increase the water
pared to those of 2 (Table 5, Entry 5). The hydroxy position solubility (Figure 2).
was then investigated, and we observed that a hydroxy
A convergent approach was used to synthesize protected
group in position R4 gives a similar result than a hydroxy azo–arene 26 by a diazonium coupling reaction between
group in position R5 (Table 5, Entries 4 and 6). A series of aniline 28 and resorcinol 30 as the key-step (Figure 3). On
disubstituted phenol substrates were then assayed (Table 5, the one hand, the amine derivative 28 was obtained from 2-
Entries 7–9). According to the literature, the results ob- amino-5-iodobenzoic acid. After amine and carboxylic acid
tained with substrates 21 and 22 were quite unexpected protection, N-Fmoc-propargylamine was introduced into
(Table 5, Entries 8 and 9).[7a] A hydroxy group in position the protected iodide derivative by Sonogashira–Hagihara
R4 appears to be not essential for an efficient azo–arene coupling reaction. The corresponding aniline 28 was ob-
reduction. We found that the combination with a methoxy tained by deprotection and reduction by hydrogen (4 steps,
group in position R4 and a hydroxy group in position R5 17% overall yield). On the other hand, a triethylene glycol
(21) was more reactive than the reverse combination (22). spacer terminated by the Boc-protected amine 29, was ob-
A similar trend was observed in the phloroglucinol series tained in 5 steps with 53% overall yield.[13] Briefly, phenol
(Table 5, Entries 10 and 11). This set of experiments clearly derivative 30 was easily prepared by alkylation of resorcinol
showed that a hydroxy group in position R5 and an alkoxy with the tosylated spacer 29. Diazotation of aniline 28 and
group in position R4 is by far the most suitable substitution reaction with phenol 30 gave the orthogonally protected
pattern of ring B. This scaffold offers a significant improve- HAZA 26 with 56% of yield. The desired ortho-alkylated
ment of the kinetic rate compared with that of a hydroxy compound was confirmed by NOESY NMR spectroscopy.
group in position R4 already described.[7a–7d] Furthermore,
the alkoxy group could be used as a straightforward conju- been chosen to exemplify the chemoselective deprotection
gation point. and derivatization sequence of HAZA 26. Piperidine treat-
Alkyne as reactive group and biotin as affinity tag have
With all these prerequisites in hand, the optimal com- ment and further coupling with N-succinimidyl-5-hexyno-
pound 25 bearing an ortho-carboxylic acid at the electron- ate gave HAZA 31 with 78% yield. Under acidic conditions,
poor arene ring A and an ortho-O-alkylresorcinol as the the Boc group was removed, and the linker derivatization
electron-rich arene ring B was synthesized (Figure 2). As ex- was achieved by coupling with biotin in 78% yield. Final
pected, this optimized compound showed a half-life Ͻ 1 s hydrolysis of the methyl ester provided the water-soluble
and a total cleavage time of Ͻ 5 s upon treatment with functionalized HAZA 32 in good yield.
6 m sodium dithionite. A decrease of the reducing agent
concentration to 1 m gave the same result and a total monitored by UV spectroscopy at 463 nm, which showed a
cleavage in Ͻ 15 s. half-life Ͻ 1 s and a total cleavage time of Ͻ 10 s with 1 m
The reduction of the final alkyne–HAZA–biotin 32 was
Starting from this scaffold, 2-(2Ј-alkoxy-4Ј-hydroxyphen- dithionite (Figure 4). Under these conditions, no side prod-
ylazo)benzoic acid (HAZA) 26 was designed with one or- ucts were observed and the total cleavage was confirmed by
thogonal protecting group on each side of the cleavable mass spectrometry.[13] The reaction of 32 with 1 m dithio-
bond. tert-Butoxycarbonyl (Boc) and (fluorenylmethoxy)- nite, in the presence of E coli cell lysate, lead to a total
carbonyl (Fmoc) groups were chosen because of their versa- cleavage time of Ͻ 10 s (Figure 4). This result confirmed
tile well-established chemistry. Selective deprotection will that dithionite is a potent bioorthogonal reagent and dem-
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Eur. J. Org. Chem. 2010, 4360–4364

Azobenzene Cleavable Linker for Proteomic Applications
Figure 3. Reaction pathway for the formation of alkyne–HAZA–biotin 32: (a) benzyl chloroformate, 0.1 mol-% β-CD, H2O, room temp.,
16 h, 71%; (b) dimethyl sulfate, K2CO3, acetone, 40 °C, microwaves, 20 min, 63%; (c) N-Fmoc-propargylamine, PdCl2(PPh3)2, CuI, TEA,
DMF, room temp., 16 h, 60%; (d) H2, Pd/C, DMF/EtOAc, room temp., 16 h, 65%; (e) K2CO3, DMF, microwaves, 120 °C, 20 min, 57%;
(f) NaNO2, HCl, H2O/acetone (1:1), 0 °C, 45 min, then Na2CO3, NaOH, H2O/acetone (1:1), 0 °C to room temp., 10 min, 54%; (g)
piperidine, DCM, room temp., 3 h; (h) 5-hexynoic acid–NHS, TEA, DMF, room temp., 16 h, 78% (for two steps); (i) TFA, DCM, room
temp., 16 h; (j) biotin, HBTU, TEA, DMF, room temp., 16 h 78% (for two steps); (k) LiOH, MeOH/H2O (4:1), 40 °C, 16 h, 61%.
onstrates the potency of alkyne–HAZA–biotin 32 as a tions of linkers,[7] alkyne–HAZA–biotin 32 can be reduced
highly efficient cleavable linker for future proteomic in Ͻ 10 s with 1 m reducing agent, and the reactivity was
studies.
500-fold increased with respect to our reference.[13] Further-
more, the presence of a carboxylic acid group and the tri-
ethylene glycol spacer provided a very good water solubility.
The application of the HAZA linker 26 for protein complex
enrichment is currently under investigation and will be re-
ported in due course.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental procedures and spectroscopic data for all com-
pounds.
[1] H. Zollinger, Color chemistry: syntheses, properties, and applica-
tions of organic dyes and Pigments, 3rd ed., Wiley-VCH,
Weinheim, 2003, p. 166.
[2] Z. Zhang, D. C. Burns, J. R. Kumita, O. S. Smart, G. A. Wool-
ley, Bioconjugate Chem. 2003, 14, 824.
[3] R. H. Kramer, J. J. Chambers, D. Trauner, Nat. Chem. Biol.
2005, 1, 360.
[4] J. Auernheimer, C. Dahmen, U. Hersel, A. Bausch, H. Kessler,
J. Am. Chem. Soc. 2005, 127, 16107.
Figure 4. Reduction kinetics of alkyne–HAZA–biotin 32 with 1 m
sodium dithionite in two different medias: phosphate buffer (solid
line) and cell lysate (dashed line).
[5] J. M. Hooker, E. W. Kovacs, M. B. Francis, J. Am. Chem. Soc.
2004, 126, 3718.
[6] a) H. Fasold, J. Klappenberger, C. Meyer, H. Remold, Angew.
Chem. Int. Ed. Engl. 1971, 10, 795; b) C. L. Jaffe, H. Lis, N.
Sharon, Biochemistry 1980, 19, 4423; c) J. B. Denny, G. Blobel,
Proc. Natl. Acad. Sci. USA 1984, 81, 5286.
[7] a) M. Bogyo, V. H. L. Stephen, F. Marko, US patent 60/
825,548, 2006; b) S. H. L. Verhelst, M. Fonovic´, M. Bogyo, An-
gew. Chem. 2007, 119, 1306; Angew. Chem. Int. Ed. 2007, 46,
1284; c) M. Fonovic´, S. H. L. Verhelst, M. T. Sorum, M.
Bogyo, Mol. Cell Proteomics 2007, 6, 1761; d) Y.-Y. Yang, J. M.
Ascano, H. C. Hang, J. Am. Chem. Soc. 2010, 132, 3640; e) F.
Conclusions
Structural optimization of the azobenzene scaffold en-
abled the development of an efficient HAZA linker. This
difunctional orthogonally protected linker can be conve-
niently conjugated with various molecular tags and affinity
probes. Most interestingly, instead of using elution with
100 m dithionite or several elutions with 25 m dithionite
to reduce N=N bond as it was needed with previous genera-
Eur. J. Org. Chem. 2010, 4360–4364
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4363

G. Leriche, G. Budin, L. Brino, A. Wagner
SHORT COMMUNICATION
Landi, C. M. Johansson, D. J. Campopiano, A. N. Hulme, Org.
Biomol. Chem. 2010, 8, 56.
[8] H.-Y. Shiu, T.-C. Chan, C.-M. Ho, Y. Liu, M.-K. Wong, C.-
M. Che, Chem. Eur. J. 2009, 15, 3839.
[9] K. D. Park, R. Liu, H. Kohn, Chem. Biol. 2009, 16, 763.
[10] a) A. Dirksen, S. Yegneswaran, P. E. Dawson, Angew. Chem.
2010, 122, 2067; Angew. Chem. Int. Ed. 2010, 49, 2023.
[11] a) S. Zbaida, A. M. Stoddart, W. G. Levine, Chem. Biol. Inter-
Chemosphere 1996, 33, 2533; g) A. Stolz, Appl. Microbiol. Bio-
technol. 2001, 56, 69; h) F. P. van der Zee, G. Lettinga, J. A.
Field, Chemosphere 2001, 44, 1169.
[12] a) S. Zbaida, W. G. Levine, J. Pharmacol. Exp. Ther. 1992, 260,
554; b) A. H. Gemeay, Dyes Pigm. 2002, 54, 201.
[13] See Supporting Information.
[14] S. Hashimoto, J. Sunamoto, K. Sato, Bull. Chem. Soc. Jpn.
1967, 40, 2860.
act. 1989, 69, 61; b) W. G. Levine, Drug Metab. Rev. 1991, 23, [15] a) C. R. Wasmuth, C. Edwards, R. Hutcherson, J. Phys. Chem.
253; c) K.-T. Chung, S. E. Stevens, C. E. Cerniglia, Crit. Rev.
Microbiol. 1992, 18, 175; d) K.-T. Chung, S. E. Stevens Jr.,
Toxicol. Environ. Chem. 1993, 12, 2121; e) S. Zbaida, Drug Me-
tab. Rev. 1995, 27, 497; f) H. An, Y. Qian, X. Gu, W. Z. Tang,
1964, 68, 423; b) M. Sokolovsky, J. F. Riordan, B. L. Vallee,
Biochem. Biophys. Res. Commun. 1967, 27, 20.
Received: April 20, 2010
Published Online: July 9, 2010
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