Synthesis of Hetero-bifunctional Azobenzene Glycoconjugates for Bioorthogonal Cross-Linking of Proteins

Article abstract of DOI:10.1002/ejoc.201600136

Modification of proteins with azobenzene derivatives allows their form and function to be controlled by photochemical E/Z isomerization of the azobenzene N=N bond. Because azobenzene glycoconjugates (ABGs) are particularly promising cross-linkers for the

Full text of DOI:10.1002/ejoc.201600136

DOI: 10.1002/ejoc.201600136
Communication
Bioorthogonal Cross-Linkers
Synthesis of Hetero-bifunctional Azobenzene Glycoconjugates
for Bioorthogonal Cross-Linking of Proteins
Anne Müller[a] and Thisbe K. Lindhorst*[a]
Abstract: Modification of proteins with azobenzene derivatives ABGs with the synthesis of hitherto unknown hetero-bifunc-
allows their form and function to be controlled by photochemi-
cal E/Z isomerization of the azobenzene N=N bond. Because
azobenzene glycoconjugates (ABGs) are particularly promising
cross-linkers for the modification of peptides and proteins, we
advanced the collection of so far known homo-bifunctional
tionalized ABGs. Alkyne and alkene, alkyne and sulfhydryl, and
azido and alkene functions were combined in one molecule
to serve as bioorthogonal reaction pairs. With this, access to a
multitude of photosensitive proteins is provided.
Introduction
dichloroacetamido-functionalized and di-O-allylated azobenz-
ene glycoconjugates.[3] However, the application of such homo-
difunctionalized cross-linkers has limitations. Especially, the pos-
sibility of controlled consecutive cross-linking is excluded. Also,
the orthogonal functionalization of peptides, modified with un-
natural amino acids, cannot be fully addressed. Therefore, it has
become our goal to advance the existing collection of homo-
bifunctional glycoazobenzene cross-linkers by the synthesis of
orthogonally bifunctionalized examples. On the basis of state-
of-the-art approaches in the field of bioorthogonal ligations,[4]
we focused on sulfhydryl, alkene, alkyne, and azido functional
groups as complementary reaction pairs (Figure 1). Sulfhydryl
groups can ligate with maleimides or alkene functionalities ac-
cording to thiol–ene coupling.[5] The thiol–ene reaction is or-
thogonal to cross-linking between azido and alkyne functional
groups, which can be ligated in a CuI-catalyzed[6] and copper-
free click reaction,[7] respectively. Also, Staudinger–Bertozzi liga-
tion[8] can be envisaged to achieve bioorthogonal peptide con-
jugation with, for example, an azido–alkene hetero-bifunctional
cross-linker.
Currently there is tremendous interest in the development of
functional molecules for the specific modification of peptides
and proteins. Important fields of application are related to the
labeling of proteins in biological environments, identification
of peptides in complex mixtures, and changing and control of
structure and function of proteins.[1]
Lately, azobenzene derivatives have been employed for intra-
molecular protein modification; this has resulted in photosensi-
tive conjugates that can be reversibly manipulated by light.
Hence, irradiation of azobenzene-modified proteins at appropri-
ate wavelengths effects E/Z isomerization of the azobenzene
unit, and concomitantly, the whole protein structure can be
switched between two different states.[2] This approach allows
modification of protein function with spatiotemporal resolution
under minimally invasive conditions.
Ideally, the azobenzene derivatives used for protein modifi-
cation should be water soluble and biocompatible to allow bio-
logical studies in a natural environment. Thus, we recently intro-
duced bifunctional azobenzene glycoconjugates for cysteine-
based cross-linking of peptides.[3] As azobenzene glycosides
have favorable photochromic properties, glycoazobenzene
cross-linkers are promising candidates for photoswitching of
protein function. In addition, carbohydrates offer a number of
advantages in protein modification. They are nontoxic, improve
water solubility, and their derivatization can be easily adjusted
to control distances between functional groups and their orien-
tation. This was exemplified in our work by the synthesis of
[a] Otto Diels Institute of Organic Chemistry, Christiana Albertina University of
Kiel,
Figure 1. Hetero-bifunctional azobenzene derivatives enable the orthogonal
inter- and intramolecular cross-linking of (modified) proteins. For cross-linking
we aimed at thiol–ene coupling between thiols and alkenes or maleimides
(a). This is compatible with a click reaction between azido and alkyne func-
tional groups as well as Staudinger–Bertozzi ligation (b). As variable, carbo-
hydrates were chosen and functionalized appropriately.
Otto-Hahn-Platz 3/4, 24118 Kiel, Germany
E-mail: tklind@oc.uni-kiel.de
www.otto-diels-institut.de/lind/index.html
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201600136.
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Results and Discussion
tional azobenzene glycoconjugate 14 (Scheme 2). Hence,
known azobenzene mannoside 8,[11] which can be obtained
from D-mannose in four steps, was deprotected with potassium
We report on the synthesis of hetero-bifunctional (glyco)azo-
benzene derivatives for bioorthogonal cross-linking of proteins
with unnatural amino acid residues.
carbonate to afford OH-free azobenzene mannoside 9[15] in
79 % yield. This was then subjected to benzylidenation under
catalysis of tetrafluoroboric acid in the presence of benzalde-
hyde dimethyl acetal to provide 4,6-O-benzylidene acetal 10 in
76 % yield. To selectively protect the hydroxy group at C3 of
diol 10, dibutyltin oxide was employed in refluxing methanol
to form a stannylidene acetal intermediate. After exchanging
the solvent to dimethylformamide the stannylidene acetal was
subsequently treated with p-methoxybenzyl chloride
(PMBCl) and tetrabutylammonium iodide to afford exclusively
3-O-PMB-protected ether 11 in 84 % yield over two steps.
Reaction with sodium hydride and propargyl bromide then
gave fully protected azobenzene mannoside 12 in 86 % yield.
Oxidative removal of the PMB group in 12 with 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (DDQ) and subsequent cleavage
of the benzylidene acetal with TFA in dichloromethane fur-
nished desired 2-O-propargylated azobenzene glycoconjugate
14.
Quite clearly, even azobenzene as such can be converted
into many variations of bifunctional derivatives.[9,10] For exam-
ple, p,p′-dihydroxyazobenzene has been used for the synthesis
of symmetrical azobenzene glycosides;[3,10,11] however, it does
not offer entry into hetero-bifunctional azobenzene derivatives.
Thus, we employed propargyloxy-functionalized azobenzene al-
cohol 4 in our earlier work,[11] but here we thought of a more
rational approach to p,p′-hetero-bifunctionalized azobenzene
derivatives (Scheme 1). The synthesis started from commercially
available N-tert-butoxycarbonyl (Boc)-aminophenol 1. A syn-
thetic sequence of propargylation to 2,[12] removal of the Boc
protecting group, and subsequent azo coupling of 3[13] with
phenol furnished azobenzene alcohol 4[11] in 61 % overall yield.
Then, allylation under classical Williamson etherification condi-
tions gave hetero-bifunctionalized azobenzene 5. Propargyl al-
cohol 4 could also be converted into thioacetate 6 with thio-
acetic acid S-(3-bromopropyl) ester under basic conditions to
provide azobenzene derivative 7 after deacetylation with so-
dium methoxide in high yield. Azobenzene derivatives 5 and 7
allow bioorthogonal cross-linking according to click chemis-
try[6,7] on the one hand and to thiol–ene coupling[5] on the
other hand. Cross-linker 7 also enables maleimide–sulfhydryl
coupling, which has been widely used in protein conjugation
and labeling.[4,14]
Carbohydrate chemistry also provides facile access to hetero-
bifunctional azobenzene glycosides that combine an azido
functional group with an orthogonal alkene functionality. For
this, 6-azido-6-deoxyglucosyl donor 18[16] can be utilized, which
is available in five steps from
-glucose.[17] Tosylate 15 was ob-
D
tained after tosylation and acetylation in one pot and under-
goes nucleophilic substitution to yield azide 16 and after
anomeric modification functionalized glucosyl donor 18
(Scheme 3). Then, BF3·OEt2-catalyzed glycosylation of recently
described allylated azobenzene derivative 19[3] was performed
in acetonitrile owing to the limited solubility of 19. This glyc-
osylation reaction led to bifunctional azobenzene glycoside 20
in moderate yield (Scheme 3). Final deprotection gave pure 21
in excellent yield. The azido group of azobenzene glycoside 21
In the next step, we wished to include carbohydrate moieties
in the azobenzene cross-linkers to improve their water solubil-
ity. In addition, if functional groups are installed at various posi-
tions of a sugar ring, the distance between two orthogonal
functional groups can be adjusted. As we recently optimized
access to 2-O-allylmannosyloxy azobenzene derivatives,[3] we
utilized this synthetic pathway to access alkene–alkyne-bifunc-
Scheme 1. Synthesis of hetero-bifunctional allyl–alkyne and alkyne–sulfhydryl azobenzene derivatives 5 and 7, respectively (TFA = trifluoroacetic acid).
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Scheme 2. Synthesis of hetero-bifunctional allyl–alkyne azobenzene glycoconjugate 14 (TBAI = tetrabutylammonium iodide).
Scheme 3. Synthesis of hetero-bifunctional allyl–azido azobenzene glycoconjugate 21 (Tos = para-tolylsulfonyl, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene).
allows copper-catalyzed and strain-promoted alkyne–azide cy- spectroscopy. The photochromic data obtained from respective
cloaddition or Staudinger–Bertzozzi ligation. Consecutive cross- azobenzene derivatives 5, 7, 14, and 21 are collected in Table 1
linking experiments with bifunctional peptides by employing (see also the Supporting Information).
allyl–azido-bifunctional azobenzene glycoside 21 are currently
Table 1. Characterization of the E and Z isomers of azobenzene derivatives 5,
7, 14, and 21.
in progress.
For any photochemical application, the photochromic prop-
Azobenzene
derivative
λmax [nm]
E/Z (PSS)[a]
τ1/2
[h]
erties of hetero-bifunctional azobenzene derivative 5, 7, 14, and
21 have to be known. Ideally, E → Z photoisomerization leads
to photostationary states (PSSs), in which almost all E isomers
have been converted. Photoirradiation was performed at room
temperature in the dark, and the process was monitored by
1H NMR and UV/Vis spectroscopy. The PSSs of the respective
azobenzene derivatives in dimethyl sulfoxide were reached
after approximately 10 min by employing a 365 nm light-emit-
ting diode (LED). The resulting E/Z ratios were determined by
E isomer
Z isomer
5
7
14
21
360
362
362
360
312, 447
312, 448
309, 447
311, 447
3:97
2:98
3:97
3:97
26.0
24.5
37.2
49.9
[a] According to the integration ratio of the aromatic proton signals of the E
and Z isomers in the 1H NMR spectra.
Azobenzene derivatives 5, 7, 14, and 21 showed favorable
integrating the 1H NMR signals of the aromatic protons. The photochromic properties. Photoirradiation of the E isomers led
half-lives τ1/2 of the Z isomers were determined by 1H NMR to almost quantitative isomerization, and all of the Z isomers
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have half-lives ranging from 1 to 2 days. Thus, the independent
investigation of both isomeric states of cross-linked peptides is
feasible.
Keywords: Glycoconjugates · Protein modifications ·
Bioorthogonality · Photochemistry
To estimate the effect of azobenzene derivatives 5, 7, 14, and
21 on the conformational properties of cross-linked peptides or
proteins, the change in their end-to-end distances upon isom-
erization were monitored by using molecular dynamics simula-
tions (see the Supporting Information). The end-to-end dis-
tance distributions are collected in Table 2. The most probable
end-to-end distances of the E isomers varies from about 15 to
about 18 Å. The respective Z isomers show a more complex
distribution with the most probable distances varying from
around 8 to around 15 Å. The separation of the distance distri-
butions between the E and Z states is characteristic for the
individual cross-linkers.
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In conclusion, we synthesized bifunctionalized (glyco)azobenz-
ene derivatives in which alkyne–alkene, alkyne–sulfhydryl, and
alkene–azido reaction pairs were combined for bioorthogonal
cross-linking of peptides and proteins. Hence, we combined
variable ligation chemistries and varied the distances between
the respective functional groups in both isomeric states (E/Z).
All synthesized representatives of these hetero-bifunctional
cross-linker molecules have favorable photochromic properties
and should thus be tested in the photoswitching of the form
and function of peptides and proteins. We hope to inspire cor-
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can now be readily accessed and varied according to the syn-
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Acknowledgments
The authors thank the Deutsche Forschungsgemeinschaft
(DFG), collaborative network SFB 677, for financial support and
Prof. Dr. F. D. Sönnichsen for fruitful discussions.
Received: February 8, 2016
Published Online: March 4, 2016
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