Building photoswitchable 3,4'-AMPB peptides: Probing chemical ligation methods with reducible azobenzene thioesters

Article abstract of DOI:10.3762/bjoc.8.101

Photoswitchable peptides were synthesized by using cysteine- and auxiliary-based native chemical ligation reactions. For this purpose, the two regioisomeric azobenzene building blocks 3,4'-AMPB thioester 1b and 4,4'-AMPB thioester 2b were employed in the

Full text of DOI:10.3762/bjoc.8.101

Building photoswitchable 3,4'-AMPB peptides:
Probing chemical ligation methods with
reducible azobenzene thioesters
Gehad Zeyat and Karola Rück-Braun*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 890–896.
Institut für Chemie, Technische Universität Berlin, Strasse des
17. Juni 135, 10623 Berlin, Germany
doi:10.3762/bjoc.8.101
Received: 28 February 2012
Accepted: 04 May 2012
Published: 18 June 2012
Email:
Karola Rück-Braun* - karola.rueck-braun@tu-berlin.de
* Corresponding author
This article is part of the Thematic Series "Molecular switches and cages".
Guest Editor: D. Trauner
Keywords:
acyl transfer auxiliary; azobenzenes; ligation; molecular switches;
peptides; redox chemistry
© 2012 Zeyat and Rück-Braun; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Photoswitchable peptides were synthesized by using cysteine- and auxiliary-based native chemical ligation reactions. For this
purpose, the two regioisomeric azobenzene building blocks 3,4'-AMPB thioester 1b and 4,4'-AMPB thioester 2b were employed in
the ligation reactions. While 4,4'-AMPB requires the 4,5,6-trimethoxy-2-mercaptobenzyl auxiliary to minimize reduction of the
diazene unit, 3,4'-AMPB can be used in combination with the 4,5,6-trimethoxy-2-mercaptobenzyl auxiliary as well as the Nα-2-
mercaptoethyl auxiliary. Thus, 3,4'-AMPB derivatives/peptides proved to be significantly less prone to reduction by aliphatic and
aromatic thiols than were the 4,4'-AMPB compounds.
Introduction
Optical switches not only offer the advantage to elucidate, but In this respect, an increased and predictable stability of azoben-
also to control biological processes with high spatial and zene building blocks under reducing conditions seems to be a
temporal resolution by using light, either in vitro or in vivo [1]. prerequisite in light of synthetic challenges, but also when
In this context, azobenzenes remain a privileged class of photo- considering the reducing intracellular environment. We recently
switches, and typical applications based on light-triggered re- reported on the synthesis, properties and applications of a series
versible conformational control of cyclic, helical or beta-hairpin of novel azobenzene ω-amino acids, with a preference for meta-
peptides have been intensively reviewed [2,3]. Current research substitution patterns. Our purpose was amongst other things (i)
in this field focuses on advanced methods and designs for the to increase flexibility, and (ii) to suppress resonance effects in
synthesis of complex azopeptides and azoproteins for ambi- order to enhance the stability of the diazene unit [4,5]. Since
tious biophysical studies, and also for intracellular applications. methods based on native chemical ligation (NCL), e.g., NCL
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Beilstein J. Org. Chem. 2012, 8, 890–896.
with Nα-acyl transfer auxiliaries, have gained considerable
interest in the past two decades [6], we reasoned that a compar-
ative ligation study with the Boc-protected azobenzene ω-amino
acids Boc-3,4'-AMPB 1a and Boc-4,4'-AMPB 2a may result in
an in-depth analysis of the redox-stability of these building
blocks under the reducing conditions of thiol-based ligation
methods. Generally, native chemical ligation allows the
coupling of two unprotected peptides in neutral aqueous solu-
tion: a C-terminal thioester peptide and either an N-terminal
cysteine peptide or Nα-auxiliary-capped peptides. However, for
elucidating the complex redox chemistry of the two azoben-
zene building blocks under the reducing conditions of ligation
methods, we solely applied the Boc-protected azobenzene
ω-amino acid thioesters 1b and 2b instead of a C-terminal
thioester peptide. We explored the conventional cysteine-based
NCL with Cys-peptide 3, and also screened the application of
the TFA-cleavable 4,5,6-trimethoxy-2-mercaptobenzyl (Tmb)
and 1-(2,4-dimethoxyphenyl)-2-mercaptoethyl auxiliaries by
using peptides 4 and 5 (Figure 1) [6-8], in order to circumvent
the need for the presence of a cysteine at the ligation site.
The peptide motif in peptides 3, 4 and 5 is based on a specific
binding sequence for PDZ recognition [9]. Numerous scaf-
folding proteins contain multiple PDZ domains for their inter-
action with PDZ-binding motifs at the C-terminus of transmem-
brane channels and receptors or other intracellular signaling
proteins. Class 1 interactions involve a (S/T)-X-(V/I/L)-COOH
sequence motif, and examples for these interactions include the
proteins PSD-95, Fas or NHERF 1 [10]. Fas (APO-1/CD95), for
example, is a cell surface receptor, belonging to the tumor-
necrosis-factor receptor superfamily, which induces apoptosis.
Fas-associated phosphatase-1 (FAP-1) is a Fas binding protein,
which interacts with 12 to 15 of the C-terminal amino acids of
the Fas receptor; however, the necessary and sufficient region
for binding consists of the three C-terminal amino acids (SLV)
Figure 1: Structures of azobenzene thioesters, Nα-ligation auxiliaries
and peptides for the application in ligation studies. AMPB =
(aminomethylphenylazo)benzoic acid.
[11-13]. PDZ-domain-containing proteins are superb examples tional NCL, we used cysteine at the ligation site, and for the
of allosteric systems built up by semirigid domains able to auxiliary mediated ligations, we decided to introduce auxiliary-
interact by means of flexible regions, and therefore they seem to glycine-conjugates at the N-terminus, due to the steric limita-
be ideally suited for biophysical function studies with photo- tions of this type of ligation reaction [6-8]. After ligation, the
switchable ligands. For instance, Fas-associated studies in SKV binding motif in the photoswitchable azopeptides should
certain cells using the tripeptide SLV suggest that this small only be accessible in the dark trans-azo state (Scheme 1),
peptide alone can induce apoptosis [14,15]. Related peptides whereas light switching to the cis-azo state should result in
containing class I C-terminal sequence motifs, e.g., SKV, are unfavorable interactions between the N-terminus, which follows
also derived from viral origins [16]. The latter specific binding behind the azo-switch, and the side chains that are critical for
motif is associated with H5N1 influenza infections. We PDZ recognition (as exemplified in Scheme 1 for the 4,4'-
reasoned that the attachment of a photoswitch next to SXV substituted azobenzene system). However, the design presented
PDZ-binding motifs would be a powerful strategy for exploring herein, in combination with an appropriate ligation method,
the function of these PDZ-binding peptide ligands in vitro and seems also to be applicable to the synthesis and evaluation of
in vivo. For the synthetic ligation studies presented herein we photoswitchable protein analogues, for example receptors with
had to place the ligation junction next to the third amino acid the photoswitch placed in the direct neighborhood of the
serine of the SKV binding motif. In the case of the conven- specific C-terminal binding motif.
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Beilstein J. Org. Chem. 2012, 8, 890–896.
Scheme 1: Structural differences between the trans- and the cis-state of azopeptides with a SKV PDZ binding motif.
Results and Discussion
The azobenzene ω-amino-acid thioesters 1b and 2b were
prepared by following and applying literature procedures
(Supporting Information File 1) [4,17]. The syntheses of the
auxiliary-linked glycine conjugates 7 and 8 were accomplished
by using literature protocols developed by Dawson, Offer and
MacMillan [7,8] (Figure 2).
Figure 2: Structure of the glycine-linked auxiliary conjugates 7 and 8.
The Cys-peptide 3 and the Nα-auxiliary peptides 4 and 5 were
assembled on Wang resin by manual Fmoc-based peptide syn-
thesis using standard amino-acid building blocks (Scheme 2)
[4]. Commercially available preloaded Wang resin was used,
and HBTU was applied as the coupling reagent in the presence
of DIPEA in NMP. Removal of the temporary Fmoc-protecting
group was achieved by using 20% piperidine in NMP. Cleavage
Scheme 2: Solid-phase synthesis of the ligation-mediating peptides
3–5.
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Beilstein J. Org. Chem. 2012, 8, 890–896.
from the resin and removal of the permanent tert-butyl and trityl conducted under normal lighting conditions, and therefore small
protecting groups was carried out with a mixture of trifluoro- amounts of the cis-azobenzene forms of the thioesters 1b/2b
acetic acid, water and triisopropylsilane. The peptides 3 and 4 and of the ligation products 10–15 were detected during HPLC
were purified by preparative RP-HPLC and isolated in 86% and monitoring (Supporting Information File 1). The results of all
56% yield, respectively. Peptide 9 with a protected thiol moiety ligation courses prior to purification by chromatography are
was treated with Hg(OAc)2 in TFA, followed by DTT summarized in Table 1.
according to the literature [8], and the deprotected peptide 5
was purified by preparative RP-HPLC and isolated in 64% In ligation reactions employing the N-terminal Cys-peptide 3,
yield.
nearly complete conversion was determined after five hours, but
stirring was nevertheless continued overnight (Table 1, entries 1
All attempts to ligate azobenzene ω-amino acid thioester 1b in and 4). For peptide 5 containing the 4,5,6-trimethoxy-2-mercap-
phosphate buffer in the presence of TCEP·HCl and guani- tobenzyl auxiliary, complete consumption of the starting ma-
dinium hydrochloride failed, because of the poor solubility of terial was detected after 23–24 h (Table 1, entries 3 and 6). In
the azobenzene thioester in the aqueous buffer as well as in ligation reactions employing the Nα-2-mercaptoethyl auxiliary
buffer/NMP mixtures. Therefore, a ligation protocol of peptide 4 reaction times were doubled for complete turnover
Danishefsky et al. was applied using DMF as the solvent [18]. (Table 1, entries 2 and 5). These results are in accordance with a
The ligations were realized by reaction of the appropriate chemoselective bimolecular thioester exchange prior to the S- to
peptide 3, 4 or 5 with an excess of the azobenzene thioester N-acyl transfer through a five-membered transition state for
(1.9 equiv), and TCEP·HCl (2.75 equiv) in the presence of Cys-peptide 3, a six-membered transition state for 4,5,6-
Na2HPO4 (5.5 equiv, pH 7.3) at room temperature, and the trimethoxy-2-mercaptobenzyl auxiliary peptide 5, and a steri-
reaction courses were monitored by analytical RP-HPLC cally more demanding five-membered transition state for
(Supporting Information File 1). The ligation reactions were racemic Nα-2-mercaptoethyl auxiliary peptide 4 [6].
Table 1: Cysteine- and auxiliary-based ligation courses with 3,4'-AMPB thioester 1b and 4,4'-AMPB thioester 2b.a
No.
Thioester
Peptide
Red-1b/2bb [%]
Product
R1
R2
Ratio of
Yieldd [%]
product/red-productc
1
2
3
4
5
6
1b
1b
1b
2b
2b
2b
3
4
5
3
4
5
–
10
11
12
13
14
15
H
CH2SH
69:31
97:3
62
43
38
44
43
45
9
Aux1
Aux2
H
H
–
H
CH2SH
H
100:0
4
14 (12)e
2
52:48
Aux1
Aux2
18:82 (21:79)e
90:10
H
aReaction conditions: 1.9 equiv of azobenzene thioester 1b or 2b, TCEP·HCl (2.75 equiv), Na2HPO4 (5.5 equiv), DMF, rt. Peptides 3–5 were
employed as the symmetric disulfides and were reduced in situ by TCEP. bConversion of 1b or 2b to the hydrazines Red-1b or Red-2b related to the
unreacted excess of 1b or 2b in the ligation mixture during HPLC-monitoring after the appropriate reaction time; – : Red-1b was not detected. cHPLC-
based ratio determined by HPLC-monitoring after work-up and lyophilization of the crude ligation mixture. dIsolated yield after preparative RP-HPLC.
eReinvestigated ratio given in brackets.
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Beilstein J. Org. Chem. 2012, 8, 890–896.
During the reaction courses employing Cys-peptide 3 and the desired peptide 11 and its hydrazine analogue Red-11 were
4,5,6-trimethoxy-2-mercaptobenzyl auxiliary peptide 5, only detected in a ratio of 97:3 by analytical RP-HPLC (Table 1,
reduction of the 4,4'-AMPB thioester 2b to the corresponding entry 2), and peptide 11 was isolated in 43% yield after prepara-
hydrazine compound Red-2b was observed, in 2–4% yield (Ta- tive RP-HPLC. In light of the reactivity of the Nα-2-mercap-
ble 1). When using the Nα-2-mercaptoethyl auxiliary peptide 4, toethyl auxiliary, reduction of the azobenzene ligation peptides
reduction of both azobenzene thioesters was detected: Forma- derived from peptide 4 can obviously only be avoided by using
tion of 9% Red-1b was observed for the 3,4'-AMPB thioester 3,4'-AMPB thioester 1b, due to the lower reactivity of the
1b, and of 14% Red-2b for 4,4'-AMPB thioester 2b. Obviously, diazene unit in this thioester owing to the meta-substitution
the excess of the azobenzene thioester prohibits reduction of the pattern.
ligation products. Additional experiments showed that the
yields of the ligation products could be improved by dilution of The ligation reaction of 3,4'-AMPB thioester 1b with the 4,5,6-
the reaction solutions with water after complete conversion of trimethoxy-2-mercaptobenzyl auxiliary peptide 5 furnished
the peptide starting materials, followed by extraction of the ligation peptide 12 in 38% yield (Table 1, entry 3). In this case,
aqueous phase with diethylether and ethyl acetate to remove no hydrazine species Red-12 were detected at all (Table 1,
4-acetamidothiophenol (Aatp), derived from the thioesters, as entry 3). In addition, the ligation reaction of 4,5,6-trimethoxy-2-
well as the excess of the azobenzene thioesters 1b and 2b. After mercaptobenzyl auxiliary peptide 5 and 4,4'-AMPB thioester 2b
lyophilization, analysis of the crude products by RP-HPLC indi- was also successfully accomplished, furnishing a ratio of 90:10
cated the formation of varying amounts of reduced ligation for 15/Red-15, and the pure ligation product 15 was isolated in
peptides in five out of six cases. Severe reduction was observed 45% yield (Table 1, entry 6). Thus, the 4,5,6-trimethoxy-2-
in ligation reactions with Cys-peptide 3 and 3,4'-AMPB mercaptobenzyl auxiliary applied in peptide 5 seems to be of
thioester 1b, with a ratio of 69:31 peptide 10/peptide Red-10, as general use for both azobenzene systems 1b and 2b, with a
well as 4,4'-AMPB thioester 2b, with a ratio of 52:48 peptide manageable tendency toward reduction in the case of the 4,4'-
13/peptide Red-13 (Table 1, entries 1 and 4). Moreover, the AMPB thioester 2b.
Nα-2-mercaptoethyl auxiliary peptide 4 in combination with
4,4'-AMPB thioester 2b gave peptide 14/peptide Red14 in an The reduction sensitivity observed for the 4,4'-AMPB-derived
18:82 ratio (Table 1, entry 5). However, reoxidation of the thioester 2b and its ligation peptides, may be rationalized by the
hydrazine ligation peptides Red-10, Red-13 and Red-14 to the following considerations [19,20]: The initial addition of a thiol
azobenzene ligation peptides 10, 13 and 14 was observed after to an electron-poor diazene double bond is a reversible reaction,
purification by preparative RP-HPLC, and is obviously initi- furnishing a zwitterionic adduct intermediate A en route to an
ated by oxygen from the air. Finally, the pure ligated azoben- N-sulfenohydrazodiarene B, with a rather labile N–S bond
zene peptides 10, 13 and 14 were isolated in yields of 62%, (Scheme 3, reaction 1). Aliphatic thiols readily undergo adduct
44% and 43%, respectively. When 3,4'-AMPB thioester 1b and formation towards the N-sulfenohydrazodiarene intermediates B
the Nα-2-mercaptoethyl auxiliary peptide 4 were used, the because of their higher nucleophilicity. Accordingly, we
Scheme 3: Reduction of the diazene unit of 4,4'-AMPB thioesters and peptides during aliphatic thiol-based Nα-auxiliary ligation strategies.
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Beilstein J. Org. Chem. 2012, 8, 890–896.
Scheme 4: Synthesis of the azopeptides 16/17 by final TFA cleavage of the Boc-protecting groups, and of the auxiliaries Aux1 and Aux2 in the liga-
tion peptides 11, 12 and 14.
observe a higher tendency toward reduction when applying the side-reactions upon application of aliphatic as well as aromatic
aliphatic Nα-2-mercaptoethyl auxiliary in comparison to the thiols in Nα-auxiliary ligation strategies using peptides 4 and 5.
aromatic 4,5,6-trimethoxy-2-mercaptobenzyl auxiliary. Further- In stark contrast, the Boc-protected 4,4'-AMPB thioester repro-
more, only 4,4'-substituted azobenzene systems, e.g., 2b and ducibly furnished peptide 15 only in combination with the
peptide 14, furnish resonance-stabilized zwitterionic intermedi- 4,5,6-trimethoxy-2-mercaptobenzyl auxiliary peptide 5 during
ates A. Finally, reduction is completed by a nucleophilic attack the ligation course and work-up, whereas the Nα-2-mercap-
of the second aliphatic thiol molecule at the N-sulfenohydrazo- toethyl auxiliary peptide 4 preferentially gave the hydrazine
diarene B, yielding the symmetric disulfide and the respective ligation peptide Red-14 besides the azobenzene ligation peptide
hydrazine thioester or hydrazine peptide (Scheme 3, reaction 2). 14. Our future efforts directed towards the synthesis of photo-
The reduction of diazene units by thiols has been intensively switchable peptides will focus on water-soluble 3,4'-AMPB
studied in the past for electron-poor azobenzenes or azodi- building blocks being applicable in native chemical ligation
carboxylates, en route to symmetrically and asymmetrically methods in aqueous media.
substituted disulfides [21-23].
Supporting Information
The final cleavage of the Boc-protecting group, the Nα-2-
mercaptoethyl auxiliary and the 4,5,6-trimethoxy-2-mercapto-
Supporting Information File 1
benzyl auxiliary in the 3,4'-AMPB ligation peptides 12 and 14
was successfully achieved with 90% TFA under standard condi-
tions furnishing peptide 16 in 94% and 89% yield, respectively,
after purification by preparative RP-HPLC (Scheme 4). Simi-
larly, the Boc-protected 4,4'-AMPB ligation peptide 14 derived
from the Nα-2-mercaptoethyl auxiliary peptide 4 was depro-
tected and purified yielding peptide 17 in 81% yield.
Experimental procedures, characterization data and copies
of spectra.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-101-S1.pdf]
Acknowledgements
We thank the Technische Universität Berlin and the Deutsche
Forschungsgemeinschaft (DFG-Cluster of Excellence 314) for
financial support.
Conclusion
In conclusion, we have demonstrated the high redox stability of
the Boc-protected 3,4'-AMPB thioester 1b when applying
Nα-auxiliary ligation methods en route to photoswitchable
peptides containing the C-terminal PDZ binding motif SKV.
The positioning of the carbonyl group relative to the diazene
unit in the 3,4'-AMPB-derived thioester 1b and its peptides
predictably decreases the reactivity towards unwanted redox
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