Azobenzene-Based w-amino acids and related building blocks: Synthesis, properties, and application in peptide chemistry

Article abstract of DOI:10.1055/s-0029-1217074

Azobenzenes are widely used as optical triggers for the development of photoresponsive materials. Starting from the literature-known w-amino acids 4,4'-APB 1a and 4,4'-AMPB 2a, metasubstituted analogues were synthesized for the incorporation into peptides

Full text of DOI:10.1055/s-0029-1217074

4256
FEATURE ARTICLE
Azobenzene-Based w-Amino Acids and Related Building Blocks: Synthesis,
Properties, and Application in Peptide Chemistry
A
zobenzene-Base
a
w
-
Amino
r
A
cids in
o
Peptide Che
l
mistrya Rück-Braun,* Stefan Kempa, Beate Priewisch, Anja Richter, Sabine Seedorff, Lukas Wallach
Institut für Chemie, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany
Fax +49(30)31428625; E-mail: karola.rueck-braun@tu-berlin.de
Received 13 July 2009; revised 26 August 2009
With the aim to develop photoswitchable Grb2-SH2 an-
Abstract: Azobenzenes are widely used as optical triggers for the
development of photoresponsive materials. Starting from the litera-
ture-known w-amino acids 4,4¢-APB 1a and 4,4¢-AMPB 2a, meta-
substituted analogues were synthesized for the incorporation into
tagonists for Ras signal transduction, we initiated a long-
term research program directed towards the development
of novel photoswitchable building blocks for the synthesis
peptides and proteins. While 4,4¢-APB 1a requires special chemis- and screening of libraries of cyclic photoswitchable pep-
try for peptide synthesis, an Fmoc/t-Bu protecting group strategy is
applicable for 3,3¢-APB 1b. The versatility of this and other novel
tide ligands with the binding motif pTyr-Val-Asn-Val. In
the past, Ettmayer and co-workers successfully demon-
azobenzene w-amino acids is demonstrated by the preparation of
strated that a b-turn conformation centered around this
cyclic photoswitchable phosphopeptides with the binding motif
binding motif in cyclic peptides is essential for high-affin-
pTyr-Val-Asn-Val. Photochromic properties of the azo subunits
ity binding to the SH2-domain.5 If this binding motif is in-
corporated into a photoswitchable cyclic peptide of
suitable ring size, it should be possible to modulate the b-
turn structural element and thereby the binding event,
through conformational changes arising from the light-
induced switching process within the photoswitchable
and of the corresponding peptides are also presented.
Key words: azo compounds, amino acids, peptides, photoswitch,
photochromism
Introduction
hinge unit.
Our program is founded on the development of novel pho-
toswitchable azobenzene-6,7 and hemithioindigo-based8–11
subunits for application in peptide synthesis. A selection
of azobenzenes from our studies is shown in Scheme 1.
The peptide library design is also based on variations in
the ring size of the cyclic peptides and in the amino acid
sequence surrounding the binding motif.10 The first step
within an iterative selection process towards photoswitch-
Photoswitchable compounds have experienced consider-
able interest for studying complex living systems using
light.1 One application is based on the modulation of the
structure and chemical dynamics of peptides and proteins
by site-specific incorporation of a photoswitchable sub-
unit.2,3 Functionalized photoswitches are also appealing
candidates to broaden the range of scaffolds and templates
for the design of peptidomimetics.4
N
CO2H
H2N
N
CO2H
H2N
N
N
1a (4,4'-APB)
peptide assembly requires silylation,
and amino acid fluorides for acylation
2a (4,4'-AMPB)
Fmoc-based peptide assembly
4,4'-APB-based peptidomimetic structural
elements derived from N-arylation
examples for meta-substitution patterns with increased flexibility
H2N H2N
N
CO2t-Bu
CO2t-Bu
N
N
Br
N
N
N
CO2H
CO2H
CO2H
12
[Cu]
1b (3,3'-APB)
2b (3,3'-AMPB)
CO2R
N
N
H2N
N
N
R2
N
H2N
N
R1
N
CO2H
1c (3,4'-APB)
2c (3,4'-AMPB)
Fmoc-based peptide assembly
Scheme 1 4,4¢-APB-based peptidomimetics and analogues of 4,4¢-APB 1a and 4,4¢-AMPB 2a derived by meta-substitution: enhancing and
suppressing resonance effects
SYNTHESIS 2009, No. 24, pp 4256–4267
x
x.
x
x
.
2
0
0
9
Advanced online publication: 22.10.2009
DOI: 10.1055/s-0029-1217074; Art ID: E24809SS
© Georg Thieme Verlag Stuttgart · New York

FEATURE ARTICLE
Biographical Sketches
Azobenzene-Based w-Amino Acids in Peptide Chemistry
4257
Karola Rück-Braun studied
Chemistry at the University of
Mainz where she received her
Diploma in 1988 and her Ph.D.
in 1992, working under the su-
pervision of Prof. Horst Kunz.
After postdoctoral studies with
Prof. Steven V. Ley at the Uni-
versity of Cambridge (UK) in
1992–1993, she obtained her
Habilitation at the University of
Mainz in 1998, and became a
Professor of Organic Chemistry
at the Technische Universität
Berlin in 2000. Amongst other
awards, she received the ADUC-
Habilitation Award in 1998 and
held a Habilitandenstipendium
of the Deutsche Forschungs-
gemeinschaft (1993–1996). Her
research interests are directed to-
wards the synthesis of heterocy-
cles and photoswitches and in-
clude metal-mediated reactions
and the development of reaction
cascades for the preparation of
functionalized
heterocycles.
Current research topics are the
development of photoswitches
for biological applications and
the design and synthesis of
photoswitch-linker-conjugates
for nanostructured surfaces.
Stefan Kempa was born in Ber-
lin in 1980. He studied Chemis-
try at the Technische Universität
Berlin and obtained his Diploma
in 2006. Currently he is carrying
out his Ph.D. studies in Prof.
Rück-Braun’s group.
Beate Priewisch was born in
Berlin in 1976. She studied
Chemistry at the Technische
Universität Berlin and the Uni-
versidad de Complutense de
Madrid, Spain, and obtained her
Diploma in 2002 and her Ph.D.
in 2006. Her research focused on
the development of photoswitch-
able amino acids for the photo-
modulation of the conformation
and activity of peptides. Since
2007 she is working as a patent
attorney trainee at Polypatent,
Overath.
Anja Richter was born in Berlin
in 1979. She completed a train-
ing as a chemical laboratory as-
sistant at the Schering AG in
2002. Thereafter she studied
Chemistry at the Technische
Universität Berlin and received
her Diploma in 2007 under the
supervision of Prof. Rück-Braun.
As a scholarship holder of the
Fond der Chemischen Industrie,
she is currently working as a
Ph.D. student in the research
group of Prof. Herbert Wald-
mann at the Max-Planck-Institut
für Molekulare Physiologie in
Dortmund, focusing on the syn-
thesis of natural products.
Sabine Seedorff was born in
Berlin in 1978. She studied
Chemistry at the Technische
Universität Berlin and obtained
her Diploma in 2006. Currently
she is carrying out her Ph.D.
studies in Prof. Rück-Braun’s
group in collaboration with the
group of Dr. Peter Schmieder at
the Leibniz-Institut für Moleku-
lare Pharmakologie (FMP) in
Berlin-Buch.
Lukas Wallach was born in
Hydebreck/Cosel, Poland, in
1981. He studied Chemistry at
the Technische Universität
Berlin and obtained his Diploma
in 2008. Currently he is carrying
out his Ph.D. studies in Prof.
Rück-Braun’s group.
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York

4258
K. Rück-Braun et al.
FEATURE ARTICLE
able cyclic phosphopeptides is directed towards the ties of a series of meta-substituted analogues of the w-ami-
choice of suitable photoswitchable subunits, driven by the no acids 4,4¢-APB 1a and 4,4¢-AMPB 2a. However,
evaluation and optimization of synthetic routes. This step during our search for alternative synthetic approaches to-
includes also the analysis of the photochromic properties wards 4,4¢-azobenzene structural motifs, particularly suit-
of the subunits and of small linear peptides incorporating able as ultra fast light-triggers for protein folding, we also
them.6,8,9 The next step is the synthesis of small libraries studied the copper-catalyzed N-arylation of a-amino acids
of cyclic photoswitchable phosphopeptides and their with the bromo-substituted azobenzene precursor system
characterization, for example, by CD spectroscopy.
12 (Scheme 1).
Herein, we describe the synthesis of cyclic peptides Finally, we report on the successful synthesis and charac-
c[-Gly-pTyr-Val-Asn-Val-Pro-Gly-Azo-] with novel azo- terization of photoswitchable phosphopeptides derived
benzene-based w-amino acids (Azo) as backbone constit- from a small library approach towards four peptides incor-
uents.10 The research program is currently being extended porating the w-amino acids 4,4¢-AMPB 2a, 3,3¢-AMPB
by an activity analysis, and will be supplemented by the 2b, 3,4¢-AMPB 2c, and 3,3¢-APB 1b. These studies also
conformational analysis of selected members of the cyclic include detailed discussions of photochromic properties
phosphopeptide library based on NMR spectroscopy.
of differently substituted azobenzenes presented herein.
Our studies towards novel azobenzene-based photoswit-
chable subunits started from the literature-known w-ami-
no acids 4,4¢-APB12,13 1a and 4,4¢-AMPB7,14,15 2a
(Scheme 1). Because of the poor nucleophilicity of the
amino group in the more rigid w-amino acid 4,4¢-APB 1a,
the typical peptide coupling reagents cannot be ap-
plied.12,13 Instead, amide bond formation requires activa-
tion of the amino group by silylation, followed by
acylation using amino acid fluorides. We herein report on
alternatives to overcome these synthetic limitations.
Synthesis and Peptide Assembling: 3,3¢-APB 1b and
Other Analogues
The preparation of 3,3¢-APB 1b, starting from 3-amino-
aniline and 3-nitrosobenzoic acid in basic medium similar
to the literature protocol13 for 4,4¢-APB 1a, gave no con-
version under various reaction conditions; redox process-
es between the reactants were observed instead. To reduce
these side-reactions, the protected precursors 3 and 4 were
employed in an alternative approach (Scheme 3), afford-
ing the amino acid derivative 5 in 53% yield. Simulta-
neous hydrolysis of the amide and the ester was achieved
by treatment of 5 with aqueous 3 N HCl at 80 °C for
48 hours furnishing 3,3¢-APB 1b, quantitatively.
In meta-substituted analogues, resonance effects of the
amino group or the carboxyl group with the diazenyl link-
age cannot operate (Scheme 1).6,11 As a consequence we
expected a higher nucleophilicity of the amino group in
3,3¢-APB 1b. Therefore, a typical Fmoc-protocol should
be applicable. Furthermore, this meta-substitution should
also result in an increased conformational freedom of the
azobenzene unit3,6,16,17 leading to additional configura-
tional isomers in both photoisomeric states. The axes for
the rotation of the differently substituted phenyl rings and
For the application in solid-phase synthesis, compound 1b
was converted into the Fmoc-protected building block 6
with FmocOSu in the presence of NaHCO3 in dioxane–
water in high yield by using a standard protocol.
their dihedral angles b and b¢ for 3,3¢-ABP 1b are shown However, an alternative synthetic route starting from ni-
in Scheme 2. These rotary joints are responsible for a trosonitrobenzenes 7 was also investigated (Scheme 4).
higher flexibility of meta-substituted azobenzenes. For Condensation of nitrosobenzene 7a with 3-aminobenzoic
3,3¢-AMPB 2b, the flexibility is further increased by the acid (8a) afforded the nitro-substituted azobenzene 9a in
methylene group as indicated by the additional axis with 93% yield. Subsequent reduction of compound 9a with
the dihedral angle a. Following these considerations, we sodium sulfide in dioxane–ethanol under reflux gave 3,3¢-
investigated the synthesis and the photochromic proper- APB 1b in 75% yield. By following this two-step synthe-
β
β'
hν
β'
N
H2N
CO2H
β
N
hν, Δ
CO2H
trans-1b
N
N
N
N
cis-1b
H2N
H2N
hν
N
N
α
CO2H
hν, Δ
CO2H
trans-2b
cis-2b
H2N
α
Scheme 2 Flexibility induced by rotary joints of azobenzene hinge units illustrated for the trans- and cis-isomers of 3,3¢-APB 1b and 3,3¢-
AMPB 2b
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York

FEATURE ARTICLE
Azobenzene-Based w-Amino Acids in Peptide Chemistry
4259
HO2C
AcHN
NH2
3
N
N
ON
NH2
1b
CO2Me
4
(S)-H-Val-Ot-Bu
AcOH, r.t., 11 d, 53%
EDC, HOBt, DIPEA, DMF
t-BuO
O
0 °C to r.t., 36 h, 82%
R1HN
O
N
N
HN
N
CO2R2
N
NH2
10
5: R1 = Ac, R2 = Me
1b: R1 = H, R2 = H
3 N HCl, 80 °C, 2 d, quant.
Boc-Gly-OH
TCTU, HOBt, DIPEA, DMF
0 °C to r.t., 26 h, 92%
FmocOSu, NaHCO3
dioxane–H2O, r.t., 36 h, 87%
t-BuO
O
6: R1 = Fmoc, R2 = H
O
HN
Scheme 3 Synthesis of Fmoc-3,3¢-APB 6 for peptide chemistry
N
N
O
tic route, 3,4¢-APB 1c and 4,3¢-APB 1d (Scheme 4) were
also successfully prepared in good yields via the interme-
diate nitro-substituted azobenzenes 9b and 9c.18
HN
11
NHBoc
Scheme 5 Peptide chain assembly of 3,3¢-APB 1b by standard pep-
tide chemistry
R1
published in the literature for aryl halides represent cata-
lytic examples of the long-known Ullmann-coupling
without the need for disadvantageously high tempera-
tures.19,20 Ma and co-workers successfully demonstrated
that these coupling procedures proceed without racemiza-
tion.19
R2
NO
H2N
R4
R3
7a: R1 = NO2, R2 = H
7b: R1 = H, R2 = NO2
8a: R3 = CO2H, R4 = H
8b: R3 = H, R4 = CO2H
1. AcOH, r.t.
Treatment of bromide 12 with 1.5 equivalents of (S)-ala-
nine in the presence of 10 mol% copper(I) iodide and 2
equivalents of K3PO4 in 2-(dimethylamino)ethanol at
80 °C gave the coupling product 13b in 65% isolated yield
(Scheme 6). During storage of this product at –18 °C
amine 14 was observed, presumably formed by decarbox-
(9a: 93%, 9b: 83%, 9c: 81%)
2. Na2S, EtOH–dioxane, 90 °C
(1b: 75%, 1c: 70%, 1d: 76%)a
R1
N
R4
R2
N
R3
1b: R1 = NH2, R2 = R4 = H, R3 = CO2H (3,3'-APB)
1c: R1 = R4 = H, R2 = NH2, R3 = CO2H (3,4'-APB)
1d: R1 = NH2, R2 = R3 = H, R4 = CO2H (4,3'-APB)
N
Br
t-BuO2C
N
12
Scheme 4 Synthesis of APB analogues 1b–d by reduction of nitro
precursors 9
(S)-Val or (S)-Ala or (S)-Pro
CuI (10 mol%), K3PO4
Me2N(CH2)2OH, 80 °C
(13a: 70 h, 67%; 13b: 139 h, 65%;
13c: 136 h, 66%)
To verify that the amino group of 3,3¢-APB 1b can be acy-
lated by standard peptide coupling reagents, the synthesis
of tripeptide 11 was carried out by solution-phase synthe-
sis (Scheme 5). 3,3¢-APB 1b was treated with 1.5 equiva-
lents of (S)-H-Val-Ot-Bu using EDC in the presence of
HOBt and DIPEA in DMF to obtain the dipeptide 10 in
82% yield. Acylation of 10 with Boc-Gly-OH using
TCTU/HOBt/DIPEA furnished tripeptide 11 in 92%
yield. According to these findings, the Fmoc-protected
3,3¢-APB building block 6 was found to be suitable for
solid-phase peptide synthesis using Fmoc-chemistry.
R1
N
N
t-BuO2C
N
R2
HO2C
13a from (S)-Val: R1 = H, R2 = i-Pr
13b from (S)-Ala: R1 = H, R2 = Me
13c from (S)-Pro: R1-R2 = (CH2)3
by-product from (S)-Ala:
N
NH2
For the synthesis of peptides or peptidomimetics contain-
ing a 4,4¢-azobenzene structural motif, we also investigat-
ed the copper(I)-catalyzed coupling procedures of
bromide 127 with a-amino acids (Scheme 6). All methods
t-BuO2C
N
14
Scheme 6 Cu-catalyzed N-arylation towards 4,4¢-APB-based pepti-
domimetic structural elements 13
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York

4260
K. Rück-Braun et al.
FEATURE ARTICLE
ylation furnishing an imine intermediate, prior to hydro-
lysis to the corresponding aldehyde and amine by-
products. Analogously, reactions of compound 12 with
(S)-valine and (S)-proline furnished 13a (67%) and 13c
(66%). Prior to an optimization of the coupling protocol
the photochromic properties of compound 13b were in-
vestigated, which are described below.
H-Pro-Cl-Trt
coupling:
Fmoc-protected amino acid
6, 17a–c (4.00 equiv)
HCTU (4.00 equiv)
DIPEA (8.00 equiv), NMP
removal of the Fmoc group:
40% piperidine–NMP
The synthesis of the two analogues, 3,3¢-AMPB 2b and
3,4¢-AMPB 2c, was carried out following our previously
published procedure (Scheme 7) for the Fmoc-protected
4,4¢-AMPB building block 17a.7 Condensation reactions
of the Fmoc-protected anilines 15a16 and 15b14 with ni-
trosoarene 16 were carried out at room temperature in
DMSO–glacial AcOH furnishing the isomeric Fmoc-pro-
tected w-amino acids 17b,c. The Fmoc-group was re-
moved by treatment with 20% aqueous NaOH–THF to
give the w-amino acids 2b,c in good overall yields.
H2N-Gly-Azo-Gly-pTyr(NMe2)2-Val-Asn(Trt)-Val-Pro-Cl-Trt
AcOH–TFE–CH2Cl2 (1:1:3)
H2N-Gly-Azo-Gly-pTyr(NMe2)2-Val-Asn(Trt)-Val-Pro-OH
18a–d
PyBOP (4.00 equiv)
HOBt (4.00 equiv)
DIPEA (8.00 equiv)
3% NMP in CH2Cl2
R1
CO2H
cyclo(Gly-Azo-Gly-pTyr(NMe2)2-Val-Asn(Trt)-Val-Pro)
R2
NH2
ON
19a–d
90% TFA
15a: R1 = CH2NHFmoc, R2 = H
15b: R1 = H, R2 = CH2NHFmoc
16
Asn
Val
Val
Pro
pTyr
NH
1. AcOH–DMSO (1:1), r.t.
(17b: 62%; 17c: 74%)
2. aq 20% NaOH–THF (1:1), r.t.
(2b: 86%; 2c: 69%)
Gly
Gly
n N
O
O
N
H
R1
N
N
20a–d
R2
N
18,19,20a: n = 1, Azo = 4,4'-AMPB
18,19,20b: n = 1, Azo = 3,3'-AMPB
18,19,20c: n = 0, Azo = 3,3'-APB
18,19,20d: n = 1, Azo = 3,4'-AMPB
CO2H
2b: R1 = CH2NH2, R2 = H (3,3'-AMPB)
2c: R1 = H, R2 = CH2NH2 (3,4'-AMPB)
Scheme 8 Solid-phase synthesis of a small library of cyclic photos-
witchable peptide ligands 20 (details are provided in the Supporting
Information)
Scheme 7 Synthesis of meta-substituted AMPB analogues 2b,c
Synthetic studies towards a small library of peptides con-
taining the pTyr-Val-Asn-Val binding motif was carried
out with the Fmoc-protected building blocks Fmoc-4,4¢-
AMPB 17a,7 Fmoc-3,3¢-AMPB 17b, Fmoc-3,4¢-AMPB
17c, and Fmoc-3,3¢-APB 6. The solid-phase synthesis of
the target phosphopeptides 20 is shown in Scheme 8.
carried out using NMP as a solvent, and all amino acids
following the phosphotyrosine building block were cou-
pled twice. Removal of the temporary Fmoc-protecting
group was achieved using 40% piperidine in NMP. Acidic
cleavage from the resin with a mixture of acetic acid, tri-
fluoroethanol, and dichloromethane afforded the linear
protected peptides 18a–d according to HPLC-ESI-MS
analysis. These side-chain-protected peptides were cy-
clized using a threefold excess of PyBOP and HOBt in the
presence of DIPEA in a solution of 3% NMP in dichlo-
romethane (c = 4·10–4 mol/L). Peptides 19a–c were final-
ly deprotected using 90% trifluoroacetic acid in water.
3,3¢-AMPB-peptide 20b and 3,3¢-APB-peptide 20c were
purified by semi-preparative RP-HPLC. The 4,4¢-AMPB-
peptide 20a could not be isolated in pure form, and we
have been unable to ascertain the reasons for this singular
purification problem. The 3,4¢-AMPB-peptide 19d was
purified after the cyclization procedure by semi-prepara-
tive RP-HPLC and finally deprotected, affording the pure
cyclic phosphopeptide 20d.
All four linear peptide precursors 18 were assembled on
solid support in an automated synthesis. Commercially
available preloaded chlorotrityl resin was used as a solid
support to enable the cleavage of the side-chain protected
peptide and to avoid diketopiperazine formation associat-
ed with proline as the starting amino acid. The Fmoc-
group was used as the temporary protecting group and
HCTU as the coupling reagent in the presence of DIPEA.
The trityl group was employed as a side-chain protecting
group for asparagine, and the dialkylamide protected
building block Fmoc-pTyr(NMe2)2-OH was used to incor-
porate the phosphotyrosine. To avoid the formation of
truncated sequences due to a back-folding of the growing
peptide chain on the solid support, induced by the b-turn
structural element, the assembly of the linear peptides was
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York

FEATURE ARTICLE
Azobenzene-Based w-Amino Acids in Peptide Chemistry
4261
Conformational Analysis by CD Spectroscopy
Table 1 Photochromic Properties of Selected Azobenzene Com-
pounds in DMSO
All three peptides 20b–d were photoresponsive molecules
undergoing cis-to-trans isomerization. However, CD
spectroscopy revealed significant structural differences
between the conformations of the dark-adapted and the ir-
radiated states only for the 3,3¢-APB-peptide 20c and the
3,4¢-AMPB-peptide 20d (Figure 1). This might be due to
a decreased flexibility of their photoswitchable subunits,
and thus a larger impact of the isomerization process onto
the peptide backbone, compared to the 3,3¢-AMPB sub-
unit of peptide 20b. The higher flexibility of the latter
peptide can be explained by three independent rotary
joints in the hinge 3,3¢-AMPB 2b (methylene group and
two meta-functionalized phenyl rings), in comparison to
two rotary joints in the hinges 3,3¢-APB 1b (two meta-
functionalized phenyl rings) and 3,4¢-AMPB 2c (methyl-
ene group and one meta-functionalized phenyl ring). For
peptide 20b the CD spectra at the best resemble the char-
acteristics of a-helix-like spectra, generally assigned to
type I b-turns, (maxima below 200 nm and negative bands
in the 220–235 nm region)21 shifted to longer wave-
lengths, which, however, are not altered upon irradiation
with l = 340 nm (cis-pss; pss = photostationary state) or
l = 405 nm (trans-pss). In the cis-form the 3,3¢-APB-pep-
tide 20c displayed the CD spectrum of a b-sheet-like
spectrum22 with a maximum at 199 nm and a strong min-
imum at 215 nm, whereas for the trans-isomer the CD
spectrum revealed no significant signals of a defined pep-
tide conformation. On the contrary, for both isomeric
forms of the 3,4¢-AMPB-peptide 20d maxima below 200
nm and minima at 219 nm were observed, also indicating
b-sheet-like spectra. Upon photoisomerization from the
trans-form to the cis-form a decrease of [Q]R,219 by 3.5·103
deg·cm2·dmol–1 was detected.
a
Compound
lmax,trans
lmax,cis
liso
eb
t1/2
(nm)
(nm)
(nm)
(h)
3,3¢-APB 1b 325, 426 432
3,4¢-APB 1c 401
4,3¢-APB 1d 333, 410 318, 436 280
265
1.28
42 (49)c
357, 447 360, 476 1.18
1.98
276, 397 1.7
265, 406 1.02
1.7
23.6
5d
319, 444 433
323, 437 427
(246)c,d
92 (205)e
37
peptide 20c
4,4¢-AMPB 2a 346, 453 290, 434 290, 406 1.74
3,4¢-AMPB 2c 326, 446 433
275, 389 1.29 129
peptide 20df 331, 440 295, 432 281, 387 1.59
81 (231)e
a lmax,cis was determined in the pss upon light illumination at 325 nm
(compound 1d), 405 nm (compound 1c), or 340 nm.
b For trans-isomers [104 L mol–1·cm–1].
c t1/2 was determined by 1H NMR spectroscopy (200 or 400 MHz).
d Ac-3,3¢-APB-OMe 5 in CH2Cl2.
e t1/2 was determined in H2O.
f H2O content in DMSO: 6%.
azobenzene-type of azoaromatics according to the classi-
fication of Rau,23 showing two clearly separated absorp-
tion maxima, for the lowest lying n-p* transition centered
around 430 nm and the p-p* transition at 320–350 nm.
The light-induced trans-to-cis isomerization is readily
achieved upon illumination at 325, 340, or 405 nm
(Table 1 and Figure 2) and shows isosbestic points. 4,4¢-
APB 1a with donor/acceptor arrangement shows the char-
acteristics of an aminoazobenzene, rather than the effects
of a pseudo-stilbene-type azo compound.24 For the trans-
isomer, a close energetic proximity of the n-p* and the p-
p*-transitions was observed.24 However, peptides derived
from 4,4¢-APB 1a more or less exhibit the classical fea-
tures of azobenzene-type azo compounds.12 Peptidomi-
metic ligands obtained by N-arylation show the
characteristics of a typical pseudo-stilbene-type azo com-
pound, as exemplified for compound 13b (Scheme 6 and
Figure 2). In dichloromethane, the absorption maximum
is located around 397 nm and is shifted to 473 nm in
DMSO, due to the increased solvent polarity. After light
Photochromic Analysis
The photochromic properties of a series of azobenzenes
presented in this study were investigated in DMSO by
1
means of UV/vis and H NMR spectroscopy. All com-
pounds in Table 1, apart from 3,4¢-APB 1c, belong to the
Figure 1 CD-evaluation of the light-induced conformational changes in the photoswitchable peptides 20b–d
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York

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K. Rück-Braun et al.
FEATURE ARTICLE
induced reversible trans-to-cis isomerization with 4,3¢-APB 1d a close proximity of the two absorption max-
l = 365, 385, or 405 nm for 1–20 minutes in dichlo- ima is concluded. Strikingly, for both compounds a de-
romethane and with l = 480 nm for 3–60 minutes in DM- crease of the intensities of the n-p* transitions upon trans-
SO, a fast relaxation was observed, but the corresponding to-cis isomerization is observed. In comparison with all
half-lives could not be determined with standard equip- other compounds (Table 1) this effect correlates with the
ment. Thus, incorporation of azobenzene hinge units by amino group in meta-position.
N-arylation breaks new ground for light-induced changes
The thermal cis-to-trans isomerization of azobenzene-
of peptide and protein conformations with very short half-
type azo compounds in solution follows first order kinet-
lives of the cis-isomeric states.
ics, and the half-lives t1/2 of the cis-isomers presented in
3,4¢-APB 1c belongs to the aminoazobenzene-type azo Table 1 were determined at 30 °C following the time-
aromatics,23 however, peptides derived by amide bond dependent absorption changes by UV/vis spectroscopy
formation should show the typical photochromic proper- (~1·10–4 M). As expected, the half-life increased in the
ties of azobenzene-type azo compounds, comparable to APB series with the amino group in meta-position
the peptides derived from 4,4¢-APB 1a.12 Furthermore, (Table 1, compounds 1c and 1b). In the AMPB series, a
from the data summarized in Table 1, for 3,3¢-APB 1b and strong increase of the half-life is also observed upon
Figure 2 Comparison of the UV/vis spectra of representative types of azobenzenes in DMSO
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York

FEATURE ARTICLE
Azobenzene-Based w-Amino Acids in Peptide Chemistry
4263
nm) or using ninhydrin stain. Column chromatography was per-
formed on silica gel (ICN Biomedicals GmbH silica; 32–63 mm,
60Å).
placement of the carboxyl group in meta-position (com-
pounds 2a and 2c), and a similar trend is seen in the APB
series (compounds 1d and 1c).
Furthermore, for 3,3¢-APB peptide 20c a half-life of
205 hours was determined in water, and for the 3,3¢-APB
derivative 5 a half-life of 246 hours was observed in
dichloromethane (Table 1). Irradiation of peptide 20c in
DMSO-d6 (5.8·10–2 M) gave a pss containing 84% of the
Methyl 3-[(3-Acetamidophenyl)diazenyl]benzoate (5)
Solid N-(3-aminophenyl)acetamide (3; 9.09 g, 60.6 mmol, 2.00
equiv) was added in one portion to a solution of 4 (5.00 g, 30.3
mmol) in glacial AcOH (300 mL). The mixture was stirred at r.t. for
11 d. H2O (300 mL) was then added, and the precipitate that formed
was separated by filtration using a suction filter, washed with H2O
(300 mL), and dried (desiccator, Sicacide). The solid was dissolved
in EtOAc (300 mL), the mixture was filtered to remove an insoluble
black-brown residue, and the filtrate was concentrated under re-
duced pressure. Recrystallization (CH2Cl2) gave 5 as an orange-
yellow solid; yield: 4.80 g (53%); mp 143 °C; Rf = 0.67 (EtOAc).
1
cis-isomer, as determined by H NMR spectroscopy at
room temperature using the NH of Gly3 as a probe. For
the 3,4¢-AMPB peptide 20d a half-life of 231 hours was
ascertained in water. Upon irradiation a pss containing
93% of the cis-isomer was reached under RP-HPLC con-
ditions. In the dark adapted state, generally only the trans- IR (ATR): 3304, 1724, 1672, 1598, 1547 cm–1.
1H NMR (400 MHz, CDCl3): d = 2.23 (s, 3 H), 3.97 (s, 3 H), 7.41–
isomers of all azobenzenes reported herein were observed.
However, for the peptides 20 also traces of the cis-isomers
have been detected in isolated cases by 1H NMR spectros-
copy.
7.83 (m, 5 H), 8.04–8.16 (m, 3 H), 8.53 (t, J = 1.7 Hz, 1 H).
13C NMR (100.6 MHz, CDCl3): d = 24.7, 52.5, 113.5, 119.8, 122.8,
124.2, 127.2, 129.3, 129.8, 131.3, 131.8, 138.9, 152.5, 153.0, 166.7,
168.7.
MS (190 °C): m/z (%) = 92 (44), 134 (100), 297 (80, [M]+).
HRMS: m/z [M]+ calcd for C16H15N3O3: 297.1113; found:
Summary and Outlook
297.1111.
In conclusion, we have developed practical synthetic
routes for a series of novel meta-substituted azobenzene-
based w-amino acid building blocks for site-specific in-
corporation into peptides. We have demonstrated that
Fmoc-3,3¢-APB 6 is well suited for an Fmoc-based pep-
tide assembly on the solid support. The methylene group
in the AMPB series and the meta-functionalized phenyl
rings in the APB and the AMPB series are effective rotary
joints for introducing flexibility. Using a library approach,
these differences in conformational freedom could suc-
cessfully be demonstrated by CD-evaluation of the light-
induced conformational changes of the peptide backbones
of four cyclic phosphopeptides with the binding motif
pTyr-Val-Asn-Val. Our future efforts will focus on addi-
tional concepts to further improve the properties of
azobenzenes for biological applications, including strate-
gies to increase solubility and redox-stability.
3-[(3-Aminophenyl)diazenyl]benzoic Acid (1b)
Method 1: Compound 5 (2.00 g, 6.72 mmol) was suspended in aq 3
N HCl (250 mL) and warmed to 80 °C. The resulting solution was
stirred at 80 °C for 2 d. The mixture was allowed to attain r.t., and
H2O (200 mL) was added. The precipitate was separated by filtra-
tion using a suction filter, washed with a small amount of cold H2O,
and dried (desiccator, Sicacide). Recrystallization (EtOAc–MeOH)
afforded 1b·HCl as an orange solid; yield: 1.74 g (quant); mp
205 °C (dec.); Rf = 0.46 (CH2Cl2–MeOH, 9:1).
IR (ATR): 3362, 2930, 1701, 691 cm–1.
1H NMR (200 MHz, DMSO-d6): d = 4.70 (br s, 3 H), 7.35 (d,
J = 7.8 Hz, 1 H), 7.56–7.65 (m, 2 H), 7.72–7.80 (m, 2 H), 8.12–8.18
(m, 2 H), 8.37 (s, 1 H).
13C NMR (100.6 MHz, DMSO-d6): d = 113.7, 121.3, 122.4, 124.5,
127.5, 130.1, 130.7, 132.2, 132.3, 137.1, 151.7, 152.4, 166.6.
MS (200 °C): m/z (%) = 65 (58), 92 (100), 121 (24), 241 (53, [M]+).
HRMS: m/z [M]+ calcd for C13H11N3O2: 241.0851; found:
241.0853.
Chemicals, including compounds 3, 8a, and 8b, were obtained from
Acros, Aldrich, Fluka, or Novabiochem and were used as supplied.
Compounds 4, 7a,25 7b,25 12, 16, and 17a were prepared by follow-
ing the literature procedure.7 Solvents were distilled prior to use:
CH2Cl2 (P2O5), EtOAc (K2CO3), pentane (P2O5), MeOH, acetone.
Solvents for chromatography were dried according to standard pro-
cedures and distilled prior to use.26All reactions were carried out un-
der N2. 1H NMR spectra were recorded at 400 MHz and 13C NMR
spectra at 100.6 MHz on a Bruker AM 400. Residual solvent pro-
tons were used as internal standards. All chemical shifts are given
in ppm relative to TMS and coupling constants in Hz. Low- and
high-resolution mass spectra were obtained on a Varian MAT 95S
spectrometer with electron ionization, using an ionization potential
of 70 eV. ESI-MS spectra were obtained on a Bruker Esquire 2000
with an ESI voltage of 4 kV and HRMS-ESI spectra on a Thermo
Fisher Scientific LTQ Orbitrap XL with an ESI voltage of 5 kV. IR
spectra were recorded on a Nicolet Avatar 360 spectrometer. CD
spectra were recorded on a JASCO J-715 spectropolarimeter. Melt-
ing points were measured with a Büchi melting point apparatus ac-
cording to Dr. Tottoli and are uncorrected. TLC was carried out on
silica gel 60 F254 (Merck) and was visualized under UV light (254
Method 2: Compound 9a (50.0 mg, 184 mmol) was suspended in
EtOH–dioxane (1:1; 3 mL). A solution of Na2S (32–38%, 140 mg,
1.79 mmol, 9.7 equiv) in H2O (1.5 mL) was added and the clear red
solution was heated to 90 °C in a pressure vessel for 9.5 h. After
cooling to r.t., EtOH and dioxane were removed under reduced
pressure. Then H2O (10 mL) was added and the solution was adjust-
ed to pH 5.5, using aq 1 N HCl. The precipitate was separated by
filtration using a suction filter, washed with aq 1 N HCl (5 mL) and
H2O (5 mL), and dried (desiccator, Sicacide). By dissolving this
precipitate in EtOAc (50 mL) an insoluble blackish brown solid
formed, which was filtered and discarded. Removal of the solvent
under reduced pressure gave 1b as an orange-yellow solid; yield: 33
mg (75%).
3-[(3-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}phenyl)di-
azenyl]benzoic Acid (6)
Solid NaHCO3 (782 mg, 9.32 mmol, 2.50 equiv) was added to a so-
lution of 3,3¢-APB-OH 1b (900 mg, 3.73 mmol) in dioxane–H2O
(1:1; 60 mL). A solution of N-(9H-fluoren-9-ylmethoxycarbonyl-
oxy)succinimide (1.45 g, 4.29 mmol, 1.15 equiv) in dioxane (12
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York

4264
K. Rück-Braun et al.
FEATURE ARTICLE
3-[(4-Aminophenyl)diazenyl]benzoic Acid (1c)
mL) was added dropwise over a period of 10 min by syringe. The
solution was stirred at r.t. for 36 h. Dioxane was removed under re-
duced pressure, and the precipitate was separated by filtration using
a suction filter, washed with H2O (100 mL) and hexane–EtOAc
(3:1, 100 mL), and dried. Compound 6 was obtained as a bright or-
ange solid; yield: 1.50 g (87%); mp 214–216 °C; Rf = 0.75
(EtOAc).
Compound 9b (600 mg, 2.21 mmol) was dissolved in EtOH–diox-
ane (1:1, 24 mL) to give a clear red solution. A solution of Na2S
(32–38%, 1.64 g, 21.0 mmol, 9.5 equiv) in H2O (12 mL) was added,
turning the solution dark green. After the mixture was heated to re-
flux for 3 h, it was cooled to r.t. and the organic solvents were re-
moved under reduced pressure. Then H2O (20 mL) was added and
the insoluble brown solid was filtered and discarded (pH 14). The
filtrate was adjusted to pH 7, using aq 1 N HCl, and the precipitate
formed was filtered and dried (desiccator, Sicacide). Additional ma-
terial was collected from the mother liquor upon addition of more
aq 1 N HCl (pH 5.5). Then the precipitates were dissolved separate-
ly in EtOAc (250 mL and 100 mL), and an insoluble blackish-
brown solid was filtered and discarded from both fractions. The
EtOAc fractions were combined and concentrated in vacuo to give
1c as an orange-brown solid; yield: 376 mg (70%); mp 207–210 °C
(dec.); Rf = 0.32 (CH2Cl2–MeOH, 9:1).
IR (ATR): 3309, 1700, 1600, 1544, 1222 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 4.34 (t, J = 6.7 Hz, 1 H), 4.54
(d, J = 6.7 Hz, 2 H), 7.32–7.79 (m, 10 H), 7.91 (d, J = 7.4 Hz, 2 H),
8.10–8.15 (m, 3 H), 8.36 (t, J = 1.7 Hz, 1 H), 10.05 (s, 1 H), 13.35
(br s, 1 H).
13C NMR (100.6 MHz, DMSO-d6): d = 46.6, 65.7, 111.0, 118.1,
120.2, 122.3, 125.2, 125.9, 126.8, 127.2, 127.7, 129.3, 129.8, 131.9,
140.2, 140.8, 143.7, 145.2, 151.7, 152.4, 153.5, 167.4.
HRMS-ESI: m/z [M + H]+ calcd for C28H22N3O4: 464.16103; found:
464.15942.
IR (ATR): 3369, 1694, 1625, 1599, 1506 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 6.20 (s, 2 H), 6.67–6.69 (m,
2 H), 7.63 (t, J = 7.79 Hz, 1 H), 7.68–7.71 (m, 2 H), 7.95–7.98 (m,
2 H), 8.24 (t, J = 1.85 Hz, 1 H).
13C NMR (100.6 MHz, DMSO-d6): d = 113.4, 121.5, 125.5, 126.6,
129.6, 129.7, 131.9, 142.7, 152.5, 153.3, 167.0.
MS (130 °C): m/z (%) = 92 (100), 121 (48), 241 (55, [M]+).
HRMS: m/z [M]+ calcd for C13H11N3O2: 241.0851; found:
3-[(3-Nitrophenyl)diazenyl]benzoic Acid (9a)
Compound 7a (4.80 g, 31.6 mmol, 2.00 equiv) was suspended in
glacial AcOH (350 mL) and the suspension was sonified until a
clear green solution was obtained. 3-Aminobenzoic acid (8a; 2.12
g, 15.8 mmol) was added, and the mixture was stirred at r.t. for 12
h. The precipitate was separated by filtration using a suction filter
and washed with H2O (300 mL). Additional material precipitated
from the mother liquor upon addition of H2O. The combined solid
was recrystallized (MeOH) and washed thoroughly with Et2O to af-
ford 9a as an orange solid; yield: 4.00 g (93%); mp 223 °C;
Rf = 0.48 (CH2Cl2–MeOH, 9:1).
241.0847.
4-[(3-Aminophenyl)diazenyl]benzoic Acid (1d)
Compound 9c (600 mg, 2.21 mmol) was suspended in EtOH–diox-
ane (1:1, 24 mL). A solution of Na2S (32–38%, 1.64 g, 21.0 mmol,
9.5 equiv) in H2O (12 mL) was added and the clear red solution was
heated to reflux for 3 h. Cooling to r.t. and removal of the organic
solvents in vacuo caused the formation of a red precipitate. After ad-
dition of H2O (20 mL), the insoluble solids were filtered and dis-
carded (pH 14). The filtrate was adjusted to pH 7, using aq 1 N HCl,
and the precipitate was filtered and dried (desiccator, Sicacide). Ad-
ditional material was collected from the mother liquor upon addi-
tion of more aq 1 N HCl (pH 3.5). Then the precipitates were
dissolved separately in EtOAc (250 mL and 100 mL), and an insol-
uble blackish-brown solid was filtered and discarded from both
fractions. The EtOAc fractions were combined and concentrated in
vacuo to give 1d as an orange-red solid; yield: 407 mg (76%); mp
227–229 °C (dec.); Rf = 0.38 (CH2Cl2–MeOH, 9:1).
IR (ATR): 3081, 1687, 1530, 1352, 759, 681 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 7.54 (t,J = 7.7 Hz, 1 H), 7.85–
7.95 (m, 2 H), 8.11 (td, J = 7.6, 1.3 Hz, 1 H), 8.33–8.47 (m, 3 H),
8.57 (t, J = 2.0 Hz, 1 H).
13C NMR (50.3 MHz, DMSO-d6): d = 115.8, 122.5, 125.8, 127.9,
130.0, 130.1, 131.2, 132.3, 132.7, 148.7, 151.5, 152.0, 166.5.
MS (180 °C): m/z (%) = 65 (100), 76 (70), 121 (94), 149 (22), 271
(26, [M]+).
HRMS: m/z [M]+ calcd for C13H9N3O4: 271.0593; found: 271.0591.
3-[(4-Nitrophenyl)diazenyl]benzoic Acid (9b)
4-Nitrosonitrobenzene (7b; 2.50 g, 16.4 mmol) was suspended in
glacial AcOH (190 mL) and the suspension was sonified until a
clear green solution was obtained. 3-Aminobenzoic acid (8a; 1.13
g, 8.22 mmol) was added, and the mixture was stirred at r.t. for 24
h. The precipitate was separated by filtration using a suction filter
and washed with H2O (100 mL). Additional material precipitated
from the mother liquor upon the addition of H2O, which was like-
wise separated by filtration using a suction filter and washed with
H2O (100 mL). Both fractions were dried (desiccator, Sicacide), and
recrystallized twice (acetone–MeOH, 5:1) to afford 9b as an orange
solid; yield: 1.84 g (83%); mp 242 °C; Rf = 0.43 (CH2Cl2–MeOH,
9:1).
IR (ATR): 3347, 3063, 1681, 1623, 1601 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 5.51 (br s, 2 H), 6.78–6.80 (m,
1 H), 7.10–7.14 (m, 2 H), 7.25 (t, J = 7.83 Hz, 1 H), 7.90 (d,
J = 8.35 Hz, 2 H), 8.12 (d, J = 8.25 Hz, 2 H).
13C NMR (100.6 MHz, DMSO-d6): d = 105.1, 112.9, 117.9, 122.3,
129.6, 130.5, 132.4, 149.7, 152.9, 154.3, 166.6.
MS (140 °C): m/z (%) = 65 (57), 92 (100), 121 (28), 241 (75, [M]+).
HRMS: m/z [M]+ calcd for C13H11N3O2: 241.0851; found:
241.0855.
IR (ATR): 1682, 1523, 1346, 762, 689 cm–1.
Dipeptide 10
1H NMR (400 MHz, DMSO-d6): d = 7.78 (t, J = 7.81 Hz, 1 H), 8.13
(d, J = 8.87 Hz, 2 H), 8.18–8.23 (m, 2 H), 8.44–8.46 (m, 3 H).
EDC (357 mg, 1.87 mmol, 1.50 equiv), HOBt (252 mg, 1.87 mmol,
1.50 equiv), DIPEA (326 mL, 241 mg, 1.24 mmol, 1.00 equiv), and
compound 1b (300 mg, 1.24 mmol) were successively added to a
solution of (S)-valine tert-butyl ester hydrochloride (392 mg, 1.87
mmol, 1.50 equiv) in DMF (25 mL) at 0 °C. Then the mixture was
allowed to attain r.t. and was stirred for 36 h. Afterwards, CH2Cl2
(50 mL) was added, and the organic layer was washed with aq 0.5
N HCl (2 × 50 mL), aq NaHCO3 (50 mL), H2O (50 mL), and brine
(50 mL), dried (MgSO4), and concentrated in vacuo. H2O (50 mL)
13C NMR (100.6 MHz, DMSO-d6): d = 122.6, 123.6, 125.0, 127.8,
130.1, 132.3, 132.9, 148.7, 151.7, 154.8, 166.4.
HRMS: m/z [M]+ calcd for C13H9N3O4: 271.0593; found: 271.0587.
Anal. Calcd for C13H9N3O4: C, 57.57; H, 3.34; N, 15.49. Found: C,
57.40; H, 3.69; N, 15.14.
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York

FEATURE ARTICLE
Azobenzene-Based w-Amino Acids in Peptide Chemistry
4265
was added to the oily residue, and a red precipitate was separated by
decanting the supernatant mother liquor. The precipitate was dis-
solved in MeOH (20 mL) and reprecipitated by the addition of H2O,
twice. Lyophilization gave compound 10 as an orange-red solid;
yield: 402 mg (82%); mp 55 °C; [a]D20 +61.2 (c = 0.49, CHCl3);
Rf = 0.69 (EtOAc–pentane, 3:2).
mL), dried (MgSO4), and concentrated in vacuo. The residue was
purified by column chromatography (silica gel).
(S)-2-(4{[4-(tert-Butoxycarbonyl)phenyl]diazenyl}phenyl-
amino)-3-methylbutanoic Acid (13a)
The crude product was purified by chromatography on silica gel
(CH2Cl2–MeOH, 40:1 to 9:1) to obtain 13a as a red solid; yield: 110
mg (67%); mp 66 °C; Rf = 0.45 (CH2Cl2–MeOH, 9:1).
IR (ATR): 3432, 2932, 1719, 1653, 1602, 1526 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.00–1.06 (m, 6 H), 1.50 (s, 9 H),
2.25–2.32 (m, 1 H), 3.87 (br s, 2 H), 4.68–4.73 (m, 1 H), 6.79–6.83
(m, 2 H), 7.22–7.26 (m, 1 H), 7.31 (t, J = 7.80 Hz, 1 H), 7.36–7.39
(m, 1 H), 7.59 (t, J = 7.78 Hz, 1 H), 7.94–7.96 (m, 1 H), 8.01–8.03
(m, 1 H), 8.29 (d, J = 1.40 Hz, 1 H).
IR (ATR): 3369, 2929, 1710, 1599, 1291, 1136 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.07–1.12 (m, 6 H), 1.61 (s, 9 H),
2.21–2.30 (m, 1 H), 4.01–4.07 (m, 1 H), 6.68 (d, J = 8.3 Hz, 2 H),
7.81 (t, J = 8.3 Hz, 4 H), 8.07 (d, J = 8.3 Hz, 2 H).
13C NMR (100.6 MHz, CDCl3): d = 18.0, 19.1, 28.2, 32.0, 57.9,
82.4, 107.6, 115.4, 118.4, 121.5, 125.6, 129.6, 130.0, 135.5, 147.4,
152.7, 153.6, 166.6, 171.4.
13C NMR (100.6 MHz, CDCl3): d = 18.6, 19.2, 28.2, 31.4, 81.3,
113.0, 122.0, 125.7, 130.4, 132.5, 145.4, 150.2, 155.4, 165.5.
MS (180 °C): m/z (%) = 120 (100), 297 (80), 351 (62), 397 (49,
MS (190 °C): m/z (%) = 57 (24), 92 (48), 224 (100), 295 (24), 396
[M]+).
(15, [M]+).
HRMS: m/z [M]+ calcd for C22H27N3O4: 397.2002; found:
397.2002.
HRMS: m/z [M]+ calcd for C22H28N4O3: 396.2161; found:
396.2162.
(S)-2-(4{[4-(tert-Butoxycarbonyl)phenyl]diazenyl}phenyl-
amino)propanoic Acid (13b)
The crude product was purified by chromatography on silica gel
(EtOAc, then EtOAc–MeOH, 19:1 to 1:1) to obtain 13b as a brown-
red solid; yield: 99.0 mg (65%); mp 63 °C (dec); Rf = 0.59 (EtOAc–
MeOH, 1:1).
Tripeptide 11
TCTU (112 mg, 315 mmol, 2.50 equiv), HOBt (43.0 mg, 315 mmol,
2.50 equiv), DIPEA (55.0 mL, 41.0 mg, 315 mmol, 2.50 equiv), and
peptide 10 (50.0 mg, 1.26 mmol) were successively added to a
stirred solution of Boc-Gly-OH (55.0 mg, 315 mmol, 2.50 equiv) in
DMF (2.50 mL) at 0 °C. Then the mixture was allowed to attain r.t.
and stirred at this temperature for 26 h. Afterwards, CH2Cl2 (10 mL)
was added, and the organic layer was washed with aq 0.5 N HCl
(2 × 10 mL), aq NaHCO3 (10 mL), H2O (50 mL), and brine (50
mL), dried (MgSO4), and concentrated in vacuo. H2O (10 mL) was
added to the oily residue, and a red precipitate was separated by de-
canting the supernatant mother liquor. The precipitate was dis-
solved in MeOH (5 mL) and reprecipitated by the addition of H2O,
twice. Lyophilization gave compound 11 as an amorphous orange-
red foam; yield: 66 mg (92%); Rf = 0.76 (pentane–EtOAc, 2:3).
IR (ATR): 3375, 2977, 1709, 1599, 1394, 1292, 1135, 1116 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.59 (d, J = 7.00 Hz, 3 H), 1.62 (s,
9 H), 4.29 (q, J = 7.0 Hz, 1 H), 5.57 (br s, 1 H), 6.68 (d, J = 8.9 Hz,
2 H), 7.83–7.88 (m, 4 H), 8.08–8.10 (m, 2 H).
13C NMR (100.6 MHz, CDCl3): d = 18.7, 28.2, 51.4, 81.3, 112.9,
122.0, 125.7, 130.3, 132.5, 145.5, 149.4, 155.3, 165.5, 178.6.
MS (170 °C): m/z (%) = 92 (100), 297 (41), 369 (22, [M]+).
HRMS: m/z [M]+ calcd for C20H23N3O4: 369.1689; found:
369.1689.
IR (ATR): 3322, 1715, 1690, 1648 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.00 (d, J = 6.6 Hz, 3 H), 1.02 (d,
J = 6.8 Hz, 3 H), 1.44 (s, 9 H), 1.45 (s, 9 H), 2.25–2.33 (m, 1 H),
4.00–4.07 (m, 2 H), 4.72 (dd, J = 8.7, 4.8 Hz, 1 H), 5.70 (t, J = 5.6
Hz, 1 H), 7.29 (d, J = 8.5 Hz, 1 H), 7.40 (t, J = 8.0 Hz, 1 H), 7.53
(t, J = 7.8 Hz, 1 H), 7.62 (d, J = 7.9 Hz, 1 H), 7.80 (d, J = 7.2 Hz,
1 H), 7.86 (s, 1 H), 7.95 (dt, J = 7.8, 1.6 Hz, 2 H), 8.32 (s, 1 H), 9.04
(br s, 1 H).
13C NMR (100.6 MHz, CDCl3): d = 18.1, 19.1, 28.1, 28.4, 31.9,
51.2, 58.1, 80.5, 82.5, 112.9, 120.3, 122.4, 122.8, 124.9, 129.5,
129.7, 130.0, 135.3, 138.7, 152.2, 152.7, 156.7, 166.9, 168.4, 171.5.
(S)-2-(4{[4-(tert-Butoxycarbonyl)phenyl]diazenyl}phenyl)pyr-
rolidine-2-carboxylic Acid (13c)
The crude product was purified by chromatography on silica gel
(EtOAc, then EtOAc–MeOH, 85:15 to 1:1) to obtain 13c as a red
solid; yield: 108 mg (66%); mp 168 °C; Rf = 0.17 (EtOAc–MeOH,
5:1).
IR (ATR): 3365, 2925, 1709, 1598, 1369 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.62 (s, 9 H), 2.05–2.22 (m, 2 H),
2.26–2.42 (m, 2 H), 3.48 (q, J = 8.3 Hz, 1 H), 3.66 (td, J = 8.6, 3.1
Hz, 1 H), 4.40 (dd, J = 8.6, 2.4 Hz, 1 H), 6.64 (d, J = 9.0 Hz, 2 H),
7.84 (d, J = 8.4 Hz, 2 H), 7.90 (d, J = 9.0 Hz, 2 H), 8.08 (d, J = 8.4
Hz, 2 H).
MS (380 °C): m/z (%) = 120 (100), 553 (3, [M]+).
HRMS: m/z [M]+ calcd for C29H39N5O6: 553.2900; found:
13C NMR (100.6 MHz, CDCl3): d = 23.7, 28.2, 30.9, 48.6, 60.6,
81.2, 112.1, 121.9, 125.6, 130.3, 132.3, 144.4, 149.4, 155.4, 165.5,
178.7.
553.2901.
Copper-Catalyzed N-Arylation of Amino Acids; General Proce-
dure
MS (200 °C): m/z (%) = 55 (100), 144 (56), 294 (26), 395 (10,
Compound 12 (150 mg, 415 mmol), the appropriate amino acid (623
mmol, 1.5 equiv), and K3PO4 (176 mg, 829 mmol, 2.0 equiv) were
suspended in 2-(dimethylamino)ethanol (420 mL). The vial was
flushed with N2, and CuI (8.00 mg, 42 mmol, 0.1 equiv) was added
under N2. The mixture was stirred and heated to 80 °C whereupon
the solids completely dissolved. Stirring was continued until no fur-
ther conversion was detected (TLC). The mixture was then allowed
to attain r.t., and H2O (15 mL) and EtOAc (15 mL) were added. The
aqueous layer was separated and extracted with EtOAc (3 × 25
mL). The combined organic layers were washed with brine (2 × 20
[M]+).
HRMS: m/z [M]+ calcd for C22H25N3O4: 395.1845; found:
395.1850.
tert-Butyl 4-[(4-Aminophenyl)diazenyl]benzoate (14)
Mp 103 °C; Rf = 0.71 (EtOAc–hexane–AcOH, 50:50:0.5).
IR (ATR): 3372, 2976, 2930, 1698, 1599, 1507 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.62 (s, 9 H), 6.75 (dt, J = 8.84,
2.43 Hz, 2 H), 7.82–7.86 (m, 4 H), 8.09 (dt, J = 8.48, 1.97 Hz, 2 H).
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York

4266
K. Rück-Braun et al.
FEATURE ARTICLE
13C NMR (100.6 MHz, CDCl3): d = 28.2, 81.2, 114.6, 122.0, 125.6,
130.3, 132.5, 145.6, 150.2, 155.3, 165.4.
3-{[(3-Aminomethyl)phenyl]diazenyl}benzoic Acid (2b); Typi-
cal Procedure
To a solution of the Fmoc-protected w-amino acid 17b (240 mg, 503
mmol) in THF (10 mL) was added 20% aq NaOH (10 mL) and the
resulting mixture was stirred vigorously at r.t. for 48 h (TLC moni-
toring). The layers were separated, the organic layer was discarded,
and the aqueous layer was acidified (pH 1) by addition of aq 1 N
HCl. The precipitate was separated by filtration using a suction fil-
ter, washed with a small amount of cold H2O and lyophilized, to af-
ford 2b as a yellow solid; yield: 55.0 mg (86%); mp 253 °C;
Rf = 0.17 (CH2Cl2–MeOH–AcOH, 50:48:2).
MS (140 °C): m/z (%) = 92 (100), 120 (57), 297 (47, [M]+).
HRMS: m/z [M]+ calcd for C17H19N3O2: 297.1477; found:
297.1480.
3-{[3-({[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}meth-
yl)phenyl]diazenyl}benzoic Acid (17b)
A suspension of 3-nitrosobenzoic acid (16; 5.50 g, 36.5 mmol) in
AcOH–DMSO (1:1, 500 mL) was sonified until a clear green solu-
tion was obtained. Then the carbamate 15a (6.28 g, 18.2 mmol) was
added as a solid, and the resulting soln was stirred at r.t. for 13 d
(TLC monitoring). H2O (700 mL) was added, and the resulting pre-
cipitate was filtered, thoroughly washed with H2O, dried under re-
duced pressure, and recrystallized several times from acetone–H2O
to afford 17b as a brown solid; yield: 5.35 g (62%); mp 171–173 °C
(dec.); Rf = 0.45 (CH2Cl2–MeOH, 9:1).
IR (ATR): 3081, 1687, 1530, 1352 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 4.17 (s, 2 H), 7.67 (t, J = 7.7
Hz, 1 H), 7.73–7.78 (m, 2 H), 7.96 (d, J = 7.9 Hz, 1 H), 8.10 (s, 1
H), 8.13–8.17 (m, 2 H), 8.40 (t, J = 1.7 Hz, 1 H), 8.57 (br s, 2 H),
13.3 (br s, 1 H).
13C NMR (100.6 MHz, DMSO-d6): d = 41.8, 122.3, 123.0, 123.1,
127.3, 129.8, 130.1, 132.1, 132.4, 132.5, 135.7, 151.80, 151.82,
166.6.
IR (ATR): 3406, 3320, 3066, 2667, 1699, 1600 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 4.25 (t, J = 6.8 Hz, 1 H), 4.32
(d, J = 6.0 Hz, 2 H), 4.38 (d, J = 6.9 Hz, 2 H), 7.31 (t, J = 7.4 Hz,
2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7.45 (d, J = 7.6 Hz, 1 H), 7.57 (t,
J = 8.2 Hz, 1 H), 7.71 (d, J = 7.3 Hz, 2 H), 7.74 (t, J = 7.8 Hz, 1 H),
7.82–7.84 (m, 2 H), 7.89 (d, J = 7.5 Hz, 2 H), 8.00 (t, J = 6.0 Hz, 1
H), 8.11–8.14 (m, 2 H), 8.39 (t, J = 1.8 Hz, 1 H).
MS (330 °C): m/z = 77 (55), 89 (22), 106 (100), 121 (46), 255 (92,
[M]+).
HRMS: m/z [M]+ calcd for C14H13N3O2: 255.1008; found:
255.1006.
13C NMR (50.3 MHz, DMSO-d6): d = 43.5, 46.8, 65.4, 120.1,
120.8, 121.8, 122.3, 125.1, 127.1, 127.4, 127.6, 129.5, 130.0, 130.6,
131.9, 132.2, 140.8, 141.5, 143.9, 151.9, 156.4, 166.7.
3-{[(4-Aminomethyl)phenyl]diazenyl}benzoic Acid (2c)
A solution of the Fmoc-protected w-amino acid 17c (200 mg, 419
mmol) in THF (16 mL) was treated with 20% aq NaOH (16 mL) for
48 h. Then aq 2 M HCl was added (pH 1), and the resulting precip-
itate was filtered. The crude material was washed with small
amounts of cold H2O and THF and dried under reduced pressure, to
afford 2c as a yellow solid; yield: 74.0 mg (69%); mp >253 °C
(dec.); Rf = 0.12 (CH2Cl2–MeOH–AcOH, 50:48:2).
MS (240 °C): m/z (%) = 165 (22), 178 (100), 196 (21), 477 (1,
[M]+).
HRMS: m/z [M]+ calcd for C29H23N3O4: 477.1689; found:
477.1697.
IR (ATR): 2920, 1691, 1626, 1592, 1529, 1388 cm–1.
3-{[4-({[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}meth-
yl)phenyl]diazenyl}benzoic Acid (17c)
1H NMR (400 MHz, DMSO-d6): d = 4.14 (s, 2 H), 7.73–7.78 (m, 3
H), 7.98 (d, J = 8.4 Hz, 2 H), 8.12–8.17 (m, 2 H), 8.39 (t, J = 1.7
Hz, 1 H), 8.59 (br s, 2 H).
13C NMR (100.6 MHz, DMSO-d6): d = 41.7, 122.2, 122.8, 127.5,
130.0, 130.1, 132.0, 132.3, 138.0, 151.6, 151.8, 166.7.
A suspension of 3-nitrosobenzoic acid (16; 4.76 g, 31.5 mmol) in
AcOH–DMSO (1:1, 580 mL) was sonified until a clear green soln
was obtained. Then the carbamate 15b (5.40 g, 15.7 mmol) was
added as a solid in several portions, and the resulting soln was
stirred at r.t. for 3 d (TLC monitoring). H2O (380 mL) was added to
the brownish-black solution and then the aqueous layer was extract-
ed with EtOAc (3 × 400 mL). The combined organic layers were
washed with brine (200 mL), dried (MgSO4), and concentrated in
vacuo. After addition of H2O, the precipitate formed was filtered,
thoroughly washed with H2O, and recrystallized several times from
acetone to afford 17c as a light orange solid; yield: 5.53 g (74%, pu-
rity >95%); mp 212–215 °C (dec.); Rf = 0.64 (EtOAc). A portion of
the product (176 mg, 369 mmol) was purified by column chromatog-
raphy on silica gel (EtOAc + 0.1% AcOH) to afford highly pure 17c
(133 mg, 76%, 278 mmol).
MS (250 °C): m/z = 65 (22), 89 (100), 106 (58), 121 (22), 255 (50,
[M]+).
HRMS: m/z [M]+ calcd for C14H13N3O2: 255.1008; found:
255.1007.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.
Acknowledgment
IR (ATR): 3312, 3065, 2971, 2635, 1700, 1604 cm–1.
We thank the Deutsche Forschungsgemeinschaft (SFB 498 and
DFG-Cluster of Excellence 314), the Volkswagen Foundation, and
the Fonds der Chemischen Industrie for financial support. We also
thank Prof. Dr. Josef Wachtveitl (Johann Wolfgang Goethe-Univer-
sität, Frankfurt am Main) and Dr. Peter Schmieder (FMP, Berlin-
Buch) for useful discussions.
1H NMR (400 MHz, DMSO-d6): d = 4.25 (t, J = 6.7 Hz, 1 H), 4.30
(d, J = 6.0 Hz, 2 H), 4.40 (d, J = 6.7 Hz, 2 H), 7.34 (t, J = 6.8 Hz, 2
H), 7.40–7.45 (m, 4 H), 7.71–7.75 (m, 3 H), 7.89 (d, J = 7.3 Hz, 2
H), 7.90 (d, J = 8.3 Hz, 2 H), 7.97 (t, J = 6.2 Hz, 1 H), 8.11–8.15 (m,
2 H), 8.39 (s, 1 H).
13C NMR (100.6 MHz, DMSO-d6): d = 43.5, 46.8, 65.3, 120.1,
122.2, 122.8, 125.1, 127.0, 127.3, 127.6, 127.9, 129.9, 131.7, 132.4,
140.8, 143.9, 144.0, 150.8, 151.9, 156.4, 166.8.
References
MS (200 °C): m/z (%) = 165 (67), 178 (100), 196 (21), 477 (<1,
(1) Szobota, S.; Gorostiza, P.; Del Bene, F.; Wyart, C.; Fortin,
D. L.; Kolstad, K. D.; Tulyathan, O.; Volgraf, M.; Numano,
R.; Aaron, H. L.; Scott, E. K.; Kramer, R. H.; Flannery, J.;
Baier, H.; Trauner, D.; Isacoff, E. Y. Neuron 2007, 54, 535.
[M]+).
HRMS: m/z [M]+ calcd for C29H23N3O4: 477.1689; found:
477.1680.
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FEATURE ARTICLE
Azobenzene-Based w-Amino Acids in Peptide Chemistry
4267
(2) Woolley, G. A. Acc. Chem. Res. 2005, 38, 486.
(3) Renner, C.; Moroder, L. ChemBioChem 2006, 7, 868.
(4) Dugave, C.; Demange, L. Chem. Rev. 2003, 103, 2475.
(5) Ettmayer, P.; France, D.; Gounarides, J.; Jarosinski, M.;
Martin, M.-S.; Rondeau, J.-M.; Sabio, M.; Topiol, S.;
Weidmann, B.; Zurini, M.; Bair, K. W. J. Med. Chem. 1999,
42, 971.
(15) Renner, C.; Cramer, J.; Behrendt, R.; Moroder, L.
Biopolymers 2000, 54, 501.
(16) Aemissegger, A.; Kräutler, V.; van Gunsteren, W. F.;
Hilvert, D. J. Am. Chem. Soc. 2005, 127, 2929.
(17) Dong, S.-L.; Löweneck, M.; Schrader, T. E.; Schreier, W. J.;
Zinth, W.; Moroder, L.; Renner, C. Chem. Eur. J. 2006, 12,
1114.
(6) Priewisch, B.; Steinle, W.; Rück-Braun, K. In Peptides
2004; Flegel, M.; Fridkin, M.; Gilon, C.; Slaninova, J., Eds.;
Kenes International: Genf, 2005, 756.
(18) Hecker, E. Chem. Ber. 1955, 88, 1666.
(19) Ma, D.; Zhang, Y.; Yao, J.; Wu, S.; Tao, F. J. Am. Chem.
Soc. 1998, 120, 12459.
(7) Priewisch, B.; Rück-Braun, K. J. Org. Chem. 2005, 70,
(20) Lu, Z.; Twieg, R. J.; Huang, S. D. Tetrahedron Lett. 2003,
2350.
44, 6289.
(8) Steinle, W.; Rück-Braun, K. Org. Lett. 2003, 5, 141.
(9) Herre, S.; Steinle, W.; Rück-Braun, K. Synthesis 2005, 3297.
(10) Cordes, T.; Weinrich, D.; Kempa, S.; Riesselmann, K.;
Herre, S.; Hoppmann, C.; Rück-Braun, K.; Zinth, W. Chem.
Phys. Lett. 2006, 428, 167.
(21) Woody, R. W. In Houben-Weyl: Methods of Organic
Chemistry, Vol. E22b; Goodman, M.; Felix, A.; Moroder,
L., Eds.; Thieme: Stuttgart, 2003, 739.
(22) Sreerama, N.; Woody, R. W. Protein Sci. 2003, 12, 384.
(23) Rau, H. In Photoreactive Organic Thin Films; Sekkat, Z.;
Knoll, W., Eds.; Academic Press: San Diego, 2002, 3.
(24) Wachtveitl, J.; Nägele, T.; Puell, B.; Zinth, W.; Krüger, M.;
Rudolph-Böhner, S.; Oesterhelt, D.; Moroder, L.
J. Photochem. Photobiol., A 1997, 105, 283.
(25) Bamberger, E.; Hübner, R. Ber. Dtsch. Chem. Ges. 1903, 36,
3803.
(11) Schadendorf, T.; Hoppmann, C.; Rück-Braun, K.
Tetrahedron Lett. 2007, 48, 9044.
(12) Behrendt, R.; Renner, C.; Schenk, M.; Wang, F.; Wachtveitl,
J.; Oesterhelt, D.; Moroder, L. Angew. Chem. Int. Ed. 1999,
38, 2771.
(13) Behrendt, R.; Schenk, M.; Musiol, H.-J.; Moroder, L.
J. Pept. Sci. 1999, 5, 519.
(26) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals; Wiley-VCH: Weinheim, 1996.
(14) Ulysse, L.; Chmielewski, J. Bioorg. Med. Chem. Lett. 1994,
4, 2145.
Synthesis 2009, No. 24, 4256–4267 © Thieme Stuttgart · New York