Chemistry and folding of photomodulable peptides - Stilbene and thioaurone-type candidates for conformational switches

Article abstract of DOI:10.1039/b812001c

Optimized synthetic strategies for the preparation of photoswitchable molecular scaffolds based on stilbene or on thioaurone chromophores and their conformationally directing properties, as studied by computations and by NMR spectroscopy, are addressed. For the stilbene peptidomimetics 1, 2 and 3, the length of connecting linkers between the chromophore and the peptide strands was varied, resulting in photochromic dipeptidomimetics with various flexibility. Building blocks of higher rigidity, based on para-substituted thioaurone (4 and 6) and meta-substituted thioaurone chromophores (5 and 7) are shown to have a stronger conformationally directing effect. Design, synthesis, theoretical and experimental conformational analyses are presented.

Full text of DOI:10.1039/b812001c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Chemistry and folding of photomodulable peptides – stilbene and
thioaurone-type candidates for conformational switches†
Ma´te´ Erde´lyi,a,b Miranda Varedian,b Christian Sko¨ld,a Ida B. Niklasson,a Johanna Nurbo,a
a
c
b
˚
Asa Persson, Jonas Bergquist and Adolf Gogoll*
Received 14th July 2008, Accepted 9th September 2008
First published as an Advance Article on the web 17th October 2008
DOI: 10.1039/b812001c
Optimized synthetic strategies for the preparation of photoswitchable molecular scaffolds based on
stilbene or on thioaurone chromophores and their conformationally directing properties, as studied by
computations and by NMR spectroscopy, are addressed. For the stilbene peptidomimetics 1, 2 and 3,
the length of connecting linkers between the chromophore and the peptide strands was varied, resulting
in photochromic dipeptidomimetics with various flexibility. Building blocks of higher rigidity, based on
para-substituted thioaurone (4 and 6) and meta-substituted thioaurone chromophores (5 and 7) are
shown to have a stronger conformationally directing effect. Design, synthesis, theoretical and
experimental conformational analyses are presented.
interconvertible stilbene and thioaurone derivatives, which are
Introduction
expected to induce turn-like conformations in their Z configu-
rations, and open, linear geometries in their E configurations.11,12
Their general synthetic strategies, suitable for the construction of
phototriggerable, functional protein- as well as peptidomimetics,
are exemplified by simple model compounds. In order to reduce
complexity and focus on the role of the chromophore, apolar,
non-aromatic amino acids were attached to the stilbene amino
acid core.
The resulting peptidomimetics were, similar to numerous
previously investigated compounds,13,14 poorly soluble in water.
Conformational studies were therefore perfomed in methanol and
dimethylsulfoxide solutions. Here, we would like to stress that
these compounds were designed to provide simplified models of
the photoswitchable core of peptides and proteins with larger
molecular weight. Their photochemical E → Z conversion as
well as conformational properties, in comparison to those of
the standard DPro-Gly13 and diphenylacetylene15 turn mimetics
(Scheme 1), is discussed.
Proteins capable of reversible photochemically driven conforma-
tional changes have attracted a lot of interest in recent years and
were predicted to bear tremendous potential in the fields of data
storage, molecular motors, protein and genetic engineering, in
vivo protein tracking and fluorescent optical microscopy. Built-
in switching units are expected to result in photoresponsive
materials,1 conformationally switchable nucleic acids,2 antibodies,3
and peptidomimetics.4 Among examples from Nature, photo-
switchable green fluorescent proteins (GFP) found in certain
marine organisms have recently attracted much interest, including
practical applications.5 The demand for photomodulable units
of improved and well-controlled properties motivated us to
develop photoswitchable units that can straightforwardly be
incorporated into functional peptides by standard automatable
methods. Azobenzene chromophores have been successfully ap-
plied to control the structure of synthetic peptides and proteins,6
however, their conformation inducing ability appeared to be
limited, which in combination with their sensitivity to reducing
agents and their unavoidable thermal isomerization make their
use in control of protein function difficult if not impossible.7
In contrast, in spite of their well-studied photochemistry8 and
successful incorporation into DNA,2 examples for the use of
stilbene derivatives in peptidomimetics are very rare.9 Likewise, the
thioaurone chromophore has only recently been proposed for this
purpose.10 Due to their greater chemical stability and resistance
against thermal isomerization, stilbenes are promising alternatives
to azobenzenes. Here, we propose a range of photochemically
Results and discussion
Synthesis
Based on computational design, meta,meta¢-disubstituted
stilbene-type turn-mimetics 1–3 (Scheme 1) encompassing methy-
lene linkers of various length ((CH2)n, n = 0, 1, 2), providing
different conformational flexibility, were prepared (Schemes 2–4).
E-Isomers were synthesized and the Z analogues were obtained
by photoisomerization of the former. Peptidomimetics 4–7, in-
corporating the thioaurone chromophore were prepared in their
thermally stable Z isomeric forms, and the E isomers were
then obtained by analogous photochemical isomerization. Since
peptides containing DPro-Gly13 or diphenylacetylene-type turn
mimetics are known to fold in well-defined b-turn conformations,15
mimetics 8–10 having identical amino acid strands to those
attached to the stilbene and thioaurone units were synthesized as
aDept. of Medicinal Chemistry, Uppsala University, Box 574, 751 23,
Uppsala, Sweden
bDept. of Biochemistry and Organic Chemistry, Uppsala University, Box
576, 751 23, Uppsala, Sweden
cDept. of Physical and Analytical Chemistry, Uppsala University, Box 599,
751 24, Uppsala, Sweden. E-mail: adolf.gogoll@biorg.uu.se; Fax: 46 18
4713818; Tel: 46 18 4713822
† Electronic supplementary information (ESI) available: NMR spectra.
See DOI: 10.1039/b812001c
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Scheme 1 Model peptidomimetics incorporating reversibly switchable chromophores (1–7) and b-turn mimetics (8–10).
reference compounds. Synthesis and structural studies of 9 have
been published earlier13 and are cited here for comparison.
The anilinic amino group15 of 13 was supposed to show reduced
nucleophilicity as compared to typical amino acids, a fact known
to cause difficulties in solid phase synthesis. Therefore, E-1 was
prepared in solution following the reaction path depicted in
Scheme 2. A fast microwave mediated Heck-coupling16 of 11
with the electron rich 3-iodoaniline resulted in a 2:18:80 mixture
of the Z:internal:E products. The E-isomer was separated using
column chromatography, its ester functionality was subsequently
hydrolysed, and the zwitterionic 13 was then subjected to PyBOP
mediated attachment of alanine methylamide, yielding 14. Under
these conditions, the anilinic amino group of 13 does not react,
and therefore the coupling could be performed selectively.
The product was isolated by precipitation from the reaction mix-
ture and then investigated by 1H NMR spectroscopy. Observation
of only one set of signals ascertained that racemization at the
valine C-a had not occurred. Obviously, a single set of signals
could also result from complete inversion at this carbon, but
this is highly unlikely. Analogous tri- or tetrapeptidomimetics are
easily incorporatable into standard solid-phase peptide synthesis
(SPPS).
The switchable units of 2 and 3 and the diphenylacetylene
moiety of 10 were synthesized in solution and could subse-
quently be used in SPPS. As shown in Scheme 3, benzylic
nucleophilic substitution17 followed by a microwave-accelerated
Stille reaction,18 was utilized for the preparation of the central
part of mimetic 2. For the efficient Heck coupling16 of 17 and
18, leading to the artificial amino acid 20, both Boc-protection
of the amino group of 16, yielding 17, and esterification of
the carboxyl group of 3-iodophenyl acetic acid to give 18 are
indispensable. Base mediated hydrolysis of the resulting methyl
In the next step, the electrone density of the amino
group of 13 was increased, by activation with N,O-bis(trime-
thylsilyl)acetamide. Now the attachment of N-acetyl-valine was
possible with HATU under mild basic conditions, yielding E-1.
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Scheme 2 Outline of the synthesis of 1: (a) CH3OH, conc. HCl, 70 ◦C, 14 h, 88%. (b) 3-Iodoaniline, Pd(OAc)2, (C2H5)3N, DMF, 150 ◦C, 20 min, 56%.
(c) 6 M NaOH (aq), 100 ◦C, 6 h, 98%. (d) 2-Amino-N-methyl-propionamide, PyBOP, DIEA, r.t., 2 h, 17%. (e) 2-Acetylamino-3-methyl-butyric acid,
N,O-bis(trimethylsilyl)acetamide, HATU, DIEA, r.t., 18 h, 49%.
Scheme 3 Outline of the synthesis of 20: (a) 1) NaN(SiMe3)2, HMDS, r.t., 24 h. 2) H , 63%. (b) Bu3SnCH CH2, Pd(PPh3)2Cl2, Et3N, DMF, 130 ◦C,
+
=
15 min, 84%. (c) O[CO2C(CH3)3]2, CH2Cl2, K2CO3 in H2O, r.t., 72 h, 68%. (d) CH3OH, conc. HCl, r.t., 24 h, 98%. (e) Pd(OAc)2, (CH3C6H4)3P, Et3N,
DMF, 90 ◦C, 15 min, 70%. (f) 6M NaOH (aq), 120 ◦C, 20 h, 34%.
ester 19 yields 20, a photochromic amino acid mimetic suitable
for direct application in standard SPPS. The harsh conditions
used for hydrolysis of 19 and also 12, were required because
of the low reactivity that is often encountered for benzylic and
in particular aromatic esters. Peptidomimetic 3 was prepared
using Knoevenagel condensation19 followed by esterification of
the carboxyl group, resulting in the a,b-unsaturated ester 22
(Scheme 4).
Since the reduction of a double bond in the presence of an aryl
halide is known to be difficult, a nickelboride catalyzed route20
was optimized for the reduction of 22 to 23, giving 68% isolated
yield and notably only 1.5% debrominated byproduct. Compound
26 was obtained by Heck coupling of 23 and 25 in a microwave
reactor.16 After several unsuccessful attempts to hydrolyse the ester
26 by conventional methods, the hydrolysis could be achieved with
K-tert-BuO in diethyl ether, yielding 27. The stilbene derivative
artificial amino acid 27 could then be directly incorporated into 3
by standard SPPS. Details of the experimental procedure for the
preparation of 27 are presented elsewhere.12
The syntheses of the thioaurone cores followed a previously
reported general scheme via thioindoxyl (Schemes 5 and 6).21
The aldehyde required for the preparation of 34 was obtained
by the oxidation of the corresponding alcohol 29.22 Boc-protected
thioaurone derivatives 33 and 39 were synthesized by an aldol
condensation reaction between thioindoxyl 32 and the aldehydes
30 and 38, respectively.10 Here, conditions and synthetic strategy
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Scheme 4 Outline of the synthesis of 27: (a) CH2(COOH)2, pyridine, piperidine, 100 ◦C, 1.5 h, 95%. (b) CH3OH, conc. HCl, r.t., 16 h, 93%. (c) Ni(OAc)2,
◦
=
NaBH4, CH3OH, EtOAc, H2 (2 bar), 20 min, r.t., 68%. (d) Bu3SnCH CH2, Pd(PPh3)2Cl2, Et3N, DMF, 130 C, 25 min, 78%. (e) O[CO2C(CH3)3]2, CH2Cl2,
K2CO3 in H2O, r.t., 24 h, 70%. (f) Pd(OAc)2, (CH3C6H4)3P, Et3N, DMF, 120 ◦C, 30 min, 43%. (g) K-tert-BuO, Et2O, 10.5 h, 99%.
Scheme 5 Outline of the synthesis of 34: (a) 1) 1,4-dioxane, 1 M NaOH, O[CO2C(CH3)3]2, 0 ◦C, 40 min. 2) HCl, 81%. (b) LAH, THF, 40 ◦C o.n., 71%.
(c) PCC, NaOAc, DCM, r.t. o.n., 72%. (d) 1) Na2CO3, Na2S2O4, reflux 30 min. 2) ClCH2COOH neutralised with Na2CO3, reflux 1 h. 3) HCl to Congo
red, 71%. (e) 1) SOCl2, reflux 2 h. 2) AlCl3, dichloroethane, r.t. o.n., 81%. (f) 1) 1% NaOH w/w, t-BuOH, 0 ◦C then reflux o.n. 2) HOAc, 81%. (g) 1) 50%
TFA in DCM, 15 min. 2) Fmoc-Cl, Na2CO3, THF, 90%.
had to be altered from the literature procedure to achieve
satisfactory results. Thus, an excess of thioindoxyl over aldehyde
was used, and the reaction time was increased. To be able to use
the thioaurone derivatives in SPPS, the protecting group had to be
changed from Boc to Fmoc. Due to the instability of thioaurones
under basic conditions, including primary but not secondary or
tertiary amines, the synthetic strategy to prepare the peptides 4–7
had to be adjusted: Instead of using Boc-chemistry and the
Kaiser oxime resin, as for 2 and 3, a resin yielding the desired
N-methylated amides after mild acidic cleavage was used, thus
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Scheme 6 Outline of the synthesis of 40: (a) Ethylene glycol, PTSA, reflux 18 h, 60% (b) LAH, THF, r.t., o.n. 40% (c) Anhyd. MeOH, TEA,
O[CO2C(CH3)3]2, r.t., o.n., 60% (d) Acetone, water, cat. PTSA, r.t. o.n., 78% (e) 1) 1% NaOH w/w, t-BuOH, 0 ◦C then reflux o.n. 2) HOAc, 45%. (f) 1)
50% TFA in DCM, 15 min. 2) Fmoc-Cl, Na2CO3, THF, 70%.
avoiding the basic conditions required for the final cleavage of the
peptidomimetic from the Kaiser oxime resin.
Reference compound 8 was prepared applying standard Boc-
SPPS methodologies. The precursor of 10 was afforded in a similar
reaction route to the one used for preparation of 2 (Scheme 7), but
using ortho-substituted starting materials and rapid Sonogashira
cross-couplings13,23 instead of a Stille and a Heck reaction. The
central part of compound 46 was obtained by benzylic nucleophilic
Scheme 7 Outline of the synthesis of 46: (a) 1) NaN(Si(CH3)3)2, hexamethyldisilazane, r.t., 40 h. 2) H+, 50%. (b) O[CO2C(CH3)3]2, CH2Cl2, K2CO3 in
H20, r.t., 72 h, 60%. (c) 1) (CH3)3Si–C CH, Pd(PPh3)2Cl2, CuI, (C2H5)2NH, DMF, 120 ◦C, 6 min, 98%. 2) KF·2H2O, r.t., 14 h, 63%. (d) CH3OH, HCl,
∫
r.t., 4 h, 99%. (e) Pd(PPh3)2Cl2, CuI, (C2H5)2NH, DMF, 120 ◦C, 6 min, 81%. (f) 6 M NaOH (aq), 120 ◦C, 2 h, 61%.
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substitution, followed by Boc-protection of the amino group of
41. It is worth mentioning that protection of the benzylamine
of 41 with Boc instead of Fmoc, was necessary to facilitate the
Sonogashira coupling. Furthermore, esterification of the carboxyl
group of 44 was also required for efficient coupling of the two
sides of 45. Base mediated hydrolysis of the protected methyl ester
yielded compound 46, appropriate for incorporation into 10 by
standard SPPS.
Photochemistry
The photochemical properties of stilbene and thioaurone deriva-
tives are well documented in the literature.8,21b The absorbance
maxima of the p–p* transitions of the trans-double bonds in
peptidomimetics 1–3 at approximately 300 nm indicated that
meta-substituents do not significantly affect the lmax of the
stilbene double bond. Photoisomerizations were performed in
acetonitrile solutions and the E/Z ratio was thereafter determined
by 1H NMR measurements. In contrast to azobenzenes24 and
thioaurones,4,21b thermal isomerization was absent for stilbene
derivatives 1–3.
Conversion between E-stilbene and Z-stilbene derivatives was
achieved by irradiation at the absorption maxima of 300 nm and
280 nm, respectively. Isomerization of thioaurones was performed
by non-selective irradiation, followed by investigation of the
E-isomers, having half-lives of several hours.
Molecular mechanics calculations
Conformational analysis using molecular mechanics was used to
predict the conformational population of the photoswitchable
compounds 1–3, 6 and 7. The GB/SA CHCl3 solvation model25
was used as implemented in the program Macromodel 9.5.26 Con-
formations within 25 kJ mol-1 of the global minimum were retained
(Scheme 8). Interstrand hydrogen bonds and distances between
Ca,Ala–Ca,Val and the capping group Cmethyl–Cmethyl were recorded
to identify folded or non-folded conformations. The presence of
˚
two hydrogen bonds (< 2.5 A distance between HNH and O, see
˚
Fig. 1) or an interstrand Ca–Ca distance of < 5.5 A was used as
criterion for beta sheet and/or folded conformations, respectively.
The populations of folded conformations were estimated using the
Boltzmann equation and are summarized in Table 1.
Scheme 8 Overlays of the most stable conformers of peptidomimetics
with stilbene and thioaurone chromophores. Conformers within 5 kJ mol-1
of the global minimum are shown.
Compared to compound 8, which has been included for
comparison as a peptide predestined to generate b-hairpin motifs,
the stilbene derivatives 1–3 show excellent preference for formation
of turn-like motifs in their Z forms whereas their E isomers
show larger interstrand distances. Compound 1 shows the most
pronounced conformational alteration, while model compounds
2 and 3 display a tendency for intramolecular interchain inter-
action in both their Z and E isomers as a result of the high
flexibility of their linker regions. It should be emphasized that
the preferred geometry of Z-2 and Z-3 does not fully resemble the
conformation of conventional turns, which is likely to be a result
of the shortness of the amino acid strands present in the model
systems. This is further supported by our previous studies which
have shown that such flexible photochromic turn mimetics can
provide a sufficient conformationally directing effect when they
are incorporated into a medium sized peptide.12 Switch candidates
6 and 7 show a pronounced difference between their respective
Fig. 1 Selected interstrand NOEs (arrows) and hydrogen bonds (dashed
lines) observed in CDCl3 solutions of 8 and 9.
Z and E isomers. As for 1, their superior rigidity is indicated by
the pronounced absence (i.e., relative amount ≤ 0.1%) of turn-like
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Table 1 Relative amounts of folded conformations of the Z and E isomers
of photoswitchable compounds 1–3, 6–7, and reference compound 8
interactions further indicates the high conformational flexibility
of the peptidomimetics.
Discussion
Our NMR investigations (measuring NOE, DdNH/DT, and
DdNH,solv) showed that the DPro-Gly nucleated peptide 8 folds
into a b-turn in methanol and chloroform solutions. However, in
DMSO or D2O:H2O (1:1) solutions, no interchain NOEs could be
detected, an observation in line with the well-known high flexibility
and low folding propensity of small peptides. The more rigid
diphenylacetylenic mimetic 9 formed a b-hairpin in both CDCl3,
DMSO, and methanol solutions.13 The chemical shift alteration
upon change of solvent suggested that compound 9 folded into
a stable b-hairpin conformation, in which NHPh and NHCH3 were
involved in intramolecular hydrogen bonding, while only a single
interchain amide hydrogen bond (NHAla) was detectable for 8
(Ddsolv = dDMSO - dCDCl3 for the amide protons, which assumed
the following values: Ddsolv(NHPh) = 0.1 (9); Ddsolv(NHCH3) =
0.01 (9), 0.6 (9); Ddsolv(NHAla) = 1.6 (9), 0.1 (8); Ddsolv(NHVal ) =
1.6 (9), 0.4 (8); Ddsolv(NHGly) = 0.8 (8)). In the DMSO solution
of the comparatively flexible non-switchable mimetic 10, a non-
folded structure was observed whereas in its methanol solution
Z
E
Ca,Ala–Ca,Val
5.5 A (%)
<
CMe–CMe
5.5 A (%)
<
Ca,Ala–Ca,Val
5.5 A (%)
<
CMe–CMe <
5.5 A (%)
˚
˚
˚
˚
1
2
3
6
7
8
98
97
99
12
100
94
25
30
12
91
0
0
7
87
0
0
—
0
92
6
0
0.1
—
97
conformations for their E isomers. An interesting feature is the
similar conformational behaviour of Z-7 and E-3, as well as
Z-6 compared to E-2, revealing that this geometry can easily
be reached from a tighter (Z-2 or Z-3) or a more extended
(E-6 or E-7) state through photochemical induction. However,
some of these turn motifs show a twisted geometry, which is also in-
dicated by the presence of “crossing” hydrogen bonds (e.g., in Z-1,
see ESI for details). Thus, depending on the need of the intended
application area a stilbene or a thioaurone-type switch should be
chosen to provide an optimal conformational modulator.
1
a complicated H NMR spectrum was obtained, indicative for
a mixture of low energy conformations. Hence, increased turn
flexibility leads to decreased folding propensity.
In agreement with our computational studies, no sign of
folding was observable for methanolic and DMSO solutions
of neither Z-1–3 (Table 1), nor for DMSO solutions of Z-6
and Z-7. Here it should be noted that these model compounds
are insoluble in chloroform, which should be taken in account
when comparing computational and experimental results. Polar,
hydrogen bond accepting and/or donating solvents are likely
to interfere with intramolecular hydrogen bonding and thereby
support the formation of unfolded conformations in solution.32
Furthermore, water would not be an alternative choice of solvent
for these compounds, since it is known to counteract the formation
of, e.g., b-turn motifs in small peptides.33
Investigations of the non-switchable model compounds 8–10
indicate that turn rigidity is a critical factor for hairpin folding.
Thus, a thermodynamically stable b-hairpin conformation with
two interchain hydrogen bonds was observed for the most rigid
diphenylacetylene derivative 9, but not for 10.
Spectroscopic conformational analysis
Conformational preferences were further investigated by NMR
spectroscopy. Structural assignment was performed using COSY,27
NOESY,28 ROESY,29 and TOCSY30 experiments. If necessary,
solvent signals were suppressed using the WET presaturation
scheme.30 Similarly to other investigations,4d,6b the Z-stilbene
isomers were studied in mixtures with the E isomers, obtained
by photoisomerization of the latter. The molecular conformation
in solution was examined by the study of amide temperature
coefficients (Table S1, ESI) and by analysis of NOE cross-peak
patterns.
The presence of b-hairpin populations of 9, and of folded
conformers of 8, was indicated by interstrand NOEs (Fig. 1).
The NOESY and Dd/DT values did not indicate folding for the
DMSO or methanol solutions of stilbene derivatives 1–3 nor of
the thioaurone derivatives 4–7. This result is in excellent agreement
with the extraordinarily high flexibility of low-molecular weight
peptides, frequently mentioned in the literature.31 Hence, small
peptides can only be expected to fold into a well-defined structure
when an exceptionally rigid conformational directing organic unit,
such as the tolan moiety in 9, is present in their sequence. The
uncapped peptidomimetics 4 and 5, having a free carboxy group,
and their capped congeners 6 and 7, showed similar chemical shifts,
temperature coefficients and NOE patterns. Since the presence
of an additional polar group would be expected to increase the
tendency to form interstrand hydrogen bonds, the absence of such
Formation of one hydrogen bond (resulting in a b-turn) was
indicated for the less rigid 8, and no folding was observed for the
flexible derivative 10. If the flexibility is high, folding is disfavoured
for entropic reasons.
We therefore conclude that the absence of any folded conformers
in solutions of stilbene derivatives 2 and 3 is due to their flexible
(CH2)n linkers. Another factor likely to affect the formation of
folded conformers is the orientation of the amide groups of the
antiparallel chains of 1 and 2. In native hairpins, hydrogen bonds
were shown to be close to planar.34 According to our computations,
the N–H–O and N–O–C angles within the existing interchain
hydrogen bonds, in partially folded single conformations, would
be tilted for mimetics 1–2 (typically N–H–O < 155◦, N–O–
C < 135 ◦). On the other hand, the high flexibility of the
(CH2)2 linkers of 3 simultaneously decreases its turn inducing
properties through entropic factors, and allows its amino acid
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strands to align themselves in optimal angles and distance for
intramolecular hydrogen bonding. Thus, incorporation of the
stilbene chromophore of 3 into a larger cyclic peptidomimetic
resulted in a system for which photoisomerization between the Z
and E configurations lead to reversible reorientation between a
b-hairpin and random coil conformations.12
column (4.6 ¥ 100 mm), or by analytical HPLC using a Chromolith
Performance RP-18e column (4.6 ¥ 100 mm). The procedure
appliedfor microwave heatingis described inref 23. Photochemical
reactions were performed on acetonitrile solutions under N2 gas
flow using an Oriel 1000 W Xe ARC light source and a 300 nm
Oriel UV filter. The emitted light intensity was determined using
a UV enhanced Si photodiode (5.8 mm2) attached to a current
meter. The thioaurone peptides 4–7 were isomerized for 15 min
in an NMR-tube with a nonselective ACE Glass Incorporated
450 W UV-lamp connected to a 7830 Power supply. UV spectra
were measured with a Varian Cary 3 spectrometer. IR spectra
were obtained on a Perkin Elemer 1600 series FTIR instrument;
recording 16 scans on 10 mol·dm-3 samples in CHCl3 solution
using a 1 mm cuvette (NaCl windows).
Conclusion
Synthetic strategies as well as conformationally directing prop-
erties of a set of small stilbene and thioaurone-based building
blocks, aimed to be applied as photoswitchable nucleating cores
of functional protein mimetics, have been investigated. Straight-
forward synthetic protocols were developed providing N-Boc and
N-Fmoc protected amino acid analogues, which can be directly
transferred to standard solid-phase peptide synthesis. Hence,
using the proposed synthetic routes, building blocks resembling
standard Fmoc- and Boc-protected amino acids can be prepared
and straightforwardly applied for the synthesis of peptide and
protein mimetics.
Mass spectra (EI, 70 eV) were obtained with a Hewlett Packard
5971 Series Mass Selective detector interfaced with a Hewlett
Packard 5890 Series II Gas Chromatograph equipped with a DB-
1 (25 m ¥ 0.20 mm) capillary column. The ESI-MS data were
obtained with a Finnigan ThermoQuest AQA mass spectrometer
(ESI-MS 30eV, probe temperature 100 ◦C) equipped with a
Gilson 322-H2 gradient pump system and an SB-C18 column.
A water-acetonitrile-formic acid (0.05%) mobile phase was used
with a gradient of 20% to 100% acetonitrile during 3–5 minutes.
High resolution MS spectra were run on an Agilent LC/MSD
TOF instrument (Agilent Technologies, Santa Clara, CA, USA).
Detection was performed in positive ion mode. The voltage applied
at the sampling capillary at the entrance of the mass spectrometer
was 4.0 kV. Nitrogen at 300 ◦C and 7 L min-1 was used as drying
gas, and nebuliser gas flow was at 15 L min-1. Voltages fixed at
fragmentor, skimmer and octopole guides were 215 V, 60 V and
250 V, respectively. The ion pulser at the TOF analyser was set up to
a measurement frequency of 0.5 cycles/s. Agilent TOF software
and Agilent QS software were used to record and analyse mass
spectra, respectively. Standard autotune of masses was performed
in the TOF-MS instrument before the experimental runs, and
typical mass errors of 1 to 3 ppm were achieved in the calibration.
NMR spectra were recorded on a Varian UNITY INOVA
spectrometer (1H at 499.9 MHz), a Jeol JNM EX400 spectrometer
(1H at 399.8 MHz, 13C at 100.5 MHz), a Varian UNITY
spectrometer (1H at 399.9 MHz, 13C at 100.6 MHz), a Varian
Mercury plus spectrometer (1H at 300.0 MHz, 13C at 75.4 MHz),
or on a JEOL JNM EX270 spectrometer (1H at 270.2 MHz,
13C at 67.9 MHz). Data are reported in the following manner:
chemical shift (integration, multiplicity, coupling constant if
appropriate, assignment). Coupling constants (J) are reported in
Hertz. Chemical shifts are referenced indirectly to TMS via the
2H lock signal and the following abbreviations are used: s, singlet;
d, doublet; t, triplet; m, multiplet; br, broad. NOE effects were
measured from NOESY and ROESY spectra with mixing times
between 0.4 and 1.5 s.
Theoretical conformational analysis indicated that these pho-
tochromic systems should be excellent conformational switch-
ing units when incorporated into a peptide environment. By
small variation of chemical structure a wide range of different
switching units were obtained. Amongst these, 1 is the most rigid,
capable of reversible alternation between a turn-like motif and an
open structure upon irradiation at 300 and 280 nm, respectively.
Compounds 2 and 3 provide the possibility of photochemical
˚
switching between turn-like (Z, Ca–Ca < 5.5 A) and open (E, Ca–
˚
Ca ~ 8.0 A) geometries. The thioaurone derivatives 6 and 7 should
provide access to more rigid open and turn-like motifs. Formation
of turns under experimental conditions was not observed, as can
be expected for small, linear peptides. However, by incorporation
into suitable petidomimetic environments, they should have the
potential to provide access to a variety of photoswitchable target
molecules. This has recently been verified using a congener of 3,12
and further studies are currently in progress.
Experimental
Starting materials were purchased from commercial suppliers and
were used without purification. Column chromatography was
performed using Merck silica gel 60 (40–63mm) or Florisil (100–
200 mesh). Thin-layer chromatography (TLC) was performed
using aluminum sheets precoated with silica gel 60 F254 (0.2 mm,
E. Merck). Chromatographic spots were visualized by UV and/or
spraying with an ethanolic solution of ninhydrin (2%), followed
by heating.
Purification of the peptides was performed on a Gilson 321
HPLC system connected to a Vydac Protein & Peptide C18
(218TP) column (10 mm, 22 ¥ 250 mm) using a gradient of MeCN
in 0.1% aq. TFA (10–85% MeCN in 75 min) at a flow rate of
5 cm3 min-1 and with detection by UV absorbance at 230 nm (LKB
2151 absorbance detector), or on a Gilson system (Gilson 231 XL
Injector, 118 UV/VIS detector, Gilson 402 Syringe pump, Gilson
333 and 334 pumps and Gilson FC204) connected to a Grace
Vydac C18 (22 ¥ 250 mm, 5 mm) with a gradient of MeCN in 0.1%
aq. TFA (20–60% MeCN in 60 min). The fractions were further
analyzed by LC-MS using a Chromolith Performance RP-18e
Melting points were determined in open capillaries using a Stu-
art Scientific melting point apparatus SMP10 and are uncorrected.
Amino acid analyses were performed at the Department of
Biochemistry and Organic Chemistry, Biomedical Centre, Upp-
sala, Sweden, on 24 h hydrolyzates with an LKB 4151 Alpha Plus
analyzer, using ninhydrin detection.
Conformational analysis was performed in MacroModel 9.526
using the OPLS-AA/L force field (OPLS 2005) and the CHCl3
solvation model using the generalized Born solvent-accessible
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surface area (GB/SA) method developed by Still et al.25 The con-
formational search was conducted using the Systematic Unbound
Multiple Minimum search method,35 with 20,000 search steps.
The number of torsion angles allowed to vary simultaneously
during each search step ranged from 1 to n-1, where n is the
total number of investigated torsion angles. Truncated Newton
conjugate gradient (TNCG) energy minimization with a maximum
Z-Stilbene derivative (Z-1)
A solution of E-1 in acetonitrile (24.00 cm3, 2.0 mmol/dm3
solution) was irradiated at 300 nm for 2 h under nitrogen gas flow.
The solution was concentrated under reduced pressure resulting in
22.0 mg of a 7:3 mixture of the cis:trans isomers. Quantum yield:
5.7%. dH (499.5 MHz; CD3OD:CH3OH (1:1)) 0.96 (6H, 2d, J 6.4,
CH3V), 1.41 (3H, d, J 7.4, CH3A), 2.01 (3H, s, CH3CO), 2.14 (1H, m,
CHbV), 2.75 (3H, d, J 4.9, CH3), 4.23 (1H, dd, J 7.3, 7.6, CHaV),
4.49 (1H, dq, J 7.0, 7.6, CHaA), 6.68 (1H, d, J 12.2, CH), 6.70 (1H,
d, J 12.2, CH), 6.96 (1H, d, J 7.8, ArH), 7.19 (1H, t, J 7.8, ArH),
7.30 (1H, t, J 8.3, ArH) 7.36 (1H, d, J 7.8, ArH), 7.37 (1H, d, J
8.3, ArH), 7.47 (1H, br s, ArH), 7.68 (1H, d, J 8.3, ArH), 7.77 (1H,
br s, ArH), 8.3 (1H, d, J 7.6, NHA), 7.93 (1H, q, J 4.9, NHCH3),
8.11 (1H, d, J 7.6, NHV), 9.84 (1H, s, NHPh); m/z (ESI-MS, 30 eV)
465 [M + H]+, 434, 255.
of 500 iterations was used with the derivative convergence criterion
-1
set to 0.05 kJ mol-1 A . Two subsequent energy minimizations
˚
were performed on the found conformations using a derivative
-1
convergence criterion of 0.001 kJ mol-1 A , first using a maximum
˚
of 500 TNCG minimization steps, followed by a maximum of
10,000 PR conjugate gradient minimization steps. Conformations
within 25 kJ mol-1 of the lowest energy minimum found were saved
in the conformational search. The relative population of the found
conformations was calculated using Eq 1,
/ RT
e-DE
i
%populationi =
¥100
(1)
/ RT
e-DE
j
E-Stilbene derivative (E-2)
Â
j
Compound 20 was incorporated into standard solid-phase pep-
tide synthesis techniques using 4-nitro-benzophenoneoxime resin
(1.1 mmol/g) on a 0.55 mmol scale. Standard Boc-Alanine or
N-acetyl-Valine and PyBOP mediated couplings were applied.
The Boc groups were deprotected with 50% trifluoroacetic acid
in dichloromethane and cleavage was performed with 2 M
methylamine in THF, purification by HPLC yielding white crystals
(17.4 mg, 7%); amino acid analysis: Ala, 1.04; Val, 0.96; UV: lmax
(CH3OH)/nm 203, 232, 297, 310, 323 nm; [a]D -5.7 (c 0.008 in
MeOH); dH (499.5 MHz; CD3OD:CH3OH (1:1)) 0.97 (3H, d, J
6.6, CH3V), 0.98 (3H, d, J 6.6, CH3V), 1.35 (3H, d, J 6.9, CH3A),
2.02 (3H, s, CH3CO), 2.10 (1H, dh, J 6.6, 7.4, CHbV), 2.73 (3H, d,
J 2.7, CH3NH), 3.61 (1H, d, J 14.5, CH2CO), 3.61 (1H, d, J 14.5,
CH2CO), 4.19 (1H, dd, J 7.4, 8.1, CHaV), 4.33 (1H, dq, J 6.6, 7.4,
CHaA), 4.40 (1H, d, J 6.0, 15.0, CH2NH), 4.43 (1H, d, J 6.0, 15.0,
CH2NH), 7.17 (1H, d, J 16.0, CH), 7.20 (1H, d, J 16.0, CH), 7.21
(2H, 2dd, J 7.5, 7.5, ArH), 7.31 (2H, 2t, J 7.5, ArH), 7.44 (2H, 2dt,
J 1.5, 7.5, ArH), 7.51 (2H, 2t, J 1.5, ArH), 7.88 (1H, br q, J 2.7,
NHCH3), 8.05 (1H, d, J 8.1, NHV), 8.28 (1H, d, J 6.6, NHA), 8.56
(1H, t, J 6.0, NHCH2); dC (75 MHz, CD3OD:CH3OH (1:1)) 17.0,
17.5, 18.5, 21.3, 25.2, 30.5, 42.2, 42.7, 49.4, 59.5, 125.0, 125.3,
125.4, 126.7, 127.1, 128.3, 128.6 (2C), 128.7 (2C), 136.0, 137.9,
139.1, 146.7, 172.3, 172.5, 174.4, 175.8; m/z (ESI-MS, 30 eV) 986
[2M + H]+, 494 [M + H]+, 258.
where DE is the relative potential energy of the conformation, R
is the universal gas constant and T is the temperature (T = 298 K
was used in the calculations). The conformations were classified
as folded or non-folded depending on interstrand hydrogen bond
pattern and distance. Hydrogen bonds were defined as an acceptor
˚
atom and amide proton at a distance below 2.5 A. The additional
criteria for optimal hydrogen bonds in a beta sheet were defined
◦
20
◦
=
as the angle N–H ◊ ◊ ◊ O > 155 and angle N ◊ ◊ ◊ O C between 135–
165◦.34 The criteria for folded conformations based on interstrand
˚
distance were C–C < 5.5 A (see Table 1 for details).
E-Stilbene derivative (E-1)
A solution of 45.0 mg of 14 (0.14 mmol) in dichloromethane
(1.0 cm3) was treated with N,O-bis(trimethylsilyl)acetamide
(0.07 cm3, 0.28 mmol) for 30 minutes. 2-Acetylamino-3-methyl-
butyric acid (0.11 g, 0.70 mmol) was dissolved in a mixture of
dichloromethane (2.0 cm3), dimethylformamide (1.00 cm3) and
N,N-diisopropylethylamine (0.24 cm3, 1.40 mmol). HATU (0.26 g,
0.70 mmol) was added and the mixture was stirred for 20 minutes.
The two solutions were combined and the mixture was stirred for
18 h. Thereafter, the mixture was poured into 0.1 M HCl (10 cm3)
and was extracted three times with dichloromethane (10 cm3). A
precipitate was filtered off from the◦organic layer yielding a white
solid (32.0 mg, 49%); mp 198–199 C; amino acid analysis: Ala,
1.00; Val, 1.00; lmax (CH3OH)/nm 246, 292; [a]D20 +13.45 (c 0.001
in MeOH); dH (499.5 MHz; CD3OD:CH3OH (1:1)) 1.03 (6H, 2d,
J 6.8, CH3V), 1.46 (3H, d, J 7.2, CH3A), 2.03 (3H, s, CH3CO), 2.14
(1H, m, CHbV), 2.76 (3H, d, J 4.0, CH3NH), 4.28 (1H, dd, J 6.8,
7.7, CHaV), 4.55 (1H, dq, J 7.0, 7.2, CHaA), 7.25 (1H, d, J 16.5,
CH), 7.26 (1H, d, J 16.5, CH), 7.31 (1H, t, J 7.9, ArH), 7.33 (1H,
m, ArH), 7.43 (1H, m, ArH), 7.46 (1H, t, J 7.7, ArH), 7.71 (1H,
m, ArH), 7.76 (1H, ddd, J 1.1, 1.6, 7.7, ArH), 7.85 (1H, dd, J
0.9, 1.5, ArH), 8.0 (1H, q, J 4.0, NHCH3), 8.10 (1H, t, J 1.6, ArH),
8.18 (1H, d, J 6.8, NHV), 8.54 (1H, d, J 7.2, NHA), 9.96 (1H, br s,
NHPh); dC (75 MHz; DMSO-d6) 18.6, 19.1, 19.8, 23.0, 26.2, 31.1,
49.6, 59.4, 117.9, 119.6, 122.2, 125.9, 127.5, 128.6, 129.2, 129.7,
129.8, 130.0, 135.1, 137.4, 137.9, 139.8, 166.4, 170.0, 171.1, 173.2;
m/z (ESI-MS, 30 eV) 465 [M + H]+, 434, 255.
Z-Stilbene derivative (Z-2)
A solution of 8.2 mg (16.60 mmol) E-2 in acetonitrile was irradiated
at 300 nm for 1.5 h under nitrogen gas flow. The solution was
concentrated under reduced pressure resulting in a 8.3:1.7 mixture
of the cis:trans isomers. Quantum yield: 3.9%. dH (499.5 MHz;
CD3OD:CH3OH (1:1)) 0.89 (6H, 2d, J 6.8, CH3V), 1.29 (3H, d, J
6.0, CH3A), 1.98 (1H, dh, J 6.8, 7.5, CHbV), 2.00 (3H, s, CH3CO),
2.72 (3H, d, J 5.0, CH3NH), 3.45 (1H, d, J 14.5, CH2CO), 3.48 (1H,
d, J 14.5, CH2CO), 4.16 (1H, dd, J 7.5, 8.5, CHaV), 4.23–4.32 (3H,
m, CH2NH, CHaA), 6.58 (1H, d, J 12.6, CH), 6.60 (1H, d, J 12.6,
CH), 7.07–7.18 (8H, m, ArH), 7.88 (1H, br q, J 5.0, NHCH3), 7.97
(1H, d, J 8.1, NHV), 8.20 (1H, d, J 6.0, NHA), 8.44 (1H, t, J 5.8,
NHCH2).
4364 | Org. Biomol. Chem., 2008, 6, 4356–4373
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E-Stilbene derivative (E-3)
(1H, br t, J 6.3, NH), 9.08 (1H, d, J 7.2, NHA), 12.66 (1H, br s,
OHA); m/z (ESI-MS, 30 eV) 523.90 [M + H]+, 540.95.
Stilbene derivative 27 was incorporated into solid-phase peptide
synthesis using 4-nitro-benzophenoneoxime resin (1.1 mmol/g) on
a 0.55 mmol scale. Boc-Ala, N-acetyl-Valine and PyBOP mediated
couplings were applied. The Boc groups were deprotected with
50% trifluoroacetic acid in dichloromethane and cleavage was
performed with 2 M methylamine in THF, purification on HPLC
yielded white crystals (33.6 mg, 12%); amino acid analysis:
Val, 1.04; Ala, 0.96; UV: lmax (CH3OH)/nm 205, 232, 298,
E-para-Thioaurone derivative (E-4)
The chemical shifts were elucidated from a spectrum with a 0.6:1
mixture of Z:E. dH (499.9 MHz; DMSO-d6) 0.85 (1H, dd, J 1.7,
V
6.9, CHg ), 1.43 (1H, d, J 7.4 CHbA), 1.89 (3H, s, CH3), 1.98 (1H,
dq, J 6.9, CHbV), 4.16 (1H, dd, J 6.9, 6.9, CHaV), 4.34 (2H, t, J 6.7,
CH2), 4.46 (1H, q, J 7.4, CHaA), 7.32 (2H, XX¢ part of AA¢XX¢,
310, 323; [a]D = -11.9◦ (c 0.004 in MeOH); dH (499.5 MHz;
20
=
ArH), 7.50 (1H, t, J 7.7, ArH), 7.59 (1H, s, C CH), 7.89 (1H, d,
CD3OD:CH3OH (1:1)) 0.88 (3H, d, J 7.0, CH3V) 0.89 (3H, d, J
7.0, CH3V), 1.25 (3H, d, J 7.0, CH3A), 1.98 (3H, s, CH3CO), 2.01
(1H, dh, J 7.0, 7.4, CHbV), 2.57 (2H, dt, J 2.5, 8.3, CH2CH2CO), 2.69
(3H, d, J 4.5, CH3NH), 2.83 (2H, t, J 7.4, CH2CH2NH), 2.94 (2H, t, J
7.4, CH2CO), 3.51 (2H, m, CH2NH), 4.09 (1H, dd, J 7.4, 8.5, CHaV),
4.29 (1H, dq, J 5.5, 7.0, CHaA), 7.12 (2H, 2d, J 8.0, ArH), 7.15 (2H,
m, CH), 7.27 (2H, 2t, J 8.0, ArH), 7.39 (2H, 2d, J 8.0, ArH), 7.42
(2H, br s, ArH), 7.64 (1H, br q, J 4.5, NHCH3), 7.94, (1H, d, J 8.5,
NHV), 8.09 (2H, m, NHA, NHCH2); dC (75 MHz; CD3OD:CH3OH
(1:1)) 17.1, 17.5, 18.7, 21.5, 25.4, 30.6, 31.5, 35.3, 37.4, 40.6, 49.1,
59.4, 124.4, 124.5, 126.5, 126.9, 127.7, 128.1, 128.5, 128.6, 128.72,
128.74, 137.80, 137.81, 139.7, 141.4, 172.1, 172.5, 173.8, 174.4;
m/z (ESI-MS, 30 eV) 1042 [2M + H]+, 521 [M + H]+, 450.
J 8.5, NHV), 8.16 (2H, AA’ part of AA¢XX¢, ArH), 8.37 (1H, dd,
J 1.0, 7.7, ArH), 8.51 (1H, br t, J 6.3, NH), 9.02 (1H, d, J 7.2,
NHA), 12.64 (1H, br s, OHA).
Z-meta-Thioaurone derivative (Z-5)
Compound 40 was incorporated into standard solid-phase
peptide synthesis techniques using 2-chlorotrityl chloride resin
(1.4 mmol/g) on 0.5 mmol scale. Standard Fmoc-protected
monomers and PyBOP mediated couplings were applied. The
Fmoc groups were deprotected with 20% piperidine in DMF
and cleavage from resin was performed with 0.5% TFA in
dichloromethane. After purification on HPLC and lyophilization
a yellow, fluffy solid was obtained (13.0 mg, 5%); amino acid
analysis: Val, 0.92; Ala, 1.08; dH (499.9 MHz; DMSO-d6) 0.85
Z-Stilbene derivative (Z-3)
V
(1H, dd, J 1.7, 6.9, CHg ), 1.46 (1H, d, J 7.4, CHbA), 1.89 (3H, s,
CH3), 1.99 (1H, dq, J 6.9, CHbV), 4.16 (1H, dd, J 6.9, 6.9, CHaV),
4.33 (1H, dd, J 5.8, 15.4, CH2), 4.37 (1H, dd, J 5.8, 15.4, CH2),
4.48 (1H, q, J 7.4, CHaA), 7.39 (1H, m, ArH), 7.55 (1H, t, J 7.6,
A solution of compound E-3 (18.1 mg) dissolved in acetonitrile
was irradiated at 300 nm for 1.5 h under nitrogen gas flow. The
solution was concentrated under reduced pressure resulting in a
49:51 mixture of the cis:trans isomers. Quantum yield: 7.8%. dH
(499.5 MHz; CD3OD:CH3OH (1:1)) 0.89 (3H, d, J 6.5, CH3V),
0.90 (3H, d, J 6.5, CH3V), 1.25 (3H, d, J 7.5, CH3A), 1.98 (3H, s,
CH3CO), 2.01 (1H, dh, J 6.5, 7.4, CHbV), 2.46 (1H, dt, J 2.5, 8.3,
CH2CH2CO), 2.70 (3H, d, J 4.5, CH3NH), 2.83 (1H, t, J 7.4, CH2CH2NH),
2.94 (2H, t, J 7.4, CH2CO), 3.44 (2H, m, CH2NH), 4.09 (1H, dd, J
7.4, 8.5, CHaV), 4.29 (1H, dq, J 5.5, 7.5, CHaA), 6.57 (1H, d, J
13.2, CH), 6.59 (1H, d, J 13.2, CH), 7.05 (4H, m, ArH), 7.09 (2H,
2t, J 1.5, 6.5, ArH), 7.13–7.15 (2H, m, ArH), 7.71 (1H, br q, J 4.5,
NHCH3), 7.94 (1H, d, J 7.2, NHV), 8.03 (1H, br t, J 5.0, NHCH2),
8.06 (1H, d, J 7.5, NHA).
ArH), 7.56 (1H, t, J 7.6, ArH), 7.66 (1H, m, ArH), 7.73 (1H, m,
V
=
ArH), 7.83 (1H, s, C CH), 7.91 (1H, d, J 8.7, NH ), 8.05 (1H, dd,
J 1.0, 7.6, ArH), 8.41 (1H, dd, J 1.0, 7.6, ArH), 8.57 (1H, br t, J
5.8 Hz, NH), 9.08 (1H, d, J 7.4, NHA), 12.66 (1H, br s, OHA); m/z
(ESI-MS, 30 eV) 523.90 [M + H]+, 540.95.
E-meta-Thioaurone derivative (E-5)
The chemical shifts were elucidated from a spectrum with a 0.3:1
mixture of Z:E. dH (499.9 MHz; DMSO-d6) 0.83 (1H, dm, J 6.9,
V
CHg ), 1.43 (1H, d, J 0.7, 7.4, CHbA), 1.87 (3H, d, J 0.9,CH3),
1.99 (1H, dq, J 6.9, CHbV), 4.16 (1H, dd, J 6.9, 6.9, CHaV), 4.32
(1H, d, J 5.8, CH2), 4.46 (1H, q, J 7.4, CHaA), 7.34 (1H, dm, J
Z-para-Thioaurone derivative (Z-4)
7.6, ArH), 7.40 (1H, t, J 7.6, ArH), 7.50 (1H, t, J 7.6, ArH), 7.57
V
=
Compound 34 was incorporated into standard solid-phase
peptide synthesis techniques using 2-chlorotrityl chloride resin
(1.4 mmol/g) on 0.5 mmol scale. Standard Fmoc-protected
monomers and PyBOP mediated couplings were applied. The
Fmoc groups were deprotected with 20% piperidine in DMF
and cleavage from resin was performed with 0.5% TFA in
dichloromethane. After purification on HPLC and lyophilization a
yellow fluffy solid was obtained (9.0 mg, 8%); amino acid analysis:
Val, 0.92; Ala, 1.08; dH 499.9 MHz; DMSO-d6) (0.85 (1H, dd, J
(1H, s, C CH), 7.89 (1H, d, J 8.7, NH ), 7.94 (1H, dt, J 1.0, 7.6,
ArH), 7.95 (1H, m, ArH), 8.10 (1H, dm, J 8.05, ArH), 8.37 (1H,
dt, J 1.0, 7.6, ArH), 8.51 (1H, br t, J 5.8, NH), 9.01 (1H, d, J 7.4,
NHA), 12.66 (1H, br s, OHA).
Z-para-Thioaurone derivative (Z-6)
Compound 34 was incorporated into standard solid-phase
peptide synthesis techniques using {3-[(methyl-Fmoc-amino)-
methyl]-indol-1-yl}-acetyl AM resin (0.63 mmol/g) on 0.3 mmol
scale. Standard Fmoc-protected monomers and PyBOP mediated
couplings were applied. The Fmoc groups were deprotected with
20% piperidine in DMF and cleavage from resin was performed
with 5% TFA in dichloromethane. After purification on HPLC
and lyophilization a yellow, fluffy solid was obtained (29.0 mg,
V
1.7, 6.9, CHg ), 1.46 (1H, d, J 7.4, CHbA), 1.89 (3H, s, CH3), 1.99
(1H, dq, J 6.9, CHbV), 4.16 (1H, dd, J 6.9, 6.9, CHaV), 4.34 (2H,
d, J 6.1, CH2), 4.48 (1H, q, J 7.4, CHaA), 7.45 (2H, XX¢ part of
AA¢XX¢, ArH), 7.55 (1H, t, J 7.7, ArH), 7.78 (2H, AA’ part of
V
=
AA¢XX¢, ArH), 7.87 (1H, s, C CH), 7.91 (1H, d, J 8.5, NH ),
8.05 (1H, dd, J 1.0, 7.7, ArH), 8.40 (1H, dd, J 1.2, 7.7, ArH), 8.55
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17%); amino acid analysis: Val, 0.88; Ala, 1.12; dH (499.9 MHz;
CH3CO-V-DP-G-A-NHCH3 (8)
V
DMSO-d6) 0.86 (1H, dd, J 2.2, 6.9, CHg ), 1.37 (1H, d, J 7.4,
The peptide 8 was prepared by standard solid-phase peptide
synthesis techniques using 4-nitro-benzophenone-oxime resin
(1.1 mmol/g) on 0.55 mmol scale with standard Boc-protected
monomers or N-acetyl-Valine and PyBOP mediated couplings.
For deprotection of the Boc groups 50% trifluoroacetic acid
in dichloromethane was applied and cleavage was performed
with 2M methylamine in THF, purification on HPLC yielding
a colorless oil (85.0 mg, 39%); amino acid analysis: Val, 1.04;
CHbA), 1.89 (3H, s, CH3), 1.99 (1H, q, J 6.9, CHbV), 2.62 (3H, d, J
4.6, CH3), 4.17 (1H, dd, J 6.9, 6.9, CHaV), 4.34 (2H, d, J 6.0, CH2),
4.48 (1H, q, J 7.4, CHaA), 7.55 (1H, t, J 7.7, ArH), 7.45 (2H, XX¢
part of AA¢XX¢), 7.78 (2H, AA¢ part of AA¢XX¢), 7.87 (1H, s,
V
C CH), 7.93 (1H, d, J 8.7, NH ), 7.94 (1H, q, J 4.6, NHcap), 8.04
=
(1H, dd, J 1.3, 7.7, ArH), 8.40 (1H, dd, J 1.3, 7.7, ArH), 8.57 (1H,
br t, J 6.0, NH), 8.93 (1H, d, J 7.4, NHA); m/z (ESI-MS, 30 eV)
537.11 [M + H]+, 554.15, 1073.18 [2M + H]+, 1090.22.
◦
20
Pro, 1.01; Gly, 0.96; Ala, 0.99; [a]D -9.97 (c 0.02 in MeOH); dH
(499.5 MHz; CD3OD:CH3OH (1:1),) 0.97 (3H, d, J 6.5, CH3V),
1.00 (3H, d, J 6.5, CH3V), 1.42 (3H, d, J 7.0, CH3A), 2.00 (3H, s,
E-para-Thioaurone derivative (E-6)
CH3CO), 2.06 (1H, dh, J 6.5, 7.0, CHbV), 2.10–2.28 (4H, m, CHb ,
P
The chemical shifts were elucidated from a spectrum of a 0.6:1
P
CHb¢P, CHg , CHg¢P), 2.70 (3H, d, J 4.0, CH3NH), 3.71 (1H, m,
mixture of Z:E. dH (499.9 MHz; DMSO-d6) 0.86 (1H, dd, J 2.2,
CHd¢P), 3.79 (1H, dd, J 6.0, 17.0, CHa¢G), 3.86 (1H, dd, J 6.0,
V
6.9, CHg ), 1.35 (1H, d, J 7.4, CHbA), 1.88 (3H, s, CH3), 1.98 (1H,
P
17.0, CHaG), 3.94 (1H, m, CHd ), 4.2 (1H, m, CHaP), 4.21 (1H,
q, J 6.9, CHbV), 2.60 (3H, d, J 4.6, CH3), 4.16 (1H, dd, J 6.9, 6.9,
dq, J 7.0, 7.5, CHaA), 4.44 (1H, dd, J 7.0, 7.5, CHaV), 7.66 (1H,
q, J 4.0, NHCH3), 7.95 (1H, d, J 7.5, NHA), 8.15 (1H, d, J 7.5,
NHV), 8.44 (1H, t, J 6.0, NHG); dC (75 MHz; CDCl3) 16.7, 19.1,
19.4, 22.4, 25.0, 26.4, 29.5, 30.4, 43.1, 48.1, 49.1, 58.2, 61.6, 170.1,
172.6, 172.7, 173.0, 173.5; m/z (ESI-MS, 30 eV) 796 [2M + H]+,
398 [M + H]+, 341.
CHaV), 4.3 (2H, d, J 6.0, CH2), 4.46 (1H, q, J 7.4, CHaA), 7.32
(2H, XX¢ part of AA¢XX¢), 7.48 (1H, t, J 7.7, ArH), 7.58 (1H, s,
V
C CH), 7.92 (1H, d, J 8.7, NH ), 7.94 (1H, q, J 4.6, NHcap), 7.94
=
(1H, dd, J 1.3, 7.7, ArH), 8.16 (2H, AA¢ part of AA¢XX¢), 8.42
(1H, dd, J 1.3, 7.7, ArH), 8.54 (1H, br t, J 6.0, NH), 8.85 (1H, d,
J 7.4, NHA).
Diphenylacetylene derivative (10)
Z-meta-Thioaurone derivative (Z-7)
Compound 46 was incorporated into standard solid-phase
peptide synthesis techniques 4-nitro-benzophenoneoxime resin
(1.1 mmol/g) on 0.55 mmol scale. Standard Boc-protected
monomers or N-acetyl-Valine and PyBOP mediated couplings
were applied. The Boc groups were deprotected with 50% triflu-
oroacetic acid in dichloromethane and cleavage was performed
with 2M methylamine in THF, purification on HPLC and by
recrystallization from a mixture of methanol, acetonitrile and
Compound 40 was incorporated into standard solid-phase
peptide synthesis techniques using {3-[(methyl-Fmoc-amino)-
methyl]-indol-1-yl}-acetyl AM resin (0.63 mmol/g) on 0.3 mmol
scale. Standard Fmoc-protected monomers and PyBOP mediated
couplings were applied. The Fmoc groups were deprotected with
20% piperidine in DMF and cleavage from resin was performed
with 5% TFA in dichloromethane. After purification on HPLC
and lyophilization a yellow, fluffy solid was obtained (29.0 mg,
11%); amino acid analysis: Val, 0.87; Ala, 1.12; dH (499.9 MHz;
trifluoroacetic acid yielding white crystals (76.0 mg, 28%); mp
246–248 ◦C; amino acid analysis: Val, 1.00; Ala, 1.00; [a]D
=
20
V
27.8◦ (c 0.001 in MeOH); dH (499.9 MHz; DMSO-d6) 0.83 (6H,
2d, J 6.5, CH3V), 1.14 (3H, d, J 7.0, CH3A), 1.86 (3H, s, CH3CO),
1.97 (1H, dh, J 6.0, 7.5, CHbV), 2.54 (3H, d, J 5.4, CH3NH), 3.74
(1H, d, J 15.5, CH2bCO), 3.79 (1H, d, J 15.5, CH2aCO), 3.94 (1H,
DMSO-d6) 0.84 (1H, dd, J 6.9, 1.0, CHg ), 1.37 (1H, d, J 7.4,
CHbA), 1.89 (3H, s, CH3), 2.01 (1H, q, J 6.9, CHbV), 2.62 (3H, d,
J 4.6, CH3), 4.15 (1H, dd, J 6.9, 6.9, CHaV), 4.37 (2H, dd, J 6.0,
15.4, CH2), 4.33 (2H, dd, J 6.0, 15.4, CH2), 4.48 (1H, q, J 7.4,
CHaA), 7.39 (1H, dm, J 7.6, ArH), 7.55 (1H, t, J 7.6, ArH), 7.67
P
m, CHd ), 4.20 (1H, m, CHaP), 4.20 (1H, dd, J 7.5, 9.0, CHaV),
4.23 (1H, dq, J 7.0, 7.2, CHaA), 4.47 (1H, dd, J 6.1, 15.5, NHCH2b),
4.52 (1H, dd, J 6.1, 15.5, NHCH2a), 7.79 (1H, q, J 5.4, NHCH3), 7.93
(1H, d, J 9.0, NHV), 8.20 (1H, d, J 7.2, NHA),8.50 (1H, t, J 6.1,
NHCH2); dC (75 MHz; DMSO-d6) 18.9, 19.1, 20.0, 26.2, 30.9, 35.0,
48.9, 58.6, 70.5, 91.4, 93.2, 121.8, 123.4, 127.4, 127.6, 127.9, 129.4,
130.6, 132.6, 132.7, 138.7, 141.1, 141.2, 169.9, 170.1, 172.1, 173.2;
m/z (ESI-MS, 30 eV) 982 [2M + H]+, 491 [M + H]+, 420.
=
(1H, m, ArH), 7.73 (1H, dm, J 7.6, ArH), 7.83 (1H, s, C CH),
7.92 (1H, d, J 8.7, NHV), 7.94 (1H, q, J 4.6, NHcap), 8.04 (1H, dd,
J 1.2, 7.6, ArH), 8.45 (1H, dd, J 1.2, 7.6, ArH), 8.57 (1H, br t, J
6.0, NH), 8.93 (1H, d, J 7.4, NHA); m/z (ESI-MS, 30 eV) 537.11
[M + H]+, 554.15, 1073.18 [2M + H]+, 1090.22.
E-meta-Thioaurone derivative (E-7)
The chemical shifts were elucidated from a spectrum of a 0.6:1
Methyl-3-vinyl-benzoate (11)
mixture of Z:E. dH (499.9 MHz; DMSO-d6) 0.83 (dd, 1H, J 6.9,
V
1.0, CHg ), 1.35 (1H, d, J 7.4, CHbA), 1.87 (3H, s, CH3), 1.99 (1H,
3-Vinyl-benzoic acid (2.00 g, 13.50 mmol) was dissolved in
methanol (100 cm3). Concentrated HCl (1.15 cm3, 13.50 mmol)
was added dropwise to the stirred solution. The reaction mixture
was refluxed for 14 h, then the solvent was removed by rotary
evaporation. The residual was dissolved in a mixture of water
(20 cm3) and diethyl ether (20 cm3) and the phases were allowed
to separate. The water phase was reextracted two times. The
combined ether phases were concentrated on a rotary evaporator
q, J 6.9, CHbV), 2.62 (3H, d, J 4.6, CH3), 4.16 (1H, dd, J 6.9, 6.9,
CHaV), 4.32 (2H, d, J 6.0, CH2), 4.46 (1H, q, J 7.4, CHaA), 7.34
(1H, dm, J 7.6, ArH), 7.40 (1H, t, J 7.6, ArH), 7.49 (1H, t, J 7.6,
V
=
ArH), 7.56 (1H, s, C CH), 7.89 (1H, d, J 8.7, NH ), 7.94 (1H, q,
J 4.6, NHcap), 7.94 (1H, dd, J 1.2, 7.6, ArH), 7.96 (1H, m, ArH),
8.10 (1H, dm, J 7.6, ArH), 8.42 (1H, dd, J 7.6, 1.2, ArH), 8.51
(1H, br t, J 6.0, NH), 8.85 (1H, d, J 7.4, NHA).
4366 | Org. Biomol. Chem., 2008, 6, 4356–4373
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giving 3-vinyl-benzoic acid methyl ester as an oil (1.94 g, 88%); dH
(270.2 MHz; CDCl3) 3.92 (3H, s, CH3), 5.31 (1H, d, J 11.0, CH2),
5.82 (1H, d, J 18.0, CH2), 6.74 (1H, dd, J 11.0, 18.0, CH), 7.39
(1H, dd, J 8.9, 9.2, ArH), 7.58 (1H, d, J 9.2, ArH), 7.92 (1H, d,
J 8.9, ArH), 8.08 (1H, br s, ArH); dC (67.9 MHz; CDCl3) 56.6,
119.5, 131.7, 133.0, 133.2, 134.8, 134.9, 140.3, 142.2, 171.4; m/z
(EI, 70 eV) 162 [M + H]+, 131, 117, 103.
E-3-[2¢-(3¢¢-Amino-phenyl)-vinyl]-N-(1¢¢¢¢-methyl-carbamoyl-
ethyl)-benzamide (14)
3-(3¢-Amino-phenylethenyl)-benzoic acid (0.20 g, 0.84 mmol) was
dissolved in DIEA (1.50 cm3) and dichloromethane (15 cm3).
PyBOP (0.87 g, 1.67 mmol) was added and the mixture was
stirred for 2 minutes. Then, a solution of 2-amino-N-methyl-
propionamide (170.0 mg, 1.67 mmol), diisopropyl-ethylamine
(0.50 cm3), dichloromethane (2.5 cm3) and dimethylformamide
(5 cm3) was added and the mixture was stirred for 2 h. The reaction
mixture was poured into 0.1 M HCl (10 cm3). The mixture was ex-
tracted three times with dichloromethane (10 cm3). The combined
organic layers were washed with conc. aqueous NaHCO3 (10 cm3),
re-extracting the aqueous phase twice with dichloromethane.
Thereafter, water (10 cm3) was added to the organic layer. The
water phase was extracted with three portions of dichloromethane
(10 cm3) before the organic layers were concentrated on a rotary
evaporator. The residue was purified by column chromatography
using ethyl acetate:diethylamine:methanol (1:0.01:0.01) eluent
mixture. The residual was precipitated from chloroform yielding
a white solid (45.00 mg, 17%); dH (270.2 MHz; CDCl3) 1.44 (3H,
d, J 6.9, CH3NH), 2.76 (3H, s, CH3CO), 4.65 (1H, q, J 6.9, CHaA),
6.62 (1H, ddd, J 1.0, 2.0, 7.9, ArH), 6.83 (1H, t, J 2.0, ArH), 6.90
(1H, ddd, J 1.0, 2.0, 7.9, ArH), 7.00 (1H, d, J 16.6, CH), 7.01 (1H,
d, J 16.6, CH), 7.11 (1H, t, J 7.9, ArH), 7.34 (1H, t, J 7.8, ArH),
7.38 (1H, br q, J 6.9 NHCH3), 7.55 (1H, ddd, J 1.2, 1.7, 7.8, ArH),
7.64 (1H, ddd, J 1.2, 1.7, 7.8, ArH), 7.68 (1H, d, J 6.9 NHA),
7.91 (1H, t, J 1.7, ArH); dC (67.9 MHz; CDCl3) 18.2; 26.0, 49.0,
113.3, 115.3, 117.7, 125.1, 125.9, 127.3, 128.7, 129.5, 129.6, 129.9,
133.8, 137.7, 137.8, 145.8, 167.3, 173.3; m/z (ESI-MS, 30 eV) 324
[M + H]+, 293, 265, 222.
E-3-(3¢-Amino-phenylethenyl)-benzoic acid methyl ester (12)
Methyl-3-vinyl-benzoate (1.00 g; 6.17 mmol), 3-iodoaniline
(0.65 cm3, 5.61 mmol), Pd(OAc)2 (50.00 mg, 0.22 mmol), triethy-
lamine (1.40 cm3, 9.81 mmol), and dimethylformamide (1.50 cm3)
was added to a Smith Process Vial and was heated to 150 ◦C
for 20 min with microwave irradiation. The procedure was
repeated two times and the reaction mixtures were combined.
The mixture was filtered through Celite to a separation funnel
with dichloromethane (25 cm3). 0.1 M HCl (25 cm3) was added
and the phases were allowed to separate. The aqueous phase was
reextracted two times. Thereafter water (25 cm3) was added to
the combined organic layers. The water phase was extracted with
three portions of dichloromethane (25 cm3) before the organic
layers were concentrated on a rotary evaporator. The residue
was purified by column chromatography using hexane:ethyl ac-
etate:dichloromethane: triethylamine (3:1:1:0.05) eluent mixture.
The fractions containing product were concentrated on a ro-
tary evaporator giving E-3-(3-amino-phenylethenyl)-benzoic acid
methyl ester as yellow solid (2.38 g, 56%); nmax (CHCl3)/cm-1 3451,
3401, 3027, 2977, 1718, 1619, 1288, 1238; UV: (CH3OH)/nm 232,
294; dH (270.2 MHz; CDCl3) 3.92 (3H, s, CH3), 6.62 (1H, ddd, J
1.0, 2.1, 7.8, ArH), 6.82 (1H, t, J 2.1, ArH), 6.92 (1H, dd, J 1.7,
7.6, ArH), 7.05 (1H, d, J 17.6, CH), 7.06 (1H, d, J 17.6, CH),
7.14 (1H, t, J 7.8, ArH), 7.39 (1H, t, J 7.7, ArH), 7.63 (1H, dt,
J 1.0, 7.8, ArH), 7.86 (1H, dt, J 1.7, 7.6, ArH), 8.12 (1H, t, J
1.7, ArH); dC (67.9 MHz; CDCl3) 52.1, 113.7, 115.6, 118.2, 127.3,
127.4, 128.3, 128.6, 129.5, 129.8, 130.3, 130.8, 137.5, 137.9, 145.1,
167.2; m/z (EI, 70 eV) 253, 220, 193, 177, 165. HRMS, m/z =
210.1248 ([M + H]+), C15H16N requires 210.1282.
3-Iodobenzylamine (15)
A mixture of 3-iodobenzyl bromide (5.00 g, 16.84 mmol), sodium
bis(trimethylsilyl)amide (9.26 g, 50.51 mmol) and hexamethyl-
disilazan (24.86 cm3, 117.87 mmol) was stirred for 72 h. The
crude mixture was extracted with conc. NaHCO3 and CH2Cl2,
re-extracting the water phase three times. Then, the combined
organic phases were extracted with aqueous HCl (5M, pH = 2).
A precipitate was formed that could be dissolved in a mixture
of CHCl3 and 1M HCl. By adding NaOH pellets, the pH of
the combined water phase was increased to 14, and thereafter
it was extracted with CH2Cl2. The organic phase was filtered
through MgSO4 and concentrated under reduced pressure yielding
a yellowish oil (2.47 g, yield: 63%); nmax (neat)/cm-1 3280, 3051,
2853, 1589, 1561, 1061, 994, 880, 769, matching literature data;36
dH (270.2 MHz; CDCl3) 1.53 (2H, s, NH2), 3.64 (2H, s, CH2),
6.92 (1H t, J 7.7, ArH), 7.12 (1H ddd, J 0.7, 1.8, 7.7 ArH), 7.43
(1H ddd, J 0.7, 0.6, 7.7, ArH), 7.54 (1H dd, J 0.6, 1.8, ArH); dC
(100.5 MHz; CDCl3) 45.8, 94.9, 126.0, 130.0, 135.8, 136.1, 145.7;
m/z (EI, 70 eV) 233 [M + H]+, 217, 127, 106, 77.
E-3-(3¢-Amino-phenylethenyl)-benzoic acid (13)
3-(3¢-Amino-phenylethenyl)-benzoic acid methyl ester (600.0 mg,
2.37 mmol) and aqueous 6M NaOH (50 cm3) were stirred in a
round bottom flask at 100 ◦C for 6 h. The heating was turned off
and the reaction mixture was stirred at room temperature for 14 h.
The mixture was poured into water in a separation funnel and the
pH was decreased to 7 using 5 M HCl (aq). A yellow solid was
filtered off and by adding additional HCl the pH was increased
to 4, yielding yellow precipitate (280.0 mg, 98%); dH (270.2 MHz;
CD3OD) 6.63 (1H, ddd, J 1.0, 2.3, 7.8, ArH), 6.90 (1H, dt, J 1.5,
7.6, ArH), 6.95 (1H, t, J 2.3, ArH), 7.08 (1H, t, J 7.8, ArH), 7.11
(1H, d, J 16.5, CH), 7.12 (1H, d, J 16.5, CH), 7.33 (1H, t, J 7.6,
ArH), 7.55 (1H, dt, J 1.0, 7.8, ArH), 7.81 (1H, dt, J 1.5, 7.6,
ArH), 8.12 (1H, t, J 1.5, ArH); dC (67.9 MHz; CD3OD) 113.1,
114.9, 116.8, 126.9, 127.7, 127.8, 127.9, 128.0, 129.0 (2C), 137.2,
138.4, 138.3, 147.6, 174.0; m/z (ESI-MS, 30 eV) 240 [M + H]+,
214. HRMS, m/z = 240.0970 ([M + H]+), C15H14NO2 requires
240.1025.
3-Vinylbenzylamine (16)
3-Iodobenzylamine (0.50 g, 2.15 mmol), tributylvinyltin (1.02 g,
3.22 mmol), PdCl2(PPh3)2 (30.1 mg, 0.04 mmol), lithium chloride
(272.8 mg, 6.44 mmol) and DMF (1.5 cm3) were mixed in a
◦
Smith Process Vial and irradiated at 130 C for 20 minutes with
microwaves, repeating the procedure five times (the total amount
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of 3-iodobenzylamine used being 2.47 g). The combined products
from five runs were filtered through Celite and extracted with conc.
aqueous NaHCO3. Thereafter, the organic phase was extracted
with 1M aqueous HCl and the water phase was kept. By adding
NaOH pellets, the pH of the aqueous solution was increased to
14, followed by extraction with CH2Cl2. The organic phase was
filtered through MgSO4 and concentrated under reduced pressure
yielding a yellowish oil (1.18 g, 84%); dH (270.2 MHz; CDCl3) 1.66
(2H, s, NH2), 2.71 (2H, s, CH2), 5.14 (1H, d, J 10.9 CH2), 5.67
(1H, d, J 18.0, CH2), 6.61 (1H, dd, J 10.9, 18.0, CH), 7.0–7.2 (3H,
m, ArH), 7.83 (1H, br s, ArH); dC (67.9 MHz; CDCl3) 45.3, 112.8,
123.9, 125.7, 127.6, 127.8, 135.8, 136.6, 142.6. m/z (ESI-MS, 30
eV) 132 [M + H]+, 115, 105, 77, 51.
the aqueous phase twice. The combined organic layers were
washed three times with water (25 cm3) and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy using hexane:ethyl acetate:dichloromethane:triethylamine,
(3:1:1:0.05) eluent mixture, yielding a yellowish oil (696.9 mg,
70%); nmax (CHCl3)/cm-1 3446, 3032, 2971, 2894, 1731, 1501, 1251,
1171; dH (399.8 MHz; CDCl3) 1.41 (9H, s, CH3Boc), 3.56 (2H, br s,
CH2NH), 3.62 (3H, s, CH3), 4.23 (2H, s, CH2CO), 5.25 (1H, br s, NH),
6.80–7.10 (2H, m, ArH), 7.09 (2H, 2d, J 6.8, ArH), 7.20–7.25 (2H,
m, ArH), 7.27–7.35 (4H, m, ArH); dC (100.5 MHz, CDCl3) 28.4
(3C), 41.1, 44.6, 52.0, 79.4, 125.4, 125.5, 125.6, 126.8, 127.5, 128.5,
128.6, 128.8, 128.9, 129.0, 134.5, 137.5, 137.6, 139.7, 156.1, 171.9;
m/z (ESI-MS, 30 eV) 763 [2M + H]+, 707, 663, 607, 382 [M + H]+,
367.
(3-Vinyl-benzyl) carbamic acid tert-butyl ester (17)
(3-{2¢-[3¢¢-(tert-Butoxycarbonylaminomethyl)-phenyl]-vinyl}-
3-Vinylbenzylamine (1.18 g, 8.91 mmol) was dissolved in 15 cm3
CH2Cl2 and was mixed with an aqueous solution of di-tert-
butyldicarbonate (2.46 g, 26.73 mmol) and potassium carbonate
(3.69 g, 26.73 mmol). The reaction mixture was stirred for 3 h, then
an additional 2.46 g di-tert-butyldicarbonate was added and the
mixture was stirred for a further 72 h. The organic phase was sepa-
rated and extracted with water, and the water phase was three times
re-extracted with dichloromethane. Thereafter the product was
purified on a Silica 60 column using hexane:ethylacetate (9:1)
eluent mixture, yielding a yellowish oil (805.9 mg, 68%); nmax
(CHCl3)/cm-1 3345, 2974, 2923, 1699, 1514, 1370, 1252, 1168;
dH (270.2 MHz; CDCl3) 1.44 (9H, s, CH3Boc), 4.25 (2H, s, CH2),
5.2 (1H, s, NH), 5.22 (1H, d, J 17.9, CH), 5.72 (1H d, J 10.9,
CH), 6.68 (1H, dd, J, 10.9, 17.9, CH), 7.15 (1H, m, ArH), 7.28–
7.30 (3H, m, ArH); dC (67.9 MHz, CDCl3) 28.1 (3C), 44.3, 79.1,
113.8, 124.8, 125.0, 126.6, 128.5, 136.4, 137.5, 139.1, 155.8; m/z
(ESI-MS, 30 eV) 467 [2M + H]+, 260, 234 [M + H]+, 219.
phenyl) acetic acid (20)
A solution of 19 (969.6 mg, 1.82 mmol) in 50 cm3 6 M aqueous
NaOH solution was refluxed for 24 h. Thereafter, the solution
was acidified to pH = 3 with 5.0 M aqueous HCl and extracted
with CH2Cl2. The organic phase was filtered through MgSO4
and concentrated under reduced pressure yielding a white solid
(230.8 mg, 0.63 mmol, 34%); nmax (CHCl3)/cm-1 3446, 3026, 2977,
2934, 1703, 1497, 1234; dH (399.8 MHz; CDCl3) 1.48 (9H, s,
CH3Boc), 3.67 (2H, s, CH2NH), 4.33 (2H, s, CH2CO), 4.94 (1H, br s,
NH), 7.08 (2H, m, 2CH), 7.18 (2H, d, J 7.2, ArH), 7.28–7.34
(2H, m, ArH), 7.37–7.43 (4H, m, ArH), 9.77 (1H, br s, COOH);
dC (100.5 MHz, CDCl3) 28.5 (3C), 41.2, 44.8, 79.8, 125.6, 125.7,
125.8, 126.9, 127.6, 128.7, 128.8, 128.9, 129.0, 129.1, 134.0, 137.6,
137.7, 139.4, 156.1, 177.1; m/z (ESI-MS, 30 eV) 757 [2M + Na]+,
735 [2M + H]+, 390 [M + Na]+, 353, 312.
4-(N-tert-Butoxycarbonylaminomethyl)benzoic acid (28)22a
Methyl-3-iodophenyl acetic acid (18)37
To
a solution of 4-(aminomethyl)benzoic acid (10.00 g,
3-Iodophenylacetic acid (2.26 g, 8.62 mmol) was stirred in a
mixture of 15 cm3 methanol and 10 drops of concentrated aqueous
HCl for 24 h. The solvent was evaporated, the residue dissolved in
dichloromethane and washed with water, then filtrated through
MgSO4 and concentrated under reduced pressure yielding a
colorless oil (2.34 g, 98%); nmax (CHCl3)/cm-1 3054, 2993, 2948,
1727, 1560; dH (399.8 MHz; CDCl3) 3.52 (2H, s, CH2), 3.64 (3H s,
CH3), 7.00 (1H, ddd, J 0.5, 7.8, 8.1, ArH), 7.21 (1H, d, J 1.2,
1.7, 7.8, ArH), 7.55 (1H, ddd, J 1.2, 2.2, 8.1, ArH), 7.61 (1H,
ddd, J 0.5, 1.7, 2.2, ArH); dC (100.5 MHz, CDCl3) dC 40.6, 52.3,
94.7, 128.8, 130.4, 136.3, 136.4, 138.3, 171.4; m/z (EI, 70 eV) 276
[M + H]+, 217, 107, 90.
66.2 mmol), 1,4-dioxan (125 cm3), water (60 cm3) and 1 M
NaOH (64 cm3, 64 mmol) were added. To the mixture◦di-tert-
butylpyrocarbonate (15.9 g, 72.8 mmol) was added at 0 C. The
mixture was stirred at 0 ◦C for 40 min, then stirred at r.t. for
another 30 min. The volume of the solution was reduced to
70 cm3, then ethyl acetate (150 cm3) was added. The mixture was
acidified to pH 2 with 5 M HCl (20 cm3). The white precipitate was
dissolved in ethyl acetate and the solution was washed with water.
The combined organic layers were dried over Na2SO4, filtered,
and concentrated under reduced pressure to yield a white solid.
Recrystalization from ethyl acetate (120 cm3) gave 28 as white
◦
colorless needles (13.5 g, 81%); mp 167–168 C (Lit.:22a 167–
168 ◦C); dH (499.9 MHz; CDCl3) 1.47 (9H, s, (CH3)3), 4.40 (2H, d, J
6.1, CH2), 4.94 (1H, br s, NH), 7.38 (2H, m, XX¢ part of AA¢XX¢),
8.07 (2H, m, AA’ part of AA¢XX¢); dC (125 MHz; CDCl3) 28.5,
44.5, 80.1, 127.4, 128.4, 130.7, 145.3, 156.1, 171.0.
Methyl-(3-{2¢-[3¢¢-(tert-butoxycarbonylamino-methyl)-phenyl]-
vinyl}-phenyl)-acetate (19)
Methyl-3-iodophenyl acetic acid (830.7 mg, 3.01 mmol), (3-vinyl-
benzyl)-carbamic acid tert-butyl ester (637.4 mg, 2.73 mmol),
Pd(OAc)2 (18.4 mg, 0.08 mmol), tri-o-tolyl-phosphine (49.9 mg,
0.16 mmol), triethylamine (1.40 cm3; 1.61 mmol), and dimeth◦yl-
formamide (1.50 cm3) were stirred in a Smith Process Vial at 90 C
for 15 min in the microwave cavity. The resulting mixture was
filtered through Celite into a separation funnel, then extracted with
dichloromethane (25 cm3) and 0.1 M HCl (25 cm3), re-extracting
4-(N-tert-Butoxycarbonylaminomethyl)benzyl alcohol (29)22a
Lithiumaluminium hydride (0.40 g, 10.35 mmol) was suspended
in dry THF (70 cm3). The protected benzoic acid 28 was slowly
added and the reaction mixture was stirred over night at 40 ◦C. The
reaction was quenched by addition of water and the pH was
adjusted to 5 by addition of acetic acid. The organic phase was
4368 | Org. Biomol. Chem., 2008, 6, 4356–4373
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separated and the remaining aqueous phase was extracted with
diethyl ether (4 ¥ 50 cm3). Unreacted carboxylic acid 28 was
removed by extracting the combined organic phases with aqueous
NaHCO3. The combined organic layers were dried over anhydrous
Na2SO4, filtrated and evaporated under reduced pressure to give a
white solid (1.09 g, 44%); mp 100–101 ◦C (Lit.:22a 100–101 ◦C); dH
(399.9 MHz; CDCl3) 1.47 (9H, s, (CH3)3); 4.31 (2H, d, J 6.4, CH2),
4.68 (2H, s, CH2OH), 4.88 (1H, br s, NH), 7.28 (2H, m, XX¢part
of AA¢XX¢), 7.34 (2H, m, AA¢part of AA¢XX¢); dC (100.5 MHz;
CDCl3) 28.6, 44.6, 79.7, 65.2, 127.4, 127.8, 138.5, 140.2, 156.1.
The reaction was quenched with ice/water and the orange mixture
was extracted with DCM (3 ¥ 50 cm3). The DCM solution was
washed with water, dried over anhydrous Na2SO4, filtrated and
evaporated to give the product as an orange solid (2.44 g, 81%);
mp 110–111 ◦C; dH (499.9 MHz; CDCl3) 8.06 (1H, dd, J 1.3, 7.7,
H-5), 7.43 (t, 1H, J 7.7, H-6), 3.83 (s, 2H, H-2), 8.54 (1H, dd, J
1.3, 7.7, H-4), dC (100.6 MHz; CDCl3) 39.9, 125.3, 128.7, 132.7,
133.2, 141.0, 158.3, 166.2, 198.9 (COCl).
2-[1¢-{4¢¢-(tert-Butoxycarbonylamino-methyl)-phenyl}-meth-(Z)-
ylidene]-3-oxo-2,3-dihydro-benzo[b]thiophene-7-carboxylic acid
(33)10b
4-(N-tert-Butoxycarbonylaminomethyl)benzaldehyde (30)22d
The alcohol 29 (1.24 g, 5.23 mmol) was dissolved in dry
dichloromethane (25 cm3). Sodium acetate (0.12 g, 1.46 mmol)
followed by PCC (0.97 g, 4.49 mmol) were added to the solution.
The black mixture was stirred in the dark for 15 h. Diethyl ether
was added (30 cm3) and the mixture was triturated and filtered
through a pad of cellite. The filtrate was washed with water (50 cm3)
and the resulting aqueous phase was extracted with ether (70 cm3).
The combined organic layers were dried over anhydrous Na2SO4,
filtrated and evaporated under reduced pressure. The resulting
greenish solid was purified by flash chromatography (20% EtOAc
to 50% in pentan◦e and 5% TEA) to give a off white solid (0.9 g,
To a mixture of thioindoxyl 32 (3.94 g, 18.53 mmol), 1% NaOH
w/w (110 cm3) and t-BuOH (19 cm3), 4-(N-tert-butoxyamino-
methyl)benzalde◦hyde 30 (2.08 g, 8.82 mmol) in t-BuOH (27 cm3)
was added at 0 C. The orange mixture was refluxed over night
under nitrogen atmosphere. The mixture was cooled to r.t. and
acidified with acetic acid (pH 1–2). The reaction mixture was
extracted with ethyl acetate (3 ¥ 50 cm3). The combined organic
phases were washed with water, dried over anhydrous Na2SO4
and concentrated in vacuo to give an orange solid. After flash
chromatography (Florisil, 1:1 EtOAc:pentane and 1% HOAc) the
thioaurone 33 was obtained as an orange solid (3.0 g, 84%); dH
(499.9 MHz; CD3OD) 1.47 (9H, s, (CH3)3), 4.30 (1H, br s, CH2),
7.33 (1H, t, J 7.5, H-5), 7.42 (2H, m, XX¢ part of AA¢XX¢), 7.81
◦
72%); mp 83–84 C (Lit.:22d 82–84 C); dH (399.9 MHz, CDCl3)
1.45 (9H, s, (CH3)3), 4.39 (2H, s, CH2NH), 5.01 (1H, br s, NH),
7.44 (2H, m, XX¢ part of AA¢XX¢), 7.84 (2H, m, AA¢ part of
AA¢XX¢), 10.0 (1H, s, CHO); dC (100.5 MHz; CDCl3) 29.0, 44.9,
80.3, 128.1, 130.5, 136.0, 146.6, 156.2, 192.6.
=
(2H, m, AA¢part of AA¢XX¢), 7.86 (1H, s, C CH), 7.94 (1H, dd,
J 1.4, 7.5, H-4), 8.27 (1H, dd, J 1.4, 7.5, H-6), dC (125 MHz;
CD3OD) 28.8, 44.9, 96.8, 126.2, 128.7, 128.9, 132.4, 132.6, 134.0,
134.3, 134.6, 134.8, 137.8, 143.7, 149.1, 153.2 (Boc-CO), 172.3
(COOH), 191.3 (CO).
o-Carboxyphenylmercaptoacetic acid (31)21a
To a solution of anhydrous sodium carbonate (20 g) in water
(150 cm3), 2,2-dithiodisalicylic acid (10 g, 32.6 mmol) and sodium
dithionite (15 g, 86.2 mmol) were added. The mixture was refluxed
for 30 min under the formation of sulfur dioxide. A solution of
chloroacetic acid (15 g, 159 mmol) was neutralized with sodium
carbonate, then added to the reaction mixture and refluxing was
continued for 1 h. The mixture was allowed to cool to room
temperature before acidified with hydrochloric acid (conc.) against
Congo red. The yellow precipitate was recrystallized from H2O,
removing the green Congo red particles by filtration of the warm
solution. Yellow powdery solid (9.77 g, 71%); mp 219–221 ◦C
(Lit.:21a 217–218 ◦C); dH (399.8 MHz; CD3OD) 3.77 (1H, s, CH2);
5.01 (2H, br s, CH2COOH), 7.21 (1H, ddd, J 1.3, 7.2, 7.8, ArH),
7.43 (1H, ddd, J 0.5, 1.3, 8.2 ArH), 7.48 (1H, ddd, J 1.6, 7.2,
8.2, ArH), 7.98 (1H, ddd, J 0.5, 1.6, 7.8, ArH); dC (100.5 MHz;
CD3OD) 34.5, 124.2, 125.7, 128.1, 131.4, 132.4, 140.8, 168.5 (CO),
172.1 (CO).
2-[1¢-{4¢¢-[(9¢¢¢H-Fluoren-9¢¢¢-ylmethoxycarbonylamino)-methyl]-
phenyl}-meth-(Z)-ylidene]-3-oxo-2,3-dihydro-benzo[b]thiophene-7-
carboxylic acid (34)38
Thioaurone derivative 33 (2.4 g, 5.8 mmol) was stirred with
50% TFA in DCM (40 cm3) for 15 min. The solution was
concentrated, yielding a dark red oily residue. The remains were
mixed with a mixture of 10% aq Na2CO3 and THF (80 cm3) and 9-
fluorenylmethyl chloroformate (1.8 g, 7.0 mmol) was added. After
three days of stirring at r.t., the pH was adjusted to pH 1 by the
addition of HCl (conc.). The mixture was extracted with DCM
and the emulsions were removed by centrifugation. The combined
organic phases were dried over anhydrous Na2SO4, filtrated and
evaporated to give an orange solid. After flash chromatography
(Florisil, gradient of CHCl3 with 0.1% MeOH to 1:1 MeOH:THF)
the product 34 was obtained yellow-orange solid (2.8 g, 90% yield
over two steps); dH (499.9 MHz; CD3OD) 4.23 (1H, t, J 7.2, Fmoc-
CH), 4.34 (2H, br s, CH2NH), 4.46 (2H, d, J 7.2, Fmoc-CH2),
7.33–7.33 (2H, m, ArH), 7.36–7.43 (5H, m, ArH) 7.67 (2H, d, J
7.6, Fmoc-ArH), 7.81 (2H, d, J 7.6, Fmoc-ArH), 7.80 (2H, m,
3-Oxo-2,3-dihydro-benzo[b]thiophene-7-carbonylchloride (32)21b
Following a general literature procedure,21b o-carboxyphenyl mer-
captoacetic acid 31 (3.00 g, 14.1 mmol) was refluxed in SOCl2
(18 cm3) under the formation of SO2. The excess of SOCl2 was
removed by distillation under reduced pressure. The resulting
gold coloured acid chloride was dissolved in 1,2-dichloroethane
(30 cm3). To the cold solution (< 0 ◦C), AlCl3 was added in 4
portions (4.70 g, 35.2 mmol). The green mixture was stirred under
nitrogen with cooling another 20 min, then stirred at r.t. for 14 h.
=
AA¢part of AA¢XX¢), 7.87 (1H, s, C CH), 8.00 (1H, dd, J 1.4,
7.6 Hz ArH), 8.28 (1H, dd, J 1.4, 7.6 ArH, H7); dC (125 MHz;
CD3OD) 45.1, 48.8 (CH2), 67.6 (OCH2), 120.9, 126.1, 126.3, 128.1,
=
128.7, 128.8, 129.0, 132.5, 132.7, 134.0 ( CH), 134.2, 134.5, 134.8,
137.8, 142.7, 143.2, 145.3, 149.1, 159.0 (CO) 172.3 (CO), 191.2
(CO).
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3-(1,3-dioxolan-2-yl)benzonitrile (35)10a
(2 cm3) and a catalytic amount of PTSA (3 mg, 15.8 mmol)
the solution was stirred over night at 30 ◦C under nitrogen.
After evaporation of the solvents, the residu was dissolved in
DCM (20 cm3) and washed with 10% w/w aq. NaHCO3. The
organic layers were washed with water, dried over anhydrous
Na2SO4, and concentrated in vacuo, yielding a slighly yellow-
ish oil. After purification by flash chromatography (silica, 1:1
EtOAc:pentane), compound 38 was obtained as a solid (0.79 g,
64%); dH (499.9 MHz; CDCl3) 1.43 (9H, s, (CH3)3), 4.35 (2H, d, J
6.0, CH2NH), 5.16 (1H, br s, NH), 7.46 (1H, m, ArH), 7.53 (1H,
m, ArH), 7.74 (2H, m, ArH), 9.96 (1H, s, CHO); dC (125 MHz;
CDCl3) 28.4 (CH3), 44.1 (CH2), 79.8 (quart. C), 128.2, 128.8,
129.3, 133.5, 136.7 (ipso C), 140.5 (ipso C), 156.0 (CO), 192.3
(CHO).
3-Cyanobenzaldehyde (2.50 g, 19.1 mmol) was dissolved in toluene
(70 cm3) and treated with ethylene glycol (7.50 cm3, 134 mmol)
followed by PTSA (2.70 g, 14.3 mmol). The solution was refluxed
in a Dean Stark apparatus for 18 h. After cooling to r.t. the solution
was washed with 5% aq NaHCO3 (50 cm3). The yellow organic
phase was separated and dried over anhydrous Na2SO4, filtered
and concentrated under reduced pressure to give a yellow oil. The
residue was purified by bulb to bulb distillation (140 ◦C, 0.7 mbar)
and the colourless oil obtained was dissolved in diethyl ether and
washed with a 10% aq sodium bisulfite solution. The organic layer
was dried over anhydrous Na2SO4, filtered and concentrated to
1
give colourless oil (2.00 g, 60%, 92% purity according to the H
NMR spectra); dH (499.9 MHz; CDCl3) 4.04–3.93 (4H, m, CH2),
5.73 (1H, s, CH), 7.41–7.38 (1H, m, ArH), 7.57–7.54 (1H, m,
ArH), 7.64–7.62 (1H, m, ArH), 7.70 (1H, m, ArH); dC (125 MHz;
CDCl3) 65.4, 102.2, 112.4, 118.5, 129.2, 130.2, 130.9, 132.6,
139.7.
2-[1¢-{3¢¢-(tert-Butoxycarbonylamino-methyl)-phenyl}-meth-(Z)-
ylidene]-3-oxo-2,3-dihydro-benzo[b]thiophene-7-carboxylic
acid (39)10a
To a mixture of thioindoxyl 32 (3.11 g, 14.6 mmol), 1%
NaOH w/w (87 cm3) and t-BuOH (14.5 cm3), t-Butyl(3-
Formylbenzyl)carbamate 38 (1.64 g, 6.97 mmol) in t-BuOH
3-(1,3-dioxolan-2-yl)benzylamine (36)10a
Lithium aluminium hydride (0.50 g, 13.3 mmol) was mixed with
dry THF (40 cm3) under nitrogen at 0 ◦C. The benzonitrile (2.00 g,
12.0 mmol) was dissolved in THF (2.5 cm3) and added dropwise
to the suspension with a speed of 0.8 cm3/h. The mixture was
stirred over night at r.t., before quenching with water (0.50 cm3),
15% aq NaOH (0.50 cm3) and water (1.50 cm3). The mixture was
extracted with diethyl ether and the aqueous phase was extracted
with more diethyl ether (3 ¥ 20 cm3). The combined organic layers
were washed with water, then brine, dried over anhydrous Na2SO4,
filtered and evaporated in vacuo to give a yellow oil (0.85 g, 40%);
dH (499.9 MHz; CDCl3) 3.82 (2H, br s, CH2NH), 4.12–3.95 (m,
4H, CH2), 5.76 (s, 1H, CH), 7.41–7.27 (m, 4H, ArH); dC (125 MHz;
CDCl3) 46.1, 65.2, 103.5, 124.8, 124.9, 127.8, 128.5, 138.1,
143.4.
◦
(14.5 cm3) was added at 0 C. The orange mixture was refluxed
over night under nitrogen atmosphere. The mixture was cooled
to r.t. and acidified with acetic acid (pH 1–2). The red precipitate
was filtrated and purified by flash chromatography (Florisil, 20%
MeOH in EtOAc) to give the thioaurone 39 as an orange solid
(1.29 g, 45%); dH (499.9 MHz; CD3OD) 1.46 (9H, s, (CH3)3), 4.30
(2H, br s, CH2), 7.35 (1H, dd, J 2.5, 7.6, ArH), 7.36 (1H, t, J 7.5,
ArH), 7.45 (1H, t, J 7.6, ArH), 7.67 (1H,br s, ArH), 7.75 (1H,
=
br d, J 7.6, ArH) 7.83 (1H, s, C CH), 7.93 (1H, dd, J 1.4, 7.5,
ArH), 8.29 (1H, dd, J 1.4, 7.5, ArH); dC (100.6 MHz; CD3OD)
29.8, 44.9, 80.4, 126.2, 129.1, 129.9, 130.1, 130.5, 131.3, 132.6,
134.0, 134.2, 134.9, 136.2, 137.9, 142.1, 149.2, 158.5 (CO), 172.4
(COOH), 191.2 (CO).
tert-Butyl[3-(1,3-dioxolan-2-yl)benzyl]carbamate (37)10a
2-[1¢-{3¢¢-[(9¢¢¢H-Fluoren-9¢¢¢-ylmethoxycarbonylamino)-methyl]-
phenyl}-meth-(Z)-ylidene]-3-oxo-2,3-dihydro-benzo[b]thiophene-7-
carboxylic acid (40)
The 3-(1,3-dioxolan-2-yl)benzylamine 36 (3.92 g, 21.9 mmol) was
dissolved in anhydrous MeOH (35 cm3), before the addition of
triethyl amine (3.1 cm3, 21.9 mmol). The yellow solution was
cooled to 0 ◦C before the addition of di-t-butyl dicarbonate (6.21 g,
28.4 mmol). The solution was stirred for a couple of days at r.t.
under nitrogen. The mixture was diluted with some diethyl ether
and the organic phase was separated and washed with saturated
NH4Cl. The organic layers were washed with water, then brine,
dried over anhydrous Na2SO4, filtered and evaporated in vacuo
to give a light yellow oil. The product 37 was recrystallized from
diethyl ether and pentane (2:1) (4.93 g, 81%); dH (499.9 MHz;
CDCl3) 1.46 (9H, s, (CH3)3), 4.15- 4.00 (4H, m, 2 x CH2), 4.32
(2H, br d, J 5.7, CH2NH), 4.86 (1H, br s, NH), 5.79 (1H, s,
CH), 7.41–7.27 (4H, m, ArH); dC (125 MHz; CDCl3) 28.9 (CH3),
45.1, 65.8 (CH2), 79.8, 103.9 (CH), 126.0 (2C), 128.6, 138.6, 138.9,
139.5, 155.9 (CO).
Thioaurone derivative 39 (1.0 g, 2.43 mmol) was stirred with
50% TFA in DCM (10 cm3) for 15 min. The solution was
concentrated, yielding a dark red oily residue. The remains were
mixed with a mixture of 10% aq Na2CO3 and THF (18 cm3) and
9-fluorenylmethyl chloroformate (0.75 g, 2.88 mmol) was added.
After three days of stirring at r.t., the pH was adjusted to pH 1
by the addition of HCl (conc.). After separation of the organic
phase, the aqueous phase was extracted with DCM. The combined
organic phases were dried over anhydrous Na2SO4, filtrated and
evaporated to give an orange solid. After flash chromatography
(Florisil, gradient of CHCl3 with 0.1% MeOH to 1:1 MeOH:THF)
the product 40 was obtained as a yellow solid (0.9 g, 70% yield
over two steps); mp 203–205 ◦C; nmax (neat)/cm-1 3326, 1599, 1403,
1262, 1046, 758; dH (499.9 MHz; 1:3 CD3OD:CDCl3) 4.20 (1H, br
t, J 6.5, Fmoc-CH), 4.37 (2H, br s, Fmoc-CH2), 4.42 (2H, d, J 6.9,
CH2), 7.26–7.23 (2H, m, ArH), 7.35–7.31 (3H, m, ArH), 7.39 (1H,
t, J 7.6, ArH), 7.43 (1H, t, J 7.6, ArH), 7.60–7.58 (2H,m, ArH),
tert-Butyl(3-Formylbenzyl)carbamate (38)10a
The carbamate 37 (1.47 g, 5.26 mmol) was dissolved in acetone
(16 cm3) to form a yellow solution. After the addition of water
7.62 (1H, br s, ArH), 7.72–7.68 (3H, m, ArH), 7.91 (1H, s, C CH),
=
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8.07 (1H, dd, J 1.2, 7.6, ArH), 8.30 (1H, dd, J 1.3, 7.6 Hz, ArH); dC
(100.6 MHz; 1:3 CD3OD:CDCl3) 46.2, 49.1 (CH2), 68.5 (OCH2),
121.6, 126.7, 127.1, 129.8, 129.4, 131.0, 131.1, 131.6, 131.9, 132.0,
132.2, 133.8, 133.9, 133.9, 136.3, 136.4, 139.0, 141.7, 143.1, 145.6,
150.5, 159.0 (CO), 169.3 (CO), 190.7 (CO).
tert-butyl ester as a brown oil (1.07 g, 98%); nmax (CHCl3)/cm-1
3450, 3016, 2976, 2401, 2154, 1713, 1505, 1218; dH (270.2 MHz;
CDCl3) 0.25 (9H, s, Si(CH3)3); 1.43 (9H, s, CH3Boc), 4.43 (2H,
br d, J 5.6, CH2), 5.13 (1H, br s, NH), 7.17–7.32 (3H, m,
ArH), 7.42 (1H, dd, J 1.3, 7.3, ArH); dC (67.9 MHz; CDCl3)
dC 0.22 (3C), 28.2 (3C), 43.1, 79.1, 99.2, 102.7, 121.7, 126.8, 127.5,
128.7, 132.2, 140.9, 155.7; m/z (ESI-MS, 30 eV) 304 [M + H]+,
248, 204. The (2-trimethylsilylethynyl-benzyl)-carbamoyl acid tert-
butyl ester (1.07 g, 3.53 mmol) was dissolved in methanol (25 cm3)
and KF·2H2O (1.00 g, 10.60 mmol) was added. The mixture was
stirred for 14 h at room temperature. The solvent was removed
and the residue was extracted with dichloromethane (20 cm3) and
water (20 cm3), re-extracting the water phase twice. The combined
organic layers were concentrated and the residue purified by
column chromatography using hexane:ethyl acetate (9:1) eluent
mixture yielding a brown solid (0.51 g, 63%); nmax (CHCl3)/cm-1
3450, 3301, 3004, 2980, 2101, 1709, 1507, 1364, 1251, 1168; dH
(270.2 MHz; CDCl3) 3.31 (1H, s, CH), 1.41 (9H, s, CH3Boc), 4.42
(2H, s, CH2), 5.14 (1H, br s, NH), 7.17 (1H, t, J 7.3, ArH), 7.25–
7.33 (2H, m, ArH), 7.44 (1H, d, J 7.3, ArH); dC (67.9 MHz;
CDCl3) 28.0 (3C), 42.9, 79.1, 81.0, 81.8, 120.6, 126.8, 127.5, 128.7,
132.5, 141.1, 155.5; m/z (ESI-MS, 30 eV) 232 [M + H]+, 217, 176,
132.
2-Iodobenzylamine (41)
2-Iodobenzyl bromide (5.00 g, 16.84 mmol), N,N-bistrimethy-
lsilylamide (9.26 g, 50.50 mmol) and hexamethyldisilazane
(25 cm3) were mixed and stirred at room temperature for 40 h.
The reaction mixture was transferred to a separation funnel with
dichloromethane (25 cm3) and was extracted three times with conc.
NaHCO3 (aq) (25 cm3). 5 M HCl (25 cm3) was added to the
dichloromethane phase and the mixture was extracted three times.
NaOH pellets were added slowly to the combined acidic layers
until pH 11. The basic aqueous layer was extracted three times
with dichloromethane (25 cm3). Thereafter the combined or-
ganic layers were concentrated on a rotary evaporator giving 2-
iodobenzylamine as brown oil (1.95 g, 50%); nmax (CHCl3)/cm-1
3051, 2957, 1588, 1467, 1432, 1011; dH (270.2 MHz CDCl3) 2.43
(2H, br s, NH2), 3.86 (2H, s, CH2), 6.94 (1 H, ddd, J 1.2, 6.7, 7.9,
ArH), 7.32 (1 H, ddd, J 2.3, 6.7, 7.9, ArH), 7.37 (1H, dd, J 2.3,
7.9, ArH), 7.82 (1H, dd, J 1.2, 7.9, ArH); dC (67.5 MHz CDCl3)
51.1, 127.9, 128.5, 128.6, 128.7, 139.4, 144.5; m/z (EI, 70 eV) 233
[M + H]+, 127, 106, 77, 51.
2-Iodophenyl-acetic acid methyl ester (44)39
To 2-Iodophenyl-acetic acid (2.50 g; 9.54 mmol) dissolved in
methanol (100 cm3) conc. aqueous HCl (0.81 cm3; 9.54 mmol)
was added dropwise and the mixture was stirred for 4 h at
room temperature. The solvent was concentrated under reduced
pressure. The residual was poured into water (20 cm3) and
extracted three times with dichloromethane (20 cm3). The organic
phase was concentrated yielding a yellow oil (2.61 g, 99%); nmax
(CHCl3)/cm-1 2948, 1741, 1436, 1218, 1167, 1016; dH (270.2 MHz;
CDCl3) 3.72 (3H, s, CH3), 3.81 (2H, s, CH2), 6.97 (1H, ddd, J 1.3,
2.5, 7.9, ArH), 7.27–7.35 (2H, m, ArH), 7.84 (1H, dd, J 1.3, 7.9,
ArH); dC (67.9 MHz; CDCl3) dC 46.0, 52.1, 100.9, 128.4, 128.8,
130.5, 137.6, 139.4, 170.8; m/z (EI, 70 eV) 276 [M + H]+, 217, 149,
121.
2-Iodobenzyl-carbamoyl acid tert-butyl ester (42)
2-Iodobenzylamine (1.75 g, 7.50 mmol) dissolved in dichloro-
methane (20 cm3) was mixed with K2CO3 (3.11 g, 22.49 mmol) in
water (20 cm3), then di-tert-butyldicarbonate (9.82 g, 44.98 mmol)
was added and stirred at room temperature for three days. The
organic phase was separated and the water phase was extracted
with dichloromethane (20 cm3) twice. The dichloromethane phase
was concentrated and purified by column chromatography yield-
ing yellowish crystals (1.49 g, 60%); nmax (CHCl3)/cm-1 3446, 3015,
1708, 1522, 1497, 1218, 1167; dH (270.2 MHz; CDCl3) 1.45 (9H, s,
CH3Boc), 4.32 (2H, s, CH2), 5.03 (1H, br s, NH), 6.96 (1H, t, J
7.9, ArH), 7.29–7.37 (2H, m, ArH), 7.81 (1H, d, J 7.9, ArH); dC
(67.9 MHz; CDCl3) 28.4 (3C), 49.3, 79.6, 128.5, 129.1 (2C), 132.7,
139.3, 140.9, 155.6; m/z (ESI-MS, 30 eV) 667 [2M + H]+, 360, 334
[M + H]+, 319, 275.
Methyl-{2-[2¢-(tert-Butoxycarbonylamino-methyl)-phenyl-
ethynyl]-phenyl}-acetate (45)
A solution of 44 (260 mg, 1.11 mmol), methyl-2-iodophenylacetate
(330 mg, 1.22 mmol), Pd(PPh3)2Cl2 (15.60 mg, 22.23 mmol), CuI
(8.50 mg, 44.63 mmol), diethylamine (1.50 cm3) and dimethylfor-
mamide (0.50 cm3) was mixed in a Smith Process Vial and stirred
at 120 ◦C for 6 min in a microwave cavity. The procedure was
repeated one time and the mixtures were combined by filtering
them through Celite into a separation funnel. The mixture was
extracted using dichloromethane (25 cm3) and conc. NaHCO3
(aq) (25 cm3) re-extracting the aqueous phase twice. The organic
phase was washed with water (25 cm3), re-extracting the water
phase three times. Then, the organic phase was concentrated under
reduced pressure yielding a yellowish solid (0.68 g, 81%); nmax
(CHCl3)/cm-1 3452, 3020, 2981, 2400, 1732, 1709, 1505, 1214,
1167; dH (270.2 MHz; CDCl3) 1.44 (9H, s, CH3Boc), 3.69 (3H, s,
CH3), 3.91 (2H, s, CH2), 4.51 (2H, s, CH2), 5.21 (1H, br s, NH),
7.24–7.36 (5H, m, ArH), 7.42 (1H, d, J 7.3, ArH), 7.55 (2H, m,
(2-Ethynyl-benzyl)-carbamoyl acid tert-butyl ester (43)
A mixture of 42 (0.40 g, 1.20 mmol), ethynyl-trimethyl-silane
(0.19 cm3, 1.32 mmol), Pd(PPh3)2Cl2 (16.80 mg, 23.94 mmol), CuI
(9.10 mg, 47.78 mmol), diethylamine (1.50 cm3) and dimethyl-
formamide (0.50 cm3) was added to a Smith Process Vial and
was stirred at 120 ◦C for 6 min in the microwave cavity. The
combined products of three runs were filtered through celite into a
separation funnel. Dichloromethane (25 cm3) and conc. NaHCO3
(aq) (25 cm3) was added and the phases were separated, re-
extracting the aqueous phase twice. Thereafter, the organic phase
was washed with water (25 cm3), re-extracting the water phase
three times. The organic phase was concentrated and purified
by flash chromatography using hexane:ethyl acetate (9:1) eluent
mixture yielding (2-trimethylsilylethynyl-benzyl)-carbamoyl acid
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ArH); dC (67.9 MHz; CDCl3) 28.4 (3C), 40.0, 43.5, 52.2, 79.5, 91.4,
91.9, 122.0, 123.3, 127.3, 128.1 (2C), 128.8 (2C), 129.9, 132.4 (2C),
135.8, 140.5, 155.9, 171.6; m/z (ESI-MS, 30 eV) 380 [M + H]+,
280, 263.
6 (a) M. Sisido, M. Harada, K. Kawashima, H. Ebato and Y. Okahata,
Biopolymers, 1998, 47, 159; (b) C. Renner, J. Cramer, R. Behrendt and
L. Moroder, Biopolymers, 2000, 54, 501; (c) M. Schutt, S. S. Krupka,
A. G. Milbradt, S. Deindl, E.-K. Sinner, D. Oesterhelt, C. Renner and L.
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Sci. USA, 2007, 104, 5383; (f) P. Hamm, J. Helbing and J. Bredenbeck,
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{2-[2¢-(tert-Butoxycarbonylamino-methyl)-phenylethynyl]-
phenyl}-acetic acid (46)
Compound 45 (1.34 g, 3.52 mmol) and 6 M NaOH (aq) (100 cm3)
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were mixed in a round bottom flask. The suspension was stirred
◦
at 120 C for 2 h. Thereafter the mixture was poured into water
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in a separation funnel and 5 M HCl (aq) was added to decrease
pH to 7, and was extracted with dichloromethane three times.
The pH of the aqueous phase was first adjusted to pH 5, then
pH 3, using 1 M HCl (aq), and the aqueous phase was extracted at
each pH three times with dichloromethane. The combined organic
layers were concentrated, filtered through a silica gel plug and
recrystallized from methanol giving a white solid (0.82 g, 61%);
mp 155–156 ◦C; nmax (CHCl3)/cm-1 3446, 3357, 3067, 2929, 1709,
1503, 1165; dH (399.8 MHz; Acetone-d6) 1.46 (9H, s, CH3Boc); 3.98
(2H, s, CH2), 4.59 (2H, d, J 6.0, CH2), 6.60 (1H, br d, J 6.0,
NH), 7.31–7.48 (6H, m, ArH), 7.60 (1H, dd, J 1.6, 7.6, ArH),
7.66 (1H, dd, J 1.7, 7.6, ArH); dC (100.5 MHz; Acetone-d6) 27.8,
39.6, 42.6, 78.2, 91.2, 92.4, 121.6, 123.6, 126.9, 127.1, 128.7, 128.8,
130,4, 132.1, 132.3, 137.1, 141.6, 156.1, 171.6; m/z (ESI, 30 eV) 366
[M + H]+, 310, 266, 214, 199. HRMS, m/z = 388.1464 ([M + Na]+),
C22H23NO4Na requires 388.1525.
9 G. Mayer and A. Heckel, Angew. Chem. Int. Ed., 2006, 45, 2900.
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