Recognition of solvent exposed protein surfaces using anthracene derived receptors

Article abstract of DOI:10.1039/b612975g

A new class of receptor is described that can selectively bind to the solvent exposed surface of proteins such as cytochrome c and lysozyme with low micromolar affinity over cytochrome c551, α-lactalbumin, myoglobin and RNase A, under physiologically relevant conditions (5 mM phosphate, pH 7.4). The use of anthracene as a hydrophobic scaffold allows the receptor to act as a selective chemosensor via fluorescence quenching or FRET. The study reveals that co-operative electrostatic interactions over a large surface area dominate binding. Further investigations reveal that the receptor binds to the solvent exposed heme edge of cytochrome c inhibiting its reaction with small reducing agents and validating the strategy for the disruption of protein function. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b612975g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Recognition of solvent exposed protein surfaces using anthracene derived
receptors†
Andrew J. Wilson,‡ Jason Hong, Steven Fletcher and Andrew D. Hamilton*
Received 11th September 2006, Accepted 2nd November 2006
First published as an Advance Article on the web 5th December 2006
DOI: 10.1039/b612975g
A new class of receptor is described that can selectively bind to the solvent exposed surface of proteins
such as cytochrome c and lysozyme with low micromolar affinity over cytochrome c551, a-lactalbumin,
myoglobin and RNase A, under physiologically relevant conditions (5 mM phosphate, pH 7.4). The use
of anthracene as a hydrophobic scaffold allows the receptor to act as a selective chemosensor via
fluorescence quenching or FRET. The study reveals that co-operative electrostatic interactions over a
large surface area dominate binding. Further investigations reveal that the receptor binds to the solvent
exposed heme edge of cytochrome c inhibiting its reaction with small reducing agents and validating the
strategy for the disruption of protein function.
Introduction
In this paper we describe the synthesis and binding properties of
a series of anthracene-derived receptors. These compounds have
the advantages, relative to our previously-reported porphyrins,
of being of lower molecular weight, virtually colourless and
exhibiting an efficient fluorescence resonance energy transfer
The design of molecular receptors capable of binding protein
surfaces and disrupting their function is an active area of research
due to the importance of protein–protein interactions in many
1,2
cellular processes. Traditionally, this disruption of function is
achieved through recognition of well-defined cavities within the
(
FRET) based quenching of fluorescence on protein binding. The
design strategy leads to selectivity in the recognition and sensing of
1
protein, while recognition of the solvent-exposed exterior is
cytochrome c and lysozyme when compared to cytochrome c551,
2
less explored. Every protein presents a unique distribution of
3
a-lactalbumin, RNase A and myoglobin.
3
hydrophobic and charged residues at its surface (Fig. 1) that
influences the selectivity and affinity with which it interacts with
4
other proteins. Recognition of small peptide fragments has been
Results and discussion
achieved in aqueous solution using combinations of hydrophobic,
electrostatic and metal–ligand interactions, an approach that has
We chose to focus upon two primary targets in this work with the
added goal of incorporating a ready readout of recognition into the
receptor design. Cytochrome c was an attractive target as we had
already shown that the unparied electrons in the ferro form could
5,6
been extended further to the recognition of proteins. Similarly,
numerous reports describing model peptides or miniature proteins
focus upon stabilizing and presenting the features of secondary
7
9a,c
structure critical to a protein–protein interface and in turn
quench fluorescence upon binding to a fluorogenic receptor.
this has inspired the development of protein surface mimetics
Lysozyme was chosen as a complementary non-metalloprotein
that might promote a different fluorescent response. Cytochrome
c is a highly basic protein (12 kDa, pI 9.79) and plays key roles in
electron transfer and apoptosis, that are mediated by complex
formation with proteins such as cytochrome c peroxidase and
8
(
proteomimetics).
In recent years, we have reported synthetic receptors that
are capable of binding to the exterior surfaces of cytochrome
c, a-chymotrypsin, platelet-derived growth factor and other
proteins through complementary electrostatic and hydrophobic
12
Apaf1. A critical recognition region on the surface of cytochrome
c involves a hydrophobic patch centered on the solvent exposed
Fe(III) heme unit that is surrounded by lysine and arginine residues.
This domain has been characterised in terms of its interaction with
9
interactions. Latterly, we have validated this approach for the
modulation of protein function by (a) disrupting protein–protein
10
11
interactions and (b) inducing denaturation of the target protein.
12b,13
In further developing these studies we sought to construct a
broader range of receptor designs with the added potential for
function in the recognition and sensing of a wider variety of
proteins.
a complementary domain of cytochrome c peroxidase.
The
interaction is dominated by large entropic and small enthalpic
13
contributions and a large negative heat capacity. Likewise the
highly basic and helical hen egg white lysozyme is a monomeric
protein (14 kDa, pI 9.80) that destroys the cell wall in certain
bacteria via cleavage of the b-glycosidic bond between the C1 of
N-acetylmuramic acid and the C4 of N-acetylglucosamine of the
Department of Chemistry, PO Box 208107, 225 Prospect Street, Yale
University, New Haven, CT 06520-8107, USA. E-mail: Andrew.Hamilton@
yale.edu
14
bacterial peptidoglycan. While there is no specific protein partner
associated with lysozyme, there are several crystal structures of
†
Electronic supplementary information (ESI) available: Further Job
analyses and titration data. See DOI: 10.1039/b612975g
Current address: School of Chemistry, University of Leeds, Woodhouse
Lane, Leeds LS2 9JT, UK.
−
5
lysozyme-antibody complexes with affinities ranging from 10 –
−
11
10
M. Additional investigations have demonstrated that the
‡
14,15
entire surface of the protein is antigenic.
In each case, the
2
76 | Org. Biomol. Chem., 2007, 5, 276–285
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electrostatic contact between the carboxylates of the receptor and
basic residues of the protein (Fig. 2a, b).
Fig. 2 Receptor 1a manually docked to cytochrome c (a) electrostatic
representation and (b) ribbon representation (colours are as for Fig. 1
except the prosthetic heme group is shown in CPK format. The receptor
is shown as yellow cylinders. The basic residues proposed as important to
the binding interaction are shown as blue cylinders).
Titration of horse heart cytochrome c into a solution of receptor
1a (5 mM phosphate, pH 7.4) resulted in efficient fluorescence
quenching (ex 330 nm, similar results are obtained upon excitation
at 285 nm) (Fig. 3a) as a result of complex formation with the
heme edge region of the protein; a well known recognition site for
9a,c,e,10b,11,12b,13
binding various substrates.
Curve fitting of this data
to a 1 : 1 binding model gave a dissociation constant of 0.66 lM,
reflecting good affinity for a first generation receptor. Job analysis
was used to confirm the predominant formation of a 1 : 1 complex
(
Fig. 3b).
Fig. 1 Electrostatic and ribbon representations of (a) horse heart cy-
tochrome c, (b) Pseudomonas aeruginosa cytochrome c551, (c) horse skele-
tal myoglobin, (d) hen egg white lysozyme, (e) bovine milk a-lactalbumin,
and (f) bovine RNase A (hydrophobic patches are represented in white
and grey, acidic patches in red and basic patches in blue. a-Helical regions
are shown in red, b-sheet blue and random coil light grey. Prosthetic heme
3
groups are shown as green cylinders).
interaction between antigen and antibody is characterised by a
large number of hydrophobic contacts coupled with critical ion
15
pair interactions.
We reasoned that a fluorescent, hydrophobic group such as
anthracene could act as a useful core around which to attach
different charged groups. The synthesis of receptor 1a was achieved
in 6 steps and was tailored to match the basic nature of the targeted
proteins. Starting with dimethyl-5-hydroxyisophthalate, alkylation
and then hydrolysis yielded the benzyl protected di-acid as
16
described previously. EDCI mediated coupling with aspartic acid
dimethyl ester followed by hydrogenation over 10% Pd(C) gave
5
-hydroxy(bisdimethylaspartate)isophthalamide in 70% yield. Di-
alkylation using 9,10-bis(bromomethylanthracene) was achieved
with K CO as base (32%) followed by ester hydrolysis to yield
receptor 1a (87%) (Scheme 1). Manual docking of receptor 1a
Fig. 3 Fluorescence spectra (ex 330 nm, 5 mM phosphate, pH 7.4) (a)
lM receptor 1a on addition of cytochrome c, inset shows the change
in signal at 401 nm over the course of the titration as a function of the
1
2
3
concentration of cytochrome c and the curve derived by regression analysis.
3,12b
to the known structure of cytochrome c
indicates that the
(
b) Job’s plot: emission of receptor 1a was monitored at 401 nm as a
anthracene scaffold could bind to the solvent exposed hydrophobic
patch of the protein with additional interactions resulting from
function of the mole fraction of cytochrome c. The total concentration of
the two species was held constant at 2 lM.
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Scheme 1 Synthesis of receptors 1–2.
This result prompted us to probe the structural properties of
the receptor that are key to protein affinity. Receptors 1b–d and
were synthesised in a similar manner to 1a (Scheme 1). Due
to weak fluorescence emission from receptor 2, the fluorescence
recovery of 1a upon titration of 2 into a 1 : 1 mixture of 1a and
cytochrome c was used to assess the binding with cytochrome
c. In a control experiment, no changes in fluorescence of 1a were
observed upon titration with 2. There is a deviation from simple 1 :
Since the readout of binding relied upon fluorescence quench-
ing, we sought to assess the versatility of the receptor design
in the recognition of other proteins. Titration of lysozyme into
a solution of receptor 1a (5 mM phosphate buffer, pH 7.4, ex
285 nm) results in fluorescence resonance energy transfer (FRET).
This is due to the overlap in emission spectra of the protein
tryptophan residues (emmax 340 nm) and absorption spectra of
the anthracene moitey (ex 354 nm) suggesting a mutual proximity
2
˚
1
stoichiometry observed for binding of receptor 1c to cytochrome
(<25 A) from complex formation (data not shown). However,
c and this is further reflected in a non-ideal 1 : 1 Job’s plot.
this approach proved unsuitable for studying the binding because
of signal overlap at higher concentrations of protein. Consistent
with this observation, titration of receptor 1a into a solution of
lysozyme causes a decrease in intensity of the protein emission due
to FRET (Fig. 4a). These data were used to derive a dissociation
constant of 0.52 lM by curve fitting to a 1 : 1 binding isotherm
and the stoichiometry of the interaction was once again confirmed
using Job analysis (Fig. 4b).
It has been proposed on several occasions that a second weak
11b
binding site on cyctochrome c exists and this may account for
the observed deviation from a simple 1 : 1 stoichiometry with
the more hydrophobic receptor 1c. A summary of the results
obtained from binding assays is shown in Table 1. Receptors 1a,
1d and 2 all have affinity for cytochrome c below 1 lM whereas
receptors 1b and c bind an order of magnitude less efficiently. This
result indicates that the dominant contribution to the binding
interaction is derived from the presence of eight appropriately
spaced carboxylate residues that act in a cooperative manner
across a large surface area. Curiously no significant increase in
affinity results from additional hydrophobic residues (1a v 1d and
Accordingly, we sought to assess the selectivity of receptor 1a for
the surface of cytochrome c and lysozyme against other proteins.
Cytochrome c551 from Pseudomonas aeruginosa has a similar
tertiary structure and performs a similar biological function to
cytochrome c yet lacks lysine residues surrounding the prosthetic
heme. Likewise a-lactalbumin, which forms a complex with
galactosyl transferase, has a similar tertiary structure to that of
9a,c
1b v 1c) as was observed for porphyrin derived receptors.
3,14a,17
lysozyme.
The protein fold and electrostatic representations
Table 1 Dissociation constants of receptors 1a–d and 2 with cytochrome
of both lysozyme and a-lactalbumin (bovine milk) highlight the
similar evolutionary origins of the two proteins (Fig. 1).
a
c
3,14a
Furthermore, a-lactalbumin contains several tryptophans that are
close to the surface suggesting its compatibility with the FRET
assay. In addition, RNase A (bovine) containing no tryptophans
and myoglobin (horse skeletal), were selected to provide a further
measure of selectivity.
The results of the binding assays are summarized in Table 2
and highlight a clear electrostatic dependence upon the binding of
receptor 1a to different proteins. Both cytochrome c551 and myo-
globin behaved well in the quenching assay and showed an affinity
Receptor
Stoichiometry
Dissociation constant
1
1
1
1
2
a
b
c
1 : 1
1 : 1
1 : 1
0.66 (± 0.048) lM
3.32 (± 0.097) lM
1.4 (± 0.192) lM
0.30 (± 0.013) lM
3
b
d
1 : 1
n/d
c
0.57 (± 0.036) lM
a
c
b
5
mM phosphate, pH 7.4. Non-ideal 1 : 1 Job’s plot (see ESI†).
Competition assay.
2
78 | Org. Biomol. Chem., 2007, 5, 276–285
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The electrophoretic mobility of a protein should in principle
be affected upon complex formation with an anionic receptor
such as 1a. A gel shift assay of lysozyme with and without
1a shows that a discernable change in electrophoretic mobility
occurs in the presence of receptor 1a. No effect is seen with a-
lactalbumin further supporting the selectivity of 1a for lysozyme
over a-lactalbumin (Fig. 5).
Fig. 4 Fluorescence spectra (ex 285 nm, 5 mM phosphate, pH 7.4) (a)
lM lysozyme on addition of receptor 1a, inset shows the change in signal
1
Fig. 5 Agarose gel shift assay: lanes 1 and 3 lysozyme, lanes 2 and 4
lysozyme and 1a, lanes 5 and 7 a-lactalbumin, lanes 6 and 8 a-lactalbumin
and 1a.
at 340 nm over the course of the titration as a function of the concentration
of receptor 1a and the curve derived by regression analysis. (c) Job’s plot:
emission of receptor 1a was monitored at 423 nm as a function of the mole
fraction of lysozyme. The total concentration of the two species was held
constant at 2 lM.
The reaction of cyctochrome c with reducing agents represents a
useful model of the reaction that occurs between cytochrome c and
its natural protein partner—cytochrome c peroxidase. Previously,
it has been shown that the pseudo first order reduction of Fe(III)
cytochrome c to Fe(II) cytochrome c with ascorbate can be
a
Table 2 Dissociation constants for binding of proteins to receptor 1a
Protein
pI
MW
Dissociation constant
8d,18
inhibited through binding of proteins or surface receptors.
b
Cytochrome c
Cytochrome c551
Myoglobin
9.8
5.7
7.4
9.8
4.7
8.6
12 kDa
11 kDa
16 kDa
14 kDa
14 kDa
14 kDa
0.66 (± 0.048) lM
b
Reduction of cytochrome c by ascorbate was clearly inhibited
in the presence of the receptor 1a Krel = 2 and 1d Krel = 4
7.36 (± 0.960) lM
b
3.56 (± 0.160) lM
c
Lysozyme
0.52 (± 0.062) lM
(Fig. 6) indicating the receptor sterically blocks approach of the
a − Lactalbumin
>5 lM
n/d
reducing agent (Fig. 2). Furthermore, the receptors both have
RNase A
affinity greater than that of the natural protein partner (2.4 lM,
a
b
12d
pH 7.4, 5 mM sodium phosphate buffer. Determined by following
5
mM phosphate, pH 7.0). Therefore, these receptors could
quenching of anthracene emission (emmax = 401 nm, ex 330 nm) upon
in principle disrupt protein function by binding to the solvent
exposed surface of cytochrome c displacing the natural protein
partner and preventing the redox reaction. The enhanced effect of
receptor 1d is presumably a result of the greater surface area (i.e.
greater steric impedence to the reducing agent) that it can provide
compared to 1a.
c
addition of protein. Determined by following the decrease in protein
emission (emmax = 340 nm, ex 285 nm) due to FRET from tryptophan of
protein (ex = 285 nm) to anthracene upon addition of receptor.
for 1a an order of magnitude lower than lysozyme and cytochrome
c reflecting their less basic nature (Table 2). At concentrations up
to 5 lM of protein, no FRET was observed between 1a and a-
lactalbumin. At higher concentrations, deviations from a linear
dependence upon fluorescence occurred for both receptor and
protein suggesting aggregation. However, Beers law was obeyed
−
4
in both cases up to at least 2 × 10 M. Furthermore, no exciplex
emission was observed for the anthracene group indicating that
inner filter effects are probably responsible for the non-linear
behaviour. This suggests that 1a binds to a-lactalbumin with K
d
>
5
lM. Only small changes in the fluorescence of 5 lM 1a were
observed upon titration with RNase A indicating that the binding
is likely quite weak. Although RNase A contains no tryptophan
residues, the high tyrosine content should still permit some FRET
to occur.
Fig. 6 Reduction kinetics of 10 lM cytochrome c by 2 mM ascorbate
(5 mM phosphate, pH 7.4) in the presence and absence of receptors 1a and
1d.
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4
−3
−1
−1
Conclusions
lactalbumin, 1.39 × 10 dm mol cm myoglobin, 0.82 ×
4
−3
−1
−1
1
0 dm mol cm RNase A) and found to be within 5–10%
In summary, the synthesis and characterization of a new class of
receptors for exterior protein surfaces represents a strategy that
allows for the detection of high affinity binding to a broad range
of proteins. We observe marked selectivity among these proteins
suggesting this strategy can form the basis for more selective
receptors with higher affinity.
of that determined by weight. The concentration of horse heart
cytochrome c and Pseudomonas aeruginosa cytochrome c551 was
determined using the molar extinction coefficient at 550 nM
4
of 2.95 × 10 after reduction using dithionite. No deviations
from Beers law were observed for any of the receptors in the
concentration ranges studied indicating they were monomeric in
solution. During the course of all titrations the guest solution
contained a concentration of host equivalent to that in the host
solution and the volume added was kept small to minimize large
changes in the concentration of guest, which were corrected for in
the regression analysis.
Experimental
All solvents were purchased from Malinkroft and all reagents
were purchased from Aldrich unless otherwise stated and used
without further purification. Yields are not optimized. Purification
by column chromatography was carried out using silica gel (230–
Eqn (1) operating in SigmaPlot was used to derive the dissocia-
tion constant for a 1 : 1 binding isotherm:
4
00 mesh size). Analytical thin layer chromatography (TLC)
√
2
f = m[(nc + x + K) − {(n*c + x + K) − 4ncx}]/2nc
(1)
was conducted using Baker 25 mm silica gel pre-coated glass
plates with fluorescent indicator active at UV245. Preparative TLC
was conducted using Analtech 1000 mm silica gel pre-coated
plates with fluorescent indicator active at UV245. Melting points
were obtained using an Electrothermal capillary melting point
f = change in relative fluorescence, m = maximum value of f , n =
stoichiometry, c = concentration of receptor, K = dissociation
constant, x = concentration of protein added.
Eqn (2) operating in SigmaPlot was used to derive the apparent
dissociation constant in the competition assay and then eqn (3)
was used to calculate the real dissociation constant
1
13
apparatus and are uncorrected. Both H and C NMR spectra
were acquired on either Bruker DPX 400 or DPX 500 series spec-
trometers at 400 and 100 MHz, or 500 and 125 MHz, respectively.
Chemical shifts are expressed as parts per million using solvent as
internal standard and coupling constants are expressed in Hz. The
following abbreviations are used: s for singlet, d for doublet, t for
triplet, q for quartet, p for pentet, m for multiplet and br for broad.
Preparative HPLC was performed on a Waters 600E controller
in conjunction with a Waters 490E multiwavelength UV detector.
Analytical HPLC was performed on a Rainin HP controller with a
Rainin UV detector, both attached to a Dell Optiplex PC running
Varian StarWorkstation software. Mass spectrometry data were
obtained by the University of Illionois at Urbana-Champaign
Mass Spectrometry Laboratory, under the guidance of Dr Steve
Mullen. CHN analyses were performed at Atlantic Microlabs
New Jersey. UV spectra were measured using an Agilent A453
spectrophotometer or a molecular devices spectramax250 plate
reader spectrophotometer. Fluorescence spectra were acquired on
a Hitachi F-4500 instrument.
√
2
f = f max*[(s + x + k
d
) − {(s + x + k
d
) − 4xs}]/2s
(2)
(3)
K
dcomp = K
d
/(1 + s/Kdrec)
f = change in fluorescence, f max = maximum change in fluo-
rescence, s = concentration of protein, x = concentration of
competitor added, K
d
= apparent dissociation constant, Kdcomp =
real dissociation constant of competitor, Kdrec = dissociation
constant of receptor as derived from eqn (1).
Electrophoretic mobility shift assay
Non-denaturing gel-shift assay was performed on thin (10 mm),
% agarose gel in 5 mM sodium phosphate gel buffer. 5 lL of
1
sample (protein = 8 lM, receptor 16 lM) were loaded on the gel,
and power (constant at 100 V) was applied for 1 hour. The gel was
fixed briefly in acetone, washed with water, and dried. The gel was
stained quickly with Coomasie blue R250 and destained in 10%
acetic acid solution.
Stock solutions of receptor were prepared to a concentration
of 0.1–0.9 mM in 5 mM phosphate buffer. Once the receptors
had been dissolved, the pH was adjusted to 7.4 via addition of
1
N sodium hydroxide or 1 N HCl. Likewise, stock solutions
UV–Vis-monitored ascorbate reduction assay
of hen egg white lysozyme, bovine milk a-lactalbumin, horse
skeletal myoglobin, bovine RNase A, horse heart cytochrome
c and Pseudomonas aeruginosa cytochrome c551 (all obtained
from Sigma and used without further purification except where
stated) were prepared to a concentration of 0.3–1.0 mM in
A solution containing cytochrome c was incubated with or without
synthetic agent in a 2 mL quartz cuvette with a pathlength of
1 cm, and the volume was adjusted to 1.9 mL by addition of 5 mM
sodium phosphate buffer (pH 7.4). After 10 min of incubation
at rt, 100 lL of a 20 mM stock solution of ascorbate in 5 mM
sodium phosphate buffer (pH 7.4) were added to the cuvette. The
final concentration of cytochrome c was 15 lM and of receptor
between 16–18 lM. The cuvette was immediately agitated with a
glass rod for <1 second, and the spectrophotometer started. The
instrument automatically measured the absorbance at 550 nm,
every 0.5 seconds, for at least 1 min. The collected data was
analyzed based on the equation derived from a pseudo first order
5
mM phosphate and the pH adjusted to 7.4 via addition
of sodium hydroxide or HCl (extinction coefficients and pI’s
were calculated using the program “ProtParam” available at
www.expasy.org). Pseudomonas aeruginosa cytochrome c551 was
subjected to ultrafiltration (5 kDa molecular cutoff) then dissolved
in 5 mM phosphate and subjected to ultrafiltration two more times
to remove undesired salts. The concentration of protein in the
stock solution was verified from the absorbance at 280 nm
in 6 M GdmHCl, 20 mM phosphate, pH 6.5 (e = 3.84 ×
−
kobs*t
rate expression: DAbs = max* (1 − e
); where DAbs = Abs −
t
4
−3
−1
−1
4
−3
−1
−1
1
0 dm mol cm lysozyme, 2.84 × 10 dm mol cm a-
Abst0 = 0, and t = time in seconds. Both max and k were floated,
This journal is © The Royal Society of Chemistry 2007
2
80 | Org. Biomol. Chem., 2007, 5, 276–285

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and were determined by regression analysis in the SigmaPlot 2001
software. The parameter max corresponds to maximal DAbs as t
approaches infinity or completion of reaction, and the parameter
kobs corresponds to the observed pseudo-first order rate constant
concentrated. The resultant crude product was passed through a
short pad of silica (1% methanol in dichloromethane) to yield the
product (0.54 g, 70%) as a white solid, mp 101.5–103.5 C (Found:
◦
C, 57.3; H, 5.3; N, 4.9. C27
5.8; N, 4.7%); d (400 MHz, DMSO-d
8.0), 2.96 (2H, dd, J 16.4 and 6.0), 3.62 (6H, s), 3.65 (6H, s), 4.84
2H, dt, J = 8.0, 6.3 Hz), 5.22 (2H, s), 7.36 (1H, d, J = 8.3 Hz),
7.42 (2H, t, J = 7.0), 7.49 (3H, d, J = 7.0), 7.66 (2H, s), 7.95
(100 MHz, CDCl ) 36.40,
9.51, 52.54, 53.36, 117.45, 118.27, 128.14, 128.71, 129.11, 135.88,
H
30
O
11
N
2
·CH
3
OH requires C, 56.9; H,
) 2.85 (2H, dd, J 16.4 and
−
1
in units of s . The relative rates of reaction were then calculated
H
6
based on the following equation Krel = K(cyt c + receptor)/Kcyt c
.
(
Benzoyloxycarbamoylphenylalanylaspartic acid diethyl ester
(
4
1
1H, s), 9.09 (2H, d, J = 7.8 Hz); d
H
3
To a stirred solution of benzoyloxycarbamoylphenylalanine
(
3.00 g, 10.0 mmol), aspartic acid hydrochloride diethyl es-
+
36.36, 159.47, 166.35, 171.48, 171.89; m/z (EI) 558.1860 (M ).
requires 558.1850).
ter (2.26 g, 10.0 mmol), DMAP (1.22 g, 10.0 mmol) in
C
27
H
30
O
11
N
2
◦
dichloromethane (150 mL) at 0 C was added EDCI (1.91 g,
1
0.0 mmol). The reaction mixture was then allowed to warm to
5
-Benzyloxy-N-bis(glycine ethyl ester) isophthalamide. 5-Ben-
room temperature and stirred overnight. The reaction mixture was
washed successively with hydrochloric acid (1 N, 25 mL), saturated
sodium bicarbonate (25 mL) and saturated sodium chloride then
dried over magnesium sulfate, filtered and concentrated to ca.
16
zyloxyisophthalic acid (0.40 g, 1.47 mmol), DMAP (0.45 g,
.67 mmol), glycine ethyl ester hydrochloride and EDCI
3.67 mmol, 0.7 g). Crude product was purified by silica gel
chromatography (1% methanol in dichloromethane) to yield the
product (0.46 g, 72%) as a clear viscous oil; d (400 MHz, CDCl
.28 (6H, t, J 7.0), 4.15 (4H, d, J 5.6), 4.21 (4H, q, J 7.0), 4.98
(2H, s), 7.32 (5H, m), 7.45 (2H, s), 7.57 (2H, t, J 5.6), 7.68 (1H, s);
(100 MHz, CDCl ) 14.55, 42.34, 53.83, 62.09, 117.40, 117.71,
28.07, 128.58, 129.03, 135.72, 136.55, 159.43, 167.13, 170.74; m/z
3
(
1
5 mL. The solution was passed through a short pad of silica
H
3
)
gel using 10% ethyl acetate in dichloromethane as eluent and
concentrated to leave the product (3.91 g, 83%) as a white solid, mp
1
◦
1
29.5–132 C (Found: C, 63.6; H, 6.4; N, 6.0. C26
H
31
O
7
N requires
) 1.27 (6H, dt, J
.0 and 7.0), 2.82 (1H, dd, J 17.1 and 4.5), 3.0 (1H, dd, J 16.9 and
.5), 3.15 (2H, m), 4.13 (2H, q, J 7.0), 4.22 (2H, q, J 7.0), 4.52
d
C
3
C, 63.8; H, 6.4; N, 5.9%); d
7
4
H
(400 MHz, CDCl
3
1
(
+
EI) 442.1741 (M ). C23
H
26
O
7
2
N requires 442.1740).
(
1H, m), 4.81 (1H, dt, J 7.8 and 4.5), 5.11 (2H, s), 5.36 (1H, d,
J 7.3), 6.89 (1H, d, J 7.8), 7.31 (10H, m); d (100 MHz, CDCl
4.46, 14.49, 36.59, 38.81, 49.08, 56.23, 61.48, 62.33, 67.42, 127.44,
C
3
)
5-Benzyloxy-N-bis(phenylalanine methyl ester) isophthalamide.
16
1
1
1
5-Benzyloxyisophthalic acid (1.00 g, 3.68 mmol), DMAP (1.12 g,
0.919 mmol) phenylalanine methyl ester hydrochloride (1.74 g,
8.09 mmol) and EDCI (1.76 g, 0.919 mmol). The reaction mixture
was purified by silica gel chromatography (30% ethyl acetate
in dichloromethane) to leave the product (1.76 g, 80%) as an
28.44, 128.58, 128.93, 129.05, 129.75, 136.42, 136.55, 156.22,
+
70.61, 171.07, 171.18; m/z (EI) 470 (M ).
Phenylalanylaspartic acid diethyl ester
amorphous white solid (Found: C, 70.8; H, 6.0; N, 4.4. C35
requires C, 70.7; H, 5.8; N, 4.7%); d (400 MHz, CDCl
2H, dd, J 13.9 and 5.8), 3.29 (2H, dd, J 13.9 and 5.8 Hz),
.77 (6H, s), 5.07 (2H, dt, J 7.6 and 5.8 Hz), 5.11 (2H, s), 6.62
2H, d, J 7.6), 7.13 (4H, d, J 8.0), 7.33 (11H, m), 7.48 (2H, s),
H
34
O
) 3.20
7
N
2
A solution of benzoyloxycarbamoylphenylalanine aspartate di-
ethyl ester (3.00 g, 6.38 mmol) and 10% palladium on carbon (0.16
g) in 1 : 1 tetrahydrofuran–methanol (25 mL) was stirred under
an atmosphere of hydrogen for 6 hours. When the reaction was
complete, the solution was filtered through celite and concentrated
H
3
(
3
(
7
1
1
.61 (1H, s); d
C
(100 MHz, CDCl ) 38.34, 52.90, 54.13, 70.90,
3
in vacuo to yield the product (2.05 g, 96%) as an off yellow glass,
17.32, 117.86, 127.71, 128.06, 128.72, 129.11, 129.68, 136.11,
36.15, 136.34, 159.53, 166.13, 172.23; m/z (EI) 594.2358 (M ).
◦
mp 207–209 C; d
H
(400 MHz, CDCl
3
) 1.25 (6H, m), 2.73 (1H, dd,
+
J 13.6 and 9.0), 2.78 (1H, dd, J 16.9 and 4.8), 3.00 (1H, dd, J 16.7
and 4.8), 3.23 (1H, dd, J 13.6 and 4.0), 3.69 (1H, dd, J = 9.0 and
C
35
H
34
O
7
2
N requires 594.2366.
4
.0), 4.13 (2H, m), 4.21 (2H, m), 4.83 (1H, dt, J 8.3 and 4.8), 7.27
5
-Benzyloxy-N-bis(phenylalanylaspartic acid diethyl ester)
(
5H, m), 8.12 (1H, d, J 8.3); d
C
(100 MHz, CDCl ) 14.50, 14.51,
3
16
isophthalamide. 5-Benzyloxyisophthalic
acid
(0.69
g,
3
6.91, 41.16, 48.71, 56.61, 61.40, 62.21, 127.26, 129.10, 129.73,
+
2.55 mmol), DMAP (0.68 g, 5.61 mmol), phenylalanylaspartic acid
diethyl ester (1.88 g, 5.61 mmol) and EDCI (1.07 g, 5.61 mmol).
Crude product was purified by silica gel chromatography (20%
137.91, 171.09, 171.13, 174.43; m/z (EI) 337 (M ).
General procedure for the synthesis of benzyl protected
bis(isophthalamides)
ethyl acetate in dichloromethane) to yield the product (1.45 g,
◦
6
3%) as a white solid, mp 160–162 C (Found: C, 64.5; H, 6.2; N,
5-Benzyloxy-N-bis(aspartic acid dimethyl ester) isophthalamide.
6.1. C49
H
57
O
13
N
4
requires C, 64.7; H, 6.2; N, 6.2%); d
H
(400 MHz,
To a stirred solution of 5-benzyloxyisophthalic acid (prepared
as described previously ) (0.40 g, 1.47 mmol), DMAP (0.45 g,
CDCl
3
) 1.15 (6H, t, J 7.0), 1.20 (6H, t, J 7.0), 2.83 (2H, dd, J 16.4
16
and 5.8), 2.92 (2H, dd, J 16.2 and 5.8), 3.18 (2H, dd, J 14.1 and
8.6), 3.29 (2H, dd, J 14.1 and 5.0), 4.70 (1H, dd, J 11.3 and 4.3),
4.81 (1H, dd, J 11.3 and 3.0), 4.89 (4H, m), 7.26 (19H, m), 7.54
3
3
.67 mmol) and aspartic acid dimethyl ester hydrochloride (0.64 g,
.23 mmol) in dimethyl formamide (25 mL) was added EDCI
(
0.70 g, 3.67 mmol). The reaction mixture was stirred overnight
then partitioned between water (150 mL) and ethyl acetate (3 ×
0 mL). The organic layer was washed successively with HCl (1
(1H, s), 7.90 (2H, brs); d
38.27, 49.40, 55.49, 61.43, 62.21, 70.48, 116.69, 118.54, 127.17,
128.27, 128.48, 128.92, 128.96, 129.61, 135.13, 136.71, 137.58,
C
(100 MHz, CDCl ) 14.45, 14.47, 36.38,
3
5
+
N, 50 mL), saturated bicarbonate (50 mL) and saturated sodium
chloride (50 mL) then dried over magnesium sulfate, filtered and
158.88, 166.23, 171.01, 171.43, 172.37; m/z (EI) 909.3925 (M ).
C
49
H
57
O
13
N
4
requires 909.3922.
This journal is © The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 276–285 | 281

View Article Online
General procedure for the debenzylation of bis(isophthalamides)
-Hydroxy-N-bis(aspartic acid dimethyl ester) isophthalamide.
A solution of 5-benzyloxy-N-bis(aspartic acid dimethyl ester)
isophthalamide (0.32 g, 0.57 mmol) and palladium on carbon (0.05
g) in tetrahydrofuran (15 mL) was stirred under an atmosphere
of hydrogen for 18 hours. When the reaction was complete, the
solution was filtered through celite and concentrated in vacuo to
bis(bromomethyl)anthracene (0.071 g, 0.194 mmol) and a few
crystals of sodium iodide in acetone (25 mL) was heated at reflux
for a period of 16 hours (caution, the product has poor solubility
in common organic solvents and can sometimes precipitate from
the reaction mixture). The reaction mixture was then filtered
and the precipitate washed several times with chloroform. The
combined organics were concentrated and dissolved in chloroform
5
(
75 mL). The solution was washed successively with hydrochloric
yield the product (0.27 g, 98%) as a clear oil, d
) 2.83 (2H, dd, J 16.4 and 8.2), 2.94 (2H, dd, J 16.4 and 6.4),
.62 (6H, s), 3.64 (6H, s), 4.82 (2H, dt, J 7.6 and 6.0), 7.38 (2H,
s), 7.74 (1H, s), 8.98 (2H, d, J 7.6), 10.06 (1H, s); d (100 MHz,
CDCl ) 36.38, 49.68, 52.63, 53.45, 117.49, 118.40, 135.49, 157.64,
67.00, 171.67, 171.96; m/z (EI) 468.1387. C20 requires
68.1380.
H
(500 MHz, DMSO-
acid (1 N 50 mL), saturated sodium bicarbonate (50 mL) and
saturated sodium chloride (50 mL) and then dried over magnesium
sulfate, filtered and concentrated. The crude material was then
purified by silica gel chromatography (2 : 10 : 100 ethanol–
acetone–chloroform) followed by size exclusion chromatography
d
6
3
C
3
1
4
H
24
O
11
N
2
(
(
sephadex LH20, 1 : 1 methanol–chloroform) to yield the product
0.0691 g, 32%) as a yellow glassy solid (Found: C, 58.6; H, 5.3;
N, 4.5. C56
H
58
O
22
N
4
·CH
) 2.98 (4H, dd, J 17.2 and 4.8), 3.12 (4H, dd, J
7.2 and 4.5), 3.68 (12H, s), 3.79 (12H, s), 5.08 (4H, dt, J 7.8 and
3
OH requires C, 58.5; H, 5.3; N, 4.8%); d
H
5
-Hydroxy-N-bis(glycine ethyl ester)isophthalamide. 5-Ben-
(400 MHz, CDCl
3
zyloxy-N-bis(glycine ethyl ester)isophthalamide (0.40 g,
.90 mmol), palladium on carbon (0.05 g). Product isolated
0.31 g, 97%) as a white solid, mp 126.5–129 C (Found: C, 54.8;
1
0
(
◦
4.5), 6.03 (4H, s), 7.39 (4H, d, J 7.8), 7.54 (4H, dd, J 6.8 and 3.0),
.73 (4H, s), 7.87 (2H, s), 8.28 (4H, dd, J 6.8 and 3.2); d (100 MHz,
CDCl ) 36.42, 49.48, 52.63, 53.44, 109.98, 117.30, 124.95,
27.05, 131.22, 136.16, 159.88, 166.38, 171.38, 171.95; m/z (FAB
7
C
H, 5.8; N, 7.9. C16
H
20
O
7
N
2
requires C, 54.5; H, 5.7; N, 7.9%);
) 1.20 (6H, t, J 7.0) 3.98 (4H, d, J 5.8),
.12 (4H, q, J 7.0), 7.40 (2H, s), 7.79 (1H, s), 8.94 (2H, t J 5.8),
0.04 (1H, s); d (100 MHz, CDCl ) 14.01, 41.93, 61.78, 116.80,
17.77, 134.71, 156.94, 167.59, 170.81; m/z (EI) 352.1270 (M ).
requires 352.1270.
d
4
1
1
H
(400 MHz, DMSO-d
6
3
1
+
mNBA matrix) 1139.3624 (M + H ). C56
1139.3621.
H
58
O
22
4
N + H requires
C
3
+
C
16
H
20
O
7
N
2
9
,10-Bis-(methyl-5-oxy-N-bis(glycine ethyl ester) isophthala-
mide) anthracene. A stirred solution of 5-hydroxy-N-bis(glycine
ethyl ester) isophthalamide (0.080 g, 0.227 mmol), potassium car-
bonate (0.033 g, 0.237 mmol), 9,10-bis(bromomethyl)anthracene
5
-Hydroxy-N-bis(phenylalanine methyl ester) isophthalamide.
-Benzyloxy-N-bis(phenylalanine methyl ester) isophthalamide
1.74 g, 2.93 mmol) and 10% palladium on carbon (0.17 g). The
product was isolated (1.56 g, 95%) as an amorphous white solid
Found: C, 66.1; H, 5.6; N, 5.4. C28 requires C, 66.7; H,
) 3.19 (2H, dd, J 13.9 and
5
(
(0.038 g, 0.103 mmol) and a few crystals of sodium iodide in
acetone (25 mL) was heated at reflux for 48 hours. The reaction
mixture was then filtered, to leave a pale yellow solid. The solid
was rinsed with water then methanol and dried thoroughly to give
the product (0.0654 g, 70%) as a pale yellow amorphous solid,
(
H
28
O
7
N
2
5
6
7
.6; N, 5.5%); d
H
(400 MHz, CDCl
3
.6), 3.27 (2H, dd, J 13.9 and 5.8), 3.76 (6H, s), 5.02 (2H, dt, J
.0 and 6.8), 5.30 (2H, s), 6.98 (2H, d, J 7.8), 7.16 (4H, m), 7.25
6H, m), 7.39 (2H, s), 7.46 (1H, s), 7.94 (1H, brs); d
d
7
6
H
(400 MHz, DMSO-d ) 1.21 (12H, t, J 7.0), 4.06 (8H, dt, J
.5 and 6.0), 4.14 (8H, q, J 7.0), 6.24 (4H, s), 7.67 (4H, dd, J
.8 and 3.0), 7.89 (4H, s), 8.10 (2H, s), 8.50 (4H, dd, J 6.8 and
6
(
C
(100 MHz,
CDCl
3
) 38.23, 52.99, 54.50, 117.03, 118.35, 127.68, 129.12, 129.63,
1
35.69, 136.14, 157.60, 166.78, 172.58; m/z (CI) 505.1959 (M +
+
3.2), 9.15 (4H, dt, J 9.8 and 5.5); d
C
(100 MHz, DMSO-d ) 14.47,
6
H ). C28
H
28
N
2
O
7
+ H requires 505.1974.
41.77, 60.88, 63.15, 116.89, 119.72, 125.27, 126.88, 129.49, 130.67,
5
-Hydroxy-N-bis(phenylalanyl aspartic acid diethyl ester)
135.71, 135.76, 159.10, 166.30, 170.15, 170.65; m/z (FAB mNBA
+
isophthalamide. 5-Benzyloxy-N-bis(phenylalanyl aspartatic acid
dimethyl ester) isophthalamide (0.505 g, 0.556 mmol), 10%
palladium on carbon (0.047 g). Product isolated (0.325 g, 71%)
as an amorphous white foam (Found: C, 61.3; H, 6.2; N, 6.6.
matrix) 907.3399 (M + H ). C H O N requires 907.3402.
4
8
50 14
4
9
,10-Bis-(methyl-5-oxy-N-bis(phenylalanine
isophthalamide)anthracene. stirred solution of 9,10-bis-
bromomethyl)anthracene (0.164 g, 0.451 mmol), 5-hydroxy-
N-bis(phenylalanine methyl ester) isophthalamide (0.500 g,
.992 mmol), potassium carbonate (0.143 g, 1.04 mmol), and
methyl
ester)
A
(
C
42
H
50
O
13
N
7
requires C, 61.6; H, 6.2; N, 6.8%); d
) 1.15 (6H, t, J 7.0), 1.16 (6H, t, J 7.0), 2.72 (2H, dd,
J 16.4 and 6.8), 2.82 (2H, dd, J 16.4 and 6.0), 2.98 (2H, dd, J
3.50 and 10.1), 3.10 (2H, dd, J 10.1 and 3.3), 4.08 (8H, m), 4.67
2H, dt, J 7.6 and 6.8), 4.74 (2H, m), 7.16 (2H, m), 7.25 (8H, m),
H
(400 MHz,
DMSO-d
6
0
a few crystals of sodium iodide in acetone (50 mL) was heated
under reflux for 16 hours. The reaction mixture was then filtered,
concentrated and dissolved in dichloromethane (75 mL). The
solution was washed successively with hydrochloric acid (1 N
1
(
7
9
3
1
.33 (2H, m), 7.62 (1H, s), 8.55 (2H, d, J 8.6), 8.60 (2H, d, J 7.8),
.91 (1H, s); d (100 MHz, DMSO-d ) 14.26, 14.32, 36.15, 37.45,
7.40, 49.04, 54.89, 60.73, 61.23, 117.35, 117.66, 126.60, 128.42,
29.46, 137.87, 138.55, 157.33, 166.21, 170.25, 170.84, 171.78; m/z
FAB mNBA matrix) 819.3454 (M + H ). C42
H
6
5
0 mL), saturated sodium bicarbonate (50 mL) and saturated
sodium chloride (50 mL) and then dried over magnesium sulfate,
filtered and concentrated. The solution was then passed down
a silica gel column using 10% ethyl acetate in dichloromethane
+
(
8
H
51
O
13
4
N requires
19.3452.
as the eluent to give the product (0.313 g, 57%) as a pale yellow
◦
9
,10-Bis-(methyl-5-oxy-N-bis(aspartic acid dimethyl ester)
solid, mp 198–200 C, d
H
(400 MHz, CDCl ) 3.24 (4H, dd, J
3
isophthalamide) anthracene. A stirred solution of 5-hydroxy-
N-bis(aspartic acid dimethyl ester) isophthalamide (0.200 g,
13.6 and 6.5), 3.36 (4H, dd, J 13.8 and 5.6), 3.83 (12H, s), 5.17
(4H, dt, J 7.6 and 6.5), 5.97 (4H, q, J 10.0), 7.10 (4H, d, J 7.6),
7.29 (20H, m), 7.58 (4H, dd, J 6.8 and 3.0), 7.67 (4H, s), 7.79
0
.427 mmol), potassium carbonate (0.062 g, 0.446 mmol), 9,10-
2
82 | Org. Biomol. Chem., 2007, 5, 276–285
This journal is © The Royal Society of Chemistry 2007

View Article Online
(
2H, s), 8.29 (4H, dd, J 6.8 and 3.0); d
8.23, 52.93, 54.40, 63.52, 117.13, 118.14, 124.93, 126.88, 127.62,
28.84, 129.08, 129.60, 131.07, 136.26, 136.34, 159.70, 166.55,
72.64.
C
(100 MHz, DMSO-d
6
)
The supernatant was decanted and the remaining pellet dissolved
in 40% acetonitrile in water and lyophilized to yield the product
(0.046 g, 87%) as a yellow fluffy solid, kmax (5 mM phosphate,
3
1
1
−
3
−1
−1
pH 7.4)/nm 300 (e/dm mol cm 5773) 354 (7051) 372 (10946)
3
8
6
93 (10617); d
H
(400 MHz, DMSO-d ) 2.73 (4H, dd, J 16.7 and
6
9
,10-Bis-(methyl-5-oxy-N-bis(phenylalanylaspartatic acid di-
.0), 2.87 (4H, dd, J 16.7 and 5.8), 4.79 (4H, dt, J 7.8 and 6.0),
.24 (4H, s), 7.66 (4H, dd, J 5.5 and 3.0), 7.86 (4H, s), 8.06 (2H,
methyl ester) isophthalamide) anthracene. A stirred solution
of 5-hydroxy-N-bis(phenylalanylaspartic acid dimethyl ester)
isophthalamide (0.1014 g, 0.124 mmol), potassium carbon-
ate (0.0179 g, 0.129 mmol), 9,10-bis(bromomethyl)anthracene
s), 8.49 (4H, dd, J 6.5 and 3.0), 8.96 (4H, d, J 7.8); d
DMSO-d ) 36.08, 49.75, 63.16, 118.83, 119.86, 125.28, 126.87,
29.47, 130.65, 135.82, 158.96, 165.76, 166.14, 172.08, 172.78; m/z
C
(100 MHz,
6
1
(
0.0205 g, 0.056 mmol) and a few crystals of sodium iodide in
(
(
MALDI-TOF a-hydroxy-4-hydroxycinnamic acid matrix) 1027
M + H ).
acetone (30 mL) was heated at reflux for a period of 16 hours. The
reaction mixture was then filtered, and the resultant precipitate
washed with water (2 × 10 mL) then dried thoroughly to yield
+
9,10-Bis-(methyl-5-oxy-N-bis(glycine) isophthalamide) anthra-
the product (0.1005 g, 97%) as a slightly yellow glassy solid, d
H
cene 1b. To a stirred solution of 9,10-bis-(methyl-5-oxy-N-
bis(glycine ethyl ester) isophthalamide) anthracene (0.020 g,
0.0221 mmol) in 1 : 1 ethanol in water (10 mL) was added sodium
hydroxide (1 N, 0.13 mL, 6 eq.). The reaction mixture was then
stirred until deprotection was complete as evidenced by reverse
phase HPLC, then concentrated and acidified with 1 N HCl to
give a cloudy suspension, which was filtered and dried thoroughly
to yield the product (0.015 g, 86%) as a yellow solid, kmax (5 mM
(
6
3
400 MHz, DMSO-d ) 1.15 (24H, m), 2.72 (4H, dd, J 16.4 and
.8), 2.82 (4H, dd, J 16.4 and 6.0), 2.98 (4H, dd, J 12.2 and 11.6),
.12 (4H, dd, J 14.4 and 3.3), 4.06 (16H, m), 4.67 (4H, dt, J 6.8
and 6.3), 4.80 (4H, m), 6.15 (2H, d, J 12.2), 6.24 (2H, d, J 12.2),
.16 (4H, m), 7.24 (8H, m), 7.35 (8H, m), 7.67 (4H, dd, J 7.0 and
.0), 7.75 (4H, s), 7.89 (2H, s), 8.46 (4H, dd, J 6.3 and 3.0 Hz),
.65 (4H, d, J 7.8), 8.74 (4H, d, J 8.0); d
) 14.26, 14.30, 36.11, 37.46, 49.07, 54.96, 60.66, 61.22, 63.05,
16.69, 120.05, 125.22, 126.62, 126.91, 128.43, 129.46, 130.65,
6
7
3
8
d
1
C
(100 MHz, DMSO-
−
3
−1
−1
6
phosphate, pH 7.4)/nm 300 (e/dm mol cm 5928) 353 (7173)
372 (10933) 393 (10582); d (400 MHz, DMSO-d ) 3.97 (8H, d,
H
6
1
35.93, 138.45, 138.54, 158.65, 165.81, 170.26, 170.76, 171.77; m/z
J = 5.8 Hz), 6.24 (4H, s), 7.66 (4H, dd, J = 7.0, 3.3 Hz), 7.87 (4H,
+
(
FAB mNBA matrix) 1839 (M + H ).
s), 8.08 (2H, s), 8.49 (4H, dd, J = 7.0, 3.3 Hz), 9.01 (4H, t, J =
5.8 Hz); d
C
(100 MHz, DMSO-d ) 49.86, 63.16, 117.33, 119.70,
6
9
,10-Bis-(methyl-5-oxy-N-bis(aspartic acid dimethyl ester)
1
25.08, 126.89, 129.50, 130.66, 135.86, 159.04, 166.51, 171.61;
isophthalamide) benzene. A stirred solution of 5-hydroxy-
N-bis(aspartic acid dimethyl ester) isophthalamide (0.200 g,
+
m/z (FAB mNBA matrix) 795.2149 (M + H ). C40
H
34
O
4
N
14
+
H requires 795.2150.
0
.427 mmol), caesium carbonate (0.152 g, 0.460 mmol), p-
xylylenedibromide (0.0513 g, 0.194 mmol) and a few crystals
of sodium iodide in acetone (25 mL) was heated at reflux for
a period of 16 hours. The reaction mixture was then filtered,
concentrated and dissolved in dichlormethane (75 mL). The
solution was washed successively with hydrochloric acid (1 N
9,10-Bis-(methyl-5-oxy-N-bis(phenylalanyl) isophthalamide) an-
thracene 1c. To a stirred solution of 9,10-bis-(methyl-5-oxy-
N-bis(phenylalanyl methyl ester) isophthalamide) anthracene
(0.244 g, 0.201 mmol) in 1 : 1 water in ethanol (15 mL) was added
sodium hydroxide (1 N, 2.01 mL, 10eq). The reaction mixture
was then stirred until deprotection was complete as evidenced by
reverse phase HPLC, then concentrated and acidified with 1 N HCl
to give a cloudy suspension, which was subjected to centrifugation.
The supernatant was decanted and the remaining oily pellet
dissolved in 30% acetonitrile in water and lyophilized to yield the
5
0 mL), saturated sodium bicarbonate (50 mL) and saturated
sodium chloride (50 mL) and then dried over magnesium sulfate,
filtered and concentrated. The crude material was then purified
by silica gel chromatography (2 : 10 : 100 ethanol–acetone–
chloroform) followed by size exclusion chromatography (sephadex
LH20, 1 : 1 methanol–chloroform) to yield the product (0.129 g,
◦
product (0.154 g, 66%) as a slightly yellow solid, mp 167–169 C;
−
3
−1
−1
6
4%) as an amorphous white solid (Found: C, 53.3; H, 5.1; N,
·2H O requires C, 53.6; H, 5.4; N, 5.2%); d
) 2.97 (4H, dd, J 17.0 and 4.4), 3.12 (4H, dd, J
7.0 and 4.7), 3.70 (12H, s), 3.79 (12H, s), 5.06 (4H, dt, J 7.8 and
.4), 5.12 (4H, s), 7.33 (4H, d, J 7.2), 7.47 (4H, s), 7.56 (4H, s), 7.78
(100 MHz, CDCl ) 36.41, 49.48, 52.61, 53.42, 117.47,
18.34, 128.48, 135.88, 136.51, 159.41, 166.33, 171.47, 171.98;
k
max (5 mM phosphate, pH 7.4)/nm 298 (e/dm mol cm 4587)
5.1. C48
H
54
O
22
N
4
2
H
355 (5277) 374 (8228) 394 (7885); d (400 MHz, DMSO-d ) 3.12
H
6
(500 MHz, CDCl
3
(4H, dd, J 13.4 and 10.6), 3.20 (4H, dd, J 13.4 and 4.0), 4.66
(4H, m), 6.21 (4H, m), 7.16 (4H, m), 7.27 (8H, m), 7.34 (8H,
m), 7.66 (4H, dd, J 6.7 and 3.0), 7.80 (4H, s), 8.00 (2H, s), 8.49
1
4
(
2H, s); d
C
3
(4H, dd, J 7.0 and 3.0), 8.96 (4H, d, J 7.8), 12.82 (8H, brs); d
(100 MHz, DMSO-d ) 36.54, 54.74, 63.11, 116.79, 119.98, 125.29,
C
1
6
+
m/z (FAB mNBA matrix) 1039.3305 (M + H ). C48
H
54
O
22
N
4
+
126.71, 126.90, 128.56, 129.43, 129.50, 130.65, 135.83, 138.55,
H requires 1039.3308.
158.81, 165.93, 166.01, 173.41; m/z (MALDI-TOF a-hydroxy-
+
4
-hydroxycinnamic acid matrix) 1178 (M + Na ).
9
,10-Bis-(methyl-5-oxy-N-bis(aspartic acid) isophthalamide) an-
thracene 1a. To a stirred solution of 9,10-bis-(methyl-5-oxy-
N-bis(aspartic acid dimethyl ester) isophthalamide) anthracene
9,10-Bis-(methyl-5-oxy-N-bis(phenylalanyl aspartic acid) isoph-
thalamide) anthracene 1d. To a stirred solution of 9,10-
bis-(methyl-5-oxy-N-bis(phenylalanyldiethylaspartate) isophtha-
lamide) anthracene (0.0725 g, 0.0394 mmol) in 1 : 1 ethanol in
water (10 mL) was added sodium hydroxide (1 N, 0.39 mL, 10
eq.). The reaction mixture was then stirred until deprotection was
complete as evidenced by reverse phase HPLC, then concentrated
(
0.0582 g, 0.0514 mmol) in 1 : 1 ethanol in water (10 mL) was added
sodium hydroxide (1 N, 0.51 mL, 10 eq.). The reaction mixture
was then stirred until deprotection was complete as evidenced by
reverse phase HPLC, then concentrated and acidified with 1 N HCl
to give a cloudy suspension, which was subjected to centrifugation.
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and acidified with 1 N HCl to give a cloudy suspension, which
was subjected to centrifugation. The supernatant was decanted
and the remaining oily pellet dissolved in 30% acetonitrile in
water and lyophilized to yield the product (0.058 g, 90%) as a
slightly yellow fluffy solid, kmax (5 mM phosphate, pH 7.4)/nm
M. A. Fazal, B. C. Roy and S. Mallik, Org. Lett., 2000, 2, 911–914;
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C. M. Goodman, T. Emrick and V. M. Rotello, J. Am. Chem. Soc.,
−
3
−1
−1
3
d
5
00 (e/dm mol cm 5685) 353 (7175) 372 (11183) 393 (10819);
(400 MHz, DMSO-d
) 2.62 (4H, m), 2.74 (4H, dd, J = 16.7,
.8 Hz), 2.96 (4H, m), 3.12 (4H, m), 4.58 (4H, q, J 6.6), 4.82 (4H,
H
6
2
004, 126, 730–743; (c) M. Braun, S. Atlick, D. M. Guldi, H. Lanig, M.
Brettreich, S. Burghardt, M. Hatzimarinaki, E. Ravanelli, M. Prato,
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m), 6.18 (4H, p, J 11.6), 7.15 (4H, m), 7.22 (8H, m), 7.34 (8H, m),
.67 (4H, d, J 7.6), 7.74 (4H, s), 7.91 (2H, m), 8.49 (8H, m), 8.71
4H, m), 12.64 (8H, brs); d (100 MHz, DMSO-d ) 36.36, 36.53,
(
d) N. O. Fischer, C. M. McIntosh, J. M. Simard and V. M. Rotello,
7
Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 5018–5023; (e) M. A. Fazal,
B. C. Roy, S. Sun, S. Mallik and K. R. Rodgers, J. Am. Chem. Soc.,
2001, 123, 6283–6290; (f) K. Leung, Z. Yang and R. Breslow, Proc.
Natl. Acad. Sci. U. S. A., 2000, 97, 5050–5053; (g) C. Roy, M. A.
Fazal, S. Sun and S. Mallik, Chem. Commun., 2000, 547–548; (h) H.
Takashima, S. Shinkai and I. Hamachi, Chem. Commun., 1999, 2345–
2346.
(
C
6
3
1
1
7.57, 39.24, 49.09, 54.96, 116.74, 125.25, 126.59, 126.92, 128.41,
29.50, 130.66, 135.85, 138.64, 158.68, 165.82, 171.44. 171.63,
72.03, 172.57; m/z (MALDI-TOF a-hydroxy-4-hydroxycinnamic
+
acid matrix) 1639 (M + Na ).
7
8
(a) A. C. Gemperli, S. E. Rutledge, A. Maranda and A. Schepartz,
J. Am. Chem. Soc., 2005, 127, 1596–1597; (b) T. L. Schneider, R. S.
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Malashkevich and P. S. Kim, Proc. Natl. Acad. Sci. U. S. A., 2002, 99,
9
,10-Bis-(methyl-5-oxy-N-bis(aspartyl) isophthalamide) ben-
zene 2. To a stirred solution of 9,10-bis-(methyl-5-oxy-N-
bis(dimethylaspartate) isophthalamide) benzene (0.0474 g,
.0457 mmol) in 1 : 1 tetrahydrofuran in water (10 mL) was added
0
1
4664–14669; (d) J. W. Chin and A. Schepartz, Angew. Chem., Int. Ed.,
lithium hydroxide (1 N, 0.54 mL, 12 eq.). The reaction mixture
was then stirred until deprotection was complete as evidenced by
reverse phase HPLC, then concentrated and acidified with 1 N HCl
and concentrated. The resulting white solid was dissolved in 1 : 1
chloroform–methanol, filtered and concentrated then redissolved
in 40% acetonitrile in water and lyophilized to yield the product
0.038 g, 89%) as a white powder, d
4H, dd, J 16.4 and 7.8 Hz), 2.85 (4H, dd, J 16.4 and 6.0), 4.74
4H, dt, J 8.0 and 6.0), 5.24 (4H, s), 7.53 (4H, s), 7.68 (4H,
2001, 40, 3806–3809; (e) I. Hamachi, Y. Yamada, T. Matsugi and S.
Shinkai, Chem.–Eur. J., 1999, 5, 1503–1511.
(a) H. Yin, G.-I. Lee, H.-S. Park, G. A. Payne, J. M. Rodriguez, S. M.
Sebti and A. D. Hamilton, Angew. Chem., Int. Ed., 2005, 44, 2704–2707;
(
b) H. Yin, G.-I. Lee, K. A. Sedey, J. M. Rodriguez, H. G. Wang, S. M.
Sebti and A. D. Hamilton, J. Am. Chem. Soc., 2005, 127, 5463–5468;
c) S. Fletcher and A. D. Hamilton, Curr. Opin. Chem. Biol., 2005, 9,
(
6
2
32–638; (d) H. Yin and A. D. Hamilton, Bioorg. Med. Chem. Lett.,
004, 14, 1375–1379; (e) J. T. Ernst, J. Becerril, H.-S. Park, H. Yin
(
(
(
H
(400 MHz, DMSO-d ) 2.72
6
and A. D. Hamilton, Angew. Chem., Int. Ed., 2003, 42, 535–539; (f) O.
Kutzki, H.-S. Park, J. T. Ernst, B. P. Orner, H. Yin and A. D. Hamilton,
J. Am. Chem. Soc., 2002, 124, 11838–11839; (g) J. T. Ernst, O. Kutzki,
A. K. Debnath, S. Jiang, H. Lu and A. D. Hamilton, Angew. Chem.,
Int. Ed., 2002, 41, 278–281; (h) P. L. Toogood, J. Med. Chem., 2002, 45,
1543–1558; (i) M. S. Cubberly and B. K. Iverson, Curr. Opin. Chem.
Biol., 2001, 5, 650–653; (j) B. P. Orner, J. T. Ernst and A. D. Hamilton,
J. Am. Chem. Soc., 2001, 123, 5382–5383.
s), 8.02 (2H, s), 8.96 (4H, d, J 7.8 Hz); d
) 36.10, 39.84, 71.72, 116.87, 120.62, 128.28, 135.71, 136.72,
58.53, 165.65, 172.06, 172.76; m/z (MALDI-TOF a-hydroxy-4-
C
(100 MHz, DMSO-
d
1
6
+
hydroxycinnamic acid matrix) 927 (M + H ).
9
(a) T. Aya and A. D. Hamilton, Bioorg. Med. Chem. Lett., 2003, 13,
2651–2654; (b) M. A. Blaskovich, Q. Lin, F. L. Delarue, J. Sun, H.-S.
Park, D. Coppola, A. D. Hamilton and S. M. Sebti, Nat. Biotechnol.,
2000, 18, 1065–1070; (c) R. K. Jain and A. D. Hamilton, Org. Lett.,
Acknowledgements
We thank the National Institutes of Health (GM35208) for
financial support of this work.
2
000, 2, 1721–1723; (d) H.-S. Park, Q. Lin and A. D. Hamilton, J. Am.
Chem. Soc., 1999, 121, 8–13; (e) Y. Hamuro, M. C. Calama, H.-S. Park
and A. D. Hamilton, Angew. Chem., Int. Ed. Engl., 1997, 36, 2680–
2
683.
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