Molecular probe for enzymatic activity with dual output

Article abstract of DOI:10.1016/j.bmc.2007.08.046

A novel molecular probe for enzymatic activity with a dual output detection-mode has been developed. The probe effectively detected the presence of the bacterial protease penicillin-G-amidase; a single cleavage by the enzyme initiated the fragmentation of

Full text of DOI:10.1016/j.bmc.2007.08.046

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7318–7324
Molecular probe for enzymatic activity with dual output
Eyal Danieli and Doron Shabat*
Department of Organic Chemistry, School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences,
Tel-Aviv University, Tel Aviv 69978, Israel
Received 17 April 2007; revised 7 August 2007; accepted 17 August 2007
Available online 29 August 2007
Abstract—A novel molecular probe for enzymatic activity with a dual output detection-mode has been developed. The probe effec-
tively detected the presence of the bacterial protease penicillin-G–amidase; a single cleavage by the enzyme initiated the fragmen-
tation of a self-immolative dendritic platform to release two reporter units. The signals of the free reporters were detected by
two different spectroscopic techniques, fluorescence and UV–vis. This is the first reported molecular probe with two different chro-
mogenic reporter units activated by a specific stimulus.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
a novel molecular probe with UV–vis and fluorescence
modes for the detection of a specific catalytic activity.
There is a constant and growing need for the detection
of enzymatic activity for imaging and diagnostics pur-
poses.1–4 Most currently used molecular probes are
based on generation of a chromogenic compound
through the catalytic activity of a specific enzyme.5–7
The chromogenic assay is usually monitored by fluores-
cence or UV–vis spectroscopy and in other examples by
generation of luminescence or precipitated dye com-
pounds.8 Each assay has its own advantages and disad-
vantages. For example, fluorescence can be detected at
very low chromophore concentration (down to nm
range). However, it cannot be used in an environ-
ment—as in the presence of certain proteins—that
quenches the fluorescent signal. A molecular probe with
dual output signals offers two detection modes allowing
use of the same probe in different environments. Here we
report the design, synthesis, and enzymatic activation of
The molecular probe is illustrated in Figure 1. The cen-
tral unit of the probe (the molecular adaptor) is linked
to an enzymatic substrate that acts as a trigger and to
two different reporter molecules. Cleavage of the enzy-
matic substrate triggers the release of the two reporters
and a consequent activation of their signals.
In order to construct a molecular probe with the re-
quired activity we have used AB2 self-immolative den-
dron 1 (Fig. 2). Self-immolative dendrimers are novel
class of molecules that can amplify a single cleavage
event received at the focal point into release of multiple
tail groups at the periphery.9–17 In dendron 1, a pheny-
lacetamide group (red), known to be a substrate for the
bacterial enzyme penicillin-G–amidase (PGA), is used as
a trigger and the reporter units are 4-nitrophenol and 6-
aminoquinoline. The reporter molecules are attached
through stable carbamate linkages that maintain the sig-
nals in an OFF position. Upon cleavage of the trigger
and release of the reporter units, the signal from each
is turned ON. The 4-nitrophenol is detected by visible
yellow color and 6-aminoquinoline by fluorescence
emission. PEG5000 is also attached to the dendritic
adaptor in order provide substantial aqueous
solubility.18
Abbreviations: ACN, acetonitrile; Boc, t-butoxycarbonyl; DCM,
dichloromethane; DIPEA, diisopropyl ethyleneamine; DMAP, dime-
thylaminopyridine; DMF, dimethyl formamide; EDC, N-(3-dimethyl-
aminopropyl)-N0-ethylcarbodiimide; EtOAc, ethylacetate; He, hexanes;
HOBT, 1-hydroxybenzotriazole; PEG, polyethylene glycol; PNP, p-ni-
trophenol; RT, retention time; TBSCl, t-butyldimethylsillyl chloride;
TBTA, tris-(1-benzyl-1H-[1,2,3]triazol-4-ylmethyl)-amine; TFA, trifluo-
roacetic acid; THF, tetrahydrofuran.
Keywords: Molecular probe; Assay; Enzyme; Dendrimer; Self-
immolative.
The disassembly pathway of molecular probe 1 is initi-
ated by catalytic cleavage of phenylacetic acid by PGA,
elimination of azaquinone-methide, decarboxylation,
*
Corresponding author. Tel.: +972 (0) 3 640 8340; fax: +972 (0) 3 640
9293; e-mail: chdoron@post.tau.ac.il
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.046

E. Danieli, D. Shabat / Bioorg. Med. Chem. 15 (2007) 7318–7324
7319
Reporter I
Reporter I
Reporter II
Enzymatic
Cleavage
Enzyme
Substrate
Molecular
Adaptor
Solublizer
Reporter II
Figure 1. A graphical illustration of a molecular probe for detection of enzymatic activity with dual output.
O
O N
O
2
N
N
O
H
O
O
N
O
NH
O
N
O
O
O
N
N
N
O
N
O
1
H
PEG 5000 N
NH
O
N
Figure 2. Chemical structure of a molecular probe with UV–vis and fluorescence outputs for penicillin-G–amidase activity. The phenylacetamide
group (red) is a substrate for PGA. The reporter units, 4-nitrophenol and 6-aminoquinoline, provide a visible signal and a fluorescence signal,
respectively, upon release.
and cyclization to release dimethylurea derivative and
phenol 1a (Fig. 3). The latter rapidly undergoes double
elimination10 to release the two reporter units and
byproduct 1b. The output of these cascade reactions
should be expressed by new absorbance and fluorescence
signals as a result of the release of 4-nitrophenol and 6-
aminoquinoline, respectively.
lease of the reporters, 6-aminoquinoline and 4-nitrophe-
nol, by fluorescence and UV–vis spectroscopy. The
fluorescence spectrum of 1 exhibited a strong emission
band at 390 nm that decreased over time as the probe
fragmented (Fig. 5a). The generation of a new band at
460 nm indicated the formation of free 6-aminoquino-
line. In order to evaluate the kinetic behavior of the
sequential fragmentation, the maximal intensities of
the band at 460 nm were plotted as a function of the
time (Fig. 5b). The enzymatic cleavage was also moni-
tored in the visible range; upon incubation of 1 in the
presence of PGA, the release of 4-nitrophenol resulted
in a significant increase in absorbance with a maximum
at approximately 405 nm (Fig. 5c). This absorbance
gradually increased as a function of time in the presence
of PGA (Fig. 5d). Importantly, no release of 6-amino-
quinoline or 4-nitrophenol was observed when com-
pound 1 was incubated in the buffer without PGA
(data not shown).
Dendritic molecule 1 was synthesized according to the
scheme shown in Figure 4. 4-Hydroxybenzoic acid was
coupled with propargyl amine to form amide 2. The amide
wasreactedwithformaldehydetogeneratedibenzylalcho-
hol 3, which was then protected with two equivalents of t-
butyldimethylsilylchloride (TBSCl) to afford phenol
derivative 4. The latter was activated with 4-nitrophenyl-
chloroformate to produce carbonate 5, which was further
reacted with mono-Boc-N,N0-dimethylethylene-diamine
toaffordcompound6.Deprotectionof6withtrifluoroace-
tic acid (TFA) gave an amine salt, which was immediately
reacted with the carbonate trigger 7 to give compound 8.
Activation of diol 8 with 4-nitrophenyl-chloroformate
affordeddoublecarbonate9, whichwasselectivelyreacted
with one equivalent of mono-Boc-N,N0-dimethylethyl-
ene-diamine to yield compound 10. Reaction of 10 with
6-aminoquinoline afforded compound 11, which was
deprotected using TFA and in situ reacted with bis(4-
nitrophenyl)-carbonate to generate compound 12. The
latter was coupled with PEG5000 azide derivative18 13
via the copper(I)-catalyzed Huisgen cycloaddition19 to
give dendritic molecule 1.
As far as we know, this is the first reported molecular
probe that includes two different types of reporter units
activated upon on a specific stimulus. The only examples
in the literature for a molecular probe with dual mode of
detection employ generation of a single reporter mole-
cule with UV and fluorescence signals.20 The other op-
tion to achieve dual detection would be to use two
separate probes. However, in this case there could be a
problem of competitive catalysis (circumstances in
which the km of the two substrate is not identical). In
our probe, 6-aminoquinoline and 4-nitrophenol, de-
tected by fluorescence and absorbance spectroscopy,
respectively, were used as reporter units. Due to the syn-
thetic flexibility of our approach, other reporter mole-
In order to test our molecular probe, we incubated com-
pound 1 in phosphate buffered saline (PBS, pH 7.4) at
37 °C (with and without PGA) and monitored the re-

7320
E. Danieli, D. Shabat / Bioorg. Med. Chem. 15 (2007) 7318–7324
O
O2N
O
N
N
O
O
O
H2O
1
NH
O
OH
N
N
O
O
N
HO
O
1a
CO2
CO2
NH
N
N
N
N
O
H
PEG 5000
HO
N
NH
O
N
NH
O
OH
1b
N
N
N
O2N
OH
N
NH2
HO
H
PEG 5000
N
O
Figure 3. Disassembly pathway of AB2 self-immolative dendritic molecule 1.
cules with different types of functional groups, like
amine or hydroxyl, can be linked to our molecular
probe. The two assays must be orthogonal each to other,
in order to prevent disturbances in the detection mea-
surement. Another advantage of the probe is the aque-
ous solubility provided by the PEG5000 tail; this tail
should guarantee sufficient solubility for most organic
reporter molecules in assay media. As demonstrated,
this probe functioned under physiological conditions
and this design should allow detection of any number
of specific bioactivities. We have used phenylacetamide
as a triggering substrate for PGA, however, many other
substrates can be applied instead. Specifically, this sys-
tem could be used for detection of proteolytic activity
by PGA, which is expressed in bacteria.22 The disassem-
bly of the probe occurred according to Figure 3. Recent
studies of our self-immolative dendritic systems have
shown that the rate-limiting step of the disassembly
takes place after the enzymatic cleavage and is depen-
dent on the nature of the substituent on the aromatic
ring.23
2. Experimental
2.1. General
All reactions requiring anhydrous conditions were per-
formed under argon or N2 atmosphere. Chemicals and
solvents were either A.R. grade or purified by standard
techniques. Thin layer chromatography (TLC): silica gel
plates Merck 60 F254; compounds were visualized by
irradiation with UV light and/or by treatment with a
solution of 25 g phosphomolybdic acid, 10 g Ce(SO )
4 2*-
H2O, 60 ml conc. H2SO4, and 940 ml H2O, followed by
heating. Flash chromatography (FC): silica gel Merck
60 (partical size 0.040–0.063 mm), eluent given in paren-
theses. H NMR: Bruker AMX 200 or 400. The chemical
shifts are expressed in d relative to TMS (d = 0 ppm) and
the coupling constants J in Hz. The spectra were re-
corded in CDCl3 or CD3OD as a solvent at room tem-
perature. All reagents, including salts and solvents,
were purchased from Sigma–Aldrich. TBTA was kindly
obtained from the Sharpless laboratory (Scripps, CA).
In summary, we have synthesized and demonstrated the
utility under physiological conditions of a novel molec-
ular probe, based on an AB2 self-immolative dendron,
that has a dual output detection-mode for enzymatic
activity. The probe was designed for detection of the
bacterial protease PGA, such that a single cleavage by
the enzyme initiated the fragmentation of the dendritic
platform to release two different types of reporter units.
Each free reporter was visualized by a different spectro-
scopic technique, fluorescence and UV–vis. The reporter
units can be readily replaced by other chromogenic mol-
ecules thereby the type of output signal desired in the
probe system. Similarly, substrates other than the
phenylacetamide trigger can be used to allow detection
2.1.1. 4-Hydroxy-N-(prop-2-ynyl)benzamide (compound
2). Commercially available 4-hydroxybenzoic acid
(2.0 g, 14.5 mmol) was dissolved in DMF. Then, EDC
(3.3 g, 17.4 mmol), HOBT (1.0 g, 7.3 mmol), and prop-
argyl amine (1.0 ml, 14.5 mmol) were added. The mix-
ture was stirred overnight and monitored by TLC
(EtOAc/Hex = 2:3). After completion of the reaction
the solvent was removed under reduced pressure. The
crude product was purified by column chromatography
on silica gel (EtOAc/Hex = 2:3) to give compound 2
(1.8 g, 70%) in the form of yellowish oil.
1H NMR (200 MHz, CDCl3)d = 7.70 (d, J = 6.8 Hz,
2H); 6.81 (d, J = 6.8 Hz, 2H); 4.11 (d, J = 2.5 Hz, 2H);
of different enzymatic activities.
2.71
(t,
J = 2.5 Hz,
1H).
13C(400 MHz,

E. Danieli, D. Shabat / Bioorg. Med. Chem. 15 (2007) 7318–7324
7321
HO
TBSO
TBSO
NH
O
NH
O
NH
O
TBSCl
HO
EDC
formaldehyde
OH
OH
OH
OH
NaOH/H2O
O
H2N
3
4
2
HO
TBSO
TBSO
TBSO
N
O
HN
4-nitrophenyl
chloroformate
O
1.TFA
2.
O
NH
O
O
NH
O
N
O
O
O
O
N
O
O
NO2
O
H
N
O
O2N
5
6
7
O
TBSO
O2N
O
O
HO
O
O
N
O
O
4-nitrophenyl
chloroformate
O
HN
NH
O
NH
N
O
H
N
O
H
O
O
N
O
N
N
N
O
O
O
O
O
8
O
O
9
HO
O2N
O
O
O
O
N
N
N
N
O
O
O
O
O
O
NH
O
H
O
O
N
6-aminoquinoline
N
NH
O
N
H
N
O
N
O
N
O
O
O
O
O
O
O
O
11
O
NH
10
O2N
N
O2N
O
N
O
13
N
O
O
O
NH
O
PEG 5000
N
3
O
NH
O
H
N
O
N
1. TFA
1
O
N
O
Cu / CuSO4
2. bis(4-nitrophenyl) carbonate
O
O
12
NH
N
Figure 4. Chemical synthesis of molecular probe 1.
CDCl3)d = 167.9, 160.6, 128.8, 124.4, 114.5, 79.5, 70.3,
28.3. MS (FAB): calculated for C10H9NO2176.0
[M+H+], found 176.0.
pressure. The crude product was purified by column
chromatography on silica gel (EtOAc/MeOH = 19:1)
to give compound 3 (1.9 g, 80%) in the form of a white
solid.
2.1.2. 4-Hydroxy-3,5-bis(hydroxymethyl)-N-(prop-2-ynyl)-
benzamide (compound 3). To a cool 12% solution of
NaOH in water (12 ml) compound 2 (1.8 g, 10.2 mmol)
was added while maintaining the temperature at 0 °C.
Formaldehyde 37% in water (10 ml) was added. The
reaction mixture was stirred at 55 °C for 3 days and
monitored by TLC (EtOAc/ MeOH = 95:5). After com-
pletion, the reaction mixture was diluted with EtOAc
and washed with saturated ammonium chloride solu-
tion. The aqueous layer was washed twice with EtOAc.
The combined organic layer was dried over magnesium
sulfate and the solvent was removed under reduced
1H NMR (200 MHz, CD3OD) d = 7.80 (s, 2H); 4.91 (s,
4H); 4.26 (d, J = 2.5 Hz, 2H); 2.70 (t, J = 2.5 Hz, 1H).
13C(400 MHz, CD3OD) d = 168.1, 156.7, 126.8, 126.0,
124.4, 79.4, 70.2, 60.3, 28.3. MS (FAB): calculated for
C12H13NO4 236.0 [M+H+], found 236.0.
2.1.3. 3,5-Bis((tert-butyldimethylsilyloxy)methyl)-4-
hydroxy-N-(prop-2- ynyl)benzamide (compound 4). Com-
pound 3 (713 mg, 3.0 mmol) was dissolved in DMF at
0 °C. Imidazole (408 mg, 6.0 mmol) and TBSCl
(910 mg, 6.0 mmol) were added. The reaction mixture

7322
E. Danieli, D. Shabat / Bioorg. Med. Chem. 15 (2007) 7318–7324
Figure 5. (a) Emission fluorescence (kex = 250 nm) of 1 [500 lM] upon addition of PGA (1.0 mg/mL). (b) Fluorescence of 1 at 460 nm in the presence
of PGA (1.0 mg/mL) as a function of time. (c) UV–vis absorbance spectra of 1 [500 lM] in the presence of PGA (1.0 mg/mL). (d) Absorbance of 1 at
400 nm after addition of PGA (1.0 mg/mL) as a function of time.
was stirred at room temperature for 2 hours and moni-
tored by TLC (EtOAc/He = 2:8). After completion, the
reaction mixture was diluted with ether and washed with
saturated ammonium chloride solution. The organic
layer was dried over magnesium sulfate and the solvent
was removed under reduced pressure. The crude product
was purified by column chromatography on silica gel
(EtOAc/Hex = 15:85) to give compound 4 (1.12 g,
80%) in the form of a colorless oil.
1H); 4.91 (s, 4H); 4.38 (dd, J = 5.0, 2.6, 2H); 2.41 (t,
J = 2.5 Hz, 1H); 1.08 (s, 18H); 0.29 (s, 12H).
13C(400 MHz, CDCl3) d = 166.4, 155.2, 149.4, 147.7,
145.5, 133.9, 132.2, 126.3, 125.3, 121.5, 79.2, 71.8,
60.3, 31.5, 25.8, 18.2, ꢀ5.5. HRMS (MALDI-TOF): cal-
culated for C31H44N2O8Si2 651.2528 [M+Na+], found
651.2562.
2.1.5. Compound 6. Compound 5 (1.5 g, 2.3 mmol) was
dissolved in DMF. N,N0-Dimethylethylenediamine-
mono-Boc10 (541 mg, 2.9 mmol) was added. The reac-
tion mixture was stirred at room temperature for 1 h
and monitored by TLC (EtOAc/He = 1:1). After com-
pletion, the solvent was removed under reduced pressure
and the crude product was purified by column chroma-
tography on silica gel (EtOAc/Hex = 2:8) to give com-
pound 6 (1.45 g, 90%) in the form of a colorless oil.
1H NMR (400 MHz, CDCl3)d = 7.57 (s, 2 H); 4.87 (s,
4H); 4.23 (dd, J = 5.0, 2.6 Hz, 2H); 2.17 (t, J = 2.5 Hz,
1H); 0.95 (s, 18H); 0.13 (s, 12H). 13C(400 MHz,
CDCl3)d = 166.7, 156.4, 126.1, 124.5, 79.6, 71.7, 62.7,
29.6, 25.8, 25.6, 18.2, ꢀ5.5. MS (FAB): calculated for
C24H41NO4Si2 464.2 [M+H+], found 464.2.
2.1.4. 2,6-Bis((tert-butyldimethylsilyloxy)methyl)-4-(prop-
2-ynylcarbamoyl)phenyl 4-nitrophenyl carbonate (com-
pound 5). Compound 4 (1.12 g, 2.4 mmol) was dissolved
in dry THF. Et3N (1.0 ml, 7.2 mmol) was added and the
mixture was cooled to 0 °C. Then, p-nitrophenyl chloro-
formate (581 mg, 2.9 mmol) dissolved in dry THF
(10 ml) was added dropwise and the reaction mixture
was stirred for 1 h at room temperature and monitored
by TLC (EtOAc/He = 2:8). After completion the reac-
tion mixture was filtered, the solvent was evaporated,
and the crude product was purified by column chroma-
tography on silica gel (EtOAc/Hex = 15:85) to give com-
pound 5(1.35 g, 90%) in the form of a colorless oil.
1H NMR (400 MHz, CDCl3)d = 7.79 (s, 2H); 6.32 (m,
1H); 4.68–4.67 (m, 4H); 4.27–4.25 (m, 2H); 3.61–3.43
(m, 4H); 3.24 (s, 2H); 3.12 (s, 1H); 2.96 (s, 3H); 2.32
(bs, 1H); 1.51–1.46 (m, 9H); 0.92 (s, 18H); 0.08 (s,
12H). 13C(400 MHz, CDCl3) d = 167.2, 153.1, 153.0,
134.6, 130.8, 125.1, 80.2, 78.8, 72.0, 59.9, 46.4, 46.1,
36.4, 35.9, 35.1, 29.8, 28.3, 25.7, 18.2, ꢀ5.5. MS
(FAB): calculated for C34H59N3O7Si2 700.4 [M+Na+],
found 700.3.
2.1.6. Compound 8. Compound 6 (1.4 g, 2.1 mmol) was
dissolved in TFA and the reaction mixture was stirred
for a few minutes. The excess of acid was removed under
reduced pressure and the crude amine salt was dissolved
in DMF (1.0 ml). Then, previously reported compound
1H NMR (200 MHz, CDCl3)d = 8.43 (d, J = 8.1 Hz,
2H); 8.02 (s, 2H); 7.63 (d, J = 8.1 Hz, 2H); 7.01 (m,

E. Danieli, D. Shabat / Bioorg. Med. Chem. 15 (2007) 7318–7324
7323
721 (4-nitrophenyl 4-(2-phenylacetamido)benzyl carbon-
ate) (1.1 g, 2.7 mmol) and Et3N (1.0 ml) were added and
the reaction mixture was monitored by TLC (EtOAc).
After completion the solvent was removed under re-
duced pressure. The crude product was purified by col-
umn chromatography on silica gel (MeOH/
EtOAc = 5:95) to give compound 8 (870 mg, 67%) as a
white solid.
(MeOH/EtOAc = 1:9). After completion, the solvent
was removed under reduced pressure. The crude product
was purified by column chromatography on silica gel
(MeOH/EtOAc = 1:9) to give compound 11 (50 mg,
62%) in the form of a white solid.
1H NMR (200 MHz, CDCl3)d = 8.75 (s, 1H), 8.05–7.55
(m, 7H), 7.39–6.97 (m, 9H); 5.04 (bs, 6H), 4.20–4.19 (m,
2H); 3.79–3.29 (m, 10H); 3.10–2.85 (m, 12H); 2.30–2.22
(m, 1H); 1.43–1.36 (m, 9H). HRMS (MALDI-TOF):
calculated for C53H60N8O12 1023.4223 [M+Na+], found
1023.4139.
1H NMR (400 MHz, CD3OD) d = 7.88 (s, 2 H); 7.52–
7.19 (m, 9H); 5.09–5.02 (m, 2H); 4.54–4.43 (m, 4H);
4.12 (s, 2H); 3.64 (s, 2H); 3.60–3.48 (s, 4H); 3.00–2.83
(s, 6H); 2.57 (s, 1H). HRMS (MALDI-TOF): calculated
for C33H36N4O8 639.2425 [M+Na+], found 639.2483.
2.1.10. Compound 12. Compound 11 (50 mg, 0.05 mmol)
was dissolved in TFA and stirred for few minutes, the
excess of acid was removed under reduced pressure
and the crude amine salt was dissolved in DMF
(0.5 ml). Then, bis-(4-nitrophenyl)carbonate (50 mg,
0.16 mmol) and Et3N (0.1 ml) were added and the reac-
tion was monitored by TLC (MeOH/EtOAc = 1:9).
After completion, the solvent was removed under re-
duced pressure. The crude product was purified by col-
umn chromatography on silica gel (MeOH/
EtOAc = 1:9) to give compound 12 (45 mg, 90%) as a
white solid.
2.1.7. Compound 9. Compound 8 (400 mg, 0.64 mmol)
was dissolved in dry THF and the reaction mixture
was cooled to 0 °C. Then, DIPEA (950 ll, 5.12 mmol)
was added followed by PNP-chloroformate (780 mg,
3.90 mmol) and pyridine (25 ll, 0.32 mmol). The reac-
tion mixture was allowed to warm up to room tempera-
ture and monitored by TLC (EtOAc/Hex = 3:1). After
completion the reaction mixture was diluted with EtOAc
and washed with saturated NH4Cl and with saturated
NaHCO3 solutions. The organic layer was dried over
magnesium sulfate. The solvent was removed under re-
duced pressure. The crude product was purified by col-
umn chromatography on silica gel (EtOAc/Hex = 7:3)
to give compound 9 (300 mg, 50%) in the form of a
white solid.
1H NMR (200 MHz, CDCl3)d = 8.60 (s, 1 H); 8.03–7.67
(m, 9H); 7.38–7.00(m, 11H); 5.13–4.94 (m, 6H); 4.00 (bs,
2H); 3.56–3.46 (m, 10H); 3.00–2.79 (m, 12H); 2.54 (s,
1H).
HRMS
(MALDI-TOF):
calculated
for
C55H55N9O14 1088.3761 [M+Na+], found 1088.3818.
1H NMR (200 MHz, CDCl3)d = 8.18 (d, J = 8 Hz, 4H);
7.85 (m, 2H); 7.30–6.98 (m, 13H); 5.15–4.93 (m, 6H);
4.19 (dd, J = 5.0, 2.6 Hz, 2H); 3.72 (s, 2H); 3.51–3.46
(m, 4H); 3.14–2.87 (m, 6H); 2.24 (t, J = 2.5 Hz, 1H).
HRMS (MALDI-TOF): calculated for C47H42N6O16
969.2545 [M+Na+], found 969.2594.
2.1.11. Compound 1. Compound 11 (25 mg, 0.025 lmol)
was dissolved in DMF. Then, previously reported com-
pound 1318) (150 mg, 0.027 lmol) was added followed
by addition of copper sulfate (3.0 mg, 19.0 lmol), TBTA
(10.0 mg, 19 lmol) and a few copper turnings. The reac-
tion mixture was stirred for overnight at room tempera-
ture. It was monitored by RP-HPLC and after
completion, the mixture was filtered and the solvent
was removed under reduced pressure. The crude product
was purified by column chromatography on silica gel
(MeOH/DCM = 2:8) to give compound 1 (130 mg,
85%) in the form of a white solid.
2.1.8. Compound 10. Compound 9 (200 mg, 0.21 mmol)
was dissolved in dry DMF and the reaction mixture
was cooled to 0 °C. N,N0-Dimethylethylenediamine-
mono-Boc (541 mg, 2.9 mmol) dissolved in 2 ml of
DMF was added dropwise. The reaction mixture was al-
lowed to warm up to room temperature and monitored
by TLC (EtOAc). After completion the solvent was re-
moved under reduced pressure. The crude product was
purified by column chromatography on silica gel
(EtOAc) to give compound 10 (90 mg, 45%) in the form
of a white solid.
2.2. Reporters’ release analysis—general protocol
PGA (56 mg/ml) was purchased from sigma and was
diluted with PBS 7.4 to give a 1.0 mg/ml solution.
Stock solution (10 mM) of compound 17 was pre-
pared in DMSO. The stock solution (10 lL) was di-
luted either with 190 lL PBS 7.4 (control) or with
190 lL of the 1.0 mg/ml PGA solution to give final
concentration of 500 lM for compound 17. All solu-
tions were kept at 37 °C and their fluorescence and
absorbance spectra were measured by SpectraMax
M2 spectrophotometer (molecular devices). Standard
coaster 96-well plates were used with sample volumes
of 200 lL. The fluorescence spectra were performed
by excitation at 250 nm. The emission fluorescence
was recorded between 330 nm and 600 nm. The
absorbance spectra were recorded between 330 nm
and 530 nm.
1H NMR (200 MHz, CDCl3)d = 8.18 (d, J = 8 Hz, 2H);
7.95 (m, 2H); 7.30–7.00 (m, 11H); 5.23–4.92 (m, 6H);
4.18 (bs, 2H); 3.71 (s, 2H); 3.45–3.32 (m, 8H); 2.95–
2.80 (m, 12H); 2.15 (s, 1H); 1.40–1.34 (m, 9H). HRMS
(MALDI-TOF): calculated for C50H57N7O15 1018.3805
[M+Na+], found 1018.3770.
2.1.9. Compound 11. Compound 10 (85 mg, 0.09 mmol)
was dissolved in DMF. Then, 6-aminoquinoline
(50 mg, 0.32 mmol) and a catalytic amount of HOBT
were added, followed by the addition of DIPEA
(40 ll, 0.12 mmol). The reaction mixture was heated to
50 °C, stirred overnight, and monitored by TLC

7324
E. Danieli, D. Shabat / Bioorg. Med. Chem. 15 (2007) 7318–7324
10. Amir, R. J.; Pessah, N.; Shamis, M.; Shabat, D. Angew.
Acknowledgments
Chem. Int. Ed. Engl. 2003, 42, 4494–4499.
11. Amir, R. J.; Popkov, M.; Lerner, R. A.; Barbas, C. F.,
3rd; Shabat, D. Angew. Chem. Int. Ed. Engl. 2005, 44,
4378–4381.
12. Amir, R. J.; Shabat, D. Chem. Commun. 2004, 1614–1615.
13. de Groot, F. M.; Albrecht, C.; Koekkoek, R.; Beusker, P.
H.; Scheeren, H. W. Angew. Chem. Int. Ed. Engl. 2003, 42,
4490–4494.
D.S. thanks the Israel Science Foundation and the Israel
Ministry of Science ‘Tashtiot’ program for financial
support.
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