General strategy for the preparation of membrane permeable fluorogenic peptide ester conjugates for in vivo studies of ester prodrug stability

Article abstract of DOI:10.1016/j.bmc.2003.11.016

To study ester prodrug stability properties in living cells we have conjugated fluorogenic esters to the cell membrane permeable peptide Arg ₉. The desired conjugates are prepared by coupling N-maleoyl amino acid esters of monoalkylated fluoresceins or fluorescein to TyrArg ₉Cys. The photophysical properties of the monoalkylated fluorescein derivatives are described.

Full text of DOI:10.1016/j.bmc.2003.11.016

Bioorganic & Medicinal Chemistry 12 (2004) 545–552
General strategy for the preparation of membrane permeable
fluorogenic peptide ester conjugates for in vivo studies of ester
prodrug stability
Xiaoxu Li and John Stephen Taylor*
Department of Chemistry, Washington University in St Louis, St. Louis, MO 63130, USA
Received 30 August 2003; accepted 18November 2003
Abstract—To study ester prodrug stability properties in living cells we have conjugated fluorogenic esters to the cell membrane
permeable peptide Arg9. The desired conjugates are prepared by coupling N-maleoyl amino acid esters of monoalkylated
fluoresceins or fluorescein to TyrArg9Cys. The photophysical properties of the monoalkylated fluorescein derivatives are described.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
stability of these phenyl esters in living cells. Rather
Prodrugs are a practical way to increase drug bioavail-
ability and selectivity. In a recent series of papers we
have described a new approach for the design of patient
specific chemotherapeutic drugs and diagnostic agents
based on nucleic acid triggered drug or probe release.1ꢀ3
The basic idea is to use a unique or overexpressed
mRNA molecule specific to the disease state to template
the association of two complementary nucleic acid
analogues, one bearing a prodrug and the other a catalytic
group that can trigger the release of the drug or probe.
Currently we have been using imidazole as the catalytic
group to release phenol-based probes and drugs by
hydrolysis of the corresponding phenyl ester. A key
requirement of this approach is that the prodrug is stable
under physiological conditions, and that it is readily
hydrolyzed by the catalytic group when held in proximity
by the disease specific nucleic acid sequence.
than use cell lysates, we have decided to develop a
general method for conjugating fluorogenic ester
prodrugs to cell membrane permeable peptides so that
their hydrolysis inside living cells can be monitored by
fluorescence microscopy and flow cytometry.
2. Results and discussion
2.1. Design of the fluorogenic ester peptide conjugates
We designed the fluorogenic ester peptide conjugates so
that they could be prepared by a versatile synthetic
strategy that would allow for variation of the membrane
permeability of the fluorophore to be released, the labi-
lity of the ester linkage, and the membrane permeability
of the peptide. These criteria could be met as described
below through the use of the N-maleimido amino acid
esters of mono-O-alkylated fluoresceins. The alkyl
group on the fluorescein could be used to modulate its
membrane permeability properties, natural and unna-
tural amino acids side chains could be used to modulate
the rate of enzymatic hydrolysis of the ester, and the
maleimido group could be used to couple different
permeation peptides through an appended cysteine
under neutral conditions.
Usually the stability of a candidate prodrug is evaluated
by monitoring the prodrug and its degradation products
in physiological buffer solutions and with purified
enzymes, or with intestinal juices, serum, and cell or
tissue homogenates or lysates.4ꢀ6 We have found that
we can dramatically enhance the stability of phenyl ester
prodrugs in human serum by substituting the a-position
with bulky groups2 but know nothing about the
We chose to make fluorogenic esters based on mono-O-
alkylated fluoresceins, because their long wavelength
absorption and fluorescence properties are similar to
Keywords: Prodrug; Fluorogenic ester; Arg9 peptide; Fluorescein.
* Corresponding author. Tel.: +1-314-935-6721; fax: +1-314-935-
4481; e-mail: taylor@wustl.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.11.016

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X. Li, J. S. Taylor / Bioorg. Med. Chem. 12 (2004) 545–552
those of fluorescein itself, which has found wide
application as a fluorescent probe in biological systems.
While mono-O-substituted and unsubstituted fluorescein
is in equilibrium with the highly fluorescent carboxylate
form under neutral and basic conditions, di-O-sub-
stituted fluorescein compounds exist in a non-fluor-
escent lactone form. As a result, di-O-substituted
fluoresceins have been widely used as fluorogenic
substrates for enzyme activity studies. For example,
mono-O-alkylated fluorescein phosphate has been used
as a fluorogenic substrate for the continuous assay of
phosphoinositide-specific phospholipase C.7,8 In
another study, N-acetyl-b-d-glucosaminides of 60-O-
alkylated fluorescein derivatives were used as substrates
for a kinetic assay of N-acetyl-b-d-glucosaminidase.9 In
these studies, enzyme activities were quantified by
analysis of the fluorescence resulting from enzymatic
formation of the fluorescent mono-O-alkylated fluor-
escein. The additional advantage of using mono-O-sub-
stituted fluoresceins as fluorescent probes is that their
solubility and membrane permeability following release
can be tailored by the nature of the appended side
chain. In this study, we have sought to replace the
previously used hydrophobic butyl substituent with
more water soluble methoxyethoxymethyl (MEM) and
ethylmethylether (EME) groups, but in principle, any
suitable side chain could be added.
of fluorescein with NaOH. The disodium salt is then
alkylated with the appropriate alkyl bromide to give the
intermediate ester 2, which can then be hydrolyzed with
base and acidified to provide the desired alkylated
fluoresceins 3. Whereas alkylation to give the butyl and
EME derivatives 3a and 3b required higher tempera-
tures and longer reaction times, alkylation to give the
MEM23 derivative 3c only required room temperature
and a 30 min reaction time. MEM fluorescein can also
be obtained directly from fluorescein upon treatment
with MEMCl.
2.3. UV–Visible absorption properties of the mono-O-al-
kylated fluoresceins
30-O-Butyl,-EME and-MEM fluoresceins have the same
absorption maxima of 450 and 475 nm in the visible
spectrum (Fig. 1), which are blue shifted relative to that
of 488 nm for fluorescein. The molar extinction coeffi-
cients, e, of the mono-alkylated fluoresceins at 475 nm
(Table 1) were 22,300, 18,200 and 16,200 cmꢀ1 Mꢀ1 for
the 30-O-butyl,-MEM and-EME derivatives of fluor-
escein, respectively, at pH 7 compared to 68,000 cmꢀ1
Mꢀ1 for fluorescein at 488 nm. The lower absorptivity
of the mono-O-alkylated fluoresceins than fluorescein
is likely due to lower electron delocalization that
results from replacement of a phenolate anion with a
phenylether.
Although some fluorescein esters have shown the ability
to disperse into cells passively10ꢀ14 a common carrier
would be necessary to transport different fluorogenic
esters into cells efficiently and quantitatively so that the
hydrolysis rates can be directly compared. We have
initially chosen Arg9 to transport the esters into cells
because polyarginine peptides show high membrane
permeability in many cell lines,15ꢀ19 and are able to
transport a variety of molecular cargoes such as drugs,
peptides, and enzymes into cells.20ꢀ22 Among oligoargi-
nines in the range 4 to 16 arginines, Arg9, which has
similar charge and amino acid composition as HIV Tat
peptide 46–57, shows both efficient permeability and
synthetic economy.15,16,19,22
2.4. Fluorescence excitation and emission spectra
The fluorescence excitation spectra of 30-O-butyl,-EME
and-MEM fluoresceins under neutral conditions have
similar excitation spectra at pH 7.0 (Fig. 2), with a
excitation maximum wavelength of 467 nm, which is
blue-shifted compared to that of 492 nm for fluorescein.
The emission spectra (Fig. 2B) show a similar emission
maximum of 512 nm, though 30-O-butyl fluorescein
shows greater fluorescence intensity. The acetyl deriv-
ative of the 30-O-butyl,-EME and-MEM fluoresceins
4a–c were not fluorescent in the 400–500 nm range
(shown for 60-O-acetyl-30-O-EME-fluorescein, 4b, Fig.
Because the fluorogenic esters are readily hydrolyzed
under basic conditions, we required a method for
coupling them to the membrane permeabilizing peptides
that takes place efficiently under neutral or acidic con-
ditions. We therefore decided to make use of the same
Michael addition of thiol to maleimide that we have
previously used to conjugate the base labile p-nitrophe-
nyl, hydroxycoumarin, and fluorescein esters to oligo-
deoxynucleotides at neutral pH1,2 and PNA,.3 The
required N-maleimido amino acid esters are
conveniently made from maleic anhydride and amino
acids in two steps, and thiolated Arg9 or other peptides can
be readily made by appending cysteine to the sequence.
2.2. Synthesis of the mono-O-alkylated fluoresceins
Figure 1. The molar extinction curves of the mono-O-alkylated fluor-
escein derivatives in comparison to fluorescein. Absorption spectra for
30-O-butyl fluorescein 3a, 30-O-EME fluorescein 3b, 30-O-MEM
fluorescein 3c and fluorescein were obtained at pH 7.0 (30 mM in 10
mM PBS). The scale on the left is for 3a–c and on the right for
fluorescein (3d).
The mono-O-alkylated fluoresceins were synthesized as
illustrated in Scheme 1 according to methods previously
reported for the butyl derivative.7,8 In the first step, the
disodium salt of fluorescein 1 is prepared by treatment

X. Li, J. S. Taylor / Bioorg. Med. Chem. 12 (2004) 545–552
547
Scheme 1.
Table 1. Photophysical properties of mono-O-alkylated fluoresceins
pH 7.0
pH 9.0
l max (nm) excitation
l max (nm) emission
e
475 (cmꢀ1Mꢀ1
)
l max (nm) excitation
l max (nm) emission
e )
475 (cmꢀ1 Mꢀ1
3a
3b
3c
467
467
467
512
512
512
2.2ꢁ104
1.6ꢁ104
1.8ꢁ104
467
467
467
512
512
512
2.6ꢁ104
1.8ꢁ104
1.9ꢁ104
1A). Emission from the mono-alkylated fluoresceins
upon excitation at 467 nm is only about 15% that of
fluorescein excited at 492 nm, which can be explained in
part by the lower absorptivity of the mono-alkylated
fluoresceins compared to fluorescein (Fig. 1).
process. In the first step, the amino acids 5a–d were
treated with maleic anhydride and the resulting N-maleoyl
amino acids were treated with thionyl chloride to effect
formation of the maleimide and simultaneously convert
the carboxylic acid to the acid chloride 6a–d (Scheme 2).24
The crude acid chlorides were then treated with the
appropriate fluorescein derivative to yield the esters 7–10
(Scheme 3). In addition to coupling the N-maleimido
amino acids to the mono-O-alkylated fluoresceins 3a–c, we
also coupled some of them to 30-O-acetyl-fluorescein 4d
and fluorescein 3d. We expect that the acetyl group of the
Arg9 conjugates of the N-maleimido amino acid esters of
30-O-acetyl-fluorescein will be rapidly hydrolyzed in vivo
to yield the mono amino acid ester of fluorescein. Though
the mono ester will be fluorescent, the fluorescence is
expected to increase almost 10-fold upon hydrolysis of the
ester linkage, the rate of which could be monitored.
2.5. pH Dependence of the fluorescence
The fluorescence emission intensity of the alkylated
fluoresceins was obtained as a function of pH (Fig. 3) so
that their pKa’s could be estimated as well as their
fluorescence efficiency under biologically relevant pHs.
The mono-O-alkylated fluoresceins all have similar pKa
values in the range of 6.0 to 6.3 (Fig. 3), and at pH 7.0
fluoresce with greater than 90% of their maximum
value. The excitation and emission spectra acquired at
pH 9.0 retain the same features as those observed at pH
7.0 except for a slight increase in intensity (data not
shown), due to the exclusive presence of the fluorescent
carboxylate form (Table 1).
2.7. Conjugation of the fluorogenic esters to TyrArg9Cys
The TyrArg9Cys peptide was synthesized by standard
Fmoc solid phase methodology and then coupled to a
four-fold excess of the maleimido esters 7–10 in metha-
nol for 2 h to give the desired conjugates 11–14 (Scheme
4). Methanol was used as a solvent instead of water
because of it was better able to solubilize the reactants,
and because the conjugated products could be readily
isolated by ether precipitation. All the conjugates were
purified by RP-HPLC and characterized by MALDI-TOF
MS (Table 2).
2.6. Synthesis of N-maleimido amino acid esters of the
monoalkylated fluoresceins
To test the effects of sterics on the stability of the esters
in vivo we prepared a series of substituted amino acid
esters in which the a-substituent was varied from
hydrogen to methyl to isopropyl to diphenylmethyl. The
N-maleimido amino acid esters of the mono-O-alky-
lated fluoresceins were synthesized by a two step

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X. Li, J. S. Taylor / Bioorg. Med. Chem. 12 (2004) 545–552
3. Conclusion
anhydride, thionyl chloride, d-valine, b-alanine, and
d,l-3-amino-2-methylpropionic acid were purchased
from Aldrich. d-3,3-diphenylalanine was from PepTech
Co. Fmoc protected amino acids, resin and other
reagents for solid phase peptide synthesis were
In summary, we have developed a general method for
synthesizing fluorogenic esters and attaching them to a
permeation peptide. These fluorogenic ester conjugates
should enable the stability of the ester linkage of
prodrugs to be evaluated in different cell lines by
fluorescence microscopy and flow cytometry, and are
currently under study. Fluorogenic ester conjugates
with labile ester linkages might also be useful for vali-
dating entry of membrane permeabilizing peptides into
cells. Other fluorogenic ester conjugates might find use
as reporter molecules for disease specific enzymes with
esterase activity.
1
purchased from NovaBiochem. H NMR spectra were
obtained on a 300 MHz Varian UnityPlus-300 or Varian
Mercury-300 spectrometer. Proton chemical shifts are
expressed in ppm from tetramethylsilane using residual
chloroform (d=7.27 ppm), methanol (d=4.87 ppm),
acetone (d=2.05) as an internal standard. Flash chro-
matography was performed on Selecto Scientific silica
gel. TLC and preparative TLC were run on precoated
254-nm fluorescent silica gel sheets manufactured by
Alltech Associates. Fluorescence spectra were recorded
on a SPEX Fluoromax instrument. UV spectral data
were acquired on a Bausch and Lomb Spectronic
1001 spectrophotometer or Varian Cary 100 Bio UV-
Visible Spectrophotometer. Matrix-assisted laser
desorption ionization (MALDI) mass spectra of peptide
conjugates were measured on PerSeptive Voyager RP
4. Experimental
4.1. General
Dichloromethane (DCM) and triethylamine (TEA) were
dried by refluxing with CaH2 overnight followed by
distillation. Fluorescein, 1-bromobutane, bromoethyl
methyl ether, 2-methoxyethoxymethyl chloride, maleic
Table 2. MALDI-TOF MS of conjugates of fluorogenic esters and
TyrArg9Cys peptide shown in Scheme 4
R1
R
Calcd. MW Observed (M+1)
11a –CH(CH(Ph)2)-CO—
12a –CH(CH(CH3)2)-CO— Butyl
12b –CH(CH(CH3)2)-CO— EME
12d –CH(CH(CH3)2)-CO—
13a –CH2CH(CH3)-CO—
13b –CH2CH(CH3)-CO—
13d –CH2CH(CH3)-CO—
Butyl
2381
2257
22582259.14
2200
2243
2244
2186
2229
2173
2382.15
2256.69
H
Butyl
EME
H
Butyl
H
COCH3
2201.38
2244.26
2243.99
2186.97
2229.80
2174.14
2216.18
14a
14d
14e
–CH2CH2-CO—
–CH2CH2-CO—
–CH2CH2-CO—
2215
Scheme 2.
Scheme 3.

X. Li, J. S. Taylor / Bioorg. Med. Chem. 12 (2004) 545–552
549
Figure 3. Relative fluorescence as a function of pH for the fluorescein
derivatives. The derivatives were excited at 467 nm and detected at 518
nm in 10 mM PBS at the indicated pH and normalized to the max-
imum intensity for each derivative.
Figure 2. Fluorescence spectra of the fluorescein derivatives in
comparison to fluorescein. A. Excitation spectra of 30-O-butyl
fluorescein 3a, 30-O-EME fluorescein 3b, 30-O-MEM fluorescein 3c
and 60-O-acetyl-30-O-EME fluorescein 4b, (1 mM in 10 mM pH 7.0
PBS, detection at 518nm) B. Emission spectra of butyl fluorescein 3a,
EME fluorescein 3b, and MEM fluorescein 3c. (1 mM in 10 mM pH
7.0 PBS, excitation at 467 nm). The emission intensity was normalized
to 100 for the maximum emission intensity of fluorescein (excitation at
492). The scale is on the left for 3a–c, and 4b, and on the right for
fluorescein (3d).
MALDI-time of flight (TOF) mass spectrometer. HR-FAB
mass spectra were obtained on a MS-50TA (Ion Spec
B126). HPLC were carried out on Beckman Coulter
System Gold 126 with RP C18semi preparative
columns.
Scheme 4.
4.1.1. 30-O-Butyl-fluorescein (3a). 1-Bromobutane (2.8
mL, 26.5 mmol) was added dropwise with stirring to a
suspension of the disodium salt of fluorescein 18 (2.5 g,
6.6 mmol) in DMF (60 mL) at RT and then heated at
90–100 ꢂC with stirring for 24 h. The DMF was
removed from the mixture by evaporation under
reduced pressure and the residue treated with 60 mL of
4% NaOH in MeOH–water (3:1, v/v) and stirred for 2
h. The solution was then acidified by dropwise addition
of 3 N aqueous HCl until a pH of about 2. The aqueous
solution was extracted with ethyl acetate (3ꢁ50 mL)
and the combined ethyl acetate fractions were dried
over Na2SO4, and evaporated to give the crude butyl
substituted fluorescein. Further purification by flash
chromatography on silica gel using hexane/ethyl acetate
(2:1) gave 1.23 g (48% yield) of 3a as a yellow powder.
1H NMR (CD3COCD3) d 0.96 (t, J=7.5 Hz, 3H), 1.43–
1.56 (m, 2H), 1.72–1.81 (m, 2H), 4.08 (t, J=6.5 Hz, 2H),
6.64–6.66 (m, 2H), 6.68–6.71 (m, 2H), 6.84–6.85 (m,
1H), 6.75 (d, J=2 Hz, 1H), 7.27(d, J=8Hz, 1H), 7.73
(td, J=7.5, 1 Hz, 1H), 7.81 (td, J=7.5, 1 Hz, 1H), 7.98
(d, J=7.5 Hz, 1H), 9.05 (s, 1H); HR FAB MS (m/z),
calculated for C24H20O5Li (M+Li+) 395.1471, found
395.1472.
4.1.2. 30-O-EME-fluorescein (3b). Bromoethyl methyl
ether (1 mL, 1.48g, 10.6 mmol) was added dropwise to
a suspension of the disodium salt of fluorescein 18 (1.0
g, 2.7 mmol) in DMF (25 mL) at RT and stirred for 8h
at 90 ꢂC. The DMF was removed under reduced pres-
sure and 20 mL of 4% NaOH in MeOH and water (3:1,
v/v) was added to the residue and stirred for 2 h. The
reaction was worked up as described for 3a to give 0.50
g (48% yield) of 3b as a yellow powder following flash

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X. Li, J. S. Taylor / Bioorg. Med. Chem. 12 (2004) 545–552
chromatography with hexane–ethyl acetate (1:1). 1H
NMR (CD3COCD3) d 3.36 (s, 3H), 3.73 (t, J=4.5 Hz,
2H), 4.21 (t, J=4.5 Hz, 2H), 6.62–6.79 (m, 5H), 6.85–
6.89 (m, 1H), 7.28 (d, J=7.5 Hz, 1H), 7.81 (t, J=7.5
Hz, 2H), 7.73 (t, J=7.5 Hz, 1H), 8.00 (d, J=7.5 Hz,
1H), 9.05 (s, 1H); HR FAB MS (m/z) calculated for
C23H18O6Li (M+Li+) 397.1263, found 397.1273.
6.77–6.95 (m, 4H), 7.02–7.08(m, 1H), 7.17–7.25 (m,
1H), 7.32–7.41 (m, 1H), 7.72–7.88 (m, 2H), 7.98–8.08
(m, 1H); HR FAB MS (m/z), calculated for C26H22O8Li
(M+Li+) 469.1475, found 469.1480.
4.1.7. 30-O-acetyl-fluorescein (4d). Acetic anhydride
(0.28mL, 0.31 g, 3 mmol) and TEA (0.30 g, 0.42 mL, 3
mmol) in 10 mL DCM were added dropwise to fluor-
escein 3d (1 g, 3 mmol) suspended in 10 mL of DCM
and stirred for 4 h at rt. The solvent was removed under
reduced pressure and the residue purified by silica gel
flash chromatography (hexane-ethyl acetate, 1:1.5) to
afford 0.45 g (40% yield) of 4d as an off yellow solid. 1H
NMR (CD3COCD3) d 2.28(s, 3H), 6.64–6.73 (m, 2H),
6.79 (d, J=2.5 Hz, 1H), 6.86–6.96 (m, 2H), 7.15–7.22
(m, 1H), 7.34 (d, J=7.5 Hz, 1H), 7.76 (td, J=7.5, 1 Hz,
1H), 7.83 (td, J=7.5, 1 Hz, 1H), 8.01 (d, J=7.5 Hz,
1H), 9.05 (s, 1H); HR FAB MS (m/z), calculated for
C22H14O6Li (M+Li+) 381.0950, found 381.0955.
4.1.3. 30-O-MEM-fluorescein (3c). MEMCl (121 mL, 1.1
mmol) was added dropwise to a suspension of the di-
sodium salt of fluorescein 18 (200 mg, 0.53 mmol) in
DMF (25 mL) and stirred for 30 min at rt. The DMF
was removed under reduced pressure and 20 mL of 4%
NaOH in MeOH and water (3:1, v/v) was added and
stirred for 2 h. The reaction was worked up as described
for 3a to give 159 mg (71% yield) of 3c as a yellow
powder following purification by flash chromatography
1
on silica gel using hexane–ethyl acetate (1.5:1–1:1). H
NMR (CD3COCD3) d 3.27 (s, 3H), 3.52 (t, J=4.5 Hz,
2H), 3.80 (t, J=4.5 Hz, 2H), 5.34 (s, 2H), 6.60–6.69 (m,
3H), 6.73–6.81 (m, 2H), 7.00 (d, J=2.5 Hz, 1H), 7.26–
7.31 (m, 1H), 7.70–7.84 (m, 2H), 7.96–8.02 (m, 1H), 9.08
(s, 1H); HR FAB MS (m/z) calculated for C24H20O7Li
(M+Li+) 427.1369, found 427.1373.
4.1.8. N-Maleoyl-D-diphenylalanine (5a). Maleic anhy-
dride (203 mg, 2.1 mmol) was added all at once to
d-diphenylalanine (500 mg, 2.1 mmol) in 4 mL of a 3:1
mixture of water and DMF, and stirred overnight at rt.
The resulting white solid was filtered, washed with water
(3ꢁ10 mL), followed by anhydrous ethanol (3ꢁ10 mL)
and then anhydrous ether (3ꢁ10 mL) to give 0.524 g
(75% yield) of 5a as a white powder. 1H NMR
(300 MHz, CD3OD) d 4.38(d, J=11 Hz, 1H), 5.29 (d,
J=11 Hz, 1H), 6.11 (d, J=13 Hz, 1H), 6.18(d, J=13
Hz, 1H), 7.11 (t, J=7.5 Hz, 2H), 7.17–7.23 (m, 4H),
7.28–7.31 (m, 4H); HR FAB MS (m/z), calculated for
C19H18O5N (M+H+) 340.1185, found 340.1186.
4.1.4. 60-Acetyl-30-O-butyl-fluorescein (4a). A mixture of
acetic anhydride–pyridine–DCM (1.5 mL, 1:1:2, v/v)
was added to 30-O-butyl-fluorescein 3a (26 mg, 0.067
mmol) in 4 mL of DCM and stirred for 4 h at rt. The
DCM was removed under reduced pressure and the
residue was purified by preparative TLC with hexane/
ethyl acetate (1:1) to afford 22 mg (76% yield) of 4a as a
1
white solid. H NMR (CD3COCD3) d 0.97 (t, J=7.5
Hz, 3H), 1.44–1.57 (m, 2H), 1.72–1.83 (m, 2H), 2.28 (s,
3H), 4.09 (t, J=6.5 Hz, 2H), 6.71–6.79 (m, 2H), 6.87–
6.93 (m, 3H), 7.16–7.19 (m, 1H), 7.34 (d, J=7 Hz, 1H),
7.76 (t, J=7 Hz, 1H), 7.83 (t, J=7 Hz, 1H), 8.02 (d,
J=7 Hz, 1H); HR FAB MS (m/z), calculated for
C26H22O6Li (M+Li+) 437.1576, found 437.1577.
4.1.9. N-Maleoyl-D,L-3-amino-2-methylpropionic acid
(5c). Prepared by the procedure previously described
1
for 5a1 giving 5c as a white solid. H NMR (300 MHz,
DMSO-d6) d 1.08(d, J=7 Hz, 3H), 2.55–2.64 (m, 1H),
3.16–3.25 (m, 1H), 3.35–3.43 (m, 1H), 6.25 (d, J=12.5
Hz, 1H), 6.42 (d, J=12.5 Hz, 1H), 9.08(t, J=6.5 Hz,
1H); HR FAB MS (m/z): calculated for C8H11O5N
(M+H+) 202.0637, found 202.0716.
4.1.5. 60-acetyl-30-O-EME-fluorescein (4b). Acetic anhy-
dride (65 mg, 0.64 mmol, 50 mL) and pyridine (51 mg,
0.64 mmol, 34 mL) were added to 30-O-EME-fluorescein
3b (25 mg, 0.064 mmol) in 3 mL DCM and stirred for 4
h at rt. The product was isolated and purified as descri-
bed for 4a to give 11 mg (39% yield) of 4b as a white
solid. 1H NMR (CD3COCD3) d 2.40 (s,3H), 3.36 (s,
3H), 3.72–3.76 (m, 2H), 4.20–4.24 (m, 2H), 6.73–6.78
(m,3H), 6.84–6.95 (m, 3H), 7.08 (s, 2H), 7.34 (d, J=7.5
Hz, 1H), 7.76 (td, J=7.5, 1 Hz, 1H), 7.83 (td, J=7.5, 1
Hz, 1H), 8.02 (d, J=7.5 Hz, 1H); HR FAB MS (m/z),
calculated for C25H20O7Li (M+Li+) 439.1369, found
439.1369.
4.1.10. 60-O-(N-Maleimido-D-diphenylalanyl)-30-O-butyl-
fluorescein (7a). N-Maleoyl-d-diphenylalanine 5a (100
mg, 0.29 mmol) was dissolved in 5 mL of thionyl chlo-
ride and heated at reflux until gas evolution had ceased
at which point the excess thionyl chloride was removed
under reduced pressure. The residual thionyl chloride
was removed by dissolving the residue in carbon tetra-
chloride and then removing the solvent under reduced
pressure. The crude acid chloride was dissolved in 5 mL
of CH2Cl2 and was slowly added to a stirred mixture of
30-O-butyl-fluorescein 3a (50 mg, 0.13 mmol) and
triethylamine (22 mL, 16 mg, 0.16 mmol) in 5 mL of
CH2Cl2 at 0 ꢂC. The reaction mixture was allowed to
warm to rt. After stirring overnight, the reaction mix-
ture was diluted with 20 mL of CH2Cl2, washed with
brine and water, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel TLC (1:2
ethyl acetate–hexane) to afford 26 mg (29% yield) of 7a
4.1.6. 60-acetyl-30-O-MEM-fluorescein (4c). 30-O-MEM-
fluorescein 3c (25 mg, 0.058mmol) was dissolved in 3
mL of DCM and Ac2O (59 mg, 0.58mmol, 43 mL)
and pyridine (46 mg, 0.58mmol, 30 mL) was added
and stirred for 4 h at rt. The desired product was
isolated and purified by a similar procedure as described
for 4a to give 22 mg (80% yield) of 4c as a white solid.
1H NMR (CD3COCD3) d 2.29 (s, 3H), 3.27 (s, 3H), 3.53
(t, J=4.5 Hz, 2H), 3.80 (t, J=4.5 Hz, 2H), 5.35 (s, 2H),
1
as white powder. H NMR (300 MHz, CD3COCD3) d
0.97 (t, J=7.5 Hz, 3H), 1.44–1.56 (m, 2H), 1.72–1.82

X. Li, J. S. Taylor / Bioorg. Med. Chem. 12 (2004) 545–552
551
(m, 2H), 4.07 (t, J=6.5 Hz, 2H), 5.17 and 5.23 (d, J=12
Hz, 1H), 5.91 and 5.97(d, J=12 Hz, 1H), 6.62–6.97 (m,
5H), 7.15–7.40 (m, 12H), 7.60–7.66 (m, 2H), 7.74 (t,
J=7.5 Hz, 1H), 7.80 (t, J=7.5 Hz, 1H), 8.00 (d, J=7.5
Hz, 1H); HR FAB MS (m/z), calculated for C43H34O8N
(M+H+) 692.2284, found 692.2259.
gel TLC (1:2 ethyl acetate–hexane) gave 52.2 mg (61%
1
yield) of 9a as a white powder. H NMR (300 MHz,
CD3COCD3) d 0.97 (t, J=7 Hz, 3H), 1.26 (d, J=7 Hz,
3H), 1.44–1.58(m, 2H), 1.73–1.83 (m, 2H), 3.06–3.18
(m, 1H), 3.72–3.98(m, 2H), 4.09 (t, J=6.5 Hz, 2H),
6.70–6.79 (m, 2H), 6.88–6.98 (m, 5H), 7.21–7.24 (m,
1H), 7.35 (d, J=7.5 Hz, 1H), 7.76 (td, J=7.5, 1 Hz,
1H), 7.83 (td, J=7.5, 1 Hz, 1H), 8.02 (d, J=7.5 Hz,
1H); HR FAB MS (m/z), calculated for C32H27O8NLi
(M+Li+) 560.1897, found 560.1906.
4.1.11. 60-O-(N-Maleimido-D-valyl)-30-O-butyl-fluores-
cein (8a). N-Maleoyl-d-valine 5b1 (153 mg, 0.71 mmol)
was treated with thionyl chloride as described for the
preparation of 7a to give the crude acid chloride which
was dissolved in 3 mL of CH2Cl2 and was slowly added
to a stirred mixture of 30-O-butyl-fluorescein 3a (200
mg, 0.52 mmol) and triethylamine (0.19 mL, 1.4 mmol)
in 5 mL of CH2Cl2 at 0 ꢂC. The reaction was worked up
as described for 7a, and the residue was flash chroma-
tographed on silica gel (1:2 ethyl acetate–hexane) to
4.1.15. 60-O-(N-Maleimido-D,L-3-amino-2-methylpropio-
nyl)-30-O-EME-fluorescein (9b). The title compound
was made from N-maleoyl-d,l-3-amino-2-methylpro-
pionic acid 5c (52 mg, 0.26 mmol) and 30-O-EME-
fluorescein 7b (100 mg, 0.26 mmol) as described for 7a.
The crude product was purified by silica gel TCL (1:5,
ethyl acetate–DCM) to afford 41 mg (29% yield) of 9b
as a white powder. 1H NMR (300 MHz, CD3COCD3) d
1.26 (d, J=7 Hz, 3H), 3.08–3.18 (m, 1H), 3.37 (s, 3H),
3.72–3.81 (m, 3H), 3.88–3.98 (m, 1H), 4.22–4.27 (m,
2H), 6.73–6.78 (m, 2H), 6.88–6.98 (m, 5H), 7.23–7.29
(m, 1H), 7.35 (d, J=7 Hz, 1H), 7.77 (td, J=7.5, 1 Hz,
1H), 7.84(td, J=7.5, 1 Hz, 1H), 8.03 (d, J=7.5 Hz, 1H);
HR FAB MS (m/z), calculated for C31H26O9N
(M+H+) 556.1608, found 556.1627.
1
afford 108mg (37% yield) of 8a as a white powder. H
NMR (300 MHz, CD3COCD3) d 0.97 (t, J=7.5 Hz,
3H), 0.93 (d, J=6.5 Hz, 3H), 1.12 (d, J=6.5 Hz, 3H),
1.43–1.56 (m, 2H), 1.72–1.82 (m, 2H), 4.08 (t, J=6.5
Hz, 2H), 4.78(d, J=7 Hz, 1H), 6.71–6.78(m, 2H),
6.84–6.94 (m, 3H), 7.05 (s, 2H), 7.08–7.13 (m, 1H),
7.31–7.35 (m, 1H), 7.76 (td, J=7.5, 1 Hz, 1H), 7.82 (td,
J=7.5, 1 Hz, 1H), 8.01 (m, 1H); high-resolution FAB
mass spectrometry (m/z) calculated for C33H29O8NLi
[M+Li+] 574.2053, found 574.2053.
4.1.16. 30-O-(N-Maleimido-D,L-2-amino-1-methylpropio-
nyl)-fluorescein (9d). The title compound was made
from N-maleoyl-d,l-2-amino-1-methylpropionic acid 5c
(60 mg, 0.3 mmol) and fluorescein 3d (100 mg, 0.3
mmol) using the same methods described for 8e. The
crude product was purified by silica gel column
chromatography (1:1–1.5:1, ethyl acetate–hexane) to afford
4.1.12. 60-O-(N-Maleimido-D-valyl)-30-EME-fluorescein
(8b). The title compound was made from N-maleoyl-d-
valine 5b1 (70 mg, 0.33 mmol) and 30-O-EME-fluor-
escein 3b (90 mg, 0.23 mmol) as described above for 7a.
The crude product was purified by silica gel TCL (1:20,
ethyl acetate–DCM) to afford 35 mg (27%) of 8b as a
white powder. 1H NMR (300 MHz, CD3COCD3) d 0.93
(d, J=6.5 Hz, 3H), 1.12 (d, J=6.5 Hz, 3H), 2.59–2.69
(m, 1H), 3.36 (s, 3H), 3.72–3.75 (m, 2H), 4.21–4.24 (m,
2H), 4.78(d, J=7 Hz, 1H), 6.74–6.78(m, 2H), 6.85–
6.98(m, 3H), 7.03–7.16 (m, 3H), 7.34 (d, J=7 Hz, 1H),
7.76 (td, J=7.5, 1 Hz, 1H), 7.83 (td, J=7.5, 1 Hz, 1H),
8.02 (d, J=7.5 Hz, 1H); HR FAB MS (m/z), calculated
for C32H28O9N (M+H+) 570.1765, found 570.1757.
1
77 mg (52% yield) of 9d as a yellow powder. H NMR
(300 MHz, CD3COCD3) d 1.26 (d, J=7 Hz, 3H), 3.06–
3.18(m, 1H), 3.72–3.79 (m, 1H), 3.98–3.96 (m, 1H),
6.65–6.73 (m, 2H), 6.80 (d, J=2 Hz, 1H), 6.88–6.96 (m,
4H), 7.21–7.23 (m, 1H), 7.35(d, J=7.5 Hz, 1H), 7.76 (t,
J=7.5 Hz, 1H), 7.83 (t, J=7.5 Hz, 1H), 8.02 (d, J=7.5
Hz, 1H), 9.12 (s,1H); HR FAB MS (m/z), calculated for
C28H20O8N(M+H+) 498.1190, found 498.1191.
4.1.13. 30-O-(N-Maleimido-D-valyl)-fluorescein (8d). The
title compound was prepared from N-maleoyl-d-valine
5b (65 mg, 0.3 mmol) and fluorescein 3d (100 mg, 0.3
mmol) as described for 7a. Silica gel column chromato-
graphy (1:1–1.5:1, ethyl acetate–hexane) gave 54 mg
4.1.17. 60-O-(N-Maleimido-ꢀ-alanyl)-30-O-butyl-fluores-
cein (10a). The title compound was prepared from N-
maleoyl-b-alanine 5d2 (50 mg, 0.27 mmol) and 30-O-
butyl-fluorescein 3a (50 mg, 0.13 mmol) as described for
7a. Silica gel TLC (1:1 ethyl acetate–hexane) gave 48mg
(71% yield) of 10a as a white powder. 1H NMR
(300 MHz, CD3COCD3) d 0.97 (t, J=7.5 Hz, 3H), 1.44–
1.57 (m, 2H), 1.73–1.82 (m, 2H), 2.88–2.96 (m, 2H),
3.85–3.96 (m, 2H), 4.09 (t, J=6.5 Hz, 2H), 6.71–6.79
(m, 2H), 6.88–6.98 (m, 5H), 7.21–7.24 (m, 1H), 7.34 (d,
J=7.5 Hz, 1H), 7.76 (td, J=7.5, 1 Hz, 1H), 7.83 (td,
J=7.5, 1 Hz, 1H), 8.02 (d, J=7 Hz, 1H); HR FAB MS
(m/z), calculated for C31H25O8NLi (M+Li+) 546.1740,
found 546.1733.
1
(35% yield) of 8d as an off yellow powder. H NMR
(300 MHz, CD3COCD3) d 0.94 (d, J=7 Hz, 3H), 1.12
(d, J=7 Hz, 3H), 2.58–2.70 (m, 1H), 4.78 (d, J=7 Hz,
1H), 6.64–6.73 (m, 2H), 6.79 (d, J=2 Hz, 1H), 6.86–
6.93 (m, 2H), 7.05 (s, 2H), 7.07–7.13 (m, 1H), 7.34 (d,
J=7.5 Hz, 1H), 7.76 (td, J=7.5, 1 Hz, 1H), 7.83 (td,
J=7.5, 1 Hz, 1H), 8.01 (d, J=7.5 Hz, 1H), 9.10 (s,1H);
HR FAB MS(m/z), calculated for C29H21O8NLi
(M+Li+) 518.1427, found 518.1423.
4.1.14. 60-O-(N-Maleimido-D,L-3-amino-2-methylpropio-
nyl)-30-O-butyl-fluorescein (9a). The title compound was
prepared from N-maleoyl-d,l-3-amino-2-methylpro-
pionic acid 5c (60 mg, 0.30 mmol) and 30-O-butyl-fluor-
escein 3a (60 mg, 0.16 mmol) as described for 7a. Silica
4.1.18. 30-O-(N-Maleimido-ꢀ-alanyl)-fluorescein (10d).
The title compound was prepared from N-maleimido-
b-alanine 5d2 (28mg, 0.15 mmol) and fluorescein 3d (50
mg, 0.15 mmol) as described for 8e. The crude product
was purified by silica gel TLC (1:1 ethyl acetate–hexane)

552
X. Li, J. S. Taylor / Bioorg. Med. Chem. 12 (2004) 545–552
to afford 25 mg (36% yield) of 10d as a yellow powder.
1H NMR (300 MHz, CD3COCD3) d 2.92–2.96 (m, 2H),
3.88–3.97 (m, 2H), 6.62–6.72 (m, 2H), 6.78–6.81 (m,
1H), 6.88–6.94 (m, 3H), 6.95–6.98 (m, 1H), 7.19–7.23
(m, 1H), 7.34 (d, J=7.5 Hz, 1H), 7.76 (tt, J=7.5, 1.2
Hz, 1H), 7.83 (tt, J=7.5, 1 Hz, 1H), 8.01 (d, J=7.5
Hz, 1H), 9.08(s, 1H); HR FAB MS ( m/z), calculated
for C27H17O8NLi (M+Li+) 490.1114, found
490.1109.
Acknowledgements
This work was financially supported by an NIH grant
(RO1-CA92477). Mass spectrometry was provided by
the Washington University Mass Spectrometry
Resource (Grant No. P41RR0954).
References and notes
4.1.19. 60-O-(N-Maleoyl-ꢀ-alanyl)-30-O-acetyl fluores-
cein (10e). The title compound was prepared from N-
maleoyl-b-alanine 5d2 (37 mg, 0.2 mmol) and 30-O-ace-
tyl-fluorescein 3d (50 mg, 0.13 mmol) as described for
7a. The crude product was purified by silica gel TLC
(1:1 ethyl acetate–hexane) to give 50 mg (74% yield) of
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10e as
a
white powder. 1H NMR (300 MHz,
CD3COCD3) d 2.29 (s, 3H), 2.88–2.96 (m, 2H), 3.89–
3.97 (m, 2H), 6.91–6.99 (m, 6H), 7.21–7.28(m, 2H), 7.41
(d, J=7.5 Hz, 1H), 7.78(t, J=7.5, 1H), 7.85 (t, J=7.5,
1H), 8.04 (d, J=7.5 Hz, 1H); HR FAB MS (m/z),
calculated for C29H19O9 NLi (M+Li+) 532.1220, found
532.1233.
8. Zaikova, T. O.; Rukavishnikov, A. V.; Birrell, G. B.;
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4.1.20. Synthesis of TyrArg9Cys. The oligopeptide H2N-
Tyr-(Arg)9-Cys-CONH2 was synthesized by standard
solid phase Fmoc peptide synthesis methodology. Fmoc
protected Rink amide resin (100 mg, substitution=0.61
mmol/g) was used as the solid support. After deprotec-
tion of the resin with 20% piperidine in DMF (v/v) and
washing with MeOH and DMF alternately, FmocNH-
Cys(Trt)-OH (2.5 equiv) was coupled on the resin in
presence of PyBOP (2.5 equiv) and DIPEA (4 equiv) in
3 mL of DMF. This was followed by nine cycles with
FmocArg(Pbf)-OH and one cycle with FmocTyr(But)-
OH. After removal of the terminal Fmoc group and
washing, the resin was dried and cleaved with 4 mL of
95% TFA containing 0.5% TIS, 2.5% EDT and 2.5%
H2O (v/v) for 12 h at rt. The resin was removed by
filtration and the crude product was precipitated from
solution by addition of 10 parts of ethyl ether. The
oligopeptide was purified by semi-preparative RP-HPLC
with a 30 min 0–40% gradient of acetonitrile containing
0.1%TFA in water containing 0.1% TFA at a flow rate
of 1.0 mL/min and detection at 270 nm. The calculated
MW of product is 1689 and the MALDI-TOF MS
found (M+1): 1689.8.
4.1.21. General method for conjugating TyrArg9Cys to
the fluorogenic esters (11–14). The maleimido fluor-
escein derivatives 7–10 (4 equiv) in 50 mL acetonitrile or
MeOH were added separately to TyrArg9Cys in 0.5 mL
of deoxygenated MeOH and then shaken under N2
for 2–4 h at rt. Ethyl ether (10 fold excess) was then
added to precipitate the products. Purification was
carried out by semi-preparative RP-HPLC with a 1.0
mL/min 0–40% 30 min gradient of B (acetonitrile
containing 0.1% TFA) 0–40% in A (water containing
0.1% TFA) with detection at 270 nm. The products
were characterized by MALDI-TOF MS (Table 2).
23. Corey, E. J.; Gras, J. L.; Ulrich, P. Tetrahedron Lett.
1976, 809.
24. Frazier, K. A. in U.S. Patent 4980482 (The Dow
Chemical Company, US), 1990 pp 4.