Development of water-soluble far-red fluorogenic dyes for enzyme sensing

Article abstract of DOI:10.1016/j.tet.2005.10.020

A series of Nile Blue analogs, 5-amino-9-dialkylamino benzo[a]phenoxazine dyes, 7-9, were prepared by condensation of N-alkyl or N-sulfo-propyl 4-arylazo-substituted 3-hydroxyaniline with 4-arylazo-substituted 1-naphthlamines or 8-amino-2-naphthalenesulfo

Full text of DOI:10.1016/j.tet.2005.10.020

Tetrahedron 62 (2006) 578–585
Development of water-soluble far-red fluorogenic
dyes for enzyme sensing
*
Nan-hui Ho, Ralph Weissleder and Ching-Hsuan Tung
Center of Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA
Received 2 March 2005; revised 5 October 2005; accepted 10 October 2005
Available online 3 November 2005
Abstract—A series of Nile Blue analogs, 5-amino-9-dialkylamino benzo[a]phenoxazine dyes, 7–9, were prepared by condensation of
N-alkyl or N-sulfo-propyl 4-arylazo-substituted 3-hydroxyaniline with 4-arylazo-substituted 1-naphthlamines or 8-amino-2-naphthalene-
sulfonic acid in the presence of perchloric acid. These fluorochromes have excitation and emission maxima near 640 and 680 nm,
respectively. The fluorescence intensity in aqueous solution increases as additional sulfonate groups are added to the benzo[a]phenoxazine
core. Compound 7, which has two sulfonate groups, is ten-times brighter than Nile Blue. Fluorogenic substrates containing this hydrophilic
far-red dye were synthesized and applied to detect enzymatic activities of two model proteases, trypsin and leucine aminopeptidase. Given
their excellent fluorogenic property, these novel far-red dyes should be useful for enzyme sensing in biological assays.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
for the development of fluorogenic dyes that have
photochemical stability, long wavelength excitation and
emission maxima, and are water-soluble for cellular as well
as, potentially, for in vivo applications.
Fluorogenic substrates using 7-amino-4-methylcoumarin
(AMC) as a reporter are the most popular probes used in
proteolytic assays today.1,2 Typically, AMC is attached to
the C-terminus of a peptide sequence that is specific for a
targeted protease. The amide linkage between the peptide
chain and AMC results in a dramatic blue shift of both
absorption and emission. Due to this unique property, when
the excitation wavelength was set for free AMC, the intact
probes emit almost no fluorescence unless the AMC group is
released. Although AMC-based probes are in widespread
use, low emission wavelength (430 nm) of AMC has
restricted their uses to cell free assays to avoid interference
with background autofluorescence from intact cells and
tissues.
In searching industrial dyes, we found that benzo[a]-
phenoxazines (BPO), which were utilized for dyeing fibers
and paper, could potentially be converted into fluorogenic
substrates. As a result of their compact and rigid
structures compared to that of cyanine dyes, BPO have
better photochemical stability. The relatively high molar
absorbance, strong fluorescence into the deep red region
(650–730 nm) and modest Stokes shifts (20–60 nm)
indicate the potential utility of these dyes as fluorophores
for biological applications. Indeed it had previously been
demonstrated that the reaction of the primary amino group
on BPO with a carboxylic acid resulted in a dramatic
reduction in fluorescence intensity.6 This is a desirable
property for creating fluorogenic enzyme probes. Unfortu-
nately, the low solubility of BPO and its tendency to
aggregate in aqueous environments have limited its
biological applications. We hypothesized that BPO
analogs with increased sulfonation would have higher
water solubility,7 improved biocompatibility and poten-
tially higher fluorescence output. In the present report, we
detail the synthesis, characterization, and preliminary
biological studies of such a new class of hydrophilic
BPO-based far-red fluorogenic probes (Scheme 1). This
type of probe should have potential use for in vivo
sensing and imaging of proteases.8
Recently, rhodamine-based fluorogenic substrates with
emission maxima near 520 nm have been reported for
cellular studies.3–5 Similar to the AMC probes, peptides are
linked to the rhodamine analogs by amide linkages through
their C-termini. However, these rhodamine analogs contain
two amino groups requiring two amino acids or peptides to
react with a single fluorophore. The requirement that both
peptide chains need to be cleaved from the core fluorophore
further limits the linear dynamic range. Thus, there is a need
Keywords: Fluorogenic; Fluorescent probe; Water-soluble; Nile Blue;
Protease; Leucine aminopeptidase.
Corresponding author. Tel.: C1 617 726 5779; fax: C1 617 726 5708;
e-mail: tung@helix.mgh.harvard.edu
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.10.020

N.-h. Ho et al. / Tetrahedron 62 (2006) 578–585
579
Scheme 1. Synthetic scheme of 7–9 and structure of Nile Blue.
2. Results and discussion
Blue with the N-(di-3-sulfonyl-propyl)amino group generates
7. Compound 9 was designed to incorporate sulfonyl groups
substituted on the aromatic ring (Scheme 1). The sulfonated
aminophenol 1 was obtained by reaction of 3-aminophenol
with propane sultone. The arylazo compounds 4–6 were
prepared by treating the corresponding N-substituted amino-
phenols 1–3 with an aryl diazonium salt. Three desired dyes
7–9wereobtainedfromthecorrespondingarylazocompounds
4–6. For compound 8, the ring system was readily formed in
5 min; however, prolonged heating was necessary for ring
condensation of 7 and 9. During the condensation reactions,
the temperature was maintained below 160 8C because
product decomposition was observed at higher temperature.
2.1. Synthesis of benzo[a]phenoxazine dyes
BPO dyes, such as Nile Blue (NB) and Nile Blue 2B, are
generally prepared by reacting the appropriate nitrosoanilines
with 1-naphthylamines or 2-naphthol in ethanol in the
presence of hydrochloric acid.9–11 However, those procedures
are not suited for the synthesis of sulfonated benzo[a]-
phenoxazines. Cyclization using sulfonated nitrosoanilines or
1-naphthlamines as building blocks is not very effective.
Sulfonation after cyclization is also not desirable because
multiple substitutions at several aryl positions produce
mixtures, which must then be separated. A recent literature
report demonstrated that the bridged benzo[a]phenoxazinium
salts could be synthesized by heating arylazo-substituted
aminophenols or naphthamines with the appropriate
1-naphthylamine or aminophenol in DMF in the presence
of perchloric acid.12 We found that this method is well-suited
for transforming sulfonated arylazo-substituted aminophenols
into sulfonated benzo[a]phenoxazines (Scheme 1).
2.2. Absorption and fluorescence properties
For biological applications, high fluorescence quantum
yield in water is essential. It is known that p-stacking of
xanthene dyes by van der Waal’s forces or hydrophobic
interactions causes serious fluorescence quenching in
aqueous solution.13–15 BPO type dyes, such as NB and
cresyl violet readily form sandwich-like H-aggregate type
of dimers or even higher aggregates in aqueous solution.
H-aggregated dimers are known to be nonfluorescent due to
Compound 8, which has no sulfonate group, was synthesized
initially. Replacement of the 9-diethylamino group of Nile
Table 1. Absorption and fluorescence properties of BPO dyes
a
lmax (nm)
Dyes
Extinction coefficient
((M cm)K1 a
FWHM (nm)
CH3OH
Relative quantum
yieldb
)
Ex
Em
H2O
Nile Blue
PYBPO 8
1SBPO 9
2SBPO 7
635
643
637
633
675
678
677
675
4000
8000
11,000
36,000
115
89
93
86
76
77
70
65
0.01c
0.03
0.03
0.10
a 1!10K5 M in PBS.
b The fluorescence intensities in water were measured from the emission curve using ‘area’ function of the fluorometer.
c Ref. 22.

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N.-h. Ho et al. / Tetrahedron 62 (2006) 578–585
the forbidden nature of transition between the lowest excited
singlet and ground states.16 To improve the fluorescence
properties and to reduce the tendency of the dyes to
aggregate in aqueous media, the hydrophobic ethyl group
at the 9-amino position of NB were replaced with
alkylsulfonate groups as in 2SBPO 7. A similar effect is
observed when a sulfonate group is introduced at one of the
aryl position of the dye as in 1SBPO 9.
dramatically increases the fluorescence intensity in aqueous
solution (Table 1). As expected, addition of sulfonate
groups also increases water solubility and reduces the
tendency of aggregate formation, thus resulting in a shift in
the monomer–dimer equilibrium. With the increase of
monomer fraction in water, the relative fluorescence
intensities increase. The relative fluorescence intensity of
1SBPO 9 in water is three-fold higher than that of NB.
Modification with two sulfonate groups increases the
relative fluorescence intensity by a factor of 10 for
2SBPO 7. Surprisingly, PYBPO 8 exhibits similar emission
property to 1SBPO 9. The increase in fluorescence intensity
of PYBPO versus NB is likely due to its less aggregated
nature. In aqueous solution, the 89 nm FWHM value of
PYBPO shows less dimerization in comparison to NB
(115 nm).
The absorption and fluorescence maxima, relative fluore-
scence intensities and extinction coefficients for compounds
7–9 are summarized in Table 1. The ‘parent dye’, NB, is
included for comparison. In general, modifications at the
9-amino and C-3 positions of the BPO have little effect on the
absorption and emission maxima. PYBPO 8 shows a slightly
red shifted absorption maximum at 643 nm when a
pyrrolidinyl group was introduced at the 9-amino position.
For dyes 7–9, the absorption maximum are pH independent in
the range of 4–8 and the Stokes’ shifts are approximately
40 nm. The extinction coefficients of 7–9 in aqueous solution
vary between 8000 and 36,000 (M cm)K1. For 2SBPO 7, a
nine-fold improvement over NB, which has a very low 3 value
around 4000 (M cm)K1 in aqueous solution, was achieved.
The higher extinction coefficient of 2SBPO 7, in comparison
to 1SBPO 9 and PYBPO 8, may be caused by the increased
water-solubility.
2.3. Water-soluble BPO-based fluorogenic substrates
Due to its superior fluorescence properties in aqueous
buffer, 2SBPO 7 was chosen for subsequent preparation of
fluorogenic substrates (10–15, Scheme 2). Various amino
acid residues activated in situ by HATU were coupled to 7
using collidine as base. The coupling reactions were
normally completed in 1–2 h at room temperature with
95–100% conversion as determined by TLC. As expected,
all amino acid-2SBPO conjugates were very soluble in
water and gave only low fluorescence signals.
It has been previously reported that in weak acidic to basic
condition, NB exists as the blue cation HNBC (absorbance
maximum at 640 nm), and a dimer (HNB)22C (absorbance
maximum at 600 nm).17 Because of the close proximity of
the absorption bands of the monomer and dimer, the full
width at half-maximum (FWHM) values in water were used
to access the equilibrium between monomer and dimer
semi-quantitatively.18,19 In methanol, sharp absorption
bands near 640 nm with FWHM values ranging from 65
to 76 nm were observed for all the dyes, indicative of
nonaggregated systems. In water, FWHM values for
compounds 7–9 and NB ranged from 86 to 115 nm. The
86 nm FWHM value of 7 indicates a reduced tendency to
dimerize in comparison to other dyes in aqueous solution.
To demonstrate the general applicability of the water-
soluble BPO-based fluorogenic protease sensors, the
fluorogenic substrate, Lys-2SBPO 13, was initially
synthesized for digestion with trypsin. The conjugate
showed a nearly 10-fold reduction in fluorescence
intensity. However, the substrate was found to be only
stable at low pH. When pHO5, spontaneous hydrolysis
was observed. In order to investigate whether the
spontaneous hydrolysis of 2SBPO-based substrates
depends on the nature of the amino groups on the
amino acid residues, we conjugated 7 to lysine with and
without the Boc protected amino groups. Overall, four
analogs, Boc-Lys(Boc)-2SBPO 10, Boc-Lys-2SBPO 11,
Lys(Boc)-2SBPO 12 and Lys-2SBPO 13, were prepared
(Scheme 2). The stabilities of these conjugates in
Comparison of relative fluorescence emission intensities of
the dyes reveal that modification with sulfonyl groups
Scheme 2. Structures of fluorogenic enzyme substrates 10–15.

N.-h. Ho et al. / Tetrahedron 62 (2006) 578–585
581
aqueous solution were then examined over the pH range
2.4. Leucine aminopeptidase
of 4–8. Free 2SBPO 7 was included in this study as a
control to verify that the fluorescence intensity of free 7
is not significantly affected within the tested pH range.
Lys-2SBPO 13 and Lys(Boc)-2SBPO 12, without
protecting the a-amino groups, were found to be stable
at pH 4 and 5, but not at higher pH (Fig. 1A). At pHO5,
compared to pH 4 and 5, there was approximately a
three-fold difference in fluorescence intensity indicating
spontaneous hydrolysis of the substrates. Importantly,
when the a-amino group was blocked with a Boc group,
the fluorescence intensities of Boc-Lys-2SBPO 11 and
Boc-Lys(Boc)-2SBPO 10 were unchanged for at least 3 h
over the pH range investigated. The experimental
evidence indicates that the free a-amino residues of
amino acids have a catalytic effect on the hydrolysis of
the amide bond.
Two BPO-based fluorogenic substrates, Boc-Leu-2SBPO
14 and Leu-2SBPO 15, were also synthesized as model
substrates to detect leucine aminopeptidase (amino-
peptidase M, AP-M). AP-M is a cytosolic exopeptidase
and catalyzes the hydrolysis of amino acids from the amino
terminus of polypeptide chains.20 Similar pH effects on the
stabilities of leucine containing substrates were observed.
At pHO5, increase in fluorescence signal from 15
is attributable to the spontaneous hydrolysis, whereas
Boc-Leu-2SBPO 14 remained stable between pH 4 and 8
(Fig. 2A). Upon incubating 14 with AP-M at neutral pH, an
increase in fluorescence emission was observed (Fig. 2B). In
contrast, without AP-M, the fluorescence intensity remained
unchanged.
Figure 2. The effects of pH on the stabilities of Boc-Leu-2SBPO 14 and
Leu-2SBPO 15 (3 h) (A) and leucine aminopeptidase proteolysis assay (B).
The fluorescence assay of hydrolysis of Boc-Leu-2SBPO 14 (4 mM) with
leucine aminopeptidase (0.7 u) in 0.1 M HEPES (pH 7.0) containing 5 mM
MgCl2 and 0.1% bovine serum albumin. The fluorescence excitation and
emission were 610 and 670 nm, respectively.
The kinetic parameters for the hydrolysis of 14 with AP-M
are listed in Table 2. The kinetic parameters of Leu-7-
amido-4-methyl-coumarin (Leu-AMC), bis-Leu-rhod-
amine-110 (Bis-(Leu)2-Rhod) and Leu-rhodamine-110
(Leu-Rhod110-NH2) from the literature4 are included in
the table for comparison. Under the conditions used, the Km
values show that 14 binds more effectively to AP-M than
coumarin- and rhodamine- based protease substrates. The
Vmax value for 14 is significantly higher than those of the
previously reported fluorogenic substrates. The linearity
of the fluorescence enhancement versus incubation time
Figure 1. Effects of pH on the stabilities of the fluorogenic substrates
containing lysine residues and trypsin proteolysis assays. (A) 2SBPO 7,
Boc-Lys(Boc)-2SBPO 10, Boc-Lys-2SBPO 11, Lys(Boc)-2SBPO 12 and
Lys-2SBPO 13 (1 mM) incubated in 0.1 M buffers (pH 4.0–8.0) at room
temperature for 3 h (nZ3). The fluorescence intensities are normalized.
(B) Fluorescence assay for the hydrolysis of Boc-Lys-2SBPO 11 (4 mM)
with trypsin (6 u) in 0.1 M sodium phosphate buffer (pH 7.4). The
fluorescence excitation and emission were 610 and 670 nm, respectively.
The fluorogenic substrate, Boc-Lys-2SBPO 11, was then
assayed with its target protease, trypsin. Figure 1B showed
the time course for the enzyme catalyzed hydrolysis at
pH 7.4. After adding trypsin to the substrate solution, a
strong fluorescent signal generated at 670 nm indicated
catalytic cleavage of the amide bond and release of highly
fluorescent free 7. A seven-fold increase in fluorescence
intensity was observed in comparison to the control reaction
without trypsin.
Table 2. Kinetic parameters of the leucine aminopeptidase fluorogenic
substrates
Substrates
Km (mM)
Vmax (nM/min)
Leu-AMCa
39.6G3.6
23.6G3.3
5.6G1.2
4.3G0.1
3.7G0.2
2.9G0.2
34G0.7
Bis-(Leu)2-Rhod110a
a
Leu-Rhod110-NH2
Boc-Leu-2SBPO 14b
2.8G0.23
a Ref. 4.
b 0.1 M HEPES, pHZ7.0, 5 mM MgCl2 and 0.1% bovine serum albumin.

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N.-h. Ho et al. / Tetrahedron 62 (2006) 578–585
(for more than 10 min) is satisfactory (Fig. 2B). The high
water solubility of the 2SBPO-based substrates allowed the
assays to be performed without using any organic solvent as
co-solvent, whereas the AMC and rhodamine-based
substrates require 1–3% dimethyl sulfoxide (DMSO) as
co-solvent.4,21
with cold methanol giving a gray solid (0.8 g, 80%).
1H NMR (DMSO-d6): d 1.94 (quintet, 2H, JZ6.6 Hz,
NCH2CH2CH2SO3H), 2.62 (t, 2H, JZ6.6 Hz, NCH2CH2-
CH2SO3H), 3.36 (t, 2H, JZ6.6 Hz, NCH2CH2CH2SO3H),
6.80 (m, 3H, H2, H4, H6), 7.24 (t, 1H, JZ7.5 Hz, H5). MS
(MALDI-TOF) calcd for C9H14NO4S (MC1)C: 232.27,
found 231.30.
3. Conclusions
4.1.2. 3,30-[(3-Hydroxyphenyl)amino]bis-1-propane-
sulfonic acid (1). A solution of 3-(3-sulfo-propyl)amino-
phenol (1 g, 4.3 mmol) and 1,3-propane sultone (0.54 g,
4.4 mmol) in 5 mL of DMF was heated to 130 8C for 2 h with
stirring. After cooling, t-butyl ethyl ether was added to
precipitate the dark oily product. The crude product was
purified on a 35 cc Sap-Pak C18 cartridge (Waters, Milford,
MA) eluting with water followed by 15% acetonitrile in water.
The solvent was removed to give an orange-red crystalline
We have developed a highly efficient synthetic strategy for
incorporation of sulfonate groups on the BPO moiety. This
methodology allows for the production of water-soluble
BPO derivatives in high yield. The mono-functional 2SBPO
has excellent fluorogenic properties and is highly fluores-
cent in aqueous solution. The 2SBPO containing substrates
have several advantages: (1) Kinetic determination of
enzymatic process can be performed in aqueous solution
without organic solvent. (2) Since the fluorescent spectra of
the dyes are not sensitive to pH, the dyes are functional over
a broad pH range. (3) Unlike coumarin and rhodamine,
water-soluble BPO fluoresces beyond 650 nm in the far-red
region and a better signal-to-noise ratio can be obtained.
This system shows promise for in vivo enzyme imaging
applications. (4) Up to a seven-fold change in fluorescence
intensity can be obtained. Finally, the novel fluorogenic
sensors could be used for in vivo sensing typically
necessitating measurements in the far-red and near-infrared
ranges.
1
solid (1.48 g, 95%). H NMR (DMSO-d6): d 1.88 (m, 4H,
NCH2CH2CH2SO3H), 2.50 (m, 4H, NCH2CH2CH2SO3H),
3.64 (m, 4H, NCH2CH2CH2SO3H), 6.20 (s, 1H, ar), 6.57,
(d, 1H, JZ9.2 Hz, ar), 7.55 (d, 1H, JZ9.2 Hz, ar), 7.86
(d, 1H, JZ9.2 Hz, ar), 8.29 (d, 1H, JZ9.2 Hz, ar).
MS (MALDI-TOF) calcd for C12H19NO7S2 (MC1)C:
354.41, found 353.19.
4.1.3. 5-(Di-3-sulfonyl-propylamino)-2-(4-nitro-pheny-
lazo)phenol (4). A solution of 4-nitroaniline (0.1 g,
0.80 mmol) was dissolved in a mixture of 0.5 mL of
concentrated hydrochloric acid and 0.5 mL of water. The
reaction flask was cooled in an ice-bath and sodium nitrite
(55 mg, 0.80 mmol) dissolved in 100 mL of water was added
slowly. The reaction mixture was stirred at 0 8C for an
additional 20 min. At the end of the reaction, the solution
turned orange. Then a solution of 1 (247 mg, 0.7 mmol) in
0.2 mL of methanol was added to the reaction flask. The
reaction mixture was stirred for 30 min at room temperature.
The red precipitate was filtered and washed with cold
ethanol. The crude product was purified on a 35 cc Sep-Pak
C18 cartridge eluting with water followed by 5% acetonitrile
in water to yield 4 as a red solid (264 mg, 75%). 1H
NMR (DMSO-d6): d 1.88 (m, 4H, NCH2CH2CH2SO3H),
2.51 (m, 4H, NCH2CH2CH2SO3H), 3.64 (m, 4H,
NCH2CH2CH2SO3H), 6.20 (s, 1H, ar), 6.57 (d, 1H, JZ
9.2 Hz, ar), 7.55 (d, 1H, JZ9.2 Hz, ar), 7.86 (d, 1H, JZ
9.2 Hz, ar), 8.29 (d, 1H, JZ9.2 Hz, ar). MS (MALDI-TOF)
calcd for C18H23N4O9S2 (MC1)C: 503.52, found 503.10.
4. Material and methods
3-Pyrrolidin-1-yl-phenol was purchased from ChemOvation
(West Sussex, UK). Aminopeptidase-M (AP-M, EC 3.4.11.2)
(porcine kidney, microsomal) was purchased from Sigma-
Aldrich (St. Louis, MO) and Roche (Indianapolis, IN).
Trypsin (porcine pancreas) and other reagents were obtained
from Sigma-Aldrich and used without further purification.
NMR spectra were recorded on either a Varian (Palo Alto,
CA) XL-200 or an Inova-400 instrument for the H–1H 2D
1
COSY and13 spectra. Absorbance spectra were measured on a
Varian Cary 50 Bio spectrophotometer. Fluorescence spectra
were carried out on either a F-4500 (Hitachi, Danbury, CT)
or a Fluorolog-3 (JOBIN YVON-SPEX, Edison, NJ)
fluorophotometer. Kinetic parameters were determined on a
TECAN (Salzburg, Austria) Genios Pro fluorescence micro
plate reader. Electrospray mass spectra were obtained on a
Platform LCT mass spectrometer (Micromass, Beverly, MA)
and MALDI mass spectra were measured on a Voyager
liner mass spectrometer (Applied Biosystems; Framingham,
MA). The HR MALDI mass spectra were acquired on a
Voyager-DE STR mass spectrometer (Applied Biosystems).
4.1.4. 2-(4-Nitro-phenylazo)-5-pyrrolidin-1-yl-phenol (5)
and 8-amino-5-(4-nitro-phenylazo)-2-naphthalenesulfonic
acid (6). Compounds 5 and 6 were synthesized using the
same procedure as 4, except 3-pyrrolidin-1-yl-phenol (2)
and 8-amino-naphthalene-2-sulfonic acid (3) were used,
respectively.
4.1. Chemical synthesis
Compound 5. The crude product was recrystallized in hot
n-butanol. After drying, 5 (190 mg, 85%) was obtained as
a purple solid. 1H NMR (DMSO-d6): d 2.00 (m, 4H,
–CH2CH2N–), 3.50 (m, 4H, –CH2CH2N–,), 5.86 (s, 1H, ar),
6.53 (d, 1H, JZ9.2 Hz, ar), 7.41 (d, 1H, JZ9.1 Hz, ar), 7.81
(d, 1H, JZ8.8 Hz, ar), 8.28 (d, 1H, JZ8.8 Hz, ar); MS
(MALDI-TOF) calcd for C16H17N4O3 (MC1)C: 313.33,
found 313.8.
4.1.1. 3-(3-Sulfo-propyl)aminophenol. To a solution of
3-aminophenol (5.0 g, 4.5 mmol) in 5 mL of n-butanol was
added 1,3-propane sultone (0.56 g, 4.6 mmol), and the
resulting mixture was refluxed with stirring for 30 min
during which a gray precipitate was formed. The reaction
mixture was stirred continuously at room temperature
overnight. The precipitate was filtered and washed

N.-h. Ho et al. / Tetrahedron 62 (2006) 578–585
583
Compound 6. The crude red solid was purified on a 35 cc
Sep-Pak C18 cartridge eluted with a stepwise gradient,
starting with water followed by 10% acetonitrile and then
20% acetonitrile in water. Pure 6 was obtained as red solid
with typical yield of 80–90%. 1H NMR (DMSO-d6): d 6.80
(d, 1H, JZ8.8 Hz, H-7), 7.88 (d, 1H, JZ8.8 Hz, H-6), 8.23
(m, 3H, ar), 8.36 (d, 2H, JZ8.8 Hz, ar), 8.49 (s, 1H, H-1),
8.84 (d, 1H, JZ8.8 Hz, H-3). MS (MALDI-TOF) calcd for
C16H14N4O5S (MC1)C: 373.37, found 373.08.
The product was purified on a 35 cc Sap-Pak C18 cartridge
eluting with 20% acetonitrile in water. After removing of
the solvent, 9 was obtained as a blue solid (90 mg, 55%).
1H NMR (DMSO-d6): d 8.76–8.79 (m, 3H, H2, H4), 8.18
(d, 1H, JZ8.0 Hz, H1), 7.86 (d, 1H, JZ9.3 Hz, H11), 7.25
(d, 1H, JZ10.5 Hz, H10), 7.0 (s, 1H, H8), 6.85 (s, 1H, H6),
3.30 (q, 4H, JZ6.0 Hz, CH3CH2N–), 1.21 (t, 6H, JZ
6.0 Hz, CH3CH2N–). HRMS (ESC/TOF): calcd for
C20H21N3O4S (M)C: 398.1169, found 398.1168.
4.1.5. 9-Di-3-sulfonyl-propylaminobenzo[a]phenoxazonium
perchlorate (2SBPO, 7). A mixture of 5-(di-3-sulfonyl-
4.1.8. 5-N-a,3-Di-t-butoxycarbonyl-L-lysinylamino-9-
di-3-sulfonyl-propylaminobenzo[a]phenoxazonium per-
chlorate (Boc-Lys(Boc)-2SBPO, 10). To a stirred solution
of (Boc)-Lys(Boc)-OH (23 mg, 0.066 mmol) in DMF
(1 mL) was added 2SBPO 0 (20 mg, 0.033 mmol), O-(7-
azabenzotriazol-1-yl)-N,N,N ,N0-tetramethyluronium PF6
(HATU) (25 mg, 0.066 mmol) and 2,4,6-collidine (43 mL,
0.33 mmol). The solution was stirred at room temperature
for 2 h. The reaction was monitored by silica gel TLC. The
crude product was precipitated with ether and purified on a
35 cc C18 cartridge (Waters) eluting with 30% acetonitrile in
propylamino)-2-(4-nitro-phenylazo)phenol
4 (86 mg,
0.17 mmol), 1-aminonaphthalene (27 mg, 0.19 mmol) and
3 mL of DMF containing perchloric acid (0.1 mL, 70%) was
heated to 155–160 8C for 15 min with stirring. The color of
the reaction mixture changed from brown to deep blue. The
reaction was monitored by silica gel TLC (methanol/ethyl
acetate, 4:5). After cooling, diethyl ether was added to
precipitate the product. The crude perchlorate salt was
purified on a 35 cc Sap-Pak C18 cartridge eluting with 10%
1
1
acetonitrile in water (88 mg, 85%). H NMR (DMSO-d6):
water to give 10 as a blue solid (25 mg, 93%). H NMR
d 1.80–2.00 (m, 4H, NCH2CH2CH2SO3H) 2.52–2.58 (m,
4H, NCH2CH2CH2SO3H), 3.70–3.80 (m, 4H, NCH2CH2-
CH2SO3H), 6.80 (s, 1H, H6), 7.10 (s, 1H, H8), 7.36 (d, 1H,
JZ8.8 Hz, H10), 7.72 (d, 1H, JZ8.8 Hz, H11), 7.80–8.0
(m, 2H, H2, H3), 8.42 (d, 1H, JZ9.1 Hz, H4), 8.74 (d, 1H,
JZ8.4 Hz, H1). Peak assignment was made using a
combination of 1H–1H 2D COSY and 1D proton NMR
spectra. 13C NMR (400 MHz, D2O): d 159.35, 153.64,
149.07, 145.82, 136.73, 132.45, 131.35, 129.70, 128.82,
127.93, 122.69, 122.40, 120.85, 114.79, 96.25, 95.81, 49.94,
47.96, 22.57. HRMS (MALDI-TOF, negative-ion mode)
calcd for C22H23N3O7S2 (MK2)K: 504.0899, found 504.093.
(DMSO-d6): d 1.1—1.42 (m, 22H, OC(CH3)3 and CH2),
1.60–1.82 (br s, 2H, CH2), 1.85–2.15 (m, 4H, NCH2CH2-
CH2SO3H) 2.45–2.65 (m, 4H, NCH2CH2CH2SO3H), 2.90
(br s, 2H, NCH2), 4.00–4.20 (m, 4H, NCH2CH2CH2SO3H),
4.40–4.60 (m, 1H, COCHN), 6.80 (s, 1H, H6), 7.35 (s, 1H,
H8), 7.60 (br s, 1H, H10), 7.85–8.15 (m, 3H, H2, H3, H11),
8.45–8.65 (m, 2H, H1, H4), 8.85 (br s, 1H, NH), 10.7 (br s,
1H, NH). Rf (MeOH/AcOEt, 2:1) 0.22; MS (MALDI-TOF),
calcd for C38H53N5O12S2 (MC1)C: 835.98, found: 836.06.
4.1.9. 5-N-a-t-Butoxycarbonyl-L-lysinylamino-9-di-
3-sulfonyl-propylaminobenzo[a]phenoxazonium per-
chlorate (Boc-Lys-2SBPO, 11). Boc-Lys-2SBPO was
obtained from Boc-Lys(Fmoc)-2SBPO by removing
the fluorenylmethoxycarbonyl (Fmoc) protecting group.
Boc-Lys(Fmoc)-2SBPO was synthesized using the same
procedure as described for Boc-Lys(Boc)-2SBPO.
Boc-Lys(Fmoc)-2SBPO was dissolved in 20% piperidine
in DMF. After removing the Fmoc protecting group, the
crude product was precipitated with diethyl ether and
purified on a 35 cc Sap-Pak C18 cartridge eluting with 20%
acetonitrile in water to give 11 (20 mg, 82%) as a blue solid.
1H NMR (DMSO-d6): d 0.86 (m, 9H, OC(CH3)3), 1.30–1.50
(m, 4H, CH2), 1.70–2.00 (m, 6H, NCH2CH2CH2SO3H and
CH2), 2.95 (br s, 2H, NCH2), 4.40 (br s, 1H, COCHN), 5.38
(br s, 1H, ar), 6.35 (s, 1H, H8), 6.44 (d, JZ8.8 Hz, 1H,
H10), 7.20 (d, JZ8.4 Hz, 1H, H11), 7.30–7.51 (m, 3H, ar),
8.23 (br s, 1H, ar), the signals for NCH2CH2CH2SO3H
and NCH2CH2CH2SO3H were not detected due to
overlap with solvent peaks. MS (MALDI-TOF), calcd for
C33H45N5O10S2Na (MCNa)C757.86, found: 757.19.
4.1.6. 9-Pyrrolidin-aminobenzo[a]phenoxazonium per-
chlorate (PYBPO, 8). To a solution of 2-(4-nitro-phenyl-
azo)-5-pyrrolidin-1-yl-phenol 5 (100 mg, 0.32 mmol) in
3 mL of DMF containing perchloric acid (0.1 mL, 70%) was
added 1-aminonaphthalene (50.4 mg, 0.35 mmol). The
reaction mixture was refluxed for 5 min and the color of
the mixture changed from brown to an intense blue. The
progress of the reaction was followed by silica gel TLC with
ethyl acetate/methanol (9:1). After cooling, the crude
product was precipitated with diethyl ether. The precipitated
salt was dissolved in a small amount of DMF and
precipitated again from diethyl ether. The crude dye was
purified on a 35 cc Sap-Pak C18 cartridge eluting with water
followed by acetonitrile/water mixture going from 5 to 10%
acetonitrile. The solvent was removed to yield 8 as a blue
solid (120 mg, 90%). 1H NMR (DMSO-d6): d 2.04 (m, 4H,
–CH2CH2N–,), 3.58 (m, 4H, –CH2CH2N–,), 6.86 (s, 2H, ar),
7.13 (d, 1H, JZ9.6 Hz, ar), 7.84–8.10 (m, 3H, ar), 8.46
(d, 1H, JZ8.2 Hz, ar), 8.81 (d, 1H, JZ7.2 Hz, ar). HRMS
(ESC/TOF): calcd for C20H19N3O MC: 316.1444, found
316.1447.
4.1.10.
5-N-3-t-Butoxycarbonyl-L-lysinylamino-9-
di-3-sulfonyl-propylaminobenzo[a]phenoxazonium per-
chlorate (Lys(Boc)-2SBPO, 12). Lys(Boc)-2SBPO
was obtained by the same procedure as described for
Boc-Lys(Boc)-2SBPO, but using Fmoc-Lys(Boc)-OH
(31 mg, 0.066 mmol) and 2SBPO (20 mg, 0.033 mmol).
Fmoc-Lys(Boc)-2SBPO was dissolved in 20% piperidine
in DMF. After 15 min, diethyl ether was added and the
precipitate was filtered off. The crude product was purified
4.1.7. 3-Sulfonyl-9-diethylaminobenzo[a]phenoxazonium
perchlorate (1SBPO, 9). A solution of 6 (0.1 g, 0.27 mmol)
and 3-(diethylamino)phenol (0.5 g, 0.3 mmol) in 3 mL of
DMF containing perchloric acid (0.1 mL, 70%) was heated
to 150 8C for 25 min. The solution turned to deep blue.
Upon cooling, the crude product was precipitated with ether.

584
N.-h. Ho et al. / Tetrahedron 62 (2006) 578–585
on a 35 cc C18 cartridge (Waters) eluting with 20%
acetonitrile in water to give 12 (18 mg, 75%) as blue solid.
1H NMR (DMSO-d6): d 1.2–1.50 (m, 13H, OC(CH3)3 and
CH2), 1.6–2.2 (m, 6H, NCH2CH2CH2SO3H and CH2) 2.4–
2.65 (m, 4H, NCH2CH2CH2SO3H), 2.95 (br s, 2H, NCH2),
4.0–4.2 (m, 4H, NCH2CH2CH2SO3H), 4.8 (br s, 1H,
COCHN), 6.50 (br s, 1H, NH), 6.80 (s, 1H, H6), 7.4–7.8
(br s, 1H, H8), 7.80–8.20 (m, 3H, H3, H11, H10), 8.40–8.6
(m, 2H, H1, H2, H4), 9.0 (br s, 1H, NH), 10.8 (br s, 1H,
NH). MS (MALDI-TOF), calcd for C33H45N5O10S2
(MC1)C735.87, found: 736.14.
with solvent peaks. MS (MALDI-TOF), calcd for
C28H36N4O8S2 (MC1)C620.76, Found: 620.27.
4.2. Optical properties of compounds 7–9
The excitation spectra (emission at 675 nm) and emission
spectra (excitation at 635 nm) of benzo[a]phenoxazine dyes
were determined in phosphate buffer, pH 7.4. at the
concentration of 1!10K5 M. The extinction coefficients
were also determined in phosphate buffer. The fluorescence
intensities in water were measured from the emission
curve using ‘area’ function of the fluorometer. Relative
fluorescence quantum yields were obtained by using Nile
Blue chloride as a reference.
4.1.11. 5-L-Lysinylamino-9-di-3-sulfonyl-propylamino-
benzo[a]phenoxazonium perchlorate (Lys-2SBPO, 13).
Lys-2SBPO was obtained from Boc-Lys(Boc)-2SBPO.
Boc-Lys(Boc)-2SBPO (25 mg, 0.03 mmol) was dissolved
in TFA (1 mL). After 15 min, diethyl ether was added and
the precipitate was filtered off. The crude product was
purified on a 35 cc Sap-pak C18 cartridge eluting with 10%
acetonitrile in water to give 13 (16 mg, 85%) as a blue solid.
1H NMR (DMSO-d6) d 1.40–1.80 (m, 6H, CH2), 1.85–2.15
(m, 6H, NCH2CH2CH2SO3H) 2.40–2.65 (m, 4H, NCH2-
CH2CH2SO3H), 2.8 (br s, 2H, NCH2), 4.00–4.20 (m, 4H,
NCH2CH2CH2SO3H), 7.40–8.50 (m, 8H, ar), 9.0 (br s, 1H,
NH), the signals for NCHCOOH were not detected due to
overlapping with solvent peaks. MS (MALDI-TOF), calcd
for C28H37N5O8S2 (MC1)C: 635.75, found: 636.13.
4.3. pH Dependent stability of compounds 10–15
Stock solutions of compounds 2SBPO 7 and 10–15 were
prepared in water at a concentration of 1!10K4 M. The stock
solutionswerethenaddedto0.1 MpH4.0(citrate–phosphate),
pH5.0(citrate–phosphate), pH6.0(citrate–phosphate), pH7.4
(PBS) and pH 8.0 (phosphate–borate) buffer. The final
concentration of the solutions was 1!10K6 M. The
fluorescence emission at 670 nm was measured with
excitation at 610 nm using a plate reader at tZ0 and 3 h.
4.4. Proteolysis of Boc-Lys-2SBPO by trypsin
A solution of trypsin (10 mL, 6 unit) was added to Boc-Lys-
2SBPO (200 mL, 4 mM) in 0.1 M sodium phosphate buffer
(pH 7.4) at 25 8C, and the increase in fluorescence emission
at 670 nm was measured using a plate reader with excitation
at 610 nm. The same conditions were applied to the control
sample solution without adding trypsin.
4.1.12.
5-N-t-Butoxycarbonyl-L-leucinylamino-9-
di-3-sulfonyl-propylaminobenzo[a]phenoxazonium per-
chlorate (Boc-Leu-2SBPO, 14). Boc-Leu-2SBPO was
obtained by the same procedure as described for Boc-
Lys(Boc)-2SBPO from Boc-Leu-OH (15 mg, 0.066 mmol)
and 2SBPO (20 mg, 0.033 mmol). The crude product was
purified on a 35 cc Sap-Pak C18 cartridge eluting with 25%
acetonitrile in water to give 14 (16 mg, 78%) as blue solid.
1H NMR (DMSO-d6): d 0.98 (d, 6H, JZ5.5 Hz, CH3), 1.41
(s, 9H, OC(CH3)3), 1.6–1.8 (m, 3H, CH(CH3) and CH2),
1.90–2.15 (m, 6H, NCH2CH2CH2SO3H and CH2) 2.4–2.65
(m, 4H, NCH2CH2CH2SO3H), 4.0–4.2 (m, 4H, NCH2CH2-
CH2SO3H), 4.4–4.6 (m, 1H, COCHN), 6.50 (br s, 1H, NH),
7.08 (s, 1H, H6), 7.4–7.8 (br s, 1H, H8), 7.80–8.20 (m, 3H,
H3, H11, H10), 8.40–8.6 (m, 3H, H1, H2, H4), 9.0 (br s, 1H,
NH), 10.8 (br s, 1H, NH). MS (MALDI-TOF), calcd for
C33H44N4O10S2 (MC1)C: 720.88, found 720.75.
4.5. Proteolysis of Boc-Leu-2SBPO by leucine
aminopeptidase
To assess the kinetic parameters for the leucine amino-
peptidase-catalyzed hydrolysis of Boc-Leu-2SBPO, aliquots
of Boc-Leu-2SBPO were prepared with concentrations
ranging from 1.0 to 16.6 mM in 0.1 M HEPES buffer
(pHZ7.0) containing 5 mM MgCl2 and 0.1% bovine serum
albumin. The reactions were monitored using a fluorescence
plate reader with excitation and emission at 610 and
670 nm, respectively. The total volumes of the reactions
were 0.2 mL. The reactions were initiated by the addition a
5 mL (0.7 U) aliquot of aminopeptidase M stock solution.
The initial velocities of the reactions were determined by the
initial fluorescence intensities extracted with average values
(nZ3). The kinetic parameters for the substrate were
obtained using the direct linear plot of the Eisenthal and
Cornish-Bowden equation. Initial velocity measurements
were converted from relative fluorescence unit (AU/min) to
nM product formed/min by using a standard curve with
authentic fluorophore and the same buffer conditions and
wavelength settings (nZ3).
4.1.13. 5-L-Leucinylamino-9-di-3-sulfonyl-propylamino-
benzo[a]phenoxazonium perchlorate (Leu-2SBPO, 15).
Leu-2SBPO was obtained from Boc-Leu-2SBPO by remov-
ing the Boc protecting group. Boc-Leu-2SBPO (20 mg,
0.024 mmol) was dissolved in TFA (1 mL). After 15 min,
diethyl ether was added and the precipitate was filtered off.
The crude product was purified on a 35 cc Sap-pak C18
cartridge eluting with 20% acetonitrile in water to give 15
1
(16 mg, 0.92) as a blue solid. H NMR (DMSO-d6): d 1.01
(d, 6H, JZ5.5 Hz, CH3), 1.65–1.85 (m, 3H, CH(CH3) and
CH2), 1.85–2.15 (m, 6H, NCH2CH2CH2SO3H and CH2)
2.40–2.65 (m, 4H, NCH2CH2CH2SO3H), 4.00–4.20 (m, 4H,
NCH2CH2CH2SO3H), 7.08 (s, 1H, H6), 7.40–7.80 (br s, 1H,
H8), 7.80–8.20 (m, 3H, H3, H11, H10), 8.40–8.60 (m, 2H,
H1, H2, H4), 7.40–8.50 (m, 8H, ar), 9.00 (br s, 1H, NH), the
signals for H2NCHCO were not detected due to overlapping
Acknowledgements
This research was supported in part by NIH P50-CA86355,
RO1 CA99385 and R21 CA114149. We thank Dr. Poguang
Wang for the HRMS analysis.

N.-h. Ho et al. / Tetrahedron 62 (2006) 578–585
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