BF2-chelated tetraarylazadipyrromethenes as NIR fluorochromes

Article abstract of DOI:10.1021/bc100051p

The synthesis and properties of a new visible red/near-infrared fluorochrome for bioconjugation via activated ester groups is described. Representative amine conjugations with amino acid lysine and proteins lysozyme and trypsin were successfully accomplis

Full text of DOI:10.1021/bc100051p

1130
Bioconjugate Chem. 2010, 21, 1130–1133
BF2-Chelated Tetraarylazadipyrromethenes as NIR Fluorochromes
Mariusz Tasior and Donal F. O’Shea*
Centre for Synthesis and Chemical Biology, School of Chemistry and Chemical Biology, University College Dublin, Belfield,
Dublin 4, Ireland. Received February 1, 2010; Revised Manuscript Received May 7, 2010
The synthesis and properties of a new visible red/near-infrared fluorochrome for bioconjugation via activated
ester groups is described. Representative amine conjugations with amino acid lysine and proteins lysozyme and
trypsin were successfully accomplished.
In recent years, there has been growing interest in near-
infrared (NIR) fluorochromes (>700 nm) for qualitative and
quantitative assays (1). Their simplicity of use coupled with
high sensitivity ensures a continual expansion of their applica-
tions for in vitro and in vivo imaging (2). The inherent advantage
of NIR fluorochromes over those that absorb in the shorter blue
and green wavelengths lies in the dramatic reduction in
background autofluorescence which leads to greatly improved
sensitivity (3). In spite of these benefits, there are limited
compound classes which have the desired absorption and
emission properties, with cyanine dyes such as Cy5 1a and
Cy5.5 1b remaining the most widely utilized visible red/NIR
fluorescent probes for bioconjugation (Figure 1) (4-8). As such,
a distinct need exists for new classes of NIR emitting fluoro-
chromes, and thus, intensive efforts have recently been put into
their development (9).
Our efforts have focused on the boron chelated tetraarylaza-
dipyrromethene class 2, as they are relatively easily synthesized,
amenable to structural modification, and exhibit excellent
Figure 1. Structures of Cy5 and Cy5.5.
spectral properties (10, 11). For example, the tetraaryl analogue
2 has an absorption λmax at 696 nm and emission at 727 nm in
formulated aqueous solutions (Figure 2) (11). These promising
photophysical characteristics have encouraged the adaption of
this class to specific functions such as fluorescent sensors (12-15)
and photodynamic therapeutic agents (16-18). In this paper,
we present the synthesis and spectral properties of the first
fluorochromes, based on this fluorophore scaffold, capable of
amine conjugations. Our strategy was to exploit the bisphenol
analogue 3 as a key synthetic starting point (19). Synthetic
manipulation of the phenolic rings by differential oxygen
alkylation would be utilized to introduce a water-solubilizing
Figure 2. BF2 Chelated Tetraarylazadipyrromethenes.
group on one ring and a conjugation group on the other
(Figure 2).
salt, were investigated; the latter would provide enhanced
aqueous solubility without introducing charge directly onto the
fluorophore.
For this first study, our approach was to impart partial aqueous
solubility to the fluorophore by introducing a polyether on one
phenol ring and a spacer-conjugatable activated ester group on
the other. It was anticipated that a single polyether alone would
not be sufficient to impart complete water solubility but perhaps
sufficient to facilitate the conjugation reaction. While other
charged solubilizing groups are currently under investigation,
this approach offers the potential to produce an overall charge-
neutral fluorochrome, which may limit the complications of
noncovalent charge interactions of fluorophore and the conjugat-
ing biomolecule. Two different activated esters, N-hydroxysuc-
cinimide and a sulfonated N-hydroxysulfosuccinimide sodium
The synthetic challenge of functionalizing the equivalent
phenols of 3 with differing substituents was achieved utilizing
simultaneous Mitsunobu coupling of 3 with two different
alcohols. It was found that rather than reacting the phenols in
successive steps conditions were optimized to achieve this in a
single operation. As such, desymmetrization of 3 was achieved
in one step by condensation with the two different alcohols,
the polyether triethylene glycol monomethyl ether and N-Boc
protected ethanolamine under standard coupling conditions
(Scheme 1). This afforded a mixture of a bis-polyether, a bis-
N-Boc amine, and the desired product monopolyether, monoam-
ine substituted product 4. Chromatographic separation of 4 from
unwanted symmetrical coupling products was achievable due
to the large differences in retention factors on silica. This
* Tel: +353-(0)1-7162425; E-mail: donal.f.oshea@ucd.ie.
10.1021/bc100051p  2010 American Chemical Society
Published on Web 06/10/2010

Communications
Bioconjugate Chem., Vol. 21, No. 7, 2010 1131
Table 1. Spectroscopic Propertiesa
entry compound
maxabs nmb ε M-1 cm-1
Scheme 1. Fluorochrome Synthesis
λ
maxemission nmc d e Φff
,
,
λ
1
2
3
4
5
7
688
691
691
95 000
85 500
74 500
714
714
714
0.23
0.20
0.22
b
c
a In ethanol. Conc 5 × 10-6 M. Conc 5 × 10-7 M. d Excitation at
640 nm. e Slit widths 5 nm. f 2 used as standard.
substituted derivative 7 (Scheme 2). Encouragingly, the spec-
troscopic properties of 7 differed very little from those of its
precursor 5 (Table 1, Figure 3).
Next, the conjugation of 6a and b with the 14.4 kDa protein
lysozyme was examined. Lysozyme was chosen as a representa-
tive substrate, as it has six potentially reactive lysine residues
in addition to the N-terminal amine (20, 21). Conjugation of
lysozyme with 5 equiv of 6a was carried out in 1:1 water/DMSO
and 4:1 water/THF mixtures and 6b was coupled in pH 8.3
NaHCO3 buffered water. Coupling was effectively achieved in
all cases and confirmed by MALDI-TOF mass spectrometry and
HPLC analysis (Supporting Information, Figure S3). Encourag-
ingly, the MALDI analysis of the conjugates identified a
distribution of mono to pentaconjugates from both 6a and 6b
with each conjugate separated by an approximate mass of 800
amu (Figure 4 and Supporting Information Figure S3). As might
be expected, the coupling of 6b was superior to that of 6a with
the triconjugated protein being observed as the most abundant
from coupling with 5 equiv of fluorochrome (Figure 4). The
high loading achievable illustrates the effectiveness of the
coupling reaction and reactive nature of the fluorochromes.
The use of greater ratios of fluorochrome 6b (10 and 20 equiv)
to protein increased the loading levels with the tri to pentacon-
jugates being the most abundant (Supporting Information, Figure
S3). A further confirmatory coupling of 10 equiv of 6b with
approach proved superior to a stepwise introduction of the
different phenolic substituents due to poorer overall conversions
and more difficult purifications. The introduction of a four-
carbon spacer unit was achieved by the Boc-deprotection of 4
with TFA in CH2Cl2 and reaction with succinic anhydride to
yield the carboxylic acid 5. Subsequently, 5 was transformed
into the activated ester 6a or 6b by coupling with N-hydroxy-
succinimide or N-hydroxysulfosuccinimide respectively (Scheme
1, Supporting Information Figure S1). Fluorochrome 6a was a
bench-stable solid, and due to the higher reactivity of 6b, it
was directly used upon generation.
Figure 3. Normalized absorption (5 × 10-6 M) and emission (5 ×
10-7 M) spectra of 4 (red line), 5 (blue line), and 7 (green line) in
EtOH.
Scheme 2. Coupling of 6a and 6b with Nε-Boc-L-lysine
Spectroscopic analysis of 4 and 5 as representative constituent
building blocks of the fluorescent probe showed very little
variance in their properties with absorbance maxima at 688 and
691 nm, respectively, and fluorescence maxima at 714 nm for
both (Table 1, Figure 3). Both had extinction coefficients in
excess of 80 000 and good fluorescence quantum yields (Table
1). Their spectral features were also relatively insensitive to
solvent polarity (Supporting Information, Figure S2).
Activated succinimide esters typically react with the N-
terminus R-amino group and the amine of lysine constituent
amino acids. Illustrative coupling of 6a in organic and 6b in
aqueous solutions with Nε-Boc-L-lysine rapidly gave amino acid

1132 Bioconjugate Chem., Vol. 21, No. 7, 2010
Communications
removed under reduced pressure and dark green residue was
chromatographed (silica, AcOEt/cyclohexane gradient of 1:1 to
4:1). Middle green fraction was isolated (Rf ) 0.35, silica,
AcOEt/cyclohexane, 4:1), evaporated and rechromatographed
(silica, AcOEt/CH2Cl2, 1:4) to give 4 (321 mg, 33%) as a dark
green solid. Mp ) 51-53 °C. δH (500 MHz, CDCl3): 1.47 (s,
9H), 3.38 (s, 3H), 3.54-3.58 (m, 4H), 3.67 (t, J ) 4.6 Hz,
2H), 3.70 (t, J ) 4.6 Hz, 2H), 3.76 (t, J ) 4.6 Hz, 2H), 3.90 (t,
J ) 4.6 Hz, 2H), 4.11 (t, J ) 4.6 Hz, 2H), 4.22 (t, J ) 4.6 Hz,
2H), 4.99 (br s, 1H), 6.97-7.06 (m, 6H), 7.39-7.48 (m, 6H),
8.04-8.10 (m, 8H). δC (125 MHz, CDCl3): δ 161.2, 160.8,
158.3, 157.8, 155.8, 145.4, 143.2, 132.4, 131.6, 129.2, 128.5,
124.5, 124.2, 118.7, 118.5, 114.8, 114.6, 79.6, 71.9, 70.9, 70.7,
70.6, 69.6, 67.6, 67.3, 59.0, 40.0, 28.4. HRMS (ES) calcd for
C46H49N4O7NaBF2 [M + Na+]+: 841.3560, found 841.3571. IR
(KBr disk) cm-1: 1504, 1603, 1710. λabs (CHCl3, ε × 10-3
)
689 (87.1), 450 (17.3), 367 (11.9), 318 (26.0) nm. λemiss (CHCl3):
717 nm, Φ 0.36 (2 used as standard Φ ) 0.36).
Figure 4. MALDI-MS of lysozyme conjugated with 5 equiv of 6b.
Diconjugate, 15 904; triconjugate, 16 704; tetraconjugate, 17 503;
pentaconjugate, 18 305 m/z.
Synthesis of 5. Compound 4 (164 mg, 200 µmol) was
dissolved in CH2Cl2 (10 mL), TFA (1 mL) was slowly added
and the reaction mixture was stirred at r.t. for 2 h. Saturated
aqueous NaHCO3 was added and the resulting suspension
extracted with CH2Cl2 (2 × 30 mL). The combined organic
phases were washed with water, dried over Na2SO4, and
evaporated to dryness. The resulting green residue was dissolved
in dry THF (10 mL), treated with succinic anhydride (24 mg,
240 µmol) and DIPEA (70 µL, 400 mmol), and stirred at r.t.
for 3 h. Solvent was removed under reduced pressure and the
residue chromatographed (silica, MeOH/CH2Cl2, 2:8) to give 5
(93 mg, 57%) as a dark green solid. Mp ) 72-74 °C. δH (500
MHz, CDCl3): 2.45 (t, J ) 6.2 Hz, 2H), 2.65 (t, J ) 6.2 Hz,
2H), 3.36 (s, 3H), 3.52-3.55 (m, 2H), 3.56-3.61 (m, 2H),
3.62-3.68 (m, 4H), 3.70-3.74 (m, 2H), 3.85 (t, J ) 4.7 Hz,
2H), 4.01 (t, J ) 4.7 Hz, 2H), 4.16 (t, J ) 4.7 Hz, 2H), 6.45 (s,
1H), 6.87-7.01 (m, 6H), 7.34-7.44 (m, 6H), 7.92-8.08 (m,
8H). δC (100 MHz, CDCl3): 26.9, 58.9, 66.5 (br), 67.5, 69.4,
70.4, 70.5, 70.7, 71.8, 114.5, 114.7, 118.4, 118.6, 124.1, 124.2,
128.3, 128.4, 129.0, 129.14, 129.17, 131.5 (br), 131.6 (br),
132.1, 132.4, 142.56, 142.59, 142.7, 145.0, 145.2, 157.5, 157.8,
160.7, 161.0. HRMS (ESI) calcd for C45H45N4O8NaBF2 [M +
Na+]+: 841.3196, found 841.3215. IR (KBr disk) cm-1: 1509,
1602, 1655. λabs (CHCl3) 689 nm. λemiss (CHCl3): 717 nm.
Synthesis of 6a. Compound 5 (27 mg, 33 µmol), N-
hydroxysuccinimide (5.2 mg, 45 µmol) and DMAP (cat.) were
dissolved in dry DCM (5 mL). 1-Ethyl-3-(3′-dimethylamino-
propyl)carbodiimide (EDCI) (8.8 mg, 45 µmol) was added at 0
°C and the resulting mixture stirred at r.t. for 16 h. The organic
phase was washed with HCl (1 M, 1 mL), then water (2 mL),
and dried over Na2SO4. Solvent was removed under reduced
pressure to give 6a (28 mg, 99%) as a dark green solid. δH
(500 MHz, CDCl3): 2.54 (t, J ) 7 Hz, 2H), 2.57 (s, 4H), 2.91
(t, J ) 7 Hz, 2H), 3.30 (s, 3H), 3.46-3.49 (m, 2H), 3.56-3.64
(m, 6H), 3.65-3.69 (m, 2H), 3.81 (t, J ) 5 Hz, 2H), 4.01 (t, J
) 5 Hz, 2H), 4.13 (t, J ) 5 Hz, 2H), 6.18 (t, J ) 5.6 Hz, 1H),
6.87-6.97 (m, 6H), 7.31-7.39 (m, 6H), 7.95-8.00 (m, 8H).
HRMS (ESI) calcd for C49H48BF2N5O10Na [M + Na+]+:
938.3360, found 938.3886. IR (KBr disk) cm-1: 1603, 3054.
Figure 5. Normalized absorption (left) and emission (right) spectra of
lysozyme (red line) and trypsin (green line) in aqueous PBS.
the 23.3 kDa protein trypsin in PBS at pH 7.5 gave a distribution
of mono and difunctionalized enzyme (Supporting Information,
Figure S4) (22).
Spectral analysis of the lysozyme and trypsin conjugates show
that the attractive spectral features of this chromophore such as
sharp absorption and emission bands are retained (Figure 5).
Importantly, the emission maxima of both were above 720 nm.
In summary, we have outlined a short synthesis to a new
NIR fluorchrome and illustrated its potential for effective amine
conjugations with amino acids and proteins. Their application
to in vivo use is currently under investigation and will be
reported upon in due course. The development of further
fluorochrome derivatives by combining different solubilizing
groups (23) and conjugation methods is also ongoing.
Synthesis of 6b. Compound 5 (5 mg, 6.11 µmol), N-
hydroxysulfosuccinimide sodium salt (1.4 mg, 6.4 µmol),
1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride
(1.2 mg, 6.2 µmol), and DMAP (cat.) were dissolved in dry in
DMSO (200 µL) were stirred at r.t. for 16 h, under nitrogen.
Monitoring the reaction by reverse-phase HPLC (C-18 column
eluting with acetonitrile water 7:3) complete consumption of
starting material. The solution was used directly for conjugation
experiments. HRMS (ESI) calcd for C49H47BF2N5O13S [M -
H+]-: 994.2952, found 994.2911.
EXPERIMENTAL SECTION
Synthesis of 4. Compound 3 (630 mg, 1.2 mmol), triethylene
glycol monomethyl ether (294 µL, 1.8 mmol), N-Boc ethanol-
amine (285 µL, 1.8 mmol), and PPh3 (1.257 g, 4 mmol) were
dissolved in dry THF (100 mL). DIAD (969 µL, 4 mmol) in
dry THF (5 mL) was slowly (20 min) added to the solution
and the reaction mixture stirred at r.t. for 16 h. Solvent was

Communications
Bioconjugate Chem., Vol. 21, No. 7, 2010 1133
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Conjugation with Nε-Boc-L-lysine, Synthesis of 7. Activated
ester 6a (11 mg, 12 µmol), Boc-lysine (6 mg, 24 µmol), and
DIEA (4.3 µL, 24 µmol) were suspended in dry acetonitrile (5
mL) and stirred for 1 h under nitrogen. Solvent was removed,
and the residue was chromatographed (silica, CH2Cl2/MeOH,
9:1 then 8:2, 7:3) affording 7 (9.5 mg, 76%) as green solid. A
solution of activated ester 6b (6.0 µmol) was added to Boc-
lysine (3 mg, 12 µmol) solution in 0.1 M NaHCO3 (1 mL, pH
) 8.3). After 15 min, the resulting mixture was acidified with
2 M HCl and extracted with AcOEt. Subsequent chromatogra-
phy (silica, CH2Cl2/MeOH, 7:3) afforded 7 as green solid (4
1
mg, 63%). H NMR (500 MHz, CDCl3) 1.26-1.46 (m, 4H),
1.39 (s, 9H), 1.55-1.65 (m, 2H), 1.70-1.80 (m, 2H), 2.25-240
(m, 2H), 2.46-2.54 (m, 2H), 3.12-3.18 (m, 2H), 3.34 (s, 3H),
3.52-3.55 (m, 2H), 3.50-3.53 (m, 2H), 3.60-3.66 (m, 4H),
3.68-3.71 (m, 2H), 3.82 (t, 2H), 3.95-4.02 (m, 3H), 4.10-4.16
(m, 2H), 5.83 (s, 1H), 6.83-6.97 (m, 6H), 7.05 (s, 1H), 7.22
(s, 1H), 7.29-7.40 (m, 6H), 7.90-8.05 (m, 8H). 13C NMR (100
MHz, CDCl3) δ: 173.2, 172.8, 161.1, 160.8, 157.8, 156.1, 145.2,
145.1, 142.9, 118.6, 114.7, 114.6, 71.8, 70.8, 70.5, 70.4, 69.5,
67.5, 66.6, 59.0, 28.5. HRMS (ESI) calcd for C56H65BF2N6O11Na
[M + Na+]+: 1069.4670, found 1069.5371. IR (KBr disk) cm-1:
1475, 1506, 1603, 1657, 3055.
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sensing properties of dimethlyamino-substituted BF2-azadipyr-
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ACKNOWLEDGMENT
We would like to thank Science Foundation Ireland and The
Irish Research Council for Science, Engineering and Technology
for financial support. Thanks to Dr. F. Paradisi for helpful
discussions and Marco Grossi for technical assistance.
(16) McDonnell, S. O., Hall, M. J., Allen, L. T., Byrne, A.,
Gallagher, W. M., and O’Shea, D. F. (2005) Supramolecular
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cytotoxic potential and influence of hypoxia. Br. J. Cancer 92,
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Supporting Information Available: Conjugation experi-
mental procedures, UV-visible and fluorescence spectra, NMR
spectra, MALDI-TOF data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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BC100051P