BF2-Chelated Tetraarylazadipyrromethenes as NIR Fluorochromes

Publication Date (Web): June 10, 2010. Copyright © 2010 American ..... Yuan Ge , Donal F. O'Shea. Chemical Society Reviews 2016 45 (14), 3846-3864 ...
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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 nearinfrared (NIR) fluorochromes (>700 nm) for qualitative and quantitative assays (1). Their simplicity of use coupled with high sensitivity ensures a continual expansion of their applications 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 fluorochromes, and thus, intensive efforts have recently been put into their development (9). Our efforts have focused on the boron chelated tetraarylazadipyrromethene class 2, as they are relatively easily synthesized, amenable to structural modification, and exhibit excellent 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 group on one ring and a conjugation group on the other (Figure 2). 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 chargeneutral fluorochrome, which may limit the complications of noncovalent charge interactions of fluorophore and the conjugating biomolecule. Two different activated esters, N-hydroxysuccinimide and a sulfonated N-hydroxysulfosuccinimide sodium * Tel: +353-(0)1-7162425; E-mail: [email protected].

Figure 1. Structures of Cy5 and Cy5.5.

Figure 2. BF2 Chelated Tetraarylazadipyrromethenes.

salt, were investigated; the latter would provide enhanced aqueous solubility without introducing charge directly onto the fluorophore. 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 bisN-Boc amine, and the desired product monopolyether, monoamine 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

10.1021/bc100051p  2010 American Chemical Society Published on Web 06/10/2010

Communications Scheme 1. Fluorochrome Synthesis

Bioconjugate Chem., Vol. 21, No. 7, 2010 1131 Table 1. Spectroscopic Propertiesa entry compound λmaxabs nmb ε M-1 cm-1 λmaxemission nmc,d,e 1 2 3

4 5 7

688 691 691

95 000 85 500 74 500

714 714 714

Φff 0.23 0.20 0.22

a In ethanol. b Conc 5 × 10-6 M. c 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 spectroscopic 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 representative 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). Encouragingly, 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 pentaconjugates 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 fourcarbon 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-hydroxysuccinimide 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. 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 Nterminus 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

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

1132 Bioconjugate Chem., Vol. 21, No. 7, 2010

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.

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.

EXPERIMENTAL SECTION Synthesis of 4. Compound 3 (630 mg, 1.2 mmol), triethylene glycol monomethyl ether (294 µL, 1.8 mmol), N-Boc ethanolamine (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

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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). 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), Nhydroxysuccinimide (5.2 mg, 45 µmol) and DMAP (cat.) were dissolved in dry DCM (5 mL). 1-Ethyl-3-(3′-dimethylaminopropyl)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. Synthesis of 6b. Compound 5 (5 mg, 6.11 µmol), Nhydroxysulfosuccinimide 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.

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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 Boclysine (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 chromatography (silica, CH2Cl2/MeOH, 7:3) afforded 7 as green solid (4 mg, 63%). 1H 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.

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. Supporting Information Available: Conjugation experimental 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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