Asymmetrical, Water-Soluble Phthalocyanine Dyes for Covalent

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Bioconjugate Chem. 2002, 13, 1244−1252

Asymmetrical, Water-Soluble Phthalocyanine Dyes for Covalent Labeling of Oligonucleotides Robert P. Hammer,* Clyde V. Owens, Seok-Hwan Hwang, Christie M. Sayes, and Steven A. Soper* Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804. Received November 30, 2001; Revised Manuscript Received July 31, 2002

Two new water-soluble, porphyrazine (Pz) dyes containing an isothiocyanate function for covalent linking have each been prepared by cross condensation of two different aromatic dinitriles, one containing carboxylates for solubilizing purposes and the other containing a nitro group for conversion into the labeling function. The initial mononitrotricarboxylato Pzs have been purified to homogeneity from the mixture of Pz congeners formed in the condensation reaction by anion exchange chromatography. The phthalocyanine dye 1 has an absorption maxima at 683 nm while the trinaphthoporphyrazine dye 2 has an absorption maxima at 755 nm, due to the increased size of the aromatic system. Both dyes were successfully conjugated to oligonucleotide primers, showing their potential for use in near-infrared-based DNA diagnostic applications.

INTRODUCTION

Most bioanalytical applications using fluorescence as the readout mode for detection typically use labeling reagents that fluoresce in the visible region (400-650 nm) of the electromagnetic spectrum (1, 2). Unfortunately, the spectroscopic range utilized by these fluorophores is susceptible to biological interferences from the sample matrix, which inevitably limits the sensitivity of the measurement. An attractive alternative is the use of near-IR fluorescence (650-1000 nm), which has recently been demonstrated to be a viable detection strategy in many bioanalytical assays, such as liquid chromatography (3-5), free solution capillary electrophoresis (6-10), and capillary gel electrophoresis for DNA sequencing and fragment analysis (11, 12). Near-IR fluorescence offers reduced background matrix interferences compared to UV or visible fluorescence resulting primarily from the fact that a limited number of components show intrinsic fluorescence in this region of the electromagnetic spectrum. In addition, lower scattering levels are also observed in the near-IR because of the λ-4 dependence of the Raman cross section on the excitation wavelength. Indeed, we have demonstrated improved limits of detection in the near-IR compared to visible excitation for oligonucleotides separated using capillary gel electrophoresis (11). Also, near-IR fluorescence allows the use of inexpensive semiconductor devices, such as diode lasers and detectors, significantly simplifying the readout hardware (13). Recently, reports on near-IR water-soluble dyes that can covalently and noncovalently label biomolecules such as amino acids, proteins and oligonucleotides, have been discussed (14-23). The dyes typically used are the tricarbocyanines, which consist of heteroaromatic structures linked by a polymethine chain containing conjugated carbon/carbon double bonds. These dyes possess large molar absorptivities (>105 cm-1 M-1) and display favorable solubility in aqueous solvents provided by the negatively charged sulfopropyl groups attached to the * To whom correspondence should be addressed. E-mail: [email protected]; [email protected].

heteroaromatic rings (24). The major limitations associated with these dyes are their poor chemical and photochemical stabilities and low quantum yields in aqueous media, therefore limiting their utility in ultrasensitive applications (25, 26). An attractive alternative to the cyanine-based nearIR fluorophores is the phthalocyanine [Pc; also know as tetraazaporphyrins or porphyrazines (Pz)] family of compounds. The Pc dyes are chemically and photochemically robust and have large extinction coefficients (>105 cm-1 M-1) as well as favorable quantum yields (27). Additionally, absorbance and fluorescence maxima can easily be altered by varying the substituents around the ring or by changing the metal center (Zn, Sn Al, Si, etc.). For example, annulation of benzene rings onto the Pc core will produce the naphthalocyanine (Npc) dyes, which have λmax red-shifted by 50-100 nm compared to the Pc dyes (28, 29). Unfortunately, the Pc and Npc dyes are very hydrophobic and essentially insoluble in water. Functionalization of the periphery of the dye with charged groups (SO3-, CO2-, PO32-, etc.) or attachment of a polar functionality to the metal center (PEG-OSi) does provide water-soluble Pc dyes (30). General routes to water-soluble Pc dyes that also contain a labeling function have not been reported to-date because of the need for preparing asymmetrical analogues, which can be very difficult to purify due to statistical formation of isomers during ring assembly. One way to approach the preparation of labeling watersoluble Pc is to put the reactive group (L) in one “quadrant” of the macrocycle (see Figure 1) and then use one or more of the remaining quadrants to incorporate water-solubilizing groups (S). Pc and Npc dyes are generally prepared by cyclo-tetramerization of aromatic 1,2-dinitriles (phthalonitriles or naphthalonitriles) using either Lewis base or acid conditions (31, 32). The simplest route to prepare nonsymmetrically substituted Pc dyes as discussed above would use one phthalonitrile that contains the (latent) labeling group L and another containing the solubilizing group S in a molar ratio of 1:3. This mixed “condensation” route is commonly used to prepare asymmetric Pc and Npc dyes, but as yet has not been applied to dyes containing a mixture of labeling

10.1021/bc0155869 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/01/2002

Phthalocyanine Dyes for Oligonucleotide Labeling

Bioconjugate Chem., Vol. 13, No. 6, 2002 1245

Figure 1. (A) Preparation of labeling, water-soluble Pc dye placing the reactive group (L) in one “quadrant” of the macrocycle with one or more of the remaining quadrants used to incorporate water-solubilizing groups (S). (B) Structure of water-soluble Pc (1) and pseudo-Npc (2) dyes containing reactive isothiocyanates for labeling.

and water-solubilizing groups we desire. Herein, we report the synthesis, spectroscopic properties, and labeling reactions of two new water-soluble near-IR dyes, Pc 1 and pseudo-Npc benzotrinaphthoporphyrazine (Tnpz) 2 (Figure 1), both of which contain a reactive isothiocyanate functional group used for covalent labeling of targets containing primary amines. EXPERIMENTAL SECTION

Reagents. 2,3-Naphthalenedicarbonitrile, 3-nitropthalonitrile, 4-nitropthalonitrile, 1,1′-thiocarbonyldi-2(1H)pyridone, palladium 10% on activated carbon, sodium thiosulfate, fumaronitrile, sodium iodide, carbon tetrachloride, 3,4-dimethylbenzoic acid, and zinc acetate dihydrate were obtained from Aldrich Chemical Co. (Milwaukee, WI). Hydrochloric acid and sulfuric acid were obtained from Fisher Scientific (Houston, TX). Potassium nitrate, sodium carbonate, sodium hydroxide, and urea were obtained from Sigma Chemical Co (St. Louis, MO). All chemicals were used as received. The dinitrile derivatives 3 and 4 used for final ring assembly were synthesized according to Kliesch et al. (33). Chromatographic Purification and Spectroscopic Characterization. The crude reaction mixtures containing the water-soluble macrocycles were assayed by a Rainin HPLC system using a Hamilton PRP-1 analytical column (0.46 × 15 cm; Hamilton, Reno, NV) using a flow rate of 2.0 mL/min and a gradient consisting of 0.1 M triethylammonium acetate/20 mM sodium phosphate buffer (pH ) 10.1) and tetrahydrofuran (THF). The absorbance detector (Shimadzu, Columbia, MD) was set at either 680 or 780 nm to monitor elution of the nearIR dyes. For typical analytical analyses, 1 mg of the crude dye mixture was dissolved in 2 mL of 20 mM sodium phosphate buffer. A 20 µL aliquot was injected into the

column, which had been equilibrated with 100% 20 mM sodium phosphate buffer. Elution was carried out by a stepwise gradient from 0 to 100% THF over a period of 25 min. Large-scale separations were carried out on a 2.15 × 25 cm Hamilton PRP-1 preparatory column (Hamilton, Reno, NV) packed with a polymeric reverse phase (C18) that was equilibrated for 10 min with 20 mM sodium phosphate buffer (pH ) 10.1). The water-soluble dye mixture (less than 1 g) was dissolved in 10 mL of buffer and injected into the preparatory column. The HPLC stepwise gradient used for analytical separations was slightly modified for preparatory work by extending the gradient step times to compensate for the increased flow rate (5 mL min-1). Dyes were recovered from collected fractions by removing the solvent (HPLC mobile phase) using a rotary evaporator. The absorption spectra were acquired on a PerkinElmer Lambda 3B UV/Vis spectrophotometer. The fluorescence spectra were obtained using a SPEX Fluorolog spectrofluorometer (SPEX, Edison, NJ) equipped with a 75 W xenon lamp. The emission gratings were blazed for 750 nm and the photomultiplier tube was a Hammamatsu R636 red-sensitive tube (Hammamatsu Corporation, Bridgewater, NJ). Synthesis of Dyes. [1-Nitro-9(10),16(17),23(24)-tris(4-carboxyphenoxy)phthalocyanato]zinc(II) (5). 4-(3,4-Dicyanophenoxy)benzoic acid (3; 0.425 g, 1.60 mmol) and 3-nitropthalonitrile (0.135 g; 0.81 mmol) were combined with 0.100 g of zinc acetate dihydrate. The reaction mixture was ground together into a fine homogeneous powder and heated at 200 °C for 20 min. Reactions with the metal salts at 200-210 °C proceeded rapidly. Further heating decreased the yield of the desired metal complex as determined by the loss in

1246 Bioconjugate Chem., Vol. 13, No. 6, 2002

Hammer et al.

Figure 2. (A) Structures of nitro-Pc congeners (Va, 5, Vb, Vb′, Vc, Vd) obtained from the reaction of 3-nitrophthalonitrile with 4-(3,4-dicyanophenoxy)benzoic acid (3). (B) Structures of the nitro-Tnpz congeners (VIIa, 7, VIIb, VIIb′, VIIc, VIId) obtained from the reaction of 4-nitrophthalonitrile with 6,7-dicyanonaphthalenecarboxylic acid (4).

absorption in the near-IR. Lowering the temperature slowed the reaction, but did not increase product yield. The dark green precipitate containing desired Pc 5 and the other Pc congeners Va-d was dispersed in boiling 1 M HCl, cooled to room temperature, and collected. The precipitate was dissolved in 1 M NaOH and filtered to remove any insoluble impurities (e.g., tetranitrocongener Vd). The filtrate was neutralized with 1 M HCl and

rotary-evaporated under vacuum to yield a green solid. This product was a mixture of at least four components, which varied in the number of water soluble groups (S) or functional labeling components (L) present in the dye (Figures 1 and 2A). The chromatographic purification/isolation involved injecting 1.0 mg of the water-soluble Pc dye into a polymeric C18 column equilibrated with 0.1 M TEAA/20


mM sodium phosphate buffer (pH ) 10.1). The components eluted in the following order: tetracarboxylate, tricarboxylate, dicarboxylate, and monocarboxylate, compounds Va, 5, Vb/b′, Vc, respectively. They were characterized by their electronic absorbance spectra and mass spectra (data not shown). Tetracarboxylate (4CO2-) Pc Va: HPLC tr ) 9.22 min; λmax (DMSO) ) 677 nm. Tricarboxylate/mononitro (3 CO2-/1 NO2) Pc 5: HPLC tr ) 11.11 min; λmax (DMSO) ) 672 nm; MALDI (m/z) 1028 (M - H)-. Dinitro/dicarboxylate (2 CO2-/2 NO2) Pc (Vb/ b′): HPLC tr ) 12.13 min; MALDI (m/z) 938 (M - H)-. Trinitro/monocarboxylate (1 CO2-/3 NO2) Pc (Vc): HPLC tr ) 17.07 min; MALDI (m/z) 847 (M - H)-. The tricarboxylate/mononitro compound 5 is the preferred compound for labeling because of its greater water solubility (3 CO2-) and its single nitro group. Crude yield is typically 20% of theory as expected for the isomeric mixture obtained from the reaction. Elemental analysis shows correct C/N ratio for this product: calculated 4.99; found 5.15. MALDI-MS calculated for C53H27N9O11Zn ) 1029, found m/z 1028 (M - H)-. (λmax (DMSO) ) 672 nm). [1-Amino-9(10),16(17),23(24)-tris(4-carboxyphenoxy)phthalocyanato]zinc(II) (6) A large pressure flask containing mononitro-Pc 5 (500 mg; 0.48 mmol) in water (20 mL) and 30 mg of 10% palladium on charcoal was pressurized to 40 psi of hydrogen and was shaken for 3 h at room temperature. The reaction product was filtered to remove the catalyst. The filtrate was concentrated in vacuo and the dark green precipitate dried under vacuum. Crude yield 275 mg (54%). Elemental analysis shows correct C/N ratio for this product: calculated 5.71; found 5.75. MALDI-MS calculated for C53H29N9O9Zn ) 999, found m/z 1000 (M + H). (λmax (DMSO) ) 702 nm). [1-(Isothiocyanato)-9(10),16(17),23(24)-tris(4-carboxyphenoxy)phthalocyanato]zinc(II) (1). Amino-Pc 6 (350 mg; 0.35 mmol) was added to a solution of 1,1′-thiocarbonyldi-2(1H)-pyridone (250 mg, 1.08 mmol) in DMF (3 mL) and stirred for 2 h. The DMF was removed in vacuo and the dark green product was taken up in HPLC grade H2O and filtered through a 0.45 µM filter to remove insoluble 1,1′-thiocarbonyldi-2(1H)-pyridone products. The filtrate was dried under vacuum to yield 86 mg (27%). Elemental analysis for C54H27N9O9SZn‚3H2O, Calculated (Found): C, 59.33 (59.58); H 3.35 (2.99); N, 11.32 (11.18). MALDI-MS calculated for C54H27N9O9SZn ) 1041, found m/z 1040 (M - H)-. (λmax (DMSO) ) 686 nm). [2-Nitro-10(11),19(20),28(29)-tricarboxybenzo[b]trinaphtho[2,3-g:2′,3′-l:2′′,3′′-q]porphyrazinato]zinc(II) (7). In a condensation reaction, 6,7-dicyano-2-naphthalenecarboxylic acid (4) (0.474 g; 1.69 mmol) and 4-nitrophthalonitrile (0.135 g; 0.78 mmol) were combined with 0.100 g of zinc acetate dihydrate (see Scheme 2). The reaction mixture was ground together into a fine homogeneous powder and heated to 200 °C for 20 min. The resulting solid black cake (Compound 7, plus other congeners, VIIa, VIIb, VIIb′, VIIc, VIId; Figure 2B) was monitored by UV absorbance for the appearance of a near-IR peak. The dark green precipitate was dispersed in boiling 1 M HCl, cooled to room temperature and collected. The precipitate was dissolved in 1 M NaOH and filtered to remove any insoluble impurities (e.g., tetranitrocongener VIId). The filtrate was then neutralized with 1 M HCl and rotary-evaporated under vacuum to yield a green solid. This product was a mixture of at least four components, which varied in the number of water soluble groups (S) or functional labeling components (L) present in the dye (Figure 1 and Figure 2B). The chromatographic purification/isolation involved injecting 1.0 mg of the water-soluble dye into a polymeric


C18 column equilibrated with 0.1 M TEAA/20 mM sodium phosphate buffer (pH ) 10.1). The components eluted in the following order: tetracarboxylate, tricarboxylate, dicarboxylate, and monocarboxylate, compounds VIIa, 7, VIIb/b′, VIIc, respectively (see Figure 2B) and were characterized by their electronic absorbance spectra and mass spectra. Tetracarboxylate (4 CO2-) Npc VIIa: HPLC tr ) 5.68 min; λmax (DMSO) ) 768 nm; MALDI (m/z) 952 (M - H)-. Tricarboxylate/mononitro (3 CO2-/1 NO2) Npc (7): HPLC tr ) 7.08 min; λmax (DMSO) ) 765 nm, 727 nm; MALDI (m/z) 905 (M - H)-. Dinitro/dicarboxylate (2 CO2-/2 NO2), Npc VIIb/b′: HPLC tr ) 16.03 min; λmax (DMSO) ) 720 nm, 689 nm; MALDI (m/z) 856 (M - H)-. Trinitro/monocarboxylate (1 CO2-/3 NO2), Npc VIIc: HPLC tr ) 18.82 min; λmax (DMSO) ) 684 nm; MALDI (m/z) 806 (M - H)-. The tricarboxylate/mononitro compound, 7, is a preferred compound for labeling because of its high number of water soluble groups (3 CO2-) and its single nitro group, which can be converted to a labeling function. The yield of 7 is typically ∼25% of theory as is expected for the mixture of compounds obtained in the initial condensation. IR (KBr) ν ) 1645 cm-1 (CdO), 1525 cm-1 (NO2), 1350 cm-1(NO2); MALDI-MS calculated for C47H21N9O8Zn ) 904, found m/z 905 (M + H)+. (λmax (DMSO) ) 765 nm, 727 nm). [2-Amino-10(11),19(20),28(29)-tricarboxybenzo[b]trinaphtho[2,3-g:2′,3′-l:2“,3”-q]porphyrazinato]zinc(II) (8). In a large pressure flask, the nitro-Tnpz 7 (0.40 g; 0.44 mmol) was dissolved in water (20 mL) and reduced as described for amino-Pc 6. Crude yield 210 mg (54%). Elemental analysis shows correct C/N ratio for this product: calculated 4.56; found 4.47. MALDI-MS calculated for C47H23N9O6Zn ) 873, found m/z 874 (M + H)+. (λmax (DMSO) ) 750 nm). [2-Isothiocyanato-10(11),19(20),28(29)-tricarboxy-benzo[b]trinaphtho[2,3-g:2′,3′-l:2“,3”-q]porphyrazinato]zinc(II) (2). Amino-Tnpz 8 (300 mg; 0.34 mmol) was added to a solution of 1,1′-thiocarbonyldi-2(1H)-pyridone (250 mg, 1.08 mmol) in DMF (3 mL) and stirred for 2 h (Scheme 2). The DMF was removed in vacuo and the dark green product was taken up in HPLC grade H2O and filtered through a 0.45 µM filter to remove insoluble 1,1′thiocarbonyldi-2(1H)-pyridone products. The yield 60 mg (28%). Elemental analysis for C48H21N9SO6Zn‚7H2O, Calculated (Found): C, 51.8, (51.76); H, 3.2 (2.86); N, 11.3 (11.29). MALDI-MS calculated for C48H21N9SO6Zn ) 916, found m/z 917 (M + H)+, (λmax (DMSO) ) 756 nm, 723 nm). Labeling and Purification of DNA Oligonucleotides with Pc and Tnpz Functionalized Dyes. An M13 universal sequencing primer (17mer) containing a 6-carbon alkyl linker terminated with an amino group on the 5′-end was derivatized with the dyes according to procedures outlined by Li-COR (34). Briefly, 50 nmol of DNA was added to 25 µL of 40 mM carbonate buffer (pH ) 9.1), 25 µL of 2 mM EDTA, and 100 µL of the appropriate dye (2.5 mM) dissolved in DMF. After the reaction was allowed to proceed at room temperature for approximately 4 h, 10 µL of 3 M sodium carbonate and 480 µL of cold EtOH were added to the reaction mixture. The solution was centrifuged for 20 min at 4 °C. The supernatant was discarded and the cold ethanol precipitation step repeated again. The DNA/dye conjugate was then dried and 200 µL of water added to the pellet. The DNA/dye conjugate was finally purified using preparatory HPLC under the following conditions: Column, C18; flow rate, 1.7 mL/min; mobile phase A, 0.1 M triethylammonium acetate, 4% acetonitrile, 96% water; mobile


Hammer et al.

Scheme 1. Synthesis of [1-(Isothiocyanato)-9(10),16(17),23(24)-tris(4-carboxyphenoxy)phthalocyanato]zinc(II) (1)

Scheme 2. Synthesis of [2-Isothiocyanato-10(11),19(20),28(29)-tricarboxy-benzo[b]trinaphtho[2,3-g:2′,3′-l:2“,3”-q]porphyrazinato]zinc(II) (2)

phase B, 0.1 M triethylammonium acetate, 80% acetonitrile, 20% water. The gradient conditions were 90/10 to 55/45 A/B over 5 min, 55/45 to 0/100 A/B over 20 min, hold 0/100 A/B for 5 min. The collected fractions were pooled and taken to dryness using a centrifugal evaporator and stored in the dark at 0 °C until required for use. RESULTS AND DISCUSSION

Several different strategies were considered for preparing the water-soluble Pc or Npc dyes containing a labeling moiety. One issue that predicated the selected scheme was the identity of the labeling group on the chromophore. In our case, we selected a labeling group to target primary amines, either an isothiocyanate or succinimide ester. Neither of these reactive groups [Figure 1A, L ) NCS, or L ) CO(OSu)] would be expected to survive Pc or Npc ring assembly even under the mildest basic or acidic conditions. Therefore, it was decided that the reactive group “L” (Figure 1A) would have to be incorporated following ring assembly from a precursor that was stable to these reaction conditions. We selected isothiocyanates as the final amine-labeling group, since they can be quantitatively prepared from amines using thiophosgene (or synthetic equivalents). However, in our hands the condensation of aminophthalonitriles (L ) NH2) to form the final ring structure was not successful (data not shown). Thus, we chose the nitrophthalonitriles (L ) NO2) as masked forms of aminophthalonitriles as these were found to be stable under our ring assembly conditions (vide infra, Schemes 1 and 2). Sulfonates are the most commonly used water-solubilizing groups and

can be incorporated into the phthalonitrile precursor or added postsynthetically by aromatic sulfonation. In our hands, postassembly sulfonation of nitro-substituted Pc or Npc analogues was unsuccessful due to decomposition of the macrocycle from harsh acidic conditions (14). Coassembly of nitrophthalonitriles and sulfonaphthalonitriles did produce near-IR products, but separation of the congeners of these reactions proved impossible either before or after the reduction of the nitro group (data not shown). We chose to use Zn as the metal center for the Pc and pseudo-Npc dyes as this was both synthetically convenient and provided highly fluorescent dyes (28). Metals such as Cu or Co incorporated into the ring center yielded chromophores with only weak fluorescence. Coassembly of the nitrophthalonitrile with the carboxynaphthalonitrile in the presence of zinc acetate provided the most successful synthetic route for preparing our asymmetrical macrocycles (Schemes 1 and 2). In general, the condensation of two different phthalonitriles, A and B, with an appropriate metal should produce a mixture of Pcs containing a statistical mixture of products: four A or B units (AAAA or BBBB), three A units and one B unit (AAAB), three B units and one A unit (BBBA), or two A units and two B units (two isomers: ABAB and AABB). To skew the product mixture toward the desired product having three watersolubilizing groups and one conjugatable group, we used a 3:1 stoichiometric ratio of the two phthalonitrile coupling partners. However, solventless condensation of 3 equiv of 4-(3,4-dicyanophenoxy)benzoic acid (3) and



Figure 4. UV absorbance profiles of crude HPLC fractions containing Tnpz congeners (VIIa, 7, VIIb/b′, VIIc; see Figure 2A) were evaluated after collection from the HPLC (concentrations are unknown).

Figure 3. (A) HPLC chromatogram of the crude mixture of Pc congers Va, 5, Vb, Vb′ and Vc. Note the uncharged tetranitroPz congener Vd is removed during the workup of the crude reaction as it is not at all soluble in water. HPLC conditions: Hamilton PRP-1 analytical column (4.6 × 15 cm; flow rate 2 mL/min) and a gradient of 0.1 M triethylammonium acetate/20 mM sodium phosphate buffer (pH ) 10.1) and THF (0-100% THF) over 25 min, detection 680 nm. (B) HPLC chromatogram of the crude mixture of Tnpz congeners (VIIa, 7, VIIb/b′, VIIc). Note the uncharged tetranitro-Tnpz congener VIId is removed during the workup of the crude reaction as it is not at all soluble in water. HPLC conditions: same as above except detection at 700 nm.

with 1 equiv of 3-nitrophthalonitrile in the presence of zinc acetate still produced a mixture of at least four congeners (as mixtures of constitutional/positional isomers) as shown by HPLC and MS analysis (see Figure 3A). The organic soluble self-condensation product of 3-nitrophthalocyanine was likely removed during extraction of the product. Similar condensation of 3 equiv of 6,7-dicyano-2-naphthalenecarboxylic with 1 equiv of 4-nitrophthalonitrile again produced four major congeners as seen in the chromatograms shown in Figure 3B. To aid in characterization of the mixtures, the individual precursors, 6,7-dicyano-2-naphthalenecarboxylic acid precursor and nitrophthalonitrile, were reacted separately with zinc acetate and monitored by UV absorbance (see Figure 4). The water-soluble tetracarboxylate zinc Npc VIIa was determined to have an absorption maximum at 768 nm, which corresponded to the absorption maximum of the first fraction eluting from the HPLC (tr ) 5.68 min, Figure 3B). The more hydrophobic trinitro zinc Pc VIIc was determined to have an absorbance maximum at 684 nm, which also corresponded to the fourth eluting component (tr ) 18.82 min) shown in the chromatogram of Figure 3B. Following HPLC purification, the isolated product was brought to dryness under reduced pressure and the

Figure 5. Reverse-phase HPLC chromatograms of (A) Pclabeled M13 primer (9) and (B) Tnpz-labeled M13 primer (10). Column: Reverse Phase-C18 50 × 4.6 mm i.d.; mobile phase: 0.1 M TEAA, pH ) 7.0 linear gradient 4 to 80% acetonitrile in 20 min; flow rate: 1.7 mL/min. UV absorbance detection at 254 nm. See Experimental Section for gradient conditions.

water-soluble nitro compound (5 or 7) was reduced to the amino group (6 or 8) by hydrogenation over palladium/ charcoal (Schemes 1 and 2). We originally attempted to reduce the nitro group using Sn(II) chloride in hydrochloric acid. However, the reaction was unsuccessful due to decomposition of the macrocycle and possible exchange of the zinc metal with tin. For conversion of the amino group to the reactive isothiocyanate (1 or 2), 1,1′thiocarbonyldi-2(1H)-pyridone was preferred over thiophosgene because of its ease of handling and higher stability, which resulted in higher product yields. After purification by reverse-phase chromatography, the analysis and structural characterization by mass


Hammer et al.

Scheme 3. Conjugation of Pc Dyes to an Oligonucleotide

spectrometry (MALDI-MS, negative mode) of the substituted derivatives exhibited the characteristic pseudomolecular ion for Pc 1 and Tnpz 2. The infrared spectra demonstrated very strong lines at 1645 cm-1 (CdO), 1525 cm1 (NO2), 1350 cm-1 (NO2) for compounds 5 and 7. Unfortunately, the infrared spectra of the amino (6, 8) and isothiocyanate (1, 2) groups in the target dyes were obscured by other absorbances. The H1 NMR spectrum provided little information due to substantial aggregation of the samples in the solvents required for NMR, which produced significant broadening in the signal peaks. The HPLC chromatograms shown in Figure 5 depict the conjugates formed following reaction of an aminemodified DNA 17mer oligonucleotide with the watersoluble Pc (1) or Tnpz (2) dyes containing the reactive isothiocyanate functional group (Scheme 3) to make DNA-dye conjugates 9 (Figure 5A) and 10 (Figure 5B), respectively. Both dyes demonstrated reasonable conjugation efficiency toward the amine-containing oligonucleotide. In the chromatogram of the Pc 1 conjugation mixture, it is apparent that the conjugation efficiency was slightly better compared to the Tnpz 2 because of the disappearance of unbound DNA in the conjugation reaction. The extinction coefficients for both dyes at 260 nm were found to be small, thereby producing little or no detectable absorbance signal in the chromatogram from free dye. We are not sure of the reason for the slightly poorer conjugation of the Tnpz dye, but it may be due to

greater aggregation of the more hydrophobic Tnpz in the conjugation solvent, which lowered its availability for conjugation to the amine of the DNA. In Figure 6A is shown the absorbance spectrum of the Tnpz dye 2 in methanol/DMF, which displayed two absorption bands at 755 and 725 nm, while the spectrum for this dye in an aqueous buffer solution showed significant broadening, indicative of extensive groundstate aggregation. In the case of the dye-oligonucleotide conjugate, the absorption maximum was found to be 735 nm, with the peak at ∼755 nm appearing as only a weak shoulder on the band at 735 nm. The fluorescence emission profile (see Figure 6B) for the Tnpz dye 2 displayed a maximum at ∼ 765 nm, while the oligonucleotide conjugate 10 showed a 15 nm red shift (λem ) 780 nm). Using procedures outlined in our previous work for determining the quantum yields of near-IR dyes (35), the quantum yield of the conjugate was found to be 0.20 with an extinction coefficient of 1.82 × 105 cm-1 M-1. The dual absorbance bands observed in the electronic spectra of pseudo-Npc 2 most likely resulted from the asymmetrical character of this type of compound. When one of the four naphtho groups along the macrocycle is replaced by a benzo group, it would be expected that only one major absorption band be observed. The presence of a symmetric, single absorption band would suggest that both the molecular asymmetry and the substituents exert a synergistic effect on the electronic properties of the



Figure 6. Absorbance (A) and emission (B) spectra of: Tnpz dye 2 in 40 mM borate buffer (- - -); Tnpz dye 2 in 70:30 methanol/ DMF (s), and Tnpz-oligonucleotide conjugate 10 in 40 mM borate buffer/30% DMF (4). Absorbance (C) and emission (D) spectra of: Pc dye 1 in 40 mM borate buffer (- - -); Pc dye 1 in 70:30 methanol/DMF (s); and Pc-oligonucleotide conjugate 9 in 40 mM borate buffer/30% DMF (4). The dye concentration for all the experiments was ∼2.5 × 10-6 M.

macrocycle derivatives. Instead, we observed two bands in the naphthalocyanine absorption region in organic solvent, which would be expected to be relatively free of aggregation. This observation is in agreement with the 2-fold orbital degeneracy of the excited electronic state of the metallomacrocycle when the group symmetry changes from pseudo-D4h (as in Zn-Pc 1) to pseudo-C2v (as in Tnpz 2) (36). The extensive broadening of the absorption spectrum of the free dye in the aqueous buffer solution resulted from extensive ground-state aggregation; however, the dye-oligonucleotide conjugate in this same solvent system displayed predominately the monomeric form, as was evident from the similarity of the electronic spectra for the conjugate in water and free dye in CH3OH/DMF, where only the monomeric form is expected to be the major component. The absorption maximum for the Pc 1 dye was found to be 683 nm (see Figure 7C) and the maximum for the 1-oligonucleotide conjugate was determined to be 694 nm, an 11 nm red shift with respect to the free dye. As with the Tnpz dye in aqueous solvents, Pc dye 1 demonstrated extensive ground state aggregation as is evident from the broad electronic spectrum. However, this aggregation effect was reduced to a large degree when the free dye was conjugated to the highly anionic DNA oligonucleotide. The fluorescence emission profile (see Figure 6D) for the free dye and dye/oligonucleotide conjugate were determined to be 700 and 692 nm, respectively. The extinction coefficient for the conjugate was found to be 1.9 × 105 cm-1 M-1 with a quantum yield of 0.26.

CONCLUSIONS

We have prepared two near-IR labeling dyes, 1 and 2 (see Figure 1), which are asymmetric Pc and Npc dyes that contain water-solubilizing groups and a labile functional group appropriate for tagging compounds containing primary amines. While most Pc and Npc dyes show poor water solubility, the excellent water solubility demonstrated by these dyes was achieved by insertion of the negatively charged carboxylate groups into the metallomacrocycle. The labeling dyes demonstrated facile conjugation to nucleic acids containing a primary amine group and yielded DNA conjugates that fluoresced strongly in the near-IR. Due to the favorable photophysical properties of these labeling dyes and their superior water solubility, they will make excellent fluorescent tags for genetic assays requiring ultrasensitive fluorescence analysis. ACKNOWLEDGMENT

The authors would like to thank the NIH (R01HG01499 and R24-CA84625) for financial support of this work. The authors would also like to express their appreciation to Dr. Tracy McCarley for assistance on obtaining the mass spectrometric measurements and Dr. Frank Zhou for his assistance with the NMR spectrometry. LITERATURE CITED (1) Wehr, E. L. (1976) Modern Fluorescence Spectroscopy, Plenum Press, New York. (2) Bright, F. V. (1988) Bioanalytical Applications of Fluorescence Spectroscopy. Anal. Chem. 60, A1031-A1039.

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BC0155869

Asymmetrical, Water-Soluble Phthalocyanine Dyes for Covalent

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