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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Org Chem. Author manuscript; available in PMC 2010 December 18.
Published in final edited form as:
PMCID: PMC2796328
NIHMSID: NIHMS159300

Mono-Amine Functionalized Phthalocyanines: Mwave-Assisted Solid-Phase Synthesis and Bioconjugation Strategies

Abstract

Phthalocyanines (Pcs) are excellent candidates for use as fluors for near-infrared (near-IR) fluorescent tagging of biomolecules for a wide variety of bioanalytical applications. Mono-functionalized Pcs, having two different types of peripheral substitutents; one for covalent conjugation of the Pc to biomolecules and others to improve the solubility of the macrocycle, ideally suit for the desired applications. To date, difficulties faced during the purification of the mono-functionalized Pcs limited their usage in various types of applications. Herein are reported a new synthetic method for rapid synthesis of the target Pcs and bioconjugation techniques for labeling of the oligonucleotides with the near-IR flours. A novel synthetic route was developed utilizing a hydrophilic, polyethylene glycol-based (PEG) support with an acid labile Rink Amide linker. The Pcs were functionalized with an amine group for covalent conjugation purposes and were decorated with short PEG chains, serving as solubilizing groups. Mwave-assisted solid-phase synthetic method was successfully applied to obtain pure asymmetrically-substituted mono-amine functionalized Pcs in a short period of time. Three different bioconjugation techniques, reductive amination, amidation and Huisgen cycloaddition, were employed for covalent conjugation of Pcs to oligonucleotides. The described μwave-assisted bioconjugation methods give an opportunity to synthesize and isolate the Pc-oligonucleotide conjugate in a few hours.

INTRODUCTION

Fluorescence-based detection is the mainstay of many bioassays including immunoassays, flow cytometry, DNA sequencing, nucleic acid diagnostics, proteomics, and other clinical chemistry applications.1,2 Increasing requirements for sensitivity and decreased scale as in capillary and microchip devices has put increasing demands on the efficiency of these assays. One method to improve fluorescence-based detection is to use of near-IR dyes due to the decreased background due scattering and native fluorescence in this region of the spectrum (650–850 nm).3 Our laboratory and a number of others have been developing phthalocyanines (Pcs) as near-IR flours for bioanalytical applications. Pcs have a number of improved properties such as narrow absorbance and emission spectra, resistance to photobleaching and tunability of spectral and photophysical properties by variation of substituents around the Pc aromatic core or the central metal.4 Applications of Pcs in bioassays included analysis of PCR products,5 single gene mutation detection6 and resonance energy transfer-based assays.7 Structural requirements of Pcs for most bioanalytical applications include the need for water-solubility and a unique functional group for attachment to the biomolecule. Asymmetrically substituted Pcs with two different types of peripheral substituents, one reactive group for conjugation and others for solubility, are ideally suited for these applications, but difficulties in the isolation of these Pcs have limited their use in biological and bioanalytical applications. Recently, we have reported solid-phase synthesis of mono hydroxylated Pcs.8 Herein we present a time efficient, μwave-assisted method for synthesis of pure “AB3-type” asymmetrically-substituted, mono-amine functionalized Pcs on solid-support along with methods for their ready conjugation to oligonucleotides.

RESULTS AND DISCUSSION

Generalized retrosynthetic analysis of a functionalized Pc-biomolecular conjugate is shown in Scheme 1. The stepwise synthesis starts with immobilization of one phthalonitrile to the polymer support via a cleavable linker. Cyclotetramerization of the polymer-bound phthalonitrile with the second type of phthalonitrile in solution yields the “AB3-type” target Pc on the polymer support. Following the cleavage of the desired mono- functionalized Pc from the polymer support, in the final step, AB3-type Pc is covalently conjugated to a biomolecule by a variety of conjugation strategies.

SCHEME 1
Generalized retrosynthetic analysis of mono-functionalized Pc-biomolecular conjugates

Scheme 2 shows the synthesis of an amine-functionalized, solid-supported phthalonitrile as the starting point for Pc synthesis. Polyethylene glycol (PEG)-based Rink Amide ChemMatrix™ resin with initial loading of 0.52 mmol/g was employed as the polymer support. Synthesis of the mono-amine functional Pcs 7b and 8b started with incorporation of the amine functionality to the solid support via coupling of 0.1 mmol Fmoc-Lys(Mtt)-OH according to standard protocols.9 The ε–amine of the lysine will provide the functional handle for bioconjugation after removal of the acid-labile 4-methyltrityl (Mtt) protecting group during the cleavage of the Pc from the polymer support. The remaining amine sites of the resin 1 were capped with an excess of Ac2O. Following the removal of the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group, carboxylate phthalonitrile 2 was anchored to the amino terminus of the polymer bound lysine to give a polymer-supported phthalonitrile 3 with 0.1 mmol/g loading (Scheme 2).

SCHEME 2
Immobilization of the amino acid and phthalonitrile to the polymer support

Due to the dynamic nature of the polymer support, the outcome of the solid-phase reactions are affected by various factors such as the reaction time and temperature, loading of the resin, linker length, solvent and the concentration of the reaction.10 Base-promoted phthalonitrile tetramerization reaction was optimized based on these criteria as summarized in Table 1. Experiments were performed in CEM MARS extraction system using fiber optic temperature and pressure probes. Early experiments showed that higher phthalonitrile loadings (0.46 mmol/g), determined based on the absorption of the Fmoc adduct, and longer linker lengths lead to the formation of other Pc congeners (A2B2, A3B, etc.) on the resin via site-site interactions (Table 1) (Scheme 3). The affect of the linker length on the outcome of the cyclotetramerization reaction was examined by incorporating commercially available Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-miniPEG-(OH)) as a spacer. (Table 1, entries1–3) (Scheme 3). As a result of an increased mobility of the reactive sites, it was not possible to have complete site isolation even at lower loadings (0.1 mmol/g). Thus, the spacer was excluded from the sequence. Solvent effect is another factor regulating site interactions on the polymer support.10 Solvents in which the resin swelling is minimum promote site isolation by limiting the mobility of the reactive sites on the resin.10 Thus, the cyclotetramerization reaction was performed in dodecanol. However, the selected solvent did not have a significant effect on the outcome of the reaction. While neither the reaction time nor the temperature affected the outcome of the cyclotetramerization reaction, at elevated reaction temperatures (≥ 180 °C) we saw the decomposition of the PEG resin. Thus, the following experiments were performed at 150 °C. The protecting group on the ε–amine of the Lys has a critical effect on the outcome of the cyclotetramerization reaction. Initially, we employed lysine with a tert-butyloxycarbonyl (Boc) protecting group on the ε–amine and MALDI-MS mass spectrometry analysis of the cleaved product showed a major ion at m/z M+100 (Table 1, entry 2) (Scheme 3), corresponding to displacement of t-butyl group of the Boc protecting group with n-butyl group of the solvent, butanol (Scheme 3). Further proof for this transition was obtained by performing the reaction in different alcohols, such as n-pentanol and 2-butanol (data not shown). Due to the inseparable mixtures obtained with Boc protected amine, Fmoc-Lys(Mtt)-OH was employed for the later experiments. The last parameter that we evaluated to optimize the base-promoted cyclotetramerization reaction was the reaction concentration. Since at high reaction concentrations molecules in solution occupy most of the reactive sites,10 high degree of site isolation is achieved. We successfully suppressed the formation of A2B2-type Pc by increasing the phthalonitrile concentration in solution from 0.3 M to 0.8 M (Table 1, entries 5 and 6). Based on these results, optimized conditions for the base-promoted cyclotetramerization reaction were determined as shown in Table 1, entry 6.

SCHEME 3
Structures of the most common Pc congeners formed during the cyclotetramerization reaction
Table 1
Optimization of Cyclotetramerization Reaction

Suspension of polymer-bound phthalonitrile 3, triethylene-glycol-substituted phthalonitrile 4,8 Zn(OAc)2 and 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) in BuOH were irradiated for 40 min. in a μwave to give polymer-bound-AB3-type ZnPc 6 and the corresponding symmetrically-substituted-B4-type Pc 7a in solution (Scheme 4). ZnPc 7a8 was removed from the mixture by washing the resin with hot BuOH. Following the cleavage of the target Pc from the polymer support, asymmetrically substituted ZnPc 7b was simply purified by filtration through LH-20 column and was obtained in 27% yield without any contamination with other Pc congeners. Water soluble-oligoethyleneglycol-subsituted ZnPc 7b exhibits sharp fluorescence emission in the near-IR region of the electromagnetic spectrum, which is highly favorable for the desired bioanalytical applications (Figure 1).

Figure 1
Absorbance-emission spectra of ZnPc 7b and bioconjugate 9. a) Black: ZnPc 7b λmax(abs)=681 nm (DMSO); b)Green: ZnPc 7b λmax(em)=691 nm (DMSO); c)Blue: Bioconjugate 9 λmax(abs)=640, 690 nm (H2O); d)Red: Bioconjugate 9 λmax ...
SCHEME 4
Solid-phase synthesis of AB3-type Pcs

The same method was employed to synthesize ZnPc 8b, decorated with six triethylene-glycol chains. Synthesis of ZnPc 8b endorsed the applicability of the method to different types of phthalonitriles (Scheme 4).

The successful synthesis of the mono-amine functionalized oligoethyleneglycol-substituted AB3-type Pcs was followed by the covalent labeling of oligonucleotides with ZnPc 7b utilizing three different bioconjugation techniques. All Pc-oligonucleotide conjugates were successfully isolated by reverse-phase ion-pair HPLC. If desired, excess of the Pcs can be recycled via simple purification to be used for the next reaction. Initially, ZnPc 7b was conjugated to two different 17-mer oligonucleotides, one of which functionalized with a carboxylic acid via a C10-linker at the 5'-end and the other one was modified with a 4-formyl N-hexylbenzamide linker at the 5'-end (Scheme 5). While bioconjugate 9 was synthesized via reductive amination by incubating the reaction in a μwave at 70 °C for 30 min. (79 % labeling efficiency calculated based on the absorbance of the unreacted oligonuclotide) as well as at room temperature overnight (Scheme 5, Figure 2), synthesis of bioconjugate 10 was achieved by amidation in a μwave at 75 °C for 30 min (35 %) (Scheme 5). Due to the competition between the hydrolysis of the ester, formed in situ, and amide bond formation, the bioconjugate 10 was obtained in low yield relative to bioconjugate 9. Comparison of the two conjugation techniques revealed that the synthesis of Pc-oligonucleotide conjugates could be achieved in relatively high yields by employing reductive amination route. From the results above it has been concluded that while μwave irradiation did not significantly affect the labeling efficiency of the oligonucleotide (Figure 2), it reduced the reaction time from hours to minutes. Thus, the following experiments were performed in a μwave.

Figure 2
Labeling of the oligonucleotide with ZnPc 7b by reductive amination. The chromatograms absorbance data were extracted at λ = 680 nm.
SCHEME 5
Conjugation of the ZnPc 7b to an oligonucleotide by reductive amination or amidation

Figure 1 shows the absorbance-emission spectra of the bioconjugate 9 in aqueous medium. Bioconjugate 9 was aggregated in water as judged by the split Q band in the absorbance spectrum. However, it had a sharp emission band at 699 nm, with 9 nm of Stokes shift, in pH 8.5 buffer.

The scope of the Pc-oligonucleotide bioconjugation techniques was extended with a Huisgen cycloaddition, so-called “click chemistry”.11 For the ease of the purification as well as to reduce the reaction time the reactions were performed on the solid support under μwave irradiation (Scheme 6). In order to remove the Mtt protecting group without cleaving the Pc from the solid support, polymer-supported ZnPc 6 was treated with 2% trifluoroacetic acid (TFA) solution to yield resin-bound ZnPc 11 (Scheme 6). Bromoacetylation of the ε-amine of the polymer-bound ZnPc 11 to yield ZnPc 12 was followed by treating the resin with NaN3 in a μwave for 45 min to give the azide functionalized resin-bound ZnPc 13 (Scheme 6).12 Azide Pc 13 was cleaved from the solid support, purified by running through LH-20 column and obtained in 19% overall yield. Conjugation of ZnPc 13 to an 18-mer oligonucleotide, bearing an hexynyl linker at the 5'-end, was carried in a μwave at 60 °C for an hour (Scheme 6).13 Pc-oligonucleotide bioconjugate 14 was obtained with 80% labeling efficiency, which was calculated based on the absorbance of the unreacted oligonucleotide.

SCHEME 6
Synthesis of Pc-oligonucleotide conjugate 14 via Huisgen cycloaddition

CONCLUSION

The presented solid-phase synthetic method is an imperative approach to synthesize mono-amine functionalized-water soluble Pcs in a short period of time. Coupling of the effective method with μwave irradiation not only improved the yield of the cyclotetramerization reaction but also reduced the reaction time from hours to minutes.8 Covalent conjugation of Pcs to oligonucleotides, modified with various types of functional groups, was successfully achieved by employing different types of bioconjugation techniques. The oligonucleotides were efficiently labeled and isolated in high yields. The discussed synthetic methods are being applied to synthesis of a wide variety of Pcs and Pc-oligonucleotide conjugates for bioanalytical applications.

EXPERIMENTAL PROCEDURES

Polymer-Supported Fmoc-Lys-(Mtt)-OH 1

Prior to the synthesis, the Rink Amide resin (0.4 g) with 0.52 mmol/g loading capacity was swelled in CH2Cl2 (20 mL) for 30 min. The solvent was filtered off. and CH2Cl2 (7 mL) was added to the well-swelled resin. Fmoc-Lys-(Mtt)-OH (0.05 mmol, 31 mg), 1-hydroxybenzotriazole (HOBt) (0.05 mmol, 6.7 mg) and O-(1-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (0.05 mmol, 19 mg) were dissolved in N,N-dimethylformamide (DMF) (1 mL). The solution was added to the resin in CH2Cl2. Diisopropylethyl amine (DIEA) (0.1 mmol, 17.4 μL) was added to the suspension and the reaction flask was placed in a shaker and kept at room temperature for 1h to give the resin 1 with 0.1 mmol/g of loading. The resin was washed with CH2Cl2 (4 × 15 mL), DMF (2 × 15 mL) and CH2Cl2 (4 × 15 mL).

Acetylation of the Polymer Support

The resin 1 was suspended in 0.28 M solution of DIEA in DMF (25 mL). Ac2O (8.4 mmol, 0.8 mL) was added to the suspension and the reaction was placed in a shaker and kept at room temperature for 3h. The resin was washed with DMF (5 × 15 mL) and CH2Cl2 (5 × 15 mL).

General Procedure for Fmoc Cleavage

The resin was suspended in 20% (v/v) solution of 4-methyl piperidine in DMF (10 mL). The reaction was placed in a shaker and kept at room temperature for 10 min. Following the filtration of the solution, the fresh cleavage cocktail (10 mL) was added to the resin and the reaction was shaken 10 more min. at room temperature. The resin was washed with DMF (5 × 15 mL), CH2Cl2 (3 × 15 mL) and was submitted to next amino acid (phthalonitrile) coupling.

Determination of Loading Capacity of the Phthalonitrile Bound Resin

Before the acetylation step, ~ 50 mg of the polymer-supported Fmoc-Lys-(Mtt)-OH (1) was placed in 5 mL syringe equipped with coarse frit and and dried in the desicator for 4–5 h. 6–7 mg of the dry resin was placed in a 3 mL syringe equipped with coarse frit and swelled in CH2Cl2 (1 mL) for 30 min. Following the filtration of the CH2Cl2, the resin was placed in 20% (v/v) 4-methyl piperidine solution in DMF (1 mL) and kept in the cleavage solution for 20 min. The solution was collected by filtration and the resin was washed with CH3OH (3 × 2 mL). Combined filtrate solution (7 mL) was detected with electronic absorption spectrometry. The loading capacity (mmol/g) of the resin was calculated using the following equation:

(UV300×106×0.007)(7800×#of mg of the resin)

Polymer-supported 4-(3,4-dicyanophenoxy)benzoic Acid 3

The resin 1 (0.4 g) with 0.1 mmol/g Lys-(Mtt)-OH loading was placed in CH2Cl2 (3 mL). HOBt (0.44 mmol, 59 mg), HBTU (0.44 mmol, 167 mg) and the carboxylated phthalonitrile 2 (0.44 mmol, 110 mg) were dissolved in DMF (1 mL). The solution and DIEA (0.88 mmol, 0.15 mL) were added to the resin and the reaction flask was placed in a shaker and kept at room temperature for 2h. (In order to have an efficient coupling yield, amino acid and/or carboxylated phthalonitrile concentration in the reaction was adjusted to 0.2 M) The resin was washed with CH2Cl2 (4 × 15 mL), DMF (3 × 15 mL) and CH2Cl2 (3 × 15 mL). FT-IR (KBr, cm−1): 3515, 2863, 2232, 1751, 1738, 1656, 1610, 1588, 1542, 1508, 1452, 1348, 1300, 1177, 1146, 1088, 1031, 991, 951, 937, 920, 864, 840, 801, 773, 751, 709, 660, 639.

General Procedure for Synthesis of AB3 Type Pcs 7b, 8b and 13

The resin 3 (0.4 g) was swelled in BuOH (2 mL) for 30 min in the μwave vessel. Corresponding phthalonitrile (5 mmol) and Zn(OAc)2 (1.4 mmol, 0.25 g) were placed in a 20 mL glass vial in BuOH (4 mL) and heated up to 90 °C in water bath. DBU (2.8 mmol, 0.38 mL) was added to the suspension and the mixture was vigorously stirred until all the metal salt was dissolved. The solution was quickly transferred to the μwave vessel, having the resin 3 with BuOH (2 mL). The reaction was irradiated in the μwave at 850 W. The temperature was ramped to 150 °C in 5 min. and was held at the temperature for 40 min. The resin was washed, until a colorless filtrate was obtained, first with hot BuOH (12 × 25 mL), and then hot CH2Cl2 (3 × 25 mL).

General Procedure for Cleavage of the AB3 type Pc 7b, 8b and 13

The resin was suspended into a solution of TFA-CH2Cl2-TIPS (triisopropylsilane) (20:78:2; 25 mL) (TFA concentration was increased to 30% for the cleavage of the ZnPc 13) and agitated in the shaker for 3h at room temperature. Filtrate was evaporated to dryness and the crude mixture was purified by filtration through a LH-20 column using CH3OH as eluent (see supporting information for ZnPc 8b).

ZnPc 7b

Obtained as a blue solid (14 mg, 27% yield). Condition B was employed to perform HPLC. tR=29.45 min. MALDI-MS: Calcd C66H75N11O15Zn+: 1325.47; found: 1324.98 λmax(abs) (DMSO): 681 ± 1 nm (logε=5.2). λmax (em) (DMSO): 691 nm. ϕf : 0.10.

General Procedure for Purification of the Symmetrically Substituted Pcs 7a and 8a

Filtrate solutions were combined and evaporated to dryness. The crude mixture was dissolved in a minimum amount of CH3OH and large excess of diethyl ether (30 X) was added to solution. The solution was kept at −20 °C overnight and the suspension was centrifuged to give Pcs 7a and 8a as a blue / green solid. If desired, the Pcs can further be purified by running thorugh LH-20 column (see supporting information for ZnPc 7a).

ZnPc 8a

Obtained as a green solid (1.9 g, 20% yield). (Yields of the asymmetrically substituted Pcs were calculated based on the phthalonitrile loading, 0.1 mmol/g, of the resin 3) Condition A was employed to perform HPLC. tR=37.90 min. ESI Calcd C88H128N8O24S8Zn+: 2003.93; found: 2003.59 λmax(abs) (DMSO): 709 nm (logε=5.5). λmax (em) (DMSO): 716 nm. ϕf: 0.13

Selective Cleavage of the Mtt Protecting Group (11)

The Pc-bound polymer support was suspended into a solution of TFA-CH2Cl2-TIPS (2:96:2; 10 mL) and agitated for 2 min. The yellow colored solution was filtered off. This step was repeated for 7 times using 10 mL of cleavage cocktail at a time. The resin 10 was washed with CH2Cl2 (10 × 10 mL) and submitted to the next step.

Bromoacetylation of Polymer-Bound ZnPc 11 (12)

0.5 g of polymer-bound ZnPc 11 with 0.1 mmol/g Pc loading, was swelled in DMF for 30 min. prior the synthesis. Following the filtration, the resin was suspended in 1 mL of DMF. Bromoacetic acid (1.1 mmol, 153 mg) and N,N'-diisopropylcarbodiimide (DIC) (1.32 mmol, 126.2 mg) were dissolved in 2 mL of CH2Cl2 - DMF (1:1) mixture and the solution was added to resin. The reaction was agitated for 45 min. The reaction was repeated twice. The resin was washed with DMF (6 × 15 mL), CH2Cl2 (3 × 15 mL), DMF (3 × 15 mL), and proceeded to the next reaction. ~ 50 mg of the resin was suspended into a solution of TFA-CH2Cl2-TIPS (20:78:2; 1 mL) for 3h at room temperature. The collected filtrate was evaporated to dryness and the crude mixture was purified by filtration through a LH-20 column using CH3OH as eluent. MALDI-MS: Calcd C68H76N11O16BrZn +H: 1449.72; found 1449.80.

Polymer–Bound Azide Pc 13

0.5 g of polymer-bound-bromoacetylated ZnPc 12 was suspended in 4 mL of DMF-H2O (5:1 v/v) solution. To the suspension, NaN3 (5.5 mmol, 357 mg) was added. μwave vessel was placed in the μwave and the reaction was irradiated at 400 W. The temperature was ramped to 120 °C in 3 min. and was held at that temperature for 45 min. Following the reaction, the resin was washed with DMF (5 × 30 mL), and MeOH −H2O (6 × 30 mL), DMF (2 × 30 mL) and CH2Cl2 (4 × 30 mL). ZnPc 13 was cleaved from the polymer support and purifed by employing general methods described above.

ZnPc 13

Obtained as a blue solid (11 mg, 19% overall yield). Condition B was employed to perform HPLC. tR=27.48 min. MALDI-MS: Calcd C66H76N14O16Zn+H: 1409.50; found: 1409.50 λmax(abs) (DMSO): 681 ± 1 nm (logε=5.6). λmax (em) (DMSO): 691 nm. ϕf: 0.09

Preparation of Oligonucleotide-Pc Conjugate 9

The sequence of the 17mer oligonucleotide is: 5'(Aldehyde C6)-GTA AAA CGA CGG CCA GT 3' To a 4.3 mM oligonucleotide solution in water (4.4 μL) in an Eppendorf tube, NaOAc buffer (pH=5.5; 50 μL), 10 mM ZnPc 7b solution in CH3OH (51.6 μL) and 10 mM NaCNBH3 solution in CH3OH (10 μL) were added in the given order. The reaction was incubated either at room temperature for 16 h or in μwave for 30 min. The Eppendorf tube was placed in a screw-capped μwave vessel containing enough water to surround the Eppendorf tube (~1.5 mL) and the vessel was tightened. The μwave vessel was equipped with temperature and pressure probes and the reaction was irradiated at 400 W. The temperature was ramped to 70 °C in 1 min. and held at the set temperature for 30 min. Following the completion of the reactions, cold ethanol (300 μL) and 3 M NaCl solution in water (10 μL) were added to reaction mixtures and the suspensions were placed in −20 °C and kept for an hour. The suspensions were centrifuged at 4 °C at 12,000 rpm for 30 min. The precipitates were reconstituted in 0.05 M triethylammonium acetate (TEAA) solution in water and isolated by HPLC in 79% labeling efficiency. (76% labeling efficiency was obtained when the reaction was incubated at room temperature) Condition B was employed to perform HPLC. tR=18.95 min. ESI-TOF Calcd (M-3): 2255.14; found: 2255.14. λmax(abs) (H2O): 640, 690 nm. λmax (em) (pH 8.5 buffer; 4 mM MgCl2, 15 mM KCl 10mM TRIS) 699 nm. Unreacted oligonucleotide tR=1.38 min.

Preparation of Oligonucleotide-Pc Conjugate 10

The sequence of the 17mer oligonucleotide is: 5'(Carboxylic acid C10)-GTA AAA CGA CGG CCA GT 3' To a 3.6 mM oligonucleotide solution in water (4.4 μL) in an Eppendorf tube, NaHCO3 buffer (pH=7.6; 60 μL), 10 mM ZnPc 7b solution in CH3OH (30 μL), 150 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution in DMSO (5 μL) and 350 mM N-hydroxysuccinimide (NHS) solution in DMSO (5 μL) were added in the given order. The Eppendorf tube was placed in a screw-capped μwave vessel containing enough water to surround the Eppendorf tube (~1.5 mL) and the vessel was tightened. The μwave vessel was equipped with the temperature and pressure probes and the reaction was irradiated at 400 W. The temperature was ramped to 75 °C in 1 min. and held at the set temperature for 30 min. Following the completion of the reaction, cold ethanol (300 μL) and 3M NaCl solution in water (10 μL) were added to reaction mixture and the suspension was placed in −20 °C and kept for an hour. The suspension was centrifuged at 4 °C at 12,000 rpm for 30 min. The precipitate was reconstituted in 0.05 M TEAA solution in water and isolated by HPLC in 35% labeling efficiency. Condition B was employed to perform HPLC. tR=19.17 min. Unreacted oligonucleotide tR=1.43 min.

Preparation of Oligonucleotide-Pc Conjugate 14

The sequence of the 18mer oligonucleotide is 5'(Hexynyl C6)- GTA-AAA-CGA-CGG-CCA-AGT To a 5 mM oligonucleotide solution in water (8 μL) in an Eppendorf tube, 10 mM ZnPc 7b solution in CH3OH (120 μL), 20 mM solution of CuSO4.5H2O in water (24 μL), freshly prepared 100 mM solution of Na ascorbate in water (24 μL) and water (4 μL) were added in the given order (Water : CH3OH; 1:2). The Eppendorf tube was placed in a screw-capped μwave vessel containing enough water to surround the Eppendorf tube (~1.5 mL) and the vessel was tightened. The μwave vessel was equipped with the temperature and pressure probes and the reaction was irradiated at 400 W. The temperature was ramped to 60 °C in 1 min. and held at the set temperature for 60 min. Following the completion of the reaction, cold ethanol (300 μL) and 3M NaCl solution in water (10 μL) were added to reaction mixture and the suspension was placed in −20 °C and kept for an hour. The suspension was centrifuged at 4 °C at 12,000 rpm for 30 min. The precipitate was reconstituted in 0.1 M TEAA solution in water and isolated by HPLC in 80 % labeling efficiency. Condition B (using 0.1M TEAA solution) was employed to perform HPLC. tR=17.04 min. Unreacted oligonucleotide tR=1.56 min.

Supplementary Material

1_si_001

ACKNOWLEDGMENT

We thank the National Science Foundation (CHE-0304833, EPS-0346411) and the National Institutes of Health (EB006639 and HG01499) for financial support.

Footnotes

Supporting Information Available: Experimental procedures for the synthesis of phthalonitriles, spectroscopic data, and HPLC chromatograms for 7–10, 13–14. This material is available free of charge via the Internet at http://pubs.acs.org

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