Solid-Supported Porphyrins Useful for the Synthesis of Conjugates

Article pubs.acs.org/bc

Solid-Supported Porphyrins Useful for the Synthesis of Conjugates with Oligomeric Biomolecules Satish Jadhav,† Cheng-Bin Yim,‡ Johan Rajander,‡ Tove J. Grönroos,§,∥ Olof Solin,†,§ and Pasi Virta*,† †

Department of Chemistry, University of Turku, FI-20014 Turku, Finland Turku PET Centre, Åbo Akademi University, FI-20520 Turku, Finland § Turku PET Centre, University of Turku, FI-20520 Turku, Finland ∥ Medicity Research Laboratory, University of Turku, FI-20520 Turku, Finland ‡

S Supporting Information *

ABSTRACT: meso-Tris(pyridin-4-yl)(4-carboxyphenyl)porphyrin and 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (Photochlor, HPPH) were amide-coupled to 1R,2S,3R,4R2,3-dihydroxy-4-(hydromethyl)-1-aminocyclopentane and immobilized via an ester linkage to long chain alkyl aminederivatized controlled pore glass (LCAA-CPG). The applicability of these supports (5 and 6) for the synthesis of porphyrin conjugates with oligomeric biomolecules was demonstrated using an automated phosphoramidite coupling chemistry. Cleavage from the support with concentrated ammonia gave the products, viz., porphyrin conjugates of oligonucleotides (7−9) and dendritic glycoclusters (10−13) and a cyclooctyne derivative (14) in 23−58% yield. In addition, the synthesized cyclooctyne derivative of meso-tris(pyridin-4-yl)(4-carboxyphenyl)porphyrin (14) was conjugated with an azidopropyl-modified hyaluronic acid (19). The hyaluronic acid−porphyrin conjugate (15) was radiolabeled with 64Cu and its (15[64Cu]) receptor binding affinity to CD44-expressing tumor cells was evaluated.

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INTRODUCTION The multifunctional porphyrin core (exhibiting excellent photophysical, metal chelating, and π−π stacking properties) is an attractive target to be conjugated with various biomolecules. In photodynamic therapy (PDT),1−3 glycoconjugated porphyrinbased photosensitizers4,5 have attracted particular interest, since the glycomoiety may offer selective targeting of cancer cells via host lectin binding. The glycoconjugation may increase the efficacy of the photosensitizers and reduce their toxicity in healthy tissues.6−10 Moreover, water solubility of the lipophilic porphyrin core increases. Stable chelation with radiometals makes porphyrins good probes for positron emission tomography (PET) studies.11 64Cu-chelated porphyrin−peptide− folate conjugate has been prepared and used as a PET probe for cancer imaging.12 Increased uptake in human serous ovarian cancer xenografts, overexpressing the folate receptor, could be detected both by PET and fluorescence imaging. Bombesin conjugate of 64Cu-labeled protoporphyrin IX, with an enhanced accumulation into GRPR (gastrin-releasing peptide receptors) positive PC-3 human prostate cancer cells, has been reported.13 Porphyrins have also high affinity to G-quadruplexes14 that serves additional binding motives in the targeting of oligonucleotides. Site-specific photocleavage of DNA by porphyrin− oligonucleotide conjugates has been reported.15 The strong π−π stacking between porphyrins and graphene may additionally be applied for the preparation of biomolecule−porphyrin-coated graphene.16 For the wide-ranging applications described above, a straightforward synthetic method that may be expanded to © XXXX American Chemical Society

the preparation of small libraries of porphyrin−biomolecule conjugates would be valuable.8,17 Immobilization of porphyrins to a solid support may allow simple and even automated tailoring of the conjugate part that facilitates, in particular, screening of new photosensitizers and PET-tracers.18 In the present study, the applicability of CPG-supported porphyrins (5 and 6, Scheme 1) for the synthesis of conjugates by automated phosphoramidite coupling chemistry was evaluated. The porphyrin structures selected for the immobilization were meso-tris(pyridin-4-yl) (4-carboxyphenyl)porphyrin (PyCPP) and HPPH (A and B in Scheme 1). HPPH is a commercially available and effective photosensitizer that has shown promising results in phase II clinical trials.7,19 In addition, it has recently been used as a bifunctional agent in tumor imaging and PDT.20 Furthermore, it may be worth mentioning that a structurally related pyropheophorbide has recently been used for the synthesis of nucleic acid photodynamic molecular beacons on a solid phase.21,22 PyCPP15,23 has been used for G-quadruplex recognition and photomodification of DNA. 14,15 Using supports 5 and 6, porphyrin conjugates of oligonucleotides (7−9) and of dendritic galactoclusters (10−13) were synthesized (Table 1). Cyclooctyne-modified porphyrin 14 was additionally prepared and conjugated with an azidemodified hyaluronic acid (HA) by the strain-promoted azide alkyne cycloaddition (SPAAC)24 (15, Table 1). 15 was Received: January 27, 2016 Revised: February 17, 2016

A

DOI: 10.1021/acs.bioconjchem.6b00051 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Scheme 1a

Reagents and conditions: (i) MeOCOCF3, MeOH, Et3N; (ii) DMTrCl, pyridine; (iii) NH2NH2·H2O, dioxane; (iv) meso-tris(pyridin-4-yl)(4carboxyphenyl)porphyrin or HPPH, PyBOP, DIEA, DMSO−DMF (1:3, v/v); (v) Ac2O, DMAP, pyridine; (vi) KOH, H2O, DMF; (vii) succinic anhydride, DMAP, pyridine; (vi) LCAA-CPG, DIEA, PyBOP, DMF; (vii) Ac2O, 2,6-lutidine, N-methylimidazol, THF; (vii) concentrated aqueous NH3; (ix) 19, DMF; (x) 0.1 mol L−1 aq NaOH, 3 h at 55 °C, concentrated aqueous NH3, 5d at 55 °C; (xi) Ac2O, H2O, MeCN. a

Table 1. Porphyrin Conjugates Synthesized

compound

R1

R3

calculated molecular mass

observed molecular massb

isolated yield

7 8 9 10 11 12 13 14 15

A B A A B A B A A

a a b c c d d e f

2616.1 2591.2 3447.6 1930.6 1905.7 4585.6 4560.7 1016.4 2256.2

2615.3 2590.7 3446.8 1930.6 1905.7 4584.2 4559.3 1016.4 2255.7

58% 31% 40% 51% 36% 25% 23% n.d.a 20%

a Crude 14 (isolated yield not determined: n.d.) has been used for the synthesis of 15. bObserved molecular masses have been calculated from the most intensive isotope combination at [M-H]−1 (14), [(M-2H)/2]−2 (7−11 and 15) and [(M-3H)/3]−3(12 and 13).

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radiolabeled with 64Cu and its (15[64Cu] Scheme 2) receptor binding properties in an in vitro assay with CD44-expressing tumor cells was evaluated.

RESULTS AND DISCUSSION Synthesis of Solid-Supported Porphyrins 5 and 6. 1R,2S,3R,4R-2,3-Dihydroxy-4-[(4,4′-dimethoxytrityloxy)B


Article

Bioconjugate Chemistry Scheme 2a

Conditions: (i) 64CuCl2, aqueous 0.5 mol L−1 NH4OAc (pH 5.5), 30 min at 60 °C. RP HPLC conditions in the chromatograms (equipped with (a) radio- and (b) UV-detector, λ = 400 nm, respectively): An analytical (C18, 250 × 4.6 mm, 5 μm) column, a gradient elution from 20% to 80% MeCN in H2O over 20 min, flow 1.0 mL min−1.

a

profiles (Figure 2 and the Supporting Information), the products (7−13) could be efficiently synthesized (isolated yields are listed in Table 1, based on the porphyrin absorption in aqueous solutions of the products). The authenticity of the conjugates (7−13) was verified by MS (ESI-TOF) (Table 1). Synthesis and Radiolabeling of HA-PyCPP-Conjugate (15) and In Vitro Receptor Affinity of 15[64Cu] with CD44Expressing Cancer Cells. Synthesis. Cyclooctyne-modified porphyrins are attractive compounds, since they may be used as reactive agents in the SPAAC-induced conjugation with azidemodified biomolecules.24 This was demonstrated by the synthesis of hyaluronic acid (HA) hexasaccharide conjugate of PyCPP (15). It may be worth mentioning that polymeric HA has previously been used as a conjugate moiety for a macrophagetargeting ROS-responsible (reactive oxygen species) activatable photosensitizer29 and for a tumor-targeting self-quenching photosensitizer.30 2-(Bicyclo[6.1.0]non-4-yn-9-yl)ethyl 2-cyanoethyl N,N-diisopropylphosphoramidite (18, a 600 s coupling time using benzylthiotetrazol as an activator) was coupled to 5 and the cyclooctyne-derived porphyrin (14) was released from the support with concentrated ammonia. Azidopropyl-modified HA (19) was synthesized as previously described31 and mixed with a 0.125 μmol aliquot of crude 14 ((ix) in Scheme 1). The protecting group manipulation [viz., (x) in Scheme 1: hydrolysis of the benzoyl protections and methyl esters of the glucuronic acid units; (xi) in Scheme 1: ammonolysis of the trichloroacetyl groups; and (xii) in Scheme 1: selective N-acetylation of the exposed glucosamino units] required for the HA moiety was carried out after the SPAAC-conjugation31 and the desired conjugate 15 was purified by RP HPLC (overall isolated yield 20%). The authenticity of the product was verified by MS (ESI-TOF) (Table 1). Radiolabeling. HA-PyCPP (15) was efficiently radiolabeled in ammonium acetate buffer at 60 °C for 20−30 min (Scheme 1, Figure 1). Notably, stabilizing agents (e.g., ascorbic acid, ethanol) were not required. The lipophilicity of 15[64Cu] was determined by vortex mixing in 1-octanol/water: logP (15[64Cu] = −1.73 ± 0.11). In Vitro Receptor Affinity. HA possesses high binding affinity for the CD44 receptors, which are highly expressed on the surface of various tumor cells, including the MDA-MB-231 and MCF-7 breast cancer cell lines.32 In the absence of any competitor (i.e., nonlabeled 15), in vitro receptor affinity experiments revealed low radiolocalization of 15[64Cu] on these tumor cells (Supporting Information). Approximately 10% of the applied radioactivity was bound or internalized into MDA-MB-231 cells after 30 min incubation. Increasing the concentration of

methyl]-1-aminocyclopentane (2) was synthesized in a straightforward manner in three steps: Temporal trifluoroacetylation of the amino group ((i) in Scheme 1), 4,4′-dimethoxytritylation ((ii) in Scheme 1) and exposure of the amino group ((iii) in Scheme 1) were carried out in one pot to give the desired compound 2 in 70% overall yield. HPPH and PyCPP (synthesized by Rothmund−Longo method15,23) were then nearly quantitatively coupled to 2 using PyBOP-induced amide coupling ((iv) in Scheme 1, 3 and 4). Compounds 3 and 4 were treated with succinic anhydride resulting in mixtures of mono- and disuccinates of 3 and 4 ((v) in Scheme 1), which were then coupled to LCAA-CPG support in the presence of PyBOP and DIEA ((vi) in Scheme 1). According to DMTr-cation assay, loadings of PyCPP and HPPH on the supports were 20 μmol g−1 (5) and 23 μmol g−1 (6), respectively. The unreacted amino and hydroxyl groups on the supports (5 and 6) were capped by acetylation ((vii) in Scheme 1) prior to the automated phosphoramidite coupling chemistry. Phosphoramidite Coupling Chemistry Using Supports 5 and 6. Supports 5 and 6 (0.5 μmol aliquots) were set on an automated DNA/RNA-synthesizer and their applicability for the synthesis of porphyrin conjugates of oligonucleotides (7−9) and of dendritic galacto clusters (10−13) was demonstrated. The synthesized compounds are described in Table 1. 7 and 8 are homothymidine derivatives used for the determination of molar absorptivities of porphyrin moieties A and B, below. 9 is an example of potential G-quadruplex-targeting oligonucleotide, complementary to single strand region of c-Myc (an oncogene that induces transcription of growth-stimulating genes in many types of human cancer25). 10−13 are examples of dendritic galactoclusters. The galactose-binding lectins (i.e., galectins) overexpress, e.g., on breast cancer cells26 and some melanomas27 for which galactosylated PDT reagents or radiotracers may show increased affinity. For the chain assembly, commercially available nucleoside phosphoramidites, trebler unit 16, and previously prepared cyanoethyl (methyl 2,3,4-tri-O-acetyl-α-D-galactopyranoside-6-O-yl)-N,N,-diisopropyl phosphoramidite (17)28 were used as building blocks (Figure 1). A manual coupling (see the details in the Experimental Procedures) was used to introduce the second generation of the trebler unit (16 for 12 and 13), but otherwise an automated assembly was applied. Benzylthiotetrazol was used as an activator with 25 s (for 2′-deoxy nucleoside phosphoramidites) and 600 s (for 16 and 17) coupling times. After construction of the conjugate moiety, the solid supported products were released by concentrated ammonia (2 h at 55 °C) and purified by RP HPLC. As may be seen in the crude RP HPLC C


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Bioconjugate Chemistry

Figure 1. Building blocks used for the synthesis of the porphyrin conjugates (7−15).

Figure 2. Example of RP HPLC profiles of crude product mixtures (12 and 13). RP HPLC conditions: An analytical column (C18, 250 × 4.6 mm, 5 μm), a gradient elution from 20% to 100% MeCN in H2O over 25 min, flow 1.0 mL min−1, detection at λ = 400 nm.

1R,2S,3R,4R-2,3-Dihydroxy-4-[(4,4-dimethoxytrityl)oxymethyl]-1-aminocyclopentane (2). (1R,2S,3R,4R)-2,3-Dihydroxy-4-(hydromethyl)-1-aminocyclopentane hydrochloride (1.0 g, 5.4 mmol) was dissolved in a mixture of MeOH (5.0 mL) and triethylamine (5.0 mL). Ethyl trifluoroacetate (1.3 mL, 11 mmol) was added and the resulting mixture was stirred overnight at room temperature ((i) in Scheme 1). The mixture was evaporated to dryness and the residue was dissolved in dry pyridine (10 mL). 4.4′-Dimethoxytrityl chloride (2.0 g, 5.9 mmol) was added and the mixture was stirred overnight at room temperature ((ii) in Scheme 1). Saturated aqueous NaHCO3 was added and the mixture was washed with ethyl acetate. The organic fractions were combined, dried over Na2SO4, filtered, and evaporated to dryness. The residue was dissolved in dioxane (10 mL) and NH2NH2·H2O (1.0 mL) was added ((iii) in Scheme 1). The mixture was stirred overnight at 50 °C, evaporated to dryness, and purified by silica gel chromatography (5% Et3N, 10% MeOH in CH2Cl2) to yield 1.7 g (70%) of the product 2 as white foam. 1H NMR (500 MHz, CDCl3) δ 7.40−7.38 (m, 2H), 7.29−7.23 (m, 6H), 7.16 (m, 1H), 6.80 (m, 4H), 3.87 (m, 1H), 3.80 (m, 1H), 3.73 (s, 6H), 3.39 (m, 1H), 3.11 (m, 1H), 3.06 (m, 1H), 2.22−2.10 (m, 2H), 1.20 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 158.4, 144.9, 136.0, 130.0, 128.1, 127.8, 126.8, 113.1, 86.1, 76.3, 73.0, 64.6, 55.8, 55.21, 55.16, 44.0, 30.1; HRMS (ESI-TOF) m/z: [M + H]+ calcd for C27H31NNaO5 472.2100, found 472.2103. Compound 3. meso-Tris(pyridin-4-yl)(4-methoxycarbonylphenyl)porphyrin (70 mg, 0.10 mmol, synthesized by Rothmund−Longo method15,23) was dissolved in a mixture of DMF and H2O (1:1, v/v, 5.0 mL) and KOH (58 mg, 1.0 mmol in

nonlabeled 15 did not significantly influence the percent cellbound fraction, which is an indication of cell binding mechanics other than selective ligand−receptor interactions, although this may be questionable considering the low cell-bound radioactivity levels.

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CONCLUSION The applicability of solid-supported porphyrins (5 and 6) for the synthesis of conjugates with oligomeric biomolecules has been demonstrated using phosphoramidite coupling chemistry. Ammonolytic cleavage gave porphyrin conjugates of oligonucleotides (7−9) and dendritic glycoclusters (10−13) in 23−58% yield. A cyclooctyne-derived porphyrin (14) was additionally synthesized and conjugated with an azide-modified HA-hexasaccharide (15). A modest affinity of the 64Cu-labeled HA−porphyrin conjugate (15[64Cu]) to CD44-expressing MDA-MB-231 and MCF-7 breast cancer cells has been observed. The described method, based on immobilized porphyrins, may allow simple and automated tailoring of the conjugate part that would facilitate screening of new photosensitizers, PET-tracers, and G-quadruplex-targeting oligonucleotides.

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EXPERIMENTAL PROCEDURES General Remarks. MeOH, DMSO, DMF, and pyridine were dried over molecular sieves (3 and 4 Å) and triethylamine over CaH2. The NMR spectra were recorded at 500 MHz. The chemical shifts in 1H and 13C NMR spectra are given in ppm from the residual signal of the deuterated solvents CDCl3 and DMSO-d6. The mass spectra were recorded using ESI ionization. D


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Bioconjugate Chemistry

dryness. The residues (mono- and disuccinates of 3 or 4, see MS (ESI-TOF) data in the Supporting Information), LCAA-CPG (0.20 g) and PyBOP (42 mg, 80 μmol in 0.1 mL DMF) were suspended in DMF (0.8 mL) and DIEA (28 μL, 0.16 mmol) was added ((vi) in Scheme 1). The suspensions were shaken overnight at ambient temperature and filtered. The supports were washed with DMF, CH2Cl2, and MeOH and then dried. The potential unreacted hydroxyl and amino groups on the supports were then capped by acetylation ((vii) in Scheme 1): Supports were suspended in a mixture of acetic anhydride, 2,6-lutidine, and N-methylimidazol in THF (5:5:8:82, v/v/v/v, for 15 min at 25 °C), filtered, washed with THF, CH2Cl2, and MeOH, and dried under vacuum. According to DMTr-cation assay of the supports, loadings of 20 μmol g−1 (5) and 23 μmol g−1 (6) were obtained. The color of the supports (5 as dark red, 6 as dark green) also indicated the immobilized porphyrins. Synthesis of Porphyrin Conjugates 7−14. Columns loaded with supports 5 and 6 (0.5 μmol) were installed in an automated DNA/RNA synthesizer and phosphoramidite coupling chemistry using benzylthiotetrazol as an activator was used for the chain elongation. For the commercially available nucleoside phosphoramidites, for the trebler phosphoramidite (16) and for the cyclooctyne phosphoramidite (19) 25, 600, and 600 s coupling times, respectively, were used. A manual coupling was used to introduce the second generation of the trebler unit for 12 and 13: After coupling of the first trebler phosphoramidite (16), the supports (0.2 μmol samples) were removed from the synthesizer and placed in microcentrifuge tubes. 80 μL of a 0.25 mol L−1 solution of trebler phosphoramidite (16) in acetonitrile and 80 μL of a 0.25 mol L−1 solution of benzylthiotetrazol in acetonitrile were added and the suspensions were mixed for 10 min under nitrogen at ambient temperature. The suspensions were filtered, the manual coupling was repeated, and then the supports were sent back to the synthesizer. Cyanoethyl (methyl 2,3,4-tri-O-acetyl-α-D-galacto-pyranoside-6-O-yl)-N,N,diisopropyl phosphoramidite (17) was prepared previously and introduced to compounds 10 and 11 using 600 s coupling time. Two repeated couplings (2 × 600 s) were used for the synthesis of 12 and 13. After the phosphoramidite coupling chemistry, the solid-supported porphyrin conjugates were suspended in concentrated aqueous ammonia (2 h at 55 °C), the suspensions were filtered, and the filtrates were evaporated to dryness. The residues of the released conjugates (7−13) were dissolved in water and purified by RP HPLC (Figure 1). Isolated yields (Table 1) of the products (7−13, determined using UV-absorption spectra of the aqueous solutions of the products, cf. below) are shown in Table 1. The authenticity of the products (7−14) was verified by MS (ESI-TOF) (Table 1). Synthesis of HA-PyCPP-Conjugate (15). SPAAC-conjugation ((ix) in Scheme 1): An aliquot (0.125 μmol) of crude 14 was dissolved in DMF (50 μL) and a mixture of azidopropyl modified HA (19, 0.25 μmol) in DMF (50 μL) was added. The mixture was shaken for 5 h at 55 °C and evaporated to dryness. Removal of benzoyl groups and methyl esters of HA ((x) in Scheme 1): The residue was dissolved in 0.1 mol L−1 aqueous NaOH (1.0 mL). The mixture was stirred for 3 h at 55 °C, neutralized by addition of 1.0 mol L−1 aqueous ammonium chloride (0.11 mL), and evaporated to dryness. Removal of trichloroacetyl groups of HA ((xi) in Scheme 1): The residue was dissolved in concentrated ammonia and the mixture was stirred for 5 d at 55 °C and evaporated to dryness. Selective acetylation of the amino groups of HA ((xii) in Scheme 1): The residue was dissolved in a mixture of H2O and acetonitrile (1:2, v/v, 300 μL) and 50 μL of

H2O, 0.5 mL) was added. The mixture was stirred at room temperature for 6 h and poured slowly (dropwise) to saturated aqueous NaHCO3. The dark red precipitate [meso-tris(4-Npyridyl)(4-carboxyphenyl)porphyrin] was separated, dried, and dissolved in a mixture of DMSO and DMF (1:3, v/v, 4.0 mL). Compound 2 (56 mg, 0.12 mmol in 0.1 mL DMF), PyBOP (66 mg, 0.12 mmol in 0.10 mL DMF) and DIEA (43 μL, 0.24 mmol) were added and the mixture was stirred overnight at ambient temperature ((iv) in Scheme 1). The mixture was poured slowly to cold diethyl ether and the dark red precipitate was separated. The precipitate was purified by silica gel chromatography (0.5% Et3N, 3−5% MeOH in CH2Cl2) to yield 0.11 g (97%) of the product (3) as dark red foam. 1H NMR (500 MHz, DMSO-d6) δ 9.02 (d, 6H, J = 5.2 Hz), 8.89 (b, 8H), 8.77 (d, 1H, J = 7.8 Hz), 8.30 (m, 4H), 8.24 (m, 6H), 7.45 (m, 2H), 7.35−7.30 (m, 6H), 7.24 (m, 1H), 6.92 (m, 4H), 4.79 (b, 1H), 4.59 (b, 1H), 4.40 (m, 1H), 3.93 (m, 1H), 3.84 (m, 1H), 3.74 (s, 6H), 3.17 (m, 1H), 3.00 (m, 1H), 2.36 (m, 1H), 2.25 (m, 1H), 1.41 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 166.5, 158.5, 149.3, 148.8, 145.8, 143.9, 136.5, 136.4, 134.9, 134.5, 132.3 (b), 130.2, 129.6, 128.3, 128.2, 127.1, 126.5, 120.7, 118.1, 117.9, 113.6, 85.6, 76.4, 72.9, 65.6, 55.5, 55.2, 43.7, 31.5; HRMS (ESI-TOF) m/z: [M + H]+ calcd for C69H57N8O6 1093.4401, found 1093.4371. Compound 4. HPPH (50 mg, 79 μmol) and 2 (42 mg, 94 μmol) were dissolved in DMF and PyBOP (49 mg, 94 μmol in 0.1 mL DMF) and (1.0 mL) DIEA (33 μL, 0.19 mmol) were added ((iv) in Scheme 1). The mixture was stirred at ambient temperature for 5 h, poured into saturated aqueous NaHCO3, and extracted with diethyl ether. The organic layers were combined, dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (1% Et3N, 5% MeOH in CH2Cl2) to yield 84 mg (quant.) of the product (4) as dark green foam. 1H NMR(500 MHz, CD3CN): δ 9.79 and 9.76 (2 × s, 1H), 8.81 and 8.79 (2 × s, 1H), 8.26 (s, 1H), 7.35−7.32 (m, 2H), 7.23−7.20 (m, 4H), 7.15−7.08 (m, 3H), 7.02−6.99 (m, 1H), 6.67−6.63 (m, 4H), 5.93 (m, 1H), 5.23 (d, 1H, J = 19.6 Hz), 4.93 (d, 1H, J = 19.6 Hz), 4.51 (m, 1H), 4.18 (m, 1H), 4.10 (m, 1H), 3.90 (m, 1H), 3.81 (m, 1H), 3.74−3.61 (m, 2H), 3.56−3.39 (m, 2H), 3.50 and 3.49 (2 × s, 3H), 3.47 and 3.46 (2 × s, 3H), 3.391 and 3.387 (2 × s, 3H), 3.29 and 3.28 (2 × s, 3H), 3.07 (m, 2H), 2.79 and 2.77 (2 × s, 3H), 2.70 (m, 1H), 2.41−2.35 (m, 2H), 2.25−2.18 (m, 2H), 2.15 and 2.14 (2 × d, J = 6.5 Hz, both), 1.82−1.73 (m, 5H), 1.54 (q, 3H, J = 7.6 Hz), 1.33−1.16 (m, 7H), 0.82−0.76 (m, 3H); 13C NMR (125 MHz, CDCl3): δ 197.0, 174.87, 174.84, 174.79, 174.75, 172.32, 172.30, 160.5, 158.3, 155.6, 150.7, 148.83, 148.81, 145.18, 145.16, 145.0, 141.72, 141.65, 141.52, 141.45, 139.73, 139.71, 137.0, 136.1, 136.04, 136.00, 135.7, 132.6, 132.5, 129.99, 129.97, 129.4, 128.1, 127.8, 127.7, 127.4, 126.6, 112.9, 105.7, 103.8, 97.9, 97.8, 92.7, 85.9, 77.9, 73.5, 72.8, 69.7, 64.5, 55.9, 55.8, 54.94, 54.91, 51.57, 51.55, 50.1, 48.0, 43.6, 32.90, 32.86, 32.81, 31.72, 31.69, 30.76, 30.72, 30.2, 29.89, 29.85, 29.81, 29.7, 24.7, 24.6, 22.9, 22.6, 22.5, 19.2, 17.3, 17.2, 13.95, 13.91, 11.3, 10.98, 10.96; HRMS (ESI-TOF) m/z: [M + H]+ calcd for C66H78N5O8 1068.5850, found 1068.5806. Preparation of Solid Supported Porphyrins 5 and 6. Compound 3 or 4 (40 μmol), succinic anhydride (16 mg, 0.16 mmol), and a catalytic amount of N,N′-dimethylaminopyridine were dissolved in pyridine (2.0 mL)((v) in Scheme 1). The mixtures were stirred overnight at ambient temperature, poured into saturated aqueous NaHCO3, and the succinylated products were extracted with ethyl acetate. The organic fractions were combined, dried over Na2SO4, filtered, and evaporated to E


Article

Bioconjugate Chemistry triethylamine and 25 μL of acetic anhydride were added. The mixture was mixed for 1 h at room temperature and the treatment was repeated twice (2 × 50 μL Et3N and 2 × 25 μL Ac2O). H2O (300 μL) was added, the mixture was evaporated to dryness, and the residue was introduced to RP HPLC purification. According to UV-absorption at 425 nm, 25 nmol of the homogenized product (20%, overall isolated yield calculated from 5) was obtained. The authenticity of the product was verified by MS (ESI-TOF) (Table 1). The RP HPLC monitoring of the each reaction step above ((ix−xii) is described in the Supporting Information. Molar Absorptivities of the Porphyrin Moieties (A and B in Scheme 1). Aqueous solutions of T6-PyCPP (7) and T6-HPPH (8) gave UV-absorption bands with absorption maxima at 265 nm (T6 of 7), 268 nm (T6 of 8), 417 nm (HPPH (B) of 8) and 425 nm (PyCPP (A) of 7). The porphyrin chromophores may electronically interact partly with adjacent thymine bases,33 but molar absorptivity of T6 (50 400 L mol−1 cm−1, http://biotools.nubic.northwestern.edu/OligoCalc.html) and absorbance ratios of the maxima (7: Abs(A)/Abs(T6) = 3.39 and 8: Abs(B)/Abs(T6) = 1.43) may be used to estimate the molar absorptivities of the porphyrin moieties. Accordingly, ε(A) = 171 000 L mol−1 cm−1 and ε(B) = 72 100 L mol−1 cm−1 were obtained. These ε-values were used to determine isolated yields of the products. Radiolabeling of HA-PyCPP Conjugate (15) with 64Cu. 64 Cu (t1/2 = 12.7 h, β+ = 17%, β− = 39%, EC = 44%) in the form of 64 [ Cu]CuCl2 was produced via the 64Ni(p,n)64Cu nuclear reaction, as previously described.34−36 64Cu was formulated as 10 MBq/μL in 0.04 M HCl-solution. To a solution of 15 (0.5 nmol in 2.5 μL H2O) in aqueous NH4OAc (90 μL, 0.5 M, pH 5.5), was added 64Cu (50 MBq, 5 μL) and the resulting reaction mixture was stirred at 60 °C for 30 min in a thermomixer. Thereafter, the tracer 15[64Cu] was isolated on an ODS Hypersil C18 column (250 × 4.6 mm) using a CH3CN/H2O gradient in triethylammonium acetate-buffered solution (pH 7). Collected product fraction (2.5 mL) was diluted in H2O (7 mL) and concentrated on a Sep-Pak tC18 Cartridge (Waters, USA), and eluted with 0.2 mL ethanol followed by 1 mL H2O. The eluate was collected in a 10 mL sealed glass vial and used immediately. Cell Culturing and Affinity Assay with 15[64Cu]. MDAMB-231 and MCF-7 breast cancer cell lines were cultured in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% (v/v) fetal bovine serum, 1% nonessential amino acids, 1% L-glutamine, and 1% streptomycin-penicillin (100 IU/mL). The cell lines were incubated in culture flasks at 37 °C, at an atmosphere of 95% humidity and 5% CO2. Cells were harvested using trypsin/ethylenediaminetetraacetic acid/PBS-solution. To aliquots of MDA-MB-231 cells (1 × 106) in DMEM (1 mL) in Eppendorf Protein LoBind tubes (1.5 mL), a series of increasing concentrations of nonlabeled 15 were added. 15[64Cu] (100 000 CPM, 100 μL) was added to each tube and the resulting mixture was incubated at 37 °C for 30, 60, and 120 min. Thereafter, the tubes were measured for radioactivity using a γ-counter, including three standards. After centrifugation and removing the medium the cell pellet was measured. The experiment was repeated for MCF-7 cells (60 min incubation). Lipophilicity of 15[64Cu]. The octanol/water partition coefficient (logP) of 15[64Cu] was measured using a previously described procedure.35,36 Briefly, approximately 100 kBq of radioactive compound was mixed vigorously with 1-octanol (500 μL) and water (500 μL). Following centrifugation (10 min at 5000 rpm),

samples from the organic and aqueous phases were analyzed using a γ-counter. LogP (−1.73 ± 0.11) for 15[64Cu] was calculated as the Briggsian logarithm of the ratio of radioactivity in the octanol phase to its value in the aqueous phase.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00051. NMR spectra for 2−4, RP HPLC profiles for 7−15, MS (ESI-TOF) spectra for 7−15, MS (ESI-TOF) spectra of the succinylated 3 and 4, RP HPLC- and MS (ESI-TOF)monitoring of the protecting groups manipulation required for 15 and Cell binding data of 15[64Cu] with MDA-MB-231 and MCF-7 tumor cell lines (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +358 2 333 6777, E-mail: pamavi@utu.fi. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Tamiko Ishizu for her technical assistance during cell experiments. Financial support from the Academy of Finland (No. 251539) is gratefully acknowledged. Part of the study was conducted within the Finnish Centre of Excellence in Cardiovascular and Metabolic Disease, financially supported by the Academy of Finland, University of Turku, Turku University Hospital, and Åbo Akademi University.

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REFERENCES

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Solid-Supported Porphyrins Useful for the Synthesis of Conjugates

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