Article pubs.acs.org/molecularpharmaceutics
Lipophilic Decyl Chain−Pterin Conjugates with Sensitizer Properties Mariana Vignoni,†,‡ Niluksha Walalawela,‡,§ Sergio M. Bonesi,∥ Alexander Greer,*,‡,§ and Andrés H. Thomas*,† †
Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), CCT La Plata-CONICET, Casilla de Correo 16, Sucursal 4, 1900 La Plata, Argentina ‡ Department of Chemistry, Brooklyn College, City University of New York, Brooklyn, New York 11210, United States § Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, 365 Fifth Avenue, New York, New York 10016, United States ∥ CIHIDECAR-CONICET, Departamento de Química Orgánica, FCEyN, Universidad de Buenos Aires, Pabellón 2, 3er Piso, Ciudad Universitaria, Buenos Aires, Argentina S Supporting Information *
ABSTRACT: A new series of decyl chain [−(CH2)9CH3] pterin conjugates have been investigated by photochemical and photophysical methods, and with theoretical solubility calculations. To synthesize the pterins, a nucleophilic substitution (SN2) reaction was used for the regioselective coupling of the alkyl chain to the O site over the N3 site. However, the O-alkylated pterin converts to N3-alkylated pterin under basic conditions, pointing to a kinetic product in the former and a thermodynamic product in the latter. Two additional adducts were also obtained from an N-amine condensation of DMF solvent molecule as byproducts. In comparison to the natural product pterin, the alkyl chain pterins possess reduced fluorescence quantum yields (ΦF) and increased singlet oxygen quantum yields (ΦΔ). It is shown that the DMF-condensed pterins were more photostable compared to the N3- and O-alkylated pterins bearing a free amine group. The alkyl chain pterins efficiently intercalate in large unilamellar vesicles, which is a good indicator of their potential use as photosensitizers in biomembranes. Our study serves as a starting point where the synthesis can be expanded to produce a wider series of lipophilic, photooxidatively active pterins. KEYWORDS: lipophilic pterins, synthesis, fluorescence, singlet oxygen, LUVs
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INTRODUCTION
lipophilic pterins. Lipoidal biopterin (Bip) and tetrahydrobiopterin (H4Bip) derivatives were synthesized to be used as prodrugs in diseases deficient in H4Bip, such as phenylketonuria, Parkinson’s disease, and Alzheimer’s disease.5 In addition, an O-alkylated 6-carbon chain at position 4 of a pterin derivative was synthesized for use as a redox-active wire targeting the pterin binding site of nitric oxide synthase.6 However, except for the lipoidal Bip, there appear to be no other reports of long-chain alkylated pterins in the literature. The conjugation of long alkyl chains to drugs offers a handle for delivery to lipophilic sites. For example, a long-chain alkylated α-glucoside inhibitor 1-deoxynojirimycin increased its lipophilicity as well as its inhibitory potency.7,8
In 2016, a report showed that the natural product pterin (Ptr) freely passes across large unilamellar vesicles (LUVs) and photoinduces the oxidation of lipids of the membrane.1 It was concluded that no direct contact existed between Ptr and the membrane, so that the photooxidation is a dynamic encounter process.1 What was highlighted in this study is that pterins are not soluble in organic solvents and are only slightly soluble in water. However, despite much research on pterins and much photochemical insight gained,2−4 solubility problems have persisted. For this reason, we now report on how an alkylation approach, using decyl chain conjugation, provides avenues for studying pterins in lipophilic environments. Below, we first give background on pterin research to provide a sense of the state of pterin science. Poor solubility has been a long-standing problem in pterin research. The synthesis of pterins can be cumbersome due to organic solvent insolubility so that column chromatography techniques are hampered. Only scarce reports exist on © XXXX American Chemical Society
Special Issue: Pharmacology by Chemical Biology Received: Revised: Accepted: Published: A
February 22, 2017 April 22, 2017 May 2, 2017 May 2, 2017 DOI: 10.1021/acs.molpharmaceut.7b00136 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
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Article
EXPERIMENTAL SECTION Chemicals. Pterin (Ptr), 1-iododecane, dimethylformamide (DMF), dichloromethane (DCM), hexane, deuterated dimethyl sulfoxide (DMSO-d6, 99.5%), and potassium carbonate (K2CO3) were obtained from Sigma and were used as received. Tris(hydroxymethyl)aminomethane (Tris) was provided by Genbiotech. L-α-Phosphatidylcholine from egg yolk (eggPC) and Sepharose CL-4B was bought from Sigma-Aldrich. Chloroform was from U.V.E., and acetonitrile and methanol were from J. T. Baker. All of them were HPLC grade. Water was purified using a deionization system. Synthesis of Pterins 1−4. To a solution of Ptr (25 mg, 0.15 mmol) in DMF (12 mL) was added potassium carbonate (22 mg, 0.15 mmol). The mixture was sonicated and sparged with argon for 20 min. Then, 1-iododecane (65 μL, 0.3 mmol) was added to the solution. The reaction mixture was placed into a water bath and was stirred at 70 °C for 20 h. The solution was cooled at room temperature, and finally, the solvent was evaporated to dryness under vacuum, providing a solid residue. This solid residue was treated with NaCl (s.s.) (10 mL) and then was extracted with DCM (3 × 10 mL). The organic layers were separated, dried over Na2SO4, filtered, and evaporated to dryness. The white solid residue obtained was worked up by silica gel column chromatography (eluent: DCM 100% followed by DCM−methanol mixtures). From the eluted fractions, the products 1−4 were isolated and characterized by means of physical and spectroscopic methods. The first eluted fraction contained a mixture of products 1 and 2, which were separated and purified by HPLC method. A Shimadzu HPLC apparatus with PDA and fluorescence detector was employed with a Synergi Polar-RP column (ether-linked phenyl phase with polar end-capping, 150 × 4.6 mm, 4 μm, Phenomenex) for product separation. The mobile phase was methanol, and the runs were carried out with a flow of 0.3 mL min−1. 4-(Decyloxy)pteridin-2-amine (1). Yield: 17.5 mg (37%), purity 99%. Rf (methanol/DCM 5:95 v/v): 0.35. 1H NMR (500 MHz, DMSO-d6): δ 8.78 (d, J = 2 Hz, 1H), 8.43 (d, J = 2 Hz, 1H), 7.28 (s, 2H), 4.46 (t, J = 7 Hz, 2H), 1.81 (m, 2H), 1.23 (m, 14H), 0.84 (t, J = 7 Hz, 3H). 13C NMR (100.6 MHz, DMSO-d6): δ 166.9, 161.6, 157.2, 150.9, 139.5, 123.4, 67.2, 31.3, 29.0, 28.9, 28.7, 28.6, 28.1, 25.4, 22.1, 14.0. HRMS (ESI): m/z calcd for C16H25N5O [M + H+] = 304.2137, found 304.2135. N′-(4-(Decyloxy)pteridin-2-yl)-N,N-dimethylformimidamide (2). Yield: 17.5 mg (31%), purity 99%. Rf (methanol/ DCM 5:95 v/v): 0.38. 1H NMR (500 MHz, DMSO-d6): δ 8.84 (s, 1H), 8.75 (d, J = 2 Hz, 1H), 8.50 (d, J = 2 Hz, 1H), 4.20 (t, J = 7 Hz, 2H), 3.26 (s, 3H), 3.13 (s, 3H), 1.65 (m, 2H), 1.24 (m, 14H), 0.85 (t, J = 7 Hz, 3H). 13C NMR (100.6 MHz, DMSOd6): δ 161.3, 159.0, 157.4, 154.4, 149.9, 140.6, 130.0, 66.4, 42.5, 41.0, 35.0, 31.3, 28.9, 28.7, 27.4, 26.4, 22.1, 14.0. HRMS (ESI): m/z calcd for C19H30N6O [M + H+] = 359.2559, found 359.2552. 2-Amino-3-decylpteridin-4(3H)-one (3). Yield: 9 mg (20%), purity 97%. Rf (methanol/DCM 5:95 v/v): 0.26. 1H NMR (400 MHz, DMSO-d6): δ 8.66 (d, J = 2 Hz, 1H), 8.36 (d, J = 2 Hz, 1H), 7.59 (s, 2H), 3.95 (t, J = 7 Hz, 2H), 1.56 (m, 2H), 1.23 (m, 14H), 0.85 (t, J = 7 Hz, 3H). 13C NMR (100.6 MHz, DMSO-d6): δ 160.7, 155.8, 153.9, 150.0, 139.1, 128.0, 41.7, 26.8, 31.3, 28.9, 28.8, 28.7, 26.0, 22.1, 14.0. HRMS (ESI): m/z calcd for C16H25N5O [M + H+] = 304.2137, found 304.2134.
In general, pterins are substituted at the 6 position (Scheme 1), bearing small substituents with 1 to 4 carbon atoms Scheme 1
(unconjugated pterins) or larger substituents, such as the paminobenzoic acid (PABA) moiety (conjugated pterins) in folic acid (PteGlu).9 Indeed, natural pterins exist in oxidized and reduced forms (Scheme 1). Pterins exhibit several dissociation equilibria, with the neutral (acid) form predominating at physiological pH (Scheme 1). Upon UVA excitation (320−400 nm), unconjugated oxidized pterins present broad emission bands centered around 450 nm, fluorescence quantum yields (ΦF) above 0.10, and fluorescence lifetimes (τF) in the range 2−14 ns.10 On the other hand, conjugated pterins such as PteGlu show weak fluorescence (ΦF < 0.01) because the PABA substituent acts as an “internal quencher” and deactivates the singlet excited states of the pterin moiety. Oligodeoxynucleotides have been covalently bound to 6,7diphenylpterin and used as photosensitizers.12 Nucleoside analogues have been synthesized as pterin derivatives.13 In the presence of oxygen, aromatic pterins act as photosensitizers through both type I (generation of radicals via electron transfer or hydrogen abstraction) and type II (production of 1O2) mechanisms;10,14 the former species (such as hydroxyl radicals) have very short diffusion distances, but the latter species 1O2 can diffuse one hundred or more nanometers in biological media.15,16 Pterins are reported to photosensitize oxidation of biomolecules, such as nucleotides,17 DNA,18 amino acids,19 peptides,20 proteins,21,22 and biomembranes.1 Relatedly, pterins have been found to induce the photokilling of cervical cancer (HeLa) cells upon UVA irradiation, and alter the integrity of the cell membranes, among other deleterious aspects.23 Finally, the photodynamic inactivation of Staphylococcus aureus induced by carboxypterin has been recently demonstrated.24 Nevertheless, synthetic methods for modifying pterins with a long alkyl chain, as has been done with drugs and dyes,25−27 to better control solubilization and localization are needed to improve their versatility. Here, we present a variation of the pterin topic to address the pterin solubility problem by the attachment of a straight chain of ten carbons. We thought a decyl−pterin conjugate would be a good target and bear a similar overall length to long-chain (C12−C16) fatty alcohols. The present study describes (1) the synthetic efficiency of pterin alkylation, (2) structural characterization and solubility, (3) pterins fluorescent quantum yields and singlet oxygen quantum yields, (4) photostability, and (5) phospholipid membrane binding data. B
DOI: 10.1021/acs.molpharmaceut.7b00136 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics N′-(3-Decyl-4-oxo-3,4-dihydropteridin-2-yl)-N,N-dimethylformimidamide (4). Yield:
