Dazhong Fan

Expanded Scope of Synthetic Bacteriochlorins via Improved Acid Catalysis Conditions and Diverse Dihydrodipyrrin-Acetals

Bacteriochlorins are attractive candidates for a wide variety of photochemical studies owing to their strong absorption in the near-infrared spectral region. The prior acid-catalysis conditions [BF(3) x O(Et)(2) in CH(3)CN at room temperature] for self-condensation of a dihydrodipyrrin-acetal (bearing a geminal dimethyl group in the pyrroline ring) typically afforded a mixture of three macrocycles: the expected 5-methoxybacteriochlorin (MeOBC-type), a 5-unsubstituted bacteriochlorin (HBC-type), and a free base B,D-tetradehydrocorrin (TDC-type). Here, a broad survey of >20 acids identified four promising acid catalysis conditions of which TMSOTf/2,6-di-tert-butylpyridine in CH(2)Cl(2) at room temperature was most attractive owing to formation of the 5-methoxybacteriochlorin as the sole macrocycle regardless of the pyrrolic substituents in the dihydrodipyrrin-acetal (electron-withdrawing, electron-donating, or no substituent). Eleven new dihydrodipyrrin-acetals were prepared following standard routes. Application of the new acid catalysis conditions has afforded diverse bacteriochlorins (e.g., bearing alkyl/ester, aryl/ester, diester, and no substituents) in a few days from commercially available starting materials. Consideration of the synthetic steps and yields for formation of the dihydrodipyrrin-acetal and bacteriochlorin underpins evaluation of synthetic plans for early installation of bacteriochlorin substituents via the dihydrodipyrrin-acetal versus late installation via derivatization of beta-bromobacteriochlorins. Treatment of the 5-methoxybacteriochlorins with NBS gave regioselective 15-bromination when no pyrrolic substituents were present or when each pyrrole contained two substituents; on the other hand, the presence of a beta-ethoxycarbonyl group caused loss of regioselectivity. The 15 new bacteriochlorins prepared herein exhibit a long-wavelength absorption band in the range 707-759 nm, providing tunable access to the near-infrared region. Taken together, this study expands the scope of available bacteriochlorins for fundamental studies and diverse applications.

Stable synthetic bacteriochlorins overcome the resistance of melanoma to photodynamic therapy

Cutaneous malignant melanoma remains a therapeutic challenge, and patients with advanced disease have limited survival. Photodynamic therapy (PDT) has been successfully used to treat many malignancies, and it may show promise as an antimelanoma modality. However, high melanin levels in melanomas can adversely affect PDT effectiveness. Herein the extent of melanin contribution to melanoma resistance to PDT was investigated in a set of melanoma cell lines that markedly differ in the levels of pigmentation;3 new bacteriochlorins successfully overcame the resistance. Cell killing studies determined that bacteriochlorins are superior at (LD50≈0.1 µM) when compared with controls such as the FDA‐approved Photofrin (LD50≈10 µM) and clinically tested LuTex (LD50≈=1 µM). The melanin content affects PDT effectiveness, but the degree of reduction is significantly lower for bacteriochlorins than for Photofrin. Microscopy reveals that the least effective bacteriochlorin localizes predominantly in lysosomes, while the most effective one preferentially accumulates in mitochondria. Interestingly all bacteriochlorins accumulate in melanosomes, and subsequent illumination leads to melanosomal damage shown by electron microscopy. Fluorescent probes show that the most effective bacteriochlorin produces significantly higher levels of hydroxyl radicals, and this is consistent with the redox properties suggested by molecular‐orbital calculations. The best in vitro performing bacteriochlorin was tested in vivo in a mouse melanoma model using spectrally resolved fluorescence imaging and provided significant survival advantage with 20% of cures (P<0.01).—Mroz, P., Huang, Y.‐Y., Szokalska, A., Zhiyentayev, T., Janjua, S., Nifli, A.‐P., Sherwood, M. E., Ruzié, C., Borbas, K. E., Fan, D., Krayer, M., Balasubramanian, T., Yang, E., Kee, H. L., Kirmaier, C., Diers, J. R., Bocian, D. F., Holten, D., Lindsey, J. S., Hamblin, M. R. Stable synthetic bacteriochlorins overcome the resistance of melanoma to photodynamic therapy. FASEB J. 24, 3160–3170 (2010). www.fasebj.org

In Vitro Photodynamic Therapy and Quantitative Structure–Activity Relationship Studies with Stable Synthetic Near-Infrared-Absorbing Bacteriochlorin Photosensitizers

Photodynamic therapy (PDT) is a rapidly developing approach to treating cancer that combines harmless visible and near-infrared light with a nontoxic photoactivatable dye, which upon encounter with molecular oxygen generates the reactive oxygen species that are toxic to cancer cells. Bacteriochlorins are tetrapyrrole compounds with two reduced pyrrole rings in the macrocycle. These molecules are characterized by strong absorption features from 700 to >800 nm, which enable deep penetration into tissue. This report describes testing of 12 new stable synthetic bacteriochlorins for PDT activity. The 12 compounds possess a variety of peripheral substituents and are very potent in killing cancer cells in vitro after illumination. Quantitative structure-activity relationships were derived, and subcellular localization was determined. The most active compounds have both low dark toxicity and high phototoxicity. This combination together with near-infrared absorption gives these bacteriochlorins great potential as photosensitizers for treatment of cancer.

Synthesis and physicochemical properties of metallobacteriochlorins.

Access to metallobacteriochlorins is essential for investigation of a wide variety of fundamental photochemical processes, yet relatively few synthetic metallobacteriochlorins have been prepared. Members of a set of synthetic bacteriochlorins bearing 0-4 carbonyl groups (1, 2, or 4 carboethoxy substituents, or an annulated imide moiety) were examined under two conditions: (i) standard conditions for zincation of porphyrins [Zn(OAc)(2)·2H(2)O in N,N-dimethylformamide (DMF) at 60-80 °C], and (ii) treatment in tetrahydrofuran (THF) with a strong base [e.g., NaH or lithium diisopropylamide (LDA)] followed by a metal reagent MX(n). Zincation of bacteriochlorins that bear 2-4 carbonyl groups proceeded under the former method whereas those with 0-2 carbonyl groups proceeded with NaH or LDA/THF followed by Zn(OTf)(2). The scope of metalation (via NaH or LDA in THF) is as follows: (a) for bacteriochlorins that bear two electron-releasing aryl groups, M = Cu, Zn, Pd, and InCl (but not Mg, Al, Ni, Sn, or Au); (b) for bacteriochlorins that bear two carboethoxy groups, M = Ni, Cu, Zn, Pd, Cd, InCl, and Sn (but not Mg, Al, or Au); and (c) a bacteriochlorin with four carboethoxy groups was metalated with Mg (other metals were not examined). Altogether, 15 metallobacteriochlorins were isolated and characterized. Single-crystal X-ray analysis of 8,8,18,18-tetramethylbacteriochlorin reveals the core geometry provided by the four nitrogen atoms is rectangular; the difference in length of the two sides is ∼0.08 Å. Electronic characteristics of (metal-free) bacteriochlorins were probed through electrochemical measurements along with density functional theory calculation of the energies of the frontier molecular orbitals. The photophysical properties (fluorescence yields, triplet yields, singlet and triplet excited-state lifetimes) of the zinc bacteriochlorins are generally similar to those of the metal-free analogues, and to those of the native chromophores bacteriochlorophyll a and bacteriopheophytin a. The availability of diverse metallobacteriochlorins should prove useful in a variety of fundamental photochemical studies and applications.

Examination of Chlorin-Bacteriochlorin Energy-transfer Dyads as Prototypes for Near-infrared Molecular Imaging Probes †

New classes of synthetic chlorin and bacteriochlorin macrocycles are characterized by narrow spectral widths, tunable absorption and fluorescence features across the red and near‐infrared (NIR) regions, tunable excited‐state lifetimes (<1 to >10 ns) and chemical stability. Such properties make dyad constructs based on synthetic chlorin and bacteriochlorin units intriguing candidates for the development of NIR molecular imaging probes. In this study, two such dyads (FbC‐FbB and ZnC‐FbB) were investigated. The dyads contain either a free base (Fb) or zinc (Zn) chlorin (C) as the energy donor and a free base bacteriochlorin (B) as the energy acceptor. In both constructs, energy transfer from the chlorin to bacteriochlorin occurs with a rate constant of ∼(5 ps)−1 and a yield of >99%. Thus, each dyad effectively behaves as a single chromophore with an exceptionally large Stokes shift (85 nm for FbC‐FbB and 110 nm for ZnC‐FbB) between the red‐region absorption of the chlorin and the NIR fluorescence of the bacteriochlorin (λf = 760 nm, Φf = 0.19, τ ∼ 5.5 ns in toluene). The long‐wavelength transitions (absorption, emission) of each constituent of each dyad exhibit narrow (≤20 nm) spectral widths. The narrow spectral widths enabled excellent selectivity in excitation and detection of one chlorin–bacteriochlorin energy‐transfer dyad in the presence of the other upon diffuse optical tomography of solution‐phase phantoms.

Imine-substituted dipyrromethanes in the synthesis of porphyrins bearing one or two meso substituents

5,15-substituted porphyrins are valuable compounds in bioorganic and materials chemistry. A new synthesis has been developed that employs 1,9-diformylation of a dipyrromethane, conversion of the diformyldipyrromethane to the bis(imino) derivative, and reaction of the bis(imino)dipyrromethane + a dipyrromethane to give the zinc-porphyrin bearing trans-AB-substituents. 1,9-diformylation was achieved via Vilsmeier reaction. Imination was achieved by treatment of the 1,9-diformyldipyrromethane with excess amine under neat conditions at room temperature. The porphyrin-forming reaction was carried out over 2 h in refluxing ethanol containing zinc acetate exposed to air. Oxidation of the intermediate porphyrinogen occurs aerobically. A complex composed of two bis(imino)dipyrromethanes and two zinc atoms was observed to form reversibly during the course of the reaction. A set of zinc-porphyrins with trans-AB-, A2-, or A-substituents has been prepared in yields of ~30% (without detectable scrambling) with straightforward purification. The reaction is applicable to A/B substituent combinations of aryl/aryl, aryl/alkyl, and aryl/H.

Chlorin–Bacteriochlorin Energy-transfer Dyads as Prototypes for Near-infrared Molecular Imaging Probes: Controlling Charge-transfer and Fluorescence Properties in Polar Media

The photophysical properties of two energy‐transfer dyads that are potential candidates for near‐infrared (NIR) imaging probes are investigated as a function of solvent polarity. The dyads (FbC‐FbB and ZnC‐FbB) contain either a free base (Fb) or zinc (Zn) chlorin (C) as the energy donor and a free base bacteriochlorin (B) as the energy acceptor. The dyads were studied in toluene, chlorobenzene, 1,2‐dichlorobenzene, acetone, acetonitrile and dimethylsulfoxide (DMSO). In both dyads, energy transfer from the chlorin to bacteriochlorin occurs with a rate constant of ∼(5–10 ps)−1 and a yield of >99% in nonpolar and polar media. In toluene, the fluorescence yields (Φf = 0.19) and singlet excited‐state lifetimes (τ∼5.5 ns) are comparable to those of the benchmark bacteriochlorin. The fluorescence yield and excited‐state lifetime decrease as the solvent polarity increases, with quenching by intramolecular electron (or hole) transfer being greater for FbC‐FbB than for ZnC‐FbB in a given solvent. For example, the Φf and τ values for FbC‐FbB in acetone are 0.055 and 1.5 ns and in DMSO are 0.019 and 0.28 ns, whereas those for ZnC‐FbB in acetone are 0.12 and 4.5 ns and in DMSO are 0.072 and 2.4 ns. The difference in fluorescence properties of the two dyads in a given polar solvent is due to the relative energies of the lowest energy charge‐transfer states, as assessed by ground‐state redox potentials and supported by molecular‐orbital energies derived from density functional theory calculations. Controlling the extent of excited‐state quenching in polar media will allow the favorable photophysical properties of the chlorin–bacteriochlorin dyads to be exploited in vivo. These properties include very large Stokes shifts (85 nm for FbC‐FbB, 110 nm for ZnC‐FbB) between the red‐region absorption of the chlorin and the NIR fluorescence of the bacteriochlorin (λf = 760 nm), long bacteriochlorin excited‐state lifetime (∼5.5 ns), and narrow (≤20 nm) absorption and fluorescence bands. The latter will facilitate selective excitation/detection and multiprobe applications using both intensity‐ and lifetime‐imaging techniques.

Synthesis and Excited-state Photodynamics of a Chlorin–Bacteriochlorin Dyad—Through-space Versus Through-bond Energy Transfer in Tetrapyrrole Arrays

Understanding energy transfer among hydroporphyrins is of fundamental interest and essential for a wide variety of photochemical applications. Toward this goal, a synthetic free base ethynylphenylchlorin has been coupled with a synthetic free base bromobacteriochlorin to give a phenylethyne‐linked chlorin–bacteriochlorin dyad (FbC‐pe‐FbB). The chlorin and bacteriochlorin are each stable toward adventitious oxidation because of the presence of a geminal dimethyl group in each reduced pyrrole ring. A combination of static and transient optical spectroscopic studies indicate that excitation into the Qy band of the chlorin constituent (675 nm) of FbC‐pe‐FbB in toluene results in rapid energy transfer to the bacteriochlorin constituent with a rate of ∼(5 ps)−1 and efficiency of >99%. The excited bacteriochlorin resulting from the energy‐transfer process in FbC‐pe‐FbB has essentially the same fluorescence characteristics as an isolated monomeric reference compound, namely a narrow (12 nm fwhm) fluorescence emission band at 760 nm and a long‐lived (5.4 ns) Qy excited state that exhibits a significant fluorescence quantum yield (Φf = 0.19). Förster calculations are consistent with energy transfer in FbC‐pe‐FbB occurring predominantly by a through‐space mechanism. The energy‐transfer characteristics of FbC‐pe‐FbB are compared with those previously obtained for analogous phenylethyne‐linked dyads consisting of two porphyrins or two oxochlorins. The comparisons among the sets of dyads are facilitated by density functional theory calculations that elucidate the molecular‐orbital characteristics of the energy donor and acceptor constituents. The electron‐density distributions in the frontier molecular orbitals provide insights into the through‐bond electronic interactions that can also contribute to the energy‐transfer process in the different types of dyads.

New stable synthetic bacteriochlorins for photodynamic therapy of melanoma

Photodynamic therapy (PDT) has been successfully used to treat many malignancies, and has afforded highly encouraging results in skin cancers such as basal cell carcinoma. However, pigmented melanoma remains a notable exception from the range of tumors treated by PDT largely due to the fact that melanin has high absorption of light in wavelength regions where most clinically approved photosensitizers (PS) absorb light (600-690 nm). Moreover, melanoma cells sequester exogenous molecules including photosensitizers inside melanosomes. The aforementioned drawbacks of the clinically used PS have motivated us to search for new classes of PS with improved spectral properties, such as bacteriochlorins (BC) to be used in PDT of melanoma. To overcome the PDT-resistance mechanisms of melanoma, particularly the high optical absorption of melanin, three near-infrared (NIR) absorbing synthetic stable BC were used in PDT treatment of melanoma. Dose and fluence dependent cell killing, intracellular localization (particularly in melanosomes), and correlation between the melanin level and cell death were examined. Intracellular melanosomes are ruptured after illumination as shown by electron microscopy. The best in vitro performing BC were tested upon delivery in micellar nanoparticles against a mouse pigmented melanoma. Two of the BC were effective at significantly lower concentrations (<0.5 μM) than common photosensitizers in present use.