Preparative purification of anti-tumor derivatives of honokiol by high-speed counter-current chromatography

In our program to synthesize a series of novel derivatives as potential analogs of honokiol for anti-tumor treatment, we have found that at least three of the derivatives of honokiol showed more potency to inhibit the proliferation of K562 leukemia cells and SPC-A1 adenocarcinoma cells. As a critical step to our further series synthesis of derivatives of honokiol, three derivatives of honokiol composed of two isomers and one compound with two formyl groups, which were hardly separated by common purification methods, needed to be rapidly separated and purified. The present work describes analytical and preparative high-speed counter-current chromatography (HSCCC) for the isolation and purification of these three C-formylation derivatives of honokiol, named 3'-formylhonokiol, 5-formylhonokiol and 3',5-diformylhonokiol, respectively. The solvent system for HSCCC separation was composed of hexane-ethyl acetate-methanol-water with the ratio of 1:0.4:1:0.4 (v/v). The one-step purification produced 157.8 mg, 121.6 mg and 21.2 mg of 3'-formylhonokiol, 5-formylhonokiol, 3',5-diformylhonokiol from crude sample of 400mg with purities of 98.6%, 99.2% and 99.6%, respectively, in an elution time of 2.5 h. The purities and structural identification were determined by HPLC, (1)H NMR, (13)C NMR and mass spectroscopy. Their anti-proliferation effects on K562, A549 and SPC-A1 cell lines were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.     

Separation of honokiol and magnolol by intermittent counter-current extraction

Recently, intermittent counter-current extraction (ICcE) has been developed and shown its advantage in improving resolution between targeted compounds. However, how to choose suitable parameters to increase the throughput has not been systematically studied yet. In present work, we first calculated theoretically the conditions to carry out ICcE elution mode. Then, honokiol and magnolol were separated as model compounds using ICcE elution mode to confirm our conclusion. After parameters like sample concentration and sample feed were optimized in analytical high-performance counter-current chromatography (HPCCC), the separation process was scaled up to preparative HPCCC successfully. 12.8 g honokiol and 16.1 g magnolol were separated from 30 g mixture with purities of 98.6% and 93.7%. And the throughput of target isolation of ICcE elution mode was at least 3.75× higher than isocratic elution mode with the same HPCCC instruments. Our results confirmed our theory calculation and demonstrated the enormous potential of ICcE on preparative separation of binary mixture.     

High-performance liquid chromatographic method for simultaneous determination of honokiol and magnolol in rat plasma

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of honokiol and magnolol in rat plasma. The plasma was deproteinized with acetonitrile which contained an internal standard (diphenyl) and was separated from the aqueous layer by adding sodium chloride. Honokiol and magnolol are extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC and ultraviolet detection. The limits of quantitation for honokiol and magnolol were 13 and 25 ng ml⁻¹ in plasma, respectively, and recovery of both analytes was greater than 93%. The assay was linear from 20 to 200 ng ml⁻¹ for honokiol and from 40 to 400 ng ml⁻¹ for magnolol. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration-time profiles of honokiol and magnolol in the plasma following rectal administration of Houpo extract at a dose of 245 mg kg⁻¹, equivalent to 13.5, 24.4 mg kg⁻¹ of honokiol and magnolol, respectively.     

Isolation of antioxidants from Psoralea corylifolia fruits using high-speed counter-current chromatography guided by thin layer chromatography-antioxidant autographic assay

A combinative method using high-speed counter-current chromatography (HSCCC) and thin layer chromatography (TLC) as an antioxidant autographic assay was developed to separate antioxidant components from the fruits of Psoralea corylifolia. Under the guidance of TLC bioautography, eight compounds including five flavonoids and three coumarins were successfully separated from the fruits of P. corylifolia by HSCCC with an optimized two-phase solvent system, n-hexane–ethyl acetate–methanol–water (1:1.1:1.3:1, v/v/v/v). The separation produced 5.91 mg psoralen, 6.26 mg isopsoralen, 3.19 mg psoralidin, 0.92 mg corylifol A, and 2.43 mg bavachinin with corresponding purities of 99.5, 99.8, 99.4, 96.4, and 99.0%, as well as three sub-fractions, in a single run from 250 mg ethyl acetate fraction of P. corylifolia extract. Following an additional clean-up step by preparative TLC, 0.4 mg 8-prenyldaidzein (purity 91.7%), 4.18 mg neobavaisoflavone (purity 97.4%) and 4.36 mg isobavachalcone (purity 96.8%) were separated from the three individual sub-fractions. The structures of the isolated compounds were identified by ¹H NMR and ¹³C NMR. The results of antioxidant activity estimation by electron spin resonance (ESR) method showed that psoralidin was the most active antioxidant with an IC₅₀ value of 44.7 μM. This is the first report on simultaneous separation of eight compounds from P. corylifolia by HSCCC.     

Completed preparative separation of alkaloids from Cephaltaxus fortunine by step-pH-gradient high-speed counter-current chromatography

Cephalotaxine-type alkaloids are the anti-cancer components in twigs, leaves, roots and seeds of Cephalotaxus fortunine. It is very important to use the limited resource by finding an efficient purification technology of the alkaloids. Separation of cephalotaxine-type alkaloids in Cephalotaxus fortunine by step-pH-gradient high-speed counter-current chromatography (step-pH-gradient HSCCC) was studied in this paper. The step-pH-gradient HSCCC was performed on a HSCCC instrument equipped with a 400-mL column, using the upper phase of ethyl acetate–n-hexane–water, with added 0.01% trifluoroacetic acid (TFA) as stationary phase, and the lower phase of ethyl acetate–n-hexane–water, with added 2% NH₄OH, 0.2% NH₄OH and 0.05% TFA as mobile phase. For each separation, 800 mg of extract of cephalotaxine-type alkaloids was separated to yield 9.3 mg of drupacine, 15.9 mg of wilsonine, 130.4 mg of cephalotaxine, 64.8 mg of epi-wilsonine, 12.8 mg of fortunine and 35.6 mg of acetylcephalotaxine with purities 81.2%, 85.7%, 95.3%, 97.5%, 89.1% and 96.2%, respectively. The recovery of each alkaloid was more than 90%. The structures of the six alkaloids were identified by electrospray ionization mass spectrum (ESI-MS) and ¹H and ¹³C NMR.     

Preparative separation of di- and trisulfonated components of Quinoline Yellow using affinity-ligand pH-zone-refining counter-current chromatography

Four positionally isomeric 2-(2-quinolinyl)-1H-indene-1,3(2H)-dionedisulfonic acids (SA) and one triSA, components of the color additive Quinoline Yellow (QY, Color Index No. 47005), were isolated from the dye mixture by affinity-ligand pH-zone-refining counter-current chromatography (CCC) through complementary use of ion-exchange and ion-pair reagents as the ligand. The added ligands facilitated the partitioning of the very polar polysulfonated components into the organic stationary phase of the two-phase solvent systems that consisted of isoamyl alcohol–methyl tert-butyl ether–acetonitrile–water (3:5:1:7), (3:4:1:7) or (3:1:1:5). Thus, separation of a 5-g portion of QY using sulfuric acid as the retainer and dodecylamine as the ligand (an ion-exchange reagent, 20% in the stationary phase), resulted in 1.21 g of 6′,5-diSA and 1.69 g of 6′,8′,5-triSA, both of over 99% purity. A minor component, 8′,4-diSA, not previously reported was also obtained (4.8 mg of over 94% purity) through a similar separation of a different batch of QY using hydrochloric acid as the retainer and 10% dodecylamine as the ligand in the stationary phase. Two components that co-eluted (0.55 g) in the 5 g separation were separated when trifluoroacetic acid was used as the retainer and tetrabutylammonium hydroxide (an ion-pair reagent) as the ligand. The separation resulted in 20.7 mg of 6′,4-diSA, not previously reported, and 111.8 mg of 8′,5-diSA, both of over 98% purity. The isolated compounds were characterized by high-resolution mass spectrometry and proton nuclear magnetic resonance with correlated spectroscopy assignments.     

Isolation of a polysaccharide with anticancer activity from Auricularia polytricha using high-speed countercurrent chromatography with an aqueous two-phase system

Polysaccharides from a crude extract of Auricularia polytricha were separated by high-speed countercurrent chromatography (HSCCC). The separation was performed with an aqueous two-phase system of PEG1000–K₂HPO₄–KH₂PO₄–H₂O (0.5:1.25:1.25:7.0, w/w). The crude sample (2.0 g) was successfully separated into three polysaccharide components of AAPS-1 (192 mg), AAPS-2 (137 mg), and AAPS-3 (98 mg) with molecular weights of 162, 259, and 483 kDa, respectively. These compounds were tested for growth inhibition of transplanted S180 sarcoma in mice. AAPS-2 had an inhibition rate of 40.4%. The structure of AAPS-2 was elucidated from partial hydrolysis, periodate oxidation, acetylation, methylation analysis, and NMR spectroscopy (¹H, ¹³C). These results showed AAPS-2 is a polysaccharide with a backbone of (1 → 3)-linked-β-d-glucopyranosyl and (1 → 3, 6)-linked-β-d-glucopyranosyl residues in a 2:1 ratio, and has one terminal (1→)-β-d-glucopyranosyl at the O-6 position of (1→3, 6)-linked-β-d-glucopyranosyl of the main chain.     

Neuroprotective glucosides of magnolol and honokiol from microbial-specific glycosylation

Thirteen new glucosides (1–13) of magnolol and honokiol were obtained from specific O-glycosylation by two filamentous fungi, Cunninghamella echinulata AS 3.3400 and Rhizopus japonicus ZW-4. The glucosides' structures were determined on the basis of extensive spectroscopic (HRESIMS, 1D and 2D NMR, and CD) analyses and a chemical method. C. echinulata appeared to transfer a glucosyl moiety to 2-OH of magnolol and honokiol, whereas R. japonicus preferred to regio-specifically transfer a glucosyl moiety to 4′-OH when honokiol was as the substrate. In addition, hydroxylation by C. echinulata and specific 6″-O-acylation of the introduced glucosyl moiety by R. japonicus were observed as minor reactions. Bioassay results indicated that glucosides 1–12 together with magnolol and honokiol at 10 μM attenuated the glutamate-induced toxicity in SK-N-SH cells to levels comparable to the results for MK-801, a positive control. However, the water-solubility of major glucosylated products (1, 8, and 11) increased greatly.     

Isolation and purification of honokiol and magnolol from cortex Magnoliae officinalis by high-speed counter-current chromatography

High-speed counter-current chromatography was used to isolate and purify honokiol and magnolol from cortex Magnoliae Officinalis (Magnolia officinalis Rehd. et Wils.), a plant used in the traditional Chinese medicine. A crude sample, 150 mg, was successfully separated with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.4:1:0.4, v/v), and the fractions were analyzed by high-performance liquid chromatography. The separation produced 80 and 45 mg of honokiol and magnolol with purities of 99.2 and 98.2%, respectively, in 2.5 h.     

New procedure of selected biogenic amines determination in wine samples by HPLC

A new procedure for determination of biogenic amines (BA): histamine, phenethylamine, tyramine and tryptamine, based on the derivatization reaction with 2-chloro-1,3-dinitro-5-(trifluoromethyl)-benzene (CNBF), is proposed. The amines derivatives with CNBF were isolated and characterized by X-ray crystallography and ¹H, ¹³C, ¹⁹F NMR spectroscopy in solution. The novelty of the procedure is based on the pure and well-characterized products of the amines derivatization reaction. The method was applied for the simultaneous analysis of the above mentioned biogenic amines in wine samples by the reversed phase-high performance liquid chromatography. The procedure revealed correlation coefficients (R²) between 0.9997 and 0.9999, and linear range: 0.10–9.00 mg L⁻¹ (histamine); 0.10–9.36 mg L^-1 (tyramine); 0.09–8.64 mg L⁻¹ (tryptamine) and 0.10–8.64 mg L⁻¹ (phenethylamine), whereas accuracy was 97%–102% (recovery test). Detection limit of biogenic amines in wine samples was 0.02–0.03 mg L⁻¹, whereas quantification limit ranged 0.05–0.10 mg L⁻¹. The variation coefficients for the analyzed amines ranged between 0.49% and 3.92%. Obtained BA derivatives enhanced separation the analytes on chromatograms due to the inhibition of hydrolysis reaction and the reduction of by-products formation.     

Preparative isolation and purification of isobenzofuranone derivatives and saponins from seeds of Nigella glandulifera Freyn by high-speed counter-current chromatography combined with gel filtration

Although the medicinal plant and food Nigella glandulifera Freyn has been researched for decades, isobenzofuranones have never been isolated before. Two isobenzofuranone derivatives and two saponins were successfully separated and purified from seeds of N. glandulifera Freyn by high-speed counter-current chromatography (HSCCC) with the optimized two-phase solvent system, n-hexane-ethyl acetate–methanol–water (7:3:5:5, v/v). Salfredin B₁₁ (22.1 mg, HPLC purity 95.3%), 5, 7-dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one (18.9 mg, HPLC purity 97.3%) and crude sample 2 (555 mg) were separated from 600 mg of ethyl acetate extract of N. glandulifera Freyn. Following a cleaning-up step by chromatography on Sephadex LH-20, hederagenin (12 mg) and 3-O-[β-d-xylopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-hederagenin (45 mg) were separated from sample 2. All of the fractions before peak II were collected and subjected to a Sephadex LH-20 column and eluted by methanol, two of triterpene saponins (12 mg of hederagenin and 45 mg of 3-O-[β-d-xylopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-hederagenin) were isolated. The structures of peak fractions were identified by IR, electron ionization MS, ¹H NMR and ¹³C NMR. 5, 7-Dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one was isolated for the first time from higher plant and salfredin B11 was isolated for the first time in this plant.     

Efficient synthesis of neurotrophic honokiol using Suzuki–Miyaura reactions

Efficient synthesis of honokiol (1) was accomplished using two kinds of Suzuki–Miyaura reactions. The first Suzuki–Miyaura reaction was employed to couple 2-bromophenol (6) with 4-hydroxyphenylboronic acid (5), giving rise to biphenol 4, and the second coupling was used to introduce allyl groups at 5- and 3′-positions of honokiol. The total synthesis of 1 was completed in 74% yield over five steps from 6, or in 83% yield over four steps from biphenol 4.     

Preparative separation of six antimycin A components from antimycin fermentation broth by high-speed counter-current chromatography

A method of using high-speed counter-current chromatography (HSCCC) was established for preparative isolation and purification of antimycin A components from antimycin fermentation broth. Six antimycin A components were successfully purified for the first time by HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (5:2:4:1, by volume). Total of 20 mg antimycin A₄(a or b), 25 mg antimycin A₃(a or b), 21 mg antimycin A₈(a or b), 34 mg antimycin A₂(a or b), 26 mg antimycin A₁(a or b) and 34 mg antimycin A₁(a or b) with the purities of 93.2, 98.6, 96.2, 94.1, 94.9 and 96.7%, respectively, determined by high-performance liquid chromatography (HPLC), were yielded from 200 mg crude sample only in one HSCCC run.     

Elution–extrusion counter‐current chromatography for the separation of two pairs of isomeric monoterpenes from Paeoniae Alba Radix

In this work, a simple and efficient protocol for the rapid separation of two pairs of isomeric monoterpenes from Paeoniae Alba Radix was developed by combining macroporous resin and elution–extrusion counter‐current chromatography. The crude extract was firstly subjected to a D101 macroporous resin column eluted with water and a series of different concentrations of ethanol. Then, effluents of 30 and 95% ethanol were collected as sample 1 and sample 2 for further counter‐current chromatography purification. Finally, a pair of isomers, 96 mg of compound 1 and 48 mg of compound 2 with purities of 91.1 and 96.2%, respectively, was isolated from 200 mg of sample 1. The other pair of isomers, 14 mg of compound 3 and 8 mg of compound 4 with purities of 93.6 and 88.9%, respectively, was isolated from 48 mg of sample 2. Their purities were analyzed by high‐performance liquid chromatography, and their chemical structures were identified by mass spectrometry and ¹H NMR spectroscopy. Compared to a normal counter‐current chromatography separation, the separation time and solvent consumption of elution–extrusion counter‐current chromatography were reduced while the resolutions were still good. The established protocol is promising for the separation of natural products with great disparity of content in herbal medicines.     

Ranking the effect of [1A(ax), 1B(eq)] versus [1A(eq), 1B(ax)] cyclohexane ring substitution on the H chemical shifts of γ-methylene cyclohexane ring protons using 2,2-disubstituted adamantanes as models

When two different substituents are placed in the nonbridgehead position of adamantane, the two [1A(ax), 1B(eq)] and [1A(eq), 1B(ax)] cyclohexane chair conformers are modeled and features of their NMR spectra can be studied from a single spectrum at 298 K. The effect of [1A(ax), 1B(eq)] and [1A(eq), 1B(ax)] cyclohexane ring substitution on the ¹H resonance separation within the γ-CH₂s of cyclohexane ring is compared for various substituent pairs; this aim is approached by measuring the ¹H chemical shift separation within the 4′,9′-H and 8′,10′-H methylenes from the ¹H NMR spectrum of the model 2A,2B-disubstituted adamantane at 298 K.     

Simultaneous qualitative assessment and quantitative analysis of flavonoids in various tissues of lotus (Nelumbo nucifera) using high performance liquid chromatography coupled with triple quad mass spectrometry

Flavonoid composition and concentration were investigated in 12 different tissues of ‘Ti-1’ lotus (Nelumbo nucifera) by high performance liquid chromatography equipped with photodiode array detection tandem electrospray ionization mass spectrometry (HPLC-DAD-ESI-MSⁿ). A total of 20 flavonoids belonging to six groups (myricetin, quercetin, kaempferol, isohamnetin, diosmetin derivatives) were separated and identified. Myricetin 3-O-galactoside, myricetin 3-O-glucuronide, isorhamnetin 3-O-glucuronide and free aglycone diometin (3′,5,7-trihydroxy-4′-methoxyflavone) were first reported in lotus. Flavonoid composition varied largely with tissue type, and diverse compounds (5–15) were found in leaf and flower stalks, flower pistils, seed coats and embryos. Flower tissues including flower petals, stamens, pistils, and, especially, reproductive tissue fruit coats had more flavonoid compounds (15–17) than leaves (12), while no flavonoids were detectable in seed kernels. The flavonoid content of seed embryos was high, 730.95 mg 100 g⁻¹ DW (dry weight). As regards the other tissues, mature leaf pulp (771.79 mg 100 g⁻¹ FW (fresh weight)) and young leaves (650.67 mg 100 g⁻¹ FW) had higher total flavonoid amount than flower stamens (359.45 mg 100 g⁻¹ FW) and flower petals (342.97 mg 100 g⁻¹ FW), while leaf stalks, flower stalks and seed coats had much less total flavonoid (less than 40 mg 100 g⁻¹ FW).     

Simultaneous measurement of trace monoadenosine and diadenosine monophosphate in biomimicking prebiotic synthesis using high-performance liquid chromatography with ultraviolet detection and electrospray ionization mass spectrometry characterization

The method for simultaneous separation and determination of trace monoadenosine and diadenosine monophosphate (i.e. 2′-AMP, 3′-AMP, 5′-AMP and 3′-5′ ApA) in biomimicking prebiotic synthesis was developed using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) identification. The separation was performed on a Supelco C₁₈ column with a gradient elution (solvent A: 10 mM NH₄Ac aqueous solution; solvent B: MeOH). The flow rate was set at 1.0 ml/min. The quantitative determination was achieved by HPLC with UV detection at 260 nm. The linearity ranged from 0.5 to 100 μg/ml for each nucleotide. The limits of detection (LODs) for the four nucleotides were less than 0.30 μg/ml. The recovery ranged from 95.2 to 100.7%. The intra-day relative standard deviations (RSDs) of the retention times were between 0.7 and 1.1%. Both full-scan ESI-MS and -MS² for the four nucleotides under both positive and negative polarity were carried out and the possible cleavage pathways of them were depicted. The specific ions, [AMP + H]⁺ at m/z 348 and [ApA + H]⁺ at m/z 597, were chosen to characterize the four nucleotides in biomimicking prebiotic synthesis between N-(O,O-diisopropyl) phosphoryl amino acid (Dipp-aa) and adenosine. Using the proposed HPLC/UV/ESI-MS method, the concentration of 2′-AMP, 3′-AMP, 5′-AMP and 3′-5′ ApA in the biomimicking prebiotic synthesis samples were determined.     

Development of fast enantioselective gas-chromatographic analysis using gas-chromatographic method-translation software in routine essential oil analysis (lavender essential oil)

The study aimed to find the best trade-off between separation of the most critical peak pair and analysis time, in enantioselective GC–FID and GC–MS analysis of lavender essential oil, using the GC method-translation approach. Analysis conditions were first optimized for conventional 25 m × 0.25 mm inner diameter (d_c) column coated with 6^I–VII-O-tert-butyldimethylsilyl-2^I–VII-3^I–VII-O-ethyl-β-cyclodextrin (CD) as chiral stationary phase (CSP) diluted at 30% in PS086 (polymethylphenylpolysiloxane, 15% phenyl), starting from routine analysis. The optimal multi-rate temperature program for a pre-set column pressure was determined and then used to find the pressures producing the efficiency-optimized flow (EOF) and speed-optimized flow (SOF). This method was transferred to a shorter narrow-bore (NB) column (11 m × 0.10 mm) using method-translation software, keeping peak elution order and separation. Optimization of the enantioselective GC method with the translation approach markedly reduced the analysis time of the lavender essential oil, from about 87 min with the routine method to 40 min with an optimal multi-rate temperature program and initial flow with a conventional inner diameter column, and to 15 min with FID as detector or 13.5 min with MS with a corresponding narrow-bore column, while keeping enantiomer separation and efficiency.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

Synthesis,characterization and fluorescence behavior of new fluorescent probe phthalocyanines bearing coumarin substituents

In this study, we report a new ligand, 6-hexyloxy-3-[p-(3′,4′-dicyanophenoxy)phenyl]coumarin, and its fluorescent tetrasubstituted phthalocyanines {M[Pc(OBzCou)₄], M = 2H, Zn(II), Co(II); Bz: Benzene}. The effect of the coumarin derivative on the intensity of the fluorescence spectra of the metal-free (H₂Pc) and zinc phthalocyanine (ZnPc) derivatives was investigated. The change of the emission properties of both the coumarin moieties and the phthalocyanine core in the presence of the metal ion and the ring-opening reaction of the coumarin were studied by means of steady-state fluorescence spectroscopy. The radiative decay of the Pcs and the treated coumarin substituents bound to the Pcs was examined. The novel chromogenic compounds were characterized by elemental analysis, ¹H NMR, ¹³C NMR, Maldi-TOF, IR and UV–Vis spectral data. The photophysical properties of the Pcs are extensively affected by their state of aggregation: in particular, dimerization and aggregation result in a remarkable modification of the absorption and emission bands and may induce significant quenching of the usually strong Pc fluorescence. The electronic spectra exhibit a band of coumarin identity together with characteristic Q and B bands of the phthalocyanine core.