Graphical abstract Circular dichroism of anthocyanidin 3-glucoside self-aggregates Raquel Gavara, Vesselin Petrov, Alexandre Quintas, Fernando Pina * The circular dichroism spectra of the six most common anthocyanidin 3-glucoside show the formation of left handed aggregates compatible with dimers. The absorption bands of the monomer split by increasing concentration according to the formation of H and J aggregates. The angle and distance between the transition moments of the two monomers in the dimer was calculated from the splitting of the 0–0 absorption band. While the angle is similar for the series the distance changes dramatically. The intensity of the CD signal is proportional to the inverse of the square of the distance. Highlights Q10 " The circular dichroism spectra of six common anthocyanins 3-glucosides was obtained. " Like 3,5-diglucoside analogous they exhibit left- handed CD signals. " J and H aggregates are formed by concentration increasing. " The distance of the transition moments correlate with the intensity of the CD signal. 1 1 2 Circular dichroism of anthocyanidin 3-glucoside self-aggregates a a b a,⇑ 3 Q1 Raquel Gavara , Vesselin Petrov , Alexandre Quintas , Fernando Pina 4 a REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829516 Monte de Caparica, Portugal 5 b Instituto Superior de Ciencias da Saude Egas Moniz, Centro de Investigação Interdisciplinar Egas Moniz, P-2829511 Monte de Caparica, Caparica, Portugal 6 7 article info a b s t r a c t 1 9 10 Self-association constants for the flavylium cations of the six most common anthocyanidin 3-glucosides 20 11 were determined by circular dichroism (CD) and UV–Vis spectroscopy. Along with previous 1H NMR 21 12 results, all measurements were consistent with a monomer–dimer model. The CD spectra of the antho- 22 13 cyanidin 3-glucosides were similar to the analogues 3,5-diglucosides. All dimers of the anthocyanidin 23 3-glucosides exhibited left-handed CD signals, with petunidin-3-glucoside and myrtillin having the most 24 14 Keywords: intense signals. In addition, the magnitude of the molar ellipticity, [h], was generally higher for the 3-glu- 25 15 Circular dichroism cosides than for the 3,5-diglucosides. For all six anthocyanins studied, the CD absorption spectra of their 26 16 Anthocyanins dimers showed evidence of the splitting of the monomer absorption into lower (J) and higher (H) energy 27 17 J and H aggregates 18 bands. The angle and the distance between the dipolar moments of the two monomers comprising the 28 dimer were obtained from the lower energy absorption band. 29 While the angle was more or less similar in all six dimers, the separation distance between the mono- 30 mer dipole moments differed dramatically. The intensity of the CD signal displayed a linear dependence 31 with the inverse square of the dipole moment distances. 32 33 34 35 36 1. Introduction anthocyanidin 3-glucosides, raises the question of self-aggregation 59 and its influence on the colour definition. 60 37 Anthocyanins are the pigments responsible for the beautiful red In a series of papers Hoshino etal. (1982) demonstrated that 61 38 to blue colours of flowers and fruits. However, when their pH anthocyanidin 3,5-diglucosides self-aggregate by stacking in a 62 39 dependent equilibrium is studied in water at low concentrations, right-handed or left-handed screw axis. While quinoidal bases of 63 40 the red flavylium cation is only stable at very acidic pH values, cyanin and pelargonin lead to right-handed adducts, peonin, 64 41 while the blue quinoidal base is a minor species (<5%) at moder- delphin and malvin form left-handed ones. On the other hand, all 65 42 ately acidic ones. Due to the fact that the pH of the vacuoles, where the respective flavylium cations lead to aggregates exhibiting 66 43 anthocyanins are located, changes roughly from 3 to 6 ( Steward left-handed CD signals, due to a super-asymmetry imposed by 67 44 Q4 et al., 1975) some kind of interactions that permit to achieve colour the oligomeric species (Rodger and Nordén, 1997). 68 45 in these conditions must take place. One solution found by Nature In a recent paper we have carried out an extended studied of the 69 46 regarding the achievement of blue colour, is the formation of self-aggregation in the six most abundant anthocyanidin 3-gluco- 70 47 supramolecular structures involving metals and flavonoids, which sides (Leydet et al., in press), by means of 1H NMR and UV–Vis 71 48 are able to stabilize the blue quinoidal base (Yoshida et al., 1995, absorption. It was verified that the rate and equilibrium constants 72 49 2009). of the network of chemical reactions involving the dyes is dramat- 73 50 In the case of red colour it was reported for raspberry the pres- ically dependent on the anthocyanin concentration. In this work 74 51 ence of high concentrations of cyanidin 3-glucoside (kuromanin, we report the circular dichroism spectra of these compounds, 75 52 ca. 2.4 mg/g of fresh fruit) and other derivatives bearing different Scheme 1, and correlate the magnitude of the CD signal with the 76 53 sugars in position 3 (Melo et al., 2000). Nature uses high concentra- angle and distance between the dipolar moments of the monomers 77 54 tions of the anthocyanin to compensate for the fact that is not in the dimer. 78 55 using the total colouring power of the flavylium cation (for pH 56 3.1 found in raspberry extract, the mole fraction of flavylium cation 2. Experimental 79 57 of the cyanidin 3-glucoside is only ca. 0.33). The use of high 58 concentration of anthocyanins (Wu et al., 2006), in particular Myrtillin chloride, oenin chloride, kuromanin chloride and 80 callistephin chloride were purchased from Extrasynthèse; peoni- 81 Q2 ⇑ Corresponding author. din-3-glucoside chloride and petunidin-3-glucoside chloride were 82 E-mail address: [email protected] (F. Pina). purchased from PhytoLab. All the reagents (P95%) were used 83 2 OH OCH3 3' OH 2' 4' OH OH 8 HO O 2 B HO O 5' + HO O 7 + + A C 6' 6 3 OGlc OGlc 5 4 OGlc OH OH OH Pelargonidin-3-glucoside (callistephin) Cyanidin-3-glucoside (kuromanin) Peonidin-3-glucoside OH OH OCH3 OH OH OH HO O HO O HO O + OH + OCH3 + OCH3 OGlc OGlc OGlc OH OH OH Delphinidin-3-glucoside (myrtillin) Petunidin-3-glucoside Malvidin-3-glucoside (oenin) Scheme 1. The six anthocyanidin 3-glucosides. 84 without any further purification. The solutions were prepared in tillin chloride, oenin chloride, kuromanin chloride, callistephin 105 85 Millipore water, and HCl 0.1 M was employed to acidify the chloride, peonidin-3-glucoside chloride and petunidin-3-glucoside 106 86 samples at pH 1. chloride) on a UV–Vis spectrophotometer Jasco V-530. 107 87 UV–Vis absorption spectra were recorded on Varian Cary 100 Molar ellipticity for each compound has been calculated using 108 88 Bio, Varian Cary 5000i and Jasco V-530 spectrophotometers. the formulae: 109 110 ½¼h h=ðC:l:10Þdeg cm2 dmolÀ1 112112 89 2.1. Circular dichroism analysis where h is the ellipticity (mdeg), C the concentration (mol lÀ1) and l 113 the path length (cm). 114 90 Myrtillin chloride, oenin chloride, kuromanin chloride, calliste- CD spectra of the appropriate blanks (HCl 0.1 M) were recorded 115 91 phin chloride, peonidin-3-glucoside chloride and petunidin-3-glu- and subtracted from the anthocyanidin 3-glucosides spectra. 116 92 coside solutions at pH 1 were prepared 12 h before the 93 experiments and kept at 4 °C. 94 Circular dichroism were performed using near-UV and visible 3. Results and discussion 117 95 (350–700 nm) CD in a Jasco J810 spectropolarimeter equipped 96 with a temperature control unit Julabo F25 using a range of con- 3.1. Circular dichroism 118 97 centration of the different 3-glucoside anthocyanins from 10À3 to 98 10À6 M. Near-UV and visible CD spectra were recorded with 0.01, The circular dichroism spectra representing the ellipticity of 119 99 0.05, 0.1 and 1 cm (linear) path length quartz cuvette at 20 °C, petunidin-3-glucoside at pH 1.0 is shown in Fig. 1A. The molar 120 100 according to the measured high tension (HT) voltage measured in ellipticity at the maximum of the first and second cotton bands 121 101 each sample. For each spectrum, three scans were averaged and is represented in Fig. 1B. The same is shown for oenin (malvidin 122 102 anthocyanidin 3-glucosides concentration was checked by absor- 3-glucoside) in Fig. 2A and B. Identical figures for the four remain- 123 103 bance at the maximum in the visible region using the molar ing anthocyanins of Scheme 1, are reported in Supplementary 124 104 absorption coefficients of each flavylium cation under study (myr- material, Fig. S1–S4. At lower concentrations the molar ellipticity 125 A B 400 4 104 300 461 nm 4 200 ) 2 10 -1 100 dmol 2 0 0 (mdeg) θ -100 ] (deg cm θ [ 4 -200 -2 10 -300 539 nm -4 104 -400 360 400 440 480 520 560 600 640 0 2 10-4 4 10-4 6 10-4 8 10-4 1 10-3 1.2 10-3 Wavelength (nm) Concentration (M) Fig. 1. (A) Ellipticity of petunidin-3-glucoside as a function of concentration (up to 10À3 M); (B) molar ellipticity at the first (539 nm) and second (461 nm) cotton bands. À1 KD = 900 M . 3 A B 150 1 104 100 492 nm 5000 ) 50 -1 0 dmol 0 2 -50 (mdeg) θ -5000 ] (deg cm -100 θ [ -150 -1 104 550 nm -200 -1.5 104 -250 360 400 440 480 520 560 600 640 0 4 10-4 8 10-4 1.2 10-3 1.6 10-3 Wavelength (nm) Concentration (M) À3 À1 Fig.