Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution.

Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution André Manuel Alves de Sousa Programa Doutoral em Química Departamento de Química e Bioquímica 2015 Orientador Victor de Freitas, Professor Catedrático, Faculdade de Ciências da Universidade do Porto Coorientador Nuno Mateus, Professor Associado com Agregação, Faculdade de Ciências da Universidade do Porto ! eressado, que a tal se compromete. This work was possible thanks to FCT (Fundação para a Ciência e Tecnologia) for a PhD grant (ref. SFRH/BD/68736/2010) and a research grant (PTDC/QUIQUI/117996/2010). Financial support was conceded to work in another institution (Faculty of Sciences and Technology, New University of Lisbon) as well as funding to international conferences. Studies presented in this dissertation were performed in the Chemistry and Biochemistry Department of Faculty of Sciences, University of Porto, in the Group of Food Chemistry (line 2) and in the Group of Physical Chemistry of Faculty of Sciences and Technology, New University of Lisbon. “Only those who attempt the absurd can achieve the impossible.” – Albert Einstein “Stay hungry. Stay foolish.” – Steve Jobs FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution vii Agradecimentos . Desta forma gostaria de expressar o meu sincero agradecimento: Ao meu orientador Professor Doutor Victor Freitas, sem o qual este tra teria sido . Obrigado pela oportunidade que me deu de trabalhar num grupo de excelência e pelo contributo que teve no meu crescimento científico, de investigação e pessoal. Ao meu co-orientador Professor Doutor Nuno Mateus, sempre disponível, interessado e bem-disposto. que me proporcionaram, os professores representarão sempre as minhas referências máximas de formação. da Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, em particular ao Professor Doutor Fernando Pina e ao Doutor Vesselin Petrov. Duas das mentes mais brilhantes que alguma vez tive o prazer de conhecer. O estágio que realizei neste grupo foi sem dúvida uma experiência extremamente enriquecedora. . . (SFRH/BD/68736/2010) e do projeto no qual este trabalho se encontra inserido, PTDC/QUI-QUI/117996/2010. 2 do Centro de Faculdade de Ciências, nomeadamente à Dra. Zélia pela ajuda técnica nas análises de LC-MS e à Mariana pela disponibilidade e apoio na realização de análises de RMN. xiv FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution xv Abstract Polyphenols assume an important role in food matrixes and namely in wines, contributing directly to their organoleptic properties such as color and flavor. Some of these compounds, such as anthocyanins and derivatives present intense colors and are relatively stable in aqueous solutions. This feature has given them the potential advantage to act as viable commercial food colorants, which has consequently led to advances in the research developed in their chemistry over the years. The purpose of this project was to investigate and study pigments with interesting color features and stability in aqueous solutions that could have potential applications in food, health and other industries. Open chalcone forms of the common anthocyanins are assumed to be crucial in reactions leading to irreversible degradation of anthocyanins, particularly under weakly acidic to weakly alkaline solution conditions. However, the natural deoxyanthocyanidins are much more stable in slightly acidic solutions than anthocyanins and anthocyanidins, which points to the potential advantage of this type of compounds as viable commercial food colorants, and justifies the research developed in the chemistry of 3deoxyanthocyanins and, in particular, the search for new colorants with significant stability. In addition, more studies have demonstrated other potential applications for these compounds, such as their use as hair dyes, laser dyes, sensitizers for solar cells, and molecular-level memory systems. Bearing this, several new pigments of the family of deoxyanthocyanidins were synthesized, isolated and structurally characterized. In the first work, the reaction between dimeric procyanidins, which are present in real wines, and cinnamic aldehydes which are extracted from wood barrels during wine aging was studied. The formation of new orange pigments that were oaklin-catechin adducts was observed and confirmed. These adducts are classified as deoxyanthocyanidins, since they are not substituted in the carbon in m-position to the positively charged oxygen of the flavylium core. The hypothetic mechanism of formation of these new pigments was elucidated and the adduct formed was structurally characterized by mass spectrometry and NMR. A new and simple synthesis procedure to obtain 3-deoxyanthocyanidins, particularly deoxypeonidin and deoxymalvidin, from the reaction between phloroglucinol and cinnamic aldehydes (coniferaldehyde and sinapaldehyde) was described. The stability of different types of deoxyanthocyanidins was investigated by UV-Vis spectroscopy, comparing their behaviour and physical-chemical properties under xvi FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution different pH conditions and light stimulus with analogous compounds such as common anthocyanins and other flavyliums. The rate and equilibrium constants of the respective pH dependent network of chemical reactions presented by these compounds were calculated and their photochemistry evaluated. The synthesis of new compounds analogous to pyanoanthocyanins (visitins) was carried out. The resulting products were designated deoxyvitisins. The color stability and the network of chemical reactions occurring in aqueous solution upon pH variations for the newly synthesized deoxyvitisins were thourougly investigated. Deoxyvitisins were found to be less acidic, thus more stable, than the corresponding pyranoanthocyanin 3-Oglucosides. With a deeper understanding of the physical-chemical properties of deoxyanthocyanidins provided by the previous works, it was investigated the contribution that other effects might have on color features of these compounds. Hence, copigmentation interactions were studied between oaklins (ring A substituted deoxyanthocyanidins), which have been previously identified in wines, and several copigments: catechin, epicatechin, chlorogenic acid, epigallocatechin, and procyanidin B3. The results showed that oaklins, like common anthocyanins, also present copigmentation interactions that further stabilize the flavylium cation in hydroalcoholic solutions. Molecular dynamic simulations were also performed to interpret the binding data, to specify the relative arrangement of the pigment and copigment molecules within the complexes, and to interpret their absorption properties in the visible range. Finally, the antioxidant and antiproliferative properties of several deoxyanthocyanidins were evaluated by DPPH, FRAP, liposomes peroxidation inhibiton assays and antiproliferative effects against cancer cell lines from stomach and colon. The results showed that all the deoxyanthocyanidins tested demonstrated antiradicalar, antioxidant and antiproliferative properties. It was very interesting to observe that one of the deoxyanthocyanidins, the oaklin guaiacylcatechinpyrylium, despite showing lower antioxidant/antiradicalar capacity in DPPH and FRAP assays when compared to an anthocyanin (cyanidin-3-glucoside), revealed a surprising higher antioxidant effect in the liposomes model. The use of liposomes constitutes a more promising method for assessing antioxidant properties relevant to human nutrition, since it uses a model biological membrane. Oaklins, which were previously shown to have interesting color features for its use as food colorants, due to their higher color stability in acidic/neutral pH solutions, also revealed antioxidant properties similar to common anthocyanins. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution xvii This research has allowed a significative scientific advance in the study of a specific anthocyanidin derived type of coumpounds, named deoxyanthocyanidins. They have been previously identified and described but there has never been a profound and extensive study about their characteristics. KEYWORDS: anthocyanins, deoxyanthocyanidins, oaklins, food colorants, wine aging, flavylium network reactions, photochemistry, copigments, antioxidante properties, UVVis spectroscopy, mass spectrometry, NMR xviii FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution xix Table of Contents Agradecimentos ........................................................................................................... vii Resumo ........................................................................................................................ xi Abstract ....................................................................................................................... xv Table of Contents ....................................................................................................... xix List of Figures ............................................................................................................. xxi List of Tables ........................................................................................................... xxvii Scientific Communications and Thesis Organization ................................................. xxxi General Introduction ................................................................................................... 33 1. Flavonoid and non-flavonoid compounds ........................................................... 36 2. Anthocyanins ...................................................................................................... 38 3. Deoxyanthocyanidins ......................................................................................... 41 4. Physico-chemical properties of anthocyanins and deoxyanthocyanidins ............ 44 4.1 Flavylium reaction network ........................................................................... 44 4.2 Deoxyanthocyanidins ................................................................................... 45 4.3 Self-association of anthocyanins .................................................................. 45 4.4 Intramolecular copigmentation ..................................................................... 46 4.5 Intermolecular copigmentation ..................................................................... 47 5. Importance of the reactions between polyphenolic compounds .......................... 48 6. Oaklins ............................................................................................................... 51 7. Antioxidant properties of anthocyanins and deoxyanthocyanidins ...................... 55 7.1 Experimental methods to measure antioxidant properties ............................ 56 7.1.1 DDPH – antiradical capacity ................................................................. 56 7.1.2 FRAP – reducing power ........................................................................ 57 7.1.3 Lipid peroxidation inhibition in liposomes .............................................. 57 8. Polyphenolic compounds in food ........................................................................ 58 Objectives ................................................................................................................... 61 Results ....................................................................................................................... 65 xx FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Chapter 1 ............................................................................................................... 67 Synthesis and Structural Characterization of Oaklin-Catechins .......................... 67 Chapter 2 ............................................................................................................... 83 A novel reaction mechanism for the formation of deoxyanthocyanidins .............. 83 Chapter 3 ............................................................................................................... 95 Thermodynamics, Kinetics and Photochromism of Oaklins: a Recent Family of Deoxyanthocyanidins ......................................................................................... 95 Chapter 4 ............................................................................................................. 117 Deoxyvitisins: a new set of pyrano-3-deoxyanthocyanidins .............................. 117 Chapter 5 ............................................................................................................. 131 Color Stability and Spectroscopic Properties of Deoxyvitisins in Aqueous Solution ........................................................................................................................ 131 Chapter 6 ............................................................................................................. 143 Evidence for Copigmentation Interactions between Deoxyanthocyanidin Derivatives (Oaklins) and Common Copigments in Wine Model Solutions ....... 143 Chapter 7 ............................................................................................................. 163 Antioxidant and antiproliferative properties of 3-deoxyanthocyanidins.............. 163 Conclusion and Final Considerations ........................................................................ 179 Literature Cited ......................................................................................................... 187 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution xxi List of Figures Fig. 1 - Subclasses of flavonoids................................................................................... 36 Fig. 2 - Base structure of flavonoid compounds (2-phenylbenzopyran, commonly named flavanic nucleous). ........................................................................................................ 37 Fig. 3 - 3-flavanol monomers (catechins and gallocatechins). ....................................... 37 Fig. 4 - Type B dimeric procyanidins. ............................................................................ 38 Fig. 5 - Anthocyanins (flavylium cation) an λmax values in acidic methanol (adapted from (Stintzing and Carle 2004)). .................................................................................. 39 Fig. 6 - Acylated esters of malvidin 3-glucoside. ........................................................... 40 Fig. 7 - Malvidin 3-glucoside pyruvic derivative. ............................................................ 41 Fig. 8 – y y ’ . ....................................................................... 42 Fig. 9 - y ’ blood resin. ................................................................................................................... 43 Fig. 10 – Flavylium network of chemical reactions. ....................................................... 44 Fig. 11 - k y ’ -association phenomenon (adapted from (Hoshino 1991)). ................................................................................................... 46 Fig. 12 - Representative scheme of the intramolecular copigmentation (adapted from (Kondo et al. 1991)). ..................................................................................................... 47 Fig. 13 - Examples of intermolecular copigmentation (adapted from Haslam, 1998). .... 48 Fig. 14 - Formation mechaninsm of malvidin 3-glucoside-alkyl/aryl-catechin adducts (adapted from (Bendz, Martenss 1967))........................................................................ 49 Fig. 15 - Anthocyanin discoloration reactions: H2O and HSO-3 addtions. ....................... 50 Fig. 16 - Hypothetic mechanism for the formation of catechin-pyrylium pigment obtained from catechin and sinapaldehyde (adapted from de Freitas, 2004). .............................. 53 xxii FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Fig. 17 - Structures of the catechinpyrylium pigments resulting from the reaction between catechin and coniferaldehyde (R = H, guaiacylcatechinpyrylium) or sinapaldehyde (R = OCH3, syringylcatechinpyrylium) (adapted from Sousa, 2005). ..................................... 54 Fig. 18 - DPPH radical structure (2,2-diphenyl-1-picrylhydrazyl). .................................. 57 Fig. 19 - Redox reaction of ferric tripyridyltriazine to ferrous tripyridyltriazine. ............... 57 Fig. 20 - HPLC chromatogram recorded at 500 nm of the model solution containing B4 and coniferaldehyde, after 5 days of reaction at pH 3.5 and 35ºC. UV/Vis spectrum of GCP-catechin (* - unidentified peaks; a - GCP-catechin). ............................................. 73 Fig. 21 - Mass spectra and respective MS2 (of the molecular ion) and MS3 (of the main fragment in MS2) fragmentations for GCP-catechin and SCP-catechin adducts. ........... 74 Fig. 22 – Fragmentation pattern of GCP-catechin (R = H) and SCP-catechin (R = OCH3) adducts in the positive ion mode. .................................................................................. 75 Fig. 23 - Hypothetical mechanism for the formation of oaklin-catechins IX obtained from the reaction between procyanidin B4 III and cinnamic aldehydes I. ............................... 80 Fig. 24 - Structure of the new synthesized 3-deoxyanthocyanidins 9 (R=H, 3deoxypeonidin; R=OCH3, 3-deoxymalvidin). ................................................................. 87 Fig. 25 - Visible spectra of 3-deoxypeonidin (A) and 3-deoxymalvidin (B) determined directly by HPLC-DAD (pH≈2). ...................................................................................... 88 Fig. 26 - Kinetic study of the reaction between phloroglucinol and coniferaldehyde. A – Influence of molar ratios (phloroglucinol:coniferaldehyde) at pH 1.5, 12% ethanol: (●) 1:1; (■ 1:5; ♦ 1:10; ▲) 5:1; (▼) 10:1; B – Influence of diferent pH values and percentage of ethanol: (●) pH 1.0 /12% ethanol ; (■ H 1.5 /12% ; ♦ H 3.5 /12% ethanol; (▲) pH 1.0/20% ethanol; (▼) pH 1.5/30 % ethanol (for a molar ratio of 1:10, phloroglucinol:coniferaldehyde). ........................................................................... 89 Fig. 27 - Mechanism proposed for the formation of 3-deoxypeonidin and 3-deoxymalvidin 9 obtained from the reaction between phloroglucinol 3 and cinnamic aldehydes 1. ....... 92 Fig. 28 - Network of chemical reactions of malvidin-3-glucoside. .................................. 98 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution xxiii Fig. 29 - The model compound Deoxypeonidin (DOP) and the derivatives Guaiacylcathechinpyrylium (GCP), and Syringylcatechinpyrylium (SCP). ..................... 99 Fig. 30 - A - Spectral variations of the compound deoxypeonidin immediately after a pH jump from 1 to the range 1.9<pH<5.3; B - the same for 5.3<pH<10; C - Fitting of the absorption at 480 nm and 550 nm as a function of pH; D - Absorption spectra of the flavylium cation, quinoidal base and ionized quinoidal bases obtained by mathematical decomposition............................................................................................................. 102 Fig. 31 - Spectra of equilibrated solutions of deoxypeonidin at different pH values, showing the equilibrium between AH+, A and Ct; inset representation of the fitting obtained at two different wavelengths (pKca=3.6±0.1).................................................. 103 Fig. 32 - A - Spectral variations after a pH jump from 1 to 4.7 (kobs=3.8x10-4 s-1) - inset trace at 495 nm, the ratio between the final absorbance and initial one gives 0.54; B - representation of the observed rate constant as a function of pH. Fitting was achieved for pKa=3.9; KhKtki=2.6×10-8; k-i=2.55×10-4 s-1; kiKt/k-h=6.65×10-6 M. ................................. 104 Fig. 33 - A - Spectral variations following the irradiation of DOP at pH=4.3 at the irradiation wavelength of 366 nm; B - flash photolysis of a solution of DOP, pH=4.4: flash photolysis showing the trace of the flavylium formation (493 nm) up and the recovery of the trans-chalcone (393 nm) down; C - representation of the rate constants of the flash photolysis process as a function of pH. ....................................................................... 106 Fig. 34 - Energy level diagram for DOP based on the equilibrium constants of Table 3. The energy levels of B and Cc were obtained through the fitting of eqs.(7) to (9) due to the lack of observation of traces corresponding to B and Cc in the reverse pH jumps experiments carried out by stopped flow. .................................................................... 108 Fig. 35 - A - Spectral variations of the compound guaiacylcatechinpyrylium (GCP) 1.6×10-4 M (10% EtOH) immediately after a pH jump from 1 to the range 1.6<pH<5.2; B - the same for 5.2<pH<9; C - Fitting of the absorption at 485 nm and 563 nm as a function of pH; D - Absorption spectra of the flavylium cation, quinoidal base and ionized quinoidal base obtained by mathematical decomposition. ........................................... 109 Fig. 36 - Spectra of equilibrated solutions of GCP (A) at different pH values, showing the equilibrium between AH+, A and Ct; inset representation of the fitting obtained at two different wavelengths (pKca=3.05±0.1). ....................................................................... 110 xxx FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution NMR – Nuclear Magnetic Resonance ROS – Reactive oxygen species Pg – Pelargonidin Pn – Peonidin Pt – Petunidin s – Singlet Scp - Syringylcatechinpyrylium rt – Retention time Trolox - 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid UV – Ultraviolet Vis – Visible δ – Chemical shift λmax – maximum absorption wavelenght FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution xxxi Scientific Communications and Thesis Organization This dissertation integrates results that were published in international scientific conferences through panel and oral communications and in international journals with known scientific recognition: Papers published on international scientific journals 1. Sousa, A., Fernandes, A., Mateus, N., de Freitas, V. (2012) Synthesis and Structural Characterization of Oaklin-Catechins. J. Agric. Food Chem. 60(6): 1528– 1534. doi:10.1021/jf204408p 2. Sousa, A., Mateus, N., de Freitas, V. (2012) A novel reaction mechanism for the formation of deoxyanthocyanidins. Tetrahedron Lett. 53(10): 1300-1303. doi:10.1016/j.tetlet.2012.01.006 3. Sousa, A., Petrov, V., Araújo, P., Mateus, N., Pina, F., de Freitas, V. (2013) Thermodynamics, Kinetics and Photochromism of Oaklinsa Recent Family of Deoxyanthocyanidins. J. Phys. Chem. B. 117(6): 1901-10. doi:10.1021/jp3110216. 4. Sousa, A., Araújo, P., Mateus, N., de Freitas, V. (2013) Deoxyvitisinsa new set of pyrano-3-deoxyanthocyanidins. Tetrahedron Lett. 54(35): 4785-4788. doi:10.1016/j.tetlet.2013.06.135 5. Sousa, A., Cabrita, L., Araújo, P., Mateus, N., Pina, F., de Freitas, V. (2014) Color stability and spectroscopic properties of deoxyvitisins in aqueous solution. New J. Chem., 38(2): 539-544. doi:10.1039/c3nj01271a 6. Sousa, A., Araújo, P., Cruz, L., Brás, N. F., Mateus, N., de Freitas, V. (2014) Evidence for copigmentation interactions between deoxyanthocyanidin derivatives (oaklins) and common copigments in wine model solutions. J. Agric. Food Chem. 62(29): 6995–7001. doi:10.1021/jf404640m 7. Sousa A., Araújo, P., Azevedo, J., Cruz, L., Fernandes, I., Mateus, N., de Freitas, V. (2015) Antioxidant properties of 3-deoxyanthocyanidins. Submitted to Food Chemistry. xxxii FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Oral communications 1. Sousa, A. . M . . “Synthesis and structural k ”. “9 Sy ’ y” 15-17 June 2011. 2. Sousa, A., Petrov, V., Araújo, P., Mateus, N., Pina, F., de Freitas, V. “Thermodynamics, Kinetics, and Photochromism of OaklinsA Recent Family of y y ”. “8 y S ” R 2-5 July 2013. Panel communications 1. Sousa, A., Fernandes, A., Mateus, N., de Freitas, V. “Sy k ” “9 Sy ’ y” 15-17 June 2011 “34 W W ” 20-27 June 2011 “XX E S ” 3-6 July 2011 “ y S ” G 20-23 July 2011 2. Sousa, A., Mateus, N., de Freitas, V. “ y y ” “XX y ” y 22-26 July 2012 3. Sousa, A., Araújo, P., Mateus, N., de Freitas, V. “ y : a new set of pyrano-3y y ” “10º E ” 4-6 September 2013 “7 W k y ” 9-11 September 2013 4. Sousa, A., Petrov, V., Araújo, P., Mateus, N., Pina, F., de Freitas, V. “Thermodynamics, Kinetics, and Photochromism of OaklinsA Recent Family of y y ”. “XX E S ” 12-14 June 2013. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 33 General Introduction 34 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution The evolution process of the Plant Kingdom has led several plants to adopt defense mechanisms against agressions of their environment, leading to changes in their ability to synthethize and use chemical compounds that control their growth and development. Some of these compounds act as toxins against pathogenic agents or herbivores and others attract symbionts and other life beings required for procreation. The need for these chemical responses to changing environments has originated over time an enormous structural diversity on the largest groups of secondary metabolites that include alkaloids, terpenoids and other polyphenolic compounds. The latter assume a great importance, playing a range of diverse roles, such as the defense against agressors, flowers and fruits coloring, attraction of symbionts and the regulation of the cellular growth and maturation. Polyphenols belong to a class of chemical compounds, which feature a benzene system with one or more hydroxyl groups, which in their turn may be methylated or glucosylated. They can be divided into several groups according to their properties, but are generically classified as non-flavonoids, including phenolic acids and other derivatives such as stilbenes, or flavonoids, which include flavonols, flavones, anthocyanins and others. Polyphenols assume an important role in food matrixes and namely in wines, contributing directly to their organoleptic properties such as color and flavor. Anthocyanins, for example, are responsible for the majority of red/violet color in vegetables, while tanins are responsible for the adstringency and bitterness in food. In wine, throughout the fermentation and aging processes, these compounds undergo major changes, quantitatively and structurally, usually leading to the formation of new products with different physical-chemical properties from their precursors. Some of these compounds, such as anthocyanins and derivatives present intense colors and are relatively stable in aqueous solutions. This feature has given them the potential advantage to act as viable commercial food colorants, which has consequently led to advances in the research developed in their chemistry over the years, in particular, the search for new colorants with significant stability. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 35 Furthermore, these compounds present interesting photochemical behaviors in certain conditions and may undergo reversible and non-reversible structural transformations, according to pH, concentration, solvents, light stimulus, etc. These recent discoveries have widen up the range of their applications which now extend to their use as hair dyes, laser dyes, sensitizers for solar cells, and molecular-level memory systems. Besides their obvious use and potential in the industry of food colorants, cosmetics, energy transformation and information systems, they also have been studied for their antioxidant properties and regarded as promising health promoting agents and of great nutritional value in diets. 36 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 1. Flavonoid and non-flavonoid compounds Phenolic compounds present as its base structure an aromatic ring, which can be attached to one or more hydroxyl groups, leading to a wide range of compounds. Polyphenols can be divided into two large groups: flavonoids, which are part of flavanols, flavonols, flavanonones, flavones and anthocyanins (Fig. 1); and non-flavonoids, which include phenolic acids (benzoic and cinnamic), and other phenolic derivatives such as stilbenes (e.g. resveratrol). Fig. 1 - Subclasses of flavonoids. Flavonoids are compounds widely distributed in the plant kingdom, are present in fruits, leaves, seeds and other plant parts in the form of glycosides and aglycones. They are compounds with a relatively low molecular weight, consisting of 15 carbon atoms arranged in a C6-C3-C6 configuration. The chemical structure of flavonoids consists of two aromatic rings, designated ring A and B, joined by three carbons which form an heterocyclic ring, called ring C (Merken and Beecher 2000) (Fig. 2). Substitutions on ring C result in important classes of flavonoids such as flavonols, flavones, flavanones, flavanols (e.g. catechins), isoflavones and anthocyanidins (Fig. 3). Substitutions in rings A and B give different compounds within each class of flavonoids. These substitutions may include oxygenation, alkylation, glycosylation, acylation and sulfation (Hollman and Katan 1999). Flavones Anthocyanidins Flavononols Flavonols Flavan-3-ols Flavanones FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 37 Fig. 2 - Base structure of flavonoid compounds (2-phenylbenzopyran, commonly named flavanic nucleous). Taking a closer look at the group of flavanols, their types of structures in nature differ in the stereochemistry of the asymmetric carbons of the piranic ring (C2 and C3) as well as in the degree of hydroxylation of the rings A and B (Fig. 3). The monomeric flavanols most common in plants, and in particular the species Vitis vinifera, are hydroxylated at positions 5 and 7 of the ring A, differing only in the number of hydroxyl groups on ring B and on the stereochemistry of carbon 3 from ring C. Fig. 3 - 3-flavanol monomers (catechins and gallocatechins). In nature, flavanols also exist in polymerized forms, called proanthocyanidins (or condensed tannins). This family of polymers differ in the nature of the constituent monomer units (procyanidins consist of catechins, while prodelphinidins consist of gallocatechins), in the type of interflavanolic bond between these units (e.g. C4-C6 or C4-C8 bonds which are characteristic of dimeric procyanidins of type B, Fig. 4) and in their degree of polymerization (dimers, trimers, oligomers and polymers) (Batesmith 1954, Haslam 1996). Flavanol R1 R2 R3 (+)-catechin OH H H (-)-epicatechin H OH H (+)-gallocatechin OH H OH (-)-epigallocatechin H OH OH 38 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Fig. 4 - Type B dimeric procyanidins. 2. Anthocyanins Anthocyanins (from the Greek anthos meaning flower and kyanos which means blue) are the most abundant and important pigments of plants, fruits and vegetables, being responsible for the extensive range of colors presented by these products. They are water-soluble pigments and are present in the vacuole of the epidermal cells of plants, generally giving them a red, violet or blue color, according to the vacuolar pH. Anthocyanins can be found in all plant tissues, leaves, roots, stems, flowers and fruits. They are not found in animals, plants or marine organisms. Their role as coloring agents constitutes an important factor in animal attraction, aiding in pollination, and having a considerable value in this co-evolution of plant-animal interactions. Besides the coloring functions in plants, they are particularly important in UV protection, defense against pathogens and insect attacks (Stintzing and Carle 2004). Its structure corresponds to the glucosylated form of anthocyanidins, which comprise the flavylium cation polyhydroxylated and/or methoxylated. The flavylium cation corresponds Flavanol R1 R2 R3 R4 B1 OH H H OH B2 OH H OH H B3 H OH H OH B4 H OH OH H B5 OH H OH H B6 H OH H OH B7 OH H H OH B8 H OH OH H FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 39 to a benzene system formed by the rings A, B and C and anthocyanidins differ in the degree of hydroxylation and/or methoxylation of the B ring (Fig. 5). Anthocyanidin R1 R2 R3 λmax (nm) Pg H H H 520 Cy H OH H 535 Df H OH OH 546 Pn H OCH3 H 532 Pt H OCH3 OH 543 Mv H OCH3 OCH3 542 Anthocyanin Pg 3-glc Glc H H 516 Cy 3-glc Glc OH H 530 Df 3-glc Glc OH OH 543 Pn 3-glc Glc OCH3 H 536 Pt 3-glc Glc OCH3 OH 546 Mv 3-glc Glc OCH3 OCH3 546 Fig. 5 - Anthocyanins (flavylium cation) and their λmax values in acidic methanol (adapted from (Stintzing and Carle 2004)). 46 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution self-association is explained by hydrophobic interactions between the aromatic rings, forming a complex with a chiral packing geometry, in which the axes of the superimposed structures form angles ranging between 0° and 90º (Hoshino 1991). Fig. 11 - k y ’ -association phenomenon (adapted from (Hoshino 1991)). In this model there is a formation of an extensive hydrophobic core surrounded by glycosidic residues that make this entire complex water-soluble, while protecting the ring of the flavylium cation from a water nucleophilic attack, thus enabling stabilization against color loss. Given that this type of interaction is very strong, in the usual concentrations of these pigments in petals and flowers, this self-association phenomenon of anthocyanins contributes significantly to the colors displayed in vivo (Hoshino 1991). 4.4 Intramolecular copigmentation Another phenomenon, which contributes to the stabilization of the color, is the intramolecular copigmentation that helps to stabilize the color of anthocyanins for slightly acidic to neutral pH values. Some anthocyanins are acylated with hydroxycinnamic acids and their phenolic groups form a sort of "sandwich" with the aromatic core of the anthocyanin (Fig. 12). This phenomenon consists of π-π hydrophobic intramolecular interactions between the two phenolic groups rich in electrons and the electron deficient core of the anthocyanin (the flavylium cation or the quinoidal base). The anthocyanin is FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 47 thus protected from the attack of water at higher pH values, contributing to a stabilization of the color (Kondo, Yoshida 1991). Fig. 12 - Representative scheme of the intramolecular copigmentation (adapted from (Kondo et al. 1991)). 4.5 Intermolecular copigmentation Anthocyanins also present other mechanisms for the stabilization of its color in the presence of copigments, which are compounds that when isolated may even have very weak staining or may be even colorless, but when added to a solution of anthocyanins may contribute significantly to color retention. Analogously to what happens in intramolecular copigmentation, this phenomenon consists of hydrophobic interactions that take place in an aqueous solution between an electron-rich system (copigments) and an electron-deficient system (anthocyanin). These interactions thus protect anthocyanins from hydration and consequent color loss. This phenomenon is however dependent on various factors such as the type of copigments, the anthocyanin involved, pH, temperature, and even the presence of metals. Examples of copigments are flavonols, such as quercetin and hydroxycinnamic esters, such as glucose (Haslam 1998) (Fig. 13). Anthocyanidin Acyl group Acyl group 48 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Fig. 13 - Examples of intermolecular copigmentation (adapted from Haslam, 1998). 5. Importance of the reactions between polyphenolic compounds Polyphenolic compounds may react with each other and with other compounds, leading to structural changes and the subsequent modification of organoleptic properties in food. Flavanols, for example, have two hydroxyl groups in carbons C5 and C7 of ring A that induce formal negative charges by mesomeric donor effect in carbons C6 and C8, which conferrs a certain nucleophilic character to this ring. This characteristic is responsible for many already described reactions that occur involving these compounds. In wine, for example, reactions occurring between anthocyanins and flavanols mediated by aldehydes, result in the formation of anthocyanin-flavanol adducts with alkyl/aryl/furanyl bridges derived from the aldehydes (Fig. 14). These aldehydes may stem from the fermentation or from the transfer of compounds from the wood during the wine aging process (Timberlake and Bridle 1976, Rivas-Gonzalo, Bravo-Haro 1995, Pissarra, Lourenco 2004, 2005). Anthocyanic core Anthocyanic core Flavonol Hydroxycinnamic ester FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 49 [-H+] [-H2O] [-H+] Fig. 14 - Formation mechaninsm of malvidin 3-glucoside-alkyl/aryl-catechin adducts (adapted from (Bendz, Martenss 1967)). Regarding the mechanism, the aldehyde in acidic solution gives the carbocation by protonation of the carbonyl group. This cation will induce an electrophilic attack to the catechin ring, preferably in the position of carbon 8, since the negative formal charge is more delocalized for this carbon compared to carbon 6 (Bendz et al. 1967). An intermediate compound is formed, which then reacts with malvidin 3-glucoside yielding new adducts. Diastereoisomers (R and S) are also formed due to the presence of an asymmetric carbon in the interflavanoid bond (Bendz et al. 1967, Sousa, Mateus 2007). These new compounds have different structural, chromatic and stability characteristics from their precursors and can somehow play an important role in the color evolution of wine aging. The structure of anthocyanins also contains an important feature: the positive charge in the oxygen of ring C. This positive charge can be delocalized between carbons at position 2 and 4, conferring an electrophilic character to the pyranic ring. This fact provides two reactions, which define the color stability of anthocyanins. The first is the hydration reaction in the C2 position with the formation of hemiketal, as mentioned before and explained in detail; the second is the addition of bisulfite in the C4 position (Cheminat and Brouillard 1986, Berke, Cheze 1998, Escribano-Bailon, Alvarez-Garcia 2001) (Fig. 15). Sulfite is widely used in wine production, acting as an antioxidant agent Aldehyde Intermediate carbocation Catechin Adduct 50 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution and inhibitor of growth of unwanted microorganisms (Bridle & Timberlake, 1966). Both reactions result in the discoloration of the solution, thus representing a very important topic of study on the stability of anthocyanin compounds. Fig. 15 - Anthocyanin discoloration reactions: H2O and HSO-3 addtions. H2O HSO3incolor product incolor product FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 51 In aqueous solution, SO2 originates the bisulfite anion that rapidly and reversibly binds to carbon 4 of the flavylium cation, giving a colorless adduct. SO2 can also bind to carbon 2 of the anthocyanin, also resulting in color loss. The equilibrium constants of the reaction between bisulfite and anthocyanins are high, which is why small amounts of sulfite can bleach large quantities of anthocyanins (Timberlake and Bridle 1976). However, acidifying the medium, the equilibrium shifts toward the formation of the flavylium cation and the solution returns to the red color. To summarize, it should be highlighted the importance of the reactions occurring between polyphenolic compounds, namely anthocyanin pigments, and its role in the evolution of food color. Particularly in wines, reactions that occur in the beginning of the winemaking process originate new pigments different from their precursors. And during the aging process other oxidation and polymerization reactions still contribute to the evolution of color of wines over the years. In this complex process, it should also be noted the importance of certain aldehydes and whose presence in wine is due to several factors, as by-products of alcoholic fermentation, resulting from the oxidation of higher alcohols, or by being extracted directly from the wood barrels where wines are stored (Wildenra.Hl and Singleto.Vl 1974, Canas, Belchior 2003, Pissarra, Mateus 2003). Wine storage also marks the importance of wood and the cooperage industry in the wine business; hence the composition of aldehydes can decisively influence the organoleptic properties of wines, especially color. 6. Oaklins The use of oak wood (e.g., barrels and chips) in wine storage during the first years of aging is a procedure widely employed by winemakers, strengthening the wine organoleptic characteristics and contributing to its stability. Nevertheless, it must be carried out with caution to obtain better quality and well-balanced wines (Sanza, Dominguez 2004, Sanza, Domniguez 2004, Sanza, Escudero 2004). Aging in oak wood allows wine to extract a series of phenolic and volatile compounds, depending upon the characteristics of the oak and the contact time between wine and wood. The factors that affect the pool of oak extractables are the species and 52 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution geographical origin of the wood (Chatonnet 1991, Miller, Howell 1992), as well as its processing, especially the method to obtain the staves, their seasoning, and the degree of oak toasting (Sefton, Francis 1993, del Alamo, Bernal 2000). Among the potential compounds extractable from oaks, furanic (e.g., furfural and hydroxymethylfurfural), benzoic (e.g., hydroxybenzaldehyde and vanillin), and cinnamic aldehydes (e.g., coniferaldehyde and sinapaldehyde) are of particular interest because they can interact with some wine components (e.g., catechins and anthocyanins), hence contributing directly or indirectly to color and other sensory changes. As mentioned before, several studies have demonstrated that different aldehydes can serve as intermediaries in reactions involving catechin and anthocyanins or even react directly with catechin, yielding a variety of pigments with different color characteristics (Timberlake and Bridle 1976, Rivas-Gonzalo et al. 1995, Es-Safi, Cheynier 2000, Mateus et al. 2003). R y w / λmax at 500 nm) catechinpyrylium-derived pigment has been shown for the first time resulting from the direct reaction between catechin and sinapaldehyde, one of the main oak wood aldehydes (Fig. 16) (de Freitas, Sousa 2004). FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 53 Fig. 16 - Hypothetic mechanism for the formation of catechin-pyrylium pigment obtained from catechin and sinapaldehyde (adapted from de Freitas, 2004). [+H+] [+H+] -[H2O] [O] 54 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution In another work, it was reported the formation of brick-red pigments resulting from the reaction between catechin and an extract solution rich in oak aldehydes, especially sinapaldehyde and coniferaldehyde (Fig. 17). Fig. 17 - Structures of the catechinpyrylium pigments resulting from the reaction between catechin and coniferaldehyde (R = H, guaiacylcatechinpyrylium) or sinapaldehyde (R = OCH3, syringylcatechinpyrylium) (adapted from Sousa, 2005). The formation of a great variety of oaklins in the model solution with catechin and the oak extract was unambiguous, indicating that these pigments could also occur in wines aged in oak barrels. The formation of syringylcatechinpyrylium and guaiacylcatechinpyrylium at pH 3.5 and 25°C was indicative of the possibility of the existence of this type of pigments in wines aged in oak barrels. To evaluate it, a 4-year-old commercial table red wine aged in oak barrels during a year was analyzed. Further fractionation of the wine was performed and the full mass chromatograms of these fractions displayed several molecular ion peaks supposedly corresponding to different oaklins, presenting molecular ions and retention times analogous to the ones corresponding to syringylcatechinpyrylium, guaiacylcatechinpyrylium, and guaiacylcatechinpyrylium/syringylcatechinpyrylium catechin adducts. However and despite the extensive purification of the wine, the major part of these molecular ion peaks could not be individually selected because other compounds present in the obtained fractions were predominant. This did not allow the analysis of the fragmentation pattern of these pigments, thereby preventing further comparison and subsequent identification of these pigments with the various oaklins detected in model solution. This could be due to presumably low amounts of these pigments in wines, easily surpassed by other pigments such as the original anthocyanins and derived FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 55 compounds. Nonetheless, one of these pigments was individually and selectively detected in the wine. These results demonstrated the existence of one of the main oaklins in a red wine. Therefore, it is expected that several oaklins can occur in different red wines aged in oak barrels. Because of their intense red/orange color and although probably not occurring in high amounts, their presence may somehow contribute to some color changes observed during wine aging in oak barrels. This preliminary study confirmed the formation in model solutions of several red/orange pigments resulting from the reaction between catechin and aldehydes extracted from oak wood, namely, coniferaldehyde and sinapaldehyde. These pigments were all found to have a distinctive catechinpyrylium core and consequent related structural features, which lead to their designation as oaklins. 7. Antioxidant properties of anthocyanins and deoxyanthocyanidins In addition to decisively influence the organoleptic properties of food, polyphenols also reveal important antioxidant properties. Among various substances classified as antioxidants, there are for example vitamins, minerals, natural pigments and other vegatable compounds, and also enzymes, which block the effect of reactive oxygen species (ROS). These species negatively affect tissues, cells and genes contributing to the development of chronic diseases. Antioxidants in food can normally act as inhibitors of radicals in chain reactions, as metal chelating agents, as inhibitors of oxidative enzymes and as cofactors of antioxidant enzymes (Huang 2005). As a definition, an antioxidant is any substance that, when present at low concentrations compared to the oxidizable substrate, significantly delays or prevents oxidation of that substrate, and that after oxidation must be sufficiently stable so as not to trigger new 62 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution The purpose of this project was to investigate and study pigments with interesting color features and stability in aqueous solution that could have potential applications in food, health and other industries. Bearing this, several new pigments of the family of deoxyanthocyanidins were synthesized, isolated and structurally characterized. Their physico-chemical properties were thoroughly investigated as well as their stability in aqueous solution in the absence/presence of copigments. Finally, their antioxidant properties were also evaluated. During the project various issues and specific objectives were addressed: Chapter 1 During wine aging in oak barrels several wood compounds (e.g. aldehydes) can interact with wine constituents like anthocyanins and catechins yielding new pigments that sometimes present interesting color features and are more stable than their precursors. This work deals with the formation of new oaklin pigments resulting from a direct reaction of a procyanidin dimer B4 ((+)-catechin4−8 - − -epicatechin) with two cinnamic aldehydes, coniferaldehyde and sinapaldehyde in hydroalcoholic solutions. The focus of this work was to evaluate the importance that dimeric procyanidins and cinnamic aldehydes could have on the color evolution of wines during storage in oak barrels. Chapter 2 Despite the interest in deoxyanthocyanidin-type of compounds, few synthetic approaches have been made toward their synthesis and the procedures described in the literature are rather complex. The goal of this work was to test a simple synthesis method to form 3deoxyanthocyanidins from the reaction between phloroglucinol with two cinnamic aldehydes, coniferaldehyde and sinapaldehyde, describing their chemical structure and mechanism of formation and evaluating the kinetics of formation of the reaction products under different conditions. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 63 Chapter 3 Two oaklin compounds (guaiacylcatechinpyrylium and syringylcatechinpyrylium), which are formed in wine aged in oak barrels and a model deoxyanthocyanidin compound (deoxypeonidin) were synthesized. Combining pH jump techniques with flash photolysis, the rate and equilibrium constants of the respective pH dependent network of chemical reactions were calculated and their photochromism was also investigated. This work would allow a global understanding and comprehensive analysis of the stability of these types of deoxyanthocyanidins, comparing their behaviour and physicochemical properties under different pH conditions and light stimulus with analogous compounds such as common anthocyanins and other flavyliums. The ultimate purpose of this work was to evaluate and identify potential applications for these types of compounds, coming from the knowledge of their physico-chemical characteristics. Chapter 4 y y y w “ ” y which makes them much more stable towards pH variations and bleaching by SO2 in comparison to the genuine anthocyanins. On the other hand, and opposite to anthocyanins, deoxyanthocyanidins also have an increased stability in slightly acidic solutions compared to anthocyanins. Bearing this, the aim of this work was to synthesize compounds that would benefit from both structural features. These compounds would be called deoxyvitisins (or pyranodeoxyanthocyanidins) and would be obtained from the reaction between deoxyanthocyanidins and the reagents pyruvic acid, vinyloxy-trimethylsilane and acetone-1,3-dicarboxylic acid in separate model aqueous solutions. Chapter 5 The aim of this work was to investigate the color stability and the network of chemical reactions occurring in aqueous solution upon pH variations for the deoxyvitisins synthesized in the previous work, comparing the results with a flavylium model 64 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 4’ 7-dihydroxyflavylium and with values reported in the literature for the corresponding glycosidic analogs, vitisins. Chapter 6 The importance and contribution of the copigmentation effect cannot be underestimated in the analysis of color changes in wine solutions. The aim of this work was thus to study the copigmentation interactions possibly occurring between the oaklin compounds guaiacylcatechin-pyrylium (GCP) and syringylcatechin-pyrylium (SCP) with common copigments such as catechin, epicatechin, chlorogenic acid, epigallocatechin, and procyanidin B3, determining the respective copigmentation constants and evaluating the relationship between copigmentation ability and the structure of the complexes formed. Chapter 7 The study of the antioxidant properties of six deoxyanthocyanidins (deoxypeonidin, deoxymalvidin, luteolinidin, apigeninidin, guaiacylcatechinpyrylium and syringylcatechinpyrylium) and an anthocyanin (cyanidin-3-glucoside) was carried out. The aim was to evaluate the relationship between the structure and the antioxidant properties of individual deoxyanthocyanidins, compared to a common derivative anthocyanin, cyanidin-3-glucoside. The ability of these compounds to inhibit lipid peroxidation in a liposome membrane system was examined by monitoring oxygen consumption and the antiradical and reducing capacities were determined using the DPPH and FRAP assay, respectively. Furthermore, the antiproliferative effects of deoxyanthocyanidins have been evaluated against two cancer cell lines from stomach (AGS, MKN-28) and one colon cancer cell (Caco-2), and compared with the effect of their anthocyanic forms. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 65 Results 66 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 67 Chapter 1 Synthesis and Structural Characterization of Oaklin-Catechins Sousa, A., Fernandes, A., Mateus, N., de Freitas, V. J. Agric. Food Chem. 2012, 60 (6), 1528–1534 68 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution In this work, the first author was responsible for undertaking all experimental activities with the assistance of MS and NMR technicians and guided by the scientific knowledge of the rest of the authors. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 69 Synthesis and structural characterization of oaklin-catechins André Sousa, Ana Fernandes, Nuno Mateus and Victor de Freitas* Departamento de Química, Faculdade de Ciências, Universidade do Porto, Centro de Investigação em Química, Rua do Campo Alegre 687, 4169-007 Porto, Portugal Condensation reactions of procyanidin dimer B4 with two representative oak wood cinnamic aldehydes (coniferaldehyde and sinapaldehyde) were conducted in wine-like model solutions. Coniferaldehyde led to the formation of guaiacylcatechin-pyryliumcatechin (GCP-catechin, 737 m/z), whereas sinapaldehyde led to the formation of syringylcatechin-pyrylium-catechin (SCP-catechin, 767 m/z). The former was also structurally characterized by 1D and 2D NMR, allowing an elucidation of the formation mechanism of these oaklin-catechin adducts and demonstrating the importance of procyanidins in the formation of colored compounds through the reaction with cinnamic aldehydes extracted from oaks during storage. KEYWORDS: Red wine; aging; oak barrels; procyanidin B4; aldehydes; oaklin; NMR; mass spectrometry  INTRODUCTION Wine storage in oak barrels during the first years of aging is a common procedure in wine industry and the extraction of volatile and non-volatile compounds from the wood influences important characteristics in matured wine, namely aroma, taste and color (Jurd 1969, Chassaing, Lefeuvre 2010). The extraction of these compounds depends on the period of contact between wine and wood and on the chemical composition of the wood, which is affected by the species and origin of the trees, the seasoning of the staves, the age of the barrel and most important by the heat treatment or toasting of barrels (Chatonnet, Boidron 1989, Sefton et al. 1993, Vivas 1995). In this procedure, macromolecular components like lignins and polysaccharides of the wood are degraded into smaller compounds such as several aldehydes (Nonier, Vivas 2006) (eg. furfural, hydroxymethylfurfural, hydroxybenzaldehyde, vanillin, coniferaldehyde, sinapaldehyde) which are of particular interest because they are aroma compounds and they can also interact with some wine compounds like anthocyanins and catechins, hence contributing to color changes (de Freitas et al. 2004, Pissarra et al. 2005, Nonier, Vivas 2007, Sousa et al. 2007, Sousa, Mateus 2010). 70 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Indeed, one of these reactions most studied is the aldehyde-mediated association of anthocyanins and catechins through a Bayer acid-catalyzed condensation, yielding new pigments with different chromatic properties than the anthocyanin precursor (Timberlake and Bridle 1976, Rivas-Gonzalo et al. 1995, Dallas, Ricardo-da-Silva 1996, EscribanoBailon, Dangles 1996, Fulcrand, Doco 1996, Es-Safi et al. 2000, Pissarra, Lourenco 2004, Sousa et al. 2007). More recently, cinnamic aldehydes have shown to react with catechin yielding a new class of brick-red catechin-pyrilium pigments (3deoxyanthocyanidin derivatives), named oaklins (de Freitas et al. 2004). The formation of these compounds was confirmed in wine-like model solutions containing oak wood extract and an oaklin derived pigment was already found in a commercial table red wine (Sousa, Mateus 2005). Analogous compounds may be formed through the reaction of oak-derived aldehydes with proanthocyanidins. These latter are extracted from grapes to wine during winemaking and contribute to the astringency of red wines (Arnold, Noble 1980, Havsteen 1983, Haslam and Lilley 1988). The present work deals with the formation of new oaklin pigments (see Fig. 4, structure IX) resulting from a direct reaction of a procyanidin dimer B4 ((+)-catechin-(4-8)-(-)- epicatechin) (III) with two cinnamic aldehydes (I), coniferaldehyde and sinapaldehyde. The newly formed compounds described herein for the first time point out the importance of procyanidins and cinnamic aldehydes in the formation of colored compounds during storage.  MATERIALS AND METHODS Samples. Coniferaldehyde and sinapaldehyde were purchased from SigmaAldrich®(Spain). Procyanidin B4 was obtained by hemisynthesis following the procedure described in the literature (Geissman and Yoshimur.Nn 1966). Study of the reaction between procyanidin B4 and cinnamic aldehydes. Procyanidin B4 (1,7 mM, 2 mg) was incubated with coniferaldehyde (2,1 mM) and sinapaldehyde (2,1 mM) separately in 2 mL of 12% (v/v) hydroalcoholic solutions at pH 3.5 with a molar ratio of 1:1.2 (phloroglucinol:cinnamic aldehyde). These model solutions were kept at a temperature of 35 ºC and protected from light. The formation of new compounds was followed over time by HPLC-DAD using a reversed phase C-18 (Merck) column (250 x 4.6 mm i. d., particle size 5 µm), at 25 ºC. Solvents were (A) water/formic acid (95:5) and (B) acetonitrile. The elution gradient was performed using a L-2130 Merck pump from 10 to 35% B for 55 min at a flow rate of 1.5 mL.min-1. LC-MS conditions. Mass spectrometry analysis was performed using a Finnigan SurVeyor series liquid chromatograph, equipped with an API source, using an FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 71 electrospray ionization (ESI) probe. Solvents were (A) aqueous 0.1% acetic acid and (B) acetonitrile. The elution conditions were as follows: 0.5 mL.min-1 flow rate; oven temperature, 35 ºC; elution began with linear gradient from 5 to 30% B in 40 min, from 30 to 40% in 10 min and from 40 to 100% in 5 min, followed by washing and reequilibration of the column. The capillary voltage was 11 V, and the capillary temperature was 200 ºC. Spectra were recorded in positive ion mode between m/z 100 and 1200. The mass spectrometer was programmed to do a series of three scans: a full mass spectrum, a MS2 spectrum of the most intense ion, and a MS3 spectrum of the most intense ion in the second scan, using a relative collision energy of 45 V. Synthesis and purification of the guaiacylcatechin-pyrylium-catechin (GCPcatechin) adduct. Procyanidin B4 (1,7 mM, 80 mg) was incubated with coniferaldehyde (8,3 mM) in 80 mL of a 12% (v/v) hydroalcoholic solution at pH 1 with a molar ratio of 1:4.8 (phloroglucinol:coniferaldehyde). Before the synthesis, several experiments at different pH values and molar ratios were tested in order to improve the yield of the reaction to a maximum of 9%. The model solution was kept at a temperature of 35 ºC and protected from light, and the formation of new compounds was followed by HPLCDAD. When the reaction was completed, the sample was applied on a silica gel C-18 reversed phase SPE cartridge in order to remove inorganic salts and other impurities and the pigments were eluted with methanol acidulated with 2% HCl. Methanol was evaporated in a rotary evaporator at 38 ºC, and the sample was freeze-dried and stored at -18 ºC until use. The sample was further applied into a 5 cm diameter medium-porosity sintered glass funnel with TSK Toyopearl gel HW-40(S) (Tosoh, Japan), connected to standard vacuum filtration glassware and gradually eluted with increasing percentages of acidified methanol (F1, 30%; F2, 40%; F3, 50%; F4, 60%; and F5, 80%). The criterion used for changing the percentages of methanol was the decrease in color intensity of the solution eluted from the column. The solvent of each fraction was partially evaporated in a rotary evaporator at 38 ºC, and the samples were freeze-dried and stored at -18 ºC until use. Semi-preparative HPLC. Semi-preparative HPLC was performed in order to further isolate and purify the oaklin-catechin aducts, eluted in fraction F2 from toyopearl gel. This fraction was injected into a reversed phase C-18 (Merck) column (250 x 4.6 mm i. d., particle size 5 µm) at room temperature (volume injected was 1 mL). Solvents were (A) water/formic acid (95:5) and (B) acetonitrile. The elution gradient was performed using a L-2130 Elite LaChrom pump from 10 to 35% B for 65 min at a flow rate of 1.5 mL.min-1 and detection was carried out at 500 nm using a L-2420 Elite LaChrom detector. 78 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Ring D, E, F 2F1 5.20* 80.2 H-2F1 2F2 5.30* H-2F2 3F1 4.85* 66.3 H-3α 3F2 4.26* H-3β 4α 1 2.86* 40.0 H-4α 1 4β 1 2.92* H-4β 1 4α 2 3.44* H-4α 2 4β 2 3.49* H-4β 2 4aF na 5D 145.0 H-6D 6D 7.02; s 115.5 H-6D 7D 145.0 H-6D 8D na 8aD na 1ʹE 128.4 H-2F, H-6ʹE 2ʹE 6.88* 115.0 H-2ʹG 3ʹE 144.8 H-2ʹE 4ʹE 145.0 H-6ʹE 5ʹE 6.88* 118.5 H-5ʹG 6ʹE 6.74* 115.4 H-6ʹG na - not attributed; s - singlet; d - doublet; brs - broad singlet; * - unresolved Carbons C-4aA and C-7A were determined from their long range 1H– 13C correlation with H-6A. The quaternary carbons C-5A and C-8aA were assigned at δ 168.8 and 153.7ppm from their long distance correlations with protons H-6A and H-9H, respectively, observed in the HMBC spectrum. These correlations as well as the lack of a long-distance correlation between carbon C8aA and the singlet at 7.17ppm (H-6A) identify unambiguously the position of the pyrylium ring H linkages onto carbons C-7A and C-8A, as they could not be observed if ring H was formed between the hydroxyl group at carbon C-5A and the carbon C-6A. The chemical shift of the remaining protons and carbons identified in Table 1 were easily established by HSQC and HMBC techniques. Formation Mechanism. The hypothetic mechanism of formation of these new pigments from the reactions between procyanidin B4 and cinnamic aldehydes is represented in Fig. 23 - Hypothetical mechanism for the formation of oaklin-catechins IX obtained from the reaction between procyanidin B4 III and cinnamic aldehydes I. By analogy with the mechanism described in the literature for the formation of monomeric FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 79 oaklins, the reaction starts with the protonation of the cinnamic aldehyde I in acidic medium, forming a carbocation in the carbonyl carbon II, followed by a nucleophilic attack of ring A of procyanidin B4 III, leading to structure IV. The A ring attack may occur from position C6 or preferentially from position C8. Indeed, the negative formal charge in ring A is expected to be higher at carbon 8, as it is well documented for flavylium compounds in the reaction leading to the formation of catechin-alkyl/aryl-anthocyanin adducts (Bendz et al. 1967, Rivas-Gonzalo et al. 1995). The putative presence of compound IV was evidenced in the mass spectra by the presence of a protonated molecular ion ([M+1]+) at m/z 757. The dehydration of the resulting protonated adduct V yields a new carbocation VI, which undergoes a rearrangement leading to carbocation VII. This carbocation suffers an intra-molecular nucleophilic attack by the hydroxyl group at carbon 7 of ring A, leading to structure VIII. The putative presence of compound VIII was evidenced in the mass spectra by the presence of a protonated molecular ion ([M+1]+ / 739. fi y IX, which has the pyrylium ring H associated with the aromatic ring A and constitutes a chromophore group. The π around 500nm. The GCP-catechin position isomer formed at pH 1.0 in lower amount may result from an initial nucleophilick attack from position C6 to the aldehyde resulting in the formation of a new pyranic ring involving position 5 and 6 of ring A. 80 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution IX -H+ [O] II III V VII VIII R= H, coniferaldehyde R= OCH3, sinapaldehyde O OH OH AC B O OH HO OH O OH OH D E HO R H3CO H H G H 2 3 4 4a 5 6 7 8 8a 1' 2' 3' 4' 5' 6' H H F H H H H H OH OH H H H H H H H 4 3 2 4a 5 6 7 8 8a 1' 2' 3' 4' 5' 6' H H H 9 10 11 12 13 14 15 16 17 CH R HO H3CO CH C O H I CH R HO H3CO CH C OH H O OH OH OH A C B HO OH HO OH O OH OH OH D F E 8 6 O OH OH OH HO OH HO OH O OH OH OH C OH HCHCHHO R H3CO O OH OH OH HO OH HO OH O OH OH OH C CH H CHHO R H3CO O OH OH OH HO OH HO OH O OH OH OH C CCHHO R H3CO H H O OH OH OH O OH HO OH O OH OH OH HO R H3CO H H +H+ +H+ -H2O Fig. 23 - Hypothetical mechanism for the formation of oaklin-catechins IX obtained from the reaction between procyanidin B4 III and cinnamic aldehydes I. I IV VI IX O OH OH OH HO OH HO OH O OH OH OH C OH2 HCHCHHO R H3CO -H+ [M+1]+ m/z 757 [M+1]+ m/z 739 [M]+ m/z 737 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 81  CONCLUSION Procyanidin B4 showed ability to react directly with cinnamic aldehydes, namely coniferaldehyde and sinapaldehyde to give orange pigments (oaklins). Oaklins monomers formed from the reaction between catechin and cinnamic aldehydes have already been detected in wine model solutions (11-guaiacylcatechinpyrylium) and wine (Sousa et al. 2005). Therefore, attending to the relative high amount of procyanidin dimers in real wines it is also expected that oaklin-catechin adducts may also occur in wine and even play a role in some color changes observed during the aging process. Nonetheless, further studies are still required to unequivocally detect the formation of oaklin-catechins in wine and to understand their overall contribution to wine properties, such as color and taste.  AKNOWLEDGEMENTS The authors thank FCT (Fundação para a Ciência e Tecnologia) for a PhD grant (ref. SFRH/BD/68736/2010). This research was also supported by a research project grant (PTDC/QUI-QUI/117996/2010) funded by FCT (Fundação para a Ciência e Tecnologia). 82 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 83 Chapter 2 A novel reaction mechanism for the formation of deoxyanthocyanidins Sousa, A., Mateus, N., de Freitas, V. Tetrahedron Lett. 2012, 53(10), 1300-1303 84 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution In this work, the first author was responsible for undertaking all experimental activities with the assistance of MS and NMR technicians and guided by the scientific knowledge of the rest of the authors. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 85 Cinnamic aldehydes: R= H, coniferaldehyde R= OCH3, sinapaldehyde Deoxyanthocyanidins: R= H, 3-deoxypeonidin R= OCH3, 3-deoxymalvidin phloroglucinol (Intermediary compounds) CH H3CO HO R CH C O H CH H3CO HO R CH C OH H HO OH OH O HO OH OCH3 R H HOH 2 3 4 AC B 4a 5 6 7 88a 1' 2' 3' 4' 5' 6' +H+ A novel reaction mechanism for the formation of deoxyanthocyanidins André Sousa, Nuno Mateus and Victor de Freitas* Departamento de Química, Faculdade de Ciências, Universidade do Porto, Centro de Investigação em Química, Rua do Campo Alegre 687, 4169-007 Porto, Portugal The synthesis of deoxyanthocyanidins from the reaction between cinnamic aldehydes (coniferaldehyde and sinapaldehyde) and phloroglucinol is reported herein. The resulting structures were characterised by visible, MS and NMR spectroscopy. KEYWORDS: deoxyanthocyanidins; aldehydes; phloroglucinol; flavonoids; NMR; mass spectrometry  INTRODUCTION Deoxyanthocyanidins are yellowish pigments found in several food plants such as corn, black tea leaves and sorghum. Sorghum is one of the most important cereal crops in the world and is rich in 3-deoxyanthocyanidins, particularly luteolinidin and apigeninidin (Coggon, Moss 1973, Sweeny and Iacobucci 1977, 1981). Deoxyanthocyanidins are considered the chemical ancestors of anthocyanins, the ubiquitous water-soluble pigments that are found in flowers and fruits and are responsible for their impressive blue and purple colors (Sweeny and Iacobucci 1977). 487 nm 3-deoxypeonidin 0 0,01 0,02 0,03 0,04 350 400 450 500 550 600 A Wavelenght (nm) 86 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Open chalcone forms of the common anthocyanins are assumed to be crucial in reactions leading to irreversible degradation of anthocyanins, particularly under weakly acidic to weakly alkaline solution conditions (Mazza and Brouillard 1987, Francis 1989, Cabrita et al. 2000, Torskangerpoll and Andersen 2005, Sadilova et al. 2007). However, the natural yellow deoxyanthocyanidins are much more stable in slightly acidic solutions than anthocyanins and anthocyanidins, which points to the potential advantage of this type of compounds as viable commercial food colourants, and justifies the research developed in the chemistry of 3-deoxyanthocyanins and, in particular, the search for new colourants with significant stability (Iacobucci and Sweeny 1983, Dangles and Elhajji 1994, Khalil et al. 2010). In addition, more studies have demonstrated other potential applications for these compounds, such as their use as hair dyes, laser dyes, sensitizers for solar cells and molecular-level memory systems (Roque et al. 2002). Despite the interest in these types of compounds, few synthetic approaches have been made towards deoxyanthocyanidins and the procedures described in the literature are complex (Pratt and Robinson 1922, 1923, Pratt et al. 1924, Pratt and Robinson 1925, Sweeny and Iacobucci 1977, Kuhnert et al. 2001). Only in recent years attemps have been made to synthesize these compounds with simpler methods (Mas 2003, Chassaing et al. 2007, Kueny-Stotz et al. 2007).  MATERIALS AND METHODS The synthesis of 3-deoxyanthocyanidins 9 (Fig. 24) from the reaction of phloroglucinol 3 with two cinnamic aldehydes 1 (Fig. 27), coniferaldehyde and sinapaldehyde, is described herein and their chemical structure and mechanism of formation elucidated. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 87 O HO OH OCH3 R H H OH 2 3 4 AC B 4a 5 6 7 8 8a 1' 2' 3' 4' 5' 6' Fig. 24 - Structure of the new synthesized 3-deoxyanthocyanidins 9 (R=H, 3-deoxypeonidin; R=OCH3, 3-deoxymalvidin). Phloroglucinol 3 (8mM) was incubated with coniferaldehyde and sinapaldehyde 1 separately under different conditions of pH, percentage of ethanol in water and molar ratios. These model solutions were kept at a temperature of 35 ºC and protected from light. The formation of new compounds was followed over time by HPLC-DAD using a reversed phase C-18 (Merck) column (250 x 4.6 mm i. d., particle size 5 µm) at 25 ºC. Solvents were (A) water/formic acid (95:5) and (B) acetonitrile. The elution gradient was performed using a L-2130 Merck pump from 10 to 35% B for 55 min at a flow rate of 1.5 mL.min-1.  RESULTS AND DISCUSSION The mass spectra of these compounds obtained by LC–DAD/ESI/MS in the positive ion mode showed a molecular ion [M]+ at m/z 285 (3-deoxypeonidin) through the reaction with coniferaldehyde, and [M]+ at m/z 315 (3-deoxymalvidin), from the reaction with sinapaldehyde. In addition, the MS2 spectrum of 3-deoxipeonidin shows a major fragment at m/z 270 (loss of a methyl group, [M-15]+), and further MS3 fragmentation of the ion at m/z 270 yielded a fragment at m/z 242 (loss of CO, [M-28]+). The other pigment formed in the reaction of phloroglucinol with sinapaldehyde followed a similar fragmentation scheme. The two compounds, 3-deoxypeonidin and 3-deoxymalvidin revealed a maximum absorption in the visible spectrum at 487 nm and at 492 nm (Fig. 25), respectively, conferring them a yellow colour. 94 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 95 Chapter 3 Thermodynamics, Kinetics and Photochromism of Oaklins: a Recent Family of Deoxyanthocyanidins Sousa, A., Petrov, V., Araújo, P., Mateus, N., Pina, F., de Freitas, V. J. Phys. Chem. B. 2013, 117(6), 1901-10 96 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution In this work, the first author actively participated in all of the experimental activities. He counted with the help of Paula Araújo in the chemical synthesis of the tested compounds and was instructed and guided by Vesselin Petrov in the pH jumps and photochromic experiments as well as in the model data treatment. He was always actively supported by the scientific expertise of the rest of the authors. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 97 Thermodynamics, Kinetics and Photochromism of Oaklins: a Recent Family of Deoxyanthocyanidins. André Sousa†, Vesselin Petrov‡, Paula Araújo†, Nuno Mateus†, Fernando Pina*,‡, Victor de Freitas*,† † Centro de Investigação em Química, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal; ‡ REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal Two oaklins Guaiacylcatechinpyrylium (GCP) and Syringylcatechinpyrylium (SCP) and a model compound Deoxypeonidin (DOP) were synthesized and the rate and equilibrium constants of the respective pH dependent network of chemical reactions were calculated. In contrast to anthocyanins, the three compounds possess a small cis-trans isomerization barrier and hence the rate of the trans-chalcone formation follows a bell shaped curve as a function of pH. The three compounds exhibit photochromism obtained by irradiation of the trans-chalcone, which, depending on pH, leads to the colored species flavylium cation and quinoidal base. The flash photolysis together with pH jumps followed by UV-Vis absorption and stopped flow is a very useful tool to achieve the rate and equilibrium constants of the network of chemical reactions followed by these molecules. Oaklin compounds which are formed in wine aged in oak barrels present physical-chemical properties more similar to simpler deoxyanthocyanidins rather than anthocyanins and may play a significant role in color changes observed in wine aging. Given their higher stability, they may be regarded as potential food colorants. KEYWORDS: oaklins, deoxyanthocyanidins, anthocyanins, flavylium network, photochemistry, food colorants, wine aging  INTRODUCTION 2-phenyl-1-benzopyrilium (flavylium) derivatives, which comprise anthocyanins, deoxyanthocyanins, anthocyanidins as well as bio-inspired synthetic flavylium compounds, have been a recurrent subject of research through more than a century (Pina, Melo 2012). This is due to the role played by anthocyanins as colorants of most 98 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution flowers and fruits and to the antioxidant properties of these compounds and the consequent potential of their applications in human health (Kumpulainen 1996). In general, independently of their natural or synthetic origin, flavylium derivatives follow the same network of chemical reactions as reported on Fig. 28 for malvidin-3-glucoside. Fig. 28 - Network of chemical reactions of malvidin-3-glucoside. Despite the apparent complexity of the pH dependent chemistry of flavylium compounds, due to the manifold of chemical reactions involving these compounds, the system can be viewed as a single acid-base equilibrium involving flavylium cation and its conjugate base CB, eq.(1) AH+ + H2O CB + H3O+ K’a=Ka+Kh+ Kh Kt+ Kh Kt Ki (1) CB, defined as the sum of the concentrations of the other species in the network, [CB]=[A]+[B]+[Cc]+[Ct] (Brouillard and Delaporte 1977, Brouillard and Dubois 1977, Brouillard, Delaporte 1978, Brouillard and Lang 1990, Pina 1998). The global equilibrium defined by eq.(1) is decomposed in their components according to eq.(2) to eq.(5). AH+ + H2O A + H3O+ Ka Proton transfer (2) AH+ + 2H2O B + H3O+ Kh Hydration (3) B Cc Kt Tautomerization (4) Cc Ct Ki Isomerization (5) “ ” y substitution pattern of the flavylium core. For example in anthocyanins B is the major FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 99 species, while in 7,4c-dihydroxyflavylium it is the trans-chalcone Ct (Pina, Melo 1998, Pina et al. 2012). It is worth noting the reversibility of all the reactions, at least in acidic medium, which makes the flavylium network a unique multi-component system. Photochemistry takes place upon irradiation of Ct leading to Cc, which spontaneously gives flavylium cation or/and quinoidal base depending on pH. The photochemical system is reversible and Ct is recovered through the thermal back reaction. In this work, the compounds Guaiacylcathechinpyrylium (GCP), syringylcatechinpyrylium (SCP) and the model compound deoxypeonidin (DOP) (Fig. 29) were synthesized and the thermodynamic and the kinetics of the respective network fully described. DOP GCP SCP Fig. 29 - The model compound Deoxypeonidin (DOP) and the derivatives Guaiacylcathechinpyrylium (GCP), and Syringylcatechinpyrylium (SCP). The formation and synthesis of the catechinpyrylium compounds SCP and GCP was previously described and these compounds named oaklins, a new class of brick-red colored pigments resulting from the reaction between catechin and wood aldehydes (de Freitas et al. 2004, Sousa et al. 2005). The formation of these pigments was also performed in a wine model solution and one of these (GCP) was also found in real wines aged in oak barrels, confirming that this type of compounds may contribute to the overall color changes observed during the aging process. Oppositely to anthocyanins, these pigments do not possess a glucose group in the flavylium core and are classified as deoxyanthocyanidins. To test and compare the properties of these compounds, a structurally simpler deoxyanthocyanidin (DOP) was also synthesized. There is interest in knowing how the structural modifications carried out in the model compound DOP to give GCP and SCP affect the network of chemical reactions reported 100 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution above, and in particular the photochromic properties, as well as to make an argument regarding the stability of these compounds and their putative role in color changes in wine aging when compared to common wine anthocyanins. Furthermore, deoxyanthocyanidins are reported to be much more stable in slightly acidic solutions than anthocyanins and anthocyanidins, which points to the potential advantage of this type of compounds as viable commercial food colorants, and justifies the research developed in the chemistry of 3-deoxyanthocyanins and, in particular, the search for new colorants with significant stability (Iacobucci and Sweeny 1983, Dangles and Elhajji 1994, Sousa, Mateus 2012).  MATERIALS AND METHODS Synthesis of GCP (guaiacylcatechinpyrylium), SCP (syringylcatechinpyrylium) and deoxypeonidin. Regarding the synthesis of GCP, catechin ((2R,3S)-2-(3,4dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol) (8mM) was incubated with coniferaldehyde ((E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enal) (8mM) in 100 mL of a 12% ethanol-water solution (v/v) at pH 1.5 at 35ºC. The formation of the compound was followed over time by HPLC–DAD using a reversed phase C-18 (Merck) column (250 x 4.6 . . 5 μ 25 º . S w w / 95:5) and (B) acetonitrile. The elution gradient was performed using L-2130 Merck pump from 10% 35% 55 fl w 1.5 -1. When the reaction was completed, after 10 days the sample was applied on a silica gel C-18 reversed phase SPE cartridge in order to remove inorganic salts and other impurities and the pigments were eluted with acidified methanol (2% v/v). Methanol was evaporated in a rotary evaporator at 38 °C, and the sample was freeze- −18 ° . ple was further applied into a 5 cm diameter medium porosity sintered glass funnel with TSK Toyopearl gel HW-40(S) (Tosoh), connected to standard vacuum filtration glassware and gradually eluted with increasing percentages of acidified methanol (F1, 30%; F2, 40%; F3, 50%; F4, 60%; and F5, 80%). The criterion used for changing the percentages of methanol was the decrease in color intensity of the solution eluted from the column. Semipreparative HPLC was performed in order to further isolate and purify the GCP compound, eluted in fraction F2 from Toyopearl gel. This fraction was injected into a reversed phase C-18 M k 250 × 4.6 . . 5 μ temperature (volume injected was 1 mL). Solvents were (A) water/formic acid (95:5) and (B) acetonitrile. The elution gradient was performed using a L-2130 Elite LaChrom pump from 10 to 35% B for 65 min at a flow rate of 1.5 mL min −1 and detection was carried out FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 101 at 500 nm using a L-2420 Elite LaChrom detector. The synthesis of syringilcatechinpyrylium (SCP) was performed using the same procedure. The synthesis of the compound 3-deoxypeonidin was followed according to procedure described in the literature (Sousa et al. 2012). Phloroglucinol (8mM) was incubated with coniferaldehyde (80mM) in a 100 mL 12% ethanol-water solution (v/v) at pH 1.0 at 35ºC. When the reaction was completed, after 4 days the sample was applied on a silica gel C18 reversed phase SPE cartridge in order to remove inorganic salts and other impurities and the pigments were eluted with methanol acidulated with 2% HCl. The sample was further applied into a 300 x 16 mm i.d. TSK Toyopearl gel HW-40(S) (Tosoh, Japan) column and eluted with 40% aqueous methanol in 4 hours. UV/Vis absorption spectra were recorded on a Varian Cary 100 Bio and Varian Cary 5000 spectrophotometers. The stopped flow experiments were conducted in an Applied Photophysics SX20 stopped-flow spectrometer provided with a PDA.1/UV photodiode array detector with a minimum scan time of 0.65 ms and a wavelength range of 200 nm to 700 nm. Flash photolysis was carried out as reported elsewhere (Pina, Melo 2001): to monitor the transient species a common spectrophotometer with a slightly modified compartment was used: (i) a slit (5 mm wide and 20 mm high) was opened on the external side of the sample holder in order to perform light excitation perpendicular to the analyzing beam; (ii) the whole sample compartment shielded (except for the slit described above) with black cardboard and black tape, to reduce as much as possible the flash light entering the exit slits. The time driven acquisition mode of the spectrophotometer was used and the traces were obtained each 5 nm or less according to the accuracy needed. From the traces, the time dependent absorption spectra were calculated.  RESULTS AND DISCUSSION Deoxypeonidin (DOP). Fig. 30 shows the spectral variations of the model compound deoxypeonidin, 5,7,4c-trihydroxy-3c-methoxyflavylium (DOP), occurring immediately after a pH jump from stock solutions at pH=1 to higher pH values (direct pH jumps). The spectral modifications are compatible with a triprotic acid with pKacs equal to 3.9, 6.5 and 8.2, Table 3. The absorption spectra of the four species were determined by mathematical decomposition, Fig. 30D. The first deprotonation is likely to occur in position 7, a behavior early reported by Jurd and Geissman (Jurd and Geissman 1963) and confirmed in other anthocyanins (Pina et al. 2012). The second deprotonation most 102 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution probably occurs at position 4 and the third deprotonation at position 5. The pKa of the first deprotonation compares with 4.1 for the analog luteolinidin (5,7,3c4ctetrahydroxyflavylium) (Melo, Moura 2000), and 4.2 for apigeninidin (5,7,3ctrihydroxyflavylium) (Brouillard et al. 1982). 0 0,15 0,3 200 300 400 500 600 700 A Wavelenght (nm) pH=1.9 pH=5.3 0 0,15 0,3 200 300 400 500 600 700 A Wavelenght (nm) pH=5.2 pH=10 0 0.05 0.1 0.15 0.2 0 1 2 4 6 8 10 A Mole fraction pH 480 nm 550 nm pKa1=3.9 pKa2=6.5 pKa3=8.2 0 0,15 0,3 200 300 400 500 600 700 A Wavelenght (nm) AH+ A2AA Fig. 30 - A - Spectral variations of the compound deoxypeonidin immediately after a pH jump from 1 to the range 1.9<pH<5.3; B - the same for 5.3<pH<10; C - Fitting of the absorption at 480 nm and 550 nm as a function of pH; D - Absorption spectra of the flavylium cation, quinoidal base and ionized quinoidal bases obtained by mathematical decomposition. A B C D FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 103 Table 3. Ionization constants of DOP, GCP and SCP. The pH dependent absorption variations taking place in equilibrated solutions of DOP are represented in Fig. 31. The data are compatible with a single acid base equilibrium, eq.(1), permitting to define pKca=3.6, a value that compares with 3.8 (Melo et al. 2000) and 4.0 (Brouillard et al. 1982), for luteolinidin and apigeninidin. The shape and position of the equilibrium species at moderately acidic medium are compatible with the presence of quinoidal base in equilibrium with trans-chalcone. The fraction of the base at the higher pH plateau is given by the ratio Ka/Kca=0.5 which is in good agreement with the data of Fig. 31. Fig. 31 - Spectra of equilibrated solutions of deoxypeonidin at different pH values, showing the equilibrium between AH+, A and Ct; inset representation of the fitting obtained at two different wavelengths (pKca=3.6±0.1). The relatively large mole fraction of the quinoidal base at the moderately acid equilibrium plateau when compared with anthocyanins (less than 5% in diluted solutions) (Pina et al. 2012, Petrov, Gavara 2013) or even 4c,7-dihydroxyflavylium (ca 10%) (Pina et al. 2012) Compound pKa1 pKa2 pKa3 DOP 4.0±0.1 6.5±0.1 8.2±0.1 GCP 3.6±0.1 7.7±0.1 - SCP 3.8±0.1 n.d. - n.d. – not determined 110 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 0 0.1 0.2 0.3 300 400 500 600 700 A Wavelenght (nm) pK' a=3.05 0 0.25 0 2 4 6 A (484) and (390) nm pH Fig. 36 - Spectra of equilibrated solutions of GCP (A) at different pH values, showing the equilibrium between AH+, A and Ct; inset representation of the fitting obtained at two different wavelengths (pKca=3.05±0.1). The kinetic processes from the initial state reported in Fig. 35 to the equilibrium, Fig. 36, are described in Fig. 37A. The spectral changes indicate that at pH=5.2 the quinoidal base initially formed through a pH jump partially disappears to give the trans-chalcone. The kinetics follows a mono-exponential decay. The rate constants measured at different pH values are reported in the inset of Fig. 37A, and follow the previously reported bellshape curve. Fitting was achieved for the following parameters KhKtki=2.5x10-7 M-1; ki=0.00028 s-1; kiKt/k-h=1.9x10-5 M. From the ratio KhKtki /(kiKt/k-h), kh=0.013 s-1. 0 0.4 0.8 1.2 200 300 400 500 600 700 A Wavelength (nm) 0 0.001 1 3 5 7 k obs pH 0.46 0.47 0.48 0.49 0.5 0.51 0 1 2 3 4 5 A (488 nm) Time (s) kobs1= 9 s-1 kobs2= 1.2 s-1 A B Cc Kt=1.5 Fig. 37 - A - Spectral variations of GCP after a pH jump from 1 to 5.2 (kobs=1.8x10-4 s-1): inset-representation of the observed rate constant as a function of pH. Fitting was achieved for pKa=3.6; KhKtki=2.5x10-7 M-1; k-i=0.00028 s-1; kiKt/kh=1.9x10-5 M. From the ratio KhKtki /(kiKt/k-h), kh=0.013 s-1; (z) direct pH jumps ({) thermal recovery of the photoproduct, see below Fig. 38; B - Reverse pH jump from solutions aged about 5 minutes at pH=5.4 and back to pH=1.2 (Kt=1.5). A B FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 111 A first pH jump on stocked solutions at pH=5.14 was carried out, the solutions kept during 5 min at this pH value and a reverse pH jump to 1.2 was performed allowing the monitoring of the flavylium appearance by stopped flow, Fig. 37B. The traces indicate that all the quinoidal base present at pH=5.4 was converted during the mixing time of the stopped flow, and the flavylium grows according to a bi-exponential kinetics. The fast process corresponds to the hydration reaction, which at pH=1.2 is faster than tautomerization due to its proton dependence. The slowest step corresponds to the formation of more flavylium cation from Cc through B and it is assigned to the rate constant k-t=1.2 (Pina et al. 2012). Moreover, from the ratio of amplitudes of both kinetics the constant Kt=1.5 was calculated, allowing to obtain kt=1.8 and Kh Ki=8.6x10-4. Similarly to DOP the compound GCP is photochromic, Fig. 38. Irradiation of the transchalcone leads to the formation of quinoidal base with a quantum yield of 0.1. The system is reversible and thus a photostationary state is achieved. The kinetics of the thermal recovery of the photoproduct was measured at two pH values and fits quite well with the bell shape curve, Fig. 37A. Flash photolysis of equilibrated solutions at pH=4.1 is similar to the parent compound. Within the lifetime of the flash Ct disappears to give Cc, which goes forward to form AH+/A or backward to recover Ct. Representation of the observed rate constants from experiments like the one of Fig. 38B at different pH values are shown in Fig. 38C. A global fitting was achieved through eq. (8) and eq.(9) and the one of the bell-shaped curve, eq.(9) leading to the rate and equilibrium constants presented in Table 4 and Table 5. 112 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 0 0.2 0.4 0.6 0.8 200 300 400 500 600 700 A Wavelength (nm) h pH=4.2 irr=366 nm -0.01 0 0.01 A (495 nm) -0.01 0 0.01 A (495 nm) -0.03 -0.015 0 0 4 8 12 A (398 nm) Time (s) -0.01 0 0.01 A (495 nm) 0 1 2 2 3 4 5 6 7 8 Kobs (s-1) pH Fig. 38 - A - Spectral variations following the irradiation of GCP at pH=4.1 at the irradiation wavelength of 366 nm; B - Flash photolysis followed at 495 nm (AH+ and A) up and 398 nm (Ct) bottom; C - observed rate constant of the flash photolysis as a function of pH. As in the case of DOP it is easy to construct an energy level diagram where the five species are relatively positioned, Fig. 39. It is worth of note the fact that similarly to DOP (and to SCP see below) the energy level of Ct and A are close and lower relatively to B and Cc, as expected from the shape and position of the absorption spectra at the equilibrium. Fig. 39 - Energy level diagram for GCP based on the equilibrium constants of Table 3. A B C FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 113 Syringylcatechinpyrylium (SCP). The absorption spectra of SCP was obtained immediately after the pH jump and a value of pKa1=3.8 was obtained in Fig. 40. 0 0.1 0.2 0.3 300 400 500 600 700 A Wavelength (nm) 0 0.1 0.2 1 2 3 4 5 6 A pH 497 nm 607 nm pKa=3.8 Fig. 40 - Absorption spectra of solutions of SCP taken immediately after a direct pH jump from stock solutions at pH=1 to higher pH values; inset representation of the fitting obtained at two different wavelengths (pKa=3.9±0.1). The further ionization constants were not determined due to the appearance of some decomposition, which can be attributed to the presence of the cathecol unit, which is known to be easily oxidized at higher pH values. The absorption spectra of the equilibrated solutions, Fig. 41, are again compatible with an equilibrium involving essentially the species AH+, A and Ct. 0 0.2 0.4 300 400 500 600 700 800 A Wavelenght (nm) pK'a=3.2 0 0.5 1 3 5 A (498 nm) pH Fig. 41 - A - Absorption spectra of equilibrated solutions of SCP; B - Bell shaped curve for the compound SCP. A B 114 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution The spectral variations upon irradiation of SCP of equilibrated solutions at pH=4.4 are shown in Fig. 42, and it can be concluded that SCP shows a behavior similar to DOP and GCP. However there are some differences, which can be easily visualized from the data of Fig. 43C, Fig. 38C and Fig. 42C. Inspection of eq.(9) used to fit the data indicate that it tends to eq.(10) when the proton concentration decreases. kobs(hydr )=kiKt 1+Kt (10) The value of eq.(10) defines the plateau reached in the above mentioned figures, respectively 0.057, 0.22 and 0.63. This is also in accordance with the data reported in Table 3 and Table 4. Flash photolysis of equilibrated solutions at pH=4.4 is similar to the parent compound and GCP. Within the lifetime of the flash Ct disappears to give Cc, which goes forward to form AH+/A or backward to recover Ct. Representation of the observed rate constants from experiments like the one of Fig. 42B at different pH values are shown in Fig. 42C. 0 0.1 0.2 0.3 0.4 300 400 500 600 700 A Wavelength (nm) h pH=4.4 =0.1 0 0.015 0 5 10 15 A (502 nm) -0.04 0 0 5 10 15 A (396 nm) Time (s) 0 1 2 2 3 4 5 6 7 kobs(Hydr) pH kobs(taut) kobs Fig. 42 - A - Irradiation of the compound SCP at pH=1.0 (full lines): the spectrum of the solution immediately after its preparation from a pH jump from pH=1.0 to pH=4.4, before being irradiated is also shown (traced line); B - flash photolysis traces at 502 nm (AH+/A) and 396 nm (Ct); C - observed rate constant of the flash photolysis as a function of pH. The energy level diagram of SCP is shown in Fig. 43 and is qualitatively similar to DOP and GCP showing the higher stability of the species A and Ct at higher pH values, much higher than B and Cc. A B C FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 115 Fig. 43 - Energy level diagram for SCP based on the equilibrium constants of Table 3. The energy levels of B and Cc were obtained through the fitting of eqs.(7) to (9) due to the lack of observation of traces corresponding to B and Cc in the reverse pH jumps experiments carried out by stopped flow.  CONCLUSION The network of chemical reactions involving anthocyanins and related compounds is pH dependent and by consequence the detailed kinetics of the system have been obtained through pH jumps, which allow us to follow the relaxation processes toward the new equilibrium. When the flavylium derived networks of chemical reactions exhibit photochemistry flash photolysis is an excellent complementary technique to collect kinetic information on the system without changing the pH. This is particularly important for flavylium derivatives possessing a low cis-trans isomerization barrier, since hemiketal, B, and cis-chalcone, Cc, are transient species not detected from the pH jump studies. In fact, when compared to common wine anthocyanins in which B (incolor) is the major species at higher pH values, sometimes at the pH of the wine, these oaklin compounds have a behavior more similar to simpler deoxyanthocyanidins, in which the equilibrium involves essentially AH+, A and Ct and the mole fraction of the quinoidal base A is approximately 50%. Furthermore the pKca values obtained for GCP and SCP (3.05 and 3.2, respectively) are higher than the ones reported for malvidin-3-glucoside (2.54 (Brouillard et al. 1978) and 2.3 (Nave, Petrov 2010)), meaning that the flavylium cation is more stable in the former. The lost of color in solution is thereby greatly diminished in oaklin compounds when compared to other anthocyanins, not only because pKca is greater but also because the mole fraction of the base is substantially higher at higher pH values. In consequence, oaklins which are formed through the reaction of catechin and oak wood aldehydes, may play an important role in some color changes observed in wine aging and may contribute to the overall color presented by some wines. Nevertheless, it is important to note that anthocyanins like malvidin-3-glucoside are still present in wines at concentration levels far higher than oaklins and their contribution to 116 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution wine color must not be underestimated, neither the effects of copigmentation (Boulton 2001). Despite some differences, the general behavior of guaiacylcathechinpyrylium and syringylcatechinpyrylium is quite similar to the model compound deoxypeonidin. Given the fact that the lost of color in solution of these compounds is lower than anthocyanins they may be regarded as potential commercial food colorants. However, further studies should be performed in order to test their stability to other conditions (temperature, sulfites, etc) as well as to study their antioxidant properties.  AKNOWLEDGEMENTS The authors thank FCT (Fundação para a Ciência e Tecnologia) for a PhD grant (ref. SFRH/BD/68736/2010) and a research grant (PTDC/QUI-QUI/117996/2010). This research was also supported by a research project grant (PTDC/QUI-QUI/117996/ 2010) funded by FCT (Fundação para a Ciência e Tecnologia). FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 117 Chapter 4 Deoxyvitisins: a new set of pyrano-3-deoxyanthocyanidins Sousa, A., Araújo, P., Mateus, N., de Freitas, V. Tetrahedron Lett. 2013, 54(35), 4785-4788 118 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution In this work, the first author was responsible for undertaking all experimental activities with the assistance of Paula Araújo in the purification of the new compounds. He also benefited from the advice of MS and NMR technicians and guidance by the rest of the authors. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 119 464-484 nm Deoxyvitisins: a new set of pyrano-3-deoxyanthocyanidins André Sousa, Paula Araújo, Nuno Mateus, and Victor de Freitas* Departamento de Química, Faculdade de Ciências, Universidade do Porto, Centro de Investigação em Química, Rua do Campo Alegre 687, 4169-007 Porto, Portugal The chemical synthesis of deoxyvitisins (pyrano-3-deoxyanthocyanidins) is reported herein for the first time. Three different types of compounds were synthesized from the reaction between two deoxyanthocyanidins (deoxypeonidin and deoxymalvidin) and pyruvic acid, vinyloxy-trimethylsilane and acetone-1,3-dicarboxylic acid. The structure of the new compounds has been characterized by means of visible, MS and NMR spectroscopy. KEYWORDS: deoxyvitisins; vitisins; deoxyanthocyanidins; anthocyanidins; aldehydes; vinyloxy-trimethylsilane; pyruvic acid; acetone-1,3-dicarboxylic acid; NMR; mass spectrometry  INTRODUCTION Pyranoanthocyanins belong to an important group of anthocyanin-derived pigments that occur essentially in fruits and processed foodstuffs like vegetable juices or red wines (Brouillard, Chassaing 2003, Fulcrand, Atanasova 2004, Rentzsch, Schwarz 2007). Formation of this kind of compounds in food matrixes result from a cycloaddition between C-4/5-OH of the anthocyanin and a double bond from another molecule such as acetaldehyde (Bakker and Timberlake 1997), pyruvic acid (Fulcrand et al. 1998), 464-484 nm (nm) Absorbance Wavelenght (nm) Deoxyvitisins: R3 = COOH, R2 = OH, deoxyvitisin A (peonidin derived) R3 = H, R2 = OH, deoxyvitisin B (peonidin derived) R3 = H, R2 = OCH3, deoxyvitisin B (malvidin derived) R3 = CH3, R2 = OH, methylpyranodeoxypeonidin Deoxyanthocyanidins: R1= H, 3-deoxypeonidin R1= OCH3, 3-deoxymalvidin 126 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Fig. 45 - Hypothetical mechanism for the formation of deoxyvitisins 3 obtained from the reaction between deoxyanthocyanidins 1 and carbonyl compounds 2 (enolic forms).  CONCLUSION These results show for the first time a new class of deoxyanthocyanidins that have interesting visible spectroscopic properties, representing a step forward towards the research for new pigments with significant stability. Nonetheless, further studies are still required to test their chromatic properties at different conditions (pH, temperature, sulfites, etc) as well as their antioxidant capability in order to precisely evaluate the usefulness of these compounds in several potential applications in the food, medical, cosmetic and/or technology industries.  AKNOWLEDGEMENTS The authors thank FCT (Fundação para a Ciência e Tecnologia) for a PhD grant (ref. SFRH/BD/68736/2010) and a research grant (PTDC/QUI-QUI/117996/2010). This research was also supported by a research project grant (PTDC/QUI-QUI/117996/ 2010) funded by FCT (Fundação para a Ciência e Tecnologia). IX O - R2H2 - R4 R1 = H: deoxypeonidin; R1 = OCH3: deoxymalvidin R2 = OH, R3 = COOH, R4 = H: pyruvic acid (enolic form) R2 = OSi(CH3)3, R3 = H, R4 = H: vinyloxy-trimethylsilane R2 = OH, R3 = CH2COOH, R4 = COOH: acetone-1,3-dicarboxylic acid (enolic form) 1 2 3 D C A B 4 5 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 127  SUPPLEMENTARY DATA Table. 8. 1H and 13C NMR data and HMBC and HSQC correlations of deoxyvitisin A (peonidin derived), determined in DMSO/TFA (90:10). Position δ 1H (ppm); J (Hz) δ 13C (ppm) HMBC HSQC Ring A, B, C 2C 169.7 H-3C, H-2ʹB, H-6ʹ 3C 8.06; s 103.3 H-9D H-3C 4C 154.7 H-3C 4aA 109.1 H-8A, H-6A, H-9D, H-3C 5A 153.0 H-6A 6A 7.09; d (1.60) 101.1 H-8A H-6A 7A 168.4 H-6A, H-8A 8A 7.27; d (1.60) 101.2 H-6A H-8A 8aA 154.0 H-8A 1ʹB 120.5 H-3C, H-5ʹB 2ʹB 7.76; d (1.72) 111.7 H-6ʹB H-2ʹB 3ʹB 149.1 OCH3, H-2ʹB, H5ʹB 4ʹB 155.2 H-2ʹB, H-5ʹB, H6ʹB 5ʹB 7.08; d (8.57) 117.0 H-5ʹB 6ʹB 7.87; dd (1.72; 8.57) 124.4 H-2ʹB, OCH3 H-6ʹB OCH3 3.94; s 56.3 OCH3 Ring D 9D 7.64; s 109.4 H-9D 10D 154.5 H-9D COOH 160.1 H-9D s - singlet; d - doublet; dd – double doublet 128 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Table 9. 1H and 13C NMR data and HMBC and HSQC correlations of deoxyvitisin B (peonidin derived), determined in DMSO/TFA (90:10). Position δ 1H (ppm); J (Hz) δ 13C (ppm) HMBC HSQC Ring A, B, C 2C 169.1 H-3C, H-2ʹB, H-6ʹB 3C 7.87; s 101.8 H-9D H-3C 4C 150.9 H-10D 4aA 109.1 H-8A, H-6A, H-9D, H-3C 5A 153.4 H-6A, H-10D 6A 7.13; d (1.38) 100.7 H-8A H-6A 7A 167.5 H-6A, H-8A 8A 7.32; d (1.38) 100.9 H-6A H-8A 8aA 154.0 H-8A 1ʹB 120.5 H-3C, H-5ʹB 2ʹB 7.76; d (1.72) 111.5 H-2ʹB 3ʹB 148.9 OCH3, H-2ʹB, H5ʹB 4ʹB 154.6 H-2ʹB, H-5ʹB 5ʹB 7.06; d (8.57) 116.8 H-5ʹB 6ʹB 7.85; dd (1.72; 8.57) 123.9 H-2ʹB H-6ʹB OCH3 3.93; s 56.2 OCH3 Ring D 9D 7.03; * 107.3 H-3C, H-10D H-9D 10D 8.47; d (5.14) 160.0 H-9D H-10D s - singlet; d - doublet; dd – double doublet, * - unresolved FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 129 Table 10. 1H and 13C NMR data and HMBC and HSQC correlations of deoxyvitisin B (malvidin derived), determined in DMSO/TFA (90:10). Position δ 1H (ppm); J (Hz) δ 13C (ppm) HMBC HSQC Ring A, B, C 2C 169.0 H-3C, H-2ʹB/H-6ʹ 3C 7.95; s 101.8 H-3C 4C 150.9 H-10D 4aA 109.1 H-8A, H-6A, H-9D, H-3C 5A 153.3 H-10D 6A 7.13; d (1.70) 100.6 H-6A 7A na 8A 7.32; d (1.70) 101.0 H-8A 8aA 154.0 H-8A 1ʹB 119.0 H-3C, H-2ʹB/H-6ʹB 2ʹB/6ʹB 7.58; s 106.6 H-6ʹB/H-2ʹB H-2ʹB/ H-6ʹB 3ʹB/5ʹB 148.9 OCH3, H-2ʹB/ H6ʹB 4ʹB 144.0 H-2ʹB/H-6ʹB OCH3 3.94; s 56.7 OCH3 Ring D 9D 7.03; d (5.33) 107.0 H-3C, H-10D H-9D 10D 8.49; d (5.33) 159.6 H-9D H-10D s - singlet; d - doublet; dd – double doublet; na – not attributed 130 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution Table 11. 1H and 13C NMR data and HMBC and HSQC correlations of methylpyranodeoxypeonidin, determined in DMSO/TFA (90:10). Position δ 1H (ppm); J (Hz) δ 13C (ppm) HMBC HSQC Ring A, B, C 2C 168.3 H-3C, H-2ʹB, H-6ʹB 3C 7.78; s 101.1 H-9D H-3C 4C 154.1 H-3C 4aA 107.6 H-8A, H-6A, H-9D, H-3C 5A 153.8 H-6A 6A 7.13; d (1.50) 100.4 H-8A H-6A 7A 167.3 H-6A, H-8A 8A 7.32; d (1.50) 100.7 H-6A H-8A 8aA 153.5 H-8A 1ʹB 120.6 H-3C, H-5ʹB, H-2ʹB 2ʹB 7.72; d (1.90) 111.5 H-6ʹB H-2ʹB 3ʹB 148.8 OCH3, H-2ʹB, H5ʹB 4ʹB 154.2 H-2ʹB, H-5ʹB, H6ʹB 5ʹB 7.06; d (8.43) 116.7 H-5ʹB 6ʹB 7.81; dd (1.90; 8.43) 123.5 H-2ʹB H-6ʹB OCH3 3.91; s 56.3 OCH3 Ring D 9D 6.91; s 105.1 H-3C, CH3 H-9D 10D 172.2 H-9D, CH3 CH3 2.57; s 21.2 H-9D CH3 s - singlet; d - doublet; dd – double doublet FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 131 Chapter 5 Color Stability and Spectroscopic Properties of Deoxyvitisins in Aqueous Solution Sousa, A., Cabrita, L., Araújo, P., Mateus, N., Pina F., de Freitas, V. New J. Chem. 2014, 38(2), 539-544 132 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution In this work, the first author was responsible for the chemical synthesis of the tested compounds, in which he counted with the assistance from Paula Araújo in their purification. Spectroscopy experiments were performed by Luís Cabrita. The first author analyzed the experimental data and compared it with spectroscopy studies from analogous compounds available in the literature. He was always guided and advised by the rest of the authors. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 133 Color Stability and Spectroscopic Properties of Deoxyvitisins in Aqueous Solution André Sousa†, Luís Cabrita‡, Paula Araújo†, Nuno Mateus†, Fernando Pina‡ and Victor de Freitas† † Centro de Investigação em Química, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal; ‡ REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal The color properties and stability of three types of 3-deoxyvitisins derived from peonidin (deoxyvitisin A, B and methyldeoxyvitisin) were studied by UV-Visible spectroscopy. Similarly to pyranoanthocyanins, the conjugated double bonds among pyranic rings C and D provide a higher electronic delocalization that prevents the nucleophilic attack of water at position 2 and the subsequent formation of the hemiketal and chalcone species. Consequently, besides flavylium cation (AH+), neutral (A) and ionized bases (An-) have been identified by increasing pH, and the respective acidity constants were determined. The acidity constant values for the formation of the neutral quinoidal base (pKa2(deoxyvit A) = 4.8, pKa1(deoxyvit B) = 4.7 and pKa1(methyldeoxyvitisin) = 5.2) are slightly higher than the ones reported in the literature for their corresponding 3-glucosyl derivatives (vitisins). Given their higher stability, these pigments may be regarded as potential food colorants. KEYWORDS: 3-deoxyvitisins, deoxyanthocyanidins, vitisins, anthocyanins, flavylium network, food colorants  INTRODUCTION It is well known that the color of anthocyanins is greatly affected by the pH of the solution. The sequence of chemical reactions was correctly established by Brouillard et al. using temperature, pressure, and pH jump experiments: at very acidic pH, the red flavylium cation (AH+) is the predominant species; when the pH is raised the flavylium cation immediately undergoes a proton transfer reaction, giving rise to the blue/purple quinoidal base (A), and simultaneously but more slowly the flavylium cation leads to the formation of colorless hemiketal (B) through the hydration reaction. The hemiketal compound further undergoes a tautomerization reaction to give the pale yellow cischalcone (Cc), which isomerizes to trans-chalcone (Ct) (Brouillard and Lang 1990). 134 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 3-Deoxyanthocyanidins are yellowish pigments commonly found in several food plants such as corn, black tea leaves and sorghum. Oppositely to anthocyanins, these pigments lack the 3-O-sugar moiety and show a significant increase in stability in slightly acidic solutions (Melo et al. 2007). It was recently demonstrated that at moderately acid pH values, while the predominant species in anthocyanins is the colorless hemiketal, in 3-deoxyanthocyanidins the equilibrium occurs between the species flavylium cation, trans-chalcone and quinoidal base. Moreover, the mole fraction of the base is approximately 50%, which contributes to the observed higher color intensity (Sousa, Petrov 2013). Several studies have also demonstrated the potential applications of these compounds for viable commercial food colorants, hair dyes, laser dyes, sensitizers for solar cells, molecular-level memory systems and health-promoting phytochemicals (Czerney et al. 1995, Cherepy et al. 1997, Roque et al. 2002, Awika et al. 2005, Shih et al. 2007). On the other hand, pyranoanthocyanins differ from their genuine precursors (antho y y y . y λmax hypsochromically shifted (between 478 and 510 nm) conferring orange colors, except for w w λmax values to more bluish hues around 580 nm (Mateus et al. 2003). Due to the additional pyranic ring, the color of these compounds is much more stable towards pH variations and bleaching by SO2 in comparison to the parent anthocyanins, because it prevents the hydration reaction to give the hemiketal and the subsequent formation of the chalcone species (SarniManchado, Fulcrand 1996). Although it has been generally accepted that anthocyanins are stable only at low pH values, it has been shown that, depending on the substitution pattern on the B-ring, some anthocyanin 3-glucosides lose 40-80% of their initial color intensity between pH 1 and pH 5-7, whereas pyranoanthocyanins practically do not change their color intensity (Fossen, Cabrita 1998, Cabrita et al. 2000, Oliveira et al. 2009, Oliveira, Petrov 2011, Oliveira, Mateus 2013). Theoretically, designing deoxyanthocyanidins with a pyranic ring would result in compounds with further increased stability. Bearing this, we recently synthesized three types of 3-deoxypyranoanthocyanidins (deoxyvitisins) from the reaction between 3deoxyanthocyanidins and pyruvic acid, vinyloxy-trimethylsilane or acetone-1,3dicarboxylic acid (Sousa, Araujo 2013). In another study, a new natural pigment was FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 135 also isolated from red Sorghum bicolor with a deoxyvitisin-type structure, containing apigeninidin as a base unit (Khalil et al. 2010). The aim of this work was to investigate the color stability and the network of chemical reactions occurring in aqueous solution upon pH variations for 3-deoxyvitisins, comparing the results with the model compound 4',7-dihydroxyflavylium and with values reported in the literature for corresponding vitisins. Fig. 46 - The model compound 4',7-dihydroxyflavylium and the 3-deoxyvitisins: methyldeoxyvitisin (R=CH3), deoxyvitisin A (R=COOH) and B (R=H).  MATERIALS AND METHODS Synthesis of 3-deoxyvitisins and the model compound 4',7dihydroxyflavylium. The synthesis of the deoxyvitisin compounds was followed according to the procedures described in the literature (Sousa et al. 2013). Initially, the synthesis of the reagent deoxypeonidin was performed by incubating phloroglucinol (8 mM) with coniferaldehyde (80mM) in a 12% ethanol aqueous solution, adjusted to pH 1.5 (Sousa et al. 2012). The deoxypeonidin was then incubated with the reagents pyruvic acid, vinyloxy-trimethylsilane and acetone-1,3-dicarboxylic acid in separate model aqueous solutions, under different conditions, as described elsewhere (He et al. 2006, Morata et al. 2007, Oliveira et al. 2009), to form the compounds deoxyvitisin A, B and methyldeoxyvitisin, respectively. The formation of new compounds was followed by HPLC-DAD using a reversed phase C-18 (Merck®) column (250 x 4.6 mm i. d., particle size 5 µm), at 25 ºC. Solvents were (A) water/formic acid (95:5) and (B) acetonitrile. The elution gradient was performed using a L-2130 Merck® pump from 10 to 35% B for 55 min at a flow rate of 1.5 mL.min-1. 4’ 7-dihydroxyflavylium was synthesized according to the method of Katritzky (Katritzky, Czerney 1998). 142 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 143 Chapter 6 Evidence for Copigmentation Interactions between Deoxyanthocyanidin Derivatives (Oaklins) and Common Copigments in Wine Model Solutions Sousa, A., Araújo, P., Cruz, L., Brás, N. F., Mateus, N., de Freitas, V. J. Agric. Food Chem. 2014, 62 (29), 6995–7001 144 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution In this work, the first author was responsible for all the experimental activities with the exception of molecular dynamic simulations, which were performed by Natércia Brás. He also counted with the help of Paula Araújo in the purification of oaklins and with Luís Cruz in the copigmentation experiments and data interprentation. Guidance and advice were also obtained from the rest of the authors, regarding project planning and data interpretation. FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 145 Evidence for Copigmentation Interactions between Deoxyanthocyanidin Derivatives (Oaklins) and Common Copigments in Wine Model Solutions André Sousa†, Paula Araújo†, Luís Cruz†, Natércia F Brás‡, Nuno Mateus† and Victor de Freitas†* † Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal; ‡ REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal The aim of this study was to investigate interactions possibly taking place in red wine between oaklins, that are deoxyanthocyanidin derivatives (guaiacylcatechin-pyrylium and syringylcatechin-pyrylium) and several copigments: catechin (CP1), epicatechin (CP2), chlorogenic acid (CP3), epigallocatechin (CP4) and procyanidin B3 (CP5). The results show that oaklins, like common anthocyanins, also present copigmentation interactions that further stabilize the flavylium cation in hydroalcoholic solutions. Molecular dynamics simulations were also performed to interpret the binding data, to specify the relative arrangement of the pigment and copigment molecules within the complexes, and to interpret their absorption properties in the visible range. KEYWORDS: oaklins; copigments; deoxyanthocyanidins; anthocyanins; aldehydes; flavylium network; food colorants; wine aging; molecular dynamics  INTRODUCTION Oaklins are catechinpyrylium compounds formed in red wines aged in oak barrels and result from the reaction between catechin and wood cinnamic aldehydes. They present a brick-red color and oppositely to anthocyanins, these pigments do not possess a glycosyloxy substituent at C3 of the flavylium core and are thus classified as deoxyanthocyanidins (de Freitas et al. 2004, Sousa et al. 2005). Despite the thermodynamic tendency of anthocyanins to form colorless hemiketals, natural colors expressed by anthocyanins are fairly stable, which evidences naturally occurring stabilization mechanisms. Indeed, copigmentation has been one of the most important mechanisms described in the literature to stabilize the colored forms. Copigmentation mainly refers to interactions between colorless phenols (copigments) 146 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution y ’ . pro y y y π−π k between the polarizable orbitals of the aromatic rings (Galland, Mora 2007, GonzalezManzano, Duenas 2009, Yoshida, Mori 2009, Cruz, Bras 2010). In a previous investigation it was concluded that when compared to common wine anthocyanins in which the hemiketal B (incolor) is the major species at wine pH (~3.5), the equilibrium of oaklins involves essentially the flavylium cation AH+, the quinoidal base A, and the trans-chalcone Ct and the % of hemiketal is very low. The behavior is similar to simpler deoxyanthocyanidins as described in the literature. Furthermore, the pK′a values obtained for oaklin compounds were higher than the ones reported for malvidin-3-glucoside, meaning that the flavylium cation is more stable in the former. The loss of color in solution is thereby greatly diminished in oaklin compounds when compared to other anthocyanins, not only because the pK′a is greater but also because the mole fraction of the base is substantially higher at higher pH values. Nevertheless, the effects of copigmentation cannot be underestimated when analyzing color changes in wine solutions (Sousa et al. 2013). It is well known the importance and contribution of the copigmentation effect on anthocyanin pigments (Asen, Stewart 1971, 1972, Mazza and Brouillard 1990). It was only recently that the copigmentation interactions between deoxyanthocyanidins with some phenolics were studied (Awika 2008). The aim of this work was thus to study the copigmentation interactions possibly occurring between the oaklin compounds guaiacylcatechinpyrylium (GCP) and syringylcatechinpyrylium (SCP) with common copigments such as catechin, epicatechin, chlorogenic acid, epigallocatechin, procyanidin B3 and determine the respective binding constants. The relationship between copigmentation ability and the structure of the complexes was also evaluated.  MATERIALS AND METHODS Samples. (+)- − -epicatechin (EC) and chlorogenic acid were purchased from Sigma-Aldrich (Madrid, Spain). − -Epigallocatechin (EGC) was purchased from Biopurify Phytochemicals Ltd. (Sichuan, China). Procyanidin B3 (PCB3) was extracted from barley and isolated according to the procedures described elsewhere (Dvorakova, Moreira 2008, Teixeira, Cruz 2013). The pigments guaiacylcatechinpyrylium (GCP) and syringylcatechinpyrylium (SCP) were obtained through the chemical synthesis between catechin and coniferaldehyde or sinapaldehyde, respectively, according to the procedure described elsewhere (Sousa et al. 2013). FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 147 Copigmentation. All solutions used were prepared in a citrate buffer solution (0.2 M) with 10% ethanol at pH 3.5, and the ionic strength was adjusted to 0.5 M by the addition of sodium chloride. Each pigment/copigment solution was prepared by mixing a volume of pigment (10-4 M) solution with an aliquot of copigment solution to give the required pigment/copigment molar ratio of 1:0, 1:1, 1:5, 1:10, 1:20, 1:30, 1:40. Each experiment was performed in triplicate. All of the solutions were left to equilibrate for 30 min before spectroscopic measurements. For GCP with the copigment epigallocatechin, the absorbance values were collected at the w λmax 498 nm) of the isosbestic point of the flavylium cation (10-4 M) and its copigmentation complex (GCP/copigment molar ratio=1:40). This parameter was determined in strongly acidic solutions (1 M H H ≈0 w y is the sole anthocyanin form. However, for the rest of the copigments and for the pigment SCP, an isosbestic point was not observed, hence the absorbance values were collected at the maximum absorption w G /S H 3.5 λmax 485 nm for GCP and 500 nm for SCP). UV−Visible Spectroscopy. − w -Tek Power Wave XS spectrophotometer at a constant temperature of 25 °C from 360 to 830 nm (1 nm sampling interval) using a 1 cm path length cell. Data Analysis. The curve fittings were carried out using the software program MicroMath (Salt Lake City, UT, USA). Curve fittings were achieved through a leastsquares regression method. Statistical analysis reported standard deviations and correlation coefficients. Molecular Dynamic Simulations. The initial geometries of the pigments (GCP and SCP) and copigments (catechin, epicatechin, epigallocatechin, B3 and chlorogenic acid) molecules were built with the GaussView software (Gaussian). To calculate the optimized geometries and electronic properties for the subsequent parameterization of these compounds, the Gaussian 09 suite of programs (Frisch 2009) was used to perform restricted Hartree-Fock calculations (RHF), with the 6-31G(d) basis set. Atomic charges were further recalculated using the RESP algorithm (Bayly, Cieplak 1993). This methodology was chosen for its consistency with that adopted in the parameterization process of AMBER 10.0 simulation package (Case 2008). MD (Molecular Dynamic) simulations were performed with GAFF (generalized amber force field for small organic molecules) (Wang, Wolf 2004) and the TIP3P model for the solute and water, respectively. Explicit solvation was included as a rectangular box with a 12Å distance between the box faces and any atom of the compounds. One counter-ion (Cl-) was employed to neutralize the positive charge of each system, which has a size of 148 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution approximately 5,700 atoms. All complex geometries were minimized in two stages: first, the solute was kept fixed and only the position of the counter-ion and water molecules was minimized (500 steps using the steepest descent algorithm and 1,500 steps carried out using conjugate gradient); second, the full system was minimized (1,000 steps using the steepest descent algorithm and 2,000 steps carried out using conjugate gradient). Subsequently, an MD simulation of 100 ps at constant volume and temperature, and considering periodic boundaries conditions was run, followed by 30 ns MD simulation with the NPT ensemble, in which Langevin dynamics was used (collision frequency of 1.0 ps-1) to control the temperature at 303.15 K (Izaguirre, Catarello 2001). All simulations were carried out using the PMEMD module, implemented in the Amber 10.0 simulation package (Case 2008). Bond lengths involving a hydrogen atom were constrained using the SHAKE algorithm, and the equations of motion were integrated with a 2 fs time-step using the Verlet leapfrog algorithm (Ryckaert, Ciccotti 1977). The Particle-Mesh Ewald (PME) method (Essmann, Perera 1995) was used to include the long-range interactions, and the nonbonded interactions were truncated with a 10Å cutoff. The MD trajectory was saved every 2 ps and the MD results were analyzed with the PTRAJ module of AMBER 10.0 (Case 2008). Calculation of Relative Binding Free Energies (ΔΔGbinding). The MM-PBSA script (Molecular Mechanics−Poisson−Boltzmann Surface Area) (Kollman, Massova 2000, Massova and Kollman 2000, Huo, Massova 2002) as implemented in Amber 10.0 simulations package (Case 2008) was used to calculate the binding free energies (ΔGbinding) for all complexes. A series of 150 geometries was extracted every 100 steps of each simulation. The internal energy (bond, angle, and dihedral), the electrostatic and the van der Waals interactions were calculated using the Cornell et al. force field (Cornell, Cieplak 1995) with no cutoffs. The electrostatic solvation free energy was calculated by solving the Poisson−Boltzmann equation with the PBSA program. The nonpolar contribution to the solvation free energy due to van der Waals interactions between the solute and the solvent and cavity formation was modeled as a term that is dependent on the solvent accessible surface area of the molecule. As these compounds possess similar structures and binding modes, the relative binding energies (ΔΔGbinding) were calculated with respect to the most stable complex.  RESULTS AND DISCUSSION The association of the oaklins GCP (guaiacylcatechinpyrylium) and SCP (syringylcatechinpyrylium) with the copigments selected (Fig. 54) was quantitatively FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 149 O O OCH3 OH OCH3 OH HO HO H HO H D E AC B H 3 O O H OH OCH3 OH HO HO H HO H D E AC B H 3 evaluated through the determination of the copigmentation binding constants (KCP) for each pigment-copigment pair. Fig. 54 - Chemical structures of the deoxyanthocyanidin derivatives - oaklins (GCP and SCP) and the copigments investigated in this work. GCP (Guaiacylcatechinpyrylium) SCP (Syringylcatechinpyrylium) Catechin Epicatechin Chlorogenic acid Epigallocatechin Procyanidin B3 150 FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution ka k-a kh k-h kt k-t ki k-i To minimize self-association effects in the intermolecular copigmentation studies, a low concentration of pigment (1 × 10−4 M) was used. Although ethanol largely reduces the copigmentation effect (Dangles and Brouillard 1992), its inclusion in the experimental conditions was crucial to prevent the precipitation during the spectroscopic analysis of the pigment and copigment compounds due to their low solubility in water. Fig. 55 - Network of chemical reactions of oaklins (R=OCH3, SCP; R=H, GCP). Copigmentation interactions occurring in oaklins. In a very acidic medium, only the flavylium cation is present and the visible absorbance is A0 = εAH+C where C is the total pigment concentration, and can be approximated to C = [AH+]. Oppositely to anthocyanins, in which the contribution of quinonoidal bases (A) is negligible in mildly acidic medium (pH 3.5) and the hemiketal is the predominant species, in deoxyanthocyanidins, particularly oaklins, the equilibrium plateau occurs between the flavylium cation, the neutral quinoidal base and the trans-chalcone, with the hemiketal and the cis-chalcone being transient species (Sousa et al. 2013). Copigmentation of the quinoidal bases could make a contribution, as those forms are also prone to stacking interactions with copigments. However, as the quinoidal bases and their copigmentation complexes absorb the visible light at higher wavelengths than their flavylium counterparts, no specific contribution is expected in the measured wavelength range (480-500 nm) (Haslam and Lilley 1988, Pina 1998). Copigmentation of quinoidal bases also promotes a high bathochromic shift (which is not observed in our case), especially with purine copigments like caffeine and theophylline (Pina et al. 2012). Quinoidal base (A) Flavylium cation (AH+) Hemiketal (B) cis-chalcone (Cc) trans-chalcone (Ct) FCUP Deoxyanthocyanidins and derivatives: physical-chemical and antioxidant properties in aqueous solution 151 Bearing this in mind, the total concentration of the pigment for deoxyanthocyanidins at mildly acidic solutions can be represented by (Malien-Aubert, Dangles 2002, Awika 2008) C = [AH+] + [A] + [B] + [Cc] + [Ct] = [AH+] + [A] + [Bc] where Bc is the pool of colorless forms in equilibrium. The apparent thermodynamic constant for the overall reactions (Fig. 55) taking place is thus given by Kca = Ka + Kh + Kh Kt + Kh Kt Ki Being so, one can also write C= + AH 1+a ' K 3 H+ O Oppositely to common anthocyanins in which 0 A=AH+ + AH 1+h K 3 H+ O , from the equations above, and in the absence of the copigment one easily obtains 0 A=AH+ C 1+a ' K 3 H+ O (eq 1) In the presence of the copigment, and assuming a 1:1 stoichiometry for the complex, the visible absorbance at pH 3.5 becomes A = εAH+ C + εAHCP+ [AHCP+] We thus have (eq 2) with