Structure determination and colour properties of a new directly linked flavanol–anthocyanin dimer Erika Salas, * Christine Le Guerneve ´, He ´le `ne Fulcrand, Ce ´line Poncet-Legrand and Ve ´ronique Cheynier INRA–UMR Sciences pour l’Oenologie, 2 Place Viala, 34060 Montpellier, France Received 20 August 2004; revised 15 September 2004; accepted 20 September 2004 Available online 5 October 2004 Abstract—The structure of catechin-(4a!8)-malvidin 3-O-glucoside obtained by reaction of taxifolin and malvidin 3-O-glucoside following a protocol adapted from proanthocyanidin dimer synthesis was determined by NMR spectrometry. Incorporation of the anthocyanin moiety into a covalent linked flavanol–anthocyanin dimer did not modify its colour properties (i.e., hydration equi- librium constant and copigmentation). Ó 2004 Published by Elsevier Ltd. Red wine is rich in phenolic compounds, which are responsible for its colour and astringency while also con- tributing to its flavour. During ageing and storage of red wine, many changes occur. Phenolic compounds in wines, particularly anthocyanins and proanthocyanidins (i.e., flavanol oligomers and polymers, also referred to as condensed tannins) are progressively converted by vari- ous reaction mechanisms to new pigment and tannin species during wine storage. These new pigments present different colour properties from those of their anthocya- nin precursors (i.e., red grape pigments). 1 Two mechanisms, leading, respectively, to anthocyanin– flavanol (A + –F) and to flavanol–anthocyanin (F–A + ) adducts, are postulated for direct reactions between anthocyanins and flavanols. During the formation of A + –F, the anthocyanin is in the flavylium form (A + ). Nucleophilic addition of the flavanol onto the flavylium cation leads to the colourless flavene (A–F), which can be oxidized to the red flavylium (A + –F) and then to a xanthylium salt. 1–3 In the formation of F–A + , proantho- cyanidins (F–F) are affected by acid-catalyzed cleavage of their interflavanic bond, releasing the intermediate carbocation F + , 4 which acts as an electrophile. Nucleo- philic addition of the anthocyanin in its hydrated hemi- ketal form (AOH) 5 yields the colourless dimer (F– AOH), which dehydrates to the red flavylium form (F– A + ). So far, flavanol–anthocyanin adducts in the flavylium form have been observed in wine 6 and recently been suc- cessfully synthesized 7 but only tentative identification based on mass spectrometry data was provided. The objective of the work was to fully elucidate the structure of the F–A + dimer, and more specifically to unambiguously determine the position and configura- tion of linkage. The dimer was synthesized according to previously pub- lished data. 7 During the reaction, catechin carbocation (catechin + ) was obtained from taxifolin after reduction into a flavan-3,4-diol followed by protonation and dehy- dration. The malvidin 3-O-glucoside (Mv3glc) added in its hydrated form reacted with catechin + to lead to two F–A + dimers. The major dimer was extracted from reac- tion medium by countercurrent chromatography. The presence of catechin–Mv3glc dimer was monitored by HPLC–ESI-MS. The mass spectrum showed a signal at m/z = 781 in the positive ion mode, which is in agree- ment with the dimeric structure. The characteristic frag- mentations obtained by MS 2 and MS 3 experiments confirmed the postulated structure. In particular the loss of a 126 fragment indicated the flavanol to be in the upper position. 7 0040-4039/$ - see front matter Ó 2004 Published by Elsevier Ltd. doi:10.1016/j.tetlet.2004.09.127 Keywords: Anthocyanins; Flavanol; Pigment. * Corresponding author. Tel.: +33 4 99 61 24 84; fax: +33 4 99 61 26 83; e-mail: salas@ensam.infra.fr Tetrahedron Letters 45 (2004) 8725–8729 Tetrahedron Letters