2 0 -Methyl and 1 0 -xylosyl derivatives of 2 0 -hydroxyflexixanthin are major carotenoids of Hymenobacter species Jonathan L. Klassen a , Ryan McKay b , Julia M. Foght a, * a Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 b National High Field NMR Centre (NANUC), University of Alberta, Edmonton, Alberta, Canada T6G 2E1 article info Article history: Received 24 February 2009 Revised 13 March 2009 Accepted 16 March 2009 Available online 21 March 2009 abstract Bacteria related to the genus Hymenobacter are colored intensely red due to their carotenoid pigmenta- tion. Using high-resolution mass spectrometry and comparison to the literature reports, we previously isolated and presumptively identified seven structurally related carotenoids from representative Hyme- nobacter-like strains. After further analysis using one- and two-dimensional 1 H NMR, UV–vis, and circular dichroism spectroscopies, we determined that the major carotenoid present in these strains is 2 0 - hydroxyflexixanthin, as proposed previously by others in a related organism. Furthermore, we have iden- tified three novel, related carotenoids abundant in these strains as 1 0 -xylosyl-2 0 -hydroxyflexixanthin, 2 0 - methoxyflexixanthin, and 3-deoxy-2 0 -methoxyflexixanthin. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Bacteria of the genus Hymenobacter (Flavobacteria, Bacteroide- tes) are often isolated from dry environments subject to intense oxidative stress, including dry soils, 2,12,13,22,25,35,37 aerosols, 8,9 and irradiated meat. 10 Habitation of these high-radiation and high-des- iccation environments requires efficient antioxidative defense mechanisms. Accordingly, all isolated Hymenobacter strains are colored bright red-pink due to the presence of carotenoids, natural pigments with notable biotechnological application as natural col- orants 21 and nutritional supplements. 11,24 Carotenoid antioxidant function is well established, especially in Deinococcus, 32,33,36 a genus that has been repeatedly co-detected with Hymenobact- er. 12,22,25 Carotenoids likely play an important antioxidative role in both genera. We have previously isolated 10 Hymenobacter-related strains from Victoria Upper Glacier, Antarctica glacial ice. 14 In these strains and nine additional reference Hymenobacter species we de- tected and isolated seven chemically distinct carotenoids using high-performance liquid chromatography (HPLC), with the exact carotenoid composition varying between strains. 14 Based on in- line HPLC UV–vis spectra, high-resolution mass spectrometry (MS), and comparison to previous synthetic work 6 claiming to rep- resent the major carotenoids of an unidentified ‘‘Taxeobacter” (now Hymenobacter 8 ) strain, these carotenoids were proposed to be 2 0 - hydroxyflexixanthin, its pentosyl-, hexosyl-, and methyl- deriva- tives and related non-ketolated precursors. Because the previous synthetic work 6 did not present the isolation and characterization of 2 0 -hydroxyflexixanthin from its natural source, the identification of this pigment as the major carotenoid in ‘Taxeobacter’ (now Hymenobacter) is considered unsubstantiated. 7 Here we report full characterization of the four most abundant carotenoids isolated from Hymenobacter-like strains VUG-A42aa and VUG-A141a using 1 H NMR, circular dichroism (CD), and UV–vis spectroscopies and previously generated high-resolution MS data. 14 High resolution MS and in-line HPLC UV–vis spectroscopy pre- viously suggested that the dominant carotenoid in all surveyed Hymenobacter strains (1; previously designated carotenoid 5 14 ) is a monocyclic ketocarotenoid containing 12 double bonds and hav- ing a molecular formula of C 40 H 54 O 4 . UV–vis spectroscopy of puri- fied 1 shows a broad peak with a central absorbance maximum at 494 nm and minimal spectral fine structure (Supplementary data Fig. S1), consistent with this proposed structure. The 1 H NMR spec- tra of 1 (Table 1, Supplementary data Figs. S2 and S3) are consis- tent with those previously reported for 2 0 -hydroxyflexixanthin, 3 with important diagnostic peaks at d1.76 (H-2; doublet of doublets split by H-3 and OH-3; see Fig. 1 for numbering scheme), d4.28 (H- 3; doublet of doublets split by H-2 ax and H-2 eq ), and d3.96 (H-2 0 ; linked in the 2D-TOCSY to H-3 0 and H-4 0 ); the latter shift clearly suggests hydroxylation at the 2 0 -position by comparison with the NMR structure of flexixanthin. 3 The CD spectrum of 1 (Supplemen- tary data Fig. S4) clearly indicates S stereochemistry at the 2 0 -hy- droxyl position by comparison to the previously published data. 3,23 No CD signal, however, was observed for any of the 2 0 - methoxy carotenoids identified in this study (see below). We therefore hesitate to apply the additivity hypothesis for carote- noids 4 to the 3-hydroxyl group of 1 and consider the stereochem- istry at this position undetermined. Based on this evidence, we identify 1 as 2 0 S-hydroxyflexixanthin (Fig. 1), as previously 0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2009.03.117 * Corresponding author. Tel.: +1 780 492 3279; fax: +1 780 492 9234. E-mail address: Julia.Foght@ualberta.ca (J.M. Foght). Tetrahedron Letters 50 (2009) 2656–2660 Contents lists available at ScienceDirect Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet