The material that was

retained inside this membrane (fraction SF-SK10-100R, 45 mg) was eluted on HPSEC as a single Dinaciclib concentration peak (Fig. 2c), with Mw 41 kDa and dn/dc=0.148. It was composed of glucose (51%), galactose (28%) and mannose (21%) and its 13C NMR spectrum (Fig. 3b) contained many C-1 signals, indicating the presence of a complex heteropolysaccharide. Methylation analysis (Table 2) indicated that all galactosyl units were present as nonreducing end-units (Galp and Galf), together with Glcp units. The Manp units were mainly 6-O-substituted, with small amounts of 2,6-di-O-substituted residues, while the Glcp units were 2-O-, 4-O-, 2,3-di-O-, 4,6-di-O- and 2,6-di-O-substituted residues. Substitution at HO-6 of the Manp and Glcp units was also shown by DEPT (Fig. 3b, inset), which provided inverted signals at δ 68.7 and 69.0. In its 13C

NMR spectrum, C-1 signals at δ105.1 and 105.6 corresponded to the nonreducing end-units of β-Galf. The signals at δ 102.6, 102.9 and 103.0 probably arose from C-1 of β-Glcp units. The anomeric configuration of these units was confirmed by their low-field C-1 resonances and also by their 1JC−1, H−1 of 161.5, BGJ398 purchase 164.2 and 160.0 Hz. The remaining C-1 signals at δ 100.7 and 100.2 belong to the α-pyranose series, due to their high-field C-1 resonances and 1JC−1, H−1 (174.4 and 171.5 Hz, respectively) (Agrawal, 1992; Duus et al., 2000; Bubb, 2003). The signals of O-substituted C-2, C-3 and C-4 could be seen at δ 87.5 (C-3), δ 84.9 and 83.3 (C-2) and δ 81.5 (C-4). The material that passed over this membrane (fraction SF-SK10-100E, 66 mg) had high glucose content (79%), with small amounts of mannose (10%) and galactose (11%), indicating the presence of a

glucan. This fraction still had Exoribonuclease a heterogeneous elution profile on gel permeation (Fig. 2c) and due to its small amount was not further purified. However, its 13C NMR spectrum (Fig. 3c) showed β-configurations, due to low-field C-1 signals at δ 103.7 and 103.0. Moreover, it is possible to observe (13)- and (16)-linked Glcp units, due to the presence of a signal at δ 86.2, characteristic of O-substituted C-3, and to the presence of inverted signals at δ 68.8 and 69.0 in the DEPT experiment (Stuelp et al., 1999; Carbonero et al., 2001; Cordeiro et al., 2003). Thus, this glucan resembles a lentinan-type β-glucan. A similar glucan was isolated from the lichen Thamnolia vermicularis var. subuliformis by Olafsdottir et al. (2003) and had a backbone of β-d-(13)-linked glucopyranosyl units branched with a single β-d-(16)-linked unit for every third unit of the backbone. In an attempt to find the isolichenan, we also analyzed the fraction SW, which was obtained in low yield. This fraction was composed of galactose (60.0%), mannose (22.5%) and glucose (17.5%).