Sample preparation for D isotopomer measurements
Dry moss biomass formed during the incubations was ground to a fine
powder at 30 Hz for 2 min using a MM 400 ball mill
(Retsch®, Haan, Germany), and 200-700 mg portions were
used as starting material for the following sample preparation for D
isotopomer measurements. Glucose-containing structural polymers were
hydrolyzed to glucose and converted to
1,2-O-isopropylidene-α-D-glucofuranose according to established
protocols (Betson et al ., 2006). To remove contamination by a
mannose derivative, which has overlapping signals with the NMR spectrum
of the glucose derivative, an oxidation step was introduced. Each sample
was dissolved in 20 mL 0.187 M phosphate buffer
(NaH2PO4, pH 4), 15.6 mg
NaClO2 was added per 100 mg sample and the pH was
adjusted to 3.5 with H3PO4. The
NaClO2 addition and pH adjustment was repeated five
times over a period of 3 days (every 12 h). The complete reaction
procedure was performed under the absence of light. 12 h after the last
addition the reaction was quenched by adding a molar equivalent amount
of Na2SO3 to the total amount of
NaClO2 added, then the mixture was neutralized with
CaCO3, vacuum-filtered and evaporated under reduced
pressure. To remove remaining salts the sample was extracted three times
with 20 mL ethanol. The derivative was subsequently converted to
3,6-anhydro-1,2-O-isopropylidene-α-D-glucofuranose following Schleucheret al . (1999). The derivative was purified by flash
chromatography using silica gel and diethyl ether. Pure fractions were
identified by thin-layer-chromatography and pooled. Diethyl ether was
evaporated, the sample was washed with amylene-stabilized chloroform and
purity was checked by 1H-NMR.