Mono and two-dimensional MR as tools to determine the structure of the monosaccharide in a diterpenic glycoside

Abstract

Nuclear Magnetic Resonance is currently the routine technique to determine the structure of organic molecules. This determination not only includes the connectivity of the atoms in the molecule, but also the relative configuration of the chiral centers that it possesses (Jacobsen, N. 2007).

Glycosides are secondary metabolites widely distributed in nature, their existence being reported in both the terrestrial and marine worlds (Cutler, S.; Cutler, H. 2002; Blunt, J. et al. 2009). They are assigned a wide variety of biological activities, ranging from antimicrobial, cardiotonic, antitumor, to anti-inflammatory, the latter being the most recently reported (Cutler, S.; Cutler, H. 2002).

The polar extracts are rich in glycosides that have between 2 and 6 units (in some cases more) of monosaccharides. Monoglycosidated aglycones, generally acetylated at one or more monosaccharide positions, are found in the hexanic or chloroform fractions of the extracts (Jones, W.; Kinghorn, D. 2006).

There are recent works that allow us to unambiguously deduce the key structural information about the position of the different carbohydrate units on an aglycon, via the analysis of chemical shifts, coupling patterns and signal multiplicity (Bagno, A. et al. 2007) .
Routinely, the chemical shift of the protons provides information about their chemical environment, relative stereochemistry of the chiral center where it is located, and even the constitution and conformation of the molecule (Bose-Basu, B. et. al. 2007).

Combining all this information and carefully analyzing it as a spectral identity, it is possible to know
the position of the glycosidated chains in the aglycon and the sequence of monosaccharides in them.

This is done with the help of the information provided by C-NMR and two-dimensional data, mainly COZY, NOESY, HMQC and HMBC (Jiménez, C. 2008).

Typically, the identity of individual monosaccharides is deduced through analysis of the respective hydrolyzate (1N HCl at 48-50 C for three hours for 0.5 mg of the glycoside) by chiral GC-MS, using pure monosaccharide standards for comparison ( Leavitt, A.; Sherman, W. 1982).

This work aims to illustrate in a simple way, how to elucidate the structure of a natural monoglycosidated diterpene, isolated from the Caribbean gorgonian Eunicea sp., which has cytotoxic properties (10-4-10-5 M against several cancer cell lines) and anti-cancer. -inflammatory at levels comparable to indomethacin (Cóbar, O. et. al. 1997).

The glycoside shows a molecular formula of C30H48O8, obtained from the HRFABMS spectrum, which indicates the existence of seven degrees of unsaturation.
The infrared spectrum shows absorptions attributable to hydroxyl groups (3438 cm-1) and ester carbonyl (1748 cm-1).

The 1H-NMR spectrum (300 MHz in CDCl3) shows at high field, the presence of six methyl groups as singlets at δ 1.22, δ 1.55, δ 1.56, δ 1.63, δ 2.05 and δ 2.11 (the last two assignable to methyl located on acetate ester groups), four methylene groups (all multiplets at δ 1.27, δ 1.65, δ 1.86 and δ 2.20) and one methine multiplet at δ 1.45.

At low field ten clearly resolved signals are observed: two methylenes at δ 4.10 (dd, J = 2.4, 12.0 Hz) and δ 4.19 (dd, J = 6.1, 12.0 Hz) and eight methines at δ 3.41 (t, J = 8.46). Hz), δ 3.59 (ddd, J = 2.5, 6.1, 9.1 Hz), δ 3.68 (t, J = 9.16 Hz), δ 4.45 (d, J = 4.5 Hz), δ 4.87 (t, J = 9.8 Hz) , δ 4.93 (t, J = 6.6 Hz), δ 5.00 (t, J = 6.7 Hz), and δ 5.07 (t, J = 6.7 Hz).