Biomolecules and Biochemical Analysis

Carbohydrate stereochemistry

Epimers are diastereomers that differ in the configuration of exactly one stereocentre, and an epimer is named by the carbon that differs, such as the C-2, C-3 or C-4 epimer. Diastereomers are stereoisomers that are not mirror images of each other, whereas enantiomers are mirror images that differ at every stereocentre. D-Mannose is the C-2 epimer of D-glucose, while D-galactose is the C-4 epimer, so the epimer position must match the carbon where the two sugars actually differ. D-Glucose and L-glucose are mirror images at every stereocentre, so they are enantiomers and not diastereomers. D-Glucose and D-galactose differ at only one carbon and are not mirror images of each other, so they are diastereomers.

In nucleosides, the anomeric carbon C-1′ of a ribofuranose ring fixes whether the glycosidic bond is α or β, and natural ribonucleosides such as adenosine have the β-configuration, with the base on the same face of the ring as the C-4′ CH_2OH group. Vorbrüggen-type glycosylation couples a silylated nucleobase with a peracetylated ribofuranose in the presence of a Lewis acid, and for adenine the new bond forms at N-9 of the purine. Acetyl esters on the sugar hydroxyls are removed by basic hydrolysis, and the same treatment cleaves an N-acetyl group on the purine, so a fully deprotected nucleoside is obtained.

DNA structure and base pairing

DNA is built from four nucleobases: the purines adenine (A) and guanine (G), and the pyrimidines cytosine (C) and thymine (T). Thymine is 5-methyluracil, whereas uracil, which lacks that methyl group, is the pyrimidine of RNA, so uracil replaces thymine in RNA and does not occur in DNA.

Complementary strands pair by hydrogen bonding, with A pairing with T through two hydrogen bonds and G pairing with C through three, and adjacent base pairs stack on one another. When two single strands associate into one double helix, the number of independent molecules and the conformational freedom of the chains both fall, so the entropy change is negative, \Delta S < 0. Hydrogen-bond formation and base stacking release energy, so the enthalpy change is negative, \Delta H < 0. The free-energy change is \Delta G = \Delta H - T\Delta S. Because -T\Delta S is positive here, the duplex is favoured only when the enthalpy term dominates, which is the case at temperatures below the melting temperature.

Beer-Lambert law absorbance

The Beer–Lambert law relates absorbance to concentration: A = \varepsilon b c, where A is dimensionless absorbance, \varepsilon is the molar absorptivity in \text{M}^{-1}\text{cm}^{-1}, b is the path length in cm, and c is the molar concentration in M. Because \varepsilon is quoted per centimetre, the path length must be converted to cm before use, and 1\ \text{dm} = 10\ \text{cm}. The equation rearranges to c = A/(\varepsilon b). For example, an absorbance of 0.20 in a 1 cm cell with \varepsilon = 4.0 \times 10^{3}\ \text{M}^{-1}\text{cm}^{-1} gives c = 5.0 \times 10^{-5}\ \text{M}. Absorbance is proportional to concentration only over a limited range, so very concentrated samples must be diluted before measurement.

Ion-exchange chromatography of peptides

In ion-exchange chromatography, charged molecules bind reversibly to oppositely charged groups fixed on the stationary phase and are released by changing the pH or the ionic strength of the eluent. A cation exchanger such as carboxymethyl (CM) cellulose carries negatively charged carboxylate groups and binds positively charged species, whereas an anion exchanger does the reverse. At a chosen pH, the molecule with the lowest net positive charge is bound least strongly and elutes first.