Nucleic Acids. The Ribose Sugar

Transcription

1 Nucleic cids adenosine triphosphate (TP) a mononucleotide Nucleic acids consist of: a ribose (aldopentose) sugar; a heteroaromatic, glycosidic base; a phosphoester. ribonucleic acid (RN) a polynucleotide The Ribose Sugar cyclizes to Numbering of rings: 1-n for base; 1 - for sugar. 5 -D-ribofuranose D-ribose (an aldopentose) cytidine (a nucleoside) glycoside formation cytosine (a base)

2 Phosphates and Phosphoesters of Ribonucleosides adenosine (a nucleoside) a phosphodiester a phosphoanhydride adenosine triphosphate (TP, a nucleotide) Bases Found in Nucleic cids pyrimidines pyrimidine purines purine cytosine uracil thymine guanine adenine cytidine () uridine (U) guanosine () adenosine ()

3 Polynucleotides: RN and DN DN uses thymidine (T), RN uses uracil (U) RN has OH groups at 2 -carbon, DN does not. ribonucleic acid (RN) deoxyribonucleic acid (DN) Polynucleotides Nucleotide units in DN and RN are linked by and oxygens. T Backbone is negatively charged. Information is stored in sequence of bases. So, this DN sequence would read: T (typically read )

4 RN Is Less Stable Than DN strand break! 2 -OH can serve as intramolecular nucleophile to cleave RN strand DN lacks a 2 -OH, so it does not self-cleave in this way. So, t ½ (RN) = minutes hours t ½ (DN) = centuries Nucleotide Bases Pair with One nother Via Hydrogen Bonding T Each pair matches one purine with one pyrimidine. Optimal organization for DN strands is antiparallel.

5 Every DN Sequence Has Unique, omplementary Sequence T T T So, the DN sequence complementary to would be -TT- -T- Strands of DN Form a Right-Handed Double Helix DN base pairs lie flat, stretch across center of helix. Each turn of the helix is 10 base pairs, 3.4 nm long. 3.4 nm 3.0 nm

6 In Nature, DN and RN re Synthesized by opying a Single-Strand Template ddition catalyzed by a DN/RN polymerase. Reaction fidelity: T > 99% Synthesizing DN in the Laboratory: Limits of Solution-Phase Synthesis typical solution-phase synthesis scheme: 1. dd reactants 2. Purify product 1. dd reactants 2. Purify product 1. dd reactants 2. Purify product T fter each synthetic step, products must be purified away from reagents, unreacted starting material, side products Weakness: Material often lost in purification steps. For long synthesis, these losses multiply. So, DN cannot be synthesized chemically by solution-phase methods.

7 Synthesizing DN in the Laboratory: Solid-Phase Synthesis lass lass cleavable linker cleavable linker dd reactants, wash dvantage: Eliminates most purification steps. Weakness: Reactions must be extremely high-yield, because losses multiply. lass cleavable linker dd reactants, wash T lass cleavable linker dd reactants, wash T 1. leave linker 2. Purify product Solid-Phase Synthesis of DN with Phosphoramidites dimethoxytrityl (DMT) protecting group acyl protecting group cyanoethyl (NEt) protecting group a phosphoramidite diisopropylamine leaving group (ipr) 2

8 Solid-Phase Synthesis of DN with Phosphoramidites lass (w/ linker) OH tetrazole (ipr) 2 lass I 2, H 2 O (oxidize P) equals lass strong H + (remove DMT) lass Solid-Phase Synthesis of DN with Phosphoramidites lass (w/ linker) equals OH tetrazole (ipr) 2 If yield from one lass cycle of synthesis is 99% (0.99), then the yield of a 50- base DN molecule is (0.99) 50 = 61%. I 2, H 2 O (oxidize P) lass strong H + (remove DMT) lass

9 Solid-Phase Synthesis of DN with Phosphoramidites remove protecting groups, cleave bead linker purify Similar chemistry used for RN. lass Stepwise yield >99%. an use to make DN/RN up to 100 bases long.