Chem 313: Nucleic Acids We have studied two of the three major kinds of biopolymers: polysaccharides and proteins.

Now we will look at the third: nucleic acids. There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA contains the genes for heredity; it must be replicated for cell division. In most organisms, the genetic information stored in DNA is transcribed into RNA. This information can then be translated into the synthesis of all the proteins needed for cellular structure and function. DNA was first isolated in 1869 from the nuclei of white blood cells. Because this material was in the nucleus and was acidic, it was called nucleic acid. Nucleotides are also involved in a number of other cellular processes such as energy storage (ATP) and cofactor (NAD/NADH) functions.
R' O O O Bn

O P O O O O O P O O
n

Bi

O B1

R'

DNA exists largely as a double helix that is composed of an alternating sugar-phosphate backbone. The polymer contains four monomers (A,C,G,T). Each unit is composed of a pentafuranose (deoxyribose), a purine or pyrimidine base, and a phosphate. The bases are hydrogen bonded to each other via Watson-Crick base pairing.
Watson-Crick Base-Pairing Hydrogen Bonds H O N O Thymine (T) N R N N R N N H H N N O N R R = sugar

H N N R

H N

O H

N H H Cytosine (C)

Adenine (A)

Guanine (G)

O N N Pyrimidine
2 1N 6

4 5

H N N
7

9 8

NH N H Uracil O

-O

Purine

O 5' P O O O3'OH

Nomenclature: The bases are often called nucleobases. Thymine, cytosine, and uracil are pyrimidines (1, 3-diazabenzenes). Gaunine and adenine are purines (fused pyrimidine and imidazole). Uracil is found in RNA in place of thymine. (Thymine is more stable than Uracil; T likely evolved from U.) A ribose (RNA) or 2'-deoxyribose (DNA) containing a base (C, A, G, T, or for RNA, U) is called a nucleoside. A unit containing sugar, base, and phosphate is called a nucleotide. Several nucleotides linked together are called oligonucleotides (oligos for short). Many nucleotides together are called nucleic acids: ribonucleic acids (RNA) or deoxyribonucleic acids (DNA). The phosphates are linked at the 3' and 5' positions of the sugars. We wont worry about the numbering system of the nucleobases, but they get numbers 1, 2, 3, etc., and in order to distinguish them from the sugar carbons, the sugar carbons get numbered 1', 2', 3', etc.
NH2 N HO N O H N N O O P O O N N O H N NH2 N

OH H Deoxyribonucleoside

OH H Deoxyribonucleotide

DNA Synthesis. Why study DNA synthesis? (1) Synthetic DNA is frequently used by chemists, biochemists, and molecular biologists. Non-natural variants of DNA are used for a variety of purposes including as catalysts, drugs, and to probe the origin of life. Enzyme mutants are often generated by incorporating synthetic DNA into plasmids. Likewise for mutation to other compounds such as membrane-bound receptors. Genetherapy usually involves use of synthetic DNA. (2) Looking at the details of DNA synthesis leads us to examine the structure and chemical properties of DNA more closely. Biosynthesis of DNA: DNA replication is a complex process that we will not discuss in detail. The double helix is opened and the complementary nucleotides are brought into position along each single strand. Over 20 enzymes are involved in the process. In the key step, the 3' hydroxyl of the growing oligo reacts with a 5'-triphosphate of an incoming nucleotide. The leaving group is a pyrophosphate.

O P O O O O O P O O O Bi

Biosynthesis of DNA O O P O O O O O P O O O O Bn+1 O P O O O B1 O R' O Bn+2 Bi O O O P O P O O O pyrophosphate

Bn+1

n
3'-hydroxyl

O O O O O P P O P O O O O O 5'-triphosphate

R'

Phosphorous nomenclature. Phospha: prefix for P-containing heterocycles. H3PO4: phosphoric acid, PO43: phosphate; PO(OR)3: phosphotriester; PO(OR)2OH: phosphodiester; PO(OR)(OH)2: phosphate ester; H3PO3: phosphorous acid; PO33: phosphite; P(OR)3: phosphite (ester); PR3: phosphine; P(OR)2NR'2: phosphoramidite. Overview of chemical synthesis of DNA: The synthesis is via solid phase, similar to peptide synthesis. A nucleotide is bound to the resin at its 3' hydroxyl. (The nucleobases are protected if needed.) Another nucleotide, protected at its 5' hydroxyl and activated at its 3' hydroxyl as a phosphoramidite, is then added, and it is coupled (linked) to the resinbound nucleotide. The newly formed phosphite group is oxidized to a phosphate, and the protecting group at the (new) 5' hydroxyl is removed. Further coupling followed by cleavage and deprotections yields the oligonucleotide. The 3' and 5' ends of the cleaved oligo can be either hydroxyls (R' = H) or phosphate esters. Well see that, as with protein synthesis, DNA biosynthesis proceeds in the opposite direction as DNA chemical synthesis. Its just that chemists dont always find natures way to be easist in lab.

HO O
R' O O O O P O O O O O P O O O B1 H O O Bi

B1
PG O

5'
O O B2

Bn+2

Resin

3' phosphoramidite

P OR N(iPr)2

PG O O O

B2

P OR O O O B1

Issues that we will try to address: How to get the first nucleotide onto the resin, protection of the bases and the hydroxyls, activation and coupling mechanism, oxidation, cleavage, and deprotection. Note that UBC has a rich history in DNA research. Gobind Khorana was at UBC 19521960. He won the Nobel Prize in 1968 for determining that, in DNA, there is a three nucleotide code for each amino acid. He also went on to do seminal work in oligonucleotide synthesis. Michael Smith (d. 2000), who spent most of his career at UBC, won the Nobel Prize in 1993 for his work in site-directed mutagenesis. Before we get to DNA synthesis, . . . . : Phosphates. The charge in phosphates is distributed evenly over the oxygens that are not bound to a metal, hydrogen, or a carbon; metals, hydrogens, and carbons localize the charge. The geometry is close to tetrahedral. The phosphoryl bond (-bonding) is O(p) P(d). ATP. The base is the recognition site for enzymes. The fourth pKa is 6.5; at physiological pH, around 7.4, ATP is mostly 4 charged, with some in the 3 form. ATP is a very good chelator (binder of metal cations), especially for Mg2+. ATP is the local energy source in cells. It is an anhydride of phosphates. Hydrolysis of ATP yields energy: Entropically, one molecule becomes two. Enthalpically: (1) Charge repulsion is reduced. 4

n
R'

O O O P O

B2

x 1) O

tion rotec p e D 2)

n idatio

Resin

1) More cycles

OR O B1

2) Cleavage and Deprotection

Resin

(2) The formation of an acid that is unstable at pH 7 pushes the equilibrium (releases energy). (3) The lone pairs on the middle oxygen are freed to participate in resonance fully with one phosphorous each. Phosphate ester hydrolysis. The rates and mechanisms of phosphate ester hydrolysis are complex. There are mono-, di-, and tri-esters, and the reactivities differ somewhat. The pH of the solution has a large effect: the charged state of the phosphate esters change with pH, and the nucleophile changes (H2O to HO). Anionic phosphate esters tend to repel nucleophiles, particularly charged ones like HO (this makes DNA fairly stable to hydrolysis at pH >7). Attack by the nucleophile can be on carbon or on phosphorous. Enzymes catalyze formation and hydrolysis of phosphate esters, and the mechanisms and rates are enzyme-dependent. Pseudorotation refers to the reorganization of the structure of a penta-coordinate phosphate: two bonds that are 180 to each other (apical) are longer than the rest (the structure is trigonal bipyramidal); the substituents at apical positions can switch the other substituents (pseudorotate). Leaving groups must be apical to PO bonds. Although DNA is stable to hydrolysis under basic conditions, RNA is easily hydrolyzed due to the proximity of the 2' OH to the 3' phosphate ester. Note that this serves nature well, as RNA is often used as a transient species that is not wanted for long periods (proteins could be over-expressed if RNA lingered too long). Back to DNA Synthesis. Protection of nucleobases: Amines need to be protected, as they would interfere with the synthesis owing to their nucleophilicity. Thymine does not need a protecting group, as it does not contain an amine. Adenine and cytosine are protected at their amino group as a benzamide. The protection reaction is an amine plus an acid chloride. Guanine is protected at its amine as an isobutrylamide. (The benzamide group is difficult to remove from a guanine.) The protection of guanine, adenine, and cytosine are often done via transient protection using TMS protection transiently. TMS is trimethylsilyl, Me3Si. Reaction of an alcohol with TMSCl goes via an SN2-like mechanism, although the Si could form a pentavalent intermediate. SiO bonds are very strong and drive the reaction selectively over formation of SiN bonds. Stronger still than SiO bonds are SiF bonds. Thus, TBAF, tetrabutylammonium fluoride, is used to remove the TMS from the ROTMS, again via an SN2-like mechanism. Once the nucleobases are protected, the 5' hydroxyl can be protected as RODMT, where DMT is 4,4'-dimethoxyltrityl. This group is very similar to trityl, but is easier to remove and is colored, which can be used to monitor reaction progress. Like trityl, DMT is selective to primary over secondary alcohols owing to its steric bulk. Next, the nucleoside with protection at its nucleobase and DMT at its 5'-hydroxyl is then attached to the resin. The resin is called CPG for Controlled Pore Glass, meaning that the pore size is controlled, either 500 or 1000 , depending on the size of the oligo to be made. Glass is silica gel, (SiO2)n. Before coupling to the next nucleoside, the DMT group must be removed. This is done using trichloroacetic acid, and proceeds via an SN1 mechanism. These conditions do not affect the other PGs, the phosphate triesters, or the linkage to the CPG resin.

The incoming nucleoside, protected at its nucleobase and at its 5'-hydroxyl is then activated at its 3'-hydroxyl as a phosphoramidite using cyanoethylchloro-N,Ndiisopropylphosphoramidite. The reaction at the phosphoryl chloride proceeds via an SN2-like mechanism on the phosphorous atom. Coupling then occurs using tetrazole as the base. Tetrazole has a pKa of 8.2 (this refers to the neutral form; the conjugate base is the anion). The charge is delocalized on all ring atoms. The coupling reaction itself likely proceeds via nucleophilic catalysis where the tetrazole anion is the nucleophilic catalyst. Following each coupling, capping is often done using acetic anhydride and 1methylimidazole. As in peptide synthesis, this makes purification easier, as most of the impurities are kept small and therefore easy to separate from the large product. Following each coupling, the enusing phosphite is oxidized to the phosphate because the phosphite is not sufficiently stable to survive repeated cycles. Once all couplings are complete, the last DMT is removed (using trichloroacetic acid). Then, the oligo is cleaved from the resin, the cyanoethyls are cleaved from the phosphate triesters, and the nucleobase protecting groups are removed, all under the same conditions: concentrated ammonium hydroxide at 65 C for one hour. The cyanoethyl removal is via a E2 reaction. The nucleobases are deprotected via amide hydrolysis. The cleavage from the resin is ester hydrolysis. Summary of solid phase DNA synthesis: (1) Link nucleotide to CPG protected at the nucleobase and the 5' OH. (2) Protect the incoming nucleotide at its nucleobase and its 5' OH. (3) Incorporate the phosphoramidite into incoming nucleotide. (4) Do the phosphoramidite coupling. (5) Cap unreated 5' hydroxyls. (6) Oxidize the phosphite to the phosphate. (7) Deprotect the new 5' OH. (8) Repeat steps 2-7 for each nucleotide. (9) Remove final DMT; then cleave oligo from the CPG and remove protecting groups from the nucleobases and the cyanoethyls from the phosphotriesters.

HO O OH

B2

1) TMSCl, pyr

DMT O O OH

B2* CN Cl P O N(iPr)2

2) PhCOCl (or isobutryl chloride) 3) TBAF 4) DMTCl, pyr B* = protected base

HO O O O

B1* O N H SiO2

DMTO O

B2*

P OR N(iPr)2 R = CH2CH2CN

1) Tetrazole DMTO O O O P O O 1) Cl3CCO2H 2) More Cycles O O OR B1* O N H SiO2 2) Capping B2* 3) I2, H2O

DMTO O O O P O

Bn+2*

HO O O 1) Cl3CCO2H 2) NH3, H2O, Heat O P O

Bn+2

OR O O O P O O
n

O O O O P O O O B1 Bi

Bi*

OR B1* O O O N H SiO2

OH

A final note about RNA: The 2' OH of the ribose must be protected. The protecting group of choice is TBDMS, t-butyldimethylsilyl, which is more stable than TMS, and is put on and removed in a similar manner (TBAF) to TMS. The protection reaction gives about a 1:1 mixture of the 2' OTBDMS and 3' OTBDMS; the compounds must be separated (you want the 2' OTBDMS).