BioOrg Unit 3
DNA Structure
• Nucleoside: nucleobase + 2’-deoxyribose
o Aromatic nucleosides cannot protonate to make a good leaving group because
the N lone pair is delocalized into the aromatic system
o Thymine is the only nucleobase lacking a nucleophilic exocyclic amine
§ Amine is a better nucleophile than hydoxyl
• Nucleotide: nucleoside + phosphate group
• Nucleic acid: nucleotide polymer
• Phosphodiester bonds are a 3’-hydroxyl group of one nucleotide combined with the 5’
phosphate group of another
• Purines
o Purines made of H
o Readily undergo alkylation due to nucleophilic nitrogens on the nucleobase
o Biosynthesis of purines involves a hypoxanthine intermediate
§ Amino acid is beginning synthesis substrate
o Deoxy purines made of 2’-deoxyribose
o Deoxy purine 5’-monophosphates made of 5’-phosphorylated 2’-deoxyribose
o Non-natural purines
§ Xanthine
§ Hypoxanthine
§ Diaminopurine
o Guanine
§ Tautomerization obstructs H bonding to Cytosine at 1 of 3 bonds
§ GC bonds are 50% stronger than AT
§ If tautomerized, the nucleobase can no longer pair with cytosine
§ Hoogsteen G-Quadruplex
• 4 Gs arranged to maximize H bonding
• G-rich regions at the end of genes + potassium cation
• Lone pair bonds on carbonyl O allow K+ to be central in the
quadruplex
• Most common in telomeres
• Pyrimidines
o Pyrimidines made of nucleobases
o Amino acid is beginning synthesis substrate
o Deoxy pyrimidines made of deoxyribonucleoside
o Deoxy pyrimidine 5’-monophosphates made of deoxyribonucleotides
o Cytosine
§ Aromatic
§ Resonance preserves the H bonds to Guanine
§ Resonance structures follow Watson and Crick base pair rules
• Grooves
, o Double helix is not symmetrical
o Lone pairs and H atoms are not taken up with aromaticity/base pairing can be
used as H bond donors/acceptors
§ N-H: H bond donor
§ C=O: H bond accepter
o Major
§ Bigger things like proteins/enzymes
• Proteins are huge
o Minor
§ Small molecules
§ Drugs and environmental toxins
• Ex: chemotherapeutics
§ DNA damage usually happens here
§ Binding and reactions
• Aromaticity
o Nitrogen lone pair in p orbitals is required for resonance
• Codons
o tRNA anticodon has limited specificity in the third position
§ can pair with more than one codon in mRNA
o mRNA can differ in the third position
o Non-specific third base is called wobble base
§ A wobble base pair shifts slightly for optimal H bonding
,DNA Synthesis
• Synthesis is directional
• CPG Resin Beads
o Solid-phase synthesis
§ Easy purification
§ Excess of reagents
§ Higher yield
§ Ability to see and manipulate reactant
o Polar solvents use non-polar resin beads
§ Like polystyrene
§ Contracted by so-called “hydrophobic” interactions
o Amino acids with a C-terminus connected to a solid-phase resin bead can have
their free N terminus combine with an activated ester (with a protected N) to
yield amide bond
o Nucleoside monomer + activated phosphate ester à SN2 to phosphodiester or
addition/elimination to amide
§ Nucleophile on a solid-phase resin bead
§ Coupling on activated ester
§ One end of phosphate ester has an activated ester, and the other has a
cleavable protecting group
§ Products
• Amide product becomes protein
• Phosphodiester product becomes nucleic acid
• These conversions happen on the solid-phase so one end is
connected to a resin bead
o A crowded nucleophile cannot do SN2
o Do not want the electrophile on the bead
§ Reduces coupling yield
• Phosphodiester Coupling
o Polymers grow out of the controlled pore glass (CPG bead)
o Phosphenyl chloride is the electrophile
§ Replace the upper P=O with a P with a lone pair
§ Phosphotidylchloride is so reactive that it reacts with water to inactivates
the incoming monomer
§ SN2 reaction
o Phosphoamidite functional group
§ Has a cleavable P-N bond
§ Highly electrophilic
§ Creates a phosphite that is easily oxidized to phosphate with iodine
o Oxidize the product under mild conditions to create the desired phosphodiester
bond
• Steps
, o Loading step, deprotection, coupling, capping, oxidation, final deprotection
§ Iterative steps between the other steps bring in limitless series of new
monomer units
§ The final step cleaves polymer from resin bead to make DNA
o Loading step
§ First pathway
• Coupling first nucleic acid monomer to CPG
o CPG might have to be adapted to the monomer
o CPG made of silicon-oxide with a mildly nucleophilic
monomer
§ Two mildly nucleophilic things will not combine, so
use n adaptor
• Addition/elimination with a carboxylic acid anhydride
o A strong electrophile
§ Electrophilicity is strengthened with fewer anions
and fewer steric interactions
o Reacts twice: once with CPG, once with the monomer
o Creates an ester
• Use DCC, DMAP with a mildly nucleophilic molecule
o DCC is not as efficient as it is with amide polymers
o DMAP is a catalyst
o Creates a product where the first monomer is linked to
CPG by an ester bond
§ Second pathway
• Nucleophilic N on DMAP attacks electrophilic C on DCC
• Creates pyridinium, a great leaving group
• Addition/elimination with a poor nucleophile attached to CPG
• Creates a DCC-activated ester and catalytic DHAP
o Can do another addition/elimination to get to the product
of the first pathway
o Deprotection
§ Highly conjugated p systems make bright colors
• Like DMT is bright orange
§ DMT is an easy to cleave protecting group
• SN1 reaction
• Any exocyclic amine must be protected
o Do not protect without exocyclic amines present
o If amines are not protected, they will be too nucleophilic
and react with many strong electrophiles during synthesis
o Protecting groups should be very electrophilic so ammonia
attacks them in the final deprotection step
• Protecting groups designed to be cleaved by same reagent that
releases the polymer chain from the resin bead
DNA Structure
• Nucleoside: nucleobase + 2’-deoxyribose
o Aromatic nucleosides cannot protonate to make a good leaving group because
the N lone pair is delocalized into the aromatic system
o Thymine is the only nucleobase lacking a nucleophilic exocyclic amine
§ Amine is a better nucleophile than hydoxyl
• Nucleotide: nucleoside + phosphate group
• Nucleic acid: nucleotide polymer
• Phosphodiester bonds are a 3’-hydroxyl group of one nucleotide combined with the 5’
phosphate group of another
• Purines
o Purines made of H
o Readily undergo alkylation due to nucleophilic nitrogens on the nucleobase
o Biosynthesis of purines involves a hypoxanthine intermediate
§ Amino acid is beginning synthesis substrate
o Deoxy purines made of 2’-deoxyribose
o Deoxy purine 5’-monophosphates made of 5’-phosphorylated 2’-deoxyribose
o Non-natural purines
§ Xanthine
§ Hypoxanthine
§ Diaminopurine
o Guanine
§ Tautomerization obstructs H bonding to Cytosine at 1 of 3 bonds
§ GC bonds are 50% stronger than AT
§ If tautomerized, the nucleobase can no longer pair with cytosine
§ Hoogsteen G-Quadruplex
• 4 Gs arranged to maximize H bonding
• G-rich regions at the end of genes + potassium cation
• Lone pair bonds on carbonyl O allow K+ to be central in the
quadruplex
• Most common in telomeres
• Pyrimidines
o Pyrimidines made of nucleobases
o Amino acid is beginning synthesis substrate
o Deoxy pyrimidines made of deoxyribonucleoside
o Deoxy pyrimidine 5’-monophosphates made of deoxyribonucleotides
o Cytosine
§ Aromatic
§ Resonance preserves the H bonds to Guanine
§ Resonance structures follow Watson and Crick base pair rules
• Grooves
, o Double helix is not symmetrical
o Lone pairs and H atoms are not taken up with aromaticity/base pairing can be
used as H bond donors/acceptors
§ N-H: H bond donor
§ C=O: H bond accepter
o Major
§ Bigger things like proteins/enzymes
• Proteins are huge
o Minor
§ Small molecules
§ Drugs and environmental toxins
• Ex: chemotherapeutics
§ DNA damage usually happens here
§ Binding and reactions
• Aromaticity
o Nitrogen lone pair in p orbitals is required for resonance
• Codons
o tRNA anticodon has limited specificity in the third position
§ can pair with more than one codon in mRNA
o mRNA can differ in the third position
o Non-specific third base is called wobble base
§ A wobble base pair shifts slightly for optimal H bonding
,DNA Synthesis
• Synthesis is directional
• CPG Resin Beads
o Solid-phase synthesis
§ Easy purification
§ Excess of reagents
§ Higher yield
§ Ability to see and manipulate reactant
o Polar solvents use non-polar resin beads
§ Like polystyrene
§ Contracted by so-called “hydrophobic” interactions
o Amino acids with a C-terminus connected to a solid-phase resin bead can have
their free N terminus combine with an activated ester (with a protected N) to
yield amide bond
o Nucleoside monomer + activated phosphate ester à SN2 to phosphodiester or
addition/elimination to amide
§ Nucleophile on a solid-phase resin bead
§ Coupling on activated ester
§ One end of phosphate ester has an activated ester, and the other has a
cleavable protecting group
§ Products
• Amide product becomes protein
• Phosphodiester product becomes nucleic acid
• These conversions happen on the solid-phase so one end is
connected to a resin bead
o A crowded nucleophile cannot do SN2
o Do not want the electrophile on the bead
§ Reduces coupling yield
• Phosphodiester Coupling
o Polymers grow out of the controlled pore glass (CPG bead)
o Phosphenyl chloride is the electrophile
§ Replace the upper P=O with a P with a lone pair
§ Phosphotidylchloride is so reactive that it reacts with water to inactivates
the incoming monomer
§ SN2 reaction
o Phosphoamidite functional group
§ Has a cleavable P-N bond
§ Highly electrophilic
§ Creates a phosphite that is easily oxidized to phosphate with iodine
o Oxidize the product under mild conditions to create the desired phosphodiester
bond
• Steps
, o Loading step, deprotection, coupling, capping, oxidation, final deprotection
§ Iterative steps between the other steps bring in limitless series of new
monomer units
§ The final step cleaves polymer from resin bead to make DNA
o Loading step
§ First pathway
• Coupling first nucleic acid monomer to CPG
o CPG might have to be adapted to the monomer
o CPG made of silicon-oxide with a mildly nucleophilic
monomer
§ Two mildly nucleophilic things will not combine, so
use n adaptor
• Addition/elimination with a carboxylic acid anhydride
o A strong electrophile
§ Electrophilicity is strengthened with fewer anions
and fewer steric interactions
o Reacts twice: once with CPG, once with the monomer
o Creates an ester
• Use DCC, DMAP with a mildly nucleophilic molecule
o DCC is not as efficient as it is with amide polymers
o DMAP is a catalyst
o Creates a product where the first monomer is linked to
CPG by an ester bond
§ Second pathway
• Nucleophilic N on DMAP attacks electrophilic C on DCC
• Creates pyridinium, a great leaving group
• Addition/elimination with a poor nucleophile attached to CPG
• Creates a DCC-activated ester and catalytic DHAP
o Can do another addition/elimination to get to the product
of the first pathway
o Deprotection
§ Highly conjugated p systems make bright colors
• Like DMT is bright orange
§ DMT is an easy to cleave protecting group
• SN1 reaction
• Any exocyclic amine must be protected
o Do not protect without exocyclic amines present
o If amines are not protected, they will be too nucleophilic
and react with many strong electrophiles during synthesis
o Protecting groups should be very electrophilic so ammonia
attacks them in the final deprotection step
• Protecting groups designed to be cleaved by same reagent that
releases the polymer chain from the resin bead
