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Sequencing by Synthesis
• Newer method to sequence whole genomes
– Uses allyl protecting group:
color
PPPO
DNA
3'-OH
O
+
B
Mixture of dNTPS
O
allyl
polymerase
O
DNA
color
O P O
O
OH
B
O
RXN STOPS!
Pd0
(30 s, deprotects
allyl moieties)
O
DNA
O P O
O
OH
OH
B
free to repeat
Sequencing by Synthesis
Ju, Jingyue et al. (2006) Proc. Natl. Acad. Sci. USA 103, 19635-19640
Copyright ©2006 by the National Academy of Sciences
DNA/RNA Analogues
• There have been several recent
reports of the modification of
oligonucleotides
• Modifications have included:
– Altering nitrogenous base
structure
– Employing different sugar
structures
– Modifying the backbone
– Or in combination
H2N
N
N
O
O
N
N
O
O
OH(H)
O P O
NH
O
O
N
O
O
O
H
N
OH(H)
N
O P O
O
O
O
N
OH(H)
N
NH2
• Why make oligo analogues?
– Structure/activity relationships (i.e., catalytic versatility)
– Antisense technology
– Insight into evolutionary process?
• Synthetic oligos can be designed to bind with itself, DNA,
RNA or all of the above
• Effects:
– H-bonding (base-pairing)
– Other types of interactions (i.e. Van de Waals)
– Overall shape (i.e., double helix? Hairpin loop?)
• How do we examine interactions?
–
–
–
–
Melting temp. Tm = 40.5 °C (DNA), 42.5 °C (RNA)
NMR (as well other spectroscopic methods)
Xray crystallography
Calculations
Antisense Technology
• Employs a synthetic oligo that is complementary (antisense)
to an mRNA sequence of interest
• One of Two main effects can occur:
– Translation arrest (no protein)
– Recruits RNAase that degrades mRNA
• Potential for therapeutic use
– Vitravene (acts on CMV virus)
Unnatural Base Pairs
• Recall the natural base pairing (Watson-Crick) in DNA:
D
A
H
H
N
O
H
N
N
N
sugar
N
sugar
A
N D
N
N
O
H
A
D
H
G
C
• If we change the number of or location of donor/acceptor
groups, what interactions can occur?
For example: Expand the “genetic alphabet”
N
•
Tm measurements vary
dramatically due to changes
in H-bonding properties &
hydrophobicity
D
D
NH2
N
N
N
N A
N
A
N
sugar
N
sugar
NH2
Adenine
D
D
H N
H
D
H N
H
A
H N
N
• DNA polymerase found to
work with some modified
bases (oligos)
H N
A
N O
N
O
N
H
D
H
D
N
N
A
N
A
sugar
A
A
NH2
NH2 D
N
D
sugar
sugar
• Such structures would be
unlikely to form under
prebiotic conditions
NH2
Cytosine
sugar
Cytosine
A
D
O
N
N
NH2
N A
N
N
N
N
sugar
NH2
D
guanine
N
sugar
A
O
A
Employing Novel Sugars
Some examples:
O
• 5- vs 6- membered ring
5'
Base
O
• Location of phosphodiester bond
1'
4'
2'
3'
• Hydroxyl groups (# & stereochemistry)
O
• Position of base
P
OH(H)
DNA/RNA
O
6'
5'
4'
O
Base
O
OH
3'
1'
5'
4'
O
Base
1'
5'
4'
3'
2'
O
P
-D-Xylopyranosyl NA
O
6'
O
3'
2'
P
Hexitol NA
(HNA)
Base
O
OH
1'
2'
O
P
6'
OH
Altropyranosyl-NA
5'
O
O
Base
1'
4'
O 3'
2'
P
Homo-NA
O
6'
5'
4'
O
O
Base
3'
2'
P
Hexitol NA
(HNA)
1'
• Adopts chair conformation in
oligo
• Forms helical duplex with RNA
(Tm = + 3°C) & with DNA (Tm =
+ 3°C)
• Adding more hydroxyl groups,
increases affinity for RNA
– Also increases thermal stability
Modification of the Backbone
• Peptide Nucleic Acid (PNA)
– Aminoethyl glycine units linked by a peptide bond
– achiral
OH
OH
NH2
NH2
R
O
HN
Base
Base
N
R
O
O
O
HO
O
NH
O
R
NH
Base
O
HN
P
-O
O
O
O
Base
R
O
O
N
NH
R
O
HO
O
Base
NH
-O
P
O
O
O
OH
O
Base
HO
N
O
CONH2
Protein
PNA
RNA
• Duplex formation
– PNA-DNA Tm = 68.8 °C (~20 °C higher than DNA-DNA!)
– PNA-RNA Tm = 72.2 °C
•  PNA has the recognition properties of DNA (can carry
genetic information) & has a higher stability than RNA
and DNA
• Stability?
– Lack of phosphate groups → neutral backbone  no
electrostatic repulsion!
– Not recognized by proteases
• Did a PNA molecule precede RNA?
– Has demonstrated numerous forms: hairpins, triplexes, etc
– Simple in structure (i.e., achiral) & stable
– Amino building blocks in primordial soup
Another Modified Nucleic Acid - GNA
OH
glycol
• Glycol nucleic acid
OH
• Can nucleic acid be made with
such a simple sugar?
– Is a ring structure important?
– Minimum # of carbons?
Synthesis of GNA
O
DMT-Cl
O
DMTO
HO
H N
each base
(protected)
O
CN
R2N
R2N P O
P
O
HO
CN
Cl
DMTO
SN2
(inversion @ least
hindered centre)
N (Base)
R & S GNA made
DNA synthesizer
DMTO
N (Base)
• Tested duplex formation:
– Duplex formation Tm = 63 °C (22 ° higher than same DNA
or RNA sequence!)
–  GNA more stable than DNA!
– Demonstrates that cyclic sugar not necessary!
• Was ribose in fact the sugar in the first pre-biotic nucleic
acids?
• Is GNA a pre-RNA candidate?
– A glycerol derivative, which is related to triose
– Recall, we looked at 3C sugars (i.e., glyceraldehyde) in relation
to the prebiotic formation of sugars
An example of chemical biology:
• Uses biological concepts, DNA structure
• Uses chemical ideas → conformation & functional
groups
• Uses chemical synthesis principles
• Makes a non-natural molecule with novel properties
• Relates those properties to the natural system
• Problem: Still difficult to predict and analyze singlestranded oligonucleotide structures
Part II:
The Protein World
•
We’ve seen how early catalysis by e.g. clay, leads to
synthesis of more complex structures
•
These in turn led to catalysis of specific reactions,
eventually leading to proteins taking over as the
“normal” catalysts
RNA world  Protein world
Questions
1) How were amino acids first formed?
2) Origin of homochirality: relationship between D-sugars
& L-amino acids?
3) How did amino acids condense to give peptides in the
prebiotic world?
4) Is this related to chemical peptide synthesis
5) Ribosomal peptide synthesis  relationships?
6) Can this knowledge be used to evolve better synthetic
strategies?
How were Amino Acids Formed?
• The Urey-Miller Experiment:
R
H2O
+
CH4
+
NH3
+
H2
OH
H2N
electrical
discharge
O
amino acid
The Urey-Miller Experiment
• CH4, NH3 & H2 atmosphere + H2O
• Water is heated to induce
evaporation
• Vapor reaches gas & sparks are
then fired through atmosphere
(simulates lightning)
• Atmosphere is then cooled
• Water and organic compounds are
trapped
• Continued experiment for 7 d, then
analyzed residue
• Results:
– Some insoluble material: likely a cyanide-aldehyde
polymer
– Aqueous residue showed that 10 -15% of carbon had been
converted to organic compounds (including amino acids)
– Glycine (R=H) was found to be most abundant (least C-C
bond forming reactions needed)
– 12 of the other proteinogenic amino acids (20 in modern
cells) were formed:
– These were  amino acids (C relative to C=O)

OH
H2N
O
• Note: there are >> 400 naturally occurring amino acids,
including both enantiomers, -amino acids, etc
For example:


H2N
OH

• Gamma-aminobutyric acid
• A  amino acid
O
GABA
• The amino acids used for protein synthesis are always L
and the  stereocentre (except gly) is has S configuration
• D amino acids do occur & are often found in nonribosomal
peptides
• Constructed on a nonribosomal peptide synthase (NRPS)
• Found primarily in bacteria and fungi
• More on these later!
Proteinogenic Amino Acids:
• Neutral Amino Acids
–
–
–
–
–
Alkyl (Ala, Ile)
The amides (Asn, Gln)
Alcohol (Ser, Thr)
Thiol (Cys)
Thioether (Met)
• H+ donors (acidic)
– Phenol (Tyr)
– Acids (Asp, Glu)
• H+ acceptors (basic)
– Amines (Lys, His, Arg)
pH dependence:
OH
H2N
O
H3N
O
O
present at isoelectric point -- zwitterion
base
acid
+
OH
H3N
O
H2N
O
O
ions -- more soluble
AA precipitates at isoelectric point
How to show amino acids are formed in the Urey-Miller
Experiment?
1)
Ninhydrin detection of amino acids separated by
electrophoresis:
Apply AA mixture
paper
+
apply
electric
potential
soak in buffer
The more –ve charge on the AA at a given buffer pH, the faster
it moves towards the cathode (+)
+ve charged AAs move toward the anode
+
-
origin: neutral
AAs remain
• After drying, paper is exposed to ninhydrin:
O
O
H2 O
OH
O
OH
O
Anhydrous -- all
3 carbons have
+ next to one
another
O
Hydrate is very
stable:
+
+
interaction relieved.
Normal form of
ninhydrin
Ninhydrin reacts with amino acids via imine formation:
O
O
O
H2N
O
H
N
CO2H
OH
O
O
O
CO2H
N
OH
+
H
O
-CO2
H+
OH
N
H
NH2
OH
OH
reacts with another
molecule of ninhydrin
O
O
N
OH
O
O
O
O
H2O
N
H+
HO
• This condensation product has an extended conjugated
system (how many resonance forms can you draw?)
• It absorbs light in the visible region (recall that in UV/VIS
spectroscopy, max depends on the length of the
conjugated system)
O
O
N
OH
O
•  if we spray ninhydrin on the paper chromatogram (or
on a TLC plate), purple spots develop
• Cannot distinguish between amino acids
• Alternatively, can separate amino acids by ion exchange
chromatography
– Separates based on charge
– Derivatize with ninhydrin as eluant comes off the column
• Subsequent studies of Urey-Miller Exp’t also showed:
– Rapid initial formation of HCN & aldehydes
– This was followed by a decrease of these products as amino
acids were slowly formed
– Hypothesized that amino acids were being formed via a
previously known method, the Strecker synthesis
• Strecker Synthesis:
– Developed by Strecker in 1850 to give racemic amino acids
– Can still be found in use, with some modifications
O
+
R
H
NH3
HCN
O
H2N
OH
R
Strecker Synthesis: mechanism
H+
O
R
+
HO
NH2
NH
IMINE
NH3
R
H
H
R
H
*** recall from prebiotic
synthesis of adenine
-CN
+
NH3
R
nitrile hydrolysis
H
COO-
NH2
H
R
CN
Racemic amino acid
Was this how amino acids formed in the prebiotic world?
• Note the pH rate profile:
+ H
O
Rate
At low pH: plenty of
H+ to increase
reactivity of aldehyde,
but no –CN or NH3
O
H
H
NH4+
NH3
HCN
-CN
At high pH: all NH3
& -CN, but no H+
to catalyze
addition of Nu: to
aldehyde
pH
4-5
 Optimum pH to carry out reaction is around 4-5 → a balance between
[catalyst] & [nucleophile]
• Note that the Strecker synthesis must give racemic
amino acids: achiral starting materials
NC-
NH
Re face
R
H
Si face
-CN
• Likewise, Urey-Miller experiment gives racemic AAs
• How to separate?
– Can perform chromatography using chiral solid phase: two
enantiomers bind differently to chiral phase because they are
diastereomeric:
AAR + SPS
AAS + SPS
AAR SPS
AAs SPS
Diastereomers
separate
•
The chemistry of imines is also important in natural
processes that are still used today:
e.g. Another cofactor, PLP, pyridoxal phosphate
(Vit B6 derivative)
O
H
O
HO
O P O
N
OH
PLP
•
Involved in amino acid metabolism (e.g. His →
histamine)
Reacts with amino group of amino acids to make imine
(see mechanism)
•
–
Fate of imine?
a)
Lose the acidic proton (), the reprotonate from the opposite face:
epimerization
N
Enz
H
COOH
H2 N
O
+
S
H
COOH
N
R
H
H
N
PLP
R
AA
:B-Enz
N
N
 Carbon is now sp2 
enzyme can add H to
either side
O
N
N - COOH
OH
H
H
R
R
H
Enz
N
O
H2 O
N
COOH
H
H
COOH
H2 N
R
R
R amino acid
hydrolysis regenerates
PLP & amino acid
H
+
N
b)
Decarboxylation: AA decarboxylases
N
N
+
PLP
O
AA
N
O H
H
N
R
H
N
-N
O
H
+
H2 N
N
H
N
H
H
R
N
H
H
H
For example:
NH2
PLP
OH
H2N
decarboxylase
H2N
NH2
O
Lysine
-
H Enz
H2 O
R
R
Cadaverine
(rotting flesh)
R
c)
Tautomerize & hydrolyze: transamination (transaminase)
N
N
PLP +
AA
:B-Enz
COOH
N
N - COOH
H
H
H
R
R
Amine of amino
acid converted to
carbonyl
N
O
N
H2O hydrolysis
O
O
OH
N
H
PLP is now
in the amine
form & can
to perform
amination
reaction
H
R
+
NH2
H
H
e.g.
O
O
O
N
OH
O
pyruvic acid
+
H
-
N
OH
R
Imine
N
O
OH
H
R
H+
OH
NH2
alanine
d)
Cleave the R group (on -C) of amino acid:
e.g. serine (serine hydroxymethyl transferase – SHMT)
N
PLP
H2N
+
COOH
HO
COOH
N
serine
H
OH
N
N
+
H
+
H2 N
COOH
O
H
glycine
H
N
COOH
H
+
H
O
•  the role of PLP is to make an imine, which stabilizes
negative charge through delocalization of charge
– Allows for cleavage of C-C and C-N bonds
N
OH
P
N - COOH
O
H
R
O
H
O
HO
O P O
N
PLP
OH
N
O
-
N
OH
H
R
The synthesis of Dilantin also involves imines (expt 7):
+
H
Ph
+
H
OH
H2N
O
+
+
H
O
H2N
O
Ph
Ph
H
N
O
Ph
OH
N
H
Note the two +
C's are adjacent
+
H
H
N
Ph
Ph
Ph
OH
H
N
O
N
H
Dilantin
Ph
O
Ph
N
H
Ph
O
N
O
+
HO
Ph
N
H
N
O
Ph
N