Origins of Sugars in the Prebiotic World One theory: the formose reaction (discovered by Butterow in...
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Transcript of Origins of Sugars in the Prebiotic World One theory: the formose reaction (discovered by Butterow in...
Origins of Sugars in the Prebiotic World
• One theory: the formose reaction (discovered by Butterow in 1861)
Mechanism?
H H
O Mineral catalysis
eg Ca(OH)2
mixture of sugars, including a small amount of ribose
formaldehyde
H H
O
H H
OO O O OH
H
OH O OH
HO-
O O O OH
H H
O
O O O OH
OH
H2O
-
slow, veryunfavorable
paraformaldehyde
*
H
H
O *n
O O O OH
OH
H depolymerise
O H
OH
-O H
OHH H
O
O H
OHOH
OH
O
OH
-OH OH
-O
OH
H H
O
OH
O
OH
OH
H
O
OH
OH
OH
glycolaldehyde:simplest sugar & a catalyst for further rxns
Ca(OH)2
ene-diloate(enol)
glyceraldehyde
viaene-diolate
dihydroxyacetone
pentoses, hexoses
viaene-diolate
erythrose/threose
Con’t
• Today, similar reactions are catalyzed by thiazolium, e.g., Vitamin B1 (TPP), another cofactor:
• Cf Exp. 7: Benzoin condensation
• e.g. N
SH
OH
Py
+
(PP)
H O
OH OH
OH
OP OH
OP
OH
OOH+
glycolaldehyde
G3PD-xylulose-5-P
Mechanism? Uses thiazolium
N
SH
OH
Py
N
SR
Py
H
O
OH
N
SR
Py
OH
OH
H
N
SR
Py
OH
OH
O
HOH
OP
N
SR
Py
OH
OH
OHOH
OP
N
SR
Py
O
OH
OHOH
OP
+
-OH
-+
carbanion: zwitterionic;stablized by +/- chargeinteraction
+
:B
N+ acidifies H
enamine
+
+ +
xylulose-5-P
thiazolium anioncatalyst regenerated
• We have seen how the intermediacy of the resonance-stablized oxonium ion accounts for facile substitution at the anomeric centre of a sugar
• What about nitrogen nucleophiles?Many examples:
Could this process have occurred in the prebiotic world?
O
OPP
OHOH
RO
O+
OHOH
RO
N
CO2H
CO2H
N
CO2H
CO2HO+
OHOH
RO
O+
OHOH
RO NH2
R = H or P
quinolinate
NADH
NH3
nucleosides
• Reaction of an oxonium ion with a nitrogenous base: NUCLEOSIDES!
• Nucleosides are quite stable:1) Weaker anomeric effect: N< O < Cl (low electronegativity of N)
2) N lone pair in aromatic ring hard to protonate
OOH
OHOH
OH
O
OHOH
OH
NH
NH
O
O
N
NH
O
OO
OHOH
OH
OO
OHOH
OH P
O
OH
O
Mineral days?
Hydrothermalvents?
Mn+
+&/orapatite (mineral
phosphate)
Activated leaving group:CATALYSIS
Thymidine (a nuclesoside)
N
NH
O
OO
OHOH
OH
O
OHOH
OHN
NH
O
O+-
Chargeseparation:unfavorable, since -ve charge is onN, a lesselectronegative group
1)
Anomeric effect: Cl > O > N (remember the glycosyl chloride prefers Cl axial
2)
N
NH
O
OO
OHOH
OH
N
NH
O
OO
OHOH
OH
H
H+
X
lone pair part of aromatic sextet
+
aromaticity destroyed(i.e., pyridine & pyrrole)
• These effects stabilize the nucleoside making its formation possible in the pre-biotic soup
• Thermodynamics are reasonably balanced• However, the reaction is reversible
– e.g. deamination of DNA occurs ~ 10,000x/day/cell in vivo– Deamination is due to spontaneous hydrolysis & by damage of
DNA by environmental factors– Principle of microscopic reversibility: spontaneous reaction
occurs via the oxonium ion
Ribonucleosides & Deoxyribonucleosides
Ribonucleosides• Contain ribose & found in RNA:
Deoxyribonucleosides• Contain 2-deoxyribose, found in DNA
N
N
OOH
OH OH
NH2
O N
N
OOH
OH OH
O
O
HN
N N
N
NH2
OOH
OH OH
N
N N
N
O
OOH
OH OH
NH2
cytidine (C) uridine (U) adenosine (A) guanosine (G)
Ribonucleosides
Deoxyribonucleosides
N
N
OOH
OH
NH2
O N
N
OOH
OH
O
O
HN
N N
N
NH2
OOH
OH
N
N N
N
O
OOH
OH
NH2
2'-deoxycytidine (dC) 2'-deoxythymidine (dT) 2'-deoxyadenosine (dA) 2'-deoxyguanosine (dG)
Important things to Note:• Numbering system:
– The base is numbered first (1,2, etc), then the sugar (1’, 2’, etc)
• Thymine (5-methyl uracil) replaces uracil in DNA• Confusing letter codes:
– A represents adenine, the base– A also represents adenosine, the nucleoside– A also represents deoxyadenosine (i.e., in DNA sequencing,
where “d” is often omitted)– A can also represent alanine, the amino acid
• Nucleoside + phoshphate nucleotide• In the modern world, enzymes (kinases) attach
phosphate groups
OOH
OH OH
AOO
OH OH
AP
O
-O
OHOO
OH OH
AP
O
O
O
P
O
OH
O
OO
OH OH
AP
O
O
O
P
O
O
O
P
O
O
OH
P
Adenosine-5'-monophosphate (AMP) Adenosine-5'-
diphosphate (ADP)
Adenosine-5'-triphosphate (ATP)
Energy source for cellCentral to metabolism
In the pre-RNA world, how might this happen?
Observation:
N
NH
O
OOOH
OH OH
clay
(apatite)
5'
2'
1'
3'
4'
5' phosphate + 3' phosphate + higher phosphates(30 % + 50%)
NUCLEOTIDES!
• Surprisingly easy to attach phosphate without needing an enzyme– One hypothesis: cyclo-triphosphate (explains preference for
triphosphate
OOH
OH OH
T
O
PO
P
OP O
OO
O
O
OATP
release of somering strain incylco-triphosphatedrives reaction?
Primary OH?sterics?
• If correct, this indicates a central role for triphosphates of nucleosides (NTPs) in early evolution of RNA (i.e., development of the RNA world)
• NTPs central to modern cellular biology
Triphosphates
• Triphosphates are reactive– Attack by a nucleophile at P, P or P gives a
good resonance stabilized leaving group (can also assisted by metal cation)
• Other examples where phosphorylation is essential include:– Glucose metabolism – Enzyme regulation: Carbohydrate
metabolism, Lipid metabolism, receptorsOO
OH OH
AP
O
O
O
P
O
O
O
P
O
O
OH
OH
O P
O
OH
O
Mg2+
+ ADP
• If the nucleophile is the 3’-OH group of another NTP, then a nucleic acid is generated: polymer of nucleotides– Oligomers (“oligos”) short length (DNA/RNA polymers of long
length)
P
O
O
O
P
O
O
O
P
O
O
OH O
Nuc
OO
OH OH
BPPPO
O
OH OH
B2O
OO
O OH
B1PPPO
PO O
+
PPP
a dinucleotide-5'-PPP
trinucleotide
"oligo" (polymer)
Mg2+
Note that nature faces some problems:
1) Nucleophilic attack required by 3’-OH, not 2’-OH
2) Specific attack on P required
3) In a mixture of NTPs, get non-specific sequence
4) Reaction rate is slow
• Nucleic acids contain a regular array of bases, spaced evenly along a backbone of phosphates & sugars
• Even spacing allows self-recognition, – i.e., RNA short stretches form in which bases complement
one another– tRNA folds into a specific conformation (more about tRNA
later)– DNA: strand I and its reverse complement form a regular
sequence with bases paired through H-bonds
Copyright 2006, John Wiley & Sons Publishers, Inc.
tRNA
Template-Directed Synthesis in the Pre-Biotic Soup
N NH
O
O
N NH
O
O
O
O
OH
PO
O
OH OH
OH
O
O
NN
N
N
NH2
NN
N
N
NH2O
O
O
O
OH
P OO
OH
OH
OH
• Template-directed synthesis in the pre-biotic world allows AMPLIFICATION due to MOLECULAR RECOGNITION & rate acceleration results: an entropic effect!
• Now, catalyzed by enzymes:– DNA polymerase makes DNA copy of a DNA template (i.e.,
replication)– RNA polymerase makes RNA copy of a DNA template
(transcription)
O
OH
ROB1
OH
O
OH
PPPOB2
H
O
O
ROB1
OH
P
O
OH
B2
H
OO
O
PP
DNA template strand DNA template strandMechanism of Chain Elongation reaction catalyzed by RNA polymerase
O
OH
ROB1
H
O
OH
PPPOB2
H
O
O
ROB1
H
P
O
OH
B2
H
OO
O
PP
template strand
Mechanism of Chain Elongation reaction catalyzed by DNA polymerase
• Viruses contain– Reverse transcriptase (RT): makes a DNA copy of RNA genome
• Template strand = RNA, Product = DNA
– RNA synthetase: makes an RNA copy of RNA• Template strand = RNA, Product = RNA
RNA as a Catalyst = Ribozymes
• Tom Cech & Sid Altman- Nobel Prize (1989)• Ribozymes that catalyze many reactions are being
discovered– i.e., cleavage of RNA (this is the reverse of synthesis)
O
O O
O
PO
O
O
B
H
-O Pb2+ O
O O
O B
P
O O
OH Pb2+
HO
C60
U59
3'5'
Yeast tRNA
17C60
U59
3'5'
17
• This reaction is specific: – Pb2+ binds to U59/C60 (if these are mutated no binding)– Cleavage is specific requires 2’-OH at B17
– One of few systems where x-ray structure is available revealing potential mechanism
• Another example: Can RNA catalyze addition of a base to a sugar? YES!
see (on website):Lau, M; Cadieux, K; Unrau, P. J. Am. Chem. Soc., 126, 15686-
15693
Chemical synthesis random sequences of RNA
a) Attach sugar, lacking base, to 3’ end
b) Few molecules react with base to make nucleotide at 3’ end
c) Sort out those with base at 3’ end
d) Amplify (PCR), enrich pool & cycle many times
Gives pure catalytic RNA!
More on Ribozymes• We have seen examples of self-cleaving ribozymes• Riboswitches represent another class of ribozymes:
– Regulate gene expression through a structural rearrangement by binding a small metabolite (from pathway)
– Small molecule can bind in “pocket”– Usually located near site of gene (protein expression)
In absence of metabolite, the initiation signal of protein synthesis is exposed Conformational change through
base pairing blocks expression
• glmS ribozyme is a riboswitch that is also self-cleaving:
OH
O
O
OO
B
H
P
O
O O
O
-O
B
O
OHOH
NH
H
OO
OO
B
H
OP
O
O O
O
O
B
P
+
Glucosamine-6-phosphate
cleavage
translation blocked
GlcN6P , GlcN6P binds ribozyme = cleavage = no synthase made
Fruc-6-P GlcN6PGlcN6P synthase
GlcN6P , GlcN6P = ribozyme is inactive = translation occurs = synthase produced Potential drug target???
Nitrogenous Bases
Prebiotic world
• HCN/CN- + NH3 ? (similarity to chemical synthesis?)
• Nicotinamide (NAD+/NADH)
Structure/Chemistry
• A, T, U, C, G
•Pyridine & pyrrole
• H-bonding
The story so far…
Sugars
StructureReactions of Sugars
• triose, tetrose, pentose, etc.
• D/L, R/S
• Projections
• Redox reactions
• Reactions with a Nu
• acetals
• Oxonium ion formation
• Anomeric effect
• Protecting groups/activating groups (i.e., AZT)
• vs
Structural determination by NMR (1D & 2D)
Prebiotic word
• formose reaction (polymers of formaldehyde)
Modern world
• thiazolium ion (cofactor)
Sugar + Nitrogenous Base = Nucleosides
Modern World
• Enzymes (later)
Prebiotic world
• Mineral cations (hydrothermal vents?)
Nucleotide + phosphate = Nucleotide
Prebiotic World
• Apatite (P)
• Template-directed synthesis
Modern World
• ATP
• DNA/RNA polymerase
Ribozymes (link?)
Chemical Synthesis of Oligo’s
• Challenge: Many different functional groups present we need to use protecting groups– Same concepts of protection & activation that we have already
seen in sugar chemistry
• Automated synthesis: allows molecular biologists to order oligo’s; made by machine
• Uses solid phase beads, which allows washing with reagents, solvents, etc.– CPG = Controlled Pore Glass
O
OH
DMTO
OH
B
O
OH
O
O
DMTO
OH
BO
Ph
OMeMeO
CPGActivate acid with DCC (see lab 6)
CPG
DMT = dimethoxytrityl
- recall trityl group in sugar chemistry- protects 1o OH
N N
NN
NH
sugar
O
Ph
N N
NN
NH2
sugar
B = Base, protected to make it non-nucleophilic (amine amide) This protection must be done prior to attachment to bead
3’ OH is ONLY nucleophile to react
O
O
DMTO
BO
O
O
OH
BO
O
O
DMTOB2
PO
NC
NN
N N
O
O
O
BO
O
O
DMTOB2
P O CN
CPG
H+
CPG(removes DMT)
CPG
+
Phosphoramidite
– relatively stable
– mild conditions for synthesis
– high selectivity of activation
O
O
O
BO
O
O
DMTOB2
P O CNO
CPG
I2
I2 + 2e- 2I
PIII - 2e- Piv
[oxidize]
Repeat Cycle:
1) Deprotect DMT with H+
2) Add B3
i. Phosphoramidite
ii. Couple 5’OH of growing chain
3) I2 oxidation to Piv
4) Add B4, etc…
• Each step goes in 1-2 mins in > 99% yield!
• Last step is H+ deprotection of DMT
• Then remove of bead (CPG), remove cyanoethyl & benzoyl (on base) NH4OH
Mechanisms?
O
O
RNH2
O
OH RCPG
NH3
CPG+
Free hydroxyl
Cleavage from the bead
OPCN
H
H+
P OH
NH3
NH
O
PhAr
NH3
Ar NH2
1)
2)
3)
• Final product is the oligo, fully deprotected, released from CPG elutes from column
• RNA synthesis: similar, need a 2’-OH protecting group:– Common one: R3Si- (“silyl”)
O
O
O
BO
O
O
DMTOB2
P O CNO
O
O
O
BO
O
O
DMTOB2
P O
CNO
O SiR3
R'-OH R3Si Cl
R3Si OR'F
CPG
DNA
CPG
RNA
Attachment:
+
Removal:
What if you need to know the sequence?
• Amplification of nucleic acids (PCR): key to molecular biology
a) heat, denature double helixb) cool, primers anneal through H-bonds
c) DNA polymerase (thermostable, allows cycles) fills the gap withreverse compliment of desire sequence
DNA + polymerase + dNTPs + 2 templates + rATP amplify selected target
• Once you amplify DNA, how do you know the sequence?
DNA (to sequence) A T GC
primer(cf PCR) polymerase begins to extend
from primer- adds dNTPs- occasionally adds a ddNTP
this terminates chain growth
A T GC
ddT
ddA
ddC
ddG
dT
dT dA
dGdT dA
separate (gel orcap. electrophoresis)
sequence of peaks
Molecules of different size, each terminated by ddN
• In order to “read” sequence, need to tag each ddNTP
• Previously, 32P was used (radioactive)
• Now, each ddNTP is tagged with a different chemical dye look at color of peak at terminating nucleotide
• Based on the synthesis of 2,3-deoxyribose (“dideoxy method”):
O
OH
TrOX
O
O
TrOX
SO
CF3
O
S
O
CF3
O
Cl O
H
TrOX
(TfCl)
NaBH4
*