Origins of Sugars in the Prebiotic World One theory: the formose reaction (discovered by Butterow in 1861) Mechanism?

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

H

O

OH

OH

OH

-OH

O H

OH

erythrose/threose

retro-aldol

glycolaldehyde:cycle back for catalysis

2x

• 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

DNA

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

*

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