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Chemical Synthesis of OligonucleotidesRichard T. Pon, Professor (adjunct),

Dept. of Biochemistry & Molecular Biology andDirector, UCDNA Services

(DNA/RNA Synthesis and DNA Sequencing)Faculty of Medicine

www.med.ucalgary.ca/dnaservices

Biochemistry 537 Nucleic Acids

10/2005

Chemical Synthesis of Oligonucleotides

Advantage of Chemical Synthesis:• no constraints on sequence or structures• modified bases, modified sugars, modified backbones• modifications can confer new or enhanced functionalityDisadvantages• less efficient than enzymes (slower and lower yield) • limited to single-stranded sequences < ~ 150 bases• cost of synthesis & purification

Synthetic oligonucleotides are very valuable as reagents, diagnostics, and DNA or RNA-based therapeutics.

“Oligo” – Prefix meaning few (~ 2-10). For oligonucleotides, this generally means anything made chemically, i.e. up to about 150 bases.

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Markets for Synthetic Oligonucleotides

Research Scale Oligo Market• PCR and sequencing primers, genetic

engineering, DNA probes, RNA interference (RNAi)• many millions of oligo’s per year• DNA > $100 million (US)/yr• RNA ~ $60 million (US)/yr → $250M/yr

Pharmaceutical Oligo Market• nucleic acid based drugs: antisense, aptamers &

speigelmers (protein binding), immune stimulating (CpG motifs), RNAi

• 2 drugs FDA approved, 2-3 dozen in clinical trials • ≥ 1 tonne/yr (when FDA approved)• potential $billion/yr markets

Oligonucleotide Building Blocks

Nucleosides can be obtained from either natural sources (i.e. salmon sperm) or chemically synthesized.

O

N

NH

OO

B

N

NH

OO

B

Phosphodiester Phosphorothioate Amide (PNA)

OO

O

B

OO

O

B

P OO H

OO

O

B

OO

O

B

P OS H

OHO

OH

B

OH

OHO

OH

BOH

OHO

OH

BF

OHO B

OOH

OHO

OH

B

OCH3

OOH

OH

B

OHN

NH2

OO

B

OHO

OH

B

DNA RNA ANA (arabino) LNA (Locked)

L-DNA 2'-O Methyl RNA FANA (2'-fluoroarabino)

PNA (Peptide Nucleic Acid)

Linkages are introduced by chemical coupling reactions

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Protecting Groups

5’-OH position is protected with a dimethoxytrityl (DMT) group• acid labile• temporary blocking group• removed at beginning of synthesis to

create a site for chain extension

Amino groups on Ade, Cyt, and Gua are protected with benzoyl (Bz) or isobutyryl (iBu) groups• kept throughout synthesis• stable to acid• removed by hydrolysis with NH4OH

NN

N N

NHC

O

NN

N N

O

NH

CO

N

NH

NH

O

CO

B =

"A(Bz)" "C(Bz)" "G(iBu)"

OHO

OH

BC

OCH3

OCH3

OO

OH

B

"DMT"

Protecting groups prevent reaction at unwanted positions and allow solubility in organic solvents

Protecting Groups

OO

O

B'"

OO

O

B"

OO

O

B'

PO O

PO O

CH2CH2CN

CH2CH2CN

OO

O

B'"

OO

O

B"

OO

O

B'

PO O

PO O

Phosphate linkages are protected as uncharged phosphotriesters using 2-cyanoethyl (CE) groups. After synthesis the CE groups are removed to yield the negatively charged phosphodiester groups of naturally occurring nucleic acids.

ODMTO

OH O

B

R

Si

CH3

CH3

CH3

CH3

CH3

R =

(TOM)Qiagen

(tBDMS)Public Domain

SiiPr

iPrIPr

CH2 O CHO (CH2)2

O (CH2)2

O CH3

O

O CH3

O

(ACE)Dharmacon

RNA has an extra OH group and requires an extra protecting group on the 2’-hydroxyl position. Three different protecting groups are currently widely used.

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Coupling Reactions

Synthesis consists of a series of coupling reactions to build up the desired sequence, followed by deprotection to remove all protecting groups. • Sequence must be in correct 5’-3’ orientation without 5’-5’, 3’-3’, (5’-2’, RNA) or

branched linkages• After deprotection, synthesis yields single-stranded DNA or RNA• Purification after deprotection is required to remove protecting group residues

and incomplete synthesis products

OHO

O

B

+O

DMTO

O

B

P ORY

OO

O

B

ODMTO

O

B

P ORO

OO

O

BP ORO

OO

O

B

ODMTO

O

B

P ORO

* * *

* - multiple steps

ODMTO

O

B

H+

Nucleoside 3’-Phosphoramidites

• Nucleosides converted to 3’-phosphoramidite 1 (commercially available)• PIII oxidation state (phosphite) is more reactive than PV (phosphate)• Beaucage discovered that P(III) compounds with P-alkylamine bonds

(phosphoramidites) were stable and easy to handle until “activated”• Activation with weak acid (tetrazole, pKa = 6.5) instantly forms highly reactive

reagent 2 (generated in situ in presence of another nucleoside)• 2 reacts rapidly (1 min) in high yield (99%) to form new phosphite linkage 3• 3 is oxidized to phosphate (PV) linkage with iodine/water after coupling • Discovery revolutionized Life Sciences → $billion economic activity since 1981

“Phosphoramidite Method”

Marvin Caruthers & Serge Beaucage

1981

OB

OH

DMTOO

B

O

DMTO

P OCE

N

OB

O

DMTO

P OCE

N

N NN

OB'

O

O

OB

O

DMTO

P OCE

1 2 3

5

Solid-Phase Synthesis Strategy

P

N1

N1P

+

+

N2

N1N1P

Wash away

+Wash away

N2

P N1 N2

Insoluble Solid-Phase Support

Wash away

Robert Merrifield (1962)• strategy for peptides, but also applicable to

oligonucleotides• insoluble polymer, silica, or glass used

as solid-phase support• target is synthesized on surface• products are covalently attached• impurities and by-products are not

attached and can be easily washed away• ease of purification is major & significant

advantage• easily automated• 1984 Nobel Prize in Chemistry

Solid-Phase Synthesis Strategy

Robert Merrifield’s peptide synthesizer (Rockefeller University)

Wash solvents

Amino acids & coupling reagents

Drum programmer

Reaction vessel

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Solid-Phase Synthesis Strategy

Oligonucleotide synthesis• controlled pore glass supports• first nucleoside chemically attached

through ester linkage to 3’-OH • extend one base at a time using four step

coupling cycle: detritylation, coupling, capping, and oxidation

• product remains attached to the surface of the support and excess, unincorporated reagents wash off

• after synthesis, linkage to support is hydrolyzed (NH4OH) to release the product

After cleavage from support, base and phosphate protecting groups must be removed and the full-length product isolated.Final product is single-stranded (mix two ssDNA’s to make dsDNA)

Phosphoramidite with 5’-DMT

DMT removed from 5’-OH

*

*

*

* - Capping, oxidation, and detritylation steps not shown

OBase1

RO

DMTO

XNH

OO

OBase1

RO

OPNCCH2CH2OO

OBase2

DMTO

R

OBase1

RO

HO

OBase1

RO

HO

+

coupling

OBase2

RO

DMTO

POCH2CH2CN(iPr)2N

+

ACTIVATOR

detritylation OBase1

RHO

OPO

OBasen

O

RO-O

PO

O-O

OBasen+1

HO

R

n

deprotection

OBase1

RO

OPNCCH2CH2OO

OBase2

DMTO

RO

oxidation

OBase1

RO

AcO

capping

cycle entry

Beaucage & CaruthersTetrahedron Letters 22, 1879 (1981)

Oligonucleotide Synthesis Cycle

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Coupling yields must be close to 100% because of the exponentialrelationship between overall yield (OY) and average coupling yield (AY). Very hard to do in practice!

Overall Yield Vs. Coupling Yield

0%

20%

40%

60%

80%

100%

0 25 50 75 100 125 150

Coupling Number

Ove

rall

Yiel

d

90% Coupling Yields 95% Coupling Yields98% Coupling Yields 99% Coupling Yields

%100100

×⎟⎠⎞

⎜⎝⎛=

nAYOY

Automated Solid-Phase Synthesis

• Packing the support into a column and passing the reagents through the column allows mechanization and automation

• Synthesis occurs inside the column on surface of support• Unreacted products are flushed out to waste

Early instruments for semi-automated oligonucleotide synthesis (~1980-81)

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Biologicals DNA/RNA Synthesizer

1981 First commercially available DNA synthesizer was developed in Canada (McGill University)K.K. Ogilvie et al, Science 214, 270 (1981)

Automated Oligonucleotide Synthesizers

ABI 380A/B ABI 394

ABI 3900

MerMade IV

3 oligos per run 4 oligos per run

48 oligos per run 192 oligos per run2 x 96-well plates

disposable synthesis column

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DNA Microarrays (Gene Chips)

Microarrays are made by:1. Robotic spotting of individual sequences

(cDNA’s or oligos) onto glass slides2. Chemical synthesis directly on the chip using

either light-directed or ink-jet synthesis technologies (Oligonucleotides are not cleaved from the surface after synthesis)

Micromax slide with array of cDNAs (1” x 3” slide)

DNA microarrays are 2-D arrays of 1,000’s or 10,000’s of sequences attached to a flat glass surface in a defined location.Identity of each spot is known.

Hybridization to DNA Microarrays

Microarrays are used as diagnostic probes• Sample DNA sequences are fluorescently labeled• Complementary sequences allowed to hybridize to chip surface• Unbound sequences washed from chip surface• Scanning fluorescence confocal microscopy locates sites of hybridization• Single-base pair mismatch discrimination possible• Arrays of up to 1 million or more different features per sq. cm possible • Used for monitoring gene expression, diagnostics or re-sequencing

256 element array 18,495 element array –HIV-1 rt and pro genes

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Light Directed Chemical Synthesis

• Replace 5’- DMT group with light sensitive 5’- protecting group• Cover surface with lithographic mask and expose to light• Sequence at every site is defined by order of masking steps• 20 μ feature size → 250,000 sequences per sq. cm.• Features down to 5 μ are possible (106 sequences/sq. cm)

Photolabile α-methyl-6-nitro-piperonyloxy-carbonyl protecting group (MeNPoc)

Affymetrix technology (1990’s) –

photolithographic masks

“Maskless” Microarray Synthesis Using Micro-Mirrors

Digital Light Processors• high contrast video

displays• eliminate need for

expensive masking steps

• Nimblegen (USA) and Febit AG(Germany)

• can produce new array designs in hours

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Creating Synthetic Genomes – First 30 yearsIf “reading” the genome was the first step, then “writing” it is next.• 1972 First total gene synthesis (77 bp, 3 yr)

by G. Khorana (Nobel Prize, 1968)• 2002 First de novo synthesis of a virus

(C332,652H492,388N98,245O131,196P7501S2340, Poliovirus, 7.5k bp) from synthetic oligonucleotides (Science 297, 1016). A molecule with it’s own life cycle.

2004. Microfluidic PicoArray gene synthesis chips (4 fmol/oligo) allow multiplex gene synthesis and assembly (15k bp)1972. The first gene synthesis was only

77 bp. It took over three years and when published occupied the entire issue of J. Mol. Biol. – 283 pages

Creating Synthetic Genomes

• 2003 Polymerase Chain Assembly (PCA) reduces time to make φX174 phage (5.4k bp) to 14 days.

• 2005 Codon Devices and Synthetic Genomics founded to exploit synthetic biology• 2005 Spanish Flu of 1918 “reconstructed”

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“Believe me, Reiser, there’s more to it than that.”

Additional MaterialReviews on solid-phase synthesis by the phosphoramidite method1. M. H. Caruthers, 1985, Gene Synthesis Machines: DNA Chemistry and Its Uses,

Science 230, 281-285.2. M. H. Caruthers, 1991, Chemical Synthesis of DNA and DNA Analogues, Acc.

Chem. Res. 24, 278-284.3. R. P. Iyer, A. Roland, W. Zhou, and K. Ghosh, 1999, Modified Oligonucleotides –

Synthesis, Properties and Applications, Curr. Opin. Molec. Therap. 1, 344-358.

An interesting personal account of the discovery of phosphoramidites by the Canadian inventor, Serge Beaucage

• “FDA’s Serge L. Beaucage on 20 years of Oligonucleotide Synthesis”, ScienceWatch, Sept./Oct. 2001, http://www.sciencewatch.com/sept-oct2001_page4.htm

Information on Synthetic Genes and Genomes1. J. Tian et al, 2004, Accurate multiplex gene synthesis from programmable DNA

microchips, Nature 432, 1050-1054.2. H. O. Smith, 2003, Generating a synthetic genome by whole genome assembly:

φX174 bacteriophage from synthetic oligonucleotides, PNAS 100, 15440-15445.