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Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es. Synthesis of pyrimidine bases. Transforma tions of pyrimidine bases. Syntheses of puri ne bases. Transforma tions of puri ne bases. Synthesis of nu c leosid es. Synthesis of nu c leosid es. - PowerPoint PPT Presentation
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Chemical syntheses of nucleobases, nucleosides, nucleotides a oligonucleotides
N
N NH
N
NH2
N
N N
N
O
OH OH
HO
NH2
N
N N
N
O
OH OH
O
NH2
PO
HOOH
N
N N
N
O
OH OH
O
NH2
PO
OOH
POPHOOH
O
OH
O
N
NN
N
NH2
O
O
OP-OO-
O
N
NH2
ON
O
O
OP-O O
N
NH2
ON
O
OH
OP-O O
Synthesis of pyrimidine bases
H2N
NH
SEtN
NH
O
SEt
R
R'
HCl, H2O
NH
NH
O
O
R
R'H2N
NH2
O
OEt
O
Br
R
R'
OEt
O
OMe
R
R'
OEt
O
O
R
R'+or or
heterocyclization
NH
N
NH2
O
R
R'H2N
NH2
O
CN
OMe
R
R'
CN
O
R
R'R'
CN
+or or
heterocyclization
Transformations of pyrimidine bases
N
NH
O
OR
N
NH
N
N
N
OR
N
NH3
N
N
NH2
OR
N
NH
O
OR
I
I2, CAN
R-M (organometallic)
catalysis N
NH
O
OR
R
Syntheses of purine bases
N
N NH
N
X
X'
N
N NH2
NH2
X
X'
HN
H2N NH
N
X
X"X'-COY X"-COY+ +heterocyclization heterocyclization
X"
OCN
OEtNH2 O
H2N NH
N
OEtHN
N NH
N
O1. NH3
H2N
HNH+ 2. HC(OEt)3
N
N N
N
NH2
R
N
N Cl
NO2
NH2
N
N NH
NO2
NH2
R
RNH2
2. HC(OEt)3
1. reduction
Transformations of purine bases
N
N N
N
NH2
R
HNO2, AcOH
HN
N N
N
O
R
POCl3, DMFN
N N
N
Cl
R
N
N N
N
I
R
HI
i-AmONO, CH2I2
NH3
R'-M
catalysis
R'-M
catalysis
N
N N
N
R'
R
O
OBz
BzO
OBz
N
N
OEt
O
O
OH
HO
OH
N
N
NH2
OCl
O
OBz
BzO
OBz
N
N
OEt
OEt
NaOH O
OH
HO
OH
N
NH
O
O
CH3CN, 0°C then reflux
NH3, MeOH
Quaternization method
Synthesis of nucleosides
O
OBz
BzO
OBz
N
NH
O
O
OAc
N
N
OTMS
OTMS
Silyl Base method
1. TMSTf (from -Cl) or SnCl4 (from -OAc)2. H2O
O
OBz
BzO
R
N
N N
N
X
Y
N
N NH
N
Y
X
ClO
OBz
BzO
R
OAcO
OBz
BzO
RN
N NH
N
Y
X
O
OBz
BzO
R
N
N N
N
X
Y
O
OBz
BzO
R
N
N N
N
X
Y
O
OBz
BzO
R
N
N N
N
X
Y
SnCl4
CH3CN, reflux
base (NaH)
CH3CN, reflux
9--
7--
7--
9--
Synthesis of nucleosides
O
OBz
BzO
OO
Ph
O
OBz
BzO
OBz
NuNu
9--
Neighboring group participation
N
NN
N
NH2
O
OHOH
OP-OO-
O
N/CH replacement
CH/N replacement; substitution
substitution
configuration; substitution
sulfa, aza, carba ... analogues 1
2345 67
8 9
• acyclic nucleoside/nucleotide analogues• cyclonucleosides• fused and bicyclic analogues• homonucleosides• modified oligomucleotides
Modifications of Nucleosides and Nucleotides
HN
N N
N
OHO
O
Didanosine, ddI
N
OHO
HN
O
O
N3
Zidovudine, AZT
N
N N
N
HO
NH2
OH OHAristeromycin
HN
N N
N
OHO
O
H2N
Acyclovir
N
N N
N
NH2
O PO
OHOH
Adefovir
N
N N
N
OHO
NH2
OH
F
OH
Fludarabine
N
N
O
NH2
AraC
antiviral antineoplastic
N
N N
N
OHO
NH2
OH OH
R
adenosine receptorsantagonists -antihypertensive
OHO
OH
OH
Biological Activity of Nucleoside Analogues
BO
OH
HO
R
POCl3
P(O)(OMe)3
H2O (1 eq.)
BO
OH
O
R
PClO
Cl
selective
H2O work-upB
O
OH
O
R
PHOO
HO
Synthesis of nucleotides
1. (NHBu3)2H2P2O7NBu3, DMF
2. TEAB, H2O
BO
OH
O
R
POO
-OPOPO
O O
O- O-
1. (PhO)2POCl or DCC,morpholine2. (NHBu3)2H2P2O7
Enzymatic: nucleoside kinase
BO
OH
HO
R
BO
OH
O
R
PHOO
HB
O
OH
O
R
PHOO
HOH3PO3
SO2 NN
NN
I2, H2O, Py
use of P(III) reagents
Synthesis of nucleotides
Synthesis of oligonucleotides
1. Phosphodiester method2. Phosphotriester method3. H-Phosphonate method4. Phosphoramidite method
N
NN
N
NH2
O
O
OP-OO-
O
N
NH2
ON
O
O
OP-O O
N
NH2
ON
O
OH
OP-O O
BO
O
O
O-P-O O
PGB
O
O
O
BO
O
OP-O O
PG
PG
BO
O
HO
PG
DCC
N C NDCC
+days30-80%
Phosphodiester method
History…
Phosphodiester method
BO
O
O
O-PO O
DMTr
NC
O
OCH3
BO
O
O
O-PO O
DMTr
N
BO
OH
ODMTr
OHNO
OCH3
OP
N
N
NNN
N
N
NN
OP
N
O
NNN
N
N
NO2
1.
2. HOCH2CH2CN3. H2O
1.
2.+ TPSCl, MeIm
3. aq. NaOH
Phosphotriester method
BO
O
O
O-PO O
PG
NC
BO
O
HO
PG
BO
O
O
BO
O
OPO O
PG
PG
NC
SO
OCl
+
Phosphotriester method
BO
O
O
O-PO O
PG
NO
OCH3
BO
O
HO
PG
BO
O
O
BO
O
OPO O
PG
PG
NO
OCH3
SO
OCl
+minutes80-99%
Phosphotriester method
BO
O
O
BO
O
OPO O
PG
PG
NO
OCH3
thiophenol
BO
O
O
BO
O
OP-O O
PG
PG
O
PO
ROO
O BOH
PO
OO
O OB
N
CH3O
PO
OO
O OB
N
CH3O
OSO2Ar
OP
OO
HO
O
ONO
OCH3
SO2R
R
R
BO
O
O
O-PH O
PGBO
OH
OPG
OPO
O
Cl
TEABtriethylammonium bicarbonate
1. PCl3/imidazole/Et3N2. hydrolysis
H-Phosphonate method
BO
O
O
O-PH O
PG
BO
O
HO
PG
BO
O
O
BO
O
OPH O
PG
PG
+seconds98-99.5%
O
Cl
H-Phosphonate method
OCl OCl
O
O
O
O
PH O
OHO
BO
O
O
BO
O
OP-O O
PG
PGB
O
O
O
BO
O
OPH O
PG
PG
I2
oxidation
H-Phosphonate method
nucleophilicsubstitution
Nu-
BO
O
O
BO
O
OPNu O
PG
PG
Nu = S, NR2, BH3
BO
O
O
PN O
PG
CN
BO
OH
OPG
ClPN O
CN
EtN(i-Pr)2
NPN O
CN
+ tetrazol
Phosphoramidite method
BO
O
O
PN O
PG
CNB
O
O
HO
PG
BO
O
O
P O
PG
BO
O
O
PG
CN
N
NN
HN
N
NN
HN
EtS N
N
H
OTf
+seconds99-99.8%
Phosphoramidite method
O
O
P OCH2CH2CN(i-C3H7)2N
H
NN
NN
O
O
P OCH2CH2CN(i-C3H7)2N
NN
NN
H
O
O
P OCH2CH2CN
NN
NN
OCH2CH2CN
O
P
OO
OHO
BO
O
O
P O
PG
BO
O
O
PG
CNO
BO
O
O
P O
PG
BO
O
O
PG
CN
BO
O
O
P OH
PG
BO
O
O
PG
O
NH3oxidation
I2, H2O, lutidine-elimination+deacetylation of bases
Phosphoramidite method
N
NN
N
HN
O
N
N
HN
O
O
NH
N
O
O
NH
N
O
O
NH
NN
N
O
NH
O
ABz CBz
GiBu
unprotected
Protection of bases
BO
OH R
HO
BO
OH R
PGO
O
BO
OH R
TrO
MMTr
O
OCH3
DMTr
BO
OH R
HO
O
OCH3
OCH3
5'
Tr
TrCl, PyH2, Pd/Cor H+
DMTr - TFA or TCA
Protection of sugar
BO
OH OH
HO
BO
OH OH
DMTrO
BO
OH OPG
RO
Cl Si O Si Cl
TBDMS
OSi
BO
O OH
O
Si
SiO
BO
OH OTBDMS
DMTrO
TFA
Bu4N+ F-
(TBAF)
N
H3CO
F
O
N
OCH3
F
BO
OH OH
DMTrO
BO
O OFpmp
O
Si
SiO
2'
TBSMDCl, imidazole
Fpmp
Ribonucleotides
+ 3'-isomer
Protection of sugar
Solid-phase oligodeoxyribonucleotides
H2N
HN O O Si CPG
O
OAc OMe
OMeB
O
O
DMTrO
O
O
O
NO2
NH
HN O O Si CPG
O
OAc OMe
OMe
BO
O
DMTrO
O
O
Attachment to solid support
CPG = controlled pore glass
10-50mol/g
Solid-phase oligodeoxyribonucleotides
OB1
DMTrO
TCAO
B1
HO5'
3'5'
3'
OB2
DMTrOP
N
OCH2CH2CN
OB1
OOB2
DMTrOP
OCH2CH2CN
5'3'
5'3'
5'3'
tetrazole
OB1
OOB2
DMTrOP
OCH2CH2CN
O
I2, H2O, pyridine
5'3'
5'3'
TCAOB1
OOB2
HOP
OCH2CH2CN
O
5'3'
5'3'
next coupling
OB1
HOAc2OO
B1
AcO
5'3'
capping
5'3'
unreacted
Solid-phase oligodeoxyribonucleotides
1. Detritylation2. Coupling with phosphoramidite3. Capping4. Oxidation5. Detritylation…..6. Deprotection and release (aq. NH3, 50°C, 5h)7. Purification (HPLC, GEP)
Total yield Yn= Yi(n-1) 20-mer 80% -> 1.4%
90% -> 13%99% -> 82%
99.8% -> 96%
REGULATION OF GENE REGULATION OF GENE EXPRESSIONEXPRESSION
ANTISENSE STRATEGYANTISENSE STRATEGYinteraction with RNAinteraction with RNA
ANTIGENE STRATEGYANTIGENE STRATEGYinteraction with DNAinteraction with DNA
APTAMER STRATEGYAPTAMER STRATEGYinteraction with proteinsinteraction with proteins
Hybrid duplexHybrid duplexm-RNA*DNA-oligomerm-RNA*DNA-oligomer
TranslationTranslationarestarest
No protein No protein synthesissynthesis
Hybrid duplexHybrid duplexm-RNA*DNA-oligomerm-RNA*DNA-oligomer
DNA-oligomerDNA-oligomer
RNase HRNase H
Products of m-RNA cleavage
O
O
O
PHO O
OO
O
B1
B2
Resistance against nucleaseResistance against nucleasecleavagecleavage
High affinity towards targetHigh affinity towards target sequences of RNA/DNAsequences of RNA/DNA
Selectivity – discrimination Selectivity – discrimination between DNA and RNA between DNA and RNA
Low non-specific binding andLow non-specific binding andhigh sensitivity to mismatch pairshigh sensitivity to mismatch pairs
REQUIREMENTS FOR MODIFIED REQUIREMENTS FOR MODIFIED OLIGONUCLEOTIDESOLIGONUCLEOTIDES
Activation of RNase H cleavageActivation of RNase H cleavageabilityability
OPO
HS
O
O
B
BS
OPO
HO
O
O
B
BS
OCH2CH2OCH3
OPO
HO
O
O
B
B
OCH2CH2OCH3
S
O B
X
OP OO
HO
O B
X
OP X
HNHO
O
O
B
BOP OO
O
O
B
BH3B
N
OB
O
N
OB
P ONCH3
CH3OP OO
CH3
O
O
B
B
!! !!
!! !!!!
MOST IMPORTANT MODIFICATIONS MOST IMPORTANT MODIFICATIONS OF INTERNUCLEOTIDE LINKAGESOF INTERNUCLEOTIDE LINKAGES
PPeptide eptide NNucleic ucleic AAcidscids
NN
NN
NN
.........N
O OO
B1
HH
OB2
H
O
H
OB3
O
........
Classical synthesis of genes (duplexes DNA)
1. Synthesis of oligonucleotide fragments (20-40-mers, cohesive ends)
2. 5’-Phospohorylation (enzymatic or chemical)3. Ligation – T4 DNA ligase
PCR (Polymerase Chain Reaction)
1. Add primers complementary to flanking sequence2. Add all nucleoside triphosphates and thermostable
DNA polymerase3. Heat 95°C 15s - strand separation4. Cool 54°C – hybridization5. Heat 72°C (optimal temp.) – DNA synthesis
DNA cloning
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