Transcript BioorgNukleoSynt

Chemical syntheses of nucleobases,
nucleosides, nucleotides a oligonucleotides
NH2
NH2
N
N
N
H
N
HO
O
HO P O
OH
N
O
N
OH OH
N
N
O
-O P O
O-
NH2
N
N
N
O
N
N
N
N
NH2
O
O
-O P O
O
OH OH
O
O
O
HO P O P O P O
OH OH OH
NH2
N
N
O
NH2
N
N
N
N
N
O
NH2
O
O
-O P O
O
N
N
O
OH
OH OH
O
Synthesis of pyrimidine bases
O
R
R'
O
NH
N
H
heterocyclization R
O
O
OEt R
or
Br
R'
R'
O
or
OMe
O
HCl, H2O
R
R'
R'
O
OEt
+
NH2
H2N
O
NH
NH
N
R
OEt
H2N
SEt
SEt
NH2
R
R'
heterocyclization
N
N
H
CN
R
CN
or
O
R'
R
CN
or
R'
OMe
R'
O
NH2
+
H2N
O
Transformations of pyrimidine bases
N
N
O
NH
N
R
NH2
N
N
H
NH3
N
O
N
R
N
N
R
O
I2, CAN
O
O
I
R-M (organometallic)
R
NH
NH
catalysis
N
R
O
N
R
O
O
Syntheses of purine bases
X
N
HN
X'-COY +
H2N
heterocyclization
N
X"
N
H
H
2
X'
N
H2N
N
H
1. NH3
N
HN
2. HC(OEt)3
NO2
Cl
N
N
N
H
N
NH2
RNH2
NH2
N
O
O
NH2
N
N
H
N
NH2
N
X"
OEt
NH2 HN
+
CN
H N
N
heterocyclization
N
X'
OEt
O
X
X
NH2
NO2
1. reduction
NH
R
2. HC(OEt)3
N
N
N
N
R
+ X"-COY
Transformations of purine bases
NH2
N
N
N
i-AmONO,
CH2I2
I
N
R
N
NH3
HNO2, AcOH
N
R
POCl3, DMF
R'-M
HI
R'
N
N
R'-M
Cl
N
N
N
R
catalysis
O
HN
N
N
N
N
N
N
R
catalysis
N
N
R
Synthesis of nucleosides
O
OEt
Quaternization method
OEt
BzO
O
N
N
BzO
OEt
N
O
HO
NaOH
O
N
O
OH OH
OBz OBz
NH3, MeOH
CH3CN, 0°C
then reflux
O
NH
N
NH2
N
Cl
HO
OBz OBz
OTMS
OAc
1. TMSTf (from -Cl)
or SnCl4 (from -OAc)
2. H2O
Silyl Base method
N
OH OH
O
N
N
O
NH
OTMS
BzO
O
N
OBz OBz
O
O
Synthesis of nucleosides
Y
Y
N
N
X
BzO
BzO
O
Cl
N
N
N
H
base (NaH)
X
BzO
N
N
O
O
N
OBz R
N
N
X
N
OBz R
CH3CN, reflux
9--
O
SnCl4
CH3CN, reflux
X
OAc
Y
OBz R
OBz R
BzO
N
N
N
H
N
Nu
BzO
O
O
Ph
Neighboring group participation
N
N
X
N
7--
Nu
OBz O
OBz R
Y
Y
O
7-- OBz R
O
O
N
N
BzO
N
Y 9--
N
N
X
BzO
BzO
9-- OBz OBz
N
Modifications of Nucleosides and Nucleotides
substitution
N/CH replacement
NH2
sulfa, aza, carba ...
analogues
O
-O P O
O-
N
8
7
9
N
5 61
4 32
N
N
O
CH/N replacement;
substitution
OH OH
• acyclic nucleoside/nucleotide analogues
configuration; substitution
• cyclonucleosides
• fused and bicyclic analogues
• homonucleosides
• modified oligomucleotides
Biological Activity of Nucleoside Analogues
antiviral
NH2
O
O
N
HN
N
O
HO
N
O
HO
F
HO
N
OH
R
HO
O OH
AraC
NH2
N
N
O
N
OH
Fludarabine
Zidovudine, AZT
O
HO
O OH
O
NH2
N
N
N
N
N3
Didanosine, ddI
N
N
HN
NH2
antineoplastic
N
OH OH
adenosine receptors
antagonists antihypertensive
HO
O
N
N
N
N
OH OH
Aristeromycin
N
HN
H2N
HO
NH2
N
N
N
N
N
N
O
O
Adefovir
Acyclovir
O
P OH
OH
Synthesis of nucleotides
B
HO
O
POCl3
Cl
Cl
O
P
O
B
O
O
H2O work-up HO P O
HO
B
O
P(O)(OMe)3
OH R
H2O (1 eq.)
selective
OH R
OH R
1. (NHBu3)2H2P2O7
NBu3, DMF
1. (PhO)2POCl or
DCC,morpholine
2. (NHBu3)2H2P2O7
2. TEAB, H2O
O
O
O
O P O P O P
O
OO- -O
Enzymatic: nucleoside kinase
B
O
OH R
Synthesis of nucleotides
B
HO
O
HO P
O
H
H3PO3
O
OH R
SO2 N
N N
N
use of P(III) reagents
B
O
OH R
O
HO P
O
HO
I2, H2O, Py
B
O
OH R
Synthesis of oligonucleotides
NH2
N
1.
2.
3.
4.
Phosphodiester method
-O
Phosphotriester method
H-Phosphonate method
Phosphoramidite method
O
P O
O-
N
N
N
NH2
O
O
-O P O
O
N
N
O
NH2
O
O
-O P O
O
N
N
O
OH
O
Phosphodiester method
History…
B
PG O
B
PG O
O
O
DCC
O
-O P O
O-
+
days
30-80%
B
HO
O
O
PG
N C N
DCC
O
-O P O
O
B
O
O
PG
Phosphodiester method
N
N
B
N N N
P
O
N
N
N
1.
B
DMTr O
N
DMTr O
O
OH
N
N
O
O P O
O-
NC
2. HOCH2CH2CN
3. H2O
O
N
B
N N N
1.
P
O
O
DMTr O
NO2
O
N
O
N
OH
2.
+ TPSCl, MeIm
OCH3
3. aq. NaOH
OCH3
O
O
O P O
O-
Phosphotriester method
O
S Cl
O
B
PG O
NC
O
O
O P O
OHO
+
B
B
PG O
NC
O
O
O P O
O
B
O
O
O
PG
O
PG
Phosphotriester method
O
S Cl
O
B
PG O
O
N
OCH3
O
O
O P O
OHO
+
minutes
80-99%
B
PG O
O
N
B
O
O
PG
OCH3
O
O
O P O
O
B
O
O
PG
Phosphotriester method
B
PG O
O
N
OCH3
B
O
PG O
O
thiophenol
O
O P O
O
B
O
O
PG
O
O P O
O
B
O
O
PG
O
R
O
R
SO2
OCH3
O
HO
O
P
O
O
N
O
RO
O
P
O
R
CH3O
CH3O
N
O
N
O
O
O
O
B
P
O
O
B
P
O
O
O
OSO2Ar
O
H
O
B
H-Phosphonate method
O
O
B
PG O
O
O
P
Cl
TEAB
triethylammonium
bicarbonate
B
PG O
OH
1. PCl3/imidazole/Et3N
2. hydrolysis
O
O
H P O
O-
H-Phosphonate method
B
PG O
O
O
Cl
O
H P O
OHO
B
+
B
seconds
98-99.5%
PG O
O
O
H P O
O
B
O
O
O
PG
O
PG
Cl
O
Cl
O
O
O
H
P O
O
HO
O
O
H-Phosphonate method
B
PG O
B
B
PG O
O
PG O
O
nucleophilic
substitution
oxidation
O
O P O
O
B
O
I2
O
H P O
O
B
Nu-
O
Nu P O
O
B
O
O
Nu = S, NR2, BH3
O
PG
O
O
PG
O
PG
Phosphoramidite method
N
Cl
P
O
B
CN
PG O
O
B
PG O
EtN(i-Pr)2
O
N
OH
O
P
O
CN
N
N
P
+ tetrazol
O
CN
Phosphoramidite method
H
N N
B
PG O
O
B
PG O
N
N
N
O
P
O
H
N N
B
O
EtS
O
PG
P O
N
CN
B
O
CN
HO
O
seconds
99-99.8%
+
O
N
N
N OTf
H
O
O
PG
O
O
O
O
(i-C 3H 7)2N P OCH 2CH 2CN
P OCH2CH2CN
N
O
N
N
N
H
HO
N
N
N
N
O
(i-C3H7)2N P OCH2CH2CN
H
N
N
N
O
N
P OCH 2CH 2CN
O
O
O
Phosphoramidite method
PG O
PG O
O
oxidation
O
P O
CN
O
O
PG
PG O
O
O
NH3
O
I2, H2O, lutidine O P O
CN
B
B
O
B
B
B
O
O
O
PG
-elimination
+deacetylation of
bases
O
O P OH
B
O
O
O
PG
Protection of bases
O
NH
N
O
O
NH
O
N
O
N
NH
N
N
GiBu
unprotected
O
HN
HN
N
N
O
N
N
N
ABz
N
CBz
O
N
H
O
Protection of sugar
OCH3
OCH3
B
PGO
5'
O
O
OH R
Tr
B
HO
O
O
O
OH R
MMTr
B
TrCl, Py TrO
O
H2, Pd/C
HO
or H+
DMTr
OCH3
B
O
OH R
OH R
DMTr - TFA or TCA
Protection of sugar
Ribonucleotides
B
RO
O OCH3
O
O
Si
2'
Fpmp
TBDMS
B
DMTrO
O
B
TBSMDCl, DMTrO
imidazole
HO
O
OH OH
Bu4N+ FDMTrO
(TBAF)
O
B
O
OH OTBDMS
+ 3'-isomer
OH OH
B
F
N
OH OPG
OH OH
B
O
B
Cl Si O Si Cl
O
Si
TFA
O
Si
H3CO
O
Si
O
OH
O
O
N
F
Si
O
OFpmp
Solid-phase oligodeoxyribonucleotides
Attachment to solid support
B
DMTrO
H2N
O
OMe
Si CPG
OMe
O
O
O
NO2
O
O
CPG = controlled pore glass
O
O
10-50mol/g
B
DMTrO
OAc
H
N
O
O
O
O
N
H
OAc
H
N
O
O
O
OMe
Si CPG
OMe
Solid-phase oligodeoxyribonucleotides
B1
2
1
B
DMTrO
3'
B
B1
TCA
O
HO
3'
tetrazole
DMTrO
next coupling
3'
3'
5'
B2
5'
5'
DMTrO
O
O P O
O P
3'
O
5'
OCH2CH2CN
OCH2CH2CN
N
5'
I2, H2O, pyridine
B
HO
2
3'
O
O P O
B1
3'
O
TCA
DMTrO
5'
5'
B
B
AcO
1
3'
5'
3'
5'
OCH2CH2CN
capping
B1
O
Ac2O
HO
3'
2
O
5'
unreacted
O
O P O
B1
3'
O
5'
OCH2CH2CN
Solid-phase oligodeoxyribonucleotides
1. Detritylation
2. Coupling with phosphoramidite
3. Capping
4. Oxidation
5. 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
EXPRESSION
ANTISENSE STRATEGY
interaction with RNA
ANTIGENE STRATEGY
interaction with DNA
APTAMER STRATEGY
interaction with proteins
Translation
arest
Hybrid duplex
m-RNA*DNA-oligomer
No protein
synthesis
DNA-oligomer
RNase H
Hybrid duplex
m-RNA*DNA-oligomer
Products of m-RNA cleavage
REQUIREMENTS FOR MODIFIED
OLIGONUCLEOTIDES
O
Resistance against nuclease
cleavage
B1
O
High affinity towards target
sequences of RNA/DNA
O
Selectivity – discrimination
between DNA and RNA
HO P O
O
O
O
B2
Low non-specific binding and
high sensitivity to mismatch pairs
Activation of RNase H cleavage
ability
MOST IMPORTANT MODIFICATIONS
OF INTERNUCLEOTIDE LINKAGES
O
B
O
!
O
HO P S
O
O
O
HO P
O
S
HS P
O
B
S
O
B
O
B
O
!
O
OCH 2CH 2OCH 3
O
B
B
X
O
HO P O
O
B
OCH 2CH 2OCH 3
O
O
H 3B P O
O
B
O
!
O
B
B
O
HN
CH3
HO P X
CH3
O
X
B
O
B
O
N
O
N P O
O
O
CH3 P O
O
B
O
N
B
O
B
O
HOOC-CH2 P X
O
B
O
B
!!
O
B
Peptide Nucleic Acids
B1
O
O
........
N
H
B2
O
O
N
N
H
B3
O
O
N
N
H
O
N
.........
N
H
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 sequence
2. Add all nucleoside triphosphates and thermostable
DNA polymerase
3. Heat 95°C 15s - strand separation
4. Cool 54°C – hybridization
5. Heat 72°C (optimal temp.) – DNA synthesis
DNA cloning