chemical syntheses of nucleobases, nucleosides, nucleotides a oligonucleotides
TRANSCRIPT
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
P
O
HO
OH
N
N N
N
O
OH OH
O
NH2
P
O
O
OH
POPHO
OH
O
OH
O
N
NN
N
NH2
O
O
OP-O
O-
O
N
NH2
ON
O
O
O
P-O O
N
NH2
ON
O
OH
O
P-O O
Synthesis of pyrimidine bases
H2N
NH
SEt
N
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
O
R
N
NH
N
N
N
O
R
N
NH3
N
N
NH2
O
R
N
NH
O
O
R
I
I2, CAN
R-M (organometallic)
catalysisN
NH
O
O
R
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"
O
CN
OEt
NH2 O
H2N NH
N
OEt
HN
N NH
N
O
1. 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
O
O
Ph
O
OBz
BzO
OBz
NuNu
9--
Neighboring group participation
N
NN
N
NH2
O
OHOH
OP-O
O-
O
N/CH replacement
CH/N replacement; substitution
substitution
configuration; substitution
sulfa, aza, carba ... analogues 1
234
5 678
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 OH
Aristeromycin
HN
N N
N
OHO
O
H2N
Acyclovir
N
N N
N
NH2
O P
O
OH
OHAdefovir
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
B
O
OH
HO
R
POCl3
P(O)(OMe)3
H2O (1 eq.)
B
O
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
B
O
OH
O
R
POO
-OPOPO
O O
O- O-
1. (PhO)2POCl or DCC,morpholine2. (NHBu3)2H2P2O7
Enzymatic: nucleoside kinase
B
O
OH
HO
R
B
O
OH
O
R
PHOO
H
B
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-O
O-
O
N
NH2
ON
O
O
O
P-O O
N
NH2
ON
O
OH
O
P-O O
B
O
O
O
O-
P-O O
PGB
O
O
O
B
O
O
O
P-O O
PG
PG
B
O
O
HO
PG
DCC
N C NDCC
+days30-80%
Phosphodiester method
History…
Phosphodiester method
B
O
O
O
O-
PO O
DMTr
NC
O
OCH3
B
O
O
O
O-
PO O
DMTr
N
B
O
OH
ODMTr
OHN
O
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
B
O
O
O
O-
PO O
PG
NC
B
O
O
HO
PG
B
O
O
O
B
O
O
O
PO O
PG
PG
NC
SO
OCl
+
Phosphotriester method
B
O
O
O
O-
PO O
PG
N
O
OCH3
B
O
O
HO
PG
B
O
O
O
B
O
O
O
PO O
PG
PG
N
O
OCH3
SO
OCl
+minutes80-99%
Phosphotriester method
B
O
O
O
B
O
O
O
PO O
PG
PG
N
O
OCH3
thiophenol
B
O
O
O
B
O
O
O
P-O O
PG
PG
O
P
ORO
O
O BOH
PO
OO
O OB
N
CH3O
PO
OO
O OB
N
CH3O
OSO2Ar
OP
OO
HO
O
ON
O
OCH3
SO2R
R
R
B
O
O
O
O-
PH O
PGB
O
OH
OPG
OP
O
O
Cl
TEABtriethylammonium bicarbonate
1. PCl3/imidazole/Et3N2. hydrolysis
H-Phosphonate method
B
O
O
O
O-
PH O
PG
B
O
O
HO
PG
B
O
O
O
B
O
O
O
PH O
PG
PG
+seconds98-99.5%
O
Cl
H-Phosphonate method
OCl OCl
O
O
O
O
PH O
OHO
B
O
O
O
B
O
O
O
P-O O
PG
PGB
O
O
O
B
O
O
O
PH O
PG
PG
I2
oxidation
H-Phosphonate method
nucleophilicsubstitution
Nu-
B
O
O
O
B
O
O
O
PNu O
PG
PG
Nu = S, NR2, BH3
B
O
O
O
PN O
PG
CN
B
O
OH
OPG
Cl
PN O
CN
EtN(i-Pr)2
N
PN O
CN
+ tetrazol
Phosphoramidite method
B
O
O
O
PN O
PG
CNB
O
O
HO
PG
B
O
O
O
P O
PG
B
O
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
N
NN
N
O
O
P OCH2CH2CN(i-C3H7)2N
N
NN
N
H
O
O
P OCH2CH2CN
N
NN
N
OCH2CH2CN
O
P
OO
OHO
B
O
O
O
P O
PG
B
O
O
O
PG
CNO
B
O
O
O
P O
PG
B
O
O
O
PG
CN
B
O
O
O
P OH
PG
B
O
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
B
O
OH R
HO
B
O
OH R
PGO
O
B
O
OH R
TrO
MMTr
O
OCH3
DMTr
B
O
OH R
HO
O
OCH3
OCH3
5'
Tr
TrCl, Py
H2, Pd/Cor H+
DMTr - TFA or TCA
Protection of sugar
B
O
OH OH
HO
B
O
OH OH
DMTrO
B
O
OH OPG
RO
Cl Si O Si Cl
TBDMS
OSi
B
O
O OH
O
Si
SiO
B
O
OH OTBDMS
DMTrO
TFA
Bu4N+ F-
(TBAF)
N
H3CO
F
O
N
OCH3
F
B
O
OH OH
DMTrO
B
O
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
B
O
O
DMTrO
O
O
Attachment to solid support
CPG = controlled pore glass
10-50mol/g
Solid-phase oligodeoxyribonucleotides
O
B1
DMTrO
TCAO
B1
HO
5'
3'
5'
3'
O
B2
DMTrOP
N
OCH2CH2CN
O
B1
OO
B2
DMTrOP
OCH2CH2CN
5'
3'
5'
3'
5'
3'tetrazole
O
B1
OO
B2
DMTrOP
OCH2CH2CN
O
I2, H2O, pyridine
5'
3'
5'
3'TCAO
B1
OO
B2
HOP
OCH2CH2CN
O
5'
3'
5'
3'
next coupling
O
B1
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
O
P
O
HS
O
O
B
B
S
O
P
O
HO
O
O
B
B
S
OCH2CH2OCH3
O
P
O
HO
O
O
B
B
OCH2CH2OCH3
S
O B
X
O
P O
O
HO
O B
X
O
P X
HN
HO
O
O
B
BO
P O
O
O
O
B
B
H3B
N
OB
O
N
OB
P ONCH3
CH3
O
P O
O
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
O
B2
H
O
H
O
B3
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