Spezielle Methoden der pharmazeutischen Chemie 3Spezielle Methoden der pharmazeutischen Chemie 3
Asymmetrische Naturstoff und Arzneistoffsynthesey y(für Diplomanden und Dissertanten im Fach Pharmazeutischen Chemie/Synthese)
Kapiteleinteilung:1 Terpenoide1. Terpenoide2. Alkaloide3. Zucker4. Nukleoside5. Oligonukleotide6 GABA A l6. GABA-Analoga
E. Urban 11
An Introduction to Antisense-Oligonucleotides• The Antisense Principle
• Synthesis of Oligonucleotides
• Biophysical Characterisation of Artificial Oligonucleotides• Biophysical Characterisation of Artificial Oligonucleotides
• Structurally Modified Antisense Oligonucleotides
• Oblimersen Modifications
E. Urban 22
The Antisense Principle
A C
P P
A G
P
T T
P PP
A G
P
G C
P PP
A G
P5' 3'
A CT G
P P
A GT C
P
T TA A
P PP
A GT C
P
G CC G
P PP
A GT C
P3' 5'
DNA-matrix strand
Transcription
Sense - m-RNAA C
P P
A G
P
U U
P PP
A G
P
G C
P PP
A G
P5' 3'
Inactivation
A C
P P
A G
P
U U
P PP
A G
P
G C
P PP
A G
P5' 3' Sense - m-RNA
U GP P
U CP
A AP PP
U CP
C GP PP
U CP3' 5' Antisense Oligonucleotide
E. Urban 33
Inhibition of the Protein Synthesis by Antisense Oligonucleotides
DNA
T i ti Inhibition of the transcription
PrimaryRNA Transcript
Transcription Inhibition of the transcriptionby triple helix formation
RNA-Transcript
Processing Blockade of the processing factors
mRNA
TransportInhibition of the transport by double strand formation
mRNA
p by double strand formation
Enzymatic degradationof double strands
Nucleus
Cytoplasma
Translation Inhibition of the translation by double strand formation
Protein
E. Urban 44
Possible Binding Positions of Antisense Oligonucleotides
Nucleusgenomic DNA
TranscriptionantisenseOligonucleotide
genomic DNA
primary Transcriptcappingregion
polyadenylationsite
exon-introntransition
RNA ProcessingantisenseOligonucleotide
t T i tribosomal
binding siteAUG
TranslationantisenseOli l tid
mature Transcriptbinding site
Oligonucleotide
CytoplasmaProtein
E. Urban 55
y
Catalytic Effect of Antisense Oligonucleotides
Nucleusgenomic DNA
Transcription
ge o c
primary Transcriptcappingregion
polyadenylationsite
exon-introntransition
RNA Processing
t T i tribosomal
binding siteAUG
mature Transcriptbinding site
antisenseOligonucleotide Degradation by RNAse H
Cytoplasma
E. Urban 66
Specific Action of Antisense Oligonucleotides
- sequence dependent inhibition of the gene sequence of the target proteinof the gene sequence of the target protein
- inhibition of genes of other proteins should be avoided g p(BLAST data bank)
~ 4.000.000.000 base pairs consist in the human genoman 18mer statistically occurs onlyone time in 68.000.000.000 base pairs
but sequence of bases is not statistical in the genomand pairing can occur even if there are 3 mismatches
E. Urban 77
Unspecific Action of Antisense Oligonucleotides
- typical action of the polyanionic backbonehaemostasis activation of the complement systemhaemostasis, activation of the complement systemoccuring on high dosage beyond the therapeutic range
- caused by special structure features:immune stimulation by the CpG motiveimmune stimulation by the CpG motiveimmune stimulation by the phosphorthioate backbone
E. Urban 88
Choice of the Target Sequence
- exclusion of sequences pairing with other mRNA (gene data bank)- not more that 2 CpG motivesp- no hairpin structures- no repetitive guanosines as targets
- no sequence as target, which is hindered by the secundary or tertiary structure of mRNAy y y
due to the matter of fact, that predictions of th d t ti t t f RNA till li blthe secundary or tertiary structure of mRNA are still unreliable the empirical method („gene walk“) is the method of choice
E. Urban 99
Synthesis of Oligonucleotides - the Phosphoroamidite Process
lstart
O
O
BDMTO
O
OBHO O
BHO
Ocleavagestart29 % NH3
O
O N
O
CPGH
O
O N
O
CPGH
O
O
BOPOOH
BDMTO
coupling3 % TCA detritylation
OBDMTO
O
HO
BOOOHPO
OP
OiPr
NiPr
O
OBOPO
O
O
O
NC + Tetrazol
O
cappingoxidation
O
O N
O
CPGH
O
CN
N
N
O
+
oxidationH
OBDMTO O
BAcO
I2 OBOP
O
OO
O N
O
CPGH
E. Urban 1010
O
O N
O
CPGH
CN
Pyrimidines for the Synthesis of Oligonucleotides
N O OO
Ph
H
MeO
N
N
N
DMTO N
N
O
H
DMTO N
N
O
H
O
Ph
O
O
NO O
O
DMTO NO O
O
DMTO NO
O
MeO
PO N
PO N
PO N
C
N CC
N TC
N U
E. Urban 1111
Purines for the Synthesis of Oligonucleotides
PhH
MeO
N DMTO N
N
N
ON
H
N
N
O
OH
Ph
O
O
NO
O
DMTO NN N
NH
O
MeO
O
PO N
O
PO N
C
N AC
N G
E. Urban 1212
Disadvantages of Natural Oligonucleotides
Unsatisfactory binding affinityUnsatisfactory binding affinity
Instability against cellular nucleasesy g
Insufficient membrane penetration
Insufficient bioavailability
Development of a suitable mode of application⇒ Development of a suitable mode of application(protection, penetration enhancement, targeting)
⇒
Structural optimization of oligonucleotides⇒
E. Urban 1313
Oligonucleotide Modifications
4'- position (O, S, C)
b difi tiX
BO
3'
base modification (artifical bases)
sugar modification
O
OZ
YP
3
5'
R1 2'- functionalization
backbone modificationO
R2
OY
B 1'- modification (α or β)
3' difi i
Y= O, SZ= S, CH3, alkyl,
R2 3'- modification3 O-alkyl or NH-alkyl
E. Urban 1414
Backbone Modified Oligonucleotides (First Generation)- Phosphorothioates
Ph h dithi t- Phosphorodithioates
- Methylphosphonatesy p p
- Phosphoric Acid Triesters
- Phosphoramidates
E. Urban 1515
Phosphorothioates
O Base-1O
O Base-1O
O
O
O
OBase-2
PO
S
HOO
Base-2P
OO
HO
OO
HO P
OBase-3O
HO
S
P
OBase-3O
HO
O
P
OO
PhosphorothioatesDNA
E. Urban 1616
Synthesis of Phosphorothioates - Sulfurization Reagents
Sulfurization TimeDescriptionReagent
N SS
S
N
15 minTetraethylthiuramdisulfide (= TETD Reagent)
S 0.5 M in anhydrous acetonitril
1 minBenzoyltetrasulfide
SS
O
SS
O0.4 M in THF or dichloromethane
y
S S 15 secTetrakis-(2-methylethyl)-thioperoxy-POO
PO
OS S
0.5 M in acetonitril
diphosphate
E. Urban 1717
Phosphorodithioates
O Base-1O
O Base-1O
O
O
O
OBase-2
PO
S
HSO
Base-2P
OO
HO
OO
HO P
OBase-3O
HS
S
P
OBase-3O
HO
O
P
OO
PhosphorodithioatesDNA
E. Urban 1818
Methylphosphonates
OO Base-1
OO Base-1
O
O
O
O
OBase-2
PO
HO
OBase-2
PO
O
HO
OO
HO P
OBase-3O
P
OBase-3O
HO
O
PHO
OO
MethylphosphonatesDNA
E. Urban 1919
Phosphoric Acid Triesters
O Base-1O
O Base-1O
O
O
O
OBase-2
PO
O
ROO
Base-2P
OO
HO
OO
HO P
OBase-3O
RO
O
P
OBase-3O
HO
O
P
OO
Phosphoric Acid TriestersDNA
E. Urban 2020
Phosphoramidates
OO Base-1
OO Base-1
O
O
O
O
OBase-2
PO
O
NO
Base-2P
OO
HO RH
OO
HO P
H
R
OBase-3O
N
O
P
OBase-3O
HO
O
PH
OO
PhosphoramidatesDNA
E. Urban 2121
Biophysical Characterisation of OligonucleotidesKonformation of the DNA-Backbone
S l Ch i i f A liStructural Characteristics of DNA-Helices
Circular Dichroism Spectra of DNA-Modificationsp
Temperature Dependence of Circular Dichroism Spectra
E. Urban 2222
Konformation of the DNA-Backbone
O O
α
--
P
OO O
PO O
O
γ
β
2´ 4´
5´
O
BH
O
O
O
B
ε
δ
1´
2
3´
4- OH
PO
O
HO
OH
H
ξ-
O
P
OOOH
H
A C3´ endo B: C2´-endo
O
A: C3 -endo B: C2 -endo
E. Urban 2323
Structural Characteristics of DNA-Helices
E. Urban 2424
Structural Characteristics of DNA-Helices
A DNA B DNA Z DNAA-DNA B-DNA Z-DNAsugar conformation C3'-endo C2'-endo C2'-endo (only dC)
i t ti i ht i ht l ftorientation right right leftdiameter (Å) 26 20 18base pairs per winding 11 10 3 – 10 6 12 (6 dimers)base pairs per winding 11 10,3 – 10,6 12 (6 dimers)rotation per nucleotide (Å) 32,7° 36° -60° (per dimer)distance per turn (Å) 28,2 33,7 45p ( )distance per base pair (Å) 2,56 3,37 3,62 – 3,72inclination of the base 20° -5,9° -7°major groove (Å; width; depth) 2,7; 13,5 11,7; 8,5 -minor groove (Å; width; depth) 11,0; 2,8 5,7; 7,5 2,7; 9,9
E. Urban 2525
Circular dichroism (CD)
Right-hand and left-hand polarised light runs through chiral media with differentRight hand and left hand polarised light runs through chiral media with different speed and is absorbed in a different degreeand is absorbed in a different degree.The absorption coefficients of right-hand polarised light (εR) and left-hand polarised light (ε ) are different! This effect is named circular dichroismus (CD)light (εL) are different! This effect is named circular dichroismus (CD).
Δε = εL - εRL R
positive CD bands: Δε > 0negati e CD bands: Δε < 0negative CD bands: Δε < 0
The difference of the absorption coefficients is determined by measuring a CD spectrum.
E. Urban 2626
a) UV spectrum measured with linear polarised light b) UV spectrum measured with left-hand polarised lightb) UV spectrum measured with left hand polarised light c) UV spectrum measured with right-hand polarised light
E. Urban 2727
Comparison of UV, ORD and CD Absorption Curves:
E. Urban 2828
Application of CD Spectroscopy:• for the deteremination of configuration of chiral natural products• for the conformation analysis of chiral compounds• for the anaysis of high molecular chiral natural products (proteins, nucleotides, carbohydrates) (p y )
• for the anaysis of interactions between drugs and receptor proteins and nucleotides
E. Urban 2929
CD Spectra of DNA-Modifications
E. Urban 3030
CD Spectra and Duplex Formation
3,0
2,0
0 0
1,0
mde
g)
-1,0
0,0
CD
(m
-2,0
-3,0
00 30 60 90 202 2 2 2 3
Wavelength (nm)
E. Urban 3131
Temperature Dependence of CD Spectra
3,0
2,0
0 0
1,0
mde
g)
-1,0
0,0
CD
(m
-2,0
-3,0
200
240
280
320
Wavelength
10° 20° 30° 40° 50° 60° 70° 80°
E. Urban 3232
Circular Dichroism Spectra and Transition Temperatures (Tm)
1,2
0 81,0,
ptio
n
0,60,8
Abs
orp
0 20,4
tive
A
0,00,2
Rel
at
-0,2
50 60 70 80
Temperature
E. Urban 3333
Fomivirsen (Isis 2922)
(P-thio)-G-C-G-T-T-T-G-C-T-C-T-T-C-T-T-C-T-T-G-C-G-desoxy-ribonucleic acid
Vitravene TM - solution for injection (6.6 mg Fomivirsen sodium / ml)j ( g )
Developer: Isis and CIBA-VisionProducer: Novartis Ophthalmics EuropeProducer: Novartis Ophthalmics EuropeApplication: CMV - Retinitis (Cytomegalovirus)Target: Major Immediate Early Region 2 (IE2) of the human CMVTarget: Major Immediate Early Region 2 (IE2) of the human CMVEC 50: 0,34 ±0,25 μM
for virus antigen production in human fibrinoblastsfor virus antigen production in human fibrinoblasts0,03 ±0,02 μM for virus antigen production in human retina pigment epithelg p p g p
E. Urban 3434
Dosage Dependence of Side Effects of Phosphorothioates
Dosage Side effects
100 mg / kg80 mg / kg
immune stimulation and hepatotoxicity in miceclotting and complement effects in monkeysg g g p yproximal tubular degradation
20 mg / kg10 mg / kg
lymphoid hyperplasia in miceinhibition of clotting timescomplement activationatrophic and regenerative changes in proximal tubules
3 mg / kg slight inhibition of clotting times
2.5 mg / kg highest dosage in humans
E. Urban 3535
Oblimersen (Augmerosen, Genasense, G3139)
(P-thio)-T-C-T-C-C-C-A-G-C-G-T-G-C-G-C-C-A-T-desoxy-ribonucleic acid
Target:Target: BCL 2 gene which codes an apoptosis blocking proteinTyp ofcancer
Trial location Status Partner drug
Target:Target: BCL-2 gene, which codes an apoptosis blocking protein
cancerMelanoma University of Vienna, Austria Phase III Dacarbazine
(DTIC)Prostate Memorial Sloan-Kettering Cancer
Center,NY, USAPhase I-II Taxol®,
Taxotere®Prostate University of Texas TX USA Phase II T t ®Prostate University of Texas, TX, USA Phase II Taxotere®Lung (Smallcell)
Ohio State University,OH, USA Phase I-II Taxotere®
Colorectal University of Texas, TX, USA Phase II Camptosar®Breast Georgetown University,
Washington DC USAPhase I-II Taxotere®
Washington, DC, USAAcuteLeucaemia
Ohio State University,OH, USA Phase I-II Fludara®
E. Urban 3636
2'-Modified Oligonucleotides (Second Generation)- 2'-O-Alkyl Oligonucleotides
2' O Eth l Oli l tid- 2'-O-Ethylenoxy Oligonucleotides
E. Urban 3737
2'-O-Alkyl Oligonucleotides
OBaseHO
OO Base-1
Base-2
HO O
BHO
O
PO
O
HOO
O
Base-2O
HO
BaseHO
O
OHO
OBase-3
PO
O
HO
HO O
OBaseHO
O
Base 4OO
HO
P
ORHO O
O
O
Base-4OHO
OR
OBaseHO
HO O
E. Urban 3838
Sequence of the Model Nucleotides
A A A A A A A A A A A AA* A A A A A A A A A A AA A A A A A A A A A A AA* A* A A A A A A A A A A
5' - A* A* A* A A A A A A A A A - 3'A* A* A* A* A A A A A A A AA* A* A* A* A A A A A A A AA* A* A* A* A* A A A A A A AA* A* A* A* A* A* A A A A A A
E. Urban 3939
Impact of the Alkyl Substituent on the Transition Temperatures
0.15M NaCl, 0.01M Tris-HCl, pH 7.0,
40Ethyl Modification
9µM
30
35y
e [°
C]
20
25
Butyl Modificationmpe
ratu
r
10
15
Butyl Modification
ion
Tem
0
5 Decyl Modification
Tran
sit
0 1 2 3 4 5 60
Number of the Modified Nucleotides
E. Urban 4040
2'-O-Ethylenoxy Oligonucleotides
OBaseHO
OO Base-1
HO OO
O
PO
O
O
Base-2O
BaseHOOHO
O
OBase-3
PO
O
HO
HO OO
O
OBaseHO
O
O P
O
HO OO
OO
O
O
Base-4OHO
OBaseHO
OHO O
OO
OO
O
E. Urban 4141
Impact of Oxygen Atoms on the Transition Temperatures
0.15M NaCl, 0.01M Tris-HCl, pH 7.0, 9µM
35
40
Butyl Modification
e [°
C]
25
30
mpe
ratu
r
10
15
20
Monoethylenglycol Modificationion
Tem
0
5
10
Tran
sit
0 1 2 3 4 5 60
Number of the Modified Nucleotides
E. Urban 4242
Impact of Oxygen Atoms on the Transition Temperatures
0.15M NaCl, 0.01M Tris-HCl, pH 7.0, 9µM
40
30
40 Triethylenglycol Modification[°
C]
20
30
D l M difi tierat
ure
10
Decyl Modification
n Te
mpe
0
Tran
sitio
0 1 2 3 4 5 6
T
Number of the Modified Nucleotides
E. Urban 4343
Impact of Oxygen Atoms on the Transition Temperatures
0.15M NaCl, 0.01M Tris-HCl, pH 7.0, 9µM
40
30
35
40Pentaethylenglycol Modification
[°C
]
20
25
30
pera
ture
15
20
Hexadecyl Modification
on T
emp
5
10
Tran
sitio
0 1 2 3 4 5 60T
Number of the Modified Nucleotides
E. Urban 4444
Zwitterionic oligonucleotides (Third Generation)- 2'-O-Aminohexyl Oligonucleotides
2' O A i h l l l Oli l id- 2'-O-Aminohexyl-L-lysyl Oligonucleotides
E. Urban 4545
2'-O-Aminohexyl Oligonucleotides
OO Base-1
OO Base-1
O
O
O
O ONH
OBase-2
PO
O
HOO
Base-2P
OO
HO
NH2
O
HO P
O
HO P
ONH2
OBase-3O
HO
O
P
OBase-3O
HO
O
P
O O ONH2
2'-O-Aminohexyl DerivativesDNA
E. Urban 4646
Sequence of the Model Nucleotides
T T T T T T T T T T T TU*T T T T T T T T T T TU T T T T T T T T T T TU*U*T T T T T T T T T T
5' - U*U*U* T T T T T T T T T - 3'U*U*U* U*T T T T T T T TU*U*U* U*T T T T T T T TU*U*U* U*U*T T T T T T TU*U*U* U*U*U* T T T T T T
E. Urban 4747
Impact of Amino Function on the Transition Temperatures
0.15M NaCl, 0.01M Tris-HCl, pH 7.0, 9µM
Aminohexyl Modification
35
40Ethyl Modification
yre
[°C
]
25
30
fmpe
ratu
r
15
20 Butyl Modification
on T
em
5
10Decyl Modification
Tran
siti
0 1 2 3 4 5 60T
Number of the Modified Nucleotides
E. Urban 4848
2'-O-Aminohexyl-L-lysyl Oligonucleotides
OO Base-1
OO Base-1
O
O
O
O ONH
O
NH2
OBase-2
PO
O
HOO
Base-2P
OO
HO
HNH2
O
O
B 3OHO P
O
B 3OHO P
O
O
NH2
NH2NH
O
O
Base-3OOO
O
Base-3OO
O
O
NHO
2' O A i h l L l l D i ti
O O
NH2
NH2NH
2'-O-Aminohexyl-L-lysyl DerivativeDNA
E. Urban 4949
Impact of Amino Function on the Transition Temperatures
-1
1
e 2/ °
C
-3
1iff
eren
ceo
A12
-T1
7
-5
ratu
re d
aris
on to
-9
-7
Tem
per
Com
pa
Aminohexyl modification
-110 1 2 3 4 5 6
in
Lysylaminohexyl Modification
Number of the Modified Nucleotides
E. Urban 5050
Circular Dichroism Spectra of Zwitterionic Oligonucleotides
2
Lysylaminohexyl modificationLysylaminohexyl modificationAminohexyl modificationAminohexyl modification
17
10 1
234
modification17
/ mde
g
0
6
510
mde
g
34
CD
-20
-1060
CD
/ 4562
modification
1
210 320250 300wave-length / nm-24
0
-10210 320250 300wave-length / nm
Experimental conditions: 9 µM, 0,15 M NaCl, 0,01 M TrisExperimental conditions: 9 µM, 0,15 M NaCl, 0,01 M Tris--HCl; pH 7,0; 0 HCl; pH 7,0; 0 °°CC
E. Urban 5151
Stability of Zwitterionic Oligonucleotides against Nuclease
200 µU 5'-Exonuclease (bovine spleen), EDTA; pH 6,0; 30 min
MgCl2, 90 °C; 10 min
RP-HPLC
(UAmin)3T9
RP HPLC
(UAmin)1T11Rate of
degradation(%)
(ULys)1T11
T12
( )T12 90
5'-(UAminohexyl)1T11-3' 15
5' (U ) T 3' 2
10 20 30 40 50 min
T12 5'-(UAminohexyl)3T9-3' 2
5'-U(Lysylaminohexyl)1T11-3' 0
E. Urban 5252
Application of Artificial Oligonucleotides
- Effectors: enhance antisense actionIntercalatorsIntercalatorsIon ChelatorsZwitterionic OligonucleotidesZwitterionic Oligonucleotides
- Modulators: modify biological effectsPh h l tiPhosphorylationInhibitors of Enzymatic Degradation
ll l li i f li l id- Detectors: allow localization of oligonucleotidesBiotinylationFluorescein-Linked OligonucleotidesRadioactive Oligonucleotides
E. Urban 5353
Oligonucleotide - Peptide - Chimäres(T-C-T-C-C-C-A-G-C-G-T-G-C-G-C-C-A-T)-Linker-(Peptide)
OO Base-1
O
PHO
OBase-2OO
O
Base-3OHO
O
P
O
HO NN
O
O
OH
R3
N
H OR1
N
O H OH
N
R2OH
E. Urban 5454
Oligonucleotide - Fluorescin - Chimäres(T-C-T-C-C-C-A-G-C-G-T-G-C-G-C-C-A-T)-Linker-(Aminofluorescin)
OO Base-1
O
PHO
OBase-2OO
O
Base-3OHO
O
PO OHHO
O
HO N
O
OO
OHO NN
O HH
O
E. Urban 5555
Synthesis of 2'-Amino-2'-desoxyuridine
O O O
O N
NH
O
O O N
N
O
(PhO)2CO/NaHCO3
85% O N
N
O
DMTCl
71%
OHHO
HOO
HO
HO O85%
HO
O ODMT71%
O NH
O
O
OO
N
N
ODMT
OO
NO
DMTCl3C-CN/TEA
77%
NaOH/EtOH/H2OO
O
N
NH
ODMT66%
HO
O O N
Cl ClCl
NH2HO
O
E. Urban 5656
Attachment of the Linker at the 2'-Amino Position
O O
O N
NH
ODMTO
O
N
NH
ODMT O
OO
HOO
NH2HO
ODMT
NH
HO
OO
O
O97%
O
O
O N
NH
O
OO OON
NH
O
O
NH
O
OO
DMT
OO
OO
O OO
70%
O
NH
HO
O
N ODMT
OO
H OHO O
H O
E. Urban 5757
Attachment of the Nucleotide to the Solid Phase
NH
O
ODMTO
N
NH
OO
O Ph HO
HN
OO
O+
NH
HO
O
O Ph HO O
On
6 mmol/g
T t G l S COOH
O
TentaGel S COOH
O N
NH
O
O
NH
O
DMTOO
O
O
O
O PhH2N
OO
n
+
237 l/
OH
O
O
237 mmol/g
TentaGel S NH2
E. Urban 5858
Oligonucleotide Synthesis
OO
O
BDMTO
Ph h idit
O NNH
OOP
O
ONC
Phosphoramidit1H-Tetrazol TETD
O
NH
O R
OO BDMTO
O NNH
O
R
HO
O NNH
OOP
O
ONC S
O
NH
O RNH
O R
O NNH
ODMTO
NH
O RCl3CCOOH Capping
E. Urban 5959
Oligonucleotide Synthesis - Yield of the Coupling Steps
100,00
90,00
80,00
in %
70,00Yie
ld i
60,00
50,001 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Coupling Step
E. Urban 6060(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
Cleavage of the Oligonucleotide
ODMTO Base-1
ODMTO Base-1
N
O
Base-xP
OO
X
O
Base-xP
OHO
XO
O
Base-xOXO
O
Base-xOXNH3 conc.N
16 16O
OBase-18O
O
X
P
O
OBase-18O
HO
X
P
O NO
O
PhH
OHO N
OH
O
HO
ON
HO
X = O,S
E. Urban 6161(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
Deprotection of the Linker by Selective DebenzylationDeprotection of the Linker by Selective Debenzylation
DMTO Base-1 DMTO Base-1O
O
NO
O
N
OBase-x
PO
O
X
N
Pd-PVP-Nanoparticles /1,4-Cyclohexadiene O
Base-xP
OO
X
N
O
Base 18OO
X
P
16O
Base 18OO
X
P
16
O
O
Base-18
NO
OX
OO
O
O
Base-18
NOH
OX
OO
O
OPhH
ON
H
OH
OH
ON
H
O
X = O,SO
E. Urban 6262(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
Synthesis of Oligonucleotide - Peptide - Chimäres
ODMTO Base-1DMTO Base-1
O
O
PHO
O
O
N
1) Pd / 1 4-Cyclohexadien
OBase-x
PO
HO
OO
Base-xP
OO
X
N16 16
1) Pd / 1,4-Cyclohexadien2) DIC / HOBt /Nε-TFA-L-Lys-Bzl (1-3x)3) NH4OH konz.
O
OBase-18O
HO
O
P
O
OBase-18O
O
X
P
16 16
NH2
O
HO NN
O
O
HH
O
O NO
O
PhH
O
O
OH
1-3O HO
ON
H
X = O,S
O 1 3
E. Urban 6363(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
Synthesis of the Lysyl Building Block
H2NOH
OpH 12.0 / 0°COF3C + OH
NH281%O
OHN
OH
O
F3CHN
O
O
F3CBzl-OHp-TSS
91%NH2O NH2O91%
E. Urban 6464
Synthesis of a Oligonucleotide Synthesis of a Oligonucleotide -- Fluorescin Fluorescin -- ChimärChimär
ODMTO Base-1DMTO Base-1
O
O
PHO
O
O
PO
N
1) Pd / 1 4 C l h di
O
O
Base-xP
OHO
OO
Base-xP
OO
X
N16
1) Pd / 1,4-Cyclohexadiene2) DIC / HOBt / 5-Aminofluorescein3) NH4OH conc.
16O
OBase-18O
HO
O
PO
O
OHHOO
OBase-18O
O
X
P
HO NN
O
O
HH
OO NO
O
O
PhH
O
H
ON
H
X = O,S
E. Urban 6565(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
Purification of the Artificial Oligonucleotides by HPLC
0,30
0,20
0,25
Vol
ts 0,15
0,05
0,10
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0,00
11,7
83
HPLC-Conditions: gradient elution, linear gradient 10-40% B in 0-30 min
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
A 0,1M triethylammonium acetate (TEAA) in WaterB 0,1M TEAA in Water / acetonitril (20:80)
E. Urban 6666(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
CD Spectra of the modified Oblimersen Derivatives
3,0
4,0
1,0
2,0
deg)
0,0
1,0
CD
(md
-2,0
-1,0
-3,0
200
230
260
290
320
Wavelength
Oblimersen free carboxy group aminofluorescein
E. Urban 6767(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
CD Spectra of the modified Oblimersen Derivatives
3,0
4,0
1 0
2,0
eg)
0,0
1,0
CD
(mde
-2,0
-1,0
-3,0
200
230
260
290
320
2 2 2 2 3
Wavelength
Oblimersen 1 lysine 2 lysine 3 lysine
E. Urban 6868(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
Transition Temperatures (Tmm)
78,780,0
69,568,4
67 4 67 6 68 270,0
75,0
pera
ture
65,5
,67,4 67,6 68,2
65,0
Tem
p
60,0
n ster
n oate
roup
cein
ine
ine
ine
Obl
imer
sen
osph
odie
s
Obl
imer
sen
osph
orth
io
carb
oxy
gr
noflu
ores
c
1 ly
s
2 ly
s
3 ly
s
OPh
o OPh
o
free
c
amin
E. Urban 6969(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
Cell Culture Test for Antisense OligonucleotidesCell Culture Test for Antisense Oligonucleotides
- Transfection reagent necessary for membrane penetration
- Measurement of the expression inhibition by determination of the target mRNA concentration (Northern Blot)the target mRNA concentration (Northern Blot)
- Measurement of the expression inhibition by determination of the target protein concentration (Western Blot)g p ( )
- Controll experiments with not pairing oligonucleotides(sense oligonucleotide, scrambled oligonucleotide, reverse
oligonucleotide)
E. Urban 7070
Cell Culture Test Cell Culture Test of the modified Oblimersen Derivatives
- human melanoma cell line 607B
t f ti ith Li f ti ( i t f k ti i li id )- transfection with Lipofectin (a mixture of kationic lipids)
- incubation for 48 hours
- protein extraction by lysis of the cells
- quantitative determination of BCL-2 by Western Blotq y
- standardisation with Actin
E. Urban 7171
Western Blot
80
607080
)
304050
CL-
2 (%
)
102030B
C
0
ence
scei
n
rsen
ysin
e
ysin
e
ysin
e
grou
p
rsed
seq
ue
min
oflu
ores
Obl
ime
1 l y
2 ly
3 ly
carb
oxy
g
reve
r
amfree
E. Urban 7272(T(T--CC--TT--CC--CC--CC--AA--GG--CC--GG--TT--GG--CC--GG--CC--CC--AA--U)U)--LinkerLinker--RR
The Research Team
P f D Ch i ti N
I tit t f Ph Ch i t I tit t f Ph Ch i t D t t f D t l
Prof. Dr. Christian Noe
Institute of Pharm. ChemistryUniversity of Frankfurt / Main
Atmaca-Abdel Aziz Serap
Institute of Pharm. ChemistryUniversity of Vienna
Bauchinger Arnulf
Department of Dermatology University of Vienna
Pehamberger HubertAtmaca-Abdel Aziz Serap Gilbert MatthiasKaufhold Lucius
Kock Michael
Bauchinger ArnulfSaadat KarminNovak Clemens
Winkler Johannes
Pehamberger Hubert
Department of Clinical PharmacologyUniversity of ViennaKock Michael
Werner DorisWinkler Johannes
Wurst Sonja
University of Vienna
Wacheck VolkerLosert Doris
E. Urban 7373