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