Selection of Peptide-bond forming fDNAzyme:

Strategy and Setup-Synthesis

Goran Rasched, 8.Nov.2001

 Overview

• Introduction
• Selection Principle for Functional DNA
• Preparation for the Selection-Setup
• Synthesis Targets
• Synthesis Routes
• Some Thoughts on the Active Sites of Proteolytic Enzymes
 Selection Cycle

Random fDNA-pool

fDNA-pool Selection-Process

dsDNA-Hybrid separation Selected fDNA

by affinity-chromatography
Amplification (PCR)

fDNA/DNA-Hybrid Regular DNA

Primer extention
with modified dNTP`s fDNA: DNA consisting
of modified dNTP‘s
 Selection Cycle (Detail)

5´ 3´ Substrate A linked fDNA-Pool

A

BA Reaction with biotin-linked B

substrate B

discarded fDNA Selection on streptavidin-column

(no catalytic properties)

Selected fDNA Amplification

 Selection Scheme II
5´ 3´ Discarded fDNA
A
A
Desired reaction B

BA Selection by affinity-
chromatography
A
h·

BA

Photocleavage

B A

A
5´ 3´
Amplification (PCR)
Primer extention with natural dNTP´s
with modified dNTP`s

Regular DNA
Mod. DNA (fDNA)
 Desired Reaction:
NH2

B N N A
O N N
O -O P O O
O-
HN NH
O O OH
H H O
H
N
S N R
H
O
O O
O P O 5´ fDNA 3´
NH2 H N
H O-
N O
O NO2

HN NH
H H O R O NO2
H H
N N
S N N O
H H H O-
O O N
O P O 5´ fDNA 3´
O O

BA
 Desired Reaction:
NH2
N N

O N N
O -O P O O
O-
HN NH
O O OH
H H O
H
N
S N R
H
O
O O
O P O 5´ fDNA 3´
NH2 H N
H O-
N O
O NO2

HN NH
H H O R O NO2
H H
N N
S N N O
H H H O-
O O N
O P O 5´ fDNA 3´
O O
 Selection Scheme II
5´ 3´ Discarded fDNA
A
A
Desired reaction B

BA Selection by affinity-
chromatography
A
h·

BA

Photocleavage

B A

A
5´ 3´
Amplification (PCR)
Primer extention with natural dNTP´s
with modified dNTP`s

Regular DNA
Mod. DNA (fDNA)
 Photocleavage:
O
h·
HN NH
H H O R O NO2
H H
N N
S N N O
H H H O-
O O N
O P O 5´ fDNA 3´
O O

HN NH
H H O R O
H H
N N
S N N OH
H H
O O

NO2

O H O-
N
O P O 5´ fDNA 3´
O O
 Selection Scheme II
5´ 3´ Discarded fDNA
A
A
Desired reaction B

BA Selection by affinity-
chromatography
A
h·

BA

Photocleavage

B A

A
5´ 3´
Amplification (PCR)
Primer extention with natural dNTP´s
with modified dNTP`s

Regular DNA
Mod. DNA (fDNA)
 Synthesis of the 5´-Substrate-modified fDNA-Pool
N(iPr) 2
A P
O(CH 2)2CN
DNA-Synthesizer

3´ 5´

A
5´ 3´
5´-Substrate modified primer Primer extention with modified dNTP´s

Seperation and purification

A 3´
5´
fDNA-Pool
 Solid-Phase Synthesis of the 5´-Substrate modified Primer

N(iPr)2
A P
A O(CH2)2CN

Last step of
The synthesis
 Target Molecules I
Substrate A:

O
O N(iPr)2
O N P
H
H2 N O OEtCN
N 5
H
NO2

Photocleaver

Substrate B:
NH2
N
N

O N N
O -O P O O
O-
HN NH
O O OH
H H O
H
N
S N R
H
O
 Substrate-B
Substrate B R=
NH2
O O
N N H2N CHC OH H2N CHC OH
CH2 CH2
O N N CH2
O -O P O O C O
O- OH
HN NH O OH Phe
O O Glu
H H H
N O O
S N R
H H2N CHC OH H2N CHC OH
O
CH2 CH2
CH2 CH2
S CH2
CH3 CH2
NH2
Met Lys
 Target Molecules II

Reference-Compound for analytical comparison by HPLC and MS

HN NH
H H O R O
H H
N N
S N OH
H n
O O
 Substrate-A Synthesis-Route
O
BocHN 1. EDC, NHS O O
OH OH
2.H2N 5 BocHN OH OH
N 5
H Br
+
NO2

O 1. EDC, NHS O
Na2CO3, OH
O OH 2.H2N OH
Aceton/H2O O N
RF BocHN O H
N 5 BocHN O
H N 5
NO2 H
NO2

O OEtCN
NCEtO O P
O N N(i-Pr)2
P Cl H
(i-Pr)2N BocHN O
N 5
DIPEA H
NO2
CH2Cl2

Oligosynthese
 Substrate-B Synthesis-Route b

O
O
HN NH O H2N
OH
O Route1 H H
O N ~40%
HN NH S
DCC/NHS
H H DMF O O
O
OH
S HN NH
O Route2 H H O
O H
O N
S OH
TSTU/DIPEA HN NH O H2N
DMF/Dioxane/ OH O
H H
O N 85%
S
O O

DCC: DIPEA: N

N C N

BF4
NHS: O
TSTU: O
N
HON
O N
N
O O
 Substrate-B Synthesis-Route c
O O

HN NH RT, 24h HN NH
DMSO/DMF O
H H O H H H O
H
N 1.) NHS/DMAP N
S OH S O N
2.) DCC
O O
85% O
O
O
HN NH
O O
H H H O RT, 48h HN NH
N H2N H H O R
O N H
S OH DMF N OH
O R S N
O H
O O
90%
NH2
N N

O N N
O -O P O O
O-
1. CDI/DMF, 15min O
HN NH O OH
O
2. AMP/Formamide H H O N N
H N N
24h, RT N
S N R
H
O CDI: 1,1`-Carbodiimidazol
60%
Some Thoughts on the Active Sites
of Proteolytic Enzymes
? ?
O NH2

HN N

H 2N N N N N
O O

-O P O -O P O O
O
O O

OH OH

mod. A
mod. G

NH2 ? O ?
N HN

O N O N
O O
-O P O O -O P O O
O O
OH OH
mod. C
mod. T
 Identifing Catalytic Aminoacid Residues in
Proteolytic Enzymes

• Frequently occuring aminoacids : His, Arg, Ser, Asp, Cys, Tyr

• Large aliphatic/aromatic aminoacid side-chains form apolar

niches for hydrophobe peptide regions of the digestion substrate

• Michaelis complex consisting of Asp, His, Ser, forms a catalytic

centre
 Literature:

Articles on Ribozyme-Selection:
B. Zhang, T. R. Cech, Nature, 1997, Vol 390, No 6
A. Jenne, M. Famulok, Chemistry&Biology, 1998, Vol 5, No 1
G. Sengele, A. Eisenführ, M. Famulok, Chemistry&Biology, 2001, Vol 8, No 5
Frauendorf, A. Jaeschke, Angew. Chem., 1998, Vol 110, No 10
G. Sengele, A. Jenne, M. Famulok, Bioorg. & Med. Chem., 2000, Vol 8

Synthesis :
W. Bannwarth, R. Knorr, Tetrahedron Letters, 1991, Vol 32, No 9, pp 1157-1160
B. Cosimelli et al., Tetrahedron, 1996, pp 11281-11290
B. Zhang, T. R. Cech, Chemistry&Biology, 1998, Vol 5, No 10
 O
N
NH

O O N N NH2
O P O
O N O
H O-
BocHN O
N 5 OH OH
H
NO2

Initiatornucleotide

O O O

N N N
NH NH O NH O
1. TMSCl, Pyridine MeO OMe
N N NH 2 2. Isobuturylchlorid N N N N N N
H HO H
HO 3. NH3 HO TSOH
O O O
DMF
OH OH OH OH O O
 Substrate-A Synthesis-Route (Initiator-Nucleotide) a

NCEtO O
O P Cl O N(i-Pr)2
O N P
OH (i-Pr)2N H
O N BocHN O O
H DIPEA N
BocHN O H 5 CN
N 5 NO2
H CH 2Cl2
NO2

O O O

N N N
NH NH O NH O
1. TMSCl, Pyridine MeO OMe
N N NH 2 2. Isobuturylchlorid N N N N N N
H HO H
HO 3. NH3 HO TSOH
O O O
DMF
OH OH OH OH O O
 Substrate-A Synthesis-Route (Initiator-Nucleotide) b

O
O
N
O N(i-Pr)2 NH O
O N P
H N
BocHN O O N N
N 5 HO H Iod
H Tetrazol
NO2 O
CN + ACN H2O,
O O Pyridin,
THF

O
N
NH

O O N N NH 2
O P O
O N O
H O-
BocHN O
N 5 OH OH
H
NO2

Initiator-Nucleotide
 Building the 5´-Substrate-modified fDNA-Pool

N(iPr) 2 Synthesis of a substrate linked amidite

A P for use in the DNA-Synthesizer
O(CH 2)2CN

A Building the substrate A linked primer on a DNA-synthesizer

A Primerextention with modified dNTP´s

A 5´-Substrate modified fDNA-Pool

 Work done so far:
O O O O
H2N
OH
HN NH HN NH O in
RT, 20h HN NH
H H H H CH2Cl2
H H O
OH DCC/NHS O N H
S S RT, 3h N
DMF OH
O O S
O
244,31 g/mol O
357,47 g/mol

80%
 Amplification and primer extention

5´ 3´ 5´ 3´ 5´ 3´
5´ 3´
5´
3´
5´ 3´

3´
5´ 3´ A

5´ 3´

3´ 5´
5´ 3´ A
5´ 3´
5´ 3´