Furan Modified DNA Building Blocks: A Toolbox for ... · 1 Furan Modified DNA Building Blocks: A Toolbox for Crosslinking, Ligation and Secondary Structure Analysis Laboratory for

1

Furan Modified DNA Building Blocks: A Toolbox for Crosslinking, Ligation and Secondary Structure AnalysisLaboratory for Organic and Biomimetic Chemistry

[email protected]

GHENT UNIVERSITY

IschiaSeptember 28th 2010

© Annemieke Madder, 2010 All Rights Reserved

Conformationally defined multipodal peptides as functional miniproteins

Estrogen receptor mimics for solid phase extraction and preconcentration of endocrine

disrupting chemicalshv spacer

O

O

NHGN3

NH

NH

O

NHGivDdeNH

NH

NHBoc

O

S c a ffo ld

Transcription factor mimicsas DNA binding ligands

EurJOC 2009Tetr 2010

NH

NHO

O

NH2

3

7

12 24

G

N396

HNQ384

A385 C386

K387

G388 K389

I390Q391

A392

L393

E394C394

SS

O

O

O Epitope mimics asPeptide vaccins

EurJOC 2007, Arkivoc 2007 (Alain Krief issue)Drug Discovery Today: Technol. 2010

J. Pep. Sci. 2007Org. Biomol. Chem. ,2009


2

Design and synthesis of serine-protease mimics

Chymotrypsin active site

Oligopeptide ApproachAngew. Chem. 2000

J. Comb. Chem. 2002 EurJOC 2006, 2007

O

O

HNN

O

O

HNN

N

NO

H2N

O

HNN

N

NO

H2N

O

O

O

NN

NH2

O

NN

N

N NH2

NN

NH2

O

HN

OH

O

O

HN

HOO

NHN

NH

O

3'

5'

5'

3'

Oligonucleotide ApproachMolecules 2007, 2009


Novel techniques for crosslinking,labeling and conjugation of biomacromolecules

Oxidation

1. On-bead labeling

2 Cleavage

O OO

Dye

N

ChemComm 2009

II. Labeling of peptides: Imaging, conjugation

OO

O5'

3' 3'

5'

3'

5'

5'

3'

a) hybridization

b) oxidation

5'

3'

cross-link

formation

ChemComm 2005 Nucl. Nucl. Nucl. Acids 2007

Nucl Acids Res. 2009JACS 2010

I. Crosslinking of oligonucleotides: Antisense, antigene, crosslink repair,decoy DNA


3

Use of crosslinked ON: study of repair processes

DNA + chemotherapeutics

Variety of lesionsIncl. DNA interstrand crosslinks

Inhibition of strand separation and thusinhibition of transcription and replication

Block cell survival

Repair enzymes

Resistance to chemotherapeutics

Short crosslinked ON as model substratesfor repair studies

P.S. Miller et al., Frontiers in Bioscience 9, 2004, 421-437.© Annemieke Madder, 2010 All Rights Reserved

Use of crosslinking ON: antisense, antigene and decoy therapy


4

Crosslinked ON: assembly of nano-sized DNA structures

Rothemund et al., Nature, 2006Sugiyama et al., Chem Eur J, 2010

• Simple yet efficient and highly selective repetitive base pairing

� nucleic acid based nanometer sized structures

e.g. DNA origami

� need for ways to covalently strengthen the assembled

structures for further manipulation

� need for efficient processes to

to immobilize proteins,

receptors and

reporting devices© Annemieke Madder, 2010 All Rights Reserved

Possible problems:

mixtures of inter-and intrastrandLow selectivity

crosslinks limited to T

labour intensive

metabolic stability

stability and selectivity issues

Existing crosslink methods

• Cisplatin and transplatinBifunctional crosslinkers:

e.g. nitrogen mustards, psoralen, ...

• �-radiolysis of thymine• crosslinked nucleoside dimers

for DNA synthesisNoronha et al.

• Disulfide linkedVerdine et al., Sigurdsson et al.

• Alkylating oligonucleotides:halocarbonyl, aziridines

Tabone et al., Coleman et al. Matteucci et al.,


5

Oligonucleotide crosslinking

modified oligonucleotide

target sequence

interstrand cross-linked duplex

+ 1) hybridization

5'

5'3'

5'

5'

3'

3'

reactive functionality

3'

2) formation of a covalent bond


The concept of inducible reactivity

Crosslinking only in duplex context through in situ generation of a reactive moiety

Sasaki et al, JOC 2005Greenberg et al. JACS 2006

Rokita et al. PNAS 2003

OTBDMS

R

OAc KF

OH

R

OAc

O

R

OH

R

Nuc

Nuc:

OO

O

N

NH

O

PhSe

O OO

O

N

HN

O

OSePhO1. Ox (NaIO4)

2. Rearrange


6

Synthesis of Macrosphelides A and B

Inspiration from natural product synthesis

Kobayashi, Journal of Organic Chemistry 2001

Vassilikogiannakis, Acc. Chem Res.

2008

Synthesis of PolyoxygenatedNatural Products from furan


Furan: toxicity via reactive metabolite

* Liver and kidney toxicant

* Metabolic activation important

* Proposed bioactivation pathway:

Peterson et al, Chemical Research in Toxicology, 2002, 15, 373-379.

O

P 450OO Toxicity and

carcinogenicity

Reaction of 2-butene-1,4-dial

• with proteins elicits toxic response

� cell proliferation � tumor formation

• with DNA to form mutagenic adducts

O


7

A possible furan-oxidation crosslink strategy ?

• Crosslinking only in duplex context

Use of furan as latent functionality for reactive aldehyde

OO

O5'

3' 3'

5'

3'

5'

5'

3'

a) hybridization

b) oxidation

5'

3'

cross-link

formation


Furan oxidation by N-bromosuccinimide

OO

O

O

R

NBS

H2O

OR Br

a

b OR Br

RO

O

base

H

a

H2O

b


8

Oligonucleotide test reactions

3’GUGACTGTCCG5’

Unmodified oligo’s are stabletowards NBS treatment

U*

3’G

GACTGTCCG5’

ara

Furan modified oligo’s givea complex mixture upontreatment with NBS !

NH

O

ONO H

N

OH

HO

O

OU*ara


NH

O

ONO

HNOH

HO

OO

NH

O

ONO H

N

OH

HO

O

O

First generation building blocks

3’G

GACTGTCCG5’

5’CACTGACAGGC3’

U*

U* Compl

U*

S. Haliala, A. Madder et al, Chem. Comm 2005

U*Compl

U* XLCompl

C219H273N85O130P20Exact Mass: 6792,21 confirmed by ES-MSXL

First proof of principle of selective interstrand crosslinking

XL


9

Site of covalent bond formation ?

N N

O

N

NN

N

O

Minor groove

Major groove

H

NH

H

HN

H

1

23

4

5

67

89

123

4

5

6

O

OH

HO

O

OH

OH

G

C

N N

O

O

H

N

NN

N

HNH

Minor groove

Major groove

12

3

4 56

7

8

9

12

34

56

O

OH

HO

O

OH

OH

R

A

T

3’G

GACTGTCCG5’

5’CACTGACAGGC3’

U*


5’- CTG ACG G X*G TGC -3’3’- GAC TGC C C ACG -5’

5’- CTG ACG A X*A TGC -3’3’- GAC TGC T T ACG -5’

5’- CTG ACG C X*C TGC -3’3’- GAC TGC G G ACG -5’

5’- CTG ACG T X*T TGC -3’3’- GAC TGC A A ACG- 5’

ACTG ACTG

ACTG ACTG

X* = orO

O O

O

OO O

or O

O

O

O

Second generation building blocks


10

Comparison of selectivity for different furan NA’s

O

O O

Selective crosslinking to complementary A and C

High yields:Up to 70 % isolated crosslinked duplex !

Severe loss of duplex stability upon incorporation

of acyclic BB

CXC

*GTG

GXG

*CA

C GXG

*CG

CG

XG*C

CC G

XG*C

TC

TXT*

AA

A TXT*

AG

ATX

T*A

CA TX

T*A

CA

AXA

*TA

T AXA

*TG

TA

XA*T

CT A

XA*T

TT

CXC

*GA

G CXC

*GG

GC

XC*G

CG

GXG CXC

AXA TX

TModified sequence

XL

ssON


Crosslink site: enzymatic degradation studies with EXO III

Enzymatic digestion of 5’- CTG ACG GXG TGC -3’3’- GAC TGC CAC ACG-5’

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

100

200

300

400

500

600

700

DAD1 B, Sig=260,16 Ref=off (M:\07-06-07\KS000003.D)

4.2

91 4

.494

4.9

17 5

.024 5.4

89

9.6

48

11.

291

11.

667

11.

892

12.

350

12.

424

12.

874

MALDI-analysis: 3301.4

5’- CTG ACG GX G T -3’

A MW : 3300.6


11


Enzymatic digestion of

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

100

200

300

400

500

600

700


4.2

91 4

.494

4.9

17 5

.024 5.4

89

9.6

48

11.

291

11.

667

11.

892

12.

350

12.

424

12.

874

5’- CTG ACG GXG TGC -3’3’- GAC TGC CAC ACG-5’



A C ACG-5’MW : 4459.3



Enzymatic digestion of

5’- CTG ACG GXG TGC -3’3’- GAC TGC CCC ACG-5’

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

200

400

600

800


3.9

87 4

.310

4.5

06 4

.91 5

5.0

10 5

.464

9.5

90

11.

904

12.

417



C MW : 3294.6



C C ACG-5’MW : 4434.8


12

CXC

*GTG

GXG

*CA

C GXG

*CG

C

GXG

*CC

C GXG

*CTC

TXT*

AA

A TXT*

AG

A

TXT*

AC

A TXT*

AC

A

AXA

*TA

T AXA

*TG

T

AXA

*TC

T AXA

*TTT

CXC

*GA

G CXC

*GG

G

CXC

*GC

G

GXG

AXA TX

T

CXC

O

OO O

Duplex stability restoredSelective crosslinking to complementary A and C

butbrominated side product

yields 10-20% lower


Modified sequence

XL

ssON


CXC

*GTG

GXG

*CA

C

GXG

*CG

C

GXG

*CC

C

GXG

*CTC

CXC

*GA

G

CXC

*GG

G

CXC

*GC

G

GXG

CXCTX

T*A

AA

TXT*

AG

ATX

T*A

CA

TXT*

AC

A

AXA

*TA

T

AXA

*TG

T

AXA

*TC

T

AXA

*TTT

AXA TX

T

O

O

O

O


More pronounced C-selectivity

also here brominated side product

XL

ssON

In collaboration with M. Hocek, Prague Acad of Sciences© Annemieke Madder, 2010 All Rights Reserved

13

Stability studies of the modified duplexes: comparison

O

OO O

O

O OO

O

O

O

Reference(fully matched duplex)

melting temperatures AXA

0

10

20

30

40

50

60

A C T G

tem

pera

ture

Tm for AXA:TYT60

50

40

30

20

10

A C T G© Annemieke Madder, 2010 All Rights Reserved

Towards biological applications ?

* Restore duplex stability avoiding brominated side products

* Alternative oxidation methods

* RNA targets

reconsider the original cyclic building blocks

�Tm ~13°C versus ~5°C

NAR, 2004, Sontheimer: ribo oriented ureido-derivative

less destabilizing in RNA A duplexes

NH

O

ON

O

OH HN

HO

OO

NH

O

ON

O

OH HN

HO

NO

O

H


14

Synthesis of the ureido analogue

N

OO

a b,cO

OH

HO N

NH

O

O

NH2

O

OH

HO N

NH

O

O

NH PO

NCN

N

OO

O

O

DMTO N

NH

O

O

NHH

NCOO

a) DMF, 1h, 96%; b) DMTrCl, pyridine;17h, kt, 79% c) 2-cyanoethyl-N,N-diisopropylchlorophosphine, DIPEA, 2.5h, 0°C, 68%

H

Incorporation into oligonucleotides via automated DNA synthesis


0,0

10,0

20,0

30,0

40,0

50,0

60,0

A A A A

5'�CTG�ACG�GXG�TGC�3' 5'�CTG�ACG�TXT�TGC�3' 5'�CTG�ACG�CXC�TGC�3' 5'�CTG�ACGAXA�TGC�3'

X=T X=amide X=ureide X=KST X=SRF

Stability of different furan-modified oligonucleotides

HO

HOO O

HO

HO O

X = T X = X = X = X = NH

O

ON

O

OH HN

HO

OO

NH

O

ON

O

OH HN

HO

NO

O

H


15

For MOB2: higher stabilitylarger mismatch discrimination

� important for SNiP detection

Stability of amido versus ureido-furan oligonucleotides

O

O O

N

OO

O

O

O N

NH

O

O

NH H

OO

O

O

O N

NH

O

O

NH

KST

MOB 1

MOB 2

X KST MOB1 MOB2

AXA 27.4 27.1 27.0TCT

AXA 30.9 38.7 42.3TAT

Representative Tm’s


Cross-linking of amido-furan oligonucleotides

OO

O

O

O N

NH

O

O

NH

MOB 1

U* =

5’-CTG ACG T U*T TGC-3’3’-GAC TGC A C A ACG-5’

Compl.XLCompl.

Modif XL

2 cross-link products formed !

* Same mass (MALDI-TOF)

* Both products are crosslinked to C (enzymatic degradation)

4 eq NBS


16

Proposed cross-linking mechanism

N

N

N

OR

OOH

dehydration N

N

N

OR

OR R

- H2O

MW - 18

N

N

NH2

O

OO

R

N

N

HN

OR

OHO

R R

min18 18.5 19 19.5 20 20.5 21 21.5

mAU

1

2

3

4

5

6

DAD1 B, Sig=260,16 Ref=off (D:\DATA\09-02-07\MO000003.D)

19.

840

min18 18.5 19 19.5 20 20.5 21 21.5

mAU

0

50

100

150

200

DAD1 B, Sig=260,16 Ref=off (D:\DATA\09-02-05\MO000009.D)

19.

618

19.

988

Overnight incubation at 50°C

ESI-MS:- 18 (- H2O)


Furan metabolite adducts

N

N

NH2

O

O

O

R

N

N

N

O

R

OHOH

R = dCR = CH3

+

cis / trans

H

H

Cfr. ‘ Characterisation of nucleoside adducts of cis-2-butene-1,4-dial,a reactive metabolite of furan’

dC >> dA > dG although in general unusual for C to be the preferential target for adduction in DNA modification reactions

Peterson et al., Chem. Res. Toxicol. , 2002, 15, 373-379.Dedon et al., JACS, 2001, 123, 2664-2665.


17

Confirmation of the proposed cross-link structure:

* Cross-linking of individual nucleosides to the dinucleotide

* Complete enzymatic degradation of the cross-linked duplex

* Coinjection on RP-HPLC

Structural characterisation of crosslinked duplex

NH

O

ONO

HNOH

HO

O

O

N

NH2

ONO

OH

HO+


Structural characterisation of crosslinked duplex: starting from individual nucleosides

NH

O

ONO

HNAcO

AcO

O

O NHO

O

N

OHN

HO

OH

O

O

OH

HO

N

N

N

O

O

OH

NHO

O

N

OHN

HO

OH

O

O

OH

HO

N

N

N

O

O

N

NH2

ONO

OAc

AcO

a b

a) i. NBS, pyridine, THF/acetone/H2O 5/4/2, overnight, rt; ii. NH3/MeOH; overnight, rt, 80% (over 2 steps); b) 0.1 M HCl, 9 days, rt, quantitative

A1 + A2

Aar


18

Structural confirmation of crosslinked duplex

A2A1

Aar


Structural confirmation of crosslinked duplex: starting from crosslinked duplex

3'

3'

5'

5'

crosslinked duplex

5'-CTG ACG TU*T TGC-3' |

3'-GAC TGC AC A ACG-5'

snake venom phosphodiesterase

crosslinked dinucleoside

=

NHO

O

N

OHN

HO

OH

O

O

OH

HO

N

N

N

O

O

alkaline phosphatase

Bar


19

Structural confirmation of crosslinked duplex

Bar

Aar + Bar


Cross-link selectivity: amido-furan oligonucleotides

OO

O

O

O N

NH

O

O

NH

MOB 1

U* =Cross-linking to A and C

Isolated yields:25-53%

TAT

TGT

GG

G

GA

G

GTG

AU

*A

CU

*C

TTT

TCT

GC

G

Modified sequence

AC

AA

GA

CA

C

CG

C

TU*T

GU

*G

CC

C

CTC AA

A

ATA

Complementary sequence

XL

ssON


20

Cross-linking with ureido-furan oligonucleotides

5’-CTG ACG TU*T TGC-3’3’-GAC TGC AC A ACG-5’

N

OO

O

O

O N

NH

O

O

NH H

MOB 2

Compl. XLCompl.Modif

1 product formed !

U* =


N

OO

O

O

O N

NH

O

O

NH H

Cross-link selectivity: ureido-furan oligonucleotides

TU*T

:AA

A

TU*T

:AC

A

TU*T

:AG

A

TU*T

:ATA

TU*T

GU

*G:C

AC

GU

*G:C

CC

GU

*G:C

GC

GU

*G:C

TC

MOB 2Complete C-selectivity !

Isolated yields:16-34%U* =

AU

*A

CU

*C

AU

*A:T

AT

AU

*A:T

CT

AU

*A:T

TT

AU

*A:T

GT

CU

*C:G

GG

CU

*C:G

AG

CU

*C:G

CG

M. Op de Beeck, A. Madder et al, JACS, 2010, accepted © Annemieke Madder, 2010 All Rights Reserved

21

Furan-probes for Adenine detection

ONH

O

O6 O T

OAGCAGAGGTGTC'3

'5 d(TCGTCTCCACAGANACATACTCCATAA) 3'

A clearly distinguished versus C through formation of fluorescent derivative

R

O

N

N

N N

N

A. Kobori et al., BMCLett., 2009, 3567

Cfr. Work of Akio Kobori et al. , Kyoto Institute of Technology,Dept of Biomolecular Engineering

Diagnosis of A related DNA mutations

5’-furan conjugated probes directed against the point mutation site in the JAK2gene, associated with chronic myeloproliferative disease


Conclusions

Incorporation of a furan moiety as a masked reactive functionality

O

OO O

O

O

O

O

C-selective

O

O OOO

O

O

O N

NH

O

O

NH

N

OO

O

O

O N

NH

O

O

NH H

into oligonucleotides:

new, efficient and high yielding ON crosslink methodusing a proximity based reaction

modular strategy: allows for stability and selectivity tuning

High Yield

22

Thanks to

Kristof Stevens

Prof. Stefan Schürch, Bern

Prof. Michal Hocek, Prague

Prof. Floris Van Delft, Nijmegen

Dr. Sami Halila

Dr. Trinidad Velasco

Marieke Op De BeeckSara Figaroli

Annelies Deceuninck

FWO-Vlaanderen

Ugent - BOF

EC (HPRN-CT-2000-00014)

Thanks for your attention !

Documents

Furan Modified DNA Building Blocks: A Toolbox for ... · 1 Furan Modified DNA Building Blocks: A Toolbox for Crosslinking, Ligation and Secondary Structure Analysis Laboratory for