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Furan Modified DNA Building Blocks: A Toolbox for Crosslinking, Ligation and Secondary Structure AnalysisLaboratory for Organic and Biomimetic Chemistry
annemieke.madder@ugent.be
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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.,
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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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
© Annemieke Madder, 2010 All Rights Reserved
Furan oxidation by N-bromosuccinimide
OO
O
O
R
NBS
H2O
OR Br
a
b OR Br
RO
O
base
H
a
H2O
b
© Annemieke Madder, 2010 All Rights Reserved
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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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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*
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
11
Crosslink site: enzymatic degradation studies with EXO III
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
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
5’- CTG ACG GXG TGC -3’3’- GAC TGC CAC ACG-5’
MALDI-analysis: 4458.8
5’- CTG ACG GX G T -3’
A C ACG-5’MW : 4459.3
© Annemieke Madder, 2010 All Rights Reserved
Crosslink site: enzymatic degradation studies with EXO III
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
DAD1 B, Sig=260,16 Ref=off (M:\07-06-07\KS000006.D)
3.9
87 4
.310
4.5
06 4
.91 5
5.0
10 5
.464
9.5
90
11.
904
12.
417
MALDI-analysis: 3294.3
5’- CTG ACG GX G T -3’
C MW : 3294.6
MALDI-analysis: 4434.6
5’- CTG ACG GX G T -3’
C C ACG-5’MW : 4434.8
© Annemieke Madder, 2010 All Rights Reserved
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
Comparison of selectivity for different furan NA’s
Modified sequence
XL
ssON
© Annemieke Madder, 2010 All Rights Reserved
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
Comparison of selectivity for different furan NA’s
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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)
© Annemieke Madder, 2010 All Rights Reserved
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.
© Annemieke Madder, 2010 All Rights Reserved
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+
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
18
Structural confirmation of crosslinked duplex
A2A1
Aar
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
19
Structural confirmation of crosslinked duplex
Bar
Aar + Bar
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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* =
© Annemieke Madder, 2010 All Rights Reserved
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
© Annemieke Madder, 2010 All Rights Reserved
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 !