1924 ;st

80~

UNIVERSITA' DEGU STUDI DI MILANODipartimento di ChimicaOrganica e Industriale

Via 6o19i19, 20133 Milan (Italy)stefano. maiorana@unimi.it

Peptide Nucleic Acids:a Challengeat the Beginningof the

Post-Genome Era

Stefano Maiorana

IASOC 2004

1

Expressionof gene-stored information

GENE=a single unit of genetic information

DNATRANSCRIPTION

RNATRANSlATION

EI~ ~ PROT N

A single mistake in the sequence of nucleobases (SingleNucleotide Polymorphism, SNP) can determine severediseases and different response to drugs.

IASOC 2004

Pharmaceutical science + knowledgeof genes and proteins

ilpharmacogenomics

anti-geneoligonucleotide

anti-sense

oligonucleotide

IASOC 2004

1

DNAdetection is based on the fact that DNAexists as a duplexofcomplementarysequences. Thus, a single stranded DNAis able topick up its complementarytarget specifically from a mixture ofgenes. If a probe DNAis labelledappropriately, the duplexformedis detected easilyand with sensitivity.

~~

Ci>signal

~

DNA forgef

Stability and sequence specificity as well as binding affinity ofnaturaI DNAare often worst than desired. Therefore much efforthas been directed to the development of DNA analogues withimprovedproperties.

IASOC 2004

PEPTIDE NUCLEIC ACIDS (PNAs):a major breakthrough

I

°\çYBoO=P-O.. I~ .Jn

IHN

(NZB

're J n

-

DNA PNA:

highbio-stability,highaffinityandsequencespecificityfor DNA

P.E. Nielsen, Science, 1991, 254, 1497.

IASOC 2004

a

~ o~C~

." T-- T - "'H H

1 -~h-N 'N', ~éf H~ ~ ,H

/11 ~~:;' ~. I .HOOgst~en,' Wats~n-CriCk

l~UPL~XP~A/ ~_NAJ l!R~~LEXJPNA2/DN~J

IASOC 2004

C-term PNA- DNA

Antiparallel

Melting temperatures of duplexes formed by PNA andDNA 15-mer TGTACGTCACAACTA.

IASOC 2004

.3

DUPLEX Tm (antiparallel)/oC Tm (parallel)/°CPNA:DNA 69.5 56.1

PNA:RNA 72.3 51.2

DNA:DNA 53.3 -

DNA:RNA 50.6 -

PEPTIDE NUCLEIC ACIDS

Potential applications in:

~ pharmacogenomics

~ anti-gene and antisense drugs

~diagnostics

IASOC 2004

PEPTIDE NUCLEIC ACIDS

Drawbacks:

~ Low solubility in physiological medium~ Low lipophilicity

~ Difficult celi up-take~ Sensitivity to some hydrolases~ Lack of relevant analytical probes within their backbone

Dmodified PNA

IASOC 2004

4

Metal-PNA conjugateso

Me~NHllN~O

~OO M = Cr. W. Fe

H2N~NrOHLnM

oMe~NH

llN~O

~OOHN~N~OH

I

LnM

LnM = -ArCr(COh, Fischer-type carbene. Ferrocene

AIMS ~ 1. lipophilic PNAs

2. analytical probes

IASOC 2004

Our work

ArCr(CO)a '-M(CO)5R/

Maiorana, S., Ucandro E., Zinzalla G., Giannini C. Syn/ett, .2004, 1044.

Licandro E., Maiorana S., Baldoli C., Vandoni, B. , Salmain, M. J. Mo/. Cata/ysis, 2003, 204. 165.

Baldoli C., Maiorana, S., Licandro E., Zinzalla G., Perdicchia, D. Org. Lett. 2002, 4, 4341.

00[

(PNA monomer ]

> ~ln = 1, 2, 3

IASOC 2004

M = Cr, W

5

Ferrocene as the analytical probe

Ruthenium, osmium, iron, rhodium, and copper complexeshave so far been proposed for the electrochemical

detection of DNA

Among them, ferro ceneis the most convenient

~~

t@

~ very stable

~ undergoes reversible one-electronoxidation

~ exhibits characteristic UV-Visabsorptions that facilitate itsdetection e.g.during HPLCpurification

IASOC 2004

Gene sensor using ferrocenyl oligonucleotideT. Ihara et al.; Chem Commun.1997,1609

I -CL

~'

1i,w'

\{."

,', p",~,-,..~ '~

Target DNA bindstwo different probe DNAs.Probe 11abelled with the Fcunit and then probe 2 which is immobilizedon the electrode

IASOC 2004

(5

Mono-ferrocene-unit PNAmonomer

oMeyJlNH

lLN~o

polymer~o polymerD N * BI

Pg-HN~ Y'OMe

""

, ;o'A o~Fe

c:pIASOC 2004

chiral

free H bondsites in

the nucleobase

thymine as a base

The Mitsunobuonthe PNA monomer

oMe..,)LNH

QN~O

~oo-""'N~MeZ-HN

,'":: OH

o..-,OHFe

\Q

....PPh3. DEAD,THF, r.t., 72 h

IASOC 2004

O

Me..,)LNH

QN~O

~OO-""'N ~MeZ-HN

'"

,...: oO Fe

y= 28'Yo ~

7

The Mitsunobuon tyrosine and backbone

~o Q...,OH

~o H N~

O

Z-HN OMe Fe Z-HN OMe 2 OMec:P HCOO"NH4.,PeIIC

" "" .."I ~ PPh.. DEAD,THF I ~ MeDH, r.t" 26 h I ~

OH r.~,18h 0""0 0""""'0F Fe

81% é 75% \Q>

H O""""""N~MeZ-HN

"I ~ ""O

Fe75% \Q>

oZ.HN..)I.H

ZnCI.,NaCNBH3,o .C, MeDH.18 h

..

~~OH

PPh,. DEAD.THFr.~,4d

H O

Z-HN""""""N ~Me

,"~ OH

70%

O

H2Nr~Me

lGlOH

oZ.HN..)I.H

ZnCI., NaCNBH3,o .C. MeDH, 15 h

IASOC 2004

The fina Istep: Fc-Iabelled PNA monomer

..

oMe0NH

l!.N~O

~ooZ-HN""-N~Me

,"78% ~ O~

Fe~

H O

Z-HN""-NI'~Me

~O"OFe

~

aMe.,)tNH

~N~O"-faOH

ecc, DhBtOH, DMFr.t.,18h.

o

DhBtOH = ~~...OHlAN<>N

IASOC 2004

8

oMe~NH

QN~O

~OO"""""'N~MeZ-HN

....

I...: 0"'0~ Fe~ c:Ir

Cyclovoltammetryof ferrocene dervs0,30

0,25

0,20

0,15

"e 0,10

i 0,05::; 0,00

-0,05

-0,10

-0,15

-0,20

-0,25 -0,2 -0,15 -0,1 -0.05 o 0,05 0,1 0,15 0,2 0,25

E ~clFe+)/V

7,5-7.8*10-' M, " clec11'ode,CMf + 0,1 M TEAP,200 ",VI. SCGllratc,

.single and quite reversible monoelectronic wave.shift of ali potentials in the positive direction with respectto ferrocene methanol

D.

. increased stability to the oxidation

.the potential shift remains almost unchanged even increasingthe molecular complexity

IASOC 2004

Analysisof limitingdetection

!B

81 $,CtIoCN'I\W>O.11f ,//

). ~~../-----

//"

o/'111

Eli

111

111. . I li !Il .,. !Il

Q ;°HFe

C

"'N

~COOM.

>'," o~

F.2

Z"'N

~C~."I ~

Fe3 -

H o

z",o""or::-"0-0"'0

F.4 c;p

r~.::.JtNH'I!...,~ .:""" L_,o :c

,~

"'"

""T ~O o ON. i,

'

,

'

,iz.«N j

~. ': ~il; 5"",,~

CV obtained on GCelectrode (r = 2 mm), 200 mVS-lscan rate, 298.15 K

IASOC 2004

tJ

New multifunctional organometallic PNA monomer

oMe"""'NH

~"J::o

~O o c=)N

~e ~

Z-HN"'-""'" <o.; o cf ~I ""- .A.N~ .t:2.

t.f .../Te'--Q.<:iiI'

.a..

~'C2o'

H2N~ '~J~~Fe

~

IASOC 2004

HO

c::::> H2NtOHHO

Tris ferro cene derivativesH

fO OHH2N

HO

Cbz.clNaHC03

~H2<).AcOEt.r.t.,24h

I QO Fe

H2Nì .~)'0 FeO ~'-O

Fe~

PdIC

HCOO"NH4 +

Il!(

CH2Ci2. MeOH.

r.t.. 24 h

90%

It Patent N. MI2004AOO1427(2004)IASOC 2004

HO

Z-HNtOH93% HO

I

Q...,..°H.a,

CF3COOH,CH~12. toluene60 "C , 45h

I -OO Fe

Z-HN+o~O Fe~-Q

73% Fe~

10

Three different possibilities

oo Me~NHMe~NH , lI L. N~ON"-O l --. ~~~OOMo 't: 'FO

~O

Z~N- ~ ~ ,.N OMe"" Z~N

I ~ OH tyrosine PNA I ~ OHPNAbackbone, ..,~. .'. .~" 1 ..'"" .'.

o oH2N~Me Z-HN~H

,"",.-: OH

tyrosineMe ester

Z = benzyloxycarbonyl

~",N~~

~IASOC2004

Synthesis of tris-ferrocene tyrosine

o

Z-HN~~Me +

lGlOH

oOCOCI Z-HN~ç NE13,CH2CI2 OMe"" 4O'atO.C

,(JrN~',.-: .. ."" o ,

2.5hr.t. ,.-: oJlo ~N0290'0

CH3CN

~ cf 119h,70.C L

H,N)'o-Ro CIr'-9'"CIr

50'0

oZ_HNt:Me ~

o O c:;,

+ I ~ O~N+o~°\ Q

37'0 Ò

9-. ~F. O o o F.CIr Jl L CIr

~Of HN N)'O~

CIr o HO CIro-J ç.F. F... ..

IASOC 2004

11

Effect of adding more ferrocene groups in thePNAmonomer

111II1I11I.

o

---(lNHI!.N~O

Yo

Z~N"""""N-(l,:".'rGl-o"Q

Fe5 ~

o~NH

l!N.I:>O"f°o

""""'N~e !'"'"9Z-HN .. o q a

" O~+o~°'""111

7.5-7.8..10-4 M, GCelectrode, CHzClz + 0.1 M TBAP, 20 rnV/s sCGn roteo

With three ferrocene groups the current density ratio is 2.8:1.Current density + to number of Fc units.

IASOC2004

Analysis of limiting detection

rn ! 3.7x10"6M

~l

I

l, '

..- f\~.~I

I

DIJl

ì 6.2x10-7 M

~

o...,)lNH

l!.N~O~OoN

tò:MeZ-HN

""

l"" AQFe

~5

o,;tNHl!N~O

'-,:.00""'N~Me r-9

Z-HN .. o o Cf

I, oj+o~

0'-111

Voltammetric patterns obtained at 298 Kon GC disk electrode (diameter =3 mm) working with extremelydiluted solutions of PNA monomers 5 and 11 in ACN + 0.1TBAP medium. CV:0.2 V S-1scan rate, DPV:0.05 V

S-I, modulation time 2 ms, modulation amplitude 25 mV, step potenti al 5 mV, interval time 0.1 sIASOC 2004

12

Fischer-type carbene complexes

X-R

(OC)5M~:/ R'",<:>

Electrophilic center

M = Cr, Mo IW

X = NR", O IS

R, R' = alkyl, aryl etc..

Aminolysis reactionRIN-R'

(OC)sW=<CH3

amino carbene

=<OCH3 HNRR'

(OC)sW .

CH3 -CHpH

Tungsten a/koxy carbeneIASOC 2004

SPS of PNA decamer: GTAGATCACT

MBHA

HBTUlDiPEA .H o

Boc"N~OH(6eq.) H I)'

NH'2'.cI-l

H o

BOC N...)l~H~),HHN\

2'.cl-l

CF3COOe odeprotection H3R.~ .A

.. N-.TFAIm-cresol H ), H

96:6 HN

Base

l

'2' .ct-l

'--fO O couplingBoc.N"""N..)lOH HBTUIDiPEA

H (6eq.)

1.capping H ---2.depro~on oc""N"""""""'N~YS'"3.couphng B

~ n- O9times oBase

~~

BoC-N-GTAGATCAC'f-Ly--8H

!

1. deprotection (TFAIm-cresol)2. capping (Ac;zO/Py/NMP

1:26:26)

H3COCHN-GTAGATCAC'H.y--8cleavage ..

TFAlTFMSAIm-cresoUtioanisole6:2:1:1

O

H;>OOHN-G'AGA"",~~"NH2J~esin

H2~ = '" MBHA,'" N~

Me '" IASOC 2004

13

Aminolysis of Fiscner-type carbene

H O

~GTAGATCAC>-~NH'

H,c H ~NH2

OCH3

(OC)SW=( 20 eqCH3

H O

~GTAGATCAC>-~~NH2

H,C H ~NH

(CO)SW=(CH3

borate buffer pH 9

CH~N.~. 22°C. 1 h

IASOC 2004

Recent reviews

1. Uhlmann.E.; Peyman.A.; Breiphol. G.;WiII,D. W. PNA: Synthetic Polyamide NucleicAcids with Unusual Binding Properties. Angew. Chem.Int. Ed (1998). 37.2792-2823NielsenPE Peptidenucleicacids: on the road to newgene therapeutic drugs.Pharmacology&toxicology (2000 Jan), 86(1), 3-7.

RayA;NordenB Peptide nucleicacid (PNA):its medicaiand biotechnicalapplicationsand promise for the future. FASEB journal : offidal publication of the Federation ofAmerican Societies for Experimental Biology (2000 Jun), 14(9), 1041-60.

Ray. Arghya; Norden. Bengt. Peptide nucleic acid (PNA): its medicai and biotechnicalapplications and promise for the future. FASEB Journal (2000), 14(9). 1041-1060.

Ganesh, Krishna N.; Nielsen. Peter E.Peptide nucleic acids: analogs and derivatives.Current Organic Chemistry (2000), 4(9), 931-943.

Koppelhus,Uffe; Nielsen, Peter E. Cellular delivery of peptide nucleic acid (PNA).AdJtf1ncedDrug Delivery Reviews (2003), 55(2), 267-280.

Kaihatsu, Kunihiro;Janowski, Bethany A.; Corey, DavidR. Recognition of chromosomalDNA by PNAs. Chemistry &Biology (2004), 11(6). 749-758.

Nielsen. P.E.Peptide Nucleic Acids: Protocols and Applications. Second Edition.(2004)Horizon Bioscience. Wymondham,UK.

2.

3.

4.

5.

6.

7.

8.

IASOC 2004

14-