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Automated Solid Phase Synthesis of Platinated Oligonucleotides via Nucleoside Phosphonates.

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[2] J. L. Oudar. D S. Chcmla. J Cherii. Phvs. 1977, 66,2664.
[3] S. R. Marder. D. N.Beratan, L -T. Cheng. Sience 1991, 252. 103.
141 D. M. Burland. R. D. Miller. C. A. Walsh. Clien?.RPL,.1994, 94, 31
[5] See also G. Wittig. W. Herwig. Chmn Ber. 1955,88.962. Synthetic procedures
for 1-4. as well as X-ray structures of 1 and 4. will be published elsewhere.
[6] S. R. Marder. C. B Gorman. F Meyers. J. W Perry. G. Bourhill. JLL Bredas.
B. M. Pierce, Science 1994, 26s. 632.
[7] The NLO properties of some hetaines have been studied earlier: B. F. Levine.
C. G Bethea, E. Wasserman. L. Leenders. J (%ern. Pizj.7. 1978, 6N. 5042; A.
Dulic. C. Flvtzanis, 001. Cummim 1978. 25. 402; M. S. Palev. J. M. Harris. J.
0 r g . Cheni. 1991, 56. 568. However, the x polarization in the compounds
studied (pyridinium phenoxides, merocyanines) is mainly caused by mesomeric
rather than inductive effects as in 1-4. This simplified view disregards the fact
that thenitrogen and boron alkyl substituentsin 1-4also may interact with the
n svstem bv, hvoerconiueation.
[8] M. Stiihelin. D. M. Burland, J. E. Rice, Chrm. Ph.v.7. Lert. 1992, I9f. 245.
[9] a) L -T. Cheng, W Tam, S. H. Stevenson, G. R. Meredith. G. Rikken. s. R.
Marder. J PI7ys. Chem. 1991,95,10631; b) L:T. Cheng. W. Tam. S. R. Marder,
A. E. Stiegman. G. Rikken. C. W. Spangler. ibid. 1991. 95. 10643.
[lo] C. Reichardt, So/wnr.! ondSo1rent €/fects in Organic Chemisrrj..2nd ed.. VCH.
Weinheim, 1990, p. 289.
(111 The AM1 cdlcula~ionswere performed with the VAMP 5.03. program 1121. An
analogue of compound 4 was calculated: the n-butyl groups were replaced by
methyl groups. The excited states were calculated at the geometry of the ground
states with the PECI = 10 1131. The UV transitions and hyperpolarizdbilities
were computed with the SCRF method in order to model the influence of the
solvent [14]. The hyperpoldrizability 8, was calculated with the SOS method at
zero and at 1.16 eV excitation energy (1064 nm) [13]. 8, is the projection of the
fl tensor on the dipole moment vector. To compare the calculated p, values with
the experimental values. they must be divided by 3, Since the ~ 0 approxima.
tion refers to ,8 based on a perturbation theory. whereas p of the experimental
reference compound is differently defined [8,15].
[12] VAMP. Version 5.01. G. Rauhut, A. Alex. J. Chandrasekhar. T. Steinke, T.
Clark, Erlangen, 1993.
1131 T. Clark. J. Chandrasekhar. Isr. J. Chenz. 1993, 33, 435.
[I41 G. Rauhut, T. Clark. T. Steinke, J Am. Chem. Soc. 1993, fl5. 9174.
[15] A. Willetts. J. E. Rice. D. M. Burland, D. P. Shelton. J. Chern. P h w 1992, 97.
[16] While ground state dipole moments are usually well reproduced by the AM1
method, there is no information available on the reliability of excited state
dipole moments calculated with the PECI method; see J. J. P. Stewart in Reviews in Compirtational Chemistr?, Vol. I (Eds.: K. B. Lipkowitz. D. B. Boyd),
VCH, Weinheim, 1991, p. 45; N. Matsuzawa. D. A. Dixon, Phw. Chem.
1992, 96. 6232; J. 0. Morley, ihid. 1994, 98. 13182.
[17] a ) A . E. Stiegmdn, E. Graham. K. J. Perry, L. R. Khundkar. L.-T. Cheng, J. W.
Perry, J. Am. Cheni. Sot. 1991,113,7658;b) N. G. Bakhshiev, M. 1. Knyazhanskii. V. I. Minkin, 0. A. Osipov, G. V. Saidov. Russ. Chem. Res. 1969,38. 740.
measured in the HRS experiment is the averaged isotropic value it
1181 Although /l
corresponds to p, in dipolar molecules where the largest fl component lies
parallel to the CT direction; see G. J. T Heesink, A. G. T. Ruiter, N. F van
Hulst, B. Bolger, Phw. Rev. Lert. 1993, 71, 999; E. Hendrickx. C. Clays, A.
Persoons, C. Dehu, J. L Bredas. J Am. Chem. Soc. 1995, 117. 3547.
[19] A description of the specific experimental set-up used in this study and the data
analysis can be found in S. Stadler, F. Feiner, C. Brauchle, S. Brandl, R.
Gompper, Chem. Phm. Leu. 1995, 245, 292. For general introductions into
HRS measurements, see K. Clays. A. Persoons, Plzy.7. Re)! Lert. 1991,66.2980;
Rev. Sci. Instrum 1992. 63. 3285; K. Clays. A. Persoons. L. De Maeyer. A h .
Chem. PIzys. 1994. 85,455. The chromophores were checked for fluorescence
prior to HRS measurements. In addition, the spectral purity of the HRS signal
was investigated with a set of narrow bdndpass filters of varying transmission
peak wavelength (500,532, and 570 nm). Only at the wavelength of the second
harmonic (532 nm) could a signal be detected. Thus, we can exclude the possibility of broadband contributions such as bot-two-photon- or three-photon-induced fluorescence. For 3 we found an unusually strong bathochromic shift of
at higher number densities (15.71 x 10" ~ m - ~For
) . 4 we found a positive deviation from a linear correlation between the SHG signal and the num~
data have been omitted in the
ber density above about 2.5 x l O " ~ m - (these
data evaluation of p). Both effects might indicate aggregation at these very high
concentrations. I t is currently not known to which extent these aggregation
effects influence the HRS signal, for example, by correlated scattering.
_. ~-
Automated Solid Phase Synthesis of Platinated
Oligonucleotides via Nucleoside Phosphonates**
Jurgen Schliepe, Ulrich Berghoff, Bernd Lippert?
and Dieter Cech*
Platinum compounds, in particular, cis-diamminedichloroPlatinum(I1) (cisplatin, CDDP) and several of its derivatives, are
widely used nowadays in the treatment of a number of turnour
diseases." 1 The cancerostatic activity of these compounds is directly related to their reaction with cell DNA, and the formation
of covalent adducts.['' On the basis of numerous studies with
model nucleobases, nucleosides, nucleotides, and short DNA
fragments, we now believe that we understand the fundamental
aspects of these interaction^.^^] Till now, studies o f the platination Of oligonucleotides have been carried out exclusively with
s a m ~ l e s . [To
~ ] our knowledge. there has been no
- report to date of any platinated oligonucleotide that was obtained by direct inclusion of a platinated nucleotide building
in an automated DNA synthesis,though this route Offers
clear advantages with respect to possible synthetic variations.
We describe herein the automated synthesis of trans-(NH,),Pt"-modified oligonucleotides via a 3'-phosphitylated platinum
nucleoside synthon. Initial studies showed that it is necessary to
carry out the phosphitylation before the platination. For reasons of greater stability, we chose to use pho~phonates[~]
in this
procedure, rather than the more usual phosphoramidites.r61Till
now, we have concentrated on the reaction of the 5'dimethoxytritylated thymidine-3'-phosphonate 1 with trans[(NH,),ptCI,)] (TDDP) (Scheme 1 ) . Cornpound 1 was synthe0
Scheme 1. DMTr = dimethyoxytrityl; a)
TDDP, N,N-dimethylformamide (DMF).
+ 1 equiv
KOH, H,O; b)
+ 1 equiv
[*I Prof. Dr. D. Cech, Dip].-Chem. J. Schliepe
Fachbereich Chemie der Humboldt-Universitlt
Hessische Strasse 1-2, D-10099 Berlin (Germany)
Fax: Int. code +(30)2397 2479
Dip1.-Chem. U. Berghoff, Prof. B. Lippert
Fachbereich Chemie der Universitat Dortmund (Germany)
[**I This work was supported by the Deutsche Forschungsgemeinschaft
8 VCH Verlagsgeseilschaftm h H , D-6945l
Weinheim, 1996
B lS.0Oi 25p
Angew. Chem. in!. Ed. Engl. 1996. 35, No. 6
sized a s p r o iously r e p ~ r t e d , ~deprotonated
with KOHl8] to
give 2 , and \ubsequently treated with tvans-[(NH,),PtC12)] to
afford the Pt complex 3.1y1Complex 3 was characterized by
elemental analysis, as well as by 'H N M R , 195PtN M R , and IR
spectroscopy.""] The lg5Pt N M R signal of 3 (6 = - 2265,
[D,DMF]) lies in the expected region for a PtN,CI coordination
sphere." ' I and its chemical shift is also clearly distinct from that
of free /r.~i~~.v-[(NH,),PtCl~)]
and its D M F solvolysis products.
The thymine H 6 resonance signal of 3 is shifted upfield compared to that of 1. as expected, and is obscured by the resonance
signals of the dimethoxytrityl group.["] However, its existence
can be unambiguously proven by 'H,'H-COSY experiments
(coupling to the C(S)CH, of the thymine). The formulation of
3 as a KCI adduct is based on the analytical data["] and is
possibly due to the tendency of N3-platinated thymine or uracil
to take up further cations at 0 4 or 0 5 , respectively, as described
several times in the past."
The modified oligodeoxyribonucleotides 5'-d(CXCA) and 5'd(ATAGTAXACAGA). with X = truns[(NH,j,Pt"]-T were
synthesized by using 3 and an automated synthesizer (Gene
Assembler. Pharmacia)
The coupling of 3 to the unmodified
nucleotides was carried out following usual synthesis protocols.
To improve the yield, 3 and the following phosphonate in the
elongation cycle were added in two- to threefold excess. The
yields were estimated to be 98.3 '4 per coupling step and 85.2%
in overall yield (by measurement of DMTr release). Following
the final cycle. the oligonucleotides were cleaved from the carrier and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) .[' The yields of platinated product
were about 30%, (estimated from the HPLC signal intensities,
Fig. 1 ) based o n the yields obtained from similar syntheses with
hydrolysis with synthetically produced [(NH,),~(N3-T)Pt]+.l1
The trans-(NH,),Pt" structure is also retained after deprotection with NaOH. The platinated 4-mer gave manoisotope peaks
of m/z 1439.8 (z = 1) and m / z 720.9 ( z = 2) in the ion spray mass
spectra. This also corresponds to the pyridine-coordinated platinum complex (mi? 1440).
The synthesized platinated oligonucleotides were investigated
by gel electrophoresis before and after deplatination.[I8]As expected, the platinated oligonucleotides showed a shift in comparison with the unmodified oligonucleotides,l'yl which was no
longer observed after treatment with cyanide (Fig. 2). Enzy-
Fig. 2. A) Gel electrophoresis ofd(CXCA) before (lane 1) and after deplatination
(lane 2). and ofd(CTCA) (lane 3); B) gel electrophoresis ofd(ATAGTAXACAGA)
before (lane 1) and after deplatination (lane 2). and of d(ATAGTATACAGA) (lane
3) [18].
2 6 0 nm
matic hydrolysis under the usual conditionsfZo1
\howed the expected total composition in the HPL chromatogram of the
oligonucleotides (Fig 3) 12'] The peaks at 27.1 and 2 6 min are
2 8 0 nm
platinummodlf led
platinummodif red
Fig 1 HPL chroili,trogrdm\ 01 the oligonucleotidea d(CXCA) dnd d(ATAGTAXACAGA) ( X = Pt T) before purification [lS]
unmodified oligonucleotides. Although the yields for the individual coupling steps and the estimated overall yield was in the
region expected for standard oligonucleotide syntheses. the low
yields of isolated product point to numerous side reactions during cleavage and workup, which still need to be optimized. Deplatination of the sensitive oligonucleotides presumably plays a
large role here. The rr~,ls-(NH,)~TPt"(py)unit which is produced during the synthetic cycle remains unchanged during deprotection with NH3; substitution by NH,, as for C1-containing
compounds, does not occur.['61Matrix-assisted laser desorption
ionization mass spectrometry (MALDI-MS) revealed the
molecular ion at 3985.0, as calculated for the platinated
12-mer; 3984.7 for rrans-[(NH,j,Py{d(ATAGTATACAGAjN3-T(7))Pt]'. The non-occurrence of the triammine complex
was confirmed by a comparison of the retention times (HPLC)
of the platinum-modified nucleoside obtained after enzymatic
3 0 rnin
Fig. 3. Enzymatic hydrolysis of the modified 12-iner ( A ) and o f the unmodified
12-mer ( B ) with 0 1 u snake venom phosphodiesterase and Z u alk,iline phosphatase.
a s described in ref [20].
an indication of the successful incorporation of the platinated
thymidine. The latter peak can be assigned to the platinated
thymidine on the basis of its polarity. However, the platinummodified material appears initially as a peak at 27.2 min., which
is degraded with time and reappears as a peak at 2.6 min. Presumably the first peak can be assigned to a platinated dinucleotide containing the incorporated platinated thymidine, that is,
the modification evidently leads to a reduction in the enzymatic
1141 P. J. Garreg. I. Lindt, T. Regberg. J. Stawinski, R. Stromberg, Terrahedrun Lett.
hydrolysis rate. Such products are known for the enzymatic
1986. 27,4051 -4054.
hydrolysis of modified oligonucleotides.[221The presence of Pt
1151 Synthesis of the 12-mer: for the unmodified monomers nucleoside phosphoin the synthetic oligonucleotides was shown qualitatively with
nates from Millipore were used. deprotection with conc. NH, (7 h 50‘C);
SnCI, (thin layer chromatography) and e l e c t r o ~ h e m i c a l l y . ~ ~ ~ ~ purification with RP-HPLC (Nucleosil 100. ( 3 8 . 7 pm; 15 - 2 5 % MeOH gradient in0.1 M NH40Acover30min)and ionexchange-HPLC. Synthesisofthe
Quantitative Pt analysis by atomic absorption spectroscopy
4-mer: PAC-dA (Pharmacia) was used as carrier. for the unmodified dC iBu(AAS) yielded a value of 1.05 Pt per tetramer or dodecamer.
dC-H-phosphonate was synthesized (by standard procedures). deprotection
The medium-term aim of our studies is to synthesize platinatwith 0 . 1 7 ~NaOH for 1 - 2 days. purification analogous to that of the 12-mer.
ed oligonucleotides that are able to react with target sequences
but using a 10-20%, MeOH gradient in 0.1 M NH,OAc over 30 min.
Acru 1982. 66, 193-204.
[16] R Pfab. P. Jandik. B. Lippert. I ~ i o r g Chbn
in D N A or R N A via the metal ion. Such reactions cannot be
1171 Synthesis of trons-[(NH,),CI(N,-T)Pt] analogous [Y], conversion of
expected with the coordinately saturated platinum oligonucle[(NH3),(N3-T)Pt]+ by reaction with N H , (conc.) for 30min. a t 90’C and
otides described here. They could then lead to the development
removal of the excess NH, (conc.) in vacuo.
of useful chemical nucleic acid probes and/or antisense and
1181 Deplatinationin0.0S~TrisHCI,pH = 8.0 w i t h 0 . 3 ~ N a C N a t S O T f o r 2 4h.
gel electrophoresis in 40% acrylamide/bisacrylamide + 7~ urea.
Received: August 18. 1995
Revised version: November 27. 1995 [Z8322IE]
German version: Angeir. Cliiwi. 1996, 108. 705 707
Keywords: cytostatics - D N A synthesis * oligonuleotides . platinum compounds
1191 Synthesis of the unmodified standard nucleotides with the Gene Assembler
(Pharmacia), using the amidite method with the corresponding standard
reagents from Pharmacia. deblocking with conc. ammonia (6 h. 50°C). desalt~ ~ Sephadex G-10. and of the 12-mer with Sephadex G-25
ing of the 4 - m with
[20] S. Schmidt, C.-D Pein, H.-J. Fritz, D. Cech. Nucleic A d s Res. 1992. 20,
2421 -2426.
[21] The number of bases of the platinated 12-mer compared as integration areas
with those of the unmodified oligonucleotides (calculated, theoretical) A (6.0,
6);C(l.l. l);G(2.1.2);T2.2,2).
1221 A. Eastman. M. M. Jennerwein, D. L. Nagel. Chem. Bid. Interact. 1988, 67,
I1 --80.
[23] E. Merck. Anfurheraugentien f i r Diinnschrcht- unil Pupier-Cliromatographie,
1980, p. 109: J. Wang. S. Zadeii, M. S. Lm, J Eli.ctrounal. Chem.
1987,237. 281 -287.
[24] 0 .Krizanovic. M. Sabat. R. Beyerle-Pfnur. B. Lippert. J. Am. Chem. Soc. 1993,
115. 5538- 5548.
M. E. Heim in Metu/Comp/ese,sin Cancer Cheniotherap~~(Ed.:
B. K. Keppler).
VCH Weinheim. 1993, pp. 9ff.
S. E. Sherman. S. J. Lippard, Clieni. Rev. 1987, X7(5). 1153-1181: W. 1.
Sundquist, S. J. Lippard. Comd. Chem. Rev. 1990, IUO. 293- 322; E. Holler in
Metal in Cancer Chemotherupj (Ed.: B. K . Keppler). VCH. Weinheim. 1993, pp. 37 ff; J. Reedijk, A. M. J. Fichtinger-Schepman. A. T. van Oosterom, P. van de Putte, Struct. Bondmg (Berlin)1987. 67. 53-89.
B. Lippert. Prog. Inorg. Chem. 1989. 37, 1-97; S . Yao. J. P. Plastaras, L. G.
Marzilli. Inorg. Chem. 1994, 33. 6061 -6077; G. Admiraal. M . Alink. C. Altona, F. J. Dijt. C. J. van Garderen. R. A. G. deGraaff, J. Reedijk. J. Am. Chein.
Soc. 1992. 114. 930- 938.
C. A. Lepre, K. G. Strothkamp. S. J Lippard, Biochemistr?. 1987. 26. 5651 5657. L. J. Naser, A. L. Pinot, S. J Lippard, J. M. Essigmann. ihid. 1988. 27.
4357-4367; C. A. Lepre, L. Chassot. C. E. Costello. S. J. Lippard. ihid. 1990,
2Y, 81 1-823; K. M. Comess, C. E. Costello, S. J. Lippard, ;hid 1990.29.21022110. D. P. Bancroft. C. A. Lepre, S. J. Lippard. J. h i . Chmi. So<..1990, 112,
6860-6871 ;V. Brabec, M. Sip, M. Leng. Biochemi.\tr?. 1993.32. 11676- 11681.
V. Brabec. M. Leng, Proc. Nut/. Acud Scf. USA 1993. 90, 5345-5349: J M.
Malinge, C. Perez. M. Leng, Nucleir’ Acfds Res. 1994, 22. 3834-3839
Tiziana Benincori, Elisabetta Brenna,
B. C. Froehler, M. D. Matteucci. Teiruhedron Lett. 1986. 27, 469-472; B. C.
Franco Sannicolo,* Licia Trimarco, Gianni Zotti,
Froehler. P. G. Ng. M. D. Matteucci. N i d e i c Acids Res. 1986. 14, 5399-5407,
Piero Sozzani
P. J Garreg. T Regberg. J. Stawinski. R. Stroemberg. Chem. Scr. 1986. 26,
M. J. Gait in An Inlroduction to Modern Methods of DNA S~nthcsi.\(Ed.: M. J.
A great deal of effort is currently being directed towards the
Gait). IRL Press, Oxford, 1984, pp. 1-22.
and realization of functionalized “intelligent” materiR. A. Jones in Preparation of Protected Drsor?riohnucleosid~s (Ed.: M. J.
als : [ I 1 the synthesis of polymers containing fullerene moieties in
Gait), IRL Press, Oxford, L984. p. 23-34; H Takaku. S. Yamakage. 0.Sakateither the main or side chains‘2.31and which show peculiar
sume. M. Ohtsuki. Client. h i t 1988. 10, 1675-1678.
Synthesis in analogy to that of thymine: C. J. L. Lock. P. Pilon, B. Lippert.
electrical‘41 and e l e c t r ~ c h e m i c a l ‘properties
is a challenging
Acta CrrstuNugr. Sect. B 1979.35.2533 2537: the Na salt analogous to 2 has
first conjugated
in the meantime also been synthesized and converted to 3 .
conducting “charm bracelet” polymer,161consisting of fullerene
Method: Compound 2 (206 mg. 0 3 mmol) was made anhydrous by repeated
moieties linked to a highly ordered polythiophene backbone.
distillation in vacua with anhydrous pyridine. and was then treated with ITUIIS[(NH,jZPtCI,] (90 mg, 0.3 mmol) in D M F (1.5 mL) ( 5 h SO C, 3 d 22 C ) .
One of the potential interests in associating a p-type conjugated
After removal of the D M F in vacuo, MeOH (6 mL) was added. filtered after
polythiophene and an n-type fullerene lies in the possibility of
24 h. and the filtrate reduced to dryness. After reprecipitation in acetone. the
transfer between these two moieties, as recently shown
product 3 (255 mg, 86 %) was used without further purification
by Heeger and Wudl et al., who, however, prepared and studied
Satisfactory elemental analysis for C. H. N. C1: Proof of Na and K by scannine
electron microscopy (SEM); IR (KBr): v(Pt-Cl) 330cm-’: *‘H N M R
heterogeneous blends of C,, and poly(3-alkylthiophenes)
([D,IDMFj: 6 ( 3 ) = 1.47 (s, 3 H. 5-CH,). 2.28-2.41 (m. 2H. H-7.H-2”). 3.26onIy.[’l
1 H . H-49, 4.95 (m, 1 H, H-3’). 6.41 (m. 1 H. H-1’). 6.79 (d.
A3’P,’H) = 577 Hz. 1 H.H-P).6.93-7.53(m, 13H. DMTr).7,50accordingto
[*] Prof. F. Sannicolo. Dr. T. Benincori. Dr. E. Brenna, Dr. L. Trimarco,
‘H-‘H-COSY (s. 1 H, H-6); ly5Pt NMR ([D,]DMF): 6 = - 2265 relative to
Prof. P. Sozzani
[PtCl,]’~. The N M R measurements were carried out on Bruker-AC 200; -AM
Dipartimento di Chimica Organica e Industriale
300- as well as -DRX-400-approaches.
Centro C N R Sintesi e Stereochimica Speciali Sistemi Organici
1111 T. G. Appleton, A. J. Bailey, K. J. Barnham. J. R. Hall. Inorg. Chml 1992. 31.
Universitli degli Studi di Milano
3077-3082 and references therein.
Via Golgi 19, 1-20133 Milano (Italy)
1121 ‘H NMR([D,]DMF):d:(l) =1.46(s.3H,5-CH3),2.44-2.47(m.2H.H-2’,H- Fax: Int. code +(Z) 2364369
Z”), 3.23-3.46 (m, 2H. H-S’,H-S‘‘), 3.80 (s, 6 H . CH,O), 4.21 (m. 1 H, H-4’).
e-mail: staclaus(a‘
4.99 (m. 1 H, H-3‘). 6.36 (m, 1 H. H-1’), 6.78 (d. J(”P,’H) = 582 Hz. l H ,
Dr. G. Zotti
H-P). 6.94-7.52 (m, 13H. DMTr). 7.65 (s, 1 H, H-6). 11.42 (s, 1 H. 3-NH).
lstituto di Pokarografiaed Elettrochimicd Preparatrvadel CNR. Padova (Italy)
114 0.Renn. 8. Lippert. 1. Mutikainen, Inorg. Chim. Acta 1994,21#, 117 -120 and
(**I This work was supported by CNR - Progetto Strategic0 Materiali Innovativi.
references therein.
The First “Charm Bracelet” Conjugated
Polymer: An Electroconducting Polythiophene
with CovaIentiy Bound Fullerene Moieties**
VCH Ver/ag.sgesellschuft ntbH, 0.69451 Weinheim,1996
3 U.OO+ .2SlO
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