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8: 31P{1H} NMR (CDCl3): d 17.0; 13C{1H} NMR (CDCl3): d 147.8 (d,
A Convergent Strategy for the Modification of
Peptide Nucleic Acids: Novel Mismatch-
Specific PNA-Hybridization Probes**
1J(P,C) 113.2 Hz, P CH N); 1H NMR (CDCl3): d 7.23 (d, 1H,
2
J(P,H) 40.0 Hz, CH N).
Received: March 10, 1999 [Z13141IE]
German version: Angew. Chem. 1999, 111, 2338 ± 2340
Oliver Seitz,* Frank Bergmann, and Dieter Heindl
Keywords: aldehydes ´ hydrazones ´ phosphorus ´ Wittig
reactions
Medicinal applications such as gene therapy as well as gene
diagnostics greatly benefit from synthetic compounds that
sequence-specifically recognize and bind nucleic acids.[1]
Peptide nucleic acids (PNAs) represent a promising class of
DNA analogues in which the entire sugar± phosphate back-
bone is replaced by a pseudopeptide backbone.[2] Their
successful use as hybridization probes fuels research that is
aimed at the development of new polyamide-based DNA
binders.[2d] However, little attention is directed towards the
elaboration of techniques to site-specifically modify PNA
oligomers, although the feasibility to serve as hybridization
probe relies on the selective introduction of reporter groups.
In all studies to date, nonstandard nucleobases were incorpo-
rated by coupling of the corresponding monomeric building
blocks.[2d, 3] A strategy in which the modified nucleobases are
coupled to an orthogonally protected backbone on solid phase
would omit the need to synthesize an entire monomer in
solution.[4] Thus, the rapid synthesis and the efficient screen-
ing of modified PNA conjugates would be greatly facilitated.
This work presents a convergent strategy to selectively
functionalize and label PNA at terminal as well as internal
positions.
[1] a) F. H. Elsner, H.-G. Woo, T. D. Tilley, J. Am. Chem. Soc. 1988, 110,
313 ± 314; J. A. Soderquist, E. I. Miranda, J. Am. Chem. Soc. 1992, 114,
10078 ± 10079.
Á
[2] Review: B. Iorga, F. Eymery, V. Mouries, P. Savignac, Tetrahedron
1998, 54, 14637 ± 14677.
[3] a) R. A. Firestone (Merck and Co., Inc), US-A 3784590, 1974 [Chem.
Abstr. 1974, 80, 60031]; b) A. Vasella, R. Voeffray, Helv. Chim. Acta
1982, 65, 1953 ± 1964; c) V. V. Moskva, V. Y. Mavrin, Zh. Obshch.
Khim. 1987, 57, 2793 ± 2794; V. V. Moskva, V. Y. Mavrin, J. Gen. Chem.
USSR Engl. Transl. 1988, 57, 2492 ± 2493; d) H. Möhrle, W. Vetter, Z.
Naturforsch. B 1988, 43, 1662 ± 1671.
[4] a) M. Mikolajczyk, S. Grzejszczak, A. Zatorski, B. Mlotkowska, H.
Gross, B. Costisella, Tetrahedron 1978, 34, 3081 ± 3088; b) M. Miko-
lajczyk, P. Balczewski, Tetrahedron 1992, 48, 8697 ± 8710; c) M.
Mikolajczyk, P. P. Graczyk, M. W. Wieczorek, J. Org. Chem. 1994, 59,
1672 ± 1693; d) R. Hamilton, M. A. McKervey, M. D. Rafferty, B. J.
Walker, J. Chem. Soc. Chem. Commun. 1994, 37 ± 38.
[5] a) T. S. Franczyk (Monsanto Co., USA), WO-A 9850391, 1998 [Chem.
Abstr. 1998, 130, 3962]; W. A. Carter (Hem Research, Inc., USA),
EU-B 286224A2, 1988 [Chem. Abstr. 1988, 111, 90415]; T. E. Rogers
(Monsanto Co., USA), US-A 4568432A, 1986 [Chem. Abstr. 1986,
104, 225049]; B. Wahren, J. Harmenberg, V. A. Sundqvist (Swed.),
EU-B 97633A1, 1984 [Chem. Abstr. 1984, 100, 82417].
The central building block for the ªon-resin synthesisº of
nonstandard PNA monomers is the orthogonally protected
aminoethylglycine 1 (Scheme 1).[5] For validation, the building
block 1 was conjugated to the allylic HYCRON linker, which
provides orthogonal stability in combination with commonly
used protecting-group strategies.[6] The aminoethylglycine ±
HYCRON conjugate 3 was synthesized by allowing the Boc/
Fmoc-protected PNA backbone 1 to react with the allylic
bromide 2 followed by the reductive removal of the phenacyl
ester moiety. The Boc/Fmoc-protected conjugate 3 was
attached to the resin using HBTU and HOBt. Treatment of
4 with DMF/morpholine liberated a compound with a
secondary amino group, which subsequently was subjected
to a coupling reaction with 5. In the presence of the allyl
scavenger morpholine, resin 6 was treated with catalytic
amounts of the Pd0 catalyst [Pd(PPh3)4]. The Boc/Z-protected
guanosine analogue 7 was obtained in a yield of 61% based on
the initial load of resin 4 with Fmoc groups. A comparison
with the 70 ± 80% yield of the corresponding solution syn-
thesis illustrates the efficiency of this on-resin synthesis of the
protected PNA-monomer 7.[7, 8]
[6] S. Goumri, Y. Leriche, H. Gornitzka, A. Baceiredo, G. Bertrand, Eur.
J. Inorg. Chem. 1998, 1539 ± 1542.
[7] T. S. Mikhailova, V. I. Zakharov, V. M. Ignat'ev, B. I. Ionin, A. A.
Petrov, Zh. Obshch. Khim. 1980, 50, 1690 ± 1702; T. S. Mikhailova,
V. I. Zakharov, V. M. Ignat'ev, B. I. Ionin, A. A. Petrov, J. Gen. Chem.
USSR Engl. Tranl. 1980, 50, 1370 ± 1382.
[8] Crystal data for 2b: C25H45N2OP, Mr 420.60, monoclinic, space group
P21/n, a 11.105(1), b 11.332(2), c 20.194(2) , b 103.57(1)8,
3
V 2470.3(5) 3, Z 4, 1calcd 1.131 Mgm
,
F(000) 928, l
0.71073 , T 173(2) K, m(MoKa) 0.129 mm 1, crystal size 0.6 Â
0.5 Â 0.1 mm, 2.078 < V< 23.268; 17819 reflections (3432 independ-
ent, Rint 0.116) were collected at low temperatures using an oil-
coated shock-cooled crystal[13] on a STOE-IPDS diffractometer. A
numerical absorption correction was employed, and the min./max.
transmissions are 0.7441 and 0.9559. The structure was solved by direct
methods (SHELXS-97[14]), and 266 parameters were refined using the
least-squares method on F 2.[15] Largest residual electron density
0.203 e 3, R1 0.047 (for F > 2s(F)) and wR2 0.121 (all data) with
R1 SjjFoj jF j j S j F j and wR (Sw(F 2 F 2)2 Sw(F 2)2)0.5. Crys-
/
/
c
o
2
o
c
o
tallographic data (excluding structure factors) for the structure
reported in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication no.
CCDC-116054. Copies of the data can be obtained free of charge on
application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax:
(44)1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).
[9] J. Kapp, C. Schade, A. M. El-Nahasa, P. von R. Schleyer, Angew.
Chem. 1996, 108, 2373 ± 2376; Angew. Chem. Int. Ed. Engl. 1996, 35,
2236 ± 2238, and references therein.
As part of our research on new assays for the real-time
detection of oligonucleotide hybridization of an oligomer, we
[10] R. B. King, N. D. Sadanani, P. M. Sundaram, J. Chem. Soc. Chem.
Commun. 1983, 477 ± 478.
[*] Dr. O. Seitz
[11] The diethyl formylphosphonate was reported to readily decompose at
Institut für Organische Chemie der Universität
Richard-Willstätter-Allee 2, D-76128 Karlsruhe (Germany)
Fax : ( 49)721-6084825
108C into dialkylphosphite and carbon monoxide.[3c]
[12] A. W. Johnson in Ylides and imines of phosphorus (Ed.: A. W.
Johnson), Wiley-Interscience, New York, 1993.
[13] D. Stalke, Chem. Soc. Rev. 1998, 27, 171 ± 178.
[14] G. M. Sheldrick, Acta Crystallogr. Sect. A 1990, 46, 467 ± 473.
[15] SHELXL-97, Program for Crystal Structure Refinement, G. M.
Sheldrick, Universität Göttingen, 1997.
Dr. F. Bergmann, Dr. D. Heindl
Roche Diagnostics GmbH, Penzberg (Germany)
[**] This work was supported by the Fonds der Chemischen Industrie.
Angew. Chem. Int. Ed. 1999, 38, No. 15
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