Phosphoramidites and oligonucleotides containing 7-deazapurines and pyrimidines carrying aminopropargyl side chains

Article abstract of DOI:10.1081/NCN-100002568

The synthesis of phosphoramidites containing 7-deazaguanine, 7-deazaadenine, uracil and cytosine carrying aminopropargyl chains is described. The corresponding oligonucleotides are stabilized in duplexes thermally as well as against degradation by exonucl

Full text of DOI:10.1081/NCN-100002568

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PHOSPHORAMIDITES AND OLIGONUCLEOTIDES
CONTAINING 7-DEAZAPURINES AND PYRIMIDINES
CARRYING AMINOPROPARGYL SIDE CHAINS
F. Seela a , N. Ramzaeva a , P. Leonard a , Y. Chen a , H. Debelak a , E. Feiling a , R.
Kröschel a , M. Zulauf a , T. Wenzel , T. Fröhlich & M. Kostrzewa
a Institut für Chemie, Universität Osnabrück, Laboratorium für Organische und
Bioorganische Chemie, Germany
Version of record first published: 07 Feb 2007.
To cite this article: F. Seela , N. Ramzaeva , P. Leonard , Y. Chen , H. Debelak , E. Feiling , R. Kröschel , M. Zulauf , T.
Wenzel , T. Fröhlich & M. Kostrzewa (2001): PHOSPHORAMIDITES AND OLIGONUCLEOTIDES CONTAINING 7-DEAZAPURINES
AND PYRIMIDINES CARRYING AMINOPROPARGYL SIDE CHAINS, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7,
1421-1424
To link to this article: http://dx.doi.org/10.1081/NCN-100002568
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1421–1424 (2001)
PHOSPHORAMIDITES AND OLIGONUCLEOTIDES
CONTAINING 7-DEAZAPURINES AND
PYRIMIDINES CARRYING AMINOPROPARGYL
SIDE CHAINS
F. Seela,∗ N. Ramzaeva,1 P. Leonard,1 Y. Chen,1
H. Debelak,1 E. Feiling,1 R. Kro¨schel,1 M. Zulauf,1
T. Wenzel,2 T. Fro¨hlich,2 and M. Kostrzewa2
1Laboratorium fu¨r Organische und Bioorganische Chemie,
Institut fu¨r Chemie, Universita¨t Osnabru¨ck, Germany
2Bruker Saxonia Analytik GmbH, Leipzig, Germany
ABSTRACT
Thesynthesisofphosphoramiditescontaining7-deazaguanine, 7-deazaadenine,
uracil and cytosine carrying aminopropargyl chains is described. The corre-
sponding oligonucleotides are stabilized in duplexes thermally as well as against
degradation by exonucleases.
INTRODUCTION
In a series of manuscripts our laboratory has reported on oligonucleotides
containing alkynyl- or aminoalkynyl side chains linked to the 7-position of 7-
deazapurines or 8-aza-7-deazapurines (purine numbering is used throughout the
manuscript) (1–3). A positively charged side chain transforms a negatively charged
oligonucleotide in a zwitterion or even a positively charged species. As a result
favorable properties are generated, such as duplex stabilization, resistance against
enzymatic degradation, or an increased sensitivity of oligonucleotide detection by
MALDI-TOF spectrometry.
∗ Corresponding author.
1421
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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SEELA ET AL.
Scheme 1.
This manuscript reports on the synthesis of the phosphoramidites 1–4
(Scheme 1) carrying an aminopropargyl side chain protected by phthaloyl residues
(4–6). This protecting group is removed with ammonia under standard conditions
(25% ammonia, 60◦C, 24 h) and is superior over the rather labile trifluoroacetyl
group (7,8) which gives rise to side reaction (9).
RESULTS AND DISCUSSION
1. Synthesis of the 7-deazapurine Phosphoramidites 1 and 2
The synthesis of the phosphoramidites 1 and 2 was performed using the iodo
compounds 5 (10) and 8 (11) as precursors (Scheme 2). While the 7-deazaguanine
Scheme 2.

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PHOSPHORAMIDITES AND OLIGONUCLEOTIDES
1423
Scheme 3.
base was protected with an isobutyryl residue, the 7-deazaadenine moiety was
acetylated. Next, the DMTr residues were introduced. Compounds 6b and 9b
were coupled with phthaloyl-propargylamine employing the Sonogashira reaction
(Pd(PPh3)4, CuI, Et3N in DMF). The fully protected nucleosides 7 and 10 gave the
phosphoramidites 1 and 2 applying standard methodology.
Table. Tm-Values and Thermodynamic Data of Base Modified Oligonucleotides
Tm
ꢀH◦
ꢀS◦
ꢀG◦298
Duplex
[◦C] [kcal/mol] [cal/mol · K] [kcal/mol]
5ꢁ-d(T-A-G-G-T-C-A-A-T-A-C-T)
3ꢁ-d(A-T-C-C-A-G-T-T-A-T-G-A)
5ꢁ-d(T-A-G-G-T-C-A-A-T-A-C-T)
3ꢁ-d(A-T-C-C-A-G∗-T-T-A-T-G∗-A)
5ꢁ-d(T-A-G∗-G∗-T-C-A-A-T-A-C-T)
3ꢁ-d(A-T-C-C-A-G∗-T-T-A-T-G∗-A)
5ꢁ-d(T-A-G-G-T-C-A-A-T-A-C-T)
3ꢁ-d(A-T-C-C-A∗-G-T-T-A∗-T-G-A)
17 50a)
18 47b)
17 54a)
19 52b)
20 54a)
19 54b)
17 55a)
21 53b)
−90
−89
−80
−95
−71
−98
−84
−85
−82
−84
−86
−91
−70
−87
−81
−84
−252
−253
−220
−268
−189
−272
−232
−237
−222
−230
−239
−254
−187
−239
−222
−234
−11.8
−10.9
−12.0
−12.0
−11.9
−13.0
−12.4
−11.8
−12.9
−12.7
−12.0
−11.8
−12.3
−13.1
−12.0
−11.8
5ꢁ-d(T-A∗-G-G-T-C-A∗-A∗-T-A∗-C-T) 22 59a)
3ꢁ-d(A-T-C-C-A∗-G-T-T-A∗-T-G-A)
5ꢁ-d(T-A-G-G-U∗-C-A-A-U∗-A-C-T)
3ꢁ-d(A-T-C-C-A-G-T-T-A-T-G-A)
5ꢁ-d(T-A-G-G-T-C∗-A-A-T-A-C∗-T)
3ꢁ-d(A-T-C∗-C∗-A-G-T-T-A-T-G-A)
5ꢁ-d(T-A-G-G-T-C∗-A-A-T-A-C∗-T)
3ꢁ-d(A-T-C-C-A-G-T-T-A-T-G-A)
21 56b)
23 53a)
18 50b)
24 57a)
25 56b)
24 54a)
18 53b)
a) 1 M NaCl, 100 mM MgCl2, 60 mM Na-cacodylate (pH 7.0). b) 100 mM NaCl, 10 mM MgCl2,
and 10 mM Na-cacodylate (pH 7.0) with 5 µM single strand concentration. The residues with a∗
represent the modified constituents.

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SEELA ET AL.
2. Synthesis of the Pyrimidine Phosphoramidites 3 and 4
The reaction sequence performed on the 7-deazapurine nucleosides was also
used for the preparation of the phosphoramidite 3 (Scheme 3). The phosphoramidite
4 was prepared in a slightly different way. The 5-iodo-2ꢁ-deoxycytidine (14) was
directly used for the cross coupling reaction yielding the derivative 15. The crude
reaction product was tritylated and benzoylated in a one-pot reaction to give the in-
termediate 16. Afterwards, the phosphoramidite 4 was prepared as described before.
3. Synthesis and Properties of Oligonucleotides
The oligonucleotides shown in the Table were prepared by solid-phase synthe-
sis in a 1-µmole scale using the phosphoramidites 1–4. The coupling yields were
always higher than 96%. The oligonucleotides were deprotected and purified as
DMTr derivatives. They were detritylated with 3% aq. trifluoroacetic acid on OPC
cartridges. In the case of the dU∗-derivatives a side product was formed during the
solid-phase synthesis when multiple residues were incorporated. The Tm-values
of the Table show that the positively charged aminopropargyl chain stabilizes the
duplexes. Also degradation with exonucleases is retarded. The favorable properties
of such oligonucleotides were used in MALDI-TOF spectrometry to increase the
oligonucleotide detection sensitivity (12).
REFERENCES
1. Seela, F.; Zulauf, M. Helv. Chim. Acta, 1999, 82, 1878–1898.
2. Ramzaeva, N.; Mittelbach, C.; Seela, F. Helv. Chim. Acta, 1997, 80, 1809–1822.
3. Seela, F.; Zulauf, M. J. Chem. Soc., Perkin Trans. 1, 1999, 479–488.
4. Ramzaeva, N.; Mittelbach, C.; Seela, F. Nucleosides Nucleotides, 1999, 18, 1439–
1440.
5. Gibson, K.J.; Benkovic, S.J. Nucleic Acids Res., 1987, 15, 6455–6467.
6. Meyer, R.B. in “Methods in Molecular Biology”, 1994, Vol 26, pp. 73–91, Humana
Press Inc. Totowa, NJ, USA.
7. Hobbs, Jr., F.W. J. Org. Chem., 1989, 54, 3420–3422.
8. Ahmadian, M.; Zhang, P.; Bergstrom, D.E. Nucleic Acids Res., 1988, 26, 3127–3135.
9. Ferrer, E.; Neubauer, G.; Mann, M.; Eritja, R. J. Chem. Soc., Perkin Trans. 1, 1997,
2051–2057.
10. Ramzaeva, N.; Seela, F. Helv. Chim. Acta, 1995, 78, 1083–1090.
11. Seela, F.; Zulauf, M. Synthesis, 1996, 726–730.
12. Sauer, S.; Lechner, D.; Berlin, K.; Lehrach, H.; Escary, J.L.; Fox, N.; Gut, I.G. Nucleic
Acids Res., 2000, 28, e13.

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