Synthesis of an oxyamino-containing phenanthroline derivative for the efficient preparation of phenanthroline oligonucleotide oxime conjugates

Article abstract of DOI:10.1016/j.tetlet.2003.09.128

A phenanthroline derivative bearing an oxyamino linker was efficiently prepared from commercial 5-nitro-1,10-phenanthroline. The subsequent reaction with an oligonucleotide containing an aldehyde either at the 5′ end or the 3′ end afforded, in good yield, the phenanthroline-oligonucleotide conjugates through oxime bond formation.

Full text of DOI:10.1016/j.tetlet.2003.09.128

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 8379–8382
Synthesis of an oxyamino-containing phenanthroline derivative
for the efficient preparation of phenanthroline oligonucleotide
oxime conjugates
Ste´phanie Deroo, Eric Defrancq,* Ce´cile Moucheron, Andre´e Kirsch-De Mesmaeker and
Pascal Dumy
Laboratoire Europe´en Associe´ Inge´nie´rie Biomole´culaire Universite´ Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France
et Universite´ Libre de Bruxelles, CP. 160/08, B-1050 Brussels, Belgium
Received 18 July 2003; revised 5 September 2003; accepted 16 September 2003
Abstract—A phenanthroline derivative bearing an oxyamino linker was efficiently prepared from commercial 5-nitro-1,10-phenan-
throline. The subsequent reaction with an oligonucleotide containing an aldehyde either at the 5% end or the 3% end afforded, in
good yield, the phenanthroline–oligonucleotide conjugates through oxime bond formation.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
age with a reactive derivative.2 To avoid disturbing the
hybridisation process between the modified oligonucleo-
tide and the RNA or DNA target, conjugation is
generally achieved at the 3%- or 5%-ends. The two most
popular modified oligonucleotides are amino and thiol
functionalised oligonucleotides.3 However, the coupling
reaction using a primary amine suffers from drawbacks
such as competing reagent hydrolysis (a large excess of
the electrophilic reporter molecule is in fact required to
achieve complete conjugation) as well as cross reactivity
with other functionality present within the oligonucleo-
tide or the reporter group.
During the last decade, modified oligonucleotides have
been of increasing interest as useful tools in molecular
biology as well as new therapeutic candidates (anti-
sense, triple helix, siRNA strategies). The conjugation
with a variety of reporter molecules including fluores-
cent tags, peptides, sugars and intercalators in order to
provide specific properties to those synthetic oligonucleo-
tides has thus been the subject of great development.1
Most of the reported methods for the conjugation
involve a post-solid-phase synthesis modification by
preliminary incorporation of a protected nucleophilic
group during automated synthesis and subsequent link-
The anchoring of phenanthroline derivatives to
oligonucleotides has received particular attention. In
fact, the phenanthroline aromatic ring can provide
interesting intercalator properties. Furthermore, artifi-
cial nucleases have been designed by exploiting the
redox properties of coordination complexes bearing one
or more phenanthroline ligands.4 For instance, the
bis(1,10-phenanthroline) cuprous chelate represents one
of the most used cleaving agents in addition with other
complexes containing a ruthenium, osmium or rhodium
metallic centre.5–9 Most of the reported methods for the
preparation of phenanthroline–oligonucleotide conju-
gates employ amino-modified oligonucleotides and an
‘activated ester’ of the phenanthroline moiety (i.e.
phenylcarbamate and iodoacetamido derivatives of the
5-aminophenanthroline).4,10 Nevertheless, an excess of
the phenanthroline derivative has to be used which
renders the purification procedure more laborious.
Figure 1. Structure of the phenanthroline derivative 1 and
aldehyde-containing oligonucleotides 2 and 3.
Keywords: chemoselectivity; conjugation; oligonucleotide; oxime;
phenanthroline.
* Corresponding author. Fax: +33(0)4.76.51.49.46; e-mail: eric.
defrancq@ujf-grenoble.fr
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.09.128

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S. Deroo et al. / Tetrahedron Letters 44 (2003) 8379–8382
compound 6 was then removed by treatment with
CH2Cl2/TFA solution (50/50, v/v) at room temperature
for 2 h to furnish the amino derivative 7 which was
used without further purification in the next step. Intro-
duction of the oxyamino group was achieved by coup-
ling the activated ester of N-Boc-O-(carboxymethyl)-
hydroxylamine 8b with the amino derivative 7. The
oxyamino moiety in 8 was protected with a Boc group
using commercial carboxymethoxyamine hemihydro-
chloride 8 leading to the acid 8a. Before the coupling
reaction, the acid 8a was reacted with N-hydroxy-
succinimide in the presence of DCC to give the corres-
ponding activated ester 8b. Reaction of 8b with the
amino phenanthroline derivative 7 was carried out in
dry DMF to afford the protected oxyamino phenan-
throline derivative 9, after purification by column
chromatography, as a white powder in 41% overall
yield from the 5-aminophenanthroline 4.15 The Boc
protecting group was then cleaved under acidic con-
ditions (CH2Cl2/TFA solution 50/50, v/v) to give
the oxyamino phenanthroline derivative 1. Taking
advantage of the high reactivity of the oxyamino moi-
ety with carbonyl derivatives, the structure of 1 was
confirmed by formation of the corresponding oxime
ether by reacting 1 with acetone.
Scheme 1. Preparation of the phenanthroline derivative 1.
Reagents and conditions: (a) N2H4, Pd/C, EtOH, 70°C, 3 h,
70%; (b) (BocNHCH2CO)2O 5, CH3CN, overnight, 60%; (c)
CH2Cl2/TFA (50/50, v/v), 2 h, 90%; (d) 8b, DMF, 1 h, 80%;
(e) CH2Cl2/TFA (50/50, v/v), 1 h, 90%; (f) (Boc)2O, NaOH/
dioxane, 2 h, 80%; (g) N-hydroxysuccinimide, DCC, CH2Cl2,
1 h, 90%.
With the aim of developing a new strategy for the
conjugation of oligonucleotides, we have investigated
the use of the oxime linkage. The high efficiency of this
oxime ligation technique for the conjugation of
fluorophore, peptides and carbohydrates with oligonu-
cleotides has been largely demonstrated.11–14 A major
advantage of this ligation technique is that it requires
neither a coupling reagent nor chemical manipulations
except mixing of the two components, namely an
oxyamine and an aldehyde derivative.
The preparation of the oligonucleotide 2 bearing an
aldehyde moiety at the 5%-end was accomplished using
the method that we previously described by incorporat-
ing the phosphoramidite 10 at the final step of the
automated DNA synthesis (Scheme 2).12,16 Subsequent
oxidative cleavage of the intermediate diol 11 with
excess NaIO4 generated the aldehyde function. HPLC
analysis (Fig. 2A) showed the exclusive formation of
the desired aldehyde-containing oligonucleotide 2 in a
very short time. Compound 2 was purified by reverse-
phase HPLC and obtained in 70% isolated yield.17 For
the introduction of the masked aldehyde at the 3%-end,
the commercial solid supported 3%-glyceryl CPG 12
bearing a 1,2-diol was chosen as starting material
(Scheme 2). This support was preferred to the previ-
ously reported 1,2-aminoalcohol-containing support18
as the efficiency of this latter support for the introduc-
tion of the 1,2-aminoalcohol decreased dramatically
after 3–4 months storage. The oligonucleotide 13 with
the 1,2-diol at the 3%-end was synthesised according to
standard b-cyanoethyl phosphoramidite chemistry
using the support 12.16 After the usual deprotection and
purification steps, the oligonucleotide 13 was treated
with NaIO4 to generate the aldehyde. Compound 3 was
obtained after purification by reverse-phase HPLC in
almost 50% isolated yield (Fig. 2C shows the HPLC
profile of crude oxidation mixture of 13, and reveals a
single major product).
We thus envisioned exploiting these favourable charac-
teristics of the oxime bond formation for the conjuga-
tion of a phenanthroline derivative either at the 5%- or
the 3%-end of the oligonucleotide by introduction of the
reactive oxyamino moiety on the phenanthroline ring
and subsequent reaction with the aldehyde-containing
oligonucleotide. In the present paper we report the
synthesis of the phenanthroline derivative 1, bearing a
glycine linker with a terminal oxyamino group, and its
subsequent reaction with the oligonucleotides 2 and 3
containing the aldehyde at the 5%- or at the 3%-end,
respectively (Fig. 1). The glycine linker was chosen as it
was easily available at low cost.
2. Results and discussion
The preparation of the phenanthroline derivative 1 was
accomplished by a straightforward route shown in
Scheme 1. Commercial 5-nitro-1,10-phenanthroline was
first reduced to the corresponding amino derivative 4
using Pd/C and hydrazine in EtOH at 70°C for 3 h. The
glycine linker was then introduced by reaction with the
corresponding N-Boc anhydride 5. This compound was
easily prepared by mixing N-Boc-glycine (2 equiv.) with
DCC in acetonitrile for 1 h and subsequent filtration to
remove the DCU by-product. Due to the inactivation
of the exocyclic amine on the phenanthroline ring, an
excess of anhydride has to be used for completion of
the reaction. The protected phenanthroline 6 was then
purified from the excess of anhydride 5 by column
chromatography. The tert-butyloxycarbonyl group on
Conjugation reactions were carried out in ammonium
acetate buffer at pH 4.5 using the oligonucleotides 2
and 3 containing an aldehyde group at the 5%- and
3%-ends, respectively, and a slight excess (2 equiv.) of the
phenanthroline derivative 1.19 The course of the reac-
tion was followed by reverse-phase HPLC and the
reaction proceeded essentially to completion within 3 h
to yield exclusively the corresponding conjugates 14

S. Deroo et al. / Tetrahedron Letters 44 (2003) 8379–8382
8381
Scheme 2. Preparation of aldehyde containing oligonucleotides 2 and 3 and corresponding conjugates 14 and 15. Reagents and
conditions: (i) automated DNA synthesis then NH4OH 28%, 55°C for 16 h; (ii) AcOH 80% for 1 h; (iii) NaIO4 in H2O; (iv)
phenanthroline derivative 1, ammonium acetate buffer (pH 4.5).
and 15 (HPLC analysis of crude mixture of conjugation
with 2 and 3 are depicted in Figure 2B and 2D,
respectively).17 Subsequent purification by reverse-
phase HPLC afforded the conjugates 14 and 15 in
almost 50% isolated yield. The conjugates 14 and 15
were characterised by ES-MS. In all cases, the experi-
mentally determined molecular weights were in excel-
lent agreement with the calculated values (Table 1).
3. Conclusion
In conclusion, we report a straightforward preparation
of the oxyamino containing phenanthroline derivative 1
from commercially available 5-nitro-1,10-phenanthro-
line. The usefulness of this phenanthroline derivative
for the efficient and improved preparation of 5%- or
3%-oligonucleotide oxime conjugates was demonstrated.
Introduction of the phenanthroline derivative 1 as a
ligand in ruthenium complexes may open the way for
efficient conjugation to oligonucleotides as well as to
peptides through oxime ligation.21 Work is currently
underway to prepare ruthenium complexes using this
oxyamino containing phenanthroline ligand for such
purposes.
Figure 2. HPLC profiles: (A) crude oxidation mixture of
5%-diol oligonucleotide 11, (B) crude reaction mixture of
oligonucleotide 2 with the phenanthroline derivative 1, (C)
crude oxidation mixture of 3%-diol oligonucleotide 13, (D)
crude reaction mixture of oligonucleotide 3 with the phenan-
throline derivative 1.17 Detection at 260 nm.
Acknowledgements
This work was supported by the CNRS (France) and
the FNRS (Belgium). The ‘Institut Universitaire de

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S. Deroo et al. / Tetrahedron Letters 44 (2003) 8379–8382
Cho, Y.-M.; Sigman, D. S. Bioconjugate Chem. 1996, 7,
Table 1. ES-MS analysis*
413–420.
Oligonucleotides
Calcd mass
Found mass
11. Tre´visiol, E.; Renard, A.; Defrancq, E.; Lhomme, J.
Nucleosides, Nucleotides and Nucleic Acids 2000, 19,
1427–1439.
12. Forget, D.; Boturyn, D.; Defrancq, E.; Lhomme, J.;
Dumy, P. Chem. Eur. J. 2001, 7, 3976–3984.
13. Stetsenko, D. A.; Gait, M. J. Bioconjugate Chem. 2001,
12, 576–586.
5%-Diol 11
3%-Diol 13
5%-Conjugate 14
3%-Conjugate 15
5304.50
5262.50
5579.70
5537.80
5304.89
5262.69
5579.51
5536.81
* The analysis was performed in the negative mode. The eluent was
50% aqueous acetonitrile and the flow rate was 8 mL/min. The
oligonucleotides were dissolved in H2O/CH3CN/NEt3, 50/50/2 (v/v/
v).
14. Forget, D.; Renaudet, O.; Defrancq, E.; Dumy, P. Tetra-
hedron Lett. 2001, 42, 7829–7832.
15. Data for 9: 1H NMR (200 MHz, d6-DMSO): l ppm=
1.39 (s, 9H, tBu), 4.16 (d, J=5.5 Hz, 2H, CH2NH), 4.28
(s, 2H, CH2-O), 7.71–7.82 (m, 2H, H-phen), 8.13 (s, 1H,
H-phen), 8.43 (m, 2H, NH+H-phen), 8.61 (d, J=8.2 Hz,
1H, H-phen), 9.04 (d, J=4 Hz, 1H, H-phen), 9.12 (d,
J=4 Hz, 1H, H-phen), 10.22 (1H, s, NH), 10.31 (1H, s,
NH). MS (DCI): m/e=425.9. Mp 135–136°C.
16. Automated DNA synthesis was performed on an Expe-
dite DNA synthesiser (Perkin–Elmer) following the manu-
facturer’s protocols for standard b-cyanoethyl nucleoside
phosphoramidite chemistry on a 1 mM scale. The support
12 was purchased from Eurogentec.
France’ and the ‘Laboratoire Europe´en Associe´ Inge´-
nierie Biomole´culaire’ are greatly acknowledged for
financial support. S.D. thanks the FRIA (Fonds pour
la Recherche dans l’Indusdrie et l’Agriculture) for a
fellowship. We also thank J. E. Cavendish for careful
reading of this manuscript.
References
17. The oligonucleotides and the conjugates were purified on
a m-bondapak C-18 column (Macherey-Nagel Nucleosil:
10×250 mm, 7 mm). The following solvent system was
used. Solvent A, 20 mM ammonium acetate buffer/
CH3CN, 95/5 (v/v); solvent B (CH3CN); flow rate, 4
mL/min; a linear gradient from 0 to 30% B in 20 min was
applied.
18. Forget, D.; Renaudet, O.; Boturyn, D.; Defrancq, E.;
Dumy, P. Tetrahedron Lett. 2001, 42, 9171–9174.
19. Reactions were carried out in slightly acidic conditions as
the optimal pH is around 4–5 for oxime bond forma-
tion.20
1. Manoharan, M. In Antisense Drug Technology, Princi-
ples, Strategies, and Applications; Crooke, S. T., Ed.; M.
Dekker: New York, Basel, 2001; pp. 391–469.
2. Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123–
6194.
3. Arimondo, P. B.; Boutorine, A. S.; Franc¸ois, J.-C. Recent
Res. Devel. Bioconj. Chem. 2002, 1, 29–53.
4. Astrom, H.; Stromberg, R. Nucleosides, Nucleotides and
Nucleic Acids 2001, 20, 1385–1388.
5. Hwang, J.-T.; Baltasar, F. E.; Cole, D. L.; Sigman, D. S.;
Chen, C. B.; Greenberg, M. M. Bioorg. Med. Chem. Lett.
2003, 11, 2321–2328.
20. Jencks, W. P. J. Am. Chem. Soc. 1959, 81, 475–481.
21. The usefulness of such complexes has been recently
reported22 as well as the interest of conjugation with a
peptide.23
6. Grimm, G. N.; Boutorine, A. S.; Lincoln, P.; Norde´n, B.;
He´le`ne, C. ChemBioChem 2002, 3, 324–331.
7. Hurley, D. J.; Tor, Y. J. Am. Chem. Soc. 2002, 124,
3749–3762.
22. Lentzen, O.; Constant, J.-F.; Defrancq, E.; Prevost, M.;
Schumm, S.; Moucheron, C.; Dumy, P.; Kirsch-De Mes-
maeker, A. ChemBioChem 2003, 4, 195–202.
8. Odom, D. T.; Dill, E. A.; Barton, J. K. Nucl. Acids Res.
2001, 29, 2026–2033.
9. Content, S.; Kirsch-De Mesmaeker, A. J. Chem. Soc.,
23. Copeland, K. D.; Lueras, A. M. K.; Stemp, E. D. A.;
Faraday Trans. 1997, 93, 1089–1094.
10. Gallagher, J.; Chen, C. B.; Pan, C. Q.; Perrin, D. M.;
Barton, J. K. Biochemistry 2002, 41, 12785–12797.