A novel solid support for the synthesis of 3′-aminoalkylated oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2003.10.166

A novel improved controlled pore glass (CPG) support based on the 2-(hydroxymethyl)-6-nitrobenzoyl (HMNB) protecting group was developed for the synthesis of 3′-aminoalkylated oligonucleotides. The release of oligonucleotides with free 3′-amino groups from the support is complete within 2h at 55°C in concentrated ammonia.

Full text of DOI:10.1016/j.tetlet.2003.10.166

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 317–320
A novel solid support for the synthesis of 30-aminoalkylated
oligonucleotides
Michael Leuck,* Rubina Giare, Matthias Paul, Nicole Zien and Andreas Wolter
Proligo Biochemie GmbH Hamburg, Georg-Heyken-Str. 14, D-21147 Hamburg, Germany
Received 4 September 2003; revised 21 October 2003; accepted 28 October 2003
Abstract—A novel improved controlled pore glass (CPG) support based on the 2-(hydroxymethyl)-6-nitrobenzoyl (HMNB) pro-
tecting group was developed for the synthesis of 30-aminoalkylated oligonucleotides. The release of oligonucleotides with free
30-amino groups from the support is complete within 2h at 55 °C in concentrated ammonia.
Ó 2003 Elsevier Ltd. All rights reserved.
A number of methods in molecular biology and DNA-
based diagnostics to amplify, detect, analyze, and
quantify nucleic acids require chemically synthesized
and modified oligonucleotides. 30-Modifications are
particularly useful to stabilize oligonucleotides against
exonucleolytic degradation1 and are required for the
synthesis of doubly-labeled probes with two different
covalently-bound reporters at their 30- and 50-termini.
Doubly labeled oligonucleotides such as molecular
beacons2 or Taqman3 hydrolysis probes are widely
employed in oligonucleotide-based diagnostic assays,
especially in real-time quantitative PCR methods.
Various solid-phase supports for the synthesis of
30-amino oligonucleotides have been described,4 but
none of these supports displays all of the desirable fea-
tures for their routine application in a high-throughput
oligonucleotide synthesis laboratory. For instance, the
synthesis of 30-amino oligonucleotides on a support
loaded with
a
cleavable 3-aminopropane-1,2-diol
linker4a resulted in the formation of 30-acetamido oli-
gonucleotides as side products.5 The 3-aminopropane-
1,2-diol structure also introduces a chiral center to the
oligonucleotides, which complicates their analysis by
HPLC. The 30-amino support based on an immobilized
phthalimido protective group4h requires long deprotec-
tion times in concentrated aqueous ammonia for the
formation of the free 30-amine (17 h at 55 °C still leaves
10–20% of the oligonucleotides attached to the support),
which reduces the throughput in oligonucleotide pro-
duction and makes the support incompatible with base-
sensitive modifications.
Oligonucleotides with 30-modifications are conveniently
prepared through standard solid-phase synthesis on
speciality 30-amino supports, which release the oligo-
nucleotides with an attached 30-aminoalkyl tail during
the deprotection process. Suitable supports must be fully
compatible with the chemistry cycle of the solid-phase
synthesis and the formation of the oligomers with free
30-amino groups should be complete under the standard
deprotection conditions for oligonucleotides. In addi-
tion, the attached amino linker should not introduce
chiral centers to the oligomer and should not be prone
to side reactions that result in dysfunctional oligo-
nucleotide modifications such as alkylated or acylated
30-amino groups.
In this communication we report on the development
of an improved 30-amino CPG support that releases
30-aminoalkyl oligonucleotides within 2h at 55 °C in
concentrated aqueous ammonia. The novel support is
based on the 2-(hydroxymethyl)-6-nitrobenzoyl (HMNB)
protecting group for the amino function. A similar
group, the 2-(acetoxymethyl)benzoyl (AMB) group
has been utilized as a very base-labile Ôprotected pro-
tecting groupÕ6 and has been employed for the exocyclic
amino groups of nucleobases in the synthesis of base-
sensitive oligonucleotides, for example, methylphospho-
nates.7
Keywords: 30-Amino modified oligonucleotides; Amino-ON CPG.
* Corresponding author. Tel.: +49-40-79-702-212; fax: +49-40-79-702-
100; e-mail: mleuck@proligo.com
0040-4039/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.166

318
M. Leuck et al. / Tetrahedron Letters 45 (2004) 317–320
Scheme 1. Synthesis of 30-amino CPG support 5 and solid-phase oligonucleotide synthesis on 5. Reagents and conditions: (a) AlCl3, Et3N, 1,2-
dichloroethane, rt, 16 h, 8%;8 (b) succinic anhydride, Et3N, DMAP, EtOAc, 50 °C, 2h; (c) 4-nitrophenol, DCC, 1,4-dioxane, rt, 16 h, 87% over two
steps;9 (d) amino modified CPG, Et3N, DMF, rt, 16 h; (e) Ac2O, pyridine, NMI, THF, rt, 2h; 10 (f) solid-phase oligonucleotide synthesis (SPOS)
employing DMT-protected dT-, dAbz-, dCbz-, and dGdmf -phosphoramidites; (g) NH4OH, rt, 40 min or AMA, rt, 5 min; (h) NH4OH, 55 °C, 2h or
AMA, 65 °C, 30 min.
The CPG support 5 was prepared in four steps
employing 6-nitrophthalide 1 and the DMT-protected
amino linker 211 as starting materials (Scheme 1). The
synthesis proceeded smoothly despite a rather poor yield
in the aminolysis of phthalide 1 with 2, which can be
attributed to the presence of the acidic catalyst AlCl3 in
the reaction medium, resulting in partial detritylation.
The resulting CPG 500 support 5 had a loading of
36.0 lmol/g.
The kinetics under various deprotection conditions were
easily monitored by HPLC analysis of the released
oligomers and by photometric measurements of the
support to determine the amount of uncleaved oligo-
nucleotides. As expected, the HMNB-protected oligo-
nucleotides were completely released into solution
within 40 min in concentrated ammonia or within 5 min
in AMA reagent12 (40% aqueous methylamine/concen-
trated aqueous ammonia 1/1, v/v) at room temperature.
The complete removal of the HMNB protecting group
from the released oligonucleotides required 2h in con-
centrated ammonia at 55 °C or 30 min in AMA reagent
at 65 °C. Complete cleavage and deprotection could also
be accomplished at room temperature within 24 h in
concentrated ammonia. In contrast, the complete
removal of the similar 2-(hydroxymethyl)benzoyl group
lacking the nitro substituent from 30-amino oligonucleo-
tides required 6 h in concentrated ammonia at 55 °C.
The novel support 5 releases oligonucleotides with free
amino groups 8 in two steps (Scheme 1). First, treatment
of support-bound oligonucleotides 6 with concentrated
ammonia cleaves HMNB-protected 30-amino oligo-
nucleotides 7 from the support. In the second step the
hydroxymethyl group in the ortho position of the aro-
matic ring removes the HMNB group through an
intramolecular attack on the amide bond. 6-Nitroph-
thalide 1 is formed as a by-product.
The utility of support 5 was demonstrated by the syn-
thesis of a poly-dT sequence on a 1:1 mixture of support
5 with a commercially available dT support. The syn-
thesizer was programmed to prepare a dT30 oligomer,
The cleavage of oligonucleotides from support 5 and the
removal of the HMNB moiety were investigated
through 30-aminoalkyl poly-dT model oligonucleotides.

M. Leuck et al. / Tetrahedron Letters 45 (2004) 317–320
319
Figure 1. RP-HPLC chromatogram of the crude oligonucleotide 12 after post-synthetic conjugation with QSY7-NHS ester. The UV–vis spectra of
the main components are displayed as inserts: (A) nonreacted oligonucleotide with 50-FAM modification, (B) 12 with 50-FAM and 30-QSY7
modifications, (C) oligonucleotide without 50-FAM, but with 30-QSY modification.
which gave a mixture of 30-aminohexyl-d(TTT TTT
TTT TTT TTT TTT TTT TTT TTT TT) 9 and d(TTT
TTT TTT TTT TTT TTT TTT TTT TTT TTT) 10 after
deprotection with concentrated ammonia for 16 h at
55 °C. The ratio of the obtained oligonucleotides 9 and
10 was determined by anion exchange HPLC as
0.96:1.00. The approximately 1:1 ratio of products 9 and
10 demonstrates that the 30-amino-CPG 5 is compatible
with standard solid-phase synthesis conditions applying
the phosphoramidite method. The yields and coupling
efficiencies are comparable to syntheses on standard
CPG supports. A repetition of the experiment with a
commercially available phthalimidyl CPG support as a
substitute for support 5 gave a ratio of 0.45:1.00 for the
oligonucleotide products 9 and 10. This result can be
explained by the stability of the phthalimido group in
concentrated ammonia and indicates that the release of
the 30-aminoalkyl-dT29 oligonucleotide 9 from the
phthalimidyl support is far from being complete under
standard deprotection conditions.
50-FAM-d(GCG AGT TTT TTT TTT TTT TTC TCG
C)-QSY7-30 12 was synthesized employing dGdmf -phos-
phoramidite for oligonucleotide synthesis and FAM
phosphoramidite for 50-functionalization. After the
deprotection with concentrated ammonia for 2h at
55 °C the crude oligonucleotide 12 was conjugated at its
30-terminus with QSY7 NHS ester. The synthesis and
the post-synthetic conjugation reaction proceeded with
high efficiency as demonstrated in Figure 1. The HPLC-
purified molecular beacon 12 (MALDI-TOF MS: calcd
8950.4; found 8939.6) displayed a sharp increase in
fluorescence upon incubation with a poly-dA sequence
under hybridization conditions.
In conclusion, the novel amino-ON CPG support 5 is
compatible with standard solid-phase oligonucleotide
synthesis conditions employing the phosphoramidite
method, and the observed coupling efficiencies and
yields are comparable to those obtained with standard
deoxynucleoside-loaded CPG supports. The application
of mild cleavage and deprotection conditions allows the
use of 5 in high throughput oligonucleotide synthesis
and the on-support preparation of labile 50-modified
oligonucleotides with 30-aminoalkyl chains.
Support 5 can be favorably applied in fast deprotection
synthetic schemes as exemplified in the preparation of
the oligonucleotide 30-aminohexyl-d(AAC TCC GAG
CGA CTC TC)-50 11. A set of dT-, dAbz-, dCbz-, and
dGdmf -phosphoramidites13 was employed in the synthe-
sis to ensure complete base deprotection while applying
limited exposure to concentrated ammonia. The crude
11 was obtained after incubation with concentrated
ammonia for 2h at 55 °C in 80% purity (MALDI-TOF
MS: calcd 5294.5; found 5294.2).
References and Notes
1. Zendegui, J. G.; Vasquez, K. M.; Tinsley, J. H.; Kessler,
D. J.; Hogan, M. E. Nucleic Acids Res. 1992, 20, 307–314.
2. Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–
308.
3. Heid, C. A.; Stevens, J.; Livak, K. J.; Williams, P. M.
Genome Res. 1996, 6, 986–994.
4. (a) Nelson, P. S.; Frye, R. A.; Liu, E. Nucleic Acids Res.
1989, 17, 7187–7194; (b) Nelson, P. S.; Kent, M.; Muthini,
S. Nucleic Acids Res. 1992, 20, 6253–6259; (c) Asseline, U.;
Thuong, N. T. Tetrahedron Lett. 1990, 31, 81–84; (d)
Kumar, P.; Gupta, K. C.; Rosch, R.; Seliger, H. Bioorg.
The 30-aminoalkylated oligonucleotides synthesized on
the CPG support 5 were successfully employed in post-
synthetic labeling experiments with dye NHS esters. For
instance, the conjugation with TAMRA NHS ester
proceeded with more than 90% labeling efficiency under
standard conditions, as determined by RP-HPLC. In
another example the molecular beacon sequence

320
M. Leuck et al. / Tetrahedron Letters 45 (2004) 317–320
Med. Chem. Lett. 1996, 6, 2247–2252; (e) Gryaznov, S. M.;
Letsinger, R. L. Tetrahedron Lett. 1993, 34, 1261–1264; (f)
mixture was filtered and concentrated to give 250 mg
(87%) of the nitrophenylester 4 as a light yellow foam,
which was used as a crude product in subsequent
reactions. 1H NMR (300 MHz, CD3CN) 8.28–8.21 (m,
3H), 7.72–7.70 (m, 1H), 7.46–7.42 (m, 2H), 7.35–7.13 (m,
10H), 6.88–6.84 (m, 4H), 5.40 (s, 2H), 3.76 (s, 6H), 3.33 (q,
J ¼ 6:6 Hz, 2H), 3.02 (t, J ¼ 6:4 Hz, 2H), 2.94–2.90 (m,
2H), 2.83–2.79 (m, 2H), 1.66–1.52 (m, 4H), 1.48–1.32 (m,
4H). MS (FAB) m=z 819 [M+H]þ.
€
Hovinen, J.; Guzaev, A.; Azhayev, A.; Lonnberg, H.
Tetrahedron 1994, 50, 7203–7218; (g) Avino, A.; Garcia,
~ꢀ
R. G.; Albericio, F.; Mann, M.; Wilm, M.; Neubauer, G.;
Eritja, R. Bioorg. Med. Chem. 1996, 4, 1649–1658; (h)
Petrie, C. R.; Reed, M. W.; Adams, A. D.; Meyer, R. B.
Bioconjugate Chem. 1992, 3, 85–87; (i) Lyttle, M. H.;
Adams, H.; Hudson, D.; Cook, R. M. Bioconjugate Chem.
1997, 8, 193–198.
ꢁ
10. CPG 500 A with an aminohexyl spacer (5.0 g) was
5. Vu, H.; Joyce, N.; Rieger, M.; Walker, D.; Goldknopf, I.;
Schmaltz Hill, T.; Jayaraman, K.; Mulvey, D. Bioconju-
gate Chem. 1995, 6, 599–607.
6. Cain, B. F. J. Org. Chem. 1976, 41, 2029–2031.
7. (a) Kuijpers, W. H. A.; Huskens, J.; van Boeckel, C. A. A.
Tetrahedron Lett. 1990, 31, 6729–6732; (b) Kuijpers, W.
H. A.; Kuyl-Yeheskiely, E.; van Boom, J. H.; van Boeckel,
C. A. A. Nucleic Acids Res. 1993, 21, 3493–3500.
suspended in DMF (13 mL) and Et3N (0.5 mL). Nitro-
phenyl ester 4 (250 mg, 305 lmol) was dissolved in DMF
(4.0 mL) and added to the suspension, which was then left
overnight on an orbital shaker at room temperature. The
CPG was washed with DMF/Et3N 9/1 (v/v), DMF, EtOH,
acetonitrile, and EtOAc in multiple 20 mL portions. The
CPG was finally treated with commercially available Cap
A (Ac2O/THF) and Cap B (1-methylimidazole/pyridine/
THF) synthesis solutions for 2h and washed with THF,
EtOH/pyridine 9/1 (v/v), EtOH, acetonitrile, and EtOAc.
The loading was determined to be 36.0 lmol/g by a
photometric (497 nm) DMT loading assay.
8. The synthesis of 3 was conducted according to Bigg, D. C.
H.; Lesimple, P. Synthesis 1992, 277–278.
9. The amide 3 (200 mg, 0.334 mmol) was reacted in 1.5 mL
EtOAc with succinic anhydride (40 mg, 0.401 mmol) in the
presence of Et3N (46 lL, 0.327 mmol) and 4-dimethyl-
aminopyridine (10 mg, 0.084 mmol) for 2h at 50 °C. The
mixture was concentrated and redissolved in CH2Cl2. The
solution was extracted with 10% aqueous citric acid and
water, dried over Na2SO4, and concentrated to a foam.
The residue was redissolved in 1,4-dioxane (3 mL) and
pyridine (70 lL) and reacted with 4-nitrophenol (50 mg,
0.358 mmol) and 1,3-dicyclohexylcarbodiimide (94 mg,
0.456 mmol) at room temperature overnight. The reaction
11. Woo, S. L.; Fung, S. Solid support reagents for the
synthesis of 30-nitrogen containing polynucleotides. PCT
Int. Appl. WO 9605215, 1996; Chem. Abstr. 1996, 125,
58993.
12. Reddy, M. P.; Hanna, N. B.; Farooqui, F. Tetrahedron
Lett. 1994, 35, 4311–4314.
13. Vu, H.; McCollum, C.; Jacobsen, K.; Theisen, P.; Vina-
yak, R.; Spiess, E.; Andrus, A. Tetrahedron Lett. 1990, 31,
7269–7272.