A novel phosphate protection for oligonucleotide synthesis: The 2-[(1- naphthyl)carbamoyloxy]ethyl (NCE) group

Article abstract of DOI:10.1016/S0040-4039(00)00911-4

The utility of the 2-(arylcarbamoyloxy)ethyl group for protection of internucleosidic phosphate linkages in oligonucleotide synthesis was studied. Of the three protecting groups tested, the 2-[(1-naphthyl)carbamoyloxy]ethyl demonstrated high coupling yields, favorable deprotection kinetics and the highest hydrolytic stability of the thymidine phosphoramidite building block. The mechanism of deprotection was confirmed by deprotecting a model phosphate triester and synthetic dodecathymidylate. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00911-4

Tetrahedron Letters 41 (2000) 5623±5626
A novel phosphate protection for oligonucleotide synthesis:
the 2-[(1-naphthyl)carbamoyloxy]ethyl (NCE) group
Andrei P. Guzaev* and Muthiah Manoharan
Department of Medicinal Chemistry, Isis Pharmaceuticals, 2292 Faraday Ave., Carlsbad, CA 92008, USA
Received 9 May 2000; accepted 26 May 2000
Abstract
The utility of the 2-(arylcarbamoyloxy)ethyl group for protection of internucleosidic phosphate linkages
in oligonucleotide synthesis was studied. Of the three protecting groups tested, the 2-[(1-naphthyl)carbamoyl-
oxy]ethyl demonstrated high coupling yields, favorable deprotection kinetics and the highest hydrolytic
stability of the thymidine phosphoramidite building block. The mechanism of deprotection was con®rmed
by deprotecting a model phosphate triester and synthetic dodecathymidylate. # 2000 Elsevier Science Ltd.
All rights reserved.
For the past three decades, oligonucleotide synthesis on solid support has made tremendous
progress from manual synthesis on a small-scale to the manufacturing of the ®rst antisense drug,
an anti-CMV 21-mer phosphorothioate oligonucleotide. Currently, oligonucleotide synthesis
employs a phosphoramidite method1 and is carried out automatically by stepwise coupling of
nucleoside building blocks to a solid support. This allows an ecient preparation of natural2 and
modi®ed3 DNA fragments on a routine basis. Phosphoramidite building blocks are most often
protected with a 2-cyanoethyl group at the phosphite moiety.4 Treatment of the oligonucleotide
with ammonia e€ects b-elimination in the 2-cyanoethyl group releasing internucleosidic phos-
phodiester moieties and acrylonitrile as a side product. Recently, acrylonitrile has been demon-
strated to alkylate nucleic bases under conditions of deprotection.5 Increasing interest in
manufacturing oligonucleotides as drugs motivated our search for alternate protecting groups,
whose removal is governed by mechanisms di€erent from b-elimination.
More than 40 years ago, Winstein et al. studied base-catalyzed cyclization of 2-bromoethyl N-
arylcarbamates to 3-aryl-1,3-oxazolidine-2-ones.6 We hypothesized that this reaction might also
occur with less ecient leaving groups e.g. the phosphodiester anion, which would provide a
novel type of protection for the internucleosidic phosphate, via the 2-(arylcarbamoyloxy)ethyl
group. Here we report the use of this protecting strategy in DNA synthesis by the phosphor-
amidite method.
* Corresponding author. Tel: 760 603 2445; fax: 760 929 0036; e-mail: aguzaev@isisph.com
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00911-4

5624
Scheme 1.
For the preparation of novel phosphoramidite building blocks, 1 was converted to a bisamidite
2.7 Without isolation, 2 was treated with 3, 4, or 58 in the presence of 1H-tetrazole to give, after
aqueous work-up, 6±8 (Scheme 1). These were isolated by column chromatography in 75±89%
yield and characterized.9 The phosphoramidites 6 and 8 were stable compounds whose shelf life
exceeded 6 months. Moreover, as measured by 31P NMR, the half-life of 8 in 95% aqueous
MeCN was 235 h, which indicated its high hydrolytic stability. In contrast, 7 was light-sensitive
and demonstrated a limited shelf life.
Next, the utility of the proposed protecting strategy for synthesis of oligonucleotides was studied.
The model oligonucleotides 9±11 were assembled on a commercial solid support (2 mmol)
employing 0.1 M of 6±8 in MeCN and the standard synthetic protocol (Scheme 1). When the
synthesis was completed, aliquots of 9±11 were treated with conc. aqueous ammonium hydroxide
at 25 or 55ꢀC. On removal of the ammonia and extraction of the aqueous solution with CH2Cl2,
the deprotection mixtures were analyzed by reverse phase HPLC±ESMS. The following obser-
vations are relevant to note. As judged by the DMTr-assay, all phosphoramidites demonstrated
high coupling eciency (>98%). Regardless of the oxidation protocol (I2 for phosphodiester and
3H-1,2-benzodithiol-3-one 1,1-dioxide10 for phosphorothioate oligonucleotides; X=O and S,
respectively), all protecting groups demonstrated excellent stability towards the conditions of the
chain assembly. Under treatment with ammonium hydroxide, the stability of the protecting
groups increased as follows: 4-dimethylaminophenyl<1-naphthyl<Ph (deprotection times of 14,
18, and 64 h at 25ꢀC; 1, 1.5, and 6 h at 55ꢀC for 10, 11, and 9, respectively). The deprotection
led exclusively to 12, with no appreciably detectable nucleic base or backbone modi®cations
occurring.
Taking into account the fact that the deprotection conditions for 10 and 11 were fairly similar
and that the phosphoramidite 7 was less stable than 8, the 2-[(1-naphthyl)carbamoyloxy]ethyl
group (NCE) was therefore considered as the protection of choice. Indeed, the deprotection time
for NCE is shorter than that for the standard base proꢀtecting groups ꢀ(6 h at 55ꢀC) and matched
that of the N,N-dimethylformamidino group (2 h at 50 C or 1 h at 65 C).11
In order to illustrate the deprotection mechanism of the NCE group, a model compound 13
was synthesized (Scheme 2). Compound 5 (1 equiv.) was reacted with diethyl chlorophosphate
(1.2 equiv.) in the presence of excess pyridine to give 13, which was isolated by column chroma-
tography in 86% yield.12

5625
Scheme 2. (i) Diethyl chlorophosphate/Py/CH2Cl2
The reactivity of 13 under basic conditions was next studied. When treated with a mixture of
conc. aqueous ammonium hydroxide and EtOH (4:1) at 55ꢀC, 13 was completely consumed in 5 h.
The reaction mixture was extracted with ethyl acetate and water, and 14 and 15 were isolated in
quantitative yields from the organic and aqueous layers, respectively.13 Similarly, when 11a was
synthesized and deprotected on a 5 mmol scale, the aqueous solution was extracted with CH2Cl2
to give 14 (10.5 mg, 90%), which was identical with the sample prepared according to Scheme 2
1
by HPLC and H NMR. In both examples, formation of 14 demonstrated that the deprotection
resulted from intramolecular cyclization of NCE where a phosphodiester group, either 15 or the
internucleosidic phosphate in 11, served as the leaving group.
To con®rm the neutral character of NCE protection towards nucleic bases, mixtures of 13 (10
equiv.) and each of the 50-DMTr protected nucleosides (dA, dC, dG, and T; 1 equiv.) were treated
with ammonium hydroxide as described above for 13 alone. As evidenced by HPLC, both 14 and
each of the nucleosides remained unchanged for at least 48 h at 55ꢀC.
In conclusion, the 2-[(1-naphthyl)carbamoyloxy]ethyl (NCE) is a novel, convenient phospho-
diester protecting group that is stable towards ancillary reagents used in DNA synthesis. NCE is
quantitatively removed under standard deprotection conditions to release an internucleosidic
phosphate and a neutral, inert by-product.
Acknowledgements
We thank Professor V. A. E®mov (Shemyakin & Ovchinnikov Institute of Bioorganic Chem-
istry, Moscow, Russia) for helpful discussion.
References
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5626
8. Ethylene glycol (6.45 g, 10.4 mmol) and 1-naphthyl isocyanate (4.40 g, 26 mmol) were reacted overnight in MeCN.
On evaporation, water (20 mL) was added, and the product was extracted with ethyl acetate (5Â20 mL), dried,
and evaporated. The residue was re-crystallized to give 5 (5.59 g 93%): m.p. 102.5±103ꢀC (toluene). H NMR
1
(DMSO-d6): ꢀ 9.59 (1H, s), 8.10 (1H, m), 7.93 (1H, m), 7.74 (1H, m), 7.6±7.4 (4H, m), 4.87 (1H, t, J=5.3 Hz), 4.16
(2H, t, J=5.2 Hz), 3.67 (2H, m). 13C NMR (CDCl3): ꢀ 155.1, 134.1, 132.3, 128.7, 126.3, 126.1, 125.8, 125.5, 120.7,
67.2, 61.5. FAB HRMS: calcd for C17H23NO6 (M+Na+): 254.0793; found: 254.0795.
9. Chloro bis[(N,N-diisopropyl)amino]phosphite (1.28 g, 4.8 mmol) in CH2Cl2 (5 mL) was added to 1 (2.18 g, 4.0
mmol) and EtN(iPr)2 (1.0 mL, 5.2 mmol) in CH2Cl2 (10 mL) at ^20ꢀC. This was kept at rt for 1 h and then treated
with 5 (1.11 g, 4.8 mmol) and 1H-tetrazole (140 mg, 2.0 mmol). After 2 h, 5% aqueous NaHCO3 and brine were
added. The product was extracted with ethyl acetate (3Â50 mL), dried, and evaporated. Separation on a silica gel
column using a gradient from 20:75:5 to 50:45:5 ethyl acetate:hexane:Et3N gave 8, fast diastereomer (0.51 g), 8,
1
slow diastereomer (0.45 g), and their mixture to total in 3.21 g (88.8%) of 8. Compound 8, fast diastereomer: H
NMR (CDCl3): ꢀ 8.89 (1H, br. s), 7.92±7.20 (17H, m), 7.07 (1H, s), 6.84±6.76 (4H, m), 6.42 (1H, m), 4.71 (1H, m),
4.45±4.16 (3H, m), 3.75 (6H, s), 3.90±3.30 (6H, m), 2.55 (1H, m), 2.36 (1H, m), 1.42 (3H, s), 1.19 (12H, d, J=5.5
Hz). 13C NMR (CDCl3): ꢀ 163.9, 158.7, 154.3, 150.4, 144.4, 135.7, 135.5, 134.1, 132.5, 130.2, 128.7, 128.2, 128.0,
127.2, 126.2, 126.0, 125.8, 125.2, 120.7, 113.3, 111.2, 86.9, 85.8, 84.9, 73.6, 73.3, 65.4, 65.3, 63.1, 61.8, 61.5, 55.3,
43.3, 43.1, 40.1, 24.7, 24.6, 11.8. 31P NMR (CDCl3): ꢀ 148.9. FAB HRMS: calcd for C50H57N4O10P (M+Na+):
1
927.3710; found: 927.3716. Compound 8, slow diastereomer: H NMR (CDCl3): ꢀ 7.94±7.76 (3H, m), 7.70±7.18
(16H, m), 6.88±6.76 (4H, m), 6.42 (1H, dd, J=7.9, 5.9 Hz), 4.67 (1H, m), 4.50±4.15 (3H, m), 3.77 (6H, s), 4.0±3.25
(8H, m), 2.63 (1H, ddd, J=13.5, 4.0, ꢁ1 Hz), 2.32 (1H, m), 1.41 (3H, s), 1.17 (6H, d, J=6.8 Hz), 1.08 (6H, d,
J=6.8 Hz). 13C NMR (CDCl3): ꢀ 163.8, 158.7, 154.5, 150.4, 144.4, 135.7, 135.5, 135.4, 134.1, 132.7, 130.1, 128.6,
128.2, 128.0, 127.2, 126.2, 126.0, 125.8, 125.2, 120.9, 113.3, 111.2, 86.9, 85.7, 85.5, 85.0, 74.1, 73.8, 65.4, 65.2, 63.4, 61.8,
61.5, 55.3, 43.2, 43.0, 40.1, 24.7, 24.5, 11.8. 31P NMR (CDCl3): ꢀ 148.5. FAB HRMS: calcd for C50H57N4O10P
(M+Na+): 927.3710; found: 927.3714.
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12. Compound 13: m.p. 52±53ꢀC. H NMR (CDCl3): ꢀ 7.88 (3H, m), 7.69 (1H, m), 7.54 (3H, m), 7.11 (1H, s), 4.44
1
(2H, m), 4.31 (2H, m), 4.13 (4H, sept., J=7.2 Hz), 1.33 (6H, t, J=7.2 Hz). 13C NMR (CDCl3): ꢀ 154.1, 134.1,
132.4, 128.7, 127.7, 126.3, 126.1, 125.8, 125.4, 120.7, 65.5, 65.4, 64.1, 16.2, 16.1. 31P NMR (CDCl3): ꢀ ^1.3. FAB
HRMS: calcd for C17H22NO6P (M+H+): 368.1263; found: 368.1262.
13. Compound 14: m.p. 125±126ꢀC (water). H NMR (CDCl3): ꢀ 7.88 (3H, m), 7.48 (4H, m), 4.66 (2H, m), 4.11 (2H,
1
m). 13C NMR (CDCl3): ꢀ 157.5, 134.7, 134.1, 130.0, 128.8, 127.0, 126.6, 125.7, 124.6, 122.4, 62.5, 49.1. FAB
1
HRMS: calcd for C13H11NO2 (M+H+): 214.0868; found: 214.0870. Compound 15 (ammonium salt): H NMR
(D2O): ꢀ 3.79 (4H, sept., J=7.2 Hz), 1.12 (6H, t, J=7.2 Hz). 31P NMR (D2O): ꢀ ^1.8.