Scaleable and efficient synthesis of 2'-deoxy-2'-N-phthaloyl nucleoside phosphoramidites for oligonucleotide synthesis.

Article abstract of DOI:10.1016/S0960-894X(02)00744-8

2'-Deoxy-2'-N-phthaloyl nucleosides were prepared from arabino nucleosides by triflate displacement with phthalimide in the presence of DBU. The corresponding phosphoramidites suitable for automated oligonucleotide synthesis were also synthesized. The scalability of described procedures was demonstrated on a 100-g scale preparation of 2'-deoxy-2'-amino-C phosphoramidite.

Full text of DOI:10.1016/S0960-894X(02)00744-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3345–3347
Scaleable and Efficient Synthesis of 20-Deoxy-20-N-phthaloyl
Nucleoside Phosphoramidites for Oligonucleotide Synthesis
Alexander Karpeisky,* David Sweedler, Peter Haeberli, Javier Read, Keith Jarvis
and Leonid Beigelman*
Department of Chemistry and Biochemistry, Ribozyme Pharmaceuticals Inc., 2950 Wilderness Place, Boulder, CO 80301, USA
Received 23 May 2002; accepted 1 August 2002
Abstract—20-Deoxy-20-N-phthaloyl nucleosides were prepared from arabino nucleosides by triflate displacement with phthalimide
in the presence of DBU. The corresponding phosphoramidites suitable for automated oligonucleotide synthesis were also synthe-
sized. The scalability of described procedures was demonstrated on a 100-g scale preparation of 20-deoxy-20-amino-C phosphor-
amidite.
# 2002 Elsevier Science Ltd. All rights reserved.
Structural modifications of oligonucleotides are becom-
ing increasingly important as their possible clinical
applications emerge.1ꢀ5 As support for a ‘New Ribo-
zyme Motifs’ program that is under development in our
laboratory we were interested in creating a quick and
scalable synthesis of 20-amino nucleoside phosphor-
amidites. We have previously shown6 that phthaloyl
protection of the 20-aminogroup during oligonucleotide
synthesis is preferable as compared to trifluoroacetyl or
Fmoc group. We also reported6 the synthesis of 20-N-
phthaloyluridine phosphoramidite starting from 20-ami-
nouridine using Nefkins’ method.7 However, this
procedure requires 20-aminonucleosides as starting
materials, which are not commercially available.
with subsequent reduction. In turn, 20-azidopurine
nucleosides were prepared by glycosylation with 20-
azido-20deoxy ribose derivatives,10 transglycosylation
with 20-amino-20-deoxyuridine,11 opening of 8,2-cyclo-
purine nucleosides with azide ion,12,13 and displacement
of the corresponding 20-arabino triflates with azide ion.14
We were interested in a large scale procedure that is
universal for purine and pyrimidine nucleosides which
involves fewer amounts of chemical steps and which
avoids the use of hazardous chemicals as lithium azide.
Earlier we reported on the preparation of 20-O-amino
ribonucleosides and their phosphoramidites utilizing
displacement of 20-O-triflyl group in the corresponding
arabino nucleosides with N-hydroxyphthalimide.15
We decided to apply the similar approach, but using
phthalimide or its derivatives.
The first preparation of 20-aminouridine was first
described by Verheyden et al.8 in 1971 by lithiumazide
opening of 2,20-O-anhydrouridine in 50% yield followed
by catalytic reduction to the corresponding amine. Sev-
eral reports elaborating this approach with minor
modifications have been published. A rather interesting
approach utilizing intramolecular cyclization of 30-O-
trichloroacetimidate of 2,20-O-anhydrouridine, followed
by acid hydrolysis has been recently published9 as an
alternative to use of azide ion. Methods for the synth-
esis of the 20-aminopurine nucleosides use the same
general strategy, namely, introduction of 20-azido group
The present communication describes syntheses of
20-amino uridine, adenosine and cytidine and their cor-
responding 20-deoxy-20-N-phthaloyl phosphoramidites.
Arabinonucleosides 1 (Fig. 1, X=Ura, or X=N4Ac-
Cyt16), 9 (Fig. 2) were protected with the tetra-
isopropyldisiloxyl group and then treated with
trifluromethanesulfonic anhydride (ara-U) or tri-
fluoromethanesulfonic chloride (ara-A or ara-CAc) to
give arabino derivatives 3 or 11. Inversion of configur-
ation by substitution with phthalimide in the presence
of DBU provided ribonucleosides 4, or 12 respectively
in 65–70% yield.17
*Corresponding author. Fax: +1-303-449-6995; e-mail: alkarp@
mindspring.com (A. Karpeisky); lnb@rpi.com (L. Beigelman)
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00744-8

3346
A. Karpeisky et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3345–3347
Figure 1. Synthesis of 20-deoxy-20-N-phthaloyl nucleosides and their phosphoramidites. Reagents and conditions: (i) TIPS-Cl/Pyr; (ii) (CF3SO2)O or
ꢁ
.
CF3SO2Cl, DMAP/CH2Cl2, 0 C, 3 h; (iii) PhthNH, DBU/MeCN; (iv) Et3NH HF/THF; (v) 40% aq MeNH2; (vi) DMT-Cl/Pyr; (vii) 2-cyanoethyl
N,N-diisopropyl chlorophosphoramidite.
Figure 2. Synthesis of 20-deoxy-20-N-phthaloyl adenosine and its phosphoramidite. Reagents and conditions: (i) TIPS-Cl/Pyr; (ii) CF3SO2Cl,
ꢁ
.
DMAP/CH2Cl2, 0 C, 3 h; (iii) PhthNH, DBU/MeCN; (iv) t-BuBzCl/Pyr, then morpholine; (v) Et3N HF/THF; (vi) DMT-Cl/Pyr; (vii) 40% aq
methylamine; (viii) 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite.

A. Karpeisky et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3345–3347
3347
Table 1. Triflate displacement with substituted phthalimides
References and Notes
Compd
Reaction conditions
Elimination
yield (%)
Yield of 4
from 2 (%)
1. Agrawal, S. Trends Biotech. 1996, 14, 376.
2. Usman, N.; Blatt, L. M. J. Clin. Inv. 2000, 106, 1197. Cris-
toffersen, R. E.; Marr, J. J. J. Med. Chem. 1995, 38, 2023.
3. Crooke, S. T.; Bennet, C. F. Annu. Rev. Pharmacol. Tox-
icol. 1996, 36, 107.
4a
60 ꢁC, 3 h, then rt.
Overnight
Rt, 20 h
70–80 ꢁC, 3 h
Rt, 20 h
Rt, 20 h
10–20
60
4b
4c
4d
4e
10–20
Traces
20
56
70
40
35
4. Gold, L. J. Biol. Chem. 1995, 270, 13581.
5. Usman, N.; Stinchcomb, D. T. In Catalytic RNA; Eckstein,
F.; Lilley, D. M. J., Eds.; Springer-Verlag: Heidelberg, 1996;
Vol.10, pp 243–264.
20
6. Beigelman, L.; Karpeisky, A.; Matulic-Adamic, J.; Haeberli,
P.; Sweedler, D.; Usman, N. Nucleic Acids Res. 1995, 23, 4434.
7. Nefkins, J. S. L.; Tesser, G. I.; Nivard, R. G. F. Recl. Trav.
Chim. Pays-Bas 1960, 79, 688.
8. Verheuden, J. P. H.; Wagner, D.; Moffat, J. G. J. Org.
Chem. 1971, 36, 250.
It is worth noting that substitutions with sodiumor
potassiumsalts of phthalimide as well as with DBU salt
in the presence or absence of DBU produced no reaction.
9. McGee, D. P.; Vaughunn-Settle, A.; Vargeese, C.; Zhai, Y.
J. Org. Chem. 1996, 61, 781.
10. Hobbs, J. B.; Eckstein, F. J. Org. Chem. 1977, 42, 714.
11. Imazawa, M.; Eckstein, F. J. Org. Chem. 1979, 44, 2039.
12. Ikehara, M.; Maruyama, T.; Miki, H.; Takatsuka, Y.
Chem. Pharm. Bull. 1977, 25, 754.
13. Ikehara, M.; Maruyama, T. Chem. Pharm. Bull. 1978, 26, 240.
14. Robins, M. J.; Hawrelak, S. D.; Hernandez, A. E.; Wnuk,
S. F. Nucleosides Nucleotides 1992, 11, 821.
15. Karpeisky, A.; Gonzalez, C.; Burgin, A. B.; Beigelman, L.
Tetrahedron Lett. 1998, 39, 1131.
The main side product on this step is the 20-deoxy-10,20-
didehydro-nucleoside,18 which is formed in competing
elimination reaction. This ‘elimination product’ can be
easily separated by crystallization. We also investigated
the effect of various substituents in the phenyl ring of
phthalimide on triflate displacement reaction (Table 1).
20-Arabino-triflate 3 (Fig. 1. X=N4-Ac-Cyt) was trea-
ted with 4,5-di-chloro-, 3,4,5,6-tetrachloro-, 3-nitro- or
4-nitro-phthalimides in the presence of DBU (1.2 equiv)
to produce corresponding 20-N-phthaloyl-cytidine deri-
vatives 4b–e. It is interesting to note that in case of tet-
rachlorophthalimide, the desired product was formed in
70% isolated yield and only traces of ‘elimination
product’ were detected in this reaction.
16. Bhat, V.; Ugarkar, B. G.; Sayeed, V. A.; Grimm, K.;
Kosora, N.; Domenico, P. A.; Stocker, E. Nucleosides
Nucleotides 1989, 8, 179.
17. Triflation and displacement with phthalimide (typical
procedure): To a solution of 50,30-tetraisopropyldisiloxyl-1-b-
d-arabinofuranosyl-N4-acetylcytosine 2 (71.5 g, 135.5 mmol),
DMAP (3 mmol) stirring at ꢀ10 ꢁC under argon in anhydrous
dichloromethane was added triflic chloride (1.2 mmol) drop-
wise via syringe. After stirring at 0 ꢁC for three h, TLC (70%
EtOAc) indicated complete reaction. Pyridine and DMAP
were removed by washing with cold 1.5% acetic acid in water
followed by aqueous sodiumbicarbonate. The organic layer
was dried over sodiumsulfate, filtered, and the filtrate evapo-
rated in vacuo. The triflate was used without further purifica-
tion. To a solution of 50,30-tetraisopropyldisiloxyl-20-O-triflyl-
The exocyclic amino group of adenosine derivative 12
(Fig. 2) was acylated with 2 equiv of t-butylbenzoyl
chloride to provide compound 13 after morpholine
hydrolysis.
.
Subsequent silyl deprotection (Et3N HF, 3 h, rt) affor-
ded 20-deoxy-20N-phthaloyl nucleosides 7 or 14 in 95%
yield. Application of the standard procedures of dime-
thoxytritylation and phosphitylation to these com-
pounds resulted in high yield (>90%) formation of the
corresponding phosphoramidites 519 and 17.19 20-Amino
nucleosides 8 and 16 were obtained by further hydro-
lysis of derivatives 7 and 14 with 40% aq methylamine
in nearly quantitative yields.
1-b-d-arabinofuranosyl-nucleoside
and,
phthalimide
(1.2 mmol) stirring at 60 ꢁC under argon in anhydrous aceto-
nitrile was added the solution of DBU (1.2 mmol) in acetoni-
trile slowly via self-equalized separatory funnel. The reaction
mixture was stirred at 60 ꢁC for 3 h and then overnight at
room temperature, at which time TLC (90% EtOAc/hexane)
indicated complete reaction. The precipitated ‘elimination
product’ was filtered off and washed with cold acetonitrile.
Combined mother liquor and washings were concentrated to a
minimal volume. The residue was diluted with dichloromethane
and washed with saturated sodiumbicarbonate solution. The
organic layer was then dried over sodiumsulfate, filtered, and
dried in vacuo. The residue was dissolved in ethyl acetate and
filtered through slicagel pad. The appropriate fractions were
combined and evaporated to dryness. The resulted product was
crystallized fromtoluene–hexane (1:2) to provide 53.4 g (60%
on compound 3) of 20-deoxy-20-N-phthaloyl-derivative 4a.
18.
In conclusion, we have identified a straightforward uni-
versal synthetic path to 20-deoxy-20-amino-nucleosides
and their phosphoramidites starting from arabinonu-
cleosides. Described synthetic procedures can be easily
scaled-up to a scale of 100 g and higher. In the case of
20-amino-C, the described process includes only one
flash chromatography step—purification of final phos-
phoramidite. All other intermediate products were
successfully
preparations in 40–50% overall yields on a 100 g scale.
crystallized,
allowing
cost-effective
Acknowledgements
Authors wish to thank Kevin Johnson for mass-spectral
analysis and Dr. Vladimir Serebryany for valuable sug-
gestions.
19. 31P NMR (CDCl3, d ppm): 5a (X=Ura): 151.395, 149.644;
5a (X=CytAc): 152.114, 150.621; 17: 151.509, 150.558.