Chimeric RNA with modified backbones: Alternating methylene(methylimino) linked phosphodiester backbone oligonucleotides with 2′-OH and 2′-OMe groups

Article abstract of DOI:10.1081/NCN-100002476

Synthesis of a novel ribo-MMI dimer with 2′-OH and 2′-OMe in 5′- and 3′-nucleosides, respectively is presented. The synthesis was accomplished by reductive coupling of 3′-deoxy-3′-C-formyluridine and 2′-O-methyl-5′-O-methylaminouridine via a thioacetal as

Full text of DOI:10.1081/NCN-100002476

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CHIMERIC RNA WITH MODIFIED BACKBONES:
ALTERNATING METHYLENE(METHYLIMINO) LINKED
PHOSPHODIESTER BACKBONE OLIGONUCLEOTIDES
WITH 2′-OH AND 2′-OMe GROUPS
Marija Prhavc a , Balkrishen Bhat a , George Just b , P. Dan Cook a & Muthiah Manoharan a
a Department of Medicinal Chemistry , Isis Pharmaceuticals, Inc. , Carlsbad, California,
U.S.A.
b Department of Chemistry , McGill University , Montreal, Quebec, Canada
Published online: 07 Feb 2007.
To cite this article: Marija Prhavc , Balkrishen Bhat , George Just , P. Dan Cook & Muthiah Manoharan (2001) CHIMERIC
RNA WITH MODIFIED BACKBONES: ALTERNATING METHYLENE(METHYLIMINO) LINKED PHOSPHODIESTER BACKBONE
OLIGONUCLEOTIDES WITH 2′-OH AND 2′-OMe GROUPS, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 995-997, DOI:
10.1081/NCN-100002476
To link to this article: http://dx.doi.org/10.1081/NCN-100002476
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 995–997 (2001)
CHIMERIC RNA WITH MODIFIED BACKBONES:
ALTERNATING METHYLENE(METHYLIMINO)
LINKED PHOSPHODIESTER BACKBONE
OLIGONUCLEOTIDES WITH 2ꢀ-OH
AND 2ꢀ-OMe GROUPS
Marija Prhavc,1,2,∗ Balkrishen Bhat,1 George Just,2
P. Dan Cook,1 and Muthiah Manoharan1
1Department of Medicinal Chemistry, Isis Pharmaceuticals,
Inc., Carlsbad, California
2Department of Chemistry, McGill University, Montreal,
Quebec, Canada
ABSTRACT
Synthesis of a novel ribo-MMI dimer with 2ꢀ-OH and 2ꢀ-OMe in 5ꢀ- and
3ꢀ-nucleosides, respectivelyispresented. Thesynthesiswasaccomplishedbyre-
ductivecouplingof3ꢀ-deoxy-3ꢀ-C-formyluridineand2ꢀ-O-methyl-5ꢀ-O-methy-
laminouridine via a thioacetal as the key intermediate for the top part of the
dimer. Incorporation of ribo- MMI dimers into oligonucleotides increased bind-
ing affinity for target RNA.
The MMI modification, a methylene group replacing the C-3ꢀ oxygen atom
and the phosphodiester group replaced by a N-methylhydroxylamine (methylene-
(methyl)imino, 3ꢀ-CH2-N(Me)-O-CH2-4ꢀ), is one of the most interesting back-
bone modifications of antisense oligonucleotides (1). The synthesis of MMI-linked
dimeric nucleosides has been studied extensively due to the favorable properties of
the oligonucleotides containing MMI and phosphodiester linkages (2). Analogs
including 2ꢀ-deoxy-, 2ꢀ-deoxy-2ꢀ-fluoro- and 2ꢀ-O-methyl ribo derivatives have
been synthesized and characterized (3) (Fig. 1). Here we report the synthesis of
∗Corresponding author.
995
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

ORDER
REPRINTS
996
HO
PRHAVC ET AL.
B
X
H
H
H
Y
H
F
OMe
Achiral
Neutral
O
X
Totally synthetic
NMe
O
Linkage is not a substrate for nuclease
Lipophilic
OMe OMe
B
MMI
F
F
F
OMe
O
Enhanced binding to RNA target
OMe F
OH
Y
Figure 1. MMI backbone.
RO
RO
T
HO
T
O
2
O
O
O
S
OH
O
4
OH
NMe
O
S
O
MeHNO
T
T
O
O
OH OMe
1
OMe
TBDPSO
3
Scheme 1. Retrosynthetic anayisis of Ribo-MMI-dimer.
HO
TIPSO
TIPSO
S
OH
iv, v
i, ii
iii
O
O
6
O
O
O
O
O
O
O
O
O
S
5
7
TBDPSO
HO
S
TBDPSO
S
T
R
ix, x
vi, vii
O
O
O
R
OH
O
OR1
R1
O
S
S
2
4
8 Ac Ac
9 T
viii
H
Scheme 2. Synthesis of 3ꢀ-deoxy-3ꢀ-C-formyl-5-methyluridine. Reagents and conditions: i. TIPSCI,
Et3N, DMAP, DMF, rt, overnight, 100%; ii. DMSO, AC2O, rt, overnight, 78%; iii. 2-TMS-1, 3-
dithiane, n-BuLi, THF, −78◦C to 0◦C, overnight, 60%; iv. TBAF, THF, 0◦C, 0.5 h, 100%; v. LiAlH4,
THF, 55◦C, 6 h, 60%; vi. TBDPSCl, Imidazole, DMF, rt, 2 h, 100%; vii. Ac2O, AcOH, CSA, 70◦C,
10 min; 80%; viii. (TMS)2T, TMSOTf, (CH2Cl)2, ꢀ, 0.5 h; ix 0.1 N NaOH, MeOH, overnight, 95%;
x. HgO (9 equiv), HgCl2 (3 equiv), 90% aq. Me2CO, ꢀ, 1d, 94%.

ORDER
REPRINTS
CHIMERIC RNA WITH MODIFIED BACKBONES
997
a ribonucleoside (Fig. 1: X = OH, Y = OMe) MMI dimer. We wanted to examine
the influence of 2ꢀ-OH on the solubility, pharmacokinetics and hybridization prop-
erties of the oligonucleotides. The 2ꢀ-OH substitution may also act as a starting
point for further modifications (e.g. alkylation).
Retrosynthetic analysis (Scheme 1) of the desired dimer 1 indicated that dithi-
ane 4 could serve as the key intermediate for the top part since thioacetals are known
as masked aldehydes (4,5).
The synthesis started from commercially available 1,2-O-isopropylidene-D-
xylose (5) (Scheme 2) which was upon 5-O silylation converted into 3-ketosugar
6. Wittig condensation of 6 with 2-(trimethylsily)-1,3-dithiane followed by hydro-
genation of ketene dithioacetal 7 with LiAlH4 yielded dithiane 4. Further acetol-
ysis (6) of 5-O-TBDPS derivative and Vorbru¨ggen coupling of diacetate 8 with
bis(trimethylsilyl)thymine followed by cleavage of 2ꢀ-O acetyl protection afforded
3ꢀ-dithianyl ribo-thymidine 9 which was successfully hydrolyzed with HgO and
HgCl2 in aqueous acetone (7) into 3ꢀ-C-formyl nucleoside 2. Compound 2 was
used for the reductive coupling with 5ꢀ-O-methylaminouridine 3 (8) into ribo-
MMI-dimer 1 which was converted into phosphoramidite and incorporated into
standard Isis sequences. Melting temperatures were measured against unmodified
RNA compliments. In all cases a substantial increase (nearly +3◦C/modification)
was observed in comparison to unmodified oligodeoxy-nucleotides.
ACKNOWLEDGMENT
M.P. was a postdoctoral fellow at McGill University on leave from National
Institute of Chemistry, Ljubljana, Slovenia, and wishes to acknowledge the financial
support from Slovenian Ministry of Science and Technology and the National
Science and Engineering Council of Canada.
REFERENCES
1. Vasseur, J-J.; Debart, F.; Sanghvi, Y. S.; Cook, P. D. J. Am. Chem. Soc. 1992, 114, 4006.
2. Bhat, B.; Swayze, E. E.; Wheeler, P; Dimock, S.; Perbost, M.; Sanghvi, Y. S. J. Org.
Chem. 1996, 61, 8186.
3. Sanghvi, Y. S.; Swayze, E. E.; Peoc’h, D.; Bhat, B.; Dimock, S. Nucleosides &
Nucleotides, 1997, 16, 907.
4. For the review see: Gro¨bel, B.-T.; Seebach, D. Synthesis, 1977, 357.
5. Bamford, M. J.; Coe, P. L.; Walker, R. T. J. Med. Chem. 1990, 33, 2494.
6. Kawai, S. H.; Chin, J.; Just, G. Carbohydr. Res. 1991, 211, 245.
7. Just, G.; Oh, H. Can. J. Chem. 1981, 59, 2729.
8. Swayze, E. E.; Sanghvi, Y. Synlett, 1997, 859.

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