A new approach for the synthesis of 3'-deoxy-3'-C-formyl-ribonucleosides and the synthesis of alternating methylene(methylimino) linked phosphodiester backbone oligonucleotides with 2'-OH and 2'-OMe groups

Article abstract of DOI:10.1016/S0040-4039(00)01814-1

A stereoselective synthesis of 3'-deoxy-3'-C-formyl-5-methyluridine 2 is described via the dithiane 4 as the key intermediate. Compound 2 was coupled with 3 into a novel ribo-MMI dimer 1. The dimer was then incorporated into antisense oligonucleotides which were found to have high binding affinity to the target RNA. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01814-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 9967–9971
A new approach for the synthesis of
3%-deoxy-3%-C-formyl-ribonucleosides and the synthesis of
alternating methylene(methylimino) linked phosphodiester
backbone oligonucleotides with 2%-OH and 2%-OMe groups
Marija Prhavc,a,b George Just,b,* Balkrishen Bhat,a P. Dan Cooka and
Muthiah Manoharana,*
aDepartment of Medicinal Chemistry, Isis Pharmaceuticals, Inc., Carlsbad, CA 92008, USA
bDepartment of Chemistry, McGill University, Montreal, Quebec, Canada H3A 2K6
Received 25 September 2000; accepted 3 October 2000
Abstract
A stereoselective synthesis of 3%-deoxy-3%-C-formyl-5-methyluridine 2 is described via the dithiane 4 as
the key intermediate. Compound 2 was coupled with 3 into a novel ribo-MMI dimer 1. The dimer was
then incorporated into antisense oligonucleotides which were found to have high binding affinity to the
target RNA. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: dithianes; nucleoside; nucleic acid analogs; antisense; oligonucleotides.
The methylene(methylimino), (MMI, 3%–CH2–N(Me)ꢀOꢀCH2–4%) modification, in which a
methylene group replaces the C-3% oxygen atom and a N-methylhydroxylamine replaces the
phosphodiester group, is a promising modification of the backbone of antisense oligonucleo-
tides.1 The synthesis of MMI-linked dimeric nucleosides has been studied extensively due to the
favorable properties of oligonucleotides containing alternating MMI and phosphodiester link-
ages,2 and analogs including 2%-deoxy-, 2%-deoxy-2%-fluoro-, and 2%-O-methyl ribo-derivatives
have been synthesized and characterized.3 Here we report the synthesis of a novel ribonucleoside
containing (2%-OH in the 5%-nucleoside end) MMI-dimer (1, Scheme 1) utilizing a unique
synthetic approach to 3%-deoxy-3%-C-formyl ribo-nucleosides. The 2%-OH may influence solubil-
ity, pharmacokinetics, and hybridization properties of the oligonucleotides. Biologically, the
native RNA structure with its 2%-OH group is crucial in determining many RNA secondary
structures (zippers, U-turns and bulges) and also in many nucleic acid–protein interactions.4 The
2%-OH may also act as a starting point for further modifications (e.g. alkylation).
* Corresponding authors.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01814-1

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Scheme 1.
Recently, a mild procedure for the synthesis of N,N,O-trisubstituted hydroxylamines via
reductive alkylation of N,O-disubstituted hydroxylamines with aldehydes has been developed
and applied to the synthesis of MMI-linked dinucleosides,5 with nucleoside aldehydes being
prepared by cis-dihydroxylation/oxidation of the corresponding olefins.5–7 In an analogous
fashion the synthesis of ribo-MMI dimer 1 through the coupling of 3%-deoxy-3%-C-formyl-5-
methyluridine (2) and 5%-O-methylaminouridine derivative 3 was envisaged (Scheme 1). Our
search for a versatile intermediate for the 5%-portion of the molecule led to the dithiane 4, since
thioacetals are masked aldehydes.8,9
The commercially available 1,2-O-isopropylidene- -xylose (5a) was quantitatively converted
D
into the corresponding 5-O-silyl ether 5b with triisopropylsilyl chloride (TIPSCl) (Scheme 2). A
20-fold excess of Ac2O/DMSO was required for the Moffat reaction10 to give the 3-ketosugar 6
in good yield (78%). Wittig condensation with 2-(trimethylsilyl)-1,3-dithiane11 afforded ketene
dithioacetal 7a in 60% yield. Attempts to reduce the double bond by catalytic hydrogenation
were unsuccessful. However, removal of the 5-O-TIPS protecting group (7b), followed by
treatment with LiAlH4 yielded the dithiane 4a exclusively in the ribo configuration12 in 80%
yield.13 The acetonide 4a was treated with Ac2O/AcOH/( )-camphor-10-sulfonic acid (CSA) at
70°C for 10 min14 to afford the triacetate 8a in 60% yield. Vorbru¨ggen coupling of 8a with
bis(trimethylsilyl)thymine15 gave the ribo-thymidine derivative 9a in quantitative yield (Scheme
3). In an alternate route to improve the moderate yield (60%) during the acetolysis of 4a, a
Scheme 2. Reagents and conditions: (i) TIPSCl, Et3N, DMAP, DMF, rt, overnight, 100%; (ii) DMSO, Ac2O, rt,
overnight, 78%; (iii) 2-TMS-1,3-dithiane, n-BuLi, THF, −78 to 0°C, overnight, 60%; (iv) TBAF, THF, 0°C, 0.5 h,
100%; (v) LiAlH4, THF, 55°C, 6 h, 80%; (vi) TBDPSCl, imidazole, DMF, rt, 2 h, 100%; (vii) Ac2O, AcOH, CSA,
70°C, 10 min; 60% for 8a, 80% for 8b

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Scheme 3. Reagents and conditions: (viii) (TMS)2T, TMSOTf, (CH2Cl)2, D, 0.5 h, 100%; (ix) 0.1N NaOH, MeOH,
rt, overnight, 95%; (x) HgO (9 equiv.), HgCl2 (3 equiv.), 90% aq. Me2CO, D, 1 day, 94%; (xi) 1 M PPTS (1 equiv.),
MeOH, 0°C, 8 M Py·BH3 (1 equiv.), 0°C–rt; 2 h, 60%; (xii) 1 M TBAF/THF, rt, 6 h, 100%; (xiii) DMTCl, Py,
DMAP, rt, overnight, 80%; (xiv) TBDPSCl, imidazole, CH2Cl2, rt, 8 h, 93%; (xv) Ac2O, Et3N, DMAP, MeCN, rt,
2 h, 85%; (xvi) TBAF(SiO2), THF, rt, 15 h, 82%; (xvii) 2-cyanoethyl-N,N,N%,N%-tetraisopropylphsphordiamidite,
diisopropylammonium tetrazolide, MeCN, rt, 22 h, 90%
5-O-TBDPS group was introduced to give 4b, which eventually increased the yield of the
reaction 4b“8b to 80%. Vorbru¨ggen coupling of 8b with bis(trimethylsilyl)thymine, followed by
cleavage of 2%-O-acetyl protection of 9b afforded the 3%-dithianyl ribo-thymidine 9c. Compound
9c was hydrolyzed in high yield (94%) to the 3%-C-formyl nucleoside 216 after a one day
treatment with HgO and HgCl2 (9 and 3 equivalents, respectively) in boiling 90% aqueous
acetone.17 The crude compound 2 was used for the reductive coupling with 5%-O-methylamino-5-
methyluridine (3).5 The resulting ribo-MMI-dimer 1a18 was isolated in 60% yield after purifica-
tion on silica column (Scheme 3). Deprotection with TBAF and dimethoxytritylation provided
the crystalline product 1c, which was further converted into MMI-phosphoramidite 1g19 in four
steps.
The ribo-MMI-dimer was incorporated into standard Isis oligonucleotide sequences (Table 1).
The oligonucleotides were synthesized with a 0.1 M CH2Cl2 solution of phosphoramidite 1g
following standard protocols with extended (3×15 min) couplings for all MMI dimers and using
(1S)-(+)-10-(camphorsulfonyl)oxaziridine as the oxidizer.20 The oligonucleotides were depro-
tected using standard conditions and purified by reverse phase HPLC. Electrospray mass
spectral measurements (ESMS) and capillary gel electrophoresis measurements confirmed the
integrity of the novel oligonucleotides. Helix to coil transition melting temperatures were
measured against RNA complements (Table 1). In all cases, substantial increase in Tm was
observed in comparison to unmodified oligodeoxyonucleotides.

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Table 1
Properties of MMI-linked chimeric (2%-OH/2%-OMe) oligonucleotides
Isis No.
Sequence (5%“3%)
Anal. HPLCa
Time (min)
18.4
Mass
Found
Tm (°C)
DTm/mod.b
Calcd
17221
17222
17223
CTC GTA CCT*TTC CGG TCC
CTC GTA CCT*TT*T CGG TCC 19.9
GCG T*TT*TT*TT*T*T GCG 20.6
5387.3 5386.8 67.9
5411.4 5411.1 67.9
4921.3 4921.1 67.2
+2.26
+3.19
+3.83
* Chimeric ribo-MMI backbone modifications.
a C-18 Waters Delta Pak (15 m; 3.8×300 mm) RP-column; solvents: (A) 50 mM triethylammonium acetate pH 7.0;
(B) MeCN; Linear gradient: 5–60% B in 60 min.
b Compared to unmodified DNA.
In summary, we have described a novel synthesis of 3%-deoxy-3%-C-formyl-5-methyluridine (2)
via the 3-deoxyribose-3-dithiane 4 as the key intermediate. This chemistry opens many avenues
in nucleoside and oligonucleotide synthesis for diagnostic and therapeutic applications. It also
allows the synthesis of a novel ribo-MMI dimer with 2%-OH and 2%-OMe in 5%- and 3%-
nucleosides, respectively. The MMI-dimer was incorporated into antisense oligonucleotides,
which have high binding affinity to the target RNA. It is already known that the MMI
backbone provides extremely high nuclease stability to antisense oligonucleotides by providing
stability to adjacent phosphate linkages. Protein binding and pharmacology of this new class of
antisense compounds will have to be evaluated.
Acknowledgements
Dr. Elena Lesnik carried out the Tm studies. 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 Research Council of Canada.
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3.30 (dd, 1H, H-3%; J3%,4%=9.4, J3%,2%=5.7 Hz), 3.95 (dd, 1H, H-5%a; J5%a,5%b=12.2, J5%a,4%=2.4 Hz), 4.26 (dd, 1H,
H-5%b; J5%b,4%=1.8 Hz), 4.79 (apparent td, 1H, H-4%), 4.90 (d, 1H, H-2%), 5.75 (s, 1H, H-1%), 7.33–7.44 and
7.59–7.65 (2m, 10H, 2Ph), 7.67 (q, 1H, H-6), 9.83 (s, 1H, CHO), 10.24 (s, 1H, NH). 13C NMR (CDCl3) l 12.00
(C5-Me), 19.45 (C6 Me3), 27.03 (CMe3), 52.63 (C-3%), 63.11 (C-5%), 77.31 (C-2%), 80.90 (C-4%), 93.26 (C-1%), 110.75
(C-5), 127.95, 128.04, 130.07, 130.17, 132.26, 133.04, 135.18, 135.44 (2Ph, C-6), 150.84 (C-4), 164.43 (C-2), 197.81
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1
18. Compound 1a: H NMR ((CD3)2CO) (U1 and U2 represent 3%- and 5%-substituted uridine moieties, respectively)
l 1.08 (s, 18H, 2 t-Bu), 1.45 (d, 3H, U1:C-5-Me; JMe,H-6=0.7 Hz), 1.74 (d, 3H, U2:C-5-Me; JMe,H-6=1.0 Hz),
2.52 (s, 3H, NMe), 2.54 (m, 1H, U1:H-3%), 2.80 (m, 1H, H-3¦a), 3.07 (dd, 1H, H-3¦b; J3¦a,3¦b=11.3, J3%,3¦b=8.2
Hz), 3.21 (s, 3H, OMe), 3.49 (t, 1H, U2:H-2%; J1%,2%=4.1 Hz), 3.67 (dd, 1H, U2:H-5%a; J5%a,5%b=11.2, J5%a,4%=4.2 Hz),
3.84 (m, 1H, U2:H-5%b), 3.89 (dd, 1H, U1:H-5%a; J5%a,5%b=11.7, J5%a,4%=4.2 Hz), 4.10–4.19 (3m, 3H, U1:H-5%b,
U1:H-4%, U2:H-4%), 4.23 (t, 1H, U2:H-3%; J3%,4%=J3%,2%=5.4 Hz), 4.46 (m, 1H, U1:H-2%), 4.75 (d, 1H, 2%-OH;
J
2%,2%-OH=2.7 Hz), 5.82 (d, 1H, U1:H-1%; J1%,2%=2.2 Hz), 5.93 (d, 1H, U2:H-1%; J1%,2%=3.7 Hz), 7.38–7.48, 7.66–7.69
and 7.71–7.77 (3m, 22H, 4Ph, 2H-6), 9.98 (s, 2H, 2NH). 13C NMR ((CD3)2CO) l 12.01, 12.40 (2C5-Me), 19.21,
19.42 (2CMe3), 26.81, 27.03 (2CMe3), 38.89 (NMe), 44.95 (U1:C-3%), 56.00 (U1:C-3¦), 57.73 (OMe), 63.51
6
(U1:C-5%), 70.36 (U2:C-5%, C-3%), 76.74 (U2:C-2%), 81.41 (U1:C-2%), 82.34, 83.38 (2C-4%), 88.77, 92.28 (2C-1%),
110.50, 110.56 (2C-5), 127.65, 127.79, 127.90, 127.94, 130.03, 130.08, 132.53, 132.77, 132.93, 133.01, 135.25,
135.42, 135.59, 135.73 (4Ph, 2C-6), 149.92, 150.71 (2C-4), 163.87, 164.09 (2C-2). ESMS: m/z 1032.7 (M+);
C55H69N5O11Si2 requires 1032.5.
19. Compound 1g: 31P NMR (CD3CN) l 151.21 and 150.95.
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.
.