Synthesis of 2′-Substituted MMI Linked Nucleosidic Dimers: An Optimization Study in Search of High Affinity Oligonucleotides for Use in Antisense Constructs

Article abstract of DOI:10.1081/NCN-120028337

The synthesis of a series of methylene(methylimino) (MMI) linked oligodeoxyribonucleotide dimers modified at the 2′-position with fluoro and/or methoxy groups and their incorporation into different sequences has been accomplished. From these dimers, bis 2

Full text of DOI:10.1081/NCN-120028337

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Synthesis of 2′‐Substituted MMI Linked Nucleosidic
Dimers: An Optimization Study in Search of High
Affinity Oligonucleotides for Use in Antisense
Constructs
Didier Peoc'h a , Eric E. Swayze a , Balkrishen Bhat a & Yogesh S. Sanghvi a
a Department of Medicinal Chemistry , Isis Pharmaceuticals, Inc. , 2292 Faraday Avenue,
Carlsbad, California, 92008, USA
Published online: 01 Jun 2007.
To cite this article: Didier Peoc'h , Eric E. Swayze , Balkrishen Bhat & Yogesh S. Sanghvi (2004) Synthesis of 2′‐Substituted
MMI Linked Nucleosidic Dimers: An Optimization Study in Search of High Affinity Oligonucleotides for Use in Antisense
Constructs , Nucleosides, Nucleotides and Nucleic Acids, 23:1-2, 411-438, DOI: 10.1081/NCN-120028337
To link to this article: http://dx.doi.org/10.1081/NCN-120028337
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 1 & 2, pp. 411–438, 2004
Synthesis of 2’-Substituted MMI Linked Nucleosidic
Dimers: An Optimization Study in Search of
High Affinity Oligonucleotides for Use in
Antisense Constructsy
*
Didier Peoc’h, Eric E. Swayze, Balkrishen Bhat, and Yogesh S. Sanghvi
Department of Medicinal Chemistry, Isis Pharmaceuticals, Inc.,
Carlsbad, California, USA
ABSTRACT
The synthesis of a series of methylene(methylimino) (MMI) linked oligodeoxyri-
bonucleotide dimers modified at the 2’-position with fluoro and/or methoxy groups
and their incorporation into different sequences has been accomplished. From these
dimers, bis 2’-OMe MMI dimer was selected for further studies based on its synthetic
accessibility and the increased thermodynamic stability conferred upon oligonucleo-
tides incorporating this modification.
Key Words: Antisense; Oligonucleotide; MMI dimer.
yIn honor and celebration of the 70th birthday of Professor Leroy B. Townsend.
*
Correspondence: Eric E. Swayze, Department of Medicinal Chemistry, Isis Pharmaceuticals,
Inc., 2292 Faraday Ave., Carlsbad, CA 92008, USA; Fax: (760) 603-4653; E-mail: eswayze@
isisph.com.
411
DOI: 10.1081/NCN-120028337
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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INTRODUCTION
Replacement of the natural phosphodiester linkage of nucleic acids with a nonionic
dephosphono linkage has proven to be a useful strategy for the preparation of antisense
oligomers exhibiting enhanced nucleolytic stability and higher binding affinity for
the complement RNA.[1–4] The prospects of improved cellular uptake with oligomeric
constructs that are more lipophilic and carry reduced net negative charge than natural
DNA and RNA has provided further stimulation for the synthesis of such surrogates.[5] Our
investigations in this area have resulted in the discovery of the methylene (methylimimo)
(MMI) linkage, which we have found to be an excellent backbone modification, as
it confers several desirable attributes on to the resulting antisense oligomer.[6]
In addition to the modification of 3’ ! 5’ linkages, modifications at the 2’-position
of nucleic acids with a variety of substituents have been successfully utilized in
antisense strategies leading to oligomeric analogs with improved affinity.[7] In certain
cases the improved affinity has translated well into increased biological activity in cell
based experiments, as well as higher efficacy in animals.[8] Therefore, a logical choice
was to combine one of our best backbone modifications (i.e. MMI) with an appropriate
2’-sugar substituent to create oligomers that may exhibit higher affinity towards their
corresponding RNA target, as well as generally improved antisense properties.
Damha et al.,[9] Grayznov et al.,[10,11] Reynolds et al.[12] and DeMesmaeker et
al.[13] have independently published on such additive and selective effects on the
affinity for complement RNA over DNA, in which a 2’-substituent was added to a
backbone modified oligomer. However we believe that a systematic optimization of the
2’-substituents has never been attempted with a series of suitable functionalities in
combination with a single nonionic backbone modification. We elected the 2’-F, 2’-
OMe, 2’-O(CH2)2OMe as the suitable 2’-functionalities for our studies based on their
high affinity profile.[14] Herein, we describe the synthesis of seven 2’-substituted MMI
linked nucleosidic dimers (1–3), their conversions to the standard phosphoramidites,
and subsequent incorporation into oligonucleotide via an automatic DNA synthesizer.
The hybridization data on doubly modified oligomers is also presented which allowed
the selection of a preferred 2’-substituent in combination with the MMI linkage.
CHEMISTRY AND DISCUSSION
Our initial investigations in the 2’-deoxynucleoside series of dimeric units linked
via an MMI bridge has resulted in two principal synthetic routes. First, a reductive
coupling procedure[15] and second, a radical coupling[16] approach. The latter route has
been published[17] covering the synthesis of various MMI linked dimers both in purine
and pyrimidine bases. This methodology worked exceedingly well toward the
preparation of the 2’-deoxy series of MMI dimers with complete stereo control of
the 3’-a- C–C bond formation. However, when this procedure was applied to the 2’-
OMe series, a nonstereoselective addition occurred (5–25% of b-isomer) with modest
yields of purine containing dimers. In view of this, we chose to pursue the synthesis of
2’-modified dimers 1–3 utilizing the reductive coupling methodology (Figure 1).
We opted to synthesize dimers 1a-c first for the following reasons. We had
previously known that the top sugar residue (with 3’-C C bond) of the 2’-deoxy MMI

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413
Figure 1. 2’-modified MMI dimers.
dimer (1, x = H) exists predominantly in an RNA-like conformation (C3’-endo)
whereas the bottom sugar residue (with 3’-C O bond) displays frequent transitions
between N () S puckered states.[18] Such unproductive motion of the sugar
conformation can be reduced via a placement of a 2’-electronegative substituent
in nucleosides and oligonucleotides.[19] It was therefore hoped that placement of a
2’-substituent on the lower sugar of an MMI linked dimer might drive adoption of a 3’-
endo conformation. This would result in a uniform RNA like structure when assembled
into an oligomer, and thereby increase the affinity for the RNA target. Thymine (5–
methyluracil) bases were utilized as placement of a methyl group at the C5 in
pyrimidines is well known to alter the hydration resulting in an enhancement of affinity
for the target RNA.[20,21] Additionally, use of a thymine residue allows incorporation
into oligonucleotides without base protection.
The synthesis of dimers 1a-c was envisaged via coupling of 3’-C-formyl nucleoside
8 with the 5’-O-amino derivative 7 to provide an oxime dimer 9 which upon reduction
followed by methylation and deprotection would give the desired dimer. The common
top piece 8 was synthesized in a facile manner using an intermolecular radical C
C
bond formation reaction reported previously.[22] The synthesis of the three-bottom
pieces 7a-c is depicted in Scheme 1. The commercial availabilitya of 5-methyluridine
4d allowed a convenient starting-point for the synthesis of 5d. Treatment of 4d under
Mitsunobo conditions (HOPhth/Ph3P/DIPAD/DMF)[25] furnished 5d (69%) in a
regioselective manner. Subsequent methylation of 5d using a modified protocol
[(Bu)2SnO/(Bu)4NI/CH3I/DMF)][26] gave 2’-OMe 5a in 70% yield. However, a small
amount (< 10%) of 3’-OMe isomer was also formed and removed via chromatography.
The 2’-OMe 5a was then silylated (TBDPSCl/imidazole/DMF) to give 3’-O-silylated 6a
(92%), which upon hydrazinolysis (H3CNHNH2) gave 5’-O-amino 7a (79%) as
crystalline product.
aPurchased from Yamasa Corporation, Summit Pharmaceuticals, 400 Kelby St., Fort Lee, NJ
07024. For a chemical synthesis see Ref. [23]. For an enzymatic synthesis see Ref. [24].

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Peoc’h et al.
Scheme 1. Reagents and Conditions: (i) N-hydroxyphthalimide/DEAD/Ph3P/DMF; (ii) dibutyltin
oxide/NaH/MeI; t-BuSiPh2Cl/imidazole/DMF; (iv) MeNHNH2/CH2Cl2. Abbreviations: T = thy-
mine; TBDPS = t-Bu(Ph2)Si.
In order to avoid the formation of minor 3’-isomer, we decided to utilize 4b[27] and
4c[28] as pure 2’-substituted nucleosides for the synthesis of 7b and 7c, respectively.
Both 4b and 4c underwent standard Mitsunobu reaction to provide 5b (70%) and 5c
(38%) which upon silylation, followed by hydrazinolysis of the products furnished 7b
and 7c, respectively, in good yield. Coupling of the three aldehyde containing bottom
pieces 7a-c to the hydroxylamine 8, was accomplished utilizing acid catalyzed coupling
conditions, which were previously found to be essentially quantitative both in solution
and solid-support.[29] In this manner, an equimolar mixture of 8 and 7a-c in CH2Cl2/
tolune/AcOH was coevaporated repeatedly until 8 was fully consumed (TLC)
(Scheme 2). The residue was then treated independently with NaBH3CN in AcOH,
followed by addition of aq. HCHO and more NaBH3CN to furnish 10a-c, respectively,
in good to excellent yields. This condensed procedure allows three reactions to be
performed in one-pot (coupling/reduction/methylation), and enables the rapid construc-
tion of such dimers.[30] Desilylation (TBAF/THF) of 10a-c furnished 1a-c, respectively,
in good yield. Diols 1a-c were dimethoxytritylated and the products (23a-c) were
phosphitylated in a standard manner[31] to provide 24a-c, respectively, in good overall
yield (Scheme 6).

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415
Scheme 2. Reagents and Conditions: (i) PhCH3/CH2Cl2/catalytic AcOH; (ii) NaBH3CN/CH2O/
AcOH; (iii) TBAF/THF.
The phosphoramidites 24a-c were then incorporated once, twice or five times into
an oligomeric sequence using a standard[32] automated DNA synthesizer. The modified
oligomers were HPLC purified and characterized by CGE and ES-MS (for experimental
details see Refs. [33,34]). The results of the Tm studies are summarized in the Table 1.
All three modifications (i.e. 2’-OMe, 2’-F, and 2’-MOE) had significant stabilizing
effects in duplex formation, with a DTm of roughly 2°C per incorporation, when
compared to MMI alone without the 2’-substituent in a heavily modified sequence
(Table 1, column A). As predicted, placement of an appropriate 2’-substituent in the
bottom sugar residue of an MMI dimer was indeed able to enhance the affinity of the
modified oligomer for its complement RNA.
These results encouraged us to further explore the effects of 2’-modifications on
the hybridization of MMI linked dimers of type 2 and 3, in which both sugar residues
have been altered. Since the effects of 2’-OMe and 2’-OCH2CH2OCH3 substituents in
the bottom residue were similar and to minimize the synthetic efforts, we chose to
synthesize and study four more dimers (2a,b and 3a,b) containing combinations of 2’-F
and 2’-OMe substituents. We elected to employ a similar reductive coupling procedure
for the synthesis of these new dimers, which requires access to nucleosides bearing a

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Peoc’h et al.
Table 1. Effects of 2’-substituent on hybridization of MMI linked oligonucleotides to RNA.
2’-Substituent
X (bottom)a
DTm per MMI dimer incorporationb
Y (top)a
Ac
Bc
Cc
Average DTm
H
H
0.13
1.83
2.27
2.13
3.27
3.74
3.13
3.71
À 0.23
1.00
1.67
1.61
2.20
3.10
2.50
2.78
1.51
À 0.15
0.87
0.47
0.89
1.60
1.56
2.31
2.93
2.39
2.78
H
F
H
OMe
MOEd
F
H
0.95
1.47
F
F
OMe
F
1.95
1.56
OMe
OMe
OMe
1.85
a2’-substituent at the top or the bottom sugar residue(s) of the MMI dimer.
bDTm per MMI dimer incorporation is the increase in Tm of the MMI containing
oligonucleotide:RNA duplex relative to an unmodified DNA:RNA duplex, normalized to the
number of MMI dimers contained within the sequence. See Ref. [15] for the experimental details on
measurement of Tms.
cSequences of modified oligos (* = MMI linked dimer, other linkages are phosphodiester):
A = GCG T*T T*T T*T T*T T*T GCG; B = CTC GTA CT*T T*TC CGG TCC; C = CTC GTA
CCT*TTC CGG TCC.
3’-C-formyl functionality and a 2’-F or 2’-OMe substituent. A literature search revealed
that Walker and his group[35] have prepared a 3’-C-functionalized nucleoside 11, which
would serve as a convenient intermediate if an inversion of the 2’-hydroxyl to a 2’-F or
2’-OMe substituent could be accomplished. Scheme 3 summarizes our attempts to
accomplish the intended inversion at the 2’-position of 11. Mesylation of 11 fur-
nished 12a in 75% yield. Traditional methodologies[36–38] [C6H5CO2H/CsF/DMF;
C2H5CO2Cs/DMF; (Bu)4N+OH/H2O; NaOMe/MeOH] of inversion failed in our hands
to provide 13a. In most of these attempts mesylate 12a was recovered unchanged.
Therefore, 11 was transformed to triflate[39]12b (70%), which was found to be fairly
stable and amenable to chromatographic purification. Again, our efforts to convert 12b
to 13a utilizing standard conditions described above failed. Our difficulties inverting
12b with a nucleophile continued with our attempts to prepare the 2’-fluoro substituted
13b. Treatment of 12b with anhydrous TBAF[38] in THF furnished 13b and 13c (1:1)
due to concomitant deprotection of 5’-O-silyl group. However, silylation (TBDPSCCl/
imidazole/DMF) of the mixture furnished 13b in 70% yield. In order to unmask the 3’-

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Scheme 3. Reagents and Conditions: (i) CH3SO2Cl/i-Pr2NEt/CH2Cl2; (ii) (CF3SO2)2/pyridine/
CH2Cl2; (iii) TBAF/THF; (iv) HgO/HgCl2/THF/H2O.
C-formyl group, 13b was treated with HgO/HgCl2 in wet THF at 0°C, furnishing a
more polar product in 75% yield. The latter product was characterized as 14 based on
1H NMR, elemental analysis and HRMS. Additionally, the spectral data for 14 agreed
with that of previously obtained uracil derivative reported by Walker and his group.[35]
Exclusive formation of 14 from 13b presumably occurs via mercuric salt catalyzed
hydrolysis to provide the desired 3-C-formyl product 13d, which subsequently
eliminates HF to furnish 14. Extensive attempts at modification of the reaction
conditions to obtain 13d were unsuccessful in our hands. The facile elimination can be
attributed to the trans juxtaposition of an acidic 3’-H portion and a 2’-fluoro substituent
as a good leaving group in the intermediate nucleoside 13d during the reaction. Taken
together, these unsuccessful attempts at preparation of 13a and 13d from 11 forced us
to explore alternate routes.
One alternative strategy to gain access to 3’-C-formyl-2’-O-methyl substituted 18
is depicted in Scheme 4. To this end, we[22] have successfully demonstrated that b-
tributylstannylstyrene (TBBS) mediated C C bond formation was able to generate 3’-
C-formyl functionality in the 2’-deoxynucleosides. Herein we now report an extension
of our study to install a C C bond in the 2’-OMe series of ribonucleosides. The
synthesis of thymine analog 18 commenced with the commercial 15a, which reacted

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Peoc’h et al.
Scheme 4. Reagents and Conditions: (i) t-BuSiPh2Cl/pyridine/DMAP; (ii) p-MePhO(CS)Cl/
DMAP/CH2Cl2; (iii) Bu3SnCH = CHPh/AIBN/PhH/D; (iv) OsO4/NaIO4/dioxane; (v) 7a or 7b/
PhCH3/CH2Cl2/catalytic AcOH then NaBH3CN/CH2O/AcOH; (vi) TBAF/THF. Abbreviations:
DMT = 4,4’-dimethoxytriphenylmethyl.
with p-tolyl chlorothionoformate and an excess of DMAP in CH3CN to furnish 16a
(85%). Reaction of 16a with TBBS under the conditions described earlier afforded the
3’-C-styryl derivative 17a (45%) and a small amount of 3’-deoxy nucleoside (ꢀ 10%).
The latter product was easily separated from the desired 17a via column
chromatography. Oxidative-cleavage of 17a furnished a viable synthesis of 2’-OMe-
3’-C-formyl nucleoside. The next consideration was the synthesis of 2’-fluoro derivative
17b (Y = F) from the 2’-fluoro 4b following a similar pathway. Silylation of 4b gave

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419
15b (98%), which upon thionoformylation gave 16b (68%). Reaction of 16b under
standard radical condition furnished a complex mixture of products. The major isolated
product was characterized as a mixture of two stereoisomers at the C3’-position bearing
an a- and b-styrene substituent. The loss of stereoselectivity in this case is not
particularly surprising because of a highly electronegative 2’-fluoro substituent that may
control the outcome of radical reaction.[17]
Coupling of 18 with 7a or 7b under standard one-pot procedure furnished 19a and
19b, respectively, with concomitant deprotection of the 5’-O-protecting group.
Desilylation of 19a and 19b furnished 2a and 2b, respectively, in good yield.
Subsequently, 2a and 2b were transformed to the corresponding 5’-O-DMT-3’-O-
amidites 24d and 24e, respectively, following the standard protocol in excellent yield.
Various MMI modified oligonucleotides were prepared via incorporations of 24d and
24e and their Tms measured. Table 1 summarizes the hybridization data. The average
DTms per modification were markedly increased via placement of a 2’-OMe substituent
in the top sugar residue in combination with 2’-OMe or 2’-F substituents in the bottom
sugar residues. This is likely due to affecting a C3’–endo conformational preorganiza-
tion with bis-2’-substituted MMI modifications which facilitates binding to the
complement RNA with high affinity. Additional factors such as reduced interstrand
Scheme 5. Reagents and Conditions: (i) 7a or 7b/PhCH3/CH2Cl2/catalytic AcOH then
NaBH3CN/CH2O/AcOH; (ii) (CF3SO2)2/pyridine/CH2Cl2; (iii) TBAF/THF.

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Peoc’h et al.
Scheme 6. Reagents and Conditions: (i) DMTCl/pyridine/DMAP; (ii) 2-cyanoethyl-N,N,N’,N’-
tetraisopropylphosphorodiamidite/diisopropylammonium tetrazolide/CH2Cl2 or 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite/(i-Pr)2NEt/THF.
phosphate repulsion due to neutral linkage and the 2’-electronegativity effects that
stabilize the C3’-endo sugar pucker may contribute significantly to the affinity for the
RNA target.
In order to further elaborate our SAR and understanding of the role of the 2’-
electronegative substituent we forged ahead with the synthesis of 3a and 3b containing
a 2’-fluoro substituent. Having failed twice in the direct synthesis of 2’-fluoro-3’-C-
formyl nucleoside, we opted to take a longer but surer route. Until recently, the
synthesis of 2’-fluoro modified oligonucleotides has been arduous due to lack of
appropriate synthetic routes to prepare the monomeric nucleosides.[40] Although our
repeated attempts to prepare monomeric building-blocks containing a 2’-fluoro-3’-C-
formyl substituents resulted in little success, the stable 2’-fluoro-3’-C-(4,5-dihydro-5-
methyl-1,3,5-dithiazin-2-yl) analog 13c was easily prepared. This prompted us to
undertake the synthesis of 3 via dimerization first, and followed by fluorination as shown
in Scheme 5. Hydrolysis (HgO/HgCl2/H2O) of 11 furnished 20 in moderate yield.
Coupling of 20 with 7a or 7b as described before furnished 21a and 21b, respectively.
The subsequent fluorination of 21a and 21b via triflates 22a and 22b provided 3a and
3b, respectively, in good overall yield. The 3’- and 5’-silyl protecting groups were
simultaneously deblocked during fluorination with TBAF in THF. The spectral (1H
NMR, FAB MS) data and the elemental analyses of 3 were consistent with the proposed
structure. Furthermore, dimers 3 displayed a characteristic very small J 1’,2’ coupling
1
constant and a large J 3’,4’ coupling in the H NMR spectra suggesting a high 3’-endo
sugar conformation for both residues. Standard procedures allowed the conversions of 3a
and 3b to the desired amidites 24f and 24g, respectively (Scheme 6). Incorporation of

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421
amidites 24 into oligonucleotides was accomplished using slightly modified standard
oligomerization conditions. However, due care must be taken during the base-
deprotection step, which was carried out with NH3/MeOH solution instead of standard
NH4OH treatment in order to avoid loss of the 2’–fluoro group during deprotection.b
The Tm data of oligonucleotides containing dimer units 3a and 3b is summarized
in Table 1. Once again, we observed a dramatic increase in the Tm when a 2’-fluoro
substituent was placed in the top sugar residue in combination with 2’-OMe or 2’-F in
the bottom units. These results are similar to that obtained with 2’-OMe modification in
the top unit as in 2. Therefore, it may be safe to assume that the contributing effects of
the two very different 2’-substituents (i.e., F and OMe) is similar when used in
combination with a 2’-F or 2’-OMe in the bottom sugar residue.
CONCLUSIONS
The synthesis of seven novel nucleosidic dimers 1–3 representing a class of doubly
modified (i.e., backbone and sugar) molecules useful as building-blocks for antisense
constructs has been accomplished.. In the process of making these dimers, several
modified nucleoside analogs (e.g., 7, 14, and 18) have been prepared. Potentially, some
of these may have interesting biological activity after deprotection of the blocking
groups and serve as an intermediate for the synthesis of modified nucleosides of general
interest (for analogs of 3’-C-branched nucleoside see Ref. [41]). The one-pot coupling/
reduction/methylation procedure provides a convenient and efficient alternative to the
standard multistep sequences that are commonly used to prepare functionalized amines.
This procedure may also have applications in conjugation chemistry.[42]
The main focus of the study was to generate an hybridization SAR in order to
assist selection of the best 2’-substitution in combination with the MMI backbone. The
thermodynamic profiles obtained from this study clearly provide additional insight
towards designing of high affinity antisense oligonucleotides. It has been suggested that
the higher affinity of 2’-O-methylated RNA is entropically driven, possibly due to
distortion in the minor groove hydration at the local and the global level, resulting in a
net increase in the degrees of the freedom for duplex formation.16b Additionally, 2’-O-
methylated RNA is conformationally preorganized due to C3’-endo sugar pucker that is
a preferred conformation for binding to RNA. As indicated earlier, the presence of a 3’-
C
C bond in the MMI linkage reduces the O4’ and C3’ gauche interaction compared
to the natural phosphodiester backbone linkage. Further gain in enthalpic stabilization
was realized by introducing neutral MMI linkages and stabilizing effects of a methyl
group at the C5 of pyrimidine residue. The effects of the foregoing attributes were
clearly visible in the Tm data generated in this study. The data unambiguously reveal
that combination of an appropriate 2’-modification with a neutral and achiral linkage
provides oligomers with high affinity for an RNA target.
bIn order to release the 2’-fluoro oligomer from the support and deblock the base labile protecting
groups CPG was placed in a screw-cap bottle with saturated (at 0° C) NH3 in MeOH and heated at
55° C (metal block) for 12 h. For more details see reference 27.

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From the synthesis and scale-up point-of-view, the bis–2’-OMe MMI construct has
advantages which overcome the small affinity advantage of the 2’–F containing
construct derived from 3a. First, 2’-O-methyl-nucleosides are available from various
commercial sources in kilo quantities at reasonable prices when compared to the 2’-
fluoro nucleosides (particularly the purines). Second, synthetic manipulations with 2’-
OMe nucleosides are much simpler and more robust compared to the 2’-F analogs, as
evidenced by the difficulties encountered herein when preparing the 2’–F dimers 3a
and 3b. Lastly, 2’-OMe analogs of antisense oligonucleotides have been recently safely
evaluated in animals and in human subjects.[43] Considering both the synthetic
advantages and the Tm data, we believe that the use of bis-2’-OMe substitution is the
ideal 2’–modification for use in combination with MMI linkage. With high affinity for
the target RNA, combined with a neutral substitution of the phosphate backbone and
subsequent complete resistance to nucleolytic degradation, this construct is a promising
candidate for use in future generations of antisense drugs.[44,45]
EXPERIMENTAL SECTION
General. Unless otherwise noted, materials were obtained from commercial
suppliers and were used as provided. Other general experimental procedures were
carried out as described previously.
General Procedures
A. One-Pot Coupling/Reduction/Methylation. An equimolar mixture of 5’-O-
amino nucleoside and 3’-deoxy-3’-C-formyl nucleoside were dissolved in 1:1 toluene/
CH2Cl2 containing several drops of acetic acid, mixed at 35°C on a rotary evaporator at
atmospheric pressure for 1 h, then concentrated to a foam under reduced pressure. This
process was repeated until TLC analysis (5% MeOH/CH2Cl2) showed the reaction to be
complete (3–6 times), with the intermediate E and Z oximes evident as the only
spot(s). The crude isomeric oxime was then dissolved in glacial acetic acid (0.1 M),
placed in a cool water bath (5°C), and sodium cyanoborohydride (3 Â 2 eq) was added
over 0.25 h. The mixture was allowed to warm to room temperature over 1 h, placed in
a cool water bath, and aqueous formaldehyde (20 eq) was added in one portion.
Sodium cyanoborohydride (3 Â 2 eq) was added over 0.25 h, and the mixture was
allowed to warm to room temperature over 1 h. Pouring into ice water (5 times
volume) with vigorous stirring terminated the reaction. The resulting residue was
collected and dried, or extracted into CH2Cl2, concentrated, azeotroped (3 times) with
toluene, and chromatographed. The column was eluted with 1 to 5% EtOH (95%)/
EtOAc, which removed several trace impurities off the column, and the desired product
was then obtained by elution with 10% MeOH/CH2Cl2, and concentration of the
appropriate fractions.
B. Desilylation. The silylated MMI dimer was dissolved in dry THF (0.1 M) and
cooled in an ice bath. A solution of tetrabutylammonium fluoride in THF (1 M, 1.5 eq
per silyl group) was added dropwise. The solution was stirred at 0°C until the reaction
was complete as judged by TLC (1–2 h), at which point silica (5 g/mmol) was added,

ORDER
REPRINTS
2’-Substituted MMI Linked Nucleosidic Dimers
423
and the mixture carefully concentrated. The silica was applied to a column packed in
5% MeOH/CH2Cl2, and eluted with 5% to 10% MeOH/CH2Cl2. The appropriate
fractions were combined and concentrated to provide the desired dimer.
C. Dimethoxytritylation. The appropriate dimer (1 eq) and N,N-dimethylami-
nopyridine (0.1 eq) was azeotroped 3 times with dry pyridine, then dissolved in the
minimum amount of dry pyridine. Triethylamine (2 eq) and 4,4’-dimethoxytrityl
chloride were added to the stirred solution at room temperature, and the mixture diluted
with dichloromethane (ca 5 mL/mL pyridine). Stirring was continued until the reaction
was complete (typically overnight) as judged by TLC (10% MeOH/CH2Cl2 + 0.1%
Et3N), methanol was added to quench the reaction, and the reaction mixture extracted
with 5% aqueous sodium bicarbonate. The organic layer was dried (trace magnesium
sulfate), filtered, and concentrated to afford a syrup, which was chromatographed on
silica gel (CH2Cl2 + 0.1% Et3N to 10% MeOH/CH2Cl2 + 0.1% Et3N). Fractions
containing only product were combined, and concentrated to afford the 5’-
dimethoxytritylated compound as hard foam.
D. Phosphitylation. The appropriate 5’-dimethoxytrityl derivative was azeo-
troped with dry acetonitrile (3 Â), then dissolved in dry CH2Cl2 (0.1 M) at rt.
Diisopropylammonium tetrazolide (0.5 eq) and 2-cyanoethyl-N,N,N’,N’-tetraisopropyl-
phosphorodiamidite (1.2 eq) was added, and the reaction allowed to stir at room
temperature until complete (typically overnight) as judged by TLC (EtOAc + 0.1%
Et3N). The reaction mixture was then directly loaded onto a column packed with 25%
EtOAc/hexane + 0.1% Et3N, and eluted with a stepwise gradient to EtOAc + 0.1%
Et3N. Fractions containing only the product were pooled and concentrated to yield hard
foam, which was lyophilized from dry 1,4-dioxane to afford the phosphoramidite as a
fine white powder.
E. Trifluoromethylsulfonylation. A solution of dry (P2O5 overnight) hydroxyl
compound (1 equiv.) in dry CH2Cl2 (25 ml/mmol) was stirred and cooled to 0–5°C. To
this mixture under an inert atmosphere dry pyridine (8.5 equiv.) was added followed by
dropwise addition of trifluoromethanesulfonic anhydride (1.65 equiv.) at 0–5°C. The
resulting mixture was stirred for 2–4 hours. The reaction was found to be complete by
TLC. In certain cases a small amount of the starting material remained, which can be
further reacted with an excess of trifluoromethanesulfonic anhydride (0.1–0.2 equiv) to
push the reaction to completion. At this time, the reaction mixture was poured into ice
water containing saturated NaHCO3 and extracted with CH2Cl2 (2 Â 50 ml/mmol). In
order to remove the excess of pyridine, combined organic layers were washed with cold
water containing 1% AcOH (2 Â 50 ml/mmol). The organic extract was dried over
anhydrous MgSO4 and concentrated under vacuum at room temperature to furnish the
crude triflate as foam. Analytically pure sample of triflate may be obtained by silica gel
column chromatography. However, in most of the examples crude triflate was carried
over to the next step without purification.
F. Fluorination/Desilylation. Dry (coevaporated with 2 Â toluene) triflate
compound (1 equiv.) was dissolved in dry THF (30 ml/mmol) and cooled to 0–5°C.
A solution of freshly prepared anhydrous (Ref.) TBAF in dry THF (10 ml/mmol of

ORDER
REPRINTS
424
Peoc’h et al.
TBAF, 3 equiv.) was added via syringe to the triflate solution at 0–5°C while stirring.
After complete addition, the resulting mixture was allowed to stir for 1–2 hours. TLC
indicated that reaction was usually complete at this time. Subsequently, in situ
desilylation was accomplished by addition of TBAF (1.0 M in THF with ꢀ 5% water,
2 ml/mmol) to the reaction mixture. The reaction mixture was then stirred until a single
polar spot was detected (TLC) and resulting orange colored solution was concentrated
to syrup. The syrup was dissolved in CH2Cl2: MeOH (96:4, v/v) and purified by silica
gel column chromatography. Elution with a mixture of CH2Cl2:MeOH furnished the
desilylated fluoro compound in 70–75% yield.
0
0
2 -O-Methyl-5 -O-phthalimido-5-methyluridine (5a). A stirred mixture of 5’-
O-phthalimido-5-methyluridine (40.0 g, 0.1 mol), dibutyltin oxide (29.76 g, 0.12 mol),
tetrabuytylammonium iodide (40.59 g, 0.11 mol) and iodomethane (64.18 ml, 1 mol)
in DMF (200 ml) was heated in a sealed flask at 50°C for 16 hours under argon
atmosphere. The reaction mixture was cooled (ꢀ 10°C) and to this another addition of
methyl iodide (2.82 g, 20 mmol) was made. The flask was sealed and heating
continued for 16 hours. The reaction mixture was cooled to room temperature and
diluted with CH2Cl2 (50 ml). The CH2Cl2 suspension was transferred onto the top of
a prepacked silica gel (CH2Cl2) column. Elution with CH2Cl2-CH2Cl2:MeOH (9:1,
v/v) furnished the desired product as homogenous material. Appropriate fractions were
pooled and concentrated to provide 0.6 g (69%) of the title compound (contaminated
with 10% of the 3’-O-methyl derivative). An analytical sample was obtained by
1
crystallization (EtOH/CH2Cl2) mp 213–214°C; H NMR (DMSO-d6) 1.71 (s, 3, CH3),
3.58 (s, 3, O CH3), 4.42 (m, 2, 5’ CH2), 4.63 (m, 1, 4’H), 5.36 (m, 1, 2’ H), 5.61 (m,
1, 3’H), 5.90 (s, 1, 1’H), 7.50 (s, 1, C6H), 7.82 (m, 4, ArH), 11.55 (br s, t, NH). Anal.
calc. for C19H19N3O8 0.5 H2O: C, 53.52; H, 4.73; N, 9.85; Found: C, 53.51; H, 4.49;
N, 9.84.
ꢁ
0
0
5 -O-Phthalimido-2 -fluorothymidine (5b). To 2’-Fluorothymidine (4b, 1.95 g,
7.5 mmol), triphenylphosphine (2.07 g, 7.88 mmol) and N-hydroxyphthalimide (1.29 g,
7.88 mmol) in dry DMF (60 mL) was added diethylazodicarboxylate (1.24 mL,
7.88 mmol) dropwise at room temperature over 0.5 h. The mixture was stirred
overnight and poured into a rapidly stirred ice cold mixture of ether (150 mL) and
water (150 mL). The aqueous layer was slowly diluted to a total volume of 350 mL
with cold water, at which point solid began forming. The ether was decanted, an
additional portion ether was added, stirred, and decanted, and the process repeated. The
solution was now diluted to a total volume of 500 mL with water, and allowed to stand
on ice for several hours. The solid was collected, and dried to afford 2.11 g (70%) of
1
5b: Rf 0.31 (5% MeOH/CH2Cl2); H NMR (CDCl3) d 8.84 (s, 1H, NH), 7.90–7.70 (m,
4H), 7.26 (s, 1H, H-6), 6.03 (dd, J = 3.0, 17.0 Hz, 1H), 5.11 (ddd, J = 1.5, 3.0, 52.8
Hz, 1H), 4.75 (m, 1H), 4.56 (m, 2H) 4.34 (m, 1H), 3.26 (br s, 1H), 1.94 (s, 3H, CH3).
Anal. Calcd for C18H16N3O7F 1/4Et2O: C, 53.84; H, 4.40; N, 9.91. Found: C, 53.52;
H, 4.60; N, 9.96.
ꢁ
0
0
2 -O-Methoxyethyl-5 -O-Phthalimido-5-methyluridine (5c). To a stirred mix-
ture of 2’-methoxyethyl-5-methyluridine (3.16 g, 10 mmol), triphenylphosphine (2.88 g,
11 mmol) and N-hydroxyphthalimide (1.79 g, 11 mmol) was added dropwise diethyl

ORDER
REPRINTS
2’-Substituted MMI Linked Nucleosidic Dimers
425
azodicarboxylate (2.17 g, 12.5 mol) over a period of 1 hour at ꢀ 5°C. After complete
addition, reaction mixture was warmed to room temperature and stirred for 16 hours.
The reaction was ꢀ 50% (TLC, CH2Cl2:MeOH, 9:1, v/v) complete at this point of
time. Therefore, another equivalent of all reagents were added in the manner described
above. After second addition, the reaction mixture was stirred at room temperature for
16 hours. The reaction was ꢀ 80% (TLC) complete at this time, therefore, solution was
concentrated under vacuum to provide a thick syrupy residue. The residue was poured
into a vigorously stirred mixture of ether:ice water (100 mL:200mL) to precipitate the
product. The precipitate was filtered, washed with water (3 Â 100 mL) and ether
1
(3 Â 50 ml), and dried over P2O5 to provide 1.75 g (38%) of 5c: H NMR (CDCl3) d
1.98 (s, 3, CH3), 3.41(s, 3, OCH3), 3.52, 3.70, 3.94 and 3.99 (m, 5H), 4.06 (t, 1, 3’
OH), 4.32 (m, 1H), 4.51(d, 2H), 4.61 (m, 1, 4’H), 6.10 (d, 1, 1’H), 7.80 (s, 1, C6H),
7.86 and 7.88 (2m, 4, ArH), 8.22 (brs, 1, NH); Anal. calc. for C21H23N3O9: C, 54.66;
H, 5.02; N, 9.10; Found: C, 54.38; H, 5.33; N, 8.77.
0
5 -O-Phthalimido-5-methyluridine (5d). To a stirred mixture of 5-methyluridine
(51.64 g, 0.2 mol), triphenylphosphine (57.64 g, 0.22 mol) and N-hydroxyphthalimide
(35.86 g, 0.22 mol) was added dropwise diethyl azodicarboxylate (43.5 g, 0.25 mol) over
a period of 4 hours at ꢀ 5°C. After complete addition, reaction mixture was warmed
to room temperature and stirred for 16 hours. The reaction was ꢀ 50% (TLC,
CH2Cl2:MeOH, 9:1, v/v) complete at this point of time. Therefore, additional quantities
of triphenylphosphine (28.8 g, 0.11 mol), N-hydroxyphthalimide (17.9 g, 0.11 mol) and
diethyl azodicarboxylate (21.7 g, 0.12 mol) were added in the manner described above.
After second addition, the reaction mixture was stirred at room temperature for 16 hours.
The reaction was ꢀ 90% (TLC) complete at this time, therefore, solution was
concentrated under vacuum to provide a thick syrupy residue. The residue was poured
into a vigorously stirred mixture of ether:ice water (500 mL: 1Lt) to precipitate the
product. The precipitate was filtered, washed with water (3 Â 100 mL) and ether
(3 Â 50 ml), and dried over P2O5 to provide 56 g (69%) of the title compound. An
analytical sample was crystallized from EtOH:CH2Cl2, mp 236–237°C; 1H NMR
(DMSO-d6) d 1.78 (s, 3, CH3), 4.12 (m, 2’, 3’, 4’ H), 4.36 (m, 2, 5’CH2), 5.45 and 5.62
(2d, 2, 3’, 4’ OH), 7.78 (s, 1, C6H), 7.90 (m, 4, ArH), 11.32 (brs, 1, NH); Anal. calc. for
C18H17N3O8 1/4 H2O:C, 53.00; H, 4.32; N, 10.30; Found: C, 53.04; H, 4.28; N, 10.19.
ꢁ
0
0
0
3 -O-tert-Butyldiphenylsilyl-2 -O-methyl-5 -O-phthalimido-5-methyluridine
(6a). A mixture of 2’-O-methyl-5’-O-phthalimido-5-methyluridine (5.5 g, 13.18 mmol),
imidazole (2.68 g, 39.54 mmol) and tert-butyldiphenylsilylchloride (7.35 g, 26.37
mmol) in DMF (50 mL) was stirred at room temperature for 12 hours under an
argon atmosphere. The reaction mixture was concentrated under vacuum to 1/4 of its
volume and poured into ice water (500 ml). The aqueous mixture was extracted with
CH2Cl2 (2 Â 250 ml) and washed with water (2 Â 100 ml) and dried (MgSO4). The
CH2Cl2 extract was concentrated and the residue was purified by silica gel
chromatography. Elution with ether: hexanes (9:1, v/v) furnished the desired product
as homogenous material. Appropriate fractions were pooled and concentrated to
1
provide 8.0 g (92.6%) of the title compound. H NMR (CDCl3) d 1.14 (s, 9, SiMe3),
1.90 (s, 3, CH3), 3.26 (s, 3, OCH3), 3.38 (m, 1, 4’H), 4.08 (m, 1, 2’ H), 4.23 (m, 2, 5’
CH2), 4.50 (m, 1, 3’ H), 6.08 (d, 1, 1’ H), 7.34–7.42 (m, 10, Ar H), 7.60 (s, 1, C6 H),

ORDER
REPRINTS
426
Peoc’h et al.
7.65–7.85 (m, 4, Ar H), 8.08 (br s, 1, NH); Anal. Calc. for C35H37N3O8 Si : C, 64.10;
H, 5.68; N, 6.40; Found: C, 63.96; H, 5.67; N, 6.16.
0
0
0
3 -O-tert-Butyldiphenylsilyl-2 -fluoro-5 -O-phthalimidothymidine (6b). Com-
pound 5b (6.24 g, 10 mmol) was silylated in the same manner as for 5a to yield
1
5.70 g (89%) of 6b: Rf 0.76 (70% EtOAc/hexane); H NMR (CDCl3) d 8.13 (s, 1H),
7.87–7.68 (m, 4H), 7.47–7.26 (m, 10H), 6.15, (dd, J = 4.2, 14.6 Hz, 1H), 4.74 (t,
1H), 4.55 (m, 1H), 4.20 (m, 2H), 4.01 (m, 1H), 1.91 (s, 3H), 1.14 (s, 9H). Anal.
Calcd for C34H34N3O7SiF 1/2 EtOAc : C, 62.87; H, 5.57; N, 6.11. Found: C, 62.90;
H, 5.54; N, 6.25.
ꢁ
dine (6c). Silylation of 5c in the same manner as for 5a provided 6c (84%). H NMR
0
0
0
3 -O-tert-Butyldiphenylsilyl-2 -O-methoxyethyl-5 -O-phthalimido-5-methyluri-
1
(CDCl3) d 1.34 (s, 9, SiMe3), 1.91 (s, 3, CH3), 3.27 (s, 3, OCH3), 3.42, 3.63, 4.02 and
4.19 (m, 7H), 4.52 (t, 1, 3’ H), 6.10 (d, 1, 1’ H), 7.26–7.43 (m, 10, Ar H), 7.62 (s, 1,
C6 H), 7.70–7.78 (m, 4, Ar H), 8.04 (br s, 1, NH).
0
0
0
5 -O-Amino-3 -O-tert-butyldiphenylsilyl-2 -O-methyl-5-methyluridine (7a).
Hydrazinolysis of 6a in the same manner as for 7b furnished 7a (79%) as white
1
foam. H NMR (CDC13) d 1.10 (s, 9, SiMe3), 1.83 (s, 3, CH3), 3.24 (m, 1, 2’H), 3.38
(s, 3, OCH3), 3.63 and 3.96 (dd, 2, 5’ CH2), 4.11 and 4.19 (2m, 2, 3’, 4’ H), 5.32 (br s,
2, ONH2), 5.93 (d, 1, 1’ H), 7.30 (s, 1, C6H), 7.37–7.50 and 7.63–7.73 (m, 10, Ar H)
and 9.22 (br s, 1, NH). Anal. Calc. for C27H35N3O6Si 1/4 H2O : C, 61.16; H, 6.74; H,
7.92; Found: C, 61.03; H, 6.56; N, 7.86.
ꢁ
0
0
5 -O-Amino-3 -O-tert-butyldiphenylsilyl-2’-fluorothymidine (7b). A solution of
6b (5.56 g, 8.65 mmol) in CH2Cl2 (90 mL) at 0°C was treated with methylhydrazine
(0.55 mL, 10.4 mmol) for 1 h with stirring, the mixture filtered, and the filtrate washed
with cold CH2Cl2. The combined organics were washed with water (3 Â), dried
(MgSO4), diluted with toluene and concentrated, and dried to provide 4.20 g (95%) of
1
pure 7b: Rf 0.42 (70% EtOAc/hexane); H NMR (CDCl3) d 8.39 (s, 1H), 7.68 (d, 4H),
7.45 (m, 6H), 7.15 (s, 1H), 5.92 (dd, J = 2.2, 17 Hz, 1H), 5.30 (s, 2H), 4.54 (dm,
J = 53 Hz, 1H), 4.35–4.06 (m, 2H), 3.91 (dd, 1H), 3.57 (dd, 1H), 1.83 (s, 3H), 1.11 (s,
9H). Anal. Calcd for C26H32N3O5SiF: C, 60.80; H, 6.28; N, 8.18. Found: C, 61.01; H,
6.05; N, 8.03.
0
0
0
5 -O-Amino-3 -O-tert-butyldiphenylsilyl-2 -O-(2-methoxyethyl)-5-methyluri-
dine (7c). Hydrazinolysis of 6c in the same manner as for 7b furnished 7c (86%) as
1
white foam. H NMR (CDC13) d 1.11 (s, 9, SiMe3), 1.82 (s, 3, CH3), 3.24 (m, 1, 2’H),
3.32 (s, 3, OCH3), 3.53, 3.58, 3.75 and 4.02 (m, 7H), 5.28 (br s, 2, ONH2), 5.90 (d, 1,
1’ H), 7.31 (s, 1, C6H), 7.38–7.50 and 7.65–7.73 (m, 10, Ar H) and 7.90 (br s, 1, NH).
Anal. Calc. for C29H39N3O9Si 0.5MeOH : C, 60.49; H, 7.06; H, 7.17; Found: C,
60.48; H, 6.96; N, 7.23.
ꢁ
0
0
5 -O-Amino-2 -fluorothymidine (7d). 5-O-Phthalimido-2’-fluorothymidine
(5b, 4.0 g, 10 mmol) was dissolved in 100 mL of 10% MeOH/CH2Cl2 with slight
warming, then cooled on ice. When the reaction mixture became cloudy, methyl-

ORDER
REPRINTS
2’-Substituted MMI Linked Nucleosidic Dimers
427
hydrazine (0.80 mL, 15 mmol) was added dropwise. The clear reaction mixture was
stirred for 1.5 h at 0°C, and the solid collected and washed with cold 10% MeOH/
CH2Cl2, then dried to provide 1.99 g (72%) of 7d (mp 165–166°C). Recrystallization
from EtOH provided needles (87% recovery): mp 168–169°C; Rf 0.32 (10% MeOH/
CH2Cl2);1H NMR (DMSO-d6) d 11.43 (s, 1H), 7.55 (s, 1H), 6.23 (s, 2H), 5.89 (dd,
J = 2.0, 19.5 Hz, 1H), 5.69 (d, 1H), 5.09 (ddd, J = 2.0, 2.5, 53.3 Hz, 1H), 4.25–3.65
(m, 4H), 1.80 (s, 3H). Anal. Calcd for C10H14N3O5F: C, 43.64; H, 5.13; N, 15.27.
Found: C, 44.00; H, 5.01; N, 15.06.
0
0
0
5 -O-tert-Butyldiphenylsilyl-3 -De(oxyphosphinico)-3 -methylene(methylimino)-
0
0
0
0
thymidylyl-(3 5 )-2 -O-methyl-5 -O-tert-Butyldiphenylsilyl-5-methyluridine
!
(10a).
A mixture of 5’-O-amino-3’-O-tert-butyldiphenylsilyl-2’-O-methyl-5-methyl-
uridine (5.25 g, 10 mmol) and 1-[5-(tert-butyldiphenylsilyl)-2,3-dideoxy-3-C-(formyl)-b-
D-erythro-pentofuranosyl]thymine[22] (4.92 g, 10 mmol) in CH2Cl2:ACOH (50:1 mL)
was stirred at room temperature for 5 minutes. The reaction mixture was then
coevaporated with toluene (3 Â 50 ml) under vacuum. The reaction was complete (by
TLC) by the third coevaporation. The residue was dissolved in AcOH (25 ml) and cooled
to ꢀ 15°C. NaCNBH3 (3 Â 250 mg, 12 mmol) was added to the stirred reaction mixture
in small portions (fume-hood). The reaction mixture was stirred for 30 min. at ꢀ 15°C
and to the cold solution aq. HCHO (30%, 5 ml) was added in one portion. The stirring
was continued for 30 minutes and additional amount of NaCNBH3 (3 Â 250 mg,
12 mmol) was added in a similar manner. After 2 hours, the reaction mixture was poured
into ice water (250 ml) and extracted with CH2Cl2 (2 Â 250 ml). The CH2Cl2 layer was
washed with water (2 Â 250 ml) and dried MgSO4). The solvent was removed and
the residue purified by silica gel column chromatography. Elution with a gradient
of CH2Cl2-CH2Cl2:MeOH (95:5, v/v) provided the desired product as homogenous
material. Appropriate fractions were pooled and concentrated to furnish 5.08 g (50%) of
1
the 3’, 5’-protected MMI dimer. H NMR (CDCl3) d 1.12 (s, 18, SiMe3), 1.62 and 1.79
(2s, 6, CH3), 2.45 (s, 3, N-CH3), 2.65 (m, 2, CH2-N-CH3), 3.30 (s, 3, O CH3), 5.82 (d, 1,
1’H), 6.17 (t, 1, 1’H), 9.04 and 9.09 (2 s, 2, NH) and other protons.
0
0
0
5 -O-tert-Butyldiphenylsilyl-3 -de(oxyphosphinico)-3 -methylene(methylimino)-
0
0 0
0
thymidylyl-(3 5 )-2 -deoxy-2 -fluoro-5-methyluridine (10b). Reaction of 5’-O-
!
amino-2’-fluorothymidine (0.54 g, 2 mmol) and 5’-O-tert-butyldiphenylsilyl-3’-deoxy-
3’-C-formylthymidine (0.98 g, 2 mmol) according to general procedure A provided a fine,
powdery solid after pouring into ice water. This material was collected, washed with
water, and dried to provide 1.45 g (97%) of product that contained an impurity (ca 10%).
This material was used directly in the desilylation step: Rf 0.44 (10% MeOH/CH2Cl2);1H
NMR (CDCl3) d 9.48 (s, 1H), 9.40 (s, 1H), 7.70–7.16 (m, 12H), 6.07 (t, J = 5.7 Hz), 5.75
(d, J = 19 Hz), 5.05 (d, J = 52 Hz), 4.40–3.65 (m, 7H), 3.35 (m, 1H), 2.68 (m, 2H), 2.62
(s, 3H), 2.31 (m, 2H), 1.89 (s, 3H), 1.78 (m, 1H), 1.65 (s, 3H), 1.08 (s, 9H).
0
0
0
5 -O-tert-Butyldiphenylsilyl-3 -De(oxyphosphinico)-3 -methylene(methylimino)-
0
0
0
0
thymidylyl-(3 5 )-2 -O-(2–methoxy)ethyl-5 -O-tert-Butyldiphenylsilyl-5-methyl-
!
uridine (10c). Reaction of 5’-O-amino-3’-O-(tert-butyldiphenylsilyl)- 2’-(2-metoxy-
ethyl)thymidine (1.31 g, 2.28 mmol) and 5’-O-tert-butyldiphenylsilyl-3’-deoxy-3’-C-

ORDER
REPRINTS
428
Peoc’h et al.
formylthymidine (1.13 g, 2.28 mmol) according to general procedure A provided a
white solid (2.12 g, 88%) after chromatography. Rf 0.45 (5% MeOH/CH2Cl2); Anal.
Calcd for C57H75N5O10Si2 NaOAc: C, 60.53; H, 6.74; N, 5.79. Found: C, 60.61; H,
6.67; N, 5.94.
ꢁ
1-[5-O-(tert-Butyldiphenylsilyl)-3-deoxy-3-C-(4,5-dihydro-5-methyl-1,3,5-dithia-
zin-2-yl)-b-D-arabino-pentofuranosyl]thymine (11). Dihydro-5-methyl-1,3,5-dithia-
zine (23.80 g, 176 mmol) was dissolved in dry THF (200 ml) and Hexa-
methylphosphoramide (HMPA) (32ml) was added. The solution was cooled to -78°C
(acetone/dry ice bath) under argon atmosphere. n-Butyllithium (2.0 M in pentane, 88
ml) was then added dropwise (over 5 min.) to produce a white precipitate. Metallation
was allowed to proceed for 1 hour. 1-(2,3-Epoxy-5-O-tert-Butyldiphenylsilyl-b-D-lyxo-
pento-furanosyl)thymine (19.15 g, 40 mmol), synthesized according to the known
procedure,[35] was dissolved in a mixture of dry THF (80 ml) and HMPA (108 ml). The
resulting solution was added dropwise to the vigorously stirred reaction mixture, while
the low temperature was maintained. After 90 min, TLC (5% MeOH/CH2Cl2)
indicated no remaining starting material and a single more polar product. The reaction
mixture was poured into water (400 ml), neutralized with 1M HCl and extracted with
ethyl acetate. The organic phase was dried (MgSO4), filtered and evaporated and the
residue chromatographed on a silica gel column with 2% MeOH/CH2Cl2 to yield
1
16.85 g (69%) of a light yellow foam. Rf 0.35 (5% MeOH/CH2Cl2); H NMR (CDCl3)
d 8.61 (bs, 1H, NH), 7.74–7.37 (m, 11H, H-6 and Ph2), 6.06 (d, 1H, J = 4.2 Hz, H-
1’), 4.74–4.62 (m, 3H, H-2’ and SCH2N), 4.49 (d, 1H, J = 6.4 Hz, SCHS), 4.37 (q,
1H, H-4’), 4.18–4.04 (m, 3H, H-5’ and SCH2S), 4.18–4.04 (m, 3H, H-5’ and SCH2N),
3.81 (dd, 1H, Ja = 2.6 Hz, Jb = 11.5 Hz, H-5’), 3.63 (d, 1H, J = 7.5 Hz, OH-2’), 2.75
(m, 1H, H-3’), 2.58 (s, 3H, N-CH3), 1.69 (s, 3H, CH3), 1.12 (s, 9H, tBu) ppm. Anal.
Calc. for C27H32N2O6Si : C, 63.76; H, 6.34; N, 5.51; Found: C, 63.62; H, 6.27;
N, 5.33.
1-[5-O-(tert-Butyldiphenylsilyl)-3-deoxy-3-C-(4,5-dihydro-5-methyl-1,3,5-dithia-
zin-2-yl)-2-O-(trifluoromethanesulfonyl)-b-D-arabino-pentofuranosyl]thymine
(12b). Ara nucleoside 11 was transformed to 12b following the general procedure E
for triflate preparation in 70% yield. Anal. Calc. for C31H38N3O7SiF3 2H2O: C, 47.62;
H, 5.41; N, 5.37; Found: C, 47.34; H, 4.86; N, 5.25.
ꢁ
1-[5-O-(tert-Butyldiphenylsilyl)-2,3-Dideoxy-3-C-(4,5-dihydro-5-methyl-1,3,5-
dithiazin-2-yl)-2-fluoro-b-D-ribo-pentofuranosyl]thymine (13b). Silylation of 13c
was accomplished in the same manner as for 5a to furnish 5’-O-silylated 13b (75%) as
1
a white foam. H NMR (DMSO-d6) d 11.40 (s, 1H, NH), 7.35–7.75 (m, ArH), 5.82 (d,
J = 22.0 Hz, 1’H), 5.66 and 5.41 (dd, J = 51.0 Hz and 4.0 Hz, 2’H), 2.52 (s, 3H,
NCH3), 1.40 (s, 3H, CH3) 1.05 (s, 9H, t-BuH) and other sugar protons.
1-[2,3-Dideoxy-3-C-(4,5-dihydro-5-methyl-1,3,5-dithiazin-2-yl)-2-fluoro-b-D-
ribo-pentofuranosyl]thymine (13c). Fluorination of 12b was achieved by the general
procedure F to furnish a mixture of two compounds. This mixture was further treated
with TBAF to deprotect the remaining 5’-OTBPS group to furnish 13c (73%) as a white

ORDER
REPRINTS
2’-Substituted MMI Linked Nucleosidic Dimers
429
foam. 1H NMR (DMSO-d6) d 11.34 (s, 1H, NH), 8.01 (s, 1H, C6H), 5.86 (d,
J = 18.2 Hz, 1’H), 5.48 and 5.23 (dd, J = 51.5 Hz and 4.2 Hz, 2’H), 5.39 (t, 1H, 5’OH),
4.66 and 4.33 (2m, 6H), 3.92 (m, 2H, 5’CH2), 2.85 (m, 1H, 3’H), 2.50 (s, 3H, NCH3),
1.76 (s, 3H, CH3). Anal. Calc. for C14H20N3O4S2F 0.75H2O: C, 45.19; H, 5.99; N,
10.20; Found: C, 45.43; H, 5.72; N, 9.78.
ꢁ
0
0
0
0
3 -De(oxyphosphinico)-3 -methylene(methylimino)-thymidylyl-(3 5’)-2 -O-
!
methyl-5-methyluridine (1a). To a stirred solution of 3’, 5’-protected MMI dimer
(2.2g, 1.88 mmol) in THF (25 ml) was added tetraloutylammonium fluoride (0.52 g,
2 mmol) at room temperature. The stirring was continued for 24 hours. The reaction
mixture was loaded on the top of a silica gel column and elution with CH2Cl2 : MeOH
(93:7, v/v). Appropriate fractions were concentrated to furnish 1.0 g (99%) of the title
1
compound as white foam. H NMR at 60°C (D2O) d 2.26 and 2.29 (2s, 6 CH3), 2.77
(m, 2, 2’ CH2), 3.10 (s, 3, N-CH3), 3.88 (s, 3, O-CH3), 6.27 (d, 1, 1’H), 6.48 (t, 1, 1’ H),
7.90 and 8.09 (2s, 2, C6H) and other protons.
0
0
0
0
0
0
3 -De(oxyphosphinico)-3 -methylene(methylimino)-thymidylyl-(3 5 )-2 -de-
!
oxy-2 -fluoro-5-methyluridine (1b). Crude 10b (1.30 g, 1.70 mmol) was desilylated
according to general procedure B to afford 0.65 g (67%) of 1b as a hard foam: Rf 0.29
(10% MeOH/CH2Cl2);1H NMR (DMSO-d6) d 11.44 (s, 1H), 11.26 (s, 1H), 7.82 (s,
1H), (7.52 s, 1H), 6.03 (t, J = 5.3 Hz, 1H), 5.88 (dd J = 2.0, 18.9 Hz, 1H), 5.71 (d,
1H), 5.08 (t, 1H), 5.12 (dm, J = 74.4 Hz, 1H), 4.2–3.4 (m, 9H), 2.72 (m, 1H), 2.59 (s,
3H), 2.15 (m, 2H), 1.80 (s, 3H), 1.77 (s, 3H). Anal. Calcd for C22H30N5O9F H2O: C,
48.44; H, 5.91; N, 12.84. Found: C, 48.80; H, 5.92; N, 12.53.
ꢁ
(2–methoxy)ethyl-5-methyluridine (1c). Dimer 10c (2.02 g, 1.93 mmol) was de-
0
0
0
0
3 -De(oxyphosphinico)-3 -methylene(methylimino)-thymidylyl-(3 5’)-2 -O-
!
silylated according to the general procedure to afford 0.85 g (75%) of a white solid: Rf
1
0.4 (10% MeOH/CH2Cl2); H NMR (DMSO-d6) d 11.35 (s, 1H), 11.23 (s, 1H), 7.82 (s,
1H), 7.53 (s, 1H), 5.99 (t, J = 5.0 Hz, 1H, H-1’), 5.82 (d, 1H, H-1@), 5.11 (d, 1H, 3@-OH),
5.07 (t, 1H, 5’-OH), 3.4–4.04 (m, 12H), 3.19 (s, 3H, OCH3), 2.62–2.75 (m, 2H), 2.56 (s,
3H, NCH3), 2.12 (m, 2H), 1.78 (s, 3H, CH3), 1.75 (s, 3H, CH3). Anal. Calcd for
C25H37N5O11 0.5H2O: C, 50.67; H, 6.46; N, 11.82. Found: C, 51.01; H, 6.45; N, 11.56.
ꢁ
1-[5-O-(tert-Butyldiphenylsilyl)-3-deoxy-3-C-formyl-b-D-glycero-pent-2-eno-
furanosyl]thymine (14). Mercuric salt catalyzed oxidation of 13b with HgO/HgCl2 in
1
wet THF at 0°C furnished the elimination product 14 in quantitative yield. H NMR
(DMSO-d6) d 11.42 (s, 1H, NH), 10.01 (s, 1H, CHO), 7.00–7.65 (m, ArH), 5.18 (s, 1H,
4’H), 4.12 (m, 2H, 5’CH2), 1.11 (s, 3H, CH3) 0.97 (s, 9H, t-BuH) and other sugar protons.
0
0
0
0
5 -O-(4,4 -dimethoxytrityl)-3 -O-(3-methylphenoxy)thiocarbonyl-2 -O-methyl-
5-methyl-uridine (16a). 5’-O-(4,4’-dimethoxytrityl)-2’-O-methyl-5-methyluridine
(14.37 g, 25 mmol) and (N,N-dimethylamino)pyridine (5eq) were azeotroped (3 times)
in dry acetonitrile, then dissolved in acetonitrile (7 ml/mmol). A solution of para-
toluylchlorothionoformate (4.63 ml, 30 mmol) in dry acetonitrile (20 ml) was added
dropwise over 15 min. The resulting solution was stirred overnight at room

ORDER
REPRINTS
430
Peoc’h et al.
temperature, then quenched with few drops of water, concentrated under vacuum,
extracted with CH2Cl2, washed with water, dried over sodium sulfate and finally
chromatographed on silica gel column with 2% MeOH/CH2Cl2 to yield 15.55 g (86%)
1
of the desired xanthate product as a white foam: Rf 0.5 (5% MeOH/CH2Cl2); H NMR
(CDCl3) d 8.20 (s, 1H, NH), 7.59 (s, 1H, H-6), 7.45–6.83 (m, 17H, H-Ar), 6.21 (d, 1H,
J = 5.5 Hz, H-1’), 5.97 (t, 1H, J = 4.6 Hz, H-3’), 4.43 (d, 1H, J = 3.9 Hz, H-4’), 4.37 (t,
1H, J = 5.4 Hz, H-2’), 3.79 (s, 6H, O-CH3), 3.55 (m, 5H, 2’-O-CH3 , H-5’, H-5@), 2.38
(s, 3H, CH3), 1.45 (s, 3H, T-CH3) ppm.
0
0
0
0
5 -O-tert-Butyldiphenylsilyl-2 –deoxy–2 –fluoro-3 -O-(4-methylphenoxy)thio-
carbonyl -5-methyluridine (16b). Compound 4b (260 mg, 1 mmol) was azeotroped
with dry pyridine, then dissolved in dry pyridine, and DMAP (1 mg) and tert-
butyldiphenylsilyl chloride (280 mg, 1 mmol) were added. The solution was stirred at rt
for 48 h, then concentrated, dissolved in EtOAc and washed with water (3 Â), brine,
then dried (MgSO4) and concentrated to yield 490 mg (98%) of crude 15b. This
material was azeotroped with CH3CN (2 Â), dissolved in CH2Cl2 (10 mL), and DMAP
(171 mg, 1.4 mmol) followed p-tolyloxychlorothionoformate (0.19 mL, 1.2 mmol,
dropwise) were added. The solution was stirred 18 h at rt, washed with water (2 Â),
brine, dried (MgSO4), concentrated, and chromatographed (30 to 50% EtOAc/hexane)
1
to afford 0.44 g (68%) of 16b: Rf 0.52 (5% MeOH/CH2Cl2); H NMR (CDCl3) d 8.27
(s, 1H), 7.88 (s, 1H), 7.75–7.60 (m, 4H), 7.50–7.30 (m, 8H), 7.22 (s, 1H),7.01 (d, 2H),
6.26 (dd, J = 4.5, 15.3 Hz, 1H), 5.95 (m, 1H), 5.46 (dt, J = 4.5, 54 Hz, 1H), 4.45 (m,
1H), 4.05 (m, 1H), 2.39 (s, 3H), 2.54 (m, 1H), 1.66 (s, 3H), 1.12 (s, 9H).
0
0
0
0
5 -O-dimethoxytrityl-2 -O-methyl-3 -deoxy-3 -C-styryl-5-methyluridine (17a).
A portion of this material was dissolved in MeOH/THF (1:1), and NaOMe was added
to bring the pH = 10 (to wet litmus paper).[22] The mixture was stirred 24 h at rt, at
which point a slightly faster moving contaminant (70% EtOAc/hexane) was converted
to a much slower moving compound. The solvent was removed, the residue partitioned
between EtOAc and water, and the organic layer dried (MgSO4), concentrated, and
chromatographed (50 to 70% EtOAc/hexane + 5 drops/L Et3N) to provide pure 17a
1
(80–90% recovery); Rf 0.66 (70% EtOAc/hexane); H NMR (CDCl3) d 8.55 (s, 1H),
8.00 (s, 1H), 7.50–7.15 (m, 14H), 6.78 (dd, 4H), 6.50 (d, J = 16 Hz, 1H), 6.17 (dd,
J = 8.7, 16 Hz, 1H), 5.93 (s, 1H), 4.28 (m, 1H), 3.91 (d, 1H), 3.80–3.15 (m, 3H), 3.72
(d, 6H), 3.62 (s, 3H), 1.36 (s, 3H). Anal. Calcd for C40H40N2O7 0.25 EtOAc: C, 72.12;
H, 6.20; N, 4.10. Found: C, 72.15; H, 6.21; N, 4.36.
ꢁ
0
0
0
0
5 -O-dimethoxytrityl-2 -O-methyl-3 -deoxy-3 -C-formyl-5-methyluridine (18).
To a solution of 17a (2.1 g, 3.17 mmol) in dioxane (80 mL) was added NaIO4
(2.95 g, 13.8 mmol) in water (30 mL), followed by OsO4 (2% w/w in water, 1.62 mL,
0.13 mmol). The mixture was stirred at rt in the dark for 5 h, and poured into EtOAc
(200 mL) and water (100 mL). The organic layer was separated, washed with water,
5% Na2SO3, water, and brine (100 mL each), then dried over MgSO4, filtered, and
concentrated. The residue was dissolved in the minimum amount of EtOAc, then
precipitated into hexane. The solid was collected and dried to provide 1.82 g (88%) of
the aldehyde, which contained 0.5 eq hexane and 1 eq water (not as hydrate of RCHO)

ORDER
REPRINTS
2’-Substituted MMI Linked Nucleosidic Dimers
431
1
1
by HNMR and elemental analysis; Rf 0.24 (5% MeOH/CH2Cl2); H NMR (CDCl3) d
9.76 (s, 1H), 7.75 (s, 1H), 7.45–7.20 (m, 9H), 6.9–6.75 (m, 4H), 5.92 (s, 1H), 4.75 (d,
1H), 4.43 (d, 1H), 3.79 (s, 6H), 3.61 (s, 3H), 3.80–3.35 (m, 3H), 1.37 (s, 3H). Anal.
Calcd for C18H16N3O7F H2O 0.5 hexane: C, 66.75; H, 6.69; N, 4.03. Found: C,
66.53; H, 6.75; N, 4.11.
ꢁ ꢁ
uridylyl-(3 5 )-2 -O-methyl-5-methyluridine (2a). A solution of 18 (200 mg,
0
0
0
3 -De(oxyphosphinico)-3 -methylene(methylimino)-2 -O-methyl-5-methyl-
0
0 0
!
0.30 mmol) and 7a (184 mg, 0.30 mmol) were coupled according to general procedure
A. The crude oxime residue was treated with Cl3CO2H (250 mg, 1.5 mmol) in CH2Cl2
(10 mL) for 10 m at rt, and then extracted with 10% NaHCO3. The organic layer was
separated, dried over MgSO4, concentrated, and then chromatographed (70% EtOAc/
hexane to 1% MeOH/EtOAc) to afford 0.17 g (71%) of the 3’-silylated oxime dimer as
a mixture of E and Z isomers (Rf 0.45, 10% MeOH/CH2Cl2). This material was
reductively methylated as described in general procedure A to yield 160 mg (94%) of
crude 3’-silylated dimer (Rf 0.55, 10% MeOH/CH2Cl2) after extractive work-up. This
material was desilylated according to general procedure B, then chromatographed (10%
MeOH/CH2Cl2), and the residue lyophilized to provide 0.10 g (83%) of 2a: Rf 0.29
1
(10% MeOH/CH2Cl2); H NMR (D2O) d 7.94 (s, 1H), 7.39 (s, 1H), 5.83 (s, 1H), 5.71
(d, 1H), 4.15–3.65 (m, 9H), 3.52 (s, 3H), 3.44 (s, 3H), 3.08 (m, 1H), 2.67 (s, 3H), 2.66
(m, 1H), 2.44 (m, 1H), 1.81 (s, 3H), 1.73 (s, 3H). Anal. Calcd for C24H35N5O11 H2O:
C, 49.06; H, 6.35; N, 11.92. Found: C, 49.01; H, 6.05; N, 11.55.
ꢁ
uridylyl-(3 5 )-2 -deoxy-2 -fluoro-5-methyluridine (2b). A solution of 18 (900
0
0
0
3 -De(oxyphosphinico)-3 -methylene(methylimino)-2 -O-methyl-5-methyl-
0
0
0 0
!
mg, 1.28 mmol) and 7b (660 mg, 1.28 mmol) were coupled according to general
procedure A. The crude oxime residue was treated with Cl3CO2H (1.0 g,6.4 mmol) in
CH2Cl2 (33 mL) for 10 m at rt, and then extracted with 10% NaHCO3. The organic
layer was separated, dried over MgSO4, concentrated, and then chromatographed (70%
EtOAc/hexane to 1% MeOH/EtOAc) to afford 0.54 g (54%) of the 3’-silylated oxime
dimer as a mixture of E and Z isomers (Rf 0.69 and 0.76, 1% MeOH/EtOAc). This
material was reductively methylated as described in general procedure A to yield the
crude 3’-silylated dimer (Rf 0.50, 10% MeOH/CH2Cl2) after extractive work-up. This
material was desilylated according to general procedure B, chromatographed (5–10%
MeOH/CH2Cl2), and the residue lyophilized to afford 0.26 g (68%) of 2b: Rf 0.26 (10%
1
MeOH/CH2Cl2); H NMR (D2O) d 7.93 (s, 1H), 7.38 (s, 1H), 5.85 (s, 1H), 5.78 (d,
J = 20 Hz, 1H), 5.10 (dm, J = 52 Hz, 1H), 4.35–3.65 (m, 8H), 3.52 (s, 3H), 3.09 (m,
1H), 2.67 (s, 3H), 2.65 (m, 1H), 2.44 (m, 1H), 1.82 (s, 3H), 1.77 (s, 3H); HRMS (CsI/
NBA) calcd for C23H32N5O10F + Cs+ 690.1188, found 690.1199.
1-[5-O-(tert-butyldiphenylsilyl)-3-deoxy-3-C-formyl-b-D-arabino-pentofurano-
syl] thymine (20). 3’-C-(4,5-dihydro-5-methyl-1,3,5-dithiazin-2-yl) nucleoside (2.76 g,
4.5 mmol) was dissolved in 15% (v/v) aqueous THF (10ml/mmol), and the resulting
solution was cooled to À 5°C under argon atmosphere. To this, with rapid stirring, was
added red mercuric oxide (2.14 g, 2.2 eq) followed by mercuric chloride (2.69 g,
2.2 eq). Stirring was continued until appearance of a white precipitate generally after

ORDER
REPRINTS
432
Peoc’h et al.
15 min. At this time, the reaction mixture was diluted with THF (80 ml) and treated
with aqueous sodium sulfide (1M, 19 ml). The black precipitate was filtered off on a pad
of celite, and the filtrate was partitioned between ethyl acetate and water. Organic phases
were combined, dried (MgSO4), filtered, and evaporated and the residue was purified by
silica gel chromatography. Elution with 3.5% MeOH/CH2Cl2 furnished the desired
product as homogenous material. Appropriate fractions were pooled, concentrated,
azeotroped (3 times) with dry acetonitrile to provide 1.25 g (55%) of white foam. Rf 0.30
(5% MeOH/CH2Cl2); 1H NMR (CDCl3) d 11.35 (s, 1H, NH), 9.73 (d, 1H, CHO), 7.69–
7.36 (m, 11H, H-6 and Ph2), 6.04 (d, 1H, J = 5.8 Hz, H-1’), 5.88 (d, 1H, J = 5 Hz, OH-
2’), 4.76 (q (t on D2O-shake), 1H, H-2’), 4.30 (q, 1H, H-4’), 4.02–3.84 (m, 2H, H-5’ and
H-5@), 3.20 (m, 1H, H-3’), 1.59 (s, 3H, CH3), 1.03 (s, 9H, tBu) ppm.
0
0
0
0
5 -O-tert-Butyldiphenylsilyl-2 –arabino–3 -De(oxyphosphinico)-3 -methylene
0
0
0
(methylimino)-5-methyluridylyl-(3 5’)-2 -O-methyl-3 -O-tert-Butyldiphenylsilyl-
!
5-methyluridine (21a). Coupling reaction of 5’-O-amino-3’-O-tert-butyldiphenylsilyl-
2’-O-methyl-5-methyluridine (1.47 g, 2.8 mmol) and 1-[5-O-(tert-butyldiphenylsilyl)-3-
deoxy-3-C-formyl-b-D-arabino-pentofuranosyl] thymine (1.42 g, 2.8 mmol) according
to the general procedure A provided the corresponding oxime dimer which was further
reduced and methylated to yield 2.85 g (99%) of a hard foam after concentration under
1
vacuo: Rf 0.45 (5% MeOH/CH2Cl2, 2 developments); H NMR (DMSO) d 11.39 and
11.32 (2s, 2H, NH), 7.68–7.35 (m, 22H, H-6 and Ph2), 5.99 (d, 1H, J = 4.9 Hz, H-1’),
5.90 (d, 1H, J = 4.7 Hz, H-1@), 5.45 (d, 1H, J = 4.9 Hz, OH-2’), 4.13 (q (t on D2O-
shake), 1H, H-2’), 3.35 (s, 3H, O-CH3), 2.42 (s, 3H, N-CH3), 1.73 and 1.54 (2s, 6H,
CH3), 1.03 and 1.01 (2s, 18H, tBu) ppm and other protons.
0
0
0
0
5 -O-tert-Butyldiphenylsilyl-2 –arabino–3 -De(oxyphosphinico)-3 -methylene
0
0
0
0
0
(methylimino)-5-methyluridylyl-(3 5 )-2 -deoxy–2 –fluoro-3 -O-tert-Butyl-
!
diphenylsilyl-5-methyluridine (21b). Coupling reaction of 5’-O-amino-3’-O-tert-
butyldiphenylsilyl-2’-fluorothymidine (1.44 g, 2.8 mmol) and 1-[5-O-(tert-butyldiphe-
nylsilyl)-3-deoxy-3-C-formyl-b-D-arabino-pentofuranosyl]thymine (1.42 g, 2.8 mmol)
according to the general procedure A provided the corresponding oxime dimer which
was further reduced and methylated to yield 2.80 g (98%) of a hard foam after
1
concentration under vacuo.Rf 0.45 (5% MeOH/CH2Cl2); H NMR (DMSO) d 11.41 and
11.31 (2s, 2H, NH), 7.66–7.35 (m, 22H, H-6 and Ph2), 5.98 (d, 1H, J = 5 Hz, H-1’), 5.90
(dd, 1H, Ja = 2.3 Hz, Jb = 18.3 Hz, H-1@), 5.43 (d, 1H, J = 4.8 Hz, OH-2’), 4.90 (dm, 1H,
J = 53.7 Hz, H-2@), 2.37 (s, 3H, N-CH3), 1.69 and 1.53 (2s, 6H, CH3), 1.03 and 1.00 (2s,
18H, tBu) ppm and other protons. Anal. Calc. for C54H66N5O10Si2 F 0.5H2O: C, 63.01;
H, 6.56; N, 6.80; Found: C, 62.97; H, 6.48; N, 6.77.
ꢁ
methyluridylyl-(3 5 )-2 -O–methyl-5-methyluridine (3a). Dimer 21a (2.64 g,
0
0
0
0
2 –Deoxy–2 –fluoro-3 -de(oxyphosphinico)-3 -methylene(methylimino)-5-
0 0
0
!
2.55 mmol) was reacted with trifluoromethane sulfonic anhydride according the
general procedure, except that the reaction mixture was directly chromatographed
without any work-up, to provide 1.60 g (54%) of the purified triflate intermediate. This
triflate dimer was fluorinated and desilylated according to the general procedures to
afford 550 mg (72%) of 3a as a white foam: Rf 0.35 (10% MeOH/CH2Cl2); H NMR
(DMSO) d 11.37 (s, 2H, NH), 7.98 and 7.56 (2s, 2H, H-6), 5.88 (d, 1H, J = 19.1 Hz,
1

ORDER
REPRINTS
2’-Substituted MMI Linked Nucleosidic Dimers
433
H-1’), 5.85 (d, 1H, J = 5.1 Hz, H-1@), 5.33 (t, 1H, OH-5’), 5.27 (dd, 1H, Ja = 3.6 Hz,
Jb = 51.9 Hz, H-2’), 5.22 (d, 1H, J = 5.8 Hz, OH-3@), 3.31 (s, 3H, O-CH3), 2.62 (s, 3H,
N-CH3), 1.81 and 1.75 (2s, 6H, CH3) ppm and other protons. MS (FAB+) m/e 558
(M + H). Anal. Calc. for C23H32N5O10F 0.2 H2O + 0.4 EtOH: C, 49.32; H, 6.05; N,
12.08; F, 3.28; Found: C, 49.19; H, 5.76; N, 11.71; F, 3.44.
ꢁ
methyluridylyl-(3 5 )-2 –deoxy–2 –fluoro-5-methyluridine (3b). Dimer 21b
0
0
0
0
2 –Deoxy–2 –fluoro-3 -de(oxyphosphinico)-3’1-methylene(methylimino)-5-
0
0
0
!
(2.80 g, 2.75 mmol) was reacted with trifluoromethane sulfonic anhydride according
the general procedure to provide 1.45 g (48%) of the triflate intermediate. This triflate
dimer was fluorinated and desilylated according to the general procedure to afford
480 mg (70%) of 3b as a white foam: Rf 0.35 (10% MeOH/CH2Cl2); 1H NMR
(DMSO) d 11.43 and 11.37 (2s, 2H, NH), 7.93 and 7.50 (2s, 2H, H-6), 5.90 (d, 1H,
J = 19 Hz, H-1’), 5.87 (dd, 1H, Ja = 2.0 Hz, Jb = 18.5 Hz, H-1@), 5.66 (m, 1H, OH-3@), 5.30
(m, 1H, OH-5’), 5.25 (dd, 1H, Ja = 3.6 Hz, Jb = 51.9 Hz, H-2’), 5.06 (dm, 1H, J = 54.1 Hz,
H-2@), 2.62 (s, 3H, N-CH3), 1.80 and 1.74 (2s, 6H, CH3) ppm and other protons. MS
(FAB+) m/e 546 (M + H). Anal. Calc. for C22H29N5O9F2 0.6H2O + 0.25EtOH: C, 47.59;
ꢁ
(methylimino)-thymidylyl-(3 5 )-2 -deoxy-2 -fluoro-5-methyluridine (23b).
H, 5.63; N, 12.33; F, 6.69; Found: C, 47.83; H, 5.26; N, 11.96; F, 6.61.
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-3 -de(oxyphosphinico)-3 -methylene
0
0
0
0
!
Compound 1b (0.65 g, 1.23 mmol) was dimethoxytritylated according to general
procedure C to afford 0.72 g (73%) of 23b: Rf 0.39 (10% MeOH/CH2Cl2 + 0.1%
Et3N);1H NMR (CDCl3) d 9.45 (s, 1H), 9.42 (s, 1H), 7.71 (s, 1H), 7.50–7.10 (m, 10H),
6.82 (m, H), 6.08 (t, J = 5.4 Hz, 1H), 5.68 (d, J = 19.5 Hz, 1H), 5.07 (dd J = 54.0, 4.2
Hz, 1H), 4.31 (m, 1H), 4.15–3.75 (m, 3H), 3.78 (s, 6H), 3.58–3.18 (m, 3H), 2.68 (m,
3H), 2.60 (s, 3H), 2.38 (m, 2H), 1.80 (s, 3H), 1.49 (s, 3H).
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-3 -de(oxyphosphinico)-3 -methylene
0
0 0
(methylimino)-thymidylyl-(3 5 )-2 -O–(2–methoxy)ethyl-5-methyluridine (23c).
!
Compound 1c (0.80 g, 1.36 mmol) was tritylated according to the general procedure
to afford 0.80 g (66%) of the dimethoxytrityl ether 23c: Rf 0.8 (10% MeOH/
1
CH2Cl2 + 0.1% Et3N); H NMR (CDCl3) d 8.99 (brs, 2H, NH), 7.71 (s, 1H, C5H),
7.2–7.43 (m, ArH), 6.8 (m, 4H, ArH), 6.14 (t, J = 5.2 Hz, 1H, H-1’), 5.78 (d, 1H, H-
1@), 3.78 (s, 6H, 2OCH3), 3.36 (s,3H, OCH3), 2.58 (s, 3H, NCH3), 1.83, 1.47 (2s, 6H,
CH3) and other protons. MS (FAB+) m/e 886 (M + H). Anal. Calcd for
C46H55N5O13 1.0H2O: C, 61.12; H, 6.36; N, 7.75. Found: C, 61.03; H, 6.20; N, 7.73.
ꢁ
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-3 -de(oxyphosphinico)-3 -methylene
0
0
0 0
(methylimino)-2 -O-methyl-5-methyluridylyl-(3 5 )-2 -O-methyl-5-methyluridine
!
(23d). Compound 2a (0.30 g, 0.53 mmol) was dimethoxytritylated according to
general procedure C to afford 0.36 g (79%) of 23d: Rf 0.68 (10% MeOH/
1
CH2Cl2 + 0.1% Et3N); H NMR (CDCl3) d 9.42 (s, 1H), 9.36 (s, 1H), 7.85 (s, 1H),
7.50–7.18 (m, 10H), 6.89–6.80 (m, 4H), 5.89 (s, 1H), 5.82 (d, J = 1.7 Hz, 1H), 4.25–
2.50 (m, 13H), 3.79 (s, 6H), 3.60 (s, 3H), 3.58 (s, 3H), 2.58 (s, 3H), 1.88 (s, 3H), 1.36
(s, 1H). Anal. Calcd for C45H53N5O13 0.5H2O: C, 61.35; H, 6.18; N, 7.95. Found: C,
61.43; H, 6.12; N, 7.93.
ꢁ

ORDER
REPRINTS
434
Peoc’h et al.
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-3 -de(oxyphosphinico)-3 -methylene
0
0
0
0
0
(methylimino)-2 -O-methyl-5-methyluridylyl-(3 5 )-2 -deoxy-2 -fluoro-5-methyl-
!
uridine (23e). Compound 2b (0.24 g, 0.43 mmol) was dimethoxytritylated according
to general procedure C to afford 0.33 g (89%) of 23e: Rf 0.46 (10% MeOH/
1
CH2Cl2 + 0.1% Et3N); H NMR (CDCl3) d 9.42 (s, 1H), 9.10 (s, 1H), 7.88 (s, 1H),
7.50–7.10 (m, 10H), 6.89–6.80 (m, 4H), 5.84 (s, 1H), 5.70 (d, J = 19 Hz, 1H), 5.07
(dd, J = 4.2, 54 Hz, 1H), 4.40–2.92 (m, 11H), 3.79 (s, 6H), 3.59 (s, 3H), 2.59 (s, 3H),
2.54 (m, 1H), 1.88 (s, 3H), 1.39 (s, 1H) ). Anal. Calcd for C44H50N5O12: C, 61.46; H,
5.86; N, 8.14. Found: C, 61.66; H, 5.89; N, 7.90.
0
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-2 –deoxy–2 –fluoro-3 -de(oxyphosphi-
0
0
0 0
nico)-3 -methylene(methylimino)-5-methyluridylyl-(3 5 )-2 -O–methyl-5-methyl-
!
uridine (23f). Dimer 3a (502 mg, 0.90 mmol) was tritylated following the general
procedure to afford 565 mg (73%) of the 5’-dimethoxytritylated dimer 23f: Rf 0.55
1
(10% MeOH/CH2Cl2); H NMR (DMSO) d 11.45 and 11.38 (2s, 2H, NH), 7.62 and
7.49 (2s, 2H, H-6), 7.43–6.88 (m, 13H, H-Ar), 5.89 (d, 1H, J = 20.5 Hz, H-1’), 5.81 (d,
1H, J = 5.1 Hz, H-1@), 5.37 (dd, 1H, Ja = 2.6 Hz, Jb = 52.6 Hz, H-2’), 5.24 (d, 1H,
J = 5.8 Hz, OH-3@), 5.05 (dm, 1H, J = 55.2 Hz, H-2@), 3.73 (s, 6H, O-CH3), 1.74 and
1.41 (2s, 6H, CH3) ppm and other protons. Anal. Calc. for C44H50N5O12F 0.3H2O: C,
61.07; H, 5.89; N, 8.09; Found: C, 61.00; H, 5.89; N, 7.88.
ꢁ
0
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-2 –deoxy–2 –fluoro-3 -de(oxyphosphi-
0
0
0 0
0
nico)-3 -methylene(methylimino)-5-methyluridylyl-(3 5 )-2 –deoxy–2 –fluoro-5-
!
methyluridine (23g). Dimer 3b (430 mg, 0.79 mmol) was tritylated following the
general procedure to afford 520 mg (78%) of the 5’-dimethoxytritylated dimer 23g: Rf
1
0.55 (10% MeOH/CH2Cl2); H NMR (DMSO) d 11.43 (bs, 2H, NH), 7.61 (s, 1H, H-6),
7.45–6.87 (m, 14H, H6 and H-Ar), 5.90 (d, 1H, J = 20.5 Hz, H-1’), 5.82 (d, 1H,
J = 19.1 Hz, H-1@), 5.68 (d, 1H, J = 6.2 Hz, OH-3@), 5.35 (dd, 1H, Ja = 2 Hz,
Jb = 52.6 Hz, H-2’), 5.05 (dm, 1H, J = 55.2 Hz, H-2@), 3.73 (s, 6H, O-CH3), 2.54 (s,
3H, N-CH3), 1.73 and 1.39 (2s, 6H, CH3) ppm and other protons. Anal. Calc. for
C43H47N5O11F2: C, 60.91; H, 5.59; N, 8.26; Found: C, 60.84; H, 5.76; N, 8.10.
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-3 -de(oxyphosphinico)-3 -methylene
0 0
0
(methylimino)-thymidylyl-(3 5 )-3 -O-(b-cyanoethyldiisopropylamino)phosphiryl-
2 -O–methyl-5-methyluridine (24a). Compound 1a was treated according to general
1
procedures C and D to provide 24a in 92% overall yield: H NMR (CD3CN) d 1.51
!
0
and 1.75 92s, 6, CH3), 5.85 (t, 1, 1’H), 6.10 (pseudo t, 1, 1’H) 9.17 (br s, 2, NH) and
other protons. 31P (NMR) d 150.9 and 151.2 ppm.
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-3 -de(oxyphosphinico)-3 -methylene
0
0 0
(methylimino)-thymidylyl-(3 5 )-3 -O-(b-cyanoethyldiisopropylamino)phosphiryl-
!
0
0
2 -deoxy-2 -fluoro-5-methyluridine (24b). Compound 23b (0.72 g, 0.9 mmol) was
phosphitylated according to general procedure D to afford 0.79 g (85%) of 24b: Rf 0.86
(EtOAc + 0.1% Et3N);1H NMR (CD3CN) d 9.39 (s, 2H), 7,55–7.15 (m, 11H), 6.84 (d,
4H), 6.09 (t, J = 5.6 Hz, 1H) 5.81 (d, J = 18.8 Hz, 1H), 5.05 (dm, J = 54 Hz), 3.60 (s,

ORDER
REPRINTS
2’-Substituted MMI Linked Nucleosidic Dimers
435
6H), 1.71 (s, 3H), 1.50 (s, 3H), and other protons; 31P NMR (CD3CN) d 151.66 (d,
J = 9.0 Hz), 151.35 (d, J = 7.9 Hz).
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-3 -de(oxyphosphinico)-3 -methylene
0
0
0
0
(methylimino)-2 -O-methyl-5-methyluridylyl-(3 5 )-3 -O-(b-cyanoethyldiisopro-
!
0
pylamino)phosphiryl-2 -O-methyl-5-methyluridine (24d). Compound 23d (0.32 g,
0.37 mmol) was phosphitylated according to general procedure D, except 10% acetone/
CH2Cl2 + 0.1% Et3N to 4% MeOH/CH2Cl2 + 0.1% Et3N was used for chromatogra-
phy, affording 0.33 g (83%) of 24d: H NMR (CD3CN) d 9.15 (bs, 2H), 7.66 (s, 1H),
7.55–7.15 (m, 10H), 6.86 (d, 4H), 5.81 (bs, 2H), 1.78 (s, 3H), 1.36 (s, 3H), and other
protons; 31P NMR (CD3CN) d 151.0.
1
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-3 -de(oxyphosphinico)-3 -methylene
0
0
0
0
(methylimino)-2 -O-methyl-5-methyluridylyl-(3 5 )-3 -O-(b-cyanoethyldiisopro-
pylamino)phosphiryl-2 -deoxy-2 -fluoro-5-methyluridine (24e). Compound 23e
(0.27 g, 0.31 mmol) was phosphitylated according to general procedure D to afford
!
0
0
1
0.28 g (85%) of 24e: H NMR (CD3CN) d 9.16 (bs, 2H), 7.66 (s, 1H), 7.55–7.15 (m,
10H), 6.86 (d, 4H), 5.80 (s, 1H), 5.76 (dd, J = 1.5, 16 Hz, 1H), 5.08 (dm, J = 55 Hz,
1H), 1.77 (s, 3H), 1.33 (s, 3H), and other protons; 31P NMR (CD3CN) d 151.8, 151.7,
151.3, 151.2.
0
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-2 –deoxy–2 –fluoro-3 -de(oxyphosphi-
0
0
0
0
nico)-3 -methylene(methylimino)-5-methyluridylyl-(3 5 )-3 -O-(b-cyanoethyldi-
isopropylamino)phosphiryl-2 -O–methyl-5-methyluridine (24f). Dimer 23f (301
!
0
mg, 0.35 mmol) was phosphitylated according to general procedure D to afford 240
mg (65%) of the phosphoramidite 24f: Rf 0.55 (7% MeOH/CH2Cl2 + 0.1% ET3N); 31P
NMR (CD3CN) d 151.29, 151.00.
0
0
0
0
0
5 -O-(4,4 -Dimethoxytriphenylmethyl)-2 –deoxy–2 –fluoro-3 -de(oxyphosphi-
0
0
0
0
nico)-3 -methylene(methylimino)-5-methyluridylyl-(3 5 )-3 -O-(b-cyanoethyldi-
isopropylamino)phosphiryl-2 –deoxy–2’–fluoro-5-methyluridine (24g). Dimer
!
0
23g (297 mg, 0.35 mmol) was phosphitylated according to general procedure D to
afford 330 mg (89%) of the phosphoramidite 24g: Rf 0.55 (7% MeOH/CH2Cl2 + 0.1%
ET3N); 31P NMR (CD3CN) d 151.68, 151.48, 151.38.
0
Automated incorporation of 2 -modified MMI Dimers/Assembly of oligonu-
cleotides. All synthesis were performed on an automated DNA synthesizer such
as Millipore Expedite or Applied Biosystem 380B utilizing the standard phosphor-
amidite protocol.
A representative protocol:
Step 1—Detritylation: 1 mmole of 5’-DMT -3’(succinyl-CPG-NMe2) nucleoside
was packed into a small column and connected to a Millipore Expedite DNA
synthesizer. TCA (3%) solution was pumped through the column according the
standard protocol used for standard amidite chemistry.

ORDER
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436
Peoc’h et al.
Step 2—Coupling (Of the 2’,2’ Modified MMI Phosphoramidite dimers): 2’,2’
Modified MMI phosphoramidite dimers were diluted in anhydrous acetonitrile
up to a concentration of 0.0673 Mol/l. Using the standard coupling step, 0.217
ml of a mixture of MMI dimer solution and 1H-tetrazole (1/1, v/v) were passed
through the column with an extended coupling wait step of 300 seconds.
Step 3—Oxidation of the phosphite triester linkage: This step was carried out using
the standard oxidizing protocol recommended for commercial deoxyribonucleo-
side phosphoramidites.
Step 4—Capping: This step was carried out using the standard capping protocol
recommended for commercial deoxyribonucleoside phosphoramidites.
Step 5—Isolation and purification: At the end of the automated synthesis, 2’,2’
Modified MMI containing oligonucleotides were concomitantly deprotected and
cleaved from the solid support by treatment with concentrated aqueous
ammonia solution (30%) at 55°C for 10 hours. The ammonia solution was then
evaporated and the full-length oligonucleotides will be separated from the
failure sequences on reverse phase HPLC. The sequences were analyzed by
HPLC, CE, and ESI–MS.
ACKNOWLEDGMENTS
Dr. Swayze thanks Prof. Townsend for his mentorship and guidance, and is
grateful for the lessons in life and science that were learned in his laboratory. We also
thank Dr. Elena Lesnik and Dr. Sue Freier for melting experiments, Dr. Richard H.
Griffey for stimulating discussions regarding sugar conformation, and Dr. P. Dan Cook
for his support of this project.
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Received August 22, 2003
Accepted October 31, 2003

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