Fmoc-protected altritol phosphoramidite building blocks and their application in the synthesis of altritol nucleic acids (ANAs)

Article abstract of DOI:10.1002/ejoc.200600868

Fmoc-protected altritol nucleoside phosphoramidite building blocks with adenine, guanine, thymine, uracil, cytosine and 5-methylcytosine as bases have been synthesized. These building blocks were used for the synthesis of altritol nucleic acid (ANA) and c

Full text of DOI:10.1002/ejoc.200600868

FULL PAPER
DOI: 10.1002/ejoc.200600868
Fmoc-Protected Altritol Phosphoramidite Building Blocks and Their
Application in the Synthesis of Altritol Nucleic Acids (ANAs)
Mikhail Abramov,[a] Guy Schepers,[a] Arthur Van Aerschot,[a] and Piet Herdewijn*[a]
Keywords: Altritol nucleoside / Fmoc protecting group / Altritol nucleic acids / Phosphoramidite chemistry
Fmoc-protected altritol nucleoside phosphoramidite building
blocks with adenine, guanine, thymine, uracil, cytosine and
5-methylcytosine as bases have been synthesized. These
building blocks were used for the synthesis of altritol nucleic
acid (ANA) and chimeric ANA-RNA oligonucleotides. Whilst
the yields of oligonucleotides were lower than those obtained
with 3Ј-O-benzoyl-protected altritol building blocks, the ex-
cellent compatibility with Pac-RNA chemistry for the synthe-
sis of chimeric oligonucleotides makes Fmoc a valuable pro-
tecting group for the secondary alcohol functions of the sugar
moieties of nucleosides for oligonucleotide synthesis.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
but was similarly unsuccessful in cases of long stretches of
ANA.
Introduction
Because of different and not always reproducible depro-
tection results for the 3Ј-O-TBDMS group in ANA oligo-
nucleotides synthesis (base modification, migration of the
phosphate linkage, and degradation have been observed by
HRMS analysis), we decided to investigate other protecting
strategies for ANA oligonucleotide synthesis.
The selection of appropriate protecting groups is a criti-
cal issue in successful solid-phase oligonucleotide synthesis.
In view of their potential interest as therapeutic or diagnos-
tic agents, we have described the synthesis and physico-
chemical properties of (4Ј–6Ј) altritol nucleic acids
(ANAs).[1] However, the synthetic problems associated with
the need to protect the additional 3Ј-hydroxy groups of al-
tritol nucleosides for oligonucleotide synthesis have slowed
down the further development of ANA.
The problems in ANA oligonucleotide synthesis have
usually been overcome by the use of the benzoyl protecting
group for the 3Ј-hydroxy group, in combination with the
phosphoramidite method.[1] However, the problem of
3ЈǞ4Ј benzoyl migration during the synthesis of the pro-
tected building blocks[2] results in difficulties for the large-
scale preparation of isomerically pure phosphoramidites,
and so we investigated the use of the 3Ј-O-TBDMS protect-
ing group in ANA oligonucleotide synthesis.[3] Although
RNA could be produced by this process, the deprotection
steps for the preparation of ANA sequences were much
more difficult than those for RNA sequences. Steric hin-
drance in the ANA amidites, such as of the axial TBDMS
groups, requires longer coupling times, which increase the
formation of side products. Base deprotection with ammo-
nia also needs longer reaction times, which might cause
internucleotide cleavage. Desilylation with TBAF is very
sensitive to water and could produce salts that must be re-
moved prior to analysis. Triethylamine–tris(hydrogen fluo-
ride) (TEA·3HF) has been used as an alternative to TBAF,
The decision to use Fmoc as the protecting group was
based on the fact that it can be removed from protected
bases and sugar moieties by treatment with aliphatic amines
such as triethylamine or piperidine,[4] oximate reagent[5] or
potassium carbonate in methanol.[6] In addition, Fmoc can
be used as a protecting group both for the heterocyclic base
and for the 3Ј-OH group.[7] We decided to use the 2-cyano-
ethyl N,N-diisopropylphosphoramidite approach[8] because
of its high yield in the internucleotide coupling reaction.
The more base-labile 2-cyanoethyl phosphate protecting
group should be released more rapidly than the Fmoc pro-
tecting group, avoiding migration reactions. Moreover, all
protecting groups (except for MMTr) can be removed by
β-elimination, which makes a one-step final deprotection
procedure possible.[8a]
Discussion
Synthesis of ANA Phosphoramidite Building Blocks
Seven phosphoramidites of -altritol nucleosides incor-
porating a 3Ј-O-(9-fluorenylmethoxycarbonyl) protecting
group – compounds 1a–7a (base moieties are adenine, gua-
nine, uracil, cytosine, thymine and 5-methylcytosine) – were
synthesized by a new strategy. The nucleosides were ob-
tained by ring opening of 1,5:2,3-dianhydro-4,6-O-benzyl-
idene--allitol,[3] which was in turn prepared in five steps
[a] Laboratory of Medicinal Chemistry, Rega Institute for Medical
Research
Minderbroedersstraat 10, 3000 Leuven, Belgium
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Fmoc-Protected Altritol Phosphoramidite Building Blocks
FULL PAPER
from commercially available tetraacetyl α--bromoglucose [N6-(9-fluorenylmethoxycarbonyl)adenin-9-yl]--altro-hexi-
(54% overall yield), basically by the procedure described by tol (1d). We tested different conditions for the selective re-
Brockway et al. (Figure 1).[9]
moval of one Fmoc group on the N6-amino group (pyri-
dine/water, ammonia/dioxane, triethylamine/dioxane), but
this resulted either in complete deprotection of hexitol 1c
(to give 1b) or in partial conversion of 1c into a mixture of
1d and the 3Ј-OH-deprotected compound 1e in low yield.
Removal of the benzylidene protecting group of 1c could
be accomplished by treatment with trifluoroacetic acid in
dichloromethane without migration of the 3Ј-O-Fmoc pro-
tecting group (and without formation of a 3Ј,4Ј-cyclic car-
bonate), giving 1f in 64% yield. Similarly, the 6Ј-O-mono-
methoxytrityl group can be introduced under common re-
action conditions (pyridine, room temperature). Synthesis
of the A(Fmoc)3 phosphoramidite 1a was accomplished by
phosphitylation of the 3Ј-O-Fmoc-6Ј-O-monomethoxytri-
tyl-protected building block 1g with the aid of 2-cyanoethyl
chloro-N,N-diisopropylposphoramidite (CEPA).[8b]
Figure 1. Structures of amidites 1a–7a containing altritol sugar
moieties.
The advantage of this approach is that a -altritol nucleo-
side with a free 3Ј-OH group and protected 4Ј-OH and 6Ј-
OH groups is obtained, thus avoiding problems with the
regioselective introduction of a protecting group in the 3Ј-
position. Different conditions were tested for the nucleo-
philic opening of the epoxide by the salts of nucleobases.
As well as the classical sodium and lithium salts,[1] a softer
base (DBU) or a phase-transfer catalyst such as tetrabu-
tylammonium chloride/potassium carbonate could be ap-
plied.[3] The preferred reaction conditions proved to be
base-dependent.
The fully protected altritol phosphoramidite incorporat-
ing an adenine base moiety was obtained in five steps.
Treatment of the DBU salt of adenine (3 equiv.) with
1,5:2,3-dianhydro-4,6-O-benzylidene--allitol in DMF at
90 °C for 6 h gave altritol 1b (Scheme 1) in 70% yield. One-
pot Fmoc protection both of the N6-amino group in the
adenine base and of the 3Ј-OH group in the hexitol moiety
was carried out by treatment with Fmoc chloride in pyri-
dine to give only 1,5-anhydro-4,6-O-benzylidene-2-[N6-
As might be expected, the guanine congener is a particu-
lar case. Previously[1] we had described epoxide opening
with the sodium salt of 2-amino-6-chloropurine in DMF in
40% yield. Besides the major product, two side compounds
were identified: the N7-substituted compound and the
bis(purinyl) nucleoside. The same reaction with the lithium
salt of N2-acetyl-2-amino-6-[2-(trimethylsilyl)ethoxy]purine
afforded the protected guanine nucleoside in 45% yield (af-
ter deacetylation), unsatisfactory for large-scale synthesis of
the altritol nucleosides, whilst the reaction in the presence
of Aliquat 336/K2CO3 in DMF gave a 45% yield of the
desired compound together with three side compounds. The
additional side compound proved to be the N9-substituted
2-amino-4-(dimethylamino)purine nucleoside. By utilising
the same phase-transfer catalyst, but in HMPA as solvent,
side-product formation could be avoided and the desired
compound was obtained in 70% yield.[3]
Direct Fmoc protection of 2b[3] (Scheme 2) did not yield
bis(9-fluorenylmethoxycarbonyl)adenin-9-yl]-2-deoxy-3-O- the desired N2,3Ј-O-bis-Fmoc-protected guanine 2e; only
(9-fluorenylmethoxycarbonyl)--altro-hexitol (1c) in 76% the 3Ј-O-Fmoc-monoprotected compound was formed, in
yield. To avoid solubility problems associated with the fully 45% yield. Transient trimethylsilylation of 2b followed by
protected building blocks in the solvent used for oligonucle- treatment with Fmoc chloride and deprotection with
otide synthesis and steric hindrance at the base moiety in TBAF, however, yielded a mixture of 2c and 2d in a 1:1
the adenine building block, we tried to remove one of the ratio, and this was treated with a twofold excess of Fmoc
Fmoc protecting groups to obtain 1,5-anhydro-4,6-O- chloride in pyridine to give tris(Fmoc)-protected 2e exclu-
benzylidene-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2- sively in a 50% yield based on 2b.
Scheme 1. Synthesis of adenine building block 1g.
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M. Abramov, G. Schepers, A. Van Aerschot, P. Herdewijn
FULL PAPER
Scheme 2. Synthesis of guanine amidite 2a.
After removal of the benzylidene group, the primary hy- ter removal of the benzylidene protecting groups, the pri-
droxy group was protected by treatment with monometh- mary hydroxy groups were each protected with a monome-
oxytrityl chloride. Finally, the phosphoramidite 2a was ob- thoxytrityl group. Finally, the T(Fmoc) phosphoramidite 4a
tained in 71% yield by phosphitylation of the protected and the U(Fmoc) phosphoramidite 5a were obtained by
building block 2g with CEPA.[8b] Unfortunately, though, phosphitylation of the 3Ј-O-Fmoc-6Ј-O-monomethoxytri-
the amidite 2a has a low solubility in acetonitrile and is not tyl-protected T (4e) and U (5e) building blocks.[8b]
suited for oligonucleotide synthesis in this solvent, and so
we synthesized the dimethylformamidine-protected (dmf- amidites were each obtained in six steps from uracil and
protected) G building block. thymine, respectively. 1,5-Anhydro-4,6-O-benzylidene-2-de-
The fully protected C (6a) and MeC (7a) phosphor-
The dmfG-protected phosphoramidite 3a was obtained by oxy-2-(uracil-1-yl)--altro-hexitol (5b) and the thymine ana-
starting from 2-amino-6-chloropurine, which was converted logue 4b were then used as starting materials (Scheme 4) for
into the guanine base 2b. This was followed by the introduc- the synthesis of the protected cytosine (6b) and 5-methylcy-
tion of the dimethylformamidine protecting group, afford- tosine (7b) congeners.[1,3] The method used was 1,2,4-triaz-
ing 3b, and the Fmoc group on 3Ј-OH, affording 3c olyl activation of the 4-positions of the uracil and thymine
(Scheme 3).
bases, followed by substitution with ammonia to yield 6b
After removal of the benzylidene group, the primary hy- and 7b.[10] In all cases investigated, it seems that the conver-
droxy group was protected with monomethoxytrityl chlo- sion of the uracil and thymine bases into the cytosine bases
ride to yield 3e. The phosphoramidite 3a was obtained in is a better way (higher yield) to obtain the 4-aminopyrim-
71% yield.[8b]
idine nucleosides than the direct opening reaction of the ep-
The fully protected U and T altritol phosphoramidites oxide ring with the salts of the corresponding nucleobases.
were each obtained in five steps, from uracil and thymine,
The N4-positions and the 3Ј-OH groups were each Fmoc-
respectively. The DBU salts of the bases were each treated protected in one step, followed by benzylidene removal and
with 1,5:2,3-dianhydro-4,6-O-benzylidene--altritol in 6-O-monomethoxytritylation. Finally, the C(Fmoc) and
DMF, yielding 1,5-anhydro-4,6-O-benzylidene-2-deoxy-2- MeC(Fmoc) phosphoramidites 6a and 7a were obtained in
(thymin-1-yl)--altro-hexitol (4b, 94%) and 1,5-anhydro- 88% yields by phosphitylation of the protected C (6e) and
4,6-O-benzylidene-2-deoxy-2-(uracil-1-yl)--altro-hexitol MeC (7e) building blocks.
(5b, 75%).[3] Introduction of the 3Ј-O-Fmoc protecting
In a preliminary effort to apply the Fmoc protecting
group was carried out by treatment with Fmoc chloride in group strategy for the synthesis of RNA we started with
pyridine and yielded 4c and 5c, respectively (Scheme 3). Af- Fmoc protection of the 2Ј-OH groups in the ribose moieties
Scheme 3. Synthesis of guanine, thymine and uracil building blocks 3e–5e.
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Fmoc-Protected Altritol Phosphoramidite Building Blocks
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Scheme 4. Synthesis of cytosine and 5-methylcytosine building blocks 6e and 7e.
of TIPS-protected rU and dmfrG. After removal of the TIPS protected oligomers and steric hindrances due to the bulky
groups with TEA·3HF in pyridine, 2Ј-O-Fmoc-protected protecting groups. Buck et al. had already experimented
rU and dmfrG were isolated in good yields. During the tri- with Fmoc-protected monomers as transient protecting
tylation of rU and dmfrG, however, 2ЈǞ3Ј Fmoc migration groups for heterocyclic bases for the synthesis of short
was observed and the desired 2Ј-O-Fmoc-protected ribonu- oligomers.[4a] For the preparation of longer oligonucleo-
cleosides were difficult to separate. New reaction conditions tides, however, they used a protection strategy in solution
for the application of 2Ј-O-Fmoc protection in RNA syn- after having assembled the oligomer on solid support with
thesis are to be investigated.
traditional building blocks (Table 1).
Table 1. Series of fully modified altritol oligomer sequences pre-
pared with amidites 1a–7a.
ANA Oligonucleotide Synthesis
Oligomer sequences (6ЈǞ4Ј)
Yield (%)[a]
With six new building blocks – 1a and 3a–7a – to hand,
both the coupling and the deprotection conditions needed
to be studied. Reactions were first tested and optimized
with the thymine analogue 4a, and decamers containing
four altritol blocks in the middle of a series of deoxythymid-
ines (5Ј-TTT-aT-aT-aT-aT-TTT-3Ј) were synthesized. In
view of the expected steric hindrance, one of the more
powerful reported coupling agents, pyridine trifluoroacetate
(PTFA),[11] was used with 10 equiv. of the amidite solution
and a 14 min coupling time, giving yields of about 70% per
a(TTTGACAAAACC) (10)
a(GAGACAACGGGT) (11)
a(CTACTACTTTTC) (12)
a(GCGCTTTTGCGC) (13)
a(AAATTTATGTCT) (14)
22[b]
18[c]
38[d]
16[e]
31[f]
[a] Overall coupling yields were calculated from MMTr cation
quantitation measurements. [b] AAAA stretch 50%. [c] GGG
stretch 25%. [d] First coupling 50%. [e] Last nine couplings 62%
overall. [f] Last nine couplings 70% overall.
As well as the suitability of the coupling, deprotection
coupling. In view of the low coupling yields, in the next was also investigated, for which a (5Ј-TTT-aG-aG-aG-TTT-
series of tests we used 14.5 equiv. of the amidite solution 3Ј) oligomer was prepared.[14] In addition, a thymidine sup-
and compared tetrazole (0.45 ), ethylthiotetrazole (0.25 ) port with the labile Q-linker (based on hydroquinone-O,OЈ-
and PTFA (0.22 ) as activators. Hardly any difference diacetic acid)[15] was used to allow rapid cleavage from the
could be seen in the detritylation graphs, with combined support. A large excess of the aG amidite 3a was used to
yields over five couplings of 82%, 91% and 90%, respec- ensure suitable coupling (22 equiv., in the presence of ETT
tively. The supports were treated with the AMA reagent[12] as the activator), resulting in an almost quantitative overall
at 30 °C for 1.5 h and analysed on a MonoQ ion-exchange yield according to detritylation measurements.
column. While detritylation yields were slightly reduced
The following deprotection conditions were compared by
with tetrazole as the activator, no differences in purity could analysis by ion-exchange chromatography under basic con-
be seen when using the different HPLC profiles as analyti- ditions (NaOH, pH = 12): (a) ammonia in methanol (2 )
cal tools. Similar results were obtained with phosphoramid- for 4 h at room temp. or (b) at 55 °C, (c) potassium carbon-
ites 1a, 3a and 5a–7a.
ate in methanol (0.05 ) for 4 h at room temp. and (d) at
A series of fully modified and mixed altritol sequences 55 °C, and finally (e) piperidine in anhydrous DMF (10%)
was then prepared on a 1 µmol scale with use of ETT and for 2 h at room temp. While conditions (a) were not satis-
14.5 equiv. of amidite (sequences 10–14). To avoid the need factory, (b) gave a more pronounced peak under the high-
for the synthesis of different altritol nucleoside containing salt eluting conditions, but with still many side products.
supports, a propanediol-modified universal support was While conditions (c) generated only broad diffuse peaks,
used, generating a 3-hydroxypropyl phosphate extension at heating as in (d) proved detrimental, with mostly shorter
the 4Ј-end.[13] Unfortunately, the synthesis turned out to be fragments being eluted from the HPLC column. Exclusive
cumbersome and HPLC profiles after deprotection showed piperidine treatment for 2 h only afforded a very small, dif-
complex mixtures of oligomers, with only small amounts of fuse and broad peak representing the desired oligonucleo-
the desired products being isolated. Overall it seems that tide, whilst additional piperidine treatment proved detri-
coupling might be reasonable, as indicated by good detrityl- mental.
ation yields, but that the procedure is not fully reproducible
We next tried single or multiple incorporations of ade-
and is sequence-dependent. The reasons for these short- nine (1a) and uracil (5a) monomers into RNA on a 1 µmol
comings might involve lower solubilities of the fully Fmoc- scale, as outlined in Table 2. Phenoxyacetyl-(Pac-)2Ј-O-silyl-
Eur. J. Org. Chem. 2007, 1446–1456
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M. Abramov, G. Schepers, A. Van Aerschot, P. Herdewijn
Table 2. Series of mixed altritol oligomer sequences prepared with amidites 1a–7a.
FULL PAPER
Oligomer sequences (5ЈǞ3Ј)
Yield (%)[a]
OD260
MS calcd.
MS found
GUA UUG ACA GCU AUaU CGA AdTdT (15)
GUA UUG ACA GCaU AUU CGA AdTdT (16)
GUA UaUG ACA GCU AUU CGA AdTdT (17)
GaUA UUG ACA GCU AUU CGA AdTdT (18)
GUA UaUG ACA GCaU AaUaU CGA AdTdT (19)
GUaA UaUG ACaA GCaU AUaU CGaA AdTdT (20)
GaUaA aUaUG aACaA GCaU aAaUaU CGaA aAdTdT (21)
UUC GAA UAG CUG UCA AaUA CdTdT (22)
UUC GAA UAG CUG aUCA AUA CdTdT (23)
UUC GAA UAG CaUG UCA AUA CdTdT (24)
UUC GAA aUAG CUG UCA AUA CdTdT (25)
UaUC GAA UAG CUG UCA AUA CdTdT (26)
53
66
68
55
55
70
34
64
62
75
60
65
18.3
24.2
28.0
25.7
11.6
8.0
6665.9
6665.9
6665.9
6665.9
6708.0
6736.0
6820.1
6625.9
6625.9
6625.9
6625.9
6625.9
6666.1
6666.7
6666.1
6666.1
6708.8
6736.1
6820.6
6626.1
6626.3
6626.4
6626.0
6626.2
9.7
32.0
22.7
18.0
17.8
13.3
[a] Yields of isolated oligonucleotides. Oligonucleotides were purified by anion-exchange HPLC and the purities and structures of chimeric
oligonucleotides were confirmed by anion-exchange HPLC and HRMS. In all cases the purities of the samples were 95% or higher.
(TBDMS-)protected RNA phosphoramidites were applied
for chimeric oligonucleotide synthesis. Amidite solution
(0.08 , 18 equiv.) was used for the altritol monomers in the
presence of ETT activator (compared to 14 equiv. for the
Experimental Section
General Procedures: Tetra-O-acetyl-α--bromoglucose was pro-
vided by Fluka; adenine, cytosine, guanine and uracil were from
ACROS. All other chemicals were provided by Aldrich or ACROS
standard silyl-protected RNA monomers), in general ensur-
ing adequate coupling. Final trityl yields, amounts of iso-
lated oligomer (OD260) and mass analyses (M+) are listed
in Table 2. Sequences 15–26 were synthesized and depro-
tected by use of AMA solution at 30 °C for 90 min, fol-
lowed by a triethylamine–tris(hydrogen fluoride) treatment
(1.5 mL NMP + 0.750 mL TEA + 1 mL TEA·3HF) for
3.5 h at 55 °C, allowing removal of the silyl protecting
groups. The crude oligonucleotides were purified by anion-
exchange HPLC and the purities and structures of the
modified oligonucleotides were confirmed by anion-ex-
change HPLC and HRMS.
and were of the highest quality. 1H and 13C NMR spectra were
determined with a 200 MHz Varian Gemini apparatus with tet-
1
ramethylsilane as internal standard for the H NMR spectra (s =
singlet, d = doublet, dd = double doublet, t = triplet, br. s = broad
signal, br. d = broad doublet, m = multiplet) and the solvent sig-
nal – [D6]DMSO (δ =39.6 ppm) or CDCl3 (δ =76.9 ppm) – for the
13C NMR spectra. For some products a Varian Unity-500 spec-
trometer (500 MHz for 1H NMR) was used. Coupling constant
values were derived by first-order spectral analysis. Exact mass
measurements were performed with a quadrupole/orthogonal ac-
celeration time-of-flight tandem mass spectrometer (qTOF2,
Micromass, Manchester, UK) fitted with a standard electrospray
ionization interface. Precoated Machery–Nagel Alugram SILG/
UV254 plates were used for TLC, and the spots were examined un-
der UV light and by use of sulfuric acid/anisaldehyde spray. Col-
umn chromatography was performed on ACROS silica gel (0.060–
0.200 mm or 0.035–0.060 mm). Anhydrous solvents were obtained
as follows: dichloromethane was stored over calcium hydride,
heated at reflux and distilled. Pyridine was heated at reflux in the
presence of potassium hydroxide pellets and distilled. Dimethyl-
formamide was dried with activated molecular sieves (4 Å). HMPA
was dried by azeotropic distillation with toluene. Methanolic am-
monia was prepared by bubbling NH3 gas through absolute meth-
Conclusions
The synthesis of six altritol nucleoside phosphoramidite
building blocks with Fmoc protection on base and sugar
moieties has been accomplished successfully. Coupling
yields are generally lower than those obtained with 3Ј-ben-
zoyl-protected building blocks (protected on the base moie-
ties with the conventional aryl protecting groups). The ex- anol at 0 °C and stored at 0 °C.
cellent compatibility with Pac-RNA chemistry for the syn-
Automated Synthesis of Oligonucleotides with 3Ј-O-Fmoc ANA
thesis of chimeric oligonucleotides points to the value of
the Fmoc protecting group for oligonucleotide synthesis, al-
though new research to select the optimal conditions for
Building Blocks: The synthesis of oligonucleotides was ac-
complished by the standard phosphoramidite method on an Exed-
ite synthesiser (Applied Biosystems) with an extended 12 min coup-
synthesis and deprotection still needs to be carried out. The ling step. A 15-fold excess of nucleobase acetonitrile solution
(200 µL of 0.07 ) and an excess of activator (200 µL) relative to
CPG-bound 5Ј-hydroxy group we used in each coupling cycle. The
synthesis scale was 1.0 µmol. Average coupling yield was monitored
by online colorimetry by colorimetric quantitation of the trityl frac-
tions. The detritylation solution was 3% TCA in DCM, capping
was performed with Ac2O/THF and 1-methylimidazole/THF/PyH
(10%) 1-methylimidazole/THF/PyH, whilst the oxidation solution
was I2 in THF/H2O/PyH (0.02 ). Tetrazole solution in acetonitrile
(0.45 ), S-ethylthioterazole solution in MeCN (0.25 ) or pyridi-
nium trifluoroacetate solution in MeCN (0.22 ) was used as an
activator.
full deprotection of the synthesized oligonucleotides is
proving to be cumbersome at this moment: this could be
due to steric hindrance and/or to low solubilities of the
growing oligonucleotides in the solvents used for synthesis
and deprotection. In conclusion, the benzoyl group is a bet-
ter protecting group than Fmoc (for the 3Ј-OH function)
for the synthesis of ANA. Further research will be focussed
on the synthesis of 3Ј-O-benzoyl-protected altritol phos-
phoramidite building blocks, avoiding migration reactions
during the synthesis.
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Deprotection and Purification of Oligonucleotides: Cleavage from Synthesis of Individual Protected Nucleosides
the support and base and 3Ј(4Ј)-OH deprotection were performed
1,5-Anhydro-2-[N6,N6-bis(9-fluorenylmethoxycarbonyl)adenin-9-yl]-
in one step with AMA at 30 °C for 1.5 h. The solution of crude
oligonucleotide was desalted on an NAP-25 column and purified
by anion-exchange HPLC. The purities and structures of modified
oligonucleotides were confirmed by anion-exchange HPLC and
HRMS.
2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-monomethoxytrityl-
-altro-hexitol (1g)
D
1,5-Anhydro-4,6-O-benzylidene-2-deoxy-2-[N6,N6-bis(9-fluorenyl-
methoxycarbonyl)adenin-9-yl]-3-O-(9-fluorenylmethoxycarbonyl)-
D-
altro-hexitol (1c): 9-Fluorenylmethoxycarbonyl chloride (4.2 g,
16.3 mmol) was added under nitrogen in four portions to a solution
of 1b[9] (1.5 g, 4.06 mmol) in dry pyridine (30 mL) and the reaction
mixture was stirred at room temperature for 1 h. The reaction was
monitored by TLC. MeOH (10 mL) was then added and the stir-
ring was continued for 30 min. The yellow solution was concen-
trated and co-evaporated to dryness with toluene (2ϫ30 mL). The
residue was subjected to silica gel flash column chromatography
with acetone in dichloromethane (2.5%) as eluent. Precipitation
from dichloromethane/hexane at –60 °C afforded the title com-
Synthesis of 1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbon-
yl)-6-O-monomethoxytrityl-D-altro-hexitol Nucleoside 4-[(2-Cyano-
ethyl)-N,N-diisopropylphosphoramidites] 1a–7a: Dry 3-O-(9-fluor-
enylmethoxycarbonyl)-6-O-(monomethoxytrityl)--altro-hexitol N-
(9-fluorenylmethoxycarbonyl)-protected nucleoside (1 mmol) was
dissolved in dry THF (5 mL). 2,4,6-Collidine (7.5 mmol) was
added, followed by N-methylimidazole (0.5 mmol). (2-Cyanoethyl)-
N,N-diisopropylphosphonamidic chloride (2.5 mmol) was then
added dropwise at room temperature over 5 min. The reaction was
complete after 1–2 h as determined by TLC. The reaction mixture
was diluted with dichloromethane (50 mL), washed with water and
dried with Na2SO4. The solvent was removed in vacuo, yielding a
viscous oil. Co-evaporation with toluene (2ϫ10 mL) afforded the
crude phosphoramidites as off-white foams or oils. The phos-
phoramidites were further purified by silica gel chromatography
and precipitated from hexane (150 mL) at –60 °C, yielding white,
fine powders in 75–85% yields.
1
pound 1c as a white powder (3.2 g, 76%). H NMR (CDCl3): δ =
3.64 (dd, J = 2.6, 9.7 Hz, 1 H, 4Ј-H), 3.74 (t, J = 10.4 Hz, 1 H,
6Јax-H), 4.10–4.70 [m, 13 H, 1Ј-H, 5Ј-H, 6Јeq-H, CH2O and 9-H
(Fmoc)], 5.00 (br. s, 1 H, 2Ј-H), 5.39 (s, 1 H, PhCH), 5.72 (br. s, 1
H, 3Ј-H), 7.19–7.50 (m, 21 H, H arom), 7.65 (m, 6 H, H arom),
7.80 (d, J = 7.7 Hz, 2 H, H arom), 8.55 (s, 1 H, 8-H), 8.94 (s, 1 H, 2-
H) ppm. HRMS: calcd. for C63H50N5O10 [MH]+ 1036.3558; found
1036.3553.
1,5-Anhydro-2-[N2-bis(9-fluorenylmethoxycarbonyl)adenin-9-yl]-2-
deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-monomethoxytrityl-D-
1,5-Anhydro-4,6-O-benzylidene-2-deoxy-3-O-(9-fluorenylmethoxy-
carbonyl)-2-[N6-(9-fluorenylmethoxycarbonyl)adenin-9-yl]-
D-altro-
altro-hexitol 4-[(2-Cyanoethyl)-N,N-diisopropylphosphoramidite]
(1a): 31P NMR (CDCl3): δ = 149.75, 152.41 ppm. HRMS: calcd.
for C85H79N7O12P [MH]+ 1420.5524; found 1420.5562.
hexitol (1d): Compound 1c (103 mg, 0.1 mmol) was dissolved in
dioxane (2 mL) and ammonia (26%, 500 µL) was added at 0 °C.
After 5 min, the solution was concentrated and co-evaporated to
dryness with toluene (2ϫ5 mL). The residue was purified by silica
gel flash column chromatography with acetone in dichloromethane
(5%) to afford the title compound 1d as a white solid (17 mg, 21%).
1H NMR (CDCl3): δ = 3.72–3.86 (m, 2 H, 4Ј-H, 6Јax-H), 4.14–
4.78 [m, 11 H, 1Ј-H, 5Ј-H, 6Јeq-H, CH2O and 9-H (Fmoc)], 4.98
(br. s, 1 H, 2Ј-H), 5.50 (s, 1 H, PhCH), 5.66 (br. s, 1 H, 3Ј-H), 7.20–
7.50 (m, 13 H, H arom), 7.65 (m, 4 H, H arom), 7.78 (d, J = 7.7 Hz,
4 H, H arom), 8.60 (s, 1 H, 8-H), 8.81 (br. s, 1 H, 2-NH), 8.90 (s,
1 H, 2-H) ppm. HRMS: calcd. for C63H50N5O10 [MH]+ 814.2877;
found 814.2883.
1,5-Anhydro-2-[N2,O6-bis(9-fluorenylmethoxycarbonyl)guanin-9-yl]-
2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-monomethoxytrityl-
D
-altro-hexitol 4-[(2-Cyanoethyl)-N,N-diisopropylphosphoramidite]
(2a): 31P NMR (CDCl3): δ = 149.46, 151.59 ppm. HRMS: calcd.
for C85H79N7O13P [MH]+ 1436.5474; found 1436.5537.
1,5-Anhydro-2-deoxy-[2-(N2-dimethylaminomethylene)guanin-9-yl]-
3-O-(9-fluorenylmethoxycarbonyl)-6-O-monomethoxytrityl-D-altro-
hexitol 4-[(2-Cyanoethyl)-N,N-diisopropylphosphoramidite] (3a): 31P
NMR (CDCl3): δ = 150.9 ppm. HRMS: calcd. for C58H64N8O9P
[MH]+ 1047.4534; found 1047.4547.
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N6,N6-
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-mono-
bis(9-fluorenylmethoxycarbonyl)adenin-9-yl]-D-altro-hexitol (1f):
methoxytrityl-2-(thymin-1-yl)-D-altro-hexitol 4-[(2-Cyanoethyl)-
Compound 1c (2.9 g, 2.8 mmol) was dissolved in dichloromethane
(30 mL) and TFA (4 mL) was added at 0 °C. The reaction was
monitored by TLC. After the mixture had been stirred at room
temperature for 1 h, ethanol (20 mL) was added and the yellow-
brown solution was concentrated and co-evaporated to dryness
with toluene (2ϫ30 mL). The residue was purified by silica gel
flash column chromatography with a stepwise gradient of methanol
(2–4%) in dichloromethane to afford the title compound 1f as a
white solid (1.7 g, 64%). 1H NMR (CDCl3): δ = 2.0–2.8 (br. s, 2
H, 4Ј-OH and 5Ј-OH), 3.83–3.95 (m, 4 H, 4Ј-H, 5Ј-H, 6Ј-H), 4.10–
4.61 [m, 11 H, 1Ј-H, CH2O and 9-H (Fmoc)], 5.00 (br. s, 1 H, 2Ј-
H), 5.72 (br. s, 1 H, 3Ј-H), 7.19–7.50 (m, 21 H, H arom), 7.65 (m,
6 H, H arom), 7.80 (d, J = 7.7 Hz, 2 H, H arom), 8.55 (s, 1 H, 8-
H), 8.94 (s, 1 H, 2-H) ppm. HRMS: calcd. for C56H46N5O10
[MH]+ 948.3245; found 948.3253.
N,N-diisopropylphosphoramidite] (4a): 31P NMR (CDCl3): δ =
150.02, 151.93 ppm. HRMS: calcd. for C55H60N4O10P [MH]+
967.4047; found 967.4099.
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-mono-
methoxytrityl-2-(uracil-1-yl)-D-altro-hexitol 4-[(2-Cyanoethyl)-N,N-
diisopropylphosphoramidite] (5a): 31P NMR (CDCl3): δ = 150.61,
152.63 ppm. HRMS: calcd. for C55H60N4O10P [MH]+ 967.4047;
found 967.4099.
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N4-(9-
fluorenylmethoxycarbonyl)cytosin-1-yl]-6-O-monomethoxytrityl-D-
altro-hexitol 4-[(2-Cyanoethyl)-N,N-diisopropylphosphoramidite]
(6a): 31P NMR (CDCl3): δ = 149.50, 151.93 ppm. HRMS: calcd.
for C70H71N5O11P [MH]+ 1188.4888; found 1188.4873.
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N4-(9-
fluorenylmethoxycarbonyl)-5-methylcytosin-1-yl]-6-O-monometh-
1,5-Anhydro-2-[N6,N6-bis(9-fluorenylmethoxycarbonyl)adenin-9-yl]-
2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-monomethoxytrityl-
oxytrityl-
D
-altro-hexitol 4-[(2-Cyanoethyl)-N,N-diisopropylphos-
D-altro-hexitol (1g): Monomethoxytrityl chloride (0.65 g,
phoramidite] (7a): 31P NMR (CDCl3): δ = 150.03, 151.96 ppm. 2.1 mmol) was added under nitrogen at room temperature to a
HRMS: calcd. for C70H71N5O11P [MH]+ 1188.4888; found stirred solution of 1f (1.6 g, 1.7 mmol) in dry pyridine (15 mL). The
1188.4873.
reaction was monitored by TLC. After the mixture had been stirred
1451
Eur. J. Org. Chem. 2007, 1446–1456
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org

M. Abramov, G. Schepers, A. Van Aerschot, P. Herdewijn
FULL PAPER
for 2 h, methanol (3 mL) was added and the solution was concen-
trated and co-evaporated to dryness with toluene (2ϫ15 mL). The
residue was purified by silica gel flash column chromatography
with acetone in dichloromethane (3%). Precipitation from dichlo-
romethane/hexane at –60 °C afforded the title compound 1g as a
white powder (1.2 g, 63%). 1H NMR (CDCl3): δ = 1.94 (br. s, 1 H,
4Ј-OH), 3.38 (dd, J = 1.1, 1.1 Hz, 1 H, 6Јax-H), 3.56 (dd, J = 1.1,
1.1 Hz, 1 H, 6Јax-H), 3.79 (s, 3 H, CH3), 3.89 (br. s, 2 H, 4Ј-H, 5Ј-
H), 4.06–4.18 (m, 2 H, 1-H), 4.25–4.69 [m, 9 H, CH2O and 9-H
(Fmoc)], 5.04 (br. s, 1 H, 2Ј-H), 5.56 (br. s, 1 H, 3Ј-H), 6.82 (d, J
= 8.9 Hz, 2 H, H arom), 7.19–7.50 (m, 24 H, H arom), 7.65 (m, 6
H, H arom), 7.80 (d, J = 7.7 Hz, 2 H, H arom), 8.79 (s, 1 H, 8-H),
8.96 (s, 1 H, 2-H) ppm. HRMS: calcd. for C76H62N5O11 [MH]+
1220.4446; found 1220.4454.
7.20–7.36 (m, 5 H, H arom), 7.33–7.41 [m, 4 H, H arom (Fmoc)],
7.73–7.80 [m, 4 H, H arom (Fmoc)], 8.08 (s, 1 H, 2-NH), 8.10 (s,
1 H, 8-H), 11.30–11.90 (br. d, 1 H, NH) ppm. HMRS: calcd. for
C33H30N5O7 [MH]+ 608.2145; found 608.2151.
1,5-Anhydro-4,6-O-benzylidene-2-[N2,O6-bis(9-fluorenylmethoxy-
carbonyl)guanin-9-yl]-2-deoxy-D
-altro-hexitol (2d): 1H NMR
(CDCl3): δ = 2.42 (br. s, 1 H, 3Ј-OH), 3.57 (dd, J = 1.8, 9.5 Hz, 1
H, 4Ј-H), 3.78 (t, J = 10.5, Hz, 1 H, 6Јax-H), 3.95–4.62 [m, 12 H,
1Ј-H, 2Ј-H, 3Ј-H, 5Ј-H, 6Јeq-H, 9-H and CH2O (Fmoc)], 5.47 (s, 1
H, PhCH), 7.14–7.48 (m, 18 H, 2-NH and H arom), 7.58–7.65 [m,
4 H, H arom (Fmoc)], 8.32 (s, 1 H, 8-H) ppm. HMRS: calcd. for
C48H40N5O9 [MH]+ 830.2826; found 830.2817.
1,5-Anhydro-4,6-O-benzylidene-2-[N2,O6-bis(9-fluorenylmethoxy-
1,5-Anhydro-2-[N2,O6-bis(9-fluorenylmethoxycarbonyl)guanin-9-yl]-
2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-monomethoxytrityl-
carbonyl)guanin-9-yl]-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-D-
altro-hexitol (2e): The mixture (2.1 g) of 2c and 2d in pyridine
(120 mL) was concentrated to 25 mL, Fmoc chloride (4.0 g,
15.4 mmol) was added in 1 g portions over 3 h, and stirring was
continued for 1 h. Methanol (10 mL) was added dropwise at 0 °C
and the reaction mixture was stirred for a further 10 min. The re-
sulting mixture was concentrated and co-evaporated with toluene
(2ϫ30 mL) under reduced pressure, and the residue was extracted
with ethyl acetate, washed with water, dried with magnesium sulfate
and purified by flash silica gel column chromatography with meth-
anol in dichloromethane (1.5%). Yield 2.2 g (42%) based on 2b. 1H
NMR (CDCl3): δ = 3.67 (dd, J = 1.8, 9.5 Hz, 1 H, 4Ј-H), 3.78 (t,
J = 10.5 Hz, 1 H, 6Јax-H), 3.85–4.50 [m, 14 H, 1Ј-H, 2Ј-H, 5Ј-H,
6Јeq-H, 9-H and CH2O (Fmoc)], 5.19 (br. s, 1 H, 3Ј-OH), 5.30 (s,
1 H, PhCH), 7.00–7.60 (m, 27 H, H arom), 7.70–7.82 [m, 2 H, H
arom (Fmoc)], 8.31 (s, 1 H, 8-H) ppm. HRMS: calcd. for
C63H50N5O11 [MH]+ 1052.3507; found 1052.3541.
D-altro-hexitol (2g)
1,5-Anhydro-4,6-O-benzylidene-2-[N2,O6-bis(9-fluorenylmethoxy-
carbonyl)guanin-9-yl]-2-deoxy- -altro-hexitol (2c): 1,5-Anhydro-
D
4,6-O-benzylidene-2-deoxy-2-(guanin-9-yl)--altro-hexitol (2b,[3]
1.95 g, 5.0 mmol) was co-evaporated with pyridine (2ϫ50 mL),
and TMSCl (6.4 mL, 50 mmol) was added dropwise at 0 °C under
argon to the resulting suspension in pyridine (30 mL). The resulting
clear solution was stirred at room temperature for 2 h; Fmoc chlo-
ride (5.2 g, 20 mmol) was added in 1 g portions over 3 h and stir-
ring was continued for 1 h. Methanol (10 mL) was added dropwise
at 0 °C and the reaction mixture was stirred for 10 min. The re-
sulting mixture was concentrated and co-evaporated with toluene
(2ϫ30 mL) under reduced pressure. The residue was extracted with
ethyl acetate, washed with water, dried with magnesium sulfate and
purified by flash silica gel column chromatography with methanol
in dichloromethane (1.5 %). Yield of 1,5-anhydro-4,6-O-benzyl-
idene-2-deoxy-2-[N2-(9-fluorenylmethoxycarbonyl)guanin-9-yl]-3-
O-trimethylsilyl--altro-hexitol 3.0 g (66%). 1H NMR (CDCl3): δ
= 0.21 (s, 9 H, CH3Si), 3.54 (dd, J = 1.8, 9.5 Hz, 1 H, 4Ј-H), 3.72
(t, J = 10.5 Hz, 1 H, 6Јax-H), 4.09–4.70 [m, 9 H, 1Ј-H, 2Ј-H, 3Ј-H,
5Ј-H, 6Јeq-H, 9-H and CH2O (Fmoc)], 5.44 (s, 1 H, PhCH), 7.20–
7.45 (m, 9 H, H arom), 7.55–7.60 [m, 4 H, H arom (Fmoc)], 7.87
(br. s, 1 H, 2-NH), 8.37 (s, 1 H, 8-H), 11.30 (br. s, 1 H, NH) ppm.
HRMS: calcd. for C36H38N5O7Si [MH]+ 680.2541; found 680.2535.
1,5-Anhydro-4,6-O-benzylidene-2-[N2,O6-bis(9-fluorenylmethoxy-
carbonyl)guanin-9-yl]-2-deoxy-3-O-trimethylsilyl--altro-hexitol
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N2-(9-
fluorenylmethoxycarbonyl)guanin-9-yl]-6-D-altro-hexitol (2f): TFA
(5 mL) was added dropwise at 0 °C to a solution of 2e (2.2 g,
2.1 mmol) in dichloromethane (30 mL), and the reaction mixture
was stirred for 30 min. Water (100 µL, 5.6 mmol) was added and
stirring was continued for 15 min, ethanol (80 %, 10 mL) was
added, and solvents were removed. The residue was co-evaporated
with toluene (2ϫ30 mL) and the crude material was subjected to
flash silica gel column chromatography with methanol in dichloro-
methane (4%) to afford the title compound as a white foam (1.0 g,
52%). 1H NMR (CDCl3): δ = 2.0–2.8 (br. s, 2 H, 4-OH and 5-
OH), 3.80–4.48 (m, 15 H, 1Ј-H, 4Ј-H, 5Ј-H, 6Ј-H), [CH2O and 9-
H (Fmoc)], 5.06 (br. s, 1 H, 3Ј-H), 6.91–7.58 [m, 23 H, 2-NH and
H arom (Fmoc)], 7.77–7.80 [m, 2 H, H arom (Fmoc)], 8.85 (s, 1 H,
8-H) ppm. HRMS: calcd. for C56H46N5O11 [MH]+ 964.3194; found
964.3174.
1
(300 mg) was isolated as a minor product. H NMR (CDCl3): δ =
0.40 (s, 9 H, CH3Si), 3.45 (dd, J = 1.8, 9.5 Hz, 1 H, 4Ј-H), 3.64 (t,
J = 10.5, Hz, 1 H, 6Јax-H), 4.09–4.70 [m, 12 H, 1Ј-H, 2Ј-H, 3Ј-H,
5Ј-H, 6Јeq-H, 9-H and CH2O (Fmoc)], 5.39 (s, 1 H, PhCH), 7.02–
7.38 (m, 18 H, 2-NH and H arom), 7.50–7.60 [m, 4 H, H arom
(Fmoc)], 8.37 (s, 1 H, 8-H) ppm. HRMS: calcd. for C51H48N5O9Si
[MH]+ 902.3221; found 902.3228. A solution of 1,5-anhydro-4,6-
O-benzylidene-2-[N2,O6-bis(9-fluorenylmethoxycarbonyl)guanin-9-
yl]-2-deoxy-3-O-trimethylsilyl--altro-hexitol (3.0 g, 3.3 mmol) was
dissolved in THF (10 mL) and Bu4NF (1  in THF, 6 mL) was
added dropwise to the resulting solution at 0 °C. After 30 min, the
solution was added slowly (dropwise) to ice-cold water (250 mL)
with stirring. The obtained solid was filtered off, dried and purified
by flash silica gel column chromatography with methanol in dichlo-
romethane (2.5%). A mixture of two products (2.1 g) was isolated.
1,5-Anhydro-2-deoxy-2-[N2,O6-bis(9-fluorenylmethoxycarbonyl)-
guanin-9-yl]-3-O-(9-fluorenylmethoxycarbonyl)-6-O-monomethoxy-
trityl-D-altro-hexitol (2g): A solution of 2f (1.0 g, 1 mmol) in pyri-
dine (50 mL) was concentrated to 10 mL and MMTrCl (620 mg,
2 mmol) was added under argon at room temperature. After 3 h,
methanol (5 mL) was added and the volatiles were removed. The
residue was co-evaporated with toluene (2ϫ20 mL) and purified
by silica gel flash column chromatography with methanol in dichlo-
romethane (2 %). Precipitation from dichloromethane/hexane at
–60 °C afforded the title compound 2g as a white powder (1.0 g,
1
1,5-Anhydro-4,6-O-benzylidene-2-deoxy-2-[N2-(9-fluorenylmethoxy- 82%). H NMR (CDCl3): δ = 2.04 (br. s, 1 H, 4Ј-OH), 3.42 (dd, J
carbonyl)guanin-9-yl]-
D
-altro-hexitol (2c): 1H NMR ([D6]DMSO): δ
= 1.1, 1.1 Hz, 1 H, 6Јax-H), 3.54 (dd, J = 1.1, 1.1 Hz, 1 H, 6Јeq-
H), 3.79 (s, 3 H, CH3), 3.89–4.62 [m, 13 H, 2Ј-H, 4Ј-H, 5Ј-H, CH2O
= 3.64 (dd, J = 1.8, 9.5 Hz, 1 H, 4Ј-H), 3.79 (t, J = 10.5, Hz, 1 H,
6Јax-H), 4.09–4.70 [m, 9 H, 1Ј-H, 2Ј-H, 3Ј-H, 5Ј-H, 6Јeq-H, 9-H and 9-H (Fmoc)], 4.96 (br. s, 1 H, 3Ј-H), 6.82 (d, J = 8.9 Hz, 2 H,
and CH2O (Fmoc)], 5.58 (s, 1 H, PhCH), 5.73 (br. s, 1 H, 3Ј-OH), H arom), 7.06–7.60 (m, 35 H, H arom and 2-NH), 7.74 [m, 2 H,
1452
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1446–1456

Fmoc-Protected Altritol Phosphoramidite Building Blocks
FULL PAPER
H arom (Fmoc)], 8.49 (s, 1 H, 8-H) ppm. HRMS: calcd. for
C76H62N5O12 [MH]+ 1236.4395; found 1236.4346.
solution of 4b[3] (1.08 g, 3 mmol) in dry pyridine (10 mL) and the
reaction mixture was stirred at room temperature for 2 h. The reac-
tion was monitored by TLC. MeOH (5 mL) was then added and
the stirring was continued for 10 min. The yellow solution was con-
centrated and co-evaporated to dryness with toluene (2ϫ10 mL),
and the residue was subjected to silica gel flash column chromatog-
raphy with methanol in dichloromethane (1.5%) as eluent. Precipi-
tation from dichloromethane/hexane at –60 °C afforded the title
1,5-Anhydro-2-deoxy-2-[(N2-dimethylaminomethylene)guanin-9-yl]-
3-O-(9-fluorenylmethoxycarbonyl)-6-O-monomethoxytrityl-D-altro-
hexitol (3e)
1,5-Anhydro-4,6-O-benzylidene-2-deoxy-[2-(N2-dimethylaminometh-
ylene)guanin-9-yl]-3-O-(9-fluorenylmethoxycarbonyl)- -altro-hexitol
D
1
(3c): A mixture of 3b (2.5 g, 5.7 mmol) and pyridine (20 mL) was
concentrated to 5 mL, Fmoc chloride (1.75 g, 6.5 mmol) was added
in 500 mg portions for 30 min, and stirring was continued for 1 h.
Methanol (5 mL) was added dropwise at 0 °C and the reaction mix-
ture was stirred for 10 min. The resulting mixture was concentrated
and co-evaporated with toluene (2ϫ30 mL) under reduced pres-
sure and the residue was extracted with ethyl acetate, washed with
water and purified by flash silica gel column chromatography with
compound 4c as a white powder (1.1 g, 63%). H NMR (CDCl3):
δ = 2.02 (s, 3 H, 5-Me), 3.76–3.88 [m, 2 H, 4Ј-H and 9-H (Fmoc)],
4.10–4.60 [m, 7 H, 6Јax-H, 1Јax-H, 5Ј-H, 6Јeq-H, 1Јeq-H, CH2O
(Fmoc)], 4.65 (t, J = 2.9 Hz, 1 H, 2Ј-H), 5.50 (br. s, 1 H, 3Ј-H),
5.64 (s, 1 H, PhCH), 7.23–7.35 (m, 5 H, H arom), 7.35–7.46 (m, 4
H, H arom), 7.62 [d, J = 7.0 Hz, 2 H, H arom (Fmoc)], 7.88 [d, 6-
H, J = 7.7 Hz, 2 H, H arom (Fmoc)], 7.88 (d, J = 1.1 Hz, 1 H, 6-
H), 8.72 (br. s, 1 H, NH) ppm. HMRS: calcd. for C34H31N2O8
[MH]+ 583.2081; found 583.2078.
1
methanol in dichloromethane (1.5%). Yield 3.0 g (80%). H NMR
(CDCl3): δ = 3.09 (s, 6 H, NMe2), 3.67–3.85 (m, 2 H, 4Ј-H, 6Јax-
H), 4.10–4.60 [m, 7 H, 1Ј-H, 2Ј-H, 5Ј-H, 6Јeq-H, 9-H and CH2O
(Fmoc)], 5.51 (s, 1 H, PhCH), 5.86 (br. t, 1 H, 3Ј-H), 7.29–7.44 (m,
9 H, H arom), 7.50–7.68 [m, 2 H, H arom (Fmoc)], 7.77–7.82 [m,
2 H, H arom (Fmoc)], 8.03 (s, 1 H, 8-H), 8.87 (s, 1 H, CH), 8.95
(br. s, 1 H, NH) ppm. HRMS: calcd. for C36H34N6O8 [M]+
662.2489; found 662.2451.
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-(thymin-
1-yl)-D-altro-hexitol (4d): Compound 4c (1.75 g, 3 mmol) was dis-
solved in dichloromethane (30 mL) and TFA (3 mL) was added at
0 °C. The reaction was monitored by TLC. After the mixture had
been stirred at room temperature for1 h, ethanol (20 mL) was
added and the yellow-brown solution was concentrated and co-
evaporated to dryness with toluene (2ϫ30 mL). The residue was
purified by silica gel flash column chromatography with methanol
in dichloromethane (5%) to afford the title compound 4d as a white
1,5-Anhydro-2-deoxy-2-[N2-(dimethylaminomethylene)guanin-9-yl]-
3-O-(9-fluorenylmethoxycarbonyl)-D-altro-hexitol (3d): TFA (2 mL)
1
was added dropwise at 0 °C to a solution of hexitol 3c (2.2 g,
3.3 mmol) in dichloromethane (20 mL) and the reaction mixture
was stirred for 30 min. Water (100 µL, 5.6 mmol) was added and
stirring was continued for 15 min. The light yellow solution was
neutralized with pyridine, washed with saturated NaCl and concen-
trated to dryness, and the crude material was precipitated from
dichloromethane/hexane at 0 °C to afford the title compound 3d as
solid (1.1 g, 74%). H NMR (CDCl3): δ = 1.80 (s, 3 H, CH3), 3.20
(br. s, 2 H, 6Ј-OH and 4Ј-OH), 3.70–3.95 (3 H, m 4Ј-H, 5Ј-H, 6Јax-
H), 3.96–4.50 [m, 7 H, 6Јeq-H, 1Јax-H, 3Ј-H, 1Јeq-H, 9-H and
CH2O (Fmoc)], 5.50 (br. s, 1 H, 3Ј-H), 7.20–7.40 (m, 4 H, H arom),
7.58 [d, J = 7.0 Hz, 2 H, H arom (Fmoc)], 7.74 [d, J = 7.7 Hz, 1
H, 6-H, H arom (Fmoc)], 7.80 (d, J = 1.1 Hz, 1 H, 6-H), 9.50 (br.
s, 1 H, NH) ppm. HMRS: calcd. for C26H27N2O8 [MH]+ 495.1768;
found 495.1765.
1
a white solid in 95% yield. H NMR ([D6]DMSO): δ = 3.02 (s, 6
H, NMe), 3.06 (s, 6 H, NMe), 3.60–3.80 (m, 4 H, 4Ј-H, 6Јax-H,
2ϫOH), 4.00–4.60 [m, 7 H, 1Ј-H, 2Ј-H, 5Ј-H, 6Јeq-H, 9-H and
CH2O (Fmoc)], 5.43 (br. t, 1 H, 3Ј-H), 7.29–7.39 (m, 4 H, H arom),
7.55–7.58 [m, 2 H, H arom (Fmoc)], 7.74–7.78 [m, 2 H, H arom
(Fmoc)], 8.08 (s, 1 H, 8-H), 8.76 (s, 1 H, CH), 11.10 (br. s, 1 H,
NH) ppm.
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-mono-
methoxytrityl-2-(thymin-1-yl)-D-altro-hexitol (4e): MMTrCl (0.95 g,
3 mmol) was added under nitrogen at room temperature to a stirred
solution of 4d (1.0 g, 2 mmol) in dry pyridine (15 mL). The reaction
was monitored by TLC. After the mixture had been stirred for 2 h,
methanol (3 mL) was added and the solution was concentrated and
co-evaporated to dryness with toluene (2ϫ15 mL). The residue was
purified by silica gel flash column chromatography with methanol
in dichloromethane (3%). Precipitation from dichloromethane/hex-
ane at –60 °C afforded the title compound 4e as a white powder
1,5-Anhydro-2-deoxy-2-[N2-(dimethylaminomethylene)guanin-9-yl]-
(9-fluorenylmethoxycarbonyl)-6-O-monomethoxytrityl-D-altro-hexi-
tol (3e): A solution of 3d (1.15 g, 2 mmol) in pyridine (10 mL) was
concentrated to 3 mL and MMTrCl (620 mg, 2 mmol) was added
under argon at room temperature. After 3 h, methanol (1.5 mL)
was added and the solution was washed with water (3ϫ50 mL)
and dried with magnesium sulfate and the solvents were evaporated
to dryness. Precipitation from dichloromethane/hexane at 0 °C af-
1
(1.2 g, 52%). H NMR (CDCl3): δ = 1.80 (s, 3 H, CH3), 3.40 (dd,
J = 1.1, 1.1 Hz, 1 H, 6Јax-H), 3.48 (dd, J = 1.1, 1.1 Hz, 1 H, 6Јax-
H), 3.78 (s, 3 H, CH3), 3.72–3.85 (m, 1 H, 4Ј-H), 4.05–4.30 [m, 4
H, 1Јax-H, 3Ј-H, 1Јeq-H, 5Ј-H, 9-H (Fmoc)], 4.40–4.50 [m, 2 H,
CH2O (Fmoc)], 4.66 (br. s, 1 H, 2Ј-H), 5.50 (br. s, 1 H, 3Ј-H), 6.82
(d, J = 8.9 Hz, 2 H, H arom), 7.22–7.40 (m, 16 H, H arom), 7.58
[d, J = 7.0 Hz, 2 H, H arom (Fmoc)], 7.75 [d, J = 7.7 Hz, 1 H, 6-
H, H arom (Fmoc)], 7.80 (d, J = 1.1 Hz, 1 H, 6-H), 9.50 (br. s, 1
H, NH) ppm. HMRS: calcd. for C46H43N2O9 [MNa]+ 767.2969;
found 767.2977.
1
forded the title compound 3e as a white powder (1.35 g, 80%). H
NMR (CDCl3): δ = 3.05 (s, 6 H, NMe2), 3.47 (m, 2 H, 4Ј-H, 6Јax-
H), 3.80 (s, 3 H, CH3), 3.90 (m, 2 H) and 4.10–4.60 (m, 5 H) [1Ј-
H, 2Ј-H, 5Ј-H, 6Јeq-H, 9-H and CH2O (Fmoc)], 5.67 (br. t, 1 H,
3Ј-H), 6.84–6.88 (m, 2 H, H arom), 7.29–7.62 (m, 16 H, H arom),
7.59–7.66 [m, 2 H, H arom (Fmoc)], 7.77–7.81 [m, 2 H, H arom
(Fmoc)], 8.07 (s, 1 H, 8-H), 8.77 (s, 1 H, CH), 9.21 (br. s, 1 H,
NH) ppm. HRMS: calcd. for C49H47N6O8 [MH]+ 847.3455; found
847.3458.
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-mono-
methoxytrityl-2-(uracyl-1-yl)-
1,5-Anhydro-4,6-O-benzylidene-2-deoxy-3-O-(9-fluorenylmethoxy-
carbonyl)-2-(uracil-1-yl)- -altro-hexitol (5c): Fmoc chloride (1.03 g,
D-altro-hexitol (5e)
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-mono-
D
methoxytrityl-2-(thymin-1-yl)-
1,5-Anhydro-4,6-O-benzylidene-2-deoxy-3-O-(9-fluorenylmethoxy-
carbonyl)-2-(thymin-1-yl)- -altro-hexitol (4c): Fmoc chloride
(1.03 g, 4 mmol) was added under nitrogen in four portions to a
D-altro-hexitol (4e)
4 mmol) was added under nitrogen in four portions to a solution
of 5b[3] (1.04 g, 3 mmol) in dry pyridine (10 mL) and the reaction
mixture was stirred at room temperature for 2 h. The reaction was
monitored by TLC. MeOH (5 mL) was then added and the stirring
D
Eur. J. Org. Chem. 2007, 1446–1456
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1453

M. Abramov, G. Schepers, A. Van Aerschot, P. Herdewijn
FULL PAPER
was continued for 10 min. The yellow solution was concentrated
and co-evaporated to dryness with toluene (2ϫ10 mL), and the
residue was subjected to silica gel flash column chromatography
with methanol in dichloromethane (1.5%) as eluent. Precipitation
from dichloromethane/hexane at –60 °C afforded the title com-
brine, and the solvents were evaporated to dryness to afford a yel-
low foam. This crude intermediate was dissolved in dioxane
(40 mL), and aqueous ammonia (25%, 15 mL) was added. After
the mixture had been stirred for 45 min, the volatiles were evapo-
rated and the solid was co-evaporated with toluene. The residue
was suspended in chloroform, co-evaporated with silica gel and
1
pound 5c as a white powder (1.1 g, 63%). H NMR (CDCl3): δ =
3.76–3.88 [m, 2 H, 4Ј-H and 9-H (Fmoc)], 4.10–4.60 [m, 7 H, 6Јax- subjected to silica gel column chromatography with a stepwise gra-
H, 1Јax-H, 5Ј-H, 6Јeq-H, 1Јeq-H, CH2O (Fmoc)], 4.65 (t, J =
2.9 Hz, 1 H, 2Ј-H), 5.51 (br. s, 1 H, 3Ј-H), 5.62 (s, 1 H, PhCH),
5.82 (dd, J = 1.8, 8.4 Hz, 1 H, 5-H), 7.23–7.35 (m, 5 H, H arom),
7.36–7.46 (m, 4 H, H arom), 7.62 [d, J = 7.3 Hz, 2 H, H arom
(Fmoc)], 7.80 [d, J = 7.7 Hz, 2 H, H arom (Fmoc)], 8.05 (d, J =
dient of methanol in dichloromethane (2–10%), to afford the title
compound 6b as a white powder (1.9 g, 55%). 1H NMR ([D6]-
DMSO): δ = 3.60 (dd, J = 2.3 and 9.6 Hz, 1 H, 4Ј-H), 3.64 (t, J =
10.2 Hz, 1 H, 6Ј-Ha), 3.91 (dd, J = 4.9 and 9.6 Hz, 1 H, 5Ј-H),
4.00 (m, 1 H, 3Ј-H), 4.00–4.26 (m, 3 H, 1Ј-Ha, 1Ј-He, 6Ј-He), 4.29
8.4 Hz, 1 H, 6-H), 9.46 (br. s, 1 H, NH) ppm. HMRS: calcd. for (m, 1 H, 2Ј-H), 5.65 (s, 1 H, Ph-CH), 5.72 (d, J = 4.2 Hz, 1 H, 3Ј-
C32H29N2O8 [MH]+ 569.1925; found 569.1924.
OH), 5.77 (d, J = 7.5 Hz, 1 H, 5-H), 7.05 and 7.19 (2ϫbr. s, 2 H,
4-NH2), 7.30–7.45 (m, 5 H, ar-H), 7.94 (d, J = 7.5 Hz, 1 H, 6-H)
ppm. 13C NMR ([D6]DMSO): δ = 57.46 (C-2Ј), 64.00 (C-1Ј), 64.87
(C-3Ј), 65.79 (C-5Ј), 68.28 (C-6Ј), 76.50 (C-4Ј), 94.09 (C-5), 101.20
(Ph-CH), 126.50 (2 C, ar-Co), 128.10 (2 C, ar-Cm), 128.95 (ar-Cp),
137.93 (ar-Ci), 143.75 (C-6), 154.98 (C-2), 165.19 (C-4) ppm.
HRMS (thgly): calcd. for C17H20N3O5 [MH]+ 346.1403; found
346.1380.
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-(uracil-1-
yl)-D-altro-hexitol (5d): Compound 5c (1.70 g, 3 mmol) was dis-
solved in dichloromethane (30 mL), TFA (3 mL) was added drop-
wise at 0 °C, and the reaction mixture was stirred for 30 min. Water
(100 µL, 5.6 mmol) was added and stirring was continued for
15 min. The light yellow solution was neutralized with pyridine,
washed with saturated NaCl and concentrated to dryness, and the
crude material was precipitated from dichloromethane/hexane at
0 °C to afford the title compound 5d as a white solid in 95% yield.
1H NMR (CDCl3): δ = 3.20 (br. s, 2 H, 6Ј-OH and 4Ј-OH), 3.70–
3.95 (3 H, m, 4Ј-H, 5Ј-H, 6Јax-H), 3.96–4.50 [m, 7 H, 6Јeq-H, 1Јax-
H, 3Ј-H, 1Јeq-H, 9-H and CH2O (Fmoc)], 5.30 (br. s, 1 H, 3Ј-H),
5.64 (d, J = 8.1 Hz, 1 H, 5-H), 7.20–7.40 (m, 4 H, H arom), 7.60
[d, J = 7.0 Hz, 2 H, H arom (Fmoc)], 7.71 [d, J = 7.7 Hz, 1 H, 6-
H, H arom (Fmoc)], 8.00 (d, J = 8.1 Hz, 1 H, 6-H), 10.0 (br. s, 1 H,
NH) ppm. HMRS: calcd. for C25H25N2O8 [MH]+ 481.1611; found
481.1611.
1,5-Anhydro-4,6-O-benzylidene-2-deoxy-3-O-(9-fluorenylmethoxy-
carbonyl)-2-[N4-(9-fluorenylmethoxycarbonyl)cytosin-1-yl]-
D-altro-
hexitol (6c): 9-Fluorenylmethoxycarbonyl chloride (5.0 g, 19 mmol)
was added under nitrogen in 1 g portions over 1 h to a stirred solu-
tion of 6b (1.5 g, 4.4 mmol) in dry pyridine (20 mL). The reaction
mixture was stirred at room temperature for 1 h, the pyridine was
removed, the residue was co-evaporated with toluene and sus-
pended in dichloromethane (50 mL), and the organic phase was
washed with water. The solvent was removed and the crude mate-
rial was subjected to flash silica gel column chromatography with
a mixture of dichloromethane/ethyl acetate (1:5) as eluent, to afford
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-6-O-mono-
methoxytrityl-2-(uracil-1-yl)-D-altro-hexitol (5e): Monomethoxytri-
1
the title compound 6c (1.75 g, 51%). H NMR (CDCl3): δ = 3.60–
3.82 [m, 2 H, 4Ј-H and 9-H (Fmoc)], 4.05–4.60 [m, 10 H, 6Јax-H,
1Јax-H, 5Ј-H, 6Јeq-H, 1Јeq-H, CH2O (Fmoc)], 4.82 (br. s, 1 H, 2Ј-
H), 5.45 (s, 1 H, PhCH), 5.59 (br. s, 1 H, 3Ј-H), 7.15–7.48 (m, 14
H, 6-H and H arom), 7.50–7.64 [m, 4 H, H arom (Fmoc)], 7.66–
7.82 [m, 4 H, H arom (Fmoc)], 7.68 [d, J = 7.7 Hz, 2 H, H arom
(Fmoc)], 7.78 [m, 4 H, H arom (Fmoc)], 7.94 (d, J = 7.8 Hz, 1 H,
6-H), 8.95 (br. s, 1 H, NH) ppm.
tyl chloride (0.95 g, 3 mmol) was added under nitrogen at room
temperature to a stirred solution of 5d (1.0 g, 2.1 mmol) in dry
pyridine (15 mL). The reaction was monitored by TLC. After the
mixture had been stirred for 2 h, methanol (3 mL) was added and
the solution was concentrated and co-evaporated to dryness with
toluene (2ϫ15 mL). Precipitation from dichloromethane/hexane at
–60 °C afforded the title compound 5e as a white powder (1.1 g,
1
72%). H NMR (CDCl3, 500 MHz): δ = 3.40 (dd, J = 1.1, 1.1 Hz,
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N4-(9-
1 H, 6Јax-H), 3.48 (dd, J = 1.1, 1.1 Hz, 1 H, 6Јax-H), 3.78 (s, 3 H,
CH3), 3.72–3.85 (m, 1 H, 4Ј-H), 4.05–4.30 [m, 4 H, 1Јax-H, 3Ј-H,
1Јeq-H, 5Ј-H, 9-H (Fmoc)], 4.40–4.50 [m, 2 H, CH2O (Fmoc)], 4.66
(br. s, 1 H, 2Ј-H), 5.50 (br. s, 1 H, 3Ј-H), 5.88 (d, J = 8.1 Hz, 1 H,
5-H), 6.82 (d, J = 8.9 Hz, 2 H, H arom), 7.22–7.40 (m, 16 H, H
arom), 7.58 [d, J = 7.0 Hz, 2 H, H arom (Fmoc)], 7.75 [d, J =
7.7 Hz, 1 H, 6-H, H arom (Fmoc)], 7.80 (d, J = 8.1 Hz, 1 H, 6-H),
8.95 (br. s, 1 H, NH) ppm. HMRS: calcd. for C45H41N2O9 [MH]+
753.2812; found 753.2812.
fluorenylmethoxycarbonyl)cytosin-1-yl]-D-altro-hexitol (6d): Com-
pound 6c (1.2 g, 1.5 mmol) was dissolved in dichloromethane
(15 mL) and cooled to 0 °C. TFA (2 mL) was then added and the
reaction mixture was stirred at room temperature for 45 min; 80%
ethanol (10 mL) was added, solvents were removed in vacuo, and
the residue was co-evaporated with toluene. The crude material was
subjected to flash silica gel column chromatography with methanol
in dichloromethane (2.5%) to afford the title compound 6d as a
1
white foam (0.75 g, 71%). H NMR (CDCl3): δ = 3.05 (br.s, 2 H,
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N4-(9-
6Ј-OH and 4Ј-OH), 3.70–3.95 (3 H, m 4Ј-H, 5Ј-H, 6Јax-H), 3.96–
4.45 [m, 9 H, 6Јeq-H, 1Јax-H, 3Ј-H, 1Јeq-H, 9-H and CH2O
(Fmoc)], 4.63 (m, 1 H, 3Ј-H), 5.49 (br. s, 1 H, 3Ј-H), 6.81 (d, J =
7.7 Hz, 1 H), 7.26–7.48 (m, 8 H, H arom), 7.55 [m, 4 H, H arom
(Fmoc)], 7.65 [m, 4 H, H arom (Fmoc)], 8.04 (d, J = 7.7 Hz, 1
H) ppm. HMRS: calcd. for C40H36N3O9 [MH]+ 702.2450; found
702.2480.
fluorenylmethoxycarbonyl)cytosin-1-yl]-6-O-monomethoxytrityl-
altro-hexitol (6e)
D-
1,5-Anhydro-4,6-O-benzylidene-2-(cytosin-1-yl)-2-deoxy-D-altro-hex-
itol (6b): Chlorotrimethylsilane (6.4 mL, 50 mmol) was added un-
der nitrogen to a stirred suspension of 5b[3] (3.6 g, 10.0 mmol) in
dry pyridine (40 mL). After 1 h, the reaction mixture was cooled
in an ice bath, 1H-1,2,4-triazole (6.9 g, 100 mmol) and phosphorus
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N4-(9-
oxychloride (1.86 mL, 20 mmol) were added, and stirring was con- fluorenylmethoxycarbonyl)cytosin-1-yl]-6-O-monomethoxytrityl-D-
tinued for 5 h. The volatiles were removed and the residue was co-
evaporated with toluene (3ϫ20 mL) and partitioned between water
and ethyl acetate. The organic layer was washed with water and
altro-hexitol (6e): Monomethoxytrityl chloride (0.46 g, 1.42 mmol)
was added at room temperature under nitrogen to a solution of 6d
(0.65 g, 0.9 mmol) in dry pyridine (6 mL). After 4 h, methanol
1454
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1446–1456

Fmoc-Protected Altritol Phosphoramidite Building Blocks
FULL PAPER
(1 mL) was added, the volatiles were removed, and the residue was
co-evaporated with toluene. The residue was subjected to flash sil-
ica gel column chromatography with acetone (2 %) in dichloro-
methane, to afford the title compound 6e as a white solid (0.55 g,
60%). 1H NMR (CDCl3): δ = 3.38 (dd, J = 1.1, 10.5 Hz, 1 H, 6Јax-
H), 3.48 (dd, J = 1.1, 10.5 Hz, 1 H, 6Јax-H), 3.78 (s, 3 H, CH3),
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N4-(9-
fluorenylmethoxycarbonyl)-5-methylcytosin-1-yl]-D-altro-hexitol
(7d): Compound 7c (1.2 g, 1.5 mmol) was dissolved in dichloro-
methane (15 mL) and cooled to 0 °C. TFA (2 mL) was then added,
and the reaction mixture was stirred at room temperature for
45 min. Ethanol (80%, 10 mL) was added, solvents were removed,
3.72–3.85 (m, 1 H, 4Ј-H), 4.05–4.30 [m, 4 H, 1Јax-H, 1Јeq-H, 5Ј- and the residue was co-evaporated with toluene. The crude material
H, 9-H (Fmoc)], 4.40–4.50 [m, 4 H, CH2O (Fmoc)], 4.66 (br. s, 1
H, 2Ј-H), 5.50 (br. s, 1 H, 3Ј-H), 5.78 (dd, J = 1.8, 8.1 Hz, 1 H, 6-
was subjected to flash silica gel column chromatography with meth-
anol in dichloromethane (2.5%), to afford the title compound 7d
H), 6.82 (d, J = 8.9 Hz, 2 H, H arom), 7.22–7.40 (m, 20 H, H as a white foam (0.70 g, 65%). 1H NMR (CDCl3): δ = 2.05 (s, 3
arom), 7.58 [m, 2 H, H arom (Fmoc)], 7.68 [m, 2 H, H arom H, CH3), 3.70–3.95 (3 H, m 4Ј-H, 5Ј-H, 6Јax-H), 3.96–4.50 [m, 9
(Fmoc)], 7.75 [m, 4 H, H arom (Fmoc)], 8.25 (d, J = 8.1 Hz, 1 H, H, 6Јeq-H, 1Јax-H, 3Ј-H, 1Јeq-H, 9-H and CH2O (Fmoc)], 4.63
6-H), 8.93 (br. s, 1 H, NH) ppm. HMRS: calcd. for C60H52N3O10
(br. s, 1 H, 3Ј-H), 5.35 (br. s, 1 H, 3Ј-H), 6.20 (br. s, 2 H, 6Ј-OH
and 4Ј-OH), 7.20–7.40 (m, 8 H, H arom), 7.58 [m, 4 H, H arom
(Fmoc)], 7.74 [m, 4 H, H arom (Fmoc)], 8.40 (d, J = 1.1 Hz, 1 H,
6-H), 9.50 (br. s, 1 H, NH) ppm. HMRS: calcd. for C41H38N3O9
[MH]+ 716.2608; found 716.2605.
[MH]+ 974.3653; found 974.3633.
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N4-(9-
fluorenylmethoxycarbonyl)-5-methylcytosin-1-yl]-6-O-monometh-
oxytrityl-
D-altro-hexitol (7e)
1,5-Anhydro-2-deoxy-3-O-(9-fluorenylmethoxycarbonyl)-2-[N4-(9-
fluorenylmethoxycarbonyl)-5-methylcytosin-1-yl]-6-O-monometh-
oxytrityl-D-altro-hexitol (7e): Monomethoxytrityl chloride (0.46 g,
1,5-Anhydro-4,6-O-benzylidene-2-deoxy-2-(5-methylcytosin-1-yl)-
D-
altro-hexitol (7b): Chlorotrimethylsilane (6.4 mL, 50 mmol) was
added under nitrogen to a stirred suspension of 4b[3] (3.6 g,
10.0 mmol) in dry pyridine (40 mL). After 1 h, the reaction mixture
was cooled in an ice bath, 1H-1,2,4-triazole (6.9 g, 100 mmol) and
phosphorus oxychloride (1.86 mL, 20 mmol) were added, and stir-
ring was continued for 5 h. The volatiles were removed and the
residue was co-evaporated with toluene (3 ϫ 25 mL) and parti-
tioned between water and ethyl acetate. The organic layer was
washed with water and brine, and the solvents were evaporated to
dryness to afford a yellow foam. This crude intermediate was dis-
solved in dioxane (40 mL), and aqueous ammonia (25%, 15 mL)
was added. After the mixture had been stirred for 45 min, the vola-
tiles were evaporated and the solid was co-evaporated with toluene.
The residue was suspended in chloroform, adsorbed on silica gel
and subjected to silica gel column chromatography with a stepwise
gradient of methanol in dichloromethane (2–10%), to afford the
title compound 7b as a white powder (2.0 g, 55%). 1H NMR ([D6]-
DMSO): δ = 1.97 (s, 3 H, CH3), 3.53 (dd, J = 2.4, 9.5 Hz, 1 H, 4Ј-
H), 3.69 (t, J = 10.4 Hz, 1 H, 6Јax-H), 3.85–4.15 (m, 7 H, 1Јax-H,
5Ј-H, 6Јeq-H, 3Ј-H, 1Јax-H, 2Ј-H and 3Ј-OH), 5.61 (s, 1 H, PhCH),
6.90 (br. s, 2 H, NH2), 7.29–7.32 (m, 3 H, H arom), 7.39–7.45 (m,
2 H, H arom), 7.75 (s, 1 H, 6-H) ppm. HMRS: calcd. for
C18H21N3O5 [MH]+ 360.1559; found 360.1554.
1.42 mmol) was added at room temperature under nitrogen to a
solution of 7d (0.65 g, 0.9 mmol) in dry pyridine (6 mL). After 4 h,
methanol (1 mL) was added, the volatiles were removed, and the
residue was co-evaporated with toluene. The residue was subjected
to flash silica gel column chromatography with acetone (2%) in
dichloromethane, to afford the title compound 7e as a white solid
(0.55 g, 60%). 1H NMR (CDCl3): δ = 1.96 (s, 3 H, CH3), 3.38 (dd,
J = 1.1, 10.5 Hz, 1 H, 6Јax-H), 3.48 (dd, J = 1.1, 10.5 Hz, 1 H,
6Јax-H), 3.78 (s, 3 H, CH3), 3.72–3.85 (m, 1 H, 4Ј-H), 4.05–4.30
[m, 4 H, 1Јax-H, 1Јeq-H, 5Ј-H, 9-H (Fmoc)], 4.40–4.50 [m, 4 H,
CH2O (Fmoc)], 4.66 (br. s, 1 H, 2Ј-H), 5.50 (br. s, 1 H, 3Ј-H), 6.82
(d, J = 8.9 Hz, 2 H, H arom), 7.22–7.40 (m, 20 H, H arom), 7.58
[m, 2 H, H arom (Fmoc)], 7.68 [m, 2 H, H arom (Fmoc)], 7.75 [m,
4 H, H arom (Fmoc)], 8.09 (d, J = 1.1 Hz, 1 H, 6-H) ppm. HMRS:
calcd. for C61H54N3O10 [MH]+ 988.3765; found 988.3796.
Acknowledgments
The authors thank the DWTC for financial support.
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(5.0 g, 19 mmol) was added under nitrogen over 1 h in 1 g portions
to a stirred solution of 7b (1.5 g, 4.2 mmol) in dry pyridine (20 mL).
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(CDCl3): δ = 2.13 (s, 3 H, 5-Me), 3.77–3.83 [m, 2 H, 4Ј-H and 9-
H (Fmoc)], 4.20–4.55 [m, 10 H, 6Јax-H, 1Јax-H, 5Ј-H, 6Јeq-H,
1Јeq-H, CH2O (Fmoc)], 4.65 (br. s, 1 H, 2Ј-H), 5.49 (br. s, 1 H, 3Ј-
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Received: October 3, 2006
Published Online: January 25, 2007
1456
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1446–1456