Solid-Phase Synthesis and Screening of Macrocyclic Nucleotide-Hybrid Compounds Targeted to Hepatitis C NS5B

Article abstract of DOI:10.1002/chem.200305402

A convergent strategy for the synthesis of cyclic nucleotide-hybrid molecules on controlled pore glass is reported. A major advantage of the approach is the lack of restrictions on the sequence and structural variation, allowing the incorporation of modif

Full text of DOI:10.1002/chem.200305402

FULL PAPER
Solid-Phase Synthesis and Screening of Macrocyclic Nucleotide-Hybrid
Compounds Targeted to HepatitisCNS5B
Michael Smietana,[a] Robert B. Johnson,[b] Q. May Wang,[b] and Eric T. Kool*[a]
Abstract: A convergent strategy for
the synthesis of cyclic nucleotide-
hybrid molecules on controlled pore
glass is reported. A major advantage of
the approach is the lack of restrictions
on the sequence and structural varia-
tion, allowing the incorporation of
modified ribonucleosides (such as 2’-
OMe-ribonucleotides), as well as threo-
ninol derivatives. This methodology
allows a fully automated assembly by
means of standard phosphoramidite
chemistry and is based on a recently
published procedure for the prepara-
tion of cyclic oligodinucleotides in the
DNA series (M. Smietana, E. T. Kool,
Angew. Chem. 2002, 114, 3856 3859;
Angew. Chem. Int. Ed. Engl. 2002, 41,
cyclic hybrid inhibitor compounds tar-
geting hepatitisC virus NS5B enzyme
(the replicating polymerase of HCV)
was generated by means of the paral-
lel-pool strategy. Screening of the li-
brary revealed that cyclic hybrid c(COME
-
3704 3707).
A
library of potential
EthenodA) was a significant inhibitor
of NS5B, with an IC50 of 40 mm. Pre-
liminary structure activity studies of
this lead compound are described.
Keywords: antiviral agents ¥ DNA
structures ¥ solid-phase synthesis ¥
structure activity relationships
Introduction
sequence[4] or it can involve an internal secondary struc-
ture.[5] Template-free methodologies on a modified solid
support have been described for the synthesis of small- to
medium-sized cyclic DNAs[6] and RNAs.[7,8]
Cyclic molecules are frequently found in nature. The discov-
ery of numerous naturally occurring cyclic metabolites has
encouraged scientists to systematically study the chemical,
physical, and biological properties of these compounds. In
this context, cyclic DNAs and RNAs play a key role reflect-
ed by their unusual chemical and biological activities.[1] Such
molecules are attractive targets for study as they combine
two favorable properties: i) reduced conformational flexibili-
ty of the backbone, which can enhance receptor selectivity
and binding affinity, and ii) lowered susceptibility to degra-
dation in biological systems.[2,3] Both aspects make such
cyclic molecules potentially good leads for drug discovery.[1]
However, macrocyclic molecules, such as cyclic oligonucleo-
tides, are more difficult to synthesize than linear congeners
because of the entropic cost of ring closure. Typically, the
preparation of large cyclic DNAs (>ꢀ30 nucleotides) in-
volves a template-directed ligation of a linear precursor oli-
gonucleotide. The template can be provided as a separate
We recently described the first solid-phase synthesis of
cyclic oligonucleotides that utilizes the standard b-cyano-
ethylphosphoramidite method,[9] which is based on a rapid
and inexpensive automated 5’-iodination method developed
in our laboratory.[10] The main advantage of this method is
that cyclization occurs spontaneously during deprotection of
the oligonucleotide, leading to cyclic compounds bearing a
5’-bridging phosphorothioate linkage with good-to-excellent
yields (Scheme 1, R = H).[9] Since the chemical diversity of
natural nucleotides is limited, we sought to increase the
chemical and structural variation by replacing natural mono-
mers with modified nucleotides and non-nucleotide moiet-
ies. We now report on the extension of this methodology to
the preparation of cyclic 2’-(OMe) RNA, and cyclic DNA-
hybrid threoninol derivatives. The preparation of a library
of such compounds targeted to viral polymerases is also de-
scribed. Screening of this library against the replicative poly-
merase of the hepatitisC virus revealed a member that is in-
hibitory at low micromolar concentrations.
[a] Dr. M. Smietana, Prof. Dr. E. T. Kool
Department of Chemistry, Stanford University
Stanford, CA 94305-5080 (USA)
Fax : (+1)650-725-0259
E-mail: kool@leland.stanford.edu
Results and Discussion
[b] R. B. Johnson, Dr. Q. M. Wang
Infectious Diseases Research, Lilly Research Laboratories
Eli Lilly Company
Our goals in this work were to investigate whether our pre-
viously developed chemistry could be adapted to the prepa-
Indianapolis IN 46285 (USA)
173
Chem. Eur. J. 2004, 10, 173 181
DOI: 10.1002/chem.200305402
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER
Scheme 1. Formation of cyclic oligonucleotides.
ration of more structurally and chemically diverse mole-
cules, and whether such molecules might be active as inhibi-
tors of viral polymerases. Although the conditions previous-
ly described work well for the preparation of cyclic DNAs,
as exemplified by the synthesis of c(QQ)[11] (Table 1,
entry 1), cyclic oligoribonucleotides, because of their greater
reactivity, are much more difficult to prepare than the corre-
sponding 2’-deoxy analogues. To circumvent the difficulties
generally encountered with the classic 2’-tert-butyldimethyl-
silyl-protected amidites, the use of the 2’-O-triisopropylsilyl-
oxymethyl (TOM) protecting group has been described.[8]
A strict application of the conditions developed in our labo-
ratory for cyclic DNAs showed us that 2’-O-methoxyribonu-
cleotides are well-iodinated on the 5’-end, furnishing high
yields of cyclic, mixed ribo- and deoxy-oligonucleotides
(Table 1, entry 2). However, the synthesis of cyclic oligo-di-
(2’-OMe)-ribonucleotides appeared more difficult to ach-
ieve. A brief investigation revealed sulfurization to be the
step that failed. Our initial conditions were based on a pro-
tocol that required a single delivery of Beaucage reagent
(entry 3) as well as cyclic oligodi-(2’-OMe)-ribonucleotides
without any restrictions on the nucleobases (entries 4 7)
(Scheme 1, R = OMe). These conditions also allow the
preparation of fully modified trimers (entry 8).
We then turned our attention toward the formation of
cyclic nucleotide-hybrid compounds. Cyclic peptide-DNA
hybrids with ring sizes of 30 40 atoms have already been de-
scribed in the literature with respect to their potential pro-
tein-binding properties.[13] In order to preserve the more
rigid and smaller 12-membered ring structure of cyclic oligo-
dinucleotides, we decided to use d-threoninol derivatives as
the hybrid backbone. Both l- and d-threoninol enantiomers
have already been synthesized and introduced into ODNs
for several purposes;[14] however, they have never been part
of cyclic structures. The d-threoninol backbone was chosen
for three reasons: i) it mimics the spacing of the traditional
ribose moiety, which has three carbons between the primary
and secondary alcohols, ii) it can be modified to be suitable
for standard phosphoramidite chemistry, and iii) the second-
ary amino group can be condensed with a series of carboxyl-
ic sidechains to provide the diversity necessary for the syn-
thesis of a library.
The synthesis of cyclic hybrids 5 is shown is Scheme 2.
The secondary amino group of d-threoninol was modified
by means of an amide linkage with a variety of carboxylic
acids, by treatment with DCC (1 equiv), HOBt (1 equiv),
and diisopropylethylamine (1.2 equiv) in DMF. For the pur-
pose of selective protection of the primary alcohol, either
4,4’-dimethoxytrityl (DMT) or 4-monomethoxytrityl (MMT)
groups were used. The latter
(0.05m, 15 equiv), followed by a waiting period of 30 s.[12]
A
series of experiments were executed in which the number of
equivalents of Beaucage reagent and the reaction times
were varied. Complete sulfurization efficiency was achieved
with two deliveries of 20 equiv of reagents, each one fol-
lowed by a waiting period of 500 s. Clearly, the requirements
for sulfurization of 2’-substituted linkages are more stringent
than for their DNA counterparts. Application of these con-
ditions allowed cyclization of mixed cyclic oligonucleotides
could be completely removed
Table 1. Synthesis and characterization of cyclic compounds.
by following the standard pro-
Entry
Product
31P NMRYield
P(S)[a]
[b] [%]
45
Mass[c]
tocol on a DNA synthesizer. Fi-
nally, the secondary hydroxyl
group was activated with bis
(diisopropylamino)-2-cyanoethyl-
phosphine. In the purification
of such products by silica-gel
column chromatography, 2%
triethylamine was typically
used to prevent decomposition.
Compounds 4 were used along
with standard nucleobases on
an automated DNA synthesizer
following the conditions used
P(O)[a]
calcd
found
1
2
3
4
5
6
7
8
c(QQ)
c(UOMeT)
c(TUOMe
c(COMeCOMe
c(COMeUOMe
c(COMeGOMe
c(COMeAOMe
c(QQCOMe
c(Ts)
c(vs)
À0.12
À0.81
20.32
20.45
19.33
19.47
19.27
19.40
19.46
17.98
19.18
636.1
638.0
638.0
652.1
653.1
692.1
676.1
952.1
579.1
565.1
616.1
618.1
637.1
638.9
638.9
653.1
654.1
693.1
677.1
953.1
580.0
588.0
616.9
619.1
85
90
70
93
77
75
38
96
37
75
80
)
À0.36
)
)
)
À0.98
À0.42
À1.04
)
À0.77
)
À0.31, À0.21
À0.55
9
10
11
12
À0.34
19.08
18.92
c(Tp)
À0.53
c(xT)[d]
À0.51, À0.35
19.65, 19.71
[a] 31P chemical shift measured with respect to 85% H3PO4 as an external standard. [b] Absolute HPLC yield
determined as a percentage of all areas displayed. [c] Electrospray MS. [d] Serinol derivative.
for
2’-O-methoxyribonucleo-
174
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Chem. Eur. J. 2004, 10, 173 181

Macrocyclic Nucleotide-Hybrid Compounds
173 181
Scheme 2. Synthesis of cyclic DNA hybrids.
tides. Deprotection of bases, release from the solid support,
and cyclization with aqueous ammonia at room temperature
overnight gave cyclic DNA-hybrids 5. Structural characteri-
zation of 5 included 31P NMRspectroscopy and ES-MS
(Table 1). Exploratory experiments showed that these deriv-
atives can be incorporated as the primary as well as the sec-
ondary substrate on the column (entries 9 11), even though
complete hybrid structures led to more complex mixtures of
products in some cases.
An exception was noted with amino acid derivatives 4v
and 4x; if incorporated as the secondary substrate, alkaline
deprotection liberates the free amino group which is ideally
placed for an intramolecular attack on the 5’-iodo inter-
mediate, leading to a six-membered cycle. To circumvent
this side reaction, these derivatives–which were synthesized
for potentially enhanced membrane permeability on account
of their ionizable N-termini–had to be placed in the pri-
mary position (entries 10 and 12), or in the middle of a
trimer. In case of compound x, prochiral serinol condensed
with phenylalanine was used in place of d-threoninol lead-
ing to two diastereomeric dimers (entry 12). Although no di-
astereomer was clearly apparent in 31P NMR(Table 1,
entry 10), or by HPLC, we cannot rule out the possibility
that some racemization might occur at the amino acid
moiety during alkaline deprotection.
sessed in a particular biological assay, and if positive, decon-
voluted by synthesis of subpools and unique members. In
this strategy, the CPG support was functionalized with
5’end-ODMT derivatives in the four separate columns of an
ABI394 DNA synthesizer yielding intermediates 6. Each
pool was then mixed in equal amounts, and split into four
batches. Each refilled column 7 was then reacted with a dif-
ferent 5’end-ODMT derivative to produce sets of supported
dimers. Treatment with ammonium hydroxide liberated a li-
brary of 256 cyclic dimers as pools of 4 members (Figure 1).
All pools were purified as a mixture by preparative
HPLC, although in most cases four well-separated chroma-
tographic peaks were visible. For more than 80% of the
With these optimized conditions, we envisioned the feasi-
bility of a library. For this purpose, the synthesis of 11 threo-
ninol-like core structures containing heteroatomic and
amino acid sidechains was achieved. Associated with stan-
dard or modified nucleobases, a set of 24 monomers was ob-
tained (Scheme 3). A parallel-pool library strategy that in-
corporates the well-known techniques of split-mix and paral-
lel combinatorial synthesis was chosen.[15] In this way, small
pools of dimers (4 members each) were synthesized through
a unique mixing and splitting step. Each pool can be as-
Figure 1. Parallel-pool synthesis of a library of cyclic dinucleotides and
nucleotide-hybrid molecules (B1 = natural and modified bases).
4
175
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E. T. Kool et al.
FULL PAPER
Scheme 3. Monomers present in the library.
pools only minor amounts of other compounds were evi-
dent, as exemplified by pool 8 (Figure 2).
Preliminary structure activity relationship studies were
carried out on second-generation analogues of this com-
pound, again by the use of our solid-supported synthesis
procedure. The results show that both the etheno and the 2’-
OMe parts of the molecule are necessary for activity
(Table 2, entries 1 3). As expected, the position of the sulfur
bridge does not influence the IC50 value (entry 4). Interest-
ingly, the IC50 value obtained with c(UOMeEthenodA)
(entry 5) suggests that hydrogen bonding with the cytosine
enhances the potency of the inhibitor. Further modification
at the 2’-O-alkyl position may be the most promising strat-
egy to provide a further increase in the biological activity of
this lead compound.
Finally, we investigated the potency of the library pools
toward the RNA-dependent RNA polymerase NS5B encod-
ed by the hepatitisC virus; this was carried out following an
elongation assay already described.[16] Each pool was indi-
vidually examined for antiviral activity. Although significant
inhibition was exhibited by some pools (Figure 3), unique
members obtained after deconvolution displayed lower ac-
tivities than the initial pool.[17] We surmise that a small un-
identified synthesis side product was active, or that multiple
compounds were active only in combination. Nevertheless,
despite this complication, we were able to identify cyclic
hybrid c(COMeEthenodA) (c(il) in Scheme 3) (Table 2) as an
active compound in our library with an IC50 value in the mi-
cromolar range (IC50 40 mm).
176
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Chem. Eur. J. 2004, 10, 173 181

Macrocyclic Nucleotide-Hybrid Compounds
173 181
methodology enables rapid preparation of hundreds of com-
pounds that may be separated and screened individually. In
doing so, we have demonstrated that members of this library
have biological activities that can serve as leads for the pos-
sible development of new therapeutic agents in the treat-
ment of chronic HCV disease.
Experimental Section
Figure 2. Analytical HPLC chromatogram of crude pool 8, containing
four discrete library members.
General: All commercially available chemical reagents were used with-
out purification. 1H and 13C NMRspectra were recorded on a INOVA
500 spectrometer at 500 and 125 MHz, respectively. The signals of the re-
sidual protonated solvent (CDCl3 or CD3OD) were used as reference
signal. 31P NMRspectra were recorded on a INOVA300 spectrometer at
121.5 MHz. 31P chemical shifts were measured with respect to 85%
H3PO4 as an external standard. Mass spectra were measured on a Finni-
gan LCQ for ESI-MS (analysis performed in the negative mode for cyclic
oligonucleotides and cyclic hybrids). HRMS analyses were performed on
a VGZAB2SE high resolution mass spectrometer (UC Riverside). Cyclic
structures were synthesized on a 1 mmol scale with a ABI394 DNA syn-
thesizer (Applied Biosystems) according to standard protocol in ™trityl-
off∫ mode. HPLC purifications were performed on a Shimadzu system.
A Hypersil BDSC18 5 mm column (250î4.6 mm, flow rate 1 mLminÀ1
,
Alltech) was used for analytical purification and a HS Hyper prep 100
BDSC18 8 m-column (250î10 mm, flow rate 3 mLminÀ1 Alltech) for
preparative purification, with a linear gradient of acetonitrile (0 20%) in
50 mm triethylammonium acetate (TEAA, pH 7.0). For determination of
oligonucleotide concentrations, the following extinction coefficients (l =
260 nm) [LmolÀ1 cmÀ1] were used: adenosine 15300, guanosine 11700, cy-
tosine 7400, uridine 9900, thymine 8400, ethenodA 4800, 2’-OMe-cyto-
sine 7000, 2’-OMe-uridine 9750. [a-32P]-Uridine triphosphate was pur-
chased from Amersham. Autoradiography was performed with a Molecu-
lar Dynamics Storm860 Phosphorimager.
Figure 3. Inhibition of NS5B by pools 1 64. Each pool contains four
cyclic compounds. Each assay was performed in duplicate at a concentra-
tion of 100 mm of each compound by pool.
Table 2. Inhibition of hepatitisC virus NS5B by cyclic dinucleotides.
Preparation of active HCVNS5B enzyme was performed by RBJ and
QMW at Lilly Research Laboratories following a procedure described
previously.[16] The library synthesis, assay of activity[16] and screening of
compounds was performed by MS and ETK at Stanford University.
Monomers synthesis
General procedure for the coupling step: DCC (1 equiv) and HOBT
(1 equiv) were dissolved in a 0.4m solution of the acid (1 equiv) in DMF.
After 1 h at room temperature, d-threoninol in DMF was added, and the
resulting mixture was stirred overnight. The mixture was then filtered,
and the DMF was removed by distillation. The crude compound was pu-
rified by silica-gel chromatography (usually AcOEt/MeOH 1:1).
[a]
[a]
Entry
B1
B2
IC50 [mm][b]
1
2
3
4
5
6
7
C(OMe)
C(OMe)
C
EthenodA
U(OMe)
5-Me-C(OMe)
2’-F-C
EthenodA
A
EthenodA
C(OMe)
EthenodA
EthenodA
EthenodA
40
>1000
>1000
40
800
>1000
1000
General procedure for the protection of the primary alcohol: 0.3 equiv
DMAP and 1.5 equiv DIPEA were added to a solution of the threoninol
derivative in dry pyridine. A solution of 4,4’-dimethoxytrityl (DMT) chlo-
ride (or 4-momethoxytrityl chloride (MMT)) (1.2 equiv) in pyridine was
added slowly over a period of 45 min. The reaction was followed by TLC
analysis, and quenched with methanol after completion. The pyridine was
evaporated, and the compound was purified by silica-gel chromatography.
[a] Corresponding to Scheme 3: C(OMe): i; EthenodA: L; C: c; U(OMe): j.
[b] The IC50 values are the mean of three separate experiments.
General procedure for the protection of the secondary alcohol: The
DMT ether (or MMT ether) was dissolved in dry dichloromethane (0.4m
solution), and DIPEA (1.2 equiv) and N,N’-diisopropylchlorophosphor-
amidite (1.5 equiv) were added. The reaction mixture was stirred for
90 min at room temperature and then quenched by the addition of a satu-
rated solution of NaCl. The organic layer was extracted, washed with
water, dried, and purified by silica-gel chromatography. The compound
was obtained as a mixture of two diastereoisomers.
Conclusion
In conclusion, we have developed a solid-phase synthesis of
cyclic DNA/RNA and d-threoninol-DNA- or -RNA hybrids.
This approach allows fully automated assembly of commer-
cial and modified phosphoramidite derivatives. More diverse
libraries are created by condensing the threoninol amino
group with a larger number of carboxylic acids. The advan-
tages in synthetic efficiency offered by the parallel-pool syn-
thesis, as well as all mixture-based synthetic combinatorial
libraries, are tempered by the false positives or additive ef-
fects that these mixtures may induce. Nevertheless, this
N-(2-Hydroxy-1-hydroxymethylpropyl)-6-methyl-nicotinamide (2n): Rf
=
0.2 (AcOEt/MeOH 8.5:1.5); yield: 92%; 1H NMR(500 MHz,
CD3OD): d = 1.20 (d, J = 6.5 Hz, 3H), 2.56 (s, 3H), 3.71 (dd, J =
6.5 Hz, 1H), 3.77 (dd, J = 5.5 Hz, 1H), 4.07 (m, 2H), 4.96 (s, 2H), 7.36
(d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.88 (s, 1H); 13C NMR
(125 MHz, CD3OD): d = 20.55, 23.89, 58.34, 62.65, 67.27, 124.56, 129.31,
137.46, 148.65, 162.43, 168.35; MS (ESI): m/z: 225.1 (100) [M+H]+
HRMS (EI): m/z: calcd for C11H17N2O3: 225.1233; found: 225.1239).
;
177
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E. T. Kool et al.
FULL PAPER
N-(2-Hydroxy-1-hydroxymethylpropyl)-1-methyl-1H-indole-3-carboxa-
mide (2o): Rf
9H-Fluoren-9-ylmethyl ester of [1-(2-hydroxy-1-hydroxymethylpropylcar-
bamoyl)-2-(1H-indol-2-yl)-ethyl]-carbamic acid (2w): Rf = 0.62 (AcOEt/
MeOH 9.5:0.5); yield: 70%; 1H NMR(500 MHz, CD 3OD): d = 0.91 (d,
J = 6.5 Hz, 3H), 3.11 (m, 1H), 3.60 (m, 2H), 3.78 (m, 1H), 3.92 (m, 1
H), 4.13 (m, 1H), 4.21 (m, 1H), 4.30 (m, 1H), 4.49 (m, 1H), 7.08 (m, 3
H), 7.28 (m, 2H), 7.37 (m, 3H), 7.55 (m, 2H), 7.66 (d, J = 7.5 Hz, 1H),
7.76 (d, J = 7.5 Hz, 2H); 13C NMR(125 MHz, CD 3OD): d = 18.84,
28.09, 47.14, 56.13, 56.64, 61.29, 65.42, 66.91, 109.90, 111.16, 118.26,
118.71, 119.71, 121.28, 123.46, 125.05, 127.01, 127.58, 136.94, 141.35,
144.06, 157.17, 173.82; MS (ESI): m/z: 514.1 (100) [M+H]+, 536.2 (30)
[M+Na]+ ; HRMS (FAB): m/z: calcd for C30H32N2O5: 514.2342; found:
514.2319.
=
0.5 (AcOEt/MeOH 8:2); yield: 66%; 1H NMR
(500 MHz, CD3OD): d = 1.23 (d, J = 6.5 Hz, 3H), 3.68 (s, 3H), 3.77 (m,
2H), 4.07 (m, 1H), 4.14 (m, 1H), 7.20 (m, 2H), 7.31 (d, J = 8.0 Hz, 1H),
7.79 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H); 13C NMR(125 MHz, CD OD): d
3
= 20.73, 33.31, 56.99, 63.04, 67.15, 110.63, 111.00, 121.53, 122.24, 123.49,
127.28, 133.40, 138.56, 168.13; MS (ESI): m/z: 263.0 (100) [M+H]+
HRMS (EI): m/z: calcd for [C14H19N2O5]+ : 263.1396; found: 263.1401.
;
N-(2-Hydroxy-1-hydroxymethylpropyl)-2-(3-methylisoxazol-5-yl)aceta-
mide (2p): Rf
=
0.3 (AcOEt/MeOH 8:2); yield: 60%; 1H NMR
(500 MHz, CD3OD): d = 1.14 (d, J = 6.0 Hz, 3H), 2.24 (s, 3H), 3.60
(dd, J = 6.5 Hz, 1H), 3.66 (dd, J = 6.0 Hz, 1H), 3.77 (d, 2H), 3.85 (m, 1
H), 3.99 (m, 1H), 4.87 (s, 2H), 6.20 (s, 1H); 13C NMR(125 MHz,
9H-Fluoren-9-ylmethyl ester of [1-(2-hydroxy-1-hydroxymethylpropylcar-
bamoyl)-2-phenylethyl]-carbamic acid (2x): Rf = 0.5 (AcOEt); yield: 75
%; 1H NMR(500 MHz, CD 3OD): d = 2.90 (m, 1H), 3.15 (m, 1H), 3.51
(m, 1H), 3.63 (d, J = 5.5 Hz, 2H), 3.91 (m, 1H), 4.15 (m, 1H), 4.36 (m,
2H), 7.31 (m, 8H), 7.59 (m, 2H), 7.80 (d, 2H); 13C NMR(125 MHz,
CD3OD): d = 12.21, 26.07, 26.75, 34.76, 54.31, 58.01, 61.59, 68.00, 120.91,
126.17, 127.75, 128.77, 129.45, 130.44, 142.54, 145.19, 174.08; MS (ESI):
m/z: 483.1 (100) [M+Na]+ ; HRMS (FAB): m/z: calcd for C27H28N2O5Na:
483.1896; found: 483.2319.
CD3OD): d
= 11.22, 20.36, 34.88, 57.68, 62.67, 66.89, 104.95, 161.57,
168.50, 169.95; MS (ESI): m/z: 229.0 (100) [M+H]+ ; HRMS (EI): m/z:
calcd for C10H17N2O4: 229.1188; found: 229.1196.
N-(2-Hydroxy-1-hydroxymethylpropyl)-2-oxo-2H-chromene-3-carboxa-
mide (2q): Rf
=
0.15 (AcOEt) Yield: 70%; 1H NMR(500 MHz,
CD3OD): d = 1.04 (d, J = 6.5 Hz, 3H), 3.58 (d, J = 5.5 Hz, 2H), 3.83
(m, 1H), 4.01 (m, 1H), 4.31 (brs, 2H), 7.24 (m, 2H), 7.53 (m, 2H), 8.68
(s, 1H); 13C NMR(125 MHz, CD 3OD): d = 19.49, 55.61, 62.14, 65.94,
116.13, 118.11, 125.06, 129.58, 134.06, 148.28, 154.03, 160.97, 162.20; MS
(ESI): m/z: 278.2 (100) [M+H]+ ; HRMS (EI): m/z: calcd for C14H16NO5:
278.1028; found: 278.1031.
N-(1-O-[4,4’-Dimethoxytrityl]-2-hydroxypropyl)-6-methyl-nicotinamide
(3n): Rf = 0.40 (AcOEt/Et3N 98:2); yield: 67%; 1H NMR(500 MHz,
CDCl3): d = 1.21 (d, J = 6.5 Hz, 3H), 2.55 (s, 3H), 3.35 (dd, J = 3.5 Hz,
1H), 3.56 (dd, J = 4.5 Hz, 1H), 3.75 (s, 6H), 4.12 (m, 1H), 4.22 (m, 1H),
6.79 (m, 5H), 7.19 (m, 6H), 7.52 (d, J = 7.0 Hz, 1H), 7.80 (d, J =
7.5 Hz, 2H), 8.31 (d, J = 7.0 Hz, 1H), 9.15 (s, 1H); 13C NMR(125 MHz,
CDCl3): d = 17.9, 20.9, 56.0, 57.2, 64.2, 68.5, 88.8, 114.6, 124.3, 126.6,
128.4, 129.0, 129.4, 135.3, 137.2, 143.0, 152.1, 157.1, 159.5, 167.6; MS
N-(2-Hydroxy-1-hydroxymethylpropyl)-1H-benzimidazole-5-carboxamide
(2r): The synthesis of this product was realized in a mixture of NMP/
DMF (1:1). Rf
=
0.4 (AcOEt/MeOH 8:2); yield: 93%; 1H NMR
(500 MHz, CD3OD): d = 1.22 (d, J = 6.5 Hz, 3H), 3.72 (dd, J = 6.0 Hz,
1H), 3.77 (dd, J = 5.5 Hz, 1H), 4.12 (m, 2H), 5.07 (s, 3H), 7.62 (d, J =
8.5 Hz, 1H), 7.80 (d, J = 8.5 Hz, 1H), 8.17 (s, 1H), 8.28 (s, 1H); 13C
NMR(125 MHz, CD 3OD): d = 20.60, 58.23, 62.79, 67.33, 115.75, 116.39,
123.17, 130.47, 138.83, 140.69, 144.68, 171.01; MS (ESI): m/z: 250.1 (100)
[M+H]+ ; HRMS (EI): m/z: calcd for C12H16N3O3: 250.1192; found:
250.1192.
(ESI): m/z: 549.2 (100) [M+H]+
C32H34N2O5Na: 549.2365; found: 549.2337.
; HRMS (EI): m/z: calcd for
N-(2-Hydroxy-1-O-[4-monothoxytrityl]-propyl)-1-methyl-1H-indole-3-
carboxamide (3o): Rf = 0.33 (AcOEt/hexane/Et3N 60:38:2); yield: 78%;
1H NMR(500 MHz, CDCl ): d = 1.27 (d, J = 6.5 Hz, 3H), 3.4 (m, 1H),
3
3.64 (m, 1H), 3.75 (s, 6H), 3.77 (s, 3H), 4.26 (m, 2H), 6.82 (m, 3H),
N-(2-Hydroxy-1-hydroxymethylpropyl)-hexa-2,4-dienamide (2s): Rf
=
7.21 7.36 (m, 10H), 7.48 (d, J = 7.0 Hz, 4H), 7.71 (s, 1H), 8.08 (d, J =
0.4 (AcOEt/MeOH 9.5:0.5); yield: 70%; 1H NMR(500 MHz, CD 3OD):
d = 1.18 (d, J = 6.5 Hz, 3H), 1.87 (d, J = 6.5 Hz, 3H), 3.65 (dd, J =
6.0 Hz, 1H), 3.70 (dd, J = 6.0 Hz, 1H), 3.93 (m, 1H), 4.05 (m, 1H), 4.96
(brs, 3H), 6.01 (m, 1H), 6.11 (m, 1H), 6.22 (m, 1H), 7.14 (m, 1H); 13C
NMR(125 MHz, CD 3OD): d = 18.65, 20.36, 57.42, 62.77, 67.08, 122.81,
8.0 Hz, 1H); 13C NMR(125 MHz, CDCl ): d = 20.04, 33.07, 53.42, 55.01,
3
65.32, 68.78, 86.84, 109.97, 110.40, 113.11, 120.02, 121.44, 122.37, 125.13,
126.93, 126.96, 127.87, 128.06, 130.16, 132.51, 134.77, 137.09, 143.76,
143.91, 158.50, 165.35; MS (ESI): m/z: 557.1 (100) [M+Na]+ ; HRMS
(FAB): m/z: calcd for C34H34N2O4Na: 557.2444; found: 557.2416.
131.12, 138.74, 142.33, 169.48; MS (ESI): m/z: 200.0 (100) [M+H]+
HRMS (EI): m/z: calcd for C10H18NO3: 200.1287; found: 200.1278.
;
N-(1-O-[4,4’-Dimethoxytrityl]-2-hydroxypropyl)-2-(3-methylisoxazol-5-
yl)-acetamide (3p): Rf = 0.5 (AcOEt/Et3N 98:2); yield: 77%; 1H NMR
(500 MHz, CDCl3): d = 1.11 (d, J = 6.5 Hz, 3H), 2.26 (s, 3H), 3.26 (dd,
J = 3.5 Hz, 1H), 3.41 (dd, J = 4.5 Hz, 1H), 3.74 (s, 2H), 3.94 (s, 6H),
3.95 (m, 1H), 4.08 (m, 1H), 6.84 (d, J = 7.0 Hz, 4H), 7.23 7.37 (m, 9H);
13C NMR(125 MHz, CDCl 3): d = 11.38, 19.90, 34.88, 53.72, 55.18, 64.85,
68.42, 86.73, 104.08, 113.26, 126.99, 127.78, 127.99, 129.82, 135.09, 135.30,
144.19, 158.56, 160.15, 165.74, 166.75; MS (ESI): m/z: 553.1 (100)
N-(2-Hydroxy-1-hydroxymethylpropyl)-benzamide (2t): Rf
=
0.6
(AcOEt/MeOH 8:2); yield: 60%; 1H NMR(500 MHz, CD 3OD): d =
1.14 (d, J = 6.5 Hz, 3H), 3.76 (d, J = 4.6 Hz, 2H), 3.94 (m, 1H), 4.14
(m, 1H), 7.32 (m, 3H), 7.74 (d, J = 8.0 Hz, 2H), 8.12 (brs, 2H); 13C
NMR(125 MHz, CD 3OD): d = 20.04, 55.48, 63.34, 67.23, 126.98, 128.36,
131.53, 133.96, 168.67; MS (ESI): m/z: 210.0 (100) [M+H]+ ; HRMS (EI):
m/z: calcd for C11H16NO5: 210.1130; found: 210.1134.
[M+Na]+
; HRMS (FAB): m/z: calcd for C31H34N2O6Na: 553.2315;
9H-Fluoren-9-ylmethyl ester of [1-(2-hydroxy-1-hydroxymethylpropylcar-
bamoyl)-2-methyl-butyl]-carbamic acid (2u): Rf = 0.34 (AcOEt); yield:
62%; 1H NMR(500 MHz, CD 3OD): d = 0.84 (m, 6H), 0.97 (d, J =
6.5 Hz, 3H), 1.12 (m, 1H), 1.41 (m, 1H), 1.74 (m, 1H), 2.50 (s, 1H),
3.28 3.46 (m, 2H), 3.63 (m, 1H), 3.92 (m, 2H), 4.21 (m, 2H), 4.61 (m, 2
H), 7.30 7.45 (m, 2H), 7.74 (m, 2H), 7.89 (d, J = 7.5 Hz, 2H); 13C NMR
(125 MHz, CD3OD): d = 11.73, 16.18, 20.71, 24.92, 37.20, 47.37, 56.30,
60.13, 60.88, 64.45, 66.32, 120.80, 126.03, 127.76, 128.34, 141.39, 144.44,
144.61, 156.71, 172.03; MS (ESI): m/z: 441.0 (100) [M+H]+ ; HRMS
(FAB): m/z: calcd for C25H33N2O5: 441.2389; found: 441.2377.
found: 553.2340.
N-(1-O-[4,4’-Dimethoxytrityl]-2-hydroxypropyl)-2-oxo-2H-chromene-3-
carboxamide (3q): Rf = 0.44 (AcOEt/hexane/Et3N 49:49:2); yield: 57%;
1H NMR(500 MHz, CDCl 3): d = 1.18 (d, J = 6.5 Hz, 3H), 3.20 (brs,
1H), 3.37 (dd, J
= 3.5 Hz, 1H), 3.52 (dd, J = 4.5 Hz, 1H), 3.77
(s, 6H), 4.18 (m, 2H), 6.84 (m, 4H), 7.20 7.41 (m, 10H), 7.69 (m, 2H),
8.89 (s, 1H), 9.51 (d, J = 8.5 Hz, 1H); 13C NMR(125 MHz, CDCl 3):
d = 20.04, 54.38, 55.11, 64.64, 68.77, 86.67, 113.16, 116.61, 118.28, 118.51,
125.20, 126.83, 127.92, 129.73, 129.93, 134.06, 135.29, 135.45, 144.24,
148.35, 154.45, 158.43, 161.09, 161.98; MS (ESI): m/z: 602.1 (100)
[M+Na]+ ; HRMS (FAB): m/z: calcd for C35H33NO7Na: 602.2155; found:
602.2131.
9H-Fluoren-9-ylmethyl ester of [1-(2-hydroxy-1-hydroxymethylpropylcar-
bamoyl)-3-methylsulfanylpropyl]-carbamic acid (2v): Rf = 0.55 (AcOEt/
MeOH 9.5:0.5); yield: 80%; 1H NMR(500 MHz, CD 3OD): d = 1.14 (d,
J = 6.5 Hz, 3H), 1.93 (m, 2H), 2.11 (s, 3H), 2.55 (m, 2H), 3.64 (m, 2H),
3.81 (m, 1H), 4.03 (m, 1H), 4.26 (m, 2H), 4.42 (m, 2H), 4.92 (brs, 4H),
7.35 (m, 2H), 7.41 (m, 2H), 7.68 (m, 2H), 7.81 (m, 2H); 13C NMR
(125 MHz, CD3OD): d = 14.10, 19.40, 30.06, 31.79, 54.66, 56.23, 61.57,
65.49, 66.78, 119.77, 125.03, 127.02, 127.63, 141.44, 143.92, 144.22, 157.34,
173.63; MS (ESI): m/z: 458.9 (100) [M+H]+ ; HRMS (FAB): m/z: calcd
for C24H31N2O5S: 459.1954; found: 459.1949.
N-(1-O-[4,4’-Dimethoxytrityl]-2-hydroxypropyl)-1H-benzimidazole-5-car-
boxamide (3r): Rf = 0.5 (AcOEt/hexane/Et3N 90:8:2); yield: 90%; 1H
NMR(500 MHz, CDCl 3): d = 1.02 (d, J = 6.5 Hz, 3H), 3.74 (s, 6H),
3.86 (m, 2H), 4.06 (m, 1H), 4.22 (m, 1H), 6.44 (d, J = 7.0 Hz, 1H), 6.62
(d, J = 7.0 Hz, 1H), 6.79 (d, J = 8.0 Hz, 4H), 6.98 7.12 (m, 4H), 7.28
(m, 3H), 7.42 (m, 1H), 7.54 (d, J = 7.0 Hz, 1H), 7.91 (s, 1H), 7.98 (s, 1
H); 13C NMR(125 MHz, CDCl 3): d = 20.33, 54.97, 55.12, 64.03, 67.96,
113.33, 127.93, 128.06, 128.63, 128.71, 129.48, 130.93, 133.00, 134.35,
178
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 173 181

Macrocyclic Nucleotide-Hybrid Compounds
173 181
158.91, 168.11; MS (ESI): m/z: 574.1 (100) [M+Na]+ ; HRMS (FAB):
m/z: calcd for C33H34N3O5: 552.2498; found: 552.2496.
3-O-[4,4’-Dimethoxytrityl]-1-methyl-2-[(6-methylpyridine-3-carbonyl)-
amino]-propyl ester of 1-(2-cyanoethyl)-N,N-diisopropylphosphoramidite
(4n): Rf
0.30 (AcOEt/hexane/Et3N 40:58:2); yield: 60%; 1HNMR
=
N-(1-O-[4,4’-Dimethoxytrityl]-2-hydroxypropyl)-hexa-2,4-dienamide (3s):
=
0.25 (AcOEt/hexane/Et3N 40:58:2); yield: 80%; 1H NMR
(500 MHz, CDCl3): d = 1.01 (d, J = 7.0 Hz, 3H), 1.13 1.31 (m, 12H),
2.38 (m, 2H), 2.61 (s, 3H), 3.32 (d, J = 5.5 Hz, 2H), 3.55 (m ; 2H), 3.77
(s, 6H), 4.38 (m, 1H), 4.43 (m, 1H), 6.81 (m, 4H), 7.28 (m, 8H), 7.43 (d,
Rf
(500 MHz, CDCl3): d = 1.13 (d, J = 6.5 Hz, 3H), 1.82 (d, J = 6.0 Hz, 3
H), 3.28 (m, 1H), 3.42 (m, 1H), 3.73 (s, 6H), 4.03 (m, 1H), 4.12 (m, 1H),
5.82 (m, 1H), 6.06 (m, 1H), 6.14 (m, 1H), 6.38 (m, 1H), 6.81 (d, J =
8.5 Hz, 4H), 7.18 7.31 (m, 7H), 7.40 (d, J = 7.5 Hz, 2H); 13C NMR
(125 MHz, CDCl3): d = 19.36, 19.78, 54.88, 60.15, 64.56, 67.84, 86.34,
112.98, 121.21, 126.66, 127.68, 127.70, 129.46, 129.69, 135.26, 135.39,
137.70, 141.25, 144.32, 158.28, 166.55; MS (ESI): m/z: 524.2 (100)
[M+Na]+ ; HRMS (FAB): m/z: calcd for C31H35NO5Na: 524.2413; found:
524.2434.
J
= 7.0 Hz, 2H), 8.01 (d, J = 8.0 Hz, 1H), 8.88 (s, 1H); 13C NMR
(125 MHz, CDCl3): d = 19.62, 20.10, 24.38, 42.97, 54.63, 55.02, 57.97,
62.49, 68.80, 85.94, 112.92, 117.66, 122.88, 126.62, 126.66, 127.25, 127.66,
127.95, 128.03, 129.86, 129.91, 135.18, 135.73, 144.60, 147.39, 158.28,
161.46, 165.31; MS (ESI): m/z: 727.1 (100) [M+H]+, 749.3 (45) [M+Na]+
; HRMS (FAB): m/z: calcd for C41H52N4O6P: 727.3625; found: 727.3595.
3-O-[(4-Monomethoxytrityl]-1-methyl-2-[(1-methyl-1H-indole-3-carbon-
yl)-amino]-propyl ester of 1-(2-cyanoethyl)-N,N-diisopropylphosphorami-
N-(1-O-[4,4’-Dimethoxytrityl]-2-hydroxypropyl)-benzamide (3t): Rf
=
1
0.45 (AcOEt/hexane/Et3N 49:49:2); yield: 67%; 1H NMR(400 MHz,
CDCl3): d = 1.21 (d, J = 6.5 Hz, 3H), 3.35 (dd, J = 3.5 Hz, 1H), 3.56
(dd, J = 4.5 Hz, 1H), 3.75 (s, 6H), 4.12 (m, 1H), 4.22 (m, 1H), 6.79 (m,
5H), 7.25 (m, 6H), 7.44 (m, 5H), 7.80 (d, J = 7.5 Hz, 2H); 13C NMR
(125 MHz, CDCl3): d = 17.2, 56.4, 57.6, 64.3, 68.5, 89.8, 126.2, 127.3,
128.4, 129.2, 131.9, 133.5, 135.3, 143.0, 159.5, 171.1; MS (ESI): m/z: 512.1
(100) [M+H]+ ; HRMS (EI): m/z: calcd for C32H34NO5: 512.2498; found:
512.2437.
dite (4o): Rf = 0.33 (AcOEt/hexane/Et3N 40:58:2); yield: 65%; HNMR
(500 MHz, CDCl3): d = 11.18 (d, J = 7.0 Hz, 6H), 1.20 (d, J = 7.0 Hz,
6H), 1.37 (d, J = 6.5 Hz, 3H), 2.36 (dt, J = 6.25 Hz, 2H), 3.28 (dd, J =
7.0 Hz, 1H), 3.43 (dd, J = 7.0 Hz, 1H), 3.52 3.62 (m, 4H), 3.79 (s, 3H),
3.82 (s, 3H), 4.59 (m, 2H), 6.41 (d, J = 9.0 Hz, 1H), 6.83 (d, J = 9.0 Hz,
2H), 7.24 (m, 3H), 7.29 7.39 (m, 5H), 7.37 (m, 3H), 7.50 (d, J = 8.5 Hz,
4H), 7.75 (s, 1H), 7.99 (d, J
=
8.0 Hz, 1H); 13C NMR(125 MHz,
CDCl3): d = 19.61, 20.03, 24.27, 25.52, 53.72, 55.06, 58.10, 68.93, 69.05,
86.21, 110.02, 110.71, 112.92, 117.66, 119.89, 121.18, 122.28, 125.02, 126.76,
127.67, 127.70, 128.37, 128.41, 130.32, 132.68, 135.42, 137.12, 144.26,
144.31, 158.35, 164.72; MS (ESI): m/z: 757.4 (100) [M+Na]+ ; HRMS
(FAB): m/z: calcd for C43H51N4O5PNa: 757.3495; found: 757.3513.
9H-Fluoren-9-ylmethyl ester of (1-{1-O-[4,4’-dimethoxytrityl]-2-hydroxy-
propylcarbamoyl}-2-methylbutyl)-carbamic acid (3u): Rf = 0.5 (AcOEt/
hexane/Et3N 40:58:2); yield: 60%; 1H NMR(500 MHz, CDCl 3): d =
0.81 0.90 (m, 6H), 1.03 (d, J = 6.5 Hz, 3H), 1.42 (m, 2H), 1.81 (m, 1H),
3.15 (m, 1H), 3.34 (m, 1H), 3.64 (s, 6H), 3.82 4.31 (m, 6H), 6.72 (d, J =
8.5 Hz, 4H), 7.14 7.29 (m, 13H), 7.43 (m, 2H), 7.66 (d, J = 7.5 Hz, 2H);
13C NMR(125 MHz, CDCl 3): d = 11.34, 14.09, 15.48, 19.93, 24.63, 37.56,
47.06, 53.63, 55.04, 59.87, 64.77, 66.95, 68.07, 86.63, 113.20, 119.85, 125.00,
126.89, 126.98, 127.59, 127.85, 127.91, 129.80, 129.83, 135.21, 135.43,
141.17, 143.63, 144.23, 156.15, 158.53, 171.38; MS (ESI): m/z: 765.2 (100)
3-[4,4’-Dimethoxytrityl]-1-methyl-2-[2-(3-methylisoxazol-5-yl)-acetylami-
no]-propyl ester of 1-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (4
p): Rf
=
0.35 (AcOEt/hexane/Et3N 60:38:2); yield: 70%; 1H NMR
(500 MHz, CDCl3): d = 11.11 (d, J = 6.5 Hz, 6H), 1.14 (d, J = 7.0 Hz,
6H), 1.18 (d, J = 6.5 Hz, 3H), 2.19 (s, 3H), 2.38 (m, 2H), 3.18 (d, J =
6.0 Hz, 2H), 3.48 (m, 4H), 3.68 (s, 2H), 3.75 (s, 6H), 4.20 (m, 1H), 4.31
(m, 1H), 6.06 (s, 1H), 6.82 (d, J = 8.5 Hz, 4H), 7.19 7.30 (m, 7H), 7.41
(d, J = 8.5 Hz, 2H); 13C NMR(125 MHz, CDCl 3): d = 11.03, 19.20,
19.92, 19.97, 23.94, 24.00, 24.30, 24.36, 34.56, 42.68, 42.78, 54.28, 54.32,
54.84, 57.80, 57.95, 62.24, 68.16, 68.28, 85.71, 103.72, 112.74, 117.79,
126.46, 127.48, 127.83, 129.75, 129.77, 135.50, 135.55, 144.44, 158.12,
159.68, 165.81, 166.18; MS (ESI): m/z: 753.3 (100) [M+Na]+ ; HRMS
(EI): m/z: calcd for C40H51N4O7PNa: 753.3393; found: 753.3395.
[M+Na]+
; HRMS (FAB): m/z: calcd for C46H50N2O7Na: 765.3516;
found: 765.3552.
9H-Fluoren-9-ylmethyl ester of (1-{1-O-[4,4’-dimethoxytrityl]-2-hydroxy-
propylcarbamoyl}-3-methylsulfanylpropyl)-carbamic acid (3v): Rf = 0.3
(AcOEt/hexane/Et3N 40:58:2); yield: 50%; 1H NMR(500 MHz, CDCl 3):
d = 1.06 (d, J = 6.5 Hz, 3H), 1.99 (m, 5H), 2.51 (t, J = 7.0 Hz, 2H),
3.00 (brs, 1H), 3.20 (m, 1H), 3.35 (m, 1H), 3.66 (s, 6H), 4.05 (m, 3H),
4.40 (m, 3H), 6.75 (d, 4H), 7.11 7.32 (m, 15H), 7.68 (d, J = 8.0 Hz, 2H);
13C NMR(125 MHz, CDCl 3): d = 15.03, 19.87, 29.90, 53.77, 54.97, 60.23,
64.36, 66.88, 67.92, 86.50, 113.11, 119.77, 124.92, 126.81, 126.92, 127.54,
127.82, 129.78, 135.19, 135.39, 141.07, 143.46, 143.76, 144.23, 155.87,
158.42, 171.33; MS (ESI): m/z: 783.2 (100) [M+Na]+ ; HRMS (FAB):
m/z: calcd for C45H48N2O7SNa: 783.3080; found: 783.3046.
3-O-(4,4’-Dimethoxytrityl)-1-methyl-2-[(2-oxo-3,8a-dihydro-2H-chro-
mene-3-carbonyl)-amino]-propyl ester of 1-(2-cyanoethyl)-N,N-diisopro-
pylphosphoramidite (4q): Rf = 0.4 (AcOEt/hexane/Et3N 30:68:2); yield:
80%; 1H NMR(500 MHz, CDCl 3): d = 1.00 1.16 (m, 15H), 2.43 2.58
(m, 4H), 3.22 (m, 1H), 3.37 (m, 1H), 3.53 (m, 2H), 3.75 (s, 6H), 4.40 (m,
2H), 6.82 (m, 4H), 7.18 7.36 (m, 8H), 7.47 (m, 2H), 7.61 (m, 2H), 8.92
(d, J = 8.5 Hz, 1H), 9.07 (m, 1H); 13C NMR(125 MHz, CDCl 3): d =
19.30, 19.92, 24.10, 24.27, 42.79, 45.93, 54.30, 54.50, 54.87, 57.70, 62.54,
68.56, 85.82, 122.81, 116.23, 117.54, 118.22, 124.95, 126.41, 127.52, 127.95,
129.48, 129.82, 133.76, 135.68, 135.82, 144.61, 148.07, 154.14, 158.11,
160.82, 161.26; MS (ESI): m/z: 802.2 (100) [M+Na]+ ; HRMS (FAB):
m/z: calcd for C44H52N3O8PNa: 802.3233; found: 802.3251.
9H-Fluoren-9-ylmethyl ester of [1-{1-O-[4,4’-dimethoxytrityl]-2-hydroxy-
propylcarbamoyl}-2-(1H-indol-2-yl)-ethyl]-carbamic acid (3w): Rf = 0.28
(AcOEt/hexane/Et3N 40:58:2); yield: 50%; 1H NMR(500 MHz, CDCl 3):
d = 0.87 (d, J = 6.5 Hz, 3H), 2.84 (brs, 1H), 3.15 3.30 (m, 4H), 3.88 (s,
6H), 4.01 (m, 2H), 4.06 (m, 3H), 4.32 (m, 2H), 4.58 (m, 1H), 6.70 (d, J
= 8.5 Hz, 4H), 7.15 7.45 (m, 19H), 7.70 (d, J = 7.5 Hz, 2H), 8.24 (s, 1
H); 13C NMR(125 MHz, CDCl 3): d = 19.47, 20.81, 28.67, 46.86, 54.07,
54.92, 60.21, 63.93, 66.95, 86.34, 110.06, 111.29, 113.02, 118.48, 119.55,
119.74, 122.03, 123.28, 124.95, 126.75, 126.91, 127.13, 127.49, 127.74,
129.79, 135.16, 135.28, 136.11, 141.03, 143.47, 143.67, 144.37, 155.88,
158.35, 171.65; MS (ESI): m/z: 838.3 (100) [M+Na]+ ; HRMS (FAB):
m/z: calcd for C51H49N3O7Na: 838.3468; found: 838.3504.
2-[(1H-Benzimidazole-5-carbonyl)-amino]-3-O-[4,4’-dimethoxytrityl]-1-
methylpropyl ester of 1-(2-cyanoethyl)-N,N-diisopropylphosphoramidite
(4r): Rf
=
0.75 (AcOEt/hexane/Et3N 60:38:2); yield: 60%; 1H NMR
(500 MHz, CDCl3): d = 1.07 1.19 (m, 15H), 2.57 (m, 4H), 3.55 (m, 4H),
3.68 (s, 6H), 4.22 (m, 2H), 6.77 (m, 4H), 7.01 7.25 (m, 10H), 7.42 7.95
(m, 3H); 13C NMR(125 MHz, CDCl 3): d = 19.10, 20.02, 24.27, 42.75,
54.49, 54.90, 58.07, 74.59, 113.06, 113.12, 115.23, 117.43, 117.57, 119.63,
122.29, 127.66, 127.80, 127.87, 129.30, 130.73, 132.93, 133.01, 134.38,
136.66, 141.06, 143.97, 145.22, 145.84, 146.64, 158.71, 166.69, 167.37; MS
9H-Fluoren-9-ylmethyl ester of (1-{1-hydroxymethyl-2-O-[(-[4-monome-
thoxytrityl]-ethylcarbamoyl}-2-phenylethyl)-carbamic acid (3x): Rf
=
0.45 (AcOEt/hexane/Et3N 80:18:2); yield: 70%; 1H NMR(500 MHz,
CDCl3): d = 3.14 (m, 2H), 3.33 (m, 1H), 3.69 (m, 1H), 3.50 (m, 1H),
3.76 (m, 4H), 4.12 (m, 1H), 4.29 (m, 1H), 4.37 (m, 1H), 4.46 (m, 1H),
4.59 (m, 1H), 6.85 (d, J = 8.5 Hz, 4H), 7.19 7.47 (m, 18H), 7.53 (d, J =
(ESI): m/z: 774.3 (100) [M+Na]+
; HRMS (FAB): m/z: calcd for
C42H50N5O6PNa: 774.3396; found: 774.3577.
3-O-[4,4’-Dimethoxytrityl]-2-hexa-2,4-dienoylamino-1-methylpropyl ester
J =
7.5 Hz, 1H), 7.79 (m, 2H); 13C NMR
of 1-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (4s): Rf
= 0.42
7.5 Hz, 2H), 7.58 (d,
(AcOEt/hexane/Et3N 30:68:2); yield: 70%; 1H NMR(500 MHz, CDCl 3):
d = 1.01 (d, J = 7.0 Hz, 3H), 1.13 1.28 (m, 12H), 1.83 (d, J = 6.5 Hz, 3
H), 2.40 (m, 1H), 2.57 (m, 1H), 3.53 (m, 4H), 3.78 (s, 6H), 4.28 4.42 (m,
2H), 5.72 5.95 (m, 2H), 6.09 (m, 1H), 6.16 (m, 1H), 6.83 (m, 4H), 7.18
7.33 (m, 7H), 7.43 (d, J = 7.0 Hz, 2H); 13C NMR(125 MHz, CDCl 3): d
= 19.87, 20.58, 20.66, 24.67, 24.80, 43.27, 43.37, 54.20, 55.44, 58.14, 68.90,
(125 MHz, CDCl3): d = 38.72, 46.76, 51.12, 54.89, 56.10, 60.24, 62.08,
62.37, 66.80, 83.35, 112.99, 119.72, 124.85, 126.69, 126.84, 127.53, 127.72,
128.03, 128.09, 128.32, 128.44, 129.09, 130.06, 134.86, 136.27, 141.00,
143.50, 143.89, 155.75, 158.39, 171.04; MS (ESI): m/z: 755.1 (100)
; HRMS (FAB): m/z: calcd for C47H44N2O6Na: 755.3113;
found: 755.3097.
[M+Na]+
179
Chem. Eur. J. 2004, 10, 173 181
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

E. T. Kool et al.
FULL PAPER
69.95, 86.33, 118.11, 118.28, 121.94, 126.99, 128.06, 128.43, 128.52, 129.99,
130.32, 130.42, 136.27, 137.92, 141.48, 145.12, 158.66, 166.50; MS (ESI):
shaker at room temperature for 25 min. The iodination reagent was then
removed and the column washed with CH2Cl2 (10 mL), CH3CN (10 mL),
and CH2Cl2 (10 mL). Cleavage and cyclization of the nucleotides was
achieved by soaking the beads in concentrated ammonia at room temper-
ature for 24 h. Cyclic compounds were purified by preparative reverse-
phase HPLC with a gradient of 0 20% of acetonitrile over 20 min at a
m/z: 724.4 (100) [M+Na]+
;
HRMS (FAB): m/z: calcd for
C40H52N3O6PNa: 724.3491; found: 724.3497.
2-Benzoylamino-3-O-[4,4’-dimethoxytrityl]-1-methylpropyl ester of 1-(2-
cyanoethyl)-N,N-diisopropylphosphoramidite (4t): Rf
= 0.45 (AcOEt/
flow rate of 3 mLminÀ1
.
hexane/Et3N 30:68:2); yield: 92%; 1H NMR(500 MHz, CDCl 3): d =
0.90 1.21 (m, 15H), 2.36 (t, J = 6.5 Hz, 2H), 2.47 (m, 2H), 3.29 (m, 1H),
3.47 (m, 3H), 4.19 (m, 2H), 7.76 (d, J = 9.0 Hz, 4H), 7.18 7.45 (m, 12
H), 7.76 (m, 2H); 13C NMR(125 MHz, CDCl 3): d = 13.18, 19.42, 23.32,
23.54, 42.07, 42.31, 53.89, 54.18, 57.14, 59.35, 61.86, 85.18, 112.20, 125.91,
125.99, 126.06, 126.93, 127.29, 127.35, 127.67, 129.24, 130.53, 133.86,
135.13, 135.25, 144.032, 157.66, 166.29; MS (ESI): m/z: 734.3 (100)
[M+Na]+ ; HRMS (FAB): m/z: calcd for C41H50N3O6PNa: 734.3335;
found: 734.3356.
HCV assay: Poly(A) was used as the template (10 mgmLÀ1), oligu(U)12 as
the primer (1 mgmLÀ1), 10 mm a-UTP and 0.025 mCi of [a-32P]UTP as the
substrate, in a total volume of 5 mL reaction mix containing 20 mm tris-
HCl pH 7.5, 5 mm MgCl2, 25 mm KCl, and purified NS5B sample
(0.04 mgmLÀ1). The reaction was started by adding the template, primer,
enzyme, and buffer at 08C for 1 h. The inhibitor and the substrate were
then added and the reaction mixture was incubated at 308C for 60 min.
Reactions were terminated by adding 1 volume of stop buffer (95% form-
amide, 20 mm EDTA (pH 8.0), 0.05% xylene cyanol a,d-bromophenol
blue), and reactions mixtures were kept 1 h at À208C.
Aliquots of each reaction mixture (2 mL) were then spotted uniformly
onto a HybondN+ nylon membrane filter, kept at ambient temperature
for 15 min, and washed with phosphate buffer (pH 7). Finally, the filter
was thoroughly dried and the amount of 32P that remained bound to the
Hybond paper was quantified by PhosphoImager analysis.
3-O-(4,4’-Dimethoxytrityl)-1-methyl-2-((N-Fmoc-isoleucine)-amino)-
propyl ester of 1-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (4u):
Rf
=
0.55 (AcOEt/hexane/Et3N 30:68:2); yield: 70%; 1H NMR
(500 MHz, CDCl3): d = 0.84 0.99 (m, 8H), 1.03 1.22 (m, 15H), 2.32 (t, J
= 6.0 Hz, 2H), 2.49 (m, 1H), 3.11 (m, 2H), 3.46 (m, 4H), 3.68 (s, 6H),
4.01 4.31 (m, 4H), 6.75 (d, J = 8.5 Hz, 4H), 7.10 7.48 (m, 15H), 7.69 (d,
J = 7.5 Hz, 2H) ; 13C NMR(125 MHz, CDCl 3): d = 11.51, 15.38, 19.79,
20.19, 24.25, 24.62, 37.74, 42.92, 43.16, 47.07, 55.07, 57.96, 59.84, 62.46,
66.91, 86.03, 112.99, 119.83, 125.07, 126.68, 127.59, 127.71, 128.15, 130.04,
135.84, 141.15, 143.96, 144.67, 156.41, 158.36, 171.15; MS (ESI): m/z:
948.2 (100) [M+H]+, 965.3 (30) [M+Na]+ ; HRMS (FAB): m/z: calcd for
C55H67N4O8PNa: 965.4594; found: 965.4598.
Acknowledgments
We thank the US National Institutes of Health (GM62658 and RR15054)
for their support. MS was supported in part by a postdoctoral fellowship
from the International Agency for Research on Cancer (IARC).
3-O-(4,4’-Dimethoxytrityl)-1-methyl-2-((N-Fmoc-methionine)-amino)-
propyl ester of 1-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (4v):
Rf
=
0.50 (AcOEt/hexane/Et3N 40:58:2); yield: 75%; 1H NMR
(500 MHz, CDCl3): d = 0.90 1.21 (m, 15H), 2.02 (m, 5H), 2.32 (t, J =
7.0 Hz, 2H), 2.50 (m, 2H), 3.13 (m, 2H), 3.44 (m, 4H), 3.69 (s, 6H),
4.10 4.33 (m, 4H), 6.77 (m, 4H), 7.12 7.50 (m, 15H), 7.70 (d, J
=
7.5 Hz, 2H); 13C NMR(125 MHz, CDCl ): d = 15.15, 19.66, 20.25, 24.16,
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3-O-[4,4’-Dimethoxytrityl]-1-methyl-2-((N-Fmoc-trypthophane)-amino)-
propyl ester of 1-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (4w):
Rf
=
0.38 (AcOEt/hexane/Et3N 40:58:2); yield: 75%; 1H NMR
(500 MHz, CDCl3): d = 0.89 1.31 (m, 15H), 2.27 (t, J = 6.0 Hz, 3.05
3.24 (m, 4H), 3.72 3.76 (s, 6H), 4.13 4.25 (m, 4H), 6.79 (m, 4H), 7.24
7.83 (m, 22H); 13C NMR(125 MHz, CDCl 3): d = 14.09, 19.07, 20.00,
24.11, 24.46, 27.92, 42.86, 45.78, 47.02, 54.00, 55.08, 57.82, 57.97, 60.29,
62.67, 67.00, 68.64, 68.77, 86.01, 111.42, 112.88, 117.94, 118.33, 119.79,
120.91, 121.97, 122.04, 123.66, 125.02, 126.71, 126.95, 127.00, 127.49,
128.22, 130.09, 135.72, 135.76, 141.11, 143.55, 143.78, 144.94, 155.89,
158.34, 171.05; MS (ESI): m/z: 1038.4 (100) [M+Na]+, HRMS (FAB): m/
z: calcd for C60H66N5O8PNa: 1038.4547; found: 1038.4576.
3-O-(4-Monomethoxytrityl)-2-(2-((N-Fmoc-phenylalanine)-amino)-propyl
ester of 1-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (4x): Rf
=
0.28 (AcOEt/hexane/Et3N 30:68:2); yield: 70%; 1H NMR(500 MHz,
CDCl3): d = 1.09 1.20 (m, 12H), 2.46 (m, 2H), 3.09 3.28 (m, 4H), 3.55
3.77 (m, 12H), 4.21 4.44 (m, 4H), 6.83 (m, 2H), 7.17 7.56 (m, 23H), 7.78
(d, J = 7.5 Hz, 2H); 13C NMR(125 MHz, CDCl 3): d = 20.08, 20.76,
24.28, 34.46, 42.67, 42.79, 46.79, 49.57, 54.85, 55.97, 57.81, 58.01, 58.19,
60.10, 60.92, 66.68, 86.10, 112.84, 117.66, 119.68, 124.89, 126.69, 126.80,
127.44, 127.58, 128.09, 128.28, 128.38, 128.44, 129.05, 130.10, 134.94,
134.99, 136.22, 140.96, 143.44, 143.56, 143.85, 155.52, 158.29, 170.84; MS
(ESI): m/z: 933.1 (100) [M+H]+, 955.4 (95) [M+Na]+ ; HRMS (FAB):
m/z: calcd for C56H61N4O7PNa: 955.4176; found: 955.4167.
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Cyclic oligonucleotide synthesis: All cyclic oligonucleotides and cyclic hy-
brids were prepared on a DNA synthesizer by means of standard phos-
phoramidite chemistry and standard synthesis cycles. After coupling the
second nucleotide (DMT-off), the columns were removed and 1 mL sy-
ringes were attached to both ends of the columns. (PhO)3PCH3I (1 mL,
0.5m in anhydrous DMF) was then passed from one syringe, through the
column, to the other syringe for 5 min before the sample was put on a
180
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2004, 10, 173 181

Macrocyclic Nucleotide-Hybrid Compounds
173 181
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181
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim