Semisynthesis of 3′(2′)-O-(aminoacyl)-tRNA derivatives as ribosomal substrate

Article abstract of DOI:10.1002/hlca.200790034

An efficient synthesis of (3′-terminally) 3′(2′)-O- aminoacylated pCpA derivatives is described, which could lead to the production of (aminoacyl)-tRNAs following T4 RNA ligase mediated ligation. The tetrahydrofuranyl (thf) group was used as a permanent p

Full text of DOI:10.1002/hlca.200790034

Helvetica Chimica Acta – Vol. 90 (2007)
297
Semisynthesis of 3’(2’)-O-(Aminoacyl)-tRNA Derivatives as Ribosomal
Substrate
by Zhiyong Cui1), and Biliang Zhang*
Laboratory of RNA Chemical Biology, Guangzhou Institute of Biomedicine and Health, Chinese
Academy of Sciences, Guangzhou Science Park , Guangzhou, Guangdong 510663, P. R. China
(phone: +86 (20) 3229-0708; fax: +86 (20) 3229-0606; e-mail: Zhang_Biliang@gibh.org)
An efficient synthesis of (3’-terminally) 3’(2’)-O-aminoacylated pCpA derivatives is described, which
could lead to the production of (aminoacyl)-tRNAs following T4 RNA ligase mediated ligation. The tet-
rahydrofuranyl (thf) group was used as a permanent protective group for the 2’-OHof the cytidine moi-
ety which can be removed during the purification of the 3’(2’)-O-aminoacylated-pCpA. This approach
allowed for a general synthesis of (3’-terminally) 3’(2’)-O-aminoacylated oligonucleotides. The fully pro-
tected pCpA 14 was synthesized by phosphoramidite chemistry and treated with NH3 solution to remove
the 2-cyanoethyl and benzoyl groups (! 15; Schemes 1 and 2). The 2’-O-thf-protected-pCpA 15 was cou-
pled with a-amino acid cyanomethyl esters, and the products 20a–c were deprotected and purified with
AcOHbuffer to afford 3 ’(2’)-O-aminoacylated pCpA 21a–c in high yields. The 3’(2’)-O-aminoacylated
pCpA were efficiently ligated with tRNA(ꢀCA) to yield (aminoacyl)-tRNA which was an active sub-
strate for the ribosome.
Introduction. – Despite decades of intensive research, the catalytic mechanism of
protein synthesis in the ribosome is still largely unknown [1–3]. The peptidyl-transfer-
ase reaction in the ribosome has never been well characterized because studies have
been hampered by technical problems associated with the complexity of the ribosome
and its substrates. The (3’-terminally) 3’(2’)-O-aminoacylated pCpA derivatives are the
universally conserved terminal sequences of (aminoacyl)-tRNA [4–7]. The enzymatic
preparation of (aminoacyl)-tRNA yields only very limited quantities and is not used as
a general approach to all natural and unnatural amino acids. Furthermore, the X-ray
crystallographic study of the ribosome requires large quantities of (aminoacyl)-
tRNAs. The development of a general chemical synthesis of (3’-terminally) 3’(2’)-O-
aminoacylated oligonucleotides is of considerable importance [8–10]. Hecht and co-
workers have developed a successful solution to this problem. They coupled tRNA,
missing the 3’-terminal pCpA moiety, with 3’(2’)-O-aminoacylated pCpA derivatives
in the presence of T4 RNA ligase [11–19]. However, the synthesis of aminoacylated
pCpA suffered several drawbacks.
Several groups have reported the synthesis of 3’(2’)-O-aminoacylated oligonucleo-
tides by either nonchemical methods [20–21] or by various protective-groups/amino-
acylating methods [15–19][22–33]. These methods lead to low aminoacylation yields,
1
)
Present address: Department of Medicine, Dartmouth Medical School, Hanover, NH 03755 USA.
ꢀ 2007 Verlag Helvetica Chimica Acta AG, Zürich

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long reaction paths, or racemization of the products. Schultz and co-workers reported
that the aminoacylation of amino acid cyanomethyl esters with unprotected pdCpAwas
an efficient method for preparing aminoacylated pdCpA [34]. Although the 2’-deoxy-
cytidine may not be important for the in vitro translation system to incorporate a non-
natural amino acid into a protein from a suppressor tRNA, it may be considerably crit-
ical for studying the peptidyl-transferase reaction in the ribosome. Literature searches
revealed that the 2’-OHof the nucleotide was either not protected during aminoacyla-
tion [15][27][28] or was protected with the tetrahydro-2H-pyran-2-yl (thp) [22], the 4-
methoxytetrahydro-2H-pyran-2-yl (mthp) [23–26], or a fluoride-labile group [33].
These methods were not satisfactory as they resulted in diacylation of the nucleotide,
a missing 5’-terminal phosphate that is required for the ligation with tRNA(ꢀCA)
[17][33], extra deprotection steps, or removal of the 2’-OHprotecting groups under rel-
atively strong acidic conditions. All these problems led to low overall yields.
Results and Discussion. – The pHrange ensuring the best stability of 3 ’(2’)-amino-
acylated oligonucleotides is pH3–4.5 [35]; thus, the ideal deprotection of the 2 ’-OH
protective group of the cytidine moiety of pCpA should be conducted within this
range. It is known that the tetrahydrofuran-2-yl (thf) group can be removed under
this condition (acetic acid, pH4). Ikehara and co-workers [36] used thf as a protective
group to synthesize oligonucleotides by the phosphotriester method. However, the thf
protective group was not widely used in nucleotide synthesis because this group was
believed to be incompatible with the deprotection condition for regular 5’-OHprotec-
tive groups like the 4,4’-dimethoxytrityl (MeO)2Tr, levulinoyl, or 4,4’,4’’-tris(4,5-
dichlorophthalimido)trityl group under relatively strong acidic conditions. We found
that the thf group was stable while (MeO)2Tr was selectively removed by catalytic
hydrogenation in MeOHunder 1 atm of H . Here, we describe an efficient route for
2
synthesizing 3’(2’)-O-aminoacylated pCpA and corresponding (aminoacyl)-tRNAs.
The thf group served as a permanent protection group for the 2’-OHof the cytidine
moiety while the 5’-OHwas protected by the (MeO) Tr group.
2
The cytidine synthon N4-benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-O-(tetrahydro-
furan-2-yl)cytidine 3’-(2-cyanoethyl diisopropylphosphoramidite) (7) was prepared as
shown in Scheme 1. Cytidine (1) was treated first with chlorotrimethylsilane in pyridine,
then with benzoyl chloride, and finally with NH4OHin a one-pot procedure to produce
N4-benzoylcytidine (2) in 94% yield. Compound 2 was treated with 1,3-dichloro-
1,1,3,3-tetraisopropyldisiloxane (iPr2Si(Cl)OSi(Cl)iPr2) in pyridine to give N4-benzo-
yl-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine (3) in 94% yield. In the
presence of 4-toluenesulfonic acid in THF 3 reacted with 2,3-dihydrofuran to produce
N4-benzoyl-2’-O-(tetrahydrofuran-2-yl)-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)cytidine (4) in 97% yield. Then, the 3’,5’-tetraisopropyldisiloxanediyl group was
removed by treatment with Bu4NF in THF generating N4-benzoyl-2’-O-(tetrahydro-
furan-2-yl)cytidine 5 in 90% yield (49% anti isomer and 41% syn isomer). Selective
protection of the 5’-OHgroup with 4,4 ’-dimethoxytrityl chloride ((MeO)2TrCl) yielded
6, which was coupled with 2-cyanoethyl diisopropylphosphoramidochloridite in the
standard manner to afford the building block 7 in 55–59% yield.
Scheme 2 shows the synthesis of the adenosine synthon and the 2’-O-thf-protected
pCpA. The N6-benzoyladenosine was obtained in the same manner as N4-benzoylcyti-

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Scheme 1. Synthesis of N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-O-(tetrahydrofuran-2-yl)cytidine 3’-
(2-Cyanoethyl Diisopropylphosphoramidite) (7)
a) 1. Chlorotrimethylsilane, pyridine; 2. benzoyl chloride, pyridine; 3. NH4OH. b)
iPr2Si(Cl)OSi(Cl)iPr2, pyridine. c) 2,3-Dihydrofuran, TsOH, THF. d) Bu4NF, THF. e) (MeO)2TrCl,
i
i
pyridine, Pr2EtN. f) 2-Cyanoethyl diisopropylphosphoramidochloridite, Pr2EtN, CH2Cl2.
dine (2) and then protected with (tert-butyl)chlorodiphenylsilane (tBuPh2SiCl) at 5’-
OHto form 9 in 95% yield. The treatment of 9 with Ac2O in pyridine yielded 10 in
quantitative yield. The removal of the (tert-butyl)diphenylsilyl group with Bu4NF pro-
duced 11 in 95% yield. The cytidine phosphoramidite 7 was coupled with 2’,3’-di-O-ace-
tyl-N6-benzoyladenosine (11) in the presence of 1H-tetrazole to yield the fully pro-
tected dinucleotide 12 (84% yield). The (MeO)2Tr group at the 5’-position of dinucleo-
tide 12 was removed by hydrogenation in MeOHover 10% Pd/C. However, the thf
group at the 2’-position of cytidine was also partially cleaved on a prolonged reaction
time. This might be caused by the accumulation of in situ formed protons in the hydro-
genation process. Thus, a base (pyridine or Et3N) was added to the hydrogenation mix-
ture to prevent thf cleavage. But the Pd/C catalyst was poisoned by these amine bases.

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Scheme 2. Synthesis of 2’-O-(Tetrahydrofuran-2-yl)cytidylyl-(3’ ! 5’)-adenosine 5’-(Dihydrogen Phos-
phate) Tris(tetrabutylammonium) Salt 15
t
a) 1. Chlorotrimethylsilane, pyridine; 2. benzoyl chloride, pyridine; 3. NH4OH; 4. BuPh2SiCl, N,N-
dimethylpyridin-4-amine (DMAP). b) Ac2O, pyridine, DMAP. c) Bu4NF, THF. d) 1. 7, 1H-tetrazole,
i
i
MeCN; 2. t-BuOOH. e) H2, Pd/C, MeOH, 2 h. f) 1. Pr2N-P(OCH2CH2CN)2, Pr2EtN, 1H-tetrazole,
MeCN; 2. t-BuOOH. g) 1. NH3, EtOH, 558; 2. ion exchange, Bu4N(OH).
Replacing the solvent MeOHby EtOHstrongly reduced the deprotecting rate. The
tBuPh2Si group was also used to protect the 5’-OHof cytidine while the 2 ’-OHgroup

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was protected by thf. But the 2-cyanoethyl group at the phosphate moiety was not sta-
ble under the cleaving conditions for tBuPh2Si by Bu4NF. However, a 70% yield of iso-
lated desired product 13 was adieved by treatment of 13 with H2 over PdC in MeOHfor
2 h. Then the 5’-HO-CpA reacted with bis(2-cyanoethyl) diisopropylphosphoramidite
[7] in the presence of 1H-tetrazole in MeCN to yield the fully protected pCpA 14
(86% yield). This was then dissolved in NH3 solution/EtOH3 :1 ( v/v) in a sealed reac-
tion vessel. The mixture was heated at 558 overnight and then exchanged by ion-
exchange chromatography (Amberlite CG-50, Bu4N+ form) to give 2’-O-(tetrahydro-
furan-2-yl)cytidylyl-(3’ ! 5’)-adenosine 5’-(dihydrogen phoshate) tris(tetrabutylam-
monium) salt 15 (89%).
The synthesis of 2’(3’)-O-[(pent-4-enoyl)amino]acylated pCpAs and {[(pent-4-
enoyl)amino]acyl}-tRNAs is shown in Scheme 3. The synthesis of [(pent-4-enoyl)-
A
treated with N-hydroxysuccinimide in the presence of N,N-dimethylpyridin-4-amine
(DMAP) and dicyclohexylcanbodiinide (DCC) to give active ester 17. The reaction
of 17 with three different a-amino acids in DMF yielded the N-(pent-4-enoyl)-L-
amino acids 18a–c. These were converted to active cyanomethyl esters of N-(pent-4-
enoyl)-L-amino acids 19a–c in 60–74% yield from 16. The spectral data were identical
with published data. The cyanomethyl esters 19a–c were then treated with (internally)
2’-O-thf-protected pCpA dinucleotide 15 in DMF to yield 20a–c. After deprotection
and purification, the [(pent-4-enoyl)amino]acylated pCpA 21a–c were obtained in
over 80% yield from 15, as determined by HPLC. The preparation of the {[(pent-4-
enoyl)amino]acyl}-tRNAs 22a–c was accomplished by T4-RNA-ligase-mediated liga-
A
tion with aminoacylated pCpAs 21a–c and Escherichia coli tRNA(ꢀCA)Phe transcripts.
The pent-4-enoyl protection group can be removed by treatment with 10 mM I2 for 20
min.
To determine the ligation efficiency, (3’-terminally) 3’(2’)-O-{N-[(biotinylamino)-
caproyl]-L-methionyl}-substituted pCpA and the corresponding {N-[(biotinylamino)-
caproyl]-L-methionyl}-tRNA were also synthesized in the same manner as described
above2). The coupling reaction of N-[(biotinylamino)caproyl]-L-methionine cyano-
methyl ester2) with the pCpA tris(tetrabutylammonium) salt 15 in anhydrous DMF
yielded 3’(2’)-O-{N-[(biotinylamino)caproyl]-L-methionyl}-substituded pCpA. This
biotinylated aminoacylated pCpA was ligated with an Escherichia coli tRNA(ꢀCA)Phe
transcript in the presence of T4 RNA ligase. The resulting {N-[(biotinylamino)caproyl]-
L-methionyl}-tRNA and ligation efficiency were determined by streptavidin-gel-shift
analysis (Fig. 1). Over 90% of tRNA(ꢀCA) was converted to {N-[(biotinylamino)-
caproyl]-L-methionyl}-tRNA within 25 min (Lane 5). The result demonstrated that
the ligation reaction of aminoacylated pCpA with tRNA(ꢀCA) was highly efficient.
The determination of the peptidyl-transferase activity of the {N-[(biotinylamino)-
caproyl]-L-methionyl}-tRNA substrate was performed with 5’-([32P]p)CpCpA-NH-
Phe as a peptidyl acceptor in the 50 S ribosome as shown Fig. 2. After 30 min., the pep-
tidyl-transferase reaction was complete (Lane 4, Fig. 2). The results demonstrated that
chemically semisynthesized peptidyl-tRNA was an active peptidyl donor for the ribo-
2
)
The systematic name of the acyl group (biotinylamino)caproyl is 6-{{5-[(3aS,4S,6aR)-hexahydro-2-
oxo-1H-thieno[3,4-d]imidazol-4-yl]-1-oxopentyl}amino}-1-oxohexyl.

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Scheme 3. Synthesis of 2’,(3’)-O-AminoacylatedpCpA andtRNA
i
a) N-Hydroxysuccinimide, DMAP, DCC, THF. b) L-Amino acid, Pr2EtN, DMF. c) Chloroacetonitrile,
iPr2EtN, MeCN. d) 15, DMF. e) 1. 50 mM NH4OAc, C18 reversed-phase chromatography; 2. 0.87M
AcOH, C18 reversed-phase chromatography. f) tRNA(ꢀCA)Phe, T4 RNA ligase.
some. We also studied the other semisynthesized (aminoacyl)-tRNAs 22a–c as the
ribosomal substrate, all of them were active for the peptidyl-transferase reaction in
the ribosome (data not shown).
Conclusion. – We have developed a general method for the synthesis of pCpA. The
advantage of the synthetic strategy is that the tetrahydrofuran-2-yl protective group is
compatible with the (MeO)2Tr group and can be removed during purification resulting
in high aminoacylation yields. Since T4 RNA ligase is a readily available and inexpen-
sive enzyme, a relatively large quantity of (aminoacyl)-tRNA can be prepared by this

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Fig. 1. Autoradiogram of the streptavidin-gel-shift analysis of ligation products [3’(2’)-O-{N-[(biotinyl-
amino)caproyl]-L-methionyl-pCpA and[5 ’-32P]-tRNA(ꢀCA)Phe]. The samples were run on 7.5M urea/
a
10% polyacrylamide gel with 1”TBE buffer at 30 W (TBE=Tris/boric acid/Na4edta).
) aa-
b
tRNA=Escherichia coli tRNA(ꢀCA)Phe
.
)
aa-tRNA={N-[(biotinylamino)caproyl]-L-methionyl}-
tRNA.
Fig. 2. Peptidyl-transferase activity of {N-[(biotinylamino)caproyl]-L-methionyl}-tRNA, determined
with ([32P]p)CpCpA-NH-Phe as peptidyl acceptor
strategy. Semisynthesized peptidyl- or (aminoacyl)-tRNA were active for the peptide
synthesis in the ribosome. This synthetic method may provide a potential way to pre-
pare (aminoacyl)-tRNA or unnatural aminoacyl-tRNA for the peptide synthesis in
the ribosome.
This work was partially supported by grants from the startup fund of the Guangzhou Institute of Bio-
medicine and Health.

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Experimental Part
General. All solvents, organic chemicals, and inorganic chemicals were purchased from Acros or
Aldrich. Solvents were dried by using standard methods. CH2Cl2, MeCN, and pyridine were refluxed
over CaH2 and freshly distilled before use. Reactions were run under Ar. TLC: precoated silica gel 60
1
F254 sheets from EM. Flash chromatography (FC): silica gel 60, 180–240 mesh, from EM. H-NMR
Spectra: Varian VNMR-400 spectrometer; CDCl3 or (D6)DMSO as solvents, with the trace-solvent signal
as reference; d in ppm, J in Hz. ESI-MS: Finnigan LCQDUO spectrometer; in m/z.
Ligation Reaction of tRNA(ꢀCA) with 3’(2’)-O-(Aminoacyl)-SubstitutedpCpA . E. coli tRNA-
(ꢀCA)Phe was prepared by in vitro transcription in the presence of T7 RNA polymerase. Then 40 mg
of 3’(2’)-O-L-aminoacyl)-substituted pCpA 21a–c or 3’(2’)-O-{N-[(biotinylamino)caproyl]-L-
methionyl}-substituted pCpA was incubated with tRNA(ꢀCA)Phe in the presence of 200 U T4 RNA
ligase in the ligation buffer (55 mM HEPES (=4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid);
pH7.5, 250 mM ATP, 15 mM MgCl2, 10% (v/v) DMSO) at 378 for 1–5 h. The ligated product was purified
by 10% polyacrylamide gel electrophoresis.
Labeling of HO-CpCpA-NH-Phe. The kinase reaction was performed with 1 nmol of 5’-HO-
CpCpA-NH-Phe, 50 mCi of [g-32P]-ATP, and 200 units of T4 polynucleokinase (PNK) in the presence
of 1”PNK buffer at 378 for 1 h. Excess ATP (100 nmol) was added into the radioactive mix, and the
labeling reaction was continued for another hour to convert all 5’-HO-CpCpA-NH-Phe into 5’-
([32P]p)CpCpA-NH-Phe which was purified by 20% polyacrylamide gel electrophoresis.
Ribosomal Reactions. Reactions were performed with 1.0 A260 unit of 50 S (36 pmol), 200 mM {N-
[(biotinylamino)caproyl]-L-methionyl} tRNA, and a trace amount of 5’-([32P]p)CpCpA-NH-Phe in the
presence of 50 mM Tris·HCl buffer (pH 7.4), 35 mM MgCl2, 100 mM NH4Cl, and 1000 mM KCl at 378.
Samples were loaded onto 24% polyacrylamide/7.5M urea gel.
N4-Benzoylcytidine (2). Rf 0.55 (20% MeOH/AcOEt). 1H-NMR ((D6)DMSO6, 400 MHz): 3.57–3.77
(m, 2 H); 3.89–4.01 (m, 3 H); 5.06 (d, J=5.47, 1 H); 5.18 (t, J=5.08, 1 H); 5.52 (d, J=5.09, 1 H); 5.79 (d,
J=2.74, 1 H); 7.31 (m, 1 H); 7.48–7.63 (m, 3 H); 7.97–7.99 (m, 2 H); 8.47 (d, J=7.42, 1 H); 11.16 (br. s, 1
H). 13C-NMR ((D6)DMSO6, 100 MHz): 60.57; 69.32; 75.23; 84.90; 90.88; 96.80; 129.14; 133.42; 133.86;
145.94; 155.22; 163.70; 168.03.
N4-Benzoyl-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine (3). Rf 0.57 (9.1% MeOH/
1
CHCl3). H-NMR (400 MHz, CDCl3): 0.94–1.11 (m, 28 H); 2.88 (br. s, 1 H); 4.01–4.05 (dd, J=13.44,
2.69, 1 H) 4.20–4.35 (m, 4 H); 5.86 (s, 1 H); 7.50–7.63 (m, 4 H); 7.87–7.89 (d, J=6.59, 2 H); 8.22 (d,
J=6.60, 1 H); 8.67 (br. s, 1 H). 13C-NMR (100 MHz, CDCl3): 12.70; 13.13; 13.15; 13.60; 17.05; 17.12;
17.21; 17.52; 17.63; 17.68; 60.23; 68.82; 75.46; 82.26; 91.80; 96.24; 127.70; 129.34; 133.49; 144.92;
162.56. ESI-MS: 590.2 ([M+H]+).
N4-Benzoyl-2’-O-(tetrahydrofuran-2-yl)-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine
(4). Compound 3 and TsOHwere dried under vacuum (oil pump) for 5 h. To a soln. of 3 (12.89 g, 21.87
mmol) and TsOH(1.46 g, 7.67 mmol) in THF (250 ml), 2,3-dihydrofuran (16.7 ml, 219.2 mmol) was
added by syringe at 08. The mixture was stirred for 3 h at 08 under Ar. Then conc. NH3 soln. (11.7 ml)
was added and the solvent evaporated at low temp. The residue was dissolved in CHCl3 (800 ml) and
washed with sat. aq. NaHCO3 soln. (100 ml) and H2O (100 ml). The aq. layers were extracted with
CHCl3 (100 ml). The combined org. phase was dried (Na2SO4) and concentrated, and the residue purified
by FC (SiO2, 3.3–75% AcOEt/hexane): 4 (14 g, 97.0%, two diastereoisomers) as a white solid. Rf 0.67
(75% AcOEt in hexane). 1H-NMR (400 MHz, CDCl3): 0.92–1.11 (m, 28 H); 1.81–2.00 (m, 4 H );
3.82–4.36 (m, 7 H); 5.64–5.74 (m, 1 H); 5.84 (d, J=8.99, 1 H); 7.45–7.61 (m, 4 H); 7.88 (d, J=7.42, 2
H); 8.30 (m, 1 H). 13C-NMR (100 MHz, CDCl3): 12.76; 12.80; 13.04; 13.23; 13.59; 13.63; 17.06; 17.11;
17.16; 17.18; 17.22; 17.30; 17.46; 17.51; 17.61; 17.68; 22.76; 23.16; 32.23; 32.74; 59.58; 59.64; 66.67;
66.77; 67.21; 68.95; 75.80; 77.80; 81.99; 82.20; 90.71; 90.85; 96.57; 102.61; 103.06; 128.02; 128.99;
133.15; 133.47; 144.34; 154.69; 162.94; 171.34. ESI-MS: 682.1 ([M+Na]+), 660.2 ([M+H]+).
N4-Benzoyl-2’-O-(tetrahydrofuran-2-yl)cytidine (5a/5b). Data of 5a: Rf 0.46 (9.1% MeOH/CHCl3).
1H-NMR (400 MHz, CDCl3): 1.84–2.05 (m, 4 H); 3.58 (d, J=5.86, 1 H); 3.80–4.09 (m, 6 H); 4.30 (m,
1 H); 4.46 (m, 1 H); 5.47 (m, 1 H); 5.78 (d, J=2.05, 1 H); 7.44–7.56 (m, 4 H); 7.86 (m, 2 H); 8.40 (d,

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J=7.32, 1 H); 9.14 (br. s, 1 H). 13C-NMR (100 MHz, (D6)DMSO): 23.51; 32.47; 60.02; 66.92; 68.21; 79.01;
84.95; 89.40; 96.77; 102.87; 129.12; 129.42; 133.42; 133.77; 145.62; 155.05; 163.82; 168.11. ESI-MS: 416.2
([MꢀH]ꢀ).
1
Data of 5b: Rf 0.39 (9.1% MeOH/CHCl3). H-NMR (400 MHz, (D6)DMSO): 1.02–1.86 (m, 4 H );
3.59–3.67 (m, 2 H); 3.88 (m, 1 H); 4.11–4.18 (m, 2 H); 5.14–5.17 (m, 2 H); 5.26 (m, 1 H); 5.93 (d,
J=4.69, 1 H); 7.31–7.61 (m, 4 H); 7.99 (d, J=7.42, 2 H); 8.39 (d, J=7.04, 1 H); 11.25 (br. s, 1 H). 13C-
NMR (100 MHz, (D6)DMSO): 23.27; 32.54; 61.19; 66.92; 69.20; 78.49; 85.90; 88.78; 96.97; 103.40;
129.12; 129.16; 133.42; 133.84; 146.28; 155.30; 163.70; 168.15. ESI-MS: 416.3 ([MꢀH]ꢀ).
N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-O-(tetrahydrofuran-2-yl)-cytidine (6b). Rf 0.36 (4.8%
MeOH/CHCl3). 1H-NMR (400 MHz, CDCl3): 1.85–2.06 (m, 4 H); 2.56 (d, J=7.03, 1 H); 3.48–3.58
(m, 2 H); 3.82 (s, 6 H); 3.87–3.97 (m, 2 H); 4.12–4.14 (m, 1 H); 4.36–4.42 (m, 2 H); 5.54 (m, 1 H );
6.27 (s, 1 H); 6.86–6.88 (m, 4 H); 7.26–7.63 (m, 13 H); 7.86 (m, 2 H); 8.35 (br. s, 1 H); 8.55 (s, 1 H).
13C-NMR (100 MHz, CDCl3): 23.46; 32.65; 55.47; 62.24; 68.05; 69.14; 79.43; 83.70; 87.29; 89.47; 97.12;
104.59; 113.56; 127.37; 127.98; 128.28; 128.50; 129.19; 130.30; 130.39; 133.27; 135.59; 135.87; 144.41;
145.39; 155.20; 158.89; 162.61. ESI-MS: 742.3 ([M+Na]+), 720.1 ([M+H]+).
Isomer 6a: Treatment of 5a (0.48 g, 1.15 mmol) and 4,4’-dimethoxytrityl chloride (0.779 g, 2.3 mmol)
1
as described for 6b gave 6a (0.78 g, 94%). Rf 0.40 (9.1% MeOH/CHCl3). H-NMR (400 MHz, CDCl3):
1.84–2.08 (m, 4 H); 3.55–3.59 (m, 3 H); 3.81 (s, 6 H); 3.84–4.09 (m, 3 H); 4.33–4.43 (m, 2 H); 5.56
(m, 1 H); 5.94 (s, 1 H); 6.86–6.89 (m, 4 H); 7.24–7.57 (m, 13 H); 7.88–7.91 (m, 2 H); 8.54 (d, J=7.42,
1 H); 8.95 (br. s, 1 H). 13C-NMR (100 MHz, CDCl3): 24.01; 32.78; 55.48; 61.32; 68.04; 68.18; 81.99;
83.40; 87.25; 90.36; 96.57; 105.36; 113.55; 127.39; 127.74; 128.28; 128.51; 129.27; 130.30; 130.39; 133.36;
135.59; 135.90; 144.39; 145.17; 155.08; 158.90; 162.38. ESI-MS: 742.3 ([M+Na]+), 720.1 ([M+H]+).
N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-O-(tetrahydrofuran-2-yl)cytidine 3’-(2-Cyanoethyl Diiso-
propylphosphoramidite) (7b). To a cooled (ice bath) soln. of 6b (0.8 g, 1.11 mmol; previously dried
under vacuum (oil pump) overnight) in CH2Cl2 (25 ml), iPr2EtN (0.794 ml, 4.56 mmol) and 2-cyanoethyl
diisopropylphosphoramidochloridite was added successively under Ar. After 20 min the mixture was stir-
red at r. t. for 4.5 h. The mixture was diluted with AcOEt (250 ml), the org. layer washed with sat. aq.
NaHCO3 soln. (25 ml) and sat. aq. NaCl soln. (25 ml) and dried (Na2SO4), the solvent evaporated, and
the residue purified by FC (SiO2, 77% AcOEt/19.2% hexane/3.8% Et3N): 7b (0.6 g, 58.5%, two diaster-
eoisomers). Yellow solid. Rf 0.59, 0.70 (4.8% AcOEt/CHCl3). 1H-NMR (400 MHz, CDCl3): 1.00–1.15 (m,
12 H); 1.76–1.94 (m, 4 H); 2.53 (t, J=6.25, 1 H); 2.61 (t, J=6.25, 1 H); 3.42–3.66 (m, 6 H); 3.77–3.76 (m,
6 H); 3.83–3.91 (m, 2 H); 4.22–4.30 (m, 1 H); 4.44–4.53 (m, 2 H); 5.44–5.48 (m, 1 H); 6.19–6.21 (m, 1
H); 6.81–6.85 (m, 4 H); 7.02 (br. s, 1 H); 7.20–7.54 (m, 13 H); 7.88–7.90 (m, 2 H); 8.26–8.36 (m, 1 H).
13C-NMR (100 MHz, CDCl3): 20.36; 20.43; 20.57; 20.63; 23.17; 23.31; 24.70; 24.77; 24.82; 24.86; 32.58;
32.73; 43.27; 43.40; 43.53; 55.41; 55.44; 58.17; 58.37; 58.71; 58.89; 61.83; 62.23; 67.50; 67.54; 70.47;
70.53; 70.61; 70.68; 82.98; 83.05; 89.34; 89.40; 97.24; 103.65; 103.82; 113.47; 113.50; 117.72; 117.92;
127.37; 127.40; 127.98; 128.21; 128.23; 128.53; 128.64; 129.07; 130.39; 130.51; 133.16; 133.67; 135.37;
135.49; 135.58; 135.70; 144.30; 144.36; 145.15; 158.91; 162.03; 171.29. 31P-NMR (162 MHz, CDCl3):
150.92; 151.38. ESI-MS: 920.1 ([M+H]+).
Treatment of 6a (0.23 g, 0.32 mmol) as described for 7b gave 7a (0.162 g, 55.1%). Light yellow solid.
Rf 0.64, 0.81 (4.8% AcOEt/CHCl3). 1H-NMR (400 MHz, CDCl3): 1.05–1.15 (m, 12 H); 1.80–1.95 (m, 4
H); 2.40 (m, 2 H); 3.40–3.80 (m, 12 H); 3.81–4.10 (m, 2 H); 4.24–4.53 (m, 3 H); 5.65–5.72 (m, 1 H );
6.05–6.11 (m, 1 H); 6.85–6.87 (m, 4 H); 7.05 (br. s, 1 H); 7.26–7.59 (m, 13 H); 7.88–7.90 (m, 2 H );
8.29–8.55 (m, 1 H). 31P-NMR (162 MHz, CDCl3): 150.58; 151.37. ESI-MS: 920.1 ([M+H]+).
N6-Benzoyladenosine. Rf 0.24 (13.3% MeOH/CHCl3). 1H-NMR (400 MHz, (D6)DMSO): 3.55–3.70
(m, 2 H); 3.96–3.99 (m, 1 H); 4.17–4.19 (m, 1 H); 4.63–4.67 (m, 1 H); 5.14 (t, J=5.50, 1 H); 5.26 (d,
J=4.77, 1 H); 5.58 (d, J=5.87, 1 H); 6.04 (d, J=5.51, 1 H); 7.53–7.64 (m, 3 H); 8.03–8.04 (m, 2 H );
8.72 (s, 1 H); 8.75 (s, 1 H); 11.22 (br. s, 1 H). 13C-NMR (100 MHz, (D6)DMSO): 62.00; 71.05; 74.33;
86.40; 88.24; 126.58; 129.18; 133.17; 133.99; 143.86; 151.09; 152.33; 152.91, 166.31.
N6-Benzoyl-5’-O-[(tert-butyl)diphenylsilyl]adenosine (9). Into a soln. of N6-benzoyladenosine (1.9 g,
5.12 mmol) and N,N-dimethylpyridin-4-amine (0.02 g, 0.164 mmol) in pyridine (100 ml), (tert-butyl)-
chlorodiphenylsilane (1.6 ml, 6.14 mmol) was injected in one portion. The mixture was stirred at r.t.
under Ar for 96 h. The solvent was evaporated, the residue diluted with EtOH(10 ml), and precipitation

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induced by adding Et2O (250 ml). The solid was filtered off and washed with H2O (2”50 ml): 9 (3.09 g,
99%). White solid. Rf 0.32 (9.1% MeOH/CHCl3). 1H-NMR (400 MHz, (D6)DMSO): 0.96 (s, 1 H );
3.78–4.09 (m, 2 H); 4.07 (m, 1 H); 4.36 (m, 1 H); 4.73 (t, J=5.14, 1 H); 6.06 (d, J=5.13, 1 H);
7.32–7.66 (m, 13 H); 8.02–8.04 (m, 2 H); 8.59 (s, 1 H); 8.66 (s, 1 H); 11.22 (br. s, 1 H). 13C-NMR (100
MHz, (D6)DMSO): 27.25; 64.62; 70.60; 73.76; 85.25; 88.46; 126.53; 128.20; 128.48; 128.53; 129.16;
129.19; 129.85; 130.59; 132.24; 133.27; 133.44; 134.00; 135.10; 135.69; 135.74; 143.78; 151.09; 152.77.
2’,3’-Di-O-acetyl-N6-benzoyl-5’-O-[(tert-butyl)diphenylsilyl]adenosine (10). To a soln. of 9 (10.6 g,
17.4 mmol) and N,N-dimethylpyridin-4-amine (0.106 g, 0.87 mmol) in dry pyridine (250 ml), Ac2O (3.6
ml, 38.26 mmol) was added slowly by syringe. The mixture was stirred at r. t. under Ar for 17.5 h, then
another portion of Ac2O (0.164 ml, 0.132 mmol) was added. The mixture was stirred for another 18 h.
H2O (1 ml) was added to quench the reaction. After evaporation of the solvent, the residue was dissolved
in CH2Cl2 (500 ml) and the soln. washed with sat. aq. NaCl soln. (2”60 ml). The org. phase was dried
(Na2SO4), the solvent evaporated, and the residue purified by FC (SiO2, 4.7–40% AcOEt/hexane): 10
1
(23.2 g, 99%). White solid. Rf 0.72 (13.3% MeOH/CHCl3). H-NMR (400 MHz, CDCl3): 1.11 (s, 9 H );
2.05 (s, 3 H); 2.15 (s, 3 H); 3.87 (dd, J=11.72, 3.12, 1 H); 3.99 (dd, J=11.72, 3.12, 1 H); 4.31 (m, 1 H );
5.72 (dd, J=5.47, 2.74, 1 H); 5.94 (dd, J=6.64, 5.47, 1 H); 6.40 (d, J=7.03, 1 H); 7.35–7.45 (m, 6 H );
7.51–7.55 (m, 2 H); 7.60–7.63 (m, 1 H); 7.66–7.68 (m, 4 H); 8.00–8.02 (m, 2 H); 8.26 (s, 1 H); 8.78 (s,
1 H); 8.92 (br. s, 1 H). 13C-NMR (100 MHz, CDCl3): 20.63; 20.92; 27.16; 63.67; 71.49; 73.72; 84.04;
84.99; 123.40; 128.06; 128.18; 128.20; 129.15; 130.25; 130.33; 132.38; 132.73; 133.08; 133.84; 135.68;
135.89; 141.11; 149.82; 152.25; 153.28; 164.74; 169.58; 169.96. ESI-MS: 694.1 ([M+H]+).
2’,3’-Di-O-acetyl-N6-benzoyladenosine (11). Bu4NF (25.4 ml, 25.4 mmol) was slowly injected into an
ice-cooled soln. of 10 (17.60 g, 25.4 mmol) in THF (300 ml). The mixture was stirred in an ice bath under
Ar for 7 h. After the solvent was evaporated, the residue was dissolved in CH2Cl2 (800 ml), the soln.
washed with H2O (2”100 ml) and sat. aq. NaCl soln. (100 ml), dried (Na2SO4), and concentrated, and
the residue purified by FC (SiO2, 0.5–2.0% MeOH/CH2Cl2) 11 (10.95 g, 94.8%). White solid. Rf 0.23
(4.8% MeOH/CHCl3). 1H-NMR (400 MHz, CDCl3): 2.01 (s, 3 H); 2.16 (s, 3 H); 3.83–4.01 (m, 2 H );
4.37 (m, 1 H); 5.68–5.69 (m, 1 H); 5.87–5.90 (m, 1 H) 6.00–6.03 (m, 1 H); 6.12 (d, J=7.6, 1 H). 13C-
NMR (100 MHz, CDCl3): 20.55; 20.98; 62.78; 72.85; 72.93; 86.54; 88.54; 124.82; 128.15; 129.16;
133.23; 133.53; 142.74; 150.56; 151.12; 152.64; 164.75; 169.24; 169.95. ESI-MS: 454.1 ([MꢀH]ꢀ).
N4-Benzoyl-P(O)-(2-cyanoethyl)-5’-O-(4,4’-dimethoxytrityl)-2’-O-(tetrahydrofuran-2-yl)cytidylyl-
(3’ ! 5’)-N6-benzoyl-2’,3’-di-O-acetyladenosine (12b). A soln. of 7b (0.32 g, 0.348 mmol) in MeCN (5 ml)
was injected into a soln. of 11 (0.144 g, 0.316 mmol) and 1H-tetrazole (0.11 g 1.58 mmol) in MeCN (8 ml).
The mixture was stirred at r. t. under Ar for 4 h. tert-Butyl hydroperoxide (0.646 ml, 3.16 mmol) was
added to the cooled mixture (ice bath), and the mixture was stirred for another 2.5 h. The mixture
was diluted with AcOEt (300 ml), the org. phase washed with sat. aq. NaHCO3 soln. (20 ml) and sat.
aq. NaCl soln. (20 ml), dried (Na2SO4), and concentrated, and the residue purified by FC (SiO2,
0.1–4% MeOH/CH2Cl2): 12b (0.377 g, 84%). Yellow solid. Rf 0.50 (9.1% MeOH/AcOEt). 1H-NMR
(400 MHz, CDCl3): 1.67–1.87 (m, 4 H); 2.01–2.11 (m, 6 H); 2.58–2.73 (m, 2 H); 2.98 (br. s, 1 H );
3.42–3.71 (m, 4 H); 3.73–3.74 (m, 6 H); 4.07–4.67 (m, 6 H); 4.62–4.67 (m, 1 H); 5.02–5.08 (m, 1 H );
5.30–5.38 (m, 1 H); 5.67–5.69 (m, 1 H); 5.86–5.90 (m, 1 H); 6.22–6.30 (m, 2 H); 6.79–6.84 (m, 4 H );
7.04 (br. s, 1 H); 7.19–7.53 (m, 16 H); 7.85–7.87 (m, 2 H); 7.96–7.98 (m, 2 H); 8.05–8.06 (m, 1 H );
8.22–8.34 (m, 1 H); 8.70–8.71 (m, 1 H). 13C-NMR (100 MHz, CDCl3): 19.71; 19.79; 20.60; 20.77;
20.80; 23.26; 23.34; 32.49; 32.62; 55.46; 55.49; 62.55; 62.59; 62.64; 62.84; 62.89; 66.96; 67.00; 67.27;
67.32; 67.81; 67.85; 70.42; 70.58; 73.18; 73.26; 76.31; 76.36; 76.90; 81.09; 81.16; 81.27; 82.56; 82.63;
85.97; 86.27; 87.44; 87.53; 87.68; 87.71; 97.40; 103.86; 103.90; 113.61; 116.71; 116.79; 123.95; 123.96;
127.49; 127.53; 127.98; 128.28; 128.32; 128.49; 128.53; 128.96; 129.11; 130.42; 130.46; 132.93; 133.33;
133.74; 135.09; 141.83; 141.91; 144.07; 145.04; 150.21; 151.90; 152.04; 153.01; 158.96; 158.99; 162.49;
165.24; 169.62; 169.64; 169.87; 169.97. 31P-NMR (162 MHz, CDCl3): ꢀ1.77; ꢀ1.61. ESI-MS: 1328.4
([M+K]+).
Treatment of 7a (0.74 g, 0.8 mmol) and 11 (0.333 g, 0.73 mmol) as described for 12b gave 12a (0.783 g,
1
83%). Light yellow solid. Rf 0.23 (4.8% MeOH/CHCl3). H-NMR (400 MHz, CDCl3): 1.90–1.96 (m, 4
H); 2.00–2.11 (m, 6 H); 2.55–2.68 (m, 2 H); 3.45–3.77 (m, 9 H); 3.79–3.96 (m, 2 H); 4.15–4.38 (m, 6
H); 4.99–5.03 (m, 1 H); 5.48–5.54 (m, 1 H); 5.65 (m, 1 H); 5.82 (m, 1 H); 6.04–6.07 (m, 1 H );

Helvetica Chimica Acta – Vol. 90 (2007)
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6.22–6.24 (m, 1 H); 6.81–6.85 (m, 4 H); 7.00 (br. s, 1 H); 7.22–7.54 (m, 16 H); 7.85–7.87 (m, 2 H );
7.97–7.99 (m, 2 H); 8.21–8.30 (m, 3 H); 8.71–8.72 (m, 1 H). 13C-NMR (100 MHz, CDCl3): 19.66;
19.72; 20.58; 20.75; 23.42; 23.55; 32.70; 55.44; 55.47; 61.07; 61.37; 62.59; 62.64; 62.81; 62.85; 67.15;
67.78; 70.47; 70.56; 73.12; 73.20; 77.34; 77.74; 81.07; 81.14; 86.12; 87.58; 89.10; 89.22; 97.30; 103.72;
103.79; 113.55; 116.81; 116.89; 124.02; 124.08; 127.51; 128.06; 128.31; 128.66; 128.89; 128.97; 130.48;
132.89; 133.18; 133.34; 133.76; 135.09; 135.24; 141.99; 142.11; 143.90; 144.62; 150.22; 151.98; 152.86;
154.92; 158.95; 162.89; 165.43; 169.63; 169.82. 31P-NMR (162 MHz, CDCl3): ꢀ1.41; ꢀ1.25. ESI-MS:
1328.4 ([M+K]+), 1312.4 ([M+Na]+).
N4-Benzoyl-P(O)-(2-cyanoethyl)-2’-O-(tetrahydrofuran-2-yl)cytidylyl-(3’ ! 5’)-N6-benzoyl-2’,3’-di-
O-acetyladenosine (13b). A suspension of 12b (0.49 g, 0.38 mmol) and 10% Pd/C (0.10 g) in MeOH(20
ml) was stirred at r. t. under H2 for 1.5 h. The catalyst was filtered off, the solvent evaporated, and the
residue purified by FC (SiO2, 0.1–5% MeOH/CH2Cl2): 13b (0.263 g, 70.1%). Yellow solid. Rf 0.36
(9.1% MeOH/CHCl3). 1H-NMR (400 MHz, CDCl3): 1.54–1.86 (m, 4 H); 2.00–2.10 (m, 6 H );
2.72–2.76 (m, 2 H); 3.12 (br. s, 1 H); 3.56–3.79 (m, 4 H) 4.19–4.28 (m, 3 H); 4.40–4.80 (m, 4 H );
5.10–5.21 (m, 2 H); 5.66–5.70 (m, 1 H); 5.86–5.94 (m, 2 H); 6.27–6.30 (m, 1 H); 7.37–7.51 (m, 7 H );
7.82–8.36 (m, 5 H); 8.36 (m, 1 H); 8.68 (s, 1 H); 9.63 (br., 2 H). 13C-NMR (100 MHz, CDCl3): 19.73;
19.78; 19.81; 19.86; 20.61; 20.80; 23.35; 23.43; 32.40; 32.47; 61.43; 62.85; 62.92; 62.98; 66.87; 67.18;
67.66; 67.72; 70.45; 70.60; 73.21; 73.34; 76.86; 81.19; 81.27; 81.39; 84.65; 84.70; 85.95; 86.21; 90.88;
97.63; 104.34; 104.45; 117.03; 117.05; 123.95; 128.12; 128.37; 128.91; 129.00; 132.98; 133.30; 133.58;
133.61; 141.96; 142.01; 150.20; 152.03; 152.09; 152.93; 163.01; 165.65; 169.72; 169.73; 170.01, 170.03.
31P-NMR (162 MHz, CDCl3): ꢀ1.69; ꢀ1.67. ESI-MS: 1026.1 ([M+K]+), 1010.2 ([M+Na]+).
Treatment of 12a (0.3 g, 0.232 mmol) with H2 over 10% Pd/C (0.1 g) as described for 13b gave 13a
(0.15 g, 65.2%). Light yellow solid. Rf 0.45 (9.1% MeOH/CHCl3). 1H-NMR (400 MHz, CDCl3):
1.79–1.95 (m, 4 H); 2.04–2.11 (m, 6 H); 2.26 (br. s, 1 H); 2.75–2.77 (m, 2 H); 3.72–3.96 (m, 4 H );
4.24–4.67 (m, 7 H); 4.90–4.99 (m, 1 H); 5.51 (m, 1 H); 5.69–5.72 (m, 1 H); 5.85–5.89 (m, 2 H );
6.30–6.31 (m, 1 H); 7.43–7.58 (m, 7 H); 7.86 (m, 2 H); 8.01 (m, 2 H); 8.34–8.41(m, 2 H); 8.75 (m, 1
H); 9.32 (br., 2 H). 13C-NMR (100 MHz, CDCl3): 19.80; 19.88; 20.62; 20.81; 23.52; 23.57; 32.66; 60.00;
62.91; 62.96; 67.08; 67.74; 67.83; 70.39; 70.49; 73.26; 73.36; 76.94; 81.22; 81.29; 83.16; 83.22; 85.94;
86.23; 91.09; 97.25; 104.14; 116.76; 116.98; 123.82; 127.96; 128.28; 128.34; 129.05; 129.13; 133.07;
133.35; 133.63; 141.75; 150.09; 152.01; 153.09; 162.83; 165.25; 169.72; 169.99; 170.04. 31P-NMR (162
MHz, CDCl3): ꢀ1.11; ꢀ0.98. ESI-MS: 1010.2 ([M+Na]+).
N4-Benzoyl-P(O)-(2-cyanoethyl)-2’-O-(tetrahydrofuran-2-yl)-cytidylyl-(3’ ! 5’)-N6-benzoyl-2’,3’-di-
O-acetyladenosine 5’-[Bis(2-cyanoethyl) Phosphate] (14b). To the soln. of 13b (0.2 g, 0.203 mmol; dried
under vacuum (oil pump) overnight) in dry MeCN (30 ml), bis(2-cyanoethyl) diisopropylphosphorami-
dite (0.082 g, 0.304 mmol) and 1H-tetrazole (0.071 g, 1.01 mmol) were added. The mixture was stirred
at r.t. for 3 h, and tert-butyl hydroperoxide (0.2 ml, 1.01 mmol) was added. After 2 h, the solvent was
evaporated, the residue dissolved in AcOEt (200 ml), the soln. washed with sat. aq. NaHCO3 soln. (20
ml) and sat. aq. NaCl soln. (20 ml), dried (Na2SO4), and concentrated, and the residue purified by FC
(silica gel, 0.1–5% MeOH/CH2Cl2): 14b (0.204 g, 86.1%). White solid. Rf 0.44 (9.1% MeOH/CHCl3).
1H-NMR (400 MHz, CDCl3): 1.67–1.92 (m, 4 H); 2.03–2.13 (m, 6 H); 2.75–2.78 (m, 6 H); 3.65–3.73
(m, 2 H); 4.26–4.67 (m, 13 H); 5.09–5.12 (m, 1 H); 5.22–5.27 (m, 1 H); 5.68–5.73 (m, 1 H); 5.83–5.91
(m, 2 H); 6.30–6.32 (m, 1 H); 7.43–7.57 (m, 7 H); 7.78–7.89 (m, 3 H); 7.99–8.02 (m, 2 H); 8.36–8.38
(m, 1 H); 8.74–8.75 (m, 1 H); 9.43 (br., 2 H). 13C-NMR (100 MHz, CDCl3): 19.84; 19.86; 19.91; 20.61;
20.63; 20.83; 23.50; 23.56; 32.51; 32.59; 62.92; 62.97; 62.99; 66.62; 67.25; 67.86; 67.91; 70.52; 70.56;
73.18; 73.29; 74.60; 74.75; 76.45; 76.56; 81.05; 81.12; 81.24; 81.31; 85.95; 86.08; 92.40; 97.44; 105.05;
117.03; 117.14; 124.07; 124.12; 128.06; 128.33; 129.00; 129.12; 133.02; 133.42; 133.71; 141.96; 142.06;
146.60; 150.21; 152.14; 154.75; 163.04; 165.38; 169.66; 169.71, 169.98. 31P-NMR (162 MHz, CDCl3):
ꢀ1.59; ꢀ1.43; ꢀ1.41. ESI-MS: 1212.1 ([M+K]+), 1196.1 ([M+Na]+).
Treatment of 13a (0.096 g, 0.0972 mmol) with bis(2-cyanoethyl) diisopropylphosphoramidite (0.039
g, 0.144 mmol) as described for 14b gave 14a (0.1g, 87.7%). White solid. Rf 0.44 (9.1% MeOH/CHCl3).
1H-NMR (400 MHz, CDCl3): 1.77–1.99 (m, 4 H); 2.02–2.14 (m, 6 H); 2.74–2.83 (m, 6 H); 3.75–3.84 (m,
2 H); 4.27–4.60 (m, 13 H); 4.85–4.90 (m, 1 H); 5.54 (m, 1 H); 5.68–5.73 (m, 1 H); 5.80–5.88 (m, 2 H );
6.29–6.33 (m, 1 H); 7.44–7.58 (m, 7 H); 7.88–7.92 (m, 2 H); 8.00–8.05 (m, 3 H); 8.38–8.41 (m, 1 H );

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Helvetica Chimica Acta – Vol. 90 (2007)
8.72–8.76 (m, 1 H); 9.37 (br., 2 H). 13C-NMR (100 MHz, CDCl3): 19.82; 19.91; 19.98; 20.57; 20.63; 20.85;
23.52; 23.57; 32.14; 32.62; 63.03; 63.06; 63.08; 63.14; 65.74; 67.38; 67.43; 67.82; 67.88; 70.48; 70.58; 73.31;
76.47; 76.51; 80.17; 80.23; 81.22; 81.29; 85.81; 86.06; 90.81; 97.28; 103.99; 104.10; 116.99; 117.02; 117.06;
117.17; 123.94; 128.01; 128.31; 128.35; 129.03; 129.15; 133.04; 133.42; 133.71; 141.80; 142.12; 144.78;
150.16; 150.22; 152.09; 153.01; 154.93; 162.95; 165.26; 169.73; 169.75; 170.00; 170.02. 31P-NMR (162
MHz, CDCl3): ꢀ1.34; ꢀ1.31; ꢀ1.19; ꢀ0.98. ESI-MS: 1196.3 ([M+Na]+).
2’-O-(Tetrahydrofuran-2-yl)cytidylyl-(3’ ! 5’)-adenosine 5’-(Dihydrogen Phosphate) Tris(tetrabutyl-
ammonium) Salt (15a). A soln. of 14a (0.098 g, 0.0835 mmol) in conc. NH3 soln. (9 ml) and EtOH(3 ml)
in a sealed pressure flask was stirred at 558 for 16 h. The reaction was shown to be complete by 31P-NMR.
After the solvent was evaporated, the residue in H2O (60 ml) was first extracted by CHCl3 (2”15 ml) and
then lyophilized. The solid was exchanged to the tetrabutylammonium salt by ion exchange (Amberlite-
CG-50 resin (100–200 mesh), Bu4N+ form). The eluate was lyophilized overnight: 15a (0.1 g, 83.3%).
White solid. Rf 0.32 (iPrOH/NH3 soln./H2O 7:1:2). 1H-NMR (400 MHz, D2O): 0.75 (m, 36 H); 1.17
(m, 24 H); 1.44 (m, 24 H); 1.65–1.81 (m, 4 H); 2.98 (m, 24 H); 3.62–3.66 (m, 1 H); 3.77–4.00 (m, 5
H); 4.19 (m, 2 H); 4.26 (m, 1 H); 4.36 (m, 1 H); 4.46–4.52 (m, 2 H); 5.28 (m, 1 H); 5.75 (d, J=7.42, 1
H); 5.84 (d, J=4.29, 1 H); 5.90 (d, J=6.25, 1 H); 7.95 (d, J=7.43, 1 H); 8.04 (s, 1 H); 8.38 (s, 1 H).
13C-NMR (100 MHz, D2O): 12.98; 19.27; 22.56; 23.22; 31.83; 58.19; 62.39; 65.05; 67.99; 70.76; 73.01;
75.29; 77.62; 84.32; 84.40; 86.89; 87.50; 96.87; 103.78; 118.71; 139.70; 141.43; 149.11; 153.00; 155.68;
157.39; 165.87. 31P-NMR (162 MHz, D2O): 3.75; ꢀ1.03. ESI-MS: 721.3 ([MꢀH]ꢀ).
Treatment of 14b (0.1 g, 0.0852 mmol) with NH3 soln. (15 ml) and EtOH(5 ml) as described for 15a
gave 15b (0.109 g, 88.6%). White solid. Rf 0.30 (iPrOH/NH3 soln./H2O 7:1:2). 1H-NMR (400 MHz, D2O):
0.73 (m, 36 H); 1.15 (m, 24 H); 1.42 (m, 24 H); 1.55–1.72 (m, 4 H); 2.96 (m, 24 H); 3.41–3.50 (m, 2 H );
3.78 (m, 2 H); 3.94 (m, 2 H); 4.18 (m, 3 H); 4.34 (m, 1 H); 4.59 (m, 2 H); 5.03 (m, 1 H); 5.87–5.90 (m, 2
H); 5.93 (d, J=6.97, 1 H); 7.86 (d, J=7.70, 1 H); 8.03 (s, 1 H); 8.37 (s, 1 H). 13C-NMR (100 MHz, D2O):
12.98; 19.25; 22.65; 23.20; 31.65; 58.16; 63.39; 65.24; 67.58; 70.93; 73.80; 74.64; 77.85; 83.99; 84.35; 86.46;
86.77; 97.03; 104.12; 118.65; 139.84; 141.93; 149.25; 153.00; 155.68; 157.72; 165.99. 31P-NMR (162 MHz,
D2O): ꢀ0.46; ꢀ4.89. ESI-MS: 721.3 ([MꢀH]ꢀ).
2’-O-(Tetrahydrofuran-2-yl)cytidylyl-(3’ ! 5’)-3’(2’)-O-[N-(1-oxopent-4-enyl)-L-methionyl]adeno-
sine 5’-(Dihydrogen Phosphate) (20a·3 H+). To a soln. of 15 (0.05 g, 0.0296 mmol) in DMF (0.61 ml), a
stock soln. of 19a [37][38] (0.0182 g, 0.0673 mmol) in DMF (0.25 ml) was slowly added in drops in several
portions within 4 h under N2. A mixture of 50 mM NH4Ac (2 ml, pH4.5) and MeCN (1 ml) was added to
quench the reaction, and the solvent was evaporated. The residue was redissolved in 50 mM NH4Ac (pH
4.5) and MeCN and applied to reversed-phase chromatography (1–50% of MeCN/50 mM NH4Ac). After
lyophilization, the residue was applied to reversed phase chromatography (1–30% MeCN/0.87M AcOH):
20a·3 H+ (0.01 g, 39%). Rf 0.45 (BuOH/AcOH/H2O 5 :2 :3). 1H-NMR (400 MHz, D2O; 2 :1 mixture of
monoacylated diastereoisomers): 1.95 (s, 3 H); 2.06–2.54 (m, 8 H); 3.90 (m, 2 H); 4.04 (m, 2 H); 4.18 (t,
J=5.13, 1 H); 4.46 (m, 1 H); 4.39–4.49 (m, 3 H); 4.83–4.96 (m, 3 H); 5.38–5.74 (m, 3 H); 6.00–6.19 (m, 2
H); 7.92 (m, 1 H); 8.20 (s, 1 H); 8.37 (s, 1 H). 31P-NMR (162 MHz, D2O): 0.03; ꢀ0.71. ESI-MS: 864.1
([MꢀH]ꢀ).
2’-O-(Tetrahydrofuran-2-yl)cytidylyl-(3’ ! 5’)-3’(2’)-O-[N-(1-oxopent-4-enyl)-L-phenylalanyl]adeno-
sine 5’-(Dihydrogen Phosphate) (20b·3 H+). Treatment of 15 (0.050 g, 0.0296 mmol) and 19b [37][38] as
described for 20a·3 H+ gave 20b·3 H+ (0.01 g, 38.5%). White solid. Rf 0.37 (BuOH/AcOH/H2O 5 :2 :3).
1H-NMR (400 MHz, D2O, 2 :1 mixture of monoacylated diastereoisomers): 1.98–2.21 (m, 4 H); 3.05 (m,
2 H); 3.91 (m, 2 H); 3.97 (m, 2 H); 4.17–4.23 (m, 2 H); 4.69–4.85 (m, 3 H); 5.27–5.58 (m, 2 H); 5.69–5.74
(m, 1 H); 5.84–6.11 (m, 2 H); 7.00–7.26 (m, 5 H); 7.90–7.92 (m, 1 H); 8.14 (s, 1 H); 8.33 (s, 1 H). 31P-
NMR (162 MHz, D2O): 0.05; ꢀ0.76. ESI-MS: 904.1 ([M+Na]+).
2’-O-(Tetrahydrofuran-2-yl)cytidylyl-(3’ ! 5’)-3’(2’)-O-[N-(1-oxopent-4-enyl)-L-leucinyl]adenosine
5’-(Dihydrogen Phosphate) (20c·3 H+). Treatment of 15 (0.050 g, 0.0296 mmol) and 19c as described for
20a·3 H+ gave 20c·3 H+ (0.012 g, 48%). White solid. Rf 0.50 (BuOH/AcOH/H2O 5 :2 :3). 1H-NMR (400
MHz, D2O, 2 :1 mixture of monoacylated diastereoisomers): 0.75 (m, 6 H); 1.57 (m, 3 H); 2.11–2.27 (m, 4
H); 3.90 (m, 2 H); 4.04 (m, 2 H); 4.18 (t, J=5.14, 1 H); 4.23 (m, 1 H); 4.36–4.49 (m, 3 H); 5.36–5.74 (m, 3
H); 6.00–6.17 (m, 2 H); 7.91–7.94 (m, 1 H); 8.19 (s, 1 H); 8.36 (s, 1 H). 31P-NMR (162 MHz, D2O): 0.03;
ꢀ0.73; ESI-MS: 870.1 ([M+Na]+).

Helvetica Chimica Acta – Vol. 90 (2007)
309
N-[(Biotinylamino)caproyl]-L-methionine Cyanomethyl Ester (=N-{6-{{5-[(3aS,4S,6aR)-Hexahy-
dro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]-1-oxopentyl}amino}-1-oxoheyl}-L-methionine Cyanomethyl
i
Ester). To a soln. of N-[(biotinylamino)caproyl]-L-methione (0.958 g, 1.96 mmol) in DMF (20 ml), Pr2-
EtN (2.4 ml, 13.72 mmol) and chloroacetonitrile (0.24 ml, 3.92 mmol) were added at 08 under N2. The
mixture was stirred at r. t. overnight. The solvent was evaporated and the residue washed with acetone
(2”6 ml), dried under vacuum overnight, and then suspended in CH2Cl2 (15 ml) for 1 h. The solid was
filtered off washed with CH2Cl2 (2”10 ml) and Et2O (2”10 ml), and then dried in vacuo overnight:
N-[(biotinylamino)caproyl]-L-methionine cyanomethyl ester (0.75 g, 72.8%). White solid. 1H-NMR
(400 MHz, (D6)DMSO): 1.52–1.64 (m, 12 H); 1.87–2.10 (m, 9 H); 2.41–2.6 (m, 3 H); 2.81 (dd,
J=12.48, 5.14, 1 H); 2.99 (m, 2 H); 3.08 (m, 1 H); 4.11 (m, 1 H); 4.28 (m, 1 H); 4.40 (m, 1 H); 4.99 (s,
2 H); 6.37 (s, 1 H); 6.43 (s, 1 H); 7.74 (t, J=5.13, 1 H); 8.36 (d, J=7.33, 1 H). 31C-NMR (400 MHz,
(D6)DMSO): 15.18; 25.57; 26.00; 26.66; 28.71; 28.91; 29.63; 30.03; 30.72; 35.47; 35.88; 38.96; 40.53;
50.11; 51.30; 56.12; 59.85; 61.71; 116.50; 163.38; 171.84; 172.46; 173.37.
Cytidylyl-(3’ ! 5’)-3’(2’)-O-{N-[(biotinylamino)caproyl]-L-methionyl}adenosine
Phosphate)=Cytidylyl-(3’ ! 5’)-3’(2’)-O-{N-{6-{{5-[(3aS,4S,6aR)-hexahydro-2-oxo-1H-thieno[3,4-d]-
imidazol-4-yl]-1-oxopentyl}amino}-1-oxohexyl}-L-methionyl}adenosine 5’-(Dihydrogen Phosphate). To a
5’-(Dihydrogen
ACHTREUNG
soln. of 15 (0.035 g, 0.0242 mmol) in DMF (0.34 ml), N-[(biotinylamino)caproyl]-L-methionine cyano-
methyl ester was added under N2. The mixture was stirred for 2 h. A mixture of 50 mM NH4Ac (2 ml,
pH4.5) and MeCN (1 ml) was added to quench the reaction, and the solvent was evaporated. The residue
was subjected to reversed-phase chromatography (1–50% MeCN/50 mM NH4Ac). After lyophilization,
the dry residue was applied to reversed-phase chromatography (1–50% of MeCN/0.87M AcOH): 3’(2’)-
O-{N-[(biotinylamino)caproyl]-L-methionyl}-substituted pCpA (5 mg, 21.4% for two steps). White solid
after lyophilization. Rf 0.32 (BuOH/AcOH/H2O 5 :2 :3). 1H-NMR (400 MHz, D2O, 2 :1 mixture of mon-
oacylated diastereoisomers): 1.12–1.48 (m, 12 H); 1.91–2.16 (m, 9 H); 2.32–2.55 (m, 3 H); 2.72–2.77 (m,
1 H); 2.93–2.98 (m, 2 H); 3.04–3.09 (m, 1 H); 3.85–4.86 (m, 12 H); 5.35–5.53 (m, 1 H); 5.67–5.72 (m, 1
H); 5.96–6.15 (m, 2 H); 7.90–7.93 (m, 1 H); 8.19–9.21 (m, 1 H); 8.35–8.42 (m, 1 H). ESI-MS: 1121.3
([MꢀH]ꢀ).
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ReceivedOctober 2, 2006