Novel fluoride-labile nucleobase-protecting groups for the synthesis of 3'(2')-O-aminoacylated RNA sequences

Article abstract of DOI:10.1002/1522-2675(20000906)83:9<2477::AID-HLCA2477>3.0.CO;2-9

With the aim to develop a general approach to a total synthesis of aminoacylated t-RNAs and analogues, we describe the synthesis of stabilized, aminoacylated RNA fragments, which, upon ligation, could lead to aminoacylated t-RNA structures. Novel RNA phosphoramidites with fluoride-labile 2'-O-[(triisopropylsilyl)-oxy]methyl (=tom) sugar-protecting and N-{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl (=tboc) base-protecting groups were prepared (Schemes 4 and 5), as well as a solid support containing an immobilized N⁶-tboc-protected adenosine with an orthogonal (photolabile) 2'-O-[(S)-1-(2-nitrophenyl)ethoxy]methyl (=(S)-npeom) group (Scheme 6). From these building blocks, a hexameric oligoribonucleotide was prepared by automated synthesis under standard conditions (Scheme 7). After the detachment from the solid support, the resulting fully protected sequence 34 was aminoacylated with L-phenylalanine derivatives carrying photolabile N-protecting groups (→42 and 43; Scheme 9). Upon removal of the fluoride-labile sugar- and nucleobase-protecting groups, the still stabilized, partially with the photolabile group protected precursors 44 and 45, respectively, of an aminoacylated RNA sequence were obtained (Scheme 9 and Fig. 3). Photolysis of 45 under mild conditions resulted in the efficient formation of the 3'(2')-O-aminoacylated RNA sequence 46 (Fig. 4). Additionally, we carried out model investigations concerning the stability of ester bonds of aminoacylated ribonucleotide derivatives under acidic conditions (Table) and established conditions for the purification and handling of 3'(2')-O-aminoacylated RNA sequences and their stabilized precursors.

Full text of DOI:10.1002/1522-2675(20000906)83:9<2477::AID-HLCA2477>3.0.CO;2-9

Helvetica Chimica Acta Vol. 83 (2000)
2477
Novel Fluoride-Labile Nucleobase-Protecting Groups
for the Synthesis of 3'(2')-O-Aminoacylated RNA Sequences
by Alfred Stutz, Claudia Hˆbartner, and Stefan Pitsch*
Laboratorium f¸r Organische Chemie, ETH-Zentrum, Universit‰tstr. 16, CH-8092 Z¸rich
Dedicated to Prof. Albert Eschenmoser on the occasion of his 75th birthday
With the aim to develop a general approach to a total synthesis of aminoacylated t-RNAs and analogues, we
describe the synthesis of stabilized, aminoacylated RNA fragments, which, upon ligation, could lead to
aminoacylated t-RNA structures. Novel RNA phosphoramidites with fluoride-labile 2'-O-[(triisopropylsilyl)-
oxy]methyl (ˆtom) sugar-protecting and N-{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl (ˆtboc) base-
protecting groups were prepared (Schemes 4 and 5), as well as a solid support containing an immobilized N6-
tboc-protected adenosine with an orthogonal (photolabile) 2'-O-[(S)-1-(2-nitrophenyl)ethoxy]methyl (ˆ(S)-
npeom) group (Scheme 6). From these building blocks, a hexameric oligoribonucleotide was prepared by
automated synthesis under standard conditions (Scheme 7). After the detachment from the solid support, the
resulting fully protected sequence 34 was aminoacylated with l-phenylalanine derivatives carrying photolabile
N-protecting groups (!42 and 43; Scheme 9). Upon removal of the fluoride-labile sugar- and nucleobase-
protecting groups, the still stabilized, partially with the photolabile group protected precursors 44 and 45,
respectively, of an aminoacylated RNA sequence were obtained (Scheme 9 and Fig. 3). Photolysis of 45 under
mild conditions resulted in the efficient formation of the 3'(2')-O-aminoacylated RNA sequence 46 (Fig. 4).
Additionally, we carried out model investigations concerning the stability of ester bonds of aminoacylated
ribonucleotide derivatives under acidic conditions (Table) and established conditions for the purification and
handling of 3'(2')-O-aminoacylated RNA sequences and their stabilized precursors.
1. Introduction. About one year ago, we introduced a concept for a total synthesis
of aminoacylated t-RNAs based on the ligation of short, aminoacylated RNA
fragments to truncated t-RNAs. For this purpose, we had developed novel RNA
phosphoramidites with photocleavable sugar- and nucleobase-protecting groups [1].
However, we soon realized that the purification and handling of such amino acid/RNA
conjugates was very difficult due to their instability towards hydrolysis. We, therefore,
decided to modify the original concept by preparing stabilized precursors that could be
transformed into the target structures by a final photolytic step1).
It is well-known that both N-acylation and the absence of a neighboring 2'-OH
group stabilize the ester bond of aminoacylated nucleotide derivatives towards
hydrolysis [4]. Our modified concept, therefore, implies the preparation of short
1
)
So far, aminoacylated t-RNA analogues were synthesized by enzymatic ligation of truncated t-RNAs with
3'-O-aminoacylated dimers. The latter compounds were prepared by condensation of a weakly activated, N-
protected amino acid with a non-protected or partially protected ribodinucleotide [2]. The preparation of a
trimeric aminoacylated RNA sequence from a partially protected RNA precursor has been reported.
Thereby, [(9H-fluoren-9-yl)methoxy]carbonyl (ˆ Fmoc), tetrahydro-4-methoxy-2H-pyran-4-yl (ˆ Mthp),
and [1-([1,1'-biphenyl]-4-yl)-1-methylethoxy]carbonyl (ˆ Bpoc) were employed as protecting groups for
the nucleobases, the sugar moieties, and the amino acid, respectively [3].

2478
Helvetica Chimica Acta Vol. 83 (2000)
aminoacylated RNA precursors that still contain two photolabile protecting groups at
these positions, which, finally, after ligation and purification, can be removed in situ
under mild conditions (Scheme 1).
Scheme 1. Retrosynthetic Analysis: Ligation of Stabilized N- and 2'-O-Protected Aminoacylated RNA
Fragments to Truncated RNAs, Followed by the Liberation of the Labile Aminoacylated t-RNAs upon Photolysis
To achieve an efficient and unambiguous (regioselective) aminoacylation, the sugar
and nucleobase moieties of the RNA precursors require protection by groups that,
ideally, are quantitatively removable in the presence of the labile ester bond (to the
amino-acid conjugate) and the remaining two photolabile protecting groups.
For this purpose, we decided to investigate fluoride-labile groups. In this work, we
describe the evaluation and synthesis of novel RNA phosphoramidites carrying
fluoride-labile 2'-O-[(triisopropylsilyl)oxy]methyl (ˆtom [5]) and N-{{[(triisopropyl-
silyl)oxy]benzyl}oxy}carbonyl (ˆtboc) groups as sugar- and base-protecting groups,
respectively, the synthesis of an immobilized adenosine nucleoside carrying an
orthogonal photolabile 2'-O-[(S)-1-(2-nitrophenyl)ethoxy]methyl (ˆ(S)-npeom) pro-
tecting group, and the assembly of a protected RNA fragment from these building
blocks, its aminoacylation, and final deprotection. Additionally, we determined the
stability of an aminoacylated model nucleotide under acidic conditions and inves-
tigated the relative influence of N-acylation and 2'-O-protection on the stability of the
ester bond between an amino acid and a nucleotide.

Helvetica Chimica Acta Vol. 83 (2000)
2479
2. Results. 2.1. Evaluation of Protecting Groups. First, we evaluated the stability of
the ester bond of an aminoacylated model nucleotide 1 under potential deprotection
conditions required for the detachment of fluoride-labile protecting groups. In 1m
Bu4NF ¥ 3 H2O in THF at room temperature, the half-life (t1/2) of compound 1 was
only ca. 2 h (!2). Upon addition of 0.5m AcOH, however, the stability of the ester
bond was significantly higher (t1/2 ˆ 24 h), and only ca. 5% were cleaved to 2 within 3 h
(Scheme 2). Under these conditions, 2'-O-tom groups were cleaved in less than 5 min.
Scheme 2. Model Investigation to Determine the Stability of Protected Aminoacylated Nucleotide Derivatives
under the Conditions Required for the Removal of Fluoride-Labile Protecting Groups
Subsequently, we evaluated fluoride-labile nucleobase-protecting groups that could
be cleaved under such conditions. Earlier, we [5] and others [6] had found that some
nitrobenzyl-alcohol derivatives are cleaved upon treatment with FÀ-ions. Therefore, we
first investigated such compounds as potential fluoride-labile nucleobase-protecting
groups. Indeed, the [(2-nitrobenzyl)oxy]carbonyl- and [(4-nitrobenzyl)oxy]carbonyl-
protected adenosine derivatives 3 and 4, respectively, were cleaved to 8 under the
conditions mentioned above (1m Bu4NF ¥ 3 H2O/0.5m AcOH in THF, 208), but were too
stable to be considered for our purpose (t1/2 of 3 h and 4 h, resp.). The isoelectronic (2-
oxopropoxy)carbonyl-protected adenosine derivative 5 was more labile (t1/2 ˆ 1h), but,
unfortunately, still too stable. We then turned our attention towards silyl-based
protecting groups. Under our conditions, the {{2-[(triisopropylsilyl)oxy]benzyl}oxy}-
carbonyl- and {{4-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl-protected adenosine de-
rivatives 6 and 7, respectively, were both deprotected within < 2 min and were,
therefore, promising candidates (Scheme 3)2).
Both adenosine derivates 6 and 7 were completely stable (for at least 2 h) under the
oxidation conditions (I2, H2O/pyridine/THF) usually employed during the chain
assembly of oligonucleotides. However, they showed completely different behaviors
under the detritylation conditions (4% CHCl2COOH/(CH2Cl)2). Whereas the ortho-
substituted derivative 6 was completely stable for at least 30 min (ˆtotal detritylation
time for 15 couplings), the para-substituted analogue 7 was removed quantitatively
within only 2 min.
Both the stability under the conditions required for the assembly of RNA sequences
and the lability towards Bu4NF encouraged us to investigate the {{2-[(triisopropyl-
silyl)oxy]benzyl}oxy}carbonyl (ˆtboc) moiety as fluoride-labile base-protecting
group.
2
)
The analogous {{4-{[(tert-butyl)dimethylsilyl]oxy}benzyl}oxy}carbonyl protecting group was recently
employed in a different context [7].

2480
Helvetica Chimica Acta Vol. 83 (2000)
Scheme 3. Model Investigation to Determine the Lability of Various Nucleobase-Protecting Groups under
Deprotection Conditions
2.2. Synthesis of Building Blocks. During the assembly of oligonucleotides, the
nucleobase of uridine derivatives usually does not require protection, since it is not
reacting with activated phosphoramidites. Recent investigations by Hayakawa and
Kataoka have shown that unprotected guanines are also not reacting under coupling
conditions [8]. We could verify these observations by performing oligoribonucleotide
syntheses with the N2-unprotected guanosine phosphoramidite 9 (see below, Scheme 5)
and our standard 2'-O-tom-protected uridine, (N4-acetylated) cytosine, and (N6-
acetylated) adenosine phosphoramidites. Crude products from such syntheses were
absolutely identical to products obtained from the standard 2'-O-tom-protected (N2-
acetylated) guanosine phosphoramidite [5]; specifically, no indication for the
formation of branched derivatives could be found. In the context of this project, we
thus decided to omit nucleobase protection of the guanosine building block, and to
introduce the tboc protecting group only in cytidine and adenosine derivatives.
In Scheme 4, the preparation of the 2'-O-tom- and N-tboc-protected adenosine and
cytidine phosphoramidites 13 and 17 is shown. The reagent 2-[(triisopropylsilyl)oxy]-
‡
benzyl alcohol (18) was obtained by treating a DMF solution of the Na salt of 2-
hydroxybenzyl alcohol with iPr3SiCl at À 708. The N6-tboc-protected adenosine building
block was prepared under the conditions we had developed earlier for the preparation
of N-[(2-nitrobenzyl)oxy]carbonyl(ˆ nboc)-protected nucleosides [1]. The regular 5'-
O-dimethoxytritylated, N6-acetylated, 2'-O-tom-protected adenosine 10 [5] was N-

Helvetica Chimica Acta Vol. 83 (2000)
2481
Scheme 4
a) MeNH2, EtOH, r.t. b) Ac2O, DMAP, Py, r.t. c) 1. COCl2, DMAP, Py/CH2Cl2; 2. 18, Et3N, r.t.; 3. NaOH in
THF, MeOH, H2O, 48. d) ClP(NiPr2)(OCH2CH2CN), iPr2NEt, CH2Cl2, r.t. e) 19, DMAP, iPr2NEt, toluene, 808.
i
f) 1. NaH in DMF, 48; 2. Pr3SiCl, À 708. g) 4-Nitrophenyl carbonochloridate, Et3N, toluene, r.t.
deacetylated with MeNH2 in EtOH and reacetylated at the 3'-OH position with Ac2O
in pyridine. The intermediate 11 was treated first with COCl2/DMAP (N,N-
dimethylpyridin-4-amine) in pyridine/CH2Cl2 ( ! formation of a reactive N6-carba-
moyl derivative [1]), and then with 18 in the presence of Et3N. After extraction, the 3'-
O-acetyl group was removed with NaOMe, and the resulting N6-tboc-protected
adenosine 12 was isolated by chromatography in 63% yield. The N4-tboc-protected
cytosine could not be prepared under the conditions that were successfully employed in

2482
Helvetica Chimica Acta Vol. 83 (2000)
the synthesis of the corresponding N4-nboc derivative [1]3). However, N-deacetylation
of the regular 5'-O-dimethoxytritylated, N4-acetylated, 2'-O-tom-protected cytosine 14
[5], followed by treatment of the resulting crude nucleoside 15 with the nitrophenyl
carbonate 19 at 808 in toluene gave the N4-tboc-protected cytidine 16 in 60% yield.
The two N-tboc-protected nucleosides 12 and 16 were finally transformed under
standard conditions into the corresponding phosphoramidites 13 and 17, respectively.
The guanosine phosphoramidite 9 was prepared from the regular 5'-O-dimethoxy-
tritylated, N2-acetylated, 2'-O-tom-protected guanosine 20 [5] by removing the N2-
acetyl group with MeNH2 in EtOH and subsequent phosphitylation of the intermediate
21 under standard conditions (Scheme 5). The phosphoramidite 9 was only poorly
soluble in MeCN, and was, therefore, used as a solution in THF. For the incorporation
of uridines, we could use the regular 2'-O-tom-protected phosphoramidite 22 [5]. For
the introduction of the terminating nucleoside, we prepared the 5'-O-tBuMe2Si
(ˆtbdms)-substituted uridine phosphoramidite 25 (Scheme 5). Uridine was silylated
with tbdms-Cl/1H-imidazole in DMF. Under our established reaction conditions
Scheme 5
a) MeNH2 in EtOH, r.t. b) ClP(NiPr2)(OCH2CH2CN), iPr2NEt in CH2Cl2, r.t. c) tbdms-Cl, 1H-imidazole, DMF,
r.t. d) 1 . BuSnCl2, iPr2NEt, (CH2Cl)2, r.t., 2. tom-Cl [5], 808
2
3
)
Reaction with 2-nitrobenzyl carbonochloridate in a biphasic mixture of aqueous Na2CO3 solution/
dichloroethane at 258. The analogous tboc-Cl was extremely sensitive and decomposed rapidly under such
conditions.

Helvetica Chimica Acta Vol. 83 (2000)
2483
[5][9], the resulting nucleoside 23 was activated with Bu2SnCl2/iPr2NEt and sub-
sequently alkylated with tom-Cl [5]. The 2'-O-tom derivative 24 was obtained in 45%
yield after chromatography (together with 30% of the corresponding regioisomeric 3'-
O-tom derivative). Phosphitylation of 24 gave the corresponding phosphoramidite 25.
Additionally, we prepared a solid support with an immobilized adenosine, protected
with a N6-tboc group and a photolabile 2'-O-[(S)-npeom] moiety. The latter was chosen
because we are using it in combination with the 2'-O-tom group successfully as an
orthogonal 2'-O-protection for ribonucleosides [5]. After activation of the 5'-O-
dimethoxytritylated, N6-benzoylated adenosine nucleoside 26 with Bu2SnCl2/iPr2NEt
and alkylation with (RS)-npeom-Cl, the two pairs of regioisomeric 2'-O- and 3'-O-
substituted derivatives (RS)-27 and (RS)-28 could be isolated (each as a diaster-
eoisomer mixture) by chromatography (Scheme 6). The 2'-O-npeom derivatives (RS)-
27 were N-debenzoylated with MeNH2 in EtOH and reacetylated at the 3'-OH position
with Ac2O in pyridine. At this stage, the two diastereoisomers could be easily separated
by column chromatography. The product (S)-29 ((S)-configuration of the npeom
group) was transformed into the corresponding N6-tboc-derivative by the conditions
Scheme 6
a) 1 . BuSnCl2, iPr2NEt, (CH2Cl)2, r.t.; 2. (RS)-npeom-Cl [5], 808. b) 1. MeNH2, EtOH, r.t.; 2. Ac2O, DMAP, Py,
2
r.t.; c) 1. COCl2, DMAP, Py/CH2Cl2; 2. 18, Et3N, r.t.; 3. NaOH, THF, MeOH, H2O, 48. d) [1,4-phenyl-
enebis(oxy)]bis[acetic acid], BOP ((1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
phosphate), iPr2NEt, DMAP, Py, r.t. e) 1. LCAA-CPG, iPr2NEt, BOP, MeCN, r.t.; 2. Ac2O/Py.

2484
Helvetica Chimica Acta Vol. 83 (2000)
described for 11 ! 12 (1. COCl2, DMAP, pyridine/CH2Cl2; 2. 18, Et3N; 3. NaOMe,
MeOH). The N6-tboc-protected adenosine 30 was isolated by chromatography in
58% yield. The 3'-O-position of nucleoside 30 was then esterified with [1,4-phenyl-
enebis(oxy)]bis[acetic acid] under conditions developed by Pon and Yu [10].
After chromatography, acid 31 was obtained in 49% yield. Its subsequent immobiliza-
tion on LCAA-CPG ((long-chain-alkyl)amino controlled-pore glass) under
peptide-coupling conditions led to the corresponding solid support 32 loaded at 20
45 mmol/g.
All compounds were characterized by their NMR and mass spectra, and all data
1
unambiguously agreed with the proposed structures. H-NMR Spectra of N-tboc-
protected nucleosides showed the typical signals of an additional 2-substituted benzyl
group (arom. H and benzylic CH2 at d ca. 5.3). The 1H- and the 13C-NMR signals of the
nucleobase moieties underwent no significant changes upon exchange of the base-
protecting groups (ac ! tboc), which illustrated the correct position of the tboc-groups.
2.3. Assembly of an Aminoacylated RNA Fragment. Under our standard conditions
[5], the phosphoramidites 9, 13, 17, 25, and the solid support 32 were assembled to a
hexameric sequence 33 (Scheme 7). According to the spectrophotometrically deter-
mined amounts of the released dimethoxytrityl cation, the individual coupling yields
were > 98%. The assembled sequence was subsequently detached from the solid support.
Due to the lability of the [1,4-phenylenbis(oxy)]bis[acetic acid] linker, very mild conditions
could be employed. The release of the sequence 34 was followed spectrophotometri-
cally by analyzing the supernatants. With 6m NH3 in H2O/EtOH, the sequence was
completely detached after 5 min, and with 10% Et3N or Et2NH in MeOH after 8 h.
After evaporation, a small portion of the crude, still partially protected sequence
344) was treated for 2 h with an excess of 1m Bu4NF ¥ 3 H2O/0.5m AcOH in THF. After
addition of an equal volume of Tris ¥ HCl buffer (pH 7.4), the mixture was desalted on
Sephadex G-10 and analyzed by reversed-phase HPLC (Fig. 1,a) which revealed that
the sequence 35 was formed as the predominating product; the HPLC trace reflects a
clean overall process, including good coupling yields, absence of side reactions, and
complete removal of all fluoride-labile protecting groups. The crude product 35 was
then photolyzed for 15 min with Pyrex-filtered light from a mercury lamp in 20 mm aq.
AcOH (pH 3.1), leading in a clean, quantitative reaction to the more polar RNA
sequence 36, as shown by HPLC (Fig. 1,b). This product was identical (MALDI-TOF
MS, HPLC5)) to the authentic RNA sequence 36, obtained with our standard 2'-O-
tom-protected phosphoramidites [5].
These results demonstrated the compatibility of our novel base-protecting groups
with an automated RNA synthesis under standard conditions. We subsequently
investigated the preparation of aminoacylated derivatives. First, we established
reaction conditions with the model nucleotide 2. The required N-nboc-protected l-
phenylalanine 39 was prepared from l-phenylalanine and 2-nitrobenzyl carbonochlor-
idate [1] and the corresponding N-[(RS)-1-(2-nitrophenyl)ethoxy]carbonyl (ˆ(RS)-
npeoc)-protected l-phenylalanine 41 from l-phenylalanine and imidazolide 40
4
)
)
Unfortunately, sequence 34 was too apolar to be analyzed by reversed-phase HPLC.
5
The co-injections were carried out on a reversed-phase column (0 ! 20% B (30 min) and on an ion-
exchange column (Nucleogel SAX, 1 0 mm phosphate (pH 6) ! 1 0 mm phosphate ‡ 0.2m NaCl (30 min)).

Helvetica Chimica Acta Vol. 83 (2000)
2485
Scheme 7
a) Automated 1.5-mmol synthesis. b) Et3N, MeOH, r.t. c) 1m Bu4NF ¥ 3 H2O, 0.5m AcOH, THF, r.t. d) Photolysis,
20 mm aq. AcOH (pH 3.1), r.t.
Fig. 1. Reversed-phase HPLC traces (measured at 260 nm) a) of the crude 2'-O-[(S)-npeom]-protected RNA
sequence 35 and b) of the crude RNA sequence 36 obtained from 35 by photolysis (see Scheme 7)
(Scheme 8). We found that activating these two l-phenylalanine derivatives 39 and 41,
respectively, with (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexa-
fluorophosphate(BOP)/iPr2NEt and performing the acylation reaction at room
temperature in pyridine and in the presence of 0.5 equiv. of 4-(pyrrolidin-1-yl)pyridine
led to the quantitative formation of the corresponding aminoacylated products 1 and 37

2486
Helvetica Chimica Acta Vol. 83 (2000)
Scheme 8
a) l-(nboc)NHCH(Bn)COOH (39; ! 1) or l-[(RS)-npeoc]NHCH(Bn)COOH (41; ! 37), BOP, iPr2NEt, 4-
t
(pyrrolidin-1-yl)pyridine, Py, r.t. b) Photolysis, BuOH, H2O, ammonium citrate (pH 3.6), r.t. c) l-Phenyl-
alanine, NaOH, H2O, r.t. d) 1 ,1'-Carbonylbis[1H-imidazole], CH2Cl2, r.t.; e) l-Phenylalanine, 1H-tetrazole,
DMF, 808.
within 1 2 h ( Scheme 8, Fig. 2,a). We observed that nucleotide 2 (protected with a 2'-
O-[(S)-npeom] group) reacted faster than the corresponding diastereoisomer (pro-
tected with a 2'-O-[(R)-npeom] group)6). Under these optimized conditions,
racemization of the N-nboc-protected l-phenylalanine was observed only to a very
small extent7). The photolysis of nucleotide 37 at pH 3.6 proceeded efficiently,
resulting in a quantitative formation of the deprotected aminoacylated nucleotide 38
without concomitant hydrolysis of the ester linkage (Fig. 2,b)8).
The partially protected, crude RNA sequence 34 (see Scheme 7) was then
aminoacylated under the same conditions, employing an excess of N-[(RS)-npeoc]-
or N-nboc-protected l-phenylalanine 39 or 41, respectively, and BOP/iPr2NEt/4-
(pyrrolidin-1-yl)pyridine in pyridine (Scheme 9). It was very important to dry the crude
sequence 34 by repeated co-evaporation from dry pyridine or DMF. After 4 h at 208,
6
)
)
Additionally, we found that the 3'-O-[(R)-npeom]-protected analogue reacted faster than nucleotide 2.
Chromatographically, the aminoacylated analogue 53 (see below, Scheme 10), containing a d-phenyl-
alanine, could be distinguished from 2. Its position on the HPLC trace is indicated in Fig. 2,a.
According to 31P-NMR analysis, two products in a 3 :1ratio were formed during the photolytic
deprotection. This is typical for phenylalanine-substituted adenosines, which adopt 3:1equilibrium
mixtures of the 3'-O- and 2'-O-substituted derivatives, respectively [11].
7
8
)

Helvetica Chimica Acta Vol. 83 (2000)
2487
Fig. 2. Reversed-phase HPLC traces (measured at 260 nm) a) of the crude N-(nboc)- and 2'-O-[(S)-npeom]-
protected aminoacylated nucleotide 1 and b) of the crude aminoacylated nucleotide 38 obtained from 1 by
photolysis (see Scheme 8). The position of the corresponding protected d-phenylalanine derivative 53 (see
Scheme 10) is indicated by the arrow.
Scheme 9
a) l-(nboc)NHCH(Bn)COOH (39; ! 42) or l-[(RS)-npeoc]NHCH(Bn)COOH (41; ! 43), BOP, iPr2NEt,
4-(pyrrolidin-1-yl)pyridine, Py, r.t.; b) 1m Bu4NF ¥ 3 H2O, 0.5m AcOH, THF, r.t. c) Photolysis, 20 mm aq. AcOH
(pH 3.1), r.t. d) 50 mm Phosphate buffer (pH 11.5), r.t.
the reaction mixtures (!42 and 43, resp.) were treated with H2O and evaporated, and
the residues were treated with 1m Bu4NF ¥ 3 H2O/0.5m AcOH in THF. An equal volume
of 1m Tris ¥ HCl buffer (pH 7.4) was added after 2 h, and the mixtures were desalted on

2488
Helvetica Chimica Acta Vol. 83 (2000)
Fig. 3. Reversed-phase HPLC traces (measured at 260 nm) of crude products obtained by aminoacylation of the
partially protected RNA sequence 34 a) with N-[(RS)-npeoc]-protected l-phenylalanine (!45) and b) with N-
nboc-protected l-phenylalanine (!44). The remaining not-aminoacylated sequence 36 is indicated (see
Scheme 9).
Sephadex G-10. The obtained aminoacylated crude products 44 and 45 were analyzed
by reversed-phase HPLC (Fig. 3).
The HPLC trace of the reaction mixture obtained with the N-nboc-protected l-
phenylalanine 39 reveals that the aminoacylation occurred to an extent of ca. 80%,
according to the integral ratio between the peak of the product 44 and the peak of the
non-acylated product 36 (see Fig. 3,b). A similar result was obtained with the N-[(RS)-
npeoc]-protected l-phenylalanine 41; however, in this case, two diastereoisomeric
sequences 45 were formed, causing a splitting of the product peak (Fig. 3,a).
The aminoacylated sequence 45 was isolated by prep. reversed-phase HPLC
(Fig. 4) and subjected to photolysis (Scheme 9), which yielded a more polar product 46
after 30 min (HPLC analysis). When this latter product was incubated at pH 11.5
(phosphate buffer), another more polar compound, identical to the authentic RNA
sequence 36 was formed within minutes (Fig. 4).
When compound 45 was subjected to a short photolysis (10 min), we were able to
detect by MALDI-TOF mass spectrometry not only the dominating signal of the
completely deprotected aminoacylated sequence 46, but additionally the signals of the
intermediates, each having lost one of the two photolabile protecting groups (Fig. 5).
According to this measurement, the 2'-O-npeom and the N-npeoc group of the partially
protected, aminoacylated sequence 45 were cleaved at a comparable rate.
2.4. Stability of Aminoacylated Nucleotides under Acidic Conditions. The ester bond
of aminoacylated ribonucleotide derivatives is an activated, energy-rich bond with a
free energy of hydrolysis comparable to the one of ATP hydrolysis [12]. Fast and
reversible migration of the amino acid occurs between the vicinal 2'-O- and 3'-O-
substituents of the ribofuranose moiety, resulting in an equilibrium mixture of 2'-O-
and 3'-O-aminoacylated species. Both processes, the hydrolysis under neutral and basic
conditions, and the transacylation process have been studied intensively9). Particularly,
9
)
This subject is thoroughly reviewed [4].

Helvetica Chimica Acta Vol. 83 (2000)
2489
Fig. 4. Reversed-phase HPLC traces (measured at 260 nm) of the purified, partially protected aminoacylated
RNA sequence 45 (top left) of the crude product 46 obtained on photolysis of 45 (top center), and of the RNA
sequence 36 obtained on hydrolysis of 46 under weakly alkaline conditions (top right) (see Scheme 9)
Fig. 5. MALDI-TOF Mass spectrum of a product mixture obtained by short photolysis of the aminoacylated
RNA sequence 45. Besides the small signal of the precursor 45 and the signal of the completely deprotected
product 46, small signals of two intermediates at m/z 2198 and 2184 (loss of the N-npeoc and the 2'-O-npeom
‡
group, resp.) are present. The signals marked by an asterisk are due to Na adducts (‡23 amu).

2490
Helvetica Chimica Acta Vol. 83 (2000)
it was found that the t-RNA structure of aminoacylated t-RNAs has no influence on the
reactivity of the ester bond, since l-valyl-t-RNA and l-valyl-AMP are hydrolyzed at a
comparable rate [13]. The nucleobase of the aminoacylated nucleotide has only a very
minor influence on the rate of hydrolysis, whereas the structure of the amino-acid
residue affects it significantly [14][15]. Absence of a (negatively charged) 5'-phosphate
group at the sugar moiety results in a higher rate of hydrolysis [13]. In their protonated
forms, aminoacylated nucleotides are hydrolyzed much faster than in their unproto-
nated forms. From pH 6 to 7.5, where the amino groups are protonated, a linear
correlation between the OHÀ concentration and the rate of hydrolysis was observed;
furthermore, the slope equal to 1indicates specific base catalysis. Consequently, esters
formed between nucleotides and amino-acid derivatives with a masked NH2 group are
significantly more stable [16]. Moreover, a similar stabilization of the ester bond is
observed upon removal of the neighboring 2'(3')-OH group [17].
In the context of this project, we wanted to explore conditions that would allow
preparation, purification, and handling of different aminoacylated oligonucleotides
without concomitant hydrolysis of the ester bond. It was known, though only in a
qualitative way, that acidic conditions stabilize such compounds compared to neutral or
alkaline conditions. Therefore, we performed a detailed study of the stability of
aminoacylated model nucleotides under potential preparative conditions.
The synthesis of the model nucleotides started from 29 (see Scheme 6), which was
detritylated under acidic conditions. The resulting alcohol 47 was transformed into the
phosphotriester 48 by treatment with (MeO)P(NiPr2)(OCH2CH2CN)/5-(benzylthio)-
1H-tetrazole, followed by oxidation with I2 in H2O/pyridine (Scheme 10). Removal of
the 3'-O-acetyl and the cyanoethyl group with MeNH2 in EtOH gave the nucleotide 2
which served as precursor of the aminoacylated nucleotides 1, 37, 52, and 53.
Analogously, we prepared from adenosine the nucleoside 49, which was phosphory-
lated (! 50), and subsequently transformed into the 2'-O-methylated nucleotide 51.
This nucleotide served as precursor for the preparation of the corresponding
aminoacylated derivative 56 (Scheme 10).
The aminoacylated nucleotides 38, 54, 55, and 57 were obtained by photolysis of the
t
corresponding protected precursors in BuOH/H2O 1:1 at pH 3.6 (40 m m ammonium
citrate) (Scheme 10). After extraction, the crude products were desalted on a plug of
reversed-phase silica gel (H2O/MeCN 4 :1( ‡20 mm AcOH)), diluted with an equal
volume of the appropriate buffer solution, and incubated at 258. Periodically, aliquots
were removed and analyzed by reversed-phase HPLC (detection at 260 nm). From the
time-dependant ratios between aminoacylated starting materials and hydrolyzed
products, individual pseudo-first-order rate constants were obtained (Table).
The pH dependence of the hydrolysis rate of the l-phenylalanyl-nucleotide 38 is
shown in Fig. 6. As expected, the stability increased dramatically at lower pH values, up
to a half-life of 250 h at pH 2.510). At a little higher value, at pH 3.6 where photolyses
usually are carried out, the half-life of the ester bond was still ca. 150 h (0.1m formate),
which translates into < 5% hydrolysis within 5 h (see Table). Concerning the influence
of the buffer concentration on the stability, both at a pH value of 7.4 and of 3.6, the rate
of hydrolysis increased with increasing buffer concentration, resulting, e.g., in
10
)
Lower pH values were not considered, because of the limited stability of RNA sequences.

Helvetica Chimica Acta Vol. 83 (2000)
2491
Scheme 10
a) CHCl2COOH, MeOH, CH2Cl2, 48. b) 1. (MeO)P(NiPr2)(OCH2CH2CN), 5-(benzylthio)-1H-tetrazole,
MeCN, r.t.; 2. I2, H2O, Py, THF, r.t. c) MeNH2, EtOH, r.t. d) 1. NaH, MeI, DMF, 48; 2. tbdms-Cl, 1H-imidazole,
r.t.; 3. BzCl, Py, r.t.; 4. HCl, H2O, THF, r.t. e) N-[(benzyloxy)carbonyl]-l-phenylalanine (! 52) or ent-39 (! 53)
or 39 (! 56), iPr2NEt, 4-(pyrrolidin-1-yl)pyridine, Py, r.t. f) Photolysis, tBuOH, H2O, ammonium citrate
(pH 3.6), r.t.
approximately a third of the half-life upon raising the formate concentration from 0.1to
1m at pH 3.6 (see Table).
We were not able to detect a difference between the hydrolysis rates of the l-
phenylalanine and the d-phenylalanine derivatives 38 and 55, respectively, at pH 7.4.

2492
Helvetica Chimica Acta Vol. 83 (2000)
Table. Hydrolysis of Aminoacylated Nucleotides 38, 54, 55, 57, and 1 a)
Amino-acid
conjugate
Buffer
pH
k
t1/2 [h] Hydrolysis Time for
[10À5
minÀ1
after 5 h
[%]
10% hydrolysis
[h]
c
b
]
38 l-Phe
2.5
3.6
4.7
5.5
6.5
7.4
0.1m citric acid, Na2HPO4
0.1m HCOOH, NaOH
0.1m AcOH, NaOH
0.1m AcOH, NaOH
0.1m NaH2PO4, NaOH
0.1m Tris ¥ HCl
4.7
7.6
24.7
50.7
240
1430
4.9
246
152
47
1.4
2.3
7.17.0
14
48
98
37
23
23
3.5
4.8
0.8
238
0.75 ˆ 45 min
0.12 ˆ 7 min
3.10.02
m
AcOH
1.4
36
3.6
3.6
3.6
7.4
7.4
7.4
0.05 m HCOOH, NaOH
0.5 m HCOOH, NaOH
1.0m HCOOH, NaOH
0.5 m Tris ¥ HCl
0.1m Tris ¥ HCl
0.1m Tris ¥ HCl
7.6
17.7
21.3
6270
1420
83.0
151
65
54
0.2
0.8
14
2.3
5.2
6.2
23
10
8.2
> 99
98
22
0.03
55 d-Phe
54 Z-l-Phe
0.12 ˆ 7 min
2.0
57 l-Phe (2'-OMe) 3.10.02
m
AcOH
2.3
3.6
191
505
325
6
< 175
1.0
43
5.0
7.4
0.1m AcOH, NaOH
0.1m Tris ¥ HCl
50
0.9
8.5
1 nboc-l-Phe
(2'-O-npeom)
7.4
0.1m Tris ¥ HCl
20.6
56
6.0
8.2
0.05 m Tris ¥ B(OH)3)
23.6
49
6.8
7.5
a) All experiments were carried out at 258. b) The individual k values were obtained from the ratios of starting
materials and products, determined by time-dependent HPLC analyses (> 5 values). The amount of hydrolysis
product was plotted against the time, and the analysis was carried out assuming pseudo-first-order kinetics.
Estimated error: Æ 5%.
Fig. 6. Rate constants k and half-lives t1/2 for the hydrolysis of the aminoacylated nucleotide 38 at room
temperature as a function of pH. Buffer concentration: 0.1m; estimated error: Æ 5% (Table).

Helvetica Chimica Acta Vol. 83 (2000)
2493
Our retrosynthetic scheme includes ligation of N- and 2'-O-protected amino-
acylated RNA-fragments to a truncated t-RNA (Scheme 1), followed by purification
and deprotection. Usually, the purification of relatively long oligonucleotides is carried
out by polyacrylamide-gel electrophoresis (PAGE) at pH 8.2 (50 mm Tris ¥ borate
buffer). Under these standard conditions, the half-life of hydrolysis of the protected
model compound 1 was ca. 50 h, corresponding to ca. 5% cleavage within 3 h (the time
usually required for a gel purification) (see Table).
As expected, the N- and 2'-O-protected compound 1 was hydrolyzed much more
slowly than the completely deprotected derivative 38. At pH 7.4, the difference
amounted to a factor of 70 (t1/2 (1) 56 h, t1/2 (38) 48 min). To investigate the relative
influence of the two separate protecting groups, we additionally prepared the
aminoacylated analogues 57 and 54, containing 2'-O-Me and N-(benzyloxy)carbonyl
(ˆ Z) groups, respectively, and determined their rates of hydrolysis at pH 7.4. It turned
out that N-protection of the amino acid resulted in a 17, and O-protection of the sugar
residue in a 7 times slower hydrolysis, respectively, compared to the unprotected 38.
In general, our results concerning the stability of various aminoacylated compounds
under acidic and neutral conditions are in good agreement with previously reported
data obtained in the neutral and alkaline pH range. The 17-fold increase in stability of
the N-Z-protected compound 54, compared to the unprotected analogue 38, at pH 7.4
and 258, is similar to the 5-fold increase found for N-acetylphenylalanyl-tRNA at
pH 8.8 and 378 [15][18]. Blocking the 2'-O group by methylation (compound 57)
resulted in a 7-fold increase in stability at pH 7.4 and 258, whereas, with a similar
aminoacylated 2'-deoxynucleotide, a 3-fold increase was found at pH 9 and 378 [17].
The known stabilization of the aminoacyl ester bond by N-acylation and 2'-O-
protection seems to be even more pronounced at lower pH values.
3. Discussion. We are interested in a general, nonenzymatic synthesis of 3'-O-
aminoacylated t-RNA analogues for structural studies related to the process of
transcription and for the ribosome-mediated incorporation of unnatural amino acids
into proteins [2]. Our retrosynthetic scheme differs from the known concepts by
excluding all enzymatic steps. Currently, artificial aminoacylated t-RNAs are obtained
by enzymatic ligation of a truncated t-RNA (produced by T7-polymerase-mediated
transcription of an appropriate gene construct) with an aminoacylated r(CA) dimer
[2]. This approach results in t-RNA analogues, containing only the four canonical
nucleosides. A total synthesis, in contrast, would allow the incorporation of any desired
modification at any site and, therefore, enable a multitude of new investigations and
applications related to the biosynthesis of proteins.
A severe limitation in the chemical synthesis of oligonucleotide analogues is the
requirement to remove the commonly used acyl-type nucleobase-protecting groups
with strong nucleophiles such as NH3 or MeNH2. A variety of modified nucleosides are
not stable under the conditions required for deprotection of the standard base-
protecting groups. Base-protecting groups, which are removable under milder
conditions, such as phenoxyacetyl [19] or (allyloxy)carbonyl [20] have been developed
and extended the range of tolerated modifications significantly. In the same context, we
have recently introduced the photolabile base-protecting nboc group [1].

2494
Helvetica Chimica Acta Vol. 83 (2000)
We now have developed novel fluoride-labile nucleobase-protecting groups that
can be cleaved under very mild conditions. Their combination with photolabile N- and
2'-O-protecting groups allowed a straightforward preparation and purification of
stabilized precursors of aminoacylated RNA sequences. Finally, these can be cleanly
deprotected in situ by photolysis.
Currently, we are about to develop methods for the template-directed ligation of
oligoribonucleotides, which would enable us to prepare any desired aminoacylated t-
RNA by joining truncated analogues with stabilized, aminoacylated RNA fragments.
This work was supported by the ETH Z¸rich. We thank Patrick A. Weiss (Xeragon AG, Z¸rich) for
providing us with reagents and an authentic RNA sequence, and Thomas Honegger for experimental
contributions.
Experimental Part
General. Reagents and solvents (Py ˆ pyridine) from Fluka, tom-Cl, nucleosides 10, 14, 20, 26, 5-
i
(benzylthio)-1H-tetrazole, −LCAA×-CPG, Pr3SiCl, and the authentic RNA sequence 36 from Xeragon AG.
Photolysis: 250-W Hg lamp, 1-cm Pyrex filter, r.t. Workup implies distribution of the reaction mixture between
CH2Cl2 and sat. aq. NaHCO3 soln., drying of the org. layer (MgSO4), and evaporation. Column chromatography
(CC): silica gel from Fluka. TLC: precoated silica-gel plates from Merck stained by dipping into a soln. of
anisaldehyde (Aldrich; 10 ml), conc. H2SO4 (10 ml), and AcOH (2 ml) in EtOH (180 ml) and subsequent
heating with a heat gun. Reversed-phase HPLC: Aquapore RP 300, 4.6 Â 220 mm (Brownlee Labs), flow 1ml/
min; eluent A: 0.1m (Et3NH)OAc in H2O (pH 7); eluent B: MeCN; detection at 260 nm, elution at 408. Ion-
exchange HPLC: Mono Q HR5/5 (Pharmacia), flow 1ml/min; eluent A: 1 0 mm sodium phosphate in H2O
(pH 11.5); eluent B: 1 0 mm sodium phosphate/1m NaCl in H2O (pH 11.5); detection at 260 nm, elution at r.t.
UV Spectra: lmax/lmin (e) in nm. NMR: chemical shift d in ppm and coupling constants J in Hz. MS: FAB, 2-
nitrobenzyl alcohol as matrix, pos. mode; ESI, neg. or pos. mode; MALDI, 3,5-dihydroxybenzoic acid as matrix,
pos. mode; MALDI-TOF, 2,4,6-trihydroxyacetophenone (diammonium citrate) as matrix, pos. mode; m/z (rel.
int. in%).
2-[(Triisopropylsilyl)oxy]benzyl Alcohol (18). At 48, NaH (55%; 4.15 g, 0.095 mol) was added to a soln. of
2-hydroxybenzyl alcohol (12.4 g, 0.1 mol) in DMF (100 ml). After 15 min, the mixture was cooled to À 708 (dry
i
ice/acetone), and Pr3SiCl (14.25 g, 0.095 mmol) was added during 30 min. Workup (AcOEt/hexane 1:1, aq.
NaHCO3 soln.) and CC (hexane/CH2Cl2 9 :1 ! 7:3, followed by hexane/AcOEt 4 :1) gave 16.2 g (64%) of 18.
Colorless viscous liquid. TLC (hexane/AcOEt 9 :1): Rf 0.35. UV (MeOH): 276 (1300), 273 (1200), 270 (1300),
240 (100), 223 (4600). 1H-NMR (300 MHz, CDCl3): 1.02 1.19 (m, 3 Me2CH); 1.24 1.42 (m, 3 Me2CH); 2.09 (t,
J ˆ 6.2, OH); 4.73 (d, J ˆ 6.2, ArCH2); 6.83 (dd, J ˆ 0.8, 7.5, 1arom. H); 6.94 ( dt, J ˆ 0.8, 7.5, 1arom. H); 7.17 ( dt,
J ˆ 1.7, 7.5, 1 arom. H); 7.31 (dd, J ˆ 1.7, 7.5, 1 arom. H). 13C-NMR (75 MHz, CDCl3): 13.0 (d, Me2CH); 18.1 (q,
i
Me2CH); 62.3 (t, ArCH2); 118.0, 121.0, 128.6, 128.8 (4d, arom. C); 131.0 (s, arom. C); 153.9 (s, Pr3SiOC).
‡
MALDI-MS: 303 (100, [M ‡ Na] ), 219 (99), 199 (82), 187 (98), 137 (60).
3'-O-Acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}adenosine (11). A soln. of 10
[5] (2.0 g, 2.33 mol) in 10m MeNH2/EtOH (20 ml) was kept for 30 min at r.t. After evaporation, the residue was
dissolved in Py (9.3 ml) and treated with DMAP (28 mg, 0.23 mmol) and Ac2O (286 mg, 2.8 mmol). Workup (1.
10% citric acid, 2. NaHCO3 soln.) after 30 min at r.t. and CC (AcOEt/hexane 3 :2 ! 9 :1 (‡2% Et3N)) gave 11
(1.76 g, 88%). Colorless foam. TLC (hexane/AcOEt 1 :9): Rf 0.57. UV (MeOH): 258 (sh, 15900), 235 (23400),
1
i
224 (19200). H-NMR (300 MHz, CDCl3): 0.88 (m, Pr3Si); 2.14 (s, MeCO); 3.43 (dd, J ˆ 6.5, 10.4, HÀC(5'));
3.52 (dd, J ˆ 5.6, 10.4, H'ÀC(5')); 3.78 (s, 2 MeO); 4.34 (m, HÀC(4')); 4.87 (s, OCH2O); 5.19 (dd, J ˆ 5.3, 6.9,
HÀC(2')); 5.51( dd, J ˆ 2.5, 5.3, HÀC(3')); 5.80 (br. s, NH2); 6.16 (d, J ˆ 6.9, HÀC(1')); 6.78 6.83 (m, 4 arom.
H); 7.20 7.45 (m, 9 arom. H); 7.97 (s, HÀC(2)); 8.25 (s, HÀC(8)). 13C-NMR (75 MHz, CDCl3): 11.7 (d,
Me2CH); 17.6 (q, Me2CH); 20.9 (q, MeCO); 55.2 (q, MeO); 63.4 (t, C(5')); 72.1, 77.0, 82.5 (3d, C(2'), C(3'),
C(4')); 86.2 (d, C(1')); 86.8 (s, Ar2C(Ph)); 89.6 (t, OCH2O); 113.2 (d, arom. C); 120.1 (s, C(5)); 127.0, 127.9,
128.2, 130.1 (4d, arom. C); 135.6 (s, arom. C); 139.2 (d, C(8)); 144.4 (s, arom. C); 150.2 (s, C(4)); 153.2 (d, C(2));
‡
155.4 (s, C(6)); 158.6 (s, MeOÀC); 170.1 (s, CO). MALDI-MS: 820 (20, [M ‡ Na] ), 516 (31), 456 (16), 303
(100).
5'-O-(4,4'-Dimethoxytrityl)-N6-{{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-2'-O-{[(triisopropylsilyl)-
oxy]methyl}adenosine (12). A soln. of 11 (956 mg, 1.2 mmol) in CH2Cl2 (3.6 ml) was added during 20 min to a

Helvetica Chimica Acta Vol. 83 (2000)
2495
suspension obtained from 1.9m COCl2 in toluene (0.95 ml, 1.8 mmol), DMAP (15 mg, 0.12 mmol) in Py (2.4 ml)
and CH2Cl2 (4.8 ml). After 15 min at r.t., 18 (1.01 g, 3.6 mmol) and Et3N (490 mg, 4.8 mmol) were added.
Workup (1. 10% citric acid, 2. NaHCO3 soln.) after 24 h at r.t. gave a residue, which was dissolved in 0.2m NaOH
in THF/MeOH/H2O 5 :4 :1(20 ml). Workup and CC (AcOEt/hexane 1:4 ! 1:1 (‡2% NEt3)) gave 12 (805 mg,
63%). Colorless foam. TLC (hexane/AcOEt 1:1): Rf 0.49. UV (MeOH): 274 (sh, 18700), 267 (21900), 252
(16800), 235 (sh, 25100). 1H-NMR (200 MHz, CDCl3): 1.05 (m, iPr3SiOCH2); 1.08 1.15 (m, 3 Me2CH); 1.25
1.40 (m, 3 Me2CH); 3.07 (d, J ˆ 4.7, OH); 3.40, 3.52 (2dd, J ˆ 3.3, 10.4, 2 HÀC(5')); 3.78 (s, MeO); 4.30 (br. q,
J ˆ 3.3, HÀC(4')); 4.56 (m, HÀC(3')); 4.97 (t, J ˆ 5.2, HÀC(2')); 4.99, 5.15 (2d, J ˆ 4.6, OCH2O); 5.39 (s,
ArCH2); 6.20 (d, J ˆ 5.4, HÀC(1')); 6.80 (d, J ˆ 9.1, 4 arom. H); 6.84 6.99 (m, 2 arom. H); 7.17 7.45 (m, 1 1
arom. H); 8.01(br. s, NH); 8.1 3 (s, HÀC(2)); 8.68 (s, HÀC(8)). 13C-NMR (75 MHz, CDCl3): 11.8, 13.0 (2d,
Me2CH); 17.8, 18.0 (2q, Me2CH); 55.2 (q, MeO); 63.3, 63.8 (2t, C(5'), ArCH2); 70.9, 82.0, 84.3 (3d, C(2'), C(3'),
C(4')); 86.6 (s, Ar2C(Ph)); 87.2 (d, C(1')); 90.8 (t, OCH2O); 113.2, 118.2, 120.8 (3d, arom. C); 122.5 (s, C(5));
125.6 (s, arom. C); 126.9, 127.9, 128.2, 128.3, 129.5, 130.1, 130.2 (7d, arom. C); 135.6, 135.7 (2s, arom. C); 141.6 (d,
C(8)); 144.5 (s, arom. C); 149.4 (s, C(4)); 150.8 (s, CO); 151.0 (s, C(6)); 152.9 (d, C(2)); 154.3 (s, arom. C); 158.5
‡
(s, MeOÀC). MALDI-MS: 1084 (5, [M ‡ Na] ), 862 (6), 736 (8), 398 (15), 303 (100), 242 (15), 101 (9).
5'-O-(4,4'-Dimethoxytrityl)-N6-{{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-2'-O-{[(triisopropylsilyl)-
oxy]methyl}adenosine 3'-(2-Cyanoethyl Diisopropylphosphoramidite) (13). A soln. of 12 (700 mg, 0.66 mmol),
ClP(NiPr2)(OCH2CH2CN) (187 mg, 0.79 mmol), and iPr2NEt (171 mg, 1.32 mmol) in CH2Cl2 (2.6 ml) was kept
for 14 h at r.t. CC (AcOEt/hexane 1:9 ! 2 :3 (‡2% Et3N)) gave 13 (704 mg, 85%; 1:1 diastereoisomer
mixture). Colorless foam. TLC (hexane/AcOEt 2 :3): Rf 0.40. UV (MeCN): 273 (sh, 21400), 267 (24000), 252
(19900), 236 (25200), 233 (24900). 1H-NMR (300 MHz, CDCl3): 0.88 (m, 1 2 H,Me2CH); 1.07 1.20 (m, 42 H,
iPr3Si); 2.38 (t, J ˆ 6.5, 0.67 H, OCH2CH2CN); 2.64 (m, 1.33 H, OCH2CH2CN); 3.32, 3.36 (2t, J ˆ 4.0, 0.67 H,
OCH2CH2CN); 3.50 3.72 (m, 4 H, 2 HÀC(5'), OCH2CH2CN, Me2CH); 3.77, 3.78 (2s, 6 H, MeO); 3.83 3.97
(m, 1.33 H, Me2CH); 4.36 (m, 0.66 H, HÀC(4')); 4.41( m, 0.44 H, HÀC(4')); 4.65 (m, 1H, H ÀC(3')); 4.91, 4.94,
4.98 (3d, 2 H, OCH2O); 5.18 (m, 1H, H ÀC(2')); 5.38 (s, 2 H, ArCH2); 6.16, 6.18 (2d, J ˆ 6.4, 1H, H ÀC(1'));
6.76 6.81( m, 4 arom. H); 6.85 (d, J ˆ 8.1, 1 arom. H); 6.94 (t, J ˆ 7.5, 1arom. H); 7.17 7.45 ( m, 11 arom H);
8.03 (br., s, 1H, NH); 8.09, 8.12 (2 s, 1H, H ÀC(2)); 8.63, 8.65 (2s, 1H, H ÀC(8)). 13C-NMR (125 MHz, CDCl3):
11.8, 13.0 (2d, Me2CH); 17.6, 18.0 (2q, Me2CH); 20.1, 20.4 (2t, J(C,P) ˆ 6, OCH2CH2CN); 24.55, 24.59, 24.65 (3q,
Me2CH); 43.2, 43.26, 43.34, 43.4 (4d, Me2CH); 55.19, 55.22 (2q, MeO); 58.0, 58.9 (2t, J(C,P) ˆ 18,
OCH2CH2CN); 62.9, 63.3 (2t, C(5')); 63.8 (t, ArCH2); 71.2 and 71.8 (2d, J(CP) ˆ 16), 77.2 (d), 77.5 and 84.1
(2d, J(C,P) ˆ 4), 84.2 (d), (C(2'), C(3'), C(4')); 86.5, 86.6 (2s, Ar2C(Ph)); 87.45, 87.53 (2d, C(1')); 89.5, 89.6 (2t,
OCH2O); 112.8, 113.1 (2d, arom. C); 117.4, 117.6 (2s, CN); 118.1, 120.8 (2d, arom. C); 122.4, 122.5 (2s, arom. C);
125.6, 125.7 (2s, C(5)); 126.90, 126.93, 127.8, 128.2, 128.26, 128.34, 129.5, 130.04, 130.05, 130.1, 130.2 (11d, arom.
C); 135.66, 135.70, 135.8 (3s, arom. C); 142.0, 142.1 (2d, C(8)); 144.4, 144.5 (2s, arom. C); 149.31, 149.33 (2s
C(4)); 150.7 (s, CO); 151.0 (s, C(6)); 152.78, 152.81 (2d, C(2)); 154.3, 158.53, 158.55 (3s, arom. C). 31P-NMR
‡
(121 MHz, CDCl3): 150.6, 151.3. MALDI-MS: 1284 (18, [M ‡ Na] ), 303 (100).
5'-O-(4,4'-Dimethoxytrityl)-N4-{{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-2'-O-{[(triisopropylsilyl)-
oxy]methyl}cytidine (16). A soln. of 14 [5] (930 mg, 1.2 mmol) in 10m MeNH2/EtOH (10 ml) was kept 15 min at
r.t., evaporated, and co-evaporated with benzene (!15). Separately, a mixture of 18 (505 mg, 1.8 mmol), Et3N
(364 mg, 3.6 mmol), and 4-nitrophenyl carbonochloridate (387 mg, 1.92 mmol) in toluene (3.6 ml) was kept for
30 min at r.t. and then filtered. The filtrate (!19) was added to the soln. of crude 15 (see above) and DMAP
(15 mg, 0.12 mmol) in toluene (2.4 ml) and kept for 5 h at 808. Workup (1. 10% citric acid, 2. NaHCO3 soln.) and
CC (AcOEt/hexane 1:9 ! 2 :3 (‡2% Et3N)) gave 16 (742 mg, 60%). Colorless foam. TLC (hexane/AcOEt 6 :4):
1
Rf 0.30. UV (MeOH): 296 (sh, 4700), 281 (6000), 265 (4700), 236 (18800), 228 (17800). H-NMR (300 MHz,
CDCl3): 1.02 1.15 (m, iPr3SiOCH2, 3 Me2CH); 1.25 1.38 (m, 3 Me2CH); 3.36 (d, J ˆ 8.4, OH); 3.53 (dd, J ˆ 2.8,
11.1, HÀC(5')); 3.60 (dd, J ˆ 1.9, 11.1, H'ÀC(5')); 3.81( s, MeO); 4.09 (m, HÀC(4')); 4.23 (d, J ˆ 4.7, HÀC(2'));
4.37 (m, HÀC(3')); 5.16, 5.28 (2d, J ˆ 4.7, OCH2O); 5.25 (s, ArCH2); 5.96 (s, HÀC(1')); 6.85 6.88 (m, 5 arom.
H); 6.91 6.96 ( m, 1arom. H); 6.94 ( d, J ˆ 7.5, HÀC(5)); 7.19 7.44 (m, 11 arom. H); 8.47 (d, J ˆ 7.5, HÀC(6)).
13C-NMR (75 MHz, CDCl3): 11.9, 13.0 (2d, Me2CH); 17.8, 18.0 (2q, Me2CH); 55.2 (q, MeO); 61.1, 64.2 (2t, C(5'),
ArCH2); 67.6, 83.3, 83.4 (3d, C(2'), C(3'), C(4')); 87.0 (s, Ar2C(Ph)); 90.1( d, C(1')); 90.8 (t, OCH2O); 94.7 (d,
C(5)); 113.3, 118.2, 120.8 (3d, arom. C); 125.0 (s, arom. C); 127.1, 128.0, 128.3, 130.0, 130.1, 130.2, 130.4 (7d,
arom. C); 135.4, 135.7, 144.1 (3s, arom. C); 144.5 (d, C(6)); 152.1 (s, CO); 154.5, 154.9 (2s, C(2), 1arom. C);
‡
158.7 (s, MeOÀC); 162.3 (s, C(4)). MALDI-MS: 1061 (17, [M ‡ Na] ), 712 (12), 440 (40), 396 (14), 303 (100).
5'-O-(4,4'-Dimethoxytrityl)-N4-{{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-2'-O-{[(triisopropylsilyl)-
oxy]methyl}cytidine 3'-(2-Cyanoethyl Diisopropylphosphoramidite) (17). A soln. of 16 (600 mg, 0.58 mmol),
i
ClP(NiPr2)(OCH2CH2CN) (166 mg, 0.7 mmol), and Pr2NEt (150 mg, 1.16 mmol) in CH2Cl2 (2.3 ml) was kept

2496
Helvetica Chimica Acta Vol. 83 (2000)
for 14 h at r.t. CC (AcOEt/hexane 1 :9 ! 2 :3 (‡2% Et3N)) gave 17 (608 mg, 85%, 1:1 diastereoisomer
mixture). Colorless foam. TLC (hexane/AcOEt 3:2): Rf 0.35, 0.42. UV (MeCN): 295 (sh, 7000), 278 (8500), 264
(6600), 236 (32400), 228 (31300). 1H-NMR (300 MHz, CDCl3): 0.96 1.17 (m, 51H, Me 2CH); 1.19 1.37 (m, 3
H, Me2CH); 2.38 (t, J ˆ 6.2, 1H, OCH 2CH2CN); 2.60, 2.61(2 t, J ˆ 6.2, 1H, OCH 2CH2CN); 3.40-3.70 (m, 6 H, 2
HÀC(5'), OCH2CH2CN, Me2CH); 3.806, 3.813 (2s, 6 H, MeO); 4.30 4.54 (m, 3 H, HÀC(2'), HÀC(3'),
HÀC(4')); 5.16 5.21 (m, 2 H, OCH2O); 5.24 (s, 2 H, ArCH2); 6.14, 6.16 (2d, J ˆ 1.9, 1 H, HÀC(1')); 6.80 6.96
(m, 7 H, 6 arom. H, HÀC(5)); 7.19 7.44 (m, 11 arom. H); 8.34 (d, J ˆ 7.8, 0.5 H, HÀC(6)); 8.45 (d, J ˆ 7.5, 0.5 H,
HÀC(6)). 13C-NMR (125 MHz, CDCl3): 12.0, 13.0 (2d, Me2CH); 17.8, 18.0 (2q, Me2CH); 20.1, 20.4 (2t, J(C,P) ˆ
6, OCH2CH2CN); 24.47, 24.53, 24.57, 24.60, 24.66 (5q, Me2CH); 43.2, 43.3 (2d, J(C,P) ˆ 9, Me2CH); 55.20, 55.23
(2q, MeO); 58.1, 58.8 (2t, J(C,P) ˆ 17, O CH2CH2CN); 61.0, 61.6 (2t, C(5')); 64.08, 64.16 (2t, ArCH2); 69.4, and
69.6 (2d, J(C,P) ˆ 13); 78.4, 78.7, and 82.3 (3d), 82.5 (d, J(C,P) ˆ 5, C(2'), C(3'), C(4')); 87.0, 87.1(2 s, Ar2C(Ph));
89.5 (t, OCH2O); 89.48, 89.55 (2d, C(1')); 94.66, 94.73 (2d, C(5)); 113.2, 113.3 (2d, arom. C); 117.4 117.7 (2s, CN);
118.7, 120.8 (2d, arom. C); 125.1 (s, arom. C); 127.17, 127.19, 128.0, 128.3, 128.5, 129.9, 130.1, 130.18, 130.22,
130.25, 130.27, 130.4, 130.1 (13d, arom. C); 135.3, 135.4, 135.6, 144.0, 144.1 (5s, arom. C); 144.76, 144.84 (2d,
C(6)); 152.1 (s, CO); 154.5 (s, arom. C); 154.86, 154.90 (2s, C(2)); 158.6 (s, arom. C); 162.1, 162.3 (2s, C(4)). 31P-
‡
NMR (121 MHz, CDCl3): 150.7, 150.3. MALDI-MS: 1260 (25, [M ‡ Na] ), 790 (5), 772 (14), 489 (10), 440 (5),
303 (100).
5'-O-(4,4'-Dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}guanosine (21). A soln. of 20 [5] (977 mg,
1.2 mmol) in 10m MeNH2/EtOH (10 ml) was kept for 1 h at r.t. and was then evaporated. CC (CH2Cl2 ! CH2Cl2/
MeOH 47:3) gave 21 (806 mg, 87%). Off-white foam. TLC (MeCl2/MeOH 9 :1): Rf 0.42. UV (MeOH): 270 (sh,
11800), 236 (25800), 223 (20900). 1H-NMR (300 MHz, CDCl3): 0.93 1.16 (m, iPr3Si); 3.16 (d, J ˆ 2.1, OH); 3.34
(dd, J ˆ 4.4, 10.4, HÀC(5')); 3.48 (dd, J ˆ 4.0, 10.4, H'ÀC(5')); 3.75, 3.76 (2s, 2 MeO); 4.25 (m, HÀC(4')); 4.54
(m, HÀC(3')); 4.84 (t, J ˆ 5.5, HÀC(2')); 4.96, 5.15 (2d, J ˆ 5.0, OCH2O); 5.89 (br. s, NH2); 5.95 (d, J ˆ 5.5,
HÀC(1')); 6.8 (d, J ˆ 8.7, 4 arom. H); 7.16 7.45 (m, 9 arom. H); 7.65 (s, HÀC(8)); 11.97 (br. s, NHÀC(1)).
13C-NMR (75 MHz, CDCl3): 11.8 (d, Me2CH); 17.8 (q, Me2CH); 55.2 (q, MeO); 63.4 (t, C(5')); 70.9, 81.8, 83.9
(3d, C(2'), C(3'), C(4')); 86.3 (s, Ar2C(Ph)); 86.5 (d, C(1')); 90.8 (t, OCH2O); 113.2 (d, arom. C); 117.7 (s, C(5));
126.9, 127.9, 128.2, 129.8, 130.1 (5d, arom. C); 135.69, 135.72 (2s, arom. C); 136.4 (d, C(8)); 144.6 (s, arom. C);
‡
151.8, 153.5 (2s, C(2), C(4)); 158.5 (s, MeOÀC); 159.1 (s, C(6)). MALDI-MS: 810 (2, [M ‡ K] ), 794 (14, [M ‡
‡
Na] ), 303 (100), 273 (6), 151 (5).
5'-O-(4,4'-Dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}guanosine 3'-(2-Cyanoethyl Diisopropyl-
phosphoramidite) (9). A soln. of 21 (750 mg, 0.97 mmol), ClP(NiPr2)(OCH2CH2CN) (166 mg, 0.7 mmol) and
iPr2NEt (251mg, 1.94 mmol) in CH 2Cl2 (3.9 ml) was kept for 14 h at r.t. CC (CH2Cl2 ! MeCl2/MeOH 9 :1
(‡2% Et3N)) gave 9 (761mg, 81%; 1:1diastereoisomer mixture). Colorless foam. TLC (CH 2Cl2/MeOH 9 :1):
Rf 0.44. UV (MeCN): 269 (sh, 12800), 237 (26400), 222 (19200). 1H-NMR (300 MHz, CDCl3): 0.88 1.22 (m, 33
H, iPr3Si, Me2CH); 2.36 (t, J ˆ 6.5, 1H, OCH 2CH2CN); 2.652 (t, J ˆ 5.6, 0.5 H, OCH2CH2CN); 2.672 (t, J ˆ 7.5,
0.5 H, OCH2CH2CN); 3.28 3.35 (m, 1H, Me CH); 3.50 3.75 (m, 4 H, 2 HÀC(5'), OCH2CH2CN, Me2CH);
2
3.749, 3.751, 3.755 (3s, 2 MeO); 3.87 3.99 (m, 1H, OC H2CH2CN); 4.31( m, 0.5 H, HÀC(4')); 4.38 (m, 0.5 H,
HÀC(4')); 4.59 4.69 (m, 1H, H ÀC(3')); 4.92 (d, J ˆ 6.0, 0.5 H, OCH2O); 4.96 (s, 1H, OCH 2O); 4.97 (d, J ˆ 6.0,
0.5 H, OCH2O); 5.04 5.11 (m, 1H, H ÀC(2')); 5.68 (br. s, NH2); 5.92 (d, J ˆ 6.2, 0.5 H, HÀC(1')); 5.95 (d, J ˆ
5.5, 0.5 H, HÀC(1')); 6.79, 6.80, 6.81(3 d, J ˆ 9.0, 4 arom. H); 7.13 7.47 (m, 9 arom. H); 7.64, 7.65 (2s, 1 H,
HÀC(8)); 12.08 (br. s, HÀN(1)). 13C-NMR (125 MHz, CDCl3): 12.9 (d, Me2CH); 18.22, 18.25 (2q, Me2CH);
20.5, 20.7 (2t, J(C,P) ˆ 7, OCH2CH2CN); 24.9, 24.93, 24.99, 25.03, 25.2 (5q, Me2CH); 44.1, 44.3 (2d, J(C,P) ˆ 13,
Me2CH); 55.4 (q, MeO); 59.4 (t, J(C,P) ˆ 19, OCH2CH2CN); 60.4 (t, J(C,P) ˆ 16, OCH2CH2CN); 64.5, 64.6 (2t,
C(5')); 72.5 (d, J(C,P) ˆ 8), 73.2 (d, J(C,P) ˆ 14), 77.8 (d, J(C,P) ˆ 5), 78.4, 84.6, 84.9 (3d, C(2'), C(3'), C(4'));
87.1, 87.2 (2s, Ar2C(Ph)); 87.28, 87.34 (2d, C(1')); 90.1, 90.4 (2t, OCH2O); 113.8 (d, arom. C); 118.2 (s, C(5));
118.4, 118.7 (2s, CN); 127.4, 128.5, 129.17, 129.23, 130.9, 130.98, 131.05 (7d, arom C); 136.73, 136.75 (2s, arom. C);
136.9 (d, C(8)); 146.0, 146.1 (2s, C(4)), 152.7 (s, C(2)); 154.8 (s, C(6)); 159.7 (s, MeOÀC). 31P-NMR (121 MHz,
‡
CDCl3): 150.9, 151.5. MALDI-MS: 994 (13, [M ‡ Na] ), 304 (22), 303 (100).
5'-O-[(tert-Butyl)dimethylsilyl]uridine (23). A soln. of uridine (1.0 g, 4.1 mmol) and 1H-imidazole (558 mg,
8.2 mmol) in DMF (16 ml) was treated at r.t. with tbdms-Cl (548 mg, 4.3 mmol). Workup after 1 h and CC
(CH2Cl2 ! CH2Cl2/MeOH 24 :1) gave 23 (1.2 g, 80%). Colorless foam. TLC (CH2Cl2/MeOH 9 :1): Rf 0.55. UV
(MeOH): 262 (9000), 231(1800). 1H-NMR (300 MHz, CDCl3): 0.11 (s, Me2Si); 0.92 (s, tBuSi); 3.63 (br. s, OH);
3.84 (dd, J ˆ 1.6, 11.5, HÀC(5')); 4.03 (dd, J ˆ 1.6, 11.5, H'ÀC(5')); 4.41( m, HÀC(4')); 4.22 4.28 (m, HÀC(2'),
HÀC(3')); 5.54 (br. s, OH); 5.65 (d, J ˆ 8.7, HÀC(5)); 5.91( d, J ˆ 2.5, HÀC(1')); 8.09 (d, J ˆ 8.7, HÀC(5));
10.54 (br. s, NH). 13C-NMR (75 MHz, CDCl3): À 5.5 (q, Me2Si); 18.4 (s, Me3CSi); 25.9 (q, Me3C); 61.7 (t, C(5'));

Helvetica Chimica Acta Vol. 83 (2000)
2497
69.1, 75.6, 84.8 (3d, C(2'), 3C('), C(4')); 90.3 (d, C(1')); 102.1 (d, C(5)); 140.6 (d, C(6)); 151.3 (s, C(2)); 164.1 (s,
‡
C(4)). MALDI-MS: 381(100, [ M ‡ Na] ), 274 (7), 251 (5), 229 (8), 213 (14), 191 (7), 177 (7), 137 (9), 127 (9).
5'-O-[(tert-Butyl)dimethylsilyl]-2'-O-{[(triisopropylsilyl)oxy]methyl}uridine (24). A soln. of 23 (800 mg,
2.23 mmol) and iPr2NEt (1.53 ml, 8.9 mmol) in (CH2Cl)2 (9 ml) was treated with Bu2SnCl2 (745 mg, 2.45 mmol)
at r.t. for 1h and then treated with tom-Cl ([5], 596 mg, 2.68 mmol) at 80 8 for 15 min. Workup and CC (hexane/
AcOEt 4 :1 ! 3 :2) gave 24 (510 mg, 45%). Colorless foam. TLC (hexane/AcOEt 1:1): Rf 0.46. UV (MeOH):
261(9000), 230 (1900). 1H-NMR (300 MHz, CDCl3): 0.12 (s, Me2Si); 0.93 (s, tBuSi); 1.01 1.14 (m, iPr3Si); 3.12
(d, J ˆ 4.7, OH); 3.83, 3.99 (2dd, J ˆ 1.9, 11.8, 2 HÀC(5')); 4.10 (dd, J ˆ 1.9, 4.7, HÀC(4')); 4.13 (t, J ˆ 4.7,
HÀC(2')); 4.24 (q, J ˆ 4.7, HÀC(3')); 4.94, 5.18 (2d, J ˆ 5.0, OCH2O); 5.68 (d, J ˆ 8.1, HÀC(5)); 6.08 (d, J ˆ 4.1,
HÀC(1')); 8.00 (d, J ˆ 8.1, HÀC(6)); 8.81(br. s, NH). 13C-NMR (75 MHz, CDCl3): À 5.56, À 5.51(2 q, Me2Si);
11.8 (d, Me2CH); 17.8 (q, Me2CH); 18.4 (s, Me3C); 25.9 (q, Me3C); 62.5 (t, C(5')); 69.4, 83.1, 85.0 (3d, C(2'),
C(3'), C(4')); 87.4 (d, C(1')); 90.6 (t, OCH2O); 102.3 (d, C(5)); 140.1 (d, C(6)); 150.2 (s, C(2)); 163.1 (s, C(4)).
‡
MALDI-MS: 567 (100, [M ‡ Na] ), 323 (6), 283 (6), 269 (10), 267 (24), 225 (5), 189 (9).
5'-O-[(tert-Butyl)dimethylsilyl]-2'-O-{[(triisopropylsilyl)oxy]methyl}uridine 3'-(2-Cyanoethyl Diisopropyl-
phosphoramidite) (25). A soln. of 24 (260 mg, 0.48 mmol), ClP(NiPr2)(OCH2CH2CN) (137 mg, 0.58 mmol),
i
and Pr2NEt (124 mg, 0.96 mmol) in CH2Cl2 (1.9 ml) was kept for 14 h at r.t. CC (hexane/AcOEt 9 :1! 7:3
(‡2% Et3N)) gave 25 (303 mg, 85%, 3 :2 diastereoisomer mixture). Colorless foam. TLC (hexane/AcOEt 7:3):
1
Rf 0.38. UV (MeCN): 259 (9500), 229 (2300). H-NMR (300 MHz, CDCl3): 0.110, 0.116, 0.119, 0.13 (4s, 6 H,
Me2Si); 0.93, 0.94 (2s, 9 H, tBuSi); 0.96 1.11 (m, 21 H, iPr3Si); 1.14 1.22 (m, 1 2 H,Me2CH); 2.61, 2.65, 2.66 (3t,
J ˆ 6.2, 2 H, OCH2CH2CN); 3.57 3.95 (m, 6 H, 2 HÀC(5'), OCH2CH2CN, Me2CH); 4.18 (m, 0.4 H, HÀC(4'));
4.31 4.42 ( m, 2.6 H, HÀC(2'), C(3'), C(4')); 4.88, 4.93, 4.97, 4.99 (4d, J ˆ 5.3, 2 H, OCH2O); 5.67 (d, J ˆ 8.1, 1 H,
HÀC(5)); 6.16, 6.18 (2d, J ˆ 5.6, 1H, H ÀC(1')); 7.84, 7.90 (2d, J ˆ 8.1, 1 H, HÀC(6)); 8.14 (br. s, 1H, NH).
13C-NMR (125 MHz, CDCl3): À 5.6, À 5.5, À 5.5 (3q, Me2Si); 11.9, 11.9 (2d, Me2CH); 17.73, 17.76, 17.78 (3q,
Me2CH); 18.4, 18.5 (2s, Me3C); 20.4, 20.5 (2t, J(C,P) ˆ 8, OCH2CH2CN); 24.53, 24.55, 24.57, 24.59, 24.63, 24.66
(6q, Me2CH); 25.9, 26.0 (2q, Me3C); 43.2, 43.4 (2d, J(C,P) ˆ 12, Me2CH); 57.6, 59.0 (2t, J(C,P) ˆ 16,
OCH2CH2CN); 63.0, 63.2 (2t, C(5')); 70.2, 71.8 (2d, J(C,P) ˆ 16), 77.2, 77.9 (d, J(C,P) ˆ 3), 84.9, 85.4 (2d,
J(C,P) ˆ 4) (C(2'), C(3'), C(4')); 86.6, 86.6 (2d, C(1')); 88.5, 88.9 (2t, OCH2O); 102.4, 102.5 (2d, C(5)); 117.6,
117.7 (2s, CN); 140.2, 140.4 (2d, C(6)); 150.1, 150.2 (2s, C(2)); 162.8, 162.9 (2s, C(4)). 31P-NMR (121 MHz,
‡
CDCl3): 150.4, 150.8. MALDI-MS: 767 (100, [M ‡ Na] ), 722 (11), 684 (7), 613 (14), 599 (15), 261 (7).
N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-{[(RS)-1-(2-nitrophenyl)ethoxy]methyl}adenosine ((RS)-27),
N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-{[(R)-1-(2-nitrophenyl)ethoxy]methyl}adenosine ((S)-28), and N6-
Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-{[(R)-1-(2-nitrophenyl)ethoxy]methyl}adenosine ((R)-28). A soln. of
i
26 (10.1 g, 15 mmol) and Pr2NEt (10.5 ml, 60 mmol) in (CH2Cl)2 (60 ml) was treated with Bu2SnCl2 (5.01g,
16.5 mmol) at r.t. for 1 h and then treated with (RS)-npeom-Cl [5] (3.2 g, 18 mmol) at 808 for 15 min. Workup
and CC (hexane/AcOEt 2 :3 ! AcOEt (‡2% Et3N)) gave (RS)-27 (4.54 g, 36%), (R)-28 (2.15 g, 17%), and
(S)-28 (2.27 g, 18%). Pale yellow foams.
Data of (RS)-27: TLC (hexane/AcOEt 9 :1): Rf 0.6. 1H-NMR (300 MHz, CDCl3): 1.35, 1.49 (2d, J ˆ 6.5, 3
H, MeCHAr); 2.52, 2.71(2 d, J ˆ 5.0, 1H, OH); 3.35 3.45 (2 dd, J ˆ 4.3, 11.0, 1 H, HÀC(5')); 3.47 3.55 (2dd,
J ˆ 3.7, 11.0, 1 H, H'ÀC(5')); 3.78 (s, 6 H, MeO); 4.20 4.26 (m, 1H, H ÀC(4')); 4.36, 4.58 (2 br. q, J ˆ 5, 1H,
HÀC(3')); 4.69, 4.72, 4.83, 4.85 (4d, J ˆ 6.8, 2 H, OCH2O); 5.01( t, J ˆ 4.7, 0.5 H, HÀC(2')); 5.03 (t, J ˆ 5.0, 0.5 H,
HÀC(2')); 5.22, 5.36 (2 br. q, J ˆ 6.5, 1H, MeC HAr); 6.12 (d, J ˆ 4.7, 0.5 H, HÀC(1')); 6.23 (d, J ˆ 5.0, 0.5 H,
HÀC(1')); 6.81( d, J ˆ 6.8, 4 arom. H); 7.19 7.64 (m, 15 arom. H); 7.87 (dd, ˆ 1.5, 8.4, 1 arom. H); 8.03 (br. d,
J ˆ 7, 2 arom. H); 8.12, 8.22 (2s, 1H, H ÀC(2)); 8.67, 8.75 (2s, 1H, H ÀC(8)); 8.96, 9.00 (2 br. s, 1H, NH).
‡
MALDI-MS: 975 (20, [M ‡ Na] ), 696 (9), 303 (100).
Data of (S)-28: TLC (hexane/AcOEt 9 :1): Rf 0.47. 1H-NMR (300 MHz, CDCl3): 1.56 (d, J ˆ 6.2,
MeCHAr); 3.15, 3.40 (2dd, J ˆ 3.7, 10.5, H'ÀC(5)); 3.77 (s, MeO); 3.95 (d, J ˆ 5.6, OH); 4.10 4.16 (m,
HÀC(4')); 4.45 (dd, J ˆ 3.5, 5.0, HÀC(3')); 4.75, 4.79 (2d, J ˆ 6.8, OCH2O); 4.96 (br. q, J ˆ 5.5, HÀC(2')); 5.36
(q, J ˆ 6.2, MeCHAr); 5.99 (d, J ˆ 5.3, HÀC(1'); 6.77 (2d, J ˆ 6.8, 4 arom. H); 7.20 7.68 (m, 15 arom. H); 7.88
(dd, J ˆ 1.2, 8.1, 1 arom. H); 8.03 (br.d, J ˆ 7.2, 2 arom. H); 8.22 (s, HÀC(2)); 8.77 (s, HÀC(8)); 8.99 (br. s, NH).
Data of (R)-28: TLC (hexane/AcOEt 9 :1): Rf 0.36. 1H-NMR (300 MHz, CDCl3): 1.46 (d, J ˆ 6.5,
MeCHAr); 3.33, 3.50 (2dd, J ˆ 4.0, 10.6, 2 HÀC(5')); 3.78 (s, MeO); 3.82 (m, OH); 4.35 4.40 (m, HÀC(4'));
4.46 4.50 (m, HÀC(3')); 4.64, 4.83 (2d, J ˆ 6.8, OCH2O); 4.73 4.81( m, HÀC(2')); 5.34 (q, J ˆ 6.5, MeCHAr);
5.98 (d, J ˆ 5.3, HÀC(1')); 6.78 (d, J ˆ 6.2, 2 arom. H); 6.79 (d, J ˆ 6.8, 2 arom. H); 7.19 7.73 (m, 15 arom. H);
7.89 (dd, J ˆ 1.2, 8.1, 1 arom. H); 8.02 (br. d, J ˆ 7, 2 arom. H); 8.20 (s, HÀC(2)); 8.75 (s, HÀC(8)); 9.00 (br. s,
NH).

2498
Helvetica Chimica Acta Vol. 83 (2000)
3'-O-Acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-{[(S)-1-(2-nitrophenyl)ethoxy]methyl}adenosine ((S)-29) and
3'-O-Acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-{[(R)-1-(2-nitrophenyl)ethoxy]methyl}adenosine ((R)-29). A soln.
of (RS)-27 (4.40 g, 5.2 mmol) in 10m MeNH2/EtOH (44 ml) was kept for 15 min at r.t. After evaporation, the
residue was dissolved in Py (20 ml) and treated with DMAP (63 mg, 0.52 mmol) and Ac2O (0.6 ml, 6.2 mmol).
Workup (1. 10% citric acid; 2. NaHCO3 soln.) after 30 min at r.t., and CC (AcOEt/hexane 3 :2 ! AcOEt (‡2%
Et3N)) gave (S)-29 (1.34 g, 33%) and (R)-29 (1.51 g, 37%). Off-white foams.
Data of (S)-29: TLC (hexane/AcOEt 1:9): Rf 0.46. UV (MeOH): 255 (20100), 235 (26600), 225 (24400).
1H-NMR (300 MHz, CDCl3): 1.08 (d, J ˆ 6.2, MeCHAr); 2.1( s, MeCO); 3.40 3.59 (2dd, J ˆ 4.0, 10.5, 2
HÀC(5')); 3.81( s, 2 MeO); 4.30 4.40 (m, HÀC(4')); 4.35, 4.69 (2d, J ˆ 7.5, OCH2O); 4.91( q, J ˆ 6.2 CHAr);
5.28 (dd, J ˆ 5.0, 7.0, HÀC(2')); 5.41( dd, J ˆ 2.0, 5.0, HÀC(3')); 5.62 (br. s, NH2); 6.17 (d, J ˆ 7.0, HÀC(1')); 6.85
(d, J ˆ 6.8, 4 arom. H); 7.24 7.57 (m, 12 arom. H); 7.87 (br. d, J ˆ 7.9, 1arom. H); 8.06 ( s, HÀC(2)); 8.37 (s,
HÀC(8)). 13C-NMR (75 MHz, CDCl3): 20.8 (q, MeCO); 23.0 (q, MeCHAr); 55.2 (q, MeO); 63.4 (t, C(5')); 69.9,
71.7, 76.9 (3d, C(2'), C(3'), MeCHAr); 82.4 (d, C(4')); 86.0 (d, C(1')); 86.9 (s, Ar2C(Ph)); 92.7 (t, OCH2O); 113.3
(d, arom. C); 120.0 (s, C(5)); 124.3, 127.0, 127.7, 128.0, 128.2, 129.5, 130.1, 133.5 (8d, arom. C); 135.4, 135.5, 138.7
(3s, arom. C); 139.2 (d, C(8)); 144.4 (s, arom. C); 148.2 (s, C(4)); 150.2 (s, arom. C); 153.5 (d, C(2)); 155.6 (s,
‡
‡
C(6)); 158.6 (s, MeOÀC); 170.2 (s, MeCO). MALDI-MS: 819 (15, [M ‡ K] ), 813 (5, [M ‡ Na] ), 791(3, [ M ‡
‡
H] ), 624 (11), 570 (22), 303 (100), 164 (38).
Data of (R)-29: TLC (hexane/AcOEt 1:9): Rf 0.34. UV (MeOH): 265 (18100), 235 (26000), 225 (23200).
1H-NMR (300 MHz, CDCl3): 1.40 (d, J ˆ 6.2, MeCHAr); 2.21( s, MeCO); 3.43 (dd, J ˆ 3.7, 10.6, HÀC(5')); 3.51
(dd, J ˆ 3.7, 10.6, H'ÀC(5')); 3.78, 3.79 (2s, MeO); 4.29 4.33 (m, HÀC(4')); 4.54, 4.71(2 d, J ˆ 7.1, OCH2O); 5.18
(t, J ˆ 5.8, HÀC(2')); 5.29 (q, J ˆ 6.2, CHAr); 5.52 (dd, J ˆ 3.1, 5.3, HÀC(3')); 5.76 (br. s, NH2); 6.06 (d, J ˆ 6.2,
HÀC(1')); 6.81( d, J ˆ 6.8, 4 arom. H); 7.19 7.58 (m, 12 arom. H); 7.83 (s, HÀC(2)); 7.86 (dd, J ˆ 1.2, 8.1, 1 arom.
H); 8.18 (s, HÀC(8)). 13C-NMR (75 MHz, CDCl3): 20.9 (q, MeCO); 23.0 (q, MeCHAr); 55.2 (q, MeO); 63.0 (t,
C(5')); 69.4, 72.1, 77.4 (3d, C(2'), C(3'), MeCHAr); 82.2 (d, C(4')); 86.4 (d, C(1')); 86.8 (s, Ar2C(Ph)); 92.7 (t,
OCH2O); 113.2 (d, arom. C); 119.9 (s, C(5)); 124.3, 127.0, 127.5, 127.9, 128.1, 128.2, 130.1, 133.6 (8d, arom. C);
135.4, 135.5, 138.8 (s, arom. C); 138.9 (d, C(8)); 144.4 (s, arom. C); 147.6 (s, C(4)); 149.8 (s, arom. C); 153.1 (d,
‡
C(2)); 155.5 (s, C(6)); 158.6 (s, MeOÀC); 170.4 (s, MeCO). MALDI-MS: 819 (4, [M ‡ K] ), 813 (10, [M ‡
‡
‡
Na] ), 791(2, [ M ‡ H] ), 624 (8), 509 (26), 303 (100), 164 (28).
5'-O-(4,4'-Dimethoxytrityl)-2'-O-{[(S)-1-(2-nitrophenyl)ethoxy]methyl}-N6-{{{2-[(triisoproysilyl)oxy]ben-
zyl}oxy}carbonyl}adenosine (30). A soln. of (S)-29 (389 mg, 0.49 mmol) in CH2Cl2 (1ml) was added during
20 min to a suspension obtained from 1.9m COCl2 in toluene (0.39 ml, 0.74 mmol), DMAP (12 mg, 0.1 mmol) in
Py (1.4 ml) and CH2Cl2 (1ml). After 15 min at r.t., 18 (412 mg, 1.47 mmol) and Et3N (198 mg, 1.96 mmol) were
added. Workup (1. 10% citric acid; 2. NaHCO3 soln.) after 18 h at r.t. gave a residue, which was dissolved in 0.2m
NaOH in THF/MeOH/H2O 5 :4 :1(10 ml). Workup after 5 min at 4 8, and CC (AcOEt/hexane 3 :7 ! 3 :2 (‡2%
Et3N)) gave 30 (300 mg, 58%). Colorless foam. TLC (hexane/AcOEt 1:1): Rf 0.17. UV (MeOH): 273 (sh,
18100), 270 (sh, 19300), 277 (20500), 252 (16800), 234 (sh, 22800). 1H-NMR (300 MHz, CDCl3): 1.12 (d, J ˆ 7.1,
1 8 H, MeCH); 1.33 (d, J ˆ 6.2, MeCHAr); 1.22 1.38 (m, 3 H, Me2CH); 2.52 (br. d, J ˆ 5.0, OH); 3.37 (dd, J ˆ
2
4.1, 10.6, HÀC(5')); 3.51( dd, J ˆ 6.8, 10.6, H'ÀC(5'); 3.78 (s, MeO); 4.21( m, HÀC(4')); 4.34 (m, HÀC(3'));
4.67, 4.81(2 d, J ˆ 6.5, OCH2O); 4.98 (t, J ˆ 5.0, HÀC(2')); 5.21( q, J ˆ 6.2, MeCHAr); 5.38 (s, ArCH2); 6.18 (d,
J ˆ 5.0, HÀC(1')); 6.80 (d, J ˆ 9.0, 4 arom. H); 6.78 6.98 (m, 2 arom. H); 7.17 7.44 (m, 12 arom. H); 7.51 7.62
(m, 2 arom. H); 7.86 (dd, J ˆ 1.3, 8.1, 1 arom. H); 8.12 (br. s, NH); 8.1 3 (s, C(2)); 8.69 (s, C(8)); 13C-NMR
(75 MHz, CDCl3): 13.0 (d, Me2CH); 18.0 (q, Me2CH); 23.2 (q, MeCHAr); 55.2 (q, MeO); 63.1, 63.8 (2t, ArCH2,
C(5')); 70.4, 70.9, 79.9, 84.1(4 d, C(2'), C(3'), C(4'), MeCHAr); 86.7 (s, Ar2C(Ph)); 87.2 (d, C(1')); 94.0 (t,
OCH2O); 113.2, 118.2, 120.8 (3d, arom. C); 122.5 (s, C(5)); 124.3 (d, arom. C); 125.6 (s, arom. C); 127.0, 127.8,
127.9, 128.2, 128.4, 129.6, 130.1, 130.2, 133.6 (9d, arom. C); 135.5, 135.6, 138.5 (3s, arom. C); 141.6 (d, C(8));
144.5, 148.0 (2s, arom. C); 149.5 (s, C(4)); 150.8, 150.9 (2s, C(6), CO); 153.0 (d, C(2)); 154.3 (s, arom. C); 158.6
‡
(s, MeOÀC). MALDI-MS: 1077 (5, [M ‡ Na] ), 855 (5), 729 (4), 535 (4), 303 (100), 242 (11).
5'-O-(4,4'-Dimethoxytrityl)-2'-O-{[(S)-1-(2-nitrophenyl)ethoxy]methyl}-N6-{{{2-[(triisopropylsilyl)oxy]benz-
yl}oxy}carbonyl}adenosine 3'-{Triethylammonium 2,2'-(1,4-Phenylenebis(oxy)]bis[acetate] (31 ¥ Et3N). A soln.
of 30 (160 mg, 0.152 mmol), [1,4-phenylenebis(oxy)bis[acetic acid] (68 mg, 0.3 mmol), DMAP (4 mg,
i
0.03 mmol), and Pr2NEt (98 mg, 0.76 mmol) in Py (1.5 ml) was treated with BOP (133 mg, 0.3 mmol) and
kept 30 min at r.t. Workup (1. 10% citric acid, 2. NaHCO3 soln.) and CC (CH2Cl2 ! CH2Cl2/MeOH 19 :1 (‡2%
Et3N)) gave 31 (103 mg, 49%). Colorless foam. TLC (CH2Cl2/MeOH 9 :1): Rf 0.49. UV (MeOH): 275 (sh,
24200), 267 (28900), 252 (22900), 236 (sh, 33300). 1H-NMR (300 MHz, CDCl3): 0.94 (d, J ˆ 6.3, MeCHAr); 1.11
(d, J ˆ 7.2, 3 Me2CH); 1.25 (t, J ˆ 7.4, 9 H, Et3N); 1.23 1.38 (m, 3 Me2CH); 3.05 (q, J ˆ 7.4, 6 H, Et3N); 3.43 (dd,
J ˆ 4.1, 10.5, HÀC(5')); 3.57 (dd, J ˆ 4.0, 10.5, H'ÀC(5')); 3.77 (s, MeO); 4.25 (d, J ˆ 7.5, 1H, OCH 2O); 4.35 (m,

Helvetica Chimica Acta Vol. 83 (2000)
2499
HÀC(4')); 4.40 (s, OCH2COO); 4.56 4.62 (m, 3 H, OCH2O, OCH2COO); 4.8 (q, J ˆ 6.3, MeCHAr); 5.38 (s,
ArCH2); 5.93 (dd, J ˆ 5.3, 7.3, HÀC(2')); 5.50 (dd, J ˆ 1.9, 5.3, HÀC(3')); 6.08 (d, J ˆ 7.3, HÀC(1')); 6.76 6.96
(m, 10 arom. H); 7.16 7.55 (m, 12 arom H); 7.82 (dd, J ˆ 1.2, 8.2, 1 arom H); 8.14 (s, HÀC(2)); 8.18 (br. s, NH);
8.70 (s, HÀC(8)). 13C-NMR (75 MHz, CDCl3): 8.5 (q, Et3N); 13.0 (d, Me2CH); 18.0 (q, Me2CH); 22.9 (q,
MeCHAr); 45.0 (t, Et3N); 55.2 (q, MeO); 63.2, 63.8, 65.8, 67.8 (4t, C(5'), ArCH2, 2 ArOCH2); 69.8, 72.5, 76.5,
77.2, 82.3 (4d, C(2'), C(3'), C(4'), MeCHAr); 86.3 (d, C(1')); 87.0 (s, Ar2C(Ph)); 92.8 (t, OCH2O); 113.3, 115.5,
115.6, 118.1, 120.8 (5d, arom. C); 122.4 (s, C(5)); 124.3 (d, arom. C); 125.6 (s, arom. C); 127.1, 127.6, 128.0, 128.1,
128.4, 129.5, 130.1, 130.2 (8d, arom. C); 133.6, 135.3 (2s, arom. C); 135.4 (d, arom. C); 138.1 (s, arom. C); 141.7
(d, C(8)); 144.3, 148.3 (2s, arom. C); 149.6 (s, C(4)); 150.7, 151.2 (2s, NCOO, arom. C); 151.7 (s, C(6)); 153.2 (s,
arom. C); 153.9 (d, C(2)); 154.3 (s, arom. C); 158.7 (s, MeOÀC); 168.5, 174.6 (2s, CH2COO). FAB-MS: 1263
‡
(44, [M ‡ H] ), 1219 (16), 338 (11), 303 (100).
Solid Support 32. A suspension of LCAA-CPG (1g), 31 (68 mg, 0.05 mmol), iPr2NEt (0.1ml, 0.58 mmol),
and BOP (22.1mg, 0.05 mmol), in MeCN (4 ml) was shaken 20 h at r.t. After filtration, the solid was washed
with MeCN and CH2Cl2, suspended in Py (3 ml) and Ac2O (1ml), and shaken for 2 h at r.t. After filtration, the
solid was washed with DMF and CH2Cl2. Loading: 43 mmol/g.
N-{[(2-Nitrobenzyl)oxy]carbonyl}-l-phenylalanine (39). A soln. of l-phenylalanine (250 mg, 1.5 mmol) in
2m aq. NaOH (0.75 ml) was treated consecutively (during 15 min) with small portions of nboc-Cl [1] (90%;
440 mg, 1.8 mmol) and 2m aq. NaOH (0.75 ml). After 2.5 h at r.t., H2O (15 ml) and Et2O (15 ml) were added;
the aq. phase was acidified with HCl, extracted with CH2Cl2, and evaporated. Crystallization (benzene) gave 39
(253 mg, 49%). Colorless crystals. TLC (CH2Cl2/MeOH 9 :1): Rf 0.2. UV (MeOH): 300 (1500), 258 (5500), 231
(2200). 1H-NMR (300 MHz, CDCl3): 3.14 (dd, J ˆ 6.0, 14.0, 1 H, HÀC(b)); 3.25 (dd, J ˆ 5.4, 14.0, 1 H,
HÀC(b)); 4.72 (br. q, J ˆ 6, HÀC(a)); 5.29 (br. d, J ˆ 8, NH); 5.52 (s, ArCH2O); 7.18 7.12 (m, 2 arom. H);
7.27 7.36 (m, 3 arom. H); 7.44 7.53 (m, 2 arom. H); 7.58 7.66 (m, 1arom. H); 8.10 ( d, J ˆ 8, 1arom. H).
13C-NMR (75 MHz, CDCl3): 37.7 (t, C(b)); 54.6 (d, C(a)); 63.7 (t, ArCH2O); 125.0, 127.3, 128.5, 128.6, 128.7,
129.4 (6d, arom. C); 132.9 (s, arom. C); 133.8 (d, arom. C); 135.4, 147.2 (2s, arom. C); 155.3 (s, OCON); 176.2 (s,
‡
COO). ESI-MS (neg.): 687.4 (53, [2M À H]À), 343.2 (100, [M À H]À). ESI-MS (pos.): 727.4 (20, [2M ‡ K] ),
‡
‡
‡
711.4 (70, [2M ‡ Na] ), 383.1(18, [ M ‡ Na] ), 367.1(100, [ M ‡ Na] ).
1-{[(RS)-1-(2-Nitrophenyl)ethoxy]carbonyl}-1H-imidazole (40). A soln. of rac-1-(2-nitrophenyl)ethanol
(2.0 g, 12 mmol) and 1,1'-carbonylbis[1H-imidazole] (2.14 g, 13.2 mmol) in CH2Cl2 (12 ml) was kept for 30 min
at r.t. Workup gave crude 40 (quant.) as an off-white solid, which was used without further purification. TLC
(hexane/AcOEt 3:7): Rf 0.30. UV (MeCN): 334 (sh, 500), 261(sh, 4700), 237 (sh, 6300). 1H-NMR (300 MHz,
CDCl3): 1.84 (d, J ˆ 6.5, MeCHAr); 6.58 (q, J ˆ 6.5, MeCHAr); 7.07 (dd, J ˆ 0.9, 1.6, 1 arom. H); 7.42 (t, J ˆ 1.5, 1
arom. H); 7.48 7.52 (m, 1arom. H); 7.65 7.69 ( m, 2 arom. H); 7.99 8.03 (m, 1arom. H); 8.15 ( t, J ˆ 0.9, 1arom.
H). 13C-NMR (75 MHz, CDCl3): 21.9 (q, MeCHAr); 72.7 (d, MeCHAr); 117.1, 124.8, 126.9, 129.3, 130.9, 134.0
(6d, arom. C); 136.1 (s, arom. C); 137.0 (d, arom. C); 147.7 (s, arom. C); 182.2 (s, CO). MALDI-MS: 284 (20,
‡
[M ‡ Na] ), 221 (53), 218 (100), 150 (83).
N-{[(RS)-1-(2-Nitrophenyl)ethoxy]carbonyl}-l-phenylalanine (41). A soln. of l-phenylalanine (1.0 g,
6.05 mmol), 40 (1.74 g, 6.66 mmol), and 1H-tetrazole (212 mg, 3.03 mmol) in DMF (6 ml) was kept for 14 h at
808. DMF was evaporated and the residue extracted (Et2O, 0.5m phosphate buffer, pH 12.5). The aq. phase was
acidified with HCl (! pH 2) and extracted with CH2Cl2: 41 (1.15 g, 53%; 1 :1 diastereoisomer mixture). Yellow
solid, which was used without further purification. TLC (CH2Cl2/MeOH 9 :1): Rf 0.37. UV (MeOH): 335 (sh,
400), 306 (sh, 900), 257 (4200), 235 (2900). 1H-NMR (300 MHz, (D6) DMSO): 1.46, 1.59 (2d, J ˆ 6.5, 3 H,
MeCHAr); 2.79 (dd, J ˆ 7.5, 9.3, 0.5 H, HÀC(b)); 2.91 3.12 ( m, 1H, H 'ÀC(b)); 3.93 4.07 (m, 1H, H ÀC(a));
4.3 (m, 1H, NH); 5.90, 5.91(2 q, J ˆ 6.5, 1H, MeC HAr); 7.10 7.29 (m, 5 arom. H); 7.42 7.96 (m, 4 arom. H).
13C-NMR (75 MHz, (D6)DMSO): 21.7 (q, MeCHAr); 36.3 (t, C(b)); 55.5, 55.6 (2d, C(a)); 67.0 (d, MeCHAr);
124.0, 126.2, 127.9, 128.0, 129.0, 129.2, 133.9 (7d, arom. C); 137.8, 137.9, 147.0, 147.1 (4s, arom. C); 154.7, 154.9 (2s,
‡
OCON); 173 (COO). MALDI-MS: 381 (29, [M ‡ Na] ), 335 (56), 313 (100), 295 (29), 150 (32), 12 (38).
3'-O-Acetyl-2'-O-{[(S)-1-(2-nitrophenyl)ethoxy]methyl}adenosine (47). At 48, a soln. of (S)-29 (600 mg,
0.76 mmol) in CH2Cl2 (14 ml) was treated with CHCl2COOH (0.56 ml). After 10 min, MeOH (2 ml) was added,
and the mixture was kept for 20 min. Workup and CC (CH2Cl2 ! CH2Cl2/MeOH 19 :1) gave 47 (306 mg, 82%).
Yellow foam. TLC (CH2Cl2/MeOH 9 :1): Rf 0.48. UV (MeOH): 257 (19200), 229 (6700). 1H-NMR (300 MHz,
CDCl3): 0.77 (d, J ˆ 6.2, MeCHAr); 2.11 (s, MeCO); 3.77 (br. d, J ˆ 13, HÀC(5')); 3.98 (br. d, J ˆ 13, H'ÀC(5'));
4.15 (d, J ˆ 7.4, 1H, OCH 2O); 4.36 (br. s, HÀC(4')); 4.56 (d, J ˆ 7.4, 1H, OCH 2O); 4.78 (q, J ˆ 6.2, MeCHAr);
5.22 (dd, J ˆ 5.0, 7.8, HÀC(2')); 5.52 (d, J ˆ 5, HÀC(3')); 5.87 (d, J ˆ 8.1, HÀC(1')); 6.2 (br. s, NH2); ca. 6.6 (br. s,
OH); 7.34 7.40 (m, 1arom. H); 7.46 (br. d, J ˆ 8.1, 1 arom. H); 7.56 (br.t, J ˆ 8.1, 1 arom. H); 7.85 (br. d, J ˆ 8.1,
1arom. H); 7.97 ( s, HÀC(2)); 8.38 (s, HÀC(8)). 13C-NMR (75 MHz, CDCl3): 20.8 (q, MeCO); 22.7 (q,

2500
Helvetica Chimica Acta Vol. 83 (2000)
MeCHAr); 63.1( t, C(5')); 68.8, 72.5, 75.8 (3d, C(2'), C(3'), MeCHAr); 86.1, 89.3 (2d, C(4'), C(1')); 91.9 (t,
OCH2O); 121.2 (s, C(5)); 124.2, 127.7, 128.3, 133.8 (4d, arom. C); 138.5 (s, arom. C); 140.9 (d, C(8)); 148.3, 148.4
‡
(2s, C(4), arom. C); 152.6 (d, C(2)); 156.3 (s, C(6)); 170.1 (s, MeCO). MALDI-MS: 511 (74, [M ‡ Na] ), 498
‡
(100, [M ‡ H] ), 332 (59), 310 (52), 136 (83).
3'-O-Acetyl-2'-O-{[(S)-1-(2-nitrophenyl)ethoxy]methyl}adenosine 5'-(2-Cyanoethyl Methyl Phosphate]
(48). A soln. of 47 (200 mg, 0.41mmol) and 5-(benzylthio)-1 H-tetrazole (175 mg, 0.9 mmol) in MeCN (2 ml)
was treated with 0.25m (MeO)P(NiPr2)(OCH2CH2CN) in MeCN (2 ml, 0.5 mmol) and kept for 20 min at r.t.
After evaporation, the residue was treated with a soln. of I2 (0.5 g) in H2O (0.4 ml), Py (4 ml), and THF (15 ml).
Workup (1. Na2S2O3 soln; 2. NaHCO3 soln.) and CC (CH2Cl2 ! CH2Cl2/MeOH 2 :3) gave 48 (164 mg, 63%, 1:1
diastereoisomer mixture). Colorless foam. TLC (MeCl2/MeOH 9 :1): Rf 0.52. UV (MeOH): 258 (18000), 229
(6300). 1H-NMR (300 MHz, CDCl3): 1.06 (d, J ˆ 6.2, MeCHAr); 2.1( s, MeCO); 2.75 (m, OCH2CH2CN); 3.80,
3.84 (2d, J ˆ 3.1, POMe); 4.24, 4.27 (2q, J ˆ 6.1, OCH2CH2CN); 4.33 (d, J ˆ 7.4, 1H, OCH O); 4.36 4.48 (m,
2
HÀC(4'), 2 HÀC(5')); 4.62 (d, J ˆ 7.4, 1H, OCH 2O); 4.93 (q, J ˆ 6.2, MeCHAr); 5.14 (m, HÀC(2')); 5.37 5.42
(m, HÀC(3')); 6.11, 6.12 (2d, J ˆ 6.5, HÀC(1')); 6.2 (br. s, NH2); 7.37 (ddd, J ˆ 1.5, 6.8, 8.1, 1 arom. H); 7.49 7.60
(m, 2 arom. H); 7.84 (dd, J ˆ 1.2, 8.1, 1 arom. H); 8.127, 8.131 (2s, HÀC(2)); 8.413, 8.416 (2s, HÀC(8)). 13C-NMR
(75 MHz, CDCl3): 19.6 (t, J(C,P) ˆ 7.6, OCH2CH2CN); 20.7 (q, MeCO); 22.9 (q, MeCHAr); 55.0 (q, J(CP) ˆ
4.1, POMe); 62.2 (t, J(C,P) ˆ 4.8, OCH2CH2CN); 66.70, 66.76 (2t, J(C,P) ˆ 6, C(5')); 69.99, 70.02, 70.71, 70.75,
76.42, 76.46 (6d, MeCHAr, C(2'), C(3')); 81.0 (d, J(C,P) ˆ 7.3, C(4')); 86.97, 87.02 (2d, C(1')); 92.85 (t, OCH2O);
116.39 (s, CN); 120.14 (s, C(5)); 124.28, 127.71, 128.31, 133.63 (4d, arom. C); 138.47 (s, arom. C); 139.54, 139.57
(2d, C(8)); 148.18 (s, C(4)); 149.82 (s, arom. C)); 153.06, 153.10 (2d, C(2)); 155.55 (s, C(6)); 170.06 (MeCO).
‡
31P-NMR (121 MHz, CDCl3): 0.026, 0.066. MALDI-MS: 636 (11, [M ‡ H] ), 471 (20), 150 (100).
2'-O-{[(1S)-1-(2-Nitrophenyl)ethoxy]methyl}adenosine 5'-(Triethylammonium Methyl Phosphate) (2 ¥
‡
Et3NH ). A soln. of 48 (120 mg, 0.19 mmol) in 10m MeNH2/EtOH (1.2 ml) was kept for 20 min at r.t., and
then evaporated. The residue was dissolved in H2O/EtOH 4 :1and extracted with CH Cl2. The aq. phase was
2
treated with Et3N, evaporated, and co-evaporated with Py: 2 (quant.). TLC (AcOEt/EtOH/H2O 5 :4 :1): Rf
1
0.31. H-NMR (300 MHz, D2O): 0.97 (d, J ˆ 6.2, MeCHAr); 1.24 (t, J ˆ 7.2, 9 H, Et3N); 3.16 (q, J ˆ 7.2, 6 H,
Et3N); 3.54 (d, J ˆ 10.9, POMe); 4.07 (br. s, 2 HÀC(5')); 4.36 (br. s, HÀC(4')); 4.49 (m, HÀC(2')); 4.55 (br. d,
J ˆ 7.1, 1 H, OCH2O); 4.67 4.75 (m, 3 H, HÀC(3'), MeCHAr, OCH2O); 6.1( d, J ˆ 7.1, H ÀC(1')); 7.33 7.45
(m, 2 arom. H); 7.57 (br. t, J ˆ 8, 1arom. H ); 7.74 (br. d, J ˆ 8, 1arom. H); 8.21( s, HÀC(2)); 8.46 (s, HÀC(8)).
13C-NMR (100 MHz, D2O): 10.9 (q, Et3N); 24.2 (q, MeCHAr); 49.3 (t, Et3N); 55.5 (q, J(C,P) ˆ 5.8, POMe); 67.8
(t, C(5')); 72.2, 73.0, 82.5 (3d, C(2'), C(3'), MeCHAr); 87.7 (d, J(C,P) ˆ 8.7, C(4')); 87.9 (d, C(1')); 95.3 (t,
OCH2O); 121.2 (s, C(5)); 126.8, 130.0, 131.3, 136.6 (4 d, arom. C); 139.4 (s, arom. C); 142.3 (d, C(8)); 150.3, 151.6
(2s, C(4), 1arom. C); 155.9 ( d, C(2)); 158.3 (s, C(6)). 31P-NMR (121 MHz, D2O): 2.269. ESI-MS (neg.): 593.3
(100, [M À H]À).
N6,N6,3'-O-Tribenzoyl-2'-O-methyladenosine (49). At 48, a soln. of adenosine (4 g, 15 mmol) in DMF
(60 ml) was treated with NaH (758 mg, 18 mmol). After 45 min at 48, MeI (3.4 g, 24 mmol) was added during
3 h [21]. At r.t., 1H-imidazole (3.0 g, 45 mmol) and tbdms-Cl (2.26 g, 15 mmol) were added. Workup (AcOEt/
hexane 1:1, NaHCO soln.) after 20 min and filtration over silica gel (CH2Cl2 ! CH2Cl2/MeOH 93 :7) gave a
3
mixture of products (2.5 g; TLC (CH2Cl2/MeOH 95 :5): Rf ca. 0.5). Part of this mixture (1.8 g) was dissolved in
CH2Cl2/Py 3 :1 (20 ml), treated with BzCl (2.64 g, 18.4 mmol), and stirred for 14 h at r.t. Workup gave a residue
(TLC (hexane/AcOEt 1:1): Rf ca. 0.7), which was dissolved in THF (90 ml), treated with 10m aq. HCl (10 ml)
and stirred for 5 min at r.t. Workup and CC (hexane/AcOEt 4 :1 ! 2 :3) gave 49 (1.9 g, 30%). Colorless solid.
TLC (hexane/AcOEt 1:1): Rf 0.32. UV (MeOH): 270 (18500), 248 (23800), 230 (28400). 1H-NMR (300 MHz,
CDCl3): 3.35 (s, MeO); 3.99 (d, J ˆ 13, HÀC(5')); 3.91( dd, J ˆ 10, 13, HÀC(5')); 4.51( s, HÀC(4')); 4.85 (dd, J ˆ
5, 8.1, HÀC(2')); 5.88 (d, J ˆ 5, HÀC(3')); 5.93 (br. d, J ˆ 10, OH); 6.05 (d, J ˆ 8.1, HÀC(1')); 7.34 7.39 (m, 5
arom. H); 7.47 7.50 (m, 3 arom. H); 7.63 (m, 1arom. H); 7.86 7.88 ( m, 4 arom. H); 8.12 8.15 (m, 2 arom. H);
8.20 (s, HÀC(2)); 8.68 (s, HÀC(8)). 13C-NMR (75 MHz, CDCl3): 59.1( q, MeO); 62.9 (t, C(5')); 72.5, 81.3, 86.7
(3d, C(2'), C(3'), C(4')); 89.8 (d, C(1')); 128.3, 128.6, 128.8 (3d, arom. H); 129.0, 129.3 (2s, C(5), arom. C); 129.5,
129.9, 133.2, 133.7 (4d, arom. C); 133.9 (s, arom. C); 145.1 (d, C(8)); 151.6 (d, C(2)); 151.8 (s, C(4)); 152.9 (s,
‡
‡
C(6)); 165.7 (s, OCOPh); 172.2 (NCOPh). MALDI-MS: 616 (100, [M ‡ Na] ), 594 (5, [M ‡ H] ), 512 (14), 472
(38), 366 (86), 344 (40), 251(71), 222 (53).
N6,N6,3'-O-Tribenzoyl-2'-O-methyladenosine 5'-(2-Cyanoethyl Methyl Phosphate) (50). A soln. of 49
(300 mg, 0.5 mmol) and 5-(benzylthio)-1H-tetrazole (211 mg, 1.1 mmol) in MeCN (1.5 ml) was treated with a
soln. of 0.25m (MeO)P(NiPr2)(OCH2CH2CN) in MeCN, (2.4 ml, 0.6 mmol) and kept for 20 min at r.t. After
evaporation, the residue was treated with a soln. of I2 (0.5 g) in H2O (0.4 ml), Py (4 ml), and THF (15 ml).
Workup (1. Na2S2O3 soln.; 2. NaHCO3 soln.) and CC (CH2Cl2 ! CH2Cl2/MeOH 2 :3) gave 48 (264 mg, 72%, 1:1

Helvetica Chimica Acta Vol. 83 (2000)
2501
diastereoisomer mixture). Colorless foam. TLC (CH2Cl2/MeOH 93 :7): Rf 0.4. UV (MeOH): 270 (20000), 248
(25900), 231(30700). 1H-NMR (300 MHz, CDCl3): 2.70 (m, OCH2CH2CN); 3.44 (s, MeO); 3.77, 3.81(2 d, J ˆ
3.1, POMe); 4.21 (m, OCH2CH2CN); 4.46 (m, 2 HÀC(5')); 4.58 (m, HÀC(4')); 4.81, 4.82 (2t, J ˆ 5.6, HÀC(2'));
5.75 (m, HÀC(3')); 6.26, 6.27 (2d, J ˆ 5.9, HÀC(1')); 7.37 (m, 4 arom. H); 7.50 (m, 4 arom. H); 7.64 (m, 1arom.
H); 7.86 (m, 4 arom. H); 8.12 (m, 2 arom. H); 8.36, 8.37 (2s, HÀC(2)); 8.699, 8.702 (2s, HÀC(8)). 13C-NMR
(75 MHz, CDCl3): 19.7 (t, J(C,P) ˆ 7.3, OCH2CH2CN); 55.1( q, J(C,P) ˆ 6.1, POMe); 59.4 (q, MeO); 62.37,
62.46 (2t, J(C,P) ˆ 4.8, OCH2CH2CN); 66.65, 66.75 (2t, J(C,P) ˆ 5.9, C(5')); 70.94, 70.98 (2d, C(2')); 81.41, 81.46
(2d, C(3')); 81.51, 81.61 (2d, J(C,P) ˆ 7.3, C(4')); 87.33, 87.46 (2d, C(1')); 116.58, 116.61 (2s, CN); 128.2 (s, C(5));
128.89, 129.02 (2d, arom. C); 129.2 (s, arom. C); 129.7, 130.2, 133.3, 134.1 (4d, arom. C); 134.3 (s, arom. C);
143.71, 143.76 (2d, C(8)); 152.4 (s, C(4)); 152.7 (br. d, C(2)); 153.2 (s, C(6)); 165.99 (s, OCOPh); 172.62 (s, 2C,
‡
NCOPh). 31P-NMR (121 MHz, CDCl3): 0.133, 0.161. MALDI-MS: 763 (100, [M ‡ Na] ), 659 (22), 588 (10),
275 (12).
‡
2'-O-Methyladenosine 5'-[Triethylammonium Methyl Phosphate] (51 ¥ Et3NH ). A soln. of 50 (200 mg,
0.27 mmol) in 10m MeNH2/EtOH (2 ml) was kept for 20 min at r.t. and then evaporated. The residue was
dissolved in H2O/EtOH 4 :1and the soln. extracted with CH 2Cl2. The aq. phase was treated with Et3N,
evaporated, and co-evaporated with Py: 51 (quant.). TLC (BuOH/AcOH/H2O 5 :2 :3): Rf 0.48. 1H-NMR
(300 MHz, D2O): 1.23 (t, Et3N); 3.16 (q, Et3N); 3.44 (s, MeO); 3.48 (d, J ˆ 10.9, POMe); 4.06 (dd, J ˆ 3.1, 4.7, 2
HÀC(5')); 4.34 (q, J ˆ 3.1, HÀC(4')); 4.47 (t, J ˆ 5.3, HÀC(2')); 4.63 (dd, J ˆ 3.7, 5, HÀC(3')); 6.14 (d, J ˆ 5.9,
HÀC(1')); 8.18 (s, HÀC(2)); 8.41( s, HÀC(8)). 31P-NMR (121 MHz, D2O): 2.18.
5'-O-[(tert-Butyl)dimethylsilyl]-2'-O-{[(triisopropylsilyl)oxy]methyl]uridylyl-(3' ! 5')-N6-{{{2-[(triisopropyl-
silyl)oxy]benzyl}oxy}carbonyl}-2'-O-{[triisopropylsilyl)oxy]methyl}adenylyl-(3'! 5')-2'-O-{[(triisopropylsilyl)oxy]-
methyl}guanylyl-(3' ! 5')-N4-{{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-2'-O-{[(triisopropysilyl)oxy]me-
thyl}cytidylyl-(3' ! 5')-N4-{{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-2'-O-{[(triisopropylsilyl)oxy]me-
thyl}cytidyl-(3' ! 5')-N4-{{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl}-2'-O-{[(S)-1-(2-nitrophenyl)ethoxy]-
methyl}-N6-{{{2-[(triisopropylsilyl)oxy]benzyl}oxy}carbonyl}adenosine (34). An automated 1.5-mmol synthesis
(−trityl-on× mode) was carried out on a Gene Assembler (Pharmacia) by the following protocol: 1) 1.5 min
detritylation with 4% CHCl2COOH in (CH2Cl)2; 2) 2.5 min coupling with the appropriate phosphoramidite
(0.12 ml of a 0.1m soln. in MeCN; 9: 0.1m in THF) and 5-(benzylthio)-1H-tetrazole (0.36 ml of a 0.35m soln. in
MeCN); 3) 1min capping with a 1:1mixture of Ac 2O/2,6-dimethylpyridine/THF 1:1:8 and 16% 1-methyl-1 H-
imidazole in THF; 4) 0.5 min oxidation with I2/H2O/Py/THF 2 :2 :20 :75. According to the trityl assay, the
average coupling yields were > 98%. The immobilized sequence (10 mg) was shaken with MeOH/Et3N 9 :1for
8 h at r.t. The solid materials were removed by filtration and washed with MeOH. The filtrate was freeze-dried,
dissolved in Py/DMF 1:1 (0.1ml), and freeze-dried again: crude 34 as an off-white solid.
Uridylyl-(3' ! 5')-adenylyl-(3' ! 5')-guanylyl-(3' ! 5')-cytidylyl-(3' ! 5')-cytidylyl-(3' ! 5')-2'-O-{[(S)-1-(2-
nitrophenyl)ethoxy]methyl}adenosine 3'-{N-{[(2-Nitrobenzyl)oxy]carbonyl}-l-phenylalaninate} (44) and Uri-
dylyl-(3' ! 5')-adenylyl-(3'! 5')-guanylyl-(3' ! 5')-cytidylyl-(3' ! 5')-cytidylyl-(3' ! 5')-2'-O-{[(S)-1-(2-nitrophe-
nyl)ethoxy]methyl}adenosine 3'-{N-{[(RS)-1-(2-Nitrophenyl)ethoxy]carbonyl}-l-phenylalaninate} (45). Crude 34
i
(obtained from 10 mg of solid support) was treated with 50 ml of a soln. prepared from Pr3NEt (0.24m), 4-
(pyrrolidin-1-yl)pyridine (0.1m), BOP (0.2m), and 39 or 41 (0.2m) in Py. After 4 h at r.t., H2O (50 ml) was added
and the mixture freeze-dried. The residue (!42 or 43, resp.) was dissolved in 0.5 ml of 1m Bu4NF ¥ 3 H2O/0.5m
AcOH in THF. After 2 h at r.t., 0.5 ml of Tris ¥ HCl buffer (pH 7.4) was added, and the mixture was desalted on a
Sephadex G-10 column (H2O). The product-containing fractions were analyzed by reversed-phase HPLC (Fig. 3).
The sequence 45 was additionally purified by reversed-phase chromatography. After desalting the pooled product-
‡
containing fractions, 6 OD260nm (20%) of pure 45 was obtained (Fig. 4). MALDI-TOF MS: 2378 ([M ‡ H] ).
Aminoacylated Nucleotides 1, 37, 52, 53, and 56: General Procedure (G.P.). The nucleotide 2 or 51 (25 mmol
for each experiment), was treated with 400 ml of a soln. prepared from iPr3NEt (0.24m), 4-(pyrrolidin-1-
yl)pyridine (0.1m), BOP (0.2m), and 39, 41, ent-39, or N-[(benzyloxy)carbonyl]-l-phenylalanine (0.2m), in Py.
After 2 h at r.t., the mixture was diluted with AcOEt and separated by CC (AcOEt ! AcOEt/EtOH 11:9 (‡1%
Et3N), then ! AcOEt/EtOH/H2O 11:9 :1): 45 50% yields.
2'-O-{[(S)-1-(2-Nitrophenyl)ethoxy]methyl}adenosine 5'-(Methyl Phosphate) 3'-{N-{[(2-Nitrobenzyl)oxy]-
carbonyl}-l-phenylalaninate} (1). According to the G.P. from 2 and 39: 55% yield. TLC (BuOH/AcOH/H2O
5 :2 :3): Rf 0.62. Reversed-phase HPLC (A ! 50% B): tR 28.1min. 31P-NMR (121 MHz, D2O/tBuOH 7:3,
ammonium citrate (pH 3.6)): 1.77. ESI-MS (neg.): 865.5 (100, [M À H]À), 343.2 (44), 269 (16), 134 (32).
2'-O-{[(S)-1-(2-Nitrophenyl)ethoxy]methyl}adenosine (5'-Methyl Phosphate) 3'-{N-{[(RS)-1-(2-Nitrophen-
yl)ethoxy]carbonyl}-l-phenylalaninate} (37). According to the G.P. from 2 and 41: 45% yield. TLC (BuOH/
AcOH/H2O 5 :2 :3): Rf 0.60. Reversed-phase HPLC (A ! 50% B): tR 28.5 min.

2502
Helvetica Chimica Acta Vol. 83 (2000)
2'-O-{[(S)-1-(2-Nitrophenyl)ethoxy]methyl}adenosine 3'-{N-[(Benzyloxy)carbonyl]-l-phenylalaninate} 5'-
(Methyl Phosphate) (52). According to the G.P., from 2 and N-[(benzyloxy)carbonyl]-l-phenylalanine: 52%
yield. TLC (BuOH/AcOH/H2O 5 :2 :3): Rf 0.65. Reversed-phase HPLC (A ! 50% B): tR 28.2 min.
2'-O-{[(S)-1-(2-Nitrophenyl)ethoxy]methyl}adenosine 5'-(Methyl Phosphate) 3'-O-{[N-{[(2-Nitrobenzyl)-
oxy]carbonyl}-d-phenylalaninate} (53). According to the G.P., from 2 and ent-39: 48% yield. TLC (BuOH/
AcOH/H2O 5 :2 :3): Rf 0.61. Reversed-phase HPLC (A ! 50% B): tR 27.6 min.
2'-O-Methyladenosine 5'-(Methyl Phosphate) 3'-{N-{[(2-Nitrobenzyl)oxy]carbonyl}-l-phenylalaninate}
(56). According to the G.P., from 51 and 39: 55% yield. TLC (BuOH/AcOH/H2O 5 :2 :3): Rf 0.48.
Reversed-phase HPLC (A ! 50% B): tR 24.0 min.
Aminoacylated Nucleotides Adenosine 5'-(Methyl Phosphate) 2'(3')-(l-Phenylalaninate) (38), Adenosine
2'(3')-{N-[(Benzyloxy)carbonyl]-l-phenylalaninate} 5'-(Methyl Phosphate) (54), Adenosine 5'-(Methyl Phos-
phate) 2'(3')-(d-Phenylalaninate) (55), and 2'-O-Methyladenosine 5'-(Methyl Phosphate) 2'(3')-(l-Phenyl-
alaninate) (57): General Procedure. The aminoacylated nucleotides 38, 54, 55, and 57 were obtained by
photolysis (30 min to 1.5 h, depending on the substrate and the concentration) of the corresponding protected
t
precursors 1 or 37, 52, 53, and 56 (c ˆ 150 250 mm), resp., in BuOH/H2O 1:1 at pH 3.4 (40 m m ammonium
citrate). After extraction with CH2Cl2, the crude products were desalted on 1g of C18-RP silica gel (H2O/
MeCN 4 :1( ‡20 mm AcOH, pH 3.1)). The product-containing fractions (UV; yields typically > 70%) were
pooled and analyzed by reversed-phase HPLC; the purity of such samples usually exceeded 95%. Aliquots of
these stock solns. were used directly for hydrolysis studies.
Additionally, a semi-prep. photolysis experiment was carried out with 1: a 2 mm soln. of 1 in D2O/tBuOH
7:3 (0.7 ml) (40 mm ammonium citrate (pH 3.4)) was photolyzed in a NMR tube. Periodically, 31P-NMR spectra
(121 MHz) and HPLCs were recorded. The deprotection was nearly finished after 3.5 h (HPLC) and gave 38 as
the only product. However, the 31P-NMR spectrum showed two signals at d 1.77 and 1.88 (ratio 3 :1), which were
attributed to the regioisomeric 3'-O- and 2'-O-aminoacylated nucleotides 38.
Hydrolysis Studies. Aliquots of the stock solns. containing each ca. 0.1m m aminoacylated nucleotide 38, 54,
55, or 57 (preparation, see above) were diluted with an equal volume of the appropriate buffer soln. and
incubated at 258. In the course of every individual experiment, 5 8 samples were removed at periodic intervals,
acidified with AcOH (! pH ca. 3), and analyzed by reversed-phase HPLC (detection at 260 nm). The integral
ratios between the signals of aminoacylated starting materials and hydrolyzed products were translated into
individual pseudo-first-order rate constants (Table).
REFERENCES
[1] A. Stutz, S. Pitsch, Synlett 1999, 930.
[2] C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, Science (Washington D.C.) 1989, 244, 182;
S. A. Robertson, J. A. Ellman, P. G. Schultz, J. Am. Chem. Soc. 1991, 113, 2722; T. G. Heckler, L. Chang, Y.
Zama, T. Naka, M. S. Chorghade, S. M. Hecht, Biochemistry 1984, 23, 1468; G. Baldini, B. Martoglio, A.
Schachenmann, C. Zugliani, J. Brunner, Biochemistry 1988, 27, 7951; M. van Cleve, in −Topics in Bioorganic
and Biological Chemistry: Bioorganic Chemistry: Nucleic Acids×, Ed. S. M. Hecht, Oxford University
Press, New York, 1996; Y. Ueno, T. Mishima, H. Hotoda, T. Hata, Chem. Lett. 1992, 595; T. Niinomi, M.
Sisido, Chem. Lett. 1993, 1305.
[3] M. D. Hagen, C. Scalfi-Happ, E. Happ, S. J. Chladek, J. Org. Chem. 1988, 53, 5040; M. D. Hagen, S. J.
Chladek, J. Org. Chem. 1989, 54, 3189.
[4] S. Chladek, in −Chemistry of Nucleosides and Nucleotides×, Vol. 3, Ed. L. B. Townsend, Plenum Press, New
York, 1994; S. Chladek, M. Sprinzl, Angew. Chem., Int. Ed. 1985, 24, 371.
[5] S. Pitsch, P. A. Weiss, X. Wu, D. Ackermann, T. Honegger, Helv. Chim. Acta 1999, 82, 1753; S. Pitsch, P. A.
Weiss, L. Jenny, −Ribonucleoside Derivative and Method for Preparing the Same×, US Patent 5,986,084 (16.
Nov. 1999); S. Pitsch, P. A. Weiss, X. Wu, L. Jenny, −Easy and Efficient Chemical Synthesis of
Oligoribonucleotides from 2'-O-{[(Triisopropylsilyl)oxy]methyl}-Protected Nucleoside Building Blocks×,
in preparation.
[6] G. R. Gough, T. J. Miller, N. A. Mantick, Tetrahedron Lett. 1996, 37, 981.
[7] R. Unno, H. Michishita, H. Inagaki, Y. Baba, T. Jomori, T. Nishikawa, M. Isobe, Chem. Pharm. Bull. 1997,
45, 125.
[8] Y. Hayakawa, M. Kataoka, J. Am. Chem. Soc. 1998, 120, 12395.
[9] S. Pitsch, Helv. Chim. Acta 1997, 80, 2286; X. Wu, S. Pitsch, Nucleic Acids Res. 1998, 26, 4315.

Helvetica Chimica Acta Vol. 83 (2000)
[10] R. T. Pon, S. Yu, Tetrahedron Lett. 1997, 38, 3327.
2503
[11] M. Taiji, S. Yokoyama, S. Higuchi, T. Miyazawa, J. Biochem. 1981, 90, 885; M. Taiji, S. Yokoyama, S.
Higuchi, T. Miyazawa, Biochemistry 1983, 22, 3220.
[12] J. Preiss, P. Berg, E. J. Ofengand, F. H. Bergman, M. Dieckmann, Proc. Natl. Acad. Sci. U.S.A. 1959, 45, 319.
[13] H. G. Zachau, Chem. Ber. 1960, 93, 1822; H. G. Zachau, W. Karan, Chem. Ber. 1960, 93, 1830.
[14] N. Coles, M. W. Bukenherper, A. Meister, Biochemistry 1962, 2, 317; M. Gatica, C. C. Allende, G. Mona,
J. E. Allende, J. Medina, Biochim. Biophys. Acta 1966, 129, 201; D. Hentzen, P. Mandel, J. P. Garel,
Biochim. Biophys. Acta 1972, 281, 228.
¬
[15] S. Chousterman, G. Herve, F. Chapeville, Bull. Soc. Chim. Biol. 1966, 48, 1295.
[16] F. Schuber, M. Pinck, Biochimie 1974, 56, 383; F. Schuber, M. Pinck, Biochimie 1974, 56, 397.
[17] M. Sprinzl, F. Cramer, Nature New Biol. 1973, 245, 3.
[18] A. Novogrodsky, Biochim. Biophys. Acta 1971, 228, 688.
¬
[19] D. Gasparutto, T. Livache, H. Bazin, A.-M. Duplaa, A. Guy, A. Khorlin, D. Molko, A. Roget, R. Teoule,
Nucleic Acids Res. 1992, 20, 5159.
[20] Y. Hayakawa, H. Kato, H. K. Uchiyama, R. Noyori, J. Org. Chem. 1986, 51, 2402; Y. Hayakawa, S.
Wakabayashi, H. Kato, R. Noyori, J. Am. Chem. Soc. 1990, 112, 1691; Y. Hayakawa, M. Hirose, M.
Hayakawa, R. Noyori, J. Org. Chem. 1995, 60, 925.
[21] E. Wagner, B. Oberhauser, A. Holzner, H. Brunar, G. Issakides, G. Schaffner, M. Cotten, M. Knollm¸ller,
C. R. Noe, Nucleic Acids Res. 1991, 19, 5965.
Received June 9, 2000