Solid-phase synthesis of peptide ribonucleic acids (PRNA)

Article abstract of DOI:10.1016/j.tet.2003.08.024

The range of available peptide ribonucleic acid (PRNA) monomers was fully expanded for the use in solid-phase synthesis of PRNA oligomers, which were designed to reversibly control the recognition and complexation behavior of the complementary DNA/RNA by external factors. A couple of PRNA 12-mers with desired purine-pyrimidine mixed sequences were prepared indeed in high yields by the solid-phase synthesis.

Full text of DOI:10.1016/j.tet.2003.08.024

Tetrahedron 59 (2003) 7871–7878
Solid-phase synthesis of peptide ribonucleic acids (PRNA)
a,b
Hirofumi Sato,a Yusuke Hashimoto,a Takehiko Wadaa and Yoshihisa Inoue
,
,
*
*
aDepartment of Molecular Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita Osaka 565-0871, Japan
bICORP Entropy Control Project, JST, 4-6-3 Kamishinden, Toyonaka 565-0085, Japan
Received 12 July 2003; revised 15 August 2003; accepted 19 August 2003
Abstract—The range of available peptide ribonucleic acid (PRNA) monomers was fully expanded for the use in solid-phase synthesis of
PRNA oligomers, which were designed to reversibly control the recognition and complexation behavior of the complementary DNA/RNA by
external factors. A couple of PRNA 12-mers with desired purine–pyrimidine mixed sequences were prepared indeed in high yields by the
solid-phase synthesis.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
The completion of the human genome sequencing project1
has driven our research interest and endeavor to the creation
of gene therapeutic drugs using the antisense and antigene
strategies. Thus, a number of nucleic acid derivatives and
analogues have been proposed for the use in gene therapy,
for which both the resistance to nuclease, and the enhanced
hybridization affinity2 are required for the oligomers as well
Chart 1.
as the resulting hybrids. Peptide nucleic acid (PNA),
originally proposed by Nielsen,3 is one of the most
successful nucleic acid analogues, which employ an
oligo[N-(2-aminoethyl)glycine] backbone instead of the
ribose–phosphate backbone in natural nucleic acid. How-
ever, these hitherto reported nucleic acid derivatives and
analogues, including PNA, have an inherent limitation or
drawback, lacking an ability to actively control the function
of these nucleic acids by external stimuli.
control of DNA recognition by external factor has not been
achieved and hence the PRNA approach is certainly useful
as a unique method to realize the ‘on-demand’ gene
therapeutic drug in the antisense strategy and may find
various biochemical and pharmaceutical applications as a
powerful and versatile tool. However, the range of available
PRNA is quite limited in variety at present, only simple
8-mers of homo-uracil sequence and of alternating uracil–
cytosine sequence having been synthesized by fragment
condensation method in solution.4 To further expand the
scope and also to demonstrate the general validity of the
PRNA strategy, it is absolutely necessary to establish
simpler-and-versatile synthetic routes not only to the PRNA
monomers of all four purine and pyrimidine nucleobases but
also to 10 to 20-meric PRNA oligomers of purine–
pyrimidine mixed sequences.2
We have recently proposed a new category 0of peptide
ribonucleic acid (PRNA), in which the 5 -amino-50-
deoxypyrimidine ribonucleoside moiety is appended to an
oligo(g-L-glutamic acid) backbone through the 50-amino
group4 (Chart 1).
Possessing improved solubility in water, longer ribose
tether, and matched helical pitch, g-PRNA 8-mers with an
isopoly(L-glutamic acid) backbone form a stable complex
with complementary DNAs. Furthermore, the recognition
and complexation behavior of g-PRNA 8-mers with target
DNAs can be controlled by borax added as an external
factor through the 20,30-borate formation.4 Such an active
In an effort to develop a simpler and more effective route to
oligo-PRNAs, we found that the solid-phase peptide
synthesis technique is applicable to the PRNA oligomer
synthesis. In this strategy, 9-fluorenemethyloxycarbonyl
(Fmoc), which is readily removable under mild basic
conditions,5 was employed as the protective group for the
N-terminus of PRNA monomer, while benzoyl (Bz) group
for the exocyclic amino residues of nucleobases.6 In this
paper, we report the syntheses of four Fmoc-protected
peptide ribonucleic acid monomers with uracil,
Keywords: peptide; L-glutamic acid; homo-uracil.
*
Corresponding authors. Tel.: þ81-668-797922; fax: þ81-668-797923;
e-mail: hiko@chem.eng.osaka-u.ac.jp
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.08.024

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H. Sato et al. / Tetrahedron 59 (2003) 7871–7878
N-Bz-cytosine, hypoxanthine, and N-Bz-adenosine nucleo-
bases, which were subsequently used in the solid-phase
syntheses of a couple of PRNA 12-mers with desired
purine–pyrimidine mixed sequences.
2.1. Preparation of 50-amino-50-deoxynucleosides
In this study, adenosine, cytidine, uridine, and inosine were
used as nucleic acid-recognizing moieties of PRNA.
Inosine, lacking the 2-amino group, is a substitute for the
guanosine nucleoside of natural nucleic acids and is also a
complementary nucleobase of cytosine. 50-Azido-50-deoxy-
nucleosides were prepared by reacting the corresponding
nucleosides with carbon tetrabromide and triphenyl-
phosphine in the presence of lithium azide.12 The exocyclic
amino group of adenosine and cytidine was protected by a
benzoyl group,6 which can survive the catalytic hydrogen-
ation to deprotect the benzyl group and also the trifluoro-
acetic acid (TFA) treatment to deprotect the Boc group, yet
be removed by the treatment with 28% aqueous ammonia.
The nucleosides with and without protected amino group
were converted to 50-azido-50-deoxy-nucleosides in
moderate to high yields (56–90%). 50-Azido-50-deoxy-
uridine (50-N3-Urd) and -inosine (50-N3-Ino) were purified
by column chromatography on silica gel, while exocyclic
amino-protected 50-azido-50-deoxycytidine (50-N3-Cyd(N 4-
Bz)) and -adenosine (50-N3-Ado(N 6-Bz)) by recrystalliza-
tion from ethanol. The azido residue of 50-azido-50-
deoxynucleoside was converted to an amino group by
catalytic hydrogenation.12 These 50-azido-50-deoxynucleo-
sides were readily hydrogenated in quantitative yields under
a mild condition. The 50-amino-50-deoxynucleosides thus
obtained were purified by reprecipitation from methanol by
adding ether (Scheme 2).
2. Results and discussion
Most of the existing antisense molecules are designed to
simply inhibit the genetic information transfer, while little
effort has been devoted to the active control of the
DNA/RNA recognition processes.2 In our previous
studies,4,7 we have proposed a new methodology and
effective tools for controlling DNA/RNA recognition by
external agent. This strategy employs a novel nucleic acid
analogue, i.e. g-peptide ribonucleic acid (g-PRNA), as the
recognition moiety with a built-in switch triggered by an
external agent. The borate ester formation of ribose’s cis-
20,30-diol and the synchronized 0 hydrogen-bonding inter-
action between the ribose’s 5 -amide proton and the
2-carbonyl oxygen of nucleobase act as the external and
internal switching devices (Scheme 1). Furthermore, we
have demonstrated for the first time that stable complexes of
g-PRNA with complementary oligonucleotides are readily
dissociated by adding borax. Thus, PRNA is certainly one of
the most promising antisense molecules of the next
generation and can be used as a powerful and versatile
tool for switching biological functions.
In this study, we applied the solid-phase peptide synthesis
technique to the construction of the peptide backbone of
PRNA oligomer. In this strategy, we employed the
Fmoc/benzotriazol-1-yloxytris (dimethylamino) phosphon-
iumhexafluoro-phosphate) (Bop), rather than tert-butyl-
oxycarbonyl (Boc)/Bop, pair for the protection of
N-termini, since the glycosyl bond of nucleoside derivatives
is acid-sensitive8 and the unprotected cis-20,30-hydroxyl
groups of PRNA is less stable under acidic condition.9 The
Fmoc protection allows us to use the newly developed resin
with a long ethylene glycol spacer (NovaSynw TRG resin),
which is known to give high coupling yields particularly in
the syntheses of bulky long peptides,10 whereas the Boc
protection requires the use of conventional, but somewhat
unstable, oxime resin as solid support, which is however
somewhat unstable and less reliable in some cases.11 We
first prepared the full set of Fmoc-protected PRNA
monomers, which are the essential building blocks for the
PRNA synthesis of desired sequence.
2.2. Preparation of N-Boc-protected g-PRNA monomers
PRNAs, in which 50-amino-50-deoxyribonucleoside moiety
is appended to each amino acid residue of the oligo(g-L-
glutamic acid) backbone, are designed not only to enhance
the hybridization affinity as well as the stability of antisense
molecule and its hybrid in cytosol, but also to reversibly
control the recognition behavior by external factors. In
PRNA monomers, 50-amino-50-deoxyribonucleoside unit is
tethered to the a-carboxyl of a-amin0o/g-carboxyl-protected
L-glutamic acid through the 5 -amino group, thus
reserving the ribose’s0 cis-20,30-diol for the cyclic borate
formation and the 5 -amide proton for the hydrogen-
bonding interaction with the pyrimidine’s 2-carbonyl.
Since the treatment with diisopropylethylamine (DIEA) is
unavoidable in the synthesis of PRNA monomer, Boc-
protected L-glutamic acid was chosen as the starting
compound (as the Fmoc protective group is unstable even
under mild basic conditionsa). In all PRNA monomer
Scheme 1. Proposed recognition control model of PRNA with complementary DNA.

H. Sato et al. / Tetrahedron 59 (2003) 7871–7878
7873
Scheme 2.
syntheses, N-Boc-L-glutamic acid g-benzyl ester (Boc-
Glu(OBzl)-OH) was condensed with 50-amino-50-deoxy-
nucleosides in the presence of Bop reagent and 1-hydroxy-
benzotriazole (HOBt) as condensation reagents. The
protected PRNA monomers showed solub0ilities in methanol
varying from highly soluble Boc-isoGln(5 U)-OBzl (3a) and
Boc-isoGln(50A(N 6-Bz))-OBzl (3d) to less soluble Boc-
isoGln(500I)-OBzl (3b), and then to slightly soluble Boc-
isoGln(5 C(N 4-Bz))-OBzl (3c). In particular, 3c could not
be subjected to open column chromatography. Hence, the
PRNA monomers containing uracil (3a), hypoxanthine
(3b), and adenine (3d) were purified by column chroma-
tography on silica gel, while the cytosine-containing PRNA
monomer (3c) was purified by reprecipitation from ether
with methanol (Scheme 3).
OBzl monomers (4a–d). Then, these PRNA-OBzl mono-
mers, possessing a free amino group at the a-position, were
reacted with N-(9-fluorenylmethyloxycarbonyloxy)
succinimide (Fmoc-OSu) to give Fmoc-protected PRNA-
OBzl monomers (5a–d). Fmoc-isoGln(50U)-OBzl 5a was
purified by reprecipitation from a methanol solution by
adding diethyl ether, while Fmoc-isoGln(50I)-OBzl (5b) and
Fmoc-isoGln(50A(N 6-Bz))-OBzl (5d) were purified by
short column chromatography. The Bzl protection of
Fmoc-PRNA-OBzl monomers was quantitatively removed
by catalytic hydrogenation over palladium/charcoal to
afford the corresponding Fmoc-PRNA monomers, carrying
a free carboxylic acid at the g-position for 0 solid-phase
peptide synthesis. Since the Fmoc-isoGln(5 C(N 4-Bz))-
OBzl (4c) showed poor solubility in DMF (although the
fully deprotected 50C(N 4-Bz)-PRNA monomer was
sufficiently soluble in DMF),0 then we employed a different
approach to Fmoc-isoGln(5 C(N 4-Bz))-OH (6c). In this
strategy, the Bzl group of 4c was first removed by catalytic
hydrogenation, and then the Fmoc group was introduced by
reaction with Fmoc-OSu to give 6c. Now, a full set of the
four Fmoc-protected PRNA monomers with a free carboxyl
group (Fmoc-isoGln(50N)-OH (6a–d) were prepared in
moderate overall yields (Scheme 4). The obtained Fmoc-
PRNA monomers were soluble and stable enough to be
handled in N-methylpyrrolidone (NMP),13 a representative
solvent employed in solid-phase peptide synthesis.
2.3. Preparation of N-Fmoc-protected g-PRNA
monomers
Fmoc, which is readily removable under mild basic
conditions, is one of the most frequently employed
a-amino-protecting groups in solid-phase peptide synthesis.
An alternative conventional solid-phase peptide synthesis
employs Boc as the protective group for the N-terminus and
trifluoroacetic acid (TFA) as the deprotecting reagent.
However, this method often causes depurination of
purinenucleoside derivatives upon treatment with TFA.7
We therefore used the Fmoc method in the solid-phase
synthesis of PRNAs.
2.4. Solid-phase synthesis of PRNA oligomers
Fmoc-protected PRNA monomers (6a–d) were prepared
from the corresponding Boc-PRNA-OBzl monomers 3a–d.
The Boc group of 3a–d was removed by the treatment with
TFA at room temperature to give the TFA salts of PRNA-
First of all, the compatibility of Fmoc/Bz-protected PRNA
monomers with the standard solid-phase peptide synthesis
protocol (Chart 2) was checked. It was thus examined
whether or not the Bz-protected exocyclic amino residue of
Scheme 3.

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H. Sato et al. / Tetrahedron 59 (2003) 7871–7878
Scheme 4. Preparation of Fmoc-protected PRNA monomers (6a–d).
nucleobase in PRNA monomers can survive the piperidine
treatment used in the Fmoc deprotection. HPLC and UV
spectral analyses indicated that both PRNA monomers,
containing N 4-benzoylcytosine and N 6-benzoyladenine, are
resistant to the piperidine treatment at ambient temperature,
and that the Bz protection is efficiently removed in
quantitative yield upon treatment with 28% aqueous
ammonia. It was further demonstrated that the conventional
solid-phase synthesis protocol does not damage the ribose’s
unprotected cis-20,30-diol of PRNA monomers appended to
the polymer support. Thus, practically no side reactions,
such as esterification, were found to occur in the oligomeric
PRNAs recovered from the solid support. These results
clearly indicate that oligomeric PRNAs can be prepared
from the Fmoc/Bz-protected PRNA monomers using the
standard solid-phase peptide synthesis protocol.
In this study to prepare a series of long-tethered oligomeric
PRNAs, we employed a newly developed solid support with
a long oxyethylene spacer (NovaSynw TRG resin), which
was shown to give high coupling yields particularly in the
synthesis of bulky or long peptides.7 Fmoc-PRNA monomer
(3 equiv.), Bop reagent (3 equiv.), HOBt (3 equiv.), and
DIEA (6 equiv.) were added to an NMP slurry of resin
(1 equiv.), and the peptide backbone was elongated step by
step (Chart 2). The Fmoc protective group was removed by
the treatment with 20–30% piperidine in NMP. In each
coupling step, the backbone elongation was confirmed by
the conventional Kaiser test and the UV detection of Fmoc–
piperidine adduct. If the result of Kaiser test was positive,
the coupling reaction was repeated 2–3 times until the
Kaiser test became negative and the Fmoc–piperidine
adduct was formed quantitatively. All of the four PRNA
monomers displayed excellent coupling efficiency and the
quantitative coupling was achieved even in the synthesis of
12-mers, as demonstrated below. In the final step of the
solid-phase synthesis, the Bz protection at the exocyclic
amino group of adenosine and cytosine was removed by the
treatment with 28% aqueous ammonia. After being
thoroughly washed with NMP and chloroform, the target
PRNA oligomer synthesized on the solid support was
cleaved and isolated from the resin by TFA containing 2.5%
water and 2.5% triisopropylsilane as scavengers. The crude
PRNA oligomers in TFA were reprecipitated by adding cold
ether to give TFA salts of PRNA oligomers as white
powder. The PRNA oligomers thus obtained were purified
by reversed phase preparative HPLC. Excellent yields of up
Chart 2. Solid-phase peptide synthesis using Fmoc protective group.

H. Sato et al. / Tetrahedron 59 (2003) 7871–7878
7875
to 98.5%/step were obtained and no byproduct was detected,
while lower coupling yields around 80% were reported for
the solution-state fragment condensation synthesis.
Similarly, the self-complementary PRNA 12-mer, NH2-III-
CCI-CII-CCC-Lys-OH (8), was obtained in good yield
(88.0%, 98.4%/step). The purity was checked with 1H NMR
and MALDI-TOF MS (positive mode; Found m/z 4535.15
(Mþ1); calcd 4533.68).
A couple of PRNA 12-mers, which contain either uracil and
hypoxanthine or uracil, adenine, and cytosine, were
synthesized indeed. Thus, PRNA 12-mer NH2-CCU-UAC-
UAU-CUC-Lys-OH (7) was prepared by the Fmoc solid-
phase synthesis on the newly developed solid support with
an amino residue content of 0.21 mmol/g. In the first step,
the solid support (100 mg) was suspended in an NMP
solution of Fmoc-Lys(Boc)-OH (29.5 mg, 0.63 mmol), Bop
reagent (28.0 mg, 0.63 mmol, 1 equiv.), and HOBt
(12.2 mg, 0.63 mmol, 1 equiv.), to which was added DIEA
(23.1 mL, 1.26 mmol, 2 equiv.), and then the resulting
solution was gently stirred at room temperature. After
15 min of stirring, the resin was washed 5 times with NMP,
and if the Kaiser test was negative Fmoc was removed by
treating the Lys(Boc)-appended resin with 30% piperidine
in NMP for 15 min. After the deprotection, the resin
was washed 5 times with NMP. The same procedure was
repeated until the PRNA 12-mer of desired sequence was
obtained by using the appropriate Fmoc-PRNA monomers,
i.e. Fmoc-isoGln(50U)-OH (37.5 mg, 0.63 mmol), Fmoc-
isoGln(500A(N 6-Bz)-OH (45.5 mg, 0.63 mmol), and Fmoc-
isoGln(5 C(N 4-Bz)-OH (44.0 mg, 0.63 mmol). After the
12-mer was reached, the resin was washed with water and
the benzoyl protection was removed by the treatment with
28% aqueous ammonia, and finally the resin was thoroughly
washed with NMP and chloroform. After drying under a
reduced pressure, the total resin weight was measured and
the crude yield of PRNA 12-mer was determined as 83.4%
(98.5%/step). A consistent yield of 82% was obtained from
the UV spectrometric determination of ammonium benzoate
solution obtained in the deprotection process of the solid-
phase PRNA synthesis. The deprotected PRNA 12-mer was
cleaved from the resin by TFA treatment and purified by
reversed phase preparative HPLC eluted with aqueous
acetonitrile. HPLC fractions, containing the PRNA 12-mer,
were collected and the combined fraction was freeze-dried
to give the target PRNA 12-mer, NH2-CCU-UAC-UAU-
CUC-Lys-OH (7), as white powder. The isolated PRNA
12-mer was analyzed by analytical HPLC on an RP18
column to show a single peak (.99% pure); see Figure 1.
MALDI-TOF mass spectrometric analysis (negative mode)
also indicated a .98% purity of PRNA 12-mer (found m/z
4436.51 (M); Calcd 4436.65).
3. Conclusion
We established the synthetic routes to a series of PRNA
monomers, carrying adenine, cytosine, hypoxanthine, and
uracil nucleobases as recognition sites. This enabled us to
fully expand the range of available PRNA monomers. We
further demonstrated that the newly synthesized Fmoc-
protected PRNA monomers are compatible with the
standard solid-phase peptide synthesis protocol, and oligo-
meric PRNAs with purine–pyrimidine mixed sequences can
be prepared. Indeed, representative PRNA 12-mers of
mixed sequences were synthesized in high yields by the
Fmoc solid-phase peptide synthesis, and were characterized
by HPLC and MALDI-TOF mass spectrometric analyses. It
is noted that the solid-phase synthesis is much more
convenient, efficient, and reliable than the conventional
fragment condensation method in solution reported pre-
viously. The present synthetic strategy should lead us to a
wide variety of PRNA oligomers with desired sequences,
which function as reversibly controllable antisense
molecules upon complexation with the complementary
DNAs. Studies along this line are currently in progress.
4. Experimental
4.1. General
Standard abbreviations for amino acids and protecting
groups are as recommended by the IUPAC-IUB Com-
mission on Biochemical Nomenclature.
All starting materials, reagents, and solvents were commer-
cially available and used without further purification.
Nucleosides were purchased from Seikagaku Co. Ltd.
(Tokyo, Japan). Boc-L-amino acids, Fmoc-L-amino acids,
and 1-hydroxybenzotriazole (HOBt) were purchased from
Peptide Institute, Inc. (Osaka, Japan). Other chemicals of
guaranteed grade were purchased Tokyo Kasei Kogyo Co.
(Tokyo, Japan) or Wako Pure Chemical Industries Co. Ltd.
(Osaka, Japan). Column chromatography was performed on
silica gel (70–230 mesh) from Kanto Kagaku. Gel filtration
was performed on Sephadex G-25 from Pharmacia. Nucleic
acid analogues were purified on an ODS column by elution
with 10% acetonitrile in water at a flow rate of 3 mL/min.
IR spectra were recorded on a JASCO FT/IR-230
spectrometer. UV spectra were recorded on a JASCO
V-550 UV/vis spectrophotometer equipped with a tempera-
1
ture controller. H NMR spectra were obtained on a JEOL
GSX-270 at 270 MHz or a Varian INOVA-600 at 600 MHz.
Chemical shifts are reported in parts per million (ppm)
relative to tetramethylsilane (dH¼0.0 ppm) as internal
standard. Mass spectral measurements were performed on
a JEOL AX-500 instrument by fast-atom bombardment
(FAB) ionization with nitrobenzyl alcohol (NBA) as a
Figure 1. HPLC trace of PRNA 12-mer (8), purified on a preparative ODS
column (acetonitrile/H2O¼9/91).

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H. Sato et al. / Tetrahedron 59 (2003) 7871–7878
matrix or on a Voyager RP from PerSeptive Biosystems
with a-cyano-4-hydroxycinnamic acid (a-CHCA) or
picolinic acid as a matrix (MALDI-TOF).
amorphous solid. To a solution of TFA·NH2-isoGln(C(N 4-
Bz))-OH (2.00 g) and 9-fluorenylmethyl N-succinimidyl
carbonate (Fmoc-OSu) (1.36 g, 4.03 mmol) in DMF
(100 mL), diisopropylethylamine (526, 4.20 mmol) was
added at 08C. The mixture was continuously stirred for
10 min at room temperature. The solvent was evaporated
under a reduced pressure and methanol was added to the
residue to precipitate Fmoc-isoGln(C(N 4-Bz))-OH as white
powder. The powder was filtered and washed with methanol
and dried under a reduced pressure to give the title
compound (1.40 g, 61%): nmax (KBr disc)/cm21 3300,
3060, 2930, 1700, 1660, 1560, 1490, 1390, 1320, 1260,
1150, and 1090; dH (270 MHz; DMSO-d6) 1.67–2.00 (2H,
m, b-CH2), 2.26, (2H, t, g-CH2), 3.40–3.55 (2H, m, 50-H),
3.79–4.13 (4H, m, fluorene–CH2–O, 30, 40), 4.16–4.31
(3H, m, a, 20, methine of fluorene0), 5.19 (1H, d, 30-OH), 5.52
(1H, d, 20-OH), 5.79 (1H, d, 1 -H), 7.25–8.22 (17H, m,
fluorene (8H), a-NHCO (1H), 6-H (1H), 5-H (1H), o, m,
p-H (5H), 50-NH (1H)), 11.27 (1H, s, 4-NHCO); HR-FAB
MS. Found m/z 698.2469 (Mþ1, error [ppm/mmu,
þ1.0/þ0.7]), calcd 698.2462 (MþH).
4.1.1. N a-Fluorenylmethyloxycarbonyl-N 5-(50-deoxy-50-
uridyl)-L-isoglutamine (Fmoc-isoGln(50U)-OBzl) (6a).
N 5-(50-Deoxy-50-uridyl)-L-isoglutamine 0 benzyl
ester
trifluoroacetic acid salt (TFA·isoGln(5 U)-OBzl) was
synthesized as described previously.4 To a solution of
TFA·isoGln(50U)-OBzl
(2.20 g,
3.81 mmol)
and
9-fluorenylmethyl N-succinimidyl carbonate (Fmoc-OSu)
(1.54 g, 4.57 mmol) in DMF (100 mL), diisopropylethyl-
amine (1.19 g, 7.62 mmol) was added at 08C. The mixture
was stirred for 10 min at room temperature. The solvent was
removed under a reduced pressure and methanol was added
to the residue to precipitate Fmoc-isoGln(50U)-OBzl (5a) as
a white precipitate, which was filtered, washed with
methanol, and dried under a reduced pressure to give the
product (2.16 g, 82.8%). A catalytic amount of 10% Pd/C
(ca. 0.1 g) was added to a solution of Fmoc-isoGln(50U)-
OBzl (2.16 g, 3.15 mmol) in methanol–DMF mixture (1:1
v/v). The mixture was stirred for 2 h under a hydrogen at-
mosphere, and then the catalyst was filtered. The filtrate was
concentrated by rotary evaporation under a reduced
pressure, to which a small amount of methanol and then
ether were added to give the title compound as white
powder (2.08 g, 3.12 mmol).
4.1.5. 50-Azido-5-deoxy-inosine (50-N3-Ino). Inosine
(890 mg,
3.0 mmol),
triphenylphosphine
(2.20 g,
8.4 mmol), and lithium azide (1.02 g, 21.0 mmol) were
suspended in dry DMF and carbon tetrabromide (2.78 g,
8.14 mmol) was added to the suspension. After stirring for
3 h at room temperature, the solvent was evaporated to
concentrate. The residue was purified by column chroma-
tography on silica gel with chloroform–methanol (5:1 v/v)
to give the title compound (775 mg, 88.0%): dH (270 MHz;
DMSO-d6) 3.51–3.72 (2H, m, 50-H), 3.94–4.27, (2H, m, 30-
H, 40-H), 4.63 (1H, q, 20-H), 5.4 (1H, d, 30-OH), 5.63 (1H, d,
20-OH), 5.9 (1H, d, 10-H), 8.08 (1H, s, 2-H), 8.33 (1H, s,
8-H). Anal. found: C, 40.95; H, 3.68; N, 33.32. Calcd for
C10H11N7O4: C, 40.96; H, 3.78; N, 33.44; FAB MS. Found
m/z 294 (Mþ1), calcd 293.09.
4.1.6. 50-Amino-50-deoxy-inosine (50-NH2-Ino) (2b). 10%
Pd/C (ca. 0.1 g) was added to a solution of 50-N3-Ino
(239 mg, 1.0 mmol) in methanol–DMF mixed solvent (1:1
v/v) (50 mL). After being stirred for 2 h under a hydro-
gen atmosphere (1 atm), the mixture was filtered and the
filtrate was evaporated to concentrate and a small amount of
methanol was added to the solution. Further, ether was
added to give the title compound as white powder (245 mg,
92%): dH (270 MHz; DMSO-d6) 2.71–2.91 (2H, m, 50-H),
3.45 (2H, bs, 50-NH2), 3.88, (1H, q, 40-H), 4.13 (1H, q, 30-H),
4.57 (1H, t, 20-H), 5.2–5.4 (2H, bs, 30-OH, 20-OH), 5.84 (1H,
d, 10-H), 8.06 (1H, s, 2-H), 8.36 (1H, s, 8-H). Anal. found: C,
44.67; H, 5.11; N, 25.91. Calcd for C10H13N5O4: C, 44.94;
H, 4.90; N, 26.21.); FAB MS. Found m/z 268 (Mþ1), calcd
267.10.
4.1.2. Fmoc-isoGln(50U)-OBzl (5a). nmax (KBr disc)/cm21
3320, 3070, 3950, 1690, 1530, 1450, 1400, 1320, 1280, and
1200; dH (270 MHz; DMSO-d6) 1.69–2.00 (2H, m, b-CH2),
2.37, (2H, t, g-CH2), 3.20–3.44 (2H, m, 50-H), 3.75–3.89
(2H, m, fluorene–CH2–O), 3.97–4.08 (2H, m, 30, 40), 4.15–
4.30 (3H, m, a, 20, fluorene methine), 5.07 (2H, s, OCH2–
Ph), 5.17–5.30 (1H, bs, 30-OH), 5.38–5.50 (1H, bs, 20-OH),
5.61 (1H, d, 5-H), 5.73 (1H, d, 10-H), 7.25–7.88 (15H, m,
fluorene0(8H), a-NHCO (1H), benzyl (5H), 6-H (1H)), 8.13
(1H, t, 5 -NH), 11.34 (1H, s, 3-NH). Anal. found: C, 63.18;
H, 5.39 N, 8.10. Calcd for C36H36N4O10: C, 63.15; H, 5.30;
N, 8.18; FAB MS. Found m/z 685 (Mþ1), calcd 684.24.
4.1.3. Fmoc-isoGln(50U)-OH (6a). nmax (KBr disc)/cm21
3320, 3060, 2940, 1700, 1540, 1450, 1390, 1260, and 1100;
dH (270 MHz; DMSO-d6) 1.67–2.00 (2H, m, b-CH2), 2.29,
(2H, t, g-CH2), 3.20–3.48 (2H, m, 50-H), 3.73–3.89 (2H, m,
fluorene–CH2–O), 3.96–4.09 (2H, m, 30, 40), 4.16–4.32
(3H, m, a, 20, methine of fluorene), 5.23–5.51 0(2H, bs, 20-
OH, 30-OH), 5.63 (1H, d, 5-H), 5.73 (1H, d, 1 -H), 7.25–
7.76 (10H, m, fluorene (8H), a-NHCO (1H), 6-H (1H)),
8.10 (1H, t, 50-NH), 11.35 (1H, s, 3-NH); HR-FAB MS.
Found m/z 595.2015 (Mþ1, error [ppm/mmu, 24.2/22.5]),
calcd 595.2040 (MþH).
4.1.4. N a-Fluorenylmethyloxycarbonyl-N 5-(50-deoxy-50-
cytidyl(N -benzoyl))-L -isoglutamine
(Fmoc-
4.1.7. N a-tert-Butyloxycarbonyl-N 5-(50-deoxy-50-inosyl)-
L-isoglutamine (Boc-isoGln(50I)-OBzl) (3b). To a solution
of N-tert-butyloxycarbonyl-L-glutamic acid g-benzyl ester
(Boc-L-Glu(OBzl)-OH) (1.32 g, 3.90 mmol), HOBt
(526 mg, 3.9 mmol), and BOP reagent (1.15 g, 3.9 mmol)
in DMF (50 mL), diisopropylethylamine (613 mg,
3.9 mmol) was added. After 30 s, 50-NH2-Ino (1.04 g,
3.9 mmol) was added and stirring was continued for 1 h at
room temperature. The solvent was removed under a
4
isoGln(50C(N 4-Bz)-OH) (6c). TFA·isoGln(50C(N 4Bz))-
OBzl was synthesized as described previously.4 A solution
of NH2-isoGln(50C(N 4Bz))-OBzl (3.20 g, 4.71 mmol) in
DMF–methanol (1:1 v/v) (500 mL) was vigorously stirred
under a hydrogen atmosphere with 10% Pd/C (ca. 0.3 g) for
6 h. The mixture was filtered and evaporated under a
reduced pressure. The residue was precipitated from ether to
give 2.00 g of TFA·NH2-isoGln(C(N 4-Bz))-OH as a white

H. Sato et al. / Tetrahedron 59 (2003) 7871–7878
7877
reduced pressure and the residue was purified by column
chromatography on silica gel with chloroform–methanol
(5:1 v/v) to give the title compound as white powder (1.56 g,
68.0%): nmax (KBr disc)/cm21 3420, 1680, 1520, 1460,
1390, 1250, and 1170; dH (270 MHz; DMSO-d6) 1.33 (9H,
s, t-Bu–H), 1.63–2.00 (2H, m, b-CH2), 2.36 (2H, g-CH2),
3.24–3.43 (2H, m, 50-H), 3.76–4.06 (3H, m, 20-H, 30-H, 40-
H), 4.53 (1H, q, a-CH2), 5.06 (2H, s, PhCH2), 5.31 (1H, d,
30-OH), 5.47 (1H, d, 20-OH), 5.83 (1H, d, 10-H), 6.98 (1H, d,
Boc-NH), 7.27–7.40 (5H, m, Ar-H), 8.00–8.12 (2H, m,
2-H, 50-NH) 8.30 (1H, s, 8-H). Found: C, 55.01; H, 6.12; N,
14.01. Calcd for C27H34N6O9: C, 55.28; H, 5.84; N, 14.33;
FAB MS. Found m/z 587 (Mþ1), calcd 586.2387.
4.1.8. N 5-(50-Deoxy-50-inosyl)-L-isoglutamine benzyl
ester trifluoroacetic acid salt (TFA-isoGln(50I)-OBzl)
(4b). Boc-isoGln(50I)-OBzl (1.01 g, 1.72 mmol) was dis-
solved in TFA (10 mL) and the solution was kept at 08C for
30 min. Ether was added to the reaction mixture to give the
title compound as an amorphous solid (1.03 g, 99%).
residue was purified by column chromatography on silica
gel with chloroform–methanol (7:1 v/v) to give the title
compound as white powder (2.11 g, 75.8%): nmax (KBr
disc)/cm21 3320, 1680, 1540, 1450, 1380, 1250, and 1170;
dH (270 MHz; DMSO-d6) 1.33 (9H, s, t-Bu–H), 1.66–2.00
(2H, m, b-CH2), 2.36 (2H, g-CH2), 3.29–3.54 (2H, m, 50-
H), 3.92–4.13 (3H, m, 20-H, 30-H, 40-H), 4.76 (1H, q, a-CH),
5.06 (2H, s, PhCH2), 5.37 (1H, d, 30-OH), 5.58 (1H, d, 20-
OH), 6.00 (1H, d, 10-H), 6.98 (1H, d, Boc-NH), 7.27–7.40
(5H, m, Ar-H), 7.56 (2H, t, Ar-m-H), 7.66 (1H, t, Ar-p-H),
8.05 (2H, d, Ar-m-H), 8.13 (1H, t, 50-NH), 8.71 (1H, s, 2-H)
8.80 (1H, s, 8-H), 11.25 (1H, s, 4-NH); Found: C, 56.66; H,
5.59; N, 14.22. Calcd for C34H39N7O9: C, 59.21; H, 5.70; N,
14.22; FAB MS. Found m/z 690 (Mþ1), calcd 689.28.
4.1.11. N 5-(50-Deoxy-50-inosyl)-L-isoglutamine benzyl
ester trifluoroacetic acid salt TFA·isoGln(50A(N 6-Bz))-
OBzl (4d). Boc-isoGln(50A(N 6-Bz))-OBzl (2.11 g,
3.03 mmol) was dissolved in TFA (10 mL) and the solution
was kept at 08C for 30 min. Ether (500 mL) was added to the
solution to give the title compound as an amorphous solid
(1.28 g, 60%).
4.1.9. N a-Fluorenylmethyloxycarbonyl-N 5-(50-deoxy-50-
inosyl)-L-isoglutamine (Fmoc-isoGln(50I)-OH) (6b). To a
solution of TFA·isoGln(50I)-OBzl (910 mg, 1.51 mmol) and
9-fluorenylmethyl N-succinimidyl carbonate (Fmoc-OSu)
(611 mg, 1.81 mmol) in DMF (50 mL), diisopropylethyl-
amine (475 mg, 3.02 mmol) was added at 08C. The reaction
mixture was continuously stirred for 10 min at room
temperature. The solvent was removed under a reduced
pressure and the residue was purified by column chroma-
tography on silica gel with chloroform–methanol (7:1 v/v)
to give Fmoc-isoGln(50I)-OBzl as white powder. The
powder was filtered and washed with methanol and dried
under a reduced pressure to give Fmoc-isoGln(50I)-OBzl
(943.8 mg, 87.7%). 10% Pd/C (ca. 0.1 g) was added to a
solution of Fmoc-isoGln(50I)-OBzl (943.8 mg, 1.32 mmol)
in methanol–DMF (1:1 v/v) (250 mL). After the reaction
mixture was stirred for 2 h under a hydrogen atmosphere
(1 atm), the catalyst was filtered and the filtrate was
evaporated to concentrate and a small amount of methanol
was added to the solution. Ether was further added and the
precipitate formed was filtered to give 880 mg of the title
compound (1.32 mmol, 87.4%): nmax (KBr disc)/cm21
3300, 3060, 2930, 1700, 1660, 1320, 1260, 1150, and
1090; dH (270 MHz; DMSO-d6) 1.63–1.97 (2H, m, b-CH2),
2.23 (2H, g-CH2), 3.33–3.46 (2H, m, 50-H), 3.91–4.07 (3H,
m, 40-H, fluorene–CH2–O), 4.16–4.30 (3H, m, 20-H, 30-H,
methine of fluorene), 4.53 (1H, t, a-CH), 5.83 (1H, d, 10-H),
7.26–7.93 (9H, m, fluolene (8H), a-NH), 8.11 (1H, s, 2-H),
8.19 (1H, t, 50-NH), 8.33 (1H, s, 8-H); HR-FAB MS. Found
m/z 619.2151 (Mþ1, error [ppm/mmu, 20.2/20.1]), calcd
619.2153 (MþH).
4.1.12. N a-Fluorenylmethyloxycarbonyl-N 5-(50-deoxy-
50-adenyl(N 6-benzoyl))-L-isoglutamine (Fmoc-iso-
Gln(50A(N 6-Bz))-OH) (6d). To a solution of TFA·
isoGln(A(N 6-Bz))-OBzl (3.60 g, 5.12 mmol) and 9-fluor-
enylmethyl N-succinimidyl carbonate (Fmoc-OSu) (2.07 g,
6.14 mmol) in DMF (100 mL), diisopropylethylamine
(1.32 g, 10.5 mmol) was added at 08C. The mixture was
continuously stirred for 10 min at room temperature. The
solvent was removed under a reduced pressure and
methanol was added to the residue to precipitate Fmoc-
isoGln(A(N 6-Bz))-OBzl as white powder. This powder was
filtered and washed with methanol and dried under a
reduced pressure (2.20 g, 62.3%). 10% Pd/C (10%, ca. 1 g)
was added to a solution of Fmoc-isoGln(A(N 6-Bz))-OBzl
(3.30 g, 4.78 mmol) in methanol–DMF mixed solvent
(30:70 v/v, 800 mL). After the reaction mixture was stirred
for 12 h under a hydrogen atmosphere (1 atm), the catalyst
was filtered and the filtrate was evaporated to concentrate
and the residue was purified by column chromatography on
silica gel with chloroform–methanol (1:1 v/v). The fraction
(Rf¼0.3 with 1:5 chloroform–methanol) was evaporated to
concentrate and the title compound was deposited as
powder (1.64 g, 49%): nmax (KBr disc)/cm21 3300, 3060,
1690, 1550, 1440, 1390, 1290, and 1100; dH (270 MHz;
DMSO-d6) 1.73–2.07 (2H, m, b-CH2), 2.38 (2H, g-CH2),
3.35–3.64 (2H, m, 50-H), 3.95–4.34 (6H, m, 40-H,
fluorene–CH2–O, 20-H, 30-H, methine of fluorene), 4.77
(1H, q, a-CH), 05.38 (1H, d, 30-OH), 5.62 (1H, d, 20-OH),
6.02 (1H, d, 1 -H), 7.22–8.05 (14H, m, fluolene (8H),
a-NH, Ar (5H)), 8.24 (1H, t, 50-NH), 8.72 (1H, s, 8-H), 8.80
(1H, s, 2-H), 11.25 (1H, s, 6-NH); HR-FAB MS. Found m/z
722.2584 (Mþ1, error [ppm/mmu, þ1.5/þ1.1]), calcd
722.2574 (MþH).
4.1.10. N a-tert-Butyloxycarbonyl-N 5-(50-deoxy-050-ade-
nyl(N 6-benzoyl))-L-isoglutamine Boc-isoGln(5 A(N 6-
Bz))-OBzl (3d). To a solution of N-tert-butyloxycarbonyl-
L-glutamic acid-g-benzyl ester (Boc-L-Glu(OBzl)-OH)
(1.40 g, 4.04 mmol), HOBt (1.36 g, 4.04 mmol), and BOP
reagent (546 mg, 4.04 mmol) in DMF (100 mL), diiso-
propylethylamine (653 mg, 4.04 mmol) was added. After
30 s, 50-NH2-Ado(N6-Bz)12 (1.5 g, 4.04 mmol) was added
and stirring was continued for 1 h at room temperature. The
solvent was removed under a reduced pressure and the
4.1.13. NH2-CCU-UAC-UAU-CUC-Lys-OH (7). nmax
(KBr disc)/cm21 3400, 1660, 1550, 1480, 1400, 1230,
1150, and 1050; dH (270 MHz; DMSO-d6) 1.34 (4H, m,
g-CH2 (Lys) and d-CH2 (Lys)), 1.76–1.90 (24H, m, b-CH2
(Glu)), 2.14 (24H, m, g-CH2 (Glu)), 2.99 (2H, m, e-CH2
(Lys)), 3.33–3.46 (24H, m, 50-H (U, C and A)), 3.91–4.07

7878
H. Sato et al. / Tetrahedron 59 (2003) 7871–7878
(12H, m, 40-H (U, C and A)), 4.16–4.30 (24H, m, 20-H, 30-H
(U, C and A)), 4.53 (13H, m, a-CH (U, C, A and Lys)),05.17
(12H, m, 30-OH (U, C and A)), 5.40–5.52 (12H, m, 2 -OH
(U, C and A)), 5.64 (5H, m, 5-H (U)), 5.72–5.83 (12H, m,
10-H (U, C and A)), 7.23 (2H, bs, e-NH2 (Lys)), 7.32 (5H, m,
5-H (C)), 7.64 (5H, m, 6-H (U)), 7.92–8.13 (14H, m, a-NH
and NH2), 8.11 (2H, s, 2-H(A)), 8.19–8.22 (17H, m, 50-NH
(U, C and A) and 6-H (C)), 8.33 (2H, s, 8-H(A)), 11.33
(12H, m, 3-NH (U), 4-NH (C), 6-NH (A)); MALDI-TOF
MS (a-CHCA). Found m/z 4436.51 (M), calcd 4436.65.
A.; Kramer, J. B.; Cook, L. L.; Fulton, R. S.; Johnson, D. L.;
Minx, P. J.; Clifton, S. W.; Hawkins, T.; Branscomb, E.;
Predki, P.; Richardson, P.; Wenning, S.; Slezak, T.; Doggett,
N.; Cheng, J.F.; Olsen, A.; Lucas, S.; Elkin, C.; Uberbacher,
E.; Frazier, M. Nature 2001, 409, 860–921. (b) Venter, J. C.;
Adams, M. D.; Myers, E. W.; Li, P. W.; Mural, R. J.; Sutton,
G. G.; Smith, H. O.; Yandell, M.; Evans, C. A.; Holt, R. A.;
Gocayne, J. D.; Amanatides, P.; Ballew, R. M.; Huson, D. H.;
Wortman, J. R.; Zhang, Q.; Kodira, C. D.; Zheng, X. H.; Chen,
L.; Skupski, M.; Subramanian, G.; Thomas, P. D.; Zhang, J.;
Gabor Miklos, G. L.; Nelson, C.; Broder, S.; Clark, A. G.;
Nadeau, J.; McKusick, V. A.; Zinder, N.; Levine, A. J.;
Roberts, R. J.; Simon, M.; Slayman, C.; Hunkapiller, M.;
Bolanos, R.; Delcher, A.; Dew, I.; Fasulo, D.; Flanigan, M.;
Florea, L.; Halpern, A.; Hannenhalli, S.; Kravitz, S.; Levy, S.;
Mobarry, C.; Reinert, K.; Remington, K.; Abu-Threideh, J.;
Beasley, E.; Biddick, K.; Bonazzi, V.; Brandon, R.; Cargill,
M.; Chandramouliswaran, I.; Charlab, R.; Chaturvedi, K.;
Deng, Z.; Di Francesco, V.; Dunn, P.; Eilbeck, K.;
Evangelista, C.; Gabrielian, A. E.; Gan, W.; Ge, W.; Gong,
F.; Gu, Z.; Guan, P.; Heiman, T. J.; Higgins, M. E.; Ji, R. R.;
Ke, Z.; Ketchum, K. A.; Lai, Z.; Lei, Y.; Li, Z.; Li, J.; Liang,
Y.; Lin, X.; Lu, F.; Merkulov, G. V.; Milshina, N.; Moore,
H. M.; Naik, A. K.; Narayan, V. A.; Neelam, B.; Nusskern, D.;
Rusch, D. B.; Salzberg, S.; Shao, W.; Shue, B.; Sun, J.; Wang,
Z.; Wang, A.; Wang, X.; Wang, J.; Wei, M.; Wides, R.; Xiao,
C.; Yan, C. Science 2001, 291, 1304–1351.
4.1.14. NH2-III-CCI-CII-CCC-Lys-OH (8). nmax (KBr
disc)/cm21 3420, 1660, 1560, 1490, 1390, 1260, 1170, and
1050; dH (270 MHz; DMSO-d6) 1.34 (4H, m, g-CH2 (Lys)
and d-CH2 (Lys)), 1.75–1.92 (24H, m, b-CH2 (Glu)), 2.11
(24H, m, g-CH2 (Glu)), 2.99 (2H, m, e-CH2 (Lys)), 3.32–
3.47 (24H, m, 50-H (C and I), 3.91–4.07 (12H, m, 40-H (C
and I)), 4.16–4.34 (24H, m, 20-H, 30-H (C a0nd I)), 4.54
(13H, m, a-CH (C, I and Lys)), 5.17 (12H, m, 3 -OH (C and
I)), 5.40–5.52 (12H, m, 20-OH (C and I)), 5.72–5.83 (12H,
m, 10-H (C and I)), 7.23 (2H, bs, e-NH2 (Lys)), 7.32 (5H, m,
5-H (C)), 7.92–8.13 (14H, m, a-NH and NH2), 8.19 (2H, s,
2-H(I)), 8.20–8.25 (17H, m, 50-NH (C and I) and 6-H (C)),
8.33 (2H, s, 8-H(I)), 11.35 (6H, m, 4-NH (C)); MALDI-TOF
MS (a-CHCA). Found m/z 4535.15 (Mþ1), calcd 4533.68.
Acknowledgements
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Financial support by a Grant-in-Aid for Scientific Research
from MEXT and Research Fellowships of the Japan Society
for the Promotion of Science for Young Scientists are
gratefully appreciated. We would like to thank Dr H. Mihara
of Tokyo Institute of Technology for kind guidance to solid
state synthesis. We also thank Dr Guy A. Hembury for
assistance in the preparation of this manuscript.
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