Synthesis and properties of aminoacylamido-AMP: Chemical optimization for the construction of an N-acyl phosphoramidate linkage

Article abstract of DOI:10.1021/jo0008338

This paper describes the design and synthesis of a new type of aminoacyl-adenylate analogue (aa-AMPN) having an N-acyl phosphoramidate linkage where the oxygen atom of the mixed anhydride bond of aminoacyl-adenylate (aa-AMP) is replaced by an amino group. This new type of aa-AMP analogue is expected to be useful as material for studies on the recognition mechanism of the aminoacylation of tRNA and other biochemical reactions. The condensation of phosphoramidite derivatives of carboxamides with nucleoside derivatives failed, because the activated phosphoramidite derivatives reacted with not only the hydroxyl groups but also another reactive species. An alternative approach was examined by the reaction of 5'-O-phosphoramidite adenosine derivatives with carboxamide derivatives. The TBTr and TSE groups were chosen for protection of the amino group of amino acid amides and the phosphate group, respectively. Detailed studies revealed that the use of 5-(3,5-dinitrophenyl)-1H-tetrazole as an activating catalyst of phosphoramidites resulted in rapid condensation within 10 min to give fully protected aa-AMPN derivatives. No side reaction occurred. Deprotection of these products via a two-step procedure gave aa-AMPN derivatives in good yields. It also turned out that aa-AMPNs thus obtained are stable under both acidic and basic conditions, such as 0.1 M HCl (pH 1.0) and 0.1 M NaOH (pH 13.0).

Full text of DOI:10.1021/jo0008338

J . Org. Chem. 2000, 65, 8229-8238
8229
Syn th esis a n d P r op er ties of Am in oa cyla m id o-AMP : Ch em ica l
Op tim iza tion for th e Con str u ction of a n N-Acyl P h osp h or a m id a te
Lin k a ge
Tomohisa Moriguchi, Terukazu Yanagi, Masao Kunimori, Takeshi Wada, and Mitsuo Sekine*
Faculty of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, J apan
msekine@bio.titech.ac.jp
Received May 31, 2000
This paper describes the design and synthesis of a new type of aminoacyl-adenylate analogue (aa-
AMPN) having an N-acyl phosphoramidate linkage where the oxygen atom of the mixed anhydride
bond of aminoacyl-adenylate (aa-AMP) is replaced by an amino group. This new type of aa-AMP
analogue is expected to be useful as material for studies on the recognition mechanism of the
aminoacylation of tRNA and other biochemical reactions. The condensation of phosphoramidite
derivatives of carboxamides with nucleoside derivatives failed, because the activated phosphora-
midite derivatives reacted with not only the hydroxyl groups but also another reactive species. An
alternative approach was examined by the reaction of 5′-O-phosphoramidite adenosine derivatives
with carboxamide derivatives. The TBTr and TSE groups were chosen for protection of the amino
group of amino acid amides and the phosphate group, respectively. Detailed studies revealed that
the use of 5-(3,5-dinitrophenyl)-1H-tetrazole as an activating catalyst of phosphoramidites resulted
in rapid condensation within 10 min to give fully protected aa-AMPN derivatives. No side reaction
occurred. Deprotection of these products via a two-step procedure gave aa-AMPN derivatives in
good yields. It also turned out that aa-AMPNs thus obtained are stable under both acidic and basic
conditions, such as 0.1 M HCl (pH 1.0) and 0.1 M NaOH (pH 13.0).
In tr od u ction
Some nucleotide antibiotics bearing a P-N bond at the
5′-hydroxyl of the adenosine derivatives have been found
to date.1-3 Phosmidosine is a new type of antifungal
antibiotic, which was isolated from a culture filtrate of a
newly isolated storeptomycete identified as Streptomyces
sp.1 This unique antibiotic exhibits specific inhibitory
activity against spore formation of Botytis cinerea, a
pathogenic fungus that causes a gray mold disease in a
variety of fruits and vegetables. It was found by mass
spectrometry and NMR spectroscopy that phosmidosine
has a novel proline-containing nucleotide type antibiotic.4
Moreover, it has an N-acyl phosphoramidate linkage that
connects a nucleoside analogue, 8-oxoadenosine, with an
L-proline residue (Figure 1).
On the other hand, Agrocin 84 is an adenine nucleotide
antibiotic that controls crown gall disease biologically.2
Its structure was determined by sequential degradation
that showed an N-acyl phosphoramidate linkage at the
5′-hydroxyl moiety of the adenosine analogue. Dinogunel-
lin is a toxic phospholipid found in the northern blenny
roe, which also has an N-acyl phosphoramidate linkage
at the 5′-hydroxyl of adenosine.4 To synthesize such
natural products having N-acyl phosphoramidate link-
age,5,6 the construction of the N-acyl phosphoramidate
linkages seems to be a key step. We have previously
(1) Uramoto, M.; Kim, C. J .; Shin-ya, K.; Kusakabe, H.; Isono, K. J .
Antibiot. 1991, 44, 375.
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Microb. Toxins, Proc. Int. Symp., 4th 1974, 2, 145. (b) Hatano, M.;
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Chem. 1993, 58, 854.
F igu r e 1. Natural products having N-acyl phosphoramidate
linkages.
reported the attempt for the synthesis of the natural
products.5,6
10.1021/jo0008338 CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/02/2000

8230 J . Org. Chem., Vol. 65, No. 24, 2000
Moriguchi et al.
It is well-known that aminoacyl-adenylates are very
important intermediates in protein biosynthesis. The
natural products have a mixed anhydride bond formed
by condensation between the phosphate group of 5′-AMP
and the carboxylic group of an amino acid. It was also
found that the mixed anhydride bond is extremely
unstable under aqueous conditions.7 From a different
point of view, natural products bearing the N-acyl phos-
phoramidate linkage described above are considered as
the analogues of aminoacyl-adenylates (aa-AMP). Since
the displacement of the oxygen atom of the mixed anhy-
dride bond of aa-AMP with an NH group gives the N-acyl
phosphoramidate derivatives, the synthesis of aa-AMP
analogues having an N-acyl phosphoramidate linkage is
important to understand the recognition mechanism of
an amino acid of aa-AMP by the cognate aminoacyl-tRNA
synthetase (ARS). The presence of a hydrogen bond
network between the aa-AMP and the cognate ARS has
been suggested by site-directed mutagenesis or crystal
structure analysis.8,9 In all cases, the oxygen atom of the
mixed anhydride bond is not recognized by the polypep-
tide residue of the cognate ARS. Therefore, it is expected
that substitution of this unrecognizable atom would not
affect the enzyme recognition ability and would show
highly specific enzyme inhibitory activity.
Various aa-AMP analogues have been reported as
stable mimics of aa-AMP to date. Some of them were
designed as inhibitors of the aminoacylation reaction of
tRNA by ARSs.10-12 Others were used for cocrystalliza-
tion with ARSs or complexes of ARS-tRNA to elucidate
the recognition mechanism of tRNAs and aa-AMPs by
the cognate ARSs.13,14 In all cases, however, the amino
acid core structure or the mixed acid anhydride bond was
changed to the stable but obviously structurally different
substrates having aminoalkyl phosphoryl10,11 and pyro-
phosphate12 linkages. Since it is likely that the P-N bond
of the N-acyl phosphoramidate linkage is stable under
physiological conditions, the aa-AMP analogue bearing
an N-acyl phosphoramidate linkage can be expected to
have activity as a potential specific inhibitor in aminoa-
cylation of tRNA.
plored. In this paper, we report the synthesis and
properties of a new type of aminoacyl-adenylate ana-
logues which have an N-acyl phosphoramidate linkage.
Resu lts a n d Discu ssion
Con str u ction of N-Acylp h osp h or a m id a te Lin k -
a ges via Ca r boxa m id e P h osp h or a m id ite Der iva -
tives. Synthesis of N-acyl phosphoramidate derivatives
15-19
has been reported previously.
The synthetic strate-
gies are classified into three types: The first involves
C-N bond forming reaction by the N-acylation of phos-
phoramidates.15,16 The second involves construction of the
P-N bond by the reaction of a carboxamide anion species
with phosphorochloridates.17,18 The last example involves
the Arbzov-type reaction of trialkyl phosphites with
N-haloamides.19 However, these methods require the use
of strong bases, such as butyllithium, and are limited to
the synthesis of only a few substrates. Recently, Grandas
et al. have reported the synthesis of DNA-peptide hybrids
connected through the N-acyl phosphoramidate linkage.20
The naturally occurring DNA-peptide hybrid, which had
been synthesized by several groups,21-25 has a base-labile
phosphodiester linkage, therefore the several problems
happen to synthesize this hybrid. According to their
paper, the construction of the N-acyl phosphoramidate
linkage was achieved by the phosphoramidite method via
condensation of carboxamides with nucleoside deriva-
tives. Two synthetic strategies are available to construct
the N-acylphosphoramidate linkage. One strategy is the
condensation of phosphoramidite derivatives of carboxa-
mide derivatives with nucleoside derivatives followed by
oxidation. The other method is the condensation of
nucleoside phosphoramidite derivatives with carbox-
amide derivatives.
To obtain the key intermediates 4a and 4d in one of
the two approaches, the N-protected phenylalaninamide
derivatives 3a and 3d were synthesized as starting
materials (Scheme 1). The N-Fmoc phenylalaninamide
3a 21 was synthesized by the amidation of the correspond-
ing carboxylic acid derivative with NH4HCO3 in the
presence of EEDQ. An attempt to synthesize the N-Tr
To achieve the synthesis of such aa-AMP analogue, the
construction of the N-acyl phosphoramidate linkage is a
key step. Therefore, a general method for the synthesis
of the N-acyl phosphoramidate linkage should be ex-
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Synthesis and Properties of Aminoacylamido-AMP
J . Org. Chem., Vol. 65, No. 24, 2000 8231
Ta ble 1. P r ep a r a tion of N-Acyl P h osp h or a m id a te
Lin k a ge in th e P r esen ce of Va r iou s Activa tor s
Sch em e 1
time
yield of 8a
entry
activators
1H-tetrazole
5-(4-nitrophenyl)-1H-tetrazole
5-(3,5-dinitrophenyl)-1H-tetrazole
N-methylimidazolium triflate
N-methylimidazolium perchlorate
o-sulfobenzimide
(min)
(%)
1
2
3
4
5
6
30
30
5
30
30
30
17
46
quant.
78
72
22
a
Conditions: (a) 0.15 mmol of 6 (82 mg), 0.1 mmol of 7 (14 mg),
0.3 mmol of activators in CH3CN (2 mL) at room temperature; (b)
b
0.5 mmol of t-BuOOH (94 µL) for 5 min at room temperature. All
yields were estimated by 31P NMR chemical shifts.
midate linkage was previously.20 In the paper, the N-acyl
phosphoramidate linkage was constructed by the reaction
of the nucleoside 3′-phosphoramidate derivatives with
carboxamide derivatives in the presence of 1H-tetrazole.
Previously, we reported the synthesis of nucleoside
N-acylphosphoramidate derivatives by use of 5-(4-nitro-
phenyl)-1H-tetrazole30 in place of 1H-tetrazole as an
activator of nucleoside 5′-O-phosphoramidite deriva-
tives.31 However, the yields of the coupling reactions were
around 40%. Therefore, to optimize the coupling reaction
of phosphoramidite derivatives and carboxamide deriva-
tives, a model reaction 6 with phenylacetamide (7) was
studied in the presence of various types of activators. The
reaction was monitored by 31P NMR.
derivative 3d in a similar manner failed. In this reaction,
the ethoxycarbonylated derivative was obtained as the
main product. This amidation reaction requires an
electron-withdrawing group for the amino protecting
group.26 Therefore, compound 3d was prepared by hy-
drogenation of the N-Cbz phenylalaninamide 3b followed
by N-tritylation.27,28
The N-carbamate-type derivative 3a was phosphityl-
ated by the reaction with 2-cyanoethyl N,N-diisopropyl-
aminophosphorochloridite to give the phosphorodiamidite
derivative 4a . The formation of the phosphorodiamidite
1
4a was confirmed by H NMR and 31P NMR (δP 118.2
These results are summarized in Table 1. The activa-
tors tested have been used for the activation of less
reactive phosphoramidite derivatives, such as phospho-
rothioamidite derivatives.32 Most of these activators were
unsatisfactory because the reaction did not complete.
While 1H-tetrazole (entry 1), which is commonly used for
the current oligonucleotides synthesis by the phosphora-
midite method, required long reaction time, it was found
that other tetrazole derivatives with an electron-with-
drawing group at C-5 of tetrazole showed better yields.
It seems that the once generated phosphorotetrazolide
intermediates should have more efficient leaving ability
on the tetrazole residues. 1H-Tetrazole has sufficient
acidity (pKa ) 4.8)33 for the activation of a phosphora-
midite derivative. However, it is likely that the phospho-
rotetrazolide intermediate derived from 6 dose not have
sufficient reactivity to effect new P-N bond formation.
A more acidic tetrazole derivative, 5-(4-nitrophenyl)-1H-
tetrazole (pKa ) 3.8)34 resulted in an improved yield
(46%). A more satisfactory result was obtained in the case
of 5-(3,5-dinitrophenyl)-1H-tetrazole (entry 3).35 The
reaction proceeded within only 10 min to give the desired
product quantitatively. Since the phosphite intermediate
ppm) after simple purification using reprecipitation from
hexane. Next, reaction of compound 4a was carried out
with N-benzoyl-2′,3′-di-O-benzoyladenosine 9 in the pres-
ence of 1H-tetrazole. However, many products were found
as suggested by 31P NMR spectra.
The reaction of the N-Tr derivative 4d with 9 in the
presence of 1H-tetrazole gave a cyclized derivative 5d .
1
The structure of 5d was determined by H, 13C, and 31P
NMR and FAB mass spectra. This compound resulted
from the reaction of the activated phosphoramidite with
the NH group of the N-Tr part of the same compound.
Recently, van Boom et al. have reported the synthesis
of a partial structure of Agrocin 84, which has an N-acyl
phosphoramidate linkage. They used the reaction of a
carboxamide phosphoramidite derivative with 5′-OH of
the adenosine derivative.29 In this case, no intramolecular
side-reaction occurred because an R-amino group was not
present in the N-acyl phosphoramidate derivative.
Con str u ction of N-Acylp h osp h or a m id a te Lin k -
a ges via Nu cleosid es 5′-P h osp h or a m id ite Der iva -
tives. Next, we attempted to construct the N-acyl phos-
phoramidate linkage by the reaction of carboxamides with
nucleoside phosphoramidite derivatives. The synthesis of
the DNA-peptide hybrids having the N-acyl phosphora-
(30) Froehler, B. C.; Matteucci, M. D. Tetrahedron Lett. 1993, 24,
3171.
(26) Nozaki, S.; Muramatsu, I. Bull. Chem. Soc. J pn. 1988, 61, 2647.
(27) Hillmann-Elies, A.; Hillmann, G.; J atzkewitz, H. Z. Naturforsch.
1953, 8b, 445.
(28) Zervas, L.; Theodoropoulos, D. M. J . Am. Chem. Soc. 1956, 78,
1359.
(29) (a) Filippov, D.; Timmers, C. M.; van der Marel, G. A.; van
Boom, J . H. Nucleosides Nucleotides 1997, 16, 1403. (b) Filippov, D.;
Timmers, C. M.; Roerdink, A. R.; van der Marel, G. A.; van Boom,. J .
H. Tetrahedron Lett. 1998, 39, 4891.
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Lett. 1998, 39, 3725.
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1991, 113, 3972.
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2291.

8232 J . Org. Chem., Vol. 65, No. 24, 2000
Moriguchi et al.
Sch em e 2
is found to be also very unstable, prolonged reaction time
promotes the hydrolysis of this intermediate. In contrast,
N-methylimidazolium derivatives36 (entries 4 and 5)
reacted with the 5′-phosphoramidite derivative to form
the phosphorimidazolide intermediate indicated in 31P
NMR (δP 130 ppm), and gave the desired product in a
moderate yield. The other acid activators, such as N-
methylanilinium salts or carboxylic acids, showed un-
satisfactory results. Formation of the N-acyl phosphora-
midate linkage requires sufficiently reactive azolide
intermediates capable of substitution with poor nucleo-
philic carboxamide derivatives.
As far as side reactions are concerned, only the
coupling efficiency was poor in the case of 1H-tetrazole
and the reagents tested in this study except for 5-(3,5-
dinitrophenyl)-1H-tetrazole and the competitive hydroly-
sis of the phosphoramidite derivatives was the main
unavoidable side reaction. In summary, 5-(3,5-dinitro-
phenyl)-1H-tetrazole satisfies this requirement for the
construction of the N-acyl phosphoramidate linkage.
Syn th esis of Am in oa cyla m id o-Ad en yla tes (a a -
AMP Ns). Fully esterified neutral N-acyl phosphorami-
date derivatives R1C(O)NH-P(O)(OR2)OR3, such as com-
pound 11a in Scheme 2, are unstable and could not be
purified by silica gel column chromatography. It was
found that dissociated N-acyl phosphoramidates are more
stable than the protected ones. Therefore, 2-(trimethyl-
silyl)ethyl (TSE)37 was used as the phosphate protecting
group, which can be removed selectively by treatment
with Bu4NF after construction of the P-N bond.
The 5′-O-phosphoramidite derivative 10 was prepared
by the reaction of N-benzoyl-2′,3′-di-O-benzoyladenosine
9 with 2-(trimethylsilyl)ethyl N,N,N′,N′-tetraisopropyl
phosphorodiamidite in the presence of diisopropylammo-
nium tetrazolide.
4,4′,4′′-tris(benzoyloxy)trityl (TBTr)38,39 was selected to
meet these criteria. The TBTr group has been previously
used for protection of the primary hydroxyl group of
deoxynucleosides38 and for exo-amino protecting group of
deoxyadenosine, deoxycytidine and deoxyguanosine in
DNA39 and RNA40 synthesis.
The starting material, N-TBTr-L-phenylalaninamide
was synthesized in 95% yield by the reaction of L-
phenylalaninamide with TBTrBr in the presence of
triethylamine at 60 °C. This N-TBTr derivative 3e was
allowed to react with the 5′-O-phosphoramidite derivative
10 in the presence of 1H-5-(3,5-dinitrophenyl)tetrazole.
After oxidation of the phosphite intermediate with tert-
butyl hydroperoxide, the selective removal of the TSE
group by treatment with an equimolar mixture of TBAF‚
H2O and acetic acid gave the desired N-acyl phosphora-
midate derivative 11b in 62% yield. Finally, all the
protecting groups were removed by treatment with
aqueous ammonia to give the desired product 12. The
aa-AMPN derivative 12 was purified by C-18 silica gel
column chromatography, and its structure was deter-
1
mined by means of H, 13C, and 31P NMR and MALDI-
TOF mass.
Another amino acid derivative was also synthesized by
a similar procedure. The N-TBTr amino acid amide
derivatives were synthesized by the reaction of the
corresponding N-unprotected amino acid amide deriva-
tives, which were synthesized in 68% yield by amidation
of N-Cbz amino acids followed by successive treatments
with H2 on Pd/C and TBTr. In the case of L-isoleucine
and L-valine derivatives, the condensation of the N-TBTr
derivatives 14i and 14v with 10, followed by oxidation
with t-BuOOH and removal of the TSE group gave the
(38) Sekine, M.; Hata, T. J . Org. Chem. 1983, 26, 3011.
(39) (a) Sekine, M.; Masuda, N.; Hata, T. Tetrahedron 1985, 41,
5445. (b) Sekine, M.; Masuda, N.; Hata, T. Bull. Chem. Soc. J pn. 1986,
59, 1781.
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J pn. 1988, 61, 1669. (b) Iwase, R.; Tsukada, Y.; Sekine, M.; Hata, T.;
Miura, K. Nucleic Acids Symp. Ser. 1988, 20, 85. (c) Iwase, R.; Sekine,
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K. Nucleic Acids Res. 1992, 20, 1643.
The amino groups of the amino acid amide derivatives
needed to be protected with a lipophilic group (conferring
higher solubility in organic solvents), yet which could
be easily removed under basic conditions. Accordingly,
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Synthesis and Properties of Aminoacylamido-AMP
J . Org. Chem., Vol. 65, No. 24, 2000 8233
Sch em e 3
Sch em e 4
condensed products 16i and 16v in 57 and 83% yields,
respectively (Scheme 3). All the protecting groups were
removed by treatment with aqueous ammonia to give the
desired products 17i and 17v in 48 and 77% yields,
respectively. In the case of the L-methionine derivative,
the Cbz group which protects the amino group could not
be removed by the catalytic hydrogenolysis. Therefore,
9-(fluorenyl)methoxycarbonyl (Fmoc) was used to protect
the amino group instead of the Cbz group. The N-
unprotected carboxamide derivative 14m was obtained
by treatment with morpholine-DMF (1:1, v/v) and was
crystallized by addition of an equimolar amount of acetic
acid to give a white crystalline acetate salt. The final
product 17m was obtained by the same procedure as
described in the case of the L-phenylalanine derivative.
The L-proline derivative, an analogue of phosmidosine,
was also synthesized by the same procedure. The cou-
pling reaction of 10 with N-TBTr prolinamide (15p ) gave
the fully protected derivative 16p in 76% yield. The
following deprotection successfully gave the L-proline
derivative in 65% yield (Scheme 4).
The functional groups on the side chain of amino acids
should be protected at the same time. For the synthesis
of tyrosine derivatives, both the L- and D-forms of which
are expected to have interesting properties, the phenolic
OH group of the tyrosine side chain must be protected
with an appropriate protecting group. Thus, the fully
unprotected tyrosinamide derivatives 14y and 14z were
synthesized from N,O-protected tyrosine derivatives.
Simultaneous protection of the amino and phenolic
groups of the unprotected tyrosinamide derivatives 14y
and 14z were attempted by the reaction with 2.5 equiv
of TBTrBr. However, these reactions gave predominantly
the mono-TBTr derivatives. Satisfactory results were
obtained by the use of 4.0 equiv of TBTrBr and prolonged
reaction time. Under these conditions, the bis-TBTr
derivatives 15y and 15z were obtained as the main
products. The TBTr acetate ester derivative was formed
when the acetate salts of amino acid amide derivatives
were used as the starting materials. This acetate ester
species did not react with the phenolic OH group of the
tyrosine side chain residue, but reacted with the amino
group. Therefore, an excess amount of TBTrBr to fully
protect the side chain of tyrosine derivatives is required.
Condensation of the N,O-bis-TBTr derivatives 15y and
15z with 10 followed by the oxidation and the selective
removal of the TSE group gave the coupled products 16y
and 16z in 60 and 61% yields, respectively. All the
protecting groups were removed by treatment with

8234 J . Org. Chem., Vol. 65, No. 24, 2000
Moriguchi et al.
p-toluenesulfonyl chloride for several hours, redistilled from
CaH2, and stored over molecular sieves 4A. 2-(Trimethylsilyl)-
ethyl N,N,N′,N′-tetraisopropylphosphorodiamidite was syn-
thesized by the method reported previously.37 4,4′,4′′-Tris-
(benzoyloxy)trityl bromide (TBTrBr) was synthesized by the
method reported previously.38 1H NMR spectra were obtained
at 270 MHz with tetramethylsilane (TMS) as an internal
standard in CDCl3 and with sodium 3-(trimethylsilyl)propi-
onesulfonate (DSS) as an external standard in D2O. 13C NMR
spectra were obtained at 67.8 MHz with TMS as an internal
standard and with DSS as an external standard in D2O. 31P
NMR spectra were obtained at 109.25 MHz using 85% H3PO4
as an external standard. MALDI-TOF mass spectra were
obtained in the positive ion mode. The MALDI matrix used
was R-cyano-4-hydroxycinnamic acid (R-CHCA, 10 mg/mL, 1:1,
H2O-MeCN, (v/v)). The calibration was performed with
R-CHCA ([M + H] ) 190.050) and its dimer ([M + H] )
379.093) as internal standards.
N -(9-F lu or e n ylm e t h oxyca r b on yl)-L-p h e n yla la n in a -
m id e (3a ). To a solution of N-(9-fluorenylmethoxycarbonyl)-
L-phenylalanine (6.5 g, 20.0 mmol) in MeCN (200 mL) were
added EEDQ (4.6 g, 22.0 mmol) and NH4HCO3 (4.0 g, 60.0
mmol), and the mixture was stirred at room temperature for
18 h. The precipitate was removed by filtration, and was
washed successively by H2O, hexane, and CHCl3. The filtrate
and washings were combined and washed twice with 5%
NaHCO3. The organic layer was collected, dried over Na2SO4,
filtered, and concentrated to dryness under reduced pressure.
The residue was applied to a silica gel column with hexane-
CHCl3 (1:9, v/v) to give 3a (3.34 g, 51%) as a white solid: 1H
NMR (DMSO-d6) δ 2.79 (1H, dd), 3.03 (1H, dd), 4.15 (4H, m),
7.05-7.88 (14H, m); 13C NMR (DMSO-d6) δ 37.43, 46.51, 55.95,
65.52, 119.91, 125.14, 126.90, 127.89, 129.09, 138.20, 140.56,
143.65, 155.67, 173.30. Anal. Calcd for C24H22N2O3: C, 74.59;
H, 5.74; N, 7.25. Found: C, 75.20; H, 5.70; N, 7.23.
L-P h en yla la n in a m id e (3c). To a solution of N-benzyloxy-
carbonyl-L-phenylalaninamide (1.82 g, 1.7 mmol) in MeOH
(200 mL) was added 10% Pd/C (700 mg), and the suspension
was vigorously stirred under hydrogen atmosphere at room
temperature for 3 h. The reaction mixture was filtered by use
of Celite, and the filtrate was concentrated to dryness under
reduced pressure. Recrystalization of the residue from ethyl
acetate-EtOH (1:1, v/v) gave 3c (1.0 g, quant) as white
crystals: 1H NMR (DMSO-d6) δ 2.82 (1H, dd, J ) 7.6 Hz, 13.5
Hz), 3.02 (1H, dd, J ) 5.9 Hz, 13.5 Hz), 3.66 (1H, dd, J ) 5.9
Hz, 7.6 Hz), 7.19-7.32 (6H, m), 7.73 (1H, br); 13C NMR
(DMSO-d6) δ 39.02, 54.86, 126.56, 128.29, 129.49, 137.16,
173.41.
N-Tr ityl-L-p h en yla la n in a m id e (3d ). To a solution of
N-Benzyloxycarbonyl-L-phenylalaninamide (1.82 g, 1.7 mmol)
in MeOH (200 mL) was added 10% Pd/C (700 mg), and the
suspension was vigorously stirred under hydrogen atmosphere
at room temperature for 3 h. The reaction mixture was filtered
by use of Celite, and the filtrate was concentrated to a small
volume under reduced pressure. The residue was dried by
repeated coevaporation with dry pyridine and dry toluene and
finally dissolved in CH2Cl2 (60 mL). To the mixture were added
trityl chloride (1.8 g, 6.6 mmol) and triethylamine (1.0 mL,
6.6 mmol), and the mixture was stirred at room temperature
for 6 h. The reaction mixture was concentrated to a small
volume under reduced pressure. The residue was diluted with
CHCl3 and washed twice with 5% NaHCO3. The organic layer
was dried over Na2SO4, filtered, and concentrated to dryness
under reduced pressure. The residue was applied to a silica
gel column with hexane-ethyl acetate (7:3, v/v) to give 3d
(2.4 g, 99%) as a colorless foam: 1H NMR (CDCl3) δ 2.32 (1H,
dd, J ) 13.5 Hz), 2.93 (1H, dd, J ) 13.5 Hz), 3.44 (1H, m),
5.03 (1H, br), 6.05 (1H, br), 7.05-7.38 (20H, m); 13C NMR
(CDCl3) δ 41.06, 59.03, 71.61, 126.77, 126.88, 128.01, 128.54,
128.9, 128.9, 129.9, 136.87,145.68, 177.30. Anal. Calcd for
aqueous ammonia to give the desired products 17y and
17z in 55 and 35% yields, respectively.
It seems possible to synthesize aa-AMPNs of all amino
acid derivatives despite natural or non-natural amino
acids, if their side chain functional groups can be masked
with appropriate protecting groups that can be removed
without damage the aminoacylamido linkage.
Ch em ica l P r op er ties of a a -AMP N Der iva tives. It
is known that the phosphoric acid derivatives (RC(O)-
NH-P(O)(OH)2) which have N-acyl phosphoramidate
linkages are extremely unstable even at 0 °C and are
difficult to store even for a few days.18 Because the car-
boxyl amide residues act as good leaving groups, these
compounds decompose via a monomeric metaphosphate
derivative, which rapidly reacts with water to give
orthophosphate, even during storage at 0 °C. Moreover,
N-acyl phosphoramidate diester derivatives (R1C(O)NH-
P(O)(OR2)(OR3)) such as phosmidosine1 are stable under
weakly acidic conditions. However, it was also reported
that the basic conditions cause decrease of the antifugal
activity of phosmidosine. Grandas et al. also reported that
2-cyanoethyl 5′-O-(dimethoxytrityl)thymidine-3′-yl N-
(phenylacetyl)phosphoramidate having the structure of
R1C(O)NH-P(O)(OR2)(OR3) is fairly unstable. On the
other hand, it was previously reported that N-acyl phos-
phoramidate monoester derivatives (R1C(O)NH-P(O)-
(OR2) (OH)) are stable under the basic conditions pre-
scribed for removal of the 2-cyanoethyl and acyl protect-
ing groups, or under acidic conditions using 80% AcOH
prescribed for removal of the DMTr group.20 Therefore,
the stability of aa-AMPN derivative 12 under acidic and
basic conditions was reexamined. As the result, this
phenylalanine derivative 12 was found to be stable to
acidic conditions of 0.1 M HCl (pH 1.0) for 24 h. Moreover,
treatment of 12 with 0.1 M NaOH (pH 13) did not affect
the N-acyl phosphoramidate monoester derivative
for 24 h.
Con clu sion
Aminoacyl-adenylates (aa-AMPs) are very important
intermediates in protein biosynthesis, and the recognition
mechanism between an amino acid and the cognate
aminoacyl-tRNA synthetase (ARS) is not clear in this
field. The lability of aa-AMPs in aqueous solution causes
difficulty in analysis of the aminoacyl transfer reaction.
The results above-mentioned show that replacement of
the oxygen atom of aa-AMP by the amino group greatly
stabilized the extremely unstable mixed anhydride bond
between the phosphate of 5′-AMP and the carboxylate
of an amino acid. The above-mentioned specific properties
of this type aa-AMP analogue would be useful for studies
of the mechanism of the aminoacylation reaction of tRNA
as well as the amino acid specific inhibitory activity in
the process of the protein biosynthesis in an amino acid-
specific manner. Therefore, the most simplified analogues
which we proposed here would provide a powerful tool
for the search of diverse biological reactions. The attempt
for the chemical synthesis of the natural products having
the N-acyl phosphoramidate linkages is now under
investigation.5,6
Exp er im en ta l Section
C
28H26N2O: C, 82.73; H, 6.45; N, 6.89. Found: C, 82.94; H,
Gen er a l P r oced u r es. CH2Cl2 and MeCN were distilled
from CaH2 after being refluxed for several hours, and stored
over molecular sieves 4A. Triethylamine was distilled
from CaH2 after being refluxed for several hours, and stored
over CaH2. Pyridine was distilled after being refluxed over
6.66; N, 6.86.
N -[4,4′,4′′-Tr is(b e n zoyloxy)t r it yl]-L-p h e n yla la n in a -
m id e (3e). L-Phenylalaninamide (3c) (96 mg, 0.60 mmol) was
dried by repeated coevaporation with dry pyridine and finally

Synthesis and Properties of Aminoacylamido-AMP
J . Org. Chem., Vol. 65, No. 24, 2000 8235
dissolved in dry pyridine (6 mL). To this solution were added
4,4′,4′′-tris(benzoyloxy)trityl bromide (492 mg, 0.72 mmol) and
triethylamine (201 µL, 1.44 mmol), and the mixture was
stirred at 60 °C for 2 h. The reaction mixture was concentrated
to a small volume under reduced pressure, diluted with CHCl3
and washed twice with 5% NaHCO3. The organic layer was
dried over Na2SO4, filtered, and concentrated to dryness under
reduced pressure. The residue was applied to a silica gel
column with hexane-ethyl acetate (7:3, v/v) to give 3e (428
mg, 95%) as a yellow foam: 1H NMR (CDCl3) δ 2.78 (1H, dd,
J ) 13.3 Hz), 3.05 (1H, m), 3.41 (1H, m), 5.10 (1H, br), 5.46
(1H, br), 7.11-8.21 (32H, m); 13C NMR (CDCl3) δ 41.40, 59.41,
70.53, 121.24, 126.88, 128.52, 128.60, 129.31, 129.52, 129.83,
130.01, 130.09, 133.59, 137.29, 143.05, 149.61, 165.09, 177.80.
Anal. Calcd for C49H38N2O7: C, 76.75; H, 4.99; N, 3.65.
Found: C, 76.30; H, 5.10; N, 3.46.
washed three times with 5% NaHCO3. The organic layer was
dried over Na2SO4, filtered, and concentrated to dryness under
reduced pressure. The residue was dissolved with CDCl3 (500
µL). The products were determined by 31P NMR, and the yield
of the product 8 was estimated on the basis of the integration
of the signals.
N-Ben zoyl-2′,3′-d i-O-ben zoyla d en osin e-5′-O-[2-(tr im e-
th ylsilyl)eth yl N,N-d iisop r op ylp h osp h or a m id ite] (10).
N-Benzoyl-2′,3′-di-O-benzoyladenosine (9) (2.89 g, 5.0 mmol)
was dried by repeated coevaporation with dry pyridine and
dry toluene and finally dissolved in dry CH2Cl2 (50 mL). To
this solution were added 2-(trimethylsilyl)ethyl N,N,N′,N′-
tetraisopropylphosphorodiamidite (2.0 mL, 6.0 mmol) and
diisopropylammonium tetrazolide (420 mg, 2.5 mmol), and the
mixture was stirred at room temperature for 8 h. The reaction
mixture was concentrated to a small volume, diluted with
CHCl3, and washed three times with 5% NaHCO3. The organic
layer was dried over Na2SO4, filtered, and concentrated to
dryness under reduced pressure. The residue was applied to
a silica gel column, and elution was performed with hexane-
ethyl acetate (5:3, v/v) containing 0.5% triethylamine. The
fractions containing 10 were combined and concentrated to
give 10 (3.3 g, 80%) as a colorless foam: 31P NMR (CDCl3) δ
147.27, 147.60; 1H NMR (CDCl3) δ 0.02 (9H, br), 1.04-1.26
(14H, m), 3.63-4.04 (6H, m), 4.65 (1H, m), 5.95-6.00 (1H, m),
6.10-6.21 (1H, m), 6.70-6.75 (1H, m), 7.15-8.06 (15H, m),
8.74 (1H, s), 8.83 (1H, br), 9.07 (1H, br); 13C NMR (CDCl3) δ
19.7, 24.3, 42.5, 60.72, 61.03, 61.33, 62.23, 62.46, 63.55, 62.80,
72.58, 72.80, 74.47, 74.61, 83.38, 83.52, 83.60, 83.72, 84.99,
85.14, 122.92, 122.97, 127.47, 127.79, 127.92, 128.12, 128.35,
128.50, 128.55, 128.59, 128.75, 129.00, 129.29, 129.61, 132.06,
133.12, 133.15, 133.29, 141.22, 141.44, 149.24, 149.40, 151.66,
151.84, 152.26, 164.31, 164.37, 164.49, 164.69, 164.89. Anal.
Calcd for C42H51N6O8PSi: C, 61.00; H, 6.22; N, 10.16. Found:
C, 60.62; H, 6.27; N, 9.85.
Tr ieth yla m m on iu m O-(N-Ben zoyl-2′,3′-d i-O-ben zoyl-
a d en osin e-5′-O-yl)-N-[N-[4,4′,4′′-Tr is(ben zoyloxy)tr ityl]-
L-p h en yla la n in e]p h osp h or a m id a te (11b). N-(4,4′,4′′-Tris-
(benzoyloxy)trityl)-L-phenylalaninamide (3e) (153 mg, 0.2
mmol) and 10 (248 g, 0.3 mmol) were dried by repeated
coevaporation with dry pyridine and dry toluene, and this
mixture was dissolved in dry MeCN (4 mL). The solution was
added to 5-(3,5-dinitrophenyl)-1H-tetrazole (142 mg, 0.6 mmol),
which was dried by repeated coevaporation with dry pyridine
and dry toluene. After being stirred at room temperature for
30 min, the mixture was added tert-butyl hydroperoxide (125
µL, 1.0 mmol) and stirred at room temperature for 10 min.
The reaction mixture was diluted with CHCl3 and washed
three times with 5% NaHCO3. The organic layer was dried
over Na2SO4, filtered and concentrated to dryness under
reduced pressure. The residue was dissolved in dry THF (10
mL), and tetrabutylammonium fluoride monohydrate (157 mg,
0.6 mmol) and AcOH (34 µL, 0.6 mmol) were added to this
mixture. After being stirred at room temperature for 20 h, the
reaction mixture was concentrated to a small volume and
diluted with CHCl3. The CHCl3 solution was washed three
times with 0.5 M triethylammonium hydrogen carbonate, dried
over Na2SO4, filtered, and concentrated to dryness under
reduced pressure. The residue was applied to a silica gel
column, and elution was performed with CHCl3-MeOH
(99:1-98:2, v/v) containing 1% triethylamine. The fractions
containing 11b were combined and concentrated under reduces
pressure to give 11b (232 mg, 77%) as a yellowish foam: 31P
NMR (CDCl3) δ -7.10; 1H NMR (CDCl3) δ 1.28 (9H, t, J ) 7.3
Hz), 2.08 (1H, m), 2.54 (1H, m), 3.05 (6H, q, J ) 7.3 Hz), 3.59
(1H, m), 4.25 (2H, m), 4.72 (1H, m), 6.07 (1H, m), 6.16 (1H,
dd), 6.71 (1H, d J ) 6.6 Hz), 7.06-8.16 (47H, m), 8.78 (1H, s),
9.11 (1H, s); 13C NMR (CDCl3) δ 8.63, 39.05, 45.50, 59.25, 65.04
(d, J ) 4.7 Hz), 71.03, 72.80, 75.14, 83.28 (d, J ) 7.6 Hz), 85.12,
120.81, 122.62, 125.14, 126.85, 127.73, 127.94, 128.07, 128.20,
128.27, 128.39, 128.57, 128.68, 128.87, 129.25, 129.47, 129.63,
129.67, 129.76, 129.97, 130.35, 130.55, 132.53, 133.44, 133.69,
135.94, 142.71, 142.84, 149.20, 149.52, 151.90, 152.49, 164.61,
164.69, 164.82, 165.12, 176.53. Anal. Calcd for C86H77N8O16P‚
H2O: C, 67.62; H, 5.21; N, 7.34. Found: C, 67.66; H, 5.42; N,
(N,N-Diisop r op yla m in o)(2-cya n oeth yl)[N-(9-flu or en yl-
m eth oxyca r bon yl)-L-p h en yla la n in a m id e]p h osp h in e (4a ).
N-(9-fluorenylmethoxycarbonyl)-L-phenylalaninamide (3a) (369
mg, 0.96 mmol) was dried by repeated coevaporation with dry
pyridine and dry toluene, and finally dissolved in CH2Cl2 (4
mL). To the mixture were added 2-cyanoethyl N,N-diisopro-
pylphosphorochloridite (415 µL, 2.0 mmol) and N,N-diisopro-
pylethylamine (522 µL, 3.0 mmol), and the mixture was stirred
at room temperature for 45 min. The reaction mixture was
diluted with CHCl3 and washed three times with 5% NaHCO3.
The organic layer was dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure. The residue
was purified by reprecipitation of its CHCl3 solution from
hexane-ether (85:15, v/v) to give 4a (241 mg, 43%) as a white
1
powder: 31P NMR (CDCl3) δ 118.18; H NMR (CDCl3) δ 1.16
(6H, s), 1.19 (6H, s), 2.59 (2H, t), 3.09 (2H, m), 3.45 (2H, m),
3.86 (2H, m), 4.18-4.41 (4H, m), 7.05-7.88 (15H, m); MS-
(FAB+) m/z calcd for C33H39N4O4P 586, obsd 587 (M + H).
(N,N-Diisop r op yla m in o)(2-cya n oeth yl)(N-tr ityl-L-p h e-
n yla la n in a m id e)p h osp h in e (4d ). N-Trityl-L-phenylalani-
namide (3d ) (121 mg, 0.30 mmol) was dried by repeated
coevaporation with dry pyridine and dry toluene, and finally
dissolved in CH2Cl2 (3 mL). To the mixture were added
2-cyanoethyl N,N-diisopropylphosphorochloridite (120 µL, 0.6
mmol) and N,N-diisopropylethylamine (150 µL, 0.9 mmol), and
the mixture was stirred at room temperature for 5 min. The
reaction mixture was diluted with CHCl3 and washed three
times with 5% NaHCO3. The organic layer was dried over Na2-
SO4, filtered, and concentrated to dryness. The residue was
dissolved in hexane, and washed three times with pyridine-
H2O (20 mL, 1:1, (v/v)) to give 4d (174 mg, 97%) as a white
powder: 31P NMR (CDCl3) δ 117.41, 119.23; 1H NMR (CDCl3)
δ 1.04-1.21 (12H, 2s), 1.97, 2.26 (1H, dd), 2.58 (2H, m), 2.93
(1H, m), 3.30 (1H, m), 3.47 (2H, m), 3.88 (2H, m), 7.03-7.35
(20H, m); 13C NMR (CDCl3) δ 20.40, 20.50, 24.4, 39.10, 39.91,
44.57, 44.67, 44.76, 44.85, 58.91, 59.48, 60.06, 60.49, 71.68,
117.73, 123.63, 125.87, 126.76, 126.81, 127.03, 127.08, 127.13,
127.48, 127.51, 127.66, 127.78, 127.94, 128.03, 128.18, 128.23,
128.46, 128.53, 128.63, 128.72, 129.78, 129.94, 130.10, 136.17,
136.53, 145.50, 177.84, 178.00.
Cyclized p r od u ct (5): 31P NMR (CDCl3) δ 126.42; 1H NMR
(CDCl3) δ 2.05 (1H, dd), 2.19 (2H, t), 2.92 (1H, dd, J ) 13.8
Hz), 3.30 (2H, m), 4.10 (1H, m), 6.96-7.45 (20H, m); 13C NMR
(CDCl3) δ 20.45, 39.28, 59.87, 60.27 (d, J ) 26.9 Hz), 60.29,
60.40, 71.59, 117.61, 126.11, 126.76, 126.84, 127.60, 127.81,
127.94, 127.99, 128.57, 128.79, 129.88, 130.53, 130.62, 160.67,
130.67, 130.97, 137.23, 143.20, 143.25, 145.66, 180.20, 180.30.
31P NMR Stu d y of th e Rea ction of Th ym id in e-5′-
p h osp h or a m id ite Der iva tive (6) w ith P h en yla ceta m id e
(7). 3′-O-Benzoylthymidine-5′-O-(2-cyanoethyl N,N-diisopro-
pylphosphoramidite) (6) (82 mg, 0.15 mmol) and phenylacet-
amide (7) (14 mg, 0.10 mmol) were dried by repeated coevapo-
ration three times with dry pyridine and twice with dry toluene
and dissolved in dry CH3CN (2.0 mL). To this solution was
added a condensing reagent (0.3 mmol) which was separately
dried by repeated coevaporation three times with dry pyridine
and twice with dry toluene, and these solutions were vigorously
stirred at room temperature. Next, tert-butyl hydroperoxide
was added to the reaction mixture, and this mixture was
stirred for 5 min. The mixture was diluted with CHCl3, and

8236 J . Org. Chem., Vol. 65, No. 24, 2000
Moriguchi et al.
7.24; MS(FAB) m/z calcd for C86H77N8O7P 1508.52, obsd 1406
(M - TEA - H), 1409 (M - TEA + H).
L-Tyr osin a m id e Aceta te (14y). Compound 14y was syn-
thesized by the general procedure A to give 457 mg (63%) as
white crystals.
O-(Ad en osin e-5′-O-yl) N-(L-p h en yla la n yl)p h osp h or a -
m id a te (12). Triethylammonium O-(N-benzoyl-2′,3′-di-O-ben-
zoyladenosine-5′-O-yl)-N-[N-(4,4′,4′′-tris(benzoyloxy)trityl)-L-
phenylalanyl] phosphoramidate (11b) (2.24 g, 1.48 mmol) was
treated with concentrated NH3-dioxane (24 mL, 1:1, (v/v)) at
room temperature for 8 h. The mixture was evaporated under
reduced pressure, and residue was dissolved in water. The
aqueous solution was washed five times with ether and
concentrated to a small volume. The residue was applied to a
C18 reversed-phase column, and elution was performed with
water, applying a gradient of MeCN (0-10%). The fractions
containing 12 were combined and lyophilized to give 12 (52%)
as a white powder: 31P NMR (D2O) δ -5.37; 1H NMR (D2O) δ
2.74-2.82 (1H, m), 2.92 (1H, dd, J ) 6.3 Hz, J ) 13.9 Hz),
3.96 (1H, br). 4.04 (2H, m), 4.32 (1H, m), 4.39 (1H dd, J ) 5.3
Hz, 4.0 Hz), 4.69 (1H, dd, J ) 5.6 Hz, 5.3 Hz), 6.03 (1H, d, J
) 5.6 Hz), 7.02-7.16 (5H, m), 8.11 (1H, s), 8.35 (1H, s); 13C
NMR (D2O) δ 38.95, 57.33 (d, J ) 9.6 Hz), 67.85, 72.82, 76.73,
86.12 (d, J ) 9.8 Hz), 89.43 120.98, 129.90, 130.19, 131.62,
142.44, 151.37, 155.27, 157.88, 183.99; MALDI-TOF mass m/z
calcd for C19H25N7O7P 494.2, obsd (M + H) 494.2.
Gen er a l P r oced u r e for th e Am id a tion of Am in o Acid
Der iva tives (13). The reaction was carried out according to
the procedure previously reported by Nozaki et al.18
To a solution of N-protected amino acid (10 mmol) in MeCN
(50 mL) were added EEDQ (2.72 g, 11 mmol) and NH4HCO3
(2.37 g, 30 mmol), and the mixture was stirred at room tem-
perature for 15 h. The precipitate was filtered, and this pre-
cipitate was washed by H2O and Et2O to give 13 as a white
solid.
N-Ben zyloxyca r b on yl-L-isoleu cin a m id e (13i). Com-
pound 13i was synthesized by the general procedure to give
2.46 g (89% yield) as a white solid.
N-(9-F lu or en ylm et h oxyca r b on yl)-L-m et h ion in a m id e
(13m ). Compound 13m was synthesized by the general
procedure to give 6.35 g (86% yield) as a white solid.
N-(9-F lu or en ylm eth oxyca r bon yl)-L-p r olin a m id e (13p ).
Compound 13p was synthesized by the general procedure to
give 4.40 g (88% yield) as a white solid.
O-Ben zyl-N-ben zyloxyca r bon yl-L-tyr osin a m id e (13y).
Compound 13y was synthesized by the general procedure to
give 4.22 g (85% yield) as a white solid.
O-Ben zyl-N-ben zyloxyca r bon yl-D-tyr osin a m id e (13z).
Compound 13z was synthesized by the general procedure to
give 4.22 g (85% yield) as a white solid.
D-Tyr osin a m id e Aceta te (14z). Compound 14z was syn-
thesized by the general procedure A to give 457 mg (63%) as
white crystals.
Gen er al P r ocedu r e for th e Syn th esis of N-TBTr Am in o
Acid Am id e Der iva tives (15). The amino acid amide deriva-
tive 14 (2 mmol) was dried by repeated coevaporation with
dry pyridine and finally dissolved in dry pyridine (20 mL). To
this solution were added 4,4′,4′′-tris(benzoyloxy)trityl bromide
(1.64 g, 2.4 mmol) and triethylamine (697 µL, 5.0 mmol), and
the mixture was concentrated to a small volume, diluted with
CHCl3 and washed twice with 5% NaHCO3. The organic layer
was dried over Na2SO4, filtered, and concentrated to dryness
under reduced pressure. The residue was applied to a silica
gel column, and elution was performed with hexane-ethyl
acetate. The fractions containing 15 were combined and
concentrated under reduced pressure to give 15 as a yellow
foam.
N-[4,4′,4′′-Tr is(ben zoyloxy)tr ityl]-L-isoleu cin am ide (15i).
The reaction was carried out according to the general proce-
dure as described in the case of 14i (189 mg, 1.0 mmol), column
chromatography was performed with hexane-ethyl acetate
(1:1, v/v) to give 15i (625 mg, 79%) as a yellow foam: 1H NMR
(CDCl3) δ 0.86 (3H, dd, J ) 7.3 Hz, 7.6 Hz), 1.00 (3H, d, J )
6.9 Hz), 1.25 (1H, m), 1.42-1.44 (1H, m), 1.69 (1H, m), 2.99
(1H, d, J ) 7.2 Hz), 3.15 (1H, br), 5.17 (1H, br), 5.42 (1H, br),
7.15 (6H, d, J ) 7.9 Hz), 7.47-7.61 (15H, m), 8.19 (6H, d, J )
7.3 Hz); 13C NMR (CDCl3) δ 12.11, 14.09, 26.83, 40.54, 60.24,
70.66, 120.95, 128.32, 129.15, 129.88, 133.39, 143.25, 149.38,
164.92, 175.02. Anal. Calcd for C46H40N2O7‚0.5H2O: C, 74.48;
H, 5.57; N, 3.78. Found: C, 74.60; H, 5.48; N, 3.60.
N-[4,4′,4′′-Tr is(b en zoyloxy)t r it yl]-L-m et h ion in a m id e
(15m ). The reaction was carried out according to the general
procedure as described in the case of 14m (416 mg, 2.0 mmol),
column chromatography was performed with hexane-ethyl
acetate (3:2, v/v) to give 15m (1.32 g, 88%) as a yellow foam:
1H NMR (CDCl3) δ 1.84-1.91 (1H, m), 1.98-2.05 (1H, m), 2.06
(3H, s), 2.34-2.47 (1H, m), 2.60-2.70 (1H, m), 3.21 (1H, br),
3.34 (1H, m), 5.33 (1H, br), 5.59 (1H, br), 7.16 (6H, d, J ) 8.6
Hz), 7.47-7.65 (15H, m), 8.17 (6H, m); 13C NMR (CDCl3) δ
15.08, 29.99, 34.02, 56.05, 70.59, 121.15, 128.45, 129.24,
129.92, 129.99, 133.53, 143.34, 149.52, 165.05, 176.41. Anal.
Calcd for C45H38N2O7S: C, 71.98; H, 5.10; N, 3.73; S, 4.17.
Found: C, 71.56; H, 5.17; N, 3.52; S, 4.01.
N-[4,4′,4′′-Tr is(ben zoyloxy)tr ityl]-L-p r olin a m id e (15p ).
The reaction was carried out according to the general proce-
dure as described in the case of 14p (228 mg, 2.0 mmol) column
chromatography was performed with hexane-ethyl acetate
(6:4-3:7, v/v) to give 15p (996 mg, 74%) as a yellow foam: 1H
NMR (CDCl3) δ 1.02-1.18 (2H, m), 1.54-1.64 (1H, m), 1.73-
1.81 (1H, m), 3.03-3.13 (1H, m), 3.33-3.43 (1H, m), 3.95 (1H,
d, J ) 8.3 Hz), 5.92 (1H, d, J ) 4.0 Hz), 7.18 (7H, d, J ) 8.6
Hz), 7.51 (6H, dd, J ) 7.3 Hz, 8.0 Hz), 7.61-7.66 (9H, m), 8.19
(6H, m); 13C NMR (CDCl3) δ 24.28, 31.36, 50.59, 64.94, 121.22,
128.55, 129.39, 130.14, 130.21, 133.62, 141.90, 149.54, 164.98,
178.72. Anal. Calcd for C45H36N2O7‚1.5H2O: C, 72.67; H, 5.28;
N, 3.77. Found: C, 72.15; H, 5.00; N, 3.73.
Gen er a l P r oced u r e for th e Syn th esis of N-Un p r otect-
ed Am in o Acid Am id e (14). P r oced u r e A. To a solution of
N-benzyloxycarbonyl amino acid amide (13) in AcOH was
added 10% Pd/C. The mixture was stirred under hydrogen
atmosphere at room temperature for 1.5 h. The mixture was
filtered by use of Celite, concentrated to dryness, coevaporated
by toluene, and recrystallized from ethyl acetate to give 14 as
white crystals.
P r oced u r e B. N-(9-Fluorenylmethoxycarbonyl) amino acid
amide (13) was treated with morpholine-DMF (1:1, (v/v)) for
1 h. To this mixture was added H2O. The resulting precipitate
was filtered, and washed with H2O. This filtrate was diluted
with H2O, and washed three times with ether, and back-
extracted with H2O. The aqueous layers were combined, and
concetrated to dryness. The residue was crysrallized by the
addition of a small amount of AcOH, and recrystallized from
ethyl acetate to give 14 as white crystals.
N-[4,4′,4′′-Tr is(ben zoyloxy)t r it yl]-L-va lin a m id e (15v).
The reaction was carried out according to the general proce-
dure as described in the case of 14p (269 mg, 1.53 mmol)
column chromatography was performed with hexane-ethyl
acetate (7:3, v/v) to give 15p (751 mg, 68%) as a yellow foam:
1H NMR (CDCl3) δ 1.01 (3H, d, J ) 6.9 Hz), 1.05 (3H, d, J )
6.9 Hz), 2.05 (1H, m), 3.04 (1H, d, J ) 4.0 Hz), 5.11 (2H, br),
7.14-7.19 (6H, m), 7.49-7.68 (15H, m), 8.18-8.22 (6H, m);
13C NMR (CDCl3) δ 17.49, 20.25, 33.52, 61.94, 70.73, 121.20,
128.57, 129.45, 130.15, 130.21, 133.62, 143.50, 149.69, 165.21,
175.26. Anal. Calcd for C45H38N2O7‚0.5H2O: C, 74.26; H, 5.40;
N, 3.85. Found: C, 74.07; H, 5.33; N, 3.69.
L-Isoleu cin a m id e Aceta te (14i). Compound 14i was syn-
thesized by the general procedure A to give 1.15 g (85% yield)
as a white.
L-Meth ion in a m id e Aceta te (14m ). Compound 14m was
synthesized by the general procedure B to give 1.57 g (70%
yield) as a white.
L-P r olin a m id e (14p ). Compound 14p was synthesized by
the general procedure B to give 991 mg (73% yield) as a white.
L-Va lin a m id e Aceta te (14v). Compound 14v was synthe-
sized by the general procedure A to give 1.0 g (quant) as white
crystals.
N ,O -B is[4,4′,4′′-t r is(b e n zoy lox y )t r it yl]-L -t y r o sin a -
m id e (15y). The reaction was carried out according to the
general procedure as described in the case of 14y (240 mg,
1.0 mmol) column chromatography was performed with hex-
ane-ethyl acetate (6:4, v/v) to give 15y was given in yield

Synthesis and Properties of Aminoacylamido-AMP
J . Org. Chem., Vol. 65, No. 24, 2000 8237
(944 mg, 68%) as a yellow foam: 1H NMR (CDCl3) δ 2.39 (1H,
dd, J ) 7.9 Hz, J ) 13.2 Hz), 2.86 (1H, d, J ) 7.3 Hz), 2.98
(1H, dd, J ) 5.3 Hz, 13.2 Hz), 3.29 (1H, dd, J ) 7.9 Hz, 5.3
Hz), 5.15 (1H, br), 5.35 (1H, br), 6.71 (2H, d, J ) 8.6 Hz), 6.84
(2H, d, J ) 8.6 Hz), 7.15 (12H, t, J ) 8.6 Hz), 7.45-7.66 (30H,
m), 8.15-8.19 (12H, m); 13C NMR (CDCl3) δ 59.12, 70.64,
89.97, 120.95, 121.22, 121.96, 128.48, 129.25, 129.33, 129.92,
130.08, 133.53, 141.42, 143.25, 149.58, 149.96, 154.82, 164.94,
165.01, 176.51. Anal. Calcd for C89H64N2O14‚2H2O: C, 75.31;
H, 4.69; N, 1.97. Found: C, 75.36; H, 4.67; N, 2.13.
N ,O-Bis[4,4′,4′′-t r is(b e n zoyloxy)t r it yl]-D -t yr osin a -
m id e (15z). The reaction was carried out according to the
general procedure as described in the case of 14z (240 mg, 1.0
mmol) column chromatography was performed with hexane-
ethyl acetate (6:4, v/v) to give 15z (1.07 g, 77%) as a yellow
foam: 1H NMR (CDCl3) δ 2.37 (1H, dd, J ) 7.9 Hz, 13.2 Hz),
2.83 (1H, d, J ) 6.9 Hz), 2.96 (1H, dd, J ) 5.0 Hz, 13.2 Hz),
3.28 (1H, dd, J ) 5.0 Hz, 7.9 Hz), 5.13 (1H, br), 5.34 (1H, br),
6.69 (2H, d, J ) 8.6 Hz), 6.82 (2H, d, J ) 8.6 Hz), 7.10-7.16
(12H, m), 7.43-7.65 (30H, m), 8.12-8.17 (12H, m); 13C NMR
(CDCl3) δ 39.98, 59.09, 70.60, 89.96, 120.94, 121.20, 121.94,
128.46, 129.22, 129.31, 129.88, 130.05, 130.66, 133.55, 141.38,
143.23, 149.56, 149.94, 154.81, 164.91, 164.98, 176.46. Anal.
out according to the general procedure as described in the case
of 15m (150 mg, 0.20 mmol), column chromatography was
performed with CHCl3-MeOH (99:1-98:2, v/v) containing 1%
triethylamine to give 16m (185 mg, 62%) as a yellow foam:
1
31P NMR (CDCl3) δ 7.28; H NMR (CDCl3) δ 1.19 (9H, t, J )
7.3 Hz), 1.92 (3H, s), 2.32 (1H, m), 2.47 (1H, m), 2.87 (6H, q,
J ) 7.3 Hz), 2.99 (1H, d, J ) 5.9 Hz), 3.39 (1H, m), 4.33 (1H,
m), 4.41 (1H, m), 4.72 (1H, m), 6.07 (1H, d, J ) 4.9 Hz), 6.18
(1H, dd, J ) 7.3 Hz, 4.9 Hz), 6.69 (1H, d, J ) 7.3 Hz), 7.18
(6H, d, J ) 8.9 Hz), 7.23-7.29 (2H, m), 7.37-7.63 (22H, m),
7.78 (2H, d, J ) 7.6 Hz), 7.97-8.01 (4H, m), 8.15 (6H, d, J )
7.3 Hz), 8.72 (1H, s), 9.09 (1H, s); 13C NMR (CDCl3) δ 8.41,
15.19, 29.54, 33.21, 45.52, 58.06 (d, J ) 4.9 Hz), 65.18, 71.11,
72.80, 75.13, 83.37 (d, J ) 8.6 Hz), 85.18, 121.20, 122.65,
127.82, 128.03, 128.18, 128.30, 128.37, 128.57, 128.84, 129.22,
129.61, 129.67, 129.92, 132.44, 133.64, 142.52, 143.00, 149.34,
149.51, 151.91. 152.42, 164.69, 164.78, 164.96, 165.10, 176.14.
Anal. Calcd for C82H77N8O16PS: C, 65.15; H, 6.33; N, 7.41; S,
2.12. Found: C, 65.05; H, 5.67; N, 7.04; S, 2.46.
Tr ieth yla m m on iu m O-(N-Ben zoyl-2′,3′-d i-O-ben zoyl-
a d en osin e-5′-O-yl)-N-[N-(4,4′,4′′-tr is(ben zoyloxy)tr ityl)-L-
p r olyl]p h osp h or a m id a te (16p ). The reaction was carried
out according to the general procedure as described in the case
of 15p (248 mg, 0.20 mmol), column chromatography was
performed with CHCl3-MeOH (99.5:0.5-98:2, v/v) containing
1% triethylamine to give 16p (221 mg, 76%) as a yellow foam:
31P NMR (CDCl3) δ -7.46; 1H NMR (CDCl3) δ 1.08-1.16 (2H,
m), 1.23 (9H, t, J ) 7.3 Hz), 1.58-1.61 (1H, m), 1.73-1.85 (1H,
m), 3.00 (6H, q, J ) 7.3 Hz), 3.02 (1H, m), 3.48 (1H, m), 3.96
(1H, d, J ) 8.6 Hz), 4.51 (2H, m), 4.76 (1H, m), 6.08 (1H, d, J
) 5.3 Hz), 6.22 (1H, dd, J ) 5.3 Hz, 6.9 Hz), 6.72 (1H, d, J )
6.9 Hz), 7.17 (6H, d, J ) 8.6 Hz), 7.24-7.69 (21H, m), 7.79-
7.81 (3H, m), 8.00-8.03 (6H, m), 8.14 (6H, d, J ) 8.6 Hz), 8.43
(1H, d, J ) 10.9 Hz), 8.68 (1H, s), 9.09 (1H, s); 13C NMR
(CDCl3) δ 8.97, 24.23, 31.61, 45.59, 50.46, 65.20, 65.86, 72.87,
75.06), 83.34 (d, J ) 9.8 Hz), 85.19, 121.08, 122.71, 127.75,
127.85, 128.19, 128.32, 128.37, 128.43, 128.66, 128.91, 129.67,
129.97, 130.19, 132.49, 133.35, 133.48, 133.66, 133.76, 142.52,
149.25, 149.36, 151.90, 152.45, 164.65, 164.78, 165.16, 177.90.
Anal. Calcd for C82H75N8O16P‚H2O: C, 66.66; H, 5.25; N, 7.58.
Found: C, 66.37; H, 5.45; N, 7.27.
Tr ieth yla m m on iu m O-(N-Ben zoyl-2′,3′-d i-O-ben zoyl-
a d en osin e-5′-O-yl)-N-[N-[4,4′,4′′-tr is(ben zoyloxy)tr ityl]-L-
va lyl]p h osp h or a m id a te (16v). The reaction was carried out
according to the general procedure as described in the case of
15v (144 mg, 0.2 mmol), column chromatography was per-
formed with CHCl3-MeOH (99:1-98:2, v/v) containing 1%
triethylamine to give 16v (292 mg, 83%) as a yellow foam: 31P
NMR (CDCl3) δ -7.05; 1H NMR (CDCl3) δ 0.86 (6H, d, J ) 6.3
Hz), 1.23 (9H, t, J ) 7.3 Hz), 2.45 (1H, m), 2.94 (6H, q, J )
7.3 Hz), 3.16 (1H, m), 4.24-4.37 (2H, m), 4.71 (1H, m), 6.06
(1H, dd, J ) 5.3 Hz, 3.6 Hz), 6.16 (1H, dd, J ) 6.6 Hz, 5.3 Hz),
6.71 (1H, d J ) 6.6 Hz), 7.18 (6H, d, J ) 8.6 Hz), 7.26-7.59
(21H, m), 7.76-7.81 (3H, m), 7.95-8.03 (6H, m), 8.15 (6H, d,
J ) 8.6 Hz), 8.72 (1H, s), 9.15 (1H, s); 13C NMR (CDCl3) δ 8.70,
17.06, 20.24, 33.66, 45.54, 63.02, 65.09, 71.32, 72.83, 75.16,
83.47, 85.23, 121.15, 122.68, 127.78, 127.92, 128.03, 128.19,
128.32, 128.39, 128.63, 128.90, 128.99, 129.27, 129.40, 129.63,
129.83, 129.96, 130.19, 130.46, 131.11, 132.45, 133.37, 133.42,
133.71, 143.07, 149.52, 151.88, 152.43, 164.67, 164.83, 165.09,
175.09. Anal. Calcd for C82H77N8O16P‚1.5H2O: C, 66.16; H,
5.42; N, 7.53. Found: C, 66.04; H, 5.67; N, 7.70.
Tr ieth yla m m on iu m O-(N-Ben zoyl-2′,3′-d i-O-ben zoyl-
a d en osin e-5′-O-yl)-N-[Bis-N,O-[4,4′,4′′-t r is(b en zoyloxy)-
tr ityl]-L-tyr osyl]p h osp h or a m id a te (16y). The reaction was
carried out according to the general procedure as described in
the case of 15y (1.38 g, 1.0 mmol), column chromatography
was performed with CHCl3-MeOH (99.5:0.5-98:2, v/v) con-
taining 1% triethylamine to give 16y (2.11 g, 60%) as a yellow
foam: 31P NMR (CDCl3) δ -7.46; 1H NMR (CDCl3) δ 1.29 (9H,
t, J ) 7.3 Hz), 2.92 (1H, m), 3.05 (6H, q, J ) 7.3 Hz), 3.07
(1H, m), 3.51 (1H, m), 4.23 (1H, m), 4.32 (1H, m), 4.69 (1H,
m), 6.05 (1H, m), 6.15 (1H, dd, J ) 6.3 Hz, 5.6 Hz), 6.61-6.71
(5H, m), 7.08-7.64 (45H, m), 7.76-8.15 (22H, m), 8.71 (1H,
s), 9.18 (1H, s); 13C NMR (CDCl3) δ 8.34, 33.17, 45.43, 62.11,
67.73, 70.94, 71.11, 72.76, 74.65, 87.58, 89.34, 89.97, 111.14,
Calcd for
Found: C, 76.17; H, 4.92; N, 2.00.
C89H64N2O14‚H2O: C, 76.16; H, 4.74; N, 2.00.
Gen er a l P r oced u r e for th e Syn th esis of th e F u lly
P r otected a a -AMP Ns (16). N-TBTr Amino acid amide (15)
(0.20 mmol) and 10 (143 mg, 0.30 mmol) were dried by
repeated coevaporation with dry pyridine and dry toluene, and
this mixture was dissolved in dry MeCN (4 mL). The solution
was added to 5-(3,5-dinitrophenyl)-1H-tetrazole (142 mg, 0.6
mmol), which was dried by repeated coevaporation with dry
pyridine and dry toluene. The mixture was stirred at room
temperature for 30 min. To the mixture was added tert-butyl
hydroperoxide (125 µL, 1.0 mmol). After being stirred at room
temperature for 10 min, the mixture was diluted with CHCl3
and washed three times with 5% NaHCO3. The organic layer
was dried over Na2SO4, filtered, and concentrated to dryness
under reduced pressure.
The residue was dissolved in dry THF (10 mL), and
tetrabutylammonium fluoride monohydrate (157 mg, 0.6 mmol)
and AcOH (34 µL, 0.6 mmol) were added to this mixture. After
being stirred at room temperature for 20 h, the reaction
mixture was concentrated under reduced pressure to a small
volume, diluted with CHCl3, and washed three times with 0.5
M triethylammonium hydrogen carbonate. The organic layer
was dried over Na2SO4, filtered, and concentrated to dryness
under reduced pressure. The residue was applied to a silica
gel column, and elution was performed with CHCl3-MeOH
containing 1% triethylamine. The fractions containing 16 were
combined and concentrated to give 16 as a yellow foam.
Tr ieth yla m m on iu m O-(N-Ben zoyl-2′,3′-d i-O-ben zoyl-
a d en osin e-5′-O-yl)-N-[N-(4,4′,4′′-tr is(ben zoyloxy)tr ityl)-L-
isoleu cyl]p h osp h or a m id a te (16i). The reaction was carried
out according to the general procedure as described in the case
of 15i (248 mg, 0.20 mmol), column chromatography was
performed with CHCl3-MeOH (99.5:0.5-98:2, v/v) containing
1% triethylamine to give 16i (167 mg, 57%) as a yellow foam:
1
31P NMR (CDCl3) δ 7.05; H NMR (CDCl3) δ 0.64 (3H, t, J )
7.3 Hz), 0.85 (4H, m), 1.23-1.30 (2H, m), 1.27 (9H, t, J ) 7.3
Hz), 2.44 (1H, d, J ) 5.3 Hz), 3.07 (6H, q, J ) 7.3 Hz), 3.24
(1H, d, J ) 5.0 Hz), 4.39 (2H, m), 4.73 (1H, m), 6.08 (1H, dd,
J ) 2.0 Hz, 5.3 Hz), 6.21 (1H, dd, J ) 5.3 Hz, 6.9 Hz), 6.68
(1H, d, J ) 6.9 Hz), 7.17-7.61 (32H, m), 7.77-8.16 (12H, m),
8.71 (1H, s), 8.71 (1H, s); 13C NMR (CDCl3) δ 8.32, 11.90, 13.77,
27.19, 40.47, 45.27, 61.65 (d, J ) 6.1 Hz), 64.87 (d, J ) 4.8
Hz), 71.34, 72.56, 74.90, 83.05 (d, J ) 8.5 Hz), 85.12, 121.01,
122.64, 124.98, 127.51, 127.69, 127.89, 128.00, 129.16, 128.21,
128.52, 128.70, 128.77, 129.15, 129.47, 129.63, 129.81, 132.35,
133.15, 133.24, 133.55, 142.59, 142.91, 149.02, 149.36, 151.77,
152.26, 164.46, 164.64, 164.89, 174.68. Anal. Calcd for
C
83H79N8O16P: C, 67.56; H, 5.40; N, 7.59. Found: C, 65.19; H,
5.64; N, 8.05.
Tr ieth yla m m on iu m O-(6-N-,2′,3′-O-Tr iben zoyla d en o-
sin e-5′-O-yl)-N-[N-(4,4′,4′′-t r is(b en zoyloxy)t r it yl)-L-m e-
th ion yl]p h osp h or a m id a te (16m ). The reaction was carried

8238 J . Org. Chem., Vol. 65, No. 24, 2000
Moriguchi et al.
120.85, 120.97, 121.17, 121.44, 121.69, 121.96, 127.71, 127.82,
127.98, 128.09, 128.16, 128.28, 128.62, 128.79, 128.86, 128.93,
129.13, 129.56, 129.68, 129.92, 130.24, 130.40, 130, 64, 131.45,
132.38, 132.72, 133.32, 141.08, 142.53, 142.75, 143.04, 149.04,
149.42, 149.79, 150.93, 152.36, 164.56, 164.71, 164.91, 165.03,
178.53. Anal. Calcd for C126H103N8O23P: C, 71.11; H, 4.88; N,
5.27. Found: C, 71.54; H, 4.42; N, 5.88.
mmol). C-18 Reversed-phase column chromatography was
performed with water, applying a gradient of MeCN (0-10%)
to give 17p (42 mg, 65%) as a white powder: 31P NMR (D2O)
1
δ -5.26; H NMR (D2O) δ 1.72-1.92 (3H, m), 2.27-2.36 (1H,
m), 3.23-3.28 (2H, m), 4.08 (2H, br), 4.28-4.42 (3H, m), 4.65
(1H, d, J ) 5.6 Hz, 5.3 Hz), 5.97 (1H, d, J ) 5.3 Hz), 8.03 (1H,
s), 8.33 (1H, s); 13C NMR (D2O) δ 26.11, 32.22, 48.98, 63.07 (d,
J ) 11.0 Hz), 67.60 (d, J ) 4.9 Hz), 72.87, 76.75, 86.22 (d, J )
8.5 Hz), 89.58, 121.17, 142.89, 151.61, 154.46, 157.46, 173.56;
MALDI-TOF mass m/z calcd for C15H23N7O7P 444.1, obsd
(M + H) 444.1.
Tr ieth yla m m on iu m O-(N-Ben zoyl-2′,3′-d i-O-ben zoyl-
a d en osin e-5′-O-yl)-N-[N,O-b is[4,4′,4′′-t r is(b en zoyloxy)-
tr ityl]-D-tyr osyl]p h osp h or a m id a te (16z). The reaction
was carried out according to the general procedure as
described in the case of 15z (277 mg, 0.20 mmol), column
chromatography was performed with CHCl3-MeOH
(99.5:0.5-98:2, v/v) containing 1% triethylamine to give 16z
(258 mg, 61%) as a yellow foam: 31P NMR (CDCl3) δ -7.37;
1H NMR (CDCl3) δ 1.29 (9H, t, J ) 7.3 Hz), 2.91 (1H,
m), 3.09 (6H, q, J ) 7.3 Hz), 3.10 (1H, m), 3.55 (1H,
m), 4.22 (1H, m), 4.36 (1H, m), 4.53 (1H, m), 6.11 (2H,
m), 6.64 (5H, m), 7.11-7.60 (45H, m), 7.73-8.24 (22H, m),
8.68 (1H, s), 9.12 (1H, s); 13C NMR (CDCl3) δ 8.93, 34.25,
45.55, 62.25, 65.03, 70.92, 72.74, 73.33, 74.00, 75.17, 85.00,
87.73, 89.70, 111.14, 120.68, 120.88, 121.18, 121.69, 121.76,
122.07, 122.70, 122.97, 123.97, 127.73, 127.84, 127.89,
128.16, 128.21, 128.37, 128.59, 128.93, 128.97, 129.25,
129.58, 128.93, 128.96, 129.25, 129.58, 129.63, 129.70,
129.97, 130.39, 132.41, 132.56, 132.94, 133.37, 133.44,
133.71, 141.22, 142.50, 142.70, 142.93, 149.24, 149.47,
149.90, 150.11, 151.90, 152.20, 152.40, 154.72, 164.65,
164.73, 164.76, 165.07, 165.25, 176.60. Anal. Calcd for
O-(Aden osin e-5′-O-yl)N-(L-valyl)ph osph or am idate (17v).
The reaction was carried out according to the general proce-
dure as described in the case of 16v (299 mg, 0.205 mmol).
C-18 Reversed-phase column chromatography was performed
with water, applying a gradient of MeCN (0-10%) to give 17v
(70 mg, 77%) as a white powder: 31P NMR (D2O) δ -5.21; 1H
NMR (D2O) δ 0.85 (3H, d, J ) 6.9 Hz), 0.92 (3H, d, J ) 6.9
Hz), 2.08-2.15 (1H, m), 3.79 (1H, d, J ) 5.0 Hz), 4.15 (2H,
m), 4.35 (1H, m), 4.47 (1H, m), 4.72 (1H, m), 6.06 (1H, d, J )
5.9 Hz), 8.14 (1H, s), 8.44 (1H, s); 13C NMR (D2O) δ 18.71,
20.34, 32.22, 61.69 (d, J ) 9.8 Hz), 67.69 (d, J ) 6.2 Hz), 72.92,
76.80, 86.27 (d, J ) 9.8 Hz), 89.52, 121.17, 142.73, 151.62,
155.02, 157.84, 173.53: MS(FAB) m/z calcd for C15H24N7O7P
445.1475, obsd (M + H) 446.1560.
O-(Ad en osin e-5′-O-yl) N-(L-t yr osyl)p h osp h or a m id a t e
(17y). The reaction was carried out according to the general
procedure as described in the case of 16y (1.27 g, 0.60 mmol).
C-18 Reversed-phase column chromatography was performed
with water, applying a gradient of MeCN (0-10%) to give 17y
(168 mg, 55%) as a white powder: 31P NMR (D2O) δ -5.01;
1H NMR (D2O) δ 2.65-2.73 (2H, m), 3.82-3.96 (3H, m), 4.18
(1H, m), 4.25 (1H, dd, J ) 5.3 Hz, 3.6 Hz), 4.53 (1H, dd, J )
5.6 Hz, 5.3 Hz), 5.89 (1H, d, J ) 5.6 Hz), 6.42 (2H, d, J ) 8.6
Hz), 6.74 (2H, d, J ) 8.6 Hz), 7.95 (1H, s), 8.16 (1H, s); 13C
NMR (D2O) δ 39.26, 57.89 (d, J ) 8.6 Hz), 67.86 (d, J ) 6.1
Hz), 72.90, 76.91, 86.20 (d, J ) 9.8 Hz), 117.91, 121.04, 128.43,
133.21, 142.46, 151.41, 155.27, 157.27, 157.95, 176.10; MALDI-
TOF mass m/z calcd for C19H25N7O8P 510.2, obsd (M + H)
510.1.
O-(Ad en osin e-5′-O-yl) N-(D-tyr osyl)p h osp h or a m id a te
(17z). The reaction was carried out according to the general
procedure as described in the case of 16z (167 mg, 0.079 mmol).
C-18 Reversed-phase column chromatography was performed
with water, applying a gradient of MeCN (0-10%) to give 17z
(14 mg, 35%) as a white powder: 31P NMR (D2O) δ -5.27; 1H
NMR (D2O) δ 2.46-2.60 (2H, m), 3.82-3.95 (2H, m), 4.02-
4.07 (1H, m), 4.15 (1H, m), 4.31 (1H, dd, J ) 4.3 Hz, 4.6 Hz),
4.59 (1H, dd, J ) 5.0 Hz, 4.3 Hz), 5.91 (1H, d, J ) 5.0 Hz),
6.48 (2H, d, J ) 7.6 Hz), 6.66 (2H, d, J ) 7.9 Hz), 7.94 (1H, s),
8.19 (1H, s); 13C NMR (D2O) δ 38.63, 57.53 (d, J ) 13.3 Hz,),
68.09 (d, J ) 3.7 Hz), 72.96, 76.39, 86.19 (d, J ) 8.6 Hz), 89.54,
118.04, 121.15, 127.73, 133.10, 142.17, 151.53, 155.33, 157.39,
157.95, 174.83; MALDI-TOF mass m/z calcd for C19H25N7O8P
510.2, obsd (M + H) 510.3.
C
126H103N8O23P: C, 71.11; H, 4.88; N, 5.27. Found: C, 70.62;
H, 4.92; N, 5.25.
Gen er a l P r oced u r e for th e Syn th esis of a a -AMP N (17).
Fully protected aa-AMPN (16) was treated with concentrated
NH3-dioxane (1:1, (v/v)) at room temperature for 8 h. The
mixture was evaporated under reduced pressure, and residue
was dissolved in water. The aqueous solution was washed five
times with ether and concentrated to a small volume. The
residue was applied to a C18 reversed-phase column, and
elution was performed with water, applying a gradient of
MeCN. The fractions containing 17 were combined and lyoph-
ilized to give 17 as a white powder.
O-(Ad en osin e-5′-O-yl) N-(L-isoleu cyl)p h osp h or a m id a te
(17i). The reaction was carried out according to the general
procedure as described in the case of 16i (197 mg, 0.134 mmol).
C-18 Reversed-phase column chromatography was performed
with water, applying a gradient of MeCN (0-5%) to give 17i
(29 mg, 48%) as a white powder: 31P NMR (D2O) δ -5.26; 1H
NMR (D2O) δ 0.58 (3H, d, J ) 6.6 Hz, 7.3 Hz), 0.70 (3H, d, J
) 6.9 Hz), 0.81-0.92 (1H, m), 1.11-1.21 (1H, m), 1.62-1.68
(1H, m), 3.67 (1H, d, J ) 5.0 Hz), 3.89-4.04 (2H, m), 4.19 (1H,
m), 4.31 (1H, dd, J ) 5.3 Hz, 3.6 Hz), 4.59 (1H, dd, J ) 5.9
Hz, 5.3 Hz), 5.93 (1H, d, J ) 5.9 Hz), 8.06 (1H, s), 8.32 (1H, s);
13C NMR (D2O) δ 13.12, 16.82, 26.09, 38.76, 61.29 (d, J ) 9.8
Hz), 67.74 (d, J ) 4.9 Hz), 72.92, 76.67, 86.25 (d, J ) 9.8 Hz),
89.31, 121.19, 142.64, 151.73, 155.52, 158.20, 173.74; MALDI-
TOF mass m/z calcd for C16H27N7O7P 460.2, obsd (M + H)
460.2.
Sta bility of com p ou n d 12. A solution of 12 (1 mL, 1 OD
unit/mL) were treated with the following reagents: (i) 0.1 M
HCl, rt; (ii) 0.1 M NH4OAc, rt; (iii) 0.1 M NaOH, rt. The
products were monitored by reversed-phase HPLC.
O-(Ad en osin e-5′-O-yl) N-(L-m et h ion yl)p h osp h or a m i-
d a t e (17m ). The reaction was carried out according to the
general procedure as described in the case of 16m (228 mg,
0.153 mmol). C-18 Reversed-phase column chromatography
was performed with water, applying a gradient of MeCN (0-
10%) to give 17m (53 mg, 73%) as a white powder: 31P NMR
Ack n ow led gm en t. This work was supported by a
Grand from “Research for the Future” Program of
the J apan Society for the Promotion of Science
(J SPS-RFTF97I00301) and a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science,
Sports and Culture, J apan. We also thank Dr. S.
Higashiya for measuring the FAB MS spectra.
1
(D2O) δ -5.36; H NMR (D2O) δ 1.91 (3H, s), 1.97-2.06 (2H,
m), 2.39 (1H, dd, J ) 7.6 Hz, 7.9 Hz), 4.07-4.14 (3H, m), 4.32
(1H, m), 4.44 (1H, dd, J ) 5.0 Hz, 4.0 Hz), 4.70 (1H, dd, J )
5.9 Hz, 4.0 Hz), 6.08 (1H, d, J ) 5.9 Hz), 8.23 (1H, s), 8.47
(1H, s); 13C NMR (D2O) δ 0.16.48, 30.53, 32.43, 55.73 (d, J )
11.0 Hz), 67.69 (d, J ) 4.9 Hz), 72.83, 76.80, 86.24 (d, J ) 9.7
Hz), 89.47, 121.24, 142.98, 151.66, 154.28, 157.39, 173.39;
MALDI-TOF mass m/z calcd for C15H25N7O7PS 478.1, obsd
(M + H) 478.2.
Su p p or tin g In for m a tion Ava ila ble: Characterization
data for all compounds obtained in Experimental Section; 1H,
13C, and 31P NMR spectra and MALDI-TOF mass spectra of
compounds 12 and 17i,m ,p ,v,y,z. This material is available
free of charge via the Internet at http://pubs.acs.org.
O-(Ad en osin e-5′-O-yl) N-(L-p r olyl)p h osp h or a m id a t e
(17p ). The reaction was carried out according to the general
procedure as described in the case of 16p (215 mg, 0.015
J O0008338