Synthesis and structure of the hypermodified nucleoside of rat liver phenylalanine transfer ribonucleic Acid.

Article abstract of DOI:10.1248/cpb.50.1318

The first synthesis of (alphaS,betaS)-beta-hydroxy-alpha-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-3-beta-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester [(alphaS,betaS)-11] has been achieved by OsO(4) oxidation of [S-(E

Full text of DOI:10.1248/cpb.50.1318

1318
Chem. Pharm. Bull. 50(10) 1318—1326 (2002)
Vol. 50, No. 10
Synthesis and Structure of the Hypermodified Nucleoside of Rat Liver
Phenylalanine Transfer Ribonucleic Acid
Taisuke ITAYA* and Tae KANAI
Faculty of Pharmaceutical Sciences, Kanazawa University; Takara-machi, Kanazawa 920–0934, Japan.
Received April 10, 2002; accepted June 26, 2002
The first synthesis of (aS,bS)-b-hydroxy-a-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-3-b-D-ribofura-
nosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester [(aS,bS)-11] has been achieved by
OsO4 oxidation of [S-(E)]-4-[4,6-dimethyl-9-oxo-3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-b-D-ribofuranosyl]-4,9-
dihydro-3H-imidazo[1,2-a]purin-7-yl]-2-[(methoxycarbonyl)amino]-3-butenoic acid methyl ester (13) followed by
successive g-deoxygenation through the cyclocarbonates, separation from the (aS,bR)-isomer by means of flash
chromatography, and deprotection. On the other hand, the minor nucleoside of rat liver tRNAPhe was isolated on
a scale of 100 mg by partial digestion of unfractionated tRNA (1 g) with nuclease P1, followed by reverse-phase
column chromatography, complete digestion with nuclease P1/alkaline phosphatase, and reverse-phase HPLC.
Comparison of this nucleoside with the synthetic one has unambiguously established its structure to be (aS,bS)-
11.
Key words b-hydroxywybutosine; minor nucleoside; rat liver tRNAPhe; fluorescent nucleoside; condensed tricyclic nucleoside
Many eukaryotic tRNAsPhe have fluorescent components
isolated the corresponding nucleoside from rat liver for the
at the position next to the 3Ј-end of the anticodon.1—5) The
fluorescent base isolated from chicken, rat, and bovine liver
tRNAsPhe was first reported to be b-hydroperoxywybutine
(3)6,7) on the basis of comparison of the UV, fluorescent, and
MS spectra as well as the chromatographic behavior with
those of wybutine (1),8,9) the structurally related precedent
from yeast tRNAPhe. The base from the plant Lupinus luteus
was also characterized as 3.10) In this case, the presence of
the hydroperoxy group was supported by a specific color test
employing Fe(SCN)2. The structure 3 was suggested to be as-
first time and determined its structure to be (aS,bS)-11, the
first synthesis of which is also described. A preliminary com-
munication of this work has been published.17,18)
Results and Discussion
Synthesis The synthesis of the bases (aS,bR)- and
(aS,bS)-2 has been accomplished by OsO4 oxidation of the
olefin 4 followed by separation of the resulting diastereomers
5 and 7 and hydrogenolysis through the cyclic carbonates 6
and 8, as shown in Chart 1.3,15,16) In the present study, we first
examined the applicability of this reaction sequence to the
nucleoside level. Thus the Heck reaction between 2Ј,3Ј,5Ј-tri-
O-acetyl-7-iodowyosine (9)19) and (Ϯ)-2-[(methoxycar-
bonyl)amino]-3-butenoic acid20) was conducted according to
the procedure reported for the synthesis of 3-b-D-ribofura-
nosylwybutine (12).19) The product was treated with
Me3SiCHN2 to give a mixture of diastereomers 10. This was
subjected to OsO4 oxidation followed by cyclocondensation
with triphosgene, catalytic hydrogenolysis, purification by
preparative TLC, and deprotection to afford a mixture of four
diastereomers 11, as shown in Chart 2. These were separated
by HPLC. Two were (aS,bR)- and (aS,bS)-11, of which un-
ambiguous syntheses are described below. The structures of
the others [(aR,bS)- and (aR,bR)-11] were assignable by
comparison of the 1H-NMR patterns of their amino acid moi-
eties with those of (aS,bR)- and (aS,bS)-11. Assuming that
2 is the correct two-dimensional expression of the structure
of the hypermodified base of rat liver tRNAPhe, one of the
four diastereomers 11 is likely the correct structure of the
corresponding nucleoside. We preferred (aS,bR)-11 and
(aS,bS)-11 as the most probable alternatives because struc-
turally related wybutosine (12) from yeast tRNAPhe had been
determined by us to have the (aS) configuration.2)
signed to the base from wheat germ tRNAPhe 10)
because it
,
had been shown to be indistinguishable from that of beef.11)
Kasai et al., however, reported that the fluorescent base from
rat liver tRNAPhe was b-hydroxywybutine (2) on the basis of
the MS spectral data as well as the negative coloring test for
the hydroperoxy group.12) Those authors proposed that 3
might be an artifact formed during storage of the sample of 2
and suggested that the base from wheat germ tRNAPhe was
also 2. Notwithstanding that report, Mochizuki et al. pre-
ferred 3 for the fluorescent base isolated from the aquatic
fungus Geotrichum candidum tRNAPhe 13)
Wiewiórowski’s
.
group also reported that the base from tRNAsPhe of wheat
germ, yellow lupine seeds, and maize seeds was 3. They de-
scribed 3 as very unstable and found that it decomposed to 2
and 114): The stability observed for 2 and 3 contradicted that
reported by Kasai et al.12) Although we achieved the synthe-
sis of (aS,bR)- and (aS,bS)-2 as the most probable alterna-
tives for the base isolated from rat liver tRNAPhe 3,15,16)
the
,
lack of a sample of the base from the tRNAPhe has hampered
its structural determination. In the present investigation, we
Because it is difficult to obtain stereochemically pure
(aS)-10 in a large quantity,19) we selected 132) as a key inter-
mediate for the stereospecific synthesis of (aS,bR)- and
(aS,bS)-11. OsO4 oxidation of the olefin 13 in the presence
of N-methylmorpholine N-oxide in acetone–phosphate buffer
(pH 6) at room temperature, followed by HPLC on silica gel
* To whom correspondence should be addressed. e-mail: itaya@mail.p.kanazawa-u.ac.jp
© 2002 Pharmaceutical Society of Japan

October 2002
1319
Chart 1
Chart 2
afforded the diols 14 and 16 in 51% and 30% yields, respec- would provide the desired monohydroxy compound 21, elim-
tively. The configurations of these compounds were assigna- ination of H2CO3 followed by hydrogenation would form the
ble by comparison of their 1H-NMR spectra with those of the dideoxy compound 23 through the olefin 22. Analogous ex-
bases 5 and 7.3) Treatment of the major isomer 14 with an ex- amples of the formation of saturated compounds and olefins
cess of triphosgene in CH2Cl2 in the presence of pyridine at have already been reported for the electrochemical reduction
0 °C afforded the cyclic carbonate 15 in 80% yield. Catalytic of cyclic carbonates of meso-hydrobenzoin, (Ϯ)-hydroben-
hydrogenolysis of 15 over Pearlman’s catalyst afforded the b- zoin, and (E)-2,3-diphenylbutane-1,2-diol.21) Although the
hydroxy compound 17 in 28% yield together with the undesirable dideoxygenation of (Ϯ)-19 could be suppressed
dideoxy compound 182) (23%). The analogous concomitant to some extent by the use of Pt instead of Pd–C,16) the Pt cat-
formation of the dideoxy compounds has already been recog- alyst was found in the present investigation to be inferior in
nized in the reaction of the model compound (Ϯ)-193,16) and view of reproducibility for hydrogenolysis of 15. Deprotec-
in the reactions involved in Charts 1, 2. As illustrated in tion of 17 was accomplished by treatment with Bu4NF in
Chart 4, hydrogenolysis of (Ϯ)-19 should produce the inter- aqueous THF in the presence of pyridine at room tempera-
mediate 20. While hydrolysis of the hydrogen carbonate 20 ture22) without cleaving the extraordinarily labile glycosyl

1320
Vol. 50, No. 10
Chart 3
Chart 4
bond to provide the desired nucleoside in 86% yield. The
correctness of the assignment of the structure (aS,bR)-11 to
this product was established by its hydrolysis with 0.1 N
1
aqueous HCl, leading to optically pure (aS,bR)-2. The H-
NMR [(CD3)2SO or (CD3)2CO] spectral patterns of signals
arising from the side chains of these two compounds closely
1
resemble each other. This is also the case with the H-NMR
spectra of 17 and (R*,S*)-1-benzyl-b-hydroxy-a-[(methoxy-
carbonyl)amino]-4,6-dimethyl-9-oxo-4,9-dihydro-1H-imi-
dazo[1,2-a]purine-7-butanoic acid methyl ester [(Ϯ)-24]16)
measured in CDCl3, as shown in Table 1. However, (aS,bR)-
employed for the preparation of 15. The starting material 16
was recovered in ca. 80% yield. This is an unbelievably
strange result in view of the positive reaction of the base 73)
(Chart 1) having the same configurations and the reaction of
the (aS,bR,gR)-nucleoside involved in the reaction sequence
shown in Chart 2. The latter case indicated what should be
done to obtain the desired carbonate 26. When a mixture of
16 and 14, accessible in 91% yield in a ratio of 1 : 2 in the
above OsO4 oxidation of 13, was subjected to the reaction
with triphosgene, 26 was obtained as a mixture with 15 in a
1
2 shows a somewhat different H-NMR spectrum from that
of 17 in CDCl3 (Table 1), suggesting that N(1)-H of (aS,bR)-
2 affects the conformation of its side chain through hydrogen
bonding in this solvent.
Contrary to the successful conversion of the diol 14 into
the carbonate 15, the minor isomer 16 with (aS,bR,gR) con-
figurations did not produce the cyclic carbonate 26 at all
upon treatment with triphosgene in a manner similar to that

October 2002
1321
Table 1. 1H-NMR Spectral Data for (aS,bR)-, (aS,bS)-2, and Related Compounds Measured in CDCl3
Chemical shift (d)
a)
Proton
(aS,bS)-2b)
Species 2
(aS,bR)-2b)
17c)
(Ϯ)-24d)
27c)
(Ϯ)-25d)
Species 1
CO2CH3
3.68 s
3.73 s
3.72
3.76
4.46
4.39
3.15
3.57
4.17
5.66
2.25
4.11
7.94
—
3.74
3.77
4.49f )
4.37
3.22
3.46
4.15
5.65f )
2.27
3.92
7.66
—
3.69 s
3.82 s
3.69 s
3.87 s
4.19 m
4.58 m
3.44 dd (15, 10.7)
3.55 dd (15, 1.5)
4.10 br
8.00 br
2.44 s
3.70
3.80
4.50
4.16
3.41
3.72
3.73
4.52 f )
4.15
3.36
3.41
3.80
5.90 f )
2.26
3.90
7.66
—
C(a)-H
C(b)-H
C(g)-H2
4.56 d (9.8)e)
4.47 m
4.78 m
4.27 m
3.10 m
3.70 m
4.78 br
6.90 br
2.26 s
3.95 s
7.93 s
11.46 br
3.19 dd (15.6, 4.4)
3.75 dd (15.6, 8.8)
4.94 d (5.9)g)
6.39 d (9.5)h)
2.32 s
C(b)-OH
C(a)-NH
C(6)-CH3
NCH3
C(2)-H
N(1)-H
3.83
5.88
2.25
4.11
7.94
—
3.97 s
3.97 s
7.89 s
13.52 br
7.90 s
11.51 si)
a) Figures in parentheses denote coupling constants (J) in Hz. b) Measued for a 2.4 mM solution. c) See Experimental for complete data. d) Taken from ref. 16.
e) Accompanied by a small broad signal at 4.35. f ) Accompanied by a minor signal. g) Accompanied by a small broad signal at 5.03. h) Accompanied by a small broad sig-
nal at 6.29. i) Accompanied by a small broad signal at 11.74.
Chart 5
Chart 6
ratio of 1 : 2.9. However, 26 was not formed when 16 was chains of 14 and 16 is important for the cyclocondensation of
added after the reaction of 14 with triphosgene was com- 16. Although separation of the mixture of 15 and 26 was dif-
pleted even in the presence of excess reagents. These results ficult, 16 underwent cyclocondensation as well with triphos-
suggest that an intermolecular interaction between the side gene in the presence of the base 5 having the (aS,bS,gS)

1322
Vol. 50, No. 10
configurations, giving a mixture of the carbonates (26 and 6) isolating these nucleosides on a scale of 70—80 mg. To deter-
as shown in Chart 5. Compound 26 was easily obtained from mine how many rats are required for isolation of a compara-
this mixture by flash chromatography in 57% yield. Hy- ble amount of the target nucleoside, we first attempted isola-
drogenolysis of 26 using Pearlman’s catalyst afforded the tion of the corresponding base as a preliminary experiment.
monohydroxy compound 27 in 39% yield together with the Unfractionated tRNA obtained from the livers of ca. 10 rats
dideoxy compound 18 (19%). As the two diastereomers 17 was subjected to mild acid treatment23) (pH 2.9 at 40 °C for
and 27 can be separated by flash chromatography, these were 16 h), and polynucleotides were removed by precipitation
more conveniently obtained by hydrogenolysis of the 1 : 2.9 with EtOH. The minor base was obtained on a scale of a few
mixture of 15 and 26 described above in 21% and 8% yields, micrograms from the soluble part by means of TLC on silica
respectively, based on the olefin 13 (Chart 6). Desilylation gel. The HPLC behavior of the base thus obtained was iden-
of 27 afforded (aS,bS)-11 in 86% yield. Hydrolysis of tical with that3) of (aS,bS)-2, and the correctness of its ab-
(aS,bS)-11 produced optically pure (aS,bS)-2 in 98% yield. solute configurations was established by chiral HPLC analy-
It should be noted that the 1H-NMR spectrum arising from sis. Furthermore, the assignable signals appearing in the H-
1
the side chain of the base (aS,bS)-2 in CDCl3 does not re- NMR spectrum [(CD3)2CO] of this compound corresponded
semble those of (Ϯ)-2516) and 27. Interestingly, (aS,bS)-2 to those of (aS,bS)-2. No trace of wybutine (1) was detected
shows two sets of signals (species 1 and 2 in Table 1) in in contrast to the results reported by Kasai et al.12) It follows
CDCl3, while the diastereomer (aS,bR)-2 exists as a single that (aS,bS)-2 is unlikely to be an artifact of hydroperoxy-
species. This complexity of the signals disappeared in the wybutine (3), because 3 was reported to decompose to 2 and
spectra taken in (CD3)2SO,3) (CD3)2CO, CD3CN, and pyri- 1.14) Thus we concluded that the formula (aS,bS)-2 is a com-
dine-d5, all of which are stronger hydrogen bond acceptors plete expression of the hypermodified base of rat liver
than CDCl3. Furthermore, the molar ratio of the two species tRNAPhe. It should be noted that (aS,bS)-2 was stable during
estimated on the basis of relative areas of the C(6)-Me sig- storage at room temperature, contrary to the contention of
nals depended on the total concentration, as shown in Table Kasai et al.12)
2. These results suggest that (aS,bS)-2 molecules associate
themselves in part through intermolecular hydrogen bond(s) next started with the same grade of tRNA (1 g) obtained from
in CDCl3.
100 rats. Because this material resisted digestion with nucle-
To isolate the nucleoside on a scale of some 50 mg, we
Compounds (aS,bR)- and (aS,bS)-11 thus obtained were ase P1, it was extracted with aqueous Me2CHOH and then
converted into their tetraacetates-d12 28 and 29 according to with H2O. The tRNA in the combined solution was purified
the strategy that we had used to determine the structures of by ion-exchange chromatography to give unfractionated
wyosine1) from torula yeast tRNA and wybutosine (12)2) tRNA (350 mg). This was partially digested with nuclease P1
from baker’s yeast tRNA. Compounds 28 and 29 could be followed successively by reverse-phase column chromatogra-
1
distinguished by comparison of their H-NMR spectra taken phy, complete digestion with nuclease P1, dephosphorylation
in CDCl3.
with alkaline phosphatase, and HPLC according to the re-
Isolation and Identification of the Minor Nucleoside ported procedure1,2) to give the target nucleoside (100 mg).
and Its Base from Rat Liver tRNA We had already deter- We did not find any trace of wybutosine (12). The HPLC be-
mined the structures of wyosine1) and wybutosine (12)2) by havior of the nucleoside was identical to that of (aS,bS)-11
but different from that of the diastereomer (aR,bR)-11, rul-
ing out the 3-b-L-ribofuranosyl structure 30 for this nucleo-
Table 2. Effect of Total Concentrations on Ratios of Two Species of
(aS,bS)-2 in CDCl3 as Determined by 1H-NMR Spectroscopy
side because (aR,bR)-11 is the enantiomer of 30. The minor
nucleoside thus obtained was converted into the tetraacetate-
Total concentration of (aS,bS)-2
Mole fraction of species 1a)
(%)
1
d12, which was identical to 29 on the basis of MS and H-
(mM)
NMR spectroscopy. Finally the tetraacetate-d12 of natural ori-
gin was treated with MeONa–MeOH and then with dilute
aqueous HCl to give the base, the identity of which with
(aS,bS)-2 was confirmed by comparison of their HPLC be-
havior and 1H-NMR spectra. The structure of the hypermodi-
fied nucleoside of rat liver tRNAPhe was hereby determined
unambiguously to be (aS,bS)-11.
0.038
0.075
0.15
0.30
0.60
1.2
81
71
63
59
52
49
46
2.4
a) Estimated on the basis of relative areas of the C(6)-Me signals of the two species.

October 2002
1323
tion was identical (by comparison of the 1H-NMR spectrum and HPLC mo-
bility) with authentic (aS,bR)-11 described below. (aR,bR)-b-Hydroxy-a-
[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-3-b-D-ribofuranosyl-4,9-di-
hydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester [(aR,bR)-11]
Experimental
General Notes Spectra reported herein were recorded on a JEOL JMS-
SX102A mass spectrometer, a Hitachi U-3010 spectrophotometer, or a
JEOL JNM-GSX-500 NMR spectrometer (measured at 25 °C with Me4Si as
an internal standard unless otherwise stated). CDCl3 for measurements of
small samples was treated with alumina according to the reported proce-
dure.1) MS measurements were performed by Dr. M. Takani and her associ-
ates at Kanazawa University. The optical rotation was measured with a
Horiba SEPA-300 polarimeter using a 10-cm sample tube. The HPLC sys-
tem employed consisted of a Tosoh CCPD pump, an injection valve unit, a
UV-8020 detector, and a Chromatocorder 21 integrator or a Waters 6000A
pump, a U6K injector, and a model 440 absorbance detector. The following
abbreviations are used: brϭbroad, dϭdoublet, ddϭdoublet-of-doublets,
dddϭdoublet-of-doublets-of-doublets, ddddϭdoublet-of-doublets-of-dou-
blets-of-doublets, ddtϭdoublet-of-doublets-of-triplets, mϭmultiplet, and
sϭsinglet.
1
was obtained from the fourth fraction, H-NMR [(CD3)2CO] d24): 2.24 [3H,
s, C(6)-Me], 3.18 [1H, dd, Jϭ14.7, 8.3 Hz, one C(g)-H2], 3.64, 3.67 [3H
each, s, overlapping with a 1H signal arising from one C(g)-H2, CCO2Me
and NCO2Me], 3.84, 3.92 [1H each, m, C(5Ј)-H2], 4.21 [1H, m, C(4Ј)-H],
4.24 (3H, s, NMe), 4.28 [1H, m, C(b)-H], 4.37 [1H, m, C(a)-H], 4.49 [2H,
m, C(5Ј)-OH and C(3Ј)-H], 4.58 [1H, br, C(b)-OH], 4.74 [1H, m, C(2Ј)-H],
4.90 [1H, br, C(3Ј)-OH], 5.25 [1H, br, C(2Ј)-OH], 6.30 [1H, d, Jϭ4.9 Hz,
C(1Ј)-H], 6.68 [1H, d, Jϭ7.8 Hz, C(a)-NH], 8.21 [1H, s, C(2)-H].
Dihydroxylation of 13 A solution of N-methylmorpholine N-oxide
monohydrate (90 mg, 0.67 mmol) in 0.5 M phosphate buffer (pH 6, 33 ml)
and a 1.1% (w/v) OsO4 solution in Me3COH (1.2 ml, 0.05 mmol) were added
to a solution of 132) (333 mg, 0.392 mmol) in Me2CO (33 ml). The resulting
mixture was stirred at room temperature for 4 h and then for a further 30 min
after the addition of Na2S2O5 (124 mg, 0.652 mmol). The mixture was con-
centrated to half the initial volume and extracted with CH2Cl2 (3ϫ20 ml).
The organic layers were combined, dried over MgSO4, and concentrated in
vacuo to leave a foam (341 mg). This was dissolved in hexane (4 ml) and the
solution was subjected to HPLC [LiChrosorb Si-60 (7 mm, 250ϫ10 mm)
(Merck); hexane–CHCl3–MeOH (50 : 48 : 2, v/v)] in eight portions, provid-
ing (aS,bS,gS)-b,g-dihydroxy-a-[(methoxycarbonyl)amino]-4,6-dimethyl-
9-oxo-3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-b-D-ribofuranosyl]-4,9-dihy-
dro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester monohydrate
(14·H2O) (176 mg, 50%), mp 117—121 °C. Recrystallization of this sample
from 90% (v/v) aqueous MeOH and drying over P2O5 at 2 mmHg and 50 °C
for 17 h afforded an analytical sample of 14·H2O as colorless needles, mp
Synthesis of Four Diastereomers of b-Hydroxy-3-b-D-ribofuranosyl-
wybutine (11) A mixture of Pd(OAc)2 (6.6 mg, 0.029 mmol), NaHCO3
(201 mg, 2.39 mmol), 919) (468 mg, 0.797 mmol), Bu4NCl (222 mg, 0.799
mmol), and Me2NCHO (12 ml) was stirred at 60 °C for 10 min. (Ϯ)-2-
[(Methoxycarbonyl)amino]-3-butenoic acid20) (191 mg, 1.20 mmol) was
added to the mixture, and the whole was stirred at 60—65 °C for 8 h. After
H2O (30 ml) was added, the resulting mixture was brought to pH 3 by the ad-
dition of 10% aqueous H3PO4 and extracted with CHCl3 (2ϫ30 ml). The or-
ganic layers were combined and extracted with saturated aqueous NaHCO3
(3ϫ20 ml). The aqueous layers were combined, brought to pH 3 with 10%
aqueous H3PO4, and extracted with CHCl3 (4ϫ20 ml). The organic layers
were combined, dried over MgSO4, and concentrated in vacuo. The oily
residue was dissolved in a mixture of MeOH (1 ml) and benzene (4 ml), and
2.0 M Me3SiCHN2 solution in hexane (0.3 ml) was added. The resulting solu-
tion was concentrated in vacuo, and the residue was purified by flash chro-
matography [AcOEt–EtOH (10 : 1, v/v)] to give 10 (115 mg, 23%) as a yel-
low glass. The diastereomeric mixture thus obtained was dissolved in
Me2CO (15 ml). After a solution of N-methylmorpholine N-oxide monohy-
drate (42 mg, 0.31 mmol) in 0.5 M phosphate buffer (pH 6, 15 ml) and a 1.6%
(w/v) OsO4 solution in Me3COH (0.4 ml) were added, the mixture was
stirred at room temperature for 5 h and then for a further 30 min after the ad-
dition of Na2S2O5 (57 mg, 0.30 mmol). The resulting suspension was con-
centrated to half the initial volume and extracted with CHCl3 (2ϫ20 ml).
The organic layers were combined, washed with saturated aqueous NaCl,
dried over MgSO4, and concentrated in vacuo to leave a colorless glass (109
mg). This was purified by flash chromatography [CHCl3–MeOH (10:1, v/v)],
providing a colorless glass (57 mg). A portion (20 mg) of this mixture of the
diols was treated with triphosgene (18 mg) in CH2Cl2 (2 ml) in the presence
of pyridine (0.03 ml) at 0 °C for 20 min. The resulting solution was washed
successively with H2O (3 ml), 5% aqueous citric acid (2ϫ3 ml), and H2O
(2ϫ3 ml), dried over MgSO4, and concentrated in vacuo, leaving a colorless
glass (7 mg). A portion (5 mg) of this material was hydrogenated over Pearl-
man’s catalyst (15 mg) in MeOH (4 ml) at 40 °C for 2 h. The catalyst was re-
moved by filtration and washed with hot MeOH (50 ml). The filtrate and
washings were combined and concentrated in vacuo. The residue was puri-
fied by TLC on silica gel [CHCl3–MeOH (10 : 1, v/v)] to give a diastere-
omeric mixture of the monohydroxy compounds (2 mg) as a colorless glass.
After a solution of this product in 0.1 M MeONa–MeOH (0.15 ml) was
stored at 0 °C for 5 min, 0.1 M aqueous NaH2PO4 (0.3 ml) was added at once.
The resulting mixture was concentrated in vacuo, and the residue was puri-
fied by TLC on silica gel [CHCl3–MeOH (5 : 1, v/v)], giving a colorless
glass (1 mg). Separation of the diastereomers thus obtained was accom-
plished by HPLC [LiChrosorb RP18 (7 mm, 250ϫ10 mm) (Merck); MeOH–
H2O (30 : 70, v/v) at the rate of 5 ml/min] in two portions. The molar ratio of
the diastereomers was 1 : 2 : 2 : 1 in the order of elution. The isomer that was
115 °C (softened) 207—209.5 °C. [a]D20 Ϫ14.2° (cϭ0.456, MeOH). FAB-
95% EtOH
MS m/z: 905 (MNaϩ), 865 (MHϩϪ18). UV l
nm (e): 240 (35600),
max
Nujol
1
297 (7000). IR n
cmϪ1: 1746, 1725, 1684 (CϭO). H-NMR (CDCl3) d:
max
Ϫ0.29, Ϫ0.02, 0.13, 0.15, 0.16, 0.17 (3H each, s, three SiMe2), 0.75, 0.95,
0.97 (9H each, s, three tert-Bu), 1.56 (s, H2O), 2.24 [3H, s, C(6)-Me], 3.44
[1H, br s, C(b)-OH], 3.66, 3.72 (3H each, s, CCO2Me and NCO2Me), 3.80
[1H, dd, Jϭ11.7, 1.5 Hz, one C(5Ј)-H2], 3.88 [1H, d, Jϭ10 Hz, C(a)-H],
3.89 [1H, dd, Jϭ11.7, 2.5 Hz, one C(5Ј)-H2], 4.13 [1H, dd, Jϭ1.5, 2.5 Hz,
C(4Ј)-H], 4.18 (3H, s, NMe), 4.20 [1H, d, Jϭ4.4 Hz, C(3Ј)-H], 4.42 [1H, dd,
Jϭ4.4, 7.8 Hz, C(2Ј)-H], 4.53 (0.1H), 4.57 (0.9H) [d each, Jϭ9.8 Hz, C(b)-
H], 4.79 [1H, dd, Jϭ9.8, 11.7 Hz, C(g)-H], 5.46 (0.1H), 5.61 (0.9H) [d each,
Jϭ10 Hz, C(a)-NH], 5.62 [1H, d, Jϭ11.7 Hz, C(g)-OH], 6.25 [1H, d, Jϭ7.8
Hz, C(1Ј)-H], 8.04 [1H, s, C(2)-H]. Anal. Calcd for C39H70N6O11Si3·H2O: C,
51.97; H, 8.05; N, 9.32. Found: C, 52.12; H, 7.94; N, 9.38. (aS,bR,gR)-b,g-
Dihydroxy-a-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-3-[2,3,5-tris-
O-(tert-butyldimethylsilyl)-b-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-
a]purine-7-butanoic acid methyl ester (16) (103 mg, 30%) was obtained
from a later fraction as a colorless glass, [a]D261 Ϫ13.9° (cϭ0.512, MeOH).
FAB-MS m/z: 905 (MNaϩ), 865 (MHϩϪ18). H-NMR (CDCl3) d: Ϫ0.23,
Ϫ0.01, 0.13, 0.14 (3H each), 0.15 (6H) (s, three SiMe2), 0.75, 0.949, 0.953
(9H each, s, three tert-Bu), 2.39 [3H, s, C(6)-Me], 3.51 [1H, br, C(b)-OH],
3.55, 3.77 (3H each, s, NCO2Me and CCO2Me), 3.79 (dd, Jϭ11.5, 1.5 Hz),
3.88 (dd, Jϭ11.7, 2.5 Hz) [1H each, C(5Ј)-H2], 4.13 [1H, dd, Jϭ1.5, 2.5 Hz,
C(4Ј)-H], 4.17 (3H, s, NMe), 4.21 [1H, d, Jϭ4.4 Hz, C(3Ј)-H], 4.22 [1H, m,
C(a)-H], 4.30 [1H, dd, Jϭ2.4, 8.3 Hz, C(b)-H], 4.44 [1H, dd, Jϭ4.4, 7.3 Hz,
C(2Ј)-H], 5.16 [1H, dd, Jϭ8.3, 11.2 Hz, C(g)-H], 5.45 [1H, d, Jϭ11.2 Hz,
C(g)-OH], 5.62 (0.1H, d, Jϭ10 Hz), 5.72 (0.9H, d, Jϭ7.8 Hz) [C(a)-NH],
6.22 [1H, d, Jϭ7.3 Hz, C(1Ј)-H], 8.00 [1H, s, C(2)-H].
(aS,4S,5S)-5-[4,6-Dimethyl-9-oxo-3-[2,3,5-tris-O-(tert-butyldimethyl-
silyl)-b-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-a]purin-7-yl]-a-
[(methoxycarbonyl)amino]-2-oxo-1,3-dioxolane-4-acetic Acid Methyl
Ester (15) A solution of 14·H2O (65 mg, 0.072 mmol) in benzene (5 ml)
was dried over MgSO4 and concentrated in vacuo. The residual foam was
dried over P2O5 at 2 mmHg at room temperature for 3 h. This was dissolved
in CH2Cl2 (2 ml), and pyridine (0.06 ml, 0.7 mmol) was added. A solution of
triphosgene (16 mg, 0.054 mmol) in CH2Cl2 (2 ml) was then added dropwise
at 0 °C over a period of 3 min, and the mixture was stirred at 0 °C for a fur-
ther 15 min. The reaction mixture was diluted with CH2Cl2 (5 ml), washed
successively with H2O (5 ml), 5% aqueous citric acid (2ϫ5 ml), and satu-
rated aqueous NaHCO3 (5 ml), dried over MgSO4, and concentrated in
vacuo, leaving a yellow glass. This was purified by flash chromatography
[hexane–AcOEt (2 : 3, v/v)] to give 15 (53 mg, 80%) as a faintly yellow
glass, [a]D18 Ϫ50.6° (cϭ0.425, MeOH). FAB-MS m/z: 909 (MHϩ). 1H-NMR
(CDCl3) d: Ϫ0.27, Ϫ0.02, 0.13 (3H each), 0.15 (6H), 0.16 (3H), (s, three
SiMe2), 0.76, 0.95, 0.96 (9H each, s, three tert-Bu), 2.38 [3H, s, C(6)-Me],
1
eluted the fastest was identical (by comparison of the H-NMR spectrum
and HPLC mobility) to the authentic (aS,bS)-11 described below. (aR,bS)-
b-Hydroxy-a-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-3-b-D-ribofu-
ranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester
1
[(aR,bS)-11] was obtained from the second fraction, H-NMR [(CD3)2CO]
d
24): 2.19 [3H, s, C(6)-Me], 3.28 (dd, Jϭ14.2, 5.5 Hz), 3.36 (dd, Jϭ14.2,
7 Hz) [1H each, C(g)-H2], 3.68, 3.69 (3H each, s, CCO2Me and NCO2Me),
3.85 (ddd, Jϭ12.2, 5.4, 2.9 Hz), 3.94 (ddd, Jϭ12.2, 4.9, 2.9 Hz) [1H each,
C(5Ј)-H2], 4.21 [1H, m, C(4Ј)-H], 4.22 (3H, s, NMe), 4.26 [1H, dd, Jϭ1.5,
9.3 Hz, C(a)-H], 4.49 [2H, m, C(5Ј)-OH, C(3Ј)-H], 4.57—4.67 [2H, m,
C(b)-OH, C(b)-H], 4.74 [1H, m, C(2Ј)-H], 4.78 [1H, br, C(3Ј)-OH], 5.13
[1H, br, C(2Ј)-OH], 6.19 [1H, d, Jϭ9.3 Hz, C(a)-NH], 6.30 [1H, d, Jϭ4.9
Hz, C(1Ј)-H], 8.21 [1H, s, C(2)-H]. The isomer obtained from the third frac-

1324
Vol. 50, No. 10
3.78, 3.79 (3H each, s, two CO2Me), 3.79 (dd, Jϭ11.7, 2 Hz), 3.87 (dd, C(a)-H], 5.88 [1H, d, Jϭ7.3 Hz, C(a)-NH], 6.21 [1H, d, Jϭ7.8 Hz, C(1Ј)-
Jϭ11.7, 2.9 Hz) [1H each, C(5Ј)-H2], 4.12 [1H, dd, Jϭ2, 2.9 Hz, C(4Ј)-H], H], 7.94 [1H, s, C(2)-H].
4.14 (3H, s, NMe), 4.20 [1H, d, Jϭ4 Hz, C(3Ј)-H], 4.42 [1H, dd, Jϭ4, 7.5
Hz, C(2Ј)-H], 4.67 [1H, dd, Jϭ1, 9 Hz, C(a)-H], 5.41 [1H, dd, Jϭ1, 7.3 Hz,
ii) Compound 26 (4 mg, 0.004 mmol) was hydrogenated over Pearlman’s
catalyst (4 mg) in MeOH (2 ml) at 40 °C for 2 h and then at 50 °C for a fur-
C(b)-H], 5.62 [1H, d, Jϭ9 Hz, C(a)-NH], 5.83 [1H, d, Jϭ7.3 Hz, C(g)-H], ther 2 h. The catalyst was filtered off and washed with hot MeOH (100 ml).
6.23 [1H, d, Jϭ7.5 Hz, C(1Ј)-H], 8.00 [1H, s, C(2)-H].
The filtrate and washings were combined and concentrated in vacuo. The
(aS,4R,5R)-5-[4,6-Dimethyl-9-oxo-3-[2,3,5-tris-O-(tert-butyldimethyl- residue was purified by TLC on silica gel [CHCl3–MeOH (40 : 1, v/v)] to
silyl)-b-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-a]purin-7-yl]-a-
[(methoxycarbonyl)amino]-2-oxo-1,3-dioxolane-4-acetic Acid Methyl
Ester (26) A solution of triphosgene (6 mg, 0.02 mmol) in dry CH2Cl2 (0.6
give 182) (0.7 mg) and 27 (1.5 mg) as a colorless glass.
(aS,bR)-b-Hydroxy-a-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-
3-b-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic
ml) was added dropwise to a solution of 16 (7.8 mg, 0.0088 mmol), 53,16) Acid Methyl Ester [(aS,bR)-11] A 1 M Bu4NF solution (0.7 ml, 0.7
(9.0 mg, 0.018 mmol), and pyridine (0.03 ml) in CH2Cl2 (0.6 ml) at 0 °C mmol) in THF was added to a solution of 17 (58 mg, 0.067 mmol) in pyri-
under N2 over a period of 3 min, and the mixture was stirred at 0 °C for a dine–THF–H2O (1 : 8 : 1, v/v) (3.3 ml), and the mixture was stirred at room
further 15 min. The reaction mixture was diluted with CH2Cl2 (5 ml), temperature for 18 h. The resulting solution was concentrated in vacuo. The
washed successively with H2O, 5% aqueous citric acid, and saturated aque- residue was purified by flash chromatography [CH2Cl2–MeOH (5 : 1, v/v)] to
ous NaHCO3 (5 ml each), dried over MgSO4, and concentrated in vacuo,
give (aS,bR)-11·H2O (31 mg, 86%), mp 173—183 °C. Recrystallization of
leaving a yellow glass. This was purified by column chromatography on sil- this compound from 80% (v/v) aqueous MeOH and drying over P2O5 at 2
ica gel (AcOEt) to give 26 (4.6 mg, 57%) as a faintly yellow glass, 1H-NMR mmHg and 50 °C for 10 h afforded colorless plates, mp 205—210 °C. These
(CDCl3) d: Ϫ0.26, Ϫ0.01, 0.13 (3H each), 0.14 (6H), 0.15 (3H), (s, three were exposed to air at room temperature until a constant weight was reached,
SiMe2), 0.76, 0.947, 0.953 (9H each, s, three tert-Bu), 2.36 [3H, s, C(6)- providing (aS,bR)-11·H2O: mp 198—210 °C (dec.). [a]D30 Ϫ72.8° (cϭ
1
Me], 3.69, 3.77 (3H each, s, CCO2Me and NCO2Me), 3.79 (dd, Jϭ11.7, 2
0.167, H2O). FAB-MS m/z: 547 (MNaϩ), 525 (MHϩ). H-NMR [(CD3)2SO]
Hz), 3.88 (dd, Jϭ11.7, 2.9 Hz) [1H each, C(5Ј)-H2], 4.12 [1H, dd, Jϭ2, 2.9 d: 2.07 [3H, s, C(6)-Me], 3.10 (dd, Jϭ14.2, 6.8 Hz), 3.16 (dd, Jϭ14.2, 7 Hz)
Hz, C(4Ј)-H], 4.13 (3H, s, NMe), 4.20 [1H, d, Jϭ4.4 Hz, C(3Ј)-H], 4.41 [1H each, C(g)-H2], 3.53 (0.3H, s), 3.59 [5.7H, s, overlapping with a 1H sig-
[1H, dd, Jϭ4.4, 7.8 Hz, C(2Ј)-H], 4.86 [1H, dd, Jϭ3.4, 8.8 Hz, C(a)-H], nal arising from one C(5Ј)-H2] (two CO2Me), 3.69 [1H, ddd, Jϭ12.2, 3.4,
5.20 [1H, dd, Jϭ3.4, 7.3 Hz, C(b)-H], 5.69 [a total of 1H with a small broad 4.9 Hz, one C(5Ј)-H2], 3.89 (0.1H, br), 3.94 (0.9H, dd, Jϭ2.4, 8.8 Hz) [C(a)-
signal at 5.35, br, C(a)-NH], 6.21 [1H, d, Jϭ7.8 Hz, C(1Ј)-H], 6.43 [1H, d, H], 3.99 [1H, ddd, Jϭ3.4, 3.4, 4.9 Hz, C(4Ј)-H], 4.03 (3H, s, NMe), 4.13
Jϭ7.3 Hz, C(g)-H], 7.98 [1H, s, C(2)-H]. Compound 63,16) (5.9 mg, 62%) [1H, ddd, Jϭ4.9, 4.9, 5.9 Hz, C(3Ј)-H], 4.41 [1H, dddd, Jϭ7, 7, 2.4, 7.8 Hz,
was obtained from the later fraction.
(aS,bR)-b-Hydroxy-a-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-
C(b)-H], 4.45 [1H, ddd, Jϭ4.9, 5.9, 4.9 Hz, C(2Ј)-H], 4.97 (0.9H), 5.01
(0.1H) [d each, Jϭ7.8 Hz, C(b)-OH], 5.12 [1H, dd, Jϭ5.4, 4.9 Hz, C(5Ј)-
3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-b-D-ribofuranosyl]-4,9-dihydro- OH], 5.32 [1H, d, Jϭ5.9 Hz, C(3Ј)-OH], 5.71 [1H, d, Jϭ5.9 Hz, C(2Ј)-OH],
3H-imidazo[1,2-a]purine-7-butanoic Acid Methyl Ester (17) Com- 6.10 [1H, d, Jϭ4.9 Hz, C(1Ј)-H], 6.63 (0.1H), 7.11 (0.9H) [d each, Jϭ8.8
pound 15 (52 mg, 0.057 mmol) was hydrogenated over Pearlman’s catalyst Hz, C(a)-NH], 8.22 [1H, s, C(2)-H]. 1H-NMR [(CD3)2CO] d24): 2.19 [3H, s,
(52 mg) in MeOH (20 ml) at 40 °C under atmospheric pressure for 3 h. The C(6)-Me], 3.28 (dd, Jϭ14.7, 5.9 Hz), 3.36 (dd, Jϭ14.7, 7 Hz) [1H each,
catalyst was filtered off and washed with hot MeOH (100 ml). The filtrate C(g)-H2], 3.68, 3.69 (3H each, s, CCO2Me and NCO2Me), 3.85 (ddd,
and washings were combined and concentrated in vacuo to leave a colorless Jϭ12.2, 5.4, 2.9 Hz), 3.94 (ddd, Jϭ12.2, 4.9, 2.9 Hz) [1H each, C(5Ј)-H2],
glass. This was purified by TLC on silica gel [CHCl3–MeOH (40 : 1, v/v)] to 4.21 [1H, m, C(4Ј)-H], 4.22 (3H, s, NMe), 4.26 [1H, dd, Jϭ1.5, 9.3 Hz,
give 182) (11.2 mg, 23%) and 17 (14.3 mg, 29%) as a colorless glass, [a]D18
C(a)-H], 4.49 [2H, m, C(5Ј)-OH, C(3Ј)-H], 4.57—4.67 [2H, m, C(b)-OH,
Ϫ28.6° (cϭ0.500, MeOH). FAB-MS m/z: 889 (MNaϩ), 867 (MHϩ). 1H- C(b)-H], 4.74 [1H, m, C(2Ј)-H], 4.78 [1H, br, C(3Ј)-OH], 5.13 [1H, br,
NMR (CDCl3) d: Ϫ0.28, Ϫ0.03, 0.12 (3H each), 0.14 (6H), 0.15 (3H) (s,
C(2Ј)-OH], 6.19 [1H, d, Jϭ9.3 Hz, C(a)-NH], 6.30 [1H, d, Jϭ4.9 Hz, C(1Ј)-
three SiMe2), 0.74, 0.94, 0.95 (9H each, s, three tert-Bu), 2.25 [3H, s, C(6)- H], 8.21 [1H, s, C(2)-H]. Anal. Calcd for C21H28N6O10·H2O: C, 46.49; H,
Me], 3.15 (dd, Jϭ15, 3.5 Hz), 3.57 (dd, Jϭ15, 8.5 Hz) [1H each, C(g)-H2], 5.57; N, 15.49. Found: C, 46.40; H, 5.38; N, 15.41.
3.72, 3.76 (3H each, s, two CO2Me), 3.79 (dd, Jϭ11.7, 1.5 Hz), 3.87 (dd,
Jϭ11.7, 2.9 Hz) [1H each, C(5Ј)-H2], 4.11 [3H, s, overlapping with a 1H
(aS,bS)-b-Hydroxy-a-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-
3-b-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic
signal arising from C(4Ј)-H, NMe], 4.17 [1H, d, Jϭ5.4 Hz, C(b)-OH], 4.19 Acid Methyl Ester [(aS,bS)-11] Treatment of 27 (37 mg, 0.043 mmol)
[1H, d, Jϭ4.4 Hz, C(3Ј)-H], 4.39 [1H, dd, Jϭ4.4, 7.5 Hz, overlapping with a with Bu4NF and purification of the product were performed in manners sim-
1H signal arising from C(b)-H, C(2Ј)-H], 4.46 [1H, d, Jϭ9 Hz, C(a)-H], ilar to those described above for the preparation of (aS,bR)-11, giving
5.66 [1H, d, Jϭ9 Hz, C(a)-NH], 6.21 [1H, d, Jϭ7.5 Hz, C(1Ј)-H], 7.94 [1H, (aS,bS)-11 (19 mg, 86%) as a colorless glass. This was subjected to HPLC
s, C(2)-H].
[LiChrosorb RP18 (7 mm, 250ϫ10 mm) (Merck); MeOH–H2O (30 : 70, v/v)
at the rate of 1.5 ml/min] in seven portions to give (aS,bS)-11·3/2H2O (12
(aS,bS)-b-Hydroxy-a-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-
3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-b-D-ribofuranosyl]-4,9-dihydro- mg, 50%), mp 183—185 °C. Recrystallization of this compound from 90%
3H-imidazo[1,2-a]purine-7-butanoic Acid Methyl Ester (27) i) Dihy- (v/v) aqueous MeOH, drying over P2O5 at 2 mmHg and 50 °C for 10 h, and
droxylation of 132) (300 mg, 0.353 mmol) was conducted in a manner similar exposure to air at room temperature until a constant weight was reached,
to that described above, and the crude product was purified by flash chro- provided an analytical sample of (aS,bS)-11·3/2H2O as colorless needles,
1
matography [CHCl3–MeOH (30 : 1, v/v)] to give a 2.1 : 1 (estimated by H- mp 189 °C (softened), 200—203 °C (dec.). [a]D29 Ϫ38.3° (cϭ0.174, H2O).
95% EtOH
NMR spectroscopy) mixture (288 mg) of 14 and 16. A portion (277 mg, FAB-MS m/z: 547 (MNaϩ), 525 (MHϩ). UV l
nm (e): 239.5
max
1
0.314 mmol) of this product was treated with triphosgene (70 mg, 0.24 (32300), 280 (sh) (5000), 298 (6500). H-NMR [(CD3)2SO] d: 2.14 [3H, s,
mmol) in a manner similar to that described above for the preparation of 15.
C(6)-Me], 3.02 (dd, Jϭ14.7, 7.5 Hz), 3.41 (dd, Jϭ14.7, 4.9 Hz) [1H each,
The crude product was purified by flash chromatography [hexane–AcOEt C(g)-H2], 3.55, 3.58 [3H each, s, overlapping with a 1H signal arising from
1
(2 : 3, v/v)] to give a 2.9 : 1 (estimated by H-NMR spectroscopy) mixture one C(5Ј)-H2, NCO2Me and CCO2Me], 3.68 [1H, ddd, Jϭ12, 3.4, 5.4 Hz,
(195 mg) of 15 and 26. The mixture (194 mg) was shaken in MeOH (77 ml) one C(5Ј)-H2], 3.99 [1H, ddd, Jϭ3.4, 3.4, 4.4 Hz, C(4Ј)-H], 4.04 (3H, s,
under H2 in the presence of Pearlman’s catalyst (194 mg) at 40 °C for 2 h. NMe), 4.09 [1H, m, C(b)-H], 4.11—4.16 [2H, m, C(a)-H and C(3Ј)-H],
The catalyst was filtered off and washed with hot MeOH (100 ml). The fil- 4.45 [1H, ddd, Jϭ4.4, 5.9, 4.9 Hz, C(2Ј)-H], 5.06 [1H, d, Jϭ5.4 Hz, C(b)-
trate and washings were combined and concentrated in vacuo. The residue OH], 5.12 [1H, dd, Jϭ5.4 Hz each, C(5Ј)-OH], 5.31 [1H, d, Jϭ5.4 Hz,
was purified by repeated flash chromatography and TLC on silica gel C(3Ј)-OH], 5.70 [1H, d, Jϭ5.9 Hz, C(2Ј)-OH], 6.11 [1H, d, Jϭ4.9 Hz,
[CHCl3–MeOH (40 : 1, v/v)], providing 182) (79 mg, 28%), 17 (61 mg, 21%), C(1Ј)-H], 6.86 (0.1H, br), 7.28 (0.9H, d, Jϭ8.3 Hz) [C(a)-NH], 8.22 [1H, s,
1
and 27 (23 mg, 8%) as a colorless glass, [a]D18 Ϫ21.5° (cϭ0.413, MeOH). C(2)-H]. H-NMR [(CD3)2CO] d24): 2.24 [3H, s, C(6)-Me], 3.18 [1H, dd,
FAB-MS m/z: 889 (MNaϩ), 867 (MHϩ). 1H-NMR (CDCl3) d: Ϫ0.27, Jϭ14.5, 8.5 Hz, one C(g)-H2], 3.65, 3.66 [3H each, s, overlapping with a 1H
Ϫ0.03, 0.12 (3H each), 0.14 (6H), 0.15 (3H) (s, three SiMe2), 0.75, 0.94, signal arising from one C(g)-H2, CCO2Me and NCO2Me], 3.84 (ddd,
0.96 (9H each, s, three tert-Bu), 2.25 [3H, s, C(6)-Me], 3.41 [2H, d, Jϭ5.9
Hz, C(g)-H2], 3.70 (3H, s, CCO2Me), 3.79 [1H, dd, Jϭ11.5, 1.5 Hz, one
Jϭ12.2, 5.4, 2.9 Hz), 3.94 (ddd, Jϭ12.2, 4.9, 2.9 Hz) [1H each, C(5Ј)-H2],
4.22 [1H, m, C(4Ј)-H], 4.24 (3H, s, NMe), 4.27 [1H, m, C(b)-H], 4.37 [1H,
C(5Ј)-H2], 3.80 (3H, s, NCO2Me), 3.83 [1H, d, Jϭ6.8 Hz, C(b)-OH], 3.87 m, C(a)-H], 4.44 [1H, br, C(5Ј)-OH], 4.49 [1H, m, C(3Ј)-H], 4.54 [1H, d,
[1H, dd, Jϭ11.5, 2.5 Hz, one C(5Ј)-H2], 4.11 [3H, s, overlapping with a 1H Jϭ5.9 Hz, C(b)-OH], 4.65 [1H, br, C(3Ј)-OH], 4.76 [1H, m, C(2Ј)-H], 5.01
signal arising from C(4Ј)-H, NMe], 4.16 [1H, m, C(b)-H], 4.20 [1H, d, [1H, br, C(2Ј)-OH], 6.29 [1H, d, Jϭ4.9 Hz, C(1Ј)-H], 6.67 [1H, d, Jϭ9.8 Hz,
Jϭ4.4 Hz, C(3Ј)-H], 4.41 [1H, dd, Jϭ4.4, 7.8 Hz, C(2Ј)-H], 4.50 [1H, m, C(a)-NH], 8.19 [1H, s, C(2)-H]. Anal. Calcd for C21H28N6O10·3/2H2O: C,

October 2002
1325
45.73; H, 5.67; N, 15.24. Found: C, 45.51; H, 5.38; N, 15.29.
Hydrolysis of (aS,bR)-11 Leading to (aS,bR)-2
(aS,bR)-11·H2O (7 mg) in 0.1 N aqueous HCl (2 ml) was stored at room
(ca. 700 ml), and the whole was stored at 4 °C overnight. The precipitate that
separated was collected by centrifugation at 4 °C and suspended in 0.3 M
aqueous AcONa (140 ml). The mixture was stirred at room temperature for
A solution of
temperature for 1 h, neutralized with 0.2 M aqueous Na2HPO4 (2 ml), and 10 min after the addition of Me2CHOH (56 ml) and centrifuged at 20 °C.
concentrated in vacuo. The residue was purified by TLC on silica gel The precipitate was again treated with 0.3 M aqueous AcONa (80 ml) and
[CHCl3–MeOH (10 : 1, v/v)], providing (aS,bR)-2 (5 mg), mp 232—235 °C Me2CHOH (40 ml), and the mixture was centrifuged at 20 °C. The combined
(dec.) [lit.3) mp 232—233.5 °C (dec.)]. 1H-NMR (CDCl3) (Table 1). 1H- supernatant was cooled with ice for 1 h after the addition of Me2CHOH (130
NMR [(CD3)2CO] d24): 2.21 [3H, s, C(6)-Me], 3.33 [2H, d, Jϭ6.8 Hz, C(g)-
ml), and the precipitate was collected by centrifugation. The precipitate thus
H2], 3.68, 3.69 (3H each, s, CCO2Me and NCO2Me), 3.88 (3H, s, NMe), obtained was washed successively with 67% (v/v) aqueous EtOH (contain-
4.19 (0.1H, br), 4.30 (0.9H, dd, Jϭ2.0, 9.3 Hz) [C(a)-H], 4.51 (0.9H, d,
Jϭ5.9 Hz), 4.58 (0.1H, br) [C(b)-OH], 4.62 [1H, ddt, Jϭ2.0, 5.9, 6.8 Hz,
ing 0.1 M NaCl–0.005 M MgCl2), EtOH, and Et2O (10 ml each), and dried
over P2O5 under reduced pressure, giving crude tRNA (184 mg). A portion
C(b)-H], 5.74 (0.1H, br), 6.17 (0.9H, d, Jϭ9.3 Hz) [C(a)-NH], 8.17 [1H, d, of this product (100 mg, 1000 A260 units) was dissolved in H2O (15 ml), and
Jϭ1.0 Hz, C(2)-H], 12.42 [1H, br, N(1)-H]. This sample was identical (by the solution (pH 8.9) was brought to pH 2.9 by the addition of dilute aque-
comparison of the IR and 1H-NMR spectra and TLC mobility) to an authen- ous HCl. The resulting mixture was stirred at 40 °C for 16 h and centrifuged
tic sample3) and optically pure on the basis of chiral HPLC analysis.16)
at 4 °C. After the addition of EtOH (30 ml) the supernatant was centrifuged
Hydrolysis of (aS,bS)-11 leading to (aS,bS)-2 Hydrolysis of at 4 °C. The supernatant was neutralized with NaHCO3, and the precipitate
(aS,bS)-11·3/2H2O (2.1 mg) and purification of the product were per- that resulted was removed by centrifugation at 4 °C. The supernatant was
formed in manners similar to those described above for the preparation of concentrated in vacuo, and the residue was purified by TLC on silica gel
(aS,bR)-2, providing (aS,bS)-2·1/2H2O (1.5 mg), mp 230—233 °C (dec.)
[CHCl3–MeOH (10 : 1, v/v)], giving the minor base. The HPLC [LiChrosorb
[lit.3) mp 233—235 °C (dec.)]. 1H-NMR (CDCl3) (Table 1). 1H-NMR Si 60 (Merck); CHCl3–MeOH (95 : 5, v/v)] and chiral HPLC16) behavior of
[(CD3)2CO] d24): 2.25 [3H, s, C(6)-Me], 3.17 [1H, dd, Jϭ14.6, 7.8 Hz, one this substance was identical to that of (aS,bS)-2. The observable H-NMR
1
C(g)-H2], 3.63, 3.65 (3H each, s, two CO2Me), 3.67 [1H, dd, Jϭ14.6, 4.4 signals [[(CD3)2CO] d24): 2.25 (s), 3.63 (s), 3.65 (s), 3.90 (s), 4.48 (d, Jϭ6.4
Hz, one C(g)-H2], 3.90 (3H, s, NMe), 4.28 [1H, dddd, Jϭ7.8, 4.4, 5.9, 6.4 Hz), 6.64 (d, Jϭ8 Hz), 8.20 (s), 12.50 (br)] were superimposable on those
Hz, C(b)-H], 4.38 [1H, dd, Jϭ5.9, 8.3 Hz, C(a)-H], 4.49 [1H, d, Jϭ6.4 Hz, obtained for a dilute solution of (aS,bS)-2.
C(b)-OH], 6.25 (0.1H, br), 6.64 (0.9H, d, Jϭ8.3 Hz) [C(a)-NH], 8.20 [1H,
d, Jϭ1.0 Hz, C(2)-H], 12.50 [1H, br, N(1)-H]. 1H-NMR [CD3CN] d25): 2.20 (1 g, 10000 A260 units) was produced in five batches of 20 rats in a manner
[3H, s, C(6)-Me], 3.08 (1H, dd, Jϭ15.1, 8.3 Hz), 3.54 (1H, dd, Jϭ15.1, 4.4 similar to that described above. This was extracted with a mixture of 0.3 M
Isolation and Identification of b-Hydroxywybutosine Crude tRNA
Hz), [C(g)-H2], 3.63, 3.65 (3H each, s, CCO2Me and NCO2Me), 3.71 [1H, d, aqueous AcONa (75 ml) and Me2CHOH (30 ml) and then with a mixture of
Jϭ6.4 Hz, C(b)-OH], 3.84 (3H, s, NMe), 4.15 [1H, m, C(b)-H], 4.29 [1H,
m, C(a)-H], 6.32 [1H, br, C(a)-NH], 7.94 [1H, s, C(2)-H], 11.09 [1H, br,
0.3 M aqueous AcONa (42 ml) and Me2CHOH (21 ml). The extracts were
combined, centrifuged, and diluted with Me2CHOH (70 ml). The resulting
precipitate was collected by centrifugation at 4 °C after cooling with ice,
1
N(1)-H]. H-NMR (pyridine-d5) d26): 2.47 [3H, s, C(6)-Me], 3.61 [1H, dd,
Jϭ14.7, 8.3 Hz, one C(g)-H2], 3.64, 3.66 (3H each, s, two CO2Me), 3.70 washed successively with EtOH and Et2O, and dried to give the first crop of
[1H, m, C(b)-H], 3.91 (3H, s, NMe), 4.14 [1H, dd, Jϭ14.7, 3.9 Hz, one tRNA (520 mg). The residue, which remained undissolved on extraction
C(g)-H2], 5.16 [1H, dd, Jϭ6.4, 7.8 Hz, C(a)-H], 8.33 [1H, s, C(2)-H], 8.57 with a mixture of aqueous AcONa and Me2CHOH, was further extracted
[1H, d, Jϭ7.8 Hz, C(a)-NH]. This sample was identical (by comparison of
with 0.3 M aqueous AcONa (2ϫ28 ml). The extracts were collected by cen-
the IR and 1H-NMR spectra and TLC mobility) to an authentic sample3) and trifugation, diluted with Me2CHOH (58 ml), and chilled with ice. The result-
optically pure on the basis of chiral HPLC analysis.16)
(aS,bR)-b-(Acetoxy-d3)-a-[(methoxycarbonyl)amino]-4,6-dimethyl-9-
oxo-3-[2,3,5-tri-O-(acetyl-d3)-b-D-ribofuranosyl]-4,9-dihydro-3H-imi-
ing precipitate was collected by centrifugation at 4 °C, providing a second
crop of tRNA (210 mg). These crops were combined and extracted twice
with the Tris buffer (20 ml and 10 ml) described above. The extracts were
dazo[1,2-a]purine-7-butanoic Acid Methyl Ester (28) Compound collected by centrifugation at 20 °C and subjected to a column packed with
(aS,bR)-11 (5 mg) was treated in a manner similar to that described below ion-exchange cellulose (Whatman DE 32) (wet volume 50 ml) which had
for the preparation of 29, giving 28 (6 mg) as a colorless glass, MS m/z: 704
(Mϩ). 1H-NMR (CDCl3) d: 2.21 [3H, s, C(6)-Me], 3.09 [1H, dd, Jϭ15.1, 9.8
Hz, one C(g)-H2], 3.71, 3.75 (3H each, s, NCO2Me and CCO2Me), 3.78
[1H, dd, Jϭ15.1, 2.9 Hz, one C(g)-H2], 4.12 (3H, s, NMe), 4.32 [2H, d,
Jϭ2.4 Hz, C(5Ј)-H2], 4.49 [1H, dt, Jϭ2.4, 2.9 Hz, C(4Ј)-H], 4.63 (0.1H, br),
4.67 (0.9H, dd, Jϭ9.8, 2.9 Hz) [C(a)-H], 5.28 (0.1H, br), 5.54 (0.9H, d,
been equilibrated with the Tris buffer. The column was washed with the Tris
buffer (140 ml) followed by elution with the Tris buffer (800 ml) containing
1 M NaCl. EtOH (1.5 l) was added to the eluate and the mixture was stored at
4 °C overnight. The resulting precipitate was collected by centrifugation at
4 °C, washed successively with 67% (v/v) aqueous EtOH (containing 0.1 M
NaCl–0.005 M MgCl2), EtOH, and Et2O (30 ml each), and dried over P2O5
Jϭ9.8 Hz) [C(a)-NH], 5.50 [1H, dd, Jϭ2.9, 5.4 Hz, C(3Ј)-H], 5.80 [1H, under reduced pressure, providing tRNA (355 mg).
ddd, Jϭ9.8, 2.9, 2.9 Hz, C(b)-H], 5.85 [1H, dd, Jϭ5.4, 6.3 Hz, C(2Ј)-H],
6.18 [1H, d, Jϭ6.3 Hz, C(1Ј)-H], 7.74 [1H, s, C(2)-H].
(aS,bS)-b-(Acetoxy-d3)-a-[(methoxycarbonyl)amino]-4,6-dimethyl-9-
oxo-3-[2,3,5-tri-O-(acetyl-d3)-b-D-ribofuranosyl]-4,9-dihydro-3H-imi-
dazo[1,2-a]purine-7-butanoic Acid Methyl Ester (29) A solution of
A portion of this product (350 mg, 5300 A260 units) was treated with nu-
clease P1 (Yamasa) (500 units) in 0.02 M acetate buffer (pH 5.35, 30 ml) at
50 °C for 3 h. The hydrolysate was subjected to column chromatography on
Cosmosil 140C18-OPN (Nacalai Tesque) (10 g) [H2O (80 ml) and then
MeOH–H2O (30 : 70, v/v)] in two portions. The MeOH–H2O fractions (80
(aS,bS)-11 (5 mg) in a mixture of Ac2O-d6 (47 mg) and pyridine (102 mg) ml) were combined and concentrated in vacuo to leave a yellow glass (210
was allowed to stand at room temperature for 1.5 h and then concentrated in A260 units). It was dissolved in H2O (3.9 ml), and a solution of nuclease P1
vacuo. The residue was purified by TLC on silica gel [CHCl3–MeOH (30 : 1,
(2000 units) in 0.02 M acetate buffer (pH 5.35, 4.7 ml) was added. The whole
was stored at 50 °C for 3 h and then 0.02 N aqueous NaOH (1.05 ml) and
Escherichia coli alkaline phosphatase (Takara Shuzo) (6.4 units) were
added. The mixture (pH 9) was stored at 50 °C for 1 h and concentrated in
vacuo. This was dissolved in H2O (8 ml) and purified by HPLC [LiChrosorb
1
v/v)], giving 29 (6 mg) as a colorless glass, MS m/z: 704 (Mϩ). H-NMR
(CDCl3) d: 2.24 [3H, s, C(6)-Me], 3.01 [1H, dd, Jϭ15.1, 8.8 Hz, one C(g)-
H2], 3.69, 3.70 (3H each, s, NCO2Me and CCO2Me), 3.89 [1H, dd, Jϭ15.1,
4.4 Hz, one C(g)-H2], 4.11 (3H, s, NMe), 4.32 [2H, m, C(5Ј)-H2], 4.50 [1H,
m, C(4Ј)-H], 4.71 [1H, dd, Jϭ5.4, 8.8 Hz, C(a)-H], 5.48 [1H, dd, Jϭ3.4, 5.4 RP18 (7 mm, 250ϫ10 mm) (Merck); MeOH–H2O (30 : 70, v/v)] in seven
Hz, C(3Ј)-H], 5.50 [1H, m, C(b)-H], 5.83 [1H, dd, Jϭ5.4, 6.4 Hz, C(2Ј)-H], portions, providing the target nucleoside (1.5 A310 unit, ca. 100 mg), of
6.12 [1H, d, Jϭ8.8 Hz, C(a)-NH], 6.19 [1H, d, Jϭ6.4 Hz, C(1Ј)-H], 7.74 which HPLC behavior was identical to that of (aS,bS)-11.
[1H, s, C(2)-H].
The nucleoside was dissolved in a mixture of Ac2O-d6 (29 mg) and pyri-
Isolation of b-Hydroxywybutine Unfractionated tRNA was obtained dine (69 mg), stored at room temperature for 1.5 h, and purified by TLC on
as described below according to the reported procedure.27) Twenty female silica gel [CH2Cl2–MeOH (30 : 1, v/v)] to afford the tetraacetate-d12, MS m/z
Wistar rats were decapitated and their livers (160 g) removed and quickly (%): 704 (Mϩ) (7), 641 (3), 609 (18), 582 (12), 550 (2), 483 (18), 437 (3),
chilled in ice. Batches of 9 g were homogenized with 88% (v/v) aqueous 374 (2), 342 (22), 315 (14), 283 (12), 268 (9), 216 (100), 142 (26). 1H-NMR
phenol (14 ml) and Tris buffer [0.02 M (HOCH2)3CNH2–HCl (pH 7.5) con-
taining 1 M NaCl–0.5 mM ethylenediaminetetraacetic acid–0.01 M MgCl2] (14
(CDCl3) d: 2.24 (3H, s), 3.02 (1H, dd), 3.697, 3.704 (3H each, s), 4.11 (3H,
s), 4.33 (2H, m), 4.50 (1H, m), 4.71 (1H, dd), 5.48 (1H, dd), 5.51 (1H, m),
ml) in a homogenizer at high speed for 2—3 min. The combined ho- 5.83 (1H, dd), 6.12 (1H, d), 6.19 (1H, d), 7.74 (1H, s). This compound was
mogenate was shaken at room temperature for 2 h and centrifuged (8000 identical (by comparison of the MS and 1H-NMR spectra) to 29.
rpm, 40 min) at 4 °C. The precipitate was mixed with the Tris buffer (150
ml) and centrifuged. EtOH (1.4 l) was added to the combined supernatant
A solution of half of the tetraacetate-d12 in 0.1 M MeONa–MeOH (0.1 ml)
was stored at 0 °C for 5 min and 0.1 N aqueous HCl (0.2 ml) was added at

1326
Vol. 50, No. 10
once. The resulting solution was stored at room temperature for 1 h, neutral-
ized by the addition of 0.2 M Na2HPO4 (0.1 ml), and concentrated in vacuo.
The residue was purified by TLC on silica gel [CHCl3–MeOH (10 : 1, v/v)],
B., J. Am. Chem. Soc., 92, 7617—7619 (1970).
9) Thiebe R., Zachau H. G., Baczynskyj L., Biemann K., Sonnenbichler
J., Biochim. Biophys. Acta, 240, 163—169 (1971).
giving the base, 1H-NMR [(CD3)2CO] d24): 2.25 (3H, s), 3.18 (1H, dd), 3.64, 10) Feinberg A. M., Nakanishi K., Barciszewski J., Rafalski A. J., Au-
3.65 (3H each, s), 3.67 (1H, dd), 3.90 (3H, s), 4.28 (m), 4.39 (1H, m), 4.47
gustyniak H., Wiewiórowski M., J. Am. Chem. Soc., 96, 7797—7800
(1H, d), 6.62 (d), 8.20 (1H, s), 12.50 (1H, br). This compound was identical
(1974).
1
(by comparison of the H-NMR spectrum and chiral HPLC16) mobility) to 11) Yoshikami D., Keller E. B., Biochemistry, 10, 2969—2976 (1971).
(aS,bS)-2.
12) Kasai H., Yamaizumi Z., Kuchino Y., Nishimura S., Nucleic Acids
Res., 6, 993—999 (1979).
Acknowledgments This work was supported in part by a grant from the 13) Mochizuki A., Omata Y., Miyazawa Y., Bull. Chem. Soc. Jpn., 53,
Japan Research Foundation for Optically Active Compounds. We are grate- 813—814 (1980).
ful to Professor Ken-ichi Miyamoto (Kanazawa University) and his associ- 14) Barciszewska M., Kaminek M., Barciszewski J., Wiewiórowski M.,
ates for providing rat livers. We are also indebted to Professor Masashi Ohba Plant Sci. Lett., 20, 387—392 (1981).
(Kanazawa University) for assignment of 1H-NMR spectra (CDCl3) of 15) Itaya T., Watanabe N., Mizutani A., Tetrahedron Lett., 27, 4043—4046
(aS,bR)- and (aS,bS)-2.
(1986).
16) Itaya T., Kanai T., Chem. Pharm. Bull., 46, 1220—1224 (1998).
17) Itaya T., Kanai T., Iida T., Tetrahedron Lett., 38, 1979—1982 (1997).
References and Notes
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(2002).
19) Itaya T., Morisue M., Shimomichi M., Ozasa M., Shimizu S., Naka-
2) Itaya T., Kanai T., Iida T., Chem. Pharm. Bull., 50, 530—533 (2002).
gawa S., J. Chem. Soc., Perkin Trans. 1, 1994, 2759—2765.
3) Itaya T., Watanabe N., Iida T., Kanai T., Mizutani A., Tetrahedron, 51, 20) Itaya T., Shimizu S., Chem. Pharm. Bull., 43, 398—402 (1995).
6419—6430 (1995).
4) Itaya T., Morisue M., Takeda M., Kumazawa Y., Chem. Pharm. Bull.,
38, 2656—2661 (1990).
21) Riley J. H., Sopher D. W., Utley J. H. P., Walton D. J., J. Chem. Res.
(S), 1982, 326—327.
22) Ogilvie K. K., Beaucage S. L., Schifman A. L., Theriault N. Y.,
Sadana K. L., Can. J. Chem., 56, 2768—2780 (1978).
23) Thiebe R., Zachau H. G., Eur. J. Biochem., 5, 546—555 (1968).
24) The residual peak at 2.09 was used as a reference.
25) The residual peak at 1.96 was used as a reference.
5) References cited in refs. 1—4.
6) Nakanishi K., Blobstein S., Funamizu M., Furutachi N., Van Lear G.,
Grunberger D., Lanks K. W., Weinstein I. B., Nature, New Biol., 234,
107—109 (1971).
7) Blobstein S. H., Grunberger D., Weinstein I. B., Nakanishi K., Bio- 26) The residual C(3)-H peak at 7.55 was used as a reference.
chemistry, 12, 188—193 (1973).
8) Nakanishi K., Furutachi N., Funamizu M., Grunberger D., Weinstein I.
27) Nishimura S., “Proceedings of Nucleic Acid Research,” Vol. 2, ed. by
Cantoni G. L., Harper and Row, New York, 1971, pp. 542—564.