A simple and efficient synthesis of puromycin, 2,2′-anhydro- pyrimidine nucleosides, cytidines and 2′,3′-anhydroadenosine from 3′,5′-O-sulfinyl xylo-nucleosides

Article abstract of DOI:10.1080/15257770600725929

Synthesis of antibiotics, puromycin and 3 ′-amino-3 ′-deoxy-N 6 ,N 6 -dimethyladenosine 11 was achieved by utilizing the cyclic sulfite 6a of the xylo -3 ′,5 ′-dihydroxy group as a new protective group. The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization of 2-α-carbamate 7 . In a similar manner, 2,2 ′-anhydro-pyrimidine nucleosides 15 , ribo -cytidines 17 and 2 ′,3 ′-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4 , 5 , and 6b , respectively. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770600725929

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A simple and efficient synthesis of
puromycin, 2,2′-anhydro-pyrimidine
nucleosides, cytidines and 2′,3′-
anhydroadenosine from 3′,5′-O-sulfinyl
Xylo-nucleosides
Ken-ichi Takatsuki a b , Sumito Ohgushi b , Shigeo Kohmoto c , Keiki
Kishikawa c & Makoto Yamamoto c
a Ichikawa Research Institute, Kobayashi Perfumery Co., Ltd. ,
Ichikawa-City, Japan
b Department of Quality Materials Science, Graduate School of
Science and Technology , Chiba University , Inageku, Chiba, Japan
c Department of Applied Chemistry and Biotechnology, Faculty of
Engineering , Chiba University , Inageku, Chiba, Japan
Published online: 24 Feb 2007.
To cite this article: Ken-ichi Takatsuki , Sumito Ohgushi , Shigeo Kohmoto , Keiki Kishikawa & Makoto
Yamamoto (2006) A simple and efficient synthesis of puromycin, 2,2′-anhydro-pyrimidine nucleosides,
cytidines and 2′,3′-anhydroadenosine from 3′,5′-O-sulfinyl Xylo-nucleosides, Nucleosides, Nucleotides
and Nucleic Acids, 25:7, 719-734, DOI: 10.1080/15257770600725929
To link to this article: http://dx.doi.org/10.1080/15257770600725929
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Nucleosides, Nucleotides, and Nucleic Acids, 25:719–734, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600725929
A SIMPLE AND EFFICIENT SYNTHESIS OF PUROMYCIN,
2,2ꢀ-ANHYDRO-PYRIMIDINE NUCLEOSIDES, CYTIDINES
AND 2ꢀ,3ꢀ-ANHYDROADENOSINE FROM 3’,5’-O-SULFINYL
XYLO-NUCLEOSIDES
Ken-ichi Takatsuki
Ichikawa Research Institute, Kobayashi Perfumery Co., Ltd.,
2
Ichikawa-City, Japan, and Department of Quality Materials Science, Graduate School of
Science and Technology, Chiba University, Inageku, Chiba, Japan
Sumito Ohgushi
Department of Quality Materials Science, Graduate School of
2
Science and Technology, Chiba University, Inageku, Chiba, Japan
Shigeo Kohmoto, Keiki Kishikawa, and Makoto Yamamoto
Department of
2
Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, Inageku,
Chiba, Japan
Synthesis of antibiotics, puromycin and 3 ꢁ-amino-3 ꢁ-deoxy-N 6,N 6-dimethyladenosine 11 was
2
achieved by utilizing the cyclic sulfite 6a of the xylo-3 ꢁ,5 ꢁ-dihydroxy group as a new protective group.
The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization
of 2-α-carbamate 7. In a similar manner, 2,2 ꢁ-anhydro-pyrimidine nucleosides 15, ribo-cytidines
17 and 2 ꢁ,3 ꢁ-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4,
5, and 6b, respectively.
Keywords
Puromycin; 2,2ꢁ-Anhydro-pyrimidine Nucleoside; 3ꢁ,5ꢁ-O-sulfinyl xylo-
nucleosides
INTRODUCTION
There has been considerable interest in nucleosides modified on the
sugar moiety as potential antiviral, anticancer, and biosynthetic inhibitor
agents; therefore, a variety of new effective protective groups for hy-
droxyl groups have been devised to design nucleoside analogue.[1] In the
Received 31 August 2005; accepted 1 February 2006.
We thank the Ichikawa Research Institute, Kobayashi Perfumery Co., Ltd., for support, Haruhiro
Yamashita, the former manager of the research institute, for his useful suggestions, and Mariko Matsuki,
the analytical chemist, for her assistance.
Address correspondence to Ken-ichi Takatsuki, Ichikawa Research Institute, Kobayashi Perfumery
Co., Ltd., 4-12-1 Ohwada, Ichikawa City 272-0025, Japan. E-mail: vej05006@nifty.com
719

720
K.-I. Takatsuki et al.
preliminary report, we exploited a new protective group, the cyclic sulfite,
which was not only an efficient protective group for 3ꢁ,5ꢁ-O-xylohydroxy
groups, but also a potent leaving group. The deprotection itself was the
key synthetic step of our nucleoside synthesis. The cyclic sulfite protective
group was eliminated either by an intramolecular nucleophilic attack with
2ꢁ-carbamoyl group (synthesis of 8) or with hydroxy group (synthesis of
15). In this article we demonstrate the effectiveness of this protecting
group in the syntheses of the puromycin and 2,2ꢁ-anhydro pyrimidines
15.
The family of 3ꢁ-amino-3ꢁ-deoxyadenosines is known to possess strong
antibacterial, anticancer, and biosynthetic inhibitory properties.[2] One of
the most important members of the family is puromycin, a metabolite
of Streptomyces alboniger, which was first isolated by Porter and cowork-
ers in 1952.[3] Puromycin has been used extensively for elucidation of
the mechanism of protein biosynthesis.[2] It has close structural simi-
larity to the aminoacyl end of aminoacyl-tRNA.[4] Consequently, it is a
strong acceptor for the peptidyl tRNA site of the ribosome. Puromycin
inhibits the growth of Gram-positive bacteria and various animal and
insect cells. In addition, the nucleoside moiety of puromycin, 3ꢁ-amino-
3ꢁ-deoxy-N 6,N 6-dimethyladenosine 11, showed activity against Trypanosoma
equiperdum.[5] Even though a number of synthetic routes to puromycin
and its analogues have been reported, most of them have problems
owing to numerous synthetic steps and low overall yields.[6] Recently,
Robins[6a] and Strazewski[6c] reported the synthesis of puromycin and its
analogues via 3ꢁ-amino-3ꢁ-deoxyadenosine starting from d-ribo-adenosine.
However, their syntheses still had problems in 3ꢁ-regioselectivity[6c] and a
usage of a pyrophoric reagent, bromodimethylborane,[6a] and an inacces-
sible reagent, N ,N ꢁ-bis[(dimethylamino)methylen]hydrazine (BDMAMH)
dihydrochloride.[6a,7] In this article, we have challenged to solve these
critical problems and developed a novel synthesis of puromycin using 3ꢁ,5ꢁ-
O-sulfinylxyloadenosine with a reasonably safe procedure.
In our previous paper, we communicated the efficacy of this pro-
tective group, 3ꢁ,5ꢁ-O-sulfinyl, in the synthesis of xylo-nucleosides, 2,2ꢁ-
anhydro-pyrimidine nucleosides and a puromycin intermediate (3ꢁ-amino-
3ꢁ-deoxyadenosine).[8] However, in the puromycin synthesis via 3ꢁ-amino-
3ꢁ-deoxyadenosine, it is inevitable to use (BDMAMH)dihydrochloride, a
N6-methylation[6a,7] reagent rather difficult to obtain. Therefore, we studied
a new short-step synthesis of puromycin and 11 from easily available N6,N6-
dimethyladenosine 3a. Herein, we report on the details of their synthesis
and the synthesis of 15 and 14 by applying a novel and efficient ribo-
stereoselective and 3ꢁ-regioselective rearrangement of 3ꢁ,5ꢁ-O-sulfinyl xylo-
nucleosides 4, 5, 6, and 7, respectively, prepared from d-xylose. Also, we pre-
sented a new type of stereospecific hydroxylation via 2,2ꢁ-anhydrocytidines
16 resulting in a formation of ribo-cytidines 17.

Synthesis of Puromycin from 3ꢁ,5ꢁ-O-Sulfinyl Xylo-Nucleosides
RESULTS AND DISCUSSION
721
Synthesis of 3 ꢁ,5 ꢁ-O-Sulfinyl Xylonucleosides (4, 5, and 6) and Property of the
3ꢁ,5ꢁ-O-Sulfinyl Group. β-d-Xylofuranosyl nucleosides 1, 2, and 3 were pre-
pared from commercially available inexpensive D-xylose according to the
literature.[9,10] N 6,N6-Dimethyl-xyloadenosine 3a was obtained from N ,N -
dimethyladenine[11] and xylofuranoside. The synthesis of 3ꢁ,5ꢁ-O-sulfinyl
xylo-nucleosides 4, 5, and 6 is shown in Scheme 1. Reactions of 1, 2,
and 3 with thionyl chloride in dry acetonitrile afforded crystalline 3ꢁ,5ꢁ-
O-cyclosulfinyl derivatives 4, 5, and 6 in 77.9–85.3% yields[12] without
chromatographic purification. Their infrared spectra showed the sharp
bands corresponding to the νS = O of the alkylated sulfinate ester at
1180–1257 cm−1. Their structures were determined based on NMR and
FAB-MS spectral analyses. The 3ꢁ,5ꢁ-O-sulfinyl group can be a good leaving
group with an assistance of a nucleophilic attack by the 2ꢁ-carbamoyl or
2ꢁ-hydroxyl group from the α-side of 3ꢁ-position affording 3ꢁ-substituted
ribo-nucleosides in a stereo-and regioselective way.
Synthesis of 3 ꢁ-amino-3 ꢁ-deoxy-N6,N6-Imethyladenosine (11) and Puromycin.
The synthesis of 11 and puromycin is shown in Scheme 2. Treatment of the
cylosulfinyl xyloadenosine 6a with benzylisocyanate at room temperature
gave the carbamate 7, keeping the sulfinyl group and the amino group
intact. Subsequent treatment of 7 with NaH in dry DMF at –30◦C for 20
h gave 3ꢁ-cyclization product 8[6a] in 60% yield after silica gel column

722
K.-I. Takatsuki et al.
chromatography with epoxide 9 similar to 13 as a minor product (less than
10% yield) detected by thin-layer chromatography. The epoxide 9 would
be formed via 6a possibly derived from the hydrolysis of 7. This minor
product was also reported by Robins.[6] Following hydrolytic decabonyla-
tion (NaOH/H2O/1,3-dioxolane) of 8 gave 3ꢁ-benzylamine derivative 10
(70.3%). Hydrogenolysis of 10[6a] with Pd(OH)2-C (Pearlman’s catalyst) as
a catalyst afforded 11 (71.4%), a key intermediate for puromycin. Com-
pletely different results were reported for this hydrogenolysis in literature.
Robins[6a] reported that the reaction was highly efficient (reaction time:
1.5 h), whereas Strazewski[6c] commented that it was an inefficient and
problematic procedure. In our hands, the hydrogenolysis was completed
in 16.0 h with the yield mentioned above. Finally, puromycin was prepared
from 11 according to the literature.[6a] Condensation of BOC-(4-methoxy-
L-phenyalanine) with 11 gave the protected aminoacyl-aminonucleoside.
The removal of BOC from the aminonucleoside with trifluoroacetic acid
completed our practical puromycin synthesis.
Synthesis of 2,2 ꢁ-Anhydro-Pyrimidine Nucleosides (15) and Their Transforma-
tion into ribo-cytidines (17). The synthetic route to 2,2ꢁ-Anhydro-Pyrimidine
nucleosides 15 is outlined in Scheme 3. Treatment of cyclosulfinyluridines
4a, 4c and cyclosulfinylthymidine 4b with sodium hydrogencarbonate in dry
DMF at 90◦C for 5 h gave 2,2ꢁ-anhydronucleosides 15a, 15c, and 15b[13]

Synthesis of Puromycin from 3ꢁ,5ꢁ-O-Sulfinyl Xylo-Nucleosides
723
in 87.3–95.9% yields, respectively. The reaction proceeded in a high yield
irrespective of the electronic nature of substituents at the 5-position.
Cyclosulfinylcytidines 5a and 5b, obtained by the same procedure as 4,
were stable at room temperature. Surprisingly, they were transformed into
ribo-cytidines 17a and 17b in 76.5 and 49.2% yields, respectively. In contrast
to the synthesis of 15, neither the formation of 2,2ꢁ-anhydrocytidines nor
arabino-cytidines was observed. In order to confirm the reaction mecha-
nisms, we carried out the reaction using an independently prepared 16a.
The reaction of 16a under the same reaction conditions as for the prepa-
ration of 17a directly from 5 afforded 17a in 80.9% yield. Consequently,
the results support the assumption that the reactions proceed via 2,2ꢁ-
anhydrocytidines 16. It is well known that the hydrolysis of 16 gives arabino-
cytidine (ara-C).[12a,14] Similar to this, cytidines were probably formed via
the intermediate 16 incorporated with a carbonate in dry DMF as shown in
Scheme 3.
Synthesis of 2 ꢁ,3 ꢁ-Anhydroadenosine (14). The base-catalyzed reaction of
6b afforded 14 in a yield of 83.0% (Scheme 3). The 2ꢁ,3ꢁ-oxirane structure
is evidently prepared by the substitution of the cyclosulfinyl group at the 3ꢁ-
position with the 2ꢁ-hydroxy group from the α-side. The results also indicate
the presence of the 2ꢁ,3ꢁ-oxirane intermediates 12 and 13 derived from 3ꢁ,5ꢁ-
sulfinyl pyrimidine nucleosides 4 and 5, respectively.

724
K.-I. Takatsuki et al.
CONCLUSION
In conclusion, we have established an efficient synthesis of puromycin,
3ꢁ-amino-3ꢁ-deoxy-N 6,N 6-dimethyladenosine, 2,2ꢁ-anhydro-pyrimidine nu-
cleosides, and 2ꢁ,3ꢁ-anhydroadenosine from 3ꢁ,5ꢁ-O-sulfinyl xylo-nucleosides
in a novel ribo-stereoselective and 3ꢁ-regioselective manner.
EXPERIMENTAL SECTION
Materials
Reagents (extra pure grade) except the mentioned below were pur-
chased from Wako Pure Chemical Industries, Ltd., and were used without
purification. Thionyl chloride was purchased from Tokyo Kasei Kogyo Co.,
Ltd. 2,2ꢁ-anhydrouridine (authentic sample of 15a), cytidinew (authentic
sample of 17a), 2,2ꢁ-anhydrocytidine hydrochloride (authentic sample of
16), BOC-4(methoxy-l-phenylalanine), Pd(OH)2-C (Pearlman’s catalyst),
and DCC were purchased from Sigma-Aldrich Co. β-d-Xylofuranosyl nucle-
osides (1a-c, 2a,b, and 3a,b) were synthesized from commercially available
d-xylose via 2,3,5-tri-O-benzoyl-β-d-xylofuranosyl-nucleosides according to
the literature.[10]
General Methods
Melting points were determined with Yamato Melting Point Apparatus
1
Model MP-21. H-NMR(400 MHz) and 13C-NMR(126.5 MHz) spectra were
recorded on JEOL LA 400 and GSX500 spectrometers, respectively. DMSO-
d6 or D2O was used as solvent and Me4Si or 3-(trimethylsilyl)propionic-
2,2,3,3-d4 acid sodium salt (TSP) as an internal standard. Chemical shifts
are reported as δ, ppm. IR spectra were recorded on a JSACO FT/IR-410
spectrometer as a KBr-pellet. High-resolution mass spectra were performed
on a JEOL JMX-HX110. Column chromatography was performed on silica
gel (230–400 mesh). Thin-layer chromatography (TLC) was performed on
silica gel (Merck, TLC grade 7749 with gypsum binder).
Synthesis of β-d-Xylofuranosyl Nucleosides[15]
9-(2,3,5-Tri-O-benzoyl-β-d-xylofuranosyl)-N6,N6-dimethyladenine, Which, was
Used to Synthesize (3a). Oil; νmax(KBr)/cm−1 1726, 1598, 1264 and
1094; 1H NMR (DMSO-d6; Me4Si) δH 3.39(6H, m, N–Me2), 4.77
(2H, m, 5ꢁ-H), 5.06 (1H, pseudo-quartet, J = 4.9 Hz, 4ꢁ-H), 6.05 (1H,
d, J = 4.9 Hz, 1ꢁ-H), 6.47 (2H, br s, 2ꢁ-H and 1ꢁ-H), 7.69–7.45 (9H, m,
ArH), 8.08–7.94 (7H, m, 8-H and ArH), 8.39 (1H, d, J = 3.1 Hz, 2-H);
13C NMR (DMSO-d6; Me4Si) δC 61.9, 75.64, 77.73, 78.48, 78.75, 79.01,
86.9, 119.5, 128.36, 128.43, 128.47, 128.52, 128.60, 128.63, 128.96, 129.01,

Synthesis of Puromycin from 3ꢁ,5ꢁ-O-Sulfinyl Xylo-Nucleosides
725
129.13, 129.20, 129.27, 129.33, 129.56, 133.2, 133.5, 133.7, 149.9, 151.8,
154.2, 134.3, 134.5, 135.2; m/z (EI) 608([M+H]+, 21%), 445(9), 201(10),
134(9), 105(100).
1
1-β-d-Xylofuranosyluracil (1a). mp 153◦C; H NMR (DMSO-d6; Me4Si)
δH 3.66 (1H, m), 3.73N (1H, m), 3.94 (2H, d, J = 20.8 Hz, 2-H), 4.09 (1H,
dd, J = 9.0 and 5.7 Hz), 4.72 (1H, s, OH), 5.40 (1H, d, J = 3.4 Hz, OH), 5.61
(1H, d, J = 7.9 Hz, 5-H), 5.66 (1H, s, OH), 5.73 (1H, d, J = 4.0 Hz, 1ꢁ-H),
7.76 (1H, d, J = 8.2Hz, 6-H), 11.25 (1H, br s, NH); 13C NMR (DMSO-d6;
Me4Si) δC 59.0, 74.5, 80.6, 83.7, 90.8, 100.8, 141.3, 150.5, 133.2; m/z (FAB)
245.0760 ([M+H]+, C9H13N2O6 requires m/z: 245.0774).
1-β-d-Xylofuranosylthymine (1b). mp 158◦C; 1H NMR (DMSO-d6; Me4Si)
δH 1.76 (3H, d, J = 0.6 Hz, Me), 3.71 (2H, m), 3.96 (2H, d, J = 14.3 Hz),
4.07 (1H, m), 4.74 (1H, br s, OH), 5.39 (1H, d, J = 2.4 Hz, OH), 5.68 (1H,
d, J = 1.8 Hz, OH), 5.72 (1H, d, J = 4.3 Hz, 1ꢁ-H), 7.66 (1H, d, J = 0.9 Hz,
6-H), 11.28 (1H, br s, NH); 13C NMR (DMSO-d6; Me4Si) δC 12.4, 59.2, 74.8,
80.6, 83.2, 90.4, 108.4, 137.1, 150.6, 133.8; m/z (FAB) 259.0938 ([M+H]+,
C10H15N2O6 requires m/z: 259.0930).
1-(β-d-Xylofuranosyl)-5-fluorouracil (1c). Oil; 1H NMR (DMSO-d6; Me4Si)
δH 3.71 (2H, m), 3.93 (1H, br s), 3.99 (1H, br s), 4.09 (1H, m), 4.78 (1H,
br s, OH), 5.50 (1H, br s, OH), 5.65 (1H, t, J 1.4 Hz, OH), 5.77 (1H, br s,
1ꢁ-H), 8.00 (1H, d, J = 7.6 Hz, 6-H), 1135 (1H, br s, NH); 13C NMR (DMSO-
d6; Me4Si) δC 59.0, 74.4, 80.4, 83.8, 90.9, 125.6 (d, J cp = 35.2Hz), 139.4
(d, J cp = 229.7 Hz), 149.0, 157.0 (d, J cp = 25.9 Hz); m/z (FAB) 263.0702
([M+H]+, C9H12N2O6F requires m/z: 263.0679).
1-β-d-Xylofuranosylcytosine (2a). mp 229◦C; 1H NMR (DMSO-d6; Me4Si)
δH 3.65 (1H, quintet, J = 5.7 Hz), 3.73(1H, quintet, J = 5.6 Hz), 3.87 (1H,
t, J = 3.5 Hz), 3.90 (1H, d, J = 4.0 Hz), 4.08 (1H, m), 4.69 (1H, t, J = 5.6
Hz, OH), 5.28 (1H, d, J = 3.7 Hz, OH), 5.63 (2H, m, 1ꢁ-H and OH), 5.68
(1H, d, J = 7.3 Hz, 5-H), 7.00 (1H, br s, NH), 7.10 (1H, br s, NH), 7.70 (1H,
d, J = 7.3 Hz, 6-H); 13C NMR (DMSO-d6; Me4Si) δC 59.2, 74.9, 80.8, 83.5,
92.1, 93.0, 142.1, 155.3, 135.7; m/z (FAB) 244.0946 ([M+H]+, C9H14N3O5
requires m/z: 244.0933).
1
1-(β-d-Xylofuranosyl)-5-fluorocytosine (2b). mp 219◦C; H NMR (DMSO-
d6; Me4Si) δH 3.68 (1H, quintet, J = 5.5 Hz), 3.74 (1H, quintet, J = 5.5
Hz), 3.89 (1H, br s), 3.92 (1H, br s), 4.09 (1H, m), 4.76 (1H, t, J = 5.5
Hz, OH), 5.37 (1H, br s), 5.62 (1H, s, OH), 5.70 (1H, d, J = 3.7 Hz, 1ꢁ-H),
7.47 (1H, br s, NH), 7.68 (1H, br s, NH), 7.85 (1H, d, J = 7.3 Hz, 6-H);
13C NMR (DMSO-d6; Me4Si) δC 59.2, 74.8, 80.6, 83.7, 91.9, 126.4 (d, J cp
=
33.1Hz), 135.5 (d, J cp = 240 Hz), 153.4, 157.3 (d, J cp = 13.5 Hz); m/z (FAB)
262.0847 ([M+H]+, C9H13N3O5F requires m/z: 262.0839).
1
9-(β-d-Xylofuranosyl)-N6,N6-dimethyladenine (3a). Oil; H NMR (DMSO-
d6; Me4Si) δH 3.50 (6H, m, N–Me2), 3.70 (1H, m, 5ꢁ-H), 3.82 (1H, m, 5ꢁꢁH),
4.09 (1H, m, 1ꢁ-H), 4.21 (1H, m, 4ꢁ-H), 4.34 (1H, s, 2ꢁ-H), 4.85 (1H, br s,
5ꢁ-OH), 5.94 (3H, br s, 1ꢁ-H, 2ꢁ-OH, and 3ꢁ-OH), 8.23 (1H, s, 8-H), 8.29 (1H,

726
K.-I. Takatsuki et al.
s, 2-H); 13C NMR (DMSO-d6; Me4Si) δC 39.0, 59.4, 75.3, 80.9, 83.5, 89.7,
119.4, 138.5, 149.4, 151.5, 154.2; m/z (EI) 296([M+H]+, 84%), 206(12),
192(13), 134(100), 157(57).
1
9-β-d-Xylofuranosyladenine (3b). mp 152◦C; (lit.16 mp 154–156◦C) H
NMR (DMSO-d6; Me4Si) δH 3.69 (1H, dd, J = 113 and 6.1 Hz, 5ꢁ-H), 3.79
(1H, dd, J = 11.5 and 4.7 Hz, 5ꢁꢁ-H), 4.07 (1H, br s, 1ꢁ-H), 4.18 (1H, m, 4ꢁ-H),
4.35 (1H, s, 2ꢁ-H), 4.81 (1H, br s, OH), 5.88 (2H, br s, OH), 5.90 (1H, d,
J = 1.8 Hz, 1ꢁ-H), 7.35 (2H, br s, NH2), 8.18 (1H, s, 8-H), 8.29 (1H, s, 2-H);
m/z (EI) 268([M+H]+, 100%), 89 (50), 77 (40); (lit.16 1H NMR (DMSO-d6;
Me4Si) δH 3.64 (1H, dd, J = 12.0 and 5.5 Hz, 5ꢁ-H), 3.82 (1H, dd, J = 12.0
and 5.5 Hz, 5ꢁꢁ-H), 4.06 (1H, dd, J = 3.5 and 1.5 Hz, 1ꢁ-H), 4.20 (1H, m,
4ꢁ-H), 4.34 (1H, m, 2ꢁ-H), 4.73 (1H, t, J = 5.5 Hz, 5ꢁ-OH), 5.9 (2H, br s,
2ꢁ-OH and 3ꢁ-OH), 5.90 (1H, d, J = 2.0 Hz, 1ꢁ-H), 7.34 (2H, br s, NH2), 8.13
(1H, s, 8-H), 8.28 (1H, s, 2-H).
Synthesis of 3ꢁ,5ꢁ-O-Sulfinyl Xylo-Nucleosides
1-(3,5-O-Sulfinyl-β-d-xylofuranosyl)uracil (4a). To a solution of thionyl
chloride (7.80 g, 65.6 mmol) in acetonitrile (41 mL) was added 1-(β-d-
xylofuranosyl)uracil 1a (4.00 g, 13.4 mmol) with vigorous stirring, and then
the temperature of the reaction mixture was maintained at 10◦C. After
stirring for an additional 3 h, the mixture was poured into a suspension
of sodium bicarbonate (22.0 g) in water (100 mL) and extracted with ethyl
acetate (2 × 200 mL). The combined extracts were dried over anhydrous
magnesium sulfate and evaporated to dryness in vacuum. Recrystallization
from 4-methyl-2-pentanone gave 3.90 g (82.0% yield) of 4a as white crystals;
mp 185◦C; νmax(KBr)/cm−1 3271, 3139, 3043, 1371, 1471, 1415, 1273, 1210
(RO-SO-OR), 1191, 1113, 1089, 1010, 833, 768, and 715; 1H NMR (DMSO-
d6; Me4Si) δH 4.21 (1H, d, J = 4.3 Hz), 4.34 (1H, d, J = 13.4 Hz), 4.39 (1H,
br s), 4.71 (1H, d, J = 2.4 Hz), 4.91 (1H, dd, J = 13.4 and 1.8 Hz), 5.71 (1H,
dd, J = 8.2 and 1.5 Hz, 1H), 5.73 (1H, s, 2ꢁ-OH), 6.27 (1H, d, J = 4.3 Hz,
1ꢁ-H), 7.61(1H, d, J = 8.2Hz, 6-H), 11.41 (1H, s, NH); 13C NMR (DMSO-d6;
Me4Si) δC 55.8, 70.5, 73.1, 78.7, 91.0, 101.5, 139.4, 150.4, 133.1; m/z (FAB)
291.0295 ([M+H]+, C9H11N2O7S requires m/z: 291.0287).
1-(3,5-O-Sulfinyl-β-d-xylofuranosyl)thymine (4b). Similar to the synthesis
of 4a, 4b was obtained from the reaction of 1b (4.23 g, 13.4 mmol) and
thionyl chloride (7.80 g, 65.6 mmol) in acetonitrile (41 mL). Recrystalliza-
tion from 4-methyl-2-pentanone gave 4.19 g (84.6% yield) of 4b as white
crystals; mp 176◦C; νmax(KBr)/cm−1 3245, 3087, 2962, 1700, 1470, 1401,
1268, 1205 (RO-SO-OR), 1104, 997, 839, and 795; 1H NMR (CDCl3; Me4Si)
δH 1.94 (3H, d, J = 0.9 Hz); 4.33 (1H, d, J = 13.1 Hz), 4.45 (1H, d, J =
2.2 Hz), 4.51 (1H, d, J = 1.8 Hz), 4.95 (1H, d, J = 2.4 Hz), 5.09 (1H, dd,
J = 13.1 and 13 Hz), 5.81 (1H, s, 2ꢁ-OH), 5.96 (1H, d, J = 3.4 Hz, 1ꢁ-H), 7.76
(1H, d, J = 1.2Hz, 6-H), 10.67 (1H, s, NH); 13C NMR (CDCl3; Me4Si) δC12.6,

Synthesis of Puromycin from 3ꢁ,5ꢁ-O-Sulfinyl Xylo-Nucleosides
727
55.7, 69.8, 75.2, 79.7, 93.2, 110.2, 135.7, 150.8, 134.8; m/z (FAB) 305.0437
([M+H]+, C10H13N2O7S requires m/z: 305.0443).
1-(3,5-O-Sulfinyl-β-d-xylofuranosyl)-5-fluorouracil (4c). In a similar man-
ner of the synthesis for 4a, 4c was obtained from the reaction of 1c (4.30 g,
13.4 mmol) and thionyl chloride (7.80 g, 65.6 mmol) in acetonitrile (41
mL). Recrystallization from 4-methyl-2-pentanone gave 4.19 g (82.9% yield)
of 4c as white crystals; mp 215◦C; νmax(KBr)/cm−1 3432, 3088, 1712, 1468,
1380, 1332, 1257 (RO-SO-OR), 1155, 1096, 1069, 1038, 1006, 887, 840, and
1
714; H NMR (DMSO-d6; Me4Si) δH 4.27 (1H, d, J = 3.7 Hz), 4.39 (1H,
d, J = 13.7 Hz), 4.42 (1H, m), 4.71 (1H, d, J = 2.5 Hz), 4.92 (1H, dd,
J = 13.1 and 1.8 Hz), 5.68 (1H, s, 2ꢁ-OH), 6.32 (1H, d, J = 4.3 Hz, 1ꢁ-H),
7.74 N(1H, d, J = 7.3 Hz, 6-H), 11.97 (1H, s, NH); 13C NMR (DMSO-d6;
Me4Si) δC 55.8, 70.3, 73.5, 78.4, 91.1, 123.6 N(d J cp = 35.2Hz), 139.7
(d J cp = 230.7 Hz), 148.8, 156.9(d, J cp = 26.9 Hz); m/z (FAB) 309.0191
([M+H]+, C9H10N2O7FS requires m/z: 309.0193).
1-(3,5-O-Sulfinyl-β-d-xylofuranosyl)cytosine Hydrochloride (5a). To a solu-
tion of thionyl chloride (7.80 g, 65.6 mmol) in acetonitrile (41 mL) and
1,3-dioxolane (20 mL) was added 1-(β-d-xylofuranosyl)cytosine 2a (3.99
g, 13.4 mmol) with vigorous stirring, and then the temperature of the
reaction mixture was maintained at 25◦C. After stirring for an additional
5 h, the solid precipitated was collected by filtration, washed twice with
acetonitrile, and dried to give 4.56 g (85.3% yield based on 2a) of 5a
as white crystals, which contained some amount of free base; mp 178◦C;
νmax(KBr)/cm−1 3377, 1731, 1377, 1543, 1399, 1331, 1275, 1186 (RO-SO-
1
OR), 1119, 1036, 1007, 956, 904, 881, 853, 834, 788, 762, and 731; H
NMR (D2O; TSP) δH 4.01–5.09 (5H + HDO, m), 5.80 (0.14H, s, 1ꢁ-H),
5.82 (0.86H, s, 1ꢁ-H), 6.21 (0.14H, pseudo-d, J = 8.0 Hz, 5-H), 6.26 (0.86H,
dd, J = 8.0 and 1.8 Hz, 5-H), 8.10 (0.14H, pseudo-d, J = 7.9 Hz, 6-H),
8.13 (0.86H, dd, J = 7.9 and 1.5 Hz, 6-H); 13C NMR (D2O; TSP) δC 56.8,
60.5, 70.8, 75.2, 76.1, 79.5, 81.0, 85.5, 93.1, 93.3, 94.9, 95.2, 144.5, 145.6,
149.0, 149.1, 130.1; m/z (FAB) 290.0475 ([M+H−HCl]+, C9H12N3O6S
requires m/z: 290.0447). The compound was pure enough for the next
step.
1-(3,5-O-Sulfinyl-β-d-xylofuranosyl)-5-fluorocytosine Hydrochloride (5b).
Similar to the synthesis of 5a, 5b was obtained as white crystals from the
reaction of 2b (4.28 g, 13.4 mmol) and thionyl chloride (7.80 g, 65.6 mmol)
in acetonitrile (41 mL) and 1,3-dioxolane (20 mL); yield: 4.39 g (77.9%
based on 2b). The product contained some amount of free base; mp 172◦C
νmax(KBr)/cm−1 3311, 2767, 1731, 1385, 1344, 1558, 1402, 1350, 1276, 1189
(RO-SO-OR), 1114, 1084, 1057, 1032, 1004, 959, 930, 913, 893, 878, 852,
1
834, 795, 778, and 742; H NMR (D2O; TSP) δH 4.02–5.10 (5H + HDO,
m), 5.78 (1H, br s, 1-H), 8.10 (0.24H, d, J = 6.4 Hz, 6-H), 8.28 (0.76H, d,
J = 6.2Hz, 6-H); 13C NMR (D2O; TSP) δC 56.8, 60.5, 70.7, 75.2, 76.3, 79.5,
81.1, 85.7, 93.3, 93.5, 128.7, 129.0, 129.9, 130.2, 135.8, 137.7, 148.8, 149.1,

728
K.-I. Takatsuki et al.
154.8; m/z (FAB) 308.0361 ([M+H − HCl]+, C9H11N3O6FS requires m/z:
308.0353). The compound was pure enough for the next step.
9-(3,5-O-Sulfinyl-β-d-xylofuranosyl)-N6,N6-dimethyladenine (6a). Thionyl
chloride (3.57 g, 30.0 mmol) was added to 3a (2.95 g, 10.0 mmol) in
acetonitrile (10.0 mL) and pyridine (2.37 g, 30.0 mmol), and the resulting
solution was stirred for 4 h at 25◦C. After stirring for an additional 4 h, the
mixture was poured into a suspension of sodium bicarbonate (10.1 g) in
water (60 mL) and extracted with ethyl acetate (2 × 50 mL). The combined
extracts were dried over magnesium sulfate and evaporated to dryness in
vacuum. Recrystallization from (4-methyl-2-pentanone/diisopropyl ether)
gave 2.56 g (75.0% yield) of 6a as white crystals; mp 180◦C; νmax(KBr)/cm−1
3140, 2952, 1308, 1482, 1349, 1299, 1227, 1181 (RO-SO-OR), 1082, 998,
1
and 836; H NMR (DMSO-d6; Me4Si) δH 3.42(6H, br s, N–Me2), 4.35 (1H,
d, J = 13.4 Hz, 5ꢁ-H), 4.50 (1H, s, 5ꢁꢁ-H), 4.58 (1H, d, J = 4.0 Hz, 2ꢁ-H), 4.84
(1H, d, J = 2.4 Hz, 1ꢁ-H), 4.96 (1H, dd, J = 13.1 and 1.2Hz, 4ꢁ-H), 6.09 (1H,
s, 1ꢁ-H), 6.45 (1H, d, J = 4.0 Hz, OH), 8.13 (1H, s, 8-H), 8.25 (1H, s, 2-H);
13C NMR (DMSO-d6; Me4Si) δC 55.8, 70.7, 73.2, 78.64, 78.74, 78.90, 89.8,
119.2, 136.4, 149.8, 152.4, 154.2; m/z (EI) 342([M+H]+, 81%), 307(23),
289(13).
9-(3,5-O-Sulfinyl-β-d-xylofuranosyl)adenine (6b). Thionyl chloride (7.80
g, 49.2 mmol) was added to 3b (4.38 g, 13.4 mmol) in acetonitrile (13.4
mL) and pyridine (3.89 g, 49.2 mmol), and the resulting solution was
stirred for 4 h at 25◦C. After being stirred for an additional 4 h, the
mixture was poured into a suspension of sodium bicarbonate (13.5 g)
in water (75 mL) and pyridine (25 mL) and extracted with ethyl acetate
(2 × 5 mL). The combined extracts were dried over anhydrous magnesium
sulfate and evaporated to dryness in vacuum. Recrystallization from 4-
methyl-2-pentanone gave 4.43 g (86.2% yield) of 6b as white crystals; mp
229◦C; νmax(KBr)/cm−1 3396, 3334, 3194, 2907, 1355, 1307, 1578, 1488,
1427, 1330, 1248, 1225, 1186 (RO-SO-OR), 1109, 1092, 1069, 1015, 970, 910,
886, 841, 797, 736, and 714; 1H NMR (DMSO-d6; Me4Si) δH 4.36 (1H, d, J =
13.1Hz, 5ꢁ-H), 4.51 (1H, d, J = 1.8 Hz, 5ꢁꢁ-H), 4.63 (1H, d, J = 2.8 Hz, 2ꢁ-H),
4.87 (1H, d, J = 2.4 Hz, 1ꢁ-H), 4.98 (1H, dd, J = 13.3 and 1.9 Hz, 4ꢁ-H),
6.09 (1H, br s, 1ꢁ-H), 6.48 (1H, d, J = 4.0 Hz, OH), 7.37 (2H, br s, NH2),
8.15 (1H, s, 8-H), 8.22 (1H, s, 2-H); 13C NMR (DMSO-d6; Me4Si) δC 55.8,
70.8, 73.1, 78.7, 89.8, 118.7, 137.7, 149.0, 152.7, 155.9; m/z (FAB) 314.0537
([M+H]+, C10H12N5O5S requires m/z: 314.0559).
Synthesis of 3ꢁ-Amino-3ꢁ-Deoxy-N6,N6-Dimethyladenosine (11) and Puromycin
9-[2-O-(N-Benzylcarbamoyl)-3,5-O-sulfinyl-β-d-xylofuranosyl]-N6,N6-dimethyl-
adenine (7). Benzylisocyanate (2.66 g, 20.0 mmol) and Et3N (1.51 g,
15 mmol) were added to the solution of 6a (3.41 g, 10.0 mmol) in
THF (50 mL) and MeCN (50 mL). The resulting solution was stirred

Synthesis of Puromycin from 3ꢁ,5ꢁ-O-Sulfinyl Xylo-Nucleosides
729
for 21 h at 25◦C. EtOH (99%, 10 mL) was added, and stirring was
continued for 30 min. Volatiles were evaporated, and the residue was
recrystallized (4-methyl-2-pentanone/diisopropyl ether) to give 4.67 g
(98.5% yield) of 7 as white crystals; mp 140◦C; νmax(KBr)/cm−1 3228,
3044, 2946, 1749, 1301, 1429, 1287, 1229, 1199 (RO-SO-OR), 1133, 1092,
1
1043, and 840; H NMR (DMSO-d6; Me4Si) δH 3.46 (6H, br s, NMe2),
4.24 (2H, m, ArCH2N), 4.35 (1H, d, J = 13.2 Hz, 5ꢁ-H), 4.45 (1H, d,
J = 1.7 Hz, 5ꢁꢁ-H), 4.96 (1H, dd, J = 13.4 and 2.0 Hz, 4ꢁ-H), 5.11 (1H, d,
J = 2.4 Hz, 1ꢁ-H), 5.44 (1H, s, 2ꢁ-H), 6.28 (1H, d, J = 1.5 Hz, 1ꢁ-H), 7.30
(5H, m, ArH), 8.20 (1H, s, 2-H), 8.26 (2H, m, NH and 8-H); 13C NMR
(DMSO-d6; Me4Si) δC 42.9, 43.9, 55.4, 69.1, 73.0, 80.4, 86.8, 119.0, 126.87,
126.91, 127.1, 128.10, 128.22, 136.4, 138.9, 149.8, 152.2, 154.1, 154.4; m/z
(EI) 475([M+H]+, 78%), 312(20), 241(14).
9-[3-(Benzylamino)-3-N,2-O-carbonyl-3-deoxy-β-d-ribofuranosyl]-N6,N6 -dimet -
hyladenine (8). To a suspension of NaH (50% in mineral oil; 112 mg, 2.32
mmol, rinsed with n-hexane) in DMF (20 mL) were added crystalline 7
(0.95 g, 2.00 mmol) and dipotassium hydrogenphosphate (0.48 g, 2.78
mmol) at –30◦C. The resulting mixture was stirred for 20 h. After stirring
for 1 h with NH4Cl (0.64 g, 12 mmol), the mixture was filtered (with a layer
of through Celite). Volatiles were evaporated, and the residue was purified
on silica gel column chromatography eluting with chloroform-methanol
(15:1, v/v) to give 0.49 g (60.0% yield based on 7) of 8 as an oil. Although
the compound contained a small amount of impurities, it was pure enough
for further synthetic use; m/z (EI) 411([M+H]+).
9-(3-Amino-3-deoxy-β-d-ribofuranosyl)-N6,N6-dimethyladenine (3 ꢁ-Amino-3 ꢁ-
deoxy-N6,N6-dimethlyladenosine) (11). To an aqueous solution of NaOH (1
M, 30 mL) was added 8 (0.82 g, 2.0 mmol based on 7) in 1,3-dioxolane (30
mL), and the solution was stirred for 96 h at ambient temperature. After the
solution was neutralized with HCl solution (1 M), volatiles were evaporated.
The residue was extracted with chloroform (40 mL × 2) and concentrated
to dryness in vacuum. The resulting syrup was purified by silica gel column
chromatography eluting with chloroform-methanol (15:1, v/v) to give 0.54
g (70.3% yield) of 10 as an oil; 1H NMR (DMSO-d6; Me4Si) 3.36–3.56 (12H,
m), 3.71–3.83 (2H, m, 5ꢁ-H), 3.93–3.97 (1H, m), 4.54 (1H, dd, J = 4.9 and
1.5 Hz), 6.02 (1H, d, J = 3.4 Hz, 1ꢁ-H), 7.28–7.36 (5H, m, ArH), 8.24 (1H,
s, 2-H), 8.34 (1H, s, 1H, 8-H); m/z (EI) 385([M+H]+).
The benzylaminocompound 10 (0.38 g, 1.0 mmol), NH4HCO2 (0.43 g,
5.1 mmol), and Pd(OH)2-C (0.07 g) were heated at reflux in H2O/MeOH
(1/10 v/v) for 13.0 h. After filtration of the residue, volatiles were evapo-
rated. The resulting syrup was purified by silica gel column chromatography
eluting with chloroform-methanol (4:1, v/v) to give 0.21 g (71.4% yield)
of 11 as white crystals; mp 213◦C, (lit.[6a] 213–215.5◦C); νmax(KBr)/cm−1
1
3398 and 3330, 2914, 2362, 2341, 1305, 1037; H NMR (DMSO-d6; Me4Si)
δH 3.1–3.7 (12H, m), 3.75–3.79 (2H, m, 5ꢁ-H), 4.28 (1H, q, J = 3.4 Hz),

730
K.-I. Takatsuki et al.
5.97 (1H, d, J = 2.4 Hz, 1ꢁ-H), 8.22 (1H, s, 2-H), 8.41 (1H, s, 8-H);
13C NMR (DMSO-d6; Me4Si) δC 52.3, 60.9, 74.7, 79.2, 85.3, 89.1, 119.6,
138.0, 149.6, 152.0, 154.2, (lit.[6a] 13C NMR (DMSO-d6; Me4Si) δC 52.3,
)
60.9, 75.0, 85.5, 89.3, 119.8, 138.2, 149.7, 152.0, 154.5); m/z (EI) 295
([M+H]+).
Puromycin. Synthesis of Puromycin was carried out according to the
literature.[6a] DCC (146 mg, 0.708 mmol) was added to a cold (0◦C) solution
of 11 (199 mg, 0.676 mmol), BOC-(4-methoxy-l-phenylalanine)(206 mg,
0.70 mmol) and N -hydroxysuccinimide (82.6 mg, 0.718 mmol) in dried
DMF (8 mL). The resulting solution was stirred for 30 min in an ice-water
bath and then for additional 24 h at 25◦C. Dicyclohexylurea was filtered off
and washed with EtOAc (17 mL), and the combined filtrate and washing
were evaporated to dryness. The residue was purified by silica gel column
chromatography eluting with chloroform-methanol (48:2, v/v) to give 320
mg (82.8% yield) of BOC-Puromycin.
The BOC-Puromycin (236 mg, 413 mmol) was stirred with trifluo-
roacetic acid (2 mL) for 10 min at 20◦C, then MeCN (60 mL) was added
and evaporated in vacuum. The residue was dissolved into chloroform (20
mL), and the solution was poured into a suspension of sodium hydrogen
carbonate (1.0 g) in water (20 mL). The organic layer was dried over
magnesium sulfate and evaporated to dryness in vacuum. The resulting solid
was purified by silica gel column chromatography eluting with chloroform-
methanol (9:1, v/v) to give Puromycin (85 mg, 43.7% deprotection yield)
as foam; νmax(KBr)/cm−1 3352, 2925, 1740, 1369, 1597, 1513, 1425, 1249,
and 1034; 1H NMR (CDCl3; Me4Si) δH 1.26(2H, br s, NH2), 2.61–2.70 (1H,
m), 2.80 (1H, dd, J = 13.8 and 7.9 Hz), 3.39–3.63 (8H, m, 5ꢁ-H and NMe2),
3.72–3.82 (4H, m), 3.99 (1H, dd, J = 13.1 and 2.1 Hz), 4.33–4.51 (2H, m,
1ꢁ-H and 4ꢁ-H), 4.69 (1H, dd, J = 5.8 and 3.7 Hz, OH), 5.87 (1H, d, J =
3.3 Hz, OH), 5.98 (1H, d, J = 1.8 Hz, 1ꢁ-H), 6.83 (2H, dd, J = 8.8 and 2.4
Hz, ArH), 7.11 (2H, dd, J = 8.8 and 2.1 Hz, ArH), 8.00 (1H, d, J = 6.7
Hz, NH), 8.13 (1H, s, 2-H), 8.17 (1H, s, 8-H), (lit.[6a] [1]H NMR (DMSO-d6;
Me4Si) δH 1.73 (2H, br s), 2.5–2.6 (1H, m), 2.91 (1H, dd, J = 13.4 and 4.9
Hz), 3.35–3.62 (8H, m), 3.62–3.78(4H, m), 3.9–4.0(1H, m), 4.4–4.5(2H, m),
5.15(1H, t, J = 5.4 Hz), 5.98(1H, d, J = 1.8 Hz), 6.15 (1H, d, J = 4.3 Hz),
6.84 (2H, d, J = 8.5 Hz), 7.15 (2H, d, J = 8.5 Hz), 8.05 (1H, s), 8.24 (1H,
s), 8.45 (1H, s,), peaks at 1.73, 5.15, 6.15 and 8.05 exchanged with D2O.);
[13]C NMR (CDCl3; Me4Si) 40.4, 50.9, 55.5, 56.9, 62.2, 74.2, 81.8, 85.1, 91.4,
114.2, 114.4, 121.2, 130.4, 130.6, 137.5, 148.9, 151.8, 155.1, 158.8, 175.2,
176.0, (lit[6a] 13C NMR (DMSO-d6; Me4Si) δC 50.1, 55.2, 56.3, 61.0, 73.3,
)
83.6, 89.7, 113.9, 119.8, 130.5, 130.6, 138.2, 149.8, 152.1, 154.5, 158.0, 175.2,
177.8); m/z (EI) 472([M+H]+).
Synthesis of 2,2 ꢁ-Anhydro-Pyrimidine Nucleosides (15), cytidines (17) and 2ꢁ,3ꢁ-
Anhydroadenosine (14). 6b (0.63 g, 2.0 mmol) and sodium bicarbonate(0.76
g, 9.0 mmol) were heated in DMF (40 mL) at 110◦C for 1h and then

Synthesis of Puromycin from 3ꢁ,5ꢁ-O-Sulfinyl Xylo-Nucleosides
731
cooled. The solid precipitated was filtrated off and washed with 5 mL of
DMF. The filtrate and washing were combined and concentrated to dryness
in vacuum. Recrystallization from ethanol gave 0.40 g (80.3% yield) of
2ꢁ,3ꢁ-anhydroadenosine; mp 181◦C; νmax(KBr)/cm−1 1385, 1308, 1340, 1303
and 1097; 1H NMR (DMSO-d6; Me4Si) δH 3.55 (2H, m, 5ꢁ-H), 4.20 (2H, m,
1ꢁ-H and 4ꢁ-H), 4.45 (1H, d, J = 2.7 Hz, 2ꢁ-H), 5.46 (1H, br s, OH), 6.22 (1H,
s, 1ꢁ-H), 7.31 (2H, br s, NH2), 8.18 (1H, s, 8-H), 8.34 (1H, s, 2-H); 13C NMR
(DMSO-d6; Me4Si) δC 57.7, 58.7, 79.1, 81.1, 82.0, 139.5, 149.1, 152.6, 156.0;
m/z (EI) 250 ([M+H]+, 100%), 89 M(57), 77 (50).
2,2ꢁ-O-Anhydro-1-(β-d-arabinofuranosyl)uracil
(15a). 4a
(1.45
g,
5.0 mmol) and sodium bicarbonate (1.89 g, 22.5 mmol) were heated
in DMF (100 mL) at 90◦C for 5 h and then cooled. The solid precipitated
was filtrate off and washed with 10 mL of DMF. The filtrate and washing
were combined and concentrated to dryness in vacuum. The resulting
syrup was purified by silica gel column chromatography eluting with
chloroform-methanol (4:1, v/v) to give 15a (0.99g, 87.3%). Further
recrystallization from 2-propanol-ethanol (5:1, v/v) gave white crystals; mp
1
246◦C, (lit.[17] 244–247◦C). Its H– and 13C-NMR spectra were identical
with those of the authentic sample (Sigma-Aldrich).
2,2ꢁ-Anhydro-1-β-d-arabinofuranosylthymine (15b). Similar to the synthesis
of 15a, 15b was obtained from the reaction of 4b (1.52 g, 5.0 mmol) and
sodium bicarbonate (1.89 g, 22.5 mmol) in DMF (100 mL); yield: 1.12 g
(93.3%). Recrystallization from 2-propanol-ethanol (5:1, v/v) gave white
1
crystals; mp 218◦C; H NMR (DMSO-d6; Me4Si) δH 1.80 (3H, s, Me), 3.17
(1H, m), 3.26 (1H, m), 4.06 (1H, t, J = 4.9 Hz), 4.37 (1H, d, J = 3.4 Hz),
4.95 (1H, t, J = 5.2Hz, OH), 5.18 (1H, d, J = 5.8 Hz, 2ꢁ-H), 5.87 (1H, d, J =
4.3 Hz, OH), 6.29 (1H, d, J = 5.8 Hz, 1ꢁ-H), 7.74 (1H, d, J = 1.2Hz, 6-H);
(lit.[13] 1H NMR (250 MHz; DMSO-d6; Me4Si) δH 1.79(3H, d, J = 0.9 Hz),
3.22 (2H, m), 4.06 (1H, m), 4.37 (1H, br s), 4.97 (1H, t, J = 5.31Hz), 5.15
(1H, d, J = 5.75 Hz), 5.88 (1H, d, J = 4.52Hz), 6.29 (1H, d, J = 5.75 Hz),
7.75 (1H, d, J = 1.33 Hz)); 13C NMR (DMSO-d6; Me4Si) δC 13.6, 60.9, 74.8,
88.6, 89.2, 90.2, 113.7, 132.3, 159.4, 171.7; m/z (FAB) 241.0819 ([M+H]+,
C10H13N2O5 requires m/z: 241.0824).
2,2ꢁ-O-Anhydro-1-(β-d-arabinofuranosyl)-5-fluorouracil (15c). Similar to
the synthesis of 15a, 15c was obtained from the reaction of 4c (1.54 g, 5.0
mmol) and sodium bicarbonate(1.89 g, 22.5 mmol) in DMF (100 mL);
yield: 1.17 g (95.9%). Recrystallization from 2-propanol-ethanol (5:1, v/v)
gave white crystals; mp 190◦C; 1H NMR (DMSO-d6; Me4Si) δH 3.24 (1H, m),
3.26 (1H, m), 4.12 (1H, m), 4.40 (1H, br s), 4.97 (1H, t, J = 4.9 Hz, OH),
5.26 (1H, d, J = 5.8 Hz, 2ꢁ-H), 5.93 (1H, br s, OH), 6.31 (1H, d, J = 5.8 Hz,
1ꢁ-H), 8.26 (1H, d, J = 4.6 Hz, 6-H); 13C NMR (DMSO-d6; Me4Si) δC 60.8,
75.0, 89.75, 89.82, 90.8, 121.4(d, J cp = 37.2Hz), 145.3 (d, J cp = 248 Hz),
155.4 (d, J cp = 256 Hz), 157.4; m/z (FAB) 245.0581 ([M+H]+, C9H10N2O5F
requires m/z: 245.0574).

732
K.-I. Takatsuki et al.
Cytidine (17a) from (5a). The hydrochloride 5a containing some
amount of free base (1.63 g, 5.0 mmol based on 5a) and sodium bicarbonate
( 2.31 g, 27.5 mmol) were heated in DMF (100 mL) at 105◦C for 3 h and
cooled. The solid precipitated was filtrated off and washed with 10 mL of
DMF. The filtrate and washing were combined and concentrated to dryness
in vacuum. The resulting syrup was purified by silica gel column chro-
matography eluting with chloroform-methanol (4:1, v/v) to give cytidine
17a (0.93 g, 76.5%). Recrystallization from methanol gave white crystals;
mp 213◦C; 1H NMR (DMSO-d6; Me4Si) δH 3.53 (1H, m), 3.65 (1H, m), 3.81
(1H, m), 3.93 (2H, m), 4.97 (1H, d, J = 4.6 Hz), 5.03 (1H, t, J = 5.2
Hz), 5.27 (1H, d, J = 4.6 Hz), 5.71 (1H, d, J = 7.6 Hz), 5.76 (1H, d,
J = 3.7 Hz), 7.10 (1H, br s), 7.13 (1H, br s), 7.84 (1H, d, J = 7.6 Hz); 13C
NMR (DMSO-d6; Me4Si) δC 60.6, 69.4, 74.0, 84.1, 89.2, 93.8, 141.5, 155.4,
135.5; m/z (FAB) 244.0953 ([M+H]+, C9H14N3O5 requires m/z: 244.0933).
[Authentic sample 17a; mp 213◦C; 1H NMR (DMSO-d6; Me4Si) δH 3.54 (1H,
m), 3.65 (1H, m), 3.81 (1H, m), 3.92 (2H, m), 4.98 (1H, d, J = 4.9 Hz, OH),
5.04 (1H, t, J = 5.2Hz, OH), 5.27 (1H, d, J = 5.2Hz, OH), 5.71 (1H, d, J =
7.3 Hz, 5-H), 5.76 (1H, d, J = 3.7 Hz, 1ꢁ-H), 7.11 (1H, br s, NH), 7.17 (1H,
br s, NH), 7.84 (1H, d, J = 7.3 Hz, 6-H); 13C NMR (DMSO-d6; Me4Si) δC
60.6, 69.4, 74.0, 84.0, 89.2, 93.8, 141.5, 155.4, 135.6]
Cytidine (17a) from (16a). Hydrochloride 16a (Sigma-Aldrich) (0.785 g,
3.0 mmol) and sodium bicarbonate (0.88 g, 10.5 mmol) were heated in
DMF (60 mL) at 105◦C for 3 h and cooled. The solid thus precipitated
was filtrated and washed with DMF (10 mL). The filtrate and washing were
combined and concentrated to dryness in vacuum. The resulting syrup
was purified by silica gel column chromatography eluting with chloroform-
methanol (2:1, v/v) to give 17a (0.59 g, 80.9%) whose spectral data were
identical with those of the authentic sample (Sigma-Aldrich).
5-Fluorocytidine (17b). The hydrochloride 5b containing some amount
of free base (0.462 g, 1.5 mmol based on 5b) and sodium bicarbonate
(0.693 g, 8.25 mmol) were heated in DMF (30 mL) at 105◦C for 3 h.
The solid precipitated after cooling was filtrated and washed with DMF
(5 mL). The filtrate and washing were combined and concentrated to
dryness in vacuum. The resulting syrup was purified by silica gel column
chromatography eluting with chloroform-methanol (2:1, v/v) to give 17b
(0.18 g, 49.2%) as an oil; 1H NMR (DMSO-d6; Me4Si) δH 3.57 (1H, m), 3.68
(1H, m), 3.82 (1H, m), 3.95 (2H, m), 4.99 (1H, d, J = 5.5 Hz, OH), 5.20
(1H, d, J = 4.9 Hz, OH), 5.33 (1H, d, J = 5.2 Hz, OH), 5.71 (1H, dd, J =
3.7 and 1.9 Hz, 1ꢁ-H), 7.49 (1H, br s, NH), 7.76 (1H, br s, NH), 8.20 (1H,
d, J = 7.3 Hz, 1H, 6-H); 13C NMR (DMSO-d6; Me4Si) δC 60.2, 69.0, 74.3,
84.1, 89.3, 125.7 (d, J cp = 32.1 Hz), 136.1 (d, J cp = 240 Hz), 153.6, 157.4 (d,
J cp = 13.4 Hz); m/z (FAB) 262.0826 ([M+H]+ C9H13N3O5F requires m/z:
262.0839).

Synthesis of Puromycin from 3ꢁ,5ꢁ-O-Sulfinyl Xylo-Nucleosides
REFERENCES
733
1. (a) Vorbru¨ggen, H.; Ruh-Pohlenz, C. Handbook of Nucleoside Synthesis, John Wiley & Sons, Inc., 2001.
(b) Ichikawa, E.; Kato, K. Current Medicinal Chemistry 2001, 8, 385–423.
2. (a) Suhadolnik, R.J. Nucleoside Antibiotics, pp. 1–50, Wiley, New York, 1970. (b) Suhadolnik, R.J.
Nucleosides as Biological Probe, pp. 96–102, Wiley, New York, 1979.
3. Porter, J.N.; Hewitt, R.I.; Hesseltine, C.W.; Krupka, G.; Lowery, J.A.; Wallace, W.S.; Bohonos, N.;
Williams, J.H. Antibiotics & Chemotherapy 1952, 2, 409–410.
4. Yarmolinsky, M.B.; De la Haba, G.L. Proceeding of the National Academy of Sciences U.S.A. 1959,
45, 1721–1729.
5. Baker, B.R.; Joseph, J.P.; Williams, J.H. Puromycin. Synthetic Studies. VII. Partial Synthesis of Amino
Acid Analogs. Journal of the American Chemical Society 1995, 77, 1.
6. (a) Robins, M.J.; Miles, R.W.; Samano, M.C.; Kaspar, R.L. Syntheses of Puromycin from Adenosine
and 7-Deazapuromycin from Tubercidin, and Biological Comparisons of the 7-Aza/Deaza Pair.
Journal of Organic Chemistry 2001, 66, 8204–8210. (b) Samano, M.C.; Robins, M.J. High-yield
synthesis of 3ꢁ-amino-3ꢁ-deoxynucleosides. Conversion of adenosine to 3ꢁ-amino-3ꢁ-deoxyadenosine.
Tetrahedron Letters 1989, 30, 2329–2332. (c) Nguyen-Trung, N.Q.; Botta, O.; Terenzi, S.; Strazewski,
P. A Practical Route to 3ꢁ-Amino-3ꢁ-deoxyadenosine Derivatives and Puromycin Analogues. Journal of
Organic Chemistry 2003, 68, 2038–2041. (d) Botta, O.; Strazewski, P. Synthesis of an alanyladenosine
analog. Nucleosides & Nucleotides 1999, 18, 721–723. (e) Gerber, N.N.; Lechevalier, H.A. 3ꢁ-Amino-
3ꢁ-deoxyadenosine, an Antitumor Agent from Helminthosporium sp. Journal of Organic Chemistry
1962, 27, 1731–1732. (f) Guarino, A.J.; Kredich, N.M. Isolation and identification of 3ꢁ-amino-
3ꢁ-deoxyadenosine from Cordyceps militaris. Biochimica et Biophyica Acta 1963, 68, 317–319. (g)
Sowa, W. Convenient synthesis of 3-amino-3-deoxy-D-ribose. Canadian Journal of Chemistry 1968,
46, 1586–1589. (h) Okruszek, A.; Verkade, J.G. 2ꢁ,3ꢁ-Bis(2-chloroethyl)aminophosphoryl-3ꢁ-amino-
3ꢁ-deoxyadenosine: A cyclic nucleotide with antitumor activity. Journal of Medicinal Chemistry 1979,
22, 882–885. (i) Ozols, A.M.; Azhayev, A.V.; Dyatkina, N.B.; Krayevsky, A.A. Aminonucleosides
and their Derivatives; VI1. A New Synthesis of 1,2,5-Tri-O-acyl-3-azido-3-deoxy-β-D-ribofuranose.
Synthesis 1980, 557–559. (j) Ozols, A.M.; Azhayev, A.V.; Krayevsky, A.A.; Ushakov, A.S.; Gnuchev,
N.V.; Gottikh, B.P. Aminonucleosides and their Derivatives; VII1. Synthesis of the 3ꢁ,5ꢁ-Dideoxy-
3ꢁ,5ꢁ-diaminonucleosides. Synthesis 1980, 559–561. (k) Saneyoshi, M.; Nishizaka, H.; Katoh, N.
Synthetic nucleosides and nucleotides. XVIII. Synthesis and cytostatic activity of 5-fluoropyrimidine
nucleosides of 3-amino-3-deoxy-β-D-ribofuranose and related compounds. Chemical and Pharmaceu-
tical Bulletin 1981, 29, 2769–2775. (l) Visser, G.M.; Schattenkerk, C.; van Boom, J.H. Recueil des
Travaux Chimique des Pays-Bas 1984, 103, 135–138. (m) McDonald, F.E.; Gleason, M.M. Asymmetric
Synthesis of Nucleosides via Molybdenum-Catalyzed Alkynol Cycloisomerization Coupled with
Stereoselective Glycosylations of Deoxyfuranose Glycals and 3-Amidofuranose Glycals. Journal of
the American Chemical Society 1996, 118, 6648–6659. (n) Baker, B.R.; Joseph, J.P.; Williams, J.H.
Puromycin. Synthetic Studies. VII. Partial Synthesis of Amino Acid Analogs. Journal of the American
Chemical Society 1955, 77, 1–7. (o) Baker, B.R.; Schaub, R.E.; Williams, J.H. Puromycin. Synthetic
Studies. VIII. Synthesis of 3-Amino-3-deoxy-D-ribofuranoside Derivatives. A Second Synthesis of
3-Amino-3-deoxy-D-ribose. Journal of the Amirican Chemical Society 1955, 77, 7–12. (p) Baker, B.R.;
Schaub, R.E.; Joseph, J.P.; Williams, J.H. Puromycin. Synthetic Studies. IX. Total Synthesis. Journal of
the American Chemical Society 1955, 77, 12–15. (q) Baker, B.R.; Schaub, R.E.; Kissman, H.M. Puromycin.
Synthetic Studies. XV. 3ꢁ-Amino-3ꢁ-deoxyadenosine. Journal of the American Chemical Society 1955, 77,
5911–5915. (r) Mengel, R.; Wiedner, H. Chemische Beichte 1976, 109, 433–443.
7. Bartlett, R.K.; Humphrey, I.R. Transaminations of NN-dimethylformamide azine. Journal of the
Chemical Society C 1967, C7, 1664–1666.
8. (a) Takatsuki, K.; Yamamoto, M.; Ohgushi, S.; Kohmoto, S.; Kishikawa, K.; Yamashita, H. A new
protecting group ‘3’,5ꢁ-O-sulfinylꢁ for xylo-nucleosides. A simple and efficient synthesis of 3ꢁ-amino-
3ꢁ-deoxyadenosine (a puromycin intermediate), 2,2ꢁ-anhydro-pyrimidine nucleosides and 2ꢁ,3ꢁ-
anhydro-adenosine. Tetrahedron Letters 2004, 45, 137–140. (b) Takatsuki, K.; Kohmoto, S.; Kishikawa,
K.; Yamashita, H.; Yamamoto, M. Discovery of a novel route to 2ꢁ-deoxy and 2ꢁ-functional pyrimidine
nucleosides via 3ꢁ,5ꢁ-O-sulfinyl xylo-nucleosides. Nucleic Acids Research Supplement 2002, 2, 137–138.
9. Rama Rao, A.V.; Gurjar, M.K. Discovery of a novel route to β-thymidine: A precursor for anti-AIDS
compounds. Journal of the Chemical Society Chemistry Communications 1994, 1255–1256.

734
K.-I. Takatsuki et al.
10. (a) Nakayama, C.; Saneyoshi, M. Synthetic nucleosides and nucleotides. XX. Synthesis of various
1-β-D-xylofuranosyl -5-alkyluracils and related nucleosides. Nucleosides & Nucleotides 1982, 1, 139–146.
(b) Gosselin, G.; Bergogne, M.-C.; Rudder, J.; De Clercq, E.; Imbach, J.-L. Systematic synthesis and
biological evaluation of α- and β-D-xylofuranosyl nucleosides of the five naturally occurring bases
in nucleic acids and related analogs. Journal of Medicinal Chemistry 1986, 29, 203–213. (c) Koshkin,
A.A.; Fensholdt, J.; Pfundheller, H.M.; Lomholt, C. A Simplified and Efficient Route to 2ꢁ-O, 4ꢁ-C-
Methylene-Linked Bicyclic Ribonucleosides (Locked Nucleic Acid). Journal of Organic Chemistry 2001,
66, 8504–8512.
11. Girgis, N.S.; Pedersen, E.B. Phosphorus Pentoxide in Organic Synthesis; II1. A New, One-Step
Conversion of Hypoxanthine into N 6-Substituted Adenines. Synthesis 1982, 480–482.
12. (a) Sowa, T.; Tsunoda, K. The Convenient Synthesis of Anhydronucleosides via the 2ꢁ,3ꢁ-O-Sulfinate
of Pyrimidine Nucleosides as the Active Intermediates. Bulletin of the Chemical Society of Japan 1975, 48,
505–507. (b) Sowa, T.; Tsunoda, K. Novel Synthesis of Anhydronucleosides via the 2ꢁ,3ꢁ-O-Sulfinate
of Purine Nucleosides as Intermediates. Bulletin of the Chemical Society of Japan 1975, 48, 3243–3245.
They synthesised 2ꢁ,3ꢁ-O-sulfinate of ribo-nucleosides using thionyl chloride in acetonitrile.
13. Murtiashaw, C.W. Eur. Pat. Appl. EP351, 126.
14. (a) Doerr, I.L.; Fox, J.J. Nucleosides. XXXIX. 2ꢁ-Deoxy-2ꢁ-fluorocytidine, 1-β-D-arabinofuranosyl-
2-amino-1,4(2H)-4-iminopyrimidine, and related derivatives. Journal of Organic Chemistry, 1967, 32,
1462–1471. (b) Kikugawa, K.; Ichino, M. Vilsmeier-Haack reaction. IV. Convenient synthesis of 2,2ꢁ-
anhydro-1-β-D-arabinofuranosyl cytosine (2,2ꢁ-cyclocytidine) and its derivatives. Journal of Organic
Chemistry 1972, 37, 284–288. (c) Wang, M.C.; Sharma, R.A.; Bloch, A. Cancer Research 1973, 33, 1265.
15. Even though the β-d-xylofuranosyl nucleosides prepared in this work are known compounds, we
have included their 1H and 13C NMR spectral data since no data available on their high-resolution
spectra due to their publication in 1960’s. The present data will be helpful for researchers in this
field.
16. Poopeiko, N.E.; Kvasyuk, E.I.; Mikhailopulo, I.A.; Lidaks, M.J. Stereospecific Synthesis of β-D-
Xylofuranosides of Adenine and Guanine. Synthesis 1985, 605–609.
17. Fox, J.J.; Miller, N.; Wempen, I. Nucleosides. XXIX. 1-β-D-Arabinofuranosyl-5-fluorocytosine and
Related Arabino Nucleosides. Journal of Medicinal Chemistry 1966, 9, 101–105.