First synthesis of tepidopterin [2′-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-threo-biopterin]

Article abstract of DOI:10.1016/j.carres.2007.06.017

N²-(N,N-Dimethylaminomethylene)-1′-O-(4-methoxybenzyl)-3-[2-(4-nitrophenyl)ethyl]-l-threo-biopterin (14) was prepared from l-xylose in an 11-step-sequence. The first synthesis of tepidopterin (3) was achieved by treatment of 14 with 3,4,6-tri-O

Full text of DOI:10.1016/j.carres.2007.06.017

Carbohydrate Research 342 (2007) 2159–2162
Rapid Communication
First synthesis of tepidopterin
[20-O-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-L-threo-biopterin]
Tadashi Hanaya,a,* Hiroki Babaa and Hiroshi Yamamotob
aDepartment of Chemistry, Faculty of Science, Okayama University, Tsushima-naka, Okayama 700-8530, Japan
bSchool of Pharmacy, Shujitsu University, Okayama 703-8516, Japan
Received 24 May 2007; received in revised form 13 June 2007; accepted 15 June 2007
Available online 26 June 2007
Abstract—N2-(N,N-Dimethylaminomethylene)-10-O-(4-methoxybenzyl)-3-[2-(4-nitrophenyl)ethyl]-L-threo-biopterin (14) was pre-
pared from L-xylose in an 11-step-sequence. The first synthesis of tepidopterin (3) was achieved by treatment of 14 with 3,4,6-tri-
O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl bromide in the presence of silver triflate and tetramethylurea, followed by
removal of the protecting groups.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Pterin glycoside; Pteridine; Tepidopterin; Glycosylation; L-threo-Biopterin
Some pterins having a hydroxyalkyl side chain at C-6,
a representative example being L-biopterin (1a), have
been found as glycosidic forms in certain prokaryotes
such as cyanobacteria and anaerobic photosynthetic
bacteria; for example, the 20-O-(a-D-glucopyranosyl)-L-
biopterin (1b) isolated from cyanobacterium, Anacystis
nidulans,1 Synechococcus sp.,2 and Spirulina platensis,3
and limipterin (1c) from a green sulfur photosynthetic
bacterium, Chlorobium limicola f. thiosulfatophilum.4
Various other glycosides consisting of different pterins
and sugar moieties have also been found in nature,
although the glycosidic linkages of some derivatives
remain unclear.5 The physiological function of the par-
ent pterins has been studied in detail: for example, 1
exhibits enzyme cofactor activity in aromatic amino acid
hydroxylation6 and nitric oxide synthesis7 as the form of
its tetrahydro derivative. By contrast, the functional
roles of pterin glycosides have remained obscure,
although some inhibitory activities against tyrosinase8
and photostabilization of photosynthetic pigments9
were reported for L-biopterin D-glucoside (1b). Despite
considerable interest from the viewpoint of their biolog-
ical activities and functions, as well as structural proof,
attempts at preparation of pterin glycosides have so far
scarcely been made.10
O
OH
O
OH
3
HN
6
2'
N
N
N
N
3'
HN
H2N
1'
OR
OH
7
H2N
N
N
2
1a R = H (L-biopterin)
2 (L-threo-biopterin)
HO
O
O
OH
1b R =
1c R =
OH
N
HN
H2N
HO
HO
O
N
N
HO
O
HO
OH
O
OH
HO
NHAc
HO
NHAc
3 (tepidopterin)
Meanwhile, tepidopterin (3) was isolated from a green
sulfur photosynthetic bacterium Chlorobium tepidum
and was characterized as the 20-O-(2-acetamido-2-
deoxy-b-D-glucopyranosyl) derivative of L-threo-bio-
pterin (2).11 The L-threo structure of 3 appears somewhat
*
Corresponding author. Fax: +81 86 251 7853; e-mail: hanaya@cc.
okayama-u.ac.jp
0008-6215/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.06.017

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T. Hanaya et al. / Carbohydrate Research 342 (2007) 2159–2162
peculiar in view of the tendency that L-biopterin deriva-
tives having an L-erythro form are common among nat-
ural pterin glycosides, and thus identifying the enzyme
from C. tepidum has been attempted.12 These facts
prompted us to develop an efficient synthetic protocol
for pterin glycosides as well as to prove the proposed
structure of the reported natural product. We describe
herein the first synthesis of tepidopterin (3) via an
appropriately protected L-threo-biopterin.
The retrosynthetic analysis for 3 is outlined in Scheme
1. The L-threo-biopterin derivative (4), whose pyrimi-
dine ring moiety and 10-hydroxy group of the side chain
are protected, can be perceived as the key precursor to
achieve the selective 20-O-glycosylation. The pteridine
ring formation of 4 would be achieved by condensation
of 2,5,6-triamino-4-hydroxypyrimidine (5) with the
pentos-2-ulose (6), which would be derived from the 3-
O-protected 5-deoxy-L-xylose (7). Taking into consider-
ation the available conditions to remove the protecting
groups of the glycoside derived from 4, we employed
p-methoxybenzyl (PMB) group for protection of
10-hydroxy, N,N-dimethylaminomethylene group for
2-amino, and 2-(4-nitrophenyl)ethyl (NPE) group for
N-3 of the ring.13
The synthesis commenced from 1,2-O-isopropylidene-
5-O-tosyl-a-L-xylofuranose (8), which was prepared
from L-xylose according to the reported procedures14
(Scheme 2). Reduction of 8 with lithium aluminum
hydride in ether afforded the 5-deoxy derivative (in
95% yield), which was then treated with p-methoxyben-
zyl chloride and sodium hydride in the presence of tetra-
butylammonium iodide to give the 3-O-PMB derivative
(9) in 98% yield. Hydrolysis of 9 in 70% acetic acid
containing catalytic hydrochloric acid afforded 5-
deoxy-3-O-PMB-L-xylofuranose (10) in 83% yield.
The selective oxidation of 2-hydroxy group of 10 with
cupric acetate15 provided the L-threo-pentos-2-ulose
derivative (11). The pteridine ring formation of 11 with
sulfate of 2,5,6-triamino-4-hydroxypyrimidine (5) under
neutral conditions afforded an inseparable mixture
(35:65) of the 6-substituted pterin (12a) and its 7-substi-
tuted isomer (12b), while the same condensation in an
aqueous sodium bicarbonate solution resulted in the
preferential formation of the desired 12a in a ratio of
75:25. These products were separated and characterized
after having been converted into the fully protected
derivatives (13a,b) by the following three steps: treat-
ment of 12a,b with N,N-dimethylformamide dimethyl
acetal in DMF, followed by acetylation of a hydroxy
group, afforded 20-O-acetyl-N2-(N,N-dimethylamino-
methylene)-10-O-PMB derivatives, whose N-3 position
was then protected with NPE group by Mitsunobu reac-
tion with 2-(4-nitrophenyl)ethanol in the presence of tri-
phenylphoshine and diethyl azodicarboxylate (DEAD)
to give 13a and 13b. These products were separated by
column chromatography on silica gel to provide the
O
N
OR3
R2
N
N
N
3
OH
R1HN
4
(R1, R2, R3 = protecting groups)
CHO
OR3
O
HO
HO
NH2
NH2
O
O
OR3
CH3
HN
+
OH
H2N
N
H
5
7
6
Scheme 1.
1) acetone
H2SO4
1) LiAlH4, ether
95%
HO
O
O
O
O
O
O
70% AcOH aq.
HO
OH
O
O
HO
OH
cat. HCl, 40 °C
83%
2) PMBCl, NaH
Bu4NI, DMF
98%
2) TsCl, Py
TsO
H3C
H3C
HO
HO
OPMB
OPMB
L-xylose
8
9
10
O
OPMB
OH
O
OPMB
OAc
N
NPE
N
N
N
1) Me2NCH(OMe)2
HN
N
N
O
N
DMF, rt, 3 h
NH2
NH2
HN
H2N
N
O
N
N
N
N
O
2) Ac2O, Py
rt, 12 h
H2N
O
Cu(OAc)2
12a
13a (52% from 11)
+
+
OH
3) NPEOH, PPh3
DEAD, THF
rt, 16 h
NaHCO3
MeOH-H2O
reflux, 3 h
MeOH-H2O
reflux, 0.5 h
49%
H3C
O
NPE
N
N
N
N
PMBO
OAc
HN
H2N
OH
11
4) separation
N
N
N
OPMB
OPMB
13b (17%)
12b
Scheme 2.

T. Hanaya et al. / Carbohydrate Research 342 (2007) 2159–2162
2161
AcO
AcO
O
N
OPMB
Br
O
NPE
N
N
N
OAc
N
O
N
OPMB
OH
NPE
N
O
NaOMe
DDQ
N
N
NPhth
N
N
13a
AcO
CF3SO3Ag, TMU
CH2Cl2, rt, 3 h
92%
MeOH, rt, 2 h
98%
CH2Cl2, rt, 2 h
89%
N
O
OAc
AcO
14
15
NPhth
O
N
OH
N
O
OH
O
N
OAc
NPE
NPE
N
N
N
N
HN
H2N
N
N
O
N
O
O
N
N
N
AcNH
1) MeNH2, MeOH
rt, 10 h
1) NH3 aq.
AcO
AcO
MeOH, rt, 12 h
HO
O
OAc
O
OAc
O
OH
2) Ac2O, Py
rt, 12 h
2) DBU, DMF
rt, 12 h
AcO
AcO
HO
NPhth
NHAc
NHAc
85%
85%
16
17
3
Scheme 3.
Table 1. 1H and 13C NMR spectral parameters for tepidopterin (3) in DMSO-d6
Chemical shifts (d)
a,b
Coupling constants (Hz)
H-10
4.59
H-20
4.01
H3-30
1.12
H2N-2
6.99
H–N(3)
11.55
HO-10
5.31
J1 ;2
J2 ;3
J1 ;OH
0
0
0
0
0
Pterin moiety
H-7
8.65
4.4
6.4
5.3
Glycosyl moietyc H-1 H-2 H-3 H-4 H-5 Ha-6 Hb-6 Ac
4.32 3.32 3.20 3.02 3.05 3.67 3.42 1.71 7.42
NH-2 J1,2 J2,3 J3,4 J4,5 J5,6a J5,6b J6a,6b J2,NH
8.3 9.9 8.6 9.5 2.0
6.1
11.7
8.8
Chemical shifts (d)
Pterin moiety
C-2
156.82
C-4
161.27
C-4a
127.63
C-6
150.72
C-7
149.12
C-8a
153.91
C-10
74.82
C-20
78.43
C-30
18.20
Glycosyl moiety
C-1
101.88
C-2
56.07
C-3
74.62
C-4
70.83
C-5
77.02
C-6
61.32
COCH3
169.58
COCH3
23.31
a The solvent peak was used as an internal standard (d 2.50 for 1H, 39.70 for 13C).
b The assignments were made with the aid of D2O exchange and 2D C–H COSY measurements.
c d 4.95 (HO-3, J3,OH = 4.8 Hz), 4.85 (HO-4, J4,OH = 5.1 Hz), 4.47 (HO-6, J6,OH = 5.8 Hz).
desired 6-substituted pterin derivative (13a) (52% overall
yield from 11) and the 7-substituted derivative (13b)
(17%).
for the latter. These assignments are supported by the
fact that H-7 signal (d 8.96) of 13a appears at a lower
field than H-6 signal (d 8.78) of 13b due to conjugation
with the 4-oxo group.
The structural assignment of 13a and 13b was made
primarily on the basis of their 1H and 13C NMR spectral
data. The signals of C-6 and C-7 of 6-alkylpteridines
generally appear at a similar field, whereas C-7 signals
of 7-alkyl derivatives shift to a lower field (ca. 20 ppm)
from those of C-6.16 Therefore, the close values of 13a
(C-6: d 151.14, C-7: d 149.35) and the distant values of
13b (C-6: d 140.24, C-7: d 160.50) indicate the 6-substi-
tuted pterin for the former and the 7-substituted pterin
Methanolysis of 20-O-acetyl-10-O-PMB-L-threo-bio-
pterin derivative (13a) in the presence of sodium methox-
ide provided the 10-O-PMB derivative (14), a versatile
precursor for the 20-O-monoglycosylation (Scheme 3).
Efficient glycosylation of 14 was attained by condensa-
tion with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimindo-b-
D-glucopyranosyl bromide17 in the presence of silver tri-
flate and tetramethylurea (TMU) in dichloromethane at
room temperature for 3 h, affording the 20-O-(b-D-
glucopyranosyl)-L-threo-biopterin derivative (15) in
92% yield.
All new compounds gave elemental analysis and/or MS (high-
resolution) consistent with their structures. Synthetic procedures for
compounds 13a,b from 10 and the NMR spectral parameters of 13–
17 are available as Supplementary data.
Removal of the protecting groups of 15 was carried
out according to the following steps: cleavage of PMB

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group of 15 was achieved by treatment with 2,3-di-
chloro-5,6-dicyano-1,4-benzoquinone (DDQ), to afford
16 (89%), followed by removal of the phthaloyl and
N,N-dimethylaminomethylene group of 16 with methyl-
amine. Then the product was acetylated with acetic
anhydride in pyridine to give the fully acetylated deriv-
ative (17) in 85% overall yield from 16. Treatment of
17 with aqueous ammonia (to cleave acetyl groups
except for 2-acetamido of the sugar) and then with
DBU (to cleave the NPE group) furnished tepidopterin
(3) in 85% overall yield. The precise parameters obtained
1
on 600 MHz H and 125 MHz 13C NMR spectra for 3
are listed in Table 1;à the spectral data of the synthetic
compound (3) were found to be essentially identical with
those reported for the natural product.11
The present work thus demonstrates the first synthesis
of tepidopterin (3) from L-xylose via 10-O-PMB-L-threo-
biopterin derivative (14). This synthetic strategy has
proved to provide a useful method that can be applied
to a series of other natural pterin glycosides and their
analogs.
10. Hanaya, T.; Soranaka, K.; Harada, K.; Yamaguchi, H.;
Suzuki, R.; Endo, Y.; Yamamoto, H.; Pfleiderer, W.
Heterocycles 2006, 67, 299–310.
Acknowledgment
11. Cho, S.-H.; Na, J.-U.; Youn, H.; Hwang, C.-S.; Lee, C.-
H.; Kang, S.-O. Biochim. Biophys. Acta 1998, 1379, 53–60.
12. Supangat, S.; Seo, K. H.; Choi, Y. K.; Park, Y. S.; Son,
D.; Han, C.; Lee, K. H. J. Biol. Chem. 2006, 281, 2249–
2256.
We are grateful to the SC-NMR Laboratory of
Okayama University for the NMR measurements.
13. Hanaya, T.; Torigoe, K.; Soranaka, K.; Yamamoto, H.;
Yao, Q.; Pfleiderer, W. Pteridines 1995, 6, 1–7.
14. Renaut, P.; Millet, J.; Sepulchre, C.; Theveniaux, J.;
Barberousse, V.; Jeanneret, V.; Vogel, P. Helv. Chim. Acta
1998, 81, 2043–2052.
15. (a) Weinstock, J. U.S. Patent 3,505,329, 1970; Chem.
Abstr. 1970, 72, 132787h; (b) Taylor, E. C.; Jacobi, P. A. J.
Am. Chem. Soc. 1976, 98, 2301–2307.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2007.06.017.
16. (a) Geerts, J. P.; Nagel, A.; Van der Plas, H. C. Org.
Magn. Reson. 1976, 8, 606–610; (b) Hanaya, T.; Toyota,
H.; Yamamoto, H. Synlett 2006, 2075–2078; (c) Hanaya,
T.; Takayama, D.; Yamamoto, H. Heterocycles 2006, 70,
355–365.
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à The complete assignments of 3 have been established by the present
work; some ambiguous parameters were included in the previous
report for the natural compound.