Design and Synthesis of Peptide Mimetics of GDP-Fucose: Targeting Inhibitors of Fucosyltransferases

Article abstract of DOI:10.1055/s-2003-44984

Novel peptide mimetics of GDP-fucose were designed and synthesized targeting inhibitors of the fucosyltransferases that transfer L-fucose from GDP-fucose to oligosaccharides, on the basis of the background that nikkomycin Z, a peptide mimetic of UDP-N-acetylglucosamine, shows potent inhibitory activity toward an N-acetylglucosamine transfer enzyme. The synthetic routes of the GDP-fucose mimetics take advantage of an enzymatic aldol reaction catalyzed by L-threonine aldolase to prepare the guanine carrying β-hydroxy-α- L-amino acid, a key synthetic intermediate.

Full text of DOI:10.1055/s-2003-44984

LETTER
243
Design and Synthesis of Peptide Mimetics of GDP-Fucose: Targeting
Inhibitors of Fucosyltransferases
Targeting Inhib
o
itors of Fuco
r
syltransf
u
erases Tanaka,a Chihiro Tsuda,a Tsuyoshi Miura,b Toshiyuki Inazu,b Shuichi Tsuji,c Shoko Nishihara,d Mitsuko
Hisamatsu,d Tetsuya Kajimoto*a
a
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588,
Japan
Fax +81(42)3778170; E-mail: kajimoto@cc.ocha.ac.jp
b
c
d
The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0003, Japan
The Glycoscience Institute, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8555, Japan
Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji-shi, Tokyo 192-8577, Japan
Received 17 October 2003
O
N
Abstract: Novel peptide mimetics of GDP-fucose were designed
and synthesized targeting inhibitors of the fucosyltransferases that
transfer L-fucose from GDP-fucose to oligosaccharides, on the basis
of the background that nikkomycin Z, a peptide mimetic of UDP-N-
acetylglucosamine, shows potent inhibitory activity toward an N-
acetylglucosamine transfer enzyme. The synthetic routes of the
GDP-fucose mimetics take advantage of an enzymatic aldol reac-
tion catalyzed by L-threonine aldolase to prepare the guanine carry-
ing b-hydroxy-a-L-amino acid, a key synthetic intermediate.
N
N
NH
O
O
NH2
Me
O
OH
O
O
O
P
P
O
OH
OH
O
O
OH OH
GDP-fucose (1)
O
N
N
N
NH
Key words: aldol reactions, amino acids, enzymes, inhibitors,
nucleotides
O2C
O
NH2
Me
O
OH
O
N
H
OH
OH
OH
A group of fucosyltransferases that transfer an L-fucose
unit from guanosine 5¢-diphospho-b-L-fucose (GDP-
fucose, 1) to D-galactose or N-acetyl-D-glucosamine
residues of glycoconjugates forms a family of glycosyl-
transfer enzymes, which utilize sugar nucleotides as the
substrates of glycosyl donors. The biological functions of
the fucosyltransferases, which afford L-fucosylated oli-
gosaccharides, e.g., Ley and Leb, have been revealed in the
last decade.1 The activity of an a-1,2-fucosyltransferase,
for instance, affects the immune responses to bacterial
infection through the mucous membrane since a glyco-
protein related to the immunity is activated to secrete into
the mucous liquid by the fucosylation.2 Moreover, the
activities of the fucosyltransferases including the above
enzyme are, in general, hereditary. Therefore, the selec-
tive inhibitors and/or inducers of the fucosyltransferases
are promising candidates targeting custom-made medi-
cine that is usable for specific patients or their families
having the same gene. However, only a few tediously
prepared inhibitors have been reported so far.3
Peptide mimetic of GDP-fucose
(2a: α-isomer, 2b: β-isomer)
O
N
OH
O
NH
HO
HO
O
O
AcHN
O
O
O
O
P
P
O
O
O
OH OH
UDP-N-acetyl-D-glucosamine (4)
O
NH
O2C
OH NH2
N
O
H
N
N
O
Me
O
HO
OH OH
nikkomycin Z (3)
Figure 1 Structure of sugar nucleotids and their mimetics
Targeting useful and easily available inhibitors of the fu-
cosyltransferases, we designed novel peptide mimetics
of GDP-fucose (2a, 2b) on the basis of the fact that an
N-acetylglucosamine transfer enzyme, which utilizes
uridine 5¢-diphospho-N-acetyl-a-D-glucosamine (UDP-
GlcNAc, 4) as the substrate, was potently inhibited by
nikkomycin Z (3),4 a mimetic of 4 (Figure 1).5
Upon designing the GDP-fucose analogs (2a, 2b), we fo-
cused on the fact that the C-terminal’s peptide bond that
also exists in the structure of 3 can be a bioisoster of the
diphosphate part of 1 as well as that of 4 in terms of having
the negative charge and high polarity. In addition, the hy-
droxyl groups at the b-position of the guanine-carrying
amino acid parts in 2a and 2b were expected to behave as
the 2¢- and/or 3¢-hydroxyl group of 1.
SYNLETT 2004, No. 2, pp 0243–0246
0
2.
0
2.
2
0
0
4
Advanced online publication: 08.12.2003
DOI: 10.1055/s-2003-44984; Art ID: U21603ST.pdf
© Georg Thieme Verlag Stuttgart · New York

244
T. Tanaka et al.
LETTER
R1
N
presence of camphor sulfonic acid to afford a mixture of
a-L-fucoside (12a) and b-L-fucoside (12b, 71%), and L-
fucofuranoside (18%) albeit obtained the yield of L-fu-
copyranoside (12a,b) was no higher than that of the
literature.9 Acetylation of the L-fucopyranoside mixture
made the separation of the two isomers possible by silica
gel chromatography (n-hexane:EtOAc = 7:1) to afford 1-
O-pentenyl 2,3,4-tri-O-acetyl-a-L-fucoside (13a)10 and 1-
O-pentenyl 2,3,4-tri-O-acetyl-b-L-fucoside (13b)11 (93%)
in the ratio of 65:35.
OEt
Cl
N
Br
N
N
7
OEt
N
N
K2CO3
58%
N
R2
N
H
H2N
H2N
8 : R1 = Cl, R2 = CH2CH(OEt)2
9 : R1 = OH, R2 = CH2CHO
6
O
N
1) Boc2O, NaOH (91%)
2) Cs2CO3, and then
BnBr (52%)
L-Threonine
aldolase
N
N
HN
NH2
CO2H
H2N
glycine
69%
3) 1 N HCl / EtOAc
(quant.)
OH
O
5a : erythro
5b : threo
4-penten-1-ol
OH
Me
O
OH
11
Me
O
O
O
OH
OH
OH
(+)-CSA
71%
OH
OH
12a, 12b
N
N
HN
HN
+
N
NH2
CO2Bn
N
NH2
H2N
N
H2N
N
O
Ac2O, pyridine
CO2Bn
Me
O
OAc
13a
separation
of anomers
10a (erythro)
10b (threo) OH
OH
OAc
OAc
and β-anomer (13b)
93%
Scheme 1 Preparation of guanine carrying amino acids 5a,b and
their benzyl esters 10a,b
Scheme 2 Preparation of 1-O-pentenyl fucosides (13a,b)
In the present paper, we report the facile synthesis of 2a
and 2b, in which the L-threonine aldolase-catalyzed reac-
tion, an enzymatic aldol reaction,6 is advantageous in
preparing the guanine carrying b-hydroxy-a-L-amino acid
with the L-erythro configuration (5a)7, a key synthetic
intermediate.
Neighboring group participation effect of the benzoyl
group at the C-2 position was used to obtain 1-O-pentenyl
b-L-fucoside derivative as a predominant product.
1a,2,3,4-Tetra-O-benzoyl-L-fucose was, namely, treated
with HBr/HOAc in acetic anhydride and dichloromethane
to afford 1-bromo-2,3,4-tri-O-benzoyl-L-fucose (14).12 4-
Penten-1-ol was L-fucosylated with 14 in the presence of
silver triflate in a relatively polar mixed solvent composed
of acetonitrile, dichloromethane, and diethyl ether (1:1:1)
to give 1-O-(4-pentenyl) 2,3,4-tri-O-benzoyl-b-L-fuco-
side (15, 80%, 2 steps). Debenzoylation with sodium
methoxide followed by acetylation gave 13b (93%).
2-Amino-6-chloropurine (6) and 2-bromo-1,1-diethoxy-
ethane (7) were heated in the presence of potassium
carbonate at 100 °C in DMF to give a-(2-amino-6-chloro-
prin-9-yl)acetaldehyde diethylacetal (8, 58%). The di-
ethyl acetal 8 was hydrolyzed with 1 M hydrochloric acid
to aldehyde 9 and the reaction mixture was diluted with
Tris-buffer (pH 7.5, 200 mM). After adding glycine (50
equiv to 9) and L-threonine aldolase solution (450 units/
200 mL) from Candida humicola (AKU 4586), the reac-
tion mixture was incubated at 30 °C for 24 hours. Heating
the reaction medium at 100 °C for 30 minutes to terminate
the reaction, followed by celite filtration, evaporation in
vacuo, and purification by column chromatography on
ODS eluted with hot water (50 °C) afforded guanine car-
rying b-hydroxy-a-L-amino acid (69%, 2 steps), albeit a
mixture of erythro (5a) and threo (5b) isomers.8 Separa-
tion of these stereoisomers was attained by converting to
benzyl esters 10a,b in 3 steps [protection of the amino
group of 5a and 5b with t-butoxycarbonyl (Boc) group,
making cesium salt of the carboxylic acid, reaction with
benzyl bromide, and treatment with 1 M HCl/EtOAc], and
the following purification by silica gel column chroma-
tography (CHCl3:MeOH:H2O = 8:1.7:0.1).
Br
4-penten-1-ol, AgOTf
Me
O
OBz
CH3CN, CH2Cl2, Et2O
(1 : 1 : 1)
OBz
OBz
14
15
80%
1) NaOMe, MeOH
O
OBz
Me
O
13b
2) Ac2O, pyridine
93%
OBz
OBz
Cl
OMe
4-penten-1-ol,
AgOTf
Cl2CHOCH3
ZnCl2
Me
Me
O
O
OBn
OBn
CH2Cl2
OBn
OBn
OBn
35%
(2 steps)
OBn
17
16
O
On the other hand, L-fucose (11) was derived to 1-O-(4-
pentenyl)-a-L-fucose (12a) and/or 1-O-(4-pentenyl)-b-L-
fucose (12b), since the terminal olefins of these L-fuco-
sides can be derived with oxidative agents to carboxylic
acid to form an amide bond with the a-amino group of
10a. L-Fucose (11) was first treated with 4-pentenol in the
1) Birch reduction
Me
O
13a
OBn
2) Ac2O, pyridine
50%
OBn
OBn
18
Scheme 3 Respective preparation of a- and b-L-fucosides (13a,b)
Synlett 2004, No. 2, 243–246 © Thieme Stuttgart · New York

LETTER
Targeting Inhibitors of Fucosyltransferases
245
Neither 2a nor 2b unfortunately showed inhibitory activi-
ty toward fucosyltransferases (FUT-3, FUT-6),19,20 how-
ever, this result and structure comparison of 2a,b with 3
provided useful information in that both the hydroxyl and
amino groups on the g-hydroxybutyric chain were found
to be indispensable for the incorporation into the glycosyl-
transferases by forming a chelate with Mn2+ or Mg2+, the
essential metal ion for glycosyltransferase-catalyzed reac-
tions. Synthesis of GDP-fucose analogs carrying the hy-
droxyl and amino groups on the g-hydroxybutyric linker
is now in progress.
O
CO2H
RuO2, NaIO4
13a
Me
Me
O
OAc
19a
CCl4, CH3CN, H2O
(2 : 2 : 3)
OAc
OAc
90%
RuO2, NaIO4
O
CO2H
O
13b
OAc
CCl4, CH3CN, H2O
(2 : 2 : 3)
OAc
OAc
19b
quant.
Scheme 4 Preparation of fucose carboxylates (19a,b)
On the other hand, 1-O-pentenyl a-L-fucoside derivative Acknowledgment
can be solely prepared by using an anomeric effect in a
We thank Profs. T. Nohara and T. Ikeda (Kumamoto University)
non-polar solvent. 4-Penten-1-ol was L-fucosylated with
1-chloro-2,3,4-tri-O-benzyl-a-L-fucose (16), prepared
from 1-O-methyl 2,3,4-tri-O-benzyl-L-fucoside (17) with
dichloromethoxy-methane/ZnCl2 in CHCl3, to afford 1-O-
(4-pentenyl) 2,3,4-tri-O-benzyl-a-L-fucoside (18, 2 steps,
50%).13 Debenzylation of 18 under Birch reduction condi-
tions followed by conventional acetylation gave 13a
(50%).
for their kindness for measuring FAB mass spectra of 20a, 20b, 2a,
and 2b.
References
(1) For example, see:Taniguchi, N.; Honke, K.; Fukuda, M.
Handbook of Glycosyltransferases and Related Genes;
Springer-Verlag, 2002, 205.
(2) Stapleton, A.; Stround, M. R.; Hakomori, S.; Stamm, W. E.
Infect. Immun. 1998, 66, 3856.
Both a- and b-L-fucosides (13a,b) were respectively treat-
ed with ruthenium tetroxide14 to afford 1-O-(1-carboxyl-
butyl-4-oxyl) 2,3,4-tri-O-acetyl-a-L-fucoside (19a) and
1-O-(1-carboxylbutyl-4-oxyl) 2,3,4-tri-O-acetyl-b-L-fu-
coside (19b) in excellent yields.
(3) Compain, P.; Martin, O. R. Bioorg. Med. Chem. 2001, 9,
3077.
(4) (a) König, W. A.; Hahn, H.; Rathman, R.; Hass, W.;
Keckeisen, A.; Hagenmaier, H.; Bormann, C.; Dehler, W.;
Kurth, R.; Zähner, H. Liebigs Ann. Chem. 1986, 407.
(b) Saksena, A. K.; Lovey, R. G.; Girijavallabhan, V. M.;
Guzik, H.; Ganguly, A. K. Tetrahedron Lett. 1993, 34, 3267.
(5) Dahn, U.; Hagenmeier, H.; Höhne, H.; König, W. A.; Wolf,
G.; Zähner, H. Arch. Microbiol. 1976, 107, 143.
(6) (a) Vassilev, V. P.; Uchiyama, T.; Kajimoto, T.; Wong, C.
H. Tetrahedron Lett. 1995, 36, 4081. (b) Vassilev, V. P.;
Uchiyama, T.; Kajimoto, T.; Wong, C. H. Tetrahedron Lett.
1995, 36, 5063. (c) Shibata, K.; Shingu, K.; Vassilev, V. P.;
Nishide, K.; Fujita, T.; Node, M.; Kajimoto, T.; Wong, C. H.
Tetrahedron Lett. 1996, 37, 2791. (d) Fujii, M.; Miura, T.;
Kajimoto, T.; Ida, Y. Synlett 2000, 1046. (e) Nishide, K.;
Shibata, K.; Fujita, T.; Kajimoto, T.; Wong, C. H.
Heterocycles 2000, 52, 1191. (f) Miura, T.; Kajimoto, T.
Chirality 2001, 577.
Finally, both of the erythro isomer of guanine carrying
amino acid (10a) corresponding to the nucleotide part,
and the sugar units (19a and 19b) were coupled with
DCC/HOBt/NMM to give fully protected GDP-fucose
analogs (20a,b)15,16 (43% and 83%, respectively). Cleav-
age of the acetyl groups in 20a and 20b with sodium meth-
oxide in methanol smoothly proceeded and was also
accompanied with ester exchange from benzyl to methyl
ester, which was quantitatively hydrolyzed in aqueous
sodium hydroxide to afford the target molecules 2a17 and
2b18.
O
N
NH
BnO2C
O
19a, DCC, HOBt, NMM
1) 0.02 N NaOMe
N
N
NH2
10a
2a
O
2) 0.01 N NaOH aq
quant.
DMF
43%
N
H
Me
O
OH
OAc
20a
OAc
OAc
O
N
NH
BnO2C
O
1) 0.02 N NaOMe
19b, DCC, HOBt, NMM
N
N
NH2
10a
2b
2) 0.01 N NaOH aq
quant.
DMF
O
OAc
Me
O
N
83%
H
OAc
OAc
OH
20b
Scheme 5 Synthetic route of peptidic mimetics of GDP-fucose (2a,b)
Synlett 2004, No. 2, 243–246 © Thieme Stuttgart · New York

246
T. Tanaka et al.
LETTER
(16) Compound 20b: 1H NMR (CD3OD): d = 1.06 (d, 3 H,
(7) Miura, T.; Fujii, M.; Shingu, K.; Koshimizu, I.; Naganoma,
J.; Kajimoto, T.; Ida, Y. Tetrahedron Lett. 1998, 39, 7313.
(8) (a) The absolute stereochemistry of the a-carbon was
determined to be L-configuration by the observation that 5a
and 5b were not substrates of the D-amino acid oxidase but
those of the L-amino acid oxidase. The erythro- and threo-
configuration was determined by converting to the
J = 6.5 Hz, Me-6), 1.76 (m, 2 H), 1.84, 1.94, 2.04 (s, each 3
H, 3 × Ac), 2.25 (t, 2 H, J = 8.0 Hz), 3.45 (dt, 1 H, J = 10.0,
6.0 Hz), 3.74 (dt, 1 H, J = 10.0, 6.0 Hz), 3.81 (br q, 1 H,
J = 6.5 Hz, H-5), 4.03 (dd, 1 H, J = 8.5, 14.0 Hz, H-g), 4.16
(dd, 1 H, J = 4.0, 14.0 Hz, H-g¢), 4.19 (m, 1 H, H-b), 4.44 (d,
1 H, J = 7.5 Hz, H-1), 4.51 (d, 1 H, J = 5.5 Hz, H-a), 4.94
(dd, 1 H, J = 7.5, 10.5 Hz, H-2), 4.98 (dd, 1 H, J = 3.5, 10.5
Hz, H-3), 5.06, 5.09 (d, each 1 H, AB type, J = 12.5 Hz,
CH2Ph), 5.11 (dd, 1 H, J = 1.0, 3.5 Hz, H-4), 7.30 (m, 5 H,
Ph), 7.56 (s, 1 H, guanine H-8). FAB MS: Calcd for
C32H40N6O13: 716.3. Found: 717.4.
corresponding oxazolidones treating with ethyl
chlorocarbonate in 1 M aq NaOH. See also:Saeed, A.; Yong,
D. W. Tetrahedron 1992, 48, 2507. (b) Kaneko, T.; Inui, T.
Bull. Chem. Soc. Jpn. 1961, 82, 1075.
(9) Udodong, U. E.; Rao, C. S.; Fraser-Reid, B. Tetrahedron
Lett. 1992, 48, 4713.
(17) Compound 2a: 1H NMR (D2O): d = 1.01 (d, 3 H, J = 6.5 Hz,
Me-6), 1.73 (m, 2 H), 2.24 (m, 2 H), 3.34 (m, 1 H), 3.54 (m,
1 H), 3.59 (dd, 1 H, J = 4.0, 10.0 Hz, H-2), 3.62 (br d, 1 H,
J = 3.5 Hz, H-4), 3.70 (dd, 1 H, J = 3.5, 10.0 Hz, H-3), 3.88
(br q, 1 H, J = 6.5 Hz, H-5), 3.91 (dd, 1 H, J = 8.0, 14.5 Hz,
H-g), 4.04 (dd, 1 H, J = 6.0, 14.5 Hz, H-g¢), 4.18 (d, 1 H,
J = 3.0 Hz, H-a), 4.37 (m, 1 H, H-b), 4.71 (d, 1 H, J = 4.0
Hz, H-1), 7.63 (s, 1 H, guanine H-8). MALDI-TOF MS:
Calcd for C19H28N6O10 + Na+: 523.2. Found: 523.1.
(18) Compound 2b: 1H NMR (D2O): d = 1.01 (d, 1 H, J = 6.5 Hz,
Me-6), 1.72 (m, 2 H), 2.23 (m, 2 H), 3.24 (dd, 1 H, J = 7.5,
10.0 Hz, H-2), 3.38 (dd, 1 H, J = 3.5, 10.0 Hz, H-3), 3.46 (dt,
1 H, J = 10.5, 6.5 Hz, A part of AB type), 3.48–3.55 (m, 2 H,
H-4 and H-5), 3.72 (dt, 1 H, J = 10.5, 6.5 Hz, B part of AB
type), 3.95–4.12 (m, 3 H, H-b and H2-g), 4.13 (d, 1 H, J = 7.5
Hz, H-1), 4.19 (d, 1 H, J = 5.5 Hz, H-a), 7.62 (s, 1 H,
guanine H-8). FAB MS: Calcd for C19H28N6O10 + Na+:
523.2. Found: 523.2.
(10) Compound 13a: 1H NMR (CDCl3): d = 1.14 (d, 3 H, J = 6.5
Hz, Me-6), 1.69 (m, 2 H), 2.00, 2.08, 2.17 (s, each 3 H, 3 ×
Ac), 2.13 (m, 2 H), 3.41 (dt, 1 H, J = 10.0, 6.5 Hz, A part of
AB type), 3.69 (dt, 1 H, J = 10.0 Hz, B part of AB type), 4.16
(br q, 1 H, J = 6.5 Hz, H-5), 4.95–5.02 (m, 2 H), 5.05 (d, 1
H, J = 4.0 Hz, H-1), 5.11 (dd, 1 H, J = 4.0, 10.5 Hz, H-2),
5.30 (dd, 1 H, J = 1.0, 3.5 Hz, H-4), 5.35 (dd, 1 H, J = 3.5,
10.5 Hz, H-3), 5.81 (ddt, 1 H, J = 10.5, 17.0, 7.5 Hz). 13
NMR (CDCl3): d = 15.8, 20.6 × 2, 20.7, 28.4, 30.0, 64.2,
67.5, 68.0, 68.2, 71.1, 96.0, 115.0, 137.7, 170.0, 170.4,
170.6.
C
(11) Compound 13b: 1H NMR (CDCl3): d = 1.23 (d, 3 H, J = 6.5
Hz, Me-6), 1.70 (m, 2 H), 2.00, 2.06, 2.18 (s, each 3 H, 3 ×
Ac), 2.11 (m, 2 H), 3.49 (m, 1 H), 3.81 (br q, 1 H, J = 6.5 Hz,
H-5), 3.92 (dt, 1 H, J = 9.5, 6.0 Hz), 4.43 (d, 1 H, J = 7.5, H-
1), 4.95–5.05 (m, 2 H), 5.02 (dd, 1 H, J = 3.0, 10.5 Hz, H-3),
5.20 (dd, 1 H, J = 7.0, 10.5 Hz, H-2), 5.24 (br d, 1 H, J = 3.0
Hz, H-4), 5.80 (ddt, 1 H, J = 10.0, 17.0, 6.5 Hz).
(19) Nishihara, S.; Nakazato, M.; Kudo, T.; Kimura, H.; Ando,
T.; Narimatsu, H. Biochem. Biophys. Res. Commun. 1993,
190, 42.
(12) Ichikawa, Y.; Sim, M. M.; Wong, C. H. J. Org. Chem. 1992,
57, 2943.
(13) Iversen, T.; Bundle, D. R. J. Org. Chem. 1981, 46, 5389.
(14) Carlsen, P. H. J.; Katzuki, T.; Martin, V. S.; Sharpless, K. B.
J. Org. Chem. 1981, 46, 3936.
(20) The assay was performed in 50 mM cacodylate buffer (pH
6.8) containing 5 mM ATP, 10 mM L-Fuc, 25 mM MnCl2,
15 mM acceptor substrate, Galb1-3GlcNAcb1-3Galb1-
4Glc-2-aminobenzamide (for FUT 3) or Galb1-4GlcNAcb1-
3Galb1-4Glc-2-aminobenzamide (for FUT 6), 75 mM donor
substrate GDP-Fuc, and 2a or 2b (0 mM for the positive
control; 0.75 mM, and 7.5 mM respectively for the
inhibitory assay). After incubation at 37 °C for 2 h in the
presence of the fucosyltransferases (FUT 3 or FUT 6), the
enzyme reaction was terminated by heating at 97 °C for 5
min followed by adding H2O. After centrifugation of the
reaction mixture, in order to detect the fucosylated products
and estimate their amounts, each supernatant was filtered
and subjected to reverse-phase HPLC analysis on TSK-gel
ODS-80Ts QA column (4.6 × 250 mm; Tosoh, Tokyo,
Japan) and eluted with 20 mM ammonium acetate buffer (pH
4.0) containing 7% MeOH at flow rate of 1.0 mL/min at
50 °C, with monitoring by a fluorescence spectrophotometer
(JASCO FP-920; Nihon Bunkoh, Tokyo, Japan).
(15) Compound 20a: 1H NMR (CD3OD): d = 0.99 (d, 3 H, J = 6.5
Hz, Me-6), 1.80 (m, 2 H), 1.85, 1.92, 2.04 (s, each 3 H, 3 ×
Ac), 2.30 (t, 2 H, J = 7.5 Hz), 3.33 (dt, 1 H, J = 10.0, 6.0 Hz),
3.60 (dt, 1 H, J = 10.0, 6.0 Hz), 4.01 (dd, 1 H, J = 9.0, 15.0
Hz, H-g), 4.07 (br q, 1 H, J = 6.5 Hz, H-5), 4.14 (dd, 1 H,
J = 3.0, 15.0 Hz, H-g¢), 4.16 (m, 1 H, H-b), 4.53 (d, 1 H,
J = 5.5 Hz, H-a), 4.88 (d, 1 H, J = 3.5 Hz, H-1), 4.94 (dd, 1
H, J = 3.5, 11.0 Hz, H-2 or H-3), 5.05, 5.08 (d, each 1 H, AB
type, J = 13.0 Hz, CH2Ph), 5.15 (dd, 1 H, J = 1.0, 3.5 Hz, H-
4), 5.20 (dd, 1 H, J = 3.5, 11.0 Hz, H-2 or H-3), 7.25 (m, 5
H, Ph), 7.53 (s, 1 H, guanine H-8). FAB MS: Calcd for
C32H40N6O13: 716.3. Found: 717.4.
Synlett 2004, No. 2, 243–246 © Thieme Stuttgart · New York