Preparation of glycosylated amino acid derivatives for glycoprotein synthesis by in vitro translation system

Article abstract of DOI:10.1016/S0968-0896(01)00304-2

General preparation of glycosylated amino acylated nucleotide for in vitro peptide synthesis was described. Both O-glycosylated amino acyl nucleotides and C-glycosylated amino acyl nucleotide were synthesized by choosing the appropriate protecting group.

Full text of DOI:10.1016/S0968-0896(01)00304-2

Bioorganic & Medicinal Chemistry10 (2002) 573–581
Preparation of Glycosylated Amino Acid Derivatives for
Glycoprotein Synthesis by In Vitro Translation System
Shino Manabe,a Kimitoshi Sakamoto,a Yoshiaki Nakahara,a,b Masahiko Sisido,c
Takahiro Hohsakac and Yukishige Itoa,*
aRIKEN (The Institute of Physical and Chemical Research) and CREST, Japan Science and Technology Corporation (JST),
2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
bDepartment of Applied Biochemistry, Tokai University, Kitakaname 1117, Hiratsuka-shi, Kanagawa 259-1292, Japan
cDepartment of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka,
Okayama 700-8350, Japan
Received 3 July2001; accepted 23 August 2001
Abstract—General preparation of glycosylated amino acylated nucleotide for in vitro peptide synthesis was described. Both
O-glycosylated amino acyl nucleotides and C-glycosylated amino acyl nucleotide were synthesized by choosing the appropriate
protecting group. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
An alternative in vitro translation system which utilizes
four-base codons, CGGG and AGGU, has been devel-
oped.5,6 Since these quadruplets has been derived from
the minor codons of arginine (CGG and AGG), com-
petition between four-base and three-base should be in
favor of the former. With a set of orthogonal four-base
codons, incorporation of two different nonnatural amino
acids was demonstrated to be possible. Considering the
success of incorporating various types of modified
amino acids into proteins, exploration of the possibility
to produce glycoproteins with four-base codon approach
seems to be highlypromising (Fig. 1).
Glycosylation is one of the most prominent post-transla-
tional modifications that controls protein conformation,
functions, stability, and intra/intercellular trafficking. Due
to increased understanding of the biological functions of
glycan chains as the recognition signals in various bio-
logical events, synthetic effort toward glycoprotein has
become a major issue in organic synthesis and chemical
biology.1 Thanks to the recent advances in peptide as
well as oligosaccharide synthesis technology, chemical
synthesis of relatively complex glycopeptide is now a
quite feasible task.2 However, it is still considered to be
a daunting challenge to produce intact glycoproteins by
a purelychemical means (Schemes 1–4).
Prerequisite to the incorporation of a tailored amino
acid residue (Xaa) byin vitro protein synthesis is the
preparation of tRNA amino acylated at the terminal A
residue (tRNA-Xaa). It has been demonstrated that
tRNA-Xaa can be prepared byenzymatic ligation of
tRNA lacking terminal pCpA [tRNA(–CA)] with amino
acylated dinucleotide pCpA-Xaa7 or pdCpA-Xaa.8 As
an active ester, cyanometyl ester has proved to be the most
effective in terms of the ease of preparation and chemose-
In vitro protein synthesis has proved to be a powerful
method to incorporate non-natural or modified amino
acids in a site-specific manner.3 For such purpose, the
use of amino acylated suppressor tRNA, which recog-
nizes amber nonsense codon, has been explored. Based
on this approach, Hecht et al. successfullyincorporated
glucosylated serine into firefly luciferase,4 albeit in
modest incorporation efficiency.
9
lective reactivitytoward ribose hydroxygroups.
We report here the preparation of three types of glyco-
sylated amino acid cyanomethyl esters 1, 2, and 3,
which were designed as keyintermediates for the in vitro
production of glycoproteins having novel carbohydrate
*Corresponding author. Tel.: +81-48-467-9430; fax: +81-48-432-4680;
e-mail: yukito@postman.riken.go.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00304-2

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S. Manabe et al. / Bioorg. Med. Chem. 10 (2002) 573–581
Scheme 1. Reagent and conditions. (i) TMSOTf, 1,2-dichloroethane,
50 ꢀC, 10 h, 64%; (ii) H2, 10% Pd/C, EtOAc, Boc2O, rt, 3 h, 91%; (iii)
NaOMe, MeOH, rt, 3 h, TBSCl, imidazole, DMF, then TESCl, imi-
dazole, DMF, 66%; (iv) LiOH, MeOH, H2O, then bromoacetonitrile,
i-Pr2NEt, CH3CN, rt, overnight, 41%.
Figure 1.
Scheme 2. Reagent and conditions: (i) AgOTf, CH2Cl2,ꢁ10 ꢀC, over-
night, 56%; (ii) 10% Pd/C, Boc2O MeOH, rt, overnight, 92%; (iii)
NaOMe, MeOH, then TESCl, pyridine, rt, overnight, 66%; (iv) LiOH,
H2O, MeOH, rt, 3 h, 84%; (v) bromoacetonitrile, i-Pr2NEt, CH3CN,
rt, overnight, 84%.
structures whose biological functions are attracting
recent attention.10
Results and Discussion
Adequate choice of protecting group for hydroxy
groups of sugar part and amino group of amino acid is
crucial for preparation of glycosylated aminoacyl
pdCpA because of the sensitivityof the ester linkage
between pdCpA and amino acid in the presence of
nucleophile.11 While Hecht et al. adopted the allyloxy-
carbonyl group for hydroxy protection, we focused our
attention on the use of acid labile groups. In this context,
the ester linkage was shown to be tolerant under N-Boc
(tert-butoxycarbonyl) deprotection conditions (TFA).12
It was then presumed that either the silyl or acetonide
group can be adopted for the protection of carbo-
hydrate components. Simultaneous removal of N- and
O-protecting groups should then be possible without
deteriorating the ester linkage.
As the glycosylated amino acid residue to be incorporated
byin vitro system, serine linked N-acetyl-d-glucosamine
(O-GlcNAc-Ser), serine linked d-mannose (O-Man-Ser),
and C-linked d-mannosyl tryptophan (C-Man-Trp)
were selected because of their biological significance.
Scheme 3. Reagent and conditions: (i) 2,2-dimethoxypropane, PPTS,
DMF, rt, 2 day s, 74%; (ii) 10% NaOH aq, EtOH, rt-reflux, overnight; (iii)
bromoacetonitrile, i-Pr2NEt, CH3CN, rt, overnight, 80% (two steps).

S. Manabe et al. / Bioorg. Med. Chem. 10 (2002) 573–581
575
the acetyl group was removed. The hydroxy groups
were protected as TBS and TES group in order that the
deprotection at the final stage can be performed under
acidic conditions. After hydrolysis of methyl ester under
alkaline conditions, the acid was converted to cyano-
methyl ester 1. Similarly, a-O-linked d-mannosyl serine
cyanomethyl ester 2 was prepared from bromide 7 in
five steps. b-Elimination during the removal of acetate
could be minimized (< 3%) bycontrolling the NaOMe
concentration (0.03%).
Mannosyl tryptophan cyanomethyl ester 3 was synthe-
sized as below. Tetraol 1015 was protected as its corre-
sponding diacetonide by2,2-dimethoxypropane in the
presence of PPTS without affecting the Boc group. After
hydrolysis of methyl ester and sulfone amide at indole
nitrogen, carboxylic acid was changed to cyanomethyl
ester 3 in 80% yield.
Subsequently, condensation of cyanomethyl ester 1, 2, 3
and nucleotide was investigated. The condensation of 1
with AMP tetrabutylammonium salt 11 as a model of
pdCpA was performed in 44% yield at 0 ꢀC to reduce
the amount of b-elimination of glycosylated amino ester
under basic conditions. Similarly, condensation of 2 and
11 was performed at 0 ꢀC in DMF to afford 13a in 50%
yield. When 3 was used as the glycosylated amino acid
source, the reaction proceeded relativelyslowlyand
required a high temperature (60 ꢀC), probablybecause
of steric hindrance of the acetonide. Deprotection of
silyl group and Boc group of 12a and 13a were per-
formed in TFA at the same time in a short period and
12b and 13b were obtained. Because TFA treatment of
14a caused decomposition of mannosyl tryptophan
moiety, acidic hydrolysis of acetonide and Boc was per-
formed in 4 M aq HCl in dioxane to afford 14b.
Conclusion
Scheme 4. Reagent and conditions: (i) DMF, 0 ꢀC, 15a, 44%, 16a,
50%, 17a, 53%; (ii) TFA for 15b, 42%, and 16b, quant for 17b, 4 M
HCl dioxane, H2O, rt, 65%.
In conclusion, glycosylated amino acid derivatives for in
vitro synthesis of glycoproteins were synthesized.
Although incorporation of a glucose residue into firefly
luciferase was demonstrated successfullybyHecht et al.,
the generalityof in vitro production of glycoproteins is
not clear. Since synthetic routes are now established for
O-linked-b-d- (1), O-linked-a-d- (2) and C-linked-a-d-
(3) glycosylated amino acids, comparative studies on
incorporation efficiencies of these extremelydifferent
structures would reveal the scope of the in vitro system
for the synthesis of biologically relevant glycoproteins.
For instance, O-Linked N-acetyl-d-glucosamine is
found in a varietyof nuclear and cytoplasmic proteins
and functions in signal transduction.13 O-Mannosyl
linkage in a-dystroglycan may play a significant role in
binding to laminin, which influences development and
regeneration of the nervous system.14 C-linked mannosyl
tryptophan is a naturally occurring novel subclass of
glycoprotein, which is synthesized by our group for the
first time.15 The biological role of mannosylated trypto-
phan is not clear at the present time, but recent study
Experimental
suggests that C-glycosylation is not a rare post-trans-
16
lational modification as previouslythought. Not
onlyfrom human, C-Mannosylated tryptophan is also
General procedures
obtained from marine ascidians.17
1H NMR spectra were taken byJEOL EX-400 as solu-
tions in CDCl3 otherwise mentioned. Chemical shifts
are expressed in ppm downfield from the signal for
Me4Si. Optical rotations were measured byJasco DIP-310
at ambient temperature. Melting points were uncorrected.
Kanto silica gel (spherical, neutral, 100–210 mm) were
Oxazoline 4 was glycosylated in the presence of
TMSOTf with serine derivative 5, which was synthe-
sized from serine methyl ester by the action of TfN3.18
After transformation of azide group to Boc in one pot,

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S. Manabe et al. / Bioorg. Med. Chem. 10 (2002) 573–581
used for column chromatography. Merck TLC (silica gel
60F254) was used for TLC analysis. Molecular sieves
were activated byheating to 180 ꢀC just before use.
off through Celite and the solvent was evaporated. The
residue was purified bysilica gel column chromato-
graphy(CHCl /MeOH=98:2) to afford Boc derivative
3
(11.1 g, 91%) as an amorphous: [a]2D5 ꢁ3.5 (c 1.1,
CHCl3); 13C NMR (100 MHz, CDCl3) d 170.6 (C),
170.4 (C), 170.3 (C), 170.1(C), 169.1 (C), 155.2 (C),
100.9 (CH), 80.1 (C), 72.1 (CH), 71.9 (CH), 69.2 (CH2),
68.4 (CH), 62.0 (CH2), 54.5 (CH), 53.9 (CH), 52.7
(CH3), 28.4 (CH3), 23.4 (CH3), 20.8 (CH3), 20.8 (CH3),
Methyl (2S)-2-azide-3-hydroxypropanoate (5). To a
mixture of NaN3 (66.7 g, 1.0 mol) in H2O (160 mL) and
CH2Cl2 (240 mL), Tf2O (66.7 g, 0.23 mol) was added
with vigorous stirring at 0 ꢀC. The mixture was stirred
for 4 h at 0 ꢀC. The organic layer was separated, and the
aqueous layer was extracted with CH2Cl2 (70 mL ꢂ 2).
The combined organic layers were washed with satd
NaHCO3 (120 mL) and H2O (120 mL). The organic
layer was dried over Na2SO4. To a solution of l-serine
1
20.7 (CH3); H NMR (400 MHz, CDCl3) d 5.74 (d, 1H,
J=8.1 Hz), 5.43 (d, 1H, J=7.6 Hz), 5.27 (dd, 1H, J=9.3,
10.5 Hz), 5.05 (t, 1H, J=9.6 Hz), 4.71 (d, 1H, J=8.3 Hz),
4.41 (m, 1H), 4.30–4.18 (m, 2H), 4.12 (dd, 1H, J=2.3,
12.3 Hz), 3.88–3.80 (m, 2H), 3.76 (s, 3H), 3.65 (m, 1H),
2.10 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H), 1.46
(s, 9H). Anal. calcd for C23H36N2O13: C, 50.36; H, 6.62;
N, 5.11. Found: C, 50.23; H, 6.61; N, 5.13.
.
methyl ester HCl salt (25.8 g, 186 mmol) and 4-(dime-
thylamino)pyridine (112 g, 0.92 mol) in MeCN (840
mL), TfN3 solution (350 mL, as prepared above) was
added at 0 ꢀC, and stirred at 0 ꢀC for 2 h. The mixture was
diluted with 1,2-dichloroethane (500 mL) and con-
centrated at 30 ꢀC in vacuo to ca. 300 mL, and filtered.
The mother liquid was subjected to silica gel column
chromatography(hexane/EtOAc=6:4) to afford 5 (15.0
g, 62%) as a pale yellow oil: [a]2D5ꢁ92.2 (c 1.0, CHCl3);
13C NMR (100 MHz, CDCl3) d 169.0 (C), 63.4 (CH),
(N-tert-Butoxycarbonyl)-3-O-[2-acetamido-2-deoxy-3,4-
bis-O-triethylsilyl-6-(tert-butyldimethylsilyl)-ꢀ-D-gluco-
pyranosyl]-S-serine methyl ester. To a solution of acetate
(8.2 g, 14.9 mmol) in dryMeOH (150 mL) was added
MeONa (0.1% wt MeOH solution, 30 mL) at room
temperature. The mixture was stirred for 3 h at room
temperature. The mixture was neutralized with Amberlyst
15-E. The resins were filtered off and the solvent was
evaporated. The material was pumped to dryness. The
residue was dissolved in DMF (36 mL) and imidazole
(6.1 g, 90 mmol) was added. To the solution was added
t-butyldimethylsilyl chloride (2.0 g, 13.0 mmol) at room
temperature. After the mixture was stirred for 2 h, trie-
thylsilyl chloride (4.6 mL, 27.5 mmol) was added at 0 ꢀC
and the mixture was stirred for 12 h at room tempera-
ture. To the solution was added MeOH (4 mL) and
stirred for 30 min to destroythe excess of silyl chloride.
The reaction mixture was diluted with EtOAc, and H2O
was added to the solution. The aqueous layer was
extracted with EtOAc. The combined organic layers
were washed with aq 5% citric acid, satd. NaHCO3 aq,
and brine. The organic layer was dried over Na2SO4.
The solvent was evaporated and the residue was purified
bysilica gel column chromatography(10–30% EtOAc
in hexane) to afford silyl ether (7.5 g, 66%, 2 steps) as a
colorless solid: mp 108–109 ꢀC; [a]D25 ꢁ9.1 (c 1.0, ben-
zene); 13C NMR (100 MHz, C6D6) d 170.8 (C), 168.9
(C), 155.6 (C), 100.8 (CH), 79.5 (C), 78.4 (CH), 73.4
(CH), 70.8 (CH), 69.6 (CH2), 63.6 (CH2), 55.0 (CH),
54.7 (CH), 52.1 (CH3), 28.7 (CH3), 26.4 (CH3), 23.7
(CH3), 18.8 (C), 7.6 (CH3), 7.5 (CH3), 5.6 (CH2),ꢁ4.5
1
62.7 (CH2), 52.9 (CH3); H NMR (400 MHz, CDCl3) d
4.11 (dd, 1H, J=4.4, 5.4 Hz), 3.94 (d, 1H, J=4.4 Hz),
3.94 (d, 1H, J=5.4 Hz), 3.85 (s, 3H), 2.11 (brs, 1H);
HRMS (FAB), m/z 146.0567 (M+H)+, (C4H8N3O3
requires 146.0566).
3-O-(2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-ꢀ-D-gluco-
pyranosyl)-(2S)-2-azide-3-hydroxypropionic acid methyl
ester (6). To a solution of oxazoline 4 (15.2 g, 46.2
mmol), azide 5 (13.4 g, 92.4 mmol) and 1,1,3,3-tetra-
methylurea (5.5 mL, 46.2 mmol) in 1,2-dichloroethane
(100 mL) was added TMSOTf (8.8 mL, 48.5 mmol)
dropwise at 0 ꢀC. After addition, the mixture was stirred
at 50 ꢀC for 10 h under N2 atmosphere. After the reac-
tion mixture was cooled to 0 ꢀC, MeOH (10 mL) and
Et3N (6.8 mL, 48.5 mmol) were added. The solvent was
evaporated, and the residue was purified bysilica gel
column chromatography(hexane/AcOEt/MeOH=8:9:0.7)
to afford 6 (14.1 g, 64%) as a colorless solid: mp 135–
137 ꢀC; [a]D25 ꢁ22.4 (c 1.0, CHCl3); 13C NMR (100 MHz,
CDCl3) d 170.3 (C), 170.3 (C), 169.0 (C), 168.1(C),
100.1 (CH), 72.0 (CH), 71.7 (CH), 68.6 (CH), 68.5
(CH2), 62.0 (CH2), 61.3 (CH), 54.3 (CH), 52.8 (CH3),
23.2 (CH3), 20.7 (CH3), 20.6 (CH3), 20.6 (CH3); 1H
NMR (400 MHz, CDCl3) d 5.52 (d, 1H, J=8.8 Hz),
5.32 (dd, 1H, J=9.3, 10.5 Hz), 5.07 (t, 1H, J=9.3 Hz),
4.83 (d, 1H, J=8.3 Hz), 4.24 (m, 1H), 4.14 (dd, 1H,
J=2.4, 12.2 Hz), 4.04 (t, 1H, J=4.0 Hz), 3.90 (dd, 1H,
J=3.7, 11.0 Hz), 3.82 (s, 3H), 3.80–3.75 (m, 1H), 3.71
(m, 1H), 2.09 (s, 3H), 2.03 (s, 3H), 2.03 (s, 3H), 1.98 (s,
3H). Anal. calcd for C18H26N4O11: C, 45.57; H, 5.52; N,
11.81. Found: C, 45.33; H, 5.45; N, 11.60.
1
(CH3),ꢁ4.9 (CH3); H NMR (400 MHz, CDCl3) d 6.63
(d, 1H, J=9.0 Hz), 5.41 (d, 1H, J=8.3 Hz), 4.61 (d, 1H,
J=2.4 Hz), 4.40 (m, 1H), 4.20 (dd, 1H, J=3.8, 9.9 Hz),
4.07–3.95 (m, 2H), 3.88 (m, 1H), 3.71 (s, 3H), 3.75–3.67
(m, 3H), 3.62 (dd, 1H, J=3.9, 10.0 Hz), 1.96 (s, 3H),
1.45 (s, 9H), 1.01–0.94 (m, 18H), 0.90 (s, 9H), 0.69–0.59
(m, 12H), 0.08 (s, 3H), 0.07 (s, 3H). Anal. calcd for
C35H72N2O10Si3: C, 54.95; H, 9.48; N, 3.66. Found: C,
55.00; H, 9.59; N, 3.74.
N-(tert-Butoxycarbonyl)-3-O-(2-acetamido-2-deoxy-3,4,6-
tri- O-acetyl-ꢀ-D-glucopyranosyl)-S-serine methyl ester.
To a solution of azide 6 (10.6 g, 22.3 mmol) and di-
tert-butyl dicarbonate (5.9 g, 27 mmol) in EtOAc (200
mL) was added a suspension of 10% Pd/C (1.0 g) in
EtOAc (20 mL). The mixture was stirred vigorously
under H2 atmosphere for 3 h. The catalyst was filtered
(N-tert-Butoxycarbonyl)-3-O-[2-acetamido-2-deoxy-3,4-
bis-O-triethylsilyl-6-(tert-butyldimethylsilyl)-ꢀ-D-gluco-
pyranosyl]-S-serine cyanomethyl ester (1). The methyl
ester (647 mg, 846 mmol) was dissolved in THF (8.4 mL)

S. Manabe et al. / Bioorg. Med. Chem. 10 (2002) 573–581
577
and the solution was cooled to 0 ꢀC. To the solution was
added ice cooled solution of LiOH (24.3 mg, 1.02 mmol)
in water (2.5 mL). The mixture was stirred at 0 ꢀC for 90
min. The reaction was quenched with aq 10% citric
acid, and aqueous layer was extracted with EtOAc three
times, then the combined organic layers were washed
with brine and dried over Na2SO4. The solvent was
evaporated, and the residue was purified bysilica gel
N-(tert-Butoxycarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-ꢁ-D-
mannopyranosyl)-S-serine methyl ester. To a solution of
9 (6.55 g, 11.22 mmol) and di-tert-butyl dicarbonate
(3.19 g, 14.62 mmol) in MeOH (150 mL) was added
10% Pd/C (800 mg). The mixture was stirred vigorously
under H2 atmosphere for 1 h. The mixture was diluted
with EtOAc (200 mL), and the catalyst was filtered off
through Celite and the catalyst was washed with EtOAc.
After the solvent was evaporated, the residue was purified
bysilica gel column chromatography(hexane/EtOAc=
6:4) to afford Boc derivative (5.65 g, 92%) as an amor-
phous: [a]2D5 48.3 (c 1.0, CHCl3); 13C NMR (100 MHz,
CDCl3) d 170.3 (C), 170.1 (C), 169.6 (C), 169.5 (C),
169.3 (C), 155.0 (C), 98.2 (CH), 80.2 (C), 69.3 (CH),
69.2 (CH2), 68.9 (CH), 68.8 (CH), 65.9 (CH), 62.2
(CH2), 53.8 (CH), 52.8 (CH3), 28.4 (CH3), 20.9 (CH3),
20.8 (CH3), 20.8 (CH3), 20.7 (CH3); 1H NMR
(400 MHz, CDCl3) d 5.47 (d, 1H, J=7.8 Hz), 5.31–5.25
(m, 2H), 5.18 (dd, 1H, J=1.7, 2.9 Hz), 4.80 (d, 1H,
J=1.5 Hz), 4.50 (m, 1H), 4.28 (dd, 1H, J=5.2, 12.3 Hz),
4.12 (dd, 1H, J=2.3, 12.3 Hz), 4.00–3.92 (m, 3H), 3.80
(s, 3H), 2.15 (s, 3H), 2.12 (s, 3H), 2.05 (s, 3H), 1.99 (s,
3H), 1.47 (s, 9H). Anal. calcd for C23H35NO14: C, 50.27;
H, 6.42; N, 2.55. Found: C, 50.33; H, 6.47; N, 2.48.
column chromatography(0–4% MeOH in CHCl ) to
3
afford carboxylic acid (311 mg, 414 mmol). The obtained
acid (152 mg, 202 mmol) and i-Pr2NEt (37 mL, 212
mmol) were dissolved in MeCN (1.0 mL), and bromo-
acetonitrile (28 mL, 404 mmol) was added at room tem-
perature. After stirring for 9 h, the solvent was
evaporated. To the residue was added water and aqueous
layer was extracted with EtOAc three times, then the
combined organic layers were washed with brine and
dried over Na2SO4. The solvent was evaporated, and
the residue was purified bysilica gel column chromato-
graphy(hexane/EtOAc=7:3) to afford caynomethly
ester 1 (135 mg, 41%, two steps) as a colorless solid: mp
116–118 ꢀC; [a]2D5 ꢁ18.9 (c 1.0, benzene); 13C NMR
(100 MHz, C6D6) d 169.4 (C), 169.2 (C), 155.6 (C), 114.4
(C), 100.8 (CH), 80.0 (C), 78.6 (CH), 73.9 (CH), 71.0
(CH), 68.7 (CH2), 63.6 (CH2), 55.4 (CH), 54.6 (CH),
48.9 (CH2), 28.6 (CH3), 26.4 (CH3), 23.7 (CH3), 18.9
(C), 7.5 (CH3), 7.5 (CH3), 5.7 (CH2), 5.7 (CH2),ꢁ4.5
N-(tert-Butoxycarbonyl)-3-O-(2,3,4,6-tetrakis-O-triethyl-
silyl-ꢁ-D-mannopyranosyl)-S-serine methyl ester. To a
solution of acetate (1.15 g, 2.09 mmol) in dryMeOH (30
mL) was added MeONa (0.1% wt MeOH solution, 2
mL) at room temperature in the presence of phe-
nolphthalein as an indicator. The mixture was stirred
for 2 h and then neutralized with Amberlyst 15E. The
resins were filtered and the solvent was evaporated. The
residue was dissolved in pyridine (30 mL) and chloro-
triethylsilane (2.5 mL, 14.63 mmol) was added at 0 ꢀC
and the mixture was stirred for 8 h at room tempera-
ture. To the solution was added MeOH (3 mL) and
stirred for 30 min to destroythe excess of silyl chloride,
and the solvent was evaporated. The residue was dis-
solved into EtOAc and satd NH4Cl. The aqueous layer
was extracted with EtOAc, and the combined organic
layers were washed with brine. The organic layer
was dried over Na2SO4. The solvent was evaporated
and the residue was purified bysilica gel column
chromatography(hexane/EtOAc=9:1) to afford silly
ether (1.48 g, 84%, 2 steps) as a colorless oil: [a]2D5 33.9
(c 1.0, benzene); 13C NMR (100 MHz, C6D6, 70 ꢀC) d
170.7 (C), 155.3 (C), 101.6 (CH), 79.6 (C), 77.3 (CH),
75.5 (CH), 73.7 (CH), 70.1 (CH), 68.4 (CH2), 63.1
(CH2), 55.0 (CH), 51.8 (CH3), 28.7 (CH3), 7.6 (CH3),
7.5 (CH3), 7.3 (CH3), 7.3(CH3), 6.2 (CH2), 6.1 (CH2),
5.9 (CH2), 5.5 (CH2); 1H NMR (400 MHz, C6D6, 70 ꢀC)
d 5.44 (brs, 1H), 4.71 (d, 1H, J=3.2 Hz), 4.50 (brs, 1H),
4.21 (t, 1H, J=7.2 Hz), 4.03–3.91 (m, 5H), 3.81 (m, 1H),
3.61 (brs, 1H), 3.38 (s, 3H), 1.44 (s, 9H), 1.13–1.02 (m,
36H), 0.83–0.68 (m, 24H). Anal. calcd for
C39H83NO10Si4: C, 55.87; H, 9.98; N, 1.67. Found: C,
55.79; H, 10.11; N, 1.76.
1
(CH3),ꢁ4.8 (CH3); H NMR (400 MHz, CDCl3) d 6.54
(d, 1H, J=9.3 Hz), 5.38 (d, 1H, J=8.3 Hz), 4.78 (s,
2H), 4.63 (d, 1H, J=3.2 Hz), 4.48 (m, 1H), 4.19 (dd,
1H, J=4.6, 9.8 Hz), 3.94 (m, 2H), 3.85–3.79 (m, 2H),
3.74–3.65 (m, 3H), 1.96 (s, 3H), 1.46 (s, 9H), 1.01–0.94
(m, 18H), 0.91 (s, 9H), 0.69–0.59 (m, 12H), 0.08 (d, 6H,
J=4.6 Hz). Anal. calcd for C36H71N3O10Si3: C, 54.72;
H, 9.06; N, 5.32. Found: C, 54.77; H, 9.18; N, 5.23.
N-(Benzyloxycarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-ꢁ-D-
mannopyranosyl)-S-serine methyl ester (9). To a suspen-
sion of l-Z-Ser-OMe 8 (7.76 g, 30.64 mmol), AgOTf
(10.34 g, 40.24 mmol) and activated molecular sieves 4A
(12.0 g) in CH2Cl2 (100 mL) was added a solution of
bromide 7 (16.47 g, 40.05 mmol) in CH2Cl2 (30 mL) at
ꢁ20 ꢀC under Ar. The mixture was stirred at ꢁ10 ꢀC
overnight. The reaction mixture was quenched with
triethylamine (8 mL). The mixture was filtered through
Celite, and the filter cake was washed with CH2Cl2 (30
mL ꢂ 2). After removal of the solvent, the residue was
directlysubjected to silica gel column chromatography
(CHCl3/EtOAc 4:1–1:1) to afford 9 (9.99 g, 56%) as an
amorphous: [a]2D5 48.2 (c 1.1, CHCl3); 13C NMR
(100 MHz, CDCl3) d 170.2 (C), 169.6 (C), 169.5 (C),
169.4 (C), 169.3 (C), 155.5 (C), 135.8 (C), 128.3 (CH),
128.0 (CH), 98.1 (CH), 69.2 (CH2), 69.1 (CH), 69.0
(CH), 68.6 (CH), 67.1 (CH2), 65.8 (CH), 62.2 (CH2),
54.2 (CH), 52.8 (CH3), 20.8 (CH3), 20.7 (CH3), 20.6
(CH3); 1H NMR (400 MHz, CDCl3) d 7.40–7.28 (m,
5H), 5.77 (d, 1H, J=7.8 Hz), 5.26–5.22 (m, 2H), 5.18
(m, 1H), 5.14 (s, 2H), 4.79 (d, 1H, J=1.5 Hz), 4.57 (m,
1H), 4.23 (dd, 1H, J=5.6, 12.2 Hz), 4.09 (dd, 1H,
J=2.2, 12.2 Hz), 4.04–3.92 (m, 3H), 3.81 (s, 3H), 2.15
(s, 3H), 2.08 (s, 3H), 2.03 (s, 3H), 1.99 (s, 3H). Anal.
calcd for C26H33NO14: C, 53.51; H, 5.70; N, 2.4. Found:
C, 53.65; H, 5.80; N, 2.23.
N-(tert-Butoxycarbonyl)-3-O-(2,3,4,6-tetrakis-O-triethyl-
silyl-ꢁ-D-mannopyranosyl)-S-serine. To a solution of
methyl ester (2.77 g, 3.31 mmol) in THF (50 mL) was
added a solution of LiOH (95 mg, 3.97 mmol) in H2O

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S. Manabe et al. / Bioorg. Med. Chem. 10 (2002) 573–581
(10 mL) at room temperature. The mixture was stirred
for 3 h, and diluted with CH2Cl2. The reaction was
quenched with aq 10% citric acid, and the aq layer was
extracted with CH2Cl2. Then the combined organic lay-
ers were washed with brine and dried over Na2SO4. The
solvent was evaporated, and the residue was purified by
silica gel column chromatography(hexane/ EtOAc 7:1–
1:1) to afford the acid (2.29 g, 84%). 13C NMR (DMSO-
d6, 100 ꢀC) d 170.5 (C), 156.9 (C), 99.6 (CH), 77.8 (C),
75.6 (CH), 73.7 (CH), 72.0 (CH), 66.7 (CH2), 61.5
(CH2), 60.6 (CH), 53.8 (CH), 27.6 (CH3), 6.1 (CH3), 6.0
(CH3), 6.0 (CH3), 5.9 (CH3), 5.9 (CH3), 5.4 (CH2), 4.5
(CH2), 4.4 (CH2), 4.3 (CH2), 3.9 (CH2), 1H NMR
(DMSO-d6, 100 ꢀC) d 5.68 (1H, bs), 4.09 (1H, d, J=2.8
Hz), 3.65 (1H, m), 3.46 (1H, t, J=7.6 Hz), 3.3–3.15 (7H,
m), 2.93 (1H, m), 0.91 (9H, s), 0.50–0.41 (36H, m), 0.18–
0.10 (20H, m),ꢁ0.01 (4H, q, J=8.0 Hz); [a]2D4 +28.3 (c
0.58, CHCl3); HRMS (FAB), m/z 824.5007
(C38H82O10NSi4 requires 824.5016).
(CH2), 64.82 (CH), 62.83 (CH2), 53 22 (CH), 52.28
(CH3), 29.05 (CH3), 28.51 (CH3), 27.27 (CH2), 26.54
(CH3), 24.35 (CH3), 19.24 (CH3), Anal. calcd for
C35H44N2O11S C, 59.99; H, 6.33; N, 4.00. Found C,
59.92; H, 6.33; N, 3.92.
N-(tert-Butoxycarbonyl)-[2,3;4,6-bis-O-(1-methylethyl-
idene)-ꢁ-D-mannopyranosyl]-tryptophan cyanomethyl ester
(3). To the suspension of the above methyl ester (107
mg, 0.153 mmol) in EtOH (10 mL), 10% NaOH aq (1.0
mL) was added at room temperature. The mixture was
stirred at room temperature for 3 h until the methyl
ester was consumed on TLC. After starting material was
consumed, the mixture became homogenous. Then the
mixture was refluxed overnight. After cooling the mixture
to room temperature, the solvent was evaporated, and
the residue was diluted with CHCl3. The mixture was
washed with aq 10% citric acid. The aq layer was
extracted with CHCl3. The combined layers were
washed with brine. After removal of solvent in vacuo,
the residue was pumped. The residue was dissolved in
CH3CN (1 mL), then i-Pr2NEt (53 mL, 0.306 mmol) and
bromoacetonitrile (21 mL, 0.306 mmol) were added at
room temperature. The mixture was stirred at room
temperature over night. The mixture was diluted with
CHCl3 and brine was added. The aq layer was extracted
with CHCl3, then the combined layers were dried over
Na2SO4. After evaporation, the residue was purified by
silica gel column chromatography(hexane/EtOAc 3:2).
The material was washed with hexane to give the cyano-
methyl ester 3 (71.2 mg, 80%) : [a]2D4 +103 (c 1.25,
PhH), 13C NMR d (C6D6 C6D6 as 128 ppm) 172.06 (C),
155.84 (C), 136.23 (C), 133.38 (C), 123.01 (CH), 120.56
(CH), 119.32 (CH), 111.58 (CH), 110.96 (C), 108.18 (C),
99.62 (C), 79.55 (C), 76.86 (CH), 76.41 (CH), 72.13
(CH), 69.18 (CH), 66.14 (CH), 63.63 (CH2), 54.45 (CH),
48.48 (CH2), 29.54 (CH3), 28.55 (CH3), 27.22 (CH3),
27.03 (CH2), 24.54 (CH3), 19.25 (CH3),1H NMR d 8.29
(s, 1H), 7.63 (d, 1H, J=8.0 Hz), 7.37 (d, 2H, J=8.0 Hz),
7.3–7.1 (m, 2H), 6.30 (d, 1H, J=4.8 Hz), 4.92–4.86 (m,
2H), 4.74–4.69 (m, 2H), 4.50–4.45 (m, 2H), 4.36–4.34 (m,
1H), 4.02 (dd, 1H, J=10.8 Hz, 6.2 Hz), 3.84–3.71 (m, 2H),
3.38 (d, 1H, J=14.0 Hz, 3.6 Hz), 3.00 (t, 1H, J=11.6 Hz),
1.71 (s, 3H), 1.71 (s, 3H), 1.60 (s, 3H), 1.49 (s, 3H), 1.40
(s, 3H), 1.34 (s, 9H). Anal. calcd for C30H39N3O9 C,
61.53; H, 6.71; N, 7.18. Found C, 61.30; H, 6.80, N, 7.00.
N-tert-Butoxycarbonyl-3-O-(2,3,4,6-tetrakis-O-triethyl-
silyl-ꢁ-D-mannopyranosyl)serine cyanomethyl ester (2).
The above prepared acid (1.99 g, 2.41 mmol) and i-
Pr2NEt (0.84 mL, 4.82 mmol) were dissolved in
6:1 MeCN/DMF (35 mL), and bromoacetonitrile (0.58
mL, 4.82 mmol) was added at 4 ꢀC. After stirring the
mixture for 1 dayat room temperature, solvent was
evaporated. To the residue was added H2O, and the
aqueous layer was extracted with EtOAc. The combined
organic layers were washed with brine and dried over
Na2SO4. The solvent was evaporated, and the residue was
purified bysilica gel column chromatography(hexane/
EtOAc=9:1–4:1) to afford cyanomethyl ester 2 (1.75 g,
84%) as a colorless oil: [a]2D5 29.7 (c 1.1, benzene); 13C
NMR (100 MHz, C6D6, 70 ꢀC) d 169.2 (C), 155.3 (C),
113.9 (C), 101.8 (CH), 80.1 (C), 77.7 (CH), 75.6 (CH),
73.3 (CH), 70.3 (CH), 68.2 (CH2), 63.2 (CH2), 54.9
(CH), 48.6 (CH2), 28.6 (CH3), 7.6 (CH3), 7.4 (CH3), 7.3
(CH3), 7.3 (CH3), 6.2 (CH2), 6.0 (CH2), 5.9 (CH2), 5.5
(CH2); H NMR (400 MHz, DMSO-d6, 80 ꢀC) d 6.83
1
(brs, 1H), 4.95 (s, 2H), 4.77 (d, 1H, J=3.2 Hz), 4.59 (d,
1H, J=3.2 Hz), 4.32 (m, 1H), 3.95 (t, 1H, J=7.3 Hz),
3.86 (dd, 1H, J=4.8, 10.6 Hz), 3.79–3.69 (m, 5H), 3.41
(m, 1H), 1.39 (s, 9H), 0.99–0.91 (m, 36H), 0.67–0.55 (m,
24H). Anal. calcd for C40H82N2O10Si4: C, 55.64; H,
9.57; N, 3.24. Found: C, 55.36; H, 9.69; N, 3.21.
N-(tert-Butoxycarbonyl)-[2,3;4,6-bis-O-(1-methylethyl-
idene)-ꢁ-D-mannopyranosyl]-(1-phenylsulfonyl)-tryptophan
cyanomethyl ester. To a solution of tetraol 10 (121.0 mg,
0.195 mmol) and 2,2-dimethoxypropane (0.25mL) in
DMF (0.7 mL), PPTS (19mg) was added at room tem-
perature. The mixture was stirred at room temperature
for 2 days. Triethylamine (0.3 mL) was added to quench
PPTS, and stirred for 15 min. After the mixture was diluted
with CHCl3, purified bysilica gel column chromatography
3-O-[2-Acetamido-2-deoxy-3,4-bis-O-triethylsilyl-6-(tert-
butyl-dimethylsilyl)-ꢀ-D-glucopyranosyl]-N-tert-butoxy-
.
carbonyl-S-serine AMP ester (12a). AMP tetra-n-butyl-
ammonium salt 11 (97.6 mg, 138 mmol) was dissolved in
DMF (1.5 mL) and cooled to 0 ꢀC under Ar atmo-
sphere. To the solution was added pre-cooled (0 ꢀC)
solution of cyanomethyl ester 1 (432 mg, 547 mmol) in
DMF (0.8 mL). The mixture was stirred for 2 h at 0 ꢀC,
and then 5% (v/v) acetic acid in water (221 mL) was
added to neutralize the mixture. The mixture was
washed with hexane (8 mL ꢂ 8) to remove the excess of
cyanomethyl ester. The DMF solution was diluted with
30% MeCN in water (12 mL), and the suspension was
subjected to C18 reverse phase column chromatography
(Waters Sep-Pak Vac C18 5 g/12 cc, MeCN/H2O=3:7!
1:1!8:2). Fractions containing target molecule were
directly(CHCl /MeOH 7:3). The material recrystallized
3
from EtOAc–hexane to afford the diacetonide (100.1
mg, 74%): [a]2D4ꢁ11 (c 0.47, CHCl3); mp 142–143 ꢀC;
13CNMR (67.8 MHz) d 172.83 (C), 155.40 (C), 133.11
(CH), 128.75 (CH), 122.90 (CH), 125.71 (CH), 123.89
(CH), 120 16 (C), 119.42 (C), 115.50 (CH), 110.63 (C),
99.36 (C), 79.77 (C), 76.50 (CH), 71.96 (CH), 70.60

S. Manabe et al. / Bioorg. Med. Chem. 10 (2002) 573–581
579
diluted with three volumes of MeCN. To the solution
was added 5% (v/v) acetic acid in water (158 mL) and
the solution was evaporated at 30 ꢀC for avoiding b-
elimination and saponification. The residue was
pumped to give 12a (95.5 mg, 44%). The ratio of 20-acyl
and 30-acyl AMP was determined by NMR as 20-acyl/30-
acyl=1:10. The peaks corresponding to 30-acyl AMP
were picked up for NMR data: 13C NMR (100 MHz,
DMSO-d6) d 169.8 (C), 169.2 (C), 155.8 (C), 155.1 (C),
152.3 (CH), 150.0 (C), 139.1(CH), 118.3 (C), 100.7
(CH), 85.0 (CH), 82.8 (CH), 78.5 (C), 76.3 (CH), 75.9
(CH), 75.7 (CH), 72.5 (CH), 71.4 (CH), 67.8 (CH2), 63.6
(CH2), 61.7 (CH2), 57.5 (CH), 55.4 (CH), 54.1 (CH),
28.1 (CH3), 25.7 (CH3), 23.2 (CH3), 23.1 (CH2), 19.2
(CH2), 18.1 (C), 13.5 (CH3), 6.9 (CH3), 6.9 (CH3), 4.9
(CH2), 4.7 (CH2),ꢁ4.8 (CH3),ꢁ5.6 (CH3); 1H NMR
(400 MHz, DMSO-d6) d 8.58 (s, 1H), 8.11 (s, 1H), 8.01
(d, 1H, J=9.3 Hz), 7.22 (br s, 2H), 6.85 (d, 1H, J=8.5
Hz), 5.98 (d, 1H, J=7.8 Hz), 5.37 (d, 1H, J=4.4 Hz),
5.11 (m, 1H), 4.49 (d, 1H, J=7.8 Hz), 4.32 (m, 1H), 4.06
(br s, 1H), 3.93 (m, 1H), 3.85 (m, 1H), 3.76 (m, 2H),
3.68 (m, 1H), 3.60 (m, 1H), 3.50 (t, 1H, J=8.5 Hz), 3.40
(m, 1H), 3.15 (m, 9H), 1.83 (s, 3H), 1.55 (m, 8H), 1.40
(s, 9H), 1.29 (m, 8H), 0.95–0.80 (m, 30H), 0.83 (s, 9H),
0.66 (q, 6H, J=8.0 Hz), 0.57 (q, 6H, J=7.8 Hz), 0.05 (s,
3H), 0.04 (s, 3H); HRMS (FAB), m/z 1080.4940
(M+H)+, (C44H83N7O16PSi3 requires 1080.4942).
mmol) was dissolved in DMF (1.6 mL) and cooled to
0 ꢀC under Ar atmosphere. To the solution was added
pre-cooled (0ꢀC) solution of cyanomethyl ester 2 (525 mg,
608 mmol) in DMF (1.0 mL). The mixture was stirred for
2 h at 0 ꢀC, and then 5% (v/v) acetic acid in water (250
mL) was added to neutralize the mixture. The mixture
was washed with hexane (8 mLꢂ2) to remove the excess
of cyanomethyl ester. The DMF solution was diluted
with 90% MeCN in water (10 mL), and the solution
was subjected to C18 reverse phase column chromato-
graphy(Waters Sep-Pak Vac C
5 g/12 cc, MeCN/
18
H2O=9:17!1:1!THF/H2O 1:1). Fractions containing
target molecule were diluted with same volume of THF
and four volumes of MeCN. To the solution was added
5% (v/v) acetic acid in water (25 mL) and the solution
was evaporated at 30 ꢀC for avoiding b-elimination and
saponification. The residue was pumped to give 13a
(110 mg, 50%). The ratio of 20-acyl and 30-acyl AMP
0
was determined byNMR as 2 -acyl:30-acyl=1:10. The
peaks corresponding to 30-acyl AMP were picked up for
NMR data: 13C NMR (100 MHz, DMSO-d6) d 169.1
(C), 155.6 (C), 154.9 (C), 152.1 (CH), 150.0 (C), 139.0
(CH), 118.2 (C), 100.5 (CH), 84.9 (CH), 83.1 (CH), 78.3
(C), 76.6 (CH), 74.4 (CH), 74.1 (CH), 73.0 (CH), 72.7
(CH), 67.2 (CH), 66.9 (CH2), 63.5 (CH2), 61.3 (CH2),
57.5 (CH2), 54.3 (CH), 28.1 (CH3), 23.1 (CH2), 19.2
(CH2), 13.5 (CH3), 6.9 (CH3), 6.9 (CH3), 6.6 (CH3), 4.9
(CH2), 4.6 (CH2), 4.6 (CH2), 4.2 (CH2); 1H NMR
(400 MHz, DMSO-d6) d 8.63 (s, 1H), 8.11 (s, 1H), 7.21
(br s, 2H), 7.01 (d, 1H, J=8.1 Hz), 6.00 (d, 1H, J=8.1 Hz),
5.32 (d, 1H, J=4.6 Hz), 5.07 (m, 1H), 4.58 (d, 1H, J=2.2
Hz), 4.43 (m, 1H), 4.00 (m, 2H), 3.90 (m, 1H), 3.83 (m,
1H), 3.82–3.70 (m, 6H), 3.37 (m, 1H), 3.15 (m, 8H), 1.55
(m, 8H), 1.40 (s, 9H), 1.38–1.25 (m, 8H), 0.95–0.83
(m, 48H), 0.65–0.50 (m, 24H); HRMS (FAB), m/z
1153.5546 (M+H)+, (C48H94N6O16PSi4 requires
1153.5541).
3-O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-S-serine
AMP ester (12b). To a test tube containing 12a (34.7
mg, 22.0 mmol) was added pre-cooled (0 ꢀC) TFA (3.0
mL), and the solution was stirred for 1 h at 0 ꢀC. TFA
was removed byblowing N gas. The pellet remained
2
was washed with ether (4 mL ꢂ 3) and dried in vacuo.
The precipitate was dissolved in 3% MeCN in water
(2.0 mL), and the solution was subjected to C18 reverse
phase column chromatography(Waters Sep-Pak Vac
C18 10 g/35 cc, MeCN/H2O=3:97 ! 1:9). To avoid
saponification, column was performed at 4 ꢀC. Frac-
tions containing target molecule were diluted with 10
volumes of MeCN and evaporated at 30 ꢀC, and the
residue was pumped to give 12b (5.9 mg, 42%). The
ratio of 20-acyl and 30-acyl AMP was determined by
NMR as 20-acyl/30-acyl=1:5. The peaks corresponding
to 30-acyl AMP were picked up for NMR data: 13C
NMR (100 MHz, DMSO-d6) d 169.8 (C), 166.7 (C),
155.7 (C), 152.5 (CH), 149.4 (C), 138.9 (CH), 118.5 (C),
100.8 (CH), 86.2 (CH), 80.9 (CH), 76.9 (CH), 75.3
(CH), 74.0 (CH), 72.2 (CH), 70.4 (CH), 66.7 (CH2), 64.1
(CH2), 60.9 (CH2), 55.1 (CH), 53.1 (CH), 23.1 (CH3);
1H NMR (400 MHz, DMSO-d6) d 8.46 (s, 1H), 8.15 (s,
1H), 8.02 (d, 1H, J=8.5 Hz), 7.32 (br s, 2H), 6.00 (d,
1H, J=6.6 Hz), 5.47 (m, 1H), 4.93 (m, 1H), 4.47 (d, 1H,
J=8.3 Hz), 4.34 (br s, 1H), 4.25 (br s, 1H), 4.13 (m,
1H), 4.05 (br d, 1H, J=9.0 Hz), 3.93 (m, 2H), 3.70 (d,
2H, J=10.3 Hz), 3.45 (m, 2H), 3.32 (dd, 1H, J=8.8, 9.8
Hz), 3.20 (m, 1H), 3.09 (dd, 1H, J=8.8, 9.0 Hz), 1.85 (s,
3H); HRMS (FAB), m/z 638.1839 (M+H)+
(C21H33N7O14P requires 638.1823).
3-O-ꢁ-D-Mannopyranosyl-S-serine AMP ester (13b). To
a test tube containing 13a (28.7 mg, 20.3 mmol) was
added pre-cooled (0 ꢀC) TFA (3.0 mL), and the solution
was stirred for 1 h at 0 ꢀC. TFA was removed byblowing
N2 gas. The pellet remained was washed with ether (4 mL
ꢂ 3) and dried in vacuo. The precipitate was dissolved in
1.5% MeCN in water (2.0 mL), and the solution was
subjected to C18 reverse phase column chromatography
(Waters Sep-Pak Vac C18 10 g/35 cc, MeCN/H2O=
1.5:98.5 !1:9). To avoid saponification, column was
performed at 4 ꢀC, fractions containing target molecule
were diluted 10 volumes of MeCN and evaporated at
30 ꢀC, and the residue was pumped to give 13b (12.1 mg,
quant). The ratio of 20-acyl and 30-acyl AMP was
0
determined byNMR as 2 -acyl/30-acyl=1:5. The peaks
corresponding to 30-acyl AMP were picked up for NMR
data: 13C NMR (100 MHz, DMSO-d6) d 167.3 (C),
155.7 (C), 152.4 (CH), 149.3 (C), 139.0 (CH), 118.6 (C),
101.0 (CH), 86.5 (CH), 80.9 (CH), 74.8 (CH), 74.0
(CH), 71.8 (CH), 70.4 (CH), 69.5 (CH), 67.0 (CH), 65.8
1
(CH2), 63.7 (CH2), 61.1 (CH2), 52.8 (CH); H NMR
(400 MHz, DMSO-d6) d 8.44 (s, 1H), 8.16 (s, 1H), 7.31
(br s, 2H), 5.98 (d, 1H, J=6.3 Hz), 5.47 (m, 1H), 4.95 (t,
1H, J=6.1 Hz), 4.69 (s, 1H), 4.36 (br s, 1H), 4.31 (d,
1H, J=3.2 Hz), 4.00–3.90 (m, 4H), 3.69 (m, 2H), 3.57
N-tert-Butoxycarbonyl-3-O-(2,3,4,6-tetrakis-O-triethylsi-
lyl-ꢁ-D-mannopyranosyl)-S-serine AMP ester (13a).
.
AMP tetra-n-butylammonium salt 11 (111 mg, 156

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S. Manabe et al. / Bioorg. Med. Chem. 10 (2002) 573–581
(m, 1H), 3.46 (m, 1H), 3.39 (m, 2H) ; HRMS (FAB), m/z
597.1551 (M+H)+, (C19H30N6O14P requires 597.1558).
7.38 (d, 1H, J=7.8 Hz), 7.30 (br s, 1H), 7.08 (t, 1H,
J=7.1 Hz), 7.04 (t, 1H, J=7.1 Hz), 5.89 (d, 1H, J=6.6
Hz), 5.39 (d, 1H, J=4.2 Hz), 4.94 (d, 1H, J=8.8 Hz),
4.79 (m, 1H), 4.28 (t, 1H, J=7.3 Hz), 4.16 (m, 1H), 3.95
-3.80 (m, 4H), 3.75–3.68 (m, 2H), 3.65 (dd, 1H, J=4.2,
11.5 Hz), 3.42 (dd, 1H, J=6.8, 14,6 Hz), 3.34 (dd, 1H,
J=8.05, 14.6 Hz), 3.15 (m, 8H), 1.55 (m. 8H), 1.30 (m,
8H), 0.92 (t, 12H, J=7.3 Hz); HRMS (FAB), m/z
696.2024 (M+H)+, (C27H35N7O13P requires 696.2030).
N-(tert-Butoxycarbonyl)-[2,3;4,6-bis-O-(1-methylethyl-
idene)-ꢁ-D-mannopyranosyl]-tryptophan AMP ester (14a).
.
To a solution of AMP tetra-n-butylammonium salt 11
(9.3 mg, 13.1 mmol) in DMF (190 mL) was added cyano-
methyl ester 3 (37.2 mg, 63.5 mmol). The mixture was
stirred at 40 ꢀC for 14 h, at 60 ꢀC for 8 h, cooled to room
temperature and poured into ether (40 mL). The pre-
cipitate was collected bycentrifuge, and washed with
ether (10 mL) twice. The pellet was dried in vacuo and
dissolved in 10% MeCN in H2O (2.0 mL). The solution
was subjected to C18 reverse phase column chromato-
graphy(Waters Sep-Pak Vac C 18 2 g/12 cc, MeCN/H2O=
2/8!3/7). The fractions containing target molecule were
diluted with five volumes of MeCN and evaporated at
30 ꢀC for avoiding saponification. Then 14a was
obtained as an amorphous (7.9 mg, 53%). The ratio of
20-acyl and 30-acyl AMP was determined by NMR as 20-
acyl/30-acyl=1:5. The peaks corresponding to 30-acyl
AMP were picked up for NMR data: 13C NMR
(100 MHz, DMSO-d6) d 171.5 (C), 155.6 (C), 154.9 (C),
149.9 (C), 139.1 (CH), 135.6 (C), 133.4 (C), 126.7 (C),
121.4 (CH), 118.5 (CH), 118.2 (CH), 118.2 (C), 111.3
(CH), 109.2 (C), 108.3 (C), 98.6 (C), 85.1 (CH), 82.9
(CH), 78.5 (C), 75.8 (CH), 75.5 (CH), 75.1 (CH), 72.5
(CH), 71.2 (CH), 67.6 (CH), 64.8 (CH2), 63.6 (CH2),
62.2 (CH2), 57.4 (CH2), 54.7 (CH), 28.9 (CH3), 28.0
(CH3), 26.9 (CH3), 24.6 (CH3), 23.1 (CH2), 19.2 (CH2),
Acknowledgements
We thank Dr. H. Koshino and Ms. T. Chijimatsu for
NMR measurement, and Dr. T. Chihara and his staff
for elemental analysis. We thank Ms. A. Takahashi for
technical assistance.
References and Notes
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1
13.5 (CH3); H NMR (400 MHz, DMSO-d6) d 11.11 (s,
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0
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