Efficient synthesis of MUC4 sialylglycopeptide through the new sialylation using 5-acetamido-neuraminamide donors

Article abstract of DOI:10.1021/jo702609p

(Chemical Equation Presented) Sialylation reactions using a new sialyl donor, diethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-β-D- glycero-D-galacto-2-nonulopyranosylonamide phosphite (Neu5Ac-1-amide-2- phosphite) derivatives, and the synthesis of the sialyl-T_N-MUC4 glycopeptide are described. The sialylation was performed in CH ₂Cl₂ solvent toward the 6-hydroxyl group of several monosugar acceptors and generated α-sialoside in good yield under low temperature and TMSOTf activation system. Amide derivatives of sialoside were easily converted into naturally occurring sialoside after hydrolysis of the amide group. Sialyl-α(2,6)-GalN₃ was also prepared by this new sialylation protocol, and then this sialoside was further converted into a Fmoc-protected sialyl-T_N serine derivative for solid-phase glycopeptides synthesis. The solid-phase glycopeptide synthesis using this sialyl-T_N serine derivative in which the sugar hydroxyl group was free afforded the target sialyl-T_N-MUC4 glycopeptide.

Full text of DOI:10.1021/jo702609p

Efficient Synthesis of MUC4 Sialylglycopeptide through the New
Sialylation Using 5-Acetamido-Neuraminamide Donors
Ryo Okamoto, Shingo Souma, and Yasuhiro Kajihara*
International Graduate School of Arts and Sciences, Yokohama City UniVersity, 22-2, Seto, Kanazawa-ku,
Yokohama, 236-0027, Japan
kajihara@yokohama-cu.ac.jp
ReceiVed December 6, 2007
Sialylation reactions using a new sialyl donor, diethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
2-O-ꢀ-D-glycero-D-galacto-2-nonulopyranosylonamide phosphite (Neu5Ac-1-amide-2-phosphite) deriva-
tives, and the synthesis of the sialyl-TN-MUC4 glycopeptide are described. The sialylation was performed
in CH2Cl2 solvent toward the 6-hydroxyl group of several monosugar acceptors and generated R-sialoside
in good yield under low temperature and TMSOTf activation system. Amide derivatives of sialoside
were easily converted into naturally occurring sialoside after hydrolysis of the amide group. Sialyl-
R(2,6)-GalN3 was also prepared by this new sialylation protocol, and then this sialoside was further
converted into a Fmoc-protected sialyl-TN serine derivative for solid-phase glycopeptides synthesis. The
solid-phase glycopeptide synthesis using this sialyl-TN serine derivative in which the sugar hydroxyl
group was free afforded the target sialyl-TN-MUC4 glycopeptide.
Introduction
these have also been found in O-linked glycopeptides as tumor-
associated glycopeptides.2 These glycoconjugates have been of
interest because of their potential as a cancer vaccine.2 Synthesis
of these O-linked glycopeptides has been carried out by chemical
and chemoenzymatic methods.3 The critical points in these
synthetic methods are how to prepare an appropriate amount
of sialyloligosaccharylamino acid and how to cleave the acid-
labile sialylglycopeptide from its solid support. Hence, an
annoying problem is the stereoselective construction of the
Glycoprotein and glycopeptide play important roles in a large
number of biological events, and these glycoconjugates fre-
quently have sialic acid (Neu5Ac) at the terminal of those
oligosaccharides.1 It is known that sialyloligosaccharides exhibit
several glycoforms, such as sialyl-TN, R(2,3)-sialyl-T, R(2,6)
sialyl-T, sialyl Lewis x, sialyl Lewis y, and sialyl Lewis a, and
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10.1021/jo702609p CCC: $40.75 2008 American Chemical Society
Published on Web 03/25/2008

Efficient Synthesis of MUC4 Sialylglycopeptide
SCHEME 1. Synthesis of Amide Donors 1-3
naturally occurring R-sialyl linkage that is a thermodynamically
unstable configuration compared with the unnatural ꢀ-sialyl
linkage, because of the endoanomeric effect.
reduces the electron-withdrawing effect comparing to that of
the ester function. During our extensive investigation using
1-amide derivatives of Neu5Ac-donors, we found an interesting
donor, 1-amide derivatives of the Neu5Ac-donor derivative,
which can afford R-selectivity in CH2Cl2 solvent. Using this
new sialylation method, we have studied the synthesis of sialyl-
TN and sialyl-TN glycopeptide derivative. Herein, we report
interesting new sialyl donors which can be prepared through a
concise synthetic route with application for the synthesis of
O-linked sialylglycopeptides.
Beyond such inherent difficulties, sialylation reactions using
a variety of sialyl donors have been reported.4 These methods
can sort out two phenomena, the solvent effect5 and neighboring
group effect.6 The solvent effect is easily obtained in acetonitrile
solvent. This method was reported by Kiso and Hasegawa5a
and has been known as an easy way to obtain R-selectivity.
Recently, modification of the acetamide group at the 5 position
(Ac2,7a TFA,7b–d N3,7e,f Troc,7g–i Phth7j group) was found to
elevate R-selectivity. On the other hand, the neighboring group
effect type has also been used to achieve R-selectivity, tradition-
ally introducing the auxiliary group at the 3-position6a–e or the
1-position.6f–h In addition, an oxazoline derivative at the 4 and
5 positions of Neu5Ac was found to be an excellent sialyl donor
which does not require the solvent effect.8
Results and Discussion
Preparation of Sialyl Donor. In order to study the capacity
of the amide group for the sialylation reaction, we chose three
different amide functional groups, namely the amide (CONH2),
monomethylamide (CONHCH3), and dimethylamide (CON-
(CH3)2) groups 1-3 shown in Scheme 1.
Each of the new sialyl donors having one of these amide
groups was synthesized from the known compound, methyl
5-acetamido-3,5-dideoxy-ꢀ-D-glycero-D-galacto-2-nonulopyra-
nosonate (Neu5Ac-1-methyl ester, 5).10 For the synthesis of
amide derivative 1, Neu5Ac-1-methyl ester 5 was treated with
NH4OH solution containing NH4HCO3 to convert into Neu5Ac-
1-CONH2 6 in 91% yield. Acetylation of compound 6 by
pyridine/Ac2O and subsequent selective de-O-acetylation at the
2 position by HClO4 in THF/acetone solvent gave pentaacetyl-
Neu5Ac-1-CONH2 8 in 80% overall yield. Compound 8 was
then equipped with a diethyl phosphite group at the 2 position
to be converted into 1-amide donor 1 (77% yield).
Synthesis of monomethyl donor 2 also started from compound
5. Treatment with 2 M CH3NH2/CH3OH solution gave Neu5Ac-
1-CONHCH3 7 in 90% yield. Using the same manner of
synthesis as in 1-amide donor 1, pentaacetyl-Neu5Ac-1-CON-
HCH3 9 was prepared in 77% yield. Monomethyl amide 9 was
then introduced as a diethyl phosphite group at the 2 position
to be converted into monomethyl amide donor 2 (85% yield).
On the other hand, the dimethyl amide group was
introduced by condensation reaction. 5-Acetamido-2,4,7,8,9-
penta-O-acetyl-3,5-dideoxy-R-D-glycero-D-galacto-2-nonu-
So far, all of the sialyl donors reported have an ester group
at the 1-position.9Therefore, we developed an interest in the
ability of the amide group at the 1-position instead of an ester
group (Scheme 1, 1-3). It is known that the amide function
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Lin, C. C. Org. Lett. 2005, 7, 4169–4172. (e) Lin, C. C.; Wong, C. H. Angew.
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Eur. J. 2005, 11, 849–862. (h) Ando, H.; Koike, Y.; Ishida, H.; Kiso, M.
Tetrahdron. Lett. 2003, 44, 6883–6886. (i) Ando, H.; Koike, Y.; Koizumi, S.;
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J. Org. Chem. Vol. 73, No. 9, 2008 3461

Okamoto et al.
TABLE 1. Sialylation Using Amide Donors 1-3 and Conventional
Methyl Ester Donor 4
FIGURE 1. Presumable reaction mechanism of the new sialylation.
product: yield
entry
donor (R)
acceptor
(%) (R:ꢀ)a
TABLE 2. Temperature Dependence of Sialylation Reaction Using
Neu5Ac-1-monomethylamide 2
1
2
3
4
5
6
7
8
1 (NH2)
1 (NH2)
1 (NH2)
a
b
c
a
b
c
b
c
a
b
c
12: 84 (7:1)
13: 79 (4.5:1)
14: 88 (4:1)
15: 90 (10:1)
16: 91 (5:1)
17: 92 (5.4:1)
18: 43 (1:6)
19: 46 (1:9)
20: 75 (4:1)
21: 80 (1:1)
22: 80 (1:1)
2 (NHCH3)
2 (NHCH3)
2 (NHCH3)
3 (N(CH3)2)
3 (N(CH3)2)
4 (OCH3)
4 (OCH3)
4 (OCH3)
9
10
11
a
1
The ratios were determined by H NMR.
losonic acid (hexaacetyl-Neu5Ac, 10)11 was treated with
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlo-
ride (EDC · HCl) and 2 M NH(CH3)2/THF solution to be
converted to compound 11. Selective de-O-acetylation at the
2 position of 11 and subsequent phosphitation afforded
1-dimethylamide donor 3 in 33% overall yield.
1
* The ratios were determined by H NMR.
Sialylation Reaction. The new sialyl donors 1-3 were
examined for the sialylation reaction with three acceptors (a-c)
having a free hydroxyl group at the 6 position, and the results
are summarized in Table 1.
amide derivative was shown to be optimum among sialyl donors
investigated in this study.
To gain insight into the origin of the stereoselectivity, we
hypothesized the intermediacy of the three-membered cyclic
intermediate generated by the interaction of the amide group
with the C(2) position to stabilize the oxocarbenium ion
intermediate (Figure 1).
Sialylation of glucoside acceptor a with amide donor 1
afforded sialoside 12 in 84% yield (R:ꢀ ) 7:1, entry 1). In the
case of a monomethyl amide donor 2 with acceptor a, it afforded
sialoside 15 in 90% yield (R:ꢀ ) 10:1, entry 4). Using acceptors
b and c, naturally occurring Neu5Ac-R(2,6)-Gal and Neu5Ac-
R(2,6)-GalNAc linkages were also obtained in good yield,
respectively. Coupling of galactoside acceptor b with amide
donor 1 provided sialoside 13 in 79% yield (R:ꢀ ) 4.5:1, entry
2), and coupling of 2-N3-galactoside acceptor c, which is a
precursor of galactosamine, with amide donor 1, provided
sialoside 14 in 88% yield (R:ꢀ ) 4:1, entry 3). Furthermore,
the monomethyl amide donor 2 exhibited better R-selectivity
and higher yield (y ) 91%, R:ꢀ ) 5:1, entry 5, and y ) 92%,
R:ꢀ ) 5.4:1, entry 6). We also examined the same reaction using
dimethylamide donor 3 toward acceptors b and c. Unexpectedly,
these reactions showed ꢀ-selectivity rather than R-selectivity
(R:ꢀ ) 1:6, entry 7, and R:ꢀ ) 1:9, entry 8). In order to evaluate
the capacity of the amide group at the C(1) position, we also
examined the sialylation reaction using a conventional methyl
ester donor 4 in CH2Cl2.5b Of those results, even the best R:ꢀ
ratio was 4:1, as shown in entry 9, which was a coupling reaction
with acceptor a, and other reactions afforded a 1:1 ratio (entries
10 and 11). As a result, we concluded that the monomethyl
This interaction may afford two possible intermediates, (a)
or (b), on the R-side or ꢀ-side. According to the aforementioned
result of the sialylation reaction, intermediate (a), which can
generate an R-sialyl linkage, may be dominant. In the case of
a dimethyl amide donor, which showed ꢀ-selectivity, approach
of the acceptor from R-side might be disturbed by the steric
hindrance of dimethyl group. For formation of this intermediate,
oxygen of the amide group may interact with the cation at the
2 position instead of nitrogen, as shown in Figure 1, but we
could not determine which atom exhibited the specific interac-
tion. We also examined the sialylation reaction dependent on
temperature using amide Neu5Ac-1-monomethylamide donor
2 (Table 2).
At -50 °C, the sialylation reaction afforded moderate
R-selectivity (R:ꢀ ) 3:1 ∼ 4.9:1). On the other hand, the
sialylation at lower temperature such as -78 or -87 °C
exhibited better R-selectivity (R:ꢀ ) 4.9:1-9:1). In terms of
these results, we hypothesize that the formation of intramolecular
cyclization having a ꢀ-configuration, as shown in Figure 1a,
may be faster than that of glycosylation. The lower temperature
conditions decrease the accessibility of acceptor to the sialyl
(11) Myers, R. W.; Lee, Y. C.; Thomas, G. H.; Reynolds, L. W.; Uchida, Y.
Anal. Biochem. 1980, 101, 166–174.
3462 J. Org. Chem. Vol. 73, No. 9, 2008

Efficient Synthesis of MUC4 Sialylglycopeptide
(sialyl-TN-MUC4 peptide).13 MUC4 is one of the tumor-
associated glycoproteins and has a variable number of tandem
repeats of 16 amino acids with aberrant glycoforms. Like the
MUC1 peptide, this tandem repeat glycopeptide is potentially
an immune response target.2,14
TABLE 3. Deamidation of 1-Amide Sialosides
In order to synthesize this target, we also prepared Fmoc-
Ser(sialyl-TN)-OH conjugate by use of this sialylation reaction
(Scheme 2).15
Sialoside 25 was esterified at the 1 position by Dowex 50W-
X8 in MeOH followed by acetylation with pyridine/Ac2O to
give compound 26 (75% yield). Conversion of the trimethyl-
silylethyl (SE) group at the 1-position of Gal-2-N3 to a
trichroloacetimidate group gave donor 28 (92% yield for
affording 27, 94% yield for affording 28). Then, glycosylation
of the benzyl-esterified serine derivative by donor 28 in the
presence of TMSOTf gave sialyl-TN derivative 29 in 69%
yield.16 Reduction of azido, benzyl ether, and benzyl ester of
compound 29 by catalytic hydrogenation over Pd followed by
acetoamidation with Ac2O afforded compound 30 (93% overall
yield). Then, the Boc-protected compound 30 was converted
into Fmoc derivative 31 through deacetylation, deprotection of
the Boc group, and introduction of the Fmoc group. As a result,
we obtained Fmoc-Ser(sialyl-TN-methyl ester)-OH 31 in 84%
overall yield, which was ready to use in solid-phase glycopeptide
synthesis.
In order to synthesize the sialyl-TN-MUC4 glycopeptide by
solid-phase peptide synthesis, we employed 31, which has a
free hydroxyl group. We did this because we have already
demonstrated synthesis of N-linked glycopeptides by the use
of an Fmoc-Asn-complex type oligosaccharide having a free
sugar hydroxyl group.17 Using this oligosaccharylamino acid
with the free hydroxyl group has the major advantage of
avoiding undesired side reactions, such as ꢀ-elimination of
amino acids during base-deprotection steps for de-O-acetylation.
However, deprotection for the ester group of O-linked sialylo-
ligosaccharide requires more careful treatment under basic
conditions. As far as we examined, the ester group of sialoside
could be removed after 15 min, and this condition was expected
to afford current synthetic target 32 in good yield. In addition,
we also have already demonstrated the resistance of esterified
sialyl-linkage to the 95% TFA condition, which is the general
condition for the cleavage from the resin (Scheme 3).17
Considering these, using the oligosaccharylamino acid with the
free hydroxyl group has the advantage over using a protecting
group because we do not need multiple deprotection steps for
several functional groups on oligosaccharide.17
donor through solvent due to intermolecular reaction. Therefore,
alcohol might attack the C(2) carbon from the R-side preferably
after the formation of the thermodynamically stable intermediate
(a) as shown in Figure 1.
We have also examined the sialylation reaction using
Neu5Ac-1-amide donors having thiophenyl glycoside (SPh) at
the 2 position. Although these donors also gave slight R-selec-
tivity in CH2Cl2 solvent (R:ꢀ ) 2:1-3:1) under general
activation conditions (NIS, TfOH), the reproducibility for
R-selectivity was found to be poor. We speculated that the SPh
group was not activated efficiently because TfOH might be
trapped by the amide group. On the basis of the obtained data,
a phosphite group was considered suitable for the amide donors.
In particular, amide and monomethylamide donors afford tiny
amounts of Neu5Ac-2-OH- and 2,3-en-Neu5Ac-type byproducts.
Therefore, purification of sialoside was very easy on a silica
gel column. In terms of sialylation toward secondary alcohol,
Neu5Ac-1-monomethylamide 2 has not afforded sialyloligosac-
charides in moderate yield. Therefore, we hope to optimize this
sialylation toward sugar secondary alcohols. We also examined
this sialylation in acetonitrile, but it afforded sialic acid
derivative incorporated acetonitrile covalently to the 2 position.
Therefore, we concluded that Neu5Ac-1-monomethylamide 2
cannot be used in acetonitrile solvent under the current
conditions.
Since we obtained sufficient R-selectivity for the sugar
primary alcohol, we examined the deamidation reaction at the
1 position by 2 M NaOH solution at 100 °C12 followed by acet-
amidation with Ac2O at the 5 position. This condition afforded
several sialosides in good yield (two steps with an overall yield
of ca. 80%, Table 3, and Supporting Information Figure S-1).
As a result, we could obtain naturally occurring sialoside
derivatives by use of amide derivatives of the Neu5Ac donor.
Synthesis of Sialyl-TN Serine Derivative and Sialylglycopep-
tide. As we succeeded in finding an interesting new sialylation,
we applied this sialylation reaction to the synthesis of naturally
occurring sialylglycopeptide. In terms of the synthetic target,
we selected MUC4 glycopeptide having the sialyl-TN epitope
The MUC4-glycopeptide synthesis was performed on poly-
(ethyleneglycol)-poly(dimethylacrylamide) copolymer (PEGA)
resin, which contains the acid-labile linker 4-(4-hydroxymethyl-
3-methoxyphenoxy)butyric acid (HMPB).17b The first residue,
aspartic acid, was introduced as Fmoc-Asp(tBu)-OH by activa-
tion with 1-(mesitylenesulfonyl)-3-nitro-1,2,4-triazole (MSNT)
and N-methylinidazole. The second to eleventh residues were
(14) (a) Dekker, J.; Rossen, J. W. A.; Büller, H. A.; Einerhand, A. W. C.
Trends. Biochem. Sci. 2002, 27, 126–131. (b) Singh, A. P.; Chaturvedi, P.; Batra,
S. K. Cancer Res. 2007, 67, 433–436.
(15) Previous synthesis of O-sialyloligosaccharyl-serine derivatives see
reference 2 and 3.
(16) Chen, X. T.; Sames, D.; Danishefsky, S. J. J. Am. Chem. Soc. 1998,
120, 7760–7769.
(17) (a) Yamamoto, N.; Ohmori, Y.; Sakakibara, T.; Sasaki, K.; Juneja, L. R.;
Kajihara, Y. Angew. Chem., Int. Ed. 2003, 42, 2537–2540. (b) Kajihara, Y.;
Yoshihara, A.; Hirano, K.; Yamamoto, N. Carbohydr. Res. 2006, 341, 1333–
1340. (c) Yamamoto, N.; Takayanagi, A.; Yoshino, A.; Sakakibara, T.; Kajihara,
Y. Chem. Eur. J. 2007, 13, 613–625.
(12) Roy, R.; Pon, R. A. Glycoconjugate. J. 1990, 7, 3–12.
(13) (a) Dziadek, S.; Brocke, C.; Kunz, H. Chem. Eur. J. 2004, 10, 4150–
4162. (b) Brocke, C.; Kunz, H. Synthesis 2004, 525–542. (c) Brocke, C.; Kunz,
H. Synlett 2004, 2052–2056.
J. Org. Chem. Vol. 73, No. 9, 2008 3463

Okamoto et al.
SCHEME 2. Synthesis of Sialyl-TN Derivative 31
SCHEME 3. Solid-Phase Synthesis of Sialyl-TN-MUC4 Glycopeptide 32
coupled by diisopropylcarbodiimide (DIPCDI) and hydroxy-
benzotriazole (HOBt) in DMF. At this point, 31 was introduced
onto the resin by DIPCDI and HOBt. The following amino acid
residues were incorporated by means of these same reagents,
but 40 mM concentration of amino acids was essential to avoid
esterfication of the sugar-hydroxyl group on sialyl-TN.17c After
construction of the glycopeptide, the glycopeptide was released
from the resin by treatment with TFA/TIPS/H2O (95:2.5:2.5)
for 2 h. As expected, the sialyl linkage was stable throughout
this treatment.
Brief saponification of the methyl ester was carried out by 200
mM NaOH solution for 10 min at 0 °C or 20 mM NaOH solution
3464 J. Org. Chem. Vol. 73, No. 9, 2008

Efficient Synthesis of MUC4 Sialylglycopeptide
mide) phosphite (3). Compound 11 (160 mg, 0.293 mmol) was
dissolved in a solution of acetone and THF (1:9, 5.8 mL) containing
5% HClO4, and this solution was stirred for 2 h at room temperature.
The mixture was allowed to cool to 0 °C. This mixture was diluted
with EtOAc, and the organic phase was washed with saturated
NaHCO3 solution and brine. The organic phase was dried over
MgSO4 and concentrated in vacuo. The residue was dissolved in
CH2Cl2 (4.9 mL, 60 mM) and the mixture was allowed to cool to
0 °C. To this solution was added diisopropylethylamine (300 µL,
1.76 mmol) and diethyl chlorophosphite (126 µL, 0.879 mmol) and
this mixture was stirred for 15 min at 0 °C. To quench the reaction,
MeOH was added to this mixture. The mixture was diluted with
CHCl3 and washed with saturated NaHCO3 solution and brine. The
organic phase was dried over MgSO4 and concentrated in vacuo.
Purification of the residue by silica gel column chromatography
for 1.5 h. As far as we analyzed by extensive HPLC analysis, ca.
5–7% of eliminated product was observed. Purification of the crude
material by a reversed-phase HPLC column afforded sialyl-TN-
MUC4 peptide 32 (50% or 61% isolated yield).
Conclusion
We found a concise sialylation reaction using Neu5Ac-1-
amide derivatives, and these donors exhibited good R-selectivity
and yield. Simple modification by an amide group at the C(1)
position of sialic acid instead of the ester group might afford
an interesting intermediate to afford R-selectivity, and this
reaction did not require acetonitrile solvent. Therefore, this
finding enabled examination of sialylation in several different
solvents and temperatures. This method was also developed for
the synthesis of glycopeptides having a sialyl-TN epitope. In
order to obtain glycopeptides for measurement of the NMR
spectrum and for bioassay, an adequate amount of Fmoc-Ser-
O-oligosaccharide is essential. Our sialylation method satisfied
this demand and afforded an appropriate amount of the MUC4
fragment having sialyl-TN. Research is in progress to synthesize
larger repetitive O-linked glycopeptides having sialyl-TN.
(5% Et3N solution of EtOAc/hexane ) 2:1) afforded compound 3
1
(60 mg, y ) 33%): [R]25 ) + 7.50 (c ) 0.8, CHCl3). H NMR
D
(400 MHz, CDCl3) δ 5.42 (d, 1H, J ) 9.47 Hz), 5.38–5.30 (m,
2H), 5.17 (ddd, 1H, J ) 2.74 Hz, J ) 6.31 Hz, J ) 6.73 Hz), 4.35
(dd, 1H, J ) 2.74 Hz, J ) 12.5 Hz), 4.04–3.82 (m, 4H), 4.30 (dd,
1H, J ) 1.83 Hz, J ) 10.6 Hz), 4.10 (dd, 1H, J ) 6.32 Hz, J )
12.5 Hz), 4.08 (ddd, 1H, J ) 10.3 Hz, J ) 10.3 Hz, J ) 10.3 Hz),
3.19 (s, 3H), 2.97 (s, 3H), 2.53 (dd, 1H, J ) 14.0 Hz, J ) 4.98
Hz), 2.29 (ddd, 1H, J ) 14.0 Hz, J ) 14.5 Hz, J ) 0.85 Hz), 2.13,
2.09, 2.03, 2.03, 1.92 (5s, 15H), 1.27 (m, 6H); 13C NMR (100 MHz,
CDCl3) δ 170.7, 170.7, 170.6, 170.5, 170.1, 166.10, 100.1, 72.2,
70.6, 69.6, 68.1, 62.7, 59.5, 58.3, 50.0, 38.3, 38.0, 37.2, 23.6, 21.3,
21.3, 21.1, 21.1, 17.3, 17.3; 31P NMR (CDCl3) δ 137.3; MALDI-
HRMS calcd for C25H41N2NaO14P [M + Na]+ 647.2193, found
647.2215.
Experimental Section
Diethyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-ꢀ-D-
glycero-D-galacto-2-nonulopyranosylonamide Phosphite (1) and
Diethyl (Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
2-O-ꢀ-D-glycero-D-galacto-2-nonulopyranosylonamide) Phos-
phite (2). Compound 8 (110 mg, 0.240 mmol) was dissolved in
CH2Cl2 (4.0 mL) at room temperature, and this solution was allowed
to cool to 0 °C. To this solution were added diisopropylethylamine
(245 µL) and diethyl chlorophosphite (104 µL), and then the mixture
was stirred for 20 min at 0 °C. To this mixture was added MeOH
(100 µL) for quenching of diethyl chlorophosphite at 0 °C. The
mixture was diluted with CHCl3 and washed with saturated
NaHCO3 solution and brine. The organic phase was dried over
MgSO4 and concentrated in vacuo. Purification of the residue by
silica gel column chromatography (5% Et3N solution of EtOAc/
hexane ) 3:1 f 5% Et3N solution of EtOAc) afforded compound
1 (107 mg, 77%). Preparation of compound 2 from compound 9
(150 mg) was also performed by the same procedure described
(Typical procedure for sialylation using amide-type phos-
phite donor 1–3). Each of donor (0.0503 mmol) and acceptor
(0.100 mmol) was individually coevaporated with dry benzene twice
and then dried up using vacuum pump. Both substrates were
dissolved in CH2Cl2 (628 µL) containing activated 4Å molecular
sieves (160 mg/1 mL) and this mixture was stirred for 2 h at room
temperature. Then this mixture was allowed to cool to -78 °C. To
this mixture was added TMSOTf (10% solution in CH2Cl2, 15 µL)
and this mixture was stirred for 1 h. The mixture was allowed to
warm to -40 °C and the mixture was stirred for additional 1 h. To
this mixture was added Et3N (15 µL) and then the mixture was
diluted with CHCl3 and then allowed to warm up to room
temperature. The mixture was filtered and the filtrate was washed
with saturated NaHCO3 and brine. The organic phase was dried
over MgSO4 and concentrated in vacuo. Purification of the residue
by gel permeation column chromatography followed by silica gel
column chromatography (EtOAc f EtOAc/MeOH ) 15:1) afforded
desired sialoside.
above (157 mg, 85%). Compound 1: [R]24 ) -22.7 (c ) 0.6,
D
CHCl3); 1H NMR (400 MHz,CDCl3) δ 6.83, (s, 1H), 5.37 (dd, 1H,
J ) 2.41 Hz, J ) 7.23 Hz), 5.36 (s, 1H), 5.33–5.23 (m, 3H), 4.34
(dd, 1H, J ) 2.68 Hz, J ) 12.6 Hz), 4.22 (dd, 1H, J ) 2.41 Hz,
J ) 11.0 Hz), 4.20–4.08 (m, 3H), 4.05 (dd, 1H, J ) 6.69 Hz, J )
12.6 Hz), 4.02–3.85 (m, 4H), 2.70 (dd, 1H, J ) 13.7 Hz, J ) 4.82
Hz), 2.00 (1H), 2.15, 2.14, 2.05, 2.03. 1.90 (5 s, 15H), 1.28 (m,
6H); 13C NMR (100 MHz, D2O) δ 171.1, 170.8, 170.7, 170.4, 170.1,
169.5, 97.7, 71.7, 69.4, 68.8, 67.4, 62.6, 59.0, 58.3, 49.3, 37.3, 23.1,
21.1, 21.0, 20.9, 20.8, 16.7, 16.7; 31P NMR (CDCl3) δ 138.2;
MALDI-HRMS calcd for C23H37N2NaO14P [M + Na]+ 619.1880,
found 619.1870. (Compound 2) [R]25D ) -24.7 (c ) 1.0, CHCl3).
1H NMR (400 MHz,CDCl3) δ 6.89 (brd, 1H, J ) 4.87 Hz), 5.37
(dd, 1H, J ) 2.4 Hz, J ) 6.84 Hz), 5.33–5.24 (m, 3H), 4.37 (dd,
1H, J ) 2.62 Hz, J ) 12.4 Hz), 4.22 (dd, 1H, J ) 2.4 Hz, J )
10.7 Hz), 4.20–4.07 (m, 3H), 4.07 (dd, 1H, J ) 6.77 Hz, J ) 12.4
Hz), 4.00–3.80 (m, 4H), 2.87 (d, 3H, J ) 4.87 Hz), 2.73 (dd, 1H,
J ) 13.6 Hz, J ) 4.87 Hz), 1.92 (1H), 2.15, 2.13, 2.06, 2.03. 1.90
(5s, 15H), 1.27 (m, 6H); 13C NMR (100 MHz,CDCl3) δ 171.0,
170.8, 170.7, 170.3, 170.1, 167.7, 97.7, 71.8, 69.7, 68.8, 67.6, 62.6,
58.9, 58.1, 49.9, 37.5, 26.0, 23.1, 21.0, 20.8, 20.7, 20.7, 16.7, 16.7;
(Typical deamidation reaction). To a solution of MeOH (450
µL) containing amide derivative of R-sialoside (0.0451 mmol) was
added NaOMe (0.0451 mmol). This mixture was stirred at room
temperature for 1 h and then the mixture was added ion-exchange
resin IR-120. The mixture was filtered and the filtrate was
concentrated in vacuo. Subsequently, the residue was dissolved in
a solution of 2 M NaOH solution (450 µL) and MeOH (100 µL)
and then this mixture was stirred for 2–8 h (depended on the
substrate) at 100 °C. Then the mixture was allowed to cool to 0 °C
and acetic anhydride was added to this mixture for acetamidation.
After finish of acetamidation, the mixture was neutralized by AcOH
and then directly concentrated in vacuo. Purification of the residue
by C-18 reverse phase column chromatography (residue was
subjected by H2O and CH3CN/H2O ) 50:50 was used as eluent)
to obtain R-sialoside. The yields were shown in Table 3.
(Compound data of sialyl-TN derivative 31). [R]21 ) +49.9
D
1
(c ) 0.95, MeOH). H NMR (400 MHz, CD3OD) δ 7.77 (d, 2H,
31P NMR (CDCl3)
δ
138.1; MALDI-HRMS calcd for
J ) 7.50 Hz), 7.65 (br, 2H), 7.39–7.28 (m, 4H), 4.79 (br, 1H),
4.43–4.39 (m, 3H), 4.23 (brt, 1H), 4.22 (dd, 1H, J ) 3.65 Hz, J )
10.83 Hz), 3.91–3.83 (m, 6H), 3.83 (dd, 1H, J ) 2.18 Hz, J )
11.69 Hz), 3.81–3.76 (m, 4H), 3.72 (dd, 1H, J ) 10.83 Hz, J )
C24H39N2NaO14P [M + Na]+ 633.2037, found 633.2017.
Diethyl (dimethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-2-O-ꢀ-D-glycero-D-galacto-2-nonulopyranosylona-
J. Org. Chem. Vol. 73, No. 9, 2008 3465

Okamoto et al.
2.81 Hz), 3.67 (dd, 1H, J ) 4.48 Hz, J ) 10.12 Hz), 3.66 (m, 1H),
3.65 (dd, 1H, J ) 5.71 Hz, J ) 11.48 Hz), 3.61 (dd, 1H, J )
10.46 Hz, J ) 1.31 Hz), 3.50 (dd, 1H, J ) 1.31 Hz, J ) 9.04 Hz),
2.67 (dd, 1H, J ) 12.87 Hz, J ) 4.66 Hz), 1.99, 1.95 (2s, 6H),
1.78 (dd, 1H, J ) 12.87 Hz, J ) 12.24 Hz); 13C NMR (100 MHz,
CD3OD) δ 175.1, 173.9, 173.6, 145.3, 142.6, 128.8, 128.2, 126.1,
121.0, 100.4, 99.9, 75.0, 72.4, 71.0, 70.3, 69.7, 69.6, 69.2, 68.6,
68.0, 64.9, 64.1, 55.9, 53.8, 53.4, 51.4, 48.5, 41.2 22.9, 22.7;
MALDI-HRMS calcd for C38H49N3NaO18 [M + Na]+ 858.2909,
found 858.2907.
Solid-Phase Peptide Synthesis. The synthesis of MUC4 was
performed on HMPB-PEGA resin (0.01 mol scale). The first amino
acid, Fmoc-Asp(tBu)-OH (21 mg, 0.05 mmol), was attached
quantitatively to the resin using MSNT (15 mg, 0.05 mol) and
N-methylimidazole (3 µl, 0.0375 mol) in CH2Cl2 (500 µL).
Coupling of amino acid from second to eleventh (T, V, P, L, P, T,
A, H, G, T) was performed by use of Fmoc-amino acid (0.05 mmol),
DIPCDI (0.05 mmol), and HOBt (0.05 mmol) in DMF (310 µL),
and 20% piperidine/DMF was used for Fmoc deprotection. Then,
coupling of compound 31 (17 mg, 0.02 mmol) was performed using
DIPCDI (4.6 µL, 0.03 mmol) and HOBt (4 mg, 0.03 mmol) in
DMF/DMSO ) 1:1 solvent (1 mL) for 15 h followed by washing
by DMF and deprotection of Fmoc by 20% piperidine/DMF for
10 min. The remaining amino acid was elongated manually by
Fmoc-amino acid (A, S, S) or Boc-amino acid (T) (0.05 mmol),
DIPCDI (0.05 mmol), and HOBt (0.05 mmol) in DMF (1.25 mL).
After completion of chain assembly, the resin was treated with
solution containing 95% TFA, 2.5% TIPS and 2.5% H2O for 2 h
at room temperature followed by filtering, and the filtrate was
evaporated in vacuo. Purification of the residue by RP-HPLC with
Vydac column C-18 (5 µm, 250 × 4.5 mm linear gradient of 9 f
49.5% containing 0.09% TFA in 0.1% TFA aqueous over 30 min)
at a flow rate of 1.0 mL/min afforded the desired sialylglycopeptide
(7.4 mg, 38% yield based on the first amino acid attached).
Saponification of Methyl Ester SialyltN-MUC4 Peptide:
Condition A. Sialylglycopeptide (5 mg, 2.4 µmol) was dissolved
in cooled 200 mM NaOH solution (400 µL) at 0 °C, and this
mixture was stirred for 10 min. After neutralization by 200 mM
HCl solution (400 µL), purification of the residue by RP-HPLC
with Vydac column C-18 (5 µm, 250 × 4.5 mm linear gradient of
13.5 f 31.5% containing 0.09% TFA in 0.1% TFA solution over
60 min at a flow rate of 1.2 mL/min) afforded the desired
sialylglycopeptide (2.5 mg, 50% yield). Condition B. Sialylgly-
copeptide (2.3 mg, 1.1 µmol) was dissolved in cooled 20 mM NaOH
solution (225 µL) at room temperature, and this mixture was stirred
for 1.5 h. After neutralization by 200 mM HCl solution (200 µL),
purification of the residue by RP-HPLC with a Vydac column C-18
(5 µm, 250 × 4.5 mm linear gradient of 13.5 f 31.5% containing
0.09% TFA in 0.1% TFA aqueous over 60 min at a flow rate of
1.2 mL/min) afforded the desired sialylglycopeptide (1.4 mg, 61%):
MALDI-HRMS calcd for C83H134N20O39 [M + H]+ 2035.9195,
found 2035.9210.
Acknowledgment. We thank Mr. Kazuhiro Fukae and Mr.
Naohiro Hayashi at Otsuka Chemical Co. Ltd. for their skillful
measurement of mass spectra. We also thank Dr. Yukishige Ito
at RIKEN, Japan, and Dr. Michio Sasaoka at Otsuka Chemical
Co. Ltd. for support and encouragement. Financial support from
the Japan Society for the Promotion of Science (Grant-in-Aid
for Creative Scientific Research No.17GS0420) is acknowledged.
Supporting Information Available: Full experimental details
for the preparation of 1-3 and 31 and NMR spectra of all new
compounds and coupling compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO702609P
3466 J. Org. Chem. Vol. 73, No. 9, 2008