A new oligosaccharide synthesis using special hydroxy protecting group

Article abstract of DOI:10.1016/j.tetlet.2004.02.032

A new hydroxy protecting group for convenient preparation of oligosaccharide was developed using uni-chemo protection (UCP) concept. The UCP group was comprised of polymerized amino acid derivatives and protecting each hydroxyl groups by ester linkage. De

Full text of DOI:10.1016/j.tetlet.2004.02.032

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 2759–2762
A new oligosaccharide synthesis using special hydroxy protecting
group
Shiro Komba,* Motomitsu Kitaoka and Takafumi Kasumi*
Enzyme Laboratory, National Food Research Institute, 2-1-12 Kannondai, Tsukuba 305-8642, Japan
Received 13 January 2004;revised 6 February 2004;accepted 9 February 2004
Abstract—A new hydroxy protecting group for convenient preparation of oligosaccharide was developed using uni-chemo pro-
tection (UCP) concept. The UCP group was comprised of polymerized amino acid derivatives and protecting each hydroxyl groups
by ester linkage. Depending on the polymerization degree, the hydroxyl groups were characterized and controlled. Using this
protecting group, two kinds of sialyl-T analogues were successfully synthesized from same sugar parts merely by repeating Edman
degradation and coupling.
Ó 2004 Elsevier Ltd. All rights reserved.
Various protecting groups for the synthesis of oligo-
saccharides have been developed and selectively used for
desired synthesis.1–4 The selection of protecting groups,
however, is quite laborious. If the oligosaccharide
library is tried to synthesize, each hydroxyl groups on
mono-saccharide have to be protected orthogonally.
The number of these orthogonal protecting groups
increase with each additional glycosidic linkage. It is not
so easy to apply these methods to the complicated oli-
gosaccharide synthesis, since there are only limited
number of such orthogonal protecting groups. On
resolving this problem, hydroxyl protecting groups for
the saccharide should be one dimensional for the syn-
theses of oligosaccharide library and complicated
branched oligosaccharide. If it is possible to apply only
one kind of protecting group and one de-protecting
method, the syntheses of oligosaccharide library and
complicated branched oligosaccharide become much
more easy. Furthermore, the headache problem for the
selection of the orthogonal protecting group can be
reduced. Recently, Les P. Miranda and Morten Meldal
reported a uni-chemo protection concept.5 Uni-chemo
protection (UCP) comprises of an amino acid polymer
that can attach and protect amino groups. Each amino
groups were protected individually different degrees of
polymerized amino acid. De-protection of this protect-
ing group is performed simply using the Edman degra-
dation method6 that removes only N-terminal
mono-amino acid from every UCP groups. Depending
on the polymerization degree of UCP groups, each
amino groups could be characterized and controlled. We
applied this idea to the hydroxyl protection for oligo-
saccharide synthesis. To protect individual hydroxyl
groups on carbohydrates, individually different degree
of polymerized amino acid derivatives were introduced
by ester linkage. Subsequently, the polymerized amino
acid protecting groups were removed one by one from
N-terminal using Edman degradation. Each hydroxyl
groups were characterized and controlled using only one
kind of protecting group and only one de-protecting
method for applied oligosaccharide synthesis.
We attempted to synthesize one of the important cell-
surface tumor antigens, sialyl-T antigen, to demonstrate
the validity of these idea. It is a common marker of
epithelial tumors (e.g., prostate and breast carcinomas)
and some blood cell tumors.7 Two kinds of sialyl-T
antigens are a (2–3) and a (2–6) sialyl-T antigen. Using
this method, both analogues were satisfactory synthe-
sized from the same sugar parts merely by repeating
Edman degradation and coupling.
N-1-Ethyl propyl glycine was chosen as a UCP group to
avoid the existing chiral center of carbon compared with
N-1-methyl propyl glycine, which was used by Miranda
and Meldal5 (Scheme 1). The NMR peaks were very
complicated despite use of the N-1-ethyl propyl group
Keywords: UCP;New hydroxy protecting group;Oligosaccharide
library;Sialyl-T.
* Corresponding authors. Tel.: +81-29-838-8071;fax: +81-29-838-
7321;e-mail addresses:
affrc.go.jp
skomba@nfri.affrc.go.jp; kasumi@nfri.
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.02.032

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S. Komba et al. / Tetrahedron Letters 45 (2004) 2759–2762
3; R1 = H, R2 = tBu
O
a
O
4; R1 = Fmoc, R2 = Bub
+
Br
O
t
2
N
NH2
O
R1
R
5; R1 = Fmoc, R2 = Hc
1
2
O
O
f
8; R = t Bu
9; R = Hg
d
6; R = t Bu
7; R = He
N
O
N
O
3+7
3+5
R
R
Fmoc
3
Fmoc
2
Scheme 1. Preparation of UCP group. Reagents and conditions: (a) THF, rt, 4 h, 98%;(b) 1.0 equiv Fmoc–Cl, 1,4-dioxane/satd aq NaHCO 3, rt, 2 h,
86%;(c) 90% formic acid, 40 °C, 7 h, 90%;(d) 2 equiv DIPEA, 1.5 equiv PyBroP, DMF, rt, 30 min, 91%;(e) 90% formic acid, 50 °C, 3 h, 93%;(f)
2 equiv DIPEA, 1.5 equiv PyBroP, DMF, rt, 30 min, 85%;(g) 90% formic acid, 50 °C, 2 h, 98%.
Ph
O
OR
O
O
RO
O
O
OTBDMS
N3
OTBDMS
HO
N
O
Fmoc
N3
O
OH
O
10
N
5
11; R = benzylidenea
N
Fmoc
OH
Fmoc
3
12; R = Hb
9
O
N
O
N
Fmoc
Fmoc
O
3
RO
Boc
2
e
O
O
O
OAc
OTBDMS
N3
N
O
O
N3
15
OTBDMS
13; R = Hc
HO
14; R = Acd
Scheme 2. Preparation of GalNAc acceptor. Reagents and conditions: (a) 1.5 equiv 5, 5 equiv cyanuric chloride, py, 40 °C, 2 h, 90%;(b) 80% aq
AcOH, 60 °C, 2 h, 79%;(c) 1.1 equiv 9, 5 equiv cyanuric chloride, py, 40 °C, 2 h, 65%;(d) Ac 2O, py, 40 °C, 2 h, 91%;(e) (i) 20% piperidine in DMF, rt,
20 min. (ii) PITC, N-methyl morpholine (pH 8–9), rt, 30 min. (iii) TFA, CH2Cl2, rt, 30 min. (iv) Boc2O, satd aq NaHCO3, DMF, rt, 30 min, 69% (four
steps).
because of the rotamers of carbamate. GalNAc residue
was designed as Scheme 2.8 The 2-position was used
with N3 group for coupling Ser or Thr linked with a
configuration for future steps. The 3-position was pro-
tected by the mono-UCP group where the Gal residue
was introduced in the next step. The 4-position was
protected by the acetyl group as permanent protection.
The 6-position was protected by the tri-UCP group
where the sialic acid residue was introduced at final step.
The Gal residue was designed as Scheme 3.9 The 2-
position was protected by the benzoyl group, which
could make the glycoside linkage to a b configuration
through neighboring effect. The 3-position was pro-
tected by the mono-UCP group where sialic acid residue
was introduced at final step. The 4 and 6 positions were
protected by the acetyl group as permanent protection.
In addition, the sialic acid residue was used as the most
common thiophenyl donor (25).10
For synthesis of selectively 3-position free GalNAc
residue (15), compound 14 was treated by one cycle of
Edman degradation (Scheme 2). The one cycle of
Edman degradation was comprised of total four steps,
first step: de-protection of Fmoc or Boc group by using
20% piperidine in DMF or TFA in CH2Cl2, respectively,
second step: coupling PITC (phenyl isothiocyanate;
Edman reagent) with basic condition to the all amino
terminal, third step: cleaving of the N-terminal phenyl
thiocarbamoyl mono-amino acid derivative from all
UCP group by using TFA in CH2Cl2, fourth step:
re-protection by Boc group to the newly appeared all
N-terminal amino group. As a footnote, the mono-UCP
group was immediately removed during PITC coupling
step. The phenyl thiocarbamoyl mono-amino acid was
not stable under the such basic condition, because of
electron withdrawing effect in phenyl thiocarbamoyl
moiety. Also, the Boc re-protecting step is important
Ph
O
17; R1 = H, R2 = Fmoc, R3 = benzylidenea
18; R1 = Bz, R2 = Fmoc, R3 = benzylideneb
OR3
O
OR1
R3O
O
O
OH
O
19; R1 = Bz, R2 = Fmoc, R3 = Hc
20; R1 = Bz, R2 = Fmoc, R3 = Acd
21; R1 = Bz, R2 = Boc, R3 = Ace
SPh
SPh
HO
R2
N
O
O
16
N
5
Fmoc
OH
Scheme 3. Preparation of Gal donor. Reagents and conditions: (a) 1.1 equiv 5, 5 equiv cyanuric chloride, py, 0 °C, 1 h, 73%;(b) 1.5 equiv BzCl, py, rt,
2 h, 97%;(c) 80% aq AcOH, 60 °C, 2 h, 65%;(d) Ac 2O, py, rt, 2 h, 76%;(e) 20% piperidine in DMF, rt, 20 min then Boc 2O, satd aq NaHCO3, rt, 1 h,
83% (two steps).

S. Komba et al. / Tetrahedron Letters 45 (2004) 2759–2762
2761
O
N
Boc
OAc
O
2
O
OAc
a
b
AcO
15 + 21
O
O
OTBDMS
O
N
O
N3
Boc
O
OBz
22
N
Boc
O
OAc
OAc
OH
O
N3
OAc
O
OAc
O
AcO
HO
AcO
AcO
c
O
OTBDMS
OTBDMS
O
O
N3
OBz
OBz
23
24
OAc
OAc
COOMe
SPh
e
AcO
d
O
OAc
25
AcHN
OAc
OAc
COOMe
O
N
Boc
O
AcO
O
OAc
OAc
O
AcHN
OAc
AcO
OAc
O
OAc
AcO
AcO
OAc
OAc
O
O
N3
O
O
OTBDMS
OBz
O
AcO
AcHN
O
N3
O
OAc
COOMe
OTBDMS
OBz
27
26
ꢀ
Scheme 4. Preparation of two kinds of sialyl-T antigen analogues. Reagents and conditions: (a) 2 equiv NIS, 0.3 equiv TfOH, MS 4 A, CH2Cl2,
)40 °C, 2 h, 89%;(b) (i) TFA, CH 2Cl2, rt, 30 min. (ii) PITC, N-methyl morpholine (pH 8–9), rt, 30 min. (iii) TFA, CH2Cl2, rt, 30 min. (iv) Boc2O, satd
aq NaHCO3, DMF, rt, 30 min, 70% (four steps);(c) (i) Ac 2O, py, rt, 2 h, 99%. (ii) TFA, CH2Cl2, rt, 30 min. (iii) PITC, N-methyl morpholine (pH 8–
ꢀ
ꢀ
9), rt, 30 min, 98% (two steps);(d) 2 equiv NIS, 0.2 equiv TfOH, MS 3 A, CH3CN, )30 °C, 48 h, 13%;(e) 2 equiv NIS, 0.3 equiv TfOH, MS 3 A,
CH3CN, )30 °C, 10 h, 67% (a:b ¼ 71:29).
step. If amino free UCP protecting group was used for
next coupling, the target di-saccharide was not obtained
(unpublished data). Furthermore, if Fmoc group was
used for amino protection for UCP group instead of Boc
group, the di-UCP group was immediately cyclized and
then, removed from the hydroxyl group during Fmoc
cleaving condition at the next step. After treating one
cycle of Edman degradation, the polymerizing degree of
all UCP group equally degraded one degree, tri-UCP on
6-position was degraded to di-UCP, mono-UCP on 3-
position was degraded to free hydroxyl group. The yield
of this one cycle of Edman degradation was totally 69%,
which was acceptable for applying oligosaccharide syn-
thesis. This selectively 3-position free GalNAc acceptor
(15) was coupled with Gal donor (21) by using NIS and
TfOH activating method, to obtain selectively b and 3-
position glycosidic di-saccharide (22;T-antigen) as 89%
yield (Scheme 4). This di-saccharide (22) was merely
treated one cycle of Edman degradation, successfully
converted to 30-position free di-saccharide acceptor (23)
as 70% yield. This acceptor (23) was for synthesis of
sialyl (2–3) T antigen. To convert the compound 23 into
selectively 6-position free di-saccharide (24) that was the
acceptor for the synthesis of sialyl (2–6) T antigen,
successive 30-position acetylation and one cycle of
Edman degradation (which exclude third step and
fourth step) were carried out. These two kinds of di-
saccharide acceptor (23 and 24) were coupled with sialic
acid donor (25) by using NIS and TfOH activating
method in acetonitrile,11 to obtain sialyl (2–3) T antigen
(26) and sialyl (2–6) T antigen (27) derivatives, respec-
tively. Unfortunately, only b-glycosidic sialyl (2–3) T
antigen analogue was obtained instead of a-glycosidic
one that was essential configuration for the natural sialyl
(2–3) T antigen. Also, for the synthesis of sialyl (2–6) T
antigen, the anomeric nature was mixed as a:b ¼ 71:29.
In spite of using acetonitrile as the solvent at both
coupling, a selectivity was lost, due to sterical problem
and high reactivity of the primary hydroxyl group,
respectively.12;13
Nevertheless, this new method for oligosaccharide syn-
thesis using UCP group is quite promising because of
selectively cleavage of desired hydroxyl group and
stepwise de-protection using only one de-protecting
method. This may be the proof, the UCP method here is
the basic technique to the oligosaccharide library syn-
thesis.
Acknowledgements
We thank Dr. H. Ono and Dr. I. Maeda for NMR
measurements and Dr. M. Kameyama and Dr. T. Mu-
rata for MS measurements.
References and notes
1. Plante, O. J.;Palmacci, E. R.;Seeberger, P. H. Science
2001, 291, 1523–1527.
2. Sears, P.;Wong, C. H. Science 2001, 291, 2344–2350.
3. Wong, C. H.;Ye, X. S.;Zhang, Z. Y. J. Am. Chem. Soc.
1998, 120, 7137–7138.

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S. Komba et al. / Tetrahedron Letters 45 (2004) 2759–2762
4. Ye, X. S.;Wong, C. H. J. Org. Chem. 2000, 65, 2410–
2431.
5. Miranda, L. P.;Meldal, M. Angew. Chem., Int. Ed. 2001,
40, 3655–3657.
6. Edman, P. Acta Chem. Scand. 1950, 4, 283–293.
7. Finn, O. J.;Jerome, K. R.;Henderson, R. A.;Pecher, G.;
Domenech, N.;Magarian-Blander, J.;Barratt-Boyes,
S. M. Immunol. Rev. 1995, 145, 61–89.
8. Grundler, G.;Schmidt, R. R. Liebigs Ann. Chem. 1984,
1826–1847.
59.99 ((CH3CH2)2CH), 60.95, 61.10 (C-6 Gal), 62.76 (C-6
GalN3), 62.96, 63.01 (C-7 SA and C-9 SA), 64.95, 65.00
(C-2 GalN3), 67.70 (C-4 SA), 68.41, 68.47 (C-4 GalN3),
69.03, 69.06, 69.51, 70.01, 70.07, 70.12, 70.19, 70.34, 70.38
(C-4 Gal, C-5 Gal, and C-3 Gal), 71.60, 71.62 (C-5
GalN3), 71.71 (C-2 Gal), 72.54 (C-8 SA), 73.16 (C-6 SA),
75.08, 75.24 (C-3 GalN3), 79.95, 80.04 (Me3CO), 97.55,
97.61 (C-1 GalN3), 98.93 (C-2 SA), 100.90, 100.93 (C-1
Gal), 128.61, 129.81, 130.14, 130.17, 133.28, 133.31
(PhCOO), 155.66, 156.30 (C@O Boc), 165.71 (C@O Bz),
166.51 (C-1 SA), 169.77, 169.84, 169.86, 169.93, 170.08,
170.27, 170.67, 170.71, 171.17, 171.25, 171.27, 172.35,
172.38 (NCH2CO and C@O 8Ac). ESI-FT-MS, calcd for
C63H93N5O29SiNaþ (M+Naþ): 1434.56177, found:
9. Ellervik, U.;Magnusson, G. J. Org. Chem. 1998, 63, 9314–
9322.
€
10. Marra, A.;Sina y, P. Carbohydr. Res. 1989, 187, 35–42.
11. (a) Murase, T.;Ishida, H.;Kiso, M.;Hasegawa, A.
Carbohydr. Res. 1988, 184, c1–c4;(b) Hasegawa, A.;
J. Carbo-
1434.56135. ½aꢀ 17.6 (c 0.5, CHCl3).
D
Nagahama, T.;Ohki, H.;Hotta, K.;Kiso, M.
hydr. Chem. 1991, 10, 493–498.
Compound 27 a configuration, 1H NMR (600 MHz,
CDCl3) d (ppm);1.88 (t, 1H, J3ax:;4 ¼ Jgem 12.7 Hz, H-3ax.
SA), 2.52 (dd, 1H, J3eq:;4 4.6 Hz, Jgem 12.7 Hz, H-3eq. SA),
3.25 (dd, 1H, J5;6 3.7 Hz, Jgem 10.1 Hz, H-6 GalN3), 3.76
12. Boons, G. J.;Demchenko, A. V. Chem. Rev. 2000, 100,
4539–4565.
13. Compound 26, 1H NMR (600 MHz, CDCl3) d (ppm);
1.39, 1.45 (2s, 9H, Boc), 1.72 (br t, 1H, H-3ax. SA), 2.43
(dd, 1H, J3eq:;4 4.6 Hz, Jgem 13.3 Hz, H-3eq. SA), 3.11, 3.12
(2s, 3H, COOMe), 3.83 (m, 2H, H-9 SA), 3.97 (m, 1H, H-5
SA), 4.47, 4.47 (2d, 1H, J1;2 7.7, 7.8 Hz, H-1 GalN3), 4.65
(dd, 1H, J5;6 10.5 Hz, J6;7 1.5 Hz, H-6 SA), 4.89, 4.89 (2d,
1H, J1;2 7.9, 8.0 Hz, H-1 Gal), 4.94, 4.95 (2dd, 1H, J2;3 10.1,
9.7 Hz, J3;4 3.5, 3.1 Hz, H-3 Gal), 5.04 (ddd, 1H,
J3ax:;4 ¼ J4;5 11.0 Hz, J3eq:;4 4.5 Hz, H-4 SA), 5.27 (m, 1H,
H-8 SA), 5.37 (m, 1H, H-7 SA). 13C NMR (150 MHz,
CDCl3) d (ppm); )5.18, )4.35 (Me2Si), 10.99, 11.01, 11.19
(2CH3CH2), 17.97 (Me3CSi), 20.52, 20.53, 20.66, 20.69,
20.83, 20.96, 20.99, 21.04, 21.06 (7CH3COO), 23.26
(CH3CON), 25.58, 25.59 (Me3CSi), 25.91, 25.96, 26.39
(2CH3CH2), 28.25, 28.40 (Me3CO), 37.26, 37.34 (C-3 SA),
43.54 (NCH2CO), 48.57 (C-5 SA), 52.40 (COOMe), 58.31,
(dd, 1H, J5;6 2.4 Hz, Jgem 10.1 Hz, H-60 GalN3), 4.00 (m, 1H,
0
H-9 SA), 4.04 (m, 1H, H-90 SA), 4.04 (m, 1H, H-5 SA), 4.45
(d, 1H, J1;2 7.7 Hz, H-1 GalN3), 4.25 (dd, 1H, J5;6 12.3 Hz,
J
6;7 2.3 Hz, H-6 SA), 4.93 (d, 1H, J1;2 7.9 Hz, H-1 Gal), 4.82
(m, 1H, H-4 SA), 5.32 (m, 1H, H-8 SA), 5.32 (dd, 1H, J6;7
2.3 Hz, J7;8 4.9 Hz, H-7 SA). 13C NMR (150 MHz, CDCl3) d
(ppm);37.63 (C-3 SA), 49.33 (C-5 SA), 52.85 (COO Me),
60.92 (C-6 Gal), 62.28 (C-6 SA and C-9 SA), 63.57 (C-6
GalN3), 65.29 (C-2 GalN3), 66.92 (C-4 Gal), 67.11 (C-7
SA), 67.63 (C-8 SA), 68.42 (C-4 GalN3), 69.04 (C-4 SA),
69.40 (C-2 Gal), 70.58 (C-3 Gal), 70.74 (C-5 Gal), 72.86 (C-
5 GalN3), 75.90 (C-3 GalN3), 97.36 (C-1 GalN3), 98.45 (C-
2 SA), 101.32 (C-1 Gal), 165.11 (C@O Bz), 167.75 (C-1
SA). ESI-FT-MS, calcd for C53H75N4O27Siþ (M+Hþ):
1227.43825, found: 1227.43891. The a to b ratio was
calculated from H-3 equatorial proton of NMR.