Di-tert-butylsilylene-directed α-selective synthesis of 4-methylumbelliferyl T-antigen

Article abstract of DOI:10.1021/ol051592z

(Chemical Equation Presented) We have succeeded in the facile synthesis of 4-methylumbelliferyl T-antigen as a substrate for endo-α-N- acetylgalactosaminidase by exploiting the combination of the di-tert-butylsilylene effect and the Mitsunobu reaction.

Full text of DOI:10.1021/ol051592z

ORGANIC
LETTERS
2005
Vol. 7, No. 20
4415-4418
Di-tert-butylsilylene-Directed
Synthesis of 4-Methylumbelliferyl
T-Antigen
r-Selective
Akihiro Imamura,† Hiromune Ando,*,‡ Hideharu Ishida,† and Makoto Kiso*,†
Department of Applied Bioorganic Chemistry, Gifu UniVersity and CREST, Japan
Science and Technology Corporation (JST), 1-1 Yanagido, Gifu 501-1193, Japan, and
Life Science Research Center, Gifu UniVersity, Gifu 501-1193, Japan
hando@cc.gifu-u.ac.jp; kiso@cc.gifu-u.ac.jp
Received July 7, 2005
ABSTRACT
We have succeeded in the facile synthesis of 4-methylumbelliferyl T-antigen as a substrate for endo-r-N-acetylgalactosaminidase by exploiting
the combination of the di-tert-butylsilylene effect and the Mitsunobu reaction.
endo-R-N-Acetylgalactosaminidase is a glycosidase of wide-
spread occurrence in the bacteria kingdom.1 The enzyme
hydrolyzes the O-glycosidic R-linkage between T-antigen
[â-D-Gal-(1f3)-R-D-GalNAc] and a serine or threonine
residue in mucin-type glycoprotein.2 To elucidate the sub-
strate specificity of this enzyme, or screen the new species
from other living organisms, sensitive synthetic fluorogenic
T-antigen probes are intensively desired.
In this paper, we report the synthesis of 4-methylumbel-
liferyl (4-MU) T-antigen 1 (Figure 1) as a sensitive fluoro-
genic probe, featuring a di-tert-butylsilylene (DTBS)-directed
R-selective Mitsunobu reaction.
4-MU glycosides have been popular type of fluorogenic
probe for hydrolases because of the potent fluorometric
property of the phenolic counterpart liberated by enzymatic
hydrolysis.3 However, the 4-MU glycoside synthesis is
generally difficult.4 In particular, the synthesis of R-gly-
Figure 1. Structure of 4-methylumbelliferyl T-antigen.
cosaminides such as the title compound is extremely arduous
in order to circumvent the participatory effects of the N-acetyl
group. In their synthesis of 4-MU-R-GalNAc, Lemieux and
co-workers utilized 2-azidogalactosyl chloride as a glycosyl
donor as it possesses a nonparticipatory group at C2.
Unfortunately, its synthesis required many laborious ma-
nipulations. Moreover, the glycosyl donor could only be
coupled to 4-methylumbelliferone (4-MU-OH) in poor yield
(33%).5 To solve this synthetic issue, we envisaged using
* To whom correspondence should be addressed.
† Department of Applied Bioorganic Chemistry, Gifu University.
‡ Life Science Research Center, Gifu University.
(1) (a) Tanaka, Y.; Takahashi, Y.; Shinose, M.; Omura, S.; I.-Karakasa,
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H.; Yamamoto, K.; Murata, T.; Usui, T.; Kumagai, H. Arch. Biochem.
Biophys. 2000, 373, 394-400.
(2) (a) Iwase, H.; Hotta, K. Methods Moll. Biol. 1993, 14, 151-159. (b)
Bhavanandan, V. P.; Codington, J. F. Carbohydr. Res. 1983, 118, 81-89.
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23-33.
10.1021/ol051592z CCC: $30.25
© 2005 American Chemical Society
Published on Web 09/01/2005

our DTBS-directing R-selective galactosylation6 for 4-MU
T-antigen synthesis.
Scheme 2. Condensation of 7 and 4-Methylumbelliferone 8
Taking the advantage of the compatibility of our R-selec-
tive galactosylation with acyl functionality on C-2 amino
groups, we designed the N-2,2,2-trichloroethoxycarbonyl
(Troc)-protected disaccharide 7 as a DTBS glycosyl donor.
Treatment of the readily accessible 2-N-Troc galactothiogly-
coside 27 with DTBS(OTf)2 in pyridine8 gave 4,6-silylated
3 in 93% yield, which was then orthogonally glycosylated
with the 1,2,3,4,6-penta-O-acetyl-â-D-galactopyranose 4
catalyzed by trimethylsilyl trifluoromethanesulfonate9 to
afford disaccharide 5 in 67% yield. The hemiacetalization
of 5 with NBS in aqueous acetone10 produced 6. Finally,
the hemiacetal 6 was converted into the corresponding
chloride 7 by the action of Vilsmeier’s reagent11 (Scheme
1).
Scheme 1. Preparation of Gal â(1f3) GalN Disaccharide
6 was reacted with 8 in the presence of various combinations
of trialkyl phosphines (TPP,12 TBP,13,15 DPPE14) and azo-
compounds (DEAD,12 ADDP,13 TMAD,15 DIAD12,14) as
summarized in Table 1. Surprisingly, the anomeric config-
Table 1. 4-Methylumbelliferylation by Mitsunobu Reaction
phosphine/azo MU-OH
% yieldc
(R/â)
entry
compdb
(equiv) solvent T (°C)
With the glycosyl chloride 7 in hand, we then subjected
it to a DTBS-directing R-glycosidation with 4-methyl-
umbelliferone. Initially, we attempted reaction of the R-chlo-
ride 7 with 4-methylumbelliferone 8 in the presence of the
silver triflate-γ-collidine complex.5 This reaction provided
the R-glycoside 9 exclusively in 24% yield together with
the hemiacetal 6 as the main byproduct (Scheme 2).
However, the yield could not be elevated any further.
Accordingly, we next investigated the utility of the
Mitsunobu reaction in this capacity.12 Thus, the hemiacetal
1d
2e
3f
4g
5
TPP/DEAD
TBP/ADDP
TBP/TMAD
DPPE/DIAD
TPP/DEAD
TBP/ADDP
TPP/DEAD
3.0
3.0
3.0
3.0
3.0
3.0
8.0
THF
THF
THF
THF
toluene
toluene
toluene
80
80
80
47:5
20:-
8:-
no reaction
74:8
80
130
130
130
6
7
62:15
80:9
a Every reaction was conducted under reflux condition. b TPP, triphen-
ylphosphine, TBP, tributylphosphine, DPPE, 1,2-bis(diphenylphosphino)-
ethane, DEAD, diethyl azodicarboxylate, ADDP, 1,1′-(azodicarbonyl)dipi-
peridine, TMAD, 1,1′-azobis(N,N′-dimethylformamide), DIAD, diisopropyl
azodicarboxylate. c Isolated yield. d See ref 12. e See ref 13. f See ref 15.
g See ref 14.
(5) Szweda, R.; Spohr, U.; Lemieux, R. U.; Schindler, D.; Bishop, D.
F.; Desnick, R. J. Can. J. Chem. 1989, 67, 1388-1391.
(6) Imamura, A.; Ando, H.; Korogi, S.; Tanabe, G.; Muraoka, O.; Ishida,
H.; Kiso, M. Tetrahedron Lett. 2003, 44, 6725-6728.
(7) Compound 2 was derived from galactosamine hydrochloride through
four-step manipulation (65% overall) according to the method of 2-N-Troc
glucothioglycoside: Yan, F.; Mehta, S.; Eichler, E.; Wakarchuk, W. W.;
Gilbert, M.; Schur, M. J.; Whitfield, D. M. J. Org. Chem. 2003, 68, 2426-
2431.
uration of 6 was mostly retained; the R-glycoside 9 pre-
dominating in all these reactions. Interestingly, the yield of
9 increased when the reaction was performed at higher
(8) Furusawa, K.; Ueno, K.; Katsura, T. Chem. Lett. 1990, 97-100.
(9) (a) Ogawa, T.; Beppu, K.; Nakabayashi, S. Carbohydr. Res. 1981,
93. C6-C9. (b) Paulsen, H.; Paal, M. Carbohydr. Res. 1984, 135, 53-69.
(10) Kaesbeck, L.; Kessler, H. Liebigs. Ann. Chem. 1997, 169-173.
(11) (a) Newman, M. S.; Sujeeth, P. K. J. Org. Chem. 1978, 43, 3, 4367-
4369. (b) Iversen, T.; Bundle, D. R. Carbohydr. Res. 1982, 103, 29-40.
(12) Mitsunobu, O. Synthesis 1981, 1-28.
(13) Tsunoda, T.; Yamamiya, Y.; Ito, S. Tetrahedron Lett. 1993, 34,
1639-1642.
(14) O’Neil, I. A.; Thompson, S.; Murray, C. L.; Kalindjian, S. B.
Tetrahedron Lett. 1998, 39, 7787-7790.
(15) (a) Tsunoda, T.; Otsuka, J.; Yamamiya, Y.; Ito, S. Chem. Lett. 1994,
539-542. (b) Tsunoda, T.; Yamamiya, Y.; Kawamura, Y.; Ito, S.
Tetrahedron Lett. 1995, 36, 2529-2530.
4416
Org. Lett., Vol. 7, No. 20, 2005

temperature (entries 5 and 6). Additionally, the use of excess
4-MU-OH (8.0 equiv) led to the optimum product yield
(80%) (entry 7).
Table 2. Various Arylations by Mitsunobu Reactiona
In contrast, in entries 1 and 2, the trichloroethoxyoxazole
10 was observed as a major byproduct. Furthermore, we
observed the transitory formation of 10 during all our
reactions according to TLC monitoring. This result implied
that the oxazole 10 is a reaction intermediate; in fact, the
isolated oxazole 10 reacted with 4-MU-OH in toluene under
reflux to afford R-glycoside 9 in 85% yield (Scheme 3).
entry
aryl alcohol
product
% yield (R/â)
1
2
MPOH
PNPOH
11
12
56:-
41:17
Scheme 3. Coupling of 10 and 8 in the Absence of Activators
a All reaction conditions are the same as entry 5 in Table 1.
ylphosphine oxide elimination, (ii) oxocarbenium ion forma-
tion then occurs, (iii) “through-space” electron-donation16
t
from axially oriented C4 or C6 hydroxyl places the Bu
moiety closer to the anomeric carbon, and (iv) attack of
4-MU-OH is then restricted to the R-face of anomeric center
due to steric hampering by the DTBS group (Scheme 4).
Finally, as depicted in Scheme 5, 4-MU glycoside 9 was
transformed into 1. Thus, deprotection of Troc group by the
action of Zn and subsequent acetylation provided acetamide
13 in 89% yield. Removal of 4,6-O-DTBS group by
TBAHF17 and sequential acetylation yielded fully acetylated
4-MU T-antigen 14 in 95% yield, which was subjected to
de-O-acetylation18 to afford free 4-methylumbelliferyl T-
antigen 1.
In conclusion, we have found a new method for forming
R-4-MU galactosaminide based upon the DTBS effect and
the Mitsunobu reaction. The synthesized 4-MU T-antigen
will serve as a powerful probe for enzymatic studies, e.g.,
seeking the unknown endo-R-N-acetylgalactosaminidase.
Significantly, we have shown that this reaction can produce
other R-aryl glycosides 11 and 12 in moderate yields (Table
2). Taking into consideration these results, we hypothesize
that the coupling reactions proceed as follows: (i) initial
formation of the oxazole intermediate results from triphen-
Scheme 4. Expected Reaction Mechanism on Aryl Glycosylation
Org. Lett., Vol. 7, No. 20, 2005
4417

Acknowledgment. This work was partly supported by
the Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) of Japan (Grant-in-Aid for Scientific
Research to M. Kiso, No. 17101007) and CREST of JST
(Japan Science and Technology Corporation.). We thank Ms.
Kiyoko Ito for technical assistance.
Scheme 5. Final Deprotections
Supporting Information Available: Full experimental
details and characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL051592Z
(16) (a) Miljkovic, M.; Yeagley, D.; Deslongchamps, P.; Dory, Y. L. J.
Org. Chem. 1997, 62, 7597-7604. (b) Bols, M.; Liang, X.; Jensen, H. H.
J. Org. Chem. 2002, 67, 8970-8974.
Now we are also undertaking the synthesis of other tumor-
associated glycan antigen probes having 4-MU.
(17) Furusawa, K. Chem. Lett. 1989, 509-510.
(18) Rothermel, J.; Faillard, H. Carbohydr. Res. 1990, 196, 29-40.
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