Rapid oligosaccharide synthesis using a fluorous protective group

Article abstract of DOI:10.1021/jo049425k

The Bfp-OH, a novel fluorous protecting reagent, was able to be easily prepared. The Bfp group was readily introduced to a carbohydrate, removed in high yield, and recyclable after cleavage. The use of the Bfp group made it possible to synthesize a pentasaccharide by minimal column chromatography purification. Each synthetic intermediate was able to be easily purified only by simple fluorous-organic solvent extraction and monitored by TLC, NMR, and MS.

Full text of DOI:10.1021/jo049425k

Ra p id Oligosa cch a r id e Syn th esis Usin g a F lu or ou s P r otective
Gr ou p
Tsuyoshi Miura,*,†,‡ Kohtaro Goto,† Hideki Waragai,† Hiroharu Matsumoto,† Yuriko Hirose,†
Masashi Ohmae,†,§ Hide-ki Ishida,† Ai Satoh,† and Toshiyuki Inazu*,†,|
The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0003, J apan, and Department of Applied
Chemistry, School of Engineering, and Institute of Glycotechnology, Tokai University, Kitakaname 1117,
Hiratsuka, Kanagawa 259-1292, J apan
tmiura@cis.ac.jp; inz@keyaki.cc.u-tokai.ac.jp
Received April 7, 2004
The Bfp-OH, a novel fluorous protecting reagent, was able to be easily prepared. The Bfp group
was readily introduced to a carbohydrate, removed in high yield, and recyclable after cleavage.
The use of the Bfp group made it possible to synthesize a pentasaccharide by minimal column
chromatography purification. Each synthetic intermediate was able to be easily purified only by
simple fluorous-organic solvent extraction and monitored by TLC, NMR, and MS.
In tr od u ction
chemistry.4,5 Curran and co-workers elaborated the fluo-
rous synthesis (fluorous-tag method) as a strategic
alternative to solid-phase synthesis.5 Recently, they have
also reported a fluorous mixture synthesis using a
fluorous silica gel.6 The fluorous protecting groups are
essential for the fluorous synthesis performance. Several
fluorous oxygen protecting groups such as acetal, silyl,
and benzyl groups have already been reported.7 Other
fluorous protecting groups for amino and carboxyl func-
tions have also been reported.8 Curran and co-workers
reported the fluorous disaccharide synthesis using the
fluorous benzyl protective group by a glycal method.7e
Unfortunately, their glycosylation method using a fluo-
rous glycosyl donor gave only the 2-deoxy disaccharides.
In addition, the yield for the reaction step to introduce
the fluorous benzyl group to the hydroxyl function was
The oligosaccharides on cell surfaces play important
roles in biological processes such as cell-cell interactions,
cell adhesion, and immunogenic recognition.1 However,
the synthesis of oligosaccharides is not easy, in contrast
to peptides and nucleotides, which are easily prepared
by a solid-phase synthesis using a commercially available
automatic synthesizer. Although the solid-phase synthe-
sis of oligosaccharides has also been actively studied,2
the usual solid-phase method suffers from some serious
disadvantages, such as reduced reactivity, the difficulty
of large-scale synthesis, and the inability to monitor the
reaction by TLC, NMR spectroscopic analysis, or mass
spectrometry.
A fluorous solvent such as perfluorohexane is insoluble
in most organic solvents and water, and three layers are
formed. A highly fluorinated (fluorous) compound exhibits
a high solubility for fluorous solvents and is readily
separated from nonfluorinated compounds by the simple
fluorous-organic solvent partition. Since Horva´th and
Raba´i used these properties to introduce the concept of
the fluorous biphasic system in 1994,3 fluorous chemistry
has been developed for use in several fields such as
combinatorial chemistry, parallel synthesis, and catalytic
(4) (a) Tzschucke, C. C.; Markert, C.; Bannwarth, W.; Roller, S.;
Hebel, A.; Haag, R. Angew. Chem., Int. Ed. 2002, 41, 3964. (b)
Nishikido, J .; Kamishima, M.; Matsuzawa, H.; Mikami, K. Tetrahedron
2002, 58, 8345. (c) Rocaboy, C.; Gladysz, J . A. Org. Lett. 2002, 4, 1993.
(d) Nakamura, Y.; Takeuchi, S.; Okumura, K.; Ohga, Y. Tetrahedron
2001, 57, 5565. (e) Barrett, A. G. M.; Braddock, D. C.; Catterick, D.;
Chadwick, D.; Henschke, J . P.; McKinnell, R. M. Synlett 2000, 847. (f)
Horva´th, I. T. Acc. Chem. Res. 1998, 31, 641 and references therein.
(5) (a) Zhang, Q.; Luo, Z.; Curran, D. P. J . Org. Chem. 2000, 65,
8866. (b) Curran, D. P. Pure Appl. Chem. 2000, 72, 1649. (c) Curran,
D. P. Angew. Chem., Int. Ed. 1998, 37, 1174 and references therein.
(6) (a) Zhang, W.; Luo, Z.; Chen, C. H.; Curran, D. P. J . Am. Chem.
Soc. 2002, 124, 10443. (b) Curran, D. P.; Furukawa, T. Org. Lett. 2002,
4, 2233. (c) Zhang, Q.; Rivkin, A.; Curran, D. P. J . Am. Chem. Soc.
2002, 124, 5774. (d) Curran, D. P. Synlett 2001, 1488. (e) Luo, Z.;
Zhang, Q.; Oderaotoshi, Y.; Curran, D. P. Science 2001, 291, 1766.
(7) (a) Wipf, P.; Reeves, J . T.; Balachandran, R.; Giuliano, K. A.;
Hamel, E.; Day, B. W. J . Am. Chem. Soc. 2000, 122, 9391. (b) Ro¨ver,
S.; Wipf, P. Tetrahedron Lett. 1999, 40, 5667. (c) Wipf, P.; Reeves, J .
T. Tetrahedron Lett. 1999, 40, 5139. (d) Wipf, P.; Reeves, J . T.
Tetrahedron Lett. 1999, 40, 4649. (e) Curran, D. P.; Ferritto, R.; Hua,
Y. Tetrahedron Lett. 1998, 39, 4937. (f) Studer, A.; Curran, D. P.
Tetrahedron 1997, 53, 6681.
* To whom correspondence should be addressed.
† The Noguchi Institute.
‡ Current address: Faculty of Pharmaceutical Sciences, Chiba
Institute of Science, J apan.
§ Current address: Department Material Chemistry, Graduate
School of Engineering, Kyoto University, J apan.
| Tokai University.
(1) (a) Varki A. Glycobiology 1993, 3, 97. (b) Dwek, R. A. Chem. Rev.
1996, 96, 683. (c) Blithe, D. L. Trends Glycosci. Glycotech. 1993, 5, 81.
(2) (a) Manabe, S.; Ito, Y. J . Am. Chem. Soc. 2002, 124, 12638. (b)
Ando, H.; Manabe, S.; Nakahara, Y.; Ito, Y. J . Am. Chem. Soc. 2001,
123, 3848. (c) Plante, O. J .; Palmacci, E. R.; Seeberger, P. H. Science
2001, 291, 1523. (d) Eichler, E.; Yan, F.; Sealy, J .; Whitfield, D. M.
Tetrahedron 2001, 57, 6679. (e) Ito, Y.; Kanie, O.; Ogawa, T. Angew.
Chem., Int. Ed. Engl. 1996, 35, 2510. (f) Douglas, S. P.; Whitfield, D.
M.; Krepinsky, J . J . J . Am. Chem. Soc. 1995, 117, 2116; and references
therein.
(8) (a) Curran, D. P.; Amatore, M.; Guthrie, D.; Campbell, M.; Go,
E.; Luo, Z. J . Org. Chem. 2003, 68, 4643. (b) Schwinn, D.; Bannwarth,
W. Helv. Chim. Acta 2002, 85, 255. (c) Filippov, D. V.; Zoelen, D. J .;
Oldfield, S. P.; Marel, G. A.; Overkleeft, H. S.; Drijfhout, J . W.; Boom,
J . H. Tetrahedron Lett., 2002, 43, 7809. (d) Pardo, J .; Cobas, A.;
Guitla´n, E.; Castedo, L. Org. Lett. 2001, 3, 3711. (e) Luo, Z.; Williams,
J .; Read, R. W.; Curran, D. P. J . Org. Chem. 2001, 66, 4261.
(3) Horva´th, I. T.; Ra´bai, J . Science 1994, 266, 72.
10.1021/jo049425k CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/03/2004
5348
J . Org. Chem. 2004, 69, 5348-5353

Rapid Fluorous Oligosaccharide Synthesis
fluorous carboxylic acid 6 (MW ) 1023) in 98% yield. We
thought that the two fluorous chains of 6 enhance the
efficiency of the liquid-liquid extraction. Some methylene
spacers might effectively block the strong electron-
withdrawing effect of the long perfluoroalkyl chain
without a decrease in the reactivity of the carboxylic
group. We named the acyl moiety of 6, the Bfp (bisfluo-
rous chain type propanoyl) group.
Among the many useful methods for glycosylation, we
selected Schmidt’s popular imidate method for oligosac-
charide synthesis.12 We first attempted to synthesize the
disaccharide 12 as shown in Scheme 2. The Bfp group
was easily introduced to the three hydroxyl functions of
the mannose derivative 713 using N,N′-dicyclohexylcar-
bodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP)
to give the fluorous compound 8. The triphenylmethyl
(Trt) group of 8 was removed by treatment with cam-
phorsulfonic acid (CSA) in MeOH-ether to afford the
flourous glycosyl acceptor 9. The fluorous disaccharide
11 was obtained by the reaction of 9 with the excess
glycosyl donor 10 in the presence of trimethylsilyl trif-
luoromethanesulfonate (TMS-OTf) in ether/EtOC4F9.14
The glycosylation yield was much improved using ether/
EtOC4F9 as the reaction solvent system.9a The fluorous
intermediates 8, 9, and 11 were each extracted with the
fluorous solvent FC-7215 by partitioning the product
mixtures between FC-72 and an organic solvent. No
further purification such as silica gel column chroma-
tography was carried out. The Bfp group of 11 was easily
removed by treatment with NaOMe in MeOH/ether to
afford the crude 12, which was extracted with MeOH
by partitioning the mixture between FC-72 and MeOH.
The methyl ester of Bfp (Bfp-OMe, 5b ) was recovered
from the FC-72 layer in 81% yield. Compound 5b was
treated with aqueous sodium hydroxide to give Bfp-OH
(6), which was reused. Finally, the pure disaccharide 12
was obtained from a single silica gel column chromato-
graphic purification step in 59% overall yield from 7 (four
steps).
Next, we synthesized the longer chain oligosaccharide
as shown in Scheme 3. The fluorous glycosyl acceptor 9
was coupled with the glycosyl donor 1316 to afford the
fluorous disaccharide 14. The TBDPS group of 14 was
removed by treatment with HF-pyridine in THF to give
the fluorous compound 15. The reaction of the glycosyl
acceptor 15 with the glycosyl donor 13 under similar
glycosylation conditions afforded the fluorous trisaccha-
ride 16. The repeat of similar deprotection and glycosy-
lation gave the fluorous tetrasaccharide 19. The fluorous
intermediates 14-19 were each extracted with the fluo-
rous solvent FC-72 by partitioning the product mixtures
between FC-72 and an organic solvent (MeOH or MeCN).
No further purification such as silica gel column chro-
F IGURE 1. Concept of fluorous oligosaccharide synthesis.
not satisfactory. Recently, we reported a method for the
fluorous oligosaccharide synthesis involving the novel
fluorous acyl protective group and the fluorous support
in a preliminary communication.9 We would like to report
the full details of the development of a novel fluorous acyl
protective group Bfp and its application to the rapid
synthesis of oligosaccharides.
Our concept of fluorous oligosaccharide synthesis is
shown in Figure 1. We adopted the introduction of a
fluorous tag to the glycosyl acceptor but not to the
glycosyl donor in order to efficiently synthesize the longer
chain oligosaccharides. The glycosyl acceptor containing
the fluorous tag couples with the glycosyl donor to afford
the fluorous disaccharide. After the partition of the
reaction mixture with fluorous and normal organic
solvents, the fluorous disaccharide and the excess amount
of the glycosyl donor are extracted by the fluorous phase
and organic phase, respectively. After selective depro-
tection, repeating this procedure gives the fluorous
oligosaccharide, which is able to be purified only by
liquid-liquid extraction without column chromatogra-
phy. Finally, the fluorous tag is removed to give the
desired oligosaccharide extracted with an organic solvent.
The fluorous tag is extracted by a fluorous solvent and
is recyclable.
Resu lts a n d Discu ssion
We designed and synthesized compound 6, which
contains two fluorous chains, as a novel fluorous acyl
protecting reagent (Scheme 1). The reaction of the
â-alanine ethyl ester (1) with a fluorous tosylate 210
provided the monoalkylating product 3 in 83% yield.
Compound 3 was coupled with perfluorooctylpropionic
acid (411) to afford compound 5a in 93% yield. Treatment
of 5a with aqueous sodium hydroxide gave the desired
(11) Hungerhoff, B.; Sonnenschein, H.; Theil, F. J . Org. Chem. 2002,
67, 1781.
(12) Schmidt, R. R.; Michel, J .; Roos, M. Liebigs Ann. Chem. 1984,
1343.
(13) Tennant-Eyles, R. J .; Davis, B. G.; Fairbanks, A. J . Tetrahe-
dron: Asymmetry 2000, 11, 231.
(14) EtOC4F9 is a commercially available fluorocarbon solvent (3M,
Tokyo), which is called Novec HFE-7200.
(15) FC-72 is a commercially available fluorocarbon solvent (3M,
Tokyo), which consists of perfluorohexane (C6F14) isomers and is called
Fluorinert FC-72. The boiling point of FC-72 is 76 °C.
(16) Nicolaou, K. C.; Pfefferkorn, J . A.; Roecker, A. J .; Cao, G.-Q.;
Barluenga, S.; Mitchell, H. J . J . Am. Chem. Soc. 2000, 122, 9939.
(9) (a) Miura, T.; Hirose, Y.; Ohmae, M.; Inazu, T. Org. Lett. 2001,
3, 3947. (b) Miura, T.; Inazu, T. Tetrahedron Lett. 2003, 44, 1819. (c)
Miura, T.; Goto, K.; Hosaka, D.; Inazu, T. Angew. Chem., Int. Ed. 2003,
42, 2047.
(10) Campo, F. D.; Laste´coue`res, D.; Vincent, J .-M.; Verlhac, J .-B.
J . Org. Chem. 1999, 64, 4969.
J . Org. Chem, Vol. 69, No. 16, 2004 5349

Miura et al.
SCHEME 1 a
a
Reagents and conditions: (a) K2CO3, MeCN, reflux, 17 h, 83%; (b) PyBOP, Et3N, CH2Cl2, rt, 3 h, 93%; (c) 1 M NaOH, dioxane, 70 °C,
4 h, 98%.
SCHEME 2 a
a
Reagents and conditions: (a) 6, DCC, DMAP, CH2Cl2, rt, 20 h; (b) CSA, MeOH-CHCl3, rt, 3 h; (c) TMS-OTf, molecular sieves 4A,
Et2O, EtOC4F9, 0 °C, 30 min; (d) NaOMe, Et2O-MeOH, rt, 22 h, then silica gel chromatography, 59% from 7.
matography was carried out. The pure fluorous tetrasac-
charide 19 was obtained from a single silica gel column
chromatographic purification step in 11% overall yield
from 7 (eight steps). Although the yields for the glyco-
sylation step to synthesize the trisaccharide 16 (50%) and
the tetrasaccharide 18 (10%) were not satisfactory in the
preliminary communication,9a the glycosylation yields
were much improved using ether/EtOC4F9 as the reaction
solvent system (up to 70-85% from 10 to 75% yield in
each glycosylation step). The Bfp groups of 19 were
removed by treatment with NaOMe in MeOH/ether to
afford the tetrasaccharide 20, which was extracted with
MeOH by partitioning the mixture between FC-72 and
MeOH.
We also measured the liquid-liquid partition coef-
ficients for the fluorous oligosaccharides (9, 14, 16, and
18) between FC-72 and two organic solvents (meth-
anol and toluene) as shown in Table 1. The fluorous
compounds show higher partition coefficients for sep-
aration by FC-72-ethanol extraction than FC-72-
toluene extraction. Although the fluorine content of the
fluorous compound 18 is low (44.9%), its partition coef-
ficient is high in contrast to other known fluorous
compounds.5a
5350 J . Org. Chem., Vol. 69, No. 16, 2004

Rapid Fluorous Oligosaccharide Synthesis
SCHEME 3 a
a
Reagents and conditions: (a) TMS-OTf, 13, molecular sieves 4A, EtOC4F9, 0 °C; (b) HF-Py, THF, rt; (c) NaOMe, Et2O, MeOH, rt,
then silica gel chromatography, 11% from 7.
SCHEME 4 a
TABLE 1. P a r tition Coefficien ts of F lu or ou s
Oligosa cch a r id e in 50/50 (v/v) of F C-72/Or ga n ic Solven ts
(P ) Cflu or ou s p h a se/Cor ga n ic p h a se)
compd
F content (wt %)
FC-72/methanol
FC-72/toluene
9
60.4
51.9
48.1
44.9
>100
85
>100
9.4
14
16
18
60
21
3.9
2.2
Next, we synthesized the galactose â-(1-6) pentamer
36, which is known as a component part of arabinoga-
lactan-proteins,17 as an all-free oligosaccharide (Scheme
5). To synthesize the galactose â-(1-6) pentamer 36, we
used compound 23 as the glycosyl donor and compound
26 as the glycosyl acceptor, which had the anomer
hydroxyl function protected by a benzyl group. The
glycosyl donor 23 was prepared as shown in Scheme 4.
The introduction of the TBDPS group to the primary
hydroxyl function of 2118 followed by treatment with
acetic anhydride afforded compound 22 in 93%onwedtion
yield. The treatment of compound 22 with Pd(PPh3)4 in
acetic acid, followed by the reaction with trichloroaceto-
nitrile gave the glycosyl donor 23 in 70% yield. The
galactose â-(1-6) pentamer 36 was synthesized using
compound 23 as a glycosyl donor as shown in Scheme 5.
The Bfp group was introduced to the three hydroxyl
a
Reagents and conditions: (a) TBDPS-Cl, Py, rt, 18 h, then
Ac2O, rt, 3 h; (b) (i) Pd(PPh3)4, AcOH, 80 °C, 1 h; (ii) Cl3CCN, DBU,
CH2Cl2, 0 °C, 1 h.
functions of the galactose derivative 2419 using DCC and
DMAP to give the fluorous compound 25. The triphenyl-
methyl (Trt) group of 25 was removed by treatment with
camphorsulfonic acid (CSA) in MeOH-CHCl3 to afford
the flourous glycosyl acceptor 26. Compound 26 was
unstable because the Bfp group (4-position) of compound
26 easily migrated to the primary hydroxyl function (6-
position). Therefore, it was difficult to isolate compound
26 by silica gel column chromatographic purification. The
fluorous glycosyl acceptor 26 was coupled with the
glycosyl donor 23 to afford the fluorous disaccharide 27.
(17) Kuroyama, H.; Tsutsui, N.; Hashimoto, Y.; Tsumuraya, Y.
Carbohydr. Res. 2001, 333, 27.
(18) Lee, R. T.; Lee, Y. C. Carbohydr. Res. 1974, 37, 193.
(19) Miyai, K.; J eanloz, R. W. Carbohydr. Res. 1972, 21, 45.
J . Org. Chem, Vol. 69, No. 16, 2004 5351

Miura et al.
SCHEME 5 a
a
Reagents and conditions: (a) 6, DCC, DMAP, CH2Cl2, rt, 2 h; (b) CSA, LiCl, CHCl3, MeOH, rt, 2h; (c) 23 TMS-OTf, molecular sieves
4A, EtOC4F9, Et2O, 0 °C, (d) HF-Py, THF, rt; (e) NaOMe, Et2O, MeOH, rt, 3 h; (f) H2, Pd/C, EtOH, H2O, rt, 18 h.
The TBDPS group of 27 was removed by treatment with
HF-pyridine in THF to give the fluorous compound 28.
The reaction of the glycosyl acceptor 28 with the glycosyl
donor 23 under similar glycosylation conditions afforded
the fluorous trisaccharide 29. The repeat of similar
deprotection and glycosylation gave the fluorous pen-
tasaccharide 33. The intermediates 25-33 were each
extracted with FC-72 by partitioning the product mix-
tures between FC-72 and an organic solvent. No further
purification such as silica gel column chromatography
was carried out. The pure fluorous pentasaccharide 33
was obtained from a single silica gel column chromato-
graphic purification step in 29% overall yield from 24
(nine steps). After treatment of 33 with HF-pyridine, the
Bfp group of 34 was removed by treatment with NaOMe
in MeOH/ether to afford the crude 35, which was
extracted with MeOH by partitioning the mixture be-
tween FC-72 and MeOH. The crude product 35 was
isolated by ODS silica gel column chromatographic
purification in 92% yield from 33 (two steps). The benzyl
group of 35 was removed by hydrogenation in the
presence of Pd/C to afford the galactose â-(1-6) pentamer
36. Compounds 28, 30, 32, and 34 were also unstable
because the acetyl group easily migrated to the primary
hydroxyl function. It is worth noting that the unstable
compounds (26, 28, 30, 32, and 34) were able to be
5352 J . Org. Chem., Vol. 69, No. 16, 2004

Rapid Fluorous Oligosaccharide Synthesis
quickly purified by partitioning the product mixtures
between FC-72 and an organic solvent without decom-
position.
fluorous compounds containing the Bfp group are some-
what broad due to the influences of the amide linkages
and the fluorous groups.7e Although the fluorous inter-
mediates could also be subjected to silica gel column
chromatography if necessary, only the final compounds
were purified by chromatography. This fluorous oligosac-
charide synthesis should be applicable to large-scale
synthesis as it is performed in the liquid phase. Thus,
oligosaccharide synthesis using the fluorous protecting
group Bfp is an attractive strategic alternative to solid-
phase oligosaccharide synthesis, and removed some of the
disadvantages of the usual solid-phase method. Further
application to the synthesis of bioactive carbohydrates
and glycoconjugates is now in progress.
Con clu sion
The use of Bfp group as a fluorous protective group
made it possible to rapidly synthesize a natural oligosac-
charide by minimal column chromatography purification.
The fluorous protecting reagent 6 (Bfp-OH) could be
easily prepared on a large scale. The Bfp group was
readily introduced to the carbohydrate hydroxyl func-
tions, removed in high yield by the usual procedure, and
was recyclable after cleavage. Only three Bfp groups
made it possible to extract the derivative of the pentasac-
charide with the FC-72 phase.20 Each fluorous synthetic
intermediate could be obtained in a straightforward
manner by simple FC-72-organic solvent extraction. The
reaction conditions for each synthetic step could be
rapidly optimized because the reactions could be moni-
tored as a single compound by TLC and mass spectrom-
etry, in contrast to the usual solid-phase reactions. The
fluorous intermediates could also be measured by NMR
spectroscopic analysis. The peaks in the NMR of the
Ack n ow led gm en t. This work was partly supported
by Grants-in-Aid for Scientific Research (C) (Nos.
11680598 and 13680680) and a Grant-in-Aid for En-
couragement of Young Scientists (No. 13771349) from
the J apan Society for the Promotion of Science, by a
grant for Hi-Tech Research from Tokai University, and
by the Takeda Science Foundation. This work was
performed through the Noguchi Fluorous Project by our
institute.
(20) The fluorous monosaccharide derivative, which was introduced
only one Bfp group into the anomeric hydroxyl function of 2,3,4,6-tetra-
O-benzyl-D-galactose, cannot be extracted with FC-72 by partitioning
between FC-72 and organic solvent.
(21) Eisenbraun, E. J . Organic Syntheses; Wiley: New York, 1973;
Collect. Vol. V, p 310.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedure and 1H NMR and 13C NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
J O049425K
J . Org. Chem, Vol. 69, No. 16, 2004 5353