Automated solution-phase synthesis of oligosaccharides via iterative electrochemical assembly of thioglycosides

Article abstract of DOI:10.1021/ol402034g

A new iterative one-pot sequential method for the solution-phase synthesis of oligosaccharides has been devised on the basis of the electrochemical oxidation of a propagating thioglycoside terminus to generate the corresponding triflate, followed by the r

Full text of DOI:10.1021/ol402034g

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Automated Solution-Phase Synthesis
of Oligosaccharides via Iterative
Electrochemical Assembly of
Thioglycosides
Toshiki Nokami,*,†,§ Ryutaro Hayashi,† Yoshihiro Saigusa,† Akihiro Shimizu,†
Chih-Yueh Liu,‡ Kwok-Kong Tony Mong,‡ and Jun-ichi Yoshida*,†
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto University, Nishikyoku, Kyoto 615-8510, Japan, and Department
of Applied Chemistry, National Chiao Tung University, 1001, Ta-Hsueh Road,
Hsinchu 300, Taiwan, ROC
tnokami@chem.tottori-u.ac.jp; yoshida@sbchem.kyoto-u.ac.jp
Received July 24, 2013
ABSTRACT
A new iterative one-pot sequential method for the solution-phase synthesis of oligosaccharides has been devised on the basis of the
electrochemical oxidation of a propagating thioglycoside terminus to generate the corresponding triflate, followed by the reaction with a
thioglycoside building block having a free hydroxyl group. A practical automated synthesizer was developed for the method and was effectively
used for assembling up to six thioglycoside building blocks to synthesize partial structures of poly-β-D-(1À6)-N-acetylglucosamine.
The iterative assembly of small building blocks1 by the
integration of reactions2 in a one-pot sequential manner
serves as a powerful method for constructing oligo-
saccharides.3 Although solid-phase oligosaccharide synth-
esis is advantageous from the viewpoint of separation of
intermediates and final products from excess reagents and
wastes derived from them,4 it often suffers from limited
scale, a high price for the solid supports, a low reactivity of
substrates on the resin-bound media, and difficulties in the
analysis of intermediates. To solve such problems, a one-
pot sequential solution-phase method has been developed.
However, at least two orthogonal activation protocols are
necessary because a glycosyl donor (precursor of reactive
intermediate) is usually activated in the presence of a
† Kyoto University
‡ National Chiao Tung University
§ Department of Chemistry and Biotechnology, Graduate School of En-
gineering, Tottori University. 4-101 Koyamachominami, 680-8552 Tottori,
Japan.
(3) (a) Seeberger, P. H.; Haase, W.-C. Chem. Rev. 2000, 100, 4349. (b)
Seeberger, P. H. Chem. Commun. 2003, 1115. (c) Seeberger, P. H. Chem.
Soc. Rev. 2008, 37, 19. (d) Boltje, T. J.; Kim, J.-H.; Park, J.; Boons, G.-J.
Nat. Chem. 2010, 2, 552. (e) Hsu, C.-H.; Hung, S.-C.; Wu, C.-Y.; Wong,
C.-H. Angew. Chem., Int. Ed. 2011, 50, 11872.
(1) (a) Ho, T.-K. Tactics of Organic Synthesis; Wiley: New York, 1994.
€
(b) Feuerbacher, N.; Vogtle, F. Top. Curr. Chem. 1998, 197, 1. (c) Yoshida,
J.; Maekawa, T.; Morita, Y.; Isoe, S. J. Org. Chem. 1992, 57, 1321. (d)
Yamago, S.; Yamada, T.; Maruyama, T.; Yoshida, J. Angew. Chem., Int.
Ed. 2004, 43, 2145. (e) Nokami, T.; Ohata, K.; Inoue, M.; Tsuyama, H.;
Shibuya, A.; Soga, K.; Okajima, M.; Suga, S.; Yoshida, J. J. Am. Chem.
Soc. 2008, 130, 10864. (f) Wang, C.; Glorius, F. Angew. Chem., Int. Ed.
2009, 48, 5240.
(4) (a) Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Science 2001,
291, 1523. (b) Werz, D. B.; Castagner, B.; Seeberger, P. H. J. Am. Chem.
Soc. 2007, 129, 2770. (c) Walvoort, M. T. C.; van den Elst, H.; Plante,
€
O. J.; Krock, L.; Seeberger, P. H.; Overkleeft, H. S.; van der Marel,
ꢀ
G. A.; Codee, J. D. C. Angew. Chem., Int. Ed. 2012, 51, 4393. (d) Ganesh,
N. V.; Fujikawa, K.; Tan, Y. H.; Stine, K. J.; Demchenko, A. V. Org.
€
Lett. 2012, 14, 3036. (e) Krock, L.; Esposito, D.; Castagner, B.; Wang,
(2) (a) Suga, S.; Yamada, D.; Yoshida, J. Chem. Lett. 2010, 39, 404.
(b) Yoshida, J.; Saito, K.; Nokami, T.; Nagaki, A. Synlett. 2011, 1189.
€
C.-C.; Bindschadler, P.; Seeberger, P. H. Chem. Sci. 2012, 3, 1617.
r
10.1021/ol402034g
XXXX American Chemical Society

glycosyl acceptor (building block).5 A method consisting
of activation of a precursor in the absence of a building
block, followed by the addition of a building block, is
beneficial because one activation protocol can be used
iteratively,1d,6 and therefore the glycosylation can be easily
repeated in a one-pot sequential manner enabling auto-
mated synthesis of oligosaccharides (Figure 1).7
direction of assembly from Seeberger method based on the
acceptor-bound solid-support approach. If the present
method would be a solid-support approach, it would be
the donor-bound version. We also report the synthesis of
partial structures of poly-β-D-(1À6)-N-acetylglucosamine
(PNAG), by assembling six thioglycosides in a one-pot
sequential manner using an automated synthesizer devel-
oped for the method.
We focused on the synthesis of oligoglucosamines,10
because PNAG has received significant research interest
as the major component of the biofilm formed by
pathogens.11 We initiated our study by optimizing the
structure of the aryl group in arylthioglycosides both
as precursors of glycosyl triflates and building blocks
(Table 1). Arylthioglycoside 1a (Ar = 4-CH3C6H4-,
Eox = 1.65 V vs SCE) was electrochemically oxidized to
generate glycosyl triflate 2 according to our previous
procedure (see the Supporting Information). The reac-
tion with building block 3a (Ar = 4-CH3C6H4-, Eox
=
1.54 V vs SCE), which has the same aryl group, gave
the corresponding disaccharide 4a in 84% yield. The 1,6-
anhydrosugar 5 was also obtained in 13% yield, which was
produced by the intramolecular glycosylation of 3a.12 To
prevent the formation of 5, the reaction with a building
block bearing a bulky aryl group (3b, Ar = 2,6-(CH3)2-
C6H3-, Eox = 1.50 V vs SCE) was examined.13 Although
the yield of 5 decreased slightly to 8%, the yield of the
desired 4b also decreased. However, the use of a building
block bearing an electron-withdrawing fluorine atom on
the phenyl ring (3c, Ar = 4-FC6H4-, Eox = 1.67 V vs SCE)
resulted in the formation of the corresponding disacchar-
ide 4c in 84% yield. Now, the 1,6-anhydrosugar 5 was
produced only in a trace amount. The structure of the
aryl group in the precursor proved important. The use of
building block 3c with precursor 1c gave the best result
among those examined (4c: 92% yield). Therefore, the
remaining syntheses of oligoglucosamines were carried out
using thioglycosides having the 4-FC6H4- group on sulfur.
Figure 1. Iterative synthesis of oligosaccharides based on the
activation of a thioglycoside donor in the absence of a glycosyl
acceptor.
Herein, we report a new method for automated solution-
phase synthesis of oligosaccharides based on electro-
chemical activation8 of thioglycosides to generate glycosyl
triflates in the absence of a building block.9 This method
enables us to make up oligosaccharides in the different
(9) (a) Nokami, T.; Shibuya, A.; Tsuyama, H.; Suga, S.; Bowers,
A. A.; Crich, D.; Yoshida, J. J. Am. Chem. Soc. 2007, 129, 10922. (b)
Nokami, T.; Tsuyama, H.; Shibuya, A.; Nakatsutsumi, T.; Yoshida, J.
Chem. Lett. 2008, 37, 942. (c) Nokami, T.; Shibuya, A.; Manabe, S.; Ito,
Y.; Yoshida, J. Chem.;Eur. J. 2009, 15, 2252. (d) Nokami, T.; Nozaki,
Y.; Saigusa, Y.; Shibuya, A.; Manabe, S.; Ito, Y.; Yoshida, J. Org. Lett.
2011, 13, 1544. (e) Nokami, T.; Shibuya, A.; Saigusa, Y.; Manabe, S.;
Ito, Y.; Yoshida, J. Beilstein J. Org. Chem. 2012, 6, 456. (f) Nokami, T.;
Saito, K.; Yoshida, J. Carbohydr. Res. 2012, 363, 1.
(5) (a) Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.;
Wong, C.-H. J. Am. Chem. Soc. 1999, 121, 734. (b) Tanaka, H.; Adachi,
M.; Tsukamoto, H.; Ikeda, T.; Yamada, H.; Takahashi, T. Org. Lett.
2002, 4, 4213. (c) Tanaka, H.; Matoba, N.; Tsukamoto, H.; Takimoto,
H.; Yamada, H.; Takahashi, T. Synlett 2005, 824.
(10) Chemical syntheses of PNAG, see: (a) Melean, L. G.; Love,
K. R.; Seeberger, P. H. Carbohydr. Res. 2002, 337, 1893. (b) Yang, F.;
He, H.; Du, Y. Tetrahedron Lett. 2002, 43, 7561. (c) Gening, M. L.;
Titov, D. V.; Grachev, A. A.; Gerbst, A. G.; Yudina, O. N.; Shashkov,
A. S.; Chizhov, A. O.; Tsvetkov, E.; Nifantiev, N. E. Eur. J. Org. Chem.
(6) (a) Crich, D.; Sun, S. J. Org. Chem. 1997, 62, 1198. (b) Crich, D.;
Sun, S. J. Am. Chem. Soc. 1997, 119, 11217. (c) Crich, D.; Sun, S. J. Am.
Chem. Soc. 1998, 120, 435. (d) Yamago, S.; Yamada, T.; Hara, O.; Ito,
Y.; Yoshida, J. Org. Lett. 2001, 3, 3867. (e) Crich, D.; Chandrasekera,
N. S. Angew. Chem., Int. Ed. 2004, 43, 5386. (f) Yamada, T.; Kinjyo, S.;
Yoshida, J.; Yamago, S. Chem. Lett. 2005, 34, 1556. (g) Yamada, T.;
Takemura, K.; Yoshida, J.; Yamago, S. Angew. Chem., Int. Ed. 2006, 45,
7575.
(7) (a) Huang, X.; Huang, L.; Wang, H.; Ye, X.-S. Angew. Chem., Int.
Ed. 2004, 43, 5221. (b) van den Bos, L. J.; Boltje, T. J.; Provoost, T.;
Mazurek, J.; Overkleeft, H. S.; van der Marel, G. A. Tetrahedron Lett.
2007, 48, 2697.
(8) For the electrochemical method in organic synthesis, see: (a)
Moeller, K. D. Tetrahedron 2000, 56, 9527. (b) Lund, H. J. Electrochem.
Soc. 2002, 149, S21. (c) Sperry, J. B.; Wright, D. L. Chem. Soc. Rev. 2006,
35, 605. (d) Yoshida, J.; Kataoka, K.; Horcajada, R.; Nagaki, A. Chem.
Rev. 2008, 108, 2265. (e) Waldvogel, S. R.; Elsler, B. Electrochim. Acta
2012, 82, 434.
ꢀ
ꢀ ꢀ
2010, 2465. (d) Fekete, A.; Borbas, A.; Gyemant, G.; Kandra, L.;
Fazekas, E.; Ramasubbu, N.; Antus, S. Carbohydr. Res. 2011, 346, 1445.
(11) Biological studies of PNAG, see: (a) Wang, X.; Preston, J. F., III;
Romeo, T. J. Bacteriol. 2004, 186, 2724. (b) Sloan, G. P.; Love, C. F.;
Sukumar, N.; Mishra, M.; Deora, R. J. Bacteriol. 2007, 189, 8270. (c) Izano,
E. A.; Sadovskaya, I.; Vinogradov, E.; Mulks, M. H.; Velliyagounder, K.;
Ragunath, C.; Kher, W. B.; Ramasubbu, N.; Jabbouri, S.; Perry, M. B.;
ꢀ
Kaplan, J. B. Microb. Pathog. 2007, 43, 1. (d) Gening, M. L.; Maira-Litran,
T.; Kropec, A.; Skurnik, D.; Grout, M.; Tsvetkov, Y. E.; Nifantiev, N. E.;
Pier, G. B. Infect. Immun. 2010, 78, 764.
(12) Glycosyl triflates work as thiophilic reagents, which can activate
thioglycoside building block to form 1,6-anhydrosugar.
(13) Li, Z.; Gildersleeve, J. C. J. Am. Chem. Soc. 2006, 128, 11612.
B
Org. Lett., Vol. XX, No. XX, XXXX

the addition of a building block (CH2Cl2 solution, 1.0 equiv,
1 min), and the reaction temperature. It takes 20 min
to change the temperature of the cooling bath from
À80 to À60 °C or vice versa (rate of temperature change
= ( 1 °C/min). The reaction with a building block was
carried out at À60 °C for 30 min.
Table 1. Optimization of the Structure of the Aryl Group in
Thioglycosides
Scheme 1. Stepwise Synthesis of Oligoglucosamines
a
a
precursor (Eox
)
building block (Eox
)
product (yield %)b
1a (1.65 V)
1a
3a (1.54 V)
3b (1.50 V)
3c (1.67 V)
3b
4a (84)/5 (13)
4b (76)/5 (8)
1a
4c (84)/5 trace
4b (76)/5 trace
1b (1.52 V)
1c (1.73 V)
c
3c
4c (92)/5 À
a Oxidation potentials V vs SCE. b NMR yields based on 1,1,2,2-
tetrachloroethane as an internal standard. c 5 was not detected by
1H NMR.
With the optimized aryl group for both the precursor
and the building block identified, we next performed the
stepwise synthesis of oligoglucosamines manually as shown
in Scheme 1. The electrochemical oxidation of thioglyco-
side precursors 4c, 6, 7, and 8 (n = 1À4) to generate the
corresponding glycosyl triflates and the subsequent reac-
tion with 1.2 equiv of building block 3c was performed to
obtain the corresponding oligoglucosamines 6À9 (n =
1À4), respectively. Oxidation potentials of oligoglucosa-
mines were around 1.7 V vs SCE, which is almost the same
as that of thioglycoside 1c (1.73 V vs SCE). Therefore, the
length of the oligoglucosamines did not affect the current
efficiency of the electrochemical oxidation and chemical
yields of the oligoglucosaminesexcept for hexaglucosamine
9.14 The successful result of manual operation prompted us
to develop an automated synthesizer for the method.
An automated synthesizer was developed by assembling
commercially available devices such as a syringe pump, a DC
power supply, a temperature controlling system, a magnetic
stirrer, an electrochemical reaction system equipped with a
divided electrolysis cell, and system controller (Figure 2).
The system controller,15 which is connected to a DC power
supply, a syringe pump, and a chiller, controls the schedule
of theelectrochemical oxidation(1.0F/mol,À80°C, 40min),
Figure 2. The automated oligosaccharide synthesizer and the
schedule of the automated process.
(14) We assume that reactivity of glycosyl triflates of pentasaccharide
might be low, because starting pentasaccharide 8 was consumed after the
electrolysis.
(15) The system controller is a Windows PC equipped with our
original program, which was written in LabVIEW (National Instrument
Corporation) to control instruments.
As shown in Table 2, several oligoglucosamines were
successfully synthesized using the automated synthesizer.
One of the advantages of solution-phase syntheses is that
Org. Lett., Vol. XX, No. XX, XXXX
C

better yields, and average yields were improved by around
10%. Although the targeted oligoglucosamines were con-
taminated with byproducts such as 1,6-anhydrosugar 5
and shorter oligoglucosamines, such byproducts were
easily separated at the end by preparative recycling gel
permeation chromatography (PR-GPC). Thus, partial
structures of PNAG were easily prepared. Repeating the
cycle, inprinciple, leadstothe synthesisofPNAGof higher
molecular weight.
Table 2. Automated Iterative Synthesis of Oligoglucosamines
In summary, we have developed an iterative method for
automated solution-phase synthesis of oligosaccharides
based on the electrochemical method. The electrochemical
method avoids the use of strong activators, which often
causes the formation of byproducts derived from them.
A practical automated synthesizer has been developed for
the method, and itwaseffectivelyusedfor assembling upto
six thioglycoside building blocks to synthesize partial
structures of PNAG. Further studies to obtain PNAGs
of higher molecular weight, and their modification for
biological tests, and the application of this method to the
synthesis of other oligosaccharides is in progress in our
laboratory.
yields %a (average yields % per cycle)
cycles oligoglucosamine
condition Ab
condition Bc
2
3
4
5
6 (n = 1)
7 (n = 2)
8 (n = 3)
9 (n = 4)
50 (71)
34 (70)
17 (64)
9 (62)
69 (82)
52 (81)
31 (75)
15 (68)
Acknowledgment. The authors gratefully acknowledge
financial support from Grants-in-Aid for Scientific
Research on Innovative Areas (No. 2105) from the
MEXT. T.N. thanks the Ube Foundation for financial
support. C.-Y.L. thanks the National Science Council,
Taiwan, and Interchange Association, Japan, for a study
visit to Japan. The authors thank Dr. Keiko Kuwata
of Kyoto University for MS analyses and Mr. Hideyo
Aoyama of The Institute of Creative Chemistry Co., Ltd.,
for the development of the automated synthesizer.
a Isolated yield. b Condition A (glycosylation temp: À60 °C, chan-
ging rate: ( 1 °C/min). c Condition B (glycosylation temp: À50 °C,
changing rate: ( 2 °C/min).
the reactions in each cycle can be easily monitored by
conventional analytical methods. Indeed, we observed
the corresponding molecular ions of oligoglucosamines
in every cycle by analyzing the reaction mixture with
MALDI-TOF MS (see the Supporting Information).
Further optimization of the reaction conditions showed
that glycosylation at slightly higher temperature (À50 °C)
afforded the corresponding oligoglucosamines 6À9 in
Supporting Information Available. Procedures and data
for all new compounds. This material is available free of
charge via Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX