On-resin real-time reaction monitoring of solid-phase oligosaccharide synthesis

Article abstract of DOI:10.1021/ja020781z

On-resin real-time monitoring by a combination of a color test of (p-nitrobenzyl)pyridine and Disperse Red was developed for oligosaccharide synthesis. Copyright

Full text of DOI:10.1021/ja020781z

Published on Web 10/03/2002
On-Resin Real-Time Reaction Monitoring of Solid-Phase Oligosaccharide
Synthesis
Shino Manabe and Yukishige Ito*
RIKEN (The Institute of Physical and Chemical Research), Hirosawa, Wako-shi, Saitama, 351-0198, Japan
Received June 3, 2002
The roles of glycoconjugates in various aspects of biological
events have been well defined in recent years.1 To gain facile access
to a synthetic oligosaccharide in significant amount, solid-phase
synthesis has received much attention in the past decade.2 However,
solid-phase reaction has an obvious drawback due to the inherent
difficulties in monitoring the reaction profile. As a result, a
significant time and trial-and-error effort are often required to
establish a practical route and optimize each reaction condition.
For monitoring solid-phase peptide synthesis, the Kaiser test
(ninhydrin test) that detects the residual amino group is widely
used.3 To attain the similar situation in solid-phase oligosaccharide
synthesis, the detection of the hydroxy group is required, which is
much more difficult. Although spectroscopic means such as high-
resolution magic angle spinning NMR,4 gated decoupling NMR
using a 13C enriched acetyl group,5 and the 19F NMR technique6
have proved to be powerful, these methods are not suitable for real-
Figure 1. The (p-nitrobenzyl)pyridine method and Disperse Red-cyanuric
chloride conjugate method monitoring cycle in solid-phase oligosaccharide
synthesis.
time monitoring. We wish to report herein a practical method for
the real-time monitoring of solid-phase oligosaccharide synthesis,
which enables side-by-side detection of glycoside bond formation
and acceptor consumption. It relies solely upon on-resin color tests
and is readily performed in any organic chemistry laboratory.
Recently, we developed the real-time monitoring method for
solution-phase polymer-supported carbohydrate synthesis, which
employs MALDI-TOF MS and a color test for the chain elongation
and selective deprotection, respectively.7
Scheme 1a
For the latter purpose, the chloroacetyl (CAc) group was used
as the “temporary” hydroxy protection, removal of which can be
achieved in a highly chemoselective manner. Its presence can be
detected with high precision by a red color generated with
(p-nitrobenzyl)pyridine (PNBP) under basic conditions.8 This
procedure was expected to be suitable for on-resin detection; the
color density increases as the CAc having a sugar unit accumulates
and fades away after deprotection of the CAc group. On the other
hand, to observe the disappearance of the hydroxy group, modified
Taddei’s method was to be adopted, which employs cyanuric
chloride-Disperse Red conjugate 2. It readily reacts with nucleo-
philes such as hydroxy or amino groups to give a persistent red
color on the resin (Figure 1).9
Our experiment commenced with the preparation of resin-
supported acceptor, which was performed as depicted in Scheme
1. The 3-position of 3 was selectively protected as TBS ether, and
subsequent benzoylation gave 4.10 Reductive ring opening of the
benzylidene group was accompanied by desilylation, and resultant
diol was converted to 5. After desilylation, the acid-withstanding
linker 811 was introduced to give 6. The latter was immobilized
onto piperazine modified TentaGel 9 in the presence of 1,3-
diisopropylcarbodiimide to give amido linked 7.
a Reagents and conditions: (i) (a) TBSCl, imidazole, DMF, quant., (b)
benzoyl chloride, DMAP, pyridine, 75%; (ii) (a) BH3‚NMe3, AlCl3, MS
4A, CH2Cl2, (b) TBDPSCl, Et3N, DMAP, CH2Cl2 (95%, two steps), (c)
chloroacetic anhydride, pyridine, CH2Cl2, 92%; (iii) (a) BnOH, NIS,
TESOTf, 97%, (b) aqueous HF, CH3CN, 85%, (c) 8, PPh3, DEAD, CH2Cl2,
99%, (d) TFA, CH2Cl2, quant.; (iv) 9, 1,3-diisopropylcarbodiimide, CH2Cl2.
At this stage, successful immobilization was clearly seen by the
color test with PNBP 1, while a positive response to Disperse Red
2 indicated the presence of the residual amino group (Figure 2).
After capping with Ac2O, we monitored the deprotection reaction
of CAc in a real-time manner. Thus, 7 was reacted with 10 equiv
of HDTC (hydrazinedithiocarbonate)12 to yield 10 in 5 min as
indicated by the PNBP color test.
Subsequently, acceptor-immobilized resin 10 was submitted to
the glycosylation reaction using 11 as a donor. Completion of the
reaction was ascertained by the Disperse Red (negative) as well as
PNBP (strongly positive) test. The quality of disaccharide 12 was
confirmed after cleavage from resin by NaOMe to afford 14 in
* To whom correspondence should be addressed. E-mail: yukito@
postman.riken.go.jp.
9
12638
J. AM. CHEM. SOC. 2002, 124, 12638-12639
10.1021/ja020781z CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
lished. The deprotection of CAc was monitored by the PNBP
method, whereas the glycosylation was monitored by the cyanuric
chloride-Disperse Red conjugate method. It is operationally simple
and quick and does not require any special equipment. Because
PNBP and Disperse Red color tests are complementary to each
other, the combination allows one to gain a clear indication of the
progress of glycosylation, even if complete conversion is not
obtained. Several examples of fluorescence or dye based screening
methods of reaction conditions are known;14 however, to our
knowledge, this is the first example for the multistep transformation
monitored on resin, except peptide synthesis. Further application
of this methodology will be reported in due course.
Acknowledgment. This work was supported by a Grant-in-Aid
for Scientific Research (No. 13480191) and for Encouragement of
Young Scientists (No. 13771350) from the Ministry of Education,
Culture, Sports, Science, and Technology, the Mizutani Foundation,
and the Presidential Fund in RIKEN. We thank Ms. Akemi
Takahashi for her technical assistance. We thank Dr. T. Chihara
and his staff for elemental analysis.
Supporting Information Available: Detailed experimental pro-
cedures for the synthesis of all compounds, general procedure, and
1
deprotection and glycosylation reactions on solid-phase, H and 13C
NMR spectra (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 2. Reaction monitoring by PNBP and Disperse Red method. (i)
(a) Ac2O, i-Pr2NEt, CH2Cl2, (b) HDTC, DMF; (ii) 11, BF3‚OEt2, CH2Cl2.
References
(1) Essentials of Glycobiology; Varki, A., Cummings, R., Esko, J., Freeze,
H., Hart, G., Marth, J., Eds.; Cold Spring Harbor Laboratory Press:
Plainview, NY, 1999.
(2) (a) Ito, Y.; Manabe, S. Curr. Opin. Chem. Biol. 1998, 2, 701-708. (b)
Seeberger, P. H.; Hasse, W.-C. Chem. ReV. 2000, 100, 4349-4394.
(3) Kaiser, E.; Colescott, R L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem.
1970, 34, 595-598.
Figure 3. TLC profile of the crude mixture after cleavage of 13 (CHCl3:
MeOH 8:1).
(4) Seeberger, P. H.; Beebe, X.; Sukenick, G. D.; Pochapsky, S.; Danishefsky,
S. J. Angew. Chem., Int. Ed. Engl. 1997, 36, 491-493.
(5) (a) Kanemitsu, T.; Kanie, O.; Wong, C.-H. Angew. Chem., Int. Ed. 1998,
37, 3415-3418. (b) Kanemitsu, T.; Wong, C.-H.; Kanie, O. J. Am. Chem.
Soc. 2002, 124, 3591-3599.
Scheme 2
(6) Mogemark, M.; Elofsson, M.; Kihlberg, J. Org. Lett. 2001, 3, 1463-
1466.
(7) Ando, H.; Manabe, S.; Nakahara, Y.; Ito, Y. J. Am. Chem. Soc. 2001,
123, 3848-3849.
(8) Kuisle, O.; Lolo, M.; Quin˜oa´, E.; Riguera, R. Tetrahedron 1999, 55,
14807-14812.
Scheme 3
(9) (a) Attardi, M. E.; Falchi, A.; Taddei, M. Tetrahedron Lett. 2000, 41,
7395-7399. (b) Attardi, M.; Taddei, M. Tetrahedron Lett. 2001, 42, 2927.
(10) Nicolaou, K. C.; Winssinger, N.; Pastor, J.; DeRoose, F. J. Am. Chem.
Soc. 1997, 119, 449-450.
(11) (a) Manabe, S.; Nakahara, Y.; Ito, Y. Synlett 2000, 1241-1244. (b)
Manabe, S.; Ito, Y. Chem. Pharm. Bull. 2001, 49, 1241-1242. (c) Wu,
X.; Grathwohl, M.; Schmidt, R. R. Org. Lett. 2001, 3, 747-750.
(12) van Boechel, C. A. A.; Beetz, T. Tetrahedron Lett. 1983, 24, 3775-
3778.
(13) (a) Tabata, K.; Ito, W.; Kojima, T.; Kawabata, S.; Misaki, A. Carbohydr.
Res. 1981, 89, 121. (b) Nishimura, K.; Nishimura, S.; Nishi, N.; Saiki, I.;
Tokura, S.; Azuma, I. Vaccine 1984, 2, 93-99. (c) Chihara, G. Cancer
Res. 1986, 85-96. (d) Schizophyllan was demonstrated to form a triple
helix with single-stranded RNA: Sakurai, K.; Shinkai, S. J. Am. Chem.
Soc. 2000, 122, 4520-4521. (e) For a recent example of solution-phase
synthesis of immuno-active â-glycan, see: Yang, G.; Kong, F. Synlett
2000, 1423-1426.
45% yield (Scheme 2) (for TLC profile, see the Supporting
Information).
Resin-bound disaccharide 12 was subjected to the second round
of dechloroacetylation-glycosylation, and the tetrasaccharide, which
corresponds to the repeating unit of the immuno-active oligosac-
charide schizophyllan,13 was constructed successfully again with
on-resin real-time monitoring. After cleavage, the tetrasaccharide
15 was obtained in a reasonably pure form (Scheme 3, Figure 3).
After chromatographic purification, 15 was isolated in 30% overall
yield based on the initiated loading of 9 (Scheme 3, Figure 3).
In summary, a practical method for the real-time reaction
monitoring on-beads for oligosaccharide construction was estab-
(14) (a) Weingarten, M. D.; Sekanina, K.; Still, W. C. J. Am. Chem. Soc. 1998,
120, 9112-9113. (b) Shaughnessy, K. H.; Kim, P.; Hartwig, J. F. J. Am.
Chem. Soc. 1999, 121, 2123-2132. (c) Copeland, G. T.; Miller, S. J. J.
Am. Chem. Soc. 1999, 121, 4306-4307. (d) Korbel, G. A.; Lalic, G.;
Shair, M. D. J. Am. Chem. Soc. 2001, 123, 361-362.
JA020781Z
9
J. AM. CHEM. SOC. VOL. 124, NO. 43, 2002 12639