A general and highly efficient solid phase synthesis of oligosacharides. Total synthesis of a heptasaccharide phytoalexin elicitor (HPE)

Full text of DOI:10.1021/ja963482g

J. Am. Chem. Soc. 1997, 119, 449-450
Scheme 1.a Preparation of Monosaccharide Units 2-4
449
A General and Highly Efficient Solid Phase
Synthesis of Oligosaccharides. Total Synthesis of a
Heptasaccharide Phytoalexin Elicitor (HPE)
K. C. Nicolaou,* Nicolas Winssinger, Joaqu´ın Pastor, and
Frederik DeRoose
Department of Chemistry and The Skaggs
Institute of Chemical Biology, The Scripps Research Institute
10550 North Torrey Pines Road
La Jolla, California 92037
Department of Chemistry and Biochemistry
UniVersity of California San Diego
9500 Gilman DriVe, La Jolla, California 92093
a Reagents and conditions: (a) MeSH (excess), SnCl4 (0.7 equiv),
CH2Cl2, -20 °C, 3 h, 85%; (b) i. PhSH (1.1 equiv), SnCl4 (0.7 equiv),
CH2Cl2, 0 °C, 4 h, 82%; ii. K2CO3 (0.3 equiv), THF/MeOH 1:1 (v/v),
25 °C, 15 h, 98%; (c) i. t-BuPh2SiCl (1.5 equiv), imidazole (2.0 equiv),
DMF, 25 °C, 3 h, 94%; ii. PhCOCl (4.0 equiv), Et3N (8.0 equiv),
4-DMAP (0.2 equiv), THF, 50 °C, 15 h, 92%; (d) PhCH(OMe)2 (2.0
equiv), CSA (0.5 equiv), benzene, reflux, 12 h, 96%; (e) t-BuMe2SiCl
(1.2 equiv), imidazole (1.5 equiv), DMF, 0 °C, 12 h, 88%; ii. PhCOCl
(1.5 equiv), Et3N (3 equiv), 4-DMAP (0.5 equiv), CH2Cl2, 15 h, 96%;
iii. BH3‚Me3N (40 equiv), AlCl3 (4 equiv), 4 Å MS, CH2Cl2/Et2O 5:2
(v/v), 0 °C, 5 h; iv. 2 N HCl in MeOH, 25 °C, 3 h, 85% (two steps);
(f) i. t-BuPh2SiCl (1.5 equiv), imidazole (2.0 equiv), DMF, 25 °C, 3 h,
96%; ii. Fmoc-Cl (3.0 equiv), pyridine, 25 °C, 1 h, 93%. 4-DMAP )
4-(dimethylamino)pyridine; CSA ) (()-10-camphorsulfonic acid;
Fmoc-Cl ) 9-fluorenylmethyl chloroformate.
ReceiVed October 4, 1996
Combinatorial chemistry imposes new demands on the solid
phase synthesis of organic molecules. Of particular interest to
glycobiology and medicine is versatile and practical methodol-
ogy for the construction of oligosaccharides of higher molecular
complexity and in a combinatorial fashion. Although several
methods for solid phase oligosaccharide synthesis have been
reported,1 new technologies that advance the field are still very
much in demand. In this paper, we describe new synthetic
technology for the construction of complex oligosaccharides on
a solid support and its application to the synthesis of the
heptasaccharide phytoalexin elicitor (HPE, 1).2,3 The described
chemistry combines (a) application of phenolic polystyrene in
solid phase oligosaccharide synthesis, (b) synthesis and utiliza-
tion of a new photolabile linker in solid phase synthesis; (c) a
number of versatile carbohydrate building blocks for potential
applications in combinatorial chemistry, and (d) the largest
branched oligosaccharide to be constructed on solid phase from
monosaccharide units and in reiterative fashion.
Scheme 2.a Synthesis of Polystyrene-Bound
Monosaccharides 13 and 15
For the purposes of the present technology, monosaccharide
building blocks 2-44 (Scheme 1) were designed to allow,
through neighboring group participation, selective â-glycoside
(1) For a review of solid phase oligosaccharide synthesis up to 1980,
see: (a) Fre´chet, J. M. J. Polymer-Supported Reactions in Organic Synthesis;
Wiley: New York, 1980, Chapter 8, p 407. For more recent advances in
solid phase synthesis of oligosaccharides, see: (b) Veeneman, G. H.;
Notermans, S.; Liskamp, R. M. J.; van der Marel, G. A.; van Boom, J. H.
Tetrahedron Lett. 1987, 28, 6695. (c) Danishefsky, S. J.; McClure, K. F.;
Randolph, J. T.; Ruggeri, R. B. Science 1993, 260, 1307. (d) Randolph, J.
T.; Danishefsky, S. J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1470. (e)
Yan, L.; Taylor, C. M.; Goodnow, R., Jr.; Kahne, D. J. Am. Chem. Soc.
1994, 116, 6953. (f) Schulster, M.; Wang, P.; Paulson, J. C.; Wong C.-H.
J. Am. Chem. Soc. 1994, 116, 1135. (g) Randolph, J. T.; McClure, K. F.;
Danishefsky, S. J. J. Am. Chem. Soc. 1995, 117, 5712. (h) Rademann, J.;
Schmidt, R. R. Tetrahedron Lett. 1996, 23, 3989. Adinolfi, M.; Barone,
G.; De Napoli, L.; Iadonisi, A.; Piccialli, G. Tetrahedron Lett. 1996, 28,
5007. For oligosaccharide synthesis on soluble polymers, see: (i) Douglas,
S. P.; Whitfield, D. M.; Krepinsky, J. J. J. Am. Chem. Soc. 1991, 113,
5095. (j) Whitfield, D. M.; Douglas, S. P.; Krepinsky, J. J. Tetrahedron
Lett. 1992, 33, 6795. (k) Verduyn, R.; van der Klein, P. A. M.; Douwes,
M.; van der Marel, G. A.; van Boom, J. H. Recl. TraV. Chim. Pays-Bas
1993, 112, 464. (l) Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. J.
Am. Chem. Soc. 1995, 117, 2116.
(2) For isolation and biological activity, see: (a) Sharp, J. K.; Valent,
B.; Albersheim, P. J. Biol. Chem. 1984, 259, 11312. Sharp, J. K.; Valent,
B.; Albersheim, P. J. Biol. Chem. 1984, 259, 11321. For a review in this
field, see: (b) Darvill, A.; Augur, C.; Bergmann, C.; Carlson, R. W.;
Cheong, J.-J.; Eberhard, S.; Hahn, M. G.; Lo´, V.-M.; Marfa`, V.; Meyer,
B.; Mohnen, D.; O’Niell, M. A.; Spiro, M. D.; van Halbeek, H.; York, W.
S.; Albersheim, P. Glycobiology 1992, 2, 181.
(3) For previous syntheses, see: (a) Ossowski, B. P.; Pilotti, Å.; Garegg,
P. J.; Lindberg, B. Angew. Chem., Int. Ed. Engl. 1983, 10, 793. (b) Ossowski,
B. P.; Pilotti, Å.; Garegg, P. J.; Lindberg, B. J. Biol. Chem. 1984, 259,
11337. (c) Fu¨gedi, P.; Birberg, W.; Garegg, P. J.; Pilotti, Å. Carbohydr.
Res. 1987, 164, 297. (d) Fu¨gedi, P.; Garegg, P. J.; Kvarnstro¨m, I.; Svansson,
L. Carbohydr. Chem. 1988, 7, 389. (e) Birberg, W.; Fu¨gedi, P.; Garegg, P.
J.; Pilotti, Å. J. Carbohydr. Res. 1989, 8, 47. (f) Lorentzen, J. P.; Helpap,
B.; Oswald, L. Angew. Chem., Int. Ed. Engl. 1991, 12, 1681. (g) Hong, N.;
Ogawa, T. Tetrahedron Lett. 1990, 31, 3179. (h) Verduyn, R.; van der Klein,
P. A. M.; Douwes, M.; van der Marel, G. A.; van Boom, J. H. Recl. TraV.
Chim. Pays-Bas 1993, 112, 464.
a Reagents and conditions: (a) i. n-BuLi (1.3 equiv), cyclohexane,
65 °C, 4 h; ii. O2, cyclohexane, 25 °C, 2 h; iii. PPh3 (2.0 equiv) THF,
25 °C, 12 h; (b) 12 (2.0 equiv), Cs2CO3 (2.0 equiv), DMF, 25 °C, 30
h, >90%; (c) 3 (1.0 equiv), 11 (1.5 equiv), DMTST (4.0 equiv), 4 Å
MS, CH2Cl2, 25 °C, 4 h, 95%; (d) 30% hydrogen fluoride‚pyridine,
THF, 25 °C, 15 h, >98%. DMTST ) (dimethylthio)methylsulfonium
triflate; TBDMS ) Si-t-BuMe2.
bond formations at positions C-1 (unit 2), C-6 (unit 3), and C-3
(unit 4). Their construction is summarized in Scheme 1.
Polystyrene (9, Scheme 2) was functionalized to phenolic
polystyrene (10) by the sequential action of n-BuLi, oxygen,
and PPh3.5,6 As a linker,7 a readily available o-nitrobenzyl ether
tether was utilized for its ease of attachment and cleavage. Thus,
commercially available 5-hydroxy-2-nitrobenzaldehyde was
reacted with 1,3-diiodopropane in the presence of Cs2CO3 in
(5) Fre´chet, J. M. J.; de Smet, M.; Farral, J. M. Polymer 1979, 20, 675.
(6) Although only p-substituted polystyrene is shown, it is estimated that
the phenolic polystyrene 10 contains both p- and m-hydroxyphenyl rings.
The functionalization was performed to the extent of 0.25-1.0 mmol/g,
and the subsequent chemistry was carried out using a 0.25 mmol/g phenolic
polystyrene.
(4) All new compounds exhibited satisfactory spectral and exact mass
data. Yields refer to spectroscopically and chromatographically homoge-
neous materials.
S0002-7863(96)03482-8 CCC: $14.00 © 1997 American Chemical Society

450 J. Am. Chem. Soc., Vol. 119, No. 2, 1997
Communications to the Editor
Scheme 3.a Solid Phase Synthesis of HPE (1)
DMF and then directly reduced with NaBH4 to afford iodo-
benzyl alcohol 11 (92% overall yield, Scheme 2), which served
admirably its intended purposes.
Glycosidation of 11 with carbohydrate unit 3 in the presence
of dimethylthiomethylsulfonium triflate (Me2S+SMeTfO+
)
DMTST)8 proceeded in 95% yield to afford exclusively â-gly-
coside 129 (Scheme 2). The monosaccharide 12 was then
attached to phenolic polystyrene (10) via its linker by the action
of Cs2CO3 in DMF at 25 °C to afford conjugate 13 in >90%
yield based on mass gain of the polymer. Efficient cleavage
of the carbohydrate fragment from the resin was demonstrated
by irradiation of 13 in THF at 25 °C to afford monosaccharide
14 in 95% yield.10
Having established the feasibility and efficiency of the solid
phase chemistry, attention was then turned to the construction
of the complex HPE oligosaccharide (1). The heptasaccharide
1 has been isolated from mycelial walls of Phytophthora
megasperma f. sp. glycinea and shown to exhibit potent
phytoalexin elicitor activity with nanomolar binding properties
toward its receptor.2 Previous syntheses3 of this compound
involve either solution chemistry or block synthesis using a
soluble polymer (PEG).3h The present strategy required not only
suitable protecting groups on each monosaccharide unit but also
a convenient glycosidation method with high degree of â-se-
lectivity. The protecting groups shown on 2-4 (Scheme 1)
served admirably in terms of ease of attachment and removal,
survivability under the reaction conditions, and orthogonality,
whereas the thioglycoside-DMTST glycosidation method8
proved both rapid and efficient.
The total synthesis of HPE (1) by the present solid phase
technology was carried out as follows. Fluoride-induced
liberation of the primary hydroxyl group in 13 furnished 15
(Scheme 2), onto which was attached monosaccharide 4 carrying
the Fmoc protecting group at the C-3 position (Scheme 3).
Removal of the latter group with Et3N in CH2Cl2 proceeded
smoothly and efficiently furnishing 1611 (>95%, calculated after
photocleavage and based on loading of 13). Coupling of 16
with monosaccharide 2 followed by fluoride-induced desilylation
gave 17, which was converted to 18 by addition of monosac-
charide 3 and desilylation. Reiteration of these procedures
allowed the growth of the branched oligosaccharide on the solid
support in the desired direction until heptasaccharide 21 was
reached via compounds 20 (addition of unit 4) and 19 (addition
of unit 2). Photolytic cleavage of the heptasaccharide from the
resin (21), followed by acetylation of the anomeric hydroxyl
group, furnished a mixture of fully protected R- and â-hep-
tasaccharides (22, ca. 20% overall yield from 13), whose HPLC,
1H NMR, and mass spectroscopic properties were consistent
with the expected structure. The targeted heptasaccharide (1,
mixture of R- and â-anomers) was obtained from 21 by
photocleavage, followed by treatment with NaOMe in MeOH
and hydrogenolysis (H2, Pd-C, MeOH, ca. 95% for two steps).
All reactions were carried out at ambient temperatures.
The rendering of HPE (1) and its variants as readily available
materials should facilitate biological investigations in the field.
a Reagents and conditions: (a) 4 (3.0 equiv), DMTST (12 equiv), 4
Å MS, CH2Cl2, 25 °C, 15 h; (b) 20% Et3N in CH2Cl2, 25 °C, 5 h; (c)
2 (4.0 equiv), DMTST (16 equiv), 4 Å MS, CH2Cl2, 25 °C, 30 h; (d)
30% hydrogen fluoride‚pyridine, THF, 25 °C, 15 h; (e) 3 (3 equiv),
DMTST (12 equiv), 4 Å MS, CH2Cl2, 25 °C, 15 h; (f) hν, THF, 25 °C,
ca. 20% overall yield from 13; (g) i. NaOMe, THF/MeOH 1:2 (v/v)
25 °C, 15 h; ii. H2 (1 atm), Pd/C cat., MeOH, 25 °C, 12 h, 95% two
steps; (h) Ac2O (10 equiv), 4-DMAP (1.0 equiv), Et3N (20 equiv), THF,
25 °C, 15 h, 100%.
Furthermore, the described solid phase chemistry and variations
thereof should prove useful in both combinatorial synthesis of
oligosaccharide libraries and complex oligosaccharide construc-
tion.
(7) (a) For a review of photolabile protecting groups, see: Pillai, R. V.
N. Organic Photochemistry Padwa, A., Ed.; Marcel Dekker: New York,
1987; Vol. 9, p 225. (b) Zehavi, U.; Amit, B.; Patchornik, A. J. Org. Chem.
1972, 14, 2281. (c) Holmes, C. P.; Jones, D. G. J. Org. Chem. 1995, 60,
2319. (d) Venkatesan, H.; Greenberg, M. M. J. Org. Chem. 1996, 61, 525.
(8) Fu¨gedi, P.; Garegg, P. J. Carbohydr. Res. 1986, 149, 9.
(9) The purity and exclusive â-stereochemistry of 12 was confirmed by
HPLC and 1H NMR spectroscopy (H-1, δ 4.99, J ) 7.5 Hz, see Supporting
Information).
(10) Typically, 5 mg of resin is sufficient to provide, after photolysis,
enough material for HPLC and mass spectroscopic analysis at each step.
(11) The homogeneity and â-stereochemistry for the 1 f 6 and 1 f 3
glycosidations was confirmed by photolytic cleavage, followed by acety-
lation, HPLC, and 1H NMR spectroscopy for the disaccharide and
trisaccharide compounds (see Supporting Information). The stereochemistry
of the higher oligosaccharides obtained by simple reiteration of the same 1
f 6 and 1 f 3 glycosidations was assumed to be the same as that of their
dimeric and trimeric counterparts, whereas their homogeneity was verified
by HPLC analysis (see Supporting Information).
Acknowledgment. We thank Drs. Dee Huang and Gary Siuzdak
for their superb NMR and mass spectroscopic assistance and the
Ministerio de Educacio´n y Ciencia (Spain) for a postdoctoral fellowship
(J.P.). This program was financially supported by the National Institutes
of Health, The Skaggs Institute for Chemical Biology, Merck, DuPont
Merck, Amgen, and Hoffmann La Roche.
Supporting Information Available: Selected procedures and data
for compounds (and/or derivatives of) 2-4, 11, 12, 14, 16 (off the
resin), 17 (off the resin), 22, and 1 (19 pages). See any current masthead
page for ordering and Internet access instructions.
JA963482G