10.1021/ol200192d r 2011 American Chemical Society Published on Web 02/18/2011
ORGANIC
LETTERS
2011
Vol. 13, No. 6
1506–1509
One-Pot Strategies for the Synthesis of the
Tetrasaccharide Linkage Region of
Proteoglycans
Teng-Yi Huang,
†,‡Medel Manuel L. Zulueta,
†and Shang-Cheng Hung*
,†,§Genomics Research Center, Academia Sinica, 128 Sec.2 Academia Road, Taipei 115, Taiwan, Department of Chemistry, National Tsing Hua University, 101 Sec. 2 Kuang-Fu Road, Hsinchu 300, Taiwan, and Department of Applied Chemistry, National Chiao Tung University, 1001 Ta-Hsueh Road, Hsinchu 300, Taiwan
schung@gate.sinica.edu.tw
Received January 21, 2011
ABSTRACT
A linker-attached tetrasaccharide corresponding to the linkage region of proteoglycans was synthesized via one-pot procedures from the silylated monosaccharide derivatives. Regioselective one-pot protection protocols were applied in generating the requisite monosaccharide building blocks whereas stereoselective one-pot glycosylation approaches were utilized to assemble the tetrasaccharide skeleton.
Proteoglycans are biologically important macromole-cules widespread on the cell surface and in the extracellular matrix.1They interact with other biomolecules through the distinct profile of the glycosaminoglycan (GAG) chains decorating the protein core.2GAG biosynthesis initiates from theβ-attachment ofD-xylose (Xyl) to a serine residue
followed by consecutive additions of twoD-galactose (Gal)
and one D-glucuronic acid (GlcA) unit in the
respec-tive β1f4, β1f3, and β1f3 manner generating the
tetrasaccharide linkage region (Figure 1).3Further chain elongation sorts GAGs into two major categories. Heparin and heparan sulfate form by the alternate additions of N-acetylD-glucosamine and GlcA whereas chondroitin sulfate
and dermatan sulfate incorporate N-acetylD-galactosamine
Figure 1. Structures of proteoglycans.
†
Genomics Research Center, Academia Sinica.
‡
Department of Chemistry, National Tsing Hua University.
§
Department of Applied Chemistry, National Chiao Tung University. (1) (a) Iozzo, R. V.; Schaefer, L. FEBS J. 2010, 277, 3864–3875. (b) Ariga, T.; Miyatake, T.; Yu, R. K. J. Neurosci. Res. 2010, 88, 2303–2315. (c) Esko, J. D.; Kimata, K.; Lindahl, U. In Essentials of Glycobiology, 2nd ed.; Varki, A., Cummings, V.; Esko, J. D.; Freeze, H. H.; Stanley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E., Eds; Cold Spring Harbor Laboratory Press: New York, 2009; pp 229-248.
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Org. Lett., Vol. 13, No. 6, 2011 1507
and GlcA.4Intermittent N-deacetylation, uronic acid 5-C-epimerization, and multiple N- and O-sulfonations deliver the mature structure responsible for biological activity.
The formation of different GAGs with high fidelity from a common protein-linked tetrasaccharide precursor has intrigued many investigators. Occasional modifications, such as 2-O-phosphorylation of Xyl and 4- and 6-O-sulfonations of Gal residues, were suggested to influence the divergent biosynthetic routes.5 Understanding the nature of these modifications and their effects on the specificity of biosynthetic enzymes require compounds that mimic the natural substrates. Synthetic methodologies that target the proteoglycan linkage region backbone have been reported.6 Notable concerns therein include the tedious generation of appropriately protected monosac-charide building blocks, particularly that of Xyl,7and the stereocontrol in the Galβ1f3Gal glycosidic bond forma-tion that often give low selectivity and yield.8We recently demonstrated one-pot strategies for regioselective protec-tion of monosaccharides and stereoselective glycosylaprotec-tion in order to simplify synthetic procedures and reduce time-and resource-consuming workup time-and purification steps.9 Drawing from these methodologies, we present herein an approach to the chemical synthesis of the linkage region tetrasaccharide 1 fitted with an amine terminated linker as an aid to deciphering the processes associated with the modification of the linkage region together with its roles in chain elongation.
Our retrosynthetic plan is depicted in Scheme 1. By typical transformations, compound 1 is accessible follow-ing the assembly of the fully protected tetrasaccharide 2 using stereoselective one-pot glycosylations of the mono-saccharide building blocks 4-6 and the linker derivative 3. The orthogonal TBS protection of the thiogalactoside 5 would facilitate chain elongation and is also expected to enhance the donor reactivity during the coupling processes.
Theβ-selectivity in forming all glycosidic bonds would rely on neighboring group assistance by the acyl groups at 2-O of 4-6; solvent effects could be exploited in pertinent cases. Compounds 4-6 would be prepared through regioselec-tive one-pot protection strategies starting from the sily-lated thioglycosides 7-9, respectively.
The nearly similar reactivities of the 2-C, 3-C, and 4-C hydroxyls ofD-xylopyranosides render their full
differen-tiation a challenging task. Delightfully, the TMSOTf-catalyzed Et3SiH-reductive benzylation of the
2,3,4-tri-O-TMS ether 9 primarily gave, after TBAF treatment, the 3-O-benzylated 10. At -40 °C with 1.1 equiv of benzaldehyde, 10 was obtained in 65% yield (Scheme 2). Its 2- and 4-OBn isomers were also isolated at 10% and 6% yields, respectively. We next focused on regioselective benzoyl group installation at the 2-O position. Treatment of 10 with BzCl in pyridine or Bz2O in the presence of
TMSOTf formed the 4-O-benzoylated isomer as the major product in 40% and 30% yields respectively. Recently, we found that Yb(OTf)3-catalyzed acylation was effective in
Scheme 1. Retrosynthesis of Compound 1
Scheme 2. Synthesis of the Thioxyloside 6
(4) Sugahara, K.; Kitagawa, H. Curr. Opin. Struct. Biol. 2000, 10, 518–527.
(5) (a) Tone, Y.; Pedersen, L. C.; Yamamoto, T.; Izumikawa, T.; Kitagawa, H.; Nishihara, J.; Tamura, J.; Negishi, M.; Sugahara, K. J. Biol. Chem. 2008, 283, 16801–16807. (b) Kitagawa, H.; Tsutsumi, K.; Ikegami-Kuzuhara, A.; Nadanaka, S.; Goto, F.; Ogawa, T.; Sugahara, K. J. Biol. Chem. 2008, 283, 27438–27443.
(6) (a) Tamura, J.; Nakamura-Yamamoto, T.; Nishimura, Y.; Mizumoto, S.; Takahashi, J.; Sugahara, K. Carbohydr. Res. 2010, 345, 2115–2123. (b) Thollas, B.; Jacquinet, J. C. Org. Biomol. Chem. 2004, 2, 434–442. (c) Tamura, J.; Nishihara, J. J. Org. Chem. 2001, 66, 3074– 3083. (d) Allen, J. G.; Fraser-Reid, B. J. Am. Chem. Soc. 1999, 121, 468– 469. (e) Neumann, K. W.; Tamura, J.; Ogawa, T. Bioorg. Med. Chem. 1995, 3, 1637–1650. (f) Rio, S.; Beau, J. M.; Jacquinet, J. C. Carbohydr. Res. 1993, 244, 295–313. (g) Goto, F.; Ogawa, T. Tetrahedron Lett. 1992, 33, 5099–5102.
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(8) (a) McGill, N. W.; Williams, S. J. J. Org. Chem. 2009, 74, 9388– 9398. (b) Jacquinet, J.-C. Carbohydr. Res. 2004, 339, 349–359. (c) Belot, F.; Jacquinet, J.-C. Carbohydr. Res. 2000, 325, 93–106.
(9) (a) Chang, K.-L.; Zulueta, M. M. L.; Lu, X.-A.; Zhong, Y.-Q.; Hung, S.-C. J. Org. Chem. 2010, 75, 7424–7427. (b) Wang, C.-C.; Kulkarni, S. S.; Lee, J.-C.; Luo, S.-Y.; Hung, S.-C. Nat. Protoc. 2008, 3, 97–113. (c) Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Kulkarni, S. S.; Huang, Y.-W.; Lee, C.-C.; Chang, K.-L.; Hung, S.-C. Nature 2007, 446, 896–899. (d) Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Fan, H.-F.; Pai, C.-L.; Yang, W.-C.; Lu, L.-D.; Hung, S.-C. Angew. Chem., Int. Ed. 2002, 41, 2360–2362.
1508 Org. Lett., Vol. 13, No. 6, 2011
the desymmetrization of myo-inositol 1,3,5-orthofor-mate.10 Benzoylation with 5 mol % Yb(OTf)3and 1.05
equiv of Bz2O provided good regioselectivity, and the
4-alcohol 6 was isolated in 81% yield. The metal ion perhaps coordinates with both 2-O and 4-O atoms forcing the1C4 conformation in the intermediate 11, enhancing
regioselective Bz group introduction at the more nucleo-philic 2-O (path I) rather than the 4-O position (path II). When benzylation (PhCHO, Et3SiH, TMSOTf) and
ben-zoylation [Bz2O, Yb(OTf)3] were carried out in one pot
from 9, product 6 was obtained in 45% isolated yield. To our knowledge, this is, by far, the most straightforward preparation of xylosyl derivatives of identical protecting group pattern.
Unlike the similar case forD-glucopyranosides where
2-O-acylations predominate,11 one-pot benzylidenation and monobenzoylation of p-methylphenyl 2,3,4,6-tetra-O-TMS-1-thio-β-D-galactopyranoside gave us the unwanted
3-O-benzoyl derivative as the major isomer (62%). Alternatively, we performed the protecting group installation through the TBDPS functionalized 8 (Scheme 3). With catalytic TMSOTf, 8 was subjected to 3,4-O-benzylidenation, 2-O-benzoylation, and 6-O-desilylation in one pot to afford the 6-alcohols 12 (68%, exo/endo = 1/1.1). Treatment of 12 with 10-camphorsulfonic acid (CSA) led to benzylidene migration quantitatively providing the 3-alcohol 13 which, in turn, was masked with the TBS group to form the fully protected 14 in 91% yield. Although 14 carries applicable protecting groups for chain assembly, our preliminary trials for theβ1f3 bond formation between the Gal residues gave low yields, which might be caused by the rigid and bulky 4,6-O-benzylidene group. Accordingly, regioselective acetal ring-opening using BH3•THF and TMSOTf was performed furnishing the
6-alcohol 15 (95%), which underwent Williamson etherifica-tion to generate the galactosyl donor 5 in 91% yield. It was noted, in this special example, that the 2-O-benzoyl group tolerated the basic conditions.
Scheme 4 illustrates the regioselective one-pot synthesis of the D-gluco β-thiopyranoside 4 from the 2,3,4,
6-tetra-O-TMS ether 7 in 77% yield. Through TMSOTf catalysis, 7 underwent 4,6-O-benzylidenation followed by reductive 3-O-benzylation to furnish the intermediate 16. In the same flask, the 6-O-ring-open-ing of the benzylidene acetal still catalyzed by TMSOTf, employing BH3•THF as the reductant,
gen-erated the 2,6-diol intermediate 17. Then, the reaction mixture was treated with Et3N to pave the way for the
2,6-di-O-acetylation in the presence of DMAP.
With the key building blocks 4-6 in hand, the construc-tion of the target sugar backbone was initiated (Scheme 5). Following N-iodosuccinimide (NIS) and TfOH treatment, selective activation of the thiogalactoside 5 in the presence
Scheme 3. Synthesis of the Thiogalactoside 5
Scheme 4. Synthesis of the Thioglucoside 4
Scheme 5. Synthesis of the Disaccharide Acceptor 20
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Org. Lett., Vol. 13, No. 6, 2011 1509
of the thioxyloside 6 was achieved at-78 °C giving the β-disaccharide 18 (65%, J10,20= 8.1 Hz). Further
condensa-tion with the linker derivative 312formed the adduct 19 (J1,2= 7.1 Hz) in 77% yield. When this sequence was done
in one pot, 19 was acquired in an isolated yield of 48%. For the necessary TBS group cleavage, acidic hydrolysis13was utilized to prevent the base-mediated Bz migration. Con-sequently, the 3-alcohol 20 (91%) was efficiently acquired after treatment of 19 with BF3•Et2O. Extension of the
one-pot procedure to the TBS deprotection via TfOH treat-ment gave the required compound 20 in 32% yield.
We next turned to the alternative reducing end-to-non-reducing end route. Taking advantage of the higher re-activity of the primary alcohol in 3, its glycosylation at-40 °C by the thioxyloside 6 delivered the β-linked 21 (J1,2=
7.0 Hz) in 71% yield. Coupling of 21 with the thiogalacto-side 5 led to compound 19 (71%). Glycosylation of 3 with 6 followed by coupling with the donor 5 in one pot at the same temperature provided 19 in 37% yield. Unfortu-nately, inclusion of the TfOH-mediated cleavage of the TBS group in the one-pot method resulted in products which are difficult to purify.
The preparation of the target tetrasaccharide is depicted in Scheme 6. The stereoselectivity of the NIS/TfOH-acti-vated coupling of the thiogalactoside 5 and the disaccharide acceptor 20 was improved from a β/R ratio of 3/1 with CH2Cl2as solvent to 14/1 when the CH2Cl2/CH3CN (1/3)
solvent mixture14was utilized; the trisaccharide 22 (J100,200=
7.7 Hz) was obtained in 79% yield. When this glycosylation, followed by acidic desilylation, was carried out in one pot, the 300-alcohol 23 was isolated in 63% yield. The thiogluco-side 4 activation with NIS and TfOH was further included in the one-pot process which led to the linkage region tetra-saccharide skeleton 2 in a one-pot yield of 37%. Removal of all ester groups in 2 using Zemplen’s deacylation provided the pentaol 24 (84%), which underwent regioselective 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation of the primary hydroxyl in theD-glucosyl unit to give the
carboxylate 25 in 73% yield.15 Global deprotection by hydrogenolysis finally furnished the target compound 1 in 98% yield. The structure of 1 was confirmed through 1D and 2D NMR and HRMS experiments.
In conclusion, we have developed regioselective one-pot preparations of the requisite monosaccharide building blocks for the synthesis of the tetrasaccharide linkage region of proteoglycans. Stereoselective one-pot glycosida-tion protocols were also advanced which further simplified
the oligosaccharide assembly. The generated linker-at-tached tetrasaccharide is designed for the examination of the biosynthetic pathway involved in the modification of the sugar units as well as its effects in chain elongation and sorting of GAGs. These explorations will be carried out in the future.
Acknowledgment. This work was supported by the Na-tional Science Council (NSC 97-2113-M-001-033-MY3, NSC 98-2119-M-001-008-MY2, NSC 98-3112-B-007-005, NSC 98-2627-B-007-008) and Academia Sinica.
Supporting Information Available. Experimental pro-cedure and NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
Scheme 6. Synthesis of Compound 1a
a
BAIB: [bis(acetoxy)iodo]benzene.
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