Synthesis of a dimer of β-(1,4)-l-arabinosyl-(2 S,4 R)-4-hydroxyproline inspired by art v 1, the major allergen of mugwort

Article abstract of DOI:10.1021/ol102112z

Nα-tert-Butoxycarbonyl-l-trans-4-hydroxyproline allyl ester (Boc-Hyp-OAll) was glycosylated with 2,3,5-tri-O-benzyl-l-arabinose p-cresylthioglycoside in 60% yield with 4:1 β:α stereoselectivity. Deprotection of N- and C-terminii independently gave a prolyl amine and prolyl carboxylate respectively that were coupled under standard conditions with 1-[bis-(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5,b]-pyridininium hexafluorophosphate 3-oxide (N-HATU) to give the dimer 1 in 46% yield. These results represent the first steps toward the production of homogeneous oligomers to determine the minimal epitope of the Art v 1 allergen.

Full text of DOI:10.1021/ol102112z

ORGANIC
LETTERS
2010
Vol. 12, No. 21
4968-4971
Synthesis of a Dimer of ꢀ-(1,4)-L-Arabinosyl-
(2S,4R)-4-hydroxyproline Inspired by Art
v 1, the Major Allergen of Mugwort
Ning Xie and Carol M. Taylor*
Department of Chemistry, Louisiana State UniVersity, Baton Rouge,
Louisiana 70803, United States
cmtaylor@lsu.edu
Received September 4, 2010
ABSTRACT
Nr-tert-Butoxycarbonyl-L-trans-4-hydroxyproline allyl ester (Boc-Hyp-OAll) was glycosylated with 2,3,5-tri-O-benzyl-L-arabinose p-cresylthiogly-
coside in 60% yield with 4:1 ꢀ:r stereoselectivity. Deprotection of N- and C-terminii independently gave a prolyl amine and prolyl carboxylate
respectively that were coupled under standard conditions with 1-[bis-(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5,b]-pyridininium hexafluo-
rophosphate 3-oxide (N-HATU) to give the dimer 1 in 46% yield. These results represent the first steps toward the production of homogeneous
oligomers to determine the minimal epitope of the Art v 1 allergen.
The pollen of mugwort (Artemisa Vulgaris) is a significant
contributor to hay fever in Europe and North America.
Altmann and co-workers have proposed a model for the
structure of the major allergen, Art v 1 (Figure 1). Detailed
degradation studies, in conjunction with molecular modeling1
and characterization by NMR and mass spectrometry,2
revealed two novel O-glycosides linked via trans-4-hydroxy-
proline (Hyp). One of these motifs, characterized by clusters
of contiguous ꢀ-L-arabinofuranosides of Hyp, was found to
be a key recognition element for antibodies generated in
response to the natural protein. The identification of the
minimal epitope and more detailed characterization requires
access to homogeneous glycopeptide fragments. Exceedingly
small quantities of Art v 1 glycoprotein can be isolated from
pollen. Alkaline hydrolysis of the protein leads to complex
mixtures of compounds containing the ꢀ-Araf-Hyp motif.
Figure 1. Model for the Art v 1 glycoprotein. Monosaccharide units
are represented by: ꢀ-D-Gal (yellow circle), R-L-Ara (turquoise star),
ꢀ-L-Ara (pink star). Copyright The American Society for Biochem-
istry and Molecular Biology, 2005.
(1) Himly, M.; Jahn-Schmid, B.; Dedic, A.; Kelemen, P.; Wopfner, N.;
Altmann, F.; Van Ree, R.; Briza, P.; Richter, K.; Ebner, C.; Ferreira, F.
FASEB J. 2003, 17, 106–18.
(2) Leonard, R.; Petersen, B. O.; Himly, M.; Kaar, W.; Wopfner, N.;
Kolarich, D.; Van Ree, R.; Ebner, C.; Duus, J. Ø.; Ferreira, F.; Altmann,
F. J. Biol. Chem. 2005, 280, 7932–7940.
The heterogeneous nature of this digest renders the isolation
of even miniscule amounts of pure amino acids and oli-
10.1021/ol102112z  2010 American Chemical Society
Published on Web 10/11/2010

gopeptides an unrealistic goal. The key to accessing useful
amounts of high-purity oligomers is via chemical synthesis.
The repetitive ꢀ-Araf-Hyp motif of Art v 1 is unprec-
edented and poses a challenge to synthesis for several
reasons. First, it is difficult to control the stereoselectivity
of glycosylation with an arabinofuranosyl donor.3 The
anomeric effect is weak due to multiple low energy
conformations for furanoses and neighboring group partici-
pation favors an R (1,2-trans) arabinoside. Second, the
hydroxy group of Hyp is a poor nucleophile, in part because
of its axial orientation in the preferred Cγ-exo conformation
of the pyrrolidine ring, and glycosylation rarely proceeds in
good yield.4 Moreover, the best precedents for the assembly
of such a glycoclustered peptide lie in the synthesis of the
mucins, wherein glycosylated threonine residues have been
coupled.5 The amalgamation of two glycosylated prolines
is inherently more difficult since the prolyl amine is
secondary and cyclic.
stereoselectivity in some instances11 and modest results in
other cases.12 To-date glycosyl acceptors have been pre-
dominantly primary and secondary alcohols of monosaccha-
rides. Differences in the stereoselectivity of D- and L-donors
has been explored by Zhu.13
Thioglycoside and sulfoxide glycosyl donors were pre-
pared according to Scheme 2. We chose to work with the
Scheme 2. Synthesis of Glycosyl Donors
Fortunately, significant progress has been made in recent
years vis-a`-vis the synthesis of ꢀ-arabinosides. Ito’s group
has used intramolecular aglycone delivery (IAD) to good
effect6 and Lowary has employed 2,3-anhydro sugars.7 We
quickly focused our attention on the independent reports of
conformationally restricted glycosyl donors from the Boons8
and Crich9 groups (Scheme 1). The 3,5-O-di-tert-butylsilyl
Scheme 1. Conformationally-Restricted Glycosyl Donor
thiocresyl glycoside, as employed by Crich.9 We obtained
mediocre yields of the silyl acetal 4 and experienced
considerable difficulty forming the benzyl ether at C2
according to the originally reported, standard conditions
(NaH, BnBr, DMF). We found that the nonbasic conditions
reported more recently by Zhu’s group13 gave more repro-
ducible results. Oxidation of the thioglycoside gave the
sulfoxide 6 as a mixture of diastereomers. We prepared this
additional glycosyl donor, since Crich had shown that better
ꢀ-selectivity was obtained via the sulfoxide method.9
Glycosylation of Boc-Hyp-OAll (7) with thioglycoside 5
and sulfoxide 6 gave complex mixtures of products (Scheme
3). Isolation of pure 8ꢀ, for characterization, required HPLC
purification. Evidence for the stereochemistry of the glyco-
sidic linkage was afforded by the coupling constant (J )
5.2 Hz) for H1 of the arabinose unit. The plethora of other
products made it difficult to determine yields and R/ꢀ ratios.
The outcome of the reaction was unsatisfactory in both
regards.
acetal protecting group locks the arabinofuranosyl ring in a
conformation10 that favors nucleophilic attack from the
ꢀ-face (Scheme 1). A number of subsequent oligosaccharide
syntheses have utilized such donors, reporting excellent
(3) Lowary, T. L. Curr. Opin. Chem. Biol. 2003, 7, 749–756.
(4) We have previously summarized these problems: (a) Taylor, C. M.;
Weir, C. A.; Jorgensen, C. G. Aust. J. Chem. 2002, 55, 135–140. (b) Shaffer,
K. J.; Taylor, C. M. Org. Lett. 2006, 8, 3959–3962.
(5) (a) Sames, D.; Chen, X.-T.; Danishefsky, S. J. Nature 1997, 389,
587–591. (b) Kuduk, S. D.; Schwarz, J. B.; Chen, X.-T.; Glunz, P. W.;
Sames, D.; Ragupathi, G.; Livingston, P. O.; Danishefsky, S. J. J. Am. Chem.
Soc. 1998, 120, 12474–12485. (c) Braun, P.; Davies, G. M.; Price, M. R.;
Williams, P. M.; Tendler, S. J. B.; Kunz, H. Bioorg. Med. Chem. 1998, 6,
1531–1545. (d) Liu, M.; Barany, G.; Live, D. Carbohydr. Res. 2005, 340,
2111–2122. (e) Matsushita, T.; Hinou, H.; Kurogochi, M.; Shimizu, H.;
Nishimura, S. Org. Lett. 2005, 7, 877–880. (f) Vepiek, P.; Hajduch, M.;
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(b) Ishiwata, A.; Munemura, Y.; Ito, Y. Eur. J. Org. Chem. 2008, 4250–
4263.
Our hypothesis was that the poor results of the glycosy-
lation reactions were due to the instability of the silyl acetal
to the reaction conditions. To test this theory, we prepared
perbenzylated thioglycoside 9 and the corresponding sul-
(7) Gadikota, R. R.; Callam, C. S.; Wagner, T.; Del Fraino, B.; Lowary,
T. L. J. Am. Chem. Soc. 2003, 125, 4155–4165.
(11) (a) Rao, Y.; Buskas, T.; Albert, A.; O’Neill, M. A.; Hahn, M. G.;
Boons, G.-J. ChemBioChem 2008, 9, 381–388. (b) Joe, M.; Bai, Y.; Nacario,
R. C.; Lowary, T. L. J. Am. Chem. Soc. 2007, 129, 9885–9901.
(12) (a) Sanki, A. K.; Boucau, J.; Ronning, D. R.; Sucheck, S. J.
Glycoconj. J. 2009, 26, 589–596. (b) Akihiro, I.; Ito, Y. Trends Glycosci.
Glycotechnol. 2009, 21, 266–289.
(8) Zhu, X.; Kawatkar, S.; Rao, Y.; Boons, G.-J. J. Am. Chem. Soc.
2006, 128, 11948–11957.
(9) Crich, D.; Pedersen, C. M.; Bowers, A. A.; Wink, D. J. J. Org. Chem.
2007, 72, 1553–1565.
(10) The crystal structure of p-tolyl 2-O-benzyl-3,5-O-(di-tert-butyl-
silanediyl)-1-thio-R-D-arabinofuranoside has been reported: Nacrio, R. C.;
Lowary, T. L.; McDonald, R. Acta Cryst. Sect. E 2007, E63, o498–o500.
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Org. Lett., Vol. 12, No. 21, 2010
4969

glycoside 11 is more robust than 8 and, downstream, a
single hydrogenolytic step will be required to remove all
carbohydrate protecting groups. The use of the sulfoxide
donor 10 gave similar yields and superior ꢀ-selectivity
(25:1), as judged by integration of the anomeric proton
signals in the 1H NMR spectrum. Unfortunately, the
chromatography to separate pure ꢀ-glycoside from as-
sorted byproducts was more tedious. This practical
consideration, in combination with the extra step to
produce the sulfoxide donor 10, led us to favor the
thioglycoside donor 9 for large-scale preparations of 11ꢀ.
With a practical synthesis of monomer 11ꢀ in hand, the
next objective became formation of the dimer (Scheme 6).
Scheme 3. Glycosylation with Donor 5
foxide 10 (Scheme 4). Lowary had previously obtained
reasonable ꢀ-selectivity with ent-9.14
Scheme 6. Dimerization of Glycoside 11ꢀ
Scheme 4. Preparation of Perbenzylated Donors
To our delight, reaction of thioglycoside 912 with
hydroxyproline acceptor 7, under standard conditions, gave
a reasonable yield of glycoside 11 (Scheme 5) with a
Scheme 5. Formation of Glycoside 11ꢀ
Selective cleavage of the Boc-group afforded secondary
amine 12. Flash chromatography of amine 12, prior to the
coupling reaction, was beneficial. Deallylation with Pd(PPh3)4
in the presence of morpholine generated the acid 13, but we
were unable to remove traces of the catalyst.
We investigated a number of peptide coupling reagents,
renowned for their effectiveness in hindered couplings,15
including HATU,16 bromo-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBroP)17 and tetramethylfluoroform-
amidinium hexafluorophosphate (TFFH).18 Our best results
to-date are given in Scheme 6. The doubly glycosylated
dipeptide exists as four conformations, presumably varying
about the two central peptide bonds, in approximately equal
typical ꢀ/R ratio of 4:1. The product mixtures were less
complex and the yields significantly improved relative to
Scheme 3. Moreover, pure ꢀ-glycoside 11ꢀ could be
obtained by flash chromatography on gram-scale. There
are further advantages to this route. The glycosyl donor
9 is available in two steps from 3 in 84% yield (compared
to three steps in only 37% yield for donor 5). The
(15) (a) Humphrey, J. M.; Chamberlin, A. R. Chem. ReV. 1997, 97,
2243–2266. (b) Han, S.-Y.; Kim, Y.-A. Tetrahedron 2004, 60, 2447–2467.
(16) Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang,
C.; Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay, C.; Mu¨gge, C.; Wenschuh,
J. K.; Beyermann, M.; Bienert, M. Angew. Chem., Int. Ed. 2002, 41, 442–
445.
(17) (a) Coste, J.; Fre´rot, E.; Jouin, P. J. Org. Chem. 1994, 59, 2439.
(b) Walker, M. A.; Heathcock, C. H. J. Org. Chem. 1992, 57, 5566.
(18) Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc. 1995, 117, 5401–
5402.
(14) Yin, H.; D’Souza, F. W.; Lowary, T. L. J. Org. Chem. 2002, 67,
892–903.
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Org. Lett., Vol. 12, No. 21, 2010

amounts. Further efforts to improve the efficiency of dimer
formation, the assembly of larger oligomers, and conforma-
tional studies are underway.
(Universita¨t fu¨r Bodenkultur Wein, BOKU, Vienna,
Austria) for helpful discussions.
Supporting Information Available: Experimental pro-
cedures and 1H and 13C NMR spectra for compounds 1 and
3-11. This material is available free of charge via the
Internet at http://pubs.acs.org.
Acknowledgment. We thank the Department of Chem-
istry at LSU for financial support, Dr. Thomas Weld-
eghiorghis (LSU NMR Facility) for his expertise in
acquiring NMR data, and Professor Friedrich Altmann
OL102112Z
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