Automated solid phase synthesis of oligoarabinofuranosides

Article abstract of DOI:10.1039/c3cc00042g

Automated solid phase synthesis enables rapid access to the linear and branched arabinofuranoside oligosaccharides. A simple purification step is sufficient to provide the conjugation ready oligosaccharides in good yield.

Full text of DOI:10.1039/c3cc00042g

Chemical Communications
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Volume 49 | Number 40 | 18 May 2013 | Pages 4427–4574
ISSN 1359-7345
COMMUNICATION
Peter H. Seeberger et al.
Automated solid phase synthesis of oligoarabinofuranosides
1359-7345(2013)49:40;1-4

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Automated solid phase synthesis of
oligoarabinofuranosides†
Cite this: Chem. Commun., 2013,
49, 4453
a
a
Jeyakumar Kandasamy,z Mattan Hurevichz and Peter H. Seeberger*ab
Received 3rd January 2013,
Accepted 21st January 2013
DOI: 10.1039/c3cc00042g
www.rsc.org/chemcomm
Automated solid phase synthesis enables rapid access to the linear and oligosaccharides such as Lewis X and Globo H demonstrated that
branched arabinofuranoside oligosaccharides. A simple purification complex carbohydrates as well as challenging glycosidic linkages
step is sufficient to provide the conjugation ready oligosaccharides can be accessed using an oligosaccharide synthesizer.21,22 To date,
in good yield.
no automated oligofuranoside syntheses have been reported. Here,
we describe the first automated synthesis of linear and branched
Arabinogalactan (AG) and lipoarabinomannan (LAM) polysac- oligoarabinofuranosides (Fig. 1).
charides are the major components of mycobacterial cell envelopes
The simple linear (1, 2) and branched (3, 4) oligoarabinofur-
and contain multiple arabinose residues.1 Arabinogalactan consists anoside targets were pursued in order to test the ability of the
of three identical oligoarabinosides attached to a galactofuranose.2–4 automated route to produce both linear and branched structures.
Each oligoarabinoside contains arabinofuranose (Araf) residues, Linear a-D-(1–5)-Araf 1 and 2 have been prepared via iterative
linked together via a-(1–5), a-(1–3) and b-(1–2) glycosidic linkages.
glycosylations using arabinose building block 7. Thioglycoside 7
Synthetic oligoarabinofuranosides are useful tools for studying was designed according to the requirements of the automated
the biosynthetic enzymes involved in the mycobacterial cell wall approach, with 9-fluorenylmethoxycarbonyl (Fmoc) as a temporary
synthesis.5–9 Access to various arabinofuranose fragments and protecting group at C-5 and benzoates at C-2 and C-3 as participating
analogues that can be immobilized on surfaces of glycan arrays protecting groups that are readily removed during cleavage from the
and particles is instrumental in identifying potential ligands for solid support. The synthesis of thioglycoside 7 was accomplished in
these enzymes and developing biological assays to study their three steps starting from thioglycoside 5.27,28 Regioselective trityl
substrate specificities.10 Several solution phase strategies for protection of the primary hydroxyl in 5 followed by the benzoyl
synthesis of arabinofuranosidic oligomers were established.11–17 protection of remaining hydroxyls and the removal of the trityl group
The groups of Lowary and Ito have accomplished the synthesis of furnished thioglycoside 6 (Scheme 1). Fmoc protection of the C-5
the arabinan dodecasaccharide via
a convergent fragment hydroxyl produced the desired thioglycoside building block 7 in
approach.18,19 These syntheses are challenging because they high yield.
require many discrete operations and multiple purification steps.
Linear a-D-(1–5)-Araf oligosaccharides 1 and 2 were assembled
Automated solid phase synthesis significantly reduces the using an oligosaccharide synthesizer (Scheme 2).25 Glycosylations
time-requirements and enables rapid access to structurally- were carried out at temperatures between ꢀ40 1C and ꢀ20 1C
defined oligosaccharides,20–24 as well as conjugation ready using thioglycoside 7 and a mixture of N-iodosuccinimide (NIS)
oligosaccharide analogues.25,26 A combination of new technologies and trifluoromethanesulfonic acid (TfOH). Each cycle involved
and synthetic methods was developed and adjusted to keep two additions of a glycosylating agent (double coupling), using
the automation technique up to date with the most recent five equivalents of the building block. Removal of the Fmoc group
developments in carbohydrate chemistry.20–26 The synthesis of with piperidine uncovered the hydroxyl group ready for elonga-
tion. The glycosylation–deprotection cycle was repeated twice for
the synthesis of disaccharide 1 and six times for the preparation
of hexasaccharide 2. Following oligosaccharide assembly on the
solid support, cleavage from the resin using sodium methoxide
solution furnished disaccharide 8 and hexasaccharide 9 that
were purified by reverse phase preparative HPLC. Hydrogenolysis
of 8 and 9 followed by a simple Sep-Pak purification afforded
disaccharide 1 and hexasaccharide 2 in overall yield of 85% and
65%, respectively, based on resin loading.
a Department of Biomolecular Systems, Max-Planck-Institute of Colloids and
¨
Interfaces, Am Muhlenberg 1, 14476 Potsdam-Golm, Germany
b
¨
Freie Universitat Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,
14195 Berlin, Germany. E-mail: peter.seeberger@mpikg.mpg.de;
Fax: +49 331 567 9302; Tel: +49 331 567 9301
† Electronic supplementary information (ESI) available: Experimental proce-
dures, spectral (1H, 13C NMR and HSQC) and analytical (HPLC and MS) data.
See DOI: 10.1039/c3cc00042g
‡ Both authors contributed equally to this work.
c
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Fig. 1 Structures and retrosynthetic analysis of a-D-(1–5), a-D-(1–3) oligoarabinofuranosides 1–4.
Scheme 1 Synthesis of 7. Reagents and conditions: (a) (i) TrCl, Py; then BzCl;
Scheme 3 Synthesis of building block 11. Reagents and conditions: (a) t-Bu2Si-
(OTf)2, 2,6-lutidine, 60%; (b) BzCl, Py, 89%; (c) (i) HFꢁPy, THF; (ii) FmocCl, Py, 85%
(two steps).
(ii) PTSA, MeOH, 77% (two steps); (b) FmocCl, Py, 96%.
Scheme 4 Automated synthesis of branched trisaccharide 3. Reagents and
conditions: (a) 11, NIS/TfOH, ꢀ40 1C to ꢀ20 1C, repeated twice; (b) 20%
piperidine in DMF; (c) 7, NIS/TfOH, ꢀ40 1C to ꢀ20 1C, repeated four times;
(d) NaOMe in MeOH; (e) Pd/C, H2, MeOH/H2O/EtOAc/AcOH.
Scheme 2 Automated synthesis of oligosaccharides 1 and 2. Reagents and
conditions: (a) 7, NIS/TfOH, ꢀ40 1C to ꢀ20 1C; (b) 20% piperidine in DMF;
(c) NaOMe in MeOH; (d) Pd/C, H2, MeOH/H2O/EtOAc/AcOH.
synthesis of oligoarabinofuranoside 3 was carried out with
In addition to the formation of a-(1–5) linkages, the synth- thioglycoside 7 using conditions established for the synthesis
esis of branched oligoarabinofuranosides 3 and 4 requires of 1 and 2 (Scheme 4). The following glycosylation to create the
introduction of a-(1–3) branching. For this purpose building a-(1–5) linkage and install a-(1–3) branching was performed
block 11 was used. Featuring two Fmoc groups, building block using thioglycoside 7. Removal of Fmoc groups and cleavage
11 enables the removal of temporary protection and glycosyla- from the resin yielded crude trisaccharide 12. A simple purifica-
tion of both C-3 and C-5 hydroxyls in a single deprotection– tion protocol involving adsorption on a pad of silica gel followed
glycosylation cycle. En route to thioglycoside 11, thioglycoside 5 by a hexane and CH2Cl2 wash and elution with a mixture of
was converted to silyl acetal 10 in two steps (Scheme 3). CH2Cl2 : MeOH was sufficient to obtain pure trisaccharide 12.
Selective removal of the silyl acetal followed by the Fmoc Hydrogenolysis of partially protected trisaccharide 12 gave
protection of the resulting diol provided building block 11 in deprotected trisaccharide 3 in 78% overall yield based on resin
85% yield.
Upon loading the resin with building block 11 using a
loading, without the need for further purification.
The automated synthesis of branched hexasaccharide 4 was
glycosylation–deprotection cycle, the first elongation in the accomplished via four glycosylation cycles using building blocks
c
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We acknowledge the Max-Planck Society and the Swiss National
Science Foundation (200020-117889) as well as a Korber Prize
(to P. H. Seeberger) for generous financial support. M.H. thanks
the Max-Planck Society for a Minerva postdoctoral fellowship. We
thank Dr I. Vilotijevic, Dr C. Rademacher, Dr S. G. Parameswarappa,
Dr J. Hudon, Dr C. Anish, Dr C. L. Pereira and Mr H. S. Hahm for
their help in editing this paper. Cover image courtesy of scientific
illustrator, Melanie Burger (melanie.burger@mail.utoronto.ca).
Notes and references
1 P. J. Brennan and H. Nikaido, Annu. Rev. Biochem., 1995, 64, 29–63.
2 G. S. Besra, K. H. Khoo, M. R. Mcneil, A. Dell, H. R. Morris and
P. J. Brennan, Biochemistry, 1995, 34, 4257–4266.
3 L. B. Shi, S. Berg, A. Lee, J. S. Spencer, J. Zhang, V. Vissa,
M. R. McNeil, K. H. Khoo and D. Chatterjee, J. Biol. Chem., 2006,
281, 19512–19526.
4 V. Briken, S. A. Porcelli, G. S. Besra and L. Kremer, Mol. Microbiol.,
2004, 53, 391–403.
Scheme 5 Automated synthesis of branched hexasaccharide 4. Reagents and con-
ditions: (a) 7, NIS/TfOH, ꢀ40 1C to ꢀ20 1C, repeated twice; (b) 20% piperidine in DMF;
(c) 11, NIS/TfOH, ꢀ40 1C to ꢀ20 1C, repeated twice; (d) 7, NIS/TfOH, ꢀ40 1C to ꢀ20 1C,
repeated four times; (e) NaOMe in MeOH; (f) Pd/C, H2, MeOH/H2O/EtOAc/AcOH.
¨
5 J. A. Zhang, A. G. Amin, A. Holemann, P. H. Seeberger and
D. Chatterjee, Bioorg. Med. Chem., 2010, 18, 7121–7131.
6 S. Khasnobis, J. Zhang, S. K. Angala, A. G. Amin, M. R. McNeil,
D. C. Crick and D. Chatterjee, Chem. Biol., 2006, 13, 787–795.
7 J. Zhang, S. K. Angala, P. K. Pramanik, K. Li, D. C. Crick, A. Liav,
A. Jozwiak, E. Swiezewska, M. Jackson and D. Chatterjee, ACS Chem.
Biol., 2011, 6, 819–828.
8 J. D. Ayers, T. L. Lowary, C. B. Morehouse and G. S. Besra, Bioorg.
Med. Chem. Lett., 1998, 8, 437–442.
9 C. Rademacher, G. K. Shoemaker, H. S. Kim, R. B. Zheng, H. Taha,
C. Liu, R. C. Nacario, D. C. Schriemer, J. S. Klassen, T. Peters and
T. L. Lowary, J. Am. Chem. Soc., 2007, 129, 10489–10502.
10 S. Fukui, T. Feizi, C. Galustian, A. M. Lawson and W. G. Chai, Nat.
Biotechnol., 2002, 20, 1011–1017.
11 F. W. D’Souza, J. D. Ayers, P. R. McCarren and T. L. Lowary, J. Am.
Chem. Soc., 2000, 122, 1251–1260.
12 F. W. D’Souza, P. E. Cheshev, J. D. Ayers and T. L. Lowary, J. Org.
Chem., 1998, 63, 9037–9044.
13 R. R. Gadikota, C. S. Callam, T. Wagner, B. Del Fraino and
T. L. Lowary, J. Am. Chem. Soc., 2003, 125, 4155–4165.
14 F. W. D’Souza and T. L. Lowary, Org. Lett., 2000, 2, 1493–1495.
15 A. Ishiwata, H. Akao and Y. Ito, Org. Lett., 2006, 8, 5525–5528.
16 K. Marotte, S. Sanchez, T. Bamhaoud and J. Prandi, Eur. J. Org.
Chem., 2003, 3587–3598.
Table 1 Time required for the synthesis of oligosaccharides 1–4a
Steps
Compound
I
II III IV
V
Total time (h) Overall yield (%)
1
2
3
4
6
16
8
6
6
6
6
6
6
3
3
16
16
16
16
3
3
3
3
37
47
36
42
85
65
78
63
14
a
Time given in hours for all steps, (I) automated synthesis (glycosylation–
deprotection); (II) cleavage from resin (NaOMe); (III) first purification
step (HPLC or silica pad); (IV) global deprotection (Pd/C, H2); (V) final
purification (Sep-Pak, optional).
7 and 11 (Scheme 5). The automated synthesis of hexasaccharide
13 required about 14 hours. Partially protected hexasaccharide
13 was purified by passing the crude mixture over a silica pad.
Hydrogenolysis provided the amine functionalized hexasaccharide
4 in 63% overall yield based on resin loading.
17 Y. J. Lee, K. Lee, E. H. Jung, H. B. Jeon and K. S. Kim, Org. Lett., 2005,
7, 3263–3266.
18 M. Joe, Y. Bai, R. C. Nacario and T. L. Lowary, J. Am. Chem. Soc.,
2007, 129, 9885–9901.
19 A. Ishiwata and Y. Ito, J. Am. Chem. Soc., 2011, 133, 2275–2291.
20 O. J. Plante, E. R. Palmacci and P. H. Seeberger, Science, 2001, 291,
1523–1527.
Automated solid phase oligosaccharide synthesis drastically
reduces the overall synthesis time from the building block to 21 K. R. Love and P. H. Seeberger, Angew. Chem., Int. Ed., 2004, 43,
602–605.
the final compound. Table 1 provides an overview of the time
required for the synthesis of oligosaccharides 1–4. The overall
22 D. B. Werz, B. Castagner and P. H. Seeberger, J. Am. Chem. Soc.,
2007, 129, 2770–2771.
¨
time to reach branched hexamer 4 was slightly shorter than 23 M. T. C. Walvoort, H. van den Elst, O. J. Plante, L. Krock,
P. H. Seeberger, H. S. Overkleeft, G. A. van der Marel and J. D. C.
that for hexamer 2 owing to the simplified purification protocol
(Table 1, step III).
´
Codee, Angew. Chem., Int. Ed., 2012, 51, 4393–4396.
24 M. T. C. Walvoort, A. G. Volbeda, N. R. M. Reintjens, H. van den Elst,
´
In conclusion, linear and branched conjugation-ready oli-
goarabinofuranosides 1–4 were assembled via automated solid
phase synthesis. This robust and reproducible route constitutes
O. J. Plante, H. S. Overkleeft, G. A. van der Marel and J. D. C. Codee,
Org. Lett., 2012, 14, 3776–3779.
¨
¨
25 L. Krock, D. Esposito, B. Castagner, C. C. Wang, P. Bindschadler and
P. H. Seeberger, Chem. Sci., 2012, 3, 1617–1622.
the first automated solid phase synthesis of oligofuranosides. 26 D. Esposito, M. Hurevich, B. Castagner, C. C. Wang and
P. H. Seeberger, Beilstein J. Org. Chem., 2012, 8, 1601–1609.
27 C. S. Callam and T. L. Lowary, J. Chem. Educ., 2001, 78, 73–74.
28 D. J. Hou, H. A. Taha and T. L. Lowary, J. Am. Chem. Soc., 2009, 131,
This method will provide straightforward access to a large
number of different arabinogalactan fragments for biological
studies.
12937–12948.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 4453--4455 4455