De novo synthesis of the bacterial 2-amino-2,6-dideoxy sugar building blocks D-Fucosamine, D-bacillosamine, and D-xylo-6-deoxy-4-ketohexosamine

Article abstract of DOI:10.1021/ol3023227

The cell-surface glycans on bacteria contain many monosaccharides that cannot be obtained by isolation from natural sources. Availability of differentially protected monosaccharides is therefore often limiting access to potential oligosaccharide vaccine antigens. D-Fucosamine, Dbacillosamine, and D-xylo-2,6-deoxy-4-ketohexosamine building blocks were prepared via a divergent de novo synthesis from L-Garner aldehyde. The route relies on a chelation-control assisted organometallic addition and an anti-selective dihydroxylation reaction.

Full text of DOI:10.1021/ol3023227

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
De Novo Synthesis of the Bacterial
2-Amino-2,6-Dideoxy Sugar Building
Blocks D‑Fucosamine, D‑Bacillosamine,
and D‑Xylo-6-deoxy-4-ketohexosamine
Daniele Leonori and Peter H. Seeberger*
Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces,
Am Mu€hlenberg 1, 14476 Potsdam, Germany, and Institute for Chemistry and
€
Biochemistry, Freie Universitat Berlin, Arnimallee 22, 14195 Berlin, Germany
peter.seeberger@mpikg.mpg.de
Received August 20, 2012
ABSTRACT
The cell-surface glycans on bacteria contain many monosaccharides that cannot be obtained by isolation from natural sources. Availability of
differentially protected monosaccharides is therefore often limiting access to potential oligosaccharide vaccine antigens. D-Fucosamine, D-
bacillosamine, and D-xylo-2,6-deoxy-4-ketohexosamine building blocks were prepared via a divergent de novo synthesis from L-Garner aldehyde.
The route relies on a chelation-control assisted organometallic addition and an anti-selective dihydroxylation reaction.
Protein glycosylation is an important post-translational
event that directly influences many proteins’ functions.1
Glycans on the surface of bacteria serve as a basis for the
development of new therapeutics.2 The ability of bacteria
to synthesize complex oligosaccharides directly influences
their ability to colonize human hosts and to cause disease.3
Oligosaccharide antigens containing unique or unusual
monosaccharides that are not found in the human body
give rise to the development of novel vaccine candidates
against bacterial infections.4 However, access to pure and
well-defined oligo- or polysaccharide antigens represents a
major obstacle toward the development of carbohydrate
vaccines.5
Vaccine candidates6 against the highly pathogenic bac-
teria Pseudomonas aeruginosa, Neisseria gonorrheae, and
Streptococcus pneumoniae would be useful in addressing a
significant medical need. These bacteria express cell-
surface glycans containing the unusual 2-amino-2,6-dideoxy
sugars D-fucosamine (Fuc),7 D-bacillosamine (Bac),8 and
D-xylo-6-deoxy-4-ketohexosamine (DKH).9 Glycans con-
taining these monosaccharide units are important for
bacterial adherence and invasion (Figure 1).
~
(1) For selected reviews, see: (a) Marino, K.; Bones, J.; Kattla, J. J.;
Rudd, P. M. Nat. Chem. 2010, 6, 713. (b) Arnold, J. N.; Wormald, M. R.;
Sim, R. B.; Rudd, P. M.; Dwek, R. A. Annu. Rev. Immunol. 2007, 25, 21.
(2) For selected reviews, see: (a) Dube, D. H.; Bertozzi, C. R. Nat.
Rev. Drug Discovery 2005, 4, 477. (b) Stallforth, P.; Lepenies, B.;
Adibekian, A.; Seeberger, P. H. J. Med. Chem. 2009, 52, 5561. (c) Dube,
D. H.; Champasa, K.; Wang, B. Chem. Commun. 2011, 47, 87. (d) Boltje,
T. J.; Buskas, T.; Boons, G.-J. Nat. Chem. 2009, 1, 6111.
Although many of these monosaccharide building
blocks can be synthesized using classical carbohydrate
routes, those methods often require lengthy synthetic
(3) For selected examples, see: (a) Banerjee, A.; Wang, R.; Supernavage,
S. L.; Ghosh, S. K.; Parker, J.; Ganesh, N. F.; Wang, P. G.; Gulati, S.; Rice,
P. A. J. Exp. Med. 2002, 196, 147. (b) Power, P. M.; Ku, S. C.; Rutter, K.;
Warren, M. J.; Limnios, E. A.; Tapsall, J. W.; Jennings, M. P. Infect. Immun.
2007, 75, 3202.
(4) For selected examples, see: (a) van Sorge, N. M.; Bleumink,
N. M.; van Vliet, S. J.; Saeland, E.; van der Pol, W. L.; van Kooyk, Y.;
van Putten, J. P. Cell. Microbiol. 2009, 11, 1768. (b) Verma, A.; Arora,
S. K.; Kuravi, S. K.; Ramphal, R. Infect. Immun. 2005, 73, 8237.
(5) For selected reviews, see: (a) Zhu, X. M.; Schmidt, R. R. Angew.
Chem., Int. Ed. 2009, 48, 1900. (b) Codee, J. D. C.; Litjens, R.; van den
Bos, L. J.; Overkleeft, H. S.; van der Marel, G. A. Chem. Soc. Rev. 2005,
34, 769. (c) Boons, G.-J.; Demchenko, A. V. Chem. Rev. 2000, 100, 4539.
(6) For recent examples from our laboratory, see: (a) Yang, Y.;
Martin, C. E.; Seeberger, P. H. Chem. Sci. 2012, 3, 896. (b) Martin, C. E.;
Weishaupt, M. W.; Seeberger, P. H. Chem. Commun. 2011, 47, 10260.
r
10.1021/ol3023227
XXXX American Chemical Society

Scheme 1. Structural and Retrosynthetic Analysis
Figure 1. Rare 2-amino-2,6-dideoxy sugars found in bacteria.
sequences, extensive protecting group manipulations, and
expensive starting materials.10 Here, we demonstrate that
our de novo synthetic approach11 can be applied to a
straightforward synthesis of orthogonally protected bac-
terial 2-amino-2,6-dideoxy monosaccharides that can be
used in the assembly of oligosaccharide antigens.12
The three target molecules bear striking elements of
similarity (Scheme 1A). Three of the four stereocenters
(C2, C3, and C5) display identical stereochemical substitu-
tion patterns. The C4 position differs in oxidation state
or stereochemical substitution. A divergent strategy is
synthetically desirable to access all targets via selective
functionalization of a common late-stage intermediate.
The retrosynthetic analysis (Scheme 1B) revealed inter-
mediate A as a suitable precursor for further elaboration.
The carbon skeleton of thisintermediatewouldbeaccessed
by organometallic addition to commercially available
L-Garner aldehyde 1.13
The synthesis commenced with the chelation-controlled
addition14 of propynyl magnesium bromide to 1(Scheme2).15
Subsequent E-selective alkyne reduction was accom-
plished using Red-Al in Et2O. With gram quantities of 3
in hand, a sequence of O-protection, acid-catalyzed acet-
onide deprotection and Dess-Martin oxidation16 yielded
the desired intermediates 6aÀc, in five steps from commer-
cially available starting materials and only two chromato-
graphic purifications. Three different hydroxyl protecting
groups, namely benzyl ether, naphthyl ether, and benzoate
ester, were installed to access a series of orthogonally
protected derivatives (Scheme 2).
(7) (a) Smedley, J. G.; Jewell, E.; Roguskie, J.; Horzempa, J.;
Syboldt, A.; Stolz, D. B.; Castric, P. Infect. Immun. 2005, 73, 7922.
For evidence that the glycosylation aids in establishment of infection in
mouse model, see: (b) Castric, P.; Cassels, F. J.; Carlson, R. W. J. Biol.
Chem. 2001, 276, 26479. (c) Chamot-Rooke, J.; Rousseau, B.; Lanternier,
F.; Mikaty, G.; Mairey, E.; Malosse, C.; Bouchoux, G.; Pelicic, V.;
Camoin, L.; Nassif, X.; Dumenil, G. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 14783. Fucosamine is also present in the glycon of Elsamicin A; see:
(e) Sugawara, K.; Tsunakawa, M.; Konishi, M.; Kawaguchi, J. J. Org.
Chem. 1987, 52, 996. Also present in Neocarzinostatin; see: (f) Myers,
A. G.; Liang, J.; Hammond, M.; Harrington, P. M.; Wu, Y.; Kuo, E. Y.
J. Am. Chem. Soc. 1998, 120, 5319.
(8) (a) Banerjee, A.; Ghosh, S. K. Mol. Cell. Biochem. 2003, 253, 179.
For evidence that the glycosylation is implicated in adherence, blocking
immune response, and spread of infection, see: (b) Power, P. M.; Seib,
K. L.; Jennings, M. P. Biochem. Biophys. Res. Commun. 2006, 347, 904.
Bacillosamine is also present in the glycan of Clycocinnamoylspermi-
dines; see: (c) Ellestad, G. A.; Cosulich, D. B.; Broschard, R. W.; Martin,
J. H.; Kunstmann, M. P.; Morton, G. O.; Lancaster, J. E.; Fulmor, W.;
Lovell, F. M. J. Am. Chem. Soc. 1978, 100, 2515.
(9) (a) Siemieniuk, R. A. C.; Gregson, D. B.; Gill, M. J. BMC Infect.
Dis. 2011, 11, 314. DKH is also present in the lypopolysaccharide of
Yerisinia pestis; see: (b) Pinta, E.; Duda, K. A.; Hanuszkiewicz, A.;
€
Kaczynski, Z.; Lindner, B.; Miller, W. L.; Hyytiainen, H.; Vogel, C.;
Borowski, S.; Kasperkiewicz, K.; Lam, J. S.; Radziejewska-Lebrecht, J.;
Skurnik, M.; Holst, O. Chem.;Eur. J. 2009, 15, 9747.
(10) For selected synthesis of fucosamine and bacillosamine starting
from galactosamine derivatives, see: (a) Liav, A.; Hildesheim, J.;
Zehavi, U.; Sharon, N. Carbohydr. Res. 1974, 33, 217. (b) Busca, P.;
Martin, O. R. Tetrahedron Lett. 2004, 45, 4433. (c) Jones, G. B.; Lin, Y.;
Xiao, Z.; Kappenb, L.; Goldberg, I. H. Bioorg. Med. Chem. 2007, 15,
784. (d) Weerapana, E.; Glover, K. J.; Chen, M. M.; Imperiali, B. J. Am.
Chem. Soc. 2005, 127, 13766. (e) Amin, M. N.; Ishiwata, A.; Ito, Y.
Carbohydr. Res. 2006, 341, 1922. For a synthesis of bacillosamine
starting from fucose, see: (f) Bedini, E.; Esposito, D.; Parrilli, M. Synlett
2006, 6, 825.
Scheme 2. Synthesis of Common Intermediates 6aÀc
(11) For selected reviews on de novo carbohydrate synthesis, see: (a)
Schmidt, R. R. Pure Appl. Chem. 1987, 59, 415. (b) Kirschning, A.;
€
Jesberger, M.; Schoning, K.-U. Synthesis 2001, 4, 507. (c) Hemeon, I.;
Bennet, A. J. Synthesis 2007, 13, 1899. For selected examples on de novo
carbohydrate synthesis, see: (d) Voigt, B.; Scheffler, U.; Mahrwald, R.
Chem. Commun. 2012, 48, 5304. (e) Crich, D.; Navuluri, C. Org. Lett.
2011, 13, 6288. (f) Lorpitthaya, R.; Suryawanshi, S. B.; Wang, S.;
Pasunooti, K. K.; Cai, S.; Ma, J.; Liu, X.-W. Angew. Chem., Int. Ed.
2011, 123, 12260. (g) Northrup, A. B.; MacMillan, D. W. C. Science
2004, 305, 1752. (h) Babu, R. S.; Zhou, M.; O’Doherty, G. A. J. Am.
Chem. Soc. 2004, 126, 3428. (i) Enders, D.; Grondal, C. Angew. Chem.,
Int. Ed. 2005, 44, 1210.
(12) For recent de novo carbohydrate syntheses from our laboratory,
see: (a) Calin, O.; Pragani, R.; Seeberger, P. H. J. Org. Chem. 2012, 77,
870. (b) Pragani, R.; Seeberger, P. H. J. Am. Chem. Soc. 2011, 133, 102.
(13) Garner, P. Tetrahedron Lett. 1984, 25, 5855.
Org. Lett., Vol. XX, No. XX, XXXX
B

With the late-stage intermediates 6aÀc in hand, the
elaboration tothe targetmonosaccharides was established.
In order to obtain D-fucosamines, a syn-dihydroxylation
anti to the allylic ether moiety had to be achieved. Ex-
posure of 6b to Upjohn oxidative conditions gave, after
peracetylation, D-fucosamine building block 8b in 81%
yield and 5:1 dr (C3ÀC4 anti/syn) whereby the diastereo-
mers are separable by column chromatography (Table 1,
entry 1).17 Decreasing the reaction temperature to 0 °C
(entry 2) or using a different Os(VIII) source (entry 3) did
not improve the selectivity. The use of Sharpless AD
conditions led to quantitative recovery of unreacted start-
configuration. Finally, treatment of monosaccharide 9a18
that was obtained in analogy to 9b, with Dess-Martin
Periodinane (DMP), gave the desired D-2,6-DKH building
block 11 in 87% yield and in seven steps from commer-
cially available starting materials. Observation of an
NOE enhancement between H3ÀH5 confirmed the
stereochemical arrangement of 11 using NMR spectro-
scopy (Scheme 3C).
For preparative purposes, the synthesis of 8b from
alcohol 5b was carried out on gram scale with only one
chromatographic purification.19
3
ing material (entry 4). A JH3ÀH4 of 3.5 Hz ensured the
formation of the desired syn cyclic product 8b.
Scheme 3. Building Blocks Synthesis
Table 1. Dihydroxylation of 6b
entry
reaction conditions
dr yield (%)a
1
2
3
cat. OsO4, NMO, acetoneÀH2O, rt
cat. OsO4, NMO, acetoneÀH2O, 0 °C
5b:1
5b:1
81
78
85
À
cat. K2(OH)2OsO4, NMO, acetoneÀH2O, rt 4b:1
4c AD-mix(R or β), t-BuOHÀH2O, rt
À
a Yield for both diastereomers. b R:β 4:1. c Addition of MeSO2NH2
was also evaluated.
When the reaction was performed using benzoylated
aldehyde 6c, compound 8c was formed in 71% as the only
detectable diastereomer (Scheme 3A). Monosaccharide 8c
was crystallized from hot n-hexane/EtOAc and the stereo-
chemical assignment was confirmed by X-ray analysis. To
access D-bacillosamine derivatives, an equatorial nitrogen
moiety had to be introduced at C4. Dihydroxylation
of aldehyde 6b, and selective anomeric acetylation, gave
sugar 9b. After activation of the C4 hydroxyl using Tf2O
and triethylamine, followed by azide displacement with
sodium azide, the desired building block 10 was obtained
over seven steps from 1 (Scheme 3B). Crystallization of
10 from boiling n-hexane/CH2Cl2 provided good quality
crystals for X-ray analysis that confirmed the absolute
In order to present these monosaccharides on glycan
microarrays or on carrier proteins, the introduction of an
alkyl-amine spacer in the anomeric position is highly
desirable.20 Placement of a C3 napthyl ether provides for
further glycosylation at this position that typically serves
as a connection to the next sugar. Building block 8b is
therefore ideal in terms of orthogonality and chemical
synthesis. Hence we decided to test its ability to undergo
anomeric functionalization and to effect glycosylation at
the C3-OH. Due to the presence of N-acetylated D-fucos-
amine residues in P. aeruginosa O-linked glycans the
strategic N-protecting group pattern needed to be evalu-
ated. The direct use of building block 8b under glycosylat-
(14) (a) Cram, D. J.; Kopecky, K. R. J. Am. Chem. Soc. 1959, 81,
2748. For a recent review, see: (b) O’Brien, A. G. Tetrahedron 2011, 67,
9639.
ing conditions proved difficult. When BF3 Et2O at À78 °C
3
(15) For selected studies on the addition of alkynes to Garner
aldehyde, see: (a) Zhang, X.; van der Donk, W. A. J. Am. Chem. Soc.
2007, 129, 2212. (b) Garner, P.; Park, J. M.; Malecki, E. J. Org. Chem.
1988, 53, 4395. For a review, see: (c) Liang, X.; Andersch, J.; Bols, M.
J. Chem. Soc., Perkin Trans. 1 2001, 2136.
(19) Oxidation of 5b under Swern conditions resulted in partial
epimerization at C2, and monosaccharide 21 was isolated.
(16) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
(17) For a discussion of stereocontrolled reactions of acyclic allylic
ethers, see: (a) Cha, J. K.; Kim, N.-S. Chem. Rev. 1995, 95, 1761. For
selected examples, see: (b) Cha, J. K.; Christ, W. J.; Kishi, Y. Tetra-
hedron Lett. 1983, 24, 3943. (c) Karjalainen, O. K.; Koskinen, A. M. P.
Org. Biomol. Chem. 2011, 9, 1231.
For a synthetic discussion of epimerizable N-protected R-amino alde-
hydes, see: Myers, A. G.; Zhong, B.; Movassaghi, M.; Kung, D. V.;
Lanman, B. A.; Kwon, S. Tetrahedron Lett. 2000, 41, 1359.
(18) The peracetylated diastereomers of 9a could not by separated by
column chromatography.
(20) Snapper, C.; Mond, J. J. Immunol. 1996, 157, 2229.
Org. Lett., Vol. XX, No. XX, XXXX
C

At this point a sequence of N-protecting group modification
and anomeric functionalization was attempted (Scheme 4B).
Direct N-Boc deprotection/N-acetylation afforded 13 in
84% yield. In this case, glycosylation with thiocresol
Scheme 4. Fuctionalization of Fucosamines 6b (A) and 13 (B)
(BF3 Et2O) resulted in the clean formation of thioglyco-
3
side 14 in 84% yield (Scheme 4B). The 3JH1‑H2 of 10.3 Hz
matched the selective formation of the expected β-anomer.
Building block 14 proved to be suitable for further mod-
ification, and after activation with NIS and TfOH, the
anomeric position was successfully functionalized with L-
serine derivative 15 or linker 17 to give glycosylated amino
acid 16 or linker-functionalized saccharide 18 as β-anomers
(3JH1‑H2 ≈ 8.3 Hz). Direct glycosylation of 17 using
donor 13 was also possible. At this point, the C3 Nap
ether was cleaved under oxidative conditions and the
corresponding alcohol was glycosylated with differentially
protected glucose building block 19.21 The desired
β-disaccharide 20 was formed in 61% yield over two steps
(β-anomer, 3JH1‑H2 of 7.8 Hz).
In conclusion, we report the first de novo syntheses of the
rare bacterial 2-amino-2,6-dideoxy sugars D-fucosamine,
D-bacillosamine, and D-xylo-2,6-DKH. Our method al-
lows for the divergent and scalable synthesis of the fully
functionalized building blocks in six to seven steps from
commercially available starting materials. The use of these
rare sugar building blocks in the synthesis of complex
bacterial glycans is currently under investigation.
Acknowledgment. The authors gratefully thank the
Max Planck Society for generous funding and Dr. A.
Hagelbach (FU) and Dr. A. Barandov (MPI) for X-ray
crystallographic analyses.
Supporting Information Available. Experimental de-
tails and characterization data for new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
was used, the intramolecular cyclization of the Boc-group
on the oxocarbonium ion could not be prevented and bi-
cyclic carbamate 12 was isolated in 79% yield (Scheme 4A).
ꢀ
(21) Ple, K.; Chwalek, M.; Voutquenne-Nazabadioko, L. Tetrahe-
dron 2005, 61, 4347.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX