Synthesis of pseudaminic acid, a unique nonulopyranoside derived from pathogenic bacteria through 6-deoxy-AltdiNAc

Article abstract of DOI:10.1016/j.tetlet.2010.11.078

Chemical synthesis of pseudaminic acid is described. Starting from N-acetylglucosamine, deoxygenation and deoxyamination with stereo-inversion afforded 6-deoxy-AltdiNAc, which is the key intermediate for the biosynthesis of pseudaminic acid. Subsequently, the elongation reaction via In-mediated allylation of 6-deoxy-AltdiNAc with bromomethacrylate ester derivative followed by ozonolysis and hydrolysis gave the desired pseudaminic acid. Furthermore, we demonstrated glycosylation with dibenzyl phosphite derivative of pseudaminic acid as the glycosyl donor to afford disaccharide.

Full text of DOI:10.1016/j.tetlet.2010.11.078

Tetrahedron Letters 52 (2011) 418–421
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of pseudaminic acid, a unique nonulopyranoside derived
from pathogenic bacteria through 6-deoxy-AltdiNAc
Yong Joo Lee a, Akemi Kubota a, Akihiro Ishiwata a, Yukishige Ito a,b,
⇑
a RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b ERATO Ito Glycotrilogy Project, JST, Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical synthesis of pseudaminic acid is described. Starting from N-acetylglucosamine, deoxygenation
and deoxyamination with stereo-inversion afforded 6-deoxy-AltdiNAc, which is the key intermediate for
the biosynthesis of pseudaminic acid. Subsequently, the elongation reaction via In-mediated allylation of
6-deoxy-AltdiNAc with bromomethacrylate ester derivative followed by ozonolysis and hydrolysis gave
the desired pseudaminic acid. Furthermore, we demonstrated glycosylation with dibenzyl phosphite
derivative of pseudaminic acid as the glycosyl donor to afford disaccharide.
Received 21 October 2010
Revised 10 November 2010
Accepted 15 November 2010
Available online 18 November 2010
Keywords:
Pseudaminic acid
Ó 2010 Elsevier Ltd. All rights reserved.
2,4-Diacetamido-2,4,6-trideoxy-
altropyranose
L-
In-mediated allylation
In contrary to the long standing belief, studies in recent years
have revealed the presence of O- as well as N-glycosylations of pro-
teins in various prokaryotes.1,2 O-Linked glycosylation by pseud-
been identified in various bacteria. Among them, 6-deoxy-AltdiNAc
is of particular interest as a biosynthetic precursor of Pse.9
In spite of its growing attention, chemistry of Pse has been
rather unexplored. There has been only a single reported synthesis
aminic acid
1 (Pse) is particularly intriguing. In flagellin
glycoproteins derived from several pathogenic bacteria such as
Campylobacter jejuni and Helicobacter pylori,2,3 Pse is directly con-
nected to their Ser/Thr residues and is known to be important for
bacteria’s motility and invasion to host cells.2 In addition, Pse has
been identified as a component of larger glycans. For instance, tri-
saccharide repeating unit of O-antigen polysaccharide from Esche-
richia coli O1364 contains Pse and Pseudomonas aeruginosa 1244
trisaccharide was revealed to have modified pseudaminic acid.5
Although its similarity to sialic acid (Neu5Ac), which exists in
various glycoconjugates of eukaryotic glycoproteins and glycolip-
ids, is obvious, Pse has structural features clearly distinct from
Neu5Ac (Fig. 1). Firstly, stereochemical configurations are opposite
of Pse,10 in which condensation of oxalacetic acid and
L-allose
derivative was employed as the key reaction. However, this reac-
tion gave the desired isomer as a minor product and the yield
was low (3%). In order to conduct systematic studies to clarify
the function of bacterial O-linked glycosylation, more practical
means to supply highly purified Pse will be required. With these
circumstances in mind, our study has aimed to establish synthetic
route to the Pse.11
OH
OH
OH
OH
9
Me
7
HO
CO2H
CO2H
O
O
AcHN
5
AcHN
at C-5, -7, and -8 between them. Unlike Neu5Ac, Pse is an L-sugar
OH
OH
HO
AcHN
and its equatorial glycoside, a naturally occurring form, is defined
as b. Furthermore, Pse has a 7,9-dideoxy structure and its C-7 posi-
tion is substituted by an N-acetyl or an N-acetimidoyl (Am) group.
On the other hand, presence of highly deoxygenated and acetam-
ido functionalized rare sugars, such as 2,4-diacetamido-2,4,6-tride-
1: Pseudaminic acid
Sialic acid
(N-Acetylneuraminic acid; Neu5Ac)
(Pse)
CO2H
CO2H
OH
OH
D
-glucopyranose (bacillosamine, Bac),6 2,4-diacetamido-2,4,6-
HO
oxy-
trideoxy-
Me
O
O
OR
OR
-galactopyranose (6-deoxy-GaldiNAc),7 and 2,4-diace-
AcHN
D
AcHN
OH
OH
HO
tamido-2,4,6-trideoxy-L
-altropyranose (6-deoxy-AltdiNAc)8 had
AcHN
Pse glycoside (β)
R: Thr/Ser or glycan
Neu5Ac glycoside (α)
R: Gal, GalNAc, Neu5Ac
⇑
Corresponding author. Tel.: +81 (0)48 467 9430; fax: +81 (0)48 462 4680.
E-mail address: yukito@riken.jp (Y. Ito).
Figure 1. Pse (1), Neu5Ac and their glycosides.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.11.078

Y. J. Lee et al. / Tetrahedron Letters 52 (2011) 418–421
419
OH
OBn
O
OBn
O
OH
AcHN
NHAc
a
b
Me
O
OBn
Me
N
OBn
8
Me
CO2H
O
Me
CO2H
AcHN
NHAc
NHAc
OH
MeO
AcHN
OH OH OH
O
9
10
1: Pse
In-mediated allylation
and
c
ozonolysis
OBn
O
OBn
O
Me
OBn
Me
OBn
R
N
OH
O
AcHN
Me
NHAc
O
R
NHAc
N
NHAc
Me
OH
NHAc
(III)Sm
AcHN
(III)Sm
A
B
OH OH
R = H or OMe
2: 6-deoxy-L-AltdiNAc
+
XCH2C(=CH2)CO2R
OH
O
OBn
O
d
Me
Me
R'HN
OH
NHAc
OBn
HO
AcHN
O
HO
NHAc
HO
OH
11 R'
AcHN
3: D-GlcNAc
2
a
b
H
Ac
Scheme 3. Reagents and conditions: (a) Dess–Martin periodinane, NaHCO3, CH2Cl2,
17 h, 94%; (b) MeONH2ÁHCl, NaHCO3, MeOH, 65 °C, 17 h, 95%, major/minor = 2:1; (c)
SmI2, MeOH, THF, 12 h, then Ac2O, pyridine, 6 h, 66%; (d) H2, Pd(OH)2, EtOH, 4 h,
Scheme 1. Retrosynthesis of Pse (1).
94%, a/b = 2:1.
HO
HO
O
a
c
e
O
b
d
HO
HO
HO
BnO
desired 2,6-dideoxy-N-acetyl-L-idosamine derivative 8 in 84% yield
AcHN
AcHN
OH
OBn
together with a minor amount (4%) of 6-deoxy-D-glucosamine type
3
4
stereoisomer (Scheme 2).
Introduction of an amino-functionality to C-4 position of the
compound 8 with stereochmeical inversion turned out to be non-
trivial. Unexpectedly, SN2-type displacements of various sulfonate
esters derived from 8 with N3 anion were not successful. As an
alternative approach, reductive amination through oxime deriva-
tive was examined. Thus, oxidation of alcohol 8 with Dess–Martin
periodinane gave ketone 9, which was treated with O-methylhydr-
oxylamine hydrochloride and NaHCO3 to afford the oxime 10 as a
2:1 mixture due to the E/Z isomerism. Since configuration of the
C@N linkage was not consequential, rigorous assignment of the
stereochemistry was not made. Subsequent reduction of 10 with
SmI213 in THF containing MeOH followed by immediate acetylation
of resultant 11a afforded the desired 4-acetamidoaltrose derivative
11b as a single isomer. We speculate that initially formed carban-
ion is under equilibration between two isomers A and B. Between
them, equatorially oriented anion B would be less favored because
of destabilization by 1,3-diaxial repulsion between steric hinder-
ance of Sm(III)13b and NHAc and formation of 11a via A was pre-
dominant.14 Finally, hydrogenolysis of benzyl ether of 11b
afforded 6-deoxy-AltdiNAc 2 (Scheme 3), which corresponds to
the biosynthetic intermediate of Pse.
Thus obtained 2 was subjected to chain elongation through
In-mediated allylation.15 Namely, it was reacted with bromometh-
acrylate ester 12 under Whitesides’ conditions to give the desired
erythro product 13 together with a slightly smaller amount of its
threo isomer 14 (77%, 13:14 = 5:4). Although all attempts to im-
prove the selectivity, including the use of Lewis acid, such as
La(OTf)3, Ba(OTf)3, or Ce(OTf)4ÁxH2O in various amounts, in the
presence or absence of chiral ligand,16 failed to improve the selec-
tivity; chromatographic separation allowed us to obtain stereo-
chemically homogeneous 13. Subsequent ozonolysis was
accompanied by spontaneous cyclization of the 2-oxo product
and cleanly afforded, after purification by a column of Iatrobeads
(CHCl3/MeOH/H2O, 65:30:5), ethyl pseudaminate 15 (85%,
I
I
O
O
TIPSO
HO
BnO
BnO
AcHN
AcHN
OBn
OBn
OBn
5
6
OBn
O
O
HO
Me
HO
BnO
AcHN
OBn
NHAc
8
7
Scheme 2. Reagents and conditions: (a) Ref.12; (b) I2, PPh3, imidazole, THF, 0 °C, 2 h,
88%; (c) TIPSOTf, 2,6-lutidine, CH2Cl2, 12 h, 92%; (d) t-BuOK, THF, 70 °C, 10 h, then
TBAF, THF, 2 h, 96%; (e) H2, RhCl(PPh3)3, benzene, EtOH, 3 h, 84%.
Outlined in Scheme 1 is our synthetic plan toward Pse, which
intended to mimic its biosynthetic pathway, as it employed 6-
deoxy-AltdiNAc 2 as the key intermediate.9 To be brief, starting
from N-acetylglucosamine 3, deoxygenation, and deoxyamination
with stereochemical inversion were expected to afford the 6-
deoxy-AltdiNAc 2. Subsequent In-mediated allylation was going
to give the framework of Pse 1 (Scheme 1).
As a precursor of 2, L-idosamine derivative 8 was prepared as
depicted in Scheme 2. To begin with, N-acetylglucosamine 3 was
converted to benzyl glycoside 4 according to the known proce-
dure.12 Iodination with I2, imidazole, and Ph3P cleanly afforded
compound 5. Subsequent b-elimination in the presence of DBU
gave 7, however, in a modest yield (45%). More practically, the
compound 5 was once converted into 4-O-trisopropylsilyl (TIPS)
ether 6, treatment of which with t-BuOK caused clean formation
of the olefin, and subsequent desilylation gave 5,6-anhydroglucos-
amine derivative 7 in 88% overall yield. Saturation of the exocyclic
olefin was conducted by catalytic hydrogenation in the presence of
Wilkinson’s catalyst. Pleasingly, it afforded stereoselectively the
a:b = 5:1). Finally, saponification of 15 under mildly basic condi-

420
Y. J. Lee et al. / Tetrahedron Letters 52 (2011) 418–421
CO2Et
AcHN
NHAc
AcHN
NHAc
AcHN
NHAc
O
Br
Me
CO2Et
Me
CO2Et
Me
H
12
+
a
OH OH OH
OH OH OH
13 (erythro)
OH OH
14 (threo)
b
b
OH
O
OH
OH
OH
OH
OH
O
Me
AcHN
OH
NHAc
Me
CO2R
Me
CO2R
O
AcHN
AcHN
OH
2
AcHN
AcHN
R
Et
H
R
Et
H
15
1
16
17
c
c
Scheme 4. Reagents and conditions: (a) In, 0.1 N HCl–EtOH (1:6), 40 °C, 12 h, 77%, 13/14 = 5:4; (b) O3, MeOH, À78 °C, 30 min, then 30% H2O2, H2O, HCO2H, 90 min, for 15:
85%,
a/b = 5:1; for 16: 86%, a/b = 3:1; (c) TEA–H2O (1:3), 0 °C, 2 h, for 1: 96%, for 17: 92%.
OBn
OBn
P
O
OH
OH
OAc
OH
OAc
a
b
Me
CO2Et
Me
CO2Et
Me
CO2Et
O
O
OAc
18
O
OAc
19
AcHN
AcHN
AcHN
AcHN
OH
AcHN
AcHN
15
OMe
BzO
O
BzO
BzO
OAc
HO
O
CO2Et
c
O
BzO
BzO
OAc
O
Me
19 +
+
BzO
AcHN
AcHN
Me
CO2Et
O
OAc
22
OCH3
AcHN
OAc
20
21
gauche
AcHN
H
Scheme 5. Reagents and conditions: (a) Ac2O, pyridine, 0 °C, 12 h, 73%,
:b = 10:1), 22: 47%] or CH2Cl2, rt, 6 h [21: 35% ( only), 22: 62%].
a/b = 8:1; (b) Et2NP(OBn)2, 1H-tetrazole, THF, 2 h, 94%; (c) TMSOTf, CH3CN, 0 °C, 3 h [21: 28%
(a
a
tions in 25% TEA–H2O17 gave desired pseudaminic acid 1.9a Its 4-
epi-isomer 17 was also obtained from exo-methylene 14 through
ethyl ester 16 by the same procedure (Scheme 4).18,19
With synthetic Pse ester 15 in hand, our effort was extended to
examine glycosylation with Pse donor. Naturally occurring Pse, so
far identified, is known to exist as a b (equatorial)-glycoside
(Fig. 1). Being aware of ample examples of glycosylation using
Neu5Ac thioglycoside, which predominantly gave equatorial glyco-
between proton of H-3ax and carbonyl carbon of the ethyl ester
being especially diagnostic. Obviously, the property Pse is mark-
edly different from Neu5Ac, preventing selective formation of
equatorial glycoside by using methods standardized in Neu5Ac
chemistry.
In conclusion, we have achieved chemical synthesis of pseud-
aminic acid from N-acetylglucosamine through 6-deoxy-AltdiNAc
as the key intermediate. Furthermore, the first example of glyco-
sylation with Pse donor was presented. Stereocontrolled formation
of b-Pse glycosides will be a subject of future study.
side (in this case, a; see Fig. 1) in CH3CN, our initial effort was de-
voted to prepare thioglycoside of Pse. However, thioglycoside
formation from the peracetate derived from 15, under the condi-
tions established for Neu5Ac,20 was extremely sluggish. We then
turned our attention to the preparation of hemiketal 18. Although
controlled acetylation under acidic conditions21 was not success-
ful, treatment of 15 with 3 equiv of Ac2O in pyridine at 0 °C gave
the desired hemiketal 18 in 73% yield. Subsequent treatment with
dibenzyl N,N-diethylphosphoramidite and tetrazole gave dibenzyl
phosphite 19 in 94% yield,22 which was tested as the Pse donor.
In fact, glycosylation with 20 in the presence of TMSOTf22
(0.6 equiv) in CH3CN afforded the disaccharide 2123 in 28% yield.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Re-
search (B) (No. 22510243, A.I.) by the Ministry of Education, Cul-
ture, Sports, Science, and Technology, Foreign Postdoctoral
Researcher Program (Y.J.L), and Incentive Research Grant (2009)
and Fund for Seeds of Collaborative Research (2010) in RIKEN
(A.I.). We thank Mrs. A. Takahashi and Mrs. S. Shirahata for techni-
cal assistance, and Dr. M. N. Amin for his effort on the unsettled
phase of this work.
However the product mainly consisted of the
:b = 10:1) and a larger amount (47%) of 2,3-dehydro derivative
22 was formed (Scheme 5). The same reaction in CH2Cl2 solely gave
the -glycoside in somewhat higher yield (35%) together with 62%
a (axial)-glycoside
(a
Supplementary data
a
Supplementary data (experimental procedures and 1H and 13C
NMR spectra of all new compounds) associated with this article
yield of 22. Stereochemistry of the newly generated glycosidic link-
age was determined by HMBC,23 presence of gauche correlation

Y. J. Lee et al. / Tetrahedron Letters 52 (2011) 418–421
421
14. In 1H NMR of compound 8, observed signals with small coupling constants
(JH1–H2 = 2, JH2–H3 and JH3–H4 = 3, JH4–H5 = ꢀ0) suggested that the predominant
can be found, in the online version, at doi:10.1016/
j.tetlet.2010.11.078.
conformation of 8 is 1C4. For general disscussion about the conformation of
a-
D-idose; see: Grindley, T. B. In Fraser-Reid, B., Tatsuta, K., Thiem, J., Eds., second
ed.; Glycoscience, Chemistry and Chemical Biology; Springer: Berlin, 2008; Vol.
1, pp 3–55.
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OCH2CH3), 1.25 (d, J = 6.9 Hz, 3H, H-9), 1.81 (t, J = 13.1 Hz, 1H, H-3a), 1.96 (s,
3H, Ac), 1.97 (s, 3H, Ac), 1.99 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.16 (dd, J = 13.1,
5.0 Hz, 1H, H-3e), 3.45 (s, 3H, OCH3), 3.71 (d, J = 3.2 Hz, 2H, H-6Glc), 3.82–3.90
(m, 1H, OCHHCH3), 3.98–4.04 (m, 1H, OCHHCH3), 4.07 (dd, J = 10.5, 1.4 Hz, 1H,
H-6), 4.24 (dt, J = 10.6, 3.2 Hz, 1H, H-5Glc), 4.55–4.61 (m, 2H, H-5, H-7), 5.14–
5.26 (m, 4H, H-4, H-8, H-1Glc, H-2Glc), 5.48 (d, J = 11.0 Hz, 1H, NH), 5.72 (t,
J = 10.1 Hz, 1H, H-4Glc), 5.87 (d, J = 9.2 Hz, 1H, NH), 6.12 (t, J = 10.1 Hz, 1H, H-
3Glc), 7.25–7.56 (m, 9H, ArH), 7.90–7.99 (m, 6H, ArH); 13C NMR (100 MHz,
CDCl3) d 14.0, 14.6, 21.1, 21.4, 23.4, 23.5, 32.2, 45.5, 50.5, 55.9, 62.1, 62.3, 67.2,
68.0, 68.8, 70.2, 70.3, 70.7, 72.4, 97.3, 98.8, 128.4, 128.6, 129.1, 129.3, 129.9,
130.1, 133.2, 133.4, 133.5, 164.9, 165.9, 166.0, 166.7, 170.0, 170.4, 170.6, 171.1.
The crosspeak between C2 and H3a could not be observed by HMBC
experiment, which suggested the formation of b-isomer. JH3a–H4 (ꢀ13 Hz)
indicated that 21
stereochemistry.
a
has chair conformation with 4-O-equatorial
13. (a) Molander, G. A. Org. React. 1994, 46, 211–367; (b) Hsu, D.-S.; Matsumoto, T.;
Suzuki, K. Synlett 2005, 801–804.