Chemoenzymatic synthesis of CMP-N-acetyl-7-fluoro-7-deoxy-neuraminic acid

Article abstract of DOI:10.1016/j.carres.2008.02.003

7-Fluoro sialic acid was prepared and activated as cytidine monophosphate (CMP) ester. The synthesis started with d-glucose, which was efficiently converted into N-acetyl-4-fluoro-4-deoxy-d-mannosamine. Aldolase catalyzed transformation yielded the corres

Full text of DOI:10.1016/j.carres.2008.02.003

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 2075–2082
Chemoenzymatic synthesis of
CMP-N-acetyl-7-fluoro-7-deoxy-neuraminic acid
Sina Hartlieb,a Almut Gunzel,b Rita Gerardy-Schahn,b Anja K. Munster-Kuhnel,b
¨
¨
¨
Andreas Kirschninga and Gerald Dragera,*
¨
aInstitut fu¨r Organische Chemie und Zentrum fu¨r Biomolekulare Wirkstoffchemie (BMWZ), Gottfried Wilhelm Leibniz
Universita¨t Hannover, Schneiderberg 1B, 30167 Hannover, Germany
bDepartment of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Received 30 October 2007; received in revised form 31 January 2008; accepted 4 February 2008
Available online 10 February 2008
Presented at Eurocarb 14th Lu¨beck, Germany, September 2007
Abstract—7-Fluoro sialic acid was prepared and activated as cytidine monophosphate (CMP) ester. The synthesis started with D-
glucose, which was efficiently converted into N-acetyl-4-fluoro-4-deoxy-D-mannosamine. Aldolase catalyzed transformation yielded
the corresponding fluorinated sialic acid which was activated as CMP ester using three different synthetases in the presence as well as
in the absence of pyrophosphatase which possesses inhibitory properties. Finally, conditions were optimized to perform a one-pot
reaction starting from fluorinated mannosamine, which yielded the 7-fluoro-7-deoxy-CMP-sialic acid by incubation with three
enzymes.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: CMP-7-F-sialic acid; Chemoenzymatic synthesis; Sugar nucleotide
1. Introduction
sized.3,11,12 Several approaches follow chemoenzymatic
routes using derivates of N-acetyl-D-mannosamine and
pyruvate or pyruvate derivatives.2,3 Also, several total
synthesis approaches of native and artificial sialic acids
were published.2,3 Many sialic acid recognizing proteins
exhibit strong interactions to the glycerol side chain of
sialic acid, which makes this position important for
modifications.3
Sialic acids are a widely distributed class of negatively
charged 9-carbon sugars with more than 40 natural
occurring derivatives in vertebrates and bacteria.1 The
most important member of this family is N-acetyl-neu-
raminic acid. The synthesis of sialic acid and analogues
has been extensively studied.2,3 The enzymatic synthe-
sis of N-acetyl-neuraminic acid (sialic acid, 1) from
N-acetyl-D-mannosamine or N-acetyl-D-glucosamine
followed by activation as CMP-ester is well estab-
lished.4–6 Multienzyme systems have been optimized for
this sequence,7–9 which includes the regeneration of
CMP to CTP.10 To access new derivatives, every position
of sialic acid was addressed during the past years1–3 and
several variations at position 7 of sialic acid were synthe-
2. Results and discussion
Here, we report an improved synthesis of CMP-N-acet-
yl-7-fluoro-7-deoxy-neuraminic acid (1)12 starting from
D-glucose. The synthesis of 1 (Scheme 1) starts with
commercially available methyl glycoside 2. After activa-
tion as triflate, azide introduction was readily accom-
plished using sodium azide in DMF following an
approach published by Moravcova and coworkers13
and Pipik and coworkers.14 In our hands, the isolated
*
Corresponding author. Fax: +49 511 762 3011; e-mail: draeger@oci.
uni-hannover.de
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.02.003

2076
S. Hartlieb et al. / Carbohydrate Research 343 (2008) 2075–2082
N3
N3
Ph
O
Ph
O
b
Ph
O
a
O
O
O
O
O
O
HO
HO
BnO
OH
OMe
OMe
3
4
c
2
OMe
OBz
N3
BnO
N3
N3
BzO
BzO
e
O
O
O
F
HO
+
BnO
BnO
OMe
F
OMe
OMe
7
8
5
d
f
OBz
N3
F
N3
N3
BzO
BzO
O
g
O
O
F
TfO
BnO
BnO
BnO
OAc
OMe
OH
OMe
11
9
6
h
NHAc
O
NHAc
BzO
HO
O
F
F
HO
HO
12
OAc
13
Scheme 1. Reagents and conditions: (a) Tf2O, pyridine, CH2Cl2, ꢀ20 °C, 2 h; then NaN3, 15-crown-5, DMF, 50 °C, 24 h, 93%; (b) BnBr, TBAI,
NaH, DMF, 0 °C?20 °C, 14 h, 93%; (c) p-TSA, MeOH, 20 °C, 3 h; then BzCl, 2,6-lutidine, CH2Cl2, 0 °C, 2 h, 99% (d) Tf2O, pyridine, CH2Cl2,
ꢀ20 °C?ꢀ8 °C, 3 h; (e) DAST, CH2Cl2, ꢀ78 °C?20 °C, 4 d, 63% for 7; 13% for 8, 11% for 5; (f) Ac2O/H2SO4 (3v%), 0 °C, 1 h, a-anomer 63%, b-
anomer 26%; (g) Pd(OAc)2, EtOAc, Ac2O, 20 °C, 5.6 bar, 4 ꢁ 24 h, 79%; (h) NaOMe, MeOH, 20 °C, 3 h.
yield of manno azide 3 could be improved from 53% to
93% by the addition of crown ether (15-c-5), which also
allowed us to reduce the amount of sodium azide and
lower the reaction temperature.
which was ring-opened by fluoride to the corresponding
manno- and ido-configured hexoses 7 and 8, respectively
(Scheme 2). DAST-induced oxirane formation was also
encountered during the synthesis of fluorinated furano-
sides by Mikhailopulo and Sivets.15 Anomeric deprotec-
tion (H2SO4 in acetic acid anhydride) gave acetate 11 as
anomeric mixture (89%; a/b = 2.4:1), which was deben-
zylated at elevated pressure to yield compound 12. Final
transesterification was performed with MeOH/NaOMe
and the resulting N-acetyl-4-deoxy-4-fluoro-D-mannosa-
mine (13) was formed and could be employed for enzy-
matic syntheses without further purification.
Studies on the enzymatic synthesis of 7-deoxy-7-flu-
oro sialic acid (14) were initiated using commercially
available sialic acid aldolase (Fluka). Reaction condi-
tions16 found for complete transformation of N-acetyl-
mannosamine into N-acetyl-neuraminic acid were
To selectively address position 4 of the hexose, benzyl
ether formation at 3-OH was carried out first. Hydroly-
sis of the benzylidene acetal 4 under acidic conditions
(pTsOH in methanol) yielded the 4,6-diol, which could
be selectively converted into benzoate 5 in 99% overall
yield. Any attempts to fluorinate triflate 6 failed; how-
ever, the reaction of alcohol 5 with DAST in dichloro-
methane gave fluorinated hexose 7 in 63% yield.
Surprisingly, product 9 which results from SN2 inversion
of configuration was not detected. In fact, we could only
isolate the 3-fluoro sugar 8. We tentatively assume that a
DAST-promoted removal of the hydroxyl group in 5
furnishes the intermediate O-benzylated oxirane 10,
Bn
OBz
N3
Et2NSF3
N3
BzO
F
N3
O
H
BzO
S O
BnO
(DAST)
-Et2NSF2OH
O
F
O
HO
O
BnO
H
a
Et2N
OMe
OMe
OMe
5
+ HF
F
a
b
10
b
OBz
N3
N3
BzO
BnO
O
+
F
O
BnO
OMe
F
OMe
7
8
Scheme 2. Proposed mechanism for the benzyloxy migration and formation of 7 and 8.

S. Hartlieb et al. / Carbohydrate Research 343 (2008) 2075–2082
2077
OH
aldolase, CSS, CTP,
OCMP
CO2H
+ Pi
HO
AcHN
NHAc
O
HO
pyrophosphatase
F
O
HO
F
HO
OH
+ pyruvate
15
13
aldolase
CTP, CSS,
pyrophosphatase
OH
OH
HO
AcHN
O
CO2H
F
HO
14
Scheme 3. Aldolase and CMP-sialic acid synthetase reaction to CMP-7-fluoro sialic acid.
applied to N-acetyl-4-fluoro-mannosamine 13 and resulted
CMP-7F-sialic acid
7F-sialic acid
4F-ManAc
in only 56% conversion to 14. Therefore, we planned to
shift the aldolase equilibrium by coupling the aldolase
step with the CMP activation using CMP-sialic acid syn-
thetase (CSS) (Scheme 3). We tested three different CSS,
which were cloned from Neisseria meningitidis sero-
group B (NmB),17 mouse (amino acid 39–267 of the
murine CSS with N-terminal NusA-StrepII-thrombin-
tag in expression vector pET43)18 and rainbow trout
(amino acid 28–255 of rainbow trout CMP-Kdn-synthe-
tase with N-terminal NusA-StrepII-thrombin-tag in
expression vector pET43.Strep).19 N-Acetyl-mannosa-
mine served again as a model substrate to find optimized
reaction conditions. Addition of pyrophosphatase was
crucial for rapid transformation although higher conver-
sion to CMP-N-acetyl-neuraminic acid was also
achieved after a prolonged incubation time in the ab-
sence of this enzyme (Fig. 1). In contrast to these results,
no formation of CMP glycoside 15 could be observed in
the absence of pyrophosphatase, and only N-acetyl-4-
fluoro-mannosamine 13 and 7-F-sialic acid 14 were pres-
ent in the reaction mixture as judged by ESIMS. In
comparison to the other CSS, the bacterial enzyme from
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Figure 2. Optimization of reaction conditions for the synthesis of
CMP-7-deoxy-7-fluoro sialic acid (percentage of the different products
using different CMP-sialic acid synthetases at pH 8.8 according to LC-
ESIMS).
NmB gave best results (Fig. 2). Thus, coupling aldolase,
CSS and pyrophosphatase in a one-pot reaction did not
improve the low efficiency of the aldolase reaction and
hexosamine 13 was still detectable along with not acti-
vated 7-F-sialic acid 14. Using purified 14 and CSS
(NmB, pyrophosphatase) resulted in complete conver-
sion to 15 while no free 7-F-sialic acid could be detected
by ES-MS.
In summary, we present a flexible and high yielding
synthesis of N-acetyl-4-fluoro-mannosamine 13 starting
from D-glucose. Aldolase-catalyzed transformation to
the corresponding 7-deoxy-7-fluoroneuraminic acid 14
was performed and three CMP-sialic acid synthetases
were compared for CMP-activation. The present study
is currently expanded to other neuraminic acid ana-
logues in our laboratories.
CMP-sialic acid
ManAc
sialic acid
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
3. Experimental
3.1. General methods
Figure 1. Optimization of reaction conditions for the one-pot two/
three-enzyme system sialic acid aldolase/CMP-sialic acid synthetase/
pyrophosphatase (PPase) (percentage of the different products with
and without pyrophosphatase at pH 8.8 according to LC-ESIMS).
Optical rotations were measured with a Perkin Elmer
341 polarimeter. NMR spectra were recorded on Bruker

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S. Hartlieb et al. / Carbohydrate Research 343 (2008) 2075–2082
ARX-400 (1H, 400 MHz; 13C, 100 MHz) and Bruker
AM-500 (1H, 500 MHz; 13C, 125 MHz) spectrometers.
All spectra were measured using standard Bruker pulse
sequences. ESI mass spectra were recorded on a LCT
mass spectrometer (Micromass) with Lock-Spray dual
ion source. The LCT-spectrometer was coupled with a
Waters Alliance 2695 HPLC unit. All solvents used were
of reagent grade and were further dried. Reactions were
monitored by thin-layer chromatography (TLC) on Sil-
ica Gel 60 F254 (E. Merck, Darmstadt) and spots were
detected either by UV-absorption or by charring with
KMnO4/NaOH in water. Preparative column chroma-
tography was performed on Silica Gel 60 (E. Merck,
Darmstadt). HPLC-purifications were carried out using
a Merck/Hitachi LaChrom unit (L-7150 pump, L-7455
DAAD, Sedere Sedex-75 ELSD). Reagents were puri-
fied and dried by standard techniques.
3.3. Methyl 2-azido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-a-D-mannopyranoside (4)
A soln of 3 (100 mg, 0.325 mmol) in DMF (1 mL) was
stirred under argon at 0 °C. Sodium hydride (19.6 mg,
0.488 mmol) was added portionwise followed by tetra-
butylammonium bromide (12.0 mg, 0.033 mmol) and
benzyl bromide (77 lL, 0.651 mmol). Stirring was con-
tinued at 0 °C for 2 h and at 20–22 °C for 16 h. The mix-
ture was terminated by the addition of methanol
(0.2 mL), followed by water (5 mL) and extracted with
Et2O (4 ꢁ 5 mL). Combined organic extracts were
washed with water (3 ꢁ 5 mL) and brine (5 mL), dried
over MgSO4 and concentrated under diminished pres-
sure. The crude product was purified by column chro-
matography (10:1–5:1 hexane–EtOAc) to give
4
(128 mg, 0.322 mmol; 99%) as an amorphous white so-
20
lid. ½aꢂD +57.2 (c 1, CHCl3); Rf 0.40 (hexane–
1
3.2. Methyl 2-azido-4,6-O-benzylidene-2-deoxy-a-D-
EtOAc = 5:1); H NMR (400 MHz, CDCl3, d CHCl3
mannopyranoside (3)13,14
7.26) d: 7.53–7.29 (m, 10H, Ph), 5.65 (s, 1H, CHPh),
4.91 (d, J = 12.1 Hz, 1H, CH2Ph), 4.76 (d, J =
12.1 Hz, 1H, CH2Ph), 4.68 (d, J = 1.7 Hz, 1H, H-1),
4.30 (dd, J = 10.0, 4.1 Hz, 1H, H-6a), 4.16–4.12 (m,
2H, H-4, H-3), 4.02–4.00 (m, 1H, H-2), 3.90–3.77 (m,
2H, H-6b, H-5), 3.37 (s, 3H, OCH3); 13C NMR (100
MHz, CDCl3, d CDCl3 77.16) d: 138.1 (CHPh C-1),
137.5 (CH2Ph C-1), 129.0–126.1 (Ph), 101.7 (CHPh),
100.2 (C-1), 79.2 (C-4), 75.8 (C-3), 73.3 (CH2Ph), 68.8
(C-6), 63.8 (C-5), 62.7 (C-2), 55.1 (OCH3).
To a soln of methyl 4,6-O-benzylidene-a-D-glucopyran-
oside (2) (5.27 g, 18.7 mmol) in CH2Cl2 (60 mL) was
added pyridine (60 mL, 37.3 mmol) under argon. At
ꢀ20 °C, trifluoromethanesulfonic acid anhydride
(3.72 mL, 22.4 mmol) was added dropwise to the mix-
ture and the temperature was kept at ꢀ20 °C for 2 h.
The reaction mixture was quenched with brine
(50 mL), washed with brine (3 ꢁ 50 mL) and dried over
MgSO4. After the removal of CH2Cl2, traces of water
were removed by azeotropic distillation with toluene
and the trifluoromethanesulfonic ester was dissolved in
DMF (50 mL) under argon atmosphere without further
purification. Sodium azide (3.64 g, 56.0 mmol) and 15-
crown-5 ether (11.2 mL, 56.0 mmol) were added and
the suspension was stirred at 50 °C for 25 h. After termi-
nating the reaction by the addition of water (100 mL),
the mixture was extracted with Et2O (4 ꢁ 50 mL). The
combined organic extracts were washed with water
(3 ꢁ 50 mL) and brine (50 mL), dried over MgSO4 and
concentrated under diminished pressure. The crude
product was purified by column chromatography
(10:1–3:1 hexane–EtOAc) to give 3 (5.3 g, 17.3 mmol;
3.4. Methyl 2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-a-
D-mannopyranoside (5)
To a soln of 4 (12.39 g, 31.19 mmol) in MeOH (150 mL)
was added p-toluenesulfonic acid (1.78 g, 9.36 mmol).
After stirring at 20–22 °C for 3 h, the mixture was neu-
tralized with NaHCO3 and concentrated under dimin-
ished pressure. Traces of water and methanol were
removed by azeotropic distillation with toluene and
the resulting diol was dissolved in anhydr CH2Cl2
(150 mL) under argon atmosphere. After the addition
of 2,6-lutidine (5.43 mL, 46.79 mmol) followed by ben-
zoyl chloride (4.35 mL, 37.42 mmol) stirring was contin-
ued at 0 °C for 2 h. The mixture was washed with water
(3 ꢁ 50 mL) and brine (50 mL). The organic extracts
were dried over MgSO4 and concentrated under dimin-
ished pressure. The crude product was purified by col-
umn chromatography (10:1–5:1 hexane–EtOAc) to
give 5 (12.71 g, 30.75 mmol; 99%) as an amorphous
20
93%) as an amorphous white solid. ½aꢂD +59.1 (c 1,
CHCl3); Rf 0.10 (hexane–EtOAc = 3:1); 1H NMR
(400 MHz, CDCl3, d CHCl3 7.26) d: 7.52–7.47 (m, 2H,
Ph), 7.42–7.35 (m, 3H, Ph), 5.56 (s, 1H, CHPh), 4.67
(d, J = 1.1 Hz, 1H, H-1), 4.27–4.22 (m, 1H, H-6a),
4.22 (ddd, J = 9.6, 4.1, 3.9 Hz, 1H, H-3), 3.91 (dd,
J = 3.9, 1.1 Hz, 1H, H-2), 3.88 (dd, J = 9.6, 9.6 Hz,
1H, H-4), 3.82–3.72 (m, 2H, H-6b, H-5), 3.38 (s, 3H,
OCH3), 2.81 (d, J = 4.1 Hz, 1H, OH); 13C NMR
(100 MHz, CDCl3, dCDCl3 77.16) d: 137.2 (C-1 Ph),
129.4 (C-4 Ph), 128.5 (C-2 Ph), 126.4 (C-3 Ph), 102.4
(PhCH), 100.2 (C-1), 79.1 (C-4), 68.9 (C-3), 68.8 (C-6),
63.7 (C-2), 63.4 (C-5), 55.3 (OCH3).
20
white solid. ½aꢂD +20.5 (c 1, CHCl3); Rf 0.34 (hexane–
EtOAc = 2:1); 1H NMR (400 MHz, C6D6, d C6D5H
7.16) d: 8.32–8.28 (m, 2H, Bz H-3), 7.42–7.39 (m, 2H,
Bn H-3), 7.31–7.13 (m, 6H, Bn and Bz), 4.78 (dd,
J = 11.9, 5.3 Hz, 1H, H-6a), 4.71 (dd, J = 11.9,
2.3 Hz, 1H, H-6b), 4.55 (d, J = 11.7 Hz, 1H, CHH0Ph),
4.47 (d, J = 11.7 Hz, 1H, CHH0Ph), 4.46 (d, J = 1.4 Hz,

S. Hartlieb et al. / Carbohydrate Research 343 (2008) 2075–2082
2079
1H, H-1), 4.09 (dd, J = 9.7, 9.4 Hz, 1H, H-4), 3.92 (dd,
J = 9.4, 3.7 Hz, 1H, H-3), 3.88 (ddd, J = 9.7, 5.3,
2.3 Hz, 1H, H-5), 3.63 (dd, J = 3.7, 1.4 Hz, 1H, H-2),
3.03 (s, 3H, OCH3); 13C NMR (100 MHz, C6D6, d
C6D6 128.06) d: 166.7 (COPh), 138.3 (C-1 Bn), 133.0
(C-4 Bz), 130.7 (C-1 Bz), 130.1 (C-3 Bz), 128.8 (C-2
Bz), 128.6–127.8 (C-2, C3, C4 Bn), 99.5 (C-1), 79.7 (C-
3), 72.6 (CH2Ph), 71.1 (C-5), 67.4 (C-4), 64.1 (C-6),
60.9 (C-2), 54.5 (OCH3); HRESIMS: calcd for
C21H23N3O6Na: 436.1490; found: 436.1485 [M+Na]+.
MgSO4 and concentrated under diminished pressure.
The crude product was purified by column chromatog-
raphy (15:1–10:1 hexane–EtOAc) to yield 7 (76 mg,
0.183 mmol; 63%, amorphous white solid), 8 (16 mg,
0.038 mmol; 13%, amorphous white solid) and 5
(13 mg, 0.032 mmol; 11%, colourless oil).
20
Compound 7: ½aꢂD +51.7 (c 1.1, CHCl3); Rf 0.54 (hex-
ane–EtOAc = 2:1); 1H NMR (400 MHz, CDCl3, d
CHCl3 7.26) d: 8.07–8.05 (m, 2H, Bz H-3), 7.59–7.52
(m, 1H, Bz H-4), 7.45–7.41 (m, 2H, Bz H-2), 7.33–7.29
(m, 5H, Bn), 4.86 (ddd, J = 51.0, 9.6, 9.1 Hz, 1H, H-
4), 4.83 (d, J = 11.8 Hz, 1H, CHH0Ph), 4.73 (d, J =
11.8 Hz, 1H, CHH0Ph), 4.72 (dd, J = 1.7, 1.7 Hz, 1H,
H-1), 4.66 (ddd, J = 12.0, 2.2, 2.1 Hz, 1H, H-6a), 4.49
(dd, J = 12.0, 5.2 and 1.1 Hz, 1H, H-6b), 4.10 (ddd,
J = 13.4, 9.1, 4.0 Hz, 1H, H-3), 4.00 (dddd, J = 9.6,
5.3, 5.2, 2.1 Hz, 1H, H-5), 3.96 (m, 1H, H-2), 3.38 (s,
3H, OCH3); 13C NMR (100 MHz, CDCl3, d CDCl3
77.16) d: 166.4 (COPh), 137.6 (C-1 Bn), 133.3 (C-4
Bz), 129.9 (C-3 Bz), 129.8 (C-1 Bz), 128.7 (C-2 Bz),
128.6–127.8 (C-2, C-3, C-4 Bn), 99.3 (d, JFꢀC = 1.2 Hz,
3.5. Methyl 2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-4-
O-trifluoromethanesulfonyl-a-D-mannopyranoside (6)
To a soln of 5 (100 mg, 0.242 mmol) in anhydr CH2Cl2
(1.5 mL) under argon atmosphere was added pyridine
(48.5 lL, 0.605 mmol). The mixture was stirred at
ꢀ20 °C for 10 min and trifluoromethanesulfonic acid
anhydride (56 lL, 0.339 mmol) was slowly added. With-
in 3 h the temperature was raised to ꢀ8 °C. The mixture
was diluted with CH2Cl2 (10 mL), washed with brine
(4 ꢁ 5 mL), dried over MgSO4 and concentrated under
diminished pressure. The crude product was purified
by column chromatography (5:1 hexane–EtOAc) to give
C-1), 88.3 (d, JFꢀC = 181.2 Hz, C-4), 76.7 (d, JFꢀC
17.4 Hz, C-3), 73.3 (t, JFꢀC = 1.7 Hz, CH2Ph), 68.3 (d,
JFꢀC = 24.3 Hz, C-5), 63.1 (C-6), 62.2 (d, JFꢀC
8.8 Hz, C-2), 55.4 (OCH3); HRESIMS: calcd for
=
=
20
6 (128 mg, 0.235 mmol; 97%) as a colourless oil. ½aꢂD
1
+64.7 (c 1, CHCl3); Rf 0.44 (hexane–EtOAc = 3:1); H
C23H25N4O5FNa:
479.1707;
found:
479.1715
NMR (400 MHz, CDCl3, d CHCl3 7.26) d: 8.10–8.08
(m, 2H, Bz H-3), 7.60–7.59 (m, 1H, Bz H-4), 7.50–7.36
(m, 7H, Ph and Bz), 5.29 (dd, J = 9.9, 9.4 Hz, 1H, H-
4), 4.82 (d, J = 11.3 Hz, 1H, CHH0Ph), 4.74 (dd,
J = 12.5, 2.0 Hz, 1H, H-6a), 4.71 (d, J = 1.4 Hz, 1H,
H-1), 4.65 (d, J = 11.3 Hz, 1H, CHH0Ph), 4.40 (dd,
J = 12.5, 4.3 Hz, 1H, H-6b), 4.17 (dd, J = 9.4, 3.6 Hz,
1H, H-3), 4.13 (ddd, J = 9.9, 4.3, 2.0 Hz, 1H, H-5),
3.84 (dd, J = 3.6, 1.4 Hz, 1H, H-2), 3.37 (s, 3H,
OCH3); 13C NMR (100 MHz, CDCl3, d CDCl3 77.16)
d: 166.1 (COPh), 136.5 (C-1 Bn), 133.4 (C-4 Bz), 129.9
(C-3 Bz), 129.6 (C-1 Bz), 128.8 (C-2 Bz), 128.7–128.6
(C-2, C3, C4, Bn), 118.5 (q, J = 319.1 Hz, CF3), 99.2
(C-1), 80.2 (C-4), 76.2 (C-3), 73.4 (CH2Ph), 68.0 (C-5),
62.2 (C-6), 61.6 (C-2), 55.6 (OCH3).
[M+Na+CH3CN]+.
20
Compound 8: ½aꢂD +2.6 (c 0.4, CHCl3); Rf 0.69
1
(hexane–EtOAc = 2:1); H NMR (400 MHz, CDCl3, d
CHCl3 7.26) d: 8.07–8.05 (m, 2H, Bz H-3), 7.59–7.52
(m, 1H, Bz H-4), 7.45–7.41 (m, 2H, Bz H-2), 7.33–7.29
(m, 5H, Bn), 4.83 (d, J = 11.9 Hz, 1H, CHH0Ph), 4.74
(ddd, J = 49.7, 8.3, 5.5 Hz, 1H, H-3), 4.70 (d, J =
5.3 Hz, 1H, H-1), 4.63 (dd, J = 12.0, 8.0 Hz, 1H, H-
6a), 4.62 (d, J = 11.9 Hz, 1H, CHH0Ph), 4.47 (dd, J =
12.0, 4.0 Hz, 1H, H-6b), 4.38 (ddd, J = 8.0, 4.7,
4.0 Hz, 1H, H-5), 3.86 (ddd, J = 16.5, 5.5, 4.7 Hz, 1H,
H-4), 3.59 (ddd, J = 13.2, 8.3, 5.3 Hz, 1H, H-2), 3.42
(s, 3H, OCH3); 13C NMR (100 MHz, CDCl3, d CDCl3
77.16) d: 166.3 (COPh), 137.1 (C-1 Bn), 133.4 (C-4
Bz), 129.8 (C-1 Bz), 129.7 (C-3 Bz), 128.7 (C-2 Bz),
128.6–128.3 (C-2, C-3, C-4 Bn), 99.7 (d, JFꢀC = 7.1 Hz,
3.6. Methyl 2-azido-6-O-benzoyl-3-O-benzyl-2,4-dideoxy-
4-fluoro-a-D-mannopyranoside (7) and methyl 2-azido-6-
O-benzoyl-4-O-benzyl-2,3-dideoxy-3-fluoro-a-D-idopyr-
anoside (8)
C-1), 92.1 (d, JFꢀC = 184.2 Hz, C-3), 75.2 (d, JFꢀC
20.9 Hz, C-4), 73.1 (d, JFꢀC = 0.7 Hz, CH2Ph), 68.9
(d, JFꢀC = 6.3 Hz, C-5), 62.6 (C-6), 62.2 (d, JFꢀC
20.1 Hz, C-2), 56.2 (OCH3); HRESIMS: calcd for
=
=
C23H25N4O5FNa:
479.1707;
found:
479.1715
In a TeflonTM flask, DAST (0.28 mL, 2.322 mmol) was
dissolved in anhydr CH2Cl2 (1.5 mL). A soln of 5
(120 mg, 0.290 mmol) in anhydr CH2Cl2 (2 mL) was
slowly added at ꢀ78 °C. The temperature was raised
to 20 °C and stirring was continued for 4 d. The mixture
was cooled to ꢀ20 °C, terminated by the addition of
methanol (1 mL) and concentrated under diminished
pressure. The residue was dissolved in CH2Cl2
(10 mL), washed with water (3 ꢁ 5 mL), dried over
[M+Na+CH3CN]+.
3.7. 1-O-Acetyl-2-azido-6-O-benzoyl-3-O-benzyl-2,4-
dideoxy-4-fluoro-a,b-D-mannopyranose (11)
Mannopyranoside 7 (750 mg, 1.805 mmol) was dis-
solved in acetic anhydride (9.9 mL) and concd H2SO4
(0.1 mL) at 0 °C and stirring was continued for 1 h.
After neutralization with phosphate buffer (100 mM,

2080
S. Hartlieb et al. / Carbohydrate Research 343 (2008) 2075–2082
pH 7.0), the mixture was extracted with EtOAc
(4 ꢁ 50 mL). The combined organic extracts were
washed with brine (2 ꢁ 50 mL), dried over MgSO4, con-
centrated under diminished pressure and co-distilled
with toluene. The crude product was purified by column
chromatography (20:1–10:1 hexane–EtOAc) to yield 11
(a-anomer: 504 mg, 1.137 mmol; 63%, and b-anomer:
210 mg, 0.474 mmol; 26%; both as amorphous white
solids).
a short pad of CeliteTM and washing with EtOAc
(50 mL), the crude product was dried under diminished
pressure. It turned out to be necessary to repeat the
whole reaction procedure four times. The product was
purified by column chromatography (silica gel; EtOAc)
to yield 12 (305 mg, 0.826 mmol; 79%) as an amorphous
20
white solid. ½aꢂD +34.83 (c 1, CHCl3); Rf 0.38 (EtOAc);
1H NMR (400 MHz, CDCl3, d CHCl3 7.26) d: 8.01–7.99
(m, 2H, Bz H-3), 7.59–7.58 (m, 1H, Bz H-4), 7.45–7.41
(m, 2H, Bz H-2), 6.27 (d, J = 7.5 Hz, 1H, NH), 6.07
(dd, J = 2.2, 2.1 Hz, 1H, H-1), 4.66 (ddd, J = 50.5,
9.4, 9.4 Hz, 1H, H-4), 4.64 (dd, J = 12.2, 2.6, 1.5 Hz,
1H, H-6a), 4.54 (dd, J = 12.2, 5.4, 1.0 Hz, 1H, H-6b),
4.49 (dddd, J = 7.5, 4.9, 2.7, 2.1 Hz, 1H, H-2), 4.38
(ddd, J = 14.5, 9.4, 4.9 Hz, 1H, H-3), 3.92 (dddd,
J = 9.4, 5.8, 5.4, 2.6 Hz, 1H, H-5), 2.13 (s, 3H, COCH3),
2.01 (s, 3H, NHCOCH3); 13C NMR (100 MHz, CDCl3,
20
a-Anomer: ½aꢂD +44.8 (c 1.1, CHCl3); Rf 0.37 (hex-
ane–EtOAc = 2:1); 1H NMR (400 MHz, CDCl3, d
CHCl3 7.26) d: 8.07–8.05 (m, 2H, Bz H-3), 7.59–7.55
(m, 1H, Bz H-4), 7.47–7.41 (m, 2H, Bz H-2), 7.43–7.41
(m, 5H, Bn), 6.07 (dd, J = 2.2, 2.2 Hz, 1H, H-1), 4.97
(ddd, J = 51.1, 9.5, 9.5 Hz, 1H, H-4), 4.89 (d,
J = 11.8 Hz, 1H, CHH0Ph), 4.75 (d, J = 11.8 Hz, 1H,
CHH0Ph), 4.63 (ddd, J = 12.2, 1.9, 1.9 Hz, 1H, H-6a),
4.49 (ddd, J = 12.2, 4.5, 1.1 Hz, 1H, H-6b), 4.14–4.07
(m, 2H, H-5, H-3), 3.94–3.92 (m, 1H, H-2), 2.11 (s,
3H, COCH3); 13C NMR (100 MHz, CDCl3, d CDCl3
77.16) d: 168.3 (COCH3), 166.3 (COPh), 137.3 (C-1
Bn), 133.3 (C-4 Bz), 129.9–127.9 (C-2, C-3, C-4 Bn,
C1, C-2, C-3 Bz), 91.8 (d, JFꢀC = 1.2 Hz, C-1), 87.8
(d, JFꢀC = 181.3 Hz, C-4), 76.1 (d, JFꢀC = 18.0 Hz,
d
CDCl3 77.16) d: 172.4 (NHCOCH3), 168.5
(OCOCH3), 166.3 (COPh), 133.5 (Bz C-4), 129.8 (Bz
C-3), 129.7 (Bz C-1), 128.6 (Bz C-2), 91.7 (d, JFꢀC
=
1.0 Hz, C-1), 88.3 (d, JFꢀC = 181.2 Hz, C-4), 70.1 (d,
JFꢀC = 24.7 Hz, C-5), 68.4 (d, JFꢀC = 18.6 Hz, C-3),
62.8 (C-6), 52.9 (d, JFꢀC = 8.4 Hz, C-2), 23.1
(NHCOCH3), 20.9 (OCOCH3); HRESIMS: calcd for
C17H20NO7FNa: 392.1122; found: 392.1138 [M+Na]+.
C-3), 73.5 (d, JFꢀC = 1.9 Hz, CH2Ph), 70.7 (d, JFꢀC
=
24.9 Hz, C-5), 62.6 (C-6), 61.3 (d, JFꢀC = 8.6 Hz, C-2),
21.0 (COCH3); HRESIMS: calcd for C22H22N3O6FNa:
466.1390; found: 466.1390 [M+Na]+.
3.9. 2-Acetamido-2,4-dideoxy-4-fluoro-a-D-mannopyr-
anose (13)
20
b-Anomer: ½aꢂD ꢀ33.0 (c 1, CHCl3); Rf 0.60 (hexane–
1
EtOAc = 2:1); H NMR (400 MHz, CDCl3, d CHCl3
Pyranosyl acetate 12 (106 mg, 0.287 mmol) was dis-
solved in anhydr MeOH (2 mL) and freshly prepared so-
dium methoxide (0.018 mL, 1.6 M, 0.029 mmol) was
added. After 3 h at 20–22 °C aqueous HCl (0.029 mL,
1 M, 0.029 mmol) was added and the product was dried
under diminished pressure. Compound 13 (Rf 0.38, 5:1
EtOAc–MeOH) was directly used for the next reaction
without further purification.
7.26) d: 8.07–8.04 (m, 2H, Bz H-3), 7.59–7.55 (m, 1H,
Bz H-4), 7.47–7.43 (m, 2H, Bz H-2), 7.40–7.31 (m, 5H,
Bn), 5.75 (d, J = 1.7 Hz, 1H, H-1), 4.86 (d,
J = 11.9 Hz, 1H, CHH0Ph), 4.85 (ddd, J = 50.6, 9.1,
9.1 Hz, 1H, H-4), 4.74 (d, J = 11.9 Hz, 1H, CHH0Ph),
4.65 (ddd, J = 12.2, 1.9, 1.9 Hz, 1H, H-6a), 4.48 (ddd,
J = 12.2, 5.3, 1.4 Hz, 1H, H-6b), 4.02–3.99 (m, 1H, H-
2), 3.87–3.80 (m, 2H, H-5, H-3), 2.17 (s, 3H, COCH3);
13C NMR (100 MHz, CDCl3, d CDCl3 77.16) d: 168.7
(COCH3), 166.3 (COPh), 137.1 (C-1 Bn), 133.4 (C-4
Bz), 130.0 (C-3 Bz), 129.8 (C-1 Bz), 128.8 (C-2 Bz),
128.6–128.0 (C2, C3, C4 Bn), 91.6 (d, JFꢀC = 1.3 Hz,
3.10. 5-Acetamido-3,5,7-trideoxy-7-fluoro-D-glycero-D-
galacto-2-nonulopyranonic acid (14)
A soln of 13 (5.0 mg, 0.022 mmol), pyruvate (24.7 mg,
0.224 mmol), dithiothreitole (3.5 mg; 0.022 mmol) and
aldolase (1 mg; EC 4.1.3.3; Fluka; 21.1 U/mg) in sodium
phosphate buffer (0.5 mL; 0.1 M; pH 7.5) was incubated
at 32 °C for 24 h. After the addition of another batch of
pyruvate (24.7 mg, 0.224 mmol), reaction was continued
at the same temperature for 24 h. The crude mixture was
freeze-dried, dissolved in formic acid (0.4 mL, 0.1%) and
purified by HPLC (column: Luna [Phenomenex] 5l
C-1), 87.6 (d, JFꢀC = 182.3 Hz, C-4), 77.5 (d, JFꢀC
=
18.0 Hz, C-3), 73.2 (t, JFꢀC = 1.9 Hz, CH2Ph), 73.0 (d,
JFꢀC = 25.4 Hz, C-5), 62.7 (C-6), 62.0 (d, JFꢀC
=
8.8 Hz, C-2), 20.9 (COCH3); HRESIMS: calcd for
C22H22N3O6FNa: 466.1390; found: 466.1398 [M+Na]+.
3.8. 2-Acetamido-1-O-acetyl-6-O-benzoyl-3-O-benzyl-
2,4-dideoxy-4-fluoro-a-D-mannopyranose (12)
˚
NH2, 100 A, 100 ꢁ 21.20 mm; mobile phase: formic acid
Pd(OAc)2 (186 mg, 0.829 mmol) was added to a soln of
11 (a-anomer: 465 mg, 1.049 mmol) and acetic anhy-
dride (985 lL, 10.49 mmol) in EtOAc (5 mL). The sus-
pension was stirred at 20–22 °C for 22 h under an
atmosphere of H2 (5–5.6 bar). After filtration through
[0.1%; 8 mL/min]; Rt 117 min) to yield 14 (3.9 mg,
0.012 mmol; 56%) as an amorphous white solid.
1H NMR (500 MHz, D2O) d: 4.36 (dd, J = 45.7,
8.5 Hz, 1H, H-7), 3.96–3.83 (m, 3H, H-4, H-6, H-8),
3.77 (dd, J = 10.3, 10.3 Hz, 1H, H-5), 3.67 (ddd,

S. Hartlieb et al. / Carbohydrate Research 343 (2008) 2075–2082
2081
J = 12.2, 2.5, 2.5 Hz, 1H, H-9b), 3.52 (ddd, J = 12.2,
5.5, 2.1 Hz, 1H, H-9a), 2.26 (dd, J = 12.3, 4.7 Hz, 1H,
H-3eq), 1.85 (s, 3H, NHCOCH3), 1.74 (dd, J = 12.3,
12.3 Hz, 1H, H-3ax); 13C NMR (100 MHz, D2O):
178.2 (COOH), 174.5 (NHCOCH3), 96.1 (C-2), 88.8
(d, J = 178.9 Hz, C-7), (d, J = 17.3 Hz, C-6), 67.5 (d,
J = 26.6 Hz, C-8), 67.1 (C-4), 62.1 (C-9), 51.7 (C-5),
38.9 (C-3), 21.8 (NHCOCH3); HRESIMS: calcd for
C11H17NO8F: 310.0938; found: 310.0945 [MꢀH]ꢀ.
acid synthetases from mouse and rainbow trout. This
work was supported by the Deutsche Forschungsgeme-
inschaft (Grants Ki 397/8-1 and Ki 397/9-1; GE 801/
6-1) and the Fonds der Chemischen Industrie.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2008.02.003.
3.11. Cytidine-50-monophospho-5-acetamido-3,5,7-tride-
oxy-7-fluoro-D-glycero-D-galacto-2-nonulopyranonic acid
(15) from 13
References
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A soln of 13 (5.0 mg, 0.022 mmol), pyruvate (24.7 mg,
0.224 mmol), dithiothreitole (3.5 mg, 0.022 mmol) and
cytidine-50-triphosphate (13.9 mg, 0.025 mmol) in Tris–
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MgCl2 (40 lL, 0.5 M), aldolase (20 U; EC 4.1.3.3; Flu-
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mg, 0.025 mmol), aldolase (1 mg; EC 4.1.3.3; Fluka;
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3.12. Cytidine-50-monophospho-5-acetamido-3,5,7-tride-
oxy-7-fluoro-D-glycero-D-galacto-2-nonulopyranonic acid
(15) from 14
A soln of 14 (1.8 mg, 5.8 lmol), dithiothreitole (0.9 mg,
5.8 lmol) and cytidine-50-triphosphate (4.2 mg, 7.5
lmol) in Tris–HCl buffer (0.5 mL; 0.1 M; pH 8.8) was
supplied with MgCl2 (20 lL, 0.5 M), pyrophosphatase
(5 U; EC 3.6.1.1; Fluka; 80 U/mg) and NmB CSS17
(5 lL, 1.21 mg/mL). The soln was incubated at 35 °C
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