Two procedures for the syntheses of labeled sialic acids and their 1,7-lactones

Article abstract of DOI:10.1016/j.tetasy.2011.02.012

The synthesis of four deuterated sialic acids and their 1,7-lactones has been performed in two ways, one based on sialic acid classical chemistry, and the other involving a direct exchange of the unlabeled acyl group of N-acetylneuraminic acid with a labe

Full text of DOI:10.1016/j.tetasy.2011.02.012

Tetrahedron: Asymmetry 22 (2011) 338–344
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Two procedures for the syntheses of labeled sialic acids and their 1,7-lactones
⇑
Pietro Allevi, Mario Anastasia, Maria L. Costa, Paola Rota
Department of Chemistry, Biochemistry and Biotechnology for the Medicine, University of Milan, via Saldini 50, Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of four deuterated sialic acids and their 1,7-lactones has been performed in two ways, one
based on sialic acid classical chemistry, and the other involving a direct exchange of the unlabeled acyl
group of N-acetylneuraminic acid with a labeled one mediated by a perfluorinated amide. The final lact-
onization is promoted by benzyloxycarbonyl chloride.
Received 5 January 2011
Accepted 10 February 2011
Available online 14 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
anhydrides, under volatilization conditions, can cause the N-trans-
acylation of the N-acetyl group,8b the dehydration of the anomeric
N-Acetylneuraminic acid 1a (Neu5Ac) and N-glycolyl neurami-
nic acid 2a (Neu5Gc) are two very important members of sialic acid
family (Sias), a group of carbohydrates, sharing the neuraminic
hydroxyl8c or other unexpected transformations.8c,f Thus, we con-
sidered the availability of authentic pure reference standards of
the lactones 3a and 4a as essential, and of their isotopologues 3b,c
and 4b,c necessary as standards in any suitable analytical protocol
based on HPLC-MS or for setting-up the appropriate and controlled
methods of derivatization.
As a result of our interest, we recently accomplished the first
synthesis of lactones Neu5Ac1,7L 3a and Neu5Gc1,7L 4a.8a Herein,
we report the synthesis of their isotopologues 3b,c and 4b,c, la-
beled with deuterium at the N-acyl group and both at the acyl
group and at the pyranose ring, prepared in two relatively simple
methods by the application of new Sias chemistry. Key intermedi-
ates for the synthesis of these compounds were four isotopologues
1b,c and 2b,c of the parent Sias prepared herein.
acid pyranosidic ring, usually linked, through an a-glycosidic bond
to the end of the carbohydrate chains present in glycolipids and
glycoproteins of bacteria and humans (Fig. 1).1 In particular, Sias
present in healthy humans derive from Neu5Ac, while those pres-
ent in bacteria and in human tumors also derive from Neu5Gc. Sias
play a central role in numerous biological recognition processes
such as lymphocite homing, tumor methastasis and pathogenic
bacteria infections.1–3 As a result of this, much effort has been
made to map them in different biological districts and thus many
conclusions with regard to their biological roles have already been
reached.
Recently, the 1,7-lactone of N-acetylneuraminic acid 3a, first
identified in the Bufo bufo4 mucins and as a ligand ofhuman interleu-
kin-4,5,6 has been described as being present in sialoglycoproteins of
the membrane of red blood cells, affected by Polycythemia vera, a
malignant disorder of haematopoietic stem cells.7 However, the
lactone 3a has not been isolated but only indirect evidences of its
structure have been reported. These are obtained in a GLC–MS
screening of Sias present in the glycoconjugated red blood cells
affected by P. vera, after acidic hydrolysis and derivatization with
perfluorinated anhydrides. Due to our interest in Sias chemistry
and biochemistry,8 we decided to search for an analytical protocol
suitable to substantiate the reported presence of Sias 1,7-lactones
3a and 4a in red blood cells affected by P. vera. In our mind the ideal
method should permit an analysis of Sias without any derivatiza-
tion, using HPLC-MS, in order to exclude any possible artefact orig-
inating during the commonly used conditions of volatilization.7
We recently found that the treatment of Sias with perfluorinated
2. Results and discussion
In our synthetic work, we planned to label the parent sialic
acids and then subject them to a lactonization procedure which
uses benzyloxycarbonyl chloride (CbzCl) as a promoter of the
1,7-lactonization8a (Scheme 1).
Using this method, we could overcome the difficulties connected
with the direct labeling of the free 1,7-lactones due to their particu-
lar lability. Moreover, since Neu5Gc 2a is not commercially available
and the reported procedures for its preparation from the congener
Neu5Ac 1a were not completely satisfactory,9 we also decided to
search for a more convenient protocol for the transformation of
Neu5Ac 1a into Neu5Gc 2a. The success of our work would allow
us to dispose of four different isotopologues of Sias (i.e., compounds
1b,c and 2b,c), of self consistent utility as standards for a possible
analytical protocol for the simultaneous evaluation of the parent
Sias and of their 1,7-lactones. Initially we solved the synthetic prob-
lem by setting-up a protocol which started from the protected neu-
raminic acid methyl ester 5 (Scheme 2).9
⇑
Corresponding author. Tel.: +39 025 031 6047; fax: +39 025 031 6040.
E-mail address: paola.rota@unimi.it (P. Rota).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.02.012

P. Allevi et al. / Tetrahedron: Asymmetry 22 (2011) 338–344
339
GcHN
HO
NHAc
OH
HO
HO
OH
R
R
OH
R
OH
R
O
O
R
OH
O
OH
O
HO
GcHN
O
COOH
O
COOH
HO
AcHN
HO
HO
R
HO
HO
OH
OH
O
O
R
R
HO
2
1
3
4
a: Ac = CH3CO; Gc = HOCH2CO, R = H
b: Ac = CD3CO; Gc = HOCD2CO, R = H
c: Ac = CD3CO; Gc = HOCD2CO, R = D
Figure 1.
NHCOR
NHCOR
O
O
OH
HO
OH
O
OCbz
O
OH
CO2H
ii
i
HO
HO
HO
HO
HO
RCONH
OH
O
OH
O
O
R = CH3 or CH2OH
HO
Sias
1,7-lactones of Sias
protected at the anomeric
hydroxyl
1,7-lactones of Sias
Scheme 1. Reagents and conditions: (i) Et3N, THF–DMF (4:3; v/v), 0 °C, then CbzCl, THF, 0–23 °C, 1 h, 70–90%; (ii) H2, Pd on C 10%, AcOEt, 23 °C, 88–96%.
According to this, the reaction of the methyl ester9 5 with tri-
deuterated acetyl chloride in methanol afforded Neu5[2H3]Ac
methyl ester methyl acetal 6 which, after a treatment with aque-
ous sodium hydroxide and then with acidic sulfonic resin, at
80 °C for 4 h, afforded the trideuterated acid Neu5[2H3]Ac 1b. This,
upon repeated treatment with NaO[2H] in [2H2]O, afforded the
pentadeuterated isotopologue 1c deriving from a keto-enolic equi-
librium of the starting keto-acid in the labeled basic medium. A
parallel procedure allows the conversion of the methyl ester 5 into
the Sias 2b and 2c. In this case, we introduced a deuterated glycolyl
group at the neuraminic acid nitrogen by reaction of the protected
methyl ester 5 with dideuterated benzyloxyacetyl chloride and
successive hydrogenolysis of the obtained benzylated glycolic acid
derivative 7. Thus, we obtained the deuterated glycolic acid 8 in a
two steps, acylation (treatment with labeled benzyloxyacetyl chlo-
ride and regeneration of the glycolic hydroxyl). This route permits
a quantitative introduction of two deuterium atoms in the methy-
lene group of the acylating agent, by simple basic treatment of the
protected glycolic acid in 2H2O, a procedure which is not practica-
ble on the free glycolic acid. On the other hand, this two step gly-
cosylation procedure via the protected acyl chloride of glycolic acid
is routinely used in our laboratory for the transformation of the
unlabeled Neu5Ac 1a into Neu5Gc 2a since, in our hands, it is more
convenient than the direct acylation of the ester 5 with deuterated
glycolic acid and DCC.9
From the methyl ester methyl acetal 8, we prepared the deuter-
ated Neu5Gc acids 2b and 2c, adopting the previously set-up pro-
cedure. However, while we accomplished the preparation of these
deuterated Sias, in our laboratory, we discovered an interesting
procedure which allows the N-transacylation of secondary acyla-
mides to different ones via perfluorinated analogues (Fig. 2).8b
Thus, we decided to adopt the disclosed N-transacylation reac-
tion to set-up a simplified procedure for the preparation of all the
obtained deuterated lactones. In effect, with double successive
transacylations, we could obtain the desired deuterated peracylat-
ed methyl esters 11 with a simplified procedure, starting from
Neu5Ac 1 (Scheme 3). We first prepared the peracylated methyl es-
ter 9, which then was transacylated to its trideuterated isotopo-
logue 11 which, by simple saponification of its protective groups
by treatment with NaOH or with NaOD, afforded the Sias 1b or
1c, the first deuterated at the acyl group, the second deuterated
both at the acyl group and position 3.
Next, we attempted the N-transacylation of the trifluoroaceta-
mide 10 reacting it with 2,2-dideuterated benzyloxyacetylchloride,
under the above reported conditions. Unexpectedly, in this case the
reaction afforded very erratic results, even when performed in a
large scale and was studied using unlabeled benzyloxyglycolyl
chloride. In the best case the desired N-transacylated compound
12b was obtained in variable but constantly poor yields (8–22%),
always being accompanied by a mixture of inseparable compounds
apparently derived from a rearrangement and polimerization of
Sias.10 Thus, we decided to discontinue this route considering also
that the deuterated glycolic acids useful for the synthesis of the de-
sired lactones 4b and 4c were conveniently available from the pre-
viously described route (Scheme 2).
In fact, with the four deuterated acids in hand, we performed
their lactonization, reacting each of them, dissolved in DMF con-
taining Et3N, with benzyloxycarbonyl chloride, at 0 °C for 1 h
(Scheme 4).8d
Under these conditions, the corresponding 2-benzyloxycarbonyl
lactones 13–16 are formed, all possessing physicochemical proper-
ties, apart from NMR and mass spectra, superimposable to those ob-
served for the unlabeled isotopologues.8d Similarly, each protected
lactone, by simple hydrogenolysis in the presence of palladium on
carbon, afforded the corresponding free 1,7-lactone showing the ex-
pected physicochemical properties and 1H and 13C NMR spectra.
3. Conclusion
In conclusion we have set-up two protocols useful to prepare la-
beled sialic acids 1b,c 2b,c and their 1,7-lactones 3b,c and 4b,c
which are now available for analytical, synthetic and biological
studies.
4. Experimental section
4.1. General methods
Melting points are uncorrected. Nuclear magnetic resonance
spectra were recorded at 298 K operating at 500.13 MHz for 1H

340
P. Allevi et al. / Tetrahedron: Asymmetry 22 (2011) 338–344
HO
HO
HO
OH
OH
OH
OH
OH
OCH3
iii
iv
HO
HO
D3CCONH
HO
D3CCONH
O
O
O
COOH
D
COOH
ii
COOCH3
D3CCONH
HO
1b
HO
HO
D
HO
6
OH
1c
OCH3
i
HO
H2N
O
Neu5Ac 1
COOCH3
HO
5
HO
OH
HO
HO
OH
OH
v
OCH3
OCH3
COOCH3
OH
COOH
iii
vi
HO
BnOCD2CONH
O
HO
HO
HOCD2CONH
O
O
COOCH3
HOCD2CONH
HO
HO
HO
2b
iv
7
8
HO
OH
OH
COOH
HO
O
D
HOCD2CONH
HO
2c
D
Scheme 2. Reagents and conditions: (i) HCl in MeOH, 100 °C, 4.5 h; then DOWEX OHꢀ, 69%; (ii) C[2H3] COCl, Et3N, MeOH, 25 °C, 2 h, 84%; (iii) NaOH/H2O, 40 °C, 0.5 h, then
DOWEX H+, 80 °C, 4 h, 72% (1b) or 74% (2b); (iv) NaO[2H]/[2H2]O, 25 °C, 3 h, 84 (1c) or 90% (2c); (v) BnOC[2H2]COCl, Et3N, MeOH 0 °C, 2 h, 83%; (vi) H2, Pd on C 10%, THF, 23 °C,
92%.
Proton and carbon assignments were established, if necessary,
R
N
R
with 1H–1H and 1H–13C correlated NMR experiments. 1H NMR data
are tabulated in the following order: multiplicity (s, singlet; d, dou-
blet; br s, broad singlet; m, multiplet), coupling constant(s) in
Hertz, number of protons assignment of proton(s). Optical rota-
tions were taken on a polarimeter equipped with a 1 dm tube;
R
NH
(CF3CO)2O
H2O
O
O
O
O
HN
O
CH3 CF3
CF3
H3C
CH3COCl
Et3N
[a]
values are given in 10ꢀ1 deg cm2 gꢀ1 and the concentration
R
D
R
R
is given in g/100 mL. Infrared (IR) spectra were recorded in Nujol.
Mass spectrometry was performed using a quadrupole ion-trap
mass spectrometer equipped with an ESI ion source. The spectra
were collected in continuous flow mode by connecting the infusion
pump directly to the ESI source. Solutions of compounds were in-
O
N
R1COCl
Et3N
O
HN
O
NH
H2O
R1 CF3
CF3
R1
R = Alkyl or aryl group
R1 = Alkyl group
fused at a flow rate of 5 lL/min. The spray voltage was set at 5.0 kV
in the positive and at 4.5 kV in the negative ion mode with a cap-
illary temperature of 220 °C. Full-scan mass spectra were recorded
by scanning a m/z range of 100–2000. Isotopical purity of all target
compounds was determined using a JEOL Accuo-ToF instrument in
positive ESI mode. All reactions were monitored by thin-layer
chromatography (TLC) carried out on 0.25 mm E. Merck silica gel
plates (60 F254) using UV light, 50% sulfuric acid or 0.2% ninhydrin
in ethanol and heat as a developing agent. All flash chromatogra-
phy was performed with normal phase silica gel, following the
general protocol of Still.11
Figure 2.
and 125.76 MHz for 13C. Chemical shifts are reported in parts for
million (ppm, d units) relative to CDCl3 signal fixed at 7.26 ppm
for 1H and at 77.0 ppm for 13C spectra, relative to CD3OD signal
fixed at 3.31 ppm for 1H and at 49.05 ppm for 13C spectra, relative
to DMSO-d6 signal fixed at 2.50 ppm for 1H and at 39.43 for 13C
spectra and relative D2O signal refer to (CH3)3COH, used as a refer-
ence standard, at 1.23 ppm for 1H and at 30.29 for 13C spectra.
1b
AcO
AcO
iv
OAc
AcO
OAc
AcO
AcO
OAc
AcO
OAc
COOMe
OAc
COOMe
OAc
COOMe
i
ii
AcO
D3CCONH
Neu5Ac 1
AcO
F3CCONH
O
iii
O
AcO
H3CCONH
O
v
11
10
1c
9
vi
AcO
OAc
OAc
AcO
BnOCD2CONH
O
COOMe
AcO
12
Scheme 3. Reagents and conditions: (i) MeOH, DOWEX H+, 23 °C, 5 h; then Ac2O, Py, 23 °C, 6 h, 89%; (ii) TFAA, Et3N, CH3CN, 135 °C, 5 min, 82%; (iii) C[2H3] COCl, DIPEA,
CH2Cl2 0 °C, 20 min, 72%; (iv) NaOH/H2O, 2.5 h, 82–86%; (v) NaO[2H]/[2H]2O, 23 °C, 5 h, 76–80%; (vi) BnOC[2H2]COCl, DIPEA, CH2Cl2, 0 °C, 20 min, 8–22%.

P. Allevi et al. / Tetrahedron: Asymmetry 22 (2011) 338–344
341
NHCOCD3
O
H-9a), 3.52 (dd, J7,8 = 6.0 Hz, 1H; H-7), 3.28 (s, 3H; OCH3), 2.35 (dd,
J3a,3b = 13.0, J3a,4 = 5.00 Hz, 1H; H-3a), 1.65 (dd, J3b,3a = 13.0 Hz,
J3b,4 = 11.5 Hz, 1H; H-3b); 13C NMR (CD3OD) d 173.9 (C-1), 171.0
(br s, CONH), 135.5, 129.5, 129.3, 129.1 (Ph), 100.5, (C-2), 74.4
(CH2Ph), 72.0 (C-6), 71.5 (C-8), 70.1 (C-7), 67.5 (t, J = 21.4 Hz, OCD2-
CO), 67.2 (C-4) 65.3. (C-9), 53.6 (C-5), 53.2 (COOCH3), 51.7 (OCH3)
41.8 (C-3); MS (ESI positive): m/z 468.1 [M+Na]+; 99% isotopically
pure. Anal. Calcd for C20H27D2NO10: C, 53.93; H+D, 7.01; N, 3.14.
Found: C, 55.90; H+D, 6.97; N, 3.12.
NHCOCD3
O
OCbz
O
OH
O
HO
HO
i
ii
HO
HO
1b
1c
OH
O
OH
O
13
3b
NHCOCD3
NHCOCD3
D
D
O
O
OH
O
OCbz
i
i
HO
HO
HO
HO
ii
D
OH O
D
O
4.4. N-[2H2]-Glycolyl-2-O-metyl-b-neuraminic acid methyl ester 8
OH
O
3c
14
N-Benzyloxy-[2H2]-acetyl-2-O-methyl-b-neuraminic acid methyl
ester 7 (650 mg, 1.46 mmol) was dissolved in THF (10 mL) and
treated with H2 in the presence of 10% Pd/C for 3 h at 22 °C. The
catalyst was filtered through a pad of Celite and the solvent was
evaporated under reduced pressure. The crude residue was then
crystallized from a solution of ethanol in water to give the deben-
zylated compound 8 (477 mg, 92%), as a white solid: mp 138–140;
NHCOCD2OH
O
NHCOCD2OH
O
OCbz
OH
HO
HO
HO
HO
ii
2b
2c
OH O
OH O
O
O
15
4b
½
a 2D5
ꢁ
¼ þ7:1 (c 1, CH3OH); 1H NMR (CD3OD) d 4.12 (m, 1H; H-4),
NHCOCD2OH
NHCOCD2OH
3.97–3.93 (overlapping, 2H; H-6 e H-5), 3.83–3.76 (overlapping,
5H; COOCH3, H-8 and H-9a), 3.62 (dd, J9b,9a = 12.0 Hz,
J9b,8 = 5.9 Hz, 1H; H-9a), 3.52 (dd, J7,8 = 6.0 Hz, 1H; H-7), 3.27 (s,
3H; OCH3), 2.35 (dd, J3a,3b = 13.1, J3a,4 = 5.00 Hz, 1H; H-3a), 1.65
(dd, J3b,3a = 13.1 Hz, J3b,4 = 11.5 Hz, 1H; H-3b); 13C NMR (CD3OD) d
173.7 (C-1), 169.8 (br s, CONH), 100.3, (C-2), 71.9 (C-6), 71.4
(C-8), 70.0 (C-7), 67.5 (t, J = 21.2 Hz, OCD2CO), 67.1 (C-4) 65.3
(C-9), 53.6 (C-5), 53.1 (COOCH3), 51.5 (OCH3) 41.7 (C-3); MS (ESI
positive): m/z 378.4 [M+Na]+; 99% isotopically pure. Anal. Calcd
for C13H21D2NO10: C, 43.94; H+D, 7.09; N, 3.94. Found: C, 43.90;
H+D, 7.02; N, 3.92.
D
O
D
O
OCbz
OH
i
HO
HO
ii
HO
HO
D
OH O
D
OH O
O
O
4c
16
Scheme 4. Reagents and conditions: (i) Et3N, THF–DMF, 0 °C, then CbzCl, THF, 0–
23 °C, 1 h, 70–78%; (ii) H2, Pd on C 10%, AcOEt, 23 °C, 89–93%.
4.2. N-[2H3]-Acetyl-2-O-methyl-b-neuraminic acid methyl ester (6)
[2H3]-Acetyl chloride (0.84 ml, 11.85 mmol) was added drop-
wise to a stirred solution of the amine 5 (700 mg, 2.37 mmol) in
anhydrous methanol containing Et3N (3.28 mL, 23.7 mmol), at
0 °C. The mixture was stirred for 2 h at 22 °C and evaporated under
reduced pressure. Purification of the crude residue by flash chro-
matography [eluting with CH2Cl2/MeOH (4/1; v/v)] afforded the
amide 6 (677 mg, 84%), as a white solid: mp 115–117 (from diethyl
4.5. N-[2H3]-Acetyl-neuraminic acid 1b
A
solution of N-[2H3]acetyl-2-O-methyl-b-neuraminic acid
methyl ester 6 (400 mg, 1.18 mmol) in H2O (7.0 mL) was treated
with an aqueous solution of NaOH (1.8 mL, 1.0 M) at 40 °C for
0.5 h. Then, DowexÒ 50WX8 (H+) resin (500 mg) was added and
the solution was warmed at 80 °C for 4 h. After filtration of the re-
sin, lyophilisation of the solution gave a crude residue which, after
crystallization, afforded the pure compound 1b (265 mg, 72%), as a
ether); ½a 2D5
ꢁ
¼ ꢀ7:5 (c 1, CH3OH); 1H NMR (CD3OD) d 4.03 (m, 1H;
H-4), 3.94–3.88 (overlapping, 2H; H-5 and H-6), 3.85 (s, 3H;
COOCH3), 3.81–3.75 (overlapping, 2H; H-8 and H-9a), 3.72 (dd,
J9b,9a = 11.8, J9b,8 = 5.3 Hz, 1H; H-9b), 3.60 (d, J7,8 = 8.7 Hz, 1H; H-
7), 3.25 (s, 3H; OCH3), 2.32 (dd, J3a,3b = 13.0, J3a,4 = 5.0 Hz, 1H; H-
3a), 1.70 (dd, J3b,3a = 13.0 Hz, J3b,4 = 12.0 Hz, 1H; H-3b); 13C NMR
(CD3OD) 174.9 (br s, CONH), 172.2 (C-1), 100.7, (C-2), 72.3 (C-6),
71.6 (C-8), 69.4 (C-7), 67.4 (C-4) 64.7 (C-9), 53.8 (C-5), 53.2
(COOCH3), 52.0 (OCH3), 41.4 (C-3), 23.4 (hept, J = 19.2 Hz, CD3CO);
MS (ESI positive): m/z 363.3 [M+Na]+, 99% isotopically pure. Anal.
Calcd for C13H20D3NO9: C, 45.88; H+D, 7.70; N, 4.12. Found: C,
45.78; H+D, 7.79; N, 4.15.
white solid: mp 178–181 (from CH3CN); ½a D25
¼ ꢀ34:0 (c 1, H2O);
ꢁ
1H NMR (D2O) d 4.12–4.04 (overlapping, 2H; H-4, H-6), 3.95 (m,
1H; H-5), 3.86 (d, J9a,9b = 11.8 Hz, 1H; H-9a), 3.77 (m, 1H; H-8),
3.64 (dd, J9b,9a = 11.8, J9b,8 = 6.5 Hz, 1H; H-9a), 3.58 (d,
J7,8 = 9.1 Hz, 1H; H-7), 2.34 (dd, J3a,3b = 13.0, J3a,4 = 2.8 Hz, 1H; H-
3a), 1.90 (t app., J3b,3a = 13.0 Hz, 1H; H-3b); 13C NMR (D2O) d 13C
NMR (D2O) d 175.5 (br s, COCD3), 173.7 (C-1), 95.8 (C-2), 71.0 (C-
6), 70.8 (C-8), 68.8 (C-7), 67.3 (C-4), 63.8 (C-9), 52.6 (C-5), 39.5
(C-3), 22.7 (hept., J = 19.3 Hz, CD3CO); MS (ESI negative): m/z
311.1 [MꢀH]ꢀ; 99% isotopically pure. Anal. Calcd for C11H16D3NO9:
C, 42.31; H+D, 7.10; N, 4.49. Found: C, 42.40; H+D, 7.00; N, 4.52.
4.3. N-Benzyloxy-[2H2]-acetyl-2-O-metyl-b-neuraminic acid
methyl ester 7
4.6. N-[2H3]-Acetyl-[3,3-2H2]-neuraminic acid 1c
Benzyloxy-[2H2]-acetyl chloride (0.9 ml, 6.33 mmol) was added
dropwise to a stirred solution of the amine 5 (700 mg, 2.11 mmol)
in anhydrous methanol containing Et3N (1.7 mL, 12.7 mmol), at
0 °C. The mixture was stirred for 2 h at 22 °C and evaporated under
reduced pressure. Purification of the crude residue by flash chro-
matography [eluting with CH2Cl2/MeOH (9/1; v/v)] afforded the
amide 7 (785 mg, 83%), as a white solid: mp 136–140 °C (from
N-[2H3]-acetyl-b-neuraminic acid 1b (150 mg, 0.48 mmol) was
treated with a solution of NaOD in D2O (0.9 mL, 1.0 M) at 22 °C
for 3 h. The solution was neutralized with acidic resin [DowexÒ
50WX8 (H+)], then the resin was filtered and the solution was
lyophilized to afford the crude acid 1c (127 mg, 84%) which, after
crystallization shows: mp 178–180 (from CH3CN); ½a D25
¼ ꢀ33:2
ꢁ
(c 1, H2O); 1H NMR (D2O) d 4.08–4.03 (overlapping, 2H; H-4 and
H-6), 3.93 (dd, J5,4 = 10.2, J5,6 = 10.2 Hz 1H; H-5), 3.84 (dd,
J9a,9b = 11.9, J9a,8 = 2.6 Hz, 1H; H-9a), 3.75 (m, 1H; H-8), 3.62 (dd,
J9b,9a = 11.9, J9b,8 = 6.3 Hz, 1H; H-9b), 3.56 (dd, J7,8 = 9.2, J7,6
<1.0 Hz, 1H; H-7); 13C NMR (D2O) d 175.6 (br s, COCD3), 173.8
diethyl ether); ½a D25
ꢁ
¼ ꢀ35:2 (c 1, H2O); 1H NMR (CD3OD) d 7.43–
7.27 (m, 5H; Ph), 4.61 (AB system, 2H; CH2Ph), 4.10 (m, 1H; H-
4), 3.98–3.9 (overlapping, 2H; H-6 e H-5), 3.84–3.78 (overlapping,
5H; COOCH3, H-8, H-9a), 3.65 (dd, J9b,9a = 12.1 Hz, J9b,8 = 6.0 Hz, 1H;

342
P. Allevi et al. / Tetrahedron: Asymmetry 22 (2011) 338–344
(C-1), 95.9 (C-2), 71.1 (C-6), 70.8 (C-8), 68.9 (C-7), 67.3 (C-4), 63.8
(C-9), 52.7 (C-5), 39.5 (m, C-3), 22.8 (hept., J = 19.4 Hz, CD3CO); MS
(ESI negative): m/z 313.3 [MꢀH]ꢀ. 99% isotopically pure.
Anal. Calcd for C11H14D5NO9: C, 42.04; H+D, 7.69; N, 4.46.
Found: C, 42.10; H+D, 7.82; N, 4.52.
(m, 1H; H-4), 5.06 (m, 1H; H-8), 4.49 (dd, J9a,9b = 12.3,
J9a,8 = 1.9 Hz, 1H; H-9a), 4.14–4.08 (overlapping, 3H; H-5, H-6,
and H-9b), 3.78 (s, 3H; COOCH3), 2.54 (dd, J3a,3b = 13.5,
J3a,4 = 4.8 Hz, 1H; H-3a), 2.14 (s, 6H; 2 ꢂ CH3COO), 2.08 (dd,
J3b,3a = 13.5, J3b,4 = 11.8 Hz, 1H; H-3b), 2.05 (s, 3H; CH3COO); 2.03
(s, 6H; 2 ꢂ CH3COO); 13C NMR (CDCl3) 170.9, 170.5, 170.2, 170.1,
168.2 (5C, COCH3), 170.3 (br s, CONH), 166.3 (C-1), 97.5 (C-2),
72.8 (C-6), 71.3 (C-8), 68.3 (C-4), 67.9 (C-7), 62.1 (C-9), 53.2
(COOCH3), 49.4 (C-5), 35.9 (C-3), 22.0 (hept, J = 19.4 Hz, CD3CO),
20.9, 20.8 (2 ꢂ COCH3), 20.7 (3 ꢂ COCH3); IR 1750, 1721,
4.7. N-[2H2]-Glycolyl-neuraminic acid 2b
A solution of N-[2H2]-glycolyl-2-O-methyl-b-neuraminic acid
methyl ester 8 (400 mg, 1.13 mmol) in H2O (10 mL) was treated
with an aqueous solution of NaOH (0.6 mL, 1.0 M) at 40 °C for
0.5 h. Then, DowexÒ 50WX8 (H+) (360 mg) was added and the
solution was warmed at 80 °C for 4 h. Filtration of the resin and
crystallization afforded the acid 2b (272 mg, 74%), as a white solid:
;
1668 cmꢀ1 MS (ESI positive): m/z 559.0 [M+Na]+, 1095.1
[2 M+Na]+; 99% isotopically pure. Anal. Calcd for C22H28D3NO14
C, 49.25; H+D, 6.39; N, 2.61. Found: C, 49.23; H+D, 6.35; N, 2.68.
:
4.10. 2,4,7,8,9-Penta-O-acetyl-5-N-benzyloxy-[2H2]-acetyl-b-
neuraminic acid methyl ester 12
mp 182–185 °C (from CH3CN); ½a D25
ꢁ
¼ ꢀ36:1 (c 1, H2O); 1H NMR
(D2O) d 4.15–4.03 (overlapping, 2H, H-4, and H-6), 3.93 (m, 1H;
H-5), 3.85 (dd, J9a,9b = 11.8, J9a,8 = 2.6 Hz 1H; H-9a), 3.66 (m, 1H;
H-8), 3.54 (dd, J9b,9a = 11.8, J9b,8 = 6.2 Hz, 1H; H-9b), 3.47 (dd,
J7,8 = 9.4, J7,6 <1.0 Hz, 1H; H-7), 2.26 (dd, J3a,3b = 13.0, J3a,4 = 4.9 Hz,
1H; H-3a), 1.82 (dd, J3b,3a = 13.0, J3b,4 = 11.7 Hz, 1H; H-3b); 13C
NMR (D2O) d 176.1 (br s, CD2CONH), 173.5 (C-1), 95.7, (C-2), 70.7
(C-8), 70.6 (C-6), 68.6 (C-4), 66.9 (C-7), 63.3 (C-9), 61.4 (t,
J = 21.2 Hz, COCD2OH), 52.2 (C-5), 39.3 (C-3); MS (ESI negative):
m/z 326.3 [MꢀH]ꢀ; 99% isotopically pure. Anal. Calcd for
C11H17D2NO10: C, 40.37; H+D, 6.47; N, 4.28. Found: C, 40.48;
H+D, 6.51; N, 4.33.
Benzyloxy-[2H2]-acetyl chloride (0.32 ml, 2.04 mmol) in CH2Cl2
(1.8 mL) was added, at 0 °C, to a solution of 2,4,7,8,9-penta-O-Acet-
yl-5-N-(2,2,2-trifluoroacetyl)-b-neuraminic acid methyl ester8f 10
(200 mg, 0.34 mmol) in CH2Cl2 (1.8 mL) containing diisopropyleth-
ylamine (1.18 mL, 6.8 mmol). After a 20 min stirring at 0 °C, the
reaction was stopped, by adding an ice cold aqueous solution of
HCl (6.0 mL, 1 M), and the organic layer was separated. The aque-
ous phase was extracted twice with dichloromethane and the col-
lected organic layers were washed in the sequence with water, an
aqueous solution of NaHCO3 and again with water. Dressing of the
solution and evaporation of the organic solvent afforded a crude
product which was purified by flash chromatography [eluting with
AcOEt/hexane (2/3; v/v)], to afford the compound 12 (17 mg, 8%),
4.8. N-[2H2]-Glycolyl-[3,3-2H2]-neuraminic acid 2c
as a white solid: mp 114–118 (form CH3CN); ½a D20
¼ ꢀ9:3 (c 1,
ꢁ
N-[2H2]-Glycolyl-neuraminic acid 2b (150 mg, 0.46 mmol) was
treated with a solution of NaOD in D2O (0.9 mL, 1.0 M) at 23 °C
for 3 h. After neutralization of the solution with acid resin [DowexÒ
50WX8 (H+)], filtration of the resin and lyophilisation of the solu-
tion, a crude acid is obtained which, after crystallization, afforded
the acid 2c (136 mg, 90%), as a white solid: mp 175–177 (from
CHCl3);1H NMR (CDCl3)
d 7.52–7.40 (m, 5H; Ph), 6.33 (d,
JNH,5 = 9.6 Hz, 1H; N–H), 5.38 (dd, J7,8 = 6.2, J7,6 = 2.3 Hz, 1H; H-7),
5.19 (m, 1H; H-4), 5.10 (m, 1H; H-8), 4.44 (dd, J9a,9b = 12.1,
J9a,8 = 2.3 Hz, 1H; H-9a), 4.15–4.06 (overlapping, 4H; H-5, H-9b,
and PhCH2), 4.03 (dd, J6,8 = 10.8, J6,7 = 2.3 Hz, 1H; H-6), 3.78 (s,
3H; COOCH3), 2.59 (dd, J3a,3b = 13.5, J3a,4 = 4.8 Hz, 1H; H-3a), 2.17
(s, 3H, CH3COO), 2.11 (s, 6H, 2 ꢂ CH3COO), 2.05 (s, 4H; CH3COO,
and H-3b); 1. 97 (s, 3H; CH3COO); MS (ESI positive): m/z 664.2
[M+Na]+; 99% isotopically pure. Anal. Calcd for C29H35D2NO15: C,
54.29; H+D, 6.13; N, 2.18. Found: C, 54.26; H+D, 6.15; N, 2.14.
CH3CN); ½a 2D5
ꢁ
¼ ꢀ35:0 (c 1, H2O); 1H NMR (D2O) d 4.17–4.04 (over-
lapping, 2H, H-4, and H-6), 3.94 (m, 1H; H-5), 3.85 (dd, J9a,9b = 11.7,
J9a,8 = 2.6 Hz 1H; H-9a), 3.66 (m, 1H; H-8), 3.53 (dd, J9b,9a = 11.7,
J9b,8 = 6.1 Hz 1H; H-9b), 3.46 (dd, J7,8 = 9.2, J7,6 <1.0 Hz, 1H; H-7);
13C NMR (D2O) d 175.9 (br s, CD2CONH), 173.3 (C-1), 95.5, (C-2),
70.6 (C-8), 70.5 (C-6), 68.4 (C-4), 66.6 (C-7), 63.1 (C-9), 61.2 (t,
J = 21.4 Hz, COCD2OH), 52.0 (C-5), 39.1 (m, C-3); MS (ESI negative):
m/z 328.3 [MꢀH]; 99% isotopically pure. Anal. Calcd for
4.11. Preparation of the Sias 1,7-lactones from the labeled acids:
general procedure
C11H15D4NO10: C, 40.12; H+D, 7.04; N, 4.25. Found: C, 40.20;
4.11.1. Lactonization
H+D, 7.09; N, 4.18.
CbzCl (3.2 mmol), dissolved in THF (1.5 mL), was added drop-
wise to a solution of the appropriate deuterated sialic acid
(0.30 mmol), previously dissolved in a mixture of anhydrous THF
(1.5 mL) and DMF (3.0 mL) and treated with triethylamine
(3.8 mmol) under stirring for 15 min. The solution was then stirred
a 0 °C for 15 min, heated to 23 °C and stirred for an additional
60 min. At this time, MeOH (0.3 mL) was added and stirring was
continued for 15 min. The solvent was then evaporated under re-
duced pressure to afford a crude product which was purified by
flash chromatography (using the reported solvent system) to afford
the desired 1,7-lactone containing a benzyloxycarbonyl group at
the anomeric carbon.
4.9. 2,4,7,8,9-Penta-O-acetyl-5-N-[2H3]-acetyl-b-neuraminic acid
methyl ester 11
[2H3]-Acetyl chloride (0.14 ml, 2.04 mmol) in CH2Cl2 (1.8 mL)
was added drop wise, under stirring, to a solution of 2,4,7,8,9-pen-
ta-O-acetyl-5-N-(2,2,2-trifluoroacetyl)-b-neuraminic acid methyl
ester8f 10 (200 mg, 0.34 mmol) containing diisopropylethylamine
(1.18 mL, 6.8 mmol) in CH2Cl2 (1.8 mL), cooled at 0 °C. After a
20 min the reaction was stopped by addition of an ice cold aqueous
solution of HCl (6.0 mL, 1 M). The organic layer was separated and
the aqueous solution was extracted twice with dichloromethane.
The organic layer was then washed in the sequence with water,
with an aqueous solution of sodium hydrogen carbonate and with
water, dressed and evaporated under reduced pressure to afford a
crude residue which was purified by flash chromatography [eluting
with AcOEt/MeOH (95/5; v/v)] to give the ester 11 (131 mg, 72%),
4.11.2. Regeneration of the anomeric hydroxy group
The protected 1,7-lactone (0.10 mmol) dissolved in ethyl ace-
tate (10 mL) was hydrogenated in the presence of Pd/C (50 mg,
10%) for 2 h. At this time, the catalyst was filtered and washed with
anhydrous THF and the solvent was evaporated under reduced
pressure to afford the free 1,7-lactone in a pure form.
as a white solid: mp 133–136 °C; ½a D20
ꢁ
¼ ꢀ18:3 (c 1, CHCl3); 1H
NMR (CDCl3) d 5.39–5.32 (overlapping, 2H; H-7, and N–H), 5.24

P. Allevi et al. / Tetrahedron: Asymmetry 22 (2011) 338–344
343
4.12. Synthesis of the N-[2H3]-acetyl-b-neuraminic acid 1,7-
lactone 3b
(DMSO-d6) d 169.1 (C-1), 168.6 (br s, CD3CONH), 90.1 (C-2), 77.2
(C-7), 71.0 (C-8), 69.9 (C-6), 65.7 (C-4), 61.6 (C-9), 50.1 (C-5),
36.7 (m, C-3), 22.1 (hept., J = 19.2 Hz, CD3CO); IR, (nujol) 3326,
1734 cmꢀ1; MS (ESI negative): m/z 295.4 [MꢀH]; 99% isotopically
pure. Anal. Calcd for C11H12D5NO8: C, 44.59; H+D, 7.48; N, 4.73.
Found: C, 44.52; H+D, 7.59; N, 4.65.
(i) Lactonization of the N-[2H3]-acetyl-b-neuraminic acid 1b
(100 mg, 0.32 mmol) according to the general procedure, provided,
after purification by flash silica gel chromatography [eluting with
AcOEt/MeOH 9:1, v/v], the 2-benzyloxycarbonyl N-[2H3]-acetyl-
neuraminic acid 1,7-lactone 13 (103 mg; 75%) as a white solid:
4.14. Synthesis of N-[2H2]-glycolyl-b-neuraminic acid 1,7-
lactone 4b
mp 123–127 °C (decomp., in sealed tube); ½a D25
ꢁ
¼ þ16:5 (c 1,
CH3OH); 1H NMR (CD3OD) d 7.36–7.30 (m, 5H; Ph), 5.15 (AB sys-
tem, 2H; CH2Ph), 4.64 (s, 1H; H-6), 4.47 (d, J7,8 = 8.9 Hz, 1H; H-7),
4.09 (m, 1H; H-4), 4.02 (br s, 1H; H-5), 3.96 (m, 1H; H-8), 3.81 (sys-
tem ABX, J9a,9b = 11.7, J9a,8 = 2.7, 1H; H-9a), 3.77 (system ABX,
J9b,9a = 11.7, J9b,8 = 4.5 Hz, 1H; H-9b), 2.26 (dd, J3a,3b = 13.6,
J3a,4 = 3.5, 1H; H-3a), 2.15 (dd, J3b,3a = 13.6, J3b,4 = 1.8, 1H; H-3b);
13C NMR (CD3OD) d 172.9 (br s, CD3CONH), 168.0 (C-1), 153.4
(PhCH2OCO), 136.2, 129.7, 129.6, 129.2 (Ph), 94.9, (C-2), 79.7 (C-
7), 73.1 (C-6), 71.9 (C-8), 71.3 (PhCH2OCO), 67.4 (C-4), 63.3 (C-9),
52.5 (C-5), 36.9 (C-3), 22.2 (hept., J = 19.4 Hz, CD3CO); IR, (Nujol)
3327, 1755 cmꢀ1; MS (ESI negative): m/z 427.2 [MꢀH]; 99% isoto-
pically pure. Anal. Calcd for C19H20D3NO10: C, 53.27; H+D, 6.12; N,
3.27. Found: C, 53.32; H+D, 6.19; N, 3.35.
(i) The N-[2H2]-glycolyl-b-neuraminic acid 2b (100 mg,
0.30 mmol) lactonized according to the general procedure, gave,
after purification by flash chromatography (eluting with AcOEt/
MeOH 9:1, v/v), the 2-benzyloxycarbonyl-N-[2H2]-glycolyl-b-neu-
raminic acid 1,7-lactone 15 (104 mg; 78%), as a white solid: mp
107–109 °C (decomp., in sealed tube; from CH3CN); ½a D25
¼ þ9:0
ꢁ
(c 1, CH3OH); 1H NMR (CD3OD) d 7.37–7.35 (m, 5H; Ph), 5.17 (AB
system, 2H; CH2Ph), 4.65 (br s, 1H; H-6), 4.47 (d, J7,8 = 9.0 Hz, 1H;
H-7), 4.11 (br m, 1H; H-4), 4.08 (br s, 1H; H-5), 3.95 (ddd,
J8,7 = 9.0, J8,9b = 4.4, J8,9a = 2.9 Hz, 1H; H-8), 3.78 (ABX system,
J9a,9b = 11.6, J9b,8 = 4.5, J9a,8 = 2.9 Hz, 2H; H-9a, and 9b), 2.22 (dd,
J3a,3b = 13.7, J3a,4 = 3.5 Hz, 1H; H-3a), 2.16 (dd, J3b,3a = 13.7,
(ii) Hydrogenation of the 2-benzyloxycarbonyl-N-[2H3]-acetyl-
b-neuraminic acid 1,7-lactone 13 (57 mg, 0.13 mmol), according
to general procedure, provided the title lactone 3b (36 mg; 91%)
J3b,4 = 2.2 Hz, 1H; H-3b); 13C NMR (CD3OD)
d 174.4 (br s,
CD2CONH), 167.7 (C-1), 153.5 (PhCH2OCO), 136.3, 129.7, 129.7,
129.5 (Ph), 94.8 (C-2), 79.6 (C-7), 73.1 (C-6), 71.9 (C-8), 71.3
(PhCH2OCO), 67.2 (C-4), 63.4 (C-9), 62.4 (t, J = 21.2 Hz, COCD2OH),
52.0 (C-5), 37.0 (C-3); IR, (Nujol) 3333, 1757 cmꢀ1; MS (ESI posi-
tive) m/z 466.3 [M+Na]+; 99% isotopically pure. Anal. Calcd for
as
a white solid: mp 110–113 °C (dec., in sealed tube);
¼ þ25:0 (c 1, THF); (DMSO-d6) 4.31 (s, 1H; H-6), 4.18 (d,
½
a 2D5
ꢁ
J
7,8 = 8.5 Hz, 1H; H-7), 3.80 (br s, 1H; H-4), 3.73 (br d, 1H; H-5),
3.59 (m, 1H; H-9a), 3.54 (m, 1H; H-8), 3.45 (dd, J9b,9a = 10.8,
J9b,8 = 5.0 Hz, 1H; H-9b), 1.93 (br dd, J3a,3b = 13.8, J3a,4 = 2.0 Hz,
1H; H-3a), 1.81 (br d, J3b,3a = 13.8 Hz, 1H; H-3b). dC (DMSO-d6)
169.2 (C-1), 168.7 (br s, CD3CONH), 90.2 (C-2), 77.3 (C-7), 71.1
(C-8), 69.9 (C-6), 65.8 (C-4), 61.7 (C-9), 50.2 (C-5), 36.8 (C-3),
22.1 (hept., J = 19.3 Hz, CD3CO); IR, (nujol) 3328, 1737 cmꢀ1; MS
(ESI negative): m/z 293.2 [MꢀH]ꢀ; 99% isotopically pure. Anal.
Calcd for C11H14D3NO8: C, 44.90; H+D, 6.85; N, 4.76. Found: C,
44.72; H+D, 6.89; N, 4.69.
C19H21D2NO11: C, 51.47; H+D, 5.68; N, 3.16. Found: C, 51.51;
H+D, 5.71; N, 3.14.
(ii) Hydrogenation of the the 2-benzyloxycarbonyl-N-[2H2]-gly-
colyl-b-neuraminic acid 1,7-lactone 15 (50 mg, 0.11 mmol),
according to the general procedure, gave the title lactone 4b
(31 mg; 89%) as a white solid: mp 111–115 °C (dec., in sealed tube)
½
a 2D5
ꢁ
¼ þ9:0 (c 1, THF); 1H NMR (DMSO-d6) d 7.56 (d, JNH,5 = 8.4 Hz,
1H; NH), 4.32 (br s, 1H; H-6), 4.24 (d, J7,8 = 6.2 Hz, 1H; H-7), 3.88–
3.83 (m, 1H; H-4), 3.79 (d, J5,NH = 8.4 Hz, 1H; H-5), 3.61–3.50 (over-
lapping, 2H; H-8, and H-9a), 3.46 (m, 1H; H-9b), 1.87–1.80 (over-
lapping, 2H; H-3a, and H-3b); 13C NMR (DMSO-d6) d 171.0 (br s,
CD2CONH), 169.0 (C-1), 90.3 (C-2), 77.2 (C-7), 71.2 (C-8), 70.0 (C-
6), 65.7 (C-4), 61.8 (C-9), 61.1 (t, J = 21.0 Hz, COCD2OH), 49.5 (C-
5), 37.0 (C-3); MS (ESI negative): m/z 308.2 [MꢀH]ꢀ. 99% isotopi-
cally pure. Anal. Calcd for C11H15D2NO9: C, 42.72; H+D, 6.19; N,
4.53. Found: C, 42.82; H+D, 6.13; N, 4.48.
4.13. Synthesis of the N-[2H3]-Acetyl-b-[3,3-2H2]-neuraminic
acid 1,7-lactone 3c
(i) The N-[2H3]-acetyl-b-[3,3-2H2]-neuraminic acid 1c (100 mg,
0.32 mmol), lactonized according to the general procedure, gave,
after purification by flash silica gel chromatography [AcOEt/MeOH
9:1, v/v], the 2-benzyloxycarbonyl-[3,3-2H2]-N-[2H3]-acetyl-b-neu-
raminic acid 1,7-lactone 14 (97 mg; 70%) as a white solid: mp 109–
4.15. Synthesis of N-[2H2]-glycolyl-b-[3,3-2H2]-neuraminic acid
1,7-lactone 4c
111 °C (decomp., in sealed tube; from CH3CN); ½a D25
ꢁ
¼ þ17:2 (c 1,
CH3OH); 1H NMR (CD3OD) d 7.37–7.32 (m, 5H; Ph), 5.17 (AB sys-
tem, 2H; CH2Ph), 4.63 (s, 1H; H-6), 4.45 (d, J7,8 = 9.0 Hz, 1H; H-7),
4.07 (br s, 1H; H-4), 4.02 (m, 1H; H-5), 3.95 (ddd, J8,7 = 9.0,
J8,9b = 4.5, J8,9a = 2.7 Hz, 1H; H-8), 3.79 (system ABX, J9a,9b = 11.8,
J9a,8 = 2.7, J9b,8 = 4.5 Hz, 2H; H-9a, and H-9b); 13C NMR (CD3OD) d
172.8 (br s, CD3CONH), 167.8 (C-1), 153.2 (PhCH2OCO), 136.1,
129.6, 129.5, 129.3 (Ph), 94.8 (C-2), 79.7 (C-7), 73.2 (C-6), 71.8
(C-8), 71.2 (PhCH2OCO), 67.3 (C-4), 63.2 (C-9), 52.5 (C-5), 36.8
(m, C-3), 22.1 (hept., J = 19.4 Hz, CD3CO); IR, (Nujol) 3326,
1753 cmꢀ1; MS (ESI negative): m/z 429.1 [MꢀH]; 99% isotopically
pure. Anal. Calcd for C19H18D5NO10: C, 53.02; H+D, 6. 55; N, 3.25.
Found: C, 53.10; H+D, 6.59; N, 3.30.
The N-[2H2]-glycolyl-b-[3,3-2H2]-neuraminic acid 2c (100 mg;
0.30 mmol), lactonized according to the general procedure gave,
after purification by flash chromatography [eluting with AcOEt/
MeOH 9:1, v/v)], the 2-benzyloxycarbonyl-N-[2H2]-glycolyl-b-
[3,3-2H2]-neuraminic acid 1,7-lactone 16 (103 mg; 76%), as a white
solid: mp 110–113 °C (decomp., in sealed tube; from CH3CN);
½
a 2D5
ꢁ
¼ þ8:4 (c 1, CH3OH); 1H NMR (CD3OD) d 7.38–7.35 (m, 5H;
Ph), 5.18 (AB system, 2H; CH2Ph), 4.65 (br s, 1H; H-6), 4.46 (d,
J7,8 = 9.1 Hz, 1H; H-7), 4.10 (br s, 1H; H-4), 4.06 (br s, 1H; H-5),
3.94 (ddd, J8,7 = 9.0, J8,9b = 4.4, J8,9a = 2.9 Hz, 1H; H-8), 3.77 (ABX
system, J9a,9b = 11.6, J9b,8 = 4.5, J9a,8 = 2.9 Hz, 2H; H-9a, and 9b);
13C NMR (CD3OD) d 174.5 (br s, CD2CONH), 167.8 (C-1), 153.4
(PhCH2OCO), 136.2, 129.7, 129.6, 129.5 (Ph), 94.7 (C-2), 79.5 (C-
7), 73.0 (C-6), 71.8 (C-8), 71.2 (PhCH2OCO), 67.1 (C-4), 63.3 (C-9),
62.3 (t, J = 21.3 Hz, COCD2OH), 51.9 (C-5), 36.9 (m, C-3); IR, (Nujol)
3330, 1753 cmꢀ1; MS (ESI positive): m/z 468.4 [M+Na]+; 99% isoto-
pically pure. Anal. Calcd for C19H19D4NO11: C, 51.23; H+D, 6.11; N,
3.14. Found: C, 51.18; H+D, 5.17; N, 3.14.
(ii) Hydrogenation of the 2-benzyloxycarbonyl lactone 14
(50 mg, 0.12 mmol), according to the general procedure, gave the
title lactone 3c (32 mg; 93%) as a white solid: mp 110–113 °C
(dec., in sealed tube); ½a D25
ꢁ
¼ þ27:0 (c 1, THF); 1H NMR (DMSO-
d6) d 4.30 (s, 1H; H-6), 4.17 (d, J7,8 = 8.5 Hz, 1H; H-7), 3.79 (br s,
1H; H-4), 3.72 (br d, 1H; H-5), 3.57 (m, 1H; H-9a), 3.52 (m, 1H;
H-8), 3.44 (dd, J9b,9a = 10.8, J9b,8 = 5.0 Hz, 1H; H-9b), 13C NMR

344
P. Allevi et al. / Tetrahedron: Asymmetry 22 (2011) 338–344
2. Schauer, R.; Kelm, S.; Reuter, G.; Roggentin, P.; Shaw, L. In Biology of the Sialic
(ii) Hydrogenation of the 2-benzyloxycarbonyl-N-[2H2]-glyco-
Acid; Rosemberg, A., Ed.; Plenum Press: NY, 1995; pp 7–67.
3. Bulai, T.; Bratosin, D.; Pons, A.; Montreuil, J.; Zanetta, J. P. FEBS Lett. 2003, 534,
185–189.
lyl-b-[3,3-2H2]-neuraminic acid 1,7-lactone 16 (45 mg, 0.10 mmol),
according to the general gave the title lactone 4c (29 mg; 92%) as a
white solid: mp 111–115 °C (dec., in sealed tube); ½a D25
¼ þ9:3 (c 1,
ꢁ
4. For a concise summary of the analyses see: Zanetta, J. P.; Pons, A.; Iwersen, M.;
Mariller, C.; Leroy, Y.; Timmerman, P.; Schauer, R. Glycobiology 2001, 11, 663–
676. and references cited therein reported.
THF); 1H NMR (DMSO-d6) d 7.56 (d, JNH,5 = 8.3 Hz, 1H; NH), 4.34 (br
s, 1H; H-6), 4.25 (d, J7,8 = 6.2 Hz, 1H; H-7), 3.87 (d, J4,5 = 10.0 Hz,
1H; H-4), 3.79 (d, J5,NH = 8.3 Hz, 1H; H-5), 3.61–3.49 (overlapping,
2H; H-8, and H-9a), 3.47 (m, 1H; H-9b); 13C NMR (DMSO-d6) d
171.0 (br s, CD2CONH), 169.0 (C-1), 90.3 (C-2), 77.2 (C-7), 71.2
(C-8), 70.0 (C-6), 65.7 (C-4), 61.8 (C-9), 61.1 (t, J = 21.1 Hz, COC-
D2OH), 49.5 (C-5), 37.0 (m, C-3); MS (ESI negative): m/z 310.4
[MꢀH]; 99% isotopically pure. Anal. Calcd for C11H13D4NO9: C,
42.44; H+D, 6.80; N, 4.50. Found: C, 42.38; H+D, 6.83; N, 4.47.
5. Cebo, C.; Dambrouck, T.; Maes, E.; Lauden, C.; Strecker, G.; Michalski, J. C.;
Zanetta, J. P. J. Biol. Chem. 2001, 276, 5685–5691.
6. Cebo, C.; Vergoten, G.; Zanetta, J. P. Biochim. Biophys. Acta 2002, 1572, 422–434.
7. Bratosin, D.; Palii, C.; Moicean, A. D.; Zanetta, J. P.; Montreuil, J. Biochimie 2007,
89, 355–359.
8. (a) Colombo, R.; Anastasia, M.; Rota, P.; Allevi, P. Chem. Commun. 2008, 5517–
5519; (b) Rota, P.; Allevi, P.; Colombo, R.; Costa, M. L.; Anastasia, M. Angew.
Chem., Int. Ed. 2010, 49, 1850–1853; (c) Rota, P.; Allevi, P.; Mattina, R.;
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Scaringi, R.; Colombo, R.; Anastasia, M. J. Org. Chem. 2010, 75, 5542–5548; (e)
Allevi, P.; Femia, E. A.; Costa, M. L.; Cazzola, R.; Anastasia, M. J. Chromatogr., A
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Acknowledgements
9. Shibayama, S.; Yoshimura, S.; Ito, M.; Shitori, Y.; Ogawa, T. US Patent 4 774 326,
1988.
10. From the experiments performed, we concluded that the problem of the N-
transacylation of the glycolylneuraminic acid, free or protected at the glycolyl
hydroxy group is part of a larger problem involving the behavior of the N-
Financial support from MiUR, Italian Ministry of University and
Research, is acknowledged.
transacylation of amides functionalized at the
currently under study in our laboratory.
a-position. This issue is
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