Stereoselective synthesis of glycosyl amides by traceless Staudinger ligation of unprotected glycosyl azides

Article abstract of DOI:10.1016/j.carres.2008.04.023

The stereoconservative Staudinger ligation of unprotected α- and β-glucosyl azides with diphenylphosphanyl-phenyl esters to afford α- and β-glucosyl amides is described.

Full text of DOI:10.1016/j.carres.2008.04.023

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1636–1643
Stereoselective synthesis of glycosyl amides by traceless
Staudinger ligation of unprotected glycosyl azides
Filippo Nisic and Anna Bernardi*
Dipartimento di Chimica Organica e Industriale and CISI, Universita’ degli Studi di Milano, Via Venezian 21, I-20133 Milano, Italy
Received 6 February 2008; received in revised form 13 April 2008; accepted 17 April 2008
Available online 22 April 2008
Abstract—The stereoconservative Staudinger ligation of unprotected a- and b-glucosyl azides with diphenylphosphanyl-phenyl
esters to afford a- and b-glucosyl amides is described.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Glycosyl amides; Glycosyl azides; Neoglycoconjugates; Stereoselective synthesis
1. Introduction
er reduction–acylation process,7–13 which consists of the
reduction of glycosyl azides with phosphines in the pres-
ence of carboxylic acids or derivatives. An aza-ylide
(iminophosphorane) is formed, which can be trapped
by the acylating agent before the free amine is formed.
However, anomerization remains a significant problem
also for Staudinger reduction intermediates and, unless
the acylation step is very fast, b-amides or mixtures of
anomers are often obtained.14
We have recently reported that the traceless Stauding-
er ligation of O-benzyl-a-glycosyl azides 1a with diphen-
ylphosphanyl-phenyl esters 2 in polar aprotic solvents
yields a-glycosyl amides with good yields and selectivity
(Scheme 1, path A).6 The phosphines employed, which
a-Glycosyl amides represent a little known class of non-
hydrolyzable monosaccharide derivatives that may find
useful application as sugar mimics and neoglycoconju-
gates. Their stereoselective synthesis has proven difficult
because glycosyl amines are not configurationally stable
and undergo easy a to b anomerization. Various meth-
ods have been proposed to get around this problem by
reducing a-glycosyl azides in the presence of acylating
agents, but only a few have been reported to afford a-
glycosyl amides, most of which require two steps and/
or have been described for a limited number of sub-
strates.1–6 Much work has been devoted to the Stauding-
OBn
O
1a PG = -Bn
BnO
BnO
A
B
O
BnO
O-PG
O
R
O
HN
O
R
PG-O
PG-O
α
+
PPh2
O
PG-O
N3
OAc
O
1
2
H
N
AcO
AcO
R
1b PG = -Ac
OAc
β
Scheme 1. Traceless Staudinger ligation of protected glycosyl azides.
*
Corresponding author. Tel.: +39 02 50314092; fax: +39 02 50314072; e-mail: anna.bernardi@unimi.it
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.04.023

F. Nisic, A. Bernardi / Carbohydrate Research 343 (2008) 1636–1643
1637
OH
O
OH
O
PPh2
1) 70 ºC, DMA, 18 h
O
HO
HO
HO
HO
+
2) H2O, 2 h
HO
N3
HO
O
NHCOMe
70%
3
2a
4a
Scheme 2. Staudinger reduction–acetylation of the unprotected a-glucosylazide 3 with 2a.
are stable to air, allow the fast intramolecular trapping
of the reduction intermediates affording direct forma-
tion of the amide link and preventing anomerization.
However, the process depends critically on the nature
of the sugar protecting groups: the same phosphines re-
act with tetra-O-acetyl-glycosyl azides 1b in a non-ste-
reoconservative fashion and afford b-glycosyl amides
(Scheme 1, path B). The dependence of the ligation ste-
reochemistry on the nature of the sugar protecting
group appeared to be related to the electron-withdraw-
ing effect of the acetates,15,16 which reduces the rate of
the acylation step and favors the anomerization. This ef-
fect enforces the use of benzyl ethers as protecting
groups in the synthesis of a-glycosyl azides.
Here we report our initial results on the Staudinger
ligation of unprotected a and b glucosyl azides with
phosphines 2. We were able to identify conditions that
allowed a clean and stereoconservative reaction to occur
in good to moderate yields, affording a or b glucosyla-
mides depending on the configuration of the starting
azide. Isolation of the resulting glucosylamides from
the reaction by-products, notably triarylphosphine oxi-
des, was remarkably simplified and could be performed
by water extraction in all the cases examined.
yield by extracting the hydrophobic by-products in
CH2Cl2 (Scheme 2). The process appeared to be totally
stereoconservative. The anomeric configuration of 4a
was assigned on the basis of the vicinal coupling constant
of the anomeric proton (at 5.45 ppm) J1,2 = 5.4 Hz.
Extension of this method to the pentanoyl phosphine
2b (Scheme 3) yielded a complex mixture of compounds
in the water fraction that were separated by reverse-
phase HPLC and characterized as the expected pentana-
mide 4b (50% yield), the a-N-pentanoyl-glucofuranosyl-
amine isomer 5 (15%) and glucose (equilibrium mixture
of anomers, 30%). The configuration of 4b was assigned
on the basis of the anomeric proton signal (5.45 ppm,
J1,2 = 5.4 Hz). The structure of 5 was assigned on the
1
basis of ESIMS and of the H NMR (D2O), which is
consistent with the spectrum of the known a-D-glucofur-
anoside.17 The anomeric configuration of 5 was further
confirmed to be a by the NOESY spectrum (D2O),
which showed a strong crosspeak for the H-1 and H-2
protons and no crosspeak for the H-1 and H-3 protons
(Scheme 3). The presence of glucose in the reaction
crude was identified by 1H NMR spectroscopy and con-
firmed upon acetylation of the crude, which allowed the
isolation of the known N-pentanoyl-tetra-O-acetyl-a-D-
glucopyranosylamine 66 together with the tetra-O-acetyl
N-furanosylamide 7, and a and b penta-O-acetyl-glu-
cose (Scheme 3).
2. Results and discussion
Furanoside 5 must clearly derive from a ring-opening
process occurring after the azide reduction step, presum-
ably from the iminophosphorane 8 (Scheme 4) to afford
the phosphinimine 9, which can undergo ring-closure to
yield 5. Hydrolysis of the same intermediate 9 accounts
for the formation of D-glucose in the reaction mixture.
The alternative hypothesis that glucose could be formed
As a first step, the reaction of 2a with the unprotected a-
glucosyl azide 3 was examined under the same conditions
optimized for the ligation of the benzyl derivative 1a.
After 18 h of reaction in dimethylacetamide (DMA) at
70 °C, the mixture was hydrolyzed by adding water
and a-N-acetylglucosylamine 4a was isolated in 70%
H5
OH
O
HO
OH
O
H
OH
H
HO
O
HO
HO
HO
HO
+ glucose
+
HO
NH
H4
HO
N3
NH
OH
H
3
4b
5
O
1) 70 ºC, DMA, 18 h
2) H2O, 70 ºC, 2 h
O
+
Ac2O
AcO
PPh2
+ penta-
H
O
-acetyl-D-glucose
H5
O
OAc
O
OAc H
O
AcO
O
+
AcO
AcO
2b
AcO
NH
H4
NH
H
OAc
O
6
O
7
Scheme 3. Staudinger ligation of 3 with phosphine 2b in DMA.

1638
F. Nisic, A. Bernardi / Carbohydrate Research 343 (2008) 1636–1643
OH
O
HO
HO
H
acyl transfer
HO
4b
HN
O
OH
O
OH
O
HO
HO
H
3 + 2b
HO
HO
H
HO
N
PPh2
N2
HO
N
PPh2
O
O
8
9
O
O
H2O
OH
O
HO
HO
H
OH
O
glucose
HO
HO
HO
HO
H
H
OH
H
O
H
HO
N
O
PPh2
NH
H
HO
N
PPh2
H
OH
O
O
O
5
O
Scheme 4. Mechanism for the formation of furanosylamide 5 and glucose in the ligation of 3 with 2b.
by direct hydrolysis of the starting azide was discarded
because 3 was recovered unaltered after treatment with
water in DMA at 70 °C for 24 h.
Eventually, the best conditions identified consisted in
using a 98:2 DMA–DMPU solvent mixture and running
the reaction at 40 °C for 4 h, before proceeding to the
hydrolysis step by adding 5% of H2O and stirring at
40 °C for an additional 2 h. Under these conditions the
a-glucopyranosylamide 4b was isolated in 65% yield
and in an excellent anomeric ratio (Scheme 5 and Table
1, entry 2). This product was exclusively contaminated
by 4% of furanosylamide 5, which could be separated
by automated or open-air reverse-phase chromatogra-
phy (see Section 4). When the same reaction conditions
were extended to phosphines 2c–f (Scheme 5, Table 1,
entries 5, 7, 9, 11) the anomeric ratios were found to
be consistently good; the yields were moderate. Variable
amounts of furanosylamide were formed, depending on
After this preliminary analysis, the reaction condi-
tions were optimized using NMR as the analytical tech-
nique. This series of experiments allowed us to draw the
following general conclusions. First, the formation of
the a-furanosylamide 5 is favored by a temperature in-
crease and can be reduced to ꢀ5% by running the liga-
tion at temperatures below 40 °C. Second, the azide
conversion is favored by an increase in solvent polarity,
which appears to accelerate the acyl transfer step. This
increase is best realized by adding 1,3-dimethyltetrahy-
dro-2(1H)pyrimidinone (DMPU, 1–2%) to the DMA
solution.
PPh2
OH
OH
O
O
R
O
HO
HO
HO
HO
+
O
HO
HO
NHCOR
N3
3
R = Me
R = Me
4a
4b
2a
2b
R = CH2CH2CH2CH3
R= CH2CH2CH2CH3
R = (CH2)3CO2Me
4c
4d
2c R = (CH2)3CO2Me
R = CH2CHCO2Me
NHCbz
2d
R = CH2CHCO2Me
NHCbz
4e
4f
2e
R = CH2CH2CHCO2Me
R = CH2CH2CHCO2Me
NHCbz
R = CHC(CH3)2
NHCbz
2f R = CHC(CH3)2
Scheme 5. Synthesis of a-glucosylamides 4 by ligation of 3 with 2 in DMA–DMPU.

F. Nisic, A. Bernardi / Carbohydrate Research 343 (2008) 1636–1643
Table 1. Synthesis of a-glucosylamides 4 by ligation of 3
1639
Entry
Phosph.
Product
Conditions
Yielda (%)
a:ba
% Furanosea
1
2
3
4
5
6
7
8
9
2a
2b
2b
2b
2c
2c
2d
2d
2e
2e
2f
4a
4b
4b
4b
4c
4c
4d
4d
4e
4e
4f
DMA, 70 °C, 18 h
98:2 DMA–DMPU 40 °C, 4 h
98:2 DMA–DMPU MW, 120 °C, 10 min
98:2 DMA–DMPU MW, 50 °C, 50 min
98:2 DMA–DMPU 40 °C, 4 h
98:2 DMA–DMPU MW, 50 °C, 50 min
98:2 DMA–DMPU 40 °C, 4 h
98:2 DMA–DMPU MW, 50 °C, 50 min
98:2 DMA–DMPU 40 °C, 4 h
98:2 DMA–DMPU MW, 50 °C, 50 min
98:2 DMA–DMPU 40 °C, 4 h
70b
70(65b)
65b
65b
56
P99:1
98:2
98:2
98:2
98:2
98:2
98:2
98:2
98:2
98:2
95:5
98:2
—
4
3
2
2
2
10
4
9
2
40b
25
35b
40
10
11
12
50b
40
40
52b
2f
4f
98:2 DMA–DMPU MW, 50 °C, 50 min
9b
a Yields and diastereomeric ratios were estimated on the basis of the 1H NMR spectrum of the crude water extract.
b Isolated yields.
OH
O
H5
OH
PPh2
HO
HO
DMA:DMPU
OH
O
H
H
HO
HO
O
O
HO
HO
+
+
HO
HN
O
HO
NH
H4
MW
N3
H
OH
O
O
7
:
1
3
2f
10
4f
Scheme 6. Reaction of 3 with 2f under microwave irradiation.
the acyl chain transferred. Only 2% of furanose was
formed together with glutaroylamide 4c (Table 1, entry
5), ꢀ10% with the aspartic and glutamic acid derivatives
4d and 4e (Table 1, entries 7 and 9), and up to 40% with
the 3-methyl-butenoate derivative 4f (Table 1, entry 11).
Compared with the results previously obtained on
2,3,4,6-tetra-O-benzyl-a-D-glucosylazide (1a)6 this ap-
proach affords a higher a/b selectivity and a much easier
purification of the amide products, which can be isolated
by water extraction from the crude reaction mixtures.
In an effort to improve the yields, the use of micro-
wave (MW) heating was explored (Table 1, entries 3,
4, 6, 8, 10, and 12).18 Initial experiments revealed that
the reaction of 3 and 2b could be completed in 10 min
under MW irradiation at 120 °C (Table 1, entry 3) with
yield and selectivity comparable to those achieved in 4 h
at 40 °C without MW (compare with Table 1, entry 2).
Furthermore, even at 50 °C under MW irradiation the
reaction was complete in only 50 min (Table 1, entry
4) and the amount of furanoside was reduced to 2%.
Similar results were obtained with phosphines 2c–e: in
all cases the amount of furanoside was consistently re-
duced to 2–4%, the a/b selectivity was unchanged and
the yields were generally increased, especially for the
most difficult transfer reactions (compare Table 1, en-
tries 7 and 8, 9 and 10). Surprisingly, MW irradiation
during the ligation of 3 with 2f favored the formation
of furanosylamide 10 (Scheme 6), which was formed in
7:1 ratio with the isomeric pyranosylamide 4f and could
be isolated in 52% yield by flash chromatography.
To probe the scope of the microwave-assisted process,
we also examined the ligation of the unprotected b-glu-
cosylazide 11 with representative phosphines (Scheme 5,
Table 2). The process efficiently affords the b-glucosyla-
mides 12, characterized by a vicinal coupling constant of
the anomeric proton J1,2 of 9.2 Hz. Yields were gener-
ally good and the selectivity for the pyranose isomer
was excellent. Also in this case, the main advantage of
Table 2. MW-assisted ligation of b-glucosylamine 11a
Entry
Phosph.
Product
R
Yb (%)
% Furanosidec
1
2
2b
2c
12b
12c
–(CH2)3CH3
–(CH2)3CO2Me
70
75
2
2
-CH2-CH-CO2Me
3
4
2d
2e
12d
12e
32
38
2
2
NHCbz
-(CH2)2-CH-CO2Me
NHCbz
a All reactions were run in 98:2 DMA–DMPU for 50 min at 50 °C under MW irradiation.
b Isolated yield.
c Estimated on the basis of the 1H NMR spectra of the crude water extract.

1640
F. Nisic, A. Bernardi / Carbohydrate Research 343 (2008) 1636–1643
OH
O
PPh2
OH
O
1) 50 ºC, DMA/DMPU
O
R
50 min., MW
HO
HO
N3
HO
HO
NHCOR
β only
+
2) H2O, 50 ºC, 15 min.
O
HO
11
HO
2
12b
R = CH2CH2CH2CH3
R = (CH2)3CO2Me
12c
12d
R = CH2CHCO2Me
NHCbz
12e
R = CH2CH2CHCO2Me
NHCbz
Scheme 7. Synthesis of b-glucosylamides by MW-assisted ligation of the unprotected b-glucosyl azide 11.
this procedure over the one described for the corre-
sponding tetra-O-acetyl azide 1b6 lies in the easy purifi-
cation of the resulting amides from the reaction mixtures
(Scheme 7).
2(1H)pyrimidinone (DMPU), chloroform, and pyridine
were dried over activated molecular sieves. Reactions
requiring anhydrous conditions were performed under
1
argon. H, 13C, and 31P NMR spectra were recorded
at 400 MHz on a Bruker AVANCE-400 instrument.
Chemical shifts (d) for 1H and 13C spectra are expressed
in ppm relative to internal Me4Si as standard. Signals
were abbreviated as s, singlet; br s, broad singlet; d, dou-
blet; t, triplet; q, quartet; m, multiplet. Mass spectra
were obtained with a Bruker ion-trap Esquire 3000
apparatus (ESI ionization). HPLC separation of 4b
and 5 was performed with an Atlantis reverse-phase col-
umn (dc 19 ꢁ 100 mm, 5 lm, ESIMS detector, Waters)
using a H2O–CN3CN gradient (from 100:0 to 70:30)
over 6 min (4b: tR = 3.6 min), 5 (tR = 4.1 min). Thin
layer chromatography (TLC) was carried out with pre-
coated Merck F254 silica gel plates. Flash chromatogra-
phy (FC) was carried out with Macherey-Nagel Silica
Gel 60 (230–400 mesh); reverse-phase open-air flash
chromatography was carried out with silica gel Cosmosil
75 C18-OPN (75 mesh Nacalai tesque).21 Reverse-phase
automated chromatography was carried out with a Bio-
tage System SP1 (25+M dc 25 ꢁ 150 mm column, C-18
silica 40–63 lm). Azides 3 and 11 were synthesized start-
ing from the corresponding known peracetylated sugars6
by sodium methoxide deprotection. Phosphines 2a–d
and 2f have been previously described.6
3. Conclusions
Elaboration of unprotected carbohydrates has been
receiving much attention19 and one method for the syn-
thesis of b-D-2-deoxy-2-N-acetyl-glucopyranosyl-aspar-
agine has been recently described.20 The results
reported here show that unprotected a or b glucosyl
azides can be stereoselectively transformed into the cor-
responding amides by traceless Staudinger ligation using
phosphines 2. Crucial to yield and selectivity was an
appropriate solvent selection; DMA–DMPU mixtures
(98:2) were found to afford the best results. Microwave
acceleration allowed shortening of the reaction time to
50 min and improvement in selectivity. Remarkably,
reaction of 3 with the 3-methyl-2-butenoyl phosphine
2f under MW irradiation led to the isolation of furano-
sylamide 10 in moderate yields. All ligation products
could be isolated from the crude reaction mixture by
simple water extraction, and were further purified by
flash chromatography either on direct or reverse-phase
silica gel. The process described here affords the best ste-
reoselectivity achieved so far in the synthesis of some a-
glycosylamides, such as the aminoacid derivatives 4d
and 4e. Work is in progress to improve the yields of such
difficult ligation reactions.
4.2. Synthesis of N-benzyloxycarbonyl-L-glutamic acid 5-
(2-diphenylphosphanyl-phenyl)ester 1-methyl ester 2e
A solution of the o-diphenylphosphinophenol6 (200 mg,
0.715 mmol, 1 equiv), the commercially available N-car-
bobenzyloxy-L-glutamicacid 1-methyl ester (255 mg,
0.86 mmol, 1.2 equiv) and N,N-dimethylaminopyridine
(8.8 mg, 0.072 mmol, 0.1 equiv) in dry CH2Cl2 (0.1 M)
was added, at room temperature and under nitrogen,
to a suspension of N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide hydrochloride (193.3 mg, 1 mmol,
4. Experimental
4.1. General
Solvents were dried by standard procedures: dichloro-
methane, methanol, N,N-diisopropylethylamine, and
triethylamine were dried over calcium hydride; N,N-
dimethylacetamide (DMA), 1,3-dimethyltetrahydro-
1.4 equiv)
and
dry
N,N-diisopropylethylamine
(172.6 lL, 1 mmol, 1.4 equiv) in dry CH2Cl2. The mix-

F. Nisic, A. Bernardi / Carbohydrate Research 343 (2008) 1636–1643
1641
ture was stirred at rt for 2 h, monitoring by TLC (1:1
4.6. N-Pentanoyl-a-D-glucopyranosylamide 4b
hexane–AcOEt). The reaction mixture was diluted with
CH2Cl2 and extracted with 10% aqueous HCl and
water; the organic layer was dried over Na2SO4 and con-
centrated. The crude product was purified by flash chro-
matography using 7:3 hexane–AcOEt as the eluant to
Crude purified by flash chromatography: 80:20:2
CHCl3–MeOH–H2O, or by open-air chromatography
on Cosmosil 75 C18-OPN: 95:5 H2O–MeOH, or by
automated chromatography (H2O–CH3CN gradient);
yield 65%. 1H NMR (400 MHz, D2O): d 5.60 (d,
1H, J1,2 = 5.4 Hz, H-1), 3.83 (dd, 1H, J1,2 = 5.4 Hz,
J2,3 = 9.4 Hz, H-2), 3.86–3.71 (m, 3H, H-3, H-6a
and H-6b), 3.53–3.49 (m, 1H, H-5), 3.46 (dd, 1H,
J3,4 = J4,5 = 9.4 Hz, H-4), 2.39 (m, 2H, –COCH2–),
1.62 (m, 2H, –CH2–CH2–CH3), 1.35 (m, 2H,
–CH2–CH3), 0.92 (t, 3H, J = 7.5 Hz, –CH3); 13C
NMR (100 MHz, D2O): d 179.37, 76.52 C-1, 73.03
C-2, 72.68 C-5, 69.35 C-3, 69.30 C-4, 60.51 C-6,
35.34 (–COCH2–), 27.51 (–CH2–CH2–CH3), 21.54
(–CH2–CH3), 13.01 (–CH3); ESIMS m/z: 286.29
[M+Na]+.
1
afford 2e in 55% yield. H NMR (400 MHz, CDCl3): d
7.42–7.29 (m, 11H), 7.17–7.08 (m, 2H), 6.86 (m, 1H),
5.40 (d, 1H, J = 7.2 Hz, NH), 5.12 (s, 1H, J = 12.3 Hz,
CH2-Ph), 4.35 (q, 1H, J = 4.8 Hz, CH), 2.35 (m, 2H,
CH2–COO), 2.07 (m, 1H, CH2–CH, Ha), 1.86 (m, 1H,
CH2–CH, Hb).
4.3. General procedure for stereoselective ligation of 3 in
DMA–DMPU (procedure A)
Phosphine (1.2 equiv) was added, at room temperature,
to a 0.1 M solution of azide 3 (1 equiv) in 98:2 N,N-
dimethylacetamide and DMPU. The solution was stir-
red for 4 h at 40 °C, then water was added and the mix-
ture was stirred for an additional 2 h at the same
temperature. The solvent was evaporated under reduced
pressure, and the residue was diluted with water and ex-
tracted with CH2Cl2. The water layer was evaporated
under reduced pressure and the crude was purified as
indicated below for each compound.
4.7. N-Pentanoyl-a-D-glucofuranosylamide 5
Purified from the ligation in pure DMA by reverse-
phase HPLC; 80:20:2 CHCl3–MeOH–H2O, Rf = 0.3.
1
[a]D +48.5 (c 0.4, MeOH); H NMR (400 MHz, D2O):
d 5.71 (d, 1H, J1,2 = 4 Hz, H-1), 4.23 (dd, 1H,
J2,3 = 0 Hz, H-3), 4.08 (dd, 1H, J3,4 = 2.4 Hz,
J1,2 = 4 Hz, H-2), 3.98 (dd, 1H, J4,5 = 9.4 Hz,
J3,4 = 2.4, H-4), 3.79 (dd, 1H, J4,5 = 9.4 Hz,
J5,6 = 6 Hz, H-5), 3.70 (dd, 1H, J5,6 = 6 Hz,
J6a,6b = 9 Hz, H-6a), 3.52 (dd, 1H, J5,6 = 6 Hz,
J6b,6a = 9 Hz, H-6b), 2.25 (m, 2H, –COCH2–), 1.48 (m,
2H, –CH2–CH2–CH3), 1.25 (m, 2H, –CH2–CH3), 0.79
(t, 3H, J = 7.5 Hz, –CH3); 13C NMR (100 MHz, D2O):
d 178.12, 80.94 C-1, 78.68 C-4, 75.55 C-3, 74.91 C-2,
68.86 C-5, 63.34 C-6, 35.40 (–COCH2–), 27.22 (–CH2–
CH2–CH3), 21.52 (–CH2–CH3), 12.92 (–CH3). ESIMS
m/z: 286.29 [M+Na]+.
4.4. General procedure for stereoselective ligation of 3 and
11 under microwave irradiation (procedure B)
Phosphine (1.2 equiv) was added, at room temperature,
to a 0.1 M solution of the azide (1 equiv), in 98:2 N,N-
dimethylacetamide and DMPU. The solution was
heated to 50 °C in the microwave oven. After 50 min
water (10%) was added and the mixture was stirred
for an additional 10 min at the same temperature in
the MW oven. The solvent was evaporated under re-
duced pressure, and the residue was diluted with
CH2Cl2 and extracted with water. The water layer
was evaporated under reduced pressure and the crude
was purified by flash chromatography as indicated
below.
4.8. N-Pentanedioic acid-a-D-glucopyranosylamide
methyl ester 4c
Purification by flash chromatography (80:20:2 CHCl3–
MeOH–H2O; Rf = 0.2) or Cosmosil 75 C18-OPN (95:5
H2O–MeOH); procedure B, yield 40%. [a]D +75.4 (c
0.1, MeOH); 1H NMR (400 MHz, D2O): d 5.52 (d,
1H, J1,2 = 5.6 Hz, H-1), 3.64 (m, 1H, J2,3 = 9.6 Hz, H-
2), 3.63 (dd, 1H, J6a,6b = 10 Hz, J5,6 = 2.4 Hz, H-6a),
3.58 (t, 1H, J4,5 = 4 Hz, H-4), 3.54 (s, 3H, OMe), 3.54
(m, 1H, H-6b), 3.34 (m, 1H, H-5), 3.27 (t, 1H,
J3,4 = 8.8 Hz, H-3), 2.32–2.25 (m, 4H, CH2–CO–N,
CH2–COOCH3), 1.75 (m, 2H, CH2–CH2–CH2); 13C
NMR (100 MHz, D2O): d 177.75 (CO–N), 176.44
(CO2Me), 80.94 C-1, 78.68 C-4, 75.55 C-3, 74.91 C-2,
68.86 C-5, 63.34 C-6, 53 (OCH3), 35.40 (–COCH2–),
34.71–32.72 (CH2COO–, CH2–CO–N), 20.40 (CH2–
CH2–CH2); ESIMS m/z: 330.3 [M+Na]+.
4.5. N-Acetyl-a-D-glucopyranosylamide 4a
Crude purified by flash chromatography: 80:20:2
CHCl3–MeOH–H2O, Rf = 0.1, 70% yield. [a]D +111.6
1
(c 0.2, MeOH); H NMR (400 MHz, D2O): d 5.48 (d,
1H, J1,2 = 5.5 Hz, H-1), 3.71 (dd, 1H, J1,2 = 5.5 Hz,
J2,3 = 10.2 Hz, H-2), 3.69–3.66 (m, 2H, H-6a and H-
6b), 3.63 (dd, 1H, J2,3 = J3,4 = 10.2 Hz, H-3), 3.43 (m,
1H, H-5), 3.34 (dd, 1H, J3,4 = J4,5 = 10.2 Hz, H-4 ),
2.01 (s, 3H, –CH3); 13C NMR (100 MHz, D2O): d
176.01, 76.51 C-1, 73 C-2, 72.62 C-5, 69.34 C-3, 69.34
C-4, 60.48 C-6, 21.96 (–CH3); ESIMS m/z: 244.1
[M+Na]+.

1642
F. Nisic, A. Bernardi / Carbohydrate Research 343 (2008) 1636–1643
4.9. Na-Benzyloxycarbonyl-Nc-a-D-glucopyranosyl-L-
asparagine-O-methyl ester 4d
D2O): d 170.05 (CO), 155.38 (C@CH), 116.87 (CH@C),
81.05 C-1, 78.72 C-4, 75.69 C-3, 75.11 C-2, 69.02 C-5,
63.47 C-6, 26.39 (CH3a), 19.56 (CH3b); ESIMS m/z:
284.1 [M+Na]+.
Purified by flash chromatography (80:20:2 CHCl3–
MeOH–H2O; Rf = 0.35) or automated reverse-phase
chromatography (H2O–CH3CN gradient); procedure
B, yield 35%. [a]D +57.2 (c 0.2, MeOH); 1H NMR
(400 MHz, D2O): d 7.42–7.28 (m, 5H, Ph), 5.48 (d,
1H, J1,2 = 5.6 Hz, H-1), 5.06 (br s, 2H, CH2–O), 4.55
(t, 1H, J = 6.4 Hz, CH–N), 3.72 (dd, 1H,
J2,3 = 5.6 Hz, H-2), 3.54–3.76 (m, 4H, H-3, H-4, H-5,
H-6a), 3.68 (s, 3H, O–CH3), 3.34 (m, 1H, H-6b), 2.88
(d, 2H, J = 6.4 Hz, CH2–CO); 13C NMR (100 MHz,
D2O): d 179.37, 128.77, 128.40, 127.64, 76.64 C-1,
72.98 C-3, 72.61 C-5, 69.24 C-2, 69.13 C-4, 67.26
(CH2–O–), 60.51 C-6, 53.14 (O–Me), 50.83 (–CH–N);
ESIMS m/z: 465.18 [M+Na]+.
4.13. N-Pentanoyl-b-D-glucopyranosylamide 12b
The compound was purified by flash chromatography
(80:20:2 CHCl3–MeOH–H2O); procedure B, yield 70%.
1H NMR (400 MHz, D2O):
d
4.88 (d, 1H,
J1,2 = 9.2 Hz, H-1), 3.70 (dd, 1H, J5,6a = 2.4 Hz,
J6a,6b = 12.4 Hz, H-6a), 3.64 (dd, 1H, J5,6b = 5.2 Hz,
J6a,6b = 12.4 Hz, H-6b), 3.50–3.49 (m, 2H, H-5, H-4),
3.28–3.37 (m, 2H, H-3, H-2), 2.34 (m, 2H, –COCH2–),
1.51 (m, 2H, –CH2–CH2–CH3), 1.33 (m, 2H, –CH2–
CH3), 0.82 (t, 3H, J = 7.5 Hz, CH3); 13C NMR
(100 MHz, D2O): d 178.75, 78.74 C-1, 76.50 C-2, 77.85
C-5, 77.53 C-3, 71.73 C-4, 69.14 C-6, 35.52 (–COCH2–),
27.24 (–CH2–CH2–CH3), 21.55 (–CH2–CH3), 12.97
(CH3); ESIMS m/z: 286.29 [M+Na]+.
4.10. Na-Benzyloxycarbonyl-Nc-a-D-glucopyranosyl-L-
glutamine-O-methyl ester 4e
Purified by flash chromatography (80:20:2 CHCl3–
MeOH–H2O; Rf = 0.33); procedure B, yield 50%. [a]D
+50.4 (c 0.3, MeOH) 1H NMR (400 MHz, D2O): d
7.42–7.28 (m, 5H, Ph), 5.52 (d, 1H, J1,2 = 5.5 Hz, H-
1), 5.10 (s, 2H, CH2–O), 4.20 (q, 1H, J = 5 Hz,
4.14. N-Pentanedioic acid-b-D-glucopyranosylamide
methyl ester 12c
The compound was purified by flash chromatography
(80:20:2 CHCl3–MeOH–H2O); procedure B, yield 75%.
JCH–CH ¼ 9 Hz, CH–N), 3.72 (m, 4H, H-2, O–CH3),
1H NMR (400 MHz, D2O):
d
4.90 (d, 1H,
2
3.54–3.76 (m, 6H, H-3, H-4, H-5, H-6a, H-6b), 2.47 (t,
2H, J = 7 Hz, CH2–CH2–CH), 2.18 (m, 1H, Ha, CH2–
CH), 1.95 (m, 1H, Hb, CH2–CH); ESIMS m/z: 457.3
[M+H]+.
J1,2 = 9.2 Hz, H-1), 3.82 (dd, 1H, J5,6a = 2.4 Hz, H-
6a), 3.68 (m, 1H, H-6b), 3.65 (s, 3H, OCH3), 3.43–3.48
(m, 2H, H-4, H-5), 3.38–3.30 (m, 2H, H-2, H-3), 2.38
(m, 2H, CH2COO–), 2.32 (m, 2H, CH2CON–), 1.88
(m, 2H, CH2–CH2–CH2); ESIMS m/z: 330.3 [M+Na]+.
4.11. N-3-Methyl-2-butenoyl-a-D-glucopyranosylamide 4f
4.15. Na-Benzyloxycarbonyl-Nc-b-D-glucopyranosyl-L-
asparagine-O-methyl ester 12d
The compound was purified by flash chromatography
(80:20:2 CHCl3–MeOH–H2O); procedure A, yield
38%. 1H NMR (400 MHz, D2O): d 5.75 (m, 1H,
CH@C(CH3)2), 5.56 (d, 1H, J1,2 = 4 Hz, H-1) 3.9–3.3
(m, 6H, H-6a, H-6b, H-5, H-4, H-3, H-2), 2.03 (d, 3H,
J = 1.6 Hz, CH3a), 1.88 (d, 3H, J = 1.6 Hz, CH3b);
ESIMS m/z: 284.1 [M+Na]+.
The compound was purified by flash chromatography
(80:20:2 CHCl3–MeOH–H2O); procedure B, yield 32%.
1H NMR (400 MHz, D2O): d 7.45–7.35 (m, 5H, Ph),
5.10 (br s, 2H, CH2–O), 4.88 (d, 1H, J1,2 = 9.2 Hz, H-
1), 4.51 (t, 1H, J = 6.4 Hz, CH–N), 3.70 (s, 3H, O–
CH3), 3.85–3.23 (m, 6H, H-2, H-3, H-4, H-5, H-6a, H-
6b), 2.98 (m, 2H, –CH2); ESIMS m/z: 465.18 [M+Na]+.
4.12. N-3-Methyl-2-butenoyl-a-D-glucofuranosylamide 10
The compound was purified by flash chromatography
(80:20:2 CHCl3–MeOH–H2O, Rf = 0.29); procedure B,
yield 52%. [a]D +71.8 (c 0.1, MeOH); 1H NMR
(400 MHz, D2O): d 5.81 (d, 1H, J1,2 = 3.8 Hz, H-1),
5.77 (m, 1H, CH@C(CH3)2), 4.28 (dd, 1H,
J2,3 = 1.4 Hz, J3,4 = 2.65 Hz, H-3), 4.14 (dd, 1H,
J2,3 = 1.4 Hz, J1,2 = 3.8 Hz, H-2), 4.03 (dd, 1H,
J4,5 = 8.8 Hz, J3,4 = 2.65 Hz, H-4), 3.84 (dd, 1H,
J4,5 = 8.8 Hz, J5,6 = 6.2 Hz, H-5), 3.75 (dd, 1H,
J5,6a = 6.2 Hz, J6a,6b = 12 Hz, H-6a), 3.57 (dd, 1H,
J5,6b = 6.2 Hz, J6a,6b = 12 Hz, H-6b), 2.02 (s, 3H,
CH3a), 1.83 (s, 3H, CH3b); 13C NMR (100 MHz,
4.16. Na-Benzyloxycarbonyl-Nc-b-D-glucopyranosyl-L-
glutamine-O-methyl ester 12e
The compound was purified by flash chromatography
(80:20:2 CHCl3–MeOH–H2O); procedure B, yield 38%.
1H NMR (400 MHz, D2O): d 7.45–7.32 (m, 5H,Ph),
5.10 (s, 2H, CH2–O), 4.91 (d, 1H, J1,2 = 9.2 Hz, H-1),
4.22 (m, 1H, CH–N), 3.70 (s, 3H, OCH3), 3.81 (dd,
1H, J5,6a = 2 Hz, J6a,6b = 14.4 Hz, H-6a), 3.67 (dd, 1H,
J5,6b = 7.2, H-6b), 3.52–3.48 (m, 2H, H-4, H-5), 3.29–
3.40 (m, 2H, H-3, H-2), 2.40 (t, 2H, J = 7 Hz, CH2-
CO), 2.17 (m, 1H, Ha, CH2–CH–N), 1.98 (m, 1H, Hb,

F. Nisic, A. Bernardi / Carbohydrate Research 343 (2008) 1636–1643
1643
CH2–CH–N); 13C NMR (100 MHz, D2O): d 178.37,
176.26, 128.83, 128.46, 127.69, 79.27 C-1, 77.52 C-5,
76.82 C-2, 76.48 C-3, 69.26 C-4, 67.25 (CH2–O–),
60.57 C-6, 53.83 (–CH–), 53.01 (O–Me), 31.76, (CH2–
CO), 26.21 (CH2-CH); ESIMS m/z: 457.3 [M+H]+.
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Acknowledgment
10. Lin, F. L.; Hoyt, H. M.; Van Halbeek, H.; Bergman, R.
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This paper was supported by funds from the
Ministero dell’Universita’ e della Ricerca (PRIN Prot.
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Supplementary data
¨
´
14. Kovacs, L.; Osz, E.; Domokos, V.; Holzer, W.; Gyo¨rgy-
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2008.04.023.
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