N-Alkyl derivatives of 2-amino-2-deoxy-D-glucose

Article abstract of DOI:10.1016/j.carres.2005.05.013

Mono- and di-N-alkylated derivatives of 1,3,4,6-tetra-O-acetyl-2-amino-2- deoxy-β-D-glucose (alkyl = methyl, ethyl, propyl, butyl, pentyl, hexyl, benzyl) were synthesised by the reductive alkylation of per-O-acetyl-d- glucosamine. (N-ethyl, N-propyl, N-bu

Full text of DOI:10.1016/j.carres.2005.05.013

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 1876–1884
Note
N-Alkyl derivatives of 2-amino-2-deoxy-D-glucose
a
a
b
Beata Liberek,a, Anna Melcer, Anna Osuch, Roland Wakiec,
*
´
b
Sławomir Milewski and Andrzej Wisniewski
a
´
a
´
´
Faculty of Chemistry, University of Gdansk, Sobieskiego 18, PL-80-952 Gdansk, Poland
´
´
80-952 Gdansk, Poland
b
Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Narutowicza 11/12,
Received 13 December 2004; received in revised form 2 May 2005; accepted 28 May 2005
Available online 24 June 2005
Abstract—Mono- and di-N-alkylated derivatives of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-b-D-glucose (alkyl = methyl, ethyl,
propyl, butyl, pentyl, hexyl, benzyl) were synthesised by the reductive alkylation of per-O-acetyl-D-glucosamine. (N-ethyl, N-propyl,
N-butyl, N-pentyl and N-hexyl)-1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-b-D-glucoses were deacetylated in order to attempt
an enzymatic phosphorylation. All products were characterised by means of IR, NMR and MS spectra. N-Ethyl- and N-pentyl-D-
glucosamines were found to exhibit weak antifungal activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: D-Glucosamine; Reductive alkylation; Deacetylation; NMR analysis; Enzymatic phosphorylation; Antifungal activity
D-Glucosamine-6-phosphate (GlcN-6-P) synthase, an
enzyme catalysing the first committed step in the chitin
biosynthesis pathway, is a potential target in antifungal
chemotherapy.1 A structural analogue of the transition
state cis-enolamine intermediate, 2-amino-2-deoxy-D-
glucitol-6-phosphate (ADGP) specifically inhibits
GlcN-6-P synthase.2 Unfortunately, despite its strong
enzyme inhibitory potency, this compound exhibits
weak antifungal activity owing to its very slow uptake.3
On the other hand, N-acyl and ester derivatives of
ADGP were found to be muchweaker inhibitors of
GlcN-6-P synthase than the parent compound. Some
of them, however, did exhibit a more pronounced
antifungal activity.4 Our molecular modelling studies
on enzyme–inhibitor interactions have indicated that
introduction of alkyl functional groups at the nitrogen
atom of ADGP should not affect the bonding of the thus
obtained N-alkyl derivatives of ADGP at the enzyme
active centre, in comparison to the parent molecule
(Wojciechowski M., et al., unpublished data). One may
therefore expect that the strong enzyme inhibitory prop-
erties of N-alkyl derivatives of ADGP could be com-
bined with good antifungal activity, because the charge
distribution is similar to and the lipophilicity better than
that of the parent ADGP molecule.
One of the pathways that we examined in order to ob-
tain N-alkyl derivatives of ADGP involved N-alkylation
of 2-amino-2-deoxy-D-glucose followed by an enzymatic
phosphorylation and reduction (Scheme 1). Previously
reported preparations of alkylated D-glucosamine deriv-
atives include the reduction of the amido function,5,6
nucleophilic cleavage of the oxazolidine ring7 or reduc-
tive alkylation.8 None of these N-alkylations satisfies
our needs. On the other hand, reductive alkylation of
amines is one of the convenient methods for secondary
and tertiary amine formation.9–12 In this method, a pri-
mary amine is treated withan aldehyde or a ketone in
the presence of a suitable reducing agent (most often
NaCNBH3). Unfortunately, application of this strategy
to the direct alkylation of 2-amino-2-deoxy-D-glucose
turned out to be impossible, since reduction of the
aldehyde group in the open-chain form of D-glucos-
amine occurred and 2-amino-2-deoxy-D-glucitol was
formed under the reaction conditions (NaCNBH3,
*
Corresponding author. Tel.: +48 58 3450344; fax: +48 58 3410357;
e-mail: beatal@chemik.chem.univ.gda.pl
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.05.013

B. Liberek et al. / Carbohydrate Research 340 (2005) 1876–1884
1877
OH
OH
OPO H
3
2
1. enzymatic
O
O
OH
N-alkylation
phosphorylati on
OH
OH
OH
OH
OH
OH
2. reduction
HO
HO
HO
NH2
NHR
NHR
N-alkyl-D-glucosamine
N-alkyl-ADGP
D-glucosamine
Scheme 1.
CH3CN–H2O ꢀ 3:1.13) To overcome this difficulty a
procedure was applied involving the reductive alkylation
of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-b-D-glucose
followed by deacetylation. NaCNBH3 was chosen as
reducing agent since the reductive alkylation of
1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-b-D-glucose with
other agents [H2/Pd, NaBH4 or NaBH(OAc)3] was
unsuccessful.
1,3,4,6-Tetra-O-acetyl-2-amino-2-deoxy-D-glucose hydro-
chloride (2, Scheme 2) was prepared from 2-amino-
2-deoxy-D-glucose hydrochloride (1) according to the
described procedure.14 Reductive alkylation of 2 withthe
appropriate aldehydes was performed using a twofold
molar excess of aldehyde and a threefold molar excess
of NaCNBH3 withrespect to 2, and was completed
within 30 min. The reaction was not selective under
these conditions and in most cases a mixture of mono-
(3, 5, 7, 9, 11, 12 and 14) and dialkylated products (4,
6, 8, 10 and 13) was obtained. However, reaction of 2
with valeric aldehyde and benzaldehyde yielded only
monoalkylated glucosamines (11 and 14). As expected,
the longer the alkyl residue of an aldehyde, the smaller
the quantity of dialkylated product (37% for 4 and 7%
for 13). Reaction of 2 withbenzaldehyde afforded a mix-
ture of the expected N-benzylamino- (14) but also the
unexpected N-benzylideneamino derivatives (15). Isola-
tion of 15 confirms the formation of an intermediate car-
binol amine, which undergoes dehydration to yield the
imine 15 following reductive alkylation.10 This also pro-
vides evidence for the much greater stability of the aro-
matic imines in comparison with the aliphatic ones.
The structures of the synthesised N-alkyl and N,N-
dialkyl derivatives of 1,3,4,6-tetra-O-acetyl-2-amino-2-
1
deoxy-b-D-glucose were confirmed by IR, H and 13C
NMR spectroscopy (Tables 1–5), as well as mass spec-
trometry. Analysis of the NMR spectra of compounds
3–14 permitted the conclusion that the length of the
alkyl chain does not affect the chemical shifts of the sugar
ring protons or carbons. These are almost identical in
the group of monoalkyl (Tables 1 and 3) or dialkyl
(Tables 2 and 4) derivatives of D-glucosamine. The only
exception can be seen in the NMR spectra of com-
pounds 3 (the smallest alkyl chain) and 14 (a sterically
and electronically different alkyl group). Interestingly,
the number of alkyl groups has a significant influence
on the resonance frequency of the sugar ring protons
1
and carbons. The H and 13C NMR spectra of mono-
and dialkyl compounds, which carry the same alkyl
group, are clearly different. The presence of two alkyl
groups causes deshielding of the H-1 (Dd ꢀ 0.15 ppm),
H-2 (Dd ꢀ 0.1 ppm) and H-3 (Dd ꢀ 0.2 ppm) protons
in comparison with the corresponding protons of the
monoalkyl products. Conversely, the second alkyl group
screens the C-1 (Dd ꢀ 2.5 ppm) and C-3 (Dd ꢀ 2.8 ppm)
OH
OAc
OAc
OAc
O
O
O
O
AcO
AcO
AcO
AcO
b
a
+
OH
OH
OAc
OAc
OAc
AcO
AcO
HO
.
NH
NHCH R
2
N(CH R)
2
HCl
2
2
.
3
5
R = -H
R = -CH
4
6
NH2
HCl
1
2
3
7
9
11
12
14
R = -CH CH
8
10
-
13
-
2
3
OAc
R = -CH CH CH
3
2
2
O
AcO
AcO
R = -CH (CH ) CH
2 2 2 3
OAc
N=CHC H
R = -CH (CH ) CH
3
2
2 3
R = -C H
6
5
6
5
15
c
OH
16 R = -CH
3
a: [14] ; b: RCHO, NaBH CN, MeCN/H O;
O
3
2
HO
17 R = -CH CH
2
3
c: K CO , CH OH / H O
2
3
3
2
18 R = -CH CH CH
3
2
2
HO
OH
19 R = -CH (CH ) CH
3
2
2 2
NHCH R
2
20 R = -CH (CH ) CH
3
2
2 3
Scheme 2.

1878
B. Liberek et al. / Carbohydrate Research 340 (2005) 1876–1884
Table 1. Chemical shifts (ppm) of the protons in the 1H NMR spectra (CDCl3) of 3, 5, 7, 9, 11, 12, 14 and 15
H-1
H-2
H-3
H-4
H-5
H-6
H-60
OAc
NCH2
—
CH2
—
CH3
Ph
—
3
5.88
(d)
3.26
(t)
5.44
(t)
5.06
(t)
3.89
(br d)
1H
4.31
(dd)
1H
4.10
(dd)
1H
2.22
2.12
2.08
2.04
(4s)
2.36
(s)
1H
1H
1H
1H
3H
12H
5
5.55
(d)
2.85
(dd)
1H
5.06
5.06
5.05
5.06
5.06
3.77
(ddd)
1H
4.31
(dd)
1H
4.07
(dd)
1H
2.16
2.08
2.03
(3s)
2.74
(dq)
2.64
(dq)
2H
—
1.00
(t)
—
—
—
—
—
(m)
2H
1H
3H
12H
7
5.55
(d)
2.84
(dd)
1H
3.77
(ddd)
1H
4.31
(dd)
1H
4.07
(dd)
1H
2.16
2.08
2.03
(4s)
2.65
(dt)
2.57
(dt)
2H
1.38
(sex)
2H
0.86
(t)
(m)
2H
1H
3H
12H
9
5.54
(d)
2.84
(dd)
1H
3.77
(ddd)
1H
4.30
(dd)
1H
4.07
(dd)
1H
2.16
2.08
2.03
(4s)
2.64
(dt)
2.59
(dt)
2H
1.31
(m)
4H
0.88
(t)
(m)
2H
1H
3H
12H
11
12
14
5.55
(d)
2.84
(dd)
1H
3.77
(ddd)
1H
4.31
(dd)
1H
4.07
(dd)
1H
2.16
2.08
2.03
(3s)
2.67
(dt)
2.58
(dt)
2H
1.31
(m)
6H
0.88
(t)
(m)
2H
1H
3H
12H
5.54
(d)
2.84
(dd)
1H
3.77
(ddd)
1H
4.31
(dd)
1H
4.07
(dd)
1H
2.16
2.08
2.03
(4s)
2.67
(dt)
2.59
(dt)
2H
1.30
(m)
8H
0.88
(t)
(m)
2H
1H
3H
12H
5.60
(d)
2.94
(dd)
1H
5.09
(t)
5.04
(t)
3.78
(ddd)
1H
4.31
(dd)
1H
4.07
(dd)
1H
2.14
2.08
2.03
2.01
(4s)
3.89
(d)
—
—
—
7.20
(m)
5H
1H
1H
1H
3.79
(d)
2H
12H
15
5.97
(d)
3.50
(dd)
1H
5.46
(t)
5.16
(t)
3.99
(ddd)
1H
4.40
(dd)
1H
4.14
(dd)
1H
2.11
2.05
2.03
1.89
(4s)
8.23
(s)
—
7.72
(d)
1H
1H
1H
1H
2H
7.44
(m)
3H
12H
carbon atoms and deshields carbon atom C-2
(Dd ꢀ 2 ppm) in comparison withteh corresponding
carbons of the monoalkyl derivatives.
fore, the signal of each proton is a doublet of quartets (5
and 6), a doublet of triplets (7–13) or a doublet (14).
In order to obtain monoalkyl derivatives of D-glucos-
amine, 1,3,4,6-tetra-O-acetyl-2-(N-alkylamino)-2-deoxy-
b-D-glucoses (5, 7, 9, 11 and 12) were subjected to
de-O-acetylation. Sucha reaction is difficult in the case
of D-glucosamine derivatives, since acetyl groups readily
migrate to the nitrogen atom and tend to stay there.
Additionally, methyl acetate, one of the products of
deprotection carried out in methanol solution, may react
Neither the length nor the number of alkyl groups has
any influence on the conformation of the alkylated
products. All of them (3–14), as well as by-product 15,
4
adopt the C1 form, as demonstrated by the respective
coupling constants (Table 5).
It is worth noting that the protons of the methylene
group bound to the nitrogen atom in N-alkyl derivatives
of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-b-D-glucose
are diastereotopic. Thus, they absorb at different fields
and are coupled withgeminal coupling constant. There-
withthe amino group of
D-glucosamine. Thus, after
deacetylation, a mixture of products was always
obtained, irrespective of whether the reaction was

B. Liberek et al. / Carbohydrate Research 340 (2005) 1876–1884
1879
Table 2. Chemical shifts (ppm) of the protons in the 1H NMR spectra (CDCl3) of 4, 6, 8, 10 and 13
H-1
H-2
H-3
H-4
H-5
H-6
H-60
OAc
NCH2
—
CH2
—
CH3
4
5.75
(d)
2.82
(dd)
1H
5.25
(dd)
1H
5.04
(t)
3.72
(ddd)
1H
4.29
(dd)
1H
4.06
(dd)
1H
2.16
2.08
2.05
2.03
(4s)
2.38
(s)
1H
1H
6H
12H
6
5.74
(d)
2.95
(dd)
1H
5.23
(dd)
1H
5.05
(t)
3.72
(ddd)
1H
4.30
(dd)
1H
4.06
(dd)
1H
2.14
2.08
2.04
2.03
(4s)
2.64
(dq)
2.55
(dq)
4H
—
0.96
(t)
1H
1H
6H
12H
8
5.72
(d)
2.97
(dd)
1H
5.25
(dd)
1H
5.03
(t)
3.73
(ddd)
1H
4.31
(dd)
1H
4.05
(dd)
1H
2.15
2.08
2.04
2.02
(4s)
2.52
(dt)
2.46
(dt)
4H
1.36
(sex)
4H
0.82
(t)
1H
1H
6H
12H
10
13
5.70
(d)
2.97
(dd)
1H
5.24
(dd)
1H
5.03
(t)
3.73
(ddd)
1H
4.31
(dd)
1H
4.06
(dd)
1H
2.14
2.08
2.03
2.02
(4s)
2.55
(dt)
2.48
(dt)
4H
1.28
(m)
8H
0.89
(t)
1H
1H
6H
12H
5.70
(d)
2.98
(dd)
1H
5.24
(t)
5.03
(t)
3.73
(ddd)
1H
4.31
(dd)
1H
4.05
(dd)
1H
2.14
2.08
2.03
2.02
(3s)
2.54
(dt)
2.48
(dt)
4H
1.28
(m)
0.88
(t)
1H
1H
1H
16H
6H
12H
carried out in NaOMe in methanol, (CH3)2NH in meth-
anol or K2CO3 in methanol–water solution.15 Applying
potassium carbonate in the de-O-acetylation reaction
enabled the desired N-alkyl-D-glucosamines withteh
best yields to be obtained within 30 min. Prolonging
the deacetylation time led to the formation of more
byproducts.
absorption at lower fields of axially oriented H-2 and
H-3 protons in comparison withthe corresponding
b
anomers (Dd ꢀ 0.4 ppm). The C-1 carbon atom of the
b anomers resonates at lower fields than the C-1 carbon
atom of the a anomers (Dd ꢀ 7 ppm). Moreover, the C-2
carbon atom of the b anomers is deshielded in compar-
ison with the corresponding C-2 carbon atom of the a
anomers (Dd ꢀ 3 ppm).
2-(N-Alkylamino)-2-deoxy-D-glucoses were obtained
as mixtures of anomers, in which the thermodynamically
more stable a stereoisomer predominated. Their struc-
The protons of the methylene group bound to the
nitrogen atom in N-alkyl-D-glucosamines (16–20) are
diastereotopic, as in the O-acetyl-N-alkyl-D-glucosam-
ines (4–14). However, in the case of 18–20, they behave
as if they were chemically and magnetically equivalent
and appear in one position as triplets.
1
tures were established on the basis of IR, H and 13C
NMR spectroscopy (Tables 6–8) and mass spectrome-
try. The a:b ratio was determined by comparison of
the areas of the H-1 peaks of the respective anomers
in the 1H NMR spectra. All the N-alkyl-D-glucosamines
(16–20) adopt the 4C1 conformation, as indicated by the
relevant coupling constants (Table 8).
Analysis of the NMR spectra of 16–20 drew our
attention to the empirical rules in the NMR spectro-
scopy of carbohydrates. Firstly, the H-1 signal of a ano-
mers appears at higher d values than the H-1 signal of b
anomers, owing to their different equatorial and axial
orientations (Dd ꢀ 0.7 ppm). Next, the axial orientation
of the anomeric OH group in the a anomers causes
Systematic attempts were made to carry out the enzy-
matic phosphorylation of 2-(N-alkylamino)-2-deoxy-D-
glucoses (16–20) in order to obtain N-alkyl-D-gluco-
samine-6-phosphates. Unfortunately, none of the expected
products were formed during incubation of com-
pounds 16–20 withATP in teh presence of yeast
hexokinase. Under the same conditions, D-glucosamine
and D-mannosamine were efficiently phosphorylated by
the enzyme but N-acetyl-D-glucosamine was not. It is
known that yeast hexokinase has a relatively broad

1880
B. Liberek et al. / Carbohydrate Research 340 (2005) 1876–1884
Table 3. Chemical shifts (ppm) of the carbon atoms in the 13C NMR spectra (CDCl3) of 3, 5, 7, 9, 11, 12, 14 and 15
C-1
C-2
C-3
C-4
C-5
C-6
OAc
NCH2/N@C
CH2
—
CH3
Ph
—
C@O
CH3
3
90.49
57.66
70.95
68.20
72.71
61.46
171.25
170.78
169.80
169.20
21.17
21.10
20.90
20.77
—
41.79
5
95.00
94.95
95.00
95.00
95.00
95.34
93.26
60.91
61.10
61.16
61.14
61.14
60.33
74.14
74.08
74.12
74.10
74.10
74.15
68.52
68.55
68.52
68.54
68.53
68.42
68.18
72.66
72.67
72.66
72.67
72.67
72.68
72.99
61.99
61.99
61.99
61.99
61.99
61.93
61.99
171.07
170.92
169.90
169.40
21.29
21.01
20.97
20.88
42.63
50.11
47.92
48.23
48.25
52.42
165.36
—
16.10
11.63
14.06
14.25
14.26
—
—
—
—
—
—
7
171.04
170.92
169.90
169.39
21.28
21.01
20.97
20.87
23.77
9
171.02
170.91
169.90
169.39
21.27
20.98
20.96
20.86
32.83
20.19
11
12
14
15
171.02
170.92
169.91
169.39
21.29
21.00
20.98
20.88
30.45
29.31
22.70
171.02
170.92
169.91
169.39
21.29
21.01
20.98
20.88
31.86
30.73
26.82
22.84
171.15
170.92
169.86
169.37
21.29
20.98
20.86
31.86
30.73
26.82
22.84
128.70
128.25
127.41
73.30
73.18
170.93
170.13
169.76
168.97
21.03
21.00
20.91
20.73
—
—
135.51
131.76
128.91
128.79
spectrum of substrate specificity,16 although D-glucose is
a primary substrate of this enzyme. Our studies show
that hexokinase-catalysed phosphorylation of N-substi-
tuted D-glucosamines is not possible. On looking for
the most likely explanation for this, it should be noted
that the charge status of the amino groups in D-glucos-
amine and N-alkyl-D-glucosamines is the same. One
may therefore assume that an additional alkyl group
in 16–20 introduces a steric hindrance and in this way
precludes the desired substrate–enzyme interactions.
Additionally, substitution of a proton by an alkyl group
in the amino group of 16–20 may result in the lack of
proper H-bonding between substrate and enzyme.
All N-alkyl-D-glucosamines were tested for in vitro
antifungal activity using the microtitre serial dilution
method. N-Ethyl- (16) and N-pentyl-D-glucosamines
(19) were found to inhibit the growth of S. cerevisiae
ATCC 9763 and C. albicans ATCC 10261 cells at
10 mg/mL. No growth inhibition was found with the
other compounds or with the commercially available
D-glucosamine. The mechanism of the observed
antifungal effect is to be the subject of our further
studies.
1. Experimental
1.1. General methods
Melting points were uncorrected. The IR spectra were
recorded as Nujol mulls witha Bruker IFS 66 spectro-
photometer. The 1H and 13C NMR spectra (CDCl3,
DMSO or CD3OD, internal Me4Si) were measured with
a Varian Mercury 400 (400.49/100.70 MHz) instrument.
Positive-ion mode MALDITOF mass spectra were
obtained using a Bruker Biflex III spectrometer with
4-cyano-4-hydroxycinnamic matrix. Thin-layer chromato-
graphy (TLC) was performed on the E. Merck Kieselgel
60 F-254 plates using the following eluent systems (v/v):
A, 1:2 toluene–AcOEt; B, 3:1 CHCl3–MeOH; C, 1:1

B. Liberek et al. / Carbohydrate Research 340 (2005) 1876–1884
1881
Table 4. Chemical shifts (ppm) of the carbon atoms in the 13C NMR spectra (CDCl3) of 4, 6, 8, 10 and 13
C-1
C-2
C-3
C-4
C-5
C-6
OAc
NCH2
—
CH2
—
CH3
C@O
CH3
4
92.03
66.16
70.82
69.10
72.60
62.06
170.94
170.37
169.96
169.17
170.94
170.24
169.95
169.14
170.97
170.29
170.02
169.25
170.97
170.25
170.02
169.21
170.96
170.22
170.02
169.19
21.49
21.20
20.96
20.87
21.35
21.10
20.98
20.90
21.37
21.15
20.98
20.88
21.35
21.10
20.97
20.88
21.35
21.12
20.95
20.86
41.77
14.99
11.64
14.27
14.27
6
92.60
92.52
92.57
92.54
62.91
63.30
63.24
63.18
71.18
71.34
71.37
71.34
69.12
69.28
69.36
69.33
72.63
72.43
72.47
72.43
62.06
62.00
62.06
62.03
42.59
53.21
51.07
51.34
—
8
22.61
10
13
31.76
20.34
32.03
29.54
26.95
22.93
Table 5. The 1H–1H coupling constants (Hz) for compounds 3–15
Sugar
Alkyl
0
0
J1,2
J2,3
9.6
J3,4
J4,5
J5,6
J5,6
J6,6
Jgem
Jvic
a
3
8.8
8.8
8.4
8.8
8.4
8.8
8.8
8.8
8.8
8.8
8.8
8.8
8.4
9.6
9.2
9.6
10.0
9.6
4.0
4.8
4.8
4.8
4.8
4.4
4.8
4.8
4.4
4.8
4.4
4.4
4.4
12.4
12.4
12.4
12.4
12.4
12.4
12.4
12.4
12.4
12.4
12.4
12.4
12.4
—
—
—
—
4
10.8
10.4
10.8
10.0
10.8
10.4
10.4
10.4
10.4
10.8
10.0
9.6
2.0
2.0
2.4
2.0
2.0
2.0
2.0
2.0
2.4
2.0
2.0
2.0
a
5
11.6
13.6
11.6
12.8
11.6
13.3
11.6
11.6
13.6
13.6
—
7.0
6.8
6.8
7.2
6.8
6.6
6.8
6.8
6.8
—
6
9.2
a
10.0
a
7
8
9.2
a
10.4
10.0
9
10
11
12
13
14
15
9.2
a
10.4
a
a
10.0
10.4
9.6
9.2
9.2
9.6
10.0
—
a Not determined.
CHCl3–MeOH, D, 5:3:1:1 i-ProOH-25% NH3aq–H2O–
AcOH. Column chromatography was performed on
MN Kieselgel 60 (<0.08 mm).
OAc); 13C NMR (DMSO): d 169.88, 169.69, 169.24,
168.56 (4C@O), 90.05 (C-1), 71.56 (C-5), 70.29 (C-3),
67.79 (C-4), 61.22 (C-6), 52.11 (C-2), 20.88, 20.79,
20.41, 20.27 (4COCH3).
1.2. 1,3,4,6-Tetra-O-acetyl-2-amino-2-deoxy-b-D-glucose
HCl (2)
1.3. General procedure for N-alkylation
Prepared according to the literature procedure.14 Spec-
tral data for 2: IR: m 3435 (N–H), 2924, 2853 (C–H),
1760, 1747 (ester C@O), 1368 (ester CH3), 1248, 1208
1,3,4,6-Tetra-O-acetyl-2-amino-2-deoxy-b-D-glucose HCl
(2) (500 mg, 1.3 mmol) and appropriate aldehyde
(2.6 mmol) were stirred in the mixture of acetonitrile
and water (3:1, 56 mL) at rt for 30 min. Then NaCNBH3
(240 mg, 3.8 mmol) was added and stirring was contin-
ued for 30 min at rt. The end of the reaction was
detected by TLC (solvent A). The mixture was diluted
(ester C–O) cmꢁ1 1H NMR (DMSO): d 8.87 (br s,
;
3H, NH3+), 5.93 (d, 1H, J1,2 8.8 Hz, H-1), 5.37 (dd,
1H, J3,4 9.6 Hz, H-3), 4.93 (t, 1H, J4,5 9.6 Hz, H-4),
0
4.19 (dd, 1H, J6,6 12.8 Hz, H-6), 4.03 (ddd, 1H, J5,6
0
4.8, J5,6 2.4 Hz, H-5), 4.00 (dd, 1H, H-60), 3.54 (dd,
1H, J2,3 10.0 Hz, H-2), 2.17, 2.03, 1.99, 1.97 (4s, 12H,
withwater (30 mL) and extracted withCHCl
(3 · 50 mL). The CHCl3 extract was dried over Na2SO4,
3

1882
B. Liberek et al. / Carbohydrate Research 340 (2005) 1876–1884
Table 6. Chemical shifts (ppm) of the protons in the 1H NMR spectra (CD3OD) of 16–20
H-1a
H-1b
H-2a
H-2b
H-3a
H-3b
H-4a
NCH2a
NCH2b
CH2a
—
CH3a
CH3b
a
16
5.32
(d)
4.57
(d)
2.79
(dd)
1H
2.39
(dd)
1H
3.72
(m)
1H
3.30
(m)
1H
2.97
(dq)
2.94
(dq)
2H
2.90
(m)
2H
1.22
(t)
1.15
(t)
1H
1H
3H
3H
17
18
5.40
(d)
4.70
(d)
2.99
(dd)
1H
2.56
(dd)
1H
3.84
(dd)
1H
3.45
(dd)
1H
3.37
(t)
3.01
(m)
2H
2.98
(m)
2H
1.69
(m)
2H
1.00
(t)
0.98
(t)
1H
1H
1H
3H
3H
a
a
a
5.35
(d)
4.60
(d)
2.85
(dd)
1H
2.42
(dd)
1H
3.77
(m)
1H
3.37
(t)
3.38
(t)
2.96
(t)
1.62
(m)
2H
0.97
(t)
0.92
(t)
1H
1H
1H
1H
2H
3H
3H
1.41
(sex)
2H
a
a
19
20
5.34
(d)
4.60
(d)
2.86
(dd)
1H
2.42
(dd)
1H
3.76
(m)
1H
3.35
(t)
2.94
(t)
1.59
(m)
2H
0.94
(t)
1H
1H
1H
2H
3H
1.37
(m)
4H
a
a
5.37
(d)
4.65
(d)
2.93
(dd)
1H
2.48
(dd)
1H
3.81
(t)
3.36
(t)
2.99
(t)
1.63
(m)
2H
0.92
(t)
1H
1H
1H
1H
2H
3H
1.35
(m)
6H
a Not determined.
Table 7. Chemical shifts (ppm) of the carbon atoms in the 13C NMR spectra (CD3OD) of 16–20
C-1
C-2
C-3, C-4, C-5
73.20–72.26
C-6
NCH
CH2
—
CH3
16
17
18
91.08 (a)
98.48 (b)
62.77 (a)
62.70 (a)
42.64 (a)
44.52 (b)
13.64 (a)
14.99 (b)
90.21 (a)
96.88 (b)
62.35 (a)
73.27–71.97
73.24–72.20
62.49 (a)
48.96 (a)
49.09 (b)
21.32
11.54 (a)
11.72 (b)
90.71 (a)
97.98 (b)
62.78 (a)
65.75 (b)
62.61 (a)
62.96 (b)
47.22 (a)
30.77 (a)
32.31 (b)
21.25 (a)
21.36 (b)
14.15 (a)
14.32 (b)
19
20
90.73 (a)
62.78 (a)
73.25–72.22
73.26–72.14
62.60 (a)
62.98 (b)
47.85 (a)
47.76 (a)
30.25 (a)
28.44 (a)
23.50 (a)
14.34 (a)
14.42 (a)
90.46 (a)
97.46 (b)
62.58 (a)
65.56 (b)
62.54 (a)
62.90 (b)
32.67 (a)
32.85 (b)
28.26 (a)
29.72 (b)
27.69 (a)
27.85 (b)
23.66 (a)
23.97 (b)
filtered off and evaporated. The residue was chromato-
graphed on silica gel (solvent A).
two products. First was 3 (6% syrup); Rf 0.16 (solvent
A); IR: m 3352 (N–H), 2927 (C–H), 1753 (ester C@O),
1370 (ester CH3), 1222 (ester C–O) cmꢁ1. MALDI-
TOF-MS: m/z 362.3 (M+H)+.
Second was 4 (37% white solid); Rf 0.47 (solvent A); IR:
m 2940, 2876, 2792 (C–H), 1750 (ester C@O), 1368 (ester
1.3.1. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-methylamino- (3)
and -2-dimethylamino-b-D-glucoses (4). Reductive
alkylation of 2 withmethanal (37% water solution) gave

B. Liberek et al. / Carbohydrate Research 340 (2005) 1876–1884
1883
Table 8. The 1H–1H coupling constants (Hz) for compounds 16–20
a Anomer
b Anomer
Alkyl
J1,2
J2,3
J3,4
J4,5
J1,2
J2,3
J3,4
Jgem
Jvic
a
a
a
a
a
16
17
18
19
20
3.6
3.6
3.6
3.2
3.2
10.0
9.6
8.0
8.4
8.4
8.0
8.0
9.2
10.0
9.6
11.4
a
7.2
7.2
7.2
7.2
7.2
10.4
a
a
a
a
10.0
10.4
10.4
9.6
9.6
9.6
a
a
10.0
a
a
9.6
9.6
a Not determined.
CH3), 1224 (ester C–O) cmꢁ1. MALDITOF-MS: m/z
1.3.6. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-hexylamino- (12)
and -2-dihexylamino-b-D-glucoses (13). Alkylation of 2
withhexanal gave two products. First was 12 (61% syr-
up); Rf 0.64 (solvent A); IR: m 3353 (N–H), 2957, 2930,
2858 (C–H), 1753 (ester C@O), 1368 (ester CH3), 1224
376.0 (M+H)+.
1.3.2. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-ethylamino- (5)
and -2-diethylamino-b-D-glucoses (6). Alkylation of 2
withethanal yielded two products. First was 5 (68% syr-
up); Rf 0.31 (solvent A); IR: m 3354 (N–H), 2969 (C–H),
1752 (ester C@O), 1368 (ester CH3), 1224 (ester C–O)
cmꢁ1. MALDITOF-MS: m/z 376.3 (M+H)+, 398.3
(M+Na)+, 414.3 (M+K)+.
Second was 6 (14% syrup); Rf 0.80 (solvent A); IR: m
2970, 2936, 2873 (C–H), 1752 (ester C@O), 1368 (ester
CH3), 1224 (ester C–O) cmꢁ1. MALDITOF-MS: m/z
404.0 (M+H)+, 425.9 (M+Na)+, 441.9 (M+K)+.
(ester C–O) cmꢁ1
.
MALDITOF-MS: m/z 432.0
(M+H)+, 454.0 (M+Na)+, 470.0 (M+K)+.
Second was 13 (7% syrup); Rf 0.86 (solvent A); IR: m
2956, 2930, 2859 (C–H), 1755 (ester C@O), 1367 (ester
CH3), 1224 (ester C–O) cmꢁ1. MALDITOF-MS: m/z
516.4 (M+H)+, 538.4 (M+Na)+, 554.4 (M+K)+.
1.3.7. 1,3,4,6-Tetra-O-acetyl-2-benzylamino- (14) and -2-
benzylideneamino-2-deoxy-b-D-glucoses (15). Alkylation
of 2 withbenzaldehyde yielded two products. First was
14 (16% syrup); Rf 0.59 (solvent A); IR: m 1751 (ester
C@O), 1367 (ester CH3), 1222 (ester C–O), 746, 701
(C6H5) cmꢁ1. MALDITOF-MS: m/z 438.0 (M+H)+,
460.0 (M+Na)+, 475.9 (M+K)+.
1.3.3. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-propylamino- (7)
and -2-dipropylamino-b-D-glucoses (8). Alkylation of 2
withpropanal led to two products. First was 7 (74% syr-
up); Rf 0.47 (solvent A); IR: m 3354 (N–H), 2961, 2936,
2875 (C–H), 1752 (ester C@O), 1368 (ester CH3), 1224
Second was 15 (21% white solid); Rf 0.69 (solvent A);
IR: m 1755 (ester C@O), 1646 (C@N), 1367 (ester CH3),
(ester C–O) cmꢁ1
.
MALDITOF-MS: m/z 390.3
(M+H)+, 412.3 (M+Na)+, 428.2 (M+K)+.
1221 (ester C–O), 759, 696 (C6H5) cmꢁ1
.
Second was 8 (9% syrup); Rf 0.81 (solvent A); IR: m
2961, 2935, 2874 (C–H), 1753 (ester C@O), 1368 (ester
CH3), 1224 (ester C–O) cmꢁ1. MALDITOF-MS: m/z
432.4 (M+H)+, 455.4 (M+Na)+, 471.4 (M+K)+.
1.4. General procedure for deacetylation
A mixture of appropriate 1,3,4,6-tetra-O-acetyl-2-(N-
alkylamino)-2-deoxy-b-D-glucose (1 mmol) and K2CO3
(552 mg, 4 mmol) in MeOH–H2O (5:2) solution
(35 mL) was stirred at rt for 30 min. The end of deacet-
ylation was detected by TLC (solvent B). Then, the reac-
tion mixture was neutralized withMeOH saturated with
HCl and concentrated under vacuum. The crude prod-
ucts were chromatographed on silica gel (solvent first
B, next C).
1.3.4. 1,3,4,6-Tetra-O-acetyl-2-butylamino- (9) and -2-
dibutylamino-2-deoxy-b-D-glucoses (10). Alkylation of
2 withbutanal yielded two products. First was 9 (59%
syrup); Rf 0.46 (solvent A); IR: m 3353 (N–H), 2958,
2933, 2873 (C–H), 1752 (ester C@O), 1368 (ester
CH3), 1223 (ester C–O) cmꢁ1. MALDITOF-MS: m/z
404.2 (M+H)+, 426.2 (M+Na)+, 442.2 (M+K)+.
Second was 10 (8% syrup); Rf 0.78 (solvent A); IR: m
2958, 2933, 2872 (C–H), 1753 (ester C@O), 1367 (ester
CH3), 1223 (ester C–O) cmꢁ1. MALDITOF-MS: m/z
460.4 (M+H)+, 482.4 (M+Na)+, 498.4 (M+K)+.
1.4.1. 2-Deoxy-2-ethylamino-D-glucose (16). Deacetyl-
ation of 5 gave 16 (58% syrup, a:b ꢀ 2:1); Rf 0.05 (sol-
vent B); IR: m 3429 (O–H), 2926 (C–H), 1028 (alcohol
C–O) cmꢁ1. MALDITOF-MS: m/z 208.2 (M+H)+.
1.3.5. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-pentylamino-b-D-
glucose (11). Alkylation of 2 withpentanal yielded 11
(57% syrup); Rf 0.55 (solvent A); IR: m 3352 (N–H),
2957, 2932, 2860 (C–H), 1753 (ester C@O), 1367 (ester
CH3), 1224 (ester C–O) cmꢁ1. MALDITOF-MS: m/z
418.1 (M+H)+.
1.4.2. 2-Deoxy-2-propylamino-D-glucose (17). Deacetyl-
ation of 7 yielded 17 (68% syrup, a:b ꢀ 1.3:1); Rf 0.09
(solvent B); IR: m 3439 (O–H), 2922, 2853 (C–H), 1066,
1030 (alcohol C–O) cmꢁ1. MALDITOF-MS: m/z 222.2
(M+H)+, 244.1 (M+Na)+.

1884
B. Liberek et al. / Carbohydrate Research 340 (2005) 1876–1884
1.4.3. 2-Deoxy-2-butylamino-D-glucose (18). Deacetyl-
ation of 9 led to 18 (58% white solid, a:b ꢀ 2.7:1); Rf
0.11 (solvent B); IR: m 3267 (O–H), 2924, 2856 (C–H),
1040 (alcohol C–O) cmꢁ1. MALDITOF-MS: m/z 236.1
(M+H)+.
Acknowledgements
This research was supported by the Polish State Com-
mittee for Scientific Researchunder Grants DS/8361-4-
0134-5 and BW/8000-5-0200-4.
1.4.4. 2-Deoxy-2-pentylamino-D-glucose (19). Deacetyl-
ation of 11 gave 19 (40% white solid, a:b ꢀ 2.4:1); Rf
0.10 (solvent B); IR: m 3277 (O–H), 2957, 2925, 2855
(C–H), 1072 (alcohol C–O) cmꢁ1. MALDITOF-MS:
m/z 250.0 (M+H)+.
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