Synthesis and antimicrobial activities of two novel amino sugars derived from chloraloses

Article abstract of DOI:10.1016/j.carres.2010.03.043

The synthesis of 5-amino-5-deoxy-1,2-O-(S)-trichloroethylidene-β-l-arabinofuranose and 6-amino-6-deoxy-1,2-O-(S)-trichloroethylidene-α-d-glucofuranose is described by a simple three- or four-step route. Antibacterial potency of the new compounds was determined using an inhibition zone diameter test. The results show that these compounds have a broad-spectrum activity against Gram-positive, Gram-negative bacteria and Candida albicans.

Full text of DOI:10.1016/j.carres.2010.03.043

Carbohydrate Research 345 (2010) 1617–1621
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis and antimicrobial activities of two novel amino sugars derived
from chloraloses
a
a
b
c
Nilgün Yenil a, , Emriye Ay , Kadir Ay , Mustafa Oskay , Jacques Maddaluno
*
a Department of Chemistry, Sciences and Arts Faculty, Celal Bayar University, Muradiye, 45030 Manisa, Turkey
b Department of Biology, Sciences and Arts Faculty, Celal Bayar University, Muradiye, 45030 Manisa, Turkey
c IRCOF, CNRS UMR 6014 & FR 3038, Université de Rouen et INSA de Rouen, 76821-Mont St Aignan Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 March 2010
Received in revised form 25 March 2010
Accepted 31 March 2010
Available online 4 April 2010
The synthesis of 5-amino-5-deoxy-1,2-O-(S)-trichloroethylidene-b-
L-arabinofuranose and 6-amino-6-
deoxy-1,2-O-(S)-trichloroethylidene- -glucofuranose is described by a simple three- or four-step route.
a-D
Antibacterial potency of the new compounds was determined using an inhibition zone diameter test. The
results show that these compounds have a broad-spectrum activity against Gram-positive, Gram-nega-
tive bacteria and Candida albicans.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Amino sugars
Antimicrobial activity
Chloraloses
Trichloroethylidene acetals
Carbohydrates and their derivatives are important synthetic
targets because of their wide range of functions in living organ-
isms.1 These derivatives bearing an amino substituent in different
ring positions on the sugar skeleton are called amino sugars2 and
are known constituents of several bio-active compounds such as
antibiotics3 and biopolymers.4 On the cell surface, amino sugars
play a key role as receptors for proteins and enzymes,5 and they
have been shown to interact with either RNA or the backbone
phosphate of DNA.6
a
-chloralose, has found applications as a surgical anesthetic, seda-
tive, and as hypnotic drug.11 Also, 1,2-O-(S)-trichloroethylidene-
a
-
D
-arabinofuranose, namely arabinochloralose, has been used for
the development of new antituberculosis drugs.12 In this paper,
we report the synthesis of novel amino sugar derivatives based
on 1,2-O-(S)-trichloroethylidene-b-
(S)-trichloroethylidene- -glucofuranose.
The synthesis of amino sugars and their derivatives is well
documented in the literature13 but there are no examples of the
family of chloraloses and their amino derivatives. We first aimed
at a simple synthesis of the 5- or 6-amino derivatives of 1,2-O-
L-arabinofuranose and 1,2-O-
a
-D
All this explains that certain series of amino sugars are regarded
as major target molecules for many years, owing to their biological
and medicinal importance.7
(S)-trichloroethylidene-b-
L-arabinofuranose (b-L-arabinochloralose)
Acetals and ketals are common protecting groups for diols, and
find numerous applications in sugar chemistry. Trichloroethylid-
ene acetal rings, known as chloraloses, can be obtained via the
reaction of sugars, preferably in their furanose form, with chloral.
These acetals have not been widely used as protecting groups in
carbohydrate chemistry,8 notwithstanding they present some real
advantages for further modifications of the sugar compounds. First,
trichloroethylidene acetals are highly crystalline compounds and
very stable under acidic and mildly basic conditions. Next, these
acetals can be easily deprotected by hydrogenation on Raney nick-
el, followed by acidic hydrolysis.9 It is also known that trichloroe-
thylidene acetals have different activities in biological systems; for
and 1,2-O-(S)-trichloroethylidene-a-D
-glucofuranose (b-chloral-
ose) from their intermediate mono-azides, obtained in relatively
good yields. These target molecules are available from a simple
sequence involving protection of primary hydroxyl groups, nucleo-
philic substitution and reduction reactions. Under the action of cat-
alytic concd H2SO4,
O-trichloroethylidene acetal of
Makinabakan and co-workers.14 b-
thesized by using this method.8a 1,2-O-(S)-Trichloroethylidene-
D
-arabinose reacts with chloral to yield the 1,2-
-arabinofuranose as described by
-Arabinochloralose 1 was syn-
D
L
a-
D
-arabinofuranose (a-D-arabinochloralose), reported earlier in the
literature14 was also re-synthesized by us in order to make a com-
parison and determination of the acetal carbon configuration of
instance 1,2-O-(R)-trichloroethylidene-
a-
D-glucofuranose, namely
compound 1. The acetal carbon signals of b-L- and a-D-arabinochl-
oraloses resonate at relatively lower fields such as 5.67 and
5.76 ppm in DMSO-d6, respectively, when compared with the dia-
stereoisomeric analogues of 1,2-O-trichloroethylidene derivatives
* Corresponding author. Tel.: +90 236 2412151/2551; fax: +90 236 2412158.
E-mail address: nilgun.yenil@bayar.edu.tr (N. Yenil).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.03.043

1618
N. Yenil et al. / Carbohydrate Research 345 (2010) 1617–1621
R configuration of acetal carbons, which are in the range of d 5.30–
5.50. Based on this strong evidence, and the relatively low field of
acetal carbon signal (d 5.50–6.00), we can confidently assume that
the acetal carbon configuration of 1 is (S)8a,9,10 (Scheme 1). Initially,
compound 1 was acetylated with acetic anhydride in pyridine to
get its diacetylated form (2) that has chemical shift values at rela-
tively low fields as expected from O-acetyl substitution.
Compound 1 was next reacted with p-toluenesulfonyl chloride
in pyridine and the resulting derivative was treated with sodium
azide to afford the corresponding 5-azido-5-deoxy-1,2-(S)-O-tri-
against test microorganisms stated in Table 1. The antimicrobial
activity was evaluated by measuring the inhibition zone diameter
observed. In addition, commercial antibiotics such as nalidixic acid
(30 lg), chloramphenicol (30 lg), and nystatin (10 lg) were used
as positive controls to determine the sensitivity of the strains. It
is obvious that our synthesized compounds showed significant
activity against the tested microorganisms with inhibition zones
ranging from 12 to 24 mm. However, the compounds differ signif-
icantly in their activity against test microorganisms. The most ac-
tive compound was 5 that showed broad-spectrum antimicrobial
activity against Escherichia coli, Bacillus cereus, Enterococcus faecalis,
and Candida albicans 22, 22, 24, and 14 mm, respectively (Table 1).
Significant antimicrobial effects, expressed as MIC of tested
compounds against selected microorganisms are presented in Ta-
ble 2. MIC values of the compounds ranging from 187 to
chloroethylidene-b-L-arabinofuranose 4, in 65% yield. The reduc-
tion of 4 was more troublesome. Neither LiAlH4 nor NaBH4 can
be used for the reduction of such sugars: the trichloroethylidene
acetal ring turns into a ketene acetal due to the elimination of
HCl.14a Milder reduction methods such as Staudinger15 or catalytic
Zn/NH4Cl in acidic media16 proved inefficient in the case of this
derivative. Finally, using dry methanol instead of the tetrahydrofu-
ran–water (4:1) generally employed in Staudinger’s method,
turned out to be the best route.17 Thus, refluxing compound 4 with
1.5 equiv triphenylphosphine in methanol for 2 h afforded the ex-
6670
feacalis with the best MIC (187
lococcus aureus was obtained with 5 and was 1458
the highest MIC was 6670 g/mL for C. albicans by the compound
10. MIC values for E. coli from 5 and 10 were 2083 and 4167 g/
mL, respectively. The results of the present investigation clearly
indicate that the compounds of b- -arabinochloralose and b-chlo-
l
g/mL. Compound 5 showed very strong activity against E.
g/mL). The lowest MIC for Staphy-
g/mL, whereas
l
l
l
l
pected amino derivative of b-L
-arabinochloralose 5.18 The IR spec-
trum of 5 exhibits the characteristic amine peaks at 3366 and
3272 cmꢀ1 as a double shoulder for both –NH2 and –OH groups.
Also, the N–H bending vibration of the primary amine is observed
at 1590 cmꢀ1. Complementarily, a broad singlet at 2.37 ppm on the
1H NMR spectrum is assigned to the –NH2 and –OH groups. As
expected, the acetylation of 5 led to the corresponding diacetylated
compound 6.
L
ralose with amino groups are also important, since they are found
in some antimicrobial active molecules.
In conclusion, we have achieved the synthesis of novel amino
sugars (5 and 10) derived from 1,2-O-trichloroethylidene furanoses
in good yields and purities. Their antimicrobial activities were
studied against various microorganisms. Both amino sugars of b-
A comparable sequence was applied to the commercially avail-
able b-chloralose (7). Thus, the tosylation of the primary hydroxyl
group, its transformation into the corresponding azide derivative 8
and reduction of the azido group led to 6-amino-6-deoxy-1,2-O-
L-arabinochloralose and b-chloralose exhibit moderate antimicro-
bial effects. The synthesis and antimicrobial activity of other ster-
eoisomers of trichloroethylidene acetals of sugars are currently
being investigated in our laboratories.
(S)-trichloroethylidene-a-D-glucofuranose (10). The IR spectrum
of 10 exhibits the characteristic amine peaks at 3467 and
3437 cmꢀ1 as a double shoulder while the –OH was observed as
a broad band at 3200 cmꢀ1. Also, the N–H bending vibration of
the primary amine group is observed at 1590 cmꢀ1 similar to com-
pound 5. Complementarily, a broad singlet at 4.91 ppm on the 1H
NMR spectrum is assigned to the –NH2 group plus the two –OH
at positions 3 and 5. Finally, treating 10 in excess acetic anhydride
led to the expected triacetylated derivative 11.
1. Experimental
1.1. General methods
Melting points were measured by using Electrothermal 9100
melting point apparatus in capillary and uncorrected. 1H NMR
(400 MHz) and 13C NMR (100 MHz) were recorded on a Varian
AS 400 NMR spectrometer. APCI positive polarity (70 eV) mass
spectra were recorded on Agilent 1100 (LC–MSD) mass spectrom-
eter. IR spectra were recorded on Perkin–Elmer Spectrum 100 FTIR
Determination of the antimicrobial activities of the new com-
pounds was carried out in vitro by the agar well diffusion method19
O
OAc
O
CCl3
O
OAc
d
H
2
d
O
b
O
O
a
O
O
c
OAc
OH
OH
OH
OH
O
O
O
O
O
CCl3
CCl3
CCl3
CCl3
CCl3
O
O
O
O
O
NHAc
N3
NH2
OH
OTs
H
H
H
H
H
1
3 (70%)
4 (65%)
5 (71%)
6 (91%)
N3
HO
TsO
HO
HO
HO
H2N
HO
AcHN
AcO
g
f
e
d
O
O
O
O
O
OH
OH
OAc
OH
OH
O
O
O
O
O
CCl3
CCl3
CCl3
CCl3
CCl3
O
O
O
O
O
H
H
H
H
H
7
8 (50%)
9 (94%)
10 (81%)
11 (98%)
Scheme 1. Synthesis of aminosugar derivatives of b-L-arabinochloralose and b-chloralose, respectively (5 and 10). Reagents and conditions: (a) p-TsCl (1.5 equiv)/Py, in ice
bath, 8 h; (b) NaN3 (5.0 equiv)/DMF, 120 °C, 3 h; (c) PPh3 (1.5 equiv)/MeOH, reflux, 2 h; (d) Ac2O/Py, rt, overnight; (e) p-TsCl (1.2 equiv)/Py, in ice bath than at rt, 48 h; (f) NaN3
(5.0 equiv)/DMF, 100 °C, 4.5 h; (g) PPh3 (3 equiv)/MeOH, reflux, 1.5 h.

N. Yenil et al. / Carbohydrate Research 345 (2010) 1617–1621
1619
Table 1
Antimicrobial activity of synthesized compounds against test microorganisms
Compounds
Microorganismsa
STYP
SAd
EC
KR
BC
BS
PV
EF
EA
ECLO
CA
5
18
18b
20
20
ND
11
22
12
26
26
ND
0
20
22
16
28
26
ND
8
18
13
30
28
ND
0
16
10
6R
40
ND
10
16
24
20
28
28
ND
0
20
22
26
12R
ND
0
22
14
10R
28
ND
0
14
12
ND
ND
22
13
10
22
10R
30
ND
10
16
NAc
12R
16
CLH
NYS
NC (EtOH)
ND
12
a
Test microorganisms: SA, Staphylococcus aureus ATCC 6538P; EC, Escherichia coli ATCC 39628; KR, Kocuria rhizophila ATCC 9341; BC, Bacillus cereus CM 99; BS, Bacillus
subtilis ATCC 6633; STYP, Salmonella typhimurium CCM 5445; PV, Proteus vulgaris ATCC 8427; EF, Enterococcus faecalis ATCC 29212; EA, Enterobacter aerogenes ATCC 13048;
ECLO, Enterobacter cloacae ATCC 13047D; CA, Candida albicans ATCC 10231.
b
Inhibition zone diameter in millimeters, including well and disc diameter (6 mm).
c
Applied compounds dose, 1200
lg/well; standard antibiotics; NA—nalidixic acid (30
lg/disc); CHL—chloramphenicol (30 lg/disc); NYS—nystatin (10 lg/disc).
d
Bacteria tested in MHA medium, yeast in PDA; mean values, n = 3; ND, not determined; R, resistant, NC, negative control (ethanol, 60
lL), , partially inhibition.
Table 2
MIC values of the tested compounds and standard antibiotics against selected microorganisms
MOa
Tested compounds and standard antibiotics (
l
g/mL)
GE
5
10
NEOb
NYS
ND
SA
BC
KR
EC
EF
1458 510c
(1250–2500)
1125 280
(625–1250)
833 323
(625–1250)
2083 645
(1250–2500)
187 70
2917 1021
(2500–5000)
2500 0.00
(2500–2500)
938 342
(625–1250)
4167 1291
(2500 –5000)
438 170
27.08 9.88
(18.75–37.5)
7.292 2471
(4.688–9.375)
3.386 1.235
(2.344–4.688)
5.730 2.067
(4.688–9.375)
6.250 2.344
(4.688–9.375)
ND
3.907 1.172
(2.344–4.688)
5.730 2.067
(4.688–9.375)
2.865 1.034
(2.344–4.688)
6.250 2.344
(4.688–9.375)
4.427 0.781
(2.344–4.688)
ND
ND
ND
ND
ND
(156–313)
5833 2041
(5000–10,000)
(313 –625)
6670 2580
(5000–10,000)
CA
0.488 0.293
(0.293–1.172)
a
b
c
MO: Microorganisms: SA—Staphylococcus aureus; BC—Bacillus cereus; EC—Escherichia coli; KR—Kocuria rhizophila; EF—Enterobacter faecalis; CA—Candida albicans.
Standard antibiotics: NEO—neomycin; GE—gentamycin; NYS—nystatin. ND—not determined.
Minimum and maximum values are shown in parentheses. Data presented as the mean value of six determinations standard deviation.
Spectrometer. Optical rotation measurements were carried out on
an Autopol II polarimeter. Elemental analyses were carried out on
LECO CHNS-932 elemental analyzer. TLC and column chromatogra-
phy were performed on precoated aluminum plates (Merck 5554)
and Silica Gel G-60 (Merck 7734), respectively. The chromatograms
were detected with 5% aqueous sulfuric acid by heating the plates
above 120 °C for about 3 min. All solvent removals were carried
syrupy diacetate 2 (1.30 g, 95%), 1H NMR d (DMSO-d6): 6.30 (d,
1H, J1,2 4.0, H-1), 5.89 (s, 1H, H-acetal), 5.09 (br s, 1H, H-3), 5.03
(d, 1H, H-2), 4.32 (t, 1H, J4,5a 6.4 Hz, J5a,5b 6.8 Hz, H-5a), 4.16 (m,
2H, H-4, H-5b), 2.05 (s, 2H, 2 ꢂ CH3); 13C NMR: 170.6, 170.2
(2 ꢂ OCOCH3) 109.1, 107.8 (HC–CCl3 and C-1), 99.9 (HC–CCl3),
86.7, 83.8, 77.2, 63.5 (C-2, C-3, C-4, C-5), 21.1, 21.0 (2 ꢂ CH3).
out under reduced pressure with rotary evaporator.
L
-Arabinose,
1.1.3. 5-O-Tosyl-1,2-O-(S)-trichloroethylidene-b-L-arabinofur-
b-chloralose, and chloral hydrate were purchased from Sigma–Al-
drich Chem. Co. and b-chloralose was purified by crystallization
from boiling MeOH. p-Toluenesulfonyl chloride, triphenylphos-
phine, and all solvents were purchased from Merck Co.
anose (3)
A solution of 1 (22.52 g, 0.0806 mol) in pyridine (100 mL) was
cooled with ice bath and p-toluenesulfonyl chloride (18.43 g,
0.0967 mol) in pyridine (40 mL) was added dropwise and stirred
for 8 h. TLC (toluene–MeOH, 9:1) showed that a very small amount
of starting sugar remained. The reaction mixture was concentrated
to half volume and poured into ice-water (600 mL). Then, it was
extracted with CH2Cl2 (3 ꢂ 300 mL). Organic phase was washed
with water and dried over anhydrous Na2SO4, filtered off, evapo-
rated under reducing pressure. The crude syrupy product was puri-
fied by column chromatography (CH2Cl2–MeOH, 100:1) giving 3
1.1.1. 1,2-O-(S)-Trichloroethylidene-b-
Compound 1 was prepared according to literature while using
dried
-arabinose (50.0 g, 0.33 mol) as a starting material.8a,14 The
L-arabinofuranose (1)
L
pure product was obtained as colorless crystals (39.6 g, 43%), mp
191–193 °C; ½a 2D1
ꢁ
+22.55 (c 1.22, MeOH); IR cmꢀ1 (KBr); 3417 (–
OH), 814 745 (CCl3). 1H NMR d (DMSO-d6); 6.17 (d, 1H, J1,2 4.0,
H-1), 5.67 (s, 1H, H-acetal), 4.84 (br s, 1H, H-3), 4.72 (d, 1H, H-2),
3.91 (t, 1H, J4,5a 6.8 Hz, J5a,5b 6.8 Hz, H-5a), 3.40 (m, 2H, H-4, H-
5b); 13C NMR; 108.8, 107.7 (HC–CCl3 and C-1), 100.4 (HC–CCl3),
89.9, 89.5, 75.0, 61.9 (C-2, C-3, C-4, C-5).
(24.47 g, 70%), as a white solid, mp 182–184 °C, ½a D24
ꢀ30.0 (c 0.4,
ꢁ
MeOH); IR cmꢀ1 (KBr); 3450 (–OH), 2854–3092 (aliphatic and aro-
matic C–H). 1H NMR d (DMSO-d6): 7.83 (d, 2H, benzene, J 8.4 Hz),
7.50 (d, 2H, benzene), 6.24 (d, 1H, J1,2 4.0, H-1), 5.42 (s, 1H, H-ace-
tal), 4.73 (d, 1H, H-2), 5.75 (d, 1H, J3,4 4.4 Hz, H-3), 4.15 (m, 1H, H-
4), 4.15 (m, 1H, H-5a), 3.94 (dd, 1H, J5a,5b 10.2, J4,5b 7.2 Hz, H-5b),
2.42 (s, 3H, CH3); 13C NMR: 145.9, 132.5, 130.9, 128.5 (6 ꢂ C, ben-
zene), 108.7, 107.9 (HC–CCl3 and C-1), 100.1 (HC–CCl3), 88.9, 85.9,
74.9, 70.1 (C-2, C-3, C-4, C-5), 21.9 (CH3).
Anal. Calcd for C7H9Cl3O5: C, 30.08; H, 3.25. Found: C, 29.93; H,
3.17.
1.1.2. 3,5-Di-O-acetyl-(S)-trichloroethylidene-b-L-arabinofur-
anose (2)
Acetylation of compound 1 (1.04 g, 0.0038 mol) in pyridine
(10 mL) with Ac2O (3.0 mL) at room temperature afforded the
Anal. Calcd for C14H15Cl3O7S: C, 38.77; H, 3.49; S, 7.39. Found: C,
39.04; H, 3.45; S, 7.28.

1620
N. Yenil et al. / Carbohydrate Research 345 (2010) 1617–1621
1.1.4. 5-Azido-5-deoxy-1,2-O-(S)-trichloroethylidene-b-
arabinofuranose (4)
L-
Anal. Calcd for C15H17Cl3O8S: C, 38.85; H, 3.70; S, 6.92. Found: C,
38.75; H, 3.63; S, 6.75.
To a solution of 3 (20.53 g, 0.0473 mol) in DMF (100 mL) was
added NaN3 (15.37 g, 0.2365 mol). The mixture was heated with
stirring in an oil bath at 120 °C for 3 h. TLC (toluene–MeOH, 9:1)
showed completion of the reaction. The reaction mixture was
poured into ice-water (500 mL). The solid product was filtered off
and dried at room temperature. The product was crystallized from
MeOH–H2O to get the pure compound 4 (9.36 g, 65%), mp 124–
1.1.8. 6-Azido-6-deoxy-1,2-O-(S)-trichloroethylidene-a-D-
glucofuranose (9)
A solution of compound 8 (10.76 g, 0.0232 mol) in DMF was re-
acted with NaN3 for 4.5 h at 100 °C as described in Section 1.1.4.
Compound 9 (7.30 g, 94%) was obtained as a white solid, mp
165–168 °C, ½a 2D1
ꢁ
ꢀ5.0 (c 0.4, MeOH); IR cmꢀ1 (KBr); 3478–3399
126 °C; ½a 2D1
ꢁ
+40.0 (c 0.4, MeOH); IR cmꢀ1 (KBr); 3474 (–OH),
(–OH), 2124 (–N3); 1H NMR d (DMSO-d6): 6.18 (d, 1H, J1,2 3.6 Hz,
H-1), 5.84 (s, 1H, H-acetal), 3.90 (br s, 2H, 2 ꢂ OH), 4.70 (d, 1H,
H-2), 5.43 (d, 1H, H-3), 5.34 (m, 1H, H-5), 4.14 (dd, 1H, H-4), 3.38
(dd, 1H, J5,6a 2.0 Hz, J6a,6b 12.4 Hz H-6a), 3.23 (m, 1H, H-6b); 13C
NMR: 108.7, 106.8 (HC–CCl3, C-1), 100.6 (HC–CCl3), 87.8, 82.2,
73.4, 67.2 (C-2, C-3, C-4, C-5), 54.9 (–CH2N3).
2852–3008 (aliphatic and aromatic C–H), 2114 (–N3). 1H NMR d
(DMSO-d6): 6.27 (d, 1H, J1,2 3.6, H-1), 5.97 (d, 1H, H-2), 5.68 (s,
1H, H-acetal), 4.77 (d, 1H, J3,4 4.4 Hz, H-3), 4.06 (m, 1H, H-4),
3.92 (dd, 1H, J4,5a 9.2 Hz, H-5a), 3.48 (dd, 1H, J5a,5b 10.2 Hz, J4,5b
5.2 Hz, H-5b). 13C NMR: 108.8, 107.8 (HC–CCl3, C-1), 100.4 (HC–
CCl3), 52.1, 75.7, 87.8, 89.1 (C-2, C-3, C-4, C-5).
Anal. Calcd for C8H10Cl3N3O5: C, 28.72; H, 3.01; N, 12.56. Found:
C, 28.82; H, 2.94; N, 11.87.
Anal. Calcd for C7H8Cl3N3O4: C, 27.61; H, 2.65; N, 13.80. Found:
C, 27.76; H, 2.62; N, 12.78.
1.1.9. 6-Amino-6-deoxy-1,2-O-(S)-trichloroethylidene-a-D-
1.1.5. 5-Amino-5-deoxy-1,2-O-(S)-trichloroethylidene-b-
L
-
glucofuranose (10)
arabinofuranose (5)
A solution of compound 9 (5.65 g, 0.0169 mol) in MeOH was re-
acted with triphenylphosphine for 2 h as described in Section 1.1.5.
The title product was obtained as a white solid (4.22 g, 81%) after
To a solution of 4 (5.30 g, 0.0174 mol) in MeOH (100 mL) was
added triphenylphosphine (6.85 g, 0.0261 mol). The reaction mix-
ture was refluxed for 1.5 h. TLC (CH2Cl2–MeOH, 9:1) showed the
completed reaction. The solvent was evaporated under reduced
pressure. The syrupy product was purified by column chromatog-
re-crystallization in boiling water, mp 163–166 °C; ½a D27
ꢀ5.14 (c
ꢁ
2.432, DMF); IR cmꢀ1 (KBr); 3467–3437 (–NH2), 3200 (–OH),
1590 (N–H); 1H NMR d (DMSO-d6): 6.19 (d, 1H, J1,2 3.6 Hz, H-1),
5.80 (s, 1H, H-acetal), 4.91 (br s, 4H, –NH2, 2 ꢂ OH), 4.70 (d, 1H,
H-2), 4.17 (br s, 1H, H-3), 3.83 (dd, 1H, H-6a), 3.72 (br s, 1H, H-
4), 2.88 (dd, 1H, J6a,6b 11.2 Hz, H-6b); 13C NMR: 108.7, 106.8 (HC–
CCl3, C-1), 100.5 (HC–CCl3), 87.8, 83.2, 83.0 (C-2, C-3, C-4), 68.6
(C-5), 44.5 (C-6); LC/MS-APCI: m/z 308 (100%, M+), 309 (9%,
M++1), 310 (87%, M++2).
raphy with eluting solvent CH2Cl2–MeOH (100:1) to give
5
(3.44 g, 71%) as a white solid, mp 172 °C (decomp.); ½a D21
ꢀ40.51
ꢁ
(c 1.233, CH3OH); IR cmꢀ1 (KBr); 3366–3272 (–NH2 and –OH),
1590 (N–H); 1H NMR d (CDCl3): 6.27 (d, 1H, J1,2 4.0 Hz, H-1), 5.58
(s, 1H, H-acetal), 4.92 (d, 1H, H-2), 4.69 (t, 1H, J4,5a 5.2, J4,5b
3.2 Hz, H-4), 4.23 (s, 1H, H-3), 2.87 (dd, 1H, J5a,5b 7.2 Hz), H-5a),
2.85 (dd, 1H, H-5b), 2.37 (br s, 3H, –NH2 and –OH); 13C NMR:
108.7, 107.6 (HC–CCl3 and C-1), 100.6 (HC–CCl3), 43.9, 75.4, 89.5,
90.5 (C-2, C-3, C-4, C-5); LC/MS-APCI: m/z 278 (100%, M+), 279
(8%, M++1), 280 (82%, M++2).
Anal. Calcd for C8H12Cl3NO5: C, 31.15; H, 3.89; N, 4.54. Found: C,
30.90; H, 3.87; N, 3.79.
1.1.10. 6-Acetamido-6-deoxy-3,5-di-O-acetyl-1,2-O-(S)-
Anal. Calcd for C7H10Cl3NO4: C, 30.18; H, 3.62; N, 5.03. Found: C,
30.39; H, 3.74; N, 4.68.
trichloroethylidene-a-D-glucofuranose (11)
Compound 10 (1.04 g, 0.0034 mol) was acetylated with Ac2O as
described in Section 1.1.6. Compound 11 was obtained (1.45 g,
98%) as syrup. It was solidified at room temperature, mp 92–
95 °C; 1H NMR d (DMSO-d6): 7.96 (t, 1H, –NHC(O)CH3), 6.33 (d,
1H, J1,2 3.6, H-1), 5.94 (s, 1H, H-acetal), 5.17 (d, 1H, J3,4 2.8 Hz, H-
3), 4.95 (d, 1H, H-2), 4.93 (br s, 1H, H-4), 3.01 (m, 1H, H-5), 4.37
(dd, 1H, J6a,6b 8.8, J5,6a 2.8 Hz, H-6a), 3.65 (dd, 1H, J5,6b 5.6 Hz, H-
6b), 1.96, 1.92, 1.76 (s, 9H, 3 ꢂ –OC(O)CH3).
1.1.6. 5-Acetamido-5-deoxy-3-O-acetyl-1,2-O-(S)-trichloroethyl-
idene-b-L-arabinofuranose (6)
A solution of 5 (1.01 g, 0.0036 mol) in pyridine (20 mL) was
acetylated with Ac2O (1.1 mL, 0.0108 mol) at room temperature
for 24 h. The usual work-up procedure and purification on a silica
gel column eluting with CH2Cl2–MeOH (100:1) gave the white so-
lid 6 (1.31 g, 91%), mp 128–130 °C; 1H NMR d (DMSO-d6): 8.06 (t,
1H, –NHC(O)CH3), 6.22 (d, 1H, H-1), 5.82 (s, 1H, H-acetal), 4.71
(d, 1H, J1,2 3.6 Hz, H-2), 4.12 (s, 1H, H-3), 3.94 (t, 1H, J4,5a 6.8, J4,5b
5.6 Hz, H-4), 3.19 (dd, 1H, J5a,5b 13.0 Hz, H-5a), 3.14 (dd, 1H, H-
5b), 3.16 (s, 6H, –OC(O)CH3, –NHC(O)CH3).
2. Microbiological procedures
2.1. Agar well diffusion assay
In vitro antimicrobial studies were carried out by the agar well
diffusion method19 against test microorganisms. Bacterial strains
grown on nutrient agar (37 °C for 24 h) and C. albicans grown on
Potato Dextrose Agar (30 °C for 48 h) were suspended in a saline
solution (0.85% NaCI) and adjusted to a turbidity of 0.5 McFarland
1.1.7. 6-O-Tosyl-1,2-O-(S)-trichloroethylidene-a-D-glucofuranose
(8)
b-Chloralose (7), commercially available, (17.62 g, 0.0569 mol)
was reacted with p-toluenesulfonyl chloride for 48 h as described
in Section 1.1.3. Compound 8 (13.19 g, 50%) was obtained as a
standards (106 Colony Forming units/mL). Then, 50
lL (microliters)
white solid, mp 145–147 °C, ½a D21
ꢁ
ꢀ20.0 (c 0.4, MeOH); IR cmꢀ1
inoculum was added to 25 mL melted Mueller Hinton Agar (MHA)
for bacteria and Potato Dextrose Agar (PDA) (Oxoid, Basingstoke,
UK) for C. albicans medium cooled at 45 °C. These were then poured
into 90 mm diameter Petri dishes and maintained for 1 h at room
temperature. 6 mm diameter wells were cut in the agar plate and
(KBr); 3507 (–OH), 1598 (–SO2–); 1H NMR d (DMSO-d6): 7.81 (d,
2H, benzene, J 8.4 Hz), 7.48 (d, 2H, benzene), 6.16 (d, 1H, J1,2
3.6 Hz, H-1), 5.81 (s, 1H, H-acetal), 4.70 (d, 1H, H-2), 4.15 (m, 1H,
H-3), 4.15 (m, 1H, H-4), 3.90 (m, 1H, H-5), 3.94 (d, 1H, J6a,6b
7.2 Hz, H-6a), 3.87 (d, 1H, H-6b), 2.36 (s,3H, CH3); 13C NMR:
145.5, 132.9, 130.8, 128.3 (6 ꢂ C, benzene), 108.7, 106.7 (HC–CCl3
and C-1), 100.5 (HC–CCl3), 87.6, 81.3, 73.7, 73.3, 65.7 (C-2, C-3,
C-4, C-5, C-6), 21.8 (CH3).
60
l
L of compound (1200
l
g/well) and pure extraction solvent as
L) were loaded individually in
a negative control (ethanol, 60
l
the wells. The dishes were preincubated at 4 °C for 2 h to uniform
diffusion into the agar. After preincubation, the plates were

N. Yenil et al. / Carbohydrate Research 345 (2010) 1617–1621
1621
incubated at 37 °C for 24 h for bacteria and 30 °C for 48 h for yeast.
The antimicrobial activity was determined by measuring the inhi-
bition zone diameter around the wells. In addition, commercial
through the project 106T410. One of the authors (E.A.) is indebted
to TUBITAK for a grant.
antibiotics such as nalidixic acid (30
lg), chloramphenicol
References
(30 g), and nystatin (10 g) were used as positive control to
l
l
1. Reist, E. J.; Spencer, R. R.; Baker, B. R. J. Am. Chem. Soc. 1960, 82, 2025–2029.
2. (a) Csuk, R. Carbohydr. Res. 1985, 140, 167–168; (b) Ermolenko, L.; Sasaki, N. A.;
Potier, P. Tetrahedron Lett. 1999, 40, 5187–5190; (c) Juan Xie, J. Carboyhdr. Res.
2003, 338, 399–406; (d) Ji, X. M.; Mo, J.; Liu, H. M.; Sun, H. P. Carbohydr. Res.
2006, 341, 2312–2320.
determine the sensitivity of the strains. Experiments were re-
peated three times, and the results were expressed as average
values.
3. (a) Werngs, W. D.; Williams, D. H.; Feeney, J.; Brown, J. P.; King, R. W. J. Chem.
Soc., Perkin Trans. 1 1972, 443–446; (b) Wang, J.; Li, J.; Tuttle, D.; Takemoto, J. Y.;
Chang, C. W. T. Org. Lett. 2002, 4, 3997–4000; (c) Nishiyama, T.; Isobe, M.;
Icikawa, Y. Angew. Chem. 2005, 117, 4446–4449.
4. Han, M. J.; Yoo, K. S.; Kim, Y. H.; Chang, J. Y. Tetrahedron Lett. 2002, 43, 5597–
5600.
5. Timmons, S. C.; Thorson, J. S. Curr. Opin. Chem. Biol. 2008, 12, 297–305.
6. (a) Semmelhack, M. F.; Jiang, Y.; Ho, D. Org. Lett. 2001, 3, 2403–2406; (b) Wu,
B.; He, J. Y.; Swayze, E. E. Org. Lett. 2002, 4, 3455–3458.
7. SanMartin, R.; Tavassoli, B.; Walsh, K. E.; Walter, D. S.; Gallagher, T. Org. Lett.
2000, 2, 4051–4054.
2.2. Determination of minimum inhibition concentration (MIC)
The microtiter broth dilution technique was performed by using
the CLSI standards.20,21 A sterile 96 round-bottomed well plate was
labeled. A volume of 100
was pipetted into the first row of the plate. To all other wells
50 L of double strength Mueller Hinton broth or Potatoes Dex-
lL of antimicrobial compound solution
l
trose broth was added. Serial dilutions were performed using a
8. (a) Anıl, H.; Yüceer, L.; Yüceer, T. Carbohydr. Res. 1983, 123, 153–156; (b) Yenil,
N.; Yüceer, L. Carbohydr. Res. 2003, 338, 2013–2016; (c) Ay, K.; Ay, E.; Yenil, N.;
Yüceer, L. World Appl. Sci. J. 2009, 6, 880–885.
9. Forsen, S.; Lindberg, B.; Silvander, B. G. Acta Chem. Scand. 1965, 19, 359–369.
10. Butler, T. C. J. Pharmacol. Exp. Ther. 1940, 69, 229–235.
11. Landolfo, G. Z.; Tronchet, J. M. J. Il Farmaco 1999, 54, 852–853.
12. Sanchez, S.; Bamhaoud, T.; Prandi, J. Eur. J. Org. Chem. 2002, 22, 3864–3873.
13. (a) Lemieux, R. U.; Nagabhushan, T. L. Can. J. Chem. 1967, 46, 401–403; (b)
Grondin, R.; Leblanc, Y.; Hoogsteen, K. Tetrahedron Lett. 1991, 38, 5021–5024;
(c) Streicher, B.; Wünsch, B. Carbohydr. Res. 2003, 338, 2375–2385.
14. (a) Gül, S. Y.; Makinabakan, O.; Yüceer, L. Tetrahedron Lett. 1994, 35, 9233–
9236; (b) Seçen, S. M.Sc. Dissertation, Celal Bayar University, 2005.
15. (a) Kovacs, L.; Osz, E.; Domokos, V.; Holzer, W.; Gyögydeak, Z. Tetrahedron
2001, 57, 4609–4621; (b) Lindsley, C. W.; Zhao, Z.; Newton, R. C.; Leister, W. H.;
Strauss, K. A. Tetrahedron Lett. 2002, 43, 4467–4470.
16. Lin, W.; Zhang, X.; He, Z.; Jin, Y.; Gong, L.; Mi, A. Synth. Commun. 2002, 32,
3279–3284.
17. Pal, B.; Jainsankar, P.; Giri, S. V. Synth. Commun. 2004, 34, 1323–1327.
18. Ay, E.; Ay, K.; Yenil, N.; Maddaluno, J. Abstracts of Papers, XXIInd National
Chemistry Congress, Gazimagusa, Oct 6–10, 2008; East Mediterranean
University: GZM, North Cyprus, 2008, p 105.
19. Perez, C.; Pauli, M.; Bazerque, P. Acta. Biol. Med. Exp. 1990, 15, 113–115.
20. Clinical And Laboratory Standards Institute (CLSI). Methods for Dilution
Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically;
Approved Standard M7-A6, 6th ed.; National Committee for Clinical
Laboratory Standards: Wayne, Pa, 2003.
micropipette (A1–A10). Tips were discarded after use such that
each well had 50
centrations. Then, 50
l
L of the test material in serially descending con-
L of broth containing bacterial suspension
l
(5 ꢂ 106 cfu/mL) or yeast (5 ꢂ 105 cfu/mL) was added to each well.
Each column of wells contained a single antimicrobial agent in pro-
gressive dilutions and was inoculated with a single microorganism.
Plates were sealed with clean film to ensure that microorganisms
did not become dehydrated. Positive and negative controls were
present on each microtiter plate. The plates were prepared in trip-
licate, and placed in an incubator set at 37 °C for 18–24 h for bac-
teria and 30 °C for 24–36 h for C. albicans, respectively. After
incubation, was added 12 lL of 0.18% TTC (2,3-5-triphenyl tetrazo-
lium chloride) solution to each well of microtitre plate and incu-
bated at 37 °C for 1 h. The color change was then assessed
visually. Any color changes from purple to pink or colorless were
recorded as positive. The lowest concentration at which color
change occurred was taken as the MIC value. The average of six val-
ues was calculated and that was the MIC for the test material and
microorganisms.
21. Clinical And Laboratory Standards Institute (CLSI). Performance Standards for
Acknowledgments
_
Antimicrobial Susceptibility Testing, 16th Informational Supplement M100-
S16. National Committee for Clinical Laboratory Standards: Wayne, Pa, 2006.
The authors would like to thank TUBITAK (Scientific and Tech-
nical Research Council of Turkey) for their financial support