Impact of glycosylation on physico-chemical and biological properties of nitrification inhibitors

Article abstract of DOI:10.1016/j.tet.2012.06.060

The lipophilic 2-mercaptobenzothiazole (MBT), known for its nitrification inhibition properties, was derivatized thanks to direct glycosylation reactions. Similar transformations were also performed starting from 2- mercaptobenzimidazole (MBI), structurally close to MBT. The resulting S-linked mono- or disaccharides derived from d-glucose or l-arabinose, and cellobiose, gentiobiose or lactose, respectively, were subsequently studied as novel nitrification inhibitors without any further formulation or physical processes, except dilution in water. Along with ecotoxicity measurements, inhibition properties of the synthesized water soluble glycoconjugates were studied in a model reactor containing nitrification bacteria. The best results were obtained for the gentiobiosyl derivatives simply dissolved in water.

Full text of DOI:10.1016/j.tet.2012.06.060

Tetrahedron 68 (2012) 7095e7102
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Impact of glycosylation on physicoechemical and biological properties
of nitrification inhibitors
a
,
,
,
,b
Daniele Pro b, Mustapha Arkoun c d, Samuel Huguet a b, Richard Daniellou a
,
ꢀ
Caroline Nugier-Chauvin a b, Jean Morvan a b, Dominique Wolbert a b, Alain Ourry c
Jean-Claude Yvin , Vincent Ferrieres
,
,
,
,
,d
e
a,b,
*
ꢀ
a
ꢁ
ꢁ ꢁ
Ecole Nationale Superieure de Chimie de Rennes, CNRS, UMR 6226, Avenue du General Leclerc, CS 50837, 35708 Rennes Cedex 7, France
b
c
ꢁ
ꢁ
Universite europeenne de Bretagne, France
Universite de Caen Basse-Normandie, UMR 950 Ecophysiologie Vegetale, Agronomie et nutritions N, C, S. Esplanade de la paix, 14032 Caen, France
ꢁ
ꢁ ꢁ
d
ꢁ ꢁ
INRA, UMR 950 Ecophysiologie Vegetale, Agronomie et nutritions N, C, S. Esplanade de la paix, 14032 Caen, France
e Timac Agro International, 27 Avenue Franklin Roosevelt, 35408 Saint Malo, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The lipophilic 2-mercaptobenzothiazole (MBT), known for its nitrification inhibition properties, was
derivatized thanks to direct glycosylation reactions. Similar transformations were also performed
starting from 2-mercaptobenzimidazole (MBI), structurally close to MBT. The resulting S-linked mono- or
Received 28 February 2012
Received in revised form 11 June 2012
Accepted 14 June 2012
disaccharides derived from D-glucose or L-arabinose, and cellobiose, gentiobiose or lactose, respectively,
Available online 21 June 2012
were subsequently studied as novel nitrification inhibitors without any further formulation or physical
processes, except dilution in water. Along with ecotoxicity measurements, inhibition properties of the
synthesized water soluble glycoconjugates were studied in a model reactor containing nitrification
bacteria. The best results were obtained for the gentiobiosyl derivatives simply dissolved in water.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Glycosylation
Nitrification inhibitor
Ecotoxicity
2-Mercaptobenzothiazole
2-Mercaptobenzimidazole
1. Introduction
Most of the potent nitrification inhibitors in soils are substituted
heterocyclic N-compounds (e.g., pyridine, pyrazole, triazole, ben-
Ammonium, formed by decomposition of organic nitrogen in
soil or added as fertilizer, is rapidly oxidized to nitrite and nitrate in
the nitrification process carried out by microorganisms. These an-
ions are mobile in soils and readily lost from plant rooting zones by
leaching or runoff, thus resulting in potential pollution of ground
and surface waters.1,2 Moreover, nitrate can also undergo de-
nitrification in the absence of oxygen by other microorganisms to
gaseous nitrogen oxides (e.g., N2O one of the greenhouse gases
involved in the stratospheric ozone layer depletion) and N2.3e5
These losses account for inefficiency in the use of applied nitro-
gen for plants. Indeed often less than 30% of the applied nitrogen
fertilizers are recovered in intensive agricultural systems.6,7 This
international concern has stimulated researches for developing
new compounds that effectively inhibit nitrification in soils in
conjunction with the use of nitrogen fertilizers (urea, ammonium
or other nitrogen sources).
zotriazole, imidazole .),1 for which little is known about the mode
of action. An ideal nitrification inhibitor should meet the re-
quirements of being (i) non-toxic to other organisms, animals and
humans, (ii) efficient at low concentrations (iii) mobile and stable in
nutrient or fertilizer formulation, (iv) persistent to stay in soils for
an adequate period (slow-and controlled-released to enhance ef-
ficiency of nutrients applied), and finally (v) cheap.8,9 Among them,
the 2-mercaptobenzothiazole (MBT) is known as a nitrification
inhibitor10,11 produced and marketed primarily in Japan whereas
the analogous 2-mercaptobenzimidazole (MBI), structurally close
to MBT, is not registered.12 However, benzimidazole has been
tested for this use and among many others unsubstituted hetero-
cyclic N-compounds has a lower effect on nitrification.1,13 Many
nitrification inhibitors are incorporated into solid N-containing
fertilizing materials. In these cases, the dissolution and suspending
process may be aided by mechanical mixing, addition of suspend-
ing agents or other means.
Nevertheless, it is well known that glycosylation is amongst the
most common process involved in nature to increase di-
versification. As a result, glycosylation provides many biological
* Corresponding author. Tel.: þ33 223 238 058; fax: þ33 223 238 046; e-mail
ꢀ
address: vincent.ferrieres@ensc-rennes.fr (V. Ferrieres).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.06.060

7096
D. Pro et al. / Tetrahedron 68 (2012) 7095e7102
functions for both macromolecules and small molecules. Some of
the latter glycoconjugates present clinical applications as bacteri-
cides, fungicides or in the treatment of cancers.14 In this context, we
propose herein to evaluate the impact of glycosylation on the
physicoechemical and biological properties of the nitrification in-
hibitor MBT and of its MBI counterpart. We expect that the
designed conjugates can maintain the inhibition activity of the
starting material and/or can be partly hydrolyzed to release the
desired activity. Thus, we describe herein the synthesis of various
mono- or diglycosylated derivatives thereof (Fig. 1), their phys-
icoechemical properties, and their ability to inhibit nitrification
without further formulation or processes, except dilution in water.
observed that the monoglucosides Glc-S-BZT(BZI) are much more
soluble in pure water than the disaccharides. This observation is
nevertheless in good agreement with water solubility of the mono-
or disaccharides.21
Subsequently, ecotoxicity measurements were performed. A
first set of measurements was performed on a consortium of
bacteria, more representative of the soil composition. The cocktail
of bacteria used for this test came from activated mud collected
from water treatment plants. The biological oxygen demand over 5
days (BOD5) quantifies the amount of oxygen necessary for the
biological degradation of organic residue in wastewater sample22
and was calculated at a concentration of 100 mM for the selected
Fig. 1. Structures of new nitrification inhibitors, i.e., glycosylated MBT and MBI.
2. Results and discussion
Gen-S-BZT and Gen-S-BZI. Although the evaluation could not be
performed for the insoluble MBT, it was previously shown that it
can be removed by biological treatment when the effluent con-
centration is below 100e150 mg/L.23 For the structurally close
MBI, the measurement of chemical oxygen demand (COD) gave
29 mg/L O2 and a ꢁ14 mg/L O2 BOD5 value. This revealed a de-
crease of the bacterial population over five days. On the other
hand, the glycosylated compounds Gen-S-BZT (COD: 68 mg/L O2,
BOD5: ꢁ4 mg/L O2) and Gen-S-BZI (COD: 62 mg/L O2, BOD5: 6 mg/
L O2) demonstrated higher chemical and biochemical demands
than that of the aglycons. This revealed a lesser impact on the
bacterial population.
The targeted glycosylated 2-mercaptobenzothiazole MBT and 2-
mercaptobenzimidazole MBI were prepared through a glycosyla-
tion-deprotection procedure starting from the readily available
peracetylated mono- or disaccharides (Scheme 1). The coupling
between the glycosyl donors 1e4 and the aromatic thiols were
catalyzed by the boron trifluouride etherate complex and the
deacylation step of the resulting thioimidates15e17 was performed
under Zemplen conditions (Table 1). Structural elucidation of the
resulting products was deduced from 1H, 13C and 2D NMR analysis.
Amongst selected data, the
b-anomeric configuration was more
particularly established on the basis of the coupling constant J1,2 for
the reducing residue. All observed coupling constants were found
close to 10 Hz, that indeed reveals a 1,2-trans orientation between
H-1 and H-2 in thioglycosides. The use of the arabinosyl counter-
parts18 was also considered since arabinose is widely distributed in
plants and microorganisms,19,20 and because this carbohydrate is
also found in a five-membered furanose form.
We further studied their solubility in water. While MBT
(<0.03 g/L) and MBI (0.25 g/L) are weakly or non hydrosoluble at
25 ꢀC, their glycosylation resulted in improved solubility in water
[Glc-S-BZT(BZI): 121(>200) g/L; Cel-S-BZT(BZI): 19(>200) g/L;
Gen-S-BZT(BZI) 21(>200) g/L; Lac-S-BZT(BZI): 5(59) g/L]. More
interestingly, this parameter showed to be strongly depending on
the nature of the carbohydrate moiety. Considering the di-
saccharides, gentiobiose and cellobiose were much more in-
teresting than lactose, whatever the aromatic aglycon. It was also
In order to strengthen the positive impact of the glycosylation of
the aromatic compounds, a second evaluation relied on the impact
of the active principles on bioluminescent Vibrio fischeri, a gram-
negative and quorum-sensing bacterium sensitive to toxic com-
pounds. The lowest EC50 were observed for MBI and MBT dissolved
in water in the presence of non toxic polyethylene glycol (PEG). It
was also shown that a significance improvement of the ecotoxicity
(high EC50) of glucosides, lactosides, cellobiosides, and more par-
ticularly gentiobiosides, whatever the nature of the aglycon (sev-
enfold better with MBI and more than 1500-fold better for the
MBT). On the basis of the solubility tests and this first evaluation of
the ecotoxicity, we further focused our attention on the gentiobosyl
derivatives.
We thus evaluated the stability of Gen-S-BZI and Gen-S-BZT in
solutions buffered at 5.5 or 8.0. These pHs were likely to mimic
those found in soils. Under these hydrolytic conditions, both the O-

D. Pro et al. / Tetrahedron 68 (2012) 7095e7102
7097
Scheme 1. Two-step preparation of glycosylated inhibitors.
failed to establish the presence of the starting glycosylated in-
hibitors in the medium, but the presence of Glc-S-BZI(BZT) was
Table 1
Results for targeted compounds synthesized according to Scheme 1
observed, even in very low concentration (1.3
BZI(BZT), versus 100 M for the starting Gen-S-BZI(BZT)). We thus
mM for Glc-S-
m
Entry
Glycosylation
Deprotection step
hypothesized that heterotrophic bacteria still present in the reactor
were responsible for the observed fast degradation of the glyco-
sides, since these bacteria are able to digest carbohydrates. Further
digestion of Gen-S-BZI(BZT) thus liberated MBI(MBT) with their
activity against the nitrification population. This experiment also
showed, thanks to the presence of heterotrophic bacteria, the pro-
inhibitor behaviour of the glycosylated compounds initially
expected.
Donor
Acceptor
Product
Yield (%)
Product
Yield (%)
1
2
3
4
5
6
7
8
1
1
2
2
3
3
4
4
MBT
MBI
MBT
MBI
MBT
MBI
MBT
MBI
5
6
7
85
89
68
76
d
Glc-S-BZT
Glc-S-BZI
Cel-S-BZT
Cel-S-BZI
Gen-S-BZT
Gen-S-BZI
Lac-S-BZT
Lac-S-BZI
100
100
100
100
28b
100
80
8
9a
10
11
12
68
49
61
100
A second set of experiments was subsequently performed by
stabilizing the bacterial population after six months in the dark, in
order to strictly select autotrophic bacteria and to also avoid pho-
tosynthesis. Results in Fig. 2 showed that all ammonium ions were
consumed after only two days without any inhibitor. This con-
sumption has been significantly slow in the presence of MBI or
MBT formulated in PEG solution, or with aqueous solutions of
glycosylated inhibitors. While the lactosyl thioimidate Lac-S-BZT
was less efficient, arabinosyl, glucosyl, cellobiosyl, and gentiobiosyl
derivatives demonstrated interesting effects since more than 15
days were required when reactions were performed in their pres-
ence. Moreover, until two days, no significant difference between
both active ingredients was noted. However, a significant difference
in efficacy was observed beyond this time and it was interesting to
note that MBI-conjugates provided better inhibition of Nitro-
somonas. On another hand, an accumulation of nitrite anions was
observed when the active part was MBT. This accumulation
reached a peak at 8e10 days before decreasing when the ammo-
nium concentration became too low. In this case, the inhibition of
Nitrobacter was more important than that of Nitrosomonas. When
the active part is MBI, there was no accumulation of nitrites. This
was also related to the best inhibition of Nitrosomonas by MBI
derivatives. As a conclusion, MBI and its conjugates appeared to be
more suitable for agricultural applications than MBT and glycosy-
lated-MBT.
a
Not isolated.
For two steps.
b
and the S-glycosidic bonds may be cleaved to yield glucose, Glc-S-
BZI(BZT), gentiobiose and MBI (MBT). The monitoring of this
degradation by LC-MS showed that Gen-S-BZI and Gen-S-BZT were
more sensitive to acidic media. However, only traces of MBT/MBI
could be detected by mass spectrometry under chemical hydrolysis
conditions until 50 days, thus highlighting the great stability of our
compounds in aqueous solutions.
Considering both the stability and the ecocompatibility of our
glycoconjugates, we further studied their behaviour in the presence
of bacteria able to consume ammonium ions and to produce nitrite
species (Nitrosomonas) and nitrate anions (Nitrobacter). Some
amounts of Maerl (1 g for 30 mL), a natural substance containing
mineral elements, were added to the media in order to avoid pH
variations. The activity of the inhibitors was compared to that of the
pure aromatic compounds MBT and MBI, each at a concentration of
100 mM. Since the latter are not soluble in water, an additive was
required. While ethanol enhance bacteria’s growth even in very
small quantities, the addition of polyethylene glycol (PEG) in
moderation has proven particularly efficient in dissolving products
without inducing adverse effects on the bacterial population.
Subsequently, a first set of experiments was achieved using glyco-
sylated derivatives and a consortium of bacteria stabilized after one
month. The results were compared to a control experiment that
contains neither heterocycles nor glycoconjugates. Surprisingly, no
consumption of ammonium was observed under such conditions.
When using Gen-S-BZI(BZT), and after two days, LC/MS analysis
3. Conclusion
The known nitrification inhibitor 2-mercaptobenzothiazole
(MBT), and its structurally related analogue 2-mercaptobenz

7098
D. Pro et al. / Tetrahedron 68 (2012) 7095e7102
Fig. 2. Effects of inhibitors (100 mM) on (a) ammonium, and (b) nitrite concentrations.
imidazole (MBI), were easily glycosylated on a multi-gram scale. As
expected, the introduction of one or two carbohydrate residues
resulted in significant increase of solubility in water. Moreover, the
gentiobiosyl conjugates showed good stability against chemical
hydrolysis over a large period of 50 days and presented higher EC50
than the other derivatives, thus suggesting an improved ecotoxicity.
As a result, the glycosylation seem not only to increase hydro-
solubility but also to induce a better environmental tolerance
compared to the native aromatic substrates, so that simplified for-
mulations of nitrification inhibitors with low impact on the envi-
ronment can be expected, even without any additives. This should
simplify the agrochemical formulations to only bioactive com-
pounds and so move towards more sustainable developments
concepts. Moreover, glycoconjugates of the known inhibitor MBT
and its structurally analogue MBI have shown similar nitrification
inhibiting properties as the native compounds. In the presence of
heterotrophic bacteria, which require organic compounds for their
growth, Gen-S-BZI(BZT) were degraded, expectedly starting from
the O-glycosidic linkage and followed by the S-glycosidic bond. This
highlighted the pro-drug potency of the designed bioconjugates.
Since glycosylation seem to provide various benefits for both phys-
icoechemical properties and ecotoxicity, experiments in soils,
which are much more complex media, will be performed in due
course.
multiplicity: s (singlet), d (doublet), t (triplet), m (multiplet), dd
(double doublet). The high resolved mass spectra (HRMS) were
measured at the ‘Centre Regional de Mesures Physiques de l’Ouest
ꢁ
(CRMPO, Universite de Rennes 1)’ with a MS/MS ZabSpec TOF
Micromass using m-nitrobenzylic alcohol as a matrix and acceler-
ated caesium ions for ionization. Microanalyses were also per-
formed by the CRMPO.
4.2. General procedure for glycosylation reaction
To a solution of per-O-acetylated donor (1 equiv) in anhydrous
dichloromethane were successively added the acceptor (MBT or
MBI, 3 equiv) and BF3.OEt2 (9 equiv). The mixture was stirred at
room temperature for 24 h. Then, the remaining acceptor was fil-
tered off. The resulting filtrate was washed successively with
a saturated solution of aqueous NaHCO3 and water. The aqueous
layers thus obtained were extracted with dichloromethane, and the
combined organic layers finally dried over MgSO4 and concentrated
under reduced pressure. Flash chromatography on silica gel affor-
ded the desired glycosyl thioimidate.
4.2.1. Data for 5. Compound 5 was synthesized according to the
general procedure starting from donor 1 (5.00 g, 12.8 mmol),
2-mercaptobenzothiazole (6.42 g, 38.4 mmol) and BF3.OEt2
(14.6 mL, 115.2 mmol). Column chromatography (9:1 Toluene/
AcOEt) gave 5 (5.42 g, 10.9 mmol) as a white solid in 85% yield. TLC
4. Experimental section
4.1. General
(4:1 toluene/AcOEt): Rf 0.4;
½
a 2D0
ꢂ
þ6.4 (c 1.25, CH2Cl2); mp
127e128 ꢀC; 1H NMR (400 MHz, CDCl3)
d
7.93 (ddd, 1H, J¼8.2 Hz,
J¼1.2 Hz, J¼0.6 Hz, Harom), 7.79 (ddd, 1H, J¼8.0 Hz, J¼1.2 Hz,
J¼0.6 Hz, Harom), 7.46 (ddd, 1H, J¼8.2 Hz, J¼7.2 Hz, J¼1.2 Hz, Harom),
7.36 (ddd, J¼8.0 Hz, J¼7.2 Hz, J¼1.2 Hz, Harom), 5.58 (d, 1H,
J1,2¼10.3 Hz, H-1), 5.35 (t, 1H, J2,3¼J3,4¼9.3 Hz, H-3), 5.23 (dd, 1H, H-
2), 5.18 (dd, 1H, J4,5¼10.0 Hz, H-4), 4.31 (dd, 1H, J6a,6b¼12.5 Hz,
J5,6a¼4.9 Hz, H-6a), 4.18 (dd, 1H, J5,6b¼2.3 Hz, H-6b), 3.93 (ddd, 1H,
H-5), 2.05, 2.05, 2.04, 2.02 (4ꢃs, 12H, CH3CO); 13C NMR (100 MHz,
Thin layer chromatography (TLC) analyses were conducted on E.
Merck 60 F254 Silica Gel non-activated plates. Anhydrous solvents
were prepared and purified according to standard techniques just
before use. Aromatic thiones, peracetylated mono- and di-
saccharides and other reagents were purchased from commercial
suppliers and used as received. Compounds were visualized by UV
(254 nm) and a 5% solution of orcinol in 5% solution of H2SO4 in
EtOH, followed by heating. For column chromatography, Geduran Si
CDCl3)
d 170.6, 170.0, 169.4, 169.3 (CO), 161.8, 152.7, 135.8, 126.4,
125.0, 122.3, 121.1 (Carom), 84.0 (C-1), 76.2 (C-5), 73.7 (C-3), 69.6
(C-2), 68.0 (C-4), 61.8 (C-6), 20.7, 20.6, 20.5 (CH3); Calcd for
C21H23NO9S2: C 50.70, H 4.66, N 2.82, S 12.89; Found C 50.53, H 4.67,
N 2.80, S 12.91.
60 (40e63 mm) Silica Gel was used. Optical rotations were mea-
sured on a PerkineElmer 341 polarimeter. 1H, 13C, COSY, HSQC and
€
TOCSY1d NMR spectra were recorded with a Bruker ARX 400
spectrometer at 400 MHz for 1H and 100 MHz for 13C. Chemical
shifts are given in
lated in Hertz (Hz). Abbreviations were used to precise signal
d
units (ppm). Coupling constants J were calcu-
4.2.2. Data for 6. Compound 6 was prepared as described starting
from donor 1 (5.00 g, 12.8 mmol), 2-mercaptobenzimidazole

D. Pro et al. / Tetrahedron 68 (2012) 7095e7102
7099
(5.81 g, 38.4 mmol) and BF3.OEt2 (14.6 mL, 115.2 mmol).
Column chromatography (7:3 Toluene/AcOEt) afforded 6 (5.47 g,
11.4 mmol) as a white solid in 89% yield. TLC (4:1 light petroleum/
deprotection step after simple work-up but without chromato-
graphic purification.
AcOEt): Rf 0.5; ½a D20
ꢂ
ꢁ22.1 (c 0.86, CH2Cl2); mp 138e139 ꢀC; 1H
4.2.6. Data for 10. Gentiobioside 10 was obtained starting from
donor 3 (2.00 g, 2.9 mmol), 2-mercaptobenzimidazole (1.33 g,
8.8 mmol) and BF3.OEt2 (3.4 mL, 26.5 mmol). Column chromatog-
raphy (3:2 Cyclohexane/AcOEt) yielded the desired compound 10
(1.54 g, 2.0 mmol, 68%) as a white solid. TLC (2:3 Toluene/AcOEt): Rf
NMR (400 MHz, CDCl3)
d 7.60 (s, 2H, Harom), 7.29e7.24 (m, 2H,
Harom), 5.27 (ddd, 1H, J3,4¼9.5 Hz, J2,3¼7.3 Hz, J1,3¼2.0 Hz, H-3),
5.12e5.06 (m, 3H, H-1, H-2, H-4), 4.38 (dd, 1H,
J6a,6b¼12.5 Hz, J5,6b¼2.3 Hz, H-6b), 4.19 (dd, 1H, J5,6a¼4.7 Hz, H-6a),
3.79 (ddd, 1H, J4,5¼10.2 Hz, H-5), 2.11, 2.08, 2.05, 2.00 (4ꢃs, 12H,
0.4; ½a 2D0
ꢂ
ꢁ5.5 (c 4.00, CH2Cl2); mp 96e97 ꢀC; 1H NMR (400 MHz,
COCH3); 13C NMR (100 MHz, CDCl3)
d
171.2, 170.0, 169.5, 169.3
CDCl3) d 7.65 (s, 2H, Harom), 7.34e7.28 (m, 2H, Harom), 5.26 (t, 1H,
0
0
0
0
0
(CO), 143.5, 123.1 (Carom), 83.4 (C-1), 76.4 (C-5), 73.4 (C-3), 70.0
(C-2), 67.9 (C-4), 61.6 (C-6), 20.9, 20.6, 20.5, 20.5 (CH3); Calcd for
C21H24N2O9S: C 52.49, H 5.03, N 5.83, S 6.67; Found C 52.46, H
5.04, N 5.84, S 6.54.
J2 ,3 ¼J3 ,4 ¼9.6 Hz, H-3 ), 5.25 (t, 1H, J2,3¼J3,4¼9.3 Hz, H-3), 5.09
0
0
(dd, 1H, J1,2¼10.1 Hz, J2,3¼9.3 Hz, H-2), 5.08 (dd, 1H, J1 ,2 ¼8.0 Hz,
H-20), 5.02 (t, 1H, J4,5¼9.8 Hz, H-4), 4.98 (d, 1H, H-1), 5.04
0
0
0
0
(t, 1H, J4 ,5 ¼9.9 Hz, H-4 ), 4.61 (d, 1H, H-1 ), 4.16 (dd, 1H,
J6 a,6 b¼12.4 Hz, J5 ,6 b¼2.7 Hz, H-60b), 4.11 (dd, 1H, J5 ,6 a¼4.6 Hz,
H-60a), 3.89 (dd, 1H, J6a,6b¼10.7 Hz, J5,6b¼2.0 Hz, H-6b), 3.79 (ddd,
1H, J5,6a¼6.5 Hz, H-5), 3.71 (dd, 1H, H-6a), 3.70 (ddd, 1H, H-50), 2.10,
2.07, 2.05, 2.04, 2.03, 2.00, 1.98 (7ꢃs, 21H, CH3CO); 13C NMR
0
0
0
0
0
0
4.2.3. Data for 7. Disaccharide 7 was prepared as described start-
ing from donor 2 (3.00 g, 4.42 mmol), 2-mercaptobenzothiazole
(2.22 g, 13.2 mmol) and BF3.OEt2 (5.0 mL, 39.8 mmol).
Column chromatography (3:2 Cyclohexane/AcOEt) gave 7 (2.37 g,
3.0 mmol) as a white solid in 68% yield. TLC (2:3 Cylohexane/
(100 MHz, CDCl3) d 170.6, 170.3,170.1, 170.0, 169.6, 169.4, 169.4 (CO),
144.2, 123.3 (Carom), 100.5 (C-10), 84.5 (C-1), 76.8 (C-5), 73.5, 72.3
(C-3, C-30), 72.2 (C-50), 70.7 (C-20), 69.8 (C-2), 68.4, 68.2 (C-4, C-40),
67.2 (C-6), 61.7 (C-60), 20.8, 20.7, 20.6, 20.6, 20.6, 20.5 (CH3); Calcd
for C33H40N2O17S: C 51.56, H 5.24, N 3.64, S 4.17; Found C 51.40, H
5.29, N 3.91, S 3.98.
AcOEt): Rf 0.6; ½a D20
ꢂ
ꢁ19.2 (c 2.60, CH2Cl2); mp 205e206 ꢀC; 1H
NMR (400 MHz, CDCl3)
d
7.93 (ddd, 1H, J¼8.3 Hz, J¼1.1 Hz,
J¼0.6 Hz, Harom), 7.78 (ddd, 1H, J¼8.0 Hz, J¼1.2 Hz, J¼0.6 Hz, Harom),
7.46 (ddd, 1H, J¼8.3 Hz, J¼7.2 Hz, J¼1.2 Hz, Harom), 7.35 (ddd, 1H,
J¼8.0 Hz, J¼7.2 Hz, J¼1.1 Hz, Harom), 5.51 (d, 1H, J1,2¼10.3 Hz, H-1),
0
0
5.31 (t, 1H, J2,3¼J3,4¼9.1 Hz, H-3), 5.15 (t, 1H, J3 ,4 ¼9.4 Hz,
4.2.7. Data for 11. Lactosyl intermediate 11 was synthesized start-
ing from donor 4 (1.00 g, 1.47 mmol), 2-mercaptobenzothiazole
(0.74 g, 4.42 mmol) and BF3.OEt2 (1.7 mL, 13.2 mmol). Chromato-
graphic purification (4:1 Toluene/AcOEt) yielded the desired com-
pound 11 (0.57 g, 0.73 mmol, 49%) as a white solid. TLC (2:3
0
0
0
0
0
J2 ,3 ¼9.2 Hz, H-3 ), 5.14 (dd, 1H, H-2), 5.07 (t, 1H, J4 ,5 ¼9.7 Hz,
H-40), 4.94 (dd, 1H, J1 ,2 ¼7.9 Hz, H-20), 4.55 (dd, 1H,
0
0
J6a,6b¼12.1 Hz, J5,6b¼1.7 Hz, H-6b), 4.52 (d, 1H, H-10), 4.38 (dd, 1H,
J6 a,6 b¼12.5 Hz, J5 ,6 a¼4.4 Hz, H-60a), 4.15 (dd, 1H, J5,6a¼4.9 Hz,
0
0
0
0
H-6a), 4.05 (dd, J5 ,6 b¼2.3 Hz, H-600b), 3.89e3.82 (m, 2H, H-4, H-5),
Toluene/AcOEt): Rf 0.7; ½a D20
ꢂ
ꢁ9.0 (c 3.1, CH2Cl2); mp 167e169 ꢀC; 1H
0
0
0
0
3.67 (ddd, 1H, J4 ,5 ¼9.7 Hz, H-5 ), 2.09, 2.06, 2.05, 2.04, 2.03,
NMR (400 MHz, CDCl3)
d
7.93 (ddd, 1H, J¼8.2 Hz, J¼1.2 Hz, J¼0.6 Hz,
2.01, 1.99 (7ꢃs, 21H, COCH3); 13C NMR (100 MHz, CDCl3)
d
170.5,
Harom), 7.78 (ddd, 1H, J¼8.0 Hz, J¼1.2 Hz, J¼0.6 Hz, Harom), 7.45 (ddd,
1H, J¼8.2 Hz, J¼7.3 Hz, J¼1.2 Hz, Harom), 7.35 (ddd, 1H, J¼8.0 Hz,
J¼7.3 Hz, J¼1.2 Hz, Harom), 5.52 (d, 1H, J1,2¼10.3 Hz, H-1), 5.36
170.2, 169.6, 169.3, 169.1 (CO), 161.9, 152.7, 135.8, 126.4, 125.0, 122.4,
120.9 (Carom), 100.8 (C-10), 83.9 (C-1), 77.2, 76.2 (C-4, C-5), 73.3
(C-3), 72.9 (C-30), 72.0 (C-50), 71.6 (C-20), 69.9 (C-2), 67.8 (C-40), 61.9
(C-6), 61.6 (C-60), 20.8, 20.7, 20.6, 20.5, 20.4 (CH3); Calcd for
C33H39NO17S2: C 50.44, H 5.00, N 1.78, S 8.16; Found C 50.31, H
5.05, N 1.73, S 8.13.
(dd, 1H, J3 ,4 ¼3.5 Hz, J4 ,5 ¼1.0 Hz, H-40), 5.33 (t, 1H,
0
0
0
0
0
0
J2,3¼J3,4¼8.6 Hz, H-3), 5.14 (dd, 1H, H-2), 5.12 (dd, 1H, J2 ,3 ¼10.4 Hz,
J1 ,2 ¼7.9 Hz, H-20), 4.96 (dd, 1H, H-30), 4.52 (dd, 1H,
J6a,6b¼12.0 Hz, J5,6b¼1.7 Hz, H-6b), 4.49 (d, 1H, H-10), 4.15 (dd, 1H,
J5,6a¼4.9 Hz, H-6a), 4.15e4.06 (m, 2H, H-60a, H-60b), 3.91e3.86 (m,
1H, H-50), 3.89 (dd, 1H, J4,5¼9.9 Hz, H-4), 3.84 (ddd, 1H, H-5), 2.16,
2.06, 2.06, 2.05, 2.04, 2.03, 1.97 (7ꢃs, 3H, CH3CO); 13C NMR
0
0
4.2.4. Data for 8. Disaccharide 8 was synthesized starting from
donor 2 (3.00 g, 4.42 mmol), 2-mercaptobenzimidazole (1.99 g,
13.2 mmol) and BF3.OEt2 (5.0 mL, 39.8 mmol). Column chroma-
tography (3:2 Cyclohexane/AcOEt) gave the desired compound 8
(2.58 g, 3.4 mmol, 76% yield) as a white solid. TLC (2:3 Cylohexane/
(100 MHz, CDCl3) d 170.3, 170.2,170.1, 170.0, 169.6, 169.5, 169.0 (CO),
161.8,152.7,135.7,126.3,125.0,122.3,120.9 (Carom),101.0 (C-10), 83.8
(C-1), 77.0 (C-5), 75.9 (C-4), 73.5 (C-3), 70.9 (C-30), 70.7 (C-50), 69.9
(C-2), 69.0 (C-20), 66.6 (C-40), 62.0 (C-6), 60.8 (C-60), 20.8, 20.7, 20.6,
20.5 (CH3); Calcd for C33H39NO17S2: C 50.44, H 5.00, N 1.78, S 8.16;
Found C 50.62, H 5.11, N 1.88, S 3.84.
AcOEt): Rf 0.4; ½a D20
ꢂ
ꢁ30.5 (c 4.85, CH2Cl2); mp 144e146 ꢀC; 1H NMR
(400 MHz, CDCl3)
d 7.64 (s, 2H, Harom), 7.32e7.26 (m, 2H, Harom),
0
0
5.22 (t, 1H, J2,3¼J3,4¼8.9 Hz, H-3), 5.16 (t, 1H, J2 ,3 ¼9.4 Hz,
0
0
0
0
0
0
J3 ,4 ¼9.6 Hz, H-3 ), 5.07 (t, 1H, J4 ,5 ¼9.6 Hz, H-4 ), 5.00 (dd, 1H,
0
0
0
J1,2¼10.0 Hz, H-2), 4.95 (d, 1H, H-1), 4.93 (dd, 1H, J1 ,2 ¼8.0 Hz, H-2 ),
4.81 (dd, 1H, J6a,6b¼12.3 Hz, J5,6b¼2.2 Hz, H-6b), 4.57 (d, 1H, H-10),
4.2.8. Data for 12. Lactosyl intermediate 12 was obtained from
donor 4 (2.39 g, 3.5 mmol), 2-mercaptobenzimidazole (1.59 g,
10.6 mmol) and BF3.OEt2 (4.0 mL, 31.7 mmol). Chromatographic
purification (7:3/1:1 Toluene/AcOEt) afforded compound 12
(1.64 g, 2.1 mmol) in 61% yield as a white solid. TLC (2:3 Toluene/
4.37 (dd, 1H, J6 a,6 b¼12.5 Hz, J5 ,6 a¼4.3 Hz, H-60a), 4.13 (dd,
0
0
0
0
1H, J5,6a¼4.1 Hz, H-6a), 4.03 (dd, 1H, J5 ,6 b¼2.2 Hz, H-60b), 3.78
(dd, 1H, J4,5¼9.3 Hz, H-4), 3.70e3.62 (m, 2H, H-5, H-50), 2.16, 2.08,
2.06, 2.02, 2.01, 2.00, 1.98 (7ꢃs, 21H, COCH3); 13C NMR (100 MHz,
0
0
AcOEt): Rf 0.5; ½a D20
ꢂ
ꢁ25.1 (c 5.3, CH2Cl2); mp 106e108 ꢀC; 1H NMR
CDCl3)
d
171.4, 170.5, 170.3, 169.8, 169.5, 169.2, 168.8 (CO), 143.2,
(400 MHz, CDCl3) d 7.59 (s, 2H, Harom), 7.27e7.22 (m, 2H, Harom),
123.4 (Carom), 100.6 (C-1), 82.9 (C-10), 77.2 (C-5), 75.6, (C-4), 72.9
(C-3), 72.8 (C-30), 72.1 (C-50), 71.6 (C-20), 70.3 (C-20), 67.6 (C-40), 61.4
(C-60), 60.9 (C-606), 21.1, 20.6, 20.5, 20.4 (CH3); Calcd for
C33H40N2O17S: C 51.56, H 5.24, N 3.64, S 4.17; Found C 51.26, H 5.27,
N 3.30, S 3.80.
5.34 (dd, 1H, J3 ,4 ¼3.4 Hz, J4 ,5 ¼1.1 Hz, H-4 ), 5.24 (ddd, 1H,
0
0
0
0
0
0
0
J3,4¼9.1 Hz, J2,3¼6.2 Hz, J1,3¼2.8 Hz, H-3), 5.11 (dd, 1H, J2 ,3 ¼10.5 Hz,
0
0
0
0
J1 ,2 ¼7.9 Hz, H-2 ), 5.03e4.96 (m, 2H, H-1, H-2), 4.98 (dd, 1H, H-3 ),
4.77 (dd, 1H, J6a,6b¼12.2 Hz, J5,6b¼2.3 Hz, H-6b), 4.54 (d, 1H, H-10),
4.13 (dd, 1H, J5,6a¼4.2 Hz, H-6a), 4.14e4.03 (m, 2H, H-60a, H-60b),
3.88 (ddd, 1H, J5 ,6 a¼7.4 Hz, J5 ,6 b¼6.4 Hz, H-50), 3.80 (dd, 1H,
J4,5¼9.9 Hz, J3,4¼9.1 Hz, H-4), 3.65 (ddd, 1H, H-5), 2.15, 2.14, 2.07,
2.04, 2.03, 2.02, 1.96 (7ꢃs, 21H, CH3CO); 13C NMR (100 MHz, CDCl3)
0
0
0
0
4.2.5. Data for 9. This intermediate was prepared starting
from peracetylated gentiobiose
3 (2.00 g, 2.9 mmol), 2-
mercaptobenzothiazole (1.48 g, 8.8 mmol) and BF3.OEt2 (3.4 mL,
26.5 mmol). The resulting product 9 was further engaged in the
d 171.3, 170.3, 170.1, 170.0, 169.8, 169.5, 168.9 (CO), 143.3, 123.0
(Carom), 100.9 (C-10), 83.0 (C-1), 77.2 (C-5), 75.5 (C-4), 73.3 (C-3),

7100
D. Pro et al. / Tetrahedron 68 (2012) 7095e7102
70.8 (C-50), 70.7 (C-30), 70.4 (C-2), 69.1 (C-20), 66.5 (C-40), 61.1 (C-6),
60.7 (C-60), 21.0, 20.7, 20.6, 20.5 (CH3); Calcd for C33H40N2O17S: C
51.56, H 5.24, N 3.64, S 4.17; Found C 51.42, H 5.49, N 3.53, S 4.00.
the desired product (0.66,1.4 mmol). TLC (3:1 CH2Cl2/MeOH): Rf 0.6;
½
a 2D0
ꢂ
ꢁ53.2 (c 2.50, MeOH); mp 159e160 ꢀC; 1H NMR (400 MHz, D2O)
d
7.63 (dd, 2H, J¼6.1 Hz, J¼3.2 Hz, Harom), 7.38 (dd, 2H, J¼6.1 Hz,
J¼3.2 Hz, Harom), 5.12 (d, 1H, J1,2¼9.9 Hz, H-1), 4.50 (d, 1H,
0
0
0
4.3. General procedure for deacetylation
J1 ,2 ¼7.9 Hz, H-1 ), 3.97 (d, 1H, J6a,6b¼12.0 Hz, H-6b), 3.90 (dd, 1H,
J6 a,6 b¼12.3 Hz, J5 ,6 b¼2.1 Hz, H-60b), 3.84 (dd, 1H, J5,6a¼2.8 Hz, H-
0
0
0
0
6a), 3.72 (dd,1H, J5 ,6 a¼5.7 Hz, H-60a), 3.71e3.68 (m, 3H, H-3, H-4, H-
0
0
To a solution of per-O-acylated derivative in anhydrous metha-
nol was added a 0.1 M solution of sodium methylate in methanol.
The mixture was stirred at room temperature until no starting
material was detected by TLC. Neutralisation was then performed
by adding Amberlite IR-120 (Hþ-form). The resin was finally filtered
off and the solvent removed under reduced pressure to afford the
desired product.
0
0
0
0
0
0
5), 3.50 (t,1H, J2 ,3 ¼J3 ,4 ¼9.1 Hz, H-3 ), 3.50e3.44 (m, 2H, H-5 , H-2),
3.41 (dd, 1H, J4 ,5 ¼9.6 Hz, J3 ,4 ¼9.1 Hz, H-4 ), 3.31 (dd, 1H, H-2 ); 13C
0
0
0
0
0
0
NMR (100 MHz, D2O) d
145.5, 138.1, 123.1, 114.3 (Carom), 102.4 (C-10),
85.4 (C-1), 79.0 (C-5), 77.9 (C-3), 75.9 (C-50), 75.5 (C-30), 75.4 (C-4),
73.0 (C-20), 71.7 (C-2), 69.4 (C-40), 60.5 (C-60), 59.9 (C-6); ESI-HRMS
for [C19H26N2O10SþNa]þ: calcd m/z 497.12059, found 497.1205.
4.3.1. Data for Glc-S-BZT. Zemplen transesterification was per-
formed from 5 (5.11 g, 10.3 mmol) using 0.1 M NaOMe/MeOH
(10.3 mL). Work-up provided the desired product (3.38 g,
10.3 mmol, 100%) as a white powder. TLC (4:1 CH2Cl2/MeOH): Rf
4.3.5. Data for Gen-S-BZT. The crude product 9 was subjected to
deprotection using 0.1 M NaOMe/MeOH (2.9 mL). The resulting
material was purified by colomn chromatography on silica gel (7:3
CH2Cl2/MeOH) to give Gen-S-BZT as a white solid in a 28% yield
0.7; ½a 2D0
ꢂ
ꢁ72.5 (c 3.6, MeOH); mp 80e81 ꢀC; 1H NMR (400 MHz,
(0.41 g, 0.83 mmol). TLC (7:3 CH2Cl2/MeOH): Rf 0.3; ½a D20
ꢂ
ꢁ81.4
D2O)
d
7.88 (d,1H, J¼8.0 Hz, Harom), 7.83 (d,1H, J¼8.1 Hz, Harom), 7.51
(c 2.85, MeOH); mp 128e131 ꢀC; 1H NMR (400 MHz, D2O)
d 7.89
(t, 1H, J¼7.7 Hz, Harom), 7.42 (t, 1H, J¼7.3 Hz, Harom), 5.17 (d, 1H,
J1,2¼9.1 Hz, H-1), 3.96 (dd, 1H, J6a,6b¼12.5 Hz, J5,6b¼2.0 Hz, H-6b),
3.79 (dd, 1H, J5,6b¼5.3 Hz, H-6a), 3.66e3.50 (m, 4H, H-2, H-3, H-4,
(d, 1H, J¼8.0 Hz, Harom), 7.84 (d, 1H, J¼8.2 Hz, Harom), 7.51 (ddd, 1H,
J¼8.2, J¼7.5, J¼0.9 Hz, Harom), 7.42 (ddd, 1H, J¼8.0, J¼7.4, J¼0.9 Hz,
0
0
0
Harom), 5.23 (d, 1H, J1,2¼9.3 Hz, H-1), 4.42 (d, 1H, J1 ,2 ¼7.6 Hz, H-1 ),
H-5); 13C NMR (100 MHz, D2O)
d 164.7, 151.2, 134.9, 129.5, 125.1,
4.19 (dd, 1H, J6a,6b¼11.9 Hz, J5,6b¼1.7 Hz, H-6b), 3.89 (dd, 1H,
J5,6a¼5.4 Hz, H-6a), 3.82 (d, 1H, J6 a,6 b¼12.2 Hz, H-60b), 3.82e3.75
0
0
121.2, 120.9 (Carom), 86.1 (C-1), 80.1 (C-5), 77.0 (C-3), 71.7 (C-2), 68.9
(C-4), 60.5 (C-6); ESI-HRMS for [C13H15NO5S2þNa]þ: calcd m/z
352.02894, found 352.0289.
0
0
0
(m, 1H, H-5), 3.62 (dd, 1H, J5 ,6a ¼3.3 Hz, H-6 a), 3.61e3.52 (m, 3H,
H-2, H-3, H-4), 3.31e3.25 (m, 3H, H-30, H-40, H-50), 3.25e3.18
(m, 1H, H-20); 13C NMR (100 MHz, D2O)
d 164.5, 151.5, 135.2, 126.8,
4.3.2. Data for Glc-S-BZI. Deacylation of 6 (5.38 g, 11.2 mmol) using
0.1 M NaOMe/MeOH (11.2 mL) gave the target compound (3.44 g,
11.0 mmol, 100%) as a white solid. TLC (4:1 CH2Cl2/MeOH): Rf 0.5;
125.4, 121.5, 121.2, (Carom), 102.3 (C-10), 86.1 (C-1), 79.4 (C-5), 76.9
(C-3), 75.8 (C-50), 75.5 (C-30), 73.0 (C-20), 71.6 (C-2), 69.4 (C-40), 68.8
(C-4), 68.0 (C-6), 60.6 (C-60); ESI-HRMS for [C19H25NO10S2þNa]þ:
calcd m/z 514.08176, found 514.0811.
½
a 2D0
D2O)
ꢂ
ꢁ53.6 (c 5.75, MeOH); mp 114e116 ꢀC; 1H NMR (400 MHz,
d
7.45e7.40 (m, 2H, Harom), 7.21e7.16 (m, 2H, Harom), 4.96
(d, 1H, J1,2¼9.9 Hz, H-1), 3.86 (dd, 1H, J6a,6b¼12.4 Hz, J5,6b¼2.2 Hz,
H-6b), 3.69 (dd, 1H, J6a,6b¼12.4 Hz, J5,6a¼5.6 Hz, H-6a), 3.52 (t, 1H,
J2,3¼J3,4¼8.9 Hz, H-3), 3.47 (ddd, 1H, J4,5¼9.8 Hz, H-5), 3.40 (dd, 1H,
4.3.6. Data for Gen-S-BZI. Deprotection of disaccharide 10 (1.47 g,
1.9 mmol) was performed with 0.1 M NaOMe/MeOH (1.9 mL) and
afforded the desired derivative Gen-S-BZI, which was chromato-
graphically purified (7:3 CH2Cl2/MeOH) and isolated as a white
solid (0.90 g, 1.9 mmol, 100%). TLC (7:3 CH2Cl2/MeOH): Rf 0.3;
H-4), 3.35 (dd, 1H, H-2); 13C NMR (100 MHz, D2O)
d 145.6, 138.1,
123.0, 110.3 (Carom), 85.5 (C-1), 80.1 (C-5), 76.9 (C-3), 71.9 (C-2), 69.0
(C-4), 60.5 (C-6); ESI-HRMS for [C13H16N2O5SþNa]þ: calcd m/z
335.06776, found 335.0673.
½
a 2D0
ꢂ
ꢁ45.8 (c 3.95, MeOH); mp 134e136 ꢀC; 1H NMR (400 MHz,
D2O)
d
7.60 (dd, 2H, J¼6.0 Hz, J¼3.2 Hz, Harom), 7.33 (dd, 2H,
J¼6.0 Hz, J¼3.2 Hz, Harom), 5.16 (d, 1H, J1,2¼9.9 Hz, H-1), 4.41 (d, 1H,
0
0
0
4.3.3. Data for Cel-S-BZT. Deprotection of 7 (2.36 g, 3.0 mmol)
using 0.1 M NaOMe/MeOH (3.0 mL) gave Cel-S-BZT (1.46 g,
3.0 mmol, 100%) as a white solid. The latter was further recrystal-
lized from MeOH/CH2Cl2 to afford the desired product (0.85 g,
J1 ,2 ¼7.2 Hz, H-1 ), 4.15 (dd, 1H, J6a,6b¼11.9 Hz, J5,6b¼1.5 Hz, H-6b),
3.90 (dd, 1H, J5,6a¼5.8 Hz, H-6a), 3.83 (d, 1H, J6 a,6 b¼12.1 Hz, H-60b),
0
0
3.74 (ddd, 1H, J4,5¼9.1 Hz, H-5), 3.58 (t, 1H, J2,3¼J3,4¼9.1 Hz, H-3),
0
0
0
3.57 (dd, 1H, J5 ,6a ¼2.9 Hz, H-6 a), 3.50 (t, 1H, H-4), 3.44 (dd, 1H,
1.7 mmol) in 58% yield. TLC (7:3 CH2Cl2/MeOH): Rf 0.5; ½a D20
ꢂ
ꢁ121.6
H-2), 3.31e3.21 (m, 4H, H-20, H-30, H-40, H-50); 13C NMR (100 MHz,
(c 1.25, MeOH); mp 141e143 ꢀC; 1H NMR (400 MHz, D2O)
d
7.88
D2O) d
145.8, 123.2 (Carom), 102.4 (C-10), 85.5 (C-1), 79.2 (C-5), 76.9
(ddd, 1H, J¼8.3 Hz, J¼1.1 Hz, J¼0.6 Hz, Harom), 7.84 (ddd, 1H,
J¼8.3 Hz, J¼1.1 Hz, J¼0.6 Hz, Harom), 7.51 (ddd, 1H, J¼8.3 Hz,
J¼7.3 Hz, J¼1.1 Hz, Harom), 7.42 (ddd, 1H, J¼8.3 Hz, J¼7.3 Hz,
J¼1.1 Hz, Harom), 5.19 (d, 1H, J1,2¼9.9 Hz, H-1), 4.53 (d,
(C-3), 75.8, 75.5 (C-30, C-50), 73.0 (C-20), 71.9 (C-2), 69.5 (C-40), 69.0
(C-4), 68.5 (C-6), 60.6 (C-60); ESI-HRMS for [C19H26N2O10SþNa]þ:
calcd m/z 497.12059, found 497.1199.
0
0
0
1H, J1 ,2 ¼7.9 Hz, H-1 ), 4.02 (d, 1H, J6a,6b¼11.7 Hz, H-6b), 3.92 (dd,
4.3.7. Data for Lac-S-BZT. Product 11 (3.55 g, 4.2 mmol) was
deacylated using 0.1 M NaOMe/MeOH (4.2 mL). The resulting ma-
terial was purified by recrystallization from water to afford Lac-S-
BZT as a white solid in an 80% yield (1.66 g, 3.4 mmol). TLC (7:3
1H, J6 a,6 b¼12.4 Hz, J5 ,6 b¼2.2 Hz, H-60b), 3.88 (dd, 1H, J5,6a¼2.9 Hz,
0
0
0
0
0
0
H-6a), 3.78e3.69 (m, 3H, H-3, H-4, H-5), 3.73 (dd, 1H, J5 ,6 a¼5.6 Hz,
H-60a), 3.62 (dd, 01H, J2,3¼8.6 Hz, H-2), 3.52 (t, 1H,
J2 ,3 ¼J3 ,4 ¼9.1 Hz, H-3 ), 3.49 (ddd, 1H, J4 ,5 ¼9.6 Hz, H-5 ), 3.42 (dd,
CH2Cl2/MeOH): Rf 0.5; ½a D20
ꢂ
ꢁ23.3 (c 3.0, DMSO); mp 186e187 ꢀC;
0
0
0
0
0
0
0
1H, H-40), 3.34 (dd, 1H, H-20); 13C NMR (100 MHz, D2O)
d
164.7,
1H NMR (400 MHz, DMSO-d6)
d
8.04 (ddd, 1H, J¼7.9 Hz, J¼1.2 Hz,
151.4, 135.2, 126.8, 125.4, 121.5, 121.1 (Carom), 102.5 (C-10), 86.0 (C-1),
79.0 (C-5), 77.8 (C-3), 76.0 (C-50), 75.5 (C-30), 75.4 (C-4), 73.1 (C-20),
71.5 (C-20), 69.4 (C-40), 60.5 (C-60), 59.8 (C-6); ESI-HRMS for
[C19H25NO10S2þNa]þ: calcd m/z 514.08176, found 514.0814.
J¼0.5 Hz, Harom), 7.88 (ddd, 1H, J¼8.1 Hz, J¼1.0 Hz, J¼0.5 Hz, Harom),
7.48 (ddd, 1H, J¼8.1 Hz, J¼7.2 Hz, J¼1.2 Hz, Harom), 7.39 (ddd, 1H,
J¼7.9 Hz, J¼7.2 Hz, J¼1.0 Hz, Harom), 5.81 (d, 1H, J¼6.3 Hz, OH-2),
5.19 (d, 1H, J1,2¼9.9 Hz, H-1), 5.11 (d, 1H, J¼4.4 Hz, OH-20), 4.88
(d, 1H, J¼1.2 Hz, OH-40), 4.80 (d, 1H, J¼5.2 Hz, OH-3), 4.66 (d, 1H,
J¼5.14 Hz, OH-6), 4.65 (dd, 1H, J¼5.4 Hz, J¼1.1 Hz, OH-60), 4.53
4.3.4. Data for Cel-S-BZI. Deprotection of 8 (2.46 g, 3.2 mmol) was
performed with 0.1 M NaOMe/MeOH (3.2 mL) and afforded the
cellobiosyl derivative Cel-S-BZI (1.49 g, 3.1 mmol, 100%) as a white
solid. The latter was further recrystallized from MeOH/AcOEt to give
(d, 1H, J¼4.6 Hz, OH-30), 4.27 (d, 1H, J1 ,2 ¼7.3 Hz, H-1 ), 3.80 (ddd,
0
0
0
1H, J6a,6b¼12.0 Hz, J¼5.5 Hz, J¼2.1 Hz, H-6b), 3.71 (ddd, 1H,
0
0
0
0
J¼6.6 Hz, J¼4.2 Hz, H-6a), 3.63 (dd, 1H, J2 ,3 ¼4.3 Hz, J3 ,4 ¼3.0 Hz,

D. Pro et al. / Tetrahedron 68 (2012) 7095e7102
7101
H-30), 3.58e3.44 (m, 6H, H-4, H-5, H-40, H-50, H-60a, H-60b),
3.39e3.29 (m, 3H, H-2, H-3, H-20); 13C NMR (100 MHz, D2O)
164.5,
Bacteria were fed continuously, and consumed each week a solu-
tion of 50 L of water containing 32 g of NH4HCO3, 30 g NaHCO3, 4 g
Na2HPO4 and a spatula tip of iron-II chloride. After one or six
months, the consortium of bacteria can be used for inhibition tests.
d
152.2, 135.0, 126.3, 124.5, 121.6, 121.3 (Carom), 103.6 (C-10), 85.9
(C-1), 79.6 (C-5), 79.2 (C-40), 76.0 (C-30), 75.5 (C-50), 73.1 (C-4), 72.2
(C-20), 70.5 (C-2), 68.1 (C-3), 60.3 (C-6), 60.0 (C-60); ESI-HRMS for
[C19H25NO10S2þNa]þ: calcd m/z 514.08176, found 514.0823.
4.7. Preparation of bacteria solution
4.3.8. Data for Lac-S-BZI. Deacylation of 12 (1.64 g, 2.1 mmol) was
performed with 0.1 M NaOMe/MeOH (2.1 mL) and afforded the
lactosyl derivative Lac-S-BZI (1.49 g, 3.1 mmol, 100%) as a white
solid. The latter was further recrystallized from MeOH/toluene
afforded the desired derivative isolated as a white solid (0.45 g,
A 500 mL of bacteria solution was taken from the reactor. After
a first decantation, and removal of the supernantant, ultrapure
water (200 mL) was added to the resulting mixture of bacteria. The
suspension was then stirred until homogenization, decanted and
the supernatant removed. This procedure was further repeated
three times more. The resulting sample was subsequently used for
the inhibition tests.
0.95 mmol, 44%). TLC (7:3 CH2Cl2/MeOH): Rf 0.4; ½a D20
ꢂ
ꢁ48.5 (c 1.65,
MeOH); mp 163e164 ꢀC; 1H NMR (400 MHz, D2O)
d
7.61 (dd, 2H,
J¼6.1 Hz, J¼3.2 Hz, Harom), 7.34 (dd, J¼6.1 Hz, J¼3.2 Hz, Harom), 5.11
0
0
0
(d, 1H, J1,2¼9.9 Hz, H-1), 4.44 (d, 1H, J1 ,2 ¼7.8 Hz, H-1 ), 3.97 (dd, 1H,
4.8. Inhibition
0
0
J6a,6b¼12.4 Hz, J5,6b¼1.4 Hz, H-6b), 3.92 (dd, 1H, J3 ,4 ¼3.4 Hz,
0
0
0
J4 ,5 ¼0.7 Hz, H-4 ), 3.83 (dd, 1H, J5,6a¼3.9 Hz, H-6a), 3.80e3.74
For each inhibitor, a 30 mL of the solution containing ammo-
(m, 2H, H-60a, H-60b), 3.73e3.68 (m, 4H, H-3, H-4, H-5, H-50), 3.66
nium chloride (100 mg/L in NH4þ), inhibitor (100
mM), and finally
0
0
0
0
(dd, 1H, J2 ,3 ¼10.0 Hz, H-3 ), 3.55 (dd, 1H, H-2 ), 3.46 (ddd, 1H,
1 mL of bacteria solution was prepared in a test tube containing 1 g
of maerl (1 g). The blank solution consisted in the same mixture but
without organic compounds. All mixtures were prepared in paral-
lel. Test tubes were stirred at 400 rpm and room temperature.
Measurements of pH, ammonium, nitrite, nitrate concentrations
and cell viability were achieved as follows.
J2,3¼6.5 Hz, J2,4¼2.3 Hz, H-2).; 13C NMR (100 MHz, D2O)
d 145.7,
123.3 (Carom), 102.8 (C-10), 85.4 (C-1), 79.0, 77.5, 75.6, 75.3 (C-3, C-4,
C-5, C-50), 72.5 (C-30), 71.7 (C-2), 70.9 (C-20), 68.5 (C-40), 61.0 (C-60),
59.9 (C-6); ESI-HRMS for [C19H26N2O10SþNa]þ: calcd m/z
497.12059, found 497.1200.
4.4. LC/MS analysis
4.9. Cell viability
Mass spectrometry analysis was carried out on a Quattro Pre-
mier (Micromass, Manchester, UK) equipped with an electrospray
source (ESI Z-sprayÔ) and a triple quadrupole. The nebulizer and
desolvation gas was nitrogen generated from the nitrogen gener-
ator NM30LA (Peak scientific, Inchinnam, UK) and the collision gas
was high grade argon (Air Liquide, Paris, France). The ESI source
parameters were as follows: capillary voltage, 3 kV; source tem-
perature, 120 ꢀC; cone gas flow, 50 L/h, desolvation gas tempera-
ture, 350 ꢀC; desolvation gas flow, 750 L/h; extraction voltage, 1 V;
RF lens voltage, 0.3 V. Table 1 summarizes the method parameters
for the molecules detection. The UPLC/MS/MS was controlled by
the software Mass LynxÔ and data processing was performed by
the program Target Lynx.
Determination of ammoniums, nitrite and nitrate concentra-
tions: The desired concentrations were further determined starting
from 1 mL of samples, taken under stirring, which were filtrated
through filter paper. The resulting filtrate was diluted with ultra-
pure water (5e100). The concentration in ammonium cations was
obtained photometrically, using the ammonium Test from Merck
(Ref 1.14,752.0001). The concentration in nitrites was determined
either photometrically at 537 nm using a diazotation reactive
(sulfanilamide and N-(1-naphtyl)ethylene diamine solution in
water), or by ion chromatography. Nitrates concentration was de-
termined by ion chromatography.
4.10. Ion chromatographic analysis
Analyte
Glucose
Ionization Mass transition Cone voltage (V) Collision energy (V)
The samples (250
mL) were injected into a DX 120 system
ESIꢁ
179/89
20
20
17
20
30
20
5
10
13
15
20
12
Gentiobiose ESIꢁ
Glc-S-BZT ESIþ
Gen-S-BZT ESIþ
342/179
330/168
492/168
313/151
475/151
(Dionex) equipped with an Ion Pac AS 18 column (4 x 250 mm, Ref
062885), a precolumn AG 18 (Ref 062887) and a suppressor ASRS
300 (50 mA, 4 mm, P/N 064554) and eluted at a flow rate of 1 mL/
min with a gradient solution of KOH generated in situ by an EGC-
KOH cartridge. The gradient was as follows: 10 mM KOH from
0 to 10 min, an increase from 10 mM to 45 mM KOH from 10 to
25 min, then 45 mM KOH until 35 min. The detection by conduc-
tivity happened after electrolytic suppression. The system was
controlled by the software Chromeleon.
Glc-S-BZI
ESIþ
Gen-S-BZI ESIþ
4.5. Ecotoxicity of (a) MBI-derivatives and (b) MBT-conjugates
Impact of the active principles was measured on bioluminescent
Vibrio fischeri. The results obtained on a Microtox Acute apparatus
are expressed in EC50, the concentration that kills half of the bac-
terial population. The survival of the remaining bacteria was
measured at 490 nm.
Acknowledgements
ˇ
The members of the network AZOSTIMER, labelled by the Pole
ꢁ
ꢁ
ꢁ
de Competitivite Mer Bretagne and supported by the Region Bre-
tagne, have to be acknowledged for many helpful discussions. A
special thank to Prof. J. M. Garcia Mina (Timac Agro International,
Spain) for his help in determining inhibition properties and to J. P.
4.6. Cell culture
The required installation was developed from a biological floc
(20 L) taken from the aeration tank of a wastewater purification
ꢁ
Guegan (ENSCR) for NMR recording and assistance to analyze data.
ꢁ
ꢁ
(Cesson-Sevigne, Ille-et-Vilaine, France), which was first sifted
throw a 0.5 mm sieve. The filtrate was decanted and the resulting
supernatant was withdrawn. The concentrated solution thus
obtained was put in an aerobic reactor, using a continuous aeration.
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