Family of thiomercuric derivatives of sugars: Synthesis, fungicidal/herbicidal activity, and application to the x-ray structure determination of the corresponding enzymes

Article abstract of DOI:10.1560/E8DM-02PK-DL0V-U7FR

A series of thiomercuric derivatives of mono-and disaccharides 1-7, in which methylmercury or phenylmercury is covalently attached to anomeric thioglycosides, were synthesized for structure-function studies of glycosidases. Thiomethylmercuryl xylobiosides 5 and 6 were found to inhibit intracellular xylanase-T6 in a competitive manner, with K_i values of 0.35 mM and 0.01 mM, respectively. These inhibitors have been co-crystallized with the enzyme and are being used for X-ray analysis. 1-(Thiomethylmercuric)-β-D-xyloside (3) affords crystals belonging to the orthorhombic space group P2₁2₁2₁ and at 293(2) K: a = 6.7510(2), b = 9.7140(2), c = 29.4770(9) A, V = 1933.08(9) A, Z = 8, R(F²) = 0.0329, and R (F²) = 0.0626. There are two molecules (A and B) in the asymmetric unit, and each one shows an almost linear S-Hg-C arrangement. Biological tests on 1-7 indicated that they exhibit potent fungicidal and herbicidal activities.

Full text of DOI:10.1560/E8DM-02PK-DL0V-U7FR

Family of Thiomercuric Derivatives of Sugars: Synthesis, Fungicidal/Herbicidal
Activity, and Application to the X-Ray Structure Determination of the
Corresponding Enzymes
C
C
VALERY BELAKHOV,a EVGENIA DOR,b JOSEPH HERSHENHORN,b MARK BOTOSHANSKY,a TSAFRIR BRAVMAN, MIRIT KOLOG,
a,
YUVAL SHOHAM,c GIL SHOHAM,d AND TIMOR BAASOV *
aFaculty of Chemistry and cFaculty of Food Engineering and Biotechnology, and the Institute of Catalysis Science and
Technology, Technion—Israel Institute of Technology, Haifa 32000, Israel
bDepartment of Weed Research, Agricultural Research Organization, Newe Yaar Research Center,
P.O.B. 1021, Ramat-Yishay 30090, Israel
dDepartment of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology,
The Hebrew University of Jerusalem, Jerusalem 91904, Israel
(Received 1 February 2001 and in revised form 27 February 2001)
Abstract. A series of thiomercuric derivatives of mono- and disaccharides 1–7,
in which methylmercury or phenylmercury is covalently attached to anomeric
thioglycosides, were synthesized for structure–function studies of glycosidases.
Thiomethylmercuryl xylobiosides 5 and 6 were found to inhibit intracellular
xylanase-T6 in a competitive manner, with Ki values of 0.35 mM and 0.01 mM,
respectively. These inhibitors have been co-crystallized with the enzyme and are
being used for X-ray analysis. 1-(Thiomethylmercuric)-β-D-xyloside (3) affords
crystals belonging to the orthorhombic space group P212121 and at 293(2) K: a =
6.7510(2), b = 9.7140(2), c = 29.4770(9) Å, V = 1933.08(9) Å, Z = 8, R(F2) = 0.0329,
and Rw(F2) = 0.0626. There are two molecules (A and B) in the asymmetric unit, and
each one shows an almost linear S–Hg–C arrangement. Biological tests on 1–7
indicated that they exhibit potent fungicidal and herbicidal activities.
INTRODUCTION
cial for substrate specificity and catalysis, there is a need
To date, structure–function studies on glycosidases to further examine the X-ray diffraction of either the
have been hampered by difficulties either with the pro- enzyme–substrate or enzyme–inhibitor complexes.
tein itself or with its specific substrate/inhibitor. Thus,
In attempts to combine these two important steps in
although a number of glycosidases have been either structure–function studies of both glycosidases and
structurally or mechanstically studied in some detail, many other carbohydrate-binding proteins, we have de-
only in a few cases have the two approaches been satis- signed and synthesized thiomercuric derivatives of car-
factorily combined.1 The most commonly used method- bohydrates 1–7. In this set of compounds, the methylm-
ology for crystallographic phase determination is the ercury or phenylmercury moiety is covalently attached
preparation of heavy-atom derivatives (with mercury, to anomeric thioglycosides. It was predicted that the
lead, etc.) of protein crystals. However, although such examination of these compounds, in the form of stable
an approach, if successful, can finally lead to the three- binary complexes with appropriate enzymes, should
dimensional structure determination of a protein, the provide both the specific binding and the heavy-atom
information gained on the active-site architecture and derivative suitable for complete X-ray analysis. The
the catalysis is still very limited. In order to provide choice of anomeric thiomercuric derivatives follows the
insight into interactions at the active site, which is cru-
Dedicated to the memory of Raymond U. Lemieux.
*Authortowhomcorrespondenceshouldbeaddressed. E-mail:
chtimor@tx.technion.ac.il
Israel Journal of Chemistry
Vol. 40
2000 pp. 177–188

178
clude biobleaching in the pulp and paper industry,7a,b
bioconversion of lignocellulose material to fermentative
products, partial hydrolysis of animal feedstock to im-
prove digestibility, and enzymatic organic synthesis. 7c,d
It is noteworthy that, in addition to their scientific
importance, they might be expected to exhibit pesticidal
activity since compounds 1–7 represent organic mer-
cury derivatives. Thus, although the use of mercury
compounds as pesticides has virtually ceased over the
past decade due to their toxic effect,8 increased resis-
tance of bacteria, fungi, and plants to existing pesticides
requires a continuous search for new compounds exhib-
iting potent biological activities.9 Since compounds 1–7
are unique structures that combine a sugar skeleton with
a stable mercury linkage, they might have some impor-
tant advantages over other families of currently used
pesticides. In the fields of pesticides and other agricul-
results obtained by Shigeta et al.2 and Fitz et al.,3 who tural or pharmaceutical chemicals, no compounds of
used thiomercuric analogues of sialic acid to determine this class have been reported in the literature, to the best
the crystallographic phases for pertussis toxin, and of our knowledge. Here, we report the synthesis and
9-O-acetylsialic acid esterase from influenza C virus, various biological tests of a series of thiomercuric de-
respectively. These groups also showed that the replace- rivatives of mono- and disaccharides.
ment of anomeric C–O linkage in the inhibitor with a
C–S–Hg linkage does not significantly affect binding to
RESULTS AND DISCUSSION
the enzyme.
Thiomercuric derivatives 1 and 2 were designed as Synthesis of Thiomercuric Derivatives
model compounds for a structure–function study of en-
All thiomercuric derivatives were prepared accord-
zymes involved in the biosynthetic formation and utili- ing to the general procedure shown in Table 1. Ano-
zation of 3-deoxy-D-manno-2-octulosonic acid (Kdo). meric chlorides or bromides of the peracetylated saccha-
This unique 8-carbon-atom sugar is a specific constitu- ride (8a–d) were treated with potassium thioacetate in a
ent of the lipopolysaccharide of most Gram-negative dichloromethane/DMF mixture to produce the corre-
bacteria and is required by them for growth and viru- sponding thioacetate (9a–d) according to published pro-
lence.4 Several groups have therefore pursued inhibition cedures.4,10 Treatment of the thioacetate (9a–d) with
of Kdo metabolism as a strategy for the development of MeONa/MeOH resulted in the S- and O-deacetylated
novel antibacterial drugs. Indeed, some of the potent products. The resulting crude material was treated in
inhibitors of CMP-Kdo synthetase have shown in vivo situ with either methylmercury(II)chloride or
antibacterial activity.5 These results have prompted us to phenylmercury(II)chloride to produce the correspond-
further design synthetic molecules exhibiting selective ing thiomercuric derivatives 1–7. The purity and struc-
activity against Gram-negative bacterial cells.6
ture of each product were established by a combination
The derivatives of xylose (3-4), xylobiose (5-6), and of 1H, 13C NMR, 2D-COSY, and mass spectral analysis.
cellobiose (7) were designed for the structural study of The observed large coupling constants (3J~9 Hz) be-
xylanases and cellulases. Xylanases (1,4-β-D-xylan tween anomeric and C2 protons confirm the desired
xylanhydrolases; E.C. 3.2.1.8) hydrolyze the 1,4-β-D- β-configuration of thiomercuric linkages in compounds
xylopyranosyl linkage of xylans. Xylan is a major com- 2–7.
ponent of the plant cell wall, and serves as the backbone
In attempts to learn more about the conformation of
of D-xylopyranosyl units linked by β-(1-4) glycosidic these unique sugar derivatives, a single-crystal X-ray
bonds. The structural characterization of xylanase-ac- analysis of 3 was made (Fig. 1). It is noted that no
tive sites is of great interest, since it can lead to a better similar structure of any carbohydrate derivative linked
understanding of their catalytic mechanism and contrib- to a S–Hg–C fragment is yet to be found in the Cam-
ute significant knowledge to the rational design of spe- bridge Crystallographic Structural Database (CCSD).11
cific oligosaccharide-binding sites through protein engi-
The X-ray analysis of 3 reveals a chair conformation
neering. Much of the interest in xylanases stems from of the sugar ring, while the thiomercuryl moiety is ori-
their potential biotechnological applications, which in- ented trans to the oxygen substitution at carbon 2, indi-
Israel Journal of Chemistry
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179
Table 1. General Scheme for the preparation of thiomercuric derivatives 1–7
Fig. 1. ORTEP drawing of Compound 3 from single-crystal X-ray analysis. Selected crystallographic data: Average bond
lengths (Å): Hg(1)–C(6) = 2.06(1), Hg(1)–S(1) = 2.349(3), S(1)–C(1) = 1.804(9), C(1)–O(1) = 1.43(1), C(1)–C(2) = 1.52(1),
C(2)–O(2) = 1.419(9), C(2)–C(3) = 1.52(1), C(3)–O(3) = 1.43(1), C(3)–C(4) = 1.54(1), C(4)–O(4) = 1.44(1), C(4)–C(5) =
1.49(1), C(5)–O(1) = 1.43(1). Average bond angles (o): S(1)—Hg(1)–C(6) = 176.5(3), Hg(1)–S(1)–C(1) = 98.7(3), S(1)–C(1)–
C(2) = 118.8(6), S(1)–C(1)–O(1) = 108.0(7), C(1)–O(1)–C(5) = 112.0(8), O(1)–C(1)–C(2) = 110.0(6), O(2)–C(2)–C(3) =
109.4(7), C(1)–C(2)–O(2) = 108.9(6), C(1)–C(2)–C(3) = 111.4(7), C(2)–C(3)–O(3) = 109.9(7), C(4)–C(3)–O(3) = 110.2(6),
C(2)–C(3)–C(4) = 110.4(8), C(5)–C(4)–O(4) = 108.0(7), C(3)–C(4)–O(4) = 109.5(8), C(3)–C(4)–C(5) = 110.4(7), C(4)–C(5)–
O(1) = 110.3(7). The esd in parentheses is in the unit of least significant digit.
Belakhov et al. / Thiomercuric Derivatives of Sugars

180
cating the β-anomeric configuration (Fig. 1). The mer- be 47.4(6)° and 145.2(6)° in A and B, respectively. The
cury atom has an almost linear stereochemistry with presence of two similar independent structures, A and
normal Hg–S and Hg–C distances and an S–Hg–C angle B, was also reported for other thiomercury derivatives.13
of 175.7(3)°. These values are in agreement with those This phenomenon was attributed to a low residual Lewis
found for the CH3–Hg–S moieties in the CCSD.11 Inter- acidity of mercury in these complexes, leading to the
estingly, an analysis of the crystallographic data also formation of “secondary bonds” that are intermediate in
reveals that compound 3 exists as two symmetry-inde- strength between covalent and van der Waals
pendent structures, A and B, within the unit cell.12 As bonds.12a,b,13 Indeed, the intramolecular Hg O1 dis-
...
illustrated in Fig. 2, most of the bond distances and tances in structures A and B of 3 were found to be 2.965
angles in A and B are very similar, although a marked Å and 3.127 Å, respectively. These distances are smaller
difference exists in the side-chain conformation. This than the sum of the van der Waals radii for the atoms
difference is clearly seen by comparing torsion angles concerned, confirming the presence of a secondary in-
O1–C1–S–Hg (see insert in Fig. 2), which were found to teraction in this part of the molecule.
Fig. 2. View of the two independent structures, A and B, of Compound 3 as they appear in the unit cell. The inserts show Neuman
projections through the C1–S bond for A and B, highlighting the difference in torsion angle O1–C1–S–Hg between structures A
and B.
Israel Journal of Chemistry
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Interaction of 5 and 6 with intracellular xylanase
An intracellular xylanase (IXT6) from Bacillus
stearothermophilus T-6 has recently been cloned, se-
quenced, and overexpressed in our laboratory
(Technion).14 The purified enzyme exhibited different
substrate specificity towards synthetic substrates com-
pared to that of the extracellular xylanase (XT6). Crys-
tallographic studies of IXT6 were initiated in order to
study the specificity and catalysis of this unique
xylanase, as well as to provide a structural basis for the
rational introduction of enhanced thermostability by
site-specific mutagenesis. We recently reported a pre-
liminary X-ray analysis of IXT6,14b and molecular re-
placement studies and multiple anomalous dispersion
(MAD) experiments are currently in progress in order to
determine the detailed three-dimensional structure of
this enzyme. However, in attempts to ascertain the ac-
tual active site architecture, we designed and synthe-
sized mercury-substituted xylobioside inhibitors of the
enzyme, 5 and 6, which can be used to form an isomor-
phous heavy-atom derivative.
A
45
30
15
0
-10
0
10
20
30
40
50
-1
1/s [mM
]
B
12
8
4
Compounds 5 and 6 were evaluated as inhibitors of
homogeneous IXT6. Inhibition was measured by using
hydrolysis of p-nitrophenyl xylobioside (PNPX2) as a
synthetic substrate, for which the Km and kcat values were
determined to be 0.1 mM and 6.9 × 103 min–1, respec-
tively. The inhibition constants were measured from
initial velocity studies, and inhibition patterns were
found to be competitive (Fig. 3). Both 5 and 6 were
found to be inhibitors with Ki values of 0.35 mM and
0.01 mM, respectively. The observed 10-fold enhanced
binding of 6 to the PNPX2 substrate (based on the
comparison of Ki of 6 with the Km of PNPX2) indicates
that replacing the aglycon with the thiomercury group
seems to be a useful strategy, not just for preparing
heavy-atom derivatives of crystals of IXT6, but also as a
general tool for crystallographic studies of glycosidases.
The inhibitors 5 and 6 have been co-crystallized with
IXT6 and are being used for X-ray analysis.
0
-5
0
5
10
15
20
1/s [mM -1]
Fig. 3. Inhibition of IXT6 by 5 (A) and 6 (B). Double-recipro-
cal plots of initial velocities are given as a function of PNPX2
concentrations. (A) The concentrations of 5 were none (G),
0.5 mM (G), 1.0 mM (I), and 2.0 mM (I). For the assays, the
enzyme (1.9 nM) was first preincuabted with various concen-
trations of 5 (0–2.0 mM) at 50 °C for 15 min, and the reactions
were initiated by adding PNPX2 (0.02–1.0 mM).
(B) The concentrations of 6 were none (G), 4.6 mM (G),
9.3 mM (I), 27.7 mM (I) and 55.4 mM (L). For the assays,
the enzyme (1.9 nM) was first preincuabted with various
concentrations of 6 (0–55.4 mM) at 50 °C for 15 min, and the
reactions were initiated by adding PNPX2 (0.082–0.33 mM).
Examination of 1–7 for Herbicidal and Fungicidal
Activities
was achieved. For example, compounds 3, 5, and 7 were
Compounds 1–7 were tested for various biological highly inhibitory toward A. palmeri and Corchorus
activities, and while displaying only moderate bacteri- olitorius seed germination, whereas compounds 2–7
cidal activity against Bacillus sp. (data not shown), they were active against Cuscuta campestris seeds. It should
exhibited very potent herbicidal and fungicidal activi- be noted that compounds that failed to inhibit complete
ties. Herbicidal activity was tested for seed germination seed germination demonstrated a high activity level on
and shoot elongation using the Amaranthus palmeri shoot development and elongation. Therefore, for po-
weed, the Cuscuta campestris parasitic plant, and tential herbicidal use, the combined effect of each com-
Corchorus olitorius (a plant grown for agriculture) (see pound on seed germination and shoot elongation should
Table 2). The data in Table 2 show different activity be considered. The observed high selectivity of this set
levels for the various thiomercuric derivatives. In addi- of compounds is very intriguing especially in terms of
tion, a high degree of selectivity among different plants their potential for further practical applications. From
Belakhov et al. / Thiomercuric Derivatives of Sugars

182
this standpoint, of all the above compounds, derivative 4 reasons for these activities are not yet clear, the stability
is perhaps the most interesting due to its high inhibitory tests of 1–7 at 50 °C and a pH range of 6–8 (followed by
activity (100% inhibition at a concentration of 0.02 mg/ TLC) showed that they are completely stable for 24 h.
mL) on the parasitic weed Cuscuta campestris, its very Thus, the thermal degradation of these compounds and
high selectivity, and the low cost of its preparation from the release of alkyl mercury as a biologically active
commercial xylose.
compound may be ruled out. This, of course, cannot rule
Fungicidal activity was tested against phytopatho- out their biological decomposition within plant or fun-
genic fungi Pythium aphanidermatum, Phytophthora gal cells.
cytrophthora, Alternaria alternata, Macrophomina
In summary, a series of thiomercuric derivatives of
phaseolina, and Fusarium oxysporum f. sp. basilici. For mono- and disaccharides, in which alkyl(aryl)mercury
comparison, three commercial fungicides that are very is covalently attached to anomeric thioglycosides, were
common today in agriculture, Delsene [2-(metho- successfully prepared and their various biological func-
xycarbonylamino)-benzimidazole],15a Rubigan [α-(2- tions examined. The data obtained from the inhibition
chlorophenyl)-α-(4-chlorophenyl)-5-pyrimidine- studies of intracellular xylanase enzyme (IXT6) as a
methanol],15b and Vectra (bromoconazole)15c, were used case study reveals that anomeric thiomercuric deriva-
as references under the same experimental conditions. tives of sugars can be successfully used as a general tool
Following preliminary tests of all new compounds for structure–function studies of glycosidases. Various
(1–7), we selected the most active thiosaccharides, 3, 4, tests of this set of compounds for herbicidal and fungi-
and 7, and studied them in greater detail. The results of cidal activities reveal that they all exhibit potent activi-
these experiments are illustrated in Table 3. The follow- ties. Although the reasons for these activities are not yet
ing points may be noted from these data. First, all three clear, the observed high herbicidal/fungicidal activity,
thiomercuric derivatives, 3, 4, and 7, show higher activ- in addition to the observed selectivity of some of the
ity than the reference fungicides. Secondly, concentra- tested derivatives, provide evidence of their potential
tions as low as 10 ppm of compounds 3 and 4 caused a commercial applications in agriculture.
100% inhibition of all five phytopathogenic fungi tested
in our experiments. For example, Alternaria alternata,
the causal agent of many distructive diseases of numer-
ous plant crops, is almost 50% inhibited at 1 ppm con-
centration of 3, 4, and 7.
The data in Tables 2 and 3 clearly illustrate the power
of these newly prepared thiomercuric derivatives of
sugars as potent herbicides and fungicides. Although the
EXPERIMENTAL SECTION
Chemical
General. 1H NMR spectra were recorded on a Bruker AM-
400 spectrometer, and chemical shifts reported (in ppm) rela-
tive to internal Me4Si (δ 0.0) with CDCl3 as solvent, and to
HOD (δ = 4.63) with D2O as solvent. 13C NMR spectra were
recorded on a Bruker AM-400 spectrometer at 100.61 MHz,
Table 2. Inhibitory effect of thiomercuric derivatives of sugars 1–7 on seed germinationa
Amaranthus palmeri Corchorus olitorius
germination shoot length germination shoot length
Cuscuta campestris
germination shoot length
compound
(%)
(mm)
(%)
(mm)
(%)
(mm)
Water
1% DMSO
75.2
2.4
32.2
0.0
1.6
0.0
11.6
0.0
0.0
40.9 6.2
13.3 9.8
3.6 0.8
—
5.5 2.1
—
2.3 2.0
—
—
93.0
98.2
28.2
81.1
0.0
85.7
5.0
77.6
0.0
34.4 4.0
35.7 4.3
3.1 0.9
4.1 1.5
—
8.3 2.0
1.5 0.9
5.1 1.9
—
76.6
47.4
57.1
6.9
0.0
0.0
13.8
0.0
0.0
50.0 8.8
22.9 5.7
7.2 2.6
1.0 0.0
—
—
3.9 0.7
—
1
2
3
4
5
6
7
—
aFor details see Experimental Section. Concentrations of inhibitors used: 1 (1 mg/mL), 2 (0.1 mg/mL), 3 (1 mg/mL),4 (0.02 mg/
mL), 5 (1 mg/mL), 6 (0.1 mg/mL), and 7 (1 mg/mL). Compounds 2, 4, and 6 were dissolved in 1% DMSO in sterile water,
compounds 1, 3, 5, and 7 were dissolved in sterile water. Percent germination and schoot length (in mm) were determined in
relation to those in control experiments (sterile water or 1% DMSO in sterile water) after 4 days.
Israel Journal of Chemistry
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2000

183
Table 3. Inhibitory effect of the most active thiomercuric derivatives of sugars 3, 4, and 7 on phytopathogenic fungia
Colony diameter (mm)
concentration
(ppm)
Pythium
aphanidermatum cytrophthora
Phytophthora
Alternaria
alternata
Macrophomina Fusarium oxisporum
compound
phaseolina
f.sp.basilici
Water
3
68.0 3.5
62.2 2.0
31.3 8.1
0.0
50.0 1.7
46.7 3.8
21.3 1.5
0.0
62.5 1.2
47.0 5.2
27.3 1.2
0.0
75.3 2.1
57.0 0.0
12.0 4.0
0.0
64.7 1.5
61.0 1.7
46.7 3.5
0.0
0.1
1
10
50
0.0
0.0
0.0
0.0
0.0
4
7
0.1
1
10
43.0 4.4
0.0
24.3 1.5
14.3 1.2
0.0
48.7 4.0
26.3 2.9
0.0
47.7 1.5
10.3 4.0
0.0
47.0 1.0
18.0 0.0
0.0
0.0
0.1
1
10
50
67.3 0.6
47.0 2.6
0.0
48.3 1.5
24.3 0.6
0.0
50.0 0.0
29.7 4.7
0.0
56.7 2.5
27.7 4.0
0.0
62.0 2.6
50.3 1.5
7.3 0.6
0.0
0.0
0.0
0.0
0.0
Delsene
Vectra
0.1
1
10
50
67.3 1.5
65.7 1.2
64.0 1.7
64.0 1.0
49.3 1.2
47.0 3.5
32.0 7.1
35.3 9.0
52.0 1.0
49.7 1.5
54.0 3.0
58.5 2.5
26.3 3.2
12.3 1.5
0.0
60.7 0.6
56.0 1.0
0.0
0.0
0.0
0.1
1
10
50
72.7 0.6
70.7 2.3
64.7 0.6
45.7 3.0
48.3 1.5
40.7 1.2
22.0 1.7
19.7 3.8
41.3 5.5
34.0 1.0
15.7 1.2
0.0
66.7 1.2
51.3 2.1
31.0 3.0
11.3 0.6
46.7 1.5
20.0 2.0
0.0
0.0
Rubigan
0.1
1
10
50
70.3 0.6
68.7 1.2
44.0 1.7
15.7 2.3
49.0 0.0
37.7 2.5
21.1 1.8
12.3 0.6
44.0 2.0
22.7 5.5
14.7 2.5
0.0
62.3 2.1
52.0 1.7
39.0 5.3
17.3 6.1
54.3 1.5
42.3 2.1
28.3 2.1
27.7 3.2
aFor details see Experimental Section. The standard fungicides and compounds 3 and 7 were dissolved in sterile water,
compound 4 was dissolved in 1% DMSO in sterile water.
and chemical shifts (in ppm) reported relative to solvent reso- were provided by Aldrich or Sigma and used without further
nance (δ = 77.00, secondary chemical shift reference) for purification, unless otherwise specified. All solvents were dried
CDCl3 solutions, or to sodium 2,2-dimethyl-2-silapentane sul- over standard drying agents17 and freshly distilled prior to use.
fonate (δ = 0.0, external primary chemical shift reference) for
D2O solutions. Mass spectra were obtained on a TSQ-70B
Methyl 4,5,7,8-tetra-O-acetyl-2-S-acetyl-2,3-dideoxy-D-
mass spectrometer (Finnigan Mat) under fast-atom bombard- manno-2-octulosonate (9a). The solution of 8a (0.61 g, 1.39
ment (FAB) conditions in glycerol matrices or by negative mmol) in 10 mL of CH2Cl2–DMF (4:1) was added to a suspen-
chemical ionization (NCI) in isobutane. Reactions were moni- sion of potassium thioacetate (0.32 g, 2.78 mmol) in 5 mL of
tored by TLC on silica gel 60 F254 (0.25 mm, Merck), and spots CH2Cl2–DMF (4:1). After stirring for 12 h at room tempera-
were visualized by charring with a yellow solution containing ture, the reaction mixture was diluted with cold ethyl acetate
(NH4)Mo7O24.4H2O (120 g) and (NH4)2Ce(NO3)6 (5 g) in 10% (100 mL), and ice-cold water (25 mL) was added. The organic
H2SO4 (800 mL). Flash column chromatography was per- layer was washed twice with water (2 × 25 mL), dried
formed on silica gel 60 (70–230 mesh). Melting points were (MgSO4), and concentrated under reduced pressure. The resi-
determined using an Electrothermal IA-9300 melting point due was purified by chromatography on silica gel column
apparatus in open capillary tubes. Optical rotations were mea- (EtOAc/hexane, 1:9) to give 9a (0.47 g, 70%) as an oil: [α]20
D
1
sured at 20 °C using a Jasco DIP-370 digital polarimeter.
= 6.5° (c 1, CHCl3); Rf = 0.47 (1:1 EtOAc–hexane). H NMR
Compounds 8a–d were prepared according to published (400 MHz, CDCl3): δ 2.27 (t, 1H, J3a-3eq =12.7 Hz, J3a-4 = 12.5
procedures.16 1,2,3,4-Tetra-O-acetate-β-D-xylose and β-D-cello- Hz, H-3a), 2.41 (dd, 1H, J3a-3eq = 12.7 Hz, J3eq-4 = 4.7 Hz,
biose octaacetate were provided by Sigma. All other chemicals H-3eq), 4.97 (ddd, 1H, J = 2.9, 3.2, and 11.9 Hz, H-4), 5.30
Belakhov et al. / Thiomercuric Derivatives of Sugars

184
(d, 1H, J = 2.7 Hz, H-5), 4.57 (d, 1H, J = 9.7 Hz, H-6), 5.10 scribed for 9a. Treatment of 8b (3.45 g, 10.18 mmol) with
(ddd, 1H, J = 2.7, 2.2, and 8.1 Hz, H-7), 4.21 (dd, 1H, J = 12.3 potassium thioacetate (2.32 g, 20.35 mmol) and chromatogra-
and 4.7 Hz, H-8), 4.39 (d, 1H, J = 12.3 Hz, H-8’), 3.60 (s, 3H, phy of the crude mixture (EtOAc/hexane, 1:9) produced pure
CO2CH3), 2.24 (s, 3H, S-Ac), 2.00, 2.02, 2.06, 2.07 (4s, 12H, 9b (2.45 g, 72%): mp 95–98 °C [α]20 = –8.4° (c 1, CHCl3);
D
1
Ac). 13C NMR (100 MHz, CDCl3): δ 20.67 (4 OAc), 30.20 Rf = 0.56 (EtOAc/hexane, 1:1). H NMR (400 MHz, CDCl3)
(S-Ac), 31.90 (CO2CH3), 52.79 (C-3), 62.50 (C-8), 64.04 δ 5.34 (d, 1H, J1,2 = 8.5 Hz, H-1), 4.99 (t, 1H, J2,3 = 7.8 Hz, H-
(C-5), 66.50 (C-4), 67.93 (C-7), 72.45 (C-6), 85.40 (C-2), 2), 5.18 (t, 1H, J3,4 = 7.8 Hz, H-3), 4.91 (ddd, 1H, J4,5a= 8.0 Hz,
169.15, 169.65, 170.27, 170.54 (4 OAc), 191.32 (CO2CH3). J4,5b = 4.8 Hz, H-4), 3.52 (dd, 1H, J5a,5b = 11.9 Hz, H-5a), 4.12
FABMS: m/z 479.1 (M++H, C19H26O12S, Calcd 478.5).
(dd, 1H, H-5b), 2.36 (s, 3H, S-Ac), 2.02, 2.03, 2.04 (3s, 9H,
Ac). 13C NMR (100 MHz, CDCl3): δ 20.62 (3OAc), 30.81 (S-
Ac), 65.69 (C-5), 68.25 (C-4), 69.08 (C-3), 71.64 (C-2), 80.34
(C-1), 169.30, 169.58, 169.71 (3 OAc). CIMS: m/z 335.2
(MH+, C13H18O8S, calcd 334.3). Anal. Calcd for C13H18O8S: C,
46.70; H, 5.43; S, 9.59. Found: C, 47.28; H, 5.62; S, 9.24.
Methyl 2-methylthiomercuric-β-D-manno-2-octulosonate
(1). A 0.5 M solution of NaOMe in methanol (1.2 mL) was
added to a stirred solution of 9a (0.64 g, 1.34 mmol) in MeOH
(15 mL) at 0 °C. After 3.5 h at 0 °C, the reaction mixture was
neutralized by the addition of Dowex (H+), the resin was
filtered, and resulting filtrate evaporated to dryness. The resi-
1-(Thiomethylmercuric)-β-D-xyloside (3). The title com-
due was dissolved in MeOH (15 mL), and methyl- pound was prepared by using the same procedure as described
mercury(II)chloride (0.37 g, 1.48 mmol) was added at 0 °C. for 1. Treatment of thioacetate 9b (0.35 g, 1.06 mmol) with
After 1.5 h at 0 °C, the reaction mixture was neutralized with NaOMe (0.5 M, 0.6 mL) was followed by treatment with
NH4OH (dilute solution in MeOH), filtered, and concentrated methylmercury(II)chloride (0.29 g, 1.16 mmol), which, after
under reduced pressure. The crude residue was purified by chromatography of the crude material on silica gel (MeOH/
chromatography on silica gel column (MeOH/CHCl3, 1:9) and CHCl3, 1:9) and crystallization from MeOH, produced 280 mg
then crystallized from MeOH to give 1 (450 mg, 69%): mp (71%) of pure 3: mp 150–151°C; [α]D20 = –20.8° (c 1, MeOH);
172–173 °C (decomp.); [α]D20 = 58.5° (c 1, MeOH); Rf = 0.35 [a]D20 = –1.8°(c 1, DMSO); Rf = 0.44 (1:4, MeOH-CHCl3). 1H
(1:4, MeOH-CHCl3). 1H NMR (400 MHz, D2O): d 1.92 (t, 1H, NMR (400 MHz, D2O): d 4.79 (d, 1H, J1,2 = 9.2 Hz, H-1), 2.99
J3a-3eq = 12.6 Hz, J3eq-4 = 12.4 Hz, H-3a), 2.36 (dd, 1H, J3a-3eq
=
(t, 1H, J2,3 = 9.0 Hz, H-2), 3.20 (t, 1H, J3,4 = 9.0 Hz, H-3), 3.48
12.6 Hz, J3eq-4 = 4.6 Hz, H-3eq), 3.51 (ddd, 1H, J = 3.4, 4.5, (ddd, 1H, J4,5a = 9.5 Hz, J4,5b = 5.5 Hz, H-4), 3.14 (t, 1H, J5a,5b
=
and 12.2 Hz, H-4), 3.78 (d, 1H, J = 2.6 Hz, H-5), 3.01 (d, 1H, 11.5 Hz, H-5a), 3.80 (dd, 1H, H-5b), 0.62 (s, 3H, CH3). 13C
J = 9.0 Hz, H-6), 3.67-3.70 (m, H-7 and H-8′), 3.47 (dd, 1H, NMR (100 MHz, D2O): δ 12.52 (CH3), 71.88 (C-5), 72.01 (C-
J = 12.0 and 6.4 Hz, H-8), 3.65 (s, 3H, CO2CH3), 0.66 (s, 3H, 4), 79.85 (C-3), 80.92 (C-2), 86.14 (C-1). CIMS: m/z 380.3
CH3). 13C NMR (100 MHz, D2O): δ 11.12 (CH3), 41.73 (C-3), (MH+, C6H12HgO4S, Calcd 380.8). Anal. Calcd for
56.22 (CO2CH3), 66.07 (C-8), 67.48 (C-5), 69.94 (C-4), 71.54 C6H12HgO4S: C,18.92; H 3.18. Found: C 19.20; H 3.06.
(C-7), 79.23 (C-6), 86.56 (C-2), 178.17 (CO2CH3). CIMS: m/z
1-(Thiophenylmercuric)-β-D-xyloside (4). The title com-
pound was prepared by using the same procedure as described
for 1. Treatment of thioacetate 9b (2.87 g, 8.5 mmol) with
483.3 (MH+, C10H18HgO7S, Calcd 482.9). Anal. Calcd for
C10H18HgO7S: C, 24.87; H 3.76. Found: C 25.04; H 3.77.
Methyl 2-phenylthiomercuric-β-D-manno-2-octulosonate NaOMe (0.5 M, 10 mL) was followed by treatment with
(2). The title compound was prepared by using the same phenylmercury(II)chloride (2.69 g, 8.60 mmol), which, after
procedure described for 1. Treatment of thioacetate 9a (0.42 g, chromatography of the crude material on silica gel (MeOH/
0.87 mmol) with NaOMe (0.5 M, 0.90 mL) was followed by CHCl3, 1:9) and crystallization from MeOH, produced 1.85 g
treatment with phenylmercury(II)chloride (0.30 g, 0.96 (53%) of pure 4: mp 157–158 °C (decomp.); [α]D20 = 8.6° (c 1,
1
mmol), which, after chromatography of the crude material on DMSO); Rf = 0.56 (MeOH/CHCl3,1:4). H NMR (400 MHz,
silica gel (MeOH/CHCl3, 1:9) and lyophylization, produced DMSO-d6): δ 4.62 (d, 1H, J1,2 = 8.8 Hz, H-1), 2.87 (dd, 1H, J2,3
357 mg (76%) of pure 2: [α]D20 = 58.5o (c 1, MeOH); Rf = 0.41 = 9.0 Hz, H-2), 4.99 (dd, 1H, J3,4 = 9.0 Hz, H-3), 3.07 (ddd, 1H,
(MeOH/CHCl3, 1:4). 1H NMR (400 MHz, D2O) δ 1.97 (t, 1H, J4,5a = 10.3 Hz, J4,5b = 5.7 Hz, H-4), 3.03 (t, 1H, J5a,5b = 11.6 Hz,
J3a-3eq = 12.5 Hz, J3a-4 = 12.4 Hz, H-3a), 2.43 (dd, 1H, J3a-3eq
=
H-5a), 3.72 (dd, 1H, H-5b), 7.18–7.41 (m, 5H, aromatic pro-
12.5 Hz, J3eq-4 = 4.6 Hz, H-3eq), 3.54 (ddd, 1H, J = 3.2, 4.3 and tons). 13C NMR (100 MHz, DMSO-d6): δ 69.71 (C-5), 70.14
12.0 Hz, H-4), 3.81 (d, 1H, J = 2.7 Hz, H-5), 3.12 (d, 1H, J = (C-4), 78.00 (C-3), 78.15 (C-2), 84.04 (C-1), 127.55, 127.75,
8.9 Hz, H-6), 3.67-3.72 (m, H-7 and H-8′), 3.43 (dd, 1H, J = 128.15, 136.79 (aromatic carbons). CIMS: m/z 443.1 (MH+,
11.7 and 6.7 Hz, H-8), 3.69 (s, 3H, CO2CH3), 7.18-7.33 (m, C11H14HgO4S, Calcd 442.9). Anal. Calcd for C11H14HgO4S: C,
5H, aromatic protons). 13C NMR (100 MHz, D2O): δ 41.30 (C- 29.83; H, 3.19. Found: C, 29.59; H, 3.47.
3), 56.51 (CO2CH3), 66.28 (C-8), 67.56 (C-5), 70.01 (C-4),
2,3,2′,3′,4′-Penta-O-acetyl-1-S-acetyl xylobioside (9c).
71.62 (C-7), 79.57 (C-6), 86.72 (C-2), 131.59, 131.77, 139.35
The title compound was prepared by using the same procedure
(aromatic carbons). CIMS: m/z 545.2 (MH+, C15H20HgO7S,
as described for 9a. Treatment of 8c (1.17 g, 2.11 mmol) with
Calcd 544.9). Anal. Calcd for C15H20HgO7S: C, 33.06; H 3.70.
potassium thioacetate (0.48 g, 4.22 mmol) and chromatogra-
Found: C 32.88; H 3.90.
phy of the crude mixture (EtOAc/hexane, 1:9) produced pure
2,3,4-Tri-O-acetyl-1-S-acetyl-β-D-xyloside (9b). The title 9c (0.76 g, 65%): mp 200–201°C [α]20D = –61.8° (c 1, CHCl3);
compound was prepared by using the same procedure as de- Rf = 0.33 (1:1 EtOAc–hexane). 1H NMR (400 MHz, CDCl3):
Israel Journal of Chemistry
40
2000

185
δ 5.22 (d, 1H, J1,2 = 9.1 Hz, H-1), 4.93 (t, 1H, J2,3 = 8.4 Hz, H- m/z 575.1 (M+ + H, C16H22HgO8S, Calcd 575.0). Anal. Calcd
2), 5.13 (t, 1H, J3,4 = 8.4 Hz, H-3), 4.86 (ddd, 1H, J4,5a = 7.7 Hz, for C16H22HgO8S: C, 33.42; H 3.63. Found: C 33.71; H 3.88.
J4,5b = 5.0 Hz, H-4), 3.45 (t, 1H, J5a,5b = 11.0 Hz, H-5a), 4.01
2,3,6,2′,3′,4′,6′-Hepta-O-acetyl-2S-acetyl cellobioside
(dd, 1H, H-5b), 4.52 (d, 1H, J1′ 2′ = 6.1 Hz, H-1′), 4.78 (t, 1H,
(9d). The title compound was prepared by using the same
J2′,3′ = 7.8 Hz, H-2′), 5.07 (t, 1H, J3′,4′ = 7.8 Hz, H-3′), 3.80
procedure as described for 9a. Treatment of 8d (1.88 g,
(ddd, 1H, J4′ ,5′ a = 8.6 Hz, J4′,5′ b = 5.2 Hz, H-4′), 3.38 (dd, 1H,
2.69 mmol) with potassium thioacetate (0.61 g, 5.38 mmol)
J 5’a,5’b = 11.6 Hz, H-5′a), 4.08 (dd, 1H, H-5′b), 2.34 (s, 3H, S-
and chromatography of the crude mixture (EtOAc/hexane,
Ac), 1.99, 2.00, 2.01, 2.02, 2.04 (5s, 15H, Ac). 13C NMR (100
20
1:9) produced pure 9d (1.08 g, 58%): mp 209–210 °C, [α]D
=
MHz, CDCl3): δ 20.61 (3 OAc), 20.70 (OAc), 20.76 (OAc),
30.83 (S-Ac), 61.68 (C-5′), 66.55 (C-2), 69.17 (C-2′), 70.42
(C-3), 70.60 (C-3′), 72.85 (C-4′), 74.30 (C-4), 80.52 (C-1),
99.63 (C-1’), 169.06, 169.48, 169.54, 169.72, and 169.90 (5
OAc). FABMS: m/z 551.0 (M+ + H, C22H30O14S, Calcd 550.5).
Anal. Calcd for C22H30O14S: C, 47.99; H, 5.49; S, 5.83. Found:
C, 48.20; H, 5.64; S, 5.71.
–14.3 (c 1, CHCl3); Rf = 0.54 (EtOAc/hexane, 3:2). 1H NMR
(400 MHz, CDCl3) δ 5.16 (d, 1H, J1,2 = 10.5 Hz, H-1), 3.76 (t,
1H, J2,3 = 8.6 Hz, H-2), 5.20 (t, 1H, J3,4 = 8.6 Hz, H-3), 5.02
(dd, 1H, J4,5 = 9.7 Hz, H-4), 3.70 (ddd, 1H, J4,5 = 9.7 Hz, J5,6a
=5.1 Hz, H-5), 4.33 (dd, J6a,6b = 12.3 Hz, J5,6a = 5.1 Hz, H-6a),
5.10 (t, 1H, H-6b), 4.46 (d, 1H, J1′,2′ = 8.2 Hz, H-1′), 4.88 (t,
1H, J2′,3′ = 8.0 Hz, H-2’), 4.07 (dd, 1H, J3’,4’ = 8.0 Hz, H-3’),
4.45 (t, 1H, J4′,5′ = 9.7 Hz, H-4’), 3.62 (ddd, 1H, J4’,5’ = 9.7 Hz,
J5′,6′a = 4.9 Hz, H-5′), 4.02 (dd, 1H, J6′a,6′b = 12.5 Hz, J5′,6′a = 4.9
Hz, H-6′a), 4.99 (t, 1H, H-6′b), 2.33 (s, 3H, S-Ac), 1.94, 1.97,
1.98, 1.99, 2.00, 2.06, 2.08 (7s, 21H, Ac). 13C NMR (100
MHz, CDCl3): δ 20.43 (6OAc), 20.70 (OAc), 30.71 (SAc),
61.81 (C-5′), 61.95 (C-5), 68.17 (C-2), 69.49 (C-2′), 71.72
(C-3), 72.11 (C-3′), 73.02 (C-4′), 73.63 (C-4), 75.97 (C-6′),
77.31 (C-6), 80.18 (C-1), 100.54 (C-1′), 168.82, 169.13,
169.36, 169.42, 170.00, 170.05, 170.29 (7OAc). CIMS: m/z
695.1 (MH+, C28H38O18S, Calcd 694.6). Anal. Calcd for
C28H38O18S: C, 48.41; H, 5.51; S, 4.62. Found: C, 48.43; H,
5.65; S, 4.64.
1-(Thiomethylmercuric)-β-D-xylobioside (5). The title
compound was prepared by using the same procedure as de-
scribed for 1. Treatment of thioacetate 9c (0.26 g, 0.47 mmol)
with NaOMe (0.5 M, 1.5 mL) was followed by treatment with
methylmercury(II)chloride (0.12 g, 0.48 mmol), which, after
chromatography of the crude material on silica gel (MeOH/
CHCl3, 1:9) and lyophylization, produced 0.15 g (65%) of
pure 5: [α]D20 = –24.6° (c 1, MeOH); Rf = 0.38 (1:3, MeOH-
CHCl3). 1H NMR (400 MHz, D2O): δ 4.81 (d, 1H, J1,2 = 9.2 Hz,
H-1), 3.26 (t, 1H, J2,3 = 9.0 Hz, H-2), 3.33 (t, 1H, J3,4 = 9.0 Hz,
H-3), 3.47 (ddd, 1H, J4,5a = 9.7 Hz, J4,5b = 5.5 Hz, H-4), 3.08 (t,
1H, J5a,5b = 11.5 Hz, H-5a), 3.95 (dd, 1H, H-5b), 4.29 (d, 1H,
J
1′,2′ = 7.7 Hz, H-1′), 3.14 (t, 1H, J2′,3′ = 7.6 Hz, H-2′), 3.22 (t,
2-(Thiomethylmercuric)-β-D-cellobioside (7). The title
compound was prepared by using the same procedure as de-
scribed for 1. Treatment of thioacetate 9d (0.49 g, 0.72 mmol)
with NaOMe (0.5 M, 5.0 mL) was followed by treatment with
methylmercury(II)chloride (0.19 g, 5.0 mmol), which, after
chromatography of the crude material on silica gel (MeOH/
CHCl3, 1:9) and crystallization from MeOH, produced 0.41 g
(67%) of pure 7: mp 159–162 °C (decomp.); [α]D20 = 23.8 (c 1,
1H, J3’,4’ = 7.6 Hz, H-3′), 3.64 (ddd, 1H, J4′,5a′ = 9.7 Hz, J4′,5′b
=
5.3 Hz, H-4′), 3.03 (t, 1H, J5′a,5′b = 11.5 Hz, H-5′a), 3.81 (dd,
1H, H-5′b), 0.63 (s, 3H, CH3). 13C NMR (100 MHz, D2O):
δ 12.44 (CH3), 67.89 (C-5’), 69.71 (C-5), 71.87 (C-2), 75.45
(C-2’), 77.90 (C-3), 78.30 (C-3’), 79.21 (C-4′), 80.93 (C-4),
86.00 (C-1), 104.47 (C-1′). FABMS: m/z 512.9 (M+ + H,
C11H20HgO8S, Calcd 513.1). Anal. Calcd for C11H20HgO8S: C,
25.76; H 3.93. Found: C 25.64; H 4.22.
1
DMSO); Rf = 0.62 (1:1 MeOH-CHCl3). H NMR (400 MHz,
D2O): δ 4.86 (d, 1H, J1,2 = 9.3 Hz, H-1), 3.05 (t, 1H, J2,3 = 8.8
Hz, H-2), 3.38 (t, 1H, J3,4 = 8.8 Hz, H-3), 3.60 (t, 1H, J4,5 = 7.3
Hz, H-4), 3.41 (ddd, 1H, J4,5 = 7.3 Hz, J5,6a = 6.0 Hz, H-5), 3.55
(dd, 1H, J6a,6b = 12.2 Hz, J5,6a = 6.0 Hz, H-6a), 3.76 (t, 1H,
1-(Thiophenylmercuric)-β-D-xylobioside (6). The title
compound was prepared by using the same procedure as de-
scribed for 1. Treatment of thioacetate 9c (0.26 g, 0.47 mmol)
with NaOMe (0.5 M, 1.5 mL) was followed by treatment with
phenylmercury(II)chloride (0.15 g, 0.48 mmol), which, after
chromatography of the crude material on silica gel (MeOH/
CHCl3, 1:9) and crystallization from MeOH, produced 0.15 g
H-6b), 4.31 (d, 1H, J1′,2′ = 7.7 Hz, H-1′), 3.12 (t, 1H, J2′,3′
8.3 Hz, H-2′), 3.31 (t, J3′,4′ = 8.3 Hz, H-3′), 3.47 (t, 1H, J4′,5′
=
=
9.2 Hz, H-4′), 3.29 (ddd, 1H, J4′,5′ = 9.2 Hz, J5′,6a′ = 5.2 Hz,
H-5′), 3.52 (dd, 1H, J6′a,6′b = 12.7 Hz, J5′,6′a = 5.2 Hz, H-6′a),
3.74 (t, 1H, H-6′b), 0.61 (s, 3H, CH3). 13C NMR (100 MHz,
D2O): δ 12.57 (CH3), 63.14 (C-5′), 63.38 (C-5), 72.24 (C-2),
75.91 (C-2′), 78.29 (C-3), 78.42 (C-3′), 78.72 (C-4′), 80.85
(C-4), 81.61 (C-6′), 81.73 (C-6), 85.16 (C-1), 105.18 (C-1′).
FABMS: m/z 573.2 (M+ + H, C13H24O10S, Calcd 572.9). Anal.
Calcd for C13H24O10S: C, 27.25; H, 4.22. Found: C, 27.12; H,
4.33.
20
(51%) of pure 6: mp 174–175 °C (decomp.); [α]D = –12.9°
1
(c 1, MeOH); Rf = 0.47 (MeOH-CHCl3, 1:3). H NMR (400
MHz, D2O) d 4.76 (1H, J1,2 = 9.3 Hz, H-1), 3.21 (t, 1H, J2,3
=
9.0 Hz, H-2), 3.31 (t, 1H, J3,4 = 9.0 Hz, H-3), 3.43 (ddd, 1H,
J4,5a = 9.6 Hz, J4,5b = 5.6 Hz, H-4), 3.07 (t, 1H, J5a,5b =11.5 Hz,
H-5a), 3.93 (dd, 1H, H-5b), 4.24 (d, 1H, J 1′,2′ = 7.8 Hz, H-1′),
3.13 (t, 1H, J2′,3′ = 8.0 Hz, H-2′), 3.19 (t, 1H, J3′,4′ = 8.0 Hz,
H-3′), 3.61 (ddd, 1H, J4′,5′a = 9.8 Hz, J4′,5′b = 5.5 Hz, H-4′), 3.03
(t, 1H, J5′a,5′b = 11.5 Hz, H-5′a), 3.77 (dd, 1H, H-5′b), 7.14-7.26 Crystallographic
(m, 5H, aromatic protons). 13C NMR (100 MHz, D2O): δ 67.74
1-(Thiomethylmercuric)-β-D-xyloside (3) was recrystal-
(C-5′), 69.67 (C-5), 71.69 (C-2), 75.27 (C-2′), 77.73 (C-3), lized from methanol. Intensity data from clear colorless trans-
78.12 (C-3′), 79.03 (C-4′), 80.73 (C-4), 85.86 (C-1), 104.30 parent plate crystals were collected at 293(2) K on a Nonius
(C-1′), 131.26, 131.47, 139.21 (aromatic carbons). FABMS: KappaCCD diffractometer. Table 4 provides crystallographic
Belakhov et al. / Thiomercuric Derivatives of Sugars

186
and data collection details, and Table 5 contains the coordi- Table 5. Atomic coordinates (× 104) and equivalent isotropic
nates of the non-hydrogen atoms. Data reduction and space displacement parameters (Å2 × 103) for 3.
group determination were performed using the DENZO18a pro-
x
y
z
U(eq)*
gram. The SHELXS-9718b program was used for crystal struc-
ture solution by application of direct methods. The SHELXL-
9718c program was used for refinement by full-matrix least
squares. The numerical absorption correction was performed
using the maXus18d program. Flack parameter (Table 4) was
determined according to Flack.19 All hydrogen atoms were
geometrically placed and refined isotropicaly as constrained to
the corresponding carbon atoms.
Hg(1A)
S(1A)
O(1A)
O(2A)
O(3A)
O(4A)
C(1A)
C(2A)
C(3A)
C(4A)
C(5A)
C(6A)
Hg(1B)
S(1B)
O(1B)
O(2B)
O(3B)
O(4B)
C(1B)
C(2B)
1885(1)
1464(4)
4930(8)
1059(8)
3630(10)
7770(8)
3327(11)
2595(11)
4287(11)
6074(12)
6625(13)
2110(16)
2357(1)
2745(4)
–822(8)
2337(8)
–1710(9)
–4358(8)
976(12)
591(11
–6902(1)
–8636(2)
–7524(6)
–7653(6)
–5549(6)
–5998(7)
–8090(9)
–7046(8)
–6625(8)
–6148(8)
–7225(10)
–5424(9)
–1562(1)
–2948(3)
–1905(6)
–3030(6)
–3486(5)
–1367(6)
–2103(8)
–2992(9)
–2471(8)
–2082(8)
–1210(8)
–490(10)
4689(1)
4142(1)
3995(2)
3138(3)
2790(2)
3078(3)
3741(3)
3395(3)
3085(3)
3367(4)
3712(4)
5193(4)
4750(1)
4103(1)
3988(2)
3051(2)
2711(2)
3069(3)
3740(4)
3323(3)
3033(4)
3336(3)
3716(4)
5345(4)
44(1)
45(1)
43(2)
39(2)
40(2)
46(2)
34(2)
31(2)
30(2)
36(2)
47(3)
54(3)
49(1)
55(1)
39(2)
46(2)
42(2)
41(2)
37(2)
35(2)
33(2)
33(2)
38(2)
59(3)
Inhibition Study
The homogeneous IXT6 (specific catalytic activity 179
U/mg) was isolated from the overproducing strain Escherichia
coli [BL21(DE3)(pET9d-xynA2)], as previously described.14
Steady-state kinetic studies were performed by following
changes in visible absorbance using a Biochrom 4060
(Pharmacia) spectrophotometer equipped with a circulating
water bath. Initial hydrolysis rates were determined by incu-
Table 4. Selected experimental details for 3
1. Crystal data
C6H12HgO4S
Mr = 380.81
Orthorhombic
Mo Kα radiation
λ = 0.71070 Å
C(3B) –1126(12)
C(4B) –2906(12)
C(5B) –2249(12)
Cell parameters from
10373 reflections
θ = 2.2–20.5°
C(6B)
2163(16)
P212121
a = 6.7510 (2) Å
b = 9.7140 (2) Å
c = 29.4770 (9) Å
V = 1933.08 (9) Å3
Z = 8
Dx = 2.617 Mg m–3
Dm not measured
µ = 16.115 mm–1
T = 293 (2) K
*U(eq) is defined as one third of the trace of the orthogonal-
ized Uij tensor.
Plate
Transparent colourless
0.19 × 0.16 × 0.05 mm
bating appropriate concentrations of the substrate PNPX216c in
a 100 mM phosphate buffer (pH 7.0) at 50 °C within the
spectrophotometer until thermally equilibrated. Following
equilibration, IXT6 (200 µL, at a final concentration of
1.9 nM) was added, and the increase in the absorbance at
420 nm was monitored as a function of time. To determine Ki
values, stock solutions of inhibitors 5 and 6 were prepared by
dissolving in small amounts of DMSO, followed by adjusting
the resulting homogeneous solution with a 100 mM phosphate
buffer (pH 7.0) to final concentrations of 0.1–1.01% DMSO in
the assay mixture. The enzyme was first preincubated with
various inhibitor concentrations at 50 °C for 15 min, and the
reactions were initiated by addition of PNPX2 substrate. Ini-
tial rates were determined by a least-squares fitting of the first
10% of the progress curve to a straight line. All samples were
assayed in triplicate, and analogous results were obtained in
two to four different experiments. The data were fitted to the
competitive model using the equation: Y = V [S]/[K (1+[I]/Ki)
+ [S]], employing the commercial GraFit software program.
The Ki values were calculated either from the above treatment
or from secondary replots of the slopes from initial double-
reciprocal plots (1/ν versus 1/[S]) versus inhibitor concentration.
2. Data collection
KappaCCD Nonius diffractometer
Phi scan
2930 reflections with
I > 2 sigma (I)
Absorption correction:
numerical
Rint = 0.0540
θ
max = 25.32o
Tmin = 0.060, Tmax = 0.447
8559 measured reflections
3286 independent reflections
none standard reflections
h = –7→ 8
k = –11→ 9
l = -35→ 27
3. Refinement
Refinement on F2
R[F2 > 2σ (F2)] = 0.0329
wR (F2) = 0.0655
S = 1.037
(∆/σ)max = 0.001
Drmax = 1.066 e Å-3
Drmax = 1.066 e Å-3
Scattering factors from
International
3286 reflections
223 parameters
Tables for
Crystallography
(Vol. C)
Flack parameter =
–0.010 (10)
Pesticidal Activity
Herbicidal activity. Herbicidal activity of tested com-
pounds was studied with the weed Amarantus palmeri, the
Israel Journal of Chemistry
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2000

187
agriculture plant Corchorus olitorius, and the parasitic weed
Cuscuta campestris. Solutions of 1, 3, 5, and 7 were prepared
by dissolving an appropriate amount of the tested compound
in sterile water to a final concentration of 1 mg/mL. Solutions
of 2, 4, and 6 were prepared in 1% DMSO with final concen-
trations of 0.1, 0.1, and 0.02 mg/mL, respectively. The solu-
tion of tested compound (1 mL) was applied on a sterile filter
disk located in the middle of a petri dish containing surface-
sterilized seeds. Two petri dishes, each containing 100 seeds,
were used for each compound. The dishes were sealed with
Parafilm covered with aluminum foil, and placed in a growth
chamber at 26 °C. The number of germinating seeds and shoot
length were determined after four days. Water or 1% DMSO
solution in water served as controls.20a
Chem. 1996, 4, 1349.
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Fungicidal activity. Fungicidal activity of tested com-
pounds was studied with phytophogenic fungi Pythium
aphanidermatum (Phymycetes, Peronosporales, Phytiaceae),
Phytophora cytrophthora (Phymycetes, Peronosporales,
Phytiaceae), Alternaria alternata (Fungi Imperfecti,
Moniliales, Sphaeriodeceae), Macrophomina phaseolina
(Fungi Imperfecti, Sphaeropsidales, Sphaeriodeceae), and
Fusarium oxysporum f. sp. basilici (Fungi Imperfecti,
Moniliales, Tuberculariaceae). Sterile aqueous solutions of 3,
4, and 7 were mixed with a potato dextrose agar medium in
petri dishes to give final concentrations of 0.1, 1, 10, and 50
ppm. An 8-mm agar disk with fungal mycelia from a 7-day-
old culture was placed in the center of the petri dishes contain-
ing the compound-amended agar. The dishes were incubated
under continuous fluorescent light at 26 °C for 1–7 days
according to the tested fungus, after which the colony diameter
was measured in two perpendicular directions.20b Petri dishes
containing potato dextrose agar alone, or embedded with the
fungicides Delsene, Vectra, and Rubigan, served as control or
reference, respectively.
(7) (a) Shoham, Y.; Schwartz, Z.; Khasim, A.; Gat, O.;
Zosim, Z.; Rosenberg, E. Biodegradation 1993, 3, 207.
(b) Viikari, L.; Kantelinen, A.; Sundquist, J.; Linko, M.
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Acknowledgments. This work was supported by the United
States–Israel Binational Science Foundation (BSF), Jerusa-
lem, Israel (Grant no. 97-00356). Y.S. and G.S. would like to
thank the Israel Science Foundation (administered by the Is-
rael Academy of Sciences and Humanities, Jerusalem) for its
partial support. Additional support was provided by the Fund
for the Promotion of Research at the Technion (to T.B. and
Y.S.), and by the Otto Meyerhof Center for Biotechnology,
established by the Minerva Foundation (Munich, Germany).
V.B. and M.B. acknowledge the financial support by the Cen-
ter of Absorption in Science, the Ministry of Immigration
Absorption, and the Ministry of Science and Arts (Kamea
Program).
(11) CCSD, Cambridge Crystallographic Structural Data-
base, April 2000 version, Cambridge Crystallographic
Data Centre, Cambridge, England.
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