2-Nitro and 4-nitro-quinone-methides are not irreversible inhibitors of bovine β-glucuronidase

Article abstract of DOI:10.1016/S0008-6215(01)00083-0

4-Benzylamino-(and 4-chloromethyl)-2-nitro-β-D-glucuronides (4, 10) and their 2-substituted-4-nitro regioisomers (7, 13) were prepared by glycosidation of the 3-nitro-4-hydroxy- and the 2-hydroxy-5-nitro-benzylic alcohol, respectively, with a glucuronyl donor. Carbonate activation followed by reaction with benzylamine or methanesulfonyl chloride afforded, after complete deprotection, the target molecules 4, 7, 10 and 13. These compounds have been synthesized to determine whether these molecules are (or not) glucuronidase inhibitors. After incubation with bovine liver β-glucuronidase, none of the cleavage products (the titled quinone-methides) showed to be irreversible inhibitors of this enzyme.

Full text of DOI:10.1016/S0008-6215(01)00083-0

www.elsevier.nl/locate/carres
Carbohydrate Research 332 (2001) 151–156
2-Nitro and 4-nitro-quinone-methides are not irreversible
inhibitors of bovine b-glucuronidase
Michel Azoulay,a Fre´de´ric Chalard,b Jean-Pierre Gesson,b Jean-Claude Florent,a
Claude Monnereta,*
aSection de Recherche, UMR 176, CNRS-Institut Curie, 26 rue d’Ulm, F-75248 Paris, France
bFaculte´ des Sciences, UMR 6514, 40 A6enue du Recteur Pineau, F-86022 Poitiers, France
Received 5 January 2001; received in revised form 14 March 2001; accepted 15 March 2001
Abstract
4-Benzylamino-(and 4-chloromethyl)-2-nitro-b- -glucuronides (4, 10) and their 2-substituted-4-nitro regioisomers
D
(7, 13) were prepared by glycosidation of the 3-nitro-4-hydroxy- and the 2-hydroxy-5-nitro-benzylic alcohol,
respectively, with a glucuronyl donor. Carbonate activation followed by reaction with benzylamine or methanesul-
fonyl chloride afforded, after complete deprotection, the target molecules 4, 7, 10 and 13. These compounds have
been synthesized to determine whether these molecules are (or not) glucuronidase inhibitors. After incubation with
bovine liver b-glucuronidase, none of the cleavage products (the titled quinone-methides) showed to be irreversible
inhibitors of this enzyme. © 2001 Published by Elsevier Science Ltd.
Keywords: ADEPT strategy; Doxorubicin; b-Glucuronidase; Quinone-methide
1. Introduction
cept of prodrug monotherapy (PMT) recently
introduced by Bosslet and co-workers.4 In
agreement with this hypothesis, superior effi-
ciency against various tumors has been noted
for HMR 1826,5 a prodrug of the well-known
anticancer agent doxorubicin.6
Further improvements of cancer chemother-
apy will certainly result from the discovery of
new anticancer drugs but also, perhaps more
efficiently, from site-specific targeting of
known drugs. In this respect, several groups
have searched for non-toxic prodrugs which
may be activated at the tumor site by enzyme-
catalyzed hydrolysis. The enzyme may be itself
targeted by covalent binding to an antibody
(ADEPT strategy)1 or present in the tumor in
higher concentration than in normal tissues.
This is the case of b-glucuronidase, a lysoso-
mal enzyme,2 which has been shown to accu-
mulate in necrotic areas surrounding the
tumor.3 This observation has led to the con-
A key feature of HMR 1826 is the introduc-
tion of a spacer group between the glu-
curonide moiety and doxorubicin, which is
essential for enzyme recognition of the pro-
drug. After glycoside hydrolysis of this Type I
derivative (Scheme 1), fast liberation of dox-
* Corresponding author. Tel.: +33-1-42346655; fax: +33-
1-42346631.
E-mail address: claude.monneret@curie.fr (C. Monneret).
0008-6215/01/$ - see front matter © 2001 Published by Elsevier Science Ltd.
PII: S0008-6215(01)00083-0

152
M. Azoulay et al. / Carbohydrate Research 332 (2001) 151–156
2. Results and discussion
orubicin (t1/2 5 min) is expected to result from
1,6-elimination leading to an electrophilic p-
quinone-methide a (Scheme 1).7 2-Nitro-4-hy-
droxymethylphenol has been characterized as
the end-product of this process.8 The same
liberation of doxorubicin was also observed,
albeit at a lower rate (t1/2 ca. 300 min), with a
Type II ortho-substituted spacer, leading to an
o-quinone-methide b. In both cases, removal
of the nitro substituent prevented drug elimi-
nation from the intermediate phenol under
physiological conditions.
In order to study this process, model com-
pounds 4 and 7 were prepared from the
known mixed carbonates 2 and 5.13 Condensa-
tion of the latter with benzylamine afforded 3
and 6 which were further fully deprotected to
give 4 and 7 in one (t-BuOK, acetone) or two
steps (MeONa, then NaOH), respectively. On
the other hand, 4-chloromethyl-2-nitrophenyl
and 2-chloromethyl-4-nitrophenyl analogs, 10
and 13, were also prepared by treating the
corresponding 4-hydroxymethyl and 2-hy-
droxymethyl derivatives 8 and 1113 by
methanesulfonyl chloride in pyridine with sub-
sequent one or two-step deprotection (Scheme
2).
Scheme 1. RNH2=doxorubicin.
On the other hand, several quinone-me-
thides have already been postulated as reactive
intermediates in enzyme-activated irreversible
glycosidase inhibition.9 This is the case of the
naturally occurring salicortin [(2-hydrox-
Scheme 2. Reagents and conditions: (a) NH2Bn (1.3 equiv),
NEt3, CH2Cl2, 45%; (b) t-BuOK 0.5 M, acetone, 50%; (c)
NH2Bn (1.5 equiv), pyridine, CH2Cl2, 60%; (d) NaOMe 1 M,
CH3OH, then NaOH 1 M, acetone 60%; (e) MsCl (1.5 equiv),
pyridine, CH2Cl2, 50%; (f) t-BuOK 0.5 M, acetone, 35%; (g)
MsCl (1.5 equiv), pyridine, CH2Cl2, 60%, (h) NaOMe 1 M,
CH3OH, then NaOH 1 M, acetone, 45%.
ymethyl-phenyl) b- -glucopyranosid], an in-
D
hibitor of Agrobacterium faecalis b-gluco-
sidase9 and of synthetic inhibitors such as
4-difluoromethyl-aryl
for b-glucosidase10, 1,1%-difluoroalkyl a-
glucopyranosides11 and 2-chloromethyl-4-ni-
trophenyl a-
-glucopyranoside12 for a-gluco-
sidase. In the latter case, Haines and co-work-
ers have shown that deletion of the nitro
substituent leads to a much less potent in-
hibitor, in agreement with a slower rate of
formation of the quinone-methide.
Taking these data into account, the ques-
tion was to determine if para- or ortho-substi-
tuted hydroxybenzyl spacers I or II are (or
not) really irreversible inhibitors of b-glu-
curonidase. A positive answer should be a
hard restriction for further development of
prodrugs incorporating such groups.
b- -glucopyranosides
D
In order to determine their inhibitory activ-
ity and the type of inhibition, compounds 4, 7,
10 and 13 were incubated with bovine b-glu-
curonidase in conditions to show a time-de-
pendent loss of activity. Surprisingly, we
observed an increase of enzyme activity when
compared to experiments without the sup-
posed inhibitors. The only way to explain this
result is to suppose that the quinone-methide
intermediate is not an irreversible inhibitor of
the b-glucuronidase from bovine liver. There-
fore, during the incubation time, compounds
4, 7, 10 and 13 should be converted to an
unstable para or ortho quinone-methide
(Scheme 1(a, b)) and subsequently to a 4-hy-
droxymethyl-2-nitrophenol (or 2-hydroxy-
D
-
D

M. Azoulay et al. / Carbohydrate Research 332 (2001) 151–156
153
methyl-4-nitrophenol) which absorbed at 405
“ In the second experiment, the enzyme was
incubated in the presence of variable con-
centrations of 4 or 7. At different times,
aliquots (50 mL) of the previous mixture
were mixed with a fixed concentration of
p-nitrophenyl glucuronide. In this instance,
the OD2 values correspond to the forma-
tion of both p-nitrophenol and 4-hydroxy-
methyl-2-nitrophenol from 4 (or the alter-
native isomer from 7).
“ Thirdly, the enzyme was also incubated in
the presence of 4 (or 7), but this time the
enzymatic test was realized without addi-
tion of p-nitrophenyl glucuronide. Thus,
OD3 corresponds to the liberation of hy-
droxymethyl-nitrophenol only (or its regio-
isomer liberation).
From these experiments, the residual activ-
ity of the enzyme is expressed by calculating
(OD2−OD3)/OD1. The resulting values and
the corresponding time-dependent log’s are
given in Fig. 1 (for 4) and Fig. 2 (for 7). In the
presence of 4 or 7, no significant variation of
enzyme activity was observed.
nm together with p-nitrophenol liberated by
the enzyme from the p-nitrophenyl b- -
D
glucuronide.
To confirm such a hypothesis, the total
enzyme activity was estimated by measuring
the absorption at 405 nm, as a function of
incubation time (15, 30, 45, 60 min), in three
different ways:
“ First, the enzyme alone was shaked at
37 °C. At various time, aliquots (50 mL)
were incubated in the presence of the
known substrate p-nitrophenyl b- -glu-
D
curonide and the enzymatic test was per-
formed as described in experimental
section. In this experiment, the amount of
p-nitrophenol liberated corresponds to the
measured OD1.
As depicted, neither 4 and 7, nor their
cleavage products, are enzyme inhibitors of
bovine b-glucuronidase. One assumption is
that, as soon as the intermediate quinone-me-
thide is formed in solution, it is immediately
quenched by nucleophilic addition of water,
producing the release of the phenol (or phen-
ate) at the enzyme active site. The commer-
cially available bovine b-glucuronidase14 was
used in this study, because it has been re-
ported that b-glucuronidase from human
placenta15 is similar to the bovine liver one.
Therefore, it seems reasonable to assume that
the HMR 1826 spacer will not interfere with
the human enzyme.
Fig. 1. Assay of time-dependent inhibition of bovine b-glu-
curonidase with compound 4 measured at different concentra-
tions and plotted as a semi-logarithmic curve. Experimental
conditions and expression of the residual activity as described
in the text.
3. Experimental
Optical rotations were determined with a
Perkin–Elmer 241 polarimeter (589 nm) at
20 °C with a concentration expressed in g/100
1
mL. H NMR spectra were recorded using a
Bruker WP-200 (200 MHz) and a Bruker AC-
300 (300 MHz). Chemical shifts are expressed
in ppm downfield from internal Me4Si with
the notation indicating the multiplicity of the
signal. For mass spectra, CI (NH3) were
Fig. 2. Assay of time-dependent inhibition of bovine b-glu-
curonidase with compound 7, as described for compound 4.

154
M. Azoulay et al. / Carbohydrate Research 332 (2001) 151–156
mg, 0.77 mmol) with benzylamine (130 mL,
1.15 mmol) by the same protocol as for 3.
After chromatography, compound 6 (285 mg,
60%) was isolated as a white solid: mp 171–
recorded with a Nermag R10-10C. Thin-layer
chromatography (TLC) was performed on Sil-
ica gel 60F254 (E. Merck). Column chromatog-
raphy was performed on SiO2 (E. Merck,
particle size 0.004–0.063 nm) using the flash
technique. Preparations of compounds 2, 5, 8
and 1113 have already been reported.
1
172 °C; [h]2D0 −57.4° (c 1, CHCl3); H NMR
(200 MHz, CDCl3): l 2.04, 2.07, 2.10 (3s,
3×3 H, OAc), 2.90 (d, 1 H, J 16 Hz, NH),
3.65 (s, 3 H, OCH3), 4.30 (d, 1 H, J 9 Hz,
H-5), 4.40 (d, 2 H, J 6 Hz, ArꢀCH2ꢀN), 5.13
(d, 2 H, J 14 Hz, ArꢀCH2ꢀO), 5.35 (m, 4 H,
H-1, H-2, H-3, H-4), 7.15 (d, 1 H, J 8 Hz, Ar),
7.31 (m, 5 H, Ar), 8.20 (m, 2 H, Ar); MS
(DCI/NH3): m/z 636 [M+ NH4]+.
Methyl
[(2-nitro-4-(N-benzylcarbamoy-
loxymethyl) phenyl) 2,3,4-tri-O-acetyl-i-
D
-
glucopyranosid] uronate (3).—To a solution of
2 (700 mg, 1.1 mmol) in CH2Cl2 (50 mL) were
successively added benzylamine (160 mL, 1.4
mmol) and Et3N (500 mL). After stirring for 4
h at rt, the crude mixture was extracted with
additional CH2Cl2 (200 mL) and the organic
layer was washed with water and brine. The
organic phase was dried, concentrated and the
residue was chromatographed (1:1, cyclohex-
ane–EtOAc) to give 3 (306 mg, 45%) as an
[2-(N-Benzylcarbamoyloxymethyl)-4-nitro-
phenyl] i- -glucopyranosiduronic acid (7).—
D
Compound 6 (200 mg, 0.32 mmol) in anhyd
MeOH (25 mL) was stirred for 4 h at 0 °C in
the presence of NaOMe (20 mg, 3.7 mmol).
Neutralization by addition of Amberlite IRC-
50H+ was followed by filtration and the
filtrate was evaporated under reduced pres-
sure. The crude product was dissolved in ace-
tone (5 mL) containing 0.5 mL of a 1 M
NaOH solution. The mixture was stirred for
15 min at rt, neutralized with a 1 M HCl
solution and evaporated. The crude product
was purified by chromatography (9:1,
MeCN–water) to give 7 (92 mg, 60%) as a
white solid: mp 149–150 °C; [h]2D0 −45.5° (c
1
oil: [h]2D0 +6.7° (c 1.8, CHCl3); H NMR (300
MHz, CDCl3): l 2.06, 2.07, 2.13 (3s, 3×3 H,
OAc), 3.70 (s, 3 H, OCH3), 4.20 (d, 1 H, J4,5
8
Hz, H-5), 4.40 (d, 2 H, J 5.6 Hz, CH2ꢀAr),
5.10 (s, 2 H, CH2ꢀOꢀCO), 5.20 (d, 1 H, J1,2
6
Hz, H-1), 5.30 (m, 3 H, H-2, H-3, H-4), 7.30
(m, 7 H, Ar), 7.50 (d, 1 H, J 8 Hz, Ar), 7.80
(br s, 1 H, NH); MS (DCI/NH3): m/z 636
[M+NH4]+.
[2-Nitro-4-(N-benzylcarbamoyloxymethyl)
1
1, CH3OH); H NMR (200 MHz, CD3OD): l
phenyl] i- -glucopyranosiduronic acid (4).—
D
3.30 (m, 2 H, H-1, H-5), 3.55 (m, 3 H, H-2,
H-3, H-4), 4.32 (s, 2 H, CH2ꢀAr), 5.13 (s, 1 H,
NH), 5.28 (s, 2 H, ArꢀCH2ꢀO), 7.00 (d, 2 H,
J 8 Hz, Ar), 8.24 (m, 6 H, Ar), 10.80 (s, 1 H,
COOH); Anal. Calcd for C21H22N2O11: C,
52.72; H, 4.64; N, 5.86. Found: C, 52.69; H,
4.74; N, 5.66.
To a stirred, ice-cooled solution of 3 (200 mg,
0.32 mmol) in acetone (10 mL) was added 1
mL of aq potassium tert-butoxide (0.5 N).
The ice-bath was removed and the reaction
mixture was stirred at rt for 3 h. The reaction
was quenched by adding HCl (1 N) until
neutralization. Evaporation of the crude
product, followed by flash chromatography
(9:1, MeCN–water) gave 4 (77 mg, 50%) as an
Methyl [(2-nitro-4-(chloromethyl) phenyl)
2,3,4-tri-O-acetyl-i- -glucopyranosid] uronate
D
(9).—To a solution of 8 (500 mg, 1 mmol) in
dry CH2Cl2 (25 mL) were added 0.4 mL (10
equiv) of methanesulfonyl chloride and 1.8
mL of pyridine. The mixture was heated under
reflux for 3 h. After cooling to rt, the crude
product was diluted with water (50 mL) and
extracted with CH2Cl2 (150 mL). The com-
bined organic layers were washed with water,
brine and evaporated under reduced pressure.
Purification by chromatography (1:1, cyclo-
hexane–EtOAc), gave 9 (250 mg, 50%) as an
1
oil: [h]2D0 +96.3° (c 0.6, water); H NMR (300
MHz, CD3OD): l 3.50 (m, 3 H, H-2, H-3,
H-4), 3.90 (d, 1 H, J4,5 8 Hz, H-5), 4.20 (d, 2
H, CH2ꢀAr), 5.00 (s, 2 H, CH2ꢀOꢀCO), 5.10
(d, 1 H, J1,2 7 Hz, H-1), 7.30 (m, 7 H, Ar),
7.50 (dd, 1 H, J 8 Hz, Ar), 7.80 (br s, 1 H,
NH); Anal. Calcd for C21H22N2O11: C, 52.72;
H, 4.64; N, 5.86. Found: C, 52.51; H, 4.94; N,
5.51.
Methyl [2-(N-benzylcarbamoyloxymethyl)-
1
oil; [h]2D0 +19° (c 0.5, CHCl3); H NMR (300
4-nitrophenyl) 2,3,4-tri-O-acetyl-i- -glucopy-
D
ranosid] uronate (6).—Prepared from 5 (500
MHz, CHCl3): l 2.08, 2.09, 2.16 (3s, 3×3 H,

M. Azoulay et al. / Carbohydrate Research 332 (2001) 151–156
155
follows: A freshly prepared solution of com-
pounds 4, 7, 10 and 13 at different concentra-
tions (100 mL; 5, 10, 20 and 40 mmol,
respectively) was added to a solution of b-glu-
curonidase (200 mg protein) in 400 mL acetate
buffer (0.07 M, pH 5); the solution was incu-
bated at 37 °C. After 15, 30, 45 and 60 min,
aliquots (50 mL) were taken from the incuba-
tion assay and added with 450 mL of p-nitro-
OAc), 3.75 (s, 3 H, COOCH3), 4.20 (d, 1 H,
J5,4 9 Hz, H-5), 4.70 (s, 2 H, CH2ꢀCl), 5.20 (d,
1 H, J1,2 7 Hz, H-1), 5.30 (m, 3 H, H-2, H-3,
H-4), 7.35 (d, 1 H, J 10 Hz, Ar), 7.55 (dd, 1
H, J 10 Hz, J 1 Hz, Ar), 7.80 (d, 1 H, J 1 Hz,
Ar); MS (DCI/NH3): m/z 521 [M+NH4]+,
504 [M+H]+.
[2-Nitro-4-(chloromethyl) phenyl] i- -glu-
D
copyranosiduronic acid (10).—Obtained from
9 (250 mg, 0.49 mmol) as described for the
preparation of 4 and after chromatography
(9:1, MeCN–water) compound 10 (63 mg,
35%) was isolated as an oily residue: [h]D20
phenyl b- -glucuronide (2.5 mM in 0.07 M
D
acetate buffer, pH 5). The mixture was shaken
at 37 °C for 15 min and the reaction was
stopped by adding 500 mL of a glycine solu-
tion (0.4 M, pH 10.8). The activity was evalu-
ated via measurement of the absorption of
released p-nitrophenolate at 405 nm as a func-
tion of incubation time.16
1
−80° (c 0.9, CH3OH); H NMR (300 MHz,
CD3OD): l 3.40 (m, 3 H, H-2, H-3, H-4), 3.80
(d, 1 H, J5,4 8 Hz, H-5), 4.60 (s, 2 H,
ArꢀCH2ꢀCl), 5.10 (d, 1 H, J1,2 7 Hz, H-1),
7.40 (d, 1 H, J 10 Hz, Ar), 7.60 (dd, 1 H, J 10,
J 1 Hz, Ar), 7.80 (d, 1 H, J 1 Hz, Ar); HRMS
(FAB positive mode) Calcd for C13H14ClNO9
[M+H]+: 364.0436. Found: 364.0439.
Acknowledgements
Methyl
2,3,4-tri-O-acetyl-i-
[2-(chloromethyl)-4-nitrophenyl)
-glucopyranosid] uronate
We thank ‘La Ligue Nationale contre le
Cancer (Comite´ de Charente-Maritime)’ for
financial support.
D
(12).—Obtained from 11 (350 mg, 0.7 mmol)
following the same protocol as for the prepa-
ration of 9, the product was purified by
column chromatography (1:1, cyclohexane–
EtOAc) yield 12 (oil, 211 mg, 60%): [h]2D0 +2°
References
1
(c 0.5, CHCl3); H NMR (200 MHz, CDCl3):
1. Bagshawe, K. D. Drug. De6elop. Res. 1995, 34, 220–230.
2. Paigen, K. Prog. Nucleic Acid Res. Molec. Biol. 1989, 37,
155–205.
3. Sinhababu, A. K.; Thakker, D. R. Ad6. Drug Deli6er.
Res. 1996, 19, 241–273.
4. Bosslet, K.; Czech, J.; Hoffmann, D. Tumor Target. 1995,
1, 45–50.
5. (a) Mu¨rdter, T. E.; Sperker, B.; Kivisto¨, K. T.; McClel-
lan, M.; Fritz, P.; Friedel, G.; Linder, A.; Bosslet, K.;
Toomes, H.; Dierkesmann, R.; Kro¨mer, H. K. Cancer
Res. 1997, 57, 2440–2445;
l 2.05, 2.06, 2.11 (3s, 3×3 H, OAc), 3.74 (s,
3 H, OCH3), 4.28 (d, 1 H, J5,4 9 Hz, H-5), 4.58
(s, 2 H, ArꢀCH2ꢀCl), 5.32 (m, 4 H, H-1, H-2,
H-3, H-4), 7.38 (d, 1 H, J 9 Hz, Ar), 7.60 (s,
1 H, Ar), 7.85 (d, 1 H, J 2 Hz, Ar).
[2-(Chloromethyl)-4-nitrophenyl] i- -glu-
D
copyranosiduronic acid (13).—Obtained from
12 (200 mg, 0.4 mmol), as described for 7.
Compound 13 (65 mg, 45%) was isolated as
(b) Bosslet, K.; Straub, R.; Blumrich, M.; Czech, J.;
Gerken, M.; Sperker, B.; Kro¨mer, H. K.; Gesson, J.-P.;
Koch, M.; Monneret, C. Cancer Res. 1998, 58, 1195–
1201.
1
an oil: [h]2D0 −27° (c 1, CH3OH); H NMR
(200 MHz, CD3OD): l 3.40 (m, 3 H, H-2,
H-3, H-4), 3.75 (d, 1 H, J5,4 8 Hz, H-5), 4.55
(s, 2 H, ArꢀCH2ꢀCl), 5.10 (d, 1 H, J1,2 8 Hz,
H-1), 7.35 (d, 1 H, J 9 Hz, Ar), 7.55 (s, 1 H,
Ar), 7.80 (d, 1 H, J 2 Hz, Ar); Anal. Calcd for
C13H14ClNO9: C, 42.93; H, 3.88; Cl, 9.75; N,
3.85. Found: C, 43.11; H, 3.63; N, 3.71.
6. Arcamone, F. Doxorubicin, Anticancer Antibiotics.
Medicinal Chemistry; Academic: New York, 1981; Vol.
17.
7. Wakselman, M. Nou6. J. Chim. 1983, 7, 439–447.
8. Thompson, D. C.; Thompson, J. A.; Sugumaran, M.;
Moldeus, P. Chem. Biol. Interact. 1993, 86, 129–162.
9. Zhu, J.; Withers, S. G.; Reichardt, P. B.; Treadwell, E.;
Clausen, T. P. Biochem. J. 1998, 332, 367–371 and
references cited therein.
10. Halazy, S.; Berges, V.; Ehrhard, E.; Danzin, C. Bioorg.
Chem. 1990, 18, 330–344.
11. Halazy, S.; Danzin, C.; Ehrhard, E.; Gerhart, G. J. Am.
Chem. Soc. 1989, 111, 3484–3485.
i-Glucuronidase inhibition assay.—p-Nitro-
phenyl b-
D
-glucuronide was purchased from
Fluka. b-
D
-Glucuronidase (EC.3.2.1.3.1) from
bovine liver (Sigma) was used in this study.
12. (a) Briggs, J. C.; Haines, A. H.; Taylor, R. J. K. J. Chem.
Soc., Chem. Commun. 1992, 1039–1041;
Activity of b-glucuronidase was measured as

156
M. Azoulay et al. / Carbohydrate Research 332 (2001) 151–156
(b) Briggs, J. C.; Haines, A. H.; Taylor, R. J. K. J. Chem.
14. (a) Himeno, M.; Hashiguchi, Y.; Kato, K. J. Biochem.
(Tokyo) 1974, 76, 1243–1252;
Soc., Chem. Commun. 1993, 1410–1411;
(c) Briggs, J. C.; Haines, A. H.; Taylor, R. J. K. J. Chem.
Soc., Perkin Trans.1 1995, 27–32.
(b) Brot, F. E.; Bell, Jr., C. E.; Sly, W. S. Biochemistry
1978, 17, 385–391.
13. Florent, J.-C.; Dong, X.; Gaudel, G.; Mitaku, S.; Mon-
neret, C.; Gesson, J.-P.; Jacquesy, J.-C.; Mondon, M.;
Renoux, B.; Andrianomenjanahary, S.; Michel, S.; Koch,
M.; Tillequin, F.; Gerken, M.; Czech, J.; Straub, R.;
Bosslet, K. J. Med. Chem. 1998, 41, 3572–3581.
15. Jain, S.; Drendel, W. B.; Chen, Z.-N.; Mathews, F. S.;
Sly, W. S.; Grubb, J. H. Nat. Struct. Biol. 1996, 3,
375–381.
16. Diez, T.; Cabezas, J. A. Eur. J. Biochem. 1979, 94,
301–311.
.