Synthesis of bromophenyl β-D-glucuronides: Hydrophilic precursors of lipophilic standards in the analysis of environmental polychlorinated biphenyls

Article abstract of DOI:10.1135/cccc20061042

A rapid PCB-screening protocol, based on a combination of extraction techniques and instrumental nuclear activation analysis (INAA), required water soluble brominated internal standard that undergoes facile hydrolysis to a lipophilic brominated counterpart. Bromophenyl glucuronic acids 2, 3, 7 were synthesized for this application. Glucuronic acids 2 and 3 were prepared by bromination of phenyl β-D-glucopyranosiduronic acid in 81 and 54%, respectively, whereas compound 7 was prepared by coupling methyl 2,3,4-tri-O-acetyl-α-D-glucopyranosyluronate bromide (5) with 2,4,6-tribromophenol. Incubation with β-glucuronidase indicated that tribromophenyl derivative 7 is an excellent substrate, with near quantitative conversion to tribromophenol within seconds of incubation with the enzyme.

Full text of DOI:10.1135/cccc20061042

1042
Tandon, Kumar, Xia, Wang, Wiebe:
SYNTHESIS OF BROMOPHENYL β-D-GLUCURONIDES: HYDROPHILIC
PRECURSORS OF LIPOPHILIC STANDARDS IN THE ANALYSIS OF
ENVIRONMENTAL POLYCHLORINATED BIPHENYLS
a3,+
Manju TANDONa1, Piyush KUMARb1,*, Haiyan XIAa2,+, Lili WANG
and
a4,b
Leonard I. WIEBE
a
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta,
1
Edmonton, Alberta, Canada T6G 2N8; e-mail: tandonmanju@hotmail.com,
2
3
4
cyxliu@yahoo.com, lwang@hebert.phar.mun.ca, leonard.wiebe@ualberta.ca
b
Department of Oncologic Imaging, Cross Cancer Institute,
11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2;
1
e-mail: piyushku@cancerboard.ab.ca
Received March 16, 2006
Accepted May 20, 2006
Dedicated to Professor Antonín Holý on the occasion of his 70th birthday in recognition of his
outstanding contributions to the area of nucleic acid chemistry.
A rapid PCB-screen in g protocol, based on a com bin ation of extraction tech n iques an d in -
strum en tal n uclear activation an alysis (INAA), required water soluble brom in ated in tern al
stan dard th at un dergoes facile h ydrolysis to
a lipoph ilic brom in ated coun terpart.
Brom oph en yl glucuron ic acids 2, 3, 7 were syn th esized for th is application . Glucuron ic
acids 2 an d 3 were prepared by brom in ation of ph en yl β-D-glucopyran osiduron ic acid in 81
an d 54%, respectively, wh ereas com poun d 7 was prepared by couplin g m eth yl 2,3,4-tri-
O-acetyl-α-D-glucopyran osyluron ate brom ide (5) with 2,4,6-tribrom oph en ol. In cubation with
β-glucuron idase in dicated th at tribrom oph en yl derivative 7 is an excellen t substrate, with n ear
quan titative con version to tribrom oph en ol with in secon ds of in cubation with th e en zym e.
Keyw ord s: Glycosides; Glucuron ides; Brom oph en yl glucuron ic acids; Brom in ation ; PCB
an alysis stan dards; In strum en tal n eutron activation an alysis (INAA); Brom oben zen es.
Occupation al studies sh ow sign ifican t in creases in can cer m ortality in
workers exposed to PCBs 1. For exam ple, can cer of th e liver, gall bladder
an d biliary tract in capacitor m an ufacturin g workers exposed to Aroclors
1254, 1242, an d 1016, an d m align an t m elan om a in workers exposed to
a2
a3
+ Presen t address:
Revere, Massach usetts, U.S.A.;
Faculty of Ph arm acy, Mem orial Un i-
versity, St. Joh n ’s, Newfoun dlan d, Can ada.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1042–1050
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20061042

Synthesis of Bromophenyl β-D-Glucuronides
1043
Aroclors 1242 an d 1016, h ave been reported2,3. Con cern for h ealth risk as-
sociated with PCBs in en viron m en tal sam ples h as n ecessitated th e develop-
m en t of rapid, cost-effective screen s for PCBs. However, routin e screen in g
of large n um bers of en viron m en tal sam ples for polych lorin ated biph en yls
(PCBs) represen ts a m ajor ch allen ge for an alytical laboratories doin g th is
work. Th e SLOWPOKE laboratory4 at th e Un iversity of Alberta, in collabora-
tion with En virotest Ltd.5, h as developed a rapid screen in g system based on
th e estim ation of ‘organ ic ch lorin e’ usin g a com bin ation of extraction tech -
n iques an d in strum en tal n eutron activation an alysis (INAA). Sin ce m ost of
th e sam ples are water-based in origin , an d because th ese sam ples in variably
con tain n on -organ ic ch lorin e, it was n ecessary to develop an in tern al stan -
dard for th e an alysis th at could easily be con verted from h ydroph ilic to h y-
droph obic form an d be assayed by INAA. Brom in e-con tain in g glucuron ides
were con sidered to be ideal can didates for use as in tern al stan dards for th is
purpose, sin ce brom in e h as a h igh th erm al n eutron activation cross sec-
tion , for h igh sen sitivity, an d sin ce O-glucuron ides un dergo facile cleavage
un der th e in fluen ce of com m ercially available en zym es such as β-glucuron -
idase.
Th is work describes th e syn th esis of m on o-, di- an d tribrom oph en yl
glucuron ic acids 2, 3 an d 7 (Sch em es 1 an d 2) an d reports th eir en zym atic
h ydrolysis at th e glycosidic bon d to yield m on o-, di-, an d tribrom oph en ols,
respectively, by β-glucuron idase en zym e un der con dition s used with in th e
assay procedure.
Br
HOOC
O
O
OH
HO
(i)
OH
HOOC
O
2
O
Br
OH
(ii)
HO
OH
Br
HOOC
O
O
OH
HO
OH
(i) Br2/H2O, 0 oC; (ii) Br2/H2O, 25 o
C
3
SCHEME 1
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1042–1050

1044
Tandon, Kumar, Xia, Wang, Wiebe:
MeOOC
OAc
MeOOC
MeOOC
OH
OAc
O
O
O
(i)
(ii)
OAc
OH
Br
AcO
AcO
HO
OAc
OAc
OH
4
5
1
Br
Br
Br
HOOC
OH
Br
(iii)
Br
Br
MeOOC
O
O
O
O
(iv)
OAc
HO
AcO
OH
OAc
7
6
(i) Pyridine/Ac2O; (ii) HBr/AcOH; (iii) Hg(CN)2/2,4,6-tribromophenol; (iv) NaOMe, MeOH
SCHEME 2
Direct brom in ation of th e com m ercially available ph en yl β-D-gluco-
pyran osiduron ic acid proceeded sm ooth ly to afford 4-brom oph en yl
β-D-glucopyran osiduron ic acid (2) an d 2,4-dibrom oph en yl β-D-gluco-
pyran osiduron ic acid (3). It was observed th at th e degree of brom in ation
depen ded on th e tem perature of th e reaction 6. Reaction of ph en yl
β-D-glucopyran osiduron ic acid with liquid brom in e at 0 °C yielded on ly 2
(81%) wh ereas, at 22 °C, on ly 2,4-dibrom oph en yl glucoside 3 (54%) was
obtain ed. In both cases, it was n ecessary to add brom in e dropwise un til th e
color of brom in e persisted.
Attem pts to syn th esize 2,4,6-tribrom oph en yl β-D-glucopyran osiduron ic
acid (7) usin g sim ilar con dition s were futile. Efforts to in corporate th ree
brom in e atom s on th e ben zen e rin g at elevated tem perature (60 °C) re-
sulted in severe decom position , with out an y eviden ce of form ation of th e
desired product. It was, th erefore, deem ed n ecessary to use a couplin g reac-
tion to syn th esize th e correspon din g 2,4,6-tribrom oph en yl glucuron ide 7.
Th erefore, 2,4,6-tribrom oph en ol was coupled with m eth yl 2,3,4-tri-O-
acetyl-α-D-glucopyran osyluron ate brom ide (5) wh ich , in turn , was obtain ed
by brom in ation of m eth yl 1,2,3,4-tetra-O-acetyl-β-D-glucopyran osiduron ate
(4) with 40% HBr in acetic acid at 22 °C. Th e syn th esis of m eth yl
(2,4,6-tribrom oph en yl 2,3,4-tri-O-acetyl-β-D-glucopyran osid)uron ate (6) in -
volved reaction of an excess of 2,4,6-tribrom oph en ol with 5 in th e presen ce
of m ercuric cyan ide7 wh ich was used h ere as a Lewis acid. Th is reaction af-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1042–1050

Synthesis of Bromophenyl β-D-Glucuronides
1045
forded 6 in 52% yield. Deprotection of 6 yielded th e desired com poun d 7
(69%) in good yield.
Th e effects of th e degree of brom in e substitution on lipoph ilicities an d
13C NMR ch em ical sh ifts of related carbon s are presen ted in Tables I an d II,
respectively. Relative lipoph ilicity, estim ated from th e HPLC reten tion
tim es of th ese products (2, 3 an d 7), in dicated th e an ticipated in crease as
th e exten t of brom in e substitution in th e m olecule in creased (Table I). A
com parison of 13C NMR ch em ical sh ifts of th ese products revealed th at an
in crease in th e n um ber of brom in e substituen ts in ben zen e rin g directly af-
fected th e electron ic en viron m en t at C-1 of th e arom atic rin g an d C-1′ of
th e glucuron ide m oiety. Sin ce th ese position s participate in th e form ation
of glycosidic bon d, th e susceptibility of glycosidic lin kage to β-glucuron -
idase in duced h ydrolysis will depen d on its in creased electron ic polariza-
tion (assisted by th e exten t of brom in e substitution on ben zen e rin g,
Fig. 2). Th us, th e sh ieldin g of C-1 of th e arom atic rin g in creased as a fun c-
tion of in creasin g brom in e substitution due to th e stron g –I effect of bro-
m in e, producin g th e largest upfield ch em ical sh ift for C-1 in th e case of
TABLE I
Reten tion tim es of com poun ds 2, 3 an d 7, th eir β-glucuron idase h ydrolysis products
(aglycon e), an d h ydrolysis data
Reten tion tim e
m in
Reten tion tim e of
Aglycon e, m in
Hydrolysis tim e
m in
Com pd
Hydrolysis
2
3
7
2.3
5.8
7.6
26.5
33.4
32.0
100
88.6
100
5
0
0
TABLE II
Selected 1H an d 13C NMR ch em ical sh ifts (δ, ppm ) of com poun ds 2, 3 an d 7
Com pd
H-1′
H-5′
C-1′
C-4
C-1
COOH
2
3
7
5.0
5.0
5.2
3.80
100.92
101.35
103.80
115.71
115.96
119.14
156.55
153.14
149.80
176.04
175.93
171.60
3.40–3.66
3.50–3.60
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1042–1050

1046
Tandon, Kumar, Xia, Wang, Wiebe:
tribrom oph en yl glucuron ide 7. Th e resultin g h igh degree of polarization of
th e glycosidic bon d in th e case of tribrom oph en yl glucuron ic acid 7 facili-
tates its virtually in stan tan eous en zym atic h ydrolysis at “tim e zero” (Fig. 1),
wh ile m on obrom in ated glucuron ide 2 is h ydrolyzed on ly partially (12%) at
“zero tim e” an d takes 5 m in for com plete h ydrolysis (Table I). Th e C-1′
(sugar carbon ) was m ore desh ielded with th e in crease in th e degree of
brom in ation in th e m olecule (Table II). It also appears th at th e substitution
on th e arom atic rin g orien ts th e ben zen e rin g in a plan e th at affected th e
electron ic en viron m en t of carboxyl group at C-5′, sin ce carbon ch em ical
sh ifts for COOH was furth est down field in 2 (m on obrom o-substituted)
wh ile it appeared at n early 4.4 ppm upfield in case of 7 (tribrom o deriva-
tive).
Com poun ds 2, 3 an d 7 were in cubated with β-glucuron idase en zym e
an d th eir h ydrolysis profiles were determ in ed. Related data are provided
in Table I. Figure 1 sh ows th e respective HPLC ch rom atograph ic traces of
th e in cubation m ixture prior to an d im m ediately after th e addition of
β-glucuron idase to tribrom oph en yl glucuron ic acids. Th ese an alyses sh ow
th at com plete h ydrolysis of tribrom oph en yl glucuron ide occurred with in
secon ds after th e en zym e was in troduced in to th e solution , th ereby, con -
firm in g th at tribrom oph en yl glucuron ic acid is an excellen t substrate for
β-glucuron idase. Sim ilar results were obtain ed for dibrom oph en yl glucuron -
ide. Th e m on obrom oph en yl glucuron ide, h owever, was con verted at a sub-
a
b
FIG. 1
HPLC ch rom atogram sh owin g th e h ydrolysis of 2,4,6-tribrom oph en yl glucuron ic acid (7).
HPLC ch rom atogram of 7: before h ydrolysis (a), im m ediately (zero h our) after addition of
25 un its of β-glucuron idase to 100 n m oles of tribrom oph en yl glucuron ide (7) (b)
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1042–1050

Synthesis of Bromophenyl β-D-Glucuronides
1047
stan tially slower rate, with 12% h ydrolysis at “zero tim e”, 64% after 1 m in
an d 100% in 5 m in (Table I). Th e slower rate of h ydrolysis in case of 2 m ay
be attributed to weaker –I effect of m on obrom oph en yl m oiety at C-1 in
com parison to 7 an d 3 wh ere addition al brom in e substitution s afforded
im m ediate h ydrolysis at glucuron ic lin kage (Fig. 2).
In sum m ary, m on o an d dibrom oph en yl glucuron ic acids were prepared
by direct brom in ation of com m ercially available ph en yl glucuron ide wh ile
th e syn th esis of tribrom oph en yl glucuron ic acids was n ot possible usin g
th is approach , th e latter bein g obtain ed on ly via a couplin g procedure. All
th ree glucuron ides were excellen t substrates for β-glucuron idase, un der
th ese in cubation con dition s, for use as in tern al stan dards for th e an alysis of
organ och lorin e in en viron m en tal sam ples.
FIG. 2
Rate of en zym atic h ydrolysis in relation to selected 13C ch em ical sh ifts (δ, ppm )
EXPERIMENTAL
Meltin g poin ts were determ in ed on a Buch i capillary apparatus an d are un corrected. Th e 1H
an d 13C NMR spectra were recorded on a Bruker AM-300 spectrom eter usin g CDCl3 or D2O
as th e solven ts; th e ch em ical sh ifts are given in ppm (δ-scale) down field from tetram eth yl-
silan e as in tern al stan dard, couplin g con stan ts (J) in Hz. Th e 1H NMR assign m en ts were con -
firm ed by selective decouplin g experim en ts. Th e 13C NMR reson an ces are assign ed by usin g
th e J m odulation spin ech o tech n ique to determ in e th e n um ber of h ydrogen atom s attach ed
to each carbon atom . Proton s an d carbon position s of th e glucuron ic acid m oiety are repre-
sen ted by a sin gle prim e (′). Th in layer ch rom atograph y (TLC) was perform ed on Wh atm an
MK6F silica gel m icro TLC plates (25 µm th ickn ess) usin g CHCl3–MeOH–H2O; 65:35:5,
v/v/v (solven t system A) an d h exan es–eth yl acetate; 60:40, v/v (solven t system B) as th e
developin g solven ts. Colum n ch rom atograph y was carried out usin g Merck 7734 silica gel
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1042–1050

1048
Tandon, Kumar, Xia, Wang, Wiebe:
(100–200 µm particle size). Th e com poun ds were ch aracterized by th eir elem en tal an alyses
for C, H an d N or by Fast atom bom bardm en t (FAB) m ass spectra usin g a sodium probe on
an AEI-MS-12 m ass spectrom eter. Ph en yl β-D-glucopyran osiduron ic acid, D-glucuron e an d
β-glucuron idase (E. coli; 1000 un its/m l) were purch ased from Sigm a Ch em ical Co. (St. Louis,
U.S.A.). 2,4,6-Tribrom oph en ol was syn th esized by a reported procedure8 (m .p. 92 °C
(reported); 94 °C (observed)). Meth yl 2,3,4-tri-O-acetyl-α-D-glucopyran osyluron ate brom ide
(5) was prepared by th e m eth od of Bowerin g an d Tim ell9 (m .p. 104.5 °C (reported); 104 °C
(observed)).
4-Brom oph en yl β-D-Glucopyran osiduron ic Acid (2)
Liquid brom in e was added dropwise to a precooled (0 to 5 °C), stirred suspen sion of ph en yl
β-D-glucopyran osiduron ic acid (0.1 g, 0.37 m m ol) in water (1 m l), un til th e color of brom in e
persisted. Th e stirrin g was con tin ued at th is tem perature for an addition al 1 h . No startin g
m aterial was detected (TLC) at th is tim e. Th e solven t was rem oved by evaporation un der
vacuum an d th e m ixture was purified on a silica gel colum n usin g solven t system A as
eluen t. Th e eluted product was recrystallized from m eth an ol to afford 105 m g (81%) of
pure 2. RF 0.60 (solven t system A); m .p. 210 °C (dec.). 1H NMR (D2O): 3.54–3.60 m , 3 H
(H-2′, H-3′ an d H-4′); 3.80 d, 1 H, J(4′,5′) = 8.7 (H-5′); 5.00 d, 1 H, J(2′,1′) = 7.5 (H-1′);
6.97 d, 2 H, J(2,3) = J(6,5) = 8.5 (H-3 an d H-5 of ph en yl rin g); 7.42 d, 2 H, J(3,2) = J(5,6) =
8.5 (arom atic H-2 an d H-6). 13C NMR (D2O): 72.37 (C-4′), 73.40 (C-2′), 75.99 (C-5′), 76.86
(C-3′), 100.92 (C-1′), 115.71 (arom atic C-4), 119.34 (arom atic C-2 an d C-6), 133.24 (arom atic
C-3 an d C-5), 156.55 (arom atic C-1), 176.04 (COOH). FAB HRMS for C12H13BrO7, M+ rela-
tive abun dan ce 5.06% (79Br) an d 5.27% (81Br).
2,4-Dibrom oph en yl β-D-Glucopyran osiduron ic Acid (3)
Ph en yl β-D-glucopyran osiduron ic acid (0.2 g, 0.74 m m ol) was suspen ded in water (2 m l),
an d liquid brom in e was cautiously added dropwise to th is m ixture wh ile stirrin g, un til bro-
m in e decolorization stopped. Th e reaction m ixture was allowed to stir at 25 °C for 3 h . At
th is tim e TLC of th e reaction m ixture sh owed com plete disappearan ce of th e glucuron ide
an d a n ew product appeared. Water was rem oved by evaporation un der vacuum an d th e re-
sultin g im pure syrup was ch rom atograph ed on a silica gel colum n usin g solven t system A as
eluen t to obtain 0.17 g (53.6%) of pure 3. RF 0.64 (solven t system A); m .p. 195 °C (dec.).
1H NMR (D2O): 3.40–3.66 m , 3 H (H-2′, H-4′ an d H-5′); 3.76 d, broad, 1 H (H-3′); 5.00 d,
1 H, J(2′,1′) = 7.5 (H-1′); 7.04 d, 1 H, J(5,6) = 9.0 (arom atic H-6); 7.38 d, 1 H, J(6,5) = 9.0
of d, J(3,5) = 1.5 (arom atic H-5); 7.72 d, 1 H, J(5,3) = 1.5 (arom atic H-3). 13C NMR (D2O):
72.35 (C-4′), 73.31 (C-2′), 76.06 (C-5′), 76.99 (C-3′), 101.35 (C-1′), 113.68 (arom atic C-2),
115.96 (arom atic C-4), 119.05 (arom atic C-6), 132.49 (arom atic C-3), 136.07 (arom atic C-5),
153.14 (arom atic C-1), 175.93 (COOH). FAB HRMS for C12H12Br2O7, M+ relative abun dan ce
0.11% (79Br), 0.13% (79/81Br) an d 0.11% (81Br).
Meth yl (2,4,6-Tribrom oph en yl 2,3,4-tri-O-acetyl-β-D-glucopyran osid)uron ate (6)
A m ixture of 2,4,6-tribrom oph en ol (4.17 g, 12.6 m m ol) an d m ercuric cyan ide (0.35 g, 1.39
m m ol) was h eated at 110 °C to form a h om ogen ous m elt. A solution of brom ide 5 (0.5 g,
1.26 m m ol) in an h ydrous dich lorom eth an e (2 m l) was added dropwise to th is m elt. After
th e addition of brom ide 5 was com plete, th e reaction m ixture was h eated at 110 °C for
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1042–1050

Synthesis of Bromophenyl β-D-Glucuronides
1049
30 m in an d th en at 50 °C for 2 h . A TLC exam in ation of th is m ixture sh owed com plete dis-
appearan ce of brom ide 5 an d form ation of a n ew product. Purification of th is im pure prod-
uct on a silica gel colum n usin g sequen tial gradien ts of eth yl acetate (up to 14%) in h exan es
afforded 0.43 g (52.8%) of pure 6. RF 0.44 (solven t system B); m .p. 180–182 °C. 1H NMR
(CDCl3): 2.04, 2.07 an d 2.13 3 × s, 3 H (COCH3); 3.84 s, 3 H (OCH3); 3.96 d, 1 H, J(4′,5′) =
9.5 (H-4′); 5.28–5.50 m , 4 H (H-1′, H-2′, H-3′ an d H-5′); 7.68 s, 2 H (arom atic H-3 an d H-5).
13C NMR (CDCl3): 20.42, 20.47, 20.77 (3 × COCH3), 52.82 (COOCH3), 69.26 (C-4′), 71.24
(C-2′), 71.90 (C-5′), 72.66 (C-3′), 100.35 (C-1′), 118.87 (arom atic C-4), 119.23 (arom atic C-2
an d C-6), 135.39 (arom atic C-3 an d C-5), 148.92 (arom atic C-1), 166.50, 169.19, 169.32 (3 ×
C=O), 170.09 (COOH). For C19H19Br3O10 (647.0) calculated: 35.27% C, 2.90% H; foun d:
35.31% C, 2.85% H.
2,4,6-Tribrom oph en yl β-D-Glucopyran osiduron ic Acid (7)
A cold (–5 to 0 °C) solution of sodium h ydroxide (0.03 g, 0.77 m m ol) in m eth an ol (2 m l)
was added to a m eth an olic solution (2 m l) of 6 (0.1 g, 0.15 m m ol) an d th e reaction m ixture
was stirred at 40 °C overn igh t. A ch rom atograph ic exam in ation of th e reaction m ixture
sh owed com plete con version of 6 to 7. Th e solution was deion ized with KA-2-(H+) ion ex-
ch an ge resin , filtered an d th e solven t was rem oved in vacuo, to provide im pure 7. Purifica-
tion on a silica gel colum n , usin g solven t system A as eluen t, yielded 54.5 m g (69.6%) of
pure 7. RF 0.65 (solven t system A); m .p. 185 °C (dec.). 1H NMR (D2O): 3.49 dd, 1 H, J(2′,3′) =
5.5, J(4′,3′) = 3.5 (H-3′); 3.52 dd, 1 H, J(3′,4′) = 3.5, J(5′,4′) = 9.5 (H-4′); 3.58 d, 1 H, J(4′,5′) =
9.5 (H-5′); 3.68 dd, 1 H, J(1′,2′) = 7.5, J(3′,2′) = 5.5 (H-2′); 5.20 d, 1 H, J(1′,2′) = 7.5 (H-1′);
7.76 s, 2 H (arom atic). 13C NMR (D2O): 72.38 (C-4′), 74.36 (C-2′), 76.13 (C-5′), 77.50 (C-3′),
103.80 (C-1′), 118.75 (arom atic C-2 an d C-6), 119.14 (arom atic C-4), 136.06 (arom atic C-3
an d C-5), 149.80 (arom atic C-1), 171.60 (COOH). FAB HRMS for C12H11Br3O7 (506.9), M+
relative abun dan ce 0.3%.
En zym atic Hydrolysis
En zym atic h ydrolysis of brom oph en yl glucuron ic acids was studied usin g β-glucuron idase
en zym e (500 un its/m l in buffer) wh ich was prepared by dissolvin g 1000 un its of β-glucuron -
idase in 2 m l of 0.1 M acetate buffer (pH 5.0). Th e brom oph en yl glucuron ide (2, 3 or 7,
1 m m ol) solution was prepared with th e sam e buffer. Th e h ydrolysis was in itiated by addi-
tion of β-glucuron idase to th e brom oph en yl glucuron ic acids’ solution s. Th e m ixture was
sam pled at selected in tervals. For an alysis th ese sam ples were m ixed with an equal volum e
of ice-cold m eth an ol an d th en vortexed. After cen trifugation at 12,800 rpm (Brin km an
Microfuge) for 2 m in , th e supern atan t was collected an d frozen un til an alysis. Th e an alysis
was perform ed by h igh perform an ce liquid ch rom atograph y (HPLC) usin g a C18 Radial-PAK
cartridge (8 m m i.d., 10 µ particle size) with UV detection at 298 n m (di- an d tri-
brom oph en yl glucuron ic acids) or 280 n m (m on obrom oph en yl glucuron ic acid). A gradien t
system of water–m eth an ol gradien ts (0–9 m in , 20% m eth an ol; 9–40 m in , 20 to 80% m eth a-
n ol, lin ear in crease; 40–60 m in , 80% m eth an ol) was used for elution .
The authors are thankful to Dr V. Somayaji for recording NMR spectra and to Mr G. Haverland for
helpful discussions of the ‘organic chlorine’ screening procedure.
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Tandon, Kumar, Xia, Wang, Wiebe:
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