Bacterial Catabolism of Biphenyls: Synthesis and Evaluation of Analogues

Article abstract of DOI:10.1002/cbic.201800231

A series of alkylated 2,3-dihydroxybiphenyls has been prepared on the gram scale by using an effective Directed ortho Metalation–Suzuki–Miyaura cross-coupling strategy. These compounds have been used to investigate the substrate specificity of the meta-cl

Full text of DOI:10.1002/cbic.201800231

Accepted Article
Title: Bacterial Catabolism of Biphenyls. Synthesis and Evaluation of
Analogues.
Authors: Victor Snieckus, Timothy Hurst, Sven Nerdinger, Eugene
Kautsjah, Lindsay Eltis, Volker Kahlenberg, and Inge Schlapp-
Hackl
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To be cited as: ChemBioChem 10.1002/cbic.201800231
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ChemBioChem
10.1002/cbic.201800231
FULL PAPER
Bacterial Catabolism of Biphenyls. Synthesis and Evaluation of
Analogues
[a]
[b]
[c]
[d]
[e]
Sven Nerdinger, Eugene Kuatsjah, Timothy E. Hurst, Inge Schlapp-Hackl, Volker Kahlenberg,
[f]
[b]
[c]
Klaus Wurst, Lindsay D. Eltis,* and Victor Snieckus*
Dedication
Abstract: A series of alkylated 2,3-dihydroxybiphenyls (DHBs) have
been prepared on gram scale using an effective Directed ortho
Metalation (DoM) – Suzuki-Miyaura cross-coupling strategy. These
compounds have been used to investigate the substrate specificity of
the meta-cleavage dioxygenase BphC, a key enzyme in the microbial
catabolism of biphenyl. Isolation and characterization of the meta-
cleavage products allows the study of related processes, including the
catabolism of lignin-derived biphenyls.
Introduction
Understanding the bacterial catabolism of aromatic compounds is
critical for developing effective biocatalysts for various
[
1]
technologies, including bioremediation and the upgrading of
lignin to commodity chemicals.[2] For example, the catabolism of
biphenyl, a paradigm for the microbial catabolism of aromatic
[
[
a]
b]
Dr. S. Nerdinger
Global Commercial Operations, Sandoz GmbH
Biochemiestraße 10
6
250 Kundl, Austria
E. Kuatsjah, Prof. Dr. L. D. Eltis
350 Health Sciences Mall
2
Life Sciences Centre
Vancouver, BC
Canada, V6T 1Z3
E-mail: leltis@mail.ubc.ca
Dr. T. E. Hurst, Prof. Dr. V. Snieckus
Department of Chemistry, Queen’s University
Chernoff Hall
Kingston, ON
Canada, K7L 3N6
[
c]
d]
E-mail: snieckus@chem.queensu.ca
I. Schlapp-Hackl
[
Institute of General, Inorganic and Theoretical Chemistry
University of Innsbruck
Center for Chemistry and Biomedicine
Innrain 80-82
A-6020 Innsbruck, Austria
Prof. Dr. V. Kahlenberg
Institute of Mineralogy and Petrography
University of Innsbruck
Innrain 52
A-6020 Innsbruck, Austria
Prof. Dr. K. Wurst
[
e]
f]
[
Faculty of Chemistry and Pharmacy
University of Innsbruck
Innrain 80-82
Figure 1. a) The transformation of dihydroxybiphenyls (DHBs) and meta-
cleavage products (MCPs) in the bacterial catabolism of DDVA (enzyme names
in blue) and biphenyl (enzyme names in black), respectively. The substituents
in light blue only occur in DDVA catabolism. b) Synthetic analogues 5-8 and
their meta-cleavage products. The carboxylate of 8 is considered to be the
enzyme’s substrate.
A-6020 Innsbruck, Austria
Supporting information for this article is given via a link at the end of
the document.
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ChemBioChem
10.1002/cbic.201800231
FULL PAPER
compounds, has been studied and engineered to degrade
PCBs.[3] More recently, the recognition that bacteria partially
deconstruct lignin has stimulated studies of enzymes involved in
depolymerizing lignin and
catabolizing the resulting
[
4]
products. Sphingobium sp. strain SYK-6 is among the best
characterized bacterial strains that grow on lignin-derived
aromatic compounds. The strain contains pathways responsible
for catabolizing a number of lignin-derived aromatics, including
2,2’-dihydroxy-3,3’-dimethoxy-5,5’-dicarboxybiphenyl (DDVA), β-
aryl ethers, phenylcoumaran-type biaryls, pinoresinols and diaryl
propanes.[5]
The catabolism of DDVA[5b] is similar to that of biphenyl in
that both proceed via a 2,3-dihydroxybiphenyl (DHB, 1) that is
subject to meta-cleavage by an extradiol dioxygenase to 2-
hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA, 2). A meta-
5 6
cleavage product (MCP) hydrolase then catalyzes the C -C bond
cleavage of HOPDA through a vinylogous retro-Claisen reaction.
In biphenyl catabolism, these two reactions are catalyzed by
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase) and BphD,
respectively. In DDVA catabolism, the corresponding enzymes
are LigZ and LigY, whose substrates are 2,2’,3-trihydroxy-3’-
methoxy-5,5’-dicarboxybiphenyl (OH-DDVA, 1b) and 4,11-
Scheme 1. Retrosynthetic analysis of model substrates 4-Me-DHB 5, 5-Me-
DHB 6 and 6-Me-DHB 7.
the OMOM derivative 15 of commercially available 2-methoxy-5-
methylphenol. Site selectivity (step 1) for the more hindered
position is expected based on the greater directing power of the
OMOM DMG compared to the weaker OMe group.[11] Subsequent
Suzuki-Miyaura cross-coupling delivers 6-Me DHB (7).
dicarboxy-8-hydroxy-9-methoxy
(DCHM-HOPDA,
2b),
respectively (Figure 1, catabolism pathway). BphD is a serine-
dependent member of the /β-hydrolase superfamily,[6] typical of
all but one of the MCP hydrolases characterized to date. The lone
2+
MCP hydrolase that differs is LigY; it is a Zn -dependent member
of the amidohydrolase superfamily.[7] It is unclear how these two
enzymes use such different catalytic machinery to catalyze similar
reactions.
Synthesis of 4-Me-DHB. The synthesis of 4-Me-DHB (5)
(Scheme 2) was initiated by the preparation of the di-MOM phenyl
catechol 17 according to our previously reported procedure.[
Treatment of 17 with n-BuLi/TMEDA followed by quenching with
iodomethane afforded 18, a reaction which was carried out on 7
gram scale (see SI). Global deprotection using methanolic HCl
delivered 3 grams of 4-Me-DHB (5).
8]
To facilitate the further study of bacterial pathways that
catabolize aromatic compounds, we now report the synthesis of a
series of alkyl substituted DHBs (5-8, Figure 1). To illustrate the
utility of these compounds in characterizing PCB- and lignin-
degrading enzymes, we used them to investigate the specificity of
BphC. The corresponding alkylated MCPs 9-12 were
characterized for studies of BphD and LigY.
Results and Discussion
The retrosynthetic analysis of Me-DHBs 5-7 conceptualized in
Scheme 1 follows a unified Directed ortho Metalation (DoM) –
Suzuki-Miyaura cross-coupling strategy analogous to that
[
8]
documented previously. Thus, 4-Me-DHB (5) is considered to
be synthesized by a ‘walk around the ring’ tactic[9] from the
methoxymethyl-protected (OMOM) derivative 13 of commercially
available 2-phenylphenol. The first OMOM directed metalation
group (DMG) serves to introduce the second OMOM DMG (step
1) which itself is then used to introduce the methyl group (step 2).
5-Me-DHB (6) is derived from MOM-protected p-cresol 14 via two
successive DoM reactions without concern of methyl group
deprotonation.[10] In step 1, boronation is followed by Suzuki-
Miyaura cross-coupling, while in step 2 the boronation is followed
by hydroxylation. The order of steps (1 before 2) is dictated by the
requirement of protection-deprotection steps of the introduced
phenolic hydroxyl group (step 2). 6-Me-DHB (7) is derived from
Scheme 2. Synthesis of 4-methyl-2,3-dihydroxybiphenyl (4-Me-DHB). [x-ray
structure added]
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Scheme 5. Alternate synthesis of 6-methyl-2,3-dihydroxybiphenyl (6-Me-DHB).
Scheme 3. Synthesis of 5-methyl-2,3-dihydroxybiphenyl (5-Me-DHB). [x-ray
structure added]
Suzuki-Miyaura coupling gave the biphenyl 22 in 84% yield, which
upon deprotection of the MOM group with HCl/MeOH followed by
demethylation with BBr afforded 6-Me-DHB (7), whose structure
3
was confirmed unambiguously by x-ray crystallography.
In an alternate approach to 6-Me-DHB (Scheme 5), site
selective iodination[8] of 2,3-dimethoxybiphenyl 23 furnished the
contiguously substituted product 24, which underwent facile
Synthesis of 5-Me-DHB. The preparation of 5-Me-DHB (6)
(
1
Scheme 3) starts from the symmetrical MOM-protected p-cresol
4 which, in a three-step sequence involving no purification of
intermediates, was subjected to metalation (s-BuLi/TMEDA/–
8 °C) followed by trapping of the resulting anion with freshly
distilled B(OMe) to give the corresponding boronic acid which,
upon direct Suzuki-Miyaura cross-coupling, afforded biphenyl 19
4 gram scale reaction, see SI). Subsequent hydroxylation was
7
3
lithium-halogen exchange on treatment with t-BuLi in Et
Trapping of the resulting lithiated species with iodomethane
followed by final deprotection with BBr delivered 6-Me-DHB (7).
Synthesis of 5-CO H-DHB. The synthesis of the final target,
H-DHB (8) (Scheme 6), was initiated by bromination of
2
O.
(
3
then achieved through a second metalation/boronation sequence
followed by oxidation to give phenol 20 which, on final
deprotection, furnished 2.5 grams of 5-Me-DHB (6).
2
5
-CO
phenol 26 as described. Suzuki-Miyaura coupling delivered 3
grams of biphenyl 28 which, upon treatment with BBr , led to
concomitant deprotection of both the methoxy and ester groups
to afford 5-CO H-DHB (8).
2
[
13]
Synthesis of 6-Me-DHB. The synthesis of 6-Me-DHB (7)
3
(
Scheme 4) begins with the OMOM protected phenol 15 which
provides DMG hierarchy (OMOM OMe) advantage
a
>
2
irrespective of the methyl group steric effect to give, upon
metalation and iodine quench, compound 21 in high yield.[12]
2
Scheme 6. Synthesis of 5-carboxy-2,3-dihydroxybiphenyl (5-CO H-DHB).
Scheme 4. Synthesis of 6-methyl-2,3-dihydroxybiphenyl (6-Me-DHB).
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Table 1. Apparent steady-state kinetic and inactivation parameters of BphC.
Table 2. Molar extinction coefficients and pKa
2
of Me-HOPDAs.
푎푝푝
푎푝푝
푎푝푝
푖푛푎푐푡
푎푝푝
푎푝푝
푘cat
-1
푘cat /퐾M
partition
ratio
푎푝푝
푖푛푎푐푡
푘
/퐾M
3
Extinction
coefficient
푎푝푝
퐾M
푘
Corresponding
HOPDA
pKa
2
of
Wavelength
(nm)
6
-1
-1
[b]
substrate
(x 10
M
3
-1
(x 10
M
DHB
DHB
(
s
)
(x 10
s
)
[a]
(
μM)
HOPDA
-
1
3
-1
-1
-1
s
)
(x 10 )
s
)
(mM cm
)
[
a]
[a]
DHB
12 (1)
36 (4)
33 (3)
6 (1)
251 (6)
116 (4)
506 (20)
53 (3)
21 (1)
84.9 (1.4)
1.7 (0.1)
1.6 (0.1)
9.2 (0.4)
3.0 (0.1)
68 (8)
0.25 (0.02)
1.9 (0.3)
10 (1)
HOPDA
7.3
7.5
10
434
428
446
402
26.6
39.7
42.4
45.5
4
5
6
-Me-DHB
-Me-DHB
-Me-DHB
3.2 (0.2)
16 (1)
4-Me-DHB
5-Me-DHB
6-Me-DHB
3-Me-HOPDA
4-Me-HOPDA
5-Me-HOPDA
320 (20)
5.9 (0.7)
8.5 (0.8)
1.0 (0.2)
7.7
[
a]
Experiments were performed using air-saturated 40 mM HEPES (I = 0.1 M), pH
.5 at 25 °C. The values in parentheses represent standard errors. [a] Kinetic
a
pK values were determined in potassium phosphate buffer titrated with
[
b]
7
either acid or base. Molar extinction coefficients determined in 0.1 M KOH,
25˚C, with freshly generated Me-HOPDAs.
푎푝푝
parameters for DHB were obtained from reference 15. [b] 푘
was calculated
푖푛푎푐푡
푎푝푝
by dividing kcat by the partition ratio.
yellow-colored dienolate anion (λmax = 434 nm) under the reaction
conditions used here (potassium phosphate (I = 0.1 M), pH 7.5,
2
Specificity of BphC. We used an oxygraph assay[14] to
investigate the ability of BphC from Burkholderia xenovorans
LB400 to catalyze the meta-cleavage of the alkylated DHBs.
Because the steady-state cleavage of the DHBs exhibited some
substrate inhibition, the kinetic parameters were determined using
5 ᵒC).[18] Under these conditions, reaction of BphC with each Me-
DHB (5 to 8) also yielded a yellow-colored product, consistent with
1
,2-cleavage to the corresponding Me-HOPDA and its occurrence
[18]
as a dienolate, as reported for Cl-DHBs. 4-Me-HOPDA rapidly
푎푝푝
DHB concentrations only up to ~5-times the estimated 퐾M . As
½
converted to a colorless species (t ~4 s) under these conditions.
The dienolate form of 4-Me-HOPDA could be regenerated under
alkaline pH, as reported for DCHM-HOPDA.[19]
summarized in Table 1, BphC catalyzed the cleavage of the Me-
푎푝푝
푎푝푝
DHBs with apparent specificities (푘푐푎푡 /퐾M ) that were 20–70%
those of unsubstituted DHB in the following order: DHB > 5-Me >
The pK
extinction coefficients in 0.1 M KOH are summarized in Table 2.
Overall, methyl substitution of the dienol moiety increased the pK
value of HOPDA. The corresponding chlorinated HOPDAs had
a
values of the Me-HOPDAs together with their molar
6-Me > 4-Me-DHB. This is similar to what was observed with
chlorinated DHBs although the order of apparent specificity was
slightly different: DHB > 6-Cl > 4-Cl > 5-Cl-DHB.[15] Indeed, the
steady-state kinetic parameters for methylated DHBs were similar
to those reported for the corresponding chlorinated DHBs with the
exception of 5-Me-DHB, for which BphC had ~7-fold higher
apparent specificity than 5-Cl-DHB. Interestingly, BphC did not
a
lower pK
the chloro substituent.
a
values, consistent with the greater electronegativity of
[19]
The identity of Me-HOPDAs arising from the cleavage of
respective Me-DHBs was confirmed by mass spectrometry and
the results are summarized in Table 3. Unlike the 3-Me- and 5-
Me-HOPDAs, the parent-ion for 4-Me-HOPDA was not detected
using our standard mass spectrometry conditions. Subsequently,
the 4-Me-HOPDA was derivatized with ammonia to produce the
more stable pyridinal derivative whose mass was used to infer the
identity of the 4-Me-HOPDA, similar to the strategy used to
2 2
detectably cleave 5-CO H-DHB. Moreover, 5-CO H-DHB at
concentrations up to 200 µM did not detectably inhibit the BphC-
catalyzed cleavage of DHB. This suggests that BphC does not
cleave the carboxylated substrate because it has low affinity for it.
Consistent with this analysis, inspection of the BphC:DHB
complex[14] revealed that the 5-position is closely bounded by
three residues (Phe187, Ile173 and Asn243), two of which are
hydrophobic. By contrast, protocatechuate 4,5-dioxygenase
accommodates the carboxylate substituent of its substrate with
hydrogen bond-forming residues.[16]
identify the MCP resulting from the cleavage of DCHM-HOPDA
by LigZ.[18]
Table 3. Mass determination of Me-HOPDAs from BphC catalyzed Me-DHB
cleavage.
We also evaluated the susceptibility of BphC to inactivation
during the cleavage of methylated DHBs. This inactivation occurs
through the adventitious oxidation of the active site iron during the
corresponding
HOPDA
Charge
state
Calculated
m/z
Observed
m/z
DHB
catalytic cycle.[17] The susceptibility to inactivation, 푘푎푝푝 /퐾M
푎푝푝
,
푖푛푎푐푡
was calculated from the partition ratio, which is the average
number of times the enzyme turns over before inactivation. Based
4
-Me-DHB
3-Me-HOPDA
233.0813
214.0868
233.0813
233.0849
214.0844
233.0849
+1
+1
+1
(
5)
(9)
푎푝푝
푎푝푝
on 푘
/퐾M , BphC was up to 40-fold more susceptible to
푖푛푎푐푡
[
a]
5
-Me-DHB
4-Me-HOPDA
inactivation by Me-DHBs in the following order: 5-Me > 4-Me > 6-
(
6)
(10)
Me > DHB. Indeed, 5-Me-DHB inactivated BphC 100-times more
푎푝푝
푖푛푎푐푡
efficiently (푘
) than DHB. These results contrast with those for
6-Me-DHB
5-Me-HOPDA
the corresponding chlorinated DHBs, which did not inactivate
BphC more potently than DHB.[15] Indeed, BphC was slightly less
susceptible to inactivation by 5-Cl DHB than DHB.
(7)
(11)
[
a]
4-Me-HOPDA was derivatized with ammonia as discussed in text.
Properties of Me-HOPDAs. BphC catalyzes the 1,2-
[
14]
cleavage of DHB to HOPDA, which exists predominantly as the
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Conclusions
supplementary crystallographic data for this paper. These data are
provided free of charge by the Cambridge Crystallographic Data Centre.
In summary, we have reported the synthesis of a series of
alkylated 2,3-dihydroxybiphenyls (DHBs), and their use in the
evaluation of the substrate specificity of the extradiol dioxygenase
BphC. The use of an effective Directed ortho Metalation (DoM) –
Suzuki-Miyaura cross-coupling strategy proved crucial in
providing rapid access to the requisite gram scale quantities of
the isomeric highly substituted arenes needed for this study.
BphC catalyzed the cleavage of the methylated DHBs with
substrate specificities similar to those reported for the
corresponding chlorinated DHBs. However, the Me-DHBs
inactivated BphC more potently than the Cl-DHBs, particularly 5-
Me-DHB. The substituted DHBs and HOPDAs will facilitate
studies of related catabolic processes, including homologous
enzymes and downstream catabolism. For example, 5-carboxy
DHB could be a useful probe for extradiol dioxygenases whose
cognate substrates have 5-carboxy substituents. Similarly, the 4-
substituted HOPDAs are of interest as analogs of DCHM-HOPDA,
the substrate of LigY.
2
2
,3-Bis(methoxymethoxy)-4-methyl-1,1'-biphenyl (18): To a solution of
,3-bis(methoxymethoxy)-1,1'-biphenyl[8] 17 (7.00 g, 25.5 mmol) in Et
O
2
(120 mL) was added TMEDA (4.44 mL, 30.6 mmol, 1.2 equiv). The solution
was stirred for 5 min then cooled to 0 °C before the dropwise addition of
n-BuLi (1.52 M solution in hexane, 20.1 mL, 30.6 mmol, 1.2 equiv). The
reaction mixture was stirred for 2.5 h at 0 °C, then cooled to –78 °C and
quenched by the rapid addition of iodomethane (6.35 mL, 102 mmol, 4.0
equiv). After warming to rt, stirring was continued for a further 1 h, then sat.
brine solution was added (50 mL), and the layers were separated. The
aqueous layer was extracted once with Et
organic layers were washed with brine (50 mL), dried over MgSO
2
O (50 mL). The combined
4
,
subjected to filtration, and the solvents were removed in vacuo. The crude
product was purified by flash chromatography on silica gel (1:9
EtOAc/hexanes) to give the title compound 18 (5.73 g, 82%) as a pale
yellow oil. IR (ATR): ṽ = 3056, 2990, 2927, 2826, 1477, 1421, 1387, 1302,
1268, 1155, 1073, 1033, 958, 919, 818, 759, 698; 1H NMR (400 MHz;
CDCl
Hz, 1 H), 7.02 (d, J = 7.8 Hz, 1 H), 6.99 (d, J = 7.8 Hz, 1 H), 5.20 (s, 2 H),
.80 (s, 2 H), 3.63 (s, 3 H), 3.01 (s, 3 H), 2.36 (s, 3 H) ppm; 13C NMR (100
MHz; CDCl ): δ = 149.1 (C), 146.9 (C), 138.6 (C), 135.0 (C), 132.1 (C),
29.6 (CH), 128.1 (CH), 126.9 (CH), 126.4 (CH), 125.7 (CH), 99.4 (CH ),
3
): δ = 7.52-7.50 (m, 2 H), 7.41-7.37 (m, 2 H), 7.31 (tt, J = 7.3 and 1.4
4
3
1
9
2
8.9 (CH
2
), 57.6 (Me), 57.0 (Me), 16.6 (Me) ppm; EI HRMS: cald for
288.1362, found 288.1355.
Experimental Section
+
C
H O
17 20 4
Synthesis
4-Methyl-2,3-dihydroxybiphenyl (4-Me-DHB, 5): To a solution of 2,3-
bis(methoxymethoxy)-4-methyl-1,1'-biphenyl 18 (5.00 g, 17.3 mmol)
General Information: All reactions were carried out in oven-dried
glassware under an atmosphere of argon unless otherwise stated. Flash
chromatography was carried out using Silicycle Silicaflash P60 silica gel.
Melting points were determined on an Electrothermal IA9100 melting point
apparatus from chromatographically homogenous compounds without
further recrystallization. IR spectra were recorded on a Bruker Alpha FTIR
spectrometer. 1H and 13C NMR spectra were recorded using a Bruker
Avance-400 or Avance-300 spectrometer operating at 400 MHz and 300
MHz respectively ( H frequency, corresponding 13C frequencies are 100
MHz and 75 MHz). In the 13C NMR spectra, signals corresponding to CH,
MeOH (30 mL) was added 3 N HCl (15 mL). The reaction mixture was
stirred at rt for 16 h, then diluted with CH
separated. The aqueous layer was extracted with CH
combined organics were washed with sat. brine solution (100 mL), dried
over MgSO , subjected to filtration, and the solvents were removed in
2
Cl
2
(100 ml) and the phases were
2
Cl (100 mL) and the
2
4
vacuo. The crude product was purified by flash chromatography on silica
gel (1:3 EtOAc/hexanes) followed by recrystallization (EtOAc/hexane) to
give the title compound 4-Me-DHB 5 (3.08 g, 89%) as pale purple crystals.
M.p. 63.5–65 °C; IR (ATR): ṽ = 3396, 3299, 3058, 2920, 1628, 1569, 1485,
14770, 1431, 1352, 1324, 1288, 1270, 1189, 1160, 1092, 1031, 944, 888,
1
CH
2
, or Me groups are assigned from DEPT. Mass spectra were recorded
805, 763, 748, 724, 693; 1H NMR (400 MHz; CDCl
H), 7.42-7.38 (m, 1 H), 6.80 (d, J = 7.9 Hz, 1 H), 6.75 (d, J = 7.9 Hz, 1 H),
5.50 (s, 1 H), 5.31 (s, 1 H), 2.32 (s, 3 H) ppm; 13C NMR (100 MHz; CDCl ):
3
): δ = 7.52-7.48 (m, 4
on a Micromass 70-250S double focusing mass spectrometer (EI) or
Waters ZQ Single Quad mass spectrometer (ESI). X-Ray crystallographic
analyses for 4-Me-DHB and 5-Me-DHB were measured with a Bruker D8
Quest diffractometer equipped with a Photon 100 CMOS detector and
molybdenum radiation (λ = 0.710713 Å) from an Incoatec microfocus tube.
For the multiscan absorption correction of the intensity data, Sadabs
3
δ = 142.5 (C), 139.4 (C), 137.1 (C), 129.3 (CH), 128.8 (CH), 127.7 (CH),
125.9 (C), 124.0 (C), 122.6 (CH), 120.5 (CH), 15.5 (Me) ppm; EI HRMS:
+
cald for C13H O
12 2
200.0837, found 200.0832.
2
014/5 was used, and the structure solutions and parameter refinement
2
-(Methoxymethoxy)-5-methyl-1,1'-biphenyl (19): To a solution of 4-
was performed with the Shelxs/l-2013.[20] X-Ray crystallographic analysis
for 6-Me-DHB was measured with a Rigaku Oxford Diffraction Gemini-R
Ultra diffractometer using graphite monochromatized Mo-Ka (l=0.71073 Å)
radiation. Data reduction including intensity integration, background
corrections as well as Lorentz and polarization correction was performed
with the CrysAlisPRO software package. The structure was solved by direct
methods using the SIR2004 program suite.[21] All non-hydrogen atoms of
the molecule were obtained from the E-map with the highest combined
(methoxymethoxy)toluene 14 (6.00 g, 39.4 mmol) in Et O (120 mL) was
2
added TMEDA (7.09 mL, 47.3 mmol, 1.2 equiv). The solution was stirred
for 5 min then cooled to –78 °C before the dropwise addition of s-BuLi
(
1.53 M solution in cyclohexane, 30.9 mL, 47.3 mmol, 1.2 equiv). The
reaction mixture was stirred for 2.5 h at –78 °C, then quenched by the rapid
addition of freshly distilled B(OMe) (17.6 mL, 158 mmol, 4.0 equiv).
Stirring was continued for 2 h at –78 °C, then the reaction mixture was
allowed to warm to rt, quenched by the addition of H O (60 mL), and then
acidified with 2 N HCl to pH 6. The aqueous layer was extracted with
CH Cl (2 × 150 mL). The combined organics were washed with H O (50
mL) and sat. brine (50 mL), dried over MgSO , subjected to filtration, and
3
2
2
figure of merit. Full-matrix least-squares refinement on F was carried out
using the SHELXL97software.[20] All hydrogen atoms (except those
belonging to the hydroxyl groups) were geometrically fixed and allowed to
ride on the corresponding carrier atoms with C–H = 0.98 – 0.99 Å and
2
2
2
4
the solvents evaporated in vacuo. The crude boronic acid was used in the
next step without further purification.
U
iso(H)= 1.2 x Ueq(C) of the attached C atom. Hydrogen atoms of the
hydroxyl groups were located in a difference Fourier map and refined
isotropically using the same numerical coupling for Uiso but without any
restraints concerning the O-H bond distances. CCDC 1842733 (4-Me-
DHB), 1844073 (5-Me-DHB) and 1839038 (6-Me-DHB) contain the
A solution of bromobenzene (4.04 g, 25.7 mmol) in 1,2-DME (140 mL) was
degassed by bubbling with argon for 15 min. Pd(PPh
3 4
) (2.97 g, 2.57 mmol,
10 mol%) was added and stirring continued for 15 min. A degassed 2 N
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800231
FULL PAPER
1
aqueous Na
2
CO
3
solution (80 mL) was added and the mixture stirred for a
694; H NMR (400 MHz; CDCl
3
): δ = 7.49-7.47 (m, 4 H), 7.42-7.37 (m, 1
further 30 min at rt, whereupon the reaction mixture became cloudy. A
solution of the arylboronic acid (7.55 g, 38.6 mmol, 1.5 equiv) in degassed
H), 6.75 (d, J = 1.6 Hz, 1 H), 6.65 (d, J = 1.6 Hz, 1 H), 5.48 (br s, 1 H), 5.18
(br s, 1 H), 2.30 (s, 3 H) ppm; 13C NMR (100 MHz; CDCl
): δ = 144.0 (C),
3
1
,2-DME (75 mL) was then added dropwise, and the reaction mixture
stirred at reflux for 16 h. The solvent was removed in vacuo and the residue
was extracted with Et O (2 × 75 mL). The combined organics were washed
with H O (75 mL), 2 N NaOH (75 mL), and sat. brine solution (75 mL),
dried over MgSO , subjected to filtration, and the solvents removed in
137.6 (C), 137.1 (C), 130.6 (C), 129.2 (CH), 128.9 (CH), 128.1 (C), 127.8
(CH), 121.9 (CH), 115.3 (CH), 20.8 (Me) ppm; EI HRMS: cald for
+
2
C H O
13 12 2
200.0837, found 200.0843.
2
4
2
-Iodo-4-methoxy-3-(methoxymethoxy)-1-methylbenzene (21):
A
vacuo. The crude product was purified by flash chromatography on silica
gel (1:4 EtOAc/hexanes) to give compound 19 (4.33 g, 74%) as pale yellow
crystals. IR (ATR): ṽ = 3026, 2951, 2923, 2898, 2824, 1601, 1503, 1488,
[22]
solution of 1-methoxy-2-(methoxymethoxy)-4-methylbenzene 15 (4.56
g, 25.0 mmol) in Et
2.25 M in hexane, 16.7 mmol, 1.5 equiv) by dropwise addition over 15 min
so that the internal temperature did not rise above 4 °C. The reaction
mixture was stirred at 0 °C for 1.5 h, then a solution of I (12.7 g, 50.0 mmol,
equiv) in THF (50 mL) was added dropwise over 30 min such that the
2
O (50 mL) at 0 °C was treated with a solution of n-BuLi
(
1443, 1397, 1309, 1264, 1227, 1188, 1154, 1138, 1076, 1045, 983, 920,
1
884, 810, 770, 735, 697; H NMR (400 MHz; CDCl
3
): δ = 7.59-7.57 (m, 2
2
H), 7.48-7.44 (m, 2 H), 7.39-7.35 (m, 1 H), 7.21-7.14 (m, 3 H), 5.12 (s, 2
H), 3.42 (s, 3 H), 2.40 (s, 3 H) ppm; 13C NMR (100 MHz; CDCl
2
3
): δ = 152.0
internal temperature did not rise above 10 °C. The reaction mixture was
allowed to warm to rt, stirred for 16 h, then quenched by the addition of sat.
(C), 138.7 (C), 131.8 (C), 131.6, 131.5, 129.5, 129.0, 127.9, 126.8, 116.0
+
(CH), 95.3 (CH
2
), 56.0 (Me), 20.6 (Me) ppm; EI HRMS: cald for C15H O
16 2
Na
3 × 150 mL), and the combined organics washed with sat. Na
solution (150 mL), dried over MgSO subjected to filtration, and
2 2 3
S O solution (150 mL). The aqueous phase was extracted with EtOAc
228.1146, found 228.1150.
(
2 2 3
S O
4
,
5
-Methyl-2,3-dihydroxybiphenyl (5-Me-DHB, 6): Method A: To a solution
concentrated in vacuo. The crude product was purified by flash column
chromatography on silica gel (0:1 to 1:19 EtOAc/hexanes) to give the title
compound 21 (6.60 g, 86%) as a pale yellow oil. IR (ATR): ṽ = 2938, 2831,
of 2-(methoxymethoxy)-5-methyl-1,1'-biphenyl 19 (4.20 g, 18.4 mmol) in
Et O (100 mL) was added TMEDA (3.31 mL, 22.1 mmol, 1.2 equiv). The
2
solution was stirred for 5 min then cooled to –78 °C before the dropwise
addition of n-BuLi (1.53 M solution in cyclohexane, 30.9 mL, 47.3 mmol,
1589, 1477, 1436, 1381, 1287, 1252, 1211, 1156, 1080, 1021, 967, 903,
1
796; H NMR (400 MHz; CDCl
3
): δ = 6.97 (d, J = 8.3 Hz, 1 H), 6.80 (d, J =
1
.2 equiv). The reaction mixture was stirred for 2.5 h at –30 °C, then
quenched by the rapid addition of freshly distilled B(OMe) (17.6 mL, 158
mmol, 4.0 equiv). The reaction mixture was allowed to warm to rt and
stirred for 3.5 h. H O (50 mL) was added rapidly, followed by 30% H
16.2 mL) and stirring was continued for a further 2 h. The layers were
separated, and the aqueous layer was extracted with Et O (50 mL). The
combined organic layers were washed with sat. brine solution (50 mL),
dried over MgSO , subjected to filtration, and concentrated in vacuo. The
8.3 Hz, 1 H), 5.16 (s, 2 H), 3.82 (s, 3 H), 3.69 (s, 3 H), 2.41 (s, 3 H) ppm;
13C NMR (100 MHz; CDCl
): δ = 149.8 (C), 145.7 (C), 134.7 (C), 124.8
(CH), 112.4 (CH), 99.9 (C), 98.7 (CH ), 58.5 (Me), 56.2 (Me). 28.0 (Me)
ppm; EI HRMS: cald for C10 307.9909, found 307.9905.
3
3
2
2
O
2 2
H
13IO +
3
(
2
3-Methoxy-2-(methoxymethoxy)-6-methyl-1,1'-biphenyl
(22):
A
mixture of 2-iodo-4-methoxy-3-(methoxymethoxy)-1-methylbenzene 21
4
(
(
0.200 g, 0.649 mmol), PhB(OH)
0.075 g, 0.065 mmol, 10 mol%) and Na
O (3.25 mL) was heated at 90 °C for 12 h.
(0.138 g, 1.13 mmol, 1.75 equiv) was added and heating
continued at 110 °C for 8 h. After cooling to rt, H O (5 mL) was added and
the aqueous phase extracted with EtOAc (3 × 20 mL). The combined
organics were dried over MgSO , subjected to filtration, and concentrated
2
(0.159 g, 1.30 mmol, 2 equiv), Pd(PPh
3 4
)
crude product was dissolved in MeOH (20 mL) and 3 N HCl (10 mL) was
added. The reaction mixture was stirred at rt for 16 h, then diluted with
CH
extracted with CH
sat. brine solution (100 mL), dried over MgSO
concentrated in vacuo. The crude product was purified by flash column
chromatography on silica gel (1:3 EtOAc/hexanes) followed by
recrystallization (EtOAc/hexane) to give the title compound 5-Me-DHB 6
2
CO (0.138 g, 1.30 mmol, 2 equiv)
3
in toluene (3.25 mL) and H
Further PhB(OH)
2
2
Cl
2
(100 mL) and the layers were separated. The aqueous phase was
Cl (100 mL) and the combined organics washed with
, subjected to filtration, and
2
2
2
2
4
4
in vacuo. The crude product was purified by flash column chromatography
on silica gel (1:19 EtOAc/hexanes) to give compound 22 (0.141 g, 84%)
as a colourless oil. IR (ATR): ṽ = 2935, 2835, 1598, 1573, 1481, 1439,
(2.54 g, 69%) as pale purple crystals.
1
396, 1296, 1252, 1209, 1154, 1124, 1079, 1032, 1017, 976, 921, 845,
1
Method B: To a solution of 2-(methoxymethoxy)-5-methyl-1,1'-biphenyl 19
2.30 g, 10.1 mmol) in Et O (50 mL) at –7 °C was added TMEDA (1.81 mL,
2.1 mmol, 1.2 equiv) followed by n-BuLi (1.94 M solution in cyclohexane,
.25 mL, 12.1 mmol, 1.2 equiv) by dropwise addition so that the internal
801, 761, 724; H NMR (400 MHz; CDCl
3
): δ = 7.42-7.39 (m, 2 H), 7.33-
(
1
6
2
7.26 (m, 3 H), 7.00 (d, J = 8.4 Hz, 1 H), 6.85 (d, J = 8.4 Hz, 1 H), 4.80 (s,
2 H), 3.86 (s, 3 H), 2.89 (s, 3 H), 2.05 (s, 3 H) ppm; 13C NMR (100 MHz;
3
CDCl ): δ = 150.6 (C), 143.3 (C), 137.7 (C), 137.0 (C), 130.3 (CH), 129.2
temperature did not rise above –2 °C. The reaction mixture was stirred for
h at –5 °C, then quenched by the rapid addition of freshly distilled
B(OMe) (4.50 mL, 40.4 mmol, 4.0 equiv). The reaction mixture was
allowed to warm to rt and stirred for 1 h. H O (17 mL) was added rapidly,
followed by 30% H (10 mL) and stirring was continued for a further 2 h.
The layers were separated, and the aqueous layer was extracted with Et
(C), 127.9 (CH), 126.7 (CH), 125.4 (CH), 111.3 (CH), 98.4 (CH
2
), 56.6 (Me),
+
2
55.9 (Me), 20.0 (Me) ppm; EI HRMS: cald for C16
H O
18 3
258.1256, found
3
258.1262.
2
2 2
O
6-Methyl-2,3-dihydroxybiphenyl (6-Me-DHB, 7): Method A: A solution of
2
O
3-methoxy-2-(methoxymethoxy)-6-methyl-1,1'-biphenyl 22 (0.120 g, 0.466
(
(
50 mL). The combined organic layers were washed with sat. brine solution
50 mL), dried over MgSO , subjected to filtration, and concentrated in
mmol) in a mixture of MeOH (2 mL) and 2 N HCl (0.62 mL, 1.85 mmol, 4.0
equiv) was stirred at rt for 2 h. The solvent was removed in vacuo, and the
4
vacuo. The crude product 20 was dissolved in MeOH (10 mL) and 3 N HCl
5 mL) was added. The mixture was stirred at rt for 16 h, then diluted with
CH Cl (50 mL) and the layers separated. The aqueous phase was
extracted with CH Cl (50 mL) and the combined organics washed with sat.
brine solution (50 mL), dried over MgSO , subjected to filtration, and
residue partitioned between H
combined organics were dried over MgSO
concentrated in vacuo to give the crude phenol which was used in the next
step without further purification.
2
O (10 mL) and EtOAc (3 × 15 mL). The
(
4
, subjected to filtration, and
2
2
2
2
4
concentrated in vacuo. The crude product was purified by flash column
chromatography on silica gel (1:3 EtOAc/hexanes) to give 5-Me-DHB 6
A solution of the phenol in CH
2
Cl
2
(5 mL) and cooled to –78 °C was treated
Cl , 0.7 mL, 0.700 mmol) by dropwise
with a solution of BBr (1 M in CH
3
2
2
(1.23 g, 61%) as a colourless solid. M.p. 117–119 °C; IR (ATR): ṽ = 3486,
addition over 2 min, and stirring was continued at –78 °C for 15 min. The
reaction mixture was allowed to warm to rt, stirred for 1 h, and then
quenched by the addition of 1 N HCl (10 mL). The aqueous phase was
3
1
448, 3319, 3029, 2914, 1595, 1524, 1506, 1488, 1444, 1414, 1383, 1327,
204, 1156, 1103, 1070, 1031, 1003, 977, 914, 867, 835, 787, 756, 738,
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800231
FULL PAPER
extracted with EtOAc (3 × 15 mL), and the combined organics were dried
5-Carboxy-2,3-dihydroxybiphenyl (5-CO
2
H-DHB, 8): To a solution of
over MgSO
4
, subjected to filtration, and concentrated in vacuo. The crude
methyl 6-hydroxy-5-methoxy-[1,1'-biphenyl]-3-carboxylate 28 (1.29 g, 5.00
mmol) in CH Cl (25 mL) at 0 °C was added a solution of BBr (1 M in
CH Cl , 25 mL, 25.0 mmol) dropwise over 20 min. Stirring was continued
product was purified by flash column chromatography on silica gel (1:19
EtOAc/hexanes) to give 6-Me-DHB (7) (0.075 g, 81%) as a colourless solid.
2
2
3
2
2
at 0 °C for 30 min, then at rt for 17 h. The reaction mixture was cooled to
0 °C and quenched by the addition of 2 N HCl (50 mL, containing ice). The
aqueous phase was extracted with EtOAc (4 × 50 mL) and the combined
organics washed with sat. brine solution (50 mL), dried over MgSO ,
4
subjected to filtration, and concentrated in vacuo. The crude product was
purified by flash column chromatography on silica gel (1:48 to 1:19
Method B: A solution of 6-iodo-2,3-dimethoxybiphenyl[8] (0.205 g, 0.600
mmol) in dry Et O (50 mL) at 2 °C was treated dropwise with a solution of
2
t-BuLi (1.7 M in pentane, 0.42 mL, 0.710 mmol, 1.2 equiv). The reaction
mixture was stirred at 2 °C for 15 min, then cooled to –78 °C and quenched
by the rapid addition of iodomethane (0.149 mL, 2.40 mmol, 4.0 equiv).
Stirring was continued at –78 °C for 2 h, then the reaction mixture was
warmed to rt and stirred for a further 30 min, evaporated to dryness in
vacuo, and the resulting solid was dissolved in EtOAc (50 mL). The organic
2 2 2
MeOH/CH Cl ) to give 5-CO H-DHB (8) (0.485 g, 42%) as a colourless
solid. M.p. 216–218 °C; IR (ATR): ṽ = 3515, 3506, 3334, 3056, 2954, 2817,
2
642, 1648, 1610, 1516, 1440, 1418, 1338, 1293, 1263, 1171, 1106, 1032,
1
968, 897, 865, 763, 715, 700, 625; H NMR (400 MHz; DMSO): δ = 12.44
layer was washed with H
2
O (2 × 30 mL) and sat. brine solution (30 mL),
(br s, 1 H), 9.98 (br s, 1 H), 9.20 (br s, 1 H), 7.53-7.51 (m, 2 H), 7.43-7.40
dried over Na SO , subjected to filtration, and the solvents were removed
2
4
(
7
m, 1 H), 7.39 (d, J = 2.0 Hz, 1 H), 7.37 (d, J = 2.0 Hz, 1 H), 7.31 (tt, J =
.3 and 1.2 Hz, 1 H) ppm; H NMR (400 MHz; MeOD): δ = 7.57 (d, J = 7.5
in vacuo. The crude product was purified by flash column chromatography
on silica gel (1.5:8.5 EtOAc/n-heptane) to give compound 25 (93 mg, 68%,
1
Hz, 2 H), 7.53 (d, J = 1.9 Hz, 2 H), 7.46 (d, J = 1.9 Hz, 2 H), 7.39 (t, J = 7.5
Hz, 2 H), 7.30 (d, J = 7.5 Hz, 2 H) ppm; 13C NMR (100 MHz; MeOD): δ =
approx. 88% purity) which was used directly in the next step without further
1
purification. H NMR (300 MHz; CD
2
Cl
2
): δ = 7.33-7.45 (m, 3H), 7.19-7.23
170.2 (C), 148.8 (C), 146.3 (C), 139.3 (C), 130.3 (CH), 129.5 (C), 129.1
(m, 2 H), 6.97 (d, J = 8.5 Hz, 1 H), 6.85 (d, J = 8.5 Hz, 1 H), 3.86 (s, 3H),
(
CH), 128.0 (CH), 125.0 (CH), 122.6 (C), 115.9 (CH) ppm; EI HRMS: cald
3.53 (s, 3 H), 1.99 (s, 3 H).
+
for C13
H O
10 4
230.0579, found 230.0571.
To a solution of compound 25 (28 mg, 0.120 mmol) in CH
2 2
Cl (30 mL) at
Enzymatic Studies
–
78 °C was added BBr (1 M in CH Cl , 0.54 mL, 0.540 mmol). Stirring
3
2
2
was continued at –78 °C for 2 h, then the reaction mixture was warmed to
rt and stirred for a further 15.5 h. After cooling to 2 °C, the reaction was
Chemicals and reagents. All reagents were of at least analytical grade
unless otherwise noted. Water for buffers was purified using a Barnstead
Nanopure DiamondTM system to a resistance of at least 18 megaohms.
BphC was produced as described elsewhere.[14]
quenched by the dropwise addition of H
warmed to rt and stirring continued for 30 min. The aqueous layer was
separated and extracted with CH Cl (50 mL). The combined organics
were washed with H O (50 mL) and sat. brine solution (30 mL), dried over
Na SO , subjected to filtration, and the solvents were removed in vacuo.
2
O (1.8 mL). The mixture was then
2
2
2
2
4
Steady-state kinetics. Kinetic assays were performed by monitoring the
The crude product was purified by flash column chromatography on silica
gel (1:33 to 1:9 EtOAc/n-heptane) to give 6-Me-DHB (7) as a colourless
solid (9.6 mg, 40%). M.p. 118.5–119.5 °C; IR (ATR): ṽ = 3506, 3310, 3057,
consumption of O
2 2
using a Clark-type polarographic O electrode OXYG1
(Hansatech, Pentney, UK) connected to a circulating water bath. Assays
were performed in 1 mL of air-saturated 40 mM HEPES (I = 0.1 M, pH 7.5)
at 25 °C, and initiated by the addition of BphC. The amount of enzyme
used in the assay was determined from the initial velocity of a standard
reaction using 80 μM of DHB and the reported value [14]. Reaction
velocities were corrected for the background reading prior to enzyme
addition. The electrode was calibrated daily by a two-point calibration using
3
1
8
036, 2953, 2926, 2858, 1728, 1623, 1597, 1575, 1490, 1460, 1434, 1370,
320, 1283, 1235, 1202, 1157, 1143, 1091, 1071, 1021, 953, 916, 880,
-
1 1
3
07, 765, 739, 699, 680 cm ; H NMR (400 MHz; CDCl ): δ = 7.53-7.48
(m, 3 H), 7.43 (tt, J = 7.5 and 1.3 Hz, 1 H), 7.31-7.29 (m, 2 H), 6.85 (d, J =
8
2
1
1
2
.2 Hz, 1 H), 6.76 (d, J = 8.2 Hz, 1 H), 5.23 (br s, 1 H), 4.80 (br s, 1 H),
.02 (s, 3 H) ppm; 13C NMR (100 MHz; CDCl
3
): δ = 141.9 (C), 140.0 (C),
air-saturated water and
2
O -depleted water via addition of sodium
35.3 (C), 130.1 (CH), 129.4 (CH), 128.2 (CH), 128.0 (C), 121.9 (CH),
hydrosulfite, according to the manufacturer’s instructions. Stock solutions
were prepared fresh daily. Enzyme stock solution was prepared
anaerobically, stored in a sealed vial on ice, and aliquoted using a gas-
tight syringe for use. The apparent steady-state kinetic parameters were
+
14.1 (CH), 19.7 (Me) ppm; EI HRMS: cald for C13
H O
12 2
200.0837, found
00.0843.
Methyl 6-hydroxy-5-methoxy-[1,1'-biphenyl]-3-carboxylate (28):
A
determined using substrate range up to approximately 5x estimated K
M
solution of methyl 3-bromo-4-hydroxy-5-methoxybenzoate[13] 27 (6.53 g,
value. Steady-state kinetic parameters were evaluated by fitting the
Michaelis–Menten equation to the data using the least-squares fitting of
LEONORA.
25.0 mmol), PhB(OH)
2
(6.10 g, 50.0 mmol, 2.0 equiv), Pd(PPh
3 4
) (1.44 g,
1.25 mmol, 5 mol%), and K
3
PO (21.2 g, 100 mmol, 4.0 equiv) in degassed
4
DMF (100 mL) was heated at 120 °C for 22 h. After cooling to rt, the
reaction mixture was partitioned between 2 N HCl (100 mL) and EtOAc (3
pK determination for Me-HOPDAs. The Me-HOPDAs were prepared in
potassium phosphate solution (I = 0.1 M), pH 7.5, by treatment of the
corresponding DHB with BphC. Aliquots of enzyme-free filtrate were
titrated with sodium hydroxide or hydrochloric acid, as measured with a pH
electrode, while keeping the concentration of Me-HOPDA constant at ~20
×
4
100 mL EtOAc). The combined organics were dried over MgSO ,
subjected to filtration, and concentrated in vacuo. The crude product was
purified by flash column chromatography on silica gel (1:9 EtOAc/hexanes)
to give the title compound 28 (2.97 g, 46%) as colourless solid. M.p. 93.5–
9
1
1
7
7
4.5 °C; IR (ATR): ṽ = 3512, 3473, 3064, 2998, 2944, 2847, 1708, 1597,
576, 1500, 1488, 1464, 1446, 1419, 1376, 1315, 1284, 1266, 1232, 1205,
μM. Absorbance spectra were recorded using
a Cary 60 UV-vis
spectrophotometer (Agilent). The nonenzymatic transformation of Me-
HOPDA as a function of pH was monitored spectrophotometrically at or
near its respective λmax and fitted using Hill equation in Origin 8.1 software
188, 1112, 1044, 1028, 1002, 985, 901, 868, 846, 806, 774, 759, 732,
1
00, 641; H NMR (400 MHz; CDCl
3
): δ = 7.76 (d, J = 1.9 Hz, 1 H), 7.63-
.71 (m, 2 H), 7.55 (d, J = 1.9 Hz, 1 H), 7.47-7.43 (m, 2 H), 7.36 (tt, J = 7.4
and 1.2 Hz, 1 H), 6.27 (br s, 1 H), 4.00 (s, 3 H), 3.91 (s, 3 H) ppm; 13C
(Northampton, MA).
NMR (100 MHz; CDCl
3
): δ = 166.9 (C), 147.1 (C), 146.5 (C), 136.8 (C),
Mass determination for Me-HOPDAs. The Me-HOPDAs, prepared as
described above, were treated with 1% (v/v) glacial acetic acid and the
heterogeneous solutions were subjected to filtration through 0.22 μm
syringe-driven filter prior to MS analysis. 4-Me-HOPDA was treated with
1
1
2
29.2 (CH), 128.3 (CH), 127.6 (CH), 127.3 (C), 125.3 (CH), 121.8 (C),
10.4 (CH), 56.4 (Me), 52.1 (Me) ppm; EI HRMS: cald for C15
58.0892, found 258.0902.
+
14 4
H O
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800231
FULL PAPER
0
.1 M ammonium carbonate prior to acidification. The samples were
resolved on an Agilent 1100 series HPLC unit equipped with an ACE Excel
C18-PFP (50 x 2.1 mm) column using a gradient of formic acid and
[6]
[7]
[8]
A. C. Ruzzini, S. Ghosh, G. P. Horsman, L. J. Foster, J. T. Bolin, L. D.
Eltis, J. Am. Chem. Soc. 2012, 134, 4615–4624.
2
E. Kuatsjah, A. C. K. Chan, M. J. Kobylarz, M. E. P. Murphy, L. D. Eltis,
J. Biol. Chem. 2017, 292, 18290–18302.
acetonitrile. The mass spectra were measured using an Applied Biosystem
Qstar mass spectrometer.
S. Nerdinger, C. Kendall, X. Cai, R. Marchart, P. Riebel, M. R. Johnson,
C.-F. Yin, N. Hénaff, L. D. Eltis, V. Snieckus, J. Org. Chem. 2007, 72,
5960–5967.
[
9]
a) M. A. J. Miah, M. P. Sibi, S. Chattopadhyay, O. B. Familoni, V.
Snieckus, Eur. J. Org. Chem. 2018, 447–454; b) C. Schneider, E. David,
A. A. Toutov, V. Snieckus, Angew. Chem. Int. Ed. 2012, 51, 2722–2726.
Acknowledgements
This research was supported in part by Natural Sciences and
Engineering Research Council of Canada (NSERC) Discovery
Grants 171359 to LDE and 05698 to VS. LDE is a Tier I Canada
Research Chair (CRC) in Microbial Catabolism and Biocatalysis.
We thank Dr. Adam M. Crowe for his expert assistance with the
mass spectrometry data. SN thanks the Boehringer Ingelheim
Foundation for a postdoctoral fellowship and the Smith Kline
Beecham Foundation for a travel grant.
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This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800231
FULL PAPER
Entry for the Table of Contents
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FULL PAPER
A series of alkylated 2,3-
Dr. Sven Nerdinger, Eugene Kuatsjah,
Dr. Timothy E. Hurst, Inge Schlapp-
Hackl, Prof. Dr. Volker Kahlenberg, Prof.
Dr. Klaus Wurst, Prof. Dr. Lindsay D.
Eltis,* Prof. Dr. Victor Snieckus*
dihydroxybiphenyls (DHBs) have been
prepared on gram scale using an
effective Directed ortho Metalation
(
DoM) – Suzuki-Miyaura coupling
strategy. These compounds have
been used to investigate the substrate
specificity of the meta-cleavage
dioxygenase BphC, a key enzyme in
the microbial catabolism of biphenyl.
Isolation of the meta-cleavage
Page No. – Page No.
Bacterial Catabolism of Biphenyls.
Synthesis and Evaluation of
Analogues
products will allow further study of the
catabolism of lignin-derived biphenyls.
This article is protected by copyright. All rights reserved.