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Angewandte
Communications
catalysts and added hydrogen gas.[12a] Bacterially produced
hydrogen can also directly reduce organic dyes[12b] and
ethylene[12c] on an analytical scale using superstoichiometric
amounts of catalyst. Though these examples provided impor-
tant precedent for the desired chemical reactivity, they did not
imply or demonstrate synthetic utility (e.g. preparative scale,
broad substrate scope). Beyond its potential use for chemical
synthesis, we also envisioned using our hydrogenation to
elucidate factors influencing the success of biocompatible
reactions.
We began by investigating whether hydrogenation could
take place in media complex enough to support the growth of
E. coli, our intended source of hydrogen (Table 1). We
incubated the water-soluble alkene caffeic acid (1a) with
platinum(IV)oxide in two types of growth media under an
atmosphere of hydrogen gas (entries 1 and 2) and found that
a defined minimal medium (M9 glucose) provided higher
conversion than a complex medium (Luria-Bertani (LB) +
0.5% glucose). We then examined the impact of bacteria on
these reactions by performing hydrogenations under an
atmosphere of hydrogen gas in growth media containing E.
coli DD-2 (optical density OD600 = 0.4). This engineered
strain produces hydrogen from glucose using an inducible
pathway consisting of a pyruvate ferredoxin oxidoreductase,
a ferredoxin, and a [Fe-Fe] hydrogenase.[13] We observed little
change in conversion with organisms in the reaction mixture
(entries 3 and 4). Finally, we incubated catalyst and substrate
in the presence of E. coli DD-2 under a nitrogen atmosphere,
thus relying on bacterial production of hydrogen gas
(entries 5 and 6). We observed 15% conversion for the
reaction run in M9 glucose, thus providing support for our
general reaction design.
Our initial optimization efforts focused on varying growth
medium and catalyst (Table 1, and see Tables S3 and S4 in the
Supporting Information). Based on the increased conversions
observed for reactions performed with added hydrogen, we
suspected that hydrogen production by E. coli DD-2 was
limiting reaction efficiency. We tested various media additives
and found that adding either iron or casamino acids to
minimal media improved conversion (Table S3). The combi-
nation of both additives provided a further increase (Table 1,
entry 7). These components may boost hydrogen generation
by increasing the amount of functional [Fe-Fe] hydrogenase
generated in cells.[14]
We screened a variety of heterogeneous hydrogenation
catalysts using our improved reaction media. While most
catalysts examined provided no reactivity (see Table S4), the
Royer palladium catalyst[15] [2.44% palladium on polyethy-
leneimine (PEI)/silica gel] proved uniquely effective.[16] Using
this catalyst, we could double the concentration of substrate
and reduce catalyst loading to 8 mol% without impacting
conversion (Table 1, entry 8). Further optimization experi-
ments were carried out with a more challenging substrate (E)-
3-(3,4,5-trimethoxyphenyl)acrylic acid (1b) (see Figure S2
and Tables S5–S11). Ultimately, we identified reaction con-
ditions that were readily scaled to hydrogenate 9 mmol (1.6 g)
of 1a (Table 1, entry 9). The ease with which we could apply
this transformation to larger scale reactions is notable, and
may indicate that this general approach is suitable for
preparative scale synthesis.
Table 1: Proof-of-concept and reaction optimization experiments.
We used these optimal reaction conditions to evaluate
functional-group compatibility, as it was unclear to what
extent the presence of living organisms would impact
substrate scope. Overall, the hydrogenation displayed broad
utility for preparative-scale reactions of water-soluble alkenes
(Scheme 1). An alkyne substrate (1k) also underwent
exhaustive hydrogenation to the corresponding alkane.
Most notably, 2-hexenedioic acid (1x) and Z,Z-muconic
acid (1y) were converted into adipic acid, an important
industrial chemical which is produced on a multimillion ton
scale annually and has been a frequent but challenging target
for metabolic engineering.[17] These results suggest that adipic
acid could be obtained directly from fermentations by
combining a biocompatible hydrogenation catalyst with
organisms that produce hydrogen and an alkene such as 1y,
which has already been generated by engineered microbes.[18]
Finally, we investigated how the biocompatible hydro-
genation takes place and its impact on E. coli. A series of
control experiments delineated the requirements for a suc-
cessful reaction (Figure 2a and Table S12). We also quantified
the hydrogen and formic acid produced in each reaction
mixture, as both metabolites could potentially contribute to
hydrogenation.[19] No reaction was observed in the absence of
catalyst, thus confirming that E. coli cannot reduce 1a. The
presence of E. coli was essential, which indicated that the
organisms contribute a key reaction component. The low
Entry Growth medium
Cells/H2
added[a]
Catalyst
(mol%)
Conv. [%][b]
1
2
3
4
5
6
7
LB + glucose
M9 glucose
LB + glucose
M9 glucose
LB + glucose
M9 glucose
M9CA glucose +
Fe[c]
no/yes
no/yes
yes/yes
yes/yes
yes/no
yes/no
yes/no
PtO2 (40)
PtO2 (40)
PtO2 (40)
PtO2 (40)
PtO2 (40)
PtO2 (40)
PtO2 (20)
15
100
6
91
0
15
56
8
9
M9CA glucose +
Fe
M9CA glucose +
Fe
yes/no
yes/no
Royer (8)[d]
Royer (8)
100
100/87[e]
Reactions were performed at a 5 mm substrate concentration in 5 mL of
growth medium containing ampicillin (50 mgmLÀ1), spectinomycin
(25 mgmLÀ1), chloramphenicol (12.5 mgmLÀ1), and isopropyl ß-D-1-
thiogalactopyranoside (IPTG; 500 mm) under an atmosphere of either
hydrogen or nitrogen in 16 mL Hungate tubes with shaking at 190 rpm.
[a] E. coli strain DD-2 was used, OD600 =0.4. [b] Determined by 1H NMR
spectroscopy. [c] M9CA glucose + Fe medium contains Fe(NH4)2(SO4)2
(50 mm) and casamino acids (5 glÀ1). [d] Royer catalyst is 2.44 wt%
palladium on polyethyleneimine/silica gel. [e] Reaction was performed
on a 9 mmol scale (1.6 g of 1a) with 8 mol% Royer catalyst at a substrate
concentration of 10 mm for 48 h (87% yield upon isolation).
2
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
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