Non-α-hydroxylated aldehydes with evolved transketolase enzymes

Article abstract of DOI:10.1039/b924144b

Transketolase mutants previously identified for use with the non-phosphorylated aldehyde propanal have been explored with a series of linear and cyclic aliphatic aldehydes, and excellent stereoselectivities observed.

Full text of DOI:10.1039/b924144b

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Organic &
Biomolecular
Chemistry
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Volume 8 | Number 6 | 21 March 2010 | Pages 1221–1480
ISSN 1477-0520
PERSPECTIVE
FULL PAPER
Marino Petrini et al.
Helen C. Hailes et al.
Synthesis of 3-substituted indoles
via reactive alkylideneindolenine
intermediates
Non--hydroxylated aldehydes with
evolved transketolase enzymes
1477-0520(2010)8:6;1-E

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Non-a-hydroxylated aldehydes with evolved transketolase enzymes†
Armando Ca´zares,a James L. Galman,a Lydia G. Crago,a,b Mark E. B. Smith,a John Strafford,b
Leonardo R´ıos-Sol´ıs,b Gary J. Lye,b Paul A. Dalbyb and Helen C. Hailes*a
Received 18th November 2009, Accepted 9th January 2010
First published as an Advance Article on the web 5th February 2010
DOI: 10.1039/b924144b
Transketolase mutants previously identified for use with the non-phosphorylated aldehyde propanal
have been explored with a series of linear and cyclic aliphatic aldehydes, and excellent stereoselectivities
observed.
CH2OH), and enhanced specificity to propanal 2a (R = CH2CH3)
Introduction
such as D469T, were identified.11 In addition, when propanal was
used with wild-type (WT) TK, the ee of the product 3a (R =
CH2CH3) was only 58% (Table 1) and therefore chiral assays were
developed toidentify mutants withimproved stereoselectivities.12,13
Notable variants leading to high stereoselectivities were D469E
(90% ee, 3S-isomer) and H26Y (88% ee, 3R-isomer), which
remarkably with a single point active site mutation reversed the
stereoselectivity.12
The use of biocatalysis as a sustainable, atom efficient strategy
in organic synthesis is of increasing importance, and is partic-
ularly attractive due to the high stereoselectivities that can be
achieved.1 Transketolase (TK) (EC 2.2.1.1) is an essential thiamine
diphosphate (ThDP) dependent enzyme, which provides a link
between the glycolytic and pentose phosphate pathways.2 In vivo
it catalyses the reversible transfer of a ketol unit to D-ribose-
5-phosphate or D-erythrose-4-phosphate.2 The TK reaction is
made irreversible using the donor b-hydroxypyruvate (HPA 1),3
which has been used with acceptors 2 such as a-hydroxyaldehydes
where it is stereospecific for the (2R)-hydroxyaldehyde to give (S)-
a,a¢-dihydroxyketones 3 (Scheme 1).4,5 The substrate tolerance
of TK towards a range a-hydroxyaldehydes has led to interest
in industrial applications.6 E. coli TK, which has been over-
expressed,7 shows increased specific activity towards 1 compared
to yeast and spinach TKs.8
The D469E mutant TK has also been reported to reduce
the acceptance of glycolaldehyde and formaldehyde,14 and the
D469 residue has been highlighted as a key residue involved
in enantioselection with a-hydroxylated aldehydes: a yeast TK
structure with the adjunct erythrose-4-phosphate, indicated it
hydrogen bonds to the C-2 hydroxy group of 2-hydroxylated
aldehydes in the active site.15 In view of the interesting substrate
tolerances exhibited by the TK mutants, a more systematic study
was carried out using linear and cyclic aliphatic aldehydes, with
the aim of understanding substrate tolerance and limitations with
selected mutants.
Results and discussion
Scheme 1 Formation of a,a¢-dihydroxy ketones (3S)-3 using TK.
Stereoselectivity of E. coli WT TK
a,a¢-Dihydroxyketone functionalities (3) are present in a range
of natural products and are also important compounds for further
conversion into other synthons, including ketosugars and 2-
amino-1,3-diols.4d,9,10 TK shows high specificity towards the donor
substrates but is more tolerant towards the acceptor aldehyde:
several non-a-hydroxylated aldehydes have been used but lower
relative rates of reaction (5–35% compared to hydroxylated
aldehydes) were noted.5
With a view to enhancing the use of TK in synthetic applications
with a wider range of aldehydes, we used saturation mutagen-
esis that was targeted to the TK active site residues. Mutants
with improved activity towards glycolaldehyde (Scheme 1, R =
Linear aliphatic aldehydes (C4–C8) and cyclopropane-,
cyclopentane- and cyclohexanecarboxaldehyde were selected
for use with WT-TK and TK mutants to determine the influence
of chain length and ring size on reaction selectivities. Initially
racemic a,a¢-dihydroxyketones 3b–3i were prepared for chiral
assay development. The commercially available aldehydes
2b–2i were converted into 3b–3i in yields of 2–35% using N-
methylmorpholine and the previously described TK biomimetic
reaction in water (Scheme 2).16 In general, lower yields were
observed with the more lipophilic aldehydes, which may reflect
poor substrate solubilities in water.
Methods were established for the determination of ees in 3
via monobenzoylation at the primary alcohol of 3g–3i and chiral
HPLC. Compounds 3b–3f required dibenzoylation for satisfactory
peak resolution by chiral HPLC. Then WT-TK and 1 were reacted
with 2b–2f (C4–C8) to determine product stereoselectivities and
yields (Table 1). As well as establishing ees via derivatisation and
chiral HPLC, the selected ketodiols 3b and 3d were coupled to (S)-
MTPACl to give the corresponding Mosher’s esters: application of
aDepartment of Chemistry, University College London, 20 Gordon Street,
London, UK WC1H 0AJ. E-mail: h.c.hailes@ucl.ac.uk; Fax: +44 (0)20
7679 7463; Tel: +44 (0)20 7679 7463
bDepartment of Biochemical Engineering, University College London, Tor-
rington Place, London, UK WC1E 7JE
† Electronic supplementary information (ESI) available: Colorimetric
assay plates for the cyclic aldehydes. See DOI: 10.1039/b924144b
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Table 1 Stereoselectivities and yields for WT-TK and TK mutant reactions using linear aliphatic and cyclic aldehydes
Aldehyde
Product WT-TK ee (yield) D469E ee (yield)
D469T ee (yield)
D469K ee (yield) D469L ee (yield) H26Y ee (yield)
2a
2b
2c
3a12
58% (3S) (36%)12 90% (3S) (70%)12 64% (3S)12 (68%)17
—
—
—
12% (3S) (nd)
88% (3R) (63%)12
92% (3R) (16%)
84% (3R) (7%)
3b
75% (3S) (36%)
84% (3S) (16%)
98% (3S) (44%)
97% (3S) (58%)
—
—
—
—
3c
2d
2e
2f
3d
3e
3f
85% (3S) (25%)
74% (3S) (7%)
66% (3S) (<3%)
97% (3S) (47%)
86% (3S) (14%)
86% (3S) (18%)
—
—
—
—
—
—
—
—
—
84% (3R) (12%)
78% (3R) (4%)
83% (3R) (21%)
2g
2h
2i
3g
3h
3i
72% (1S) (<3%)
0% (<3%)
>99% (1S) (10%) 99% (1S) (10%)
>99% (1S) (40%) 99% (1S) (30%)
99% (1S) (<3%)
25% (1S) (10%)
25% (1S) (<3%)
99% (1S) (<3%)
no reaction
no reaction
30% (1R) (<3%)
no reaction
0% (<3%)
97% (1S) (10%)
99% (1S) (<3%)
no reaction
Scheme 2 Reagents and conditions: (i) N-methylmorpholine, pH 8, H2O; (ii) TK, ThDP, Mg2+, pH 7.
a recently reported NMR method indicated the major enantiomers
formed were (3S)-3b and (3S)-3d.13
As in previous work using propanal, WT-TK leads to the
formation of the (3S)-isomer as the major enantiomer, and the ees
are given in Table 1: by analogy, the major isomers of 3c, 3e and 3f
were assigned as (3S). Interestingly, the degree of stereoselectivity
of the transformation varied with the length of the aliphatic chain
of the aldehyde, reaching a maximum of 84% and 85% ee for
pentanal and hexanal (Fig. 1), which are similar in size to the in
vivo substrates of TK. In addition, increasing lipophilicity of the
aldehyde acceptor resulted in lower conversion yields, probably
due to decreasing aqueous solubilities.
WT-TK was then used with the cyclic aldehydes 2g–2i. The
conversion yields were low (<3%) and for the cyclopropane
analogue 3g the ee was 72%. The major isomer formed was
determined using the modified Mosher’s ester method as again
the (1S)-isomer.13 However, for the cyclopentane and cyclohexane
analogues 3h and 3i, racemic products were formed. The substrate
Fig. 1 Variation in stereoselectivities for linear aliphatic aldehydes using
WT-TK, D469E-TK and H26Y-TK.
cyclopropane carboxaldehyde may be able to adopt a similar
conformation to butanal in the active site leading to a similar
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level of stereoselectivity in the product; however, the larger-ringed
cyclic analogues cannot.
found to dock in two different conformations, with the first similar
to that for C3, and the second similar to the C5–C8 aldehydes. This
transition of docking conformations at C4 in the series from C3 to
C8 mirrors the observed increase in ee from 58% for C3, through
75% for C4 to 84% and 85% for C5 and C6 respectively. However, the
increased ee resulting from the alternative conformations adopted
by the C4–C8 aldehydes is not easily rationalised. One possibility
is that as the aldo O-atom moves closer to H26 and H261 during
the reaction, and the Van der Waals contacts between the aliphatic
chains and the lipophilic region of the active site are weakened,
allowing the chain to move further into the active site and more
freedom to rotate the carbonyl with the Re-face of the carbonyl
exposed to the ThDP-enamine Ca atom. The decrease in ee
observed for C7 and C8 may be due to an increase in the Van
der Waals interactions with the active-site wall and therefore, less
flexibility in the re-orientation of the substrate.
Finally docking of the cyclic aldehydes 2g–2i was carried out;
however, they docked preferentially in a lip at the entrance to the
active site in non-productive conformations, which could explain
the low yielding reactions observed. Only the cyclopropyl analogue
2g docked in a similar conformation to propanal 2a, which gave
3g in 72% ee. Modelling with the mutant TKs was not performed
as we do not have the crystal structures of the mutants at present,
and modelling both the protein mutation and the substrate binding
simultaneously would be less informative.
Molecular modelling: WT TK
With a view to rationalising the stereoselectivities with WT-
TK and the linear aliphatic aldehydes, which demonstrated a
clear experimental relationship between chain length and ee in
the product, molecular docking experiments were carried out
using the X-ray crystal structure of WT-TK (PDB ID: 1QGD).18
Initially, in order to validate the method, direct docking of D-
erythrose-4-phosphate, a natural substrate of TK, was carried
out using AutoDock.19 The predominant conformations obtained
were compared with an existing X-ray crystal structure of a
noncovalent complex of E. coli TK with the aldose (PDB ID:
1NGS) and co-factor.20 Most docked conformations coincided
with the conformation of the ligand in the crystal structure in
terms of hydrogen bonding and consequently orientation. Despite
the dynamic nature of proteins, the conformation generated in
silico reproduced the experimental findings suggesting a molecular
docking method would be a useful model to adopt.
Direct docking of the linear aliphatic aldehydes (C3–C8) was
performed into the TK active site containing a modelled ThDP-
enamine intermediate and the results compared, firstly in terms
of cluster populations, and then the hydrogen bonding and
orientation. The conformations obtained populated one cluster
preferentially in all cases, except for butanal, which occupied two
energetically similar conformations. For all the C3–C8 aldehydes,
docks were obtained in which the aldo oxygen atom is bound to
two histidine residues (His26 and His261), bringing the substrates
into close proximity with the coenzyme for nucleophilic attack.
The longer C5–C8 aldehydes docked with the aliphatic chain
bound to a hydrophobic region around the lip of the entrance
to the ThDP-containing cavity (Fig. 2, magenta model). This
presented neither face of the aldehyde preferentially towards the
ThDP enamine intermediate. Interestingly, the smallest aliphatic
chain (C3) of 2a docked in an alternative orientation with the
aliphatic chain occupying the narrow entrance to the cofactor
containing cavity (Fig. 2, white model), marginally rendering the
Re-face of the carbonyl exposed to the ThDP-enamine Ca atom.
Stereoselectivity of selected single-point TK mutants
In previous mutant screening studies using propanal as the ac-
ceptor, two active-site single-point mutant TKs exhibited marked
improved and reversed stereoselectivity, Asp469Glu (D469E) and
His26Tyr(H26Y). VariantD469E hadledtotheformationof(3S)-
3a in 90% ee, while H26Y gave (3R)- 3a in 88% ee (Table 1).12 With
the aim of understanding substrate tolerances, and enhancing
and reversing stereoselectivities compared to WT-TK with the
C4–C8 linear aldehydes, reactions were carried out using these
high performing mutants: others screened with propanal were not
explored due to the lower stereoselectivities achieved. D469E gave
products in 14–58% isolated yield, with decreasing yield upon
increasing chain length, but enhanced yields compared to WT-
TK. Notably, high stereoselectivities (97–98% ee) were observed
when using butanal, pentanal and hexanal, and the highest ees
were when using aldehydes of a similar size to in vivo substrates for
TK. As shown in Fig. 1, the greatest enhancement in ee between
WT and D469E was for the C3 and C4 aldehydes. The use of
H26Y gave products in generally lower yields (4–21%), with the
highest (3R)-stereoselectivities observed in 3b using butanal (92%
ee), decreasing to 78% ee for 3e. The reversal in ee was observed
across the whole series of linear aldehydes used, although a smaller
change in ee was observed in going from C3 to C8 (Fig. 1). The
lower yields with some longer chain length analogues reflect a
lower turnover of substrate and may be due to steric interactions
in the active site.
Fig. 2 Aldehydes 2a (grey) and 2d (magenta) docked into the TK
active site containing the modelled ThDP-enamine intermediate (green).
A surface plot is shown with D469, H26 and H261 highlighted as sticks
beneath the surface.
The selection of TK-mutants for use with cyclic aldehydes
was less clear, and very low yields were observed using WT-TK.
A recently developed tetrazolium red-based colorimetric assay
was therefore used with the three substrates 2g–2i against the
D469X library, which had led to the identification of several
improved mutants when using propanal.12,21 From this, three
This is reflected in the low ee (58%) in favour of the (3S)-isomer
product for reaction with 2a. The slightly larger C4 aldehyde was
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Table 2 Initial relative rates for selected reactions with the aldehyde
mutants were selected, which were able to accept one or more
of the cyclic aldehydes, D469T, D469K and D469L. These were
used together with mutants D469E and H29Y, which gave high
selectivities with the linear series. The results indicated that high
stereoselectivities could be achieved with D469E: the cyclopropane
and cyclopentane adducts, 3g and 3h, were formed in >99% ee, and
cyclohexane analogue 3i formed in 97% ee (by HPLC) (Table 1).
In all cases, the major isomer was the (1S)-product. When using
D469T similar results were observed, with all products 3g-3i
formed in 99% ee (the (1S)-isomer). Similar yields were observed
with both mutants, with 3g and 3i formed in 3–10% yields, while
the cyclopentane product was formed in a higher 30–40% yield.
This possibly reflects the comparable ring size of 2h and the
natural substrate, ribose-5-phosphate (furanose form). Mutants
D469K and D469L gave the cyclopropane product in 99% ee in
low yields, but when using 2h and 2i either low ees (D469K) or
no reaction (D469L) were observed. The use of H26Y-TK gave
no product with the cyclopropane and cyclohexane aldehydes,
but when using cyclopentanecarboxaldehyde, 3h was formed in
30% ee, and the major isomer was the (1R)-product. Overall,
these results suggest that when using cyclic substrates D469E and
D469T may be suitable mutants to achieve bioconversions. The
smallest cyclic substrate 2g appeared to have greater activity with
a larger range of mutants (leading to high ees), presumably because
it causes less steric problems. Most mutants gave (1S)-products,
other than the low ee for the (1R)-isomer observed with H26Y-
TK. The generally lower yields for the cyclic series may reflect
poor substrate solubilities and/or product inhibition.
indicated
Initial rate/ TK/
Spec. activity/ Initial rel.
Aldehyde Mutant mM h-1
mg mL-1 mmol mg-1 min-1 ratea
D469E 8.14
0.45
0.45
0.45
0.30
0.08
0.03
1
D469E 2.14
D469E 0.89
0.3
0.1
H26Y 8.00
H26Y 0.41
D469T 12.5
D469T 7.45
1.01
1.01
0.30
0.30
0.13
0.007
0.69
0.41
1
0.05
1
0.6
D469T 4.47
D469T 3.46
0.30
0.30
0.25
0.19
0.4
0.3
Initial relative rates were also determined for selected reactions
using D469E, H26Y and D469T for the cyclic series (Table 2).
Relative activities are given compared to propanal for each partic-
ular mutant: for comparison the wild-type specific activity towards
glycolaldehyde was previously reported as 0.65 mmol mg-1min-1.11
Previous work highlighted that when using propanal, higher
relative activities for D469E, H26Y and D469T compared to
WT were observed. Here, relative rates decreased with increasing
aldehyde size in all cases, although was most marked for H26Y.
Interestingly, butanal had a higher initial rate with D469E than
pentanal, although the isolated yield for the reaction was higher
for the pentanal-derived product 3c. This may be due to product
inhibition by 3b, limiting the overall reaction. For the cyclic
aldehydes and D469T, increasing ring size also had less of an
effect on initial rates, than increasing the linear chain length with
D469E, although it decreased slightly as the ring size increased.
This may result from lower conformational flexibilities of cyclic
substrates, compared to aliphatic substrates, resulting in a smaller
and more favourable entropy loss on binding. The small increase
in conformational flexibility as the ring size increases may also
lead to a less favourable loss of entropy upon binding.
a Relative rates are given for substrates compared to propanal with the
selected mutant.
under way to investigate strategies to improve yields with lipophilic
aldehyde acceptors.
Experimental
General methods
Unless otherwise noted, solvents and reagents were reagent grade
from commercial suppliers (Sigma-Aldrich) and used without
further purification. Dry CH2Cl2 was obtained using anhydrous
alumina columns.22 All moisture-sensitive reactions were per-
formed under a nitrogen or argon atmosphere using oven-dried
glassware. Reactions were monitored by TLC on Kieselgel 60
F254 plates with detection by UV, potassium permanganate and
phosphomolybdic acid (PMA) [PMA hydrate (12 g) and ethanol
(250 ml)] stains. Flash column chromatography was carried
out using silica gel (particle size 40-63 mm). 1H NMR and
13C NMR spectra were recorded at the field indicated using
Bruker AMX300 MHz, AMX400 Avance-500 MHz and Avance-
600 MHz machines. Coupling constants are measured in Hertz
(Hz) and unless otherwise specified, NMR spectra were recorded
at 298 K. Mass spectra were recorded on a Thermo Finnegan
MAT 900XP and Micro Mass Quattro LC electrospray mass
spectrometers VG ZAB 2SE. Infrared spectra were recorded on
Conclusions
Ideally, for use in synthesis, enzymes should have good substrate
tolerance and demonstrate high stereoselectivities. In this work
transketolase mutants able to accept lipophilic longer chain
and cyclic aldehydes have been identified, which gives signif-
icant potential for further synthetic applications. With several
substrate/mutant combinations very high stereoselectivities were
observed, with moderate to low conversion yields. Future work is
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1
a Shimadzu FTIR-8700 and Perkin Elmer Spectrum 100 FTIR
spectrometer. Optical rotations were recorded on a Perkin Elmer
model 343 polarimeter at 589 nm, quoted in deg cm2 g-1 and conc
(c) in g/100 mL. Chiral HPLC analysis was performed on a Varian
Prostar instrument equipped with a Chiracel AD chiral column
(Daicel; Chiral Technologies Europe, France) 25 cm ¥ 0.46 cm.
Lithium hydroxypyruvate was synthesised as previously
described.2 1,3-Dihydroxypentan-2-one 3a was prepared as pre-
viously described.12
2872, 1720; H NMR (500 MHz; CDCl3) d 4.49 (1H, d, J 19.4,
CHHOH), 4.39 (1H, d, J 19.4, CHHOH), 4.31 (1H, dd, J 7.9
and 3.9, CHOH), 2.89 (br, OH), 1.80 (1H, m, 4-HH), 1.20–1.75
(5H, m, 4-HH, (CH2)2CH3), 0.91 (3H, t, J 6.6, CH3); 13C NMR
(125 MHz; CDCl3) d 211.7 (C-2), 75.0 (C-3), 65.6 (C-1), 34.0 (C-
4), 26.8 (C-5), 22.5 (C-6), 13.9 (C-7); m/z (CI) 147 (MH+, 100%),
129 ([MH - H2O]+, 35), 85 (94); Found (HRCI) MH+ 147.10288.
C7H14O3 requires 147.10212. Racemic 3c was dibenzoylated and
HPLC analysis (97 : 3, 1 mL min-1) gave retention times of 16.6 min
(R-isomer) and 20.0 min (S-isomer).
Synthesis of racemic a,a¢-dihydroxyketones. The correspond-
ing aldehyde (3.00 mmol) was added to a solution of Li-1 (336 mg,
3.00 mmol) and N-methylmorpholine (330 mL, 3.00 mmol) in
water (60 mL) at pH 8 (adjusted with 10% HCl). The reaction
was stirred for 24–48 h at rt and monitored by TLC analysis.
Upon concentration in vacuo, the crude material was dry loaded
and purified using flash silica chromatography.
1,3-Dihydroxyoctan-2-one (3d). The reaction was carried out
for 24 h and the product purified using flash silica chromatography
(EtOAc–hexane, 1 : 1) to give 3d as a white powder (11 mg,
3%). Mp 106–110 ◦C (EtOAc); nmax(neat)/cm-1 3406, 2956, 2925,
1
2858, 1720; H NMR (500 MHz; CDCl3) d 4.49 (1H, d, J 19.4,
CHHOH), 4.38 (1H, d, J 19.4, CHHOH), 4.31 (1H, dd, J 7.9 and
3.9, CHOH), 2.89 (br, OH), 1.77 (1H, m, 4-HH), 1.58 (1H, m,
4-HH) 1.20–1.34 (6H, m, 5-H2, 6-H2, 7-H2), 0.89 (3H, t, J 6.9,
CH3); 13C NMR (125 MHz; CDCl3) d 211.7 (C-2), 75.0 (C-3),
65.6 (C-1), 34.3 (C-4), 31.6 (C-5), 24.4 (C-6), 22.5 (C-7), 14.0 (C-
8); m/z (CI) 161 (MH+, 100%); Found (HRCI) MH+ 161.11823.
C7H14O3 requires 161.11777. Racemic 3d was dibenzoylated and
HPLC analysis (97 : 3, 1 mL min-1) gave retention times of 15.5 min
(R-isomer) and 19.9 min (S-isomer).
Chiral HPLC analysis of 3b–3i to determine ees. Compounds
3b–3f were dibenzoylated (monobenzylated compounds were
not separable by chiral HPL columns used) and the products
analysed by chiral HPLC to determine ees. Ketodiols 3g–3i were
monobenzoylated at the primary alcohol for chiral HPLC analysis.
HPLC analysis was carried out using a Chiralpak AD column and
the hexane–2-propanol solvent system given.
Monobenzoylation procedure. To the ketodiol (30 mmol) in
CH2Cl2 (5 mL) triethylamine (1.5 eq, 45 mmol) and benzoyl
chloride (1.5 eq, 45 mmol) were added. The reaction was stirred for
2 h at rt, quenched by the addition of saturated NaHCO3 solution
(5 mL), and the organic phase was dried (MgSO4). Upon removal
of the solvent in vacuo, the resulting monobenzoylated material
was dissolved in hexane–2-propanol (1 : 1) to a final concentration
of 1 mg mL-1.
1,3-Dihydroxynonan-2-one (3e). The reaction was carried out
for 24 h and the product purified using flash silica chromatography
(EtOAc–hexane, 1 : 1) to give 3e as a white powder (22 mg,
4%). Mp 104–107 ◦C (EtOAc); nmax(neat)/cm-1 3406, 2955, 2925,
1
2857, 1718; H NMR (500 MHz; CDCl3) d 4.49 (1H, d, J 19.4,
CHHOH), 4.38 (1H, d, J 19.4, CHHOH), 4.30 (1H, dd, J 7.8, 3.9,
CHOH), 2.96 (br s, OH), 1.78 (1H, m, 4-HH), 1.57 (1H, m, 4-HH),
1.27–1.50 (8H, m, 5-H2, 6-H2, 7-H2, 8-H2), 0.88 (3H, t, J 6.9, CH3);
13C NMR (125 MHz; CDCl3) d 211.7 (C-2), 75.0 (C-3), 65.6 (C-1),
34.3 (C-4), 31.7 (C-5), 29.0 (C-6), 24.7 (C-7), 22.6 (C-8), 14.1 (C-
9); m/z (CI) 175 (MH+, 100%), 139 (62), 113 (97), 97 (100); Found
(HRCI) MH+ 175.13360. C9H19O3 requires 175.13342. Racemic 3e
was dibenzoylated and HPLC analysis (97 : 3, 1.2 mL min-1) gave
retention times of 11.6 min (R-isomer) and 15.4 min (S-isomer).
Dibenzoylation procedure. To the ketodiol (30 mmol) in CH2Cl2
(5 mL) triethylamine (5 eq, 150 mmol) and benzoyl chloride (5 eq,
150 mmol) were added. The reaction was stirred for 2 h at rt,
quenched by the addition of saturated NaHCO3 solution (5 mL),
and the organic phase was dried (MgSO4). Upon removal of the
solvent in vacuo, the resulting dibenzoylated material was dissolved
in hexane–2-propanol (1 : 1) to a final concentration of 1 mg mL-1.
1,3-Dihydroxydecan-2-one (3f). The reaction was carried out
for 24 h and the product purified using flash silica chromatography
(EtOAc–hexane, 1 : 1) to give 3f as a white powder (2%, 3 mg).
Mp 100–103 ◦C (EtOAc); nmax(neat)/cm-1 3420, 2959, 2928, 2873,
1721; 1H NMR (500 MHz; CDCl3) d 4.49 (1H, d, J 19.4,
CHHOH), 4.38 (1H, d, J 19.4, CHHOH), 4.31 (1H, m, CHOH),
2.93 (br s, OH), 1.77 (1H, m, 4-HH), 1.20–1.61 (11H, m, 4-HH,5-
H2, 6-H2, 7-H2, 8-H2, 9-H2), 0.88 (3H, t, J 7.0, CH3); 13C NMR
(125 MHz; CDCl3) d 211.7 (C-2), 75.0 (C-3), 65.6 (C-1), 34.3
(C-4), 31.8 (C-5), 29.3 (C-6), 29.1 (C-7), 24.7 (C-8), 22.7 (C-9),
14.1 (C-10); m/z (CI) 189 (MH+, 100%), 153 (93), 127 (97); Found
(HRCI) MH+ 189.14971. C10H21O3 requires 189.14907. Racemic 3f
was dibenzoylated and HPLC analysis (97 : 3, 1.2 mL min-1) gave
retention times of 10.5 min (R-isomer) and 13.9 min (S-isomer).
1,3-Dihydroxyhexan-2-one (3b). The reaction was carried out
for 24 h and the product purified using flash silica chromatography
(EtOAc–hexane, 1 : 1) to give 3b as a white powder (47 mg,
12%). Mp 109–112 ◦C (EtOAc); nmax(neat)/cm-1 3413, 2960, 2935,
1
2874, 1719; H NMR (500 MHz; CDCl3) d 4.49 (1H, d, J 20.1,
CHHOH), 4.38 (1H, d, J 20.1, CHHOH), 4.31 (1H, dd, J 8.0
and 3.9, CHOH), 2.70 (br, OH), 1.75 (1H, m, 4-HH), 1.57 (1H,
m, 4-HH), 1.46 (2H, m, CH2CH3), 0.95 (3H, t, J 7.3, CH3); 13C
NMR (125 MHz; CDCl3) d 211.9 (C-2), 74.8 (C-3), 65.6 (C-1),
36.3 (C-4), 18.1 (C-5), 13.8 (C-6); m/z (CI) 133 (MH+, 100%),
115 ([MH - H2O]+, 30), 85 (47); Found (HRCI) MH+ 133.08611.
C6H12O3 requires 133.08647. Racemic 3b was dibenzoylated and
HPLC analysis (97 : 3, 1 mL min-1) gave retention times of 20.4 min
(R-isomer) and 24.5 min (S-isomer).
1-Cyclopropyl-1,3-dihydroxy-2-propanone (3g). The reaction
was carried out for 48 h and the product purified using flash
silica chromatography (EtOAc) to give 3g as an oil (0.013 g,
10%). Rf 0.50 (EtOAc). nmax(KBr)/cm-1 3380, 3007, 2924, 1724; 1H
NMR (300 MHz; CDCl3) d 4.50 (1H, d, J 19.3, CHHOH), 4.37
1,3-Dihydroxyheptan-2-one (3c). The reaction was carried out
for 24 h and the product purified using flash silica chromatography
(EtOAc–hexane, 1 : 1) to give 3c a white powder (152 mg, 35%).
Mp 110–125 ◦C (EtOAc); nmax(neat)/cm-1 3430, 3263, 2956, 2929,
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(1H, d, J 19.3, CHHOH), 4.30 (1H, d, J 4.2, CHOH), 2.19 (1H,
m), 1.30–1.80 (4H, m, 2 ¥ CH2); 13C NMR (75 MHz; CDCl3)
d 211.4 (C-2), 77.0 (CHOH), 65.9 (CH2OH), 43.0, 29.1, 25.8
and 25.6; m/z (CI) 161 (MH+, 100%); m/z (FTMS) found [M
+ NH4]+ 148.0969. C6H14O3N requires 148.0968. Racemic 3g was
monobenzoylated and HPLC analysis (97 : 3, 1 mL min-1) gave
retention times of 31.1 min (S-isomer) and 33.6 min (R-isomer).
was added to the enzyme solution and the mixture stirred at rt for
24 h. During this time, the pH was maintained at 7.0 by addition of
1 M HCl using a pH stat (Stat Titrino, Metrohm) and the reactions
were followed by TLC analysis. Silica was added and the reaction
mixture concentrated to dryness, dry loaded onto a flash silica gel
column, and purified as before.
Determination of initial rates for selected reactions
1-Cyclopentyl-1,3-dihydroxy-2-propanone (3h). The reaction
was carried out for 48 h and the product purified using flash silica
chromatography (EtOAc–hexane, 1 : 1) to give 3h as a white solid
(0.119 g, 25%). Rf 0.21 (EtOAc–hexane, 1 : 1). Mp 110–112 ◦C
(EtOAc–hexane, 1 : 1); nmax(KBr)/cm-1 3411, 2953, 2870, 1718;
1H NMR (300 MHz; CDCl3) d 4.50 (1H, d, J 19.4, CHHOH),
4.36 (1H, d, J 19.4, CHHOH), 4.30 (1H, m, CHOH), 3.04
(2H, s, 2 ¥ OH), 2.18 (1H, m), 1.31–1.75 (8H, m, 4 ¥ CH2);
13C NMR (75 MHz; CDCl3) d 211.5 (C-2), 76.9 (CHOH), 65.8
(CH2OH), 42.9, 29.0, 25.7 and 25.6; m/z (FTMS) found [M +
NH4]+ 176.1281. C8H18O3N requires 176.1281. Racemic 3h was
monobenzoylated and HPLC analysis (97 : 3, 1 mL min-1) gave
retention times of 32.1 min (R-isomer) and 34.8 min (S-isomer).
Linear series. Data for selected compounds was determined
as detailed below. Aliquots of the biotransformation reaction (200
mL) were collected and diluted with 0.1% TFA (200 mL) and stored
at -20 ◦C after various reaction times (0, 0.25, 0.50, 0.75, 1, 1.5, 2,
3, 4, 12, 24 and 48 h). Samples were centrifuged at 13,000 rpm for
5 min and the supernatant transferred to a clean microcentrifuge
tube. The samples were diluted accordingly for the absorbance to
be within the range of the calibration curve and then analysed
by HPLC. Separation of the biotransformation components was
achieved by HPLC (Dionex, UK) using a Bio-Rad Amines HPX-
87H reverse phase column (300 ¥ 7.8 mm) and 0.2% TFA in water
as the mobile phase at 60 ◦C. Detection of the products was carried
out via UV absorption at 210 nm. Retention times for 3a, 3b, and
3c were 15.4, 18.5 and 23.4 min, respectively.
1-Cyclohexyl-1,3-dihydroxy-2-propanone (3i). The reaction
was carried out for 48 h and the product purified using flash
silica chromatography (EtOAc–hexane, 1 : 1) to give 3i as a white
solid (0.088 g, 17%). Rf 0.21 (EtOAc–hexane, 1 : 1). Mp 115–
Cyclic series. Data was determined via application of the
colorimetric assay21 as poor separation was observed using HPLC
methods. D469T TK lysate, 60 mL was incubated with 100 mL
cofactor solution (TPP (2.4 mM), and MgCl2 (9 mM)) for 20 min at
20 ◦C. Li-1 (7.4 mg, 50 mmol) and aldehyde (50 mmol) were added
to the reaction mixture. Aliquots (50 mL) were taken at hourly
intervals and the reaction was quenched with 50 mL methanol.
The aliquots were transferred onto a microwell plate containing
10 mg MP-Carbonate resin (Biotage AB) and incubated at 20 ◦C
for 3 h. 50 mL of the quenched reaction sample was transferred
without resin into a microwell plate. The colour assay was
performed as described previously:21 50 mL of each concentration
was diluted with 100 mL of water. Automated injection of 20 mL
of tetrazolium red solution (0.2% of 2,3,5-tripheyltetrazolium
chloride in methanol) and 10 mL 3 M NaOH (aq) with shaking by
FLUOstar Optima plate reader (BMG Labtechnologies GmbH),
was followed by immediate measurement at OD485nm.
◦
118 C (EtOAc–hexane, 1 : 1); nmax(KBr)/cm-1 3429, 2940, 1712;
1H NMR (300 MHz; CD3OD) d 4.45 (1H, d, J 19.3, CHHOH),
4.34 (1H, d, J 19.3, CHHOH), 3.96 (1H, d, J 4.4, CHOH), 1.45–
1.87 (6H, m), 1.16–1.30 (5H, m); 13C NMR (75 MHz; CD3OD) d
214.3 (C-2), 80.7 (CHOH), 67.4 (CH2OH), 43.0, 30.3, 27.7, 27.3
(signals superimposed); m/z (CI) 173 (MH+, 100%), 155 (45), 137
(86), 95 (100); Found (HRCI) MH+ 173.11736. C9H17O3 requires
173.11722. Racemic 3i was monobenzoylated and HPLC analysis
of the product (97 : 3, 1 mL min-1) gave retention times of 17.0 min
(R-isomer) and 18.5 min (S-isomer).
Biocatalytic synthesis of 3b-3i
TK Cell-free lysate preparation. Cells from a single colony of
Escherichia coli XL10 transformed with the plasmid containing the
appropriate TK mutant gene were inoculated in 10 mL of sterile
Luria-Bertani medium containing ampicillin (150 mg mL-1). The
culture was incubated overnight (14 h) with orbital shaking at 200
rpm and 37 ◦C. The overnight culture was inoculated in 90 mL of
fresh medium and incubated until the absorbance at 600 nm was
greater than 4. The culture was pelleted by centrifugation at 4000
Protein concentrations were determined using determined using
a combined Bradford and SDS-PAGE densitometry method as
previously described.11a
TK formation of 1,3-dihydroxyhexan-2-one (3b). WT-TK gave
3b (47 mg, 36%) in 75% ee (3S-isomer) by HPLC (97 : 3,
1.0 mL min-1). D469E-TK gave 3b (58 mg, 44%) in 98% ee
(3S-isomer) by HPLC (97 : 3, 1.0 mL min-1); [a]D20 -16.3 (c 1.4,
CH3OH). H26Y-TK gave 3b (21 mg, 16%) in 92% ee (3R-isomer)
by HPLC (97 : 3, 1.0 mL min-1); [a]D20 +14.2 (c 1.6, CH3OH). The
absolute stereochemistry of 3b generated using D469E-TK was
determined using the Mosher’s derivatisation method.13
◦
rpm for 20 min at 4 C and then resuspended in cold phosphate
buffer (5 mM, pH 7) to a final suspension of 1 g cell paste/10 mL
◦
buffer. The suspension was sonicated for 3 min in total at 4 C,
and centrifuged at 4500 rpm for 20 min at 4 ◦C. The supernatant
cell-free lysate was aliquoted and stored at -20 ◦C until needed.
TK conversions. ThDP (22 mg, 48 mmol) and MgCl2·6H2O
(39 mg, 180 mmol) were dissolved in H2O (10 mL) and the pH
adjusted to 7 using 0.1 M NaOH. To this stirred solution, at
25 ◦C, was added TK clarified lysate (2 mL) and the mixture
stirred for 20 min. In another flask, Li-1 (110 mg, 1.00 mmol)
and the aldehyde (1.00 mmol) were dissolved in H2O (8 mL)
and the pH adjusted to 7 with 0.1 M NaOH. Following the
20 min enzyme/cofactor pre-incubation, the 1–aldehyde mixture
(2R,3¢S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid 3¢-
hydroxy-2¢-oxo-hexyl ester. The reaction was carried out under
anhydrous conditions. Triethylamine (20 mL, 0.13 mmol) and (S)-
MTPA chloride (5 mL, 0.03 mmol) were added to a stirred solution
of 3b from the D469E-TK reaction (0.018 g, 0.07 mmol) in CH2Cl2
(2 mL) and the reaction was stirred for 2 h at rt. The product was
dry loaded onto silica gel and purified using flash chromatography
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(3S-isomer) by HPLC (97 : 3, 1.2 mL min-1); [a]D20 -13.3 (c 1.1,
CH3OH). H26Y-TK gave 3f (39 mg, 21%) in 83% ee (3R-isomer)
by HPLC (97 : 3, 1.2 mL min-1); [a]D20 +9.1 (c 0.6, CH3OH).
(hexane–EtOAc, 10 : 1) to afford the Mosher’s derivative as a
colourless oil (4.6 mg, 43%). 1H NMR (600 MHz; CDCl3) d 7.64
(2H, m, Ph), 7.45 (3H, m, Ph), 5.21 (d, J 18.0, CHHO (2R,3¢R-
trace)), 5.11 (1H, d, J 18.0, CHHO (2R,3¢S)), 5.07 (1H, d, J 18.0,
CHHO (2R,3¢S)), 4,96 (1H, d, J 18.0, CHHO (2R,3¢R-trace)), 4.33
(1H, m, CHOH), 3.66 (3H, s, OCH3), 2.90 (1H, d, J 5.4, OH),
1.79 (1H, m, CHH), 1.46–1.65 (3H, m, CHH and CH2), 0.97 (3H,
t, J 7.0, CH2CH3); 13C NMR (150 MHz; CDCl3) d 204.3 (C-2¢),
TK formation of 1-cyclopropyl-1,3-dihydroxy-2-propanone (3g).
WT-TK gave 3g (3 mg, 2%) in 72% ee (1S-isomer) by HPLC (97 : 3,
1.0 mL min-1). D469E-TK gave 3g (13 mg, 10%) in >99% ee (1S-
isomer) by HPLC (97 : 3, 1.0 mL min-1); [a]D20 +70.0 (c 0.3, CHCl3).
H26Y-TK gave no reaction. D469T-TK gave 3g (14 mg, 10%) in
99% ee (1S-isomer), D469K-TK gave 3g (3 mg, 2%) in 99% ee (1S-
isomer), and D469L gave 3g (2 mg, 2%) in 99% ee (1S-isomer).
The absolute stereochemistry of 3g generated using D469E-TK
was determined using the Mosher’s derivatisation method.13
=
166.2 (C O ester), 131.7, 129.9, 128.5, 127.5, 123.0 (q, JCF 290,
CF3), 75.1 (CHOH), 67.0 (CH2OH), 55.8 (OCH3), 36.0, 29.7, 13.8
(CH2CH3); 19F NMR (282 MHz; CDCl3) d -72.2; m/z (ES+) 371
(MNa+, 100%); Found (HRES) MNa+ 371.1096. C16H19O5F3Na
requires 371.1106.
(2S,3¢S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid 3¢-
cyclopropyl-3¢-hydroxy-2¢-oxo-propyl ester. The reaction was car-
ried out under anhydrous conditions. To a stirred solution of 3g
from the D469E-TK reaction (0.010 g, 0.08 mmol) in CH2Cl2
(1 mL) was added triethylamine (34 mL, 0.25 mmol) and (R)-
MTPA chloride (10 mL, 0.04 mmol) in CH2Cl2 (2 mL) and the
reaction was stirred for 12 h at rt. The product was dry loaded
onto silica gel and purified using flash chromatography (hexane–
EtOAc, 4 : 1) to afford the Mosher’s derivative as a colourless
oil (0.013 g, 87%). Rf 0.45 (hexane–EtOAc; 1 : 1); [a]2D0 +45.0 (c
TK formation of 1,3-dihydroxyheptan-2-one (3c). WT-TK gave
3c (24 mg, 16%) in 84% ee (3S-isomer) by HPLC (97 : 3,
1.0 mL min-1). D469E-TK gave 3c (84 mg, 58%) in 97% ee
(3S-isomer) by HPLC (97 : 3, 1.0 mL min-1); [a]D20 -38.5 (c 1.4,
CH3OH). H26Y-TK gave 3c (10 mg, 7%) in 84% ee (3R-isomer)
by HPLC (97 : 3, 1.0 mL min-1); [a]D20 +18.6 (c 1.4, CH3OH).
TK formation of 1,3-dihydroxyoctan-2-one (3d). WT-TK gave
3d (40 mg, 25%) in 85% ee (3S-isomer) by HPLC (97 : 3,
1.0 mL min-1). D469E-TK gave 3d (75 mg, 47%) in 97% ee
(3S-isomer) by HPLC (97 : 3, 1.0 mL min-1); [a]D20 -32.5 (c 1.4,
CH3OH). H26Y-TK gave 3d (20 mg, 12%) in 84% ee (3R-isomer)
by HPLC (97 : 3, 1.0 mL min-1); [a]D20 +16.0 (c 1.4, CH3OH). The
absolute stereochemistry of 3d generated using D469E-TK was
determined using the Mosher’s derivatisation method.13
1
0.2, EtOH); nmax(KBr)/cm-1 3420, 2930, 2855, 1734; H NMR
(300 MHz; CDCl3) d 7.63 (2H, m, Ph), 7.43 (3H, m, Ph), 5.37
(1H, d, J 15.0, CHHO (2S,3¢S)), 5.17 (d, J 18.0, CHHO (2S,3¢R-
trace)), 5.05 (d, J 18.0, CHHO (2S,3¢R-trace)), 4.95 (1H, d, J 15.0,
CHHO (2S,3¢S)), 4.33 (1H, m, CHOH), 3.60 (3H, s, OCH3), 3.09
(1H, d, J 4.3 Hz), 1.03 (1H, m), 0.71 (2H, m), 0.55 (2H, m);
13
(2R,3¢S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid 3¢-
hydroxy-2¢-oxo-octyl ester. The reaction was carried out under
anhydrous conditions. To a stirred solution of 3d from the D469E-
TK reaction (0.015 g, 0.06 mmol) in CH2Cl2 (2 mL) was added
triethylamine (20 mL, 0.13 mmol) and (S)-MTPA chloride (5 mL,
0.03 mmol) and the reaction was stirred for 2 h at rt. The
product was dry loaded onto silica gel and purified using flash
chromatography (hexane–EtOAc, 10 : 1) to afford the Mosher’s
derivative as a colourless oil (3.2 mg, 28%). 1H NMR (600 MHz;
CDCl3) d 7.64 (2H, m, Ph), 7.45 (3H, m, Ph), 5.20 (d, J 16.8,
CHHO (2R,3¢R-trace)), 5.10 (1H, d, J 16.8, CHHO (2R,3¢S)),
5.07 (1H, d, J 16.8, CHHO (2R,3¢S)), 4,96 (1H, d, J 16.8, CHHO
(2R,3¢R-trace)), 4.32 (1H, dd, J 8.0 and 3.9, CHOH), 3.66 (3H, s,
OCH3), 2.85 (1H, br s, OH), 1.05–1.80 (8H, m, 4 ¥ CH2), 0.89 (3H,
t, J 7.3, CH2CH3); 13C NMR (150 MHz; CDCl3) d 204.3 (C-2¢),
=
C NMR (125 MHz; CDCl3) d 203.0 (C-2¢), 166.3 (C O ester),
131.8, 129.9, 128.5, 127.6, 78.1 (CHOH), 66.9 (CH2OH), 55.8
(OCH3), 14.7, 2.9 and 2.8; 19F NMR (282 MHz; CDCl3) d -72.3;
m/z (FTMS) found [M + NH4]+ 364.1364. C16H21F3O5N requires
364.1366.
TK formation of 1-cyclopentyl-1,3-dihydroxy-2-propanone (3h).
WT-TK gave 3h (2 mg, 1%) as a racemate. D469E-TK gave
3h (63 mg, 40%) in >99% ee (1S-isomer) by HPLC (97 : 3,
1.0 mL min-1); [a]D20 +33.0 (c 0.5, CHCl3). H26Y-TK gave 3h
(3 mg, 2%) in 30% ee (1R-isomer), D469T-TK gave 3h (47 mg,
30%) in 99% ee (1S-isomer) and D469K-TK gave 3h (16 mg,
10%) in 25% ee (1S-isomer). The absolute stereochemistry of 3h
generated using D469E-TK was determined using the Mosher’s
derivatisation method.13
=
166.2 (C O ester), 131.7, 129.9, 128.5, 127.5, 75.3 (CHOH), 67.0
(2S,3¢S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid 3¢-
cyclopentyl-3¢-hydroxy-2¢-oxo-propyl ester. The reaction was car-
ried out under anhydrous conditions. Triethylamine (34 mL, 0.25
mmol) and (R)-MTPA chloride (10 mL, 0.04 mmol) in CH2Cl2
(2 mL) were added to a stirred solution of 3h from the D469E-
TK reaction (0.010 g, 0.05 mmol) in CH2Cl2 (1 mL) and the
reaction was stirred for 12 h at rt. The product was dry loaded
onto silica gel and purified using flash chromatography (hexane–
EtOAc, 4 : 1) to afford the Mosher’s derivative as a colourless
oil (0.018 g, 78%). Rf 0.50 (hexane–EtOAc; 4 : 1); [a]2D5 -60.0 (c
(CH2OH), 55.8 (OCH3), 33.9, 32.0, 29.5, 22.7, 14.0 (CH2CH3);
19F NMR (282 MHz; CDCl3) d -72.2; m/z (ES+) 399 (MNa+,
100%); Found (HRES) MNa+ 399.1338. C18H23O5F3Na requires
399.1395.
TK formation of 1,3-dihydroxynonan-2-one (3e). WT-TK gave
3e (13 mg, 7%) in 74% ee (3S-isomer) by HPLC (97 : 3,
1.2 mL min-1). D469E-TK gave 3e (25 mg, 14%) in 86% ee
(3S-isomer) by HPLC (97 : 3, 1.2 mL min-1); [a]D20 -22.7 (c 1.1,
CH3OH). H26Y-TK gave 3e (6 mg, 4%) in 78% ee (3R-isomer) by
HPLC (97 : 3, 1.2 mL min-1); [a]D20 +36.7 (c 0.9, CH3OH).
1
0.1, CHCl3); nmax(KBr)/cm-1 3429, 2930, 2855, 1733; H NMR
(600 MHz; CDCl3) d 7.62 (2H, m, Ph), 7.43 (3H, m, Ph), 5.20
(1H, d, J 16.9, CHHO (2S,3¢S)), 4.92 (1H, d, J 16.9, CHHO
(2S,3¢S)), 4.31 (1H, d, J 3.6, CHOH), 3.65 (3H, s, OCH3), 2.91
(1H, m), 2.23 (1H, m), 1.27–1.85 (10H, m, 5 ¥ CH2), no (2S,3¢R)
TK formation of 1,3-dihydroxydecan-2-one (3f). WT-TK gave
3f (3 mg, 2%) in 66% ee (3S-isomer) by HPLC (97 : 3,
1.2 mL min-1). D469E-TK gave 3f (34 mg, 18%) in 86% ee
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detected; 13C NMR (150 MHz; CDCl3) d 204.0 (C-2¢), 166.3 (C O
ester), 131.9, 130.0, 128.6, 127.6, 123.2 (q, JCF 287, CF3), 84.7 (q,
J
˚
=
of sides 80 A. Defaults were used for docking each substrate except
for the following: the maximum number of energy evaluations was
increased to 1 million, the number of genetic algorithm runs was
CF 27, CCF3), 77.2 (CHOH), 67.4 (CH2OH), 55.9 (OCH3), 42.9,
29.1, 25.8 (signals superimposed); 19F NMR (282 MHz; CDCl3)
d -72.2; m/z (FTMS) found [M + NH4]+ 392.1678. C18H25F3O5N
requires 392.1679.
˚
increased from 10 to 200, and the grid spacing used was 0.375 A.
The enamine-ThDP intermediate structure obtained in yeast TK23
was first docked into the E. coli TK, prior to docking of the
aldehyde substrates.
(2R,3¢S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid 3¢-
cyclopentyl-3¢-hydroxy-2¢-oxo-propyl ester. The reaction was car-
ried out under anhydrous conditions. Triethylamine (34 mL, 0.25
mmol) and (S)-MTPA chloride (10 mL, 0.04 mmol) in CH2Cl2
(2 mL) were added to a stirred solution of 3h from the D469E-
TK reaction (0.010 g, 0.05 mmol) in CH2Cl2 (1 mL), and the
reaction was stirred for 12 h at rt. The product was dry loaded
onto silica gel and purified using flash chromatography (hexane–
EtOAc, 4 : 1) to afford the Mosher’s derivative as a colourless oil
(0.020 g, 87%). Rf 0.50 (hexane–EtOAc; 4 : 1); [a]2D5 +30.0 (c 0.1,
Acknowledgements
CONACYT is thanked for a PhD studentship to A.C. and L.R.S.
The UK Engineering and Physical Sciences Research Council (EP-
SRC) are thanked for DTA studentships to J.L.G. and L.G.C, and
for support of the multidisciplinary Bioconversion Integrated with
Chemistry and Engineering (BiCE) programme (GR/S62505/01).
Financial support from the 12 industrial partners supporting
the BiCE programme is also acknowledged. J. S. was supported
on a Biotechnology and Biological Sciences Research Council
(BBSRC) studentship (BBS/S/A/2004/10920). We thank the
EPSRC National Mass Spectrometry Service Centre, Swansea
University, for the provision of some high resolution MS data.
1
CHCl3); nmax(KBr)/cm-1 3430, 2930, 1732; H NMR (600 MHz;
CDCl3) d 7.63 (2H, m, Ph), 7.43 (3H, m, Ph), 5.08 (1H, d, J 16.9,
CHHO (2R,3¢S)), 5.05 (1H, d, J 16.9, CHHO (2R,3¢S)), 4.31 (1H,
t, J 4.2, CHOH), 3.64 (3H, s, OCH3), 2.90 (1H, d, J 4.2, OH),
2.23 (1H, m), 1.34–1.78 (10H, m, 5 ¥ CH2), no (2R,3¢R) detected;
C NMR (150 MHz; CDCl3) d 204.0 (C-2¢), 166.3 (C O ester),
131.9, 130.0, 128.6, 127.6, 123.2 (q, JCF 287, CF3), 84.7 (q, JCF
27, CCF3), 77.0 (CHOH), 67.4 (CH2OH), 55.9 (OCH3), 42.8,
29.1, 25.8 (signals superimposed); 19F NMR (282 MHz; CDCl3) d
-72.2.
13
=
Notes and references
1 A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts and B.
Witholt, Nature, 2001, 409, 258.
2 E. Racker, in The Enzymes, ed. P. D. Boyer, H. Lardy and K. Myrzback,
Academic Press, New York, 1961, Vol. 5 pp. 397.
3 P. Srere, J. R. Cooper, M. Tabachnick and E. Racker, Arch. Biochem.
Biophys., 1958, 74, 295.
TK formation of 1-cyclohexyl-1,3-dihydroxy-2-propanone (3i).
WT-TK gave 3i (1 mg, 1%) as a racemate. D469E-TK gave
3i (17 mg, 10%) in 97% ee (1S-isomer) by HPLC (97 : 3,
1.0 mL min-1); [a]D20 +33.0 (c 0.5, CHCl3). H26Y-TK gave no
reaction. D469T-TK gave 3i (5 mg, 3%) in 99% ee (1S-isomer)
and D469K-TK gave 3i (5 mg, 3%) in 25% ee (1S-isomer). The
absolute stereochemistry with D469E-TK was determined using
the Mosher’s derivatisation method.13
4 (a) J. Bolte, C. Demuynck and H. Samaki, Tetrahedron Lett., 1987, 28,
5525; (b) F. Effenberger, V. Null and T. Ziegler, Tetrahedron Lett., 1992,
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Chem., 1992, 57, 5899; (e) R. K. Mitra, J. M. Woodley and M. D. Lilly,
Enzyme Microb. Technol., 1998, 22, 64.
5 (a) C. Demuynck, J. Bolte, L. Hecquet and V. Dalmas, Tetrahedron
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Process Res. Dev., 2006, 10, 605; (d) J. Shaeri, I. Wright, E. B. Rathbone,
R. Wohlgemuth and J. M. Woodley, Biotechnol. Bioeng., 2008, 101, 761.
7 M. D. Lilly, R. Chauhan, C. French, M. Gyamerah, G. R. Hobbs, A.
Humphrey, M. Isupov, J. A. Littlechild, R. K. Mitra, K. G. Morris, M.
Rupprecht, N. J. Turner, J. M. Ward, A. J. Willetts and J. M. Woodley,
Recombinant Dna Biotechnology Iii: the Integration of Biological and
Engineering Sciences, 1996, 782, 513.
(2S,3¢S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid 3¢-
cyclohexyl-3¢-hydroxy-2¢-oxo-propyl ester. The reaction was car-
ried out under anhydrous conditions. To a stirred solution of 3i
from the D469E-TK reaction (0.010 g, 0.05 mmol) in CH2Cl2 (1
mL) was added triethylamine (34 mL, 0.25 mmol) and (R)-MTPA
chloride (10 mL, 0.04 mmol) in CH2Cl2 (2 mL) and the reaction
was stirred for 12 h at rt. The product was dry loaded onto silica gel
and purified using flash chromatography (hexane–EtOAc, 4 : 1) to
afford the Mosher’s derivative as a colourless oil (0.018 g, 78%). Rf
1
0.45 (hexane–EtOAc; 4 : 1); [a]2D0 -26.0 (c 0.2, CHCl3); H NMR
(300 MHz; CDCl3) d 7.63 (2H, m, Ph), 7.43 (3H, m, Ph), 5.19
(1H, d, J 17.0, CHHO (2S,3¢S)), 5.07 (d, J 17.0, CHHO (2S,3¢R-
trace)), 5.03 (d, J 17.0, CHHO (2S,3¢R-trace)), 4.90 (1H, d, J 17.0,
CHHO (2S,3¢S)), 4.16 (1H, dd, J 5.2, 3.3, CHOH), 3.65 (3H, s,
OCH3), 2.80 (1H, d, J 5.2, OH), 1.11–1.76 (11H, m); 13C NMR
8 G. A. Sprenger and M. Pohl, J. Mol. Catal. B: Enzym., 1999, 6,
145.
9 D. C. Myles, P. J. Andrulis, III and G. M. Whitesides, Tetrahedron Lett.,
1991, 32, 4835.
10 (a) C. U. Ingram, M. Bommer, M. E. B. Smith, P. A. Dalby, J. M.
Ward, H. C. Hailes and G. J. Lye, Biotechnol. Bioeng., 2007, 96, 559;
(b) U. Kaulmann, K. Smithies, M. E. B. Smith, H. C. Hailes and
J. M. Ward, Enzyme Microb. Technol., 2007, 41, 628; (c) K. Smithies,
M. E. B. Smith, U. Kaulmann, J. L. Galman, Ward and H. C. Hailes,
Tetrahedron: Asymmetry, 2009, 20, 570.
11 (a) E. G. Hibbert, T. Senussi, S. J. Costelloe, W. Lei, M. E. B. Smith,
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Ward, H. C. Hailes and P. A. Dalby, J. Biotechnol., 2008, 134, 240.
12 M. E. B. Smith, E. G. Hibbert, A. B. Jones, P. A. Dalby and H. C.
Hailes, Adv. Synth. Catal., 2008, 350, 2631.
=
(125 MHz; CDCl3) d 204.2 (C-2¢), 166.2 (C O ester), 131.9, 129.9,
128.5, 127.5, 79.7 (CHOH), 67.8 (CH2OH), 55.8 (OCH3), 41.9,
29.6, 26.4, 25.9 (signal overlap), 25.5; 19F NMR (282 MHz; CDCl3)
d -72.2; m/z (FTMS) found [M + NH4]+ 406.1831. C19H27F3O5N
requires 406.1836.
Modelling of substrate binding in WT-TK. The open source
Autodock software19 was used for all substrate docking models in
the E. coli TK structure 1qgd.pdb with a cubic grid in the active site
1308 | Org. Biomol. Chem., 2010, 8, 1301–1309
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