Stereoselectivity of an ω-transaminase-mediated amination of 1,3-dihydroxy-1-phenylpropane-2-one

Article abstract of DOI:10.1016/j.tetasy.2009.03.012

The stereoselectivity of a recently isolated ω-transaminase from Chromobacterium violaceum in the amination of 1,3-dihydroxy-1-phenylpropan-2-one has been determined. The enzyme is not enantioselective towards a racemic mixture of 1,3-dihydroxy-1-phenylpr

Full text of DOI:10.1016/j.tetasy.2009.03.012

Tetrahedron: Asymmetry 20 (2009) 570–574
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Stereoselectivity of an
x-transaminase-mediated amination
of 1,3-dihydroxy-1-phenylpropane-2-one
Kirsty Smithies a, Mark E. B. Smith a, Ursula Kaulmann b, James L. Galman a,
John M. Ward b, Helen C. Hailes a,
*
a Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, UK
b Research Department of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The stereoselectivity of a recently isolated
x-transaminase from Chromobacterium violaceum in the ami-
Received 15 December 2008
Accepted 16 March 2009
Available online 22 April 2009
nation of 1,3-dihydroxy-1-phenylpropan-2-one has been determined. The enzyme is not enantioselective
towards a racemic mixture of 1,3-dihydroxy-1-phenylpropan-2-one but is highly stereoselective forming
(2S)-2-amino-1-phenyl-1,3-propanediols in >99% ee.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
version is 50%. Biocatalytic asymmetric syntheses of amines are
therefore highly desirable and a deracemization approach using
The chiral aminodiol motif is present in many pharmacologi-
cally important compounds such as antiviral glycosidase inhibi-
tors1,2 and sphingolipids.3,4 Specifically, the broad spectrum
antibiotics chloramphenicol 1 and thiamphenicol 2 contain the
2-amino-1-phenyl-1,3-propanediol functionality.5,6
racemic amines has been reported.9 Recently, the use of
x-trans-
aminases (TAm) to generate chiral amines has received increasing
attention.10–12 Practical difficulties relating to unfavourable ther-
modynamic parameters have been addressed and a coupled en-
zyme system incorporating
x-transaminase, a-amino acid
dehydrogenase and formate dehydrogenase has been reported.13,14
As part of a multidisciplinary project aiming to develop biocata-
lytic routes to pharmacologically interesting targets, we are cur-
rently investigating the biocatalytic and chemoenzymatic
synthesis of aminodiols. We have recently reported the one-pot
synthesis of aminoalcohols using an engineered de novo transke-
tolase (TK) and b-alanine TAm pathway in Escherichia coli.15 We
have also reported Chromobacterium violaceum DSM30191
OH
OH
OH
O
OH
O
HN
HN
MeO2S
O2N
CHCl2
CHCl2
x
-transaminase (C. violaceum TAm) that accepts a range of ketones
chloramphenicol 1
thiamphenicol 2
and aldehydes as well as a ketoalcohol as substrates.16 Our aim
was now to establish the stereoselectivity of C. violaceum TAm
when using an
a,
a0-dihydroxyketone, notably the aromatic
The development of efficient, stereoselective methodology to
chiral aminodiols remains a major challenge. There are a wide
range of synthetic strategies to chiral amines, however it has re-
cently been highlighted that green chemistry approaches to chiral
amines are highly desirable, particularly via ketones or aldehydes.7
Biocatalytic routes to chiral amines predominantly focus on the ki-
netic resolution of racemic mixtures. In particular, N-acyl amides
may be resolved using amidases, lipases or peptidases.8 Although
much of this methodology has been developed for use on an indus-
trial scale, it is limited by the fact that the maximum yield of con-
substrate 1,3-dihydroxy-1-phenylpropan-2-one 3 to give 2-ami-
no-1-phenyl-1,3-propanediol 4 (Scheme 1, four possible diastereo-
isomers shown). Previous work had indicated that when
C. violaceum TAm was used with 3 to generate 4, two products
were formed which when analysed by HPLC and LC/MS were be-
lieved to be the syn- and anti-isomers. Based upon previous work
describing the selectivity of
x-TAms where (S)-a-methylbenzyl-
amine was the amine donor, these were postulated to be the
(1S,2S)- and (1R,2S)-isomers 4a and 4d. However, the 1,3-dihydr-
oxyketone could adopt an alternative orientation in the active site
via hydrogen-bonding interactions, or a non-stereoselective ami-
nation with TAm could give 4b and 4d. These studies set out to
establish the absolute stereochemistries resulting from the biocon-
* Corresponding author. Tel.: +44 (0)20 7679 4654; fax: +44 (0)20 7679 7463.
E-mail address: h.c.hailes@ucl.ac.uk (H.C. Hailes).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.03.012

K. Smithies et al. / Tetrahedron: Asymmetry 20 (2009) 570–574
571
OH
OH
OH
OH
NH2
NH2
OH
C. violaceum TAm
4a
4b
(1R,2R)-isomer
(1S,2S)-isomer
OH
OH
OH
amine donor
O
3
OH
OH
NH2
4c (1S,2R)-isomer
NH2
4d (1R,2S)-isomer
Scheme 1.
x-TAm-catalysed conversion of 3 to diastereoisomers 4a–4d.
version, which is crucial for synthetic applications with this en-
zyme. Notably, it was important to determine whether one of the
enantiomers of 3 is non-stereoselectively transformed to 4, or if
both enantiomers of 3 are stereoselectively converted to 4.
substrate which would generate an isomer of 4, the 1-amino-2,3-
diol.
For synthesis of the triacetylated 2-amino-1,3-diol 5, the reduc-
tive amination of 3 was achieved using sodium cyanoborohydride
to give compound 7 in 63% yield, which was converted to the
hydrochloride salt. Hydrogenation of the hydrochloride salt gave
the desired four diastereoisomers mixture of 4 as a 2:5 mixture
of syn-4a and syn-4b: anti-4c and anti-4d isomers. These were di-
rectly acetylated as before to give the mixture 5a–5d, which was
used for chiral HPLC method development.
To establish the absolute stereochemistry of reaction prod-
ucts, synthesis of one of the anti-isomers was also required. Com-
pound 4d, the (1R,2S)-isomer was synthesised (Scheme 3) via
modification of a procedure described by Nicolaou et al.20 Sharp-
less asymmetric dihydroxylation of ethyl cinnamate yielded the
syn-diol (2S,3R)-ethyl-2,3-dihydroxy-3-phenylpropanoate. This
was selectively converted to the mononosylate (2S,3R)-8 using
stoichiometric quantities of nosyl chloride. Although some
epimerisation and generation of the (2R,3R)-isomer of 8 was ob-
served (70% de by NMR spectroscopy) this was not problematical
as the material was required for analytical purposes. Subsequent
SN2 displacement with NaN3 yielded the azide (in 70% de), then
simultaneous reduction of both azide and ester functionalities
using LiAlH4 gave the desired anti-isomer 4d in 44% yield over
the four steps and in 70% de (1R,2R-diastereoisomer 4b also pres-
ent). A sample of 5d-(1R,2S) was obtained by acetylation in
70% de.17
2. Results and discussion
In order to establish the selectivity of the C. violaceum TAm,
chemically derived analytical standards were required. Both 4a
(the (1S,2S)-isomer) and 4b (the (1R,2R)-isomer) are commercially
available and samples of both were readily triacetylated in quanti-
tative yield to give (1S,2S)-5a and (1R,2R)-5b for chiral HPLC meth-
od development.17
A sample of all four of the possible
diastereoisomers 4a–4d was synthesised which could be conve-
niently prepared from the dihydroxyketone 3 (Scheme 2). Racemic
3 was synthesised via the tertiary amine-catalysed condensation of
benzaldehyde with lithium hydroxypyruvate (LiHPA) which has
been reported recently in our laboratory.18 In addition it was noted
that if extended reaction times were used, or the product 3 was
stored in water for several days, a new product was generated
which was postulated to be 6, formed by the tautomerisation of
3. The rearranged product was confirmed to be 2,3-dihydroxy-1-
phenylpropan-1-one 6, which was readily prepared via a crossed
acyloin condensation between glycolaldehyde and benzaldehyde
with catalytic 3-benzyl-5-(2-hydroxyethyl)-4-methyl thiazolium
chloride.19 It was therefore decided to investigate the regioselec-
tivity of the TAm reaction by establishing whether 6 was also a
OH
OH
O
c
a
OH
OH
Ph
H
NHBn
O
3
7
b
O
d—f
OAc
OAc
OH
OAc
OAc
OH
NHAc
NHAc
6
5a
5b
(1S,2S)-isomer
(1R,2R)-isomer
OAc
OAc
OAc
OAc
NHAc
NHAc
5a
5b
(1S,2R)-isomer
(1R,2S)-isomer
Scheme 2. Synthesis of the four diastereoisomers of 4. Reagents and conditions: (a) LiHPA, 3-(4-morpholino)propanesulfonic acid (MOPS), 25%; (b) extended reaction time
for (a) or slow conversion in water; (c) BnNH2, NaBH3CN, AcOH, MeOH, 17 h, 63%; (d) 1 M HCl, MeOH, 100%: (e) H2, Pd/C, MeOH, 48 h, 71%; (f) Ac2O, pyridine, 100%.

572
K. Smithies et al. / Tetrahedron: Asymmetry 20 (2009) 570–574
OH
OH
CO2Et
CO2Et
a,b
OH
c,d
ONos
NH2
8
4d
Scheme 3. Synthesis of 4d. Reagents and conditions: (a) AD-b, MeSO2NH2, tBuOH/H2O (1:1), rt, 18 h, 72%; (b) NosCl, Et3N, CH2Cl2, 0 °C, 5 h, 82%; (c) NaN3, DMF, 55 °C, 17 h,
75%; (d) LiAlH4, THF, reflux, 5 h, 100%.
Using the standards synthesised, both standard reverse-phase
(RP)-HPLC and chiral HPLC assays were established using 4 (syn-
and anti-isomers) and 5a–5d. Transamination reactions were
performed, using C. violaceum TAm and cofactor pyridoxal 5’-
with enantiomeric precursors such as for two-enzyme step TK-
TAm applications.15 Indeed we have recently reported TK single-
point active site mutants that have improved substrate specificities
towards propanal, and generate (3R)- and (3S)-1,3-dihydroxypen-
tan-2-one in high ees.22,23 Such substrates could be used with TAm
to give syn- and anti-aminodiols in high optical purities. Further
studies are now underway using TAm with alternative substrates.
phosphate (PLP), with ketodiols 3 and 6, and (S)-(
benzylamine as the amine donor. Interestingly, no conversion of
to 1-amino-2,3-dihydroxy-1-phenylpropane was observed,
a)-methyl-
6
highlighting the regioselectivity of the transaminase for 1,3-dihy-
droxy-1-phenylpropan-2-one 3 rather than for 2,3-dihydroxy-1-
phenylpropan-1-one 6. This was also interesting because
acetophenone is aminated, and suggested that the 2,3-hydroxy
group is unable to fit into one of the binding pockets.
4. Experimental
4.1. General information
Compound 3 was readily biotransformed and the reactions
were monitored by RP-HPLC and LC–MS. After 24 h, the reaction
was complete and two products were evident by RP-HPLC in a
1:1 ratio. A series of RP-HPLC experiments with the synthesised
analytical standard 4d and commercially available 4a and 4b con-
firmed that the two products were syn- and anti-4. The transami-
nase reaction mixture was purified by HPLC to give a sample of
syn- and anti-4. Triacetylation as previously described gave 5,
and analysis by chiral HPLC and comparison to 5a, 5b and 5d
showed the TAm products to be exclusively (1S,2S)-5a and
(1R,2S)-5d, establishing the TAm products as 4a and 4d (Scheme
4). No 5b or 5c was detectable by chiral HPLC. This indicated that
the transaminase could accept either the (R)- or (S)-1,3-dihydroxy
ketone but formed the (2S)-amine (in >99% ee by HPLC) in 4. This is
consistent with previous work describing the stereoselectivity of a
pyruvate TAm, for which a two-binding site model was proposed,
Unless otherwise noted, solvents and reagents were of reagent
grade from commercial suppliers (Sigma–Aldrich) and were used
without further purification. Benzaldehyde was distilled prior to
use. Dry THF was obtained using anhydrous alumina columns.24
All moisture-sensitive reactions were performed 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 stains. Flash
column chromatography was carried out using silica gel (particle
size 40–63 l
m). 1H NMR and 13C NMR spectra were recorded at
the field indicated using a Bruker AMX300 MHz and AMX500 MHz
machine. 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 spectrom-
eter. Infrared spectra were recorded on a Shamadazu FTIR-8700
infrared spectrophotometer. Optical rotations were recorded on an
Optical Activity Limited PolAAR2000 polarimeter at 589 nm, and
were quoted in deg cm2 gꢀ1 and conc (c) was quoted in g/100 mL.
Lithium hydroxypyruvate was synthesised as previously
where (S)-a
-methylbenzylamine was the amine donor.21
3. Conclusion
described.25 The
x-transaminase from C. violaceum was cloned,
Using chemically derived standards we have shown that a
over-expressed in E. coli and obtained as a clarified lysate as reported
elsewhere.16 Compounds 4a and 4b were commercially available.
The acetylation of compound 4 was carried out as previously de-
scribed.17 Compounds 3,18 619 and (2S,3R)-ethyl-2,3-dihydroxy-3-
phenylpropanoate26 were prepared as previously reported.
recently reported
x-transaminase, isolated from C. violaceum,
exhibits very high (>99% ee) (S)-stereoselectivity in transforming 1,
3-dihydroxy-1-phenylpropan-2-one 3 to 2-amino-1-phenyl-1,3-
propanediol 4. The enzyme was not enantioselective for either
(R)- or (S)-3, but both enantiomers were readily bioconverted to
give a mixture of 4a and 4d. The TAm was also regioselective for
3 rather than for 6. These results are important for applications
4.2. (1S,2S)- and (1R,2R)-2-Acetamido-1-phenylpropane-1,3-
diyl diacetates 5a and 5b
of the
x-TAm with other a,
a0-dihydroxyketones.
The versatility of C. violaceum to accept either 1,3-dihydroxyke-
tone enantiomer will also enhance its potential use in applications
Compounds 4a and 4b were acetylated as previously de-
scribed17 to give 5a and 5b in quantitative yield for HPLC analysis.
OH
OH
C. violaceum ω-TAm
OH
3
PLP, HEPES (pH 7.5)
OH
OH
OH
+
O
NH2
4a
NH2
Me
O
1: 1
4d
(1S,2S) :
(1R,2S)
NH2
Scheme 4. Formation of 4a and 4d from 3 using C. violaceum TAm, PLP and (S)-(a)-methylbenzylamine as the amine donor.

K. Smithies et al. / Tetrahedron: Asymmetry 20 (2009) 570–574
573
Compound 5a ½a 2D0
ꢁ
¼ þ47:1 (c 2.0, CHCl3); 5b ½a D20
ꢁ
¼ ꢀ61:1 (c 2.0,
fied using flash silica chromatography (EtOAc/petroleum ether 60-
80, 1:4 to 1:1 gradient) to give the desired compound (6.53 g, 82%).
CHCl3); 1H NMR (300 MHz; CDCl3) d 1.92 (3H, s, NHAc), 2.01 (3H, s,
CH2OAc), 2.06 (3H, s, CHOAc), 3.82 (1H, dd, J 11.5 and 5.0, CHHOAc),
4.03 (1H, dd, J 11.5 and 4.8, CHHOAc), 4.62 (1H, m, CHNHAc), 5.90
(1H, d, J 7.1, CHOAc), 6.03 (1H, d, J 9.2, NH), 7.30 (5H, m, Ph); 13C
NMR (75 MHz; CDCl3) d 20.6, 20.9 and 23.0 (OAc and NHAc), 52.0
(CHNHAc), 63.1 (CH2OAc), 74.1 (CHOAc), 126.7, 128.6, 136.5,
149.4, 169.9 (C@O), 170.2 (C@O), 170.6 (C@O); m/z (+HRFAB)
[MNa] calcd for C15H19NNaO5, 316.11609; found 316.11550.
½
a 2D4
ꢁ
¼ ꢀ42:5 (c 3.9, CHCl3); 1H NMR (300 MHz; CDCl3) d 1.19 (3H,
t, J 7.2, CH3), 2.58 (1H, d, J 5.8, OH), 4.18 (2H, q, J 7.2, CH2), 5.02 (1H,
d, J 3.9, CHONs), 5.22 (1H, dd, J 5.8 and 3.9, CHOH), 7.22 (5H, m,
ArH), 7.81 (2H, d, J 9.0, ArH), 8.20 (2H, d, J 9.0, ArH); 13C NMR
(125 MHz; CDCl3) d 13.9 (CH2CH3), 62.7 (CH2CH3), 73.6 (CHOH),
82.4 (CHONs), 124.2, 126.2, 128.5, 128.7, 129.1, 137.6, 141.5,
150.6, 166.5 (C@O); m/z (+HRCI) [MC2H5] calcd for C19H22NO8S,
424.10661; found 424.10582.
4.2.1. 2-Benzylamino-1-phenyl-1,3-propanediol 7
Compound 318 (85 mg, 0.512 mmol) and benzylamine (112
lL,
4.5. (1R,2S)-2-Amino-1-phenyl-1,3-propanediol (1R,2S)-4d
1.024 mmol) were dissolved in MeOH (5 mL). NaCNBH3 (96 mg,
1.536 mmol) was added, the pH was adjusted to 6 with acetic acid
and the reaction mixture was stirred at rt for 17 h. The reaction
mixture was concentrated to dryness in vacuo and the residue
was partitioned between CH2Cl2 (50 mL) and NaHCO3 (satd)
(50 mL). The product was extracted with further CH2Cl2
(2 ꢂ 50 mL), dried (MgSO4) and concentrated, and was then puri-
fied using flash silica chromatography (EtOAc) to yield 7 (83 mg,
63%) as a colourless oil. The product was obtained as a 2.6:1 mix-
ture of anti:syn27 isomers. 1H NMR (300 MHz; CDCl3) d 2.65 (3H, br
s, NH and OH), 2.80 (0.28H, dt, J 7.1 and 3.7, CHCH2OH), 2.86
(0.72H, m), 3.38 (0.28H, dd, J 11.2 and 3.7, CHHOH), 3.50 (0.72H,
dd, 11.2 and 4.1, CHHOH), 3.60 (0.72H, dd, 11.2 and 5.3, CHHOH),
3.67 (0.28H, m, CHHOH), 3.83 (2H, s, CH2Ph), 4.66 (0.28H, d, J 7.1,
CHOHPh), 4.88 (0.72H, d, J 5.0, CHOHPh), 7.31 (10H, m, 2 ꢂ Ph);
13C NMR (75 MHz; CDCl3) d 51.3 and 51.5 (CH2Ph), 60.0 and 60.3
(CH2OH), 62.7 and 64.0 (CHCH2OH), 73.4 and 73.6 (CHOHPh),
125.9, 126.6, 127.3, 127.7, 127.8, 128.1, 128.5, 128.6, 139.6,
139.7, 141.3, 141.9; m/z (+HRFAB) [MH] calcd for C16H19NO2,
258.149400; found 258.149645.
Nosylate 8 (8.94 g, 22.6 mmol) was dissolved in DMF (90 mL)
and sodium azide was (2.20 g, 33.9 mmol) added. The mixture
was stirred at 55 °C for 17 h and was then cooled, diluted with
water (200 mL) and extracted with EtOAc (3 ꢂ 200 mL). The com-
bined organics were washed with water (200 mL) and then with
saturated NaCl solution (200 mL) and were dried (MgSO4). Con-
centration in vacuo yielded a residue which was purified using
flash silica chromatography (EtOAc/petroleum ether 40–60, 5:95
to 1:4 gradient) to give the azide as a yellow oil (4.01 g, 75%)
which was directly used in the next step. ½a D24
¼ þ9:4 (c 11.1,
ꢁ
CHCl3); 1H NMR (300 MHz; CDCl3) d 1.24 (3H, t, J 7.1, CH3),
3.21 (1H, br s, OH), 4.08 (1H, d, J 6.9, CHCO2Et), 4.22 (2H, q, J
7.1, CH2CH3), 5.00 (1H, dd, J 6.9 and 4.7, CHOH), 7.33 (5H, m,
ArH); 13C NMR (75 MHz; CDCl3) d 14.0, 62.2, 66.8, 74.1, 126.7,
128.6, 128.7, 139.1, 168.9 (C@O). To lithium aluminium hydride
(0.820 g, 21.7 mmol) in THF (100 mL) was added the azide
(0.850 g, 3.61 mmol) in THF (50 mL) cautiously. The reaction mix-
ture was heated under reflux for 5 h before being quenched with
water and filtered. The solvent was removed in vacuo to give 4d
as a yellow oil (0.600 g, 100%). ½a D24
ꢁ
¼ ꢀ14:9 (c 2.0, MeOH); 1H
4.3. Diastereoisomeric mixture of 5a–d
NMR (300 MHz; D2O) d 3.04 (1H, m, CHNH2), 3.51 (1H, dd, J
11.2 and 6.8, CH2OH), 3.70 (1H, dd, J 11.2 and 3.4, CH2OH),
4.46 (1H, d, J 7.8, CHOHPh); 13C NMR (75 MHz; D2O) 56.9
(CHNH2), 63.4 (CH2OH), 75.2 (CHPh), 126.9, 128.7, 129.1, 141.1;
m/z (+HRES) [MNa] calcd for C9H13NO2Na, 190.0844; found
190.0917.
Diol 7 (83 mg, 0.323 mmol) was dissolved in MeOH (5 mL) and
the pH was adjusted to 1 by the addition of 1 M HCl. The mixture
was concentrated to dryness in vacuo to yield a foam which was
redissolved in fresh MeOH (5 mL). Pd/C (50 mg, 10% wt) was added
and the mixture was subjected to hydrogenation for 2 days. The
catalyst was removed by filtration through Celite and the organics
were concentrated to yield crude 2-amino-1-phenyl-1,3-propane-
diol hydrochloride salt which was used without further purifica-
tion. The crude salt was dissolved in pyridine (3 mL) and Ac2O
(1 mL) and then DMAP (cat.) was added. The reaction mixture
was stirred at rt for 17 h, and was then concentrated to dryness
in vacuo. The residue was partitioned between EtOAc (20 mL)
and 0.3 M KHSO4 (20 mL), washed with NaHCO3 (satd; 20 mL)
and dried (MgSO4). Concentration in vacuo gave a residue which
was purified using flash silica chromatography (EtOAc/hexane,
1:1) to yield 5a–d as a colourless oil (67 mg, 71% over 3 steps).
The product was obtained as a 2.6:1 mixture of anti:syn isomers
and the spectral data were in agreement with those reported for
5 above and in the literature.17b
4.6. (1R,2S)-2-Acetamido-1-phenylpropane-1,3-diyl diacetate
5d
Compound 4d was acetylated as described above to give 5d in
quantitative yield.
½
a 2D0
ꢁ
¼ ꢀ19:4 (c 1.0, CHCl3); 1H NMR
(300 MHz; CDCl3) d 1.88 (3H, s, NHAc), 2.01 (3H, s, CH2OAc), 2.10
(3H, s, CHOAc), 3.98 (1H, dd, J 11.6 and 4.1, CH2OAc), 4.32 (1H,
dd, J 11.6 and 6.4, CH2OAc), 4.65 (1H, m, CHNHAc), 5.80 (1H, d, J
9.1, NH), 5.88 (1H, d, J 5.8, CHOAc);17b 13C NMR (75 MHz; CDCl3)
d 20.8, 21.0, 23.2 (OAc and NHAc), 51.6 (CHNHAc), 62.4 (CH2OAc),
74.6 (CHOAc), 126.6, 128.6, 136.5, 141.8, 169.7, 169.8, 171.0
(C@O); Found (+HRFAB) MNa+, 316.11654. C15H19NNaO5 requires
316.11609.
4.7. RP HPLC assay for TAm-mediated synthesis of 4
4.4. (2S,3R) Ethyl 3-hydroxy-2-(4-nitrophenylsulfonyloxy)-3-
phenylpropanoate 8
The transaminase reaction was analysed using an ACE 5 C18 re-
verse phase column (150 mm ꢂ 4.6 mm, 5
lm particle size; Ad-
To a solution of (2S,3R) ethyl-2,3-dihydroxy-3-phenylpropano-
ate (4.23 g, 20.1 mmol) in CH2Cl2 (100 mL) at 0 °C were added tri-
ethylamine (5.60 mL, 40.2 mmol) and then 4-nitrobenzenesulfonyl
chloride (4.45 g, 20.1 mmol). The reaction mixture was stirred for
5 h and was then quenched with the addition of saturated NH4Cl
(18 mL). The organic layer was washed with 1 M HCl (35 mL),
water (35 mL) and saturated NaCl solution (35 mL) and was dried
(MgSO4). Concentration in vacuo yielded a residue which was puri-
vanced Chromatography techniques, Aberdeen). A gradient was
used from 5% CH3CN/95% 0.1% (v/v) TFA in water to 20% CH3CN/
80% 0.1% (v/v) TFA in water, over 8 min followed by a re-equilibra-
tion step for 2 min (oven temperature 30 °C, flow rate 1 mL/min).
UV detection was carried out at 210 and 250 nm. The retention
times were: 4a/4b 5.73 min; 4c/4d 5.25 min. All samples were
quenched with 0.2% TFA and were briefly centrifuged prior to HPLC
analysis.

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2. Liang, P. H.; Cheng, W. C.; Lee, Y. L.; Yu, H. P.; Wu, Y. T.; Lin, Y. L.; Wong, C. H.
4.8. Chiral HPLC assay for 5
ChemBioChem 2006, 7, 165–173.
3. Hannun, Y. A.; Obeid, L. M. J. Biol. Chem. 2002, 277, 25847–25850.
4. Nakamura, T.; Shiozaki, M. Tetrahedron 2001, 57, 9087–9092.
5. Cutler, R. A.; Stenger, R. J.; Suter, C. M. J. Am. Chem. Soc. 1952, 74,
5475–5481.
6. Hajra, S.; Karmakar, A.; Maji, T.; Medda, A. K. Tetrahedron 2006, 62,
8959–8965.
7. Constable, D. J. C.; Dunn, P. J.; Hayler, J. D.; Humphrey, G. R.; Leazer, J. L.;
Linderman, R. J.; Lorenz, K.; Manley, J.; Pearlman, B. A.; Wells, A.; Zaks, A.;
Zhang, T. Y. Green Chem. 2007, 9, 411–420.
HPLC analysis was performed on a Varian Prostar instrument
equipped with
a
Chiracel AD chiral column (Daicel,
L; mo-
25 cm ꢂ 0.46 cm). HPLC conditions: injection volume, 10
l
i
bile phase, PrOH–hexanes, 3:97; flow rate, 0.6 mL/min; detection,
210 nm. Retention times of 5: (1R,2R)-5b, 74.4 min; (1S,2S)-5a,
77.1 min; (1R,2S)-5d, 99.8 min; (1S,2R)-5c, 105.4 min.
8. Bornscheur, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis, 2nd ed.;
Wiley-VCH: Weinheim, 2006.
4.9. Transaminase-mediated synthesis of 4
9. Turner, N. J. Curr. Opin. Biotechnol. 2003, 14, 401–406.
10. Shin, J. S.; Kim, B. G. Biosci. Biotechnol. Biochem. 2001, 65, 1782–1788.
11. Shin, J. S.; Kim, B. G. Biotechnol. Bioeng. 1999, 65, 206–211.
12. Koszelewski, D.; Lavandera, I.; Clay, D.; Rozzell, D.; Kroutil, W. Adv. Synth. Catal.
2008, 350, 2761–2766.
13. Hohne, M.; Kuhl, S.; Robins, K.; Bornscheuer, U. T. ChemBioChem 2008, 9, 363–
365.
14. Koszelewski, D.; Lavandera, I.; Clay, D.; Guebitz, G. M.; Rozzell, D.; Kroutil, W.
Angew. Chem., Int. Ed. 2008, 120, 9477–9480.
1,3-Dihydroxy-1-phenylpropan-2-one18 (33 mg, 0.20 mmol)
and (S)-a-methylbenzylamine (24 mg, 0.20 mM) were dissolved
in HEPES buffer containing pyridoxal-50-phosphate (7.5 mL, HEPES
100 mM, PLP 0.2 mM, pH 7.5). To this was added C. violaceum TAm
clarified lysate (2.5 mL, TAm 8.5 mg/mL, HEPES 50 mM, PLP
0.2 mM, pH 7.5) and the reaction mixture was incubated for 24 h
at 37 °C without agitation. A portion of the product mixture was
purified by C18 RP-HPLC to yield the desired product as a white so-
lid (1 mg) (m/z (+HRCI) [MH] calcd for C9H14NO2, 168.10245; found
15. Ingram, C. U.; Bommer, M.; Smith, M. E. B.; Dalby, P. A.; Ward, J. M.; Hailes, H.
C.; Lye, G. J. Biotechnol. Bioeng. 2007, 96, 559–569.
16. Kaulmann, U.; Smithies, K.; Smith, M. E. B.; Hailes, H. C.; Ward, J. M. Enzyme
Microb. Technol. 2007, 41, 628–637.
17. (a) Boruwa, J.; Borah, J. C.; Gogoi, S.; Barua, N. C. Tetrahedron Lett. 2005, 46,
1743–1746; (b) Effenberger, F.; Gutterer, B.; Syed, J. Tetrahedron: Asymmetry
1995, 6, 2933–2943.
168.10212). The product 4 was redissolved in pyridine (600
lL) and
Ac2O (200 L) and then DMAP (cat.) was added. After 17 h, the sol-
l
18. Smith, M. E. B.; Smithies, K.; Senussi, T.; Dalby, P. A.; Hailes, H. C. Eur. J. Org.
Chem. 2006, 5, 1121–1123.
19. Heck, R.; Henderson, A. P.; Kohler, B.; Retey, J.; Golding, B. T. Eur. J. Org. Chem.
2001, 14, 2623–2627.
vent was removed in vacuo and the sample of 5 was analysed by
chiral HPLC without further purification. The product was shown
to be exclusively a 1:1 mixture of (1S,2S)-5a and (1R,2S)-5d.
20. Nicolaou, K. C.; Boddy, C. N. C.; Li, H.; Koumbis, A. E.; Hughes, R.; Natarajan, S.;
Jain, N. F.; Ramanjulu, J. M.; Brase, S.; Solomon, M. E. Chem. Eur. J. 1999, 5,
2602–2621.
Acknowledgements
21. Shin, J. S.; Kim, B. G. J. Org. Chem. 2002, 67, 2848–2853.
22. Pangborn, A. B.; Giargello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J.
Organometallics 1996, 15, 1518–1520.
23. Hibbert, E. G.; Senussi, T.; Smith, M. E. B.; Costelloe, S. J.; Ward, J. M.; Hailes, H.
C.; Dalby, P. A. J. Biotechnol. 2008, 134, 240–245.
24. Smith, M. E. B.; Hibbert, E. G.; Jones, A. B.; Dalby, P. A.; Hailes, H. C. Adv. Synth.
Catal. 2008, 350, 2631–2638.
25. Dickens, F. Biochem. Prep. 1962, 9, 86–91.
26. (a) Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.; Hartung, J.;
Jeong, K. S.; Kwong, H. L.; Morikawa, K.; Wang, Z. M.; Xu, D.; Zhang, X. L. J. Org.
Chem. 1992, 57, 2768–2771; (b) Kuang, Y. Q.; Zhang, S. H.; Wei, L. L. Synth.
Commun. 2003, 33, 3545–3550.
The authors would like to thank the UK Engineering and Phys-
ical Sciences Research Council (EPSRC) for support (K.S., M.E.B.S.,
U.K.) of the multidisciplinary Bioconversion Integrated with Chem-
istry and Engineering (BiCE) programme. Financial support from
the 13 industrial partners is also acknowledged. The EPSRC is also
thanked for a DTA studentship to J.L.G.
References
27. Rozwadowska, M. D. Tetrahedron 1997, 53, 10615–10622.
1. Hughes, A. B.; Rudge, A. J. Nat. Prod. Rep. 1994, 11, 135–162.