Enantioselective biooxidation of racemic trans-cyclic vicinal diols: One-pot synthesis of both enantiopure (S,S)-cyclic vicinal diols and (R)-α-hydroxy ketones

Article abstract of DOI:10.1002/adsc.201300301

Highly regio- and enantioselective alcohol dehydrogenases BDHA (2,3-butanediol dehydrogenase from Bacillus subtilis BGSC1A1), CDDHPm (cyclic diol dehydrogenase from Pseudomonas medocina TA5), and CDDHRh (cyclic diol dehydrogenase from Rhodococcus sp. Moj-3449) were discovered for the oxidation of racemic trans-cyclic vicinal diols. Recombinant Escherichia coli expressing BDHA was engineered as an efficient whole-cell biocatalyst for the oxidation of (±)-1,2-cyclopentanediol, 1,2-cyclohexanediol, 1,2-cycloheptane-diol, and 1,2-cyclooctanediol, respectively, to give the corresponding (R)-α-hydroxy ketones in >99% ee and (S,S)-cyclic diols in >99% ee at 50% conversion in one pot. Escherichia coli (BDHA-LDH) co-expressing lactate dehydrogenase (LDH) for intracellular regeneration of NAD⁺ catalyzed the regio- and enantioselective oxidation of (±)-1,2-dihydroxy-1,2,3,4- tetrahydronaphthalene to produce the corresponding (R)-α-hydroxy ketone in >99% ee and (S,S)-cyclic diol in 96% ee at 49% conversion. Preparative biotransformations were also demonstrated. Thus, a novel and useful method for the one-pot synthesis of both vicinal diols and α-hydroxy ketones in high ee was developed via high Copyright

Full text of DOI:10.1002/adsc.201300301

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DOI: 10.1002/adsc.201300301
Enantioselective Biooxidation of Racemic trans-Cyclic Vicinal
Diols: One-Pot Synthesis of Both Enantiopure (S,S)-Cyclic
Vicinal Diols and (R)-a-Hydroxy Ketones
Jiandong Zhang,a Tingting Xu,a and Zhi Lia,*
a
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4,
Singapore 117576
Fax : (+65)-6779-1936; e-mail: chelz@nus.edu.sg
Received: April 12, 2013; Revised: September 9, 2013; Published online: November 4, 2013
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201300301.
Abstract: Highly regio- and enantioselective alcohol
dehydrogenases BDHA (2,3-butanediol dehydro-
oxidation of a racemic trans-vicinal diol to simultane-
ously produce both enantiopure vicinal diol and a-hy-
droxy ketone that are highly valuable chiral auxilia-
ries, ligands, or templates in asymmetric reactions as
well as important chiral building blocks for the syn-
thesis of fine chemicals and pharmaceuticals.[1,2] How-
ever, the use of this type of enzymatic reaction for
one-pot preparation of two enantiopure products re-
quires extremely high enantioselectivity. Thus far, the
reported biocatalysts for the oxidations of racemic
trans-vicinal diols showed low enantioselectivity, thus
being unable to produce both a-hydroxy ketone and
diol in high ee at 50% conversion. Glycerol dehydro-
genase catalyzed the oxidation of racemic 1,2-butane-
diol, but with an enantioselectivity factor (E) of only
20.[3] Bacillus stearothermophilus diacetyl reductase
was able to oxidize racemic trans-1,2-cyclopentanediol
and 1,2-cyclohexanediol, respectively, giving the cor-
responding (S)-a-hydroxy ketones in high ee but with
low yield (16–18%).[4] Gluconobacter oxydans (ATCC
621) catalyzed the oxidization of trans-cyclohexane-
diol to afford (R)-a-hydroxycyclohexanone in 96% ee
at pH of 3.5 and (S)-a-hydroxycyclohexanone in 97%
ee at pH of 8.0, respectively, but with unsatisfied
enantioselectivity (E <100) and low yield (5–18%).[5]
Here we report the development of highly enantiose-
lective biocatalysts for the oxidation of racemic trans-
vicinal diols, as the first example, for one-pot synthe-
sis of both vicinal diols and a-hydroxy ketones in high
ee.
genase
from
Bacillus
subtilis
BGSC1A1),
CDDHPm (cyclic diol dehydrogenase from Pseudo-
monas medocina TA5), and CDDHRh (cyclic diol
dehydrogenase from Rhodococcus sp. Moj-3449)
were discovered for the oxidation of racemic trans-
cyclic vicinal diols. Recombinant Escherichia coli
expressing BDHA was engineered as an efficient
whole-cell biocatalyst for the oxidation of (Æ)-1,2-
cyclopentanediol, 1,2-cyclohexanediol, 1,2-cyclohep-
tane-diol, and 1,2-cyclooctanediol, respectively, to
give the corresponding (R)-a-hydroxy ketones in
>99% ee and (S,S)-cyclic diols in >99% ee at 50%
conversion in one pot. Escherichia coli (BDHA-
LDH) co-expressing lactate dehydrogenase (LDH)
for intracellular regeneration of NAD+ catalyzed
the regio- and enantioselective oxidation of (Æ)-1,2-
dihydroxy-1,2,3,4-tetrahydronaphthalene to produce
the corresponding (R)-a-hydroxy ketone in >99%
ee and (S,S)-cyclic diol in 96% ee at 49% conver-
sion. Preparative biotransformations were also dem-
onstrated. Thus, a novel and useful method for the
one-pot synthesis of both vicinal diols and a-hy-
droxy ketones in high ee was developed via highly
regio- and enantioselective oxidations of the race-
mic vicinal diols.
Keywords: alcohol dehydrogenase; enantioselectivi-
ty; enzyme catalysis; a-hydroxy ketones; oxidation;
vicinal diols
AHCTUNGTREG(NNNU S,S)-1,2-Cyclopentanediol 1, (S,S)-1,2-cyclohexane-
diol 2, (S,S)-1,2-cycloheptanediol 3, (S,S)-1,2-cyclooc-
tanediol 4, and (S,S)-1,2-dihydroxy-1,2,3,4-terahydro-
naphthalene 5 were selected as the target vicinal
diols, and (R)-a-hydroxycyclopentanone 6, (R)-a-hy-
Enzyme catalysis has become an important tool for droxycyclohexanone 7, (R)-a-hydroxycycloheptanone
green and sustainable synthesis of chemicals, especial- 8, (R)-a-hydroxycyclooctanone 9, and (R)-a-hydroxy-
ly enantiopure compounds. An interesting and poten- tetralone 10 were chosen as the target a-hydroxy ke-
tially useful enzymatic reaction is the enantioselective tones (Scheme 1). These compounds are very useful
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Jiandong Zhang et al.
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Scheme 1. Enantioselective oxidation of trans-cyclic vicinal diols 1–5 with E. coli (BDHA) to simultaneously produce enan-
tiopure (R)-a-hydroxy ketones 6–10 and (S,S)-cyclic vicinal diols 1–5.
and valuable chiral building blocks and pharmaceuti- lectivity for the oxidation of (Æ)-1–3 with an E of
cal intermediates,[6–12] and the enantioselective oxida- >287. The specific activity was 3.5–8.6 U/g cdw. In
tions of racemic cyclic vicinal diols are also very chal- comparison, E. coli (CDDHPm) gave much higher
lenging.
specific activity, with 17–59 U/g cdw for the oxidation
An initial screening of microorganisms collected in of (Æ)-1–4. The enantioselectivity was also very high
our lab led to the identification of Bacillus subtilis (E of >363) with (Æ)-2–3 as substrates. The best bio-
BGSC1A1 as a highly enantioselective catalyst for the catalyst was E. coli (BDHA) which showed an E of
oxidation of (Æ)-2 to give both (R)-7 and (S,S)-2 in >1000 for the oxidation of (Æ)-1–4 and a high specif-
99% ee at 49.9% conversion (see the Supporting In- ic activity of 36–57 U/g cdw. For the oxidation of (Æ)-
formation). Based on the reported genome se- 5 shown in Table 2, both E. coli (BDHA) and E. coli
quence[13] of this strain, nine predicted alcohol dehy- (CDDHPm) gave 100% regioselectivity and very high
drogenases (ADHs) genes were cloned and expressed enantioselectivity with an E of >824. The specific ac-
in Escherichia coli. After testing, enzyme BDHA tivity was 22–30 U/g cdw.
(2,3-butanediol dehydrogenase) was found to show
Based on the high enantioselectivity and specific
high enantioselectivity for the oxidation of (Æ)-2. activity, E. coli (BDHA) was selected for further de-
BDHA is known in Bacillus subtilis for the produc- velopment. Conditions for cell growth and the expres-
tion of 2,3-butanediol,[14] but no synthetic application sion of BDHA were optimized. As shown in Fig-
of BDHA for the oxidation reaction was reported.
ure 1A, E. coli (BDHA) cells reached a cell density
To discover more ADHs for the enantioselective of 6.5 g cdw/L at 12 h, with an activity of 58 U/g cdw
oxidation of diols, 8 predicted putative ADH genes for the oxidation of (Æ)-2. Figure 1B showed clearly
from Sphingomonas sp. HXN-200,[15] Pseudomonas the high expression level of BDHA in E. coli
medocina TA5,[16] and Rhodococcus sp. Moj-3449[17] (BDHA) after 10 h induction with IPTG. Time cours-
(the genome DNA of these strains were sequenced by es of biooxidations of (Æ)-1–4 with resting cells of E.
our lab) were cloned, expressed, and examined for coli (BDHA) were recorded. As shown in Figure 1C
the oxidations. While Sphingomonas sp. HXN-200 for the biooxidation of (Æ)-3, the reaction was much
could oxidize vicinal 1,2-diols,[15a] no cloned ADHs faster in the first 30 min and become slower after-
from this strain demonstrated activity for such a type wards. This is partially due to the less availability of
of oxidations. On the other hand cyclic diol dehydro- (R,R)-3 in the late stage and the extremely low reac-
genases (CDDHs) from P. medocina TA5 tion rate for the oxidation of (S,S)-3. In practice, this
(CDDHPm) and from Rhodococcus sp. Moj-3449 phenomenon is good for controlling the reaction to
(CDDHRh) were found to be highly enantioselective stop at 50% conversion. During the slow-reaction
for the oxidation of (Æ)-2. Based on multiple align- period, the ee of the unreacted substrate (S,S)-3 in-
ments of the enzymes (Supporting Information, Fig- creased to a final value of >99.0% at 2 h with a con-
ure S2), CDDHPm and CDDHRh showed 42% and version of 49.9%. The ee of the product (R)-8 was
49% amino acid identity with BDHA, respectively.
very high at the beginning and remained at >99.0%
Biooxidations of (Æ)-1–5 were further investigated until the end of the reaction. Time courses of biooxi-
with the resting cells of the recombinant E. coli dation of (Æ)-1, (Æ)-2, and (Æ)-4 (Supporting Infor-
strains expressing the ADH. As shown in Table 1, E. mation) are similar to Figure 1. In all cases, both
coli (CDDHRh) demonstrated very high enantiose-
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Enantioselective Biooxidation of Racemic trans-Cyclic Vicinal Diols
Table 1. Enantioselective oxidations of cyclic diols (Æ)-1-4 with resting cells of E. coli (CDDHRh) (Cat A), E. coli
(CDDHPm) (Cat B), and E. coli (BDHA) (Cat C).[a]
Catalyst Substrate Concentration Time Activity
Conversion Unreacted Substrate Product Product
E[e]
[mM]
[h]
[U/g cdw][b] [%][c]
Substrate
ee [%][d]
ee [%][d]
Cat A
Cat A
Cat A
Cat A
Cat B
Cat B
Cat B
Cat B
Cat C
Cat C
Cat C
Cat C
(Æ)-1
(Æ)-2
(Æ)-3
(Æ)-4
(Æ)-1
(Æ)-2
(Æ)-3
(Æ)-4
(Æ)-1
(Æ)-2
(Æ)-3
(Æ)-4
10
10
10
10
10
10
10
10
20
20
20
20
8
6.5
7.1
8.6
3.5
38
17
55
59
57
43
50
36
27.4
30.6
40.0
7.4
52.3
37.3
50.0
51.1
49.9
49.9
49.9
49.9
N
37.4
43.7
65.1
7.2
85.9
59.0
>99.0
>99.0
>99.0
>99.0
>99.0
>99.0
(R)-6
(R)-7
(R)-8
(R)-9
(R)-6
(R)-7
(R)-8
(R)-9
(R)-6
(R)-7
(R)-8
(R)-9
>99.0
>99.0
>99.0
90.7
>287
>309
>392
22
12
24
4
4
12
3
5
4
8
4
78.1
22
>99.0
>99.0
92.5
>99.0
>99.0
>99.0
>99.0
>363
>1000
>118
>1000
>1000
>1000
>1000
6
[a]
Biotransformation was performed at a cell density of 10 g cdw/L in 10 mL Tris-HCl buffer (pH 8.0, 100 mM) at 308C and
250 rpm.
Specific activity was determined at 10 min with cell density of 1 g cdw/L. For Cat A, specific activity was determined at
30 min with a cell density of 2 g cdw/L.
[b]
[c]
[d]
[e]
Conversion was determined by GC analysis. Error limit: 0.5% of the state values.
ee was determined by chiral GC analysis. Error limit: 0.1% ee.
E=ln
[eePA
ACHTGNURETN(NUNG eeP+eeS)]/lnCAHUTNGTREN[NGU eePACHTNUEGRTGUN(NN 1+eeS)/ACHTNGUTRENUN(NG eeP+eeS)].
Table 2. Regio- and enantioselective oxidation of (Æ)-5 with resting cells of E. coli (CDDHPm) (Cat B), E. coli (BDHA)
(Cat C), and E. coli (BDHA-LDH) (Cat D).[a]
Catalyst (Æ)-5 Concentra- Cell density [g Pyruvate Time Activity [U/g Conversion
(S,S)-5 ee (R)-10 ee E[e]
ACHTUNGTRENNUNG
tion [mM]
cdw/L]
[mM]
[h]
cdw][b]
[%][c]
[%][d]
[%][d]
Cat B
Cat C
Cat D
Cat D
Cat D
Cat D
10
10
10
10
10
20
15
15
10
10
20
20
0
0
0
20
100
100
12
8
5
6
10
24
22
30
33
41
52
57
29.4
33.6
24.5
48.3
49.0
48.3
41.2
50.2
32.5
93.4
96.0
93.4
99.7
99.6
99.6
99.7
99.8
99.9
999
824
686
>1000
>1000
>1000
[a]
Biotransformation was performed in 10 mL Tris-HCl buffer (pH 8.0, 100 mM) at 308C and 250 rpm.
Specific activity was determined at 10 min with cell density of 1 g cdw/L.
Conversion was determined by HPLC analysis. Error limit: 0.5% of the state values.
ee was determined by chiral HPLC analysis. Error limit: 0.1% ee.
[b]
[c]
[d]
[e]
E=ln
G
G
ACHTGNURETN(NUNG eeP+eeS)]/lnCAHUTNGTREN[NGU eePACHTNUEGRTGUN(NN 1+eeS)/ACHTNGUTRENUN(NG eeP+eeS)].
(S,S)-1–4 and (R)-6–9 were obtained in >99.0% ee at of the cofactor NAD+. E. coli resting cells may be
49.9% conversion.
able to regenerate NAD+ via the remaining metabo-
As shown in Table 2, biooxidations of (Æ)-5 with lism and possible oxidation of NADH, however, their
resting cells of E. coli (BDHA) gave only 33.6% con- capacity is very limited. Moreover, based on thermo-
version. The low conversion is possibly mainly due to dynamics, the equilibrium for the dehydrogenase-cat-
the poor availability of the necessary cofactor. Biooxi- alyzed reaction with NAD+ as electron acceptor is ex-
dations of (Æ)-5 (10 mM) with the cell-free extract of pected to lie on the side of the diols. Thus, an efficient
E. coli (BdhA) suggested NAD+ as the cofactor of NAD+ regeneration is necessary to supply sufficient
BdhA and produced (S,S)-5 in 98% ee and (R)-10 in co-factor for the oxidation of diol and drive the reac-
>99% ee with 49% conversion in the presence of ex- tion to the direction of ketone formation. E. coli
cessive NAD+ (Supporting Information). For the bio- (BDHA-LDH) co-expressing BDHA and lactate de-
oxidations using resting cells, NAD+ inside the cells hydrogenase (LDH) from Bacillus subtilis was thus
contributed to the initial reaction. But the reactions engineered as a more efficient and practical catalyst
became very slow after the initial fast reaction period. for the oxidation via intracellular recycling of the co-
This is possibly due to the lack of a sufficient amount factor.[18] Figure 1B demonstrated that both BDHA
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&
^
Figure 1. A: Time course of cell growth and specific activity for biooxidation of (Æ)-2 of E. coli (BDHA), : cell density,
:
specific activity. B: SDS-PAGE analysis of the cell-free extracts of E. coli strains. Lane 1: E. coli (BDHA) with no induction,
lane 2: E. coli (BDHA) after 2 h induction, lane 3: E. coli (BDHA) after 10 h induction, lane M: protein marker (in kDa),
lane 4: E. coli (his-tag LDH), lane 5: E. coli (BDHA); lane 6: E. coli (BDHA-LDH) after 10 h induction. C: Time course of
&
^
enantioselective oxidations of (Æ)-3 (10 mM) with resting cells of E. coli (BDHA) (10 g cdw/L), : conversion of (Æ)-3.
:
ee of (S,S)-3, &: ee of (R)-8. D: Time course of regio- and enantioselective oxidation of (Æ)-5 (10 mM) with resting cells of
&
^
E. coli (BDHA-LDH) (20 g cdw/L) in the presence of sodium pyruvate (100 mM). : conversion, : ee of (S,S)-5, &: ee of
(R)-10.
and LDH were successfully co-expressed. The cell- these compounds in >99% purity and 31.2–40.0%
free extract of the recombinant strain showed 3.9 U/ isolated yield. Similarly, oxidations of (Æ)-5 with E.
mg protein for the regeneration of NAD+. As shown coli (BDHA-LDH) in the presence of sodium pyru-
in Table 2, the use of E. coli (BDHA-LDH) in the vate gave 49.0% conversion and produced pure (R)-
presence of 20–100 mM sodium pyruvate afforded 10 in 99.8% ee and pure (S,S)-5 in 96.0% ee, with an
much higher conversion than that with E. coli isolated yield of 35.5% and 40.0%, respectively.
(BDHA). Finally, 49.0% conversion was achieved to
ACHTUNGTREN(NGNU S,S)-trans-Vicinal diols 1–5 can also be prepared
give (S,S)-5 in 96.0% ee and (R)-10 in 99.8% ee. The by other methods. Enzyme-catalyzed kinetic resolu-
time course of the biotransformation of (Æ)-5 tion via hydrolysis or esterification gave (S,S)-1–3[19]
(10 mM) with resting cells (20 g cdw/L) of E. coli with an E of 3–52. Kinetic resolution via esterification
(BDHA-LDH) was shown in Figure 1D.
using organocatalysts afforded (S,S)-1–5[20] with an E
The synthetic potential was demonstrated in 100- of 8–50. In comparison, our method is much more
mL scale biotransformations. As summarized in enantioselective and thus gives higher yield. Hydroly-
Table 3, oxidations of (Æ)-1–4 with E. coli (BDHA) sis of cyclopentene oxide or cyclohexene oxide with
reached 49.8–50.2% conversion and gave both (S,S)- epoxide hydrolase gave (R,R)-1 in 90% ee and (R,R)-
1–4 and (R)-6–9 in >99.0% ee. Simple work-up and 2 in 87–96% ee.[21] They are the opposite enantiomers
purification by column chromatography afforded of the diols produced by our method.
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Enantioselective Biooxidation of Racemic trans-Cyclic Vicinal Diols
Table 3. Preparation of (R)-6–10 and (S, S)-1–5 by enantioselective oxidation of (Æ)-1–4 with resting cells of E. coli (BDHA) (Cat C)
and regio- and enantioselective oxidation of (Æ)-5 with resting cells of E. coli (BDHA-LDH) (Cat D), respectively.[a]
Substrate Substrate Catalyst Time Conversion Product Product Isolated Product Unreacted Substrate Isolated Substrate
[mg]
[h]
[%][b]
ee
yield
[mg]
Substrate
ee [%][c]
yield
[mg]
[%][c]
[%][d]
[%][d]
(Æ)-1
(Æ)-2
(Æ)-3
(Æ)-4
(Æ)-5
280
232
260
288
200
Cat C
Cat C
Cat C
Cat C
Cat D
2
12
3
4
24
49.8
50.0
50.0
50.2
49.0[e]
(R)-6
(R)-7
(R)-8
(R)-9
>99.0 33.9
>99.0 40.0
>99.0 36.5
>99.0 31.2
95
88
95
90
71
N
>99.0
>99.0
>99.0
>99.0
96.0[f]
32.5
38.8
38.5
38.2
40.0
91
90
100
110
80
(R)-10 99.8[f]
35.5
[a]
Biotransformation was performed in 100 mL Tris-HCl buffer (pH 8.0, 100 mM) at 308C and 250 rpm. For E. coli (BDHA) (Cat C):
10 g cdw/L; for E. coli (BDHA-LDH) (Cat D): 20 g cdw/L, with 100 mM sodium pyruvate.
Conversion was determined by GC analysis. Error limit: 0.5% of the state values.
ee was determined by chiral GC analysis. Error limit: 0.1% ee.
[b]
[c]
[d]
[e]
[f]
Error limit: 0.1%.
Conversion was determined by HPLC analysis. Error limit: 0.5% of the state values.
ee was determined by chiral HPLC analysis. Error limit: 0.1% ee.
Chiral a-hydroxy ketones can be prepared by LDH and explore other regeneration systems. More-
chemical methods.[22–24] The best one is the Sharpless over, we will develop immobilized enzymes for in
asymmetric dihydroxylation of enol ethers, giving (R)- vitro oxidations with co-factor recycling for even
7 in 90% ee, (R)-8 in 92% ee, and (R)-10 in 97% ee, more efficient biotransformations. Future work will
respectively.[23] However, the metal catalyst is toxic, also include the exploration of the application scope
and the product ee is still not high enough for direct of the developed alcohol dehydrogenases and the dis-
application in chiral pharmaceutical syntheses. Asym- covery of new enzymes for this type of enantioselec-
metric a-aminoxylation of ketone with nitrosoben- tive syntheses.
zene using (S)-proline as catalyst followed by the
treatment with CuSO4 gave (R)-7 in >99% ee,[25] but
the synthesis requires 20 mol% catalyst and a stoichio-
metric amount of the oxidation regent. Enzymatic
Experimental Section
preparations[26] of chiral cyclic a-hydroxy ketones
Representative Procedures for the Synthesis of (R)-a-
have been thus far not very enantioselective: kinetic
Hydroxy Ketones and (S,S)-Cyclic Vicinal Diols by
resolution via enantioselective acetylation to give (S)-
Enantioselective Oxidation of the Corresponding
10 showed an E of 16–35;[27] and reductive kinetic res-
Racemic Cyclic Vicinal Diols with E. coli (BDHA)
olution afforded (R)-6 with an E of 10.[3] Thus, our
or E. coli (BDHA-LDH)
method for the preparation of (R)-6–10 has significant
advantages over other reported methods.
Preparation of (R)-7 and (S,S)-2 with E. coli ACTHNUTRGNEUG(N BDHA) as
catalyst: E. coli (BDHA) was grown at 378C in TB (terrific
broth) medium (50 mL) containing kanamycin (50 mgmLÀ1)
for 2 h to reach an OD600 of 0.6. IPTG (0.5 mM) was then
added to induce the expression of protein, and the cells
were grown at 228C for another 10 h to reach an OD600 of
16.3 (cell density of 6.5 g cdw/L). The cells were harvested
by centrifuge (8,500 g, 5 min), and the cell pallets were re-
suspended in 100 mL Tris-buffer (100 mM, pH 8.0) to 10 g
cdw/L. (Æ)-2 (232 mg) was added, and the mixture was
In conclusion, highly regio- and enantioselective al-
cohol dehydrogenases BDHA, CDDHPm, and
CDDHRh were discovered for the oxidations of race-
mic trans-cyclic vicinal diols (Æ)-1–5. E. coli (BDHA)
and E. coli (BDHA-LDH) were developed as effi-
cient whole-cell biocatalysts for these oxidations, ena-
bling one-pot synthesis of both (R)-a-hydroxy ketones
6–10 and (S,S)-cyclic vicinal diols 1–5 in very high ee
at 50% conversion. A preparative biotransformation shaken at 250 rpm and 308C for 12 h to reach 50.0% conver-
sion. The products (R)-7 and (S,S)-2 were extracted with
ethyl acetate, and the organic solvent was dried over
Na2SO4 and filtered. Evaporation at reduced pressure af-
forded the crude products (R)-7 and (S,S)-2. Purification by
flash chromatography on a silica gel column with EtOAc:n-
hexane (1:1) as eluent gave 88 mg (R)-7 and 90 mg (S, S)-2,
with an isolated yield of 37.9% and 38.8%, respectively.
Both compounds are in >99.0% purity and >99.0% ee.
was demonstrated. This method is better than other
reported methods for the synthesis of these enantio-
pure diols and a-hydroxy ketones. It represents the
first example of using this type of reaction for simul-
taneous synthesis of both vicinal diols and a-hydroxy
ketones in high ee. A potential limitation of the cur-
rent whole-cell system for practical application is the
use of a high cell density to achieve the ideal 50%
conversion. To solve this problem, we will further im-
Preparation of (R)-10 and (S, S)-5 with E. coli
ACHTUNGTRNE(NUNG BDHA-
LDH) as catalyst: E. coli (BDHA-LDH) was grown under
prove the current NAD+-regenerating system with the same conditions as those for E. coli (BDHA) except
Adv. Synth. Catal. 2013, 355, 3147 – 3153
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asc.wiley-vch.de
3151

Jiandong Zhang et al.
COMMUNICATIONS
that both kanamycin (50 mgmLÀ1) and ampicillin
(100 mgmLÀ1) were used here. The freshly harvested cells
were resuspended in 100 mL Tris-buffer (100 mM, pH 8.0) to
a density of 20 g cdw/L. (Æ)-5 (200 mg) and sodium pyru-
vate (1.1 g) were added, and the mixture was shaken at
250 rpm and 308C for 24 h to reach 49.0% conversion. The
same work-up and purification procedures as described
above were used to give 71 mg (R)-10 in >99.0% purity,
99.8% ee, and 35.5% isolated yield and 80 mg (S,S)-5 in
>99.0% purity, 96.0% ee, and 40.0% isolated yield.
cuero, G. de Gonzalo, R. Brieva, V. Gotor, Eur. J. Org.
Chem. 2006, 4224–4230; c) C. M. Bellucci, A. Bergami-
ni, P. G. Cozzi, A. Papa, E. Tagliavini, A. Umani-
Ronchi, Tetrahedron: Asymmetry 1997, 8, 895–902.
[13] F. Kunst, N. Ogasawara, I. Moszer, A. M. Albertini, G.
Alloni, V. Azevedo, M. G. Bertero, P. Bessiꢂres, A. Bo-
lotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M.
Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bru-
schi, B. Caldwell, V. Capuano, N. M. Carter, S.-K. Choi,
J.-J. Codani, I. F. Connerton, N. J. Cummings, R. A.
Daniel, F. Denizot, K. M. Devine, A. Dꢃsterhçft, S. D.
Ehrlich, P. T. Emmerson, K. D. Entian, J. Errington, C.
Fabret, E. Ferrari, D. Foulger, C. Fritz, M. Fujita, Y.
Fujita, S. Fuma, A. Galizzi, N. Galleron, S.-Y. Ghim, P.
Glaser, A. Goffeau, E. J. Golightly, G. Grandi, G. Gui-
seppi, B. J. Guy, K. Haga, J. Haiech, C. R. Harwood, A.
Hꢄnaut, H. Hilbert, S. Holsappel, S. Hosono, M.-F.
Hullo, M. Itaya, L. Jones, B. Joris, D. Karamata, Y. Ka-
sahara, M. Klaerr-Blanchard, C. Klein, Y. Kobayashi, P.
Koetter, G. Koningstein, S. Krogh, M. Kumano, K.
Kurita, A. Lapidus, S. Lardinois, J. Lauber, V. Lazarev-
ic, S.-M. Lee, A. Levine, H. Liu, S. Masuda, C. Mauꢅl,
C. Mꢄdigue, N. Medina, R. P. Mellado, M. Mizuno, D.
Moestl, S. Nakai, M. Noback, D. Noone, M. OꢆReilly,
K. Ogawa, A. Ogiwara, B. Oudega, S.-H. Park, V.
Parro, T. M. Pohl, D. Portetelle, S. Porwollik, A. M.
Prescott, E. Presecan, P. Pujic, B. Purnelle, G. Rapo-
port, M. Rey, S. Reynolds, M. Rieger, C. Rivolta, E.
Rocha, B. Roche, M. Rose, Y. Sadaie, T. Sato, E. Scan-
lan, S. Schleich, R. Schroeter, F. Scoffone, J. Sekiguchi,
A. Sekowska, S. J. Seror, P. Serror, B.-S. Shin, B. Soldo,
A. Sorokin, E. Tacconi, T. Takagi, H. Takahashi, K.
Takemaru, M. Takeuchi, A. Tamakoshi, T. Tanaka, P.
Terpstra, A. Tognoni, V. Tosato, S. Uchiyama, M. Van-
denbol, F. Vannier, A. Vassarotti, A. Viari, R. Wam-
butt, E. Wedler, H. Wedler, T. Weitzenegger, P. Win-
ters, A. Wipat, H. Yamamoto, K. Yamane, K. Yasumo-
to, K. Yata, K. Yoshida, H.-F. Yoshikawa, E. Zumstein,
H. Yoshikawa, A. Danchin, Nature 1997, 390, 249–256.
[14] W. L. Nicholson, Appl. Environ. Microbiol. 2008, 74,
6832–6838.
[15] a) X. Jia, Y. Xu, Z. Li, ACS Catal. 2011, 1, 591–596;
b) D. Chang, B. Witholt, Z. Li, Org. Lett. 2000, 2, 3949–
3952; c) D. Chang, H. J. Feiten, K. H. Engesser, J. B.
van Beilen, B. Witholt, Z. Li, Org. Lett. 2002, 4, 1859–
1862; d) Z. Li, H. J. Feiten, D. Chang, W. A. Duetz,
J. B. van Beilen, B. Witholt, J. Org. Chem. 2001, 66,
8424–8430; e) Z. Li, H. J. Feiten, J. B. Van Beilen,
W. A. Duetz, B. Witholt, Tetrahedron: Asymmetry
1999, 10, 1323–1333; f) D. Chang, H. J. Feiten, B. With-
olt, Z. Li, Tetrahedron: Asymmetry 2002, 13, 2141–
2147.
[16] Y. Chen, F. Lie, Z. Li, Adv. Synth. Catal. 2009, 351,
2107–2112.
[17] M. Binazadeh, I. A. Karimi, Z. Li, Enzyme. Microbiol.
Technol. 2009, 45, 195–202.
[18] S. Q. Pham, P. Gao, Z. Li, Biotechnol. Bioeng. 2013,
110, 363–373.
[19] a) D. H. G. Crout, V. S. B. Gaudet, K. Laumen, M. P.
Schneider, J. Chem. Soc. Chem. Commun. 1986, 808–
810; b) R. Seemayer, M. P. Schneider, J. Chem. Soc.
Chem. Commun. 1991, 49–50.
Acknowledgements
This work was supported by the Science & Engineering Re-
search Council of A*STAR, Singapore, through a research
grant (project No. 1021010026).
References
[1] K. C. Bhowmick, N. N. Joshi, Tetrahedron: Asymmetry
2006, 17, 1901–1929.
[2] C. Palomo, M. Oiarbide, J. M. Garcꢁa, Chem. Soc. Rev.
2012, 41, 4150–4164.
[3] L. G. Lee, G. M. Whitesides, J. Org. Chem. 1986, 51,
25–36.
[4] O. Bortolini, E. Casanova, G. Fantin, A. Medici, S. Poli,
S. Hanau, Tetrahedron: Asymmetry 1998, 9, 647–651.
[5] P. Adlercreutz, Appl. Microbiol. Biotechnol. 1989, 30,
257–263.
´
[6] E. Wojaczynska, J. Skarz˙ewski, Tetrahedron: Asymme-
try 2008, 19, 593–597.
[7] a) K. Kato, H. Suemune, K. Sakai, Tetrahedron Lett.
1992, 33, 247–250; b) K. Kato, H. Suemune, K. Sakai,
Tetrahedron Lett. 1994, 35, 3103–3104; c) K. Sakai, H.
Suemune, Tetrahedron: Asymmetry 1993, 4, 2109–2118.
[8] a) M. Tanaka, M. Oba, K. Tamai, H. Suemune, J. Org.
Chem. 2001, 66, 2667–2673; b) M. D. Groaning, B. J.
Rowe, C. D. Spilling, Tetrahedron Lett. 1998, 39, 5485–
5488.
[9] a) C. H. Senayake, F. E. Roberts, L. M. DiMichele,
K. M. Ryan, J. Liu, L. E. Fredenburgh, B. S. Foster,
A. W. Douglas, R. D. Larsen, T. R. Verhoeven, P. J.
Reider, Tetrahedron Lett. 1995, 36, 3993–3996; b) C. M.
Belluci, A. Bergamini, P. G. Cozzi, A. Papa, E. Taglia-
vini, A. Umani-Ronchi, Tetrahedron: Asymmetry 1997,
8, 895–902; c) I. W. Davies, L. Gerena, D. Cai, R. D.
Larsen, T. R. Verhoeven, P. J. Reider, Tetrahedron Lett.
1997, 38, 1145–1148.
[10] a) D. Takada, H. Suemune, K. Sakai, Chem. Pharm.
Bull. 1990, 38, 234–235; b) R. S. Robinson, R. J. K.
Taylor, Synlett 2005, 6, 1003–1005; c) J. P. Marino, G.
Cao, Tetrahedron Lett. 2006, 47, 7711–7713.
[11] a) S. C. Gatling, J. E. Jackson, J. Am. Chem. Soc. 1999,
121, 8655–8656; b) M. J. Brown, T. Harrison, P. M.
Hemnton, M. H. Hopkins, K. D. Hutchinson, P. Mishra,
L. E. Overman, J. Am. Chem. Soc. 1991, 113, 5365–
5378.
[12] a) F. Orsini, G. Sello, A. M. Manzo, E. M. Lucci, Tetra-
hedron: Asymmetry 2005, 16, 1913–1918; b) V. Re-
3152
asc.wiley-vch.de
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2013, 355, 3147 – 3153

Enantioselective Biooxidation of Racemic trans-Cyclic Vicinal Diols
[20] a) C. E. Mꢃller, L. Wanka, K. Jewell, P. R. Schreiner,
Angew. Chem. 2008, 120, 6275–6278; Angew. Chem.
Int. Ed. 2008, 47, 6180–6183; b) R. Hrdina, C. E.
Mꢃller, P. R. Schreiner, Chem. Commun. 2010, 46,
2689–2690.
[23] B. F. Marcune, S. Karady, P. J. Reider, R. A. Miller, M.
Biba, L. DiMichele, R. A. Reamer, J. Org. Chem. 2003,
68, 8088–8091.
[24] M. Koprowski, J. Quczak, E. Krawczyk, Tetrahedron
2006, 62, 12363–12374.
[21] a) L. S. Zhao, B. Han, Z. Huang, M. Miller, H. Huang,
D. S. Malashock, Z. Zhu, A. Milan, D. E. Robertson,
D. P. Weiner, M. J. Burk, J. Am. Chem. Soc. 2004, 126,
11156–11157; b) D. L. Chang, Z. S. Wang, M. F. Herin-
ga, R. Wirthner, B. Witholt, Z. Li, Chem. Commun.
2003, 960–961; c) S. Wu, A. Li, Y. S. Chin, Z. Li, ACS.
Catal. 2013, 3, 752–759.
[25] A. Bøgevig, H. Sunden, A. Cordova, Angew. Chem.
2004, 116, 1129–1132; Angew. Chem. Int. Ed. 2004, 43,
1109–1109.
[26] a) W. Adam, M. Lazarus, C. R. Saha-Mçller, P. Schrei-
er, Acc. Chem. Res. 1999, 32, 837–845; b) P. Hoyos, J. V.
Sinisterra, F. Molinari, A. R. Alcꢇntara, P. Domꢁnguez
de Marꢁa, Acc. Chem. Res. 2010, 43, 288–299.
[27] W. Adam, M. T. Dꢁaz, R. T. Fell, C. R. Saha-Mçller,
Tetrahedron: Asymmetry 1996, 7, 2207–2210.
[22] B. Plietker, Tetrahedron: Asymmetry 2005, 16, 3453–
3459.
Adv. Synth. Catal. 2013, 355, 3147 – 3153
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
3153