Biocatalytic dynamic kinetic reductive resolution with ketoreductase from: Klebsiella pneumoniae: The asymmetric synthesis of functionalized tetrahydropyrans

Article abstract of DOI:10.1039/c9ob01681c

Ketoreductase from growing cells of Klebsiella pneumoniae (NBRC 3319) acts as an efficient reagent for converting racemic α-benzyl/cinnamyl substituted-β-ketoesters to the corresponding β-hydroxy esters with excellent yields and stereoselectivities (ee and de >99 %). The reactions described herein followed a biocatalytic dynamic kinetic reductive resolution (DKRR) pathway, which is reported for the first time with such substrates. It was found that the enzyme system can accept substituted mono-aryl rings with different electronic natures. In addition, it also accepts a substituted naphthyl ring and heteroaryl ring in the α-position of the parent β-ketoester. The synthesized enantiopure β-hydroxy esters were then synthetically manipulated to valuable tetrahydropyran building blocks.

Full text of DOI:10.1039/c9ob01681c

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Page 1 of 37
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BiocatalyticDynamic Kinetic Reductive Resolution withketoreductase from
Klebsiellapneumoniae: Asymmetric synthesis of functionalized tetrahydropyrans
RasmitaBarik, JoydevHalder and Samik Nanda*
Department of Chemistry, IIT Kharagpur, Kharagpur, 721302
snanda@chem.iitkgp.ac.in
Abstract: Growing cell of ketoreductase from Klebsiella pneumoniae (NBRC 3319) act as an
efficient reagent for converting racemic α-benzyl/cinnamyl substituted-β-ketoesters to its
corresponding β-hydroxy esters with excellent yield and stereoselection (eeand de>99%). The
reaction described herein followed a biocatalytic dynamic kinetic reductive resolution (DKRR)
pathway and reported for the first time with such substrates. It was found that the enzyme system
can accept substituted mono-aryl ring with different electronic natures. In addition, it also accepts
a substituted naphthyl ring and heteroaryl ring in the α-position of parent β-ketoesters. The
synthesized enantiopure β-hydroxy esters were then synthetically manipulated to valuable
tetrahydropyran building blocks.
Introduction
Whole-cell bioreductions with different pure strains of microorganism (available from culture
collections worldwide) of a prochiral carbonyl compounds for the synthesis of corresponding
enantiopure alcohol was regarded as one of the classical functional group interconversion (FGI)
nowadays. 1 The whole cells are self-replicating and require simple and cheap nutrients for their
growth. Hence a large number of whole cells (growing and resting) can be accumulated in a short
time.2 The problems associated with costly co-factor (NADH or NADPH) regeneration can be
avoided as the whole cell can integrate the biosynthesis of the cofactors in its cellular machinery
nicely.3 Whereas reduction of carbonyl systems with isolated whole-cell ketoreductases (KR) or
alcohol dehydrogenases (ADH) and recombinant enzymes were also equally explored with the
advancement of enzymology and molecular biology protocol.4. Such biocatalytic reduction system
consisting of pure KR/ADH always requires another enzymatic system for co-factor regeneration
(e.g., Formate dehydrogenase or Glucose-6P-dehydrogenase being the most popular) are
operationally simple, but care must be taken during the scale-up procedure.5 In addition, the overall
cost of such a biocatalytic reduction process with pure KR/ADH might be on the higher side as
many of the enzymes are not accessible to a classical synthetic chemist. Whereas growing or
resting cell of microbial KR/ADH (available easily) can easily be implemented in a general

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laboratory set up and high space-time yield (20-200 g/L in 3-4 day operation) of enantiopure
alcohols can be accessed with proper medium and fermentation optimization.6 From a strategical
viewpoint of synthetic organic chemist (retrosynthetic biocatalysis with KR/ADH),7
biocatalytically enantiopure secondary alcohols can be accessed by (i) reduction of a prochiral
carbonyl, (ii) deracemization via stereoinversion (single step or two-step; sometimes referred as
oxidative kinetic resolution), (iii) simultaneous stereoinversion and (iv) dynamic kinetic reductive
resolution (DKRR) pathway, this was recently documented elegantly by Bommarius and co-
workers (Figure-1).7Among the reductive strategies, biocatalytic DKRR seemed to be unique in
its own merit. Certain structural prerequisite in the starting precursor was required as delineated in
Figure-1, and such a reductive DKRR pathway was explored in recent past by few research groups
including ours.8
Dynamic Kinetic Reductive Resolution (DKRR)
OH
O
OH
KR or ADH
EWG
EWG
EWG
+
R1
R1
R1
R2
R2
R2
APKR-anti
PKR-syn
X
X
No epimerization
EWG
OH
OH
KR or ADH
O
EWG
+
R1
R1
EWG
R1
R2
PKR-anti
R2
APKR-syn
R2
Prerequisite for efficient DKRR process
* High kinetic acidity of the stereocenter is required
* The racemisation or epimerization is so fast two enantiomers are always in dynamic equilibrium
* The racemisation must be fast enough than the reduction process
* Once reduction is completed the racemisation is diminished due to reduced acidity
ERG: electron withdrawing group; PKR: Prelog Keto Reductase; APKR: Anti Prelog Keto Reductase
* Assuming combined size of EWG and R2 is greater than R1; KR: Ketoreductase; ADH: Alcohol dehydrogenase
Fig-1:Biocatalyticdynamic kinetic reductive resolution (DKRR) strategies for obtaining
enantiopure secondary alcohols
The substrate scope of such biocatalytic DKRR pathway with α-substituted-β-ketoesters (having
alkyl, allyl, propargyl and allyl appendages at α-position) have been explored but not extensively.
During our past investigations of such biocatalytic DKRR pathway with the growing cell of K.
pneumoniae, we found that this enzyme system is very particular in its substrate scope and
produced the PKR (Prelog Keto Reductase) syn product having (2R, 3S) absolute configuration in

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the stereocenter. Variation at the α-position of the parent β-ketoester (with a certain limit) is only
allowed with the enzyme. Whereas any change in the γ-position and in the ester moiety is not
tolerated by the enzyme system. Based on our extensive investigations, we have summarized the
substrate scope of KR from K. pneumoniae and were presented in Scheme1 (A detailed substrate
mapping of K. pneumoniae for this DKRR can be found in the electronic supporting information).
OH
O
O
O
K.pneumoniae
Me
OEt
Me
OEt
2-7 days
R
R
ee and de >99%
space time yield: 20-100 g/L
Works in PKR fashion and 2R,3S syn
alcohol was obtained as a sole product
Variation in appendage
at -positionis only tolarated
O
O

R'
O
Substitutions are not
tolerated


R
Substitutions are well accepted
by K.pneumoniae
R = alkyl, allyl, propargyl, benzyl
O
O
O
O
O
O
OEt
OEt
OEt
n
n
n = 0, 1, 2
n = 0, 1, 2
Not reactive at all
n = 0, 1, 2
n
Well accepted by
K.pneumoniae
Substitutions are
not accepted
Scheme 1: Substrate scope of KR from K. pneumoniae for biocatalytic DKRR pathway.
It was found that slight variations at γ-position of the parent β-keto-ester (any other group than –
Me) were not accepted by the enzyme system. Few cyclic α-substituted-β-ketoesters were accepted
by the KR system, but any variation in the γ-position resulted in similar findings (usually those
substrates remain intact after 7 days incubation with growing cells of K. pneumoniae). In this
current context, we argued that to explore the synthetic potential of K. pneumoniae other variations
at α-position (by changing different appendage) can be the best option. Allyl substitution at the α-
position in the parent β-ketoester was explored by us and yielded the corresponding PKR product

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(syn-2R,3S) exclusively.8g Similarly, a benzyl and 4-methoxybenzyl substitution at α-position of
the parent β-ketoester was also accepted by the enzyme system as shown by us in an earlier
report.8g We are curious to investigate how structurally different benzyl systems at α-position
(having electron releasing and withdrawing group present in the aryl ring, naphthyl ring) of the
parent β-ketoester respond to the enzymatic DKRR pathway for the synthesis of corresponding
enantiopure β-hydroxy esters. In the same way, a cinnamyl appendage at the α-position is
structurally related to the allyl group, but the presence of an aryl moiety may have a different steric
and electronic parameter in the starting precursor. It would be quite interesting to investigate
whether α-cinnamyl substituted-β-ketoesters will be accepted by K. pneumoniae for a DKRR
pathway. Such substrates were never tested for any KR or ADH platform through a biocatalytic
DKRR strategy (Scheme 2). In addition, we also speculate that the synthesized enantiopure α-
cinnamyl-β-hydroxy esters can be synthetically transformed to other structurally interesting chiral
small molecular scaffolds.

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Earlier work
OH
O
PKR-syn (2R, 3S): CPADH, TesADH 5l, 5o
O
O
PKR or APKR
APKR-syn (2S, 3R) = LBADH, LKADH 5j
PKR-anti (2S,3S) = Tyl-KR1 5n
OEt
OEt
With cofactor
regeneration system
APKR-anti (2R, 3R) = S. cerevisiae 5h, 5m
(±)
* All the enzymes are recombinant and reactions were
usually carried out in analytical scale (mg amount)
Few selected earlier works were given
This work
OH
O
O
O
K.pneumoniae
(a)
OEt
OEt
Ar
Ar
(±)
Ar = Monosubstituted aryl ring system with ERG and EWG,
and napthyl ring; ERG: Cl, Br, Me; EWG: NO2
EtO2C
I
OH
O
Further synthetic
manipulation
O
Ar
O
O
(b)
K.pneumoniae
OEt
Ar
or
OEt
Ar
OH
I
(±)
O
Ar
Ar = Monosubstituted aryl ring system with ERG and EWG,
napthyl ring, thiophene; ERG: Cl, Br, Me, OMe; EWG: NO2
* Such substrates were never tested before
* Whole cell mediated reaction
* Cofactor regeneration system is not required
* Reactions are carried out in preparative scale (~ 20 g scale)
Scheme 2: Biocatalytic DKRR and further synthetic manipulation presented in this article
Results and Discussion:
With this background information, initially, we have initiated the synthesis of all the precursors
required for the biotransformation. The synthetic step was outlined in Scheme 3, in general,
suitably substituted benzyl bromides were reacted with ethyl acetoacetate in the presence of NaH
in refluxing THF to furnish the corresponding α-benzyl substituted β - ketoesters (1a-1h). For the
synthesis of other sets of precursors, suitable aromatic aldehyde was subjected to HWE olefination
9 with triethylphosphonoacetate to furnish the corresponding α,β-unsaturated esters in good yield

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(2a-2n; in favor of “E” isomer). The α,β-unsaturated esters were then reduced to corresponding
allylic alcohol (3a-3n) by reduction with DIBAL-H in reasonably good yield. The allylic alcohols
were then converted to the corresponding bromides (4a-4n) by treatment with PBr3 in reasonably
good yield. The allylic bromides were then coupled with ethyl acetoacetate in the presence of NaH
to furnish the racemic α-cinnamyl-β-ketoesters in good yield (5a-5n; Scheme 3).
1a
1b
1c
X = 4-Me;
X = 4-Br;
X = 4-Cl;
O
O
NaH, THF
O
O
Br
+
OEt
X
OEt
reflux, 6-24 h
~78%
1d
1e
X = 3-NO2;
X = 4-NO2,
(±)
X
X = 4-Me, 4-Br, 4-Cl, 3-NO2, 4-NO2,
1-napthyl, 2-napthyl, 2-thienyl
1f
1g
1h
X = 1-napthyl;
X = 2-napthyl;
X = 2-thienyl;
1a-1h
(8 precursors)
O
O
DIBAL-H
NaH, THF
68-90%
TPP, CBr4
CO2Et
P
Ar
+
Ar-CHO
Ar
OH
EtO
OEt
O
DCM, -78oC
88-97%
OEt
DCM, rt, ~90%
exclusively "E"
3a-3n
2a-2n
O
O
NaH, THF
O
Ar
Br
+
OEt
Ar
-10oC,4-6 hr,78%
OEt
4a-4n
(±)
5a-5n
(14 precursors)
Ar = differently substituted mono aryl, napthyl, heteroaryl
Scheme 3: Synthesis of racemic α-benzyl/cinnamyl-substituted-β-ketoesters for biocatalytic
DKRR reaction
To test the feasibility of such substrates towards DKRR, initially compound 1a (100 mg, 0.42
mmol) was added to the growing cell of K. pneumoniae(grown for 2 days), and the reaction was
continued in an incubator shaker for further 2-3 days. Periodical analysis of the reaction mixture
was routinely done with the help of TLC analysis (by withdrawing aliquots and extracting them
with EtOAc). To our delight, we found that the complete conversion of compound 1a to its
corresponding reduced product (6a) took place within 3 days. The product 6a was purified through
chromatography (82% isolated yield) and characterized with NMR analysis. Formation of single
stereoisomer was confirmed through HPLC analysis. The assignment of absolute configuration for
compounds (6a-6h) was done based on our prior report of related compounds.8fBased on the
stereopreference of K. pneumoniae, we reasoned that PKR-syn product would be the sole

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stereoisomer hence it should have (2R, 3S) configuration in the newly created stereocenters. We
have then focused our attention for substrate scope of this biocatalytic DKRR reaction with several
structurally related α-benzyl-substituted-β-ketoesters (1b-1h) as shown in Scheme 4. Substrates
having monosubstituted aryl groups containing electron releasing group (-Br and –Cl; 1b and1c)
are well accepted by K. pneumoniae and produced the corresponding PKR-syn products (6b and6c)
with excellent stereoselection and reasonably accepted yield. Substrates having electron
withdrawing group in the aryl ring (such as –NO2; 1d and1e) were also accepted by the enzyme
system and furnished the corresponding reduced products (6d and6e) in good yield with excellent
stereoselection (ee and de > 99%). It was observed that substrates with electron withdrawing group
in the aryl ring (1d and1e) took more time for complete conversion (7 days) when compared to
substrates having electron releasing group in the aryl ring (1a-1c) in the biocatalytic DKRR with
K. pneumoniae. Next substrates 1f and 1g having a sterically bulky naphthyl group in the α-
position of the parent β - ketoesters were subjected to biocatalytic DKRR reaction with growing
cells of K. pneumoniae. Both the substrates were well accepted by the enzyme system and yielded
the alcohols (6f and6g) in good yield with excellent stereoselection after 5 days of incubation.
Presence of heterocyclic moiety in the α-position of the parent β-ketoester (1h; 2-thienyl) was also
accepted by the enzyme system for corresponding biocatalytic DKRR reaction. Complete
conversion of the starting material was observed for the compound 1h, and excellent
enantioselectivity and diastereoselectivity was also observed for the product alcohol. In general, it
was noticed that KR from K. pneumoniae could tolerate several structural variations in the α-
position of the β-ketoesters for DKRR reaction. The yields (isolated) and stereoselectivity (ee and
de) for all the synthesized α-benzyl substituted-β-hydroxy esters were summarized in Scheme 4.
Preparative scale biotransformation can also be efficiently carried out with few substrates such as
1a, 1e, 1f and 1h (in 20.0 g scale; approximately 0.07 mol), and after usual extractive workup,
corresponding alcohols (7a, 7h, 7j, and 7n) were obtained with reasonably good yield and
excellent stereoselection. As the detailed active site mapping of this enzyme was not available at
present, we are unable to predict its general substrate scope, but our previous and present
investigation will enable us to ascertain a qualitative structure-activity relationship model for such
system.

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OH
O
O
O
K.pneumoniae, rt
OEt
OEt
3-7 days
R
R
6a-6h
; PKR syn product
1a-1h
(±)
OH
O
OH
O
OH
O
OH
O
OEt
OEt
OEt
OEt
NO2
Cl
; yield = 74%;
Br
; yield = 70%;
ee = 99% ; de = 99%
OH
Me
6d
; yield = 72%;
ee = 99% ; de = 99%
6c
6a
; yield = ; 82%
6b
ee = 99% ; de = 99%
ee = 99% ; de = 99%
O
OH
O
OH
O
OH
O
OEt
OEt
OEt
S
OEt
NO2
6e
; yield = 76%;
ee = 99% ; de = 99%
6h
; yield = 80%;
ee = 99% ; de = 99%
6g
; yield = 78%;
ee = 99% ; de = 99%
6f
; yield = 72%;
ee = 99% ; de = 99%
Scheme 4: Substrate scope for α-benzyl-substituted-β-ketoesters for K. pneumoniae mediated
DKRR reaction (yield = isolated yield after purification. Ee and de was determined through chiral
HPLC analysis)
We have then focused our attention for substrate scope of this biocatalytic DKRR reaction with
several structurally related α-cinnamyl-substituted-β-ketoesters as shown in Scheme 5. In the
beginning, compound 5a was subjected to the biocatalytic DKRR reaction with growing cell of
K.pneuminiae as described earlier. To our delight, we have noticed that corresponding PKR syn-
product (7a) was obtained after 3 days of incubation. Subsequently, substrates having
monosubstituted cinnamyl unit containing electron releasing group (-Me, -OMe, -Br, -Cl), e.g.,
5b-5e are found to be well accepted by K. pneumoniae and produced the corresponding PKR-syn
products with excellent setereoselection and reasonably accepted yield. Substrates having 3,5-
dimethoxy group in the aryl ring (5f) was also accepted nicely by the enzyme system and furnished
the corresponding alcohol (7f) as shown in Scheme 5. In general complete conversions was
observed after 3 days of incubation with the growing cell of K. pneumoniae for substrates having
electron releasing groups present in the aryl ring (5a-5f). Substrates having electron withdrawing

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group in the aryl ring (such as 3-NO2 and 4-NO2; 5g and5h) were also accepted by the enzyme
system and furnished the corresponding reduced products (7g and 7h) in good yield with excellent
stereoselection (ee and de > 99%), but it took 7 days for complete conversion. Probably the
electronic nature of the aryl ring has little effect on the overall conversion, but the stereoselection
remains unaffected as shown in Scheme 6. Next, we have used substrates containing sterically
bulky naphthyl ring as precursors for DKRR reaction with K. pneumoniae. All the substrates were
well tolerated by the enzyme system and furnished the corresponding stereochemically pure
alcohols in good yield, as shown in Scheme 6. Though the conversion and stereoselectivity were
not affected, usually it took little more time (7-10 days) for complete conversion of substrates
containing the naphthyl ring. Precursors 5i and 5j having 1-naphthyl and 2-naphthyl cinnamyl
substitutions were well accepted by the enzyme systems, and corresponding reduced products (7i
and7j) were obtained in 80% and 76% yields respectively with excellent stereoselection.
Substrates (5k) with an -OMe substitution in the aryl ring (2-methoxy-1-naphthyl) was also
accepted nicely by the enzyme system. Other substrates such as 4-methoxy-1-naphthyl- cinnamyl
(7k) and 6-methoxy-2-naphthyl-cinnamyl (7l) were also tolerated by the enzyme system and
furnished the corresponding reduced products in good yield with excellent stereoselection. It also
clearly demonstrates that substitution in the o, -m and –p position of the aryl rings were well
tolerated in the benzyl system as well as in the cinnamyl system. Substrate (5n) with a 2-thienyl
unit acted as an excellent substrate for the K. pneumoniae mediated DKRR reaction, as
corresponding alcohol (7n) was obtained in 82% yield after 2 days with excellent stereoselection
(Scheme 5). The yields (isolated) and stereoselectivity (ee and de) for all the synthesized α-
cinnamyl-substituted-β-hydroxy esters were summarized in Scheme 5. Preparative scale
biotransformation can also be efficiently carried 20.0 g scale (approximately 0.07 mol), and after
usual extractive workup, corresponding alcohols (7a, 7h, 7j, and 7n) were obtained with
reasonably good yield and excellent stereoselection. The assignment of absolute configuration for
compounds (7a-7n) was done based on our prior report of related compounds.8e-g We have also
established the absolute configuration of 7a by preparing it through another way as shown in
Scheme 5. The known compound (R)-ethyl 2-((S)-1-hydroxyethyl)pent-4-enoate (synthesized by
us through K. pneumoniae mediated DKRR reaction 8e) was reacted with styrene in the presence
of G-II catalyst, the cross metathesis (CM) reaction proceeded well and produced compound 7a in

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70% yield. Comparison of spectral and optical data of both the samples of 7a confirmed the
absolute configuration of the product in the present DKRR reaction.
OH
O
O
O
K.pneumoniae, rt
OEt
OEt
Ar
3-7 days
Ar
; PKR syn product
5a-5n
(±)
7a-7n
OH
O
OH
O
OH
O
OH
O
OH
O
OEt
OEt
OEt
OEt
OEt
Cl
ee = 99% ; de = 99%
Br
OMe
Me
7e
; yield = 75%;
7a
7c
; yield = 70% ;
; yield = 78%;
7d
; yield = 74%;
7b
; yield = 75%;
ee = 99% ; de = 99%
ee = 99% ; de = 99%
ee = 99% ; de = 99%
ee = 99% ; de = 99%
OH
O
OH
O
OH
O
OH
O
OH
O
OEt
OEt
OEt
OEt
OEt
NO2
OMe
NO2
OMe
7h
; yield = 75%;
ee = 99% ; de = 99%
7g
; yield = 78%;
ee = 99% ; de = 99%
7j
; yield = 76%;
ee = 99% ; de = 99%
7i
; yield = 80%;
ee = 99% ; de = 99%
7f
; yield = 60%;
ee = 99% ; de = 99%
OH
O
OH
O
OH
O
OH
O
OEt
OEt
OEt
OEt
S
MeO
OMe
OMe
7n
; yield = 82%;
ee = 99% ; de = 99%
7m
; yield = 72%;
ee = 99% ; de = 99%
7k
7l
; yield = 75%;
; yield = 74%;
ee = 99% ; de = 99%
ee = 99% ; de = 99%
Absolute configuration assignment through synthetic manipulation
OH
O
OH
O
G-II (5 mol%), DCM,
24 h, 70%
*
OEt
*
*
OEt
*
7a
(R)-ethyl 2-((S)-1-hydroxyethyl)pent-4-enoate
(absolute configuration is known from earlier report 8e
)
[]D25 = 27.90 (c 1.0, CHCl3); prepared by DKRR
[]D25 = 28.52 (c 1.0, CHCl3); prepared by CM
Scheme 5: Substrate scope for α-cinnamyl-substituted-β-ketoesters for K. pneumoniae mediated
DKRR reaction (yield = isolated yield after purification. Ee and de was determined through chiral
HPLC analysis)

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Next, we have focused our attention on exploring the synthetic utility of biocatalylytically derived
enantiopure alcohols 7a-7n for the synthesis of substituted tetrahydropyran derivative. Substituted
tetrahydropyran (THP) core was found in many bioactive natural products. KalihinolA is a
kalihinane type diterpenoid exhibits potent antimalarial activity against P. falciparum having a
2,6-disubstituted THP core in its structure.10 Zampanolide a potent anti-tumor compound
(microtubule stabilizer) contained a cis-2,6-disubstituted THP core in its structure.11 Whereas
dactylolide another naturally occurring macrolide having potent cytotoxic activity also contains a
cis-2,6-disubstituted THP framework in its structure.12A group of naturally occurring macrolides
known as bryostatins also contain three 2,6-disubstituted THP core in their
structure.13Construction of substituted THP unit was also important in terms of the significance of
C-alkyl nucleosides (such as ambruticin-S) in recent days.14 We speculate that biocatalytically
derived precursors having a cinnamyl appendage can lead to enantiopure THP frameworks through
transformation driven approach (Scheme -6). We anticipate that through a stereospecific
halocyclization reaction (6-endo-tet mode) substituted tetrahydropyrans can be accessed as shown
in Scheme 6. Competitive 5-exo-tet mode of cyclization will not be feasible mainly due to the
presence of aryl ring at the terminus (mainly due to enhanced stability of benzylic cation). Two
different modes of intramolecular cyclization by utilizing interception of the initially formed
iodonium ion through primary and secondary hydroxyl group (as nucleophile) present in the
biocatalytically derived α-cinnamyl substituted β-hydroxyesters.
NC
H
H
O
O
O
OH
HO
H
OHC
O
O
O
O
NC
N
H
O
Kalihinol A
H
H
H
H
O
O
Cl
O
(-)-Zampanolide
O
HO2C
(+)-Dactylolide
(+)-Ambruticin S
(C-alkyl glycoside)
OH
OH
I
OP
Intramolecular
iodoetherification
EtO2C
I
Intramolecular
iodoetherification
OH
O
Ph
OH
Ar
H
OEt
Ar
O
O
Ph
6-endo-tet
6-endo-tet
OP

Organic & Biomolecular Chemistry
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DOI: 10.1039/C9OB01681C
Scheme 6: Substituted tetrahydropyran containing natural products and proposed intramolecular
iodoetherification strategy for the construction of enantiopure THP frameworks
The proposed intramolecular iodoetherification of compound 7a was next attempted with
I2/NaHCO3 in a mixed solvent (Et2O: water = 1:4). A single diastereomeric substituted iodoether
was obtained, and the structure was assigned to a 2,6-disubstituted tetrahydropyran core mainly
based on assuming a 6-endo-tet mode of cyclization (seems to be much more favorable due to
1
enhanced stability of benzylic cation). Close H-NMR inspection of compound 8a reveals that
proton associated with 6-position will be more deshielded due to the presence of aryl ring at 6-
position (nullifies the formation of 2,5-substituted tetrahydrofuran core originated through the 5-
exo-tet mode of cyclization). The intramolecular iodocyclization reaction (6-endo-tet mode) of
compound 7a was also attempted with other reagent systems (Table 1).15 It was found that superior
result was obtained with I2-NaHCO3 reagent system (entry 1) in terms of the isolated yield of the
isolated tetrahydropyran products. Reagents system consisting of NIS (N-iodosuccinimide; entry
3-6) did not provide
a
satisfactory yield of the substituted THP framework.
[Bis(2,4,6-trimethylpyridine)iodine(I) in
perchlorate]16
Iodoniumdicollidineperchlorate
acetonitrile solvent was also used for the desired cyclization reaction (entry 7), but the desired
product was obtained only in 20% isolated yield. Exploration of other electrophilic iodinating
reagents, such as DIH (1,3-diiodo-5,5-dimethylhydantoin; entry 8),17 N-iodophthalimide (entry 9)
and N-iodosaccharin (entry 10)18 for the above cyclization reaction also met with limited success.

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Organic & Biomolecular Chemistry
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DOI: 10.1039/C9OB01681C
Table 1: Iodoetherification reaction of compound 7a
OH
O
I+
Table 1
EtO2C
6-endo-tet
I
EtO2C
OEt
Ph
Me
OH
Ph
(entry 1)
O
Ph
H
7a
8a
O
O
I
O
N
N
N
I
N
I
O
N
I
N
S
O
O
O
I
N-iodophthalimide
DIH
ClO4
N-iodosachharin
[(coll)2IClO4]
Entry
1
Reagent condition
Yield (%)
82
I2, NaHCO3, Et2O: water (1:4)
I2, NaHCO3, THF:water (1:4)
I2, NaHCO3, THF:water (1:4)
NIS, CH2Cl2
2
45
3
30
4
25
5
NIS, MeCN
No product
18
6
NIS, Toluene
7
NIS, NaHCO3, MeCN
[(coll)2IclO4]
22
8
<10
9
1,3-diiodo-5,5-dimethylhydantoin (DIH)
N-iodophthalimide
<10
10
<10
a: Isolated yield for compound 8a.
For further synthetic manipulation iodo compound 8a was subjected to reductive deiodination with
AIBN/nBu3SnH to furnish 2,3,6-trisubstituted tetrahydropyran compound 9a in 80% yield. The
2D-NOESY NMR analysis of compound 9a confirms the syn stereochemistry of the 2-Me and 6-
Ph group. By applying a similar intramolecular iodoetherification/deiodination sequence similar
2,3,6-trisubstituted tetrahydropyrans (9b-9e) were accessed (Scheme 7).

Organic & Biomolecular Chemistry
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DOI: 10.1039/C9OB01681C
OH
O
EtO2C
I+
EtO2C
6-endo-tet
I
I2, NaHCO3
EtO2C
OEt
Ph
I
Me
OH
Ph
Et2O:H2O(1:4),
80%
O
Ph
O
8a
Ph
H
7a
5-exo-tet
(not formed)
nBu3SnH
AIBN
 for H at C6 ~ d(4.5 ppm) with J = 8.0 Hz for THP structure
 for H at C5 ~ m(4.1-4.3 ppm) for THF structure
EtO2C
Ph
O
O
Ph
Me
H
H
H
9a
EtO2C
H
2D-NOESY
EtO2C
EtO2C
I
EtO2C
I
EtO2C
I
I
S
O
O
O
O
H
H
H
H
8c
H
8d
H
H
H
Cl
Br
8b
8e
EtO2C
EtO2C
EtO2C
EtO2C
S
O
O
O
O
H
H
H
H
9c
H
9d
H
H
H
Cl
Br
9b
9e
Scheme 7: Intramolecular iodoetherification reaction of enzymatically derived α-cinnamyl
substituted β-hydroxyesters for the synthesis of chiral 2,3,6-trisubstituted tetrahydropyrans.
Next, we became interested in exploring the similar kind of intramolecular iodoetherification
reaction by employing the primary hydroxy functionality as a nucleophilic partner for the synthesis
of 2,3,5-trisubstituted tetrahydropyran derivatives. For that purpose, the free secondary hydroxyl
functionality in compound 5a was protected as its –TBDPS ether by treatment with imidazole and
TBDPS-Cl to furnish 10a in 90% yield. Reduction of ester functionality in 10a with DIBAL-H
furnished alcohol 11a in 85% yield. Compound 11a was next subjected to the intramolecular
iodoetherification reaction by adopting a similar reaction condition as reported by Liu and Wang.19
Thus when compound 11a was subjected to NIS treatment in the presence of 10 mol% of DMAP
in DCM solvent, tri substituted tetrahydropyran compound 12a was obtained as a major product
(Scheme 8). Formation of the corresponding tetrahydrofuran derivative 13a was also observed as
a minor product. We have tried the intramolecular iodoetherification reaction of γ,δ unsaturated
alcohol (11a) by adopting similar reaction conditions as depicted in Table-1. But all the reagent

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DOI: 10.1039/C9OB01681C
system listed in Table-1 provided less yield of the desired compounds. The reagents system
comprising of NIS with a catalytic amount of DMAP is found to be superior in terms of yield as
well as in favour of the desired tetrahydropyran derivative (12a). It was thought that initially NIS
was activated by DMAP and then the iodoniumspeciesattacks to the olefinic unsaturation of the
γ,δ-unsaturated alcohol (11a) to furnish the iodonium intermediate (Scheme 8). Deprotonation of
the primary hydroxyl functionality by the succinimide anion and subsequent intramolecular
nucleophilic attack to cyclic iodonium intermediate enforce the formation of the compound 12a.
By employing a similar reaction sequences compound 12b and 12c were also synthesized. The
formation of 12a (6-endo-tet) as a major product can be explained by the presence of the electron-
rich aromatic system in the δ-position, which can sufficiently stabilize the developing benzylic
cation.
OH
OTBDPS
OH
OTBDPS
CO2Et
CO2Et
NIS, DMAP
Imidazole, TBDPS-Cl
DCM, rt, 90%
DIBAL-H, DCM
-78oC, 85%
DCM, rt, 80%
11a
Ph
I
Ph
10a
Ph
7a
I
Ph
H
Ph
I+
H
O
+
6-endo-tet
TBDPSO
H
12a (major)
13a (minor)
O
Ph
b
a
OH
path a
5-exo-tet
OTBDPS
OTBDPS
H
13a (minor); 10%
12a (major); 90%
path b
S
I
Cl
Cl
I
I
TBDPSO
I
I
S
H
Ph
H
O
H
H
O
H
O
H
H
O
H
O
OTBDPS
OTBDPS
OTBDPS
OTBDPS
12b
13b
13c
12c
Scheme 8: Intramolecular iodoetherification reaction of enzymatically derived α-cinnamyl
substituted β-hydroxyesters for the synthesis of chiral 2,3,5-trisubstituted tetrahydropyrans.
Conclusion:
In conclusion, the substrate spectrum of ketoreductase from K. pneumoniae was investigated in
this article. It was found that α-cinnamyl substituted-β-ketoesters act as a new class of substrates
for biocatalytic dynamic kinetic reductive resolution reaction of ketoreductase from K.

Organic & Biomolecular Chemistry
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pneumoniae. A various range of substrates (having different functionality present in the aryl ring)
is well tolerated by the enzyme system and yield the corresponding PKR syn product in good yield
with excellent stereocontrol. In addition, it was also observed that few new α-benzyl substituted-
β-ketoesters could also act as very good substrates for the DKRR reaction. The enzymatically
derived enantiopure α-cinnamyl substituted-β-hydroxyesters were then synthetically manipulated
to structurally novel tetrahydropyran derivative in stereoselective fashion.
Experimental Section:
General procedures: All oxygen and/or moisture-sensitive reactions were carried out Under N2
atmosphere in glassware that had been flame-dried under vacuum (ca. 0.5 torrs) and purged with
N2 prior to use. Unless otherwise stated, materials were obtained from commercial suppliers and
used without further purification.THF and diethyl ether were distilled from sodium benzophenone
ketyl. Dichloromethane (DCM) and hexane were distilled from calcium hydride. Ketoreductase
strain of K. pneumoniae (NBRC 3319) was obtained from NBRC, Japan. Reactions were stirred
magnetically using Teflon-coated magnetic stirring bars. Teflon-coated magnetic stirring bars and
syringe needles were dried in an oven at 120 °C for at least 12 h prior to use, then cooled in a
desiccator cabinet over Drierite. Reactions were monitored by thin-layer chromatography (TLC)
carried out on 0.25 mm silica gel plates with UV light and ethanolicanisaldehyde as a visualizing
agent. Silica gel 100–200 mesh was used for column chromatography. Yields refer to
chromatographically and spectroscopically homogeneous materials unless otherwise stated. NMR
spectra were recorded on 600,400,500 and 200 MHz spectrometers at 25 °C in CDCl3 using TMS
13
As the internal standard. Chemical shifts are shown in δ. C NMR spectra were recorded in a
complete proton decoupling environment. Coupling constants (J) are reported in hertz (Hz), and
the resonance multiplicity abbreviations used are s, singlet; d, doublet; dd, doublet of doublet; t,
triplet; q, quartet; m, multiplet; and comp, overlapping multiplets of magnetically non-equivalent
protons. The mass spectrometric analysis was performed in the CRF, IIT-Kharagpur (TOF
analyzer). Chiral HPLC analysis was performed with Daicel Chiral Pak OD-H, IC, IA (25 cm ×
0.46 cm ∅) as the stationary phase.
General procedure for the synthesis of α-substituted-β-keto esters: To a suspension of NaH
(1eq) in THF, ethyl acetoacetate (1eq) was added sequentially at 0°C and stirred for 45 minutes,

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after that time the reaction vessel was settled at -10°C and then properly substituted benzyl
bromides and cinnamyl bromides were added into the reaction mixture at -10°C and left the
reaction 4-6 h at the same temperature. The low temperature is crucial for the successful coupling
reaction, as carrying out the reaction at room temperature and above a substantial amount of
dialkylated products were obtained. After completion of the reaction, the reaction solution was
quenched with saturated NH4Cl, and the organic phase was extracted with EtOAc. The organic
extracts were washed with brine and dried over anhydrous Na2SO4. The solvent was then
evaporated under reduced pressure, and the product was purified by flash column chromatography
(EtOAc/hexane = 1: 10) to furnish compounds 1a-1h and 5a-5n in good yield.
Ethyl 2-(4-methylbenzyl)-3-oxobutanoate (1a): Yield = 85%; Rf = 0.4 (EtOAc/ hexane = 1:10);
1H NMR (400 MHz, CDCl3) δ 7.04-7.09 (m, 4H), 4.15 (q, J = 7.1 Hz, 2H), 3.75 (t, J = 7.6 Hz,
1H), 3.12 (d, J = 7.5 Hz, 2H), 2.30 (s, 3H), 2.18 (s, 3H), 1.21 (t, J = 7.1 Hz, 3H).13C NMR (100
MHz, CDCl3) δ 202.6, 169.2, 136.2, 135.0, 129.2, 128.6, 61.4, 33.6, 29.6, 21.0, 14.0; HRMS (ESI)
m/z: for C14H18O3Na[M + Na]+, calculated:257.1154; found: 257.1158.
Ethyl-2-(4-bromobenzyl)-3-oxobutanoate (1b): Yield =85%; Rf = 0.42 (EtOAc/ hexane =
1:10);1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 4.14 (q, J
= 7.1 Hz, 2H), 3.72 (t, J = 7.6 Hz, 1H), 3.10 (d, J = 7.2 Hz, 2H), 2.19 (s, 3H), 1.20 (t, J = 7.2 Hz,
13
3H). C NMR (100 MHz, CDCl3) δ 201.9, 168.8, 137.2, 131.6, 130.6, 120.6, 61.6, 61.1, 33.2,
29.6, 14.0; HRMS (ESI) m/z: for C13H15BrO3Na[M + Na]+, calculated:321.0102; found: 321.0105,
323.0098.
Ethyl-2-(4-chlorobenzyl)-3-oxobutanoate (1c): Yield = 84%; Rf= 0.43(EtOAc/ hexane = 1:10);
1H NMR (400 MHz, CDCl3) δ 7.22 (d, J = 7.8 Hz, 2H), 7.12 (d, J = 7.4 Hz, 2H), 4.14 (q, J = 7.2
Hz, 2H), 3.72 (t, J = 7.6 Hz, 1H), 3.12 (d, J = 6.8 Hz, 2H), 2.19 (s, 3H), 1.20 (t, J = 7.2 Hz,
3H).).13C NMR (100 MHz, CDCl3) δ 202.0, 168.8, 136.7, 132.5, 130.2, 128.7, 61.6, 61.1, 33.2,
29.6, 14.0; HRMS (ESI) m/z: for C13H15ClO3Na[M + Na]+, calculated:277.0607; found: 277.0609.
Ethyl-2-(3-nitrobenzyl)-3-oxobutanoate (1d): Yield =78%;Rf = 0.41 (EtOAc/ hexane = 1:10);
1H NMR (400 MHz, CDCl3) δ 8.08 – 8.06 ( d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.5 Hz, 1H), 7.45 (t, J
= 7.4 Hz, 1H), 4.16 (q, J = 6.9 Hz, 2H), 3.80 (t, J = 7.6 Hz, 1H), 3.24 (d, J = 8.2 Hz, 2H), 2.24 (s,
3H), 1.21 (t, J = 7.2 Hz, 3H). ).13C NMR (100 MHz, CDCl3) δ 201.3, 168.5, 148.3, 140.3, 135.3,

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129.5, 123.7, 121.9, 61.9, 60.8, 33.2, 29.5, 14.0; HRMS (ESI) m/z: for C13H15NO5Na[M + Na]+,
calculated:288.0848; found: 288.0851.
Ethyl-2-(4-nitrobenzyl)-3-oxobutanoate (1e): Yield =79%, Rf = 0.41 (EtOAc/ hexane = 1:10),1H
NMR (400 MHz, CDCl3) δ 8.11 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 4.15 (q, J = 7.2, 2H),
3.78 (d, J = 7.0 Hz, 1H), 3.29-3.18 (m, 2H), 2.22 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 201.3, 168.5, 146.8, 146.1, 129.8, 123.7, 61.9, 60.6, 33.4, 29.5, 14.0; HRMS (ESI)
m/z: for C13H15NO5Na[M + Na]+, calculated:288.0848; found: 288.0847.
Ethyl-2-(naphthalen-1-ylmethyl)-3-oxobutanoate (1f): Yield =75%; Rf = 0.44 (EtOAc/ hexane
= 1:10); 1H NMR (400 MHz, , CDCl3) δ 7.99 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.74
(d, J = 7.9 Hz, 1H), 7.52 (m, 2H), 7.40 – 7.29 (m, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.96 (t, J = 7.3
Hz, 1H), 3.71-3.58 (m, 2H), 2.17 (s, 3H), 1.18 (t, J = 7.1Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
202.5, 169.4, 134.1, 133.9, 131.5, 129.0, 127.6, 127.2, 126.3, 125.7, 125.4, 123.2, 61.5, 60.1, 31.0,
29.7, 14.0; HRMS (ESI) m/z: for C17H18O3Na[M + Na]+ , calculated:293.1154; found: 293.1151.
Ethyl-2-(naphthalen-2-ylmethyl)-3-oxobutanoate (1g): Yield = 74%; Rf = 0.44 (EtOAc/ hexane
= 1:10); 1H NMR (400 MHz, , CDCl3) δ 7.80 – 7.76 (m, 3H), 7.63 (s, 1H), 7.47 – 7.41 (m, 2H),
7.31 (d, J = 8.5 Hz, 1H), 4.16 (q, J = 7.3 Hz, 2H), 3.88 (t, J = 7.6 Hz, 1H), 3.33 (d, J = 7.6 Hz,
2H), 2.20 (s, 3H), 1.19 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 202.4, 169.1, 135.7,
133.5, 132.3, 128.2, 127.6, 127.6, 127.4, 127.0, 126.1, 125.6, 61.5, 61.3, 34.1, 29.6, 14.0; HRMS
(ESI) m/z: for C14H18O3Na[M + Na]+ , calculated: 293.1154; found: 293.1150.
Ethyl-3-oxo-2-(thiophen-2-ylmethyl)butanoate (1h): Yield =88%; Rf = 0.6 (EtOAc/ hexane =
1:10); 1H NMR (400MHz, CDCl3) δ 7.13 (d, J = 5.2 Hz, 1H), 6.90 (dd, J = 5.1, 3.5 Hz, 1H), 6.81
(d, J = 3.4 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.81 (t, J = 7.5 Hz, 1H), 3.38 (d, J = 7.5 Hz, 2H),
2.24 (s, 3H), 1.25 (t, J = 7.2 Hz, 3H).13C NMR (150MHz, CDCl3) δ 201.9, 168.7, 140.3, 126.9,
125.9, 124.2, 61.7, 61.5, 29.7, 28.1, 14.0; HRMS (ESI) m/z: for C11H14O3SNa[M + Na]+,
calculated: 249.0561; found: 249.0564.
(E)-ethyl-2-acetyl-5-phenylpent-4-enoate (5a): Yield = 90%; Rf = 0.5(EtOAc/ hexane = 1:10);
1H NMR (400 MHz, CDCl3) δ 7.39 – 7.14 (m, 6H), 6.46 (d, J = 15.7 Hz, 1H), 6.16-6.08 (m, 1H),
4.20 (q, J = 7.2 Hz, 2H), 3.59 (t, J = 7.4 Hz, 1H), 2.75 (t, J = 7.3 Hz, 2H), 2.26 (s, 3H), 1.26 (t, J=
7.2 Hz, 3H).13C NMR (100 MHz, CDCl3) δ 202.4, 169.2, 137.0, 132.7, 128.5, 127.4, 126.2, 125.7,
61.5, 59.6, 31.5, 29.2, 14.1; HRMS (ESI) m/z: for C15H18O3Na[M + Na]+, calculated: 269.1154;
found: 269.1157.

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(E)-ethyl-2-acetyl-5-p-tolylpent-4-enoate (5b): Yield = 88%; Rf = 0.51(EtOAc/hexane = 1:10);
1H NMR (400 MHz, CDCl3)δ 7.21 (d, J = 8.2 Hz, 2H), 7.09 (d, J = 8.1 Hz, 2H), 6.42 (d, J = 15.8
Hz, 1H), 6.11 – 6.00 (m, 1H), 4.20 (q, J = 6.9 Hz, 2H), 3.61 – 3.55 (t, J = 7.3 Hz 1H), 2.73 (t, J =
7.4 Hz, 2H), 2.32 (s, 3H), 2.25 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H). 13C NMR (50 MHz, CDCl3) δ
202.5, 169.3, 137.2, 134.2, 132.6, 129.2, 126.1, 124.6, 61.45, 59.69, 31.57, 29.22, 21.14, 14.14;
HRMS (ESI) m/z: for C16H20O3Na[M + Na]+, calculated:283.1310; found: 283.1312.
(E)-ethyl-2-acetyl-5-(4-methoxyphenyl)pent-4-enoate (5c):Yield = 70%; Rf = 0.54(EtOAc/
hexane = 1:10); 1H NMR (500 MHz, CDCl3) δ 7.27 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H),
6.42 (d, J = 15.8 Hz, 1H), 6.02-5.96 (m, 1H), 4.22 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H), 3.59 (t, J =
7.4 Hz, 1H), 2.76 (t, J = 7.6 Hz, 2H), 2.28 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ 202.6,169.3, 159.1, 132.1, 129.8, 127.3, 123.4, 113.9, 61.5, 59.8, 55.3, 31.6, 29.2,14.2;
HRMS (ESI) m/z: for C16H20O4Na[M + Na]+, calculated:299.1259; found: 299.1262.
(E)-ethyl-2-acetyl-5-(4-bromophenyl)pent-4-enoate (5d):Yield = 75%; Rf = 0.52 (EtOAc/
hexane = 1:10); 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 7.3 Hz, 2H), 7.18 (d, J = 6.9 Hz, 2H),
6.39 (d, J = 15.7 Hz, 1H), 6.15 – 6.09 (m, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.58 (t, J = 7.3 Hz, 1H),
13
2.73 (t, J = 7.3 Hz, 2H), 2.26 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H). C NMR (150 MHz, CDCl3) δ
202.2, 169.1, 135.9, 131.6, 131.5, 127.7,126.7, 121.0, 61.5, 59.3, 31.4, 29.2, 14.1; HRMS (ESI)
m/z: for C15H17 BrO3Na[M + Na]+ , calculated: 347.0259 and 349.0238; found: 347.0262 and
349.024.
(E)-ethyl-2-acetyl-5-(4-chlorophenyl)pent-4-enoate (5e):Yield = 78%; Rf = 0.54 (EtOAc/
hexane = 1:10);1H NMR (400 MHz, CDCl3) δ 7.26-7.21 (m, 4H), 6.39 (d, J = 15.6 Hz, 1H), 6.14
– 6.04 (m, 1H), 4.19 (q, J = 7.3, 2H), 3.57 (t, J =7.2, 1H), 2.73 (t, J = 7.3 Hz, 2H), 2.25 (s, 3H),
1.27 (t, J =7.3, 3H). 13C NMR (100 MHz, CDCl3) δ 202.2, 169.1, 135.5, 133.0, 131.5, 128.6,
127.3, 126.5, 61.5, 59.4, 31.4, 29.2,14.1; HRMS (ESI) m/z: for C15H17ClO3Na[M + Na]+,
calculated:303.0764; found: 303.0761.
(E)-ethyl-2-acetyl-5-(3,5-dimethoxyphenyl)pent-4-enoate (5f):Yield = 75%; Rf = 0.48 (EtOAc/
hexane = 1:10); 1H NMR (400 MHz, CDCl3) δ 6.47 (s, 1H), 6.40 (s, 1H), 6.36 (s, 1H), 6.35 (d, J
= 2.1 Hz, 1H), 6.14 – 6.08 (m, 1H), 4.21 (q, J = 7.6, 2H), 3.78 (s, 6H), 3.58 (t, J =, 7.2,1H), 2.72
(t, J = 7.3 Hz, 2H), 2.25 (s, 3H), 1.26 (t, J = 6.1 Hz, 3H).13C NMR (100 MHz, CDCl3) δ 202.4,
169.2, 160.9, 139.0, 132.7, 126.3, 104.3, 99.7, 61.5, 59.5, 55.3, 31.4, 29.3, 14.1; HRMS (ESI) m/z:
for C17H22O5Na[M + Na]+, calculated:329.1365; found: 329.1362.

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(E)-ethyl-2-acetyl-5-(3-nitrophenyl)pent-4-enoate (5g): Yield = 78%; Rf = 0.52 (EtOAc/ hexane
= 1:10); 1H NMR (400 MHz , CDCl3) δ 8.16 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 7.7 Hz,
1H), 7.45 (t, J = 8.0 Hz, 1H), 6.51 (d, J = 15.6 Hz, 1H), 6.35 – 6.20 (m, 1H), 4.23 (q, J = 7.9 Hz,
2H), 3.61 (t, J = 5.0 Hz, 1H), 2.78 (t, J = 7.2 Hz, 2H), 2.27 (s, 3H), 1.26 (t, J = 7.3 Hz, 3H).13C
NMR (100 MHz, CDCl3) δ 201.9, 167.0, 148.6, 138.7, 132.0, 130.5, 129.4, 129.4, 122.0,
120.7,61.6, 59.1, 31.3, 29.2, 14.1; HRMS (ESI) m/z: for C15H17NO5Na[M + Na]+,
calculated:314.1004; found: 314.1002.
(E)-ethyl-2-acetyl-5-(4-nitrophenyl)pent-4-enoate (5h):Yield = 77%; Rf = 0.52 (EtOAc/ hexane
= 1:10); 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 8.7 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H), 6.52 (d,
J = 15.8 Hz, 1H), 6.40 – 6.28 (m, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.61 (t, J = 7.2 Hz, 1H), 2.79 (t, J
= 7.3 Hz, 2H), 2.27 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H).13C NMR (100 MHz, CDCl3) δ 201.9, 168.9,
146.8, 143.4, 131.1, 130.8, 126.6, 124.0,61.7, 59.0, 31.4, 29.2, 14.1; HRMS (ESI) m/z: for
C15H17NO5Na[M + Na]+ , calculated:314.1004; found: 314.1001.
(E)-ethyl-2-acetyl-5-(naphthalen-1-yl)pent-4-enoate (5i): Yield = 82%; Rf = 0.54 (EtOAc/
hexane = 1:10); 1H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 8.0 Hz, 1H), 7.87 – 7.78 (m, 1H), 7.74
(d, J = 8.1 Hz, 1H), 7.57 – 7.36 (m, 4H), 7.20 (d, J = 15.5 Hz, 1H), 6.21 – 6.02 (m, 1H), 4.19 (q, J
= 7.4, 2.1 Hz, 2H), 3.66 (t, J =7.3Hz, 1H), 2.86 (t, J = 7.4 Hz, 2H), 2.25 (s, 3H), 1.26 (t, J = 7.2
Hz, 3H).13C NMR (100 MHz, CDCl3) δ 202.4, 169.3, 134.8, 133.5, 131.0, 130.1, 129.0, 128.5,
127.8, 126.0, 125.7, 125.6, 123.8, 123.7, 61.4, 59.6, 31.9, 30.1, 14.1; HRMS (ESI) m/z: for
C19H20O3Na[M + Na]+ , calculated:319.1310; found: 319.1309.
(E)-ethyl-2-acetyl-5-(naphthalen-2-yl)pent-4-enoate (5j):Yield = 80%; Rf = 0.54(EtOAc/
hexane = 1:10); 1H NMR (600 MHz, CDCl3) δ 7.79 (dd, J = 14.6, 8.9 Hz, 4H), 7.56 (d, J = 8.6 Hz,
1H), 7.49 – 7.44 (m, 2H), 6.64 (d, J = 15.7 Hz, 1H), 6.28 (dt, J = 15.6, 7.2 Hz, 1H), 4.21 (q , J =
7.6, 2H), 3.66 (t, J = 7.3 Hz, 1H), 2.84 (t, J =7.2, 2H), 2.30 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H).13C
NMR (150 MHz, CDCl3) δ 202.5, 169.3, 134.4, 133.6, 132.9, 132.8, 128.2, 127.9, 127.6, 126.3,
126.1, 126.0, 125.8, 123.5, 61.6, 59.6, 31.7, 29.3, 14.2. HRMS (ESI) m/z: for C19H20O3Na[M +
Na]+ , calculated:319.1310; found: 319.1314.
(E)-ethyl-2-acetyl-5-(4-methoxynaphthalen-1-yl)pent-4-enoate (5k):Yield = 77%; Rf = 0.55
(EtOAc/ hexane = 1:10); 1H NMR (600 MHz,CDCl3) δ 7.33-7.27 (m, 2H), 7.25 (d,J = 7.9 Hz, 1H),
6.91 (t, J = 7.7 Hz, 2H), 6.83 (d,J = 6.5 Hz, 1H), 6.34 (d, J = 15.7 Hz, 1H), 5.87 (dt, J =14.4, 6.9
Hz, 1H), 4.22 (q, J = 6.8 Hz, 2H), 3.83 (s, 3H), 2.89 (t, J = 6.5 Hz, 1H), 2.51 – 2.39 (m, 2H),

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2.25(s,3H),1.30 (t, J = 6.9 Hz, 3H).13C NMR (150 MHz,CDCl3 ) δ 200.6, 168.4, 165.4, 154.2,
132.9, 131.6, 126.5, 126.2, 125.5, 124.8, 122.8, 122.7, 103.5, 102.8, , 62.8, 59.5, 55.4, 32.0, 28.2,
14.0; HRMS (ESI) m/z: for C20H22O4Na[M + Na]+ , calculated:349.1416; found: 349.1418.
(E)-ethyl-2-acetyl-5-(6-methoxynaphthalen-2-yl)pent-4-enoate (5l):Yield = 76%; Rf = 0.56
(EtOAc/ hexane = 1:10); 1H NMR (400 MHz, CDCl3) δ 7.69 – 7.64 (m, 2H), 7.60 (s, 1H), 7.49 (d,
J = 8.4 Hz, 1H), 7.13 – 7.09 (m, 2H), 6.58 (d, J = 15.7 Hz, 1H), 6.22 – 6.17 (m, 1H), 4.22 (q, J =
7.1 Hz, 2H), 3.90 (s, 3H), 3.62 (t, J = 7.5 Hz, 1H), 2.79 (t, J = 7.3 Hz, 2H), 2.27 (s, 3H), 1.27 (t, J
= 7.1 Hz, 3H). ). 13C NMR (100 MHz, CDCl3) δ 202.6, 169.3 , 157.7 , 134.0 , 132.8 , 132.4 , 129.4
, 129.0 , 127.0 , 125.8 , 125.0 , 124.0 , 119.0 , 105.8 , 61.5 , 59.6, 55.2 , 31.7 , 29.2 , 14.2 ; HRMS
(ESI) m/z: for C20H22O4Na[M + Na]+ , calculated:349.1416; found: 349.1419.
(E)-ethyl-2-acetyl-5-(2-methoxynaphthalen-1-yl)pent-4-enoate (5m):Yield = 70%; Rf = 0.54
(EtOAc/ hexane = 1:10); 1H NMR (600 MHz, CDCl3) δ 8.09 (d, J = 8.7 Hz, 1H), 7.78 (m, 2H),
7.47 (t, J = 7.8 Hz, 1H), 7.37 – 7.34 (m, 1H), 7.29 (d, J =8.1Hz,1H), 6.85 (d, J = 16.0 Hz, 1H),
6.18 – 6.12 (m, 1H), 4.27 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 3.73 (t, J = 7.6 Hz, 1H), 2.96-2.92 (m,
2H), 2.34 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 202.8, 169.4, 154.2,
132.5, 132.1, 129.2, 128.6, 128.2, 126.4, 125.7, 124.2, 123.5, 120.3, 113.2, 61.5, 59.8, 56.4, 32.7,
29.3, 14.2; HRMS (ESI) m/z: for C20H22O4Na[M + Na]+, calculated:349.1416; found: 349.1412.
(E)-ethyl-2-acetyl-5-(thiophen-2-yl)pent-4-enoate (5n):Yield = 90%; Rf = 0.6 (EtOAc/ hexane
= 1:10); 1H NMR (400 MHz, CDCl3) δ 7.11 (d, J = 5.0 Hz, 1H), 6.93 (dd, J = 5.1, 3.4 Hz, 1H),
6.88 (d, J = 3.4 Hz, 1H), 6.58 (d, J = 15.7 Hz, 1H), 5.98-5.91 (m, 1H), 4.21 (q, J = 7.1 Hz, 2H),
3.57 (t, J = 7.4 Hz, 1H), 2.71 (t, J = 7.5 Hz, 1H), 2.26 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H).13C NMR
(100 MHz, CDCl3) δ 202.3, 169.1, 142.0, 127.3, 125.9, 125.4, 125.2, 123.9, 61.5, 59.4, 31.3, 29.3,
14.1; HRMS (ESI) m/z: for C13H16O3SNa[M + Na]+, calculated:275.0718; found: 275.0722.
Growing condition for Klebsiellapneumoniae(NBRC 3319) and synthesis of enantiopureα-
substituted β-hydroxy esters:
The dried cells obtained from the culture collection are moistened with the rehydration fluid (10 g
peptone, 2 g yeast extract, 1 g MgSO4.7H2O, 1 L distilled water, pH 7.0). It was streaked into
several petridishes (containing the same components with agar 15.0 g/L is added) and then
incubated at 35 °C in an incubator for 24 h.For the biotransformation purpose with NBRC 3319, a

Organic & Biomolecular Chemistry
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liquid medium (40.0 g glucose, 5.0 g meat extract, 5.0 g NaCl, 10.0 g peptone, 15.0 g CaCO3 in
1L of distilled water) was prepared without agar, and then the grown cells of K. pneumoniae was
transferred to this medium through an inoculating loop. The content was incubated in an incubator
shaker for 48 h, and after that, the parent β-ketoester was directly added to the growing culture
medium. The reaction was occasionally monitored by TLC analysis. After completion of the
reaction, the product was isolated by extraction with EtOAc several times. It was purified through
silica gel chromatography (1: 5, EtOAc/hexane) to afford the product alcohols (6a-6h and 7a-7n)
in 60-82% yield in respective cases.
(2R,3S)-ethyl-3-hydroxy-2-(4-methylbenzyl)butanoate (6a): Yield = 82%; Rf = 0.3 (EtOAc/
hexane = 1:5); [α]D25 = 34.86 (c 1.0, CHCl3); IR(neat), ν = 3470, 2980, 2950, 1740 cm-1; 1H NMR
(600 MHz, CDCl3) δ 7.11 – 7.08 (m, 4H), 4.09 – 4.04 (m, 3H), 2.97 – 2.94 (m, 3H), 2.33 (s, 3H),
1.28 (d, J = 6.3 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 174.5, 136.0,
135.8, 129.1, 128.7, 68.1, 60.5, 54.4, 33.2, 21.0, 20.4, 14.1; HRMS (ESI) m/z: for C14H20O3Na[M
+ Na]+ , calculated: 259.1310;found: 259.1312.
(2R,3S)-ethyl-2-(4-bromobenzyl)-3-hydroxybutanoate (6b):Yield = 70%; Rf = 0.33(EtOAc/
hexane = 1:5); [α]D25 = 38.45 (c 1.0, CHCl3); IR(neat), ν = 3478, 2980, 2950, 1742 cm-1; 1H NMR
(600 MHz, CDCl3) δ 7.41 (d, J = 6.7 Hz, 2H), 7.08 (d, J = 6.8 Hz, 2H), 4.09 – 4.04 (m, 3H), 2.95
(d, J = 7.7 Hz, 2H), 2.75 – 2.70(m, 1H), 1.28 (d, J = 4.6 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H).13C NMR
(150 MHz, CDCl3) δ 174.2,138.2,131.5,130.6,120.2,68.0, 60.7, 54.2, 32.9, 20.5, 14.1; HRMS
(ESI) m/z: for C13H17BrO3Na[M+Na]+,calculated: 323.0259 and 325.0238; found:
323.0258,325.0240.
(2R,3S)-ethyl-2-(4-chlorobenzyl)-3-hydroxybutanoate (6c):Yield = 74%; Rf = 0.33(EtOAc/
hexane = 1:5); [α]D25 = 28.09 (c1.0,CHCl3); IR(neat),ν = 3470,2987,2950,1747 cm-1; 1H NMR (600
MHz, CDCl3) δ 7.25 (d, J = 8.3 Hz, 2H), 7.13 (d, J = 8.3 Hz, 2H), 4.06 (q, J = 6.8 Hz, 2H), 3.90-
3.86 (m,1H), 2.96 (d, J = 9.1 Hz, 2H), 2.73-2.69 (m, 1H), 1.27 (d, J = 6.4 Hz, 3H), 1.13 (t, J = 7.1
Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 174.2, 137.7, 132.1, 130.2, 128.5, , 68.0, 60.7, 54.3, 33.0,
20.6, 14.1; HRMS (ESI) m/z: for C13H17ClO3Na[M + Na]+, calculated: 279.0764; found: 279.0767.
(2R,3S)-ethyl-3-hydroxy-2-(3-nitrobenzyl)butanoate (6d): Yield = 72%; Rf = 0.31(EtOAc/
hexane = 1:5); [α]D25 = 33.36 (c 1.0, CHCl3); IR(neat), ν = 3415, 3000, 2930, 1752,1660,1530,1350

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DOI: 10.1039/C9OB01681C
cm-1; 1H NMR (600 MHz, CDCl3) δ 7.07 (t, J = 7.5 Hz, 1H), 6.96 – 6.88 (m, 1H), 6.59 – 6.52 (m,
2H), 4.18 (q, J = 7.3 Hz, 2H), 3.79-3.76 (m, 1H), 3.73-3.67 (m, 1H), 3.09 (d, J = 7.6 Hz, 2H), 1.26-
1.22(m, 6H).13C NMR (150 MHz,CDCl3) δ 171.0, 146.5, 139.4, 129.5, 129.1, 124.9, 122.8, 61.5,
61.2, 34.0, 29.8, 17.0, 14.0; HRMS (ESI) m/z: for C13H17NO5Na[M + Na]+ , calculated: 290.1004;
found: 290.1007.
(2R,3S)-ethyl-3-hydroxy-2-(4-nitrobenzyl)butanoate (6e): Yield =76%;Rf =0.31(EtOAc/
hexane = 1:5); [α]D25 = 34.20 (c1.0,CHCl3); IR(neat), ν = 3410, 3000, 2930, 1759, 1660,1530,1350
cm-1; 1H NMR (600 MHz, CDCl3) δ 8.16 (d, J = 8.6 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 4.11 – 4.03
(m, 3H), 3.13 – 3.06 (m, 3H), 1.31 (d, J = 6.3 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H).13C NMR (150
MHz, CDCl3) δ 173.6, 147.3, 129.8,123.7, 115.3,68.1, 60.8, 54.0, 33.4, 20.1, 14.1. HRMS (ESI)
m/z: for C13H17NO5Na[M + Na]+ , calculated: 290.1004; found: 290.1006.
(2R,3S)-ethyl3-hydroxy-2-(naphthalen-1-ylmethyl)butanoate (6f):
Yield = 72%; Rf= 0.34(EtOAc/hexane = 1:5);[α]D25 = 42.15(c1.0,CHCl3); IR(neat),ν = 3467, 3034,
2810, 1750,1627 cm-1; 1H NMR (600 MHz, CDCl3) δ 8.05 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 7.6 Hz,
1H), 7.75 (d, J = 8.1 Hz, 1H), 7.57 – 7.53 (t,J=7.2Hz, 1H), 7.50 (t, J = 7.4 Hz, 1H), 7.38 (t, J = 7.6
Hz, 1H), 7.34 (d, J = 7.4Hz, 1H), 4.21 (q, J = 7.0 Hz, 2H), 3.98-3.94 (m,1H), 2.96-2.88 (m,2H),
2.53 – 2.42 (m, 1H), 1.38 (d, J = 6.3 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz,CDCl3
) δ 174.7, 135.2, 133.9, 131.8, 129.0, 128.9, 128.6, 127.3, 127.0, 125.6, 123.5, , 68.3, 60.7, 53.4,
30.7, 20.6, 13.9; HRMS (ESI) m/z: for C17H20O3Na[M + Na]+, calculated: 295.1310; found:
295.1315.
(2R,3S)-ethyl-3-hydroxy-2-(naphthalen-2-ylmethyl)butanoate (6g): Yield = 78%;Rf = 0.34
(EtOAc/ hexane = 1:5); [α]D25 = 39.16 (c 1.0, CHCl3); IR (neat),ν = 3445, 2980,1730 cm-1; 1H
NMR (600 MHz, CDCl3) δ 7.88 – 7.73 (m, 3H), 7.65(s 1H), 7.49 – 7.44 (m, 2H), 7.37 – 7.33 (m,
1H), 4.20 (q, J = 7.1 Hz, 1H), 4.11 (q, J = 6.5 Hz, 1H ), 4.06-4.02 (m, 1H), 3.17 (d, J = 6.1 Hz,
2H), 2.91 – 2.86 (m, 1H), 1.30 (d, J = 6.8 Hz, 3H), 1.06 (t, J = 7.1 Hz, 3H).13C NMR (150
MHz,CDCl3) δ 174.5, 136.7, 133.5, 128.6, 128.0, 127.6, 127.5, 127.3, 127.2, 126.0, 125.4, 77.2,
77.0, 76.8, 68.1, 60.6, 54.2, 33.7, 20.5, 14.0; HRMS (ESI) m/z: for C17H20O3Na[M + Na]+,
calculated: 295.1310; found: 295.1314.

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(2R,3S)-ethyl-3-hydroxy-2-(thiophen-2-ylmethyl)butanoate (6h):Yield = 80%; Rf = 0.42
1
(EtOAc/ hexane = 1:5); [α]D25 = 37.85(c 1.0, CHCl3); IR (neat),ν = 3460, 2990,1740 cm-1; H
NMR (400 MHz, CDCl3) δ 7.12 (d, J = 5.1 Hz, 1H), 6.89 (dd, J = 5.1, 3.4 Hz, 1H), 6.81 (d, J =
2.2 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 4.08-4.01 (m, 1H), 3.28 – 3.14 (m, 2H), 2.79-2.74 (m, 1H),
1.26 (d, J = 6.3 Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 173.9, 141.6,
126.7, 125.5, 123.8, 68.0, 60.7, 54.8, 27.9, 20.5, 14.1; HRMS (ESI) m/z: for C11H16O3SNa[M +
Na]+, calculated: 251.0718; found: 251.0719.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-phenylpent-4-enoate (7a):Yield = 78%;Rf = 0.4 (EtOAc/
hexane = 1:5); [α]D25 = 27.90 (c 1.0, CHCl3);IR (neat),ν= 3400, 3050, 3010-2970, 1742 cm-1; 1H
NMR (400 MHz, , CDCl3) δ 7.30 (q, J = 8.1 Hz, 4H), 7.20 (t, J = 7.1 Hz, 1H), 6.44 (d, J = 15.8
Hz, 1H), 6.20 – 6.15 (m, 1H), 4.16 (q, J = 7.2 Hz, 2H), 4.09 – 4.04 (m, 1H), 2.63 – 2.54 (m, 3H),
1.27-1.21 (m, 6H).13C NMR (100 MHz, CDCl3) δ 174.5, 137.3, 132.0, 128.5, 127.2, 127.2, 126.1,
67.9, 60.6, 52.3, 31.1, 20.4, 14.3; HRMS (ESI) m/z: for C15H20O3Na[M + Na]+ , calculated:
271.1310; found: 271.1311.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-p-tolylpent-4-enoate (7b):Yield
=
75%; Rf
=
0.42(EtOAc/ hexane = 1:5); [α]D25 = 31.68 (c1.0,CHCl3); IR(neat), ν = 3480, 2980, 2940, 1745 cm-
1. 1H NMR (600 MHz,CDCl3) δ 7.24 (d, J = 7.8 Hz, 2H), 7.12 (d, J = 7.9 Hz, 2H), 6.43 (d, J = 15.9
Hz, 1H), 6.16 – 6.12 (m, 1H), 4.19 (q,J = 7.1Hz, 2H), 4.11-4.07 (m, 1H), 2.62 – 2.56 (m, 3H), 2.34
(s, 3H), 1.30-1.25 (m, 6H).13CNMR (150 MHz,CDCl3) δ 174.5, 136.9, 134.6, 132.2, 131.8, 129.2,
126.0, 67.93, 60.6, 52.3, 31.0, 21.1, 20.4, 14.3; HRMS (ESI) m/z: for C16H22O3Na[M + Na]+ ,
calculated: 285.1467; found: 285.1465.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-(4-methoxyphenyl)pent-4-enoate (7c):Yield = 70%; Rf=
0.39 (EtOAc/ hexane = 1:5); [α]D25 = 19.56 (c 1.0, CHCl3); IR (neat),ν = 3410, 2995,1730 cm-1;1H
NMR (600 MHz, CDCl3) δ 7.25 (d, J = 7.3 Hz, 2H), 6.84 (d, J = 9.1 Hz, 2H), 6.34 (d, J = 15.6 Hz,
1H), 5.87 (dt, J = 14.7, 7.2 Hz, 1H), 4.24 – 4.18 (m, 3H), 3.83 (s, 3H), 2.51 – 2.34 (m, 3H), 1.31-
1.28 (m, 6H).13C NMR (150 MHz,CDCl3) δ 173.0, 158.9, 131.9, 128.1, 127.1, 124.7, 114.0, 64.3,
61.4, 55.3, 46.7, 32.0, 30.2, 14.1; HRMS (ESI) m/z: for C16H22O4Na[M + Na]+, calculated:
301.1416; found: 301.1414.

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(R,E)-ethyl-5-(4-bromophenyl)-2-((S)-1-hydroxyethyl)pent-4-enoate (7d):Yield = 74%; Rf =
1
0.43(EtOAc/ hexane = 1:5); [α]D25 = 22.80 (c 1.0, CHCl3); IR (neat),ν = 3420, 3002,1742 cm-1; H
NMR (400 MHz, CDCl3) δ 7.40 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.1 Hz, 2H), 6.37 (d, J = 15.8 Hz,
1H), 6.17 (dt, J = 14.7, 5.3 Hz, 1H), 4.16 (q,J=7.2Hz, 2H), 4.09-4.03 (m, 1H), 2.69-2.57 (m, 3H),
1.29 – 1.21 (m, 6H).13C NMR (100 MHz, CDCl3) δ 174.4, 136.2, 131.6, 130.8, 128.1, 127.6, 120.9,
67.9, 60.7, 52.1, 30.9, 20.4, 14.3; HRMS (ESI) m/z: for C15H19BrO3Na[M + Na]+ , calculated:
349.0415 and 351.0395; found: 349.0417 and 351.0398.
(R,E)-ethyl-5-(4-chlorophenyl)-2-((S)-1-hydroxyethyl)pent-4-enoate (7e):Yield = 75%; Rf
=0.43(EtOAc/ hexane = 1:5); [α]D25 = 24.86 (c 1.0, CHCl3); IR (neat),ν = 3430, 3010,1740 cm-1;
1H NMR (400 MHz,CDCl3) δ 7.25 (d, J = 7.7 Hz, 4H), 6.38 (d, J = 15.6 Hz, 1H), 6.23 – 6.07 (m,
1H),4.16 (q, J = 6.9, Hz, 2H), 4.09-4.00 (m,1H), 2.95-2.87 (m,1H), 2.57 (t, J = 5.4 Hz, 2H), 1.29-
1.20 (m, 6H).13C NMR (100 MHz,CDCl3) δ 174.0, 137.7, 135.8, 132.8, 130.8, 130.2, 128.6, ,
127.9, 127.3, 67.9, 60.7, 52.2, 31.0, 20.5, 14.3. HRMS (ESI) m/z: for C15H19ClO3Na[M + Na]+,
calculated: 305.0920; found: 305.0921.
(R,E)-ethyl-5-(3,5-dimethoxyphenyl)-2-((S)-1-hydroxyethyl)pent-4-enoate (7f): Yield = 60%;
Rf = 0.38 (EtOAc/ hexane = 1:5); [α]D25 = 33.04 (c1.0, CHCl3); IR (neat),ν = 3428, 2988,1740
cm-1; 1H NMR (600 MHz, CDCl3) δ 7.34 (q, J = 16.9 Hz, 1H), 6.51 (s, 2H),
6.37(d,J=15.6Hz.1H),6.20 – 6.13 (m, 1H), 4.19 (q, J = 7.4 Hz, 2H),4.10-3.99 (m, 1H), 3.81 (s,
6H), 2.62 -2.57 (m, 3H), 1.29 – 1.26 (m, 6H).13C NMR (150 MHz, CDCl3) δ 174.4, 160.9, 139.4,
132.0, 127.8, 104.2, 99.5, 67.9, 60.6, 55.3, 52.2, 30.9, 20.4, 14.3; HRMS (ESI) m/z: for
C17H24O5Na[M + Na]+ , calculated: 331.1521; found: 331.1523.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-(3-nitrophenyl)pent-4-enoate (7g):Yield = 78%, Rf
=0.38 (EtOAc/ hexane = 1:5) [α]D25 = +39.13 (c 1.0, CHCl3); IR (neat),ν = 3430, 2990,1730,1525,
1
1490, 1350 cm-1; H NMR (600 MHz, CDCl3) δ 8.20 (s, 1H), 8.08 (d, J = 8.3 Hz, 1H), 7.63 (d, J
= 7.8 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 6.52 (d, J = 15.3 Hz, 1H), 6.37 (dt, J = 15.6, 6.9 Hz, 1H),
4.20 (q, J = 6.9, 3.5 Hz, 2H), 4.14 – 4.09 (m, 1H), 2.66 – 2.61 (m, 2H), 2.46-2.36 (m, 1H), 1.29 –
1.25 (m, 6H).13C NMR (150 MHz, CDCl3) δ 174.2, 148.6, 139.0, 131.9, 130.9, 129.8, 129.4, 121.8,
120.6, 67.9, 60.8, 52.0, 30.9, 20.6, 14.3; HRMS (ESI) m/z: for C15H19NO5Na[M + Na]+ ,
calculated: 316.1161; found: 316.1163.

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(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-(4-nitrophenyl)pent-4-enoate (7h):Yield = 75%; Rf =
25
0.38 (EtOAc/ hexane = 1:5); [α]D = 42.64 (c 1.0, CHCl3); IR (neat), ν = 3430, 2990,1740,
1510,1480,1352 cm-1; 1H NMR (600 MHz, CDCl3)) δ 8.19 (d, J = 8.8Hz, 1H), 7.47 (d, J = 8.6Hz,
1H), 7.36 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 8.1Hz, 1H), 6.53 (d, J= 15.9 Hz, 1H), 6.45 – 6.40 (m,
1H), 4.19 (q, J = 7.1Hz, 2H), 4.13 – 4.09 (m, 1H), 2.70 – 2.61 (m, 3H), 1.29(d, J = 7.1 Hz, 3H) ,
1.25 (t, J = 6.9 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 174.2, 146.7, 143.7, 132.7, 129.4, 128.6,
127.3, 126.6, 124.0, 67.9, 60.8, 51.9, 31.1, 20.6, 14.3; HRMS (ESI) m/z: for C15H19NO5Na[M +
Na]+, calculated: 316.1161;found: 316.1162.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-(naphthalen-1-yl)pent-4-enoate (7i):Yield = 80%;Rf=
1
0.42(EtOAc/ hexane = 1:5); [α]D25 = 14.26(c 1.0, CHCl3); IR (neat),ν = 3446, 2992,1735 cm-1; H
NMR (600 MHz, CDCl3) δ 8.10 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 8.2 Hz,
1H), 7.52 (dq, J = 15.0, 7.2 Hz, 3H), 7.45 (t, J = 7.7 Hz, 1H), 7.21 (d, J = 15.6 Hz, 1H), 6.23 (dt,
J = 14.5, 6.9 Hz, 1H), 4.22 – 4.11 (m, 3H), 2.75-2.69 (m, 3H), 1.32 (d, J = 6.3 Hz, 3H), 1.27 (q, J
= 6.9 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 174.5, 135.2, 133.6, 131.1, 130.5, 129.3, 128.5,
127.6, 125.9, 125.7, 125.6, 123.8, 123.7, 68.0, 60.7, 52.4, 31.4, 20.5, 14.3; HRMS (ESI) m/z: for
C19H22O3Na[M + Na]+, calculated: 321.1467; found; 321.1469.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-(naphthalen-2-yl)pent-4-enoate (7j):Yield = 76%;Rf =
0.42 (EtOAc/hexane = 1:5); [α]D25 = 28.87 (c 1.0, CHCl3); IR (neat),ν = 3428, 3000,1738 cm-1; 1H
NMR (600 MHz, CDCl3) δ 7.87 (dq, J = 7.4, 3.2 Hz, 4H), 7.53 – 7.49 (m, 3H), 6.64 (d, J = 15.7
Hz, 1H), 6.35 – 6.29 (m, 1H), 4.24 – 4.12 (m, 3H), 2.67 – 2.64 (m, 3H), 1.34 – 1.25 (m, 6H). 13C
NMR (150 MHz, CDCl3) δ 174.8, 167.4, 166.0, 132.5, 128.8, 128.3, 128.1, 127.7, 126.3, 126.1,
125.7, 125.4, 124.1,67.9, 59.5, 33.1, 29.5, 21.6, 14.0; HRMS (ESI) m/z: for C19H22O3Na[M + Na]+,
calculated: 321.1467; found; 321.1468.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-(4-methoxynaphthalen-1-yl)pent-4-enoate (7k): Yield =
25
75%; Rf = 0.39 (EtOAc/hexane = 1:5); [α]D = 17.80 (c 1.0, CHCl3); IR (neat),ν = 3450,
2990,1738 cm-1; 1H NMR (600 MHz, CDCl3) δ 7.35-7.24 (m,2H), 7.24 (d, J = 7.9 Hz, 1H), 6.91
(t, J = 7.7 Hz, 2H), 6.53 (d,J =7.2Hz, 1H), 6.34 (d, J = 15.7 Hz, 1H), 5.91 – 5.81 (dt, J = 15.9, 5.6
Hz, 1H), 4.21 (q, J = 7.2 Hz,2H), 3.93 – 3.86 (m, 1H), 3.83 (s, 3H), 2.51-2.41 (m, 1H), 2.38 – 2.31
(m, 2H), 1.30 (t, J = 3.4 Hz, 3H ), 1.24 (d, J = 6.2 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 173.0,

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167.2, 159.4, 158.9, 134.5, 131.9, 130.2, 129.0, 128.1, 127.1, 124.7, 114.0, 113.9, 64.3, 61.4, 55.3,
46.7, 32.0, 22.4, 14.1.HRMS (ESI) m/z: for C20H24O4Na[M + Na]+, calculated: 351.1572; found:
351.1574.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-(6-methoxynaphthalen-2-yl)pent-4-enoate (7l): Yield =
74%; Rf = 0.4(EtOAc/hexane = 1:5); [α]D2 5= 18.20 (c 1.0, CHCl3); IR (neat),ν = 3448, 2990,1744
cm-1; 1H NMR (600 MHz, CDCl3) δ 7.86 (dd, J = 6.3, 2.9 Hz, 1H), 7.52 – 7.49 (m, 1H), 7.33 (d,
J = 7.5 Hz, 2H), 7.25 (d, J = 7.7 Hz, 2H), ), 6.47 (d, J = 15.9 Hz, 1H), 6.32 (dt, J = 15.9, 5.6 Hz,
1H), 4.23-4.18 (m 3H), 3.89 (s,3H), 2.52 – 2.38 (m, 3H), 1.29 (t, J= 3.6 Hz, 3H), 1.25 (d, J = 6.3
Hz, 3H).13C NMR (150 MHz,CDCl3 ) δ 176.7, 157.5, 140.0, 136.8, 133.4, 131.6, 129.4, 128.6,
127.3, 126.6, 124.5, 119.4, 116.3, 67.6, 59.9, 55.3, 51.1, 29.7, 19.8, 14.1; HRMS (ESI) m/z: for
C20H24O4Na[M + Na]+ , calculated: 351.1572; found: 351.1575.
(R,E)-ethyl2-((S)-1-hydroxyethyl)-5-(2-methoxynaphthalen-1-yl)pent-4-enoate(7m): Yield
25
=72%,Rf = 0.41 (EtOAc/hexane = 1:5); [α]D = 21.20 (c 1.0, CHCl3); IR (neat),ν =
3452,2998,2972,1740 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 8.7 Hz, 1H), 7.77 (dt, J =
17.0, 8.6 Hz, 3H), 7.44 (t, J = 7.8 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 6.81 (d, J = 16.0 Hz, 1H), 6.17
(dt, J = 15.5, 7.0 Hz, 1H), 4.18 (m, 3H), 3.93 (s, 3H), 2.79 – 2.69 (m, 3H), 1.30 (t, J = 6.4 Hz, 3H),
1.26 (d, J = 2.7 Hz, 3H).13C NMR (150 MHz,CDCl3 ) δ 174.6, 154.1, 146.6, 133.7, 128.5, 128.2,
126.3, 125.1, 124.3, 123.4, 113.2, 68.2, 60.7, 56.4, 52.4, 29.7, 20.5, 14.1; HRMS (ESI) m/z: for
C20H24O4Na[M + Na]+ , calculated: 351.1572; found: 351.1573.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-(thiophen-2-yl)pent-4-enoate (7n):Yield = 82%; Rf = 0.5
(EtOAc/hexane = 1:5); [α]D25 = 45.92 (c 1.0, CHCl3); IR (neat), ν = 3440,2998,2972,1748,1437
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.10 (d, J = 5.0 Hz, 1H), 6.95 – 6.90 (t, J = 4.7Hz,1H), 6.87
(d, J = 3.5 Hz, 1H), 6.56 (d, J = 15.6 Hz, 1H), 6.05 – 5.97 (m, 1H), 4.16 (q, J = 7.1Hz, 2H), 4.08
– 4.03 (m, 1H), 2.60 – 2.51 (m, 3H), 1.26(t, J = 6.9Hz, 3H),1.24 (d, J = 7.1Hz, 3H).13C NMR (100
MHz, CDCl3) δ 174.4, 142.4, 127.2, 127.0, 125.2, 124.8, 123.6, 67.8, 60.7, 52.1, 30.8, 20.4, 14.3;
HRMS (ESI) m/z: for C13H18O3SNa[M + Na]+, calculated: 277.0874; found: 277.0876.
(R,E)-ethyl-2-((S)-1-hydroxyethyl)-5-phenylpent-4-enoate (7a) (prepared through cross
metathesis reaction): Styrene (100 mg, 0.096 mmol) and (R)-ethyl 2-((S)-1-hydroxyethyl)pent-
4-enoate (82mg, 0.048mmol) were added in a two necked round bottom flask in dry DCM (3 mL).

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The reaction mixture was purged with argon to remove any trace amount of dissolved oxygen.
Grubbs 2nd Generation catalyst (5mol% with reference to styrene, 5 mg) was added to the mixture
and the reaction mixture was stirred at 40°C for 24 h until the aliphatic olefin consumed
completely. The remaining solvent was then evaporated under reduced pressure and the crude
material was purified through flash column chromatography (EtOAc/hexane = 1: 10) to afford the
compound 7a as colourless liquid in 70 % yield with respect to the aliphatic olefin. [α]D25 = 28.52
(c 1.0, CHCl3).
General procedure for iodoetherification reaction for 7a and its analogues :To a solution of
compound 7 (1eq) in Et2O:H2O (1:4), I2( 1.2eq) and NaHCO3 (2eq) was added at -10 °C, and the
reaction mixture stirred at same temperature for 3 h, and then it was quenched with saturated
Na2SO3and extracted with ether. The organic layer washed with brine and dried over anhydrous
Na2SO4. The solvent was evaporated under reduced pressure, and the product was purified by flash
column chromatography (EtOAc/hexane = 1: 60) to furnish 8a and related compounds.
(2S,3R,5S,6R)-ethyl-5-iodo-2-methyl-6-phenyltetrahydro-2H-pyran-3-carboxylate
(8a):Yield = 75%;Rf = 0.2 (EtOAc/ hexane = 1 :60); [α]D25 = -49.98 (c 1.0, CHCl3); IR (neat), ν =
3036, 2872, 1742, 1132 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.37-7.34 (m, 5H), 4.53 (d, J = 10.5
Hz,1H), 4.20 – 4.10 (m, 3H), 3.95 – 3.88 (m, 1H), 2.86-2.80 (m,1H), 2.57 (dd, J = 11.0, 8.7 Hz,
2H), 1.29 (t, J = 7.1 Hz, 3H), 1.23 (d, J = 6.1 Hz, 3H).13C NMR (100 MHz, CDCl3) δ 171.5, 139.8,
128.7, 128.3, 127.6, 85.9, 75.7, 60.9, 52.3, 41.2, 29.7, 20.1, 14.2; HRMS (ESI) m/z: for
C15H19IO3Na[M + Na]+ , calculated: 397.0277; found: 397.0278.
(2S,3R,5S,6R)-ethyl-6-(4-chlorophenyl)-5-iodo-2-methyltetrahydro-2H-pyran-3-carboxylate
(8b):Yield =73%; Rf =0.23 (EtOAc/ hexane = 1 :60); [α]D25 = -28.76 (c 1.0, CHCl3); IR (neat),ν =
3032, 2878, 1744, 1136 cm-1;1H NMR (400 MHz, CDCl3) δ 7.34 – 7.29 (m, 4H), 4.50 (d, J = 10.5
Hz, 1H), 4.17 (q, J = 7.1 Hz, 2H), 4.07 – 4.01 (m, 1H), 3.93-3.86 (m, 1H), 2.84 – 2.81 (m, 1H),
2.55 (t, J = 8.9 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H), 1.22 (d, J = 6.0 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δ 171.4, 138.4, 134.4, 129.0, 128.5, 85.1,75.8, 60.9, 52.2, 41.1, 29.3, 20.1, 14.2; HRMS
(ESI) m/z: for C15H18ClIO3Na[M + Na]+, calculated: 430.9887; found: 430.9888.
(2S,3R,5S,6R)-ethyl-6-(4-bromophenyl)-5-iodo-2-methyltetrahydro-2H-pyran-3-
25
carboxylate (8c):Yield = 70%;Rf = 0.23 (EtOAc/ hexane = 1 :60); [α]D = -25.00 (c 1.0,

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CHCl3);IR (neat),ν = 3038, 2865, 1734, 1135 cm-1; 1H NMR (600 MHz, CDCl3) δ 7.51 (d, J = 8.3
Hz, 2H), 7.29 (d,J = 7.8Hz, 2H), 4.52 (d, J = 10.5 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 4.08 – 4.04
(m, 1H), 3.94 – 3.90 (m, 1H), 2.87 – 2.84 (m, 1H), 2.58 (dd, J = 22.1, 11.5 Hz, 2H), 1.31 (t, J =
7.2 Hz, 3H), 1.24 (d, J = 6.2 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 171.4, 138.8, 131.4, 129.3,
122.6, 85.2, 75.8, 60.9, 52.2, 41.1, 29.2, 20.1, 14.2; HRMS (ESI) m/z: for C15H18BrIO3Na[M +
Na]+ , calculated: 474.9382; found: 474.9384.
(2S,3R,5S,6R)-ethyl-5-iodo-2-methyl-6-(naphthalen-1-yl)tetrahydro-2H-pyran-3-
carboxylate (8d): Yield = 68%;Rf= 0.24 (EtOAc/ hexane = 1 :60); [α]D25 = -36.30 (c 1.0, CHCl3);
IR (neat),ν = 3034, 2868, 1738, 1130 cm-1; 1H NMR (600 MHz, CDCl3) δ 7.90 – 7.85 (m, 3H),
7.58 – 7.56 (m, 3H), 7.51 (d, J = 8.3Hz, 1H), 5.02 (d, J = 17.1 Hz, 1H), 4.25-4.20 (m, 3H), 4.11-
4.06 (m, 1H), 2.99 – 2.96 (m, 1H), 2.73 (t, J = 12.2 Hz, 2H), 1.36-1.31 (m, 6H).13C NMR (150
MHz, CDCl3) δ 171.6, 139.3, 129.4, 128.9, 126.1, 125.9, 125.6, 124.1, 123.5, 115.9, 114.1,76.1,
60.9, 52.5, 41.6, 29.7, 29.7, 20.2, 14.2; HRMS (ESI) m/z: for C19H21IO3Na[M + Na]+ , calculated:
447.0433; found: 447.0436.
(2S,3R,5S,6R)-ethyl-5-iodo-2-methyl-6-(thiophen-2-yl)tetrahydro-2H-pyran-3-carboxylate
(8e) :Yield = 80%; Rf = 0.26(EtOAc/ hexane = 1 :60);[α]D25 = -47.98 (c 1.0, CHCl3); IR (neat),ν =
3036, 2865, 1730, 1120 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 5.1 Hz, 1H), 7.11 (d, J=
3.3 Hz, 1H), 6.97 (dd, J = 5.2, 3.5 Hz, 1H) , 4.84 (d, J = 10.4 Hz, 1H), 4.19 – 4.10 (m, 3H), 3.95–
3.88 (m, 1H), 2.84 – 2.81 (m, 1H), 2.55 (dd, J = 11.2, 8.7 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H), 1.23
(d, J = 6.1 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 171.4, 142.8, 127.1, 126.2, 125.4, 81.2, 75.9,
60.9, 52.0, 41.2, 30.5, 20.1, 14.2; HRMS (ESI) m/z: for C13H17IO3SNa[M + Na]+, calculated:
402.9841; found: 402.9843.
General procedure for reductive deiodination reaction for 8a and its analogues
To a solution of compound 8a (1eq) in toluene, AIBN(0.2eq) and n-Bu3SnH (2eq) was added
sequentially and the reaction mixture stirred at 115°C for 30 min, and then it was quenched with
solid KF and filtered with ether. The combined organic layer was dried over anhydrous Na2SO4.
The solvent was evaporated under reduced pressure, and the product was purified by flash column
chromatography (EtOAc/hexane = 1: 60) to furnish compounds9a-9e.
(2S,3R,6S)-ethyl-2-methyl-6-phenyltetrahydro-2H-pyran-3-carboxylate (9a): Yield = 68%;
Rf = 0.23 (EtOAc/ hexane = 1 :60); [α]D25 = -35.76 (c1.0, CHCl3); IR (neat),ν = 3034, 2870, 1738,