Biocatalytic Asymmetric Reduction of γ-Keto Esters to Access Optically Active γ-Aryl-γ-butyrolactones

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

FULL PAPER

DOI: 10.1002/adsc.201 XXX.

Biocatalytic Asymmetric Reduction of γ-Keto Esters to Access

Optically Active γ-Aryl-γ-butyrolactones

a,

a

b

Paweł Borowiecki, * Natalia Telatycka, Mateusz Tataruch, Anna Żądło-

c

d

e

b

Dobrowolska, Tamara Reiter, Karola Schühle, Johann Heider, Maciej Szaleniec and

d

Wolfgang Kroutil

a

Warsaw University of Technology, Faculty of Chemistry, Department of Drugs Technology and Biotechnology,

Koszykowa 3, 00–664 Warsaw, Poland.

E-mail: pawel_borowiecki@onet.eu; pborowiecki@ch.pw.edu.pl

Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS, Niezapominajek 8, 30–239 Krakow, Poland.

Institute of Organic Chemistry Polish Academy of Sciences, Kasprzaka 44/52, 01–224 Warsaw, Poland.

Institute of Chemistry, University of Graz, NAWI Graz, BioTechMed Graz, Heinrichstrasse 28, 8010 Graz, Austria.

b

c

d

e

Laboratory of Microbiology, LOEWE Center for Synthetic Microbiology, Philipps University of Marburg, Marburg.

Manuscript Received: XXX; Version of record online: XXX.

Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201XXX.

Abstract: An efficient stereoselective syntheses of a series Biotransformations catalyzed by both enzymes, followed by

of functionalized optically active γ-aryl-γ-butyrolactones is TFA-catalyzed cyclization of the resulting γ-hydroxy esters,

achieved by enzymatic asymmetric reduction of the furnished the respective (S)- and (R)-configured products

corresponding sterically demanding γ-keto esters employing with exquisite optical purity (up to >99% ee). The synthetic

wild-type and recombinant alcohol dehydrogenases. The best value was demonstrated on preparative scale for the

stereoselectivities for the reduction via hydrogen transfer asymmetric bioreduction of the model compound, methyl

was obtained with two short chain dehydrogenases (SDRs) 4-oxo-4-phenylbutanoate, affording optically pure (S)-γ-

of complementary stereospecificity from Aromatoleum phenyl-γ-butyrolactone (>99% ee) in 67–74% isolated yield

aromaticum, namely the Prelog-specific NADH-dependent at 89–95% conversion depending on the applied scale.

(

S)-1-phenylethanol dehydrogenase [(S)-PED] and the anti-

Prelog-specific

dehydrogenase [(R)-HPED], respectively.

(R)-1-(4-hydroxyphenyl)-ethanol Keywords: Biocatalysis; Biotransformations; Stereo-

selective

Bioreductions;

Alcohol

Dehydrogenases;

Optically Active Aromatic γ-Butyrolactones

isolated from the heartwood of Centrolobium

[6]

Introduction

robustum

and from the stem of Brosinum

[7]

potabile. Moreover, several functionalized non-

racemic aromatic γ-butyrolactone derivatives serve as

key intermediates in the syntheses of active

pharmaceutical ingredients (APIs), often providing

critical sources of asymmetry. Representative

compounds reaching blockbuster status in

pharmaceutical industry are selective serotonin-

The dihydrofuran-2(3H)-one (γ-butyrolactone, GBL

or γ-BL) framework is a very common structural

motif in a plethora of naturally occurring products

displaying a broad scope of pharmacological

activities (i.e. antimicrobial, antifungal, anthelmintic,

antiviral, antitumor, cytostatic, anti-inflammatory

etc.), which makes them interesting lead structures

for drug development.^[In turn, optically active γ-

aryl-γ-butyrolactones are considered as an important

class of GBL-related compounds that serves as a

valuable chiral synthons for the preparation of more

complex structures of high therapeutic importance.

Exemplarily, the privileged structural motif of this

type is the core of many pharmacologically relevant

molecules (Figures 1), among which the most

prominent examples are: potent antitumor

[8]

reuptake inhibitors (SSRIs), such as sertraline IV

1]

[9]

and fluoxetine

V, which are common

antidepressants also used in alleviation of the

symptoms of obsessive-compulsive disorder (OCD).

Among the approved drugs originating from a γ-aryl

GBL moiety are also nonsedating histamine H1-

[

10]

receptor antagonists (H1-RA), such as terfenadine

[11]

VI and fexofenadine VII, which are both used for

the symptomatic management of allergic

rhinoconjunctivitis and chronic urticaria. In addition,

the aryl GBL derived scaffold is found in the

structures of two other synthetic medications, namely

macrocyclic depsipeptides [(+)-cryptophycin A I and

+)-cryptophycin 52 II],^[both isolated from strains

2]

(

[

12]

of the terrestrial cyanobacterial genus Nostoc (ATCC

the antiarrhythmic agent trecetilide hemi-fumarate

371891^[and GSV 2242, respectively), and the

3]

[4]

VIII, which was developed for chronic treatment of

reentrant cardiac arrhythmias, and the antipsychotic

5

[5]

anti-Leishmania agent [(–)-centrolobine III]

1

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

Figure 1. Examples of natural products (I–III) and pharmaceuticals (IV–IX) containing chiral γ-aryl-γ-butyrolactone core.

drug flutroline^[¹³^]IX, which is clinically used in the

treatment of hospitalized schizophrenic patients. Due

to the importance of the optically active aromatic γ-

substituted γ-butyrolactones, painstaking studies have

been undertaken by numerous chemo- and

biocatalytic approaches aiming to prepare these

compounds in enantiomerically pure form. In the last

decade, the most common approaches toward the

synthesis of non-racemic γ-aryl-γ-lactones was

asymmetric hydrogenation and/or asymmetric

transfer hydrogenation (ATH) of γ-keto esters/acids.

Several elegant examples regarding the significance

of both these methods have been introduced

previously through various catalytic systems, mostly

yeast species.^[²⁷^]Furthermore, recombinant alcohol

[28]

dehydrogenases (ADHs)

have been successfully

employed. Moreover, biocatalytic methods that

employ lipases or esterases as catalysts for kinetic

resolutions such as lactonization of γ-aryl-γ-hydroxy

[

29]

[30]

esters,

hydrolysis of γ-phenyl-γ-butyrolactone,

and transesterification (acetylation) of 1-aryl-butane-

1,4diols

[31]

or

32]

N-methyl-4-hydroxy-4-

[

arylbutanamide deserve attention. Less studied, but

also very promising in terms of efficient preparation

of 4-aryl-GBLs enantiomers, seems to be utilization

of ancestral hydrolytic enzymes, such as

hydroxynitrile lyase (HNL 1-NJ) and esterase (Rs

[33]

EST).

[14]

based on ruthenium complexes as well as classical

reagents such as organoboranes^[¹⁵^]and silanes. The

arsenal of other asymmetric chemical synthetic

Although enormous progress has been made since

the first chemo- or biocatalytic approaches, the

synthesis of enantiomerically enriched 4-aryl-GBLs

is still considered as a challenging transformation in

biocatalytic synthetic chemistry. This is also due to

the fact that the most common substrates used for

their preparation, namely 4-oxo-4-arylbutanoates, are

ketones known to be very difficult to reduce, because

of the presence of two bulky substituents of the

ketone moiety (''bulky-bulky'' structure), which

impedes access of the substrate to the active site of

the enzyme. In this study, taking into account all the

above challenges as well as the demand for ‘greener’

and more sustainable methods for the preparation of

enantiomeric γ-aryl-γ-butyrolactones without using

hazardous chemical reagents, we report on the

development of catalytic methods based on whole-

[16]

methods

used

encompasses:

sequential

[

17]

hydroboration-oxidation,

the

[18]

epoxidation and Baeyer–Villiger oxidation (BVO),

single-step BVO of prochiral 3-substituted

cyclobutanones,^[oxidation of racemic lactols,

19]

[20]

allylation of arenecarbaldehydes,^[²¹^]coupling of

propargyl ethers with benzylic alcohols followed by

sequential transfromation of γ-hydroxy (Z)-enol

[22]

silanes, as well as hydroacylation of aryl 1,4-keto

alcohols.^[The alternative synthetic routes towards

enantiomeric γ-aryl-GBL derivatives catalyzed by

chiral catalysts embrace cyclopropane ring

23]

[

24]

reorganization via homoaldol reaction

nucleophilic cyclopropane ring-opening.

or H O-

2

[25]

In addition to organo- and metalorgano chemical

methods, highly selective biocatalytic approaches

have been developed in this field. Noteworthy among

them are stereoselective bioreductions of γ-aryl-γ-

keto acids/esters using microbial cells, including

cell

biocatalysts

harboring

overexpressed

recombinant alcohol dehydrogenases (ADHs) of

different origin and/or pure (R)- and (S)-specific 1-(4-

hydroxyphenyl)-ethanol dehydrogenases [(R)-HPED

and (S)-HPED].

[26]

industrial baker's yeast strains

as well as other

2

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

Scheme 1. Chemoenzymatic synthesis of non-racemic γ-aryl-γ-butyrolactones (S)-(–)-4a–g and (R)-(+)-4a–g. Reagents

and conditions: (i) 1a–g (8.14 mmol), AcCl (1.2 equiv), MeOH (1 mL/0.8 mmol of substrate 1a–g, ca. 10 mL) and PhCH

in the case of 1g), room temp., 12 h; (ii) 2a–g (final concentrations 10 mM), wet cells of E. coli/(S)-PED (220 mg wet

3

(

mass, 49 mg after drying, 0.638 U assayed for 2a), 0.5 mM NADH, 0.1 M MES-KOH (pH 5.5)/2-PrOH (1000 µL, 40:60,

v/v), 20–40 h, 30 ºC, 250 rpm (laboratory shaker); (iii) 2a–g (final concentrations 10 mM), solution of pure (R)-HPED (60

µL, 0.169 U assayed for 2a), 0.5–1.0 mM NADH, 0.1 M MES-KOH (pH 5.5)/2-PrOH (1000 µL, 90:10, v/v), 40 h, 30 ºC,

2

2 2

50 rpm (laboratory shaker); (iv) TFA(cat.), CH Cl , 1 h at 30 °C.

Results and Discussion

Herein, we report the chemoenzymatic synthesis of

enantiomerically enriched (S)- and (R)-γ-aryl-γ-

butyrolactones (S)- and (R)-4a–g, whereby the key

step is an enzyme-catalyzed stereoselective reduction

of methyl 4-oxo-4-arylbutanoates 2a–g (Scheme 1).

This was accomplished by employing Escherichia

para-position of the benzene ring 2e–f, revealed to be

less reactive under the established conditions.

Therefore, they required both higher molar excess of

the reducing agent as well as elongated reaction time

to be fully converted into the desired products.

coli

cells

containing

recombinant

alcohol

Biocatalytic reduction of 2a using wild-type

dehydrogenases (E. coli/ADHs and E. coli/(S)-PED)

or by using purified enzymes from A. aromaticum

microorganisms

and

recombinant

alcohol

dehydrogenases (ADHs)

[

(R)-HPED and (S)-HPED] as biocatalysts,

respectively.

The most prevalent biocatalyst used to

stereoselectively reduce prochiral carbonyl groups in

the early times of biocatalysis was baker’s yeast (BY),

Synthesis of the Substrates

[34]

Saccharomyces cerevisiae.

Unfortunately, as it

The synthesis of the prochiral ketones 2a–g as well as

the racemic γ-hydroxy esters rac-3a–g and γ-

butyrolactones rac-4a–g was performed using

standard synthetic methodologies (Scheme 1), in a 1–

turned out that S. cerevisiae cells led to sluggish

bioreduction of 2a with low to modest enantiomeric

excesses (23–83% ee, see Supporting Information for

details), the task was to find more potent biocatalysts

possessing a wider substrate specificity and improved

stereoselectivity. Consequently, reactions were run

with γ-keto ester 2a as model substrate using eight

wild-type microbial strains (used as lyophilized

powders) as well as six recombinant alcohol

dehydrogenases (ADHs) overexpressed in E. coli

strains [BL21(DE3) or DH5α] and employed either as

lyophilized cells or wet E. coli/(S)-PED cells or in

purified form [(S)- and (R)-HPED] (Table 1).

2

-step reaction sequence, starting from cheap

commercially available 4-oxo-4-arylbutanoic acids

a–g. After chemical esterification the products 2a–g

were isolated in 76–94% yield. Subsequently, by

employing 0.50–0.75 equiv of NaBH depending on

1

4

the substrate used, racemic γ-hydroxy esters rac-3a–g

and the corresponding γ-butyrolactones rac-4a–g

were obtained in 10–60% and 10–46% yield ranges,

respectively. Noteworthy is the fact that those

compounds, which contain the bulky pyrenyl moiety

2g, as well as those with electro-donating groups in

3

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

Table 1. Analytical-scale stereoselective reduction of 2a (10 mM) using wild-type microorganisms (freeze-dried) or

recombinant defined enzymes.

Conv.^[^b^]

Yield^[^b^][%]

(3a/4a)

0/0

ee

[c]

[%]

p

Entry

Biocatalyst

Strain

Method^[^a^]

(Config. ^d^])

N.D.

[

%]

[

e]

1

2

3

4

5

6

7

8

9

Komagataella phaffi/Pichia pastoris

Pseudomonas sp.

Arthrobacter sp.

isolate Actinomyces sp. SRB-AN040

isolate Actinomyces sp. SRB-AN053

isolate Actinomyces sp. ARG-AN024

isolate ARG-AN025

isolate USA-AN012

E. coli/Lk-ADH-Lica

E. coli/RasADH

E. coli/SyADH

ATCC 76273

DSM 6978

DSM 7325

FCC025

A

B

C

5

24

63

89

4

50

82

69

>99

92

>99

81

>99

46

[

e]

0/9

6/58

0/6

0/0

0/3

86 (S)

[

e]

N.D.

77 (S)

89 (S)

90 (S)

>99 (S)

49 (R)

99 (R)

FCC027

FCC014

FCC015

FCC021

0/0

0/3

–

46/54

[

f]

1

0

1

2

3

4

4/85

17/83

[

g]

E. coli/(S)-PED (gene ped/c1A58)

(S)-HPED (gene ebA309)

(R)-HPED (gene Hped/ebA307)

66/15

80/20

37/9

[g]

[

g]

[

a]

Reaction conditions. Method A: lyophilized biocatalyst (10 mg), 20 mM glucose, 0.5 mM NADH, 0.5 mM NADPH, 0.1

M Tris-HCl buffer (pH 7.5)/2-PrOH (500 µL, 90:10, v/v), 20 h, 30 ºC, 250 rpm (laboratory shaker) – anaerobic

conditions. Method B: wet cells (220 mg wet cells, 49 mg after drying, 0.638 U assayed for 2a), 0.5 mM NADH, 0.1 M

MES-KOH (pH 5.5)/2-PrOH (1000 µL, 40:60, v/v), 20 h, 30 ºC, 250 rpm (laboratory shaker) – aerobic conditions.

Method C: solution of pure enzyme (60 µL, 0.075 U and 0.169 U for (S)- and (R)-HPED assayed for 2a), 0.5 mM

NADH, 0.1 M MES-KOH (pH 5.5)/2-PrOH (1000 µL, 90:10, v/v), 20 h, 30 ºC, 250 rpm (laboratory shaker) – aerobic

conditions.

[

b]

Conversion (%) (i.e., consumption of substrate 2a) and products yields (i.e., formation of 3a/4a) were determined by

GC analyses using calibration curve.

[

c]

Determined for 4a by HPLC analysis using a Chiralcel OD-H or (S,S)-Whelk-01 columns.

Absolute configuration of optically active γ-phenyl-γ-butyrolactone (4a) was established by comparison of HPLC peaks

d]

elution order with our previous experiments concerning BY-mediated reactions as well as literature data.^[

28a and 35]

Major

enantiomer is shown in parentheses.

Not determined.

[

e]

f]

Traces (ca. 3%) of the hydrolyzed keto acid derivative 1a were observed.

To establish % ee for 4a the lactonization of γ-hydroxy ester 3a into γ-butyrolactone 4a was performed by the treatment

g]

[

15b]

of crude mixture with catalytic amount of TFA in CH

2

Cl

2

according to a procedure reported by Ramachandran et al.

We focused our investigations toward those

and (R)-HPED (gene Hped/ebA307).^[⁴³^]

microorganisms/enzymes, which have already been

shown to be able to reduce ''bulky-bulky'' aryl alkyl

ketones with high stereoselectivity. In the study we

All the enzymatic reactions were performed with a

10 mM solution of substrate 2a containing catalytic

amounts of 0.5 mM NADH and incubated on a rotary

shaker (250 rpm) at 30 °C for 20 h. For the

[36]

employed wild-type microorganisms, recombinant

[37]

ADHs, such as: Ralstonia sp. ADH (RasADH),

SyADH (originating from Sphingobium

yanoikuyae), a variant of Lactobacillus kefir DSM

biocatalytic

hydrogen

transfer

experiments

employing lyophilized wild-type microorganisms and

E. coli/ADHs preparations, 0.5 mM NADPH and 20

mM glucose in a 0.1 M Tris-HCl buffer (pH 7.5)

have been employed. In the case of PED/HPED-

mediated biotransformations, the reaction medium

was 0.1 M MES-KOH buffer (pH 5.5) and 0.5 mM

NADH. In both employed strategies, the regeneration

of NADH cofactors was realized by a ''sacrificial

[38]

39]

[40]

2

0587^[named Lica ADH, SDRs isolated from

the denitrifying bacterium A. aromaticum (formerly

named Azoarcus sp. EbN1)^[⁴¹^]employed either as

whole-cell biocatalyst, e.g. E. coli/(S)-PED (gene

ped/c1A58)^[or as isolated enzymes, e.g. (S)-HPED

42]

(

gene ebA309)

4

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

substrate-coupled'' approach assay using 2-PrOH as

the hydride donor at concentrations of 10 or 60%

small -CH

CH CH CO

3

group in acetophenone by the larger -

2

CH in 2a apparently led to reversed

3

(

v/v), respectively. An additional advantage of

substrate binding preference in (S)-HPED, which then

presents predominantly the si-face of the prochiral 2a

towards hydride transfer from NADH.

employing 2-PrOH is that it increases substrate 2a

solubility in aqueous media and is responsible for

inactivation of many undesired enzymes present in

the cells as well. The highest applicable concentration

of 2-PrOH (60% v/v equals to 10 M) was used for E.

coli/(S)-PED as this biocatalyst is highly resistant to

All enzymes from A. aromaticum catalyzed the

reduction of 2a furnishing the mixtures of respective

products (S)-3a/(S)-(–)-4a in favour of the γ-hydroxy

ester (S)-3a formation. As the undesired uncyclized

compound could interfere with the cyclized product

during HPLC analysis, an additional lactonization

with catalytic amount of TFA was performed.

Moreover, apart from (R)-HPED preparation all of

the tested wild-type microbial resting cells and

recombinant E. coli/ADHs followed the Prelog rule

with the hydride of NADH attacking from the re-face

of the prochiral ketone 2a. This has been confirmed

by comparing the elution order of the respective

peaks on HPLC equipped with the same chiral

column as in the literature,

of the optical rotation data with literature values (see

Supporting Information).

It has already been stated above that reduction of

2a and 2d has been investigated previously

employing recombinant RasADH and SyADH

enzymes overexpressed in E. coli. Interestingly, the

performed reduction afforded preferentially optically

active γ-hydroxy esters instead of the respective γ-

lactones. This is probably due to different reaction

conditions employed (less concentrated 50 mM Tris-

[

42b]

inactivation by organic solvents.

The results of the

screening tests are summarized in Table 1.

Evaluation of the selected panel of wild-type

microorganisms revealed that Arthrobacter sp. DSM

7

325 was capable of reducing 2a, while the

remaining other seven metabolized the model ketone

a into a complex mixture of unknown compounds

2

with total substrate conversions reaching up to 89%

value. Aforementioned Arthrobacter sp. catalyzed the

reduction of 2a with 63% conv. leading to optically

active (S)-(–)-4a in 58% GC-yield and with 86% ee,

which in fact was almost 2-fold better than obtained

for BY-mediated biotransformations (see Table S1,

[28a and 35]

and by correlation

entry

8

in Supporting Information). In the

[28c]

experiments with lyophylized E. coli preparations

containing overexpressed ADHs (Table 1, entries 9–

1

1) significantly better conversions (92–100% conv.)

and optical purities (89–90% ee) were obtained with

RasADH and SyADH (Table 1, entry 10 and 11).

Furthermore, although the engineered E. coli/Lk-

ADH-Lica also proved to be very active toward 2a,

catalyzing asymmetric bioreduction with >99% conv., HCl buffer (pH 7.5) and glucose/GDH cofactor-

it led to the formation of an almost equimolar mixture

of optically active products (S)-3a/(S)-(–)-4a (46/54%

GC-yield) with moderate 77% ee (Table 1, entry 9).

Undoubtedly, the most efficient biotransformations in

terms of stereoselectivity were achieved with E.

coli/(S)-PED cells and pure (R)-HPED enzyme

regeneration system instead of 2-PrOH). According

to the publication,

[28c]

the γ-hydroxy esters were

obtained with (R)-configurations, which is in

contradiction to our results. However, it has to be

pointed out that the assignment of stereochemistry of

the obtained optically active compounds is

inconsistent since the assigned absolute configuration

of (R)-γ-(4-bromophenyl)-γ-butyrolactone [(R)-4d]

based on polarimetric analysis performed therein

(

Table 1, entry 12 and 14) with exquisite

stereoselectivity in both cases (99 to >99% ee).

Lower conversion was observed in the case of (R)-

HPED (46%), whereas E. coli/(S)-PED preparation

achieved 81% conv. during screening experiment

2

0

([α]

D

= −24.7 (c 1.23, CHCl ), see page: 392, line:

3

1) is in stark contrast with all of the previously

reported data in literature. For example, for 4d the

dextro (+) rotation is attributed to its (R)-

(

while >95% conv. was achieved in the up-scaling

test – see Table 4). Notably, both enzymes exhibit

complementary stereopreference since E. coli/(S)-

PED afforded the (S)-configured product, while (R)-

HPED yielded its mirror image. In contrast, pure (S)-

HPED enzyme showed excellent results in terms of

substrate conversion (>99%), but unexpectedly

produced the (R)-configured product (R)-(+)-4a,

albeit at low enantiomeric excess. Although (S)-

HPED was confirmed to be stereoselective for (S)-

configured alcohols in case of physiological

[

23]

enantiomer, while the levo (–) optical rotatory sign

[22 and 24c]

is characteristic for (S)-enantiomer.

Thus, the

previous data in the literature concerning the absolute

configurations of enantiomeric lactones 4a and 4d

obtained via reduction of the respective ketones

catalyzed by RasADH and/or SyADH are incorrect.

Next, we investigated the catalytic activity of the

most potent wild-type biocatalyst (Arthrobacter sp.)

and E. coli/ADHs (Lk-ADH-Lica, RasADH,

SyADH) in the asymmetric reduction of 2a, with

particular focus on omission of external cofactor

addition and cofactor recycling (Table 2). Whole-cell

systems often do not need addition of the external

cofactors because they are already present in the cells.

Furthermore, whole-cell biocatalysts in freeze-dried

(lyophilized) form greatly simplify the handling.

Consequently, we examined different reaction modes:

substrates such as acetophenone, (S)-1-phenylethanol

or para-hydroxyacetophenone,^[

43c,d]

it converted 2a

preferentially to (R)-(+)-4a with 49% ee (Table 1,

entry 13). The reversed stereopreference of (S)-HPED

observed toward 2a may be explained by assuming

different modes of substrate binding in the active site

of this enzyme than the other tested SDRs, which did

not show a similar reversal and retain their excellent

stereospecifity also in reducing 2a. Exchanging the

5

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

Table 2. Optimization of asymmetric reduction of 2a (10 mM) using whole-cell biocatalyst preparations.

[c]

NADH

NADPH

Glucose

Conv.^[^b^]Yield^[^b^][%]

ee

p

Entry

Biocatalyst^[^a^]

t [h]

[

mM]

0.5

–

[mM]

0.5

–

[mM]

20

–

20

–

20

–

20

–

[%]

>99

36

(3a/4a)

0/90

0/97

0/93

0/30

46/54

6/87

9/91

9/85

4/85

5/91

3/93

5/81

17/83

5/84

4/80

11/63

[%]

84

77

76

80

89

85

89

92

90

86

91

92

1

2

3

4

5

6

7

8

9

Arthrobacter sp. DSM 7325

48

–

E. coli/Lk-ADH-Lica

E. coli/RasADH

E. coli/SyADH

20

0.5

–

0.5

–

0.5

–

0.5

–

>99

96

>99

97

–

0.5

–

92

96

1

0

1

2

3

4

5

6

–

0.5

–

>99

95

–

88

[

a]

Reaction conditions: lyophilized biocatalyst (10 mg), 0.5 mM NADH (or without), 0.5 mM NADPH (or without), 20

mM glucose (or without), 0.1 M Tris-HCl buffer (pH 7.5)/2-PrOH (500 µL, 90:10, v/v), 20–48 h, 30 ºC, 250 rpm

(

laboratory shaker) – anaerobic conditions.

Conversion (%) (i.e., consumption of substrate 2a) and products yields (i.e., formation of 3a/4a) were determined by

[

b]

GC analyses using calibration curve.

c]

Determined for 4a by HPLC analysis using a Chiralcel OD-H column or (S,S)-Whelk-01 columns.

(

i) with the addition of NADH, NADPH and glucose,

ii) without glucose, (iii) without glucose and

Biocatalytic reduction of 2a–f using recombinant

alcohol dehydrogenases (ADHs)

NADPH, and finally (iv) without all aforementioned

compounds. In addition, for the bioreduction of 2a

catalyzed by Arthrobacter sp. DSM 7325 the time of

In the next step, to demonstrate the utility of the

library of the selected enzymes as biocatalysts in

the reaction was elongated 2-fold to reach >99% conv. asymmetric reduction of γ-aryl-γ-keto esters, we have

Inspection of Table 2 clearly shows that in almost all

cases external addition of cofactors and glucose was

not improving the process, except in case of

Arthrobacter sp. DSM 7325, for which addition of

extended the substrate scope to other ''bulky-bulky''

compounds 2b–f possessing different substitution

patterns in para-position of the benzene ring (Table

3). Moreover, to verify if these biocatalysts are able

to perform transformations of sterically highly

demanding ketones, reduction of the substrate with

pyrenyl moiety 2g was also investigated. Then

biotransformations were conducted with: E.

coli/RasADH, E. coli/SyADH, E. coli/(S)-PED and

(R)-HPED, using three different synthetic procedures

as applied before (Methods A–C) or the protocol with

isolated (R)-HPED enzyme, but after minor

modification (Method D). The assignment of the

stereochemistry to all the obtained enantiomeric

products of ADHs- and PED/HPED-catalyzed

bioreductions was accomplished by comparing

elution profiles on HPLC with a chiral phase using

the reference materials obtained in the preparative-

scale BY-mediated biotransformations of 2a and 2c–

d (see Supporting Information).

0

.5 mM NADH increased conversion from 36% to

>

99%. However, due to spontaneous lactonization of

the reduced 3a to 4a, Arthrobacter sp. DSM 7325

turned out to be the only full step-economical

biocatalyst yielding enantiomeric lactone (S)-(–)-4a

in a one-pot operation strategy in 90–97% yield and

84% ee, respectively. Furthermore, in terms of the

stereoselectivity of bioreduction, the differences were

also negligible (2–7% ee), albeit in favor of the

reactions carried out without additives, what only

proved that this transformations can be carried out

under NAD(P)H-free conditions. Undoubtedly, the

lyophylized E. coli cells containing RasADH and

SyADH turned out to be the most potent biocatalysts

with regard to optical purity of the obtained lactone

(

S)-(–)-4a (92% ee) (Table 2, entry 12 and 16), and

thus were selected along with E. coli/(S)-PED and

pure (R)-HPED for further investigations.

6

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

Table 3. Analytical-scale studies on (ADHs)-catalyzed reductions of ''bulky-bulky'' γ-aryl-γ-keto esters 2a–g (10 mM).

Conv.^[^b^]Yield^[^b^][%]

ee

[c]

[%]

Entry Substrate

R

H

ADHs

Method^[^a^]t [h]

p

[

d]

[

%]

(3a/4a)

5/81

(Config. )

92 (S)

>99 (S)

99 (R)

82 (S)

7 (S)

99 (S)

98 (R)

55 (S)

53 (R)

>99 (S)

93 (R)

62 (S)

58 (R)

>99 (S)

74 (R)

76 (R)

76 (S)

50 (S)

46 (S)

76 (R)

77 (R)

74 (S)

52 (S)

75 (S)

1

2

3

4

5

6

7

8

9

2a

C

6

5

E. coli/RasADH

E. coli/SyADH

E. coli/(S)-PED

(R)-HPED

A

B

20

40

20

40

20

40

20

40

20

40

20

40

20

96

88

90

57

67

98

96

>99

84

96

90

98

80

98

95

>99

36

44

>99

96

35

40

87

55

74

0

11/63

68/8

36/21

47/20

8/90

C

D

A

B

2b

2c

2d

4-F-C

6

H

4

E. coli/RasADH

E. coli/SyADH

E. coli/(S)-PED

(R)-HPED

E. coli/RasADH

E. coli/SyADH

E. coli/(S)-PED

(R)-HPED

E. coli/RasADH

E. coli/SyADH

E. coli/(S)-PED

(R)-HPED

8/88

86/14

71/13

9/87

C

1

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

4-Cl-C

6

H

4

A

B

1

2

3

0/81

72/26

60/20

0/91

C

4-Br-C

6

H

4

A

B

0/85

68/32

25/11

28/16

0/98

C

D

A

B

2e

4-CH

3

-C

6

H

4

E. coli/RasADH

E. coli/SyADH

E. coli/(S)-PED

(R)-HPED

0/98

78/18

24/11

30/10

0/87

0/50

50/24

0/0

C

D

A

B

2f

4-CH

3

O-C

6

H

4

E. coli/RasADH

E. coli/SyADH

E. coli/(S)-PED

(R)-HPED

E. coli/RasADH

E. coli/SyADH

E. coli/(S)-PED

(R)-HPED

[

e]

C/D

A

B

N.D.

4-Pyrenyl^[^f^]

0

0/0

[e]

2g

[

e]

0

0/0

0

0/0

C

0

0/0

[

a]

Reaction conditions. Method A: 2a–g (final concentrations 10 mM), lyophilized biocatalyst (10 mg), 0.1 M Tris-HCl

buffer (pH 7.5)/2-PrOH (500 µL, 90:10, v/v), 20 h, 30 ºC, 250 rpm (anaerobic). Method B: 2a–g (final concentrations

1

(

0 mM), wet cells (220 mg wet cells, 49 mg after drying, 0.638 U assayed for 2a), 0.5 mM NADH, 0.1 M MES-KOH

pH 5.5)/2-PrOH (1000 µL, 40:60, v/v), 20–40 h, 30 ºC, 250 rpm (aerobic). Method C: 2a–g (final concentrations 10

mM), solution of pure enzyme (60 µL, 0.169 U (R)-HPED assayed for 2a), 0.5 mM NADH, 0.1 M MES-KOH (pH

.5)/2-PrOH (1000 µL, 90:10, v/v), 20–40 h, 30 ºC, 250 rpm (aerobic). Method D: 2a, d–g (final concentrations 10

mM), solution of pure enzyme (60 µL, 0.169 U (R)-HPED assayed for 2a), 1.0 mM NADH, 0.1 M MES-KOH (pH

5

.5)/2-PrOH (1000 µL, 90:10, v/v), 40 h, 30 ºC, 250 rpm (aerobic).

[

b]

Conversion values (%) (i.e., consumption of substrate 2a–f) and products yields (i.e., formation of 3a–f/4a–f) were

determined by GC analyses using calibration curves.

c]

Determined for optically active γ-aryl-γ-butyrolactones 4a–g by chiral HPLC analysis using (S,S)-Whelk-01 or

Chiralcel OD-H columns, respectively.

d]

Absolute configuration of 4a–f established by comparison of HPLC peaks elution order and optical rotation signs with

[28a and 35]

literature data.

Not determined.

Major enantiomer is shown in parentheses.

[

e]

f]

Additional portion of DMSO (30 µL) was added in order to increase substrate 2g solubility.

7

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

Detailed analysis of the data revealed that the most

potent enzyme preparation in terms of the conversion

as well as stereoselectivity was E. coli/(S)-PED. The

reductions of γ-aryl-γ-keto esters 2a–f catalyzed by

these cells provided (S)-configured γ-aryl-γ-hydroxy

esters (S)-3a–f, which after treatment with TFA

produced the corresponding lactones (S)-(–)-4a–f

with moderate (46–52% ee) to mostly excellent

a moderate stereoselective manner (50–92% ee). We

were puzzled by the result of diminished optical

purity value for (S)-(–)-4b (7% ee) obtained from the

reaction catalyzed by SyADH especially as fluorine is

a bioisostere of the hydrogen atom. The unexpected

low stereoselectivity of this reaction for 2b was

ascertained by three independent experiments under

the same conditions. Explanation of this finding is

rather difficult, although it is possible that greater

lipophilicity of fluorine as well as its strong

electronic features may play a detrimental role in

specific interactions in a catalytic cavity of the

SyADH impeding efficient stereorecognition.

Nevertheless, the observed huge difference in

stereochemical outcome of transformation of this

particular ketone 2b can be ascribed by distinguished

substrate specificity of the tested SyADH as well.

Notably, a remarkable drop in stereoselectivity was

found in the bioreduction of para-chloro- 2c and

para-bromo-substituted 2d derivatives using both E.

coli/ADHs, together with a different selectivity

pattern as it turned out that these biocatalysts

displayed opposite stereopreference on compounds

2c–d leading to the formation of (S)-(–)-4c (55% ee)

and (S)-(–)-4d (62% ee) in the case of RasADH, and

(R)-(+)-4c (53% ee) and (R)-(+)-4d (58% ee) in the

case of SyADH.

optical

purity

(99

to

>99%

ee).

For

biotransformations of model substrate 2a as well as

derivatives 2b–d possessing electron-withdrawing

groups (–F, –Cl, and –Br) situated in the para-

position of the phenyl substituent almost no traces of

the (R)-enantiomers were detected by HPLC, which

demonstrates the excellent selectivity exerted by E.

coli/(S)-PED. Out of six ketones 2a–f, three of them,

namely 2a and 2c–d, were transformed into optically

pure lactones (S)-(–)-4a and (S)-(–)-4c–d, while in

the case of para-fluoro-substituted derivative 2b the

enantiomeric purity of (S)-(–)-4b was found to be

9

9%. For substrates substituted with electron

withdrawing groups (2b–d) E. coli/(S)-PED gave the

target products with >98% conv. after 20 h. For the

reference substrate 2a, we observed 81% conv. after

2

0 h and 90% conv. after 40 h and 98% in the up-

scaling experiment. On the other hand, lower

conversion and especially diminished

stereoselectivity were observed for compounds 2e–f

bearing electron-donating groups (–CH and –OCH

Table 3, entries 21 and 26). This result is consistent

with previous studies on E. coli/(S)-PED, where

Interestingly, the results with 2a–f revealed that the

reactions mediated by E. coli/RasADH and E.

coli/SyADH provided predominantly enantiomeric γ-

lactones 4a–g over the respective γ-hydroxy esters

3a–g. Currently, we are lacking a plausible

explanation for this observation as the lactone

formation rate was reported to be largely pH-

3

)

(

bioreduction of para-OH and para-NH

acetophenones resulted with

2

substituted

decreased

[

43a]

stereoselectivity (respective ee of 90 and 0%)

as

[44]

well as relative specific activities in bioreduction of

acetophenone and 4-hydroxyacetophenone for

HPEDs.^[

dependent,

and its promotion may be expected

when acidic conditions are applied. In this case, when

we employed MES-KOH buffer of slightly acidic pH

5.5 as the medium for PED/HPED-based reactions, it

turned out that hydroxyl products are formed in favor,

whereas in Tris-HCl buffer pH 7.5 designed for E.

coli/ADHs-based reactions, the desired lactones were

observed almost as sole products. This may suggest

that it is not the pH of the buffer, which plays a

decisive role, but maybe the local pH in the cells or

interaction with another enzyme that promotes

lactonization. Investigation of the substrate range also

revealed that all of the tested enzyme preparations are

completely inactive towards the sterically demanding

ketone 2g (Table 3, entries 28–31). The bulkiness of

the pyrenyl substituent likely prevents fitting the

substrate into the enzymes active sites, and thus

suitable variants may have to be identified to

overcome this circumstances in the future.

Having an optimal biocatalytic system in hand, we

performed further scale-up experiments with 2a (0.3

mmol, 0.6 mmol and 4.2 mmol) in order to prove that

E. coli/(S)-PED cells displayed not only high

selectivity toward prochiral γ-aryl-γ-keto esters, but

also sufficient productivity (Table 4). The

preparative-scale trials showed that under the best

reaction conditions identified, the E. coli/(S)-PED

biocatalyst allowed high level of consumption of

43d]

Subsequently, the use of (R)-HPED provided the

opposite (R)-enantiomers of the respective mixtures

of γ-aryl-(-keto esters/-lactones) (R)-3a–f/(R)-(+)-4a–

f with mostly excellent stereoselectivity allowing to

obtain exquisite enantiomeric excesses of up to 99%

for (R)-(+)-4a and 98% for (R)-(+)-4b after additional

TFA-catalyzed lactonisation procedure. While

substrates 2a–b were transformed with high

stereoselectivity, the other substrates led to the

formation of the target lactones (R)-(+)-4c–e with

enantiomeric purities between 74 and 93% ee. From

the panel of screened substrates, 2b and 2c were

reduced with >80% conv. after 40 h while in other

cases the conversion was lower. Interestingly, (R)-

HPED failed to catalyze the reduction of 2f. As the

six ketones 2a–f are of comparable size, the loss of

reactivity was probably due to unfavorable electronic

effects of the methoxy group present in 2f.

In

contrast

to

PED/HPED-mediated

biotransformations of 2a–f, the reductions catalyzed

by E. coli cells harboring the respective RasADH and

SyADH recombinant enzymes were less efficient.

Despite very high 87–100% conv. accomplished in a

reasonable time scale (20 h), the formation of

enantiomerically enriched lactones 4a–f proceeded in

8

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

Table 4. Preparative-scale E. coli/(S)-PED-catalyzed bioreduction of methyl 4-oxo-4-phenylbutanoate (2a).

Entry Scale [mmol]^[^a^]

Conv.^[^b^][%]

GC-yield^[^c^][%] (3a/4a)

Isolated yield^[^c^][%]

ee

p

[d]

[%]

98

>99

1

2

3

4

0.3

0.6

2.1

4.2

96

95

66/30

70/25

62

74

N.D.

[

e]

[f]

>98

89

N.D.

>99

75/14

67

[

a]

Reaction conditions for 0.3–0.6 mmol scale. 2a (0.3 mmol or 0.6 mmol), E. coli/(S)-PED wet cells (6.6 g, activity 19.14

U relative to 2a or 13.2 g, activity 38.28 U relative to 2a)), 0.5 mM NADH, 0.1 M MES-KOH (pH 5.5)/2-PrOH (30 mL

2 2

or 60 mL, 40:60, v/v), 40 h, 30 ºC, 250 rpm (laboratory shaker) – aerobic conditions; TFA(cat.), CH Cl (15 mL or 30

mL), 1 h at 30 °C. Reaction conditions for 2.1-4.2 mmol scale (fed-batch mode). initial loading of 2a (2.1 mmol), 2a

addition after 20 h (2.1 mmol), E. coli/(S)-PED wet cells (46.2 g, activity 133.98 U relative to 2a), 0.5 mM NADH, 0.1

M MES-KOH (pH 5.5)/2-PrOH (210 mL, 40:60, v/v), 80 h (total time), 30 ºC, 250 rpm (laboratory shaker) – aerobic

conditions; TFA(cat.), CH

2

Cl

2

(50 mL), 5 h at 30 °C.

[

b]

Conversion (%) (i.e., consumption of substrate 2a) and products yields (i.e., formation of 3a/4a) were determined by

GC analyses using calibration curve.

[

c]

2

Isolated yield after column chromatography on SiO .

d]

e]

f]

Determined for optically active (S)-(–)-4a by chiral HPLC analysis using (S,S)-Whelk-01 column.

Conversion (%) determined by HPLC analysis from the samples directly withdrawn from crude reaction mixture.

Not determined.

the starting material 2a (89–96% conv.) with GC-

yields reaching a range of 66–75% for (S)-3a and 14–

Additionally, (iv) purified oxidoreductive

enzymes were employed. From the above-listed

biocatalysts, the most outstanding selectivity in

hydrogen transfer to the carbonyl group was obtained

with E. coli cells containing recombinant NADH-

3

0% for (S)-(–)-4a, respectively. After TFA-

catalyzed lactonization of the crude mixtures, the

product isolation led to 30 mg (62% isolated yield),

72 mg (74% isolated yield) and 457 mg (67% isolated

dependent

(S)-selective

1-phenylethanol

yield) of (S)-(–)-4a depending on the applied scale. It

is worth noting that the bioreduction of 2a conducted

on a 4.2 mmol scale (807 mg of the substrate 2a) was

performed in a fed-batch mode for 80 h of total

reaction time. This attempt allowed us to clarify

reliability of the up-scaling procedure as well as led

to a significant drop in overall employed biocatalyst

to substrate ratio. The most important fact is that 2a

was reduced with perfect enantioselectivity, thus (S)-

dehydrogenase from the denitrifying bacterium A.

aromaticum strain EbN1, namely E. coli/(S)-PED.

Depending on the type of substitution pattern in the

tested prochiral substrates, significant differences in

the stereochemical outcome were observed. Using a

one-pot two-step direct bioreduction-lactonization

reaction sequence catalyzed by a system composed of

E. coli/(S)-PED biocatalysis and TFA-dependent

chemical conversion, enantiomeric (S)-(–)-γ-aryl-γ-

butyrolactones have been obtained as targets with 67–

100% conv., and mostly with excellent

stereoselectivities (i.e. reaching 99 to >99% ee for

phenyl-like as well as para-fluoro, para-chloro and

para-bromo derivatives). Moderate optical purities

(

–)-4a being obtained in highly enantiopure form

form 98% ee to >99% ee), accurately preserving

stereoselectivity of the E. coli/(S)-PED from the

analytical-scale reactions.

(

46–75% ee) were obtained with para-methyl and

Conclusion

para-methoxy compounds. The preparative-scale

studies with 4-oxo-4-phenylbutanoate (0.6–4.2

mmol) accomplished the optically pure (S)-γ-phenyl-

γ-butyrolactone (>99% ee) in high 67–74% isolated

yields. The second enzyme preparation investigated

that was identified to be highly useful for

stereoselective reduction of the γ-keto esters is (R)-

HPED, which allowed to obtain (R)-configured γ-

phenyl-γ-butyrolactone and its derivative with a

fluorine atom at the para-position with nearly

excellent enantioenrichment (98–99% ee). For the

other substrates studied (R)-HPED led to

In order to expand the bioorganic synthesis toolbox

for the preparation of enantiomerically pure γ-aryl-γ-

butyrolactones, various whole-cell biocatalysts,

including: (i) baker's yeast, (ii) wild-type

microorganisms with carbonyl reductase activity as

well as (iii) E. coli cells harboring recombinant

alcohol dehydrogenases designed for ''bulky-bulky''

substrates have been tested in the asymmetric

reduction of the respective γ-aryl-γ-keto esters.

9

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

(

30 °C) manner. Optical rotations ([α]) were measured

enantiomeric excesses between 76% and 93% (i.e.

with a PolAAr 32 polarimeter in a 2 dm long cuvette using

93% ee for para-Cl, 76% ee for para-Br, and 77% ee

the sodium D line (λ=589 nm); the units of the specific

for para-Me), while for the para-OMe derivative the

reaction was fully suppressed. Interestingly, in the

case of bioreductions catalyzed by E. coli/RasADH

1

13

rotation are: (deg×mL)/(g×dm). H NMR (500 MHz),

C

19

NMR (126 MHz) and F NMR (470 MHz) spectra were

recorded on

a

Varian NMR System 500 MHz

1

13 19

spectrometer; H, C and F chemical shifts (δ) are

reported in parts per million (ppm) relative to the solvent

signals [CDCl , δ (residual CHCl ) 7.26 ppm, δ 77.16

and

stereochemistry was found only when methyl 4-(4-

chloro-phenyl)- and 4-(4-bromo-phenyl)-4-

oxobutanoates were employed as substrates. In these

cases, E. coli/RasADH preserved Prelog

E.

coli/SyADH

a

mutual

reversed

3

H

3

C

3

ppm] or internal CFCl reference set at 0 ppm. Chemical

shifts are quoted as s (singlet), d (doublet), dd (doublet of

doublets), t (triplet), q (quartet), m (multiplet), and br s

(

broad singlet); coupling constants (J) are reported in Hertz

stereospecificity leading to (S)-configured lactones,

whereas E. coli/SyADH catalyzed the formation of

the respective (R)-configured counterparts.

(Hz). Mass spectrometry was recorded on Micro-mass ESI

Q-TOF spectrometer with MSI concept 1H (EI, 70eV

ionization) for MS analysis and on Q Exactive Hybrid

Quadrupole-Orbitrap Mass Spectrometer, ESI source:

electrospray with spray voltage 4.00 kV for FTMS

analysis; all samples were prepared by dilution with

MeOH (0.5 mL) and additives of mixtures of

Experimental Section

CH

each.

3 2

CN/MeOH/H O (50:25:25, v/v/v) + 0.5% formic acid

General experimental methods: Reagents and solvents

were purchased from various commercial sources (Sigma

Aldrich, Alfa Aesar, POCH) and were used without further

purification. High-performance liquid chromatography

HPLC-grade solvents (n-hexane, 2-PrOH and EtOH) were

purchased from POCH (Poland). Whole cell biocatalysts

General procedure for the synthesis of methyl 4-oxo-4-

arylbutanoates 2a–g

To a solution of the appropriate 4-oxo-4-arylbutanoic acid

1a–g (8.14 mmol) in MeOH (1 mL/0.8 mmol of substrate,

ca. 10 mL) acetyl chloride (1.2 equiv) was added dropwise

and the reaction mixture was stirred at room temperature

for 12 h. [Attention: in the case of 1g additional portion of

[

36]

(

WCBs) including cells of wild type strains and E. coli

cells containing overexpressed recombinant alcohol

dehydrogenases (ADHs) Ralstonia sp. ADH (RasADH,

[

37c]

pEG 105),

SyADH (originating from Sphingobium

[38]

yanoikuyae, pEG 53) have been prepared as previously

reported. All 1-(4-hydroxyphenyl)-ethanol dehydrogenases

from Aromatoleum aromaticum were from the Jerzy Haber

Institute of Catalysis and Surface Chemistry, PAS and

have been prepared following the methodology previously

3

PhCH (20 mL) was added to increase solubility of the

substrate]. After completion of the reaction [according to

TLC indications developed in hexane/AcOEt (1:1 v/v) as a

mobile phase], the crude mixture was concentrated under

vacuum to remove MeOH, the obtained slurry was

[

42a]

described [see: E. coli/(S)-PED

(

(also known as PED),

dissolved in CH

saturated NaHCO

respectively. Next, an aqueous phase was back-extracted

with CH Cl (1 × 50 mL), and the combined organic layer

was dried over anhydrous MgSO . After filtration of the

2

Cl

2

(50 mL), washed with portions of

[

43c]

[43d]

S)-HPED

(also known as EbA309) and (R)-HPED

3

(2 × 30 mL) and brine (2 × 30 mL),

also known as Hped or ChnA)]. Evaporation of the

solvent residues was performed at reduced pressure by

means of Büchi rotary evaporator and high-vacuum oil

pump at p=0.05 mmHg. Melting points, uncorrected, were

determined with a commercial apparatus (Thomas-Hoover

2

4

drying agent, the permeate was concentrated under reduced

pressure to obtain the corresponding methyl 4-oxo-4-

arylbutanoate 2a–g in an excellent purity allowing them to

be used in the next step without further purification. In the

case of crude 2g additional purification procedure

employing recrystalization from the mixture of

hexane/AcOEt (1:1 v/v) was necessary to obtain a pure

product.

"

UNI-MELT" capillary melting point apparatus) on

samples contained in rotating glass capillary tubes open on

one side (1.35 mm inner diam. and 80 mm length).

Analytical thin-layer chromatography was carried on TLC

aluminum plates (Merck) covered with silica gel of 0.2

mm thickness film containing a fluorescence indicator

green 254 nm (F254), and using UV light as a visualizing

agent. Preparative separations were carried out by column

chromatography using thick-walled glass columns and

silica gel (230–400 mesh) with grain size 40–63 μm

purchased from Merck, Germany. The chromatographic

Methyl 4-oxo-4-phenylbutanoate (2a). Yield 84% (1.31

f

g); light yellow liquid; R [hexane/AcOEt (1:1 v/v)] 0.84;

1

3

H NMR (500 MHz, CDCl ): δ 2.75 (t, J=6.6 Hz, 2H), 3.31

(

7

t, J=6.6 Hz, 2H), 3.69 (s, 3H), 7.42–7.48 (m, 2H), 7.52–

13

analyses (GC) were performed with

a

Agilent

.58 (m, 1H), 7.94–8.00 (m, 2H); C NMR (126 MHz,

Technologies 6890N instrument equipped with a flame

ionization detector (FID) and fitted with HP-50+ (30 m)

semipolar column (50% phenyl–50% methylpolysiloxane);

Helium (2 mL/min) was used as carrier gas; retention times

CDCl

3

): δ 28.0, 33.4, 51.8, 128.0, 128.6, 133.2, 136.5,

+

1

73.3, 198.0; MS (ESI-TOF) m/z: [M+H] Calcd for

+

C

11

H

13

O

3

m/z: 193.0860, Found 193.0706; FTMS (ESI-

+

TOF) m/z: [M+H] Calcd for C11

H

13

O

3

m/z: 193.08592,

R

(t ) are given in minutes under these conditions. The

R

Found 193.08623; GC [200–260 (10 °C/min)]: t =2.120

min.

enantiomeric excesses (% ee) of kinetic resolution products

were determined by HPLC analyses performed on

Shimadzu CTO-10ASV chromatograph equipped with

STD-20A UV detector and chiral columns as follows:

Chiralcel OD-H (4.6 mm × 250 mm, coated on 5 µm silica

gel grain size, from Daicel Chemical Ind., Ltd.) or

Chiralpak AD-H (4.6 mm × 250 mm, coated on 5 µm

silica gel grain size, from Daicel Chemical Ind., Ltd.) or

Methyl 4-(4-fluorophenyl)-4-oxobutanoate (2b). Yield

[45]

9

5

2% (1.57 g); white solid; mp 52–54 °C (CH Cl ) [lit.

2 2

1

1–52 °C (CH

2

Cl

2

)]; R

f

[hexane/AcOEt (1:1 v/v)] 0.82; H

NMR (500 MHz, CDCl

3

): δ 2.76 (t, J=6.6 Hz, 2H), 3.28 (t,

J=6.6 Hz, 2H), 3.70 (s, 3H), 7.09–7.16 (m, 2H), 7.97–8.03

13

(

1

m, 2H); C NMR (126 MHz, CDCl

3

): δ 28.0, 33.3, 51.8,

(

S,S)-Whelk-O 1 (4.6 mm × 250 mm, coated on 5 µm

15.8 (d, JC–F=21.5 Hz), 130.7 (d, JC–F=8.8 Hz), 133.0 (d,

C–F=2.9 Hz), 165.8 (d, JC–F=254.1 Hz), 173.3, 196.4;

silica gel grain size, from Regis Pirkle Technologies, INC.)

all of them equipped with a pre-column (4 mm × 10 mm, 5

µm) using mixtures of n-hexane/2-PrOH or n-

hexane/EtOH as mobile phase in the appropriate ratios

given in experimental section [both the mobile phase

composition as well as the flow rate were fine tuned for

each analysis (see Table S3)]; the wavelength of UV

detection was set at 220 nm for γ-hydroxybutyrates rac-

19

J

F

NMR (470 MHz, CDCl

3

): δ –105.05 (tt, J=8.39, 5.3 Hz);

+

MS (ESI-TOF) m/z: [M+H] Calcd for C11

H

12FO

3

m/z:

2

11.0765, Found 211.0766; FTMS (ESI-TOF) m/z:

+

[

M+H] Calcd for C11

H

12FO

3

m/z: 211.07650, Found

2

11.07671; GC [200–260 (10 °C/min)]: t =1.928 min.

R

3

a–g and 210 nm for γ-butyrolactones rac-4a–g; the HPLC

Methyl 4-(4-chlorophenyl)-4-oxobutanoate (2c). Yield

[46]

analyses were executed in an isocratic and isothermal

2 2

94% (1.73 g); white solid; mp 52–55 °C (CH Cl ) [lit.

1

0

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

5

1.6–52.8 °C (Et

2

O)]; R

H NMR (500 MHz, CDCl

t, J=6.6 Hz, 2H), 3.70 (s, 3H), 7.39–7.47 (m, 2H), 7.88–

f

[hexane/AcOEt (1:1 v/v)] 0.64;

yellowish oil, which was chromatographed on silica gel

1

3

): δ 2.76 (t, J=6.6 Hz, 2H), 3.27

2 2

eluting with the appropriate mixture of CH Cl /AcOEt

(

(90:10, v/v) as an eluent to afford the respective γ-hydroxy

ester rac-3a–f and γ-butyrolactone rac-4a–f. [Attention: in

the case of 2g the purification procedure was slightly

modified. After the completion of the reaction (6 h) the

precipitated dark-yellowish solid was filtered off under

1

3

7

5

.95 (m, 2H); C NMR (126 MHz, CDCl ): δ 27.9, 33.3,

3

1.9, 128.9, 129.4, 134.8, 139.7, 173.2, 196.8; MS (ESI-

+

TOF) m/z: [M+H] Calcd for C11

H

12ClO

3

m/z: 227.0470,

+

Found 227.0470; FTMS (ESI-TOF) m/z: [M+H] Calcd for

C

2

+

11

H

12ClO

3

m/z: 227.04695, Found 227.04716; GC [200–

3

suction and washed with portion of PhCH (70 mL) thus

60 (10 °C/min)]: t =2.969 min.

R

yielding pure γ-butyrolactone rac-4g. In turn, the

remaining filtrate was quenched with cold 1 % aqueous

3 2

solution of NaHCO (100 mL), extracted with Et O (3 × 50

Methyl 4-(4-bromophenyl)-4-oxobutanoate (2d). Yield

[

47]

mL), and the combined organic layer was additionally

washed with H O (100 mL), dried over anhydrous MgSO

4

8

5

6% (1.89 g); white solid; mp 49–53 °C (CH

1–52 °C (H O)]; R [hexane/AcOEt (1:1 v/v)] 0.78; H

): δ 2.76 (t, J=6.6 Hz, 2H), 3.27 (t,

Cl ) [lit.

2 2

1

2

f

2

and chromatographed on SiO in accordance to the above-

NMR (500 MHz, CDCl

3

mentioned procedure affording γ-hydroxy ester rac-3g and

residual γ-butyrolactone rac-4g].

J=6.6 Hz, 2H), 3.70 (s, 3H), 7.58–7.63 (m, 2H), 7.81–7.86

13

(

m, 2H); C NMR (126 MHz, CDCl

3

): δ 27.9, 33.3, 51.9,

1

28.4, 129.5, 131.9, 135.2, 173.2, 197.0; MS (ESI-TOF)

+

m/z: [M+H] Calcd for C11

H

12BrO

3

m/z: 270.9965, Found

Methyl 4-hydroxy-4-phenylbutanoate (rac-3a). Yield

+

2

70.9892; FTMS (ESI-TOF) m/z: [M+H] Calcd for

32% (321 mg); colorless oil; R [CH Cl /AcOEt (90:10

f 2 2

+

1

C

11

H

12BrO

3

m/z: 270.99643, Found 270.99646; GC [200–

v/v)] 0.31; H NMR (500 MHz, CDCl

3

): δ 1.99–2.16 (m,

2

60 (10 °C/min)]: t =3.718 min.

R

2H), 2.34–2.52 (m, 2H), 3.66 (s, 3H), 4.65–4.81 (m, 1H),

1

3

7

.24–7.30 (m, 1H), 7.31–7.37 (m, 4H); C NMR (126

MHz, CDCl ): δ 30.4, 33.8, 51.7, 73.5, 125.7, 127.6, 128.5,

144.0, 174.3; MS (ESI-TOF) m/z: [M+H] Calcd for

3

Methyl 4-(4-methylphenyl)-4-oxobutanoate (2e). Yield

+

[

48]

8

5

2 2

1% (1.37 g); white solid; mp 46–47 °C (CH Cl ) [lit.

+

C

11

H

15

O

3

m/z: 195.1016, Found 195.1558, [M+Na] Calcd

3

for C11H14NaO m/z: 217.0841, Found 217.1475, [M+H–

+

1–53 °C (AcOEt/petroleum ether)]; R

f

[hexane/AcOEt

): δ 2.40 (s, 3H),

1

(

1:1 v/v)] 0.70; H NMR (500 MHz, CDCl

3

+

H

2

O] Calcd for

C

11

H

13

O

2

m/z: 177.0911, Found

+

2

7

.75 (t, J=6.9 Hz, 2H), 3.29 (t, J=6.9 Hz, 2H), 3.69 (s, 3H),

13

177.0959; FTMS (ESI-TOF) m/z: [M+H–H

2

O] Calcd for

m/z: 177.09101, Found 177.09114; GC [200–

260 (10 °C/min)]: t =2.06 min; HPLC [n-hexane-i-PrOH

(90:10, v/v); f=0.8 mL/min; λ=220 nm (Chiralcel OD-H)]:

R

t =13.193 and 13.775 min.

.22–7.28 (m, 2H), 7.85–7.90 (m, 2H); C NMR (126

): δ 21.6, 28.1, 33.3, 51.8, 128.1, 129.3, 134.1,

+

11 13 2

C H O

MHz, CDCl

3

+

R

1

44.0, 173.4, 197.6; MS (ESI-TOF) m/z: [M+H] Calcd for

+

C

12 15 3

H O m/z: 207.1016, Found 207.0889; FTMS (ESI-

+

TOF) m/z: [M+H] Calcd for C12

H

15

O

3

m/z: 207.10157,

R

Found 207.10163; GC [200–260 (10 °C/min)]: t =2.640

min.

5-Phenyldihydrofuran-2(3H)-one (rac-4a). Yield 23%

192 mg); colorless oil; R [CH Cl /AcOEt (90:10 v/v)]

.71; H NMR (500 MHz, CDCl ): δ 2.08–2.25 (m, 1H),

2.55–2.74 (m, 3H), 5.49 (dd, J=7.8, 6.4 Hz, 1H), 7.28–7.46

(

0

f

2

1

3

Methyl 4-(4-methoxyphenyl)-4-oxobutanoate (2f). Yield

[

49]

8

4

6% (1.56 g); white solid; mp 51–53 °C (CH

8.1–49.4 °C (MeOH/AcOEt)]; R [hexane/AcOEt (1:1

): δ 2.74 (t, J=6.9

2 2

Cl ) [lit.

1

3

(

m, 5H); C NMR (126 MHz, CDCl

3

): δ 29.0, 30.9, 81.2,

f

1

125.3, 128.4, 128.8, 139.4, 176.9; MS (ESI-TOF) m/z:

v/v)] 0.76; H NMR (500 MHz, CDCl

3

+

[

M+H] Calcd for C10

163.1138, [M+Na] Calcd for C10

Found 185.0833; FTMS (ESI-TOF) m/z: [M+H] Calcd for

11 2

H O

m/z: 163.0754, Found

H10NaO m/z: 185.0578,

2

+

Hz, 2H), 3.26 (t, J=6.9 Hz, 2H), 3.69 (s, 3H), 3.86 (s, 3H),

1

3

6

.90–6.95 (m, 2H), 7.93–7.98 (m, 2H); C NMR (126

): δ 28.1, 33.0, 51.8, 55.5, 113.7, 129.6, 130.3,

MHz, CDCl

3

+

C

10

H

11

O

2

m/z: 163.07536, Found 163. 07545; GC [200–

260 (10 °C/min)]: t =2.26 min; HPLC [n-hexane-i-PrOH

(85:15, v/v); f=0.8 mL/min; λ=210 nm (Chiralcel OD-H)]:

=16.033 (R-isomer) and 17.239 (S-isomer) min or [n-

hexane-i-PrOH (90:10, v/v); f=0.8 mL/min; λ=210 nm

Chiralcel OD-H)]: t =20.658 (R-isomer) and 22.349 (S-

1

63.6, 173.5, 196.5; MS (ESI-TOF) m/z: [M+H] Calcd for

+

R

C

12 15 4

H O m/z: 223.0965, Found 223.0919; FTMS (ESI-

+

TOF) m/z: [M+H] Calcd for C12

H

15

O

4

m/z: 223.09649,

t

R

Found 223.09660; GC [200–260 (10 °C/min)]: t =3.907

min.

(

R

isomer) min or [n-hexane-i-PrOH (80:20, v/v); f=1.5

Methyl 4-oxo-4-(pyren-1-yl)butanoate (2g). Yield 76%

1.98 g); yellow solid; mp 111–111.5 °C (hexane/AcOEt);

mL/min; λ=210 nm (S,S-Whelk-O 1)]: t

R

=5.823 (R-

(

isomer) and 6.637 (S-isomer) min.

1

R

f

[hexane/AcOEt (1:1 v/v)] 0.80; H NMR (500 MHz,

DMSO-d ): δ 1.94 (t, J=6.5 Hz, 2H), 2.65 (t, J=6.5 Hz, 2H),

.77 (s, 3H), 7.22–7.27 (m, 1H), 7.31–7.36 (m, 1H), 7.39–

.44 (m, 2H), 7.45–7.52 (m, 3H), 7.65–7.70 (m, 1H), 7.85–

6

Methyl 4-(4-fluorophenyl)-4-hydroxybutanoate (rac-3b).

Yield 56% (617 mg); colorless oil; R [CH Cl /AcOEt

(90:10 v/v v/v)] 0.31; H NMR (500 MHz, CDCl ): δ 1.98–

.07 (m, 2H), 2.38–2.45 (m, 2H), 3.66 (s, 3H), 4.73 (t,

2

7

3

1

f

2

1

3

6

.91 (m, 1H); C NMR (126 MHz, DMSO-d ): δ 28.2,

2

6.8, 51.5, 123.5, 124.0, 124.4, 124.4, 126.0, 126.5, 126.6,

26.7, 127.1, 128.2, 129.3, 129.4, 129.9, 130.6, 132.0,

1

3

J=6.4 Hz, 1H), 6.99–7.05 (m, 2H), 7.28–7.33 (m, 2H);

NMR (126 MHz, CDCl ): δ 30.4, 34.0, 51.9, 73.0, 115.4 (d,

JC–F=21.5 Hz), 127.5 (d, JC–F=7.9 Hz), 139.9 (d, JC–F=2.9

C

+

3

33.1, 173.0, 202.8; MS (ESI-TOF) m/z: [M+H] Calcd for

+

C

21

H

17

O

3

m/z: 317.1173, Found 317.0753; FTMS (ESI-

19

+

Hz), 162.3 (d, JC–F=245.6 Hz), 174.4; F NMR (470 MHz,

+

TOF) m/z: [M+H] Calcd for C21

H

17

O

3

m/z: 317.11722,

3

CDCl ): δ –115.48 (m); MS (ESI-TOF) m/z: [M+H] Calcd

+ +

Found 317.11728; GC [260 (const.)]: t

R

=Not Found.

for C11

Calcd for C11

FTMS (ESI-TOF) m/z: [M+H–H

m/z: 195.08158, Found 195.08168; GC [200–260

10 °C/min)]: t =1.97 min; HPLC [n-hexane-i-PrOH

(90:10, v/v); f=0.8 mL/min; λ=220 nm (Chiralcel OD-H)]:

R

t =10.445 and 11.624 min or [n-hexane-i-PrOH (95:5,

H

14FO

3

m/z: 213.0922, Not Found, [M+H–H

2

O]

+

H

12FO m/z: 195.0816, Found 195.0816;

2

General procedure for the synthesis of racemic γ-

hydroxy esters rac-3a–g and γ-butyrolactones rac-4a–g

+

2

O] Calcd for C11H12FO

2

(

R

To a solution of the appropriate γ-keto ester 2a–g (5.20

mmol) in a mixture of Et

2

O/MeOH (18 mL, 1:1 v/v) was

added a solution of NaBH

4

(0.50 equiv for 2a–d and 0.75

O (400 µL) with ice cooling.

Attention: in the case of 2g additional portion of PhCH

10 mL) was added to increase solubility of the substrate].

v/v); f=0.8 mL/min; λ=220 nm (Chiralcel OD-H)]:

equiv for 2e–g) in H

2

t

R

=17.542 and 20.095 min.

[

(

3

5

-(4-Fluorophenyl)dihydrofuran-2(3H)-one

(rac-4b).

Cl /AcOEt

): δ 2.10–

.23 (m, 1H), 2.61–2.70 (m, 3H), 5.48 (dd, J=8.3, 5.9 Hz,

After stirring at 0–5 °C for 2 h (for 2a–d) and 6 h (for 2e–

Yield 21% (199 mg); colorless oil; R

f

[CH

2

f), additional portion of Et

excess of NaBH was decomposed with cold 1 % aqueous

solution of NaHCO (30 mL). The reaction mixture was

extracted with Et O (3 × 30 mL), washed with H O (100

mL), and dried over anhydrous MgSO . After removal of

the drying agent, the solvent was evaporated to give a

2

O (20 mL) was added and the

1

(

90:10 v/v)] 0.75; H NMR (500 MHz, CDCl

3

4

2

1

3

13

H), 7.04–7.11 (m, 2H), 7.28–7.35 (m, 2H); C NMR

): δ 29.1, 31.1, 80.8, 115.8 (d, JC–F=21.5

2

(

126 MHz, CDCl

3

4

Hz), 127.3 (d, JC–F=8.8 Hz), 135.2 (d, J=2.9 Hz), 161.8 (d,

19

3

J=247.9 Hz), 176.7; F NMR (470 MHz, CDCl ): δ –

1

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

+

1

13.80 (m); MS (ESI-TOF) m/z: [M+H] Calcd for

2.10 (m, 2H), 2.14 (br. s., 1H), 2.34 (s, 3H), 2.42 (t, J=7.3

+

C

10

H

10FO

2

m/z: 181.0660, Found 181.0973; FTMS (ESI-

Hz, 2H), 3.66 (s, 3H), 4.64–4.76 (m, 1H), 7.12–7.19 (m,

+

13

TOF) m/z: [M+H] Calcd for C10

H

10FO

2

m/z: 181.06593,

=2.17

min; HPLC [n-hexane-i-PrOH (85:15, v/v); f=0.8 mL/min;

λ=210 nm]: t =14.876 (R-isomer) and 16.038 min (S-

isomer) or [n-hexane-i-PrOH (90:10, v/v); f=0.8 mL/min;

λ=210 nm (Chiralcel OD-H)]: t =19.536 (R-isomer) and

2H), 7.20–7.26 (m, 2H); C NMR (126 MHz, CDCl

3

): δ

Found 181.06602; GC [200–260 (10 °C/min)]: t

R

21.2, 30.6, 33.9, 51.8, 73.5, 125.8, 129.3, 137.5, 141.2

+

174.5; MS (ESI-TOF) m/z: [M+H] Calcd for C12

H

17

O

3

+

R

m/z: 209.1173, Not Found, [M+H–H

2

O] Calcd for

+

C H O m/z: 191.1067, Found 191.1080; FTMS (ESI-

12 15 2

+

R

TOF) m/z: [M+H–H

191.10666, Found 191.10677; GC [200–260 (10 °C/min)]:

=2.45 min; HPLC [n-hexane-i-PrOH (90:10, v/v); f=0.8

mL/min; λ=220 nm (Chiralcel OD-H)]: t =12.132 and

2.910 min.

2

O] Calcd for C12

H

15

O

2

m/z:

2

1.294 (S-isomer) min or [n-hexane-i-PrOH (80:20, v/v);

f=1.5 mL/min; λ=210 nm (S,S-Whelk-O 1)]: t

isomer) and 6.730 (S-isomer) min.

R

=6.065 (R-

t

R

1

Methyl 4-(4-chlorophenyl)-4-hydroxybutanoate (rac-3c).

Yield 52% (620 mg); colorless oil; R

f

[CH

2

Cl

): δ 1.98–

.05 (m, 2H), 2.42 (td, J=7.2, 2.5 Hz, 2H), 2.56 (br. s., 1H), (CH

.66 (s, 3H), 4.73 (t, J=6.4 Hz, 1H), 7.25–7.33 (m, 4 H); [CH

2

/AcOEt

5-(4-Methylphenyl)dihydrofuran-2(3H)-one

(rac-4e).

1

(

90:10 v/v)] 0.36; H NMR (500 MHz, CDCl

3

Yield 18% (164 mg); white solid; mp 70–71 °C

[

52]

2

3

2

Cl

2

/AcOEt)

[lit.

70–71°C

(CH

2

Cl

2

)];

R

f

1

2

/AcOEt (90:10 v/v)] 0.76; H NMR (500 MHz,

): δ 2.13–2.25 (m, 1H), 2.36 (s, 3H), 2.57–2.70 (m,

13

C NMR (126 MHz, CDCl

3

): δ 30.4, 33.9, 51.9, 72.9,

CDCl

3

1

3

1

27.3, 128.7, 133.4, 142.7, 174.5; MS (ESI-TOF) m/z:

3H), 5.45–5.50 (m, 1H), 7.17–7.25 (m, 4H); C NMR

(126 MHz, CDCl ): δ 21.2, 29.1, 31.0, 81.4, 125.5, 129.5,

136.4, 138.4, 177.1; MS (ESI-TOF) m/z: [M+H] Calcd for

+

[

M+H] Calcd for C11

H

14ClO

2

O] Calcd for C11 12ClO m/z: 211.0521, Found

+

3

m/z: 229.0626, Not Found,

3

+

M+H–H

2

H

+

2

11.0645; FTMS (ESI-TOF) m/z: [M+H–H

2

O] Calcd for

m/z: 211.05203, Found 211.05206; GC [200–

=3.09 min; HPLC [n-hexane-i-PrOH

C H O m/z: 177.09101, Found 177.1155; FTMS (ESI-

11 13 2

+

C

11

H

12ClO

2

TOF) m/z: [M+H] Calcd for C11

Found 177. 09114; GC [200–260 (10 °C/min)]: t

min; HPLC [n-hexane-i-PrOH (90:10, v/v); f=0.8 mL/min;

λ=210 nm (Chiralcel OD-H)]: t =16.246 (R-isomer) and

H

13

O

2

m/z: 177.09101,

2

60 (10 °C/min)]: t

R

=2.68

(

90:10, v/v); f=0.8 mL/min; λ=220 nm (Chiralcel OD-H)]:

t

R

=10.863 and 12.669 min.

R

1

7.978 (S-isomer) min or [n-hexane-i-PrOH (80:20, v/v);

f=1.5 mL/min; λ=210 nm (S,S-Whelk-O 1)]: t

R

=5.648 (R-

5

-(4-Chlorophenyl)dihydrofuran-2(3H)-one

(rac-4c).

isomer) and 6.773 (S-isomer) min.

Yield 10% (101 mg); white solid; mp 54–56 °C

[

50]

(

[

CH

2

Cl

2

/AcOEt) [lit.

51 °C (petroleum ether)]; R

f

1

2

/AcOEt (90:10 v/v)] 0.76; H NMR (500 MHz,

Methyl 4-(4-methoxyphenyl)-4-hydroxybutanoate (rac-

CDCl

3

): δ 2.07–2.22 (m, 1H), 2.58–2.73 (m, 3H), 5.43–

3f). Yield 22% (262 mg); white solid; mp 40–42 °C

13

[53]

5

.51 (m, 1H), 7.24–7.30 (m, 2H), 7.33–7.40 (m, 2H);

C

(CH

[CH

3 2

CDCl ): δ 1.99–2.11 (m, 3H, CH and OH), 2.39–2.44 (m,

2

Cl /AcOEt) [lit. 41–43°C (Et O/petroleum ether)];

2

1

NMR (126 MHz, CDCl

3

): δ 29.0, 31.0, 80.5, 126.8, 129.1,

R

f

2 2

Cl /AcOEt (90:10 v/v)] 0.22; H NMR (500 MHz,

+

1

34.4, 138.0, 176.63; MS (ESI-TOF) m/z: [M+H] Calcd

+

for C10

H

10ClO

2

m/z: 197.0364, Found 197.0844; FTMS

2H), 3.66 (s, 3H), 3.80 (s, 3H), 4.69 (dd, J=7.8, 5.4 Hz,

+

13

(

ESI-TOF) m/z: [M+H] Calcd for C10

H

10ClO

2

m/z:

1H), 6.86–6.90 (m, 2H), 7.24–7.30 (m, 2H); C NMR

1

97.03638, Found 197.03657; GC [200–260 (10 °C/min)]:

=3.40 min; HPLC [n-hexane-i-PrOH (98:2, v/v); f=1.2

(126 MHz, CDCl ): δ 30.6, 33.9, 51.8, 55.4, 73.3, 114.0,

3

+

t

R

127.2, 136.3, 159.3, 174.4; MS (ESI-TOF) m/z: [M+H]

+

mL/min; λ=210 nm (Chiralcel OD-H)]: t

R

=33.745 (R-

17 4

Calcd for C12H O m/z: 225.1122, Not Found, [M+H–

+ +

isomer) and 35.919 (S-isomer) min or [n-hexane-i-PrOH

80:20, v/v); f=1.5 mL/min; λ=210 nm (S,S-Whelk-O 1)]:

=6.038 (R-isomer) and 6.799 (S-isomer) min.

H

2

O] Calcd for

C

12

H

15

O

3

m/z: 207.1016, Found

+

(

207.1030; FTMS (ESI-TOF) m/z: [M+H–H

2

O] Calcd for

m/z: 207.10157, Found 207.10168; GC [200–

60 (10 °C/min)]: t =3.53 min; HPLC [n-hexane-i-PrOH

95:5, v/v); f=0.8 mL/min; λ=220 nm (Chiralpak AD-H)]:

R

=31.816 and 32.984 min.

+

t

R

12 15 3

C H O

2

(

R

Methyl 4-(4-bromophenyl)-4-hydroxybutanoate (rac-

t

3

d). Yield 60% (851 mg); colorless oil; R

f

[CH

2

Cl

): δ 2.00–

.09 (m, 2H), 2.37 (br. s., 1H), 2.44 (td, J=7.1, 3.2 Hz, 2H), 5-(4-Methoxyphenyl)dihydrofuran-2(3H)-one (rac-4f).

.68 (s, 3H), 4.69–4.80 (m, 1H), 7.21–7.25 (m, 2H), 7.45– Yield 20% (196 mg); white solid; mp 58–59 °C

2

/AcOEt

1

(

90:10 v/v)] 0.36; H NMR (500 MHz, CDCl

3

2

3

7

5

1

3

[53]

.50 (m, 2H); C NMR (126 MHz, CDCl

2.0, 73.0, 121.5, 127.6, 131.8, 143.2, 174.4; MS (ESI-

3

): δ 30.4, 33.9,

(CH

[CH

CDCl ): δ 2.14–2.25 (m, 1H), 2.56–2.68 (m, 3H), 3.81 (s,

3H), 5.45 (dd, J=8.3, 6.1 Hz, 1H), 6.89–6.93 (m, 2H),

2

Cl /AcOEt) [lit. 55–57°C (Et O/petroleum ether)];

2

1

R

f

2 2

Cl /AcOEt (90:10 v/v)] 0.71; H NMR (500 MHz,

+

TOF) m/z: [M+H] Calcd for C11

H

14BrO

3

m/z: 273.0121,

3

+

Not Found, [M+H–H

2

O] Calcd for C11

H

12BrO

2

m/z:

13

2

55.0016, Found 255.0375; FTMS (ESI-TOF) m/z: [M+H–

7.23–7.28 (m, 2H); C NMR (126 MHz, CDCl

3

): δ 29.3,

+

H

2

O] Calcd for C11

H

12BrO

2

m/z: 255.00152, Found

=3.86 min;

31.0, 55.4, 81.5, 114.2, 127.1, 131.3, 159.9, 177.0; MS

+

2

55.00162; GC [200–260 (10 °C/min)]: t

R

(ESI-TOF) m/z: [M+H] Calcd for C11

193.0860, Found 193.0984, [M+Na] C11

13 3

H O + m/z:

HPLC [n-hexane-i-PrOH (90:10, v/v); f=0.8 mL/min;

λ=220 nm (Chiralcel OD-H)]: t =11.642 and 13.443 min.

H

12NaO

3

m/z:

R

215.0684, Found 215.1066; FTMS (ESI-TOF) m/z:

+

[

M+H] Calcd for C11

93.08610; GC [200–260 (10 °C/min)]: t

HPLC [n-hexane-EtOH (99:1, v/v); f=1.0 mL/min; λ=210

nm (Chiralcel OD-H)]: t =52.851 (R-isomer) and 55.599

H

13

O

3

+ m/z: 193.08592, Found

1

R

=3.89 min;

5

-(4-Bromophenyl)dihydrofuran-2(3H)-one

(rac-4d).

Yield 11% (134 mg); white solid; mp 89–90 °C

[

51]

R

(

CH

ether)]; R

MHz, CDCl

Cl

2 2

/AcOEt) [lit.

3

81.5–82.5°C (CHCl /petroleum

1

(S-isomer) min or [n-hexane-i-PrOH (80:20, v/v); f=1.5

f

[CH Cl

2

/AcOEt (90:10 v/v)] 0.75; H NMR (500

): δ 2.08–2.21 (m, 1H), 2.61–2.72 (m, 3H),

.43–5.49 (m, 1H), 7.18–7.24 (m, 2H), 7.49–7.56 (m, 2H);

3

mL/min; λ=210 nm (S,S-Whelk-O 1)]: t

R

=9.183 (S-isomer)

3

and 11.228 (R-isomer) min.

5

1

C NMR (126 MHz, CDCl

3

): δ 29.0, 31.0, 80.5, 122.5,

+

1

27.1, 132.1, 138.6, 176.6; MS (ESI-TOF) m/z: [M+H]

Methyl 4-hydroxy-4-(pyren-1-yl)butanoate (rac-3g).

+

Calcd for C10

H

10BrO

2

m/z: 240.9859, Found 241.0618,

BrNaO m/z: 262.9684, Found

63.0383; FTMS (ESI-TOF) m/z: [M+H] Calcd for

Yield 10% (163 mg); brownish solid; mp 65–66 °C

1

[

M+Na] Calcd for C10

H

9

2

(CH

Cl

2 2

/AcOEt); R

f

[CH

Cl

2 2

/AcOEt (90:10 v/v)] 0.36; H

+

2

3 2

NMR (500 MHz, CDCl ): δ 2.22–2.39 (m, 3H, CH and

+

C

10

H

10BrO

2

m/z: 240.98587, Found 241.98605; GC [200–

60 (10 °C/min)]: t =4.25 min; HPLC [n-hexane-i-PrOH

80:20, v/v); f=1.5 mL/min; λ=210 nm (S,S-Whelk-O 1)]:

=6.146 (R-isomer) and 6.945 (S-isomer) min.

OH), 2.48–2.69 (m, 2H), 3.69 (s, 3H), 5.84 (dd, J=8.2, 4.0

Hz, 1H), 7.97–8.11 (m, 4H), 8.13–8.22 (m, 4H), 8.27–8.35

2

R

13

(

3

(d, J=9.3 Hz, 1H); C NMR (126 MHz, CDCl ): δ 30.6,

t

R

33.8, 51.9, 70.3, 122.4, 123.2, 124.9, 125.1, 125.2, 125.4,

1

26.1, 127.4, 127.5, 127.6, 127.9, 130.8, 130.8, 131.5,

+

1

37.7, 174.6; MS (ESI-TOF) m/z: [M+H] Calcd for

Methyl 4-(4-methylphenyl)-4-hydroxybutanoate (rac-

e). Yield 34% (366 mg); colorless oil; R [CH Cl /AcOEt

90:10 v/v)] 0.45; H NMR (500 MHz, CDCl ): δ 2.00–

+

C

H O m/z: 319.1329, Not Found, [M+H–H O] Calcd

21 19 3 2

3

(

f

2

+

1

for C21

H

17

O

2

m/z: 301.1224, Found 301.1976; FTMS

+ +

3

(

2 17 2

ESI-TOF) m/z: [M+H–H O] Calcd for C21H O m/z:

1

2

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

3

01.12231, Found 301.12220; GC [260 (const.)]: t

R

=Not

Procedures for Method B and C: The thus composed

biocatalytic reaction systems was shaken in sample tube

for 20 h at 30 ºC and 250 rpm. After this time, the reaction

was stopped by extraction with AcOEt (3 × 2 mL). The

Found; HPLC [n-hexane-i-PrOH (78:22, v/v); f=1.0

mL/min; λ=220 nm (Chiralpak AD-H)]: t

9

R

=8.371 and

.088 min.

4

organic layer was separated, dried (MgSO ), the drying

agent was filtered off, the filtrate was additionally

centrifuged (5 min, 6000 rpm), and only then the

supernatant was transferred into a vial to perform GC

analysis in order to establish the % conv. After evaporation

of the solvent (AcOEt) under reduced pressure, the crude

5

-(Pyren-1-yl)oxolan-2-one (rac-4g). Yield 46% (685

[54]

mg); yellow solid; mp 170–171 °C (CH

2

Cl

2

/AcOEt) [lit.

1

f 2 2

78 °C (AcOH)]; R [CH Cl /AcOEt (90:10 v/v)] 0.78; H

NMR (500 MHz, CDCl

3

): δ 2.27–2.42 (m, 1H), 2.67–2.83

(

m, 2H), 2.90–3.02 (m, 1H), 6.52 (t, J=7.2 Hz, 1H), 8.01–

1

3

2 2

reaction mixture was re-dissolved in CH Cl (0.5 mL),

8

.12 (m, 6H) 8.15–8.23 (m, 3H); C NMR (126 MHz,

): δ 28.9, 31.0, 79.1, 121.7, 121.8, 124.8, 124.9,

25.1, 125.5, 125.8, 126.3, 127.1, 127.5, 127.8, 128.4,

30.6, 131.3, 131.4, 132.6, 177.3; MS (ESI-TOF) m/z:

followed by the addition of 1-2 drops of trifluoroacetic

acid (TFA). The stirring for 1 h at 30 °C completed the

lactonization process, and the reaction was further worked-

CDCl

3

1

[

+

up with saturated aqueous solution of NaHCO

3

(3 × 1.0

Cl (1.0

mL), washed with brine (1.0 mL) and dried over anhydrous

MgSO . Afterwards, the drying agent was filtered off, the

M+H] Calcd for C20

H

15

O

2

m/z: 287.1067, Found

+

mL). Next, the organic layer was diluted with CH

2

87.1706; FTMS (ESI-TOF) m/z: [M+H] Calcd for

+

C

20

H

15

O

2

m/z: 287.10666, Found 287.10654; GC [260

=Not Found; HPLC [n-hexane-i-PrOH (78:22,

v/v); f=1.0 mL/min; λ=254 nm (Chiralpak AD-H)]:

=9.673 and 10.268 min.

4

(

const.)]: t

R

filtrate was centrifuged (5 min, 6000 rpm), and the

resulting supernatant was concentrated under vacuum to

yield the respective optically active γ-butyrolactone 4a.

After evaporation of the volatiles at reduced pressure by

means of rotary evaporator and high-vacuum oil pump (at

p=0.05 mmHg), the crude mixture was re-dissolved in 2-

PrOH (1.0 mL) and subjected directly to chiral HPLC to

establish % ee-values. For additional data see Table 1.

t

R

General procedure for the analytical-scale studies on

stereoselective bioreduction of methyl 4-oxo-4-

phenylbutanoate (2a) with different whole-cell

biocatalysts – screening procedure

Method A: Each of the lyophilized whole-cell biocatalysts

including wild type microorganisms and/or E. coli with

overexpressed recombinant ADHs (10 mg) were

suspended in the reaction solution (500 μL), containing 20

mM glucose, catalytic amounts of the respective cofactors

General procedure for the analytical-scale studies on

stereoselective bioreduction of methyl 4-oxo-4-

phenylbutanoate (2a) with different whole-cell

biocatalysts – optimization procedure

[

0.5 mM NADH, 0.5 mM NADPH], and 10 mM substrate

a in a mixture of 0.1 M Tris-HCl buffer (pH 7.5)/2-PrOH

500 μL; 90:10, v/v). Biotransformations were conducted

in a glass vials (V = 1.5 mL) without air access for 20 h at

Lyophilized cells (10 mg) containing overexpressed ADHs

were rehydrated in 0.1 M Tris-HCl buffer (pH 7.5, 300 μL)

and the solution of 0.5 mM NAD(P)H in the same aqueous

buffer (50 μL each) for 30 min at 30 ºC and 250 rpm on a

rotary shaker in an glass vial (V = 1.5 mL). Then, 20 mM

glucose in Tris-HCl buffer solution (50 μL) and 10 mM

substrate 2a solution in 2-PrOH (50 μL) were added.

2

(

3

0 °C using a laboratory shaker (250 rpm). After this time,

the reaction was stopped by extracting the content of the

vial with AcOEt (3 × 1 mL), the combined organic phase

4

was dried over anhydrous MgSO , the filtrate was

[

Attention: in the case of the reactions carried out without

additionally centrifuged (5 min, 6000 rpm), and only then

the supernatant was transferred into a HPLC vial. After the

determination of the conversion using GC analyses the

solvent was evaporated under reduced pressure, the oil

residue was re-dissolved in HPLC-grade 2-PrOH (1.5 mL),

and finally, to establish enantiomeric excesses of optically

active product 4a the samples were analyzed by HPLC on

a chiral stationary phase (see Supporting Information,

Table S5). For additional data see also Table 1.

addition of NADPH or without both NADPH and glucose

or without both cofactors and glucose the missing volumes

of the reaction media were supplemented with 50 μL or

1

00 μL or 450 μL of 0.1 M Tris-HCl buffer (pH 7.5),

respectively]. The thus composed biocatalytic reaction

system was shaken at 30 ºC and 250 rpm for 48 h (in the

case of: Arthrobacter sp. DSM 7325) and for 20 h [in the

case of: E. coli/Lk-ADH-Lica, E. coli/RasADH, and E.

coli/SyADH]. After this time, the reaction was stopped by

extraction with AcOEt (3 × 1 mL), the organic layer was

Method B: To a suspension of wet E. coli/(S)-PED cell

mass (220 mg wet mass, 49 mg after drying, 2.9 mU/mg

assayed with 2a) in the mixture of 0.1 M MES-KOH buffer

4

combined and washed with brine (1 mL), dried (MgSO ),

the drying agent was filtered off, and the filtrate was

additionally centrifuged (5 min, 6000 rpm). Finally, the

supernatant was transferred into a HPLC vial and the rest

of the manipulations for the determination of reaction

conversion and % ee-values of optically active product 4a

were the same as described above. For additional data see

Table 2.

(

pH 5.5)/2-PrOH (850 µL, 40:60, v/v) a concentrated

solution of NADH in MES buffer/2-PrOH mixture (100 µL,

0:60, v/v) was added. The reaction was initiated by

4

adding a concentrated substrate 2a solution in 2-PrOH (50

μL). The final concentration of the substrate 2a and NADH

in the reaction mixture were 10 mM and 0.5 mM,

respectively. The rest of the manipulations are presented

below in section named: ‘Procedures for Method B and C’.

General procedure for the analytical-scale studies on

stereoselective bioreduction of methyl 4-oxo-4-

arylbutanoates (2a–g) with E. coli/RasADH and E.

coli/SyADH

Method C: The solutions of pure enzymes from

Aromatoleum

aromaticum,

that

are:

(S)-HPED

(

concentration: 3.7 mg/mL, activity 14.5 ±0.6 U/mL

To a suspension of the respective freeze-dried E. coli cells

containing the recombinant ADH (E. coli/RasADH, and E.

coli/SyADH, 10 mg each) in 0.1 M Tris-HCl buffer (pH

assayed with acetophenone, 1.25 ±0.03 U/mL assayed with

2

8

a) and (R)-HPED (concentration: 4.4 mg/mL, activity

1.7 ±5.2 U/mL assayed with acetophenone; 2.82 ±0.03

7

.5, 450 μL), the appropriate stock solution of substrate

a–g in 2-PrOH (50 μL) to reach 10 mM final

U/mL assayed with 2a) were used for testing reactions.

The respective enzyme solution (60 μL) was added to 0.1

M MES-KOH buffer (pH 5.5, 740 μL). Next, a

concentrated solution of NADH in the same MES aqueous

buffer (100 μL each) and portion of 2-PrOH (50 μL) were

added at once. The reaction was initiated by adding the

concentrated substrate 2a solution in 2-PrOH (50 μL). The

final concentration of 2a and NADH in the reaction

mixture were 10 mM and 0.5 mM, respectively. The rest of

the manipulations are presented below in section named:

2

concentration was added and the reaction was shaken at 30

ºC and 250 rpm for 20 h (see Method A above). The

following experimental procedures were the same as

before. For additional data see Table 3.

General procedure for the analytical-scale studies on

stereoselective bioreduction of methyl 4-oxo-4-

arylbutanoates (2a–g) with E. coli/(S)-PED or (R)-

HPED

‘Procedures for Method B and C’.

1

3

Advanced Synthesis & Catalysis

10.1002/adsc.201901483

The following experimental procedures were the same as

for the reactions catalyzed by E. coli/(S)-PED (see Method

B above) or (R)-HPED (see Method C above). The only

difference is that the (R)-HPED-mediated bioreductions of

Procedure for 4.2 mmol scale fed-batch E. coli/(S)-

PED)-catalyzed bioreduction of methyl 4-oxo-4-

phenylbutanoate (2a)

2

a–g were performed for longer time period (40 h) than in

A solution of methyl 4-oxo-4-phenylbutanoate (2a, 404.7

mg, 2.1 mmol) in 2-PrOH (3 mL) was added to a

suspension of wet cells of E. coli/(S)-PED (46.2 g, 2.9

mU/mg assayed with 2a, 910.6 mU/mg assayed with

acetophenone) and NADH (74.5 mg, 105.0 μmol, final

concentration 0.5 mM) in the mixture of 0.1 M MES-KOH

buffer (pH 5.5)/2-PrOH (210 mL, 40:60, v/v). The reaction

mixture was shaken at 30 ºC and 250 rpm. The

concentration of the substrate 2a was directly monitored

using HPLC in 20 h intervals. After 20 h of the reaction we

observed almost complete depletion of the substrate 2a

the previous experiments, and in two variants – with 0.5

mM NADH final concentration (Method C) or with 1.0

mM NADH final concentration (Method D). The analyses

of the samples were performed on GC and HPLC after

routine work-up of the crude reaction mixtures using

AcOEt and subsequent derivatization toward the respective

2 2

optically active lactone by means of TFA/CH Cl -based

procedure. For additional data see Table 3.

Enzyme activity

(

0.15 mM remained). Therefore, a second portion of

methyl 4-oxo-4-phenylbutanoate (2a, 404.7 mg, 2.1 mmol)

in 2-PrOH (3 mL) was added. The reaction was continued

for additional 60 h (i.e. 80 h of total reaction time), after

which the substrate 2a concentration reached 1.58 mM

according to HPLC indications. Finally, the reaction was

terminated by extraction with AcOEt (3 × 400 mL). The

combined organic layers were dried over anhydrous

The enzyme activity unit (U) was defined as the amount of

the enzyme which converts μmol of substrate

1

(

acetophenone or γ-keto ester 2a) into the respective

product per minute at 30 °C. For details see Supporting

Information (section 1.10).

Enzyme concentration measurement

4

MgSO , and after filtering off the drying agent under

suction and evaporation of the volatiles, the residual oil

Protein concentrations were determined by the Coomassie

2 2

was re-dissolved in CH Cl (50 mL), followed by the

[

55]

dye binding assay according to Bradford protein assay

addition of 10 drops of trifluoroacetic acid (TFA). The

stirring for 5 h at 30 °C completed the lactonization

process, and the reaction mixture was further worked-up

[

56]

using Sigma Aldrich Bradford protocol. For details see

Supporting Information (section 1.11).

with saturated aqueous solution of NaHCO

3

(3 × 70 mL).

Cl (80 mL),

washed with brine (70 mL) and dried (MgSO ). After

evaporation of the solvent on rotavapor the crude product

683 mg) was chromatographed on SiO (50 g) using

Next, the organic layer was diluted with CH

2

HPLC measurements

4

The reagent concentrations were determined with HPLC

using Agilent 1100 system equipped with a DAD detector.

(

2

2 2

gradient of CH Cl /AcOEt (100, 98:2, 95:5, v/v) mixture

®

The separations were performed on Ascentic RP-Amide

as an eluent to afford optically active γ-phenyl-γ-

butyrolactone (S)-(–)-4a (457 mg, 2.82 mmol, 67% yield,

Express column (75 mm × 4.6 mm, 2.7 μm) at 40 ˚C with

2 3

the isocratic mobile phase composed of H O/CH CN

>

99% ee) as yellowish oil. For additional data see Table 4.

(

65:35, v/v), the flow rate of 1 ml/min and injection

volumes of 5 μL. The quantitation of substrate was

conducted at 205 nm against external standard calibration.

Retention time (t ) of 2a was 2.93 min.

R

Acknowledgements

Representative procedure for 0.6 mmol scale E. coli/(S)-

PED-catalyzed bioreduction of methyl 4-oxo-4-

phenylbutanoate (2a)

This work was financially supported by Warsaw University of

Technology (Faculty of Chemistry) in the frame of “Dean's

Funding Program for Young Investigators 2019”. Mateusz

Tataruch and Maciej Szaleniec acknowledge financial support of

ICSC PAS Statutory Fund. The European Union Erasmus+

programme (project number: 2018-1-PL01-KA103-047692) is

acknowledged for providing financial support for the research.

A solution of methyl 4-oxo-4-phenylbutanoate (2a, 115.3

mg, 0.6 mmol, final concentration 10 mM) in 2-PrOH (3

mL) was added to a suspension of wet cells of E. coli/(S)-

PED (13.2 g, 2.9 mU/mg of wet cell mass of E. coli/(S)-

PED assayed with 2a, 910.6 mU/mg assayed with

acetophenone) and NADH (21.3 mg, 30.0 μmol, final

concentration 0.5 mM) in the mixture of 0.1 M MES-KOH

buffer (pH 5.5)/2-PrOH (57 mL, 40:60, v/v). [Attention:

substrate 2a and NADH were added from the appropriately

prepared stock solutions in 2-PrOH or aqueous buffer,

respectively]. The thus composed biocatalytic reaction

system was shaken at 30 ºC and 250 rpm for 40 h. After

this time, the reaction was stopped by extraction with

AcOEt (3 × 120 mL), the combined organic layers dried

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FULL PAPER

Biocatalytic Asymmetric Reduction of γ-Keto

Esters to Access Optically Active γ-Aryl-γ-

butyrolactones

Adv. Synth. Catal. Year, Volume, Page – Page

a,

a

Paweł Borowiecki, * Natalia Telatycka, Mateusz

b

c

Tataruch, Anna Żądło-Dobrowolska, Tamara

d

e

Reiter, Karola Schühle, Johann Heider, Maciej

b

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Szaleniec and Wolfgang Kroutil

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Biocatalytic Asymmetric Reduction of γ-Keto Esters to Access Optically Active γ-Aryl-γ-butyrolactones

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