Efficient synthesis of the ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer

Article abstract of DOI:10.1016/j.bioorg.2018.07.010

The ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer were prepared in a short, operationally simple synthetic sequence from racemic β-butyrolactone. Enantioselective hydrolysis of β-butyrolactone with immobilized Candida antarctica lipase-B (CAL-B) results in (R)-β-butyrolactone and (S)-β-hydroxybutyric acid, which are easily converted to (R) or (S)-ethyl-3-hydroxybutyrate and reduced to (R) or (S)-1,3 butanediol. Either enantiomer of ethyl-3-hydroxybutyrate and 1,3 butanediol are then coupled, again using CAL-B, to produce the ketone body ester product. This is an efficient, scalable, atom-economic, chromatography-free, and low cost synthetic method to produce the ketone body esters.

Full text of DOI:10.1016/j.bioorg.2018.07.010

Bioorganic Chemistry 80 (2018) 560–564
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Efficient synthesis of the ketone body ester (R)-3-hydroxybutyryl-(R)-3-
hydroxybutyrate and its (S,S) enantiomer
⁎
Noah Budin, Erin Higgins, Anthony DiBernardo, Cassidy Raab, Chun Li, Scott Ulrich
Department of Chemistry, Ithaca College, 953 Danby Rd, Ithaca, NY 14850, United States
A B S T R A C T
The ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer were prepared in a short, operationally simple synthetic sequence from
racemic β-butyrolactone. Enantioselective hydrolysis of β-butyrolactone with immobilized Candida antarctica lipase-B (CAL-B) results in (R)-β-butyrolactone and (S)-
β-hydroxybutyric acid, which are easily converted to (R) or (S)-ethyl-3-hydroxybutyrate and reduced to (R) or (S)-1,3 butanediol. Either enantiomer of ethyl-3-
hydroxybutyrate and 1,3 butanediol are then coupled, again using CAL-B, to produce the ketone body ester product. This is an efficient, scalable, atom-economic,
chromatography-free, and low cost synthetic method to produce the ketone body esters.
1
. Introduction
hydroxybutyryl-(R)-3-hydroxybutyrate, was recently shown to safely
induce serum ketone body concentrations similar to prolonged fasting
or the ketogenic diet when taken orally by humans on a normal diet
[10,11]. It is proposed that this KE is converted to two equivalents of
βHB by hydrolysis of the ester followed by liver oxidation of the re-
sulting (R)-1,3 butanediol, a known metabolic precursor of βHB
(Fig. 2) [12].
Ketosis via ingestion of KEs produces a unique metabolic state in
which glucose and ketone bodies, normally mutually exclusive, exist
simultaneously in the bloodstream. This unusual metabolic state was
shown to enhance human athletic performance. Indeed, it was found
that ingestion of this KE improved performance in a cycling time-trial
test compared to subjects who consumed the equivalent number of
calories as carbohydrate [11]. This and similar ketone body esters also
have significant promise to replace the ketogenic diet for treating the
medical conditions that respond to ketosis.
The ketone bodies (R)-β-hydroxybutyrate (βHB) and acetoacetate
(
AcAc) are produced from fatty acids in the liver under glucose depri-
vation conditions, such as fasting or adherence to a high fat/low car-
bohydrate “ketogenic” diet (Fig. 1). The brain relies on carbohydrate as
a fuel source, so in its absence, ketone body production serves as an
alternative, fat-derived fuel source for the brain that is converted to
acetyl CoA and enter the citric acid cycle [1].
There is evidence that the ketogenic state is therapeutically useful
to treat a variety of neurological diseases. The ketogenic diet is an
effective and widely used epilepsy treatment [2]. Ketosis is being
explored for a variety of other neurological conditions as well. The
brain of patients with Alzheimer’s disease shows impaired glucose
uptake and utilization, which correlates with disease progression
[
3,4]. However, ketone body uptake and utilization is unaffected,
making it reasonable to suspect that ketosis is useful to treat Alz-
heimer’s disease [5]. Similarly, ketone bodies have been shown to be
protective of neurons in Parkinson’s disease [6,7]. Clinical trials of
the ketogenic diet are ongoing for these and other medical condi-
tions, including as an adjuvant to chemotherapy for cancer treatment
The synthetic scheme by which this KE was originally made is a
one-step, Candida antarctica lipase-B (CAL-B) mediated transester-
ification of (R)-ethyl-3-hydroxybutyrate with (R)-1,3 butanediol [13].
Our interest in ketone body esters led us to seek an efficient and
scalable synthesis of this KE which begins with inexpensive, achiral
starting materials. Considering that one dose of this KE in humans may
be 20–30 g of material, cost becomes an issue for longitudinal studies
of its effects on ketosis-treatable diseases, athletic performance, and
the emerging signaling properties of βHB [14]. Synthesis of the (S,S)
enantiomer is also of interest due to its less-well understood meta-
bolism. It is plausible that it is also converted to the natural ketone
bodies AcAc and (R)-βHB with different kinetics and may well be a
useful tool to achieve ketosis.
[
8].
Despite the utility and promise of the ketogenic diet, adherence to
the diet is difficult. It is uncomfortable, has a social cost, and side
effects. Ketone body esters (KE) are an alternative approach to induce
ketosis. Ketone body esters are taken as nutritional supplements─
they release ketone bodies directly after simple metabolic reactions,
and avoid the salt/acid load if βHB or AcAc were ingested directly
[
9]. The most well-known and effective ketone body ester, (R)-3-
⁎
Corresponding author.
E-mail address: sulrich@ithaca.edu (S. Ulrich).
https://doi.org/10.1016/j.bioorg.2018.07.010
Received 19 May 2018; Received in revised form 10 July 2018; Accepted 11 July 2018
0045-2068/ © 2018 Elsevier Inc. All rights reserved.

N. Budin et al.
Bioorganic Chemistry 80 (2018) 560–564
considerably slower hydrolysis of the (R) enantiomer (Fig. 4).
Washing the reaction mixture with saturated aqueous sodium bi-
carbonate easily removed (S)-3-hydroxybutyric acid and allowed re-
covery of pure (R)-β-butyrolactone in high yield and > 99% ee. (S)-3-
Hydroxybutyric acid can be recovered by acidifying the aqueous bi-
carbonate solution to pH 2 followed by continuous liquid-liquid ex-
traction with diethyl ether. We found that both the MTBE solvent and
CAL-B enzyme can be recycled.
Fig. 1. Chemical structures of the ketone bodies β-hydroxybutyrate and acet-
oacetate.
2.1. Synthesis of (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate
With pure (R)-β-butyrolactone in hand, we found that it underwent
smooth reduction with sodium borohydride in ethanol, unusual re-
activity presumably due to ring strain. The resulting (R)-1,3 butanediol
was purified by extraction into 2-propanol from water containing 25%
Fig. 2. Proposed metabolism of the KE (R)-3-hydroxybutyryl-(R)-3-hydro-
xybutyrate to two molar equivalents of βHB by ester hydrolysis and oxidation of
2 4
K HPO [16]. (R)-β-Butyrolactone was easily converted to (R)-ethyl 3-
1,3 butanediol.
hydroxybutyrate by treatment with catalytic sulfuric acid in ethanol.
The synthesis was completed using the previously reported transester-
ification of (R)-ethyl 3-hydroxybutyrate with (R)-1,3 butanediol cata-
lyzed by CAL-B under reduced pressure and solvent-free conditions to
yield the ketone body ester product (R,R)-3-hydroxybutyryl-3-hydro-
xybutyrate (Scheme 1) [13].
2. Results and discussion
Our synthetic approach towards (R)-3-hydroxybutyryl-(R)-3-hy-
droxybutyrate and its (S,S) enantiomer is based on having both halves
of the ester and both stereocenters originate from racemic β-butyr-
olactone. It was previously reported that immobilized Candida antarc-
tica lipase B (CAL-B) can enantioselectively trans-esterify racemic β-
butyrolactone with methanol to give (S)-methyl-3-hydroxybutyrate and
2.2. Attempted stereochemical inversion
We attempted to convert (S)-3-hydroxy butyric acid into (R)-ethyl-
-hydroxy butyrate, the starting material to generate the more valuable
(
R)-β-butyrolactone with quantitative conversion and > 99% ee [15].
3
We found that altering this protocol for enzymatic hydrolysis of racemic
β-butyrolactone with immobilized CAL-B and 0.6 M equivalents of
water in MTBE yielded (R)-β-butyrolactone and (S)-3-hydroxybutyric
acid with identical enantioselectivity as the previously reported me-
thanolysis reaction (Fig. 3).
(
R,R) ketone body ester. This route involves esterification, mesylation
of the hydroxyl group followed by S 2 inversion by cesium acetate
Scheme 2) [17]. In our hands, this route resulted in substantial
n
(
amounts of the acrylate elimination product, requiring chromatography
to recover the (R) ester. As such, these steps did not conform to our goal
of accessing the ketone body ester product in an operationally simple,
scalable manner.
Monitoring the reaction via chiral GC–MS showed that (S)-butyr-
olactone was converted to (S)-3-hydroxybutyric acid in < 8 h with
Fig. 3. Enantioselective hydrolysis of racemic β-butyrolactone by CAL-B. Chiral GC–MS chromatogram of the reaction prior to CAL-B addition (left) and after
overnight incubation (right).
1
.2
1.2
1
1
0
0
0
0
.8
.6
.4
.2
0.8
0.6
0
0
.4
.2
0
0
0
100
200
300
0
50
100
150
Time (minutes)
Time (hours)
Fig. 4. Kinetics of CAL-B hydrolysis of (S) and (R) β-butyrolactone.
561

N. Budin et al.
Bioorganic Chemistry 80 (2018) 560–564
Scheme 1. Synthesis of (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate from (R)-β-butyrolactone.
Scheme 2. Attempted route to convert (S)-3-hydroxy butyric acid into (R)-ethyl-3-hydroxy butyrate.
2.3. Synthesis of (S)-3-hydroxybutyryl-(S)-3-hydroxybutyrate
(
S)-3-Hydroxybutyrate from the original chiral resolution can be
converted to (S)-3-hydroxybutyryl-(S)-3-hydroxybutyrate using similar
chemistry. First, esterification with ethanol gives (S)-ethyl-3-hydro-
xybutyrate which allows subsequent reduction to (S)-1,3 butanediol
with lithium borohydride [18]. The resulting (S)-1,3 butanediol can be
coupled to (S)-ethyl-3-hydroxybutyrate with CAL-B under reduced
pressure and solvent-free conditions to give (S)-3-hydroxybutyryl-(S)-3-
hydroxybutyrate (Scheme 3).
Scheme 4. Acetal formation of (R) 1,3 butanediol with 4-chloro benzaldehyde.
3. Conclusion
We developed a low cost, efficient method to produce the ketone
body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S)
enantiomer from racemic β-butyrolactone. The enzyme CAL-B catalyzes
enantioselective hydrolysis of racemic β-butyrolactone, and the re-
sulting (R)-β-butyrolactone and (S)-3-hydroxybutyric acid can each be
easily converted to (R) or (S) ethyl-3-hydroxybutyrate and reduced to
(R) or (S) 1,3 butanediol. Both enantiomers of ethyl-3-hydroxybutyrate
and 1,3 butanediol can be joined to form the two enantiomeric KE
products again using immobilized CAL-B. Surprisingly, while CAL-B has
a pronounced enantioselectivity for hydrolysis of (S) versus (R) β-bu-
tyrolactone, it is effective in catalyzing transesterification of either
enantiomer of ethyl-3-hydroxybutyrate and 1,3 butanediol to make the
(R,R) or (S,S) final ketone body esters. En route, we developed efficient
methods to prepare the useful chiral synthons (R)-β-butyrolactone, (R)
or (S) 1,3 butanediol and (R) or (S)-ethyl-3-hydroxybutyrate. This
synthetic method is scalable, involves no column chromatography, uses
mild reaction conditions, and is atom-economic. This method should
help provide cost-effective access to large amounts of this important
ketone body ester for research into its effects on medical conditions that
2
.4. Confirmation of stereochemistry
The stereocenters of the ketone body esters all derive from the
stereoselectivity of CAL-B hydrolysis of racemic β-butyrolactone. In
order to confirm the stereochemistry of hydrolysis by CAL-B, we gen-
erated the acetal of presumed (R)-1,3 butanediol with 4-chlor-
obenzaldehyde to generate solid, crystalline material to determine the
absolute stereochemistry by X-ray crystallography. The formation of the
acetal creates a second stereogenic carbon, but we hoped that the
equilibrium conditions of acetal formation would produce the cis dia-
stereomer allowing both substituents to be equatorial (Scheme 4).
Recrystallization of the acetal from hexane generated needle-like
crystals that were subjected to X-ray crystallography. Indeed, the con-
figuration of the stereogenic carbon from 1,3 butanediol was (R), giving
us confidence that CAL-B hydrolysis of racemic β-butyrolactone is se-
lective for the (S) enantiomer (Fig. 5).
Scheme 3. Synthesis of (S,S)-3-hydroxybutyryl-3-hydroxybutyrate from (S)-3-hydroxybutyric acid.
562

N. Budin et al.
Bioorganic Chemistry 80 (2018) 560–564
conversion of the lactone. Upon completion (> 48 h), a small amount of
solid sodium bicarbonate was added and the solvent removed by rotary
evaporation. The residue was taken up in dichloromethane and washed
with water. The water layer was extracted twice with ethyl acetate. The
pooled organic layers were dried with magnesium sulfate, which was
then filtered and the solvent removed by rotary evaporation to yield
(
R)-ethyl-3-hydroxybutyrate as a clear oil (20.2 g, 66%).
1H NMR (400 MHz, CDCl
): δ = 4.1–4.2 (m, 3H), 2.35–2.48 (m,
3
Fig. 5. X-ray crystal structure of the acetal of (R) 1,3 butanediol and 4-chlor-
obenzaldehyde.
2H), 1.20 (d, J = 6.4 Hz, 3H), 1.25 (t, J = 6.9 Hz, 3H)
1
3
C NMR: (100 MHz, CDCl
2.48, 14.24.
3
): δ = 173.04, 64.30, 60.84, 42.89,
2
are treatable by nutritional ketosis as well as the effects of ketosis on
human athletic performance.
4.3. (R)-1,3 butanediol
(
R)-β-Butryrolactone (18.1 g, 210 mmol) was dissolved in 100%
4. Materials and methods
ethanol (100 mL) in an Erlenmeyer flask and cooled in an ice bath.
Sodium borohydride (17 g, 450 mmol) was added in small portions over
Candida antarctica Lipase B immobilized on Immobead 150 was
30 min with stirring and allowed to warm to room temperature over-
purchased from Sigma. Racemic β-butyrolactone was purchased from
TCI. Sodium borohydride was purchased from EMD-Millipore. Lithium
borohydride was purchased from Fluka. MTBE was purchased from
VWR. All reagents and solvents were used without further purification.
Chiral GC–MS was carried out on a Shimadzu GC-2010/QP2010S with
an Agilent J&W HP-CHIRAL-10B column. NMR spectra were recorded
on a 400 MHz JEOL spectrometer. X-ray crystallography data was re-
corded on a Bruker single crystal X-ray diffractometer. Accurate mass
measurement/HRMS was carried out by the mass spectrometry facility
of the University of Iowa.
night. The reaction was quenched by dropwise addition of 10% HCl
until effervescence ceased and the solvent removed by rotary eva-
poration. The residue was taken up in 200 mL 25% aqueous K HPO
2 4
and extracted twice with 250 mL isopropanol. The isopropanol layers
were combined and solvent was removed by rotary evaporation to yield
a cloudy oil. The material was suspended in dichloromethane, dried
over anhydrous sodium sulfate, filtered, and the solvent removed by
rotary evaporation to yield (R)-1,3 butanediol as a clear oil. (15.2 g,
79%).
1H NMR (400 MHz, CDCl
): δ = 4.02–4.12 (m, 1H), 3.84–3.92 (m,
3
4.1. (R)-β-Butyrolactone and (S)-β-hydroxybutyrate
1H), 3.75–3.83 (m, 1H), 1.64–1.74 (m, 2H), 1.20–1.25 (d,
J = 6.4 Hz, 3H).
Racemic β-butryrolactone (50 g, 0.58 mol) was dissolved in MTBE
13
C NMR: (100 MHz, CDCl
.4. (S)-Ethyl-β-hydroxybutyrate
S)-3-Hydroxybutyric acid (9.6 g, 92 mmol) was dissolved in 100 mL
3
): δ = 68.30, 61.59, 39.96, 23.81.
(
0
3 L) in a 5 L round-bottomed flask charged with a stir bar. Water (6.3 g,
.35 mol) was added, followed by immobilized CAL-B (3.5 g, 7000 U).
4
The reaction mixture was capped and stirred at room temperature.
Aliquots (150 μL) were removed at indicated times and added to 1.0 mL
of methanol for chiral GC–MS analysis. After complete conversion of
(
ethanol with 10 drops of sulfuric acid and stirred in an Erlenmeyer
flask. Aliquots (100 μL) were added to 1.0 mL of methanol and analyzed
by GC–MS for conversion to product. Upon completion, a small amount
of solid sodium bicarbonate was added and the solvent removed by
rotary evaporation. The residue was taken up in dichloromethane and
washed with water. The water was extracted once with additional di-
chloromethane and the pooled organic layers dried over magnesium
sulfate, filtered, and the solvent removed by rotary evaporation to yield
(
S)-β-butyrolactone, the beads were filtered, washed with MTBE, and
the solvent volume reduced to 1 L by rotary evaporation. The reaction
mixture was extracted with 400 mL of saturated sodium bicarbonate.
The organic phase was dried over anhydrous sodium sulfate which was
then filtered and washed with MTBE. The solvent was removed by ro-
tary evaporation to yield (R)-β-butyrolactone as a clear oil (23 g, 92%).
Both the CAL-B and MTBE from this step can be recovered and reused.
(
S)-ethyl-3-hydroxybutyrate as a clear oil (8.9 g, 73%).
1H NMR: (400 MHz, CDCl
J = 16.0, 6.0, 1.4 Hz, 2H) 3.05 (ddd, J = 16.5, 4.2, 1.4 Hz, 2H) 1H),
.56 dd J = 6.1 Hz, 1.8 Hz, 3H).
): δ = 4.64–4.72 (m, 1H), 3.55 (ddd,
3
1H NMR (400 MHz, CDCl
3
): δ = 4.1–4.2 (m, 3H), 2.35–2.48 (m,
1
2
H), 1.20 (d, J = 6.4 Hz, 3H), 1.25 (t, J = 6.9 Hz, 3H)
C NMR: (100 MHz, CDCl ): δ = 173.04, 64.30, 60.84, 42.89,
1
3
C NMR: (100 MHz, CDCl
3
): δ = 168.2, 68.0, 44.4, 20.8 Hz.
13
3
2
2.48, 14.24.
.5. (S)-1,3 butanediol
S)-Ethyl-β-hydroxybutyrate (2.0 g, 15 mmol) was dissolved in die-
The aqueous phase was carefully acidified to pH 2 with conc. HCl
and subjected to continuous liquid-liquid extraction with 500 mL die-
thyl ether for 18 h. The ether layer was dried with magnesium sulfate,
filtered, and the solvent removed by rotary evaporation to yield (S)-3-
hydroxybutyric acid as a clear oil (19 g, 63%).
4
(
thyl ether (80 mL) and methanol (1 mL) in a covered Erlenmeyer flask
and placed in an ice bath. Lithium borohydride (0.66 g, 30 mmol) was
added, allowed to warm to room temperature and stirred overnight.
The reaction was quenched by addition of a small amount of water ice
followed by dropwise addition of 10% HCl until effervescence ceased.
1H NMR: (400 MHz, CDCl
H), 1.25 (d, J = 6.4 Hz, 3H)
): δ = 4.19–4.27 (m, 1H), 2.44–2.58 (m,
3
2
1
3
C NMR: (100 MHz, CDCl
3
): δ = 177.69, 64.39, 42.57, 22.46
2 4
Aqueous 25% K HPO (50 mL) was added and extracted twice with
4
.2. (R)-Ethyl-3-hydroxybutyrate
50 mL isopropanol. The isopropanol layers were combined and solvent
was removed by rotary evaporation to yield a cloudy oil. The material
was suspended in dichloromethane, dried over anhydrous sodium sul-
fate, filtered, and the solvent removed by rotary evaporation to yield
(S)-1,3 butanediol as a clear oil. (1.0 g, 74%).
(
R)-β-Butryrolactone (20 g, 230 mmol) was dissolved in 500 mL
ethanol with 1.0 mL H SO and stirred at room temperature. Aliquots
100 μL) were added to 1.0 mL of methanol and analyzed by GC–MS for
2
4
(
563

N. Budin et al.
Bioorganic Chemistry 80 (2018) 560–564
1H NMR (400 MHz, CDCl
): δ = 4.02–4.12 (m, 1H), 3.84–3.92 (m,
26.1
3
1H), 3.75–3.83 (m, 1H), 1.64–1.74 (m, 2H), 1.20–1.25 (d,
J = 6.4 Hz, 3H).
C NMR: (100 MHz, CDCl ): δ = 68.30, 61.59, 39.96, 23.81.
2
2
3
7.0
9.1
1.3
1
3
3
4
3
.6. (R,R)-3-Hydroxybutyryl-3-hydroxybutyrate/(S,S)-3-hydroxybutyryl-
-hydroxybutyrate
(
R)-Ethyl 3-hydroxybutyrate (2.1 g, 16 mmol) and (R)-1,3 butane-
diol (1.0 g, 11 mmol) were combined and incubated with CAL-B (0.2 g,
00 U) at 80 torr without solvent in a rotary evaporator. The reaction
4
was monitored by withdrawing 5 μL portions of the reaction mixture,
which were dissolved in 1.0 mL methanol for analysis by GC–MS. Upon
consumption of the diol, the reaction mixture was taken up in di-
chloromethane, the beads were filtered and washed, and the solvent
removed by rotary evaporation. Excess (R)-ethyl 3-hydroxybutyrate
was removed by heating to 60 deg C under reduced pressure (1 torr).
The residue was suspended in ethyl acetate, treated with activated
carbon and filtered to yield (R)-3-hydroxybutyryl-(R)-3-hydro-
xybutyrate as a clear oil (1.2 g, 62%).
5. Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bioorg.2018.07.010.
1H NMR (400 MHz, CDCl
): δ = 4.30–4.39 (m, 1H), 4.13–4.21 (m,
H), 3.84–3.93 (m, 1H), 2.78 (br s, 2H), 2.36–2.50 (m, 2H),
.66–1.84 (m, 2H), 1.21 (d, J = 6.4 Hz, 6H)
3
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[
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