Lipase-catalyzed stereoresolution of long-chain 1,2-alkanediols: A screening of preferable reaction conditions

Article abstract of DOI:10.1016/j.molcatb.2015.03.006

Scalable lipase-catalytic method for the kinetic resolution of long-chain 1,2-alkanediol enantiomers via stereoselective cleavage of esters was developed. The influence of lipase, reaction medium, nucleophile, temperature and the structure of the acyl group on the reaction velocity, the stereopreference and the stereoselectivity of the deacylation was studied. In addition, the rate of the spontaneous intramolecular migration of different acyl groups was determined for the intermediate 2-monoesters. The acyl group migration may diminish the apparent stereoselectivity of the two-step process if fast migrating acyl groups are used. It was found that the migration rate of different acyl groups differs by up to two orders of magnitude, being faster for acetyl and isobutyryl and much slower for butyryl and benzoyl groups. The best results were obtained by the sequential methanolysis of bis-butyryl-1,2-alkanediols in an acetonitrile/methanol mixture catalyzed by Candida antarctica lipase B (CALB) at 20 °C, affording (S)-1,2-alkanediols. Stereo- and chemoselective crystallization of the deacylated (S)-1,2-alkanediols from the reaction mixture complements the enzymatic process improving the stereochemical purity to up to ee > 99.8%. (R)-1,2-Alkanediol 2-monoesters were separated from the mother liquor and enriched stereochemically by repeated incubation with CALB, then separated, hydrolyzed with alkali and crystallized to afford (R)-alkanediols of ee > 99.8%.

Full text of DOI:10.1016/j.molcatb.2015.03.006

Journal of Molecular Catalysis B: Enzymatic 116 (2015) 60–69
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Lipase-catalyzed stereoresolution of long-chain 1,2-alkanediols:
A screening of preferable reaction conditions
Jaan Parvea, Indrek Reileb, Tiina Aidc, Marina Kudrjasˇovac, Aleksander-Mati Müüriseppc,
Imre Vallikivid, Ly Villoc,∗, Riina Aavc, Tõnis Pehkb, Lauri Varesa, Omar Parvec
a Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia
b National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia
c Department of Chemistry, Tallinn University of Technology, Ehitajate tee 5, 19086 Tallinn, Estonia
d TBD-Biodiscovery, Tiigi 61b, 50410 Tartu, Estonia
a r t i c l e i n f o
a b s t r a c t
Article history:
Scalable lipase-catalytic method for the kinetic resolution of long-chain 1,2-alkanediol enantiomers via
stereoselective cleavage of esters was developed. The influence of lipase, reaction medium, nucleophile,
temperature and the structure of the acyl group on the reaction velocity, the stereopreference and the
stereoselectivity of the deacylation was studied. In addition, the rate of the spontaneous intramolecular
migration of different acyl groups was determined for the intermediate 2-monoesters. The acyl group
migration may diminish the apparent stereoselectivity of the two-step process if fast migrating acyl
groups are used. It was found that the migration rate of different acyl groups differs by up to two orders
of magnitude, being faster for acetyl and isobutyryl and much slower for butyryl and benzoyl groups.
The best results were obtained by the sequential methanolysis of bis-butyryl-1,2-alkanediols in an
acetonitrile/methanol mixture catalyzed by Candida antarctica lipase B (CALB) at 20 ◦C, affording (S)-
1,2-alkanediols. Stereo- and chemoselective crystallization of the deacylated (S)-1,2-alkanediols from
the reaction mixture complements the enzymatic process improving the stereochemical purity to up to
ee > 99.8%. (R)-1,2-Alkanediol 2-monoesters were separated from the mother liquor and enriched stere-
ochemically by repeated incubation with CALB, then separated, hydrolyzed with alkali and crystallized
to afford (R)-alkanediols of ee > 99.8%.
Received 14 November 2014
Received in revised form 6 March 2015
Accepted 9 March 2015
Available online 18 March 2015
Keywords:
1,2-Alkanediols
Diesters
Stereoselective methanolysis
Lipases
Spontaneous acyl migration
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
In lipase-catalytic separations, enantiomeric diols with pro-
tected primary OH-groups have been obtained [13–16]. A chemical
Enantiomers of long-chain 1,2-alkanediols are highly valuable
chiral building blocks for the synthesis of biologically active nat-
ural products like insect sex pheromones, acetogenins, etc [1].
Enantiomeric alcohols of this type have been previously prepared
by hydrolytic kinetic resolution of terminal epoxides using asym-
metric chemical catalysis [2–7] as well as enzymatically, using
epoxide hydrolases [8,9]. Asymmetric hydroxylation of olefins [10]
and diboration–oxidation of olefins and alkynes [11,12] have given
good results as well.
catalytic resolution [17] as well as an enzymatic oxidation of ␤-
hydroxy ketones [18] have afforded enantiomeric 1,2-alkanediols
with protected secondary hydroxyl groups. Enzymatic hydrolysis
of diol diesters has afforded diols with high stereochemical purity
[19–22]. However, sequential alcoholysis [23–25] has given even
better results. A stereoselective lipase-catalyzed transesterification
process has been used quite successfully for the resolution of 1,2-
alkanediol enantiomers as well [26,27].
The analysis of the highly enantiomerically pure [28] 1,2-
alkanediols necessitates derivatization for the introduction of a
suitable chromophore prior to analyzing in HPLC. For HPLC using
chiral stationary phase, derivatization of alcohols with non-chiral
agents is the usual practice, while for NMR, on the contrary, the
use of chiral derivatizing agents (CDAs) and studying the diastereo-
meric derivatives is preferred. 1,2-Alkanediols may be selectively
derivatized as acetals [29] or boronates [30,31] involving both of
the hydroxyl groups. Conventional chiral derivatizing agents [32]
∗
Corresponding author. Tel.: +372 620 4382; fax: +372 620 2828.
E-mail addresses: jnparve@gmail.com (J. Parve), indrek.reile@kbfi.ee
(I. Reile), tiina.aid@ttu.ee (T. Aid), marinak@chemnet.ee (M. Kudrjasˇova),
spectro@chemnet.ee (A.-M. Müürisepp), imreva@mac.com (I. Vallikivi),
ly.villo@ttu.ee (L. Villo), riina.aav@ttu.ee (R. Aav), pehk@kbfi.ee (T. Pehk),
lauri.vares@ut.ee (L. Vares), omar@chemnet.ee (O. Parve).
http://dx.doi.org/10.1016/j.molcatb.2015.03.006
1381-1177/© 2015 Elsevier B.V. All rights reserved.

J. Parve et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 60–69
61
like methoxyphenylacetic acid [33,34] or mandelic acid [35] can be
used to derivatize all hydroxyl groups of a diol or polyol compound.
Named derivatization would allow NMR spectroscopic determina-
tion of stereoisomeric homogeneity and absolute configuration of
the stereogenic centres by the differential shielding effects in NMR
spectra [35,36]. A regioselective tosylation of the primary OH group
of 1,2-diols for HPLC analysis over chiral stationary phase has been
proposed [37].
Regardless of the above-mentioned separation results, long-
chain enantiomeric 1,2-alkanediols remain too expensive for
several synthetic applications. The objective of our research was
to develop a scalable lipase-catalytic method for the separation of
pure stereoisomers, preferably without resorting to chromatogra-
phy. Our recent work involved a molecular dynamics modeling,
mass spectrometric and 1H and diffusion NMR (DOSY) studies
of the aggregation of enantiomeric 1,2-alkanediols with the aim
to develop a method for the stereo- and chemoselective crys-
tallization of the target alcohol enantiomers from the reaction
mixtures. These results offered a detailed and novel insight into
the aggregation of such diols and provided an efficient protocol for
stereo- and chemoselective crystallization of (S)-1,2-dodecanediol
and related compounds from the crude bioconversion mixtures
[38].
neutralization the water layer was separated, and the organic layer
washed twice with brine, dried over anhydrous MgSO4, filtered,
concentrated and purified by flash chromatography over silica gel.
(NB! 1,2-Alkanediol bisbutyrates were used in preparative-scale
lipase-catalyzed cleavage without previous purification.) The tar-
get products were gained in 91–96% yields.
2.2.2. General protocol B. The lipase-catalyzed stereoselective
cleavage of the diesters
A diester (0.35 mmol) was dissolved in 4.8 ml of solvent fol-
lowed by the addition of the nucleophile and the immobilized
enzyme preparation and the mixture was shaken until a suitable
conversion was identified by TLC. Then the enzyme was filtered off
and the reaction mixture was concentrated and analyzed quan-
titatively by NMR. After the analysis the relevant components
were separated from the crude product by column chromatog-
raphy and the esters hydrolyzed with NaOH in EtOH. Finally, the
resulting diols were 1-tosylated and analyzed by HPLC over a
chiral stationary phase. (The analytical results are presented in
Table 1).
2.2.3. General protocol C. The alkaline hydrolysis of the
1,2-alkanediol esters
The current article presents detailed results of the screening for
more preferable reaction conditions for the lipase-catalyzed stere-
oresolution by varying reaction components such as the solvent,
the nucleophile, the lipase and the acyl groups. The semiprepara-
tive separation of both enantiomers of 1,2-dodecanediol with high
stereochemical purity has been demonstrated.
The mono- or diesters of 1,2-alkanediols were dissolved in EtOH,
1 M NaOH solution in EtOH (2.5 eq.) was added and the mixture
was stirred for 30 min. The completion of the reaction was veri-
fied by TLC. EtOH was removed on a rotary evaporator, aqueous
NaHCO3 was added and the product was extracted with 1/1 mixture
of PE and EtOAc. The extract was washed with an additional portion
of aqueous NaHCO3 and twice with brine; dried over anhydrous
MgSO4, filtered, evaporated and tosylated for the HPLC analysis or
refined by recrystallizing from CHCl3.
2. Experimental
2.1. Materials and general methods
1H and 13C NMR spectra were recorded in CDCl3 solutions on 800
or 400 MHz Bruker Avance spectrometers. All signals were refer-
enced relative to the residual solvent signal. 2D FT methods were
used for the full assignment of NMR spectra. Column chromatogra-
phy was performed on Merck silica gel 60 (230–400 mesh). Thin
layer chromatography (TLC) was performed using DC-Alufolien
Kieselgel 60 F254 (Merck) silica gel plates and stains were visual-
ized by heating with anisaldehyde solution (3 mL of anisaldehyde,
10 mL of conc. H2SO4 in 90 mL of EtOH). HPLC determination of the
enantiomeric ratio (er) was performed using an IA column (Daicel
Chiralpak® IA; 0.46 cm Ø/25 cm); eluent – 10/90 iPrOH/n-hexane;
flow rate – 1.0 ml/min; detection – UV 254 nm. Commercial racemic
1,2-octanediol and 1,2-dodecanediol were used without additional
purification. Immobilized enzymes were donated by the producer:
Candida antarctica lipase B (Novozym® 435; Batch no.: LC200210;
Novozymes®); Rhizomucor miehei lipase (RML) (Lipozyme® RM IM;
Batch no.: LUX00205; Novozymes®).
2.2.4. General protocol D. The synthesis of 1-tosylates of the
1,2-alkanediols
An 1,2-alkanediol (0.5 mmol) was dissolved in dichloromethane
(10 mL) and triethylamine (0.55 mmol) was added while stirring at
RT. Then, the tosyl chloride (0.55 mmol) was added, followed by a
catalytic amount of dibutyltin oxide (5 mg) and the mixture was
stirred at RT for 16 h. Subsequently, EtOAc (60 ml) was added and
the resulting solution was washed with aqueous NaHCO3 and brine,
dried over anhydrous Na2SO4, filtered and concentrated on a rotary
evaporator. The crude product was purified by chromatography
over silica gel to afford target 1-tosylate in 75–85% yield.
2.3. The synthesis of the diester substrates
2.3.1. Synthesis of (rac)-1,2-dodecanediol bisacetate (1)
The synthesis was performed following the General protocol
A (Section 2.2.1). Racemic 1,2-dodecanediol (3) (1.01 g; 5 mmol;
1 eq.) in pyridine (6 ml) and PE (14 ml) was acylated with acetyl
chloride (0.85 ml; 12 mmol; 2.4 eq.). The target product (1) was
gained in 94% yield (1.346 g).
2.2. General synthetic protocols
2.2.1. General protocol A. The acylation of 1,2-alkanediols with
acid chloride
1: 1H NMR (800 MHz, CDCl3) ı 5.06 (1H, dddd, J = 3.3, 5.9, 6.6,
7.4 Hz, H-2), 4.21 (1H, dd, J = 3.3, 11.9 Hz, H-1), 4.02 (1H, dd, J = 6.6,
11.9 Hz, H-1), 2.06 and 2.05 (2 × 3H, 2s, Ac-1,2), 1.56 (2H, m, H-
1,2-Alkanediol was dissolved in pyridine on slight heating;
petroleum ether (PE) was added and the mixture was shaken until
homogenization. Acid chloride was added dropwise on efficient
magnetic stirring. The mixture was stirred at RT for 0.25–16 h
depending on the target compound. Reaction was monitored by
TLC. Methanol (0.5 ml) was added to the mixture when excess
of acid chloride was used and stirring was continued for addi-
tional 10 min. The reaction mixture was diluted with ethyl acetate
(EtOAc) and aqueous NaHCO3 (10%) was added. Following the
3), 1.30–1,23 (16H, m, H-4–H-11), 0.87 (3H, t, J = 7.1 Hz, H-12). 13
C
NMR (201 MHz, CDCl3) ı 170.76 (Ac-1), 170.56 (Ac-2), 71.55 (C-2),
65.09 (C-1), 31.85 (C-10), 30.65 (C-3), 29.52, 29.47, 29.37, 29.32,
29.26 (C-5–C-9), 25.06 (C-4), 22.63 (C-11), 21.03 (Ac-2), 20.73 (Ac-
1), 14.05 (C-12). MS (m/z): 287, 227, 166, 153, 138, 128, 124, 117,
116, 111, 110, 109, 101, 100, 97, 96, 95, 86, 83, 82, 81, 73, 72, 71, 68,
67, 58, 57, 43. IR (neat, cm−1): 606, 961, 1048, 1226, 1371, 1466,
1746, 2856, 2926. Anal. Calcd. for C16H30O4 (286.46): C, 67.08; H,

62
J. Parve et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 60–69
Table 1
Lipase-catalyzed kinetic resolution of enantiomers of 1,2-alkanediols. Reaction conditions: 0.35 mmol of the substrate and 0.1 g of the immobilized lipase preparation were
incubated with 0.2 ml of nucleophile in 4.8 ml of solvent (see General protocol A, Section 2.2.1).
Run no. Substrate Lipase Solvent Nucleophile Temp. (◦C) Incubation time (h) Conversion of substrate (%) Components in crude product
Product no. Configuration Amount (%) eea
1
2
3
4
1
1
1
1
CALB
CALB
CALB
CALB
CH3CN CH3OH
CH3CN CH3OH
CH3CN H2O
20
55
20
55
48
18
44
48
85.2
93.4
41.5
76.6
1
2
4
3
1
2
4
3
1
2
4
3
1
2
4
S
R
14.8
50.5
0.6
34.1
6.6
45.4
10.9
37.1
58.5
26.3
8.2
66.4
86.2b
S
98.3
ndc
nd
nd
93.4
51.2
S
S
R
86.3b
7
nd
46.4
70.6b
CH3CN H2O
S
R
23.4
40.9
9.1
3
1
2
S
26.6
98.0
2.0
83.7
nd
nd
5
6
1
1
RML
RML
CH3CN CH3OH
CH3CN H2O
20
20
168
120
2
3
1
2
4
Not detected
70.9
29.1
<1
30
R
S
31.6
76.5
nd
3
1
2
4
Not detected
72.0
27.3
0.7
7
1
1
1
5
RML
CH3CN H2O
55
20
55
20
168
264
24
28
R
S
30.9
76.0
nd
3
1
2
4
3
1
2
4
3
5
8
11
3
3
6
9
12
3
Not detected
8.7
44.2
6.4
8
CALB
CALB
CALB
C6H6
C6H6
CH3OH
CH3OH
89.2
87
nd
nd
nd
93.6
nd
nd
nd
91.4
nd
nd
S
S
40.7
13.0
37.5
20.5
29.0
15.0
52.3
Not detected
32.7
37.0
70.9
24.1
3.5
9
10
CH3CN CH3OH
16
85.0
S
S
99.7
85.1
nd
89.4b
11
12
5
6
CALB
CALB
CH3OH CH3OH
CH3CN H2O
20
20
120 days
528
95d
29.1
R
1.5
nd
13
6
RML
CH3CN H2O
20
168
72
40.8
6
9
12
3
15
16
15
16
7
10
13
14
R
S
59.2
36.1
4.7
54.0
79.0b
Not detected
67.2
32.8
82.6
17.4
14.5
50.4
3.3
14
15
16
15
15
7
CALB
CALB
CALB
CH3CN CH3OH
CH3CN H2O
20
20
20
32.8
17.4
85.5
S
R
S
nd
90.5
20.2
91.5
nd
nd
nd
60
days
16
R
CH3CN CH3OH
S
31.8
>99.8
a
The enantiomeric excess for 1,2-alkanediols was determined in the form of 1-monotosylates by HPLC over chiral stationary phase [38].
ee was determined for the mixture of regioisomeric monoesters (which was isolated, hydrolyzed and the diol 1-tosylated).
nd, not determined.
b
c
d
Estimated by isolated unreacted diester.
10.58. Found: C, 67.02; H, 10.54. TLC: Rf = 0.20 (EtOAc/PE: 0.2/10).
gained in 96% yield (1.641 g). The characterization of the product 5
Flash chromatography eluent: EtOAc/PE: 0.5/10.
has been reported [38].
2.3.3. Synthesis of (rac)-1,2-dodecanediol bisisobutyrate (6)
The synthesis was performed following the General protocol
A (Section 2.2.1). Racemic 1,2-dodecanediol (3) (0.404 g; 2 mmol;
1 eq.) in pyridine (5 ml) and PE (15 ml) was acylated with isobu-
tyryl chloride (0.52 ml; 5 mmol; 2.5 eq.). The target product (6) was
gained in 96% yield (0.655 g).
2.3.2. Synthesis of (rac)-1,2-dodecanediol bisbutyrate (5)
The synthesis was performed following the General protocol
A (Section 2.2.1). Racemic 1,2-dodecanediol (3) (1.01 g; 5 mmol;
1 eq.) in pyridine (4 ml) and PE (12 ml) was acylated with butyryl
chloride (1.25 ml; 12 mmol; 2.4 eq.). The target product (5) was

J. Parve et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 60–69
63
6: 1H NMR (800 MHz, CDCl3) ı 5.09 (1H, dddd, J = 3.3, 6.2, 6.9,
6.9 Hz, H-2), 4.22 (1H, dd, J = 3.3, 11.8 Hz, H-1), 4.03 (1H, dd, J = 6.9,
11.8 Hz, H-1), 2.54 (2 × 1H, hept, J = 7.0 Hz, Bu-2), 1.56 (2H, m, H-3),
1.29–1.24 (16H, m, H-4–H-11), 1.162, 1.158, 1.152, 1.150 (4 × 3H,
d, J = 7.0 Hz, Bu-3), 0.81 (3H, t, J = 6.9 Hz, H-12). 13C NMR (201 MHz,
CDCl3) ı 176.71 (1-Bu-1), 176.54 (2-Bu-1), 71.13 (C-2), 64.98 (C-
1), 34.10 and 33.91 (Bu-2), 31.86 (C-10), 30.73 (C-3), 29.53, 29.47,
29.38, 29.32, 29.28 (C-5–C-9), 25.00 (C-4), 22.64 (C-11), 18.94,
18.94, 18.88 and 18.85 (Bu-3), 14.07 (C-12). MS (m/z): 343, 255,
254,241, 211, 184, 183, 166, 153, 132, 138, 129, 128, 124, 111,
110, 109, 97, 96, 95, 83, 82, 71. IR (neat, cm−1): 1155, 1192, 1257,
1387, 1470, 1740, 2856, 2927. Anal. Calcd. for C20H38O4 (342.58): C,
70.12; H, 11.20. Found: C, 70.16; H, 11.23. TLC: Rf = 0.36 (EtOAc/PE:
0.3/10). Flash chromatography eluent: EtOAc/PE: 0.2/10.
of the monoacetate (R)-2 contaminated with a small quantity of 1-
monoacetate 4 and also an additional portion of (S)-3 – 110 mg
(11%).
The mixture of the monoesters was incubated additionally with
CALB under methanolytic conditions (in CH3CN/CH3OH 96/4 mix-
ture; with 1.0 g of Novozym 435). The process was complete in 48 h;
the additional 48 h of incubation improved the already high ee of
the product only slightly (Run 2, Table 3).
After 96 h of incubation the residual (R)-2 was isolated from the
mixture by column chromatography to afford 524 mg (2.15 mmol)
of the target compound that was dissolved in 10 ml of EtOH and
hydrolyzed promptly (in accordance with the General protocol C;
in Section 2.2.3.) by adding 1.5 eq. of NaOH (3.2 ml of 1 M NaOH
in EtOH). The crude target (R)-3 obtained was recrystallized from
1.5 ml of CHCl3 to afford 273 mg of the refined product.
(S)-1,2-dodecanediol ((S)-3) [38,39]: 1H NMR (800 MHz, CDCl3,
30 ◦C) ı 3.71 (1H, m, H-2), 3.66 (1H, dd, J = 2.9, 11.2 Hz, H-1), 3.44
(1H, dd, J = 7.8, 11.2 Hz, H-1), 2.30 (2H, bs, OH), 1.43 (3H, m, 2 × H-
3, H-4), 1.33–1.25 (15H, m, H-4–H-11), 0.88 (3H, t, J = 7.2 Hz, H-12).
13C NMR (201 MHz, CDCl3, 30 ◦C) ı 72.33 (C-2), 66.79 (C-1), 33.10
(C-3), 31.88 (C-10), 29.64 (C-5), 29.59 (C-8), 29.58 (C-7), 29.55 (C-
2.3.4. Synthesis of (rac)-1,2-dodecanediol bisbenzoate (15)
The synthesis was performed following the General protocol
A (Section 2.2.1). Racemic 1,2-dodecanediol (3) (1.01 g; 5 mmol;
1 eq.) in pyridine (5 ml) and PE (15 ml) was acylated with ben-
zoyl chloride (1.13 ml, 9.8 mmol, 2 eq.). The target product (15) was
gained in 91% yield (1.867 g).
20
6), 29.32 (C-9), 25.55 (C-4), 22.67 (C-11), 14.11 (C-12). [˛]D −14
15: 1H NMR (400 MHz, CDCl3) ı 8.08 (2H, dm, J = 8,3 Hz, H-oꢀꢀ),
8.03 (2H, dm, J = 8,3 Hz, H-oꢀ), 7.57 (1H, tm, J = 7.4 Hz, H-pꢀꢀ), 7.55
(1H, tm, J = 7.4 Hz, H-pꢀ), 7.45 (2H, m, H-mꢀꢀ), 7.42 (2H, m, H-mꢀ),
5.53 (1H, m, H-2), 4.58 (1H, dd, J = 3.3, 11.9 Hz, H-1), 4.39 (1H, dd,
J = 6.8, 11.9, Hz, H-1), 1.87 and 1.80 (2H, m, H-3), 1.46 (2H, m, H-
(1.0; EtOH); IR (KBr; cm−1): 530, 582, 662, 720, 838, 871, 972, 992,
1006, 1032, 1053, 1073, 1105, 1135, 1311, 1335, 1470, 2850, 2919,
3240, 3323, 3478. Anal. Calcd. for C12H26O2 (202.38): C, 71.21; H,
12.98. Found: C, 71.20; H, 13.01. TLC: Rf = 0.14 (3/5 EtOAc/PE). Flash
chromatography eluent: EtOAc/PE 1/2. Mp = 68–70 ◦C.
4), 1.38–1.23 (14H, m, H-5–H-11), 0.89 (3H, t, J = 7.2 Hz, H-12). 13
C
(R)-2-Acetyl-1,2-dodecanediol ((R)-2): 1H NMR (800 MHz,
CDCl3) ı 4.89 (1H, dddd, J = 3.1, 6.2, 6.3, 7.5 Hz, H-2), 3.70 (1H, dd,
J = 3.3, 11.2 Hz, H-1), 3.60 (1H, dd, J = 6.3, 11.2 Hz, H-1), 2.39 (1H,
bs, OH), 2.09 (3H, s, Ac-2), 1.55 (2H, m, H-3), 1.27–1.22 (16H, m,
H-4–H-11), 0.86 (3H, t, J = 7.1 Hz, H-12). 13C NMR (201 MHz, CDCl3)
ı 171.50 (Ac-2), 75.64 (C-2), 64.66 (C-1), 31.85 (C-10), 30.44 (C-3),
29.53, 29.50, 29.41, 29.40, 29.27 (C-5–C-9), 25.26 (C-4), 22.63 (C-
11), 21.16 (Ac-2), 14.06 (C-12). MS (m/z): 245, 244, 215, 214, 158,
NMR (101 MHz, CDCl3) ı 166.28 (C-1ꢀ), 166.08 (C-1ꢀꢀ) 133.02 (C-
pꢀ), 132.96 (C-pꢀꢀ), 130.01 (C-sꢀ), 129.62 (C-sꢀꢀ), 129.58 (C-oꢀ, C-oꢀꢀ),
128.31 (C-mꢀ), 128.30 (C-mꢀꢀ), 72.10 (C-2), 65.68 (C-1), 31.81 (C-10),
30.84 (C-3), 29.50, 29.46, 29.36, 29.35, 29.26 (C-5–C-9), 25.12 (C-4),
22.62 (C-11), 14.10 (C-12). MS (m/z): 410, 288, 241, 190, 166, 138,
123, 118, 106, 105, 96. IR (neat, cm−1): 687, 711, 1027, 1070, 1109,
1176, 1265, 1315, 1452, 1603, 1723, 2855, 2926. Anal. Calcd. for
C26H34O4 (410.60): C, 76.05; H, 8.36. Found: C, 76.10; H, 8.32. TLC:
157, 156, 155, 139, 108, 93, 92, 91, 59. [˛]20 +1.53 (c 5.9; EtOAc);
D
Rf = 0.18 (EtOAc/PE: 0.2/10).
ee > 99.4%. IR (neat, cm−1): 713, 1053, 1242, 1375, 1466, 1739, 2855,
2926, 3436. Anal. Calcd. for C14H28O3 (244.42): C, 68.79; H, 11.57.
Found: C, 68.75; H, 11.60. TLC: Rf = 0.13 (EtOAc/C6H6 1/5). Flash
chromatography eluent: EtOAc/PE 3/10.
Flash chromatography eluent: EtOAc/PE: 0.4/10.
2.3.5. Synthesis of (rac)-1,2-octanediol bisbutyrate (7)
The synthesis was performed following the General protocol A
(Section 2.2.1) and the synthesis described in Section 2.3.2 [38].
Starting from racemic 1,2-octanediol (14) (1.46 g, 10 mmol) the
target bisbutyrate 7 was obtained in 96% yield (2.756 g). The char-
acterization of the product 7 has been reported [38].
(R)-1,2-Dodecanediol ((R)-3): For the NMR see the data pre-
20
sented for (S)-3. [˛]D
+13.6 (c 1.0; EtOH). Anal. Calcd. for
C12H26O2 (202.38): C, 71.21; H, 12.98. Found: C, 71.23; H, 13.02.
Mp = 69–71 ◦C.
2.4.2. Semipreparative methanolysis of rac-1,2-dodecanediol
bisbutyrate (5) catalyzed by CALB (Run 3, Table 3)
2.4. The lipase-catalyzed stereoselective cleavage of the racemic
diesters
The substrate – bisbutyrate of racemic 1,2-dodecanediol (5)
(17.12 g; 50 mmol) – was dissolved in 96 ml of CH3CN, MeOH
(4 ml) was added followed by 4 g of Novozym 435. The mix-
ture was shaken for 96 h at RT. The enzyme was filtered off, the
solution was evaporated and the residual crude product recrystal-
lized from CHCl3 (20 ml). The target (S)-3 (2.33 g) was gained in
23% yield. The components in mother liquor were separated by
column chromatography over silica gel to afford 8.35 g (49%) of
(R)-2-butyryl-1,2-dodecanediol (R)-8, which was incubated again
with Novozym 435 (3 g) at RT for 96 h. The product was chro-
matographed, hydrolyzed with NaOH in EtOH and the target (R)-3
was recrystallized from 15 ml of CHCl3 to afford 2.43 g of the refined
product with very high enantiomeric purity.
2.4.1. Methanolysis of rac-1,2-dodecanediol bisacetate (1)
catalyzed by CALB (Run 1, Table 3)
The substrate – bisacetate of racemic 1,2-dodecanediol (1.432 g;
5 mmol) was dissolved in acetonitrile (38.4 ml), methanol (1.6 ml)
and 3 g of the immobilized CALB preparation Novozym 435 was
added. The mixture was shaken at RT for 16 h. The enzyme was
filtered off, the solution was evaporated and the residual crude
product was recrystallized from 2 ml of CHCl3. Recrystallization
needs some heating for the complete dissolution of the material
and stirring of the solution on an ice bath for a couple of minutes in
order to allow (S)-1,2-dodecanediol to crystallize. The target (S)-3
was gained in 26% yield (266 mg). (See Run 1, Table 3 for conversion
and ee data.)
(S)-1,2-dodecanediol ((S)-3) characterization of the compound
is presented in Section 2.4.1.
Solvent was evaporated from the mother liquor (residual crude
product) and the residue chromatographed over silica gel. The com-
ponents were eluted with a EtOAc/PE gradient (from 0.5/10 to 1/2
ratio) to afford 64 mg (5%) of the starting diester, 1.627 g (yield 51%)
(R)-2-Butyryl-1,2-dodecanediol ((R)-8): 1H NMR (400 MHz,
CDCl3) ı 4.94 (1H, dtd, J = 3.3, 2 × 6.3, 7.1 Hz, H-2), 3.73 (1H, ddd,
J = 3.3, 5.3,12.0 Hz, H-1), 3.63 (1H, ddd, J = 5.3, 6.3, 12.0 Hz, H-1),
2.34 (2H, t, J = 7.4 Hz, Bu-2), 2.12 (1H, t, J = 5.3 Hz, OH), 1.69 (2H,

64
J. Parve et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 60–69
hex, J = 7.4 Hz, Bu-3), 1.60 (2H, m, H-3), 1.35–1.25 (16H, m, H-4–H-
11), 0.98 (3H, t, J = 7.4 Hz, Bu-4), 0.88 (3H, t, J = 6.9 Hz, H-12). 13C
NMR (101 MHz, CDCl3) ı 174.17 (Bu-1), 75.39 (C-2), 64.86 (C-1),
36.38 (Bu-2), 31.86 (C-10), 30.50 (C-3), 29.54, 29.50, 29.42, 29.40,
29.28 (C-5–C-9), 25.26 (C-4), 22.64 (C-11), 18.50 (Bu-3), 14.07 (C-
12), 13.60 (Bu-4). MS (m/z): 274, 273, 241, 201, 172, 171, 153, 131,
124, 111, 110, 109, 103, 102, 97, 96, 95, 89, 88, 87, 83, 82, 74, 73,
of (S)-14 were recently reported [38]; in this trial the residual
(R)-2-monobutyrate (R)-10 was separated from the mother liquor
by column chromatography and incubated additionally under
methanolytic conditions (analogously to the process described
in Section 2.4.1.). Stereochemically enriched (R)-10 (470 mg;
2.17 mmol, ee = 99.6%) was gained after column chromatography
over silica gel. The monoester was hydrolyzed in 10 ml of EtOH with
NaOH (1.5 eq.). The target (R)-14 was recrystallized from 1.5 ml of
CHCl3 by storing at −15 ◦C overnight, 196 mg of (R)-14 (y.: 27%;
ee = 99.8%) was gained.
20
72, 71, 70, 69, 58, 57. [˛]D +4.0 (c 12, EtOAc), ee > 99%. IR (neat,
cm−1): 1090, 1185, 1260, 1381, 1465, 1736, 2855, 2926, 3447. Anal.
Calcd. for C16H32O3 (272.48): C, 70.52; H, 11.86. Found: C, 70.47; H,
11.89. TLC: Rf = 0.34 (EtOAc/PE 1/5). Flash chromatography eluent:
EtOAc/PE 1/5.
(S)-1,2-Octanediol ((S)-14): 1H NMR (400 MHz, CDCl3) ı 3.68
(1H, dddd, J = 2.9, 5.3, 7.0, 7.9 Hz, H-2), 3.63(1H, dd, J = 2.9, 11.2 Hz,
H-1), 3.40 (1H, dd, J = 7.9, 11.2, Hz, H-1), 3.26 (1H, bs, OH), 1.41
(3H, m, 2 × H-3, H-4), 1.32–1.25 (7H, m, H-4, H-5–H-7), 0.88 (3H,
t, J = 6.8 Hz, H-8). 13C NMR (101 MHz, CDCl3) ı 72.35 (C-2), 66.74
(C-1), 33.08 (C-3), 31.73 (C-6), 29.30 (C-5), 25.52 (C-4), 22.56 (C-7),
2.4.3. Hydrolysis of rac-1,2-dodecanediol bisisobutyrate (6)
catalyzed by RML (Run 13, Table 1)
The substrate, bisisobutyrate of racemic 1,2-dodecanediol (6)
(343 mg, 1 mmol) was dissolved in acetonitrile (9.6 ml), H2O
(0.4 ml) was added followed by Lipozyme RM IM (0.5 g). The
mixture was shaken at RT for 120 h. Subsequently, the enzyme
was filtered off, the solvent evaporated and separated by column
chromatography over silica gel to afford (S)-2-isobutyryl-1,2-
dodecanediol (S)-9 (106 mg; 39%, ee = 78.4%).
14.02 (C-8). [˛]D −14.8 (c 1.0; MeOH), ee > 99.8%. mp = 44–46 ◦C.
20
IR (KBr, cm−1): 492, 537, 581, 653, 861, 881, 988, 1018, 1044, 1092,
1135, 1215, 1334, 1469, 2859, 2929, 3315, 3477. Anal. Calcd. for
C8H18O2 (146.26): C, 65.69; H, 12.43. Found: C, 65.65; H, 12.39.
TLC: Rf = 0.08 (EtOAc/PE 6/10). Flash chrom. eluent: EtOAc/PE 7/10.
(R)-2-Butyryl-1,2-octanediol ((R)-10): 1H NMR (400 MHz,
CDCl3) ı 4.90 (1H, dddd, J = 3.4, 2 × 6.3, 7.0 Hz, H-2), 3.68 (1H, dd,
J = 3.4 12.0 Hz, H-1), 3.60 (1H, dd, J = 6.3, 12.0 Hz, H-1), 2.31 (2H, t,
J = 7.4 Hz, Bu-2), 1.65 (2H, hex, J = 5 × 7.4 Hz, Bu-3), 1.59–1.54 (2H,
m, H-3), 1.33–1.23 (8H, m, H-4–H-7), 0.94 (3H, t, J = 7.4 Hz, Bu-4),
0.86 (3H, t, J = 6.9 Hz, H-8). 13C NMR (101 MHz, CDCl3) ı 174.12 (Bu-
1), 75.34 (C-2), 64.75 (C-1), 36.36 (Bu-2), 31.60 (C-6), 30.49 (C-3),
29.04 (C-5), 25.19 (C-4), 22.47 (C-7), 18.47 (Bu-3), 13.95 (C-8), 13.56
(Bu-4). MS (m/z): 217, 185, 145, 131, 128, 115, 114, 103, 102, 97,
(S)-2-Isobutyryl-1,2-dodecanediol ((S)-9): 1H NMR (400 MHz,
CDCl3) ı 4.89 (1H, dtd, J = 3.3, 6.3, 6.3, 7.1 Hz, H-2), 3.70 (1H, dd,
J = 3.3, 12.0 Hz, H-1), 3.61 (1H, dd, J = 6.3, 12.0 Hz, H-1), 2.58 (1H,
hep, J = 7.0 Hz, Bu-2), 2.30 (1H, bs, OH), 1.57 (2H, m, H-3), 1.31–1.25
(16H, m, H-4–H-11), 1.18 1.17 (2 × 3H, d, J = 7.0 Hz, Bu-3), 0.87
(3H, t, J = 6.9 Hz, H-12). 13C NMR (101 MHz, CDCl3) ı 177.66 (Bu-
1), 75.33 (C-2), 64.93 (C-1), 34.17 (Bu-2), 31.90 (C-10), 30.53 (C-3),
29.58, 29.53, 29.46, 29.42, 29.32 (C-5–C-9), 25.26 (C-4), 22.68 (C-
11), 19.12, 18.95 (Bu-3), 14.10 (C-12). MS (m/z): 273, 171, 132,
20
87, 74, 71, 58, 55, 43. [˛]D +4.85 (c 7.2; EtOAc), ee > 99%. IR (neat,
131, 111, 110, 103, 102, 97, 87, 83, 82, 74, 71, 58, 43. [˛]D −1.6
cm−1): 1089, 1187, 1260, 1384, 1465, 1735, 2852, 2929, 3445. Anal.
Calcd. for C12H24O3 (216.36): C, 66.61; H, 11.20. Found: C, 66.65; H,
11.16. TLC: Rf = 0.18 (EtOAc/PE 1/5). Flash chromatography eluent:
20
(c 6.0, EtOAc), ee = 79%. IR (neat, cm−1): 1075, 1161, 1198, 1388,
1469, 1735, 2856, 2926, 3447. Anal. Calcd. for C16H32O3 (272.48): C,
70.52; H 11.86. Found: C, 70.48; H, 11.82. TLC: Rf = 0.28 (EtOAc/C6H6
1/5). Flash chromatography eluent: EtOAc/PE 1/5.
EtOAc/PE 2/5.
20
(R)-1,2-Octanediol ((R)-14): [˛]D
+14.6 (c 1.0; MeOH);
mp = 44–45 ◦C. Anal. Calcd. for C8H18O2 (146.26): C, 65.69; H, 12.43.
Found: C, 65.66; H, 12.38. Other characteristics were identical to
those determined for (S)-14.
2.4.4. Methanolysis of rac-1,2-dodecanediol bisbenzoate (15)
catalyzed by CALB (Run 14, Table 1)
The substrate – racemic bisbenzoate 15 – was dissolved in
acetonitrile (9.6 ml); methanol (0.4 ml) was added followed by
Novozym 435 (0.3 g). The mixture was shaken at RT for 64 h.
The enzyme was filtered off, the solvent evaporated and the
product separated by chromatography to afford 111 mg of the (R)-
2-benzoyl-1,2-dodecanediol (R)-16 (y.: 36%; ee = 90.2%).
2.5. The synthesis of the reference 1-monoesters
2.5.1. The synthesis of (rac)-1-acetyl-1,2-dodecanediol (4)
(R)-2-benzoyl-1,2-dodecanediol ((R)-16): 1H NMR (800 MHz,
CDCl3) ı 8.07 (2H, m, Ph-o), 7.58 (1H, m, Ph-p), 7.46 (2H, m, Ph-
m), 5.17 (1H, dddd, J = 3.2, 5.4, 6.3 7.9 Hz H-2), 3.84 (1H, ddd, J = 3.2,
5.4, 12.0 Hz, H-1), 3.77 (1H, dt, J = 2 × 5.4, 12.0 Hz, H-1,), 2.19 (1H,
bt, J = 2 × 5.4 Hz, OH), 1.76 and 1.71 (2H, m, H-3), 1.36–1.22 (16H, m,
H-4–H11), 0.88 (3H, t, J = 6.9 Hz, H-12). 13C NMR (201 MHz, CDCl3)
ı 166.95 (C-1ꢀ), 133.08 (Ph-p), 130.10 (Ph-s), 129.65 (Ph-o), 128.37
(Ph-m), 76.43 (C-2), 64.99 (C-1), 31.86 (C-10), 30.64 (C-3), 29.54,
29.51, 29.44, 29.42, 29.29 (C-5–C-9), 25.33 (C-4), 22.65 (C-11), 14.11
(C-12). MS (m/z): 309, 308, 307, 184, 169, 166, 165, 137, 136, 135,
Racemic 1,2-dodecanediol (3) (202 mg; 1 mmol) was dissolved
in EtOAc (4 ml), vinyl acetate (2 ml) and Novozym 435 (200 mg)
were added and the mixture was shaken at RT for 12 h, until the
non-stereoselective acetylation of the primary hydroxyl group was
complete by TLC. Then the enzyme was filtered off, the solution
concentrated and the product purified by flash column chromatog-
raphy over silica gel to afford 225 mg (y.: 92%) of the target
1-monoacetate (4) that was used as a standard compound in quan-
titative analysis of bioconversion crude products.
4: 1H NMR (800 MHz, CDCl3) ı 4.15 (1H, dd, J = 2.8, 11.4 Hz, H-
1), 3.94 (1H, dd, J = 7.7, 11.4 Hz, H-1), 3.85 (1H, m, H-2), 2.11 (3H,
s, Ac), 2.09 (1H, bs, OH), 1.46 (3H, m, 2 × H-3, H-4), 1.33 (1H, m,
H-4), 1.28–1.23 (14H, m, H-5–H-11), 0.87 (3H, t, J = 7.1 Hz, H-12).
13C NMR (201 MHz, CDCl3) ı 171.21 (Ac), 69.96 (C-2), 68.78 (C-1),
33.30 (C-3), 31.88 (C-10), 29.57, 29.55, 29.53, 29.50, 29.30 (C-5–C-
9), 25.34 (C-4), 22.66 (C-11), 20.89 (Ac), 14.09 (C-12). MS (m/z): 245,
227, 172, 171, 152, 138, 127, 126, 125, 124, 123, 111, 103, 97, 96,
95, 85, 83, 82, 81, 75, 74, 71, 69, 58, 57, 55, 43. IR (neat, cm−1): 1240,
1463, 1738, 2852, 2925, 3440. Anal. Calcd. for C14H28O3 (244.42): C,
68.79; H, 11.57. Found: C, 68.74; H, 11.62. TLC: Rf = 0.44 (EtOAc/PE
1/2). Flash chromatography eluent: EtOAc/PE 1/5.
20
123, 118, 106, 105, 93, 92, 91. [˛]D +23.3 (c 5.8; EtOAc). IR (neat,
cm−1): 712, 1027, 1070, 1116, 1177, 1275, 1315, 1452, 1603, 1719,
2855, 2926, 3435. Anal. Calcd for C19H30O3 (306.49): C, 74.45; H,
9.89. Found: C, 74.40; H, 9.93. TLC: Rf = 0.42 (EtOAc/PE 3/10). Flash
chromatography eluent: EtOAc/PE 1/5.
2.4.5. Methanolysis of rac-1,2-octanediol bisbutyrate (7)
catalyzed by CALB
The semi-preparative separation of (S)-1,2-octanediol (S)-14
[40] from racemic mixture was performed starting from 0.730 g
(5 mmol) of the racemic bisbutyrate (7); details of the separation

J. Parve et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 60–69
65
2.5.2. The synthesis of (R)-1-butyryl-1,2-dodecanediol ((R)-11)
The synthesis was performed following the General protocol
A (Section 2.2.1). (R)-1,2-dodecanediol ((R)-3) (202 mg, 1 mmol;
1 eq.) in pyridine (2 ml) and PE (6 ml) was acylated with butyryl
chloride (0.11 ml, 1 mmol, 1 eq.). The reaction time was 15 minutes
and the target product ((R)-11) was gained in 93% yield (253 mg).
(R)-11: 1H NMR (400 MHz, CDCl3) ı 4.07 (1H, dd, J = 3.2, 11.4 Hz,
H-1), 3.90 (1H, dd, J = 7.3, 11.4 Hz, H-1), 3.76 (1H, m, H-2), 2.26 (2H,
t, J = 7.4 Hz, Bu-2), 2.11 (1H, bs, OH), 1.60 (2H, hex, J = 7.4 Hz, Bu-
3), 1.40 (2H, m, H-3), 1.30–1.22 (16H, m, H-4–H-11), 0.88 (3H, t,
J = 7.4 Hz, Bu-4), 0.81 (3H, t, J = 6.9 Hz, H-12). 13C NMR (101 MHz,
CDCl3) ı 173.85 (Bu-1), 70.01 (C-2), 68.49 (C-1), 36.39 (Bu-2), 33.35
(C-3), 31.85 (C-10), 29.53, 29.50, 29.42, 29.40, 29.27 (C-5–C-9),
25.26 (C-4), 22.63 (C-11), 18.50 (Bu-3), 14.05 (C-12), 13.59 (Bu-4).
MS (m/z): 273, 255, 241, 225, 211, 201, 184, 171, 166, 153, 131, 124,
C, 74.45; H, 9.89. Found: C, 74.38; H, 9.92. TLC: Rf = 0.54 (EtOAc/PE
3/10). Flash chromatography eluent: EtOAc/PE 1.5/10.
2.5.5. The synthesis of (R)-1-butyryl-1,2-octanediol ((R)-13)
The synthesis was performed following the General protocol A
(Section 2.2.1). (R)-1,2-octanediol ((R)-14) (146 mg, 1 mmol; 1 eq.)
in pyridine (2 ml) and PE (6 ml) was acylated with butyryl chloride
(0.11 ml, 1 mmol, 1 eq.). The reaction time was 15 min and the target
product ((R)-13) was gained in 94% yield (203 mg).
(R)-13: 1H NMR (400 MHz, CDCl3) ı 4.13 (1H, dd, J = 3.2, 11.4 Hz,
H-1), 3.96 (1H, dd, J = 7.2, 11.4 Hz, H-1), 3.83 (1H, m, H-2), 2.34 (2H,
t, J = 7.4 Hz, Bu-2), 2.26 (1H, bs, OH), 1.67 (2H, hex, J = 5 × 7.4 Hz,
Bu-3), 1.46 (2H, m, H-3), 1.33–1.25 (8H, m, H-4–H-7), 0.97 (3H,
t, J = 7.4 Hz, Bu-4), 0.87 (3H, t, J = 6.9 Hz, H-8). 13C NMR (101 MHz,
CDCl3) ı 173.91 (Bu-1), 69.98 (C-2), 68.51 (C-1), 36.07 (Bu-2), 33.36
(C-3), 31.71 (C-6), 29.22 (C-5), 25.33 (C-4), 22.57 (C-7), 18.42 (Bu-
3), 14.04(C-8), 13.64 (Bu-4). MS (m/z): 217, 199, 185, 173, 155, 145,
20
102, 87, 71,69, 43,41. [˛]D +2.64 (c 3.8; EtOAc), ee > 99%. IR (neat,
cm−1): 410, 457, 475, 558, 718, 749, 840, 876, 946, 991, 1019, 1078,
1109, 1143, 1213, 1269, 1312, 1377, 1413, 1426, 1449, 1471, 1705,
2849, 2873, 2921, 2960, 3507. Anal. Calcd. for C16H32O3 (272.48): C,
70.52; H, 11.86. Found: C, 70.45; H, 11.91. TLC: Rf = 0.49 (EtOAc/PE
3/10). Flash chromatography eluent: EtOAc/PE 1.5/10.
20
131, 115, 102, 97, 87, 74, 71, 69, 55, 43. [˛]D +2.85 (c 4.4; EtOAc).
IR (neat, cm−1): 725, 998, 1097, 1183, 1261, 1381, 1460, 1740, 2859,
2932, 2959, 3454. Anal. Calcd. for C12H24O3 (216.36): C, 66.61; H,
11.20. Found: C, 66.67; H, 11.15. TLC: Rf = 0.44 (EtOAc/PE 3/10).
Flash chromatography eluent: EtOAc/PE 2/5.
2.6. The synthesis of 1-tosyl-1,2-alkanediols
2.5.3. The synthesis of (R)-1-iso-butyryl-1,2-dodecanediol
((R)-12)
The synthesis of (S)-1-tosyl-1,2-dodecanediol (S)-18 and (S)-
1-tosyl-1,2-octanediol (S)-19 was performed in accordance with
the General protocol D; it has recently been described along with
characterization of the products [38]. The corresponding racemic
standards of these tosylates for use as HPLC references as well as
corresponding (R)-configured 1-tosylates to be analyzed for stereo-
chemical purity were synthesized in an analogous manner.
HPLC determination of the ee of 1,2-dodecanediols (S)-3 and
(R)-3 as well as (S)-14 and (R)-14 gave the retention times of the
1-tosylates as follows: (R)-18: 8.69 min; (S)-18: 10.50 min; (R)-19:
10.33 min; (S)-19: 12.50 min [38] (eluent – 10/90 iPrOH/n-hexane;
flow rate – 1.0 ml/min).
The synthesis was performed following the General protocol
A (Section 2.2.1). (R)-1,2-dodecanediol ((R)-3) (202 mg, 1 mmol;
1 eq.) in pyridine (2 ml) and PE (6 ml) was acylated with iso-butyryl
chloride (0.11 ml, 1 mmol, 1 eq.). The reaction time was 15 min and
the target product ((R)-12) was gained in 91% yield (248 mg).
(R)-12: 1H NMR (800 MHz, CDCl3) ı 4.13 (1H, dd, J = 3.2, 11.4 Hz,
H-1), 3.96 (1H, dd, J = 7.2 and 11.4 Hz, H-1), 3.83 (1H, m, H-2), 2.59
(1H, hep, J = 7.0 Hz, Bu-2), 1.44 (2H, m, H-3), 1.29–1.23 (16H, m,
H-4–H-11), 1.15 and 1.14 (2 × 3H, d, J = 7.0 Hz, Bu-3), 0.84 (3H, t,
J = 7.2 Hz, H-12). 13C NMR (201 MHz, CDCl3) ı 177.33 (Bu-1), 69.84
(C-2), 68.38 (C-1), 33.62 (Bu-2), 33.18 (C-3), 31.77 (C-10), 29.48,
29.44, 29.41, 29.29, 29.22 (C-5–C-9), 25.27 (C-4), 22.56 (C-11), 18.85
and 18.77 (Bu-3), 14.02 (C-12). MS (m/z): 273, 255, 211, 201, 184,
20
171, 166, 153, 131, 124, 102, 87, 71, 69, 43, 41. [˛]D +5.16 (c 4.0;
3. Results and discussion
EtOAc), ee > 99%. IR (neat, cm−1): 457, 479, 506, 564, 722, 762, 840,
880, 916, 965, 984, 998, 1050, 1085, 1108, 1135, 1170, 1218, 1247,
1295, 1322, 1355, 1391, 1453, 1470, 1709, 2849, 2920, 2957, 3514.
Anal. Calcd. for C16H32O3 (272.48): C, 70.52; H 11.86. Found: C,
70.46; H, 11.83. TLC: Rf = 0.52 (EtOAc/PE 3/10). Flash chromatogra-
phy eluent: EtOAc/PE 1.5/10.
The screening for the preferable reaction conditions for the ste-
reoselective lipase-catalysed cleavage of long-chain 1,2-alkanediol
diesters was based on previous empirical results. An efficient
lipase-catalytic process based on the use of CALB in CH3CN/CH3OH
(96/4, v/v) mixture at RT has been used for the kinetic resolution
of enantiomers of 1-phenyl-1,2-ethanediol, involving sequential
cleavage of both of the acetyl groups from the corresponding
bisacetate [23]. This was chosen as a lead process because our pre-
liminary trials showed the ability of this methodology to produce
deacetylated (S)-1,2-dodecanediol (S)-3 from racemic bisacetate
1 at a satisfactory rate and considerably high stereoselectivity
(Scheme 1). Examples of the use of acetonitrile (which is an atypi-
cal reaction medium for lipases) for the regioselective hydrolysis of
some deoxysugar diesters catalyzed by CALB have been previously
reported [41,42].
The points to be addressed by the screening trials herein were:
(i) possibility to enhance the reaction rate while retaining an
acceptable stereoselectivity; (ii) clarification of the influence the
spontaneous intramolecular migration of the acyl group (in the
initially formed intermediate 2-monoester 2 and related interme-
diates; Scheme 1) has on the “apparent” stereoselectivity of the
overall process and how to optimize it in this regard; (iii) devel-
opment of a method allowing to prepare both of the alkanediol
enantiomers in high stereochemical purity.
2.5.4. The synthesis of (R)-1-benzoyl-1,2-dodecanediol ((R)-17)
The synthesis was performed following the General protocol
A (Section 2.2.1). (R)-1,2-dodecanediol ((R)-3) (202 mg, 1 mmol;
1 eq.) in pyridine (2 ml) and PE (6 ml) was acylated with benzoyl
chloride (0.12 ml, 1 mmol, 1 eq.). The reaction time was 15 minutes
and the target product ((R)-17) was gained in 91% yield (279 mg).
(R)-17: 1H NMR (800 MHz, CDCl3) ı 8.06 (2H, m, Ph-o), 7.59 (1H,
m, Ph-p), 7.46 (2H, m, Ph-m), 4.40 (1H, dd, J = 3.2 and 11.6 Hz, H-1),
4.23 (1H, dd, J = 7.2 and 11.6 Hz, H-1), 3.99 (1H, m, H-2), 2.18 (1H, bs,
OH), 1.57 (2H, m, H-3), 1.51 and 1.40 (2H, m, H-4), 1.35–1.23 (14H,
m, H-5–H-11), 0.89 (3H, t, J = 6.9 Hz, H-12). 13C NMR (201 MHz,
CDCl3) ı 166.73 (CO), 133.16 (Ph-p), 129.81 (Ph-s), 129.62 (Ph-o),
128.41 (Ph-m), 70.11 (C-2), 69.22 (C-1), 33.40 (C-3), 31.88 (C-10),
29.58, 29.56, 29.55, 29.52, 29.31 (C-5–C-9), 25.40 (C-4), 22.67 (C-
11), 14.12 (C-12). MS (m/z): 307, 289, 275, 259, 245, 231, 218, 204,
184, 169,165, 141, 136, 123, 105, 92, 71, 55, 41. [␣]D20 + 0.90 (c
14; EtOAc). IR (neat, cm−1): 534, 689, 709, 918, 960, 1028, 1074,
1106, 1131, 1147, 1182, 1250, 1304, 1326, 1410, 1450, 1471, 1586,
1603, 1696, 2849, 2915, 3493. Anal. Calcd. for C19H30O3 (306.49):
Screening of the reaction conditions along with the correspond-
ing results are listed in Table 1. The tested modifications of the

66
J. Parve et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 60–69
Scheme 1. CALB catalyzed sequential methanolysis of the diester 1 for the kinetic resolution of 1,2-dodecanediol enantiomers involves a minor spontaneous acetyl migration
reaction which diminishes the apparent stereoselectivity of the process.
Scheme 2. Lipase-catalysed stereoselective deacylation of 1,2-alkanediol diesters.
Scheme 3. Stereoselective methanolysis of 1,2-dodecanediol bisbenzoate 15 catalysed by CALB; (R)-17 is produced by the acyl migration.
Scheme 4. Synthesis of 1,2-alkanediol 1-tosylates.
process were: (i) for lipase – RML vs. CALB; (ii) for nucleophile –
H2O vs. MeOH, (iii) for temperature – 55 ◦C vs. 20 ◦C; (iv) for the
reaction medium – C6H6 or MeOH vs. CH3CN and (v) for the struc-
ture of the acyl group of the substrate – butyryl or iso-butyryl or
benzoyl vs. acetyl (Schemes 2 and 3).
enantiomers of 2-monoester 2 occurring after the first step
(Scheme 1).
RML in acetonitrile/methanol (96/4) system was found to be
almost inactive towards bisacetate 1 (Table 1, Run 7). In contrast,
RML in acetonitrile/water (96/4) cleaves the primary ester group
with modest stereoselectivity preferring molecules with S config-
uration (Table 1, Runs 5 and 6). In conclusion, our screening results
have shown CALB to be preferable for the stereoselective cleavage
of the diols’ diesters.
Two nucleophiles, water and methanol were compared in ace-
tonitrile for CALB and, somewhat unexpectedly, methanol is clearly
preferable. (Runs 1–4; Table 1)
Regarding reaction temperature, in most cases performing reac-
tions at higher temperatures (e.g. at 55 ◦C vs. 20 ◦C) leads to a higher
rate, but lower stereoselectivity of the process (Runs 1–5, Table 1).
However, there were two examples where stereoselectivity of the
process was independent from temperature, one concerned CALB
in benzene/methanol (Runs 8, 9; Table 1) and the other RML in
acetonitrile/water (Runs 6 and 7, Table 1).
As for the solvent, CALB in acetonitrile/methanol utilizing 1,2-
dodecanediol bisbutyrate 5 as a substrate afforded (S)-3 with
exceptional stereoselectivity (ee = 99.7% determined for the diol
The crude products were analyzed by 1H NMR to afford exact
percentages of all of the four possible components in the mixtures
(Fig. 1). The ee was determined by HPLC analysis of the 1,2-
alkanediol 1-tosylates over a chiral stationary phase (Scheme 4),
derived from relevant components of the crude products (the ester
components were separated, hydrolyzed and tosylated).
Comparing the lipases’ catalytic performance, CALB first cleaves
the ester of the primary hydroxyl group of the bisacetate 1 with
a slight preference for R-enantiomer of 2-monoacetate, leaving
remaining diester 1 mainly in S configuration (Table 1, Run 1). Simi-
lar selectivity has been described by Virsu et al. in the methanolysis
of 1-phenylethan-1,2-diol bisacetate [23]. The second step of the
sequential methanolysis catalyzed by CALB is clearly the S-specific
one – affording (S)-3 in high enantiomeric purity (ee > 98%). We
attribute the formation of up to one percent of opposite (R)-
enantiomer in product 3 to the side-reaction of intramolecular
spontaneous acetyl migration in the non-racemic mixture of

J. Parve et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 60–69
67
Fig. 1. The NMR signals of the hydrogen atoms attached to C-1 and C-2 atoms of 1,2-dodecanediol and its acetates which were used for the quantification of the components
in the crude products of enzymatic cleavage of the diester 1.
Table 2
Migration rate (at RT in acetonitrile) of the acyl groups within 1,2-alkanediol 2-monoesters, estimated by NMR.
Starting 2-monester
Product, 1-monoester
Migrating acyl group
Time (hours or days)
Conversion (%)
Initial rate (% × 24 h−1
)
2
8
9
10
16
4
11
12
13
17
Acetyl
72 h
70 days
72 h
70 days
108 days
10.9
5.4
6.1
1.8
1.2
3.6
0.08
2.0
0.03
0.02
Butyryl
Iso-butyryl
Butyryl
Benzoyl
Table 3
The lipase-catalyzed kinetic resolution of 1,2-dodecanediol enantiomers in semi-preparative scale. (The reactions were carried out in CH3CN/CH3OH at RT with CALB, other
details are given in Experimental: for Runs 1–2 in Section 2.4.1 and for Run 3 in Section 2.4.2).
Run no.
Substrate
Incubation time (h)
Conversion of substrate (%)
Components in crude product
Product no
Configuration
Amount (%)
eea
1
16
94.6
1
2
4
3
S
R
5.2
48.7
5.7
ndb
82.6
nd
1 (5.0 mmol)
S
40.4
98.1d
99.4e
92.8f
99.3
99.5
99.8e
69.4f
99.9h
2
3
48g
96g
96
2
2
3
R
R
S
(R)-2 (2.5 mmol)
5 (50 mmol)
90c
23i
nd
24i
3
R
a
The ee were determined for the corresponding 1-monotosylates by HPLC over chiral stationary phase [38].
nd, not determined.
Estimated by isolated unreacted diester.
ee of (S)-3 in the crude product prior to crystallization.
ee of (S)-3 crystallized from the crude product.
ee of (S)-3 remaining in the mother liquor.
b
c
d
e
f
g
(R)-2 was gained from Run 1; in the Run 2 it was further incubated with CALB under methanolytic conditions in order to enrich the sample stereochemically; ee was
determined after 48 h and 96 h of incubation.
h
The separation of (R)-3 was performed analogously to the one described in Run 1/Run 2 followed by alkaline hydrolysis of (R)-2 and recrystallization of resulting (R)-3.
The yield of the recrystallized product.
i

68
J. Parve et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 60–69
as it formed, prior to crystallization; Run 10, Table 1). The use
of CALB in benzene/methanol affords (S)-1,2-dodecanediol with
lower stereoselectivity (ee = 90%). Diester cleavage catalyzed by
CALB in neat methanol occurs at a very low rate with modest stere-
oselectivity (Run 11, Table 1).
The influence of the acyl group on the stereoselectivity of the
process emerges via two basic mechanisms: (i) firstly, the degree
of the steric fit of the structure of the acyl group to the enzyme, and
(ii) secondly, the rate of the spontaneous intramolecular migration
of a certain acyl group in 2-monoesters (which is an equilibrium
process). As mentioned above, the acyl migration diminishes appar-
ent stereoselectivity of the process (Scheme 1). A special study –
the estimation of the spontaneous acyl group migration rate was
performed and the rates were found to differ up to two orders of
magnitude under identical conditions (Table 2).
Acknowledgements
We thank the Estonian Ministry of Education and Science for
financial support (SF0140133s08; SF0690034s09; SF0180073s08;
SF0140060s12; ETF8289; ETF8880; AR12171; IUT23-7; IUT23-9;
IUT19-9; PUT692 and TAR8103). This project has received fund-
ing from the European Union’s Seventh Framework Programme
for research, technological development and demonstration under
grant agreement no 621364 (TUTIC Green) and is greatly acknowl-
edged. We also thank the Archimedes Foundation (project no.
3.2.0501.10-0004) and Tallinn University of Technology (grant B35)
for financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2015.03.006.
Acetyl and iso-butyryl were found to be fast migrating
acyl groups while butyryl and benzoyl migrate very slowly
(Schemes 2 and 3). The use of slowly migrating acyl groups (e.g.
butyryl or benzoyl) is exceptionally important if stereoisomers of
a polyol compound (triols, tetrols) are to be sterically resolved,
as random acyl migration produces unwanted ester regioisomers
that may be cleaved with “wrong” stereopreference. Thus, nei-
ther acetyl nor iso-butyryl groups should be the first option in
this situation. In conclusion, butyryl group is clearly preferable
compared to acetyl due to being cleaved by CALB with the same
stereopreference, albeit at a higher rate and higher (apparent)
stereoselectivity.
The simultaneous stereoselectivity of the chemoselective crys-
tallization of (S)-3 from the crude product (Run 17, Table 1) has
been demonstrated by determining the ee for three samples of
(S)-3: (i) as it formed, (ii) for (S)-3 which crystallized from the
crude product and (iii) for the (S)-3 from the mother liquor. In
order to gain the opposite enantiomer (R)-3 in high stereoisomeric
purity, the corresponding (R)-2-monoacetate (R)-2 was separated
from the mother liquor by column chromatography over silica
gel and subsequently allowed to incubate again with CALB under
methanolytic conditions in order to stereochemically enrich the
product (Run 2, Table 3). Then the (R)-2 was separated by col-
umn chromatography and hydrolyzed with NaOH in EtOH to
afford (R)-3 which was recrystallized to afford a product in high
ee.
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