A facile lipase-catalyzed KR approach toward enantiomerically enriched homopropargyl alcohols

Article abstract of DOI:10.1016/j.bioorg.2019.01.050

Compounds possessing propargylic (prop-2-ynylic) system are very important building blocks for organic chemistry. Among them, preparation of enantiomeric homopropargyl alcohols (but-3-yn-1-ols) constitutes a key-challenge for asymmetric synthesis and thus

Full text of DOI:10.1016/j.bioorg.2019.01.050

BioorganicChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
A facile lipase-catalyzed KR approach toward enantiomerically enriched
homopropargyl alcohols
⁎
Paweł Borowieckia, , Maciej Drankab
a Warsaw University of Technology, Faculty of Chemistry, Department of Drugs Technology and Biotechnology (Biocatalysis Laboratory), Koszykowa St. 3, 00-664
Warsaw, Poland
b Warsaw University of Technology, Faculty of Chemistry, Department of Inorganic Chemistry and Solid State Technology (X-ray Crystallography Laboratory), Koszykowa
St. 3, 00-664 Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Paweł Kafarski on the occasion of his 70th
birthday and Ryszard Ostaszewski on the
occasion of his 60th birthday as well as in
honor of their profound contribution to the
field of polish biocatalysis.
Compounds possessing propargylic (prop-2-ynylic) system are very important building blocks for organic
chemistry. Among them, preparation of enantiomeric homopropargyl alcohols (but-3-yn-1-ols) constitutes a key-
challenge for asymmetric synthesis and thus drawn tremendous attention from the synthetic community in the
last few decades. In this work, the catalytic performance of a set of commercial lipases has been investigated for
enantioselective transesterification of 1-phenylhomopropargylic alcohols under kinetically–controlled condi-
tions. Lipase from Burckholderia cepacia (BCL) immobilized either on ceramic (Amano PS-C II) or diatomaceous
earth (Amano PS-IM) turned out to be the most active and enantioselective enzyme preparations (E ≫ 500)
furnishing both resolution products of the racemic 1-phenylbut-3-yn-1-ol in highly enantiomerically enriched
form (up > 99% ee). Variable reaction parameters, such as the acyl-group donor reagent as well as solvent,
were additionally screened to establish their impact on the stereochemical outcome. For optimal biocatalytic
systems established with model substrate, the enzymatic transformations were extended toward preparative-
scale KR of 8 other differently para-phenyl-substituted homopropargylic sec-alcohols, which resulted in the
synthesis of (S)-alcohols (96–100% ee) and the respective (R)-acetates (92–100% ee) in 19–44% yield, ac-
cordingly. Additionally, the crystal structure of (1R)-1-(4-nitrophenyl)but-3-yn-1-yl acetate has been evaluated
for the first time and helped to assess stereopreference of the studied BCL.
Keywords:
Biocatalysis
Lipases
Kinetic resolution
Chiral alcohols
Homopropargyl alcohols
Barbier reaction
1. Introduction
Stevens, Sonogashira, Cadiot-Chodkiewicz, etc.), (vi) cyclotrimeriza-
tions, (vii) silylformylation-allylsilylations, (viii) eneyne metathesis,
The acetylenic derivatives play a fundamental role in organic
chemistry as they constitute extremely valuable building blocks for
synthesis of architecturally more complex compounds possessing broad
spectrum of applications. The terminal ethylenic functional group is
very reactive and highly useful in plethora of modern synthetic en-
deavors [1]. In this context, organic compounds bearing an acetylene
unit are prone to undergo vast number of chemical transformations
including: (i) additions of various molecules to carbon–carbon triple
bond [i.e. halogens, hydrogen halides or cyanide, boranes, water in the
presence of acids or Hg(II) salts, azides under copper(I)-catalyzed 1,3-
dipolar cycloaddition (CuAAC) conditions], (ii) selective reductions of
alkyne functionality into Z- or E-olefins or full reductions to paraffins,
(iii) ozonolysis into carboxylic acids and classical (iv) alkylations and/
or substitution with electrophyllic partners. Moreover, toward the
propargyl moiety such reactions as: (v) crosscouplings (i.e. Castro-
and (ix) ring annulations can be accomplished as well.
Interestingly, terminal acetylenic functional group are a common
structural motif found in many natural products originating from bac-
teria [mycomycin (I) [2–4]; cepacin B (II) [5]; smenothiazole B (III)
[6,7]; apramides: A (IV) and G (V) [8]; jamaicamide B (VI) [9]; dra-
gonamides: A (VII) [10,11], B [12] and E [13]; spongidepsin (VIII)
[14–17]], plants [sterculynic acid (IX) [18]], marines [chondriol (X)
[19]; rhodophytin (XI) [20]; trans-kumausyne (XII) [21,22]; dideox-
ypetrosynol A (XIII) [23,24]] and other nautical organisms such as
cephalaspidean mollusk [kulolide (XIV) [25]] (Fig. 1). Since their
biological activities are very interesting and well-documented, over the
past few decades, there have been many elegant methodologies devel-
oped for the synthesis of the above-mentioned derivatives.
Moreover, among terminal acetylenic functionalities especially the
propargylic moiety seems to be very important from the view-point of
⁎ Corresponding author.
E-mail addresses: pawel_borowiecki@onet.eu, pborowiecki@ch.pw.edu.pl (P. Borowiecki).
https://doi.org/10.1016/j.bioorg.2019.01.050
Received 14 December 2018; Received in revised form 24 January 2019; Accepted 25 January 2019
0045-2068/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:Borowiecki,P.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.01.050

P. Borowiecki, M. Dranka
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Fig. 1. Examples of natural products possessing terminal acetylenic functionality I–XIV.
biological activity. This stems from the fact that 2-propynyl group
constitutes a privileged motif for numerous synthetic pharmaceuticals
(Fig. 2, XV–XIX) and agrochemicals (Fig. 2, XX–XVI).
valuable pharmaceutical intermediates, such as: 5-substituted dihy-
drofuran-3-ones (via gold-catalyzed reaction methodology) [33] or 2,3-
dihydrofurans (via molybdenum-mediated cycloisomerization) [34]
and 3,4-allenols (via Crabbé homologation) [35]. The optically active
homopropargylic alcohols have been mostly synthesized through
asymmetric addition of various nucleophilic regents [either a me-
talloallenes (i.e. allenyltri-n-butylstannanes [36–39], allenylboronic
esters [40–44], allenyltrichlorosilane [45], 10-TMS-9-borabicyclo
[3.3.2]decanes [46], allenylzinc [47]) or propargyls (i.e. propargyl
halides in the presence of low-valent metal such as chromium [48,49]
or indium [50,51], propargyl borolane [52], propargyldiisobutylalu-
minium [53])] to aldehydes in the presence of chiral catalysts or aux-
iliaries. Among chiral catalysts for enantioselective carbonyl pro-
pargylations the Lewis acids [i.e. BINOL–Ti(IV) complexes], Lewis bases
(i.e. helical chiral 2,20-bipyridine N-monoxides) and Brønsted acids
[i.e. BINOL–derived phosphoric acids (BINOL–PAs) and carboxylic
acids] are the most common (see papers cited above [36–53]). In turn,
catalysts composed of chiral aminoalcohols (i.e. pseudoephedrine) and
diamines [i.e. (S)-1-[(1-methyl-2-pyrrolidinyl)methyl]piperidine] are
willingly applied as well since especially aminoalcoholic ligands are
low-cost, easy accessible and highly modular. A few other catalysts also
proved to be effective for this transformation [i.e. tethered bis(8-qui-
nolinato) (TBOx) chromium complex, Cr(II)-carbazole tridentate ligand
complex, etc.] (also see the papers cited above [36–53]).
The most prominent examples reaching the blockbuster status in the
pharma industry are the specific selective irreversible inhibitors of the
mitochondrial isoenzymes of monoamine oxidase: type A (MAO-A)
[clorgiline, XVI (Clorgilinum®)] and type B (MAO-B) [selegiline, XV
(Emsam®, Eldepryl®, Zelapar®, etc.); pargyline, XVII (Eutonyl®); rasagi-
line, XVIII (Azilect®)], which are mainly used as medicines to treat
symptoms in early Parkinson's disease. Besides the above-mentioned
therapeutic agents, worth mentioning is the late-stage non-selective
muscarinic acetylcholine receptor agonist [talsaclidine, XIX (WAL-
2014)] developed for the treatment of Alzheimer's disease. The pro-
pargyl group can also be found in the chemical structures of various
commercialized protoporphyrinogen-IX oxidase (PPO) inhibitors used
worldwide to combat pathogens of arable crops. Among them so-called
oomycetes fungicides [mandipropamid, XX (Revus®)] and peroxidizing
herbicides [flumioxazin, XXI (Pestanal®); flumipropyn, XXII (S
23121®); oxadiargyl, XXIII (Topstar®); azafenidin, XXIV (Azafenidin®);
thidiazimin, XXV (UNII-Z1USL6USPO®); pyrazogyl, XXVI (AEB
172391®)] deserve special attention.
Approximately more than 50% of all pharmaceuticals and agro-
chemicals comprise at least one stereogenic center [26–30]. Therefore,
the current market of chiral drugs dramatically requires not only an
efficient tool box (advanced ‘chiral technologies’) to synthesize en-
antiomeric APIs in a facile manner on an industrial scale, but also a
wide array of non-racemic building blocks obtainable in consistency
with the restrictions imposed by regulatory agencies (i.e. FDA, EMA,
etc.) [31,32]. One of such potentially useful chiral synthons for the
synthesis of medicines are homopropagylic alcohols containing phenyl
substituent. In turn, these compounds can be transformed into other
Nevertheless, the chirality inducement in the case of asymmetric
prop-2-ynylation of aldehydes is often highly unsatisfactory (< 90% ee)
and still remains challenge. Notable, most of the reported methods are
also restricted by limitations concerning use of reagents that are rela-
tively expensive, low-reactive, difficult to prepare, sensitive to air and/
or moisture and often need post-reaction treatment of the crude mixture
to remove some covalent additives (i.e. TMS ethers by TBAF, boranes by
2

P. Borowiecki, M. Dranka
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Fig. 2. Representative chemical structures of compounds with propargylic moiety used as pharmaceuticals (XV–XIX) and agrochemicals (XX–XVI).
H2O2/NaOH or BF3·OEt2) from the propargylated products leading to
tedious purifications. The difficulties associated with regioselectivity of
propargylations concerning competing allenylation of carbonyl sub-
strates is also arduous to circumvent. Moreover, employing chiral cat-
alysts based on highly toxic metals largely deny the possibility of using
thus-prepared synthons in manufacturing of pharmaceuticals.
Therefore, the choice of lipases as catalysts for asymmetric functiona-
lization of homopropargylic alcohols rests not only on practical con-
siderations (particularly the ease of handling, stable to air/water and
highly selective in action relative to the metal-based catalysts), but also
on safety of usage (non-toxic and fully biodegradable).
2.1. Synthesis
2.1.1. General procedure for the synthesis of racemic homopropargyl
alcohols, rac-3a–i
The appropriate aldehyde 1a–i (10 g) was added to H2O (100 mL)
containing KI (1.5 equiv), SnCl2 dehydrate (1.5 equiv) and propargyl
bromide (2) as 80 wt% in toluene solution (1.5 equiv). Next, after
10 min of stirring saturated aqueous NH4Cl (70 mL) solution was added.
The stirring was continued for 15 h at 35 °C. Further extraction with
Et2O (4 × 250 mL) gave an organic phase which was washed with H2O
(600 mL), brine (600 mL), and subsequently dried over anhydrous
MgSO4 and concentrated. The oily residue was chromatographed on
silica gel by using two independent columns sequentially. At first, traces
of the aldehydic substrate were removed by eluting with CH2Cl2/
hexane (60:40, v/v) in accordance to dry column vacuum chromato-
graphy (DCVC) technique [54], and then pure product was obtained by
eluting the second batch with a mixture of CHCl3/acetone (99:1, v/v) as
an eluent in a conventional column chromatography manner. The de-
sired alcohols rac-3a–i were thus synthesized with 8–40% yield, re-
spectively.
In view of the considerable attention, which the above-mentioned
alkynols have received from chemists all over the world as well as our
continual interests in expanding the repertoire of biocatalytic ap-
proaches toward preparation of small-molecule chiral synthons valu-
able for pharmaceutical sciences, here we wish to report our new results
on lipase-catalyzed enantioselective synthesis of non-racemic homo-
propargylic alcohols with excellent level of enantiomeric enrichment
obtainable using simple kinetic resolution (KR) methodology.
2. Experimental section
2.1.2. General procedure for the synthesis of racemic homopropargyl esters,
rac-4a–i
General details concerning reagents, solvents, catalysts (enzymes)
and methods used as well as analytical data for the obtained com-
pounds are available in Supporting Information.
To a solution of the racemic propargyl alcohol rac-3a–i (500 mg) in
dry CH2Cl2 (5 mL), Et3N (1.5 equiv) and catalytic amount of DMAP
(15 mg) were added. Next, the mixture was cooled to 0–5 °C in the ice
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bath, and one of the appropriate acyl chloride (1.5 equiv) dissolved in
dry CH2Cl2 (2 mL) was added dropwise. Afterward, the cooling bath
was removed, and the resulting mixture was stirred at room tempera-
ture for 12 h. The crude mixture was diluted with CH2Cl2 (10 mL),
subsequently quenched with H2O (20 mL), the water phase was ex-
tracted with CH2Cl2 (3 × 10 mL), and the combined organic layer was
washed with saturated water solution of NaHCO3 (40 mL), brine
(40 mL), and dried over anhydrous MgSO4. After filtering of the drying
agent, and evaporation of the residuals of solvent under reduced pres-
sure the crude product was purified by double column chromatography
on silica gel, at first, using mixture of CHCl3/acetone (99:1, v/v), then
using n-hexane/AcOEt (80:20, v/v) as eluent, thus obtaining desired
esters rac-4a–i, rac-5a and rac-6a with 19–93% yield, respectively.
crystal of sufficient quality for a structure analysis with conventional X-
ray diffraction (XRD) method was prepared by dissolving (R)-(+)-4f
(20 mg, 96% ee) in boiling PhCH3 (1 mL). After refluxing the content of
the flask for additional 5 min, the hot solution was slightly cooled, then
transferred into glass vial (4 mL), tightly twisted, and left to cool to
room temperature. Next, a screw cap was replaced by Parafilm “M”®
appropriately perforated by needle to obtain single tiny hole in the
center of its’ surface. The system was stored at room temperature, and
crystal growth was allowed to proceed for 14 days by slow evaporation
of the solvent.
2.1.6.2. Crystal structure determination of (R)-(+)-4f. Single crystal of
(R)-(+)-4f, suitable for X-ray diffraction studies were selected under
polarizing microscope and measured with mirror monochromated
CuKα radiation on an Oxford Diffraction κ-CCD Gemini A Ultra
diffractometer. Cell refinement, data collection and data reduction
were performed with the CRYSALISPRO software [55]. Using Olex2
[56], the structure was solved with the ShelXT [57] structure solution
program and refined with the SHELXL − 2018 [58] refinement package
using Least Squares minimization. All non-hydrogen atoms were refined
with anisotropic displacement parameters. Hydrogen atoms attached to
carbon atoms were added to the structure model at geometrically
idealized coordinates and refined as riding atoms. An absolute
configuration (R) for the compound molecule (+)-4f was determined
using anomalous dispersion effects. Flack parameter [59] calculated
from 694 selected quotients (Parsons' method) [60] equals − 0.09(18).
Further analysis of the absolute structure was performed using
likelihood methods with PLATON [61]. A total of 842 Bijvoet pairs
(coverage of 1.00) were included in the calculations. The resulting
value of the Hooft parameter [62] was −0.01(16). Crystal Data for
2.1.3. General procedure for analytical-scale lipase-catalyzed KR of rac-3a
In a typical procedure, the model racemic alcohol rac-3a (25 mg,
0.17 mmol) was dissolved in organic solvent (500 µL), and subse-
quently, vinyl acetate (44 mg, 0.51 mmol, 47 µL) and the respective
lipase preparation (10 mg, 40%, w/w) were added. Thus composed
reaction mixture was stirred in thermo-stated glass vial (V = 4 mL)
placed in anodized aluminum reaction block at 30 °C and 800 rpm. The
progress of enzymatic kinetic resolution (EKR) process was monitored
by GC and chiral HPLC analyses until the required conversion was
achieved (ca. 50%). The samples were prepared by withdrawing the
suspension (50 µL) from the reaction mixture, dilution it with portion of
the respective organic solvent (500 µL), and centrifugation of the en-
zyme using laboratory centrifuge (6000 rpm). After evaporation of the
volatiles from taken supernatant, the crude oil was dissolved in n-
hexane-i-PrOH (1.5 mL, 3:1, v/v) and analysed by GC and HPLC. The
same procedure was repeated with vinyl butanoate (59 mg, 0.51 mmol,
65 µL) and vinyl decanoate (102 mg, 0.51 mmol, 115 µL), but using
catalytic system composed of Amano PS-IM (10 mg) suspended in
PhCH3 (500 µL).
C12H11NO4 (M = 233.22 g/mol): orthorhombic, space group P212121,
a = 7.1787(3) Å, b = 8.0720(3) Å, c = 20.0382(7) Å, V = 1161.16(7)
Å3, Z = 4, T = 293.0(1) K, μ(CuKα) = 0.853 mm−1, Dcalc = 1.334 g/
cm3, 6067 reflections measured (11.82° ≤ 2Θ ≤ 134.192°), 2072
unique (Rint = 0.0594, Rsigma = 0.0508) which were used in all
calculations. The final R1 was 0.0457 (I > 2σ(I)) and wR2 was
0.1236 (all data). CCDC 1,880,174 contains the supplementary
crystallographic data for compound (R)-(+)-4f. This can be obtained
free of charge on application to CDC, 12 Union Road, Cambridge
CB21EZ, UK (Fax: (+44)1223-336-033; email: deposit@ccdc.cam.ac.
uk). The Crystal data and structure refinement parameters for (R)-
(+)-4f can be found in Table S1 in Supplementary material.
2.1.4. General procedure for preparative-scale lipase-catalyzed KR of rac-
3a–i
To a solution of the respective racemic alcohol rac-3a–i (100 mg) in
PhCH3 (2 mL), vinyl acetate (3 equiv) and lipase Amano PS-IM (40 mg,
40%, w/w) were added at once. The enzymatic system was stirred in
thermo-stated glass vial (V = 4 mL) placed in anodized aluminum re-
action block at 30 °C and 800 rpm. The progress of EKR process was
monitored by GC and chiral HPLC analyses and proceeded until the
required conversion was achieved (ca. 50%). After removal of the en-
zyme by filtration on Schott funnel, washing it with portion of PhCH3
(3 mL), and evaporation of the solvent under vacuum, the crude reac-
tion mixture was purified by column chromatography on SiO2 using
mixture of CHCl3/acetone (99:1 v/v) as an eluent to afford the re-
spective EKR optically active products (S)-(−)-3a–i and (R)-(+)-4a–i.
3. Results and discussion
In this work, our principal desire was to broaden the scope and
generality of lipase-catalyzed kinetic resolution (KR) of 1-para-sub-
stituted-phenylbut-3-yn-1-ols. In this regard, our efforts focused on
chemoenzymatic synthesis of enantiomerically enriched homo-
propargylic alcohols 3a–i consisting of KR methodology as a key step.
The synthetic pathway is outlined below (Scheme 1).
2.1.5. General procedure for hydrolysis of (R)-(+)-4b to establish its
enantiomeric excess
To
a solution of optically active acetate (R)-(+)-4b (20 mg,
0.09 mmol, > 99% ee) in MeOH (2 mL) anhydrous K2CO3 (24 mg,
0.17 mmol) was added in one portion. The resulting mixture was stirred
for 2 h at room temperature. Next, the volatiles were evaporated under
vacuum, and the crude oil was diluted with CH2Cl2 (2 mL) and rinsed
with H2O (2 × 2 mL). The combined organic phase was dried over
anhydrous MgSO4, the drying agent was filtered off, and the permeate
was concentrated under vacuum. Finally, the remaining crude oil was
subjected to a short-pad SiO2 column chromatography and purified
using mixture of CHCl3/acetone (99:1, v/v) as the eluent yielding en-
antiomeric (R)-(+)-3b (14 mg, 0.07 mmol, 86%, > 99% ee) as colorless
oil.
3.1. Synthesis of the racemic starting materials, rac-3a–i
In the first step, the requisite racemic alkynols rac-3a–i were
planned to be prepared through routine coupling of propargyl bromide
(2) with a suitably functionalized benzaldehyde derivatives 1a–i pro-
moted by the respective metal. Hitherto so-called Barbier-type pro-
pargylation of the carbonyl compounds has been thoroughly studied
and even reported in a comprehensive review article [63], however,
each year novel improved modifications of this reaction appear. Hence,
from a wide repertoire of available synthetic protocols, we have in-
itially limited our investigations toward those, which employs zinc as
the mediator. This was due to the fact that zinc is significantly less
harmful for environment and human life when compared to lead, in-
dium, tin, aluminum, gallium or other metals commonly used in such
2.1.6. X-ray structure
2.1.6.1. Procedure of crystal growth of (R)-(+)-4f. Colourless single
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BioorganicChemistryxxx(xxxx)xxx–xxx
Scheme 1. Lipase-catalyzed KR of racemic
homopropargyl alcohols rac-3a–i. Reagents
and conditions: (i) 2 as 80 wt% in PhCH3
solution (1.5 equiv), KI (1.5 equiv), SnCl2
(1.5 equiv), H2O, 10 min at RT, aq.
NH4Clsat.
, then 15 h at 35 °C; (ii) acyl
chloride (1.5 equiv), Et3N (1.5 equiv),
DMAP (cat.), dry CH2Cl2, 10 min at 0–5 °C,
then 12 h at RT; (iii) vinyl ester (3 equiv),
lipase (40% w/w), organic solvent (1 mL/
50 mg of substrate), 4–24 h at 30 °C,
800 rpm (magnetic stirrer).
Table 1
Synthesis of rac-3a via Barbier-type propargylation of benzaldehyde (1a) performed at 1 g-scale.
Propargyl bromidea
Znb
SnCl2
Additive
Solvent
t [h]
T [°C]
Yieldd [%]
Ref.
c
Entry
1
2
3
4
5
1 eq
1 eq
5 eq
3 eq
–
–
aq. NH4Clsat.
THF
THF
THF
H2O
H2O
4
RT
RT
RT
50
35
20
[64]
[56]
[65]
[66]
[67]
1.5 eq
1.3 eq
1.5 eq
1.5 eq
–
aq. NH4Clsat.
24
1
56e
69e
12
–
aq. CaCl2sat. + aq. NH4Clsat.
TBABrf (0.3 eq)
1.5 eq
1.5 eq
8
–
aq. NH4Clsat. + KI (1.5 eq)
15
38
a
80 wt% in toluene.
b
c
d
e
f
Granulated zinc was used.
Anhydrous SnCl2 was used.
Isolated yield after column chromatography.
Contaminated in ca. 10% (w/w) with forming an allenic by-product in accordance to gas chromatography (GC) indications.
Tetrabutylammonium bromide.
couplings. In this regard, we examined a few Zn-mediated Barbier-type
conditions toward model benzaldehyde 1a using propargyl bromide (2)
as 80 wt% in PhCH3 solution and various additives in THF as solvent
(see Table 1, entries 1–3). Unfortunately, in the first attempt using
equimolar amounts of all reagents, the product rac-3a was isolated in
very low 20% yield. After detailed examination of the available lit-
erature, we came across that the low reaction yields with granulated
zinc might be due to the fact that a surface process is involved.
Therefore, to overcome this inconvenience, we decided to increase
molar excess of zinc metal and propargylating agent 2 up to 5 equiv and
1.5 equiv, respectively. Successfully, this simple stoichiometric ratio
adjustment as well as elongation of the reaction time up to 24 h re-
vealed some improvement in the reaction yield (56%). However, it
turned out that the desired Barbier adduct rac-3a was contaminated
with an allenic by-product, which is inseparable via the convenient
SiO2-column chromatography, as the close proximity of these two al-
cohols induce similar physical properties furnishing the same Rf -value.
In the next attempt to obtain rac-3a, we have followed procedure
reported by Bieber et al. [65]. A slightly decreased amounts of both 2
and metallic zinc as well as additional utilization of saturated aqueous
CaCl2 solution have led to obtain product rac-3a in good 69% yield after
just 1 h. Unfortunately, in this case the propargylation conditions also
rendered very low regioselectivity because the corresponding allenol
was detected at similar level of contamination as before. Since a sig-
nificant quantities of hard-to-eliminate allenic alcohol were formed
during the above-mentioned attempts, next we adopted purification
procedure elaborated by Fu et al. [68]. This simple method is based on
selective precipitation of homopropargylic alcohol in the presence of
corresponding allenol accomplished by using equimolar mixture of
AgNO3 and CaCO3 in aqueous acetone. In our hand, the separation
protocol was only partially efficient as after treatment of the filtered-off
precipitant with 1 M HCl the silver acetylide species hydrolysed to rac-
3a, however, traces of allenic alcohol still remained in the solution. It
was probably because of allenol being trapped in the precipitate or
wrongly adjusted stoichiometry toward side-product. After repeating
two-fold precipitation procedure the overall reaction’s yield dropped
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significantly (approx. by half of the initial value), and thus we finally
abandoned (AgNO3-CaCO3)-based purification method. This dis-
appointing observation led us to change our synthetic strategy once
again. In aiming to suppress the metallotropic rearrangement between
propargyl and allenyl species (responsible for the formation of allenic
by-product) we have tested stannous chloride (SnCl2) as the mediator
instead of Zn and excluded organic solvent from the reaction medium.
Inspired by paper published by Masuyama and co-workers [66], in the
first trial we used equimolar quantities of 2 and anhydrous SnCl2 in
presence of 0.3 equiv of TBABr. In the course of this reaction the
starting material 1a was totally converted into the corresponding al-
cohol rac-3a after 8 h of stirring the mixture at 50 °C. However, the
yield of propargylation was very poor (12%) as the complex mixture of
by-products was formed. Nevertheless, contribution of allenic alcohol
in overall contamination was marginal, and thus at that point, we
turned our efforts toward modifications of SnCl2-mediated Barbier re-
action. Hopefully, when the reaction was assisted by addition of 1.5
equiv of KI and aq. NH4Clsat. instead of TBABr/H2O, the desired pro-
pargylic adduct rac-3a (completely free from allenic alcohol !!!) could
be isolated in an improved 38% yield. As this method turned out to be
superior to others especially in terms of reaction’s regioselectivity,
therefore, a set of racemic phenyl-1-alkynols rac-3a–i having varying
functional group diversity, such as: eiPr, etBu, eBr, eCl, eNO2, eF,
eCF3 and eOCH3, at the para-position of the phenyl ring have been
synthesized at 10 g-scale using the commercially available and cheap
arenecarbaldehydes 1a–i as starting material (Table 2). Gratifyingly,
the conducted reactions resulted in a similarly high selectivity albeit
with poor to moderate isolated yields (8–40%) of the racemic alcohols
rac-3a–i. It turned out that especially synthesis of rac-3f is challenging
as in this case a parallel SnCl2-mediated reduction of the nitro group
was observed. In conclusion, although the efficiency of the employed
method is significantly limited due to the formation of ca. 14-compo-
nent reaction mixture (according to GC) that hinders isolation of de-
sired products rac-3a–i, we did not optimize it further as it was not our
major goal.
Table 3
Synthesis of the racemic esters rac-4a–i, rac-5a and rac-6a.
Entry
Product
R1
R2
Yielda [%]
1
rac-4a
rac-5a
rac-6a
rac-4b
rac-4c
rac-4d
rac-4e
rac-4f
rac-4 g
rac-4 h
rac-4i
H
CH3
C3H7
C9H19
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
26
33
28
30
19
20
70
49
83
93
20
2
H
3
H
4
iPr
tBu
Br
5
6
7
Cl
8
NO2
F
9
10
11
CF3
OCH3
a
Isolated yield after column chromatography.
rac-3a–i by standard DMAP-catalyzed acylation using 1.5 equiv of acyl
chlorides, excess of Et3N as a base trapping the hydrochloric acid
formed, and dry CH2Cl2 as the solvent. The representative results are
summarized in Table 3.
Surprisingly, most of the esterification reactions gave ester products
in poor-to-moderate yields (19–49%), and therefore it was postulated
that homopropagylic compounds are likely to be base-sensitive and/or
prone to be acylated at terminal acetylenic group as well. Only three
derivatives bearing the following eCl, eF and eCF3 substituent seemed
to be resistant toward dual amine mixture (Et3N/DMAP) as good-to-
excellent yields (70–93%) in their cases could be obtained. It is worth to
underline that most of the prepared esters (i.e. rac-6a and rac-4b–i) had
never been synthesized.
With a set of racemic alcohols rac-3a–i and esters rac-4a–i, rac-5a
and rac-6a in hand, our next task was to elaborate analytical methods
(GC and HPLC) for enzymatic experiments. In particular, it was im-
portant to set up a reliable chiral HPLC method, able to follow the re-
actions directly, and to improve the synthetic performances of lipase-
catalyzed reactions. In this context, we developed peculiar chiral HPLC
methods (giving a good baseline separation) for model substrate rac-3a
and its corresponding esters rac-4a, rac-5a and rac-6a in order to re-
solve the enantiomers directly from the crude mixtures in a single run,
allowing to define the enantiomeric excess, and fully characterize en-
antioselectivity of the performed reactions very fast (see Supplementary
material). Fortunately, no interference was observed at the retention
times of interest among model substrate rac-3a and its esters. For the
rest of the substrates rac-3b–i and their acetates rac-4b–i we elaborated
chiral-stationary-phase HPLC conditions for individual racemates and
not for the mixtures of both pairs of respective alcohol and ester en-
antiomers. It is worth to mention that one of the synthesized racemic
acetates, that is: 1-[4-(propan-2-yl)phenyl]but-3-yn-1-yl acetate (rac-
4b), had to be hydrolyzed before HPLC analysis due to lack of peaks
separation of this derivative in the chromatograms when using avail-
able by us chiral columns [Chiralcel OD-H and Chiralpak AD-H].
3.2. Synthesis of the racemic analytical standards, rac-4a–i
As per our planned strategy, the synthesis of racemic esters required
as an analytical standards for enzymatic reactions was performed. The
respective racemic acetates rac-4a–i as well as single butyrate rac-5a
and decanoate rac-6a were obtained from the corresponding alcohols
Table 2
Synthesis of the racemic homopropargyl alcohols rac-3a–i performed at 10 g-
scale.
Entry
Product
R1
Yielda [%]
1
2
3
4
5
6
7
8
9
rac-3a
rac-3b
rac-3c
rac-3d
rac-3e
rac-3f
rac-3g
rac-3h
rac-3i
H
33
11
20
40
23
8
iPr
tBu
Br
3.3. KR of rac-3a–i using lipase-catalyzed transesterification
Cl
NO2
F
Although lipases are the catalysts of choice when ones desire to
proceed kinetic enantiomeric resolution of chiral alcohols, very little is
reported with regard to lipase-catalyzed separation of the racemic
homopropargylic sec-alcohols. To the best of our knowledge, the report
by Burgess and co-workers [69] is the only example of the use of lipases
31
29
13
CF3
OCH3
a
Isolated yield after column chromatography.
6

P. Borowiecki, M. Dranka
BioorganicChemistryxxx(xxxx)xxx–xxx
toward synthesis of enantiomerically enriched phenyl-substituted al-
kynyl alcohols, and is limited only to kinetic resolution (KR) of racemic
1-phenylbut-3-yn-1-ol (rac-3a), which resulted in moderate level of
enantioselectivity (E = 28) yielding optically active alcohol (S)-(−)-3a
in > 95% ee and the respective acetate (R)-(−)-4a in moderate 72% ee.
In this case it was native lipase from Pseudomonas fluorescens (Amano
AK) that was used as biocatalysts for the studied transformations of rac-
3a. The other enzymatic example of enantiomeric resolution of 1-phe-
nylbut-3-yn-1-ol (rac-3a) was published by Prof. Herradon’s research
group [70]. However, this report concerns enantioselective transestri-
fication of rac-3a using acylase I (AA-I) as biocatalyst instead of the
lipase. Nevertheless, this method was also far from optimal (E = 89)
allowing to obtain enantioenriched alcohol (S)-(−)-3a in 95% ee and
the corresponding optically active butyrate (R)-(−)-4a in 92% ee. The
rest of the literature reports turned to lipases’ performances were fo-
cused rather on 2-heteroaryl substituted homopropargyl alcohols
[71–73] or their aliphatic derivatives [74]. In example, Takano et al.
[75] reported (lipase PS)-catalyzed resolution of 4-hydroxy-5(4-meth-
oxyphenoxy)-1-pentyne, however, regardless of high optical purity of
both KR products obtained, disadvantages of this reaction which limits
this procedure of being preparative are hazardous CH2Cl2 used as the
solvent and long reaction time (4 days).
assays were regularly traced by GC and HPLC analyses and finally ar-
rested either upon 50% conversion was reached or 24 h have passed.
These reaction conditions allowed us a clear characterization of the
behaviour of the different biocatalysts of various bacterial, fungal and
mammalian origin.
From the first set of experiments it turned out that limited number
of the studied enzymes were suitable for the enantioselective acetyla-
tion of rac-3a. Unfortunately, most of them were completely inactive
(i.e. Lipozyme RM IM and Amano Lipase M) or remained a residual
activity (i.e. Chirazyme L-2, C-2, Chirazyme L-2, C-3, TL-Immobead
150, Amano Lipase F-AP15, Lipase AY Amano 30, and PLE). In turn,
lipases from Candida antartica type B immobilized on acrylic resins
(Novozym 435 and Lipozyme 435) catalyzed rac-3a enantiomers’ re-
solution with excellent enantioselectivity (E > 200) resulting in the
formation of almost enantiomerically pure acetate (R)-(+)-4a (99%
ee), nevertheless, the rates of reactions were very poor as the conver-
sion reached only approx. 15% after 24 h, and thus the remaining al-
cohol (S)-(−)-3a was isolated with significantly diminished en-
antiomeric excesses (16–17% ee). An improved activity toward the
racemic substrate rac-3a, albeit still moderate, exhibited the subsequent
enzyme preparations: Lipozyme TL IM, PS-Immobead 150, Amano PS,
and Amano AK. It is worth to mention that among them especially PS-
Immobead 150 and Amano PS catalyzed KR of rac-3a with superb en-
antioselectivity (E > 250) leading to isolation of enantiomerically pure
ester (R)-(+)-4a (> 99% ee), however, only unsatisfactory 20–26%
conversions could be afforded in those cases, and thus the slower re-
acting enantiomer (S)-(−)-3a was afforded with moderate 24–35% ee.
Surprisingly, in contrary to the results reported in the literature [69], in
our hands Amano AK turned out to be slightly more efficient in terms of
enantioselectivity (E = 57) than in the paper published by Burgess et al.
(E = 28), however, enantiomeric excesses of KR products were still
unsatisfactory. To our delight, the enzyme screening procedure re-
vealed that other two immobilized preparations based on lipase from
Burckholderia cepacia (BCL) and one native lipase from Alcaligenes sp.
(ASL) were the most promising in terms of reaction rate and en-
antioselectivity. In example, BCL immobilized on diatomaceous earth
(Amano PS-IM) exhibited high activity reaching 49% conversion after
24 h, thus allowing isolation of both KR products in highly en-
antiomerically enriched forms (96–98% ee). Notably, BCL immobilized
on ceramic (Amano PS-C II) was even more efficient in the en-
antioselective acylation of rac-3a allowing 50% conversion to be
achieved after amazingly short reaction time (just 4 h) almost without
any decrease of the enantiomeric selectivity in KR manner. In this
context, (Amano PS-C II)-catalyzed KR of rac-3a yielded homochiral
alcohol (S)-(−)-3a (> 99% ee) and the forming optically active acetate
(R)-(+)-4a (98% ee) with close to the highest enantioselectivity value
(E = 922). In turn, Chirazyme L-10 although catalyzed resolution of
model rac-3a with moderate enantioselectivity (E = 27), it allowed to
obtain (S)-(−)-3a with 98% ee. As the highest activity and stereo-
selectivity discrimination during the transesterification process of rac-
3a was observed in the case of Amano PS-C II, Amano PS-IM and
Chirazyme L-10, both BCL and Chirazyme L-10 lipase preparations were
selected for further optimization studies. It is also interesting to note
that all the enzymatic reactions proceeded very cleanly without the
formation of propargyl–allenyl isomerization by-products.
Therefore, in this paper we turned our attention to novel and more
efficient lipase-catalyzed kinetic resolution of titled homopropargylic
alcohols rac-3a–i. We decided to perform those enzymatic transfor-
mations in a transesterification mode using vinyl esters as the acylating
agents. It was made by us deliberately since non-aqueous variant of KR
process is more desirable from industrial point of view due to less
harmful isolation procedure. In the first step of analytical-scale enzy-
matic studies we performed an extensive screening of 17 commercial
enzyme preparations including lipases and one esterase (pig liver es-
terase, PLE) to identify a lead candidate biocatalyst for the en-
antioselective acetylation (Table 4). The enzymes were tested in a
model reaction comprising a racemic substrate rac-3a used at 25-mg
scale in a solution of 3 equiv of vinyl acetate as an acetyl group donor
and tert-butyl methyl ether (TBME) as solvent frequently used in lipase-
catalyzed reactions, and stirred at 30 °C using a magnetic stirrer. All the
Table 4
Enzyme screening for enantioselective transesterification of rac-3a under KR-
conditions in TBME.
Entry Enzyme preparationa
t [h] Conv.b [%] ees [%] eep [%] Ed
c
c
1
Novozym 435
24
24
24
24
24
24
24
24
24
4
14
16
99
233
2
Lipozyme 435
15
17
99
235
3
Chirazyme L-2, C-2
Chirazyme L-2, C-3
TL-Immobead 150
Lipozyme TL IM
Lipozyme RM IM
PS-Immobead 150
Amano PS-IM
< 5
< 5
< 1
30
N.D.e
N.D.e
N.D.e
43
N.D.e
N.D.e
N.D.e
98
N.D.e
N.D.e
N.D.e
151
4
5
6
7
0
N.D.e
24
N.D.e
> 99
98
N.D.e
252
8
20
9
49
96
392
10
11
12
13
14
15
16
17
Amano PS-C II
Chirazyme L-10
Amano PS
50
99
98
922
24
24
24
24
58
98
72
27
26
35
> 99
N.D.e
94
280
Amano Lipase M
Amano AK
0
N.D.e
56
N.D.e
57
37
Amano Lipase F-AP15 24
< 1
< 1
< 3
N.D.e
N.D.e
N.D.e
N.D.e
N.D.e
N.D.e
N.D.e
N.D.e
N.D.e
In general, the nature of the reaction medium has been known to
hold strong influences on the catalytic performance of the enzymes
mainly due to modulation of biocatalysts’ enantioselectivity and stabi-
lity. Therefore, evaluation of the solvent impact on reaction rate and
stereochemical outcome should always be taken into account as one of
the most critical factor for biocatalysis. Similarly to the reaction con-
ditions used in our previous contribution, the effect of the solvent on
the BCL- and ASL-catalyzed enantioselective transesterification of
model racemic substrate rac-3a was studied in detail (Table 5). In this
regard, several organic solvents characterized by different logP
(0.20–3.00) values were employed. To obtain comparable results we
Lipase AY Amano 30
PLE
24
24
a
Conditions: rac-3a 25 mg, lipase 10 mg, TBME 500 µL, vinyl acetate 44 mg,
53 µL (3 equiv), 30 °C, 800 rpm (magnetic stirrer).
b
Based on GC, for confirmation the % conversion was calculated from the
enantiomeric excess of the unreacted alcohol (ees) and the product (eep) ac-
cording to the formula conv. = ees/(ees + eep).
c
d
Determined by chiral HPLC analysis by using a Chiralcel OD-H column.
Calculated according to Chen et al. [76], using the equation: E = {ln
[(1 − conv.)(1 − ees)]}/{ln[(1 − conv.)(1 + ees)]}.
e
Not determined due to a low conversion rate.
7

P. Borowiecki, M. Dranka
BioorganicChemistryxxx(xxxx)xxx–xxx
Table 5
Solvent screening for enantioselective transesterification of rac-3a with vinyl acetate catalyzed by immobilized lipases from Burckholderia cepacia (Amano PS-IM or
Amano PS-C II) and native lipase from Alcaligenes sp. (Chirazyme L-10) under KR conditions.
Lipasea
Solvent (log P)b
t [h]
Conv.c [%]
ees [%]
eep [%]
Ee
d
d
Entry
1
2
3
4
5
6
7
Amano PS-IM
Acetone (0.20)
THF (0.40)
24
24
24
24
24
24
24
25
24
49
48
51
48
50
33
> 99
> 99
98
275
272
392
637
348
637
420
32
TBME (0.96)f
96
t-Amyl alcohol (1.09)
Cyclohexane (2.50)
PhCH3 (2.52)
91
99
> 99
91
97
> 99
Hexane (3.00)
97
98
8
Amano PS-C II
Chirazyme L-10
Acetone (0.20)
4
4
4
4
4
4
4
24
24
24
24
24
24
24
14
16
50
38
50
46
51
24
28
58
55
70
59
66
16
19
99
60
97
84
99
> 99
> 99
98
233
240
922
368
207
532
259
43
39
27
18
11
26
15
9
THF (0.40)
f
10
11
12
13
14
15
16
17
18
19
20
21
TBME (0.96)
t-Amyl alcohol (1.09)
Cyclohexane (2.50)
PhCH3 (2.52)
> 99
96
> 99
Hexane (3.00)
96
Acetone (0.20)
30
94
93
72
72
42
68
51
THF (0.40)
36
TBME (0.96)f
98
t-Amyl alcohol (1.09)
Cyclohexane (2.50)
PhCH3 (2.52)
89
> 99
99
Hexane (3.00)
> 99
a
b
c
Conditions: rac-3a 25 mg, lipase 10 mg, organic solvent 500 µL, vinyl acetate 44 mg, 47 µL (3 equiv), 30 °C, 800 rpm (magnetic stirrer).
Logarithm of the partition coefficient of a given solvent between n-octanol and water according to ChemBioDraw Ultra 13.0 software indications.
Based on GC, for confirmation the % conversion was calculated from the enantiomeric excess of the unreacted alcohol (ees) and the product (eep) according to the
formula conv. = ees/(ees + eep).
d
e
f
Determined by chiral HPLC analysis by using a Chiralcel OD-H column.
Calculated according to Chen et al. [76], using the equation: E = {ln[(1 − conv.)(1 − ees)]}/{ln[(1 − conv.)(1 + ees)]}.
The results were taken from Table 1 for comparison.
decided to carry out the KR reactions deliberately for 24 h (in the case
of Amano PS-IM and Chirazyme L-10) and 4 h (in the case of Amano PS-
C II), respectively. Of all these solvents used, different non-polar, water-
immiscible solvents such as already tested TBME as well as 2-methyl-2-
butanol (t-amyl alcohol), cyclohexane, toluene (PhCH3) and hexane
gave the best conversion of starting material rac-3a. In turn, when both
biocatalysts were suspended in polar and/or water-miscible solvents
[acetone or tetrahydrofuran (THF)] then lipases maintained modest
activity (barely 14–28% conversion could be achieved depending on KR
conditions), thus furnishing slower-reacting enantiomer (S)-(−)-3a
with inferior ee-values (up to 33%). As shown in Table 5, lipase Amano
PS-C II suspended in TBME appeared to be superior to the majority of
other tested catalytic systems as furnishing astonishing enantiomeric
resolution of the model racemate rac-3a resulting in almost single en-
antiomers: (S)-(−)-3a (> 99% ee) and (R)-(+)-4a (98% ee). Interest-
ingly, Amano PS-C II was also very efficient in cyclohexane and hexane
while both resolution products could be afforded with excellent en-
antiomeric excess (96–99%).
Although one can see that Amano PS-IM catalyzed transestrification
of rac-3a very selectively in TBME, cyclohexane, and hexane leading to
isolation of the remaining alcohol (S)-(−)-3a in 96–100% ee range and
the forming acetate (R)-(+)-4a in 97–98% ee, however, it was t-amyl
alcohol and PhCH3, which furnished the most enantioselective
(E = 637) transformations. Moreover, reactions conducted in PhCH3
showed a slight increase of enantiomeric excess of (R)-(+)-4a (> 99%
ee) compared to that performed in t-amyl alcohol, in which the for-
mation of optically active acetate (R)-(+)-4a was afforded with 99%
ee. Taking into account all the above results as well as utility in DKR
approach, we found PhCH3 more suitable as the reaction medium for
the further screening experiments and potential DKR application.
Another variable parameter, which often play a pivotal role on
enantioselectivity and reaction rate is the type of the acyl group donor
reagent. Among vinyl esters highly applicable in KR approaches, those
which acidic parts consist of long aliphatic (fatty) chain is desirable as
their employment often leads to an impressive enhancement of the
catalytic activity and stereoselectivity of lipases in transestrification
reactions. Therefore, in the next set of experiments three different vinyl
esters were used: vinyl acetate, vinyl butanoate, and vinyl decanoate
(Table 6).
Although Amano PS-C II turned out to be ultra-selective catalyst
toward resolution of rac-3a enantiomers, we did not chose this lipase
for further enzymatic investigations, as it has been already withdrawn
from the offer of commercial suppliers, and optimization of the pre-
parative-scale reactions based on it would be useless from the view-
point of potential industrialization of such process. Moreover, if we
considered seriously DKR approach, very active biocatalysts such as
Amano PS-C II were not favorable. This obviously stems from the fact
that for successful DKR, the enantiomers’ resolution rate should never
exceed the racemization rate too much, to avoid depletion of the re-
solved enantiomer as this could result in an decrease of the ester re-
action product’s enantiomeric purity. In turn, Chirazyme L-10 was ex-
cluded from further enzymatic studies from similar reason as the
Amano PS-C II lipase (it is no longer commercially available) as well as
because it furnished aggravated results in terms of enantioselectivity
values (E = 11–43).
The influence of alteration of chain length in the acylating agent’s
acidic moiety on the course of the racemic alcohol rac-3a transestrifi-
cation was examined using 3 equiv of the appropriate vinyl ester under
catalysis of Amano PS-IM suspended in PhCH3 as solvent. Consideration
of the results summarized in Table 6 leads to the conclusion that re-
activity and enantioselectivity were hardly affected by the nature of
vinyl ester. All the examined acyl group donors behaved in a similar
manner except that the reaction was slightly slower in the case of vinyl
butanoate affording only 43% conversion. Moreover, as evidenced by
HPLC the performance of Amano PS-IM in PhCH3 under influence of
various vinyl esters remained constant and mostly lead to highly se-
lective transformations (E = 354–637) of the racemic starting material
rac-3a into optically active esters (R)-(+)-4a, (R)-(+)-5a and (R)-
8

P. Borowiecki, M. Dranka
BioorganicChemistryxxx(xxxx)xxx–xxx
Table 6
the optimized reaction conditions. As can be seen from the results
collected in Table 7, in almost all cases an excellent E-values can be
reached regardless of the variety of the substrate used, thus allowing
the isolation of non-racemic acetates in very high [92–97% ee for (R)-
(+)-4a, d, e, f, g, i], an excellent [99% ee for (R)-(+)-4b] and total
[ > 99% ee for (R)-(+)-4c and (R)-(+)-4h] enantiopurity. On the other
hand, using the established KR methodology a recovery of the re-
maining optically active alcohols could be obtained with very high
[96% ee for (S)-(−)-3d and (S)-(−)-3f (for 50% conv.)], excellent
[98–99% ee for (S)-(−)-3b, e, g, and i] and perfect [ > 99% ee for (S)-
(−)-3a, (S)-(−)–3c, (S)-(−)-3f (for 51% conv.) and (S)-(−)–3h] op-
tical purity. Under our standard reaction conditions, good yields of both
resolution chiral products can be afforded from racemic starting ma-
terials.
The acyl donor reagent screening for (Amano PS-IM)-catalyzed KR of rac-3a in
PhCH3 after 24 h.
Entry
b
b
Acyl donor reagent
Conv.a [%]
ees [%]
eep [%]
Ec
d
1
2
3
Vinyl acetate
48
43
49
91
76
94
> 99
> 99
98
637
459
354
e
f
Vinyl butanoate
Vinyl decanoate
a
Based on GC, for confirmation the % conversion was calculated from the
enantiomeric excess of the unreacted alcohol (ees) and the product (eep) ac-
cording to the formula conv. = ees/(ees + eep).
b
c
Determined by chiral HPLC analysis by using a Chiralcel OD-H column.
Calculated according to Chen et al. [76], using the equation: E = {ln
[(1 − conv.)(1 − ees)]}/{ln[(1 − conv.)(1 + ees)]}.
d
Conditions: rac-3a 25 mg, Amano PS-IM 10 mg, PhCH3 500 µL, vinyl
It is important to mention that in all cases except the reaction car-
ried out with non-substituted derivative rac-3a the KRs required to be
conducted for at least 72 h to achieve ca. 50% conversion value as para-
substituted derivatives rac-3b–i were enzymatically less reactive. The
performed experiments revealed that this extremely enantioselective
Amano PS-IM lipase has an acyl binding site which offers an optimum
environment to host homopropargylic alcohols bearing phenyl rings
with electron-donating bulky groups such as: –iPr and –tBu or with a
smaller electron-withdrawing –CF3 substituent. In those cases, kinetic
resolution process proceeded with the highest possible enantioselec-
tivity values (E = 1057) allowing to isolate both stereoisomeric forms
with excellent enantiomeric purities (> 99% ee). A marginally lower
enantioselectivities (E = 246–348) were observed in the cases of those
derivatives bearing simple phenyl ring with hydrogen atoms rac-3a or
which para-position in the phenyl ring was occupied by eOCH3 (rac-3i)
and eNO2 (rac-3f) groups, respectively. All these shown to be suitable
substrates here, and gave the desired resolution products in a very high
95–99% ee range. In turn, racemic compound possessing hydrogen
bioisostere (fluorine atom) at the para-position (rac-3g) as well as those
with electron-withdrawing groups, such as eBr (rac-3d) and eCl (rac-
3e) were slightly worse substrates for (Amano PS-IM)-catalyzed KR
approach (E = 126–194). Noteworthy, in order to obtain en-
antiomerically pure (S)-(−)-3a (> 99% ee) and (S)-(−)-3f (> 99% ee),
time of the preparative-scale reactions had to be slightly elongated up
to 26 h and 74 h (Table 7, entries 3 and 9), respectively.
acetate 44 mg, 47 µL (3 equiv), 30 °C, 800 rpm (magnetic stirrer). The results
were taken from Table 5 for comparison.
e
Conditions: rac-3a 25 mg, Amano PS-IM 10 mg, PhCH3 500 µL, vinyl bu-
tanoate 59 mg, 65 µL (3 equiv), 30 °C, 800 rpm (magnetic stirrer).
f
Conditions: rac-3a 25 mg, Amano PS-IM 10 mg, PhCH3 500 µL, vinyl de-
canoate 102 mg, 115 µL (3 equiv), 30 °C, 800 rpm (magnetic stirrer).
(+)-6a of very high enantiomeric purity (98–100% ee) leaving thereby
slower reacting stereoisomer (S)-(−)-3a in lower enantioenrichment
(76–94% ee). Thus, basically any of them can be used, however, for
convenience and simplicity of product isolation as well as for reasons of
the price of acyl donors, for the rest of KR studies we decided to employ
vinyl acetate.
In order to establish the scope and limitation of the developed en-
antiomer separation methodology, the optimized biocatalytic condi-
tions were applied for the rest of the synthesized homopropargylic al-
cohols rac-3b–i with different electronic and steric properties (Table 7).
Moreover, at this step of investigations in order to inspect if the de-
veloped enzymatic process is susceptible to scaling up, we decided to
examine the respective 4-fold linear enlargement of all the previously
set parameters including the amounts of racemic substrates rac-3a–i,
vinyl acetate concentration, lipase quantities and solvent volume under
Table 7
The (Amano PS-IM)-catalyzed preparative-scale KR of rac-3a–i in PhCH3.
Entry Substratea t [h] Conv.b [%] ees [%]/Yieldd eepc [%]/Yieldd Ee
3.4. Assignment of the stereochemistry of the EKR products
c
[%]
[%]
The stereochemistry of the obtained chiral non-racemic products
was determined by comparison of their specific rotation signs with re-
levant data reported in literature (see Table S2 in Supplementary ma-
terial). This assignment leads to a conclusion that Amano PS-IM follows
the empirical rule formulated by Kazlauskas [77], according to which
the (R)-enantiomer of a secondary alcohols possessing two much dif-
fering in size substituents attached to the asymmetric centre is pre-
ferentially transformed into ester when the lipases are utilized as the
catalysts. Unexpectedly, the specific rotation sign for optically active
para-nitro derivative 3f was in contrary to the rest of the enantiomeric
alcohols synthesized in the same enzyme-catalyzed kinetic resolution
manner and measured as a methanolic solution. Such result was con-
tradictive to the so-called Tschúgaeff rule [78], which assumes that
optically active compounds with a single asymmetric atom belonging to
homologous series should have the same sign of optical rotation.
Moreover, the stereochemical outcome of the reaction conducted with
rac-3f was uncertain since inconsistent results were obtained when
compared with literature data concerning specific rotation signs of that
particular non-racemic alcohol 3f [42]. This finding surprised us be-
cause it was unlikely that toward only one compound from the same
homologous series the enantioselectivity was reversed in the case of
Amano PS-IM. In addition, our uncertainty was strengthen when com-
paring the HPLC retention times for enantiomeric 3f and the rest of
optically active alcohols obtained in the lipase-catalyzed kinetic
1
rac-3a
24
25
26
72
72
72
72
72
74
72
72
72
49
51
51
50f
50
50
51
50
51
52
50
51
95/36
97/35
95/36
95/35
99g/32
> 99/29
96/25
95/34
96/42
97/43
92/44
> 99/20
96/22
246
206
206
1057
1057
194
180
194
348
126
1057
259
2
99/35
3
> 99/34
99/34
4
rac-3b
rac-3c
rac-3d
rac-3e
rac-3f
5
> 99/34
96/34
6
7
98/39
8
96/43
9
> 99/29
99/33
10
11
12
rac-3g
rac-3h
rac-3i
> 99/30
99/19
a
Conditions: rac-3a–i 100 mg, Amano PS-IM 40 mg, PhCH3 2 mL, vinyl
acetate (3 equiv), 30 °C, 800 rpm (magnetic stirrer).
b
Based on GC, for confirmation the % conversion was calculated from the
enantiomeric excess of the unreacted alcohol (ees) and the product (eep) ac-
cording to the formula conv. = ees/(ees + eep).
c
Determined by chiral HPLC analysis by using a Chiralcel OD-H or Chiralpak
AD-H column, respectively.
d
Isolated yield after column chromatography using mixture of CHCl3/
acetone (99:1, v/v) as eluent.
e
Calculated according to Chen et al. [76], using the equation: E = {ln
[(1cconv.)(1 − ees)]}/{ln[(1 − conv.)(1 + ees)]}.
f
g
Based on GC.
Determined by chiral HPLC after K2CO3-mediated hydrolysis of the re-
mainder acetate (R)-(+)-4b into the corresponding alcohol (R)-(+)-3b.
9

P. Borowiecki, M. Dranka
BioorganicChemistryxxx(xxxx)xxx–xxx
this regard, a series of racemic para-substituted 1-phenylbut-3-yn-1-ols
have been synthesized via Barbier-type propargylation of the respective
arenecarbaldehydes using 3-bromo-1-propyne (propargyl bromide, 2)
and SnCl2 assisted by KI in saturated aqueous NH4Cl solution.
Subsequently, the model substrate (1-phenylbut-3-yn-1-ol, rac-3a) was
tested with the collection of 17 different hydrolytic enzymes suspended
each in a solution of vinyl acetate as acyl donor in TBME, appropriately.
Using enantioselective transesterification synthetic variant carried out
under kinetically-controlled conditions at analytical scale, it turned out
that the lipases from Burkolderia cepacia (i.e. Amano PS-IM and Amano
PS-C II) exhibited excellent enantioselectivity (E > 200 at 30 °C).
Further screening of reaction media on stereochemical outcome re-
vealed that Amano PS-C II was superior to Amano PS-IM in terms of
catalytic activity and selectivity. However, due to criterion of avail-
ability of both commercial preparations, it was BCL immobilized on
diatomite (Amano PS-IM), which was selected for investigations on the
acyl-group donors’ effect as well as up-scaling studies. The examined
Amano PS-IM demonstrated great catalytic flexibility toward the sub-
strate scope as the variation of the substitution pattern on the phenyl
ring was very well-tolerated by this enzyme. The synthesized propargyl
alcohols with electron-donating or electron-withdrawing functional
groups are all suitable substrates for this methodology allowing to
achieve excellent enantiomeric enrichment (92–100% ee) and fair to
very good 19–44% isolated yields for both resolution products, re-
spectively. Nevertheless, the collected experimental data concerning
preparative-scale reactions clearly indicate that enantioselectivities
were the best for derivatives possessing –iPr (rac-3b), –tBu (rac-3c), and
–CF3 (rac-3h) groups in the para position, thus reaching the highest
possible E-value for lipase-catalyzed KR approach. The enantiomers of
all of the tested substrates with (R)-configuration were transesterified
with vinyl acetate preferentially by Amano PS-IM. Noteworthy, the
developed method provides significantly increased enantioselectivity
for the secondary homopropargylic alcohols compared to literature and
might be considered as highly useful starting-point for other synthetic
campaigns. Currently, DKR methodology based on the elaborated cat-
alytic system toward homopropargyl alcohols is under development in
our laboratories.
Fig. 3. An ORTEP plot of (R)-(+)-4f enantiomer. Thermal ellipsoids were
drawn at 50% probability and hydrogen atoms omitted for clarity (C black, H
gray, N blue, O red). The following crystal structure has been deposited at the
Cambridge Crystallographic Data Centre and allocated the deposition number
CCDC-1880174.
resolution of rac-3a–h. The HPLC chromatograms clearly showed the
same tendency in the order of the peaks among derivatives studied on
Chiralcel OD-H column. In all cases, the first minor peak consequently
represents the residual traces of (R)-enantiomer, while the second
major peak obviously corresponded to opposite (S)-enantiomer (see
Supplementary material). However, when compared retention para-
meters for acetate possessing nitro group (+)-4f with other chiral esters
resolved on the same HPLC column, the tendency was reversed. As we
were strongly puzzled with these results, therefore we decided to per-
form the polarimetric measurement again using this time CHCl3 (ac-
cording to Jain and co-workers [42]) instead of MeOH in which whole
homologous series was done. We suspected that it might be the solvent
that influences the sign of optical rotation. To our surprise, when we
have repeated the polarimetric measurement for a solution of homo-
chiral 3f (> 99% ee) in CHCl3 it turned out that the sign of optical
rotation was reversed {[α]D22 = –33.33 (c 1.05, CHCl3)} when com-
pared with the corresponding methanolic solution {[α]D23 = +4.76 (c
1.05, MeOH)} of the same alcohol’s sample, which confirmed our as-
sumption that it was the solvent effect. Moreover, as we could not be-
lieve in unexpected inversion of the Amano PS-IM enantiomeric pre-
ference toward para-nitro-substituted derivative rac-3f, therefore to
confirm if the absolute configuration was properly attributed by us, a
single-crystal X-ray analysis was additionally performed for con-
troversial optically active derivative. However, from the pair of en-
zymatically resolved enantiomeric products, we could prepare single
crystal of sufficient quality only for acetate (+)-4f. Moreover, despite
the lack of heavy atom in molecule of (+)-4f, Flack parameter value
was enough to determine properly the configuration. To our delight the
performed XRD inspection undoubtedly proved that (+)-4f is (R)-
configurated at the stereogenic carbon atom (Fig. 3), so the alcohol
(−)-3f must be (S)-configurated, and thus the Amano PS-IM stereo-
preference toward rac-3f is consequently consistent with Kazlauskas’
rule [77]. The ORTEP drawing of (R)-(+)-4f was prepared using free-
ware crystallographic software (ORTEP-3 for Windows) developed by
Louis J. Farrugia [79].
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This research was supported by the Warsaw University of
Technology, Faculty of Chemistry (statute work). In addition, we would
like to extend our sincere gratitude and appreciation for all of the hard
work and dedication provided by undergraduate students Marta
Wiśniewska, Dariusz Wieczorkowski and Klaudia Stachnik.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.01.050.
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