Chemoenzymatic process for the preparation of (S)-7-((tert-butyldiphenylsilyl)oxy)hept-1-yn-4-ol in a continuous packed-bed reactor, a key intermediate for eribulin synthesis

Article abstract of DOI:10.1021/acs.oprd.0c00308

A practical chemoenzymatic process has been developed for the preparation of optically pure (S)-7-((tertbutyldiphenylsilyl) oxy)hept-1-yn-4-ol, (S)-4. This was prepared via enantioselective acylation of racemic homopropargylic alcohol (+/-)-4 catalyzed by

Full text of DOI:10.1021/acs.oprd.0c00308

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Full Paper
Chemo-enzymatic process for the preparation of (S)-7-((tert-
butyldiphenylsilyl)oxy)hept-1-yn-4-ol in a continuous packed-
bed reactor (PBR), a key intermediate for eribulin synthesis.
Avula Shiva Krishna, Shalini Basetty, Rajesh Nasam, Samuel L. Ralte, Chada
Raji Reddy, Gangarajula Sudhakar, Srihari Pabbaraja, Srivari Chandrasekhar,
Prathama S. Mainkar, Thenkrishnan Kumaraguru, and Subhash Ghosh
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00308 • Publication Date (Web): 22 Sep 2020
Downloaded from pubs.acs.org on September 22, 2020
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Chemo-enzymatic
process
for
the
preparation
of
(S)-7-((tert-
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butyldiphenylsilyl)oxy)hept-1-yn-4-ol in a continuous packed-bed reactor
(PBR), a key intermediate for eribulin synthesis.
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Avula Shiva Krishna,a,b Shalini Basetty,a,b Rajesh Nasam,a,b Samuel L. Ralte,a,b Chada Raji
Reddy,a,b Gangarajula Sudhakar,a,b Srihari Pabbaraja,a,b Srivari Chandrasekhar,a,b Prathama S.
Mainkar,a,b Thenkrishnan Kumaraguru a,b and Subhash Ghosh a,b
aDepartment of Organic Synthesis and Process Chemistry, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India
bAcademy of Scientific and Innovative Research (AcSIR), Ghaziabad 201 002, India
*Corresponding Authors
E-mail: kumaraguru@iict.res.in & subhash@iict.res.in
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ABSTRACT: A practical chemo-enzymatic process has been developed for the preparation of
optically pure (S)-7-((tert-butyldiphenylsilyl)oxy)hept-1-yn-4-ol, (S)-4. This was prepared via
enantioselective acylation of racemic homopropargylic alcohol (+/)-4 catalyzed by Amano
lipase from Pseudomonas fluorescens. The acylation reaction was studied in a packed bed
reactor for the production of optically pure alcohol (S)-4 in a continuous mode. The estimated
kinetic parameters were Vmax 3.46 (0.17) mM/h/g and apparent Km value, Km,app= 582.10 (
29.10) mM (correlation coeff. r =0.96). At substrate concentration of 100 mg/mL (272 mM) and
flow rate of 0.1 mL/min, both the isomers are obtained in > 95% theoretical yield and >99 % e.e
at steady state in a packed reactor with 3.6 g enzyme. The advantage of this protocol is that the
enzyme can be recycled many times. Finally, this was converted to (R)-tert-butyl((4-chloro-6-
iodohept-6-en-1-yl)oxy)diphenylsilane, 3 which is key building block for the synthesis of C14-
C19 intermediate of eribulin.
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KEYWORDS: biocatalysis, continuous flow, lipase, kinetic parameters, resolution, eribulin.
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INTRODUCTION
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Eribulin, a truncated version of marine natural product halichondrin B is used for the treatment
of metastatic breast cancer. Currently Esai sells this drug in name Halaven or E7389. Several
strategies have been developed for the synthesis of eribulin and its intermediates.1a-k4-Methylene
tetrahydrofuran compound 1 is an essential building block (C14-C26) of eribulin (Figure 1)2 and
enantiomerically pure homopropargyl alcohol (S)-4 is used for its synthesis. From commercial
point of view, synthesis of both the enantiomers of 4 in enantiomerically pure form in less
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number of steps is an attractive option.
OMe
O
N
TfO
OMs
OTBS
2
CHO +
O
OH
I
Cl
OPiv
OTBDPS
OTBDPS
1
4
3
(S)-7-((tert-butyldiphenylsilyl)oxy)hept-1-yn-4-ol
Figure 1. Retro synthesis of C14-C26 building block of eribulin.
Cr- mediated asymmetric propargylation of similar aldehyde substrate using carbazole-
based ligands with 55% yield and 58% e.e has been reported.3 Another general process for the
preparation of optically pure homopropargylic alcohol is to add allenylzinc reagent generated in
situ in presence of a chiral amino alcohol ligand.4 Few other general methods are known in the
literatures for the synthesis of chiral homopropargyl alcohols.5ag These reported processes suffer
from major disadvantages such as use of highly expensive toxic metal-based reagents, low e.e,
large amounts of catalysts, cryogenic conditions, long reaction time, emulsion formation during
work-up and using column chromatography for the isolation of products. It is important to
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provide an alternative greener economically advantageous process for the preparation of
optically pure homopropargylic alcohol (S)-4.
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Earlier, preparation of (S)-4 has been carried out by CrBr3 mediated asymmetric
propargylation of an aldehyde with a propargyl halide with 78% yield and 90% e.e.2 The
enantiomeric purity was further improved by enantioselective hydrolysis of corresponding
acetate derivate catalyzed by Amano lipase at 50 C over a period of 43 h. This reaction was
carried out with the enzyme dissolved in aqueous buffer from which enzyme recovery and
recycle is difficult. Herein, we report the preparation of (S)-4 in a continuous mode by
enantioselective enzymatic acylation of substrate rac-homopropargyl alcohol, (+/-)-4 in organic
solvent in 95% yield with e.e. >99% (Scheme 1). The reaction was scaled up to 300 g. The
unwanted isomer (R)-4 was converted into required (S)-4 through Mitsunobu inversion.
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Scheme 1. Chemo-enzymatic preparation of (S)-4 and its conversion to homopropargyl chloride
(R)-8.
i). TBDPSCl, imidazole
DMF, rt, 12h.
O
propargyl bromide, Zn dust
OH
OTBDPS
H
ii). O3, CH2Cl2, -78 oC
30 min., Ph3P, rt, 12 h
92% over two steps.
sat. NH4Cl, THF, - 15 oC
12 h., 75%.
6
5
OAc
OH
(R)-4
enzyme, vinyl acetate
OH
OTBDPS
OTBDPS
+
OTBDPS
(+/-) 4
(S) 7
-
MTBE, 24h, 96%.
K2CO3, MeOH,
0 oC, 97%
1. DEAD,TPP,
OH
Cl
p-nitrobenzoic acid,
THF:Toluene (1:1)
2. K2CO3, MeOH,
90% over two steps.
Cl3CCONH2, PPh3
OTBDPS
OTBDPS
Py, CH2Cl2, rt, 24h,
90%.
(S)-4;
(R)-8
e.e > 99%
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RESULTS AND DISCUSSION
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Preparation of rac-homopropargylic alcohol (4). The rac-homopropargyl alcohol (+/-)-4 was
prepared from 1-pentenol in three steps as shown in scheme 1. TBDPS protection of 5 followed
by ozonolysis gave analdehyde that on propargylation with propargyl bromide in presence of
activated Zn dust and catalytic amount of saturated solution of ammonium chloride gave (+/-)-4
in 69% over three steps.6
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Screening of enzymes. Several commercially available enzymes were screened for the
resolution of rac-homopropargylic alcohol 4 by using vinyl acetate as an acyl donor in MTBE.
The results are given in Table 1.
Table 1. Screening of enzymes for enantioselective acylation
Conversion
S.No
Enzyme
% e.e
(HPLC, %)
Amano Lipase from Pseudomonas fluorescens (534730)a
Amano lipase PS, from burkholderiacepacia (534641)a
Lipase B Candida antarctica immobilized on Immobead 150
(54326)a
1
2
90.8
15.3
45.4
7.7
3
4
93.8
13.5
46.9
6.8
Lipase immobilized on Immobead 150 from
Thermomyces lanuginosus (76546)a
5
6
Lipase from porcine pancreas (L3126)a
9.8
4.7
4.9
2.3
Lipase from candida rugosa (62316)a
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8
9
CAL-B lipase immobilized on acrylic resin (Novozym 435)b
Lipase from Pseudomonas cepacia (62309)a
Lipase from Mucor miehei (62298)a
25.8
10.0
30.0
27.3
25.7
3.9
26.3
23.3
5.7
3.1
3.1
1.7
2.3
12.9
5.0
15.0
13.6
12.8
1.9
13.2
11.7
2.8
1.6
1.5
0.8
1.2
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Lipase from Aspergillus niger (62301)a
Lipase from candida cylindracea (62316)a
Lipase from Rhizopus niveus (62310)a
Lipase from Rhizopus oryzae (62305)a
Pseudomonas cepacia lipase (IMMABC-T2-150)c
Candida antarctica lipase A (IMMCALA -T2-150)c
Rhizopus oryzae lipase (IMMARO-T2-150)c
Aspergillus niger lipase (IMMANA-T2-150)c
Mucor javanicus lipase (IMMAMJ-T2-150)c
Candida cylindracea lipase (IMMCCMO-T2-150)c
Candida rugosa lipase (IMMCRL-T2-150)c
Pseudomonas fluorescens lipase (IMMAPF-T2-150)c
Thermomyceslanuginosa lipase (IMMTLL-T2-150)c
Rhizopus niveus lipase (IMMRNA-T2-150)c
2.3
22.3
3.1
1.2
11.3
1.6
4.1
2.0
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Rhizomucormiehei lipase (IMMRML-T2-150)c
3.9
11.6
4.1
0.5
3.6
1.2
2.9
9.5
1.9
5.8
2.1
0.2
1.8
0.6
1.4
4.7
Resinase HT lipase (IMMRES-T2-150)c
Lipex 100L lipase (IMMLIPX-T2-150)c
Novozymes 51032 lipase (IMML51-T2-150)c
Alcaligenes sp. lipase (IMMASMQ-T2-150)c
Bacillus subtilis lipase (IMMAULI-T2-150)c
Candida antarctica lipase B (IMMCALB-T2-150XL)c
Candida antarctica lipase B (IMMCALBY-T2-150)c
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Reaction conditions: rac-homopropargylic alcohol (50 mg, 0.14 mmol), Vinyl acetate (31 µL,
0.33 mmol) MTBE 1 ml, enzyme 10 mg, shaken for 24h at room temperature. Source: Sigma
Aldrich,a Novozyme,b Chiral vision,c catalogue number in bracket.
From the above table, two enzymes namely Amano lipase from Pseudomonas fluorescens (entry
1) & Lipase B Candida antarctica (CAL-B) immobilized on Immobead 150 from sigma Aldrich
(entry 3) were found suitable for the resolution of rac-homopropargylic alcohol 4. However,
immobilized CAL-B from Chiral Vision (entry 30 & 31) showed very less conversion. This may
be due to differences in immobilization methods, nature of the polymeric supports7af and water
content.7g,h Lipase from Pseudomonas fluorescens was selected for further studies due to its high
selectivity, robustness and cost effectiveness. In the present case, we measured the total water
content of the reaction mixture is >0.1% (w/w) and we have not observed any serious changes in
enzyme activity up to 0.2% (w/w). The competing hydrolysis reaction and the agglomeration of
enzyme powder were found to become a serious problem at >0.4%.
Choice of solvent for acylation. To find out the most appropriate solvent as reaction medium,
enzymatic reaction was performed in different solvents such as MIBK, Toluene, Acetonitrile,
THF, MTBE, DIPE and t-BuOH. Generally, lipase activity is higher in solvents of high log P
value by suitably hydrated and preserving its active conformation.8 MTBE, a solvent of low
water miscibility was chosen for further studies based on the higher solvent hydrophobicity (log
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P = 1) in comparison with ACN (log P = -0.34). Further, it was also observed that a high rate of
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conversion and enantioselectivity in this solvent (Figure 2).
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Cyclo
hexan
e
1,4
Dioxan
e
Toluen
e
t-
BuOH
MTBE
DIPE
MIBK
ACN
DCM
98.6
49.3
87.7
43.9
94.2
47.1
60.2
30.1
87.7
43.9
98.0
49.0
91.5
45.7
12.4
6.4
37.9
19.0
e.e (%)
Conversion (%)
Figure 2. Effect of different solvents on resolution of rac-homopropargylic alcohol. Reaction
conditions: [Substrate 4] = 50 mg, 0.14 mmol, [vinyl acetate] = 31 µL, 0.34 mM, MTBE = 1ml,
Amano Lipase from Pseudomonas fluorescens = 10 mg (10% w/w), shaken at 27 C. Reaction
°
period 24 h.
Enzymatic reaction in a stirred tank reactor. Initially, the reaction was carried out in a 250
mL jacketed vessel maintained at 27 C.9 Reactants were continuously stirred with an overhead
mechanical stirrer at 150 rpm. The progress of the reaction was monitored by taking samples at
regular intervals and analyzed through chiral HPLC. The reaction was continued till the reaction
was completed (1624 h). The enzyme kinetics follows a typical first-order exponential path
(Figure 3) with apparent kobsd = 0.0279 h-1 (correlation coeff. r = 0.9519).
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Time(h)
Figure 3. Kinetics of the reaction: [Substrate 4] 5g (13.64 mmol), vinyl acetate = 3.14 mL (34.06
mmol), MTBE = 50 ml, enzyme = 500 mg (10 % w/w) stirred at 27 C.
Advantages of a continuous packed-bed reactor over stirred tank reactor. The major
advantage of the resolution reaction carried out in packed bed reactor is to increase productivity
by shortening the reaction time. It will also circumvent the problems associated with mechanical
disruption of the biocatalysts, loss of enzyme & its activity during handling such as filtration,
washing and drying at commercial scale.10
Packed-bed reactor. Continuous kinetic resolution was carried out in a jacketed stainless steel
reactor at 27 C with circulating water bath. After draining excess solvent, mixture of the
substrate 4 and vinyl acetate dissolved in the MTBE was pumped from the bottom of the reactor
at 27 C and product stream was collected from the top at steady state (Figure 4).
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Lipase
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Product stream
OH
Jacketted SS column
id 4 mm & l 250 mm
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OAc
OTBDPS
OTBDPS
(S) 7
(R) 4
Water circulation
OTBDPS
OH
(+/-) 4
Pump
Substrate & acyl donor
in organic solvent
Figure 4. Schematic representation of packed-bed reactor.
Reactor performance. In a packed bed reactor the flow is in axial direction. Assuming uniform
velocity with no radial variation in reaction rate, the reaction rate is given by its performance
equation (eqn 1) at steady state condition.11
풅퐗퐀
-rA =
……..eqn (1)
풅퐖/퐅퐀ퟎ
Where, XA =Fraction of product converted; W = Weight of catalyst required; FA0= Volumetric
flow rate.
Effect of flow rate on conversion. Effect of flow rate was studied to investigate reactor
performance. Reaction is carried out using 2 g enzyme in packed bed reactor. At fixed substrate
concentration (0.14 M), the reactants were pumped at different flow rates (0.1 to 0.5 ml/min) and
the product from PBR was analyzed using HPLC. As expected for a PBR, increase in flow rate
caused decrease in conversion due to lower residence time.
The enzymatic reaction is of first order and at steady-state; the product formation in packed
reactor is given by eqn (2)12
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kobsd. τ = -ln(1-X) …….eqn (2)
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where 'τ' is residence time defined as 'τ' = reactor volume/flow rate; X is the mole fraction of the
product and 'kobsd' is the observed first-order rate constant.
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The plot of '' vs mole fraction of product, [X], followed an exponential curve expected for a
first-order reaction with kobsd = 0.028 h-1 0.0014 min-1 (Figure 5).
0.6
100
0.5
80
0.4
Mole fraction
60
of product
0.3
0.2
0.1
0.0
e.e
40
20
0
4
6
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10 12 14 16 18 20 22 24 26
Residence Time (min)
Figure 5. Effect of residence time on conversion and e.e at 27 C. Reaction Condition [Substrate
4] 0.14 M in MTBE, 1 eq vinyl acetate, 2 g Amano lipase from Pseudomonas fluorescens (bed
volume 2.5ml).
Effect of substrate concentration on reaction. Effect of substrate concentration on rate of the
reaction at steady state was studied in the packed bed reactor with fixed amount of lipase (2.0 g)
and fixed flow rate of 0.1 mL/min. As expected for an enzymatic reaction in a tubular reactor,
increasing substrate concentration (from 50 mg/mL, 0.14 M, to 500 mg/mL, 1.0 M) causes
increase in the rate of the reaction in the outflow (Figure 6). Analysis of the data using
Michaelis-Menten equation provided the apparent Vmax value, Vmax, app = 3.46 mM/h/g and
apparent Km value, Km, app = 0.58 M (correlation coeff. r = 0.96).
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2.5
2.0
1.5
1.0
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0.0
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0
500
1000
1500
Substrate 4 (mM)
Figure 6. Effect of substrate concentration for resolution of rac-homopropargylic alcohol by
using 2 g of Amano Lipase from Pseudomonas fluorescens in PBR.
Reaction conditions: Substrate (+/-)-4 concentration (0.14 M1.1 M) in MTBE at 27 C at 0.1
ml/min flow rate.
From the Michaelis-Menten kinetic parameters (assuming isothermal reactor without pressure
drop and catalyst inhibition), a theoretical model can be used to calculate the weight of the
catalyst required to achieve 50% conversion at different substrate concentrations (Figure 7).
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3
2
0
50
100 150 200 250 300 350 400 450
Substrate 4 (mg/ml)
Figure 7. Theoretical requirement of Amano Lipase from Pseudomonas fluorescens for
resolution of rac-homopropargylic alcohol (+/-)-4 at various substrate concentrations (50-400
mg/ml).
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The model was tested with packed bed reactor consisting of two columns of dimensions 250
mm×4 mm connected in series. Total weight of enzyme in the packed bed was 3.6 g. 10 g of
substrate (272 mM) dissolved in 100 mL MTBE and 2.77 mL of vinyl acetate (30 mM) was
pumped from bottom using dosing pump at 27 C. The flow rate was maintained at 0.1 mL/min.
Analysis of the sample from top of the reactor showed e.e of >99%. The enzyme filled in the
reactor was used continuously for one week without significant change in productivity. The
productivity achieved by us is 1.12 mM/h/g with advantage of using enzyme in a continuous
reactor which can provide the mixture of products (10.2 g, >96%) of high enantiomeric purity (>
99% e.e).
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Product separation. After completion of the enzymatic reaction, the reactor was washed twice
with MTBE. The filtrate contained optically pure homopropargyl alcohol, (R)-4 and acylated (S)-
7. Isolation of the products without chromatography was achieved by preparing succinyl
derivative of the hydroxyl product. Direct succinylaion with succinic anhydride in MTBE gave
only 10% poduct after 16 h probably due to insolubility of succinic anhydride. Addition of ACN
as a cosolvent (0.5% v/v) to solubilize it improved the conversion to 60% but created an
additional problem of solvent separation and recovery. Thus MTBE was first recovered by
distillation and different solvens were tested for dissolution of the oily reaction mass and
succinylation. 2-Methyltetrahydrofuran gave 5% conversion, ACN gave 45% conversion and the
complete conversion was observed in DCM (>99%). Hence, the oily reaction mass was diluted
with DCM followed by succinylation of the (R)-4 to form its half-ester. Unreacted acyl isomer
(S)-7 was extracted with hexane and the half-ester of (R)-4 was hydrolysed with 4N NaOH to
give (R)-4 which was converted to the required (S)-4 via Mitsunobu inversion. The acylated
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isomer (S)-7 was treated with K2CO3 in MeOH to give alcohol (S)-4. Thus, the products were
separated with 90% overall yield and e.e >99% purity (HPLC analysis).
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Enzyme recyclability. After completion of every batch, a sample of enzyme was collected (50
mg) and its activity was assayed after washing with MTBE. The reactor was used continuously
for one week for 5 batches without significant change in productivity and change in activity of
the enzyme. Since the product was highly soluble in the solvent, we have not observed any
product accumulation on the surface of the enzyme matrix.13
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CONCLUSIONS
We have developed a sustainable and improved process for the preparation of optically pure
homopropargyl alcohol (S)-4 in >95% overall yield with >99% e.e in a large scale. The
resolution was carried out in packed bed reactor to increase the productivity and avoid the
enzyme deactivation by rupture and prolonged contact with substrate, product and solvents. Both
isomers were utilized to produce optically pure homopropargyl chloride (R)-8, the C14 - C19
building block of halichondrins and E7389.
EXPERIMENTAL SECTION
General Remarks. Jacketed stainless steel columns with dimensions (250mm* 4.6 mm) were
used as packed bed reactors. HPLC pump (Shimadzu LC 20AD) is used to pump substrate
solution through the packed column. All other chemicals and reagents were obtained from Sigma
1
Aldrich, Himedia. IR spectra were recorded on a Perkin- Elmer RX-1 FT-IR system. H NMR
(300 MHz) and 13C NMR (75 MHz) spectra were recorded on Bruker Avance-300 MHz
spectrometer. HPLC analyses were carried out on Shimadzu HPLC Unit LC 20 AD with diode
array detector. Chiral HPLC columns were obtained from Daicel Chemical Corporation, Japan.
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Optical rotations were measured with a Horiba-SEPA-300 digital polarimeter. Mass spectra were
recorded on a Q STAR mass spectrometer (Applied Biosystems, USA).
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Determination of lipase activity. The lipase activity of Pseudomonas fluorescens was carried
out by following our reported literature procedure14 in order to check the enzyme activity and
recyclability at any given point of time. The enzyme showed lipase activity of 19,896 units/g
(according to the supplier ≥ 20,000 U/g) by using tributyrin as a substrate. The enzyme activity
was calculated by using the following equation (3),
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퐒퐩퐞퐜퐢퐟퐢퐜 퐚퐜퐭퐢퐯퐢퐭퐲 (퐔퐧퐢퐭퐬/퐠) = {[ 퐕퐨퐥.퐨퐟 퐍퐚퐎퐇 퐜퐨퐧퐬퐮퐦퐞퐝 퐢퐧 ퟏퟎ퐦퐢퐧] × [퐍퐨퐫퐦퐚퐥퐢퐭퐲 퐨퐟 퐍퐚퐎퐇] × ퟏퟎퟎퟎ × ퟏퟎퟎ}
------ eqn (3)
[퐦퐠 퐨퐟 퐞퐧퐳퐲퐦퐞 퐮퐬퐞퐝]
4-((tert-Butyldiphenylsilyl)oxy)butanal, 6. To a stirred solution of pent-4-en-1-ol, 5 (50 g, 581
mmol) in DMF (500 mL, 10% w/v) at 0 C, imidazole (59.2 g, 870 mmol) and DMAP (7 g, 57
mmol) were added followed by TBDPS-Cl (149 ml, 852 mmol) under nitrogen atmosphere. The
reaction mixture was allowed to stirring for 16 hrs. Then reaction mixture was quenched with
ice-cold saturated aqueous NH4Cl (150 mL) solution and extracted with EtOAc (3 x 500 mL).
The combined organic extracts were washed with water (500 mL) followed by brine (400 mL),
dried over anhydrous Na2SO4 and evaporated under reduced pressure to obtain 179 g of TBDPS
protected alcohol as a colourless oil. To a solution of crude compound obtained above (120 g,

370 mmol), DCM (960 mL) was added and maintained at -78 C. Ozone was bubbled until the
solution turned blue. Then oxygen was bubbled for 10 minutes and triphenylphosphine (120 g,
458 mmol) was added portion-wise at -78 C. The reaction mixture was allowed to warm up
slowly to room temperature and for 16 h. The solvent was removed under reduced pressure and
the residue was purified by flash chromatography (SiO2, 8% EtOAc/hexane) afforded the title
compound 6 as colourless oil (117 g, 97%). Rf = 0.5 (SiO2, 10% EtOAc/hexane).1H NMR (500
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MHz, CDCl3):δ 9.77 (d, J = 1.0 Hz, 1H), 7.72–7.66 (m, 4H), 7.48–7.39 (m, 6H), 3.76-3.71 (m,
2H), 2.61-2.54 (m, 2H), 1.96–1.89 (m, 2H), 1.09 (s, 9H); 13C NMR (100 MHz, CDCl3):δ 202.36,
135.44, 133.49, 129.60, 127.62, 62.84, 40.65, 26.76, 25.17, 19.11.
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(±)-7-((tert-Butyldiphenylsilyl)oxy)hept-1-yn-4-ol (4). To a stirred solution of aldehyde, 6 (100
g, 306 mmol) in dry THF (750 mL) was added propargyl bromide (79.2 mL, 918 mmol)
followed by activated Zn dust (80 g, 1224 mmol) at -15 C. To the reaction mixture saturated
aqueous ammonium chloride solution was added dropwise over a period of two hours (until
effervescence were stopped). After completion of the reaction (monitored by TLC) the crude was
filtered through celite bed and the filtrate was concentrated under reduced pressure. The crude
residue was extracted with ethyl acetate (2x500 mL) and the combined organic extracts were
washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure to
furnish racemic alcohol, 4 (84.5 g, 75 %).
Enzymatic resolution of (+/)-4 in a stirred tank reactor (STR). To a jacketed vessel (250
mL) equipped with an overhead stirrer added (+/)-homopropargylic alcohol 4 (5 g, 14 mmol),
vinyl acetate (3.1 mL, 34 mmol), MTBE (50 mL, 10% w/v), and Amano lipase PS (0.5 g, 10%
w/v). The reaction mixture was stirred at 27 C for 16–24 h. The progress of the reaction was
monitored by TLC. The optical purity of homopropargylic alcohol (R)-4 and acetate (S)-7 thus
obtained was confirmed to be > 99 % from chiral HPLC analysis. The optical purity of acetate
was checked after deprotection with K2CO3. The isolated yields were calculated after column
purification of both the isomers [(R)-4 = 2.48 g, 99% yield, > 99 % e.e & (S)-7 = 2.3 g, 92 %
yield, > 99 % e.e)].
Kinetic resolution of (+/)-4in a packed bed reactor (PBR). The flow chemistry set-up, as
illustrated in Fig. 4 was built using a jacketed SS column [2× (250 mm x 4 mm)] which was
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filled with the lipase from Pseudomonas fluorescens (3.6 g). Temperature of the packed bed
reactor was maintained at 27 C with circulating water bath. (+/-)- homopropargylic alcohol 4
(10g, 272 mM) and vinyl acetate (2.8 mL, 30 mM) dissolved in MTBE (100 mL, 10 % w/v) were
then pumped at a fixed flow rate from the bottom of the reactor using an HPLC pump (Shimadzu
LC 20AD) with 0.1 mL/min flow rate. The product stream was collected from the top and
analyzed by HPLC at steady state (30 mins). After completion of the reaction, the enzyme in the
reactor was washed with MTBE and reused for next batch.
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Product separation.
Step 1. Isolation of (S)-7 by semi ester formation of (R)-4. The combined MTBE fraction
obtained from above step was evaporated under reduced pressure. The residue (approx.10.2 g)
was diluted with DCM (100 mL) and treated with Et3N (5.9 mL, 42 mmol), succinic anhydride
(1.5 g, 15 mmol) and DMAP (332 mg, 2.7 mmol) at 0 C. The reaction mixture was stirred at
027 C for 16 h. After completion of the semi ester formation, the reaction mixture was
evaporated under reduced pressure. The residue was extracted twice with hexane (2 x 15 mL)
and washed with water (2 x 10 mL) followed by brine solution (10 mL). The organic layer was
evaporated under reduced pressure to obtain optically pure acetyl product (5.1 g, > 92%
theoretical yield, > 99% e.e). The optical purity of acetate was checked after deprotection.
1H NMR (500 MHz, CDCl3): δ 7.71–7.65 (m, 4H), 7.46-7.36 (m, 6H), 4.96 (m, 1H), 3.74–3.65
(m, 2H), 2.50-2.46 (m, 2H), 2.06 (s, 3H), 2.0 (m, 1H), 1.87 (m, 1H), 1.8 (m, 1H), 1.76 (m, 1H),
1.65 (m, 1H), 1.57 (m, 1H), 1.07 (s, 9H);13C NMR (100 MHz, CDCl3):δ170.56, 135.54, 133.86,
129.57, 127.61, 79.63, 71.54, 70.42, 63.35, 29.38, 28.22, 26.84, 23.91, 21.10, 19.19.
Step 2. Isolation of (R)-4 by base hydrolysis of semi ester. The residue obtained from step-1
was diluted with water and basified with 4N NaOH till pH reaches 12-14 and stirred for 30 mins
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to 1 hr. The progress of the reaction was monitored by TLC. After complete hydrolysis of semi-
ester, the reaction mixture was extracted twice with EtOAc (2 x 50 mL). The organic layer was
dried over anhydrous Na2SO4 and concentrated under reduced pressure to give an isolated
optically pure homopropargylic alcohol (4.9 g, 98% theoretical yield, > 99% e.e). 1H NMR (500
MHz, CDCl3):δ 7.70-7.65 (m, 4H), 7.46-7.36 (m, 6H), 3.80 (m, 1H), 3.73-3.68 (m, 2H), 2.57 (br,
OH), 2.43 (ddd, J = 8.3, 5.5, 2.6 Hz, 1H), 2.36 (ddd, J = 16.8, 5.5, 2.6 Hz, 1H), 2.04 (t, J = 2.7
Hz, 1H), 1.80-1.68 (m, 2H), 1.68-1.59 (m, 2H), 1.05 (s, 9H); 13C NMR (100 MHz,
CDCl3):δ135.55, 133.57, 129.65, 127.66, 81.01, 70.60, 69.75, 63.97, 33.12, 28.68, 27.29, 26.82,
19.15.
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Step 3. Enzyme recyclability. After completion of the reaction, small quantity of enzyme
(50mg) was collected from the reactor and the activity was tested by titration. The used enzyme
was found to be recyclable for up to 5 times without significant change in productivity and
selectivity of the enzyme. The recovered enzyme was used for next 5 batches.
(S)-7-((tert-Butyldiphenylsilyl)oxy)hept-1-yn-4-ol [(S)-4] To a stirred solution of compound
(R)-7-((tert-Butyldiphenylsilyl)oxy)hept-1-yn-4-ol, 4 (1 g, 2.7 mmol) in THF:toluene (16 ml 1:1
ratio) was added p-nitrobenzoic acid (0.7 g, 4.1 mmol) and triphenylphosphine (1.1 g, 4.1 mmol)
at rt. Then the reaction mixture was cooled to 0 C and treated with DIAD (0.8 mL, 4.1 mmol).
After stirring for 12 hours the reaction mixture was quenched with saturated solution of NaHCO3
and extracted with ethyl acetate (50 mL). The combined organic extracts were washed with
water (50 mL), brine (50 mL), dried over Na2SO4and concentrated under vacuum to give p-nitro
benzoate (1.3 g).
To a stirred solution of crude ester compound of p-nitro benzoate (1.3 g, 2.2 mmol) in dry DCM
o
(6.5 ml) added DIBAl-H (1M, 8.9 ml) slowly at -78 C and allowed to stir for 1 hour and
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reaction mixture brought to 0 C, allowed stirring for 1 hour and after completion of the reaction
(monitored by TLC) the reaction mixture was quenched with methanol and ice-cold water
allowed stirring for 2 hours and extracted with ethyl acetate, concentrated under vacuum.
Purification of the residue by flash chromatography (SiO2, 4% EtOAc/hexane) afforded (S)-7-
((tert-Butyldiphenylsilyl)oxy)hept-1-yn-4-ol, (S)-4 as colourless oil (900 mg, 92% over two
steps).Rf = 0.3 (SiO2, 10% EtOAc/hexane).
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(R)-tert-Butyl((4-chlorohept-6-yn-1-yl)oxy)diphenylsilane [(R)-8]. To
a
solution of
homopropargyl alcohol, (S)-4 (1 g, 2.7 mmol) in CH2Cl2 (10 mL, 10% w/v) was sequentially
added pyridine (0.6 mL, 7.4 mmol), triphenylphosphine (1 g, 3.8 mmol) and trichloroacetamide
(619 mg, 3.8 mmol) at 27 C. After stirring for 24 h, the reaction mixture was washed with brine
(3x10 mL). The organic layer was separated, dried overanhydrous Na2SO4 and concentrated
under reduced pressure and purified by column chromatography to give (R)-6 (0.95 g, 90%) as
1
light yellow oil. H NMR (500 MHz, CDCl3):δ 7.70-7.64 (m, 4H), 7.46-7.36 (m, 6H), 4.01 (m,
1H), 3.75 – 3.64 (m, 2H), 2.69 (ddd, J = 17.1, 6.0, 2.7 Hz, 2H), 2.63 (ddd, J = 17.1, 6.0, 2.7 Hz,
2H), 2.15-2.04 (m, 2H), 1.88 – 1.76 (m, 2H), 1.68 (m, 1H), 1.06 (s, 9H);13C NMR (100 MHz,
CDCl3):δ 135.53, 133.77, 129.60, 127.63, 79.88, 70.96, 63.01, 59.70, 33.62, 29.20, 28.63, 26.85,
19.19.
Chiral HPLC Analysis. Resolution of rac-homopropargylic alcohol was followed by chiral
HPLC. Column Chiralcel OD-H (250 x 4.6 mm, Particle size 5 µm), Daicel Chemical Industries,
Japan. Mobile phase, 2% IPA in Hexane. Flow rate, 0.4 ml/min. Detection wavelength 220nm.
Retention times: 4 (R)-OH = 22.5 mins; 4 (S)-OH = 23.95 mins; 7 (S)-OAc = 11.26 mins; 8 (R)-
Cl = 9.8 mins; 8 (S)-Cl = 10.23 mins.
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The authors declare no competing financial interests.
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ACKNOWLEDGEMENTS
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The authors thank the Council of Scientific and Industrial Research (CSIR), Ministry of Science
and Technology, Government of India for funding this project under FTT grant (MLP-0025).
S.B. gratefully acknowledges DST for WOS-A Fellowship (SR/WOS-A/ET-16/2018). The IICT
Communication Number for this article is IICT/Pubs./2020/166
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