Efficient enzymatic process for the production of (2S)-4,4-difluoro-3,3- dimethyl-N-Boc-proline, a key intermediate in the synthesis of HIV protease inhibitors

Article abstract of DOI:10.1021/op060004+

(2S)-4,4-Difluoro-3,3-dimethyl-N-Boc-proline (3) is a key intermediate for the synthesis of HIV protease inhibitors. Here, several approaches for the preparation of enantiopure 3 and its analogues are disclosed. Among these methods, one strategy relies on

Full text of DOI:10.1021/op060004+

Organic Process Research & Development 2006, 10, 650−654
Efficient Enzymatic Process for the Production of
(2S)-4,4-Difluoro-3,3-dimethyl-N-Boc-proline, a Key Intermediate in the
Synthesis of HIV Protease Inhibitors
Shanghui Hu,*,† Carlos A. Martinez,† Billie Kline, Daniel Yazbeck, Junhua Tao, and David J. Kucera
Chemical Research & DeVelopment, Pfizer Global Research and DeVelopment, La Jolla Laboratories,
10578 Science Center DriVe, San Diego, California 92121, U.S.A.
Abstract:
(2S)-4,4-Difluoro-3,3-dimethyl-N-Boc-proline (3) is a key inter-
mediate for the synthesis of HIV protease inhibitors. Here,
several approaches for the preparation of enantiopure 3 and
its analogues are disclosed. Among these methods, one strategy
relies on resolving the racemic methyl ester of 3 through a
protease-catalyzed enantioselective hydrolysis. Despite the fact
that this resolution was applied to prepare kilogram quantities
of optically pure acid 3 for clinical trials, this process suffered
from low efficiency, high cost and difficulties in improvement
by medium engineering. An alternative much more efficient and
Figure 1. HIV protease inhibitors.
cost-effective enzymatic process was therefore developed by
switching the protective group of the proline esters from a Boc
known,2a-d there are no good synthetic methods available
to a benzyl moiety. This new process has a much higher
for the preparation of difluoroproline 3. Thus, to prepare
throughput (6.3 mmol/h/L vs 0.11 mmol/h/L), and the cost of
compounds 1 and 2 on a large scale for clinical trials, it is
the process was also dramatically reduced to only 5% of the
protease resolution process.
essential to develop an efficient, scalable, and cost-effective
process for the production of enantiopure 3 or its derivatives
with high optical purity. We disclose here several efficient
enzymatic preparations of optically pure acid 3. The strategies
for improving the efficiency and the throughput of the
enzymatic process, i.e., by switching the protective groups
of substrates, will be described in detail.
Introduction
Ongoing treatment of HIV-infected patients with com-
mercially available protease inhibitors has led to the develop-
ment of mutant viruses that possess proteases that are
resistant to the inhibitory efficacy of these agents. Thus, there
continues to be a need for developing new broad-spectrum
HIV protease inhibitors against the newly emerging mutant
strains of the viruses.1a-c Compounds 1 and 2 fall into this
category of inhibitor and are currently entering human
clinical trials. Both of the molecules contain a chiral
difluoroproline moiety (bracketed, Figure 1) and 3-amino-
2-hydroxy-4-phenylbutanoic acid (AHPBA) that are essential
for their inhibitory activity.
Results and Discussion
The initial synthetic approach to 3 is composed of nine
steps starting from N-Boc-glycine, which included a difficult
and expensive fluorination step together with one chroma-
tography purification (Scheme 1).1c,d Optically pure 3 is
obtained through the resolution of any one of racemic 4 (or
4′)-6 (Scheme 1) by enzymatic hydrolysis or diastereomeric
resolution.
Here, an automated enzyme screening protocol recently
developed in our lab was used to identify the ideal enzymes.3
After screening 94 hydrolases including lipases, proteases,
esterases, and acylases, an alkaline protease from Bacillus
licheniformis (BL), commonly known as subtilisin Carlsberg
was found to catalyze the enantioselective hydrolysis of the
(S)-esters of racemic mixtures 4-6. In all three cases, the
4,4-Difluoro-3,3-dimethyl-N-Boc-proline 3 is a key in-
termediate for the synthesis of protease inhibitors 1 and 2.
Although numerous efficient syntheses of AHPBA are
* Author for correspondence. Telephone: (860)-686 2157. Fax: (860)-441-
4109. E-mail: shanghui.hu@pfizer.com.
† Current address: Pfizer Global Research and Development, Chemical
Research and Development, Eastern Point Road, Groton, CT 06340.
(1) (a) Mimoto, T.; Kato, R.; Takaku, H.; Nojima, S.; Terashima, K.; Misawa,
S.; Fukazawa, T.; Ueno, T.; Sata, H.; Shintani, M.; Kiso, Y.; Hayashi, H.
J. Med. Chem. 1999, 42, 1789. (b) Yoshimura, K.; Kato, R.; Yusa, K.;
Kavlick, M. F.; Maroun, V.; Nguyen, A.; Mimoto, T.; Ueno, T.; Shintani,
M.; Falloon, J.; Masur, H.; Hayashi, H.; Erickson, J.; and Mitsuya, H. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 8675. (c) Hammond, J. L.; Patick, A. K.
U.S. Pat. Appl. 2005/0171038. (d) Kucera, D. J.; Scott, R. W. U.S. Pat.
Appl. 2005/0171038.
(2) (a) Reetz, M. T.; Drewes, M. W.; Harms, K.; Reif, W. Tetrahedron Lett.
1988, 29, 3295. (b) Herranz, R.; Castro-Pichel, J.; Vinuesa, S.; Garcia-
Lopez, M. T. J. Chem. Soc., Chem. Commun. 1989, 938. (c) Hamashima,
Y.; Sawada, D.; Kanai, M.; Shibasaki, M. J. Am. Chem. Soc. 1999, 121,
2641. (d) Hayashi, Y.; Kinoshita, Y.; Hidaka, K.; Kiso, A.; Uchibori, H.;
Kimura, T.; Kiso, Y. J. Org. Chem. 2001, 66, 5537-5544 and references
therein.
(3) Yazbeck, D. R.; Tao, J.; Martinez, C. A.; Kline, B. J.; Hu, S. AdV. Synth.
Catal. 2003, 345, 524.
650
•
Vol. 10, No. 3, 2006 / Organic Process Research & Development
10.1021/op060004+ CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/17/2006

Scheme 1
Scheme 3
Scheme 4
Table 1. Subtilisin Carlsberg-catalyzed enantioselective
hydrolysis of esters 4-6a
substrate
time (h)
yield (%)
ee (%)
E
config
4
5
6
51
16
260
42
45
40-45
91.4
98
96
37
S
S
S
>200
74
However, when multikilogram quantities of 3 were
required for further clinical trials, it became very difficult
to provide such large amounts of material using the discovery
route due to the low efficiency and extremely high cost of
this process (<3% overall yield, Scheme 1). A modified
process based on this route was subsequently developed
(Scheme 3, 10-15% overall yield),1c,d and one of major
improvements was to use a much more efficient fluorination
process to produce benzyl-protected proline ester 8. As the
issues for chemical steps were resolved, the enzymatic step
became the bottleneck for the entire synthesis.
a The yields, ee and configurations are reported for the produced acids.
Scheme 2
To address these issues, initially we attempted to improve
the CLEC-BL resolution process through further optimization
(Scheme 4), i.e., adding different cosolvents, tuning reaction
pH, changing the buffer or the temperature. Unfortunately,
after an extensive investigation of a variety of parameters,
only a slight improvement was achieved for the process.
Under the optimized conditions, the resolution reached 45%
conversion with 20 g/L of substrate concentration in 10%
DMSO and Tris-buffer mixture at 40 °C for 6 days. This
process not only has a low throughput (0.18 mmol/h/L), but
it also is still very expensive and not feasible for the
production of optically pure acid at multikilogram scale in
an efficient and economic manner.
At this stage, the development of an alternative strategy
for the efficient production of enantiopure acid 3 was
becoming the most urgent and challenging task. Although
directed enzyme evolution is a promising approach for the
improvement of enzyme activity,4 it is time-consuming and
difficult to obtain mutated enzymes within the desired time
frame. Thus, we had to look for a simple, fast, and efficient
solution to meet the delivery timeline for the clinical trials.
It is well-known that in many cases the activity and the
stereoselectivity of enzymes can be improved through the
modification of substrates. Fine-tuning of substrate structures,
i.e., by switching the protective groups of the substrates, may
improve the enzyme activity and selectivity dramatically.5
After a thorough study of the modified route (Scheme 4),
desired (S)-acids were obtained in high enantioselectivity and
good yields (Table 1).
Alternatively, the racemic acid 4′ was resolved using (S)-
(-)-2-phenylglycinol as the diastereomeric resolving agent
to afford the desired (S)-acid 4′ in 40% isolated yield and
>98% ee (Scheme 2). This classical resolution process gives
a higher optical purity than the enzymatic resolution of 4.
Moreover, the classic resolution also cuts off the chemical
esterification step from 4′ to ester 4.
Although the enzymatic or classical resolution of the early
intermediates 4, 4′, and 5 is more efficient than the BL
protease resolution of 6, concerns regarding the loss of
chirality during downstream, harsh chemical steps and the
development of other more efficient route(s) prohibited us
from further optimization and scale-up of these resolutions.
We have thus chosen to scale-up the resolution of the proline
intermediate 6 using a cross-linked form of BL commercial-
ized as CLEC-BL by Altus. Compared to the solution form
of the enzyme, CLEC-BL showed several advantages for the
hydrolysis of 6. These included the recyclability of the
enzymes and the increased rate of reaction as compared to
the solution form of the same enzyme. In the production of
1 kg of optically pure acid 3 for clinical trials, this enzymatic
process worked fairly well, and the relatively high cost of
the enzyme was not the major issue compared to the issues
of the fluorination step. After 10-12 days of reaction with
25% v/v of CLEC-BL suspension, acid 3 was obtained with
98% optical purity and 35% isolated yield. At this stage,
the quick delivery of high-quality material was more
important than the allocation of resources to optimize the
process and the exploration of other resolution processes.
(4) (a) Carter, P.; Abrahmsen, L.; Wells, J. A. Biochemistry 1991, 30, 6142.
(b) Martinez, P.; Van dam, M. E.; Robinson, A. C.; Chen, K.; Arnold, F.
H. Biotech. Bioeng. 1992, 39, 141. (c) Morley, K. L.; Kazlauskas, R. Trends
Biotechnol. 2002, 23, 231.
(5) Pohl, T.; Waldmann, H. Tetrahedron Lett. 1995, 36, 2963.
Vol. 10, No. 3, 2006 / Organic Process Research & Development
•
651

Table 2. Enzymatic hydrolysis of proline esters with
Scheme 5
different protective groupsa
substrate
enzyme
rate
E
config
6 R ) Boc
8 R ) Benzyl
9 R ) CBz
10 R ) H
BL
1
72
S
S
-
R
-
of PLE significantly reduced the emulsion issue during the
downstream separation of the remaining ester from the
produced acid by extraction. The PLE process was success-
fully used to resolve multikilograms of 8, and a significant
cost savings was achieved via this new process (Scheme 5).
Additionally, the resolution of other intermediates, i.e.,
4-oxo-substituted proline 7 through enzymatic hydrolysis,
and racemic acid 3 by diastereomeric resolution, may provide
alternative efficient pathways for the preparation of optically
pure acid 3. Clearly, the process efficiency can also be
improved significantly through the development of an
efficient racemization process for the wrong enantiomers of
3, 6, or 8.
PLE
PLE
BL
>100
>200
45
8
5
1
>100
>100
PLE
a The experiments were performed under the enzymatic screening conditions.3
PLE represents the most useful esterase for the preparation
of optically pure fine chemicals.6 One of the major reasons
for cautious use of enzymes from mammalian sources is the
concern that they might be contaminated with infectious
pathogens of BSE. As a result, mammal enzymes are
generally used to prepare early pharmaceutical intermediates
rather than late and final active pharmaceutical ingredients.
Recently, recombinant PLE was successfully cloned and
functionally expressed in the methylotropic yeast Pichia
pastoris.7 It is possible that commercialized PLE free from
mammal sources will be available in the coming years. This
will clearly broaden the application of PLEs in pharmaceuti-
cal manufacturing processes.
In conclusion, an efficient and cost-effective enzymatic
process was developed for the production of (2S)-4,4-
difluoro-3,3-dimethyl-N-Boc-proline and its analogues in
high yield and excellent optical purity. The issues of low
throughput and high enzyme cost for the protease resolution
process were overcome by switching the protective groups
of the substrates. This simple strategy led to an efficient,
cost-effective, and scalable enzyme process for the produc-
tion of (2S)-4,4-difluoro-3,3-dimethyl-N-Boc-proline at a
multikilogram scale.
Figure 2. pH effect on PLE hydrolysis of benzyl proline
ester 8.
any of the proline derivatives 8-10 were examined as
potential substrates for enzymatic hydrolysis.
After screening our hydrolase library against substrates
8-10, the benzyl-protected proline 8 was identified as the
best substrate, and in this case, pig liver esterase (PLE) was
found to be the most active and enantioselective enzyme
(Table 2). The rate of the reaction with PLE was dramatically
increased compared to that of CLEC-BL. Further experiments
indicated that the substrate concentration was readily in-
creased to 100 g/L, and in this case the reaction rate was
comparable to that of lower concentration of substrate (45%
conversion in 24 h). It was also found that the appropriate
pH of the reaction medium was crucial for the enzyme
activity (Figure 2). Higher pH values from 7.7 to 9.0 gave
a much faster rate than lower pH values from 7.0 to 7.5
(40-45% vs 10-20% in 24 h). It is difficult to explain this
unexpected phenomenon from a molecular level since the
crystal structure of PLE is not available. However, it is well-
known that in many cases the pH change will impact
significantly enzyme kinetics, so the optimization of pH will
be essential for improving an enzymatic process.6
The PLE process showed a much higher efficiency and
throughput than the CLEC-BL process. The acid production
was 6.3 mmol/h/L for the resolution of 8 vs 0.18 mmol/h/L
for the CLEC-BL resolution of 6. Under the optimal
conditions, the ratio of the enzyme to the substrate was
significantly reduced to 2.5:1 from 10:1. The cost of the PLE
process was thus dramatically reduced to only 5% of that of
CLEC-BL resolution of 6. Moreover, a lower concentration
Experimental Section
Enzyme Screening. A screening kit was thawed for 5
min, and 80 µL of potassium phosphate buffer (0.1 M, pH
7.0) was then dispensed into each well via a multichannel
pipet. A 10 µL amount of ACN solution of the substrate
(6) (a) Bornscheur, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis-
Regio- and StereoselectiVe Biotransformations; Wiley-VCH: Weinheim,
1999. (b) Faber, K. Biotransformation in Organic Chemistry, Springer:
Berlin, 2000. (c) Roberts, S. M. Biocatalysts for Fine Chemicals Synthesis;
John Wiley & Sons: Chichester, 1999. (d). Sousa, H. A.; Afonso, C. A.
M.; Crespo, J. G. J. Chem. Technol. Biotechnol. 2000, 75, 707.
(7) Lange, S.; Musidlowska, A.; Schmidt-Dannert, C.; Schmitt, J.; Bornscheuer,
U. T. ChemBioChem 2001, 2, 576.
652
•
Vol. 10, No. 3, 2006 / Organic Process Research & Development

(10-20 mg of substrate/mL) was then added to each well,
and the plate was incubated at 23 °C on a thermomixer (900
rpm). The reactions were quenched with 100 µL of aceto-
nitrile after 5 h. The 96-well plate was then centrifuged, and
the supernatant was transferred from each well into another
96-well plate and analyzed with automated HPLC using a
short C18 column (30 × 4.6 mm, 3 µm) and ACN/H2O
containing 0.1% TFA as the solvent system.
Method A: Preparation of (2S)-4,4-Difluoro-3,3-di-
methyl-N-Boc-proline (3) by Enzymatic Hydrolysis of 6.
To a 50-L reactor equipped with a pH electrode, an overhead
stirrer, a heating coil, and a base addition line2, was added
the CLEC-BL solution (7 L of fresh CLEC + 5 L of recycled
CLEC (80% of the initial activity) and 24 L of di-water).
The pH of the suspension was adjusted to 8.0 by addition of
20 mL of 2 N NaOH. Then, the racemic ester I solution
(400 g, 1.36 mol, 1.00 equiv, in acetonitrile, 3.6 L) was
added. The suspension was then stirred at 30 °C for 262 h.
The pH of the solution was maintained at 8.0 by adding 2 N
NaOH. Reaction was followed by RP-HPLC for both
conversion and % ee of the product, and stopped after 45-
50% starting material had been consumed. The % ee of the
acid was measured as 95.5% (after 262 h under these
conditions, 246 mL of base were added).
The mixture was extracted 3× with 16 L of MTBE each,
and the combined organic layers were dried with Na2SO4
and concentrated under vacuum to afford 220 g of crude
(R)-enriched ester 4. The remaining aqueous slurry was
filtered (to remove the CLEC-BL) through Whatman paper
1. CLEC paste was removed from the paper and stored at 4
°C.3 The remaining aqueous solution was acidified to pH
5.5 and extracted twice with 16 L of MTBE each (pH was
set up to 5.5 after first extraction). Two more extractions
were performed, one at pH 5.0 and subsequently one at pH
4.0 (It was determined by HPLC that three extractions were
sufficient to remove most of the acid). The acid fractions
were pooled and concentrated under vacuum. The solid
residue was then suspended in hot tap water (1000 mL) and
allowed to cool overnight. The slurry was filtered and the
crystals dried in a vacuum oven at 40 °C overnight. A white
solid was obtained (133 g, 98% ee, 69.8% yield based on
(S)-enantiomer, >98% HPLC pure).4 1H NMR (300 MHz,
CDCl3): δ 7.9 (bs, 1H), 4.10 (d, 1H), 3.89 (dd, 2H), 1.5 (s)
+1.45 (s) (9H), 1.3 (s, 3H), 1.15 (s, 3H). HPLC Methods:
(A) Nonchiral HPLC conditions: detector wavelength 200
nm; Luna C-18, 4.6 mm × 30 mm; flow rate 1.5 mL/min:
injection volume 10 µL; mobile phases: (a) 25 mM KH2-
PO4 pH 2.5, (b) acetonitrile; gradient 35-70% ACN in 5
min. (B) Chiral HPLC for acid 3: detector wavelength 195
nm; Chiralcel OJ-R, 3 µm, C-18, 4.6 mm × 150 mm; flow
rate 0.5 mL/min; injection volume 10 µL; mobile phases:
(a) 25 mM KH2PO4 pH 2.0, (b) acetonitrile, (c) HPLC grade
H2O. Isocratic 75% A and 25% B for 17 min, then 75% B
and 25% C for 3 min, and finally 75% A and 25% B for 15
min. Retention times: acid 3 14.85 (R) and 15.84 (S).
Method B: Enzymatic Resolution of Racemic 4,4-
Difluoro-3,3-dimethyl-N-benzyl-proline Methyl Ester (8).
To 7.85 L of phosphate buffer (pH 8.0, 100 mM) was added
0.4 L of PLE ammonium sulfate solution (Biocatalytics, Inc,
CA, USA; 2 × 106 units/L of solution), and the mixture was
stirred at 700 rpm. Then 1.0 kg of benzyl ester 8 was added.
The pH of the reaction was controlled at 8.0 with a titrator
by continuously adding 1 N NaOH. The reaction was
monitored by HPLC (achiral and chiral methods). After the
conversion reached ∼50% in 24 h, 15% of NaCl (1.6 kg,
w/w) was added, and the mixture was stirred for 5-10 min.
After the addition of toluene (0.5 v, 5.0 L) the mixture was
stirred for 15-30 min. After allowing the mixture to settle
for 30 min, the toluene layer was removed. The aggregated
enzyme formed was quickly filtered by vacuum filtration
and was washed with distilled water (0.5 L). The above ex-
traction procedure was repeated once to remove the residual
(R)-ester. The pH of the aqueous layer was adjusted to 3.5
by the addition of 36% HCl slowly. MTBE (0.5 V, 5.0 L)
was added, and the mixture was gently stirred for 30 min.
After settling for 30 min, the organic layer was collected,
and the aqueous layer was extracted twice with MTBE. The
combined organic layer was dried over Na2SO4, and after
the removal of the organic solvent the pure acid 8′ was
obtained in high yield and excellent optical purity (398 g,
1
44%, >99% ee). Compound 8′: ESI [M - H]- 268.1. H
NMR (300 MHz, CDCl3): δ 7.26-7.40 (m, 5H), 3.94 (d, J
) 12.9 Hz, 1H), 3.66 (d, J ) 12.9 Hz, 1H), 3.32-3.57 (m,
2H), 3.05 (m, 1H), 1.26 (s, 3H), 1.11 (s, 3H). 13C NMR (75
MHz, CDCl3) δ 171.10, 136.19, 129.16, 128.68, 127.73,
74.83, 60.67, 56.49, 53.75, 46.48, 20.40, 18.77. Chiral HPLC
methods: compound 8′, Chiralcel AD-RH (4.6 mm × 100
mm, 3 µm); flow rate 0.6 mL/min; injection volume 5 µL;
mobile phases ACN/H2O (20:80), detection at 254 nm.
Compound 8: Chiralcel OD-RH (4.6 mm × 100 mm, 3 µm);
flow rate 0.6 mL/min; injection volume 5 µL; mobile phases
ACN/H2O (60:40), detection at 254 nm.
Method C: Preparation of (S)-3,3-Dimethyl-N-Boc-
vinylglycine by Enzymatic Hydrolysis of 4. To a 5-L three
neck flask equipped with a pH electrode, an overhead stirrer,
a heating mantle, and a titrator was added the racemic ester
4 (78 g, 0.3 mol, 1.00 equiv) in acetonitrile (280 mL). A
mixture of Alcalase (350 mL from a 5× concentrated crude
solution from a commercial solution) and distilled water (2.8
L) was then prepared, and the pH of the solution was set to
7.0. The enzyme solution was charged into the reaction flask.
The suspension was then stirred at 30 ° C for 51 h. The pH
of the solution was maintained at 7.0 by adding 1 N NaOH.
Reaction was followed by RP-HPLC for both conversion
and % ee of the product and was stopped after 45% starting
material had been consumed (after 51 h under these condi-
tions, 95.8 mL of base was added). The mixture was
extracted 3× with 1.75 L of MTBE each, and the combined
organic layers were dried with MgSO4 and concentrated
under vacuum to afford 50.81 g of crude (R)-enriched ester
4 (>55% yield, approximately 56% ee). This crude mixture
contained some carboxylic acid <7%, which was recovered
later by acid-base extraction. The remaining aqueous
solution was passed through a Pellicon 2 tangential flow
filtration equipped with an Ultracel cellulose membrane.
During this step, most of the enzyme is removed from the
Vol. 10, No. 3, 2006 / Organic Process Research & Development
•
653

aqueous solution. The remaining solution was acidified to
pH 4.0 and extracted three times with 1.75 L of MTBE each.
The acid fractions were pooled, dried with sodium sulfate,
and concentrated under vacuum. Pale-yellow oil of 4′ was
obtained (31 g, 91.4% ee, 42% yield, >98% HPLC pure).
1H NMR (300 MHz, CDCl3): δ 10.69 (s, 1H), 5.78 (dd,
2H), 5.02 (m, 2H), 4.96 (s, 1H), 4.09 (d, 1H), 1.36 (s, 9H),
1.06 (s, 6H). Chiral HPLC method for acid 4′: detector
wavelength 200 nm; Chiralcel OJ-R, 3 µm, C-18, 4.6 mm
× 100 mm; flow rate 0.5 mL/min; injection volume 10 µL;
mobile phases (A) 25 mM NaH2PO4 pH 2.0; (B) acetonitrile;
isocratic 25% B for 55 min, 3 min postrun; retention times:
acid 16.33 (R) and 17.97 (S); ester 50.40 (S), 51.30 (R).
Chiral HPLC method for ester 4: Chiralcel OD-RH, 150 mm
× 4.6 mm; flow rate 0.8 mL/min; mobile phase 30% ACN
and 70% H2O; wavelength 205 nm.
Method D: Preparation of (S)-4′ by Chemical Resolu-
tion with (S)-Phenylglycinol. To a 2-L jacketed flask
equipped with an overhead stirrer was added the racemic
acid 4′ (93.3 g, 386.2 mmol), (S)-phenylglycinol (52.6 g,
386.2 mmol), methanol (200 mL) and acetonitrile (1800 mL).
The resulting slurry was stirred and heated to 70-80 °C or
until fully dissolved. The solution was allowed to crystallize
by allowing the solution to cool to room temperature slowly
(cooling rate: 10 °C/h) with continuous stirring. The solution
was then filtered, and the crystalline salt (containing the
desired (S)-enantiomer) was washed with 100 mL of cold
acetonitrile to wash any residual filtrate from the white
crystals. The crystals were then collected and analyzed by
HPLC. The acid was then isolated from the salt. The salt
was dissolved in 250 mL of ethyl acetate (or MTBE). Water
(250 mL) was then added, and the pH of the solution was
adjusted to 3 with 5% HCl. The organic layer containing
the acid was separated from the remaining aqueous solution,
which was extracted once again with 200 mL of ethyl acetate
(or MTBE) to recover any remaining acid. The extract was
then dried with sodium sulfate and concentrated under
vacuum. The acid was isolated as a clear oil, and following
vacuum-drying overnight, (S)-4′ was obtained as a white
solid (37.4 g, >98% ee, 40.1% isolated overall yield, >98%
pure).
Recycling the (S)-Phenylglycinol. The aqueous layer
from the above step was adjusted to pH 8.0 with 2.0 N
NaOH, and the solution was extracted with 300 mL of ethyl
acetate (or MTBE). The extract was then dried with sodium
sulfate and concentrated under vacuum. The product was
isolated as white solid crystals (12.3 g, >98% pure). Note
that recovery was only from 40% of material; the remaining
resolving agent can be recovered from the filtrate.
Chiral HPLC methods: detector wavelength 205 nm;
column: Chiralcel OJ-RH, 3 µ, C-18, 4.6 mm × 100 mm;
flow rate 0.6 mL/min; injection volume 10 µL; mobile phases
(A) acetonitrile (0.1% TFA): (B) 75% H2O (0.1% TFA);
isocratic 25% A: 75% B 18 min; retention times: acid:
11.69 (R) and 12.9 (S)/5.3 R-phenylglycinol. Note that in
cases where only 90% ee is obtained after the first crystal-
lization, a second crystallization can easily be carried to
improve the ee to >98% by adding a fresh batch of
acetonitrile and methanol (only 7 volumes).
Acknowledgment
We thank Xiaobing Xiong for chiral method development,
Van Martin and Kim Albizati for general discussions, and
KNC Lab for providing substrates.
Received for review January 4, 2006.
OP060004+
654
•
Vol. 10, No. 3, 2006 / Organic Process Research & Development