Chemo-enzymatic synthesis of chiral 2-substituted succinic acid derivatives

Article abstract of DOI:10.1016/S0957-4166(99)00330-4

Prochiral discrimination by the biocatalyst Alcalase, an enzyme preparation of subtilisin Carlsberg, was used to effect enantio- and regioselective monohydrolysis of a variety of (RS)-2-substituted succinate diesters to afford the corresponding half esters in modest to excellent enantiomeric excesses (>99%). Exploitation of malonate chemistry, as well as recycling of the unhydrolyzed isomer from the enantioselective hydrolysis, has resulted in a process which is both practical and economical.

Full text of DOI:10.1016/S0957-4166(99)00330-4

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3285–3295
Chemo-enzymatic synthesis of chiral 2-substituted succinic acid
derivatives
Murray D. Bailey,∗ Ted Halmos, Dan Adamson, Josée Bordeleau and Chantal Grand-Maître
Boehringer Ingelheim (Canada) Ltd., Bio-Méga Research Division, 2100 Cunard Street, Laval (Québec) H7S 2G5, Canada
Received 3 June 1999; accepted 22 July 1999
Abstract
Prochiral discrimination by the biocatalyst Alcalase®, an enzyme preparation of subtilisin Carlsberg, was used to
effect enantio- and regioselective monohydrolysis of a variety of (RS)-2-substituted succinate diesters to afford the
corresponding half esters in modest to excellent enantiomeric excesses (>99%). Exploitation of malonate chemistry,
as well as recycling of the unhydrolyzed isomer from the enantioselective hydrolysis, has resulted in a process
which is both practical and economical. © 1999 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction
The use of hydrolytic enzymes in organic synthesis, for the preparation of enantiomerically pure
carboxylic acids, has received increased attention over the last 10 years.1 This is, in part, due to the ability
of these enzymes to catalyze the hydrolysis of both natural and unnatural substrates. Many groups have
realized the potential of using enantiomeric hydrolysis of esters as a practical and cost-effective means
of producing important synthetic building blocks in enantiomerically pure form.2–4 Attractive features
of this approach include the readily available racemic materials and the adaptability of the enzymatic
hydrolysis step to multi-gram scales.
The use of substituted succinic acid derivatives as peptidomimetics has found a number of applications
in medicinal chemistry.5–9 We have successfully utilized these fragments for the introduction of a
succinamide unit in the development of BILA 2157 BS, a highly bioavailable and potent renin inhibitor10
shown in Fig. 1. In particular, we needed an efficient method to prepare the required 2(R)-(2-amino-4-
thiazolyl)methyl succinyl core of our lead compound. A number of approaches to chiral succinic acid
derivatives have been reported in the literature including catalytic asymmetric reduction,8 the use of chiral
auxiliaries,5,6,9,10 or the chemical resolution by recrystallization.7,11 While some of these approaches
work well, they suffer from disadvantages of costly auxiliaries, lengthy preparations of chiral ligands,
∗
Corresponding author. E-mail: mbailey@lav.boehringer-ingelheim.com
0957-4166/99/$ - see front matter © 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00330-4

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Figure 1. Renin inhibitor BILA 2157 BS
or the lack of generality. In addition, a number of groups have made use of hydrolytic enzymes such
as lipases12 and esterases13,14 to prepare enantiomerically enriched materials. We have explored this
latter approach to find a cost-effective alternative to the use of chiral auxiliaries in the preparation
of enantiomerically pure 2-substituted succinic acid derivatives by enantioselective and regioselective
hydrolysis of (RS)-2-substituted succinate diesters.15 An initial screen of various enzymes led us to select
a serine protease, subtilisin Carlsberg, as the enzyme of choice. Recently, the use of subtilisin Carlsberg
has been reported for the enantioselective hydrolysis of diethyl 2-isobutyl succinate.16 We wish to report
our findings that subtilisin Carlsberg efficiently resolves a variety of 2-substituted succinate diesters with
varied functionalities in high enantiomeric excesses. In particular, we have focused on the preparation
and enantioselective hydrolysis of 2-(RS)-(2-bromo-2-propenyl) succinate diester 4, which is a key and
versatile intermediate for the preparation of a number of interesting functional groups. For example,
this vinyl bromide side chain is readily transformed in a ‘one-pot’ process into the desired 2-amino-4-
thiazolylmethyl moiety17 required in our renin inhibitors.
2. Results and discussion
2.1. Preparation of (RS)-2-substituted succinate diesters
The required racemate (RS)-4 was readily prepared by a protocol involving sequential alkylation
of dimethyl malonate and demethoxycarbonylation as shown in Scheme 1. The first alkylation was
carried out with 2,3-dibromopropene utilizing an excess of dimethylmalonate 1 (1.5 equiv.) to decrease
dialkylation. The monoalkylated malonate 2 was readily isolated by distillation under reduced pressure
in 59% yield. The preparation of dialkylated product 3 was easily accomplished by, first, deprotonation
of 2 with sodium bis(trimethylsilyl)amide, followed by treatment with tert-butyl bromoacetate. For
convenience, sodium bis(trimethylsilyl)amide was used as the base to facilitate slow addition; however, a
number of bases worked equally well including potassium tert-butoxide. Purification by distillation under
reduced pressure afforded 3 in 92% yield, although the crude product was sufficiently clean to be used
directly in the next step without purification. Demethoxycarbonylation with lithium bromide in aqueous
DMF was carried out on triester 3, involving mono-saponification and decarboxylation in a ‘one-pot’
protocol. This gave directly racemate 4 in 45% yield after purification, along with a minor contaminant
arising from transesterification of the tert-butyl ester to the corresponding methyl ester (<6%). The
yields for this operation can be improved by a two-step process involving mono-saponification (aqueous
potassium hydroxide) and decarboxylation.18 This straightforward approach to (RS)-4 makes use of
relatively inexpensive reagents, no cryogenic conditions, and requires minimal purification. Racemate 4
was prepared in a practical fashion on a multi-gram scale (270 g). Further optimizations of this sequence
for adaptation to multi-kilogram quantities have been carried out and will be reported elsewhere.18

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3287
Scheme 1. (a) NaOMe in MeOH, 2,3-dibromopropene, 0°C to rt (1.5 h); (b) NaHMDS in THF, t-butyl bromoacetate, 0°C to rt
(15 min); (c) LiBr and H2O in DMF, 135°C (8 h)
2.2. Enantio- and regioselective hydrolysis of (RS)-2-substituted succinate diesters
A number of potential hydrolytic enzymes were screened for their suitability as catalysts for the
enantioselective hydrolysis of racemic vinyl bromide 4 (Scheme 2), including α-chymotrypsin, papain
and pig liver esterase. While no conversion was observed with papain, α-chymotrypsin produced a
low conversion (30%) and poor enantioselectivity (70% ee). The enzyme which produced the desired
enantioselective hydrolysis was found to be subtilisin Carlsberg, a serine protease. We also determined
that a crude and inexpensive preparation of this enzyme, Alcalase® 2.4L,19 performed equally well at a
fraction of the cost.
Scheme 2. aThe extent of enantioselective hydrolysis was determined by esterification of the half ester with diazomethane and
subsequent analysis by HPLC using a Chiralpak® AS column, 0.25% ethanol in hexane
Treatment of a suspension of (RS)-4 in acetone:water (1:7) with a catalytic amount of Alcalase® under
controlled NaOH conditions using a pH autotitrator resulted in rapid hydrolysis to give the desired
carboxylic acid (S)-5 in 47% yield and the unhydrolyzed ester (R)-4 in 53% yield as illustrated in
Scheme 2. The progress of the enzymatic reaction was readily monitored by following the amount
of NaOH solution consumed with time. The reaction initially progressed rapidly and then slowed
considerably as the concentration of the substrate was depleted. The hydrolysis was complete after
consumption of 0.5 equivalents of the NaOH (0.4 M) solution. Stabilization of the pH at this point
was usually diagnostic that enantioselective hydrolysis had occurred. Once completed, the components
of the reaction were separated using a simple acid–base extraction protocol to afford the diester (R)-4 and
the half ester (S)-5. An aliquot of half ester (S)-5 was derivatized to the methyl ester (S)-4 by treatment
with diazomethane and the enantiomeric purity determined to be >99% ee by HPLC analysis on a chiral
support (ChiralPak® AS, 4.6×250 mm, eluents: 0.25% ethanol in hexane).
The enzymatic reaction, in addition to being enantioselective, was completely regioselective since the
tert-butyl ester was unaffected during the hydrolysis. The absolute configuration of the resolved material
was confirmed by independent synthesis20 of the vinyl bromide (S)-4 using Evans’ alkylation method.21

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The authentic material was identical (optical rotation and HPLC analysis on a chiral support) to the
resolved succinate (S)-4, confirming the natural amino acid configuration at the α-center as expected. In
this approach, the unhydrolyzed ester antipode was also available in high optical purity if the reaction was
allowed to go to theoretical completion. Hence, (R)-4 was determined to be 97% ee by HPLC analysis
(chiral support).
The practicality and efficiency of the enzymatic resolution route was further improved by converting
the unhydrolyzed enantiomer (R)-4 back to the racemate through base catalyzed racemization (DBU in
refluxing toluene, 18 h) and could subsequently be retreated under the enzyme reaction conditions. In
order to demonstrate the improvement in overall efficiency of the resolution sequence, recycling of the
unhydrolyzed enantiomer (R)-4 in two additional cycles resulted in a combined yield of 82% of (S)-
4 in high purity (98% ee). The feasibility of doing this reaction on large scale was also investigated.
Utilizing the same protocol as outlined above (molarity of the NaOH solution increased to 1 M), the
enantioselective resolution was carried out on a preparative scale (100 g of (RS)-4) which gave the desired
(S)-4 after 48 h in 46% yield and 98% ee after one cycle. The use of an autotitrator for the controlled
addition of the sodium hydroxide solution in the hydrolysis step, particularly on large scale, is practical
and provided excellent reproducibility between different runs. On small scales, however, the autotitrator
was not critical in performing the reaction. For example, Alcalase® treatment of a suspension of the
versatile vinyl bromide intermediate (RS)-4 in phosphate buffer solution (pH 7.2) produced similarly
high enantiomeric excesses and yields.
2.3. Effect of ester size and temperature on the enzymatic hydrolysis
The rate of the enzymatic hydrolysis was found to be dependent on the size of the ester group to be
hydrolyzed. While all esters studied were hydrolyzed with good enantioselectivities to the half esters, the
optimal group under our reaction conditions was the methyl ester analog with relative conversion rates
of two to five times faster than for other esters (Me>Et>n-Pr>Bn) (Table 1). The effect of temperature
was also examined. As shown in Table 2, the selectivity was not affected between 23°C and 37°C. The
enantioselectivity of the hydrolysis, however, decreased at 55°C. It is not clear whether this observed
decrease is due to increased competition with the uncatalyzed process (direct NaOH hydrolysis) or a loss
in specificity of the enzyme.
2.4. Effect of the 2-substitutent on the enzymatic hydrolysis
The generality of the enzymatic hydrolysis was evaluated with a variety of 2-substituted succinate
diesters (Table 3). These analogs were prepared in analogous fashion to the vinyl bromide derivative
4, and were subjected to the identical enzymatic hydrolysis conditions. The hydrolyzed materials were
derivatized to the methyl ester (diazomethane treatment) and analyzed by HPLC using a chiral support
as before. As can be seen from Table 3, the nature of the C-2 side chain had a significant impact on the
degree of selectivity that was obtained. When the group was small (R=Me), a 4.6:1 ratio of enantiomers
was obtained. Changing the nature of the group as in entries 2 and 3 resulted in improved ratios of 9:1 and
15:1, respectively. Interestingly, this enzyme also tolerated fairly large heterocyclic groups with different
functionality (entries 4 and 5). Hence utilizing this approach, the 2(R)-(2-amino-4-thiazolyl)methyl
succinic acid moiety needed for BILA 2157 BS can be accessed by either enantioselective hydrolysis
of the heterocycles (RS)-12 and (RS)-13), or the (RS)-4 vinyl bromide precursor. Finally, introduction
of a benzyl group (entry 6) was also very well tolerated in the specificity pocket of the enzyme and

M. D. Bailey et al. / Tetrahedron: Asymmetry 10 (1999) 3285–3295
3289
Table 1
Effect of various ester groups on the enantioselective hydrolysis reaction
Table 2
Temperature effect on enzymatic hydrolysis of (R,S)-4
produced essentially optically pure material (129:1). Based on reaction scales of 1 mmol, these reactions
were complete between a few hours (entry 1) to 24 h for the most demanding substrate (entry 5).
3. Conclusions
Subtilisin Carlsberg is known to be somewhat of a permissive enzyme which tolerates a variety
of different functionalized substrates to bind in the specificity pocket (S1 binding pocket).22 Taking
advantage of this feature of the enzyme, a variety of (RS)-2-substituted succinate diesters can be
hydrolyzed with modest to excellent enantiospecificity, depending on the nature of the substituent.

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Table 3
Enantioselective hydrolysis reaction with various succinate diesters
Furthermore, an inexpensive source of this enzyme (Alcalase® 2.4L) was shown to be equally effective.
The enzymatic hydrolysis can be carried out on scales ranging from 0.5 g to 100 g without loss of
enantioselectivity or regioselectivity as demonstrated in the case of the vinyl bromide derivative. Further
improvements on this approach were realized by racemization of the ‘unhydrolyzed’ enantiomer and
recycling. The enzymatic hydrolysis was shown to be an efficient and practical method for the preparation
of a variety of different homochiral succinic acid derivatives. In particular, the vinyl bromide analog (RS)-
4 was effectively resolved in 82% yield after two additional enzymatic cycles. The rate of the enzymatic
reaction was shown to be influenced by the nature of the ester group to be hydrolyzed (Me>Et>n-Pr>Bn)
with no loss of selectivity between 23°C and 37°C. The simplicity and cost effectiveness of this strategy
for the preparation of large amounts of these fragments will be reported shortly.
4. Experimental
All reagents, solvents and starting materials were obtained from commercial sources and used without
further purification. Column chromatography was performed on silica gel, Merck grade 60 (230–400
mesh). Optical rotations were measured on a Perkin–Elmer 241 polarimeter. NMR spectra were obtained
at 400 MHz for 1H and 100 MHz for 13C, on a Bruker AMX 400 spectrometer. All chemical shifts are
reported in ppm in deuterochloroform with TMS as an internal standard. IR spectra were recorded on

M. D. Bailey et al. / Tetrahedron: Asymmetry 10 (1999) 3285–3295
3291
a Perkin–Elmer 781 spectrometer. FAB mass spectra were recorded on an Autospec, VG spectrometer
and CI mass spectra were obtained on a Kratos MS50 spectrometer. The pig liver esterase (EC 3.1.1.1),
α-chymotrypsin (EC 3.4.21.1) and papain (EC 3.4.22.2) were purchased from Sigma. Alcalase® 2.4L
(food grade) was obtained from Novo Nordisk Biochem., Inc. Enzymatic reactions were conducted using
a pH autotitrator. The enantiomeric purities were determined using an analytical HPLC chiral column,
Chiralpak® AS column (4.6×250 mm); mobile phase, 0.25% ethanol in hexane, 0.5 mL/min, isocratic
(condition A).
4.1. 2-(2-Bromo-2-propenyl)-1,3-propanedioic acid dimethyl ester 2
To a 5 L three-necked flask equipped with a mechanical stirrer was added anhydrous methanol (1.8 L)
followed by freshly cut metallic sodium (31 g, 1.35 g-atom) portionwise over 1.5 h. To the clear solution
was added dimethyl malonate (267 g, 2.0 mol, 1.5 equiv.) in anhydrous methanol (250 mL) over 1 h.
The solution was cooled to 0°C before 2,3-dibromopropene (300 g, ∼90% technical grade, 1.3 mol)
was added in anhydrous methanol (200 mL) over 3 h. The resulting yellow solution was stirred at room
temperature for 14 h until neutral to litmus paper. The methanol was removed under reduced pressure and
the residue was taken up in ether (800 mL) and washed with distilled water (800 mL). The aqueous phase
was re-extracted with ether (300 mL) and the combined organic phases washed with saturated brine, and
dried over anhydrous MgSO4. The solution was filtered and concentrated to give a yellow oil which
was purified by vacuum distillation. After the initial forerun, the mono-alkylated product 2 distilled at
1
101–106°C (1 mm Hg) as a clear colorless oil (201 g, 59%). Rf=0.54 (4:1 hexane:EtOAc); H NMR δ
5.69 (m, 1H, olefinic), 5.48 (d, J=2.0 Hz, 1H, olefinic), 3.81 (t, J=7.5 Hz, 1H), 3.75 (s, 6H), 3.02 (dd,
J=1.0, 7.5, 2H); 13C NMR δ 168.3, 129.2, 119.7, 52.6, 50.3, 40.4; IR (neat, cm−1) ν 3000 (w), 2955 (m),
2840 (w), 1740 (s), 1625 (m); MS (FAB) m/z: 253 (MH+2)+, 251 (MH)+. Anal. calcd for C8H11BrO4: C,
38.27; H, 4.42; found: C, 38.21; H, 4.44.
4.2. 2-(2-Bromo-2-propenyl)-2-(methoxycarbonyl)-1,4-butanedioic acid 4-(1,1-dimethyl-ethyl) 1-methyl
ester 3
The mono-alkylated product 2 (201 g, 0.8 mol) was dissolved in anhydrous THF (800 mL) and cooled
to 0°C. Sodium bis(trimethylsilyl)amide (881 mL, 1 M solution in THF) was cannulated into the reaction
mixture over 45 min. After a further 30 min, tert-butyl bromoacetate (172 g, 130 mL, 0.9 mol) was added
in anhydrous THF (200 mL) over 1 h. Stirring was continued for 3.5 h (room temperature) before the
reaction mixture was diluted with hexane (500 mL) and filtered through Celite. Concentration in vacuo
gave a brownish oil which was purified by vacuum distillation at 140–145°C (1 mm Hg) to give 271 g
1
(92%) of a colorless oil. Rf=0.60 (4:1 hexane:EtOAc); H NMR δ 5.64 (bs, 1H), 5.59 (bs, 1H), 3.75 (s,
6H), 3.35 (s, 2H), 3.07 (s, 2H), 1.43 (s, 9H); 13C NMR δ 169.4, 168.4, 127.2, 122.3, 81.3, 54.6, 52.9,
43.5, 37.2, 27.9; IR (neat, cm−1) ν 2980 (s), 2950 (s), 1760 (vs), 1640 (m); MS (FAB) m/z: 367 (M+Na)+,
365 (MH)+. Anal. calcd for C14H21BrO6: C, 46.04; H, 5.80; found: C, 45.65; H, 5.77.
4.3. 2-(RS)-(2-Bromo-2-propenyl)-1,4-butanedioic acid 4-(1,1-dimethylethyl) 1-methyl ester 4
The dialkylated malonate derivative 3 (52.6 g, 0.14 mol) was dissolved in DMF (20 mL) and added to
a solution of lithium bromide (12.51 g, 0.14 mol, 1 equiv.) in H2O (5.2 mL, 0.29 mol, 2 equiv.) and DMF
(20 mL). The clear solution was heated to 135°C for 8 h before being concentrated under high vacuum.
The residue was taken up in EtOAc (120 mL) and washed with water (1×60 mL). The extraction was

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M. D. Bailey et al. / Tetrahedron: Asymmetry 10 (1999) 3285–3295
repeated twice more with EtOAc (2×60 mL). The combined organic phase was washed with saturated
brine (100 mL) and dried (MgSO4), filtered and concentrated to give 27.6 g (63%) of a pale yellow oil. A
sample of this material (9.55 g) was further purified by flash chromatography eluting with hexane:ethyl
acetate (20:1) to provide 6.8 g of pure 2-(RS)-4 (45%); 1H NMR δ 5.64 (s, 1H), 5.48 (d, J=1.5 Hz, 1H),
3.71 (s, 3H), 3.14 (m, 1H), 2.85 (dd, J=14.5, 6.5 Hz, 1H), 2.61 (dd, J=14.5, 8 Hz, 1H), 2.57 (dd, J=16.5,
8 Hz, 1H), 2.48 (dd, J=16.5, 6.5 Hz, 1H), 1.44 (s, 9H); 13C NMR δ 173.90, 170.38, 130.46, 119.55,
80.91, 51.87, 42.75, 39.75, 35.82, 27.96; IR (neat, cm−1) ν 2980 (s), 1730 (s), 1630 (m); MS (EI) m/z:
309 (MH+2)+, 307 (MH)+; HRMS (FAB) calcd for C12H20BrO4 (MH)+, 307.0545. Found, 307.0538.
Anal. calcd for C12H19BrO4: C, 47.05; H, 6.26; found: C, 46.84; H, 6.36.
4.4. 2-(RS)-Methyl-1,4-butanedioic acid 4-(1,1-dimethylethyl) 1-methyl ester (RS)-9
Using the same protocol as above and methyl iodide as the alkylating reagent, (RS)-9 was obtained in
33% overall yield. 1H NMR δ 3.69 (s, 3H), 2.92–2.4 (m, 1H), 2.64 (dd, J=16.2, 8.26 Hz, 1H), 2.33 (dd,
J=16, 6.0 Hz, 1H), 1.44 (s, 9H), 1.20 (d, J=7.3 Hz, 3H); MS (EI) m/z: 203 (MH)+. Optical rotation for
acid of (R)-9: [α]25 +1.6 (c=2.5, MeOH), lit. value23 [α]25 +3.0 (c=0.64, MeOH).
D
D
4.5. 2-(RS)-[2-(Dimethylamino)-2-oxoethyl]-1,4-butanedioic acid 4-(1,1-dimethylethyl) 1-methyl ester
(RS)-10
Using the same protocol as above and 2-chloro-N,N-dimethylacetamide as the alkylating reagent, (RS)-
10 was obtained in 28% yield overall. 1H NMR δ 3.70 (s, 3H), 3.32–3.25 (m, 1H), 3.01 (s, 3H), 2.94 (s,
3H), 2.85–2.76 (m, 1H), 2.67–2.50 (m, 3H), 1.44 (s, 9H); MS (FAB) m/z: 274 (MH)+. Optical rotation
25
for the acid of (R)-10: [α]25 +6.14 (c=3.76, MeOH), [α]
+23.14 (c=3.76, MeOH).
D
365
4.6. 2-(RS)-[2-(Methylthio)ethyl]-1,4-butanedioic acid 4-(1,1-dimethylethyl) 1-methyl ester (RS)-11
Using the same protocol as above and 2-chloroethyl methyl sulfide as the alkylating reagent, (RS)-11
1
was obtained in 30% yield overall. H NMR δ 3.70 (s, 3H), 2.98–2.88 (m, 1H), 2.78–2.60 (m, 1H),
2.55–2.47 (m, 2H), 2.45–2.37 (m, 1H), 2.10 (s, 3H), 2.02–1.92 (m, 1H), 1.84–1.73 (m, 1H), 1.44 (s, 9H);
MS (FAB) m/z: es−: 247 (M−H)−. Optical rotation for the acid of (R)-11: [α]25 +13.11 (c=3.18, MeOH),
D
25
[α]
+46.2 (c=3.18, MeOH).
365
4.7. 2-(RS)-[(2-Amino-4-thiazolyl)methyl]-1,4-butanedioic acid 4-(1,1-dimethylethyl) 1-methyl ester
(RS)-1
The vinyl bromide (RS)-4 (5.00 g, 16.3 mmol) was dissolved in acetonitrile (20 mL) and H2O (7 mL)
before being treated with N-bromosuccinimide (NBS) (4.06 g, 23.0 mmol) in one portion. The reaction
mixture was stirred 35 min before the excess NBS was quenched with 2-methoxypropene (0.63 mL, 6.5
mmol) with the disappearance of color. Thiourea (1.50 g, 19.6 mmol) was added all at once and the
reaction stirred 1 h before the acetonitrile was removed in vacuo. The residue was partitioned between
saturated aqueous NaHCO3 and EtOAc. The organic phase was washed with saturated brine and then
dried (MgSO4), filtered and concentrated in vacuo. The product was purified by flash chromatography
1
(5% MeOH/CHCl3) to give 2.7 g (55%). H NMR δ 6.17 (s, 1H), 4.89 (bs, 2H), 3.68 (s, 3H), 3.2 (m,
1H), 2.95 (m, 1H), 2.55–2.8 (m, 2H), 2.45 (m, 1H), 1.43 (s, 9H); MS (FAB) m/z: 301 (MH)+; HRMS

M. D. Bailey et al. / Tetrahedron: Asymmetry 10 (1999) 3285–3295
3293
(FAB) calcd for C13H21N2O4S (MH)+, 301.1222. Found, 301.1232. Optical rotation for the methyl ester
(R)-12 (derivatized by diazomethane addition to the resolved acid): [α]25 +2.75 (c=0.8, MeOH).
D
4.8. 2-(RS)-[2-[[(1,1-Dimethylethoxy)carbonyl]amino]-4-thiazolylmethyl]-1,4-butanedioic acid 4-(1,1-
dimethylethyl) 1-methyl ester (RS)-13
To (RS)-12 (0.76 g, 2.5 mmol) in anhydrous THF (20 mL) was added NaH (70 mg, 2.8 mmol). This
mixture was stirred for 30 min before Boc anhydride (0.72 g, 3.3 mmol) was added. The reaction was
stirred 48 h before careful addition of wet THF. The mixture was partitioned between EtOAc (100 mL)
and H2O (50 mL). The organic phase was washed with saturated brine, dried (MgSO4), filtered and
concentrated in vacuo. Purification by chromatography gave the mono-Boc derivative (R,S)-13 (0.215 g,
1
21%). H NMR δ 8.85 (bs, 1H), 6.55 (s, 1H), 3.66 (s, 3H), 3.17 (m, 1H), 3.05 (m, 1H), 2.84 (m, 1H),
2.57 (m, 1H), 2.41 (m, 1H), 1.6 (s, 9H), 1.43 (s, 9H); MS (FAB) m/z: 401 (MH)+; HRMS (FAB) calcd for
C18H29N2O6S (MH)+, 401.1746. Found, 401.1757. Specific rotation for the acid of (R)-13: [α]25 +5.16
D
(c=0.31, MeOH).
4.9. 2-(RS)-Benzyl-1,4-butanedioic acid 4-(1,1-dimethylethyl) 1-methyl ester (RS)-14
Using the above procedure and benzyl bromide as the alkylating reagent, (RS)-14 was obtained in
1
46% yield overall. H NMR δ 7.32–7.25 (m, 2H), 7.24–7.19 (m, 1H), 7.18–7.14 (m, 2H), 3.66 (s, 3H),
3.11–2.99 (m, 2H), 2.74 (dd, J=13, 8 Hz, 1H), 2.58 (dd, J=16.5, 9 Hz, 1H), 2.33 (dd, J=16.5, 5 Hz, 1H),
1.41 (s, 9H); MS (FAB) m/z: 279 (MH)+. Specific rotation for the acid of (R)-14: [α]25 +8.3 (c=5.2,
D
CHCl3), lit. value24 [α]25 +6.4 (c=0.11, CHCl3).
D
4.10. Typical procedure for the kinetic resolution of succinate derivatives using subtilisin Carlsberg.
Kinetic resolution of 2-(RS)-(2-bromo-2-propenyl)-1,4-butanedioic acid 4-(1,1-dimethylethyl) 1-methyl
ester 4
Racemate 4 (5.01 g, 16.3 mmol) was dissolved in acetone (5 mL) and diluted with deionized H2O (30
mL). The system was equipped with an automatic pH titrator and a peristaltic pump connected to a 0.4
M aqueous NaOH solution. The pH of the rapidly stirred suspension was adjusted to 7.5 before a crude
preparation of subtilisin Carlsberg (0.2 g) (Alcalase® 2.4L, ‘food grade’ enzyme preparation) was added.
The automatic titrator setting was adjusted to between pH 7.2 and pH 7.6 and later readjusted after 12 h to
pH 7.5 and pH 7.8 until the pH stabilized. The acetone was removed under reduced pressure and saturated
aqueous NaHCO3 (10 mL) was added. The unreacted (R)-ester was extracted with EtOAc (2×80 mL),
washed serially with H2O and brine, dried (MgSO4) and concentrated under reduced pressure to give
a colorless oil (2.67 g). This material was analyzed by HPLC using a chiral support (condition A)
and was determined to be the (R)-4 isomer predominating in a ratio of 68:1 (97% ee); [α]25 −1.24
D
(c=1.37, CHCl3), [α]23655 −1.61 (c=1.37, CHCl3); MS (CI) m/z: 309 (M+2)+, 307 (M)+. Anal. calcd for
C12H19BrO4: C, 46.92; H, 6.23; found: C, 47.20, H, 6.21.
The basic aqueous phase from above was rendered acidic by addition of 10% HCl (ca. 20 mL) and then
extracted with (1:1) EtOAc:Et2O (2×80 mL). The combined extracts were serially washed with brine and
dried (MgSO4). Concentration under reduced pressure followed by drying the residue under high vacuum
gave (S)-5 as a colorless oil (2.25 g, 47% yield). [α]25 −4.58 (c=1.0, MeOH), [α]25 −1.08 (c=2.69,
D
D
CHCl3), [α]23565 −2.38 (c=2.69, CHCl3); Rf=0.28 (30% EtOAc:hexane); 1H NMR δ 10.98 (bs, 1H), 5.66
(s, 1H), 5.51 (d, J=1.5 Hz, 1H), 3.16 (m, 1H), 2.89 (dd, J=14.5, 6.0 Hz, 1H), 2.65 (dd, J=14.5, 8.5 Hz,

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M. D. Bailey et al. / Tetrahedron: Asymmetry 10 (1999) 3285–3295
1H), 2.57 (dd, J=17, 7.5 Hz, 1H), 2.52 (dd, J=17, 5.5 Hz, 1H), 1.44 (s, 9H); 13C NMR δ 179.7, 170.4,
130.2, 119.9, 81.3, 42.3, 39.7, 35.4, 28.0; IR (neat, cm−1) ν 3660–2400 (s), 1730–1700 (s), 1630 (m);
MS (CI) m/z: 295 (M+2)+, 293 (M)+. Anal. calcd for C11H17BrO4: C, 45.20; H, 5.87; found: C, 45.43, H,
5.87. An aliquot of this material was reacted with diazomethane to give the corresponding methyl ester
(S)-4 which was then analyzed by HPLC using a chiral column (condition A) which determined a ratio of
214:1 of (S)- to (R)-enantiomers (>99% ee). [α]25 +1.7 (c=0.98, CHCl3); MS (CI) m/z: 309.3 (M+2)+,
D
307.3 (M)+. Anal. calcd for C12H19BrO4: C, 47.05; H, 6.26; found: C, 47.17; H, 6.39.
4.11. Racemization of 2-(R)-(2-bromo-2-propenyl)-1,4-butanedioic acid 4-(1,1-dimethyl-ethyl) 1-methyl
ester (R)-4
To the unreacted ester (R)-4 (1.64 g, 5.3 mmol), which is a mixture of the (R)- and (S)-enantiomers in
a ratio of 68:1, was added toluene (15 mL) and DBU (0.85 mL, 5.5 mmol). This mixture was heated at
reflux for 18 h and then diluted with H2O and extracted with EtOAc. The organic phase was washed
serially with 5% aqueous HCl and saturated brine, dried (MgSO4) and concentrated under reduced
pressure to give material identical to the racemate 4 (1.57 g, 96%). This material was shown by HPLC
using a chiral support (condition A) to be a mixture of the (R)- and (S)-enantiomers in a ratio of 1:1.
4.12. Preparation of (RS)-acid 5
To racemate 4 (10.4 g, 33.9 mmol) in THF (30 mL) was added H2O (15 mL) and aqueous 2 M NaOH
(19 ml, 38 mmol) at room temperature. After 5 h, the aqueous phase was acidified with aqueous 1N HCl
to pH 2.5 and the mixture diluted with EtOAc (50 mL). The organic phase was dried (MgSO4), filtered
and concentrated to give the corresponding acid (R,S)-5 (8.81 g, 89%) as a white solid. 1H NMR δ 5.66
(bs, 1H), 5.51 (bs, 1H), 3.22–3.13 (m, 1H), 2.89 (dd, J=14.5, 6.0 Hz, 1H), 2.67–2.61 (dd, J=14.5, 9 Hz,
1H), 2.58–2.54 (m, 2H), 1.44 (s, 9H).
4.13. Preparation of ester derivatives: preparation of (R,S)-2-(2-bromo-2-propenyl)-1,4-butanedioic
acid 4-(1,1-dimethylethyl) 1-benzyl ester (RS)-8
Acid (RS)-5 (4.00 g, 13.6 mmol) was dissolved in anhydrous DMF (60 mL) under a nitrogen
atmosphere and treated with solid K2CO3 (4.15 g, 30.0 mmol). To this slurry was added benzyl bromide
(2.44 mL, 20.5 mmol) over 5 min. After stirring at rt (16 h), the DMF was removed under reduced
pressure and the residue partitioned between EtOAc (100 mL) and H2O (125 mL). The aqueous phase
was extracted with EtOAc (2×50 mL) and the combined extracts washed with saturated brine, dried
(MgSO4), filtered and concentrated. The crude oil was purified by flash chromatography to give the
desired benzyl ester (RS)-8 (4.55 g, 87%). 1H NMR δ 7.38–7.31 (m, 5H), 5.58 (bs, 1H), 5.44 (d, J=1.5
Hz, 1H), 5.16 (d, J=12.5 Hz, 1H), 5.12 (d, J=12.5 Hz, 1H), 3.23–3.15 (m, 1H), 2.86 (ddd, J=14.5, 6.5,
1 Hz, 1H), 2.66–2.56 (m, 2H), 2.50 (dd, J=16.5, 5 Hz, 1H), 1.41 (s, 9H); MS (FAB) m/z: 385 (MH+2)+,
383 (MH)+.
4.14. 2-(R,S)-(2-Bromo-2-propenyl)-1,4-butanedioic acid 4-(1,1-dimethylethyl) 1-ethyl ester (RS)-6
Using the above procedure and EtI as the alkylating reagent, (RS)-6 was obtained in quantitative yield.
1H NMR δ 5.63 (bs, 1H), 5.49 (d, J=1.5 Hz, 1H), 4.16 (dq, J=7.0, 1.5 Hz, 2H), 3.16–3.08 (m, 1H), 2.85
(ddd, J=14.5, 6.5, 1 Hz, 1H), 1.44 (s, 9H), 1.26 (t, J=7 Hz, 3H); MS (FAB) m/z: 323 (MH+2)+, 320.8
(MH)+.

M. D. Bailey et al. / Tetrahedron: Asymmetry 10 (1999) 3285–3295
3295
4.15. 2-(RS)-(2-Bromo-2-propenyl)-1,4-butanedioic acid 4-(1,1-dimethylethyl) 1-propyl ester (RS)-7
Using the same procedure and propyl iodide as the alkylating reagent, (RS)-7 was obtained in 83%
1
yield. H NMR δ 5.62 (bs, 1H), 5.47 (d, J=1.65 Hz, 1H), 4.05 (t, J=7.0 Hz, 2H), 3.21–3.05 (m, 1H),
2.90–2.77 (m, 1H), 2.67–2.48 (m, 3H), 1.75–1.55 (m, 1H), 1.42 (s, 9H), 0.93 (t, J=7.5 Hz, 3H); MS
(FAB) m/z: 337 (MH+2)+, 335 (MH)+.
Acknowledgements
We wish to thank Colette Boucher and Nancy Shore of our analytical department for developing the
HPLC method used in the enantiomeric excess determinations. We would also like to acknowledge Dr.
Pierre Beaulieu, Dr. Bruno Simoneau and Dr. Montse Llinas-Brunet in the preparation of this manuscript.
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