Use of hydrolases for the synthesis of cyclic amino acids

Article abstract of DOI:10.1016/j.tet.2003.10.116

The synthesis of several cyclic amino acids that have all the necessary structural features to make them ideal scaffolds for use in medicinal chemistry is described. A key step in each synthesis is the use of hydrolase enzymes to define a chiral centre. I

Full text of DOI:10.1016/j.tet.2003.10.116

Tetrahedron 60 (2004) 717–728
Use of hydrolases for the synthesis of cyclic amino acids
†
Richard C. Lloyd, Michael C. Lloyd, Mark E. B. Smith, Karen E. Holt, Jonathan P. Swift,
Philip A. Keene, Stephen J. C. Taylor and Raymond McCague
*
Dowpharma, Chirotech Technology Ltd, Cambridge Science Park, Milton Road, Cambridge CB4 0WG, UK
Received 19 August 2003; revised 26 September 2003; accepted 17 October 2003
Abstract—The synthesis of several cyclic amino acids that have all the necessary structural features to make them ideal scaffolds for use in
medicinal chemistry is described. A key step in each synthesis is the use of hydrolase enzymes to define a chiral centre. In order to elaborate
these building blocks into more complex molecules, crystallization and asymmetric hydrogenation were used to define further stereocentres.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
The reasons for the popularity of these enzymes are well
documented, as for example in ‘Hydrolases in Organic
Synthesis’ by Uwe Bornscheuer and Romas Kazlauskas.1
Some of these reasons include their relative commercial
availability, the reasonable number that are available for
screening, a lack of need for expensive or demanding
cofactors, versatility for synthetic or hydrolytic usage, and
the ability to perform reactions in aqueous or solvent based
media. From an industrial perspective they often admirably
meet the essential criteria for ‘industrial readiness’, such as
availability, performance (turnover, selectivity, volume
efficiency) and freedom from intellectual property issues.
However, the main area of use of hydrolase enzymes has
been in kinetic resolution chemistry, and a drawback of this
approach is the maximum 50% theoretical yield obtained of
the desired enantiomer. In recent years, approaches have
One can analyse the mainstream chemical literature to
determine the habits of practicing chemists in their
application of enzymes for (predominantly) chiral synthetic
methodology. In doing this, one is immediately drawn to the
conclusion that hydrolases dominate this field; they did in
1990 and continue to do so today. Scanning a few sample
years of Tetrahedron: Asymmetry for use of different
enzymes illustrates this point clearly, as shown in Figure 1
below. Since the mid-1990’s hydrolases have been used at
over twice the rate of non-hydrolases.
In particular, it is the lipase and esterase family of enzymes
that dominate the hydrolases, and a more detailed snapshot
of enzyme usage in 2002 is given in Figure 2.
Figure 1. Reported use of hydrolase enzymes versus non-hydrolase enzymes. (Source: papers published in Tetrahedron: Asymmetry in years specified).
Keywords: Biocatalysis; Amino acids; Stereochemistry.
*
†
Corresponding author. Tel.: þ44-1223-728010; fax: þ44-1223-506778; e-mail address: rlloyd@dow.com
Present address: Solexa Ltd, Chesterford Research Park, Little Chesterford, Essex CB10 1XL, UK.
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.116

718
R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
Figure 2. Breakdown of biocatalysis papers published in Tetrahedron: Asymmetry by enzyme class in 2002.
been made to overcome this, and hydrolases have now been
employed in dynamic kinetic resolutions2 in conjunction
with racemase enzymes,3 organometallic hydrogen transfer
catalysts4 or a more judicious selection of substrate.5
(2S,4S,5R)-1, is a cyclic amino acid with three stereocentres
isolated in various glycosylated forms from Pseudomonas
acidophila and Pseudomonas mesoacidophila.11 Its rigid
structure and multiple functionality make it an ideal
candidate for use as a scaffold around which compound
libraries may be designed: when used in conjunction with
b-lactam antibiotics the natural glycosylated forms
(bulgecins) enhance the antibiotic effect. Previous workers
identified the cis-allylic alcohol S-2 (Scheme 1) as a vital
intermediate for the synthesis of 1.12 However, the
functionality on the side-chain of S-2 makes it a challenging
synthetic target that has previously only been accessed using
chemistry that we did not think would be scaleable. We
targeted the racemic N-acetyl amino acid 3 as the key
intermediate. It was anticipated that 3 would be a suitable
substrate for resolution using acylases, which would provide
a mild environment to introduce the chirality whilst
protecting the functionality of the molecule. Following
resolution, the key intermediate S-2 would then be readily
available, and the route could be carried out on 100 g
scale.13
It is against this background that we have continued to
develop and exploit hydrolases generally for chiral synthetic
routes to more complex, conformationally rigid molecules,
where these molecules are expected to usefully contribute in
medicinal chemistry. Although Lipinski’s rules6 do not
mention rigidity as a desirable function in drug-like
molecules, recent studies have indicated that the number
of rotatable bonds is a crucial parameter that should be
considered.7 However, putting extra chiral complexity into
molecules means that one must call upon additional skills
and approaches, such as discovery and development of new
hydrolase enzymes, and the practice of complementing
biocatalysis with other chiral technologies such as crystal-
lisation and chemocatalysis. In this paper we describe some
of our work that has allowed us to elevate the use of
hydrolases for more sophisticated molecules.
The N-acetyl amino acid 3 was prepared from the readily
available 2-butyne-1,4-diol as shown in Scheme 2. Sequen-
tial use of L-acylase followed by D-acylase allowed both
enantiomers of the N-Boc amino acid 8 to be prepared in a
one pot procedure. Lindlar hydrogenation14 of 8 was used to
access both enantiomers of 2. Halolactonisation of S-2 gave
the lactone (2S,4S,5S)-9 with good diastereoselectivity,12
and the modified conditions of Oppolzer15 were then used to
convert lactone (2S,4S,5S)-9 to N-Boc-(2)-bulgecinine,
N-Boc-(2S,4S,5R)-1 (Scheme 3). Elaboration of R-2 in a
similar fashion gave the N-Boc-(2R,4R,5S) diastereomer. It
can be seen that the two additional chiral centres introduced
by cyclisation are both induced by the stereochemistry of
2. Resolution of a-amino acids using acylases and
subsequent elaboration to more complex molecules
2.1. Bulgecinine
Acylases are commonly used for the synthesis of enantio-
pure amino acids.8 We have successfully used proprietary
Thermococcus litoralis L-aminoacylase9 (extremophile) and
Alcaligenes sp. D-aminoacylase10 enzymes for the synthesis
of a wide range of unnatural a-amino acids. When
approaching the synthesis of cyclic amino acids, however,
this approach has not been so successful. (2)-Bulgecinine,
Scheme 1.

R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
719
Scheme 2. Reagents and conditions: (i) PhCOCl (1 equiv.), Py (1 equiv.), DCM, 10 8C to RT, o/n; (ii) Et3N (1.1 equiv.), DMAP (0.025 equiv.), MesCl
(1.1 equiv.), DCM, 0–5 8C, 2 h; (iii) LiBr (2 equiv.), acetone, 10–15 8C, 1.5 h; (iv) Diethyl acetamidomalonate (1.4 equiv.), KOtBu (1.4 equiv.), THF, reflux,
22 h; (v) NaOH (3 equiv.), MeOH, reflux, 3.5 h, then acidify to pH 3.5 with conc. HCl, reflux, 22 h; (vi) L-acylase (200 U/g substrate), 30 mM KH2PO4, pH 7,
65 8C, 2.5 h, then Boc2O (0.5 equiv. cf. racemic N-Ac acid), THF, 5 M NaOH to maintain pH at 10. RT, 3.5 h; (vii) D-Acylase (40 U/g substrate), aqueous
NaOH to adjust to pH 8, RT, 21 h, then Boc2O (0.5 equiv. cf. racemic N-Ac acid), THF, 5 M NaOH to maintain pH at 10, RT, o/n; (viii) 10 wt% Lindlar catalyst
(5 wt% Pd on CaCO3 poisoned with Pb), MeOH, 1 bar hydrogen, 20 8C, 1.5 h.
Scheme 3. Reagents and conditions: (i) NBS (1.1 equiv.), THF, 210–0 8C, 20 min; (ii) 40% EtOAc/60% heptane slurry, RT, 15 min–0.5 h; (iii) pTsOH·H2O
(2 equiv.), EtOAc, RT, 1 h; (iv) H2O, 1 M LiOH to pH 9, RT, 16 h; (v) Boc2O (1 equiv.), THF, 1 M LiOH to maintain pH 9, RT, 4.5 h.

720
R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
Figure 3. Source and use of 4-hydroxypipecolic acids.
the amino acid 2, which was, in turn, introduced to the
synthesis by the choice of acylase enzyme.
Previous work had identified the enantiomers of allyl-
glycine, an ideal acylase substrate, as starting materials for
the synthesis of the pipecolic acid core.21 The racemic
starting material, N-acetylallylglycine 11 (Fig. 3) was
prepared from diethylacetamidomalonate and allyl
bromide,22 and proved to be an excellent substrate for
both of our proprietary L- and D-acylase enzymes allowing
for efficient resolution of both enantiomers (Scheme 4). The
next key step in the synthesis, the acyliminium ion
cyclisation of the methyl ester of 14,23 gave the 1:1
mixtures of diastereomers at C-4, 16 (from S-14) and 17
(from R-14), which had previously only been separated by
chromatography, which we deemed unsatisfactory for a
scaleable synthesis.
2.2. 4-Hydroxypipecolic acids
A similar approach was applied to the synthesis of all four
diastereoisomers of 4-hydroxypipecolic acid 10.16 The
(2S,4RS)-diastereomers are naturally occurring non-
proteinogenic amino acids that have been isolated from
the leaves of Calliandra pittieri, Strophantus scandeus and
Acacia oswaldii.17 Much like isomers of bulgecinine, these
compounds possess all the necessary criteria for use as
scaffolds in medicinal chemistry programmes. Indeed,
molecules derived from both (2S,4S)-10 and (2S,4R)-10
have been demonstrated to possess biological activity: the
naturally occurring sulphate 1218 is a NMDA receptor
agonist19 and (2S,4R)-10 is a constituent of the synthetic
HIV protease inhibitor palinavir 13 (Fig. 3).20
Selective hydrolysis of one of the formate esters in the
mixtures 16 and 17 using lipases allowed the differentiation
of the diastereomers in such a way they could then be
Scheme 4. Reagents and conditions (i) t-BuOK, allyl bromide (ii) NaOH (iii) HCl (iv) L-acylase, pH 8.0, 65 8C (v) Cbz-Cl, pH 8.0 (vi) D-acylase, pH 8.0
(vii) SOCl2, MeOH (viii) paraformaldehyde, HCO2H.

R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
721
Scheme 5.
separated.16 Lipase AY30 (Candida rugosa, Amano)
selectively hydrolysed the formate ester of the (2S,4S)-
diastereomer of mixture 16 (Scheme 5) to yield the
4-hydroxypipecolic acid methyl ester (2S,4S)-18 (90%
d.e.) and the 4-formyloxypipecolic acid methyl ester
(2S,4R)-19 (81% d.e.). The components of this mixture
were separated by in situ derivatisation of (2S,4S)-18 to the
hemiphthalate ester derivative (2S,4S)-20, and partitioning
between toluene and saturated ammonium carbonate
solution.24 Concentration of the toluene layer gave
(2S,4R)-19. Thehemiphthalate ester(2S,4S)-20was recovered
by acidification of the aqueous layer and extraction into
toluene. A similar approach was used for the separation of
mixture 17, using Chirazyme L9 (Mucor miehei, Roche) in
the biotransformation, which preferentially hydrolysed the
formate ester of the (2R,4S)-diastereomer of the mixture.
Although the lipases were able to separate the dia-
stereomers, the products still lacked the required optical
purity, which was gained using crystallisation. Optically
pure cis-4-hydroxypipecolate (2S,4R)-21 was obtained via
(2S,4R)-18 (Scheme 6), a known crystalline intermediate.25
In the preparation of trans-4-hydroxypipecolic acid (2S,4S)-
10, no suitable crystalline intermediate could be found, and
thus was crystallised to diastereomeric purity as the N-Boc
benzylamine salt (2S,4S)-22. Identical routes were used to
prepare the enantiomeric compounds (2R,4S)-21 and
(2R,4R)-22 from (2R,4S)-19 and (2R,4R)-20, respectively.
The syntheses of all four compounds demonstrate the
usefulness of crystallisation as a dovetailing technology that
greatly augments the power of the previously used
chemistry. In this example, neither biocatalysis nor crystal-
lisation alone was sufficient to separate the pairs of
Scheme 6. Reagents and conditions (i) K2CO3, MeOH (ii) recrystallise (iii) H2, 10% Pd/C, Boc2O (iv) 2 M HCl, then Amberlite IRA-93 (v) Boc2O, Et3N
(vi) BnNH2, recrystallise.

722
R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
Scheme 7.
Scheme 8.
diastereomers; however, used in tandem they have provided
an effective solution to this problem.
amino acids. For example, a number of enantiopure cyclic
b-amino acids have been prepared using lipase-catalysed
kinetic resolutions of suitably protected b-lactams.27 When
condensed to form b-peptides, their properties differ greatly
from a-peptides, especially when the Ca–Cb bond is
constrained in a small ring.28 In addition to this, b-peptide
bonds appear far more stable than a-peptide bonds in a
physiological environment as they are more resistant to
protease enzymes.29 We prepared 3-aza-tricyclo[4.2.1.02,5]-
non-7-en-4-one 23 and 7-aza-bicyclo[4.2.0]oct-4-en-8-one
3. Use of lactamases for the preparation of b-and
g-amino acids
Many lactams possess biological activity, most notably the
b-lactam class of antibiotics.26 In addition to their own
medicinal potential, lactams are a very useful source of
Scheme 9.

R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
723
Scheme 10. Reagents and conditions: i, NBS, THF–H2O (10:1), RT, 18 h, 95%; ii, KOH, MeOH–H2O (1:1), 90 8C, 3 days; iii, Boc2O, H2O–THF (5:1), pH
10.5, 5 8C to RT, 18 h, 84% (overall for steps ii and iii); iv, MeOCOCl, Et3N, MeOH, 5 8C then RT, 18 h, 81%; v, H2 (35 psi), Pd/C, MeOH, RT, 18 h, (de 76%,
98%); vi, tert-butylamine, MTBE, recrystallise; vii, MeOCOCl, Et3N, MeOH, 5 8C then RT, 18 h, 86%; viii, H2 (75 psi), [(R,R)-Me-DuPHOS)-Rh(COD)]BF4,
MeOH, RT, (de .97%, 100%); ix, MeSO2Cl, Et3N, DMAP, CH2Cl2, 5 8C, 3 h, 97%; x, KOAc, DMF, 60 8C, 48 h, 85%; xi, NaOMe, MeOH, 5 8C, 7 h, 98%;
xii, KOAc, DMF, 60 8C, 8 days, 88%.
24 as racemates from chlorosulphonylisocyanate and either
2,5-norbornadiene or 1,3-cyclohexadiene, respectively.30
These were successfully resolved using a lactamase present
in the whole cells of Rhodococcus globerulus (NCIMB
41042). The resolutions were carried out in phosphate buffer
at pH 7 for 24 h, yielding (2)-23 of .98% e.e. and (1R,6S)-
24 of .95% e.e. (Scheme 7). This stereochemistry was
assigned by comparison of the optical rotation with that of
its enantiomer.27b
(Candida rugosa, Sigma) as a highly competent differ-
entiating agent that preferentially hydrolysed the ester of the
cis-isomer. Interestingly, during the synthesis of the
opposite enantiomer, (1R,4R)-29, we found that Lipase
Type VII was again the enzyme of choice for the separation
of the diastereomer mix 32, but this time preferentially
hydrolysing the trans-isomer. This demonstrates the very
high selectivity the enzyme displays for the C-1 chiral
centre: in each case it is the R-centre that is hydrolysed, in
spite of the steric constraints placed on the molecule by both
the change in relative stereochemisty and the steric bulk of
the other pendant functional group, the N-Boc.
We have previously reported the resolution of (^)-2-
azabicyclo[2.2.1]hept-5-en-3-one 27 using a cloned lacta-
mase31 in a process that has been used to produce tonne
quantities of (2)-27, an intermediate for the industrial
synthesis of carbocyclic nucleosides, for example abacavir,
28 (Scheme 8).
The products of the bioresolution of (^)-27 have also been
used as precursors of the cyclopentane series of scaffolds
33a–33h shown in Figure 433 Starting from the N-Boc
protected amino acid methyl ester (1S,4R)-30, treatment
with NBS yielded the oxazolidinone 34, which upon
treatment with base, eliminated and rearranged to give the
allylic alcohol 35 in excellent yield (Scheme 10). Palladium
on charcoal hydrogenation of 35 gave an all cis-compound
in 76% d.e. which could be crystallised to diastereomeric
purity as the tert-butylamine salt. Cracking of this salt
followed by esterification gave 33a. If, however, 35 was
esterified, it became an excellent substrate for hydrogen-
ation using the [(R,R)-Me-DuPHOS)-Rh(COD)]BF4
catalyst, which gave 33e directly (.97% d.e.). A mesyl-
ation-acetate inversion sequence on 33a and 33e provided
the diastereomers 33b and 33f, respectively.
Derivatives of the products of these resolutions of 27 can be
used in the synthesis of a range of compounds with
biological activity. An example of this is the synthesis of
both enantiomers of trans-4-aminocyclopent-2-ene-1-car-
boxylic acid, (1S,4S)-29 (as the methyl ester (1S,4S)-30) and
(1R,4R)-29. It was previously shown that the racemic
amino acid inhibited the uptake of GABA in rat brain
(Scheme 9).32
Treatment of the Boc-protected cis-amino acid methyl ester
(1R,4S)-30 with catalytic sodium methoxide in methanol at
0 8C led to an epimerisation at C-1 to give the 1:1 mixture of
diastereomers 31. Although separation of these compounds
was not possible by crystallisation, a screen of 31 against 5
commercially available lipases revealed Lipase Type VII
The other four diastereomers were obtained from the
enantiomeric starting material (1R,4S)-30. The only
Figure 4. The eight diastereomers of 3-tert-butoxycarbonylamino-4-hydroxy-cyclopentanecarboxylic acid methyl ester.

724
R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
difference in the sequence was that to obtain 33g, the [(S,S)-
Me-DuPHOS)-Rh(COD)]BF4 catalyst was used for the
hydrogenation. These scaffolds can be used for the synthesis
of molecules with potential biological activity, for example
in structures that are analogues of natural products.34,35
t, J¼2 Hz), 2.97 (1H, br s). NMR also showed presence of
,8 mol% dibenzoate ester. 13C NMR (CDCl3) 166.4, 134.2,
130.9, 130.2, 128.8, 85.4, 81.4, 53.1, 51.5. m/z 190 (Mþ),
173, 105, 77, 51.
5.1.2. 4-Benzoyloxybut-2-yne-1-ol, methanesulphonate
ester 5.37 4 (278 g, 1.46 mol) in CH2Cl2 (2.1 L) was cooled
to 0 8C under N2 Triethylamine (224 mL, 1.61 mol) and
DMAP (4.45 g, 36 mmol) were added, followed by
methanesulphonyl chloride (125 mL, 1.61 mol) in CH2Cl2
(100 mL) dropwise over 30 min period, keeping the reaction
temperature below 5 8C. The reaction was stirred at 0 8C for
2 h, when H2O (1.5 L) was added. After stirring for 5 min,
the layers were separated, and the organic layer washed with
sat. NaHCO3 (1.5 L) and 1 M HCl (1.5 L), dried (MgSO4)
and concentrated under reduced pressure to give 5, a brown
oil (350 g, 90%). 1H NMR (CDCl3) 8.04 (2H, m), 7.57 (1H,
m), 7.45 (2H, m), 4.96 (2H, m), 4.89 (2H, t, J¼2 Hz), 3.12
(3H, s). 13C NMR (CDCl3) 166.1, 134.1, 130.6, 130.1,
128.8, 84.5, 79.3, 57.9, 53.0, 39.5. m/z No Mþ, 173, 105, 77,
51.
4. Conclusion
In the examples discussed in this paper, hydrolase enzymes
have been used to resolve enantiomers and differentiate
diastereomers such that they can be easily separated and
provide a mild environment to enable manipulation of
highly functionalised molecules. The nature of such
chemistry allows for use on large scale. This broad spectrum
of use clearly demonstrates why they are so widely used in
synthetic chemistry today. However, to elaborate simple
compounds into the more complex, rigid compounds
described has emphasised the necessity for combining
chiral technologies. In most of the syntheses described,
although the biocatalytic step has been crucial for introduc-
ing at least one of the chiral centres in the target compound,
others have been introduced by a compatible technology.
5.1.3. 1-Bromo-4-benzoyloxybut-2-yne 6.37 A solution of
5 (345 g, 1.29 mol) in acetone (1.7 L) was cooled to 10 8C
under N2. LiBr (223 g, 2.57 mol) was added portionwise,
keeping the temperature below 15 8C, and the reaction
stirred at this temperature for 90 min. The mixture was
filtered through celite, and the filtrate concentrated under
reduced pressure. EtOAc (2 L) was added to the residue, and
this solution washed with H2O (2£1 L), dried (MgSO4) and
concentrated under reduced pressure to yield 6, a brown oil
(303 g, 90%). 1H NMR (CDCl3) 8.07 (2H, m), 7.57 (1H, m),
7.46 (2H, m), 4.99 (2H, m), 3.96 (2H, m). 13C NMR
(CDCl3) 166.2, 133.8, 130.2, 129.9, 128.7, 82.2, 81.1, 53.1.
m/z No Mþ, 173, 105, 77, 51.
5. Experimental
5.1. General
All reagents and solvents were from commercially available
sources and used as received. Proton NMR spectra were
obtained on a Bruker Avance 400 spectrometer operating at
400 MHz for proton (1H) and 100 MHz for carbon (13C).
Chemical shifts are reported in ppm using Me4Si or residual
nondeuterated solvent as reference. Coupling constants (J)
are measured in Hz. GC-MS data were obtained using a HP
5890 Series 2 GC fitted with a J and W Scientific DB5
column, attached to a HP 5972 series Mass Selective
Detector using electron impact ionisation. GC d.e. and e.e.
data of final compounds were obtained using a Perkin Elmer
Autosystem Gas Chromatograph fitted with a Varian
Chirasil-Dex CB column (25 m£0.25 mm). HPLC data
were obtained using a Gilson HPLC system fitted with a
Luna Phenylhexyl column. HPLC e.e. data were recorded
using a Phenomenex D-penicillamine (150£4.6 mm)
column. Optical rotation data were obtained using a Perkin
Elmer Polarimeter 341 instrument.
5.1.4. 1,1-Dicarboethoxy-1-acetamido-5-benzoyloxy-
pent-3-yne 7. A suspension of diethylacetamidomalonate
(258 g, 1.19 mol) and KOtBu (146 g, 1.30 mol) in THF
(3 L) was heated to reflux for 1 h. To this refluxing mixture,
a stream of 6 (300 g, 1.19 mol) in THF (500 mL) was added
over a 10 min period. After 2.5 h, the reaction had not gone
to completion, so a further 100 g (0.47 mol) diethyl-
acetamidomalonate and KOtBu (58 g, 0.47 mol) were
added and the mixture refluxed overnight. The reaction
mixture was cooled and filtered through celite, and the
filtrate concentrated under reduced pressure to a volume of
1 L. EtOAc (2 L) was added and the solution washed with
0.1 M HCl (1 L) and H2O (1 L). The organic phase was
concentrated under reduced pressure to yield 7, a brown oil
(365 g, 79%). 1H NMR (CDCl3) 8.05 (2H, m), 7.58 (1H, m),
7.46 (2H, m), 6.96 (1H, br s), 4.85 (2H, t, J¼2 Hz), 4.25
(4H, m), 3.33 (2H, t, J¼2 Hz), 2.04 (3H, s), 1.24 (6H, m).
13C NMR (d6-DMSO) 170.0, 166.6, 165.3, 134.0, 129.6,
129.5, 129.2, 81.8, 77.8, 65.6, 62.4, 53.0, 22.4, 14.1. m/z 389
(Mþ), 345, 316, 274, 194, 174, 152, 124, 105, 77, 51.
5.1.1. 4-Benzoyloxybut-2-yne-1-ol 4.36 2-Butyne-1,4-diol
(500 g, 5.81 mol) was dissolved in CH2Cl2 (1 L) and
pyridine (470 mL, 5.81 mol) and cooled to 5 8C under N2.
Benzoyl chloride (608 mL, 5.25 mol) in CH2Cl2 (1 L) was
added over a 3 h period, keeping the reaction temperature
below 10 8C. The resultant yellow suspension was allowed
to warm to room temperature and stirred overnight. The
reaction mixture was washed with 1 M H2SO4 (3£600 mL)
and H2O (2£600 mL) and organic phase concentrated to
900 mL volume. EtOH (750 mL) was added, and the
solution placed in a freezer overnight. Any precipitate was
removed by filtration and washed with EtOH (100 mL). The
filtrate was concentrated under reduced pressure to give 4,
an orange oil (519 g, 52%). 1H NMR (CDCl3) 8.06 (2H, m),
7.57 (1H, m), 7.44 (2H, m), 4.96 (2H, t, J¼2 Hz), 4.34 (2H,
5.1.5. N-Acetyl-(4-hydroxybut-2-ynyl)glycine 3. To a
stirred solution of 7 (300 g, 0.77 mol) in EtOH (1.5 L) and
H2O (1 L) NaOH (92.5 g, 2.31 mol) was added in H2O
(500 mL) as a thin stream. The mixture was heated to reflux
for 4 h, cooled and the EtOH removed under reduced
pressure. The resultant aqueous solution was washed with

R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
725
EtOAc (1 L), and then carefully acidified to pH 3.5 using
conc. HCl, and heated to reflux for 24 h. After cooling, the
solution was acidified to pH 2.5 with conc. HCl and
extracted with EtOAc (1.5 L). The pH of the aqueous phase
was then adjusted to 6.0 with 46–48% NaOH solution and
concentrated under reduced pressure. The residue was
slurried in MeOH (500 mL) and filtered through celite, after
which decolourising charcoal (25 g) was added to the filtrate
and this mixture heated to 50 8C for 20 min. After filtration
through celite, the filtrate was concentrated under reduced
pressure to give 3, an orange oil (149 g, 100% crude yield).
1H NMR (D2O) 4.27 (1H, t, J¼6 Hz), 4.15 (2H, t, J¼2 Hz),
2.67 (2H, m), 2.01 (3H, s). 13C NMR (d6-DMSO) 172.5,
168.6, 82.7, 80.9, 53.3, 49.6, 23.3, 22.8.
The cells were then spun off by centrifuge (10 min,
3400 rpm) and the pellet was washed with H2O (20 mL)
and respun. The combined supernatants were extracted with
EtOAc (3£50 mL) and the organic extracts dried over
MgSO4. Removal of solvent under reduced pressure yielded
1
(2)-23 (770 mg, 38%, e.e. (GC) .98%), a white solid. H
NMR (CDCl3) 6.19–6.30 (1H, m), 6.00–6.18 (2H, m),
3.44–3.53 (1H, m), 2.98–3.10 (1H, m), 2.91–2.96 (1H, br
s), 2.82–2.91(1H, br s), 1.81 (1H, d, J¼10 Hz), 1.65 (1H, d,
J¼10 Hz). 13C NMR (CDCl3): 171.0, 138.6, 136.3, 58.6,
53.5, 44.0, 41.1, 39.1. m/z No Mþ, 107, 91, 70, 66. [a]D20 (c
0.01, MeOH) 291.
5.1.9. Resolution of racemic 7-aza-bicyclo [4.2.0]oct-4-
en-8-one 24. 24 (1.49 g, 12 mmol) and a whole cell
preparation of Rhodococcus globerulus NCIMB 41042
(1.4 g, 1 wt equiv.) were suspended in 50 mM KH2PO4, pH
7 (40 mL). The resulting mixture was stirred at 30 8C for
22 h. The cells were then spun off by centrifuge (10 min,
3400 rpm) and the pellet was washed with H2O and respun.
The combined supernatants were extracted with EtOAc
(3£50 mL) and the organic extracts dried over MgSO4.
Removal of solvent under reduced pressure yielded (1R,6S)-
24, (460 mg, 31%, e.e. (GC) .95%), an off-white solid. 1H
NMR (CDCl3): 6.19–6.10 (1H, m), 6.07 (1H, br s), 5.98–
5.90 (1H, m), 4.03 (1H, t, J¼5 Hz), 3.55–3.48 (1H, m),
2.16–2.02 (3H, m), 1.68–1.55 (1H, m). 13C NMR (CDCl3)
172.3, 135.0, 126.3, 50.2, 44.7, 22.2, 22.1. m/z no Mþ, 103,
80, 79. [a]2D5 (c 0.13, CHCl3) 2154.
5.1.6. Resolution of (6)-3. 3 (125 g, 0.67 mol) in 30 mM
KH2PO4 (1.7 L) at pH 7 was treated with L-acylase solution
(200 U/g substrate) and the solution stirred at 65 8C and pH
1
7 until H NMR of an evaporated sample showed .40%
conversion (44 h). Boc2O (47 g, 0.27 mol) in THF (200 mL)
was then added at RT, and the reaction maintained at pH 10
until the pH was static. The mixture was extracted with
MTBE (500 mL), then the remaining aqueous acidified to
pH 3 with KHSO4 and extracted with EtOAc (3£1 L). The
EtOAc portions were combined, dried (MgSO4), filtered and
evaporated in vacuo to give (S)-N-Boc-(4-hydroxybut-2-
ynyl)glycine S-8, a pale yellow oil (44 g, 36%, 98% e.e.). 1H
NMR (d6-DMSO) 12.6 (1H, br s), 7.05 (d, 1H, J¼8 Hz),
5.07 (1H, br), 4.03 (2H, m), 2.55 (2H, m), 1.37 (9H, s). 13C
NMR (d6-DMSO) 172.6, 155.6, 80.7, 78.6, 53.1, 54.0, 49.4,
28.5, 21.8. The remaining aqueous solution from above was
then adjusted to pH 8 with 46/48% NaOH, and D-acylase
solution added (40 U/g substrate). The reaction mixture was
stirred at RT and pH maintained at pH 8 until the reaction
was complete by 1H NMR (21 h). The pH was then adjusted
to 10 and the mixture treated with Boc2O (54 g, 0.31 mol)
and worked up as above to give (R)-N-Boc-(4-hydroxybut-
2-ynyl)glycine R-8, an orange oil (48 g, 34%, 95% e.e.). 1H
NMR (d6-DMSO): 12.6 (1H, br s), 7.05 (d, 1H, J¼8 Hz),
5.07 (1H, br), 4.03 (2H, m), 2.55 (2H, m), 1.37 (9H, s). 13C
NMR (d6-DMSO) 172.6, 155.6, 80.7, 78.6, 53.1, 54.0, 49.4,
28.5, 21.8.
5.1.10. Epimerisation (1R,4S)-N-Boc-4-aminocyclopent-
2-ene-1-carboxylic acid, methyl ester, (1R,4S)-30. A
solution of (1R,4S)-30 (1.82 g, 7.55 mmol) in MeOH
(30 mL) was cooled to 0 8C and NaOMe (25 wt% in
MeOH, 2 mL, pre-cooled to 0 8C) was added. The resulting
mixture was stirred for 120 min, maintaining the tempera-
ture at 0 8C throughout. After this time, it was quenched
using glacial acetic acid (4 mL). The solvent was removed
under reduced pressure to yield a yellow oil (1.6 g) which
solidified on standing. GC–MS analysis indicates that the
solid consists of a 1:1 mixture of (1R,4S) (cis) and (1S,4S)
(trans) epimers (mixture 31) in addition to a small amount
(,10%) of the conjugated adduct. GC–MS: Retention time
16.59 min (cis-epimer, m/z: 185, 168, 141, 126, 82),
17.16 min (trans-epimer m/z: 185, 168, 141, 126, 82),
17.91 min (conjugated adduct).
5.1.7. S-N-Boc-(Z-4-hydroxybut-2-enyl)glycine S-2.38
Lindlar’s catalyst (3.0 g) was added to a degassed solution
of S-8 (30 g) in MeOH (600 mL), and the resultant mixture
sealed in a 2 L Parr pressure vessel. After purging with N2
and H2, the vessel was charged to 1 bar with H2 and the
reaction mixture stirred at 20 8C until the H2 pressure above
remained constant. The remaining H2 was released, and the
vessel purged with N2. The reaction mixture was filtered
through celite and concentrated under reduced pressure to
give S-2 (30 g, 100%). This material can be purified by flash
chromatography (eluent 5:4:1 EtOAc/heptane/AcOH) if
required. 1H NMR (d6-DMSO): 7.02 (1H, d, J¼8 Hz), 5.55
(1H, m), 5.34 (1H, m), 3.94 (2H, d, J¼6), 3.86 (1H, m), 2.35
(2H, m), 1.36 (9H, s).
5.2. Screening for selective hydrolysis of mixtures 31 and
32
50 mg of substrate (either 31 or 32) was placed into a
scintillation vial with 50 mM KH2PO4, pH 7 (5 mL) and
enzyme (20 mg) and the vial agitated at 26 8C in a water
bath/shaker After 18 h the reaction was extracted with
EtOAc (5 mL). The organic extracts were dried over
MgSO4 and analysed by GC–MS. Enzymes used: Porcine
pancreatic lipase (Sigma), Lipase Type VII (Sigma), Lipase
PS (Amano), Chirazyme L9 (Roche) and Chirazyme L2
(Roche). Analysis revealed that Lipase Type VII gave the
greatest selectivity for both mixtures, hydrolysing the cis-
epimer of 31 and the trans-epimer of 32.
5.1.8. Resolution of 3-aza-tricyclo [4.2.1.02,5] non-7-en-4-
one (6)-23. (^)-23 (2 g, 15 mmol) and a whole cell
preparation of Rhodococcus globerulus NCIMB 41042 (2 g,
1 wt equiv.) were suspended in 50 mM KH2PO4, pH 7
(40 mL). The resulting mixture was stirred at 25 8C for 24 h.
5.2.1. Scale up of the separation of mixture 31. 31 (0.6 g,

726
R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
2.5 mmol) was suspended in 50 mM phosphate buffer, pH 7
(60 mL) and Lipase type VII (250 mg) added The mixture
was left to shake in a water bath/shaker at 26 8C for 24 h and
extracted with EtOAc (3£50 mL). The combined organic
extracts were washed with saturated NaHCO3 (50 mL) and
dried over MgSO4. The solvent was removed under reduced
pressure to yield (1S,4S)-30, an off-white solid (260 mg,
43%). GC-MS confirmed the structure of the recovered
material. To recover the cis-epimer, the aqueous phase was
acidified with 1.2 M HCl and extracted with EtOAc
(3£50 mL). The combined extracts were dried over
MgSO4 and concentrated to yield a colourless oil
(180 mg, 32%) that solidified on standing. 1H NMR
(CD3OD) major (cis) diastereomer39 5.81 (1H, m), 5.71
(1H, m), 4.53 (1H, br s), 3.40 (1H, m), 2.42 (1H, dt, J¼14,
8 Hz), 1.70 (1H, m), 1.34 (9H, s). 13C NMR (CD3OD)
177.9, 158.0, 135.5, 132.9, 80.6, 57.7, 50.8, 35.8, 29.2;
minor (trans) diastereomer 5.81 (1H, m), 5.71 (1H, m), 4.63
(1H, br m), 3.56 (1H, m), 2.42 (1H, m), 1.70 (1H, m), 1.34
(9H, s). 13C NMR (CD3OD) 177.5, 157.9, 135.1, 132.9,
80.1, 57.6, 50.8, 35.7, 28.8. Comparison of relative
integrations of dispersed resonances show ratio of cis:-
trans-diastereomers to be 4:1.
tion from EtOAc/heptane yielded 34 as white crystals
(110 g, 50%). A further crop of crystals was obtained from
the liquors though it was contaminated with a small amount
of succinimide (49 g, 23%). 1H NMR (CDCl3) 5.79 (1H, br
s,), 5.15 (1H, dd, J¼7, 2 Hz), 4.79 (1H, m), 4.43 (1H, app dt,
J¼7, 2 Hz), 3.75 (3H, s), 3.23 (1H, app quintet, J¼7 Hz),
2.52 (1H, dt, J¼14, 7 Hz), 2.42 (1H, dm, J¼14 Hz). 13C
NMR (CDCl3) 171.2, 158.1, 87.8, 56.1, 53.3, 52.5, 51.5,
35.2. m/z 265, 263 (1:1, Mþ), 184, 156, 140, 124, 115, 80.
5.2.4. (3S,4R)-4-tert-Butoxycarbonylamino-3-hydroxy-
cyclopent-1-enecarboxylic acid 35. KOH (43 g,
0.76 mol) in H2O (150 mL) was added to a solution of 34
(36 g, 0.19 mol) in MeOH (150 mL). The mixture was
heated under reflux at 90 8C for 2 days and allowed to cool.
MeOH was removed under vacuum, the mixture diluted
with H2O (100 mL) and the pH of the reaction was adjusted
to pH 10.5 on addition of 1 M HCl (aq.). The solution was
cooled to 10 8C and a solution of Boc2O (42 g, 0.19 mmol)
in THF (60 mL) was added dropwise and the mixture
allowed to warm to room temperature. The reaction was
then stirred for 14 h, the layer of THF removed and the
aqueous solution adjusted to pH 3 with 6 M HCl (aq). The
acidic solution was extracted with EtOAc (3£200 mL) and
the combined organic washings dried over MgSO4.
Filtration then concentration under vacuum gave 35 as a
white solid (40 g, 86%). 1H NMR (d6-DMSO) 6.58 (1H, m),
6.40 (1H, d, J¼7 Hz), 4.57 (1H, m), 4.05 (1H, m), 3.41 (1H,
br s), 2.65 (1H, app dd, J¼16, 7 Hz), 2.41 (1H, ddt, J¼16, 6,
1 Hz), 1.46 (9H, s). 13C NMR (CDCl3) 168.5, 158.5, 142.8,
139.8, 80.7, 75.3, 54.2, 37.3, 29.1.
5.2.2. Scale up of the separation of mixture 32. 32 (1.0 g,
4.15 mmol) was suspended in 50 mM phosphate buffer, pH
7 (100 mL) and Lipase type VII (400 mg) added. The
mixture was left to shake in a water bath/shaker at 26 8C for
24 h and extracted with EtOAc (3£50 mL). The combined
organic extracts were washed with saturated NaHCO3
(50 mL) and dried over MgSO4. The solvent was removed
under reduced pressure to yield (1S,4R)-30, an off-white
solid (220 mg, 22%). GC-MS confirmed the structure of the
recovered material. To recover the trans-epimer, the
aqueous phase was acidified with 1.2 M HCl and extracted
with EtOAc (3£50 mL). The combined extracts were dried
over MgSO4 and concentrated to yield a white solid
(270 mg, 29%) that solidified on standing. 1H NMR
(CD3OD) major (trans) diastereomer 5.81 (1H, m), 5.71
(1H, m), 4.63 (1H, br m), 3.56 (1H, m), 2.42 (1H, m), 1.70
(1H, m), 1.34 (9H, s). 13C NMR (CD3OD) 177.5, 157.9,
135.1, 132.9, 80.1, 57.6, 50.8, 35.7, 28.8; minor (cis)
diastereomer 5.81 (1H, m), 5.71 (1H, m), 4.53 (1H, br s),
3.40 (1H, m), 2.42 (1H, dt, J¼14, 8 Hz), 1.70 (1H, m), 1.34
(9H, s). 13C NMR (CD3OD) 177.9, 158.0, 135.5, 132.9,
80.6, 57.7, 50.8, 35.8, 29.2. Comparison of relative
integrations of dispersed resonances show ratio of trans:-
cis-diastereomers to be 3:1.
5.2.5. (3S,4R)-4-tert-Butoxycarbonylamino-3-hydroxy-
cyclopent-1-enecarboxylic acid methyl ester 36. Methyl
chloroformate (29 mL, 0.37 mol) was added dropwise to a
cooled solution (0 8C) of 35 (83 g, 0.34 mol) and triethyl-
amine (52 mL, 0.37 mol) in MeOH (600 mL). The reaction
was stirred for 1 h at 0 8C and then allowed to warm to room
temperature. After stirring for 14 h it was apparent that the
reaction was incomplete. After cooling to 0 8C, triethyl-
amine (30 mL, 0.22 mol) was added then methyl chloro-
formate (15 mL, 0.19 mol) added dropwise. The reaction
was complete after a further 2 h. The reaction mixture was
concentrated under vacuum and the residue redissolved in
CH2Cl2 (400 mL). The organic phase was washed sequen-
tially with 1 M KHSO4 (aq.) (2£200 mL), sat. NaHCO3
(2£200 mL) and brine (200 mL) before drying over MgSO4.
Following filtration, the solvent was removed to yield an
orange oil which was redissolved in MeOH (300 mL) and
cooled to 5 8C. NaOMe (0.25 mL, 25 wt% solution in
MeOH) was then added and the reaction mixture cooled and
stirred for 4 h. The reaction was quenched with glacial
AcOH, concentrated under vacuum and the residue redis-
solved in CH2Cl2 (300 mL). The organic phase was washed
with sat. NaHCO3 (100 mL) then brine (100 mL) and dried
over MgSO4. Following filtration, the solvent was removed
5.2.3. (3aR,5R,6R,6aR)-6-Bromo-2-oxo-hexahydro-
cyclopentaoxazole-5-carboxylic acid methyl ester 34.
N-Bromosuccinimide (164 g, 0.92 mol) was added in
portions to a solution of (1S,4R)-30 (200 g, 0.83 mol) in
THF (670 mL) and water (67 mL). The N-Bromosuccini-
mide was seen to completely dissolve over a 3 h period
during which a mild exotherm was evident. The reaction
was stirred for a further 14 h after which time it was
concentrated to dryness under vacuum. The residue was
redissolved in dichloromethane (1.1 L) and washed sequen-
tially with 1 M HCl (aq.) (500 mL), saturated Na2SO3 (aq.)
(500 mL) and brine (500 mL) before drying over MgSO4.
Following filtration, concentration of the organic under
vacuum yielded 238 g of a light brown solid. Recrystallisa-
1
under vacuum to yield 36 as an orange oil (76 g, 87%). H
NMR (CDCl3) 6.71 (1H, m), 5.26 (1H, d, J¼7 Hz), 4.77
(1H, br s), 4.25 (1H, br s), 3.77 (3H, s), 3.04 (1H, br s), 2.92
(1H, dd, J¼17, 7 Hz), 2.50 (1H, m), 1.46 (9H, s). 13C NMR
(CDCl3) 165.1, 156.1, 141.0, 137.6, 79.5, 74.7, 52.5, 51.8,
36.5, 28.3. m/z No Mþ, 201, 183, 151, 137, 125, 106, 96, 78,
57.

R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
727
5.2.6. (1S,3R,4S)-3-tert-Butoxycarbonylamino-4-
hydroxy-cyclopentanecarboxylic acid methyl ester 33a.
10% Pd/C (1 g) was added under N2 to a solution of 35
(150 g, 0.29 mol) in MeOH (300 mL) The reaction mixture
was transferred to a bomb and after purging with N2 and
then H2, a H2 pressure of 2 bar was applied and the reaction
stirred for 18 h. (Periodically, additional hydrogen was
added to the reaction to re-establish the initial reaction
pressure.) The pressure was released and the bomb purged
with N2. The reaction mixture was cautiously filtered
through celite and then concentrated. The residue was
dissolved in EtOAc (1.6 L) and tert-butylamine (77 mL,
0.73 mol) added dropwise. After stirring for 2.5 h, the
precipitate was recovered by filtration and washed with
MTBE (200 mL). This material was recrystallised from 1:3
MeOH/MTBE (700 mL) to give the tert-butylamine salt as a
white solid (91 g). This salt was dissolved in H2O (350 mL),
and the pH adjusted to 3 with 6 M HCl. The solution was
stirred for 1 h and extracted with CH2Cl2 (3£350 mL). The
combined organic extracts were dried over MgSO4 and
concentrated to give a free acid as a white solid (64 g). This
was dissolved in MeOH (350 mL) and cooled to 5 8C. Et3N
(44 mL, 0.31 mol) was added followed by methyl chloro-
formate (25 mL, 0.31 mol) dropwise. The reaction mixture
was stirred at 5 8C for 1 h, then allowed to warm to room
temperature overnight. The solvent was removed in vacuo,
and the residue taken up in CH2Cl2 (300 mL). This solution
was washed with 1 M KHSO4 (2£200 mL), sat. NaHCO3
(200 mL) and sat. brine (200 mL) and dried over MgSO4.
Concentration of this material yielded 33a, an orange oil
before drying over MgSO4. Following filtration, concen-
tration under vacuum yielded a brown solid which was
taken up in hot MTBE (80 mL) and stirred with decolouris-
ing charcoal for 30 min. After filtration, heptane was added
to yield 33b as white crystals (18.6 g, 71%). 1H NMR
(CDCl3) 5.26 (1H, d, J¼5 Hz), 4.13 (1H, m), 3.96 (1H, br s),
3.79 (1H, br s), 3.70 (3H, s), 3.07 (1H, m), 2.40 (1H, dt,
J¼13, 8 Hz), 2.14 (1H, m), 1.99 (1H, m), 1.71 (1H, m), 1.44
(9H, s). 13C NMR (CDCl3) 177.4, 156.7, 80.3, 78.6, 60.2,
52.4, 40.2, 36.5, 33.7, 28.7. m/z 241 (Mþ), 207, 185, 154,
140, 126, 116, 100, 83, 53. [a]2D5 (c 1.0, MeOH) 225.9.
5.2.8. (1R,3R,4S)-3-tert-Butoxycarbonylamino-4-
hydroxy-cyclopentanecarboxylic acid methyl ester 33e.
((1,2-Bis(2R,5R)-2,5-dimethylphospholano)benzene)(cyclo-
octadiene)rhodium(I) tetrafluoroborate (23 mg, 1 mol%) was
added to a degassed solution of 36 (1 g, 3.9 mmol) in MeOH
(5 mL). The reaction mixture was transferred to a bomb and
after purging with N2 and then H2, an H2 pressure of 5 bar was
applied and the reaction stirred for 14 h. The pressure was
released and the bomb purged with N2. Concentration of the
reaction mixture gave a residue which was redissolved in
CH2Cl2 (5 mL). Addition of silica (0.5 g) with stirring
removed the catalyst from the reaction and filtration and
concentration of the organic gave 33e as an off-white solid of
98% d.e. in quantitative yield. 1H NMR (CDCl3) 4.91 (1H, d,
J¼7 Hz), 4.27 (1H, m), 3.99 (1H, br s), 3.67 (3H, s), 3.10
(1H, m), 2.26 (1H, m), 2.05 (2H, m), 1.85 (1H, dt, J¼13,
10 Hz), 1.43 (9H, s). 13C NMR (CDCl3) 176.8, 156.2, 80.0,
73.0, 55.0, 52.3, 39.5, 36.7, 32.9, 28.8. m/z No Mþ, 202, 185,
172, 154, 140, 126, 116, 99, 87, 57. [a]2D5 (c 1.0, MeOH)
þ36.4.
1
(59 g, .98% d.e., 38% from 35). H NMR (CDCl3) 5.17
(1H, br s), 4.09 (1H, m), 3.92 (1H, br s), 3.71 (3H, s), 2.91
(2H, m), 2.39 (1H, app dq, J¼17 Hz), 2.12 (1H, m), 1.98
(1H, app dq, J¼14 Hz), 1.74 (1H, app dq, J¼17 Hz), 1.42
(9H, s). 13C NMR (CDCl3) 179.1, 156.2, 79.9, 73.4, 55.9,
52.8, 39.6, 36.0, 33.5, 28.8. m/z 241 (Mþ), 202, 185, 172,
154, 140, 126, 116. [a]2D5 (c 1.0, MeOH) þ3.3.
5.2.9. (1R,3R,4R)-3-tert-Butoxycarbonylamino-4-
hydroxy-cyclopentanecarboxylic acid methyl ester 33f.
1
Prepared in an identical manner to 33b. H NMR (CDCl3)
4.67 (1H, br), 4.23 (1H, br s), 4.00 (1H, m), 3.76 (1H, m),
3.70 (3H, s), 2.91(1H, m), 2.43 (1H, m), 2.34 (1H, m), 1.91
(1H, m), 1.68 (1H, m), 1.45 (9H, s). 13C NMR (CDCl3)
176.2, 157.3, 80.6, 79.1, 59.3, 52.5, 39.3, 36.4, 33.2, 28.7.
m/z 241 (Mþ), 202, 185, 172, 154, 140, 126, 116, 83, 57.
[a]2D5 (c 1.0, MeOH) 29.7.
5.2.7. (1S,3R,4R)-3-tert-Butoxycarbonylamino-4-
hydroxy-cyclopentanecarboxylic acid methyl ester 33b.
Methanesulfonyl chloride (17.0 mL, 0.22 mol) was added
dropwise to a solution of 33a (43.9 g, 0.18 mol), triethyla-
mine (50 mL, 0.36 mol) and DMAP (1.1 g, 9 mmol) in
CH2Cl2 (500 mL) at 0 8C. After 45 min the reaction was
washed with 1 M citric acid (2x200 mL), sat. NaHCO3
(200 mL), H2O (200 mL) and brine (100 mL) and then dried
over MgSO4. After filtration, concentration of the organic
under vacuum gave the mesylate as an off-white solid
(55.6 g, 96% crude), which was immediately dissolved in
DMF (250 mL) and KOAc (105 g, 1.05 mol) added. The
stirred mixture was heated at 60 8C for 3 days, cooled and
diluted with CH2Cl2 (700 mL). The mixture was washed
with H2O (1 L), sat. NaHCO3 (500 mL), H2O (500 mL) and
brine (300 mL) before drying over MgSO4. Following
filtration, concentration under vacuum gave the acetate as a
brown solid which was recrystallised from MTBE and
hexanes. NaOMe (0.7 mL, 25 wt% solution in MeOH) was
added to a solution of the recrystallised acetate (29.2 g,
0.10 mol) in MeOH (300 mL) at 0 8C. After 7 h the reaction
was quenched on addition of glacial AcOH and the solvent
removed under vacuum. The residue was redissolved in
CH2Cl2 (500 mL) and washed with H2O (200 mL), sat.
NaHCO3 (200 mL), H2O (100 mL) and brine (200 mL)
References and notes
1. Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic
Synthesis; Wiley-VCH: Weinheim, 1999.
2. (a) Azerad, R.; Buisson, D. Curr. Opin. Biotechnol. 2000, 11,
565–571. (b) Stecher, H.; Faber, K. Synthesis 1997, 1–16.
3. Strauss, U. T.; Faber, K. Tetrahedron: Asymmetry 1999, 10,
4079–4081.
4. (a) Dijksman, A.; Elzinga, J. M.; Li, Y.-X.; Arends, I. W. C.
E.; Sheldon, R. A. Tetrahedron: Asymmetry 2002, 13,
879–884. (b) Kim, M.-J.; Choi, Y. K.; Choi, M. Y.; Kim,
M. J.; ParK, J. J. Org. Chem. 2001, 66, 4736–4738.
¨
(c) Persson, A. B.; Huerta, F. F.; Backvall, J.-E. J. Org.
Chem. 1999, 64, 5237–5240.
5. Chang, C.-S.; Tsai, S.-W.; Kuo, J. Biotechnol. Bioeng. 1999,
64, 120–126.
6. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Adv. Drug Delivery Rev. 1997, 23, 3–25.

728
R. C. Lloyd et al. / Tetrahedron 60 (2004) 717–728
7. Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.;
Ward, K. W.; Kopple, K. D. J. Med. Chem. 2002, 45,
2615–2623.
24. Boaz, N. W. U. S. Patent 5,312,950, 1994..
25. Hays, S. J.; Malone, T. C.; Johnson, G. J. Org. Chem. 1991, 56,
4084–4086.
8. Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J. Am. Chem.
Soc. 1989, 111, 6354–6364.
26. The Organic Chemistry of b-Lactams; Georg, G. I., Ed.;
Verlag Chemie: New York, 1993.
9. Toogood, H. S.; Hollingsworth, E. J.; Brown, R. C.; Taylor,
I. N.; Taylor, S. J. C.; McCague, R.; Littlechild, J. A.
Extremophiles 2002, 6, 111–122.
´ ´
27. (a) Csomos, P.; Kanerva, L. T.; Bernath, G.; Fu¨lop, F.
¨
´
Tetrahedron: Asymmetry 1996, 7, 1789–1796. (b) Kaman, J.;
´
´
Forro, E.; Fu¨lop, F. Tetrahedron: Asymmetry 2000, 11,
1593–1600.
¨
10. Taylor, S. J. C.; Brown, R. C. PCT patent application WO 00/
23598 A1, 2000..
28. (a) Wang, X.; Espinosa, J. F.; Gellman, S. H. J. Am. Chem.
Soc. 2000, 122, 4821–4822. (b) Lee, H.-S.; Syud, F. A.;
Wang, X.; Gellman, S. H. J. Am. Chem. Soc. 2001, 123,
7721–7722. (c) Appella, D. H.; Barchi, J. J.; Durell, S. R.;
Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309–2310.
29. (a) Gademann, K.; Hintermann, T.; Schreiber, J. V. Curr. Med.
Chem. 1999, 6, 905–925. (b) Marastoni, M.; Guerrini, R.;
Balboni, G.; Salvadori, S.; Fantin, G.; Fogaggnolo, M.;
Lazarus, L. H.; Tomatis, R. Arzneim.-Forsch./Drug. Res.
1999, 49, 6–12.
11. Shinagawa, S.; Maki, M.; Kintaka, K.; Imada, A.; Asai, M.
J. Antibiot. 1985, 38, 17–23.
12. Ohfune, Y.; Hori, K.; Sakaitani, M. Tetrahedron Lett. 1986,
27, 6079–6082.
13. Holt, K. E.; Swift, J. P.; Smith, M. E. B.; Taylor, S. J. C.;
McCague, R. Tetrahedron Lett. 2002, 43, 1545–1548.
14. Pelter, A.; Ward, R. S.; Little, G. M. J. Chem. Soc., Perkin
Trans. 1 1990, 2775–2790.
15. Oppolzer, W.; Moretti, R.; Zhou, C. Helv. Chim. Acta 1994,
77, 2363–2380.
30. Moriconi, E. J.; Crawford, W. C. J. Org. Chem. 1968, 33,
370–378.
16. Lloyd, R. C.; Smith, M. E. B.; Brick, D.; Taylor, S. J. C.;
Chaplin, D. A.; McCague, R. Org. Proc. Res. Dev. 2002, 6,
762–766.
31. Taylor, S. J. C.; Brown, R. C.; Keene, P. A.; Taylor, I. N.
Bioorg. Med. Chem. 1999, 7, 2163–2168.
17. (a) Romeo, J. T.; Swain, L. A.; Bleeker, A. B. Phytochemistry
1983, 22, 1615–1618. (b) Schenk, V. W.; Schutte, H. R. Flora
1963, 153, 426–443. (c) Clark-Lewis, J. W.; Mortimer, P. I.
J. Chem. Soc. 1961, 189–201.
32. Allan, R. D.; Dickenson, H. W.; Fong, J. Eur. J. Pharmacol.
1986, 122, 339–348.
33. Smith, M. E. B.; Lloyd, M. C.; Derrien, N.; Lloyd, R. C.;
Taylor, S. J. C.; Chaplin, D. A.; Casy, G.; McCague, R.
Tetrahedron: Asymmetry 2001, 12, 703–705.
18. Evans, S. V.; Shing, T. K. M.; Aplin, R. T.; Fellows, L. E.;
Fleet, G. W. J. Phytochemistry 1985, 24, 2593–2596.
19. Pellicciari, R.; Natalini, B.; Luneia, R.; Marinozzi, M.;
Marinella, R.; Rosato, G. C.; Sadeghpour, B. M.; Snyder,
J. P.; Monahan, J. B.; Moroni, F. Med. Chem. Res. 1992, 2,
491–496.
34. Taylor, S. J. C.; Ruecroft, G. Chem. Br. 2002, 38, 46–49.
35. Lai, Y.-S.; Mendoza, J. S.; Jagdmann, G. E.; Mendalino, D. S.;
Biggers, C. K.; Heerding, J. M.; Wilson, J. W.; Hall, S. E.;
Jiang, J. B.; Janzen, W. P.; Ballas, L. M. J. Med. Chem. 1997,
40, 226–235.
´
20. (a) Anderson, P. C.; Soucy, F.; Yoakim, C.; Lavallee, P.;
36. Frazer, M. M.; Raphael, R. A. J. Chem. Soc. 1955,
4280–4283.
Beaulieu, P. L. U. S. Patent 5,545,640.. (b) Lamarre, D.;
Croteau, G.; Bourgon, L.; Thibeault, D.; Wardrop, E.;
Clouette, C.; Vaillancourt, M.; Cohen, E.; Pargellis, C.;
Yoakim, C.; Anderson, P. C. Antimicrob. Agents Chemother.
1997, 41, 965–971.
37. Shirokova, E. A.; Tarussova, N. B.; Shipitsin, A. V.;
Semizarov, D. G.; Krayevsky, A. A. J. Med. Chem. 1994,
37, 3739–3748.
38. Burk, M. J.; Allen, J. G.; Kiesman, W. F. J. Am. Chem. Soc.
1998, 120, 657–663.
21. Rutjes, F. P. J. T.; Veerman, J. J. N.; Meester, W. J. N.;
Hiemstra, H.; Schoemaker, H. E. Eur. J. Org. Chem. 1999,
1127–1135.
39. Taylor, S. J. C.; McCague, R.; Wisdom, R.; Lee, C.; Dickson,
K.; Ruecroft, G.; O’Brien, F.; Littlechild, J.; Bevan, J.;
Roberts, S. M.; Evans, C. T. Tetrahedron: Asymmetry 1993, 4,
1117–1128.
22. Brace, N. O. J. Org. Chem. 1967, 32, 430–434.
23. Esch, P. M.; de Boer, R. F.; Hiemstra, H.; Boska, I. M.;
Speckamp, W. N. Tetrahedron 1991, 47, 4063–4076.