The synthesis of diastereo- and enantiomerically pure β-aminocyclopropanecarboxylic acids

Article abstract of DOI:10.1021/jo005541l

The synthesis of diastereo- and enantiomerically pure β-aminocyclopropanecarboxylic acids (β-ACCs) is described. Starting from pyrrole, (rac)-4 is readily obtained, which was kinetically resolved by enzymatic hydrolysis. Subsequent oxidation of (-)-4 and deformylation gives rise to the cis-β- ACC derivative (ent)-9, while (+)-19 was converted to the trans-β-ACC derivative 8. Both 8 and (ent)-9 and their benzyl esters 13 and 16, being conformationally restricted β-alanine or γ-amino-butyric acid (GABA) derivatives, represent useful building blocks for peptides containing unnatural amino acids.

Full text of DOI:10.1021/jo005541l

8960
J . Org. Chem. 2000, 65, 8960-8969
Th e Syn th esis of Dia ster eo- a n d En a n tiom er ica lly P u r e
â-Am in ocyclop r op a n eca r boxylic Acid s
Raphael Beumer,† Christian Bubert,† Chiara Cabrele,† Oliver Vielhauer,‡
Markus Pietzsch,*,‡ and Oliver Reiser*,†
Department of Organic Chemistry, University of Regensburg, Universita¨tsstr. 31,
D-93053 Regensburg, Germany, and Department of Biochemical Engineering, University of Stuttgart,
Allmandring 31, D-70569 Stuttgart, Germany
Oliver.Reiser@chemie.uni-regensburg.de
Received J une 13, 2000
The synthesis of diastereo- and enantiomerically pure â-aminocyclopropanecarboxylic acids (â-
ACCs) is described. Starting from pyrrole, (rac)-4 is readily obtained, which was kinetically resolved
by enzymatic hydrolysis. Subsequent oxidation of (-)-4 and deformylation gives rise to the cis-â-
ACC derivative (ent)-9, while (+)-10 was converted to the trans-â-ACC derivative 8. Both 8 and
(ent)-9 and their benzyl esters 13 and 16, being conformationally restricted â-alanine or γ-amino-
butyric acid (GABA) derivatives, represent useful building blocks for peptides containing unnatural
amino acids.
In tr od u ction
â-Amino acids have also attracted considerable atten-
tion,6 most spectacular as constituents of the newly
emerging class of â-peptides.7,8 There have been quite a
few efforts toward the synthesis of â-aminocyclopropane
carboxylic acids (â-ACCs),9 being conformationally re-
stricted â-alanine or γ-aminobutyric acid (GABA) deriva-
tives, and their subsequent incorporation into peptides.10
However, since â-ACC derivatives are vicinally substi-
tuted with donor-acceptor moieties,11 they are extremely
R-Aminocyclopropanecarboxylic acids (R-ACCs) have
been proved to be important because of their inherent
biological activity1,2 and because of their application as
structural restricted building blocks for peptides.3
A
number of elegant strategies toward the synthesis of
R-ACCs have emerged during recent years.4,5
* To whom correspondence should be addressed. Tel: Int-49-941-
9434631. Fax Int-49-941-9434121.
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D. Synlett 1994, 575-583. (c) Stammer, C. H. Tetrahedron 1990, 46,
2231.
(5) (a) Burgess, K.; Ho, K.-K. J . Org. Chem. 1992, 57, 5931-36. (b)
Aufranc, P.; Ollivier, J .; Stolle, A.; Bremer, C.; Es-Sayed, M.; de
Meijere, A.; Salau¨n, J . Tetrahedron Lett. 1993, 34, 4193-4196. (c)
Dorizon, P.; Su, G.; Ludvig, G.; Nikitina, L.; Paugam, R.; Ollivier, J .;
Salau¨n, J . J . Org. Chem. 1999, 64, 4712-24. (d) Es-Sayed, M.;
Gratkowski, C.; Krass, N.; Meyers, A. I.; de Meijere, A. Synlett 1992,
962-964. (e) de Meijere, A.; Ernst, K.; Zuck, B.; Brandl, M.; Kozhush-
kov, S. I.; Tamm, M.; Yufit, D. S.; Howard, J . A. K.; Labahn, T. Eur.
J . Org. Chem. 1999, 3105, 5-3115.
† University of Regensburg.
‡ University of Stuttgart.
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Commun. 1985, 1946. (d) Hill, R. K.; Prakash, S. R. J . Am. Chem. Soc.
1984, 106, 795. (e) Adams, D. O.; Yang, S. F. Proc. Natl. Acad. Sci.
1979, 76, 170. (f) Sakamura, S.; Ichihara, A.; Shiraishi, K.; Sato, H.;
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10.1021/jo005541l CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/29/2000

â-Aminocyclopropanecarboxylic Acids
J . Org. Chem., Vol. 65, No. 26, 2000 8961
Sch em e 1
prone toward ring opening. Special requirements are
therefore necessary for their synthesis and subsequent
use as building blocks.12
We report here a synthetic strategy for the preparation
of diastereo- and enantiomerically pure 3-aminocyclo-
propane-1,2-dicarboxylic acids starting from N-Boc-pyr-
role (3a ) that allows the subsequent incorporation of both,
the cis- and the trans-â-ACC structure into peptides. The
key intermediate 2 may be transformed into 1 with
chemical differentiation of the ester groups to allow their
individual use in subsequent synthetic applications.
Sch em e 2
Resu lts a n d Discu ssion
Syn th esis of Ra cem ic â-Am in ocyclop r op a n eca r -
boxylic Acid Der iva tives. The cyclopropanation of
N-carbomethoxypyrrole (3b) with methyl diazoacetate
has been reported by Fowler using copper(I) bromide as
the catalyst at 80 °C to yield 2 in 17% yield.13 We were
able to improve this reaction by using catalytic amounts
of copper(II) triflate, activated by phenylhydrazine. This
way, the cyclopropanation of 3a proceeds smoothly at
room temperature, yielding (rac)-4 in 39% yield along
with the 2-fold cyclopropanated adduct (rac)-5 (3%) and
recovered starting material (36%). (rac)-4 is obtained as
a single diastereomer, having the ester group oriented
at the convex face of the bicyclic structure. However, we
have been unable to find a chiral catalyst so far that
would achieve the synthesis of optical active 4 with
preparative useful enantioselectivities. Therefore, we
investigated the possibility of an enzymatic resolution of
(rac)-4 as well as of the aminocyclopropane dimethylester
(rac)-7, which is conveniently obtained from (rac)-4 by
ozonolysis followed by deprotection of the N-formyl group
(Scheme 1).
Kin etic En zym a tic Resolu tion of (r a c)-7. The di-
ester (rac)-7 contains two nonequivalent ester functions;
therefore, the hydrolysis of a single ester bond of (rac)-7
could lead to four different stereoisomers (Scheme 2).
Consequently, the synthesis of one enantiomer is only
possible if the biocatalyst shows high enantioselectivity
and regioselectivity at the same time.
(8) (a) Apella, D.; Christianson, L. A.; Karle, I. L.; Powell, D. R.;
Gellman, S. H. J . Am. Chem. Soc. 1996, 118, 13071-72. (b) Huck, B.
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Applequist, J .; Bode, K. A.; Appella, D. A.; Christianson, L. A.; Gellman,
S. H. J . Am. Chem. Soc. 1998, 120, 4891. (d) Krautha¨user, S.;
Christianson, L. A.; Powell, D. R.; Gellman, S. H. J . Am. Chem. Soc.
1997, 119, 11719. (e) Appella, D. H.; Christianson, L. A.; Klein, D.;
Powell, D. R.; Huang, X.; Barchi, J . J .; Gellman, S. H. Nature 1997,
387, 381. (f) Seebach, D.; Abele, S.; Gademann, K.; J aun, B. Angew.
Chem. 1999, 111, 1700-02. (g) Gademann, K.; Ernst, M.; Hoyer, D.;
Seebach, D. Angew. Chem. 1999, 111, 1302-1304. (h) Seebach, D.;
Metthews, J . L. Helv. Chim. Acta 1997, 80, 173-182. (i) Seebach, D.;
Overhand, M.; Ku¨hnle, F. N. M.; Martinoni, B. Helvetica Chimica 1996,
79, 913-932. (j) Seebach, D.; Overhand, M.; Ku¨hnle, F. N. M.;
Martinoni, B.; Oberer, L.; Hommel, U.; Widmer, H. Hev. Chim. Acta
1996, 1996, 913-41.
(9) (a) Cannon, J . G.; Garst, J . E. J . Org. Chem. 1975, 40, 182-
184. (b) Shroff, C. C.; Stewart, W. S.; Uhm, S. J .; Wheeler, J . W. J .
Org. Chem. 1971, 22, 3356-3361. (c) Paulini, K.; Reissig, H.-U. Liebigs
Ann. Chem. 1991, 455-461. (d) Bubert, C.; Voigt, J .; Biasetton, S.;
Reiser, O. Synlett 1994, 675-677. (e) Vilsmaier, E. In The Chemistry
of the Cyclopropyl Group; Rappoport, Z., Ed.; VCH-Wiley: New York,
1987; p 1341.
(10) (a) Paulini, K.; Reissig, H.-U. Liebigs Ann. Chem. 1994, 549-
554. (b) Beck-Sickinger, A. G.; Hoffmann, E.; Paulini, K.; Reissig, H.-
U.; Willim, K.-D.; Wieland, H. A.; J ung, G. Biochem. Soc. Trans. 1994,
22, 145-149. (c) Hibbs, D. E.; Hursthouse, M. B.; J ones, I. G.; J ones,
W.; Malik, K. M. A.; North, M. Tetrahedron 1997, 53, 17417-24. (d)
Voigt, J .; Noltemeyer, M.; Reiser, O. Synlett 1997, 202-204. (e) Bubert,
C.; Cabrele, C.; Reiser, O. Synlett 1997, 827-29.
The screening for enzymes capable of the hydrolysis
of (rac)-7 was carried out either in a buffered aqueous
suspension or in a two-phase system consisting of buffer/
toluene. From the enzymes screened (Table 1) only pig
liver esterase (PLE, E-1 and E-2) and lipase from Mucor
miehei (L-9) were able to hydrolyze the substrate in
significant amounts while the other enzymes showed only
low activity or were inactive at all.
(11) Reissig, H. U. Top. Curr. Chem. 1988, 144, 73.
(12) A racemic synthesis of E- and Z-cyclo-Asp without occurrence
of ring opening was reported: Kraus, G. A.; Kim, H.; Thomas, P. J .;
Metzler, D. E.; Metzler, C. M.; Taylor, J . E. Synth. Commun. 1990,
20, 2667. We have never been able to synthesize a â-ACC derivative
with an unprotected amino group. The closest we ever got has been
the synthesis of the diester A (R ) Bn or Me),10e but already the
corresponding acid (R ) H) proved to be unstable and could also not
be observed in situ by NMR spectroscopy.
The nearly complete conversion observed in the cases
of L-9 and PLE indicated that only poor enantiospecificity
had occurred. Furthermore, the product isolated from a
preparative scale using PLE A as the biocatalyst showed
in addition poor regioselectivity as determined by 1H
NMR spectroscopy. The data revealed that approximately
40% of the cis-amino acid 9 and 60% of the trans-amino
acid 8 had been produced, suggesting that the resolution
(13) Tanny, S. R.; Grossman, J .; Fowler, F. W. J . Am. Chem. Soc.
1972, 94, 6495-6501.

8962 J . Org. Chem., Vol. 65, No. 26, 2000
Beumer et al.
Ta ble 1. Scr een in g for Ester a ses a n d Lip a ses for th e
Ta ble 2. Scr een in g for Ester a ses a n d Lip a ses w ith
Activity for th e Hyd r olysis of (r a c)-4
Hyd r olysis of (r a c)-7
screening
investigated
period of time (h)
screening
investigated
period of time (h)
enzymea
conditionsb
conversionc
enzymea
conditionsb
conversionc
complete
PLE A
E-1
E-2
PPL
L-1
L-2
L-3
L-5
L-6
suspension
suspension
suspension
suspension
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
120
24
24
nearly complete
nearly complete
>50%
PLE A
E-1
E-2
L-1
L-2
L-3
L-5
L-6
L-7
L-8
L-9
A2
A4
A6
A8
A9
A10
A11
suspension
suspension
suspension
24
24
24
240
72
complete
nearly complete
∼50%
120
165
165
165
165
165
165
165
165
traces
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
water/toluene
no conversion
no conversion
traces
no conversion
traces
no conversion
traces
nearly complete
complete
240
240
240
240
240
240
187
187
187
187
187
187
187
no conversion
no conversion
∼50%
no conversion
no conversion
,50%
L-7
L-8
L-9
no conversion
traces
traces
no conversion
traces
no conversion
no conversion
a
For enzyme abbreviations see Table 6 in the Supporting
b
Information. See the Experimental Section. c Determined by
qualitative TLC.
step at the level of the cyclopropane diester (rac)-7 is
impossible with the enzymes available to us. The enan-
tioselectivity of the reaction products was therefore not
investigated in detail.
a
For enzyme abbreviations see Table 6 in the Supporting
b
Information. See the Experimental Section. c Determined by
qualitative TLC.
The lack of discrimination between the cis- and trans-
ester group for (rac)-7 has been surprising in light of
PLE-catalyzed hydrolyses of a variety of cyclopropane
dicarboxylic esters being described in the literature14 and
the models which have consequently been developed for
such processes.15 It should be kept in mind that com-
mercially available PLE consists of a mixture of iso-
enzymes which can behave more or less differently in
regard to substrate- and enantiospecificity.14 For ex-
ample, E-1 and E-2 used in our screening experiments
represent different fractions of the isoenzymes of PLE.
Kin etic En zym a tic Resolu tion of (r a c)-4. We fo-
cused next on the kinetic enzymatic resolution of the
bicyclic adduct (rac)-4 which is used as a direct precursor
for â-ACC derivatives. From the enzymes screened (Table
2) pig liver esterase (PLE A, E-1 and E-2) and lipases
(L-1, L-2, L-6) were shown to carry out the desired
transformation with significant conversion while other
enzymes showed only low activity (L-9) or were inactive
at all.
Encouragingly, L-1, L-6, and L-9 indeed were capable
of hydrolyzing (rac)-4 without complete conversion even
after 10 d of reaction time. Furthermore, although the
reaction with L-2 was complete, the rate decreased
considerably during the course of the reaction in differ-
ence to the esterases, suggesting that resolution with L-2
might also be possible.
Next, kinetic measurements were carried out combined
with determination of the enantioselectivities being
obtained with the enzymes PLE A, L-1, L-2, and L-6 in
the hydrolysis of (rac)-4. L-9 was not considered for
further experiments since its activity was too low in order
to be useful for preparative transformations. For each
enzyme the change of substrate and product concentra-
tion with time was recorded (Figures 1a-4a) and the
enantiomeric excess of the substrate 4 was plotted versus
the conversion (Figures 1b-4b). In the latter diagrams,
Ta ble 3. Activity a n d Selectivity of En zym es for th e
Kin etic Resolu tion of (r a c)-4
activityc preferentially
reaction
(µmol/min
hydrolyzed
enantiomer
enantiomeric
ratio E d
enzymea conditionsb
× mg)
PLE A
L-1
A
A
B
A
B
A
B
0.46
(+)-4
n.d.
(-)-4
(+)-4
(+)-4
n.d.
1.7
n.d.
3.5
6.3 × 10-3
4.0 × 10-5
0.33
L-2
L-6
4.6
6.9 × 10-3
6.9 × 10-3
3.8 × 10-5
36.0
n.d.
8.5
(+)-4
a
For enzyme abbreviations see Table 6 in the Supporting
b
Information. A: 0.1 M phosphate buffer pH 7.5, 25 °C, automatic
buret. B: toluene/0.1 M phosphate buffer pH 7.4, ambient tem-
perature. c Specific enzyme activity (initial activity, total conver-
d
sion of both enantiomers of (rac)-4). According to Chen et al.16
the solid lines represent simulations for the determina-
tion of the enantiomeric ratio (E value) according to the
model described by Chen et al.16 and fit well the experi-
mental data.
On the basis of these studies, the determination of the
activity and selectivity of the enzymes tested for the
kinetic resolution of (rac)-4 became possible (Table 3).
PLE A shows the highest activity, but also the lowest
enantioselectivity. From the three lipases investigated
L-2 displayed the highest activity and, what is more
important, also the highest enantioselectivity. Interest-
ingly, L-1 was found to hydrolyze (-)-4 preferentially,
whereas (+)-4 was hydrolyzed faster by all the other
enzymes. The specific activity of the lipases in aqueous
buffer is approximately 2 orders of magnitude higher
than in a biphasic system consisting of toluene and
buffer.
Lipase L-2 was chosen for further optimization of the
reaction conditions because of its most promising char-
acteristics. The other enzymes revealed major drawbacks
either in low activity (L-1 and L-6) and/or enantioselec-
tivity (PLE A and L-1).
(14) Gais, H. J . Hydrolysis of Carboxylic acid esters. In Enzyme
Catalysis in Organic Synthesis; Drauz, K. H., Waldmann, H., Eds.;
VCH-Wiley: Weinheim, 1995; Vol. 1, Chapter B.1.1.1.1, pp 178-195.
(15) (a) Toone, E. J .; Werth, M. J .; J ones, J . B. J . Am. Chem. Chem.
Soc. 1990, 112, 4946-4952. (b) Walser, P.; Renold, P.; N’Goka, V.;
Hosseinzadeh, F.; Tamm, C. Helv. Chim. Acta 1991, 74, 1941-1952.
(c) Provencher, L.; J ones, J . B. J . Org. Chem. 1994, 59, 2729-2732.
To minimize the amount of enzyme used in the
screening experiments and to enhance the enantioselec-
(16) Chen, C. S.; Fujimoto, Y.; Sih, C. J . J . Am. Chem. Soc. 1982,
104, 7294-7299.

â-Aminocyclopropanecarboxylic Acids
J . Org. Chem., Vol. 65, No. 26, 2000 8963
F igu r e 1. PLE A catalyzed kinetic resolution of (rac)-4 (screening). (a) Time course of the concentrations of substrate and product
enantiomers. (b) Enantiomeric excess of (-)-4 (ee(S)) versus conversion. (0 (+)-4; 9 (-)-4; 4 (+)-10; b (-)-10).
F igu r e 2. Lipase L-1 catalyzed kinetic resolution of (rac)-4 (screening). (a) Time course of the concentrations of substrate and
product enantiomers. (b) Enantiomeric excess of (+)-4 (ee(S)) versus conversion. (0 (+)-4; 9 (-)-4; 4 (+)-10; b (-)-10).
tivity, the reaction conditions (cosolvent, amount of
enzyme, addition of Triton X-100; for details see Table 4
in the Supporting Information) were varied. All attempts
failed to enhance the selectivity factor E above 36, which
was initially found. However, the optimization experi-
ments revealed two important developments. The amount
of enzyme needed for the transformation could be reduced
by a factor of approximately 100 (from 13 to 0.12 g
enzyme per gram substrate). As a minor drawback the
selectivity factor E dropped from 36 to 13.5. On the other
hand the addition of Triton X-100 simplified homoge-
neous sampling by stabilizing the reaction emulsion
leaving the enantioselectivity of the enzyme almost
unchanged.
Using the optimized reaction conditions, the kinetic
resolution (rac)-4 could be carried out on a gram scale.
For example, starting with 1 g of (rac)-4 to the point of
complete depletion of (+)-4, 335 mg of (-)-4 (33%, 99.6%
ee) and 462 mg (49%, 43.3% ee) of (+)-10 were isolated.
From these data, the selectivity factor calculated (E )
13.4) is in excellent agreement with the one determined
during the optimization process (E ) 13.5, cf. Figure 5).
It was not possible to obtain enantiopure acid (+)-10
in preparative useful amounts at the E value achieved
during the synthesis of enantiopure (-)-4. Therefore, the
reaction conditions were optimized further. Using cyclo-
hexane as a cosolvent, the pH was varied between 5.0
and 8.0. Below and above these pH values, substrate and
product were unstable. The optimum of the enantio-
selectivity is found at pH 5.0, whereas the activity was
highest at alkaline pH. For comparison, in Figure 5b the
enantiomeric excess of 10 is shown versus the conversion
for the initial optimization pH 7.4 and the optimal pH of
5.0.
Consequently, using lipase L-2 and the optimized
reaction conditions (cf. experimental part and Table 5,
Supporting Information) 303 mg of (+)-10 (30% yield,
91% ee) was obtained by kinetic resolution of 1.067 g
(rac)-4. Subsequent recrystallization yielded (+)-10 in
enantiomerically pure form (>96% ee).
Syn th esis of cis- a n d tr a n s-â-Am in ocyclop r op a n e-
ca r boxylic Acid Der iva tives. Having (-)-4 in enan-
tiomerically pure form in hand, it can be readily trans-
formed into the cis-â-ACC derivative 13 (Scheme 3):

8964 J . Org. Chem., Vol. 65, No. 26, 2000
Beumer et al.
F igu r e 3. Lipase L-2 catalyzed kinetic resolution of (rac)-4 (screening). (a) Time course of the concentrations of substrate and
product enantiomers. (b) Enantiomeric excess of (-)-4 (ee(S)) versus conversion. (0 (+)-4; 9 (-)-4; 4 (+)-10; 2 (-)-10).
F igu r e 4. Lipase L-6 catalyzed kinetic resolution of (rac)-4 (screening). (a) Time course of the concentrations of substrate and
product enantiomers. (b) Enantiomeric excess of (-)-4 (ee(S)) versus conversion. (0 (+)-4; 9 (-)-4; 4 (+)-10; 2 (-)-10).
Ozonolysis of (-)-4 followed by reductive workup with
dimethyl sulfide gave rise to the amino aldehyde 11. Due
to the two electron withdrawing protecting groups at
nitrogen, 11 is perfectly stable and has already proved
in racemic form to be a valuable building block for further
synthetic transformations.17
Oxidation of 11 with NaClO2/H2O2 proceeded smoothly
to the carboxylic acid 12, followed by deprotection of the
N-formyl group to give rise to (ent)-9. Although a variety
of bases are able to readily facilitate this deformylation,
special care is necessary in this step; e.g., deprotection
with potassium bicarbonate proceeded with good chemi-
cal yield (85%) but led to complete epimerization on the
amino substituted carbon atom. Fortunately, deprotection
with amines such as piperidine, morpholine or especially
2-diethylaminoethylamine (DEAEA)18 took place without
any detectable scrambling on any of the stereocenters in
high yields (87-95%). Finally, esterification with benzyl
bromide gave rise to 13, which can be incorporated into
peptides as the cis-â-ACC structure as has been demon-
strated by us before.10e Moreover, (ent)-9 was converted
to the amide 14, from which a X-ray structure was
obtained in order to unambiguously establish the abso-
lute stereochemistry of all â-ACC derivatives described
here.
Starting from (+)-10 the â-ACC derivative 16 can be
readily obtained (Scheme 4) by similar transformations
as depicted for the synthesis of 13. Thus, ozonolysis and
workup with Ac2O/NEt3 resulted in 15 which was directly
(17) (a) Bubert, C.; Reiser, O. Tetrahedron Lett. 1997, 38, 4985-
88. (b) Bo¨hm, C.; Schinnerl, M.; Bubert, C.; Zabel, M.; Labahn, T.,
Parisini, E.; Reiser, O. Eur. J . Org. Chem. 2000, 2955-65.
(18) Grehn, L.; Gunnarson, K.; Ragnarsson, U. Chem. Commun.
1985, 1317-18.

â-Aminocyclopropanecarboxylic Acids
J . Org. Chem., Vol. 65, No. 26, 2000 8965
F igu r e 5. Lipase L-2 catalyzed kinetic resolution of (rac)-4 (preparative scale). (a) Enantiomeric excess of (-)-4 (ee(S)) versus
conversion. (b) Enantiomeric excess versus conversion for the product of the reaction 10 before (pH 7.4) and after optimization
(pH 5.0).
Sch em e 3
Sch em e 4
In summary, a short synthesis to diastereo- and
enantiomerically pure â-ACC derivatives 8, (ent)-9, 13,
and 16 has been developed which are suitable building
blocks for peptides. Especially, the incorporation of the
cis-â-ACC structure seems to be interesting as a turn
mimic, and first results in the synthesis of NPY-mimics
having (ent)-9 incorporated show promising selectivites
to specific Neuropeptide Y receptors.19
Exp er im en ta l Section
Gen er a l Meth od s. GC analysis was performed on a Hep-
takis-(2,3-di-O-methyl-6-O-thexyldimethylsilyl)-â-cyclodex-
trine capillary column (15 m × 0.25 mm) prepared and
supplied by Prof. Ko¨nig (Institute of Organic Chemistry,
University of Hamburg, Germany). Helium was used as carrier
gas at a pressure of 40 kPa. Temperature program: 100 °C, 4
min isotherm, 3 °C/min to 128 °C, 13 min isotherm, 6 °C/min
to 180 °C, 10 min isotherm, 6 °C/min to 100 °C. Retention
times: (+)-4: 22.258 min, (-)-4: 23.471 min, R ) 1.05.
deformylated with DEAEA to yield 8 (66% from (+)-10).
The sequence was completed with the synthesis of the
benzyl ester 16. Both, 8 or 16 can be used as a starting
point for incorporation of the trans-â-ACC structure into
peptides complementing (ent)-9 and 13 in which the cis-
â-ACC structure can be utilized.10e
(19) Beumer, R.; Cabrele, C.; Beck-Sickinger, A.; Reiser, O. Unpub-
lished results.

8966 J . Org. Chem., Vol. 65, No. 26, 2000
Beumer et al.
Ch ir a l-HP LC. The separation of the enantiomers of 4, 7
and 10 was accomplished using a ET 200/4 Nucleodex â-PM
column (Macherey-Nagel, Du¨ren, Germany). Solvent: 40 mM
sodium phosphate buffer pH 3.7/MeOH 60:40, flow: 0.2 mL/
min, pressure: 48 bar. 4 and 10 were detected at 249 nm, and
7 at 210 nm. Retention times: (+)-4: 100.2 min, (-)-4: 117.1
min, R ) 1.17; 7 (first peak): 38.7 min, 7 (second peak) 46.6
min, R ) 1.20; (rac)-5: 25.4 min; (rac)-6: 23.1 min; (+)-10:
72.9 min, (-)-10: 78.9 min, R ) 1.08. The conversion of the
hydrolysis of (rac)-4 to 10 was calculated according to the
equation c ) ees/(ees + eep),16 where c means the conversion,
ees the enantiomeric excess of (-)-4, and eep the enantiomeric
excess of (+)-10.
((r a c)-6). A solution of (rac)-4 (478 mg, 2.0 mmol), 100 mg of
NaHCO3, and 1.0 mL of MeOH in CH2Cl2 (35 mL) was treated
with ozone at -78 °C until the solution maintained a blue
color. After removal of the excess ozone by passing oxygen
through the solution, Ac2O (0.5 mL) and NEt3 (1.0 mL) were
added slowly. The solution was allowed to warm to room
temperature and stirred for another 6 h. The reaction solution
was extracted with water and dried over Na2SO4. The solvent
was evaporated and the residue was purified by flash chro-
matography (hexanes/ethyl acetate 5:1, Rf ) 0.23) to yield 469
mg (78%) (rac)-6 as a colorless oil, which crystallized from a
mixture CH2Cl2/cyclohexane at -20 °C as a white solid (mp )
64 °C): 1H NMR (250 MHz, CDCl3) δ 1.51 (s, 9H), 2.61-2.64
(m, 2H), 3.18 (dd, J ) 6.7, 6.0 Hz, 1H), 3.68 (s, 3H), 3.75 (s,
3H), 9.11 (s, 1H); 13C NMR (62.9 MHz, CDCl3) δ 27.82, 28.08,
29.01, 35.45, 52.35, 52.54, 84.83, 151.96, 163.27, 168.53,
170.19; MS (EI(70 eV)) 301 (1) [M+], 57 (100) [C4H9+]. Anal.
Calcd for C13H19NO7: C, 51.82; H, 6.36; N, 4.65. Found: C,
52.03; H, 6.30, N, 4.72.
(1R*,2R*,3R*)-N-ter t-Bu t oxyca r b on yl-3-a m in ocyclo-
p r op a n e-1,2-d ica r boxylic Acid Dim eth yl Ester ((r a c)-7).
To a solution of (rac)-6 (301 mg, 1.0 mmol) in MeOH (5 mL)
were added KHCO3 (120 mg, 1.2 mmol) and water (3 mL). The
mixture was stirred for 3 h at room temperature. After
addition of water (5 mL), the mixture was extracted with
CH2Cl2. The combined organic layers were dried over MgSO4,
and the residue was recrystallized from CH2Cl2/Et2O to yield
231 mg (85%) of (rac)-7 as a colorless solid (mp ) 86 °C): 1H
NMR (250 MHz, CDCl3) δ 1.39 (s, 9H), 2.22 (dd, J ) 5.0, 5.0
Hz, 1H), 2.43 (dd, J ) 8.3, 5.0 Hz, 1H), 3.66 (s, 3H), 3.70 (s,
3H), 3.72 (br s, 1H), 5.53 (br s, 1H); 13C NMR (62.9 MHz,
CDCl3) δ 26.11, 28.15, 28.58, 37.33, 52.21, 52.33, 80.19, 155.29,
170.07; MS (EI(70 eV)) 273 (1) [M+], 57 (100) [C4H9+]. Anal.
Calcd for C12H19NO6: C, 52.74; H, 7.01; N, 5.13. Found: C,
52.29; H, 7.20; N, 5.11.
Gen er a l P r oced u r es for En zym e Scr een in g for th e
Hyd r olysis of (r a c)-4 a n d (r a c)-7. (a ) Ester a ses (Aqu eou s
Su sp en sion ). Substrate suspensions: Approximately 8 mg (29
µmol) of finely powdered (rac)-7 was suspended in 2.0 mL of
potassium phosphate buffer (0.1 M, pH 7.4) by ultrasonication.
(rac)-4 could neither be powdered nor suspended in the
aqueous solvent. To prepare a homogeneous and stable
suspension approximately 7 mg of (rac)-4 (29 µmol) were
dissolved in 20 µL of acetone, and 2.0 mL of potassium
phosphate buffer (0.1 M, pH 7.4) was added. Finally, the
mixture was vigorously shaken and ultrasonicated.
Bioconversions: About 10 U of esterase (see Tables 1 and
2) was added to each of the substrate suspensions, and the
resulting reaction mixtures were stirred at ambient temper-
ature. During the investigated period of time samples of 100
µL were taken and acidified with 12 µL of 1.0 M phosphoric
acid to a pH of 2.5-3.5 and immediately extracted with 200
µL of ethyl acetate. The conversion of the reaction was
monitored by analysis of the organic phase after centrifugation
with qualitative TLC.
All chemicals were reagent grade and were used without
further purification. Enzymes are commercially available; for
details, see the Supporting Information.
Assa ys for En zym a tic Activity. Standard assays for
enzyme activities were carried out as described by Horgan et
al. for PLE21 and as described by the supplier for lipases.20
One unit (U) is defined as the amount of enzyme required to
catalyze the hydrolysis of 1 µmol substrate per minute.
2-Azabicyclo[3.1.0]h ex-3-en e-2,6-dicar boxylic Acid 2-ter t-
Bu tyl Ester 6-Meth yl Ester ((r a c)-4) a n d 5-Aza tr icyclo-
[4.1.0.02,4]h ep ta n e-3,5,7-tr ica r boxylic Acid 5-ter t-Bu tyl
Ester 3,7-Dim eth yl Ester ((r a c)-5). To a solution of N-tert-
butoxycarbonylpyrrole 3a (22.0 g, 131.7 mmol) in CH2Cl2 (100
mL) were added copper(II) triflate (100 mg, 0.28 mmol, 0.2
mol %) and phenylhydrazine (100 µL of a solution (1 wt %) in
CH2Cl2) at room temperature under a nitrogen atmosphere.
After being stirred for 20 min, a solution of methyl diazoacetate
(16.50 g, 165 mmol) in CH2Cl2 (200 mL) was added slowly via
a dropping funnel (ca. 1 drop/2 s). After the addition was
complete (6 h), the mixture was further stirred for 12 h at room
temperature. To remove the catalyst the mixture was filtered
through silica gel, and the silica gel was washed with CH2Cl2.
After evaporation of the solvent, the residue was purified by
flash chromatography (hexanes/ethyl acetate 20:1 (ca. 1 L)
followed by 10:1 to yield I: 3a (8.80 g, 36%, Rf ) 0.65 in
hexanes/ethyl acetate 10:1). II: (rac)-4 (14.2 g, 45%, Rf ) 0.26)
containing dimethyl fumarate (ca. 6%) as impurity, which was
removed by sublimation (40 °C, 0.01 Torr, 6 h) to yield 12.30
g (39%) of pure (rac)-4 as a colorless oil which solidifies after
prolonged storage at -20 °C (mp ) 48 °C): 1H NMR (250 MHz,
CDCl3, signal doubling because of rotamers) δ 0.92 (br s, 1H),
1.46 (s, 9H), 2.77 (br s, 1H), 3.62 and 3.65 (s, 3H), 4.26-4.40
(m, 1H), 5.28-5.36 (m, 1H), 6.40-6.55 (br s, 1H); 13C NMR
(62.9 MHz, CDCl3, signal doubling because of rotamers) δ 22.62
and 22.76, 28.11, 30.91 and 32.16, 44.06 and 44.19, 51.72,
81.61, 109.78, 129.55 and 129.74, 150.84 and 151.14, 173.23
and 173.50; MS (DCI(NH3)) 257 (100) [M+ + NH3 + H]. Anal.
Calcd for C12H17NO4: C, 60.24; H, 7.16. Found: C, 60.46; H,
7.17. III: mixture of (rac)-5 and dimethyl maleate (3.72 g, Rf
) 0.09). This mixture was treated with cyclopentadiene (2.5
mL) in CH2Cl2 (10 mL) for 12 h and purified by flash
chromatography (hexanes/ethyl acetate 10:1) to yield 1.37 g
(3%) pure (rac)-5 as a colorless oil, which slowly crystallized
from a mixture CH2Cl2/cyclohexane at -20 °C (mp ) 90 °C):
1H NMR (250 MHz, CDCl3) δ 1.40 (s, 9H), 1.71-1.73 (m, 2H),
2.28-2.32 (m, 2H), 3.31-3.33 (m, 1H), 3.45-3.48 (m, 1H), 3.58
(s, 3H), 3.62 (s, 3H); 13C NMR (62.9 MHz, CDCl3) δ 26.62,
27.40, 27.58, 28.12, 28.85, 42.03, 51.77, 80.92, 154.14, 170.50,
170.75; HRMS (DCI(NH3)) calcd for [C15H21NO6]+ 311.1373.
Found 311.1369.
(b) Scr een in g for Lip a ses (Bip h a sic System ). 10 mM
solutions of (rac)-4 and (rac)-7, respectively, in toluene were
used. 800 µL of substrate solution, 1200 µL potassium phos-
phate buffer (0.1 M, pH 7.4) and 25 mg lipase (see Tables 1
and 2) were stirred at ambient temperature. During the
investigated period of time three samples of each 250 µL were
taken out of the reaction mixture. After centrifugation the
toluene phase was separated, 75 µL of the aqueous phase was
acidified with 9 µL of 1.0 M phosphoric acid to pH 2.5-3.5
and immediately extracted with 150 µL of ethyl acetate. The
conversion of the reaction was monitored by analysis of the
organic phase with qualitative TLC.
Gen er a l P r oced u r e for th e Deter m in a tion of th e
Sp ecific Activity a n d En a n tiom er ic Ra tio (E Va lu e) of
Active En zym es for th e Hyd r olysis of (r a c)-4 a n d (r a c)-
7. (a ) R ea ct ion s Ca r r ied ou t in Aq u eou s Su sp en sion
(Ester a ses). Substrate suspensions: 42.0 mg (154 µmol) of
finely powdered (rac)-7 was suspended with stirring in 20.0
mL of potassium phosphate buffer (0.1 M, pH 7.4). 45.0 mg
(188 µmol) of (rac)-4 was dissolved in 125 µL of acetone, and
For large-scale preparations (rac)-4 can also be obtained in
pure form by distillation (10-4 Torr, 70-80 °C) after removal
of the catalyst by filtration on silica gel.
(1R*,2R*,3R*)-N-ter t-Bu toxyca r bon yl-N-for m yl-3-a m i-
n ocyclop r op a n e-1,2-d ica r b oxylic Acid Dim et h yl E st er
(20) Boehringer Mannheim GmbH. Instructions to Chirazyme lipases
& esterases screening set 1996.
(21) Horgan, D. J .; Stoops, J . K.; Webb, E. C.; Zerner, B. Biochem-
istry 1969, 8, 2000-2006.
(22) Merck, E. Anfa¨rbereagenzien fu¨r Du¨nnschicht und Papier-
Chromatographie; E. Merck: Darmstadt, 1970; p 29.

â-Aminocyclopropanecarboxylic Acids
J . Org. Chem., Vol. 65, No. 26, 2000 8967
500 µL of a solution of Triton X-100 in water (10 g/L) was
added to stabilize the emulsion. 2.5 mL of potassium phosphate
buffer (1.0 M, pH 7.4) and water were added to a final volume
of 25 mL (final buffer concentration 0.1 M). This mixture was
vigorously shaken and then ultrasonicated for 15 min. During
ultrasonication, (rac)-4 precipitated to form a white colored,
stable suspension.
Bioconversions: 20 mL of the substrate suspensions was
kept at 25 °C, and the reactions were started by addition of
the investigated enzyme. For the hydrolysis of (rac)-4 (i) 150
U of PLE A, (ii) 70000 U of L-1, (iii) 1440 U of L-2, and (iv)
80000 U L-6 were used. 150 U of PLE A were used for the
hydrolysis of (rac)-7.
The pH of the reaction mixtures was maintained at 7.5 by
pH-stat-controlled addition of 0.1 M aqueous NaOH. The
conversion was calculated from the amount of base consumed
during the reaction.
The enantioselectivity of the kinetic resolution of (rac)-7,
catalyzed by PLE and L-2, was investigated after extracting
500 µL samples with the same amount of cyclohexane. The
organic layer was analyzed by chiral GC.
The product mixture 8 and 9 of the PLE-catalyzed hydroly-
sis of (rac)-7 (100% conversion) was isolated as follows: After
adjusting the pH to 8.0 by addition of 0.1 M aqueous NaOH
the reaction mixture was extracted with Et2O. The aqueous
solution was acidified to pH 2.5 by addition of 1.0 M phosphoric
acid. After saturation with NaCl, the mixture was extracted
with Et2O (3x), and the combined organic layers were extracted
with brine and water. The organic layer was dried over MgSO4
and concentrated in vacuo to yield 36.3 mg (91%) of a mixture
of 8 and 9 (60:40 by NMR and HPLC) as a slightly yellow oil
which crystallized during storage for several weeks at room
temperature.
together, and water was added to a final volume of 500 mL
(final buffer concentration 0.1 M).
En zym e Solu tion . 120 mg of L-2 was thoroughly sus-
pended in 2 mL of 0.1 M potassium phosphate buffer (pH 7.4)
and left overnight at 4 °C to form a clear solution.
2-Azabicyclo[3.1.0]h ex-3-en e-2,6-dicar boxylic Acid 2-ter t-
Bu tyl Ester 6-Meth yl Ester ((-)-4). In a 1 L round-bottom
flask 360 mL of buffer and a solution of 1.0 g (4.18 mmol) of
(rac)-4 in 40 mL of toluene were mixed together and vigorously
stirred. The reaction was started by addition of the enzyme
solution prepared above, and the resulting mixture was stirred
at ambient temperature with protection from sunlight. Samples
of 200 µL were diluted with 200 µL of toluene and the organic
phase was analyzed by Chiral-GC. After 196 h (+)-4 was
completely hydrolyzed. The reaction mixture was acidified to
pH 3.0 by addition of 1.0 M phosphoric acid followed by
addition of NaCl (125 g). The mixture was immediately
extracted with Et2O (4×). The combined organic layers were
extracted with brine and several times with 1 M KHCO3
solution until TLC analysis indicated the absence of 10 in the
organic layer. The organic layer was washed with water, dried
over MgSO4, and purified by filtration using a short column
of silica gel (20 g, Et2O as eluent) to yield 335 mg (33%) of
(-)-4 as an amber-colored oil that crystallized upon storage
at -20 °C, ee ) 99.6% (HPLC, GC, for conditions and retention
times see general experimental part): [R]21D ) -254.2 (c 1.007,
CH2Cl2). For further analytical data cf. preparation of (rac)-4.
The combined aqueous layers were extracted with cyclo-
hexane, adjusted carefully to pH 3.0 by addition of aqueous
HCl (2.5 M) and 1.0 M phosphoric acid, and the resulting
suspension was extracted with Et2O. The combined organic
layers were dried and concentrated to yield 462 mg (49%) (+)-
10 as a colorless solid, ee ) 43.3% (HPLC, for conditions and
The specific activity of PLE A for the conversion of (rac)-7
was determined to be 0.21 U/mg.
retention times see the general methods), [R]25 ) +107.3 (c
D
0.122, CHCl3).
(b) Rea ction s Ca r r ied ou t in a Bip h a sic System (Li-
p a ses). A 10 mM solution of (rac)-4 in toluene and a solution
of 62.5 mg lipase (L-1, L-2, L-6) in 3000 µL potassium
phosphate buffer (0.1 M, pH 7.4) were prepared. Eight aliquot
starting mixtures, each consisting of 300 µL of lipase solution
and 200 µL of substrate solution ((rac)-4 in toluene), were
stirred at ambient temperature and worked up after different
reaction times as follows: The reaction mixture was diluted
with 1000 µL of toluene and acidified to pH 2.5-3.5 by addition
of 30 µL of 1.0 M phosphoric acid. After vigorous shaking and
subsequent centrifugation the toluene layer was analyzed by
quantitative TLC for the determination of the conversion and
by GC for the determination of the enantiomeric excess of 4.
Op tim iza tion of th e Rea ction Con d ition s for th e L-2-
Ca ta lyzed Hyd r olysis of (r a c)-4. General procedure: (for
detailed amounts and solvents see the Supporting Information,
Table 4): 5 mg (21 µmol) of (rac)-4 was dissolved in the
respective solvent. An aqueous solution of Triton X-100 in
water (10 g/L), potassium phosphate buffer (pH 7.4) and a
freshly prepared solution of L-2 in 0.1 M phosphate buffer,
pH 7.4 (60 mg/mL) were added. After homogenization by
vigorous shaking, the reaction mixture was stirred at ambient
temperature. During the investigated period of time samples
of 100 µL each were taken out of the freshly homogenized
reaction mixture. After addition of 500 µL of Et2O and 10 µL
of 1.0 M phosphoric acid, the resulting mixture was shaken
vigorously and then centrifugated. 100 µL of the resulting Et2O
layer was transferred to a HPLC-vial and the solvent was
removed by passing over a gentle stream of nitrogen. The
resulting semi-dry sample was dried in vacuo (14 mbar) and
1000 µL of HPLC solvent (see the general methods) were added
subsequently. After shaking for 1 h at room temperature on a
thermostatic mixer the sample was analyzed by chiral HPLC
for determination of the conversion and the enantiomeric
excesses of 4 and 10. For the optimization of the pH, phosphate
buffers of pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 were used
instead of the phosphate buffer, pH 7.4.
P r ep a r a tive Biocon ver sion of (r a c)-4 by L-2. Su bstr a te
solu tion : 1.067 g of (rac)-4 was dissolved in 43 mL of
cyclohexane.
En zym e solu tion : 60 mg of L-2 was thoroughly supended
in 1000 µL of 0.1 M potassium phosphate buffer, pH 7.0, and
left for 1 h at 4 °C to form a clear solution.
2-Azabicyclo[3.1.0]h ex-3-en e-2,6-dicar boxylic Acid 2-ter t-
Bu tyl Ester ((+)-10). The substrate solution was added to
384 mL of 0.1 M potassium phosphate buffer, pH 5.0 and the
biphasic mixture was stirred vigorously in a 1 L round-bottom
flask. The reaction was started by addition of the enzyme
solution and the reaction vessel was protected from sunlight
and stirred at ambient temperature. Samples of 100 µL were
diluted with 200 µL of toluene, 15 µL of 1 M KHCO3 solution
were added, and the organic phase was analyzed by GC (which
is faster than HPLC). After 19.5 h 4 displayed an enantiomeric
excess of eeS ) 48.4% corresponding to an calculated eeP of
90.3% (E ) 31.8). The reaction was stopped by addition of 10
mL of 1.0 M phosphoric acid and 300 mL of Et2O. The layers
were separated and the organic phase was washed with
saturated NaCl solution and extracted with Et2O. The com-
bined organic layers were extracted several times with 50 mL
portions of 1 M KHCO3 solution until TLC indicated absence
of acid 10 in the organic phase. The ether phase was finally
washed with water, dried over MgSO4 and concentrated to
yield 711 mg (67%) (-)-4 as an amber-colored oil: ee ) 48.5%
(GC, HPLC); [R]25 ) -114.5 (c 0.220, CHCl3).
D
The combined KHCO3 layers were washed with Et2O,
acidified to pH 3.5 by addition of aqueous HCl (2.0 M) and to
pH 3.0 by addition of 1.0 M phosphoric acid. The resulting
aqueous solution of acid (+)-10 was subsequently extracted
with Et2O. The combined ether layers were dried over MgSO4
and concentrated to yield 303 mg (30%) (+)-10 as a colorless
solid. 91.0% ee (GC, determined as methyl ester (+)-4 after
derivatization with trimethylsulfonium hydroxide). Upon re-
crystallization of 100 mg of (+)-10 from hexane/ethyl acetate
18 mg (+)-10 crystallized with 60% ee, leaving 82 mg of
P r epar ative Biocon ver sion of (r a c)-4 Usin g L-2. Bu ffer .
50 mL of 1.0 M potassium phosphate buffer (pH 7.4) and 11.1
mL of an aqueous solution of Triton X-100 (10 g/L) were mixed
enantiomerically pure (+)-10 (>96% ee) in the mother liquor:
1
[R]25 ) +255.1 (c 0.127, CHCl3); H NMR (250 MHz, CDCl3,
D
signal doubling because of rotamers) δ 0.92 (br s, 1H), 1.48

8968 J . Org. Chem., Vol. 65, No. 26, 2000
Beumer et al.
and 1.51 (s, 9H), 2.87 (br s, 1H), 4.31-4.50 (m, 1H), 5.36-
5.40 (m, 1H), 6.45-6.61 (m, 1H), 11.87 (br s, 1H); 13C NMR
(62.9 MHz, CDCl3, signal doubling because of rotamers) δ
22.95, 28.15, 31.87 and 33.10, 44.77 and 45.00, 82.02, 109.83,
129.73 and 129.98, 150.96 and 151.15, 179.18 and 179.75;
HRMS (DCI(NH3)) calcd for [C11H15NO4]+ 225.1006, found
225.1001.
+ H]. Anal. Calcd for C12H17NO6: C, 53.13; H, 6.32; N, 5.16.
Found: C, 53.02; H, 6.29; N, 5.02; [R]21 ) -48.7 (c 1.004,
D
CH2Cl2).
(1S,2S,3S)-N-ter t-Bu t oxyca r b on yl-N-for m yl-3-a m in o-
cyclop r op a n e-1,2-d ica r boxylic Acid 2-Mon om eth yl Ester
(12). A solution of 11 (2.25 g, 8.23 mmol) in CH3CN (25 mL)
was cooled to 0 °C, and under rigorous stirring a solution of
666 mg KH2PO4 in 6.7 mL of water and 0.84 mL of H2O2 (30%)
were added. After addition of a solution of 1.67 g of NaClO2
(technical, 80%) in 16.7 mL water, the reaction solution stirred
until ending of gas evolution (ca. 2 h). Then, 830 mg of Na2SO3
was added to destroy excess NaClO2 and the solution stirred
for 1 h. After addition of 20 mL of 1 M KHSO4, the solution
was extracted with ethyl acetate. The combined organic layers
were dried over Na2SO4. Evaporation of the solvent afforded
2.28 g (7.92 mmol, 96%) 12 as a colorless oil: 1H NMR (250
MHz, CDCl3) δ 1.51 (s, 9H), 2.59 (dd, J ) 7.7, 5.9 Hz, 1H),
2.65 (dd, J ) 5.9, 5.1 Hz, 1H), 3.22 (dd, J ) 7.7, 5.1 Hz, 1H),
3.77 (s, 3H), 9.12 (s, 1H); 13C NMR (62.9 MHz, CDCl3) δ 27.74,
27.82, 29.52, 35.83, 52.68, 85.19, 151.87, 163.33, 169.89,
173.47; HRMS (DCI(NH3)) calcd for [C12H18NO7]+ 288.1076,
(1R,2R,3S)-N-ter t-Bu t oxyca r b on yl-3-a m in ocyclop r o-
p a n e-1,2-d ica r boxylic Acid 2-Mon om eth yl Ester (8). To
a solution of (+)-10 (461 mg, 2.05 mmol) in MeOH (35 mL)
was added NaHCO3 (344 mg, 4.1 mmol), and the mixture was
stirred for 25 min at room temperature (gas evolution) before
treatment with ozone at -78 °C until the solution maintained
a blue color. After removal of the excess ozone by passing
oxygen through the solution, the solution was allowed to warm
to room temperature overnight. The solvent was evaporated,
and to the residue was added benzene (2 × 20 mL) before
evaporating again. The residue was dissolved in CH2Cl2 (35
mL), cooled to -78 °C, and Ac2O (1.0 mL) and NEt3 (2.0 mL)
were added. The solution was allowed to warm to room
temperature overnight again, diluted with CHCl3 and ex-
tracted with 1 M KHSO4 solution. The organic layer was dried
over Na2SO4 and concentrated. The residue was dissolved in
CH3CN (10 mL) and DEAEA (0.83 mL, 5.8 mmol) was added.
The solution stirred for 24 h at room temperature. The solvent
was evaporated and the residue was dissolved in 20 mL ethyl
acetate. After acidification with 20 mL of 1 M KHSO4 solution,
the aqueous layer was extracted with ethyl acetate. The
combined organic layers were dried over Na2SO4 and concen-
trated to yield 352 mg (66%) 8 as a colorless solid (> 96% ee
according to chiral HPLC analysis; mp ) 147 °C): 1H NMR
(250 MHz, CDCl3) δ 1.42 (s, 9H), 2.19 (br s, 1H), 2.46-2.52
(m, 1H), 3.74 (s, 3H), 3.83 (br s, 1H), 5.78 (br s, 1H), 11.34 (br
s, 1H); 13C NMR (62.9 MHz, CDCl3) δ 25.80, 28.17, 28.95,
37.38, 52.53, 80.94, 156.10, 170.60, 173.61; MS (DCI(NH3)) 277
(100) [M+ + NH3 + H]. Anal. Calcd for C11H17NO6: C, 50.96;
found 288.1083; [R]21 ) -66.9 (c 0.999, CH2Cl2).
D
(1S,2S,3R)-N-ter t-Bu t oxyca r b on yl-3-a m in ocyclop r o-
p a n e-1,2-d ica r boxylic Acid 1-Ben zyl Ester 2-Meth yl Es-
ter (13). To a solution of (ent)-9 (1.91 g, 7.35 mmol) in DMF
(37 mL) were added NaHCO3 (1.24 g, 14.7 mmol) and benzyl
bromide (1.38 g, 8.09 mmol). After being stirred for 48 h at
room temperature, the solution was diluted with 100 mL of
ethyl acetate and 100 mL of water. The organic layer was
separated, and the aqueous layer was extracted with ethyl
acetate. The combined organic layers were extracted with
water and dried over MgSO4. The solvent was evaporated and
the residue was purified by flash chromatography (hexanes/
ethyl acetate 5:1, Rf ) 0.38) to afford 2.46 g (96%) 13 as a
colorless solid (mp ) 93 °C): 1H NMR (250 MHz, CDCl3) δ
1.43 (s, 9H), 2.28 (dd, J ) 5.0, 5.0 Hz, 1H), 2.51 (dd, J ) 8.3,
5.0 Hz, 1H), 3.68 (s, 3H), 3.85 (br s, 1H), 5.11 (d, J ) 12.2 Hz,
1H), 5.20 (d, J ) 12.2 Hz, 1H), 5.55 (br s, 1H), 7.36 (s, 5H);
13C NMR (62.9 MHz, CDCl3) δ 26.24, 28.17, 28.66, 37.48, 52.24,
67.22, 80.21, 128.26, 128.44, 128.57, 135.03, 155.27, 169.70,
170.01; MS (DCI(NH3)) 367 (100) [M+ + NH3 + H]. Anal. Calcd
for C18H23NO6: C, 61.88; H, 6.64. Found: C, 61.93; H, 6.67.
H, 6.61. Found: C, 51.06; H, 6.72. [R]21 ) +48.7 (c 0.980,
D
CH2Cl2).
(1S,2S,3S)-N-ter t-Bu t oxyca r b on yl-3-a m in ocyclop r o-
p a n e-1,2-d ica r boxylic Acid 2-Mon om eth yl Ester ((en t)-
9). To a solution of 12 (1.13 g, 3.93 mmol) in CH3CN (30 mL)
was added DEAEA (1.15 mL, 8.06 mmol). The reaction was
stirred for 24 h at room temperature. The solvent was
evaporated, and the residue was dissolved in ethyl acetate (15
mL). The solution was acidified with 15 mL of 1 M KHSO4
and extracted with ethyl acetate. The combined organic layers
were dried over Na2SO4 and concentrated to yield 889 mg
(87%) (ent)-9 as a colorless solid (mp ) 139 °C): 1H NMR (250
MHz, CDCl3, signal doubling because of rotamers) δ 1.46 (s,
9H), 2.33 (dd, J ) 4.9, 4.9 Hz, 1H), 2.41-2.46 (m, 1H), 3.42
and 3.88 (br s, 1H), 3.71 (s, 3H), 5.54 and 6.81 (br s, 1H), 11.61
(br s, 1H); 13C NMR (62.9 MHz, CDCl3) δ 26.78, 28.18, 29.44,
37.50, 52.34, 82.37, 158.30, 170.46, 172.06; MS (DCI(NH3)) 536
(22) [2M+ + NH3 + H], 277 (100) [M+ + NH3 + H]. Anal. Calcd
for C11H17NO6: C, 50.96; H, 6.61. Found: C, 51.17; H, 6.56.
[R]21 ) -8.9 (c 1.000, CH2Cl2).
D
(1S,2S,3S)-N-ter t-Bu t oxyca r b on yl-2-[1′(S)-(4′′-ch lor o-
p h en yl)et h ylca r b a m oyl]-3-a m in ocyclop r op a n eca r b ox-
ylic Acid Meth yl Ester (14). To a solution of (ent)-9 (100
mg, 0.39 mmol) in THF (10 mL) were added pentafluorophe-
nole (142 mg, 0.77 mmol), (S)-p-chlorophenylethylamine (66
µL, 0.43 mmol), and EDC (148 mg, 0.77 mmol). The mixture
was stirred for 20 h at room temperature. The reaction mixture
was diluted with ethyl acetate and then washed with saturated
NaHCO3, 1 M KHSO4, and saturated NaHCO3 and dried over
Na2SO4. The solvent was evaporated, and the resulting residue
was purified by flash chromatography (hexanes/ethyl acetate
3:1, Rf ) 0.14) to provide 14 (147 mg, 95%) as a colorless solid
(mp ) 149 °C): 1H NMR (250 MHz, CDCl3) δ 1.40 (s, 9H),
1.47 (dd, J ) 6.9, 3H), 2.22-2.32 (m, 2H), 3.68 (s, 3H), 3.73
(br s, 1H), 5.02-5.11 (m, 1H), 5.89-5.92 (m, 1H), 6.50-6.53
(m, 1H), 7.21-7.32 (m, 4H); 13C NMR (62.9 MHz, CDCl3) δ
21.74, 27.55, 27.68, 28.28, 37.72, 48.80, 52.23, 80.09, 127.44,
128.88, 133.27, 141.30, 155.75, 167.55, 171.17; MS (DCI(NH3))
414 (38) [M+ + NH3 + H], 397 (100) [M+ + H], 793 (39) [2M+
+ H]. Anal. Calcd for C19H25N2O5Cl: C, 57.51; H, 6.44; N, 7.11.
[R]23 ) -22.5 (c 1.021, CH2Cl2).
D
(1S,2S,3S)-N-ter t-Bu toxyca r bon yl-N-for m yl-2-for m yl-
3-a m in ocyclop r op a n eca r boxylic Acid Meth yl Ester (11).
A solution of (-)-4 (800 mg, 3.34 mmol) in CH2Cl2 (50 mL)
was treated with ozone at -78 °C until the solution maintained
a blue color. After removal of the excess ozone by passing
oxygen through the solution, dimethyl sulfide (1.04 g, 16.7
mmol) was added and the solution was stirred for 12 h at room
temperature. Caution: It is important that the time of reduc-
tion is sufficiently long, otherwise the reduction is incomplete.
After evaporation of the solvent the residue was purified by
flash chromatography (hexanes/ethyl acetate 5:1, Rf ) 0.19)
to yield 827 mg of a colorless oil, which was crystallized from
Et2O (1.1 mL)/cyclohexane (2.9 mL) to afford 717 mg (79%)
11 as a white solid (mp ) 59 °C): 1H NMR (250 MHz, CDCl3)
δ 1.52 (s, 9H), 2.75 (dd, J ) 6.0, 4.8 Hz, 1H), 2.96 (ddd, J )
8.2, 6.0, 2.3 Hz, 1H), 3.20 (dd, J ) 8.2, 4.8 Hz, 1H), 3.75 (s,
3H), 9.07 (s, 1H), 9.54 (d, J ) 2.3 Hz, 1H); 13C NMR (62.9 MHz,
CDCl3) δ 27.68, 27.77, 34.86, 36.56, 52.50, 85.29, 151.83,
163.31, 169.91, 193.02; MS (DCI(NH3)) 289 (100) [M+ + NH3
Found: C, 57.50; H, 6.35; N, 7.06. [R]21 ) -112.4 (c 1.020,
D
CH2Cl2).
(1R,2R,3R)-N-ter t-Bu t oxyca r b on yl-3-a m in ocyclop r o-
p a n e-1,2-d ica r boxylic Acid 1-Ben zyl Ester 2-Meth yl Es-
ter (16). To a solution of 8 (86 mg, 0.332 mmol) in DMF (2
mL) were added NaHCO3 (31 mg, 0.365 mmol) and benzyl
bromide (0.58 g, 0.332 mmol). After being stirred for 48 h at
room temperature, the solution was diluted with ethyl acetate
and water. The organic layer was separated, and the aqueous
layer was extracted with ethyl acetate. The combined organic
layers were extracted with water and dried over Na2SO4. The
solvent was evaporated and the residue was purified by flash

â-Aminocyclopropanecarboxylic Acids
J . Org. Chem., Vol. 65, No. 26, 2000 8969
chromatography (hexanes/ethyl acetate 5:1, Rf ) 0.38) to afford
85 mg (74%) 16 as a colorless solid (>98% ee according to chiral
HPLC analysis mp ) 80 °C): 1H NMR (250 MHz, CDCl3) δ
1.43 (s, 9H), 2.28-2.32 (m, 1H), 2.49 (dd, J ) 8.4, 5.2 Hz, 1H),
3.73 (s, 3H), 3.88 (br s, 1H), 5.08 (d, J ) 12.2 Hz, 1H), 5.14 (d,
J ) 12.2 Hz, 1H), 5.53 (br s, 1H), 7.29-7.39 (m, 5H); 13C NMR
(62.9 MHz, CDCl3) δ 26.18, 28.18, 28.75, 37.48, 52.43, 67.13,
80.24, 128.37, 128.43, 128.57, 135.13, 155.30, 169.53; MS (DCI-
(NH3)) 367 (100) [M+ + NH3 + H]. Anal. Calcd for C18H23NO6:
C, 61.88; H, 6.64; N, 4.01. Found: C, 61.77; H, 6.68; N, 3.98.
Note Ad d ed in P r oof: After the submission of this
manuscript, a study on the enzymatic resolution of
cyclopropane-cis-1,2-dicarboxylic methyl ester followed by
Curtius degradation to arrive at cis-2-aminocyclopropane-
carboxylic acid derivatives has been published: Martin-
Vila, M.; Muray, E.; Aguada, G. P.; Alvarez-Larena, A.;
Branchadell, V.; Minguillon, C.; Giralt, E.; Ortuno, R. M.
Tetrahedron: Asymmetry 2000, 11, 3569.
[R]21 ) +10.2 (c 1.000, CH2Cl2).
D
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra of all new compounds together with X-ray crystal-
lographic details for 14. Detailed information on sources for
enzymes and on optimization protocols for kinetic resolution
of (rac)-4. This material is available free of charge via the
Internet at http://pubs.acs.org.
Ack n ow led gm en t. This work was supported by the
Deutsche Forschungsgemeinschaft (RE948-1/4) and the
Fonds der Chemischen Industrie. Dr. M. Zabel, Depart-
ment of Inorganic Chemistry, University of Regensburg,
is gratefully acknowledged for carrying out the X-ray
analysis of 14. We thank Boehringer Mannheim for
their generous gift of enzymes
J O005541L