Enantiopure trans -3-arylaziridine-2-carboxamides: Preparation by bacterial hydrolysis and ring-openings toward enantiopure, unnatural D -α-amino acids

Article abstract of DOI:10.1021/jo101377j

Several racemic trans-3-arylaziridine-2-carboxamides were prepared and then resolved by Rhodococcus rhodochrous IFO 15564-catalyzed hydrolysis. The resulting enantiopure (2R,3S)-3-arylaziridine-2-carboxamides are adequate substrates to undergo fully stereoselective nucleophilic ring-openings at the C-3 ring position to finally yield enantiopure, unnatural d-α- aminocarboxylic acids. Experimental evidence is provided that suggests the fate of the (2S,3R)-3-arylaziridine-2-carboxylic acids concomitantly formed during the resolution processes. In this context, the similar bacterial resolution of racemic 1-arylaziridine-2-carboxamides and -carbonitriles, previously investigated by our research group, has been partially re-examined.

Full text of DOI:10.1021/jo101377j

pubs.acs.org/joc
Enantiopure trans-3-Arylaziridine-2-carboxamides: Preparation
by Bacterial Hydrolysis and Ring-Openings toward Enantiopure,
Unnatural D-r-Amino Acids†
ꢀ
Roberto Moran-Ramallal, Ramon Liz, and Vicente Gotor*
ꢀ
´ ´
Departamento de Quımica Organica e Inorganica and Instituto Universitario de Biotecnologıa de Asturias,
ꢀ
Universidad de Oviedo, E-33071 Oviedo, Spain
ꢀ
vgs@fq.uniovi.es
Received July 13, 2010
Several racemic trans-3-arylaziridine-2-carboxamides were prepared and then resolved by Rhodococcus
rhodochrous IFO 15564-catalyzed hydrolysis. The resulting enantiopure (2R,3S)-3-arylaziridine-2-carbox-
amides are adequate substrates to undergo fully stereoselective nucleophilic ring-openings at the C-3 ring
position to finally yield enantiopure, unnatural D-R-aminocarboxylic acids. Experimental evidence is
provided that suggests the fate of the (2S,3R)-3-arylaziridine-2-carboxylic acids concomitantly formed
during the resolution processes. In this context, the similar bacterial resolution of racemic 1-arylaziridine-
2-carboxamides and -carbonitriles, previously investigated by our research group, has been partially
re-examined.
Introduction
antitumor, or other biological activities.1b,e Moreover, enantio-
pure or enantioenriched aziridines have recently received a great
deal of attention as precursors of bioactive nitrogen-containing
molecules, mainly through their varied regio- and stereoselective
ring-opening reactions,1a,b,d,6 some of which meet most of the
stringent criteria for a process to earn click chemistry status.7
As to their ring-opening processes, aziridines are usually
classified as activated (those bearing an electron-withdraw-
ing N-substituent such as acyl, sulfonyl, and phosphoryl) or
unactivated (mainly the NH-, N-alkyl-, and N-arylaziridines).
Ring-opening reactions of activated aziridines are easier and
hence much more widely investigated than those of their unacti-
vated counterparts, but suffer the drawback of requiring a
further deprotection step of the amide or carbamate moiety
present in their open products. Among recent examples of
The current interest in aziridine chemistry is reflected in a
series of reviews published during the past 16 years, ranging from
those dealing with synthesis and/or reactivity1 to other, more
recent and specialized reviews covering areas such as nitrogen
transfer reactions,2 parallel- and solid-phase synthesis,3 and
C-heteroatom-substituted aziridines4 (including the C-lithiated
varieties5). Several aziridine compounds exhibit antibiotic,
†
ꢀ
ꢀ
Dedicated to the memory of the late Professor Jose Manuel Concellon.
(1) (a) Tanner, D. Angew. Chem., Int. Ed. Engl. 1994, 33, 599–619.
(b) Osborn, H. M. I.; Sweeney, J. Tetrahedron: Asymmetry 1997, 8, 1693–
1715. (c) Stamm, H. J. Prakt. Chem. 1999, 341, 319–331. (d) McCoull, W.;
Davis, F. A. Synthesis 2000, 1347–1365. (e) Sweeney, J. B. Chem. Soc. Rev.
€
2002, 31, 247–258. (f) Muller, P.; Fruit, C. Chem. Rev. 2003, 103, 2905–2919.
(g) Hu, X. E. Tetrahedron 2004, 60, 2701–2743. (h) Zhang, Y.; Lu, Z.; Wulff,
W. D. Synlett 2009, 2715–2739. (i) Sweeney, J. Eur. J. Org. Chem. 2009,
4911–4919. (j) Lu, P. Tetrahedron 2010, 66, 2549–2560.
(2) Watson, I. D. G.; Lily, Y; Yudin, A. K. Acc. Chem. Res. 2006, 39,
194–206.
(5) Hodgson, D. M.; Bray, C. D.; Humphreys, P. G. Synlett 2006, 1–22.
(6) (a) Cardillo, G.; Gentilucci, G.; Tolomelli, A. Aldrich. Acta 2003, 36,
39–50. (b) Lee, W. K.; Ha, H.-J. Aldrich. Acta 2003, 36, 57–63. (c) Pineschi,
M. Eur. J. Org. Chem. 2006, 4979–4988.
(3) Olsen, C. A.; Franzyk, H.; Jaroszewski, J. W. Eur. J. Org. Chem. 2007,
1717–1724.
(4) Singh, G. S.; D’hooghe, M.; De Kimpe, N. Chem. Rev. 2007, 107,
2080–2131.
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2001, 40, 2004–2021.
6614 J. Org. Chem. 2010, 75, 6614–6624
Published on Web 09/02/2010
DOI: 10.1021/jo101377j
r
2010 American Chemical Society

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Moran-Ramallal et al.
JOCArticle
enantiopure unactivated aziridine’s ring-openings, those of
the following substrates are worth noting: a sizable number
of N-[(R or S)-(1-phenylethyl)]-2-substituted aziridines, fre-
quently converted into bioactive products by Ha, Lee, and
co-workers;6b,8 several (10S,2S)-N-alkyl-2-[1-(dibenzylamino)-
SCHEME 1. Our Previous Results in the Bacterial Resolution
of Aziridines 1 and 2
alkyl]aziridines opened with a set of heteronucleophiles by
ꢀ
9
Concellon and co-workers; a 2-(2-pyridyl)aziridine derived
from (S)-valinol also opened with a number of heteronucleo-
philes;10 and two 2-arylaziridines that underwent formal [3þ2]
cycloaddition processes.11 Several groups have also investi-
gated intramolecular ring-openings that are in fact aziridine
expansion processes.6a,12
Our interest in this chemistry has recently been demon-
strated by the preparation of a number of enantiopure
N-alkyl- and N-arylaziridine-2-carboxamides by bacterial
resolution and their further ring-openings, as well as those of
some of their derivatives, with external13 and internal14 nucleo-
philes. Two weeks after our first paper, M.-X. Wang and co-
workers also reported similar bacterial resolution of N-aryl-
aziridine-2-carbonitriles and subsequent ring-openings of the
resulting N-arylaziridine-2-carboxamides;15 while about eight
months later they extended their work mainly to trans-3-aryl-
N-methylaziridine-2-carbonitriles and -carboxamides.16 After
comparing our work, carried out using the commercial bacterium
Rhodococcus rhodochrous IFO 15564,17 and that of Wang et al.,
who use the commercially unavailable strain Rhodococcus
erythropolis AJ270,18 several coincidences emerge, but also
an important discrepancy (see first paragraph in the Results
and Discussion section).
In this paper we shall extend our work to trans-3-aryl-
aziridine-2-carboxamides, which, besides having similar sizes
to our first substrates, lack the N-substituent, which would
lend greater versatility to the possible enantiopure products
resulting from their resolutions and further ring-openings.
Moreover, with the aim of clarifying the cited discrepancy,
some of our first substrates13 will be again resolved and then
worked up in an almost identical way to that of Wang et al.16
previous results.13,14 The kinetic resolution of the amides 1 and
the nitriles 2 by hydrolysis in the presence of R. rhodochrous
IFO 15564 affords very good yields of the enantiopure amides 1,
although the corresponding aziridine-2-carboxylic acids, 3,
were never observed or isolated, in agreement with several
prior observations.19 However, besides the enantiopure or
almost enantiopure amides 1, Wang et al. claimed to obtain,
in their R. erythropolis AJ270-mediated hydrolysis of a
number of 1-arylaziridine-2-carbonitriles 2,20 very good yields
of optically active (85 to >99.5% ee) methyl 1-arylaziridine-
2-carboxylates, which proceed from the in situ treatment of
the corresponding acids 3 with diazomethane.15
As our experimental method and that of Wang and co-
workers are not identical, we detected the following differ-
ences that might perhaps explain the previously mentioned
divergent results: (1) Our substrate:whole cells wet weight ratio
was approximately 2 mmol/1 g, whereas that of Wang et al.
was 1 mmol/1 g. (2) In the isolation of the enantiopure
amides 1, we continuously extracted the supernatant liquids
resulting from the biotransformations (CH2Cl2, 40 °C, 6 h),
whereas Wang et al. extracted them manually (AcOEt, rt).
(3) For access to the amino acids 3, Wang et al. freeze-dried
the remaining aqueous phases, whereas we concentrated
them in vacuo with slight heating (<40 °C). Our slight
heating in the above steps 2 and 3 could accelerate a possible
decomposition of acids 3. Additionally, our higher substrate:
whole cells ratio, together with an apparent lower activity of
our strain, implies longer biotransformation times in our case,
which could favor a hypothetical metabolism of 3 by the
bacterium.
Results and Discussion
Bacterial Resolution of 1-Alkyl- and 1-Arylaziridine-2-
carboxamides 1 and -carbonitriles 2. Scheme 1 shows our
(8) (a) Yoon, H. J.; Kim, Y.-W.; Lee, B. K.; Lee, W. K.; Kim, Y.; Ha,
H.-J. Chem. Commun. 2007, 79–81. (b) Kim, Y.; Ha, H.-J.; Yun, S. Y.; Lee,
W. K. Chem. Commun. 2008, 4363–4365.
ꢀ
ꢀ
(9) Concellon, J. M.; Bernad, P. L.; Suarez, J. R.; Garcı
az, M. R. J. Org. Chem. 2005, 70, 9411–9416, and their own references
therein.
(10) Savoia, D.; Alvaro, G.; Di Fabio, R.; Gualandi, A. J. Org. Chem.
´
a-Granda, S.;
Dı
´
2007, 72, 3859–3862.
We therefore decided to repeat the R. rhodochrous IFO
15564-mediated resolution of one of our common nitrile
substrates, (()-2d, in such a way that the incubation time was
similar and the workup identical to those of Wang et al.
Thus, 1.8 g wet weight of our bacterium added to 50 mL of
potassium phosphate buffer (0.10 M, pH 7.0) produced a
suspension (A650 = 15.1)21 able to transform (28 °C, 45 min)
(11) Zhu, W.; Cai, G.; Ma, D. Org. Lett. 2005, 7, 5545–5548.
(12) (a) Hori, K.; Nishiguchi, T.; Nabeya, A. J. Org. Chem. 1997, 62,
3081–3088. (b) Ferraris, D.; Drury, W. J., III; Cox, C.; Lectka, T. J. Org.
Chem. 1998, 63, 4568–4569. (c) Tomasini, C.; Vecchione, A. Org. Lett. 1999,
1, 2153–2156. (d) Lu, Z.; Zhang, Y.; Wulff, W. D. J. Am. Chem. Soc. 2007,
129, 7185–7194.
ꢀ
(13) Moran-Ramallal, R.; Liz, R.; Gotor, V. Org. Lett. 2007, 9, 521–524.
ꢀ
(14) Moran-Ramallal, R.; Liz, R.; Gotor, V. Org. Lett. 2008, 10, 1935–
1938.
(15) Wang, J.-Y.; Wang, D.-X.; Zheng, Q.-Y.; Huang, Z.-T.; Wang,
M.-X. J. Org. Chem. 2007, 72, 2040–2045.
(16) Wang, J.-Y.; Wang, D.-X.; Pan, J.; Huang, Z.-T.; Wang, M.-X.
J. Org. Chem. 2007, 72, 9391–9394. The trans stereodescriptor refers to the
substituents at the C-2 and C-3 positions.
(19) (a) Lambert, C.; Viehe, H. G. Tetrahedron Lett. 1985, 26, 4439–4442.
ꢀ
(b) Martres, M.; Gil, G.; Meou, A. Tetrahedron Lett. 1994, 35, 8787–8790.
(c) Kumar, H. M. S.; Rao, M. S.; Chakravarthy, P. P.; Yadav, J. S.
Tetrahedron: Asymmetry 2004, 15, 127–130.
(17) Available from: (a) Institute for Fermentation, Osaka, Japan, IFO
ꢀ
~
15564 strain; (b) Coleccion Espanola de Cultivos Tipo, Universidad de
Valencia, Burjassot, Spain, CECT 5949 strain.
(18) Both strains show nitrile hydratase and amidase activities.
(20) R = Ph, 4-F-C6H4, 4-Cl-C6H4, 4-Br-C6H4, 4-MeO-C6H4, 4-Me-C6H4,
3-Me-C6H4.
(21) A650: absorbance or optical density measured at 650 nm.
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SCHEME 2. CAL-B-Catalyzed Hydrolysis of Ethyl (()-trans-
1-(p-Tolyl)aziridine-2-carboxylate
hydrolyses of (()-1a and (()-2a, both in the presence and in
the absence (blank reaction) of the IFO 15564 strain. A
conventional workup after the blank process leads to a 9:1
molar mixture of benzylamine and 5a, whereas the workup
after the former reaction yields benzylamine and several
unidentified products, none of which were detected (NMR
analysis) in the crude materials obtained after the standard
biotransformations of 1a and 2a.
Synthesis and Bacterial Resolution of trans-3-Arylaziridine-
2-carboxamides 8. Several failed attempts of resolving
1,2,3-trisubstituted aziridines [e.g., trans-1-benzyl-3-(trifluoro-
methyl)aziridine-2-carboxamide and cis-1-benzyl-3-phenyla-
ziridine-2-carbonitrile],23 which were recovered unchanged
after more than one week of standard incubation with
R. rhodochrous IFO 15564,13 convinced us that they are too
bulky to be accepted by the bacterial enzymes. Thus, we
turned our attention to the 2,3-disubstituted NH-aziridines
6-9. Besides their interesting features already pointed out
for 8, these substrates closely resemble 3-aryloxirane-2-carbo-
nitriles and -carboxamides, which were successfully hydrolyzed
in the presence of the Rhodococcus AJ270 strain.24 Moreover,
when our work was already in progress, the aforementioned
resolution with this last strain of trans-3-aryl-1-methylazir-
idine-2-carbonitriles and -carboxamides was reported, being
only slightly bulkier than our intended new substrates, as was
that of trans-3-phenylaziridine-2-carboxamide, 8a,16 a set of
processes thatwill allow us toestablish new points of compari-
son between the behaviors of the two bacterial strains.
0.30 mmol of 2d and to produce, after conventional room-
temperature extraction of the supernatant liquid, the corre-
sponding almost enantiopure amide (1R,2S)-1d (25% yield,
95% ee), virtually exactly as reported by Wang et al.15 The
remaining aqueous phase was lyophilized (-50 to -60 °C,
2 ꢀ 10-4 atm) and the resulting residue powdered and treated
with a freshly prepared ethereal, yellow diazomethane solu-
tion (20 min, below -15 °C). No discoloring of this solution
was observed and, finally, no residue was obtained after
filtration and further solvent removal. A similar process
starting from the amide (()-1c produced (1R,2S)-1c (40%
yield, 96% ee) and a very small amount [6% yield, probably
(1S,2R)] of the enantiopure methyl ester derived from 3c.
Consequently, and given that no or very limited amounts of
methyl esters were obtained, we conclude that amino acids 3,
which necessarily have to be formed due to bacterial amidase
activity, are essentially unstable under the conditions of our
biotransformations.
On the other hand, besides the data in the literature,19 we
can adduce clear evidence for the inherent instability of
amino acids 3. In fact, we carried out the Candida antarctica
lipase catalyzed (CAL-B) hydrolysis (28 °C, 3 h) of the
racemic ethyl ester 3c-Et (0.50 mmol) in THF (4 mL) in the
presence of a minimal amount of water (53 μL), thus avoid-
ing the extraction and freeze-drying steps of bacterial bio-
transformations. The crude residue obtained by further
in vacuo concentration at room temperature was treated with
ethereal diazomethane without discoloring. After chromato-
graphic purification, we were able to isolate the optically
active ethyl ester (20% yield, 78% ee), but not the methyl
ester 3c-Me proceeding from the concomitantly formed
amino acid 3c (Scheme 2). Moreover, this methyl ester was
not observed (1H NMR) in the crude residue or in other
chromatographic fractions.
Although we do not know the decomposition route of the
amino acids 3, it seems clear that it is different from the well-
established pathway for some related oxirane-2-carboxylic
(glycidic) acids.22 Should it be so (eq 1), they would produce
imines 5 or their hydrolysis products, which was not the case.
To prove that these putative products would survive after
our biotransformations, we prepared N-ethylidenebenzyl-
amine, 5a (R = PhCH2), and exposed it to the same experi-
mental conditions, and even reaction times, as our bacterial
In principle, any of the racemic substrates 6-9 would
have been adequate to begin the study. However, owing to
the well-known low enantiodiscrimination displayed by the
nitrile hydratase of R. rhodochrous IFO 15564,13 we prepared
only two nitrile substrates, (()-6a and (()-7a (Ar = Ph).25
As expected, our attempts at resolving 6a during the nitrile
hydratase-catalyzed step (6a f 8a) were disappointing, the
reaction being so fast that it was necessary to employ a
bacterial suspension in potassium phosphate buffer of
A650 = 0.3 instead of the standard suspension (A650 =3.0).
(23) Stereodescriptors cis/trans describe here the relative positions of the
C-2 and C-3 substituents.
(24) Wang, M.-X.; Lin, S.-J.; Liu, C.-S.; Zheng, Q.-Y.; Li, J.-S. J. Org.
Chem. 2003, 68, 4570–4573.
(25) By addition of bromine to cinnamonitrile followed by Gabriel-
Cromwell reaction between the obtained 2,3-dibromo-3-phenylpropanenitrile
and ammonia (solution in methanol), diastereomers 6a and 7a are easily
separable by flash column chromatography.
(22) Singh, S. P.; Kagan, J. J. Org. Chem. 1970, 35, 2203–2207, and
references therein. However, this pathway does seem to work in the bacterial
hydrolysis of 3-arylaziridine-2-carboxamides; see later.
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SCHEME 3. Preparation of (()-trans-3-Arylaziridine-2-car-
boxamides 8
TABLE 1. Bacterial Preparation of Enantiopure trans-3-Arylaziridine-
2-carboxamides 8
product
Ar
time (h)
yielda (%)
(2R,3S)-8a
(2R,3S)-8b
(2R,3S)-8c
(2R,3S)-8d
(2R,3S)-8e
(2R,3S)-8f
(2R,3S)-8g
Ph
15
12
37
17.5
14.5
18.5
6.25
46
46
44
47
42
40
40
4-F-C6H4
3-F-C6H4
2-F-C6H4
4-Cl-C6H4
4-Br-C6H4
4-Me-C6H4
aIsolated yield after flash column chromatography.
Under these conditions, and stopping the hydration step after
27% conversion of nitrile, the amide (2S,3R)-8a was obtained
with an enantiomeric excess of only 56%, which is indicative
of a poorly enantioselective process (enantiomeric ratio,26
E = 4).27 The hydration step of the cis-nitrile 7a was much
slower and required an A650 6.0 bacterial suspension for it to
progress adequately within a reasonable reaction time. Even
so, its enantiomeric ratio was also very low (E = 12). These two
disappointing experiments counseled us to finally discard the
nitrile hydratase-catalyzed biotransformation of the nitrile
substrates 6 and 7 as a valid resolution method.
A Staudinger reaction32 of the β-azido alcohols 11 led to the
trans-aziridine esters 12 through cis-1,3,2-oxazaphospholidine
intermediates;33 the retention of the trans geometry in com-
pounds 12 relies on the fact that both chiral centers in 10
undergo inversion during the first (C-3) and the second step
(C-2). Finally, a conventional ester ammonolysis gave rise to
the amides 8. The trans nature of all aziridinecarboxamides
(()-8 was confirmed by means of the invaluable coupling
constants between their aziridinic protons, the 1H NMR
signals of which are invariably broad singlets, whereas those
of similar protons for cis-3-phenylaziridine-2-carboxamide,
(()-9a, are doublets with a coupling constant of 7.0 Hz.
Under conditions as particularly mild as those for sub-
strates 1 and 2 (0.10 M phosphate buffer suspensions of
R. rhodochrous IFO 15564, A650 = 3.0, 28 °C, pH 7.0),13
trans-3-arylaziridine-2-carboxamides (()-8a-g underwent
efficient hydrolytic resolutions within hours (Table 1). The
reactions were stopped after chiral HPLC analysis showed
the presence of only one out of the two enantiomers of the
corresponding aziridinecarboxamide, 8. All the enantiopure
amides were obtained in this way with good yields, given that
the upper limit for any kinetic resolution is 50% yield. Only
the reaction with the 3-(p-anisyl) substrate, (()-8h, failed:
in the presence of phosphate buffer, and even in the absence
of the bacterium, it led to a complex mixture of products,
from where only one product could be identified (see later).
Bearing in mind the results of our first study,13 no efforts
were made to resolve the corresponding trans-3-arylaziridine-
2-carbonitriles (()-6 through the consecutive actions of the
bacterial nitrile hydratase and amidase, as we were convinced
thatthefinalyieldswouldnotbeimproved. ComparingScheme1
and Table 1, it can seemingly be deduced that there was an
inversion of the enantiopreferences shown by the R. rhodochrous
IFO 15564 amidase toward amides 1 (2R preference) and
As to the racemic cis-amides 9, we only prepared (()-9a
(Ar= Ph) by means of a somewhat unexpected method,
namely, the exposure of an A650 6.0 suspension of R. rhodochrous
IFO 15564 to the cis-nitrile (()-7a during five hours, after
which the cis-amide 9a was obtained with 89% yield as a
racemate, asprovenby chiral HPLC analysis. Thisexperiment
demonstrates that the cis-amide 9a is not accepted as a
substrate by the amidase of R. rhodochrous IFO 15564,28
a
feature that this microorganism shares with R. erythropolis
AJ270.16 We hence assumed that other cis-amides 9 would not
be resolved by our bacterium.
The access to racemic trans-amides 8 is shown in Scheme 3.
The methyl or ethyl trans-3-arylglycidates (()-10 are com-
mercially available or easily accessible through a fully dia-
stereoselective Darzens reaction between methyl chloroacetate
and the corresponding substituted benzaldehyde.29 The ring-
opening of epoxides 10 with sodium azide30 took place with
complete regioselectivity at the C(3) benzylic position.31
(26) (a) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
€
Soc. 1982, 104, 7294–7299. (b) Faber K.; Honig, H.; Kleewein, A. http://
www.cis.TUGraz.at/orgc.
(27) This calculation is only approximate, as a small fraction of the
obtained amide disappears as a result of the amidase activity of the
bacterium.
(28) Of course, the direct exposure of cis-amide (()-9a to R. rhodochrous
IFO 15564 led to the quantitative recovery of the substrate after one week.
(29) Tranchant, M.-J.; Dalla, V. Tetrahedron 2006, 62, 10255–10270.
(30) Boruwa, J.; Borah, J. C.; Kalita, B.; Barua, N. C. Tetrahedron Lett.
2004, 45, 7355–7358.
(32) Lin, F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.; Bertozzi,
C. R. J. Am. Chem. Soc. 2005, 127, 2686–2695.
(33) Xiong, C.; Wang, W.; Cai, C.; Hruby, V. J. J. Org. Chem. 2002, 67,
1399–1402.
(31) Legter, J.; Thijs, L.; Zwanenburg, B. Tetrahedron Lett. 1989, 30,
4881–4884.
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SCHEME 5. Proposed Pathway for the Conversion of (2S,3R)-
13g into 14g
FIGURE 1. Coincidental geometries of the enantiomers of 1 and 8
transformed (not isolated) by the R. rhodochrous IFO 15564 amidase.
8 (2S preference). However, a 180° rotation around the axis
that bisects the N1-C2-C3 angle in aziridines (2S,3R)-8
clearly shows that the amidase actually selected enantiomers
with an identical geometry in both cases (Figure 1), a feature
that Wang et al. have also highlighted with respect to the
behavior of their AJ270 amidase.16 Although the topology of
the active site of the IFO 15564 amidase remains unknown,
this observation, together with its inability to accept cis-amide
9a and trans-1-benzyl-3-(trifluoromethyl)aziridine-2-carbox-
amide, suggests that it contains an elongated rather than a
globular pocket and that the factors that control the fixation
of the substrates in it are mainly steric in nature.
As in the case of aziridinecarboxamides 1, in the biotransfor-
mations of substrates 8 we were not able to isolate the expected
amino acids 13 either. Nevertheless, on this occasion we did
obtain some evidence regarding one of their possible decom-
position pathways. In fact, the residue resulting from the fastest
biotransformation, that of (()-trans-3-(p-tolyl)aziridine-2-
carboxamide, (()-8g, consisted of a 71:29 molar mixture of
(2R,3S)-8g and 2-(p-tolyl)ethanol, 14g, which were finally iso-
lated with 40% and 17% yields, respectively (Scheme 4).
of Rhodococcus,35 and also R. rhodochrous different from the
IFO 15564 strain,36 catalyze the reduction of ketones. More-
over, R. rhodochrous IFO 15564 itself is able to enantioselec-
tively oxidize secondary alcohols,37 thus making its opposite
reductase activity conceivable. To corroborate the last step
proposed in Scheme 5, we therefore incubated 0.308 mmol of
commercial phenylacetaldehyde 16a [the maximum desir-
able amount according Scheme 5 after a standard biotrans-
formation of 0.616 mmol of (()-8a] with our bacterium
under the same conditions as the cited standard biotransfor-
mations. In fact, the aldehyde disappeared after 3.5 h and
2-phenylethanol, 14a, was finally isolated in 65% yield (eq 2).
SCHEME 4. Bacterial Resolution of (()-8g
It should be emphasized that we observed only the forma-
tion of a 2-arylethanol, 14g, at the end of the fastest (6.25 h)
bacterial resolution (that of the aziridinecarboxamide 8g,
Scheme 4). From the complex mixture obtained after only six
hours in the unsuccessful resolution of (()-trans-3-(p-anisyl)-
aziridine-2-carboxamide, (()-8h, we were also consistently
able to isolate the corresponding 2-(p-anisyl)ethanol, 14h. All
these data hint that 2-arylethanols 14 are metabolized by the
IFO 15564 bacterium, a process that might ultimately be
responsible for the usual elusiveness of the amino acids 13.
Reality, however, seems to be somewhat more complex. In
fact, the standard incubation during 24 h of 0.308 mmol of
commercial 2-phenylethanol, 14a, with R. rhodochrous IFO
15564 resulted in the recovery of 0.222 mmol of 14a as the
sole reaction product, which implies that approximately one-
quarter of the alcohol was metabolized. This metabolic
activity is very similar to that observed when 14a was added
in 14 successive portions to the bacterial suspension, an
experiment designed to mimic the supposed progressive
formation of 14a during the bacterial hydrolysis of (()-
trans-3-phenylaziridine-2-carboxamide, 8a. However, the
apparent metabolic activity of the IFO 15564 strain versus 14a
Seeing as the sum of both yields exceeds the 50% threshold,
which is the maximum for the enantiopure amide, the unexpected
alcohol must proceed from the elusive acid (2S,3R)-13g. This
process can be rationalized assuming a decomposition path-
way (Scheme 5) similar to that described for some glycidic acids,22
a routefound tobeineffectivefor the above-mentioned acids3
(eq 1). The resulting unstable imine 15g would hydrolyze
in the phosphate buffer to p-tolylacetaldehyde, 16g, which
would in turn be converted into the alcohol 14g by the action
of a reductase from R. rhodochrous IFO 15564.
To the best of our knowledge, no reductase activity has
been previously reported for the IFO 15564 cells, even in the
presence of suitable substrates such as β-keto nitriles and
β-keto amides.34 However, it is well known that other species
(35) (a) Stampfer, W.; Kosjek, B.; Moitzi, C.; Kroutil, W.; Faber, K.
Angew. Chem., Int. Ed. 2002, 41, 1015–1017. (b) Stampfer, W.; Kosjek, B.;
Faber, K.; Kroutil, W. J. Org. Chem. 2003, 68, 402–406. (c) Matsuda, T.;
Yamanaka, R.; Nakamura, K. Tetrahedron: Asymmetry 2009, 20, 513–557.
(36) Patel, R. N.; Robison, R. S.; Szarka, L. J.; Kloss, J.; Thottathil, J. K.;
Mueller, R. H. Enzyme Microb. Technol. 1991, 13, 906–912.
(37) Ohtsuka, Y.; Katoh, O.; Sugai, T.; Ohta, H. Bull. Chem. Soc. Jpn.
1997, 70, 483–491.
(34) Gotor, V.; Liz, R.; Testera, A. M. Tetrahedron 2004, 60, 607–618.
6618 J. Org. Chem. Vol. 75, No. 19, 2010

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SCHEME 6. Assignment of the Absolute Configuration of 8a
SCHEME 7. Ring-Opening of (2R,3S)-8a with Methanol
deduced from eq 2 would be much higher (35% of material
disappearing after only 3.5 h). This observation implies that
our bacterium not only reduces the aldehyde 16a but also
metabolizes it to a certain extent. Moreover, in the overall
process of decomposition of amino acids 13, it should be
conceivable that the imines 15 and even the amino acids
themselves were also partially metabolized. Thus, the excep-
tional observation of the alcohol 14g after the standard
bacterialhydrolysis of 8gcouldbe attributed to a combination
of fast hydrolysis and (in all probability) slow metabolism.
Interestingly enough, another fact worth noting is that the amino
acid 13a was not observed after the R. erythropolis AJ270-catalyzed
hydrolysis of (()-8a, nor were the expected corresponding amino
acids in the AJ270-mediated hydrolysis of a number of racemic
3-aryl-1-methylaziridine-2-carboxamides.16 Therefore, after consid-
ering the aforementioned numerous similarities between our strain
and the AJ270 bacterium, we lack a plausible explanation for the
different fates of amino acids 3 in the resolutions of the
amides 1 and nitriles 2 catalyzed by both bacteria.
of interesting properties and applications such as, inter alia,
their incorporation to semisynthetic proteins40 and ther-
apeutic peptides.41 Although ring-openings of 3-unsubsti-
tuted aziridinecarboxamides 1 occur at the N-C(2) or N-
C(3) bond depending on the used nucleophile and reaction
conditions,13,15 it is to be expected that those of (2R,3S)-8a
mainly or exclusively take place with N-C(3) breaking
owing to the benzylic nature of the C-3 aziridinic position
and in close analogy with the ring-openings of 1-methyl-
3-phenylaziridine-2-carboxamide.16 Should this be so, the
R configuration of the C-2 chiral center will be retained,
which would smooth the way for access to (R)-2-amino-
carboxylic acids, i.e., D-R-amino acids, as will be shown next.
These increasingly attractive types of nearly unnatural amino
acids, whose consistently low abundance in human tissues
has been related to renal and neurodegenerative diseases,42
have been used, for instance, in the construction of short
all-D antimicrobial peptides with considerable resistance to
enzymatic degradation, in the synthesis of anti-HIV drugs,43
and in the replacement of L-amino acid residues in depsi-
peptides to enhance their activity against colon cancer.44
Ring-Opening of (2R,3S)-8a with Methanol. The use of this
nucleophile at 55 °C, in the presence of 1.1 equiv of boron
trifluoride-diethyl ether (1:1), resulted in the fully regio-
selective attack at the C-3 position, which led quantitatively to
(2R,3R)-2-amino-3-methoxy-3-phenylpropanamide, (2R,3R)-
17 (Scheme 7).45 The enantiopurity of (2R,3R)-17 was con-
firmed by chiral HPLC analysis of its N-Boc derivative,
(2R,3R)-18. To reach the corresponding D-R-amino acid,
The (2R,3S)-absolute configuration of 8a was assigned
by its chemical correlation with methyl (2R,3S)-3-phenyl-
aziridine-2-carboxylate, (2R,3S)-12a, whose [R]20D value is -290
(c 1.1, CHCl3).38 With this aim in view, (()-12a (R =
Me; prepared as shown in Scheme 3) was enzymatically
resolved by CAL-B-catalyzed aminolysis with benzylamine,
which affords an optically active sample of 12a (R = Me)
showing [R]20 = -63.0 (23% ee), i.e., (2R,3S)-configured
D
(Scheme 6). A further conventional ammonolysis of the ester
function in (2R,3S)-12a (R = Me) necessarily led, without
racemization, to (2R,3S)-8a, with a [R]20 value of -61.0.
D
Given that the amide 8a obtained by bacterial hydrolysis was
also levorotatory, its (2R,3S) configuration is thus con-
firmed. The R configuration of the C-2 chiral center in the
enantiopure aziridine 8g will be established later (Scheme 12);
moreover, its trans geometry fixes its entire (2R,3S) configu-
ration. In view of the close similarity between 8a,g and the
remaining trans-aziridinecarboxamides 8, we also assume
the same (2R,3S) configuration for all of them.
Ring-Openings of Enantiopure Aziridinecarboxamide 8a.
Previous reports13,15,16 have shown that ring-openings of
aziridine-2-carboxamides constitute efficient ways to access
unnatural amino acids,39 a compound class with a wide profile
(40) (a) Wang, L.; Schultz, P. G. Angew. Chem., Int. Ed. 2005, 44, 34–66.
(b) Pellois, J.-P.; Muir, T. W. Curr. Opin. Chem. Biol. 2006, 10, 487–491.
(c) Parrish, A. R.; Wang, W.; Wang, L. Curr. Opin. Neurobiol. 2006, 16, 585–
592.
(41) Sato, A. K.; Viswanathan, M.; Kent, R. B.; Wood, C. R. Curr. Opin.
Biotechnol. 2006, 17, 638–642.
(42) Hamase, K.; Morikawa, A.; Zaitsu, K. J. Chromatogr., B: Anal.
Technol. Biomed. Life Sci. 2002, 781, 73–91.
(43) (a) Wakayama, M.; Yoshimune, K.; Hirose, Y.; Moriguchi, M.
J. Mol. Catal. B: Enzym. 2003, 23, 71–85. (b) Nakatani, S.; Hidaka, K.;
Ami, E.; Nakahara, K.; Sato, A.; Nguyen, J.-T.; Hamada, Y.; Hori, Y.;
Ohnishi, N.; Nagai, A.; Kimura, T.; Hayashi, Y.; Kiso, Y. J. Med. Chem.
2008, 51, 2992–3004.
(44) Otrubova, K.; Styers, T. J.; Pan, P.-S.; Rodrıguez, R.; McGuire,
´
K. L.; McAlpine, S. R. Chem. Commun. 2006, 1033–1034.
(45) The regiochemistry of all the open products in this paper was
elucidated by NMR-HMBC experiments (see the Supporting Information).
For instance, a three-bond correlation was observed between the ortho
carbon atoms and the C-3 proton in 17, but not between the ortho carbon
atoms and the C-2 proton.
(38) Gentilucci, L.; Gijzen, Y.; Thijs, L.; Zwanenburg, B. Tetrahedron
Lett. 1995, 36, 4665–4668.
ꢀ
ꢀ
(39) Boto, A.; Gallardo, J. A.; Hernandez, D.; Hernandez, R. J. Org.
Chem. 2007, 72, 7260–7269, and references therein.
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SCHEME 8. Ring-Opening of (2R,3S)-8a with Water
SCHEME 9. Ring-Opening of (2R,3S)-8a with Sodium Azide
(3R)-β-methoxy-D-phenylalanine, (2R,3R)-19 (75% overall
yield), the amide and carbamate functions in (2R,3R)-18
were hydrolyzed, with further treatment with propylene
oxide. The optical rotation of the obtained 19, [R]20
=
Ring-Opening of (2R,3S)-8a with Sodium Azide. The ring-
opening of enantioenriched aziridines with the azide anion is
a useful way for obtaining optically active vicinal diamines.
The preparation of these compounds is currently a goal of
outstanding importance because of their biological proper-
ties, medicinal interest, and versatility in organic synthesis.50
Owing to the lack of N-substituent, the azide attack to
(2R,3S)-8a will generate, after reduction of the azide group,
the same open structure (2,3-diamino-3-phenylpropanamide)
irrespective of the C-2 or C-3 point of attack. However, the
formation of D-R-amino acid derivatives requires the configu-
ration of the C-2 chiral center to be retained, i.e., the attack at
C-3. Our previous reported methodology for the ring-opening
of the 3-unsubstituted aziridinecarboxamide (1R,2S)-1a with
azide anion afforded, in the presence of aluminum trichloride, a
2.5:1 mixture of the C-2 and C-3 opening products,13 which
would be inappropriate for our current interest. Then, although
the existence of a 3-phenyl substituent in (2R,3S)-8a could
favor the C-3 attack, we decided to assay other reaction
conditions for enhancing C-3 regioselectivity in (1R,2S)-1a.
The treatment of this aziridine with sodium azide (3 equiv) in
refluxing acetonitrile and in the presence of 1.1 equiv of boron
trifluoride-diethyl ether (1:1) thus led exclusively to the C-3
opening product.51 By applying this methodology to (2R,3S)-
8a,52 we obtained (2R,3R)-2-amino-3-azido-3-phenylpropana-
mide, (2R,3R)-22, with a very good yield (Scheme 9). A further
reaction of this intermediate product with di-tert-butyl dicar-
bonate afforded the azido(carbamoyl)carbamate (2R,3R)-23, a
synthetic equivalent of an orthogonally protected vic-diamine,
as proven by its reversion into (2R,3R)-22 via acid hydrolysis
and its hydrogenolysis to (2R,3R)-3-amino-2-(tert-butoxy-
carbonylamino)-3-phenylpropanamide, (2R,3R)-24, which
was finally obtained with an overall yield of 78%. The enantio-
purity of the carbamate (2R,3R)-23 (HPLC) proved that the
initial ring-opening took place fully stereoselectively, which
is consistent with an SN2 attack of the azide anion.
D
-37.3 (c 0.60, H2O), compared with that reported for
D
(2S,3S)-19, [R]20 = þ34.5 (c 0.6, H2O),46 together with
the obligatory retention of configuration of the C-2 aziridinic
chiral center, allows us to assign the 2R,3R configuration to
19, which is consistent with an inversion of configuration at
C-3 as a result of an SN2 attack.
Ring-Opening of (2R,3S)-8a with Water. The reaction of
compounds 8 with water constitutes an evident way for
accessing β-amino alcohol derivatives. The β-amino alcohol
(also known as 1,2-amino alcohol) moiety is a common
structural component in a vast group of natural products,
synthetic pharmacologically active molecules, and ligands or
chiral auxiliaries.47 Moreover, β-amino alcohols are suitable
intermediates for the synthesis of unnatural amino acids,
β-blockers, insecticidal agents, and antibiotics.48
The ring-opening of (2R,3S)-8a with water failed in the
presence of boron trifluoride-diethyl ether (1:1) or trifluo-
roacetic acid, but was successful when employing p-toluene-
sulfonic acid (1.1 equiv) as activating agent at 55 °C(Scheme8).
To facilitate the purification process, the crude material
was treated in situ with di-tert-butyl dicarbonate, after which
only the product proceeding from an initial nucleophilic
attack at the C-3 ring position, (2R,3R)-2-(tert-butoxy-
carbonylamino)-3-hydroxy-3-phenylpropanamide, (2R,3R)-20,
was isolated with a 70% overall yield. The enantiopurity of
this carbamate (HPLC) demonstrates that the ring-opening
was completely stereoselective. Finally, simultaneous hydro-
lysis of the amide and carbamate moieties in (2R,3R)-20 led to
the (3R)-β-hydroxy-D-phenylalanine hydrochloride, (2R,3R)-
21, whose absolute configuration was assigned by comparison
of the sign of its specific rotation, [R]20D = -59.3 (c 1.0, 6 M
HCl), with a recently reported value in the literature.49
ꢀ
(46) Hansen, D. B.; Joullie, M. M. Tetrahedron: Asymmetry 2005, 16,
3963–3969. These authors also describe the diastereomer (2S,3R)-19, whose
[R]20D = -18.4 (c 0.4, H2O), considered in its absolute value, reinforces our
assignment of configuration.
(47) (a) Ager, D. J.; Prakash, I.; Schaad, D. R. Chem. Rev. 1996, 96, 835–
875. (b) Bergmeier, S. C. Tetrahedron 2000, 56, 2561–2576.
(50) (a) Lucet, D.; Le Gall, T.; Mioskowski, C. Angew. Chem., Int. Ed.
1998, 37, 2580–2627. (b) Kim., H.; So, S. M.; Kim, B. M.; Chin, J. Aldrich.
Acta 2008, 41, 77–88.
(48) (a) Corey, E. J.; Zhang, F.-Y. Angew. Chem., Int. Ed. 1999, 38, 1931–
1934. (b) Bose, D. S.; Narsaiah, A. V. Bioorg. Med. Chem. 2005, 3, 627–630.
(c) Yadav, J. S.; Reddy, A. R.; Narsaiah, A. V.; Reddy, B. V. S. J. Mol. Catal.
A: Chem. 2007, 261, 207–212. (d) Clerici, A.; Ghilardi, A.; Pastori, N.; Punta,
C.; Porta, O. Org. Lett. 2008, 10, 5063–5066.
ꢀ
(51) Moran Ramallal, R. Ph.D. dissertation, University of Oviedo, 2009.
(52) If NaN3 is previously dissolved in the minimum amount of water, the
process is much faster, with no competition of water as nucleophile. The
reaction was carried out at 55 °C, seeing as it was very slow at room
temperature; on the other hand, it afforded a complex mixture of products
in refluxing MeCN.
(49) Davies, F. A.; Srirajan, V.; Fanelli, D. L.; Portonovo, P. J. Org.
Chem. 2000, 65, 7663–7666. [R]20D = -63.2 (c 0.65, 6 M HCl).
6620 J. Org. Chem. Vol. 75, No. 19, 2010

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SCHEME 10. Assignment of the Absolute Configuration of 22
The (2R,3R)-configuration of the open product 22 was
assigned by chemical correlation between the monoprotected
diamino amide (2R,3R)-24 and its corresponding methyl ester,
(2R,3R)-27 (Scheme 10), assuming that, as in other similar
transformations, the configurations of the chiral centers not
involved in the successive reactions in Schemes 9 and 10 are
retained. The first step in Scheme 10 is the same as in Scheme 6,
although the resulting ester showed a slightly lower ee value in
this case. The azide ring-opening of the nonracemic aziridinic
methyl ester, accomplished this time in the presence of alumi-
num trichloride, occurred exclusively at the N-C(3) bond to
generate (2R,3R)-25; the inversion of configuration at the C-3
chiral center will be made evident forthwith. The protection of
this amino ester led to carbamate 26, whose retained ee value
demonstrated that no racemization took place until this point.
Given that the following azide hydrogenolysis cannot affect the
chiral center’s configurations either, it must produce the mono-
protected diamino ester (2R,3R)-27. This hypothesis was confirmed
by its specific rotation, [R]20D = -7.5 (c 0.6, CHCl3), the sign of
which is opposite that previously reported for its enantiomer.53
Finally, conventional ammonolysis of the ester function in
(2R,3R)-27 has to necessarily produce the amide (2R,3R)-24,
whose levorotatory nature coincides with that of the same amide
in Scheme 9, thus completing the initial objective of this paragraph.
Ring-Opening of (2R,3S)-8a,g by Catalytic Hydrogenolysis.
Among the reported catalytic hydrogenolyses of aziridines,
those of compounds 28 and 29 (Figure 2), by Ha, Lee, and co-
workers,6b are worthy of note. These researchers ascribe their
observed differences to the presence of an electron-withdrawing
group at the C-2 position [N-C(2) breaking in 28] or to the
absence of such a substituent [N-C(3) breaking in 29]. Our
aziridines 8 bear a weaker electron-withdrawing group at C-2,
the hydrogenolysis of (2R,3S)-8a takes place exclusively with
N-C(3) breaking.16 Quite logically, the catalytic hydrogeno-
lyses of 2-arylaziridines1g and, specifically, those of 3-arylazir-
idine-2-carboxylic acid esters 3054 occur at their correspond-
ing benzylic positions.
In our hands, however, under virtually the same reaction
conditions (balloon pressure of hydrogen, 10% Pd/C, room
temperature) as those employed by Wang and co-workers,
the crude material obtained starting from (2R,3S)-8a con-
sisted mainly of the expected product, (R)-2-amino-3-phenyl-
propanamide, (R)-31a, though accompanied by a slight amount
of 3-phenylpropanamide, 33a55 (25:1 molar ratio, respectively;
Scheme 11). As it is not conceivable that 33a might proceed from
(R)-31a, it is necessary to accept its formation via the unexpected
and not observed open product (R)-32a, the substituted benzyl-
amine nature of which means that it may be easily hydro-
genolyzed under our reaction conditions.
Two further steps (amine protection and double hydrolysis)
successively converted (R)-31a into the carbamate (R)-34a
(which proved to be enantiopure by chiral HPLC analysis) and
then into D-phenylalanine hydrochloride, (R)-35a. The R con-
figuration of the latter product, established by means of its
specific rotation, [R]20D = þ8.8 (c 1.1, H2O),56 together with
the enantiopurity of (R)-34a, proves that the majority ring-
opening of (2R,3S)-8a took place with full retention of config-
uration at C-2, a final point of divergence with Wang et al., who
reported obtaining (R)-31a with only 90% ee.16 This observa-
tion suggests that we tentatively assign the R configuration to the
non-observed direct product of the minority ring-opening, 32a.
A methodology identical to that contained in the above
scheme was applied to the (2R,3S)-trans-3-(p-tolyl)aziridine-
2-carboxamide, (2R,3S)-8g (Scheme 12). On this occasion,
3-(p-tolyl)propanamide, 33g, proceeding from the minority
N-C(2) breaking was observed only as nonquantifiable trace
amounts in the crude material after hydrogenolysis and has,
subsequently, been excluded from Scheme 12. The overall pro-
cess allows us to obtain another D-R-amino acid, β-(p-tolyl)-D-
alanine hydrochloride, (R)-35g, [R]20D = þ10.0 (c 1.0, H2O),57
FIGURE 2. Catalytic hydrogenolysis of several aziridines, arrows
denoting the broken bonds. (The stereochemistry of the chiral
centers is not shown.)
(54) Davis, F. A.; Deng, J.; Zhang, Y.; Haltiwanger, R. C. Tetrahedron
2002, 58, 7135–7143, and references therein.
(55) The identity of this byproduct was established by comparison with an
authentic sample prepared by (1) catalytic hydrogenation of methyl cinnamate
and (2) conventional ammonolysis of the ester function.
and moreover, their C-3 position is benzylic in nature. It is
therefore not surprising that Wang et al. have reported that
(56) The value [R]20D = -8.1 (c 0.91, H2O) has been previously reported
for (S)-35a: Barfoot, C. W.; Harvey, J. E.; Kenworthy, M. N.; Kilburn, J. P.;
Ahmed, M.; Taylor, R. J. K. Tetrahedron 2005, 61, 3403–3417.
(57) The value [R]20 = -6.5 (c 0.1, 0.5 M HCl) has been previously
D
(53) Capone, S.; Guaragna, A.; Palumbo, G.; Pedatella, S. Tetrahedron
2005, 61, 6575–6579. [R]20D = þ29.0 (c 0.9, CHCl3) for (2S,3S)-27.
reported for (S)-35g: Zhuze, A.; Lost, K.; Kasafirek, E.; Rudinger J. Collect.
Czech. Chem. Commun. 1964, 29, 2648–2662.
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SCHEME 11. Catalytic Hydrogenolysis of (2R,3S)-8a
Experimental Section58
SCHEME 12. Catalytic Hydrogenolysis of (2R,3S)-8g
Biotransformations of (()-8a-g with Rhodococcus rhodochrous
IFO 15564. A solution of the corresponding substrate (()-8a-g
(100 mg; 0.42-0.62 mmol) in 2.0 mL of EtOH (some substrates
required a slight heating for total solution) was added to a bacterial
suspension (A650 = 3.0) in fresh 0.10 M potassium phosphate buffer
pH 7.0 (100 mL). Incubation (rotary shaker, 200 rpm, 28 °C) was
started, 500 μL aliquots being extracted (AcOEt) from time to time
and analyzed by chiral HPLC until one of the two enantiomers of
the corresponding amide 8 disappeared. The cells were then dis-
carded by centrifugation (5000 rpm, 3 min). The supernatant liquid
was filtered through a pad of diatomaceous earth and then con-
tinuously extracted with CH2Cl2 (6 h). After drying the organic
phase, low-pressure removal of the solvent yielded the correspond-
ing, essentially pure white solid (2R,3S)-8, which was further puri-
fied by silica gel column chromatography (1:3 hexane-AcOEt as
eluent). To the best of our knowledge, only the amide (2R,3S)-8a has
been previously prepared.16
in 79% overall yield. As the initial configuration of the C-2
aziridinic chiral center remains unchanged throughout the pro-
cess, this result, together with the trans geometry of the starting
8g, proves the (2R,3S) configuration of this aziridine, as was
already anticipated elsewhere in this paper.
(2R,3S)-3-(p-Chlorophenyl)aziridine-2-carboxamide [(2R,3S)-8e]:
yield 42%; white solid; mp 178.8-180.5 °C. [R]20D = -212.2 (c 0.6,
MeOH). ee>99.5%. IR (CH2Cl2): 3450, 3367, 1665 cm-1
.
1H NMR (CD3OD, 400.13 MHz): 7.36 (d, Hc þ He, 3J = 8.5 Hz),
3
7.31 (d, Hb þ Hf, J = 8.5 Hz), 3.19 (br s, H3), 2.63 (br s, H2).
In summary, a mild bacterial resolution method of a series of
N-unsubstituted trans-3-arylaziridine-2-carboxamides has been
developed that produces good yields of the slower reacting
enantiomers in enantiopure form. The faster reacting enan-
tiomers were converted into unstable aziridine carboxylic
acids, the decomposition pathway of which, elucidated in
some cases, implies a previously unknown reductase activity
of the used bacterial strain, Rhodococcus rhodochrous IFO
15564. The ring-opening reactions of the enantiopure azir-
idine amides with methanol, water, and azide anion, as well
as by catalytic hydrogenolysis, not only proceed with full
stereoselectivity and practically complete regioselectivity at
their C-3 position but also lead to enantiopure high added
value products, unnatural D-R-amino acids. The absolute
configurations of the hydrolysis and ring-opening reac-
tion products have been unequivocally assigned by means
of chemical correlations and complementary experiments.
Moreover, a retrospective experiment, the enzymatic hydro-
lysis of a 1-arylaziridine-2-carboxylic acid ethyl ester, demon-
strates that 1-arylaziridine-2-carboxylic acids also are
inherently unstable.
13C NMR (CD3OD, 100.63 MHz): 173.5 (CO), 138.5 (Cd), 134.4
(Ca), 129.5, 128.9 (Cb þ Cf, Cc þ Ce), 40.8 (C2), 39.5 (C3). ESI-MS
(m/z, %):197.0[(Mþ H)þ, 100]. EI-HRMS: calcd for C9H9ClN2O,
196.0403; found, 196.0403.
Ring-Opening of (2R,3S)-8a with MeOH-BF3 Et2O. BF3
3
3
Et2O (0.34 mmol, 43 μL) was added under nitrogen atmosphere
at 0 °C to a stirred solution of (2R,3S)-8a (0.31 mmol, 50 mg) in
anhydrous MeOH (2.0 mL). The mixture was heated at 55 °C for
8 h and then cooled in an ice bath. Aqueous 3 M NaOH was
added until basic, and the mixture was extracted with AcOEt
(3 ꢀ 10 mL). The combined organic layers were dried and the
solvents removed under reduced pressure to quantitatively obtain
(3R)-β-methoxy-D-phenylalaninamide [(2R,3R)-2-amino-3-methoxy-
3-phenylpropanamide], (2R,3R)-17 (60 mg).
(3R)-β-Methoxy-D-phenylalaninamide [(2R,3R)-17]: white solid;
D
mp 97.0-98.5 °C. [R]20 = -71.2 (c 1.0, MeOH). IR (CH2Cl2):
3405, 3300, 1676 cm-1. 1H NMR (CD3OD, 400.13 MHz): 7.50-
3
7.30 (m, Ph), 4.42 (d, H3, J = 6.6 Hz), 3.68 (br s, H2), 3.26
(s, OCH3). 13C NMR (CD3OD, 100.63 MHz): 177.0 (CO), 138.7
(Cipso), 129.5, 128.9 (2Cortho, 2Cmeta, Cpara), 86.2 (C3), 60.6 (C2), 57.2
(OCH3).ESI-MS(m/z, %): 389.1 [(2M þ H)þ, 60], 357.1 [(2M þ H-
MeOH), 20], 195.1 [(M þ H)þ, 100].
Ring-Opening of (2R,3S)-8a with H2O-p-TsOH and Sub-
sequent N-tert-Butoxycarbonylation. A mixture of (2R,3S)-8a
(0.31 mmol, 50 mg), p-TsOH H2O (0.34 mmol, 65 mg), and
(58) The keys for NMR assignments are included in the Supporting
Information, General.
3
7:1 MeCN-H2O (1.7 mL) was reacted at 55 °C for 8 h. After
6622 J. Org. Chem. Vol. 75, No. 19, 2010

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Moran-Ramallal et al.
JOCArticle
removal of MeCN under reduced pressure, aqueous 3 M NaOH
was added until basic, subsequently extracting the mixture with
AcOEt (3 ꢀ 10 mL). The combined organic layers were dried
and concentrated in vacuo, and the obtained residue was then
treated overnight at room temperature with Boc2O (0.32 mmol,
70 mg) in MeOH (4.0 mL). After removal of MeOH under
reduced pressure, the resulting crude material was purified by
flash column chromatography [eluent, hexane-AcOEt: (1) 3:1;
(2) 1:3] to yield (3R)-N2-Boc-β-hydroxy-D-phenylalaninamide
{tert-butyl N-[(1R,2R)-1-carbamoyl-2-hydroxyethyl]carbamate},
(2R,3R)-20 (60 mg, 70% overall yield).
General ProcedureforN-tert-Butoxycarbonylationofr-Amino-
amides (2R,3R)-17, (2R,3R)-22, and (R)-31a,g. Each R-amino-
amide (0.31 mmol) was treated overnight at room temperature
with Boc2O (0.39 mmol, 84 mg) in MeOH (5.0 mL). After removal
of MeOH under reduced pressure, the resulting crude material was
purified by flash column chromatography [eluent, hexane-AcOEt:
(1) 3:1; (2) 1:1], yielding the following tert-butyl carbamates:
(3R)-N2-Boc-β-methoxy-D-phenylalaninamide [(2R,3R)-18]:
yield 83%; white solid; mp 200.2-201.4 °C. [R]20 = -61.9
D
(c 0.8, MeOH). ee > 99.5%. IR (CH2Cl2): 3391, 3354, 1685,
1660 cm-1 1H NMR (DMSO, 300.13 MHz): 7.41 (br s,
.
(3R)-N2-Boc-β-Hydroxy-D-phenylalaninamide [(2R,3R)-20]:
CONHH), 7.35-7.21 (m, Ph), 7.01 (br s, CONHH), 6.57 (d,
NHBoc, 3J = 9.4 Hz), 4.27 (d, H3, 3J = 9.0 Hz), 4.13 (t, H2, J =
9.2 Hz), 3.06 (s, OCH3), 1.19 [s, (CH3)3C]. 13C NMR (DMSO,
75.48 MHz): 172.3 (CONH2), 154.7 (NHCOOBut), 138.6
(Cipso), 128.0, 127.9 (2Cortho, 2Cmeta, Cpara), 82.9 (C3), 78.1
(OCMe3), 58.5 (C2), 56.6 (OCH3), 28.2 [C(CH3)3]. ESI-MS
(m/z, %): 489.2 [(2MþH-C4H8-CO2)þ, 60], 295.1 [(MþH)þ,
18], 195.1 [(M þ H - C4H8 - CO2)þ, 100]. ESI-TOF-HRMS:
calcd for C15H22N2O4, 295.1652; found, 295.1644.
white solid; mp 183.6-184.9 °C. [R]20D = -29.5 (c 1.0, MeOH).
ee > 99.5%. IR (CH2Cl2): 3503, 3393, 3346, 1685, 1663 cm-1
.
1H NMR (CD3OD, 300.13 MHz): 7.49-7.28 (m, Ph), 4.89
(superimposed d, H3), 4.35 (d, H2, 3J = 7.7 Hz), 1.34 [s, (CH3)3C].
13C NMR (CD3OD, 75.48 MHz): 175.9 (CONH2), 157.2
(NHCOOBut), 142.2 (Cipso), 129.1, 128.8, 128.2 (2Cortho, 2Cmeta
,
Cpara), 80.6 (OCMe3), 75.2 (C3), 60.6 (C2), 28.6 [C(CH3)3]. ESI-MS
(m/z, %): 303.1 [(M þ Na)þ, 100], 203.1 [(M þ Na - C4H8 -
CO2)þ, 50].
(3R)-β-Azido-N2-Boc-D-phenylalaninamide [(2R,3R)-23]: yield
85%; white solid; mp 183.3-184.7 °C. [R]20 = -126.3 (c 1.0,
Ring-Opening of (2R,3S)-8a with NaN3-BF3 Et2O. BF3 Et2O
D
3
3
MeOH). ee > 99.5%. IR (CH2Cl2): 3388, 3346, 1686, 1660 cm-1
.
(0.34 mmol, 43 μL) was added under nitrogen atmosphere at 0 °C
to a solution of (2R,3S)-8a (0.31 mmol, 50 mg) in anhydrous
MeCN (1.5 mL), and the mixture was stirred at 0 °C during 10 min.
A solution of NaN3 (0.93 mmol, 60 mg) in the smallest amount of
water was then added, and the mixture was heated to 55 °C for
12 h. After cooling (ice bath), aqueous 3 M NaOH was added until
basic, and the mixture was extracted with AcOEt (3 ꢀ 10 mL). The
combined organic layers were dried and concentrated in vacuo
to obtain (3R)-β-azido-D-phenylalaninamide [(2R,3R)-2-amino-
3-azido-3-phenylpropanamide], (2R,3R)-22 (58 mg, 92%).
1H NMR (DMSO, 300.13 MHz): 7.63 (br s, CONHH),
7.42-7.25 (m, Ph), 7.23 (br s, CONHH), 6.87 (d, NHBoc, 3J =
9.3 Hz), 4.77 (d, H3, 3J = 9.3 Hz), 4.30 (t, H2, 3J = 9.3 Hz), 1.20
[s, (CH3)3C]. 13C NMR (DMSO, 75.48 MHz): 171.5 (CONH2),
154.7 (NHCOOBut), 136.4 (Cipso), 128.6, 128.5, 128.3 (2Cortho
,
2Cmeta, Cpara), 78.4(OCMe3), 65.5 (C3), 57.0 (C2), 28.1 [C(CH3)3].
ESI-MS (m/z, %): 511.2 [(2M þ H - C4H8 - CO2)þ, 100], 427.1
(85), 306.1 [(M þ H)þ, 10], 279.0 [(M þ H - CHN)þ, 38], 206.0
[(M þ H - C4H8 - CO2)þ, 70].
N2-Boc-D-Phenylalaninamide [(R)-34a]: yield 85%; white solid;
(3R)-β-Azido-D-phenylalaninamide [(2R,3R)-22]: white solid;
mp 127.0-128.0 °C. [R]20D = -172.5 (c 1.0, MeOH). IR (CH2Cl2):
3377, 3055, 1689 cm-1. 1H NMR (CD3OD, 400.13 MHz): 7.52-
7.30 (m, Ph), 4.86 (partially superimposed d, H3, 3J = 7.0 Hz), 3.66
mp 153.1-154.3 °C (lit.:60 141-143 °C). [R]20 = -12.8 (c 1.0,
D
MeOH) [lit.: -14.5 (c 1.0, EtOH)]. ee > 99.5%. IR (CH2Cl2):
1
3392, 3346, 1678, 1657 cm-1. H NMR (CDCl3, 400.13 MHz):
3
(d, H2, J = 7.0 Hz). 13C NMR (CD3OD, 100.63 MHz): 176.7
7.35-7.17 (m, Ph), 6.24 (br s, CONHH), 6.03 (br s, CONHH),
5.31 (br s, NHBoc), 4.44 (br s, H2), 3.04 [br s, C(3)H2], 1.38
[s, (CH3)3C]. 13C NMR (CDCl3, 100.63 MHz): 174.0 (CONH2),
155.4 (NHCOOBut), 136.6 (Cipso), 129.2, 128.5 (2Cortho, 2Cmeta),
126.8 (Cpara), 80.0 (OCMe3), 55.2 (C2), 38.5 (C3), 28.2 [C(CH3)3].
ESI-MS (m/z, %): 429.2 [(2M þ H - C4H8 - CO2)þ, 100], 165.0
[(M þ H - C4H8 - CO2)þ, 37]. EI-HRMS: calcd for C14H20N2O3,
220.1338 (M - CONH2)þ; found, 220.1339. In a similar way N2-
Boc-β-(p-tolyl)-D-alaninamide [(R)-34g] (83% yield) was prepared.
Hydrolysis of the Carbamoylcarbamates (2R,3R)-18, (2R,3R)-20,
and (R)-34a,g. A suspension of the corresponding carbamoylcarba-
mate(0.14mmol) in6MHCl(2.6mL) wasrefluxedfor3handthen
cooled at room temperature. After concentrating to nearly dryness
under reduced pressure, the obtained residue was washed with
AcOEt (2 ꢀ 7.5 mL), with in vacuo removal of the solvent after
each washing. The resulting solid was dried in vacuo over P2O5 (10 h,
room temperature) to yield the corresponding D-R-amino acid
hydrochloride. The D-R-amino acid hydrochloride (0.13 mmol,
30 mg) proceeding from (2R,3R)-18 was further transformed (for
identification purposes) into the free amino acid by treatment with
propylene oxide (31 mmol, 2.2 mL) in EtOH (10 mL) at 90 °C for
1 h.61 Finally, the solvent and volatile compounds were removed
under reduced pressure to yield (3R)-β-methoxy-D-phenylalanine
[(2R,3R)-19].
(CO), 137.1 (Cipso), 129.9, 129.2 (2Cortho, 2Cmeta, Cpara), 69.7 (C3),
60.0 (C2).ESI-MS(m/z, %):206.1[(Mþ H)þ, 100], 163.1 [(M þ H-
CONH2)þ, 23].
Ring-Opening of (2R,3S)-8a by Catalytic Hydrogenolysis.
(2R,3S)-8a (0.31 mmol, 50 mg) and 10% Pd/C (48 mg) were
placed in a 50 mL round-bottomed flask. The flask was stop-
pered with a rubber septum and evacuated (rotary pump). A
hydrogen-filled balloon was then connected via a needle, and
MeOH (6.0 mL) was added via a syringe. The mixture was
stirred at room temperature for 2.5 h and then filtered through a
pad of diatomaceous earth. After washing the pad with MeOH,
the combined methanol solutions were concentrated in vacuo to
yield a crude material, which was finally purified by flash
column chromatography (MeOH as eluent) to obtain D-phenyl-
alaninamide [(R)-2-amino-3-phenylpropanamide], (R)-31a
(46 mg, 90%). The aziridineamide (R)-8g (0.284 mmol, 50 mg) was
hydrogenolyzed in a similar way, except that no chromatographic
purification was required, to yield almost quantitatively β-(p-tolyl)-
D-alaninamide [(R)-2-amino-3-(4-methylphenyl)propanamide],
(R)-31g.
D-Phenylalaninamide [(R)-31a]: white solid; mp 131.0-132.5 °C
D
(lit.:59 135-137 °C). [R]20 = -12.5 (c 1.0, MeOH) [lit.: -9.0
(c 1.0, MeOH), ee 77%]. IR (CH2Cl2): 3295, 3200, 3054,
1681 cm-1. 1H NMR (CD3OD, 400.13 MHz): 7.37-7.20 (m, Ph),
3.60 (dd, H2, 3J = 7.6, 3J = 6.0 Hz), 3.06 [dd, C(3)HH, 2J = 13.4,
3J=6.0Hz], 2.83[dd, C(3)HH, 2J= 13.4, 3J=7.6Hz]. 13CNMR
(CD3OD, 100.63 MHz): 179.5 (CO), 138.9 (Cipso), 130.4 (2Cortho),
129.5 (2Cmeta), 127.7 (Cpara), 57.4 (C2), 42.5 (C3). ESI-MS (m/z, %):
329.1 [(2M þ H)þ, 15], 165.0 [(M þ H)þ, 100].
(3R)-β-Methoxy-D-phenylalanine [(2R,3R)-19]:yield 90%; white
D
solid; mp 190.0-192.0 °C. [R]20 = -37.3 (c 0.6, H2O) [lit.:46
þ34.5 (c 0.6, H2O) for (2S,3S)-19]. IR (Nujol): 3131, 3048, 1614
1
cm-1. H NMR (D2O, 300.13 MHz): 7.40-7.17 (m, Ph), 4.79
(d, H3, 3J = 3.5 Hz), 4.05 (d, H2, 3J = 3.5 Hz), 3.26 (s, OCH3). 13
C
(60) Pozdnev, V. F. Tetrahedron Lett. 1995, 36, 7115–7118.
(61) Jefford, C. W.; McNulty, J. Helv. Chim. Acta 1994, 77, 2142–2146.
J. Org. Chem. Vol. 75, No. 19, 2010 6623
(59) Wang, M.-X.; Lin, S.-J. J. Org. Chem. 2002, 67, 6542–6545.

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NMR (D2O, 100.63 MHz): 174.3 (CO), 135.5 (Cipso), 128.8, 128.7,
127.2 (2Cortho, 2Cmeta, Cpara), 82.4 (C3), 59.5 (C2), 56.6 (OCH3).
ESI-MS (m/z, %): 196.1 (Mþ, 100).
syringe. The mixture was stirred at room temperature for 12 h
and then filtered through a pad of diatomaceous earth. After
washing the pad with MeOH, the combined methanol solutions
were concentrated in vacuo to quantitatively obtain (3R)-β-amino-
N2-Boc-D-phenylalaninamide {tert-butyl N-[(2R,3R)-2-amino-
1-carbamoyl-2-phenylethyl]carbamate}, (2R,3R)-24 (32 mg).
(3R)-β-Amino-N2-Boc-D-phenylalaninamide [(2R,3R)-24]: color-
less oil. [R]20D = -13.8 (c 1.0, MeOH). IR (film): 3400, 3362, 1680,
1656 cm-1. 1H NMR (CD3OD, 400.13 MHz): 7.45-7.22 (m, Ph),
4.35 (poorly resolved d, H2, 3J = 6.4 Hz), 4.20 (poorly resolved d,
H3), 1.36 [s, (CH3)3C]. 13C NMR (CD3OD, 100.63 MHz): 175.8
(CONH2), 157.4 (NHCOOBut), 142.4 (Cipso), 129.4, 128.6, 128.5
(2Cortho, 2Cmeta, Cpara), 80.6 (OCMe3), 60.9 (C3 or C2), 58.5 (C2 or
C3), 28.6 [C(CH3)3]. ESI-MS (m/z, %): 280.1 [(M þ H)þ, 100],
180.1 [(M þ H - C4H8 - CO2)þ, 5].
(3R)-β-Hydroxy-D-phenylalanine hydrochloride [(2R,3R)-21]:
yield 95%, white solid; mp 171.5-172.6 °C (dec) [lit.:49 174 °C
(dec)]. [R]20D = -59.3 (c 1.0, 6 M HCl) [lit.: -63.2 (c 0.65, 6 M
HCl)]. IR (Nujol): 3424, 3315, 3123, 3032, 1735 cm-1. 1H NMR
(D2O, 400.13 MHz): 7.42-7.27 (m, Ph), 5.29 (d, H3, 3J = 3.8Hz),
4.29 (d, H2, 3J = 3.8 Hz). 13C NMR (D2O, 100.63 MHz): 169.4
(CO), 136.9 (Cipso), 129.0, 128.9 (2Cortho or 2Cmeta, Cpara), 126.1
(2Cmeta or 2Cortho), 70.8 (C3), 58.9 (C2). ESI-MS (m/z, %): 182.1
(Mþ, 100).
β-(p-Tolyl)-D-alanine hydrochloride [(R)-35g]: yield 95%;
white solid; mp 264.0-265.1 °C. [R]20 = þ10.0 (c 1.0, H2O)
D
[lit.:57 -6.5 (c 0.1, 0.5 M HCl) for (S)-35g]. IR (Nujol): 3399,
3142, 2923, 1738 cm-1. 1H NMR (D2O, 300.13 MHz): 7.16 (d,
2Hortho or 2Hmeta, 3J = 8.1 Hz), 7.11 (d, 2Hmeta or 2Hortho, 3J =
8.1 Hz), 4.18 (dd, H2, 3J = 7.6, 3J = 5.5 Hz), 3.19 [dd, C(3)HH,
Acknowledgment. The authors gratefully thank Dr. Francisca
Rebolledo, University of Oviedo, for prior scientific criticism
of the manuscript. R.M.-R. wishes to thank the Ministry of
Education and Science of Spain (MEC) for a predoctoral
fellowship. This work was supported by the MEC (Project
CTQ2007-61126).
3
2
3
2J = 14.5, J = 5.5 Hz], 3.07 [dd, C(3)HH, J = 14.5, J =
7.6 Hz], 2.22 (s, CH3). 13C NMR (D2O, 75.48 MHz): 172.1 (CO),
138.4, 131.1 (Cipso, Cpara), 130.0, 129.6 (2Cortho, 2Cmeta), 54.7
(C2), 35.5 (C3), 20.4 (CH3). ESI-MS (m/z, %): 180.0 (Mþ, 100).
In a similar way D-phenylalanine hydrochloride [(R)-35a] (95%
yield) was prepared.
Hydrogenolysis of the Azido Group in the Azido(carbamoyl)-
carbamate (2R,3R)-23. (2R,3R)-23 (0.115 mmol, 35 mg) and
10% Pd/C (18 mg) were placed in a 50 mL round-bottomed
flask. The flask was stoppered with a rubber septum and
evacuated (rotary pump). A hydrogen-filled balloon was then
connected via a needle, and MeOH (2.2 mL) was added via a
Supporting Information Available: Experimental proce-
dures, characterization data, assignment of absolute configura-
1
tions, and copies of H, 13C, and HMBC NMR spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
6624 J. Org. Chem. Vol. 75, No. 19, 2010