Synthesis of (R)-3,4-diaminobutanoic acid by desymmetrization of dimethyl 3-(benzylamino)glutarate through enzymatic ammonolysis

Article abstract of DOI:10.1021/jo026254f

Lipase B from Candida antarctica is shown to be a highly efficient catalyst for the desymmetrization of dimethyl 3-(benzylamino)glutarate (1) through aminolysis and ammonolysis reactions. Using this procedure, enantiopure monoamides are obtained in high yield. The synthetic value of these compounds is demonstrated by the preparation of an enantiopure nonnatural amino acid, i.e. (R)-3,4-diaminobutanoic acid [(+)-12].

Full text of DOI:10.1021/jo026254f

Chiral derivatives of 1 are valuable substrates for the
synthesis of biologically active compounds, for example
(R)- and (S)-3-aminoglutaric acid monoalkyl esters are
starting materials in the synthesis of 4-substituted-2-
azetidinones,4 (+)-Negamycin,5 and various cis- and
trans-Carbapenem antibiotics.6,7 Moreover, products de-
rived from the aminolysis and ammonolysis of 1 could
serve as versatile chiral synthons of biologically active
amino acids and amino alcohols. To illustrate the syn-
thetic value of these compounds, we also introduce herein
a new chemoenzymatic method for the synthesis of (R)-
3,4-diaminobutanoic acid [(+)-12] starting with amido
ester (-)-3c obtained through ammonolysis of 1. Optically
active 4-aminobutanoic acid (GABA) analogues, such as
(R)-GABOB, (R)-baclofen (4-p-chlorophenyl-GABA), and
(R)-4-phenyl-GABA, are used clinically for different
purposes.8 These observations support that (R)-3,4-di-
aminobutanoic acid could be of interest not only as a
chiral building block but also as a nonnatural amino acid
itself.
Starting material 1 was prepared by a Michael-type
addition of benzylamine to dimethyl glutaconate. We
chose CALB as the biocatalyst, since it has exhibited high
efficiency in both the desymmetrization of dimethyl
3-hydroxyglutarate3 and the resolution of chiral esters9
as well as chiral monoamines and diamines10 through
aminolysis and ammonolysis processes. The enzymatic
reactions were run in dry 1,4-dioxane, which is known
to be a good solvent for this type of transformation, using
an equimolar amount of diester 1 and amines 2a and 2b,
Syn th esis of (R)-3,4-Dia m in obu ta n oic Acid
by Desym m etr iza tion of Dim eth yl
3-(Ben zyla m in o)glu ta r a te th r ou gh
En zym a tic Am m on olysis
Mo´nica Lo´pez-Garc´ıa, Igancio Alfonso, and
Vicente Gotor*
Departamento de Quı´mica Orga´nica e Inorga´nica, Facultad
de Quı´mica, Universidad de Oviedo, J ulia´n Claverı´a, 8,
33071 Oviedo, Spain
vgs@sauron.quimica.uniovi.es
Received J uly 29, 2002
Abstr a ct: Lipase B from Candida antarctica is shown to
be a highly efficient catalyst for the desymmetrization of
dimethyl 3-(benzylamino)glutarate (1) through aminolysis
and ammonolysis reactions. Using this procedure, enan-
tiopure monoamides are obtained in high yield. The syn-
thetic value of these compounds is demonstrated by the
preparation of an enantiopure nonnatural amino acid, i.e.
(R)-3,4-diaminobutanoic acid [(+)-12].
The enzymatic desymmetrization of meso and prochiral
compounds has been widely recognized and constitutes
an elegant approach to the synthesis of enantiomerically
pure compounds.1 This methodology avoids the inherent
50% yield limit and the difficult separations often en-
countered in the resolution of racemates. The desymme-
trization strategy has been successfully utilized in the
synthesis of biologically active compounds, such as
pharmaceuticals, pesticides, flavors, and fragrances.1
Hydrolysis, transesterification, or lactonization pro-
cesses have been largely applied to prochiral diesters and
diols to prepare chiral synthons of high optical purity.1,2
However, the potential of enzymes, especially lipases, to
catalyze the aminolysis and ammonolysis of prochiral
substrates has been recently reported by us.3 Lipase B
from Candida antarctica (CALB) has been shown to
catalyze the aminolysis and ammonolysis of dimethyl
3-hydroxyglutarate. The ammonolysis product is a useful
starting material for the synthesis of (R)-4-amino-3-
hydroxybutanoic acid [(R)-GABOB].3a
(4) Ohno, M.; Kobayashi, S.; Iimori, T.; Wang, Y.-F.; Izawa, T. J .
Am. Chem. Soc. 1981, 103, 2405.
(5) Wang, Y.-F.; Izawa, T.; Kobayashi, S.; Ohno, M. J . Am. Chem.
Soc. 1982, 104, 6465.
(6) Okano, K.; Izawa, T.; Ohno, M. Tetrahedron Lett. 1983, 24, 217.
(7) Iimori, T.; Takahashi, Y.; Izawa, T.; Kobayashi, S.; Ohno, M. J .
Am. Chem. Soc. 1983, 105, 1659.
(8) See for example: (a) Otsuka, M.; Obata, K.; Miyata, Y.; Yaneka,
Y. J . Neurochem. 1971, 18, 287. (b) Otsuka, M.; Miyata, Y. Advances
in Biochemical Psychopharmacology; Raven: New York, 1972; Vol. 6,
p 61. (c) Brehm, L.; J acobsen, P.; J ohansen, J . S.; Krogsgaardlarsen
P. L. J . Chem. Soc., Perkin Trans. 1 1983, 1459. (d) Allan, R. D.; Bates,
M. C.; Drew, C. A.; Duke, R. K.; Hambley, T. W.; J ohnston, G. A. R.;
Mewett, K. N.; Spence, I. Tetrahedron 1990, 46, 2511. (e) Moglioni, A.
G.; Brousse, B. N.; AÄ lvarez-Larena, A.; Moltrasio, G. Y.; Ortun˜o, R.
M. Tetrahedron: Asymmetry 2002, 13, 451 and references cited in these
papers.
(9) See for example: (a) Garc´ıa, M. J .; Rebolledo, F.; Gotor, V.
Tetrahedron: Asymmetry 1992, 3, 1519. (b) Garc´ıa, M. J .; Rebolledo,
F.; Gotor, V. Tetrahedron: Asymmetry 1993, 4, 2199. (c) Zoete, M. C.;
Kock-van Dalen, A. C.; Rantwijk, F.; Sheldon, R. A. J . Chem. Soc.,
Chem. Commun. 1993, 1831. (d) De Zoete, M. C.; Ouwehand, A. A.;
Rantwijk, F.; Sheldon, R. A. Recl. Trav. Chim. Pays-Bas 1995, 114,
171. (e) Hacking, M. A. P. J .; Wegman, M. A.; Rops, J .; Rantwijk, A.;
Sheldon, R. A. J . Mol. Catal. B: Enzym. 1998, 5, 155. (f) Kanerva, L.
T.; Csomo´s, P.; Sundholm, O.; Berna´th, G.; Fu¨lo¨p, F. Tetrahedron:
Asymmetry 1996, 7, 1705. (g) Sa´nchez, V.; Rebolledo, F.; Gotor, V.
Tetrahedron: Asymmetry 1997, 8, 37. (h) Starmans, W. A. J .; Doppen,
R. G.; Thijs, L.; Zwanenburg, B. Tetrahedron: Asymmetry 1998, 9, 429.
(i) Garc´ıa-Urdiales, E.; Rebolledo, F.; Gotor, V. Tetrahedron: Asym-
metry 1999, 10, 721. (j) Conde, S.; Lo´pez-Serrano, P.; Mart´ınez, A.
Tetrahedron: Asymmetry 2000, 11, 2537.
Following on from these studies, we set out to examine
the aminolysis and ammonolysis of dimethyl 3-(benzyl-
amino)glutarate (1). This substrate was chosen in view
of potential further applications of the resulting products.
(1) Schoffers, E.; Golebiowski, A.; J ohnson, C.-R. Tetrahedron 1996,
52, 3769.
(2) For typical examples see: (a) Toone, E. J .; Werth, M. J .; J ones,
J . B. J . Am. Chem. Soc. 1990, 112, 4946. (b) Shapira, M.; Gutman, A.
L. Tetrahedron: Asymmetry 1994, 5, 1689. (c) Guanti, G.; Banfi, L.;
Narisano, E. Tetrahedron: Asymmetry 1990, 1, 721. (d) Guanti, G.;
Banfi, L.; Narisano, E. J . Org. Chem. 1992, 57, 1540. (e) Tsuji, K.;
Terao, Y.; Achiwa, K. Tetrahedron Lett. 1989, 30, 6189. (f) Gutman,
A. L.; Zuobi, K.; Bravdo, T. J . Org. Chem. 1990, 55, 3546. (g) Boaz,
N.-W. Tetrahedron: Asymmetry 1999, 10, 813. (h) Takabe, K.; Iida,
Y.; Hiyoshi, H.; Ono, M.; Hirose, Y.; Fukui, Y.; Yoda, H.; Mase, N.
Tetrahedron: Asymmetry 2000, 11, 4825.
(10) See for example: (a) Astorga, C.; Rebolledo, F.; Gotor, V. J .
Chem. Soc., Perkin Trans. 1 1994, 829. (b) Garc´ıa, M. J .; Rebolledo,
F.; Gotor, V. Tetrahedron 1994, 50, 6935. (c) Reetz, M. T.; Dreisbach,
C. Chimia 1994, 48, 570. (d) Chamorro, C.; Gonza´lez-Mun˜iz, R.; Conde,
S. Tetrahedron: Asymmetry 1995, 6, 2343. (e) Reetz, M. T.; Schimossek,
K. Chimia 1996, 50, 668. (f) Iglesias, L. E.; Sa´nchez, V.; Rebolledo, F.;
Gotor, V. Tetrahedron: Asymmetry 1997, 8, 2675. (g) Alfonso, I.;
Astorga, C.; Rebolledo, F.; Gotor, V. Chem. Commun. 1996, 2471.
(3) (a) Puertas, S.; Rebolledo, F.; Gotor, V. J . Org. Chem. 1996, 61,
6024. (b) Sanchez, V.; Rebolledo, F.; Gotor, V. J . Org. Chem. 1999, 64,
1464.
10.1021/jo026254f CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/17/2002
648
J . Org. Chem. 2003, 68, 648-651

SCHEME 1
SCHEME 2 a
a
Reagents and conditions: (i) CbzCl, Na2CO3, H2O, DCM, 86%;
TABLE 1. CALB “Novozym 435”-Ca ta lyzed Am in olysis
a n d Am m on olysis of 1
(ii) PIDA, BnOH, THF, rt, 61% 7; (iii) PIDA, H2O, THF, rt, 86%;
(iv) PIFA, H2O, MeCN, rt, 100%.
time
product
R
(days) yield (%)a
[R]20
ee (%)b
D
tion conditions, facile product isolation, high yields, and
avoidance of the use of heavy metal-based reagents,
which are frequently used to carry out the rearrange-
ment.3a
(-)-3a
(-)-3b
(-)-3c
Bn
Bu
H
2
2
3
92
79
85
-14.5 (c 1.1, CHCl3)
-14.2 (c 1.0, CHCl3)
-11.5 (c 1.0, CHCl3)
>99
>99
>99
a
b
After column chromatography. Determined by chiral HPLC.
First, amido ester (-)-3c was protected with benzyl
chloroformate (Scheme 2) to eliminate the posibility of
an intramolecular nucleophilic addition of the amine
functionality to the isocyanate, which would give rise to
a cyclic urea. Reaction of (-)-6 with I,I-bis(acetoxy)-
iodobenzene (PIDA) in THF in the presence of benzyl
alcohol led to a mixture of benzyl carbamate (+)-7 (61%)
and chiral urea (+)-8 (accounting for the mass balance).
This byproduct is obtained when the intermediate iso-
cyanate and the amine that is subsequently formed from
it react under catalysis of the acetic acid liberated from
PIDA. Substituted ureas have received considerable
attention due to their wide range of applications, includ-
ing the use as herbicides, corrossion inhibitors, artificial
receptors, antioxidants in gasoline, and pharmaceuti-
cals.14 Encouraged by this, we tried to obtain compound
(+)-8 as the main product replacing benzyl alcohol with
an equimolar amount of water. Under these conditions,
we were able to prepare the C2 symmetric urea in 86%
yield. Coming back to our initial goal, we tested some
other conditions, this time, in an attempt to decrease the
yield of (+)-8. However, formation of the urea seems to
be highly favored in the presence of acetic acid. Hence,
we considered changing the iodoorganic compound to I,I-
bis(trifluoroacetoxy)iodobenzene (PIFA). The solvent ef-
fect on the yield of reactions with this reagent has been
studied by Longqin Hu et al., finding that acetonitrile is
the solvent of choice.13d Thus, we carried out the reaction
with PIFA in acetonitrile which afforded product (+)-9
in quantitave yield. The trifluoroacetic acid released
during the course of the reaction can both catalyze the
attack of water onto the isocyanate and protonate the
resulting amine, thereby preventing it from participating
in the formation of the urea.
or a freshly prepared solution of ammonia at 30 °C
(Scheme 1) until the conversion reached its maximum
(as seen by 1H NMR). With these conditions in hand,
three different CALB preparations, namely “Chirazyme
L-2, c-f, lyo”, “Chirazyme L-2, c-f, C3, lyo”, and CALB
“Novozym 435” were tested, of which the latter turned
out to be the best catalyst for the asymmetric aminolysis
and ammonolysis of 1 giving enantiopure monoamides
(-)-3a -c in high yield (Table 1). The smooth differences
in yields are due to different percent conversion depend-
ing on the nucleophile. In all cases, the reaction stopped
at the stage of the amido ester and diamides were not
detected.
Independent of the nucleophile used, CALB “Novozym
435” only reacted with the pro-R ester group of 1, yielding
amido esters 3 with the S configuration. In the case of
(-)-3c, the absolute stereochemistry was determined by
its transformation into amino acid (+)-12, whose specific
rotation matched the literature value of (R)-(+)-12.11 The
absolute configuration of the enzymatically prepared
amido esters (-)-3a ,b has been tentatively assigned on
the basis of three convergent criteria: (a) the enantiotopic
preference displayed by CALB in the aminolysis and
ammonolysis of dimethyl 3-hydroxyglutarate,3 as well as
in the ammonolysis of 1 (vide supra); (b) using Mosher’s
method for primary amines12 (compounds (-)-3a ,b and
(()-3a ,b were first N-deprotected using HCO2H/Pd-black
in MeOH), the MTPA amides derived from compounds
(()-3a ,b showed two well-resolved peaks for the methyl
1
ester group in their H NMR spectra, whereas the MTPA
amides derived from products (-)-3a ,b showed only the
high-field signal; and (c) when eluted from a chiral
column, “Chiralcel OD-H”, the enantiomer obtained
through enzyme catalysis, exhibited the longer retention
time in all cases (see the Experimental Section).
The synthesis of (R)-3,4-diaminobutanoic acid (+)-12
from compound (-)-3c involved a Hofmann rearrange-
ment of the amide function as the key step. After
surveying the literature, we decided to use polyvalent
iodoorganic compounds8i,13 to promote the rearrangement.
These reagents have several advantages, e.g. mild reac-
(13) (a) Radhakrishna, A. S.; Parham, M. E.; Riggs, R. M.; Loudon,
G. M. J . Org. Chem. 1979, 44, 1746. (b) Waki, M.; Kitajima, Y.;
Izumiya, N. Synthesis 1981, 266. (c) Boutin, R. H.; Loudon, M. J . Org.
Chem. 1984, 49, 4277. (d) Yu, C.; J iang, Y.; Liu, B.; Hu, L. Tetrahedron
Lett. 2001, 42, 1449. (e) Zhang, L.-H.; Kauffman, G. S.; Pesti, J . A.;
Yin, J . J . Org. Chem. 1997, 62, 6918.
(14) See for example: (a) Smith, P. J .; Reddington, M. V.; Wilcox,
C. S. Tetrahedron Lett. 1992, 33, 6085. (b) Funabashi, Y.; Tsubotani,
S.; Koyama, K.; Katayama, N.; Harada, S. Tetrahedron 1993, 49, 13.
(c) Tsopmo, A.; Ngnokam, D.; Ngamga, D.; Ayafor, J . F.; Sterner, O.
J . Nat. Prod. 1999, 62, 1435. (d) Getman, D. P.; Decrescenzo, G. A.;
Heintz, R. M.; Reed, K. L.; Talley, J . J .; Bryant, M. L.; Clare, M.;
Houseman, K. A.; Marr, J . J .; Mueller, R. A.; Vazquez, M. L.; Sheih,
H. S.; Stallings, W. C.; Stegeman, R. A. J . Med. Chem. 1993, 36, 288.
(e) Sijbesma, R. P.; Beijer, F. H.; Brunsveld, L.; Folmer, B. J . B.;
Hirschberg, J . H. K.; Lange, R. F. M.; Lowe, J . K. L.; Meijer, E. W.
Science 1997, 278, 1601.
(11) Misiti, D.; Santaniello, M.; Zappia, G. Synth. Commun. 1992,
22, 883.
(12) Kusumi, T.; Fukushima, T.; Ohtani, I.; Kakisawa, H. Tetrahe-
dron Lett. 1991, 32, 2939.
J . Org. Chem, Vol. 68, No. 2, 2003 649

SCHEME 3 a
Meth yl (S)-(-)-3-(Ben zyla m in o)-4-(N-ben zylca r ba m oyl)-
bu ta n oa te [(-)-3a ]. Yield, 92%; oil; [R]20D -14.5 (c 1.1, CHCl3);
1
IR (neat) 3285, 1734, 1646 cm-1; H NMR (CDCl3, 300 MHz) δ
2.31 (br s, 1H), AB portion of an ABX system (δA 2.41, δB 2.54,
J AB ) 15.9 Hz, J AX ) 7.8 Hz, J BX ) 4.2 Hz, 2H), 2.61 (d, J ) 5.8
Hz, 2H), 3.37-3.45 (m, 1H), 3.69 (s, 3H), AB system (δA 3.73, δB
3.81, J AB ) 12.6 Hz, 2H), AB portion of an ABX system (δA 4.41,
δB 4.46, J AB ) 14.8 Hz, J AX ) 5.7 Hz, J BX ) 5.6 Hz, 2H), 7.13-
7.38 (m, 10H), 8.25 (br s, 1H); 13C NMR (CDCl3, 75 MHz) δ 37.4,
39.1, 43.0, 50.2, 51.2, 51.5, 127.0, 127.1, 127.6, 128.0, 128.3,
128.4, 138.2, 138.8, 170.8, 171.9; MS (%) m/z 379 [(M + K)+, 8],
363 [(M + Na)+, 12], 341 [(M + H)+, 100]. Elemental Anal. for
a
Reagents and conditions: (i) HCOOH, “Pd-black”, MeOH; (ii)
MeOH, 1 M NaOH, rt; (iii) 6M HCl, 85 °C. 74% over 3 steps.
Hydrogenation and subsequent hydrolysis of rear-
ranged product (+)-9 led to a mixture of diamino acid 10
and γ-lactam 11 (Scheme 3). When this residue was
treated with 6 M hydrochloric acid at 85 °C for 12 h, the
γ-lactam was converted into the desired amino acid,
which was isolated as its dihydrochloride salt (+)-12.
In summary, the desymmetrization of dimethyl 3-
(benzylamino)glutarate by enzymatic aminolysis and
ammonolysis has been accomplished using lipase B from
Candida antarctica as the biocatalyst. This procedure
gives access to valuable enantiopure polyfunctionalized
synthons in high yield. Hofmann rearragement of the
ammonolysis product allows the preparation of an opti-
cally pure C2 symmetric urea and a novel chemoenzy-
matic synthesis of (R)-3,4-diaminobutanoic acid.
C
20H24N2O3: C, 70.57; H, 7.11; N, 8.23. Found: C, 70.36; H, 7.28;
N, 8.14.
Meth yl (S)-(-)-3-(Ben zyla m in o)-4-(N-bu tylca r ba m oyl)-
bu ta n oa te [(-)-3b]. Yield, 79%; oil; [R]20D -14.2 (c 1.0, CHCl3);
1
IR (neat) 3296, 1737, 1642 cm-1; H NMR (CDCl3, 300 MHz) δ
0.92 (t, J ) 7.2 Hz, 3H), 1.27-1.39 (m, 2H,), 1.42-1.52 (m, 2H),
2.25 (s, 1H), AB portion of an ABX system (δA 2.35, δB 2.49, J AB
) 15.6 Hz, J AX ) 7.4 Hz, J BX ) 4.2 Hz, 2H), 2.60 (d, J ) 6.1 Hz,
2H), 3.23 (q, J ) 6.6 Hz, 2H), 3.35-3.43 (m, 1H), 3.69 (s, 3H),
AB system (δA 3.79, δB 3.85, J AB ) 12.8 Hz, 2H), 7.26-7.38 (m,
5H), 7.66 (br s, 1H); 13C NMR (CDCl3, 75 MHz) δ 13.6, 20.0,
31.4, 37.5, 38.7, 39.2, 50.3, 51.3, 51.6, 127.2, 128.0, 128.4, 139.0,
170.9, 172.1; MS (%) m/z 354 [(M + K)+, 3], 329 [(M + Na)+,
15], 307 [(M
17H26N2O3: C, 66.64; H, 8.55; N, 9.14. Found: C, 66.49; H, 8.82;
N, 8.91.
+
H)+, 100]. Elemental Anal. Calcd for
C
Meth yl (S)-(-)-3-(Ben zyla m in o)-4-ca r ba m oylbu ta n oa te
[(-)-3c]. Ammonia was bubbled through 1,4-dioxane at 5-10
°C for 30 min under nitrogen. Then, 8 mL of this solution were
added to a mixture of dimethyl 3-(benzylamino)glutarate (2
mmol) and CALB (240 mg). The reaction mixture was shaken
at 30 °C and 250 rpm for 3 days, whereupon the enzyme was
filtered and washed with dichloromethane and the combined
organic solvents were evaporated. Purification by column chro-
matography on silica using ethyl acetate as the eluent gave the
Exp er im en ta l Section
Gen er a l. Lipase B from Candida antarctica (CALB) “No-
vozym 435” was donated by Novo Nordisk Co. and the prepara-
tions “Chirazyme L-2, c-f, lyo” and “Chirazyme L-2, c-f, C3, lyo”
were obtained from Roche Diagnostics. All reagents were
purchased from commercial suppliers and used without further
purification. Tetrahydrofuran and 1,4-dioxane were freshly
distilled from sodium and benzophenone ketyl under nitrogen
atmosphere. Methanol and acetonitrile were dried by distillation
from calcium hydride under nitrogen atmosphere. Thin layer
chromatography was performed on aluminum plates coated with
Merck silica gel 60F254. Column chromatography was carried out
on Merk silica gel 60/230-400 mesh at increased pressure (hand-
held bellows). Melting points are uncorrected. Chemical shifts
are quoted in ppm on the scale using tetramethylsilane as
internal standard. Coupling constants are given in hertz. Mass
spectra were recorded using electrospray ionization. The enan-
tiomeric excess was determined by chiral HPLC analysis using
a “Chiralcel OD-H” column.
Dim eth yl 3-(Ben zyla m in o)glu ta r a te (1). Benzylamine (15
mmol) was added to dimethyl glutaconate (15 mmol) under
nitrogen and the reaction mixture was stirred at room temper-
ature for 3 days. The crude residue was purified by column
chromatography on neutral silica gel using DCM as the eluent
affording the title compound as a colorless oil in 68% yield: IR
(neat) 3350, 1736 cm-1; 1H NMR (CDCl3, 200 MHz) δ 1.95 (br s,
1H, NH), 2.60 (d, J ) 6.2 Hz, 4H), 3.40-3.52 (m, 1H), 3.69 (s,
6H), 3.82 (s, 2H), 7.20-7.47 (m, 5H); 13C NMR (CDCl3, 75 MHz)
δ 38.3, 50.6, 50.9, 51.4, 126.8, 127.9, 128.2, 139.7, 172.0; MS (%)
m/z 288 [(M + Na)+, 25], 266 [(M + H)+, 100]. Elemental Anal.
Calcd for C14H19NO4: C, 63.38; H, 7.22; N, 5.28. Found: C, 63.26;
H, 7.43; N, 5.12,.
title compound as a colorless oil in 85% yield: [R]20 -11.5 (c
D
1.0, CHCl3); IR (neat) 3335, 3193, 1727, 1666 cm-1
;
1H NMR
(CDCl3, 200 MHz) δ AB portion of an ABX system (δA 2.35, δB
2.50, J AB ) 15.7 Hz, J AX ) 6.9 Hz, J BX ) 4.6 Hz, 2H), 2.37 (s,
1H), 2.59 (d, J ) 6.2 Hz, 2H), 3.32-3.44 (m, 1H), 3.67 (s, 3H),
3.79 (s, 2H), 6.18 (br s, 1H), 7.20-7.37 (m, 5H), 7.61 (br s, 1H);
13C NMR (CDCl3, 75 MHz) δ 37.5, 38.9, 50.4, 51.1, 51.5, 127.0,
127.9, 128.3, 139.0 172.0 173.8; MS (%) m/z 289 [(M + K)+, 14],
273 [(M + Na)+, 26], 251 [(M + H)+, 100]. Elemental Anal. Calcd
for C13H18N2O3: C, 62.38; H, 7.25; N, 11.19. Found: C, 62.54;
H, 7.13; N, 11.32.
Deter m in a tion of th e En a n tiom er ic Excess of (-)-3a -
c. (a ) Gen er a l P r oced u r e for th e Syn th esis of Ra cem ic
Com p ou n d s (()-3a ,b. A small amount of (()-3a ,b could be
obtained when the corresponding amine, 2a or 2b (0.5 mmol),
was added to a suspension of dimethyl 3-(benzylamino)glutarate
(0.5 mmol) in MeOH (1 mL) under nitrogen atmosphere, and
the mixture was stirred at room temperature for 4 days.
Afterward, the solvent was evaporated. Compounds (()-3a ,b
were purified by column chromatography using ethyl acetate as
the eluent.
A sample (20 µL) of a solution of (()-3a or (-)-3a (0.5 mg) in
a mixture of hexane/EtOH (4/1) (2 mL) was analyzed by HPLC
(Chiralcel OD-H, 0.5 mL/min, 35 °C, 18 kgf/cm2, 210 nm),
showing two peaks (tR 13.479 and 22.906) for (()-3a and one
peak (tR 22.949) for (-)-3a .
A sample (20 µL) of a solution of (()-3b or (-)-3b (0.5 mg) in
a mixture of hexane/EtOH (19/1) (2 mL) was analyzed by HPLC
(Chiralcel OD-H, 0.5 mL/min, 30 °C, 15 kgf/cm2, 210 nm),
showing two peaks (tR 21.875 and 24.169) for (()-3b and one
peak (tR 24.197) for (-)-3b.
(b) Gen er a l P r oced u r e for th e Syn th esis of Ra cem ic
Com p ou n d (()-3c. Ammonia was bubbled through 1,4-dioxane
at 5-10 °C for 30 min under nitrogen. A small amount of (()-
3c could be obtained when 1 mL of the previous solution was
added to dimethyl 3-(benzylamino)glutarate (0.5 mmol) under
nitrogen atmosphere and the mixture was stirred at 5-10 °C
Gen er a l P r oced u r e for th e Am in olysis of Dim eth yl
3-(Ben zyla m in o)glu ta r a te. The corresponding amine, 2a or
2b (0.5 mmol), was added to a suspension of dimethyl 3-(benzyl-
amino)glutarate (0.5 mmol) and CALB (60 mg) in 1,4-dioxane
(2 mL) under nitrogen atmosphere. Solvent was previously dried
to avoid the competitive enzymatic hydrolysis. The mixture was
shaken at 30 °C at 250 rpm for 2 days. Afterward, the enzyme
was filtered off and washed with DCM and the combined
organics were evaporated under reduced pressure. Products (-)-
3a ,b were purified by column chromatography on silica using
ethyl acetate as the eluent.
650 J . Org. Chem., Vol. 68, No. 2, 2003

for 2 days. The solvent was evaporated and compound (-)-3c
was purified by flash chromatography using ethyl acetate as the
eluent.
[(M + Na)+, 50], 739 [(M + H)+, 100]. Elemental Anal. Calcd for
41H46N4O9: C, 66.65; H, 6.28; N, 7.59. Found: C, 66.48; H, 6.49;
N, 7.34.
Meth yl (R)-(+)-4-Am in o-3-[(N-ben zyl-N-ben zyloxyca r bo-
C
A sample (20 µL) of a solution of (()-3c or (-)-3c (0.5 mg) in
a mixture of hexane/EtOH (9/1) (2 mL) was analyzed by HPLC
(Chiralcel OD-H, 0.5 mL/min, 35 °C, 15 kgf/cm2, 210 nm)
showing two peaks (tR 27.218 and 30.487) for (()-3c and one
peak (tR 30.468) for (-)-3c.
n yl) a m in o]bu ta n oa te a s Its TF A Sa lt [(+)-9]. To a solution
of compound (-)-6 (0.4 mmol) in CH3CN (4 mL) were added I,I-
bis(trifluoroacetoxy)iodobenzene (0.56 mmol) and H2O (0.8
mmol). After being stirred for 12 h, the mixture was heated at
60 °C for 30 min to decompose the remaining PIFA. The solvent
was evaporated under reduced pressure, and the residue was
filtered through silica and washed with ethyl acetate to remove
iodobenzene. Then, the desired product was eluted from the silica
with MeOH. Removal of the solvent gave the title compound as
Meth yl (S)-(-)-3-[(N-Ben zyl-N-ben zyloxycar bon yl)am in o]-
4-ca r b a m oylb u t a n oa t e [(-)-6]. Compound (-)-3c (1 mmol)
was dissolved in water (2 mL) and Na2CO3 (1.2 mmol) was
added. Immediately after this, the mixture was cooled to 0 °C
and vigorously stirred. Then, benzyl chloroformate (1.2 mmol)
was added dropwise. The reaction mixture was allowed to warm
to room temperature and stirred for 12 h, followed by extraction
of the aqueous solution with DCM. The combined organics were
dried over Na2SO4, filtered, and evaporated in vacuo. Column
chromatography of the residue, on silica using ethyl acetate as
the eluent, gave the title compound as a white solid in 86%
yield: mp 103-105 °C; [R]20D -5.5 (c 1.0, CHCl3); IR (KBr) 3427,
a white solid in quantitative yield: mp 139-141 °C; [R]20 +6.9
D
(c 1.1, CH3OH); IR (KBr) 3450, 1731, 1688 cm-1; 1H NMR (CD3-
OD, 300 MHz, T ) 50 °C) δ AB portion of an ABX system (δA
2.63, δB 2.73, J AB ) 16.8 Hz, J AX ) 7.1 Hz, J BX ) 6.8 Hz, 2H), A
portion of an ABX system (δA 3.17, J AB ) 13.3 Hz, J AX ) 5.1 Hz,
1H), B portion of an ABX system (δB 3.34, 1H), 3.54 (s, 3H),
4.32-4.41 (m, 1H), AB system (δA 4.52, δB 4.62, J AB ) 15.9 Hz,
2H), AB system (δA 5.21, δB 5.26, J AB ) 12.3 Hz, 2H), 7.29-
7.37 (m, 10H); 13C NMR (CD3OD, 75 MHz, T ) 50 °C) δ 36.4,
1
3178, 1731, 1691, 1653 cm-1; H NMR (CD3CN, 300 MHz, T )
65 °C) δ 2.56 (d, J ) 7.0 Hz, 2H), AB portion of an ABX system
(δA 2.64, δB 2.72, J AB ) 15.6 Hz, J AX ) 6.2 Hz, J BX ) 8.1 Hz,
2H), 3.56 (s, 3H), 4.54 (s, 2H), 4.55 (m, 1H), 5.19 (s, 2H), 5.73
(br s, 2H) 7.26-7.39 (m, 10H); 13C NMR (CD3CN, 75 MHz, T )
65 °C) δ 38.9, 40.1, 51.9, 52.2, 54.2, 68.1, 128.2, 128.8, 128.9,
129.0, 129.5, 129,. 138.3, 140.0, 157.2, 172.5, 173.3; MS (%) m/z
423 [(M + K)+, 100], 407 [(M + Na)+, 64], 385 [(M + H)+, 44] ].
Elemental Anal. Calcd for C21H24N2O5: C, 65.61; H, 6.29; N, 7.29.
Found: C, 65.37; H, 6.13; N, 7.17.
42.5, 51.9, 52.2, 54.8, 69.0, 118.2 (q, 2J CF ) 293 Hz, CF3), 128.7,
3
128.9, 129.3, 129.5, 129.7, 137.4, 134.0, 157.9, 163.1 (q, J CF
)
33 Hz, COCF3), 172.3; MS (%) m/z 379 [{(M - HCO2CF3) + Na}+,
10], 357 [{(M - HCO2CF3) + H}+, 100]. Elemental Anal. Calcd
for C22H25N2O6F3: C, 56.17; H, 5.36; N, 5.95. Found: C, 56.23;
H, 5.28; N, 5.78.
(R)-(+)-3,4-Dia m in obu ta n oic a cid d ih yd r och lor id e [(+)-
12]. To compound (+)-9 (0.2 mmol) was added 18 mL of a 4%
HCO2H solution in MeOH (purged with nitrogen prior to use),
followed by Pd-black (100 mg), and the mixture was stirred
vigorously. After 40-60 min (TLC monitoring) the reaction
mixture was filtered through Celite and washed with MeOH.
The solvent was evaporated and the crude residue was dissolved
in MeOH (1.3 mL) under nitrogen, and subsequently treated
with NaOH (1 N, 0.5 mL). After being stirred for 13 h, the
solvent was removed and 2 mL of 6 M HCl was added. The
mixture was heated at 85 °C for 12 h. The solvent was
evaporated under reduced pressure and 2 mL of H2O was added.
The mixture was filtered and the filtrate purified on an ion-
exchange column (Dowex 50WX 4-400). The desired product was
eluted with a 4% aqueous ammonia solution and the fractions
were evaporated to dryness. The residue was resuspended in
H2O (2 mL) and then acidified with 3 M HCl. Finally, the solvent
was removed yielding the title compound as a white hygroscopic
H ofm a n n R ea r r a n gem en t of Met h yl (S)-3-[(N-Ben zyl-
N-ben zyloxycar bon yl)am in o]-4-car bam oylbu tan oate. Meth -
yl (R)-(+)-3-[(N-Ben zyl-N-ben zyloxyca r bon yl)a m in o]-4-(N-
b en zyloxyca r b on yla m in o)b u t a n oa t e
[(+)-7].
I,I-bis-
(acetoxy)iodobenzene (1.4 mmol) and benzyl alcohol (2.0 mmol)
were added to a solution of amido ester (-)-6 (1 mmol) in THF
(10 mL) under nitrogen atmosphere at room temperature. After
being stirred for 12 h, the mixture was heated at 60 °C for 30
min to decompose the remaining PIDA. Afterward, the solvent
was evaporated. Column chromatography of the residue on silica
using hexane/ethyl acetate (3/2) as the eluent gave the corre-
sponding title compound as a colorless oil in 61% yield: [R]20
D
+6.8 (c 0.7, CHCl3); IR (neat) 3344, 1692-1738 cm-1; H NMR
1
(CD3CN, 300 MHz, T ) 65 °C) δ AB portion of an ABX system
(δA 2.61, δB 2.74, J AB ) 15.9 Hz, J AX ) 6.1 Hz, J BX ) 7.8 Hz,
2H), AB portion of an ABX system (δA 3.37, δB 3.45, J AB ) 13.8
Hz, J AX ) 6.4 Hz, J BX ) 6.9 Hz, 2H), 3.58 (s, 3H), 4.33-4.43 (m,
1H), AB system (δA 4.49, δB 4.59, J AB ) 16.1 Hz, 2H), 5.10 (s,
2H), 5.22 (s, 2H), 5.49 (br s, H) 7.29-7.42 (m, 15H); 13C NMR
(CD3CN, 75 MHz, T ) 65 °C) δ 36.9, 44.3, 51.7, 52.4, 56.7, 67.4,
68.3, 128.4, 128.85, 128.97, 129.04, 129.08, 129.1, 129.7, 138.4,
138.7, 140.1, 157.6, 172.5; MS (%) m/z 529 [(M + K)+, 100], 513
[(M + Na)+, 79], 491 [(M + H)+, 12]. Elemental Anal. Calcd for
C28H30N2O6: C, 68.56; H, 6.16; N, 5.71. Found: C, 68.32; H, 6.35;
N, 5.56.
solid in 74% yield: [R]20 +12.2 (c 0.9, H2O, pH 7.0); IR (KBr)
D
1
3423, 3023, 1618 cm-1; H NMR (D2O, 300 MHz) δ AB portion
of an ABX system (δA 2.92, δB 3.02, J AB ) 18.3 Hz, J AX ) 7.0
Hz, J BX ) 5.4 Hz, 2H), 3.43 (d, J ) 6.3 Hz, 2H), 3.98-4.07 (m,
1H); 13C NMR (D2O, 75 MHz) δ 34.4, 40.7, 46.0, 172.9; MS (%)
m/z 141 [{(M - 2HCl) + Na}+, 5], 119 [{(M - 2HCl) + H}+,
100]. Elemental Anal. Calcd for C4H12N2O2Cl2: C, 25.15; H, 6.33;
N, 14.66. Found: C, 25.01; H, 6.57; N, 14.39.;
Ack n ow led gm en t. We express our appreciation to
Novo Nordisk Co. for the generous gift of the CA lipase.
Financial support of this work by the Spanish Ministerio
de Ciencia y Tecnolog´ıa (Project PPQ-2001-2683) and
by Principado de Asturias (Project GE-EXP01-03) is
gratefully acknowledged. M.L.-G. thanks the Ministerio
de Educacio´n, Cultura y Deporte for a predoctoral
fellowship. We also thank Thorsten Genski for the
English language revision of the manuscript.
Dim eth yl (3R,3′R)-(+)-3,3′-Bis[N-ben zyl-N-(ben zyloxy-
ca r bon yl)a m in o]-4,4′-u r eylen ed ibu ta n oa te [(+)-8]. To a
solution of compound (-)-6 (0.2 mmol) in THF (2 mL) were added
I,I-bis(acetoxy)iodobenzene (0.28 mmol) and H2O (0.4 mmol).
After being stirred for 12 h, the mixture was heated at 60 °C
for 30 min to decompose the remaining PIDA. Afterward the
solvent was evaporated. Column chromatography of the residue
(ethyl acetate as eluent) gave the title compound as a colorless
oil in 86% yield: [R]20D +25.9 (c 1.0, CHCl3); IR (neat) 3378, 1737,
1694 cm-1; 1H NMR (CD3CN, 300 MHz, T ) 65 °C) δ AB portion
of an ABX system (δA 2.54, δB 2.67, J AB ) 15.9 Hz, J AX ) 6.0
Hz, J BX ) 8.1 Hz, 4H, 2CH-CH2-CO2Me), 3.26-3.32 (m, 4H),
3.54 (s, 6H), 4.26-4.34 (m, 2H), AB system (δA 4.46, δB 4.54,
J AB ) 16.1 Hz, 4H), 4.83 (br s, 2H) 5.19 (s, 4H), 7.26-7.38 (m,
20H); 13C NMR (CD3CN, 75 MHz, T ) 65 °C) δ 36.9, 43.4, 51.4,
52.3, 57.1, 68.2, 128.3, 128.8, 129.0, 129.1, 129.6, 129.7, 138.4,
140.3, 157.6, 159.0, 172.7; MS (%) m/z 777 [(M + K)+, 19], 761
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra of 1, (-)-3a , (-)-3b, (-)-3c, (-)-6, (+)-7, (+)-8, (+)-9,
and (+)-12. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O026254F
J . Org. Chem, Vol. 68, No. 2, 2003 651