Straightforward synthesis of (R,S)-β-methyleneaspartic acid, an inhibitor of glutamate-aspartate transaminase

Article abstract of DOI:10.1007/s00706-006-0439-7

A Baylis-Hillman adduct of methyl acrylate and ethyl glyoxalate was converted into the trichloroacetimidate that in the presence of DABCO rearranged to the corresponding trichloroacetamide. Eventually, hydrolysis under acidic conditions, led to the hydrochloride of racemic β-methyleneaspartic acid. Springer-Verlag 2006.

Full text of DOI:10.1007/s00706-006-0439-7

Monatshefte f€ur Chemie 137, 357–363 (2006)
DOI 10.1007/s00706-006-0439-7
Straightforward Synthesis of (R,S)-b-
Methyleneaspartic Acid, an Inhibitor
of Glutamate-Aspartate Transaminase
ꢀ
Roberta Galeazzi, Gianluca Martelli, Mario Orena ,
and Samuele Rinaldi
ꢀ
Dipartimento di Scienze dei Materiali e della Terra – Universita Politecnica
delle Marche – Via Brecce Bianche, 60121 Ancona, Italy
Received July 6, 2005; accepted (revised) August 12, 2005
Published online February 27, 2006 # Springer-Verlag 2006
Summary. A Baylis-Hillman adduct of methyl acrylate and ethyl glyoxalate was converted into the
trichloroacetimidate that in the presence of DABCO rearranged to the corresponding trichloroacet-
amide. Eventually, hydrolysis under acidic conditions, led to the hydrochloride of racemic ꢀ-methyl-
eneaspartic acid.
Keywords. Amino acid; Enzymes; Rearrangement; Baylis-Hillman; Dehydroamino acids.
Introduction
The synthesis of conformationally constrained analogues of natural amino acids is
of large interest, since their insertion into peptides can cause significant changes in
their conformation, which in turn may affect the ability of the peptide to fit to a
receptor and they are a valuable tool for probing activity changes in a peptide [1, 2].
In addition, these compounds can act as enzymatic inhibitors, and interesting
informations about the structure at the active site can be obtained by considering
the conformational restrictions of the guest molecule.
Among these compounds, (R,S)-ꢀ-methyleneaspartic acid (1) [3] was recog-
nized as selective inhibitor of glutamate-aspartate transaminase (Scheme 1), having
higher activity than vinylglycine that irreversibly inactivates this enzyme [4].
In fact, this transaminase gained increasing interest due to its high potential for
industrial production of amino acids [5], although the use is hampered by the low
equilibrium constant of the catalyzed reaction [6]. Thus, a better knowledge of the
structure at the active site can lead to changes in the protein sequence directed at
increasing the reaction rate.
ꢀ
Corresponding author. E-mail: m.orena@univpm.it

358
R. Galeazzi et al.
Scheme 1
Results and Discussions
Within a programme dealing with the preparation of non-proteinogenic amino
acids with conformational restrictions starting from Baylis-Hillman adducts [7],
we directed our attention towards the adduct 2 and devised to introduce a nitro-
gen-containing functionality in place of the hydroxy group. Although 2 can be
obtained starting from methyl acrylate and ethyl glyoxalate [8], a side reaction
was invariably observed leading to substantial amounts of diastereomeric com-
pounds 3. Thus, in order to avoid the formation of these products, the Baylis-
Hillman reaction was carried out in the presence of water (5 equiv) and we were
pleased to obtain 2 in higher yield (Scheme 2) [9].
Then, according to a procedure earlier developed in our laboratory [7], we first
treated 2 with trichloroacetyl isocyanate to give the corresponding trichloroacetyl
carbamate 4 in quantitative yield (Scheme 3). However, the subsequent reaction
with DABCO in DCM failed to give the expected trichloroacetylamino derivative 5,
that was obtained in very low yield, whereas trichloroacetamide was formed as the
major product. Thus, as an alternative route, we considered that the amide 5 could
be obtained starting from the trichloroacetimidate 6. However, the procedures
reported in literature did not allow to obtain 6 in satisfactory yield [10]. Accord-
ingly, a modified approach to give 6 was devised using trichloroacetonitrile as both
solvent and reactant [11]. The subsequent treatment of 6 with a small amount of
Scheme 2

Synthesis of (R,S)-ꢀ-Methyleneaspartic Acid
359
Scheme 3
DABCO in DCM allowed to obtain in nearly quantitative yield the trichloroacetyl-
amino derivative 5 that was eventually converted into the hydrochloride of (R,S)-
ꢀ-methyleneaspartic acid (1) by reaction in refluxing 6 M HCl (Scheme 3) [3c].
The rearrangement leading to the trichloroacetamide 5 starting from the tri-
chloroacetimidate 6 probably involves attack of DABCO to the conjugate double
bond followed by elimination of the anion of trichloroacetamide via an0SN0 reaction
(Scheme 4, path a). In turn, this anion attacks at C-2 by a further SN reaction to
give the trichloroacetamide 5, DABCO being the leaving group (Scheme 4, path b).
Eventually, it is worth noting that when 6 was treated with an equimolar
amount of DABCO for 1 h, a diastereomeric mixture of 1-ethyl 4-methyl (E)-3-
methyl-2-trichloroacetylamino-2-butendioate (7a) and its (Z)-isomer (7b) was
Scheme 4

360
R. Galeazzi et al.
Scheme 5
obtained in 88% overall yield and 67:33 ratio. Their configurations were assigned
on the basis of 1H NMR data and confirmed by NOE experiments. In fact the signal
for (3-CH3) in the (E)-diastereomer is more deshielded (2.01 ppm) than the corre-
sponding signal in the (Z)-diastereomer (1.87 ppm). Moreover, the methyl group at
2.01 ppm in the (Z)-diastereomer afforded a positive NOE (5.1%) over the adjacent
methylene of the ethoxycarbonyl group, suggesting a syn relationship, whereas this
effect was missing in the (E)-diastereomer for the methyl group at 1.87 ppm
(Scheme 5).
Considerable interest in recent years has been directed toward the synthesis of
ꢁ,ꢀ-dehydroamino acids [12], which are constituents of a variety of biologically
important peptides. They are also valuable intermediates for the preparation of both
natural and unnatural amino acids. Thus, compounds 7a and 7b can provide useful
precursors for both the enantiomerically pure forms of 3-methyl aspartic acid by
hydrogenation of the double bond [13], carried out in the presence of an appro-
priate chiral catalyst, and work directed towards this goal is currently underway in
our laboratory.
Experimental
IR spectra were recorded in CHCl3 on a Nicolet FT-IR 20-XS spectrophotometer. Unless otherwise
noted, NMR spectra (200 MHz for 1H, 50 MHz for 13C, chemical shifts as ppm in the ꢂ scale, coupling
constants J in Hz) were recorded at 25ꢁC on a Varian Gemini 200 spectrometer. Diastereomeric ratios
were determined by glc analysis by using a Chrompack 9001 gas-chromatograph equipped with a
capillary column Chrompack 7720 (50 mꢂ0.25 mm id; stationary phase CP-Sil-5 CB). EIMS and CI-
MS analyses were carried out on a Hewlett-Packard spectrometer model 5890, series II. Elemental
analyses (C, H, N) were conducted using a Carlo-Erba 1106 elemental analyzer, and their results were
found to be in good agreement (ꢃ0.2%) with the calculated values. Column chromatography was
performed using Kieselgel 60 Merck (230–400 mesh ASTM).
1-Ethyl 4-methyl 2-hydroxy-3-methylenebutanedioate (2, C8H12O5) and 1-Ethyl 4-methyl
2-(1-ethoxycarbonyl-2-methoxycarbonylallyloxy)-3-methylenebutanedioates (3, C16H22O9)
Method A. To a mixture containing 20.4g ethyl glyoxalate (50% solution in toluene, 100mmol) and
8.6 g methyl acrylate (100 mmol), 1.12 g DABCO (10 mmol) were added. After stirring for 12h at rt,
the oil was purified by silica gel chromatography (cyclohexane:ethyl acetate ¼ 80:20) to give first

Synthesis of (R,S)-ꢀ-Methyleneaspartic Acid
361
13.2g (70%) 2 [8] and then a diastereomeric mixture 70:30 of 1-ethyl 4-methyl 2-(1-ethoxycarbonyl-2-
methoxycarbonylallyloxy)-3-methylenebutanedioates (3), as colorless oils.
1
2: IR (CHCl3): ꢃꢁ¼ 3490, 1732, 1636 cmꢄ1; H NMR (200MHz, CDCl3): ꢂ ¼ 1.24 (t, J ¼ 7.2 Hz,
3H, OCH2CH3), 3.51 (d, J ¼ 6.2 Hz, OH), 3.76 (s, 3H, OCH3), 4.22 (q, J ¼ 7.2 Hz, 2H, OCH2CH3),
4.83 (d, J ¼ 6.2 Hz, 1H, 2-CH), 5.92 (s, 1H, vinyl), 6.34 (s, 1H, vinyl) ppm; 13C NMR (CDCl3):
ꢂ ¼ 14.0, 52.0, 62.2, 71.2, 128.8, 138.1, 165.6, 172.2ppm; EIMS (70 eV): m=z (%) ¼ 188 (Mþ, 2), 143
(22), 116 (40), 84 (100).
1
3: IR (CHCl3): ꢃꢁ¼ 1743, 1720 cmꢄ1; H NMR (200MHz, CDCl3): ꢂ ¼ 1.22 (t, J ¼ 7.2 Hz, 6H,
OCH2CH3, 70%), 1.26 (t, J ¼ 7.2 Hz, 6H, OCH2CH3, 30%), 3.75 (s, 6H, OCH3, 30%), 3.78 (s, 6H,
OCH3, 70%), 4.18 (q, J ¼ 7.2 Hz, 4H, OCH2CH3, 70%), 4.20 (q, J ¼ 7.2 Hz, 4H, OCH2CH3, 30%),
4.97 (br s, 2H, CH–O), 6.02 (s, 2H, vinyl, 30%), 6.12 (s, 2H, vinyl, 70%), 6.42 (s, 2H, vinyl, 30%),
6.45 (s, 2H, vinyl, 70%) ppm; 13C NMR (50 MHz, CDCl3): ꢂ ¼ 13.6 (70%), 13.7 (30%), 51.6, 61.1
(30%), 61.5 (70%), 70.6, 76.0 (30%), 76.5 (70%), 128.2, 135.5 (30%), 135.6 (60%), 165.0, 165.3,
168.5 (70%), 168.6 (30%), 171.8 ppm; EIMS (70 eV): m=z (%) ¼ 358 (Mþ, 8), 343 (25), 185 (16), 136
(32), 84 (100).
Method B. To a mixture containing 21.4g ethyl glyoxalate (50% solution in toluene, 105 mmol)
and 9.1 cm3 methyl acrylate (100mmol) in 35 cm3 THF and 15cm3 DMSO containing 9.0 cm3 H2O
(500 mmol), 5.6 g DABCO (50 mmol) were added. After stirring for 12h at rt, the oil was purified by
silica gel chromatography (cyclohexane:ethyl acetate¼ 80:20) to give 16.0g (85%) 2 [8] as a colorless
oil. EIMS (70 eV): m=z (%) ¼ 188 (Mþ, 2), 143 (22), 116 (40), 84 (100).
1-Ethyl 4-methyl 2-trichloroacetylaminocarbonyloxy-3-methylenebutanedioate
(4, C11H12Cl3NO7)
To a solution containing 1.9 g 2 (10 mmol) in 50cm3 dry DCM, 1.9 g trichloroacetylisocyanate
(15 mmol) dissolved in 10cm3 dry DCM were added at 0ꢁC and the mixture was stirred at rt for
2 h [5]. The solvent was removed under reduced pressure and the residue was chromatographed on
silica gel (cyclohexane:ethyl acetate ¼ 80:20) to give 3.7 g (97%) 4 as a viscous oil. IR (neat):
1
ꢃꢁ¼ 3355, 1741, 1724 cmꢄ1; H NMR (200MHz, CDCl3): ꢂ ¼ 1.24 (t, J ¼ 7.2 Hz, 3H, OCH2CH3),
3.79 (s, 3H, OCH3), 4.22 (q, J ¼ 7.2 Hz, 2H, O–CH2CH3), 5.98 (s, 1H, 2-CH), 6.11 (s, 1H, vinyl), 6.55
(s, 1H, vinyl), 8.78 (s, 1H, NH) ppm; 13C NMR (50 MHz, CDCl3): ꢂ ¼ 13.9, 52.4, 62.3, 72.4, 91.4,
132.3, 133.7, 148.5, 157.5, 164.5, 166.7 ppm; EIMS (70 eV): m=z (%) ¼ 377–375 (Mþ, 2), 143 (22),
116 (40), 84 (100).
1-Ethyl 4-methyl 2-trichloroacetylamino-3-methylenebutanedioate (5, C10H12Cl3NO5)
To a solution containing 1.9 g 4 (5.0mmol) in 20cm3 DCM at 0ꢁC, 65mg DABCO (0.5mmol) were
added and the mixture was stirred for 15 min at 0ꢁC. After dilution with 150 cm3 ethyl acetate, the
organic layer was washed first with 30 cm3 1 M HCl and then with 100 cm3 brine. After drying
(Na2SO4), the solvents were removed under reduced pressure and the residue was purified by silica
gel chromatography (cyclohexane:ethyl acetate ¼ 80:20), to give 0.35g (20%) 5 as a colorless oil,
1
followed by 0.6 g trichloroacetamide. IR (neat): ꢃꢁ¼ 3351, 1722, 1668 cmꢄ1; H NMR (200MHz,
CDCl3): ꢂ ¼ 1.26 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 3.80 (s, 3H, OCH3), 4.25 (q, J ¼ 7.1 Hz, 2H,
OCH2CH3), 5.25 (d, J ¼ 7.9 Hz, 1H, 2-CH), 6.10 (s, 1H, vinyl), 6.47 (s, 1H, vinyl), 7.81 (d, J ¼ 7.9 Hz,
Hz, 1H, NH) ppm; 13C NMR (50 MHz, CDCl3): ꢂ ¼ 13.9, 52.3, 56.0, 62.5, 92.0, 131.7, 134.1, 161.2,
165.3, 168.2 ppm; EIMS (70 eV): m=z (%) ¼ 334–332 (MHþ, 4), 318–316 (12), 260 (22), 198 (18),
158 (44), 99 (100).
1-Ethyl 4-methyl 2-trichloroacetiminoxy-3-methylenebutanedioate (6, C10H12Cl3NO5)
To a solution of 9.5 g 2 (50 mmol) in 25cm3 CCl3CN (250mmol) and 25cm3 dry THF, 375 mm3 DBU
(3.5mmol) dissolved in 0.5cm3 dry THF were added in 1 min at ꢄ15ꢁC under vigorous stirring. After
1 h the cooling bath was removed and temperature raised to rt. The mixture was directly purified by
silica gel chromatography (cyclohexane:ethyl acetate¼ 95:5) to give 12.1g (73%) 6 as a colorless oil.

362
R. Galeazzi et al.
1
IR (neat): ꢃꢁ¼ 3339, 1720, 1671 cmꢄ1; H NMR (200 MHz, CDCl3): ꢂ ¼ 1.27 (t, J ¼ 7.3 Hz, 3H,
OCH2CH3), 3.82 (s, 3H, OCH3), 4.25 (q, J ¼ 7.3 Hz, 2H, OCH2CH3), 6.12 (s, 1H, vinyl), 6.13 (s,
1H, 2-CH), 6.55 (s, 1H, vinyl), 8.53 (br s, ¼NH) ppm; 13C NMR (50 MHz, CDCl3): ꢂ ¼ 13.7, 53.0,
61.5, 73.4, 90.3, 129.7, 134.1, 160.8, 164.5, 166.8ppm; EIMS (70 eV): m=z (%) ¼ 334–332 (MHþ, 5),
318–316 (6), 170 (32), 161 (24), 144 (100).
1-Ethyl 4-methyl 2-trichloroacetylamino-3-methylenebutanedioate (5, C10H12Cl3NO5)
Following method B, to a solution containing 1.7g 6 (5.0mmol) in 20 cm3 DCM at 0ꢁC, 61 mg
DABCO (0.5mmol) were added and the mixture was stirred for 2 min at 0ꢁC. Then the mixture
was diluted with 150 cm3 ethyl acetate and the organic layer washed with 30cm3 3 M HCl and
100 cm3 brine. After drying (Na2SO4) the solvents were removed under reduced pressure and the
residue was purified by silica gel chromatography (cyclohexane:ethyl acetate ¼ 80:20), to give 1.7 g
(quantitative yield) 5 as a colorless oil. EIMS (70 eV): m=z (%) ¼ 334–332 (MHþ, 4), 318–316 (12),
260 (22), 198 (18), 158 (44), 99 (100).
(R,S)-Methyleneaspartic acid hydrochloride (1 ꢅ HCl)
To 1.0 g 5 (3.0mmol) 20cm3 6 M HCl were added and the mixture was refluxed for 24h. The reaction
mixture was concentrated at reduced pressure leaving 0.44 g (78%) 1 ꢅ HCl [3c] as a white foam.
1H NMR (200MHz, D2O, DSS): ꢂ ¼ 4.89 (s, 1H, 2-CH), 6.28 (s, 1H, vinyl), 6.67 (s, 1H, vinyl) ppm;
13C NMR (50 MHz, D2O): ꢂ ¼ 54.1, 131.9, 135.7, 167.0, 169.7ppm; MS (CI): m=z (%) ¼ 146 (Mþ, 2),
116 (40), 84 (100).
Ethyl 4-methyl (E)-3-methyl-2-trichloroacetylamino-2-butendioate (7a, C10H12Cl3NO5)
and its (Z)-diastereomer (7b, C10H12Cl3NO5)
To a solution containing 1.7 g 6 (5.0mmol) in 20 cm3 DCM at 0ꢁC, 0.61g DABCO (5.0mmol) were
added and the mixture was stirred for 1 h at rt. Then the mixture was diluted with 150 cm3 ethyl acetate
and the organic layer washedwith 30 cm3 3 M HCl and 100 cm3 brine. After drying (Na2SO4) the solvents
were removed under reduced pressure and the residue was purified by silica gel chromatography (cyclo-
hexane:ethyl acetate ¼ 80:20) to give 1.5g (88% overall yield, dr¼ 67:33) of 7a and 7b as colorless oils.
(E)-Diastereomer 7a: IR (CHCl3): ꢃꢁ¼ 3345, 1719, 1660 cmꢄ1
;
1H NMR (200MHz, CDCl3):
ꢂ ¼ 1.31 (t, J ¼ 7.3 Hz, 3H, OCH2CH3), 1.87 (s, 3H, 3-CH3), 3.78 (s, 3H, OCH3), 4.33 (q, J ¼ 7.3 Hz,
Hz, 2H, OCH2CH3), 12.09 (br s, 1H, NH) ppm; 13C NMR (50 MHz, CDCl3): ꢂ ¼ 13.3, 13.7, 52.6,
62.3, 91.4, 110.6, 138.1, 159.0, 162.5, 169.4 ppm; EIMS: m=z (%) ¼ 334–332 (MHþ, 6), 274–272
(12), 186 (21), 113 (65), 85 (100).
(Z)-Diastereomer 7b: IR (CHCl3): ꢃꢁ¼ 3341, 1722, 1665cmꢄ1
;
1H NMR (200MHz, CDCl3):
ꢂ ¼ 1.29 (t, J ¼ 7.3 Hz, 3H, OCH2CH3), 2.01 (s, 3H, 3-CH3), 3.79 (s, 3H, OCH3), 4.25 (q, J ¼ 7.3 Hz,
2H, OCH2CH3), 8.05 (br s, 1 H, NH) ppm; 13C NMR (50 MHz, CDCl3): ꢂ ¼ 13.8, 16.5, 52.5, 62.4,
91.9, 126.6, 134.2, 159.4, 162.3, 168.0ppm; EIMS: m=z (%) ¼ 334–332 (MHþ, 6), 274–272 (12),
186 (21), 113 (65), 85 (100).
Acknowledgements
We thank M.I.U.R. (Roma, Italy) for financial support within the framework Cofin 2004.
References
[1] Hanessian S, McNaughton-Smith G, Lombart HG, Lubell WD (1997) Tetrahedron 53: 12789; for
lead references, see also: Hruby VJ, Soloshonok VA, Eds. (2001) Asymmetric Synthesis of Novel
Sterically Constrained Amino Acids; Symposium-in-Print, Tetrahedron 57: 6329
[2] a) Gante J (1994) Angew Chem Int Ed Engl 33: 1699; b) Hruby VJ, Balse PM (2000) Curr Med
Chem 7: 945; c) Palczewski K, Kumassaka T, Hori T, Behnke CA, Motoshima H, Fox BA,

Synthesis of (R,S)-ꢀ-Methyleneaspartic Acid
363
Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M (2000) Science
289: 739
[3] a) Cooper AJL, Fitzpatrick SM (1982) J Am Chem Soc 104: 332; b) King S, Phillips AT (1978)
J Neurochem 30: 1399; c) Dowd P, Kaufman C (1979) J Org Chem 44: 3956
[4] Dowd P, Kaufman C, Kaufman P (1985) J Org Chem 50: 882
[5] a) Stirling DI (1992) The use of aminotransferases for the production of chiral amino acids and
amines. In: Collins AN, Sheldrake GN, Crosby J (Eds) Chirality in Industry, John Wiley & Sons,
New York, p 209; b) Taylor PP, Pantaleone DP, Senkpeil RF, Fotheringham IG (1998) Trends
Biotechnol 16: 412
[6] a) Okamoto A, Ishii S, Hirotsu K, Kagamiyama H (1999) Biochemistry 38: 1176; b) Kawaguchi
S, Kuramitsu S (1998) J Biol Chem 273: 18353; c) Hayashi H, Inoue K, Mizuguchi H,
Kagamiyama H (1996) Biochemistry 35: 6754; d) Okamoto A, Nakai Y, Hayashi H, Hirotsu
K, Kagayamiya H (1998) J Mol Biol 280: 443
[7] Ciclosi M, Fava C, Galeazzi R, Orena M, Sepulveda-Arques J (2002) Tetrahedron Lett 43: 2199
[8] a) Fikentscher R, Hahn E, Kud A, Oftring A, Ger Offen DE 3,444,097, June 05 1986; (1987)
Chem Abstr 107: 7781s; b) Brown JM, Rose M, Knight FI, Wienand A (1995) Recl Trav Chim
Pays-Bas 114: 242
[9] Patra A, Roy AK, Joshi BS, Roy R, Batra S, Bhaduri AP (2003) Tetrahedron 59: 663
[10] a) Overman LE (1974) J Am Chem Soc 96: 597; b) Overman LE (1976) J Am Chem Soc 98:
2901; c) Mehmandust M, Petit Y, Larcheveque M (1992) Tetrahedron Lett 33: 4313; d) Martin C,
Bortolussi M, Bloch R (1999) Tetrahedron Lett 40: 3735; e) Nishikawa T, Asai M, Ohyabu N,
Isobe M (1998) J Org Chem 63: 188; f) Kang SH, Kim GT, Yoo YS (1997) Tetrahedron Lett 38:
603; g) Kang SH, Kim JS, Youn JH (1998) Tetrahedron Lett 39: 9047
[11] Galeazzi R, Martelli G, Orena M, Rinaldi S (2004) Synthesis 2560
[12] For a review, see: Schmidt U, Lieberknecht A, Wild J (1988) Synthesis 159
[13] Imamoto T, Oohara N, Takahashi H (2004) Synthesis 1353