Decomposition of copper-amino acid complexes by oxalic acid dihydrate

Article abstract of DOI:10.1139/v2012-032

A facile approach to the synthesis of some side-chain-protected amino acids via oxalic acid dihydrate as the copper sequestering reagent is presented. The copper in the amino acid complex reacted with oxalic acid dihydrate to form insoluble cupric oxalate, with the free amino acid released. Compared with conventional methods, this method is convenient, inexpensive, and environmentally friendly.

Full text of DOI:10.1139/v2012-032

557
Decomposition of copper – amino acid complexes
by oxalic acid dihydrate
Yi Liu, Genguang Jia, Xin Ling, Nuo Lan, Youguang Zheng, Sai Li, Ling Zhang,
Ling Liu, Rongli Zhang, and Yunsheng Xue
Abstract: A facile approach to the synthesis of some side-chain-protected amino acids via oxalic acid dihydrate as the cop-
per sequestering reagent is presented. The copper in the amino acid complex reacted with oxalic acid dihydrate to form in-
soluble cupric oxalate, with the free amino acid released. Compared with conventional methods, this method is convenient,
inexpensive, and environmentally friendly.
Key words: side-chain-protected amino acid, oxalic acid dihydrate, cupric oxalate, synthesis.
Résumé : On a mis au point une approche simple à la synthèse des divers acides aminés à chaîne latérale protégée impli-
quant le dihydrate de l’acide oxalique comme réactif de séquestration de du cuivre. Le cuivre du complexe avec l’acide
aminé réagit avec le dihydrate de l’acide oxalique pour former l’oxalate cuivrique insoluble et la libération de l’acide aminé
libre. Par comparaison avec les autres méthodes conventionnelles, cette méthode est pratique, peu coûteuse et écologique.
Mots‐clés : acide aminé à chaîne latérale protégée, dihydrate de l’acide oxalique, oxalate cuivrique, synthèse.
[Traduit par la Rédaction]
is a low yield of protected amino acids, with the cost greatly
increased. Similarly, in the case of copper removal using so-
dium borohydride, issues such as heat generation during bulk
production of protected amino acids also result. Despite the
fact that recent research has confirmed the efficiency of so-
dium sulfide in the detachment of copper from the com-
plexes, its serious hazards to the environment are noteworthy.
Given the limitations of the methods concerning the cop-
per(II) deprotection discussed previously, it is difficult to
realize the industrialization. Several studies have shown that
oxalic acid can be preferable for its inexpensiveness and
friendliness to the environment and can form complexes with
copper.11–13 In this paper, we report a highly convenient and
efficient method in which oxalic acid dihydrate is employed
to detach the copper(II) complexes, to prepare the side-chain
amino-protected acetyl, Boc, Fmoc, and Z (benzyloxy car-
bonyl) ornithines,and lysines, respectively.
Introduction
Since the emergence of glycylglycine synthesis from a di-
ketopiperazine by Fischer and Fourneau1 in 1901, peptide
synthesis has undergone tremendous progress and it is now
possible to routinely synthesize a protein consisting of over
200 amino acids. However, when the amino acids involved
are trifunctional, such as lysine, ornithine, etc., it is necessary
to protect their side-chain functional groups. This is accom-
plished by first reacting the primary amino acids with cupric
ions to form the stable chelate complex, followed by a reac-
tion with acylation reagents to facilitate selective protection
of the side-chain functional group.
To recover the side-chain-protected amino acids, it is
necessary to decompose the copper – amino acid complexes,
for which several methods are available involving several
important reagents including EDTA,2,3 potassium cyanide,4
8-quinolinol,5 hydrogen sulfide,6,7 thioacetamide,8 sodium
borohydride,9 and sodium sulfide.10 EDTA is one of the
most widely employed copper-sequestering reagents. How-
ever, the resultant copper–EDTA complex during the course
of the reaction is water-soluble, which can lead to effluent
problems with the water-soluble side-chain-protected amino
acids. When copper is removed from the complexes using
KCN, H2S, or thioacetamide, there is a marked drawback in
that these reagents are highly toxic and, thioacetamide in par-
ticular, is a potential cancer-causing chemical. When copper
is removed from the complexes employing 8-quinolinol, there
Results and discussion
The behavior of copper in oxalic acid solutions has been
well-studied. The results have shown that copper ions with
oxalate can easily form complexes; however, the complexa-
tion ability in the solution depends on pH (1–1.5).13 In con-
tinuation of our efforts towards the decoppering reaction, we
employed oxalic acid dihydrate to sequester copper from the
copper – amino acid complexes. In general, cupric oxalate is
insoluble in water or diluted hydrochloric acid. To accelerate
Received 9 February 2012. Accepted 20 April 2012. Published at www.nrcresearchpress.com/cjc on 6 June 2012.
Y. Liu,* G. Jia,* X. Ling, N. Lan, Y. Zheng, S. Li, L. Zhang, L. Liu, R. Zhang, and Y. Xue. School of Pharmacy, Xuzhou Medical
College, 84 West Huaihai Road, Xuzhou, 221002 Jiangsu, P.R. China.
Corresponding author: Yi Liu (e-mail: cbpe200902@163.com).
*Yi Liu and Genguang Jia are co-first authors.
Can. J. Chem. 90: 557–559 (2012)
doi:10.1139/V2012-032
Published by NRC Research Press

558
Can. J. Chem. Vol. 90, 2012
Table 1. Preparation of the side-chain-protected amino acids.
chloric acid (30 mL, 0.01 mol/L) was added, followed by
the addition of oxalic acid dihydrate (10.3 mmol), and the re-
sulting mixture was stirred at 80 °C (the pH of the solutions
was ∼1.1 at this time). At the end of stirring for 1–3 h, the
consequent cupric oxalate precipitate was filtered. The filtrate
was neutralized with dilute NaOH. The resulting precipitate
was isolated by filtration and then rinsed with distilled water
(50 mL × 4) to obtain the side-chain-protected amino acids
a–f. The water-soluble products (g–h) were isolated by the
removal of the solvent and extraction of the residue with ace-
tone.
Entry
Producta
H-Orn(Z)-OH
Yield (%)
Purity (%)
98.5
98.3
97.9
98.8
97.7
98.0
97.1
96.9
1
2
3
4
5
6
7
8
a
b
c
d
e
f
g
h
70
65
78
74
69
65
59
63
H-Orn(Fmoc)-OH
H-Orn(acetyl)-OH
H-Lys(Z)-OH
H-Lys(Fmoc)-OH
H-Lys(acetyl)-OH
H-Orn(Boc)-OH
H-Lys(Boc)-OH
1
aAll compounds were characterized by H NMR and elemental analysis.
H-Orn (Z)-OH (compound a)
the reaction, diluted hydrochloric acid was selected as the ve-
hicle, with the concentrations of the acid investigated. It was
observed that, with the acid maintained at a concentration of
0.01 mol/L, the method for the removal of copper ions by
oxalic acid dihydrate was effective and could completely
eradicate the sulfide problem during the course of the reac-
tion (the pH of the solutions is ~1.1). The results in Table 1
show that the oxalic acid dihydrate-mediated detachment of
copper ions from all the copper amino acid complexes re-
sulted in protected amino acids of high purity with no race-
mization.
20
White crystals (70%), mp 248–250 °C, ½aꢀD +17.6° (lit.18
20
mp 228–230 °C, ½aꢀD +17.5°, c 0.01, water/acetone = 1:1,
1
and equal volumes of hydrochloric acid). H NMR d: 7.35–
7.45 (m, 5H, Ar-H), 5.08 (s, 2H, Ar–CH2), 3.37–3.44 (m,
1H, a-H), 3.19–3.23 (m, 2H, d-H), 1.81–1.86 (m, 2H, g-H),
1.65–1.70 (m, 2H, b-H). Anal. calcd for C13H18N2O4: C
58.63, H 6.81, N 10.52; found: C 58.57, H 6.84, N 10.48.
H-Orn (Fmoc)-OH (compound b)
White crystals (65%), mp 151–153 °C (lit.19 mp 152–154 °C).
1H NMR d: 3.41–3.46 (m, 1H, a-H), 3.17–3.23 (m, 2H, d-H),
1.78–1.84 (m, 2H, g-H), 1.61–1.67 (m, 2H, b-H), 4.53–4.69 (m,
2H, COOCH2), 4.31–4.37 (m, 1H, Fmoc-9H), 7.29–7.80 (m,
8H, Ar-H). Anal. calcd for C20H22N2O4: C 67.76, H 6.26, N
7.91; found: C 67.83, H 6.31, N 7.87.
The copper complexes of the following amino acids were
3
prepared in accordance with the methods in literature: N -
d
3
d
Boc–lysine,14 N -Boc–ornithine,15 N -Z–lysine, N -Z–ornithine,8
N -Fmoc–lysine, N -Fmoc–ornithine,16 N -acetyl–ornithine,
3
d
d
and N -acetyl–lysine.17
3
The copper – amino acid complexes were treated with ox-
alic acid dihydrate in diluted hydrochloric acid for 1–2 h at
80 °C to afford side-chain-protected amino acids a–j in
yields of 55%–80% (Scheme1 and Table 1). The isolated
H-Orn (Acetyl)-OH (compound c)
White crystals (78%), mp 246–249 °C (lit.20 mp 248–250 °C).
1H NMR d: 3.38∼3.45 (m, 1H, a-H), 3.18–3.24 (m, 2H, d-H),
1.81–1.87 (m, 2H, g-H), 1.58–1.65 (m, 2H, b-H), 2.01 (s, 3H,
CH3). Anal. calcd for C7H14N2O3: C 48.27, H 8.01, N 16.08;
found: C 48.20, H 8.09, N 16.01.
1
products were characterized by H NMR and elemental anal-
yses.
Conclusion
In conclusion, we have confirmed that oxalic acid dihy-
drate is a useful reagent in removing copper from amino
acid copper complexes during the preparation of several
side-chain-protected amino acids. This method may also be
applicable to bulk production of side-chain-protected amino
acids.
H-Lys (Z)-OH (compound d)
White crystals (74%), mp 245–248 °C (lit.8 mp 250 °C). 1H
NMR d: 7.31–7.68 (m, 5H, Ar-H), 5.11 (s, 2H, Ar-CH2),
3.13–3.22 (m, 2H, 3-H), 3.41–3.46 (m, 1H, a-H), 1.77–
1.82 (m, 2H, b-H), 1.38–1.45 (m, 2H, g-H), 1.57–1.64 (m,
2H, d-H). Anal. calcd for C14H20N2O4: C 59.99, H 7.19, N
9.99; found: C 59.90, H 7.24, N 10.00.
Experimental
H-Lys (Fmoc)-OH (compound e)
Melting points were measured in open capillaries and are
uncorrected. Optical rotations were measured on a PerkinElmer
model 341 automatic polarimeter. Elemental analysis was
performed on a PerkinElmer CHNS analyzer. 1H NMR
spectra were recorded in CD3SOCD3 on a Bruker Avance
500 spectrometer; chemical shifts (d) are reported in parts
per million (ppm) relative to tetramethylsilane (TMS), serv-
ing as an internal standard. Solvents and reagents were pur-
chased from respective suppliers and used without further
purification.
White crystals (69%), mp 209–210 °C (lit.19 mp 210–
212 °C). 1H NMR d: 3.41–3.47 (m, 1H, a-H), 3.17–3.24
(m, 2H, 3-H), 1.32–1.39 (m, 2H, g-H), 1.51–1.59 (m, 2H,
d-H), 1.79–1.85 (m, 2H, b-H), 4.74 (d, J = 3.21 Hz, 2H,
OCH2), 4.42 (t, J = 3.21 Hz, 1H, Fmoc-9H), 7.26∼7.80
(m, 8H, Ar-H). Anal. calcd for C21H24N2O4: C 68.46, H
6.57, N 7.60; found: C 68.38, H 6.49, N 7.67.
H-Lys (Acetyl)-OH (compound f)
1
White crystals (65%), mp 245–249 °C (dec). H NMR d:
General procedure for the preparation of side-chain-
protected amino acids a–h
To dissolve the copper – amino acid complex (10.0 mmol)
in a capped flask at ambient temperature, diluted hydro-
3.40–3.46 (m, 1H, a-H), 3.18–3.22 (m, 2H, 3-H), 1.30–1.38
(m, 2H, g-H), 1.53–1.61 (m, 2H, d-H); 1.73–1.85 (m, 2H, b-
H), 2.00 (s, 3H, CH3). Anal. calcd for C8H16N2O3: C 51.05,
H 8.57, N 14.88; found: C 51.00, H 8.64, N 14.77.
Published by NRC Research Press

Liu et al.
559
Scheme 1.
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676. doi:10.1246/bcsj.38.676.
H-Orn (Boc)-OH (compound g)
20
White crystals (59%), mp 210–213 °C, ½aꢀD +15.5° (lit.21
20
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mp 209.75 °C, ½aꢀD +15.2°,17 c 1.0, acetic acid). H NMR
d: 3.35–3.46 (m, 1H, a-H), 3.19–3.25 (m, 2H, d-H), 1.81–
1.92 (m, 2H, g-H), 1.58–1.65 (m, 2H, b-H), 1.44 (s, 9H,
3 × CH3). Anal. calcd for C10H20N2O4: C 51.71, H 8.68, N
12.06; found: C 51.66, H 8.60, N 12.11.
1
H-Lys (Boc)-OH (compound h)
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20
White crystals (63%), mp 247–249 °C, ½aꢀD +17.1° (c 1,
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2004, 45 (50), 9297. doi:10.1016/j.tetlet.2004.10.060.
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1
acetic acid). H NMR d: 3.37–3.44 (m, 1H, a-H), 3.14–3.21
(m, 2H, 3-H), 1.32–1.38 (m, 2H, g-H), 1.50–1.58 (m, 2H, d-
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Acknowledgements
The authors are cordially indebted to these financial sup-
ports: “Qing-Lan” Project of Jiangsu Province, the Industrial-
ization of Scientific Research Promotion Projects of
Universities and Colleges in Jiangsu Province (Grant No.
2011-16), the Natural Science Fund for Universities and Col-
leges in Jiangsu Province (11KJB350005), the Project of Pro-
vincial Key Laboratory in Universities in Jiangsu Province
(Grant Nos. JSBL0803, C0903, and C0904), the Science and
Technology Plan Projects of Xuzhou (Grant Nos. XF11C037,
XF11C062, and XF11C065), the Superiority Academic Dis-
cipline Construction Project of Jiangsu Higher Education In-
stitutions, and the Six-Major-Talent-Summit Project of
Jiangsu Province.
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