Synthesis of o,p-EDDHA and its detection as the main impurity in o,o-EDDHA commercial iron chelates

Article abstract of DOI:10.1021/jf025727g

Ethylenediamine-N,N′bis(o-hydroxyphenyl) acetic acid (o,o-EDDHA) is one of the most efficient iron chelates employed to relieve iron chlorosis in plants. However, the presence of positional isomers of EDDHA in commercial iron chelates has been recently demonstrated, and among them, it has been claimed that ethylenediamine-N(o-hydroxyphenylacetic)-N′(p-hydroxyphenylacetic) acid (o,p-EDDHA) is the main impurity present in EDDHA fertilizers. Here we report the preparation of o,p-EDDHA, a compound whose synthesis had not been previously reported. The synthetic o,p-EDDHA is able to form ferric complexes, and it has been used as a standard in the analysis of the impurities of commercial iron fertilizers. The presence of o,p-EDDHA/Fe³⁺ in commercial samples has been unambiguously demonstrated by HPLC.

Full text of DOI:10.1021/jf025727g

J. Agric. Food Chem. 2002, 50, 6395−6399 6395
Synthesis of o,p-EDDHA and Its Detection as the Main Impurity
in o,o-EDDHA Commercial Iron Chelates
MAR GOÄ MEZ-GALLEGO,† MIGUEL A. SIERRA,*,† ROBERTO ALCAÄ ZAR,†
PEDRO RAMIÄREZ,† CARMEN PIN˜ AR,† MARIÄA JOSEÄ MANCHEN˜ O,†
#
SONIA GARCIÄA-MARCO,# FELIPE YUNTA,# AND JUAN JOSEÄ LUCENA
Departamento de Qu´ımica Orga´nica, Facultad de Qu´ımica, Universidad Complutense,
28040 Madrid, Spain, and Departamento de Qu´ımica Agr´ıcola, Facultad de Ciencias,
Universidad Auto´noma de Madrid, 28049 Madrid, Spain
Ethylenediamine-N,N′bis(o-hydroxyphenyl)acetic acid (o,o-EDDHA) is one of the most efficient iron
chelates employed to relieve iron chlorosis in plants. However, the presence of positional isomers of
EDDHA in commercial iron chelates has been recently demonstrated, and among them, it has been
claimed that ethylenediamine-N(o-hydroxyphenylacetic)-N′(p-hydroxyphenylacetic) acid (o,p-EDDHA)
is the main impurity present in EDDHA fertilizers. Here we report the preparation of o,p-EDDHA, a
compound whose synthesis had not been previously reported. The synthetic o,p-EDDHA is able to
form ferric complexes, and it has been used as a standard in the analysis of the impurities of
commercial iron fertilizers. The presence of o,p-EDDHA/Fe3+ in commercial samples has been
unambiguously demonstrated by HPLC.
KEYWORDS: o,o-EDDHA; o,p-EDDHA; iron chelates; fertilizers
INTRODUCTION
Iron chlorosis is a nutritional disorder in plants that affects
their development and decreases the yield of many crops.
Chlorosis results in a decrease in the amount of chlorophyll
and is manifested in a gradual disappearance of the green
coloring of the plants (1, 2). The origins of this complex problem
are diverse, ranging from nutritional disorders to infections
caused by fungus, bacteria, insects, etc. Today, fertilization with
synthetic iron chelates is the most common agricultural practice
to relieve this problem, and ethylenediamine-N,N′bis(o-hydroxy-
phenyl)acetic acid (o,o-EDDHA) (1 in Figure 1) is among the
most efficient iron chelating agents used (3). This compound
has two phenol groups on a diaminocarboxylic acid backbone
and is able to form ferric complexes (2a and 2b in Figure 1)
of high stability in neutral and in alkaline solutions. The
structural characterization of the Mg-(rac-Fe(III)-EDDHA)2 salt
shows that this class of chelate has an octahedral disposition,
with the o,o-EDDHA ligand hexacoordinated to the Fe nucleus
(4). The [6,5,6] arrangement of rings across the Fe-center having
the phenolic groups in equatorial positions is considerably more
favored than the alternative [5,5,5] arrangement. Nevertheless,
it has been calculated that 0.5% of the [5,5,5] complex is present
in the racemic mixture (5).
Figure 1. Chelating agents and chelates described in the text.
Most commercial iron fertilizers are based on ligands that
have phenol groups on a polyamine-carboxylic acid backbone,
and several synthetic methods have been developed to obtain
this type of compound. The synthesis of o,o-EDDHA (1 in
Figure 1) originally reported by Kroll (6) in 1957, is a Strecker
reaction on the imine derived from ethylenediamine and
salicylaldehyde. Although the procedure can be employed to
obtain other symmetrically substituted EDDHAs (7, 8), the
* Corresponding author. E-mail: sierraor@quim.ucm.es. Fax: 34-91-
3944103.
† Universidad Complutense.
# Universidad Auto´noma.
10.1021/jf025727g CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/20/2002

6396 J. Agric. Food Chem., Vol. 50, No. 22, 2002
Go´mez-Gallego et al.
drawback is the need of liquid HCN during the industrial
process. Other approaches to the synthesis of ethylenediamine-
bis(o-hydroxyphenyl)acetic acids are based on a Mannich-like
reaction between phenol (or substituted phenols), ethylenedi-
amine, and glyoxylic acid (9, 10). This method is used for the
preparation of all of the EDDHA currently in the market.
The use of commercial chelates has dramatically increased
in the last years, not only in agricultural chemistry but also in
other fields (2, 11, 12). Some analytical methods for the quality
control of such products have been developed, but generally
they have focused on determining the content of chelated metal
in the product (13-15). However, other important aspects
related to the purity of the ligands employed in the commercial
formulations have been neglected. This is a very significant
matter because, as we have commented before, o,o-EDDHA is
prepared from ethylenediamine, sodium glyoxylate, and excess
of phenol. This method produces mixtures of three regioisomeric
products, namely, o,o-EDDHA, o,p-EDDHA, and p,p-EDDHA
(1, 3, and 4, respectively, in Figure 1) in variable amounts.
The lack of purity of commercial iron chelates of o,o-EDDHA
(16) and the presence of positional isomers of the phenol group
in commercial o,o-EDDHA/Fe3+ samples has been recently
addressed by us (17).
It has been claimed that the main impurity present in o,o-
EDDHA commercial iron chelates is the ethylenediamine-N(o-
hydroxyphenylacetic)-N′(p-hydroxyphenyl)acetic acid (o,p-
EDDHA) isomer (3 in Figure 1) (16, 17). However, as far as
we are aware, the intentional synthesis and purification of o,p-
EDDHA has not been previously reported, and hence, the
presence of o,p-EDDHA as an impurity in commercial fertilizers
has not been unambiguously confirmed to date. This fact excited
our interest in the design of synthetic routes for the preparation
of o,p-EDDHA and by extension to any other related unsym-
metrically substituted chelating agents. This class of compounds
cannot be obtained by any of the reported methods that are
directed to the synthesis of symmetrical o-hydroxyarylacetic acid
derivatives. In addition, the ability of o,p-EDDHA to form ferric
complexes is unknown, and it would be interesting to determine
their structure and properties, thereby establishing whether the
iron chelates derived from 3 could be useful as fertilizers. This
paper is a part of our ongoing research to establish the factors
that affect the efficacy of pure and commercial iron chelates
used with agricultural purposes. Here we report two comple-
mentary routes to prepare o,p-EDDHA 3. The procedures
described below could be employed to synthesize other unsym-
metrical ethylenediamine-bis(hydroxyphenyl)acetic acids, which
opens the uses of chelates in agriculture and other fields to new
possibilities. The obtained o,p-EDDHA will be used to confirm
the presence o,p-EDDHA/Fe3+ complexes in commercial prod-
ucts.
Figure 2. Synthetic route for o,p-EDDHA starting from arylglycinates and
glyoxal.
NaHCO3 solution followed by extraction with CH2Cl2 or generated in
situ by reaction with Et3N. Monoprotected ethylenediamine was
prepared by a modification of the reported procedure (19). The full
experimental data of all the compounds synthesized in this study are
depicted in the Supporting Information.
The o,p-EDDHA product was evaluated for its ability to complex
iron(III) and copper(II) by photometric titration and potentiometric
titrations, respectively. The end points in both potentiometric and
photometric titrations were calculated by the second derivate smoothed
of the original data (20) and by Grant equation, respectively. The molar
extinction coefficient for the iron chelate was also determined at 480
nm in order to compare it with that of the o,o-EDDHA.
For preparation of the o,p-EDDHA/Fe3+ complex, the chelating agent
was dissolved in NaOH (1:3 molar ratio). Then, an amount of FeCl3
that was calculated to be 5% in excess of the molar amount of ligand,
was added. The pH was adjusted between 6 and 8 during the addition,
and finally, the o,p-EDDHA/Fe3+ solution (pH 7) was left to stand
overnight and then filtered through 0.45-µm membranes. The solution
of o,p-EDDHA/Fe3+ complex and a solution of a commercial Fe-chelate
were compared using a Waters Symmetry C18 150 × 3.9 mm column
and an HPLC with a Waters 2690 Separation Module (Alliance). The
absorption spectra (200-600 nm) were recorded with a Waters 996
photodiode array detector and Millenium 2010 chromatography data
system. All commercially available compounds were used without
further purification.
RESULTS AND DISCUSSION
MATERIALS AND METHODS
We have designed two complementary routes to synthesize
o,p-EDDHA. The first approach requires arylglycinates and
glyoxal as reagents, whereas the second is based on the Strecker
reaction starting from substituted benzaldehydes and ethylene-
diamine.
Prior to the synthesis, the feasibility of the different steps of
the first approach was tested by preparing ethylenediamine-bis-
phenylacetic acid starting from commercially available phen-
ylglycine (Aldrich). The full experimental procedure is detailed
as Supporting Information Material.
All the products obtained in the synthesis of o,p-EDDHA were
characterized by spectroscopic techniques. 1H NMR and 13C NMR
1
spectra were recorded on a Bruker 200-AC (200.13 MHz for H and
50.03 for MHz13C) spectrometer. Chemical shifts are given in ppm
relative to the corresponding deuterated solvent. IR spectra were taken
on a Perkin-Elmer 781 spectrometer. Merck silicagel (230-400 mesh)
was used as the stationary phase for purification of crude reaction
mixtures by flash column chromatography. The synthesis of o- and
p-methoxyphenylglycines was done following the procedure of Stein
et al. (18). The corresponding methyl glycinates were obtained as
hydrochlorides by the standard procedure, refluxing the amino acids
with SOCl2 in MeOH. Free methyl o- and p-methoxyphenylglycinates
were obtained from their hydrochlorides by neutralization with saturated
As the first approach to synthesize o,p-EDDHA, methyl
o-methoxyphenylglycinate (5 in Figure 2) was employed as the
starting material. Thus, equimolar amounts of glyoxal mono-

Synthesis of o,p-EDDHA
J. Agric. Food Chem., Vol. 50, No. 22, 2002 6397
Figure 3. Synthetic route for o,p-EDDHA starting from substituted
benzaldehydes and ethylenediamine.
acetal (6 in Figure 2) (60 wt. % in H2O) and glycinate (5 in
Figure 2) were reacted in dry MeOH, and the imine thus
obtained (79%) was subsequently hydrogenated to form ami-
noacetal (7 in Figure 2) in quantitative yield. Because of the
known instability of R-aminoaldehydes (21), the formylation
of the nitrogen atom (Ac2O/HCOOH) (22, 23) had to be effected
prior to the deprotection of the acetal group in 7. Treatment of
the obtained formamide with FeCl3‚6H2O, in boiling Cl2CH2
(24) yielded aldehyde (8 in Figure 2) (82%) that was condensed
with p-methoxyphenylglycinate (9 in Figure 2) to give the
expected imine in the next step. The imine was subsequently
transformed into diester (10 in Figure 2) by catalytic hydro-
genation (65%, two steps). Finally, o,p-EDDHA was obtained
from diester (10 in Figure 2) by acid hydrolysis and further
treatment with concentrated HBr. The best results were obtained
by performing the sequential hydrolysis-deprotection of the
methoxy group in this order. In fact, the removal of the methoxy
groups in 10 with BBr3 occurred, yet in very low yields (29%).
Through this route, o,p-EDDHA was isolated as dihydrobromide
and as a 1:1 mixture of diastereoisomers (Figure 2). The
Figure 4. Photometric plot (A) where the end points have been calculated
by the linear segments intersection calculated with the minimum of the
second smoothed derivated (B). (C) Potentiometric titration with Cu(II)
solution. Grant equation was used to calculated the end point.
14 was achieved by formylation of the amino groups and
subsequent treatment with concentrated hydrochloric acid. In
this way, o,p-methoxy-EDDHA (15 in Figure 3) isolated as
dihydrochloride, and as a 1:1 mixture of diastereoisomers was
obtained in 60% yield. On the basis of 1H and 13C NMR
measurements, this compound was identical to that resulting
from the hydrolysis of compound 10 (Figure 2) with concen-
trated HCl. Therefore, the two approaches to the synthesis of
o,p-EDDHA 3 were successfully accomplished (Table 1).
The titrimetric purity of the synthetic o,p-EDDHA 3 obtained
by potentiometric titration with Cu(II) solution (69.7%) is
slightly higher than that obtained by photometric titration with
Fe(III) solution (59.7%) (Figure 4). This difference could be
due to the presence in the sample of a small amount of impurities
having amino group, able to complex copper but not iron.
Despite this fact, the photometric plot is very clean and clearly
indicates that the product may be used as standard compound.
The molar extinction coefficient (ꢀ) at 480 nm obtained for
o,p-EDDHA/Fe3+ was 2130. This value is almost half of the ꢀ
observed for the o,o-EDDHA/Fe3+ complex at the same
wavelength (4814) (26). At 480 nm, the light is being absorbed
structure of the product is fully consistent with the 1H and 13
NMR data.
C
The second approach started with the condensation between
the mono BOC-derivative of ethylenediamine (11 in Figure 3)
and o-anisaldehyde. The resulting imine was reacted with
trimethylsilyl cyanide (TMSCN) (25) in absence of catalyst, to
form R-aminonitrile (12 in Figure 3) in very high yield (93%).
Formylation of the amino group in 12 (HCOOH/Ac2O) followed
by treatment with HCl (gas) in dry MeOH, resulted in the
hydrolysis of the nitrile and the BOC groups, to give amide
(13 in Figure 3) (55%). This compound was next condensed
with p-anisaldehyde to yield the expected imine (62%), which
was transformed into nitrile (14 in Figure 3) (72% yield) with
TMSCN. Although Strecker adducts are generally stable under
neutral conditions, they may undergo rapid decomposition under
either acidic or basic conditions via a retro-Strecker reaction
(22). To avoid this undesirable process, the hydrolysis of nitrile

6398 J. Agric. Food Chem., Vol. 50, No. 22, 2002
Go´mez-Gallego et al.
Table 1. 1H and 13C Signal Assignments for Compound 3 in D O/Na2CO a
2
3
proton
δH
carbon
δC
CH (aromatics)
6.96−6.72 (4H)
6.54−6.34 (4H)
CH (aromatics)
C ipso (aromatics)
COOH
131.2, 130.6, 129.9, 129.2, 118.3, 117.8
159.3, 127.0, 124.0
179.9, 177.7
CHNH
4.17 (1H), 3.7 (1H)b
CHNH
66.9, 64.3
CH
2
2.56−2.39 (4H)
CH
2
45.4, 45.0
a In units of ppm (1H 200.13 MHz, 13C 50.03 MHz). b Meso/racemic 1:1 ratio.
presence of o,p-EDDHA ferric complexes in commercial
samples is reported. The quantitative determination of o,p-
EDDHA/Fe3+ present in commercial chelates is currently being
studied in our laboratories.
In conclusion, we have developed two alternative routes to
the synthesis of o,p-EDDHA and by extension to any other
unsymmetrical structurally related chelating agents. To the best
of our knowledge, this is the first synthesis of o,p-EDDHA
reported in the literature. Furthermore, having obtained a pure
sample of o,p-EDDHA we have been able to confirm unam-
biguously that o,p-EDDHA/Fe3+ complex is actually present
in considerable amounts in commercial samples of o,o-EDDHA
iron chelates. The structure and stability, and the potential use
of the chelates derived from o,p-EDDHA as a source of iron,
are currently under investigation in our laboratories.
ABBREVIATIONS USED
o,o-EDDHA, ethylenediamine-N,N′bis(o-hydroxyphenyl-
acetic) acid; o,p-EDDHA, ethylenediamine-N(o-hydroxyphenyl-
acetic)-N′(p-hydroxyphenylacetic) acid.
Supporting Information Available: Full experimental details
and spectroscopic and analytical data for the compounds in
Figures 2, 3, and 6. This material is available free of charge
via the Internet at http://pubs.acs.org.
Figure 5. 480 nm Chromatograms of a typical Fe-EDDHA commercial
chelate (I), Fe-o,p-EDDHA (II), and standard Fe-o,o-EDDHA (III). UV−
visible spectra for peaks at 1.78 min are also included for I and II.
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Received for review June 7, 2002. Revised manuscript received July
26, 2002. Accepted July 26, 2002. Support for this work under Grant
2FD97-0314-CO2 from the CICYT (MEC-Spain) and the European
Commission is acknowledged. Grants to P. Ram´ırez (CAM), F. Yunta
(MEC), and S. Garc´ıa-Marco (CAM) are also gratefully acknowledged.
JF025727G