Radioiodination of humic substances via azocoupling with 3-[ 125I]iodoaniline

Article abstract of DOI:10.1021/es702671v

A new method is described for radiolabeling humic substances (HS) with iodine radioisotopes. The method radiolabels the electron-rich aromatic moieties of HS with the 3-[¹²⁵I]iodobenzenediazonium ion via azocoupling. The method uses four steps: (i) 3-aminobenzenetrimethylstannane is synthesized and isolated by using a silica gel column, (ii) 3-[¹²⁵I]iodoaniline is synthesized and isolated by HPLC, with radiochemical yields of up to 60%, (iii) 3-[¹²⁵I]iodobenzenediazonium chloride is synthesized, and the reaction mixture from this step is used in step iv to radioiodinate HS with radiochemical yields of up to 95% (with reference to 3-[¹²⁵I] iodoaniline). The advantage of this method is that it is selective radiolabeling, placing the radiolabel in a specific site (the 3-position of the phenyl ring) within HS molecules, which minimizes unwanted secondary chemical interactions. Investigations of the stability of the radiolabel and the effect of photoreductive dehalogenation showed that there was a negligible release of ¹²⁵I. The production of radiolabeled HS using this method allows the sensitive detection of HS in laboratory and field studies. In addition, the method offers the possibility of using different iodine radioisotopes simultaneously in investigations using HS.

Full text of DOI:10.1021/es702671v

Environ. Sci. Technol. 2008, 42, 4083–4087
have not been experimentally defined, but models describe
Radioiodination of Humic
Substances via Azocoupling with
3-[125I]Iodoaniline
K A R S T E N F R A N K E , * , † J Ö R G T . P A T T , †
H E R M A N N K U P S C H , † A N D
P E T E R W A R W I C K ‡
Institute of Interdisciplinary Isotope Research,
Permoserstrasse 15, 04318 Leipzig, Germany, and
Loughborough University, Department of Chemistry,
Leicestershire, LE11 3TU, United Kingdom
HS as having a supramolecular-like structure with a sample
of HS containing a mixture of molecular weights (16, 17).
Because of the ease and sensitivity of the detection of
radioactivity, investigations of the behavior of HS are made
easier by using radiolabeled HS. Radioactive elements have
mostly been used to investigate the reactions of metals with
HS (10, 18-21). However, these complexes may be of limited
use in some investigations because of their relative instability.
Producing radiolabeled HS in which the radiolabel is co-
valently bound to HS molecules should produce a more stable
radiolabel. Whichever method is used, it should not change
the inherent properties of the molecules and should produce
radiolabeled molecules in high yield and high specific activity.
A number of different radiolabeling methods have been
reported (23, 24) but, although they are useful, they have
limitations in terms of authenticity, specific activity, yields,
handling, or stability. In addition to these methods, radio-
iodination via an electrophilic H/I substitution on aromatic
moieties has been reported, which is based on a widely used
and reliable mild method of radiolabeling proteins and other
biomolecules (22). The advantages of this method are ease
of handling, high labeling yield, and the preservation of the
properties of the humic material. The covalent bonding of
iodine on humic substances has been reported (25) but may
be complicated because of secondary interactions with thiol-
and imidazole-like structures and because of the complex
aqueous chemistry of iodine (26, 27). These complications
may lead to radiolabeling at different sites within the HS
molecules (i.e., nonspecific radiolabeling), resulting in ra-
diolabels that have different stabilities with respect to pH,
Eh, etc. These unwanted interactions are considered, and
somewhat overcome, by suitable quenching and purification
methods reported in ref 22. Secondary reactions may be
avoided by using a method that was reported for radioha-
logenating amino acids and peptides (28). The method was
used to radiolabel the carbon backbone of HS with 18F via
azocoupling using a precursor (4-[18F]fluorobenzenediazo-
nium chloride) (29). The 18F labeled group (Figure 1) can
react with the polyphenolic groups in the HS. The increased
effort required to make the precursor is compensated for by
the production of a more selective and specific label.
However, the disadvantage with using 18F is its short half-
life (109.7 min), which limits its use to experiments of short
time scales. A similar method was used for the 14C labeling
of HS (30), but the ꢀ- decay may limit the use of the
radiolabeled HS.
Received October 23, 2007. Revised manuscript received
March 4, 2008. Accepted March 6, 2008.
A new method is described for radiolabeling humic substances
(HS) with iodine radioisotopes. The method radiolabels the
electron-rich aromatic moieties of HS with the 3-[125I]iodoben-
zenediazonium ion via azocoupling. The method uses four
steps: (i) 3-aminobenzenetrimethylstannane is synthesized and
isolated by using a silica gel column, (ii) 3-[125I]iodoaniline is
synthesized and isolated by HPLC, with radiochemical yields of
up to 60%, (iii) 3-[125I]iodobenzenediazonium chloride is
synthesized, and the reaction mixture from this step is used in
step iv to radioiodinate HS with radiochemical yields of up
to 95% (with reference to 3-[125I]iodoaniline). The advantage of
this method is that it is selective radiolabeling, placing the
radiolabel in a specific site (the 3-position of the phenyl ring)
within HS molecules, which minimizes unwanted secondary
chemicalinteractions. Investigationsofthestabilityoftheradiolabel
and the effect of photoreductive dehalogenation showed that
therewasanegligiblereleaseof125I. Theproductionofradiolabeled
HS using this method allows the sensitive detection of HS in
laboratory and field studies. In addition, the method offers the
possibility of using different iodine radioisotopes simultaneously
in investigations using HS.
Introduction
Humic substances are important because they (i) are part of
the global carbon cycle (1), (ii) influence transport processes,
which affects plant nutrition (2), (iii) affect pharmacological
potency (3), (iv) affect the availability of chemotoxic pollutants
(4-6), (v) need to be included in estimates of risk assessments
in the disposal of radioactive wastes (7, 8), and (vi) are
important in carbon sequestration of atmospheric CO2 (1, 9).
Humic substances have therefore been studied for many
years, and their properties and behavior have been inves-
tigated using a wide range of different analytical methods
and techniques, ranging from in situ measurements of HS
in the field to the use of advanced spectrometric techniques
in the laboratory (10-15). The aims of these investigations
were to identify and characterize HS at the molecular level
and to investigate the bulk behavior of the rather complicated
and heterogeneous HS molecules. The structure(s) of HS
The limitations can be overcome by the use of radioiso-
topes of iodine (123I (t1/2 ) 13.2 h), 124I (t1/2 ) 4.17 days), 125
I
(t1/2 ) 60.14 days), and 131I (t1/2 ) 8.04 days)), and a promising
precursor for azocoupling with HS is 3-[125I]iodoanaline. This
paper describes the synthesis of this precursor, the optimi-
zation of the azocoupling reaction to radiolabel HS, and the
investigations of the radiolabeled HS. Two different humic
acids (HA) were radiolabeled, but the method can be used
to radiolabel fulvic acid.
Materials and Methods
Chemicals and solvents were obtained in reagent or analytical
quality from VWR. 125I was purchased from Amersham
Bioscience as sodium iodide in NaOH solution (pH 8.0-12.0).
The certified specific activity was >0.6 TBq/mg iodide with
a radionuclidic purity of at least 99.9% 125I.
* Corresponding author phone: +49 (0)341 235 2004; fax: +49
(0)341 235 2731; e-mail: franke@iif-leipzig.de.
† Institute of Interdisciplinary Isotope Research.
‡ Loughborough University.
Thin layer chromatography (TLC) was carried out using
plates (Polygram SIL-G/UV254) purchased from Macherey-
9
10.1021/es702671v CCC: $40.75
 2008 American Chemical Society
VOL. 42, NO. 11, 2008
/
ENVIRONMENTAL SCIENCE & TECHNOLOGY 4083
Published on Web 04/24/2008

FIGURE 1. Preparation of 4-[18F]fluorobenzenediazonium ions.
FIGURE 3. Synthesis of nca 3-[125I]iodoaniline.
FIGURE 2. Synthesis of 3-aminobenzenetrimethylstannane.
Nagel. TLC plates were contacted with imaging plates (BAS-
MS2325, Fujifilm) and afterward scanned with an IP-Reader
(BAS-1800II, Fujifilm). The images were recorded and evalu-
ated with the AIDA software (Raytest Isotopenmessgera¨te).
High performance liquid chromatography (HPLC) was
conducted using an Agilent HP1100, which was equipped
with a quaternary HPLC pump, a vacuum degasser, an
autosampler, a multiwavelength UV/vis absorption detector,
and a NaI(Tl)-detector. HPLC analysis was controlled by using
Chemstation software for LC systems (Agilent). Two different
HPLC columns were used: (i) LiChrospher 100CN 5 µm, 250
mm × 10 mm and (ii) TOSOH Bioscience G3000 PWxl, 300
mm × 7.8 mm for high performance size exclusion chro-
matography (HPSEC).
FIGURE 4. Synthesis of nca 3-[125I]iodobenzenediazonium ion.
TABLE 1. Buffer Solutions
buffer
tris
description
0.2 M tris(hydroxymethyl)-aminomethane
(250 mL)
0.1 M HCl (315 mL)
H2O (435 mL)
phosphate
borate
0.667 M KH2PO4 (31 mL)
0.667 M Na2HPO4 × 2H2O (969 mL)
0.05 M Na2B4O7 (559 mL)
0.1 M HCl (441 mL)
Radioiodinated HS was purified by using ultrafiltration
(Microsep Centrifugal Devices; PALL Corporation; filter
media: modified polyethersulfone on polyethylene substrate,
MWCO 3000 Da) at the recommended centrifugation pa-
rameters (centrifugal force: 7500g, time: 60 min, and maxi-
mum sample volume: 3.5 mL) or by precipitation by the
addition of acid. The radioactive yield of radiolabeled HS
was calculated from measurements of the 125I radioactivity
in an Aktivimeter ISOMED 1000 (Nuklear-Medizintechnik
Dresden GmbH). The following paragraphs describe the
preparation of radiolabeled HS.
Step 1: Synthesis of 3-Aminobenzenetrimethylstan-
nane (2). Tetrakis(triphenylphosphin)-palladium(0) (50 mg),
3-bromoaniline (100 µg), and hexamethyldistannane (250
mg) in toluene (2 mL) were mixed in a 10 mL vial (Figure 2)
(31). The mixture was stirred for 12 h at 105 °C under an
atmosphere of N2. After cooling, the black precipitate was
filtered off and washed with EtOAc. The remaining EtOAC
was evaporated. The residue was redissolved in petroleum
ether-ethyl acetate, 80:20 (v/v). The synthesis of 3-ami-
nobenzenetrimethylstannane was monitored by TLC against
3-bromoaniline. The product (2) was isolated from 1 by
chromatographic separation (petroleum ether-ethyl acetate,
80:20 (v/v)) using a silica gel filled glass column (180 mm ×
35 mm). The progress of elution was monitored by TLC. The
fraction containing the product (2) was evaporated to dryness.
Step 2: Synthesis of 3-[125I]iodoaniline (3). 3-Aminoben-
zenetrimethylstannane (1 mg) and IODOGEN (50 µg) were
dissolved in dichloromethane (550 µL) and then added to an
aqueous solution (500 µL) containing [125I]NaI (0.75 MBq)
(Figure 3). The mixture was stirred for 30 min. The organic
phase was then separated and evaporated. The residue was
dissolved in the HPLC mobile phase and injected into an
HPLC column (LiChrospher 100CN, eluent: petroleum ether-
ethyl acetate, 95:5, v/v and mobile phase flow rate: 5 mL
min-1). 3-Iodoaniline was used to determine the retention
time (13.05 min) of 3. The fraction containing the product
was acidified (1 M HCl, 100 µL) and then evaporated.
Step 3: Synthesis of 3-[125I]Iodobenzenediazonium Chlo-
ride (4). The 1 M sodium nitrite (20 µL) and 1 M HCl (830
µL) were added to 3-[125I]iodoaniline in 1 M HCl (100 µL)
(Figure 4). After 10 min at 0 °C, 1 M urea (50 µL) was added
to the mixture at 0 °C for 20 min.
Step 4: Preparation of 125I Radiolabeled HA (6). Two
different HAs were radiolabeledsHA purchased from Aldrich
(Aldrich HA) and peat bog humic acid (peat bog HA), which
was extracted from the ombrotrophic raised bog Kleiner
Kranichsee, situated in the upper Ore Mountains (Erzgebirge),
Saxony, Germany (32). The peat bog HA was extracted and
isolated using the procedure reported by Aiken (33). Different
buffers (Table 1) were used for the preparation of a 200 mg/L
HA stock solution (0.02 M KCl).
A total of 100 µL of the reaction mixture from step 3,
containing 3-[125I]iodobenzenediazonium chloride (4) in 1
M HCl, 1 M sodium hydroxide (100 µL), and tris buffer (300
µl), was added to 500 µL of HA stock solution (5) (Figure 5).
After an incubation time of 20 min at 0 °C, [125I]I-[125I]I-HA
(6) was separated by ultrafiltration or precipitation (addition
of 50 µL of 1 M HCl).
Results and Discussion
Optimization and Yields. Step 2. A total of 70-80% of the
125I radioactivity from Na125I was found in the organic phase
after extracting the radioactive product from the aqueous
phase. After evaporation of the organic phase and redissolving
it in the HPLC mobile phase, 3-[125I]iodoaniline was further
purified by HPLC (Figure 6) to remove organic impurities
that might (i) interfere with the diazotization process and/or
(ii) be incorporated in the HA structure, thereby changing
the properties of the HA. The radiochemical yield after HPLC
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FIGURE 5. Reaction scheme for the preparation 125I radiolabeled HAs.
FIGURE 6. Chromatographic separation of 3-[125I]iodoaniline
(radioactivity: 125I, gray line and UV absorption (254 nm): black
line). Retention time of the product: 13.05 min.
FIGURE 8. Influence of HA (peat bog HA) concentration on
radiolabeling yield after precipitation.
interactions). With these considerations in mind, the use of
phosphate and borate buffers was excluded (36). The
following studies were therefore conducted using tris buffer,
which would enable the use of HPSEC and investigations of
metal interactions.
was up to 75%, and step 2 was in total 60%. The diazotization
reaction is reported extensively in the literature (29, 30, 34, 35).
A yield can not be given for the product (4) because of the
high instability of this intermediate. However, a high excess
of nitrite ensured a quantitative chemical reaction (Fig-
ure 6).
Both purification methods (ultrafiltration and precipita-
tion), which were used in step 4 to separate [125I]I-HA from
the reaction mixture, showed comparable radiolabeling
yields. Ultrafiltration is gentler than precipitation, but
precipitation is a fast alternative (but cannot be used with
fulvic acid). A partial loss of small size fractions of humic
material is possible due to filtration, but suitable filters with
small molecular weight cutoffs (500 to 100 Da) and a mild
centrifugation would minimize the loss at the expense of
time. Alteration of HA caused by precipitation is unlikely
since precipitation is widely used for its isolation. The
radiochemical yields differ with the origin of HA and the
purification method (Figure 7). As expected, a higher ra-
diolabeling yield was obtained with increasing concentrations
of peat bog HA because of the increasing number of binding
sites (Figure 8). Almost a 100% yield was achieved with
concentrations of 20 mg/L and above.
The radiolabeling of HA (6) was optimized with respect
to (i) the buffer used, (ii) the concentration of HA, and (iii)
the method used to purify the radiolabeled HA. According
to ref 29, radiolabeling should be carried out at pH 8.
Nevertheless, different buffer systems (borate buffer, phos-
phate buffer, tris buffer, pH adjusted by means of NaOH/
HCl) were tested to determine the optimum radiolabeling
conditions. Two purification procedures (ultrafiltration and
precipitation) were used to purify the radiolabeled HA.
Depending on the purification step used, radiolabeling yields
of between 50 and 95% were obtained (Figure 7). Purification
by precipitation resulted in similar high yields for Aldrich
HA and peat bog HA using tris buffer. However, using tris
buffer and ultrafiltration, higher yields were observed from
peat bog HA than from Aldrich HA. In addition to producing
a high yield, a buffer must be noncomplexing (i.e., should
not complex with metals if the aim of the experimental work
is to investigate metal-HS interactions as well as HS
Stability of the Radiolabel. The stability of the radiolabel
with respect to changes in pH and to physical stress was
tested by repeated precipitation and ultrafiltration. When
FIGURE 7. Dependence of radioabeling yield on HA origin (black: peat HA and white: Aldrich HA), used buffer system (1: borate
buffer, 2: phosphate buffer, 3: NaOH/HCl, and 4: tris buffer), and purification method.
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VOL. 42, NO. 11, 2008

FIGURE 9. Radiochemical yield dependence on the number of
precipitation steps of [125I]I-HA (peat bog HA).
FIGURE 11. Release of 125I caused by photoreductive dehalo-
gination after radiation with white light as a function of time
(three parallel experiments with ) peat bog HA and 0 Aldrich
HA).
FIGURE 12. Comparison of radioactivity (125I, gray line) and UV
absorption (254 nm) (black line) chromatograms of radio-
iodinated peat bog HA.
FIGURE 10. Longer term stability of the radiolabeled HA () peat
bog HA and 0 Aldrich HA) tested by ultrafiltration.
was observed in repeated investigations. If necessary, samples
should be kept in the dark, which is common laboratory
practice to avoid alterations in humic material.
investigating precipitation (Figure 9), there was a loss of
approximately 5% of 125I after the fifth precipitation. The loss
was probably because of incomplete precipitation of HA.
When investigating ultrafiltration (Figure 9), nearly 45% of
the total amount of 125I was detected in the <3000 Da fraction
after five filtrations. The loss was probably because of a partial
temporary break of the interaction forces within the mo-
lecular humic assembly, which resulted in HA passing
through the filter membrane.
HPSEC Investigation. HPSEC was used to compare the
radiochromatogram of [125I]I-HA with the UV absorption (254
nm) chromatogram to investigate as to whether the complete
range of molecular weights in the peat bog HA was radio-
labeled (Figure 12). Similar results were obtained for Aldrich
HA. Figure 12 clearly shows that 125I coelutes with the UV
absorbing HA constituents, demonstrating that radiolabeling
was not selective to a particular fraction of molecular weights
but that all molecular weights and sizes of HA molecules
were radiolabeled by this method. Nonselectivity is essential
if radiolabeled HA is to be used in investigations where the
bulk behavior of HA is to be investigated.
The combination of radiolabeling at a specific site in a
molecule, nonselectivity toward molecules or fractions of
molecules, and the possibility of using a number of different
radioisotopes of iodine in the same experiment offers an
excellent and reliable tool for geochemical investigations.
The simultaneous use of different radioisotopes allows the
study of complex exchange processes of HA in coagulation,
flocculation, transport, and sorption processes.
Longer Term Stability of the Label. Radiolabeled samples
of peat bog HA and Aldrich HA were prepared and purified
by precipitation. The longer term stabilities of the radiolabels
were tested over a period of 45 days (arbitrarily chosen period
of time) (Figure 10) by storing the samples in the dark at 4
°C and taking samples at regular intervals for ultrafiltration
to determine the release of radioiodine. Results showed that,
although there was a constant loss of approximately 5%, there
was no significant time dependent release of radioiodine.
Since the release of 125I was not time dependent, the loss was
probably caused by 125I remaining on the filter and surfaces
or to HA passing through the filter. It can be concluded that
the [125I]I-HA species were stable during these investigations.
Photoreductive Dehalogination. Photoreductive deha-
logenation of halophenols is well-known (37, 38), and this
was investigated using white light (40 W light bulb held 50
cm from the sample) to simulate the working conditions in
a laboratory. Radiated and nonradiated [125I]I-HA stock
solutions were kept at 0 °C over a period of 110 h. At regular
intervals, aliquots were taken from these solutions, and the
possible release of iodine was measured by using ultrafil-
tration. The release of 125I caused by photoreductive deha-
logination is shown in Figure 11. After 110 h of radiation,
3.8% of total 125I was released from [125I]I-HA. No significant
difference between radiolabeled peat bog HA or Aldrich HA
Acknowledgments
We thank Linda Sands and Dr. Nick Evans for their expert
support during work at the Department of Chemistry of
Loughborough University. We also thank Doris Ro¨ꢀler and
Dr. Alexander Mansel for fruitful discussions and their helpful
comments.
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