Organomercury compounds have a variety of applications despite their toxicity. This document discusses two recent research topics on the use of organomercury catalysts: 1) The development of organomercury haptens for enzyme-linked immunoassays to detect inorganic and organic mercury. Antibodies were successfully generated that could bind both mercury ions and organometallic forms of mercury. 2) A direct method for derivatizing disulfide bonds in proteins using organic mercury in alkaline medium without chemical pre-reducing agents. p-Hydroxymercurybenzoate was shown to effectively label disulfide groups in lysozyme, allowing for their one-step determination. Overall, the document

1. 1
CURRENT STATE OF RESEARCH ON ORGANO MERCURY CATALYSTS
Abstract
Organomercury compounds have been extensively employed as fungicides in
agriculture and in the paper industry, and in human medicine as diuretics, but their use in
this role is limited by toxicity and environmental concerns. Organomercury compounds
are versatile synthetic intermediates due to the well controlled conditions under which
they undergo cleavage of the Hg-C bonds. Organomercurials are commonly used in
transmetallation reactions with lanthanides and alkaline-earth metals.
Despite high toxicity, organomercury compounds accommodate all functional
groups and their remarkable chemical and thermal stability have made them particularly
attractive as synthetic intermediates. These organic mercury compounds have also gained
an interest in the field of bio-analytical chemistry with number of applications.
In present review we give a brief description about the uses and toxicity of
Organomercury compounds along with their applications in different aspects. The current
research on the use of Organomercury catalyst is discussed by presenting a brief review
on recent two research topics.

2. 2
Introduction
Commercially, organometallic compounds have been used extensively over the
past 50 years and in many of these uses there is a direct interaction with the natural
environment [1]. Their use include as pesticides (organomercury or organotin
compounds) [2], gasoline additives (methyl- and ethylleads), polymers (organosicons)
and other additives and catalysts [3]. In addition there is an increasing realization that
organometallics also exist as natural products in the environment (e.g. arsenic species)
[1]. This has led to intensive research into their biological properties, toxicities, pathways
and transformations in the environment and to their ultimate fate and disposal.
The toxicity of organomercury compounds is often greater than that of the mercury
in them; the volatile alkylmercury class is particularly dangerous. The results of
organomercury poisoning with respect to brain function is rapid penetration in to the
blood–brain barrier leads to sensory disturbance, tremor, ataxia, visual and hearing
difficulties [4]. Methylmercury is lipid soluble, rapidly diffuses through cell membranes,
and once it enters the cell is quickly bound by sulphydryl groups inhibiting protein and
RNA synthesis and causes particular damage to the developing brain [5].
Much study has been devoted to organomercury compounds, particularly in the
search for compounds of pharmacological value [6]. They find wide use in horticulture
and correspondingly a very large number of preparative methods have been developed
covering widely varying types of organomercury compounds [2].

3. 3
Organomercury compounds have only slight reactivity. A generally applicable
method of preparing dialkyl- and diaryl-mercury compounds is by treatment of alkyl or
aryl halides or alkyl sulfates with sodium amalgam [7].
2RX + 2Na + Hg HgR2 + 2NaX Eq. 1
However, most of the simple dialkyl and diaryl mercury derivatives are
conveniently prepared by reaction of mercury (n) chloride with the appropriate Grignard
reagent or other reactive organometallic compounds [7].
RMgX + HgCl2 RHgCl + MgXCl Eq. 2
RMgX + RHgCl HgR2 + MgXCl Eq. 3
Organomercury compounds such as RHgX (X= Cl, Br, I), or pseudo halide (CN,
SCN), or other anions such as OH, etc. are solid compounds and are soluble in various
organic solvents such as methanol, ethanol etc. They are thermally and photochemically
not very stable and should be stored in dark. They are toxic, particularly lower dialkyls
such as Me2Hg, Et2Hg etc. and develop appreciable vapor pressure. Diarylmercury
compounds such as Ph2Hg are less toxic. The Hg-C bond or Hg-X bonds in
organomercury compounds undergo a variety of reactions. Organomercury compounds
are not very reactive towards oxygen, water, alcohols, carbonyl compounds, and simple
alkyl halides. Organomercury compounds undergo alkylation, arylation and acylation
reactions. It may be pointed out that organomercurials with simple organic groups have
low nucleophilic character towards organic halides. The electrophilic alkylating reagents
such as triarylmethyl halides react with nucleophilic organomercurials [30].

4. 4
The early development of organomercurial chemistry as compared to other
organometallic compounds can be attributed to the high stability of organomercury
compounds in air and moisture. Organomercury compounds are very useful for the
preparation of other organometallic compounds of other metals by transmetallation
process. This method is a classical synthetic route and has been used conveniently for the
synthesis of organometallic compounds of other metals. The equations 4 and 5 have been
used for metals of group IA, IIA, IIIA, or transition metals [7].
R2Hg + R′−Μ RHgR′ + R-M Eq. 4
R2Hg + 2M Hg + 2R-M Eq. 5
Perhalogeno alkyl derivatives, Hg(CF3)2, Hg(CCl3)2 are useful reagents for transferring
CX3, CX2 and CX groups to other elements. Organomercury compounds are also used in
organic synthesis, in seed dressings, and as fungicides.
Taking into consideration the various properties of Organomercury compounds, they
are employed in a variety of applications despite of their toxicity. In this review we
discuss the application of organomercury haptens for enzyme-linked immunoassay of
inorganic [8] and use of organic mercury in alkaline medium for a direct, simple
derivatization of disulfide bonds in proteins without any chemical pre-reducing agents
[9].

5. 5
Methods
(i) Synthesis and application of organomercury haptens for enzyme-linked
immunoassay of inorganic and organic mercury.
Mercury metal, mercury salts, and organomercury compounds have played an
important role in the field of bioanalytical chemistry. Mercury metal has aided electron
microscopy characterization of proteins and has been incorporated into fluorescent
peptide and protein tags [10]. More recently, chemical and fluorescent sensors have been
fabricated to detect mercury and other metal species [11, 22]. As these compounds
associated with health hazards to humans, mammals, and aquatic systems, made them a
keen interest [23].
Despite of availability of various sophisticated analytical techniques that are
capable of detecting heavy metals at exceedingly low concentrations [12], there is always
an interest in the development of efficient, cost-effective, sensitive mercury detection and
easy-to-use immunoassays. Metal-containing bioconjugates for the purpose of generating
antibodies capable of binding metals and metal ions has been the focus of numerous
research efforts [24]. Although some of these earlier bioconjugates did not yield
antibodies with adequate binding specificity for analytical assays, metal chelate
bioconjugates have found practical applications as metal-labeled therapeutic antibodies
[13, 14]. One recent extension of the EDTA chelation methodology has been the
synthesis of a fluorescent optical sensor designed to detect Hg2
+ ions, [15, 25] in which
Hg2
+ ions are trapped in a cage-like structure arising from a porphyrin dimer, showed that

6. 6
the chemical sensor is capable of detecting Hg2
+ at concentrations of 10-4– 10-7 M with
high degrees of specificity preferentially over other divalent metal ions.
Current study focused on the design and preparation of synthetic organomercury
hapten amenable to protein conjugation methods, with the goal of generating an antibody
capable of binding both Hg(II) metal and organometallic forms of mercury in a variety of
sample media. Organomercury hapten synthesized by oxymercuration is readily linked
covalently to carrier proteins, provides a unique chelation methods compared to other
methods. In this study, the highly versatile mouse anti-mercury antibody afforded by
applying the chelation method was rigorously evaluated through various immunoassay
formats. The results from this work provided the impetus to pursue mAb production
which indicates that mAbs derived from the organomercury hapten will be particularly
potent, resulting in a versatile and highly sensitive mercury detection system.
Materials and synthesis:
All the required reagents and materials were bought from various vendors. Haptens and
bioconjugates were prepared by different methods. Those are as followed:
Crude heptane (1), tert-Butyl allyl carbamate (2), tert-Butyl N-[2-(chloromercurio)-3-[(1-
hydroxypentane-2-yl)oxy]propyl carbamate (3), tert-Butyl N-{3-[(1-hydroxypentane-2-
yl)oxy] propyl} carbamate (4), 2-[3-Amino-2-(chloromercurio)propoxy] pentane-1-ol (5)
BS3 bioconjugates and SMCC bioconjugates were prepared from the haptens and
Bovine serum albumin (BSA). Chicken IgG was chosen as the immunogen carrier to
minimize nonspecific binding. Then Plate coating and ELISA were performed as a part of
analysis.

7. 7
Results and discussion:
Cross-linking reaction parameters were studied to determine the optimal coupling
conditions prior to preparing bioconjugates by covalently linking hapten 1 to protein
carriers and the extent of benzylamine coupling to BSA was consistent regardless of the
incubation times that were studied (30–120 min) Fig (1)
Fig1. Bioconjugation of organomercury hapten 1 to BSA via BS3 and SMCC cross-
linking reagent [8]
When tryptic fragments of the BSA–BS3–hapten 1 bioconjugates were characterized
by MALDI–MS [26], it was found that 8 mol of hapten was covalently attached to 1 mol
of protein. Mercury was readily apparent by its characteristic isotopic MS pattern. The
mercury concentration found in the two IgG conjugates was greater than 250 ppm, and
both the unconjugated BSA and chicken IgG controls had negative mercury

8. 8
concentration values when compared with the standard curve, when conjugates, including
the chicken IgG–hapten 1immunogen, were analyzed using ICP spectrometry.
After the positive results with the organomercury heptanes immunization experiments
were performed on the mice with BSA–BS3–hapten 1 and the results proposed that the
hydrophilic nonpeptide character of the hapten, coupled with the rigid shape imparted by
the cyclopentane backbone, yields antibodies demonstrating high degrees of sensitivity
and specificity. It was perceived that hapten 1 is a logical choice for immunization
because the availability of mercury seemed to be more favorable for eliciting an immune
response than haptens with less rigid structures. The BSA–SMCC–hapten 1 conjugate
was then tested parallel to BS3 conjugate using six antiserum dilutions added to plates in
triplicate, and two HRP-labeled antibody concentrations (80 and 40 ng/ml) were used to
evaluate antibody binding to each solid phase (Fig. 2) At the higher detection antibody
concentration (80 ng/ml), the two curves are quite similar. However, the SMCC
conjugate demonstrated improved dynamic range (25%) compared with the BS3
conjugate when the HRP-labeled detection antibody concentration was reduced to
40ng/ml.

9. 9
Fig2. Mouse anti-mercury antibody binding response to solid-phase conjugates prepared
by coupling hapten 1 to BSA via two different cross-linking reagents (BS3vs. SMCC).
Both of the solid-phase conjugates were coated at 10µg/ml. Antibody response to both
solid-phase conjugates was measured using 80 and 40 ng/ml peroxidase-labeled goat
anti-mouse IgG. [8]
An additional organomercury hapten was synthesized (hapten 5 in Fig. 3) to
further evaluate the anti-mercury antibody selectivity.
Fig3. Synthesis of organomercury hapten 5 and coupling to BSA via SMCC [8]
BSA–SMCC–hapten 5 and BSA–SMCC– hapten 1 are compared for the
antibody binding with two structurally different organomercury substrates at 40 and 20
ng/ml HRP-labeled detection antibodies. Overall, antibody binding to hapten 5 was

10. 10
higher than the binding displayed toward hapten 1. The dynamic range displayed by both
solid-phase conjugates increased when using the lower HRP-labeled detection antibody
concentration (20ng/ml) relative to 40ng/ml (Fig. 4). Hapten 5 coupled more efficiently
to BSA because it is a more polar hapten than hapten 1, affording a hapten/protein ratio
higher than 8:1. These results are encouraging because the anti-mercury antibody
generated in this work displays unique binding capability to a variety of solid-phase
configurations, offering a high degree of flexibility in assay format design.
Fig4. Antibody response to haptens 1 and 5. Both haptens were coupled to BSA via
SMCC and coated onto microtiter plates at 10µg/ml. Antibody response to both haptens
was measured with 40 and 20 ng/ml peroxidase-labeled goat anti-mouse IgG. [8]
A preliminary competitive inhibition study was conducted using BSA–BS3–hapten
1-coated plates using various dilution of mouse serum in PBS, preincubated with
mercuric acetate (Hg(OAc)2) at 20 ppm, mercuric nitrate (Hg(NO3)2) at 20 ppm, and 10.0

11. 11
ppm of unconjugated hapten 1. Hapten 1 did not block the solid-phase binding capability
of the antibody as efficiently as the mercury salts. Perhaps unconjugated hapten 1 is less
available for antibody binding because there is greater conformational flexibility and less
steric impedance when freely soluble compared with the rigid conformation that results
when the hapten–BSA conjugate is adsorbed to the plate surface. (Fig 5)
Fig5. Preliminary competitive inhibition assay results using plates coated with BSA–
BS3–hapten 1 and Hg(OAc)2, Hg(NO3)2, and hapten 1 as inhibitors [8]
These results from this study showed the synthetic strategies applied for preparing
unique organomercury-based hapten and developing a highly sensitive immunoassay
capable of detecting mercury contamination in a variety of environmental conditions. The
resulting antibody demonstrated high degrees of mercury binding under a variety of assay
formats and was capable of binding to both inorganic (Hg2
+) and organic forms of
mercury.

12. 12
(ii) Direct, simple derivatization of disulfide bonds in proteins with organic mercury
in alkaline medium without any chemical pre-reducing agents.
Disulfide bonds present in the most of cysteine proteins influences the
thermodynamics of the protein folding. The changes in the RSH/RSSR ratio are
considered indices of oxidative damage and an abnormal sulfhydryl redox ratio often
related to many diseases. Several analytical methods using liquid chromatography
combined with different detection techniques have been developed for the analysis of
RSH and RSSR groups in proteins [29]. These methods are generally based on the
derivatization by suitable probes of -SH groups of the proteins and of disulfide bonds
after their chemical reduction to -SH groups. [16, 17]
Many reducing agents like dithiothreitol (DTT) or 2-mercaptoethanol, Sodium and
potassium borohydride, Tris-(2-carboxyethyl) phosphine (TCEP) and Dithionite (DT,
sodium hydrosulfite) were used for derivatization, but each had their own disadvantage.
Disulfide bond of cystine was hydrolyzed by treatment with HgCl2 is used in the strong
alkaline media. Studies were made on the interaction between p-
Hydroxymercurybenzoate (pHMB) with reduced -SH functional groups on proteins [18,
19,], which were found to be more effective with GSSG converting GSSG into GS-
pHMB.
In the present work the reaction of pHMB with oxidized thiolic proteins were
studied. Derivatization of lysozymes by pHMB in alkaline medium without any
preliminary reduction steps was followed by the determination of lysozymes-pHMB
complex by size exclusion chromatography (SEC) coupled to CVG–AFS [27], reporting

13. 13
the one-step determination of disulfide groups in the proteins by mercurial labeling in an
alkaline medium.
Materials and method:
All the required chemicals like pHMB (4-hydroxymercuric benzoic acid, sodium salt
were bought from various vendors. The phosphate buffer solutions (PBS) were prepared
from monobasic monohydrate sodium phosphate and dibasic anhydrous potassium
phosphate. pHMB–protein complexes were obtained by incubating the proteins with a
five-fold excess of pHMB at room temperature(21±1ºC) and analysed after 150 min if not
differently specified.
An HPLC–MW/UV combined reactor with CVG–AFS [27, 28] detection system was
used for all the measurements.
Results and discussion:
Optimization of the labeling procedure:
Lysozyme with a molecular weight of 18.4 kDa, four disulfide bonds and no
reduced cysteine was chosen as model protein to optimize derivatization reaction with
pHMB in alkaline medium.
Fig. 1 shows the normalized AF signal of lysozyme–pHMB complex (1:5 molar
ratio) after 180 min reaction time at 21ºC at various pH (6–14) with various concentration
of NaOH (pH 12–14). The fraction of mercury bound to the protein is calculated by AF
mercury-specific chromatograms, where (pHMB) is the concentration of pHMB
complexed with protein, calculated on the basis of the peak area of protein in the AF

14. 14
chromatogram. For pH ˂ 10 no signal was observed. For pH > 10 the lysozyme-pHMB
signal increased and reached a plateau for pH > 13.5. The plateau value ranged between
82 and 98%.
Fig1. Normalized AF signal of lysozyme/pHMB complex as a function of the pH of the
medium. The AF signal reported on the y-axis is considered proportional to the obtained
derivatization yields.
Complexed species strongly interacts with the stationary phase of the column and that
the derivatized thiolic groups are mainly located on the outside of the protein structure,
promoting the interaction between complexed pHMB and the silica stationary phase. The
pHMB itself elutes after the included volume of the column indicating a significant
interaction of pHMB with the SEC stationary phase Fig. 2 shows the SEC–CVG–AFS
[27] chromatogram of lysozyme–pHMB complex obtained in the optimized reaction
conditions.

15. 15
Fig2. AF chromatograms of lysozyme–pHMB complex in 0.1 mol L-1 PBS pH 6.8, 0.15
mol L-1 NaCl (line b red, AF signal in mV), UV absorbance chromatograms at 280 nm of
lysozyme–pHMB complex (line a black, UV signal in AU) and UV absorbance
chromatograms at 280 nm of uncomplexed lysozyme (line c blue, UV signal in AU).
Calibration for lysozyme was evaluated by analyzing three replicates of standard
lysozyme–pHMB solutions at 0.03–0.05– 0.1–0.2 µmol L-1 concentration levels of
lysozyme. The slope of the lysozyme–pHMB curve corresponds to 98±4% of the slope of
pHMB calibration curve multiplied for eight times. This is in agreement with the number
of -SH groups titrated in the protein. Fig. 3 shows the response surface obtained from the
optimization study.

16. 16
Fig3. Response surface obtained from a central composite experimental design; pH value
was varied between 12 and 14 and the reaction time between 1 and 4 h.
Possible role of pHMB in lysozyme complexation:
It is known that glutathione disulfide in strong alkaline media gets split according to
reaction (1) followed by reaction (2) [20].
GSSG + OH- G- (1)
2GSOH GSH + GSO2H (2)
Thus the complete stoichiometric reaction (3) is:
2GSSG + 4OH- 3GS- + GSO-2 + 2H2O (3)
According to the reaction 3 maximum yield of derivatization for GSSG was
supposed to be 75%, corresponding to the quantitative complexation of 3/4 of the total
thiolic groups. In this work a quantitative titration of the thiolic groups of lysozyme
yielded 98%. The reactions were hypothesized as:

17. 17
RSSS + OH- RS- + RSOH (4)
RS-+ Rʹ-HG+ R-S-Hg-Rʹ (5)
Organomercury reagent may be able to complex the sulfenate group as well as
the thiolic groups. In strong alkaline media we can advance the hypothesis that the
following reactions (6) and (7) take place:
RSOH + OH- RSO- + H2O (6)
RSO- + Rʹ _ Hg+ RSO _ Hg _ Rʹ (7)
The specificity of pHMB derivatization reactions in alkaline medium was also
investigated. These results confirm the hypothesis that the AF mercury specific signal
obtained in the chromatogram at 24 min (Fig. 2) is due only to the thiolic groups of
lysozyme complexed with pHMB and that the process involving the protein is due to
reaction (4).
Application to other proteins:
The reaction of alpha lactalbumin, beta lactoglobulin A, aprotinin, human serum
albumin and cytochrome c with pHMB in 0.3 mol L-1 NaOH under the same operating
conditions optimized for lysozyme reaction was studied by SEC–CVG–AFS. Table 1
shows the percentages of derivatization of disulfides and thiolic groups of proteins by
pHMB in alkaline medium and in neutral conditions calculated with respect to the
theoretical number of cysteine in the proteins sequence

18. 18
Table2. Percentage of derivatization of disulfides by pHMB in neutral and alkaline
solutions
All the proteins investigated the yield of derivatization of S-S bonds in alkaline medium
ranges between 75 and 93%, except for cytochrome c which is only 14 ± 3% because the
protein has a single disulfide bond and has a compact, “hard”, globular structure [21].
This likely makes difficult its derivatization by the organic probe, which cannot access
the site for complexation.
In summary, the strongly alkaline environment (i) hydrolyses disulfide bonds; (ii)
deprotonates thiolic and sulfenic acid groups formed and (iii) contributes to denature
protein's structure. With this method we can derivatise proteins disulfide bonds in one
step, without using chemical reducing agents and denaturing agents.

19. 19
Conclusion
Organomercury compounds with Hg-C bond or Hg-X undergo a variety of
reactions which makes them an important group in the organometallics. Organomercury
compounds are the most toxic forms of mercury. They are harmful to animal and human
health. Despite of this they are found have various applications like transmetallation
process for preparing other organometallic compounds, in pesticides, as catalysts and
pharmaceutics. In this review we have discussed the application of organomercury
compounds as analytical tool.
In the first method organomercury-based hapten and holds promise for
developing a highly sensitive immunoassay capable of detecting mercury contamination
in a variety of environmental conditions. This is due to the versatile oxymercuration
reaction to synthesize a stable, water-soluble organomercury hapten, resulting the
antibody that demonstrated high degrees of mercury binding under a variety of assay
formats and was capable of binding to both inorganic (Hg2
+) and organic forms of
mercury.
Second study showed how proteins disulfide bonds can be directly derivatized
by pHMB in strong alkaline medium. The reaction was studied for lysozyme as model
protein and the lysozyme–pHMB complex was determined by SEC–CVG–AFS.
Lysozyme was converted into lysozyme–pHMB complex with a yield of 98±2% in a
reaction time of 150 min and its concentration did not change during the working day.
The reaction has been successfully applied to other thiolic proteins with derivatization
yields of more than 75%. The method proposed is advantageous because it (i) decreases

20. 20
the number of steps necessary for the analysis, (ii) is cleaner as it avoids the addition of
reducing agents and (iii) does not suffer of cross-reactions with labeling agents or the
need of removing the chemical reduction agents before the protein derivatization step.

21. 21
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